17 in t e r n a l m e d ic in e issn 2413-6077. ijmmr 2019 vol. 5 issue 1 doi 10.11603/ijmmr.2413-6077.2019.1.10155 death due to cardiac angiosarcoma: autopsy case report f. eren, n.t. inanır, m.s. gurses, b. eren*, u.n. gundogmus, b. ioan tokat gaziosmanpasa university, tokat, turkey background. primary tumors of the heart are rarely detected at autopsy, especially angiosarcomas which are primary malignant one. objective. we presented autopsy case of cardic angiosarcoma with morphologic findings. methods. we described adult man died in emergency service of the hospital. results. reported case was 33 year-old-man who was died in emergency service of hospital where he was taken when he was ill after leaving home. according the prosecution documents, and the expressions of family, it was reported that he had a heart disease; his symptoms repeated 3 day ago before he died, he thought to attend the cardiology clinic due to his symptoms. at autopsy on macroscopic internal examination, mass with rough surface in the right atrium, hematoma at the posterior of the right atrium, blood in the pericardia, nodular lesions in hemorrhagic appearance in the sections of lung, liver and spleen were detected. in histopathologic examination; in the heart angiosarcioma as primary malign heart tumor and metastatic masses in the liver, spleen and lung were detected. conclusions. we aimed to discuss cardiac angiosarcoma case with autopsy and histopathologic findings in the aspect of medico legal literature. keywords: angiosarcomas; heart; metastatic tumors; autopsy. *corresponding author: bulent eren, md, pathologist, forensic medicine specialist, associate professor, tokat gaziosmanpaşa university school of medicine, department of forensic medicine, kaleardı mahallesi, 60030 tokat, turkey. phone: +903562149444 / 7206, e-mail: drbulenteren@gmail.com introduction primary tumors of heart are rare and they are revealed with prevalence between 0.0017 and 0.19 percent at autopsy [1]. while 75 percent of primary tumors of heart are benign, 25 percent are malign tumors [2]. angiosarcomas, which are 35-40 percent of primary malign tumors, are the most common [3, 4]. primary tumors are mostly developing in the right atrium and pericardium [5]. diagnosis of patients is delayed until tumors become untreatable and many systemic metastases are present [4, 6]. the research is aimed to study the autopsy and histopathologic findings of the case in medical and legal aspect. case report the case involves a 33 year-old-man reported to pass away in the emergency department of hospital, where he was admitted, when he got ill after leaving home. in the analysis of the prosecution documents and according to the words of family members, it was reported that he had a heart disease; chest pain repeated 3 days before he died, he thought to attend the cardiology clinic due to the symptoms. in autopsy external examination, there were no pathologic findings, except for ecchymosis due to catheters on the dorsum of the right and left hands and left inguinal line. in internal examination revealed mass on the rough surface of the right atrium (fig. 1), hematoma at the posterior of the right atrium, 500 cc free blood in pericardia, inhemorrhagic nodular lesions in the sections of lungs, liver and spleen (fig. 2). histopathologically, in sections of heart, tumoral proliferation of atypical cells, which had a high mitotic index and spindle-oval nucleus and infiltrates between myocardial fibers, were observed. there was diffuse hemorrhagic necrosis in the myocardial muscle fibers around tumoral tissue. in lungs, a group of atypical spindle tumoral cells, which had thin-walled vessels and were separated from lung parenchyma with irregular borders, were observed. in liver, many extravasated erythrocytes and tumoral lesions consisting of spindle cells were evidenced. in spleen, many extravasated erythrocytes and tumoral lesions consisting of spindle cells were present. the histopathologic examinationrevealed angiosarcioma in the heart as primary malign heart tumor and metastatic tumors in the liver, spleen and lung. the toxicological investigations were absolutely negative. the cause of death was reported as cardiac angiosarcoma. international journal of medicine and medical research 2019, volume 5, issue 1, p. 17-20 copyright © 2019, tnmu, all rights reserved f. eren et al. 18 in t e r n a l m e d ic in e issn 2413-6077. ijmmr 2019 vol. 5 issue 1 discussion primary tumors of the heart are rare and are revealed with prevalence between 0.0017 and 0.19 percent at autopsy [1]. approximately three-quarters of primary heart tumors are benign with atrial myxomas comprising threequarters of them [2]. angiosarcoma is the most common of malignant cardiac tumors [7]. cardiac angiosarcomas can be found in right atrium, left atrium, pulmonary artery, but most commonly in the right atrium [8], as in our case. angiosarcoma is established to be as twofold in men, typically between the third and fifth decade [4, 10] and our case is consistent with the literature. a study reported that a small series of heart sarcomas were subjected to a specific molecular study that proved the presence of k-ras mutations at codon 13 [11]. in human study mutation of the p53 tumor suppressor gene was present in cardiac angiosarcomas [12]. cardiac sarcomas are rare but lethal disease. it is associated with a poor prognosis and median survival time from 1 to 81 months [6]. angiosarcoma is associated with a high rate of hematogenous metastases, most of this tumors produce systemic metastases at the time of its detection, most commonly to the lungs, in addition to the liver, brain, adrenal glands, and bone [6, 5, 13]. in our case lung, liver metastases were present compliant with the literature. in addition, in our case there were splenic metastasis different from the literature. patients often have non-specific symptoms. so, it is difficult to diagnose cardiac angiosarcoma. initial findings may include chest pain, dyspnea, fatigue, cough, heart murmur of unclear mechanisms, superior vena cava syndrome, constitutional symptoms, pericardial effusion, pericardial tamponade, arrhythmias, non-specific st changes on ecg [6, 13, 14] echocardiography, magnetic resonance image (mri) and computed tomography (ct) are used for diagnosis and systematic evaluation of cardiac angiosarcoma [15, 16, 17]. echocardiography, transesophageal or transthoracic, is the main tool for describing the localization and size of a mass [18]. there is no specific tissue density that is why the diagnosis of angiosarcoma is challenging. the histological examination of fluid taken by pericardiosentesis, revealed malignant cells. the definitive diagnosis of angiosarcoma is biopsy [15, 16]. several studies established that infiltration of the myocardium by spindle cells, hyperchromatic nuclei, mitotic figures in various stages, giant cells, including tumor necrosis and hemorrhage in common areas, negative for cytokeratin, vimentin and fviii were revealed as positive staining by histopathological examination [6, 15, 16]. ge et al. reported that the tumor cells in cardiac angiosarcomas were strongly positive for cd31, cd34, fli-1, and wt-1 but negative for ae1/3, d2-40, human herpesvirus 8, and epidermal growth factor receptor by immunohistochemical method [6]. the rareness of this disease limits gaining experience for adequate treatment. the therapeutic approach for primary cardiac angiosarcoma is surgery, chemotherapy and radiotherapy, alone or in combination. the initial treatment is surgery [6, 19]. conclusions primary tumors of the heart are mostly originating in the right atrium and rarely observed at autopsy. angiosarcomas are most commonly seen primary malign tumors. we discussed cardiac angiosarcoma case with autopsy and histopathologic findings in the aspect of medico legal literature. conflict of interest the authors declare no conflict of interest. fig. 1. the arrow shows the mass on the rough surface of the right atrium (arrow). fig. 2. the arrow shows hemorrhagic nodular metastatic lesions in the sections of spleen (arrow). f. eren et al. 19 in t e r n a l m e d ic in e issn 2413-6077. ijmmr 2019 vol. 5 issue 1 раптова смерть внаслідок ангіосаркоми серця: результати аутопсії f. eren, n. turkmen, i.m. serdar gurses, b. eren, u.n. gundogmus, b. ioan tokat gaziosmanpasa university, tokat, turkey вступ. первинні пухлини серця вкрай рідко зустрічаються при аутопсіях, особливо ангіосаркоми, які належать до злоякісних новоутворів. мета роботи привернути увагу та ознайомити широке коло читачів з результатами аутопсії та морфологічними даними при ангіосаркомі серця. методи. описано та проаналізовано результати аутопсії дорослого чоловіка, який помер у відділенні невідкладної допомоги. клінічний випадок. 33-річний чоловік помер у відділенні невідкладної допомоги, куди був доставлений з дому. зі слів рідних у нього була хвороба серця, а перед смертю протягом трьох днів турбував біль у серці, з приводу якого він планував звернутися у лікарню. при аутопсії виявлено новоутвір з шорсткою поверхнею у правому передсерді, гематому на задній стінці правого передсердя, 500 мл крові у перикарді, також було виявлено вузлові утвори при розтині легень, печінки та селезінки. за результатами гістологічного дослідження встановлено діагноз ангіосаркоми серця, як первинної пухлини, з метастазами у легені, печінку та селезінку. висновки. описано рідкісний клінічний випадок ангіосаркоми серця, виявлений посмертно на аутопсії. ключові слова: ангіосаркома; серце; метастази; ауатопсія. information about the authors filiz eren – md,pathologist specialist, council of forensic medicine of turkey, bursa morgue department, bursa, turkey. orcid 0000-0003-1542-8694, e-mail: filizeren2010@gmail.com nursel türkmen i̇nanır – md, forensic medicine specialist, professor, uludağ university, medical faculty, forensic medicine department, bursa, turkey. orcid 0000-0002-4047-6455, e-mail: nursel_turkmen@hotmail.com murat serdar gürses – md, forensic medicine specialist, council of forensic medicine of turkey, bursa morgue department, bursa, turkey. orcid 0000-0002-9982-0476, e-mail: dr.muratgurses@gmail.com bülent eren – md, pathologist, forensic medicine specialist, associate professor, tokat gaziosmanpaşa university school of medicine, department of forensic medicine, tokat, turkey. orcid 0000-0002-8296-5484, e-mail: drbulenteren@gmail.com ümit naci gündoğmuş – md, i̇stanbul university, cerrahpaşa forensic medicine institute, forensic medicine department, i̇stanbul, turkey. orcid 0000-0001-7981-4725, e-mail: ugundogmus@yahoo.com beatrice ioan – md, forensic pathologist, professor, department of forensic medicine, grigore t.popa university of medicine and pharmacy, institute of forensic medicine, iasi, romania. orcid 0000-0002-0005-9139, e-mail: ioanbml@yahoo.com references 1. reynen k. cardiac myxomas. n engl j med. 1995;333(24):1610-7. doi: 10.1056/nejm199512143332407 2. centofanti p, di rosa e, deorsola l, dato gm, patanè f, la torre m, et al. primary cardiac tumors: early and late results of surgical treatment in 91 patients. ann thorac surg. 1999;68(4):1236-41. doi: 10.1016/s0003-4975(99)00700-6 3. klima u, wimmer-greinecker g, harringer w, mair r, grob ch, brucke p. cardiac angiosarcoma a diagnostic dilemma. cardiovasc surg 1993;674-6. 4. putnam jb, sweeney ms, colon r, lanza la, frazier oh, cooley da. primary cardiac sarcomas. ann thorac surg 1991;51:906-10. doi: 10.1016/0003-4975(91)91003-e 5. burke ap1, cowan d, virmani r. primary sarcomas of the heart. cancer. 1992 jan 15;69(2): 387-95. doi: 10.1002/1097-0142(19920115)69: 2<387::aidcncr2820690219>3.0.co;2-n 6. ge y, ro jy, kim d, kim ch, reardon mj, blackmon s, et al. clinicopathologic and immunohistochemical characteristics of adult primary cardiac angiosarcomas: analysis of 10 cases. ann diagn pathol. 2011;15(4):262-7. doi: 10.1016/j.anndiagpath.2011.02.007 7. simpson l, kumar sk, okuno sh, schaff hv, porrata lf, buckner jc, et al. malignant primary cardiac tumors: review of a single institution experience. cancer. 2008;112(11):2440-6 doi: 10.1002/cncr.23459 f. eren et al. 20 in t e r n a l m e d ic in e issn 2413-6077. ijmmr 2019 vol. 5 issue 1 8. blackmon sh, patel a, reardon mj. management of primary cardiac sarcomas. expert rev cardiovasc ther. 2008;6(9):1217-22. doi: 10.1586/14779072.6.9.1217 9. reardon, m.j. and smythe, w.r. cardiac neoplasms. in: cohn, l.h. and edmunds, jr. l.h., eds., cardiac surgery in the adult, mcgraw-hill, new york, 2003. 10. vander salm tj. unusual primary tumors of the heart. semin thorac cardiovasc surg. 2000;12:89100. doi: 10.1053/ct.2000.5080 11. garcia jm, gonzalez r, silva jm, dominguez g, vegazo is, gamallo c, et al. mutational status of k-ras and tp53 genes in primary sarcomas of the heart. br j cancer. 2000;82(6):1183-5. doi: 10.1054/bjoc.1999.1060 12. zu y, perle ma, yan z, liu j, kumar a, waisman j. chromosomal abnormalities and p53 gene mutation in a cardiac angiosarcoma. appl immunohistochem mol morphol 2001;9(1):24-8. doi: 10.1097/00022744-200103000-00006 13. kim js, song sg, ko ws, park yh, kim jh, chun kj, et al. a case of primary right atrial angiosarcoma manifested with cardiac tamponade. j korean soc echocardiogr. 2004;12:36-8. doi: 10.4250/jkse.2004.12.1.36 14. rha sw, shim wj, park sm, park sw, lim ds, kim yh, et al. a case of right atrial sarcoma complicated with hemopericardium and cardiac tamponade. j korean soc echocardiogr 2002;10:69-73. doi: 10.4250/jkse.2002.10.1.69 15. herrmann ma, shankerman ra, edwards wd, shub c, schaff hv. primary cardiacangiosarcoma: a clinicopathologic study of six cases. j thorac cardiovasc surg.1992;103(4):655-64. 16. terada t, nakanuma y, matsubara t, suematsu t. an autopsy case of primary angiosarcoma o f t h e p e r i c a r d i u m m i m i c k i n g m a l i g n a n t mesothelioma. acta pathol jpn. 1988;38(10):1345-51 doi: 10.1111/j.1440-1827.1988.tb02285.x 17. deetjen ag, conradi g, möllmann s, hamm cw, dill t. cardiac angiosarcoma diagnosed and characterized by cardiac magnetic resonance imaging. cardiol rev. 2006 mar-apr;14(2):101-3. doi: 10.1097/01.crd.0000174802.61576.5e 18. meng q, lai h, lima j, tong w, qian y, lai s. echocardiographic and pathologic characteristics of primary cardiac tumors: a study of 149 patients. int j cardiol. 2002 jul;84(1):69-75. doi: 10.1016/s0167-5273(02)00136-5 19. erpolat op, icli f, dogan ov, gokaslan g, akmansu m, erekul s, et al. primary cardiac angiosarcoma: a case report. tumori. 2008 novdec;94(6):892-7. doi: 10.1177/030089160809400624 received 26 may 2019; revised 04 june 2019; accepted 15 june 2019. this is an open access article distributed under the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. f. eren et al. 26 p e d ia t r ic s issn 2413-6077. ijmmr 2021 vol. 7 issue 2 doi 10.11603/ijmmr.2413-6077.2021.2.12474 vitamin d3 in treatment of osteopenic syndrome in children with growth disorders n.yu. shcherbatiuk i. horbachevsky ternopil national medical university, ternopil, ukraine background. the efficacy of vitamin d3 agents for impaired growth of children of different cause was studied in the research. a positive effect on bone mineralization and calcium-phosphorus metabolism was evidenced. the treatment and prophylaxis charts using this drug for osteopenic syndrome are developed for children with hypothalamic-pituitary dwarfism, subdwarfism and dwarfism of constitutional genesis. objective. the aim of the research is to study the effect of calcimine and vitamin d3 in cases of impaired bone mineral density in children aged 6-18 years old with growth retardation of the hypothalamic-pituitary genesis, subdwarfism and dwarfism of constitutional origin. methods. the study involved 25 children, aged 6-18 years old with growth retardation: hypothalamic-pituitary (10 children), subdwarfism (5 children) and dwarfism of constitutional origin (10 children). the control group consisted of 20 children. the structural and functional state of bone tissue was studied using a two-photon x-ray densitometer “lunar”. in the study of calcium-phosphorus homeostasis, the level of calcium and the level of phosphorus in the blood serum were determined. serum alkaline phosphatase levels were also studied as a marker for bone formation. results. in the examined children, there was a significant calcium deficiency at the level of the vertebrae l1–l4. the calcium content in l1 was 72.8%, l2 – 75.7%, l3 – 81.2%, l4 – 80.1%, which significantly differed from bone density in healthy children of a similar age and body weight. it was also revealed that in children diagnosed with growth retardation of the pituitary genesis, bone rarefaction was diagnosed in 100% of cases, while with subdwarfism and dwarfism of constitutional genesis these indicators were 43% and 24%, respectively. conclusions. a differentiated choice of vitamin d3 treatment, depending on the degree of osteopenia in children, allows adjusting the calcium metabolism and preventing osteoporosis in children with stunted growth. vitamin d3 in the complex treatment of osteoporosis in children with growth impairment of different genesis normalizes calcium-phosphorus homeostasis, which increases bone mineral density and, therefore, reduces osteoporotic changes by stopping leaching of calcium from bones. keywords: bone mineralization; calcium-phosphorus metabolism; dwarfism; vitamin d3. n.yu. shcherbatiuk international journal of medicine and medical research 2021, volume 7, issue 2, p. 26-29 copyright © 2021, tnmu, all rights reserved *corresponding author: shchebatiuk nataliia, associate professor, i. horbachevsky ternopil national medical university, ternopil, ukraine. e-mail: sherbatyuk_nu@tdmu.edu.ua introduction violation of the density and structure of bone tissue in childhood is caused by damage to the hypothalamic-pituitary system. normally, restructuring of bone tissue is characterized by the advantage of bone formation over resorption until reaching the “peak of bone mass”. then insufficient somatotropic function of the pituitary gland causes delayed bone development: ossification nuclei develop with a significant delay and growth zones are open for a long time or close only in adulthood [1, 2]. this causes changes in bone density and impaired mineral metabolism in children with growth retardation. the situation is aggravated by the fact that the average alimentary daily vitamin d3 supply for children is more than 1.5 times less than required. to a large extent, this is due to the insufficient amount of foods containing calcium in the diet of children, socioeconomic factors, as well as diverse information about the norms of daily calcium intake. however, endocrinological disorders accompanying growth retardation and a decrease in bone density with impaired mineral metabolism are crucial in this metabolic imbalance. the non-controllable intake of medications also affects, above all the complexes of micro elements in the multivitamin compounds or individual preparations, i.e. magnesium, as its increase in the blood causes decreased calcium absorption. environmental factors are also important, for example, strontium in the ter27 p e d ia t r ic s issn 2413-6077. ijmmr 2021 vol. 7 issue 2 ritories contaminated with radionuclides, entering the child’s body acts as a calcium antagonist. iodine deficiency territories also have negative impact not only in the formation of calcium-phosphorus homeostasis, but also in the disharmony of physical development, in particular, delayed sexual development, which due to hormonal imbalance affects the level of calcium absorption and osteogenesis. the aim of the study was to study the effect of vitamin d3 on changes in bone mineral density in children with growth retardation of hypothalamic­pituitary genesis, subdwarfism and dwarfism of constitutional genesis. methods 25 children aged 6-18 years old with growth retardation of hypothalamic-pituitary genesis (10 children), subdwarfism (5) and dwarfism of constitutional genesis (10) were exemined. the control group consisted of 20 children. when collecting a history of children with osteoporosis it was established that glucocorticosteroids, anticonvulsants, chemotherapeutic substances, antibiotics (tetracyclines, cyclosporins), antacids were administered. there were also: prema­ turity, fetal hypoxia, malnutrition, placental pathology, multiple pregnancy, short time between births, chronic diseases of women, drugs and alcohol, smoking during childbirth. the study of the structural and functional state of the bone tissue was carried out using a twophoton x-ray densitometer “lunar”. the following parameters were used for the study: age matched, % – the percentage deviation of bone mineral density (bmd) in the patient, the average population indicator of the identical race, sex and age at the level of the lumbar vertebrae l1–l4; age matched, z-criterion – standardized deviation from the same indicator. when analyzing the results obtained, we used reference data on the indicators of the structural and functional state of the skeletal system in children and adolescents according to v.v. povoroznyuka et al. statistical analysis was performed using the microsoft excel and statistica 5.0 software packages. in the study of calcium-phosphorus homeostasis, the level of calcium in the blood serum was determined using titrometric and photometric methods; the level of phosphorus in the blood serum was determined using a unified method for the reduction of phosphorus. the level of alkaline phosphatase in blood serum as a marker of bone formation was also studied by a biochemical method. results in the examined children, there was a significant calcium deficiency at the level of the l1–l4 vertebrae. the calcium content in l1 was 72.8%, l2 – 75.7%, l3 – 81.2%, l4 – 80.1%, which significantly differed from bone density in healthy children of a similar age and body weight. it was also revealed that in children diagnosed with growth retardation of pituitary genesis bone loss was diagnosed in 100% of cases, while with subdwarfism and dwarfism of constitutional genesis these indicators were 43% and 24%, respectively, according to the deviation (standardized deviation of the bone tissue strength index of the average population index of the z­criterion) and the classification of osteopenia and osteoporosis in children and adolescents by a.p. krys-pugach [3]. three degrees of osteopenia were identified at the l1–l4 vertebrae. it was at the first step 1.0­1.5; the second and third, respectively, 1.6-2.0; 2.12.5. these children had a tendency to hypocalcemia and to increase in alkaline phosphatase levels. thus, the content of the trace element calcium in the blood was reduced and fluctuated within 1.76-2.09 mmol/l. the analysis of the level of alkaline phosphatase in the blood as a marker of bone formation showed that its content was 1.54±0.09 μmol/g/l that also in­ dicated bone loss. when comparing the activity of alkaline phosphatase and the concentration of calcium in the blood serum, it was assumed that hypocalcemia under such conditions contribute to further leaching of calcium from the bone tissue. therefore, creating a vicious circle, in the treatment of such children in addition to etiotropic hormonal treatment, drugs should be used for prevention of leaching of the calcium from the bones before balancing the composition of the diet, which is consistent with age-related requirements for phosphorus and calcium. vitamin d3 in the form of the drug of cholecalciferol increases the absorption of calcium in the intestine [4] and improves the reabsorption of phosphorus in the renal tubules, helps to maintain the proper level of calcium and phosphorus in the blood, helps to optimize calcium-phosphorus homeostasis and contributes to the normal formation and growth of bone tissue that is especially important for stunted growth in children. for grade 1 osteopenia, cholecalciferol was prescribed in courses of 2000 iu per day for 30 days, 2 times a year with an interval of 5 months between courses. in case of degree 2 osteopenia, cholecalciferol was prescribed in courses of n.yu. shcherbatiuk 28 p e d ia t r ic s issn 2413-6077. ijmmr 2021 vol. 7 issue 2 4000 iu per day for 40 days, 3 times a year with a break of 3 months between courses. in case of degree 3 osteopenia, cholecalciferol was prescribed in courses of 5000 iu per day for 45 days, 3 times a year with a break of 3 months between courses. the effectiveness of treatment was assessed by repeated densitometric examination of the lumbar spine, 3 months after the first course of treatment, analyzing the z-criterion. there was a tendency to an increase in the density of mineral tissue according to the z-score by 4.34-20% in each of the lumbar vertebrae. the effectiveness of the treatment was assessed by the parameters of the content of calcium and phosphorus in the blood serum, the level of alkaline phosphatase during the second examination one month after the first course of treatment. thus, normalization of the level of calcium and phosphorus, as well as a decrease in the level of alkaline phosphatase to the upper limit of the norm was evidenced (table 1). table 1. dynamics of blood biochemical parameters in a group of 25 children (m±m) after the introduction of cholecalciferol into treatment index before treatmentn=25 after treatment n=25 p serum calcium (mmol/l) 1.98±0.11 2.37±0.13 <0.05 serum phosphorus (mmol/l) 0.93±0.05 1.1±0.06 <0.05 discussion the results of the study proved a positive effect of vitamin d3 in the children with decrease of growth of different genesis. according to the literature, growth hormone is one of the most important components of the hormonal system for regulating bone tissue metabolism and phosphorus-calcium metabolism. in norm the restructuring of bone tissue is characterized by the advantage of bone formation over resorption until reaching the “peak of bone mass”. with insufficient somatotropic function of the pituitary gland bone development is delayed: ossification nuclei appear with a sig­ nificant delay, and growth zones remain open for a long time or close only in adulthood. this causes changes in bone density and impaired mineral metabolism in children with growth retardation [1,2]. cholecalciferol normalized the level of calcium and phosphorus as well as decreased the level of alkaline phosphatase to the upper limit of the norm that prevents osteoporosis and complies the results of studies by other authors [5, 6]. conclusions the differentiated approach in prescribing cholecalciferol depending on the degree of osteopenia in children allows correcting calcium metabolism and thereby ensures prevention of osteoporosis in children with stunted growth. this is caused by the pharmacological action of cholecalciferol, i.e. regulation of calcium-phosphorus metabolism, normal formation of the bone skeleton and teeth in children and preservation of the structure of bones. conflicts of interest author declare no conflict of interest застосування вітаміну d в лікуванні остеопорозу у дітей з затримкою росту різного генезу н.ю. щербатюк тернопільський національний медичний університет імені і. я. горбачевського моз україни, тернопіль, україна вступ. у статті наводяться результати застосування препаратів вітаміну d у дітей із затримкою росту різного ґенезу. продемонстровано їх позитивний вплив на мінеральну щільність кісткової тканини та кальцій-фосфорний гомеостаз. наводяться схеми лікування та профілактики остеопенічного синдрому у дітей з гіпоталамо-гіпофізарним нанізмом, субнанізмом та нанізмом аліментарноконституційного ґенезу. доведено ефективність їх використання. n.yu. shcherbatiuk 29 p e d ia t r ic s issn 2413-6077. ijmmr 2021 vol. 7 issue 2 мета – дослідження впливу використання вітаміну d при змінах мінеральної щільності кісткової тканини у дітей 6-18 років із затримкою росту гіпоталамо-гіпофізарного генезу, субнанізмом та нанізмом конституційного ґенезу. методи. обстежено 25 дітей 6-18 років із затримкою росту гіпоталамо-гіпофізарного ґенезу (10 дітей), субнанізмом (5) та нанізмом конституційного ґенезу (10). контрольну групу становили 20 дітей. дослідження структурно-функціонального стану кісткової тканини проводили за допомогою рентгенівського двофотонного денситометра "lunar". при дослідженні кальцій-фосфорного гомеостазу визначали рівень кальцію та рівень фосфору у сироватці крові. також вивчали рівень лужної фосфатази у сироватці крові як маркера формування кістки. результати. у обстежених дітей мав місце суттєвий дефіцит кальцію на рівні хребців l1–l4. вміст кальцію в l1 становив – 72,8%, l2 – 75,7%, l3 – 81,2%, l4 – 80,1%, що суттєво відрізняється від кісткової щільності у здорових дітей аналогічного віку та маси тіла. також було виявлено, що у дітей, яким було ви ставлено діагноз затримки зростання гіпофізарного ґенезу у 100% випадків діагностовано розрідження кісткової тканини, тоді як при субнанізмі та нанізмі конституційного ґенезу ці показники становили відповідно 43% та 24%. висновки. диференційований підхід у призначенні вітаміну d залежно від ступеня остеопенії у дітей дозволяє скоригувати кальцієвий обмін та забезпечити тим самим профілактику остеопорозу у дітей із затримкою росту. включення вітаміну d у комплексне лікування остеопорозу у дітей із затримкою росту різного генезу нормалізує кальцій-фосфорний гомеостаз, що сприяє посиленню мінеральної щільності кісткової тканини, а отже, зменшує остеопорозні зміни за рахунок припинення вимивання кальцію з кісток. ключові слова: мінеральна щільність кісткової тканини; кальцій-фосфорний гомеостаз; затримка росту; вітамін d3. information about the authors nataliia yu. shchebatiuk – phd, associate professor, i. horbachevsky ternopil national medical university, ternopil, ukraine. orcid: 0000­0003­2155­7329. e­mail: sherbatyuk_nu@tdmu.edu.ua references 1. golounina oo, runova ge, fadeyev vv. osteomalacia in practice of endocrinologist: etiology, pathogenesis, differential diagnosis with osteo porosis. osteoporosis and bone diseases. 2019;22(2):23­ 31. [in russian] https://doi.org/10.14341/osteo12117 2. bilezikian jp, bouillon r, clemens t, et al, eds. primer on the metabolic bone diseases and disorders of mineral metabolism. 1st ed. wiley; 2018. https://doi.org/10.1002/9781119266594 3. kris-pugach ap, kinchaya-polishchuk ta, gayko og. violation of the density and structure of bone tissue in childhood and adolescence. problems of osteology. 2002;3;22-5. 4. thakker rv. rickets and osteomalacia. medicine. 2009;37(9):483­8. https://doi.org/10.1016/j. mpmed.2009.06.004 5. whyte mp, povoroznyuk vv. osteoporosis in the population of ukraine: risk factors, clinic, diag­ nosis, prevention and treatment: abstract. dis .... doctor. honey. sciences. k., 1998.47p. [in ukrainian] 6. reginato a j, coquia ja. musculoskeletal manifestations of osteomalacia and rickets. best pract res clin rheumatol. 2003;17(6):1063­80. https://doi.org/10.1016/j.berh.2003.09.0041. received 22 november 2021; revised 28 november 2021; accepted 8 december 2021. this is open access article distributed under the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. n.yu. shcherbatiuk 47 su r g ic a l d is e a se s issn 2413-6077. ijmmr 2021 vol. 7 issue 2 doi 10.11603/ijmmr.2413-6077.2021.2.12556 rehabilitation of patients after cardiac surgery t.v. romaniuk, v.s. moroz, s.m. maslii, z.v. vivchar i. horbachevsky ternopil national medical university, ternopil, ukraine background. patients undergoing cardiac surgery are under a high risk of post-operative reductions in respiratory muscle strength and pulmonary function as well as lowered functional capacity. in-hospital physical inactivity and inappropriate rehabilitation increases the chances for development of these complications and affects patients’ future independence in daily routine. cardiac rehabilitation is crucial in preventing complications and assisting the early function recovery. however, despite the evidences for its benefits and strong guideline recommendations, the uptake of cardiac rehabilitation remains poor. objective. this study is aimed at promotion of cardiac rehabilitation and sharing successful experience of using it. methods. post-surgical treatment and rehabilitation of 387 patients with cardiovascular pathology in the department of vascular and cardiac surgery of municipal non-commercial enterprise “ternopil regional clinical hospital” of ternopil regional council has been analysed. results. the experience of successful post-surgical treatment and rehabilitation allowed establishing the basic approaches to perioperative management of patients with cardiovascular surgical pathology. key principles include minimized icu stay and artificial ventilation time, early verticalization and beginning of physical activities, early removed drainage tubes, tracheobronchial tree drainage, nutritional balance, lymphatic drainage massage, application of elastic jersey on the lower extremities, early transferring to the surgery ward and returning to the regular daily activities, circumstantial health education, complex work of a multidisciplinary team. conclusions. this set of rehabilitation measures helps prevent complications after cardiac surgery as well as provide a faster patient’s daily routine. keywords: cardiac surgery; cardiac rehabilitation; exercise; recovery of function; health education. *corresponding author: taras romaniuk, associate professor of the department of surgery no.2, i. horbachevsky ternopil national medical university, ternopil, 46001, ukraine. e-mail: romaniuk_tv@tdmu.edu.ua international journal of medicine and medical research 2021, volume 7, issue 2, p. 47-50 copyright © 2021, tnmu, all rights reserved t.v. romaniuk et al. introduction the increasing prevalence of risk factors such as age, obesity, diabetes, hypertension and dyslipidaemia in patients undergoing cardiac surgery causes a significant rise of possible complications [1]. those patients commonly experience reductions in respiratory muscle strength and pulmonary function [2-3]. these complications lead to longer hospital stay, higher rehospitalisation risk, reduced health-related quality of life and higher costs for healthcare [4-6]. also, makeable reduction in functional capacity can occur, which only gets worse if patients spend the majority of their time sitting or in a supine position due to the inappropriate care [7-8]. in-hospital physical inactivity causes muscle weakness and aerobic capacity decrease, which can seriously affect patients’ independence in daily routine [9-10]. cardiac rehabilitation is crucial in prevention of these complications and assisting the early function recovery [11]. cardiac rehabilitation is a complex intervention that includes postoperative management, exercise training, physical activity promotion, health education and psychological support [12]. special em phasis in the latest guidelines is made on involving a multidisciplinary team to the rehabilitation complex [13]. in addition to cardiac surgeons and icu anaesthesiologists it should include cardiologists, nursing specialists, physiotherapists, nutritionists and psychologists, trained in the core competencies of a comprehensive cardiac rehabilitation programme. however, despite the evidence for its benefits and strong guideline recommendations, the uptake of cardiac rehabilitation is poor [14]. therefore, nowadays promotion of cardiac rehabilitation and sharing the successful experience in this field is essential, as the success of heart surgery depends not only on its type or the patient’s 48 su r g ic a l d is e a se s issn 2413-6077. ijmmr 2021 vol. 7 issue 2 health before the surgery, but also on proper rehabilitation, both in the early and late postoperative period. methods post-surgical treatment and rehabilitation of 387 patients with cardiovascular pathology in the department of vascular and cardiac surgery of municipal non-commercial enterprise “ternopil regional clinical hospital” of ternopil regional council has been analysed in this study. results the experience of successful post-surgical treatment and rehabilitation allowed establishing the basic approaches to perioperative management of patients with cardiovascular surgical pathology. we are supporters of the fast and early activation concept or “fast track”. during the icu stay we adhere to the following principles: time of intubation and artificial ventilation is minimized; physical activity usually begins 12-15 hours after the surgery. the patients are verticalized – helped to sit and stand under doctor’s supervision. at the same time, we help patients to perform a small walk on place. this improves blood flow to the right heart chambers. walking also helps to restore motor function of the intestine. verticalization of the patient helps to restore vascular tone, and is also one of the mechanisms of hypertension correction. tracheobronchial tree drainage is performed by hyperventilation of the patient, as well as by forced coughing along with vibrating chest massage. this improves oxygenation and pulmonary function. after the first activity session the stability of haemostasis in the chest is checked and if no signs of bleeding are observed, drainage tubes are immediately removed. delay in this can lead to the patient’s prolonged icu stay, immobilization in bed, respiratory function limitations and development of arrhythmias caused by irritation of the heart reflex zones. one more vital thing is the nutritional balance. thus, before the operation, the patient’s diet includes high-calorie food rich in protein, vitamins and microelements that provide a good “pool” for rapid recovery of the patient in the post-operative period and high reparative potential for wound healing. in the postoperative period along with the restoration of peristalsis, the patient begins to drink, and after a short time to eat liquid, easily digestible food. this contributes to the full recovery of the gastrointestinal tract and improves the overall psychoemotional state of the patient. lymphatic drainage massage, application of elastic jersey on the lower extremities has a good therapeutic effect on the swelling caused by congestive heart failure. if no major complications take place by the middle of the first post­operation day the pa­ tient is transferred from the icu to the surgery ward. it also helps motivate our patients for early returning to their regular daily activities. they are trained to do exercises and are informed about all the restrictions for protection of the breastbone during physical activities and sleep time. also, we provide some health education consultations in order to inform the patients about their state and the following rehabilitation programme, which may last up to 6 months. discussion the efficacy and safety of complex cardiac rehabilitation in patients provided with different kinds of surgical procedures was evaluated in the study. a short-term impact of different procedures on patients might differ significantly, as valvular surgery, cabg, surgery on thoracic aorta and their various combinations are provided in our clinic. post-isolated cabg patients might therefore respond differently to the rehabilitation complex compare to those who underwent a complicated combined surgical procedure. we tried to develop a unified complex of rehabilitation measures according to the international guidelines and our own experience. not separating and analyzing patients as separate groups might therefore be a limitation to this study. while current guidelines of the european society of cardiology [15] emphasize mainly on exercise-based cardiac rehabilitation after heart surgery, our findings coincide more with the cochrane systematic review by abraham et al., who recognized that rehabilitation interventions complex may also need to include breathing and coughing exercises and vocational evaluation advice in addition to the physical exercises [16]. conclusions key principles include minimized icu stay and artificial ventilation duration, early ver­ ticalization and beginning of physical activities, early removed drainage tubes, tracheobronchial tree drainage, nutritional balance, lymphatic drainage massage, application of elastic jersey t.v. romaniuk et al. 49 su r g ic a l d is e a se s issn 2413-6077. ijmmr 2021 vol. 7 issue 2 on the lower extremities, early transferring to the surgery ward and returning to the regular daily activities, circumstantial health education, complex work of a multidisciplinary team. this set of rehabilitation measures helps prevent complications after cardiac surgery as well as provide faster patient’s daily routine. conflict of interests authors declare no conflict of interests. acknowledgements this paper and the research behind it would not have been possible without the everyday great dedication and hard work of a great multidisciplinary team of the department of vascular and cardiac surgery of municipal noncommercial enterprise “ternopil regional clinical hospital” of ternopil regional council. author's contributions volodymyr moroz, taras romaniuk – conceptualization, methodology, formal analysis, writing – original draft, writing – reviewing and editing; sofiia maslii, zoriana vivchar – data curation, writing – reviewing and editing, investigation, formal analysis. особливості реабілітації пацієнтів після кардіохірургічної операції т.в. романюк, в.с. мороз, с.м. маслій, з.в. вівчар тернопільський національний медичний університет імені і. я. горбачевського моз україни, тернопіль, україна вступ. пацієнти, які перенесли операцію на серці, мають високий ризик післяопераційного зниження сили дихальних м’язів і функції легень, а також зниження функціональних резервів. відсутність належної фізичної активності у ранньому післяопераційному періоді та невідповідна реабілітація збільшують ймовірність розвитку цих ускладнень і впливають на майбутні можливості пацієнтів щодо їх повсякденної діяльності. вирішальну роль у попередженні ускладнень та сприянні ранньому відновленню функцій організму відіграє кардіологічна реабілітація. однак, незважаючи на її доказові переваги і сильні рекомендації світових гайдлайнів, якість кардіологічної реабілітації залишається слабкою. мета. метою цієї роботи є популяризація кардіологічної реабілітації та обмін успішним досвідом у цій сфері. методи. проаналізовано післяопераційне лікування та реабілітацію 387 пацієнтів із кардіохірургічною патологією відділення судинної хірургії з кардіохірургією комунального некомерційного підприємства «тернопільська обласна клінічна лікарня» тернопільської обласної ради. результати. проведений аналіз успішного досвіду післяопераційного лікування та реабілітації пацієнтів із кардіохірургічною патологією дозволив нам встановити основні принципи їх периопераційного ведення. вони включають мінімізацію часу штучної вентиляції легень та перебування у реанімаційному відділенні, ранню вертикалізацію та початок фізичних навантажень, раннє видалення дренажних трубок, дренаж трахеобронхіального дерева, збереження нутрітивного балансу, лімфодренажний масаж, накладення еластичного трикотажу на нижні кінцівки, раннє переведення в палату хірургічного відділення та повернення до звичайних повсякденних справ, ретельне навчання пацієнтів, комплексна робота багатопрофільної команди. висновки. цей комплекс реабілітаційних заходів допомагає запобігти ускладненням після кардіохірургічних операцій, а також забезпечити швидке повернення пацієнта до нормального життя ключові слова: кардіохірургія; кардіологічна реабілітація; фізичні вправи; відновлення функції; медична освіта. information about the authors taras v. romaniuk – associate professor of the department of surgery no. 2, i. horba chevsky ternopil national medical university, ternopil, ukraine. orcid 0000­0001­9279­3711, e­mail: romaniuk_tv@tdmu.edu.ua volodymyr s. moroz – phd student of the department of surgery no. 2, i. horbachevsky ternopil national medical university, ternopil, ukraine. orcid 0000­0001­6880­5110, e­mail: moroz_vs@tdmu.edu.ua sofia m. maslii – assistant professor of the department of internal medicine no. 3, i. horbachevsky ternopil national medical university, ternopil, ukraine. orcid 0000­0001­5849­7791, e­mail: maslii@tdmu.edu.ua t.v. romaniuk et al. 50 su r g ic a l d is e a se s issn 2413-6077. ijmmr 2021 vol. 7 issue 2 references 1. hartog j, blokzijl f, dijkstra s, dejongste m, reneman m, dieperink w et al. heart rehabilitation in patients awaiting open heart surgery targeting to prevent complications and to improve 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2019;l2191. https://doi.org/10.1136/bmj.l2191 13. ambrosetti m, abreu a, corrà u, davos c, hansen d, frederix i et al. secondary prevention through comprehensive cardiovascular rehabilitation: from knowledge to implementation. 2020 update. a position paper from the secondary prevention and rehabilitation section of the european association of preventive cardiology. european journal of preventive cardiology. 2020;28(5):460­495. https://doi.org/10.1177/2047487320913379 14. taylor r, dalal h, mcdonagh s. the role of cardiac rehabilitation in improving cardiovascular outcomes. nature reviews cardiology. 2021. https://doi.org/10.1038/s41569­021­00611­7 15. pelliccia a, sharma s, gati s, bäck m, börjesson m, caselli s, collet jp, corrado d, drezner ja, halle m, hansen d, heidbuchel h, myers j, niebauer j, papadakis m, piepoli mf, prescott e, roos-hesselink jw, graham stuart a, taylor rs, thompson pd, tiberi m, vanhees l, wilhelm m; esc scientific document group. 2020 esc guidelines on sports cardiology and exercise in patients with cardiovascular disease. eur heart j. 2021:1;42(1):17­96. https://doi.org/10.1093/eurheartj/ehaa605 16. abraham ln, sibilitz kl, berg sk, tang lh, risom ss, lindschou j, taylor rs, borregaard b, zwisler a-d. exercise-based cardiac rehabilitation for adults after heart valve surgery. cochrane database of systematic reviews. 2021:5. art. no.: cd010876. https://doi.org/10.1002/14651858.cd010876. pub3. received 16 november 2021; revised 29 november 2021; accepted 10 december 2021. this is open access article distributed under the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. zoriana v. vivchar – intern of the department of surgery, faculty of postgraduate education, i. horbachevsky ternopil national medical university, ternopil, ukraine. orcid 0000­0002­2609­1583, e­mail: vivchar_zovo@tdmu.edu.ua t.v. romaniuk et al. 15 in t e r n a l m e d ic in e issn 2413-6077. ijmmr 2019 vol. 5 issue 2 doi 10.11603/ijmmr.2413-6077.2019.2.10456 primary laryngeal aspergillosis in an immunocompetent patient (case report) k.s. rajmohan1, *i.d. khan1, u. kapoor2, s.a. hashmi1, r.m. gupta3, s. sen3, g.l. nair1, k.k. singh2, k. tandel4, m.malik4 1 – army college of medical sciences and base hospital, new delhi, india 2 – command hospital (nc), udhampur, india 3 – armed forces medical college, pune, india 4 – army hospital research and referral, new delhi, india background. aspergillus is an inherently ubiquitous, weakly pathogenic fungus causing opportunistic infections. it is very rarely localized in the larynx, although laryngeal aspergillosis may develop in the immunocompromised patients including those with leukaemia and severe aplastic anaemia. objective. the aim of the research was to explore the primary laryngeal aspergillosis in an immunocompetent patient thru a case report. methods. a case report of primary laryngeal aspergillosis in an immunocompetent patient is presented. results. a male patient of 40 years old, presenting with chronic worsening hoarseness, was found to have a smooth, white spheroid submucosal growth on left vocal cord with preserved bilateral cord movements on videostroboscopy. histopathological examination of vocal cord growth revealed squamous epithelium containing septate hyphae with acute angle dichotomous branching pattern consistent with aspergillus. voice improved after a four-week course of oral itraconazole 200 mg/day. post therapy follow up of 24 months was unremarkable. conclusions. primary laryngeal aspergillosis develops in the immunocompetent patients. iatrogenic, vocal abuse, occupation and lifestyle factors may be contributory. optimal diagnosis and management mandates a high index of suspicion. key words: primary laryngeal aspergillosis; videostroboscopy. *corresponding author: dr. inam danish khan, associate professor, clinical microbiology and infectious diseases, army college of medical sciences and base hospital, new delhi 110010, india. e-mail: titan_afmc@yahoo.com introduction aspergillus is an inherently ubiquitous, weakly pathogenic fungus causing opportunistic infections [1]. it is very rarely localized in the larynx [2], although laryngeal aspergillosis may develop in the immunocompromised patients including those with leukaemia and severe aplastic anaemia [2-4]. current healthcare is witnessing a surge in emerging fungal infections due to multiple exposure to antimicrobials, which can further antimicrobial resistance [511]. invasive aspergillosis of glottic, subglottic and epiglottis have been described following steroid and radiation therapy [12-15]. there are diagnostic challenges in resource-limited facilities due to overlapping presentation [16, 17]. we report a case of primary laryngeal aspergillosis in an immunocompetent patient. case report a 40-year-old male patient presenting to the otolaryngology clinic (army college of medical sciences and base hospital, new delhi 110010, india) with complaints of chronic worsening hoarseness for two months. he was found to have a smooth, white spheroid submucosal growth on the anterior surface of left vocal cord with preserved bilateral cord movements on videostroboscopy. no history of vocal abuse, laryngeal trauma, sore throat, cough, dyspnea, fever or prolonged antimicrobial intake was present. there was no history of generalized immune deficiency, leukaemia, malig­ nant disease, diabetes mellitus or use of immunosuppressive drugs and corticosteroids. no history of diabetes, tuberculosis or malig nancy was present. neither history of tobacco abuse nor history of social drinking was pre sent. general, systemic examination and chest x-ray revealed insignificant changes. oral cavi ty, oro­ pharynx and neck were normal. hiv se ro logy was negative. hemoglobin was 13.5 gm/dl, total leucocytes 7800/mm3 with normal differential count. renal and liver function tests were in norm. histopathological examination of vocal cord growth revealed squamous epithelium containing septate hyphae with acute angle international journal of medicine and medical research 2019, volume 5, issue 2, p. 15-19 copyright © 2019, tnmu, all rights reserved k.s. rajmohan et al. 16 in t e r n a l m e d ic in e issn 2413-6077. ijmmr 2019 vol. 5 issue 2 dichotomous branching pattern consistent with aspergillus (fig. 1). the findings were seconded by pas (periodic acid-schiff) stain, grocott’s (grocott methenamine silver (gms)) stain and koh (potassium hydroxide) mount, wherein hyaline hyphae with narrow angle branching were seen. aspergillus fumigatus was isolated from the vocal cord tissue consecutively on sabouraud’s dextrose agar at 22 °c and 37 °c after incubation for 48 hours. lactophenol cotton blue tease mount from blue-green suede like surface revealed septate hyaline hyphae with columnar smooth walled uniseriate conidiophores bearing phialides and conidia over flask shaped vesicles. thermotolerance at 55 °c was observed. this was confirmed by amplification of internal transcribed spacer (its) of 5.8s rdna using its1 and its4 primers. blood cultures were negative. voice improved after a four-week course of oral itraconazole 200 mg/day. the patient was explained about good vocal practices and oral hygiene. post therapy follow up for 24 months revealed no significant changes. discussion approximately 50 cases of primary laryngeal aspergillosis have been reported, half of them involved immunocompetent patients [18-24]. aspergillus is non-pathogenic, or very weakly pathogenic, and causes opportunistic infections. aspergillosis occurs due to deficient host's defense rather than fungal pathogenicity [21]. isolated laryngeal aspergillosis may follow colonization of larynx, which may be furthered, by local factors, rather than systemic immu nosuppression. further, systemic immunodeficiency may not contribute to the development of isolated laryngeal aspergillosis. iatrogenic factors such as radiation therapy, inhaled steroids and laser treatment may, also, be contributory [12-15]. vocal abuse and oral sex may impair local protective barrier provided by healthy mucosal covering and allow colonization and subsequent invasive aspergillosis [21]. laryngeal aspergillosis can even occur in a true vocal-fold cyst or laryngocoele. systemic factors include previous prolonged antimicrobial therapy, which is implicated in altering local flora and disturbing the ecological balance between bacteria and fungi, thus allowing the growth of aspergillus. occupation, avocation and lifestyle may be contributory to exposures in healthy patients. farmers and carpenters may be at higher risk as aspergillus is a soil saprophyte. aspergillosis had a shift in its host range as a higher incidence in males since the beginning of the 21st century has now turned to a higher incidence in females aged 20-40 years old [21]. opportunistic infections mandate clinical intrepidity, diagnostic efficiency, appropriate timely therapy and prognostication for favourable results [25-31]. primary aspergillosis is often reported late due to prolonged low-grade infection and delayed onset of clinical features. fungal growth in tissues may not corroborate to clinical presen tation leaving scope for further complications such as invasion of adjacent tissues, abscess development and dissemination, for which radical surgery and aggressive antifungal therapy may be required. fatal invasive aspergillosis of larynx has also been reported [32]. conclusions primary laryngeal aspergillosis develops in immunocompetent patients. iatrogenic, vocal abuse, occupation and lifestyle factors may be contributory. optimal diagnosis and management mandates high index of suspicion. funding this research received no external funding. conflict of interests the authors declare no conflict of interest. author contributions rajmohan k.s. – conceptualization, formal analysis, khan i.d. – conceptualization, formal analysis, investigation, writing – original draft, review & editing, kapoor u. – investigation, data curation, hashmi s.a. – conceptualization, gupta r.m. – writing – original draft, review & editing, sen s. – writing – original draft, review and editing, nair g.l. – data curation, singh k.k. – data curation, tandel k. – formal analysis, malik m. – formal analysis. fig. 1. photomicrograph h&e, x400. vocal cord growth. squamous epithelium containing septate hyphae with acute angle dichotomous branching pattern consistent with aspergillus. k.s. rajmohan et al. 17 in t e r n a l m e d ic in e issn 2413-6077. ijmmr 2019 vol. 5 issue 2 первинний аспергільоз гортані у імунокомпетентного пацієнта (клінічний випадок) k.s. rajmohan1, i.d. khan1, u. kapoor2, s.a. hashmi1, r.m. gupta3, s. sen3, g.l. nair1, k.k. singh2, k. tandel4, m.malik4 1 – army college of medical sciences and base hospital, new delhi, india 2 – command hospital (nc), udhampur, india 3 – armed forces medical college, pune, india 4 – army hospital research and referral, new delhi, india вступ. aspergillus – один з найпоширеніших видів грибів з низькою патогенністю, котрий може спричиняти опортуністичні інфекції. він надзвичайно рідко локалізується у гортані, однак може викликати аспергильоз гортані у осіб з ослабленою імунною системою, до прикладу при лейкемії чи апластичній анемії. мета. дослідити особливості перебігу первинного аспергильозу гортані у імунокомпетентного пацієнта на прикладі клінічного випадку. методи дослідження. описано та проаналізовано клінічний випадок первинного аспергильозу гортані у імунокомпетентного пацієнта. результати. 40-річний чоловік звернувся до лікаря зі скаргами на хронічну прогресуючу захриплість. при огляді виявлено гладкий, білого кольору, сфероїдної форми утвір на лівій голосовій зв’язці. рухливість зв’язок збережена білатерально, що підтверджено на відеостробоскопії. гістологічне дослідження утвору голосової зв’язки виявило септований міцелій грибів роду aspergillus (плоскоклітинний епітелій з септами з гострим кутом дихотомічного розгалуження, що відповідає аспергильозу). покращення голосу спостерігалося після чотирьохтижневого курсу ітраконазолу, перорально в дозі 200 мг/день. при обстеженні через 24 місяці після лікування жодних відхилень не виявлено. висновки. первинний аспергильоз гортані може розвиватися у імунокомпетентних пацієнтів. ятрогенні чинники, перевантаження голосових зв'язок, професійні шкідливі впливи та певний спосіб та стиль життя можуть сприяти його розвитку. необхідно бути настороженим щодо можливого розвитку таких опортуністичних інфекцій для їх успішного діагностування та лікування. ключові слова: первинний аспергильоз гортані; гістологічні зміни; відеостробоскопія. information about the authors dr. k.s. rajmohan, professor pathology, neuropathologist and head of department, army college of medical sciences and base hospital, new delhi 110010, india, orcid 0000-0003-0047-3265, e-mail drksrajmohan24@gmail.com dr. inam danish khan, associate professor, clinical microbiology and infectious diseases, army college of medical sciences and base hospital, new delhi 110010, india, orcid 0000-0002-9824-8711, e-mail titan_afmc@yahoo.com. dr. umesh kapoor, professor and head pathology, command hospital (nc), udhampur 182101, india orcid 0000­0003­1432­1254, e­mail drukapoor@gmail.com dr. syed asif hashmi, assistant professor pathology, army college of medical sciences and base hospital, new delhi 110010, india orcid 0000­0002­7432­9058, syedasifhashmi@gmail.com dr. alpana gupta, assistant professor pathology, army college of medical sciences and base hospital, new delhi 110010, india orcid 0000­0003­3335­9821, e­mail dralpanangupta@gmail.com dr. sourav sen, professor and head microbiology, armed forces medical college, pune 411040, india orcid 0000-0001-9117-2235 dr. gl nair, assistant professor pathology, army college of medical sciences and base hospital, new delhi 110010, india, orcid 0000-0002-2301-7302, e-mail lakshminairg@gmail.com dr. samresh kumar singh, pathologist and senior resident nephropathology, all india institute of medical sciences, new delhi, india orcid 0000­0002­8624­8052, e­mail drsksingh78@gmail.com dr. kundan tandel, assistant professor microbiology, army hospital research and referral, new delhi 110010, india, orcid 0000­0001­7478­2656, e­mail tandel.kundan@yahoo.co.in dr. muqtadir malik, resident microbiology, army hospital research and referral, new delhi 110010, india, orcid 0000-0003-2865-5773, e-mail drmuqtadirmalik@gmail.com k.s. rajmohan et al. 18 in t e r n a l m e d ic in e issn 2413-6077. ijmmr 2019 vol. 5 issue 2 references 1. agarwal p, yadav rp, upadhyay sn. a new method of detection and differentiation of pathogenic from non-pathogenic aspergillus species. journal of medical microbiology. 2001 jul;50(7):653­4. doi: 10.1099/0022­1317­50­7­653 2. athanassiadou f, kourti m, papageorgiou t, danielidis j. invasive 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in a teenaged renal transplant recipient. journal of basic and clinical medicine. 2015 jul 30;4(1):37­9. 29. khan id, lall m, sen s, ninawe sm, chandola p. multiresistant elizabethkingia meningoseptica infections in tertiary care. med j armed forces india. 2015 jul;71(3):282­6 doi: 10.1016/j.mjafi.2014.02.002 30. khan id, mukherjee t, gupta s, haleem s, sahni ak, banerjee s, konar j. ochrobactrum anthropic sepsis in intensive tertiary care. journal of basic and clinical medicine. 2014 nov 18;3(1):18­20. 31. khan id, sahni ak, bharadwaj r, anwar i, jain v, khan s, chowdhury a, dwivedi ak, gupta ak, zaman s, alam s. cerebral toxoplasmosis diagnosed by stereotactic brain biopsy leading to detection of hiv infection. journal of basic and clinical medicine. 2016 apr 5;5(1):5­7. 32. williams ms, ali n, nonaka d, bloor a j, somervaille tc. fatal invasive aspergillosis of the larynx. eur j haematol. 2013 apr;90(4):354. doi: 10.1111/ejh.12054 received 24 september 2019; revised 05 november 2019; accepted 15 december 2019. this is open access article distributed under the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. k.s. rajmohan et al. 68 in t e r n a l m e d ic in e issn 2413-6077. ijmmr 2021 vol. 7 issue 1 doi 10.11603/ijmmr.2413-6077.2021.1.12109 a cross sectional study of lipid profile in non-diabetics with stroke in urban chitradurga s.b. vijeth, *v. mangasuli, a.m. amrutha, n. bhoovanchandra, b. sidenur basaveshwara medical college and hospital, chitradurga, india background. the amount of evidence regarding the relation between serum lipids, lipoproteins and cerebrovascular accident is not adequate. the atherogenecity of diabetics and non-diabetics are different. therefore, non-diabetic patients were included in the study. objective. to study lipid abnormalities in non-diabetic stroke patients in our setup. methods. the study was carried out at the department of general medicine, bmch, chitradurga, during the period from june 2020 to december 2020. the lipid profile and the fasting blood sugar rates of 50 stroke patients without diabetes were studied. their serum samples were assessed for fasting blood glucose (fbg), total cholesterol (tc), triglycerides (tg), low density lipoprotein cholesterol (ldl) and high-density lipoprotein cholesterol (hdl) by using standard biochemical methods. results. the age distribution of the subjects was from 19 to 72 years with a mean age of patients 54.8±15.75 years. among patients 31 (62%) were males and 19 (38%) were females. among the study subjects 58% were hypertensive, 76% were smokers, 32% were alcoholics and 34% had family history of cerebrovascular accident. among ischemic stroke group, the most common deranged value in the ischemic group was decreased hdl deranged in 54.1% of patients; the second most common deranged value – increased vldl deranged in 40.5%. among the hemorrhagic group the most common deranged value was also decreased hdl, which was deranged in 46.1% of patients and the second most common deranged value – increased total cholesterol, which was deranged in 53.8% patients. conclusion. lipid profile should be considered while predicting the risk of stroke. keywords: lipid profile; dyslipidemia; non-diabetic stroke; serum cholesterol. international journal of medicine and medical research 2021, volume 7, issue 1, p. 68-73 copyright © 2021, tnmu, all rights reserved s.b. vijeth et al. *corresponding author: vijayalaxmi mangasuli, assistant professor, basaveshwara medical college and hospital, chitradurga, karnataka, 577501, india. e-mail: dr.vijugokak@ gmail.com introduction as per world health organization, stroke is defined as a clinical syndrome consisting of rapidly developing clinical signs of focal (at times global) disturbance of cerebral function, with duration lasting more than 24 hours or leading to death with no apparent cause other than that of vascular origin [1]. stroke is the second leading cause of death worldwide causing 6.2 million deaths in 2011 [2]. stroke is a medical emergency which is an acute neurological injury that occurs due to vascular pathology and presents as a brain infarction or haemorrhage. the modification of risk factors in stroke has brought down both mortality and morbidity of stroke remarkably in the last 30 years. dyslipidemia is a major risk factor for stroke. it has been established that reduction of total cholesterol, ldl cholesterol, trigycerides, vldl cholesterol and increasing hdl cholesterol by drugs decreases the incidence of stroke. in our study, lipid profile was studied in non-diabetic patients with stroke. diabetes itself is associated with hyperlipidemia and increased atherosclerosis which makes it an undisputed risk factor for stroke. the atherogenecity of diabetics and non-diabetics are different. with this background, we conducted the study of lipid profile in the non-diabetics with stroke in our setup. methods patients with stroke and non-diabetic attending opd and ipd of the department of general medicine of basaveshwara medical college and hospital, chitradurga, were enrolled in the study. patients with haemorrhagic strokes, embolic strokes, past/present h/o diabetes mellitus or history of head injury or usage of anti-coagulant drugs were excluded from the study. the study took place over the period of 6 months from june 2020 to december 2020. 69 in t e r n a l m e d ic in e issn 2413-6077. ijmmr 2021 vol. 7 issue 1 sample size estimation was performed using open epi software version 2.3.1 at 95% confidence level, 80% power of the study, proportion of cases with stroke with raised lipid parameters as 70% [3]. sample size estimated was 37 inflated to 50. a brief personal history and medical history, including brief histories about co-existing disease states, family history of diabetes mellitus, and history of hypertension, past and present illnesses, dietary pattern, addiction and medication were recorded in self prepared questionnaire. detailed general physical examination, systemic examination and neurological examination were performed to all the patients and were recorded. the investigations done for the patients at presentation included fasting lipid profile (serum total cholesterol, serum triglycerides, serum high density lipoproteins, serum very low density lipoproteins, serum low density lipoproteins) and hba1c levels [4]. data was entered in excel sheet and analysed using the statistical package for the social sciences 20 (spss inc. chicago). results were presented in tabular and graphical forms. mean, median, standard deviation and ranges were calculated for quantitative data. the chi square analysis was used in testing for significant differences between proportions and frequencies. the t-test was used in testing for significant differences between two means. the confidence interval was set at 95% limit, with level of significance, p≤0.05. results 50 patients were enrolled in the study. the age distribution of the subjects was between 19 to 72 years with mean age of patients 54.8±15.75 years. 31 (62%) were males and 19 (38%) were females. it was established that 58% of our study subjects were hypertensive, 76% were smokers, 32% were alcoholics and 34% had family history of cva (table 1). analysis of lipid profile among both the groups of stroke, i.e., hemorrhagic and ischemic stroke, revealed that the most common rate deranged in the ischemic group was decreased hdl, which is deranged in 54.1% of patients and the second most common rate deranged was increased vldl, which was deranged in 40.5%. among the hemorrhagic group the most common deranged rate was also decreased hdl, which was deranged in 46.1% of patients, and the second most common deranged rate was increased total cholesterol, which was deranged in 53.8% of patients (table 2). our study concluded a significant association between serum total cholesterol, triglyceride, ldl level, vldl level and risk of stroke. high levels of total cholesterol, trigly cerides, ldl cholesterol were associated with higher risk of stroke. lowered hdl cholesterol levels were not significantly associated with stroke. the ratio of hdl/ldl cholesterol, tc/hdl cholesterol for males and females was eva luated. however, the association with risk of stroke was not found (table 3). discussion association of total cholesterol to the non-diabetics with stroke our study involved 50 subjects and total cholesterol was elevated in the non-diabetics with stroke. the serum total cholesterol levels in cases of either ischaemic or haemorrhagic table 1. risk factors of stroke among study participants variables hemorrhagic stroke(n=13) ischemic stroke (n=37) chi-square test gender male 9 (69.2) 22 (59.5) 0.4 p=0.5female 4 (13.8) 15 (40.5) age in years <30 1 (7.6) 2 (5.4) 1.4 p=0.530-60 6 (46.2) 24 (64.9) >60 6 (46.2) 11 (29.7) hypertension present 9 (69.2) 20 (54.1) 0.9 p=0.3absent 4 (30.8) 17 (45.9) smoking yes 8 (61.5) 30 (81.1) 2.01 p=0.2no 5 (38.5) 7 (18.9) alcoholic yes 5 (38.5) 11 (29.7) 0.34 p=0.6no 8 (61.5) 26 (70.3) family h/o cva present 5 (38.5) 12 (32.4) 0.16 p=0.7absent 8 (61.5) 25 (67.6) s.b. vijeth et al. 70 in t e r n a l m e d ic in e issn 2413-6077. ijmmr 2021 vol. 7 issue 1 stroke were high (10%) (total cholesterol >240 mg% according to the adult treatment panel (atp) – the 3rd guidelines of national cholesterol education program (ncep)). similar studies by sreedhar k et al. [5] benfante et al. [6], di mascio et al. [7] showed both ischemic and hemorrhagic stroke were associated with increased cholesterol levels. contrary to our study, iso et al. [8] emphasized an inverse association between serum cholesterol level and hemorrhagic stroke. there was no correlation between serum cholesterol and risk of stroke in a study by harmsen et al. [9] rastenyte et al. [10] and hart cl et al. [11] association of triglycerides to the nondiabetics with stroke it was established that serum triglycerides were high in 23.1% of hemorrhagic stroke patients and 13.5% of ischemic stroke patients (>200 mg% according to atp – 3rd guidelines). sreedhar k et al. [5] in his study showed 80% of non-diabetic stroke patients with s.triglyceride >200mg/dl had ischemic stroke and the other 20% had hemorrhagic stroke. tilvis r.s et al. [12] in his study showed serum triglyceride was higher in ischemic stroke. farid et al. [13] also had similar results in his study in 1972. hachinski et al. [14] showed a positive association of triglycerides in patients with atherothrombotic stroke and transient ischemic attacks compare to the control subjects. albucher j.k et al. [15] 2000 showed serum triglycerides in normal range in the study on stroke. association of serum hdl cholesterol to the non-diabetics with stroke in the study of hdl cholesterol in stroke patients, it was found out that the ischemic group (54.1%) patients had greater abnormal levels (<40mg% according to atp – 3rd guidelines) than the haemorrhagic group (46.1%). simons et al. [16] study revealed that hdl cholesterol had protective effect on ischemic stroke. alok mohankar et al. [17] in 1993 showed that increased ldl levels and low hdl levels were associated with atherosclerosis. albucher et al. [15] study clearly indicated hdl – cholesterol as the only lipid associated with stroke risk. it emphasised the need for management of low hdl cholesterol in young patients regardless of atherosclerosis. table 2. lipid profile among cases and controls variables hemorrhagic stroke(n=13) ischemic stroke (n=37) chi-square test total cholesterol <200 7 (53.8) 24 (64.8) 0.6 p=0.5*200-240 5 (38.5) 9 (24.3) >240 1 (7.7) 4 (10.9) serum triglyceride <150 6 (46.1) 21 (56.8) 0.7 p=0.5*150-199 4 (30.8) 11 (29.7) 200-499 3 (23.1) 5 (13.5) ldl cholesterol <100 7 (53.8) 17 (45.9) 0.5 p=0.5*100-130 1 (7.7) 9 (24.4) 131-160 3 (23.1) 5 (13.5) >160 2 (15.4) 6 (16.2) hdl cholesterol <40 6 (46.1) 20 (54.1) 0.24 p=0.6≥40 7 (53.8) 17 (45.9) vldl cholesterol ≤30 6 (46.1) 22 (59.5) 0.7 p=0.4>30 7 (53.8) 15 (40.5) note. * – fisher’s exact test. table 3. comparison of lipid profile between types of stroke lipid component mean value in ischemic stroke mean value in hemorrhagic stroke p value tc 171.5 214.7 0.001 serum tg 138.2 163.7 0.018 hdl 43.4 45.8 0.822 ldl 100.5 136.1 0.004 vldl 27.6 32.7 0.174 s.b. vijeth et al. 71 in t e r n a l m e d ic in e issn 2413-6077. ijmmr 2021 vol. 7 issue 1 association of serum ldl cholesterol to the non-diabetics with stroke among ischemic stroke cases, 16.2% had high ldl cholesterol and this was 15.4% among haemorrhagic stroke cases. sreedhar k et al. [5] showed that high levels of serum ldl cholesterol had significant risk of ischemic stroke in the non-diabetics. botet et al. [18] and hachinski et al. [14] showed positive correlation between ldl cholesterol levels and risk of stroke. kurth t et al. [19] 2007 showed remarkable increase in serum ldl levels in ischemic stroke patients. association of serum vldl cholesterol to the non-diabetics with stroke in our study, among ischemic stroke cases 40.5% had increased vldl cholesterol and this was 53.8% among haemorrhagic stroke cases. bidyadhar et al. [20] 1984 showed that vldl was increased in their study on stroke. sreedhar k et al. [5] in the study showed that high vldl was not associated with risk of stroke in nondiabetic patients. conclusions according to our study the conclusion can be drawn that the most common type of lipid abnormality were abnormal triglycerides, abnormal vldl, abnormal ldl. so, these parameters should be considered while predicting the risk of stroke in a dyslipidemic patient. stroke patients with dyslipidemia need a comprehensive health care approach involving dietician, physician and good bio chemistry back up. in indian scenario, where majority of the patients belong to the low socio-economic status, life style modification plays a more important role in prevention and management of stroke and dyslipidemia in contrast to high cost of lipid lowering agents. conflict of interests authors declare no conflict of interest. author’s contributions dr. vijeth s.b., dr. vijayalaxmi mangasuli, dr. am rutha a.m., dr. bhoovanchandra n., dr. bhagyalaxmi sidenur – conceptualization, methodology, formal analysis, writing – original draft, writing – reviewing and editing; dr. vijeth s.b., dr. vijayalaxmi mangasuli, dr. amrutha a.m. – investigation, formal analysis. поперечне досілдження показників ліпідного профілю у пацієнтів-недіабетиків, котрі перенесли інсульт, міста читрадурга s.b. vijeth, *v. mangasuli, a.m. amrutha, n. bhoovanchandra, b. sidenur basaveshwara medical college and hospital, chitradurga, india вступ. зв’язок між рівнем сироваткових ліпідів, ліпопротеїнів та частотою розвитку порушень мозкового кровообігу не є достатньо доведеним. оскільки рівень атерогеності діабетиків та недіабетиків є різним, тому в дослідження були включені пацієнти без діабету. мета. вивчення порушень ліпідного обміну у пацієнтів з недіабетичним інсультом у місцевій лікарні. методи. дослідження проводилося у general medicine department, bmch, chitradurga протягом періоду з червня 2020 року по грудень 2020 року. вивчався ліпідний профіль та показники глікемії натще у 50 пацієнтів з інсультом без діабету. визначалися вміст глюкози крові натще (fbg), загального холестерину (tc), тригліцеридів (tg), ліпопротеїдів низької щільності (ldl) та ліпопротеїдів високої щільності (hdl) за допомогою стандартних біохімічних методів. результати. віковий розподіл пацієнтів складав від 19 до 72 років із середнім віком 54,8±15,75 років. чоловіки становили 62% (31), жінки – 38% (19). серед досліджуваних 58% мали гіпертонічну хворобу, 76% були курцями, 32% – алкоголіки, а 34% мали сімейний анамнез із порушенням мозкового кровообігу. серед групи ішемічного інсульту найчастіше спостерігалося зниження ліпопротеїдів високої щільності (54,1%), другим найпоширенішим відхиленням було збільшення ліпопротеїдів дуже низької щільності – у 40,5%. серед групи з геморагічним інсультом найчастіше спостерігалося також зниження ліпопротеїдів високої щільності, яке виявлялося у 46,1% пацієнтів, другим найпоширенішим відхиленням було збільшення загального холестерину, яке спостерігалося у 53,8% пацієнтів. висновки. під час прогнозування ризику інсульту слід враховувати ліпідний профіль. ключові слова: ліпідний профіль; дисліпідемія; недіабетичний інсульт; сироватковий холестерин. s.b. vijeth et al. 72 in t e r n a l m e d ic in e issn 2413-6077. ijmmr 2021 vol. 7 issue 1 references 1. world health organization. the world health report 2002: reducing risks, promoting healthy life. world health organization; 2002. 2. smith ws, johnston sc, hemphill iii jc. cerebrovascular diseases. in: kasper dl, hauser sl, jameson jl, fauci as, longo dl, loscalzo j eds. harrison’s principle of internal medicine. 20th ed. mcgraw hill; 2020:2559-86. 3. kar a, dash c, murmu m, kp km. study of lipid profile in cases of non-diabetic stroke. international journal of advances in medicine. 2018 jan 18;5(1): 82-90. doi: 10.18203/2349-3933.ijam20180063 4. nigam pk. serum lipid profile: fasting or nonfasting. indian j clin biochem 2011;26(1):96-7. doi: 10.1007%2fs12291-010-0095-x 5. sreedhar k, srikant b, joshi l, usha g. lipid profile in non-diabetic stroke-a study of 100 cases. j assoc physicians india. 2010;58:547-51. doi: 10.18203/2349-3933.ijam20180063 6. benfante r, yano k, hwang lj, curb jd, kagan a, ross w. elevated serum cholesterol is a risk factor for both coronary heart disease and thromboembolic stroke in hawaiian japanese men. implications of shared risk. stroke. 1994;25(4):814-20. doi: 10.1161/01.str.25.4.814 7. dimascio r, marchiolli r, vituollo f, dipasquale a, cavasinni l, tognoni g. serum cholesterol and risk of ischemic stroke: results of a case-control study. prev med. 1995;24(2):128-33. doi: 10.1006/pmed.1995.1025 8. iso h, jacobs dr, wentworth d, neaton jd, cohen jd, mrfit research group. serum cholesterol levels and six-year mortality from stroke in 350,977 men screened for the multiple risk factor intervention trial. n engl j med. 1989;320(14):904-10. doi: 10.1056/nejm198904063201405 9. harmsen p, rosengren a, tsipogianni a, wilhelmsen l. risk factors for stroke in middle-aged men in göteborg, sweden. stroke. 1990;21(2): 223-9. doi: 10.1161/01.str.21.2.223 10. rastenyte d, tuomilehto j, domarkiene s, cepaitis z, reklaitiene r. risk factors for death from stroke in middle-aged lithuanian men. stroke. 1996;27(4):672-6. doi: 10.1161/01.str.27.4.672 11. hart cl, hole dj, smith gd. risk factors and 20-year stroke mortality in men and women in the renfrew/paisley study in scotland. stroke. 1999; 30(10):1999-2007. doi: 10.1161/01.str.30.10.1999 12. tilvis rs erkinjunti j ,sulkava r et al.serum lipids and fatty acids in ischemic stroke.american heart journal1987;113;615-619. doi: 10.1161/01.str.26.5.778 13. farid n. r, anderson. j: cerebrovascular disease and hyper-lipoproteinemias. lancet 1, 1398; 1972. doi: 10.1016/s0140-6736(72)91136-1 14. hatchinski v graffagnino c,beaudry,bernier g, buck,donner a, spenced,doidg& wolfebmf1996. lipids and stroke a paradox resolved,arch neuro53; 303-308 15. albucherjf,ferrieros j et al.serum lipids in young patients with ischemic stroke,a case control study.j neurology neurosurgery psychiatry 2000; 69(1);29-33. doi: 10.1136/jnnp.69.1.29 16. simons la, mccallum j, friedlander y, simons j. risk factors for ischemic stroke: dubbo study of the elderly. stroke 1998;29:1341-1346. doi: 10.1161/01.str.29.7.1341 17. alok mohankar, ravindrakumar garg et al. serum lipids and stroke.neurology india 1993. information about the authors dr. vijeth s.b., associate professor, basaveshwara medical college and hospital, chitradurga, karnataka, india orcid: 0000-0002-6882-2604, e-mail: vijethsb71@gmail.com dr. vijayalaxmi mangasuli, assistant professor, basaveshwara medical college and hospital, chitradurga, karnataka, india orcid: 0000-0002-2861-2012, e-mail: dr.vijugokak@gmail.com dr. amrutha a.m., assistant professor, basaveshwara medical college and hospital, chitradurga, karnataka, india orcid: 0000-0003-4958-031x, e-mail: amrutha.angadi89@gmail.com dr bhoovanchandra n., postgraduate, basaveshwara medical college and hospital, chitradurga, karnataka, india orcid: 0000-0008-2112-3265, e-mail:bhoovan2@gmail.com dr bhagyalaxmi sidenur, assistant professor, basaveshwara medical college and hospital, chitradurga, karnataka, india orcid: 0000-0001-6012-3166, e-mail: bhagyasidenur@gmail.com s.b. vijeth et al. 73 in t e r n a l m e d ic in e issn 2413-6077. ijmmr 2021 vol. 7 issue 1 18. botet pj, senti m, nogues x, rubies-prat j, roquer j, d’olhaberriague l et al. lipoprotein and apolipoprotein profile in men with ischemic stroke: role of lipoprotein(a), triglyceride-rich lipoproteins, and apolipoprotein e polymorphism. stroke 1992; 23:1556-62. doi: 10.1161/01.str.23.11.1556 19. kurth t.everett et al,lipid levels and the risk of ischemic stroke in women.neurology 2007;68;556562. doi: 10.1212/01.wnl.0000254472.41810.0d 20. bidyadhar s et al. plasma lipids and lipoproteins in patients with premature ihd and icd. japi 1984;32;11. doi: 10.2337/diacare.27.6.1496 received 19 may 2021; revised 12 jun 2021; accepted 13 jun 2021. this is open access article distributed under the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. s.b. vijeth et al. issn 2413-6077. ijmmr 2021 vol. 7 issue 220 p u b l ic h e a lt h a n d e p id e m io l o g y r. kumar et al. doi 10.11603/ijmmr.2413-6077.2021.2.12423 epidemiology and seasonal variation of appendicitis – a single center retrospective study from north-east india *r. kumar1, t.b. singh2, r. pandey1, s. bhalla1, o. singh3 1 – base hospital tezpur, assam, india 2 – military hospital ambala, haryana, india 3 – command hospital, kolkata, west bengal, india background. acute appendicitis is the most common surgical emergency presenting in emergency department, and appendectomy is one of the most common surgeries. there is a wide variation in the incidence of acute appendicitis reported for different countries, different regions, race, sex, age and also seasons. objective. the aim of this study was to understand the epidemiological trend and the seasonal variation of appendicitis in the northeast region of india. methods. it was a retrospective hospital-based study conducted in jan 2016 – dec 2020 at a tertiary care teaching hospital in the northeast region of india. the patient data were obtained from the medical record department of the hospital and the data regarding the weather was obtained from the indian meteorological department (imd) and the website www.worldweather.com. the statistical analysis was done using spss software version 24.0, and the seasonal variation was studied using kruskal wallis test. results. acute appendicitis affected both the sexes equally with a marginal higher incidence in males. it also had higher peak during the second and third decade of life in both the sexes. the incidence of acute appendicitis was high in the pre-monsoon and monsoon season with peak in the monsoon season. conclusions. acute appendicitis is more common during the pre-monsoon and monsoon season, a period known for humidity, high incidence of bacterial and viral infections. keywords: acute appendicitis (aa), pre-monsoon season, monsoon season, indian meteorological department (imd). international journal of medicine and medical research 2021, volume 7, issue 2, p. 20-25 copyright © 2021, tnmu, all rights reserved *corresponding author: ranjan kumar, assistant professor, department of surgery, base hospital tezpur, assam, india. e-mail: drranjansurgeon@gmail.com introduction acute appendicitis (aa) is the most common surgical emergency presenting in emergency department, and appendectomy is one of the most common surgeries [1]. aa has an increased incidence among the males as compared to the females. the lifetime risk of developing appendicitis is 8.6% for males and 6.7% for females [2, 3]. the aetiology of appendicitis is still unclear, hence various possible causes for aa are elucidated that include mechanical obstruction, inadequate dietary fiber, smoking, air pollution and familial susceptibility [3, 4, 5, 6, 7, 8, 9]. aa presents throughout the year but incidence is increased in some particular months [10, 11, 12, 13, 14, 25, 26]. various studies were performed to determine the seasonal variation of acute appendicitis but with variable results. some of them have concluded increased incidence in a particular month with no clear rationale behind it. although numerous epidemiological studies on appendicitis, most focused on western populations and relatively few epidemiological studies on the asian populations. these studies were mainly concerned with the monthly variation in the incidence of acute appendicitis and the volume-outcome relationship of acute appendicitis. there is very sparse research regarding seasonal variation of acute appendicitis in indian sub-continent and more so from the north-east region. the rationale of our study was to understand the epidemiological trend and the seasonal variation of aa in the northeast region of india. methods study design a retrospective hospital-based study was conducted to assess the demographic profile, incidence and seasonal variation in all patients admitted with diagnosis of acute appendicitis, who underwent operative management, in jan 2016 – dec 2020, at a tertiary care teaching hospital in the northeast region of india. the issn 2413-6077. ijmmr 2021 vol. 7 issue 2 21 p u b l ic h e a lt h a n d e p id e m io l o g y r. kumar et al. pathologically proven negative appendectomy patients were excluded from this study. data source and statistical analysis patient data including demographic details, operative notes and final histopathology were obtained at the medical record department of the hospital. the data by indian meteorological department (imd) and the website www. worldweather.com was used to obtain information regarding the weather of the place where our study was conducted. according to the imd there are four seasons in north-eastern region of india, i.e. winter (jan–feb), premonsoon (mar–may), monsoon/rainy season (jun–sep) and post-monsoon (oct–dec). the raw data was entered into microsoft excel worksheet and analysis was performed using spss 24.0 software. the seasonal variation was studied regarding males and females, and inter-seasonal variation was assessed using kruskal wallis test. p-value less than 0.05 was taken as significant seasonal variation. results during the study period a total of 405 patients underwent emergency appendectomy; all the cases were confirmed histologically as aa. there were 240 males (59.26%) and 165 females (40.74%) with male to female ratio of 1.45:1 (table 1). the age­specific incidence of aa followed a similar pattern for both the sexes, but males had a higher rate at virtually all ages with the highest male to female ratio in the third decade; gradually this ratio declined and was equal by the 7th decade (fig 1). the incidence of acute appendicitis was the highest in both the sexes in the third decade (fig 1). the overall mean age was 25.05 years (males – 24.68 years and females – 25.42 years). only 6% of the cases were recorded in the first decade of life, while 65.9% occurred in the age group of 11-30 years old (table 1). the combined incidences of the disease were higher in the pre-monsoon and monsoon season (67.9 %, p<0.0078 as per kruskal wallis test, h statistics was 16.8029), with peaks in june, july, and august (monsoon season) corroborating with the maximum rainfall and the maximum temperature of the year (fig. 2, 3) the incidence of the disease started declining from october (post monsoon period), with the lowest in december, followed by gradual minimal increment from january (winter season). table 1. demographic trend of acute appendicitis age group (years) number of cases percentage of the total cases male cases (m) female cases (f) m:f ratio 0-10 19 4.69 11 8 1.38 11-20 114 28.15 69 45 1.53 21-30 138 34.07 84 54 1.55 31-40 101 24.94 58 43 1.35 41-50 16 3.95 9 7 1.29 51-60 9 2.22 5 4 1.25 >60 8 1.98 4 4 1.00 fig. 1. age related distribution of acute appendicitis. 19 114 138 101 16 9 8 0 20 40 60 80 100 120 140 160 0-10 yrs 11-20 yrs 21-30 yrs 31-40 yrs 41-50 yrs 51-60 yrs >60 yrs males females total issn 2413-6077. ijmmr 2021 vol. 7 issue 222 p u b l ic h e a lt h a n d e p id e m io l o g y discussion aa is a common clinical condition and appendectomy counts for about 1% of all the surgical operations [1]. it is estimated that in about 6-7% of the general population appendicitis will develop during their lifetime [15]. the incidence of appendicitis varies substantially by country, geographical region, race, sex, age, and seasons. the predisposing factors for appendicitis are multifactorial and include inadequate dietary, smoking, air pollution, infections, stress, vascular disorders and inadequate childhood breast feeding [6, 8, 9, 14, 17, 18, 19, 27]. the obstruction of the lumen due fecalith or hypertrophy of lymphoid tissue or tu morigenesis is proposed as the main etiologic factor in acute appendicitis [3, 4, 5, 6, 11, 17, 19]. the obstructive pathology in age group <40 years is due to regional lymph node enlargement secondary to infections while in patients >40 years luminal obstruction is more likely to be caused by fecalith or neoplasia because lymphoid tissue atrophies with age [4, 5, 11]. acute appendicitis is relatively rare in infants and becomes increasingly common in childhood and early adult life [20]. the highest incidence of appendicitis is found in the second and third decades of life [2, 3]. after 30 years of age, the incidence declines, but appendicitis can occur in individuals of any age [21]. thus, it was established that the maximum incidence of the disease is in the third decade of life. acute appendicitis has an increased incidence among the males compare to the females. the lifetime risk of appendicitis is 8.6% for males and 6.7% for females [2, 3]. among the teenagers and young adults, the male to female ratio is about 3:2. after 25 years old, the ratio gradually declines until the sex ratio is equal by the mid­30s [21]. similar findings were noted in our study cohort. there is no definite explanation for high incidence of appendicitis in males but, genetically determined differences in the immune function as well as the variation in the concentration of sex hormones can account for this difference [22]. hormonal differences, however, do not explain the gender differences of the incidence of the disease in preadolescents and among the elderly [22]. 20 21 21 34 50 29 52 66 44 26 24 18 0 10 20 30 40 50 60 70 j a n f e b m a r a p r m a y j u n e j u l y a u g s e p o c t n o v d e c total cases fig. 3. seasonal variation of the incidence of acute appen dicitis. fig. 2. monthly incidence of acute appendicitis in jan 2016 – dec 2020. jan-feb (winter) 15% mar-may (pre-monsoon) 28% jun-sep (monsoon) 40% oct-dec (post-monsoon) 17% r. kumar et al. issn 2413-6077. ijmmr 2021 vol. 7 issue 2 23 p u b l ic h e a lt h a n d e p id e m io l o g y the seasonal variation in the incidence of acute appendicitis has been discussed in many studies but there is no definite explanation for it. the heterogeneous extrinsic factors like humidity, sun radiation, bacterial and viral infections are important in the etiogenesis of appendicitis. appendicitis is more common during the rainy reason, a period of high humidity which is the peak period of bacterial intestinal infestations and parasitic infestation. these intestinal infestations cause intestinal lymphoid hyperplasia that leads to obstruction of the appendix lumen. the reduction of sun radiation and vast fluctuations in air temperature has also been postulated in the incidence of appendicitis [3, 12, 23, 25, 26]. air pollution has also been found to have a significant effect on the incidence of appendicitis in the summer months [9, 11]. the high ambient temperature has been found to contribute to air pollution and hence short-term exposure to air pollution may trigger appendicitis [9, 11]. the allergic reaction to pollen from flowers, palm and maize may also account for some cases of appendicitis, as the pollens trigger immunological response in the form of lymphoid hyperplasia [24]. we have found higher incidence of aa in the monsoon and pre-monsoon season in the north-east region of india, the seasons of maximum rainfall and temperatures (fig 4). fig. 4. www.worldweather.com data showing weather of tezpur, assam, during the study period. (a) average temperature graph, (b) maximum, minimum and average temperature, (c) average rainfall graph, (d) average rainfall amount. conclusions acute appendicitis is still one of the most common abdominal emergencies treated by general surgeons. it affects both the sexes equally with a marginal higher incidence in the males. the disease has a higher peak during the second and third decade, the period of highly responsive lymphoreticular system. appendicitis is more common during the premonsoon and monsoon season, a period known for humidity, high incidence of bacterial and viral infections. limitation of the study the study population was not a true representation of the society as most of the patients were military background and the study was conducted in the north-east region of india. a larger prospective study needs to be undertaken across different geographical locations in india in order to confirm our findings of seasonal variation of aa in this sub­ continent. conflict of interests authors declare no conflict of interests. author’s contributions ranjan kumar, biswajit singh thokchom – conceptualization, methodology, formal analysis, writing – original draft, writing – reviewing and editing; rahul pandey – data curation, writing – reviewing and editing; sandeep bhalla, onkar singh – investigation, formal analysis. r. kumar et al. issn 2413-6077. ijmmr 2021 vol. 7 issue 224 p u b l ic h e a lt h a n d e p id e m io l o g y епедеміологія та сезонність апендициту – ретроспективне дослідження з північно-східної індії *r. kumar1, t.b. singh2, r. pandey1, s. bhalla1, o. singh3 1 – base hospital tezpur, assam, india 2 – military hospital ambala, haryana, india 3 – command hospital, kolkata, west bengal, india вступ. гострий апендицит є найбільш частим хірургічним невідкладним станом у відділенні невідкладної допомоги, а апендектомія є однією з найпоширеніших операцій. захворюваність на гострий апендицит, зареєстрована в різних країнах та різних регіонах, у людей різної раси, статі, віку, а також у різних сезонах, значно відрізняється. мета. метою цього дослідження була оцінка епідеміологічних тенденцій та сезонних змін захворюваності на апендицит в північно-східному регіоні індії. методи. у статті описується ретроспективне лікарняне дослідження, яке проводилося з січня 2016 року по грудень 2020 року в університетській лікарні третинного рівня медичної допомоги в північно-східному регіоні індії. дані про пацієнтів були отримані з медичного архіву лікарні, а дані про погоду – з індійського метеорологічного відділу (imd) та веб-сайту www.worldweather.com. статистичний аналіз проводили за допомогою програмного забезпечення spss версії 24.0, а сезонні зміни досліджували за допомогою тесту краскела-уолліса. результати. гострим апендицитом хворіли в однаковій мірі обидві статі з незначно вищою захворюваністю у чоловіків. він також мав більш високий пік переважно на другому-третьому десятку років у обох статей. захворюваність на гострий апендицит була високою в передмусонний сезон і сезон мусонів з піком у сезон мусонів. висновки. гострий апендицит частіше зустрічається під час передмусонного сезону та сезону мусонів, у період з характерною високою вологістю та високим рівнем захворюваності бактеріальними та вірусними інфекціями. ключові слова: гострий апендицит (aa); сезон перед мусонами; сезон мусонів; індійський метеорологічний відділ (imd). information about the authors ranjan kumar – assistant professor, department of surgery, base hospital tezpur, assam, india. orcid 0000­0002­4573­2446, e­mail: drranjansurgeon@gmail.com thokchom biswajit singh – assistant professor, department of surgery, military hospital ambala, haryana, india. orcid 0000­0001­5849­5999, e­mail: bishwajitsingh6068@gmail.com rahul pandey – assistant professor, department of surgery, base hospital tezpur, assam, india. orcid 0000­0003­2624­5113, e­mail: rahuladviksimpy@gmail.com sandeep bhalla – consultant community medicine and commandant, base hospital tezpur, assam, india. orcid 0000­0001­9027­0001, e­mail: bhallasap@gmail.com onkar singh – professor, command hospital kolkata, west bengal, india. orcid 0000­0001­7806­0263, email: onkarnoor@yahoo.co.in references 1. sihrah bh, shirah ha, shirah ha et al. challenges in the management of subhepatic acute appendicitis in the emergency setting. int j cur res rev 2016 mar;8(6) 2. addiss dg, shaffer n, fowler bs, tauxe rv. the epidemiology of appendicitis and appendectomy in the united states. am j epidemiol. 1990 nov;132(5): 910-925. https://doi.org/10.1093/oxfordjournals.aje. a115734. 3. brunicardi fc, anderson dk, billiar tr. the appendix. in: liang mk, anderson re, jaffe bm et al. schwartz’s principle of surgery.10th ed. mc graw hill education medical. 2015. p. 1241-1262. 4. jones ba, demetriades d, segal i, et al. the prevalence of appendiceal fecaliths in patients with and without appendicitis: a comparative study from canada and south africa. ann surg. 1985 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2021. this is open access article distributed under the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. r. kumar et al. 122 b io m e d ic a l s c ie n c e s issn 2413-6077. ijmmr 2019 vol. 5 issue 2 doi 10.11603/ijmmr.2413-6077.2019.2.10310 role of endogenic intoxication in muscle injury in experiment *v.h. dzhyvak, o.i. khlibovska, i.m. klishch i. horbachevsky ternopil national medical university, ternopil, ukraine background. endogenous intoxication is a multicomponent complex process due to the endogenous biological products or dysfunction of systemic natural detoxification. objective. the aim of the research was to study the dynamics of indices of endogenous intoxication in rats with traumatic muscle damage in the experiment. methods. the experiment was performed on 45 non-linear white rats, which were modeled with traumatic muscle damage. the level of endogenous intoxication was assessed by the content of medium plasma molecules (mmm), leukocyte and erythrocytic index of intoxication (lii and eii). the research was conducted on the 1st, 3rd, 7th, 14th days after the injury. results. it was found that traumatic muscle damage causes endotoxemia. manifestations of endogenous intoxication are: the increase of mmm1 in 2.3 times, mmm2 in 2.8 times compare to the intact animals. the level of this indicator slightly decreased in 7 days. simultaneously with an increase in the mct level in the post-traumatic period, the total toxic effect on the erythrocyte membrane also increased, which was manifested by a significant increase in eii in all terms of observation. conclusions. traumatic damage of the muscles is accompanied by the growth of molecules of average mass in upto 7 days of observation, which significantly differ from the indicators of the intact group. the results of our research prove that traumatic muscle damage causes endotoxicosis development evidenced by accumulation of endotoxins in the animals’ body that is proved by significant changes in endogenous intoxication indices: i.e. erythrocytic and leukocytic indexes of intoxication and content of medium mass molecules. key words: endogenous intoxication; traumatic muscle damage; middle mass molecule. *corresponding author: dzhyvak v.h., ph.d. student of the department of pathological physiology, i. horbachevsky ternopil national medical university, ternopil, ukraine. e-mail: volodymyrdzh@gmail.com introduction the increase in injuries all over the world is still one of the topical socio-economic issues of today [1,3,4]. every year in ukraine, about 10% of the population gets an injury of varying degrees of severity. mortality from accidents and injuries in ukraine increases by an average of 1% annually [2]. according to the world health organization, the traumatic damages are the third among the causes of mortality, and among the population under 40 years – the first. despite the fact that the patients with polytrauma make up 8-10% of all inpatient cases, they account for 68% of fatal cases. according to the data of the european commission's newsletter in 2019, about five people are seriously injured with consequences in lifethreatening road accidents. serious injuries are often more expensive for society through longterm rehab and medical needs [1]. at the same time, mortality from accidents and injuries is constantly increasing: an average of 1% an­ nually [2], which is evidence of the ineffectiveness of medical care provided to these patients. endogenous intoxication is a complex multicomponent process due to the pathological biological activity of endogenous products or dysfunction of systemic natural detoxification [5,6]. when the body is injured, there are significant general and local changes that are considered within the traumatic disease [2]. the morphological substrate of traumatic illness is the damage of organs and tissues of various localization and character, which arise with excessive mechanical influence [2, 4]. at the moment of injury, tissue elements are destroyed or damaged, receptor fields are changed, and the integrity of blood and lymphatic vessels is violated. releasing physiologically active substances, in particular, proteolytic enzymes and biogenic amines causes secondary damage. oxidative stress causes damage to the body of biomacromolecules that leads to accumulation of products of oxidative modification of proteins, degradation of lipid international journal of medicine and medical research 2019, volume 5, issue 2, p. 122-127 copyright © 2019, tnmu, all rights reserved v.h. dzhyvak et al. 123 b io m e d ic a l s c ie n c e s issn 2413-6077. ijmmr 2019 vol. 5 issue 2 v.h. dzhyvak et al. components, nucleotides, pigments, and formation of a significant number of middle mass molecules (mmm) [7]. to date, there are three stages of development of the syndrome of systemic inflammatory response: the stage of local production of mediators in response to injury, which can be regarded as a protective response (healing of wounds, protection of cells from pathogenic microorganisms); a stage of ejection of a small number of mediators into the bloodstream to support homeostasis; the stage of generalization of the inflammatory reaction, in which the regulatory systems are not able to provide the homeostasis of the organism [8]. the above mediators exhibit destructive functions, primarily in the system of endothelial cells. one of the systems of the body that is undergoing significant changes in trauma is the detoxification system. this system dysfunc­ tion leads to development of endogenous intoxication syndrome (eis), which accompanies diseases and complications associated with increased tissue disintegration, increased catabolism, internal organs insufficiency [9]. markers of endogenous intoxication are molecules of average mass, erythrocyte index of intoxication, which is established and fast in execution [9,10]. to date, the age-related mecha nisms of the development of endogenous intoxication syndrome is still unclear, there are no perfect pathogenetic approaches to explaining and predicting the early and late effects of traumatic lesions. methods the experiment was performed on 45 nonlinear white rats, males, weighing 180-200 g, which were kept on a standard vivarium diet. the rats were kept and all experiments were performed following the provisions of the “european convention for the protection of vertebrate animals used for experiments and other scientific purposes” (strasbourg, 1986); the general ethical principles of animal experiments adopted by the first national congress on bioethics (kyiv, 2001), the helsinki declaration of the world medical association (2000). the animals were divided into 2 groups: 1 – the intact (12 animals), 2 – the controls with traumatic muscle damage (33 animals). the injury was modeled by a dose-fetched foot thigh, equivalent to the animals by the severity of the injury in thiopental-sodium general anesthesia (40 mg/kg). animals on the 1st, 7th (early), 14th (intermediate), 21st (late) days of the posttraumatic period were taken from the experiment. euthanasia of rats was performed by decapitation under thiopental anesthesia with subsequent total hemorrhaging. the level of endogenous intoxication was assessed by the content of medium-mass molecules in plasma, defining of leukocyte index of intoxication (lii) and erythrocytic indices of intoxication (eii). the content of middle mass molecules was determined in accordance with the method [7]. an acid-soluble fraction was isolated from the blood serum, which was obtained by adding 1.8 ml of a 10% solution of trichloroacetic acid to 0.2 ml of serum. the middle mass molecules content was determined at wavelengths of 254 (chain amino acids were determined) and 280 nm (aromatic amino acids were determined), then the coefficient kc (mmm 280 nm / mmm 254 nm) was calculated. the leukocyte index of intoxication is the ratio of blood cell populations that indirectly allows evaluating the predominance of response to pro-or anti-inflammatory cytokines. the leukocyte index of intoxication was determined according to the formula suggested by ya. kalfkalif in modification by b.a. reis [10]. endogenous intoxication was determined by a technique based on the idea of erythrocytes as a universal adsorbent, which allows assessing the level of endogenous intoxication by changing the sorption capacity of erythrocytes of the polar, practically not penetrating through their methylene blue membrane. the amount of absorbed color (in percentages) [7] was calculated using the formula: а=100–с×100/в, where a – the amount of absorbed dye, %; b – the optical density of the initial solution, conditional units of extinction; c – the optical density of the dye solution after incubation with erythrocytes, conditional units of extinction. statistically significant differences between the control and experimental groups were estimated using mann-whitney’s non-parametric criterion. the differences were considered significant at the probability of a zero hypothesis less than 5% (p<0.05). results according to the results of the case study, the mmm1 and mmm2 indices that reflected the content of chain and aromatic amino acids in the medium-sized peptides respectively as well as their decomposition products increased by the 1st day after trauma (table 1). thus, the 124 b io m e d ic a l s c ie n c e s issn 2413-6077. ijmmr 2019 vol. 5 issue 2 content of mmm1 in the blood of the affected rats increased in 2.3 times compare to the intact animals. in the same experiment period, the content of mmm2 in rat blood increased accordingly in 2.8 times in relation to the intact animals. the most significant changes in the content of both mmm fractions were observed in 24 hours after the injury. in 7 days after the injury, there was a slight tendency to decrease in the mmm content. thus, under the influence of the injury, the concentration of blood concentration in both mmm1 and mmm2 was noted. moreover, the mmm method was more significant for the blank mmm2, indicating the severity of the aromatic amino acids of the average molecule. since mmms were the markers of endotoxicosis, the significant chan­ ges in their content evidenced the peak of the development and generalization of the syndrome. the features of mmm are their clearly expressed high biological activity. the accu mulation of mmm is not only a marker of endotoxicosis; in the future, they increase the course of the pathological process, acquiring the role of secondary toxins, affecting the livelihoods of all systems and organs. the level of mmm is considered the main biochemical marker, which reflects the level of pathological protein meta­ bolism. it does not only accompany acute and chronic pathology, but is an important factor in their pathogenesis, determines the course and consequences of the disease. ac cording to the results of the research (table 2), simultaneously with the increase in the mmm level in the post-traumatic period, the total toxic effect on the erythrocyte membrane also increased, which was manifested by a significant increase in endogenous intoxication in all terms of observation. it is established that the degree of destruction of membranes of erythrocytes during the experiment was the highest. changes are obviously repeated by the fact that when injuries are caused to the body, energy metabolism and transport of substances in erythrocytes are disturbed, the permeability of their membrane progressively increases. based on the analysis of blood cell parameters, the leukocyte index of intoxication was calculated at the beginning of the experiment and in its different terms. as a result of the study, an ambiguous response of the leukocyte relating the pathological processes development was established in a day: the leukocyte index of intoxication increased in 2.7 times (table 3). leukocyte index of intoxication by ya. calfcaliph in b.a. reis modification increased and amounted to (0.75±0.01)% on the 7th day of the post-traumatic period and (0.25±0.01)% in the control. discussion according to the case results, mmm1 and mmm2 increased in up to 1 day after trauma. the content of mmm1 in the blood of the affected rats increased in 2.3 times (0.575±0.031) compare to the intact animals (0.250±0.014), and the mmm2 content in the rat blood increased accordingly in 2.8 times (0.484±0.011) table 1. dynamics of the content of medium mass molecules (mmm) in the serum of the rats with traumatic muscle damage (m ± m) indicator animal groups intact (n=12) terms of observation 1st day (n=30) 7th day (n=27) 14th day (n=26) 21st day (n=25) mmm1, (conditional units) 0.250±0.014 0.575±0.031* 0.514±0.026* 0.414±0.026* 0.341±0.026* mmm2, (conditional units 0.173±0.001 0.484±0.011* 0.450±0.021* 0.350±0.021* 0.270±0.021* coefficient mmm2/ mmm1 0.692 0.841* 0.875* 0.845* 0.791 note: * – p<0,05 – the probable differences compared with the intact animals. table 2. dynamics of erythrocytic index of intoxication (eii) (%) in the rat blood (m ± m) indicator animal group intact (n=12) terms of observation 24 hours (n=30) 7th day (n=27) 14th day (n=26) 21st day (n=25) (eii) % 33.1±3.8 62.1±1.1* 73.5±8.0* 47.72±2.0* 40.20±1.0* note: * – p<0.05 – the probable differences compared with the animals of the intact. v.h. dzhyvak et al. 125 b io m e d ic a l s c ie n c e s issn 2413-6077. ijmmr 2019 vol. 5 issue 2 v.h. dzhyvak et al. relative to the intact animals (0.173±0.001). the most significant changes in the increase of mmm were observed in 24 hours after the injury. in 7 days after the beginning of the experiment, there was a slight tendency to decrease in the content of mmm (mmm1 0.514±0.026, mmm2 0.450±0.021). intoxication syndrome is caused by trauma and is accompanied by increased tissue breakdown, increased catabolic processes, due to the accumulation of excessive amounts of biologically active substances, deformed protein metabolites and other toxic substances of endogenous origin. the results of the research proved that simultaneously with the increase in the mmm level in the posttraumatic period, the total toxic effect on the erythrocyte membrane also increased that was manifested by a significant increase in endo­ genous intoxication in all terms of observation. as a result of the research, an ambiguous re action of the leukocyte response to the development of pathological processes was established in a day: the leukocyte index of intoxication increased in 2.7 times 0.25±0.02, and the increase on the 7th day of the post-traumatic period was significant 0.75±0.01 compared with the control 0.25±0.01. the increase of this indicator may evidence the activation of inflam­ matory reactions in the area of traumatic muscle damage and the activation of reparative processes. conclusions traumatic damage of the muscles is accompanied by the growth of molecules of average mass up to 7 days of observation, which sig_ nificantly differ from the indicators of the intact group. the results of our research indicate that traumatic muscle damage causes endotoxicosis development, which is evidenced by accu mulation of endotoxins in the body of animals that is proved by significant changes in endogenous intoxication indices: erythrocyte and leukocyte indexes of intoxication and content of medium mass molecules. funding this research received no external funding. conflict of interests the authors declare no conflict of interest. authors contributions dzhyvak v.h. – investigation, formal analysis, writing – original draft; khlibovska o.i. – investigation, formal analysis, validation, writing – original draft; klishch i.m. – conceptualization, supervision, writing – review and editing. table 3. dynamics of leukocyte index of intoxication (%) in the blood of rats (m+m) indicator animal groups intact (n=12) terms of observation 24 hours (n=30) 7th day (n=27) 14th day (n=26) 21st day (n=25) leukocyte index of intoxication (%) 0.25±0.02 0.67±0.01 0.75±0.01 0.41±0.01 0.27±0.01 note: * – p<0.05 – the probable differences compared with the animals of the intact. маркери ендогенної інтоксикації при травматичному ураженні м’язів в експерименті в.г. дживак, о.і. хлібовська, і.м. кліщ тернопільський національний медичний університет імені і.я. горбачевського, тернопіль, україна вступ. ендогенна інтоксикація – це складний багатокомпонентний процес, зумовлений патологічною біологічною активністю ендогенних продуктів або дисфункцією систем природної детоксикації, що викликає як загальні, так і локальні зміни. мета дослідження. вивчити динаміку показників ендогенної інтоксикації у щурів з травматичним ушкодженням м'язів. 126 b io m e d ic a l s c ie n c e s issn 2413-6077. ijmmr 2019 vol. 5 issue 2 методи дослідження. експеримент проводили на 45 нелінійних білих щурах. нанесення травми відбувалося в умовах тіопентало-натрієвого знечулення (40 мг/кг) шляхом дозованого удару по стегну. рівень ендогенної інтоксикації оцінювали за вмістом молекул середньої маси в плазмі, визначення лейкоцитарного індексу інтоксикації (ліі) і еритроцитарного індексу інтоксикації (eii). дослідження проводили на 1, 3, 7, 14 день після травми. результати. було виявлено, що в результаті травматичного ураження м’язів розвивається ендотоксикоз, свідченням чого є накопичення ендотоксинів в організмі тварин. проявами ендогенної інтоксикації є зростання мсм1 у 2,3 рази, мсм2 у 2,8 рази відносно інтактних тварин. рівень даного показника незначно зменшувався на 7 добу. одночасно із збільшенням у посттравматичному періоді рівня mcm, зростав і сумарний токсичний вплив на мембрани еритроцитів, який проявлявся в достовірному підвищенні eii у всі терміни спостереження. висновки. травматичне ушкодження м’язів супроводжується зростанням молекул середньої маси до 7 доби спостереження які достовірно відрізняються від контрольних показників. результати експериментального дослідження свідчать, що при травматичному ушкодженні м’язів розвивається ендотоксикоз, свідченням чого є нагромадження ендотоксинів в організмі тварин, на що вказують виражені зміни показників ендогенної інтоксикації – еритроцитарного та лейкоцитарного індексів інтоксикації та вмісту молекул середньої маси. ключові слова: ендогенна інтоксикація; травматичне ушкодження мязів; молекули середньої маси. відомості про авторів дживак володимир георгійович – аспірант кафедри патологічної фізіології тернопільського національного медичного університету імені і.я. горбачевського. хлібовська оксана іванівна – кандидат медичних наук, доцент кафедри акушерствата гінекології нніпо тернопільського національного медичного університету імені і.я. горбачевського. кліщ іван миколайович – доктор біологічних наук, професор кафедри функціональної і лабораторної діагностики тернопільського національного медичного університету імені і.я. гор­ бачевського. information about the authors dzhyvak v.h. – ph.d. student of the department of pathological physiology, i. horbachevsky ternopil national medical university, ternopil, ukraine. orcid: 0000­0002­4885­7586, e­mail: volodymyrdzh@gmail.com khlibovska o.i. – md, phd, associate professor of the obstetrics and gynecology department i. horbachevsky ternopil national medical university, ternopil, ukraine. orcid 0000­0003­3293­0010, e­mail: chlibovska@tdmu.edu.ua klishch i.m. – prof., dsc, phd, md professor of department of functional and laboratory diagnostics i. horbachevsky ternopil national medical university, ternopil, ukraine. orcid 0000­0001­6226­4296, e­mail: klishch@tdmu.edu.ua references 1. utriainen r, pöllänen m, liimatainen h. road safety comparisons with international data on seriously injured. transport policy. 2018 aug 1;66:138­45. doi: 10.1016/j.tranpol.2018.02.012 2. gaiko gv, derkach rv. cause analysis and study of the causes of influenza mortality rate in car accidents with injuries to the musculoskeletal system. science and practice. 2014; 1. 82­6. 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[in ukrainian]. 6. bakalyuk oy, punchyshyn nya, dziga sv. endogenous intoxication syndrome, mechanism of origin, methods of identification. bulletin of scientific research. 2000; 1: 11­3. 7. karyakina ev, belova sv. medium mass molecules as an integral index of metabolic disorders (lite rature review). clinic­lab diagnostics. 2004; 3: 4­8. v.h. dzhyvak et al. 127 b io m e d ic a l s c ie n c e s issn 2413-6077. ijmmr 2019 vol. 5 issue 2 v.h. dzhyvak et al. 8. tidball jg. inflammatory processes in muscle injury and repair. american journal of physiologyregulatory, integrative and comparative physiology. 2005 feb;288(2):r345­53. doi: 10.1152/ajpregu.00454.2004 9. speransky i.i., samoilenko g.e., lobacheva mv. general analysis of blood all its possibilities are exhausted? integral indices of intoxication as criteria for assessing the severity of the course of endogenous intoxication, its complications and the effectiveness of the treatment being performed. the health of ukraine. 2009; 6 (19): 51­7. (in ukrainian). 10. togaybaev a.a. kurguzkin a.v., rikun i.v. method of diagnosis of endogenous intoxication. laboratornoe delo. 1988; 9: 22­4. (in russian). received 12 september 2019; revised 19 october 2019; accepted 27 october 2019. this is open access article distributed under the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. 52 p h a r m a c y issn 2413-6077. ijmmr 2020 vol. 6 issue 2 doi 10.11603/ijmmr.2413-6077.2020.2.12013 quality control measurement and in vitro bioequivalence of valsartan and atenolol tablets marketed in ukraine k.y. peleshok i. horbachevsky ternopil national medical university, ternopil, ukraine background. the urgent issue of hypertension is determined by its high population incidence, significant burden of the disease, risk of disability and impact on life expectancy. rational combinations of drugs of different pharmacological groups in case of ineffectiveness of monotherapy to achieve the clinical effect of pharmacotherapy are clearly recommended in the world and national recommendations for diagnosis and treatment of hypertension. therefore, innovative pharmaceutical development of a combination of antihypertensive drugs and creation of domestic drugs with antihypertensive action is an urgent task of contemporary pharmacy. objective. the aim of this study was to perform the quality control measurements and evaluation of dissolution tests for different brands of valsartan and atenolol tablets available in ukraine. methods. the concentrations of valsartan and atenolol in samples (drug content and dissolution study) were determined by the proposed hplc method. results. the results of the tests conducted for evaluation of the tablets were found to be in acceptable limits for all the selected brands. the correlation coefficient (r2) was 0.9991 and the regression equation was y=61.39x+0.3117. it has been established that the equivalence of dissolution profiles for all recommended dissolution media is observed (рн 1.2, 4.5, and 6.8) for the studied drugs. in all three dissolution media, the release rates of valsartan and atenolol of all dosage forms are more than 85% in 15 min. the dissolution profile of all the selected brands was within the standard limits and was acceptable. conclusions. analytical method development is an integral part of the quality control measurements and evaluation of dissolution tests. our previously developed hplc method is essential for quality control of a large number of samples in short time intervals. therefore, the method developed by our group is suitable for a routine quality control analysis of any pharmaceutical preparation containing two tested drugs with the suggested chromatographic method advantages for checking quality during dissolution studies of their dosage forms. keywords: hypertension; valsartan; atenolol; high-performance liquid chromatography; in vitro bioequivalence; dissolution. *corresponding author: peleshok kateryna, assistant professor, i. horbachevsky ternopil national medical university, ternopil, 46001, ukraine. e-mail: peleshok@tdmu.edu.ua international journal of medicine and medical research 2020, volume 6, issue 2, p. 52-58 copyright © 2020, tnmu, all rights reserved k.y. peleshok introduction valsartan (fig. 1) is chemically described as (2s)-3-methyl-2-[pentanoyl-[[4-[2-(2h-tetrazol5-yl)phenyl]phenyl]methyl]amino]butanoic acid. valsartan is an orally active nonpeptide triazole-derived antagonist of angiotensin (at) ii with antihypertensive properties. valsartan selectively and competitively blocks the binding of angiotensin ii to the at1 subtype receptor in vascular smooth muscle and the adrenal gland, preventing at ii-mediated vasoconstriction, aldosterone synthesis and secretion, and renal reabsorption of sodium, and results in vasodilation, increased excretion of sodium and water, reduction of plasma volume, and reduction of blood pressure. therefore, analytical methods for their separation and quantification in pharmaceutical formulations and inhuman plasma are needed for quality control and therapeutic drug monitoring, respectively. several techniques have been reported in the literature for determination of valsartan individually and combination with other drug other than atenolol [1-16] in pharmaceutical dosage forms or human serum samples. fig. 1. chemical structure of valsartan. 53 p h a r m a c y issn 2413-6077. ijmmr 2020 vol. 6 issue 2 atenolol (fig. 2) is a synthetic isopropylaminopropanol derivative used as an antihypertensive, hypotensive and antiarrhythmic. atenolol is chemically known as 2-[4-[2-hydroxy-3-(propan2-ylamino)propoxy]phenyl]acetamide. atenolol acts as a peripheral, cardioselective beta blocker specific for beta-1 adrenergic receptors, without intrinsic sympathomimetic effects. it reduces exercise heart rates and delays atrioventricular conduction, with overall oxygen requirements decrease. numerous analytical methods were reported [17-27] for determination of atenolol in bulk and combination with other drugs other than valsartan. suspicious generics is the prime challenge to our health department and quality control units. this research assists highlighting the pharmaceutical products which are found to be spurious, of inferior quality and dangerous to the users. the objective of the research was to perform the quality control measurements and evaluate dissolution tests of different brands of valsartan and atenolol tablets available in ukraine. methods valsartan (purity 99.9%) was purchased from jubilant generics limited (india); atenolol (purity 98.9%) was purchased from sigmaaldrich (switzerland). the methanol and acetonitrile used in experiments was hplc gradient grade and ammonium acetate and tetramethylammonium hydroxide were of ph.eur.reagent grade and was purchased from merck darmstdat, germany. analytical balance mettler toledo mpc227, ph–metter metrohm 827, demineralized water by tka micro system, with final conductivity less than 0.05µs/cm, were used. ika orbital shaker ks4000i was used for sample agitation. the nylon and regenerated cellulose rc 0.45um syringe filters were purchased from agilent technologies. dionex ultimate 3000 uhplc system controlled by chromeleon version 6,80, composed of quaternary lpg pump ultimate 3000, autosampler ultimate 3000, ultimate 3000 column compartment, four channel uv-vis detector ultimate 3000 rs. shimadzu nexera xr uplc system with lpg quaternary pump lc-20ad with degasser dgu-20a5r, autosempler sil-20ac, pda detector m20-a, column oven and controller cbm-20a controlled by lab solutions version 5,97. the column discovery c18 (4.6 mm i.d.×150 mm, 5 μm), purchased from sigma-aldrich supelco, was used. sample preparation twelve tablets of each preparation were studied to obtain statistically significant results. the tablets of different pharmaceutical manufacters with declared contents of 80 mg valsartan and 100 mg of atenolol were purchased from local drug store, pharmacy. the tablets were put in 100 ml measuring flasks and dissolved in 50 ml 50% v/v methanol, ultrasound crushed and treated for 2 minutes and shake 15 minutes with orbital shaker. after that measuring flasks were filled to mark of 100 ml, the final concentrations were 1mg/ml for atenolol and 0.8 mg/ml for valsartan. the samples fig. 2. chemical structure of atenolol. the urgent issue of hypertension is determined by its high population incidence, significant burden of the disease, risk of disability and impact on life expectancy. rational combinations of drugs of different pharmacological groups in case of ineffectiveness of monotherapy to achieve the clinical effect of pharmacotherapy are clearly recommended in the world and national recommendations for diagnosis and treatment of hypertension. therefore, innovative pharmaceutical development of a combination of antihypertensive drugs and creation of domestic drugs with antihypertensive action is an urgent task of contemporary pharmacy. in the last few decades, the cost of medications is increasing a lot and it is quite challenging to afford lifelong medications for hypertension treatment. different strategies have been planned by the healthcare systems to reduce the medication costs, improve treatment efficacy and safety and patient compliance with pharmacotherapy [28]. the results of quality control testing, such as friability, weight variation, hardness, percentage purity, and disintegration, suggests the level up to which the gmp guidelines had been followed during the manufacturing of these generic products. when the generic and the innovator brand would have comparable dissolution profile then the in vivo bioequivalence test of the generics can be waived. it was reported that fewer generics in the market were counterfeit and of inferior quality than the innovators. so, identifying these fake and k.y. peleshok 54 p h a r m a c y issn 2413-6077. ijmmr 2020 vol. 6 issue 2 were filtered with rc 0.45um syringe filters and injected. in vitro dissolution of twelve tablets containing valsartan and atenolol was performed using buffer solutions (ph 1.2; 4.5; 6.8) as the dissolution media at 50 rpm by the usp apparatus ii. the dissolution was studied in a 900 ml volume of buffer solution at 37 °c (±0.5) using the paddle method. one ml of sample was withdrawn and replaced with fresh dis solution medium at the time intervals of 5, 15, 30, 45 minutes [29-32]. the concentrations of valsartan and atenolol in the samples (drug content and dissolution investigation) were determined by the suggested hplc method. results previously, we have made method development of valsartan and atenolol in dosage forms. the optimum mobile phase composition was composed of 20% acetonitrile, 80% of 0.16% ammonium acetate and 0.2% of 1.5 m tetramethylammonium hydroxide (v/v), pumped with 1.0 ml/min at 30 °c set temperature of column oven, with uv detector set to 225 nm and 237 nm wavelength. analyses were performed by means of the column discovery c18 (4.6 mm i.d.×150 mm, 5 μm) (fig. 3). the results of percentage purity of all the brands are shown in table 1. the drug content was determined to be highest for valsartan-1 and atenolol-4. the drug content was assessed once and compared with the calibration curve. the correlation coefficient (r2) was 0.9991 and the regression equation was y = 61.39x+0.3117. dissolution test is used to determine the quality of the drug. comparative dissolution kinetics test is used at all stages of the drugs life cycle. in the development of dosage form for comparative dissolution kinetics test allows assessing the technological correctness of techniques, and thereby increase the probability of positive results for future studies of bioequivalence. in addition to routine quality control tests, comparative dissolution tests are used to waive bioequivalence requirements (biowaivers) for lower strengths of a dosage form. dissolution study is an important parameter used to predict the bioavailability and in vivo drug release performance. dissolution study is very significant in determining the release of drug from different dosage forms including tablets. the active absorption of oral dosage forms depend on adequate release of drug. comparative dissolution profiles are shown in table 2. the point of 15 min is critical and decisive. medicine is considered very quick soluble when fig. 3. chromatogram obtained using shimadzu nexera xr uplc system and mobile phase 20% acetonitrile, 80% of 0.16% ammonium acetate and 0.2% of 1.5 m tetramethylammonium hydroxide (v/v), column discovery c18 (4.6 mm i.d.×150 mm, 5 μm) at 2 wavelengths 225 nm and 237 nm. table 1. drug content of different brands of valsartan and atenolol tablets brand code drug content (%)n=20 valsartan-1 (innovator) 99.82±2.24 valsartan-2 98.12±3.65 valsartan-3 99.11±2.74 valsartan-4 98.01±3.64 atenolol-1 (innovator) 99.11±1.89 atenolol-2 97.62±3.67 atenolol-3 98.55±1.95 atenolol-4 99.92±2.73 k.y. peleshok 55 p h a r m a c y issn 2413-6077. ijmmr 2020 vol. 6 issue 2 at least 85% of the active substance dissolves in 15 minutes, quickly soluble – when at least 85% of the active substance dissolves in 30 minutes. according to the obtained data, the equivalence of dissolution profiles for all recommended dissolution media has been established (рн 1.2, 4.5, and 6.8) for the studied drugs. in all three dissolution media, the releases of valsartan and atenolol of all dosage forms were more than 85% in 15 min (table 2). the dissolution profile of all the selected brands was within the standard limits and was acceptable. conclusions analytical method development is an integral part of the quality control measurements and evaluation of dissolution test. our previously developed hplc method was essential for quality control of a large number of samples in short time intervals. unavailability and price of innovator brand urges patients to go for alternate options including generic brands. the selected brands were evaluated and compared with those of reference or innovator brand to assure the potential for cure of the disease. moreover, the results of dissolution studies of all the brands were within the standard limits. this suggested that the proper gmp guidelines were followed during the manufacturing of these brands that proved a good quality. hence, these generics may be considered to be a substitute for innovator brand in case of unavailability. thus, all the brands selected for the study complied with the standard specifications and the definite observations on similar efficacy of these generics may be obtained after performing the in vivo studies. therefore, the method developed by our group is suitable for the routine quality control analysis of any pharmaceutical preparation containing two tested drugs with the suggested chromatographic method with advantages for checking quality during dissolution studies of their pharmaceutical preparations. conflict of interests authors declare no conflict of interest. funding author is grateful to the ministry of health of ukraine fund for providing scholarship for studies related to solutions for development of original combinations of antihypertensive agents, their analysis and standardization (№ 509 24.02.2020). table 2. comparative dissolution data of valsartan and atenolol in selected brands brand code medium % dissolved15 min % dissolved 30 min valsartan-1 ph 1.2 95.18 94.59 ph 4.5 93.84 93.02 ph 6.8 92.43 93.67 valsartan-2 ph 1.2 89.98 87.37 ph 4.5 87.38 89.84 ph 6.8 85.19 86.38 valsartan-3 ph 1.2 85.28 86.58 ph 4.5 87.68 90.01 ph 6.8 86.84 88.67 valsartan-4 ph 1.2 85.89 85.98 ph 4.5 85.96 85.78 ph 6.8 86.94 86.05 atenolol-1 ph 1.2 93.96 95.95 ph 4.5 92.56 93.36 ph 6.8 90.93 92.58 atenolol-2 ph 1.2 91.94 92.67 ph 4.5 89.49 93.06 ph 6.8 87.56 90.94 atenolol-3 ph 1.2 91.82 93.37 ph 4.5 90.01 91.65 ph 6.8 86.05 89.95 atenolol-4 ph 1.2 95.96 96.03 ph 4.5 92.94 94.56 ph 6.8 91.46 93.14 k.y. peleshok 56 p h a r m a c y issn 2413-6077. ijmmr 2020 vol. 6 issue 2 визначення контролю якості та in vitro біоеквівалентності таблеток валсартану та атенололу ринку україни к. пелешок тернопільський національний медичний університет імені і. я. горбачевського, тернопіль, україна вступ. актуальність проблеми артеріальної гіпертензії визначається її високою популяційною частотою, значним тягарем хвороби, ризиком інвалідизації та впливом на тривалість життя людини. у світових і вітчизняних рекомендаціях з діагностики та лікування артеріальної гіпертензії чітко рекомендовано раціональні комбінації препаратів різних фармакологічних груп при неефективності монотерапії для досягнення клінічного ефекту фармакотерапії. тому, інноваційна фармацевтична розробка комбінації антигіпертензивних засобів та створення вітчизняних лікарських засобів антигіпертензивної дії є актуальним завданням сучасної фармації. мета. здійснити контроль якості та оцінити тест розчинення різних марок таблеток валсартану та атенололу, які є представленими на ринку україни. методи. концентрації валсартану та атенололу у зразках (вміст в лікарських формах та тест розчинення) визначалися запропонованим методом верх. результати. результати випробувань, проведених для кількісного визначення визначення афі таблеток, є прийнятними для всіх обраних марок. було встановлено, що коефіцієнт кореляції (r2) становить 0.9991 та рівняння регресії y=61.39x+0.3117. доведено, що для досліджуваних препаратів спостерігається еквівалентність профілів розчинення для всіх рекомендованих середовищ розчинення (рн 1.2, 4.5 та 6.8). у всіх трьох середовищах розчинення вивільнення валсартану та атенололу всіх лікарських форм перевищують 85% за 15 хв. оцінка розчинення всіх вибраних марок в межах стандартних лімітів та є прийнятною. висновки. розробка аналітичної методики є невід'ємною частиною контролю якості та оцінки тесту розчинення. розроблена нами методика верх є важливою для контролю якості великої кількості зразків за короткі проміжки часу. тому метод розроблений нашою групою, підходить для рутинного аналізу якості будь-якого лікарського препарату, що містить два випробувані афі із запропонованими перевагами хроматографічного методу для перевірки якості під час досліджень розчинення їх лікарських форм. ключові слова: гіпертензія; валсартан; атенолол; високо ефективна рідинна хроматографія; in vitro біоеквівалентність; розчинення information about the author peleshok kateryna – assistant professor, i. horbachevsky ternopil national medical university, ternopil, ukraine. orcid 0000-0002-1816-6530, e-mail: peleshok@tdmu.edu.ua references 1. gupta kr, wadodkar ar, wadodkar sg. uv-spectrophotometric methods for estimation of valsartan in bulk and tablet dosage form. international journal of chemtech research. 2010;2:98589. 2. rao gs, rao gv, vardhan s, ramachandran d. development and validation of new uv-spectrophotometric assay method for valsartan in pure and in formulations. journal of chemical and phar maceutical research. 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hydrochlorthiazide using internal standard by rp-hplc. j chromat separation techniq. 2013;4:1-5. 28. gradman ah, basile jn, carter bl, bakris gl. american society of hypertension writing, g., combination therapy in hypertension. journal of the american society of hypertension. 2010;4:90-98. doi: https://doi.org/10.1016/j.jash.2010.03.001 k.y. peleshok 58 p h a r m a c y issn 2413-6077. ijmmr 2020 vol. 6 issue 2 29. hanson r, gray v. handbook on dissolution testing. 3rd ed. publisher: dissolution technologiesgies, usa, 2004. 199 р. 30. guidance for industry: waiver of in vivo bioavailability and bioequivalence studies for immediaterelease solid oral dosage forms based on a bio pharmaceutics classification system / u.s. department of health and human services, food and drug administration (hhs-fda). center for drug evaluation and research (cder). 2017. url: https://www.fda. gov/media/70963/download doi: http://academy.gmp-compliance.org/ guidemgr/files/ucm070246.pdf. 31. popy fa, dewan i, parvin mn, islam sma. evaluation of in vitro equivalence for tablets containing the poorly water-soluble compound atorvastatin. dissol technol. 2012;19(4):30-33. doi: https://doi.org/10.14227/dt190412p30 32. costa p, lobo jms. modeling and comparison of dissolution profiles. eur j pharm sci. 2001;13:123– 33. d o i : h t t p s : / / d o i . o r g / 1 0 . 1 0 1 6 / s 0 9 2 8 0987(01)00095-1 received 27 nov 2020; revised 21 dec 2020; accepted 23 dec 2020. this is open access article distributed under the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. k.y. peleshok 16 in t e r n a l m e d ic in e issn 2413-6077. ijmmr 2020 vol. 6 issue 2 doi 10.11603/ijmmr.2413-6077.2020.2.11693 diagnostic utility of leukocyte parameters in the patients with acute myocardial infarction *d.v. zhehestovska, m.v. hrebenyk i. horbachevsky ternopil national medical university, ternopil, ukraine background. inflammation is one of the key players in acute myocardial infarction (ami). one of the ways to evaluate it indirectly is to analyze leukocyte parameters of complete blood count (cbc), which is a routine and affordable method of diagnosis. leukocyte counts can provide additional information about the course, as well as a potential prognosis for complications of ami. we suggest that the dynamic changes of cbc during the treatment of the patients with ami may be of value to assess the prognosis of the course of the disease, and therefore require further study. objective. the aim of the study to evaluate the diagnostic and prognostic potential of leukocyte indexes of cbc, in particular the levels of leukocytes, lymphocytes, neutrophils and n/l, wbc/mpv, plt/l ratios in the patients with ami at the time of hospitalization and on the 7th day of hospital stay. methods. the study involved 204 individuals: 152 patients with ami (group 1), 30 patients with stable coronary heart disease (group 2) and 24 healthy volunteers (group 3). hemogram parameters and their ratios, in particular the levels of leukocytes, lymphocytes, neutrophils, esr, as well as the ratios of n/l and plt/l were studied. results. levels of leukocytes, neutrophils, lymphocytes, as well as n/l, wbc/mpv, mpv/l ratios were significantly higher in the patients with ami compared to other groups. the best diagnostic value had such indicators as the total number of leukocytes (sensitivity 71.7%, specificity 69.7%, auc 0.794), the absolute number of granulocytes (sensitivity 81.7%, specificity 77.4%, auc 0.803), the n/l ratio (sensitivity 75.0%, specificity 71.7%, auc 0.791) and the wbc/mpv ratio (sensitivity 76.7%, specificity 62.3%, auc 0.760). the plt/l ratio calculated on the 7th day of hospital stay correlated with the risk of in-hospital (r=0.369, p=0.002) and 6-month mortality (r=0.338, p=0.004) according to the grace score. conclusions. leukocytes, granulocytes, n/l and wbc/mpv ratios had a fairly high diagnostic value for the patients with ami. regarding the prognostic potential assessment, only the plt/l ratio on the 7th day of hospitalization correlated with the risk of in-hospital and 6-month mortality. this proves the importance of assessing cbc parameters not only at the time of hospitalization, but also in the dynamics of ami. keywords: inflammation; acute myocardial infarction; complete blood count; leukocytes. introduction inflammation plays a key role in the pathophysiology of acute myocardial infarction (ami), both at the time of its development and later on [1, 2]. leukocytes are the main contributors in the formation and subsequent destabilization of fibroatheroma. at the same time, they are closely related to activation of platelets increasing their prothrombotic potential [3, 4]. different white blood cell (wbc) subtypes play their own role in this process. there are several studies that prove not only the diagnostic but also the prognostic value of assessment of leukocyte indexes and their ratios in management of the patients with ami *corresponding author: diana zhehestovska, phd student, i. horbachevsky ternopil national medical university, ternopil, 46000, ukraine. e-mail: zhehestovska_dv@tdmu.edu.ua international journal of medicine and medical research 2020, volume 6, issue 2, p. 16-21 copyright © 2020, tnmu, all rights reserved d.v. zhehestovska et al. [5–7]. however, most of these works are focused on the complete blood count (cbc) parameters only at the time of hospitalization. we suggest that the dynamic changes of cbc during treatment of the patients with ami may be of value to assess the prognosis of the disease course and therefore require further study. methods the study involved 152 inpatients with ami – group 1. besides, two more study groups were formed: a comparison group, which included patients with stable ischemic heart disease (sihd) (n=30) – group 2, and a control group of healthy volunteers (n=28) – group 3. the study was carried out at ternopil city municipal hospi tal no. 2 (ukraine) in october 2018 – june 2019. 17 in t e r n a l m e d ic in e issn 2413-6077. ijmmr 2020 vol. 6 issue 2 the diagnosis of ami was verified following the clinical guidelines [8, 9]. clinical symptoms of ami, in particular the duration of angina pectoris and symptom-to-balloon time, the results of biochemical blood tests, angiographic protocols were evaluated. in addition, the risk of complications for acute coronary syndrome was assessed using the grace score [10, 11]. blood sampling was performed from the ulnar vein in the first hour and on the 7th day after hospitalization. cbc was performed using an automated hematology analyzer; in addition, a manual count of leukocyte fractions in peripheral blood was performed, as well as the erythrocyte sedimentation rate measurement (esr). statistical data was processed using the application package spss v25.0, as well as microsoft excel spreadsheets. the data were pre sented in the format of “mean ± standard error of the mean”. the significance of the diffe rences between the two independent samples in the case of normal data distribution was determined using the student’s t-test; in the case of abnormal data distribution – the non-parametric mann-whitney test. to compare three or more independent samples, one-factor analysis of variances was used – the anova (analysis of variance), in the case of abnormal data distribution – the kruskal-wallis ranking criterion. the roc analysis was used to identify the diagnostic value of individual zak indicators. such parameters as an area under the curve (auc), as well as the sensitivity and specificity of the test, were analyzed. the pearson’s correlation analysis was used to assess the relationships between the studied indicators with a normal distribution of data, and the spearman’s correlation analysis – in the case of abnormal distribution. the null hypothesis was denied at p<0.05. the correlation coefficient was evaluated according to the following criteria: r<0.3 – weak relationship; r=0.30–0.49 – moderate; r=0.50–0.69 – significant; r=0.70 – 0.89 – strong; r>0.90 – very strong, close to the functional association. results a mean age of the patients with ami was (62.91±10.90) years old, among them the male predominated (n=116, 76.3%). more than half of the subjects had concomitant pathology in the form of hypertension (n=134, 88.16%) and diabetes mellitus (n=29, 19.08%). comparing the parameters of the cbc between the three groups (table 1) it was established that in the group of patients with ami the level of leukocytes was significantly higher than in the control and comparison groups. there was no significant difference in table 1. comparison of cbc parameters between the patients with ami, sihd and healthy volunteers group 1 (n=152) group 2 (n=30) group 3 (n=24) р-value (anova) р value age, years 63.11±0.89 56.13±1.66 45.75±3.30 <0.001 0.01а,b,c men, n (%) 116 (76.3) 24 (77.4) 17 (70.8) ns ns wbc, 109/l 9.10±0.24 6.89±0.26 6.45±0.35 <0.001 <0.001a,b lymphocytes, 109/l 1.85±0.13 2.14±0.12 2.40±0.18 0.038 0.023b monocytes, 109/l 0.51±0.06 0.64±0.16 0.78±0.20 ns ns granulocytes, 109/l 7.01±0.41 5.29±0.94 3.66±0.33 0.001 <0.001b n/l ratio 5.54±0.64 2.53±0.31 1.64±0.17 <0.001 <0.001а,b 0.015c wbc/mpv ratio 1.03±0.05 0.76±0.03 0.78±0.04 <0.001 <0.001а 0.002b mpv/l ratio 7.00±0.66 4.63±0.36 5.13±0.31 0.001 0.002а <0.001b plt/l ratio 161.59±11.86 113.99±9.07 114.36±7.83 0.004 0.02а 0.001b esr, mm/hour 11.20±0.75 8.26±0.81 7.71±0.92 0.048 <0.05а,b notes: а – comparing groups 1 and 2, b – comparing groups 1 and 3, с – comparing groups 2 and 3. ns – not significant wbc – white blood cells, n/l ratio – neutrophils to lymphocytes ratio, wbc/mpv ratio – white blood cells to mean platelet volume ratio, mpv/l ratio – mean platelet volume to lymphocyte ratio, plt/l ratio – platelet count to lymphocytes ratio, esr – erythrocyte sedimentation rate. d.v. zhehestovska et al. 18 in t e r n a l m e d ic in e issn 2413-6077. ijmmr 2020 vol. 6 issue 2 the absolute number of lymphocytes and granulocytes between the ami and comparison groups, but such differences were found when comparing the ami patients and healthy volunteers: in the ami group, there was a significantly lower lymphocyte count and higher granulocytes. the n/l ratio differed between the three groups and was the highest in the patients with ami (p<0.001). the ratios that simultaneously reflected two links in the pathogenesis of coronary heart disease: inflammation and platelet activation, such as plt/l, wbc/mpv and mpv/l, were of particular interest. all of them were significantly higher in the patients with ami, compared with the control and comparison groups. taking into account the obtained results of average values comparison, the diagnostic value was determined, as well as the sensitivity and specificity of certain parameters of the cbc in the patients with ami (fig. 1). as seen in table 2, the indicators such as total leukocyte count, absolute granulocyte count, n/l ratio, and wbc/mpv ratio were of the best diagnostic value. a negative correlation was found between symptom-to-balloon time and the absolute level of lymphocytes (r=-0.38; p=0.008), while the positive correlation was determined with the following indicators: the n/l (r=0.370; p=0.07), plt/l (r=0.380; p=0.06) and mpv/l (r=0.351; p=0.011) ratios. also relationships were found between the duration of angina, i.e. the duration of ischemia and the number of granulocytes (r=0.366; p=0.004), the n/l (r=0.370; p=0.004) and plt/l (r=0.260; p=0.045) ratios and the number of lymphocytes (r=0.268; p=0.038). the relationship between the level of cpk mb and the n/l ratio (r=0.567; p<0.001), as well as hematocrit (r=0.406; p=0.004) was also established. any significant relationships between cbc and prognostic markers of ami, such as the grace score, have not been established at this stage. the dynamics of general blood test in the patients with ami were further analyzed (table 3). a decrease in the levels of leukocytes and neutrophils was evidenced (p<0.001 and p=0.012). instead, the esr increased significantly. discussion high levels of neutrophils in the patients with ami found in our study are associated with fig. 1. receiver operating characteristics analysis of leukocyte parameters in the ami patients. table 2. diagnostic sensitivity and specificity of the cbc parameters in the ami patients auc sensitivity, % specificity, % cut-off point wbc, 109/l 0.794 71.7 69.7 7.61 granulocytes, 109/l 0.803 81.7 77.4 4.99 n/l ratio 0.791 75.0 71.7 2.40 plt/l ratio 0.654 65.0 52.8 108.49 wbc/mpv ratio 0.760 76.7 62.3 0.85 mpv/l ratio 0.051 66.7 64.2 4.44 notes: auc – area under the curve. wbc – white blood cells, n/l ratio – neutrophils to lymphocytes ratio, wbc/mpv ratio – white blood cells to mean platelet volume ratio, mpv/l ratio – mean platelet volume to lymphocyte ratio, plt/l ratio – platelet count to lymphocytes ratio, esr – erythrocyte sedimentation rate. d.v. zhehestovska et al. 19 in t e r n a l m e d ic in e issn 2413-6077. ijmmr 2020 vol. 6 issue 2 the formation of platelet-leukocyte aggregates in the lumen of the vessel that leads to their increase in the area of myocardial infarction. also, neutrophils can affect platelet function by direct adhesion or by secretory factors. in contrast, the number of lymphocytes in ami tends to decrease. we were able to confirm this pattern when comparing patients with ami and healthy volunteers, at the same time there were no differences in the group of patients with stable coronary heart disease. this can be explained by the fact that the decrease in the number of lymphocytes is associated with physiological stress, which leads to increased cortisol levels and activates the process of apoptosis in the lymphocytes [12]. thus, in the case of early admission to the hospital, this index may simply not have time to decrease. this is confirmed by the correlation we found, according to which the longer the angina pectoris lasted, the lower the patient's lymphocyte level was. given that neutrophils and lymphocytes are the cells with the opposite effect in the context of vascular inflammation, it is important to assess not only their absolute numbers but also the balance between them. one of the potential ways to do this is to evaluate the n/l ratio, which is considered to be an indicator of systemic inflammation [13]. according to our results, it not only significantly differed in the patients from the study and comparison groups, but also proved a significant positive correlation with the marker of myocardial necrosis – cpk mb. when assessing the hemogram on the 7th day, a significant decrease in the level of leukocytes, due to neutrophils, and an increase in esr were noted. this phenomenon is also known as the “scissors symptom” or “crossover symptom” and is an evidence of the necroresorptive syndrome, denoted by a systemic inflammatory response to the entry of myocardial breakdown products into the bloodstream. thus, the number of neutrophils, which is traditionally the highest on the 1st-3rd day after the development of ami, dets to norm on the 5th-7th days due to active phagocytosis of dead neutrophils in the infarct area provided by macrophages [10]. the increase of esr is caused by the fact that normally the charge of the erythrocyte membrane is negative and this allows them to repel each other. at the same time, pro-inflammatory proteins, in particular fibrinogen and c-reactive protein, have a positive charge and can significantly affect the state of the erythrocyte membrane causing their further aggregation [11]. when assessing the prognostic potential of individual indicators of cbc, we were able to identify it only in relation to the plt/l ratio on the 7th day of ami. the fact that the plt/l ratio correlated with the risk of mortality only in 1 week is most likely explained by the platelet resistance to antiplatelet therapy during the treatment course, as well as to the stimulating effect of inflammation on megakaryocyte proliferation [14, 15]. the advantage of plt/l ratio, compared to individual leukocyte and platelet indices, is that it reflects two interdependent processes of inflammation and platelet activation and has already proven to be a good prognostic marker of ami, in particular regarding the prediction of left ventricular (lv) thrombus, remodeling of lv in the postinfarction period, as well as all-case mortality [14, 16–18]. table 3. comparison of cbc parameters in the ami patients at the time of hospitalization and on the 7th day after mi day 1 day 7 р value wbc, 109/l 9.34±0.32 8.07±0.27 <0.001 lymphocytes, 109/l 2.10±0.19 2.09±0.17 ns monocytes, 109/l 0.57±0.11 0.71±0.17 ns granulocytes, 109/l 6.74±0.50 5.30±0.49 0.012 n/l ratio 4.28±0.63 2.92±0.29 ns wbc/mpv ratio 1.06±0.8 0.88±0.05 ns mpv/l ratio 7.16±1.02 4.58±0.28 0.015 plt/l ratio 141.45±14.50 145.31±12.05 ns esr, mm/hour 12.34±1.21 22.88±1.45 <0.001 notes. wbc – white blood cells, n/l ratio – neutrophils to lymphocytes ratio, wbc/mpv ratio – white blood cells to mean platelet volume ratio, mpv/l ratio – mean platelet volume to lymphocyte ratio, plt/l ratio – platelet count to lymphocytes ratio, esr – erythrocyte sedimentation rate. d.v. zhehestovska et al. 20 in t e r n a l m e d ic in e issn 2413-6077. ijmmr 2020 vol. 6 issue 2 conclusions levels of leukocytes, granulocytes, lymphocytes, as well as n/l, wbc/mpv ratios, are available and informative markers that provide additional information about the processes of inflammation during the acute phase of myocardial infarction. we were unable to establish a link between cbc indexes at the time of hospitalization and prognostic factors of ami. instead, the parameters that were obtained on the 7th day of treatment, in particular the plt/l ratio was associated with the risk of in-hospital and 6-month mortality. thus, we emphasize the importance of assessing the cbc indexes and ratios in the patients with ami in the dynamics as an inexpensive and informative method for post-mi risk stratification. conflicts of interest authors declare no conflict of interest. author's contributions diana v. zhehestovska – investigation, conceptualization, data curation, formal analysis, writing – original draft. marian v. hrebenyk – data curation, writing – reviewing and editing. діагностичні можливості лейкоцитарних показників серед пацієнтів з гострим інфарктом міокарда д.в. жегестовська, м.в. гребеник тернопільський державний медичний університет імені і. я. горбачевського, тернопіль, україна вступ. запалення відіграє визначальну роль в процесі розвитку та перебігу гострого інфаркту міокарда (гім). аналіз лейкоцитарних показників загального аналізу крові (зак) є одним із способів його опосередкованої оцінки, це рутинний і доступний метод діагностики. ми припускаємо, що динамічні зміни параметрів зак в процесі лікування пацієнтів з гім можуть становити цінність, зокрема і для оцінки прогнозу подальшого перебігу захворювання, а тому потребують подальшого вивчення. мета. оцінити діагностичний та прогностичний потенціал лейкоцитарних індексів зак, зокрема кількісних рівнів лейкоцитів (wbc), лімфоцитів (l), нейтрофілів (n) та співвідношень n/l, wbc/mpv (середнього рівня тпромбоцитів, mpv), plt/l (співвідношення числа тромбоцитів до кількості лімфоцитів) у пацієнтів з гім на момент госпіталізації та на 7-му добу перебування в стаціонарі. матеріали та методи. в дослідженні взяло участь 204 особи (152 пацієнтів з гім (група 1), 30 пацієнтів з стабільною іхс (група 2) та 24 здорові добровольці (група 3)). досліджували показники гемограми та їх співвідношення, зокрема рівні лейкоцитів, лімфоцитів, нейтрофілів, шое, а також співвідношень n/l ratio та plt/l ratio. результати. рівні лейкоцитів, нейтрофілів, лімфоцитів, а також відношення n/l, wbc/mpv, mpv/l були достовірно вищими серед пацієнтів з гім порівняно із іншими групами. найкращою діагностичною цінністю володіли такі показники, як загальна кількість лейкоцитів (чутливість 71,7%, специфічність 69,7%, аuc 0,794), абсолютна кількість гранулоцитів (чутливість 81,7%, специфічність77,4%, auc 0,803), відношення n/l (чутливість 75,0%, специфічність 71,7%, аuc 0,791) та wbc/mpv (чутливість 76,7%, специфічність 62,3%, аuc 0,760). співвідношення plt/l вирахуване на 7-ий день перебування в стаціонарі корелювало з ризиком госпітальної (r = 0,369, р = 0,002) та 6-ти місячної смертності (r = 0,338; р = 0,004). висновки. показники лейкоцитів, гранулоцитів та співвідношення n/l і wbc/mpv володіли доволі високою діагностичною цінністю серед пацієнтів з гім. стосовно оцінки прогностичного потенціалу, то лише відношення plt/l на 7-ий день госпіталізації корелювало з ризиком госпітальної та 6-ти місячної смертності. це вказує на важливість оцінки зак не лише на момент госпіталізацій, а й в динаміці перебігу гім. ключові слова: запалення; гострий інфаркт міокарда; загальний аналіз крові; лейкоцити. information about the authors diana v. zhehestovska, phd student, i. horbachevsky ternopil national medical university, ternopil, ukraine orcid 0000-0001-6305-4143, e-mail: zhehestovska_dv@tdmu.edu.ua maryan v. hrebenyk, professor, head of the department of therapeutics and family medicine, i. horbachevsky ternopil national medical university, ternopil, ukraine orcid 0000-0002-8118-5742, e-mail: hrebenyk@tdmu.edu.ua d.v. zhehestovska et al. 21 in t e r n a l m e d ic in e issn 2413-6077. ijmmr 2020 vol. 6 issue 2 references 1. frangogiannis ng, smith cw, entman ml. the inflammatory response in myocardial infarction. cardiovasc res. 2002;53:31–47. 2. ferrari jp, lueneberg me, da silva rl, fattah t, gottschall cam, moreira dm. correlation between leukocyte count and infarct size in st segment elevation myocardial infarction. arch med sci atheroscler dis. 2016;1:e44–e48. 3. italiano je, hartwig jh. chapter 2 – megakaryocyte development and platelet formation. in: michelson adbt-p (third e (ed). academic press; 2013. p. 27-49. 4. korniluk a, koper-lenkiewicz om, kamińska j, kemona h, dymicka-piekarska v. mean platelet volume (mpv): new perspectives for an old marker in the course and prognosis of inflammatory conditions. mediators of inflammation. 2019 oct;2019. doi: https://doi.org/10.1155/2019/9213074 5. lassale c, curtis a, 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ratio predict left ventricular systolic dysfunction in patients with non-st elevated acute coro nary syndrome? anatol j cardiol. 2015;15:385–90. 18. akdag s, akyol a, asker m, ozturk f, gumrukcuoglu ha. the relation of platelet-lymphocyte ratio and coronary collateral circulation in patients with non-st segment elevation myocardial infarction. postep w kardiol interwencyjnej. 2016;12:224–30. received 28 oct 2020; revised 9 dec 2020; accepted 14 dec 2020. this is open access article distributed under the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. d.v. zhehestovska et al. 5 o b st e t r ic s a n d g y n e c o l o g y issn 2413-6077. ijmmr 2020 vol. 6 issue 2 *corresponding author: iryna kulyk, phd student, department of obstetrics and gynecology, i. horbachevsky ternopil national medical university, 1 maidan voli, ternopil, 46001 ukraine. email: kulyk_iryna1@ukr.net international journal of medicine and medical research 2020, volume 6, issue 2, p. 5-10 copyright © 2020, tnmu, all rights reserved i.i. kulyk et al. doi 10.11603/ijmmr.2413-6077.2020.2.12011 endometriosis-associated infertility: the role of hormones and its correction *i.i. kulyk, s.v. khmil i. horbachevsky ternopil national medical university, ternopil, ukraine background. endometriosis-associated infertility (eai) has a number of specific features, which are crucial in the choice of medical treatment. objective. the aim of the study is to analyze endocrine profile in women with eai before and after sclerotherapy and pregravid preparation (pp), which includes a vitamin complex ft 500 plus with inositol and vitamin d3. methods. the study involved 70 women aged 21-40 years with endometriosis-associated infertility. the comparison group included 30 women with tuboperitoneal infertility. elisa was used to determine concentrations of anti-mullerian hormone (amh), follicle stimulating hormone (fsh), luteinizing hormone (lh), progesterone and estradiol in blood serum using a standard kit by diagnostic systems laboratories, inc (usa). during two menstrual cycles the ft 500 plus was prescribed once a day from the 2nd/3rd day of the cycle, vitamin d3 was prescribed at the dose of 2,000 iu for women without its deficiency and in therapeutic doses in case of hypovitaminosis. the sclerotherapy with 95% ethanol solution was performed on the 6th-8th day of menstrual cycle. results. it was established that in women with eai undergoing pp and sclerotherapy the level of amh was lower (by 12.90%) as well as progesterone (by 9.84%), while fsh (by 14.47%), lh (by 21.14%) and estradiol (by 35.55%) was higher compare to the comparison group. at the same time, fsh (by 21.98%), lh (by 32.89%) and estradiol (by 32.23%) concentrations were significantly lower compare to their primary indices before sclerotherapy. conclusions. sclerotherapy and pp with a vitamin complex, inositol and vitamin d3 has a positive effect on endocrine profile in women with endometriosis-associated infertility keywords: infertility; endometriosis; hormones; sclerotherapy; inositol; vitamin d3. introduction genital endometriosis is one of the most common diseases in women of reproductive age. it attracts attention of scientists and practitioners around the world. according to the literature, every tenth woman of reproductive age suffers from endometriosis that is 176 million women (world population projection tables by country and group, 2010). the main symptoms of endometriosis are infertility, menorrhagia, dysmenorrhea, dyspareunia, chronic pelvic pain. in 20-25% of women, this disease is asymptomatic [1]. endometriosis-associated infertility is characterized by a number of specific features that must be considered when choosing treatment methods. in each case, treatment should be individual, taking into account all clinical factors, as well as the impact of the disease and the effect of treatment on the quality of woman’s life [2, 3]. the classic surgical treatment of endometriosis is necessary in cases of cyst capsule; according to the literature, it facilitates the lowest number of recurrences and the highest number of clinical pregnancies [4-7]. however, it should be taken into account that besides the capsule, a significant part of healthy ovarian tissue with antral follicles is removed [8]. therefore, the search for less traumatic and more effective treatment, i.e. puncture of endometriomas and sclerotherapy, is carried on [9]. to date, the choice of treatment for women with endometriosis-associated infertility for assisted reproductive technology (art) programs is still controversial. according to evidencebased medicine it is established that in women with endometriosis-associated infertility, the balance between intracellular trace elements is disturbed; free radical oxidation processes are activated, which leads to oxidative stress, which consecutively may disturb the effectiveness of art. reactive oxygen species are usually significant in a number of reproductive tract functions, but their overproduction has a negative effect on estrogen levels, altering 6 o b st e t r ic s a n d g y n e c o l o g y issn 2413-6077. ijmmr 2020 vol. 6 issue 2 steroidogenesis, thereby preventing oocyte maturation and ovulation [10, 11]. therefore, we consider using antioxidants during pregravid preparation of patients with endometriosisassociated infertility before cos protocol reasonable. the aim of the study was to analyze concentrations of the reproductive hormones in women with endometriosis-associated infertility before and after sclerotherapy and pregravid preparation with a complex vitamin medication comprising inositol and vitamin d3. methods the study involved 70 women aged 21-40 years with endometriosis-associated infertility, who were treated in the medical center “clinic of professor stefan khmil” in 2015-2020. the comparison group included 30 women of the same age with tuboperitoneal infertility, diagnosed by laparoscopy or echosalpingography. exclusion criteria were for patients with endometriosis, stage 3-4, polycystic ovary syndrome, uterine fibroids (submucosal, symptomatic subserosal or intramural with a diameter of more than 2 cm), who were not included into the study groups. external genital endometriosis was verified by laparoscopy and confirmed by histopathological examination (endometrioid cyst of an ovary or both ovaries). the patients with endometriosis-associated infertility were divided into 2 groups. group 1 (n=34) involved women of reproductive age (21-40 years old) with endometrioid cysts of up to 6.5 cm in diameter, who underwent sclerotherapy and pregravid preparation with a complex vitamin medication comprising inositol and vitamin d3; group 2 (n=36) comprised women, who underwent sclerotherapy. the presence of cysts was confirmed by ultrasound and bimanual examination. determination of hormone indicators was performed in different phases of the cycle before and after sclerotherapy and pregravid treatment. determination of concentrations of reproductive hormones was performed in the certified laboratory of the medical center “clinic of professor stefan khmil”. elisa was used to determine the concentrations of anti-mullerian hormone (amh), follicle stimulating hormone (fsh), luteinizing hormone (lh), progesterone and estradiol (e2) in blood serum according to manufacturer’s instructions using a standard set of reagents provided by diagnostic systems laboratories, inc. (usa), by means of the statfax analyzer. the vitamin complex ft 500 plus for women of group 1 was prescribed at the dose of 1 sachet once a day from the 2nd or 3rd day of the cycle, after taking blood test for hormones, i.e. amh, fsh and lh, during 2 menstrual cycles (cycle in which sclerotherapy was performed and subsequent cycle). vitamin d3 was prescribed at prophylactic doses of 2,000 iu to those patients who were not diagnosed with a deficiency of this vitamin and at medium therapeutic doses in cases of hypovitaminosis during 2 menstrual cycles. the procedure of sclerotherapy was performed in a sterile surgery room with or without general anesthesia on the 6th-8th day of the menstrual cycle by transvaginal puncture of the cyst and aspiration of its contents under the control of transvaginal ultrasound, introduction of sclerosing solution into the cyst capsule (without violating its integrity). 76 % ethanol solution was used as a sclerosant installed in a volume of 50%-90% of the cyst size under ultrasound control with its subsequent aspi ration. statistical analysis of the results was performed using microsoft office excel and statistica 7.0 software. the choice of the data analysis method was based on the number of the groups, their distribution, as well as the equality of variances. all data were verified by the one sample kolmogorov-smirnov test to confirm normality. abnormal distribution data was analyzed by a nonparametric test (the mann-whitney u test and the kruskal-wallis test). the indicators with abnormal distribution were defined as me (q25; q75) (medians and q25 and q75 quartiles). the biochemical findings were analyzed by repeated non-parametric anova test for multiple comparisons. a probability level of less than 0.05 was considered to be statistically significant. results study of the reproductive hormones of the women with endometriosis-associated infertility proved a significantly lower level of anti-mullerian hormone (amh) in women before and after sclerotherapy compare to the comparison group. it was established that sclerotherapy in women with endometriosis-associated infertility caused a decrease of fsh by 20.31% compare to the indices before the therapy for endometriomas but was still statistically significantly higher compare to the comparison group (by 20.75%). also, a decrease of the lh concentration by 16.96% was evidenced in the patients after i.i. kulyk et al. 7 o b st e t r ic s a n d g y n e c o l o g y issn 2413-6077. ijmmr 2020 vol. 6 issue 2 sclerotherapy compare to the indices before the therapy for endometriomas. it was statistically significantly higher than those of the comparison group (by 39.02%). the level of estradiol in blood serum of the women with endometriosis-associated infertility after sclerotherapy was significantly lower by 26.63% compared to that before surgery, but was still significantly higher compare to the comparison group (by 41.11%). similar changes were observed for progesterone levels on the 19th-21st day of menstrual cycle (d.m.c.) in blood serum of the women with endometriosis-associated infertility after sclerotherapy; specifically, it increased by 14.94% compare to the indices before sclerotherapy, but was by 25.11% lower compare to the comparison group. please note that sclerotherapy did not affect the level of prolactin (table 1). the women with endometriosis-associated infertility, who underwent pregravid preparation with a complex vitamin medication comprising inositol and vitamin d3 as well as sclerotherapy had significantly lower levels of amh (by 1 2 . 9 0 % ) , p r o g e s t e r o n e ( b y 9 . 8 4 % ) a n d significantly higher levels of fsh (by 14.47%), lh (by 21.14%) estradiol (by 35.55%) compare to the comparison group. at the same time, significantly lower levels of fsh (by 21.98%), lh (by 32.89%) and estradiol (by 32.23%) were evidenced compare to those before sclerotherapy (table 2). table 1. concentration of reproductive hormones in the women with endometriosis-associated infertility before and after sclerotherapy and no pregravid preparation (me (q25; q75)) indicators comparison group (n=30) before sclerotherapy (n=36) after sclerotherapy (n=36) амh, pmol/l 1.75 (1.56; 1.92) 1.50* (1.30; 1.73) 1.50* (1.30; 1.73) fsh, iu/l 7.95 (6.93; 8.50) 11.55* (10.90; 12.43) 9.60* (8.78; 10.60) lh, iu/l 6.15 (5.45; 6.80) 10.00* (9.30; 10.70) 8.55* (7.98; 9.23) estradiol on the 2nd-3rd d.m.c., pmol/l 36.85 (33.70; 40.08) 65.85* (60.40; 73.90) 52.00* (48.63; 55.53) progesterone on the 19th-21st d.m.c. nmol/l 13.95 (12.63; 15.58) 9.70* (8.18; 11.43) 11.15* (9.63; 12.23) prolactin, ng/ml 10.95 (10.40; 12.28) 10.40 (9.60; 11.40) 10.30 (9.50; 11.08) notes. statistically significant difference (p<0.05) compared to: * – the comparison group. me (q25; q75) (median, q25 and q75 quartiles). table 2. concentration of reproductive hormones in the women with endometriosis-associated infertility before and after sclerotherapy, who underwent pregravid preparation with a complex vitamin medication comprising inositol and vitamin d3 (me (q25; q75)) indicators comparison group (n=30) before sclerotherapy (n=34) after sclerotherapy (n=34) амh, pmol/l 1.75 (1.56; 1,92) 1.55* (1.40; 1.78) 1.50* (1.30; 1.80) fsh, iu/l 7.95 (6.93; 8.50) 11.10* (10.50; 12.40) 9.10* (8.30; 9.88) lh, iu/l 6,15 (5.45; 6.80) 9.90* (8.65; 11.00) 7.45*# (6.93; 8.65) estradiol on the 2nd-3rd d.m.c., pmol/l 36.85 (33.70; 40.08) 66.05* (61.50; 74.10) 49.95* (43.73; 55.38) progesterone on the 19th-21st d.m.c. nmol/l 13.95 (12.63; 15.58) 10,45* (8.95; 12.48) 12.70*# (11.13; 13.50) prolactin, ng/ml 10.95 (10.40; 12.28) 11.00 (9.53; 12.18) 10.80 (10.00; 12.00) note. statistically significant difference (p<0.05) compared to: * – the comparison group, # – data before sclerotherapy. me (q25; q75) (median, q25 and q75 quartiles). i.i. kulyk et al. 8 o b st e t r ic s a n d g y n e c o l o g y issn 2413-6077. ijmmr 2020 vol. 6 issue 2 in order to compare the data, we analyzed the effect of pregravid preparation on the concentration of reproductive hormones in women with endometriosis-associated infertility after sclerotherapy, so the value of hormones before this surgical manipulation, we considered 100%. it was found that the use of a complex vitamin medication comprising inositol, as well as vitamin d3 caused a decrease of lh level (by 10.25%), as well as an increase of progesterone level (by 6.58%). discussion the results of the study proved a positive effect of sclerotherapy on the endocrine profile of women with endometriosis-associated infertility. according to the literature, sclerotherapy of endometriomas is recommended for women with recurrent endometrioid cysts [12] to preserve the ovarian reserve avoiding surgical excision [13]. it was established that the recurrence rate after sclerotherapy ranged from 12.9% to 20% that almost did not differ from the indicators after laparoscopic cystectomy [14, 15]. it was found out that the number of antral follicles was increasing in the patients who underwent sclerotherapy for recurrent endometriomas [16, 17]. the amh blood level was an indirect marker of ovarian reserve. in our study, there were no significant changes in the amh level in blood serum before and after sclerotherapy that is consistent with other studies [18]. sclerotherapy can improve ovarian blood supply and follicle development [16], which consecutively may increase the probability of future fertility in the women undergoing sclerotherapy. the effect of sclerotherapy on the reproductive hormones level is still doubtful. thus, wang xiaotao et al. established decreased levels of e2, fsh and lh [19]. in a study of saeed alborzi et al., the effect of sclerotherapy was associated with ovarian activity; in particular, a decrease of the fsh level as well as an increase of the amh level and antral follicles [20]. on the other hand, aflatoonian a et al. did not prove any significant difference between the studied hormones (fsh, lh) before and after sclerotherapy. the difference of the results may be due to different sizes of cysts in the studied female patients (our study involved women with endometrioid cysts of up to 6.5 cm in diameter), presence of cystectomy in medical history, localization (unilateral or bilateral endometrioma), and recurrence of endometrioid cysts. pregravid preparation with a complex vitamin medication comprising inositol and vitamin d3 had an effect. inositol is a component of phospholipids and is a part of the cytoplasmic membranes as a phosphoinositide. inositol binds ca2+ channels and stimulates intracellular release of ca2+. increased intracellular ca2 + level is significant for oocyte maturation, fertilization, and embryonic development. a sufficient concentration of inositol in the follicular fluid reduces the free radicals’ level and membrane protein damage and increases the number of good quality oocytes [21]. 1.25-dihydroxyvitamin-d3 (vitamin d), except calcium-phosphate homeostasis, has a significant immunomodulatory effect (affects the local immune environment in an autocrine/paracrine way), affects the processes of cell proliferation and differentiation. studies have shown that vitamin d reduces concentration of anti-inflammatory cytokines: il-6, interferon-g, il-2, and tnf-a [21]. the combination of vitamin d3 multivitamin complex comprising inositol leads to normal ovarian activity, improves metabolic and hormonal profile, in particular, controls increased level of luteinizing hormone and oxidative stress [22]. conclusions sclerotherapy and pregravid preparation with a complex vitamin medication comprising inositol and vitamin d3 has a positive effect on the endocrine profile in women with endometriosis-associated infertility. conflicts of interest authors declare no conflict of interest. authors’ contributions iryna kulyk – investigation, conceptualization, data curation, formal analysis, writing – original draft. stefan khmil – data curation, writing – reviewing and editing. i.i. kulyk et al. 9 o b st e t r ic s a n d g y n e c o l o g y issn 2413-6077. ijmmr 2020 vol. 6 issue 2 особливості гормонального фону жінок з безпліддям на фоні ендометріозу та методи їх корекції і.і. кулик, с.в. хміль тернопільський національний медичний університет імені і.я. горбачевського, тернопіль, україна вступ. безпліддя на фоні генітального ендометріозу характеризується низкою специфічних особливостей, які необхідно врахувати при виборі методів лікування. мета дослідження – проаналізувати концентрацію гормонів репродуктивної системи у жінок з безпліддям на фоні ендометріозу до та після склеротерапії із застосуванням прегравідарної підготовки комплексним вітамінним препаратом з інозитолом та вітаміном d3. методи дослідження. у дослідження увійшло 70 жінок віком від 21 до 40 років з ендометріозасоційованим безпліддям та 30 жінок з трубно–перитонеальним фактором, як група порівняння. визначення концентрації гормонів (антимюллерового гормону (амг), фолоікулостимулюючого гормону (фсг, лютеїнізуючий гормон (лг), прогестерон та естрадіол) проводили за допомогою іфа з використанням наборів «diagnostic systems laboratories, inc.» (сша) на аналізаторі «statfax». препарат ft 500 plus для жінок 1групи призначали в дозуванні 1 саше – 1 раз в день з 2-го або 3-го дня циклу протягом 2-х менструальних циклів (цикл у якому проводилась склеро терапія та наступний цикл). вітамін d3 призначався у профілактичних дозах 2000 од жінкам, у яких не було діагностовано дефіциту даного вітаміну та в середньотерапевтичних дозах при гіповітамінозі протягом 2-х менструальних циклів. процедура склеротерапії проводилась в умовах стерильної операційної на 6–8-й день менструального циклу шляхом трансвагінальної пункції кісти та аспірації її вмісту під контролем трансвагінального узд, введення в капсулу кісти 95 % розчину етанолу. результати. у жінок з безпліддям на фоні ендометріозу, яким призначали прегравідарну підготовку комплексним вітамінним препаратом з інозитолом та вітаміном d3 та проводили склеротерапію, встановлено вірогідно нижчий рівень амг (на 12,90 %) та прогестерону (на 9,84 %) і вірогідно вищий рівень фсг (на 14,47 %), лг (на 21,14 %) та естрадіолу (на 35,55 %) відносно групи порівняння. при цьому встановлено вірогідно нижчі показники фсг (на 21,98 %), лг (на 32,89 %) та естрадіолу (на 32,23 %) проти їх значень до проведення склеротерапії. висновки. у кожному конкретному випадку лікування має бути індивідуальним, повністю враховувати всі клінічні фактори, а також вплив захворювання та ефект лікування на якість життя жінки. проведення склеротерапії із застосуванням прегравідарної підготовки комплексним вітамінним препаратом з інозитолом та вітаміном d3 у жінок з ендометріоз-асоційованим безпліддям має позитивний вплив на гормональний фон. ключові слова: безпліддя; ендометріоз; гормони; склеротерапія; інозитом; вітамін d3. information about the authors kulyk i.i. – phd student, department of obstetrics and gynecology, i. horbachevsky ternopil national medical university, ternopil, ukraine. orcid: https://orcid.org/0000-0003-3766-0259. e-mail: kylyk_iryna1@ukr.net khmil s.v. – professor of the department of obstetrics and gynecology, i. horbachevsky ternopil national medical university, ternopil, ukraine. orcid: https://orcid.org/0000-0003-0892-9861. e-mail: klinika_khmil@ukr.net references 1. abbas s, ihle p, koster i, schubert i. prevalence and incidence of diagnosed endometriosis and risk of endometriosis in patients with endometriosisrelated symptoms: findings from a statutory health insurance-based cohort in germany. eur j obstet gynecol reprod biol 2012;160:79–83. 2. giudice lc. clinical practice: endometriosis. n engl j med 2010; 362: 2389-98. doi: https://doi.org/10.1056/nejmcp1000274 3. bulun se. endometriosis. n engl j med 2009;360(3): 268-79. doi: https://doi.org/10.1056/nejmra0804690 4. shestakova ig, ipastova id. 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(in ukrainian). received 30 oct 2020; revised 26 nov 2020; accepted 02 dec 2020 this is open access article distributed under the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. i.i. kulyk et al. 76 b io m e d ic a l s c ie n c e s issn 2413-6077. ijmmr 2021 vol. 7 issue 2 doi 10.11603/ijmmr.2413-6077.2021.2.12657 evaluation of immunoblot results for determination of antibodies to lyme disease pathogens in children of ternopil region *s.o. nykytyuk, s.i. klymnyuk, i.m. klishch, s.s. levenets i. horbachevsky ternopil national medical university, ternopil, ukraine background. lyme disease (ld) is a multisystem disorder caused by borrelia burgdorferi and other similar tick-borne borrelia. objective. the aim of the research was to compare the results of the serological examination of children with different forms of lyme disease. methods. we observed the group of children (n=178) aged 1 to 14 years who were bitten by ticks. the control group consisted of 30 healthy children. ticks were identified using a stereomicroscopic seo system which included a stereomicroscope, a colour digital camera and a photoadapter. b. burgdorferi sensu lato (sl) (b. burgdorferi sensu stricto, b. afzeliі and b. garinii), b. miyamotoi, a. phagocytophilum dna in blood were determined by realtime pcr. baseline investigations related to clinical and immunological studies, including elisa and immunoblot, were performed. results. the survey covered 178 child parents bitten by ticks. borrelia burgdorferi sensu lato (b. afzelii, b. burgdorferi sensu stricto and b. garinii), b. miyamotoi and a. phagocytophilum were identified. serological results in children with different forms of lyme disease were compared. conclusions. it is established that b. burgdorferi sensu lato; b. miyamotoi; and a. phagocytophilum are pathogens that cause erythema migrans in children. the presence of specific igg (only positive results) to b. burgdorferi s.l. by immunoblot was confirmed in 83.8% of individuals who had positive and intermediate results in the elisa test. keywords: lyme disease; borreliosis; elisa; immunoblot; tick bite. *corresponding author: svitlana nykytyuk, md, phd, associate professor, i. horbachevsky ternopil national medical university, ternopil, 46001, ukraine. e-mail: androx@tdmu.edu.ua international journal of medicine and medical research 2021, volume 7, issue 2, p. 76-82 copyright © 2021, tnmu, all rights reserved introduction lyme disease (ld) is a multisystem disorder caused by borrelia burgdorferi and other similar tick-borne borrelia. this acute systemic disease often occurs in children and is characterized by the presence of erythema migrans (em), and in some untreated patients of inflammatory arthritis, erythema migrans as well. lyme disease, caused by borrelia burgdorferi, is the most common vector-borne disease. in western europe it is caused by b. afzelii та b. garinii, [2] whereas in the united states – by b.burgdorferi [3]. the epidemics of lyme disease is challenging in poland and ukraine. in western ukraine, b. burgdorferi s.l were revealed in 14.217.2% of adult ixodes scapularis ticks [4]. in 2020-2021, the disease incidence in ukraine and ternopil region was 10.62 and 20.05 per 100 thou sand population, respectively (public health center.org.ua). on the basis of mkh-10 allocate “lyme disease” a69.2. clinical manifestations of lyme disease can be divided into three stages: the early localized stage characterized by erythema migrans at the site of the tick bite, multiple erythema migrans, borrelia lymphocytoma; early disseminated form with early symptoms confined to peripheral nervous system, carditis or arthritis, late disseminated form – late symptoms confined to peripheral nervous and central nervous system, manifestations of late arth ri tis, cardiac complications. the pathogenesis, ecology, and epidemiology of lyme disease are well described; the developed and suggested antimicrobial treatment is very effective [2, 5]. the study aimed to assess the incidence of clinical suspicion of ld among children in ternopil region (western ukraine) by serological examination of children bitten by ticks. methods the children’s blood tests were studied in the laboratory of the centre for the study of s.o. nykytyuk et al. 77 b io m e d ic a l s c ie n c e s issn 2413-6077. ijmmr 2021 vol. 7 issue 2 s.o. nykytyuk et al. lyme disease and other tick-borne infections of i. horbachevsky ternopil national medical university. ticks were identified using a stereo­ microscopic seo-imaglab system. special defining tables were used for iden­ tification of ticks [6]. databases regarding the incidence of ld in children of ternopil region in 2017-2021 were used to evaluate ret rospective results. this study consists of two parts : the first part describes the data from the questionnaire and clinical examination of the patients, episodes of tick bites, and the second part is serological examination of the blood by elisa and immunoblot. the study involved 178 children aged 1 to 18 years, who visited to the centre for study of lyme disease of ternopil national medical university after being bitten by ticks. the control group consisted of 30 healthy boys and girls living in ternopil and ternopil region. they were not bitten by ticks and did not suffer from ld previously. the age and sex distribution in the control group corresponded to that in the control group. ticks were identified for transmissible in­ fections. dna of b. burgdorferi sensu lato (sl) (b. burgdorferi sensu strictо, b. afzeliі and b. garinii), b. miyamotoi, a. phagocytophilum were determined by real-time pcr using vector-best production test system (germany). the serological examination of the children with ld was made by two-stage diagnosis procedures, primarily using elisa and immunoblot for confirmation of the secondary re­ sults. antibodies to antigens of the b. burgdorferi s.l. complex in blood serum were determined by elisa using test systems by euroimmun ag (germany): class of igm­test system anti­bor­ relia burgdorferi elisa (igm), igg – anti-borrelia plus vlse elisa (igg). the results were evaluated quantitatively. the indicator > 22u/ml was considered positive, 16-22 u/ml – intermediate, < 16 u / ml – negative. to detect only igm against borrelia antigens, a specific line of the rn­at system was used, which contained natural purified ospc borrelia antigens of three species (b. afzelii, b. burgdorferi ss, b.garinii) and antigens p 39, p 41 and viѕe. to diagnose specific igg a line of the rn­at system was used, which contained vlse antigens of borrelia of three species (b. afzelii, b. burg dorferi s. s, and b. garinii) and other specific antigens: p18, 19, p20, p21, p58, ospc (p25), p39, p83, lipid ba, lipid bb. statistical processing of the results was performed using the methods of parametric and nonparametric statistics by computer programs microsoft office excel and statistica, estimating the absolute (n) and relative amount (%) of the indicators. the analysis of frequency was performed using pearson’s test χ2 and two-sided fisher’s exact test, the statistical significance of which was p<0.05. all the studies were performed according to the conclusion of the commission on bioethics of i. horbachevsky ternopil national medical university, dated september 1, 2021 (minutes no. 65). results erythema migrans was observed in 113 (63.4%), and arthritis in 15 (8.4%) individuals. nervous system disorders were present in 30 (16.8%) children; 18 (9.5%) children had an erythema-free form of the disease; 2 children (1.1%) complained about the cardiovascular disorders. all children bitten by ticks were divided into the following age groups (table 1). the largest age group was children of 8-12 years old (42.1% of the surveyed). among the surveyed, there were 83 (46.6%) boys and 95 (53.4%) girls. only 143 (80.3%) children had a tick bite, while others did not remember the bite. the examination of patients with an erythematous form of ld took into account the presence of a tick bite in the anamnesis and the accompanying intoxication­inflammatory syndrome, the pre­ sence of lesions of various organs and systems. in the clinical diagnosis of an erythematous form of ld the prevalent symptoms were: primary skin lesion, which was manifested by the erythema migrans, and the epidemiological history. subsequent examination revealed the presence of various pathogens in this category of patients. in children with erythema migrans, i.e. in 24.7% of 113 people, tick-borne infection with identified pathogens was confirmed (fig. 1), and in children with the disseminated form of the disease only the effects of the bite were observed. table 1. the age groups of children affected by ticks age categories of children (years) categories 1–3 4–7 8–12 13–18 number of children 18 39 75 46 78 b io m e d ic a l s c ie n c e s issn 2413-6077. ijmmr 2021 vol. 7 issue 2 when using the western immunoblot during the first 4 weeks of the disease (an early form of ld), both immunoglobulin m (igm) and immunoglobulin g (igg) were determined. since the probability of a false-positive test result for current infection is high, a positive igm test result is not recommended when determining the active phase of the disease in people who are sick for longer than 1 month. verification of the presence of specific igm antibodies was performed in the sera of 179 patients, 71 of whom had positive (56 people) or intermediate (15) results when tested by the elisa test. (table 2). it was found that in 56 patients with positive results, using the method of immunoblot (euroline borrelia rn-at) also found positive results in 17 (30.4%) persons, while intermediate was not detected. in children living in ternopil region, a positive elisa test was confirmed in 56 children (31.3%), an intermediate test in 15 (8.4%), and a negative test in 108 (60.3%) individuals. the results of blood screening for the presence of igg in elisa were positive in 28 subjects (15.6%), intermediate – in 3 (1.7%), negative – in 148 (82, 7%) (table 2). subsequently, these results were confirmed by immunoblotting. thus, from the examined group of patients (179 people) the immunoblot confirmed the total (igm + igg) absolute number of positive blood results in (20 + 26) 46 children (25.7%). to determine the etiological structure of ld, the presence of igm antibodies to the immuno genic external surface protein ospc (a marker of the early immune response) of three species was determined: b. garinii, b. burgdorferi, b. afzelii separately in patients of both groups (table 3). antibodies of this class to ospc b. afzelii were found in the sera of 11 (55%) of the 20 subjects, to ospc b. garinii, respectively, in 11 (55%), to ospc b. burgdorferi s.s. – in 6 (30%) patients. igm antibodies to antigens p41, p39, and vlse were also determined in the sera of the examined patients. it was found that antibodies to antigens p41 were detected in 17 (85%) patients, to p39 antigen – in 4 (20%), respectively, to vlse – not detected in any of the examined groups of children (table 3). simultaneously, the presence of igg antibodies (only positive results) to vlse (recombinant highly immunogenic lipoprotein of the outer membrane, variable like sequence expressed) of borrelia of different genes in the sera of 31 patients with me ld was determined with a positive result in all children. to determine the etiological structure of ld, the presence of igg antibodies to the immunogenic external fig. 1. frequency of detection of infectious agents in children with erythema migrans. bbs – b. burgdorferi sensu lato; brm – b. miyamotoi; aph – anaplasma рhagocytophilum. table 2. the content of igm and igg to b. burgdorfery s.l. determined by different methods in children living in ternopil region igm igg elisa euroline borrelia rn-at elisa euroline borrelia rn-at result total (n=179) result total (n=71) result total (n=179) result total (n=31) absolute value % absolute value % absolute value % absolute value % positive 56 31.3 positive 17 30.4 positive 28 15.6 positive 25 89.3 intermediate 0 0 intermediate 0 0 negative 39 69.6 negative 3 10.7 intermediate 15 8.4 positive 3 20.0 intermediate 3 1.7 positive 1 33.3 intermediate 0 0 intermediate 0 0 negative 12 80.0 negative 2 66.7 negative 108 60.3 negative 148 82.7 9,70% 12,40% 0,80% 1,70% bb аph brm bb+а s.o. nykytyuk et al. 79 b io m e d ic a l s c ie n c e s issn 2413-6077. ijmmr 2021 vol. 7 issue 2 s.o. nykytyuk et al. surface protein vlse (a marker of the early immune response) of three species was deter mined: b. garinii, b. burgdorferi, b. afzelii separately in the patients of both groups (table 4). osc b. garinii antigens of igm immunoglobulin predominated over ospc b. afzelii, ospc b. burgdorteri in ternopil region. vlse b.burgdorteri antigens of immunoglobulin igg prevailed over vlse b.afzelii, vlse b. garinii in ternopil region. discussion lyme disease (ld) is an endemic disease in many countries. in europe, north america, and asia, it is the most common vector disease [8,9]. it is caused by b. burgdorferi sensu lato and is transmitted to humans by ticks of the ixodes ricinus mite complex; up to 20% of them are infected with this bacterium. only 2-4% of bites are clinically manifested that is one of the diagnostic challenges [10, 11]. in the presence of erythema migrans, there was a significant difference in the elisa results for immunoglobulin m, in particular a significant predominance of negative igm values. at the same time, the erythematous form of me was characterized by positive results of igm. in a small amount of igm to flagellin (41 kb) and membrane protein ospc borrelia begin to appear in the first days of the disease. their titres increase within 4-6 weeks, and a little longer in untreated patients. during the generalization of the infectious process, igg antibodies appear against several proteins, e.g. p39 and p58 [7]. the frequency of various pathogens that caused erythema migrans was established: the leading pathogen was anaplasma phagocytophilym in (12.4%) cases, b. burgdorferi sensu lato in 9.7% of cases. it is proved that if erythema migrans develops in a patient after a tick bite in an endemic area [12], treatment tactics should be suggested immediately. however, if the diagnosis of lyme disease is uncertain, it is recommended to first determine the sensitivity of the elisa reaction. only elisa­positive cases should be confirmed with a more specific immunoblot results [7]. table 4. antigenic load to в. burgdorferi igg antigens absolutely value (igg-31) relative value (%) ospc b. afzelii 0 0 vlse b. burgdorferi ss 16 51,6 vlse b. afzelii, 13 41,9 vlse b. garinii 12 38,7 p39 0 0 p41 21 67,7 lipid ba 4 12,9 lipid bb 2 6,4 p21 4 12,9 p18 3 9,6 p58 3 9,6 fig. 2. average rate of antigenic load to anti-b. burgdorferi igm antibodies in ternopil region. table 3. antigenic load to anti-b. burgdorferi igm antigens absolute value (ig m-20) relative value (%) ospc bg (b. garinii) 11 55 ospc bb (b. burgdorferi) 6 30 ospc ba (b. afzelii) 11 55 p39 4 20 p41 17 85 vlse 0 0 80 b io m e d ic a l s c ie n c e s issn 2413-6077. ijmmr 2021 vol. 7 issue 2 it is established that the differentiation between septic arthritis and lyme arthritis in endemic areas can be a difficult task, and therefore it causes serious consequences for the treatment of the patient [14]. in the acute and late stages, lyme disease can be difficult to distinguish from other painful processes . to establish a prediction algorithm for the differentiation of septic arthritis from lyme disease in children with knee pain and exhaustion [13], a two-stage diagnosis is recommended. the main surface antigens of ospa, ospb, ospc proteins, which determine the difference of individual strains [14], can vary significantly; thus determining the possibility of long-term (for many years) persistence of the pathogen in the human body [14, 15]. many antigenic determinants of the outer shell of borrelia of different species are similar to each other and even to some bacteria of other genera, which explains the possibility of cross-immune reactions [7, 16]. serum samples from children with disseminated or late stage ld almost always have a strong igg response to borrelia burgdorferi antigens [7, 8, 15]. in the evaluation and interpretation of serological test results, both the class of antibodies to specific b. burgdorferi antigenic proteins and the type of bacterial antigen, for which these antibodies are produced, are important [14]. external surface proteins (osp) are important in the immune response to infection because they are highly immunogenic. the aantibodies to ospc are characteristic of recent infection. according to the manufacturer’s recommendations, the presence of specific igm antibodies was considered positive, intermediate, or negative, depending on the combinations of ospc antigens of three species of borrelia (b. afzelii, b. burgdorferi ss, and b. garinii), p39, and vlse bb. at the same time, the presence of igg was considered positive or negative depending on the combinations of vlse antigens of three species of borrelia (b. afzelii, b. burgdorferi ss, and b. garinii) and other specific antigens: p18, p19, p20, p21, p58, ospc (p25 ), p39, p83, lipid ba, lipid bb. in our immunological study, the genotype b. burgdorferi sensu stricto was detected in children with erythema migrans, arthritis, and neurolaim, which have statistically significant results (table 4). the diagnosis of lyme disease should be established by a laboratory (serological tests (elisa and western blot) investigations, indicating the presence of specific anti­b. burgdorferi igm / igg antibodies), which confirms clinical manifestations of the disease. this is very important because physicians often seek serological evidence of b. burgdorfery infection in patients with undefined diffuse complaints [15,18]. according to the list of symptoms compatible with lyme disease, the most common symptoms in children of ternopil region were erythema migrans (84.9%). this corresponds to the results of other studies [19, 20]. conclusions it was estanlished that b. burgdorferi sensu lato; b. miyamotoi; and a. phagocytophilum are pathogens that cause erythema migrans in children of ternopil region. the presence of specific igg (only positive results) to b. burgdorferi s.l. was confirmed by immunoblotting in 83.8% of individuals who had positive and intermediate results in the elisa test. 30,7% 36,1% 26,8% 0,0% 5,0% 10,0% 15,0% 20,0% 25,0% 30,0% 35,0% 40,0% vlse borrelia afzelii (vlse-ba) vlse borrelia burgdorferi (vlse-bb) vlse borrelia garinii (vlse-bg) vlse borrelia afzelii (vlse-ba) vlse borrelia burgdorferi (vlse-bb) vlse borrelia garinii (vlse-bg) fig. 3. average rate of antigenic load to anti-b. burgdorferi igg antibodies in ternopil. s.o. nykytyuk et al. 81 b io m e d ic a l s c ie n c e s issn 2413-6077. ijmmr 2021 vol. 7 issue 2 s.o. nykytyuk et al. conflict of interests authors declare no conflict of interests. acknowledgements laboratory staff of the centre for the study of lyme disease and other tick-borne infections of i. horbachevsky ternopil national medical university. author’s contributions svitlana oleksiivna nykytyuk – formal analysis, writing – original draft, writing – reviewing and editing; sergiy ivanovych klymnyuk – conceptualization, writing – original draft, writing – reviewing and editing; ivan mykolayovych klishch – methodology, writing – reviewing and editing; sofia sergiivna levenets – investigation, formal analysis. оцінка результатів імуноблоту для визначення антитіл до патогенів хвороби лайма у дітей тернопільської області *с.о. никитюк, с.і. климнюк, і.м. кліщ, с.с. левенець тернопільський національний медичний університет імені і. я. горбачевського моз україни, тернопіль, україна вступ. вступ. лайм бореліоз (ld) є мультисистемним захворюванням, спричиненим borrelia burgdorferi та іншими подібними кліщовими borrelia. мета. визначити і порівняти серологічних результатів крові при різних формах хвороби лайма у дітей. методи. під нашим спостереженням знаходилась група дітей (n=178) у віці від 1 до 14 років, укушених кліщами. контрольна група становила 30 здорових дітей. кліщів ідентифікували за допомогою стереомікроскопічної системи seo та визначника. фрагменти днк b. burgdorferi sensu lato (sl) (b. burgdorferi sensu strictо, b. afzeliі та b. garinii), b. miyamotoi, a. phagocytophilum визначали у крові методом плр в реальному часі. кліщів ідентифікували за допомогою стереомікроскопічної системи seo. були проведені базові дослідження, пов’язані з клінічними та імунологічними дослідженнями, включаючи дані elisa та immunoblot. результати. опитування охопило 178 батьків дітей, на яких напали кліщі. виявлено borrelia burgdorferi sensu lato (b. afzelii, b.burgdorferi sensu stricto та b. garinii), b. miyamotoi та a. phagocytophilum. проведено порівняння серологічних результатів крові при різних формах хвороби лайма у дітей. висновки. встановлено, що b. burgdorferi sensu lato; b. miyamotoi; та a. phagocytophilum є збудниками, які викликають у дітей мігруючу еритему. наявність специфічних антитіл igg (тільки позитивні результати) до b. burgdorferi s.l. імуноблотинг був підтверджений у 83,8% осіб, які мали позитивні та проміжні результати в тесті іфа. ключові слова: хвороба лайма; бореліоз ; іфа; імуноблот; укус кліща. information about the authors svitlana oleksiivna nykytyuk – md, phd, associate professor, i. horbachevsky ternopil national medical university, ternopil, ukraine orcid: 0000­0003­3146­9664, e­mail: androx@tdmu.edu.ua sergiy ivanovych klymnyuk – md, professor, i. horbachevsky ternopil national medical university, ternopil, ukraine. orcid: 0000­0002­1308­3250, e­mail: klymnyuk@yahoo.com ivan mykolaiovych klishch – professor, i. horbachevsky ternopil national medical university, ternopil, ukraine. orcid: 0000­0001­6226­4296, e­mail: klishch@tdmu.edu.ua sofia sergiivna levenets – phd, associate professor, i. horbachevsky ternopil national medical university, ternopil, ukraine. orcid: 0000­0001­5678­0008, e­mail: levenetsss@tdmu.edu.ua 82 b io m e d ic a l s c ie n c e s issn 2413-6077. ijmmr 2021 vol. 7 issue 2 references 1. weber k. aspects of lyme borreliosis in europe. eur j clin microbiol infect dis. 2001;20(1):6­13. https://doi.org/10.1007/s100960000412. 2. esposito s, bosis s, sabatini c, tagliaferri l, principi n. borrelia burgdorferi infection and lyme disease in children. int j infect dis. 2013;17(3):153­ 158. https://doi.org/10.1016/j.ijid.2012.09.014. 3. centers for disease control and prevention. reported cases of lyme disease by year, united states, 2002-2011; 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[lime disease. the current state of the problem (literature review)]. eumj. 2020;8(2):230­241. in ukrainian 19. engstrom sm, shoop e, johnson rc. immunoblot interpretation criteria for serodiagnosis of early lyme disease. j clin microbiol. 1995;33(2):19­22. https://doi.org/10.1128/jcm.33.2.419­427.1995. 20. nykytyuk s, klymnyuk si, levenets s. laboratory diagnostics of lyme borreliosis in children with ticks bites in ternopil region. georgian medical news. 2019;11(296):32­33. received 1 november 2021; revised 24 november 2021; accepted 8 december 2021. this is open access article distributed under the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. s.o. nykytyuk et al. 74 b io m e d ic a l s c ie n c e s issn 2413-6077. ijmmr 2020 vol. 6 issue 1 doi 10.11603/ijmmr.2413-6077.2020.1.11503 inflammation and impact of vincristine and enterosorption use in chemically induced colon carcer in rats o.i. kachur, *l.s. fira, p.h. lykhatskyi i. horbachevsky ternopil national medical university, ternopil, ukraine background. the increasing incidence of colon malignant tumors is one of the most urgent matters of contemporary medicine. in the study of carcinogenesis of the colon the attention is paid to the state of the body’s immune system and activation of inflammatory processes in experimental animals. objective. the aim of the study was to estimate the level of markers of inflammation in the serum of experimental animals with chemically induced carcinogenesis and their dynamics in case of administration of the cytostatic vincristine secondary to aut-m carbon enterosorbent. methods. the study was performed on white male rats. animals were modeled for colon cancer by administration of 1.2-dimethylhydrazine hydrochloride at a dose of 7.2 mg/kg body weight for 30 weeks. aut-m enterosorbent was administered intragastrically daily during 7 and 21 days after modeling of carcinogenesis at a dose of 1 ml of suspension (corresponding to 0.2 g of drug weight) per 100 g of animal body weight. the antitumor drug was administered to the animals with induced carcinogenesis intragastrically daily during 14 days at a dose of 0.23 mg/kg of body weight after a 21-day detoxification therapy. the activity of inflammatory processes was evaluated by the content of pro-inflammatory interleukin 6 and anti-inflammatory interleukin 4, c-reactive protein in the serum of experimental animals. results. it was established that introduction of 1.2-dimethylhydrazine hydrochloride in the rats caused changes in the cytokine profile and the content of c-reactive protein. in the affected animals an increase in the content of pro-inflammatory interleukin 6, c-reactive protein, as well as a decrease in the content of antiinflammatory interleukin 4 was evidenced in all periods of the study. aut-m enterosorbent contributed to normalization of these parameters. the cytostatic vincristine had a negligible effect on development of inflammatory processes in the studied animals. conclusions. in cases of induced carcinogenesis, an imbalance in the content of proand anti-inflammatory cytokines, an increase in the content of acute-phase c-reactive protein was established. the positive effect of the cytostatic vincristine secondary to a previous detoxification therapy with auт-m sorbent during a progressive development of inflammatory processes in the presence of modeled carcinogenesis was evidenced. key words: inflammatory processes; proinflammatory interleukins; anti-inflammatory interleukins; aut-m sorbent; cytostatic vincristine. international journal of medicine and medical research 2020, volume 6, issue 1, p. 74-80 copyright © 2020, tnmu, all rights reserved *corresponding author: l.s. fira, phd, dsc, professor, head of the department of pharmacy of the academic and research institute of postgraduate education, i. horbachevsky ternopil national medical university, ternopil, ukraine; e-mail: fira@ tdmu.edu.ua o.i. kachur et al. introduction malignant neoplasms of the colon are still one of the urgent matters of contemporary medicine. recently the incidence of colorectal cancer among young and middle-aged people has been increasing constantly [1]. it is established that the transformation of a normal cell into a malignant one is accom panied by a homeostasis disorders and changes in all types of metabolism. in the body of the tumour carrier, free radical processes activate, the integrity of plasma membranes is disturbed in cases of destructive changes. as the cancer develops, excessive toxins accumulate and endogenous intoxication intensifies. all these factors cause evolution of inflammatory reaction of the body [2, 3]. markers of the systemic inflammatory response in the evolution of carcinogenesis are the acute proteins: ceruloplasmin (cp), haptoglobin and especially c-reactive protein (crp), cytokines. c-reactive protein (crp) is one of the most sensitive and early markers of the acute phase of the inflammatory process. an increase in the concentration of crp in the serum evidences development of a pathological process and destructive changes in the body [4]. the synthesis of crp of the acute phase of inflammation is triggered and regulated primarily by cytokines, namely il-6, which is formed directly in the inflammatory focus and acts as a primary activator of genes involved in it and tumor transformation [5]. due to the increasing frequency and prevalence of colorectal cancer in modern society, the issue of finding and researching effective 75 b io m e d ic a l s c ie n c e s issn 2413-6077. ijmmr 2020 vol. 6 issue 1 and low-toxic cytostatics of plant origin is still topical. vincristine is a herbal cytostatic, it is an important component of the combined treatment regimen for malignant neoplasms. however, in the present literature there are some publications on the use of periwinkle alkaloids in the chemotherapeutic treatment of colorectal cancer [6]. enterosorption therapy is one of the promising methods for control of inflammation. aut-m carbon sorbent with a large specific pore area exhibits physicochemical properties of a sorbent substance capable of binding and excreting toxins of exogenous and endogenous origin [7, 8]. the research is aimed to evaluate the level of inflammation markers in the serum of experimental animals with chemically induced carcinogenesis and their dynamics with introduction of the cytostatic vincristine secondary to aut-m carbon enterosorbent. methods the study was performed on 76 white male rats. the initial body weight of animals was 200-250 g. simulation of carcinogenesis of the colon was carried out by subcutaneous weekly injection into the interscapular area of asymmetric 1.2-dimethylhydrazine (1.2-dmg, made by sigma-aldrich chemie company, japan) for 30 weeks. the carcinogen 1.2-dmg was previously diluted with isotonic sodium chloride solution, dose 7.2 mg/kg [9]. after modeling of the cancer process for 30 weeks, the affected animals underwent enterosorption therapy for 21 days. aut-m carbon sorbent was administered intragastrally daily, daily dose 1 ml of suspension (corresponding to 0.2 g of net weight of the drug) per 100 g of body weight of the animal [10]. the anticancer drug vincristine was administered intragastrically daily at a dose of 0.23 mg/kg of animal body weight for 14 days, starting immediately after 7-month modeling of the cancer process and 21-day enterosorption therapy. the dose of cytostatics was chosen according to the instructions for use of the drug and taking into account sensitivity of the animals (recalculation was carried out according to yu. r. rybolovlev, 1979) [11]. the experimental animals were divided into 4 groups: group 1 – the control involved the animals administered with isotonic sodium chloride solution, group 2 – the animals affected by 1.2-dmg, group 3 – the animals affected by 1.2-dmg, managed by aut-m enterosorbent for 21 days after a 30-week carcinogen administration, group 4 – the animals administered with the cytostatic vincristine for 14 days after enterosorption therapy. euthanasia of animals was performed by bloodletting under thiopental-sodium anesthesia monthly for 7 months and on the 14th and 21st days of administration of the aut-m enterosorbent after carcinogenesis modeling, as well as on the 14th day after administration of the cytostatic vincristine. the serum was used for studies. all manipulations with experimental rats were performed following the rules in accordance with the european convention for the protection of vertebrate animals used for research and other scientific purposes [12]. to study the development of inflammatory processes in cases of induced carcinogenesis of the colon the concentration of proinflam matory and anti-inflammatory interleukins in the serum of rats was determined by the elisa test [13]. the content of c-reactive proteins was deter mined by immunoturbidimetric method [14]. the obtained experimental results were statistically processed by the method of variation statistics using a standard package of statistica 6.0 statistical program [15]. the standard deviations, variance and other statistical parameters were determined, and the statistically significant differences were determined using student’s t-test. the differences were statistically significant at p≤0.05. results inflammatory reactions of the body are a non-specific part of the defense, the formation of which prevents the spread of the pathogen and is realized by attracting granulocytes, macrophages and lymphocytes to the site of inflammation. the first stage of such reactions is associated with the activation of pro-inflammatory cytokines. they provide chemotraction of the inflammatory cell [16]. in the dynamics of 1.2-dmg administration, the content of pro-inflammatory cytokine il-6 in the serum of affected animals probably (p ≤ 0.05) increased during the 1st month of 1.2-dmg administration (by 62% compare to the control group). intermediate results point to similar changes: in 5 months of dmg affection (increased by 182%) and in 7 months (by 251%) of adenocarcinoma modeling (fig. 1). the corrective dose of the aut-m sorbent helped to reduce the content of il-6 in the o.i. kachur et al. 76 b io m e d ic a l s c ie n c e s issn 2413-6077. ijmmr 2020 vol. 6 issue 1 serum of the affected animals. it was established that il-6 probably decreased in the serum by 138% (p≤0.05) after a 14-day sorbent therapy, the maximal changes were evidenced in 21 days of sorbent management (decreased by 168%) compare to the same group of the affected animals. cytostatic correction performed for 14 days leads to a slight decrease in il-6 in the serum of rats. the studied parameter decreased by 186% in comparison with the affected animals. toxic affection of the animals during 30 weeks causes an imbalance of the cytokine profile in their body. the functional antagonists of pro-inflammatory il-6 include anti-inflammatory il-4. this cytokine has an antitumor effect; it is a strong growth factor for b-lymphocytes, which promotes their differentiation, activation and reproduction [17]. statistically significant differences in the content of il-4 were found later after beginning of 1.2-dmg administration. the rate decreased by 29% in 5 months of carcinogenesis modeling. after a 30-week dmg affection, the content of il-4 in the serum of the rats decreased by 45% compare to the control (fig. 2). after a 21-day detoxification therapy, the level of il-4 increased by 30% compare to the group of animals with no detoxification therapy. in the group of animals undergoing cytostatic therapy, an increase in the content of anti-inflammatory il-4 by 35% was evidenced compare to the animals with carcinogenesis. with administration of 1.2-dmg into the experimental animals, changes of acute phase proteins activity were evidenced. thus, the content of crp was likely to increase starting the third month of carcinogenesis modeling and exceeded the level of control in 1.5 times. a similar trend was proved in the subsequent affections, in 5 months after the beginning of the experiment, the content of crp increased in 1.73 times, in 7 months – in 2.1 times compare to the control rats (table 1). the obtained results may indicate an intensification of the inflammatory process in the body of experimental animals affected by 1.2 dmg. the detoxification therapy (for 21 days with the aut-m sorbent) contributes to the probable decrease on this parameter in the serum of the affected animals. in 21 days of administration of this sorbent, the content of crp decreased in 1.6 times compare to the group of affected animals. during the chemotherapy treatment of the affected animals (with previous sorbent correction), a slight increase in the crp content was evidenced. in 14 days of vincristine administration, the parameter increased in 1.3 times compare to the animals modeled for colon carcinogenesis for 30 weeks. thus, a 30-week dmg affection of the rats caused an imbalance in the content of proinflammatory and anti-inflammatory cytokines with further development of inflammatory fig. 1. the level of il-6 in the serum of rats in the dynamics of the affection by 1.2-dmg and after administration of aut-m sorbent and cytostatic vincristine, %. notes: * – statistically significant difference between the parameters of the control group animals and the animals affected by 1.2-dmg; ** – statistically significant difference between the parameters of the carcinogenic animals and the animals administered with enterosorbent; *** – statistically significant difference between the parameters of carcinogenic animals after enterosorption therapy (for 21 days) and the animals administered with cytostatics (for 14 days); # – statistically significant difference between the parameters of carcinogenic animals (in 7 months) and the animals administered with cytostatic (for 14 days) secondary to enterosorption therapy (for 21 days). o.i. kachur et al. 77 b io m e d ic a l s c ie n c e s issn 2413-6077. ijmmr 2020 vol. 6 issue 1 processes that was confirmed by an increase in early marker of inflammation in the serum: c-reactive protein and interleukin 6. the use of sorption therapy had a positive effect on inflammation in the body of rats throughout the experiment. no significant side effects after administration of the cytostatic vincristine were evidenced. discussion the search for effective methods of correction of oncological pathologies is topical issue of contemporary medicine. the use of low-toxic and affordable drugs is important. our research on the efficacy of cytostatic vincristine secondary to enterosorption therapy in cases of simulated carcinogenesis is proved by reduced fig. 2. the content of il-4 in the serum of rats in the dynamics of the affection by 1.2-dmg and after administration of aut-m sorbent and cytostatic vincristine, % notes: * – statistically significant difference between the parameters of the control group animals and the animals affected by 1.2-dmg; ** – statistically significant difference between the parameters of the carcinogenic animals and the animals administered with enterosorbent; *** – statistically significant difference between the parameters of the carcinogenic animals after enterosorption therapy (for 21 days) and the animals administered with cytostatics (for 14 days); # – statistically significant difference between the parameters of the carcinogenic animals (in 7 months) and the animals administered with cytostatics (for 14 days) secondary to enterosorption therapy (for 21 days). table 1. the content of c-reactive protein in the serum of rats (mg/l) with induced oncogenesis and after administration of cytostatic vincristine secondary to detoxification therapy, (m±m) animal group/duration of dimethylhydrazine affection serum control, (n=6) 2.45±0.18 1 month, (n=7) 2.78±0.21 2 months, (n=7) 3.15±0.29 3 months, (n=7) 3.66±0.34* 4 months, (n=7) 4.12±0.38* 5 months, (n=7) 4.25±0.39* 6 months, (n=7) 5.10±0.49* 7 months, (n=7) 5.25±0.50* 7 months of dmg + aut-m (for 14 days), (n=7) 4.55±0.40 7 months of dmg + aut-m (for 21 days), (n=7) 3.80±0.36** 7 months of dmg + aut-m (for 21 days) + vincristine (for 14 days), (n=7) 4.01±0.16# notes: * – statistically significant difference between the parameters of the control group animals and the animals affected by 1.2-dmg; ** – statistically significant difference between the parameters of the carcinogenic animals and the animals administered with enterosorbent; *** – statistically significant difference between the parameters of the carcinogenic animals after enterosorption therapy (for 21 days) and the animals administered with cytostatic (for 14 days); # – statistically significant difference between the parameters of the carcinogenic animals (in 7 months) and the animals administered with cytostatic (for 14 days) secondary to enterosorption therapy (for 21 days). o.i. kachur et al. 78 b io m e d ic a l s c ie n c e s issn 2413-6077. ijmmr 2020 vol. 6 issue 1 inflammatory manifestations and control of the studied parameters. in cases of induced pathological process, a violation of the cytokine profile is observed, as the content of the pro-inflammatory cytokine il-6 increases, the content of the anti-inflammatory cytokine il-4 decreases. the obtained results point to an imbalance of the cytokine profile with an advantage towards il-6. it is established that il-6 is one of the main inducers of the acute phase response in inflammatory process. obviously, these changes may evidence of development of chronic inflammation and tumor progression [18]. the system of immunoregulation in the body of the tumor carrier along with the cytokine cascade is followed by changes in the content of crp. in cases of cytokine profile imbalance in the affected animals, an increase in the crp content was evidenced. it is established that crp is an acute marker of inflammation, so the results point towards development and progression of inflammatory processes [19]. therefore, it has been established that the affection of the experimental animals with 1.2dmg for 7 months leads to an imbalance of the cytokine profile, an increase in the content of acute phase crp proteins. no significant side effects of the cytostatic vincristine are evidenced that proves a positive effect of detoxification therapy with the aur sorbent during the progressive inflammatory process in cases of modeled oncological process. conclusions a long-term increase in the content of proinflammatory interleukin 6 in addition to reduced production of anti-inflammatory interleukin 4, as well as an increase in c-reactive protein in the rats affected with 1.2-dimethylhydrazine hydrochloride for 30 weeks that evidences development of inflammatory processes in the carcinogenesis body. after detoxification therapy with aut-m carbon sorbent a decrease in inflammatory pro cesses activity in the animals with experimental carcinogenesis was present. this is proved by a decrease in the content of proinflam matory interleukin 6, c-reactive protein and an increase in the content of anti-inflammatory interleukin 4. the positive effect of vincristine, a cytostatic drug of plant origin, on the activity of inflammatory processes in the rats in cases of experimental carcinogenesis has been established. it has been experimentally proved that this cytostatic has no side effects obviously as a result of previous enterosorption therapy. funding this research received no external funding. conflict of interest the author declares no conflict of interest. author contributions kachur o.i. – resources, investigation, formal analysis, writing – original draft; fira l.s. – conceptualization, formal analysis, writing – review & editing; lykhatskyi p.h. – conceptualization, project administration, supervision. розвиток запальних процесів у щурів із індукованим канцерогенезом товстої кишки після застосування цитостатику вінкристин на тлі ентеросорбції о.і. качур, л.с.фіра, п.г.лихацький тернопільський національний медичний університет імені і. я. горбачевського, тернопіль, україна вступ. проблема зростання частоти розвитку злоякісних пухлин товстої кишки – одна з найбільш актуальних у медицини. об’єктом уваги при вивченні канцерогенезу товстої кишки є стан імунної системи організму та активація запальних процесів в експериментальних тварин. перспективним є проведення хіміотерапії рослинними препаратами на тлі ентеросорційної корекції. мета дослідження – оцінити рівень маркерів запалення в сироватці крові експериментальних тварин із хімічно індукованим канцерогенезом та їх динаміку при введенні цитостатика вінкристин на тлі вуглецевого ентеросорбенту аут-м. методи дослідження. дослідження проводили на білих щурах самцях. тваринам моделювали рак товстої кишки шляхом введення 1,2 диметилгідразин гідрохлориду в дозі 7,2 мг/кг маси тіла протягом 30 ти тижнів. ентеросорбент аут-м вводили інтрагастрально щоденно впродовж 21 ї доби після o.i. kachur et al. 79 b io m e d ic a l s c ie n c e s issn 2413-6077. ijmmr 2020 vol. 6 issue 1 моделювання канцерогенезу в дозі 1 мл завису (що відповідає 0,2 г чистої маси препарату) на 100 г маси тіла тварини. протипухлинний препарат вводили тваринам з індукованим канцерогенезом внутрішньошлунково щоденно протягом 14-ти днів у дозі 0,23 мг/кг маси тіла після 21-денної детоксикаційної терапії. активність запальних процесів оцінювали за вмістом прозапального інтерлейкіну 6 та протизапального інтерлейкіну 4, с реактивного протеїну в сироватці крові експериментальних тварин. результати. встановлено, що введення щурам 1,2 диметилгідразин гідрохлориду супроводжується зміною цитокінового профілю та вмісту с реактивного протеїну. в уражених тварин спостерігали збільшення вмісту прозапального інтерлейкіну 6, с реактивного протеїну, а також зменшення вмісту протизапального інтерлейкіну 4 в усі терміни дослідження. застосування ентеросорбенту аут сприяло нормалізації цих показників. введення цитостатика вінкристин незначно вплинуло на розвиток запальних процесів у досліджуваних тварин. висновки. за умов індукованого канцерогенезу встановлено дисбаланс у вмісті прота протизапальних цитокінів, збільшення вмісту гострофазового с-реактивного протеїну. відмічено позитивний вплив цитостатика вінкристин на тлі попередньо проведеної детоксикаційної терапії̈ сорбентом аут під час прогресуючого розвитку запальних процесів за умов змодельованого канцерогенезу. ключові слова: запальні процеси; прозапальні інтерлейкіни; протизапальні інтерлейкіни; сорбент аут-m; цитостатик вінкристин. відомості про авторів качур о.і. – асистент кафедри загальної хімії, тернопільський ̆національний ̆медичний ̆університет імені і.я.горбачевського моз україни, тернопіль, україна фіра л.с. – доктор біологічних наук, професор, завідувач кафедри фармації нні післядипломної освіти,тернопільський національний медичний університет імені і.я. горбачевського моз україни, тернопіль, україна лихацький п.г. – доктор біологічних наук, професор кафедри медичної біохімії. тернопільський національний медичний університет імені і.я. горбачевського моз україни, тернопіль, україна information about the authors kachur o.i. – assistant professor, department of general chemistry, i. horbachevsky ternopil national medical university, ternopil, ukraine. orcid http://orcid.org/0000-0002-8997-4797, e-mail: kachur_oi@tdmu.edu.ua fira l.s. – phd, dsc, professor, head of the department of pharmacy of the academic and research institute of postgraduate education, i. horbachevsky ternopil national medical university, ternopil, ukraine. orcid http://orcid.org/0000-0002-5325-0973, e-mail: fira@tdmu.edu.ua lykhatskyi p.h. – phd, dsc, professor of the department of medical biochemistry, i. horbachevsky ternopil national medical university, ternopil, ukraine. orcid http://orcid.org/0000-0002-0021-782x, e-mail: luhatsky@tdmu.edu.ua references 1. bray f, ferlay j, soerjomataram i, et al. global cancer statistics 2018: globocan estimates of incidence and mortality worldwide for 36 cancers in 185 countries. ca cancer j. clin. 2018;68:394–424. doi: 10.3322/caac.21492 2. karmazina is, kulinich va. correlation analysis of protein metabolism indices at carcinogenesis and inflammation. natural almanac.2015;12: 112-8. 3. zhang x, liu s, zhou y. circulating levels of c-reactive protein, interleukin-6 and tumor necrosis factor-α and risk of colorectal adenomas: a metaanalysis. oncotarget. 2016 sep 27;7(39):64371-79. doi: 10.18632/oncotarget.11853 4. gross d, tolba r. ethics in animal-based research. eur. surg. res. 2015;55(1-2):43–57. doi: 10.1159/000377721 5. zhou b, shu b, yang j, et al. c-reactive protein, interleukin-6 and the risk of colorectal cancer: a meta-analysis. cancer causes control, 2014;25(10): 1397-1405. doi: 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mar 2020; revised 10 apr 2020; accepted 02 june 2020. this is open access article distributed under the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. o.i. kachur et al. 43 su r g ic a l d is e a se s issn 2413-6077. ijmmr 2020 vol. 6 issue 1 doi 10.11603/ijmmr.2413-6077.2020.1.11008 necrotizing pancreatitis: the ways of improvement of surgical treatment o.v. rozenko donetsk national medical university, mariupol, ukraine background. in connection with a steady increase of patients with destructive forms of acute pancreatitis, the proportion of which takes from 10-15 to 20-30%, despite of the wide range of treatments for non-biliary necrotizing pancreatitis, the rate of mortality reaches 80-90% in case of infected forms and needs further improvement of therapies. objective. the purpose of this study is to improve the results of treatment of patients with nonbiliary necrotizing pancreatitis, by optimizing surgical tactics. methods. the study examined the cases of 120 patients, whose age ranged from 22 to 83 years, including patients under the age of 50 years old who accounted for 60.0%. there were 80 males (66.7%) and 40 females (33.3%). disease duration up to 24 hours was found in 36 (30.0%) patients, from 25 to 72 hours in 25 (20.8%) individuals, more than 72 hours in 49 (49.2%) patients. results. the research claims in non-necrotizing pancreatitis, left-sided retroperitoneal phlegmon often develops in 64.2% (mortality rate 26.0%) of patients, right-sided in 24.2% (mortality rate 6.8%) of patients, and bilateral in 11, 6% (mortality rate 64.2%) individuals. moreover, the highest mortality was observed with a combination of retroperitoneal phlegmon cellulose and abscess of the pancreas and/or omental bursa 39.1%. conclusions. the use of various surgical interventions depending on the involvement in the pathological process of various sections of the abdominal cavity/ retroperitoneal space, which made it possible to reduce mortality and hospitalization time of patients in the hospital by 1.5-2 times is proposed. key words: necrotizing pancreatitis; lumbotomy; parapancreatic fiber; phlegmon of the retroperitoneal tissue; laparotomy; omentobursostomy. *corresponding author: oleg v. rozenko, md, ph.d., associate professor, department of surgery, endoscopy, otorhinolaryngology and reconstructive surgery, donetsk national medical university, e-mail: oleg.rozenko@gmail.com. o.v. rozenko international journal of medicine and medical research 2020, volume 6, issue 1, p. 43-49 copyright © 2020, tnmu, all rights reserved introduction during the last decade, a steady increase in patients with destructive forms of acute pancreatitis has been observed, the proportion of which takes from 10-15 to 20-30% at the pathology of the abdominal organs. according to many authors, the main causes of high mortality rate are the duration of the disease, the prevalence of pancreatic necrosis and parapancreatic fiber, inadequate selection of the method of surgical and ane sthesia, and drug therapy [1, 2, 3]. the variety of surgical treatment (from traditional lapa rotomy, lumbotomy to puncture-draining operations under the ultrasound control, using various methods of drainage of the abdominal cavity and retroperitoneal tissue) is determined by the clinical form of infected pancreatitis (abscess of the pancreas and/or the lesser sac, the abscess of the abdominal pancreatitis and/ or the lesser sac, the abscess of the abdominal pancreatitis and/or the omental purulent peritonitis). extraperitoneal access to infected foci of pancreatic necrosis is one of the options for surgical treatment of necrotizing pancreatitis, the advantages of which are less trauma, the absence of infection of the abdominal cavity, a reduction in the number of complications (in testinal fistula, bleeding), the possibility of epidural anesthesia, faster recovery of intestinal function and early en teral nutrition [4, 5, 6]. the peak of surgical activity occurs during 2-3 weeks of illness when necrosis of the pancreas and parapancreatic fiber has already developed certain limits (the prevalence of pancreatic necrosis), the process of biological sequestration is completed and the possibility of removal of sequestered and necrotized tissues has appeared. during the primary operation, it is not always possible to visualize the boundaries of healthy and necrotic modified pancreatic tissue and parapancreatic fiber which limits necral and sequestrectomy due to possible damage to the parenchyma of the 44 su r g ic a l d is e a se s issn 2413-6077. ijmmr 2020 vol. 6 issue 1 gland and the threat of bleeding that makes it impossible for one-time sanitation and adequate drainage of all purulent-necrotic cavities. therefore, a number of authors propose to leave non-invested tissues, to drain the affected section of the retroperitoneal tissue, suggesting further programmatic step-by-step revisions and sequestrectomy. the clinical meaning of the division of sanations into stages is also due to the non-simultaneous maturation of sequesters, the secondary progression of the purulent process according to the type of leakages, the danger of septic shock with the simultaneous opening of extensive retro peritoneal ulcers [7, 8, 9]. despite the wide range of treatments for infected necrotizing pancreatitis, the rate of mortality remains quite high. when analyzing the causes of deaths, inadequate drainage of the pancreatic area with necrosis of parapancreatic fiber, the formation of phlegmon or multiple abscesses in the retroperitoneal space is almost always established [10, 11, 12]. the objectives of this study are to improve the results of treatment of patients with nonbiliary necrotizing pancreatitis, by opti mizing surgical tactics (use of lumbotomy), depending on the prevalence of necrosis in the pancreas and retroperitoneal tissue. methods the study examined the cases of 120 patients who were hospitalized at the mariupol city pancreatic center at the department of surgery, endoscopy and reconstructive surgery at donetsk national medical university from 2014-2018. the age of patients ranged from 22 to 83 years, including patients under the age of 50 years old who accounted for 60.0%. there were 80 males (66.7%) and 40 females (33.3%). disease duration up to 24 hours was found in 36 (30.0%) patients, from 25 to 72 hours – in 25 (20.8%) individuals, more than 72 hours – in 49 (49.2%) patients. the study was conducted in accordance with the principles of bioethics, patients had given informed consent. the causes of acute non-biliary necrotizing pancreatitis were the following ones: unilateral feeding (excessive intake of predominantly fatty foods) – in 62 (51.7%) patients and intake of alcohol (alcoholic excess) or its surrogates – in 58 (48.3%) individuals. limited pancreatic necrosis (less than 30% necrosis of pancreatic parenchyma) was found in 10 (8.3%) patients, the spread one (from 30 to 50% necrosis of pancreatic parenchyma) – in 68 (56.7%) individuals, and subtotal-total (more than 50% of necrosis of the pancreatic parenchyma) – in 42 (35.0%) patients. retroperitoneal cellulose phlegmon with diffuse purulent peritonitis due to involvement in the inflammatory process of the peritoneum was detected in 9 (7.5%) patients, retroperitoneal cellulitis without peritonitis – in 90 (75.0%) individuals, and a combination of retroperitoneal phlegmon and pancreatic abscess and/or omen tal patient bag – in 21 (17.5%) cases. left-side retroperitoneal phlegmon was detected in 77 (64.2%) patients, right-side retroperitoneal phlegmon in 29 (24.2%), and bilateral retroperitoneal phlegmon – in 14 (11.6%). verification of phlegmon of the retroperitoneal tissue was carried out on the basis of data obtained during the ultrasound examination, com puter tomography, and surgery (macroscopic eva luation of retroperitoneal tissue – seques tration, detritus), as well as bacte riologically. all patients received intensive therapy, including hunger, gastric drainage, cathe terization of the central vein and bladder, infusiontransfusion therapy in a volume of not less than 40 ml / kg body weight with a ratio of colloidal and crystalloid solutions of 1:4; analgesia: epidural blockade at the level of th7-9 (method of choice: continuous infusion of 0.2% lidocaine solution with a speed of 6-12 ml/hour), nonsteroidal anti-inflammatory drugs (ketoprofen 100 mg 3 times a day); antisecretory therapy (the optimal period is the first three days of the disease): the drugs of choice are sandostatin (octreotide) 100 mg 3 times a day subcutaneously and omeprazole 40 mg 2 times a day iv; reserve drugs 5 – fluorouracil (5% – 5 ml i.v.) and quamel 40 mg 2 times a day i.v. antienzyme therapy (optimal duration – the first 5 days of the disease): contraction of at least 50,000 units/day, pride at least 500,000 units/day; prevention of purulent complications: pefloxacin, 400 mg 2 ti mes a day, i.v. + metronidazole, 500 mg, 3 times a day, i.v., with signs of secondary infection: cefoperazone / sulbactam, cefepime, imipenem, meropenem. with the ineffectiveness of the above activities, resorted to extracorporeal de toxification methods (lympho-sorption, plasma pheresis, etc.). however, the effect was temporary and unstable, which required the need for rehabilitation of extensive purulentnecrotic foci with laparotomy followed by closed drainage of the abdominal cavity and retroperitoneal space. o.v. rozenko 45 su r g ic a l d is e a se s issn 2413-6077. ijmmr 2020 vol. 6 issue 1 results a total of 31 (25.8%) patients died after surgery. the causes of death in 29 patients were sepsis and multiple organ failure, in 1 case – sepsis and multiple intestinal fistulas, in 1 case – erosive bleeding from the splenic artery. the severity of necrotizing pancreatitis during the first 24 hours was assessed according to the criteria of systemic inflammatory response syndrome (2 or more signs) and additional risk factors: patient age, body mass index (over 30), hematocrit, apache ii score (more than 8 points), c-reactive protein indicator (more than 120 mg/l). and after 24 hours – on the scales of assessment of marshall’s multi-organ dysfunction (mods) or sofa. lumbotomy was performed under general anesthesia or epidural anesthesia. the lumbotomy of the left mini-access was performed as follows: below the arc of the xii rib, the skin and subcutaneous tissue is dissected 2-3 cm from the middle axillary to the back axillary line, pushing the muscle mass apart, the parietal peritoneum is exposed. paracolous fiber is reached with the fingers, exfoliating the peritoneum medially, focusing on the lower pole of the spleen and the left kidney. the abscess was opened by means of an electric suction, the pus was removed, the retro peritoneal fiber affected by enzymatic aggression (free-lying sequesters). necrectomy is not performed due to the risk of damage to the “healthy” parenchyma of the pancreas, parapancreatic fiber and large ves sels. the purulent necrotic cavity was sanitized and drained by elastic silicone drains. the lumbotomy of the right mini-approach is similar to the method described above, but the guidelines for advancing in the retroperitoneal space are the lower edge of the right lobe of the liver (its posterior surface), the right kidney, the head of the pancreas, the hepatoduodenal ligament. when the purulent-necrotic lesion spreads to the paracolous tissue, the dissection and drainage of the abscesses was performed through the front mini-accesses in the left or right iliac regions, respectively, using the volkovich incision up to 4-5 cm long. the iliac regions were moved apart. the parietal peritoneum with the mesentery of the sigmoid (cecum) intestine inwardly exfoliated by stupid fingers and wet tupffers. next, the surgeon with his fingers penetrates into the paracollate tissue of the retroperitoneal regions. tissue separation should occur easily and almost bloodless. flow-through drainage of phlegmon on the left was performed by two approaches: lumbotomy with a mini-access to the left and anterior mini-access in the iliac region. separation of retroperitoneal tissue from the anterior approach on the right should be made to the level of the pancreatic head. the purpose of surgical intervention (lumbotomy) in case of infected parapancreatitis is adequate opening and drainage (by elastic silicone drains) of the abscess with destruction of cell bridges, leakages, removal of purulent exudate, loosely sequestered, creating a single well-drained cavity for the next year. in an emergency procedure, about retroperitoneal phlegmon and diffuse purulent peritonitis, caused by the involvement of the parietal peritoneum in the inflammatory process, 9 (7.5%) patients were operated on. all of them performed laparotomy, dissection of phlegmon, sanitation and drainage of the stuffing box and abdominal cavity. in 6 patients, the operation was completed by imposing a laparostomy and in the postoperative period, programmed rehabilitation of the abdominal cavity was performed, and in 1 patient the left lumbotomy was also performed. 3 patients died with left-sided localization of the abscess; postoperative mortality was 22.2%. the cause of death was sepsis and multiple organ failure. discussions retroperitoneal cellulose phlegmon without peritonitis was an indication for performing surgical interventions in 88 (73.3%) patients. 20 patients died; the mortality rate was 22.7%. the cause of death was sepsis and multiple organ failure. in 40 (45.4%) patients, laparotomy, dissection and drainage of retroperitoneal phlegmon, sanitation and drainage of the abdominal cavity were used as a surgical aid. in 3 (7.9%) patients, surgery was supplemented with lumbotomy. 14 patients died, including 8 patients with leftsided retroperitoneal cellulitis, 2 individuals with right-sided and 4 patients with bilateral ones. mortality rate was 32.5%. the cause of death was sepsis and multiple organ failure. in 24 (27.3%) patients, puncture-draining surgical interventions under the control of ultrasound (pod-uz) were applied. in addition, 9 patients underwent lumbotomy and 1 – laparotomy, sequestrectomy, omento burso stomy due to the localization of sequesters in the pancreatic head region. 4 (16.6%) patients died, sepsis and pon were the causes of death, and in 1 patient was with bleeding from acute gastric and duodenal ulcers. o.v. rozenko 46 su r g ic a l d is e a se s issn 2413-6077. ijmmr 2020 vol. 6 issue 1 in 24 (27.3%) patients with phlegmon of the retroperitoneal tissue in the form of “purulent cells”, lumbotomy, sequestrectomy, and drainage of the retroperitoneal tissue were per formed. 3 patients underwent laparotomy, sequest rectomy, omentobursostomy in connection with the localization of sequestrum in the pancreatic head region; 2 patients died. the rate of mortality was 8.3%. in 1 case, there was a left-sided lesion of retroperitoneal fat, in 1 patient – bilateral one. approximately 23 (19.2%) patients were operated on the combination of retroperitoneal phlegmon with abscess of the pancreas and/ or omental bursa. after surgery, 9 patients died, the mortality rate was 39.1%. in 8 (30.4%) patients, laparotomy, necropsy and drainage of abscesses, sanitation and drainage of the abdominal cavity were used as surgical aids. 5 patients died, mortality was 62.5%. all the dead individuals had a left-sided lesion of retroperitoneal fat. the cause of death in 4 patients was sepsis and multiple organ failure, in 1 – sepsis and multiple intestinal fistulas. a combination of various surgical procedures was used in 15 (65.2%) patients with retroperitoneal phlegmon cellulose. in 13 patients, a pancreas abscess and/or omental bursa was performed with pdo-uz, and about retroperitoneal phlegmon – lumbotomy, sequestrectomy, drainage of retroperitoneal fat. including 2 patients, a laparotomy and an omentobursostomy were performed by the third stage in order to remove large sequesters located in the region of the head and body of the pancreas. in 2 patients at the first stage, pdo-uz about the retroperitoneal phlegmon in the form of fluid accumulation were performed, followed by laparotomy, omentobursostomy, opening of the stuffing box abscess, sequestrectomy. in total, 4 patients died in this subgroup, the mortality rate was 26.7%. in 2 patients, leftsided localization of retroperitoneal phlegmon cellulose was detected, in 2 patients, bilateral. the causes of death were sepsis and multiple organ failure – in 3 patients, bleeding from the arrosive splenic artery – in 1 patient. the analysis of the data suggests that leftsided retroperitoneal phlegmon often develops in 64.4% of patients with infected non-biliary necrotizing pancreatitis. right-sided retrope ritoneal phlegmon was found in 25% of patients, and bilateral – in 10.6%. at the same time, the highest mortality rate is observed with bilateral phlegmon of retroperitoneal tissue – 63.6%, with the left-sided it was – 26.9%, and with the right-sided – 7.7%. moreover, the highest mortality is observed with a combination of retroperitoneal phlegmon cellulose and abscess of the pancreas and/or omental bursa – 41.2%. with phlegmon of the retroperitoneal fiber and diffuse purulent peritonitis caused by the in volvement of the parietal peritoneum in the inflammatory process, it was 28.6%, and with the abscess of the retroperitoneal fat without peritonitis – 22.5%. the analysis of the results of the use of various surgical interventions in the case of phlegmon of the retroperitoneal tissue suggests that the use of laparotomy in the case of the phlegmon of the retroperitoneal tissue is accompanied by the highest mortality rate – 34.9%. in this regard, this operation should not be used to treat retroperitoneal phlegmon with the exception of patients with retroperitoneal cellulitis and diffuse purulent peritonitis, due to the involvement of the parietal peritoneum in the inflammatory process. somewhat better results were obtained from the use of puncture – draining operations under the control of ultrasound – 16.7%. however, according to our data, they were effective in 28.6% of patients with retroperitoneal phlegmon cellulose in the form of free fluid accumulation. mortality after lumbotomy was 13.3%. this operation was performed in 71.4% of patients with retroperitoneal phlegmon cellulose in the form of “purulent cells”, incl. in 21.4% of patients in whom the use of pdo-ultrasound was ineffective. thus, the opening of the retroperitoneal tissue from the mini-accesses (lumbotomy, sequestrectomy, drainage of the retroperitoneal tissue) is the most popular surgery for retroperitoneal phlegmon. however, as our experience has shown one opening of retroperitoneal phlegmon cellulose is not enough. in the absolute majority of patients, there was a need to perform stepby-step rehabilitation of retroperitoneal fat (step-down sequestrectomy). comparing the results of our patients’ treatment with similar indicators of other medical institutions, similar methods of surgical interven tions were used, we established certain features (table 1). in the most severe category of patients, where diffuse purulent peritonitis and retro peritoneal phlegmon were combined, the rates of postoperative mortality did not differ sig nificantly among themselves, they also did not have significant differences in patients with isolated phlegmon of retroperitoneal tissue without peritonitis. o.v. rozenko 47 su r g ic a l d is e a se s issn 2413-6077. ijmmr 2020 vol. 6 issue 1 but when using puncture-draining operations of the pancreas abscesses and opening the phlegmon of retroperitoneal tissue from a mini-access of our patients, it made it possible to reduce slightly the mortality rates in comparison with other clinical hospitals. this figure of our patients was 39.1% as in other clinics reached 62.5-48.2% [1, 3]. also, significant differences in the results of treatment of our patients were obtained with the early use of puncture-draining operations for fluid accu mulation in the retroperitoneal tissue and isolated abscess of the pancreas and/or omental bursa – 27.6% in our clinic and 36,6-42.2 in other medical institutions [11, 6]. at the same time, it is recommended to use pdo-ultrasound for abscesses of the pancreas and/or omental bursa, and laparotomy, sequestrectomy and otobursostomy to remove sufficiently large sequesters located in the head and body of the pancreas. conclusions in cases of retroperitoneal fat phlegmon and diffuse purulent peritonitis caused by involvement of parietal peritoneum in the inflammatory process, the preferred treatment is laparotomy, sanitation, and drainage of abdominal, extraperitoneal phlegmon dissection, and retroperitoneal fiber drainage draining followed by with the subsequent programmed sanitation of the abdominal cavity and retroperitoneal tissue. in cases of postnecrotic infected pancreatic and/or parapancreatic fluid accumulations in the retroperitoneal tissue (abscess of the retroperitoneal tissue) in the form of purulent soaking of cellulose spaces of the “honeycomb” type, the operation of choice is the direct surgical intervention on the pancreas and retrope ritoneal tissue from the mini-access (extraperitoneal access lumbotomy), followed by staged sequestrectomy. puncture-draining surgical interventions under the ultrasound control can only be used with retroperitoneal phlegmon cellulose in the form of free fluid accumulation. in the presence of sequesters of large size, which are located in the head and body of the pancreas, the operation of choice is a lapa rotomy, sequestrectomy, omentobursostomy. table 1. comparative results of treatment of patients with the traditional method and with the use of puncture-draining operations patient categories indicators of treatment repeated surgical interventions (progressive diseases) postoperative mortality average length of hospital stay types of surgical interventions puncturedraining operations and/or lumbotomy with mini-access traditional interventions laparotomy and/or lumbotomy puncturedraining operations and/or lumbotomy with mini-access traditional interventions laparotomy and/or lumbotomy puncturedraining operations and/or lumbotomy with mini-access traditional interventions laparotomy and/or lumbotomy isolated abscess and/or phlegmon-retroperitoneal tissue (purulent necro tic phase) 3,3% 6,5% 39,1% 48,2-62,5% 32,5±2,5 48±3,6 phlegmon-retroperitoneal tissue in the form of an accumulation of free fluid and/or an abscess of the pancreas of the omental bursa 2,8% 5,4% 26,7% 36,5-42,2% 28,7±2,5 46±2,8 o.v. rozenko 48 su r g ic a l d is e a se s issn 2413-6077. ijmmr 2020 vol. 6 issue 1 acknowledgements the author is gratefully acknowledged to olena sydorenko (phd in philology, associate professor) with the help in translation of this article. funding this research received no external funding. conflict of interests the author declares no conflict of interests. некротичний панкреатит: шляхи удосконалення хіругічного лікування о.в. розенко донецький національний медичний університет, україна вступ. у зв’язку з неухильним зростанням кількості пацієнтів із деструктивними формами гострого панкреатиту, питома вага якого складає від 10-15 до 20-30%, незважаючи на широкий спектр способів лікування небіліарного некротичного панкреатиту, летальність досягає у випадку інфікованих форм 80-90% і потребує подальшого дослідження та поліпшення методів лікування. мета. метою дослідження є поліпшення результатів лікування хворих на небіліарний некротичний панкреатит шляхом оптимізації хірургічної тактики. методи. до дослідження залучено 120 пацієнтів віком від 22 до 83 років, причому пацієнти віком до 50 років склали 60,0%. чоловіків було 80 (66,7%), жінок – 40 (33,3%). тривалість захворювання до 24 годин визначено у 36 (30,0%) хворих, від 25 до 72 годин – у 25 (20,8%), більше ніж 72 години – у 49 (49,2%). результати. установлено, що у випадку небіліарного некротичного панкреатиту частіше розвивається лівобічна заочеревинна флегмона – у 64,2% (летальність 26,0%) пацієнтів, правобічна виявлена у 24,2% (летальність 6,8%) пацієнтів, а двобічна – у 11, 6% (летальність 64,2%). також найбільш високу летальність засвідчено в поєднанні флегмони заочеревинної клітковини й абсцесу підшлункової залози і/або сальникової сумки – 39,1%. висновки. автори дослідження запропонували різні варіанти хірургічного втручання залежно від залучення до патологічного процесу різних відділів черевної порожнини/заочеревинного простору, що дозволило знизити летальність і час перебування пацієнтів у стаціонарі в 1,5-2 рази. ключові слова: небіліарний панкреатит; люмботомія; парапанкреальна клітковина; флегмона заочеревинної клітковини; лапаратомія; оментобурсостомія. інформація про автора розенко олег володимирович – канд. мед. наук, доцент кафедри хірургії, ендоскопії, оториноларингології та реконструктивно-відновлювальної хірургії, донецький національний медичний університет. information about the author oleg v. rozenko – md, ph.d., associate professor, department of surgery, endoscopy, otorhinolaryngology and reconstructive surgery, donetsk national medical university orcid https://orcid.org/0000-0003-3434-6221, e-mail: oleg.rozenko@gmail.com references 1. baron th, dimaio cj, wang ay, morgan ka. american gastroenterological association clinical practice update: management of pancreatic necrosis. gastroenterology. 2020 jan 1;158(1):67-75. 2. lu jd, cao f, ding yx, wu yd, guo yl, li f. timing, distribution, and microbiology of infectious complications after necrotizing pancreatitis. world journal of gastroenterology. 2019 sep 14;25(34):5162. o.v. rozenko 49 su r g ic a l d is e a se s issn 2413-6077. ijmmr 2020 vol. 6 issue 1 3. ocampo c, zandalazini h, alonso f, canullan c, stagnaro g, leyton v, chiappetta l. a multimodal approach for the first-line treatment of infected pan creatic necrosis. pancreas. 2020 jul 1;49(6):757-62. 4. bugiantella w, rondelli f, boni m, stella p, polistena a, sanguinetti a, avenia n. necrotizing pancreatitis: a review of the interventions. international journal of surgery. 2016 apr 1;28:s163-71. 5. halkic n, pezzetta е, abdelmoumene a. indications and results of retroperitoneal laparostomy in the treatment of infected acute necrotizing pancreatitis. 2003; 58 (1): 97-99. 6. amano hj. therapeutic intervention and surgery of acute pancreatitis. j. hepatobiliary pancreat 2010; 17 (1): 57-59. 7. bartholdy a, werge m, novovic s, hadi a, nøjgaard c, borch a, feldager e, gluud ll, schmidt pn. endoscopic treatment with transmural drainage and necrosectomy for walled-off necrosis provides favourable long-term outcomes on pancreatic function. united european gastroenterology journal. 2020 jun;8(5):552-8. 8. lan l, luo j, yang x, yang d, li m, chen f, zeng n, zhou x. association between timing of surgical intervention and mortality in 15,813 acute pancreatitis. computational and mathematical methods in medicine. 2020 may 16;2020. 9. connor s. surgery in the treatment of acute pancreatitis – minimal access pancreatic necrosectomy. scand j. surg. 2005; 94 (2): 135-42. 10. tu y, jiao h, tan x, sun l, zhang w. laparotomy versus retroperitoneal laparoscopy in debridement and drainage of retroperitoneal infected necrosis in severe acute pancreatitis. surgical endoscopy. 2013 nov 1;27(11):4217-23. 11. sharma p, sharma s, yadav a, rotem e. ct guided percutaneous drainage in necrotizing pancreatitis-highly successful in appropriately selected patients-single center experience. journal of the pancreas. 2019 jan 1;20(1):24-9. 12. luckhurst cm, el hechi m, elsharkawy ae, eid ai, maurer lr, kaafarani hm, thabet a, forcione dg, fernández-del castillo c, lillemoe kd, fagenholz pj. improved mortality in necrotizing pancreatitis with a multidisciplinary minimally invasive step up approach: comparison to a modern open necrosectomy cohort. journal of the american college of surgeons. 2020 apr 3. received 30 march 2020; revised 18 may 2020; accepted 09 june 2020. this is open access article distributed under the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. o.v. rozenko issn 2413-6077. ijmmr 2021 vol. 7 issue 2 51 d e n t is t r y doi 10.11603/ijmmr.2413-6077.2021.2.12488 dental assistance to military personnel of the armed forces of ukraine *o.ya. lavrin, o.v. avdeev, n.ye. romanjuk, o. a. bedenyuk i. horbachevsky ternopil national medical university, ternopil, ukraine background. at present, the formation of military units of the armed forces of ukraine during mobilization takes place with underlying high dental morbidity of the population of the country. objective. the aim of this research was to study dental health of the personnel of military units and the problem of providing dental care to military personnel according to the analysis of modern scientific literature. methods. the following research methods were used to analyse dental care for military personnel, namely: bibliographic, analytical, systems approach. results. hygiene of the oral cavity is mandatory in military units and should be routinely carried out. it was found that the main reason for seeking dental care among military personnel was treatment of major dental diseases. a low number of military personnel seeking for dental preventive examination was noted that proved a low level of primary prevention among this category of people. conclusions. therefore, the obtained data on the state of dental health of military personnel require a purposeful approach to organization of prophylaxis and prevention of complications through professional hygiene and early prosthetics. keywords: military personnel; dental diseases; prevention; professional hygiene of the oral cavity. *corresponding author: oksana ya. lavrin, phd student, i. horbachevsky ternopil national medical university, ternopil, 46001, ukraine. e-mail: lavrin_oia@tdmu.edu.ua international journal of medicine and medical research 2021, volume 7, issue 2, p. 51-57 copyright © 2021, tnmu, all rights reserved o.ya. lavrin et al. introduction the current socio-political situation is characterized by a large number of local military conflicts in many countries, including ukraine. it is true that military conflicts lead to mass disability and death of people, including military personnel performing their duty at the territories of combat operations [1]. a large number of publications in the military medical literature aimed at improving organization of dental care due to specific living conditions and combat effectiveness of military personnel of the armed forces of ukraine, the peculiarities of the military profession, as well as factors influencing the course of pathological processes of the dentoalveolar apparatus, methods of their prevention and treatment [2]. at present, formation of military units of the armed forces of ukraine during mobilization takes place with a high dental morbidity of the population in the country [3, 4]. after special missions, military personnel often complain of a sharp deterioration in dental health, which is associated with the inability to receive qualified assistance at the place of temporary deployment of the unit. as a rule, this is due to the lack of dentist or appropriate materials and equipment, the difficulty of trans­ porting a serviceman to the place of timely, sometimes emergency dental care [5]. the level of dental health of organized military contingents depends on the motivation of the personnel, social and hygienic factors, which include ecological situation in the region, as well as on the development of dental services, principles of organization of medical care, and prevalence of dental diseases [4, 6]. military service and combat missions by personnel is invariably accompanied by high emotional and psychological stress, various kinds of disorders in their health, including dental health. therefore, the measures for prevention of dental diseases before military missions is very important. unfortunately, organization of dental care is insufficient. therefore, the objective of the research was to study the level of dental health of the personnel of military units and the problems of providing dental care to military personnel according to the analysis of modern scientific literature. issn 2413-6077. ijmmr 2021 vol. 7 issue 252 d e n t is t r y review a large number of publications in the military medical literature are aimed at improving the organization of the dental care system. this is due to the specific living conditions and combat effectiveness of the military personnel of the armed forces of ukraine, due to the peculiarities of the military profession, as well as factors influencing the course of pathological processes of the dentoalveolar apparatus, requiring methods of prevention and treatment. diseases of the teeth and their complications reduce the working and combat efficiency of military personnel, so the provision of dental care to this category is a problematic issue [7]. naumenko k.e., belikov o.b. (2018) after examining military personnel for dental morbidity, it was found that the prevalence of caries among conscripts was 100%, and among contract soldiers – 93.41%. when assessing the indicators of the need for military personnel in various types of outpatient dental care based on the analysis of the structure of the kpu indices, the authors found that 32.83% of the conscripts needed caries treatment, 16.41% – its complications, 17.91% – tooth removal. the amount of already filled teeth was about 22.38%, and removed – 10.44%. among the contract servicemen, 23.10% had teeth with caries, 39.29% – filled, 16.78% – removed. there is also a tendency that the need for treatment increases with age due to the number of removed teeth [8]. authors analysed the dental morbidity rate among military personnel of various branches of the chernivtsi garrison based on the analysis of medical reports and outpatient journals. among the nosological units of dental morbidity in 35.20% of cases uncomplicated caries was found, in 44.89% – its complicated forms, in 13.26% – periodontal disease, 5.35% – deformities and occlusion anomalies, and only 1.27% were inflammatory diseases of the oral mucosa. 63.25% of servicemen needed therapeutic treatment, 19.77% – surgical, 15.42% – orthopaedic and 1.56% – orthodontic care [2, 8]. among the military personnel who need therapeutic treatment, acute forms of periodontitis were the most often in 39.11%, acute and chronic medium caries – 35.08%, acute forms of pulpitis – 25.80%. among periodontal diseases, periodontitis of the first degree prevailed – 42.30% and the second degree– 36.53%, catarrhal gingivitis – 21.15% [9, 10]. according to the researchers, the prevalence of dentoalveolar anomalies among pre-conscripts is 63.3%. at the same time, almost 2% of these young people have absolute restrictions on military service. in addition, defects in the dentition is revealed in 22.6% of people aged 18-22 years, who study in military schools in kiev [9]. according to the results of dental examinations of conscript cadets aged 18-22, it was found that of the total number of conscripts routinely surveyed, 52.4% needed dental care, and in the western region of our country this number was 80-85%. the authors associate this first of all with the extremely low level of dental care just before the conscription of this category of the population in medical and preventive institutions of the ministry of health of ukraine [9, 10]. the above materials indicate a fairly high level of dental morbidity among the military personnel of ukraine, an extremely low level of dental care for this category of the population. the authors also found that the increase in the need for orthopaedic treatment depended on the age of the military personnel. the ca tegory of 19-24 years old required the restoration or manufacture of artificial crowns, the category of 25-30 and 30-35 years old – crowns and bridges, and 35 years old and older – bridges and partial removable structures [9]. lyshchyshyn m.z. and co-authors (2020) after examining military personnel for periodontal tissue diseases, found that among conscripts, gingivitis was revealed in 23.33%, and periodontitis – in 13.33%. in soldiers of the contract form of service, on average, 32.76% of the surveyed had periodontitis and 34.76% – gingivitis. at the same time, the worst indicators were in the age groups of 19-24, 30-35 and 35 years old and older [10, 11]. rachinsky s.v. and co-authors analysing oral hygiene among both conscripts and contract soldiers found out that the level of hygiene was “satisfactory” and “unsatisfactory”, and only in some categories of servicemen 11.50% it was “good”. this indicates on a lack of sanitary and educational work [12]. the main tasks of the dental service of the armed forces of ukraine are to provide certain medical assistance to the military for each level, dental care to the wounded and sick (dental care) and medical rehabilitation of combatants [13]. thanks to the signed memorandum of cooperation between the military medical department of the ministry of defence and the association of dentists of ukraine, dental o.ya. lavrin et al. issn 2413-6077. ijmmr 2021 vol. 7 issue 2 53 d e n t is t r y offices have been established at all training grounds and centres [13, 14]. most of the military returning from the zone of hostilities in the eastern ukraine need medical rehabilitation, including modern dental care and dental prosthetics. garrison dentists are important for dental assistance to the military. badiuk m.i. (2020), analysing the organization of dental care for soldiers of the armed forces of ukraine, established an increase in the number of maxillofacial wounded in the overall sanitary losses. the authors emphasize the importance of restoring mobile dental offices to improve the level of dental care for soldiers [16, 17]. mobile dental office (mdo) is a unit of the medical service designed to provide outpatient dental care to the personnel of units and subdivisions that do not have regular dental offices [14]. the mobile dental office is used to provide dental care in the field and outside of military garrisons during the conduct of hostilities, conducting exercises, field training sessions, and eliminating the consequences of emergencies [18]. mobile dental office is part of the clinics of maxillofacial surgery and dentistry of military medical clinical centres of the regions, the central dental clinic of the ministry of defence of ukraine and the ukrainian military medical academy (as an education unit) [14, 18]. the mobile dental office is deployed in a special modular unit based on a truck. the design and complete set of the mobile dental office gives the chance to provide out­patient dental services autonomously, in full and in comfortable conditions [14, 19]. the dental care to the personnel of the military unit is provides by a dentist of the medical centre, who is subordinate to the head of the medical centre, and on special issues – to the garrison dentist and follows their instructions. the duties of a dentist of a military unit are to provide outpatient dental care to the personnel of the unit as well as first aid for injuries of the maxillofacial area and to ensure timely hospitalization and treatment of dental patients [20]. in a separate medical battalion (omedb) the unit has a dental office, which includes a dentist (the head of the dental office omedb) and a nurse. in the context of reforming of the medical system, still there are a number of issues on organization of therapeutic and prophylactic dental services in the armed forces of ukraine. belikov a. (2017) analysed the level of dental care for servicemen according to the survey data. the author claims that the main reason for seeking dental care among the respondents was the treatment of major dental diseases. a low percentage of military personnel who consulted a dentist for a preventive examination was recorded that proved a low level of primary prevention among this category of population [19]. dentists of medical companies provide dental care to subordinate personnel only as emergency care (removal, disclosure of subperiosteal abscesses, disclosure of root canals, etc.), as complete equipment is not used. therefore, in the field dental care is provided sporadically and as an emergency care for acute cases (acute pulpitis, exacerbation of periodontitis, and trauma). it has been established that dentists of medical brigades perform their duties, mainly in the field and outside of their permanent locations. it has been proved that the main burden of providing dental care to military personnel in the combat zone is borne by the dental offices of military mobile hospitals. since 2014, the work of regular mobile dental offices has been resumed. at the same time, the office is constantly changing its location and provides dental care directly at the locations of various departments. analysis of the work of this cabinet shows high efficiency of its use in various military units [21]. dental care for maxillofacial wounded is provided within the general structure of medical and evacuation measures of the medical services of the armed forces of ukraine, including by regular dentists. specialized treatment of such wounded is carried out in specialized clinics of maxillofacial surgery, dentistry of military medical clinical centres and dental departments of hospitals. the dental care to military personnel is carried out with a very high dental morbidity among the personnel of the units. therefore, the full-time dentists of the brigades face a significant burden as they often treat servicemen in difficult field conditions during hostilities. most of this assistance is provided in the form of emergency dental care. the main burden of providing dental care is assigned to the dental service of the military mobile hospitals [20]. mobile dental offices were introduced into these hospitals, which made it possible to o.ya. lavrin et al. issn 2413-6077. ijmmr 2021 vol. 7 issue 254 d e n t is t r y significantly improve the quality of dental care for military personnel and bring it closer to the deployment sites of military units. today, a new model of providing dental care to military personnel has been developed and is successfully functioning in ukraine, re presented by a network of stationary and mobile offices. depending on the peculiarities of the functioning, provision and volume of care for the wounded and dental patients, the specified model provides for division of the involved dental offices into 3 types by “categories”. in the dental departments of military hospitals and spe cialized clinics of military medical clinical centres, dental care for military personnel is provided in full, including orthopaedic care [14, 21]. some scientists compared the level of dental care in the units of the armed forces of ukraine and nato countries. the health protection measures for the military personnel of nato countries are an integral element in the system of combat readiness of troops. document ajp-3.14 “allied joint doctrine for forse protection” defines health care as a very important part of their protection [22]. nato national military and allied commands emphasize the importance of ensuring an adequate level of medical, dental and mental health for troops. the experience of the alliance countries asserts that the presence of dental diseases reduces the combat readiness of troops. routine dental examinations and standardized treatment programs ensure that personnel remain healthy during troop deployments in the war zone [15]. according to the stanag 2466 standard “dental fitness standards for military personnel and a dental fitness classification system”, which regulates monitoring of dental health of military personnel even before the deployment of medical units in the combat zone, nato has introduced a system of comprehensive prevention of dental morbidity among military personnel [16]. the routine of planned dental examinations, preventive measures and unified standardized treatment programs minimizes the incidence of acute forms of oral diseases in military personnel within the combat zone. therefore, for improvement of dental care for servicemen of the armed forces of ukraine, this should be taken into account and brought in line with the requirements of nato standards. implementation of nato standards facilitates improvement of the level of dental care for military personnel, which is one of the key state tasks in improvement of defence of ukraine. significant attention is paid to dental care as a component of medical support for troops in the armies of the nato countries. that is why following the euro-atlantic standards by military dentistry in ukraine is a serious tool for further improvement of dental care for military personnel. among all the medical standards of the euro-atlantic alliance, there are three that should be singled out, they directly relate to the dental care of military personnel. all standards provide for the use of forces and means in the zone of hostilities. the first is preventive, or stanag 2466 “dental fitness standards for military personnel and a dental fitness classification system” [23]. it has been established that the armies of the nato countries have introduced a system of comprehensive prevention of dental morbidity among military personnel. the system of planned dental examinations, preventive measures and unified standardized treatment programs excludes the entry of military personnel with acute forms of dental diseases into the zone of hostilities. this standard contains a unified classification of the dental health of military personnel, provides 4 groups or classes [10, 23]. analysing the situation, it should be noted that, unfortunately, in ukraine, dental prevention programs have been curtailed both at the state level and in the ukrainian armed forces. there is a clear tendency towards an increase in dental morbidity among military personnel in a special period, which indicates the low quality of the military medical examination. the second stanag 2453 amedp-35 “the extent of dental and maxillofacial treatment at role 1­3” [24] defines the staffing, logistics and volume of medical care for servicemen with dental diseases and injuries of the maxillofacial area at three levels, deployed in the area of military action. the third dental standard – stanag 2464 amedp-3.1 “military forensic dental identification” [25] provides for participation of the dentist in the forensic medical examinations and is based on modern international protocols and procedures for identifying people by examining the remains of the oral cavity. in ukraine, in order to meet the requirements of this standard, it is necessary to introduce o.ya. lavrin et al. issn 2413-6077. ijmmr 2021 vol. 7 issue 2 55 d e n t is t r y legislative changes for the possible admission of military dentists to the process of forensic medical examination in the combat zone. to create a single electronic database of orthopantomograms for all military personnel, it is necessary to provide all hospitals with modern digital dental x-ray equipment. this will ensure not only implementation of the direct use of stanag 2464 amedp­3.1, but also significantly increase the clinical and diagnostic capabilities of military dental units in ukraine [10]. conclusions diseases of the teeth and oral cavity negatively affect the combat effectiveness of military personnel and worsen the course of existing concomitant diseases. therefore, professional hygiene of the oral cavity is mandatory in military units and should be routinely carried out. it is established that the professional hygiene of the oral cavity depends on the precise organization of dental care in military units. it should be noted that the domestic military dentistry requires further improvement. the increase in the number of military personnel with requests for dental care is associated with a decrease in the number of preventive examinations, a decrease in the number of sanitized people and an increase in those who need dental hygiene. this proves insufficient medical care for this category of the population. thus, the attained data on the state of dental health of military personnel require a purposeful approach to the organization of prophylaxis, prevention of complications through professional hygiene and early prosthetics. therefore, new approaches to organizing and ensuring the provision of dental care to military personnel taking into account nato standards should be developed in ukraine. this ensures a high level of dental care for this category of population. conflict of interests authors declare no conflict of interest. acknowledgements the research has not been funded or supported by any research grant. authors contributions oksana ya. lavrin – writing – original draft, writing – reviewing and editing; oleksandr v. avdeev – writing – reviewing and editing; nina ye. romanjuk – data collection, investigation, formal analysis; oleksandr a. bedenyuk – conceptualization, methodology, formal analysis. особливості надання стоматологічної допомоги військовослужбовцям збройних сил україни о.я. лаврін, о.в. авдєєв, н.є. романюк, о.а. беденюк тернопільський національний медичний університет імені і.я. горбачевського моз україни, тернопіль, україна вступ. в умовах сьогодення формування бойових підрозділів збройних сил україни під час мобілізації відбувається на тлі високої стоматологічної захворюваності населення держави. мета роботи вивчення рівня стоматологічного здоров'я особового складу військових підрозділів та проблеми надання стоматологічної допомоги військовослужбовцям згідно аналізу сучасної наукової літератури. методи. для аналізу стану стоматологічної допомоги військовослужбовцям використано такі методи дослідження, а саме: бібліографічний, аналітичний, системний підхід. результати. санація порожнини рота є обов’язковою у військових частинах та повинна проводитися в плановому порядку. встановлено, що основною причиною звернень за стоматологічною допомогою серед військовослужбовців було лікування основних стоматологічних захворювань. відмічено низький відсоток військовослужбовців, які звернулися до стоматолога з метою профілактичного огляду, що свідчить про низький рівень первинної профілактики серед вказаної категорії осіб. висновок. отже, отримані дані щодо стану стоматологічного здоров’я військовослужбовців потребують цілеспрямованого підходу до організації профілактики, попередження ускладнень шляхом санації і раннього протезування. ключові слова: військовослужбовці; стоматологічні захворювання; профілактика; санація ротової порожнини. o.ya. lavrin et al. issn 2413-6077. ijmmr 2021 vol. 7 issue 256 d e n t is t r y references 1. rachinsky sv, schneider sa, labunets ov, dieva tv, labunets va, nomerovskaya ee. justification of the need for a differentiated approach in the organization and planning of dental orthopedic care for people of military age in ukraine. colloquiumjournal 2021;4(91):11­5/. [in ukrainian]. 2. naumenko ky, belikov ob. the prevalence of major dental diseases and the need of the military in orthopedic treatment (review of literature). bmh journal 2017;1(81):211­14. [in ukrainian]. 3. hasiuk p, hasiuk n, kindiy d, ivanchyshyn v, kalashnikov d, zubchenko s. characteristics of cellular composition of periodontal pockets. inter ventional medicine and applied science 2016;8(4):172­7. 4. kalashnikov dv, hasiuk pa, vorobets ab, rosolovska so, kindiy dd, hrad ao, et al. features of the course of enamel biomineralization processes in various anatomical areas of the tooth. wiadomosci lekarskie 2020;73(5):864­7. 5. rachinskyi sv, schneider sa, labunets ov, diieva tv, labunets va, diieva oe. the prevalence and intensity of development of dental orthopedic morbidity among men of military age in the south of ukraine. innovation in stomatology 2019;1:50­4. [in ukrainian]. 6. rachinskij sv, shnajder sa, labunec va, labenec ov, dieva tv. morbidity and condition of dental orthopedic care in pre-conscripts and military personnel of ukraine (literature review). stomatological bulletin 2019;4(109):57­61. [in ukrainian]. 7. bilyy vya, badyuk mi, verba av, zarutsky yal, kazmirchuk ap, savitsky vl, et all. a look at the system of medical support of the defense forces of ukraine. military medicine of ukraine 2019;3(19):5­ 14. [in ukrainian]. 8. naumenko k, belikov a. the сharacteristic of the dental diseases in the military personnel of the chernivtsi boundary group. ukrainian journal of medicine, biology and sport 2018;1(18):201­6. [in ukrainian]. 9. rachinskij sv, shnajder sa, labunec ov, dijeva tv, labunec va. the need and provision of young people of military age in dental orthopedic care. visnyk stomatologii 2020;2(36):59­63. [in ukrainian]. 10. lyshchyshyn mz, kovalenko vv. status and prospects of development of military dentistry in ukraine. medicni perspective 2020;25(1):9­17. [in ukrainian]. 11. lyshchyshyn m, pavlovskyi l. central dental clinic of the ministry of defenseof ukrainian in the historical retrospective review. militari historical bulletion 2020;1(35):131­44. [in ukrainian]. 12. rachinsky sv, schneider sa, labunets ov, dieva tv, labunets va. the forecast of the prevalence and intensity of the development of dental orthopedic morbidity in people of military age in the western region of ukraine. stomatological bulletin 2020; 3(112):65­9. [in ukrainian]. 13. chernov d. assestment of the health status of conscripts. health and ecology issues 2021;18(1):5­ 13. [in ukrainian]. 14. nidzelsky mya, pisarenko oa, tsvetkova nv. organization of dental orthopedic care in military units. poltava: gontar ov; 2019. 108 р. [in ukrai nian]. 15. kovalenko vv, lyshchyshyn mz. euro-atlantic prospects of ukrainian dentistry. novini stomatologii 2017;2(91):24­7. [in ukrainian]. 16. badiuk mi, kovalenko vv, solyaryk tv, yarosh tv. improvement of dental care for military servicemen of the armed forces of ukraine in accordance with nato standards. ukrainian journal of military medicine 2020;3(1):36­44. [in ukrainian]. 17. badiuk mi, mykyta oo, shvets av, sereda ik, kovyda dv, hubar am. analysis of the work of the medical service on the priority areas of development and implementation of standards of medical support of the armed forces of ukraine for a special period. military health problems: coll. sci. paper. umma 2016;45:9­16. [in ukrainian]. information about the authors oksana ya. lavrin – phd student, i. horbachevsky ternopil national medical university, ternopil, ukraine orcid 0000­0001­8231­2945, lavrin_oia@tdmu.edu.ua oleksandr v. avdeev – professor, i. horbachevsky ternopil national medical university, ternopil, ukraine orcid 0000­0001­7703­5242, avdeev@tdmu.edu.ua nina ye. romanjuk – associate professor, i. horbachevsky ternopil national medical university, ternopil, ukraine orcid 0000­0001­6333­2828, fedchyshyn@tdmu.edu.ua oleksandr a. bedenyuk – associate professor, i. horbachevsky ternopil national medical university, ternopil, ukraine orcid 0000­0002­9644­1809, bedenyukoa@tdmu.edu.ua o.ya. lavrin et al. issn 2413-6077. ijmmr 2021 vol. 7 issue 2 57 d e n t is t r y 18. zhakhovskiy vo, livinskiy vg, kudrenko nv, slabkiy go. functioning of medical forces of the armed forces of ukraine in the conditions of reforming the healthcare system in ukraine. ukraine nation’s health 2020;4(62):24­33. [in ukrainian]. 19. naumenko k, belikov a. questioning of the military personnel of the chernivtsi boundary group concerning the level of rendering the dental care. ukrainian journal of medicine, biology and sport 2017;1(10):221­4. [in ukrainian]. 20. kazmirchuk ap. organization of the provision of specialized (highly specialized) medical care to servicemen of the armed forces of ukraine (according to the national military medical clinical center “gvkg”). military medicine of ukraine 2017;1(17):24­ 33. [in ukrainian]. 21. kalchuk av. ways of improving the medical support of the armed forces of ukraine and other military formations during the anti-terrorist operation. problems of military health care 2017;47:303­ 10. [in ukrainian]. 22. a jp-3.14. allied joint doctrine for forse protection. edition a, version 1. with uk national elements; 2015. 97 р. 23. stanag 2466. dental fitness standards for military personnel and a dental fitness classification system; 2014. 112 p. 24. stanag 2453/amedp­35: the extent of dental and maxillo-facial treatment at role 1-3; 2017. 148 p. 25. stanag 2464/amedp­31: military forensic dental identification; 2014. 88 p. received 11 november 2021; revised 23 november 2021; accepted 13 december 2021. this is open access article distributed under the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. o.ya. lavrin et al. 25 in t e r n a l m e d ic in e issn 2413-6077. ijmmr 2019 vol. 5 issue 2 doi 10.11603/ijmmr.2413-6077.2019.2.10459 some metabolic processes in the patients with long-term consequences of mild traumatic brain injury ye. lekomtseva institute of neurology, psychiatry and narcology of the national academy of medical sciences of ukraine, kharkiv, ukraine background. mild traumatic brain injury (mtbi) leads to disturbance of various metabolic processes significant in pathogenesis of the maintaining of long-term consequences after it. the objective of the research was to analyse changes in the activity of some membrane-associated enzyme markers, which are involved in different redox reactions, reflecting main metabolic processes. methods. forty-seven patients with long-term consequences of mtbi, thirty controls were enrolled. the levels of aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase, lactate dehydrogenase (ldh), gamma-glutamyl transpeptidase were evaluated in sera by gas-liquid chromatograph and calorimetric methods. results. the study revealed significant changes in metabolic processes observed for alkaline phosphatase and ldh, which were the indicators of membrane and redox processes disturbances, acidosis severity and impaired energy cell metabolism. the averages of ldh level was 662.7 versus 381.9 u/l, in the controls. the disease progression was followed by directly proportional ldh increase reaching very high values in the patients with disease duration more than 15 years (mean ±sd 144.6±16.3 versus 82.6±8.4 u/l, controls p<0.05). the long-term consequences of mtbi were characterized by statistically significant decrease of alkaline phosphatase and positive dependence (p<0.05) of it (r=+0.48) on the disease duration with the averages of alkaline phosphatase level of 152.5±11.21 versus 212.6±9.63 u/l, controls (p<0.01). the significance of changes in membrane-associated enzymes serum levels correlated with development of oxidative stress and metabolic processes dysfunction. conclusion. in the patients with long-term consequences of mtbi, dysregulation of enzymes activity was detected that might be a marker of nervous system energy impairment and membranes destruction. key words: long-term consequences of mtbi; membrane-associated enzymes; metabolic processes. corresponding author: yevgeniya lekomtseva, md, ph.d., state institute of neurology, psychiatry and narcology of the national academy of medical sciences of ukraine, department of neurology, department of functional neurosurgery and paroxysmal states, 46 academic pavlov str, kharkiv, 61068, ukraine. e-mail: yevgeniyalekomtseva@gmail.com introduction mild traumatic brain injury is a leading cause of disability in young people [2, 21]. neurotrauma leads to disturbance of various types of metabolic processes that are significant in the pathogenesis of maintaining of long-term consequences after it [9, 12, 15]. it is established that metabolic disturbances are based on biochemical reactions proceeding with participation of enzymes located in various parts of the cell [3, 7, 22]. most of these enzymes are inside the cell or mitochondria and appear in blood or cerebral spinal fluid only when cells are damaged, and this is of great diagnostic and prognostic importance. thus, the changes in any parameters of membrane-associated enzymes evidence for disturbances of metabolic processes of biochemical redox reactions, cell or mitochondria destruction or development of membrane pathology [1, 4, 18, 20]. enzymes are proteins, each type has a unique function facilitating catalyzation of routine and vital chemical reactions in the organisms [5, 17, 19]. among the most sensitive and widely used membrane-associated enzymes are aspartate aminotransferase (ast) and alanine aminotransferase (alt); in addition to them, alkaline phosphatase and lactate dehydrogenase (ldh) are the other interesting enzymes. ldh is not specific to the liver and can be elevated in many other diseases related to inflammation in tissues [16, 18]. human studies have shown that serum levels of autoantibodies (anti-nuclear antibody, anti-smooth muscle antibody, and anti-liver and kidney microsomal antibody) are elevated in patients with autoimmune hepatitis and some rare muscle neurodegenerative disorders [6, 8, 10]. symptoms of international journal of medicine and medical research 2019, volume 5, issue 2, p. 25-31 copyright © 2019, tnmu, all rights reserved ye. lekomtseva 26 in t e r n a l m e d ic in e issn 2413-6077. ijmmr 2019 vol. 5 issue 2 long-term consequences after mild traumatic brain injury can or cannot be present in the patients with mild increase of metabolic enzymes [4, 13, 21] and symptoms of posttraumatic period are not specific, thus the membrane­ associated enzyme abnormalities can sometimes provide us with the useful clarification of a cause of the condition. according to these issues, the objective of research was to analyze the dynamics of changes in the activity of some indicators of membrane-associated enzymes, which are involved into certain redox reactions, reflecting the main metabolic processes in the patients with long-term consequences of mild traumatic brain injury. methods forty-seven patients with long-term consequences of mild traumatic brain injury (mtbi) of the average age of 47.41±9.37 years old (16 women, 34.05% and 31 men, 65.95%) and the average disease duration (mean±sd, 12.67±8.92 years old) were enrolled into the study. the definition of mtbi was consistent with the world health organization’s international statistical classification of diseases and related health problems (icd­10; 1992) [2]. thirty healthy volunteers (control; mean±sd, 35.6±9.21 years) without neurological and psychiatry diseases were involved as well. in the anamnesis, the investigated patients suffered from concussion (n=18; 38.29% among them there were eleven women, 61.11% and twenty men, 38.9%) and mild brain contusion (n=29; 61.71%, 17.24% women and 82.76% men). the patients were treated with symptomatic therapy. inclusion criteria involved the patients of above 18 years of age with nonpenetrating head injury and initial glasgow coma scale score more than 7 (mean±sd, 10.37±2.14). exclusion criteria were craniectomy and sepsis in the anamnesis, pregnancy, preexisting neurologic diseases, acute cardiovascular diseases, respiratory failure, any acute or chronic liver diseases. clinical data of each case were retrieved from the patients’ history. physical and neurological examinations were performed for all patients, ct and/or mri, eeg recording were retrieved as well. neurological examination revealed focal neurological signs, indicating mesencephalic and brainstem structures lesions; the most frequent were tendon reflexes increase in 23 patients (41.07 %), coordination disturbances – 21 patients (37.5 %), ataxia – 15 patients (26.78 %), horizontal nystagmus – 11 patients (19.64 %), pathological foot reflexes – 8 patients (14.28 %) and rotator nystagmus – 5 patients (8.93 %). aspartate aminotransferase (ast), alanine aminotransferase (alt), alkaline phosphatase, lactate dehydrogenase (ldh), creatine kinase and gamma-glutamyl transpeptidase (ggt) were detected in sera by the gas-liquid chromatography and the calorimetric methods according to the standard protocols provided by the manufacture using commercially available human elisa kits (clinical biochemistry department, kharkiv national medical university). controls and individual sampling were performed in duplicate manner with a coefficient of variation of <10 % (p values ≤0.05); all pro­ tocols were approved by the local ethics committee; written informed consents were obtained from all patients. age and disease duration were compared between the groups by the χ2 test; parametric tests were used for normally distributed data; nonparametric tests were used for abnormal distributed data; kruskal­wallis and mann­ whitney u tests were applied in prism regarding the differences between groups, the multivariate analysis considering covariates was performed. results the results of all patients examinations have revealed the significant changes in mem­ brane-associated enzymes serum levels manifested as the increase of creatine kinase (ck) serum levels by 41.4%, ldh by 73.5% and decrease of alkaline phosphatase serum levels to 28.3% and a tendency of ggt serum level decrease to 18.7% in the general clinical group (table 1). analysis of the dynamics of the membraneassociated enzymes markers in the patients with long-term consequences of mtbi proved that the most severe and statistically significant changes were observed for two enzymes: alkaline phosphatase and ldh, which are the indicators of cell or mitochondria membrane damage, acidosis severity, isolation of redox processes and phosphorylation, impaired energy metabolism in cells. metabolic changes in the examined patients evidenced that the content of serum ldh, a key glycolysis enzyme, was increased in almost all patients, and this increase was in a direct proportion with the disease progression or longer posttraumatic period (p<0.05; r=+0.36). we found the elevated ldh level in serum ye. lekomtseva 27 in t e r n a l m e d ic in e issn 2413-6077. ijmmr 2019 vol. 5 issue 2 samples of the investigated patients compare to hc (p=0.01, t=5.08); in the general patient group the medians of total ldh level was 662.7 u/l and 381.9 u/l, in controls, respectively. to evaluate the clinical prognostic value of ldh changes we further segregated among the different disease duration groups. when comparing the ldh serum level in the patients with different duration of the post-traumatic period after mtbi, it was proved that the patients with the disease duration no longer than 5 years (n=14; 29.78%) were characterized by increase of ldh serum levels by 27.8% (p>0.05). in the patient group with the disease duration from 5 to 15 years (n=11; 23.4%), the ldh was increased to 78.6% (p=0.05) with average level of 682.4±24.8 u/l, and in the patients with the disease duration more than 15 years (n=22; 46.8%), the ldh was increased to 105.2% (p=0.01) with average level of 783.6±31.4 u/l. the revealed increase of ldh serum level, in our opinion, was a marker of increased membrane destruction as a result of metabolic disturbances. it should be noted that in the patients with long-term consequences for more than 15 years, ldh serum level was higher compare to hc and average group indicators, which, in our opinion, was a poor prognostic sign. when comparing the alkaline phosphatase serum level in the patients with different duration of post-traumatic period after mtbi, it was found that the patients with the disease duration no longer than 5 years was characterized by 8.5% decreasing (p>0.05) of alkaline phosphatase serum level. in the patients with the disease duration between 5 and 15 years, the mean serum level of alkaline phosphatase was decreased by 24.1% (p>0.05); in the patients with the disease duration more than 15 years, alkaline phosphatase level was decreased by 27.6% (p<0.01) in sera. at the same time, further disease progress or longer posttraumatic period was accompanied by directly proportional increase of ck serum levels: the values remained higher than the controls with an average of (mean ±sd) 116.8±19.4 u/l (p<0.05) in the general group and reached statistically significant values in the patients with the disease duration more than 15 years (mean ±sd 144.6±16.3 u/l versus 82.6±8.4 u/l controls; p<0.05). however, long­ term consequences of mtbi revealed quite significant increase in ck level by 50.3% com­ pare to the patients of the subgroup with the duration of the posttraumatic period less than 5 years. this correlated positively with a higher frequency of various neurological syndromes (p<0.05; r= +0.63) for long­term consequences of mtbi and might be a biochemical marker of the latter neurological deficit. table 1. the dynamics of the markers of membrane-associated enzymes serum levels in the patients with long-term consequences after mild traumatic brain injury data all patients(general group) dependence from the post-traumatic period duration up to 5 years (stage 1) from 5 to15 years (stage 2) more than 15 years (stage 3) ast, (u/l) 29.6±3.3 p>0.05 26.6±2.9 p>0.05 27.3±4.8 p>0.05 30.3±7.1 p>0.05 controls 25,2±1,9 alt, (u/l) 22.8±3.9 p>0.05 21.8±4.2 p>0.05 21.4±2.1 p>0.05 23.5±3.7 p>0.05 controls 20,5±1,4 alkaline phosphatase, (u/l) 152.5±11.21 p<0.01 194.6±6.7 p>0.05 161.3±15.3 p>0.05 153.9±9.8 p<0.01 controls 212,6±9,63 ldh, (u/l) 662.7±21.9 p=0.01 488.3±36.3 p>0.05 682.4±23.1 p=0.05 783.6±18.4 p=0.01 controls 381.9 ±28,1 ck, (u/l) 116.8±19.4 p<0.05 109.5±27.1 p>0.05 126.4±22.6 p>0.05 144.6±16.3 p<0.05 controls 82,6±8,4 ggt, (u/l) 14.8±6.2 p>0.05 19.6±3.5 p>0.05 15.9±9.3 p>0.05 14.1±4.2 p>0.05 controls 18,2±1,4 note. p – significance compared to the control group. ye. lekomtseva 28 in t e r n a l m e d ic in e issn 2413-6077. ijmmr 2019 vol. 5 issue 2 the same dynamics was evidenced with the respect to ggt serum level but without statistically significant changes: the ggt serum level at stage 1 and stage 2 practically did not differ from the control. in the remaining group of the patients, there was a decrease of ggt serum levels compare to hc (mean ±sd 14.1±4.2 versus 18.2±1.4 u/l) but also without any statistically significant changes (p>0.05). discussion the attained data complied with the literature, in our opinion reflecting the effect of the increasing deficiency of oxidative reactions in the body on aminoacid metabolism in this category of patients [11, 12, 14]. the decrease in some markers of membrane-associate enzymes activity could be a marker of impaired metabolic processes following long-term con sequences after mtbi. the revealed increase of membraneassociate markers enzymes in the patients with long-term consequences after mtbi was directly proportional to the severity of neurological deficit (p<0.05; r= +0.63) as a result of the de­ velop ment of membrane cell or mitochondrial membrane pathology and that might be a marker of the degree of nervous system damage. in turn, the destruction of cell membranes was pathoge netically associated with the development of oxidative stress [6, 15, 16, 18]. the long-term consequences after mtbi was characterized by the statistically significant decrease of alkaline phosphatase serum level and positive dependence of alkaline phosphatase (p<0.05; r=+0.48) on the disease dura­ tion reflecting the specificity of the revealed changes in its activity in cases of cell energy deficiency in this category of the patients, which was an important diagnostic criterion. the decrease in alkaline phosphatase activity according to the previous literature [7, 10] might be a marker of impaired intracellular metabolism in long-term consequences of mtbi. in the general patient group, there was a significant increase in ck serum level compare to controls with the maximal biochemical values in the third clinical group with long-term consequences of mtbi more than 15 years and combined in 18.2% of the patients (n=4) with partial epileptic seizures. in our opinion, the direct proportional increase in ck activity with the progression and increase of posttraumatic period was associated (p<0.05; r= +0.63) with development of destructive processes in cells as a result of oxidative stress and disturbance of intracellular bioenergetic processes. it was noteworthy that the ck increase in cases of posttraumatic encephalopathy in the patients with long-term consequences more than 15 years did not reach the same value (according to the literature) as in hereditary neuromuscular diseases and this might be a differential diagnostic criterion for posttraumatic ence phalopathy. slightly different dynamic was observed analysing the information about another enzyme involved into exchange of amino acids and peptides – ggt. at the first and second stages, a tendency of ggt serum level decrease was evidenced, which could not be significant in the patients with disease duration more than 15 years compare to the control. this data complied with the literature on ggt serum level figure 1. analysis of the dynamics of lactate dehydrogenase and creatine kinase levels in the patients after traumatic brain injury with different duration of posttraumatic period. note. p – significance compared to the control group. when comparing the alkaline phosphatase serum level in the patients with different duration of post-traumatic period after mtbi, it was found that the patients with the disease duration no longer than 5 years was characterized by 8.5% decreasing (p>0.05) of alkaline phosphatase serum level. in the patients with the disease duration between 5 and 15 years, the mean serum level of alkaline phosphatase was decreased by 24.1% (p>0.05); in the patients with the disease duration more than 15 years, alkaline phosphatase level was decreased by 27.6% (p<0.01) in sera. at the same time, further disease progress or longer posttraumatic period was accompanied by directly proportional increase of ck serum levels: the values remained higher than the controls with an average of (mean ±sd) 116.8±19.4 u/l (p<0.05) in the general group and reached statistically significant values in the patients with the disease duration more than 15 years (mean ±sd 144.6±16.3 u/l versus 82.6±8.4 u/l controls; p<0.05). however, long-term consequences of mtbi revealed quite significant increase in ck level by 50.3% compare to the patients of the subgroup with the duration of the posttraumatic period less than 5 years. this correlated positively with a higher frequency of various neurological syndromes (p<0.05; r= +0.63) for long-term consequences of mtbi and might be a biochemical marker of the latter neurological deficit. 488,3 682,4 783,6 381,9 109,5 126,4 144,6 82,6 0 100 200 300 400 500 600 700 800 900 posttraumatic period <5 years posttraumatic period 5-15 years posttraumatic period >15 years control u /l lactate dehydrogenase creatine kinase figure 1. analysis of the dynamics of lactate dehydrogenase and creatine kinase levels in the patients after traumatic brain injury with different duration of posttraumatic period. the same dynamics was evidenced with the respect to ggt serum level but without statistically significant changes: the ggt serum level at stage 1 and stage 2 practically did not differ from the control. in the remaining group of the patients, there was a decrease of ggt serum levels compare to hc (mean ±sd 14.1±4.2 versus 18.2±1.4 u/l) but also without any statistically significant changes (p>0.05). discussion ye. lekomtseva 29 in t e r n a l m e d ic in e issn 2413-6077. ijmmr 2019 vol. 5 issue 2 decrease in the patients, who suffered from severe tbi [2, 19] reflecting, in our opinion, the developing deficiency of oxidative reactions on amino acid metabolism in these patient groups and/or associated with long treatment with different painkillers and/or antiepileptic drugs. at present, there is still no effective pharmacological treatment that stops the progression of secondary brain injury; the pathogenetic mechanisms of secondary traumatic brain injuries development are still unclear and urgent. thereby, the further research on any other related markers and membrane-associated enzymes involved in internal redox reactions in the patients with long-term consequences of traumatic brain injury is topical. conclusion thus, in the patients with long-term consequences of mtbi, dysregulation of enzymes activity was detected that might be a marker of nervous system energy processes impairment and membranes destruction. the severity of the changes in the serum levels of membrane-associated enzymes markers correlated with the development of oxidative stress and metabolic processes dysfunction. the revealed dysregulation of enzymes activity might be caused by dystrophic cell changes, their disfunction and metabolic changes reflecting the severity of neurological symptoms. acknowledgement author would like to thank enormously gorbach t.v., ph.d. (kharkiv national medical university, clinical biochemistry department) for support in this research. funding this research received no external funding. conflict of interests the author declares no conflict of interest. стан метаболічних процесів у хворих з віддаленими наслідками легкої закритої черепно-мозкової травми є.в. лекомцева ду "інститут неврології, психіатрії та наркології національної академії медичних наук україни", харків, україна вступ. легка закрита черепно-мозкова травма (зчмт) призводить до розвитку різноманітних порушень метаболічних процесів, що відіграють певну роль у розвитку її віддалених наслідків. метою роботи було вивчення аналізу активності деяких маркерних мембрано-зв’язаних ферментів, що приймають участь у різних окислювально-відновлювальних реакціях, метаболічних та енергетичних процесах. методи. у сорока семи хворих із віддаленими наслідками легкої зчмт та тридцяти здорових осіб були проведені обстеження та визначення активності аланінамінотрансферази, аспартатамінотрансферази, лужної фосфатази та лактатдегідрогенази (лд) у сироватці крові хроматографічним та колорометричним методами. результати. дане дослідження виявило значущі зміни вмісту лужної фосфатази та лд, які є маркерами ушкодження клітинних мембран, важкості ацидозу, роз’єднання окислювальновідновлювальних процесів та порушення енергетичного обміну у клітинах. середній рівень лд був 662,7 мо/л vs 381,9 мо/л у контролі. виявлене збільшення рівню лд прямо пропорційно корелювало з прогресуванням захворювання, досягаючи найбільш значущих змін у групі хворих з тривалістю захворювання більш 15 років (mean±sd 144,6±16,3 мо/л vs 82,6±8,4 мо/л; p<0,05). віддалені наслідки після перенесеної легкої зчмт також характеризувалися статистично значущим зниженням активності лужної фосфатази (p<0.05) та позитивною кореляційною залежністю (r=+0.48) від тривалості посттравматичного періоду, при цьому загальний рівень лужної фосфатази був 152,5±11,21 мо/л vs 212,6±9,63 мо/л у здорових (p<0,01). ступінь вираженості змін активності мембрано-зв’язаних ферментів був пов’язаний з розвитком оксидативного стресу та дисметаболічних процесів. висновки. у хворих з віддаленими наслідками легкої зчмт було виявлено дисрегуляцію ферментативних процесів, що є маркером порушення різних видів енергетичного обміну у нервової системі та деструкції клітинних мембран. ключові слова: віддалені наслідки; легка закрита черепно-мозкова травма; мембранозв’язані ферменти; метаболічні процеси. ye. lekomtseva 30 in t e r n a l m e d ic in e issn 2413-6077. ijmmr 2019 vol. 5 issue 2 інформація про автора євгенія лєкомцева – канд. мед. наук, провідний науковий співробітник відділення функціональної нейрохірургії з групою патоморфології, лікар­невропатолог, лікар­психіатр, державна установа “інститут неврології, психіатрії та наркології національної академії медичних наук україни”, вул. академіка павлова, 46, 61068, м. харків information about the author yevgeniya lekomtseva, md, ph.d., state institute of neurology, psychiatry and narcology of the national academy of medical sciences of ukraine, department of neurology, department of functional neurosurgery and paroxysmal states, 46 academic pavlov str, kharkiv, 61068, ukraine. orcid 0000­0003­0961­1636, e­mail: yevgeniyalekomtseva@gmail.com references 1. bazan ng. second messengers derived from excitable membranes are involved in ischemic and seizure-related brain damage. patologicheskaia fiziologiia i eksperimental'naia terapiia. 1992(4):11­6. 2. brigode w, cohan c, beattie g, victorino g. alcohol in traumatic brain injury: toxic or therapeutic?. journal of surgical research. 2019 dec 1;244:196­204. doi.org/10.1016/j.jss.2019.06.043 3. brownlee m. biochemistry and molecular cell biology of diabetic complications. nature. 2001 dec;414(6865):813­20. doi: 10.1038/414813a 4. halliwell b, gutteridge jc. lipid peroxidation, oxygen radicals, cell damage, and antioxidant therapy. the lancet. 1984 jun 23;323(8391):1396­7. 5. huang 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10.1016/j.brainresbull.2013.10.013 received 03 october 2019; revised 1 november 2019; accepted 13 november 2019. this is open access article distributed under the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. ye. lekomtseva issn 2413-6077. ijmmr 2021 vol. 7 issue 1 5 p u b l ic h e a lt h a n d e p id e m io l o g y *corresponding author: оlena lanovenko – assistant professor, kherson state university, 73 000, ukraine. e-mail: lanovenko2708@gmail.com international journal of medicine and medical research 2021, volume 7, issue 1, p. 5-11 copyright © 2021, tnmu, all rights reserved o. lanovenko doi 10.11603/ijmmr.2413-6077.2021.1.12465 dynamics of frequency and peculiarities of the structure of congenital malformations in south ukraine (monitoring study) o. lanovenko kherson state university, kherson, ukraine background. in ukraine, the unfavorable demographic situation makes monitoring of the birth rate of children with congenital malformations urgent issue to identify regional features of epidemiology and develop methods for prenatal diagnosis and prognosis. objective. objective of this study is to characterize the frequency dynamics, to identify structural features of congenital malformations of newborns in kherson region over a 20-year period (2000-2019) and to compare the prevalence of various nosological forms of malformations in the region, in ukraine and in european countries. methods. research methods: epidemiological, medical-statistical. results. in kherson region, the average frequency of congenital malformations over the past 20 years is: for newborns – 31.57±1.25‰; for live births – 31.38±1.11‰; for stillborns – 197.7±0.65 per 10,000. in the structure of defects, cardiovascular malformations are leading (31.77%), musculoskeletal malformations (25.14%), genital malformations (17.5%). increased prevalence of developmental anomalies in the region is mainly associated with an increase in the frequency of model malformations recorded by eurocat (r=0.69, p<0.05). the increase in the total frequency of congenital malformations is caused by increased number of births of children with cardiovascular defects (by 4.67‰), genital defects (by 1.21‰), other congenital malformations (by 1.55‰), multiple malformations (by 0.37‰). conclusion. monitoring results showed an increase in congenital malformations incidence in kherson region over a 20-year period by 7.94‰ possibly caused by population decline due to negative natural and mechanical growth. the prevalence of hereditary defects is at the same level. the frequency of some nosological forms significantly exceeds in the region compare to that in ukraine and europe: cardiovascular defects – in 1.5 times, genital malformations – in nearly 3 times, musculoskeletal defects – almost twice. keywords: congenital malformations; chromosomal pathology; population; newborns. introduction an unfavorable demographic situation in ukraine (decrease in birth rate, high mortality and disability, negative natural population growth) necessitates monitoring of the genetic load of human populations, which averages 50-70 per 1000 newborns (5-7%): congenital malformations account for 2-5%, hereditary diseases – 1.5% (chromosomal – 0.5%, genetic – 1%), diseases with a significant hereditary predisposition – 3-3.5% [1]. monitoring the birth rate of children with congenital malformations is necessary for identifying the epidemiology of its various nosological forms and for development of methods for prenatal diagnosis and prognosis [2]. according to current concepts, congenital malformations are defects in morphogenesis in the early period of fetal life of genetic and/or epigenetic etiology [3]. the spectrum of mutations in genes that control the form-developing processes in embryogenesis may be ethno specific. therefore, during monitoring of the prevalence and structure of malformations, the influence of ethnic factors should be taken into account, as well as not only epidemiological, but also genetic and demographic aspects [4,5]. thus, the aim of the study was to define the dynamics of frequency, to identify the structural features of congenital malformations of newborns in kherson region over a 20-year period (2000-2019) and to compare the prevalence of various nosological forms of congenital malformations in the region, ukraine and european countries. methods the study was conducted in kherson region, covering all 18 territorial-administrative regions. for the analysis, the 20-year observation period (2000-2019) was divided into five-year issn 2413-6077. ijmmr 2021 vol. 7 issue 16 p u b l ic h e a lt h a n d e p id e m io l o g y periods (2000-2004, 2005-2009, 2010-2014, 2015-2019). the objects of the study were newborns (live and stillborn) with isolated and multiple congenital malformations, which were born to residents of kherson region during the study period. the obtained material was analyzed using the registration of all forms of malformations included in the list of icd-10 of section q in order to assess their frequency and structure. when conducting a comparative analysis of the incidence of malformations in populations, it is necessary to take into account standard markers – those forms of them that are often encountered and are unambiguously diagnosed by doctors of all specialties in one age cohort. such forms have a well-defined phenotype and are well diagnosed at birth. these are: multiple congenital malformations and compulsory model defects (q00-q99). long-term monitoring of populations for such markers makes it possible to assess the prevalence and dynamics of malformations and conduct their comparative analysis in populations, which is one of the tasks of the european international register eurocat (european surveillance of congenital anomalies). since the number of abortions is not large enough to have a significant impact on the prevalence of defects, calculations for this indicator were carried out in a cohort of newborn children. most cases of malformations among all pregnancies occur in children born alive; therefore, it is the assessment of the dynamics of their prevalence in this cohort, and not all births, that is the most acceptable form of analysis of epidemiological data [6]. the prevalence of malformations was calculated as the ratio of the number of newborns with congenital malformations to the total number of newborns multiplied by 1000 (‰). similar calculations were carried out in cohorts of live and stillborn children. the data for a comparative analysis of the frequencies of congenital malformations were taken from the open official website of the european register of the international organization eurocat [http://www.eurocat-network.eu]. the theoretical indicator of the number of hereditary congenital malformations was calculated as the sum of cases of chromosomal pathology of living children and half of the remaining part of congenital malformations (without chromosomal diseases), since it is established that about half of cases of congenital malformations (regardless of clinical manifestation) are caused by genetic factors [7]. statistical processing of the research results was carried out using ms excel: determining of the average value, error of the average value, growth rate. using the statistica 6.0 software package, the statistical connection of features was determined by the spearman rank cor relation method. results in total, in 2000-2019, 222,191 newborn babies were born in kherson region; 220,067 of them were live-born and 2,124 still-born. congenital malformations were found in 7015 newborns, including 6973 live births and 42 stillborns. the obtained data allowed estimating the population frequency of congenital malformations in children of kherson region, which is: for newborns – 31.57±1.25‰; for live births – 31.38±1.11‰; for stillbirths – 197.7±0.65 per 10,000. based on the data of the regional department of statistics in kherson region, the average annual number of live births in the region for the last 20 years has been calculated, which is 11,003 children (with an average statistical deviation of up to 5%). theoretical calculations of the prevalence of congenital malformations in the region show that approximately 556 newborns (5%) may be born with signs of congenital pathology. since the contribution of the genetic component to the structure of malformations is on average about 50% (the other half is a consequence of teratogenesis) [8], thus for kherson region the hereditary component of malformations is expected at the level of 278 people per year (2.5% of the number of newborns). actual calculations of the prevalence of congenital malformations are presented in table 1. according to table 1, with the decrease in the birth rate in the region, the prevalence of congenital malformations is rising (from 20.6‰ in 2004 to 37.6‰ in 2017) and on average over a 20-year period was 31.4‰. among all congenital malformations, the actual number of cases of hereditary forms (181 people per year) turned out to be lower than the theoretically calculated indicator (278 people per year); it did not undergo significant changes in recent years and averaged within 52% of all anomalies; this testifies the stable level of mutation process in the region. but in a long-time tendency, the share of hereditary forms is gradually increasing: in 2000 it was 1.29%, in 2019 it was 1.62%. there is a gradual increase in the o. lanovenko issn 2413-6077. ijmmr 2021 vol. 7 issue 1 7 p u b l ic h e a lt h a n d e p id e m io l o g y frequency of multiple malformations: from 0.29 to 0.66‰. since the incidence of neonatal malformations and spontaneous miscarriages correlates with their prevalence among live births, we analyzed the dynamics of nosological forms of congenital anomalies in the last cohort (table 2). according to table 2 in the structure of congenital malformations malformations of the cardiovascular system are leading (31.77% of all cases, an average annual increase of 9.1%), the frequency of which is constantly increasing from 5.1 to 9.93‰ on average over the past 20 years. congenital malformations of the musculoskeletal system (25.14% of all cases, the prevalence slightly decreases from 7.93 to 6.13 and averages 7.35) are the second. stably high frequency of malformations of the genital organs (on average 5.23‰; in the structure – 17.5% of the total number of cases). there is a gradual increase in the frequency of multiple malformations – from 0.29 to 0.66‰, and a slight increase in their share in the overall structure of anomalies (by 0.8%). the prevalence of chromosomal abnormalities (1.0-1.17‰) is characterized by relative stability, which confirms the previous assumption about the constancy of the level of the mutation process in the region. the greatest lethality is caused by defects of the central nervous system (q00-q07), in particular anencephaly, multiple congenital mal formations (q89.7) and chromosomal abnor malities (q90-q99). then a comparative analysis of the prevalence of congenital malformations in kherson region was carried out regarding ukraine and european countries (according to the international register eurocat) (table 3). it has been noted that there is a significant increase in the frequency of malformations of the cartable 1. dynamics of congenital malformations prevalence (kherson region, 2000-2019) years number of live births congenital malformations total frequen-cy, ‰ genetically determined congenital malformations: cases of chromosomal pathology total genetically determined part, % q90q99 of them: from congenital malformations from live birthsds ps еs тs 2000 10 184 255 25.0 8 8 131.5 51.6 1.29 2001 9 757 243 24.9 9 9 126.0 51.9 1.29 2002 9 972 280 28.1 11 8 1 145.5 52.0 1.46 2003 10 502 216 20.9 15 14 115.5 53.5 1.10 2004 10 363 201 20.6 8 5 104.5 52.0 1.00 2005 10 150 307 30.2 11 7 1 1 159.0 51.8 1.57 2006 11 475 397 34.6 13 13 205.0 51.6 1.83 2007 11 570 444 38.4 21 18 3 232.5 52.4 1.79 2008 12 473 556 44.6 18 16 1 1 287.0 51.6 2.30 2009 12 323 384 31.2 21 18 1 1 202.5 52.7 1.64 2010 12 388 391 31.6 18 13 1 204.5 52.3 1.65 2011 12 085 418 34.6 15 9 1 216.5 51.8 1.79 2012 12 643 428 33.8 16 14 222.0 51.9 1.76 2013 12 300 388 31.5 15 15 201.5 51.9 1.64 2014 12 308 433 35.2 11 10 222.0 51.3 1.80 2015 11 372 352 31.0 17 13 2 184.5 52.4 1.62 2016 10 769 350 32.5 11 9 2 180.5 51.6 1.68 2017 9 964 375 37.6 4 3 189.5 50.5 1.90 2018 9 095 293 32.2 13 12 1 153.0 52.2 1.68 2019 8 374 262 31.3 9 9 135.5 51.7 1.62 м 11003.4 348.65 31.4 13.2 11.2 0.4 0.4 0.1 180.9 51.9 1.62 m 281.48 20.15 1.28 1.02 0.9 10.36 0.13 0.065 note: ds – down syndrome; ps – patau syndrome; еs – edwards syndrome; ts turner syndrome. o. lanovenko issn 2413-6077. ijmmr 2021 vol. 7 issue 18 p u b l ic h e a lt h a n d e p id e m io l o g y table 2. dynamics of the frequency and structure of congenital malformations of compulsory registration (q00-q99) among live births in kherson region form cm (patient’s medical record-10) units monitoring periods (years) on average over 20 years rank average increase rate, % 20002004 20052009 20102014 20152019 congenital malformations: nervous system (q00-q07) abs. ‰ % 7.8 0.77 3.40 10,6 0,93 2,62 7,8 0,64 2.01 7.2 0.73 2.34 8.35 0.76 2.59 8 +0.55 + 0.05 0.81 ear. face and neck (q10-q18) abs. ‰ % 1.8 0.18 0.78 5.0 0.44 1.24 5.6 0.46 1.44 4.4 0.44 1.43 4.2 0.38 1.22 11 + 2.4 + 0.2 + 0.44 congenital heart defects (q20-q28) abs. ‰ % 52.0 5.10 22.67 130.0 11.35 32.13 150.6 12.27 38.83 102.8 10.37 33.46 108.9 9.93 31.77 1 + 56.9 + 4.67 + 9.1 respiratory (q30q34) abs. ‰ % 4.0 0.39 1.74 5.2 0.45 1.29 5.4 0.44 1.39 3.0 0.30 0.98 4.4 0.40 1.35 10 + 0.4 + 0.01 0.39 cleft lip with or without palate (q35-q37) abs. ‰ % 10.0 0.98 4.36 11.8 1.03 2.92 10.2 0.83 2.63 9.2 0.93 3.0 10.3 0.94 3.23 7 + 0.3 0.04 1.13 other congenital malformations of the digestive system (q38-q45) abs. ‰ % 10.0 0.98 4.36 19.0 1.66 4.70 12.4 1.01 3.20 10.0 1.0 3.26 12.85 1.17 3.88 6 + 2.85 + 0.18 0.48 genital (q50-q56) abs. ‰ % 41.0 4.02 17.87 61.4 5.36 15.18 64.6 5.26 16.66 62.2 6.27 20.25 57.3 5.23 17.50 3 + 16.3 + 1.21 0.37 limb (q65-q79) abs. ‰ % 80.8 7.93 35.22 99.6 8.70 24.62 81.2 6.62 20.94 60.8 6.13 19.79 80.6 7.35 25.14 2 0.2 0.58 10.08 other congenital malformations (q80-q89) abs. ‰ % 8.8 0.86 3.84 34.8 3.04 8.60 33.8 2.75 8.72 29.4 2.97 9.57 26.7 2.41 7.68 4 + 17.9 + 1.55 + 3.84 multiple malformations (q89.7) abs. ‰ % 3.0 0.29 1.31 10.6 0.93 2.62 8.2 0.67 2.11 7.4 0.75 2.41 7.3 0.66 2.11 9 + 4.3 + 0.37 + 0.80 chromosomal abnormalities (q90-q99) abs. ‰ % 10.2 1.0 4.45 16.6 1.43 4.10 14.0 1.18 3.61 10.8 1.08 3.52 12.9 1.17 3.92 5 + 2.7 + 0.17 0.53 of which down syndrome (q90) abs. ‰ % from chrom. 8.8 0.90 90.2 14.4 1.26 86.7 12.2 0.99 72.7 9.2 0.93 85.2 11.15 1.02 83.7 + 2.35 + 0.12 6.5 on average per year (q00-q99) abs. ‰ % 229.4 22.51 100.0 404.6 35.33 100.0 393.8 31.60 100.0 307.2 31.0 100.0 333.8 30.11 100.0 + 104.4 + 7.6 other congenital malformations abs. ‰ % 9.0 0.88 3.67 17.2 1.50 4.08 25.8 2.10 6.24 23.6 2.38 7.13 18.9 1.72 5.28 4 + 9.9 + 0.84 + 1.61 in total all congenital malformations abs. ‰ 238.4 24.06 421.8 36.83 419.6 33.70 330.8 33.36 352.7 32.0 + 114.3 + 7.94 note: аbs. – the number of cases of birth of a child with congenital malformation of this nosology over 5 years (monitoring period) on average o. lanovenko issn 2413-6077. ijmmr 2021 vol. 7 issue 1 9 p u b l ic h e a lt h a n d e p id e m io l o g y diovascular system of children born alive (from 5.86‰ in 2001-2005 to 10.48‰ in 2013-2017) among the populations of kherson region. moreover, these indicators are significantly higher than the prevalence of this nosological form in ukraine (7.2 and 7.88 respectively) and in european countries (7.47 and 6.96‰ respectively). a similar trend is observed in the dy na mics of the frequency of congenital malformations of the genital organs: an increase in the average annual indicator in kherson region from 3.81 to 6.12‰ (in ukraine – from 0.74 to 2.37‰), although in european countries this indicator has stabilized at the level of 2.092.11‰. discussion among all developmental anomalies the frequency of model forms and multiple defects is a sign that largely reflects the intensity of the mutation process in populations. genetic factors and the mutational component (of at least 40%) largely contributes to the etiology of multiple congenital malformations [9]. the prevalence of malformations in the populations of kherson region is inversely correlated with the prevalence of spontaneous abortions (r = 0.52 ± 0.12; tr = 2.4 > t05 = 2.12), which indicates the “sifting” effect of natural selection that eliminates nonviable genotypes in the embryonic period of ontogenesis [10]. the increase in the prevalence of developmental anomalies in the region is mainly due to the increase in the frequency of model malformations recorded by the eurocat (r=0.69, p<0.05). fetal malformations that can affect the development of intrauterine and infant mortality and lead to human disability are called model congenital malformations or strict accounting malformations. they are highlighted by the eurocat for greater objectivity of research. the prevalence of chromosomal abnormalities (1.0-1.17‰) is characterized by relative stability, which confirms the previously stated assumption about the constancy of the level of the mutation process in the region. among the nosological forms of chromosomal defects the table 3. dynamics of the frequency of various nosological forms of congenital malformations among live births [https://eu-rd-platform.jrc.ec.europa.eu/eurocat/eurocat-data/prevalence_en] nosological form of congenital malformations kherson region. ‰ ukraine. ‰ eurocat. ‰ years: 2001-2005 2013-2017 2001-2005 2013-2017 2001-2005 2013-2017 nervous system 0.87 0.93 1.006 (0.657-1.475) 1.85 (1.615-2.111) 1.274 (1.232-1.317) 1.162 (1.126-1.199) ear, face and neck 0.24 0.41 1.277 (0.879-1.794) 0.126 (0.07-0.207) 0.458 (0.433-0.484) 0. 157 (0.144-0.171) congenital heart defects 5.86 10.48 7.20 (6.203-8.311) 7.878 (7.383-8.398) 7.473 (7.371-7.576) 6.96 (6.872-7.050) respiratory 0.38 0.35 0.039 (0.00-0.219) 0.117 (0.064-0.197) 0.261 (0.2430.281) 0.334 (0.315-0.354) cleft lip with or without palate 1.00 0.97 1.161 (0.783-1.658) 0.804 (0.651-0.981) 0.854 (0.820-0.889) 0.726 (0.697-0.755) digestive system 1.00 0.85 1.084 (0.720-1.566) 1.348 (1.148-1.573) 1.627 (1.579-1.675) 1.558 (1.516-1.601) genital 3.81 6.12 0.735 (0.443-1.149) 2.369 (2.102-2.662) 2.090 (2.036-2.145) 2.108 (2.059-2.157) limb 7.74 6.51 4.103 (3.360-4.962) 4.638 (4.260-5.041) 4.781 (4.699-4.863) 3.691 (3.627-3.757) including polydactyly 1.09 1.15 1.123 (0.752-1.612) 1.407 (1.202-1.636) 0.859 (0.825-0.894) 0.915 (0.883-0.948) chromosomal abnormalities 1.05 0.97 1.123 (0.752-1.612) 1.591 (1.373-1.834) 1.767 (1.717-1.817) 1.689 (1.646-1.733) teratogenic syndromes with malformations 0.50 0.85 0.426 (0.212-0.762) 0.553 (0.427-0.703) 0.105 (0.093-0.118) 0.109 (0.098-0.12) total 24.75 31.4 17.109 (15.55-18.27) 22.061 (21.22722.920) 22.740 (22.562– 22.920) 20.520 (20.36820.674) o. lanovenko issn 2413-6077. ijmmr 2021 vol. 7 issue 110 p u b l ic h e a lt h a n d e p id e m io l o g y largest specific weight is of down syndrome (83.7%) and a frequency of 0.9-1.02‰. the frequency of malformations of the musculoskeletal system in kherson region is 6.51-7.74‰ (in ukraine and europe 4.1-4.64‰ and 4.1-3.37‰, respectively). in addition, an alarming trend was revealed of increased prevalence of multiple congenital malformations (from 0.5‰ to 0.85‰); their frequency significantly exceeds these indicators not only in ukraine (0.43-0.55 ‰) but also in european countries (0.069-0.109 ‰). the cluster of nosological forms of defects with a stable prevalence in kherson region, which does not exceed the corresponding figure in ukraine and european countries, should include malformations of the nervous system, congenital malformations of the face, neck, ears, cleft, lip and palate, other malformations of the digestive system, polydactyly (1.09‰ in 2001-2005 and 1.15‰ in 2013-2017; in ukraine – 1.12 and 1.41‰; in european countries – 0.86 and 0.92‰, respectively), chromosomal abnormalities (in kherson region – 1.05‰ and 0.97‰; in ukraine – 1.12 and 1.59‰; in european countries – 1.77 and 1.69‰ respectively). the prevalence of malformations of the respiratory system in kherson region (0.38‰ in 2001-2005 and 0.35 ‰ in 2013-2017, respectively) significantly exceeds this level in ukraine for the same period (0.04‰ and 0.12‰, respectively) and is at the level of prevalence in european countries (0.26‰ and 0.33‰, respectively). conclusion the frequency of congenital malformations in kherson region over the past 20 years is: for newborns – 31.57±1.25‰; for live births – 31.38±1.11‰; for stillbirths – 197.7±0.65 per 10.000. the increase in the prevalence of congenital malformations in the region is mainly due to an increase in the frequency of model malformationsб which are subject to mandatory registration by the eurocat system (r=0.69, p<0.05). in the structure of congenital malformations defects of the cardiovascular system (31.77%), of the musculoskeletal system (25.14%) and of genital organs (17.5%) are leading. the frequency of malformations of the cardiovascular system, genitals and musculoskeletal system, multiple congenital malformations in kherson region significantly exceeds the prevalence of these nosological forms in ukraine and european countries. among hereditarily determined forms the frequency of chromosomal abnormalities (1.01.17‰) is characterized by relative stability, which confirms our hypothesis about the constancy of the level of mutation process in the region. among the nosological forms of chromosomal defects the largest share is for down syndrome (83.7%) with a frequency of 0.9-1.02. reducing the burden of congenital anomalies in the population is possible through integration of research in epidemiology, genetic demography and epigenetics. conflict of interests author declares no conflict of interest regarding this study. acknowledgments the author expresses sincere gratitude to svetlana yevhenievna onishchenko, a geneticist at kherson children’s clinical hospital, for assistance in collecting primary medical and statistical material. динаміка частоти та особливості структури вроджених вад розвитку на півдні україни (моніторингове дослідження) о. лановенко херсонський державний університет. херсон. україна вступ. в україні в умовах несприятливої демографічної ситуації актуальним є моніторинг народжуваності дітей з вродженими вадами розвитку для виявлення регіональних особливостей епідеміології та розробки методів пренатальної діагностики та прогнозування. мета. мета дослідження – охарактеризувати динаміку частоти і виявити особливості структури вроджених вад розвитку новонароджених дітей у херсонській області за 20-річний період (2000-2019 рр.), порівняти поширеність різних нозологічних форм вад розвитку в регіоні, в україні і в країнах європи. o. lanovenko issn 2413-6077. ijmmr 2021 vol. 7 issue 1 11 p u b l ic h e a lt h a n d e p id e m io l o g y методи. методи дослідження: епідеміологічний, медико-статистичний. отримані результати. у херсонській області середня популяційна частота вроджених вад розвитку за останні 20 років становить: для новонароджених – 31,57±1,25‰; для народжених живими – 31,38±1,11‰; для мертвонароджених – 197,7±0,65 на 10 000. у структурі провідне місце займають вади розвитку серцево-судинної системи (31,77%). опорно-рухового апарату (25,14%). статевих органів (17,5%). збільшення поширеності вроджених вад розвитку в регіоні в основному пов'язано зі збільшенням частоти модельних вад, що реєструються eurocat (r=0.69. p<0.05). підвищення загальної частоти вроджених вад відбувається за рахунок збільшення кількості народжених дітей з вадами серцевосудинної системи (на 4,67‰), статевих органів (на 1,21‰), інших вад розвитку (на 1,55‰), множинних вад (на 0,37‰). висновки. результати моніторингу свідчать про зростання поширеності вроджених вад розвитку в херсонській області за 20-річний період на 7,94‰. причиною такого явища може бути скорочення чисельності населення через негативний природний і механічний приріст. поширеність спадково обумовлених вад залишається на постійному рівні. у регіоні частота деяких нозологічних форм вад значно перевищує цей показник в україні і країнах європи: вад серцево-судинної системи – в 1,5 рази, статевих органів – майже в 3 рази, опорно-рухового апарату – майже в 2 рази. ключові слова: вроджені вади розвитку; хромосомна патологія; популяція; новонароджені діти. information about the author оlena lanovenko – assistant professor, kherson state university, ukraine orcid: 0000-0003-1462-0967. e-mail: lanovenko2708@gmail.com references 1. bochkov np. clinical genetics. 2th ed. moskva: geotar-med; 2001. 448 p. [in russian]. 2. zhuchenko la, letunovskaya ab, demikova ns. the frequency and dynamics of congenital malformations in children in the moscow region, according to the register of congenital malformations for the period 2000-2005. rossiyskiy vestnik perinatologii i pediatrii. 2008;53(2):30-8 [in russian]. 3. hobbs ca, chowdhury s, cleves ma. genetic epidemiology and nonsyndromic structural birth defects from candidate genes to epigenetic. jama pediatrics. 2014; 168(4):371-77. doi: 10.1001/jamapediatrics.2013.4858 4. lanovenko og. dynamics of congenital development disorders prevalence in ukrainian regions and their structure in kherson region . 2018; 12(2):45–2. [in ukrainian]. doi: 10.30970/sbi.1202.561 5. egbe а, uppu s, lee s, stroustrup a, ho d, srivastava s. congenital malformations in the newborn population: a population study and analysis of the effect of sex and prematurity. pediat rics&neonatology.2015; 56(1):25–30. 6. mikitenko da. ukraine: regional aspects of the genetic load of congenital and hereditary pathology. sibirskiy meditsinskiy zhurnal. 2013; (3):18-3. [in russian]. 7. community genetic services in europe. report on a survey: who. copenhagen: european services;1991. no 38. 147 p. 8. minkov ip. monitoring of congenital malformations: their prenatal diagnosis, role in pathology in children and ways of prevention. perinatology and pediatrics. 2000; (1): 8-3. [in russian]. 9. lanovenko og. differentiation of local and rural populations of the kherson region for the frequency of growth and development of reproductive costs. visnyk vinnytsʹkoho natsionalʹnoho medychnoho universytetu. 2018; 22(1):10-3. [in ukrainian]. received 31 may 2021; revised 20 jun 2021; accepted 23 jun 2021 this is open access article distributed under the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. o. lanovenko 61 b io m e d ic a l s c ie n c e s issn 2413-6077. ijmmr 2020 vol. 6 issue 1 doi 10.11603/ijmmr.2413-6077.2020.1.10893 staphylococcus warneri clinical isolate susceptibility to antibiotics and its modification by emoxypine *n.a. bobrova, g.a. loban, e.m. vazhnichaya, m.o. faustova, m.m. ananieva ukrainian medical stomatological academy, poltava, ukraine background. s. warneri is a common commensal organism, but it can cause serious infections. one of the ways to increase a susceptibility of this microorganism to antibiotics is their combining with adjuvant remedies. objectives. the aim of the research is to study the susceptibility of s. warneri clinical isolate to antibiotics and its modification by emoxypine 2-ethyl-6-methyl-3-hydroxypyridine hydrochloride. methods. samples of purulent wound exudation were obtained from a patient with infective complications after a bone fracture and osteosynthesis by metal plates. susceptibility of s. warneri clinical isolate to antibiotics and their combinations with emoxypine (1000 μg/disk) was studied by a standard disc diffusion technique. in the case of microbial resistance, the susceptibility of the investigated isolate to such antibiotic was determined by a serial broth dilutions method without or with emoxypine and evaluated according to the minimum inhibitory concentration (mic). results. by the disk diffusion method, s. warneri isolate was susceptible to all tested antibiotics, except chloramphenicol. the growth inhibition zones also were formed around disks containing emoxypine, but the susceptibility of s. warneri to this agent was low. applying of emoxypine on the disks with antibiotics resulted in the increase of s. warneri growth inhibition in all cases, except using the amikacin, gentamicin, and fusidic acid. the most significant changes were evidenced for a composition chloramphenicol / emoxypine. using chloramphenicol alone in the liquid medium, the mic was over a cut-off point. adding of emoxypine (209 μg/ml) decreased the mic of the antibiotic and restored the susceptibility s. warneri to chloramphenicol. conclusions. the susceptibility of s. warneri clinical isolate to antibiotics can be increased by combining with emoxypine, which among other overcomes the resistance of the studied microorganism to chloramphenicol. key words: s. warneri, susceptibility, antibiotics, 2-ethyl-6-methyl-3-hydroxypyridine (emoxypine). international journal of medicine and medical research 2020, volume 6, issue 1, p. 61-67 copyright © 2020, tnmu, all rights reserved *corresponding author: nellya o. bobrova, ukrainian medical stomatological academy, 23 shevchenko street., poltava 36011, ukraine. e-mail: nelbobrova52@gmail.com. n.a. bobrova et al. introduction coagulase-negative staphylococci (cons) were taken as insignificant contaminant earlier, but now they are regarded as a cause of many multidrug resistant infections. the significant change in the patients profile, that is an increased number of premature newborns, elderly patients, chronically ill patients, and immunocompromised patients along with greater use of indwelling or implanted foreign bodies has made cons a predominant nosocomial pathogen [1]. s. warneri is a catalasepositive, oxidase-negative, and cons, and is a common commensal organism found as part of the skin flora in humans and animals [2]. this pathogen can cause abortion in domestic animals and humans [3]. in humans, it causes discitis, urinary tract infections, and meningitis [4, 5, 6]. orthopedic infections due to s. warneri also have been reported [7, 8]. over the last two decades, similarly to other cons species, s. warneri has been reported as a new emerging pathogen, capable of causing serious infections usually in association with the presence of implant materials [9, 10]. very often s. warneri strains isolated from clinical material are resistant to one-two antibiotics or even all tested antimicrobials (to 18% of clinical isolates) [11]. one of the ways to increase a susceptibility of the microorganism to antibiotics is their use in a combination with adjuvant remedies. synthetic antioxidant 2-ethyl-6-methyl-3-hydroxypyridine hydrochloride (emoxypine) is used in clinic, has its own antimicrobial activity and is able to enhance microbial susceptibility to antibiotics in the experiments with etalon strains of microorganisms [12, 13, 14]. for imple mentation of these experimental data into the anti-infective therapy, it is interesting to evaluate the influence of 2-ethyl-6-methyl-3hydroxypyridine hydrochloride on the clinical isolate of s. warneri as a representative of cons. the aim of the research is to study the susceptibility of s. warneri clinical isolate to anti62 b io m e d ic a l s c ie n c e s issn 2413-6077. ijmmr 2020 vol. 6 issue 1 biotics and its modification by 2-ethyl-6-methyl3-hydroxypyridine hydrochloride (emoxypine). methods some samples of purulent wound exudation were obtained from a 57-year old female patient with infective complications developed on the 7th day after the bone fracture and osteosynthesis by metal plates without subsequent antibiotic therapy. it was used for microbiological assessments and diagnosis. the patient has given an informed consent for sampling of wound exudation and the use of these samples for investigation. after collection, the sample was transferred to a microbiological laboratory in less than 1 hour. bacteriological culture was collected in sterile ball vials. gram staining was made on the first day. cultivation was carried out in thioglycolic medium manufactured by the mechnikov ilya institute of vaccines and serums. 1.5% of agar “dyfko”, 5% of blood and 0.5-1% of yeast hydrolysate was added to promote growth of microorganisms, followed by isolation of pure cultures of facultative anaero bic bacteria in presence of 5-10% co2. the medium was observed daily for microbial growth. final identification was performed by the automatic bacteriological analyzer vitek 2 staphylococcus cards (biomérieux®, france). the research was performed in the medical laboratory bright-bio (license ав no. 526132, dated february 4, 2010) and partly in the research laboratory of the department of microbiology, virology and immunology of ukrainian medical stomatologcal academy. the experiments were approved by the commission on bioethics of ukrainian medical stomatologcal academy. susceptibility of the tested isolate to antibiotics was determined using disk diffusion method [15]. the following disks with antibiotics were used: penicillin g (10 μg), oxacillin (30 μg), ampicillin (30 μg), cefoxitin (30 μg), norfloxacin (10 μg) erythromycin (15 μg), tetracycline (30 μg), amikacin (30 μg), gentamicin (10 μg), co-trimoxazole (1.25/23.75 μg), chloramphenicol (30 μg), and fusidic acid (10 μg). all the antibiotic disks were procured by himedia laboratories pvt ltd., india. substance of 2-ethyl-6-methyl3-hydroxypyridine hydrochloride (emoxypine) was produced by the plc lubnyfarm (ukraine). it was dissolved in water for injections and applied on clean sterile paper disks (himedia laboratories pvt ltd., india) or disks with antibiotics to the amount of 1000 μg/disk. these disks were introduced into the disk by diffusion method in the same way as the standard disks with antibiotics. in 18 hours of incubation at 37 oc the petri plates were examined, and the diameters of inhibition zones were assessed. the results were classified as susceptible or resistant according to the approved guidelines [16] in the case of microbial resistance, the susceptibility of the investigated clinical isolate to such antibiotic also was determined in a liquid medium without and with emoxypine as adjuvant. standard macromethod of serial broth dilutions was used [15]. substance of such antibiotic (chloramphenicol) was produced by the biopharma company (ukraine). mother solutions contained 312 μg/ml of the antibiotic (0,031% solution), 10000 μg/ml of emoxypine (1% solution), or 312 μg of the antibiotic and 10000 μg of the potential adjuvant per 1 ml. twofold serial dilutions of the antibiotic, adjuvant and their combination were prepared and dispensed into the laboratory tubes. a growth control tube and a sterility (uninoculated) tube also were prepared. the inoculum was prepared using the direct colony suspension method. an 18-hour old culture of s. warneri was grown on blood agar. for inoculation micro bial suspension equivalent to 1.0 by mcfarland equivalence standards, diluted 1/100 in saline, was used, and then the concentration of microorganisms in this suspension was 3 x 1010 cfu/cm3. laboratory tubes with macrodilutions were incubated for 24 hours at 37 oc. the amount of growth in the tubes containing the antimicrobial agent was compared with the amount of growth in the growth control tubes. the results were registered visually. the susceptibility of the tested microorganism was evaluated according to the minimum inhibitory concentration (mic). every determination was repeated five times and digital data was processed by the computer programs statistica for windows 6.0. the average mean (m) and its standard error (m, se) were calculated. the lines of variants were checked for normal values. since in all the cases there was a normal distribution of data, the difference between the groups was evaluated by the student’s t-test. results gram positive cocci were found in the samples of purulent exudation at the first stage of bacteriological investigation. they were identified as s. warneri by automatic microbiological analysis. n.a. bobrova et al. 63 b io m e d ic a l s c ie n c e s issn 2413-6077. ijmmr 2020 vol. 6 issue 1 routine disk diffusion method has demonstrated that this clinical isolate is susceptible to majority of antibiotics (table 1). zones of growth inhibition around the disks with all antimicrobials, except chloramphenicol, ranged between 24-32 mm that was charac teristic to the susceptible strain of cons [16]. at the same time, a diameter of growth inhi bition zone around a disk with chloramphenicol was less than 18 mm, a cut-off value of susceptibility that testified the resistance to this antibiotic. the growth inhibition zones also were formed around disks containing emo xy pine, but their diameters confirmed low sus ceptibility of s. warneri culture to this synthetic antioxidant. for all antibiotics, except chlora m phenicol, zones of growth inhibition were more than the same for emoxypine with a credibility from t=11.18 p<0.001 to t=6.47 p<0.005. diameter of the test culture growth inhibition around disks with chloramphenicol did not differ from this parameter around disks with emoxypine. applying of emoxypine on the standard disks with antibiotics resulted in the increase of growth inhibition zones of s. warneri test culture as compared to appropriate antibiotic in all cases, except the use of amikacin, gentamicin, and fusidic acid (table 1). diameter of the growth inhibition zone for a combination of emoxypine with benzylpenicillin was greater by 9.2 mm (t=4.03, p<0.02), oxacillin – by 9.6 mm (t=7.0, p<0.005), ampicillin – by 6.8 mm (t=2.92, p<0.05), cefoxitin – by 7.0 mm (t=4.43, p<0.02), norfloxacin – by 5.2 mm (t=2.83, p<0.05), erythromycin – by 4.5 mm (t=2.95, p<0.05), tetracycline – by 6.8 mm (t=3.03, p<0.05), cotrimoxazole – by 4.2 mm (t=3.88, p<0.02), chloramphenicol – by 15.4 mm (t=11.32, p<0.001), rifampicin – by 5.5 mm (t=2.78, p<0.05) compare to antibiotic itself. the most significant changes of growth inhibition were observed for a composition of chloramphenicol/emoxypine. distinguishes between the growth inhibition of the s. warneri isolate caused by a combination of amikacin, gentamicin, and fusidic acid with an adjuvant were not significant as compared to the control administered with the standard antibiotic. at the same time, all zones of growth inhibition by combinations of antibiotics with emoxypine were more than the same of the adjuvant drug alone (a credibility from t=22.03, p<0.001, to t=8.10, p<0.002). chloramphenicol was only one antibiotic from the tested antimicrobials for which s. warneri isolate was resistant in cases of the disk diffusion method and for which effect of emoxypine was the most as compared to other compositions. that is why it also was investigated by a serial broth dilutions method. results of mic determination are represented in fig.1. mic was over the cut-off point for cons susceptibility, when chloramphenicol was used table 1. zones of growth inhibition of s. warneri test culture under the influence of antibiotics and their combinations with emoxypine, m±m (n=5) antimicrobial agent diameter of growth inhibition zone, mm antibiotic or adjuvant (emoxypine) combination of antibiotic and adjuvant (emoxypine) emoxypine (1000 μg) 10.6 ±1.1 – benzylpenicillin (10 μg) 27.4±2.0* 36.6±1.1*, # oxacillin (30 μg) 28.8±1.2* 38.2±0.6*, # ampicillin (30 μg) 26.6±2.0* 33.4±1.2*, # cefoxitine (30 μg) 26.8±1.5* 33.8±0.5*, # norfloxacine (10 μg) 20.8±1.6* 26.0±0.9*, # erythromycin (15 μg) 23.5±1.3* 28.0±0.8*, # tetracycline (30 μg) 23.2±1.2* 30.0±1.9*, # amikacin (30 μg) 25.0±1.8* 28.0±1.6* gentamicin (10 μg) 25.4±1.0* 27.2±1.3* co-trimoxazole (1.25/23.75 μg) 19.8±0.9* 24.0±0.6*, # chloramphenicol (30μg) 7.8±0.8* 23.2±1.1*, # fusidic acid (10 μg) 25.2±1.0* 29.0±1.2* rifampicin (5 μg) 28.0±1.4* 33.4±1.9*, # notes: * – p<0.05 compare to growth inhibition zone for emoxypine; # – p<0.05 compare growth inhibition zone for this antibiotic without emoxypine. n.a. bobrova et al. 64 b io m e d ic a l s c ie n c e s issn 2413-6077. ijmmr 2020 vol. 6 issue 1 alone [16] (fig. 1a). with adding of emoxypine mic of the antibiotic decreased from average 52 μg/ml to 6.5 μg/ml (t=3.47, p<0.05) and the susceptibility of s. warneri clinical strain to chloramphenicol restored. at the same time, mic of emoxypine alone was 416 μg/ml (average); it proved low susceptibility of the studied isolate to this agent (fig. 1b). a concentration of emoxypine complied with mic of a composition chloramphenicol/emoxypine was 209 μg/ml, that was 2 times less as compared to mic of emoxypine (t=3.0, p<0.05). discussion the investigated s. warneri clinical isolate was susceptible to the antibiotics of the penicillins, cephalosporins, fluoroquinolones, aminoglycosides, macrolides, tetracyclines, rifampicin groups and steroids, which are recommended for screening of cons susceptibility to antimicrobial agents [16]. a high susceptibility of this isolate could be explained by the short history of the hospital treatment and the absence of previous antibiotic therapy in a patient. at the same time, the clinical isolate was resistant to chloramphenicol that was evidenced by a small growth inhibition in cases of disk diffusion method and high mic in a serial broth dilutions method as compared to cut-off values susceptibility / resistance for cons [16]. this can be explained by the nature of chloramphenicol. as it is an ancient microbial metabolite, genetic elements conferring resistance against this drug are retained by and are frequently dispersed in microbial communities [17]. response of the tested microorganism to emoxypine as a potential adjuvant of antimicrobials was similar to the results of our previous investigations [14, 18]. it was also consistent with other experimental results concerning etalon strains of gram negative rods [12, 13], as well as the retrospective computer prognosis of the biological activity for ethylmettylhydroxypyridine structure that showed a possible antimicrobial activity [19]. emoxypine’s mic in s. warneri test culture was less than this parameter of other cons (an etalon strain s. epidermidis атсс 14990) [14]. there are no reference for susceptibility or resistance of bacteria to emoxypine, so we cannot conclude is clinical isolate susceptible to this agent or not, but it is possible to think that s. warneri isolates susceptibility to emoxypine is low and the use of this preparation as an adjuvant of antimicrobial therapy will be more perspective. the mechanism of emoxypine antibacterial action is still unclear and the most likely it is due to modification of the bacterial cell membrane typical to antioxidants. other mechanisms, e.g. protein synthesis inhibition, may also play a part in the antimicrobial action of this 3-hydroxypyridne derivative [19]. combining of antibiotics with emoxypine led to an increase of growth inhibition zones of s. warneri test culture that proved enhance of the clinical isolate susceptibility to these agents. the effect observed was similar to the changes of susceptibility of the etalon strains of s. aureus and e. coli under the influence of combinations of antibiotics of different classes and relative fig. 1. minimum inhibitory concentration of chloramphenicol (a) and emoxypine (b) alone and in a combination against s. warneri clinical isolate. note. * – p<0.05 as compared with antimicrobial or adjuvant themselves. 0 10 20 30 40 50 60 m ic , μ g/ m l chloramphenicol chloramphenicol+emoxypine chloramphenicol’s cut-off point 0 50 100 150 200 250 300 350 400 450 m ic , μ g/ m l emoxipine emoxipine with chloramphenicol * * а в n.a. bobrova et al. 65 b io m e d ic a l s c ie n c e s issn 2413-6077. ijmmr 2020 vol. 6 issue 1 preparation: 2-ethyl-6-methyl-3-hydroxipyrydine succinate (mexidol) [14]. in the case of the resistance of s. warneri isolate to chloramphenicol, adding of emoxypine restored the susceptibility of the tested microorganism that was registered both in the disk diffusion and serial broth dilutions methods. it is established that resistance to chloramphenicol is mainly present due to the production of a specific inactivating chloramphenicol acetyltransferase, reduced drug uptake or increased drug efflux [17, 20]. that is why it is reasonably that emoxypine effect is caused by its influence on all or some of the mentioned mechanisms, especially on the membrane permeability and efflux pumps because membrane modification is a fundamental property of 2-ethyl-6-methyl3-hydroxypyridine structure [19]. overcoming resistance of the clinical isolate s.warneri to chloramphenicol occurred with significant mic of emoxypine in the medium, which is much higher than the maximum concent ration of this substance or its salt with succinic acid in the blood plasma after the intravenous administration [21]. therefore, despite the fact that the description of this phenomenon opens up new possibilities in controlling the susceptibility of cons, a matter of the ratio of the concentration of emoxypine in the in vitro experiments with a dose of this potential antimicrobial adjuvant in clinic is still unclear. the prospects of the research pre sented should cover the research of dose/con cen tration range of emoxypine adjuvant ac tivity. conclusions s. warneri isolate, susceptible to penicillin g, oxacillin, ampicillin, cefoxitin, norfloxacin, erythromycin, tetracycline, amikacin, gentamicin, cotrimoxazole, and fusidic acid, but resistant to chloramphenicol, was isolated from the clinical material. the susceptibility of this clinical isolate to all antibiotics listed, except amikacin, gentamicin, and fusidic acid, can be increased significantly when combined with 2-ethyl-6-methyl-3-hydroxypyridine hydrochloride (emoxypine), which, among other things, restores the susceptibility of the studied microorganism to conventional rates at its concentration of 209 μg/ml. funding this research received no external funding. conflict of interests the authors declare no conflict of interest. author contributions nellya o. bobrova – methodology, investigation; galina a. loban – project administration, supervision; elena m. vazhnichaya – conceptualization, writing – review and editing; mariia o. faustova – formal analysis, writing the initial draft; maiia m. ananieva – investigation, visualization. acknowledgments the authors would like to thank the staff of the bright-bio medical laboratory, who were involved in the laboratory studies of the patient’s biomaterial. чутливість клінічного штаму staphylococcus warneri до антибіотиків та її модифікація емоксипіном н. о. боброва, г. а. лобань, о. м. важнича, м. о. фаустова, м.м. ананьєва українська медична стоматологічна академія, полтава, україна вступ. s. warneri звичайний коменсальний організм, але він може спричинити серйозні інфекції. одним із способів підвищення чутливості цього мікроорганізму до антибіотиків є їх поєднання з ад'ювантними засобами. мета. метою роботи є вивчення чутливості клінічного ізоляту s. warneri до антибіотиків та її модифікації етилметилгідроксипіридину гідрохлоридом (емоксипіном). методи. зразки гнійного раневого ексудату були одержані від хворої з з інфекційними ускладненнями після перелому кістки і операції остеосинтезу металевими пластинами. чутливість клінічного ізоляту s. warneri до антибіотиків та їх комбінацій з емоксипіном (1000 мкг/диск) вивчали диск-дифузійним методом. у разі резистентності мікроорганізму чутливість досліджуваного штаму до такого антибіотика визначали методом серійних розведень без або з емоксипіном та оцінювали відповідно до мінімальної пригнічуючої концентрації (mпк). результати. у диск-дифузійному методі ізолят s. warneri був чутливим до всіх випробуваних антибіотиків, крім хлорамфеніколу. зони пригнічення росту також утворювались навколо дисків, що n.a. bobrova et al. 66 b io m e d ic a l s c ie n c e s issn 2413-6077. ijmmr 2020 vol. 6 issue 1 містять емоксипін, але чутливість s. warneri до цього засобу була низькою. нанесення емоксипіну на диски з антибіотиками призвело до посилення пригнічення росту s. warneri у всіх випадках, за винятком застосування амікацину, гентаміцину та фузидової кислоти. найбільш виражені зміни спостерігалися для комбінації хлорамфенікол / емоксипін. при використанні самого хлорамфенікол у рідкому середовищі, mпк була вище точки відсічення. додавання емоксипіну (209 мкг/мл) зменшило mпк антибіотика та відновило чутливість s. warneri до хлорамфеніколу. висновки. чутливість клінічного ізоляту s.warneri до антибіотиків можна збільшити, поєднуючи їх з емоксипіном, який, крім іншого, здатний подолати резистентність досліджуваного мікроорганізму до хлорамфеніколу. ключові слова: s. warneri; чутливість; антибіотики; 2-етил-6-метил-3-гідроксипіридин (емоксипін). відомості про авторів боброва нелля олександрівна – кандидат біологічних наук, викладач кафедри мікробіології, вірусології та імунології української медичної стоматологічної академії, м. полтава. лобань галина андріївна – доктор медичних наук, професор, завідувачка кафедри мікробіології, вірусології та імунології української медичної стоматологічної академії, м. полтава. важнича олена митрофанівна – доктор медичних наук, професор, професор кафедри експериментальної та клінічної фармакології української медичної стоматологічної академії, м. полтава. фаустова марія олексіївна – кандидат медичних наук, доцент кафедри мікробіології, вірусології та імунології української медичної стоматологічної академії, м. полтава. ананьєва майя миколаївна – кандидат медичних наук, доцент кафедри мікробіології, вірусології та імунології української медичної стоматологічної академії, м. полтава. information about the authors bobrova n.o. – ph.d., lecturer of the department of microbiology, virology and immunology, ukrainian medical stomatological academy, poltava, ukraine. orcid https://orcid.org/0000-0002-6449-6231, e-mail: nelbobrova52@gmail.com loban’ g.a. – md, ph.d., dsc, professor, head of the department of microbiology, virology and immunology, ukrainian medical stomatological academy, poltava, ukraine. orcid https://orcid.org/000-0003-0055-7696, e-mail: galina.loban@gmail.com vazhnichaya e.m. – md, ph.d., dsc, professor of the department of experimental and clinical pharmacology, ukrainian medical stomatological academy, poltava, ukraine. orcid https://orcid.org/0000-0002-5022-2352, e-mail: vazhnichaya@ukr.net faustova m.o. – phd, associate professor of the department of microbiology, virology and immunology, ukrainian medical stomatological academy, poltava, ukraine. orcid https://orcid.org/0000-0001-5327-6324, e-mail: mashafaustova@ukr.net ananieva m.m. – md, phd, associate professor of the department of microbiology, virology and immunology, ukrainian medical stomatological academy, poltava, ukraine. orcid https://orcid.org/0000-0001-9435-7622, e-mail: anfila@ukr.net references 1. singh s, dhawan b, kapil a, kabra sk, suri a, sreenivas v, et al. coagulase-negative staphylococci causing blood stream infection at an indian tertiary care hospital: prevalence, antimicrobial resistance and 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(in russian) 20. infectious diseases. 4th ed. cohen j, powderly wg, opal sm, editors. elsevier, 2017. doi: 10.1016/c2013-1-00044-3 21. medicines for the treatment of cardiovascular system diseases produced by rue belmedpreparaty (reference note). trukhacheva tv, editor. minsk, paradox llc, 2013. https://www.bsmu.by/downloads/vrachu/lekarstva/serdce.pdf (in russian) received 25 mar 2020; revised 09 apr 2020; accepted 02 june 2020. this is open access article distributed under the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. n.a. bobrova et al. 22 in t e r n a l m e d ic in e issn 2413-6077. ijmmr 2020 vol. 6 issue 2 doi 10.11603/ijmmr.2413-6077.2020.2.11509 effects of hemotoxic snake bite envenomation on haematological parameters variability in predicting complications m.p. harikrishnan, *c.r. anil kumar, m.k. anand, j. earali jubilee mission medical college and research institute, thrissur, india background. snake bite envenomation is a major public health problem in india with a high mortality rate. the major complications following a hemotoxic snake bite are disseminated intravascular coagulation (dic), shock, acute kidney injury (aki), acute respiratory distress syndrome (ards) and coagulopathy. the study explores a possible correlation of the haematological parameters studied to complications like dic, aki, acute renal failure (arf), ards, shock and gastrointestinal (gi) bleed. objective. the aim of the study was to find out the effect of snakebite envenomation on the major haematological parameters of the body in relation to complications. methods. this cross-sectional study was conducted during a period of 18 months. 150 patients were included in the study after obtaining their informed consents. data collection was done using a proforma. the study also compared clotting time (ct) by capillary tube method and whole blood clotting time at 20 minutes (wbct20). spss software was used for statistical analysis. results. among the people with complications, the majority (52%) of participants had aki, 26% of them requiring dialysis, 16.7% participants had gi bleed, 11.3% participants had shock and 10% participants had dic. conclusions. a prolonged bleeding time was found to be one of the most helpful haematological parameters in predicting shock and aki. clotting time by both capillary tube and wbct20 methods were comparable in predicting complications. keywords: hemotoxic snake bite; acute kidney injury; shock; clotting time; bleeding time. *corresponding author: anil kumar cr, associate professor, jubilee mission medical college and research institute, thrissur, 680005, india. e-mail: anilkumarcr3005@gmail.com international journal of medicine and medical research 2020, volume 6, issue 2, p. 22-29 copyright © 2020, tnmu, all rights reserved m.p. harikrishnan et al. introduction snakebite envenomation is a major public health problem in india with a high mortality rate [1]. the who estimates that there are 81000 snake envenomation in india per annum with a mortality of 11000 (13.5%) [2]. it has been reported that there are 5 million snake bites with 2.5 million envenomation and 125,000 fatalities worldwide annually [3]. india is reported to have the highest number of snake bites (81,000) and deaths (11,000) per year [3]. snakebite is considered as one of the neglected tropical diseases by the who [4]. the mortality due to snakebites in india is due to various socioeconomic, cultural and environmental causes [5]. among the 52 species of venomous snakes in india, 4 species are responsible for greatest number of snake bites which are medically significant. these are referred to as the big four, bungarus caeruleus (common krait), daboia russelii (russel’s viper), echis carinatus (saw scaled viper), naja naja (indian cobra). but recently it has been discovered that another species, the hump-pit viper (hypnale hypnale), is capable of causing lethal envenomation [6]. the venomous snakes found in india can be classified per the type and action of venom as neurotoxic (cobra, krait) and hemotoxic (vipers). then main complaints are pain and swelling at the site of snake bite [7–11]. the major complications following hemotoxic snake bite are disseminated intravascular coagulation (dic), shock, acute kidney injury (aki), acute respiratory distress syndrome (ards) and coagulopathy [12]. aki is one of the serious complications developing after snake bite [13,14]. early identification of hemotoxic envenomation and anti-snake venom (asv) administration may help decrease morbidity and mortality [15]. the study tries to find out the common complications after hemotoxic snake bites and to explore a possible correlation of the haematological parameters to complications. the study also compares clotting time by capillary tube method and wbct20. asv administration is currently based on prolongation of wbct20. capillary tube method for estimating ct could be used in a resource poor setting, if the 23 in t e r n a l m e d ic in e issn 2413-6077. ijmmr 2020 vol. 6 issue 2 predictability of the test is comparable to that of wbct20. in a study by ratnayake et al [16] the diagnostic utility of wbct20 was determined at the time of admission and it was found out that it had got sensitivity of 82% and specificity of 98% in detecting venom induced consumption coagulopathy (vicc). the aim of the study was to assess the value of haematological parameters, i.e. bleeding time (bt), clotting time (ct), prothrombin time (pt), activated partial thromboplastin time (aptt) in predicting the complications. the primary objective was to assess the effect of snake bite on the major haematological parameters of the body in relation to complications. methods this cross-sectional study was conducted over a period of 18 months after obtaining clearance from the institutional ethics committee. a total of 150 patients, who were above 18 years of age, had given written informed consent and had clinical features of hemotoxic envenomation or increase of any of the evaluated haematological parameters as bleeding time (bt), clotting time (ct), prothrombin time (pt), activated partial thromboplastin time (aptt), were included in the study. convenient sampling was done. the patients who had not given written informed consent and the patients with bites other than hemotoxic snakebites were excluded from the study. any patient who is a known case of bleeding disorder, chronic alcoholic, with acute or chronic liver disease, pregnant females and on anticoagulation therapy were excluded from the current study. after admission a complete history was taken and a thorough clinical examination was conducted. as per the protocol followed in our institute, all patients bitten by poisonous snakes received polyvalent anti snake venom (asv), within 15 minutes of reaching the hospital, provided they had clinical features of envenomation. the patients with no initial features of envenomation were administered with asv as soon as they exhibited first signs of envenomation. initial blood samples collected from the patients admitted with hemotoxic envenomation were sent for complete hemogram, random blood sugar (rbs), renal function tests (rft), liver function tests (lft), serum electrolytes and important coagulation parameters including bt, ct (both wbct20 and capillary tube methods), pt with inr, d-dimer and a ptt. all patients with features of envenomation were administered with 10 vials of asv. the response was monitored clinically by doing wbct20 and clotting time (ct) using the capillary tube method, with 2 hour intervals if prolonged initially, otherwise with 6 hour intervals. if the features of envenomation were persistent or if the wbct20 was prolonged after 6 hours of first dose, a repeated dose of 10 vials of asv was given. blood parameters like bt, ct, pt, aptt and d-dimer were investigated as per the protocol. the first sample of blood was taken on admission for evaluating bt, ct, pt with inr, and aptt. bt was subsequently estimated 2 times with a 5 minute interval, then in 2 hours, 4 hours, and 6 hours after admission. then every 6 hours if prolonged. once normal, it was repeated once daily. ct was estimated at time of admission. if in norm, ct was repeated with 6 hour intervals for 1 day to test for requirement of asv. if prolonged, it was tested with 2 hour intervals till the normal rate. ct was estimated by 20-minute whole blood clotting method (wbct20) and capillary tube method for comparison study. aptt was done on the 1st day and 7th day, but pt was done at the time of admission and daily. d-dimer was done when there was prolongation of ct on 3 consecutive occasions. asv was given according to the standard protocol with administration of asv every 6 hours till normal coagulation profile. a bite was considered hemotoxic, if the culprit snake is identified as hemotoxic or by coagulopathy as suggested by haematological parameters. aki was defined as the increase of serum creatinine ≥0.3 mg/dl within 48 hours or increase in serum creatininein ≥1.5 times from baseline. aki staging was performed according to the “kidney disease: improving global outcomes” (kdigo) clinical practice guidelines [17]. shock was defined by a systolic blood pressure of <90 mmhg with evidence of tissue hypoperfusion (<0.5 ml/kg/h decrease in urine output, cold skin, and or requiring inotropic drugs). the data was collected, occurrence of complications and derangement in haematological parameters were studied and analysed. the data collected was recorded in a standard data collection sheet as per study proforma and later transferred to microsoft excel spread sheet for statistical analysis. complications like aki, shock were considered as primary outcome variables. ct by capillary tube and wbct20 at different time periods, bt, pt, aptt etc. were considered as explanatory variables. m.p. harikrishnan et al. 24 in t e r n a l m e d ic in e issn 2413-6077. ijmmr 2020 vol. 6 issue 2 descriptive analysis was carried out by frequency and proportion for categorical variables. categorical outcomes were compared between the study groups using the chi square test / the fisher's exact test (if the overall sample size was <20 or if the expected number in any one of the cells was <5, fisher's exact test was used). p value <0.05 was considered statistically significant. ibm spss version 22 was used for statistical analysis [18]. results in the study it was observed that the snake bites were more common in males. out of 150 subjects in our study, 24 (16.0%) cases, were bites by russel’s viper, 4 (2.7%) cases by krait, 2 (1.3%) cases by hump nosed pit viper and 120 (80.0%) were unidentified. the patients included in the study, who had any comorbidity, accounted for 36.0% of the study population (table 1). among the complications, the majority (52%) of participants had aki, 26% of them required dialysis; 16.7% participants had gi bleed; 11.3% participants had shock and 10% participants had dic (table 2). association between aki and shock with various parameters have been shown in the table below (table 3). at the time of admission, 11 patients (47.8%) presenting with aki reported prolonged ct (capillary tube). however, in 6 hours 22 (100%) participants with prolonged ct had aki. a majority of 36 (60%) participants reported aki with normal wbct20 at the time of admission. the occurrence of aki was evidenced in 30 (81%) participants with prolonged wbct20 in 6 hours. the occurrence of aki was found in 17 (58.6%) participants with pt >16.50 seconds. all participants with aptt >35 reported aki (table 3). wbct20 and ct by ca pilla ry t ube met hod in 6 hours were comparable in predicting aki. table 1. summary of baseline parameters in study population (n= 150) parameter summary (number, %) age 18-30 37 (24.7%) 31-40 30 (20%) 41-50 29 (19.3%) 51-60 27 (18%) 61-65 27 (18%) gender male 98 (65.3%) female 52 (34.7%) place of bite indoor 1 (0.7%) outdoor 149 (99.3%) snake type russel’s viper 24 (16%) krait 4 (2.7%) hump nosed pit viper 2 (1.3%) unidentified 120 (80%) classical bite 43(28.7%) local reaction 65 (43.3%) cellulitis 27 (18%) gangrene 8 (5.3%) ecchymoses/bleeding 14 (9.3%) comorbidities 54 (36%) pain at bite site 128 (83.3%) numbness 125 (83.3%) sweating 98 (65.3%) periorbital oedema 71 (47.3%) vomiting 64 (42.7%) facial puffiness 48 (32%) parameter summary (number, %) abdominal pain 45 (30%) neurological signs 28 (18.7%) respiratory distress 25 (16.7%) low back ache 20 (13.3%) bleeding from any site 15 (10%) respiratory failure 15 (10%) ards 11 (7.3%) hematemesis 4 (2.7%) comorbidities diabetes mellitus 25 (16.7%) hypertension 24 (16%) ischemic heart disease 0 (0%) respiratory failure 0 (0%) bronchial asthma 11 (7.3%) chronic obstructive lung disease 23 (15.3%) ct prolonged at the time of admission 23 (15.3%) 2 hours 36 (24%) 4 hours 29 (19.3%) 6 hours–12 hours 49 (32.03%) day 2 4 (2.7%) wbc t20-prolonged at the time of admission 90 (60%) 2 hours 61 (40.7%) 4 hours 48 (32%) 6 hours–12 hours 86 (57.3%) day 2 15 (10%) m.p. harikrishnan et al. 25 in t e r n a l m e d ic in e issn 2413-6077. ijmmr 2020 vol. 6 issue 2 the occurrence of shock was found in 6 (26.1%) participants with prolonged ct at the time of admission (p value of 0.039). however, 11 (50%) participants had shock with prolonged ct in 6 hours (p value <0.001). the occurrence of shock was found in 11 (12.2%) participants with normal wbct20 at admission. 17 (42%) participants with prolonged wbc t20 in 6 hours reported shock. the occurrence of shock was found to be statistically significant with p value of <0.001 and the proportion of shock was 8 (66.7%) with bt >7 seconds. the occurrence of shock was found to be significant with a p value of <0.001 in 7 (30.4%) participants with >35 aptt (table 4). table 2. summary of the complications observed (n=150) complications summary (number, %) aki 78 (52%) acute renal failure (aki requiring dialysis) 39 (26%) dialysis done 39 (26%) gi bleed 25 (16.7%) shock 17 (11.3%) dic 15 (10%) ards 11 (7.3%) reaction to asv 6 (4%) mortality 2 (1.3%) intra cerebral bleed 0 (0%) hypopituitarism 0 (0%) guillain barre syndrome 0 (0%) peripheral neuropathy 0 (0%) table 3. association of different parameters with aki parameter aki p value present absent ct capillary tube at the time of admission prolonged (n=23) 11(47.8%) 12 (52.2%) 0.663 normal (n=127) 67 (52.8%) 60 (47.2%) ct capillary tube in 6 hours prolonged (n=22) 22 (100%) 0 (0%) * normal (n=120) 48 (40%) 72 (60%) wbct20 at admission prolonged (n=90) 42 (46.7%) 48 (53.3%) 0.109 normal (n=60) 36 (60%) 24 (40%) wbct20 at 6 hours prolonged (n=37) 30 (81%) 7 (19%) <0.001 normal (n=105) 40 (38%) 65 (62%) bt >7 (n=12) 12 (100%) 0 (0%) * ≤7 (n=138) 66 (47.8%) 72 (52.2%) pt >16.50 (n=29) 17 (58.6%) 12 (41.4%) 0.319 ≤16.50 (n=116) 56 (48.3%) 60 (51.7%) aptt >35 (n=23) 23 (100%) 0 (0%) * ≤35 (n=123) 51 (41.5%) 72 (58.5%) *no statistical tests were applied due to 0 values in one of the cells. m.p. harikrishnan et al. 26 in t e r n a l m e d ic in e issn 2413-6077. ijmmr 2020 vol. 6 issue 2 discussion in this study, shock was seen in 11.3% of the total cases, aki in 52 %, arf (aki requiring dialysis) in 26% and gastro intestinal bleed in 16.7%. a comparatively higher incidence of shock was reported by harshavardhan et al. [19] in their study on hemotoxic snake bite cases (36%). but a comparable value (10.38%) to our study was reported by kumar et al. [20]. acute kidney injury (aki) was established in 52% of the cases in the present study. a somewhat similar value was observed (30.71%) in a previous study [21]. bhalla et al studied the clinical profile of snake bite in a tertiary centre and observed aki in 47.6% of the cases [22]. similar study on outcomes of snakebite poisoning in a tertiary centre by meenakshi b et al. reported aki in 44% of cases. proteins are an important constituent of snake venoms and they are encoded by polyadenylated mrna in the venomous glands (12s and 20s) [21]. besides proteins, the other components present in snake venom include lipids, polysaccharides, nucleotides, nucleosides, free amino acids, riboflavin, serotonin and histamine. pharmacologically active substances of the venom are enzymes and low molecular weight peptides. the main sites of action of these enzymes are cell membranes, the vascular wall and the blood coagulation cascade [20, 22]. snake venom, mainly those of viperidae, contain also molecules that act on the four interconnected blood systems (i) the coagulation system, (ii) fibrinolytic system, (iii) complement and (iv) the kinin system [22]. shock is consistently the single most important factor in mortality due to indian viper bite poisoning. the pattern of shock observed shows early transient shock, early sustained shock and late onset shock. shock is caused by reduction in the circulating blood volume due to a venom-mediated generalized increase in endothelial permeability. apart from this, massive limb oedema can lead to considerable compartmentalization of blood and plasma. pulmonary intravascular coagulation, pulmonary oedema caused by increased pulmonary vascular permeability, and cardiotoxic effects of the venom may also be contributory. the kidney is a highly vascularized organ with excretory function, so it is prone to venom toxicity. aki, the most significant of all the renal table 4. association of different parameters with shock parameter shock p value present absent ct capillary tube at the time of admission prolonged (n=23) 6 (26.1%) 17 (73.9%) 0.039 normal (n=127) 11 (8.7%) 116 (91.3%) ct capillary tube at 6 hours prolonged (n=22) 11 (50%) 11 (50%) <0.001 normal (n=120) 6 (5%) 114 (95%) wbct20 at admission prolonged (n=90) 11 (12.2%) 79 (87.8%) 0.875 normal (n=60) 6 (10%) 54 (90%) wbct20 at 6 hours prolonged (n=37) 17 (46%) 20 (54%) * normal (n=105) 0 (0%) 105 (100%) bt >7 (n=12) 8 (66.7%) 4 (33.3%) <0.001 ≤7 (n=138) 9 (6.5%) 129 (93.5%) pt >16.50 (n=29) 12 (41.4%) 17 (58.6%) * ≤16.50 (n=116) 0 (0%) 116 (100%) aptt >35 (n=23) 7 (30.4%) 16 (69.6%) <0.001 ≤35 (n=123) 6 (4.9%) 117 (95.1%) *no statistical tests were applied due to 0 values in one of the cells. m.p. harikrishnan et al. 27 in t e r n a l m e d ic in e issn 2413-6077. ijmmr 2020 vol. 6 issue 2 manifestations, has been reported to be of varying frequency in different studies [23]. many studies have appeared in literature on snake bite-induced aki over a decade. phospholipase a2 is the most widespread and extensively studied of all venom enzymes. it damages mitochondria, red blood cells, leucocytes, platelets, skeletal muscle, vascular endothelium, and other membranes and leads to auto pharmacological release of histamine and anti-coagulants. viper bites cause various systemic symptoms such as coagulopathy, haemolysis, aki, a generalized increase in capillary permeability and rhabdomyolysis [23]. haemoglobinuria caused by intra-vascular haemolysis and myoglobinuria resulting from rhabdomyolysis contributes to development of aki. haemorrhage, hypotension, disseminated intravascular coagulation, intravascular haemolysis, and rhabdomyolysis enhance renal ischemia leading to renal failure. enzymatic activities of snake venoms account for direct nephrotoxicity and immunologic mechanism is insignificant [24]. a variety of factors contribute to shock like fright, hypovolemia (due to extravasation of fluids and blood loss), myocardial depression, haemorrhage into the adrenals and pituitary. conclusions a prolonged clotting time as obtained by capillary tube method in 6 hours is helpful in predicting development of shock. however, since aki occurred even in those patients having normal clotting time and wbct20, these parameters may not be useful as predictors of aki. prolonged ct by capillary tube method and wbct20 in 6 hours were comparable in predicting aki. prolongation of more than 16.5 seconds in prothrombin time was helpful in predicting the development of shock. however prolonged prothrombin time (>16.50) was not useful in predicting aki. a prolonged aptt more than 35 seconds was helpful in predicting the onset of shock and aki. a prolonged bleeding time was found to be one of the most helpful haematological parameters in predicting shock and aki. limitations of the study a comparatively low number of 150 cases may have strained the generalizations derived by the study. parameters like ct by capillary tube method did not have a control test measured in patients with non-poisonous snake bite envenomation. a degree of individual variability was inevitable in the evaluation of ct both by capillary tube and wbct20 methods as well as that of bt. the duration of envenomation prior to admission has not been taken into account in this study, possibly affecting the correlation between haematological parameters and development of complications. conflicts of interest authors declare no conflict of interest. authors’ contributions harikrishnan m.p. – data curation; investigation; methodology; supervision; writing – original draft; writing – review & editing. anil kumar c.r . – data curation; investigation; supervision; project administration; methodology; supervision; writing – original draft; writing – review & editing. anand m.k. – data curation; investigation; methodology; supervision; writing – original draft; writing – review & editing. jerry earali – data curation; investigation; methodology; supervision; writing – original draft; writing – review & editing. варіабельність гематологічних параметрів як предиктор розвитку ускладнень після потрапляння в організм гемотоксичної отрути внаслідок укусу змії m.p. harikrishnan, *c.r. anil kumar, m.k. anand, jerry earali jubilee mission medical college and research institute, thrissur, india вступ. одна з найбільших проблем закладів охорони здоров’я в індії – укуси змій, які супроводжуються високою летальністю. найбільш часті ускладнення, які розвиваються внаслідок потрапляння в організм гемотоксичної зміїної отрути, це синдром дисемінованого внутрішньосудинного згортання (двз), шок, гостре ураження нирок (гун), гострий респіраторний дистрес синдром (грдс) та кооагулопатія. m.p. harikrishnan et al. 28 in t e r n a l m e d ic in e issn 2413-6077. ijmmr 2020 vol. 6 issue 2 мета дослідження – дослідити можливий взаємозв’язок між частотою розвитку ускладнень як двз, гун, гостра ниркова недостатність (гнн), грдс, шок та кишково-шлункові кровотечі. методи. це перехресне дослідження проводилося впродовж 18 місяців. було залучено 150 пацієнтів, які підписали поінформовану згоду. збір даних проводили за допомогою спеціально розробленої форми. також досліджували швидкість згортання крові за допомогою капіляра та 20-хвилинного тесту на згортання цільної крові (англ. 20wbct, показники коагулограми. для статистичної обробки отриманих даних використовували програмне забезпечення ibm spss statistics. результати. серед покусаних людей, у яких розвинулися ускладнення, у більшості (52%) розвинулося гун, внаслідок чого 26% серед них потребували діалізу, у 16,7% учасників дослідження були кишковошлункові кровотечі, у 11,3% розвинувся шок, у 10% розвинувся двз. висновки. серед досліджуваних гематологічних параметрів для прогнозування розвитку ускладнень (гострого ураження нирок та шоку) після укусу змії найбільш інформативними був подовжений час згортання крові. обидва методи дослідження згортання крові і за допомогою капіляра і wbct20 були релевантні та корелювали при прогнозуванні ускладнень. ключові слова: гемотоксична зміїна отрута; укус змії; гостре ураження нирок; шок; час згортання крові; час кровотечі. information about the authors harikrishnan m.p. – senior resident, md general medicine, department of medicine, jubilee mission medical college and research institute, thrissur. orcid: 0000-0003=3452-5588 email: harikrishnapuram@gmail.com anil kumar c.r. – md general medicine, associate professor, department of medicine, jubilee mission medical college and research institute, thrissur. orcid: 0000-0002-6397-2005 email: anilkumarcr3005@gmail.com anand m.k. – md general medicine, assistant professor, department of medicine, jubilee mission medical college and research institute, thrissur. orcid: 0000-0002-0164-8402 email: anandmk92@yahoo.in jerry earali – md general medicine, professor, department of medicine, jubilee mission medical college and research institute, thrissur. orcid: 0000-0003-2880-1259 email: 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105-112 copyright © 2019, tnmu, all rights reserved *corresponding author: nikolay ya. golovenko, a.v. bogatsky physico-chemical institute of the national academy of sciences of ukraine, lyustdorfskaya road, 86, odesa, 65080, ukraine. e-mail: n.golovenko@gmail.com doi 10.11603/ijmmr.2413-6077.2019.2.10900 anti-inflammatory effects of propoxazepam on different models of inflammation *n.ya. golovenko, t.a. kabanova, s.a. andronati, o.i. halimova, v.b. larionov, а.s. reder a.v. bogatsky physico-chemical institute of the national academy of sciences of ukraine, odesa, ukraine background. propoxazepam, 7-bromo-5-(2-chlorophenyl)-3-propoxy-1h-benzo[e][1,4]diazepin-2(3h)-one, is a promising analgesic and anticonvulsant and is on preclinical trial. objective. the aim of the research was to study the anti-inflammatory and analgesic action of propoxazepam. methods. the anti-inflammatory action was evaluated by carrageenan induced rat paw edema, formalininduced paw licking response in mice and bradykinin-induced pain response in rat models. results. it was established for the first time that the administration of propoxazepam caused a significant anti-inflammatory activity when tested in different in vivo chemical experimental models of induced inflammation, i.e. carrageenan-, bradykininand formalin-induced inflammation tests. conclusions. propoxazepam significantly reduced acute and sub-acute inflammation and proved its efficacy and similar to anti-inflammatory action. key words: propoxazepam; diclofenac sodium; anti-inflammatory effect; carrageenan; formalin. introduction benzodiazepines are a large and still expanding group of synthetic heterocyclic derivatives. the wide spectrum of the pharmacological effects exhibited by these compounds makes them one of the most versatile class of drugs used in psychopharmacology. classical 1.4-benzodiazepine (bdz) drugs have sedative, anxiolytic and anticonvulsant properties, enhancing the gamma – aminobutyric acid (gaba)-ergic neurotransmission through binding to the specific bdz recognition sites, within gabaa receptor-ion channel complex, and allosterically modulate its activity [1]. almost since their introduction, there has been interest to the therapeutic use of the benzodiazepines for management of pain. as regarding many other drugs, initially developed and studied for indications other than pain, conclusive data regarding the analgesic activity of bdz is lacking. a relevant aspect of neuroplastic changes in inflammatory and neuropathic conditions is reduction in inhibitory glycinergic and gabaergic control of dorsal horn neurons: a reduction in the gabaa-mediated endogenous inhibitory control within the central nervous system leads to exaggerated pain and hyperalgesia [2]. potentiation of gabaa receptormediated synaptic inhibition by benzodiazepines reverses pathologically increased pain sensitivity in animal studies. though bdz and barbiturates have discriminating effects on pain, their pharmacological actions on the cns are mediated through the gaba induced chloride currents [3]. in contrast to phenobarbitone, diazepam has also been reported to have anti­inflammatory action [4] and anti­ pyretic action [5,6]. a number of 3-substituted 1.4-benzo diazepines have been synthesized at the physicochemical institute of the national academy of sciences of ukraine and their structure – activity relationships, have been studied as well. their pharmacological effect was unusual, because unlike most classical bds, in the models of nociceptive and neuropathic pain these substances showed significant analgesic activity [7]. one of them, propoxazepam, 7-bromo-5(2-chlorophenyl)-3-propoxy-1h-benzo[e][1,4] diazepin-2(3h)-one, is a promising drug and is on preclinical trials [8]. like gabapentin and pregabalin, which are well-known drugs used in general medical practice for treatment of neuropathic pain [9], propoxazepam also has n.ya. golovenko et al. 106 b io m e d ic a l s c ie n c e s issn 2413-6077. ijmmr 2019 vol. 5 issue 2 an anticonvulsant effect [10, 11], which explains the analgesic component of the pharmacological spectrum. an inflammatory response and development of pain are interdependent processes. the uncontrolled inflammatory reaction triggers the process of chronic pain generation, which is associated with the phenomena of central sensitization and neuroplasticity. it becomes obvious that the main direction of complex analgesic therapy should be the targeted use of pharmacological agents possessing antiinflammatory potential [4­6]. it has been established that acute diazepam (a bdz derivative drug) treatment suppresses cell proliferation in rat thymus, decreases interleukin release from mouse macrophages [12] and decreases macrophage and neutrophil activities [13], most probably by affecting the peripheral-type benzodiazepine receptor (pbr) present in these immune/inflammatory cells. high doses of diazepam (10.0–20.0 mg/kg) have proved to reduce the volume of acute inflam­ matory paw edema in rats as a response to carrageenan administration [14]. this effect was associated with an action of diazepam on the pbr present in the adrenal and/or immune/ inflammatory cells [15]. therefore, the objective of the study was to investigate the possible anti-inflammatory effects of the innovative derivative of bdz propoxazepam on acute and chronic inflam­ matory responses in the rats exploring the possible underlying mechanisms involved in these anti­inflammatory effects. methods animals and injection procedures male wistar rats (180-210 g in weight) and white mice (20-24 g in weight), took from institute of pharmacology and toxicology of the nams of ukraine, kept at the local animal facility, were used. the animals were exposed to a 12-hour light-dark cycle and were provided with food and water ad libitum. all experiments were conducted during the light part of the day (9.00-14.00). the experiments were carried out according to the recommendations of the committee for research and ethical issues of the iasp (1983) and were approved by the local ethical committee for animal research. all manipulations were performed to minimize animal suffering and to reduce the number of animals used. the test compound was suspended in tween 80 (1%) emulsion, and the control animals received corresponding amount of vehicle (1% tween 80). drugs and chemicals propoxazepam was synthesized according to the method described in [16]. the structure of the substance was determined and approved by a complex of physicochemical methods (ir and mass spectroscopy, as well as x-ray diffraction analysis) [17]. сhemical purity was confirmed by elemental analysis (99%). sodium carrageenan and bradykinin were obtained from sigma chemical co., usa. diclofenac sodium salt (merck). various other chemicals and reagents were of analytical grade and got from local firms. the doses of propoxazepam for experiments on animals were chosen according to previous bioscreening and pharmacodynamic data, where the analgesic properties of propoxazepam were estimated. for rat tests the mean effective dose (ed50) in the tail flick test was 1.83 mg/kg, so in this species of animals for paw edema tests the doses were raised in 1.6 (for 3 mg/kg) and 5 times (for 10 mg/kg) in order to achieve expected 82 % (1 σ) and 95 % (2 σ) effect. mice doses were derived according to allometric relations with regard to species and body weight coefficients. carrageenan-induced rat paw edema three groups of eight rats each were administered with the vehicle (tween 80 (1%) emulsion, p.o.), the test compound (3 or 10 mg/kg, p.o.), and diclofenac sodium (10 mg/kg, p.o.). one hour following the treatment with various agents, edema was induced by a subplantar injection of 0.1 ml of 1% of freshly prepared suspension of carrageenan into the right hind paw of each animal. the volume of the injected paws was estimated at 0; 2 and 4 h following carrageenan injection utilizing a plethysmometer (plethismometer, ugo basil, italy). the edema development was determined by the increase in paw volume. the increase in percentage inhibition was calculated utilizing the following equations: percentage inhibition = (δvк–δvд/δvк)·100%, where: δvк – mean paw size in the control group. δvд – mean paw size in the treated group. formalin-induced paw licking response in mice mice were divided into four groups (n=6). test drugs propoxazepam (0.01, 0.1 and 3.0 mg/kg p.o.), diclofenac sodium (10 mg/kg p.o.) and control vehicle (1% tween 80, p.o.) were ad ministered 1 h before formalin injection into the n.ya. golovenko et al. 107 b io m e d ic a l s c ie n c e s issn 2413-6077. ijmmr 2019 vol. 5 issue 2 n.ya. golovenko et al. animals in the first set (early phase) and 40 min before formalin injection into the animals in the second set (late phase), respectively. mice were subcutaneously injected with 20 μl of formalin (1% in normal saline) into the right dorsal hind paw. the amount of time that the animal spent licking the injected paw was measured during the first 5 min (phase 1, corresponding to the direct chemical stimulation of nociceptors) and 20-25 min after formalin injection (phase 2, inflammatory). bradykinin-induced pain response in rat the possible contribution of bradykinin receptors in the antinociceptive effect of propoxazepam was evaluated by using the method described by chau et al [18]. bradykinin (0.01% solution) was injected to the subplantar area of rat right hind paw 0.1 ml/animal. propoxazepam (1.83 mg/kg) was administered 2 hours prior to bradykinin injection. the degree of hyperalgesia was determined using a dolorimeter (dolorimeter baseline, usa) by determination of the threshold of pain sensitivity (tps) – the minimum pressure on the lower surface of the rat’s foot (g/mm2), which caused pain in the animal (vocalization and/or withdrawal of the foot). each animal was given 5 attempts; the threshold value was taken with such a pressure force, which caused a positive response in at least one attempt. the tps was compared on intact and damaged limbs on the 14th day after tying (pathology without treatment), as well as on the injured limb in 2 hours (peak of action) after the drugs administration. data analysis data are expressed as a mean and standard error mean (sem) and a student's t-test was used to compare the data of the control and standard groups. probabilities (p) of <0.05 were considered statistically significant. statistical analysis was performed using the standard statistical package of ms excel. results effects of propoxazepam on acute inflammation a single sub-plantar injection of carrageenan induced an increase in the paw thickness within 24 hours. the rat group pretreated with propoxazepam had a significantly reduced (p<0.05) increase in the paw thickness in 2 hours with 36.7%, (3.0 mg/kg) and 47.0% (10 mg/kg) reduction of the paw edema and in 4 hours – 25.0% (3.0 mg/kg) and 26.7% (10 mg/kg). while rats of the group pretreated with diclofenac sodium showed 47.0% and 26.7% (p<0.0001) reduction of the paw edema, respectively (table 1). effect of propoxazepam on formalininduced inflammation as shown in figure 1, the i.p. treatment with propoxazepam at the doses of 0.01, 0.10 and 3.0 mg/kg significantly inhibited the licking time in of both neurogenic (0-5 min), by 91.9% (p<0.001), 76.0% (p<0.001), and 15.0%, and inflammatory (15­30 min), by 98.4% (p<0.001), 54.0% (p<0.001), and 32.1% (p<0.01), phases of formalin-induced paw-licking test compare to the control group. diclofenac sodium (10,0mg/kg, i.p.) significantly inhibited both phases of the test by 51.0 % and 53.6% (p<0.001), respectively. propoxazepam antinociceptive action on the bradykinin-induced hyperalgesia bradykinin injection to the rats induced statistically significant tps decrease by 71.7% (fig. 2). under these conditions propoxazepam induced prominent antibradykinin effect, since on the background of its administration bradykinin induced tps decrease was threefold less that of in the control group (23.8% and 71.7% respectively). discussion the study proves the anti­inflammatory and analgesic effect of propoxazepam on different models of inflammation. the carrageenaninduced rat paw edema model is widely used to investigate mechanisms of inflammatory processes and to screen potential anti­inflam­ matory agents. this model has been extensively studied in the assessment of anti­inflammatory action of steroidal and non-steroidal drugs involving several chemical mediators such as histamine, serotonin, bradykinin and prostaglandins. the edema and inflammation induced by carrageenan is established to be mediated by histamine and 5­ht during the first hour, after which increased vascular permeability is maintained by the release of kinins up to 2.30 hours and from 2.30 to 6 hours, the mediators are found to be the prostaglandins, the release of which is closely associated with migration of leucocytes into the inflamed site [19, 20]. in carrageenan-induced paw edema test, the highest inhibitory activity was exhibited at the dose of 10 mg/kg compared to the other dose. this simply depicts that the higher the dose, the more the inhibition of the edema. hence, an inhibitory activity of propoxazepam is dose-dependent (table 1). the results of the study have revealed that propoxazepam as well as diazepam has a sig108 b io m e d ic a l s c ie n c e s issn 2413-6077. ijmmr 2019 vol. 5 issue 2 table 1. change in paw size by carrageenan-induced paw edema in albino rats treated with increased doses of propoxazepam treatment dose, mg/kg percentage inhibition (%) after 2 hours percentage inhibition (%) after 4 hours propoxazepam 3.0 10.0 36.7±4.1** 47.0±4.1*** 25.00±2.7* 26.70±5.4* diclofenac sodium 10.0 43.0±7.0*** 57.10±8.3*** notes. values are expressed as mean±sem of eight rats, * – p<0.05, ** – p<0.001, *** – p<0.0001 (student’s t-test). nificant anti­inflammatory effect on the diffe­ rent experimental models of inflammation either acute or chronic. in the acute inflam­ matory study, diazepam caused significant reduction of carrageenan-induced paw edema and serum nox levels in rats. while in the chronic inflammatory study, diazepam led to significant reduction of the paw thickness, significant reduction in the serum c­reactive protein, significant increase in serum albumin and significant increase in the serum corti­ costerone level [21, 22]. similar pharmacological actions have been reported for chlordiazepoxide while, alprazolam is reported to be devoid of *** *** *** *** *** *** ** 0 20 40 60 80 100 140 d 3,00 0,10 0,01 dose, mg/kg i phase ii phase % of effect 120 (paw licking) figure 1. effect of propoxazepam and diclofenac sodium on the paw licking induced by a formalin injection in mice. results are presented as mean±sem (n=6). ** – p<0.01, *** – p<0.001, compared to the control. figure 2. effect of propoxazepam (2 mg/kg p.o.) on bradykinin-induced hyperalgesia in rats. pressure induced hind paw withdrawal were evaluated before (baseline), and in 1 min after intraplantar bradykinin (0.01%) administration. two hours before examination the control group received vehicle, and the experimental group received propoxazepam. data are presented as a mean±sem, n=7. * – p<0.05 versus baseline, # – p<0.05 versus vehicle. 0 50 100 150 200 250 300 350 400 450 control group experimental group baseline bradykinin injection n.ya. golovenko et al. 109 b io m e d ic a l s c ie n c e s issn 2413-6077. ijmmr 2019 vol. 5 issue 2 n.ya. golovenko et al. analgesic activity [23]. these reports clearly indicate that various benzodiazepines differ individually in their pharmacological profiles despite of the common chemical structure. furthermore, the results of chronic inflam­ matory study showed that diazepam signifi­ cantly increased serum albumin level and sig nificantly decreased serum c­reactive pro­ tein, which is an acute phase protein commonly used to assess the disease activity in inflam­ matory rheumatic diseases, and this effect of diazepam could be explained by the fact that it can sup press secretion of pro­inflammatory cytokines from mouse macrophages such as tnf­α, il­1, and il­6, which is the main sti­ mulator of acute phase protein synthesis in acute and chronic inflammation [24]. struc­ turally propoxazepam is closer to diazepam and chlordiazepoxide, therefore their mechanisms of action coincide. the formalin-induced licking response is used as a model for evaluation of new analgesic. this definitely proves whether the licking is genuinely due to formalin injected into the paw, because at times the animals lick the forepaw under normal physiological conditions. however, formalin test is sensitive to non-steroidal anti­inflammatory drugs and other mild anal­ gesics. the test possesses two distinct phases, possibly reflecting different stages of pain. the early phase reflects a direct effect of formalin on nociceptors (non­inflammatory pain), whe­ reas the late phase reflects inflam matory pain [25]. moreover, formalin-induced nociception is also associated with direct action on a member of transient receptor potential family (trp) of cation channels denoted as trpa1 receptor located in c fibers [26]. the results of the study show that the i.p. administration of propoxazepam significantly and dose­depen­ dently attenuates the nociceptive response in both neurogenic and inflammatory phases of the formalin-induced paw-licking test in mice at the level reference drug diclofenac sodium. the effect of diazepam is very similar to the effect of propoxazepam in formalin-induced licking model [27]. the effect of diazepam is confined to increase the pain scores during the periods in 10, 15, and 20 min after formalin. one of the cardinal features of inflammatory states is that normally innocuous stimuli produces pain [28]. bradykinin, one of the peptide kinins, is an important inflammatory mediator. the main function of bradykinin is to increase the sensation of pain. a secondary function of bradykinin is to promote the production of histamine that is of increasing blood flow into the involved area by dilation of arteries and increased capillary vessel permeability [29]. bradykinin diversely influences on the pathophysiological processes accompanying pain and inflammation. its biological action is mediated by two established g-protein coupled receptors named b1 and b2. the bradykinin b2 receptor is constitutively expressed in most cell types and evokes acute pain responses following tissue injury, whereas the bradykinin b1 receptor is induced during inflammatory insults or painful stimuli [30]. the attained results show that propoxazepam in this experiment reduces hyperalgesia on the in the model of bradykinininduced edema. an additional argument for possible interaction of propoxazepam with bradykinin receptors is the study [31] of compound influence on the maximal normalized speed of bradykinin-induced contraction of the rat stomach smooth muscles in the presence of gadolinium ions and verapamil. for propoxazepam the statistically significant changes of the before-mentioned indicator have been shown as it is able to additionally inhibit the bradykinin-induced contraction in the presence of gd3+ and verapamil by 19.3% and 32.0% respectively, and demonstrates the effects similar to those of des-arg9-bradykinin-acetate (b2-bradykinin receptors concurrent anta gonist) that proves either interaction with receptor or influence on signal transduction pathways. additionally, the mechanisms and antinociceptive effects of propoxazepam were studied on animal models of acute and chronic pain [7, 8]. the effects of propoxazepam on pain responses were examined using tail­flick test (tft) in rats, streptozotocin-induced rat model (spz) and sciatic nerve injury (sni)-induced hyperalgesia in rats. propoxazepam (3 mg/kg)) proved statistically significant analgesic effect compare to the control and ketorolac values after acute application in tft and sni-induced hyperalgesia in rats. propoxazepam (2 mg/kg) compare to gabapentin (5 mg/kg) in greater degree after both single and chronic administrations showed analgesic action in spzdiabetic rats. propoxazepam administration reduced bradykinin-induced (0.01 %) hyperalgesia. at a low dose (1 mg/kg) flumazenil diminished propoxazepam antinociceptive effect while at a higher dose (10 mg/kg) had nearly no influence, possibly due to gabaa­ receptor complex stabilization. this suggests 110 b io m e d ic a l s c ie n c e s issn 2413-6077. ijmmr 2019 vol. 5 issue 2 that propoxazepam causes both nociceptive and neuropathic analgesia in rats and gabaareceptor and bradykinin b-receptor are key sites of analgesic action of propoxazepam. conclusions the study established the anti­inflammation effects caused by oral administration of a novel synthetic 1,4-benzodiazepine analogue, 7-bromo-5-(2-chlorophenyl)-3-propoxy-1h-benzo[e] [1,4]diazepin-2(3h)-one (propoxazepam) into the rats and mouse models with induced inflammation and nociception and explored potential mechanisms of its action. it was proved for the first time that the administration of propoxazepam had significant anti­inflam­ matory action when tested in different in vivo chemicals experimental models of induced inflammation, namely carrageenan­, bradykinin­ and formalin­induced inflammationtion tests. the mechanism of propoxazepam action might be the inhibition of synthesis or release of inflammatory mediators. as a result of the notable biological activity of propoxazepam it would be applicable to conduct additional research to implement it into medical practice. funding this research received no external funding. conflict of interests the authors declare no conflict of interest. author contributions n.ya. golovenko – conceptualization, project administration, writing (review and editing), t.a. kabanova – investigation, resourses, writing (original draft), s.a. andronati – conceptualization, project administration, writing (review and editing), o.i. halimova – investigation, resourses. v.b. larionov – formal analysis, visualization writing, data curation. а.s. reder – conceptualization, resourses, supervision протизапальні ефекти пропоксазепаму на різних моделях запалення м.я. головенко, т.а. кабанова, с.а. андронаті, о.і. халімова, в.б. ларіонов, а.с. редер фізико-хімічний інститут ім. о.в. богатського національної академії наук україни, одеса, україна вступ. пропоксазепам, 7-бром-5(2-хлорфеніл)-3-пропоксі-1н-бензо[e][1,4]діазепін-2(3н)-он розглядається, як перспективний анальгетик і антиконвульсант, і станом на сьогодні проходить доклінічні випробування. мета. вивчити протизапальні властивості та аналгетичну активність пропоксазепаму. методи. протизапальна активність була визначена на моделі карагінан-індукованого набряку лапи щурів, викликаної формаліном відповіді лизання у мишей та брадикінінової моделі болю у щурів. результати. вперше було продемонстровано, що застосування пропоксазепаму викликало значний протизапальний ефект у різних тестах in vivo викликаного хімічними речовинами запалення, а саме карагінану, брадикініну та формаліну. висновки. пропоксазепам значно зменшує інтенсивність гострого та підгострого запалення та проявляє протизапальну активність, співставну з референтними препаратами. ключові слова: пропоксазепам; диклофенак натрію; протизапальна дія; карагінан; формалін. інформація про авторів микола головенко – академік національної академії медичних наук україни, доктор біологічних наук, професор, головний науковий співробітник лабораторії фізико­хімічної фармакології відділу медичної хімії фізико­хімічного інституту імені о.в. богатського нан україни. тетяна кабанова – кандидат біологічних наук, старший науковий співробітник відділу медичної хімії фізико­хімічного інституту імені о.в. богатського нан україни. сергій андронаті – академік національної академії наук україни, доктор хімічних наук, професор, директор фізико­хімічного інститут імені о.в. богатського нан україни. олена халімова – кандидат біологічних наук, науковий співробітник відділу медичної хімії фізико­хімічного інституту імені о.в. богатського нан україни. віталій ларіонов – доктор біологічних наук, завідувач лабораторії фізико­хімічної фармакології відділу медичної хімії фізико­хімічного інституту імені о.в. богатського нан україни. анатолій редер – кандидат хімічних наук, старший науковий співробітник фізико­хімічного інституту імені о.в. богатського нан україни. n.ya. golovenko et al. 111 b io m e d ic a l s c ie n c e s issn 2413-6077. ijmmr 2019 vol. 5 issue 2 n.ya. golovenko et al. information about the authors mykola golovenko – ph.d., dsc, academician of national academy of medical sciences of ukraine, chief researcher of laboratory of physico-chemical pharmacology of medicinal chemistry department, a.v. bogatsky physico-chemical institute of nas of ukraine, odesa, ukraine. orcid 0000­0003­1485­128x, e­mail: n.golovenko@gmail.com tatyana kabanova – ph.d., senior researcher of medicinal chemistry department of a.v. bogatsky physico-chemical institute of nas of ukraine, odesa, ukraine. sergiy andronati – ph.d., dsc in chemistry, academician of national academy of sciences of ukraine, director of a.v. bogatsky physico-chemical institute of nas of ukraine, odesa, ukraine. orcid 0000-0001-8451-6327 olena halimova – ph.d., researcher of medicinal chemistry department of a.v. bogatsky physicochemical institute of nas of ukraine, odesa, ukraine. vitalii larionov – ph.d., dsc, head of laboratory of physical-chemical pharmacology of medicinal chemistry department of a.v. bogatsky physico-chemical institute of nas of ukraine, odesa, ukraine. orcid 0000-0003-2678-4264 anatoliy reder – ph.d., senior researcher of a.v. bogatsky physico-chemical institute of nas of ukraine, odesa, ukraine. orcid 0000-0002-1801-8378 references 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66 b io m e d ic a l s c ie n c e s issn 2413-6077. ijmmr 2019 vol. 5 issue 1 doi 10.11603/ijmmr.2413-6077.2019.1.10307 efficacy of granulocyte colony-stimulating factor and enterosorption in melphalan-induced bone marrow suppression in guerin carcinoma grafted rats o.o. shevchuk1*, i.m. todor2, n.yu. lukianova2, n.k. rodionova3, v.g. nikolaev2, v.f. chekhun2 1 – i. horbachevsky ternopil national medical university, ternopil, ukraine 2 – r.e. kavetsky institute of experimental pathology, oncology and radiobiology of the national academy of sciences of ukraine, kyiv, ukraine 3 – institute for nuclear research of the national academy of sciences of ukraine, kyiv, ukraine background. side effects of antineoplastic agents (especially leukopenia and neutropenia) could be the main limiting factors for efficient treatment. objective. the research is aimed at the study of myeloprotective capability of biosimilars of granulocyte colony stimulating factor (g-csf) and granular carbon oral adsorbent c2 in melphalan-induced bone marrow suppression in guerin carcinoma-grafted rats. methods. melphalan at the dose of 5.5 mg/kg was used to promote bone marrow suppression in the guerin carcinoma grafted rats. to fight myelosuppression, we used filgrastim and its analogue, designed and produced by iepor, a recombinant granulocyte colony-stimulating factor (r-gcsf). carbon granulated enterosorbent c2 was used for enteral sorption therapy (bulk density γ=0.18 g/cm3, diameter of granules 0.15-0.25 mm, bet pore surface – 2162 m2/g). all rats were sacrificed on the 17th day after carcinoma cells inoculation or on the 8th day after melphalan injection. results. alkylating cytostatic agent caused severe leukopenia (by 95.7%), neutropenia (by 73.9%), and thrombocytopenia (by 84.9%) in the experimental rats. mortality rate was 57%. filgrastim and enterosorption with carbon oral adsorbent c2 increased the studied indices, but the most prominent results were observed when combination of both factors was used. studied means did not affect the anti-tumor efficacy of melphalan alone and in combination. conclusions. our results are perspective for further investigation of the efficacy of the combination of carbon oral adsorbents and hematopoietic cytokines in cases of ameliorate anti-cancer chemotherapy side effects, and its implementation into clinics. key words: melphalan; guerin carcinoma; rats; granulocyte colony stimulating factor; enterosorption. *corresponding author: shevchuk oksana, 1 maidan voli, i. horbachevsky ternopil national medical university, ternopil, ukraine 46001. e-mail: shevchukoo@tdmu.edu.ua. introduction conventional dose-dense and dose-intense chemotherapy with radiation therapy and surgery promote significantly to the treatment of malignancies. unfortunately, the side effects of antineoplastic agents are the main limiting factors for their efficiency [1–3]. the cells and tissues with high speed of division are the most sensitive. bone marrow suppression, damage of gastrointestinal mucosa, gonadal toxicity, loss hair, dysfunctions and/or structural changes of kidney and liver are typical side effects of tumoricidal chemotherapy [2,4,5]. the incidence rates of myelotoxicity varies from 30 up to 60% [6]. the mortality rates due to febrile neutropenia among the patients undergoing chemotherapy are around 5-11% in adults and 2-6% in children [7,8], and up to 20% in case of comorbidity [6]. so, amelioration of the negative effects and saving of the tumoricidal activity of chemotherapy are topical issues of contempo rary oncology. only complete courses of treat ment may guarantee survival [7,9]. granulocyte colony-stimulating factor (g-csf) and a granulocyte-macrophage colonystimulating factor (gm-csf) are an irreplaceable part of supportive care in oncology and are used to reduce the incidence of severe leukopenia [10]. it has been proved in the cancer patients receiving multiple cycles of chemotherapy [11]. international journal of medicine and medical research 2019, volume 5, issue 1, p. 66-77 copyright © 2019, tnmu, all rights reserved o.o. shevchuk et al. 67 b io m e d ic a l s c ie n c e s issn 2413-6077. ijmmr 2019 vol. 5 issue 1 endogenous intoxication is another component that makes the side effects of chemotherapy more severe. these syndrome compounds are the products of tumor metabolism, and the consequence of treatment (surgery, chemotherapy cause the damage of tumor tissues and increase in the quantity of toxic metabolites as well as its derivatives) [12]. sorption detoxification is a common and wellknown method for reducing toxic effects of chemotherapy [13–15]. some of the main types, which are widely used in medicine today, are hemoperfusion (when blood is filtered through the column with activated carbon), enterosorption – enteral use of oral adsorbents of different types, and application-sorption therapy – use of carbon dressing for healing of burns and wounds. our previous experiments proved a significant bone marrow protection in melphalaninduced myelosuppression in the healthy rats [16,17]. but a topical issue of any additive supportive therapy in oncology is the impact on a tumor growth, not only the amelioration of anti-cancer chemotherapy side effects. so, the objective of our investigation is the study of myeloprotective capability of biosimilars of granulocyte colony stimulating factor (g-csf) and enteral sorption therapy with carbon oral adsorbent c2 in melphalan-induced bone marrow suppression in the guerin carcinoma grafted rats. methods melphalan (alkeran, glaxosmithkline, uk) was used to promote the bone marrow suppression. carbon granulated enterosorbent c2 (produced by r.e. kavetsky institute of experimental pa thology, oncology and radiobiology, iepor) was used for enteral sorption the rapy. the parameters of enterosorbent c2 are: bulk density γ=0.18 g/cm3, diameter of granules 0.15–0.25 mm, bet pore surface – 2162 m2/g. filgrastim (neupogen, hoffman la roche) and g-csf-analogue of iepor production – recombinant granulocyte colony-stimulating factor (r-gcsf) were used to increase the white blood cell (wbc) count. r-gcsf was designed within the state grant no. 487/2011 from 29.09.2011 “development of technology for synthesis of human recombinant granulocyte colony stimulating factor and medication on its basis”. the experiments were performed using mature wistar female rats, 200±20 g of body mass, which were got from the vivarium of iepor (kyiv, ukraine). guerin carcinoma cells were taken from the bank of cell lines from human and animal tissues of the abovementioned institute. all procedures were performed according to the rules and requirements of european convention for the protection of vertebrate animals used for experimental and other scientific purposes and a local ethic committee of iepor. the tumors were implanted by subcutaneous injection (dorsally into the left flank) of 2.2×106 guerin carcinoma cells suspended in 0.5 ml of sterile physiological solution. all animals were randomly divided into 6 groups (n=7). the 1st group was a tumor control group. all rats of groups 2-6 were administered with melphalan: the 3rd group involved the rats, which were administered with filgrastim injections (melphalan + filgrastim) in addition to the cytostatic agent; the 4th – enteral sorption therapy with c2 (melphalan + c2), the 5th group – the rats, which were administered with both agents (melphalan + filgrastim + c2), and the 6th group – the animals, which received r-csf and carbon oral adsorbents c2 (melphalan + r-csf + c2). on the 10th day after guerin carcinoma grafting, melphalan was injected intravenously (into the tail vein) one time at the dose of 5.5 mg/kg to the rats of groups 2-6. beginning from the following day and for four next days, filgrastim or r-csf was injected subcutaneously at the dose of 50 mcg/kg. a suspension of carbon enterosorbent (c2 dosage of 5 ml/kg of animals’ body weight, or 900 mg of dry mass of enterosorbent) in appropriate quantity of distilled water was introduced via the tube into rat stomach during 3 days before the melphalan injection and during 7 days after it (once a day). the rats’ mortality rate and dynamics of tumor growth was studied as well. the rats were weighted, and blood was taken from the heart under ketamine hydrochloride general anesthesia on the 8th day after melphalan injection (the 17th day after guerin carcinoma cells inoculation). automatic hematology analyzer particle counter e120 (erma inc., japan) was used for evaluation of complete blood cell count. statistical analysis. since data were not normally distributed in all groups (shapiro-wilk normality test), non-parametric tests (mannwhitney u-test and one-way anova test) were used in data analysis (statistical significance at p<0.05). the data were expressed as the mean± standard error of the mean (m±se). all statistical calculations were performed using the origin 7.5 software (originlab corporation, usa). o.o. shevchuk et al. 68 b io m e d ic a l s c ie n c e s issn 2413-6077. ijmmr 2019 vol. 5 issue 1 results all rats survived in the tumor control group on the 17th day after guerin carcinoma cells inoculation, while melphalan injection caused the death of 4 rats (the 2nd group). in the group of rats, which received carbon oral adsorbent, 2 rats died. in all other groups one rat died in each group till the end of experiment (figure 1). so, we evidenced the mortality rate of 57.1 % in the melphalan group, while the combination of both agents of correction (g-csf and carbon oral adsorbent) reduced this number to 14.3 %. it was established that a single injection of cytostatic agent melphalan caused significant leukopenia from (13.9±1.9) to (0.6±0.1)×109/l (figure 2). the decreasing of wbc count by 95.7% explained a high mortality rate in the melphalan group (the 2nd group). course of filgrastim promoted 2.3-fold increase of wbc count (or by 133.3%) compare to the melphalan group. enteral sorption therapy also increased this index in 1.5 times (or by 50.0%). the use of combination of both g-csf drugs and enterosorbent had the tendency to be more effective compare to the administration of each drug alone. the wbc count increased in 2.8 times (or by 183.3%) in the melphalan+filgrastim+c2 group and in 2.7 times (or by 166.7%) in the melphalan+r-gcsf+c2 group compare to the melphalan group. as for the wbc formula, the results of the experiment are presented in figure 3. single melphalan injection at the dose of 5.5 mg/kg-1 significantly decreased the granulocytes percentage in the peripheral blood by 73.9%, monocytes were absent at all. the figure 1. survival and mortality rates among the guerin carcinoma grafted rats, which received melphalan, biosimilars of g-csf and enterosorbent c2 on the 17th day after tumor cells inoculation. figure 2. white blood cell (wbc) count in the guerin carcinoma grafted rats, which received melphalan, biosimilars of g-csf and enterosorbent c2 on the 17th day after tumor cells inoculation. notes: * – p<0.05 compare to the tumor control group; ** – p<0.05 compare to the melphalan group. o.o. shevchuk et al. 69 b io m e d ic a l s c ie n c e s issn 2413-6077. ijmmr 2019 vol. 5 issue 1 increased number of lymphocytes in the pe ripheral blood was evidenced that can be explained by critically low wbc count and violation of cells composition. the administration of filgrastim and carbon oral adsorbent c2 alone did not influence significantly; a slight tendency of increase of the indices was evidenced. the combination of both factors led to normalization of granulocytes percentage in the peripheral blood, while the number of leukocytes was still quite low (figure 2). so, granulocytes number increased by 254.8% (in 3.6 times) in the the melphalan+filgrastim+c2 group and by 197.6% (approximately in 3 times) in the melphalan+r-gcsf+c2 group compare to the melphalan group. as for the red blood cell (rbc) count and hemoglobin, no significant impact of any drug used in the experiment was evidenced. but a 6.6-fold decrease of platelets count was observed in the rats (or by 84.9%), which were administered with melphalan compare to the tumor control group (table 1). the same tendency as for the granulocytes percentage was evidenced concerning the platelets count: only the tendency for amelioration of low platelets level. the combination of both drugs: officinal filgrastim or specially designed analogue r-gcsf together with carbon oral adsorbent c2, caused significant increase of thrombocytes number in peripheral blood. thus, platelets count increased in 4.2 times (or by 319.6%) in the melphalan+filgrastim+c2 group, and approximately in 3 times (or by 198%) in the melphalan+r-gcsf+c2 group compare to the 2nd melphalan group. the tumor growth dynamics in case of the administration of alkylating cytostatic agent and g-csf analogues as well as enterosorption was a significant aim of the study. melphalan was injected on the 10th day after guerin carcinoma cells inoculation, when a tumor was formed (table 2). on the 17th day of the experiment a significant slow of tumor growth compare to the tumor control group was evidenced. there was no negative effect of figure 3. white blood cell (wbc) formula of the peripheral blood in guerin carcinoma grafted rats, which received melphalan, biosimilars of g-csf and enterosorbent c2 on the 17th day after tumor cells inoculation. notes: * – p < 0.05 compare to the tumor control group; ** – p < 0.05 compare to the melphalan group. table 1. rbc and platelets count as well as hemoglobin level in peripheral blood in the guerin-carcinoma grafted rats in case of melpahaln, g-csf-analogues and enteral sorption therapy, m±se indices group rbc count, ×1012/l platelets count, ×109/l hb, g/l tumor control group, (n=7) 7.73±0.51 325.6±35.9** 152.0±8.0 melphalan group, (n=3) 8.70±2.65 49.0±11.9* 130.0±30.0 melphalan + filgrastim group, (n=6) 10.57±1.19 74.0±9.5* 211.0±25.0 melphalan + c2 group, (n=5) 8.06±0.68 60.0±8.6* 153.0±15.0 melphalan + filgrastim + c2 group, (n=6) 10.21±1.00 205.6±86.2** 188.0±18.0 melphalan + r-gcsf + c2 group, (n=6) 7.91±0.81 146.0±36.8*,** 146.0±17.0 notes: * – p<0.05 compare to the tumor control group; ** – p<0.05 compare to the melphalan group. o.o. shevchuk et al. 70 b io m e d ic a l s c ie n c e s issn 2413-6077. ijmmr 2019 vol. 5 issue 1 g-csf drugs or enterosorbent c2 on the anticancer action of melphalan. on the 17th day the tumors sizes decreased by 41.6 % in the melphalan group, by 43.4 % in the melphalan+ filgrastim group, by 46.0 % in the melphalan+c2 group, and by 46.9 % in the groups of rats, which received combination of enterosorption and hematostimulating cytokines. we assessed the tumors size in all groups of rats. discussion typical side effects of anti-cancer chemotherapy, and leukopenia as the most common among them, are tightly related to the clinical outcomes [1, 18]. more and more attention is paid to this issue, especially after the success of modern techniques such as a stem cell transplantation and cytokines treatment to re store hematopoietic functions. different che motherapy regimens are classified to develop a high risk (more than 20%), an intermediate risk (from10 to 20%) or a low risk (less than 10%) of febrile neutropenia [6]. all of them require administration of different doses of hema topoietic cytokines to prevent and treat com plication of tumoricidal therapy. g-csf in fluences cellular proliferation, differentiation, maturation, and lineage commitment in the bone marrow not only of neutrophils but of shortterm hematopoietic stem cells, colony forming units granulocyte erythroid macrophage megakaryocyte, colony-forming units granulocyte macrophage and colony forming units granulocyte as well [19]. the american society of clinical oncology (asco) recommends primary prophylaxis with g-csf or gm-csf for the expected incidence of neutropenia of ≥40% [20]. also, pegfilgrastim (sustained duration form of filgrastim, g-csf) is recommended for ad ministration for at least 1 day after chemotherapy [21]. g-csf drugs are mostly well tolerated. one of the most common reported side effects is bone pain (which is not treatment-limiting), neutrophilic dermatoses, exacerbation of psoriasis and isolated anaphylactic reactions as well as coagulation abnormalities also may a p p e a r . tra n s i e n t re n a l a n d b i o l o g i c a l disturbances are reported [22,23]. dyspnea, p a i n i n c h e s t a n d h y p oxe m i a , n a u s e a , diaphoresis, anaphylactic reactions, syncope and flushing are evidenced [24]. unfortunately, g-csf is helpless to fight other than leukopenia side effects [25]. also, it does not penetrate through the alveolar barrier and cannot prevent lung injury, especially during the concomitant radiation therapy [26]. so, it means we may enhance the efficacy of g-csf therapy in the patients with malignancies during chemotherapy courses and sorption detoxification is a promising issue. first positive results for myeloprotective effect of enterosorption were published in 1980-90th [15]. since that time there are a lot of evidences for successful use of enterosorption in oncology practice [12, 13, 27–29]. our previous experiments in healthy rats demonstrated advantages of combination of g-csf and enterosorption to ameliorate side effects of melphalan and cisplatin [16,30]. this study has proved that such combination has no negative impact on tumoricidal activity of alkylating cytostatic agent melphalan and promotes animals’ survival: mortality rate decreased from 57 to 14%. conclusions the side effects of anti-cancer drugs, espe_ cially bone marrow suppression and neutro_ penia, are the main limiting factors for full courses of chemotherapy, which is crucial for treatment efficacy. melphalan at single injection at the dose of 5.5 mg/kg caused significant leukopenia and granulocytopenia in the guerin carcinoma grafted rats. mortality rate was 57.1%. the filgrastim (recombinant granulocyte colony table 2. dynamics of the guerin carcinoma growth in the experimental groups (volume, cm3, m±se, n=7) group day after tumor’s inoculation 10th day 17th day tumor control group 3.7±0.2 11.3±0.2 melphalan group 3.8±0.3 6.6±0.2 * melphalan + filgrastim group 3.7±0.2 6.4±0.3 * melphalan + c2 group 3.5±0.2 6.1±0.2 * melphalan + filgrastim + c2 group 3.8±0.3 6.0±0.4 * melphalan + r-gcsf + c2 group 3.7±0.4 6.0±0.3 * notes: * – p<0.05 compare to the tumor control group. o.o. shevchuk et al. 71 b io m e d ic a l s c ie n c e s issn 2413-6077. ijmmr 2019 vol. 5 issue 1 stimulating factor) or enteral sorption therapy with carbon oral adsorbent c2 (bulk density γ=0.18 g/cm3, diameter of granules 0.150.25 mm, bet pore surface – 2162 m2/g) for correction of melphalan-induced myelosuppression increased white blood cell count, but the most prominent results were evidenced only when combination of both factors was used, especially regarding the granulocytes number. also, only in the group of rats, which received both filgrastim or r-gcsf, produced by iepor, and enterosorption, the restoration of platelets count was observed. the use of both factors of correction did not ameliorate the anti-tumor efficacy of alkylating cytostatic agent. thus, the results are perspective for further study of the use of combination of carbon oral adsorbents and hematopoietic cytokines to ameliorate anti-cancer chemotherapy side effects, as well as their implementation into clinical practice. conflicts of interest authors declare no conflict of interest. ефективність застосування препаратів гранулоцитарного колонієстимулюючого фактору та ентеросорбції при мелфалан-індукованій мієлосупресії у щурів з перевивною карциномою герена о.о. шевчук1, і.м. тодор2, н.ю. лук’янова2, н.к. родіонова3, в.г. ніколаєв2, в.ф. чехун2 1 – тернопільський національний медичний університет імені і.я. горбачевського, тернопіль, україна 2 – інститут експериментальної патології, онкології і радіобіології імені р.є. кавецького нан україни, київ, україна 3 – інститут ядерних досліджень нан україни, київ, україна вступ. побічні ефекти протипухлинних лікарських засобів (найчастіше лейкопенія та нейтропенія) слугують основним лімітуючим фактором ефективного лікування. наші попередні дослідження на здорових тваринах продемонстрували виражену ефективність при мелфалан-індукованій мієлосупресії, однак питання впливу будь яких додаткових чинників, які застосовуються у онкологічній практиці, на ріст та розвиток пухлини надзвичайно важливе. мета роботи: дослідити дію біосимілярів гранулоцитарного колонієстимулюючого фактора (г-ксф) та гранульованого вуглецевого ентеросорбента с2 при мелфалан-індукованій мієлосупресії у щурів з перевивною карциномою герена. методи. для індукування мієлосупресії у щурів з перевивною карциномою герена мелфалан вводили у дозі 5,5 мг/кг. для корекції викликаних змін використовували філграстим та аналог виробництва іепор – рекомбінантний гранулоцитарний колонієстимулюючий фактор (р-гксф). для проведення ентеросорбції використано гранульований вуглецевий ентеросорбент c2 з питомою вагою γ=0,18 г/см3, діаметром гранул 0,15–0,25 мм, поверхня пор за bet – 2162 м2/г). тварин виводили з експерименту на 17-у добу після перещеплення пухлини (на 8-у добу після введення мелфалану). результати. алкілуючий цитостатик викликав глибокі лейкопенію (падіння на 95,7%), нейтропенію (зниження на 73,9%) та тромбоцитопенію (падіння показника на 84,9%) у дослідних тварин. летальність склала 57%. застосування препаратів г-ксф та ентеросорбента с2 покращувало досліджувані показники, однак найбільш виражене покращення спостерігалося лише при введенні обох чинників разом. введення обох чинників не зменшувало протипухлинну активність мелфалану як при моновведенні, так і в комбінації. висновки. отримані нами результати свідчать про перспективи подальшого вивчення ефективності застосування комбінації ентеросорбції та гемостимулюючих цитокінів для пом’якшення побічних ефектів протипухлинної хіміотерапії та їх впровадження у клінічну практику. ключові слова: мелфалан; карцинома герена; щури; гранулоцитарний колонієстимулюючий фактор; ентеросорбція. o.o. shevchuk et al. 72 b io m e d ic a l s c ie n c e s issn 2413-6077. ijmmr 2019 vol. 5 issue 1 references 1. xing c, liang b, wu j, yang q, hu g, yan y, et al. prognostic significance of leukopenia during the induction phase in adult b cell acute lymphoblastic leukemia. cancer manag res. 2018;10:625-35. doi: 10.2147/cmar.s158359 2. al-ansari s, zecha jaem, barasch a, de lange j, rozema fr, raber-durlacher je. oral mucositis induced by anticancer therapies. curr oral heal reports. 2015 dec 19;2(4):202-11. doi: 10.1007/s40496-015-0069-4 3. turgeman o, medical r, blumenfeld z, medical r. minimizing the doxorubicin-induced gonadotoxicity by sphingosine-1-phosphate analogue fty720 minimizing the doxorubicin-induced gonadotoxicity by. am j clin exp obs gynecol. 2015;2(1):24-33. 4. wang y, probin v, zhou d. cancer therapyinduced residual bone marrow injury-mechanisms of induction and implication for therapy. curr cancer ther rev. 2006 aug 1;2(3):271-9. doi: 10.2174/157339406777934717 5. gaducci a, gargini a, palla e, fanucchi a, genazzani ar. neutropenic enterocolitis in an advanced epithelial ovarian cancer patient treated with paclitaxel/platinum-based chemotherapy: a case report and review of the literature. anticancer res. 2005;(25):2509-14. available from: https://pdfs.semanticscholar. org/e16b/eea03afd2a93686821eff80a60696893301f. pdf 6. lalami y, klastersky j. impact of chemotherapyinduced neutropenia (cin) and febrile neutropenia (fn) on cancer treatment outcomes: an overview about well-established and recently emerging clinical data. crit rev oncol hematol. 2017;120(june):163-79. doi: 10.1016/j.critrevonc.2017.11.005 відомості про авторів: шевчук оксана олегівна – канд. мед. наук, доцент каф. фармакології з клінічною фармакологією, тернопільський національний медичний університет імені і. я. горбачевського, тернопіль, україна. тодор ігор миколайович – д-р біол. наук, старший науковий співробітник лабораторії механізмів медикаментозної резистентності, інститут експериментальної патології, онкології і радіобіології імені р.є. кавецького нан україни, київ, україна. лук’янова наталія юріївна – д-р біол. наук, старший науковий співробітник, зав. лабораторії механізмів медикаментозної резистентності, інститут експериментальної патології, онкології і радіобіології імені р. є. кавецького нан україни, київ, україна. родіонова наталія констянтинівна – канд. мед. наук, старший науковий співробітник відділу радіобіології та радіоекології, інститут ядерних досліджень нан україни, київ, україна. ніколаєв володимир григорович – чл.-кор. нан україни, д-р мед. наук, професор, заслужений діяч науки і техніки україни, лауреат державної премії срср, зав. відділу засобів та методів сорбційної терапії, інститут експериментальної патології, онкології і радіобіології імені р. є. кавецького нан україни, київ, україна. чехун василь федорович – академік нан україни, д-р мед. наук, професор, заслужений діяч науки і техніки україни, лауреат державної премії україни в галузі науки і техніки, інститут експериментальної патології, онкології і радіобіології імені р.є. кавецького нан україни, київ, україна. information about authors: shevchuk o.o. – md, ph.d., associate professor, department of pharmacology and clinical pharmacology, i. horbachevsky ternopil national medical university, ternopil, ukraine. orcid 0000-0003-2473-6381, e-mail: shevchukoo@tdmu.edu.ua todor i.m. – ph.d., dsc, r.e. kavetsky institute of experimental pathology, oncology and radiobiology of the national academy of sciences of ukraine, kyiv, ukraine. e-mail: todor@nas.gov.ua lukianova n.yu. – md, ph.d., dsc., r.e. kavetsky institute of experimental pathology, oncology and radiobiology of the national academy of sciences of ukraine, kyiv, ukraine. rodionova n.k. – md, ph.d., institute for nuclear research of the national academy of sciences of ukraine, kyiv. e-mail: oncom@onconet.kiev.ua nikolaev v.g. – md, ph.d., dsc, professor, corr. member of the nas of ukraine, r.e. kavetsky institute of experimental pathology, oncology and radiobiology of the national academy of sciences of ukraine, kyiv. e-mail: aosiepor2@gmail.com chekhun v. f. – md, ph.d., dsc, professor, academician of the nas of ukraine, r.e. kavetsky institute of experimental pathology, oncology and radiobiology of the national academy of sciences of ukraine, kyiv. e-mail: chekhun@onconet.kiev.ua o.o. shevchuk et al. 73 b io m e d ic a l s c ie n c e s issn 2413-6077. ijmmr 2019 vol. 5 issue 1 7. denduluri n, patt da, wang y, bhor m, li x, favret am, et al. dose delays, dose reductions, and relative dose intensity in patients with cancer who received adjuvant or neoadjuvant chemotherapy in community oncology practices. jnccn j natl compr 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sv, sandeman sr, howell ca, phillips gj, nikolaev vg. biomedical applications of o.o. shevchuk et al. 74 b io m e d ic a l s c ie n c e s issn 2413-6077. ijmmr 2019 vol. 5 issue 1 carbon adsorbents. in: novel carbon adsorbents. elsevier; 2012. p. 639-69. doi: 10.1016/b978-0-08-097744-7.00021-1 29. ponomariova ov, pivniuk vm, nosko mm, sakhno lo, dekhtiar tv, nikolaev vg, et al. prophylaxis with carbon enterosorbent of acute and delayed emetogenic toxicity of chemotherapy treatment in oncologic patients. onkologia. 2008;10(3):370-3 [in ukrainian]. available from: http://dspace.nbuv.gov.ua/ handle/123456789/11944 30. sakhno la, yurchenko ov, maslenniy vn, bardakhivskaya ki, nikolaeva vv, ivanyuk aa, et al. enterosorption as a method to decrease the systemic toxicity of cisplatin. exp oncol. 2013;35(1):45-52. available from: http://dspace.nbuv.gov.ua/ handle/123456789/13911 received 01 april 2019; revised 29 april 2019; accepted 10 may 2019. this is an open access article distributed under the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. o.o. shevchuk et al. 69 p sy c h ia t r y issn 2413-6077. ijmmr 2019 vol. 5 issue 2 corresponding author: mysula yuriy, md, ph.d., associate professor of the department of psychiatry, narcology and medical psychology, i. horbachevsky ternopil national medical university, ternopil, ukraine. e-mail: yuramysula@gmail.com doi 10.11603/ijmmr.2413-6077.2019.2.10895 primary episode of bipolar affective disorder yu.i. mysula i. horbachevsky ternopil national medical university, ternopil, ukraine background. bipolar affective disorder (bad) is a topical issue of contemporary psychiatry. the features of the primary episode (pe) of the disease are extremely important for prognosis, treatment and rehabilitation measures of bad. individual psychological features of the patients with pe of bad are still unexplored that complicates development of new methods of prediction, treatment and prevention of bad. objective. the aim of the study was to investigate individual psychological features of the patients with a primary episode of bipolar affective disorder, taking into account the gender factor and clinical variant of the bad debut. methods. 153 patients (65 men and 88 women) with a primary episode of bipolar affective disorder were examined. the patients were divided into three groups according to the clinical variant of the course of pe of bad: depressive variant, manic variant and mixed variant. the examination was carried out using the standardized multifactor method of personality research (smmpr). statistical processing of the data was performed using the non-parametric mann-whitney test. results. the most significant differences in the quantitative indicators of smmpr were found when comparing depressive and manic, as well as depressive and mixed variants of pe of bad, and lesser – when comparing manic and mixed variants. most of all, these differences were expressed in terms of pessimism, impulsiveness, individualism and optimism. conclusions. some peculiar features of male and female patients with depressive, manic and mixed variants of pe of bad promoting to search for new methods of prediction, treatment and prevention of bad have been defined. key words: bipolar affective disorder; individual-psychological features; depressive, manic and mixed variants. introduction bipolar affective disorder (bad) is one of the most topical matters of contemporary psychiatric science and practice. its medical and social significance is associated with its rela­ tively high prevalence: from 0.6% to 1.0% [1], relative stability over a long period of time [2, 3] and the presence of serious lifelong problems [4], i.e. severe affective disorders [5], cognitive disorders [6] and high mortality rates [7]. the features of the primary episode of the disease are extremely important for prog nosis, treatment and rehabilitation measures of bad; however, the initial characteristics of the disease are still poorly defined and the low prognostic value of the existing predictors requires improvement of prodromal identifi­ cation means [8]. in many cases several years pass from the first manifestations of the disease to the diagnosis of bad, despite the extreme importance of pe for prediction of the disorder severity, its functional consequences and therapeutic perspectives [9, 10]. in recent years, much attention was paid to the study of individual and psychological characteristics of the patients with bad; these features are important predictors of the disease clinical course, treatment and rehabilitation perspectives [11]. it is established that bipolar disorder is associated with low self-esteem, paranoia and obsession [12]. at the same time, the individualpsychological features of the patients with pe of bad are unexplored that complicates development of new methods of prediction, treatment and prevention of bad. the objective is to investigate the individual psychological features of the patients with a primary episode of bipolar affective disorder, taking into account gender factor and clinical variant of the bad debut. methods the study was performed in a continuous manner by examining all patients with fitting diagnoses, who sought medical care within a international journal of medicine and medical research 2019, volume 5, issue 2, p. 69-75 copyright © 2019, tnmu, all rights reserved yu.i. mysula 70 p sy c h ia t r y issn 2413-6077. ijmmr 2019 vol. 5 issue 2 specified period. 153 patients (65 men and 88 women) with a primary episode of bipolar affective disorder treated at ternopil regional psychoneurological hospital in 2011–2016 were examined; according to biomedical ethics the patients’ informed consent were received. the patients with other diagnoses were excluded from the study. the examination was performed using the standardized multifactor method of personality research (smmpr) [13]. statistical data was processed using microsoft excel 2013 and statistica 13 software packages. the statistical analysis included assessment of traits distribution for quantitative variables using the shapiro-wilk’s w test, kolmogorov-smirnov test and lilliefors test. the shapiro-wilk’s w test was a reference. the non-parametric mann-whitney u test was used to analyze the differences between the groups taking into account the nature of the distribution (other than normal). the statistical significance of differences over 95.0% (p<0.05) was acceptable. the mean age of the examined patients at the time of symptomatic onset was 21.3±6.5 years old (average 19.0 years, interquartile range 17.0­22.0 years): men 20.5±5.8 years old (18.0 years, 17.0-21.0 years), women 21.9±6.9 years old (18.5 years, 18.5­22.5 years); the age at the time of seeking medical advice and examination was 21.4± 6.4 years old (19.0 years, 18.0-22.0 years): 20.7±5.7 years old (18.0 years, 17.0­21.0 years) and 22.0±6.9 years old (19.0 years, 18.522.5 years), respectively. the examined men and women were divided into three groups depending on the clinical variant of the course of pe of bad: with the prevalence of depressive symptoms (depressive variant), 119 people (44 men and 75 women); with prevalence of manic or hypo ma­ nic symptoms (manic variant), 23 persons (15 men and 8 women); and with simultaneous presence of depressive and manic symptoms, or with rapid phase change (mixed version), number of 11 persons (6 men and 5 women). results individual­psychological profile of patients with depressive variant of pe of bad is characterized by dominance of signs of depression in combination with symptoms of anxiety and fatigue (fig. 1). the average values of the indicators were: by the scale of overcontrol – 67.61±8.56 points: 65.30±9.51 points in men and 68.97±7.69 points in women (p<0.05); pessimism – 83.40±4.08 po ints; 82.32±3.87 points and 84.04±4.09 points (p<0.05), respectively; emotional lability – 59.33±7.88 points: 57.84±8.25 points and 60.20±7.57 points (p<0.05) respec tive ly; im­ pulsivity – 55.92±6.12 points: 54.82±5.27 points and 56.57±6.51 points (p>0.05) respectively; masculinity­femininity – 46.48±8,88 points: 56.05±2.97 points and 40.87±5.86 points (p<0.01) respectively; rigidity – 57.84±5.44 points: 59.41±5.04 points and 56.92±5.49 points (p<0.05) respectively; anxiety – 77.25±6.50 points: 74.86±7.85 points and 78.65±5.12 points (p<0.05) respectively; individuality – 72.09±1.03 points: 71.82±0.84 points and 72.25±1.10 points (p>0.05) respectively; optimism – 39.27±5.91 points: 40.98±9.42 points and 38.27±1.13 points fig. 1. standardized multifactor method of personality research profiles for men and women with depressive primary episode of bipolar affective disorder. (depressive variant), 119 people (44 men and 75 women); with prevalence of manic or hypomanic symptoms (manic variant), 23 persons (15 men and 8 women); and with simultaneous presence of depressive and manic symptoms, or with rapid phase change (mixed version), number of 11 persons (6 men and 5 women). results individual-psychological profile of patients with depressive variant of pe of bad is characterized by dominance of signs of depression in combination with symptoms of anxiety and fatigue (fig. 1). fig. 1. standardized multifactor method of personality research profiles for men and women with depressive primary episode of bipolar affective disorder. the average values of the indicators were: by the scale of overcontrol 67.61±8.56 points: 65.30±9.51 points in men and 68.97±7.69 points in women (p <0.05); pessimism – 83.40±4.08 points; 82.32±3.87 points and 84.04±4.09 points (p <0.05), respectively; emotional lability – 59.33±7.88 points: 57.84±8.25 points and 60.20±7.57 points (p <0.05) respectively; impulsivity – 55.92±6.12 points: 54.82±5.27 points and 56.57±6.51 points (p >0.05) respectively; masculinityfemininity – 46.48 ± 8,88 points: 56.05 ± 2.97 points and 40.87±5.86 points (p <0.01) respectively; rigidity – 57.84±5.44 points: 59.41±5.04 points and 56.92±5.49 35 45 55 65 75 85 1 2 3 4 5 6 7 8 9 0 va lu es , т -p oi nt s 1 – overcontrol; 2 – pessimism; 3 – emotional lability; 4 – impulsivity; 5 – masculinity-femininity; 6 – rigidity; 7 – anxiety; 8 – individuality; 9 – optimism; 0 – introversion all men women yu.i. mysula 71 p sy c h ia t r y issn 2413-6077. ijmmr 2019 vol. 5 issue 2 yu.i. mysula (p<0.05) respectively; introversion – 65.48±3.99 points: 63.95±4.01 points and 66.37±3.72 points (p<0.01) respectively. fig. 1 shows that the profiles of men and women with the depressive variant of pe of bad are similar, although statistical and mathematical analysis of indicators proves significant differences on the main scales. the profiles of both men and women are dominated by 2 scales (‘pessimistic’), with its average values exceeding 80 t-points, which indicates a psychotic level of depressive manifestations. high values on the pessimistic scale are depressive manifestations, expressiveness of affi lia ti ve need with its frustration, blocking of activity with refusal of self-realization. it should be noted that high scores on the pessimistic scale are not only an indicator of the current depressive state of a situational or endogenous character, but also as a predictive factor for a depressive response as a universal pattern of psychological response of an individual in situations of stration, distress or dis adaptation. tendencies revealed by high quantitative values on the pessimistic scale are exacerbated by high (over 70 t-points) values on the scale 7 (‘anxiety’) and extremely low (about 40 t-points) indicators on the scale 9 (‘optimism’). such a profile can be interpreted as a manifestation of anxious-thinking tendencies against the acutely reduced mood (in this contingent – endogenous character) with inhibition of activity, desire to stop all activities, lack of energy and increase of feeling of exhaustion and fatigue. the parallel rise on the scale 8 (‘individuality’) fits into the psychological pattern of hypoactivity, with a predominance of concentration on internal experiences and feelings over external activity, internal tension and anxiety, fixation on problems and the expectation of deterioration. it is also worth mentioning a rather high (over 65 t-points) indicators on the scale 1 (‘overcontrol’), which reflect the fixation on unpleasant somatic sen­ sations, which quite often accompanies depression development. in the profile of patients with depressive variant of pe of bad the described tendencies are combined with high rates on the scale 0 (‘introversion’) that evidences of hypostenic manifestations, passivity of personal position, fixation on internal experiences, decrease of involvement in social environment, decrease in the number of social contacts; high scores on this scale can also be an indicator of response to current difficulties and displaying escapism. low rates on scale 4 (‘impulsivity’) of the profile of patients with depressive variant appropriately reflects the decrease of motivation achievement, developing manifestations of inhibition, reducing overall energy potential, and demonstrates intensive formation of vital apathy, depressive and asthenic-depressive pattern rather than agitated or dysphoric. in the profile of patients with depressive pe of bad, they are combined with low scores the scale 3 (‘emotional lability’), which reflects the staticness of depressive tendencies, desire to decrease activity focus on internal experiences, general inhibition. increasing rates on the scale 5 (‘masculinity-femininity’) in men can be interpreted as suppression of sexual activity under the influence of depression, sentimen­ tality, sensitivity, vulnerability. in women with depressive pe of bad, the smmpr scores on the scale 5 are reduced, indicating a decrease in libido, high sensitivity, desire to be protected, asthenic-depressive mood. the smmpr profile of patients with the manic variant of pe of bad is significantly different from that inherent in the patients with a depressive variant (fig. 2). the mean values of the indicators on the scales were: over control – 55.22±3.29 points, 53.93±2.49 points and 57.63±3.38 points (p<0.05) respec tively; pessimism – 42.30±6.10 points, 40.40±5.46 points and 45.88±5.91 points (p<0.05) respectively; emotional lability – 55.61±4.60 points, 53.93±3.20 points and 58.75±5.37 points (p<0.05) respectively; impulsivity – 78.48±4.45 points, 79.87±4.02 points and 75.88±4.26 points (p<0.05) respectively; masculinity­femininity – 55.43±14.94 points, 45.13±3.68 points and 74.75±4.53 points (p<0.01) respectively; rigidity – 67.61±5.69 points, 69.60±5.80 points and 63.88±3.18 points (p<0.05) respectively; anxiety – 52.87±4.41 points, 51.47±4.09 points and 55.50±3.96 points (p<0.05) respectively; indi­ viduality – 53.52±9.46 points, 53.87±9.33 points and 52.88±10.32 points (p>0.05) respectively; optimism – 75.57±3.89 points, 76.73±3.49 points and 73.38±3.85 points (p<0.05) respectively; introversion – 52.83±8.49 points, 50.33±7.20 points and 57.50±9.21 points (p<0.05) respectively. profile analysis proves predominance of individually-psychological characteristics of the patients with manifestations of mania and impulsivity. high (70 t­points) figures on the scales of optimism and impulsiveness reflect elevated mood, bright emotions high level of activity (usually chaotic and spontaneous), 72 p sy c h ia t r y issn 2413-6077. ijmmr 2019 vol. 5 issue 2 inflated self-esteem, ease decision making promiscuous intercourse, arrogance in commu nication, impaired control, easy conflict, reduction of criticality to one's condition, readiness to act impulsively and hastily. such a profile is associated with coping strategies for displacing unpleasant information, combined with intolerance and aggression towards its source, with difficulty in self-control. it should be noted that in the examined patients high scores on the scales of impulsivity and optimism are combined with high (over 60 points) indicators on the scale 6 (‘rigidity’) that might be an indicator of explosive type of reaction with a tendency to uncontrolled reactions of aggression, domination, rejection of opposition from the environment, beliefs in their own right and exclusivity up to a delusion of grandeur. in the profiles of patients with a manic variant of pe of bad, the peak increase of indicators on the scales of impulsivity, optimism and rigidity is accompanied by a significant decrease in indicators on the scales of pessimism, overcontrol, anxiety, emotional lability and introversion. relatively, in the psychological pattern of response, the signs of sensitivity to other people’s thoughts are suppressed, attention to one's own state of health is not typical (even to the complete disregard of existing problems), lengthy reflections with situation analysis, reflection and introspection. such trends create a favorable ground for deviant behavior, first of all addictive and delinquent ones. the decreased scores on the scale 5 for men is a manifestation of sexual fig. 2. standardized multifactor method of personality research profiles for men and women with manic primary episode of bipolar affective disorder. aggravation and increased sex drive associated with a manic state, indiscriminate sexual contact, ignoring commonly accepted moral rules and regulations. moreover, the individual psychological profile in the patients with mixed variant of pe of bad is characterized by originality and differs from the profiles of patients with depressive and manic variants (fig. 3). the average values on the scales in the patients with mixed variant of pe of bad were: over control – 60.36±10.98 points, 54.67±10.33 points and 67.20±7.82 points (p>0.05) respectively; pessimism – 68.09±8.25 points, 62.50±2.81 points and 74.80±7.56 points (p<0.05) respectively; emotional lability – 60.91±6.61 points, 56.67±4.59 p o i n t s a n d 6 6 . 0 0 ± 4 . 8 5 p o i n t s ( p < 0 . 0 5 ) respectively; impulsivity – 66.09±7.08 points, 70.50±3.56 points and 60.80±6.72 points (p<0.05) respectively; masculinity­femininity – 54.91±13.88 points, 46.17±5.88 points and 65.40±13.65 points (p<0.05) respectively; rigidity – 60.82±5.33 points, 64.33±4.50 points and 56.60±2.19 points (p<0.05) respectively; anxiety – 63.27±11.23 points, 56.67±7.20 points and 71.20±10.31 points (p<0.05) respectively; individuality – 65.82±6.71 points, 66.67±6.59 points and 64.80±7.46 points (p>0.05) respectively; optimism – 56.64±14.38 points, 65.17±9.33 points and 46.40±12.97 points (p<0.05) respectively; introversion – 58.27±9.55 points, 52.33±8.33 points and 65.40±4.98 points (p<0.05) respectively. in the profile of men with mixed pe of bad impulsiveness (4 scales) predominates, fig. 2. standardized multifactor method of personality research profiles for men and women with manic primary episode of bipolar affective disorder. profile analysis proves predominance of individually-psychological characteristics of the patients with manifestations of mania and impulsivity. high (70 t-points) figures on the scales of optimism and impulsiveness reflect elevated mood, bright emotions high level of activity (usually chaotic and spontaneous), inflated self-esteem, ease decision making promiscuous intercourse, arrogance in communication, impaired control, easy conflict, reduction of criticality to one's condition, readiness to act impulsively and hastily. such a profile is associated with coping strategies for displacing unpleasant information, combined with intolerance and aggression towards its source, with difficulty in self-control. it should be noted that in the examined patients high scores on the scales of impulsivity and optimism are combined with high (over 60 points) indicators on the scale 6 (‘rigidity’) that might be an indicator of explosive type of reaction with a tendency to uncontrolled reactions of aggression, domination, rejection of opposition from the environment, beliefs in their own right and exclusivity up to a delusion of grandeur. in the profiles of patients with a manic variant of pe of bad, the peak increase of indicators on the scales of impulsivity, optimism and rigidity is accompanied by a significant 35 45 55 65 75 85 1 2 3 4 5 6 7 8 9 0 va lu es , т -p oi nt s 1 – overcontrol; 2 – pessimism; 3 – emotional lability; 4 – impulsivity; 5 – masculinity-femininity; 6 – rigidity; 7 – anxiety; 8 – individuality; 9 – optimism; 0 – introversion all men women yu.i. mysula 73 p sy c h ia t r y issn 2413-6077. ijmmr 2019 vol. 5 issue 2 yu.i. mysula although the quantitative values of the indicators are smaller than in the manic variant of pe of bad. in men, increased (more than 65 t-points) indicators on the impulsivity scale are combined with increased indicators on the scales of individuality, hypomania and rigidity. such a pattern is a manifestation of the instability of the emotional state, a tendency to mood swings, unconformity, originality and non-template reactions to external challenges, a tendency to impulsiveness, conflict, psychopa­ thization of personality traits. at the same time, the combination of contradictory traits is a constant source of intrapsychic conflicts, which is manifested by high excitability and dynamism combined with inertness and instability of behavior. high activity in such individuals is combined with rapid fatigue and self-doubt, which, under certain conditions, can create an addictive predisposition. they are characterized by increased irritability and offensiveness, hostility combined with conflict and hostility to others, especially expressed in frustration of urgent needs. in the men’s profiles with the mixed version of pe of bad, repressive positions occupy the scales of masculinity-femininity, overcontrol, emotional lability, anxiety and introversion, which are indicators of sexual behavior disorders, inhibition, loss of control over impulsivity, weakness of the emotional state. in general, it can be argued that the individual­psychological profiles of smmpr in men with mixed pe of bad are more closely related to the profiles of the patients with manic variant than with depressive one, although they have specific features. discussion in recent years, much attention is paid to the restoration of social functioning and the quality of life of the mentally ill. quality of life, which reflects the main aspects of the mental, social and physical functioning of the patient, is a key criterion for evaluation of the effectiveness of the health care in psychiatry [14]. thus, longitudinal studies revealed a significant decrease in quality of life in the patients with bad; it is established that the patients with 25 years of disease without adequate treatment can lose 9 years of life [15]. numerous studies have established significant social disadaptation of the patients with bad, i.e. reduced levels of social functioning, reduced professional status and material level, difficulties in personal and professional life [16]. a thorough study is needed for the clinical and psychopathological phenomenology of the primary episode of bad to determine the features of different variants of its course and to develop a system for predicting its clinical course. i m p rov i n g t h e s y s t e m o f d i a g n o s t i c measures in the primary episode of bad in order to improve the quality of treatment, ensure a stable remission and reduce disease recurrence, restore social functioning and quality of life for patients is of a topical scientific and practical matter [17]. thus, the most significant differences in the quantitative indicators of smmpr are established when comparing depressive and manic, as well as depressive and mixed variants of pe of bad, and lesser – when comparing manic fig. 3. standardized multifactor method of personality research profiles for men and women with mixed primary episode of bipolar affective disorder. fig. 3. standardized multifactor method of personality research profiles for men and women with mixed primary episode of bipolar affective disorder. in the profile of men with mixed pe of bad impulsiveness (4 scales) predominates, although the quantitative values of the indicators are smaller than in the manic variant of pe of bad. in men, increased (more than 65 t-points) indicators on the impulsivity scale are combined with increased indicators on the scales of individuality, hypomania and rigidity. such a pattern is a manifestation of the instability of the emotional state, a tendency to mood swings, unconformity, originality and non-template reactions to external challenges, a tendency to impulsiveness, conflict, psychopathization of personality traits. at the same time, the combination of contradictory traits is a constant source of intrapsychic conflicts, which is manifested by high excitability and dynamism combined with inertness and instability of behavior. high activity in such individuals is combined with rapid fatigue and self-doubt, which, under certain conditions, can create an addictive predisposition. they are characterized by increased irritability and offensiveness, hostility combined with conflict and hostility to others, especially expressed in frustration of urgent needs. in the men’s profiles with the mixed version of pe of bad, repressive positions occupy the scales of masculinity-femininity, overcontrol, emotional lability, anxiety and introversion, which are indicators of sexual behavior disorders, inhibition, loss of control over impulsivity, weakness of the emotional state. in general, it can be 35 45 55 65 75 1 2 3 4 5 6 7 8 9 0 va lu es , т -p oi nt s 1 – overcontrol; 2 – pessimism; 3 – emotional lability; 4 – impulsivity; 5 – masculinity-femininity; 6 – rigidity; 7 – anxiety; 8 – individualism; 9 – optimism; 0 – introversion all men women 74 p sy c h ia t r y issn 2413-6077. ijmmr 2019 vol. 5 issue 2 and mixed variants. most of all, these differences are expressed in pessimism, impulsiveness, individualism and optimism. understanding personal features of the patients with a primary episode of bar is an effective way of solving the problem of bar and is of great scientific and practical importance for its predicting, treating and preventing. conclusions patients with primary episode of bipolar affective disorder have some individual psychological characteristic features that have significant gender differences, as well as differ in different variants of the debut of the disease. in the depressive variant of primary episode of bipolar affective disorder, individually psychological pattern is characterized by a tendency to asthenic variant of reaction with prevalence of affiliation, anxiety, pessimism, desire for escapism and minimization of activity. in the manic version of primary episode of bad, the personality profile is characterized primarily by impulsivity, low level of reflectivity, aggressi­ veness and intolerance to the opinion of others. the mixed variant is characterized by the most complex and contradictory characteristics, reflecting the instability of emotional state with predominance of manifestations of impul siveness and rigidity in men, and depressive and anxious traits in women. gender differences are more significant in women with signs of pessimism, emotional lability, anxiety and introversion, and in men – of impulsiveness, rigidity and optimism. funding this research received no external funding. conflict of interests the author declares no conflict of interest. індивідуально-психологічні особливості хворих з первинним епізодом біполярного афективного розладу ю.і. мисула тернопільський національний медичний університет імені і. я. горбачевського, тернопіль, україна вступ. біполярний афективний розлад (бар) – надзвичайно актуальна проблема сучасної психіатрії. особливості первинного епізоду (пе) захворювання надзвичайно важливі для прогнозування та планування лікувально-реабілітаційних заходів бар. індивідуально-психологічні особливості хворих на пе бар залишаються невивченими, що ускладнює розробку сучасних методів прогнозування, лікування та профілактики даного розладу. мета. дослідити індивідуально-психологічні особливості хворих з первинним епізодом біполярного афективного розладу з урахуванням гендерного фактору і клінічного варіанту дебюту бар. методи. обстежено 153 пацієнта (65 чоловіків і 88 жінок) з пе бар. обстежуваних пацієнтів розділили на три групи залежно від клінічного варіанту перебігу пе бар: депресивний, маніакальний варіант та змішаний варіант. обстеження проведено з використанням стандартизованого багатофакторного методу дослідження особистості (смдо). статистична обробка отриманих даних проводилася з використанням непараметричного тесту манна-уїтні. результати. найбільші відмінності при порівнянні кількісних показників смдо виявлені при порівнянні депресивного і маніакального, та депресивного і змішаного варіантів пе бар, і менші – при порівнянні маніакального та змішаного варіантів. найбільшою мірою ці відмінності виражені для проявів песимістичності, імпульсивності, індивідуалістичності та оптимістичності. висновки. були знайдені особливості пацієнтів чоловіків та жінок з депресивними, маніакальними та змішаними варіантами пе бар, які можуть допомогти знайти методи прогнозування, лікування та профілактики бар. ключові слова: біполярний афективний розлад; індивідуально-психологічні особливості; депресивний, маніакальний та змішаний варіанти. yu.i. mysula 75 p sy c h ia t r y issn 2413-6077. ijmmr 2019 vol. 5 issue 2 yu.i. mysula references 1. mental health: strengthening our response (2018). information bulletin of world health organisation. geneva, who. 1-28. 2. patel r, shetty h, jackson r, broadbent m, stewart r, boydell j, et al. delays before diagnosis and initiation of treatment in patients presenting to mental health services with bipolar disorder. plos one. 2015;10:126­9. doi: 10.1371/journal.pone.0126530 3. chakrabarty t, alamian g, kozicky jm, ivan jt, lakshmi ny. cognitive functioning in first episode bipolar i disorder patients with and without history of psychosis. journal of affective disorders. 2018;227:109­16. doi: 10.1016/j.jad.2017.10.003 4. sam sp, nisha a, varghese pj. stressful life events and relapse in bipolar affective disorder: a cross-sectional study from a tertiary care center of southern india. indian journal of psychological medicine. 2019; 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population. ebiomedicine. 2015;2:1405­19. doi: 10.1016/j.ebiom.2015.09.006 received 11 september 2019; revised 23 october 2019; accepted 29 october 2019. this is open access article distributed under the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. відомості про автора мисула юрій ігорович, кандидат медичних наук, доцент кафедри психіатрії, наркології та медичної психології тернопільського національного медичного університету імені і.я. горбачевського моз україни, м. тернопіль, україна. information about author mysula yuriy, md, phd, associate professor of the department of psychiatry, necrology and medical psychology, i. horbachevsky ternopil national medical university, ternopil, ukraine. orcid 0000­0001­7443­5304, e­mail: yuramysula@gmail.com issn 2413-6077. ijmmr 2020 vol. 6 issue 2 59 p u b l ic h e a lt h a n d e p id e m io l o g y doi 10.11603/ijmmr.2413-6077.2020.2.11600 quality of life and well-being of population at the end of third phase of lockdown in india against the covid-19 pandemic h. rathi1, m. biyani2, m. malik3, *p. rathi4 1 – ruhs college of medical sciences, jaipur, india 2 – swai man singh medical college, jaipur, india 3 – pandit deendayal upadhyaya medical college, churu, india 4 – mahatma gandhi medical college and hospital, jaipur, india background. on march 24, 2020, a nationwide lockdown for 21 days was ordered by the government of india which was then extended till may 31, 2020. researchers have predicted lockdown is a necessary step to prevent covid-19 spread. however, others have also stated that it could cause serious damage to the economic, mental, social, and physical well-being of the people. objective. the aim of the study is to evaluate the impact of lockdown on the quality of life and well-being of the indians. methods. it is a cross sectional prospective web-based questionnaire study. a link (https://forms.gle/ px25vuahp5nxt88qa) was created. total 426 responses were received via that link and the data was included in the statistical analysis. results. our study revealed that during the lockdown 61.5% of the respondents were performing physical activities lesser than before. more than half responded they had a reduced financial satisfaction. most answers on emotional well-being and social-family wellbeing were also positive, but some responses showed disturbing too, like 22% felt anxious and nervous over half of the days. it was found in the study that physical, financial, emotional, mental, social and family wellbeing were disturbed during the lockdown and quality of life was also hampered. conclusion. though, may be nationwide lockdown was the most required action at that point of time to prevent virus spread, but our study revealed that uncertainty regarding its cure and management guidelines like lockdown and social distancing has badly affected quality of life and wellbeing of the population. keywords: pandemic; lockdown; covid-19; anxiety; well-being. *corresponding author: dr. priyanka rathi, associate professor, mahatma gandhi medical college and hospital, jaipur, 302022, india. e-mail: drpriyankarathi@gmail.com international journal of medicine and medical research 2020, volume 6, issue 2, p. 59-66 copyright © 2020, tnmu, all rights reserved h. rathi et al. introduction in december 2019, several cases of a disease having similar symptoms of pneumonia were reported in wuhan city of china [1]. world health organisation (who) defined this disease as covid-19. genetically this virus is similar to severe acute respiratory syndrome corona virus (sars-cov). sars cov-2 strain is the causative agent for covid-19. patients of covid-19 commonly present with symptoms of fatigue, cough, fever, myalgia, and diarrhoea. after china, this virus started spreading to the rest of the world. its mode of transmission is inhalation of infectious aerosol. reports revealed that covid-19 transmission is possible through infected human contact. due to interhuman transmission, soon it has become global health emergency worldwide. because of its spread in 144 countries across five continents, the world health organisation declared covid-19 as pandemic disease on march 12, 2020 [2]. by the end of november 2020 this pandemic has infected 70 million people worldwide and the number is increasing day by day. like the rest of the world covd-19 has been reported in india too. in india 9.3 million of population has been diagnosed with covid-19 positive by november 2020. meanwhile no drug therapy has been established for its prevention, control and cure till now. so, to deal with this pandemic strict quarantine and lockdown are considered to be a highly effective and important preventive measure by almost the whole world. following the footsteps, india also took the help of lockdown due to increasing number of cases of covid-19. nation underwent 4 phases of lockdown for nearly 70 days. issn 2413-6077. ijmmr 2020 vol. 6 issue 260 p u b l ic h e a lt h a n d e p id e m io l o g y the 1st phase of lockdown started on march 24, 2020, for 21 days. then, an increasing number of cases and severity of the disease forced government to further extend it into phase 2 for 19 days, and phase 3 for 14 days. from may 18 phase 4 was announced which was planned to end on may 31, 2020. strict guidelines were formulated to prevent its spread. only essential services like medical and groceries services were allowed to keep open. apart from the mentioned services everything else was closed. based on the number of the cases in particular region, country was divided into 3 zones during the lockdown: green, orange, red. green zone covered the areas with zero confirmed cases till date or no confirmed cases in the past 21 days. orange zone involved the areas, which reported a limited number of cases in the past and no surge of positive cases in recent times. red zone is for the areas or hotspots classified as those with the highest caseload. although, the lockdown was considered necessary to prevent covid-19 spread. during the period of lockdown indian residents are advised to stay at home. it hampered resident’s life style very much. some researchers stated this caused serious damage to emotional [3] economic, mental, psychological [4], social and physical well-being of the population. due to a prolonged lockdown and business closure, people experienced negative emotions, stress, aggressiveness and anxiety symptoms. so, this study was aimed to evaluate the impact of lockdown on the quality of life and wellbeing of indians. methods a cross-sectional web-based online survey was conducted for a period of two weeks starting just after the completion of third phase of lockdown in india, from may 25 to june 1, 2020. a survey link (https://forms.gle/ px25vuahp5nxt88qa) was created through a web-based google application of ‘google form’. all indian citizens above the age of 18 years old, who gave an informed consent for participation in the study, were included while nri and foreign citizens were excluded. participants were recruited by sending the survey link through various social network channels such as whatsapp, linkedin, insta gram, and facebook. the final sample was obtained using the snowball technique wherein each participant was requested to further circulate the survey link among their respective family members, friends, and colleagues. the obtained data were analysed. study tools counted in a pre-validated 47item online questionnaire, which was validated for relevance, clarity, simplicity, and ambiguity by using 4and 5-point content validity index. an informed consent document comprising the participant information sheet and informed consent form in hindi and english was suggested in the beginning of the questionnaire and only those participants, who gave their consents, were allowed further access to the questionnaire. the questions were in both languages in the questionnaire. the variables and instruments included in the questionnaire comprise the following: 1. section 1 with 13 questions on demographics of the participants including age, gender, marital status, edu cational and professional details, area of residence and its covid zone, and present state of health. 2. section 2 with 34 questions for the assessment of physical (02 questions), psy chological (09 questions), financial (07 questions), emotional (06 questions), and social and family well-being (05 questions) of the participants and their quality of life (05 questions). questions related to physical wellness were generated ad hoc. for psychological well-being among the participants two tools were used, i.e. the patient health questionnaire-2 (phq-2) [5] to screen for depression, and the generalized anxiety disorder scale (gad-7) [6] to screen for anxiety. both the tools consisted of 2 likert type questions, each with 4 response options ranging from 0-3. the phq-2 score ranged from 0-6 with 3 as the optimal cut point while the gad-7 score ranged from 0-21 with a score of 10 or higher indicates significant anxiety. financial well-being was evaluated using a modified cost-facit (version 2) consisting of 7 questions, 6 which were likert type questions with 5 response alternatives ranging from 0-4. facit-sp (version 4) was used to assess the physical, social/family and emotional well-being of the participants. for emotional well-being 6 likert type questions and for social and family well-being 5 likert type questions were asked, each with 5 response options ranging from 0-4. the who (five) well-being index (1998 version) consisting of 5 likert type questions having 6 possible options was used to evaluate the quality of life of the study participants during the lockdown. the raw score ranged from 0 to 25; 0 representing the worst possible and 25 representing the best possible quality of life. h. rathi et al. issn 2413-6077. ijmmr 2020 vol. 6 issue 2 61 p u b l ic h e a lt h a n d e p id e m io l o g y results a total of 426 responses were received v i a t h e s t u d y l i n k ( h t t p s : / / f o r m s . g l e / px25vuahp5nxt88qa) of ‘google form’. 421 participants gave their consent for participation and were included in the survey. their demographic details are depicted in table 1. 4.8% of the participants responded that they were suffering from chronic health problems, the details are depicted in fig. 1. physical well-being: 23.5% participants responded that during the lockdown, they were able to perform their routine physical activities as they used to do before the starting of lockdown, while 15% responded that they were not able to do so at all, and 61.5% could perform their routine physical activities lesser than before. health related problems due to changes in daily routine, like drowsiness, weight gain, etc. were experienced by 28.8% participants. financial wellbeing (cost facit (version 2)): regarding satisfaction with their current financial situation consequent to lockdown, majority of the participants (57%) responded table 1. demographic details of the study participants variables no. of responses (percentage) gender males females 267 (63%) 154 (36.3%) age 18-45 years old 45-60 years old above 60 years old 328 (97.32%) 8 (2.3%) 1 (0.38%) marital status unmarried married divorced/widowed 261 (61.5%) 158 (37.4%) 04 (1.1%) education graduate post-graduate high school intermediate 232 (54.8%) 154 (36.4%) 5.2% (22) 3.6% (15) occupation student service business housewife 190 (45.8%) 122 (29.3%) 53 (12.7%) 33 (7.9%) type of service not applicable private government 188 (46.2%) 126 (31%) 93 (22.9%) residence urban rural 295 (70.1%) 126 (29.9%) covid zone of the area of residence red zone orange zone green zone don’t know 186 (44.1%) 132 (31.1%) 97 (23.1%) 08 (1.7%) during lockdown, living with family initially stuck away then able to live with family away from family 296 (69.7%) 68 (15.8%) 59 (14.4%) whether profession is related to covid frontline fighting yes no 79 (19.2%) 343 (80.8%) whether suffering from any chronic health problem yes no 19 (4.8%) 402 (95.2%) preferred to stay home during lockdown because of fear of strict government action fear of getting infected pressure from family 30 (7.3 %) 342 (83%) 40 (9.7%) state of health at present excellent very good good fair poor 76 (18.4%) 145 (34.2%) 140 (33%) 51 (11.8%) 12 (2.3%) h. rathi et al. issn 2413-6077. ijmmr 2020 vol. 6 issue 262 p u b l ic h e a lt h a n d e p id e m io l o g y that they have a reduced satisfaction, 29.4% were satisfied, and 13.6% felt financially stressed. fig. 2 illustrates the responses of the participants to the cost facit likert questions on financial well-being. fig. 3 illustrates the responses of the participants to the facit-sp (version 4) likert questions on emotional wellbeing. fig. 4 illustrates the responses of the participants to the facit-sp (version 4) likert social and family well-being. psychological well-being: responses regarding mental well-being are shown in table 2. table 3 depicts the scores for phq-2, gad-7, and who well-being index. fig. 5 illustrates the responses of the participants on the quality of life (who well-being index (1998 version)). discussion the study population consisted of 426 participants: 421 participants were fulfilling inclusion and exclusion criteria. the present fig. 1. distribution of various chronic health problems among the study participants. • don’t know 08 (1.7%) during lockdown, living with • family • initially stuck away then able to live with family • away from family 296 (69.7%) 68 (15.8%) 59 (14.4%) whether profession is related to covid frontline fighting • yes • no 79 (19.2%) 343(80.8%) whether suffering from any chronic health problem • yes • no 19 (4.8%) 402 (95.2%) preferred to stay home during lockdown because of • fear of strict government action • fear of getting infected • pressure from family 30 (7.3 %) 342 (83%) 40 (9.7%) state of health at present • excellent • very good • good • fair • poor 76 (18.4%) 145 (34.2%) 140 (33%) 51 (11.8%) 12 (2.3%) 4.8% of the participants responded that they were suffering from chronic health problems, the details are depicted in fig. 1. fig. 1. distribution of various chronic health problems among the study participants. physical well-being: 23.5% participants responded that during the lockdown, they were able to perform their routine physical activities as they used to do before the starting of lockdown, while 15% responded that they were not able to do so at all, and 61.5% could perform 17,70% 23,60% 5,90% 11,80% 11,80% 17,60% asthma diabetes mellitus ophthalmic problem hypertension systemic lupus erythematosus heart disease fig. 2. participants responses (0 – not at all, 1 – a little bit, 2 – somewhat, 3 – quite a bit, 4 – very much) to statements on financial well-being in cost facit (version 2) their routine physical activities lesser than before. health related problems due to changes in daily routine, like drowsiness, weight gain, etc. were experienced by 28.8% participants. financial wellbeing (cost facit (version 2)): regarding satisfaction with their current financial situation consequent to lockdown, majority of the participants (57%) responded that they have a reduced satisfaction, 29.4% were satisfied, and 13.6% felt financially stressed. fig. 2 illustrates the responses of the participants to the cost facit likert questions on financial well-being. fig. 2. participants responses (0 – not at all, 1 – a little bit, 2 – somewhat, 3 – quite a bit, 4 – very much) to statements on financial well-being in cost facit (version 2) fig. 3 illustrates the responses of the participants to the facit-sp (version 4) likert questions on emotional wellbeing. 11,20% 14,10% 22,20% 16,70% 14,70% 21,80% 23,90% 29,30% 24,30% 21,70% 24,60% 26,90% 29,60% 23,50% 28,20% 30% 33,30% 28,90% 25,50% 18,20% 16,40% 15,70% 16,90% 13,80% 9,80% 14,90% 8,90% 15,90% 10,40% 8,50% 0% 10% 20% 30% 40% i think lockdown has affected the financial situation of household i worry about the finacial problem that i will have in the future as a result of the lockdown due to covid i feel frustrated that i cannot work or contribute as much as i usually do i have been concerned about keeping my job or study and income i feel in control of my financial situation the lockdown due to covid has been a financial hardship to me and my family 4 3 2 1 0 h. rathi et al. issn 2413-6077. ijmmr 2020 vol. 6 issue 2 63 p u b l ic h e a lt h a n d e p id e m io l o g y fig. 3. participants responses (0 – not at all, 1 – a little bit, 2 – somewhat, 3 – quite a bit, 4 – very much) to statements on emotional well-being in facit-sp (version 4). fig. 4. participants responses (0 – not at all, 1 – a little bit, 2 – somewhat, 3 – quite a bit, 4 – very much) to statements on social and family well-being in facit-sp (version 4). fig. 3. participants responses (0 – not at all, 1 – a little bit, 2 – somewhat, 3 – quite a bit, 4 – very much) to statements on emotional well-being in facit-sp (version 4). fig. 4 illustrates the responses of the participants to the facit-sp (version 4) likert social and family well-being. 28,90% 14,40% 43,10% 39,60% 50% 44,60% 30,50% 18,30% 19% 21,60% 17,70% 21,80% 21% 27,90% 21,20% 23,33% 19,90% 21,10% 14,30% 23,10% 12,50% 11% 8,90% 7,90% 5,30% 16,30% 4,10% 4,60% 3,60% 4,60% 0% 10% 20% 30% 40% 50% 60% i feel sad i am satisfied with how i am coping with lockdown i am losing hope in the fight against covid i feel nervous i worry about dying i worry that my condition will get worse 4 3 2 1 0 fig. 4. participants responses (0 – not at all, 1 – a little bit, 2 – somewhat, 3 – quite a bit, 4 – very much) to statements on social and family well-being in facit-sp (version 4). psychological well-being: responses regarding mental well-being are shown in table 2. table 2. generalized anxiety disorder scale (gad-7) not at all (0) several days (1) over half the days (2) nearly every day (3) feeling nervous, anxious or on edge 37.8 % 34% 22% 6.2% not being able to stop or control worrying 35.2% 33.5% 22% 9.3% worrying too much about different things 35.2% 32.3% 23% 9.6% trouble relaxing 39.1% 31.2% 22.8% 7% being so restless that it’s hard to sit still 46.4% 26.8% 21.5% 5.3% becoming easily annoyed or irritable 37.2% 30.9% 23.7% 8.2% feeling afraid as if something awful might happen 38.8% 33.1% 20.1% 7.9% table 3 depicts the scores for phq-2, gad-7, and who well-being index. table 3. population distribution of phq-2, gad-7, and who well-being index score 10,10% 3,40% 2,90% 2,70% 6,10% 21,90% 12,50% 13,50% 13,30% 13,50% 32,70% 21,90% 28% 22,90% 25,10% 21,90% 23,30% 28,40% 23,40% 22,60% 13,50% 38,90% 27,20% 37,70% 32,70% 0,00% 10,00% 20,00% 30,00% 40,00% 50,00% i feel close to my friends i get emotional support from my family i get support from my friends i am satisfed with my family communication i feel close to my partner (or the person who is my main support) 4 3 2 1 0 study emphasizes to all types of well-being (physical, psychological, financial, emotional, social, family and quality of life) of indian population during the lockdown due to spread of covid-19. although, the lockdown was thought to be the most effective way to prevent the spread of covid-19, it has also negatively affected the quality of life and well-being of population. people faced a lot of physical health related problems during this period. 61.5% study population responded that they were not able to perform their physical activities as before, 28.8 % of the participants felt physical changes in their body. majority of the participants (39.1%) felt lesser interest in doing things for several days. similarly, 33.7% felt down depressed for hopeless for several days during h. rathi et al. issn 2413-6077. ijmmr 2020 vol. 6 issue 264 p u b l ic h e a lt h a n d e p id e m io l o g y this period. this pandemic crisis did not only affected the physical well-being but also people were more worried about the financial situation and its impact on their financial status in future. related to financial well-being, 57% of the population responded that they had a reduced satisfaction with their financial situation and 13.6% felt financially stressed because of this. out of total, 15.9 % were worried about keeping their job or study which was not only harmful for their financial well-being but also more likely to affect their mental well-being. majority (28.9%) agreed on the statement that it was somewhat of financial hardship to them and their families. table 2. generalized anxiety disorder scale (gad-7) not at all (0) several days (1) over half the days (2) nearly every day (3) feeling nervous, anxious or on edge 37.8 % 34% 22% 6.2% not being able to stop or control worrying 35.2% 33.5% 22% 9.3% worrying too much about different things 35.2% 32.3% 23% 9.6% trouble relaxing 39.1% 31.2% 22.8% 7% being so restless that it’s hard to sit still 46.4% 26.8% 21.5% 5.3% becoming easily annoyed or irritable 37.2% 30.9% 23.7% 8.2% feeling afraid as if something awful might happen 38.8% 33.1% 20.1% 7.9% table 3. population distribution of phq-2, gad-7, and who well-being index score name of score score range no. of responses (percentage) phq-2 3–6 0–2 90 (21.12%) 336 (78.88%) gad-7 10 or higher below 10 135 (31.66%) 291 (68.34%) who well-being index 13 or higher below 13 275 (64.56%) 151 (35.44%) fig. 5. participants responses (0 – at no time, 1 – some of the time, 2 – less than half of the time, 3 – more than half of the time, 4 – most of the time, 5 – all the time) to statements on quality of life (who well-being index (1998 version)). name of score score range no. of responses (percentage) phq-2 36 0-2 90 (21.12%) 336 (78.88%) gad-7 10 or higher below 10 135 (31.66%) 291 (68.34%) who well-being index 13 or higher below 13 275 (64.56%) 151 (35.44%) fig. 5 illustrates the responses of the participants on the quality of life (who well-being index (1998 version)). fig. 5. participants responses (0 – at no time, 1 – some of the time, 2 – less than half of the time, 3 – more than half of the time, 4 – most of the time, 5 – all the time) to statements on quality of life (who well-being index (1998 version)). discussion 0 0% 0% 0% 0% 12,70% 12,10% 10,10% 9,90% 20,40% 16,10% 22,50% 19% 21,90% 32,90% 28,30% 30,80% 31% 29,50% 26,40% 26,30% 24,50% 24% 11,80% 10,90% 15,60% 13,20% 0 0,05 0,1 0,15 0,2 0,25 0,3 0,35 i have felt cheerful and in good spirits i have felt calm and relaxed i have felt active and vigorous i woke up feeling fresh and rested my daily life has been filled with things that interest me0 1 2 3 4 5 10,10% 10,80% 29,30% h. rathi et al. issn 2413-6077. ijmmr 2020 vol. 6 issue 2 65 p u b l ic h e a lt h a n d e p id e m io l o g y in our study the most positive responses were observed on emotional well-being. most answers on emotional well-being were having zero score which indicates population had great control on emotional situations. in similar way, the sections of social and family wellbeing were also answered very positively. the participants (38.9%) were very much happy with the support by their families and 37.7% felt satisfied with their communication with families. 32.7% felt very much close to their partners. emotional social and family wellbeing were responded positively by the majority of the participants, but the responses by some of the participants to several questions for disturbing too: 22% felt anxious and nervous over half of the days. they were not able to stop worrying. nearly same percentage of the population felt too much worrying and they were feeling so restless that it was hard for them to sit or relax. almost the same percentage of the population (23.7%) became easily annoyed or irritable. they (20.1%) felt afraid as a something awful might happen. however, these types of responses were given by 22% of population but still it is disturbing because it may have led to mental stress and mental health related issues to them. out of total population, 29 to 32% felt cheerful, calm, relaxed, active, vigorous and fresh for more than half of the time. nevertheless, the results are not encouraging in terms of overall wellbeing of the population. our findings indicate the need of serious attention on the quality of life and wellbeing of the population due to changes in lifestyle during the covid-19 lockdown. it would be a huge challenge for not only the individuals to regain their physical, financial, emotional, mental, social and family wellbeing again but also for the government of india to re-establish the financial condition of the country by coping this pandemic crisis. limitations. although, our study tried to involve po pulation of all social economic and educational status, due to web-based study it wasn’t feasible for the individuals of all socioeconomic status to take part in this study. apart from this, shorter time span was also its one of the limitations. so, studies including larger sample size can be conducted. conclusion our findings suggest that lockdown has affected various aspects of life of each and every individual of the country. people are dealing this pandemic with all their efforts but an xiety regarding future is making them weaker. uncertainty of prevention and treatment of sars-cov2 is the major drawback to keep up the good spirit. the lockdown has the transient benefit for prevention of spread but not a permanent solution to this problem. conflict of interests authors declare no conflict of interest regarding this study. funding this research received no external funding. acknowledgements there had been no funding supports for carrying this research. the total cost of completing the research work was carried by authors’ own finance. author’s contributions heena rathi, priyanka rathi – conceptualization, methodology, formal analysis, writing – original draft, writing – reviewing and editing; heena rathi, mohit biyani – data curation, writing – reviewing and editing; manisha malik, mohit biyani – investigation; manisha malik – formal analysis. якість життя населення індії в кінці третього локдауну, спричиненого пандемією covid-19 h. rathi1, m. biyani2, m. malik3, *p. rathi4 1 – ruhs college of medical sciences, jaipur, india 2 – swai man singh medical college, jaipur, india 3 – pandit deendayal upadhyaya medical college, churu, india 4 – mahatma gandhi medical college and hospital, jaipur, india вступ. 24 березня 2020 року уряд індії впровадив загальнодержавний локдаун на 21 день, який потім було продовжено до 31 травня 2020 року. дослідники передбачають, що обмеження пересування є необхідним кроком для запобігання поширенню covid-19. однак також відомо, що це може завдати серйозної шкоди економічному, психічному, соціальному та фізичному благополуччю людей. h. rathi et al. issn 2413-6077. ijmmr 2020 vol. 6 issue 266 p u b l ic h e a lt h a n d e p id e m io l o g y мета. завдання цього дослідження – оцінити вплив локдауну на якість життя та добробут населення індії. методи. дослідження проводилося методом проспективного поперечного перерізу шляхом вебопитування. було створено посилання (https://forms.gle/px25vuahp5nxt88qa). всього було отримано 426 відповідей за посиланням, і ці дані були проаналізовані. результати. дослідження показало, що 61,5% респондентів мали менше фізичне навантаження під час локдауну. ще 57% відповіли, що не задоволені своїм фінансовим становищем. відповідь більшості мала позитивне забарвлення щодо емоційного благополуччя та соціально-сімейного благополуччя, але відповіді деяких опитаних мали тривожний характер: 22% відчували занепокоєння та нервозність протягом майже всього періоду локдауну. наше дослідження виявило, що фізичне, фінансове, емоційне, психічне, соціальне та сімейне благополуччя порушуються під час локдауну, також страждає якість життя. висновок. незважаючи на те, що загальнодержавний локдаун, можливо і був найбільш необхідною дією для запобігання розповсюдженню вірусу, але наше дослідження показало, що невизначеність відносно лікування інфекції та рекомендацій щодо локдауну та соціального дистанціювання, мала значний вплив на якість життя та добробут населення. ключові слова: пандемія; локдаун; covid-19; тривога; якість життя. information about the authors heena rathi, b.d.s, msc. (med-pharmacology) 3rd year, ruhs college of medical sciences, jaipur, india orcid 0000-0002-8056-8768, e-mail: drheenarathi@gmail.com mohit biyani, m.b.b.s, m.d (anaesthesia) 2nd year, swai man singh medical college, jaipur, india orcid 0000-0002-5543-6480, e-mail: mohit2.ncity@gmail.com manisha malik, associate professor, pandit deendayal upadhyaya medical college, churu, india orcid 0000-0001-8288-4206, e-mail: drmanishamalik@gmail.com priyanka rathi, associate professor, mahatma gandhi medical college and hospital, jaipur, india orcid 0000-0002-6400-6888, e-mail: drpriyankarathi@gmail.com references 1. maugeri g, castrogiovanni p, battaglia g, pippi r, d’agata v, palma a, di rosa m, musumeci g. the impact of physical activity on psychological health during covid-19 pandemic in italy. heliyon. 2020 jun 1;6(6):e04315. doi: https://doi.org/10.1016/j.heliyon.2020. e04315 2. lesser ia, nienhuis cp. the impact of covid-19 on physical activity behavior and well-being of canadians. 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internal medicine. 2006 may 22;166(10):1092-7. doi: https://doi.org/10.1001/archinte.166. 10.1092 received 14 dec 2020; revised 23 dec 2020; accepted 28 dec 2020. this is open access article distributed under the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. h. rathi et al. 33 r a d ia t io n m e d ic in e a n d o n c o l o g y issn 2413-6077. ijmmr 2019 vol. 5 issue 1 doi 10.11603/ijmmr.2413-6077.2019.1.10305 significance of additional chromosomal abnormalities for the outcomes after the second line nilotinib therapy in the chronic myeloid leukemia patients i.v. dmytrenko*, zh.m. minchenko, v.v. fedorenko, i.s. dyagil national research center for radiation medicine of the national academy of medical sciences of ukraine, kyiv, ukraine background. there is limited information about impact of additional chromosome aberrations (aca) on the efficacy of the 2nd line nilotinib therapy. objective. the aim of the study was to analyze significance of acas for the outcome after second line tyrosine kinase inhibitors (tki) therapy with nilotinib in the chronic myeloid leukemia (cml) patients, who experienced previous imatinib therapy failure. methods. the cml patients in chronic phase treated with nilotinib after imatinib failure were analyzed for outcomes. results. among a total of 114 patients, 18 patients (15.8%) had acas at the beginning of the 2nd line therapy with nilotinib. seven patients (38.9%) of 18 had variant translocations and 11 patients (61.1%) had other chromosomal abnormalities in addition to t(9;22), known as clonal evolution. complete cytogenetic response (ccr) at 12 months was achieved in 37.5%, 42.8% and 45.5% (p=0.842) of patients with classic t(9;22) translocation, variant translocations and acas respectively. in the patients with variant translocations t(9;v;22) or clonal evolution treated with nilotinib after the imatinib failure, the ccr and major molecular response (mmr), event free survival (efs), progression free survival (pfs) and overall survival (os) rates did not differ from those in the cml patients with t(9;22) only. at the same time quantitative characteristics of leukemic and aca clones had prognostic value for ccr. the increased number of ph-positive cells and the number of cells with the aca at the start of nilotinib therapy reduced the probability of ccr. conclusions. higher nilotinib inhibitory activity compare with imatinib allows us to overcome imatinib resistance in the cml patients regardless of the aca presence at the beginning of nilotinib therapy. key words: additional chromosomal aberrations; chronic myeloid leukemia; nilotinib; second line of tki therapy; prognosis; resistance. *corresponding author: iryna v. dmytrenko, national research center for radiation medicine of the national academy of medical sciences of ukraine, 119/121 peremoha avenue, kyiv 03115, ukraine. e-mail: iryna.v.dmytrenko@gmail.com introduction reciprocal translocation t(9;22)(q34;q11.2), also known as ph-chromosome, leads to the formation of the bcr/abl1 fusion gene, which is considered to be the main pathogenetic event of chronic myeloid leukemia (cml) [1]. however, additional chromosomal abnormalities along with the classical translocation t(9;22) have been described in some patients with cml. these are usually either variant translocations or additional chromosome aberrations (acas). in case of variant translocation (t(9;v;22)), more than two chromosomes are involved in the formation of t(9;22). acas include any numerical or structural chromosome aberrations in addition to t(9;22) and are called clonal evolution. at the time of diagnosis, the frequency of acas is 5-10% in the patients with chronic phase cml and increases up to 30% and 80% in the patients with acceleration phase or blast crisis [2, 3]. a de-novo gain of aca during the therapy is also possible. it is established that acas confer a bcr/abl-independent mechanism of resistance to the therapy with tyrosine kinase inhibitors (tki) [4]. although, currently, there is no final consensus about the predictive value of acas, most authors point out that the outcome of imatinib-treated patients with acas is significantly lower than of those without acas [5, 6]. it has been proved that nilotinib (the second generation tki) is effective in patients with cml after imatinib failure [7, 8]. but the influence of nilotinib on leukemic clones with additional chromosomal abnormalities has not been determined. the objective of this study was to analyze the significance of acas for the outcome after second line tki therapy with nilotinib in the cml international journal of medicine and medical research 2019, volume 5, issue 1, p. 33-39 copyright © 2019, tnmu, all rights reserved i.v. dmytrenko et al. 34 r a d ia t io n m e d ic in e a n d o n c o l o g y issn 2413-6077. ijmmr 2019 vol. 5 issue 1 patients, who experienced previous imatinib therapy failure. methods a retrospective study of 114 cml patients in chronic phase treated with nilotinib after imatinib failure was conducted. all patients were monitored at the department of radiation onco-hematology and stem cell transplantation of the institute of clinical radiology at the national research center for radiation medicine from 2008 to 2018. written informed consents to use biomaterial for the research purposes were obtained from all patients. among a total of 114 patients, 49 (43%) were males and 51 (57%) – females. imatinib for the first line tki therapy was administrated at a dose of 400 mg/d with later escalation to 800 mg/d. the second line tki therapy was implemented with nilotinib dose 400 mg twice a day. cytogenetic analysis was conducted at the time of imatinib resistance conformation. karyo typing with standard gtg-banding was performed after a 24-hour bone marrow cells culture with no stimulation. chromosomes were classified according to the international system for human cytogenetic nomenclature [9]. molecular response was assessed in 3, 6 12 months and then every 6 months during nilotinib therapy by real-time polymerase chain reaction as previously reported [10]. a complete cytogenetic response (ccr), major molecular response (mmr) and deep molecular response (mr4) were defined as bcrabl1/abl ratios 1%, 0.1%, and 0.01% respectively, on the international scale. the first and second line tki therapy responses and resistance were defined according to the elnet 2013 recommendations [6]. the χ2 test and fisher’s exact test were used to compare categorical variables and the mann–whitney u-test – to compare continuous variables. the probability of overall survival (os), progression free survival (pfs), and event free survival (efs) were estimated by the kaplan-meier method and compared by the log-rank test. pfs was defined as survival without signs of progression (transformation into acceleration phase or blast crisis). efs was evaluated from the beginning of the 2nd line therapy with nilotinib to the loss of achieved ccr or mmr, progression or death for any cause. os was assessed from the beginning of the 2nd line therapy till the death of any cause during the study. the cumulative probability of ccr and mmr was evaluated by the kaplan– meier method from the beginning of the 2nd line therapy to the time of response. in this case, the data about the patients, who had not received any adequate response, with progressed disease or died, were censored at the latest observation date. univariate analyses (cox regression) were performed to evaluate the prognostic significance of additional chromosomal abnormalities. statistical analysis was performed using the spss for windows statistical software package (version 20.0). the results were considered significant at p<0.05. results the baseline characteristics of the patients are summarized in table 1. among 114 patients, who started the second line therapy with nilotinib, 101 (88.9%) were resistant to previous imatinib therapy. resistance was defined as failure to achieve ccr on the first line of therapy. twelve patients (10.5%) achieved only ccr, while 1 (0.9%) patient achieved mmr with previous imatinib therapy. any achieved therapy response was completely lost in all patients with ccr and mmr by the start of nilotinib. pathologic clone with classical t(9:22)(q34;q11) was detected in 5-100% of cells of all patients at the time of the table 1. cml patients’ characteristics at the start of the 2nd line therapy with nilotinib patient characteristics patients with only t(9;22) (n=96) patients with t(9;v;22) (n=7) patients with aca (n=11) р age, median (range), years 38 (18–66) 38 (34–50) 35 (20 – 52) 0.512 sex, men, n (%) 40 (41.7) 3 (42.9) 6 (54.5%) 0.950 median of prior imatinib treatment, (range), months 44.0 (4.0–106.0) 39,0 (13.0–48.0) 45.0 (5.0–106.0) 0.578 median of follow-up on the 2nd line therapy with nilotinib, (range), months 52.0 (4.0–106.0) 42.0 (5.0–97.0) 32.0 (1.0–79.0) 0.194 notes: cml – chronic myeloid leukemia; aca – additional chromosomal aberration i.v. dmytrenko et al. 35 r a d ia t io n m e d ic in e a n d o n c o l o g y issn 2413-6077. ijmmr 2019 vol. 5 issue 1 second-line therapy initiation. the median number of ph-positive cells with variant translocation or with an aca was 15% (range of 5-100%). the median duration of prior imatinib treatment was 43 months (range of 4 to 106 months), while the median duration of the second-line therapy monitoring was 47 months (range of 1 to 106 months). among a total of 114 examined patients, 18 patients (15.8%) had acas at the beginning of the 2nd line therapy with nilotinib. seven patients (38.9%) of 18 had variant translocations and 11 patients (61.1%) had other chromosomal abnormalities in addition to t(9;22). among the other 11, 8 patients had a single chromosomal abnormality in addition to t(9;22), while 3 patients had multiple chromosomal abnormalities. in the majority of patients with acas (7 out of 11, 63.6%), an additional translocation was a second ph-chromosome (table 2). the patients with an isolated t(9;22) translocation, variant translocations or aca were not significantly different by age, sex, and length of treatment before the beginning of the secondline therapy. in order to evaluate the effect of acas on response to the second-line tki therapy, the extent of tumor clone reduction in 12 months of therapy, the probability of ccr and mmr, as well as long-term outcomes (efs, pfs and os) were evaluated in all 114 patients with cml, treated with nilotinib after imatinib failure. the presence of acas and variant translocations at the beginning of the 2nd line therapy did not affect the rate of the tumor clone reduction. the ccr in 12 months (optimal response according to elnet2013) was achieved in 37.5%, 42.8% and 45.5% (p=0.842) of patients with classic t(9;22) translocation, variant translocations and acas respectively. in 12 months, about 50% of patients were resistant to the second line tki therapy (bcr/abl>10% in 12 months of the therapy according to elnet2013). the rate of resistance did not differ between the groups: 51.0%, 57.1% and 54.5% in the patients with classic t(9;22) translocation, variant translocations and acas, respectively (table 3). no differences in the rates of disease progression and lethal outcomes were found table 2. karyotypes of bone marrow cells with variant translocations and additional chromosomal aberrations in the cml patients at the beginning of nilotinib therapy (n=18) № sex number of ph+ metaphases, % number of metaphase with aca, % karyotype 1 f 95 100 46,хх,t(9;22;?)(q34;q11;?)[19] 46,хх[1] 2 f 100 100 46,хх,t(5;9;22)( q31;q34;q11)[20] 3 f 100 5 46,хх,t(9;22)(q34;q11)[19]/idem,+der(22)t(9;22) [1] 4 m 73 53,3 46,xy,t(9;22)(q34;q11)[3]/idem,+der(22)t(9;22)[8]/46,xy[4] 5 m 100 100 46,xy,t(6;9;22)(р22;q34;q11)[20] 6 f 100 100 46,хх,der(9)t(9;?;22)(q34;?;q11)[20] 7 m 85 5 46,хy,t(9;22)(q34;q11)[5]/ idem,+der(22)t(9;22) [1]/46,хy[14] 8 m 100 70 46,хy,t(9;22)(q34;q11)[6]/idem,+der(22)t(9;22)[11]/ idem,+der(22)t(9;22),+7[3] 9 m 100 100 47,xy,t(9;22)(q34;q11),+der(22)t(9;22)[14]/ idem,+12[2] 10 f 50 42,5 47,xx,t(9;22)(q34;q11),+der(22)t(9;22)[17]/ 46,xx,t(9;22) (q34;q11)[13]/46,xx[20] 11 m 30 30 46, хy,t(3;9;22)(р25;q34;q11)[6] /46,хy[14] 12 f 100 30 47,хх,t(9;22)(q34;q11),+3[6]/ 46,хх,t(9;22)(q34;q11)[14] 13 m 20 100 46~48,xy,t(9;22)(q34;q11)[4],del(10)(p11.2)[5], +12[2],+13[12],+14[2]+15[4] [cp20] 14 f 100 100 46,хх,t(3;21)(q26.2;q22),t(9;22)(q34;q11)[20] 15 m 100 100 46,хy,t(9;21;22;)(q34; q22.2;q11)[20] 16 f 100 5 46,хх,t(9;22)(q34;q11)[19]/idem,+der(22)t(9;22)[1] 17 m 90 5 46,хy,t(9;22)(q34;q11)[17]/ idem,+der(22)t(9;22)[1]/46,xy[2] 18 f 100 100 46,хх,t(1;9;22)(q21.2;q34;q11)[20] notes: aca – additional chromosomal aberration. i.v. dmytrenko et al. 36 r a d ia t io n m e d ic in e a n d o n c o l o g y issn 2413-6077. ijmmr 2019 vol. 5 issue 1 between the patients with classic t(9;22) translocation, variant translocations and acas (table 3). for the whole follow-up period, the only case of progression to accelerated phase with a lethal outcome took place in one patient with variant translocation of unknown origin t(9;?;22)(q34;?;q11) and in one patient with three related clones: 30% metaphases with isolated ph-chromosome, 55% metaphases with an additional ph-chromosome, and 15% of metaphases with two ph-chromosomes and an extra chromosome 7. karyotype 46,хy,t(9;22) ( q 3 4 ; q 1 1 ) [ 6 ] / i d e m , + d e r ( 2 2 ) t ( 9 ; 2 2 ) [ 1 1 ] / idem,+der(22)t(9;22),+7[3]. kaplan-meier analysis of the long-term outcomes of the 2nd line nilotinib therapy proved an absence of significant influence of the aca and variant translocation status at the beginning of the 2nd line nilotinib therapy on the three-year efp, pfs and os (table 4). despite the acas at the beginning of the 2nd line therapy did not affect the nilotinib efficacy, quantitative characteristics of the leukemic clone did have prognostic value. a positive correlation between the amount of ph-positive clone before the beginning of the 2nd line nilotinib therapy and development of resistance (bcr/abl1>10%) in 12 months of the subsequent therapy (spearman correlation coefficient r=0.423, p<0.001) was found. the increase in the number of ph-positive cells at the beginning of the nilotinib therapy and increase in size of the additional clone (percent metaphases with aca) was associated with the decreased efficiency of tumor clone reduction during the therapy, or more specifically, statistically significant decrease in the rate of ccr (p<0.001, p=0.049). in other words, the efficacy of the second line tki therapy in the imatinib-resistant cml patients was associated with the level of reduction of the tumor clone and the size of the clone with additional aberrations before the beginning of the second-line therapy (table 5). discussion studies of the aca prognostic value both in the era before the tki and after approving of the first tki imatinib indicate that the aca table 3. response to the 2nd line nilotinib therapy in the cml patients depending on the presence of additional chromosomal aberrations characteristics patients with only t(9;22) (n=96) patients with t(9;v;22) (n=7) patients with aca (n=11) р response in 12 months of the 2nd line nilotinib therapy: ccr (bcr/abl1≤1%), n (%) mmr (bcr/abl1≤0.1%), n (%) bcr/abl1>10%, n (%) 36 (37.5) 13 (13.5) 49 (51.0) 3 (42.8) 1 (14.3) 4 (57.1) 5 (45.5) 3 (27.3) 6 (54.5) 0.842 0.516 0.938 secondary resistance: mmr loss, n (%) ccr loss, n (%) progression to ap or bc, n (%) 4/30 (13,3) 4/46 (8.7) 19 (19.8) 0/2 (0) 0/4 (0) 1 (14.3) 1/5 (20) 0/5 (0) 2 (18.2) 0.829 0.656 0.934 number of lethal outcomes, n (%) 17 (17.7) 1 (14.3) 1 (9.1) 0.756 notes: cml – chronic myeloid leukemia; aca – additional chromosomal aberration; ccr – complete cytogenetic response; mmr – major cytogenetic response, ap – acceleration phase; bc – blast crisis. table 4. long-term outcomes of the 2nd line nilotinib therapy in the cml patients depending on the presence of additional chromosomal aberrations outcomes patients with only t(9;22) (n=96) patients with t(9;v;22) (n=7) patients with aca (n=11) р 3-year еfs, % (95% cі) 80.1 (71.5–88.7) 80.0 (45.5–100.0) 74.1 (42.5–100.0) 0.468 3-year pfs, % (95% cі) 86.4 (79.1–93.7) 80.0 (45.5–100.0) 76.2 (47.2–100.0) 0.936 3-year оs, % (95% cі) 90.7 (84.6–96.8) 80.0 (45.5–100.0) 85.7 (59.8–100.0) 0.975 notes: cml – chronic myeloid leukemia, aca – additional chromosomal aberration, ci – confidence interval, efs – event free survival, pfs – progression free survival, os – overall survival. i.v. dmytrenko et al. 37 r a d ia t io n m e d ic in e a n d o n c o l o g y issn 2413-6077. ijmmr 2019 vol. 5 issue 1 presence in the therapy worsens the treatment results and decreases patient survival [11–13]. s o m e d i s a g r e e m e n t s a r e o b s e r v e d i n assessment of the aca significance at the time of diagnosis. alhuraiji a., et al. in the prospective clinical study on 603 cp cml patients pointed out that the presence of aca at the time of diagnosis was not associated with worse prognosis [14]. fabarius, et al. consider that o n l y m a j o r ro u t e , b u t n o t b a l a n c e d o r unbalanced minor route aca has a negative impact on prognosis of cml [13]. wang et al. also underline that variability in outcomes is related to the type of aca, but they suggest another aca stratification system. ‘poor prognosis’ for aca including i(17)(q10), -7/ del7q, and 3q26.2 rearrangements as well as presence of 2 or more acas conferred a worse survival irrelevant to the emergence phase and time. in contrast, acas in group of ‘good prognosis’ including trisomy 8, -y, and an extra copy of ph-chromosome had no adverse impact on survival, when they emerged at the chronic phase or at the time of cml diagnosis [15]. nilotinib is more potent and selective atpcompetitive inhibitor of bcr/abl1 tyrosine kinase than imatinib and it is more active against imatinib-resistant mutant forms of enzyme [16, 17]. multicenter trials and a real-life longitudinal proved nilotinib efficacy in the cml patients with imatinib failure [8, 18]. our report presents outcomes after the second line therapy with nilotinib in the cml patients with imatinib failure depending on the presence of variant translocations or clonal evolution at the beginning of the 2nd line therapy with nilotinib. among a total of 114 examined patients, 18 patients (15.8%) had clonal evolution at the beginning of nilotinib. seven patients (38.9%) of 18 had variant translocations and 11 patients (61.1%) had c l o n a l ev o l u t i o n – a d d i t i o n a l t o t ( 9 ; 2 2 ) chromosomal abnormalities. our results suggest that for the patients with variant translocations t(9;v;22) or clonal evolution treated with nilotinib after imatinib failure, the ccr and mmr response, efs, pfs and os rates do not differ from those of the cml patients with t(9;22) only. at the same time quantitative characteristics of leukemic and aca clones have a significant impact on ccr. the greater number of ph-positive cells and the number of cells with the aca are revealed at the beginning of nilotinib, the less – for ccr probability. efs is also worse with increasing number of phpositive cells at the beginning of nilotinib. the significance of certain types of aca for longterm outcomes has not been investigated because of small groups of patients with different acas. conclusions the presence of aca provides the tumor with heterogeneous properties that determine its sensitivity to therapy. but higher inhibitory activity of nilotinib compare to the imatinib allows overcoming imatinib resistance in the cml patients regardless of the aca presence and achieves ccr in about 50 % of patients in 12 months of nilotinib therapy. investigation of prognostic value of the combination aca with other tumor clone features advantages development of criteria for the individual selection of tki in the resistant patients. conflicts of interest authors declare no conflict of interest. table 5. estimation of the significance of aca clones quantitative characteristics in the cml patients (n=18) at the beginning of the 2nd line nilotinib therapy for the probability of ccr, efs, pfs and os by the univariate cox regression analyses characteristics ccr еfs рfs os exp(b) (95% ci) р exp(b) (95% ci) р exp(b) (95% ci) р exp(b) (95% ci) р % ph+ metaphases 0.981 (0.971–0.990) <0.001 1.027 (1.00–1.054) 0.039 1.037 (1.000–1.076) 0.052 1.062 (0.997–1.132) 0.062 % metaphases with aca 1.041 (1.000–.083) 0.049 1.028 (0.979–1.080) 0.268 1.031 (0.979–1.085) 0.248 1.025 (0.973–1.080) 0.354 notes: cml – chronic myeloid leukemia; aca – additional chromosomal aberration; ci – confidence interval; efs – event free survival; pfs – progression free survival; os – overall survival. i.v. dmytrenko et al. 38 r a d ia t io n m e d ic in e a n d o n c o l o g y issn 2413-6077. ijmmr 2019 vol. 5 issue 1 значення додаткових хромосомних аберацій у формуванні відповіді на другу лінію терапії нілотинібом у хворих на хронічну мієлоїдну лейкемію і.в. дмитренко, ж.м. мінченко, в.г. федоренко, і.с. дягіль ду “національний науковий центр радіаційної медицини національної академії медичних наук україни”, київ, україна вступ. існує обмежена інформація про вплив додаткових хромосомних аберацій (дха) на ефективність терапії нілотінібом другої лінії у хворих з хронічною мієлодною лейкемією (хмл). мета: проаналізувати значущість дха щодо відповіді на терапію інгібіторами тирозинкіназ (ітк) другої лінії (нілотинібом) у хворих на хмл, у яких терапія іматинібом була неефективною. методи. аналізували ефективність терапії нілотинібом у хворих на хмл в хронічній фазі, у яких терапія іматинібом була неефективною. результати. серед 114 пацієнтів 18 пацієнтів (15,8%) мали дха на початку 2-ї лінії терапії нілотінібом. сім пацієнтів (38,9%) з 18 мали варіантні транслокації, а 11 пацієнтів (61,1%) мали інші хромосомні аномалії, крім t(9;22), тобто ознаки клональної еволюції. повна цитогенетична відповідь (пцв) через 12 місяців терапії нілотинібом була досягнута у 37,5%, 42,8% і 45,5% (p=0,842) пацієнтів з класичною транслокацією t(9;22), варіантними транслокаціями та клональною еволюцією відповідно. у пацієнтів з варіантнимии транслокаціями t(9;v;22) та клональною еволюцією швидкість пцв та великої молекулярної відповіді (вмв), безподійна виживаність (efs), виживаність без прогресії (pfs) та загальна виживаність (оs) не відрізнялися від таких у хворих на хмл, у яких виявлялась тільки транслокація t(9;22). в той же час, кількісні характеристики лейкемічного клону та клону з дха мали прогностичне значення щодо досягнення пцв. збільшення кількості ph-позитивних клітин та клітин з дха на початку терапії нілотинібом зменшувало вірогідність досягнення пцв. висновки. більш висока інгібуюча активність нілотинібу в порівнянні з іматинібом дозволяє подолати резистентність до іматинібу у пацієнтів з хмл незалежно від наявності дха на початку терапії нілотинібом. ключові слова: додаткові хромосомні аберації; хронічна мієлоїдна лейкемія; нілотиніб; друга лінія терапії ітк (інгібітори тирозинкіназ); прогнозування; резистентність. інформація про авторів дмитренко ірина віталіївна – канд. біол. наук, старший науковий співробітник лабораторії імуногенетики відділу гематології та трансплантології, ду “національний науковий центр радіаційної медицини національної академії медичних наук україни”. мінченко жанна миколаївна – д-р біол. наук, професор, завідувач лабораторії імуногенетики відділу гематології та трансплантології, ду “національний науковий центр радіаційної медицини національної академії медичних наук україни”. федоренко віра григорівна – молодший науковий співробітник лабораторії імуногенетики відділу гематології та трансплантології, ду “національний науковий центр радіаційної медицини національної академії медичних наук україни”. дягіль ірина сергіївна – д-р мед. наук, завідувач відділення радіаційної онкогематології та трансплантації стовбурових клітин відділу гематології та трансплантології, ду “національний науковий центр радіаційної медицини національної академії медичних наук україни”. information about the authors iryna v. dmytrenko – ph.d., senior researcher, immunogenetic laboratory, hematology and transplantology department, state institution “national research center for radiation medicine of the national academy of medical sciences of ukraine”, kyiv, ukraine. orcid 0000-0002-2136-5442, e-mail: iryna.v.dmytrenko@gmail.com zhanna m. minchenko – doctor of biological science, professor, head of the immunogenetic laboratory, hematology and transplantology department, state institution “national research center for radiation medicine of the national academy of medical sciences of ukraine”, kyiv, ukraine. vira v. fedorenko – researcher, immunogenetic laboratory, hematology and transplantology department, state institution “national research center for radiation medicine of the national academy of medical sciences of ukraine”, kyiv, ukraine. iryna s. dyagil – doctor of medical science, head of the radiation oncohematology and transplantology department, state institution “national research center for radiation medicine of the national academy of medical sciences of ukraine”, kyiv, ukraine. i.v. dmytrenko et al. 39 r a d ia t io n m e d ic in e a n d o n c o l o g y issn 2413-6077. ijmmr 2019 vol. 5 issue 1 references 1. faderl s, talpaz m, estrov z, o'brien s, kurzrock r, kantarjian hm. the biology of chronic myeloid leukemia. n engl j med. 1999;341:164-172. doi: 10.1056/nejm199907153410306 2. zaccaria a, testoni n, valenti am, luatti s, tonelli m, marzocchi g et al. chromosome abnormalities additional to the philadelphia chromosome at the diagnosis of chronic myelogenous leukemia: pathogenetic and prognostic implications. cancer 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kantarjian hm, giles fj, bhalla kn, pinillaibarz j, larson ra, gattermann n et al. nilotinib is effective in patients with chronic myeloid leukemia in chronic phase after imatinib resistance or intolerance: 24-month follow-up results. blood. 2011;117(4):1141-1145. doi: 10.1182/blood-2010-03-277152 received 05 may 2019; revised 19 may 2019; accepted 05 june 2019. this is an open access article distributed under the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. i.v. dmytrenko et al. 26 in t e r n a l m e d ic in e issn 2413-6077. ijmmr 2020 vol. 6 issue 1 doi 10.11603/ijmmr.2413-6077.2020.1.11268 systemic oxidative stress as a surrogate of coronary atherosclerotic plaque instability and rupture predictor z. lominadze1, *k. chelidze2, l. chelidze2, e. lominadze2 1 – ltd clinic-lj, kutaisi, georgia 2 – tbilisi state medical university, tbilisi, georgia background. oxidative stress is crucial in developing broad spectrum of diseases, including atherosclerosis and related life-threatening conditions, such as acute coronary syndrome (acs) mainly caused by atherosclerotic plaque vulnerability. objective. to clarify the relation between oxidative stress and plaque instability we decided to compare oxidative profiles of patients with acute coronary syndrome (acs) and with chronic coronary syndrome (ccs), evaluated at admission to the coronary care unit (ccu) of ltd clinic-lj (kutaisi, georgia) in april 2018 june 2019, who underwent successful primary percutaneous coronary intervention (pci). methods. 191 patients were enrolled (100 patients with acs in group 1 and 91 patients with ccs in group 2) into the study. using the cr3000 form plus (callegari srl, catellani group, italy) – callegari point of care instrument we evaluated free oxygen radical test (fort), free oxygen radicals defense (ford), calculated redox index and the overall profile of oxidative stress. results. the mean/median concentration of free oxygen radicals was significantly higher in the patients with acs (404.37±9.83 fort units/2.36 mmol/l h2o2 eq. vs 282.34±9.83 fort units/2.36 mmol/l h2o2 eq., p<0.0001). significant correlation was found between advanced oxidative stress and acute coronary syndrome (or 14.42 95% ci (7.08-29.4), rr 3.26 95% ci (2.31-4.60) with high diagnostic characteristics (sensitivity of 82% and specificity of 92.3%; positive predictive value of 92% and positive likelihood ratio of 11). conclusion. oxidative stress is crucial in life-threatening acute coronary events. measurement of overall oxidative stress profile, as a surrogate of plaque instability and rupture predictor, could help the clinicians in risk stratification and prevention of acute coronary syndrome (acs). key words: acute coronary syndrome (acs); chronic coronary syndrome (ccs); oxidative stress; free oxygen radical test (fort); free oxygen radicals defense test (ford); redox index; oxidative stress profile; vulnerable plaque; plaque rupture. *corresponding author: kakhaber chelidze, the first university clinic of tbilisi state medical university (tsmu), 4 gudamakari str. tbilisi. 0141. e-mail: k.chelidze@tsmu.edu z. lominadze et al. introduction coronary heart disease (chd) has remained the leading cause of death globally in the last two decades. according to the world health organization global health observatory (gho) data ihd is the world’s biggest killer, accounting for 9.33 million deaths in 2016 [1]. acute coronary syndrome (acs) is a clinical manifestation of chd with variable consequences range from unstable angina (ua) to non-st elevation myocardial infarction (nstemi), stelevation myocardial infarction (stemi), and sudden cardiac death. acute coronary syndrome (acs) mainly as a result of plaque disruption in coronary arteries is responsible for one-third of total deaths in people older than 35 [2]. the main therapeutic approach to acs is focused on interventional techniques designed to restore blood flow in hemodynamically compromised coronary arteries. however, this reactive strategy has a weak preventive effect on future coronary events. although phar macotherapy with antiplatelet agents and statins does not have a dramatic risk-lowering effect as well [3,4]. in the last 10 years, a major improvement has been made in an effort to understand one of the main mechanisms of acs: the concept of vulnerable plaque as a cause of major ischemic events [5]. the term “vulnerable plaque” was originally used to define a plaque prone to rupture. however, apart from rupture the term “vulnerability” includes other types of lesion, such as plaque erosion, and plaque calcification [6]. the vulnerable plaque is made of a large lipid core (foam cells, apoptotic/necrotic cells, and debris) [7] which is separated from the lumen by a fibrous cap (mainly comprising collagen, proteoglycans, and smooth muscle cells) [8, 9]. weakening of the fibrous cup under different stressors and a lack of healing results international journal of medicine and medical research 2020, volume 6, issue 1, p. 26-34 copyright © 2020, tnmu, all rights reserved 27 in t e r n a l m e d ic in e issn 2413-6077. ijmmr 2020 vol. 6 issue 1 z. lominadze et al. in plaque fissuring, thrombus formation and, therefore, in acute coronary syndrome [10, 11]. many clinical studies support a crucial role for oxidative stress in cardiovascular diseases [12,13]. oxidative stress (imbalance between enhanced production of reactive oxygen species (ros) and enzymatic/nonenzymatic antioxidative potential accountable for oxidative modification of low-density lipoprotein cholesterol (ldlc) and cell damage (including endothelial cells of vessels) initiate fatty streak formation, lesion progression, and plaque rupture [14, 15]. it is very important to identify reliable surrogates of plaque instability and predict the highest risk of rupture. the present study intended to determine the oxidative status in patients with acute coronary syndrome (acs) and to compare with oxidative/antioxidative parameters in patients with stable ischemic heart disease (sihd). these findings could help assess the risk of stratification and prevent acute coronary syndrome (acs). materials and methods study patients the study sample consisted of 191 patients who were divided into two groups: group 1 – 100 patients with acute coronary syndrome (asc) and group 2 – 91 patients with chronic coronary syndrome (ccs) admitted to the coronary care unit (ccu) of ltd clinic-lj (kutaisi, georgia) in april 2018 june 2019, who under_ went successful primary percutaneous coronary intervention (pci). in the patients with chronic coronary syndrome (ccs) invasive coronary angiography with revascularization was performed in case of high clinical likelihood of obstructive coronary artery disease (ocad) and severe symptoms refractory to optimal medical treatment, or typical angina at a low level of exercise and clinical prediction of high-risk of events, or left ventricular dysfunction suggestive of cad. patients with a history of coronary revascularization, or with hemodynamically compromised severe myocardial infarction; those recovering cardiopulmonary arrest, decompensated heart failure; and those with valvular heart disease, cardiomyopathy, severe supraventricular/ventricular arrhythmias (including atrial fibrillation) and conductivity disturbances, end-stage renal disease (esrd), chronic inflammatory conditions, active cancer, type 1 diabetes mellitus (dm) or decompensated type 2 diabetes mellitus (dm); pregnancy; those on hormone replacement therapy (hrt) or oral contraceptive assumption were excluded from the study. no corrections or changes had been made in the ongoing pharmacotherapy of the patients. all essential laboratory tests and fort/ford assays were performed during the first hour of admission. the study was approved by the ethics committee (ec) of tbilisi state medical university (tsmu) and local ec of ltd clinic-lj and a written informed consent was provided by each study participants. assay of oxidative stress the enrolled patients’ oxidative status was assessed by measuring ros damage index (fort test) and antioxidant capacity (ford test), and redox index calculation using the cr3000 form plus (callegarisrl, catellani group, italy) – callegari point of care instrument with following technical characteristics: parameters measured and specificity: (i) free oxygen radical test (fort): overall organic radicals, e.g. hydroperoxides, (roohs)/reactive oxygen species (ros); (ii) free oxygen radicals defense (ford): plasmatic antioxidant compounds including vitamin c; proteins (e.g. albumin and ceruloplasmin); bilirubin; thiol groups (e.g. glutathione); polyphenolic compounds (e.g. flavonoids and tannins); (iii) oxidativereductive balance (redox index): overall score of the oxidation-reduction state. the index was expressed as a number (from 0 to 100) identifying 5 specific profiles (a-e). assay principle: (i) free oxygen radical test (fort): colorimetric method based on the fenton reaction; (ii) free oxygen radicals defense (ford): colorimetric method based on the quen ching of the color. reference range: (i) free oxygen radical test (fort): up to 310 fort units/2.36 mmol/l h2o2 eq; (ii) free oxygen radicals defense (ford): 1.07-1.53 mmol/l trolox eq.; sample type: whole blood; technique: point of care analysis via ready to use, wet, disposable reagents; wavelength: 505 nm. the five profiles of oxidative stress were determined by basal fort and ford values and redox index calculation: (i) profile a (ideal/ normal values): redox index: 0-25; fort <300 units/2.36 mmol/l h2o2 eq.; ford ≥1.08 mmol/l trolox eq; (ii) profile b (latent oxidative stress): redox index: 25-50; fort <300 units/2.36 mmol/l h2o2 eq; ford ≤1.07 mmol/l trolox eq.; (iii) profile c (compensated oxidative stress): redox index: 50-58.3; 300 < fort <330 units/2.36 28 in t e r n a l m e d ic in e issn 2413-6077. ijmmr 2020 vol. 6 issue 1 mmol/l h2o2 eq; ford ≥1.08 mmol/l trolox eq.; (iv) profile d (at risk of oxidative stress): redox index: 58.3-66.6; 300< fort <330 units/2.36 mmol/l h2o2 eq; ford ≤1.07 mmol/l trolox eq.; (v) profile e (oxidative stress in progress): redox index: 66.6-100; fort ≥331 units/2.36 mmol/l h2o2 eq; 0.25< ford <3.00 mmol/l trolox eq. statistical analysis the data were analyzed using ibm spss statistics version 26.0 (ibm corp., armonk, ny, usa). to identify oxidative status differences between two groups with abnormal distribution nonparametric tests were used: (i) mann– whitney u test for 2 samples; (ii) median test for k samples to compare medians across the groups, and (iii) moses extreme reaction for 2 samples to compare ranges across the groups. odds ratio (or) and relative risk (rr) assessment was used to quantify the strength of the association between oxidative stress and acute coronary syndrome (acs). for assessment of sensitivity/specificity, positive predictive value (ppv), negative predictive value (npv), likelihood ratio, youden’s j statistic, and prevalence crosstabulation analysis were used. the 95% confidence interval (ci) was used to estimate the precision of the or. the p value of 0.05 was considered significant. results study population characteristics clinical characteristics of the overall study population are shown in table 1. there was no statistically significant difference between the study population charac teristics, such as age, male gender, bmi, hypertension, dyslipidemia, ongoing smoking, type 2 dm, and medications, such as beta-blockers, calcium channel blockers, aceis or arbs, and statins. nitrates consumption was much higher in the patients with chronic coronary syndrome (p<0.0001). admission oxidative stress parameters fig. 1 depicts the level of baseline oxidative stress parameters evaluated in whole blood of group 1 of the patients with acute coronary syndrome (acs) and group 2 of the patients with chronic coronary syndrome (ccs). the concentration of free oxygen radicals (fort, fort units/2.36 mmol/l h2o2 eq) evaluated in two groups was as follows: in the patients with acs, 404.37±9.83 fort units/2.36 mmol/l h2o2 eq, and in the patients with ccs, 282.34±9.83 fort units/2.36 mmol/l h2o2 eq (p<0.0001). free oxygen radicals defense capacity evaluated by the ford test in the group 1 and group 2 were 1.37±0.035 mmol/l trolox eq. and 1.5±0.045 mmol/l trolox eq., respectively (p=0.03). the distribution analysis of the calculated redox index in the patients with acs was 69.2±1.47, and in the patients with ccs was 1.5±0.45 (p<0.0001). a nominal equivalent of stress profile in both groups distributed as follows: 4.56±0.1 and 1.92±0.13, respectively in the group 1 and group 2 (p<0.0001). there are reported results of nonparametric analysis of ros and antioxidative potential across the groups of patients with acs and ccs (fig. 2). th difference between all oxidative parameters was statistically significant (p<0.0001), except free oxygen radicals defense ford test (p=0.1). table 1. characteristics of study population group 1 (patients with acs) n=100 group 2 (patients with ccs) n=91 p value age (years) 51.8±0.78 49.1±1.02 0.236 male gender, n (%) 74 (74) 52 (57) 0.874 bmi 27.76±0.35 27.81±0.36 0.924 hypertension, n (%) 51 (51) 41 (45) 0.413 dyslipidemia, n (%) 62 (62) 56 (61.5) 0.948 smoking, n (%) 52 (52) 45 (49.5) 0.726 type 2 dm, n (%) 44 (44) 38 (41.8) 0.755 bb, n (%) 31 (31) 27 (29.7) 0.842 ccb, n (%) 36 (36) 28 (30.8) 0.446 aceis/arbs, n (%) 42 (42) 38 (41.8) 0.973 statins, n (%) 42 (42) 45 (49.5) 0.238 nitrates, n (%) 22 (22) 42 (46.2) <0.0001* notes. * statistically significant difference; bmi body mass index, bb beta-blockers, ccb calcium channel blockers, acseis angiotensin converting enzyme inhibitors, arbs angiotensin receptor blockers. z. lominadze et al. 29 in t e r n a l m e d ic in e issn 2413-6077. ijmmr 2020 vol. 6 issue 1 the evaluation results of association between an exposure (oxidative stress) and an outcome (acute coronary syndrome) are shown in table 2. fig. 3 depicts the stratification of patients in the two groups in line with systemic oxidative stress profile. a cross-tabulation analysis was performed to assess the sensitivity and specificity, positive and negative predictive values (ppv and npv, respectively), and positive and negative likefig. 1. distribution of baseline oxidative stress parameters across the groups by the independent-samples mann–whitney u test. notes. group 1, the patients with acute coronary syndrome (asc). group 2, the patients with chronic coronary syndrome (ccs). (a) distribution of fort, free oxygen radical test results across the groups (mann–whitney u=919, wilcoxon w=5105, test statistic=919, standard error=381.448, standardized test statistic=-9.519, asymptotic sig. (2-sided test) =.000); (b) distribution of ford, free oxygen radicals defense test result across the groups (mann–whitney u=5376, wilcoxon w=9562, test statistic=5376, standard error=381.480, standardized test statistic=2.165, asymptotic sig. (2-sided test) =.030); (c) distribution of calculated ri, redox index across the groups (mann-whitney u=751.5, wilcoxon w=4937.5, test statistic=751.5, standard error=381.173, standardized test statistic=-9.965, asymptotic sig. (2-sided test) =.000); (d) profile distribution across the groups (mann–whitney u=821, wilcoxon w=5007, test statistic=821, standard error=355.1, standardized test statistic=-10.501, asymptotic sig. (2-sided test) =.000). 6 fig. 1. distribution of baseline oxidative stress parameters across the groups by the independentsamples mann-whitney u test. notes. group 1, the patients with acute coronary syndrome (asc). group 2, the patients with chronic coronary syndrome (ccs). (a) distribution of fort, free oxygen radical test results across the groups (mann-whitney u=919, wilcoxon w=5105, test statistic=919, standard error=381.448, standardized test statistic=-9.519, asymptotic sig. (2-sided test) =.000); (b) distribution of ford, free oxygen radicals defense test result across the groups (mann-whitney u=5376, wilcoxon w=9562, test statistic=5376, standard error=381.480, standardized test statistic=2.165, asymptotic sig. (2-sided test) =.030); (c) distribution of calculated ri, redox index across the groups (mannwhitney u=751.5, wilcoxon w=4937.5, test statistic=751.5, standard error=381.173, standardized test statistic=-9.965, asymptotic sig. (2-sided test) =.000); (d) profile distribution across the groups (mann-whitney u=821, wilcoxon w=5007, test statistic=821, standard error=355.1, standardized test statistic=-10.501, asymptotic sig. (2-sided test) =.000). there are reported results of nonparametric analysis of ros and antioxidative potential across the groups of patients with acs and ccs (fig. 2). th difference between all oxidative parameters was statistically significant (p<0.0001), except free oxygen radicals defense ford test (p=0.1). a b d c lihood ratios (lr+ and lr-, respectively) for systemic oxidative stress in the patients with acute coronary syndrome (fig. 4). an infor medness of sensitivity and specificity data was represented by positive youden’s index (j). there were following diagnostic characteristics of systemic oxidative stress for acs: sensitivity of 82%, and specificity of 92.3%; positive predictive value (ppv) of 92% versus negative predictive value (npv) of 82%; positive likelihood ratio (lr+) of 11 versus negative likelihood ratio (lr-) of 0.2. z. lominadze et al. 30 in t e r n a l m e d ic in e issn 2413-6077. ijmmr 2020 vol. 6 issue 1 table 2. the strength of association between oxidative stress and acute coronary syndrome odds ratio (or) relative risk (rr) 14.42 3.26 standard error (sr) 0.36 0.18 lower 95% confidence interval (ci) 7.08 2.31 upper 95% confidence interval (ci) 29.4 4.60 7 fig. 2. independent-samples median test of baseline oxidative stress parameters across the groups. notes. group 1, patients with acute coronary syndrome (asc). group 2, patients with chronic coronary syndrome (ccs). (a) fort, free oxygen radical test results across the groups (median=325, test statistic=99.317, degree of freedom=1, asymptotic sig. (2-sided test) =.000; yates’s continuity correction chisquare=96.447, degree of freedom=1, asymptotic sig. (2-sided test) =.000); (b) ford, free oxygen radicals defense test result across the groups (median=1.470, test statistic=3.198, degree of freedom=1, asymptotic sig. (2-sided test) =.074; yates’s continuity correction chi-square=2.7, degree of freedom=1, asymptotic sig. (2-sided test) =.1); (c) ri, redox index across the groups (median=56, test statistic=104.393, degree of freedom=1, asymptotic sig. (2-sided test) =.000; yates’s continuity correction chi-square=101.454, degree of freedom=1, asymptotic sig. (2-sided test) =.000); (d) profile across the groups (median=3.0, test statistic=105.183, degree of freedom=1, asymptotic sig. (2-sided test) =.000; yates’s continuity correction chi-square=102.229, degree of freedom=1, asymptotic sig. (2-sided test) =.000). c d a b fig. 2. independent-samples median test of baseline oxidative stress parameters across the groups. notes. group 1, patients with acute coronary syndrome (asc). group 2, patients with chronic coronary syndrome (ccs). (a) fort, free oxygen radical test results across the groups (median=325, test statistic=99.317, degree of freedom=1, asymptotic sig. (2-sided test) =.000; yates’s continuity correction chi-square=96.447, degree of freedom=1, asymptotic sig. (2-sided test) =.000); (b) ford, free oxygen radicals defense test result across the groups (median=1.470, test statistic=3.198, degree of freedom=1, asymptotic sig. (2-sided test) =.074; yates’s continuity correction chi-square=2.7, degree of freedom=1, asymptotic sig. (2-sided test) =.1); (c) ri, redox index across the groups (median=56, test statistic=104.393, degree of freedom=1, asymptotic sig. (2-sided test) =.000; yates’s continuity correction chi-square=101.454, degree of freedom=1, asymptotic sig. (2-sided test) =.000); (d) profile across the groups (median=3.0, test statistic=105.183, degree of freedom=1, asymptotic sig. (2-sided test) =.000; yates’s continuity correction chi-square=102.229, degree of freedom=1, asymptotic sig. (2-sided test) =.000). discussion oxidative stress is one of the key pathogenetic factors for various diseases, including life-threatening conditions, such as acute coronary syndrome (acs) [16] mainly caused by atherosclerotic plaque instability [14,15]. available scientific data extend our understanding of the biology of plaque vulnerability z. lominadze et al. 31 in t e r n a l m e d ic in e issn 2413-6077. ijmmr 2020 vol. 6 issue 1 8 the evaluation results of association between an exposure (oxidative stress) and an outcome (acute coronary syndrome) are shown in table 2. table 2. the strength of association between oxidative stress and acute coronary syndrome odds ratio (or) relative risk (rr) 14.42 3.26 standard error (sr) 0.36 0.18 lower 95% confidence interval (ci) 7.08 2.31 upper 95% confidence interval (ci) 29.4 4.60 fig. 3 depicts the stratification of patients in the two groups in line with systemic oxidative stress profile. fig. 3. doughnut chart of systemic oxidative stress profile frequency in the two groups. notes. a. group 1, patients with acute coronary syndrome (acs); b. group 2, patients with chronic coronary syndrome (ccs); profile a, (ideal/normal values): redox index: 0-25; fort <300 units/2.36 mmol/l h2o2 eq; ford ≥1.08 mmol/l trolox eq. profile b, (latent oxidative stress): redox index: 25-50; fort <300 units/2.36 mmol/l h2o2 eq; ford ≤1.07 mmol/l trolox eq. profile c, (compensated oxidative stress): redox index: 50-58.3; 300 < fort <330 units/2.36 mmol/l h2o2 eq; ford ≥1.08 mmol/l trolox eq. profile d, (at risk of oxidative stress): redox index: 58.3-66.6; 300< fort <330 units/2.36 mmol/l h2o2 eq; ford ≤1.07 mmol/l trolox eq. profile e, (oxidative stress in progress): redox index: 66.6-100; fort ≥331 units/2.36 mmol/l h2o2 eq; 0.25< ford <3.00 mmol/l trolox eq. p value <.0001. a b fig. 3. doughnut chart of systemic oxidative stress profile frequency in the two groups. notes. a. group 1, patients with acute coronary syndrome (acs); b. group 2, patients with chronic coronary syndrome (ccs); profile a, (ideal/normal values): redox index: 0-25; fort <300 units/2.36 mmol/l h2o2 eq; ford ≥1.08 mmol/l trolox eq. profile b, (latent oxidative stress): redox index: 25-50; fort <300 units/2.36 mmol/l h2o2 eq; ford ≤1.07 mmol/l trolox eq. profile c, (compensated oxidative stress): redox index: 50-58.3; 300 < fort <330 units/2.36 mmol/l h2o2 eq; ford ≥1.08 mmol/l trolox eq. profile d, (at risk of oxidative stress): redox index: 58.3-66.6; 300< fort <330 units/2.36 mmol/l h2o2 eq; ford ≤1.07 mmol/l trolox eq. profile e, (oxidative stress in progress): redox index: 66.6-100; fort ≥331 units/2.36 mmol/l h2o2 eq; 0.25< ford <3.00 mmol/l trolox eq. p value <.0001. fig. 4. the likelihood ratio (lr) nomograms of combination of laboratory and clinical findings in patients with acute coronary syndrome (acs) prior probability (odds): 52% (1.1). positive test: positive likelihood ratio 11, 95% confidence interval [5.20,22]; posterior probability (odds) 92% (12.1), 95% confidence interval: [85%,96%]; (~1 in 1.1 with positive test are sick). negative test: negative likelihood ratio 0.20, 95% confidence interval [0.13,0.30]; posterior probability (odds): 18% (0.2), 95% confidence interval [12%,25%] (~ 1 in 1.2 with negative test are well). and suggest the important role of ros as a plaque stability regulator and mediator of acute coronary events [17]. it appears that ros production might be considered as a marker of atherosclerotic plaque instability. the main goal of the present study was to compare the oxidative stress profile between two groups of patients with acute coronary syndrome (acs) and chronic coronary syndrome (ccs). these findings could help assess risk of stratification and prevent acute coronary syndrome (acs). recent studies have shown the usefulness of the free oxygen radical test (fort)/free oxygen radicals defense (ford) novel colorimetric assay for evaluating oxidative stress [18-22]. z. lominadze et al. 32 in t e r n a l m e d ic in e issn 2413-6077. ijmmr 2020 vol. 6 issue 1 using the cr3000 form plus (callegarisrl, catellani group, italy) – callegari point of care instrument we have assessed oxidative stress profile in 191 patients divided into group 1 and group 2 with 100 patients with acute coronary syndrome (acs), and 91 patients with chronic coronary syndrome (ccs), respectively. the characteristics of the study population were well matched between groups (table 1), except nitrates consumption – it was much higher in the group 2 in a cohort of patients with chronic coronary syndrome (p<0.0001). the blood concentration of free oxygen radicals (fort) at admission in the coronary care unit (ccu) of ltd clinic-lj (kutaisi, georgia) was much higher in the patients with acute coronary syndrome in comparison of the patients with chronic coronary syndrome: 404.37±9.83 fort units/2.36 mmol/l h2o2 eq, and 282.34±9.83 fort units/2.36 mmol/l h2o2 eq., respectively (p<0.0001). the analysis of an antioxidant defense potential (measured by the ford test) across the groups have shown higher concentration of free oxygen radicals in cases of acute coronary syndrome (mean value of ford 1.37±0.035 mmol/l trolox eq. for acs versus 1.5±0.045 mmol/l trolox eq. for ccs). the calculated redox index in the patients with acs (group 1) was significantly higher than in the patients with ccs (69.2±1.47 vs 1.5±0.45, p<0.0001). the analysis of overall oxidative status (derived from fort/ford values and calculated redox index) across groups has indicated to significant correlation of uncontrolled systemic oxidative stress (profile e, oxidative stress in progress) with acute coronary syndrome (4.56±0.1 for group 1 vs 1.92±0.13 for group 2, p<0.0001). the incidence of profile e (oxidative stress in progress) in the patients with acute coronary syndrome was 82/100 (82%) versus 7/91 (8%) in the patients with chronic coronary syndrome. a cross-tabulation analysis has shown high diagnostic characteristics of systemic oxidative stress measurement test in cases of acute coronary syndrome (sensitivity of 82%, and specificity of 92.3%; positive predictive value (ppv); positive likelihood ratio (lr+) of 11). finally, we have recognized three main limitations to the presents study. first, this was case-control design study. second, this was not a multi-center study. third, the sample size was small. therefore, we are not able to extrapolate the findings of the present study to the general population. conclusions an extremely strong association between absolute oxidative stress and acute coronary syndrome (or 14.42 95% ci (7.08-29.4), rr 3.26 95% ci (2.31-4.60), high sensitivity and specificity (82% and 92.3%, respectively), and strong positive prediction and likelihood (positive predictive value of 92% and positive likelihood ratio of 11) indicate to pivotal role of oxidative stress in the development of life-threatening acute coronary events. it seems to be a reliable surrogate of plaque instability and rupture predictor. the findings of our study could help in risk stratification and prevention of acute coronary syndrome (acs) in clinical setting. conflict of interests the authors declare no conflict of interest. author contributions zaza lominadze – data curation, formal analysis, funding acquisition, investigation, resources and writing original draft; kakhaber chelidze – conceptualization, formal analysis, methodology, project administration, supervision, validation and review and editing of original draft; levan chelidze – formal analysis, software, visualization and review and editing of original draft; ekaterine lominadze – formal analysis, visualization and review and editing of original draft. funding the study was supported by ltd clinic-lj (kutaisi, georgia) funding. z. lominadze et al. 33 in t e r n a l m e d ic in e issn 2413-6077. ijmmr 2020 vol. 6 issue 1 оксидативний стрес – як сурогат нестабільності коронарної атеросклеротичної бляшки та предиктор її розриву z. lominadze1, k. chelidze2*, l. chelidze2, e. lominadze2 1 – ltd clinic-lj, kutaisi, georgia 2 – tbilisi state medical university, tbilisi, georgia вступ. оксидативний стрес відіграє важливу роль у патогенезі багатьох захворювань, в тому числі атеросклерозу та його ускладнень, як гострий коронарний синдром (гкс), що переважно зумовлений нестабільністю атеросклеротичної бляшки. мета – з’ясувати взаємозв’язок ступенем оксидативного стресу та нестабільністю бляшки шляхом порівняння оксидативного профілю пацієнтів з гкс та хронічним коронарним синдромом (хкс). дослідження проводили у кардіологічному відділені ltd клініки-lj (кутаїсі, грузія) в період з квітня 2018 року до червня 2019 року серед пацієнтів, котрим успішно було проведено первинні перкутанні коронарні втручання (пкв). методи. до дослідження було залучено 191 пацієнта (100 пацієнтів з гкс (група 1) та 91 пацієнт з хкс (група 2). досліджували такі показники: тест для визначення інтенсивності вільнорадикальних реакцій (free oxygen radical test, fort); тест для визначення антиоксидантної здатності (free oxygen radicals defense test, ford); визначали redox індекс та профіль оксидативного стресу за допомогою cr3000 form plus (callegari srl, catellani group, italy). результати. концентрація вільних радикалів кисню була достовірно вищою у пацієнтів з гкс (404,37±9,83 vs 282,34±9,83 fort од/2.36 ммоль/л h2o2 екв., p<0,0001). між інтенсивністю оксидативного стресу та розвитком гострого коронарного синдрому встановлено вірогідні кореляційні зв’язки (or 14,42 95% ci (7.08-29.4), rr 3,26 95% ci (2,31-4,60) з високими діагностичними характеристиками (чутливість 82,0% та специфічність 92,3%; позитивна прогностична роль 92,0% та позитивний коефіцієнт вірогідності 11). висновки. оксидативний стрес відіграє вирішальну роль при гострих коронарних подіях, що загрожують життю. вимірювання оксидативного профілю, як сурогату нестабільності атеросклеротичної бляшки та предиктора її розриву, може допомогти лікарю у стратифікації ризику коронарних подій для попередження гострого коронарного синдрому (гкс). ключові слова: гострий коронарний синдром; хронічний коронарний синдром; оксидативний стрес; тест для визначення інтенсивності вільнорадикальних реакцій (free oxygen radical test, fort); тест для визначення антиоксидантної здатності (free oxygen radicals defense test, ford); redox індекс; профіль оксидативного стресу; нестабільна бляшка; розрив бляшки. information about authors zaza lominadze – md. ltd clinic-lj. director. kutaisi, georgia orcid: https://orcid.org/0000-0003-3537-1365, e-mail: lozano_vaneli@yahoo.com kakhaber chelidze – md, ph.d., professor, tbilisi state medical university (tsmu). the tsmu first university clinic. department of internal medicine. tbilisi, georgia. orcid: https://orcid.org/0000-0002-3906-9967, e-mail: k.chelidze@tsmu.edu levan chelidze – md. tbilisi state medical university (tsmu). department of internal medicine. tbilisi, georgia. orcid: https://orcid.org/0000-0003-4200-300x, e-mail: levachello@gmail.com ekaterine lominadze – md. tbilisi state medical university (tsmu). department of internal medicine. tbilisi, georgia. orcid: https://orcid.org/0000-0002-6152-2852, e-mail: eka@lominadze.ge references 1. global health observatory (gho) data. world health organization 2018. https://www.who.int/ gho/mortality_burden_disease/causes_death/ top_10/en/. 2. zègre-hemsey jk, asafu-adjei j, fernandez a, brice j. characteristics of prehospital electro cardiogram use in north carolina using a novel linkage of emergency medical services and emergency department data. prehospital emergency care. 2019 nov 2;23(6):772-9. doi: 10.1080/10903127.2019.1597230 3. finn av, nakano m, narula j, kolodgie fd, virmani r. concept of vulnerable/unstable plaque. arteriosclerosis, thrombosis, and vascular biology. 2010 jul 1;30(7):1282-92. doi: 10.1161/atvbaha.108.179739 z. lominadze et al. 34 in t e r n a l m e d ic in e issn 2413-6077. ijmmr 2020 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10.1371/journal.pone.0149927 received 30 mar 2020; revised 13 apr 2020; accepted 02 june 2020. this is open access article distributed under the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. z. lominadze et al. 61 o b st e t r ic s a n d g y n e c o l o g y issn 2413-6077. ijmmr 2019 vol. 5 issue 2 doi 10.11603/ijmmr.2413-6077.2019.2.10388 risk factors for female infertility at a tertiary health facility in akure, south-west nigeria t.a. irinyenikan university of medical sciences teaching hospital complex, akure, ondo state, nigeria background. the inability of couples to achieve pregnancy is a major cause of psycho-social problems in family relationship that could lead to marital disharmony. objective. the aim of this study was to find out the possible risk factors for female infertility. methods. a case-control design and a sample size of 400 (200 cases of infertility and 200 controls) were used in the study. cases and controls were selected at random at the infertility and family planning clinic of the university of medical sciences teaching hospital complex, akure and were subjected to a predesigned interviewer administered questionnaire to collect the data. the cases were classified into primary and secondary infertility; binary and stepwise logistic regressions were used to generate the odds ratio and 95% confidence interval of the possible risk factors and the level of significance was set at p<0.05. results. the mean age of the women with infertility was 28.5±5.43 years and the mean age of those in the control group was 29.1±5.62 years. among the cases, 155 (77.5%) had secondary infertility, while 45 (22.5%) had primary infertility. significant risk factors for female infertility included presence of fibroids, having had fibroid operation, multiple sexual partners, previous abortion, polycystic ovary syndrome (pcos), sexually transmitted infection (sti) and post abortion sepsis. conclusion. the study showed that secondary infertility is still the most prevalent and the risk factors were multi factorial. efforts should be intensified to reduce infertility due to preventable causes. key words: female infertility; risk factors; fibroids; polycystic ovary syndrome; sexually transmitted infection. corresponding author: dr. theresa azonima irinyenikan, consultant obstetrician and gynaecologist, department of obstetrics and gynaecology, faculty of clinical sciences, university of medical sciences/university of medical sciences teaching hospital complex, akure, ondo state, nigeria. e-mail: tirinyenikan2017@gmail.com introduction infertility is the inability of a couple to achieve pregnancy over an average period of one year despite adequate, regular unprotected sexual intercourse [1]. who in 1991 estimated that between 8 and 12% of couples experienced some form of infertility during their reproductive lives, thus affecting 50 to 80 million worldwide, out of which 20-35 million couples in africa expected to experience this problem [2]. this can be extrapolated to 3-4 million nigeria couples suffering from infertility [3]. according to ogunniyi in 1995, the prevalence of infertility in sub-saharan africa was reported as ranging between 20-60% [4]. however, the estimate of infertile couples in ile-ife has been put at 19% by okonofua in 1995 [5], although authors in previous studies in the other parts of nigeria presented different ranging estimates. causes of infertility could be due to female factors, male factors or both. estimates show that in 35-40% of cases a man is infertile and in 35-40% of cases a woman is infertile while in 20-30% of cases, it is related to the combination of other factors [6]. causes of female infertility include conditions that may damage the fallopian tubes, interfere with ovulation or cause hormonal imbalance [7]. these conditions include pelvic inflammatory disease resulting from sexually transmitted infections, endometriosis, polycystic ovarian syndrome, premature ovarian failure, uterine fibroids and envi ro nmental factors [7]. other causes of infertility in females include post abortion sepsis, puerperal sepsis and age-related factors [8]. the risk factors for infertility can therefore be classified into: genital, endocrinal, develop­ mental and general factors [9]. infertility can be primary, if a woman has never conceived before, or it can be secondary, if a woman has at least once conceived but may or may not have carried the pregnancy to term [10]. in resource-rich countries primary inferinternational journal of medicine and medical research 2019, volume 5, issue 2, p. 61-68 copyright © 2019, tnmu, all rights reserved t.a. irinyenikan 62 o b st e t r ic s a n d g y n e c o l o g y issn 2413-6077. ijmmr 2019 vol. 5 issue 2 tility is much more common than secondary infertility, however the reverse is the case in sub-saharan africa (ssa) [11]. in some african regions, the prevalence of secondary infertility is more than 30% [12]. the high secondary infertility rate in ssa is thought to be due to sexually transmitted infections (stis) and medical interventions under unhygienic conditions, particularly during delivery or induced abortions [13]. infertility is a global problem affecting people around the world, which cause and importance may vary according to the geo graphical location and socio­eco nomic condition; assessing risk factors for female infertility should be geographical considering the variations that could arise from different regions. the aim of this study was to evaluate the risk factors for female infertility among cases of infertility presenting to a tertiary facility in akure, south-west nigeria, and the type of infertility prevalent among women there. this will further help to highlight the burden of this condition and subsequently help in policy making. methods study design the study used a matched case control design and was conducted at the infertility clinic and family planning clinic of the university of medical sciences teaching hospital complex, akure over a period of 2 years (july 2017june 2019). sample size calculation 3 𝑛𝑛𝑛𝑛 = � 𝑟𝑟𝑟𝑟 + 1 𝑟𝑟𝑟𝑟 � (�̅�𝑝𝑝𝑝)(1 − �̅�𝑝𝑝𝑝)(𝑍𝑍𝑍𝑍𝛽𝛽𝛽𝛽 + 𝑍𝑍𝑍𝑍𝛼𝛼𝛼𝛼2)2 (𝑝𝑝𝑝𝑝1 − 𝑝𝑝𝑝𝑝2)2 where n proportion in each group; p2 proportion exposed in the control group = 20%; p1 proportion of cases exposed; p average proportion of cases exposed; r=1 (equal number of cases and controls), using 80% power, zβ= 0.84 and for 5% significance level using an odd ratio of 2.0, z∝=1.96 p1=2.0(.20)/(.20)(2.0-1)+1=0.40/1.20=0.33, p =(.33+.20)= 0.265 𝑛𝑛𝑛𝑛 = 2 (. 256)(1 − .265)(.84 + 1.96)2 (.33 − .20)2 = 181 sample size = 2 x 181= 362, this was rounded up to 400. a total of 200 cases and an age matched 200 controls enrolled into the study. consent: informed consent was obtained from all the clients who participated in this study. ethical approval: the ethical approval for the study was obtained from the ethics committee of the university of medical sciences teaching hospital complex akure, ondo state. protocol number 015, 12th may 2017. inclusion criteria:cases were infertile women who have attended the infertility clinic at least twice and the controls were age matched women attending the family planning clinic of the same hospital that gave their consent to participate in the study. exclusion criteria: women who had medications or surgery to induce pregnancy among the controls and those who did not give consent. sampling technique cases (infertile women) were selected at random from the infertility clinic of the hospital through a weekly visit till the required sample size was obtained. controls involved fertile women, who attended the family planning clinic of the same hospital. the cases and controls were subjected to a predesigned interviewer administered questionnaire to collect sociodemographic data such as age, occupation, educational level, age at marriage; menstrual history such as age of menarche, regularity of menses, family history of infertility; relevant medical history such as diabetes mellitus, thyroid diseases, hypertension were also assessed; surgical history such as abdominal/pelvic surgeries were obtained. information on probable gynaecological conditions that could cause infertility such as history of endometriosis, polycystic 2 21 2 /2 )(p )z)(1)(( ) 1 ( p zpp r r n − +−+ = αβ 181 )20.33.( )96.184)(.265.1)(265(. 2 2 2 = − +− =n where n – proportion in each group; p2 – proportion exposed in the control group = 20%; p1 – proportion of cases exposed; p̄ – average proportion of cases exposed; r=1 (equal number of cases and controls), using 80% power, zβ= 0.84 and for 5% significance level using an odd ratio of 2.0, zα=1.96 p1=2.0(.20)/(.20)(2.0-1)+1=0.40/1.20=0.33, p̄=(.33+.20)= 0.265 3 𝑛𝑛𝑛𝑛 = � 𝑟𝑟𝑟𝑟 + 1 𝑟𝑟𝑟𝑟 � (�̅�𝑝𝑝𝑝)(1 − �̅�𝑝𝑝𝑝)(𝑍𝑍𝑍𝑍𝛽𝛽𝛽𝛽 + 𝑍𝑍𝑍𝑍𝛼𝛼𝛼𝛼2)2 (𝑝𝑝𝑝𝑝1 − 𝑝𝑝𝑝𝑝2)2 where n proportion in each group; p2 proportion exposed in the control group = 20%; p1 proportion of cases exposed; p average proportion of cases exposed; r=1 (equal number of cases and controls), using 80% power, zβ= 0.84 and for 5% significance level using an odd ratio of 2.0, z∝=1.96 p1=2.0(.20)/(.20)(2.0-1)+1=0.40/1.20=0.33, p =(.33+.20)= 0.265 𝑛𝑛𝑛𝑛 = 2 (. 256)(1 − .265)(.84 + 1.96)2 (.33 − .20)2 = 181 sample size = 2 x 181= 362, this was rounded up to 400. a total of 200 cases and an age matched 200 controls enrolled into the study. consent: informed consent was obtained from all the clients who participated in this study. ethical approval: the ethical approval for the study was obtained from the ethics committee of the university of medical sciences teaching hospital complex akure, ondo state. protocol number 015, 12th may 2017. inclusion criteria:cases were infertile women who have attended the infertility clinic at least twice and the controls were age matched women attending the family planning clinic of the same hospital that gave their consent to participate in the study. exclusion criteria: women who had medications or surgery to induce pregnancy among the controls and those who did not give consent. sampling technique cases (infertile women) were selected at random from the infertility clinic of the hospital through a weekly visit till the required sample size was obtained. controls involved fertile women, who attended the family planning clinic of the same hospital. the cases and controls were subjected to a predesigned interviewer administered questionnaire to collect sociodemographic data such as age, occupation, educational level, age at marriage; menstrual history such as age of menarche, regularity of menses, family history of infertility; relevant medical history such as diabetes mellitus, thyroid diseases, hypertension were also assessed; surgical history such as abdominal/pelvic surgeries were obtained. information on probable gynaecological conditions that could cause infertility such as history of endometriosis, polycystic 2 21 2 /2 )(p )z)(1)(( ) 1 ( p zpp r r n − +−+ = αβ 181 )20.33.( )96.184)(.265.1)(265(. 2 2 2 = − +− =n sample size = 2×181= 362, this was rounded up to 400. a total of 200 cases and an age matched 200 controls enrolled into the study. consent: informed consent was obtained from all the clients who participated in this study. ethical approval: the ethical approval for the study was obtained from the ethics committee of the university of medical sciences teaching hospital complex akure, ondo state. protocol number 015, 12th may 2017. inclusion criteria:cases were infertile women who have attended the infertility clinic at least twice and the controls were age matched women attending the family planning clinic of the same hospital that gave their consent to participate in the study. exclusion criteria: women who had medications or surgery to induce pregnancy among the controls and those who did not give consent. sampling technique cases (infertile women) were selected at random from the infertility clinic of the hospital through a weekly visit till the required sample size was obtained. controls involved fertile women, who attended the family planning clinic of the same hospital. the cases and controls were subjected to a predesigned interviewer administered questionnaire to collect sociodemographic data such as age, occupation, educational level, age at marriage; menstrual history such as age of menarche, regularity of menses, family history of infertility; relevant medical history such as diabetes mellitus, thyroid diseases, hypertension were also assessed; surgical history such as abdominal/pelvic surgeries were obtained. information on probable gynaecological conditions that could cause infertility such as history of endometriosis, polycystic ovarian syndrome, presence of fib­ roids, sexually transmitted infections, genital infection following childbirth and previous abortion were obtained. the cases were classified into primary or secondary infertility. statistical analysis the collected data were analysed using the computer package spss version 23. descriptive tables were generated and binary logistic regression was used to generate the odds ratio and the 95% confidence interval of the possible risk factors for infertility. these factors were further subjected to analysis using a stepwise logistic regression to identify the main predictors of female infertility. the statistical significance was set at p<0.05. results during the study, 200 cases of female infertility were registered and 200 age-matched controls. the mean age of the women with infertility was 28.5±5.43 years old and the mean age of those in the control group was 29.1±5.62 years old. among the cases, 155 (77.5%) pat.a. irinyenikan 63 o b st e t r ic s a n d g y n e c o l o g y issn 2413-6077. ijmmr 2019 vol. 5 issue 2 t.a. irinyenikan tients suffered from secondary infertility, while 45 (22.5%) had primary infertility. the mean ages of those with secondary and primary infertility were 28.7±4.4 years old and 29.1±3.4 years old respectively. as presented in table 1, 96 (61.9%) cases of secondary infertility and 70 (45.2%) of their control were traders, the risk of secondary infertility was statistically signifi­ cantly higher among the traders compare to their controls [or=0.17, 95% ci=0.06-0.48, p=0.001]. meanwhile, there was no statistically significant difference between the occupation of those with primary infertility and their control. the majority of the subjects practiced christian religion, were married and had tertiary level of education with no statistically significant difference (p>0.05) in both groups. table 2 showed the distribution of female infertility and the controls according to their gynaecological history. most of the women in both groups were already married at the age of over 18 years old. among those with secondary infertility, 94 (60.6%) had no living children but had abortions/ miscarriages in the past compare to their controls, where 92 (59.4%) already had ≥3 child­ ren, this was statistically significant, p=0.000. similarly, the 45 (100%) of those with primary infertility have never been pregnant compare to their controls, where 31 (68.9%) had ≥3 living children. this was also statistically significant, p=0.000. among those with secondary infertility, 31 (20.0%) had more than one sexual partner compare to 4 (2.6%) of the control, while 7 (15.6%) of those with primary infertility and 0 (0%) of the control had more than one sexual partner; this was also statistically significant, p=0.000. among the cases, 91 (58.7%) of those with secondary infertility started their menses after the age of 15 years compare to 88 (56.8%) of their control, delayed menses was not a significant risk factor for infertility [or=0.92, ci=0.59-1.45, p=0.730]. only 10 (6.5%) of the cases of secondary infertility and 3 (6.7%) of their control reported family history of infertility, this was not a significant risk factor for infertility [or=0.89, ci=0.35-2.26, p=0.813]. the analysis of cases of female infertility and the control according to the medical or surgical history showed that none of the subjects have ever had diabetes, thyroid disease or tuberculosis. though, 10 (6.5%) of the cases of secondary infertility and 7 (4.5%) of their control reported to have had hypertension while 3 (6.7%) of the cases of primary infertility and none of their control reported to table 1. socio-demographic characteristics of cases of female infertility and control subjects demographic factors 2o infertility cases (n=155) no (%) control (n=155) no (%) or (95% ci) p value 1o infertility cases (n=45) no (%) control (n=45) no (%) or (95% ci) p value age 15-24 25-34 ≥35 0(0.0) 78(50.3) 77(49.7) 3(1.9) 73(47.1) 79(51.0) 0.203 2(4.4) 26(57.8) 17(37.8) 1(2.2) 19(42.2) 25(55.6) 0.34 (0.06-1.96) 0.49 (0.27-0.91) 0.229 occupation civil/public servants trading/ business housewives/ applicants® 54(34.8) 96(61.9) 5(3.2) 64(41.3) 70(45.2) 21(13.5) 0.28 (0.10-0.79) 0.17 (0.06-0.48) 0.001 15(33.3) 26(57.8) 4(8.9) 22(48.9) 22(48.9) 1(2.2) 5.86 (0.59-57.78) 3.38 (0.35-32.55) 0.177 religion christian islam 148(95.5) 7(4.5) 147(94.8) 8(5.2) 0.86 (0.31-2.45) 0.791 44(97.8) 1(2.2) 42(93.3) 3(6.7) 0.32 (0.06-1.62) 0.306 marital status single married 3(1.9) 152(98.1) 1(0.6) 154(99.4) 0.3 3 (0.03-3.19) 0.314 0(0.0) 45(100) 2(4.4) 43(95.6) – 0.153 educational level primary secondary tertiary ® 13(8.4) 46(29.7) 96(61.9) 7(4.5) 38(24.5) 110(71.0) 0.47 (0.18-1.23) 0.72 (0.43-1.20) reference 0.173 3(6.7) 12(26.7) 30(66.7) 2(4.4) 13(28.9) 30(66.7) 0.67 (1.10-4.28) 1.08 (0.42-2.75) reference 0.887 ® = reference category 64 o b st e t r ic s a n d g y n e c o l o g y issn 2413-6077. ijmmr 2019 vol. 5 issue 2 have had hypertension, this was not statistically significant [or=0.69, ci=0.25­1.85, p=0.454]. however, 15 (9.7%) of secondary infertility cases have had fibroid operation compare with 1 (0.6%) of the control and the risk of secondary infertility was significantly higher than the control [or=0.06, 95% ci=0.01-0.47, p=0.007]. also, 5 (11.1%) of those with primary infertility and none of the control have had fibroid operation, the risk of primary infertility was significantly higher than the control, p=0.021 (table 3). table 2. distribution of cases of female infertility and controls according to their gynaecological history factors 2o infertility cases (n=155) no (%) control (n=155) no (%) or (95%ci) p value 1o infertility cases (n=45) no (%) control (n=45) no (%) or (95%ci) p value age at marriage <18 ≥18 none 2(1.3) 151(97.4) 2(1.3) 0(0.0) 155(100) 0(0.0) – 0.132 0(0.0) 45(100) 0(0.0) 1(2.2) 43(95.6) 1(2.2) – 0.360 parity 1 2 ≥3 none 46(29.7) 13(8.4) 2(1.3) 94(60.6) 20(12.9) 43(27.7) 92(59.4) 0(0.0) – 0.000 0(0.0) 0(0.0) 0(0.0) 45(100) 2(4.4) 12(26.7) 31(68.9) 0(0.0) – 0.000 age at 1st menses <15® ≥15 64(41.3) 91(58.7) 67(43.2) 88(56.8) 0.92 (0.59-1.45) 0.730 23(51.1) 22(48.9) 17(37.8) 28(62.2) 0.58 (0.32-1.05) 0.203 family history of infertility yes no 10(6.5) 145(93.5) 9(5.8) 146(94.2) 0.89 (0.35-2.26) 0.813 3(6.7) 42(93.3) 0(0.0) 45(100) – 0.078 no sexual partner 1 >1 124(80.0) 31(20.0) 151(97.4) 4(2.6) 9.43 (3.24-27.46) 0.000 38(84.4) 7(15.6) 45(100.0) 0(0.0) – 0.006 table 3. distribution of cases of female infertility with controls according to their medical/surgical history medical conditions 2o infertility cases (n=155) no (%) control (n=155) no (%) or (95%ci) p value 1o infertility cases (n=45) no (%) control (n=45) no (%) or (95%ci) p value diabetes yes no 0(0.0) 155(100) 0(0.0) 155(100) – – 0(0.0) 45(100) 0(0.0) 45(100) – – hypertension yes no 10(6.5) 145(93.5) 7(4.5) 148(95.5) 0.69 (0.25-1.85) 0.454 3(6.7) 42(93.3) 0(0.0) 45(100) – 0.078 thyroid yes no 0(0.0) 155(100) 0(0.0) 155(100) – – 0(0.0) 45(100) 0(0.0) 45(100) – – tuberculosis yes no – 155(100) – 155(100) – – – 45(100) – 45(100) – – fibroid operation yes no 15(9.7) 140(90.3) 1(0.6) 154(99.4) 0.06 (0.01-0.47) 0.000 5(11.1) 40(88.9) 0(0.0) 45(100) – 0.021 other operation yes no 1(0.6) 154(99.4) 0(0.0) 155(100) – 0.317 0(0.0) 45(100) 0(0.0) 45(100) – – t.a. irinyenikan 65 o b st e t r ic s a n d g y n e c o l o g y issn 2413-6077. ijmmr 2019 vol. 5 issue 2 t.a. irinyenikan table 4 presents the analysis of cases of infertility and the control according to their gynaecological conditions. the gynaecological conditions in the subjects included fibroids, which was present in 46 (29.7%) cases of secondary infertility compare to 11 (7.1%) of their control, the risk of secondary infertility among them was significantly higher than their control [or=0.18, 95% ci=0.09-0.37, p=0.00]. similarly, among the cases of primary infertility, 15 (33.3%) had fibroids compare to only 1 (2.2%) of their control, the risk of primary infertility was significantly higher than the control [or=0.07, 95%ci=0.008-0.64, p=0.00]. also, among the cases of secondary infertility, 50 (32.3%) had endometriosis, 35 (22.6%) of their control had similar condition, while in the cases of primary infertility 15 (33.3%) had endometriosis and 10 (22.2%) had the condition, there was no statistically significant difference in both groups at p>0.05. among the 2 groups, 114 (73.5%) of those with secondary infertility had previous abortion compare to 53 (34.2%) of their control while none was reported in the cases of primary infertility. the risk of secondary infertility was significantly higher than the control [or=0.19, 95% ci=0.12-0.30, p=0.000]. post abortion sepsis was reported in 30 (19.4%) of cases of secondary infertility and only 10 (6.5%) of the control with a statistically significant higher risk among the cases [or=0.29, 95% ci=0.14-0.61, p=0.001]. another gynaecological condition prevalent was polycystic ovary syndrome (pcos), which was reported in 53 (34.2%) cases of secondary infertility and 7 (4.5%) of their control, the risk of pcos among those with secondary infertility was significantly higher than their control, [or=0.09, 95% ci=0.04-0.21, p=0.000] while in the cases of primary infertility, it was reported in 18 (40.0%) and 2 (4.4%) of their control and the risk of pcos was also significantly higher than the control [or=0.01, 95% ci=0.01-0.52, p=0.000]. sexually transmitted infection was reported in 47 (30.3%) cases of secondary infertility and 18 (11.6%) of their control with a significantly higher risk of this condition among these cases [or=0.30, 95% ci=0.17-0.55, p=0.000), while table 4. analysis of cases of female infertility and the controls according to their gynaecological conditions gynaecological conditions 2o infertility cases (n=155) no (%) control (n=155) no (%) or (95%ci) p value 1o infertility cases (n=45) no (%) control (n=45) no (%) or (95%ci) p value endometriosis yes no 50(32.3) 105(67.7) 35(22.6) 120(77.4) 0.61 (0.37-1.02) 0.056 15(33.3) 30(66.7) 10(22.2) 35(77.8) 0.79 (0.17-3.69) 0.239 fibroid diagnosis yes no 46(29.7) 109(70.3) 11(7.1) 144(92.9) 0.18 (0.09-0.37) 0.000 15(33.3) 30(66.7) 1(2.2) 44(97.8) 0.07 (0.008-0.64) 0.000 previous abortion yes no 114(73.5) 41(26.5) 53(34.2) 102(65.8) 0.19 (0.12-0.30) 0.000 0(0.0) 45(100) 16(35.6) 29(64.4) – 0.000 pcos yes no 53(34.2) 102(65.8) 7(4.5) 148(95.5) 0.09 (0.04-0.21) 0.000 18(40.0) 27(60.0) 2(4.4) 43(95.6) 0.01 (0.01-0.52) 0.000 menstrual cycle regular irregular 141(91.0) 14(9.0) 147(94.8) 8(5.2) 0.55 (0.22-1.35) 0.184 37(82.2) 8(17.8) 41(91.1) 4(8.9) 0.83 (0.10-6.71) 0.215 genital infection yes no 8(5.2) 147(94.8) 10(6.5) 145(93.5) 1.26 (0.48-3.30) 0.627 0(0.0) 45(94.8) 4(8.9) 41(91.1) – 0.061 sti yes no 47(30.3) 108(69.7) 18(11.6) 137(88.4) 0.30 (0.17-0.55) 0.000 6(13.3) 39(86.7) 7(15.6) 38(84.4) 1.11 (0.21-5.89) 0.764 post abortion sepsis yes no 30(19.4) 125(80.6) 10(6.5) 145(93.5) 0.29 (0.14-0.61) 0.001 0(0.0) 45(100) 3(6.7) 42(93.3) 0.71 (0.0-0.0) 0.078 66 o b st e t r ic s a n d g y n e c o l o g y issn 2413-6077. ijmmr 2019 vol. 5 issue 2 among the cases of primary infertility 6 (13.3%) and 7 (15.6%) of their control reported to have had this condition, this was not statistically significant, p>0.05. among the cases of secon­ dary infertility, 8 (5.2%) and 10 6.5%) of the control had genital infection, there was no statistically significant difference in both groups. menstrual irregularity was found in 14 (9.0%) cases of infertility compare to 8 (5.2%) of their control and 8 (17.8%) of primary in fertility compare to 4 (8.9%) of the control, this was not statistically significant. table 5 showed various predictors of secondary and primary infertility using stepwise logistic regression analysis of the various independent risk factors for infertility. table 5. stepwise logistic regression to identify predictors of female infertility independent variable b s.e. (standard error) test p. value or 95% ci model (1): for secondary infertility fibroid diagnosis 1.113 0.426 6.838 0.009 3.04 1.32-7.01 fibroid operation 1.621 1.151 1.984 0.159 5.06 0.53-48.21 pcos 2.213 0.45 24.174 0.00 9.14 3.78-22.08 sti 0.746 0.359 4.325 0.038 2.11 1.04-4.26 post abortion sepsis -0.271 0.473 0.329 0.57 0.76 0.30-1.93 previous abortion 1.544 0.294 27.564 0.000 4.68 2.63-8.33 constant -12.393 2.50 24.487 model (2):for primary infertility fibroid diagnosis 2.508 1.118 5.034 0.025 12.28 1.37-109.86 pcos 2.884 1.13 6.489 0.01 17.88 1.94-164.51 sti -0.052 0.84 0.004 0.951 0.949 0.18-4.94 constant -12.393 2.50 24.487 0.000 in model 1, among the various significant factors detected by the binary logistic regression analysis for secondary infertility only the presence of fibroids [or=3.04, 95% ci=1.32­7.01, p=0.009], polycystic ovary syndrome [or=9.14, 95% ci=3.78-22.08, p=0.00], sexually transmitted infection [or=2.11, 95% ci=1.04-4.26, p=0.038] and previous abortion [or=4.68, 95% ci=2.638.33, p=0.000] were predictors of secondary infertility. in model 2, the presence of fibroids [or=12.28, 95% ci=1.37-109.86, p=0.025] and polycystic ovary syndrome [or=17.88, 95% ci=1.94-164.51, p=0.01] were the predictors of primary infertility. discussion the study showed that secondary infertility was commoner among the women (77.5%) than primary infertility (22.5%), which is in keeping with studies carried out in other parts of nigeria [11,12,13]. it further highlights the burden of secondary infertility which is more prevalent in sub-saharan african countries compared to the western world [14].the demographic characteristics of the subjects showed that most of the women were already married and possibly in a sexual relationship to enable one ascertain if there are issues concerning their fertility. other factors such as the woman’s age, religion, educational level had no impact on fertility as against a similar study which reported a decrease in a woman’s fertility with increasing age [15]. these factors therefore were not risk factors for infertility among our women. menstrual irregularity was seen in some of the women with secondary infertility though not statistically significant and therefore suggest that this has no strong effect as a risk factor for infertility among our women. however, having multiple sexual partners was a significant risk factor for infertility among the women, which further confirmed the increase in sexually transmitted infection which was also reported among the women with secondary infertility. sexually transmitted infections are transmitted through sexual activity with an infected partner and a major cause of secondary infertility in sub-saharan africa [6]. this study also highlights gynaecological conditions such as the presence of fibroids and previous abdominal surgeries for fibroids as risk factors for female infertility which is in keeping with another study [16]. large fibroids may cause infertility by impairing the uterine lining, blocking the fallopian tube, distorting the shape of the uterine cavity or altering the t.a. irinyenikan 67 o b st e t r ic s a n d g y n e c o l o g y issn 2413-6077. ijmmr 2019 vol. 5 issue 2 t.a. irinyenikan position of the cervix. also, following pelvic surgery, postsurgical or post infective uterine or abdominal adhesions that result may restrict the movement of ovaries and fallopian tubes and cause infertility. previous abortion and post abortion sepsis were significant risk factors for secondary infertility in this study. this finding has also been reported in another study in nigeria where induced abortion and post abortion sepsis were found to affect future fertility [17]. this may be as a result of repeated injuries to the uterine lining from multiple dilatation and curettage which can cause adhesions within the uterus thus leading to secondary amenorrhea and infertility. however, genital infection did not have any significant effect on the cause of infertility, this could have been due to increase access to health care and the presence of skilled birth attendants during delivery. this study showed ovarian dysfunction as a result of polycystic ovary syndrome to be a significant cause of both primary and secondary infertility among the women, similar studies have shown that nearly 10% of infertile women are diagnosed with reduced ovarian dysfunction and polycystic ovary syndrome implicated as a common cause of ovula tion disorder in women of childbearing age [18,19,20]. in this study the main predictors of female infertility were the presence of fibroids, poly­ cystic ovary syndrome, sexually transmitted infections and previously induced abortions for unwanted pregnancies. these findings have further revealed the great impact of reproductive infections and hormonal imbalance as major causes of female infertility. conclusion female infertility is still a major public health issue and its cause could be multi factorial. secondary infertility remains the most prevalent type in the region mostly due to tubal damage as a result of increase in sexually transmitted infections and previous induced abortions. though, hormonal cause such as polycystic ovary syndrome may not be under our control, reproductive infections from sexual activity could be curtailed by preventing unsafe sex and prompt treatment of diseases resulting from sexually transmitted infections. efforts should be intensified to prevent unsafe abortions which could lead to infertility in the future. acknowledgements. i acknowledge the consultant obstetricians and gynaecologists of the university of medical sciences teaching hospital complex akure for allowing to use their patients for the study, the research assistants, who helped in the collection of the data and the statistician, who analysed the data. funding this research received no external funding. conflict of interests the author declares no conflict of interest. фактори ризику розвитку жіночого безпліддя на третинному рівні надання медичної допомоги у акуре, південно-західна нігерія t.a. irinyenikan university of medical sciences teaching hospital complex, akure, ondo state, nigeria вступ. неможливість пари народити спільних дітей є однією з головних психо-соціальних проблем, що призводить до дисгармонії подружніх стосунків. мета дослідження встановити можливі фактори ризику розвитку жіночої неплідності. методи. у дослідження було залучено 400 жінок (200 – з неплідністю та 200 осіб склали групу контролю). жінок було обрано методом випадкової вибірки у клініці планування сім’ї при навчальнолікувальному комплексі університету медичних наук, акуре. усі досліджувані заповнювали спеціально розроблений опитувальник. усі випадки було класифіковано на первинне та вторинне непліддя. застосовано статистичний регресійний аналіз: бінарну та непряму логістичну регресію, рівень значущості p<0.05. результати. середній вік жінок з неплідністю склав 28,5±5,43 роки, контрольної групи – 29,1±5,62 роки. у 155 (77,5%) випадках мала місце вторинна неплідність, і лише у 45 (22,5%) – первинне безпліддя. статистично значущими факторами ризику були наявність фібротичних змін, оперативні втручання з їх приводу, кілька сексуальних партнерів, попередні аборти, синдром полікистозних яйників, хвороби, що передаються статевим шляхом та постабортний сепсис. 68 o b st e t r ic s a n d g y n e c o l o g y issn 2413-6077. ijmmr 2019 vol. 5 issue 2 references 1. vander bm, wyns c. fertility and infertility definition and epidemiology. clin biochem.2018; 62:2­10. 2. ombelet w. global access to infertility care in developing countries: a case of human rights, equity, and social justice. fvv obgyn 2012; monograph: 7–16. 3. mascarenhas mn, cheung h, mathers cd, stevens ga. measuring infertility in populations: constructing a standard definition for use with demographic and reproductive health surveys. popul health metr 2012;10:1–11. 4. adeyemi as, adekanle da, afolabi af. pattern of gynaecological consultations at ladoke akintola university of technology teaching hospital. niger j clin pract 2009;12:47­50. 5.scarneciu i, lupu s, scarneciu c. smoking as a risk factor for the development of erectile dysfunction and infertility in men; evaluation depending on the anxiety levels of these patients. soc behav sci. 2014; 12. 6. olooto o, wasiu e, amballi a, adebayo a, banjo b, taiwo a. a review of female infertility; important etiological factors and management j. microbiol. biotech. res., 2012,2(3):379­85. 7. ombelet w, devroey p, gianaroli l, te velde e (eds). developing countries and infertility. spec issue human reprod 2008;1–117. 8. nachtigall rd. international disparities in access to infertility services. fertil steril 2006;85:871–5. 9. rutstein so, shah ih. infecundity, infertility, and childlessness in developing countries. dhs comparative reports no. 9. world health organization, geneva, switzerland, 2004. 10. sedgh g, singh s, shah ih, ahman e, henshaw sk, bankole a. induced abortion: incidence and trends worldwide from 1995 to 2008. lancet 2012; 379:625–32. 11. sule jo, erigbali p, eruom l. prevalence of infertility in women in a south western nigerian community. african journal of biomedical research 2008; 11: 225­7. 12. orhue a, aziken m. experience with a comprehensive university hospital-based infertility program in nigeria. int j gynaecol obstet 2008; 101(1):11­15. 13. abubakar ap, yusuf ts. the profile of infer­ tility in a teaching hospital in north west nigeria. sahel medical journal 2014;17: 1. 14. okonofua fe. infertility in sub-saharan africa. in: okonofua fe, odunsi k, editors. contemporary obstetrics and gynaecology for developing countries. publishers: women’s health and action research center. benin city: 2003:128­56. 15. sabarre ka, khan z, whitten an, remes o, phillips kp. a qualitative study of ottawa university students’ awareness, knowledge and perceptions of infertility, infertility risk factors and assisted reproductive technologies (art). reprod health j. 2013; 10(41):1­10. doi:10.1186/1742­4755­10­41 16. palihawadana ts, wijesinghe ps, seneviratne hr. aetiology of infertility among females seeking treatment at a tertiary care hospital in sri lanka. ceylon med j. 2012; 57(2):79­83. doi:10.4038/cmj.v57i2.4461 17. orji eo. comparative study of the impact of past pregnancy outcome on future fertility. singapore med j 2008; 49:1021­24. 18. samiha m, hassan ah, nehad m, fayek e, gehan s. risk factors for primary and secondary female infertility in alexandria: a hospital based case control study. journal of the medical research institute jmri 2006; 27(4):255­61. 19. pastore lm, karns lb, ventura k, clark ml. longitudinal interviews of couples diagnosed with diminished ovarian reserve undergoing fragile x permutation testing. j genet couns. 2014;23(1): 97-107. doi:10.1007/s10897­013­9616­0 20. tsai yh, wang tw, wei hj, et al. dietary intake, glucose metabolism and sex hormones in women with polycystic ovary syndrome (pcos) compared with women with nonpcos-related infertility. br j nutr. 2013;109(1):2190­8. doi:10.1017/s0007114512004369 received 13 september 2019; revised 11 october 2019; accepted 24 october 2019. this is open access article distributed under the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. висновки. дослідження показало, що вторинне безпліддя все ще переважає у структурі захворюваності, фактори ризику – мульфакторіальні. зусилля, спрямовані на зменшення розвитку непліддя через фактори, які можна попередити, повинні бути посилені. key words: жіноче безпліддя; фактори ризику; фіброз; синдром полікистозних яйників; хвороби, що передаються статевим шляхом. information about the author dr. theresa azonima irinyenikan – consultant obstetrician and gynaecologist, department of obstetrics and gynaecology, faculty of clinical sciences, university of medical sciences/university of medical sciences teaching hospital complex, akure, ondo state, nigeria. orcid 0000­0002­5594­037x, e­mail: tirinyenikan2017@gmail.com t.a. irinyenikan 137 b io m e d ic a l s c ie n c e s issn 2413-6077. ijmmr 2019 vol. 5 issue 2 doi 10.11603/ijmmr.2413-6077.2019.2.10902 gender-specific differences of cardiac vegetative control in adrenaline-induced necrosis and light deprivation h.o. bezkorovaina, i.m. klishch, *m.r. khara, v.ye. pelykh i. horbachevsky ternopil national medical university, ternopil, ukraine background. cardiovascular disease is the main cause of morbidity predominantly in males. stress is one of the crucial factors, especially with light desynchronosis. objective of the study was to assess gender-specific characteristics of cardiac vegetative control in myocardial necrosis in cases of light deprivation. methods. cardiac vegetative control in adrenaline-induced myocardial necrosis (aimn) in a setting of light deprivation (ld) was assessed in 72 mature white rats of both sexes. the animals were divided into 2 groups: g1 – the animals kept under day/night cyclic balance (12 hours/12 hours); g2 – the animals kept at ld (illumination 0.5-1 lx) for 10 days. on day 11, aimn caused by adrenaline (0.5 mg/kg) and heart rate variability (hrv) was assessed in 1 hour and 24 hours. results. the development of aimn at ld in the ♂g2 led to hrv increase that was caused by augmentation of parasympathetic and reduction of sympathetic cardiac effects. in cases of aimn, changes of cvc in the ♀g2 were similar to the ♀g1. however, in 1 hour of aimn, parasympathetic cardiac effects were more significant than in the ♀g1. while the ♀g2 aimn animals experienced balanced sympathetic and parasympathetic actions, the predominance of the sympathetic component was evidenced in the ♀g1 aimn animals. conclusions. light deprivation has different effects on baseline sympathetic/parasympathetic balance in males and females, i.e. increased parasympathetic control of heart rhythm in males and maintenance of sympathetic/ parasympathetic balance in females. key words: myocardial necrosis; heart rhythm variability; light deprivation; gender. *corresponding author: khara mariya, md, ph.d., dsc, professor of the department of pathophysiology, i. horbachevsky ternopil national medical university, ternopil, ukraine. e-mail: khara@tdmu.edu.ua introduction according to the who data, cardiovascular disease is the most frequent cause of death in the european population; moreover, it has frequent severe sequelae, leads to disability and reduces the quality of life. ukraine is on the list of countries in eastern europe, where coronary artery disease (cad) mortality in people of 55-60 years of age is higher than that in french subjects 20 years senior [1]. the leading causes of increased mortality include ageing population and lifestyle factors, especially smoking and suffering from overweight [2]. males are more frequently affected by coronary artery disease than females. this difference, however, is found in the middleaged persons. at menopause, the number of females in the population with coronary epi sodes and myocardial infarction increases greatly. this might be associated with deficiency of oestrogens and their diminished cardioprotective and vasoprotective activity [3]. important risk factors of myocardial necrosis include impaired circadian rhythms and functional disorders of the pineal gland [4, 5]. this is related to professional activity, sleep disorders and difficulty falling asleep [6, 7]. similar to all other systems in the body, the cardiovascular system is controlled by the pineal gland. its activity changes depending on the phase of the circadian cycle (light/darkness), as suggested by fluctuations in blood pressure and heart rhythm variability [5, 8]. however, the effects of melatonin synthesised by the pineal gland vary in males and females that is associated not only with the ability of this hormone to regulate the synthesis of sex hormones [4, 9, 10], but also to its capability to change the activity of sympathetic and parasympathetic components of the autonomic nervous system (ans) [11, 12]. murine experiments have demonstrated different ans responses to melatonin in males and females [11]. a higher basal tone of the parasympathetic component in female ans contributes to a better cardioprotective effect of melatonin when adrenalineinduced myocardial necrosis is modelled in a international journal of medicine and medical research 2019, volume 5, issue 2, p. 137-144 copyright © 2019, tnmu, all rights reserved h.o. bezkorovaina et al. 138 b io m e d ic a l s c ie n c e s issn 2413-6077. ijmmr 2019 vol. 5 issue 2 setting of melatonin [13]. a similar trend was found in humans. analysis of ans status (as reported by a 24-hour monitoring of heart rhythm variability) suggests reduced parasympathetic tone and increased sympathetic activity as predictors of fatal arrhythmias and sudden death in subjects with myocardial infarction [14]. the benefits of heart rhythm variability analysis and its diagnostic and prognostic value are beyond doubt. the use of this method allows assessment of cardiac vegetative control in a study of pathogenetic role of light desynchronosis as a contributor to myocardial necrosis. however, the available data do not provide a full understanding of the role of ans in adaptation of heterosexual organisms to abnormal light conditions, including the development of cardiac disease under these conditions. the objective of the study was to assess gender­specific characteristics of cardiac vege­ tative control in adrenaline-induced myocardial necrosis in rats in cases of light deprivation. methods the experiments were conducted according to ethical guidelines approved by the european convention for the protection of vertebrate animals used for experimental and other scientific purposes (strasbourg, 1986), reso­ lution of the first national congress on bioethics (kyiv, 2001) and the order of the ministry of health of ukraine no. 690, dated september 23, 2009. the tests were performed on 72 nonlinear male and female white rats (body weight: 220­270 g; age: 5­5.5 months). the rats were provided with a standard laboratory pellet diet and water and were housed in plastic cages at constant ambient temperature and humidity. the rats of the control group (group 1, 18♂, 18♀) were kept under a natural day/night cycle (light 12 hours, 500 lx/night 12 hours, 0.5-1 lx). the rats of the experimental group (group 2, 18♂, 18♀) were kept in permanent darkness (0.5-1 lx) for 10 days. necrosis of myocardium was modelled by administration of adrenaline (intramuscular, 0.5 mg/kg of body weight) on day 11. in 1 and 24 hours after adrenaline administration, ekg was captured (2 standard leads) using a cardiolab-ce computer-based complex (kharkiv, ukraine). the durations of 1000 consecutive cardiac r-r intervals accurate to 0.001 second were assessed. in order to evaluate the regulatory activity of sympathetic and parasympathetic ans components on the heart rhythm, the following parameters were registereg: mo (sec) = mode, i.e. the most frequently captured duration of the r­r interval; amo (%) = the amplitude of mode, i.e. the percentage of cardiac intervals meeting the mo value; ∆x (sec) = the difference between the largest and the smallest r­r value; hsi (abs. value) = heart strain index reflecting the degree of centralisation of heart rhythm control, this parameter is obtained from the following expression: hsi=amo/(2·∆x·mo); vbi (abs. value) = vegetative balance index reflecting the ratio between the activities of the sympathetic and parasympathetic nervous system, this parameter is obtained from the following expression: vbi=amo/∆x; vri (abs. value) = vegetative rhythm index reflecting the activity of the autonomic circuit of heart rhythm control, this parameter is obtained from the following expression: vri =1/mo·∆x, and iarp (abs. value) = index of adequate regulation processes reflecting the balance between the activity of the sympathetic component of the autonomic nervous system and the predominant functional level of the sinus node. by matching against heart rhythm (hr), this parameter allows judgment on excessive or insufficient centralisation of heart rhythm control; this parameter is obtained from the following expression: iarp=amo/mo. statistical analysis of the results was performed using parametric method of variation statistics based on established normal distribution of data in the rows compared, with n=6 in each of the rows. the following parameters were determined by the arithmetic mean (m), standard deviation (ō) and student’s t-test (t). the difference between the arithmetic means was statistically significant at t value not less than 2.228 (p≤0.05). microsoft excel xp (biostat pro 6.7.1.0) software (us) was used for calculations. results the results attained in the animals of the control group (group 1), which were under c o n d i t i o n s o f b a l a n c e d l i g h t / d a r k n e s s (12 hours/12 hours), are presented in table 1. the development of adrenaline-induced myo cardial necrosis within 1 hour after adrenaline administration was found to cause a significant increase in hr (by 12.5% in the ♂, by 13% in the ♀, p<0.05). this process was accompanied by a predictable reduction in mo values (by 12% in the ♂, by 13% in the ♀). amo values were increased in the ♂ (by 87%, p<0.05) and the ♀ animals by (34%, p<0.05). the δx h.o. bezkorovaina et al. 139 b io m e d ic a l s c ie n c e s issn 2413-6077. ijmmr 2019 vol. 5 issue 2 h.o. bezkorovaina et al. values significantly decreased in the animals of either sex (by 97% in the ♂, by 67% in the ♀, p<0.05). hsi, which is an integral parameter, increased in the animals of either sex (a 3.7-fold increase in the ♂, a 2.4-fold increase in the ♀, p<0.05). such changes reflected the increased role of the sympathetic ans component and the reduced influence of the parasympathetic component in heart rhythm control. these findings were confirmed by increases in vbi (a 3.3-fold increase in the ♂, a 2.1-fold increase in the ♀, p<0.05), vri (a 2.0-fold increase in the ♂, a 1.8-fold increase in the ♀, p<0.05) and iarp (a 2.1-fold increase in the ♂, a 1.5-fold increase in the ♀, p<0.05). in 24 hours after adrenaline administration (i.e. the peak of focal necrosis), the type of ans response to development of the abnormal process changed somewhat. in the ♂ animals, the findings of hr, mo, δx, vbi and vri recovered to the control levels, amo decreased (by 35%, p<0.05), while iarp were above control values (by 43%, p<0.05). in the ♀ rats, hr was higher (by 10%, p<0.05) and mo was lower (by 10%, p<0.05). the following parameters were higher than the respective control values: amo (by 38%, p<0.05), hsi (by 78%, p<0.05), vbi (by 61%, p<0.05), vri (by 45%, p<0.05) and iarp (by 34%, p<0.05). gender­specific analysis showed mo in the group 1 lower in the ♂ than in the ♀ (by 8%, p<0.05). in 1 hour of necrotic process development, mo was lower than in the ♀ (7%, p<0.05), amo was higher (by 31%, p<0.05) and iarp was also higher (by 40%, p<0.05). in 24 hours after adrenaline administration, the δx in the ♂ was higher than in the ♀ (by 45%, p<0.05) and hsi, vbi and vri were lower (by 37%, 41% and 45%, p<0.05, respectively), reflecting gender­specific differences of cardiac adjustment mechanisms in cases of ans-mediated damage. analysis of parameters in the animals of the experimental group (group 2) that were kept in permanent darkness for 10 days (light deprivation) showed that in 1 hour of myocardial necrosis development, ♂ animals had lower hr (by 9%, p<0.05), higher mo (by 9%, p<0.05) and lower δx (by 23%, p<0.05) (table 2). as for other parameters, no significant changes were evi­ denced. when the ♀ animals were exposed to identical conditions, hr increased (by 6%, p<0.05), while mo and δx decreased (by 6% and 29%, respectively, p<0.05). under these conditions, the increase in hsi, vbi and vbi were quite predictable (by 65%, 55% and 40%, respectively, p<0.05). iarp values did not change. in 24 hours after adrenaline administration, the ♂ hr was lower than in the controls (by 16%, p<0.05), mo was higher (by 16%, p<0.05) and iarp was lower (by 34%, p<0.05). in the ♀ at this phase of myocardial necrosis, hr was higher (by 6%, p<0.05), mo was lower (by 6%, table 1. parameters of heart rhythm variability in the rats developing adrenaline-induced myocardial necrosis under the intact day/night cycle (12 hours/12 hours), m±ō parameter gender controls(n=6) 1 h post-adrenaline (n=6) 24 h post-adrenaline (n=6) hr ♂ 472±21# 531±17* 491±29 ♀ 438±19# 496±27* 481±12* mo (sec) ♂ 0.127±0.006# 0.113±0.002*# 0.122±0.007 ♀ 0.137±0.006 # 0.121±0.007*# 0.125±0.003* amo (%) ♂ 31.7±3.8 59.2±5.8# 43.7±7.5* ♀ 33.8±4.5 45.2±5.0*# 41.2±3.1* δx (sec·10-2) ♂ 0.65±0.22 0.35±0.08* 0.58±0.04# ♀ 0.55±0.14 0.33±0.05* 0.40±0.06*# hsi (abs. value) ♂ 21621±9813 79493±23420* 30953±6342# ♀ 23875±7223 57384±10855* 42514±9872*# vbi (abs. value) ♂ 5425±2245 17873±4936* 7527±1391# ♀ 6578±2211 13783±2142* 10598±2484*# vri (abs. value) ♂ 1333±470 2716±935* 1418±190# ♀ 1419±442 2537±419* 2051±347*# iarp (abs. value) ♂ 0.250±0.040 0.525±0.058*# 0.358±0.061* ♀ 0.247±0.038 0.375±0.056*# 0.330±0.024* note: * – a statistically significant (p≤0.05) differences relative to the controls, # – relative to the animals of the opposite sex. 140 b io m e d ic a l s c ie n c e s issn 2413-6077. ijmmr 2019 vol. 5 issue 2 p<0.05), hsi and vbi were higher (by 70%, 62%, p<0.05), and amo, δx, vri and iarp did not differ from the respective baseline values in this group. gender­specific analysis showed that hr and iarp in the control group was higher in the ♂ than in the ♀ (by 18%, 37%, p<0.05); the mo values were lower (by 18%, p<0.05). there were no differences between the animals in terms of other parameters. in 1 hour of myocardial necrosis, δx in the ♂ was higher than in the ♀ (by 27%, p<0.05); hsi, vbi and iarp were lower (by 38%, 40% and 10%, p<0.05). in 24 hours of myocardial necrosis, δx ♂ was significantly higher (by 50%, p<0.05), and hsi, vbi and vri were lower (in 2.1 times, 2.0 times and 1.7-times, respectively, p<0.05). in terms of ans responses, effects of light deprivation in development of myocardial necrosis have shown that staying in darkness for 10 days had different influences on vegeta­ tive control of heart rhythm in male and female animals. in ♂, hr increased by 8.5% (p<0.05), mo decreased by 85% (p<0.05). other parameters remained unchanged and were not statistically different from those in animals of group 1. in the ♀ cohort, light deprivation did not cause any changes in investigational para meters. under conditions of a necrotic process (1 and 24 hours after administration of adrenaline), hr ♂ was lower than the respective values in group 1 (13% and 11%, respectively, p<0.05); in the meantime, mo was higher (13% and 11%, respectively, p<0.05), amo was lower (73% and 32%, respectively, p<0.05) and δx was higher (63% and 29%, respectively p<0.05). all integral parameters were significantly lower than comparative ones; in part, this was true of hsi (in 3.3 times and 1.9 times, p<0.05), vbi (in 2.9 times and 1.7 times, p<0.05), vri (in 1.9times and 1.4times, p<0.05) and iarp (in 2.0 times and 1.5 times, p<0.05). the differences between group 1 and group 2 in the females in 1 hour after adrenaline administration were for hr (by 7% lower, p<0.05), mo (by 7% higher, p<0.05) and δx (36% higher, p<0.05); the values of hsi, vbi and vri were lower (in1.7 times, 1.6 times and 1.4 times, respectively, p<0.05). in 24 hours after adrenaline administration, no significant differences were evidenced regarding the parameters. discussion analysis of heart rhythm variability is performed by a non-invasive method of functional diagnostics, which is used not only in a clinical setting, but also in experimental studies. it allows assessing of cardioregulatory ans activity, determining the balance between activities of sympathetic and parasympathetic components as they affect the heart rhythm, making a conclusion about the predominant table 2. parameters of heart rhythm variability in the rats with developing adrenaline-induced myocardial necrosis in cases of light deprivation, (m±ō) parameter gender controls (n=6) 1 hours post-adrenaline (n=6) 24 hours post-adrenaline (n=6) hr ♂ 512±14^# 469±21*^ 443±29*^ ♀ 435±11# 463±11*^ 461±22* mo (sec) ♂ 0.117±0.003^# 0.128±0.006*^ 0.136±0.009*^ ♀ 0.138±0.003# 0.130±0.003*^ 0.130±0.006* amo (%) ♂ 38.3±7.8^ 34.3±3.7^ 33.0±2.7^ ♀ 32.7±4.4 38.3±5.2 40.5±11.6 δx (sec·10-2) ♂ 0.70±0.13 0.57±0.08*^# 0.75±0.08^# ♀ 0.58±0.04 0.45±0.08*^# 0.50±0.14# hsi (abs. value) ♂ 24730±9622 24464±6557^# 16398±2345^# ♀ 20535±3552 33835±7738*^# 34836±17417*# vbi (abs. value) ♂ 5672±2123 6223±1446^# 4458±846^# ♀ 5642±939 8732±1891*^# 9118±4656*# vri (abs. value) ♂ 1257±234 1405±244^ 996±118^# ♀ 1256±90 1762±299*^ 1657±517# iarp (abs. value) ♂ 0.326±0.060# 0.268±0.039^# 0.243±0.010*^ ♀ 0.238±0.036# 0.296±0.043# 0.309±0.078 note: * – statistically significant (p≤0.05) differences relative to the controls, ^ – relative to the findings in the group 1 animals during the same observation period, # – relative to the animals of the opposite sex. h.o. bezkorovaina et al. 141 b io m e d ic a l s c ie n c e s issn 2413-6077. ijmmr 2019 vol. 5 issue 2 h.o. bezkorovaina et al. component, and assessing the stress upon regulatory systems as part of adjustment to adverse influences [15]. analysis of histograms reflecting the distribution of rr intervals (va­ riational pulsography) was used in the study. assessment of the results involved use of a cybernetic double-circuit model for heart rhythm regulation as suggested by r.m. bayevsky: the central circuit (cerebral cortex, higher autonomic centres and the cardiovascular centre) and the autonomic circuit (sinus node, lungs and the respiratory centre) [16]. the patterns of changes in experimental findings with time allowed drawing a conclusion that significant difference in ans effects on the heart rhythm occurred between male and female animals. in males, development of adrenaline-induced myocardial necrosis under normal day/night balance was accompanied by an increased impact of sympathetic ans component, as evidenced by increased levels of hsi, vbi, vri and iarp. a more significant increase in test parameters in 1 hour of the experiment was predictable due to a presence of hypercatecholaminemia caused by administration of adrenaline. the effects of adrenaline were not limited to the sinus node, but also extended to myocardial contractility. this was also true regarding its metabolite, adrenochrome [17]. the amount of adrenochrome increased significantly under our experiment as a result of catabolism and an active part of myocardial damage through stimulation of free radical processes [18]. under the circumstances, ans response to hypercatecholaminemia-induced oxidative stress was also a matter of discussion. this was confirmed by ipsidirectional but less significant changes in ans responses in the females. in this model, the severity of myocardial damage was substantially lower in the females [19]. in this case, the increase in amo reflected strengthening of the central circuit of heart rhythm regulation, and the decrease in δx reflected the reduced involvement of the autonomic circuit, i.e. the role of the vagus nerve. this data is consistent with the literature on using a model with adrenaline-induced myocardial necrosis. it was established that not only hypercatecholaminemia, but also sex hormones were crucial in the capacity of ans to develop other adaptive effects [20, 21]. in 24 hours after adrenaline administration, the primary effects of adrenaline diminished according to the pattern described. in the males, all of the parameters were normal again, which was proved by a recovery of the baseline balance between ans components. in the females, all pulsographic parameters demonstrated retention of increased sympathetic activity, which was the principal difference between the males and females. the males of group 2 responded to a 10-day stay in darkness with increased hr and with an accordingly reduced mo. this reflected the state of stress and the increased involvement of humoral adaptive mechanisms, which were implemented by the adrenal glands [22]. the maintenance of regulatory balance under such conditions was attained at the expense of reduced activity of the central regulatory circuit, as confirmed by a reduction in amo. therefore, adjustment of the males in group 2 to permanent darkness caused a moderate activation of sympathetic cardiac effects. in such a situation, myocardial necrosis developed under activation of the parasympathetic component and predominance of the latter in heart rhythm control. this was confirmed by higher (compare to the group 1 animals) δx values and by lower values of hsi, vri, vbi and iarp. lower amo values have demonstrated that the predo minance of parasympathetic ans component was also facilitated by the reduced activity of sympathetic ans component. a 10-day stay of the group 2 females under conditions of permanent darkness did not cause any functional changes in the ans, as confirmed by the absence of significant dif­ ferences in all parameters in the group 1 and group 2 females. in 1 hour after administration of adrenaline, hr increased and mo decreased in the females of group 2, which reflected an increase in adrenergic effects of ans in the heart, with the effects implemented through the humoral channel (mainly the adrenal glands). the reduction in δx suggested a de­ crease in vagal cardiac effects. as a natural result, hsi, vbi and vri values increased. the constancy of iarp reflected a preserved balance between the central and the autonomic circuits of heart rhythm control. in 24 hours after adrenaline administration, hr was still in creased, and mo was lower than in the controls of this group. to a greater degree than in the controls, the hsi and vbi values demonstrated a predominance of sympathetic ans effects in heart rhythm control, but the stability of iarp suggested a preserved balance between the activities of the central and the autonomic control circuits. it is important that under developing myocardial necrosis, the changes of parameters with time were similar to those 142 b io m e d ic a l s c ie n c e s issn 2413-6077. ijmmr 2019 vol. 5 issue 2 in the group 1, although less significant. in particular, mo was higher in the group 2 females compare to those of group 1, which suggested a less active involvement of adrenal glands under the simulated conditions [22]. in this case, there was no involvement of the central control circuit, as confirmed by the ab­ sence of significant amo changes. this demonstrated a lack of response of cerebral cortex to hyperadrenalinemia. despite the increases in hsi, vbi and vri values in 1 hour of myocardial necrosis (which reflected a phase of hyperadrenalinemia), these values were significantly lower than in the group 1 females. to a degree greater than in the group 1 animals, the δx value reflected a more active involvement of parasympathetic mechanisms in cardiac adjustment to damage, which maintained a stable iarp and preserved a baseline balance between central and autonomic control circuits, unlike the group 1 females, where the control balance shifted towards predominance of sympathetic ans activity. the development of necrotic process in the myocardium of the group 2 males was accompanied by activation of cholinergic mechanisms of cardiac control, which was more significant than in the females. this proved that the males and females employed different mechanisms of adjustment to adverse effects of adrenaline under light deprivation. these differences may be associated with different levels of melatonin synthesis by the pineal gland under conditions of permanent darkness [9, 23]. the females synthesises more melatonin at night-time [10] and their melatonin synthesis during sleep peaked sooner than in the males [24]. considering that all animals in the experiment were under identical conditions, higher melatonin levels in the females could be asserted. however, a clearer understanding of the significance of endogenous melatonin within a framework of adaptive responses with ans involvement in modelling of adrenaline-induced myocardial necrosis might yield the results attained in modelling of cardiac disease under permanent lighting, which was the prospect of the research. in summary, it should be noted that the attained data have proved that the males and females have different ans responses to developing adrenaline-induced myocardial necrosis under light deprivation that suggests the necessity to specify the gender of animals in the conclusions and the incorrectness of extrapolating the established patterns to animals of other sex. conclusions a 10-day light deprivation activates the heart rhythm impact of the sympathetic component of ans in the male rats and does not alter the baseline balance of sympathetic and parasympathetic components of ans in the females. the development of adrenalineinduced myocardial necrosis under 10-day light deprivation is characterised by a higher (compared today/night cycle) involvement of the parasympathetic ans component in heart rhythm control in the males and by development of bradycardia. in the females, light deprivation facilitates the maintenance of sympathetic/ parasympathetic balance in cases of adrenalineinduced myocardial necrosis. this differs from the females under the day-night cycle, where an increase in the sympathetic component of the ans was evidenced in cases of developing myocardial necrosis. funding this research received no external funding. conflict of interests the authors declare no conflict of interest. authors contributions bezkorovaina h.o. – conceptualization, formal analysis, investigation, methodology, visualization, writing – original draft; klishch i.m. – conceptualization, supervision, resources; khara m.r. – formal analysis, investigation, methodology; pelykh v.ye. – investigation. статева відмінність вегетативної регуляції серця щурів при адреналін-індукованому некрозі на тлі світлової депривації г.о. безкоровайна, і.м. кліщ, м.р. хара, в.є. пелих тернопільський національний медичний університет імені і.я. горбачевського, тернопіль, україна bступ. серцево-судинні захворювання є головною причиною захворюваності у світі. у когорті хворих на ішемічну хворобу серця переважають чоловіки. серед факторів ризику головним залишається стрес, в тому ж числі на ґрунті світлового десинхронозу. h.o. bezkorovaina et al. 143 b io m e d ic a l s c ie n c e s issn 2413-6077. ijmmr 2019 vol. 5 issue 2 h.o. bezkorovaina et al. мета дослідження. вивчити гендерні особливості вегетативної регуляції серця при адреналініндукованому некрозі міокарда в щурів на тлі світлової деривації. методи дослідження. у 72 білих статевозрілих самців і самиць щурів досліджували варіабельність серцевого ритму (вср) при адреналін-індукованому некрозі міокарда (анм) на тлі світлової депривації (сд). тварин поділили на 2 групи: г1 – тварини були в умовах циклу день/ніч (12 год/12 год); та г2 – щури перебували 10 днів в умовах світлової депривації (сд). на 11-й день моделювали анм (адреналін в/м, 0.5 мг/кг) і вср вивчали через 1 та 24 год. результати. розвиток анм у ♂г1 і ♀г1 викликав зменшення варіабельності серцевого ритму (вср), посилення симпатичних впливів на серце, що було суттєвішим в ♂. сд викликала посилення симпатичних впливів на серце в ♂г2. розвиток анм в ♂г2 викликав суттєве збільшення вср та значне зменшення чсс. це було результатом посилення парасимпатичних впливів на серце та зменшення симпатичних. у ♀г2 в умовах анм динаміка показників вср була аналогічною до такої в ♀г1. проте, на 1 год анм активність парасимпатичних впливів на серце була більшою, ніж в ♀г1. якщо в ♀г2 в умовах анм зберігався баланс між активністю симпатичної та парасимпатичної ланок, то в ♀г1 в умовах анм переважала активність симпатичної ланки. висновки. світлова депривація посилює симпатичні впливи на ритм серця в самців щурів і не змінює вихідного балансу активності симпатичної та парасимпатичної ланок в самиць. розвиток адреналін-індукованого некрозу міокарда на тлі світлової депривації характеризується більшою, ніж за збереженого балансу день/ніч, активністю парасимпатичної ланки в регуляції ритму серця самців та викликає розвиток брадикардії. у самиць світлова депривація сприяє підтриманню балансу між активністю симпатичної та парасимпатичної ланок в умовах адреналін-індукованого некрозу міокарда, на відміну від самиць, що перебували в умовах зміни циклу день/ніч і демонстрували посилення активності симпатичної ланки анс при розвитку некрозу міокарда. kлючові слова: некроз міокарда; варіабельність серцевого ритму; світлова депривація, стать відомості про авторів безкоровайна галина олександрівна – лікар, здобувач кафедри патологічної фізіології тернопільського національного медичного університету імені і.я горбачевського. кліщ іван миколайович – доктор біологічних наук, професор кафедри функціональної і лабораторної діагностики тернопільського національного медичного університету імені і.я горбачевського. хара марія романівна – доктор медичних наук, професор кафедри патологічної фізіології тернопільського національного медичного університету імені і.я горбачевськогою пелих володимир євгенович – кандидат медичних наук, доцент кафедри патологічної фізіології тернопільського національного медичного університету імені і.я горбачевськогою information about the authors bezkorovaina h.o. – md, ph.d. student, i. horbachevsky ternopil national medical university, ternopil, ukraine. e­mail: 111ya111@ukr.net. klishch i.m. – md, ph.d., dsc, professor, functional and clinical diagnostics department, i. horbachevsky ternopil national medical university, ternopil, ukraine. orcid 0000­0001­6226­4296, e­mail: klishch@tdmu.edu.ua. khara m.r. – md, ph.d., dsc, professor, pathophysiology department, i. horbachevsky ternopil national medical university, ternopil, ukraine. orcid 0000­0002­6028­9876, e­mail: khara@tdmu.edu.ua pelykh v.ye. – md, phd, associate professor of pathophysiology department, i. horbachevsky ternopil national medical university, ternopil, ukraine. orcid 0000­0003­3468­858x, e­mail: pelykh_ve@tdmu.edu.ua references 1. townsend n, wilson l, bhatnagar p, wickramasinghe k, rayner m, nichols m. cardiovascular disease in europe: epidemiological update 2016. european heart journal. 2016 aug 14;37(42):3232­45. 2. roth ga, forouzanfar mh, moran ae, barber r, nguyen g, feigin vl, naghavi m, mensah ga, murray cj. demographic and epidemiologic drivers of global cardiovascular mortality. new 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[in russian] 23. talaei sa, sheibani v, salami m. light deprivation improves melatonin related suppression of hippocampal plasticity. hippocampus. 2010;20(3): 447-55. doi: 10.1002/hipo.20650 24. cain sw, dennison cf, zeitzer jm, guzik am, khalsa sb, santhi n, schoen mw, czeisler ca, duffy jf. sex differences in phase angle of entrainment and melatonin amplitude in humans. j biol rhythms. 2010;25(4):288­96. doi: 10.1177/0748730410374943 received 05 september 2019; revised 13 october 2019; accepted 23 october 2019. this is open access article distributed under the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. h.o. bezkorovaina et al. 37 r a d ia t io n m e d ic in e a n d o n c o l o g y issn 2413-6077. ijmmr 2021 vol. 7 issue 2 doi 10.11603/ijmmr.2413-6077.2021.2.12253 comparative study of concurrent chemoradiation using paclitaxel in two histopathological subtypes (squamous cell carcinoma/adenocarcinoma) of unresectable non-small cell lung cancer d.l. nongrum1, *y.s. devi2, s. mohanty3, l.j. singh2, k. baidya2, d. chyrmang2, h.k. rai2 1 – civil hospital, shillong, meghalaya, india 2 – department of radiation oncology, regional institute of medical sciences, imphal, manipur, india 3 – department of medical oncology, kilpauk medical college, chennai, tamil nadu, india background. lung cancer is still a global burden and with rising population and increasing life expectancy the incidence of lung cancer is still on the rise. objective. to compare the treatment response and toxicity of weekly paclitaxel in locally advanced unresectable non-small cell lung cancer (nsclc), when administered concurrently with external beam radiation to the chest in two different histopathological types – adenocarcinoma and squamous cell carcinoma. methods. a prospective randomised control trial was conducted in 60 nsclc patients who were divided into two arms; adenocarcinoma and squamous cell carcinoma arm. all patients were treated with chemoradiation with concurrent paclitaxel 60 mg/m2. data were evaluated with spss version 21.0 for windows with p-value <0.05. results. haematological toxicity was the most common side effects evident from the third week of chemotherapy. at the end of 1 month of treatment, two (6.7%) patients had complete response in arm a and one (3.3%) patient had complete response in arm b. one (3.3%) patient had disease progression in arm a and two patients progressed in arm b. at 7 months post treatment three (10%) patients had complete response in both arm a and arm b. four (13.3%) patients had disease progression in arm a and ten (33.4%) patients progressed in arm b. conclusions. paclitaxel can be used as an alternative chemotherapeutic agent to the standard cisplatin. however, further studies with larger sample size are required to confirm the findings. keywords: unresectable; concurrent; adenocarcinoma; squamous cell. *corresponding author: yumkhaibam s. devi, assistant professor, department of radiation oncology, regional institute of medical sciences, imphal, manipur, postal code: 795004, india. e-mail: ysobita@gmail.com international journal of medicine and medical research 2021, volume 7, issue 2, p. 37-46 copyright © 2021, tnmu, all rights reserved d.l. nongrum et al. introduction lung cancer is one of the commonest cancers and the most common cause of cancer related mortality all over the world [1]. lung cancer comprises two main histopathological groups non-small cell lung cancer (nsclc) and small cell lung cancer (sclc). the major histologic subclasses of nsclc are adenocarcinoma (50%), squamous cell carcinoma (30-40%) and undifferentiated large cell carcinoma (10%) [2]. approximately 80% of cases of non-small cell lung cancer (nsclc) in men and 50% of these neoplasms in women worldwide are directly attributable to cigarette smoking. other contributing factors includes passive smoking, genetic predisposition to this disease, occu pational and environmental exposures including asbestos and silica fibres and ionizing radia­ tion [3]. more than 70% of patients diagnosed with lung cancer present with advanced stage disease (stage iii or iv) that is usually beyond surgical intervention [4]. according to the 7th edition a jcc staging classification, stage iii nsclc is often defined as locally advanced nsclc. stage iiia (t1-3 n2, t3-t4 n1, t4 n0) disease involves hilar or mediastinal lymph nodes limited to the ipsilateral mediastinum and a subset of these patients are amenable to surgery. however, stage iiib (t1-4 n3, or t4 n2) involves lymph node metastasis in the contralateral thorax or supraclavicular fossa and/ or an unresectable primary tumour, making patients with this disease not ideal candidates 38 r a d ia t io n m e d ic in e a n d o n c o l o g y issn 2413-6077. ijmmr 2021 vol. 7 issue 2 for surgical resection [5]. concurrent chemoradiation is the mainstay of treatment in patients with locally advanced, unresectable, non-small-cell lung cancer which improved survival by reducing local tumour burden and also delaying the emergence of metastatic disease [6]. long-term outcomes are poor, with baseline 5-year overall survival (os) of 15%-35% for stage iiia and 5%-10% for stage iiib [7]. the platinum-based combination regimens are considered to be the standard treatment. but due to high incidence of platinum induced chemotoxicities and platinum resistance in many cases, a third-generation chemo therapeutic agent taxens e.g. paclitaxel were tried; they possess good activity as single agent in cases of nsclc resulting in the arrest of cells in the g2-m phase of the cell cycle, which is particularly responsible for much of the radiosensitizing ability of paclitaxel [8]. even though both adenocarcinoma and squamous cell carcinoma are grouped as nsclc, both the subtypes differ in many aspects. the monolithic treatment approach to both types of nsclc has dramatically changed over last few years with the advent of molecular subtyping and novel histology specific targeted therapies [9]. though, several studies have established the role of paclitaxel in nsclc, the relative outcome response in different histopathologies (adenocarcinoma/squamous cell carcinoma) is still not clear. hence, the present study will be aiming to compare treatment response and treatment toxicity patterns between unre sectable adenocarcinoma and squamous cell carcinoma of lung treated with concurrent chemoradiation using paclitaxel. methods a randomized control trial had been un dertaken in the department of radiation oncology, regional institute of medical sciences, imphal, manipur, over a period of 2 years starting from august 2017 to july 2019. the permission of the research ethics board rims, imphal, manipur, was obtained to conduct the study. initial 18 months was for patient accrual; the study and result analysis were performed after allowing minimum of 6 months follow-up for the patients. the patients, who were histopathologically confirmed cases of unresectable non­small cell lung cancer (adenocarcinoma/squamous cell carcinoma) reporting to the department of radiation oncology, rims, imphal, manipur, with karnofsky performance status (kps) ≥60%, age below 80 years, without any major comorbidity and willing to give consent, were included in the study. the sample size is calculated using the formula: 𝑁𝑁𝑁𝑁 = (𝑢𝑢𝑢𝑢 + 𝑣𝑣𝑣𝑣)2 [{𝑝𝑝𝑝𝑝1 (100 − 𝑝𝑝𝑝𝑝1)}] + [{𝑝𝑝𝑝𝑝2 (100 − 𝑝𝑝𝑝𝑝2)}] 𝑝𝑝𝑝𝑝1 − 𝑝𝑝𝑝𝑝2 where n is the size per group, u=0.84 at 80% power, v=1.645 at 90% level of significance p1= proportion in one group =100, p2= proportion in another group =81 [10]. therefore, sample size of 60 (30 patients in each arm) will be considered for the study. patients were distributed into two separate arms – arm a (adenocarcinoma) and arm b (squamous cell carcinoma). both arms received external beam radiation therapy (ebrt) by cobalt-60 teletherapy machine (theratron 780­c. model number: a112109­101) with a source to skin distance (ssd) of 80 cm to a total tumour dose of 60 gy over 30 fractions five days in a week for six weeks by two opposing postero­anterior fields. spinal cord was spared after 46gy/23 fractions. concurrent chemotherapy with injection paclitaxel at a dose of 60 mg/m2 in 500 ml 0.9% normal saline over 3 hours intravenous weekly before radiotherapy for 6 weeks was administered along with all the necessary pre-medications. during concurrent chemoradiation (ccrt), the patients were evaluated weekly for development of any skin, pulmonary or oesophageal toxicity. acute treatment toxicity was evaluated weekly during course of treatment and late treatment toxicity was evaluated monthly till the end of the treatment in accordance with rtog criteria [11]. the early treatment response was assessed at 1 month and it was assessed again at 7 months following completion of ccrt, in accordance with recist criteria [12]. after completion of ccrt, patients were followed up monthly for a minimum period of 6 months and thereafter every 2 months. statistical analysis: descriptive data like age was presented in terms of mean and standard deviation. data like sex, stage, response and toxicity profile was presented in terms of percentages and proportions. data entry and statistical analysis was conducted using ibm spss statistics 21 for windows (ibm corp, 1995, 2012). statistical significance was analysed using the chi square and fisher’s exact test and p-value of <0.05 was considered statistically significant. d.l. nongrum et al. 39 r a d ia t io n m e d ic in e a n d o n c o l o g y issn 2413-6077. ijmmr 2021 vol. 7 issue 2 results it is established that most of the patients fall in the age range of 61-70 years with 46.7% in arm a and 50% in arm b. we can also appreciate that in younger age group 51-60 years adenocarcinoma lung are more than squamous cell carcinoma (40% vs 30%) while in older age group squamous cell carcinoma are slightly more than adenocarcinoma in this study. out of 30 patients in arm a, 17 (56.7%) patients were female and 13 (43.3%) patients were male. in arm b, 19 (59.4%) patients were male and 11 (39.3.%) were female. the sex-wise distribution shows that adenocarcinoma is more common in females whereas squamous cell carcinoma is more common in males. majority of the patients had 80% kps where 15 (43.3 %) patients were in arm a and 16 (53.3%) patients in arm b. in arm a adenocarcinoma patients, cough was the most common presentation followed by dyspnoea, chest pain, and haemoptysis. in arm b squamous cell cancer patients, cough also was the most common presentation followed by haemoptysis, dyspnoea and chest pain. in arm a, 46.7% disease were found in the right lung whereas 53.3% of disease were found in the left lung. in arm b, 43.3% disease were found in the left lung whereas 56.67% of disease were found in the right lung. in stage iiia, 8 (26.6%) patients were in arm a and 10 (33.3%) in arm b. while in stage iiib, 22 (73.3%) and 20 (66.7%) were in arm a and arm b respectively. this distribution shows the p-value of 0.389 which is statistically insignificant (fig. 1). early toxicities particularly nausea/vomiting and haematological parameters were assessed after each cycle of chemotherapy. during radiation treatment lung and oesophageal toxicity were assessed every week for 6 weeks. the most common side effects during ct were anaemia (63.33% in arm a and 53.33% in arm b) during the third week, neutropenia (36.66% and 33.33% in arm a and arm b respectively) seen mostly during the 3rd week, thrombo cytopenia (16.66% and 40% in arm a and arm b respectively) during the 4th cycle of ct and were mostly grade 1. none of the patients in both arms experienced peripheral neuropathy. the side effects of rt were mostly seen from the 3rd week after starting of treatment in both arms and the most common toxicity experienced was grade 1 lung and esophagus toxicity (table 1). in 1 month of treatment the result shows a significant improvement of the symptoms in both the arms. arm a shows most significant improvement in dyspnoea and chest pain with the p-value of 0.487 and 0.471, respectively, which was statistically not significant. whereas arm b shows improvement in cough with the p-value of 0.128 and haemoptysis with the p-value of 0.487, which were statistically not significant (table 2). the median follow-up duration of patients was estimated to be 17±2.87 months in arm a and 17±3.73 months in arm b (p-value 0.634). all the 60 patients (in both arms) were available for assessment at the end of the 1st month. two complete responses and 24 partial responses were obtained in arm a (adenocarcinoma arm). one complete response and 23 partial responses were obtained in the arm b (squamous cell carcinoma arm). the response rates were 86.66% with arm a and 79.99% with arm b (all assessable patients). the differences were statistically significant (p=0.000). the disease fig. 1. stage distribution in both the arms. 0 5 10 15 20 25 stage iiia stage iiib 8 22 10 20 arm a arm b d.l. nongrum et al. 40 r a d ia t io n m e d ic in e a n d o n c o l o g y issn 2413-6077. ijmmr 2021 vol. 7 issue 2 stabilized in three patients in arm a and in four patients in the arm b. early progression (during therapy) occurred in 1 patient in treatment arm a (adenocarcinoma) and 2 patients in arm b (squamous cell carcinoma). in adenocarcinoma arm, 77.77% of the patients in stage iiia and 90.47% of the patients in stage iiib had a good response to the treatment, whereas about 80% of the patients in both stage iiia and stage iiib had a good response to the treatment in squamous cell carcinoma arm (table 3). the late side effects of treatment were assessed as per rtog criteria [11]. in both arm a and arm b, toxicities were assessed monthly table 1. early radiation toxicity symptom week grade arm a arm b cough week 3 1 20 (66.67%) 21 (70%) week 4 1 21 (70%) 21 (70%) week 5 1 23 (76.7%) 23 (76.7%) week 6 1 23 (76.7%) 23 (76.7%) esophagitis week 3 1 18 (60%) 19 (63.3%) 2 6 (20%) 4 (13.33%) week 4 1 16 (53.33%) 11 (36.66%) 2 7 (23.33%) 4 (13.33%) week 5 1 19 (63.3%) 19 (63.3%) 2 6 (20%) 3 (10%) 3 1 (3.33%) 0 (0%) week 6 1 18 (60%) 18(60%) 2 5 (16.7%) 3 (10%) 3 3 (10%) 0 (%) nausea/vomiting week 3 1 3 (10%) 4 (13.33%) week 4 1 6 (20%) 2 (6.66%) week 5 1 2 (6.66%) 2 (6.66%) week 6 1 2 (6.6%) 3 (10%) haemoglobin week 3 1 19 (63.33%) 16 (53.33%) 2 0 (0%) 3 (10%) week 4 1 14 (46.66%) 13 (43.33%) 2 5 (16.66%) 1 (3.33%) week 5 1 16 (53.33%) 11 (36.66%) 2 3 (10%) 1 (3.33%) week 6 1 16 (53.33%) 5 (16.66%) 2 3 (10%) 0 (0%) tlc week 3 1 11 (36.66%) 10 (33.33%) 2 0 (0%) 2 (6.66%) week 4 1 6 (20%) 9 (30%) 2 5 (16.66%) 3 (10%) week 5 1 6 (20%) 9 (30%) 2 5 (16.66%) 3 (10%) week 6 1 9 (30%) 9 (30%) 2 3 (10%) 3 (10%) platelet week 3 1 9 (30%) 10 (33.33%) 2 0 (0%) 2 (6.66%) week 4 1 5 (16.66%) 12 (40%) 2 0 (0%) 1 (3.33%) week 5 1 5 (16.66%) 11 (36.66%) 2 0 (0%) 1 (3.33%) week 6 1 5 (16.66%) 6 (20%) 2 0 (0%) 1 (3.33%) peripheral neuropathy – – d.l. nongrum et al. 41 r a d ia t io n m e d ic in e a n d o n c o l o g y issn 2413-6077. ijmmr 2021 vol. 7 issue 2 after completion of treatment for 6 months. the most common late side effects of treatment were grade 1 lung fibrosis (50% in arm a and 46.7% in arm b) at the 6th month, grade 1 oesophageal toxicity (50% in arm a and 40% in arm b) at the 6th month and grade 1 cardiac toxicity (16.7% and 3.3% in arm a and arm b respectively). none of the patients in both arms experienced myelitis or nephrotoxicity (table 4). at the end of the 7th months in arm a, 17 (56.6%) patients had partial response, 6 (20%) patients had stable disease, 3 (10%) – complete response and 4 (13.3%) – progression of the diseases. about 23.3% of the patients in stage iiia and 43.3% of the patients in stage iiib had a good response to the treatment. in arm b, 3 (10%) patients had complete response, 12 (40%) patients – partial response, 5 (16.6%) patients – stable disease and 10 (33.4%) – pro gression of the disease. late treatment res pon ses were statistically significant (p­value 0.000) (table 5). discussion the two study groups were formed by histopathology – arm a adenocarcinoma, arm b squamous cell carcinoma, but all other patient characteristics in both groups were well balanced without statistically significant diffe­ rences in age, stage, kps. in this study, adenocarcinoma lung was more than squamous cell carcinoma (40% vs 30%) in the younger age group, while in the older age group squamous cell carcinoma was slightly more than adenocarcinoma. this is in consistent with the study conducted by a.l. rich et al [13]. the sex wise distribution was similar to the study by price pw et al, where adenocarcinoma was more common in women than in men (41% versus 31%, p<0.0001) and squamous cell carcinoma more common in men than women (43% versus 31%, p<0.0001) [14]. toxicity profile (acute toxicities) in this study, during 6 weeks of treatment it was observed that toxicities were mostly seen at the 3rd week after treatment starting with grade 1 lung and esophageal toxicity being the most common in both the arms. this was similar to a study conducted by huber rm et al, table 3. early treatment response at the end of the 1st month, arm a versus arm b treatment response treatment arm p-value arm a arm b cr 2 (6.66%) 1 (3.33%) 0.000* pr 24 (80%) 23 (76.66%) sd 3 (3.33%) 4 (13.33%) pd 1 (3.33%) 2 (6.66%) total 30 30 note. * – fisher’s exact test. table 2. symptomatic response before and after treatment symptoms grade pre-treatment symptoms symptoms after 1 month of treatment p-value arm a arm b arm a arm b cough 1 20 (66.66%) 9 (30%) 13 (43.33%) 12 (40%) 0.128*2 5 (16.66%) 13 (43.33%) 5 (16.66%) 0 (0%) 3 0 (0%) 0 (0%) 0 (0%) 0 (0%) chest pain 1 10 (33.33%) 4 (13.33%) 8 (26.66%) 7 (23.33%) 0.471*2 5 (16.66%) 4 (13.33%) 0 (0%) 2 (6.66%) 3 0 (0%) 0 (0%) 0 (0%) 0 (0%) dyspnoea 1 19 (63.33%) 8 (26.6%) 6 (20%) 5 (16.66%) 0.487*2 4 (13.33%) 0 (0%) 2 (6.66%) 0 (0%) 3 0 (0%) 0 (0%) 0 (0%) 0 (0%) haemoptysis 1 6 (20%) 9 (30%) 5 (16.66%) 6 (20%) 0.487*2 1 (3.33%) 5 (16.66%) 0 (0%) 2 (6.66%) 3 0 (0%) 0 (0%) 0 (0%) 0 (0%) note. * – fisher’s exact test. d.l. nongrum et al. 42 r a d ia t io n m e d ic in e a n d o n c o l o g y issn 2413-6077. ijmmr 2021 vol. 7 issue 2 table 4. side effects of treatment, assessed monthly for 6 months post treatment adverse effects arm a arm b p-value lung fibrosis month 1 grade 1 15 (50%) 16(53.3%) 0.000*grade 2 2 (6.7%) 2 (6.7%) grade 3 0 (0%) 0 (0%) month 2 grade 1 15 (50%) 19 (63.3%) 0.000*grade 2 2 (6.7%) 2 (6.7%) grade 3 0 (0%) 0 (0%) month 3 grade 1 18 (60%) 17 (56.7%) 0.000*grade 2 3 (10%) 3 (10%) grade 3 0 (0%) 0 (0%) month 4 grade 1 17 (56.7%) 17 (56.7%) 0.000*grade 2 3 ( 10%) 3 (10%) grade 3 1 (3.3%) 2 (6.7%) month 5 grade 1 19 (63.3%) 17 (56.7%) 0.000*grade 2 5 (16.7%) 3 (10%) grade 3 2 (6.7%) 2 (6.7%) month 6 grade 1 15 (50%) 14 (46.7%) 0.000*grade 2 8 (26.7%) 7 (23.3%) grade 3 2 (6.7%) 3 (10.0%) dysphagia month 1 grade 1 5 (16.7%) 4 (13.3%) 0.000*grade 2 0 (0%) 0 (0%) grade 3 0 (0%) 0 (0%) month 2 grade 1 7 (23.3%) 6 (20%) 0.000*grade 2 0 (0%) 0 (0%) grade 3 0 (0%) 0 (0%) month 3 grade 1 7 (23.3%) 7 (23.3%) 0.000*grade 2 2 (6.7%) 1 (3%) grade 3 0 (0%) 0 (0%) month 4 grade 1 9 (30%) 10 (33.3%) 0.000*grade 2 3 (10%) 2 (6.7%) grade 3 0 (0%) 0 (0%) month 5 grade 1 9 (30%) 11 (36.7%) 0.000*grade 2 5 (16.7%) 3 (10%) grade 3 0 (0%) 0 (0%) month 6 grade 1 15 (50%) 12 (40%) 0.000*grade 2 6 (20%) 3 (10%) grade 3 0 (0%) 0 (0%) cardiac toxicity grade 1 5 (16.7%) 1 (3.3%) 0.000*grade 2 0 (0%) 0 (0%) grade 3 0 (0%) 0 (0%) myelitis – – nephrotoxicity – – note. * – fisher’s exact test. table 5. treatment response at the end of the 7th months late treatment response p-value cr pr sd pd 0.000arm a 3 (10%) 17 (56.6%) 6 (20%) 4 (13.3%) arm b 3 (10%) 12 (40%) 5 (16.6%) 10 (33.4%) d.l. nongrum et al. 43 r a d ia t io n m e d ic in e a n d o n c o l o g y issn 2413-6077. ijmmr 2021 vol. 7 issue 2 where grade 3 esophageal toxicity was seen in 13% [15], but was much lower than rtog 94-10 trial, where grade 3 esophageal toxicity with ccrt was seen in 22% [16]. haematological toxicities were also present and comparable in both arms. other studies also showed that neutropenia was a common toxicity with paclitaxel [17,18]. however, in this study, in both arms none of the patients experienced peripheral neuropathy. this may be due to the low dose of injection paclitaxel (60 mg/m2) used in the study [19]. nausea and vomiting were not common and grade 1 nausea was present in only 10% and 13.33% in arm a and arm b, respectively, at week 3. this may be due to low emetogenic potential of paclitaxel that has also been proven by other studies [20,21]. tumour response rate after treatment the response rates in both the arms were similar to the rates in other trials [22,23]. the findings of this study are consistent with that by choy h et al, where 86% of overall response rate have been achieved: adenocarcinoma having 100% partial response and squamous cell carcinoma having 86% partial response. patients with stage iiib disease responded equally to stage iiia disease (82% and 92%, respectively; p=0.62) [10]. in this study, since the response rates achieved in both arms are comparable, a conclusion could be drawn that re s p o n s e ra t e s o f w e e k l y p a c l i t a xe l i n adenocarcinoma lung and squamous cell carcinoma lung are similar. symptomatic response post treatment buccheri g et al conducted a single institute study on lung cancer clinical presentation and found that the most alarming symptoms with adenocarcinoma lung was cough (18.4%) followed by chest pain (13.7%), bloody sputum (13.4%) and dyspnoea (11.7%). the alarming symptoms of squammous cell carcinma were bloody sputum (24%) followed by cough (19%), chest pain (10.7%) and dyspnoea (10.4%) [24]. this was almost similar with our study. regarding assessment of symptom response, in 1 month significant improvement of all the symptoms in both the arms was evidenced that is similar to the studies by barwal kv et al [25] and langendijk et al [26]. treatment response (at the end of the 7th months): at the end of the 7th months, 4 patients in arm a and 10 patients in arm b had disease progression in this study. bone is the most frequent site of distant metastasis followed by liver and brain. in a study conducted by liew sm et al locoregional, contralateral relapses, and distant metastases were observed in 34 (45%), 16 (21%), and 47 (63%) patients, respectively. among the 47 patients with late relapse, bone metastases were observed in 16 (34%) patients and were the most frequent site of distant metastases. this was followed by liver (n=13.28%), brain (n=12.26%), and adrenal (n= 4.9%) [27]. those with adenocarcinoma showed adrenal metastases in 54% of cases followed by liver metastases (27%). squamous cell carcinoma spread to the liver in 67% of cases as well as to adrenal glands and bones (33% each) [28]. toxicity assessment (late toxicities) in this study after 6 months of treatment grade 1 lung fibrosis was evidenced in 50% of the patients in arm a and 46.7% of the patients in arm b after radiological assessment with chest x-ray and/or ct scan thorax. this was much higher than the old rtog data, where the average incidence of pneumonitis (grade 2 and above) and fibrosis was 14.6% and 28%, respectively, after 2drt [29]. grade 1 cardiac toxicity was seen in 16.7% of patients in arm a and 3.3% of patients in arm b. arm a had a slightly higher percentage of cardiac toxicity. this might have been due to greater number of left sided lung tumours in arm a (53.33%) compare to arm b (43.33%), where portion of heart could not be avoided in the radiation. in this study grade 1 oesophageal toxicity was another radiation toxicity with no grade 3 or higher esophageal toxicity evidenced in both arms. a study conducted by curran jw et al found that in ccrt arm grade 3 esophageal toxicity was present in 3% patients and grade 4 esophageal toxicity – in 1% patients only [16]. conclusions locally advanced non-small cell lung cancer (la nsclc) comprises the most heterogeneous group of patients. over the years even with continuous evolution of treatment strategies, overall survival (os) is still low and management of stage iii la nsclc is still a challenge today. the results after 1 month showed significant improvement of symptoms in both the arms and the responses were comparable in both the arms. this study is one of the first ones on comparison of efficacy of paclitaxel as a radio­ sensitiser in ccrt in two main histopathological types of non-small cell lung cancer (adenocarcinoma and squamous cell carcinoma) of this region with limitations of small sample size d.l. nongrum et al. 44 r a d ia t io n m e d ic in e a n d o n c o l o g y issn 2413-6077. ijmmr 2021 vol. 7 issue 2 and the short study duration for which survival benefit could not be analysed. further studies have to be done on a larger population and over a longer study period to confirm the findings of this study. conflict of interests authors declare no conflict of interest. acknowledgements we would like to express our sincere gratitude to the head of the department of radiation oncology, rims, imphal, manipur, respected faculties and above all our patients for their kind cooperation and participation in the study. author’s contributions daffilyne lyngdoh nongrum, yumkhaibam sobita devi, laishram jaichand singh – conceptualization, methodology, formal analysis, writing – original draft; daffilyne lyngdoh nongrum, srigopal mohanty, kishalay baidya – data curation, writing – reviewing and editing; deiwakor chyrmang, hari krishna rai – investigation, formal analysis. порівняльне дослідження одночасної хіміопроменевої терапії з використанням паклітакселу у двох гістопатологічних підтипах (плоскоклітинний рак/аденокарцинома) нерезекційного недрібноклітинного раку легенів d.l. nongrum1, *y.s. devi2, s. mohanty3, l.j. singh2, k. baidya2, d. chyrmang2, h.k. rai2 1 – civil hospital, shillong, meghalaya, india 2 – department of radiation oncology, regional institute of medical sciences, imphal, manipur, india 3 – department of medical oncology, kilpauk medical college, chennai, tamil nadu, india вступ. рак легень все ще є глобальною проблемою і з ростом населення та збільшенням тривалості життя захворюваність на рак легень продовжує зростати. мета. порівняння відповіді на лікування та токсичність тижневої терапії паклітакселом при локально розповсюдженому нерезектабельному недрібноклітинному раку легенів (ндрл), що застосовується одночасно із зовнішнім променевим випромінюванням у грудну клітку при двох різних гістопатологічних типах аденокарциноми та плоскоклітинної карциноми. методи. проспективне рандомізоване контрольне дослідження було проведено у 60 пацієнтів з ндрл, які були розділені на дві групи: а (аденокарцинома) та б (плоскоклітинна карцинома). всім пацієнтам проводили хіміопроменеву терапію одночасно з паклітакселом 60 мг/м2. дані були оцінені за допомогою статистичного пакету spss версії 21.0 для windows з p-значенням <0,05. результати. гематологічна токсичність була найпоширенішим побічним ефектом, який проявлявся на третьому тижні хіміотерапії. наприкінці 1 місяця лікування у двох (6,7%) пацієнтів була повна відповідь у групі а, а у одного (3,3%) пацієнта була повна відповідь у групі б. у одного (3,3%) пацієнта прогресувала хвороба у групі а, а у двох пацієнтів було прогресування у групі б. через 7 місяців після лікування три (10%) пацієнти мали повну відповідь як у групі a, так і в групі б. у чотирьох (13,3%) пацієнтів прогресування захворювання в групі а було у десяти (33,4%) пацієнтів. висновки. паклітаксел можна використовувати як альтернативний хіміотерапевтичний засіб стандартному цисплатину. однак для підтвердження результатів необхідні подальші дослідження з більшим розміром вибірки. ключові слова: нерезектабельний; одночасна терапія; аденокарцинома; плоскоклітинний рак. information about the authors daffilyne lyngdoh nongrum – consultant, department of radiation oncology, civil hospital, shillong, meghalaya, india, orcid 0000­0002­9210­6683, e­mail: daffilynelnongrum@gmail.com yumkhaibam sobita devi – assistant professor, department of radiation oncology, regional institute of medical sciences, imphal, manipur, india, orcid 0000­0001­7151­5667, e­mail: ysobita@gmail.com d.l. nongrum et al. 45 r a d ia t io n m e d ic in e a n d o n c o l o g y issn 2413-6077. ijmmr 2021 vol. 7 issue 2 references 1. bray f, ferlay j, soerjomataram i, siegel rl, torre la, jemal a. global cancer statistics 2018: globocan estimates of incidence and mortality worldwide for 36 cancers in 185 countries. ca cancer j clin 2018;68(6):394–424. https://doi.org/10.3322/caac.21492. 2. collins lg, haines c, perkel r, enck re. lung cancer: diagnosis and management. am 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cancer med 2013;2(6): 916–24. https://doi.org/10.1002/cam4.142. 28. milovanovic is, stjepanovic m, mitrovic d. distribution patterns of the metastases of the lung carcinoma in relation to histological type of the primary tumor: an autopsy study. ann thorac med 2017;12(3):191–8. https://doi.org/10.4103/atm.atm_276_16. 29. emami b, graham gv: lung. in: perez ca, brady dw (eds): principles & practice of radiation oncology (3rd ed). philadelphia, pa, lippincottraven, 1997;1181-1220. received 5 september 2021; revised 28 october 2021; accepted 3 november 2021. this is open access article distributed under the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. d.l. nongrum et al. 42 in t e r n a l m e d ic in e issn 2413-6077. ijmmr 2021 vol. 7 issue 1 doi 10.11603/ijmmr.2413-6077.2021.1.12122 significance of detection of free/total psa ratio and other biochemical parameters in patients with bph, carcinoma prostate and its clinicopathologic correlation *s. joshi1, m.a.tilak2 , s. jadhav2 1 – bharati vidyapeeth medical college, pune, india 2 – dr. d. y.patil medical college, hospital and research centre, pune, india background. benign prostatic hyperplasia (bph) can raise prostate-specific antigen (psa) levels two to three times higher than the normal level. an increased psa level does not indicate prostate cancer (pca), but the higher the psa level, the higher the chance of having pca. detection and treatment have been profoundly affected by the advent of free/total psa ratio testing. objectives. the aim of the study was to estimate free, total psa levels and its ratio for serum levels of calcium, acid phosphatase and alkaline phosphatase in patients with bph and pca; to correlate clinical, biochemical and histopathological findings in the above patients. methods. psa levels were detected by chemiluminescent assay; serum calcium – by modified arsenazo method; serum acid phosphatase – by doumas et al method; and alkaline phosphatase – by lowry et al method. results. present study found high levels of total psa in bph and pca. levels of free psa were high in bph as compared to pca rate. free/total psa ratio is reduced considerably in pca as compared to bph. serum acid phosphatase and alkaline phosphatase were considerably higher in pca as compared to bph. serum calcium levels did not show significant difference in control and study groups. conclusions. it was established that patients with pca have a greater fraction of bound psa and a lower percentage of free psa than in those without pca. therefore, in clinical practice free/total psa ratio helps clinicians to decide if a biopsy is necessary. keywords: benign prostatic hyperplasia (bph); prostate cancer (pca); prostate-specific antigen (psa); free/total psa. international journal of medicine and medical research 2021, volume 7, issue 1, p. 42-50 copyright © 2021, tnmu, all rights reserved *corresponding author: dr. shilpa joshi, assistant professor, bharati vidyapeeth medical college, pune, 411043, india. e-mail: joshishilpa891@gmail.com introduction benign prostatic hyperplasia (bph), also called prostate enlargement, is a noncancerous increase in size of the prostate gland. symptoms may include frequent urination, trouble starting to urinate, weak stream, inability to urinate, or loss of bladder control. complications can include urinary tract infections, bladder stones, and chronic kidney problems [1, 2]. the clinical diagnosis of bph typically depends on a history of luts (lower urinary tract symptoms), digital rectal examination. the degree of luts does not necessarily correspond to the size of the prostate. on rectal examination, significant finding of symmetric and smooth enlarged prostate gland is suggestive diagnosis of bph. however, if the prostate gland feels asymmetrical, firm, or nodular, this raises concern for prostate cancer (pca) [2, 3]. urinalysis is typically performed when luts are present and bph is suspected to evaluate for signs of a urinary tract infection, glucose in the uri ne (suggestive of diabetes), or protein in the urine (suggestive of kidney disease). blood tests including kidney function tests and prostate specific antigen (psa) are often ordered to evaluate for kidney damage and pca, respec tively. a total psa test measures all the psa, including both the bound and the free-floating antigens. a free psa test, on the other hand, only measures psa that is floating freely in the bloodstream and not bound to a different protein. comparing the two results can help them understand the risk of pca being present [1, 3]. bph and pca are both capable of in creasing blood psa levels and psa elevation cannot differentiate these two conditions well. if psa levels are tested and are high, then further investigation is warranted. measures including psa density, free psa, rectal exa mination, and transrectal ultra sono gra phy may be helpful in determining whether a psa increase is due to bph or pca [2, 4, 5]. ultrasound examination of the testes, prostate, and kidneys is often performed, again to rule out cancer and hydronephrosis [2-4]. us food and drug association s. joshi et al. 43 in t e r n a l m e d ic in e issn 2413-6077. ijmmr 2021 vol. 7 issue 1 (fda) approved psa as an auxiliary diagnostic test in the management of the patients diagnosed with pca. since its clinical introduction to the present time, evaluation of serum psa level is one of the most widely used tests in urology practice [5, 6, 7]. in conclusion serum psa level is very important for urologists. introduction of psa test into clinical practice has enabled early diagnosis of pca, and provided important information about its staging, and postoperative follow-up period. the present study was performed with the aim to study free and total psa levels in patients with bph and pca. to aid in the early detection and diagnosis of pca. the study was carried out with the following objectives: to estimate free, total psa levels and calculate free/total psa ratio in patients with bph and pca, to study the serum levels of calcium, acid phosphates and alkaline phosphatase in patients with bph and pca and to correlate clinical, biochemical and histopathological findings in these patients. methods the present prospective study was conducted in the department of biochemistry at dr d. y. patil medical college hospital and research centre, pune-18. study period: july 2012–september2014. study design: the study was designed as a prospective case – control study. ethics statement written informed consents were obtained from all patients and healthy controls. study protocols were approved by the institutional ethics committee of dr. d. y. patil vidyapeeth pune. an informed consent was also obtained from the study population which consisted of two groups aged over 50 years old. study group and control 1. control group comprises 30 healthy adults above the age of 50 years. 2. study group comprises 35 cases of benign prostatic hyperplasia and 35 cases of carcinoma prostate. inclusion criteria: male patients above the age of 50 years who presented with urinary complaints and showed prostatic enlargement on usg. exclusion criteria: patients, who were diabetic, hypertensive and had a history of any major surgery, were excluded. collection of blood samples: under all aseptic precautions about 5 ml of venous blood was collected in a plain bulb and allowed to clot for one hour at room temperature, centrifuged at 2000 rpm for 10 min. serum was separated and analysed immediately for free psa, total psa, serum calcium, serum acid posphatase and alkaline phosphatase methods utilized: free and total psa levels: chemiluminescent assay. serum calcium: modified arsenazo method. serumacid phosphatase: doumas et al method. alkaline phosphatase: lowry et al method. measurement of total psa (chemilu minescence immunoassay method). electro-chemiluminescence immunoassay (eclia) for the quantitative determination of total psa in human serum and plasma [6,7,8,9,10,11]. principle: a chemical moiety is disclosed which comprises a chemical, biochemical, or biological substance attached to one or more electrochemiluminescent organometallic compounds. substances of interest are attached to one or more ruthenium containing or osmium containing labels or other electrochemiluminescent labels. methods for detecting small amounts of chemical moiety using chemiluminescent, electrochemiluminescent and photoluminescent means. the chemiluminescent reaction for the detection of the reaction complex is initiated by applying a voltage to the sample solution resulting in a precisely controlled reaction. electroche miluminescent technology can accommodate many immunoassay principles while providing superior performance [6,7]. measurement of free psa (chemilu minescence method [8,9]. electro-chemiluminescence immunoassay (eclia) for the quantitative determination of free psa in human serum and plasma. measu rement of free psa is used to determine the free/total psa ratio (% free psa) which can be clinically useful in evaluating the need for prostate biopsy in two ways: a) individual risk assessment: the % free psa is significantly lower in patients having prostate cancer than those with benign disea ses or normal controls. the probability of finding prostate cancer with total psa and with decreasing % free psa. therefore % free psa improves the sensitivity and specificity in patients with total psa values in this “gray zone”. b) alternatively a single cut-off may be used for men in all age groups. it was found that the s. joshi et al. 44 in t e r n a l m e d ic in e issn 2413-6077. ijmmr 2021 vol. 7 issue 1 proportion of unnecessary biopsies can be reduced by 20 % when using a threshold for % free psa of 25 %. serum calcium: modified arsenazo method (end point method) [10,11]. system reagent for the quantitative determination of calcium concentrations in human serum, plasma and urine on beckman coulter au analysers. principle: calcium reacts with a dye arsenazo at specific ph to form bluish purple coloured complex. the intensity of colour formed is directly proportional to the amount of calcium present in the sample. this calcium procedure is based on calcium i o n s ( c a 2 +) r e a c t i n g w i t h a r s e n a z o i i i (2,2’-[1,8-dihydroxy-3,6-disulphonaphthylene2,7-bisazo]bisbenzenear-sonic acid) to form an intense purple coloured complex. magnesium does not significantly interfere in calcium determination using arsenazo iii. in this method the absorbance of the ca-arsenazo iii complex is measured bichromatically at 660/700 nm. the resulting increase in absorbance of the reaction mixture is directly proportional to the calcium concentration in the sample. quality control: during operation of the beckman coulter au analyser at least two levels of an appropriate quality control material should be tested a minimum of once a day. in addition, controls should be performed after calibration with each new lot of reagent, and after specific maintenance or troubleshooting steps described in the appropriate user’s guide. quality control testing should be performed in accordance with regulatory requirements and each laboratory’s standard procedure. appropriate qualified urine controls should be established and utilized during urine analysis. results are automatically printed out for each sample in mg/dl at 37°c. for si units (mmol/l) the results must be multiplied by 0.25. serum acid phosphatase: doumas et al method [12-15]. methodology: kinetic method. acid phosphatase α-naphtyl phosphate. kinetic quantitative determination of acid phosphatase. principle: hillmann method – acid phosphatase activity present in the sample is determined according to the modified method of hillmann. α-naphtyl-phosphate+h2o → acp α-naphtol +phosphate α-naphtol + fast red tr → azo dye α-naphtol reacts with a diazoted compound forming a colour with a maximum of absorbance at 405 nm. tartrate is used as specific of the prostatic fraction. quality control: sera control are recommended to monitor the performance of assay procedures. if control values are found outside the defined range, check the instrument, reagents and technique for problems. each laboratory should establish its own quality control scheme and corrective actions if controls do not meet the acceptable tolerances. alkaline phosphatase assay [16-18]. alkaline phosphatase assay on the architect c systems™ and the aeroset system. the alkaline phosphatase assay is used for the quantitation of alkaline phosphatase in human serum or plasma. principle of procedure: several substrates have been used to measure alkaline phosphatase activity such as glycerophosphate, phenyl phosphate and p-nitrophenyl phosphate. bowers and mccomb improved the method of bessey et al. to include a kinetic measurement. tietz et al. optimized this method to include a chelated metal-ion buffer of zinc, magnesium, and hedta. this alkaline phosphatase procedure is a modification of this method. alkaline phosphatase in the sample catalyses the hydrolysis of colourless p-nitrophenyl phosphate (p-npp) to give p-nitrophenol and inorganic phosphate. at the ph of the assay (alkaline), the p-nitrophenol is in the yellow phenoxide form. the rate of absorbance increase at 404 nm is directly proportional to the alkaline phosphatase activity in the sample. optimized concentrations of zinc and magnesium ions are present to activate the alkaline phosphatase in the sample. statistical analysis: at the end of study all data was compiled and analysed statistically using annova method, tukey’s test. results serum free psa, total psa, free/total psa ratio along with serum calcium, acid phosphatase, alkaline phosphatase were estimated in 30 controls (group i) and 35 cases each of benign prostatic hyperplasia (group ii) and carcinoma prostate (group iii). the results showed that total psa levels, calcium, acid phosphatase, alkaline phosphatase were higher in group iii as compared to group i and ii. free psa levels and free/total psa ratio was decrea sed in group iii as compared to group ii. (table 1). s. joshi et al. 45 in t e r n a l m e d ic in e issn 2413-6077. ijmmr 2021 vol. 7 issue 1 the chart depicts the mean±sd distribution into controls (group i), cases (group ii) and group (iii). number of controls were 30 and cases were 35 each in group ii and group iii. age of patients was considerably higher in group iii as compared to group ii (table 2). table 3 shows comparison of mean±sd values of serum free psa in group i, ii and iii. levels of mean±sd values of free psa in group iii were significantly lower as compared to group ii. table 4 shows distribution of mean±sd values of total psa in group i, ii and iii. mean±sd values of total psa were found to be increased in group iii as compared to group i and ii. table 5 is depicting free/total psa ratio in terms of mean±sd in groups i, ii and iii. mean values of serum calcium in patients with carcinoma was considerably higher as compared to benign prostatic hyperplasia and control group. test results obtained were significant (p<0.05) (table 6). chart depicts intergroup comparison of serum alkaline phosphatase between cases and controls. mean±sd values of alkaline phosphatase were higher in group iii as compared to groups i and ii. table 1. comparison of age in study groups group number age (yrs.) f value p value mean sd group i (control) 30 68.07 7.95 5.04 <0.01 group ii (control) 35 64 4.92 group iii (control) 35 68.26 5.82 note: gr. i vs gr. ii: p<0.05 & gr. i vs gr.iii: p<0.05 p<0.05: significant p<0.01: highly significant p>0.05 and p>0.01 not significant table 2. comparison of free psa in study groups group number age (yrs.) f value p value mean sd group i (control) 30 0.68 0.56 39.56 <0.0001 group ii (control) 35 2.43 1.10 group iii (control) 35 1.59 0.55 gr. i vs gr. ii: p<0.0001andgr. i vs gr.iii: p<0.0001 and gr.ii vs gr.iii: p<0.0001 table 3. comparison of total psa in study groups group number age (yrs.) f value p value mean sd group i (control) 30 1.95 1.24 35.93 <0.0001 group ii (control) 35 8.51 5.20 group iii (control) 35 66.83 58.29 gr. i vs gr. ii: p<0.0001 & gr. i vs gr.iii: p<0.0001 table 4. comparison of free/total psa ratio in study groups group number age (yrs.) f value p value mean sd group i (control) 30 0.34 0.15 95.29 <0.0001 group ii (control) 35 0.32 0.09 group iii (control) 35 0.04 0.03 gr. i vs gr. ii: p<0.0001 & gr. i vs gr.iii: p<0.0001 s. joshi et al. 46 in t e r n a l m e d ic in e issn 2413-6077. ijmmr 2021 vol. 7 issue 1 discussion pca is a major cancer and leading cause of death in men. as with any cancer, early detection followed by treatment increases the disease-free survival rate significantly. early diagnosis is very important in management of any type of cancer and infections. after introduction of serum psa measurements into clinical use, the incidence rate of early diagnosis has increased, and a shift between stages was achieved. nowadays, 70-80% of the diagnosed cancers are organ-confined [18,19]. serum psa level was also helpful in the staging of pca. serum psa values between 0-4 ng/ml can detect 80% of the organ-confined disease, while psa values between 4-10 ng/ml, and above 10 ng/ml were found in 70% and 50% of the cases with organ-confined disease. serum psa levels also provide us helpful information about lymph node involvement. lymph node involvement has been also reported in men with serum psa levels of <10 ng/ml (5%), 10-20 ng/ml (18%), and >20 ng/ml (20%) in respective percentages. therefore, if serum psa level is below 25 ng/ml, there is no need to per form ct or mri, and bone scanning is not required for men with serum psa levels below 20 ng/ml. besides, it has been demonstrated that in men with gleason score ≤6, clinical stage t1/t2, and serum psa levels below 10 ng/ml, lymph node dissection is not required [1,3,4, 12,18,19]. t h e c u r r e n t p r o s p e c t i v e s t u d y w a s conducted with the aim to estimate free psa, total psa, free/total psa ratio in patients with benign bph and pca and correlate with the histopathology report. in the present study age wise distribution in control group is 68.07±7.95, while in bph it was 64±4.92 and in pca 68.26±5.82. there was no significant difference in the age wise distribution among all the three groups. mean values of free psa levels in benign prostatic hyperplasia were 2.43±1.10 ng/ml, in carcinoma prostate – 1.59±0.55 ng/ml as compared to 0.68±0.56 ng/ml in the control group. the result was statistically highly significant (p<0.0001). in the present study, free/total psa ratio is reduced in carcinoma prostate as compared to benign prostatic hyperplasia. histological grades of prostate table 7. comparison of serum alkaline phosphatase in study groups group number age (yrs.) f value p value mean sd group i (control) 30 51.90 13.09 42.20 <0.0001 group ii (control) 35 71.20 29.58 group iii (control) 35 112.49 33.6 gr. i vs gr. ii: p<0.0001andgr. i vs gr.iii: p<0.05 and gr.ii vs gr.iii: p<0.0001 table 6. comparison of serum acid phosphatase in study groups group number age (yrs.) f value p value mean sd group i (control) 30 4.65 0.93 36.31 <0.0001 group ii (control) 35 5.30 1.51 group iii (control) 35 18.52 12.67 gr. i vs gr. ii: p<0.001andgr. i vs gr.iii: p<0.0001 table 5. comparison of serum calcium in study groups group number age (yrs.) f value p value mean sd group i (control) 30 9.19 0.41 4.20 <0.05 group ii (control) 35 9.18 0.48 group iii (control) 35 9.52 0.71 gr. i vs gr. ii: p<0.05 & gr. i vs gr.iii: p<0.05 p<0.05: significant p<0.01: highly significant p>0.05 and p>0.01 not significant s. joshi et al. 47 in t e r n a l m e d ic in e issn 2413-6077. ijmmr 2021 vol. 7 issue 1 biopsy showed a negative correlation with free/ total psa ratio in our study. lakhey m et al (2010) reported serum free psa is elevated marginally in patients with bph without inflammation, determined the relationship between free psa and histological findings in biopsy specimens of patients with prostate disease. active inflammation and highgrade lesions are associated with free psa level more than 5 ng/ml [18]. ari adamy et al (2011) reported that with prostate specific antigen included in progression criteria prostate specific antigen at confirmatory biopsy (hr 1.29, 95% ci 1.14-1.46, p<0.0005) and positive confirmatory biopsy (hr 1.75, 95% ci 1.01-3.04, p=0.047) were independent predictors of progression [19]. measurement of serum psa levels is very important during post-treatment monitorization of pca. besides based on preoperative serum psa levels, a biochemical recurrence can be seen within 10 years after surgery. accordingly, recurrence rates of 10, 20, and 50% are anticipated in men with preoperative serum psa levels of <2.6 ng/ml, 2.6-10 ng/ml, and above 10 ng/ml, respectively. serum psa levels are important in patients who had undergone androgen suppression therapy because of metastatic disease. if serum psa level does not drop below 4 ng/ml 7 months after androgen suppression therapy, median life-expectancy of these patients is only one year. if serum psa levels of these patients drop below 0.2 ng/ml, then median life-expectancy longer than 6 years can be predicted. if after radical prostatectomy or radiotherapy, serum psa levels of the patients without radiological metastases rise above 0.2 ng/ml within the first 8 months of androgen suppression, prostate cancer mortality increases 20-fold. still psa doubling time shorter than 3 months is a very bad prognostic finding. increase in serum psa levels after prostatectomy aids in differentiation between local and systemic recurrences. if psa doubling time increases after the first 2 postoperative years, and psa doubling time is longer than 11 months, then local recurrence should be conceived with an 80% probability. however, if psa levels rise within the first postoperative year, and psa doubling time is 4-6 months, the systemic disease should be thought of with a 80% probability. similar results were obtained in a prospective study by muhittin serdar et al (2002) reported patients with serum total psa between 4.19.9 ng/ml, if none of the psa-based parameters is positive, biopsy can be postponed and the patients should be followed-up; on the other hand, patients with three positive parameters should be biopsied. if only one or two of the parameters are positive, the patient’s age, race, and clinical findings should be considered in decision-making. hence, the combined use of all markers can increase sensitivity and specificity [20]. cut off value of free/total psa ratio is 0.14 (14%) in the present study which has sensitivity of 94.3% and specificity of 99%. patients with less than 0.14 ratio had carcinoma prostate as compared to those above with cut off which showed benign prostatic hyperplasia. most psa in the blood is bound to other proteins. normally more than 23% of serum psa is in its free form (free psa). benign prostatic tumours may be associated with elevated psa, especially free psa. with prostate carcinoma, however, the proportion of free psa decreases. one theory hypothesizes that prostate carcinoma produces not only psa but also proteins to which the psa is bound. as a result, portion of free psa decreases. histological grades showed a negative correlation between free/total psa ratio and histological grade of carcinoma. a lower ratio suggests prostate carcinoma while higher ratio suggests bph by borros arneth [21]. in our study, total psa and free/ total psa ratio were in accordance with study by d weckermann et al in which the patients with prostate cancer showed high total psa and reduced free/total psa ratio as compared to bph patients. mione r et al found that percent free psa was more effective than total psa in the differential diagnosis between carcinoma prostate and bph. percent free psa is superior to total psa in distinguishing between primary carcinoma prostate from bph, in patients with total psa between 2-30 ng/ml [22]. according to manabu kuriyama et al [23] the diagnostic significance of free/total psa has been reported to improve diagnostic accuracy of prostate cancer in the group with considerably elevated serum psa values. as serum psa values increased, f/t was lower in the carcinoma prostate 56 group than in non-prostate carcinoma group. however, f/t values were lower in prostate carcinoma group than in non-prostate carcinoma group. the positive predictive value for prostate cancer increased to 54% in total psa alone. cut off value of free/total was 0.155 for obtaining relatively high specificity. in the present study, sensitivity was 85% and specificity was 56.5%. s. joshi et al. 48 in t e r n a l m e d ic in e issn 2413-6077. ijmmr 2021 vol. 7 issue 1 levels of calcium in serum were significantly correlated with serum levels of free psa. it is unlikely that serum psa acts to increase serum calcium, whose serum levels are under strict homeostatic control. specifically, if serum levels of psa increased serum levels of calcium, the men with advanced prostate cancer would develop hypercalcemia. in this study probability estimates for prostate cancer in the percent free psa ranges of ≤10 ng/ml is 58.3%. although diagnostic improvements have been made through the use of psa quotient, the diagnosis of prostate carcinoma can be made only after tumour biopsy and histopathology examination. since the introduction of prostate-specific antigen testing over two decades ago, there has been a steep increase in the prostate cancer incidence, especially the incidence of localized low risk disease. use of the percentage free psa can enhance the specificity of psa testing and decrease the number of unnecessary biopsies. however, the cut off free/total psa in our case was 18.0%. use of percent free psa has been shown to improve specificity in the detection of prostate (4.1-10 ng/ml). in our study 17 (22%) patients had total psa levels between 4-10 ng/ml. of these one of the patients had psa level 6.6 ng/ml, which on biopsy was reported as carcinoma prostate. remaining 16 patients were reported as bph on biopsy. other parameters like serum calcium showed mean values of 9.91±0.41mg/dl in the control group. in bph and pca mean values of serum calcium were 9.18±0.48 mg/dl and 9.52±0.71 mg/dl, respectively. in the present study acid phosphatase values were higher in group iii (carcinoma) as compared to group i and ii. however, with advent of prostate specific antigen (psa) measurement of acid phosphatase in the diagnosis, staging and monitoring of prostate cancer has taken a back stage. human prostatic acid phosphatase is a prostate epithelium specific differentiation antigen found in large amounts initially in seminal fluid. in our study 5 (14.28%) out of 35 patients were found to have slightly elevated acid phosphatase levels. only one patient showed bone metastases. similar study by gutman and his colleagues made the critical observation that serum prostate acid phosphatase activity was significantly higher in prostate cancer patients, particularly those with bone metastases. limitation of the study is that in the present study per rectal examination was not done, as it was not advised by clinicians. conclusions present study found high levels of total psa in bph and pca. the levels of free psa were high in bph as compared to pca rate. free/total psa ratio was reduced considerably in pca as compared to bph. serum acid phosphatase and alkaline phosphatase were considerably higher in pca as compared to bph. serum calcium levels did not show significant difference in the control and study groups. we concluded that the patients with pca have a greater fraction of bound psa and a lower percentage of free psa than in those without pca. there was a negative correlation found between free/total psa ratio and the histopathologic findings. the lower is the ratio, the higher is the grade of malignancy. therefore, in clinical practice free/total psa ratio helps clinicians to decide if a biopsy is necessary. conflict of interests the authors declare no conflict of interest. acknowledgements the authors acknowledge all technical support by the technical staff of biochemistry department of dr. d.y. patil medical college hospital and research centre pimpri, pune. author’s contributions dr. shilpa joshi – сonceptualization, methodology, practical work, formal analysis; dr. mona tilak – guidance in research work, investigation and analysis; dr. savita jadhav – writing original draft, reviewing and editing draft, finalization of draft. s. joshi et al. 49 in t e r n a l m e d ic in e issn 2413-6077. ijmmr 2021 vol. 7 issue 1 клініко-патологічна кореляція та роль співвідношення вільного/загального простатоспецифічного антигену та інших біохімічних показників у пацієнтів з доброякісною гіперплазією передміхурової залози та карциномою простати *s. joshi1, m.a.tilak2, s. jadhav2 1 – bharati vidyapeeth medical college, pune, india 2 – dr. d. y.patil medical college, hospital and research centre, pune, india вступ. доброякісна гіперплазія передміхурової залози (дгпз) може супроводжуватися відвічі-втричі підвищеним рівнем (пса). підвищений рівень пса не вказує на рак передміхурової залози (рпз), чим вищий рівень пса, тим більша ймовірність виникнення рпз. поява тесту на співвідношення вільного/загального пса мала значний вплив на частототу виявлення та вибір тактики лікування рпз. мета. оцінити співвідношення вільного/загального пса, рівень сироваткового кальцію, кислої та лужної фосфатаз у пацієнтів з дгпз та рпз. провести кореляцію клінічних, біохімічних та гістопатологічних даних у вищевказаних пацієнтів. методи. визначення пса проводилося за допомогою хемілюмінесцентного аналізу; сироватковий кальцій визначався модифікованим методом арсеназо. кисла фосфатаза сироватки визначалася методом за doumas et al., лужна фосфатаза – за lowry et al. результати. дослідження виявило високі рівні загального пса при дгпз та рпз. рівні вільного пса були вищими при дгпз порівняно з рпз. співвідношення вільного/загального пса значно знижується при рпз порівняно з дгпз. рівні сироваткової кислої та лужної фосфатаз значно підвищуються при рпз порівняно з дгпз. рівень кальцію в сироватці крові не відрізнявся в контрольній та досліджуваній групах. висновки. пацієнти з рпз мають більшу частку зв'язаного пса та менший відсоток вільного пса, порівняно з пацієнтами без рпз. тому в клінічній практиці співвідношення вільного/загального пса може допомогти клініцистам вирішити чи потрібна біопсія. ключові слова: доброякісна гіперплазія передміхурової залози (дгпз); рак передміхурової залози (рпз); простатоспецифічний антиген (пса); вільний/загальний пса. information about the authors dr. shilpa joshi, assistant professor, bharati vidyapeeth medical college, pune, india orcid: 0000-0003-0331-6425, email: joshishilpaa891@gmail.com dr. mona tilak, professor, head of the department, dr. d.y.patil medical college, hospital and research centre pimpri, pune, india orcid: 0000-0002-1734-8295, email: drmonatilal@dpu.edu.in dr. savita jadhav, professor, d.y.patil medical college, hospital and research centre pimpri, pune, india orcid: 0000-0003-3439-9462, email: patilsv78@gmail.com references 1. avci s, onen e, caglayan v, kilic m, sambel m, oner s. free prostate-specific antigen outperforms total prostate-specific antigen as a predictor of prostate volume in patients without prostate cancer. arch ital urol androl. 2020 apr 6;92(1):1-6. doi: 10.4081/aiua.2020.1.1. pmid: 32255313. 2. mao q, zheng x, jia x, wang y, qin j, yang k, bai y, xie l. relationships between total/free prostate-specific antigen and prostate volume in chinese men with biopsy-proven benign prostatic hyperplasia. int urol nephrol. 2009 dec;41(4):761-6. doi: 10.1007/s11255-009-9533-1. epub 2009 feb 18. pmid: 19224388. 3. coban s, doluoglu og, keles i, demirci h, turkoglu ar, guzelsoy m, karalar m, demirbas m. age and total and free prostate-specific antigen levels for 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density as diagnostic tools for improved prostate can cer detection. biomed res int. 2020 jul 21;2020: 9872146. doi: 10.1155/2020/9872146. pmid: 32775459; pmcid: pmc7396080. 14. sasaki m, ishidoya s, ito a, saito h, yamada s, mitsuzuka k, kaiho y, shibuya d, yamaguchi t, arai y. low percentage of free prostate-specific antigen (psa) is a strong predictor of later detection of prostate cancer among japanese men with serum levels of total psa of 4.0 ng/ml or less. urology. 2014 nov;84(5):1163-7. doi: 10.1016/j.urology.2014.04.055. epub 2014 oct 24. pmid: 25443923. 15. huang, yan mda; li, zhen-zhen mda; huang, s. joshi et al. 35 su r g ic a l d is e a se s issn 2413-6077. ijmmr 2020 vol. 6 issue 1 international journal of medicine and medical research 2020, volume 6, issue 1, p. 35-42 copyright © 2020, tnmu, all rights reserved *corresponding author: nazar i. herasymiuk, m.d., phd, associate professor of the department of surgery no 2, i. horbachevsky ternopil national medical university, ternopil, ukraine. e-mail: herasymiuknazar@gmail.com. i.k. venher et al. doi 10.11603/ijmmr.2413-6077.2020.1.11047 nonspecific dysplasia of the connective tissue – a factor of venous thromboembolic complications in endoprosthetics of hip joints i.k. venher, *n.i. herasymiuk, s.ya. kostiv, i.i. loyko, d.v. khvalyboha i. horbachevsky ternopil national medical university, ternopil, ukraine background. important part of orthopedic surgery is endoprosthetics of hip joints, which eliminates pain syndrome, restores the amplitude of movement and the support ability of lower limbs. but some complications usually take place; venous thromboembolism is leading among them. objective. the aim of the study was to investigate endothelial dysfunction and activity of the hemocoagulation system at different levels of vtec risks and thus to work out the strategy of thromboprophylaxis in patients with osteoarthrosis of the hip joint and femoral neck fracture combined with non-specific dysplasia of the connective tissue. methods. 219 patients of a mean age of 64.7±3.8 years old underwent surgery. in 137 (62.1%) cases, a total cement hip replacement was performed for osteoarthritis. 82 (37.4%) patients underwent total and unipolar cement hip replacement for cervical femoral neck fractures. results. clinical manifestations of non-specific connective tissue dysplasia were detected in 83 (37.9%) patients that was confirmed by the laboratory determination of the level of general, bound and free oxyproline. in the postoperative period, the thrombotic process in the venous system of the inferior vena cava was diagnosed in 23 (10.5%) cases. the level of indicators of endothelium status dysfunction was much more significant in the patients in cases of nonspecific dysplasia of connective tissue. operative intervention on the hip joint in the patients with nonspecific dysplasia of connective tissue in 11 (13.3%) cases was complicated by development of venous thrombosis. in the patients without non-specific connective tissue dysplasia, postoperative thrombosis in the system of the inferior vena cava was diagnosed in 12 (8.8%) cases. conclusions. patients with osteoarthrosis of the hip joint and the femoral neck fracture accompanied by the non-specific dysplasia of the connective tissue are characterized by high levels of endothelial dysfunction and increased activity of the blood-coagulation system. keywords: endoprosthetics; thromboembolism; endothelial dysfunction; dysplasia. abbreviations at iii ctd dvt fba fdp fpa ivc lmwh nctd prt scfm tfa tpa vegf ivc vtec antithrombin ііі non-specific connective tissue dysplasia deep vein thrombosis fibrinolytic blood activity fibrin degradation product fibrinopeptide а inferior vena cava low molecular weight heparins non-specific connective tissue dysplasia plasma recalcification time soluble complexes of fibrin-monomers total fibrinolytic activity thromboembolism of the pulmonary artery vascular endothelial growth factor inferior vena cava venous thromboembolic complications introduction significant occurrence of diseases of the lower extremities joints leads to the search for effective methods of treatment. among them endoprosthetics of the joints is foremost. it allows eliminating pain syndrome, restoring the amplitude of movement and the support ability of the lower limb [1]. during endoprosthetics, a number of complications often occur; vtec is leading among them [2]. without vte prophylaxis, the overall vte incidence in medical and general surgery of the hospitalized patients is in the range of 1040%, while it ranges up to 40-60% in major orthopaedic surgery. with routine vte prophylaxis, fatal pulmonary embo lism is uncommon in orthopaedic patients and the rates of symptomatic vte within three months are in the range of 1.3-10% [3]. vte preventive treatment methods are divided into mechanical and pharmacological. the mechanical include mobilization, graduated com pression stockings, intermittent pneumatic compression device and venous foot pumps; the pharmacological include aspirin, unfractionated heparin, low molecular weight heparin (lmwh), adjusted dose vitamin k antagonists, synthetic 36 su r g ic a l d is e a se s issn 2413-6077. ijmmr 2020 vol. 6 issue 1 pentasaccharide factor xa inhibitor and newer oral anticoagulants. lmwh seems to be more efficient overall compared with the other available agents. we are still sceptical about the use of aspirin as a sole method of prophylaxis in total hip and knee replacement and hip fracture surgery, while controversy still exists regarding the use of vte prophylaxis in knee arthroscopy, lower leg injuries and upper extremity surgery [3]. the factors that predispose a high risk of venous thromboembolism in the patients undergoing endoprosthetics of the joints are taken into account [4]. but there is no significant decrease of frequency of vtec development [5]. the aim of the study was to investigate endothelial dysfunction and activity of the hemocoagulation system at different levels of vtec risks and thus to work out the strategy of thromboprophylaxis in patients with osteoarthrosis of the hip joint and femoral neck fracture combined with non-specific dysplasia of the connective tissue. methods 219 patients of a mean age of 64.7±3.8 years old underwent surgery. in 137 (62.1%) cases, total cement hip replacement for osteoarthritis was performed. 82 (37.4%) patients underwent total and unipolar cement hip replacement for cervical femoral neck fractures. operative intervention was carried out under subdural anaesthesia with bupivacaine. clinical manifestations of non-specific connective tissue dysplasia (nctd) were detected in 83 (37.9%) patients. this was confirmed by laboratory determination of the level of general, bound and free oxyproline. the method of p.n. sharaev for a calibrated curve was used. mechanical prophylaxis of vtec by elastic compression of the lower extremities was used in all cases. pharmacological prevention of vtec was performed using lmwh (enoxaparin). the study included 136 patients (group i) with hip joint osteoarthrosis and femoral neck fracture and 83 patients (group ii) with osteoarthrosis of the hip joint and femoral neck fracture and associated nctd. in the postoperative period, the thrombotic process in the venous system of the inferior vena cava (ivc) was diagnosed in 23 (10.5%) cases. operative intervention on the hip joint in the patients with nctd in 11 (13.3%) cases was complicated by the development of venous thrombosis. in the patients without nctd, postoperative thrombosis in the venous system of ivc was diagnosed in 12 (8.8%) cases. in 21 patients, postoperative thrombosis was de tected in the deep venous system of ivc and only in two cases in the superficial venous system. monitoring of the thrombotic process in the venous system of the inferior vena cava was performed by duplex ultrasound scans of the vessels of the lower extremities by sonoscape s8exp with a frequency of 5-12 mhz and 2.5-4 mhz from the first day of the postoperative period. in the first day of the postoperative period, in two patients the thrombotic processes in the deep veins of the ivc system was diagnosed, on the 2nd day – in 5 cases, on the 3rd day – in one patient, on the 6th, 7th and 8th days – in 3, 5 and 2 cases, respectively, and on the 11th and 12th day – in one and two patients, respectively. in the superficial venous system of the lower limb, the thrombotic process was de tected in two cases on the 6th and 10th day of the postoperative period. 8 (34.9%) cases of the venous thrombotic processes were diagnosed within the first three days of the early postoperative period in the patients with nctd. to choose the treatment tactics for venous thrombosis of the ivc system, the determination of thrombogenicity of thrombotic masses was performed. the elastographic properties of the venous thrombus were studied by the ultrasonic system siemens acuson s2000 (germany). at the shear wave expansion speed of 2.5-2.6 m/s there is a high risk of embologenity of the throm bi, at the shear wave expansion speed of 2.7-2.9 m/s there is a moderate risk of embologenity of the thrombi, at the shear wave expansion speed of 3.0 m/s and more there is no threat of embolism. in two cases on the second day of the postoperative period the propagation of the shear wave at 2.5-2.6 m/s in the thrombotic mass of the vein was established using the ultra sonic system siemens acuson s2000 (germany), which is a significant threat to the development of pulmonary embolism. this was an indication for urgent surgical intervention to eliminate the threat of tpa. in all other cases anticoagulant therapy was performed. the rate of coagulation and fibrinolytic system functioning was determined: fibrinogen was determined by gravimetric method according to r.a. rutberg; activity of the fibrin stabilizing factor (f xiiі) – by means of the “set for determination of fba-xiii” of the scientific and production firm simko ltd (lviv); thromboplastic activity of blood – by b.a. kudryashov and i.k. venher et al. 37 su r g ic a l d is e a se s issn 2413-6077. ijmmr 2020 vol. 6 issue 1 p.d. ulytina method; plasma recalcification time – by bergerhof and rock method; plasmin, plasminogen, total fibrinolytic activity (tfa) – by v.a. monastyrska et al. method (1988); time of lysis of euglobin clots – using the “set for the determination of fibrinolytic blood plasma activity” by the scientific and production firm simko ltd (lviv). d-dimer was determined by immunoanalytical method using a coagulometer. the soluble fibrin-monomer complexes were determined using the tablet method. the state of the endothelial system was evaluated by determining the level of endothelial dysfunction markers: the concentration of p-selectin, e-selectin, tissue plasminogen activator (t-pa), type 1 vascular endothelium adhesion molecule (svca m-1) was determined using the bender medsystems (austria) for immune-enzyme analysis. endothelin-1 concentration was determined using the biomedica (canada) kits for immune-enzyme analysis. the reaction was evaluated on a sunrise microplate semimetric photometer (tecan, austria) using the hydroflex washing station (tecan, austria). to determine circulating endothelial cells (cec) the j. hiadovec and n.n. pertishchev et al. method (2001) was used. the level of nitrogen oxide (no) metabolites, vascular endothelial growth factor (vegf) was determined by the immune enzyme method using the khgo111vtgf analyser and the griess reagent. results the state of the endothelial system reflects the pathology of the organism. in the patients with osteoarthrosis of the hip joint and femoral neck fracture an increased level of all endothelial system markers was noted. at the same time, the level of indicators of endothelium status was more significant in the patients (group ii) with pathology of hip joint in the presence of ncdt (table 1). thus, at the preoperative stage of treatment, the blood level of circulating endo thelial cells was higher in 1.8 times (р<0.001), endothelin-1 level was increased in 2.2 times (р<0.001), p-selectin and e-selectin levels increased in 1.5 times (р<0.001) and 1.2 times (р<0.05) respectively, compare to in dicators of healthy persons. there was a 1.8 fold (р<0.001) increase of blood no levels and a slight 1.2 fold (р<0.05) increase of the vegf blood contents. operative intervention in the patients of group ii contributed to development of severe endothelial dysfunction. this was especially noticeable at the twelfth hour of the postoperative period. the latter occurred due to a 1.5 fold increase (р<0.05) in the contents of circulating endothelial cells in blood and a 1.5 fold increase (р<0.05) in endothelin-1, a 1.2 fold decrease (р<0.05) and a 1.5 fold decrease (р<0.05) of p-selective and e-selectin levels respectively, to compare with the preoperative period. there was also a slight increase of no contents in blood and a 1.3 fold (р<0.05) increase of vegf contents in blood. at the stage of preoperative preparation of patients, activation of the hemostasis system was observed (table 2). this was more significant among the patients of the group ii. the indicators that characterize the condition of the coagulation system of patients with nctd were in 1.2 times higher than those of the patients without nctd. in the patients with nctd in the preoperative period the content of fibrinogen was 5.07±0.35 g/l, (in healthy patients – 3.52+0.34 g/l (p<0.05). at the same time, an increase of the contents of scfm in blood was determined as of 0.57±0.11 extr. units (р<0.05). monomers that were formed as a result of sepa ration of fibrinopeptides a and b from fibrinogen under the influence of thrombin, form macromolecular degradation products of fibrin with it. trombinemia was confirmed by the increase up to 2.61±0.23 ng/l (p<0.05) of fpa content. at the same time, an increased up to 6.92±2.09 μg/l (norm 4.71±1.58 μg/ml) content of the fdp was detected. it took place on the background of minimal changes of the fba and in the absence of any changes in the level of at iii and prt. significant changes of coagulation system occurred during the intraoperative period of surgical treatment of the patient. at the traumatic stage of surgical intervention, a hypercoagulative condition of blood developed. during that period, the fibrinogen level in the blood exceeded the preoperative level by only 10.2%, then the blood contents of scfm was increased in 1.4 times (p<0.05), the contents of fpa was increased in 1.7 times (р<0.001), the contents of fdp was increased in 1.9 times (р<0.001). it took place on the background of reduced fibrinolytic activity of blood, reduced contents of at iii in blood, decrease of prt (table 3). the maximum level of hypercoagulation reached at 6 h. of early postoperative period. at that time, the blood fibrinogen level exceeded the preoperative rate in 1.3 times (p<0.05), and the contents of scfm in blood was in creased in 2.1 (р<0.001) times, the content of fpa and fdp – in 2.7 and 2.8 (р<0.001) times, respectively. it took place at a 1.3 fold decrease i.k. venher et al. 38 su r g ic a l d is e a se s issn 2413-6077. ijmmr 2020 vol. 6 issue 1 ta bl e 1. c h ar ac te ri st ic s of t h e en do th el ia l s ys te m in t h e pa ti en ts w it h p at h ol og y of t h e h ip jo in t in de x ci c, n u m be r of ce lls × 10 4 / l sv ca m 1, n g/ m l рse le ct in , n g/ m l еse le ct in , n g/ m l tpa , n g/ m l en do th el in -1 , fm ol /m l n o , µ m ol /l v eg f, p k/ m l g ro up n or m al in di ce s 4. 04 ±1 .0 9 22 9. 43 ±3 5. 31 15 4. 82 ±1 9. 54 40 .7 9± 10 .1 3 3. 68 ±0 .7 3 4. 12 ±0 .3 5 21 .3 9± 4. 71 51 ,4 3± 5, 59 g ro up i pr eo pe ra tiv e 5. 56 ±1 . 2 3* 25 7. 83 ±2 6. 61 17 5. 10 ±2 3. 35 49 .6 5± 8. 17 * 3. 10 ±0 .3 0 5. 53 ±0 .3 7 34 .1 6± 5. 54 57 ,0 5± 5, 35 po st op er at iv e, 6 hr s. 7. 87 ±2 .8 9* * 35 7. 68 ±2 9. 85 ** 17 3. 21 ±1 7. 94 42 .2 7± 8. 32 2. 53 ±0 .3 8 7. 51 ±0 .4 1 34 .2 9± 5. 65 59 ,1 2± 5, 76 po st op er at iv e, 12 h rs . 8. 13 ±3 .5 2* * 36 3. 61 ±3 1. 27 ** 17 9. 18 ±1 8. 69 35 .6 9± 6. 14 ** 2. 25 ±0 .4 7 6. 77 ±0 .4 5* * 37 .1 3± 5. 13 58 ,7 5± 8, 54 g ro up ii pr eo pe ra tiv e 7. 35 ±1 . 5 1 * 29 3. 57 ±2 8. 62 * 23 5. 16 ±2 5. 72 * 48 .4 3± 6. 46 2. 58 ±0 .3 5* 9. 11 ±0 .4 5 * 39 .2 1± 5. 43 * 60 ,1 1± 6, 85 po st op er at iv e, 6 hr s. 9. 15 ±3 .7 1* * 39 7. 14 ±3 4. 18 19 9. 16 ±7 1. 59 30 .6 7± 7. 16 ** 2. 45 ±0 .2 8 12 .5 8± 0. 56 * * 43 .6 2± 5. 17 62 ,2 7± 6, 61 po st op er at iv e, 12 h rs . 10 .8 1± 3. 61 ** 41 5. 17 ±4 5. 23 ** 19 4. 58 ±6 7. 27 ** 32 .7 4± 6. 36 ** 2. 31 ±0 .2 5 13 .4 1± 0. 54 ** 45 .8 7± 5. 39 68 ,2 1± 6, 97 n ot es : * – a s ig ni fic an t d iff er en ce b et w ee n th e no rm al in di ce s an d in di ce s be fo re th e su rg er y in th e pa tie nt s of g ro up s i a nd ii . ** – a s ig ni fic an t d iff er en ce b et w ee n th e in di ce s of th e pa tie nt s of g ro up s i a nd ii b ef or e th e su rg er y an d th e in di ce s in 6 h rs . a nd 1 2 hr s. a ft er th e su rg er y. ta bl e 2. t h e st at e of h em os ta si s in t h e pa ti en ts w it h p at h ol og y of t h e h ip jo in t in de x pr eo pe ra ti ve g ro u p i pr eo pe ra ti ve g ro u p ii n or m al v al u es fi br in og en , g /l 4. 16 ±0 .1 8 5. 07 ±0 .3 5* 3. 52 ±0 .3 4 so lu bl e co m pl ex es o f fi br in m on om er s (s cf m ), ex tr . u ni ts 0. 48 ±0 .0 6 0. 57 ±0 .1 1* 0. 42 ±0 .0 4 fi br in op ep tid e а, n g/ m l 2. 04 ±0 .2 8 2. 61 ±0 .2 3* 1. 82 ±0 .2 4 fi br in d eg ra da tio n pr od uc t, m cg /m l 5. 26 ±2 .1 4 6. 92 ±2 .0 9* 4. 71 ±1 .5 8 fi br in ol yt ic b lo od a ct iv ity , % 50 .6 3± 0. 54 54 .4 9± 0 .5 3 50 .6 3± 0 .4 6 an tit hr om bi n іі і, % 93 .8 3± 7. 56 89 .7 7± 8 .0 9 96 .7 2± 6. 22 pl as m a re ca lc ifi ca tio n tim e, с 10 4. 42 ±9 .6 8 91 .6 7± 10 .3 1 11 0. 15 ±8 .5 7 n ot e: * – a s ig ni fic an t d iff er en ce b et w ee n th e no rm a nd le ve ls in th e pa tie nt s of g ro up s i a nd ii . i.k. venher et al. 39 su r g ic a l d is e a se s issn 2413-6077. ijmmr 2020 vol. 6 issue 1 (p<0.05) of fibrinolytic activity of blood, a decrease of the contents of at iii, and a slight decrease of prt. operative intervention significantly influenced the aggregation state of patients’ blood. so, at the traumatic stage of surgical intervention and up to 6 hours of the early postoperative period, the platelet aggregation rate significantly increased with a slight decrease in platelet aggregation time. in the same period, there was a decrease in the activity of the fibrinolytic blood system. this level of anticoagulant system with a tendency to exacerbation was maintained until the 3rd h. of early postoperative period. from the 6th hour of early postoperative period a gradual increase in the activity of the fibrinolytic blood system was evidenced. the results of the study of hemostasis in the patients with nctd at the intraoperative stage of surgical intervention at the increase of hypercoagulative properties of blood, stren gthening its aggregation ability with a depressed state of the fibrinolytic blood system indicated development of conditions for thrombotic process formation in the venous system. it could be promoted by a high level of endothelial dysfunction, which increased in the conditions of surgical intervention. discussion a number of complications often occurs in cases of endoproathetics. vtec among them is still urgent [1]. patients with surgical interventions on the large joints of the lower extremities require anticoagulant prophylaxis [2]. both the classical factors of the virchow’s triad, and the specific factors, which are specific for the endoprosthetics of the hip joint [5], as well as individual factors of the patients risk and pathology of large joints are taken into account [5]. however, in thromboprophylaxis the incidence of thrombosis of deep veins after the hip replacement is 0.8-9.0%, pulmonary embolism – from 1.4 to 6.0% [6]. the latter gives the right to suppose that during the thrombo prophylaxis the factors with different etiopathogenetic origin are not taken into account, but there is a direct or indirect activation of procoagulant fbas in their presence. thus, much attention should be paid to nonspecific dysplasia of the connective tissue [7]. the patients with hip osteoarthrosis, femoral neck fracture with nctd are characterized by increased degree of endothelial dysfunction, a greater activity of the haemocoagulation system compared to the patients without nctd. thus, in the preoperative stage, the blood level of markers of endothelial dysfunction in the patients with nctd is in 1.5-2.2 times (p <0.001) higher compare to the patients without nctd. it causes a 1.2 fold higher activity of the blood hemocoagulation system of these patients. the patients with pathology of the hip joint with nctd experience an increased degree of endothelial dysfunction at the intraoperative stage which leads to hypercoagulation. these changes of the endothelial system and the hemocoagulation system create favourable conditions for vtec. endoprosthetics of the hip joint is a highrisk group of vtec and requires anticoagulation prophylaxis. a number of present recommendations offer pharmacological prophylaxis for prevention of vtec in cases of endoprosthetics of large joints. the use of low molecular heparins, synthetic inhibitors of xa and iia factors of blood coagulation or antagonists of vitamin k is recommended for this [8]. however, the rate of vtec development in the postoperative period is trill at the previous level. in order to reduce the postoperative dvt, attention should be paid to intraoperative changes in the haemocoagulation state of the operated patients. [9] there are reports [10] table 3. the intraoperative state of hemostasis in the patients with hip arthritis and nctd index preoperative traumatic stage 3hr. p/о 6hr. p/о 12 hr. p/о fibrinogen, g/l 5.07±0.35 5.59±0.46 5.82±0.49 6.54±0.48* 6.43±0.47* scfm, units.extr. 0.57±0.11 0.81± 0.11* 1.23±0.19** 1.24±0.18** 0.97±0.21** fpa, ng/ml 2.61±0.23 4.51±0.36* 7.05±0.35* 6.94±0.43* 6.39±0.48* fdp, mcg/ml 6.92±2.09 13.47±3.56* 18.25±4.41* 19.12±4.38* 14.89±4.78* fba, % 54.49±3.53 43.75±4.13 42.19±4.16* 43.41±4.23* 45.72±4.27* at ііі, % 89.77+6.09 84.12+6.55 81.24+5.49 84.54+5.75 85.56+5.68 prt, sec 91.67±7.31 80.57±7.33 82.65±7.72 84.59±7.37 89.28±8.19 notes: * – p<0.05 in comparison with preoperative indices; ** – p<0.001 in comparison with preoperative indices. i.k. venher et al. 40 su r g ic a l d is e a se s issn 2413-6077. ijmmr 2020 vol. 6 issue 1 about the diagnosis of the thrombotic process in the ivc system after the end of the surgical intervention. during the surgical intervention for the pathology of the hip joint from the second half of the traumatic phase and during the first 2-3 hours of the early postoperative period, an increase of the level of hypercoagulation due to fibrin-monomeric complexes is observed. it is the unfractionated heparin that has a predominant influence on the iia factor (thrombin – fibrinogen) of the hemocoagulation cascade. and thromboprophylaxis should be started straight after the end of the surgical intervention with the prescription of non-fractional heparin and should be continued until the first injection of lmwh, which has a predominant influence on the xa factor of the blood coagulation system [11]. the use of oral form of unfractionated heparin is promising [12]. this has become possible due to the combination of unfractionated heparin and the molecule n [8 (2-hydroxy benzene) amine] carrier – sodium caprylate. the third phase of coagulation cascade study showed that oral heparin reduces the incidence of postoperative thrombotic formation. conclusions the patients with osteoarthrosis of the hip joint and femoral neck fracture and non-specific dysplasia of the connective tissue are characterized by expressed levels of endothelial dysfunction and increased activity of the hemocoagulation system. in the postoperative period after endoprosthetics of hip joints, in the patients with osteoarthrosis of the hip joint and femoral neck fracture and non-specific dysplasia of the connective tissue, vtec was diagnosed in 13.3% of cases, in the patients with osteoarthrosis of the hip joint and femoral neck fracture in the absence of nonspecific dysplasia of the connective tissue vtec was revealed in 8.8% of cases. the thromboprophylaxis of vtec in the patients with endoprosthetics of hip joints should be started straight after the start of surgical intervention with the prescription of non-fractional heparin, which has a predominant effect on the iia fba of the hemocoagulation cascade and should be continued until the first injection of lmwh, which has a dominant influence on ha factor of the hemocoagulation system. funding this research received no external funding. conflict of interests the authors declare no conflict of interest. author contributions venher i.k. – conceptualization, project administration, supervision; herasymiuk n.i. – resources, investigation, writing – original draft, writing – review & editing; kostiv s.ya. – methodology, formal analysis; loyko i.i. – validation, writing – original draft; khvalybo ha d.v. – investigation. неспецифічна дисплазія сполучної тканини – фактор ризику венозних тромбоемболій при ендопротезуванні кульшового суглоба і.к. венгер, н.і. герасимюк, с.я. костів, і.і. лойко, д.в. хвалибога тернопільський національний медичний університет імені і.я. горбачевського, тернопіль, україна вступ. важливою складовою ортопедичної хірургії є ендопротезування тазостегнових суглобів, що усуває больовий синдром, відновлює амплітуду рухів та підтримуючу здатність нижньої кінцівки. але ці втручання супроводжуються рядом ускладнень, серед яких провідне місце займає венозна тромбоемболія. мета. дослідити ендотеліальну дисфункцію та активність системи гемокоагуляції на різних рівнях ризиків венозного тромбемболізму та, таким чином, розробити стратегію тромбопрофілактики у пацієнтів з остеоартрозом з переломом шийки тазостегнового суглоба та стегнової кістки у поєднанні з неспецифічною дисплазією сполучної тканини. методи. оперовано 219 пацієнтів із середнім віком 64,7±3,8. у 137 (62,1%) спостереженнях проведено тотальне цементне ендопротезування стегна з остеоартритом. 82 (37,4%) пацієнтів отримали тотальну та однополярну заміну стегна при переломах шийки стегнової кістки. i.k. venher et al. 41 su r g ic a l d is e a se s issn 2413-6077. ijmmr 2020 vol. 6 issue 1 результати. клінічні прояви неспецифічної дисплазії сполучної тканини були виявлені у 83 (37,9%) пацієнтів, що було підтверджено лабораторним визначенням рівня загального, зв’язаного та вільного оксипроліну. у післяопераційному періоді в 23 (10,5%) спостереженнях діагностували тромботичний процес у венозній системі нижньої порожнистої вени. рівень показників ендотеліальної дисфункції був суттєво вираженіший у пацієнтів за наявності неспецифічної дисплазії сполучної тканини. оперативне втручання на тазостегновому суглобі у пацієнтів з неспецифічною дисплазією сполучної тканини у 11 (13,3%) випадках ускладнилось розвитком венозного тромбозу. у пацієнтів без неспецифічної дисплазії сполучної тканини післяопераційний тромбоз у системі нижньої порожнистої вени був діагностований у 12 (8,8%) спостереженнях. висновки. пацієнти з остеоартрозом тазостегнового суглоба та переломом шийки стегнової кістки, що супроводжуються неспецифічною дисплазією сполучної тканини, характеризуються вираженим рівнем ендотеліальної дисфункції та підвищеною активністю системи згортання крові. ключові слова: ендопротезування; тромбемболія; ендотеліальна дисфункція; дисплазія. інформація про авторів венгер і.к. – доктор медичних наук, професор, завідувач кафедри хірургії № 2, тернопільський національний медичний університет імені і.я. горбачевського, тернопіль, україна. герасимюк н.і. – кандидат медичних наук, доцент кафедри хірургії № 2, тернопільський національний медичний університет імені і.я. горбачевського, тернопіль, україна. костів с.я. – доктор медичних наук, професор, професор кафедри хірургії № 2, тернопільський національний медичний університет імені і.я. горбачевського, тернопіль, україна. лойко і.і. – кандидат медичних наук, доцент кафедри хірургії № 2, тернопільський національний медичний університет імені і.я. горбачевського, тернопіль, україна. хвалибога д.в. – аспірант кафедри хірургії № 2, тернопільський національний медичний університет імені і.я. горбачевського, тернопіль, україна. information about the authors venher i.k. – md, ph.d., professor, department of surgery no. 2, i. horbachevsky ternopil national medical university, ternopil, ukraine. orcid https://orcid.org/0000-0003-0170-1995, e-mail: vengerik@tdmu.edu.ua herasymiuk n.i. – md, ph.d., professor, department of surgery no. 2, i. horbachevsky ternopil national medical university, ternopil, ukraine. orcid https://orcid.org/0000-0002-5476-268x, e-mail: herasymyuk_n@tdmu.edu.ua kostiv s.ya. – md, ph.d., professor, department of surgery no. 2, i. horbachevsky ternopil national medical university, ternopil, ukraine. orcid https://orcid.org/0000-0002-2991-3263, e-mail: kostivsj@tdmu.edu.ua loyko i.i. – md, ph.d., professor, department of surgery no. 2, i. horbachevsky ternopil national medical university, ternopil, ukraine. orcid https://orcid.org/0000-0003-2967-1054, e-mail: loikoii@tdmu.edu.ua khvalyboha d.v. – md, postgraduate student, department of surgery no. 2, i. horbachevsky ternopil national medical university, ternopil, ukraine. phylaxis for hospitalized and nonhospitalized medical pa tients. blood advances. 2018 nov 27;2(22):3198225. doi: 10.1182/bloodadvances.2018022954 3. flevas da, megaloikonomos pd, dimopoulos l, mitsiokapa e, koulouvaris p, mavrogenis af. thrombo embolism prophylaxis in orthopaedics: an update. efort open reviews. 2018 apr;3(4):136-48. doi: 10.1302/2058-5241.3.170018 4. yasunaga h, tsuchiya k, matsuyama y, ohe k. high-volume surgeons in regard to reductions in references 1. lychagin av, cherepanov vg, petrov pi, vyazankin ia, brkich ge. pre-surgery planning of lower limbs major joints arthroplasty. open access macedonian journal of medical sciences. 2019 sep 15;7(17):2838-43. doi: 10.3889/oamjms.2019.690 2. schünemann hj, cushman m, burnett ae, kahn sr, beyer-westendorf j, spencer fa, rezende sm, zakai na, bauer ka, dentali f, lansing j. american society of hematology 2018 guidelines for mana gement of venous thromboembolism: proi.k. venher et al. 42 su r g ic a l d is e a se s issn 2413-6077. ijmmr 2020 vol. 6 issue 1 operating time, blood loss, and postoperative 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committee. oral thrombin inhibitor dabigatran etexilate vs north american enoxaparin regimen for prevention of venous thromboembolism after knee arthroplasty surgery. the journal of arthroplasty. 2009 jan 1;24(1):1-9. doi: 10.1016/j.arth.2008.01.132 9. liew nc, alemany gv, angchaisuksiri p, bang sm, choi g, de ds, hong jm, lee l, li yj, rajamoney gn, suviraj j. asian venous thromboembolism guidelines: updated recommendations for the prevention of venous thromboembolism. international angiology: a journal of the international union of angiology. 2017 feb;36(1):1-20. doi: 10.23736/s0392-9590.16.03765-2 10. van py, schreiber ma. contemporary thromboprophylaxis of trauma patients. current opinion in critical care. 2016 dec 1;22(6):607-12. doi: 10.1097/mcc.0000000000000365 11. diamantouros a, kiss a, papastavros t, da vid u, zwarenstein m, geerts wh. the toronto thromboprophylaxis patient safety initiative (topps): a cluster randomised trial. research in social and administrative pharmacy. 2017 sep 1;13(5):997-1003. doi: 10.1016/j.sapharm.2017.05.015 12. zhang j, xu j, zhang w, jiang m, liu j, xu l, liu g, zhao z. quality appraisal of guidelines on cancer-associated thrombosis using agree ii instrument and analysis of current status of new oral anticoagulants. clinical and applied thrombosis/ hemostasis. 2019 apr 26;25:1076029619846562. doi: 10.1177/1076029619846562 received 23 march 2020; revised 15 may 2020; accepted 11 june 2020. this is open access article distributed under the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. i.k. venher et al. 88 p h a r m a c y issn 2413-6077. ijmmr 2019 vol. 5 issue 2 doi 10.11603/ijmmr.2413-6077.2019.2.10897 methods of metoprolol analysis in drugs and biological fluids: review and suggestions m.m. horyn, *l.s. logoyda i. horbachevsky ternopil national medical university, ternopil, ukraine background. analytical method is increasingly implemented into fundamental pharmaceutical chemistry and analysis, considering their high sensitivity, accuracy, specificity and expressiveness. objective. metoprolol’s analytical method development was the research goal. methods. the sources were world recognized journals (1990-2019) and key words used as filter were “metoprolol”, “spectrophotometry” “high-performance liquid chromatography, hplc”, “quantitative analysis”, “validation”. results. chromatographic methods of analysis have the highest specificity and objectivity and allow qualitative and quantitative determination of active pharmaceutic ingredient (api) in combined dosage forms and biological fluids without prior components separation. the main disadvantage of the described api analysis methods is long terms from the beginning of chromatography to api release and specific solvents used as the mobile phase in hplc. new methods development and selection such chromatographic conditions that provide high speed and high efficiency at lower pressure of the system are essential. also, the reduction of analysis time is achieved by simplifying the conditions for sample preparation. conclusions. analysts are constantly working on developing new analysis methods and their optimization in order to save time and consumables, which also ensures the efficiency of the developed method. there is no monograph on the substance or dosage forms of metoprolol in sphu. therefore, some of the developed methods should be suggested for the sphu monograph, which is important for ensuring pharmacopoeial quality control of medicines in ukraine. key words: metoprolol; spectrophotometry; high-performance liquid chromatography; quantitative analysis; method development; validation. corresponding author: logoyda l.s., ph.d., dsc, head of department of pharmaceutical chemistry, i. horbachevsky ternopil national medical university, 1 maidan voli, ternopil, ukraine. e-mail: logojda@tdmu.edu.ua introduction the quality of treatment depends much on the bioavailability of the medicinal product, which in turn is influenced by the correct and effective pharmaceutical development. antihypertensive drugs are currently the most widely used medicines, which is associated with increase in the population with high blood pressure and those at risk for stroke and other serious diseases of the cardiovascular system. considerable demand for antihypertensive drugs in tablet form is a prerequisite for inclusion of a large number of generic drugs in the range of industrial enterprises. the main task of pharmaceutical development for such drugs is to develop a composition that will provide bioavailability comparable to the original. the efficacy and correctness of pharmaceutical development must be confirmed by bioe qui­ valence studies by in vivo and in vitro methods. for these purposes, effective and reliable methods for detection of api in biological fluids should be developed. it is generally accepted to use chromatographic methods for this purpose that can provide sufficient selectivity and accuracy of determination even at relatively low doses. it should be noted that today the requirements for bioanalytical techniques are framework and too broad to be universal. therefore, making proposals for the study of individual validation characteristics for pharmacokinetic purposes is very important. there are currently no systematic studies of metoprolol. metoprolol succinate is a beta1-selective (cardioselective) adrenoceptor blocking agent, for oral administration, available as extendedrelease tablets. metoprolol succinate extendedrelease tablet has been formulated to provide a controlled and predictable release of metoprolol for once-daily administration. the tablets international journal of medicine and medical research 2019, volume 5, issue 2, p. 88-97 copyright © 2019, tnmu, all rights reserved m.m. horyn et al. 89 p h a r m a c y issn 2413-6077. ijmmr 2019 vol. 5 issue 2 m.m. horyn et al. comprise a multiple unit system containing metoprolol succinate in a multitude of controlled release pellets. each pellet acts as a separate drug delivery unit and is designed to deliver metoprolol continuously over the dosage interval. the tablets contain 23.75, 47.5, 95 and 190 mg of metoprolol succinate equivalent to 25, 50, 100 and 200 mg of metoprolol tartrate, usp, respectively. its chemical name is (±)1-(isopropylamino)-3-[p-(2-methoxyethyl) phenoxy]­2­propanol succinate (2:1) (salt) (fig.1). metoprolol is a propanolamine that is 1-(propan-2-ylamino)propan-2-ol substituted by a 4-(2-methoxyethyl)phenoxy group at position 1. it acts as a beta-adrenergic antagonist, an antihypertensive agent, a xenobiotic and an environmental contaminant. it is a propanolamine, an aromatic ether, a secondary alcohol and a secondary amino compound. metoprolol is used for a number of conditions, including hypertension, angina pectoris, acute myocardial infarction, different types of tachyarrythmia, congestive heart failure, and prevention of migraines. due to its selectivity, metoprolol is also prescribed for off-label use for anxiety disorders. metoprolol blocks β1 drenergic receptors in cardiomyocytes, thereby decreasing the slope of phase 4 in the nodal action potential (reducing na+ uptake) and prolonging repolarization of phase 3 (slowing down k+release). it suppresses the norepinephrine-induced increase in the sarcoplasmic reticulum ca2+ leak and the spontaneous sr ca2+ release, which are the major triggers for atrial fibrillation. fig. 1. chemical structure of metoprolol. within the uk, metoprolol is classified as a prescription-only drug in the beta blocker class and is regulated by the medicines and healthcare products regulatory agency (mhra). the mhra is a government body set up in 2003 and is responsible for regulating medicines, medical devices, and equipment used in healthcare. in the u.s. use of beta blockers such as metoprolol was approved by the food and drug administration (fda) in 1967 for the treatment of cardiac arrhythmias, hypertension, migraines, and others. prescribers may choose to prescribe beta blockers for other treatments if there is just cause even though it is not approved by the fda. drug manufacturers, however, are unable to advertise beta blockers for other purposes that have not been approved by the fda. since the fda does not regulate the practice of medicine after the drug has been approved, it is legal to prescribe beta blockers for other treatments such as performance anxiety. in man, absorption of metoprolol is rapid and complete. plasma levels following oral administration of conventional metoprolol tablets, however, approximate 50% of levels following intravenous administration, indicating about 50% first­pass metabolism. metoprolol crosses the blood-brain barrier and has been reported in the csf in a concentration 78% of the simultaneous plasma concentration. plasma levels achieved are highly variable after oral administration. only a small fraction of the drug (about 12%) is bound to human serum albumin. metoprolol is a racemic mixture of rand s-enantiomers, and is primarily metabolized by cyp2d6. when administered orally, it exhibits stereoselective metabolism that is dependent on oxidation phenotype. elimination is mainly by biotransformation in the liver, and the plasma half-life ranges from approximately 3 to 7 hours. less than 5% of an oral dose of metoprolol is recovered unchanged in the urine; the rest is excreted by the kidneys as me ta bolites that appear to have no betablocking activity. following intravenous administration of metoprolol, the urinary recovery of unchanged drug is approximately 10%. the systemic availability and half-life of metoprolol in patients with renal failure do not differ to a clinically significant degree from those in normal subjects. consequently, no reduction in dosage is usually needed in patients with chronic renal failure. metoprolol is metabolized predominantly by cyp2d6, an enzyme that is absent in about 8% of caucasians (poor metabolizers) and about 2% of most other populations. cyp2d6 can be inhibited by a number of drugs. concomitant use of inhibiting drugs in poor metabolizers will increase blood levels of metoprolol several-fold, decreasing metoprolol’s cardioselectivity. metoprolol has a very low melting point; around 120 °c (248 °f) for the tartrate, and around 136 °c (277 °f) for the succinate. because of this, metoprolol is always manufactured in a salt-based solution, as drugs with low melting points are difficult to work with in a manufacturing environment. the free base exists as a waxy white solid, and the tartrate salt is finer crystalline material. 90 p h a r m a c y issn 2413-6077. ijmmr 2019 vol. 5 issue 2 metoprolol contains a stereocenter and consists of two enantiomers. metoprolol succinate is a white crystalline powder with a molecular weight of 652.8. it is freely soluble in water; soluble in methanol; sparingly soluble in ethanol; slightly soluble in dichloromethane and 2­propanol; practically insoluble in ethyl­ acetate, acetone, diethylether and heptane. inactive ingredients: silicon dioxide, cellulose compounds, sodium stearyl fumarate, polyethylene glycol, titanium dioxide, paraffin. physico-chemical analysis methods are in creasingly introduced into fundamental pharmaceutical research and pharmaceutical analysis practice, taking into account their high sensitivity, accuracy, specificity and expressiveness. objective was analytical method development for metoprolol. methods literature survey has been done in range of years 1990-2019 to make the review updated and comprehensive and to show the new approaches to development of the methods of metoprolol analysis. the sources were world recognized journals and key words used as filter were metoprolol, spectrophotometry, highperformance liquid chromatography, quantitative analysis, method development, validation [1-5]. results pharmacopeian methods of analysis of metoprolol are presented in fig. 2. the state pharmacopoeia of ukraine (sphu) has not developed a monograph on the substance of metoprolol or on the prepared medical form yet. however, the united states pharmacopoeia regulates the determination of metoprolol succinate in extended-release tablets. for identification, ir­spectroscopy and hplc are suggested; for quantitative determination of metoprolol succinate in tablets in assay and dissolution test – hplc, respectively. chromatographic conditions for determination of metoprolol succinate in tablets are specified in the monograph of the united states pharmacopoeia, which are used the chromatographic column 4­mm×12.5­cm; 5­µm packing l7 and mobile phase consisting of acetonitrile and buffer (25:75). mobile phase rate – 1 ml/min, detection wavelength – 280 nm, tailing factor – nmt 2.0, relative standard deviation – nmt 2.0%. the suggested method of the united states pharmacopeia requires a long sampling. the european pharmacopeia has a monograph on metoprolol tartrate tablets. identification of metoprolol tartrate of the european pharmacopeia regulates the absorption spectrophotometry in the infrared region, uv-spectrophofig. 2. pharmacopeian methods of analysis of metoprolol. 4 fig. 2. pharmacopeian methods of analysis of metoprolol. historical development of methods for the quantitative determination of metoprolol in substances and drugs is related to the development of analytical methods only and pharmaceutical analysis in general. nowadays the literature contains a large number of scientific papers devoted to the quantitative determination of metoprolol and other apis in one medical form, since metoprolol is used in combination with different apis for the treatment of hypertension. according to the literature data, spectrophotometry [6-11], hplc [12-21], gas chromatography [22] are the most widely used techniques for the determination of metoprolol tartrate. mustafa cesme et al. suggested spectrophotometric determination of metoprolol tartrate in pharmaceutical dosage forms on complex formation with cu(ii). spectrophotometric method has been developed for the assay of metoprolol tartrate, which is based on the complexation of drug with copper(ii) [cu(ii)] at ph 6.0, using britton-robinson buffer solution, to produce a blue adduct. the latter has a maximum absorbance at 675 nm and obeys beer’s law within the concentration range 8.5-70 μg/ml. regression analysis of the calibration data showed a good correlation coefficient (r = 0.998) with a limit of detection of 5.56 μg/ml. the suggested procedure has been successfully applied to the determination of this drug in its tablets. in addition, the spectral data and stability constant for the binuclear copper(ii) complex of metoprolol tartrate (cu2mpt2cl2) have been reported [6]. two simple and selective spectrophotometric methods are described for determination of metoprolol tartar as base form metoprolol in bulk drug, and in tablets and capsules by nabil a.f., eman m.s. the methods are based on the molecular charge transfer complexation of metoprolol base metoprolol with bromothymol blue (btb) or 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (ddq). the yellow and orange colored radical anions formed on dissociation, are quantitated at 413 nm (bpb method) or 457 nm (ddq method). the assay conditions were optimized. beer’s law is obeyed in the concentration ranges 2.0-40.0 μg ml-1 in bpb method and 5.0-25.0 μg.ml-1in ddq method, with respective molar absorptivity values of 5.78 × 103 and 4.05 × 103 l mol-1 cm-1. the reaction stoichiometry in both methods was evaluated by job’s method of continuous variations pharmacopeian methods of analysis united states pharmacopoeia regulates the determination of metoprolol succinate in extendedrelease tablets. for identification, ir-spectroscopy and hplc are proposed. for quantitative determination of metoprolol succinate in tablets in assay and dissolution test – hplc, respectively the state pharmacopoeia of ukraine (sphu) does not have a monograph on the substance of metoprolol or on the prepared medical form european pharmacopeia has a monograph on metoprolol tartrate tablets. identification of metoprolol tartrate of the european pharmacopeia regulates the absorption spectrophotometry in the infrared region, uvspectrophotometry and hplc, quantitative determination – hplc/uv. m.m. horyn et al. 91 p h a r m a c y issn 2413-6077. ijmmr 2019 vol. 5 issue 2 m.m. horyn et al. tometry and hplc, quantitative determination – hplc/uv. as a solvent, a mixture of methanol and 0.1 m hydrochloric acid is used, mobile phase – solution of 1-pentanesulfonic acid sodium salt (monohydrate), anhydrous sodium acetate in mixture of methanol and water and adding glacial acetic acid. mobile phase rate – 1.0 ml/min, detection wavelength – 254 nm. historical development of methods for the quantitative determination of metoprolol in substances and drugs is related to the development of analytical methods only and pharmaceutical analysis in general. nowadays the literature contains a large number of scientific papers devoted to the quantitative determination of metoprolol and other apis in one medical form, since metoprolol is used in combination with different apis for the treatment of hypertension. according to the literature data, spectrophotometry [6-11], hplc [12-21], gas chromatography [22] are the most widely used techniques for the determination of metoprolol tartrate. mustafa cesme et al. suggested spectrophotometric determination of metoprolol tartrate in pharmaceutical dosage forms on complex formation with cu(ii). spectrophotometric method has been developed for the assay of metoprolol tartrate, which is based on the complexation of drug with copper(ii) [cu(ii)] at ph 6.0, using britton-robinson buffer solution, to produce a blue adduct. the latter has a maximum absorbance at 675 nm and obeys beer’s law within the concentration range 8.5­70 μg/ml. regression analysis of the calibration data showed a good correlation coefficient (r=0.998) with a limit of detection of 5.56 μg/ml. the suggested procedure has been successfully applied to the determination of this drug in its tablets. in addition, the spectral data and stability constant for the binuclear copper(ii) complex of metoprolol tartrate (cu2mpt2cl2) have been reported [6]. two simple and selective spectrophotometric methods are described for determination of metoprolol tartar as base form metoprolol in bulk drug, and in tablets and capsules by nabil a.f., eman m.s. the methods are based on the molecular charge transfer complexation of metoprolol base metoprolol with bromothymol blue (btb) or 2,3-dichloro-5,6-dicyano-1,4benzoquinone (ddq). the yellow and orange colored radical anions formed on dissociation, are quantitated at 413 nm (bpb method) or 457 nm (ddq method). the assay conditions were optimized. beer’s law is obeyed in the concentration ranges 2.0­40.0 μg ml-1 in bpb method and 5.0­25.0 μg·ml-1in ddq method, with respective molar absorptivity values of 5.78×103 and 4.05×103 l mol-1·cm-1. the reaction stoichiometry in both methods was evaluated by job’s method of continuous variations and was found to be 1:1 mpt­bpb, mpt­ddq. the developed methods were successfully applied to the determination of metoprolol in pure form and commercial tablets with good accuracy and precision. statistical comparison of the results was performed using student’s t-test and fratio at 95% confidence level and the results showed no significant difference between the reference and suggested methods with regard to accu racy and precision. further, the accuracy and reliability of the methods were confirmed by recovery studies via standard addition technique [7]. ukrainian scholars anastasiia donchenko and svitlana vasyuk suggested spectrophotometric determination of metoprolol tartrate in pure and dosage forms. this method is based on the reaction between metoprolol tartrate and 2,3-dichloro-1,4-naphthoquinone in dimethylformamide (dmf) medium to form the colored reaction product with maximum absorption at 493 nm. optimum conditions to carry out the reaction such as concentration of reagent, temperature and heating time were carefully studied and optimized. beer’s law was performed at the concentration range of 18.0028.00 mg/100 ml. the suggested method is valid according to the validation requirements of sphu [8]. turkish scientist yilmaz b. developed determination of metoprolol in pharmaceutical preparations by zero­, first­, second­ and third­ order derivative spectrophotometric method. zero­, first­, second­ and third­order derivative spectrophotometry methods were developed for determination of metoprolol in pharmaceutical preparations. in zero order spectrophotometry, absorbance values were measured at 276 nm in zero order spectra of solution of metoprolol in methanol in the range of 240310 nm. in the first derivative spectrophotometry, absorbance values were measured at 265, 278 and 285 nm. in the second derivative spectrophotometry, absorbance values were measured at 276, 279, 287 and 282 nm. in the third derivative spectrophotometry, absorbance values were measured at 275, 278 and 281 nm. parameters such as linearity, precision, accuracy, specificity, stability, limit of detection and limit of quantitation were studied according to the international conference on harmonization guide92 p h a r m a c y issn 2413-6077. ijmmr 2019 vol. 5 issue 2 l i n e s . a l l t h e m e t h o d s d ev e l o p e d w e re successfully applied to two tablet formulation and the results were compared statistically with each other [9]. jadhav a.s. et al. suggested uv spectrophotometric methods for estimation of metoprolol succinate from bulk and tablet formulation in phosphate buffer 6.8. the drug obeyed the beer’s law with correlation coefficient 0.9999 and 0.9979 respectively for method i and method ii. it showed absorption maxima at 223 nm and 226 nm respectively for method i and method ii; in phosphate buffer 6.8. the linearity was observed between 5 and 25 µg/ml. the results of analysis were validated by recovery studies, accuracy, precision, lod, loq and ruggedness. the method was found to be simple, accurate, precise, economical and robust [10]. rahman n. et al. have developed validated kinetic spectrophotometric method for determination of metoprolol tartrate in pharmaceutical formulations. the method is based on reaction of the drug with alkaline potassium permanganate at 25+/-1 degrees c. the reaction is followed spectrophotometrically by measuring the change in absorbance at 610 nm as a function of time. the initial rate and fixed time (at 15.0 min) methods are utilized for constructing the calibration graphs to determine the concentration of the drug. both the calibration graphs are linear in the concentration range of 1.46×10-6-8.76×10-6 m (10.0-60.0 microg per 10 ml). the calibration data resulted in the linear regression equations of log (rate)=3.634+ 0.999 log c and a=6.300 x 10-4+6.491×10-2 c for initial­rate and fixed time methods, respectively. the limits of quantitation for initial rate and fixed time methods are 0.04 and 0.10 microg ml-1, respectively. the activation parameters such as e(a), deltah(double dagger), deltas(double dagger) and deltag(double dagger) are also evaluated for the reaction and found to be 90.73 kjєmol-1, 88.20 kj mol-1, 84.54 j·k-1·mol-1 and 63.01 kj·mol-1, respectively. the results are validated statistically and through recovery studies. the method has been successfully applied to the determination of metoprolol tartrate in pharmaceutical formulations. statistical comparison of the results with the reference method shows excellent agreement and indicates no significant difference in accuracy and precision [11]. the hplc method is widely used in the analysis of metoprolol both in substance and in monoand combined drugs. simple, precise, sensitive methods are developed today: liquid chromatography, combination of tlc with densitometric determination, hplc/uv, hplc/ dmd, hplc/ms. a reverse phase hplc method has been developed for the quantitative estimation of amlodipine besylate and metoprolol tartrate in tablet by hussain s. et al. the quantification was carried out using rp stainless steel column ods c18 250×4.6× 5 µ l1 packing in isocratic mode with mobile phase containing 0.03 m phosphate buffer and acetonitrile in the ratio of 32: 68 (ph 3.5). flow rate of 1.2 ml/min and the detection wavelength were set at 230 nm and the linearity was found to be in the range of 8-12 ìg/ml for amlodipine besylate and metoprolol tartrate. the suggested method was found to be simple, precise, accurate, and reproducible for the estimation of amlodipine besylate and metoprolol tartrate [12]. singh brijesh et al. suggested reversephase hplc method for simultaneous analysis of metoprolol succinate and hydrochlorothiazide in a tablet formulation. the drugs were analyzed by a reverse phase c-18 column using 50 mm di­sodium hydrogen phospha te:me­ thanol:acetonitrile in a ratio of 525:225:250 as mobile phase. the flow rate was 1 ml/min and the compounds were detected by a uv-detector at 222 nm at a column temperature of 24±2 ºc. the method was statistically validated for linearity and accuracy. the retention time and drug content of metoprolol succinate and hydrochlorothiazide were 5.38 min, 96.05% and 3.04 min., 97.64%, respectively [13]. simultaneous estimation of metoprolol tartrate and chlorthalidone by using rp-hplc and method development as per ich guidelines has been suggested by p. hari prasad et al. the chromatographic analysis was performed on a c18column grace smart rp18 (250×4.6 mm, 5 µm) in isocratic mode, the mobile phase consisted of methanol, acetonitrile and 0.05 m phosphate buffer (adjusted toph 4.5 with orthophosphoric acid) at a ratio of 60:20:20 v/v/v, and a flow rate of 1.0 ml/min and the aspd detector was used. the eluents were monitored at 254 nm. the retention time of lamivudine and stavudine were found to be 2.50 min and 4.25 min, respectively. the linear ranges were found to be 10­602 2 µg/ml (r=0.9992) for lamivudine and 10­60 µg/ml (r=0.999) for stavudine. the suggested method is also found to be accurate, precise and robust. the method could be applied to routine quality control of pharmaceutical formulations containing metoprolol tartrate and chlorthalidone [14]. m.m. horyn et al. 93 p h a r m a c y issn 2413-6077. ijmmr 2019 vol. 5 issue 2 m.m. horyn et al. a simple, rapid, accurate, precise, selective, and reproducible stability-indicating hplc method has been developed for simultaneous estimation of metoprolol succinate and telmisartan using a mobile phase consisting mixture of methanol: 10 mm potassium dihydrogen phosphate buffer: 10 mm hexane sulphonic acid (80:10:10,v/v/v) at the flow rate of 1 ml/min and detection wavelength at 223.0 nm by s. p. mahaparale et al.. hiq sil c18 (250×4.6 mm, 5 µm) column was used as stationary phase. the retention time for metoprolol succinate and telmisartan were 3.067 min and 5.653 min, respectively. linearity was observed in the concentration range of 5­80 µg/ml for meto­ prolol succinate and 5­60 µg/ml for telmisartan. the coefficient of correlation for metoprolol succinate and telmisartan was found to be 0.9990 and 0.9980, respectively. the results of analysis have been validated statistically and by recovery studies. both the drugs were subjected to acid, alkali and neutral hydrolysis, oxidation, dry heat, and photolytic degradation. the degradation products of telmisartan and metoprolol succinate were well resolved from the pure drugs with significant differences in the retention time values. this method can be successfully implemented for simultaneous quantitative analysis of metoprolol succinate and telmisartan in bulk drugs and formulations [15]. braza a.j. et al. suggested development, validation and analytical error function of two chromatographic methods with fluorometric detection for determination of bisoprolol and metoprolol in human plasma, which describes two high-performance liquid chromatographic methods for the individual determination of bisoprolol and metoprolol in human plasma. analytical methods involve two different liquidliquid extractions of human plasma, with diethyl ether for bisoprolol and with dichloromethane for metoprolol, coupled with a similar nucleosil c(18) reversed-phase hplc column. fluorimetric detection was used to identify both betablockers. retention times for bisoprolol and metoprolol were 8.7 and 3.2 min, respectively. linear regressions for the calibration curves were linear at a concentration range of 6.25200 ng/ml. intraand inter-day precision coefficients of variations and accuracy bias were acceptable (within 15%) over the entire range for both drugs. average recovery was 89% for metoprolol and 98% for bisoprolol. once the methods had been validated, analytical error functions were established as standard deviation (sd; ng/ml)=2.216+3.608×10-4c2 (c=theoretical concentration value) and sd-(ng/ml)= 0.408+0.378×10-1c for bisoprolol and metoprolol, respectively. the developed methods and their associated analytical error functions would be suitable for pharmacokinetic studies and for determination of plasma concentration if posology individualization of these drugs is needed [16]. chiu f.c. et al. have developed efficient high-performance liquid chromatographic assay for the simultaneous determination of metoprolol and two main metabolites in human urine by solid­phase extraction and fluorescence detection. the simultaneous analysis of the zwitterionic metoprolol acidic metabolite (iii, h117/04) with the basic metabolites alphahydroxymetoprolol (ii, h119/66), metoprolol (i) and guanoxan (iv, internal standard) was achieved employing solid-phase extraction and isocratic reversed-phase hplc. the analytes were extracted from urine (100 microliters) using c18 solid-phase extraction cartridges (100 mg), and eluted with aqueous acetic acid (0.1%, v/v)­methanol mixture (40:60, v/v, 1.2 ml). the eluents were concentrated (250 microliters) under vacuum, and aliquots (100 microliters) were analyzed by hplc with fluorescence detection at 229 nm (excitation) and 309 nm (emission) using simple isocratic reversed-phase hplc (novapak c18 radial compression cartridge, 4 microns, 100×5 mm i.d.). acetonitrile-methanol-tea/phosphate buffer ph 3.0 (9:1:90, v/v) was employed as the eluent (1.4 ml/min). all components were fully resolved within 18 min, and the calibration curves for the individual analytes were linear (r2≥0.996) within the concentration range of 0.25-40.0 mg/ml. recoveries for all four analytes were greater than 76% (n=4). the assay method was validated with intra-day and inter-day variations less than 2.5% [17]. scientists johnson r.d. and lewis r.j. have developed a liquid chromatography with mass spectrometric detection (lc/ms) method for the simultaneous quantitation of three commonly prescribed beta-blockers, atenolol, metoprolol and propranolol. one advantage of the lc/ms method is the specificity provided by an ion trap ms. utilizing an ion trap ms were able to conduct ms/ms and ms/ms/ms on each analyte. this method also eliminates the timeconsuming and costly derivitization step necessary during gc/ms analysis. additionally, by utilizing this novel method, any concerns about beta-blocker metabolite and/or sample matrix 94 p h a r m a c y issn 2413-6077. ijmmr 2019 vol. 5 issue 2 interference are eliminated. the limits of detection for this method ranged from 0.39 to 0.78 ng/ml and the linear dynamic range was generally 1.6­3200 ng/ml. the extraction effi­ ciencies for each analyte ranged from 58% to 82%. this method was successfully applied to postmortem fluid and tissue specimens obtai­ ned from victims of three separate aviation accidents [18]. a stereoselective liquid chromatographytandem mass spectrometry assay was developed and validated for quantification of s­ and r-metoprolol at concentrations of 0.5-50 microg/l in human plasma by jensen b.p et al. metoprolol was extracted from plasma by liquid-liquid extraction with ethyl acetate (82% recovery). chromatographic separation of the enantiomers was achieved on a chiral chirobiotic t column using an isocratic mobile phase consisting of methanol/acetic acid/ammonia (100/0.15/0.15, v/v/v). an ion trap mass spectrometer with an electrospray interface was used for detection in the positive mode, monitoring the m/z transition 268-->191 for me toprolol. standard curves for sand r-metoprolol fitted quadratic functions (r(2)>or=0.9995) over the range of 0.5-50 microg/l in plasma, with 0.5 microg/l representing the limit of quantification. in this range, relative standard deviations were <6% for intra-day precision and <10% for inter-day precision. the accuracy was within the range of 92-105% [19]. a simple, rapid, sensitive and specific liquid chromatography-tandem mass spectrometry method has been developed and validated for quantification of metoprolol tartrate and ramipril in human plasma by gowda k.v. et al. both the drugs were extracted by liquid-liquid extraction with diethyl ether-dichloromethane (70:30, v/v). the chromatographic separation was performed on a reversed-phase c8 column with a mobile phase of 10 mm ammonium formate­methanol (3:97, v/v). the protonated analyte was quantitated in positive ionization by multiple reaction monitoring with a mass spectrometer. the method was validated over the concentration range of 5-500 ng/ml for metoprolol and ramipril in human plasma. the precursor to product ion transitions of m/z 268.0-103.10 and m/z 417.20-117.20 were used to measure metoprolol and ramipril, respectively [20]. albers s. et al. have developed hplc quantification of metoprolol with solid­phase extraction for the drug monitoring of pediatric patients. chromatographic analysis was performed on a spherisorb c(6) column (5 microm particle size) at ambient temperature and fluorimetric detection with an excitation wave­ length of 225 nm, and emission wavelength of 310 nm. the mobile phase [30% acetonitrile and 70% 0.25 m potassium acetate buffer (ph 4)] was pumped with 1 ml/min. metoprolol recovery was determined at 73.0 +/20.5%, and the limit of quantitation was 2.4 ng/ml. precision values of intraand inter-assay were below 15.5% and those for accuracy were between 90 and 110%. this method was developed for monitoring and determination of pharmacokinetic parameters of metoprolol in pediatric patients and therefore metoprolol plasma concentrations in a 2-year-old child with ventricular tachycardia were reported [21]. angier m.k. et al. suggested gas chromatographic-mass spectrometric differentiation of atenolol, metoprolol, propranolol, and an interfering metabolite product of metoprolol. atenolol, metoprolol, and/or propranolol, with their possible metabolite(s), were re-extracted from the selected case specimens, derivatized with pentafluoropropionic anhydride (pfpa), and analyzed by gas chromatography-mass spectrometry (gc-ms). the ms spectra of these three antihypertensives and a metoprolol metabolite were nearly identical. all of the pfpa derivatives had baseline gc separation, with the exception of a metoprolol metabolite product, which co-eluted with atenolol. there were four primary mass fragments (m/z 408, 366, 202, and 176) found with all of the pfpabeta-blockers and with the interfering metabolite product. however, atenolol had three unique fragments (m/z 244, 172, and 132), metoprolol had two unique fragments (m/z 559 and 107), propranolol had four unique fragments (m/z 551, 183, 144, and 127), and the metoprolol metabolite product had two unique fragments (m/z 557 and 149). these distinctive fragments were further validated by using a computer program that predicts logical mass fragments and performing gc-ms of deuterated pfpa-atenolol and pfpa-propranolol and of the pfpa-alpha-hydroxy metabolite of metoprolol. by using the unique mass fragments, none of the pilot fatality cases were found to contain more than one beta-blocker. therefore, these mass ions can be used for differentiating and simultaneously analyzing these structurally similar beta-blockers in biological samples [2224]. from the above-mentioned, chromatographic methods of analysis amongst others m.m. horyn et al. 95 p h a r m a c y issn 2413-6077. ijmmr 2019 vol. 5 issue 2 m.m. horyn et al. have the greatest specificity and objectivity and allow qualitative and quantitative determination of api in combinate dosage forms and biological fluids without prior separation of the compo­ nents. we can conclude that analysts are constantly working on developing new methods of analysis and their optimization in order to save time and consumables, which also ensures the efficiency of the developed method. the main disadvantage of the described methods of api analysis can be considered long term from the beginning of chromatography to api release and specific solvents used as the mobile phase in hplc. it is necessary to develop methods and to select such chromatographic conditions that provide high speed and high efficiency at lower pressure of the system. this reduces the amount of used mobile phase, which reduces cost analysis accordingly, while at the same time providing the necessary specificity, accuracy and reproducibility of the results of the analysis during quality control. also, the reduction of analysis time is achieved by simplifying the conditions for sample preparation. conclusions thus, analysts are constantly working on developing new methods of analysis and their optimization in order to save time and consumables, which also ensures efficiency of the developed method. there is no monograph on the substance or dosage forms of metoprolol in sphu. therefore, it would be appropriate to recommend some of the developed methods for the sphu monograph, which is important for ensuring pharmacopeial quality control of medicines in ukraine. funding this research received no external funding. conflict of interests the authors declare no conflict of interest. author contributions horyn m.m. – formal analysis, investigation, writing – original draft, logoyda l.s. –conceptualization, supervision, writing – review & editing. методи аналізу метопрололу у лікарських засобах та біологічних рідинах: огляд літератури та можливі шляхи застосування м.м. горин, л.с. логойда тернопільський національний медичний університет імені і. я. горбачевського, тернопіль, україна вступ. розробка аналітичних методів все більше впроваджується у основні практики фармацевтичної хімії та фармацевтичного аналізу з урахуванням їх високої чутливості, точності, специфічності та відтворюваності. мета роботи. критеріями пошуку була розробка аналітичного методу метопрололу. методи. огляд літератури було проведено упродовж 1990–2019 років, щоб зробити огляд оновленим та всеосяжним та показати нові підходи до розробки методів аналізу метопрололу. джерелами були визнані всесвітньо журнали, а ключовими словами, які використовувались як фільтр, були метопролол, спектрофотометрія, високоефективна рідинна хроматографія, кількісний аналіз, розробка методів, валідація. результати. хроматографічні методи аналізу, серед інших, мають найбільшу специфіку та об'єктивність та дозволяють якісно та кількісно визначити активний фармацевтичний інгредієнт (aфi) в комбінованих лікарських формах та біологічних рідинах без попереднього розділення компонентів. можна зробити висновок, що аналітики постійно працюють над розробкою нових методів аналізу та їх оптимізацією з метою економії часу та витратних матеріалів, що також забезпечує ефективність розробленого методу. основним недоліком описаних методів аналізу aфi можна вважати тривалий час від початку хроматографування до виходу aфi та специфічні розчинники, що використовуються як мобільна фаза в верх. необхідно розробити методи та підібрати такі хроматографічні умови, які забезпечать високу швидкість та високу ефективність при нижчому тиску системи. це зменшує кількість використовуваної мобільної фази, що відповідно зменшує аналіз витрат, одночасно забезпечуючи необхідну специфічність, точність та відтворюваність результатів аналізу під час контролю якості. також скорочення часу аналізу досягається шляхом спрощення умов підготовки проби. висновки. можна зробити висновок, що хіміки-аналітики постійно працюють над розробкою нових методик аналізу та їх оптимізацією з метою економії часу та витратних матеріалів, що також 96 p h a r m a c y issn 2413-6077. ijmmr 2019 vol. 5 issue 2 забезпечує ефективність розробленого методу. на даний час не має монографії на субстанцію або лікарські форми метопрололу в дфу. тому було б доцільно рекомендувати деякі розроблені методики для монографії дфу, що є важливим для забезпечення фармакопейного контролю якості лікарських засобів в україні. ключові слова: метопролол; спектрофотометрія; високоефективна рідинна хроматографія; кількісний аналіз; розробка методу; валідація. відомості про авторів горин мар’яна миронівна – студентка 5 курсу фармацевтичного факультету тернопільського національного медичного університету імені і.я. горбачевського, тернопіль, україна. логойда лілія святославівна – доктор фармацевтичних наук, доцент, завідувач кафедри фармацевтичної хімії тернопільського національного медичного університету імені і.я. горба­ чевського, тернопіль, україна. information about authors horyn m.m. – 5th year student of pharmaceutical faculty i. horbachevsky ternopil national medical university, ternopil, ukraine. logoyda l.s. – phd, dsc, head of department of pharmaceutical chemistry i. horbachevsky ternopil national medical university, ternopil, ukraine. orcid 0000­0002­5252­8806, e­mail: logojda@tdmu.edu.ua references 1. metoprolol succinate extended-release 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metabolite product of metoprolol. j anal toxicol. 2005;29:517­21. doi: 10.1093/jat/29.6.517 23. logoyda l. analysis of approaches to the development and validation of the methods of analysis of some active pharmaceutical ingredients from the group of calcium channel blockers in drugs and biological liquids. international journal of applied pharmaceutics. 2019;11(3):26­34. doi: 10.11603/mcch.2410­681x.2019.v.i3.10566 24. logoyda l. analysis of approaches to the development and validation of the methods of analysis of some active pharmaceutical ingredients from the group of angiotensin converting enzyme inhibitors in drugs and biological liquids. international journal of applied pharmaceutics. 2019;11(4):1­7. doi: 10.22159/ijap.2019v11i4.32420 received 07 september 2019; revised 19 october 2019; accepted 25 november 2019. this is open access article distributed under the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. 67 b io m e d ic a l s c ie n c e s issn 2413-6077. ijmmr 2020 vol. 6 issue 2 doi 10.11603/ijmmr.2413-6077.2020.2.11414 histomorphometric estimation of age from bone samples of nigerians *c.d. orupabo1, l.e. oghenemavwe1, t.e. diamond2 1 – department of human anatomy, university of port harcourt, port harcourt, nigeria 2 – department of orthopaedics and surgery, university of port harcourt teaching hospital, port harcourt, nigeria background. age estimation is crucial in creating the biological profile of unknown skeletal remains and recently there is emphasis on the need to develop population specific forensic baseline data for easy identification of these remains. objective. the aim of this study is to estimate age from the histomorphometric features of the bones of nigerians. methods. fragments of non-pathologic bone samples were collected during orthopaedic procedures. ground sections were prepared using modified frost’s manual method of bone preparation to determine the following histologic parameters; haversian canal diameter (hcd), primary and secondary osteons, number of osteon fragments and non-haversian canal as well as haversian canal area (hca). 29 subjects aged 35 to 85 years old were used for the study. bone fragments included samples from the femur, tibia, humerus, and the vertebrae. data obtained were subjected to descriptive statistics, pearson’s correlation, bivariate regression equation, student t-test and analysis of variance (anova). results. the mean age for our population was 58.86 years old. anova showed significant variation in the average hcd for the various regions: humerus=8.45±2.48, femur=7.09±4.06, tibia=8.70±2.52 and vertebrae=3.69±0.73. there was a strong inverse relationship between age and primary osteons. the total number of osteon fragments increased with age while total number of primary osteons and average hcd decreased with age. the hca, though statistically insignificant, also decreased with age. conclusion. our findings show that three histomorphometric parameters showed significant correlation with age: osteon fragments (os-f), primary osteons (os-p) and hcd. the histomorphometric parameters were therefore relevant in age estimation. keywords: age estimation; histomorphometric parameters; nigerians; forensics. *corresponding author: clinton david orupabo, department of human anatomy, faculty of basic medical sciences, college of health sciences, university of port harcourt, east-west road, pmb 5323, choba, rivers state, nigeria. e-mail: clinton.orupabo@ust.edu.ng international journal of medicine and medical research 2020, volume 6, issue 2, p. 67-76 copyright © 2020, tnmu, all rights reserved c.d. orupabo et al. introduction age estimation from the macrostructure of bones is an aged-long anatomical practice, which has progressed into studying the micro structural features. hence, it is an important tool in physical anthropology as well as in forensic medicine in the identification of skeletal remains. once age and sex are estimated, the identity of the skeletal remains is established for about 80% [1, 2, 3]. there are few methods used by forensic scientists whenever the issue of age assessment arises and as such several approaches have been developed. these methods include the use of dental eruptions periods [4, 5, 6], time of epiphyseal fusion of some bones and microscopic or histological features of cortical bones [7, 8, 9], morphology of the pubic symphysis and auricular surface of the ilium [10, 11, 12] the skull and length of long bones [13, 6, 7], sternal ends of the 3rd, 4th and 5th ribs [14, 15]. these foremost macrostructural approaches have lots of limitations as only some bones like the pelvic bone and some long bones give reliable results [16]. these appro aches also require the researcher to employ wide age ranges during age assessment of the subjects that would introduce major difficulties in age estimation of specimen [16, 17]. in order to overcome the limitations posed by the macroscopic methods, in 1965 kerley developed the quantification of histological features of cortical bone for age estimation [18, 19, 20, 21]. since bone growth, development and changes occurs with age, it was presumed that the study of the histological features of 68 b io m e d ic a l s c ie n c e s issn 2413-6077. ijmmr 2020 vol. 6 issue 2 bones could be important in the estimation of age for human populations [22, 23, 21, 24, 25]. according to de boer and maat (2003) [26] and thomas et al. (2000) [27], the histological findings must be combined with gross anatomical and radiological findings to obtain a conclusive diagnosis or to shorten a list of differential diagnosis. singh and gunberg (1970) [22] and wolf et al. (2017) [28] demonstrated the use of fragmentary remains of bone to determine age from histology. some studies from different populations have used the quantification of osteon in age estimation [29, 30, 31]. a common finding of these studies is that the number of osteon and diameter of the haversian canal changes with age [2, 18, 32, 33]. steyn et al. (2004) [33] also observed that age estimation was easy in children compared to adults due to bone remodeling and degeneration, and became more difficult in older ages. currently, it has been established that there is need to generate forensic data for age, sex and stature estimation for different populations, as anatomical landmarks are influenced by diet, disease, genes, sex and racial peculiarities [16, 34]. this study therefore seeks to generate baseline data for age estimation from bone histology among nigerians. methods this is a descriptive and correlational study involving life subjects. fragments of bone samples were collected during orthopedic procedures with data on the age, sex, tribe and type of pathology or clinical diagnosis of the subjects. bone samples collection and tissue preparation took a period of about six months. samples were collected from the orthopedic departments of the university of port harcourt teaching hospital (upth) and rivers state university teaching hospital (rsuth), as well as rehoboth specialist hospital port harcourt and twin towers specialist hospital port harcourt. ethical approval ethical approval was sought from the university of port harcourt research ethics committee and was granted. a consent form was issued to each patient and an informed consent was obtained after thorough guidance and counselling of the patient. sample was drawn from orthopedic patients with no prior or background metabolic disease. bone fragments were collected from amputations and other orthopedic procedures where bone fragments could be harvested. a total number of 29 subjects with samples from 12 females and 17 males aged 35 to 85 years old were used for study. bone fragments included samples from the femur, tibia, humerus, and the vertebrae. the samples included healthy and strong bone fragments without any periosteal disruption. diseased bone fragments shattered during grinding were excluded during processing. the sampling technique involved a convenience sampling method. the bone fragments were collected and ground sections were prepared using the modified frost’s manual method of bone preparation [35, 36, 16]. bone fragments were collected in properly labeled plain containers during operations and immersed in water for about 1 week to enable soft tissue removal. some of the soft tissues were manually removed gently to prevent distortion of the periosteal layer of the bones. the samples were subsequently fixed in 10% formalin for one more week. the volume of fixative is 10-20х the size of the bone to allow adequate penetration of fixative. with the help of a hacksaw, thin cross sections of bone fragments were made. a glass slab coated with vaseline was prepared. a p220 sand paper was placed on the slab. the application of vaseline helps prevent moisture and allows close and smooth adherence of the sand paper to the glass plate. this would allow proper and concurrent thinning of all edges of the bones during subsequent grinding. the rectangular glass slab measured 16cm×12cm and the sand paper was cut to a little more than the size of the glass slab. this would prevent water from escaping into the underside of the paper. a drop of water was dropped on the sand paper and the modified frost’s manual method of bone preparation was adopted. this method was implemented owing to the distortion of micro architecture of tissues with decalcification before routine histology. gentle grinding of bone sections was done by moving the pulp of the finger on the bone over the sand paper in a cyclical fashion. this would enable thinning uniformity of the bone edges. this would also prevent easy cracking and breaking of sections as they get thinner. water was added continually during grinding progresses to enable lubrication and reduce of friction. physical observation of sections for thinness was done by means of a tweezer and a fine brush. with the help of frost’s holder, very thin sections were held for grinding until required degree of thinness was attained. during the grinding process, utmost care was taken to avoid scratching away of the c.d. orupabo et al. 69 b io m e d ic a l s c ie n c e s issn 2413-6077. ijmmr 2020 vol. 6 issue 2 periosteum. hence bone sections were turned from one surface to another to allow for evenness of thinning. grinding was done until bone sections appeared opaque, and then transparent and even easily bendable using the fine brush. ready specimens were placed in a beaker containing distil water and a little drop of detergent in order to properly rinse out dirt and stains from the specimens. washing was performed by the tweezer and the fine brush. rinsed specimens were then placed on a filter paper in a petri dish to allow for drying of the specimen. the filter paper was gently roughened before use. this allowed for ease of pick of the section with the fine brush after drying. glass slides were then cleaned with absolute alcohol and then placed on a glass slab having a dark background. this was achieved by placing a black sheet of paper or polythene behind the glass slab. a drop of dpx mountant was placed on the glass slides. the dried specimen was placed on a top of the mountant. another drop of the moun tant was immediately dropped on the specimen to allow proper immersion of the specimen. glass cover slips washed in xylene were placed over the specimen. the mountant was seen to spread evenly from under the cover slip. air bubbles trapped under the cover slip in some specimens were removed by gentle pressure of the cover slip with the tweezer. ready slides were allowed to dry and correctly labelled. they were kept to dry in a horizontal position for about 24 hours before loading into glass boxes. ready slides were mounted under a photomicroscope for viewing and analysis. the leica icc 50e photomicroscope was used to view and demonstrate the histological fea tures. photomicrographs of fields adjudged by two researchers to have more osteon density were taken. this was done after thorough re view and examination of the entire field under study. results table 1 shows the various histomorphometric parameters studied. their mean distribution as well as the minimum and maximum values for each parameter is also presented. the average age distribution for sample population is shown as well. table 2 shows the mean age, average hcd, standard deviation and variance of the haversian canal diameter of various bones studied. the average hcd for the various regions is as follows: humerus=8.45±2.48, femur=7.09±4.06, tibia=8.70±2.52 and vertebrae=3.69±0.73. table 3 demonstrates the use of anova in determining the degree of variation in the hcd of the various bones studied. the f value is greater than the f critical for an alpha level of 0.05. this proves that there is a significant variation between the mean of the various haversian canal diameter of the bones studied. the p value obtained here (p=0.00) is also less than the alpha level chosen (0.05). this evidences that the variation in the haversian canal diameter of the various bones is very significant. table 4 presents the regression equation derived for age estimation using the various table 1. descriptive statistics of age and bone histomorphometric variables parameters mean standard error of mean standard deviation variation minimum variation maximum variation age 58.86 3.52 16.50 272.12 35.00 85.00 os-p 2.14 0.58 2.71 7.36 0.00 11.00 os-s 2.45 0.83 3.89 15.12 0.00 12.00 os-f 7.00 1.29 6.04 36.48 0.00 22.00 n-hc 2.32 0.47 2.19 4.80 0.00 7.00 area hc 54.13 4.70 68.71 3446.74 3.53 534.35 hcd 7.53 0.28 3.51 12.31 2.19 26. 08 notes: os-p=primary osteon, os-s=secondary osteon, os-f=osteon fragment, n-hc=non haversian canal, hc=haversian canal, hcd=haversian canal diameter. table 2. the average haversian canal diameter for the different bones groups mean age (years) counts sum average (microns) standard deviation humerus 67 31 262.06 8.453484 2.48 femur 60.71 41 356.51 7.090278 4.06 tibia 55.33 72 510.50 8.695366 2.52 vertebrae 51 11 40.60 3.690909 0.73 c.d. orupabo et al. 70 b io m e d ic a l s c ie n c e s issn 2413-6077. ijmmr 2020 vol. 6 issue 2 histomorphometric parameters. it also shows the correlation coefficient and coefficient of determination as well as the t and p values of the variables. discussion our study considered the correlation between age and histological parameters such as primary osteons (os-p), secondary osteons (os-s), and osteon fragments (os-f), nonhaversian canals (n-hc), haversian canal area (hca) and haversian canal diameter (hcd). the mean age for our total population was 58.86 years old (table 1). the average hcd also appeared to vary with the region of bone chosen as we obtained 8.45 microns for humerus, 8.70 microns for tibia, 7.09 microns for femur and 3.69 microns for the vertebra (table 2). it is also possible that bone density contributed to the size of haversian canal as demonstrated with the vertebrae. although the mean ages for tibia and vertebrae are similar, the variation in hcd is large, and more so when compared with the femur and humerus. anova test also shows that there is a significant va riation between the mean of the various haversian canal diameter of the bones studied (f value > f crit. at alpha level 0.05) (table 3). the p-value (p=0) at alpha level 0.05 is also less than the alpha level (table 3). this proves that variation in the hcd of the various bones is significant (p<0.05). these differences seen with different regions could be due to varying bone activity and bone density. it is supposed therefore that age and aging could not be the major reason for these regional differences. the vertebra is not exposed to too much stress and activity as compared to the long bones, especially the tibia, which is a major weight bearing bone. whether these findings could differ with occupation and especially in gymnasts is yet to be ascertained. according to keough (2007) [16], the average haversian canal diameter (hcd) ranges at 30-70 microns. this is quite at variance with our values as our study obtained a range of approximately 2.2–26.1 microns. the reasons also could be the type of sample used. keough used samples from femoral mid-shaft from a predominantly black south african population and being within a similar mean age to ours. also, singh and gunberg (1970) [22] in a study on bone fragments of male american population of similar age group discovered that hcd varies with region of bone chosen for the study and obtained average hcd of 63.44 microns for the mandible fragments, 43.24 microns for the femur and 45.54 microns for the tibia. both previous studies used skeletal collections from cadavers of a white and some black population whereas our study used skeletal remains of live humans from nigerian population. whether the wide variation is related to the tribe or the type of sample used is yet to be determined. however, it is obvious table 3. test of variation in hcd using anova source of variation ss df ms f value p–value (p<0.05) f critical between groups 258.1242 3 86.0414 7.905552 0.00** 2.664504 within groups 1643.434 151 10.88367 total 1901.558 154 notes: hcd – haversian canal diameter, ** – very significant. table 4. correlation, coefficient of determination and regression equation for age versus bone histomorphometric variables parameters correlation coefficient® coefficient of determination t value p value regression equation os-p 0.37 0.14 1.78 0.09 y=-2.29x+63.75 os-s 0.17 0.03 0.77 0.45 y=0.71x+60.59 os-f 0.30 0.09 1.41 0.17 y=0.812x+53.18 n-hc 0.10 0.01 0.45 0.66 y=0.8926x+50.79 area hc 0.24 0.056 1.10 0.28 y=-0.0568x+58.896 hcd 0.26 0.069 1.21 0.24 y=-1.0554x+63.768 notes: os-p=primary osteon, os-s=secondary osteon, os-f=osteon fragment, n-hc=non haversian canal, hc=haversian canal, hcd=haversian canal diameter, y=predicted age, x=parameter under consideration. c.d. orupabo et al. 71 b io m e d ic a l s c ie n c e s issn 2413-6077. ijmmr 2020 vol. 6 issue 2 that nigerians may have far lower average hcd than other populations studied. our study discovered that hcd decreases as age increases (fig. 1). this agrees with singh and gunberg, (1970) [22] who studied american population and reported that hcd decreases as age increases. they also established strong correlation with age. they proved that between age of 40-45 years old the hcd could range between 85-92 microns, and could fall to a range of 51-58 microns at age of 70-80 years old. though we obtained smaller hcd for the nigerian population, our sample was collected largely from an older age group of between 55-85 years old, hence the reason to have obtained small hcd for age. whether ethnicity, socioeconomic status, diet or undiagnosed disease may have an impact, this outcome is yet to be ascertained as it is not within the scope of this study. landeros and frost (1964) [37] also proved that closure of the haversian canal continues as age increases, hence consenting to a reduced diameter with age. however, keough (2007) [16] reported a slight positive correlation (r= 0.1377) with age and supposed males showed highest correlation with age (r=0.9964). he also noted that this parameter could not be a strong estimator of age. likewise sobol et al, (2014) [9] established that hcd increased with age and assumed it was one of the best predictors of age. but barer and josey, (1967) [38] reported little or no age-related changes in the size of the haversian canal. as age increases, the hc area decreases (fig. 2). the coefficient of determination (r2= 0.056) was quite small (table 4), showing that only 5% variation in age of the subjects can be explained by the total haversian canal area of the samples. hence though there is a correlation with age, the coefficient of determination shows that this parameter may be weak in age estimation. the hcd thus has a stronger correlation with age compared to the haversian canal area. the hcd is therefore a better estimator of age compared to the haversian canal area. fig. 3 shows a strong inverse relationship between age and primary osteons (os-p). the younger age groups appear to have more number of primary osteons. this is in consent with enlow (1963) [39] and keough, (2007) [16] who have established that primary osteons is more in the younger age g ro u p . n o n h a v e r s i a n c a n a l s h ow s n o correlation with age (r= 0.10) (fig. 4). this finding also contradicts the reports of many researchers who have proved that n-hc decreases with age [32, 30, 16]. for instance, ericksen (1991) [32] established that n-hc decreased with age for both sexes. also, keough (2007) [16] noted that after 55 years of age, the presence of non haversian canals ceased almost completely. it is also important to note that tersigni’s (2005) report agrees with our data where he wrote that n-hc showed no significant variation with age [40]. both primary osteons and non-haversian canals make up the total number of unmodeled bone. bone remodeling occurs in response to stressors and graded amount of activity, and fig. 1. scatter plot of age versus hcd. fig. 2. scatter plot of age versus area-hc. 8 haversian canal diameter (hcd) ranges at 30-70 microns. this is quite at variance with our values as our study obtained a range of approximately 2.2-26.1 microns. the reasons also could be the type of sample used. keough used samples from femoral mid-shaft from a predominantly black south african population and being within a similar mean age to ours. also, singh and gunberg (1970) [22] in a study on bone fragments of male american population of similar age group discovered that hcd varies with region of bone chosen for the study and obtained average hcd of 63.44 microns for the mandible fragments, 43.24 microns for the femur and 45.54 microns for the tibia. both previous studies used skeletal collections from cadavers of a white and some black population whereas our study used skeletal remains of live humans from nigerian population. whether the wide variation is related to the tribe or the type of sample used is yet to be determined. however, it is obvious that nigerians may have far lower average hcd than other populations studied. our study discovered that hcd decreases as age increases (fig. 1). fig. 1. scatter plot of age versus hcd this agrees with singh and gunberg, (1970) [22] who studied american population and reported that hcd decreases as age increases. they also established strong correlation with age. y = -1,0554x + 63,768 r² = 0,0693 0 10 20 30 40 50 60 70 80 90 0 5 10 20 25 30 a ge hcd age 15 9 they proved that between age of 40-45 years old the hcd could range between 85-92 microns, and could fall to a range of 51-58 microns at age of 70-80 years old. though we obtained smaller hcd for the nigerian population, our sample was collected largely from an older age group of between 55-85 years old, hence the reason to have obtained small hcd for age. whether ethnicity, socioeconomic status, diet or undiagnosed disease may have an impact, this outcome is yet to be ascertained as it is not within the scope of this study. landeros and frost (1964) [37] also proved that closure of the haversian canal continues as age increases, hence consenting to a reduced diameter with age. however, keough (2007) [16] reported a slight positive correlation (r= 0.1377) with age and supposed males showed highest correlation with age (r=0.9964). he also noted that this parameter could not be a strong estimator of age. likewise sobol et al, (2014) [9] established that hcd increased with age and assumed it was one of the best predictors of age. but barer and josey, (1967) [38] reported little or no age-related changes in the size of the haversian canal. as age increases, the hc area decreases (fig. 2). fig. 2. scatter plot of age versus area -hc the coefficient of determination (r2= 0.056) was quite small (table 4), showing that only 5% variation in age of the subjects can be explained by the total haversian canal area of the samples. hence though there is a correlation with age, the coefficient of determination shows y = -0,0568x + 58,896 r² = 0,0562 0 10 20 30 40 50 60 70 80 90 0 100 200 300 400 500 600 a ge area hc age (age ) c.d. orupabo et al. 72 b io m e d ic a l s c ie n c e s issn 2413-6077. ijmmr 2020 vol. 6 issue 2 this therefore increases with age. therefore, finding more primary osteons in the younger age group is justifiable as proved in our research findings. ingraham (2004) [30] confirmed our findings when he reported that percentage of unremodeled bone is higher in the younger age with 53.4% for 18 years of age and 2.2% for 69 years of age. fig. 6 reveals a photomicrograph taking under a magnification of ×100 for two males at ages of 67 and 35 years old. the features seen were predominantly secondary osteons for the older age and unremodeled bone for the younger individual. these findings were expected as bone remodeling increases with age. these findings however are not consistent with all ages in our study; hence the reasons could be for variable underlying factors like sex, tribe, genetics, undiagnosed metabolic disease, exposure to various levels of stress, as well as variation in graded level of activity for different individuals. osteon fragment in our study has a weak positive correlation with age (r=0.30) and statistically insignificant (p>0.05) (table 4) (fig. 5). 10 that this parameter may be weak in age estimation. the hcd thus has a stronger correlation with age compared to the haversian canal area. the hcd is therefore a better estimator of age compared to the haversian canal area. fig. 3 shows a strong inverse relationship between age and primary osteons (os-p). the younger age groups appear to have more number of primary osteons. fig. 3. scatter plot of age versus os-p this is in consent with enlow (1963) [39] and keough, (2007) [16] who have established that primary osteons is more in the younger age group. non haversian canal shows no correlation with age (r= 0.10) (fig. 4). y = -2,2873x + 63,75 r² = 0,1415 0 10 20 30 40 50 60 70 80 90 0 5 10 15 a ge os-p age (age) fig. 3. scatter plot of age versus os-p. fig. 4. scatter plot of age versus n-hc. 11 fig. 4. scatter plot of age versus n-hc this finding also contradicts the reports of many researchers who have proved that n-hc decreases with age [32, 30, 16]. for instance, ericksen (1991) [32] established that n-hc decreased with age for both sexes. also, keough (2007) [16] noted that after 55 years of age, the presence of non haversian canals ceased almost completely. it is also important to note that tersigni’s (2005) report agrees with our data where he wrote that n-hc showed no significant variation with age [40]. both primary osteons and non-haversian canals make up the total number of unmodeled bone. bone remodeling occurs in response to stressors and graded amount of activity, and this therefore increases with age. therefore, finding more primary osteons in the younger age group is justifiable as proved in our research findings. ingraham (2004) [30] confirmed our findings when he reported that percentage of unremodeled bone is higher in the younger age with 53.4% for 18 years of age and 2.2% for 69 years of age. fig. 6 reveals a photomicrograph taking under a magnification of x100 for two males at ages of 67 and 35 years old. the features seen were predominantly secondary osteons for the older age and unremodeled bone for the younger individual. these findings were expected as bone remodeling increases with age. these findings however are not consistent with all ages in y = 0,8926x + 56,794 r² = 0,0141 0 10 20 30 40 50 60 70 80 90 0 2 4 6 8 a ge n-hc age (age) fig. 5. scatter plot of age versus os-f. 12 our study; hence the reasons could be for variable underlying factors like sex, tribe, genetics, undiagnosed metabolic disease, exposure to various levels of stress, as well as variation in graded level of activity for different individuals. osteon fragment in our study has a weak positive correlation with age (r=0.30) and statistically insignificant (p>0.05) (table 4) (fig. 5). fig. 5. scatter plot of age versus os-f keough (2007) [16] documented that osteon fragments showed significant correlation with age (r=0.55). kerley’s (1965) [18] novel research on bone histology proved our findings and stated that osteon fragments were best predictors of age using the fibula. this has been proved by our research and by several authors using various other segments of bones. keough (2007) [16] also established that four of the histomorphometric parameters studied showed significant correlation with age: total osteon count (r=0.53), % unremodeled bone (r=0.53), total n-hc (r=0.55) and % osteon fragment (r=0.55). in his study, total osteon count and percentage osteon fragments increased with age while percentage of unremodeled bone and non haversian canal decreased with age. our findings proved that three histomorphometric parameters showed positive correlation with age: osteon fragments (os-f), primary osteons (os-p) and the haversian canal diameter (hcd). in our study, the total number of osteon fragments increased with age while total number of primary osteons and average haversian canal diameter decreased with age. the area of y = 0,812x + 53,18 r² = 0,0884 0 10 20 30 40 50 60 70 80 90 0 5 10 15 20 25 a ge os-f age (age) keough (2007) [16] documented that osteon fragments showed significant correlation with age (r=0.55). kerley’s (1965) [18] novel research on bone histology proved our findings and stated that osteon fragments were best predictors of age using the fibula. this has been proved by our research and by several authors using various other segments of bones. keough (2007) [16] also established that four of the histomorphometric parameters studied showed significant correlation with age: total osteon count (r=0.53), % unremodeled bone (r=0.53), total n-hc (r=0.55) and % osteon fragment (r=0.55). in his study, total osteon count and percentage osteon fragments increased with age while percentage of unremodeled bone and non haversian canal decreased with age. our findings proved that three histomorphometric parameters showed positive correlation with age: osteon fragments (os-f), primary osteons (os-p) and the haversian canal diameter (hcd). c.d. orupabo et al. 73 b io m e d ic a l s c ie n c e s issn 2413-6077. ijmmr 2020 vol. 6 issue 2 in our study, the total number of osteon fragments increased with age while total number of primary osteons and average haversian canal diameter decreased with age. the area of haversian canal, though statistically insignificant, also decreased with age. the number of non-haversian canal also increased with age but was statistically not significant as well. we can therefore infer that the haversian canal area, the non-haversian canals and the secondary osteons show no significant variation with age. purves et al. (2011) [41], stated in their study that osteon number was very reliable in age estimation but influenced by nutrition, disease, population and sex. whether these factors had a great impact on our findings is yet to be ascertained as it is not within the scope of this study. our samples were obtained from clinically healthy subjects. however about 69% of nigerians live far below the poverty level according to the 2019 global multidimensional poverty index report by undp/ophi [42], it is not without doubt that nutrition could be a factor. steyn (2004) [33] in her research on adult age estimation reported that age estimation was difficult in adults and more difficult in older ages. hence, she advised use of multifactorial approach. this would therefore combine both histological and macroscopic findings, especially where both can be readily available. in the absence of whole skeletal collections, the use of fragmentary remains becomes the main stay. however, in order to make this more robust and accurate, any possible finding on culture, environment and data on dna can make a whole lot of difference. study limitations the availability and access to bone samples is one foremost limitation to this study. skeletal remains and bone samples are handled with much pessimism in our culture for fear of ritualists; hence processes for obtaining samples were quite laborious and challenging. another major limitation to our study is the inability to use cadavers for the research. our use of cadavers may have allowed access to lots of samples but nonetheless most of our cada vers are not profiled unlike what is obtainable in other countries. conclusions the histomorphometric parameters are therefore relevant in age estimation and sex identification. most forensic case identification without choice needs skeletal remains in order to investigate their victim. forensic investigation of nigerians would therefore need the analysis of the primary osteons, the osteon fragments and the haversian canal diameter as proved in our research. thus, since histomorphometric variations with population are influenced by environment, diet and genetics, age and aging of an individual is therefore not a major factor in influencing the histological changes seen, especially for individuals who are within a similar age bracket. hence any forensic case investigation should, if possible and if the data are available, consider the multifactorial approach. acknowledgements most sincere thanks to my supervisor and others who rendered one support or the other. special thanks to the acting vice chancellor of rivers state university whose financial support aided my travel to south africa in order to study the technicalities that eased the execution of this work. the authors also thank twin towers and rehoboth specialist hospitals as well as the university of port harcourt teaching hospital where our samples were obtained. conflict of interest the authors declare no conflict of interest. funding this research received no external funding. author’s contributions loveday oghenemavwe, clinton david orupabo – conceptualization, methodology, formal analysis, writing – original draft, writing – reviewing and editing; loveday oghenemavwe, clinton david orupabo, tamunokuro diamond – data curation, investigation. c.d. orupabo et al. 74 b io m e d ic a l s c ie n c e s issn 2413-6077. ijmmr 2020 vol. 6 issue 2 оцінка віку за гістоморфометричними особливостями кісток нігерійців c.d. orupabo1, l.e. oghenemavwe1, t.e. diamond2 1 – department of human anatomy, university of port harcourt, port harcourt, nigeria 2 – department of orthopaedics & surgery, university of port harcourt teaching hospital, port harcourt, nigeria вступ. оцінка віку має вирішальне значення для створення біологічного профілю невідомих скелетних останків, і останнім часом робиться наголос на необхідності розробки специфічних для певної когорти населення судово-медичних даних для легкої ідентифікації цих останків. мета. метою цього дослідження була оцінка віку за гістоморфометричними особ ливостями кісток нігерійців. методи. фрагменти непатологічних зразків кісток збиралися під час ортопедичних процедур. зрізи були підготовлені за допомогою модифікованого ручного методу підготовки кісток для визначення наступних гістологічних параметрів: діаметр каналу гаверса (hcd), первинний та вторинний остеони, кількість фрагментів остеону та негаверсових каналів, а також площа каналу гаверса (hca). для дослідження було використано 29 досліджуваних у віці від 35 до 85 років. осколки кісток включали зразки стегнової, гомілкової, плечової кісток та хребців. отримані дані піддавали описовій статистиці, кореляції пірсона, двовимірному рівнянню регресії, t-критерію стьюдента та дисперсійному аналізу (anova). результати. середній вік для нашого населення становив 58,86 років. anova демонструє значні коливання середнього показника hcd для різних регіонів: плечова кістка = 8,45±2,48, стегнова кістка = 7,09±4,06, гомілка = 8,70±2,52 та хребці = 3,69±0,73. між віком та первинними остеонами існує сильний зворотний зв’язок. загальна кількість фрагментів остеону зростала з віком, тоді як загальна кількість первинних остеонів та середнє значення hcd зменшувались із віком. hca, хоча і статистично незначний, також зменшувався з віком. висновок. отримані нами результати показують, що три гістоморфометричні параметри продемонстрували значну кореляцію з віком: фрагменти остеонів (os-f), первинні остеони (os-p) та hcd. тому гістоморфометричні параметри є важливими для оцінки віку. ключові слова: оцінка віку; гістоморфометричні параметри; нігерійці; криміналістика. information about the authors clinton david orupabo, lecturer 1, department of human anatomy, university of port harcourt, port harcourt, nigeria orcid 000-0002-4784-4273, e-mail: clinton.orupabo@ust.edu.ng loveday ese oghenemavwe, supervisor/reviewer/senior lecturer, department of human anatomy, university of port harcourt, port 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196700520-00020 39. enlow dh. the canal system in bone. in: xxxxx (ed), principles of bone remodeling. cc thomas, springfield, 1963; pp. 60-74. 40. tersigni ma. serial long bone histology: interand intra-bone age estimation. phd diss., university of tennessee, 2005. 41. purves s, woodley l and hackman l. age determination in the adult. in: black s, ferguson e (eds). forensic anthropology 2000-2010. 2011; pp 29-60. doi: https://doi.org/10.1201/b10727-3 42. united nations development programme/ oxford poverty & human development initiative. 2019 global multidimensional poverty index. www. ng.undp.org/content/nigeria/en/home/presscenter/pressrelease/2019/new-data-challenges (2019, accessed 12 december 2019) 43. thompson dd and gunness-hey m. bone mineral-osteon analysis of yupik-inupiaq skeletons. american j physical anthro.1981; vol. 55, pp. 1-7. doi: https://doi.org/10.1002/ajpa.1330550102 44. abdullah h, jamil mma, ambar r, et al. bone histology: a key for human sex determination after death. j. phys.: conf. ser. 2018; 1019: 012010 . doi: https://doi.org/10.1088/1742-6596/1019/ 1/012010 received 30 sep 2020; revised 25 nov 2020; accepted 3 dec 2020. this is open access article distributed under the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. c.d. orupabo et al. 50 b io m e d ic a l s c ie n c e s issn 2413-6077. ijmmr 2020 vol. 6 issue 1 doi 10.11603/ijmmr.2413-6077.2020.1.11501 chromosomal diseases in the human pathology (review) *t.v. bihunyak, yu.i. bondarenko, o.o. кulyanda, s.m. charnosh, a.s. sverstiuk, k.o. bihuniak i. horbachevsky ternopil national medical university, ternopil, ukraine background. chromosomal diseases are the cause of 45-50 % of multiple birth defects. basic research on mutations is performed using genomic technologies to identify a correlation between genotype and phenotype in aneuploidies and to understand its pathogenesis. objective. the aim of the research is to study the etiology, pathogenesis of symptoms and diagnostics for patients with down, klinefelter, turner syndromes and double aneuploidies by 21 and sex chromosomes. methods. a literature review by the keywords “down syndrome”, “klinefelter syndrome”, “turner syndrome”, “double aneuploidy” for the period of 2000-2020 was carried out. results. down, klinefelter and turner syndromes are the most common aneuploidy among viable newborns. frequency of meiotic non-disjunction events causing these aneuploidies increases with the age of a woman. identified genes are responsible for pathogenesis of symptoms in trisomy 21, turner and klinefelter syndromes. diagnostics of chromosomal diseases includes prenatal screening programs and postnatal testing. conclusions. cytogenetic variants of down syndrome are simple complete trisomy 21, translocation form and mosaicism. trisomy 21 is associated with advanced maternal age. phenotypic manifestations of down syndrome are associated with the locus 21q22. the maternal and parental nondisjunction of x-chromosomes in meiosis causes klinefelter and turner syndromes. these chromosomal diseases are variants of intersexualism with intermediate chromosomal sex. down-klinefelter and down-turner syndromes are double aneuploidies. patients have a down syndrome phenotype at birth, and signs of klinefelter and turner syndromes occur during puberty. diagnosis of aneuploidy is based on the cytogenetic investigation (karyotyping), dna analysis, ultrasonography and biochemical markers of chromosomal pathology. key words: down syndrome; klinefelter syndrome; turner syndrome; double aneuploidy. international journal of medicine and medical research 2020, volume 6, issue 1, p. 50-60 copyright © 2020, tnmu, all rights reserved *corresponding author: bihunyak t.v., md, ph.d., associate professor, department of pathophysiology, i. horbachevsky ternopil national medical university, ternopil, ukraine. e-mail: bihunyak@tdmu.edu.ua. t.v. bihunyak et al. introduction changes in the number and structure of chro mosomes take place in 1% of newborns. they are the cause of 45-50% of multiple birth defects, about 36% of cases of mental retardation, 50% of primary amenorrhea in women, and 10% of sterility in men [1]. among all cases of birth defects in the life of stillbirths in ukraine for 2002-2015, almost every 2nd case referred to chromosomal pathology. the main clinical chromosomal syndromes include: down syndrome (ds) – trisomy 21 – prevalence 1:400-1:1,500 newborns, klinefelter syndrome (ks) – karyotype 47, xxy with a frequency of 1:500-1,000 newborn boys, turner syndrome (ts) – karyotype 45,x with a frequency of 1:2,000-5,000 newborn girls [2-6]. among the viable newborns, 1 of 400 boys and 1 of 650 girls have different forms of aneuploidy by sex chromosomes. ks and ts are most common among them [7]. double aneuploidy is a rare chromosomal ab normality detected after birth since most reported cases are presented in the form of one lethal aneuploidy and end in early miscarriage. it is extremely rare to find living newborn cases with double autosomal trisomy or autosomal trisomy with sex chromosome monosomy or trisomy [8]. the most frequent co-occurrence is that of ds and ks (coincidence 0.098%), because they are common and relatively well to le rated chromosome abnormalities in humans [9]. the frequency of mutations that causes chromosomal diseases does not depend on race or country. the dynamics of the spread of chromosomal diseases over the past two decades in the world has changed due to the discovery of new cases of double aneuploidy in viable newborns. early prenatal diagnosis of trisomy 21 as well as postnatal aneuploidy by sex chromosomes is urgent. basic research on aneuploidy is conducted using genomic technologies to identify a correlation between genotype and phenotype in chromosomal diseases and to understand its pathogenesis. for diagnosis of hereditary dise51 b io m e d ic a l s c ie n c e s issn 2413-6077. ijmmr 2020 vol. 6 issue 1 ases, the medical genetics methods are used, in particular cytogenetic, genealogical, dermatoglyphics, biochemical, and dna molecular analysis. the cytogenetic method (karyotyping) is widely used for diagnosis of human chromosomal diseases. it is important to be aware of diagnostic possibilities of prenatal testing for early detection of fetal chromosomal pathology such as screening programs for women at early pregnancy. the more deeply the nature of human heredity is analyzed, the more it is realized regarding the methods of diagnostics, treatment and prevention of diseases. the aim of the research is to study the cytogenetic mechanisms of occurrence, pathogenesis of changes in the phenotype and medical genetic counseling for patients with ds, ks and ts. methods a literature review by the keywords “down syndrome”, “klinefelter syndrome”, “turner syndrome”, “double aneuploidy” for the period of 2000-2020 was carried out. review according to the literature, in 1866 english physician john landon down identified a group of patients with dementia and peculiar somatic abnormalities and called the disease “mongoloid idiopathy”. the hypothesis of hereditary origin of the syndrome was considered questionable, although in 1932 the ophthalmologist and geneticist waardenburg suggested that the basis of the pathology may have been “chromosomal insufficiency” [2-3]. despite this, the cause of this syndrome was clarified only in 1959 by a french scientist lejeune. he stated that in the karyotype of those patients there were 47 chromosomes, and a small acrocentric chro mosome in the group g (21 or 22) was superfluous. as a result of development of the fluorescence chromosome identification me thod in 1971, it was possible to differentiate 21 of 22 chromosomes and establish the true karyotype in ds. the incidence of patients’ birth with trisomy 21 is not affected by sexual, racial, geographical or population differences. the incidence of children with ds depends on the age of the mother (table 1) [1]. more than 80% of children with trisomy 21 are born to women under 35 years of age [3]. hence, prenatal screening and diagno sis of trisomy 21 for a fetus are important for pregnant women aged 35 years and older [10]. cytogenetic variants of ds are diverse. however, the main part (94-95%) is the cases of simple complete trisomy 21 as a consequence of the nondisjunction of chromosomes to the opposite poles of the cell in the anaphase i or anaphase ii of the meiosis. the contribution of maternal nondisjunction is 95 %, and the father’s only 5 %. no dependence on father’s age was found in cases of ds where the additional chromosome originated from father. about 75% of maternal nondisjunction occur during meiosis i, and 25% – during meiosis ii, but about 25% of paternal nondisjunction occur during meiosis i, and 75% – during meiosis ii [14]. as a result, gametes (oocyte or sperm) have 22 chromosomes (-21) or 24 chromosomes (+21). when a normal gamete that has 23 chromo somes fuses with gametes with 24 (+21) chromosomes, then a zygote 47,+21 is formed, and a child with ds may be born. the karyotype of patients with this will be 47,xx,+21 or 47,xy,+21 [11]. about 2-5% of patients with ds have mosaic forms due to the mitotic nondisjunction of the chromosomes after fertilization (post zygotically), in which one part of the cells has a normal amount of chromosomes (46), and the other is aneuploid (47). the karyotypes of patients with 47,xx,+21/46 or 47,хy,+21/46 [11, 13]. the frequency of gonadal mosaicism in trisomy 21, according to some researchers, is actually larger. available for tissue analysis (blood lymphocytes, skin fibroblasts) have a low mosaic level, while an abnormal line is present in gonads. study of qrq polymorphism of chromosomes in 151 families of down children showed that in 8 families there is gonadal mosaicism in trisomy 21, with all cases of mothers younger than 35 years old. the frequency of carriage of mosaicism in young families was 6.5% (8/123). approximately 4% of patients with ds have a translocation form. the patients have a diploid chromosome set, but their morphology is disturbed. this form occurs, as a rule, as a result of the robertson translocation of surplus table 1. the risk of childbirth with down syndrome in women of different age groups age of a woman at the time of childbirth, years empirical risk of having a baby with down syndrome till 19 1:1640 20-24 1:1986 25-29 1:1319 30-34 1:603 35-39 1:217 40-44 1:84 45 1:31 t.v. bihunyak et al. 52 b io m e d ic a l s c ie n c e s issn 2413-6077. ijmmr 2020 vol. 6 issue 1 21 chromosomes between the acrocentric cells (d/21 and g/21). 25% of translocation forms are inherited from native carriers, and 75% of the translocations are formed de novo [14-15]. the translocation form of the ds is not phenotypically different from the classic trisomy 21. the occurrence of this pathology does not depend on the age of parents, while the parents can be phenotypically healthy carriers of translocation (45,-21,t(15q21q)). consequently, people with a well-balanced translocation have a high risk of having a child with translocation form of ds (1:3). at normal karyotype of parents, the risk of having a baby with trisomy 21 corresponds to a simple trisomy. if a patient has a translocation variant of ds, then the parent’s karyotype must be explored. in young parents, who have children with ds, balanced chromosomal aberrations (1.95 %) are found in 8 times more often than in the other population. when translocation type 13/21 is revealed in the father, the risk of birth of a sick child is 2.4%, and in the mother – about 10%. if one of the parents has a translocation type 21/21, the risk of having a baby with down syndrome is 100%. about 10% of mothers of the children with ds have mosaicism. in the case of parents’ mosaicism, genetic risk should be considered about 30% [1]. other forms of trisomy 21 are 1% of ds. they can be caused by: a) a terminal rearrangement of chromosome 21 around the telomeric region, the final chromosome having two centromeres and satellites on both ends; b) as a component of a double aneuploidy (for example, 48,xyy,+21 or 46,x,+21) [14, 16]. chromosome 21 is the smallest human chro mosome and contains 200 to 300 genes. analysis of the chromosome revealed 127 established genes, 98 predicted genes and 59 pseudogenes [14]. the most acceptable theory for the pathogenesis of trisomy 21 is the genedosage hypothesis, which declares that all changes are due to the presence of an extra copy of chromosome 21 within region 21q22. molecular studies are aimed at determining the region of 21 chromosomes, which would code for phenotypic manifestations of ds. these gene products include morphogens, cell adhesion molecules, components of multi-subunit proteins, ligands and their receptors, trans cription regulators and transporters. in particular, it has been found that the dyrk gene (21q22.13) is responsible for mental retardation. surplus of this gene in mice causes defects in learning and memory. the third copy of the other app gene (21q21.2|21q21.3) encoding the β-amyloid synthesis causes alzheimer’s syndrome in the patients with trisomy 21 of over 40 years of age. if as a result of mutation this gene in the patients with ds is absent, the signs of alzheimer’s syndrome are not manifested [1, 3]. gart-gene is also detected in the 21 chromosome which in trisomy causes an increased level of purines in the blood of patients that is one of the causes of mental retardation [17]. by the results of expression of this gene you can control the occurrence of ds even in the embryonic period. the complex of congenital abnormalities specific for ds causes the clinical picture “all children from one family”. pregnancy, from which patients are born, often is accompanied by toxicosis, the risk of miscarriage. in sick mothers there is an unfavorable obstetric anam nesis (miscarriages, stillborn). the average length of pregnancy is slightly lower than normal. children with ds are born with moderate pronounced hypoplasia (mass 8-10% below average values) [18]. craniofacial dysmorphia is manifested by brachycephaly (81%), flat occiput (78%), flattened face, flat bridge of nose (52%), small short nose (40%), large fis sured tongue (50%), open mouth (65%), hypoplasia of the upper jaw, high palate (58%), small lowly placed deformed ears, mongolism, epicanthus (80%). incorrect growth of the teeth, diastema, transverse striae on the lips are typical. in 68% of patients, there are spots of brushwild (light spots on the iris), 32.2% – cataract. in many patients with trisomy 21, there is a short neck (45%), newborn neck folds, broad fingers and palms, broad toes (70%) [1]. approximately 4050% of the affected children have congenital heart diseases (chd), one of the major causes of morbidity and mortality [19-20]. in western european countries and the usa, endocardial cushion defect (43%), which results in ventricular septal defect (vsd) (32%), secundum atrial septal defect (asd) (10%), tetralogy of fallot (6%) and isolated patent ductus arteriosus (4%) are present in this group of patients [14]. in asia, isolated vsd has been reported to be the most common cardiac defect (40%) [21]. a study from korea showed that asd was the most common defect accounting for 30.5 % of ds, followed by vsd (19.3%), patent duct arteriosus (17.5%) and atrioventricular septal defect (9.4%) [22]. the second type of asd was the most common cardiac lesion in latin america [23-24]. in libya, the most common isolated cardiac lesion was the asd, found in 23% of the patients [25]. molecular studies of rare individuals with chd and partial duplications of chromosome t.v. bihunyak et al. 53 b io m e d ic a l s c ie n c e s issn 2413-6077. ijmmr 2020 vol. 6 issue 1 21 established candidate gene dscam, which was manifested in the heart during cardiac development [26]. about half of the atrioventricular septal defects avsds occur in the patients with ds [27]. in the patients with ds and complete avsd potentially damaging variants in six genes: col6a1, col6a2, creld1, fbln, frzb, gata5 were identified [28]. immune problems are also typical in the form of immunodeficiency. laboratory abnormalities can be revealed in both humoral and cellular immunity. ds is manifested by high incidence of leukemia. however, the children with ds have a 20-fold greater risk for acute lymphoid leukemia and a 50-fold higher risk for myeloid leukemia compare to the children without ds [29]. development of ds brain is associated with reduction in the neuronal number and abnormal neuronal differentiation. it has been previously reported that ds neuron degenerate subsequently and undergoes apoptosis [4]. the mental retardation with full trisomy 21 is found in almost all patients, and it is basically oligophrenia at the stage of imbecility (65-90%). the average iq of children 10 years old and over is only 24. in a mosaic variant of ds, moderate mental retardation is much more common, and normal intelligence is also possible. in the united states ds is 10% of all cases of mental retardation. ds is associated with epilepsy in 10% of cases. hearing loss and anomalies of the vision organs is often observed in child patients, in particular, cataracts in 66% of the children over 8 years of age [1]. the patients with ds gain more weight compare to other population, and most adults with ds are overweight. antithyroid antibodies predispose to disturbance of gametogenesis and nondisjunction of the 21 pair of chromosomes. the hypofunction of the thyroid gland in parents is a risk factor. nowadays, cardiac surgery, vaccinations, antibiotics, thyroid hormones, leukemia therapies, and anticonvulsive drugs (e.g, vigabatrin) have significantly improved the quality of life of individuals with ds. actually, life expectancy that was hardly 30 years in the 1960s is now increasing – more than 60 years of age [11, 30]. oral care by dentists for the children with ds from the first months of life involve palatal plates that aim at activating and stimulating different functions, mainly for the orofacial motor function and ex pression, including language. this is comple mentary for that of speech therapists, physiotherapists, psycho-motor therapists, and pediatricians [31-32]. reproductive capacity in ds is described in women, with 40% being ovulatory disorders. men with this syndrome are sterile. women with ds produce half normal eggs and half with 24 chromosomes. consequently, the probability of zygote with trisomy 21 in women with ds is 50%. as about 75% of these pregnancies are completed with spontaneous abortion, the risk to birth of a sick child is less than 50%. trisomy 21 among aborted embryos is found at a frequency of 1:40. the cytogenetic examination in children with ds is indicated with suspicious of trisomy 21 and with clinically established diagnosis, as the patient’s cytogenetic characteristics from parents and their relatives are needed to predict the health of future children [1]. diagnostics of ds includes prenatal and postnatal testing. for pregnancies, the high risk of ds is evaluated by fetal sample analysis after invasive chorionic villus sampling (cvs) and amniocentesis, and by laboratory techniques such as conventional karyotype analysis, fluorescence in situ hybridization, quantitative fluorescence-polymerase chain reaction, multi plex ligation probe assay and array comparative genomic hybridization, which are common techniques used for prenatal diagnosis of ds and each of them presenting with advantages and disadvantages. there is also a noninvasive technique for detection of trisomy 21 by analysis of extracted cell-free fetal dna screening from maternal plasma samples [14]. the most common screening method contains the measurement of a combination of factors: advanced maternal age, multiple second trimester serum markers and second trimester ultrasonography. the first reported marker associated with ds was the thickening of the neck area. 40-50% of the affected fetuses have a thickened nuchal fold measuring ≥6 mm in the second trimester. when screening by nuchal translucency (nt), about 83% of trisomy 21 pregnancies were identified in the first trimester. later, it was revealed that screening by a combination of maternal age, nt and bitest (papp-a with second trimester β-hcg) or tri-test [alpha-fetoprotein (afp), estriol and free β-hcg] has a potential sensitivity of 94% for a 5% false positive rate [4, 33]. however, ruei-yu lan et al, reported about multicenter study of 29,137 cases that enrolled the chromosomal abnormality screening from taipei city. the mean maternal age of screen-positive group was 34±4.2 years old. the first trimester had 891 cases screening positive with a detection rate of 97.5% for fetal trisomy 21 and false t.v. bihunyak et al. 54 b io m e d ic a l s c ie n c e s issn 2413-6077. ijmmr 2020 vol. 6 issue 1 positive rate of 3.5%. second-trimester had 334 cases screening positive, the detection rate and false positive rate were 33.3% and 6.4% respectively for trisomy 21 [10]. more recent advances in genomics and related technologies have resulted in development of a noninvasive prenatal screening (nips) test using cell-free fetal dna sequences isolated from a maternal blood sample. almost 5-10% of dna in maternal serum are of fetal origin. fetal trisomy detection by dna-analysis from maternal blood has been done using massively parallel shotgun sequencing (mpss). by next generation sequencing platforms, millions of amplified genetic fragments can be sequenced in parallel. mpss detects higher relative amounts of dna in maternal plasma from the fetal trisomy chromosome compare with reference chromosomes. platforms differ according to whether amplified regions throughout the genome, chromosome-specific regions, or single nucleotide polymorphisms (snps) are the targets for sequencing [34]. although studies are hopeful and exhibit high sensitivity and specificity with low false-positive rates, there are drawbacks to nips. specificity and sensitivity are not consistent for all chro mo somes; this is due to different content of cytosine and guanine nucleotide pairs. false-positive screening results take place because the sequences derived from nips are derived from the placenta, like in cvs they may not reflect the true fetal karyotype. therefore, currently invasive testing is recommended for confirmation of a positive screening test and should remain as option for patients seeking a definitive diagnosis. nips began as a screen for only trisomy 21 (t21) and rapidly developed to include other common aneuploidies for chromosomes 13 (t13), 18 (t18), x, and y [35, 36]. amniocentesis is the most conventional invasive prenatal diagnostic method accepted in the world. it is mostly performed to acquire amniotic fluid for karyotyping from 15 weeks onwards. actually, amniocentesis performed before 15 weeks of pregnancy is referred to as early amniocentesis. cvs is usually performed between 11 and 13 weeks of gestation and includes aspiration or biopsy of placental villi. amniocentesis and cvs are quite reliable but increase the risk of miscarriage up to 0.5 to 1% compare with the background risk [33]. a number of european countries have estab lished a network of oscar clinics (one step clinic for evaluation of risk for fetal abnormalities). clinics of “one visit” allow calculating the individual risk of a child’s birth with chromosomal pathology regarding of the age of pregnancy, ultrasound and biochemical markers of chromosomal pathology. in particular, among the biochemical markers in the first trimester, the content of plasma protein-associated protein (papp-a) and β-chorionic gonadotropin (β-hcg) levels are determined [18]. for non-invasive prenatal diagnosis, studies of fetal trophoblastic cells from the cervical canal of women in the first trimester of pregnancy are also used [37]. constantly conventional karyotype from peripheral blood in postnatal period is performed to confirm diagnosis for all patients suspected with ds [14]. polysomy by sex chromosomes has less significant pathological manifestations than aneuploidy by autosomes. it may be explained by the fact that the y chromosome contains little genes, and additional x chromosomes may be heterochromatic. genetic intersexualism is the result of sex chromosome aneuploidies. ts and ks are variants of intersexualism with an intermediate sex [7]. ts (45,x) is a single form of monosomy in the liveborns. monosomy x was discovered by henry turner in 1938 (earlier, in 1930, the description was made by otto ullrich). cytogenetic syndrome 45,x was discovered by se ford in 1959 [5]. clinically ts manifests itself in three directions: 1) hypogonadism, underdeveloped genital organs and secondary sexual characteristics; 2) congenital defects; 3) short stature. ts is one of the clinical forms of sexual differentiation disorders – agenesis with somatic malfor mations. in the reproductive system there is a lack of sexual glands (agenesis of gonads), hypoplasia of the uterus and fallopian tubes, primary amenorrhea, inadequate hair follicles in the pubic and axillary areas, underdevelopment of the mammary glands, deficiency of estrogens, and excess of pituitary gonadotropins. in children with ts, congenital malformations of the heart, vessels and the kidneys make up to 25%. in newborns, the neck is short with excess skin and wing-like folds, with low neck hair growth. these patients have lymphatic edema of the feet, legs, hands and forearms. at school age, especially at the puberty period, there is a lag in the growth and development of secondary sexual characteristics. in 16-50% of cases the patients with ts are mentally retarded, prone to the lymphedema and hashimoto’s thyroiditis. it is established that if a ts patient received an x-chromosome from their father, they would have a higher iq and better adaptation in sot.v. bihunyak et al. 55 b io m e d ic a l s c ie n c e s issn 2413-6077. ijmmr 2020 vol. 6 issue 1 ciety than that received from the mother’s sex chromosome. chromosomal anomalies in women with ts are quite different: 50% of these patients have 45,x karyotype in blood lymphocytes, 30-40% are mosaics in most cases with a karyotype 45,х/46,хх, less than 45,х/46,xy; 45,х/47,ххx. among liveborn limited placental mosaicism is often observed. the more normal cells a fetus has the greater chance of its survival. about 10-20% of patients with ts have structural changes in the x-chromosome, which include deletion of short or long arms (46,x,xp-; 46,x,xq-), isochromosomes [46,x,i(xq); 46,x,i(xp)], ring chromosomes [46,x,r(x)]. these varieties of chromosomal aberrations stipulate for the phenotype of patients. molecular studies have shown that 60-80% of monosomies x are due to the disappearance of the paternal sex chromosome that occur either in early mitosis during embryogenesis or in the process of meiosis. a repeated risk of birth of a sick child with ts in a family with normal parents karyotype does not exceed 1% [7]. molecular analysis also allowed precise determining of specific genes responsible for the phenotype of ts. for example, the mutation in the shox gene causes low growth. this gene is localized in the distal segment of the x chromosome (xp22.33) and in the short arm of the y chromosome. consequently, this gene undergoes normal transcription being in two copies of a man and a woman. so, as the patients with ts have only one copy of this gene, they have a high probability of short stature development. according to linglart et al. [38], hormone reposition in the patients with ts may reduce likelihood of short stature development. webbed neck, lymphedema or coarctation of the aorta in infancy should prompt a peripheral blood karyotype to rule out ts [39]. the standard 30-cell karyotype is recommended and can detect 10% mosaicism with 95% confidence [5]. multiple sequences adjacent to the y-centromere should be amplified using polymerase chain reaction techniques to detect cryptic y-material. when y-chromosome material is present in the standard karyotype or on such additional testing (incidence of 5-12%), prophylactic gonadectomy is still recommended by expert consensus, albeit at a lower quality of evidence, due to an increased risk (around 10%) of gonadoblastoma [39-40]. in ts fetal ovaries develop normally in the early stages of embryogenesis, but the absence of a second x-chromosome leads to an accelerated loss of oocytes within 2 years. spontaneous puberty has been reported in 14% of ts patients with monosomy x and up to in one third of patients with mosaicism [5]. young ts women with normal ovarian function should be counseled about fertility preservation options. gonadotropins (especially follicle-stimulating hormone) should be monitored annually starting at about 11 years of age to confirm hypergonadotrophic hypogonadism prior to pubertal induction [6]. anti-müllerian hormone (amh) and inhibin b measurements have also been shown to predict ovarian insufficiency when found to be low, and amh is perhaps the best indicator of ovarian reserve [41]. however, the majority of the girls with ts require induction of puberty and estrogen/progestin replacement therapy to achieve adequate breast development, uterine maturation and peak bone mass. transdermal 17-β estradiol (tde) is now the preferred treatment starting at age around 11-12 years old. compared with oral estrogens, tde is thought to be more physiologically delivery since it will avoid the first-pass effect in the liver with improved bioavailability. a recent metaanalysis showed improved whole-body bone mineral density, fasting glucose and total cholesterol with tde therapy compare with oral estrogens [42]. ks is the most extensive major anomaly of sexual differentiation in men. the disease is described in 1942 by harry klinefelter in males with sexual retardation. in 1959, jacobs and strang showed the presence in these patients of a pathological karyotype. the most common cause of ks is nondisjunction of xx homologues for meiosis. meiotic non-disjunction events causing ks are inherited maternally in 50-56% of cases (36% occur in 1st and 20% in 2nd meiosis), and paternally in 44-50% (100% 1st meiosis) [43, 44]. with the age of the mother, as with ds, the probability of birth of a baby with this syndrome increases [1]. in 40-year-old mothers, ill children are born by 2-3 times more often than in 30-year-olds. preliminary, it is established that half of cases with a karyotype 47, xxy end with spontaneous abortions. mosaic among liveborn is observed in 15% of cases [7]. the presence of the y chromosome determines the formation of a male phenotype. genetic imbalance due to excess x-chromosome is manifested clinically during puberty as underdevelopment of the testicles and secondary male sexual characteristics. the presence of an extra chromosome can cause higher levels of gene expression and gene products in amounts affected by regulation at different levels, prot.v. bihunyak et al. 56 b io m e d ic a l s c ie n c e s issn 2413-6077. ijmmr 2020 vol. 6 issue 1 tein degradation and modification [45]. also, the interaction pattern of the proteins encoded by the additional chromosome can influence the severity of a trisomy. in the case of ks, x chromosome inactivation (xci) counterweights the extra amount of x chromosome genes (about 2000). still, roughly 15 % of x-linked genes escape xci, including genes in the two pseudoautosomal regions (pars). a total of 22 x chromosome genes were deregulated in ks compare to the controls: 16 were upregulated (akap17a, asmtl, csf2ra, eif1ax, eif2s3, gpr82, gtpbp6, il3ra, plcxd1, ppp2r3b, prkx, rp11epo6o15.3, sept6, slc25a6, tmsb4x and xist) and six were down-regulated (bend2, bex1, cox7b, foxo4, nhs and tfe3). some of these genes were expressed from par1: akap17a, asmtl, csf2ra, gtpbp6, il3ra and plcxd1, but no differentially expressed genes were observed encoded from par2. xci compensates extensively for the extra x chromosome gene dosa ge in ks males and probably increases their chances of survival compare to other aneuploidies [17]. thus, in the patients with ks have a high stature, eunuchoidism, narrow arms and wide pelvis, subcutaneous fat development by female type, absent hair growth on the face, in the axillary areas and in the area of the pubis, sparse hair, underdeveloped testicles and their endocrine function (production of androgens) is reduced. the prevalence of androgen deficiency in the patients with ks is 79%. the patients in most cases are sterile (due to azoospermia, oligospermia). among men, who have infertility, more than 10% have an additional x-chromosome. approximately one in three patients with this syndrome has gynecomastia and an increased risk of breast cancer development. this risk can be reduced by having a mastectomy. gynecomastia and obesity with fat deposits for the female type are a result of relative hyperestrogenemia [7]. in the ks pa tients this may lead to an increased incidence of female predominant autoimmune disorders [46]. the system biology approaches together pointed to novel aspects of ks phenotypes including perturbed jak-stat pathway, dys regulated genes important for disturbed immune system (il4), energy balance (pomc and lep) and erythropoietin signaling in ks. sex chromatin is detected in 80% of the patients with ks. modified laboratory parameters: decreased testosterone in the blood and urinary excretion of 17-ketosteroids; an increase in the amount of estrogen in the plasma, as well as their excretion with urine; high levels of gona dotropins in plasma, especially follitropin, are observed. biopsy of testicles should be used due to the inaccuracy of laboratory parameters (hyalinosis of the walls of seminal tubules, aplasia of the generative epithelium, aspermia). usually ks males present a normal growth hormone – insulin-like growth factor igf-i axis and increased luteinizing hormone and folliclestimulating hormone levels due to their androgen deficiency [17]. however, early diagnosis of sex chromosome syndromes may provide better development and quality of life for patients with these disorders. samango-sprouse et al. [47] observed significant advantages in the cognition, language, intellectual, and psychomotor functions of the patients with ks treated with hormone therapy at 3-6 years of age compare to a group of untreated patients. the double aneuploidy 48,xxy,+21 was first described in 1959, and the incidence is reported to be 0.4 to 0.9 per 10,000 male births [9, 47, 48]. most commonly, double aneuploidy occurs when two meiotic nondisjunction events happen from the same or different parental origins. the coincidence rate of ds and ks in the same patient is 0.098 % in newborns, and it has been found to dependent on parental age. advanced maternal age is one of the main risk factors for double aneuploidy, as it is for single chromosomal aneuploidies caused by parental nondisjunc tion during gametogenesis [49-51]. the nonrandom aspect of double aneuploidy provides evidence that a hereditary predisposition to nondisjunction exists, with one chromosomal imbalance increasing the risk of another to occur [52]. the phenotype of 48,xxy,+21 double aneuploidy is characterized by classical ds features in children, who then develop additional ks symptoms from the age of puberty [9, 52]. since the sexual development primarily depends on the presence of the y chromosome, the 48,xxy,+21 individuals have male phenotype. ks is a form of primary testicular failure with testicular hypotrophy and elevated gonadotropin plasma levels, and it is the most common form of male hypogonadism [53]. in addition, the spectrum of different mental problems could be wider, and their risk could be higher in this group of patients. in cases of combined ds and ks the incidence of congenital heart disease could be higher compare to the isolated forms of these aneuploidies. sevcan tug bozdogan and atil bisgin reported of downklinefelter syndrome with accompanying both heart defects and hypothyroidism [49]. t.v. bihunyak et al. 57 b io m e d ic a l s c ie n c e s issn 2413-6077. ijmmr 2020 vol. 6 issue 1 the combination of ds and ts is very rare (1 in 2,000,000) [15, 54]. evren gumus observed a two-year old girl with karyotype from the peri pheral lymphocytes using g-bandings “mos45,x[41]/47,xx,+21[59]”. she had stig mata’s such as up slant palpebral fissures, brachycephaly and epicanthus, renal hypoplasia and hepatomegaly. in this case, at early ages, the clinical picture is completely compatible with the autosomal chromosome aneuploidy, but the phenotypic effects of ts will become dominant in post pubertal period [15]. therefore, down-klinefelter and down-turner syndromes are rare chromosomal abnormalities. a patient present with physical characte ristics of ds at birth but ks or ts features during puberty. it is important to counsel fa milies about recurrence risk for future pregnancies. a clinical geneticist evaluation is also re commended. conclusions cytogenetic variants of down syndrome are simple complete trisomy 21, translocation form and mosaicism. trisomy 21 is associated with advanced maternal age. phenotypic manifestations of down syndrome are associated with the locus 21q22. the maternal and parental nondisjunction of x-chromosomes in meiosis causes klinefelter and turner syndromes. these chromosomal diseases are variants of intersexualism with intermediate chromosomal sex. down-klinefelter and down-turner syndromes are double aneuploidies. patients at birth have a down syndrome phenotype, and signs of klinefelter and turner syndromes appear during puberty. diagnosis of aneuploidy is based on the cytogenetic investigation (karyotyping), dna analysis, ultrasonography and biochemical markers of chromosomal pathology. funding this research received no external funding. conflict of interests the authors declare no conflict of interest. author contributions bihunyak t.v. – conceptualization, writing – original draft; bondarenko yu.i. – supervision; кulyanda o.o. – writing – review and editing; charnosh s.m. – formal analysis; sverstiuk a.s. – software; bihuniak k.o. – validation. хромосомні захворювання в патології людини (огляд літератури) *т.в. бігуняк, ю.і. бондаренко, о.о. кулянда, с.м. чарнош, а.с. сверстюк, к.о.бігуняк тернопільський національний медичний університет імені і.я. горбачевського, тернопіль, україна вступ. хромосомні захворювання є причиною 45-50 % множинних вроджених вад. основні дослідження мутацій – це використання геномних технологій для виявлення співвідношення генотипу та фенотипу при анеуплоїдіях та розуміння їх патогенезу. мета. метою дослідження є вивчення етіології, патогенезу симптомів та методів діагностики пацієнтів із синдромами дауна, клайнфельтера, тернера та подвійними анеуплоїдіями 21 та статевих хромосом. методи. був проведений пошук за ключовими словами "синдром дауна", "синдром клайнфельтера", "синдром тернера", "подвійна анеуплоїдія" публікацій за період 2000-2020 років. результати. синдроми дауна, клайнфельтера та тернера є найпоширенішими анеуплоїдіями серед життєздатних новонароджених. частота мейотичних нерозходжень, які спричиняють ці анеуплоїдії, зростає з віком жінки. ідентифіковані гени, які відповідають за патогенез симптомів при синдромах трисомії 21, тернера та клайнфельтера. діагностика хромосомних захворювань включає програми пренатального скринінгу та постнатальне тестування. висновки. цитогенетичні варіанти синдрому дауна – це проста повна трисомія 21, транслокаційна та мозаїчна форми. трисомія 21 асоціюється із збільшенням віку матері. фенотипові прояви синдрому дауна пов'язані з локусом 21q22. материнське та батьківське нерозходження х-хромосом при мейозі спричиняє синдроми клайнфельтера та тернера. ці хромосомні захворювання є варіантами інтерсексуалізму з проміжною хромосомною статтю. синдроми дауна-клайнфельтера та дауна-тернера є подвійними анеуплоїдіями. пацієнти при народженні мають фенотип синдрому дауна, а ознаки синдромів клайнфельтера та тернера з’являються у період статевого дозрівання. діагностика анеуплоїдій грунтується на цитогенетичному дослідженні (каріотипуванні), аналізі днк, ультрасонографії та біохімічних маркерах хромосомної патології. ключові слова: синдром дауна; синдром клайнфельтера; синдром тернера; подвійна анеуплоїдія. t.v. bihunyak et al. 58 b io m e d ic a l s c ie n c e s issn 2413-6077. ijmmr 2020 vol. 6 issue 1 інформація про авторів бігуняк тетяна володимирівна – кандидат медичних наук, доцент кафедри патологічної фізіології тернопільського національного медичного університету імені і.я. горбачевського моз україни бондаренко юрій іванович – доктор медичних наук, професор кафедри патологічної фізіології тернопільського національного медичного університету імені і.я. горбачевського моз україни кулянда олена олегівна – кандидат медичних наук, доцент кафедри патологічної фізіології тернопільського національного медичного університету імені і.я. горбачевського моз україни чарнош софія михайлівна – кандидат медичних наук, доцент кафедри патологічної фізіології тернопільського національного медичного університету імені і.я. горбачевського моз україни сверстюк андрій степанович – кандидат технічних наук, доцент кафедри медичної інформатики тернопільського національного медичного університету імені і.я. горбачевського моз україни бігуняк катерина олегівна – студентка 6 курсу медичного факультету тернопільського національного медичного університету імені і.я. горбачевського моз україни information about the author bihunyak t.v. – md, ph.d., associate professor, department of pathophysiology, i. horbachevsky ternopil national medical university, ternopil, ukraine. orcid https://orcid.org/0000-0002-4985-5443, e-mail: bihunyak@tdmu.edu.ua bondarenko yu.i. – md, ph.d., professor, department of pathophysiology, i. horbachevsky ternopil national medical university, ternopil, ukraine. orcid https://orcid.org/0000-0003-2681-5526, e-mail: bondarenkoui@tdmu.edu.ua кulyanda o.o. – department of pathophysiology, i. horbachevsky ternopil national medical university, ternopil, ukraine. orcid https://orcid.org/0000-0001-6197-9046, e-mail: kulyanda_olol@tdmu.edu.ua charnosh s.m. – md, ph.d., associate professor, department of pathophysiology, i. horbachevsky ternopil national medical university, ternopil, ukraine. orcid https://orcid.org/0000-0001-9827-8024, e-mail: charnoshsm@tdmu.edu.ua sverstiuk a.s. – phd, associate professor, department of medical informatics, i. horbachevsky ternopil national medical university, ternopil, ukraine. orcid https://orcid.org/0000-0001-8644-0776, e-mail: sverstyuk@tdmu.edu.ua bihuniak k.o. – 6th year student of the faculty of мedicine, i. horbachevsky ternopil national medical university, ternopil, ukraine. references 1. bihunyak tv current views on the causes and phenotypic manifestations of down syndrome. the achievements of clinical and experimental medicine. 2008;1(8):7-11. 2. protsyuk ov, lynchak ov, pokanevich tm prevalence of chromosomal abnormalities among live births and stillbirths in ukraine. obstetrics. gynecology. genetics. 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distributed under the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. t.v. bihunyak et al. 21 in t e r n a l m e d ic in e issn 2413-6077. ijmmr 2019 vol. 5 issue 1 doi 10.11603/ijmmr.2413-6077.2019.1.10157 pulmonary and intracranial radiographic presentations of langerhans cell histiocytosis d. mohammed*, s.b. patel saint james school of medicine, park ridge, il, usa background. langerhans cell histiocytosis is a rare disease that affects 1 to 2 adults per million worldwide and often consists of systemic manifestations including pulmonary, intracranial and osteolytic lesions and endocrinologic abnormalities such as diabetes insipidus. objective. the objective of this case report was to expand the medical literature of this rare disease. methods. a case report of a 51-year-old female patient presenting with systemic symptoms as a result of langerhans cell histiocytosis is presented. results. a 51-year-old female presented with epistaxis, fatigue, polydipsia, polyuria, headaches and dyspnea. after initial x-rays showed multiple lung and liver nodules and the patient suffered subsequently from a unilateral pneumothorax, an open lung biopsy was recommended. on a pathological basis, the patient was diagnosed with langerhans cell histiocytosis. this report focuses on the radiological presentations of the manifestations of langerhans cell histiocytosis, particularly the presentations in the lung and intracranial regions. conclusions. langerhans cell histiocytosis is an incredibly rare disease that presents systemically. recognizing and differentiating radiographic presentation of these patients is important to determine the need for confirmation by biopsy and early chemotherapeutic intervention. key words: langerhans cell histiocytosis; fdg-pet (positron emission with 18f-2-deoxy-d-glucose (fdg); mri (magnetic resonance imaging); x-ray. *corresponding author: denelle mohammed, saint james school of medicine, 1480 renaissance drive, suite 300, park ridge, il 60068, usa. e-mail: dmohammed@mail.sjsm.org introduction langerhans cell histiocytosis is a rare disorder that affects approximately 1 to 2 adult patients per million worldwide [1]. langerhans cell histiocytosis is the accepted terminology as past references of diffuse reticuloendotheliosis, histiocytosis x, hand-schullerchristian disease and letterer-siwe disease are no longer used to refer to this pathology. the birbeck granule is a characteristic feature of the langerhans cell, which has several origins including monocytes, langerin positive dendritic cells, monocytes and langerin positive dendritic cells found in the lymphoid tissues [2]. these cells go through an induction process where they resemble the langerhans cell phenotype [3]. evidence for the neoplastic argument lays in the map2k1 and braf mutations in the mapk pathway [4]. it is a group of disorders of idiopathic pathology although there has been much medical debate as to whether this pathology is neoplastic in nature or inflammatory. the pulmonary form of langerhans cell histiocytosis is prevalent in approximately 10% of cases. in addition, pulmonary langerhans cell histiocytosis expresses greater levels of cd207, s100 and cd1a than other pulmonary diseases [5]. the most common variants of the pulmonary langerhans cell histiocytosis include braf v600e and mapk2k1 [5]. objective. this case report details clinical evidence and radiographic findings of both the common intracranial and rarer pulmonary form of langerhans cell histiocytosis and is an important work for recognizing presentations of the disease. case report a 51-year-old native american female presented to the emergency department with a three-month history of severe headaches, unresolving epistaxis, weakness, mood swings, lower back pain, fatigue, dyspnea, polyuria, polyphagia and polydipsia. the patient’s past medical history was positive for depression, nausea, vitiligo and hypothyroidism. she used synthroid, folic acid, bupropion and metoclopramide with allergies to metformin. family history was positive for hypothyroidism, hypertension, diabetes, congestive heart failure, copd and coronary artery disease. surgical history was positive for a hysterectomy, double international journal of medicine and medical research 2019, volume 5, issue 1, p. 21-25 copyright © 2019, tnmu, all rights reserved d. mohammed et al. 22 in t e r n a l m e d ic in e issn 2413-6077. ijmmr 2019 vol. 5 issue 1 ovarian cystectomy, tonsillectomy and two c-sections. the patient’s social history was positive for 25 pack years of tobacco smoking and social alcohol consumption. physical examination revealed a pulse rate of 88 beats per minute, respiration was 18 breaths per minute, temperature was 37.5 degrees celsius and blood pressure was 113/75 mmhg. the decision was made to admit her to the hospital and complete testing secondary to some of the symptoms she was experiencing. x-rays were completed for dyspnea and lower back pain. the chest x-ray visualised multiple liver and lung nodules and mri of both areas was recommended. the lung nodules were then biopsied endoscopically. approximately two days after the lung nodule biopsy, the patient suffered a pneumothorax and a needle aspiration was performed. upon stabilization and subsequent examination by the pulmonologist, an open lung biopsy was recommended to be sent for pathological review to a tertiary care centre. the pathological result showed cd1a, cd207 and braf v600e positive tissue. she was diagnosed with langerhans cell histiocytosis and diabetes insipidus secondary to the primary diagnosis. an mri of the brain both with and without contrast was performed where t1 isointense and t2 hyperintense signal areas in the hypothalamus and pituitary stalk were visualized. a 4.5 mm x 4.5 mm x 4.5 mm mass was found in the hypothalamus with an extension into the pituitary stalk. a t2 weighted image showed hyperintensity in the pons area and a thickened and enhanced pituitary stalk. a pet scan was done where diffuse and patchy hypermetabolic activity was found in the liver and spleen consistent with the pathologically confirmed diagnosis of langerhans cell histiocytosis. an mri of the abdomen showed an intense t2 signal in the portal triads. centrilobular pulmonary nodules and hypermetabolic activity with lytic lesions in the sacrum and pelvis were found consistent with the diagnosis of langerhans cell histiocytosis confirmed by open lung biopsy. there was the presence of 4 mm stellate nodules, both reticular and nodular opacities and honeycombing at the periphery of the lungs. discussion the hallmarks of langerhans cell histiocytosis are well known. these include lytic bone lesions in particular, which is the most common manifestation [1]. the jaw, vertebra, ribs, digits and skull are commonly involved. there can also be a papular and erythematous rash in the abdominal and groin area that can resemble a candida infec tion, found particularly in pediatric pa tients [1]. oral manifestations of this pathology include ulcers of the soft mucosa, gingivitis and oral cavity masses [6]. additional symptoms include lymphadenopathy, cystic hepatic lesions, splenomegaly and sclerosing cholangitis [7, 8]. manifestations of the central nervous system include diabetes insipidus secondary to infiltration of the neurohypophysis, paraventricular or supraoptic nuclei as well as neurodegenerative issues affecting cognition and ataxia [9, 10]. diabetes insipidus is the most common endocrinological presentation of langerhans cell histiocytosis found in up to half of all cases [11]. this is closely followed by the deficiency of growth hormone [11]. the pituitary seems to be affected in a multitude of ways including vasopressin antibodies or invasion by histiocytes [10]. diabetes insipidus can occur months and even years before a formal diagnosis of langerhans cell histiocytosis is given, similar to this patient’s case presentation. the intracranial radiographic changes are important to note in langerhans cell histiocytosis as symptoms such as diabetes insipidus can occur for years before diagnosis. hence, the onus is on the physician to determine the need for further clinical testing based on the radiographic presentation of this disease and implement swift chemotherapeutic interventions. other cranial radiographic changes to consider include an empty sella, hypertrophied and atrophied, asymmetrical and hyperintense enhancement of the anterior pituitary [12]. the posterior pituitary can show t1w1 hyperintensity while the hypothalamus can show enhancement and lesions [12]. in this particular patient, t2 hyperintense signal areas and t1 isointense areas were seen in the hypothalamus in particular (fig. 2). these hyperintensities also extended into the pituitary stalk and were consistent with the patient’s diagnosis of langerhans cell histiocytosis (fig. 2). other radiographic changes in the posterior pituitary in particular, include thickening of the pituitary stalk and also enhancement of that area with an absent t1w1 shortening visible [13]. studies have shown that manifestations of langerhans cell histiocytosis in the brain tend to occur in those areas without a blood brain barrier such as choroid plexuses, the pineal gland, meninges and circumventricular organs [14]. d. mohammed et al. 23 in t e r n a l m e d ic in e issn 2413-6077. ijmmr 2019 vol. 5 issue 1 neuroendocrine cells of the hypothalamus can project outside of the brain and hence can fall outside the realm of the blood brain barrier even though the actual hypothalamus is protected by one [14]. t h i s c a s e re p o r t i s i m p o r t a n t s i n c e pulmonary langerhans cell histiocytosis is uncommon and expanding the medical literature on this topic is important for radiologists to determine a possible differential for seemingly common features seen in other diseases such as honeycombing, reticular and nodular opacities. lung involvement such as this patient’s recurrent spontaneous pneumothorax and lung nodules are common in those with langerhans cell histiocytosis [14]. centrilobular pulmonary nodules with pulmonary cysts as seen in this patient’s chest x-rays are radiographic findings that are consistent with an already pathologically diagnosed langerhans cell histiocytosis (fig. 1). pulmonary involvement of langerhans cell histiocytosis is a differential when features such as recurrent pneumothorax along with diabetes insipidus and bone pain co-occur [15]. features specific to pulmonary langerhans cell histiocytosis include nodular and reticular opacities, cysts in the upper lung zone along with honeycombing, normal lung volume, no involvement of the costophrenic angle and poorly defined or stellate nodules that may be up to 10 mm in size [16]. stellate nodules, honeycombing and nodular and reticular opacities were also see in this patient upon histological examination. high resolution ct of the chest can also show interstitial thickening, thin-walled cysts and a general progression from non-cavitating nodules to cavitating nodules and subsequently cystic lesions [17]. further radiologic presentation of langerhans cell histiocytosis in fdg-pet form is seen by increased uptake in the lungs and nodular radiographic patterns [18]. conclusions this case report is important for understanding the simple radiographic presentations of an already rare disease. in conclusion, langerhans cell histiocytosis is an uncommon disease with wide presentations in various organ systems. radiographic recognition of this pathology can be beneficial to provide further testing and induction of a swift and proper therapeutic regimen. conflict of interest the authors declare no conflict of interest. fig. 1. chest x-ray exhibiting centrilobular pulmonary nodules and pulmonary cysts with prominence in the bilateral upper lobes and reticular and nodular opacities consistent with langerhans cell histiocytosis. fig. 2. a 4.5 mm x 4.5 mm x 4.5 mm enhancing hypothalamic mass with extension into the pituitary stalk consistent with langerhans cell histiocytosis. d. mohammed et al. 24 in t e r n a l m e d ic in e issn 2413-6077. ijmmr 2019 vol. 5 issue 1 легеневі та інтракраніальні радіографічні ознаки гістіоцитозу з клітин лангерганса d. mohammed, s.b. patel saint james school of medicine, park ridge, il, usa вступ. гістіоцитоз з клітин лангерганса – рідкісне захворювання, що уражає 1-2 дорослих на мільйон, і часто має системний характер, включаючи остеолітичні прояви та нецукровий діабет серед багатьох інших клінічних симптомів. мета. ознайомити читачів та привернути їх увагу до цього рідкісного захворювання методи. описано та проаналізовано клінічний випадок гістіоцитозу з клітин лангерганса з системними проявами. результати. 51-однорічна жінка поступила зі скаргами на носові кровотечі, постійну втому, спрагу, полідипсію та поліурію, головний біль, задишку. при першому рентгенологічному обстеженні виявлено численні вузлики у легенях та печінці, а згодом у пацієнтки розвинувся однобічний пневмоторакс. після цього було рекомендовано провести біопсію легень. за результатами морфологічного обстеження встановлено діагноз гістіоцитозу з клітин лангерганса. описаний нами клінічний випадок фокусує увагу на проявах хвороби, які виявляються при променевих методах обстеження, особливо легень та голови (інтракраніальних ділянок). висновки. гістіоцитоз з клітин лангерганса уражає усі органи та системи організму. типовими проявами є ураження кісток, розвиток нецукрового діабету внаслідок ураження гіпоталамо-гіпофізарної області, та наявність вузликів у легенях. зміни, які виявляються променевими методами обстеження, надзвичайно важливі для встановлення потреби у проведенні біопсії та призначення хіміотерапевтичного лікування. ключові слова: гістіоцитоз з клітин лангерганса; позитронно-емісійна томографія (пет) із 18f-фдг (18-фтордезоксиглюкоза); мрт (магнітно-резонансна томографія); рентгенографія. information about the author denelle mohammed – department of medicine, saint james school of medicine, 1480 renaissance drive, suite 300, park ridge, il 60068, usa. orcid 0000-0002-1639-2008, e-mail: dmohammed@mail.sjsm.org samarth b. patel – department of medicine, saint james school of medicine, 1480 renaissance drive, suite 300, park ridge, il 60068, usa. orcid 0000-0003-0633-6376, e-mail: spatel2@mail.sjsm.org references 1. baumgartner i, von hochstetter a, baumert b, luetolf u, follath f. langerhans'-cell histiocytosis in adults. med pediatr oncol. 1997;28(1):9-14. d o i : 1 0 . 1 0 0 2 / ( s i c i ) 1 0 9 6 911x(199701)28:1<9::aid-mpo3>3.0.co;2-p 2. merad m, ginhoux f, collin m. origin, homeostasis and function of langerhans cells and other langerin-expressing dendritic cells. nat rev immunol. 2008;8(12):935-47. doi: 10.1038/nri2455 3. egeler rm, van halteren ag, hogendoorn pc, laman jd, leenen pj. langerhans cell histiocytosis: fascinating dynamics of the dendritic cell-macrophage lineage. immunol rev. 2010;234(1):213-32. doi: 10.1111/j.0105-2896.2009.00883.x 4. brown na, furtado lv, betz bl, kiel mj, weigelin hc, lim ms, elenitoba-johnson ks. high prevalence of somatic map2k1 mutations in braf v600e-negative langerhans cell histiocytosis. blood. 2014;124(10):1655-8. doi: 10.1182/blood-2014-05-577361 5. roden ac, yi es. pulmonary langerhans cell histiocytosis: an update from the pathologists' perspective. archives of pathology & laboratory medicine. 2016;140(3):230-40. doi: 10.5858/arpa.2015-0246-ra 6. annibali s, cristalli mp, solidani m, ciavarella d, la monaca g, suriano mm, lo muzio l, lo russo l. langerhans cell histiocytosis: oral/periodontal involvement in adult patients. oral diseases. 2009 nov;15(8):596-601. doi: 10.1111/j.1601-0825.2009.01601.x 7. braier jl, rosso d, latella a, chantada g, ozuna b, ripoli m, scopinaro m. importance of multilineage hematologic involvement and hypoalbuminemia at diagnosis in patients with “risk-organ” multi-system langerhans cell histiocytosis. journal of pediatric hematology/oncology. 2010 may 1;32(4):e122-5. doi: 10.1097/mph.0b013e3181d7acc5 d. mohammed et al. 25 in t e r n a l m e d ic in e issn 2413-6077. ijmmr 2019 vol. 5 issue 1 8. grois n, fahrner b, arceci rj, henter ji, mcclain k, lassmann h, nanduri v, prosch h, prayer d. central nervous system disease in langerhans cell histiocytosis. the journal of pediatrics. 2010 jun 1;156(6):873-81. doi: 10.1016/j.jpeds.2010.03.001 9. kaltsas ga, powles tb, evanson j, plowman pn, drinkwater je, jenkins pj, monson jp, besser gm, grossman ab. hypothalamo-pituitary abnormalities in adult patients with langerhans cell histiocytosis: clinical, endocrinological, and radiological features and response to treatment. the journal of clinical endocrinology & metabolism. 2000 apr 1;85(4):1370-6. doi: 10.1210/jcem.85.4.6501 10. mittheisz e, seidl r, prayer d, waldenmair m, neophytou b, pötschger u, minkov m, steiner m, prosch h, wnorowski m, gadner h. central nervous system-related permanent consequences in patients with langerhans cell histiocytosis. pediatric blood & cancer. 2007 jan;48(1):50-6. doi: 10.1002/pbc.20760 11. maghnie m, genovese e, bernasconi s, binda s, arico m. persistent high mr signal of the posterior pituitary gland in central diabetes insipidus. ajnr am j neuroradiol. 1997;18:1749-52. http://www.ajnr.org/content/ajnr/18/9/1749. full.pdf 12. prayer d, grois n, prosch h, gadner h, and barkovich aj. mr imaging presentation of intracranial disease associated with langerhans cell histiocytosis. ajnr am j neuroradiol. 2004;25:880-91. http://www.ajnr.org/content/25/5/880.long 13. ganong wf. circumventricular organs: definition and role in the regulation of endocrine and autonomic function. clin exp pharmacol physiol. 2000;27:422-7. doi: 10.1046/j.1440-1681.2000.03259.x 14. odame i, li p, lau l, doda w, noseworthy m, babyn p, weitzman s. pulmonary langerhans cell histiocytosis: a variable disease in childhood. pediatric blood & cancer. 2006 dec 1;47(7):889-93. doi: 10.1002/pbc.20676 15. samanci ns, ayer m. multisystem involvement of langerhans cell histiocytosis in an adult. the indian journal of medical research. 2019;149:78. doi: 10.4103/ijmr.ijmr_1726_17 16. kulwiec el, lynch da, aguayo sm, schwarz mi, king jr t. imaging of pulmonary histiocytosis x. radiographics. 1992;12:515-26. doi: 10.1148/radiographics. 12.3.1609142 17. castoldi mc, verrioli a, de juli e, vanzulli a. pulmonary langerhans cell histiocytosis: the many faces of presentation at initial ct scan. insights into imaging. 2014;5:483-92. doi: 10.1007/s13244-014-0338-0 18. krajicek bj, ryu jh, hartman te, lowe vj, vassallo r. abnormal fluorodeoxyglucose pet in pulmonary langerhans cell histiocytosis. chest. 2009;135:1542-9. doi: 10.1378/chest.08-1899 received 30 may 2019; revised 11 june 2019; accepted 18 june 2019. this is an open access article distributed under the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. d. mohammed et al. 31 su r g ic a l d is e a se s issn 2413-6077. ijmmr 2022 vol. 8 issue 1 doi 10.11603/ijmmr.2413-6077.2022.1.12674 case report on rare outcome of a retroperitoneal mass: management challenges and lessons learnt v. v. nair, p. sharma, *n. rajendran, s. raja, p. p. rao, r. mehta armed forces medical college, pune, maharashtra, india background. gastrointestinal stromal tumours (gist) are non-epithelial mesenchymal solid neoplasm with varied presentation. the study reports the case of a retroperitoneal gist in a 21-year-old male presented with an abdominal lump for six months. the lesion was initially thought to be a retroperitoneal sarcoma. exploratory laparotomy revealed an abdominopelvic mass covering the entire right side of abdomen and pelvis. the tumour was adherent to the terminal ileum and ascending colon. there were dense adhesions between the retroperitoneum with involvement of the middle third of the right ureter. the tumour was resected with right hemicolectomy with ileotransverse anastomosis. post-operative histopathology revealed it as high-grade spindle cell type gist. the patient is presently on post-operative chemotherapy with imatinib mesylate. objective. atypical presentations of gist are seldom discussed but frequently encountered in clinical practice. this article depicts different challenges the surgeon has to face while diagnosing such atypical entity. methods. case report of atypical gist presenting as retroperitoneal lump. results. the patient underwent surgical resection and is presently on post-operative chemotherapy with good overall outcome for a one year follow up. conclusions. gists presenting as retroperitoneal lumps are very rare, they should be considered in their differential diagnosis of an atypical retroperitoneal mass. keywords: gist; atypical presentation; retroperitoneal tumour; abdominal lump. *corresponding author: nagamahendran rajendran, clinical tutor, department of surgery, armed forces medical college, pune, maharashtra, 411040, india. e-mail: nagaa.mahendran@gmail.com international journal of medicine and medical research 2022, volume 8, issue 1, p. 31-36 copyright © 2022, tnmu, all rights reserved introduction gastrointestinal stromal tumours (gist) are intra-abdominal benign mesenchymal neoplasia arising from the interstitial cell of cajal. the most common sites are the stomach and the intestine. mostly gist presents as a slowgrowing intra-abdominal mass appreciated as a lump abdomen. they can also present as gastrointestinal bleed or intestinal obstruction. however, there are many incidences where they present atypically. this article is about the diagnostic challenges and analysis of various rare presentations of gist. case report a 21-year-old male presented with a 6-month history of lump abdomen in the umbilical and right lumbar region. the lump was insidious in onset and gradually progressive. there was no history of vomiting, constipation or obstipation. he also denies any history of trauma to the abdomen. there were no hepatic or urinary complaints. on clinical examination a nontender, smooth surfaced firm 20×15 cm lump was palpated in the right lumbar region extending to the umbilicus crossing 3 cm lateral to the midline. the lump was fixed to retro­ peritoneum. there was no palpable hepatosplenomegaly, and digital rectal examination was normal. the patient underwent extensive preoperative investigation for diagnosis. on admission his haemoglobin was 13.5 g%, total leukocyte count was 6.200/mm3 with a normal differential count, platelet count and coagulogram. the random blood sugar was 98 mg%. the renal and liver function tests were normal. the ab domen ultrasonogram revealed large hetero geneous mass of 16×11.4 cm in the pelvis region on the right side. possibility of lymphoma / lymphoproliferative disorder should be ruled out. mild fullness of pelvicalyceal system on right side and mild hepatosplenomegaly pre sen ted. keeping a working diagnosis of a retro peritoneal tumour, a contrast enhanced compu terised v. v. nair et al. 32 su r g ic a l d is e a se s issn 2413-6077. ijmmr 2022 vol. 8 issue 1 tomography scan revealed 13.2×9.9×13.4 cm round to oval, predominantly isodense lesion in lower abdomen and pelvis (fig. 1). on the right side, it appeared to involve the mesentero-peritoneal space. patchy mesenteric fat stranding was seen. mass effect with displacement of bowel loops and indentation of uri nary bladder was evidenced. another 3.2×3.1×9.7 cm iso to hyperdense lesion was present predominantly in iliopsoas muscle. an ill­defined isodense lesion of 2.2×1.3×1.2 cm was revealed in subcutaneous tissue of anterior abdominal wall in right paraumbilical region. multiple sub centimetric sized mesenteric lymph nodes was seen. posterior end of left lower ribs appeared thickened and showed mild deformity. the tumour was locally aggressive with multiple intramuscular, and subcutaneous enhancing nodular lesion was revealed. non enhancing hyperdense band like lesions was evidenced in iliacus and paraspinalis muscles. there was an expansile lesion of the 7th and 8th ribs on the left side. the differential diagnosis of desmoplastic fibroma/fibrous dysplasia of the retroperitoneum was contemplated. fine needle aspiration cytology from the lesion showed no evidence of malignant cells. a ct angiogram further revealed the same locally aggressive lesion in right lumbar and iliac region with extension into pelvis. the arterial supply of the lesion was seen to arise from hypertrophic ileo-colic-right colic trunk. the diameter of ileo colic – right colic trunk at origin was 4 mm. a ct guided trucut biopsy revealed histomorphological features suggestive of spindle cell tumour of borderline malignant potential (fig. 2). on exploratory laparotomy there was an intrabdominal mass covering the entire right side of abdomen and right side of pelvis (fig. 3). fig. 1. gist ** cect abdomen and pelvis showing predominantly solid abdominopelvic mass measuring 10×15×16 cm with heterogeneous enhancement. right sided grade iii hyd roureteronephrosis noted. fig 2. gist ** a&b: h&e stained section shows interlacing benign spindle cells embedded in myxoid stroma; c&d: immunohistochemical staining with c kit antibody showing cystoplasmic positivity. v. v. nair et al. 33 su r g ic a l d is e a se s issn 2413-6077. ijmmr 2022 vol. 8 issue 1 v. v. nair et al. the tumour was adherent to the terminal ileum and ascending colon. the tumour was resected with right hemicolectomy and ileotransverse anastomosis. there was dense adhesion between the retroperitoneum with involvement of right middle ureter. the involved part of right ureter was resected and reanastomosed with double j stent (fig. 4). on the 5th postoperative day, the patient developed increased frequency of stools, which subsided over next few days. he was discharged in 10 days with initiation of chemotherapy with tablet imatinib mesylate 400 mg once daily. the post-operative specimen histopathology showed gastrointestinal stromal tumour, spindle cell type, high grade, pt4n0mx. on immunohistochemistry dog 1 and cd 17 were positive. cd 34, desmin, s100 and sma were negative. ki 67 score was 8 to 10%. cut end of the left ureter showed small focus of tumour abutting the adventitia wall. on the follow up in 30 days, the abdominal wall wound healed well and chemotherapy was tolerated without any side effects. dj stent was removed in six weeks. review ultrasound examinations over three and sixth month showed no recurrence of growth. presently the individual has got back to the normal life activities and is perusing a master’s course in university. discussion the largest series of retroperitoneal gist reported in literature comes from miettinen et al. [1]. they analysed data for a period of 26 years and studied 112 cases of retroperitoneal gist. equal incidence was evidenced in both sexes and more common in the elderly population. avast majority of these tumours were from extraintestinal site with 35 cases from fig 3. gist ** a: preoperational image showing abdominopelvic lump in lateral view; b: intra pic showing 13×13 cm hard lump; c: intra op pic showing right ureter with hydronephrosis; d: resected specimen showing terminal ileum and transverse colon along with the lump. fig 4. post op x-ray pelvis showing right sided double j stent in place. 34 su r g ic a l d is e a se s issn 2413-6077. ijmmr 2022 vol. 8 issue 1 unspecific sites. all tumours were positive for immunohistochemistry markers for gist. regarding the analysis of the unusual presentation of gist, many articles are available in the literature. raja et al. reported a case of gist presenting as intra peritoneal bleed in a 31-year-old male [2]. the highly malignant nature of the gist caused ulceration and bleed into the peritoneal cavity and presenting as hemo peritoneum. sometimes gist arising from the retroperitoneum presents with nonspecific symp­ toms of vomiting, weight loss, low grade fever, anaemia and asthenia. there will be no mass/ lump palpable in the abdomen. thus, imaging technique and biopsy will reveal mass retroperitoneum classified as extraintestinal gist (e-gist). yan et al. reported one of the rare occurrences of pancreatic gist [3]. this was one of the first two such cases reported in the literature. the diagnosis was made by tissue biopsy using endoscopic ultrasound. a malignant gist was similarly diagnosed by rao et al. as a growth in the head of pancreas in a 40-yearold patient [4]. similarly, harindhanavudhi et al. [5] reported gist presenting as haemorrhagic cyst in the head of pancreas. another rare location with varied presentation for retroperitoneal gist is duodenum. the largest series was by vassos et al. [6]. in their 1-year review of 13 patients this site was amenable to surgical resection. both local excision and pancreaticoduodenectomy showed comparable results in experience centres. this article also stressed the use of tki imatinib mesylate in overall long-term prognosis. gorelik et al. [7] reported another rare presentation of gist. the patient presented in bacteraemia with liver abscess. the unusual presentation arose from a fistula between the small intestine and the tumour. similarly virgilio et al. [8] studied 15 cases of gist presenting as mass in the inguinal canal. finally, in the review of literature by gupta [9] 9 cases of gall bladder gist were reported. these symptoms may be presumed as a sign of mimic cholangitis and are often seen in women. almost all of these tumours are malignant and require urgent surgical excision. finally, a similar case was reported by ashoor et al. [10] as an incidental finding in a 67-year-old man reported for benign prostrate hyperplasia. the tumour measuring 15 cm was arising from the retroperitoneal aspect of right inguinal fossa. this is the last presentation of incidental findings in radiological evaluation for other problems in this study. table 1. some of the varied presentations of gist are as tabulated further down no. year topic author presentation a) 2014 atypical presentation of gastrointestinal stromal tumours: a case report kalpana raja et al. hemangioma b) 2020 atypical presentation of gastrointestinal stromal tumor as multiple intra-abdominal cysts: a case report ram prakash gurram et al. gastric cyst c) 2004 atypical presentation of gastrointestinal stromal tumor as multiple intra-abdominal cysts: a case report ke kang sun et al. intra-abdominal cyst d) 2015 atypical presentation of gastrointestinal stromal tumor masquerading as a large duodenal cyst: a case report ameet kumar et al. duodenal cyst e) 2019 unusual presentation of a large gist in an extraintestinal site: a challenging diagnosis dilemma arwa ahmed ashoor et al. jejunal mesentry f) 2018 an ulcerated ileal gastrointestinal stromal tumor disguised as acute appendicitis ashish lal shrestha et al. ileal gist presenting as appendicitis g) 2017 ileal gist presenting with bacteremia and liver abscess: a case report and review of literature gorelik et al. bacteraemia with liver abscess h) 2021 inguinal gist: a systematic literature review of primary and metastatic cases virgilio et al. mass in the inguinal canal i) 2014 malignant extra-gastrointestinal stromal tumor of the pancreas: report of two cases and review of the literature yan et al. pancreatic gist j) 2019 gallbladder gist: a review of literature gupta et al. gall bladder gist v. v. nair et al. 35 su r g ic a l d is e a se s issn 2413-6077. ijmmr 2022 vol. 8 issue 1 v. v. nair et al. клінічний випадок рідкісного заочеревинного утворення: труднощі лікування та набутий досвід v. v. nair, p. sharma, *n. rajendran, s. raja, p. p. rao, r. mehta armed forces medical college, pune, maharashtra, india вступ. шлунково-кишкові стромальні пухлини (шксп) — це неепітеліальні мезенхімальні солідні новоутворення з різноманітними проявами. у цьому дослідженні описано випадок заочеревинної шксп у 21-річного чоловіка, у якого протягом шести місяців спостерігалася припухлість в ділянці живота. утворення спочатку вважали заочеревинною саркомою. діагностична лапаротомія виявила черевнотазову пухлину, що охоплювала всю праву частину живота та таза. пухлина була спаяна з термінальним відділом клубової та висхідної ободової кишки. виникли щільні спайки між заочеревинним простором із ураженням середньої третини правого сечоводу. пухлину видалено з правосторонньою геміколектомією з ілео-поперечним анастомозом. післяопераційна гістопатологія показала, що це веретеноклітинна шксп високого ступеня диференціювання. зараз пацієнт проходить післяопераційну хіміотерапію іматинібу мезилатом в таблетках. мета. атипові прояви шксп рідко обговорюються, але часто зустрічаються в клінічній практиці. ця стаття описує різні проблеми, з якими доводиться зустрічатися хірургу під час діагностики такого нетипового утворення. методи. опис випадку атипового шксп, який проявлявся у вигляді заочеревинної припухлості. результати. пацієнту була проведена хірургічна резекція, зараз він отримує післяопераційну хіміотерапію з хорошим загальним результатом після року спостереження. висновки. шксп, які проявляються у вигляді заочеревинних утворень, зустрічаються дуже рідко, їх слід враховувати при диференціальній діагностиці атипової заочеревинної пухлини. ключові слова: шксп; атипове положення; заочеревинна пухлина; ущільнення в животі. information about the authors vipin v. nair, associate professor, armed forces medical college, pune, maharashtra, india https://orcid.org/0000­0001­6903­6368, vipinvenugopalnair@gmail.com pawan sharma, professor, armed forces medical college, pune, maharashtra, india https://orcid.org/0000­0003­4984­0708, drpawansharma55@gmail.com nagamahendran rajendran, clinical tutor, armed forces medical college, pune, maharashtra, india https://orcid.org/0000­0002­9854­7236, nagaa.mahendran@gmail.com santosh raja, resident, armed forces medical college, pune, maharashtra, india https://orcid.org/0000­0002­0332­0742, santoshrajasarali@gmail.com pankaj p. rao, professor and head, armed forces medical college, pune, maharashtra, india https://orcid.org/0000­0002­1420­7622, surgeonrao@gmail.com ritu mehta, professor, armed forces medical college, pune, maharashtra, india https://orcid.org/0000­0003­1149­3632, doctorritumehta@gmail.com conclusions gastrointestinal stromal tumours are mostly benign neoplasm arising from gastrointestinal tissues. they seldom become malignant and metastasis is rare. there are many atypical variations in clinical presentation for these tumours. the surgeon always needs to keep this diagnosis in mind while evaluating atypical cases of intra-abdominal or retro peritoneal mass. limitations single centre study. conflict of interests all authors declare no conflict of interest. author’s contributions vipin v. nair, pawan sharma, nagamahendran r. – conceptualization, methodology, formal analysis, writing – original draft, writing – re­ viewing and editing; santosh raja, pankaj p. rao – data curation, writing – reviewing and editing; ritu mehta – investigation, formal analysis. 36 su r g ic a l d is e a se s issn 2413-6077. ijmmr 2022 vol. 8 issue 1 references 1. miettinen m, felisiak-golabek a, wang z, inaguma s, lasota j. gist manifesting as a retroperitoneal tumor: clinicopathologic immunohistochemical, and molecular genetic study of 112 cases. am j surg pathol. 2017 may;41(5):577­85. https://doi.org/10.1097/pas.0000000000000807. 2. raja k, dev b, santosham r, santhosh j. atypical presentation of gastrointestinal stromal tumours-a case report. indian j surg. 2013;75 (suppl 1):185­7. https://doi.org/10.1007/s12262­012­0557­x 3. yan bm, pai rk, van dam j. diagnosis of pancreatic gastrointestinal stromal tumor by eus guided fna. jop. 2008 mar 8;9(2):192­6. 4. rao rn, vij m, singla n, kumar a. malignant pancreatic extra-gastrointestinal stromal tumor diag nosed by ultrasound guided fine needle as­ piration cytology. a case report with a review of the literature. jop. 2011 may 6;12(3):283­6. 5. harindhanavudhi t, tanawuttiwat t, pyle j, silva r. extra-gastrointestinal stromal tumor presenting as hemorrhagic pancreatic cyst diagnosed by eus­fna. jop. 2009 mar 9;10(2):189­91. 6. vassos n, perrakis a, hohenberger w, croner rs. surgical approaches and oncological outcomes in the management of duodenal gastrointestinal stromal tumors (gist). j clin med. 2021 sep 28;10(19):4459. https://doi.org/10.3390/jcm10194459. 7. gorelik m, sabates b, elkbuli a, dunne t. ileal gist presenting with bacteremia and liver abscess: a case report and review of literature. int j surg case rep. 2018;42:261­5. https://doi.org/10.1016/j.ijscr.2017.12.033. 8. virgilio e, annicchiarico a, pagliai l, morini a, romboli a, montali f, costi r. inguinal gist: a systematic literature review of primary and metastatic cases. anticancer res. 2021 jan;41(1):21­5. https://doi.org/10.21873/anticanres.14748. 9. gupta a. gallbladder gist: a review of literature. pol przegl chir. 2019 nov 5;92(1):34­7. https://doi.org/10.5604/01.3001.0013.5550. 10. ashoor aa, barefah g. unusual presentation of a large gist in an extraintestinal site: a challenging diagnosis dilemma. bmj case rep. 2020 feb 6; 13(2):e229839. https://doi.org/10.1136/bcr­2019­229839. received 18 january 2022; revised 20 may 2022; accepted 13 may 2022. this is open access article distributed under the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. v. v. nair et al. 11 in t e r n a l m e d ic in e issn 2413-6077. ijmmr 2020 vol. 6 issue 2 doi 10.11603/ijmmr.2413-6077.2020.2.11512 pleural endometriosis: bloody pleural effusion in a 28-year-old female with primary infertility (case report) s. saheer, a. paul, p. james, r. palak christian medical college and hospital, tamil nadu, india background. endometriosis is defined as presence of endometrial glands outside the uterine cavity and it most commonly involves the structures within the pelvic cavity. thoracic endometriosis syndrome usually presents as pneumothorax, hemoptysis, hemothorax or pulmonary nodules. endometriosis presenting as hemorrhagic pleural effusion is rarely reported. objectives. the aim of the study was to describe pleural endometriosis presenting as hemorrhagic pleural effusion and to insist on the role of medical thoracoscopy in making the diagnosis with the help of a case report. methods. a case report of pleural endometriosis as a non-resolving hemorrhagic pleural effusion is presented. results. a 28-years old female on treatment for her primary infertility presented with non-resolving bloody pleural effusion and she was on empirical anti-tubercular drugs for the last four months. medical thoracoscopy revealed flat brownish grey plaques over the diaphragmatic pleura and the histology of pleural tissue revealed pleural endometriosis. she was initiated on gonadotropin releasing hormone-leuprolide and there was some clinico-radiological improvement. conclusions. thoracic endometriosis should be considered as one of the differentials for pleural effusion in female patient especially on treatment for infertility. medical thoracoscopy should be the investigation of choice for evaluating pleural effusions before initiating empirical treatments. keywords: pleural endometriosis; catamenial pleural effusion; thoracic endometriosis syndrome; hemorrhagic pleural effusion. *corresponding author: dr. akhil paul, department of pulmonary medicine, christian medical college and hospital, tamil nadu, india. email: akhil_pauli@yahoo.com international journal of medicine and medical research 2020, volume 6, issue 2, p. 11-15 copyright © 2020, tnmu, all rights reserved s. saheer et al. introduction endometriosis is defined as presence of endometrial glands outside the uterine cavity and it most commonly involves the structures within the pelvic cavity. thoracic endometriosis syndrome usually presents as pneumothorax, h e m o p t y s i s , h e m o t h o ra x o r p u l m o n a r y nodules. pleural endometriosis is characterized by presence of functional endometrial tissue in the pleura [1]. malignancy and tuberculosis are common causes of hemorrhagic pleural ef fusion. endometriosis presenting as hemorrha gic pleural effusion are rarely reported [2]. here we report a case of this rare entity in a 28-years old female. case report a 28-years old non-smoker female patient was referred to our department with moderate pleural collection after presenting to emergency department with cough and exertional breathlessness. her cough was dry in nature and her effort tolerance was around 500 meters. there was no history of fever, chest pain, hemoptysis, loss of appetite and weight loss. the patient had significant past history, as she was being treated for her primary infertility for the last two years and has received multiple doses of clomiphene citrate. as she failed in medical management, she was advised intrauterine insemination. during the work up for the above she was incidentally diagnosed to have right moderate pleural collection. diagnostic thoracocentesis revealed straw colored exudative lymphocytic fluid, ultrasound guided pleural biopsy was done with abram needle and the histopathology revealed lymphohistiocytic infiltrates. based on the above results she was initiated on four antitubercular drugs of rifampicin, isoniazid, ethambutol and pyrazinamide according to body weight. she attained menarche at the age of seventeen and had dysmenorrhea in her each cycle, which lasts for 8-10 days. she had no past history of diabetes, hypertension, asthma or tuberculosis. there was no history suggestive of connective tissue disease. patient was born from a non-consanguineous marriage; both 12 in t e r n a l m e d ic in e issn 2413-6077. ijmmr 2020 vol. 6 issue 2 her parents and siblings were healthy. her general examination, cardiovascular, abdominal and neurological system examinations were unremarkable. respiratory examination revealed reduced chest expansion, stony dull note and reduced breath sounds in right mammary, interscapular and infrascapular areas. her blood investigations revealed hemoglobin of 12.8 gm/dl, platelets of 2,86,000/μl and total leukocyte count 8,500/μl with differentials (neutrophil 62%, lymphocyte 37%, and eosinophils 1%). she had normal renal and liver function (protein and albumin 6.4 and 4.5 g/dl respectively) tests. her sputum smears were negative for acid fast bacilli. underlying connective tissue and vasculitis were ruled out by appropriate investigations. admission chest x ray showed moderate pleural effusion on the right side and computed tomography revealed normal lung parenchyma with moderate pleural effusion on the right side. diagnostic thoracocentesis revealed hemorrhagic lymphocytic exudative pleural effusion (total cell count: 480/μl mainly lymphocytic [78%]); proteins: 4.5g/dl; glucose: 107 mg/dl; ldh: 455 iu/l; adenosine deaminase: 18.6 u/l). pleural fluid gram stain, tubercular cultures were sterile and cytological examination was negative for malignant cells. bronchcoscopy done ruled out endobronchial lesions. medical thoracoscopy revealed normal visceral pleura, minimal adhesions in the posterior costophrenic recess and flat brownish gray plaques with hematoma were noticed in parietal pleura (fig. 1). biopsies were obtained from parietal and diaphragmatic pleura and they revealed fragments of endometrial glands lined by columnar cells surrounded by endometrial stroma along with hemosiderin-laden macrophages (fig. 2). fig. 1. medical thoracoscopy revealing flat brownish gray plaques (black arrows) with hematoma over diaphragmatic pleura. fig. 2. microphotograph of the biopsy specimen showing foci of endometrial glands surrounded by endometrial stroma. (h&e ×20). the pleural tissue stained positive for estrogen receptor and cd 10 immunostains (fig. 3). as the patient presented with pleural effusion the possible etiology considered was for: ȃ tuberculosis; ȃ malignancy; ȃ secondary to connective tissue disease; ȃ ovarian hyperstimulation syndrome secondary to clomiphene. tuberculosis and malignancy were ruled out by histopathology of pleural tissue obtained by medical thoracoscopy. similarly, ovarian hyperstimulation was ruled out clinically by absence of features like weight gain, abdominal pain, vomiting, ascites and bilateral pleural effusion. connective tissue disease was ruled out by absence of symptoms, clinical features and serology. patient was diagnosed to have pleural endometriosis and was started on leuprolide (gonadotropin releasing hormonal analogue) after getting expert opinion from gynecologist. post second day lung thoracoscopy showed adequate expansion and intercostal drainage was removed. she was discharged on leuprolide with the advice to review after three months. the patient and her family were educated about the nature of disease; treatment options and the need of regular follow up. after three months on follow up the patient showed good clinical and radiological improvement. s. saheer et al. 13 in t e r n a l m e d ic in e issn 2413-6077. ijmmr 2020 vol. 6 issue 2 discussion maurer in 1958 for the first time described thoracic endometriosis; it is associated with growth of endometrial tissue in the bronchial tree, lung parenchyma and pleura [1, 3]. thoracic endometriosis is a rare uncommon entity and usually it affects right hemi-thorax [3] in 92% and left in 5% of cases. pleural cavity is involved more commonly than lung parenchyma. the disease usually affects nulliparous women in their child bearing ages with a peak incidence between 20-30 years of age. the common presentations of thoracic endometriosis syndrome (tes) are pneumothorax (70%), hemothorax (12%), hemoptysis (7%) and lung nodules (6%) [4]. chest pain, hemoptysis and breathing difficulty are common clinical manifestations of tes and symptoms typically present in 48-72 hours after start of menstruation. the exact etiopathogenesis of tes is unknown, but many hypotheses have been suggested for presence of endometrial tissue in the thoracic cavity. pleural tes is explained by ivanoff’s metaplasia theory (local metaplasia of cholemic epithelium) and sampson’s retrograde menstruation theory (trans diaphragmatic pass of endometrial tissue and implantation in thoracic cavity). hematogenous migration of endometrial tissue via vascular and lymphatic micro embolization after gynaecological procedures like caesarean and curettage have been suggested as causes for parenchymal tes (hematogenous migration theory/microembolization theory) [1]. tes is diagnosed clinically based on presence of symptoms during menstruation [1, 5]. histopathological confirmation is needed for definitive diagnosis. thoracoscopy is increasingly used as the diagnostic modality for obtaining tissue for histology. as in our case, pleural and diaphragmatic implants can be seen in thoracoscopy and to increase the yield it is better to perform the procedure during the menstruation [6]. medical treatment of tes includes hormonal manipulation and it involves suppression of ectopic endometrium with oral contraceptive pills, danazol, progestins and gonadotropin releasing hormonal analogues [7]. surgical intervention is reserved of patients in whom the disease recurs after hormonal the rapy. pleurodesis and tissue resection are the treatment modalities surgically performed [7]. conclusion pleural endometriosis and ovarian hyper stimulation syndrome should be among the differentials for pleural effusion especially in females on infertility treatment. medical thoracoscopy should be considered as the investigation of choice for evaluating pleural effusions before initiating on empirical treatments. conflict of interests the authors declare no conflict of interest. funding this research received no external funding. acknowledgements the departments of pulmonary medicine and pathology of christian medical college, vellore, where the study was done. author’s contributions saheer s, akhil paul, prince james – formal analysis, investigation, methodology; saheer s, akhil paul – drafting and manuscript revision; rayani palak – investigation. fig. 3. immunohistochemical analysis demonstrating positive staining estrogenic receptor and cd 10 stains (original magnification ×20×digital magnification). s. saheer et al. 14 in t e r n a l m e d ic in e issn 2413-6077. ijmmr 2020 vol. 6 issue 2 плевральний ендометріоз: геморагічний плевральний випіт у 28-річної жінки з первинною безплідністю (клінічний випадок) s. saheer, a. paul, p. james, r. palak christian medical college and hospital, tamil nadu, india вступ. ендометріоз характеризується наявністю ендометрію поза порожниною матки і зазвичай він виявляється в органах малого тазу. ектрагенітальна його форма – ендометріоз легень зазвичай маніфестує у вигляді пневмотораксу, кровохаркання, гемотораксу або легеневих вузликів. вкрай рідко зустрічаються повідомлення про ендометріоз легень з клінічними проявами геморагічного плеврального випоту. мета. метою дослідження було описати ендометріоз легень, що маніфестував як геморагічний плевральний випіт та показати важливість медичної торакоскопії у постановці діагнозу. методи. описано клінічний випадок рідкісної форми ендометріозу легень геморагічного плеврального випоту, який не розсмоктувався. результати. представлено клінічний випадок 28-річної жінки, яка лікувалася з приводу первинного безпліддя, з геморагічним плевральним випотом, який не розсмоктувався. через підозру на туберкульоз протягом останніх чотирьох місяців вона приймала призначені лікарем чотири протитуберкульозних препарати. медична торакоскопія виявила плоскі коричнево-сірі бляшки над діафрагмальною плеврою, а гістологія плевральної тканини виявила ендометріоз плеври. пацієнтці було призначено лейпроліду ацетат (синтетичний агоніст гонадотропін-рилізинг гормону), після чого спостерігалося клінікорентгенологічне покращення. висновки. при проведенні диференціальної діагностики плеврального випоту у пацієнток, особливо при лікуванні безпліддя обов’язково слід розглядати ймовірність ендометріозу легень. медична торакоскопія повинна бути методом вибору при оцінці плеврального випоту перед початком емпіричного лікування. ключові слова: ендометріоз легень; катаменіальний плевральний випіт; геморагічний плевральний випіт. information about the authors dr saheer s, assistant professor, department of pulmonary medicine, christian medical college and hospital, tamil nadu, india orcid 0000-0002-4737-7812, e-mail: drssaheer@gmail.com dr akhil paul, post-graduate student, department of pulmonary medicine, christian medical college and hospital, tamil nadu, india orcid 0000-0002-4938-0636, e-mail: akhil_pauli@yahoo.com dr prince james, professor, department of pulmonary medicine, christian medical college and hospital, tamil nadu, india orcid 0000-0002-7347-5723, e-mail: drprincej@gmail.com dr rayani palak, assistant professor, department of pathology, christian medical college and hospital, tamil nadu, india orcid 0000-0002-2303-9106, e-mail: palak.raiyani@gmail.com references 1. alifano m, trisolini r, cancellieri a, regnard jf. thoracicendometriosis: current knowledge. ann thorac surg. 2006; 81:761-9. doi: https://doi.org/10.1016/j.athoracsur.2005. 07.044 2. sevinç s, unsal s, oztürk t et al. thoracic endometriosis syndrome with bloody pleural effusion in a 28 year old woman. j pak med assoc. 2013 jan; 63:114-6. 3. rived x, sebastián f, rodríguez ji et al. endometriosispleural complicada. neumotórax catamenial. cir esp. 2002; 67:114-5. 4. joseph j, sahn sa. thoracic endometriosis syndrome: new observations from an analysis of 110 cases. j med. 1996;100:164-70. doi: https://doi.org/10.1016/s0002-9343(97) 89454-5 s. saheer et al. 15 in t e r n a l m e d ic in e issn 2413-6077. ijmmr 2020 vol. 6 issue 2 5. channabasavaiah ad, joseph jv. thoracic endometriosis: revisiting the association between clinical presentation and thoracic pathology based on thoracoscopic findings in 110 patients. medicine (baltimore). 2010;89:1838. doi: https://doi.org/10.1097/md.0b013e3181df67d5 6. bagan p, le pimpec barthes f, assouad j et al. catamenial pneumothorax: retrospective study of surgical treatment. ann thorac surg. 2003;75:37881. doi: https://doi.org/10.1016/s0003-4975(02) 04320-5 7. alifano m, jablonski c, kadiri h et al. catamenial and noncatamenial, endometriosis-related or nonendo metriosis-related pneumothorax referred for surgery. am j respir crit care med. 2007;176:104853. doi: https://doi.org/10.1164/rccm.200704587oc received 21 nov 2020; revised 18 dec 2020; accepted 22 dec 2020. this is open access article distributed under the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. s. saheer et al. 91 b io m e d ic a l s c ie n c e s issn 2413-6077. ijmmr 2021 vol. 7 issue 2 doi 10.11603/ijmmr.2413-6077.2021.2.12516 the effect of microcrystalline cellulose on the microflora of the colon d.b. koval, *h.r. malyarchuk, o.o. levenets i. horbachevsky ternopil national medical university, ternopil, ukraine background. nowadays, much attention is paid to enterosorption methods that allow cleansing the internal organs and removing extraneous substances out of the body of a sick person. objective. the aim of the research was to study the effect of microcrystalline cellulose on the microflora of the large intestine. methods. the study was performed on 50 white laboratory wistar rats weighing 180-270 g, which were divided into control and experimental groups. the experimental group was daily administered with microcrystalline cellulose at a dose of 500 mg/kg. the study followed ethical standards and recommendations for the humanization of work with laboratory animals according to the “european convention for the protection of vertebrate animals used for experimental and other purposes” (strasbourg, 1986, 2010), as well as the requirements of the commission on bioethics of i. horbachevsky ternopil national medical university (minutes no. 66, dated november 01, 2021). the first group involved the intact rats on standard diet, the second – the rats, which received normal feeding of microcrystalline cellulose. results. in the feces of the experimental white rats treated with microcrystalline cellulose, the level of escherichia coli in the large intestine decreased by 22 and 25%. the number of these microorganisms increased by 20% in 7 days and by 14% in 14 days. the content of epidermal staphylococci in the stool decreased by 10% on the 7th day of administration. microcrystalline cellulose increased the number of staphylococcus aureus by 12%, but decreased the number of enterococci in the feces by 28%. in 7-14 days of the experiment, the content of these bacteria did not change significantly in the colon. in relation to anaerobic microorganisms – bacteroides and clostridia, this supplement caused a slight increase in the number of bacteroides – by 8.64% and the number of clostridia – by 11.54% on the 14th day. the content of fungi of the candida genus on the 7th and 14th day increased by 8.3%. conclusions. in the 2nd period of the study, the microbiome of intestinal contents worsened: the process of dysbacteriosis increased, which was manifested by a significant increase in the number of proteus spp., staphylococcus aureus, anaerobes (bacteroides, clostridia) and candida spp., as well as decreased escherichia coli and enterococci. keywords: enterosorption; microcrystalline cellulose; colon microflora; enterosorbents. *corresponding author: hanna r. malyarchuk, phd., senior assistant professor, i. horbachevsky ternopil national medical university, ternopil, 46001, ukraine e-mail: malyarchuk@tdmu.edu.ua international journal of medicine and medical research 2021, volume 7, issue 2, p. 83-90 copyright © 2021, tnmu, all rights reserved d.b. koval et al. introduction these days the incidence of the large intestine disorders due to various micro organisms is increasing. therefore, it is important to study the mechanisms of its development, features of early diagnosis, prevention and treatment. the large intestine is the main reservoir of the human microbiota in general and the digestive tract in particular; it is always dominated by characteristic groups of microorganisms, the number of species of which is small, but in quantitative terms they form the basis of the biocenosis [1,4]. the main microflora of the colon contains obligate anaerobic bacteria of the bifidobacterium, bacteroides, lactobacillus, propionibacterium, peptostreptococcus genera, as well as additional anaerobic and aerobic bacteria of the escherichia, enterococcus genera. additional colon microflora includes anaerobic bacteria of the peptococcus, clostridium, eubacterium, fusobacterium genera and additional anaerobic and aerobic bacteria of the staphylococcus, citrobacter, proteus, enterobacter, edwardsiella, klebsiella and other genera [1,5]. enterosorption is an important method based on the binding and excretion of toxic substances from the gastrointestinal tract for therapeutic and prophylactic purposes. microcrystalline cellulose is of particular interest. the supplement 92 b io m e d ic a l s c ie n c e s issn 2413-6077. ijmmr 2021 vol. 7 issue 2 gently cleanses the intestinal wall, absorbs toxins, poisons and other dangerous accu mulations. for active sorption of toxins, re-administration of enterosorbents should last at least two weeks, so an important factor for their effective and safe use is the preservation or restoration of physiological parameters of gastrointestinal function, in particular normal intestinal microbiocenosis [6,9]. the aim of the research was to study the effect of microcrystalline cellulose on the microflora of the large intestine. methods the study was performed on 50 white laboratory wistar rats weighing 180-270 g, which were divided into control (20 rats) and experimental (30 rats) groups. the experimental group was daily administrated with microcrystalline cellulose at a dose of 500 mg/kg. the study was carried out following ethical standards and recommendations for the humanization of work with laboratory animals according to the “european convention for the protection of vertebrate animals used for experimental and other purposes” (strasbourg, 1986, 2010), as well as requirements of the com mittee on bioethics of i. horbachevsky ternopil national medical university (minutes no. 66, dated november 1, 2021). the study protocol was approved by the university review boards and ethics committees of the participating centers. sampling was performed from the rectum with a disposable sterile cotton swab to remove feces. disposable cotton swabs by copan (italy) were used. before and after material collection, the tampon was weighed on torsion scales. the difference in weight was taken as the mass of stool. then the swab was placed in 1 ml of 0.1% solution of triton x-100 in 0.075 m phosphate buffer ph 7.9, and shaken thoroughly for 10-15 minutes. the prepared ten-fold dilutions of the material, inoculated on nutrient media of mpa, ysa, endo, sabouraud, blaurock, mrs, agaredds, was incubated at the optimum temperature of 37 °c. in 24-96 hours of incubation the number of colonies was counted and the result was expressed by the number of colony-forming units per 1 gram of feces according to the formula: x1 = 20 · m · n / t, where x1 is the number of cfu/ml; 20 is the constant coefficient, when 0.1 ml of the sample is inoculated; m is the number of colonies that grew; n is the dilution (in 10, 100, 1000 times etc.); t is the mass of feces. since the number of microbes per unit can reach tens of thousands, we used the decimal logarithm of this indicator – log cfu/m. the arithmetic mean and standard error (m±m) were used to describe the data in the normal distribution. since the data obtained as a result of the clinical study had deviations from the normal distribution of the variation series, nonparametric statistical methods to compare groups – the mann – whitney u-test (for indepen dent groups) was used. the software and mathematical complex for the personal computer microsoft excel 2016 was used for processing of the results of the study. results the groups were statistically comparable at the beginning of the study. during the experiment, no significant changes of the microbiome of feces of the control rats on a fiber­free diet were evidenced, when comparing the two terms of the study (tables 1 and 2). however, in the control group of animals, the composition of feces showed degree 1 dysbacteriosis (the concentration of normal microflora was below standard levels). microcrystalline cellulose reduced the level of escherichia coli in the large intestine of the experimental rats up to 5.3±0.2 and 5.2±0.2 log cfu/g (by 22 and 25%, respectively), by 20% in 7 days, and by 14% in 14 days. on the 7th day of microcrystalline cellulose administration, the content of epidermal staphylococci in feces decreased by 10%. however, the microcrystalline cellulose increased the number of staphylococcus aureus by 12%. microcrystalline cellulose reduced the number of enterococci in the feces by 28%. in contrast, after taking microcrystalline cellulose in 7-14 days of the experiment the content of these bacteria did not change significantly in the colon. in relation to anaerobic micro organisms: bacteroides and clostridia, this supple­ ment caused a slight increase in the number of bacteroides – up to 8.8±0.3 log cfu/g (8,64%) and a more significant increase in the number of clostridia – 8.7±0.2 log cfu/g (11.54%) on the 14th day. after taking microcrystalline cellulose, the content of fungi of the candida genus on the 7th and 14th day slightly increased by 8,3%. discussion the effect of crystalline microcellulose on the intestinal microbiota has not been studied comprehensively. according to the literature, d.b. koval et al. 93 b io m e d ic a l s c ie n c e s issn 2413-6077. ijmmr 2021 vol. 7 issue 2 table 1. effect of microcrystalline cellulose on the microflora of the large intestine of white rats, log cfu/g (on the 7th day of the study) types of microorganisms control(n=20) microcrystalline cellulose (n=30) escherichia coli 6.79±0.2 5.3±0.2 p<0.01 enterobacteriaceae spp. 5.34±0.49 6.4±0.2 p<0.01 staphylococcus epidermidis 5.44±0.2 4.9±0.3 p<0.05 staphylococcus aureus 5.0±0.0 5.5±0.34 p<0.05 streptococcus faecalis 5.53±0.2 4.0±0.2 p<0.002 lactobacillus spp. 6.10±0.4 6.2±0.3 p<0.1 bifidobacterium spp. 6.3±0.23 6.4±0.3 p<0.1 bacteroides spp. 7.3±0.22 8.4±0.3 p<0.02 clostridium spp. 7.3±0.12 7.4±0.3 p<0.1 candida spp. 3.61±0.2 3.91±0.26 p<0.05 proteus vulgaris 2.5±0.1 3.0±0.1 p<0.01 table 2. effect of microcrystalline cellulose on the microflora of the large intestine of white rats, log cfu/g (on the 14th day of the study) types of microorganisms control(n=20) microcrystalline cellulose (n=30) escherichia coli 6.9±0.2 5.2±0.2 p<0.01 enterobacteriaceae spp. 5.6±0.3 6.4±0.2 p<0.02 staphylococcus epidermidis 5.4±0.2 4.9±0.3 p<0.05 staphylococcus aureus 5.0±0.2 5.6±0.34 p<0.05 streptococcus faecalis 5.5±0.2 4.2±0.2 p<0.01 lactobacillus spp. 6.1±0.4 6.2±0.3 p<0.1 bifidobacterium spp. 6.3±0.2 6.4±0.3 p<0.1 bacteroides spp. 8.1±0.2 8.8±0.3 p<0.05 clostridium spp. 7.8±0.2 8.7±0.2 p<0.05 candida spp. 3.6±0.2 3.9±0.26 p<0.05 proteus vulgaris 2.5±0.1 3.5±0.1 p<0.001 d.b. koval et al. 94 b io m e d ic a l s c ie n c e s issn 2413-6077. ijmmr 2021 vol. 7 issue 2 concomitant therapy in patients with various pathologies shows significant effectiveness [10, 11]. the results of various studies prove a positive effect of enterosorbents on the manifestations of oxidative stress; it normalizes prooxidant-antioxidant balance [12,13]. however, no studies have been presented on the effects of enterosorbents on the microflora of the colon, especially in its prolonged administration. enterosorbents are biomaterials that “work” in the lumen of the gastrointestinal tract and have only local pharmacokinetics. however, in cases of sorption detoxification of the body, they act not only locally, but also have remote long-term effects [14]. the sorption of toxic metabolites and compounds relieves the main organs of metabolism and excretion. this explains the positive effect of enterosorbent in many comorbid pathologies. the effect of enterosorption on the intestinal microflora is also positive [15]. conclusions microcrystalline cellulose entering the stomach has the ability to swell when absorbing fluid and is used to mechanically achieve a feeling of satiety and suppress appetite. mechanical cleaning of the intestinal mucosa and effective absorption of harmful substances when passing through the gastrointestinal tract allows using it in poisoning by chemicals, toxins and salts of heavy metals. however, in the 2nd period of the study, the microbiome of intestinal contents worsened: dysbacteriosis developed, which was manifested by a significant increase in the number of proteus vulgaris, staphylococcus aureus, anaerobes (bacteroides spp., clostridia spp.) and candida spp., as well as decreased intestinal and intestinal bacteria. limitations of the study are the restricted number of laboratory animals and the limited range of methods for studying the large intestine microflora. conflict of interests authors declare no conflict of interest. authors contributions dmytro b. koval, hanna r. malyarchuk – conceptualization, methodology, formal analysis, writing – original draft, writing – reviewing and editing; dmytro b. koval, hanna r. malyarchuk – data curation, writing – revie wing and editing; dmytro b. koval, hanna r. malyarchuk, olexandr o. levenets – investigation, formal analysis. вплив мікрокристалічної целюлози на мікрофлору товстої кишки д.б. коваль, *г.р. малярчук, о.о. левенець тернопільський національний медичний університет імені і. я. горбачевського моз україни, тернопіль, україна вступ. у наш час велика увага надається методам ентеросорбції, які дозволяють очищати внутрішнє середовище та виводити з організму хворої людини чужорідні речовини. метою дослідження було вивчення впливу мікрокристалічної целюлози на мікрофлору товстого кишечника. методи. дослідження проведено на 50 білих лабораторних щурах лінії вістар масою 180-270 г, які були розділені на контрольну і експериментальну групи. в експериментальній групі шляхом щоденного введення мікрокристалічної целюлози з розрахунку 500 мг/кг. роботу із піддослідними тваринами виконували згідно з правилами європейської конвенції про гуманне ставлення до лабораторних тварин (страсбург, 1985). першу групу склали інтактні щури, годування яких було стандартним, другу – щури, які при звичайному годуванні отримували мікрокристалічну целюлозу. результати. у випорожненнях дослідних білих щурів, які отримували мікрокристалічну целюлозу знижувався рівень кишкової палички в товстому відділі кишечнику на 22 і на 25%. збільшилася кількість цих мікроорганізмів на 20% через 7 днів і на 14% – через 14 днів. зменшувався вміст епідермальних стафілококів у випорожненнях на 10% після 7-го дня прийому. прийом мікрокристалічної целюлози d.b. koval et al. 95 b io m e d ic a l s c ie n c e s issn 2413-6077. ijmmr 2021 vol. 7 issue 2 збільшував кількість золотистих стафілококів на 12%, проте зменшував кількість ентерококів у фекаліях на 28%. в товстій кишці через 7-14 днів експерименту зміст цих бактерій не зазнав помітних змін. по відношенню до анаеробних мікроорганізмів – бактероїдів і клостридій цей препарат викликав незначне збільшення кількості бактероїдів – на 8,64% і кількості клостридій – на 11,54% після 14-го дня. вміст грибів роду candida після 7-го та 14-го дня збільшувалася на 8,3%. висновки. в 2-й термін дослідження мікробіологічний краєвид кишкового вмісту погіршувався: збільшувався процес дисбактеріозу, що виражалося в істотному збільшенні кількості протея, золотистих стафілококів, анаеробів (бактероїдів, клостридій) і грибів роду candida, а також зниженням змісту кишкової палички та ентерококів. ключові слова: ентеросорбція; мікрокристалічна целюлоза; мікрофлора товстого кишечника; ентеросорбенти. information about the authors dmytro b. koval – student, i. horbachevsky ternopil national medical university, ternopil, ukraine. orcid 0000­0002­8958­1731, e­mail: koval_dmybog@tdmu.edu.ua hanna r. malyarchuk – assistant professor, i. horbachevsky ternopil national medical university, ternopil, ukraine. orcid 0000-0003-2647-2703, e­mail: malyarchuk@tdmu.edu.ua olexandr o. levenets – student, i. horbachevsky ternopil national medical university, ternopil, ukraine. orcid 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of anthracyclines cardiotoxicity experimental oncology 2012;34(4):314­22. 15. nikolaev vg. sorption therapy with the use of activated carbons: effects on regeneration of organs and tissues hemoperfusion, plasmaperfusion and other clinical uses of general, biospecific, immuno and leucocyte adsorbents 2017:221­43. https://doi.org/10.1142/9789814749084_0007 received 27 november 2021; revised 1 december 2021; accepted 10 december 2021. this is open access article distributed under the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. d.b. koval et al. 56 su r g ic a l d is e a se s issn 2413-6077. ijmmr 2019 vol. 5 issue 2 doi 10.11603/ijmmr.2413-6077.2019.2.10894 ultrasound thromboelastagraphy for the choice of treatment of patients with postoperative venous thrombosis s.ya. kostiv, *d.v. khvalyboha, i.k. venher, o.i. zarudna, o.i. kostiv i. horbachevsky ternopil national medical university, ternopil, ukraine background. the frequency of venous thromboembolic complications in surgery practice is rather high. in many cases, it is the cause of a fatal pulmonary embolism. one of the actual tasks of ultrasonic diagnostics of acute venous thrombosis is the visual assessment of the substrate of the disease because it determines angiosurgical tactics and surgical prophylaxis of pulmonary embolism. the objective was to prevent the development of pulmonary embolism in patients with postoperative venous thrombosis of the inferior vena cava system. methods. vena cava system investigation and the determination of the sonoelastographic properties of the venous thrombus were carried out with the siemens acuson s2000 ultrasound system. the localization and prevalence of the thrombotic process were established. at the end of the topical diagnosis of a venous thrombus, the sonoelastographic properties of the thrombus were studied by determining the speed of propagation of the acoustic wave. results. the work is based on the results of examination and surgical treatment of 729 patients, of which 205 (28.12%) had operative interventions on the musculoskeletal system, 378 (51.85%) – on the abdominal organs, 146 (20.01%) – reconstructive surgery on the aorta and the main arteries of the lower extremities. conclusions. embolodengerous thrombi are those venous thrombi of the inferior vena cava system which at ultrasonoelastography of the proximal segments of the venous thrombus are characterized by the acoustic wave propagation velocity within 2.5–2.8 m/s. the detection of embolic venous thrombosis is an indication for surgical methods prevention of pulmonary embolism. key words: pulmonary embolism; postoperative deep vein thrombosis; inferior vena cava. *corresponding author: dymytrii v. khvalyboha, i. horbachevsky ternopil national medical university, maidan voli, 1, 46000, ternopil, ukraine. e-mail: varjh2@gmail.com introduction. the frequency of venous thromboembolic complications ranges from 10 to 40% in patients with a surgical profile [1]. postoperative venous thrombosis in 5-10% is the cause of fatal pulmonary embolism (pe) [2, 3]. one of the actual tasks of ultrasonic diagnostics of acute venous thrombosis (avt) is a visual assessment of the substrate of the disease since the results obtained determine angiosurgical tactics of treatment and method of surgical prophylaxis of pe if it is necessary [4]. the risk of developing lower extremity deep vein thrombosis and pulmonary artery thrombo embolism is higher in patients with surgical diseases. however, more than half of intra-hospital fatal episodes of pulmonary artery thromboembolism are recorded in patients with a nonsurgical profile. according to the framingham study, pulmonary embolism accounts for 15.6% of all in-hospital mortality, with surgical patients accounting for 18% and 82% of patients with therapeutic pathology. longterm mobility limitation of neurologic patients, inoperable cancer and hematologic diseases, complex pathology in elderly patients, and other risk factors are no less threatening predictors of venous thromboembolism than surgery. along with this, ultrasound examination in patients with suspected avt can establish a correct diagnosis only in case of typical manifestations of the disease, while the frequency of diagnostic errors reaches 50% [5]. in several cases, fatal pe after the ultrasound in patients with postoperative avt in the basin of inferior international journal of medicine and medical research 2019, volume 5, issue 2, p. 56-60 copyright © 2019, tnmu, all rights reserved s.ya. kostiv et al. 57 su r g ic a l d is e a se s issn 2413-6077. ijmmr 2019 vol. 5 issue 2 s.ya. kostiv et al. vena cava (ivc) [6], which encourages the search for more effective methods of diagnosis of embologenic venous thrombosis. the objective of the study is to prevent the development of pe in patients with postoperative venous thrombosis of the inferior vena cava system. methods the investigation of the ivc system and the determination of the sonoelastographic proper ties of the venous thrombus were carried out with the siemens acuson s2000 ultrasound system (germany). in the ultrasound examination of ivc system, localization and prevalence of the thrombotic process were established. at the end of the topical diagnosis of a venous thrombus, the sonoelastographic properties of the thrombus were studied by determining the speed of propagation of the acoustic wave. at a speed of acoustic wave propagation in the range 2.5-2.6 m/s there is a high risk of em bolism, while at a speed of propagation of an acoustic wave within 2.7-2.9 m/s there is a moderate risk of embolism, and at the acoustic wave propagation of 3.0 m/s and higher, the patient does not experience any risk of embo lism. all participants signed written informed consent. the trial was approved by the ethical committee of i. horbachevsky ternopil national medical university. results the work is based on the results of examination and surgical treatment of 729 patients, of which 205 (28.12%) had operative interventions on the musculoskeletal system, 378 (51.85%) – on the abdominal organs, 146 (20.01%) – reconstructive surgery on the aorta and the main arteries of the lower extremities. according to the j. caprini (2012) scale, 316 (43.35%) patients had a very high risk of developing thromboembolic complications, and 413 (56.64%) – a high risk. thromboprophylaxis to patients was carried out in accordance with the provisions of the accp (2016). postoperative thrombosis in the ivc system was diagnosed in 118 (16.19%) cases. the throm botic process in the deep vein system was diagnosed in 106 (88.89%), and varico thrombophlebitis – in 12 (10,17%) observations. in 4 (3.77%) patients the thrombotic process in the deep veins was diagnosed at the end of the second day of the postoperative period. in 22 (20.76%) patients thrombotic process was recorded on the 3rd day after surgery, in 36 (33.96%) patients – on the 4th day, in 29 (27.36%) observations – on the 5th day, 15 (14,15%) patients – on the 6-7th day of the postoperative period. postoperative varicothrombophlebitis in 4 cases was diagnosed on day 4 after surgery, in 7 cases – on day 5 and in 1 case – on day 6 of the postoperative period. at the ultrasonoelastography of the flotation segment of the ileum-femoral venous thrombus, the acoustic wave propagation velocity was 2.5­2.6 m/s (1 observation), the flotation seg­ ment of the common femoral vein was 2.52.6 m/s (4 observations). the proximal segment of 2.6 to 2.9 cm in length of the femoropopliteal venous thrombus was characterized by the acoustic wave propagation velocity at the level of 2.7-2.8 m/s (3 out of 45 observations). thrombosis of soleus and fibular sinuses with continuation into the popliteal vein was characterized by a velocity of acoustic wave propagation of 2.5–2.6 m/s (2 observations). the proximal segment with a length of 1.21.5 cm of the tibia-popliteal thrombus was characterized by a velocity of acoustic wave propagation within the limits of 2.7-2.8 m/s (2 out of 49 observations). embolic forms of postoperative deep vein thrombosis in ivc system were diagnosed on the 3rd (2 observations), 4th (7 cases) and 5th (3 observations) postoperative days. embolic dangerous postoperative thrombi of the deep veins of ivc system in 7 cases were found in patients after surgical interventions on the musculoskeletal system, in 4 cases – after surgery on the abdominal organs and in 1 case – after reconstructive surgery on the aorta and the main arteries of the lower limbs. in 7 cases at ultrasonoelastography of the postoperative venous thrombus of deep vein, the propagation velocity of the acoustic wave was 2.5-2.6 m/s, which indicated a high risk of embolism of the thrombus. in 5 cases at ultrasonogastography of a venous thrombus, the acoustic wave propagation velocity was 2.7-2.8 m/s, a moderate risk of embolism of the thrombus. in all 12 (11.32%) cases of embolic forms of deep vein thrombosis, with the aim of preventing pe, urgent surgical procedures were performed. in one of 12 cases of postoperative varicothrombophlebitis on the 4th day after surgery, the proximal segment of the thrombotic process was localized at the level of the saphenofemoral anastomosis. the propagation velocity 58 su r g ic a l d is e a se s issn 2413-6077. ijmmr 2019 vol. 5 issue 2 of the acoustic wave of this segment of phlebothrombosis ranged from 2.7 to 2.8 m/s. operative intervention in the form of a crossectomy and short stripping of a large saphenous vein was performed. 105 patients with postoperative venous thrombosis were prescribed anticoagulant therapy with low molecular weight heparins. discussion 316 (43.35%) patients underwent surgical treatment, according to the j. caprini scale these patients had a very high risk of developing thromboembolic complications, 413 (56.65%) patients had a high risk of developing thromboembolic complications. thromboprophylaxis at surgical interventions was carried out in accor dance with the provisions of the accp [7]. in 118 (16.19%) cases, the development of postoperative venous thrombosis was established. studies by several authors [8,9] state that, despite all the efforts of thromboprophylaxis measures, postoperative venous thrombosis in 5–10% is a source of pe that is fatal in 0,3–3,7% [10]. one of the actual tasks of ultrasound diagnosis at acute venous thrombosis is a visual assessment of the substrate of the disease. during the ultrasound examination, localization, the prevalence of the thrombotic process, and the shape of the apex of thrombotic masses were established [11]. the greatest danger in the development of pe is caused by two types of embolic venous thrombi: segmental floating and widespread occlusive thrombi with a floating tip. when they are detected, indications for the operative treatment of avt become obligatory [12]. ultrasound examination in patients with suspected avt allows the diagnostics primarily at typical manifestations of the disease [10, 11]. diagnostic errors may occur in the presence of fresh thrombotic masses that are not fixed to the venous wall, when the thrombotic process spreads from the veins of the tibia to the popliteal vein, with the dissemination of the thrombotic process from the soleus and peroneal sinuses into the popliteal vein. these examples of the thrombotic process in the venous system are dangerous in terms of the development of venous thromboembolic complications [8; 12]. to establish the embolism of thrombus, a technique of determining the density of thrombotic masses is used [7]. the method does not allow to reliably estimate the results of the study since in addition to venous thrombosis the surrounding tissues are placed in the research interest zone. a more objective and reliable method of diagnosing an embolic thrombus is the tech nique for determining the speed of propagation of an acoustic wave in a thrombotic mass by the so noelastography system siemens acuson s2000 (germany). at a speed of acoustic wave propagation in the range 2.5-2.6 m/s there is a high risk of embolism, at a speed within 2.7-2.9 m/s – moderate risk of embolism, at a speed of 3.0 m/s and higher there is no threat of embolism. in 7 cases, the acoustic wave propagation velocity was established at a level of 2.5-2.6 m/s, which indicated a high risk of embolism. in 5 cases, it was determined that the acoustic wave propagation velocity was 2.7-2.8 m/s – moderate risk of embolism of the thrombus. in all 12 (11.32%) cases of embolic forms of postoperative venous thrombosis, with the aim of preventing pe, urgent surgical procedures were performed in order to prevent pe. a sonoelastographic method of determining the embolism of postoperative venous thrombosis was used, and when it was established, urgent surgical procedures were performed. it was possible to prevent the development of pe after surgical treatment in 729 patients with a very high (43.35 %) and high (56.64 %) risk of developing tromoboembolic complications. conclusions pulmonary embolism is one of the most common causes of death from cardiovascular disease. clinical assessment using ultrasonoelastography helps to identify patients with clinical probability of venous thromboembolism. the results of our research demonstrated that embolodengerous thrombi of the proximal segments of the venous thrombus are characterized by the acoustic wave propagation velocity within 2.5-2.8 m/s. the detection of embolic venous thrombosis is an indication for conducting surgical methods for the prevention of pulmonary embolism. funding this research received no external funding. conflict of interests the authors declare no conflict of interest. author contributions kostiv s.ya. – data curation, methodology, writing – review and editing; khvalyboha d.v. – formal analysis, investigation, resources, writing – original draft; venher i.k. – conceptualization, supervision; zarudna o. i. – validation; kostiv o.i. – software. s.ya. kostiv et al. 59 su r g ic a l d is e a se s issn 2413-6077. ijmmr 2019 vol. 5 issue 2 s.ya. kostiv et al. ультразвукова тромбоеластографія у виборі лікування хворих з післяопераційним венозним тромбозом с. я. костів, д. в. хвалибога, і. к. венгер, о. і. зарудна, о. і. костів тернопільський національний медичний університет імені і. я. горбачевського моз україни вступ. частота венозних тромбоемболічних ускладнень в хірургічній практиці досить висока. у багатьох випадках це причина летальної тромбоемболії легеневої артерії. одним з актуальних завдань ультразвукової діагностики гострого венозного тромбозу є візуальна оцінка субстрату захворювання, оскільки він визначає ангіохірургічну тактику та хірургічну профілактику тромбоемболії легень. метою дослідження було запобігти розвитку тромбоемболії легеневої артерії у пацієнтів із післяопераційним венозним тромбозом системи нижньої порожнистої вени. методи дослідження. дослідження системи порожнистої вени та визначення соноеластографічних властивостей венозного тромбу проводили за допомогою ультразвукової системи siemens acuson s2000. встановлено локалізацію та поширеність тромботичного процесу. наприкінці актуального діагнозу венозного тромбу вивчали соноеластографічні властивості тромбу шляхом визначення швидкості поширення акустичної хвилі. результати й обговорення. робота заснована на результатах обстеження та хірургічного лікування 729 пацієнтів, з них 205 (28,12%) оперативних втручань на опорно-руховому апараті, 378 (51,85%) – на органах черевної порожнини, 146 (20,01%) – реконструктивна хірургія на аорті та основних артеріях нижніх кінцівок. висновки. емболонебезпечні тромби – це венозні тромби системи нижньої порожнистої вени, які при ультрасоноеластографії проксимальних сегментів венозного тромбу характеризуються швидкістю поширення акустичної хвилі в межах 2,5–2,8 м / с. виявлення емболічного венозного тромбозу є показанням до хірургічних методів профілактики тромбоемболії легеневої артерії. ключові слова: тромбоемболія легеневої артерії; післяопераційний тромбоз глибоких вен; нижня порожниста вена. відомості про авторів костів с. я. – доктор медичних наук, професор, професор кафедри хірургії № 2, тернопільський національний медичний університет імені і. я. горбачевського моз україни, м. тернопіль хвалибога д. в. – аспірант кафедра хірургії № 2, тернопільський національний медичний університет імені і. я. горбачевського моз україни, м. тернопіль венгер і. к. – доктор медичних наук, професор, завідувач кафедри хірургії № 2, тернопільський національний медичний університет імені і. я. горбачевського моз україни, м. тернопіль зарудна о. і. – кандидат медичний наук, доцент, доцент кафедри клінічної імунології, алергології та загального догляду за хворими, тернопільський національний медичний університет імені і. я. гор­ бачевського моз україни, м. тернопіль костів о. і. – кандидат медичний наук, доцент, доцент кафедри анестезіології та інтенсивної терапії, тернопільський національний медичний університет імені і. я. горбачевського моз україни, м. тернопіль information about the authors kostiv s. ya. – md, ph.d., dsc, professor of the department of surgery № 2, i. horbachevsky ternopil national medical university, ternopil, ukraine. orcid 0000­0002­7963­5425, e­mail: kostivsj@tdmu.edu.ua khvalyboha d. v. – postgraduate student of the department of surgery № 2, i. horbachevsky ternopil national medical university, ternopil, ukraine. orcid 0000­0003­0168­0848, e­mail: varjh2@gmail.com venher i. k. – dsc, md, ph.d., professor, head of the department of surgery № 2, i. horbachevsky ternopil national medical university, ternopil, ukraine. orcid 0000­0003­0170­1995, e­mail: vengerik@tdmu.edu.ua zarudna o. i. – md, ph.d., associate professor of the department of clinical immunology, allergology and general patient care, i. horbachevsky ternopil national medical university, ternopil, ukraine. orcid 0000­0002­9374­3991, e­mail: zarudna@tdmu.edu.ua kostiv o. i. – md, ph.d., associate professor of the department of anesthesiology and intensive care, i. horbachevsky ternopil national medical university, ternopil, ukraine. orcid 0000­0001­5421­9532, e­mail: kostivo@tdmu.edu.ua 60 su r g ic a l d is e a se s issn 2413-6077. ijmmr 2019 vol. 5 issue 2 references 1. cunningham r, murray a, byrne js, hammond l, barry m, mehigan d, sheehan s. venous thromboembolism prophylaxis guideline compliance: a pilot study of augmented medication charts. irish journal of medical science. 2015 jun 1;184(2):469­74. doi: 10.1007/s11845­014­1148­6 2. decousus h, quéré i, presles e, becker f, barrellier mt, chanut m, gillet jl, guenneguez h, leandri c, mismetti p, pichot o. superficial venous thrombosis and venous thromboembolism: a large, prospective epidemiologic study. annals of internal medicine. 2010 feb 16;152(4):218­24. doi: 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[in ukrainian]. received 09 september 2019; revised 17 october 2019; accepted 28 october 2019. this is open access article distributed under the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. s.ya. kostiv et al. 37 in t e r n a l m e d ic in e issn 2413-6077. ijmmr 2020 vol. 6 issue 2 doi 10.11603/ijmmr.2413-6077.2020.2.12012 endothelial dysfunction and its management in patients with acute myocardial infarction combined with metabolic syndrome i.o. yastremska i. horbachevsky ternopil national medical university, ternopil, ukraine background. oxidative stress, endothelial dysfunction, dyslipidemia and low-grade inflammation induce the disorders of energy metabolism and ischemic damage to cardiomyocytes. it is an essential issue in pathogenesis of acute coronary syndrome/myocardial infarction (acs/mi). objective. the aim of the study was to improve the existing pharmacological therapy in patients with acs/ mi combined with metabolic syndrome (ms). methods. the study enrolled 95 patients with acute myocardial infarction. the patients were divided into 2 groups depending on concomitant metabolic syndrome. all groups were divided to subgroups, where patients received typical standard treatment according to the ukrainian unified and modified treatment regimen with addition of l-arginine and l-carnitine. results. in 79.2% of patients with acs/mi + ms the course of underlying disease was associated with various complications: pericarditis epistenocardica was diagnosed in 39.8% of patients; arrhythmias were present in 35.5% of patients of the main group; left ventricular aneurysm was present in 15.9% of patients. at the same time, significant changes in the indicators of vascular endothelial function in patients with acs (mi) were revealed (the level of endothelin-1 in the blood plasma was in 2.1 times higher than the reference norm) that was the justification for inclusion of l-arginine and l-carnitine in the complex therapy of comorbid patients. conclusions. the multi-modality treatment with inclusion of l-arginine and l-carnitine facilitated restoration of energy supply of myocardial contractility, endothelial function of blood vessels, and antioxidant protection of the body and ultimately resulted in a more favorable course of this comorbid problem. keywords: myocardial infarction; metabolic syndrome; endothelial dysfunction; l-arginine; l-carnitine. corresponding author: yastremska iryna, phd student of the department of emergency and medical care, i. horbachevsky ternopil national medical university, ternopil, ukraine. e-mail: yastremskaio@tdmu.edu.ua international journal of medicine and medical research 2020, volume 6, issue 2, p. 37-43 copyright © 2020, tnmu, all rights reserved i.o. yastremska introduction the rupture of the atheromatous plaque and formation of coronary clot, which causes progressive stenosis, is considered the main cause of acute myocardial infarction (mi). however, the recently discussed pathogenetic factors include peroxide stress and endothelial dysfunction, dyslipidemia and low-intensity systemic inflammation underlying the disorders of energy metabolism and ischemic damage to cardiomyocytes. the aforementioned pathogenetic mechanism behind the development of acute coronary syndrome/myocardial infarction (acs/mi) provides a possibility of management of these pathogenetic processes with metabolic and cytoprotective pharmacological therapy. in addition to that, the main risk factors for coronary heart disease and myocardial infarction as its life-threatening manifestation are obesity, hypertension, dyslipidemia, insulin resistance and diabetes, which as a whole can be components of metabolic syndrome (ms), the latter diagnosed in 28-35% patients with myocardial infarction [1, 4, 13]. the causes of ms, including insulin resistance, hyperinsulinemia and chronic inflammation simultaneously trigger and maintain high levels of atherogenesis, endothelial dysfunction and evoke coronary plaque instability as well as processes of thrombogenesis [6, 7, 8]. that is why a differentiated approach to treatment of patients with acs/mi (with due consideration for comorbidities) is a priority at present stage. both l-carnitine and l-arginine have been proved to be very promising metabolic drug products, which occur naturally in human body. they manifest their effects as active regulators of intermediary metabolism and energy-supplying processes [7, 12, 14]. however, their main physiological role includes 38 in t e r n a l m e d ic in e issn 2413-6077. ijmmr 2020 vol. 6 issue 2 regulation of functional state of blood vessels and maintaining adequate microcirculation in the organs and tissues of the body [9, 11]. the above facts have become a rationale for studies of clinical efficacy and possibilities for correction of metabolic and endothelial disorders in patients with acs/mi. the aim of the study was to improve the existing therapeutic programs for acs/mi combined with metabolic syndrome (ms). methods the study enrolled 95 patients with acute myocardial infarction. the patients were divided into 2 groups depending on whether they had metabolic syndrome. the main study group involved 53 patients with acs/mi+ms. the control group included 42 patients with acs/mi, who were not diagnosed with ms; 32 patients in the main group and 22 patients in the control group received standard treatment for myocardial infarction according to the unified protocols of the ministry of health of ukraine [5]. other 21 patients in the main group and 20 patients in the control group received a modified treatment regimen with addition of l-arginine 4.2 g and l-carnitine 2.0 g supplied as solution for infusions 100 ml administered intravenously once a day as a 5-day course. most of study participants were males (87.5%) of productive age, 57.54±8.02 years old on average. the diagnosis of acute mi was verified according to the 2017 esc guidelines [2] in the presence of a typical anginal attack, mi-specific ecg changes with time (reciprocal st displacement) and the signs of resorption-necrosis syndrome. the diagnosis was confirmed by means of laboratory tests, ecg and imaging tests. quantitative determination of troponin t was performed using an electro chemiluminescent biochemical analyzer elecsys 2010 by roche/нitachi (switzerland). troponin test results above the reference value of 14.0 ng/ml were assessed as positive. patient assessments included general clinical examination, laboratory tests (hematology, mb fraction of creatine phosphokinase (cpkmb), troponin t, oxygen saturation of arterial blood (spo2), ecg in 12 standard leads, etc. and b-mode cardiac ultrasound imaging on aloka ssd – 2000 unit (usa) with determination of linear and volumetric parameters of the left ventricle, as well as global (assessed by ejection fraction, ef) and local myocardial contractility using the simpson’s biplane method. the diastolic function of the left ventricle was assessed by the isovolumic relaxation time (ivrt) of the left ventricle, delay time for early left ventricular filling (dt), maximal early filling velocity (е) and maximal atrial systole filling velocity of the left ventricle (а). plasma level of endothelin-1 (ет-1) was evaluated by the elisa kits by amersham pharmacia biotech; the stable no metabolites content was determined by the griess reagent reaction [2]. the number of nitrites was measured using a calibration graph. the abovementioned tests were repeated on day 14 and day 28 and tracked as they changed with time. statistical data analysis was performed using the statistica statistical package (by statsoft, usa, v 6.0). results uncomplicated course of myocardial infarction was established in 17 patients (40%) of the control groups. in 42 patients with mi+ms (79.2%), the course of their underlying disease was accompanied by various complications. thus, the patients of the test group were substantially more likely to develop pericarditis epistenocardica (21 (39.8%) patients) than those in the control group (8 (19%) patients). conduction disorders or arrhythmias mani fested as paroxysmal tachyarrhythmia, transient atrioventricular block and bundle branch blocks; extrasystole arrhythmia were also more frequent in the patients of the main group (78.5%) and significantly less frequent in the patients with mi without ms (52%). these results are presented in fig. 1. acute left ventricular failure accompanied the course of mi in all patients of both groups; however, killip i and killip ii acute heart failure (according to the killip-kimball classification, 1972) was substantially more frequent in the patients of the control group. impaired systolic and diastolic functions of the left ventricle were simultaneously observed in the patients of the main group and the control group. that is, there was a significantly more significant reduction in myocardial contractility (ef=42.12±1.13%) compared to the control group (ef=48.23±1.16%). these changes were closely related to the more pronounced processes of left ventricular remodeling during the acute period of myocardial infarction in the patients of the main group. in particular, the increase in left ventricular internal diastolic dimension (the left ventricular enddiastolic diameter, lvedd) was 5.62±0.21 cm in the main group and 4.71±0.24 cm in the control group (р<0.05). the developing diastolic dysfunction was suggested by changes in the i.o. yastremska 39 in t e r n a l m e d ic in e issn 2413-6077. ijmmr 2020 vol. 6 issue 2 isovolumic relaxation times (ivrt) in the main group (63.23±0.41 msec) and the control group (68.12±0.36 msec), as well as by delay time of early diastolic transmitral flow (dt) in the compared groups (178.11±0.54 msec and 182.43±0.37 msec, respectively). after completion of a standard mi treatment, the patients of the control group experienced significant improvement of hemodynamic parameters. relatively, there was an increase in left ventricular ejection fraction, a reduction in lvedd and improvement in left ventricular diastolic function parameters, i.e. ivrt and dt, compared to the mi+ms patients (р<0.05). however, the patients with myocardial infarction combined with ms experienced more frequent complications in a setting of standard treatment. this is because of special characteristics of carbohydrate metabolism in the patients with ms, which are manifested by insulin resistance and compensatory hyperinsulinemia (which activates the sympathoadrenal system), as well as by a combination of this pathogenetic mechanism with other pathogenetic factors of ischemia (an increase in lipid peroxidation and endothelial dysfunction) that is accompanied by a more prolonged activation of oxidative processes and inhibited activity of enzymes in the antioxidant protection system. the abnormal processes in the heart enhanced causing a significant reduction of myocardial contractility and substantial changes in electrolyte stability of the heart that contributed to life-threatening disorders of rhythm and conductivity against the backdrop of unstable hemodynamics [9, 11]. as a consequence, l-arginine and l-carnitine are promising treatments. in addition to their pronounced metabolic and energy-producing activity, these substances restore endothelial function of blood vessels, exert antioxidant effects and prevent irreversible ischemic and reperfusion damage, limit the zone of myocardial necrosis, improve cellular adjustment to functioning under hypoxia and inhibit abnormal cardiac remodeling. one of the mechanisms behind such therapeutic effect is the ability of l-arginine and l-carnitine to inhibit the formation of radicals during breakdown of fatty acids thereby reduce the damaging effects of peroxidation byproducts on the functional status of ion channels in the cells [1, 3, 17]. thus, the results of this study have demonstrated that a multi-modality therapeutic program with inclusion of l-arginine and l-carnitine in the patients of the main group had a substantial effect on restoration of systolic-diastolic function of the heart: the parameters of systolic and diastolic function of the heart returned to normal already within 28 days of treatment. that is, the linear dimensions of cardiac cavities changed insignificantly; thus, we may assume that the positive changes in ef, lvedd, ivrt and dt compared to their respective baseline levels were driven by remodeling and restoration of functional status in zones of myocardial ischemia and hibernation [10]. fig 1. frequency of arrhythmias and conduction disorders in the patients with acute coronary syndrome (myocardial infarction). 16 27 30 3 5 11 5 4 10 9 1 1 2 1 0 5 10 15 20 25 30 35 av blockade supraventricular extrasystole sinus tachycardia sinus bradycardia ventricular fibrillation paroxysm of atrial fibrillation ventricular tachycardia control group experimental group i.o. yastremska 40 in t e r n a l m e d ic in e issn 2413-6077. ijmmr 2020 vol. 6 issue 2 the changes in the parameters of endothelial function of blood vessels were also assessed in the patients with acs/mi as part of the study. the baseline levels of these parameters did not differ in between. however, they were significantly changed compared to the reference values. thus, plasma levels of endothelin-1 in the patients with acs/mi at hospitalization were in 2.1 times above the reference value (0.96±0.04 ng/ml and 0.46±0.03 ng/ml, respectively). there were no significant changes in endothelin-1 (ет-1) activity immediately after emergency angioplasty of the coronary vessel and its stenting. that is, the levels of endothelin were substantially reduced in the control group patients who received per-protocol pharmacological treatment. however, by the end of the in-patient treatment period, the levels of endothelin have not reached those of healthy individuals (0.66±0.06; р>0.05). in contrast, the patients of the test group, whose multi-modality treatment included the course of therapy with l-arginine and l-carnitine during the next 10 days of the in-patient treatment period, had a 33.3% reduction in ет-1 activity (р<0.05), and after one month of treatment its plasma levels significantly decreased by additional 43% and reached the level of healthy individuals (0.52±0.05 ng/ml; р>0.05). hence, the use of combination drug therapy with inclusion of l-arginine and l-carnitine in patients with acs/mi provided for a rapid (over 10 days) reduction and complete restoration (over 28 days) of plasma endothelin-1 levels in these patients. along with changes in endothelin-1 activity in patients with acs/mi during exacerbation of the disease, there was an abrupt reduction in metabolites of nitric oxide (noε=17.75±0.42 μmol/l, the normal level of 36.92±0.37 μmol/l), which could suggest pronounced microcirculatory disorders in these patients. thus, there was an almost two-fold baseline decrease in plasma levels of nitrates and nitrites in the patients of both groups, i. e. their total plasma levels were reduced by 45.0%. however, conventional per-protocol treatment did not lead to complete restoration of endothelial function of blood vessels in this group of patients with acs/mi; the total plasma level of metabolites of nitric oxide in these patients was by 19% below the reference value (р<0.05). at the same time, the use of combination drug therapy with inclusion of l-arginine and l-carnitine had a substantial effect on plasma levels of nitrites and nitrates in the patients of the test group. the levels of these substances were significantly increased already before 10 days of treatment; after completion of the in-patient phase of combination drug therapy they reached the reference level. discussion therefore, since patients with acs/mi + ms were diagnosed with pronounced baseline changes of morphological and functional parameters of the heart, post-infarction remodeling with compromised systolic and diastolic functions of the heart, as well as development of heart failure and endothelial dysfunction (which were retained immediately after emergency percutaneous procedures), these findings provided a rationale for inclusion of parenteral l-arginine and l-carnitine to the per-protocol therapeutic program. standard therapy was established to lack sufficient hemodynamic efficacy in this patient cohort. only enhancing standard therapy by adding l-arginine and l-carnitine caused significant ef and dt increases, lvedd reduction and e/a ratio reduction and improved post-infarction cardiac remodeling, ultimately evident as a significant improvement of ef, myocardial contractility and diastolic dysfunction. in our opinion, the positive effect of the suggested treatment for inotropic heart function and a significant reduction in incidence and severity of reperfusion arrhythmias were achieved precisely by means of the cardiometabolic effect of l-carnitine, which, as reported by many researchers, is significant in energy metabolism in the myocardium by transferring free fatty acids from the cytosol inside the mitochondria thereby ensuring bio-availability of the highenergy substrate for oxidative metabolism in the cardiomyocyte [8, 16]. in addition, by facilitating oxidation of long-chain fatty acids and by modulating the coa/coa-sh ratio, this compound is taking part in binding of acyl residues in peroxisomes and mitochondria and has a positive effect on amino acid metabolism by assimilating the pool of free radical compounds. this ensures stabilization of organelles and cellular membranes and prevents accumulation of fatty acid esters in the cytoplasm of cardiomyocytes, which may lead to fatal ventricular arrhythmias [15]. during the study it was also established that patients of the test group, whose multi-modality treatment included additional l-arginine and l-carnitine, had significantly lower ет-1 activity, while plasma levels of nitric oxide metabolites increased reai.o. yastremska 41 in t e r n a l m e d ic in e issn 2413-6077. ijmmr 2020 vol. 6 issue 2 ching the levels of healthy individuals (р>0.05). in other words, such multi-modality treatment in patients with acs/mi contributed to a rapid and complete recovery of the study parameters of endothelial function of blood vessels. the treatment outcomes are associated with the use of l-arginine as a principal substrate for the synthesis of nitric oxide. the fundamental physiological role of nitric oxide is to regulate the functional status of blood vessels and to provide an adequate level of microcirculation in organs and tissues of the body [9, 11, 14]. thus, the conclusion may be drawn that the presence of a concomitant metabolic syndrome has a substantial impact on the course of acute myocardial infarction and is accompanied by a significantly higher complication rate. these patients have more severe disorders in the lipid peroxidation system and a reduced activity of antioxidant defense, which disrupts endothelial functions of blood vessels and causes deterioration of microcirculation. at the same time, significant reductions in systolic and diastolic functions of the myocardium have been observed in patients with mi+ms as a result of impaired remodeling of cardiac chambers and changes in their linear and geometric parameters. the combination treatment of mi+ms patients with inclusion of l-arginine and l-carnitine facilitates restoration of energy supply of myocardial contractility, endothelial function of blood vessels and antioxidant defense of the body, which results in a more favorable course of this comorbid problem. conclusions the patients with myocardial infarction combined with metabolic syndrome were likely to develop disorders of central and peripheral hemodynamics and endothelial function of blood vessels. these circumstances significantly exaggerate the clinical course of the underlying morbidity, additionally enhance the disorders of systolic and diastolic cardiac function and cause more frequent complications of acs/mi. a comprehensive therapeutic program for patients with acs/mi+ms enhanced by inclusion of l-arginine and l-carnitine facilitates restoration of endothelial function of blood vessels and antioxidant defense of the body and increases the energy supply of cardiomyocytes, which is accompanied by improved myocardial contractility. acknowledgements the author acknowledges mykola shved (md, ph.d., dsc, professor) for assistance in preparation of this article. funding this research received no external funding. conflict of interests the author declares no conflict of interest. ендотеліальна дисфункція та шляхи її корекції у хворих на гострий інфаркт міокарда в поєднанні з метаболічним синдромом. ястремська і.о. тернопільський національний медичний університет імені і.я. горбачевського, тернопіль, україна вступ. окислювальний стрес, дисфункція ендотелію, дисліпідемія та низькоінтенсивне запалення спричиняють порушення енергетичного обміну та ішемічне ураження кардіоміоцитів, що є важливою ланкою у патогенезі гострого коронарного синдрому / інфаркту міокарда (гкс / ім). мета дослідження – удосконалити існуючі лікувальні програми гкс(ім) у поєднанні з метаболічним синдромом (мс). методи. у дослідженні взяли участь 95 пацієнтів з гострим інфарктом міокарда. пацієнтів розподілили на 2 групи залежно від наявності супутнього метаболічного синдрому. усі групи були розділені на підгрупи, де пацієнти отримували стандартне протокольне лікування згідно уніфікованого протоколу моз україни та модифікована схеми лікування з додаванням l-аргініну та l-карнітину. результати дослідження. у хворих на ім із супутнім мс основне захворювання перебігало з різними ускладненнями у 79,2 %: епістенокардитичний перикардит був у 39,8 %; порушення ритму або i.o. yastremska 42 in t e r n a l m e d ic in e issn 2413-6077. ijmmr 2020 vol. 6 issue 2 провідності наявні у 35,5 %; аневризма лівого шлуночка – у 15,9 % хворих. одночасно виявлено суттєві зміни у показниках ендотеліальної функції судин у хворих на гкс(ім), що стало обґрунтуванням для включення в комплексну терапію коморбідних хворих курсу l-аргініну та l-карнітину. висновки. комплексне лікування хворих на ім в поєднанні з мс із включеням l-аргініну та l-карнітину сприяло відновленню енергозабезпечення скоротливості міокарда, ендотеліальної функції судин, антиоксидантного захисту організму, що в результаті забезпечувало більш сприятливий перебіг даної коморбідної патології. ключові слова: інфаркт міокарда; метаболічний синдром; ендотеліальна дисфункція; l-аргінін; l-карнітин. information about the authors yastremska iryna – phd student of the department of emergency and medical care, i. horbachevsky ternopil national medical university, ternopil, ukraine. orcid: 0000-0001-6884-6705, e-mail: yastremskaio@tdmu.edu.ua references 1. batushkin vv. [cytoprotection in acute myocardial infarction after the discovery of infarctdependent artery: new horizons]. liky ukrainy. 2017;8(214):33–40. (in ukrainian) 2. thiele h, desch s, de waha s. acute myocardial infarction in patients with st-segment elevation myocardial infarction: esc guidelines 2017. herz. 2017 dec 1;42(8):728-38. 3. parkhomenko om, kozhukhov sn, lutay yam. [rationale and design of multicenter randomized study protect – study of the effectiveness and safety of quercetin in patients with acute myocardial infarction]. ukrainian journal of cardiology. 2016;(3): 31–6. (in ukrainian) 4. selyuk mm, kozachok mm, levkin im, selyuk ov. selection of the optimal combination of metabolic drugs for the treatment of patients with cardiovascular pathology. family medicine. 2017; 2(70):60–4. (in ukrainian) 5. acute coronary syndrome with st segment elevation: unified clinical protocol of emergency, primary, secondary and tertiary care. – ministry of health of ukraine. 2017: 78. (in ukrainian) 6. shved mi, tsuglevich lv, geryak sm. clinical efficacy of cardiocytoprotective therapy in patients with acute coronary syndrome (myocardial infarction), who underwent balloon angioplasty and coronary artery stenting. archive of clinical medicine.2019;(1):31-7. doi: https://doi.org/10.21802/acm.2019.1.2 7. astashkin ei, glezer mg. role of l-carnitine in energy metabolism cardiomyocytes and treatment of diseases of cardiovascular system. cardiology and cardiovascular surgery. 2012;6(2):58-65. 8. dinicolantonio jj, lavie cj, fares h, menezes ar, o'keefe jh. l-carnitine in the secondary prevention of cardiovascular disease: systematic review and meta-analysis. in mayo clinic proceedings 2013 jun 1 (vol. 88, no. 6, pp. 544-551). elsevier. 9. celermajer ds. endothelial dysfunction: does it matter? is it reversible?. journal of the american college of cardiology. 1997 aug;30(2):325-33. doi: https://doi.org/10.1016/s0735-1097(97) 00189-7 10. colonna p, iliceto s. myocardial infarction and left ventricular remodeling: results of the cedim trial. american heart journal. 2000 feb 1;139(2): s124-30. doi: https://doi.org/10.1067/mhj.2000.103918 11. haynes wg, webb dj. endothelin as a regulator of cardiovascular function in health and disease. journal of hypertension. 1998 aug 1;16(8):1081-98. d o i : h t t p s : / / d o i . o r g / 1 0 . 1 0 9 7 / 0 0 0 0 4 8 7 2 199816080-00001 12. george j, shmuel sb, roth a. l-arginine attenuates lymphocyte activation and antioxidized ldl antibody levels in patients undergoing angioplasty. atherosclerosis. 2004;174: 323-327. doi: https://doi.org/10.1016/j.atheroscle rosis. 2004.01.025 13. ibanez b, james s, agewall s. 2017 esc guidelines for the management of acute myocardial infarction in patients presenting with st-segment elevation: the task force for the management of i.o. yastremska 43 in t e r n a l m e d ic in e issn 2413-6077. ijmmr 2020 vol. 6 issue 2 acute myocardial infarction in patients presenting with st-segment elevation. eur heart j. 2018;2: 119-177. doi: https://doi.org/10.1093/eurheartj/ehx393. 14. opie lh. role of carnitine in fatty acid metabolism of normal and ischemic myocardium. am heart j. 1997;97 (3):375-88. doi: https://doi.org/10.1016/0002-8703(79) 90440-x 15. rizzon p, biasko g, biase mdi. high doses of l-carnitine in acute myocardial infarction: metabolic and antiarrhythmic effects. eur heart j.1989;10 (6):502-8. doi: https://doi.org/10.1093/oxfordjournals. eurheartj.a059519 16. shug a, thomsen j, folts j. changes in tissue levels of carnitine and other metabolites during myocardial ischemia and anoxia. arch biochem biophys. 1998;187(1):25-33. doi: https://doi.org/10.1016/0003-9861(78) 90003-6 17. hicks jj, montescortes dh, cruz-dominguez mp. antioxidants decrease reperfusion induced arrhythmias in myocardial infarction with st-elevation. frontiers in bioscience. 2007;12:2029-37. doi: https://doi.org/10.2741/2208 received 7 oct 2020; revised 11 nov 2020; accepted 1 dec 2020. this is open access article distributed under the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. i.o. yastremska 81 p h a r m a c y issn 2413-6077. ijmmr 2021 vol. 7 issue 1 doi 10.11603/ijmmr.2413-6077.2021.1.12100 influence of thick extract from maitake mushrooms on signs of inflammatory process in experimental toxic hepatitis *i.i. herasymets, l.s. fira, i.i. medvid i. horbachevsky ternopil national medical university, ternopil, ukraine background. the priority of the contemporary pharmaceutical industry is to create effective, safe and inexpensive drugs to ensure the highest quality of care and optimal use of available raw materials. objective. the aim of our study was to investigate anti-inflammatory properties of the maitake mushrooms thick extract in the experiment on rats with paracetamol(acetaminophen)-induced hepatitis. methods. 60 white male rats, weighing 180-210 g, randomized into 10 groups of 6 animals in each, were used for the experiment. paracetamol hepatitis was simulated by acetaminophen intragastric administering in a dose of 1250 mg/kg 1 time per day (for 2 days) as a suspension in 2% starch gel solution. maitake mushrooms thick extract, which was administered intragastrically 2 hours before the administration of acetaminophen and daily after the lesion in a dose of 150 mg/kg of the animal’s body weight, was used for the toxic lesion correction. “silibor” was selected as the comparison drug, which was administered according to the same scheme as the investigated extract in a dose of 20 mg/kg of the animal’s body weight. euthanasia was conducted on the 3rd, 7th and 10th day of the experiment with sodium barbamyl. liver homogenate and animal serum were used for the studies. the development of inflammatory processes was studied by the content of pro-inflammatory and antiinflammatory cytokines, as well as c-reactive protein in the serum of rats with toxic hepatitis and after the application of maitake mushroom extract and the comparison drug. results. it was found that the introduction of acetaminophen to animals for the acute hepatitis simulation is accompanied by changes in the cytokine profile, i.e. an increase in the level of il-6 and a decrease in the level of il-4 in the serum of rats. inflammatory development is evidenced by the content of c-reactive protein increase in the blood of the affected animals. the application of maitake mushroom extract facilitated bringing the studied indicators almost to the level of intact control. conclusions. reduction of inflammation signs in rats with the simulated paracetamol hepatitis under the influence of maitake mushrooms thick extract confirms its anti-inflammatory properties. keywords: maitake mushrooms, paracetamol, acute hepatitis, inflammation. *corresponding author: iryna herasymets, associate professor, i. horbachevsky ternopil national medical university, ternopil, 46002, ukraine. e-mail: irunaherasymets@gmail.com international journal of medicine and medical research 2021, volume 7, issue 1, p. 81-86 copyright © 2021, tnmu, all rights reserved introduction due to the serious consequences of hepatitis, there is a need for its earliest diagnosis and appropriate pharmacotherapy. the search for effective hepatoprotectors, which can influence the initial stages of initiation and development of inflammatory processes in the liver without side effects, is an important task of contemporary medicine [1, 2] maitake mushroom has properties for which the japanese have valued it for centuries: the ability to lose weight, to reduce discomfort and problems associated with menopause in women, to gently eliminate the unpleasant sensations of premenstrual syndrome, to lower blood sugar level, to reduce the effects of inflammation, to increase immunity due to b polysaccharides, available in the composition of the fungus [3, 4, 5, 6, 7, 8, 9]. the aim of our study was to investigate the anti-inflammatory properties of the maitake mushrooms thick extract in the experiment on rats with acute hepatitis induced by paracetamol (acetaminophen). methods the material for the experimental work was a thick extract of maitake mushrooms (teomm), obtained by scientists from the department of chemistry of natural compounds of the national university of pharmacy. the experiments were performed on white outbred male rats, weighing 180-210 g, kept on the standard diet of the vivarium of i. horbachevsky ternopil national medical university. all studies were conducted in compliance with the rules of bioethics in accordance with the i.i. herasymets et al. 82 p h a r m a c y issn 2413-6077. ijmmr 2021 vol. 7 issue 1 “european convention for the protection of vertebrate animals, which are used for experimental and other scientific purposes” [10]. intoxication with acetaminophen, which was administered intragastrically in a dose of 1250 mg/kg 1 time per day for 2 days as a suspension in 2% starch gel solution, was a model of toxic lesions in rats [11, 12]. teomm was administered intragastrically 2 hours before administration of the toxic agent and daily after the lesion in a dose of 150 mg/kg of the animal’s body weight, which, in our previous studies, was found to be conditionally therapeutic for this extract [5]. “silibor” (the active basis is silymarin) was chosen as the comparison drug. it was administered according to the same scheme as the investigated extract in a dose of 20 mg/kg (in terms of silymarin) of the animal’s body weight. the value of the dose of the comparison drug was chosen according to the instructions for its application and using the coefficients of species sensitivity by rybolovlev yu. r. and his method of converting the dose for humans to the dose for rats [12, 13]. the animals were randomized into 10 groups, 6 rats each: the 1st group – the animals of intact control; the 2nd, 3rd and 4th – the animals affected by acetaminophen on the 3rd, 7th and 10th days of the study, respectively; the 5th, 6th and 7th groups – the rats, which were injected with an extract of maitake mushrooms in a dose of 150 mg/kg of body weight on the 3rd, 7th and 10th days of the experiment, respectively; the 8th, 9th and 10th groups of animals that were intragastrally administered with the drug silibor on the 3rd, 7th and 10th days of the study, respectively. the rats were removed from the experiment by euthanasia under sodium barbamyl anesthesia. euthanasia was conducted on the 3rd, 7th and 10th days of the experiment. the study was subjected to liver homogenate and animal serum. blood was taken from the heart of the animals. the concentration of pro-inflammatory and anti-inflammatory interleukins in the serum of rats was determined by enzyme-linked immuno sorbent assay using commercial kits (gehealth care: amersham, uk) [14]. the content of c-reactive protein (crp) was determined by immunoturbidimetric method [15, 16]. the obtained data were statistically processed by the program statistica 12. the significance of intergroup differences was determined using the criterion of rank sums of the wilcoxon and the mann-whitney tests. p values lower than 0.05 were considered to be statistically significant [17, 18]. results c-reactive protein is a non-glycosylated protein with a pentameric structure, which belongs to β-globulins. due to its affinity to phosphorylcholine, which is a component of the cell walls of some bacteria and unicellular fungi, crp is able to bind the relevant microbial cells and opsonize them for phagocytosis or lysis with complement [16, 19]. crp acts as a pro-inflammatory “trigger” that stimulates monocytic synthesis of cytokines such as tumour necrosis factor-α, interleukin-1 and interleukin-6. thus, crp performs an immunoregulatory function: it stimulates protective reactions and activates immunity [14]. crp is determined in almost all pathological processes and diseases regarding tissue damage. the increased levels of c-reactive protein are one of the earliest laboratory signs of inflammation or tissue damage. increased protein production by the liver begins 6 hours after the onset of inflammation [20]. we found out that in rats with acute hepatitis induced by acetaminophen, the serum crp increased in 1.9, 2.4 and 2.7 times on the 3rd, 7th and 10th days of the study, respectively, according to the group of intact control. after correction with teomm there was a probable decrease in the content of crp in the serum of rats in 1.2, 1.5 and 2.1 times on the 3rd, 7th and 10th days of the experiment, respectively, in the animals of control pathology. when using silibor, the studied indicator probably (p≤0.05) decreased in 1.4 and 1.9 times on the 7th and 10th days of the experiment, respectively, for the animals with toxic hepatitis (tab. 1). the obtained results are a confirmation of the inflammatory processes development in the body of rats with acute hepatitis, which is caused by the action of acetaminophen. cytokines, a group of hormone-like proteins, peptides and mediators of inflammation, play a significant role in the pathogenesis of development and course of inflammatory processes. imbalance in the cytokine system can have a significant effect on the course of inflammatory reaction [21]. hepatocytes are very sensitive to the action of cytokines, as they contain a number of specific receptors on their surface, through which the regulation of protein synthesis, proliferation, differentiation, specialized functioning and apoptosis of liver cells is carried out. proi.i. herasymets et al. 83 p h a r m a c y issn 2413-6077. ijmmr 2021 vol. 7 issue 1 inflammatory cytokines: il-1, il-6, il-8, il-12, tnf-, ifn-γ are involved in the specific immune response triggering, while anti-inflammatory cytokines: il-4, il-10, il-13, tgf are involved in the development of reactions of the anti-inflammatory process and inhibit the synthesis of pro-inflammatory interleukins [14, 15, 20, 21]. our further research was to study the content of pro-inflammatory and anti-inflammatory cytokines, il-4 and il-6, in the blood serum of rats with simulated acetaminophen hepatitis, as well as to study the corrective effect of teomm and silibor on them. numerous studies confirm that violation of normal proportions of pro-inflammatory and anti-inflammatory cytokines synthesis can lead to disruption of regulation and development of vital immune reactions and, above all, inflammatory reactions. in the case of violations of lo cal protective reactions, inflammation spreads, cytokine synthesis increases, then they enter the bloodstream and have their effect on the systemic level, i.e. have their effect on almost all organs and systems of the body [19, 20, 22]. it was experimentally established that during the formation of acute hepatitis in rats on the 3rd day of the study, the content of il-6 increased by 82% compare to the intact animals. on the 7th and 10th days of the experiment, the content of the studied indicator increased by 126% and 150%, respectively, compare to the intact control animals (tab. 2). teomm application for correction the detected disorders in toxic hepatitis led to a probable decrease in the content of il-6 on the 7th day of the study compare to control. the comparison drug silibor had a positive effect on this indicator, although slightly inferior to our studied extract. local inflammatory process, after introduction of a pro-inflammatory agent, is important for healing and protection of the body from infection. however, excessive accumulation of pro-inflammatory cytokines in the blood leads to generalized sepsis and multiorgan failure. it is anti-inflammatory interleukins, which include il-4 and il-10, are able to reduce inflammation and cause a cessation of the inflammatory response [14, 15]. hence, it was advisable to study the content of anti-inflammatory cytokines, in particular il-4, in the serum of rats with toxic lesion. after affection of animals with aceta minophen in the serum, the il-4 content decrease in 1.4, 1.5 and 1.6 times was observed on the 3rd, 7th and 10th days of the experiment, respectively, compare to the group of intact control animals (tab. 3). teomm caused a probable increase in the il-4 content by 30% and 46% on the 7th and 10th days of the study, respectively, in the animals with toxic hepatitis. when using the comparison drug, a probable (p≤0.05) increase in the cytokine content on the 7th day in 1.3 times and in 1.4 times on the 10th day of the study compare to the control pathology group was noted. the obtained results suggest that the use of teomm in rats at a dose of 150 mg/kg for 10 table 1. the content of c-reactive protein in the serum (mg/l) of the rats affected by acetaminophen, and after application of the maitake mushrooms extract (m±m; n=60) groups of animals indicators 3rd day 7th day 10th day іc 3.51±0.28 3.51±0.28 3.51±0.28 cp 6.83±0.23* 8.47±0.24* 9.54±0.32* cp+silibor 6.10±0.20 5.93±0.29** 5.08±0.23** cp+teomm 5.71±0.18** 5.63±0.28** 4.49±0.3** notes. here and in the following tables * – probable changes between the rate of the control and paracetamol-affected animals, ** – probable changes between the rates of the paracetamol-affected and treated animals. table 2. the content of il-6 in the serum (pg/l) of the rats affected by paracetamol, and after application of the maitake mushrooms extract (m±m; n=60) groups of animals indicators 3rd day 7th day 10th day іc 2.96±0.22 2.96±0.22 2.96±0.22 cp 5.38±0.30* 6.69±0.34* 7.39±0.27* cp+silibor 4.93±0.21 4.51±0.26** 4.35±0.29** cp+teomm 4.85±0.16 4.22±0.22** 3.98±0.26** i.i. herasymets et al. 84 p h a r m a c y issn 2413-6077. ijmmr 2021 vol. 7 issue 1 days has a positive effect on the initial stages of the inflammatory reaction. discussion the inflammatory process caused by immune mechanisms is significant in the pathogenesis of diseases. crp is a marker of systemic inflammation. therefore, the experimentally revealed increase in its level in the serum of animals with toxic lesion indicates an inflammatory process in the body. crp is one of the activators of the complement system – a compound of complex proteins involved in the formation of the body’s immune response [16]. cytokines are a major factor in the interaction between immune cells and somatic cells. determination of their concentration in the blood provides information about the functional activity of different types of immunocompetent cells, severity of the inflammatory process, its transition to the systemic level, prognosis of the disease. excessive production of cytokines and other mediators of inflammation disrupts regulatory function of the immune system, their uncontrolled release takes place, as well as the imbalance between pro-inflammatory and antiinflammatory cytokines with a predominance of pro-inflammatory. as a result, the mediators of inflammation from the factors that protect the body become damaging for it [15]. the study of the content of the pro-inflammatory cytokine il-6 showed a probable increase throughout the experiment. at the same time, the serum content of the anti-inflammatory cytokine il-4 decreased. this indicates an imbalance of pro-inflammatory and anti-inflammatory cytokines caused by toxic liver damage by acetaminophen. we established an anti-inflammatory effect of the studied extract, which consists in changing the cytokine profile, in particular in reducing the dynamics of the pro-inflammatory il-6 content and increase in the anti-inflammatory cytokine il-4 content compare to the group of intact control rats. conclusions it was established that in cases of acute hepatitis in the rats induced by acetaminophen, the concentration of crp increased in the blood serum, the content of pro-inflammatory il-6 probably increased and the content of antiinflammatory cytokine il-4 decreased, which indicated the development of inflammatory processes in the affected animals. the application of a maitake mushrooms thick extract had a positive effect on the content of c-reactive protein and cytokines in the serum of the animals with acute hepatitis, which indicated its anti-inflammatory properties and the relevance of further study to creation of effective drugs. funding this research received no external funding. conflict of interest the authors declare no conflict of interests in this study. author contributions herasymets i.i. – conceptualization, data curation, formal analysis, investigation, visualization, article writing; fira l.s. – con cep tualization, supervision, validation, methodology; medvid i.i. – formal analysis, investigation, software. table 3. the content of il-4 in the serum (pg/l) of the rats affected by paracetamol, and after application of the maitake mushrooms extract (m±m; n=60) groups of animals indicators 3rd day 7th day 10th day іc 1.57±0.08 1.57±0.08 1.57±0.08 cp 1.14±0.05* 1.03±0.07* 0.96±0.09* cp+silibor 1.22±0.07 1.29±0.08** 1.34±0.06** cp+teomm 1.29±0.05 1.34±0.06** 1.40±0.08** i.i. herasymets et al. 85 p h a r m a c y issn 2413-6077. ijmmr 2021 vol. 7 issue 1 вплив густого екстракту з грибів майтаке на ознаки запального процесу за експериментального токсичного гепатиту і.і. герасимець, л.с. фіра, і.і. медвідь тернопільський національний медичний університет імені і.я. горбачевського, тернопіль, україна вступ. пріоритетним завданням сучасної фармацевтичної галузі є створення ефективних, безпечних та доступних лікарських засобів, щоб забезпечити максимально високу якість надання медичної допомоги та оптимального використання наявних сировинних ресурсів. мета роботи. метою нашого дослідження було вивчити протизапальні властивості густого екстракту з грибів майтаке в експерименті на щурах із гепатитом, індукованим парацетамолом (ацетамінофеном). методи. для проведення експерименту було використано 60 білих щурів-самців, масою 180-210 г, рандомізованих на 10 груп по 6 тварин у кожній. парацетамоловий гепатит моделювали шляхом введення ацетамінофену інтрагастрально у дозі 1250 мг/кг 1 раз на добу у вигляді суспензії в 2 % розчині крохмального гелю протягом 2 діб. для корекції токсичного ураження використовували густий екстракт грибів майтаке, який вводили інтрагастрально за 2 години до введення ацетамінофену та щоденно після ураження в дозі 150 мг/кг маси тіла тварини. як препарат порівняння обрали “силібор” (виробник – тов «фармацевтична компанія «здоров’я»), який вводили за тією ж схемою, що і екстракт майтаке в дозі 20 мг/кг маси тіла тварини. на 3-тю, 7-му та 10-ту добу експерименту здійснювали евтаназію щурів із використанням барбамілу натрію. для досліджень брали гомогенат печінки та сироватку крові. розвиток запальних процесів вивчали за вмістом прота протизапальних цитокінів, а також с-реактивного протеїну у сироватці крові щурів із токсичним гепатитом та після застосування екстракту грибів майтаке та препарату порівняння. результати. встановлено, що введення тваринам ацетамінофену для моделювання гострого гепатиту супроводжується змінами цитокінового профілю, а саме, зростанням рівня il-6 та зменшенням рівня il-4 у сироватці крові щурів. про розвиток запального процесу свідчить підвищення вмісту с-реактивного протеїну в крові уражених тварин.застосування екстракту грибів майтаке сприяло наближенню досліджуваних показників до рівня інтактного контролю. висновки. зменшення ознак запального процесу у щурів при модельованому парацетамоловому гепатиті під впливом густого екстракту грибів майтаке підтверджує його протизапальні властивості. ключові слова: гриби майтаке, парацетамол, гострий гепатит, запалення. information about the authors herasymets i.i. – phd in pharmacy, associate professor, department of pharmacology and clinical pharmacology, i. horbachevsky ternopil national medical university. orcid0000-0001-7108-3251, e-mail: irunaherasymets@gmail.com fira l.s. – doctor of biological sciences, professor, head of the department of pharmacy of the institute of postgraduate education, i. horbachevsky ternopil national medical university. orcid 0000-0002-5325-0973, e-mail: ludafira@ukr.net medvid i.i. – phd in medicine, assistant professor, department of microbiology, virology and immunology, i. horbachevsky ternopil national medical university. orcid 0000-0003-4703-4438, e-mail: medvid_ii@meta.ua references 1. beger rd, bhattacharyya s, yang x, et al. translational biomarkers of acetaminophen-induced acute liver injury. arch toxicol. 2015;89(9):1497-522. 2. caparrotta tm, antoine dj, dear jw.: are some people at increased risk of paracetamol-induced liver injury? a critical review of the literature. eur j clin pharmacol. 2017;74(2):147-160. 3. xiao c, wu q, xie y, et al. hypoglycemic effects of grifola frondosa (maitake) polysaccharides f2 and f3 through improvement insulin resistance in diabetic rats. food and function. 2015; issue 11: 29. 4. ji h-y, yu 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in any medium, provided the original work is properly cited. i.i. herasymets et al. 14 in t e r n a l m e d ic in e issn 2413-6077. ijmmr 2020 vol. 6 issue 1 doi 10.11603/ijmmr.2413-6077.2020.1.10978 disseminated histoplasmosis leading to haemophagocytic lymphohistiocytosis in an immunocompetent patient (case report) *i.d. khan1, m. brijwal2, i. joshi3, b. singh1, b. poonia1, g. gonimadatala1, s. mangalesh1, a. yadav1, h. rajput1, n. bhuttay1 1 – army college of medical sciences and base hospital, new delhi, india 2 – all india institute of medical sciences, new delhi, india 3 – vellore institute of technology, vellore, india background: emerging fungal infections can pose a serious threat in contemporary healthcare due to host variations, clinical presentation and emerging resistance. histoplasma capsulatum is a thermally dimorphic fungus, which acts as a trojan horse by residing inside macrophages. histoplasmosis is an emerging infection and its association with hemophagocytic lymphohistiocytosis (hlh) in immunocompetent patients has been scantily reported in the literature. objective. the aim of the study was to explore disseminated histoplasmosis with the help of case report. methods: a case report of histoid leprosy is presented. results: a male patient of 47 yearsof age, under treatment for chronic obstructive pulmonary disease for five years and diabetes mellitus type-ii for two years, presented with fever of unknown origin (fuo) with evidence of hlh in the bone marrow. core biopsy of the liver and spleen showed a dense tissue infiltrate with vacuolated histiocytes containing histoplasma capsulatum, eosinophils, some lymphocytes and plasma cells. conclusion: histoid leprosy is a discrete infrequent form of multibacillary leprosy with distinctive clinical, bacteriological and histomorphological features. histopathologic examination with modified fite stain remains the mainstay of diagnosis. key words: histoplasmosis; hemophagocytic lymphohistiocytosis. *corresponding author: dr. inam danish khan, associate professor, department of clinical microbiology and infectious diseases, army college of medical sciences and base hospital, new delhi 110010, india. e-mail: titan_afmc@yahoo.com i.d. khan et al. introduction histoplasma capsulatum is a thermally dimorphic fungus, which acts as a trojan horse by residing inside macrophages [1]. most individuals with intact cellular immunity are asymptomatic. a patient presented with fever of unknown origin (fuo) with evidence of hemophagocytic lymphohistiocytosis (hlh) in the bone marrow. emerging fungal infections can pose a serious threat in contemporary health care due to host variations, clinical presentation and emerging resistance [2-8]. there are diagnostic stringencies in resource limited facilities [9, 10]. histoplasmosis is an emerging infection and its association with hlh in immunocompetent patients has been scantily reported in the literature [11-16]. case report a male patient of 47 years old, under treatment for chronic obstructive pulmonary disease for five years and diabetes mellitus type-ii for two years, presented with rashes around knee for 6 months, fever for 2 weeks and black stools for 3 days. pallor, pedal edema, hepatomegaly 7 cm below right subcostal margin and splenomegaly 9 cm below left subcostal margin were evidenced. investigated for fuo, hemoglobin was 5.5-8.3 gm/dl, total leucocytes 36004200/cumm with normal differential, serum ferritin was 306 ng/dl, while other tests were non-contributory towards diagnosis. ct thorax and abdomen revealed hepatosplenomegaly, retroperitoneal lymphadenopathy with mild ascites and solitary lesion in spleen. colonoscopy was non-contributory. bone marrow aspirate and biopsy showed evidence of hlh (fig. 1). while on treatment, the patient succumbed to his illness. consented post-mortem needle biopsy from liver, spleen, lungs and kidneys was done. core biopsy of the liver and spleen showed a dense tissue infiltrate with vacuolated histiocytes containing histoplasma capsulatum, eosinophils, some lymphocytes and plasma cells. discussion histoplasmosis is an endemic infection in most of the usa, asia and africa caused by international journal of medicine and medical research 2020, volume 6, issue 1, p. 14-18 copyright © 2019, tnmu, all rights reserved 15 in t e r n a l m e d ic in e issn 2413-6077. ijmmr 2020 vol. 6 issue 1 i.d. khan et al. infectious bat and birdexcretions. disseminated histoplasmosis, classically described in the immunocompromised, can occur in immunocompetent patients. clinical presentations vary depending on the size of the inoculum, host’s immune status and presence of underlying lung disease. overt symptoms occur in 5% healthy individuals after low-level exposure; however 75% may get affected with heavy exposure. mostly asymptomatic, mild flu-like illness, fever, chills, sweating, cough, chest and joint pain may occur. acute histoplasmosis may last 1-5 days whereas chronic histoplasmosis, mostly associated with lung infections, can last 10-21 days and is associated with weight loss, persistent fatigue and night sweats. associated acute or subacute pulmonary disease, progressive disseminated disease, pericarditis, arthritis, mediastinitis, hepatomegaly, splenomegaly and bone marrow suppression may occur [17-19]. the patient presented with fever, fatigue, polyarthralgia, skin rashes and black stools with underlying chronic obstructive pulmonary disease. along with anemia and hepato splenomegaly, the clinical picture fits histoplasmosis [11-16]. hlh is an overwhelming inflammatory response, resulting in cytokine storm and activation of monocyte-macrophage system resulting in multiorgan dysfunction. it is a secondary phenomenon to infections, malignancies, autoimmune disorders and drug reaction. hlh is associated with high mortality although successful treatment has been registered [11,14,15]. diagnosis of histoplasmosis involves staining, isolation, serology and antigen detection. bone marrow gives the highest diagnostic yield. antigen detection in urine and serum by radioimmunoassay is useful in an immunocompromised patient when antibody production may be impaired. other laboratory abnormalities include anemia, leukopenia, pancytopenia, elevated liver enzymes, increased ferritin and lactate dehydrogenase. our diagnosis was based on clinical presentation and histopathology of liver and spleen [6], when culture was negative. a high index of suspicion is required as 100% mortality seen in untreated histoplasmosis, can fall to 70% when adequately treated with amphotericin-b [4, 20-25]. risk factors for acquiring acute or chronic histoplasmosis in immunocompetent patients are for farmers and travelers having prolonged contact with rural or endemic environment, speleologists/spelunkers coming in contact with bat guano, and farmers coming in contact with soil enriched with bird guano. heavy infective inoculum leads to acute presentation whereas low inoculum may lead to asymptomatic or chronic infection. prolonged exposure to endemic area in brazil led to histoplasmosis in 43.9-82.9% of immunocompetent patients [2629]. conclusion disseminated histoplasmosis needs to be differentiated from common diseases like tuberculosis, lymphoma or metastatic malignancy. emerging opportunistic resistant infections warrant a high degree of clinical intuition and mental mobility for optimal management. funding this research received no external funding. conflict of interests the authors declare no conflict of interest. author’s contributions i.d. khan – study concept/design, conduct of study, drafting and manuscript revision, final approval of manuscript; m. brijwal – study concept/design, final approval of manuscript; i. joshi – study concept/design, final approval of manuscript; b. singh – conduct of study; b. poonia, g. gonimadatala, s. mangalesh, a. yadav – statistical analysis, drafting and manuscript revision; h. rajput – drafting and manuscript revision, n. bhuttay – drafting and manuscript revision. fig. 1. photomicrograph: hpe 400x; bone marrow aspirate with histoplasma, increased histiocytes and evidence of hemophagocytosis. 16 in t e r n a l m e d ic in e issn 2413-6077. ijmmr 2020 vol. 6 issue 1 information about authors dr inam danish khan – associate professor, clinical microbiology and infectious diseases, army college of medical sciences and base hospital, new delhi 110010, india. orcid https://orcid.org/0000-0002-9824-8711, e-mail: titan_afmc@yahoo.com dr megha brijwa – associate professor, clinical microbiology and infectious diseases, all india institute of medical sciences, new delhi 110029, india orcid https://orcid.org/0000-0003-4907-7036, e-mail: megha.brijwal@yahoo.com ishitta joshi – research scholar, vellore institute of technology, vellore, india orcid https://orcid.org/0000-0003-2298-201x, e-mail: ishittajshi@yahoo.co.in bhagwat singh – mbbs student, army college of medical sciences, new delhi 110010, india. orcid https://orcid.org/0000-0002-0337-6754, e-mail: bhagwatsinghacms@gmail.com bindu poonia – mbbs student, army college of medical sciences, new delhi 110010, india. orcid https://orcid.org/0000-0003-2916-3797, e-mail: bindupooniaacms@gmail.com geethanjali gonimadatala – mbbs student, army college of medical sciences, new delhi 110010, india. orcid https://orcid.org/0000-0002-9372-4002, e-mail: geethugacms78@gmail.com mangalesh sridhar – mbbs student, army college of medical sciences, new delhi 110010, india. orcid https://orcid.org/0000-0002-2645-39949824-8711, e-mail: mangaleshsacms@gmail.com akanksha yadav – mbbs student, army college of medical sciences, new delhi 110010, india. orcid https://orcid.org/0000-0003-2087-8366, e-mail: akiacms@gmail.com himanshu rajput – mbbs student, army college of medical sciences, new delhi 110010, india. orcid https://orcid.org/0000-0003-1240-8444, e-mail: himanshurajput@gmail.com nehal bhuttay – mbbs student, army college of medical sciences, new delhi 110010, india. orcid https://orcid.org/0000-0002-1207-2648, e-mail: nehalbhuttay56acms@gmail.com дисемінований гістоплазмоз як етіологічний чинник гемофагоцитарного лімфогістіоцитозу у імунокомпетентного пацієнта (клінічний випадок) *i.d. khan1, m. brijwal2, i. joshi3, b. singh1, b. poonia1, g. gonimadatala1, s. mangalesh1, a. yadav1, h. rajput 1, n. bhuttay1 1 – army college of medical sciences and base hospital, new delhi, india 2 – all india institute of medical sciences, new delhi, india 3 – vellore institute of technology, vellore, india вступ. зростання кількості та частоти грибкових інфекцій може становити серйозну загрозу сучасній системі охорони здоров’я через варіативність клінічного перебігу, симптомів та ознак захворювання, резистентність до лікування. histoplasma capsulatum – це диморфний гриб, який поводиться як «троянський кінь» проникаючи всередину макрофагів. гістоплазмоз – захворювання, що останнім часом все частіше діагностується, однак про його взаємозв’язок з гемофагоцитарним лімфогістіоцитозом у імунокомпетентних осіб надзвичайно мало інформації. мета роботи на прикладі клінічного випадку дослідити перебіг дисемінованого гістоплазмозу. методи. описано клінічний випадок гістоїдної лепри у пацієнта. результати. чоловік, 47 років, котрий останніх п’ять років лікувався від хозл та цукрового діабету другого типу, звернувся до лікаря зі скаргами на лихоманку невідомого генезу. було діагностовано гемофагоцитарний лімфогістіоцитоз кісткового мозку. на препаратах печінки та селезінки посмертної біопсії органів знайдено щільний тканинний інфільтрат з вакуолізованими гістіоцитами, які містили histoplasma capsulatum, еозинофіли, невелику кількість лімфоцитів та плазмоцитів. висновки. гістоїдна лепра – рідкісна форма мультибацилярної лепри з характерними клінічними, бактеріологічними та гістомофологічними ознаками. проведення гістологічного дослідження дозволяє діагностувати захворювання. ключові слова: гістоплазмоз; гемофагоцитарний лімфогістіоцитоз. i.d. khan et al. 17 in t e r n a l m e d ic in e issn 2413-6077. ijmmr 2020 vol. 6 issue 1 references 1. wheat lj, conger ng. histoplasmosis. in: hospenthal dr, rinaldi mg, eds. diagnosis and treatment of human mycoses. new jersey: humana press, 2008:318-29. 2. khan id, sahni ak, sen s, et al. outbreak of prototheca wickerhamii algaemia and sepsis in a tertiary care chemotherapy oncology unit. medical journal armed forces india. 2018 oct 1;74(4):358-64. doi: 10.1016/j.mjafi.2017.07.012 3. khan id, rajmohan ks, gupta rm, et al. invasive cerebral and pulmonary mucormycosis in an immunocompromised patient. j basic & clin med 2017, 6(2): 9-11. 4. khan id, sahni ak, basu a, et al. trichosporonasahii urinary tract infection in immunocompetent patients. medical journal armed forces india. 2015 oct 1;71(4):373-6. doi: 10.1016/j.mjafi.2014.08.013 5. danish khan i, makkar a, malik a, khan s, mehdi i, arif s, aden d, somayaji p, roomi k. curvularia keratomycosis after cataract surgery. journal of archives in military medicine. 2017(in pre). doi: 10.5812/jamm.57331 6. gupta ak, khan id, shaw sc, faisal fa, sahu s, khan s, brijwal m, shende t, kundu n, khalil s, bhat s. candida parapsilosis neonatal sepsis. journal of basic and clinical medicine. 2016 apr 13;5(1):13-5. 7. khan id, rajmohan ks, jindal ak, gupta rm, khan s, shukla m, singh s, mustafa s, tejus a, narayanan s. panresistant superbugs: are we at the edge of a ‘microbial holocaust’. international journal of medicine and medical research. 2017 dec 29;3(2):39-44. doi: 10.11603/ijmmr.2413-6077.2017.2.8012 8. danish khan i, mohan gupta r, sen s, rajmohan ks, kumar jindal a, makkar a, rahman razi f, banerjee p, panda p, lakshmi nair g, kulhari k. emerging antimicrobial resistance and evolving healthcare: dangerous crossroads for the community and the military. journal of archives in military medicine. 2017(in pre). doi: 10.5812/jamm.12097 9. khan id, gupta n, rangan nm, singh r, sharma ak, khurana a, rudra p, krushnarao ms. evaluation of pre and post analytical variables in clinical microbiology services in multidisciplinary icu of a medical college and tertiary care hospital. journal of basic and clinical medicine. 2016 apr 5;5(1):2-4. 10. khan id. challenges and opportunities in diagnosis and management of infectious diseases in developing country healthcare system. journal of basic and clinical medicine. 2016 apr 5;5(1). 11. lo mm , m o jq, dixon bp, cz ec h ka. disseminated histoplasmosis associated with hemophagocytic lymphohistiocytosis in kidney transplant recipients. american journal of transplantation. 2010 mar;10(3):687-91. doi: 10.1111/j.1600-6143.2009.02969.x 12. khan id, sahni ak, bharadwaj r, lall m, jindal ak, sashindran vk. emerging organisms in a tertiary healthcare set up. medical journal armed forces india. 2014 apr 1;70(2):120-8. doi: 10.1016/j.mjafi.2013.09.005 13. van koeveringe mp, brouwer re. histoplasma capsulatum reactivation with haemophagocytic syndrome in a patient with chronic lymphocytic leukaemia. neth j med. 2010 dec;68(12):418-21. 14. phillips j, staszewski h, garrison m. successful treatment of secondary hemophagocytic lymphohistiocytosis in a patient with disseminated histoplasmosis. hematology. 2008 oct;13(5):282-5. doi: 10.1179/102453308x316013 15. sanchez a, celaya ak, victorio a. histoplasmosis-associated hemophagocytic syndrome: a case report. aids read. 2007 oct;17(10):496-9. 16. wang z, duarte ag, schnadig vj. fatal reactive hemophagocytosis related to disseminated histoplasmosis with endocarditis: an unusual case d i a g n o s e d a t a u t o p s y . s o u t h m e d j . 2 0 0 7 feb;100(2):208-11. doi: 10.1097/smj.0b013e31802b2812 17. huang am. hemophagocytic lymphohistiocytosis and disseminated histoplasmosis. blood. 2014 apr 17;123(16):2449. doi: 10.1182/blood-2014-01-550657 18. hegerova lt, lin y. disseminated histoplasmosis: a cause of hemophagocytic syndrome. inmayo clinic proceedings 2013 oct 1 (vol. 88, no. 10, p. e123). elsevier. doi: 10.1016/j.mayocp.2013.04.030 19. joshi sa, kagal as, bharadwaj rs, kulkarni ss, jadhav mv. disseminated histoplamosis. indian journal of medical microbiology. 2006 oct 1;24(4):297-8. doi: 10.4103/0255-0857.29393 20. khan id, sahni ak. bacterial infections and emerging resistance in renal transplant recipients. ban gladesh journal of medical science. 2015;14(1): 14-21. doi: 10.3329/bjms.v14i1.16306 21. khan id, basu a, kiran s, trivedi s, pandit p, chattoraj a. device-associated healthcare-associated infections (da-hai) and the caveat of multiresistance in a multidisciplinary intensive care unit. medical journal armed forces india. 2017 jul 1;73(3):222-31. doi: 10.1016/j.mjafi.2016.10.008 22. khan id, dogra pm, ramphal sk, khan s, konar j, palit a, srivastava n, aggarwal p, haleem s, alam s. polymicrobial infections in a teenaged renal transplant recipient. journal of basic and clinical medicine. 2015 jul 30;4(1):37-9. 23. khan id, lall m, sen s, et al. multiresistant elizabethkingia meningoseptica infections in tertiary care. mjafi. 2014;71(3):66-7. doi: 10.1016/j.mjafi.2014.02.002 24. khan id, mukherjee t, gupta s, haleem s, sahni ak, banerjee s, konar j. ochrobactrum anthropi sepsis in intensive tertiary care. journal of basic and clinical medicine. 2014 nov 18;3(1):18-20. 25. khan id, sahni ak, bharadwaj r, anwar i, jain v, khan s, chowdhury a, dwivedi ak, gupta ak, zaman s, alam s. cerebral toxoplasmosis diagnosed i.d. khan et al. 18 in t e r n a l m e d ic in e issn 2413-6077. ijmmr 2020 vol. 6 issue 1 by stereotactic brain biopsy leading to detection of hiv infection. journal of basic and clinical medicine. 2016 apr 5;5(1): 5-7. 26. faiolla rc, coelho mc, santana rd, martinez r. histoplasmosis in immunocompetent individuals living in an endemic area in the brazilian southeast. revista da sociedade brasileira de medicina tropical. 2013 aug;46(4):303-6. doi: 10.1590/0037-8682-0124-2013 27. garcía-marrón m, garcía-garcía jm, pajíncollada m, álvarez-navascués f, martínez-muñiz ma, sánchez-antuña aa. chronic pulmonary histoplasmosis diagnosed in a nonimmunosuppressed patient 10 years after returning from an endemic area. archivos de bronconeumología ((english edition)). 2008 jan 1;44(10):567-70. doi: 10.1016/s1579-2129(08)60103-2 28. staffolani s, buonfrate d, angheben a et al. acute histoplasmosis in immunocompetent travelers: a systematic review of literature. bmc infect dis 18, 673 (2018). doi: 10.1186/s12879-018-3476-z 29. zhu ll, wang j, wang zj, wang yp, yang jl. intestinal histoplasmosis in immunocompetent adults. world j gastroenterol. 2016 apr 21;22(15): 4027-33. doi: 10.3748/wjg.v22.i15.4027 received 25 mar 2020; revised 05 apr 2020; accepted 02 june 2020. this is open access article distributed under the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. i.d. khan et al. 83 p h a r m a c y issn 2413-6077. ijmmr 2019 vol. 5 issue 2 doi 10.11603/ijmmr.2413-6077.2019.2.10896 youden's test for chromatographic determination of enalapril in pharmaceuticals l.s. logoyda i. horbachevsky ternopil national medical university, ternopil, ukraine background. robustness tests were firstly introduced for avoiding problems in interlaboratory studies and identifying the factors potentially responsible. a robustness test performing in late validation procedure involves the possibility that when the method is established not robust, it should be redeveloped and optimized. at this stage much effort has been made and money spent for optimization and validation, and therefore avoiding this would be great. objective. the aim of the study was to consider the robustness of hplc determination of enalapril (in tablets) by the youden’s test. methods. youden’s test was chosen as an efficient method to assess the robustness among all analytical methods that is by means of an experiment design, which involved seven analytical parameters combined in eight tests. in previous studies, we evaluated the chromatographic method robustness to quantify enalapril (in tablets) by youden’s test. results. according to the youden’s test criteria, hplc method proved to be greatly robust regarding the enalapril content in introduction of variation of seven analytic parameters. the lowest variation in enalapril content was 0.91 %, when grace platinumр c8 eps column (4.6 mm i.d. x 250 mm, 5 μm) was used. a holistic approach concerning simultaneous innovations in particle technology and instrument design was endeavored for the first time to meet and tackle the analytical laboratory issues. this was aimed at promoting success of analytical scientists as well as profitability and productiveness of business. conclusion. the youden’s test has been proved to be an efficient and useful tool for evaluation of robustness of enalapril hplc assay. key word: enalapril; high-performance liquid chromatography; robustness; quantitative analysis; youden’s test. corresponding author: liliya logoyda, phd, dsc, department of pharmaceutical chemistry i. horbachevsky ternopil national medical university, 1 maidan voli, ternopil, 46001, ukraine. e-mail: logojda@tdmu.edu.ua introduction recently, robustness testing is best known and most commonly used in the pharmaceutics because of the stringent regulations in the domain set by regulatory authorities that requires extensively validated methods. therefore most definitions and existing metho­ dologies, e.g. those from the ich, are found in the field, as stated before. though, this has no implications for robustness testing of analytical methods in other domains and therefore this guideline is not confined to pharmaceutical methods [1]. evaluation of robustness of chromatographic method is a laborious, complex and straining process, taking into account a great number of analytical parameters considered while carrying out the test. some authors consider specific analytical parameters presenting small variations in the nominal conditions; statistical analysis is made using the student’s t-test or anova test. other alternative for evaluation of robustness of analytical methods is the youden’s test. this test assesses not only the robustness of the method but also determines the each analytical parameter effect on final results. the main idea of the youden’s test is not studying one alteration at time but introducing several changes all together in this way that the effects of individual chan ges can be determined [2, 3, 4]. enalapril maleate is a maleate salt of enalapril, the ethyl ester of a long-acting angiotensin converting enzyme inhibitor, enalaprilat. enalapril maleate is chemically defined as (s)­1­[n­ [1-(ethoxycarbonyl)-3-phenylpropyl]-l-alanyl]l­proline, (z)­2­butenedioate salt (1:1). enalapril, after hydrolysis to enalaprilat, inhibits angiotensin-converting enzyme (ace) in humans and animals. ace is a peptidyl dipeptidase that catalyzes the conversion of angiotensin i to the international journal of medicine and medical research 2019, volume 5, issue 2, p. 83-87 copyright © 2019, tnmu, all rights reserved l.s. logoyda 84 p h a r m a c y issn 2413-6077. ijmmr 2019 vol. 5 issue 2 vasoconstrictor substance, angiotensin ii. angio tensin ii also stimulates aldosterone secretion by the adrenal cortex. enalapril in hypertension and heart failure beneficially effects primarily from suppression of the reninangiotensin-aldosterone system. inhibition of ace leads to decrease of plasma angiotensin ii that results in decrease of vasopressor activity and decrease of aldosterone secretion [5]. the aim of the research was to determine the robustness of hplc (high-performance liquid chromatography) method for evaluation of enalapril by means of youden’s test, and define the analytical parameters that have greater influence on the final analysis. methods enalapril maleate was presented by farmak pharmaceuticals (kiev, ukraine). hplc grade acetonitrile, sodium dihydrophosphate dihydrate, phosphoric acid were got from merck pharamaceticals. instrumentation and chromatographic conditions agilent 1260, grace platinumр c8 eps column (4.6 mm i.d. x 250 mm, 5 μm). chroma­ tographic separation was carried out at ambient temperature (22-25 °c). the compound was separated isocratically with a mobile phasе consisting of acetonitrile and buffer solution ph 2.2 (25/75, v/v), at a flow rate 2.0 ml/min with injection volume 50 μl. column tempe­ rature was 50 °c. the effluent was monitored spectrophotometrically at a wavelength 215 nm. preparation of mobile phase to prepare buffer solution ph 2.2.: 3.59 g of sodium dihydrophosphate dihydrate was dissolved in 1800 ml of water, the ph of the solution was fixed with phosphoric acid to the value (2.2±0.05), and then the volume of the solution with water r to 2000.0 ml was added and mixed. stock standard solutions 20 mg of the standard sample of enalapril maleate was dissolved in a solvent, added 0.5 ml of a solution of enalaprilat with a concentration of 0.4 mg/ml and 2.0 ml of enalapril diketopiperazine solution at a concentration of 0.4 mg/ml was adjusted to a volume of 100.0 ml with the same solvent. procedures the standard solutions were prepared by dilution of the stock standard solution of mobile phase. triplicate 50.0 μl injections were made for each concentration and chromatographed under the conditions described above. the peak area of each concentration was plotted against the corresponding concentration to obtain the calibration graph and regression equation was computed [6]. results the robustness assessment of hplc method for enalapril quantitation was performed by the method suggested by youdene steiner. for the nominal values of the method, seven analytical parameters were chosen and minor va riations were induced. after, eight runs were completed in order to determine the effect of each parameter on the final result. the seven analytical parameters as well as the variations are presented in table 1. the analytical circumstances of the nominal values are defined by capi tal letters and of the small variation – by lowercase letters. the seven parameters and their respective variations were joined into eight assays or chromatographic runs randomly performed. the factorial combination of parameters for the youden’s test is presented in table 2. the results of the analyses are defined by the letters table 1. analytical parameters and variations for the robustness evaluation of hplc method for enalapril quantitation parameter nominal condition variation a/a acetonitrile in mobile phase 25 – a 35 – a b/b buffer solution ph 2.2 in mobile phase 75 – b 65 – b c/c ph of buffer solution in mobile phase 2.2 – c 2.7 – c d/d column temperature, °c 50 – d 40 – d e/e mobile phase flow rate, ml/min 2.0 – e 1.0 – e f/f column supplier grace platinumр c8 eps – f nucleosil c18 – f g/g chromatograph model agilent 1290 – g hp 1100 – g l.s. logoyda 85 p h a r m a c y issn 2413-6077. ijmmr 2019 vol. 5 issue 2 l.s. logoyda from s to z. hence, when combination 1 was assessed, the result was s, for combination 2 the result was t, and so on. three injections of each sam ple and stan_ dard solutions at the normal concentration were administered for each combination. a 30-minute pause for system stabilization took place after alteration of chroma tographic column or mobile phase composition. in each combination the assessed re sults were for a peak area, retention time (rt), tailing factor (t), theoretical plates number (n) and captopril content. the following equation was used for evaluation of the effect of the column temperature on the final analyses results: effect c/c=(s+u+w+y)/4–(t+v+x+z)/4eq the youden’s test allows definite estab­ lishing of the parameters, which have a greater influence on the results of the analyses, and control more rigorously the eventual variations of these parameters that may arise during a routine analysis. discussion in this research, the first trials were aimed to find optimal chromatographic conditions. the objective of the chromatographic method development was achievement of a peak tailing factor <1.5, retention time of between 4 and 5 minutes in consort with well resolution [7-17]. in both equipment (agilent 1290 and hp1100), the analyses of the robustness evaluation of chromatographic method were carried out simultaneously. the results were attained in eight runs to enalapril sample and standard solutions. the effects of the parameter variations on the analysis results are presented in table 3. by means of the youden’s test criteria, hplc method proved to be significantly robust as regards the content of enalapril in case of introduced variations of seven analytical parameters [18]. the lowest variation in enalapril content was 0.91 %, when column grace platinumр c8 eps column (4.6 mm i.d.×250 mm, 5 μm) was used. a holistic approach concerning simultaneous innovations in particle technology and instrument design was endeavored for the first time to meet and tackle the analytical laboratory issues. this was aimed at promoting success of analytical scientists as well as profitability and productiveness of business. the platinum™ column advantage controlled silica exposure is the dissimilarity that makes platinum™ columns unique. instead of thorough covering of the silica with bonded phase to hide the silica, the exposure of the silica in platinum™ columns is controlled to provide a dual mode separation table 2. factorial combination of the analytical parameters for robustness evaluation analytical parameter factorial combination acetonitrile in mobile phase a a a a a a a a buffer solution ph 2.2 in mobile phase b b b b b b b b ph of buffer solution in mobile phase c c c c c c c c column temperature d d d d d d d d mobile phase flow rate e e e e e e e e column supplier f f f f f f f f chromatograph model g g g g g g g g result s t u v w x y z table 3. effects of the analytical parameters on content and retention time (rt) for enalapril hplc quantitation effect content (%) rt (min) acetonitrile in mobile phase 0.15 -0.26 buffer solution ph 2.2 in mobile phase 0.16 -0.27 ph of buffer solution in mobile phase 0.12 0.05 column temperature -0.05 0.05 mobile phase flow rate -0.03 0.05 column supplier 0.91 -2.05 chromatograph model -0.04 0.11 86 p h a r m a c y issn 2413-6077. ijmmr 2019 vol. 5 issue 2 with both polar and non-polar sites exposed to the samples. this extends polar selectivity well beyond the other reversed-phase columns and gives separations that other columns cannot. conclusion youden’s test proved to be an efficient and useful tool for the robustness evaluation of hplc method for assay of enalapril in pharmaceuticals. therefore, youden’s test can be successfully used for the ro bustness evaluation for validation process of analytical methods. funding this research received no external funding. conflict of interests the author declares no conflict of interest. юден тест хроматографічного визначення еналаприлу в лікарських засобах л.с. логойда тернопільський національний медичний університет імені і. я. горбачевського, тернопіль, україна вступ. випробування на робасність спочатку були введені, щоб уникнути проблем у міжлабораторних дослідженнях та виявити потенційно відповідальні фактори. виконання перевірки надійності в кінці процедури валідації передбачає ризик того, що, коли виявиться, що метод не є надійним, його слід переробити і оптимізувати. на цьому етапі вже було витрачено багато зусиль і грошей на оптимізацію і перевірку, і тому хочеться цього уникнути. мета дослідження – визначити робасність хроматографічного визначення еналаприлу в таблетках з використанням тесту юдена. методи дослідження. ефективний метод оцінки надійності аналітичних методів за допомогою тесту юдена шляхом розробки експерименту, який включає сім аналітичних параметрів, об'єднаних у восьми тестах. у дослідженнях ми оцінювали надійність хроматографічного методу для кількісного визначення еналаприлу в таблетках з використанням тесту юдена. результати. використовуючи критерії випробування юдена, метод верх показав високу надійність щодо вмісту еналаприлу при введенні варіації семи аналітичних параметрів. найнижча зміна вмісту еналаприлу становила 0,91%, коли використовувалася колонка grace platinumr c8 eps-колони (4,6 мм i.d. x 250 мм, 5 мкм). вперше розроблено цілісний підхід, що передбачає одночасне впровадження інновацій у технології частинок та проектування приладів. це було зроблено для того, щоб зробити вчениханалітиків більш успішними, а підприємства більш прибутковими та продуктивними. висновки. тест юдена виявився ефективним і корисним інструментом для оцінки робастності для аналізу еналаприлу методом верх. ключові слова: еналаприл; високоефективна рідинна хроматографія; робасність; кількісний аналіз; юден тест. відомості про автора логойда лілія святославівна – доктор фармацевтичних наук, доцент, завідувач кафедри фармацевтичної хімії тернопільського національного медичного університету імені і.я. гор­ бачевського, тернопіль, україна. information about the author logoyda l.s. – phd, dsc, head of department of pharmaceutical chemistry i. horbachevsky ternopil national medical university, ternopil, ukraine. orcid 0000­0002­5252­8806, e­mail: logojda@tdmu.edu.ua l.s. logoyda 87 p h a r m a c y issn 2413-6077. ijmmr 2019 vol. 5 issue 2 l.s. logoyda references 1. faulon jl, bender a. handbook of chemoinformatics algorithms. crc press; 2010 apr 21:423. doi: 10.1201/9781420082999 2. youden wj, steiner eh. statistical analysis. statistical manual of the association of official analytical chemists, association of official analytical chemists arlington. 1975:33­83. 3. césar id, pianetti ga. robustness evaluation of the chromatographic method for the quantitation of lumefantrine using youden's test. brazilian journal of 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web-based 5-dimensional electronic portfolio (5dep) as a competency-based assessment tool in post-graduate medical training *z. bokhua1, k. chelidze1,2, k. ebralidze1 1 – tbilisi state medical university (tsmu), tbilisi, georgia 2 – the first university clinic of tsmu, tbilisi, georgia background. new challenges of permanently changing context of healthcare system requires new methods of medical education and new assessment tools, as well. competency-based medical education (cbme), framework which has been adopted as a new approach in medical education, needs appropriate assessment tool such as portfolio. portfolio is learner-centered assessment instrument which evaluates learner’s progression towards outcomes and enables both residents and teachers to engage in a process of learning through assessment. objective. in this paper we aim to share our successful experience of an effective use of web-based 5-dimensional electronic portfolio (5dep) as an assessment tool in the pilot group. methods. pilot group of sixteen residents (six first year residents of obstetrics/gynecology and ten first year residents of internal medicine. tbilisi state medical university institute of postgraduate medical education and continuous professional development) and twelve mentors (four obstetrics/gynecology mentor and eight internal medicine mentors) reported some feedback about 5-dimensional electronic portfolio (5dep) as a new assessment tool. results. feedback about 5-dimensional electronic portfolio (5dep) as a new assessment tool from mentors and students demonstrated efficiency of the program. it enables assessment within a framework of transparent and declared criteria and learning objectives; provides a model for lifelong learning and continuing professional development; increases competence in a wider context with benefits to both professional and personal roles; improves organizing skills. conclusions. 5dep have been recognized as an extremely effective assessment tool. key words: competency-based medical education (cbme); postgraduate education; electronic portfolio; feedback. international journal of medicine and medical research 2020, volume 6, issue 1, p. 81-87 copyright © 2020, tnmu, all rights reserved *corresponding author: zaza bokhua md. ph.d., associate professor of reproductive health department, director of institute of postgraduate medical education and continuous professional development, tbilisi state medical university, tbilisi, georgia. e-mail: z.bokhua@tsmu.edu z. bokhua et al. introduction the fundamental changes in health care and the complexity of health problems have radically changed the role of physicians in the health care system and mounted different challenges in terms of their underand postgraduate education, and continuous professional development. over the last two decades, competency‐ based medical education (cbme) frameworks have been internationally adopted as the primary educational approach in medicine. cbme is a learner-centered, active, and lifelong experience that incorporates feedback between the teacher and the learner to fulfill the desired competency outcomes [1]. despite standardized core competencies of medical education, there are no standardized assessment methods to determine learner's achievement in all the core competencies prior to completion of residency training [1]. competency-based education needs continuous, comprehensive feedback and assessment systems [2]. the use of one of most popular assessment instruments, portfolio can facilitate the above-mentioned goals of assessment by collecting information about the learner’s progression towards outcomes [3]. portfoliobased assessment tool is at the apex of miller’s pyramid, because it provides performancebased assessment in real context by analysis of actions [4, 5]. smith and tillema made four different types of portfolios [6]: 1. the dossier portfolio, containing mandated records of achievement. 2. the training portfolio, containing a mandated collection of acquired skills and competencies, in a fixed format, with some reflective comments on selected evidence. 82 m e d ic a l e d u c a t io n issn 2413-6077. ijmmr 2020 vol. 6 issue 1 3. the reflective portfolio, containing a purposeful collection of evidence for personal growth and development, and 4. the personal development portfolio, containing reflective and personal evaluations of progress in time, enabling discussion and valuing of these activities. in this paper, we aim to provide guidance to program directors for implementation of an electronic portfolio (e-portfolio) by reporting our successful experience of an effective use of web-based e-portfolio system in internal medicine and obstetrics/gynecology residency programs of tbilisi state medical university, where the 5-dimensional electronic portfolio (5dep) was introduced with support of united nations population fund (unfpa) in 2015. methods the pilot group of sixteen residents (six first year residents of obstetrics/gynecology and ten first year residents of internal medicine. tbilisi state medical university institute of postgraduate medical education and continuous professional development) and twelve mentors (four obstetrics/gynecology mentor and eight internal medicine mentors) reported some feedback about 5-dimensional electronic portfolio (5dep) as a new assessment tool. design and structure of 5-dimensional electronic portfolio (5dep) the new web-based 5-dimensional electronic portfolio (5dep) is designed in simple and intuitive manner for users with minimal experience and skills with information technologies. the three modules with different levels of access by individual username and password are designed for (fig.1): • resident (module 1, access level a), to: � upload/confirm evidences of performance/ achievements during training course, approved by the mentors � view of training course assessment form (tcaf) and � view of final portfolio assessment form (fpaf) and narratives • mentor (module 2, access level b), for resident training place assessment (tpaf); • program director/experts panel (module 3, access level c), for monitor of training process and final assessment of portfolio (ipaf/fpaf). module 1 – collected evidences (ce) this module covers all the four types of smith’s & tillema’s stratification [6] and is intended for residents to: • upload evidences collected during all training courses: � achievement of learning outcomes; � clinical (inpatients/outpatients) cases, write-ups and shift records; � acquired essential skills; � self-evaluation of progress in time. • view training course assessment form (see below) after completion each training course; • self-monitor of dynamics on each dimension during the residency training process. module 2 – training course assessment form (tcaf) this module is intended for mentors (clinical trainers) to assess resident on following five dimensions at the end of each training course: • communication – by evaluation of a resident’s interpersonal communication skills, ability to work as integral part of a multidisciplinary medical team, to prevent and manage of communication barriers. this dimension also looks at the strengths of resident’s medical record keeping skills; • self-development – by evaluation of resident’s attitude and responsibility for his/ her own personal development, ability to take active part in learning opportunities, and effective use of reflection; this dimension also looks at the resident’s ability to follow patient/staff/ self-safety and security policy; • professionalism – by evaluation of re sident’s attitude to work, professional appearance, timekeeping, professional boundaries, quality assurance; ability to maintain patient confidentiality, and recognize and respect people’s diversity, preferences, choices and beliefs; • assessment and planning – by evaluation resident’s ability to use basic knowledge and analytical skills in patient assessment and clinical reasoning, collaborate with patient in decision making process and elaborate appropriate management plan; • intervention and treatment – by evaluation resident’s ability to identify specific precautions/contraindications to the intervention/treatment, deliver intervention/treatment with skill and care, and take appropriate actions in case of complications. each dimension contains marking guide table with detailed graded descriptors. choosing specific descriptor that mostly accurately describes the resident’s performance (excellent: 5 points; very good: 4 points; good: 3 points; z. bokhua et al. 83 m e d ic a l e d u c a t io n issn 2413-6077. ijmmr 2020 vol. 6 issue 1 average: 2 points; below average: 1 point) the module calculates final score for specific dimension and turns the score into an appropriate grade with percentage mark (a –excellent: 91-100% of the maximum grade; b – very good: 81-90% of the maximum grade; c – good: 71-80% of the maximum grade; d – satisfactory: 61-70% of the maximum grade; e – sufficient: 51-60% of the maximum grade; fx – fail: 41-50% of the maximum grade and f – fail: >50% of the maximum grade). at the end of the training course the mentor should complete and approve (without further permission of editing) the training course assessment form (tcaf) with the net diagram (d-net) for marks and grades visualization figure 1. 5-dimensional electronic portfolio (5dep). design and structure. ce: collected evidences (form training courses 1, 2 and x). tcaf: training course assessment form (made by the tutor 1, 2 and x of training courses 1,2 and x, respectively); fpaf&n: final portfolio assessment form and narratives. solid line: to upload/edit/confirm; dotted line: to view; long dashed line: to approve/ confirm. 3 figure 1. 5-dimensional electronic portfolio (5dep). design and structure. ce: collected evidences (form training courses 1, 2 and x). tcaf: training course assessment form (made by the tutor 1, 2 and x of training courses 1,2 and x, respectively); fpaf&n: final portfolio assessment form and narratives. solid line: to upload/edit/confirm; dotted line: to view; long dashed line: to approve/confirm. ce-1 resident tcaf-1 mentor 1 ce-2 ce-xtcaf-2 mentor 2 tcaf-x mentor x training course xtraining course 2training course 1 program director/assigned expert ipaf/fpaf&n z. bokhua et al. 84 m e d ic a l e d u c a t io n issn 2413-6077. ijmmr 2020 vol. 6 issue 1 (fig. 2). after completion of the training course mentor will meet with the resident to discuss his/her overall performance. during the meeting should be discussed in which areas the resident has been deficient and why. module 3 – interim portfolio assessment form/final portfolio assessment form (ipaf/ fpaf) this module is designed for the program director to perform consistent monitoring of training process and provide: • interim portfolio assessment at the end of each residency year • final portfolio assessment at the end of residency course chairing the experts panel • report with analysis of resident’s performance and progression towards outcomes during the training courses and final d-net. results the pilot group of sixteen residents (six first year residents of obstetrics/gynecology and ten first year residents of internal medicine. tbilisi state medical university institute of postgraduate medical education and continuous professional development) and twelve mentors (four obstetrics/gynecology mentor and eight internal medicine mentors) reported some feedback about 5-dimensional electronic portfolio (5dep) as a new assessment tool. the mentors found that 5dep: • is customized to meet the needs of the residency programs; • is easy to understand and efficient to use; • deals with the inherent deficiencies of traditional system of evaluation; • enables assessment within a framework of transparent and declared criteria and learning objectives; • evaluates and stimulates progress towards educational and professional outcomes; • evaluates learning outcomes not easily assessed by other methods; • provides summative assessment of progress and formative evaluation; • improves mentor awareness of student’s need and support required for students; • enhances of interactions between residents and mentors; • focusses on resident’s personal attributes; • motivates mentors to focus on the training objectives; 5 figure 2. example of d-net. the resident’s excellent performance in all dimensions is visualized by d-net, except “self-development”. module 3 – interim portfolio assessment form/final portfolio assessment form (ipaf/fpaf) this module is designed for the program director to perform consistent monitoring of training process and provide: • interim portfolio assessment at the end of each residency year • final portfolio assessment at the end of residency course chairing the experts panel • report with analysis of resident’s performance and progression towards outcomes during the training courses and final d-net. results the pilot group of sixteen residents (six first year residents of obstetrics/gynecology and ten first year residents of internal medicine. tbilisi state medical university institute of postgraduate medical education and continuous professional development) and twelve mentors (four obstetrics/gynecology mentor and eight internal medicine mentors) reported some feedback about 5-dimensional electronic portfolio (5dep) as a new assessment tool. the mentors found that 5dep: • is customized to meet the needs of the residency programs; • is easy to understand and efficient to use; • deals with the inherent deficiencies of traditional system of evaluation; • enables assessment within a framework of transparent and declared criteria and learning objectives; • evaluates and stimulates progress towards educational and professional outcomes; • evaluates learning outcomes not easily assessed by other methods; • provides summative assessment of progress and formative evaluation; figure 2. example of d-net. the resident’s excellent performance in all dimensions is visualized by d-net, except “self-development”. figure 2. example of d-net. the resident’s excellent performance in all dimensions is visualized by d-net, except “self-development”. z. bokhua et al. 85 m e d ic a l e d u c a t io n issn 2413-6077. ijmmr 2020 vol. 6 issue 1 • can accommodate evidence of learning from a range of different contexts; • allows a range of learning styles to be used according to the preferences of the student; • provides a model for lifelong learning and continuing professional development. the students found that 5dep: • allows to understand learning objectives, using specific targets; • enables the consolidation of the connection between theory and practice, and apply abstract principles to practical contexts; • recognizes and encourages the autonomous and reflective learning that is an integral part of professional education and development; • enhances self-learning and self-development; • enables to correct errors and remedies deficiencies; • focusses on resident’s personal attributes; • evaluates and stimulates progress towards educational and professional outcomes; • enhances of interactions between residents and mentors; • motivates both residents to focus on the training objectives; • helps to update of knowledge and skills in existing and new areas of practice; • increases competence in a wider context with benefits to both professional and personal roles; • improves organizing skills; • helps to estimate potential to progress to other levels or courses. discussion the use of portfolio as an assessment instrument enables residents and teachers to engage in a process of learning through assessment. the use of portfolio broadens the scope of assessment and introduces several educational benefits [7, 8, 9, 10]: • contribution to: � the assessment of learning outcomes � the provision of evidence of performance � the representation of evidence collected over a period � resident progression towards the learning outcomes � summative and formative assessment • focus on personal attributes • enhancement of interactions between residents and teachers • stimulation of the use of reflective strategies • encouragement of a holistic and integrative approach to medical practice. the portfolio assessment remains relatively unknown among clinical teachers and residents [11, 12, 13, 14, 15]. conclusions 5-dimensional electronic portfolio (5dep) have been recognized as an extremely effective assessment too that could provide motivation to study, can assist students in forming positive attitudes toward learning, to enable students to individualize and personalize their learning by supporting and encouraging active participation. conflict of interest the authors declare no conflict of interest. funding this work was supported by united nations population fund (unfpa). author’s contribution zaza bokhua – contributed to the data curation, formal analysis, funding acquisition, investigation, resources and writing original draft; kakhaber chelidze – contributed to the con ceptualization, formal analysis, methodology, project administration, supervision, visualization, validation and review and editing of original draft; ketevan ebralidze – contributed to the formal analysis, and review and editing of original draft. п’ятивимірне електронне портфоліо (5dep) як інструмент оцінки компетентностей у післядипломному медичному навчанні *z. bokhua1, k. chelidze1,2, k. ebralidze1 1 – tbilisi state medical university (tsmu), tbilisi, georgia 2 – the first university clinic of tsmu, tbilisi, georgia вступ. сучасні виклики у системі охорони здоров’я, зумовлені постійними її змінами, вимагають новітніх методів навчання та інструментів їх оцінки. компетентнісний підхід до навчання, котрий було взято за основу сучасних стандартів професійної медичної освіти, потребує відповідних методів оцінки, таких як портфоліо. портфоліо – це інструмент, сконцентрований на простежуванні прогресу z. bokhua et al. 86 m e d ic a l e d u c a t io n issn 2413-6077. ijmmr 2020 vol. 6 issue 1 набутих знань особою, що навчається, який дозволяє зацікавити і залучити до процесу не лише резидента, а й викладача. мета – поділитися успішним досвідом застосування 5-вимірного електронного портфоліо (5dep) у якості інструменту оцінки набуття професійних компетентностей у пілотній групі. методи. пілотну групу склали 16 резидентів (шість з них – резиденти першого року навчання зі спеціальності акушерство/гінекологія, ще десять – першого року навчання зі спеціальності внутрішня медицина інституту післядипломної медичної освіти та безперервного професійного розвитку, тбіліський державний медичний університет) та дванадцять менторів-наставників (чотири з акушерства/гінекології та вісім – з внутрішньої медицини). учасники пілотного проекту ділилися відгуками щодо оцінки застосування 5-вимірного електронного портфоліо (5dep) у якості інструменту оцінки набуття професійних компетентностей результати. отримані у процесі застосування 5-вимірного електронного портфоліо (5dep) відгуки та зворотній зв'язок від учасників пілотної групи (менторів та резидентів) продемонстрували ефективність програми як інструменту оцінки набуття професійних компетентностей. портфоліо забезпечує прозорі та чіткі критерії оцінки знань та компетентностей, забезпечує модель безперервного професійного розвитку на “навчання протягом усього життя”, підвищує рівень як професійних так і особистісних компетентностей, покращує організаційні навики. висновки. 5dep – надзвичайно ефективний інструмент оцінки набутих знань та компетентностей. ключові слова: компетентнісно орієнтована медична освіта (cbme); післядипломна освіта; електронне портфоліо; зворотній зв’язок. information about the authors zaza bokhua – md, ph.d., associate professor of reproductive health department, director of institute of postgraduate medical education and continuous professional development, tbilisi state medical university, tbilisi, georgia. orcid https://orcid.org/0000-0003-4974-4513, e-mail: z.bokhua@tsmu.edu kakhaber chelidze – md, ph.d., professor, chair of internal medicine in the first university clinic of tsmu, the head of department of internal medicine, tbilisi state medical university, tbilisi, georgia. orcid https://orcid.org/0000-0002-3906-9967, e-mail: k.chelidze@tsmu.edu ketevan ebralidze – md, ph.d., director of internal medicine residency program, head of the organizational service of institute of postgraduate medical education and continuous professional development, invited teacher of department of physiology, tbilisi state medical university, tbilisi, georgia. orcid https://orcid.org/0000-0003-3880-3733, e-mail: k.ebralidze@tsmu.edu references 1. nejm knowledge+ team. what is competencybased medical education? nejm knowledge+. june 15th, 2017. doi: 10.1093/clinchem/63.12.1912 2. holmboe es, sherbino j, long dm, swing sr, frank jr, 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received 26 mar 2020; revised 09 apr 2020; accepted 12 june 2020. this is open access article distributed under the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. z. bokhua et al. 40 in t e r n a l m e d ic in e issn 2413-6077. ijmmr 2019 vol. 5 issue 2 doi 10.11603/ijmmr.2413-6077.2019.2.10886 gout and nonalcoholic fatty liver disease: effect of enterosorption’s addition to common treatment u.o. mudra i. horbachevsky ternopil national medical university, ternopil, ukraine background. gout is still one of the major health problems despite significant advances in treatment in recent years. it has been proved that pathogenetic mechanisms of development and progression of gout are associated with nonalcoholic fatty liver disease. complex pathogenic treatment of patients aimed at different parts of the pathological process has recently been supplemented with the enterosorbents. objective. the aim of the research is to study the clinical features of gout with concomitant nonalcoholic fatty liver disease (nafld) and to evaluate the effect of carbon enterosorbent on its course. methods. 123 patients were involved in the study. they were divided into 2 groups: group 1 included patients with gout without liver damage, and group 2 included patients with concomitant nafld. each of these groups was divided into subgroups, in which the patients received carbon enterosorbent carboline plus basic treatment. the control group consisted of 30 healthy persons. anamnesis, physical examination, uric acid (ua), c-reactive protein (crp) content, erythrocyte sedimentation rate (esr) in serum were determined. gout activity was evaluated using the gout activity score (gas). results. basic treatment in combination with carbon enterosorbent contributed to faster cure of intoxication, pain and joint syndromes, as well as decrease of the inflammatory process activity. conclusions. the course of gout in the patients with concomitant nafld is more severe. adding of carbon granular enterosorbent carboline in the complex treatment of patients with gout with or without concomitant nafld in the exacerbation phase contributes to a faster cureing dynamics of clinical and laboratory manifestations of the disease. key words: gout; nonalcoholic fatty liver disease; enterosorbent; treatment. *corresponding author: mudra uliana, department of internal medicine propedeutics and phthisiology, 1 maidan voli, i. horbachevsky ternopil national medical university, ternopil, ukraine, 46001. e-mail: heliosmuo@gmail.com introduction pathology of the articular apparatus is one of the most frequent and common human diseases. salt arthropathy accounts for a large share, among which gout is the most common. gout is a chronic progressive disease associated with impaired uric acid metabolism, which is clinically manifested by recurrent arthritis, tophi formation and internal organs damage [1]. gout is certainly an urgent health issue and a significant social and economic burden for the country. according to the ministry of health of ukraine, the number of gout patients in ukraine has increased in recent years. thus, in 2013, the prevalence of this disease was 167.6 per 100 thousand population, whereas in 2017 this figure reached 190.4 per 100 thousand population, and 113.9 – among the able-bodied population. the cost of treating new cases of acute gouty arthritis in the united states is estimated at $ 27.4 million annually [2, 3]. pain and swelling of the joints during exacerbations of gout can lead to a significant deterioration and disability, which affects the patient’s productivity and social activity. according to edwards n.l. et al., a patient with gout, who is under 65 years of age, has an average of 25.1 working days a year due to the illness [4]. uncontrolled administration of medicines for comorbid diseases, as well as malnutrition, alcohol abuse, hypodynamia, physical or psycho-emotional overload play an important role in increasing the incidence [5]. in recent years, researchers have paid particular attention to the multifaceted clinical manifestations of gout, which is not limited to damage of the musculoskeletal system and kidneys. a great number of patients suffer from obesity, disorders of lipid and carbohydrate metabolism, hypertension, which are integral parts of the metabolic syndrome [6, 7]. international journal of medicine and medical research 2019, volume 5, issue 2, p. 40-46 copyright © 2019, tnmu, all rights reserved u.o. mudra 41 in t e r n a l m e d ic in e issn 2413-6077. ijmmr 2019 vol. 5 issue 2 u.o. mudra according to the literature, metabolic syndrome is often aggravated by fatty liver infil tra­ tion, which can significantly affect the course of the underlying disease. nonalcoholic fatty liver disease is the most common liver disease in the world (46% of the population, and 74% among diabetes patients) [8, 9]. thus, the increase of gout incidence and development of concomitant pathology, which complicates its course, leads to a decrease in the effectiveness of treatment, development of complications and mortality, is an urgent issue at present that requires improved diagnosis and treatment. treatment of the patients with gout should be comprehensive, and include non-pharmacological and pharmacological methods. physicians should take into account the level of uric acid (with subsequent monitoring), the form and stage of gout, radiological stage and degree of functional failure of the joints, patient’s age, concomitant pathology and features of its pharmacotherapy. a key aspect of the treatment is strict diet, weight loss in obesity [10]. one of the current treatments is enterosorption, which is increasingly used in the treatment of acute and chronic diseases. the objective of the study is to investigate clinical features of gout with concomitant nonalcoholic fatty liver disease (nafld) and to evaluate the effect of carbon enterosorbent on its course. methods the study involved 123 patients with gout during the exacerbation period, including 118 (95.9%) men and 5 (4.1%) women. the average age was (57.73±1.01). the diagnosis of gout was based on acr/eular 2015 criteria and the order of the ministry of health of ukraine no. 676, dated october 12, 2006. the diagnosis of nafld was established according to the criteria of the unified clinical protocol of primary, secondary (specialized) medical care “nonalcoholic fatty liver disease” (order of the ministry of health of ukraine no. 826, dated november 6, 2014). the history of the disease (duration of the disease, frequency of attacks for the last 12 months) was taken into account in clinical examination of the patients. they also underwent physical examination (determination of pain intensity by a visual analogue scale (vas), evaluation of the number of tophi, affected joints, uric acid content (ua), c-reactive protein (crp), erythrocyte sedimentation rate (esr) in the blood serum. to assess the effectiveness of therapy, the patients of both groups were monitored for complaints (pain and swelling of joints, restriction of movement in them, etc.), evaluated for joint syndrome development, dynamics of the level of ua, crp and esr. gout activity was assessed using the gout activity score (gas) suggested by sciere c.a. et al.: gas = 0.09 × the number of attacks for the last 12 months + 1.01 × √ua + 0.34 × patient’s vas + 0.53 × ln (1 + number of tophi); where ua – uric acid (mg/dl), vas – visual analogue pain scale (cm) [2, 11]. all the patients were divided into two groups. group 1 included 65 patients with gout without liver damage, group 2 – 58 patients with concomitant nafld. the control group consisted of 30 healthy individuals of the same age. all the patients voluntarily agreed to participate in the study. the patient safety rules and the ethical standards and procedures for research involving human beings (2000) were followed during the whole study. statistical processing of the results was performed using statistica 10.0 software package (statsoft, usa) and microsoft office excel 2016 statistical software package (microsoft corp., usa). the statistical significance of differences between the groups was evaluated using the nonparametric wilcoxon u (mannwhitney) method. the differences were considered statistically significant at p<0.05. results according to the medical history, the average duration of gout in all examined patients was (8.75±0.50) years. clinical and laboratory characteristics of patients with gout are presented in table. 1. tophi were detected in 80 (69 %) persons, in group 1 – in 36 (45 %) patients, in group 2 – 44 (55 %). it is worth noting that patients with nafld were predominant with multiple tophi. when assessing the frequency of exacerbations of gout per year, a statistically significant increase in 1.5 times among the patients with concomitant nafld, compare to those without liver damage (p<0.05) should be emphasized. the analysis of the joints involved in the pathological process in the patients with gout without liver damage proved the following joints (3.75±0.30) disorders: monoarthritis was detected in 24.6%, oligoarthritis – in 29.2%, 42 in t e r n a l m e d ic in e issn 2413-6077. ijmmr 2019 vol. 5 issue 2 polyarthritis – in 46.2%. in the patients with concomitant nafld, oligoand polyarthritis were observed in the vast majority (96.5 ) and monoarthritis in only 3.5% of those surveyed. the average number of the affected joints in the group was (5.36±0.38). the patients in group 2 were characterized by an increase in the number of patients with severe disease course – 18 (31%) and a decrease in the percentage with mild course – 7 patients (12%) and the average – 33 (57%). at the same time, in group 1, a severe course was diagnosed in 7 (11%), medium – in 41 (63%), mild – in 17 (26%) patients. the intensity of pain according to vas in the patients of group 2 was in 1.2 times higher than in group 1 (p<0.05). the gas scale was used to evaluate gout activity. it was found that the majority of patients had a high disease activity and, on average, in the patients with concomitant nash it was in 1.3 times higher than in the patients without liver damage. in 36 (55.4%) patients of group 1 moderate activity (gas 4.5-7.4) was present, and 29 (44.6%) had a high (gas>7.4) disease activity. 45 (77.6%) patients of group 2 had high disease activity and 13 (22.4%) had moderate activity (p<0.05). in the control group, the level of ua in the serum was (309.13±14.16) μmol/l, crp – (3.39±0.17) mg/l, esr – (6.47±0.65) mm/h. the analysis of laboratory parameters revealed that the examined group 2 experienced an increase in the level of ua in the blood in 1.4 times compare to the patients in group 1, re s p e c t i v e l y , u p t o ( 6 2 1 . 5 7 ± 1 3 . 4 7 ) a n d (459.72±11.44) μmol/l (p<0.05). the inflam­ matory process, by the crp level, was more significant in 1.7 times in the patients with con­ comitant nafld than in group 1. an increase in esr, as a criterion for the activity and severity of the inflammatory process, occurred among all patients, but mostly in the patients with concomitant liver damage (29.12±1.04) mm/h, which was 1.5 times higher than the group 1 parameters (p<0.05). according to the treatment received by the patients, they were divided into subgroups. table 1. clinical and laboratory characteristics of the patients with gout parameter units group 1 (patients without nafld) (n=65) group 2 (patients with nafld) (n=58) age, years м±m 59.55±1.33 55.68±1.5 number of patients with tophi n (%) 36 (45) 44 (55) number of tophi: 1-3 4-9 10 and more n (%) 7 (19.4) 19 (52.8) 10 (27.8) 14 (31.8) 16 (36.4) 14 (31.8) number of attacks for the last 12 months: м±m 3.03±0.23 4.67±0.19* 0 1-2 3-5 6-10 more than 10 n (%) 8 (12.3) 14 (21.5) 38 (58.5) 5 (7.7) – – 4 (6.9) 41 (70.7) 13 (22.4) – number of affected joints: м±m 3.75±0.30 5.36±0.38* monoarthritis oligoarthritis (2-3 joints) polyarthritis 4 and more joints n (%) 16 (24.6) 19 (29.2) 30 (46.2) 2 (3.5) 17 (29.3) 39 (67.2) course: mild medium severity severe n (%) 17 (26) 41 (63) 7 (11) 7 (12) 33 (57) 18 (31) visual analogue scale (vas), mm м±m 56.34±1.00 69.21±1.04* gout activity score (gas) м±m 7.53±0.28 9.63±0.35* uric acid, μmol/l м±m 459.72±11.44 621.57±13.47* c-reactive protein, mg/l м±m 11.35±0.71 19.58±1.62* erythrocyte sedimentation rate (esr), mm/h м±m 19.35±0.90 29.12±1.04* note: * – statistically significant differences in the parameters between the groups of patients without and with nafld (р<0.05). u.o. mudra 43 in t e r n a l m e d ic in e issn 2413-6077. ijmmr 2019 vol. 5 issue 2 u.o. mudra group 1 – 27 people (subgroup 1a) received treatment in accordance with the order of the ministry of health of ukraine no. 676, dated october 12, 2006: hypopuricemic therapy with allopurinol according to the scheme, antiinflammatory and analgesics (basic therapy); 38 (subgroup 1b) – additionally enterosorbent carboline 1 teaspoon 3 times a day for 10 days 2 hours before or after meals or medication. among the patients of group 2 – 27 (subgroup 2 a) received basic therapy, 35 (subgroup 2 b), except basic treatment – carboline according to the scheme. the patients of group 2 were also treated according to the unified clinical protocol of primary, secondary (specialized) medical care “nonalcoholic fatty liver disease” (order of the ministry of health of ukraine no. 826, dated november 6, 2014). pain, as one of the manifestations of articular syndrome, was the main complaint of all patients before treatment (table 2). swelling of the affected joint was present in 53 (81.5%) patients of group 1 and 54 (93.1%) of group 2, with redness of the skin in 45 (69.2%) and 56 (97%), respectively, local temperature rise in 50 (77%) and 54 (93.1%) individuals, body temperature rise to subfebrile numbers took place in 11 (17%) and 13 (22.4%) of both groups. table 2. the frequency of clinical symptoms in the patients with gout symptoms patients with gout group 1, n=65 group 2, n=58 joint pain and change in joint motion 65 (100 %) 58 (100 %) swelling 53 (81.5 %) 54 (93.1 %) redness 45 (69.2 %) 56 (97 %) local temperature rise 50 (77 %) 54 (93.1 %) body temperature rise 11 (17 %) 13 (22.4 %) clinical manifestations of the disease and improvement of personal well-being decreased in the dynamics of treatment of the patients of both groups. in the patients of group 1 a significant improvement of the general condition was observed on the 2nd-3rd day, and in the patients with concomitant liver damage – swelling, redness of the joint and the intensity of pain decreased on the 3rd-4th day of the disease, according to vas, the body temperature was normal. thus, in the patients of subgroup 1a after the treatment, the intensity of pain according to vas decreased from (55.19±1.4) mm to ( 1 8 . 4 1 ± 3 . 2 1 ) m m ( p < 0 . 0 0 1 ) , a n d f r o m (57.16±1.4) mm to (11.66 ±2.12) mm – in the subgroup 1b (p<0.001). before the treatment, the examined of group 2 complained of severe pain in the affected joints that significantly influenced the functional activity of the patients. after baseline treatment, the pain intensity according to vas i n t h e s u b g r o u p 2 a d e c r e a s e d f r o m (69.04±1.67) mm to (30.48±4.68) mm (p<0.001), and in the subgroup 2b (in cases of enterosorbent ad ministration) – from (69.31±1.34) mm to (21.20±2.82) mm (p<0.001). the dynamics of ua regression in blood serum during the course of treatment in the patients of group 1 was significantly better compare to those of group 2. in cases of the baseline treatment, the level of ua gradually d e c r e a s e d i n t h e s u b g r o u p 1 a f r o m (444.01±18.57) to (389.03±14.55) μmol/l, p<0.001, and in the subgroup 1b with additional carboline administration – from (470.87±14.38) to (353.01±7.96) μmol/l, p<0.001. compare to the control group, this parameter was higher in 1.26 and 1.14 times, respectively. in the subgroup 2a, the level of ua decreased from (657.03±11.64) to (529.27±12.32) μmol/l, p < 0 . 0 0 1 , i n t h e s u b g r o u p 2 b – f r o m (598.29±20.14) to (473.95±7.75) μmol/l, p<0.001, however, it was higher than in the control group in 1.71 and 1.53 times. regarding the inflammatory process acti­ vity (esr, crp), there was a decrease in the level of esr (p<0.001) and crp (p<0.001) in the dynamics of treatment in both groups. it should be noted that in the patients of subgroup 1b after enterosorbent administration the crp concentration decreased from (12.06±1.01) to (3.51±0.37) mg/l (p<0.001), and esr – from (18.47±1.14) to (7.95±0.59) mm/h (p<0.001), compare to the control group these values became normal (p>0.05). in the patients with con comitant nafld, in cases of the basic treatment in combination with enterosorbent there was a decrease of crp – from (19.94±1.92) to ( 7 . 7 9 ± 0 . 3 6 ) m g / l ( p < 0 . 0 0 1 ) , e s r – f ro m (30.51±1.37) to (11.94±0.42) mm/h (p<0.001), but they were in 2.3 and 1.8 times higher than in the almost healthy individuals (p<0.001). 44 in t e r n a l m e d ic in e issn 2413-6077. ijmmr 2019 vol. 5 issue 2 discussion comorbidity is still an important problem in medicine, since studying the manifestations of the combined pathology of different body systems can help uncover the mechanisms of disease formation an d development of pathogenetically profound therapy [12]. at present gout is an urgent problem because its prevalence has doubled in the last decades. this disease becomes even more significant due to its comorbidity. frequent combination of gout with concomitant pathology leads to a decrease in the effectiveness of treatment and progression of complications [13]. currently, there is a high morbidity of nafld in the world, and there is a tendency to increasing incidence of this chronic liver disease in the population, development of severe complications in the disease progression that is an urgent issue of contemporary medicine. the presence of nafld, as a comorbid condition, enhances the inflammatory response in the body and significantly complicates the course of the underlying disease [14]. it is indisputable fact that the main link of gout pathogenesis is ua metabolism. the determination of the dynamics of ua levels in the blood in gout and concomitant nafld is increasingly significant as a risk factor for cardiovascular events [15]. in most such patients, prior to treatment, an increase in crp and esr is proved. it is established that in this pathology an inflam­ matory process develops, which is not only limited to local changes in the joints, but also causes a response of the whole body. the increase in crp in the patients with gout in combination with nafld confirms the presence of chronic subclinical inflammation in them, and with the progression of the disease there is an increase in the level of crp [16]. treatment of the patients with gout should be aimed at eliminating the acute attack of arthritis and inflammatory changes in the joints, reducing the content of ua compounds in the body, eliminating extra-articular lesions associated with gout, restoring the function of the musculoskeletal system, etc. [17]. it should be noted that the use of enterosorption together with basic therapy has a positive effect on the clinical course of the disease, promotes faster regression of intoxication, pain and articular syndromes, as well as is accompanied by a decrease of inflammatory process activity in the body (crp, esr) and uric acid level in blood serum. conclusions the course of gout in the patients with concomitant nonalcoholic fatty liver disease is more severe than in its absence. with this combination, the number of the affected joints, tophi, acute conditions increases for the period of a year. there is an increase of the intensity of pain according to visual analogue scale, as well as its activity by the gout activity score. the inflammatory process intensifies. the inclusion of carbon granulated enterosorbent carboline in the complex treatment of the patients with gout or without concomitant nonalcoholic fatty liver disease, contributes to a faster reverse dynamic of clinical manifestations of the disease, accompanied by a more significant de­ crease in c-reactive protein, erythrocyte sedimentation rate and uric acid in blood serum. funding this research received no external funding. conflict of interests the author declares no conflict of interest. подагра і неалкогольна жирова хвороба печінки: ефект додаткового застосування ентеросорбції в загальному лікуванні у.о. мудра тернопільський національний медичний університет імені і.я. горбачевського, тернопіль, україна вступ. незважаючи на те, що за останні роки досягнуто значних успіхів у лікуванні, подагра залишається однією із серйозних проблем охорони здоров'я. особливістю сучасних хронічних захворювань є коморбідність. доведена спільність патогенетичних механізмів у розвитку і прогресуванні подагри u.o. mudra 45 in t e r n a l m e d ic in e issn 2413-6077. ijmmr 2019 vol. 5 issue 2 u.o. mudra та неалкогольного стеатогепатиту. комплексне патогентичне лікування хворих, спрямоване на різні ланки патологічного процесу, останнім часом доповнене застосуванням ентеросорбентів. мета дослідження. вивчити клінічні особливості перебігу подагри при супутньому неалкогольному стеатогепатиті (насг) й оцінити вплив вуглецевого ентеросорбенту на її перебіг. методи дослідження. обстежено 123 хворих на подагру, які перебували на стаціонарному лікуванні. їх було поділено на 2 групи. до 1-ї групи ввійшли пацієнти з подагрою без ураження печінки, до 2-ї – хворі із супутнім насг. кожну із цих груп було розділено на підгрупи, пацієнти однієї з яких, окрім базоваго лікування, отримували вуглецевий ентеросорбент карболайн. контрольну групу склали 30 практично здорових осіб. в клінічному обстеженні хворих враховували скарги, дані анамнезу, проводили фізикальний огляд, визначали вміст сечової кислоти (ск), с-реактивного білка (срб), швидкості осідання еритроцитів (шое) в сироватці крові. активність подагри оцінювали за допомогою шкали gout activity score (gas). результати. застосування базового лікування в поєднанні з вуглецевим ентеросорбентом сприяло швидшій регресії інтоксикаційного, больового й суглобового синдромів, а також показників активності запального процесу. висновки. перебіг подагри у хворих з супутнім насг тяжчий, ніж за його відсутності. включення вуглецевого гранульованого ентеросорбенту карболайн в комплексне лікування хворих на подагру з супутнім насг чи без нього у фазі загострення, сприяє швидшій зворотній динаміці клінічних і лабораторних проявів захворювання, супроводжується більш значним зниженням рівня срб, шое та ск у сироватці крові. ключові слова: подагра; неалкогольний стеатогепатит; ентеросорбент; лікування. відомості про автора мудра уляна олегівна – аспірант кафедри пропедевтики внутрішньої медицини та фтизіатрії тернопільського національного медичного університету імені і.я. горбачевського. information about the author uliana o. mudra – ph.d. student, department of internal medicine propedeutics and phthisiology of i. horbachevsky ternopil national medical university, ternopil, ukraine. orcid 0000­0001­8160­4962, e­mail: heliosmuo@gmail.com references 1. herasymenko si, poluliakh mv, duda ms, pshenychnyi tie. current aspects of conservative treatment of acute gouty arthritis. chronicle of traumatology and orthopedics. 2013;1­2:204­7. 2. orlova iv. influence of disease activity on quality of life of patients with gout. ukrainian journal of medicine, biology and sport. 2018;3(5):127–31. doi: 10.26693/jmbs03.05.127 3. aung t, myung g, fitzgerald jd. treatment approaches and adherence to urate-lowering therapy for patients with gout. patient prefer adherence. 2017;11:795–800. doi:10.2147/ppa.s97927 4. edwards nl, sundy js, forsythe a, blume s, pan f, becker ma. work productivity loss due to flares i n p a t i e n t s w i t h c h ro n i c g o u t re f ra c t o r y t o conventional therapy. j med econ. 2011;14(1):10­5. doi: 10.3111/13696998.2010.540874 5. svintsitskyi as. current views on the diagnosis and treatment of gout. health of ukraine. rheu matolog. 2013 february: 70­2. 6. barskova vg, eliseev ms, denisov is, eliseeva me, belikov oa, fadienko gr, i dr. the frequency of metabolic syndrome and concomitant diseases in patients with gout. data from a multicenter study. scientific and practical rheumatology. 2012;6(50):15­8. 7. bardin t, richette p. impact of comorbidities on gout and hyperuricaemia: an update on pre­ valence and treatment options. bmc med. 2017; 15(1): 123. doi:10.1186/s12916­017­0890­9 8. skliarov yeya., aksentiichuk khb., kurliak nv. monitoring of liver function disorders in patients with non-alcoholic fatty liver disease on the background of metabolic syndrome. hepatology. 2015;1: 34­41. 9. tkachuk so, bashta hv, lapovets lie, zaletskyi mp. changes in carbohydrate and lipid metabolism and c-reactive protein in patients with coronary heart disease and fatty hepatosis. medical and clinical chemistry. 2016;18(4):29­33. doi: 10.11603/mcch.2410­681x.2016.v0.i4.7252 10. kondratiuk vie, tarasenko om. hyperuricemia and gout: the current state of the problem. ukrainian rheumatology journal. 2016;3:30–7. 11. scirè c, carrara g, viroli c, cimmino m, taylor w, manara m, et al. development and first validation of a disease activity score for gout. arthritis care & research. 2016; 68 (10): 1530­7. doi: 10.1002/acr.22844 12. abrahamovych oo, faiura op, abrahamovych uo. comorbidity: a modern perspective on the 46 in t e r n a l m e d ic in e issn 2413-6077. ijmmr 2019 vol. 5 issue 2 problem; classification (message second). lviv clinical bulletin. 2016;1:31­9. 13. svintsitskyy as, kozak np, ostafiichuk as. pecularities of gouts’ course in association with the metabolic syndrome. ukrainian rheumatology journal. 2011;4:46. 14. younossi zm. non-alcoholic fatty liver disease – a global public health perspective. j. hepatol. 2019;70(3):531­44. doi: 10.1016/j.jhep.2018.10.033 15. pillinger mh, goldfarb ds, keenan rt. gout and its comorbidities. bull nyu hosp jt dis. 2010;68(3):199­203. 16. orlovsky vf, murenets no. chronic inflam­ mation in nonalcoholic fatty liver disease. pathologia. 2010;7(3):99­102. 17. svіntsіtskiy as. diagnosis and treatment of rheumatic diseases: manual. kiev: the publishing house of the medknyga; 2017. 372 р. received 06 september 2019; revised 11 october 2019; accepted 21 october 2019. this is open access article distributed under the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. u.o. mudra 20 in t e r n a l m e d ic in e issn 2413-6077. ijmmr 2021 vol. 7 issue 1 doi 10.11603/ijmmr.2413-6077.2021.1.11814 clinical outcomes and adverse drug reactions in covid-19 patients treated with hydroxychloroquine and azithromycin alone or combined *h. rathi1, p. rathi2, m. biyani3 1 – rajasthan university of health sciences college of medical sciences, jaipur (rajasthan), india 2 – mahatma gandhi medical college, jaipur (rajasthan), india 3 – sawai man singh medical college, jaipur (rajasthan), india background. use of hydroxychloroquine with or without azithromycin is repurposed in sars-cov-2 in the absence of definitive treatment. objective. to evaluate the association between the use of hydroxychloroquine and azithromycin when given alone or in combination on clinical outcomes and adverse drug reactions among lab confirmed sars cov-2 positive patients admitted in a covid tertiary care hospital of a university medical college. methods. a retrospective observational comparative study was conducted. covid-19 positive patients admitted in study hospital for management of covid-19 were enrolled into the study. the patients were categorized into 4 treatment groups based on having received the following treatment during hospitalization: (a) hydroxychloroquine with azithromycin, (b) hydroxychloroquine without azithromycin (hydroxychloroquine alone), (c) azithromycin alone, and (d) neither drug, defined as no receipt of either hydroxychloroquine or azithromycin in the record; other medications may have been dispensed. results. 800 patients were enrolled. mean±standard deviation of duration of hospital stay (in days) for study group a was 11.37±7.11, for group b was 8.37±4.77, for group c was 18.22 ± 5.69 and for group d was 6.12±2.97. mortality in group a was 29.74%, group b – 33.16%, group c – 0% and in group d – 1.32%. conclusion. among hospitalized patients with covid-19 treatment, group c was associated with good clinical outcome. however, the interpretation of these findings may be limited by the observational design. keywords: covid-19; hydroxychloroquine; azithromycin; sars cov-2 international journal of medicine and medical research 2021, volume 7, issue 1, p. 20-27 copyright © 2021, tnmu, all rights reserved *corresponding author: heena rathi, b.d.s, msc. (med-pharmacology) 3rd year, ruhs college of medical sciences, jaipur (rajasthan), 302033, india. e-mail: drheenarathi@gmail.com introduction in december 2019, several cases of pneumonia like disease were reported in the wuhan city of china [1, 2]. the world health organisation (who) named this disease covid-19. the causative agent for covid-19 is the severe acute respiratory syndrome coronavirus-2 (sars-cov2). sars-cov-2 is the newest of the family and is currently the cause of covid-19 across the world [3]. within months after its onset in china, this virus had spread involving most of the countries of the world. in march 2020, who declared it as a pandemic [4]. according to the recent available data by the end of december 2020, approximately 95 million populations worldwide and 10 million individuals in india have been diagnosed as covid-19 positive. until december 2020, the recovery rate in indian population was 95.77% while the mortality rate was 1.45 %. the sources of infection of sars-cov-2 are reported to be infected animal hosts and infected humans. bats [7] are considered to be initial hosts of this virus strain [5]. main modes of transmission for interhuman spread of sars cov-2 are respiratory droplets and contact transmission. patients of covid-19 commonly present with symptoms like fatigue, cough, fever, myalgia, and diarrhoea. most of the people infected with the virus experience mild-to-moderate respiratory illness and recover without requiring any special treatment. however, elderly and those with underlying medical problems or diseases are more likely to develop serious illness. research published till date has shown evidence that covid-19 cause cytokine storm [6]. some reports also revealed that patients of covid-19 are associated with hyper inflammation and increased production of cytokines such as interleukin (il)-1, 2, 6, 8, 10, and 17 [4, 7]. this may be the reason of tissue damage in the lungs of moderate to severely infected patients. reports have also suggested that cytokine storm may h. rathi et al. 21 in t e r n a l m e d ic in e issn 2413-6077. ijmmr 2021 vol. 7 issue 1 cause cellular demise and tissue injury in cardiac system which may lead to cardiovascular arrest [5]. conditions like ards and cardiac arrest require an emergent medical attention in an intensive care unit. since no drug therapy has been specifically and conclusively established for the prevention, control, and cure at the time of its onset. so, several drugs have been repurposed to manage the rapidly deteriorating public health situation. many initial researches published during the early months of 2020 had suggested that hydroxychloroquine is highly effective in both prophylaxis and treatment of covid-19 positive patients. in vitro studies have demonstrated that chloroquine and hydroxychloroquine can inhibit viral replication at multiple points in the initial phase of viral infection [8]. it is postulated to exert a direct antiviral activity by increasing intracellular ph resulting in decreased phagolysosome fusion, impairing viral receptor glycosylation [9]. various other studies showed the potential role of azithromycin in treatment of covid-19 patients. azithromycin is a potent immunomodulator with significant antiviral properties. azithromycin, a macrolide antibiotic, has invitro antiviral properties such as decreased viral replication, blocking entrance into host cells, and a potential immunomodulating effect [10]. according to other researches, combination of both the drugs may be more effective in curing the disease but some have also contradicted this line of treatment and several studies have even supported the statement that combination of both is harmful to the patient because the combination may add or increase the severity of their adverse effects. all the above reasons led us to carry out this pilot study including drugs hydroxychloroquine and azithromycin. the aim of this study was to evaluate clinical outcome and adverse drug reactions among hospitalised laboratory confirmed covid-19 positive patients treated with hydroxychloroquine and azithromycin either given alone or in combination. methods this study received ethical approval from institutional ethics committee. study design: a retrospective, observational, comparative study. study population: all laboratory confirmed covid-19 positive patients admitted in study hospital from march 2020 to july 2020. sample size: covid-19 positive patients have been admitted in study hospital for management of covid-19. out of these, all those patients, who matched the inclusion and exclusion criteria, were enrolled in the study. inclusion and exclusion criteria inclusion criteria: 1. all laboratory confirmed covid-19 positive patients admitted in ruhs-hms between march-july 2020. 2. patients of both genders and above the age of 12 years old. exclusion criteria: 1. patients whose hospital stay was less than 5 days (due to any reason). 2. incomplete case files. study groups: patients were categorized into 4 treatment groups based on having received the following treatment during hospitalization: (a) hydroxychloroquine with azithromycin, (b) hydroxychloroquine without azithromycin (hydroxychloroquine alone), (c) azithromycin alone, and (d) neither drug, defined as no receipt of either hydroxychloroquine or azithromycin in the record; other medications may have been dispensed. results a total of 800 case records of lab confirmed covid-19 positive patients admitted to ruhs hospital of medical sciences from march to july were reviewed and enrolled in the study. all the study groups were compared via anova test. this comparison included the parameters such as age, systolic blood pressure, diastolic blood pressure, spo2, and duration of hospital stay. mean±standard deviation of each parameter of all groups were calculated. the obtained results of anova test for all the study groups has p value less than 0.05, which represents a higher significance for the study. observations and results of this test is shown in table 1. 1. gender distribution of patients as per groups results and observations obtained are presented in fig. 1. 2. spo2 distribution as per group mean±standard deviation of spo2 for study group a was 89.6±7.1, for group b was 90.24±7.44, for group c was 91.63±5.34 and for group d was 96.12±3.62. results and observations obtained are presented in fig. 2. h. rathi et al. 22 in t e r n a l m e d ic in e issn 2413-6077. ijmmr 2021 vol. 7 issue 1 3. duration of hospital stay distribution as per group mean±standard deviation of duration of hospital stay (in days) for the study group a was 11.37±7.11, for group b – 8.37±4.77, for group c – 18.22±5.69 and for group d – 6.12±2.97. results and observations obtained are presented in fig. 3. 4. severity of illness in study population severity of illness in study population in the study is classified as: asymptomatic – flu-like symptoms, patients are not hospitalized, and recover at home. mild symptoms – runny nose, sore throat, congestion, and dry cough. moderate symptoms – high fever, tiredness and fatigue, and chest pain. patients with severe symptoms – respiratory distress syndrome (shortness of breath, increased blood pressure, and decreased oxygen saturation). patients in the critical stage – severe acute respiratory syndrome (sars) (high fever, chest pain, and breathlessness). patients with mild-moderate symptoms were categorized in one group and patients with severe symptoms and patients with critical stage were categorized in another group. chi-square test was applied for evaluation which was 72.73, and p value for this was table 1. anova of the groups (n=800) parameters anova significance age 12.46 0.000001 systolic bp 3.07 0.0271 diastolic bp 3.01 0.02971 spo2 (in %) 19.01 0.000001 hospital stay 103.03 0.000001 fig. 1. gender distribution of patients as per groups (n=800). results and observations obtained are presented in fig. 1. fig. 1. gender distribution of patients as per groups (n=800). 2. spo2 distribution as per group mean ± standard deviation of spo2 for study group a was 89.6 ± 7.1, for group b was 90.24 ±7.44, for group c was 91.63 ± 5.34 and for group d was 96.12 ± 3.62. results and observations obtained are presented in fig. 2. fig. 2. mean and sd of spo2 (in %). 3. duration of hospital stay distribution as per group 69,0% 70,2% 56,6% 63,2% 31,0% 29,8% 43,4% 36,8% 0,0% 10,0% 20,0% 30,0% 40,0% 50,0% 60,0% 70,0% 80,0% group a group b group c group d male female fig. 2. mean and sd of spo2 (in %). results and observations obtained are presented in fig. 1. fig. 1. gender distribution of patients as per groups (n=800). 2. spo2 distribution as per group mean ± standard deviation of spo2 for study group a was 89.6 ± 7.1, for group b was 90.24 ±7.44, for group c was 91.63 ± 5.34 and for group d was 96.12 ± 3.62. results and observations obtained are presented in fig. 2. fig. 2. mean and sd of spo2 (in %). 3. duration of hospital stay distribution as per group 69,0% 70,2% 56,6% 63,2% 31,0% 29,8% 43,4% 36,8% 0,0% 10,0% 20,0% 30,0% 40,0% 50,0% 60,0% 70,0% 80,0% group a group b group c group d male female h. rathi et al. 23 in t e r n a l m e d ic in e issn 2413-6077. ijmmr 2021 vol. 7 issue 1 0.0000001, which was highly significant for our study. severity of illness was highly significant with p-value 0.000001 (chi-square=72.73). results and observations obtained are presented in fig. 4. 5. comparison of the clinical outcome of all the groups on the basis of illness severity and presence or absences of co-morbidity (table 2). 6. adverse events observed during study total numbers of adverse events observed in this study were 3. two patients experienced itching over the body and they were associated to group a and 1 patient of group b experienced diarrhoea. observations and results are shown in table 3. discussion for the evaluation of the treatment efficacy in our study two variables were examined. the first was the duration of hospital stay and the second was outcome (discharged or death). in the present study, duration of hospital stay for group a was 8 days (iqr: 5-16), group b – 7 days (iqr: 5-9), group c – 18 days (iqr: 15-20), and group d – 5 days (iqr: 5-6). the mean±sd for group a, b, c, d was 11.37±7.11, 8.37±4.77, 18.22±5.69, and 6.12±2.97 respectively. p value was 0.000001 and was highly significant. group d had the shortest duration of hospital stay. possible reasons for this could be that in group d, 98.68% were mild-moderately ill. also, spo2 which was an important clinical feature in covid-19 was maximum for group d (median 97.5, iqr: 95-99, and mean±sd 96.12±3.62) with a p value of 0.000001. therefore, we can infer that milder disease severity and least deranged clinical features in group d may have resulted in faster recovery and a shorter duration of hospital stay. another reason can be that in group d only 14.47% patients were having underlying comorbidities. this could also be important factor since presence of co-morbidities is now known to adversely affect the course of illness. fig. 3. mean and sd of hospital stay (in days). mean ± standard deviation of duration of hospital stay (in days) for the study group a was 11.37 ± 7.11, for group b – 8.37 ± 4.77, for group c – 18.22 ± 5.69 and for group d – 6.12 ± 2.97. results and observations obtained are presented in fig. 3. fig. 3. mean and sd of hospital stay (in days). 4. severity of illness in study population severity of illness in study population in the study is classified as: asymptomatic – flu-like symptoms, patients are not hospitalized, and recover at home. mild symptoms – runny nose, sore throat, congestion, and dry cough. moderate symptoms – high fever, tiredness and fatigue, and chest pain. patients with severe symptoms – respiratory distress syndrome (shortness of breath, increased blood pressure, and decreased oxygen saturation). patients in the critical stage – severe acute respiratory syndrome (sars) (high fever, chest pain, and breathlessness). patients with mild-moderate symptoms were categorized in one group and patients with severe symptoms and patients with critical stage were categorized in another group. fig. 4. distribution of illness severity (n=800). chi-square test was applied for evaluation which was 72.73, and p value for this was 0.0000001, which was highly significant for our study. severity of illness was highly significant with p-value 0.000001 (chi-square = 72.73). results and observations obtained are presented in fig. 4. fig. 4. distribution of illness severity (n=800). 5. comparison of the clinical outcome of all the groups on the basis of illness severity and presence or absences of co-morbidity: table 2. comparison of clinical outcome among all groups a b c d hosp ital stay reco very dea th hosp ital stay reco very dea th hosp ital stay reco very de ath hosp ital stay reco very de ath mildmoder ate comorb id patient s 16.1 27.03 % (n=30 ) 0% (n= 0) 8.3 13.67 % (n=29 ) 0% (n= 0) 18.94 88.46 % (n=69 ) 0% (n= 0) 6.9 13.33 % (n=10 ) 0% (n= 0) 111 212 78 75 121 174 28 1 0 50 100 150 200 250 group a group b group c group d mild-moderate severe h. rathi et al. 24 in t e r n a l m e d ic in e issn 2413-6077. ijmmr 2021 vol. 7 issue 1 table 2. comparison of clinical outcome among all groups a b c d h os pi ta l st ay re co ve ry d ea th h os pi ta l st ay re co ve ry d ea th h os pi ta l st ay re co ve ry d ea th h os pi ta l st ay re co ve ry d ea th mild-moderate comorbid patients 16.1 27.03% (n=30) 0% (n=0) 8.3 13.67% (n=29) 0% (n=0) 18.94 88.46% (n=69) 0% (n=0) 6.9 13.33% (n=10) 0% (n=0) mild-moderate patients without comorbidity 10.6 72.97% (n= 81) 0% (n=0) 8.4 86.32% (n=183) 0% (n=0) 8.25 11.53% (n=9) 0% (n=0) 6.3 86.66% (n=65) 0% (n=0) severe comorbid patients 11.97 33.88% (n=41) 42.14% (n=51) 7.7 16.66% (n=29) 56.32% (n=98) 21.04 100% (n=28) 0% (n=0) 14 0% (n=0) 100% (n=1) severe patients without comorbidity 7.9 9.09% (n=11) 14.8% (n=18) 8.2 9.77% (n=17) 17.24% (n=80) 0 0% (n=0) 0% (n=0) 0 0% (n=0) 0% (n=0) table 3. adverse events observed during study adr group a group b group c group d diarrhoea 0 1 0 0 itching 2 0 0 0 nil 230 385 106 76 total 232 386 106 76 the longest duration of hospital stay was in group c which was having median 18, iqr: 15-20 and mean±sd for 18.22±5.69. despite of having borderline spo2 (median 92, iqr:88-96 and mean±sd 91.63 ±5.34) with 73.58% patients of this group being mild-moderately ill, still this group had the longest duration of hospital stay. possible reasons could be that 92.45% patients of this group were having underlying comorbidities. second variable which was observed in our study was clinical outcome. two possible clinical outcomes were considered; recovered and discharged – meaning a good clinical outcome, and death -denoting a poor clinical outcome. samia arshad et. al [9] found in a retro spective observational study that overall mortality was 18.1%, 20.1% by treatment with the combination of hydroxychloroquine with azithromycin, 13.5% with hydroxychloroquine alone, 22.4% with azithromycin alone and 26.4% with neither drug. according to their results hydroxychloroquine provided 66% hazard ratio reduction and hydroxychloroquine with azithromycin 71% compared to neither treatment (p<0.001). in the present study good clinical outcome was observed in group c. in group c 100% of the patients were discharged whether in group a only 66.37% were discharged. various factors could cause this. chi square test was applied to measure the significance of illness severity in the population. results of this test showed p=0.000001, it means severity of illness was highly significant. in group c 98.68% patients were mild moderately ill and only 1.34% were severely ill. another reason could be that spo2 as spo2 was 91.63±5.34 (p value 0.000001), which showed high significance. it may have led to decreased mortality in this group. to validate this overall result sub-group analysis was performed. thus it was established that in the patients, who were mild to moderately ill with comorbidities, maximum recovery or good clinical outcome were observed in group c (88.46%). similarly, when severely ill comorbid patients were evaluated again maximum recovery or good clinical outcome were observed in group c (100%). it was also observed that in both groups of comorbidities (mild-moderate and severe) the longest duration of hospital stay was in group c. in group c the mean of h. rathi et al. 25 in t e r n a l m e d ic in e issn 2413-6077. ijmmr 2021 vol. 7 issue 1 hospital stay for mild-moderate comorbid patients was 18.24 and for severely ill co-morbid patients it was 21.04. it proved that in our study effective treatment in the co-morbid patients was observed in group c among all patients but it took more time to recover comparatively to other groups. however, in patients without co-morbidities maximum recovery was evidenced in group b. for group b patients, who were mild-moderately ill with no co-morbidities, recovery was 86.32% and for severely ill without co-morbidity, recovery was 9.77%. in group d recovery in mildmoderate non-co-morbid patients was 86.6%, which was slightly higher than in group b, but for severely ill non-comorbid patients, recovery rate was higher in group b. so, the overall recovery for non-comorbid patients was higher in group b. the duration of hospital stay for mild-moderately ill group b patients with comorbidities was 8.3 and for patients without comorbidities it was 8.4. for severely ill group b patients with comorbidities it was 7.7 and for patients without comorbidities – 8.2. this data also revealed that patients of group b without comorbidities had longer duration of hospital stay than the non-co-morbid patients. poor clinical outcome was measured by number of deaths. the highest mortality was observed in group b with 33.16% death. factors which influenced this result could be that 70.2% of patients of this group were males. males were more prone to lung infection due to their habits like smoking. smoking habit is associated with males in indians comparative to females. therefore, this could be a reason for a greater number of deaths. in this group 45.07% patient were severely ill. so, risk was higher for them compare to mild-to-moderate ill patients. 40.9% patients with underlying comorbidities were present in this group which can have led to poor clinical outcome. sub-group analysis revealed that there were no deaths in mild-moderately ill patients in both co-morbid and non-co-morbid groups. all the deaths were associated with severely ill patients. the highest mortality among all severely co-morbid patients was associated with group d, which was 100%. there were no patients in group d with non-co-morbidity. after that, the maximum deaths of non-cocomorbid patients were seen in group b (17.24%). results of our study revealed that group c treatment in comorbid patients were more effective and similar treatment in group d was not safe. recovery ratein mild-moderately ill comorbid patients was higher (27.03%) in the group a compare to group b (13.67%). however, in mild-moderately ill non-co-morbid patients, higher recovery was observed in group b (86.32%) compared to group a (72.97%). in severely ill co-morbid patients decreased mortality (42.14%) and increased recovery (33.88%) was seen in group a compare to group b, where mortality was 56.32% and recovery was 16.66%. in non-co-morbid severely ill patients, recovery in both groups (a – 9.09% and b – 9.77%) was almost the same but mortality was lesser in group a (14.80%) compare to group b (17.24%). these findings revealed that on the whole group c treatment was the best among all and among groups a and b, group a was better than b. similar results have been shown in the study by matthieu million et. al [11]. they also conducted a retrospective analysis of early treatment of covid-19 patients with hydroxychloroquine and azithromycin. a poor clinical outcome was observed in 4.3%. they concluded that this combination was safe and associated with low fatality rate in patients. adverse events distribution in the study in the present study safety of the treatment was also observed by adverse events. very few adverse events were reported and they were mild which were associated with group a and b. reason for this is because of retrospective study, we are unable to analyse all those events which were not mentioned in case record files. conclusion the present study concluded that no deaths were observed in mild-moderately ill patients with or without comorbidity. among four groups, treatment in group c (azithromycin 500 mg od for 5 days) had better results. between groups a (hydroxychloroquine with azithromycin) and b (alone hydroxychloroquine 400 mg bd on day 1 followed by 200 mg bd on day 2 to 5), treatment in group a had better outcome. the duration of hospital stay was longer for comorbid patients compare to patients with no comorbidity. limitations though, we did our best to make this study without any blemish but several limitations make the scope for future study. it was a retrospective observational study. therefore, regarding the data we had to rely on the case records and the mentioned records. there might be a few findings which were not h. rathi et al. 26 in t e r n a l m e d ic in e issn 2413-6077. ijmmr 2021 vol. 7 issue 1 mentioned in the records but may have been present in the patients. we could not note them. time constraint was the major limitation. so, we were unable to analyse the data on the basis of each sub-group. the data were incomplete for some patients because services were overwhelmed. ct scans, ecg and potential cofounders such as inflammatory markers associated with severity of the disease were not frequently measured/recorded. mortality was limited to in-hospital death and patients, who were discharged or referred, were assumed to still be alive during study period. because of retrospective analysis, we were not able to record all adverse events. therefore, the evaluation of the safety was not adequate. for this, prospective randomized controlled trials may have been conducted at that time. conflict of interest authors declare no conflict of interest. acknowledgment there had been no funding supports for this research. the total cost of completing the research work was carried by authors own finance. author’s contribution heena rathi, priyanka rathi – conceptualization, methodology, formal analysis, writing – original draft; heena rathi, mohit biyani – data curation, investigation; heena rathi, priyanka rathi, mohit biyani – writing, reviewing and editing. наслідки та побічні реакції при лікуванні пацієнтів з covid-19 гідроксихлорохіном та азитроміцином окремо та в комбінації *h. rathi1, p. rathi2, m. biyani3 1 – rajasthan university of health sciences college of medical sciences, jaipur (rajasthan), india 2 – mahatma gandhi medical college, jaipur (rajasthan), india 3 – sawai man singh medical college, jaipur (rajasthan), india вступ. застосування гідроксихлорохіну самостійно або у комбінації з азитроміцином – одна з опцій терапії sars-cov-2 за відсутності чітко визначеного лікування. мета. оцінити вплив застосування гідроксихлорохіну та азитроміцину у якості монотерапії чи у комбінації на клінічні результати та частоту розвитку побічних реакцій серед пацієнтів з лабораторно підтвердженої інфекцією sars-cov-2, які були госпіталізовані до спеціалізованої covid-лікарні при медичному коледжі університету. методи. було проведене ретроспективне спостережне порівняльне дослідження. у дослідженні брали участь госпіталізовані пацієнти з лабораторно підтвердженим діагнозом covid-19. пацієнти були розділені на 4 групи, базуючись на лікуванні яке вони отримували під час госпіталізації: (a) гідроксихлорохін з азитроміцином, (b) гідроксихлорохін без азитроміцину (лише гідроксихлорохін), (c) лише азитроміцин та (d) жоден з препаратів не призначався; могли застосовуватися інші ліки. результати. було залучено 800 пацієнтів. середнє значення±стандартне відхилення тривалості перебування в лікарні (у днях) для досліджуваної групи а становило 11,37±7,11, для групи в – 8,37±4,77, для групи с – 18,22±5,69 та для групи d – 6,12±2,97. смертність у групі a становила 29,74%, групі b – 33,16%, групі c – 0%, а групі d – 1,32%. висновок. серед госпіталізованих пацієнтів з covid-19 лікування у групі с (лише азитроміцин) було пов'язане з позитивними клінічними результатами. однак інтерпретувати висновки остаточно неможливо через обмеження і рамки проведеного дослідження. ключові слова: covid-19; гідроксихлорохін; азитроміцин; sars-cov-2. information about the authors heena rathi, b.d.s, msc. (med-pharmacology) 3rd year, rajasthan university of health sciences college of medical sciences, jaipur, india orcid 0000-0002-8056-8768, e-mail: drheenarathi@gmail.com priyanka rathi, associate professor, mahatma gandhi medical college and hospital, jaipur, india orcid 0000-0002-6400-6888, e-mail: drpriyankarathi@gmail.com mohit biyani, m.b.b.s, m.d (anaesthesia) 3rd year, swai man singh medical college, jaipur, india orcid 0000-0002-5543-6480, e-mail: mohit2.ncity@gmail.com h. rathi et al. 27 in t e r n a l m e d ic in e issn 2413-6077. ijmmr 2021 vol. 7 issue 1 references 1. lai cc, shih tp, ko wc, tang hj, hsueh pr. severe acute respiratory syndrome coronavirus 2 ( s a r s c o v 2 ) a n d c o ro n a v i r u s d i s e a s e 2 0 1 9 (covid-19): the epidemic and the challenges. int j antimicrob agents 2020 feb 17:105924. doi: 10.1016/j.ijantimicag.2020.105924. 2. wang ls, wang yr, ye dw, liu qq. a review of the 2019 novel coronavirus (covid-19) based on current evidence”. int j antimicrob agents 2020. 3. david n. juurlink. safety considerations with chloroquine, hydroxychloroquine and azithromycin in the management of sars-cov-2 infection. canadian medical association journal 2020;192 (17). 4. wen zhang et. al. the use of anti-inflammatory drugs in the treatment of people with severe coronavirus disease 2019 (covid-19): the perspectives of clinical immunologists from china. clinical immunology 2020; 214: 108393. 5. shi et al. an overview of covid-19. j zhejiang univ-sci b (biomed & biotechnol) 2020;21(5):343-60. 6. yamauchi k, shibata y, kimura t, abe s, inoue s, osaka d, et al. azithromycin suppresses interleukin-12p40 expression in lipopolysaccharide and interferon-gamma stimulated macrophages. int j biol sci 2009;5(7):667-78. 7. huang c, wang y, li x, ren l, zhao j, hu y, et al. clinical features of patients infected with 2019 novel coronavirus in wuhan, china. lance 2020; 395(10223):497–506. doi: 0.1016/s0140-6736(20) 30183-5. 8. silvano esposito, silvana noviello, pasquale pagliano. update on treatment of covid-19: ongoing studies between promising and disappointing results. le infezioni in medicina 2020; 2:198-211. 9. samia arshad et.al. treatment with hydroxychloroquine, azithromycin, and combination in patients hospitalized with covid-19. international journal of infectious diseases 2020;97:396-403. 10. tran dh, sugamata r, hirose t, suzuki s, noguchi y, sugawara a, et al. azithromycin, a 15-membered macrolide antibiotic, inhibits influenza a(h1n1) pdm09 virus infection by interfering with virus internalization process. j antibiot 2019;72 (10):759–68. doi:10.1038/s41429-019-0204-x. 11. matthieu million et.al. early treatment of covid-19 patients with hydroxychloroquine and azithromycin: a retrospective analysis of 1061 cases in marseille, france. travel medicine and infectious disease 2020:1-8. received 23 feb 2021; revised 20 apr 2021; accepted 20 may 2021. this is open access article distributed under the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. h. rathi et al. issn 2413-6077. ijmmr 2021 vol. 7 issue 258 d e n t is t r y doi 10.11603/ijmmr.2413-6077.2021.2.12486 current diagnosis, prevention and treatment of dry socket (literature review) n.s. hutor i. horbachevsky ternopil national medical university, ternopil, ukraine background. tooth extraction is the most common surgical intervention in the outpatient practice of a dental surgeon. in most cases, bone wound healing is uncomplicated. however, in some patients the course of healing is complicated by acute inflammatory process of dry socket (alveolar osteitis), the frequency of which increases with increasing trauma of surgery. in dentally healthy patients with typical tooth extraction, dry socket occurs in 2.55%, with difficult – in 9.84%, with atypical extraction of the lower wisdom tooth – in 31.03% of cases. in the presence of diabetes, the incidence of dry socket with typical removal is much higher and according to various authors it is 9.7-13.5%. objective. the aim of the research was to study the publications and their analysis on the current diagnosis, prevention and treatment of patients with dry socket. methods. scientific sources were the articles in world periodicals on dental surgery and therapy, microbiology as well as some electronic resources and patents. results. at present, many techniques, methods, schemes, compositions for treatment of various forms of dry socket are established. most often, antibacterial drugs or their combinations with other drugs are used. however, antibacterial drugs do not always have a pronounced anti-inflammatory effect due to the high resistance of the oral microflora. in these cases, contemporary drugs in the form of a composition with long-term antiseptic, anti-inflammatory and analgesic action are rational. conclusions. the action of a flupetsal composition, which contains antimicrobial and immunomodulatory drug flurenizide, was clinically proven for treatment of patients with dry socket; the properties of the available ingredients provide a high disinfectant effect, prevent development of inflammation in the tissues and its transition to purulence. a contemporary effective method of treating patients with dry socket is carried out according to the developed method. keywords: dry socket; treatment; compositions; medicines; flupetsal. n.s. hutor international journal of medicine and medical research 2021, volume 7, issue 2, p. 58-65 copyright © 2021, tnmu, all rights reserved *corresponding author: nataliia hutor, phd, md, associate professor of the department of surgical dentistry, i. horbachevsky ternopil national medical university, 3 chehova str., 46027 ternopil, ukraine. e-mail: hutor@tdmu.edu.ua introduction outpatient dental care is one of the most popular types of medical care [1]. tooth ext raction is the most common surgical dental surgery performed at outpatient clinic [2]. improving the quality of dental surgery depends on the qualifications of a dental surgeon and effective drugs for treatment after reducing complications. improvements in dental treatment methods, recent dental equipment and the latest filling materials have significantly limited the indications for tooth extraction surgery. indications for tooth extraction are pathological processes caused by complicated caries, which cannot be eliminated by other methods of treatment. sometimes tooth preservation is not possible due to the large resorption of alveolar tissue caused by periodontal disease. improperly erupted teeth are removed only when orthodontic treatments are inef fective. removal of overcomplete teeth that erupted outside the dental arch also is possible. a simple or atypical method of hollowing out and alveolotomy are often used. frequently these methods are combined, an experienced doctor chooses the least traumatic. the surgery can be very simple or technically complex, long, sometimes requiring hospitalization of the patient. surgical intervention in the removal of teeth is the rupture of periodontal tissues associated with simultaneous damage to the bone tissue of the alveolar processes of the jaws resulting in a wound of varying degrees of infection [2]. during the surgery, it is necessary to remember to treat the tissues sparingly, to avoid unnecessary injuries and ruptures of the mucous membrane. in the case of tooth extraction, the peculiarities of the structure of the dental-maxillary system, general diseases of the patient and local pathological processes issn 2413-6077. ijmmr 2021 vol. 7 issue 2 59 d e n t is t r y are taken into account. in other cases, postoperative pain continues for a long time after tooth extraction due to a violation of tooth extraction techniques and improper treatment of the cavity. anaesthesia is a very important stage of the surgery. anaesthetic effect and effective anaesthesia provide for surgical manipulations, allow avoiding tissue injuries [2, 3]. the wound is healed by the type of secondary tension under the cover of a blood clot. in dental practice, it is important to eliminate the negative impact of psycho-emotional state on the patient’s cns after surgery and the feeling of post-extraction pain [4]. currently, foreignmade painkillers are used in various dosage forms, which are produced using the latest pharmaceutical technologies [5]. in recent years, dentists have used ketones (syn. tromethamine, ketorolac) – a non-steroidal non-narcotic analgesic that relieves pain of various origins. its positive effect on hemodynamics and oxygenation of blood is proved. anaesthesia, proper selection of tools for removal, compliance with the rules of asepsis and antiseptics, timely treatment and prevention minimize the possible complications after tooth and root removal [6]. review after the tooth is removed, the hole is filled with a blood clot. the formation of a blood clot in the hole is the main condition for the healing of the postoperative wound by primary tension. the blood clot should be properly formed with a sterile tissue. the first stage of healing is the formation of a blood clot in the hole with fibrin deposition, which occurs within an hour after the tooth extraction operation. for the next 4-5 days after the tooth extraction surgery, the blood clot is replaced with granulation tissue. then the granulation tissue is replaced by connective tissue rich in collagen fibres. at this time, the epithelium at the edges of the hole begins to proliferate and grow rapidly towards the centre of the hole to completely cover it in 24-35 days after tooth extraction. in about 1 month, a net of spongy beams appears in the area of the hole; in 2-2.5 months the bone structure of the alveoli becomes almost identical to the surrounding bone tissue; in 3-4 months the formation of bone tissue is finally completed. during this period, the alveolar edges are resorbed and rebuilt, they become lower and thinner than before tooth extraction [6]. one of the most common complications associated with direct tooth extraction is bleeding from the hole. bleeding may be from vessels of the mucous membrane, the periosteum of the hole, the granulation tissue left in the hole, or from damaged branches of the interdental artery during the curettage of the hole. these types of bleeding depend on the degree of injury to the soft and bone tissues that develop during the surgery. in some cases, bleeding may occur later due to purulent melting of the clot that closes the lumen of the vessel. the cause should be established and the bleeding is stopped by stitching the soft tissues, tamponade of the hole with iodoform turund or haemostatic sponge, hemophobin [7]. if there is pain after extraction in the absence of a blood clot, it is necessary to scrape the hole with a sharp surgical spoon after anaesthesia, clean it of the softened bone and form a “fresh” full­fledged blood clot, but do not always completely scrape the hole. inaccuracies in the treatment of periodontal and periodontal diseases in the absence of radiographs were observed in more than 75% of patients [8]. thus, the qualification of the dentist­sur­ geon, preliminary x-ray examination, choice of analgesic, high quality haemostasis and management after the extraction wound is the key to the prevention of dry socket. dry socket (dolores post extractionem) is inflammation of the walls of the alveoli of the tooth with suppuration of the bone wound after its removal [9]. dry socket is significant among the complications after tooth extraction surgery. according to the authors, dry socket occurs from 33.2% to 35% of the number of cases of all complications after tooth extraction [10]. in the monograph by o.o. timofeev it is established that 33.2% of removed teeth are complicated by dry socket. treatment of dry socket should be preceded by x-ray examination of the tooth removal site, in 92.3% of cases the radiograph reveals bone fragments, tooth root and others that are important in choosing treatment tactics. if examination of a patient with dry socket does not reveal a blood clot in the hole, after anaesthesia it is necessary to cure the hole, clean it of the softened bone and form a “fresh” full­fledged blood clot. if a blood clot is partially preserved in the tooth cavity, the cavity should be rinsed with a warm solution of antiseptic n.s. hutor issn 2413-6077. ijmmr 2021 vol. 7 issue 260 d e n t is t r y (furacillin, chlorhexidine, givalex, flupetsal, etc.) after anaesthesia the alveolar bone fragments, food debris and decay products should be removed. then an excavator or a sharp surgical spoon is used to remove the surface layer of the blood clot and fill the hole with the drug (iodoform turunda, solcoseril gel, gelevin, oxycelodex, dermazin cream) [11]. the cause of dry socket is caused by objective and subjective factors. objective causes of dry socket occur after removing of retained teeth, including third molars of the mandible, tooth removal surgeries, traumatic tooth extraction without a blood clot in the hole, after prolonged bleeding, repeated removal of adjacent tooth roots [12]. anaesthesia, proper selection of tools for removal, compliance with the rules of asepsis and antiseptics, timely treatment and prevention measures minimize the possible complications after tooth and root removal [13]. currently, the issue of haemostasis after tooth extraction is still topical, despite the sufficient number of haemostatic agents. bleeding after tooth extraction is stopped by local haemostatic agents. studies have shown the advantage of collagen sponge over hemophobin and oxycelodex [14]. an antiseptic sponge with kanamycin is promising for prevention of complications after tooth extraction. antiseptic sponge with kanamycin has not only pronounced haemostatic, antimicrobial properties, but also stimulates reparative processes in the wound leading to its high therapeutic efficacy [14]. patients with hemophilia a should have their teeth removed in the haematology department. patients with pathology of the hepatobiliary system are allowed local targeted haemostatic agents [15]. the results of research prove that the causes of bleeding after tooth extraction might be mild and latent forms of coagulopathies and thrombocytopathies, which have certain clinical and laboratory features and require appropriate treatment [16, 17]. clinical observations in patients with ventricular haemorrhage caused by bone trauma due to difficult tooth or atypical root removal, as well as the experience in use of gelevin, oxycelodex, traumacil and honsuride dental pins have been described [18]. although the risk of inflammatory complications in such interventions is not significant, it is necessary to provide antimicrobial treatment of the wound with local antiseptics. the choice of a drug for local preoperative prevention of infectious complications in tooth extraction surgeries is determined by the nature of surgery, its duration, trauma, degree of wound infection, individual sensitivity to a drug, availability and other general and local factors [19, 20]. it is established that the oral cavity is under the influence of both the body and the envi­ ronment. clinical, epidemiological, microbiological studies have shown that the violation of the microbial biocenosis is one of the main factors in the emergence and development of dry socket [21-24]. the diagnosis of giardiasis in the oral cavity are described in some research. yu.v. lakhtin [25], studying the morphobiological features of oral protozoa, suggested the most rational methods of diagnosis and treatment of generalized periodontitis in the invasion of oral protozoa. thus, the literature suggests that, one of the reasons for development of dental diseases is a wide range of microorganisms. the use of microbiological technologies allows effective treatment and prevention of dental diseases [26, 27]. it is established that the main condition for rational antimicrobial therapy is the isolation of pathogens from the source of infection, their identification and determination of antibiotic susceptibility. currently, due to the lack of bacteriological results on the day of drug administration, the choice of antibacterial drug, dosage and duration of its reception is determined mainly empirically [28]. the selected drug does not always adequately affect the microorganisms of the lesion, which does not improve the patient’s condition and leads to significant disruption of microbiocenosis, re­ duced local and general immunological reactivity, deficiency or elimination of obligate microflora of the oral cavity. the irrational, often uncontrolled and widespread use of antibiotics at high rates of bacterial reproduction leads to acceleration of resistant strains, and, consequently, reduces the effectiveness of drugs used. thus, bacteriological research is urgent for the final diagnosis and treatment planning, justification of indications for antimicrobial therapy and evaluation of its effectiveness [29, 30]. it is proved that the microflora of the serous or purulent contents of the post-extraction area is quite diverse that should be taken into account in combined antimicrobial therapy n.s. hutor issn 2413-6077. ijmmr 2021 vol. 7 issue 2 61 d e n t is t r y aimed at various components of microbial associations (including fungi). the variety of pathogens of nonspecific and specific inflam­ matory processes, their different sensitivity to antibiotics have determined a large number of antibacterial drugs that are offered in clinical practice. however, the widespread use of antibacterial therapy has led to an increase in the number of resistant strains and polyresistance. this is due to both chromosomal and non-chromosomal mechanisms (r-plasmids that provide the transfer of genetic information during recombination). r-plasmid genes control various mechanisms of resistance to antibacterial drugs, primarily the bacterial synthesis of enzymes such as β­lactamases, which destroy antibiotics. there are different ways and methods of administration of antibacterial drugs. however, in the treatment of dry socket local administration of drugs are used in the form of solutions for short-term washing of the hole, water-soluble ointments – as part of complex drugs, antibiotics are introduced for electrophoresis etc. administration of the main drugs may be combined with the of antibiotics in injections or tablet form. the method of administration of the antibacterial drug into the body depends on its solubility, toxicity, irritant effect, ability to be absorbed through the gastrointestinal tract, and in some cases taking into account patient’s condition. it is established that all antibacterial agents are divided into drugs for the most common use (drugs of choice), for severe cases and support. maxillofacial infections require a similar treatment; antibacterial drugs, both for local and general treatment, can be used. antibacterial therapy to obtain data on the composition of the microflora and its sensitivity is chosen taking into account the literature on the highest frequency and structure of pathogens depending on the nosological form of the disease [30, 31]. according to the literature, main drugs for treatment (furacillin, lysozyme, balis-2) should be used. the sharp decrease in their antibacterial activity is caused by developed resistance to pathogens of purulent­inflammatory diseases of the maxillofacial area and neck (especially odontogenic); thus, their use is impractical. treatment and prevention of dry socket involves various antimicrobial drugs depending on the sensitivity of the isolated microorganisms. since chemotherapeutic drugs are administered through the oral cavity to treat diseases of various organs and systems of the body, it is necessary to study their influence on the micro­ biocenosis of various human habitats, possible development of resistance of microorganisms to drugs. the study of the physiological microbial system, microbial landscape and identification of oral microorganisms during various dental diseases provides not only objective data on the nature of the pathological process, but can significantly assist in diagnosis, choice of anti­ biotics, treatment plans, oral hygiene procedures, and formation of a protective physiological microbial system [31]. nevertheless, the volume of primary medical care is large and a mass microbiological research in dental pathology is quite complex and economically unprofitable, it can be useful and justified. various microorganisms can be detected using microbiological research methods such as bacterioscopic, bacteriological, serological, genetic and immunological. the material for microbiological examination in dry socket is the contents of the hole, nose, throat, saliva, scrape from the tongue, especially in its posterior parts. it must be collected in the morning on an empty stomach with a sterile swab. therefore, the reasons for the development of dry socket are violation of microbiocenosis of the oral cavity towards the predominance of aggressive microflora, the complexity of micro­ biological methods, insufficient effectiveness of antibacterial drugs that necessitates the search for new methods of diagnosis and treatment. the problem of prevention of postoperative complications, purulent­inflammatory diseases of the maxillofacial area is a topical issue for scientists and practitioners. in order to prevent postoperative complications in outpatient surgeries on the jaw, it is suggested to use drugs that contain chlorhexidine bigluconate [32-34]. chlorhexidine bigluconate 0.05%, 0.2% aqueous solution in the form of irrigation, rinsing, applications, affects bacteroids, actinomycetes, is effective in the treatment of dry socket. chlorhexidine is established to have not only antimicrobial but also anti­inflammatory effect. for the favourable course of the first phase of the wound process using the film diplendent hd for isolation of the bone wound in the first phase of healing of purulent­in­ flammatory diseases of the maxillofacial area new combined drugs were used: chlorhexidine + n.s. hutor issn 2413-6077. ijmmr 2021 vol. 7 issue 262 d e n t is t r y calcium carbonate called elgidium “pierre fabre medicament”, chlorhexidine + metronidazole “metrogil dent”, chlorhexidine + lidocaine called lidochlor by unique pharmaceutical laboratories; chlorhexidine + thyrotricin + lidocaine under the trade name “trachisan” [35, 36]. for prevention of infectious­inflammatory complications and dry socket, restoration of microbiocenosis in surgical dentistry, phyto rinsing of the oral cavity “naturosept” is used for patients after tooth extraction that reduces development of opportunistic microflora in the tooth cavity by 20%. [37, 38]. the drugs used for prevention: elgidium (toothpaste), elugel (gel, 0.2%), eludril (solution, 0.1%). this preparation allows heeling the wound in the oral cavity as a “clean” wound and preventing micro bial infection during surgery. antiseptic and disinfectant “d08a” are the drugs to improve trophism, “d0zan” to stimulate regeneration processes. it is recommended to use doxidine, gramicidin, furagin, chloramphenicol in various combinations: in the form of solution, ointment, gel [38]. in outpatient surgical and dental practice, antibiotic prophylaxis is prescribed in two cases: high risk of postoperative infection, secondary (opportunistic) infection, which develops on the background of a burdensome history and is a direct threat to the patient’s life. general requirements for the choice of antibiotic for dental surgeries, i.e.: the spectrum of action of the antibiotic should cover microflora of the patient, the drug should at least induce resistance of microflora, the antibiotic should easily penetrate into the tissues in the area of surgery and excrete antibiotic fluid, gums in tissues, wounds, should exceed the minimum inhibitory concentration for possible pathogens during surgery, the antibiotic should be characterized by minimal side effects (do not interact with anaesthetics, analgesics and other drugs). for preoperative prevention, the antibiotic is chosen according to the peculiarities of its pharmacodynamics, pharmacokinetics and spectrum [39-42]. givalex is one of the remedies that can be used for application and irrigation with preventive and curative effects in dry socket. the antimicrobial actiion of givalex is caused by hexitidine, which has antibacterial effect on gram-positive, gram-negative microorganisms. givalex has antifungal and weak bactericidal action [41]. a new antiseptic drug “gorosten” is promising in prevention of dental diseases [43-47]. the antiseptic “natursept” of antiseptic, wound­healing and anti­inflammatory action, is quite effective [37]. in contemporary dental practice, ukrainianmade dimexid is most often used in combination with well-known antiseptic, disinfectant, antibacterial, anti-inflammatory, anaesthetic agents in order to expand the spectrum of action on the resistant microflora of the oral cavity, to achieve the best clinical effect. the use of dimexid with various drugs in the complex treatment of dental patients in outpatient dental practice has proved to have positive results. the day reduction or control of pain, facial oedema, cessation of discharge, normalization of body temperature, appetite, sleep, full recovery were evidenced. the authors confirmed significant antiseptic and anti­in­ flammatory effect of dimexid and the feasibility of its use in treatment of various inflammatory diseases in dental patients [48]. in addition to antimicrobial action, dimexid has a local anesthetic, anti­inflammatory, desensitizing, anti­ fungal, high penetrating action, activates and potentiates the effect of drugs. the largest arsenal of drugs is used in the postoperative period, especially widely represented means for processing the postoperative hole. thus, parasept, autopack, and septopack bandages are effective for interventions on the alveolar process. sodium mefenamate and methyluracil are administered to reduce the treatment duration. the effectiveness of “aloroma” and trichopol, “alvostaz” and “povisep”, “alvogyl” and polybiolin-curiosone mixture for treatment of dry socket has been proven [49, 50]. currently, according to the law of ukraine “on medicinal products”, a new drug flurenizide has been widely used [51-55]. “flupetsal”, the composition based on flureniside, has antimicrobial and immunomodulatory properties and significant therapeutic effect [56-58]. conclusions thus, the contemporary drug composition of “flupetsal” according to the developed method is quite effective, affordable, low-cost for treatment of patients with dry socket. conflict of interests authors declare no conflict of interest. n.s. hutor issn 2413-6077. ijmmr 2021 vol. 7 issue 2 63 d e n t is t r y сучасні підходи щодо діагностики, профілактики та лікування альвеоліту (огляд літератури) н.с. гутор тернопільський національний медичний університет імені і.я. горбачевського моз україни, тернопіль, україна вступ. екстракція зуба є найчастішим оперативним втручанням в амбулаторній практиці хірургастоматолога. у більшості випадків загоєння кісткової рани проходить без ускладнень. однак у частини пацієнтів перебіг загоєння ускладняються гострим запальним процесом – альвеолітом, частота виникнення якого зростає по мірі росту травматичності оперативного втручання. у соматично здорових пацієнтів при типовому видаленні альвеоліт виникає у 2,55%, при складному – у 9,84%, при атиповому видаленні нижнього зуба мудрості – у 31,03% випадків. при наявності діабету відсоток виникнення альвеоліту при типовому видаленні значно вищий і за даними різних авторів складає 9,7-13,5%. мета. метою нашого огляду було дослідження опублікованої інформації та її аналіз щодо застосування сучасної діагностики, профілактики та лікування у хворих на альвеоліт. методи. науковими джерелами були статті у зарубіжних періодичних виданнях з хірургічної та терапевтичної стоматологій, мікробіології, електронні ресурси, патенти. результати. на сьогоднішній день відомо багато методик, методів, схем, композицій для лікування різних форм альвеоліту. найчастіше при цьому використовуються антибактеріальні препарати або їх комбінації з іншими лікарськими препаратами. однак антибактеріальним препаратам не завжди притаманний виражений протизапальний ефект через високу резистентність мікрофлори ротової порожнини. раціональним в цих випадках є місцеве застосування лікарських препаратів в вигляді композиції з тривалою антисептичною, протизапальною та анальгезуючою дією. висновки. клінічно підтверджено дію композиції під назвою „флупетсаль”, яка містить протимікробний та імуномодуляційний лікарський засіб флуренізид для лікування хворих на альвеоліт, властивості наявних інгредієнтів забезпечують високий знезаражувальний ефект, запобігають розвитку процесу запалення у тканинах та його переходу в гнійно-некротичну фазу. сучасний, ефективний метод лікування хворих на альвеоліт здійснюють згідно з розробленою методикою. ключові слова: альвеоліт; лікування; композиції; лікарські засоби; „флупетсаль”. information about the authors natalia s. hutor – phd, md, associate professor of the department of surgical dentistry, i. horbachevsky ternopil national medical university, ternopil, ukraine. orcid 0000­0002­9975­8369, e­mail: hutor@tdmu.edu.ua references 1. varava gn, tereshina tp, raiswich oe, et.al. the organization of the work of oral hygiene offices for the prevention of dental diseases: guidelines. – kyiv: 2008;42. 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[in ukrainian]. received 23 november 2021; revised 30 november 2021; accepted 10 december 2021. this is open access article distributed under the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. n.s. hutor issn 2413-6077. ijmmr 2021 vol. 7 issue 2 5 p u b l ic h e a lt h a n d e p id e m io l o g y *corresponding author: priyanka rathi, professor, department of pharmacology, mahatma gandhi medical college and hospital, jaipur, 302033, india. email: drpriyankarathi@mgumst.org international journal of medicine and medical research 2021, volume 7, issue 2, p. 5-14 copyright © 2021, tnmu, all rights reserved g. soni et al. doi 10.11603/ijmmr.2413-6077.2021.2.12636 current trends in prescribing pattern of anti-migraine drugs in patients of migraine at a tertiary care teaching hospital g. soni, a. vohra, s. jain, *p. rathi, p. goswami mahatma gandhi medical college and hospital, jaipur, india background. an inappropriate prescribing pattern of antimigraine drugs by doctors may often encourage inappropriate self-medication by patients because of the asymmetry of medical information. objective. the study is aimed to assess the current trends in prescribing patterns of anti-migraine drugs, rationality of prescription, and pattern of migraine severity in patients of migraine. methods. a cross sectional study was conducted at a tertiary care teaching hospital. all the prescribing details including patient’s demographic details, diagnosis, details of drug therapy (drug name, dose, duration, and frequency) were recorded. rationality of prescription was assessed using the who core drug prescribing indicators and the pattern of severity of migraine was assessed using midas scoring system. results. out of 85 patients, 71 were female (83.5%), mostly around 21-30 years of age, and 27 (31.7%) patients had other comorbidities. naproxen was the most commonly used nsaid for termination of acute migraine attack (15.3 %). the most common drugs prescribed for prophylaxis included beta adrenergic blockers (propranolol, 14.66%), antidepressants (amitriptyline, 9.33% and fluoxetine 3.33%), and antipsychotics (prochlorperazine, 4.66%). domperidone (17.30%) was the most commonly prescribed antiemetic. prescription of triptans was low (2.66%) with rizatriptan as the most commonly prescribed triptan. conclusions. the current study revealed that further improvements are required in prescribing practices especially in average number of drugs per prescription, prescription of drugs with generic names, and prescription of drugs of the essential drugs list. keywords: daly; midas score system; migraine; who prescribing indicators. introduction migraine is a primary, heterogeneous headache disorder characterized by recurrent, very painful and long duration headache that is moderate to severe as compared to other types of headache [1]. typically, migraine is unilateral, pulsatile, and lasts for a few hours to 3 days. associated symptoms may include nausea, vomiting, and increased sensitivity to light, sound or smell. the pain is generally worsened by physical activity. up to one-third of the affected people experience the aura, which is a short period of visual disturbance, signals that headache will occur soon. occasionally, it occurs with little or no headache following it [2]. though tension headache is the most common type of headache, migraine is the commonest headache complaint that is presented in clinical practice and is the leading cause of headache related disability in the world. it affects approximately 13% of adults in the us and its prevalence ranges between 12% and 20% in various countries around the world [3]. being more common in females than males, 19% and 7% prevalence, respectively [4], it's disability adjusted life year (daly) showed the maximum burden among women in the age of 30-34 years [5]. over 70% of migraine sufferers have a positive family history, and approximately 75% of the precipitating factors include environmental exposure, travel, education, or use of contraceptives [6]. while managing any case of headache, specific type must be ascertained using the in­ ternational classification of headache disorders (ichd) criteria before assessing the response or refractoriness to any specific treatment [7]. for termination of acute attack of migraine, the commonly used pharmacotherapeutic options are nonsteroidal anti-inflammatory drugs (nsaids), 5ht 1b/1d receptor agonists, and dopamine receptor antagonists. for prophylaxis, beta blockers, antidepressants, anticonvulsant, and flunarizine are used. in addition to these issn 2413-6077. ijmmr 2021 vol. 7 issue 26 p u b l ic h e a lt h a n d e p id e m io l o g y g. soni et al. there are the new drugs approved by the fda in 2018 including monoclonal antibodies that target the cgrp pathway like erenumab, fremanezumab, and galcanezumab; and ubrogepant which is a calcitonin generelated peptide receptor antagonist. ubrogepant is used for immediate treatment of migraine with or without aura. because of a boost in marketing of new drugs and variations in pattern of prescribing and consumption of drugs there is an increasing concern about delayed adverse effects, costeffectiveness of drugs, and volume of prescription [8]. therefore, prescription patterns for treatment of migraine needs to be moni tored. further, inappropriate drug use has occurred for as long as medicines have been available. several choices of therapy are available in treating patients with modern medicines which might be one reason to increase the number of irrational medicine treatment encounters and ultimately, poor patient outcomes. common drug use problems include: choosing incorrect medicines, polypharmacy, prescribing the incorrect dose, prescribing medicines that cause adverse drug reactions (adrs) or drug interactions, and prescribing drugs by brand names which increases the cost of treatment [9]. an inappropriate prescribing pattern by doctors often encourages inappropriate self-medication by patients because of the asymmetry of medical information [10]. the attitude today is the root on which the future grows. hence, it is generally agreed that the teaching hospitals have a special responsibility to society to promote rational prescribing by their staff and, through them – the future generations of doctors [11]. further, to increase prescription quality and improve the rationality of drug use, we need to investigate the subjective and objective factors that affect doctors’ prescription patterns [12] therefore, the present study was planned to assess the current trends in the prescribing pattern of anti-migraine drugs and to evaluate the distribution of severity of migraine among patients of migraine attending the outpatient neurology department at a tertiary care private hospital in jaipur, rajasthan. methods a cross sectional study was conducted in the department of pharmacology in collaboration with the department of neurology in mahatma gandhi medical college and hospital, jaipur, rajasthan in june 2020 – march 2021. the study was undertaken after approval from the institutional ethics committee and an informed consent was obtained from the participants before enrolling them into the study. all patients of any gender and age >18 years attending the neurology opd and diagnosed with migraine according to the international headache society classification characterized by presence of any two of the following criteria: at least unilateral pain, throbbing pain aggravated by movement, moderate or severe intensity, or accompanied by either nausea/ vomiting or photophobia/phonophobia, were included in the study. patients with recent history of cns infection or any major medical illness such as malignancy, autoimmune disorder or co-existent neurological disorder, or a case where an attending physician believes any other non-migraine diagnosis to be more likely, or a patient not willing to give an informed written consent were excluded. data collection all the prescribing details from each prescription were recorded in the case history form. the information included patient’s demographic details (name, age, sex), diagnosis, details of drug therapy (drug name, dose, duration, and frequency). study tools 1) rationality of prescription was done by using the who prescribing indicators. the prescribing indicators include average number of drugs per encounter, percentage of drugs prescribed by generic name, percentage of encounters with antibiotics prescribed, percentage of encounters with an injection prescribed, percentage of drugs prescribed from essential drug list or formulary. 2) the migraine disability assessment test (midas): the midas questionnaire was put together to assess the impact headaches have on one’s life. table 1 details the midas scoring. data was collected and tabulated using ms excel 2007 and was checked for normalcy before analyzing. qualitative data was presented as percentages and proportions. table 1. midas grade, definition, and midas score midas grade definition midas score i little or no disability 0-5 ii mild disability 6-10 iii moderate disability 11-20 iv severe disability 21+ issn 2413-6077. ijmmr 2021 vol. 7 issue 2 7 p u b l ic h e a lt h a n d e p id e m io l o g y results in the present study, 85 patients with a diagnosis of migraine were recruited. most of the patients were female (83.5%), around 21-30 years of age (31.7%), and 29.4% had a co-morbidity. the description of the study participants is presented in table 2. the severity of migraine was assessed using midas scoring system (migraine disability assessment) in which 45 (52.94%) were found to have moderate degree of migraine and 18 (21.17%) had aura. description of severity of migraine among the study population is presented in fig. 1a and 1b regarding the presence or absence of aura among the study population. out of 85 patients, little disability was observed in 8.23% patients, mild in 21.12%, moderate in 52.94%, and severe in 17.71% patients. table 2. description of the study participants (n=85) variables frequency (n) percent (%) gender male 14 16.4% female 71 83.5% age (in years) 10-20 7 8.2% 21-30 27 31.7% 31-40 24 28.2% 41-50 20 23.5% 51-60 5 5.8% 61-70 2 2.35% >70 0 0% comorbidity yes 25 29.4% no 60 70.5% pattern of co-morbidities benign paroxysmal positional vertigo [bppv] 1 4% agitated depression 4 16% allodynia 3 12% depression 6 24% diabetes mellitus 2 8% head injury 4 16% hypertension 3 12% hyperprolectenimia 1 4% lumbar prolapsed intervertebral disc [pivd] 1 4% a b fig. 1. a) severity of migraine and b) presence of aura among the study participants (n=85). g. soni et al. issn 2413-6077. ijmmr 2021 vol. 7 issue 28 p u b l ic h e a lt h a n d e p id e m io l o g y out of 85 patients, 29.4% patients had comorbidities which included depression in 24%, head injury in 16%, agitated depression in 16%. alodynia (12%), hypertension (12%), diabetes mellitus, benign paroxymal positional vertigo [bppv], hyperprolectenimia, and lumbar prolapsed intervertebral disc [pivd]. the distribution of co-morbidities among the study participants is presented in fig. 2. table 3 and fig. 3 describes the pattern of prescription of drugs for managing migraine among the study participants. the main drug class prescribed for controlling acute attack was nsaids (17%), while propranolol (14.66%) was the mainstay of migraine prophylaxis. the most common nsaid used was naproxem followed by etoricoxib. antiemetics were prescribed to 17.3% patients and included d2 receptor antagonist, domepridone in all cases. in 8% patients, antipsychotics were prescribed; prochlorperazin in 4.66% and olanzapine in 3.33% patients. antidepressants were prescribed in 12.66% patients (amitryptyline in 9.33%, and fluoxetine in 3.33%), antiepileptics in 5.33% (pregabalin in 4%, topiramate in 0.60%), proton pump inhibitors (omeprazole) in 2% patients, calcium channel blockers (flunarazine) in 7.33 %, and anxiolytics (clonazepam) in 2% of patients. other miscellaneous drugs prescribed to the study patients included calcium citrate+vitamin d3 (8%), cyproheptadine+sorbitol+tricholine citrate (0.60%), thiocolchicoside (2%), and magnisium+riboflavin (0.60%). the observed values of the core drug prescribing indicators as compared with the who standard values are presented in fig. 4. the observed average number of drugs per encounter were 3.67; the percentage of drugs prescribed by generic name and percentage of encounters with antibiotics prescribed were 0% each; 50.6% of the prescribed drugs were from the essential list, and 0.003% of the encounters were with an injection prescribed. fig. 2. description of co-morbidities among the study participants (n=25). fig. 3. prescription pattern of drugs for migraine among the study participants. g. soni et al. issn 2413-6077. ijmmr 2021 vol. 7 issue 2 9 p u b l ic h e a lt h a n d e p id e m io l o g y table 3. prescription pattern of drugs for migraine among the study participants s. no. drug class drugs number individual % total total % 1 nsaids naproxen 69 15.30% 78 17.30 etoricoxib 9 2% 2 antiemetics: d2 receptor antagonist domperidone 78 17.30% 78 17.30 3 β­adrenergic blocker propranolol 66 14.66% 66 14.66 4 antipsychotics prochlorperazine 21 4.66% 36 8 olanzapine 15 3.33% 5 proton pump inhibitors omeprazole 9 2% 9 2 6 antiepileptics pregabalin 18 4% 24 5.33 topiramate 3 0.60% divalproex sodium 3 0.60% 7 antidepressant amitriptyline 42 9.33% 57 12.66 fluoxetine 15 3.33% 8 triptans rizatriptan 12 2.66% 12 2.66 9 histamine analogue betahistine 6 1.33% 6 1.33 10 corticosteroid methylprednisolone 1 0.22% 4 0.82 prednisolone 3 0.60% 11 anxiolytic clonazepam 9 2% 9 2 12 calcium channel blocker flunarizine 33 7.33% 33 7.33 13 miscellaneous calcium citrate + vitamin d3 21 4.66% 36 8 cyproheptadine + sorbitol + tricholine citrate 3 0.60% thiocolchicoside 9 2% magnesium + riboflavin 3 0.60% fig. 4. who prescribing indicators. g. soni et al. issn 2413-6077. ijmmr 2021 vol. 7 issue 210 p u b l ic h e a lt h a n d e p id e m io l o g y discussion migraine is a headache disorder characterized by throbbing pain or a pulsating sensation, usually on one side of the head. it is often accompanied by nausea, vomiting, and extreme sensitivity to light and sound. migraine attacks can last for hours to days, and the pain can be so severe that it interferes with daily activities. the prevalence of migraine is commonly high in females of age group 21-30 years. the study was aimed to assess the current trends in the prescribing pattern of anti-migraine drugs and distribution of severity of migraine among the patients of migraine attending the outpatient neurology department at a tertiary care private hospital in jaipur, rajasthan. it was established that the majority of the patients were females. women reported episodic pain for a longer time and more frequent chronic pain than men. this may often result due to changes in estrogen levels. since estrogen controls chemicals in the brain that affect the pain sensation, therefore a drop in its level can trigger a headache. hormone levels change for a variety of reasons including menstrual cycle, pregnancy, menopause, use of oral contraceptive pills, and hormone replacement therapies. in the study, the most common comorbidity among the study patients was depression or agitated depression. migraine without an aura was present in two-third patients of this study. migraine without aura is the commonest presentation in both indian and western studies. however, in a study conducted by mukhopadhyay et al [2] the prevalence of migraine with and without aura was reported to be 68% and 30%, respectively. the mean midas score in the study suggested that the majority of the study participants were suffering from mild to moderate migraine (grade 2 and grade 3). this is contrary to a study by jawed et al [13], which reported that the majority of migraine patients had severe disability. many studies have proved that the midas score often correlates with a physician’s assessment of migraine and is a useful tool for establishing the level of care and treatment required. its use may improve physician-patient communication about headache-related disability and may favorably influence health­ care delivery for migraine patients. the midas score indicated that in the present study, moderate disability was present in the majority of the study patients. genetic, hormonal, psychological and other lifestyle factors might underlie these differences in midas score between different families and individuals in the same family [14]. epidemiological factors are also known to play a significant role and midas scores variations have been observed across different educational levels and socio_ economic status. an increased awareness may result in avoiding the precipitating triggers and seeking appropriate and timely treatment in the educated people. also, low socioeconomic status is likely to cause more stress and a difficult living and working environment, thus triggering an acute episode of migraine. psychological factors were also significant in migraine disability as observed in a study showing midas scores to be worse in depressed patients [2]. nasids were the most common class of drugs to terminate acute attack in the study and naproxen was the most commonly prescribed analgesic among nsaids. these findings are similar to khan et all [15]. similarly, a study by sumelathi et al [1] also states that nsaids are predominantly effective in both acute and chronic attack of migraine. nasids are the most commonly used due to their wide availability as over the counter drugs and their pharmaco-economic advantages. kefee et al [16] reported in their study that paracetamol was primarily used in karachi because they provide an early symptomatic effect, thus improving the treatment acceptability by patients. however, nsaids may cause some serious adverse effects, most common gastro intestinal like gas, feeling bloated, heartburn, stomach pain, nausea, vomiting, diarrhea and/or constipation. these gastrointestinal symp toms can generally be prevented by taking the drug with food, milk, or antacids. nsaids also can induce different forms of renal injury and hepatic side effects. long term use and an inappropriate high dose may cause high risk to all these adverse drug reactions. however, triptans are most commonly used in developed countries for termination of acute and chronic attack of migraine (germany, usa, australia) [1]. triptans are considered as first­line therapy for moderate to severe migraine and also mild to moderate migraine in selective cases [4]. it was established that the most commonly prescribed triptan was rizatriptan. however, only 2.66% triptans were prescribed in this study. according to us national surveillance studies (2009), triptans are the primary preg. soni et al. issn 2413-6077. ijmmr 2021 vol. 7 issue 2 11 p u b l ic h e a lt h a n d e p id e m io l o g y scribed drugs and rizatriptan showed the major share among all triptans. the findings of the present study are also similar to these observations except for low rates of triptan use. shao et al [8]. suggested that reduced effectiveness in patients with late attacks may have played a role in lower frequency of triptans use. additionally, given that sumatriptan is contra indicated in common conditions including cardiovascular disease and pregnancy, the appropriateness of its use may be narrower than was expected previously. shao et al [8] also believed that frequent occurrences of adverse effects in more than half of the patients after triptan administration may have led the physicians to avoid their use. individual factors such as previously reported poor response to triptans by patients, physician’s unfamiliarity with medication and their high cost may also contribute to low use. the characterization of such details in future studies will further elucidate the extent to which triptan prescription practices are deviated from guidelines, potentially allowing a more targeted solution to this problem. further observations on whether opiates are being prescribed as first line treat­ ment or rescue therapy and noting the eligibility of patient populations when commenting on levels of triptan therapy may also be helpful. beta adrenergic blockers, antipsychotics, and tricyclic antidepressants were the frequently prescribed medicines after nsaids. depression is commonly present as one of the commonly associated factors of migraine. hence, these medications are considered as better choices after nsaids. these findings are consistent with the guidelines that recommend beta-blockers to be prescribed to patients with both migraine and hypertension, and antidepressants – to patients with both migraine and depression [7]. in this study, antiemetics have also been prescribed along with nsaids. the literature indicates that patients of migraine are hypersensitive to dopamine that is important in causing some of the premonitory symptoms of migraine such as nausea and vomiting. it is established that dopamine receptor over sensitivity also is significant in pathogenesis of migraine. this may explain the use of dopamine receptor blockers like domperidone in the present study. domperidone is a peripherally acting dopamine antagonist used for gastric motility disorder and nausea. it is less expensive, easily tolerated and is safe and efficacious in migraine treatment. other drugs like metoclopramide and prochlorperazine can also be used as monotherapy for acute migraine headaches as these are also dopamine receptor antagonists and have antiemetics action. in this study, an average of 3.6 medicines were prescribed per prescription. this finding is greater than the standard value recommended by the who, which is less than 2 medicines per prescription. similar findings were reported in a study by aravamuthan et. al [17]. this observation may be due to the fact that most of the developing countries, including india are experiencing an epidemiological shift in the disease burden of both communicable and chronic diseases. consequently, poly-pharmacy has become more prevalent since healthcare professionals have to treat several diseases concurrently in a patient. however, this shift towards polypharmacy may result in an increased risk of drug interaction, low adherence to treatment, dispensing errors, and increase therapy cost. a relationship has been established between polypharmacy and chronic poly pharmacy that is prescription of multiple medicines is a predisposing factor to adding further drugs. the prescription of drugs in generic names ensures rational use of drugs and reduces the cost of treatment. in this study, no drug was prescribed with generic names. this is very low as compared to other studies and is even lower than ghana, lebanon, nepal and pakistan (2.9% to 65.0%) and much higher indicators (7599.8%) of generic prescription have been reported from bangladesh, cambodia, ethiopia, and tanzania [18]. prescription of drugs in generic name may help in controlling drug costs in the healthcare service and decrease the influence of medical marketing on pre­ scription. the findings from this study show that no antibiotics were prescribed as compared to the who standard that ranged 20-26.8%. there is huge variation in this finding among other studies in india by hazra et al. (72.8%) [18]. percentage of encounters prescribed with injection were 0.003%. this is less than the standard who range (13.4-24.1%). since the present study was done for out patients who were otherwise in optimal state health and most of the patients had mild-moderate migraine, this may be the reason for low rate of injection usage. these findings are com­ parable to that of hazra et al. (8%) [18] but are very low as compared to other regions, south ethiopia (38.1%) and uganda (48%) [19]. the lower rate of injections use would reduce the incidence of blood borne pathogenic infection, g. soni et al. issn 2413-6077. ijmmr 2021 vol. 7 issue 212 p u b l ic h e a lt h a n d e p id e m io l o g y reduce the transmission of hiv infection, and reduce the cost of treatment, which in turn decreases the economic burden on the patient and eventually improves compliance of the patient to the treatment. thus, the study reported that the median value of the percentage of medicines prescribed from the essential medicine list was 50.6% that was half of the standard value suggested by the who. these values are also lower as compared to the other countries such as ethiopia (99%), south ethiopia (99.6%), and nepal. this indicator helps in measuring the degree to which practices conform to the current national list of essential medicine (nlem) of 2019. following the essential drug list guarantees treatment of the principal diseases of the population besides controlling overall cost of medications. conclusions the current trends in prescription pattern o f a n t i ­ m i g ra i n e d r u g s o b s e r v e d w e re : naproxen was the most commonly used nsaid for termination of an acute migraine attack and for prophylaxis; beta adrenergic blockers (propranolol), antidepressants (amitriptyline), and antipsychotics (prochlorperazine) were prescribed the most. domperidone was the most common ly prescribed ant iemet ic. prescription of triptans was low (2.66%) with rizatriptan as the most commonly prescribed triptan. this study revealed that further improvements are required in prescribing practices especially in average number of drugs per prescription, prescription of drugs with generic names, and prescription of drugs of the essential drugs list. recommendations: continuous education and training of physicians regarding rational use of drugs should be implemented and monitored so that the required changes in prescribing become sustainable. further prospective analytical studies should be conducted on a large population with strict observation and follow up focusing largely on quality of life, adverse effect profiles of the drugs, compliance, and cost of treatment. limitations since the study was conducted during covid pandemic, the number of cases attending the opd was less than expected and only a cross-sectional study was carried out. therefore, follow-up data, comparative analysis and tests of significance could not have been applied. we enrolled only adult patients from neurology opd; further research may include patients of all ages and from other departments as well. author’s contributions gopesh soni, anusha vohra, priyanka rathi – conceptualization; gopesh soni, anusha vohra, prerana shiv goswami, priyanka rathi – methodology; gopesh soni, anusha vohra, prerana shiv goswami, shipra jain, priyanka rathi investigation, formal analysis, data curation, writing – original draft, writing – reviewing and editing. сучасні тенденції призначення протимігренозних препаратів пацієнтам з мігренню в університетській лікарні третинного рівня g. soni, a. vohra, s. jain, *p. rathi, p. goswami mahatma gandhi medical college and hospital, jaipur, india вступ. невідповідна схема призначення протимігренозних препаратів лікарем часто може спонукати пацієнтів до самолікування через асиметричність доступної медичної інформації. мета дослідження – оцінити сучасні тенденції в призначенні протимігренозних препаратів, раціональність їх призначення та тяжкість перебігу мігрені у пацієнтів. методи. перехресне дослідження було проведено в університетській лікарні третинного рівня. вивчалися всі відомості про призначення, включаючи демографічні дані пацієнта, діагноз, деталі медикаментозної терапії (назва препарату, доза, тривалість та частота прийому). раціональність призначення ліків оцінювалася з використанням основних індикаторів призначення ліків вооз, а характер тяжкості мігрені – за допомогою системи оцінки midas. g. soni et al. issn 2413-6077. ijmmr 2021 vol. 7 issue 2 13 p u b l ic h e a lt h a n d e p id e m io l o g y references 1. sumelahti ml, mattila k, sillanmaki l, sumanen m. prescription patterns in preventive and abortive migraine medication. int. j headache society. 2011;31(16):1659­63. https://doi.org/10.1177/0333102411427602 2. mukhopadhyay k, joseph ne, barkondaj b, mukherjee s, chatterjee c, chakraborty s. a clinical study on prescribing features and prescription pattern for migraine in a tertiary care hospital of eastern india. j of the ind.med asso. 2020;118(4):23­26. 3. lipton rb, stewart wf, scher ai. epidemiology and economic impact of migraine. curr med res opin. 2001;17(suppl 1):s4­s12. https://doi.org/10.1185/0300799039117005 4. rozen td, swanson jw, stang pe, mcdonnell sk, rocca wa. increasing incidence of medically recognized migraine headache in a united states popu lation. neurology. 1999;53(7):1468­ 73. https://doi.org/10.1212/wnl.53.7.1468 5. d’antona l, matharu m. identifying and managing refractory migraine: barriers and opportunities? the j of headache and pain. 2019;20(1):79­89. https://doi.org/10.1186/s10194­019­1040­x 6. goadsy pj, schoenen j, ferrari md, silberstein sd, dodick d. towards a definition of intractable headache for use in clinical practice and trials. int. j cephalgia. 2006;26(9):1168­70. h t t p s : / / d o i . o r g / 1 0 . 1 1 1 1 / j . 1 4 6 8 ­ 2 9 8 2 . 2006.01173.x 7. silberstein sd, dodick dw, pearlman s. defining the pharmacologically intractable headache for clinical trials and clinical practice. int. j of headache. 2010;50(9):1499­1506. https://doi.org/10.1111/j.1526­4610.2010. 01764.x 8. emily shao, james hughes, rob eley. the presenting and prescribing patterns of migraine in an australian emergency department: a descriptive exploratory study. world j emerg med. 2017;8(3): 170-76. https://doi.org/10.5847/wjem.j.1920­8642. 2017.03.002 9. fmhaca. manual for medicines good prescribing practice. addis ababa: food, medicine and healthcare administration and control authority of ethiopia; 2012. available from: http://apps. результати. з 85 пацієнтів 71 були жінки (83,5%), переважно у віці 21-30 років, а 27 (31,7%) пацієнтів мали інші супутні захворювання. напроксен був найбільш часто використовуваним нпзп для припинення гострого нападу мігрені (15,3 %). найпоширенішими препаратами, які призначали для профілактики, були бета-адреноблокатори (пропранолол, 14,66%), антидепресанти (амітриптилін, 9,33% і флуоксетин 3,33%) та нейролептики (прохлорперазин, 4,66%). домперидон (17,30%) був найбільш поширеним протиблювотним засобом. призначення триптанів було низьким (2,66%), при цьому найчастіше призначали різатриптан. висновки. дане дослідження показало, що необхідні подальші вдосконалення практики виписування рецептів, особливо щодо середньої кількості ліків за рецептом, призначення ліків із загальними назвами та призначення ліків із переліку основних лікарських засобів. ключові слова: daly; система оцінювання midas; мігрень; індикатори призначення вооз. information about the authors gopesh soni – b.sc. (nursing), m.sc (medicine) part-ii student, department of pharmacology, mahatma gandhi medical college and hospital, jaipur, india. orcid 0000­0002­6368­4679, e­mail: gopeshsoni@gmail.com anusha vohra – md (pharmacology), professor, department of pharmacology, mahatma gandhi medical college and hospital, jaipur, india. orcid 0000­0003­3113­5402, e­mail: drvohraanusha1979@gmail.com shipra jain – md (pharmacology), professor, department of pharmacology, mahatma gandhi medical college and hospital, jaipur, india. orcid 0000­0003­4450­473x, e­mail: drshiprajain@mgumst.org priyanka rathi – md (pharmacology), professor, department of pharmacology, mahatma gandhi medical college and hospital, jaipur, india. orcid 0000­0002­6400­6888, e­mail: drpriyankarathi@mgumst.org prerana shiv goswami – mbbs, second year post-graduate resident, department of pharmacology, mahatma gandhi medical college and hospital, jaipur, india. orcid 0000­0003­1797­1287, e­mail: preranashivgoswami@gmail.com g. soni et al. issn 2413-6077. ijmmr 2021 vol. 7 issue 214 p u b l ic h e a lt h a n d e p id e m io l o g y who.int/medicinedocs/documents/s22353en/ s22353en.pdf. 10. shankar r, partha p, shenoy n. prescribing patterns of drugs among patients admitted with cardiovascular disorders in the internal medicine ward: prescribing patterns in inpatients. internet j pharmacol. 2001;1(2). https://doi.org/10.5580/18a7 11. desalegn aa. assessment of drug use pattern using who prescribing indicators at hawassa university teaching and referral hospital, south ethiopia: a cross­sectional study: bmc health services res. 2013;13:170. available at http://www. biomedcentral.com/1472-6963/13/170. https://doi.org/10.1186/1472­6963­13­170 12. wang h, li n, zhu h, xu s, lu h, feng z. prescription pattern and its influencing factors in chinese county hospitals: a retrospective cross­ sectional study. plos one. 2013;8(5):e63225. https://doi.org/10.1371/journal.pone.0063225 13. jawed s, ali w, yaqoob u, shah s, uddin smm, haq a. effect of migraine headache on productivity of patients according to migraine disability assessment score: a cross­sectional study. pain ther. 2019;8(2):233­38. doi:10.1007/s40122­019­0130­4. https://doi.org/10.1007/s40122­019­0130­4 14. van de ven rc, kaja s, plomp jj, frants, rr, van den maagdenberg amjm, ferrari md. genetic models of migraine. arch neurol. 2007;64:643­46. https://doi.org/10.1001/archneur.64.5.643 15. kamran khan, mudassar iqbal arain, muhammad ali ghoto, abdullah dayo, mehrukh zehravi, ahad abdul rehman. prescribing trends of antimigraine drugs amongst general physicians and neuro physicians in southern pakistan: a comparative approach. isra med j. 2019;11(4): 275­80. 16. huda kafeel, ramsha rukh. prevalence of headache in general population of karachi, pakistan. j app pharm. 2017; 6:308 ­13. 17. aravamuthan a, arputhavanan m, subramaniam k, sam johnson udaya chander j. assessment of current prescribing practices using world health organization core drug use and complementary indicators in selected rural community pharmacies in southern india. j of pharm policy and pract. 2017;10(1). https://doi.org/10.1186/s40545­016­0074­6 18. hazra a, tripathi sk, alam ms. prescribing and dispensing activities at the health facilities of a non-governmental organization. natl med j india. 2000;13(4):177­82. 19. desalegn aa. assessment of drug use pattern using who prescribing indicators at hawassa university teaching and referral hospital, south ethiopia: a cross-sectional study. bmc health services res. 2013;13:170. available at http://www.biomedcentral. com/1472-6963/13/170. https://doi.org/10.1186/1472­6963­13­170 received 17 november 2021; revised 29 november 2021; accepted 3 december 2021. this is open access article distributed under the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. g. soni et al. 12 p e d ia t r ic s issn 2413-6077. ijmmr 2021 vol. 7 issue 1 doi 10.11603/ijmmr.2413-6077.2021.1.12468 vitamin d and urolithiasis in children *sh.a. yusupov1, a.m. shamsiev2, j.a. shamsiev3, p.a. pulotov4 1, 2, 3, 4 – samarkand state medical institute, samarkand, republic of uzbekistan background. urolithiasis is currently one of the topical issues of contemporary urology and medicine in general. this is primarily due to the high prevalence of urolithiasis; according to several population studies it ranges from 3.5 to 9.6%. at the same time, there is a steady increase in its incidence. therefore, the matter of urolithiasis is one of the most urgent in present-day medicine. objectives. the aim of the research was to study the content of a polymorphic genetic marker of the vitamin d receptor gene related to development and relapse of urolithiasis in children. methods. the content of a polymorphic genetic marker of the vitamin d receptor gene related to development and relapse of urolithiasis in 100 children was investigated. results. the results of the study prove that the vitamin d receptor gene assists in revealing disorders that promote urolithiasis development. conclusion. comparative analysis of the frequency of distribution of fok1 genotypes of the vitamin d receptor gene polymorphism showed that statistical significance of the association (p=0.02) of f allele according to the dominant inheritance model (total ff+ff genotypes) was established in the group of patients with urolithiasis compare to the corresponding indicator of the control group (63%). keywords: vitamin d, urolithiasis, vdr gene, gene polymorphism, dysuria. international journal of medicine and medical research 2021, volume 7, issue 1, p. 12-19 copyright © 2021, tnmu, all rights reserved sh.a. yusupov et al. *corresponding author: yusupov shukhrat, phd, dsc, head of the department of paediatric surgery, samarkand state medical institute, samarkand, republic of uzbekistan. e-mail: shuchrat_66@mail.ru introduction it is established that urolithiasis is polyetiological. the final step of stone formation is salts crystallization in a urine supersaturated solution. however, a variety of mechanisms take part in pathogenesis before this, i.e. disorders of renal circulation, disturbance of urinary tract urodynamics, and inflammation in them [2, 3]. urolithiasis is defined as a metabolic disease that is caused by several endogenous and/or exogenous factors and is characterized by occurrence of stones in the urinary system [3]. it is clear that stones in the urinary tract are a clinical manifestation of the disease; removal of the stone does not stop its progression, and it develops further if the causes are not eliminated [19]. calcium stones are the most common, that is more than 80% of cases. correspondingly, most of the calcium stones (about 85-90%) are calcium oxalates, 1-10% – calcium phosphates [7]. recently, a tendency to decrease in the incidence of phosphate stones in the world structure was noticed. probably, this is caused by a decrease in the occurrence of infected stones because of advances of minimally invasive surgery for urolithiasis and use of the latest antibacterial therapy [1,14]. thus, metabolic disorders are significant in the pathogenesis of urolithiasis. ever more, there are uric acid stones (up to 10% of all urinary stones); they include uric acid and its salts, along with mixed stones (up to 5% of all calcium stones) that contain calcium salts together with uric acid and/or its salts [18]. violation of calcium metabolism is one of the risk factors for occurrence and relapse of urolithiasis; its regulation in the human body is a complex process. three major hormones: parathyroid hormone (pth), calcitonin, and metabolite of vitamin d, 1,25-dihydroxycholecalciferol-1,25 – the most important, are involved in calcium homeostasis maintenance [12]. vitamin (hormone) d is a controlling anabolic hormone having antioxidant properties and an exclusive systemic metabolic effect [12]. then again, seasonal fluctuations in vitamin d rate (high levels in summer and autumn and low – in winter and spring, similarly to annual testosterone cycles) take place [19]. on the other hand, regulation of the expression of 13 p e d ia t r ic s issn 2413-6077. ijmmr 2021 vol. 7 issue 1 hormone d metabolism genes variates according to the androgens level. hence, androgen deficiency intensifies adverse health effects caused by vitamin d deficiency [10]. the receptor of vitamin d is encoded by the vitamin d receptor gene characterized by genetic polymorphism, i.e. different allelic variations of this gene in humans [4]. bsm i, fok i, taq i are the most important polymorphisms of the vitamin d receptor gene involved in progress of diseases [18, 19]. several studies associate vdr gene polymorphism with urolithiasis. some publications prove the significance of occurrence of the apalaa genotype that determines sensitivity to vitamin d in the formation of calcium stones in the urinary organs [7, 14]. it is also established that in patients with urolithiasis the hla b13, b22 and b35 genes are more common than in healthy individuals [2]. the huge range of biological effects of vitamin d, its involvement in carbohydrate, fat, purine metabolism, as well as its anabolic, antiproliferative, immunomodulatory effects, the investigation of cases of its deficiency is essential. methods to perform the tasks set, a clinical examination of 100 children with urolithiasis and 100 practically healthy children was conducted, as well as the collection and analysis of sta tistical data on urolithiasis in children of different age groups to identify the gender, age-related and family frequency of urolithiasis, nutrition and lifestyle features, as well as the seasonality of this pathological process. ultrasound and x-ray examinations of the urinary organs of patients were performed. the selection of patients was carried out on the basis of the diagnosis established in the clinic with the written consents of the probands. blood samples were collected from the patients with urolithiasis (100 samples) and a control group of practically healthy children (100 samples). venous blood (1 ml) was kept in 0.5 ml of sodium citrate solution at -20 °c. the material for the research was provided by the republican specialized scientific and practical medical center of pediatric surgery of samarkand. table 1 presents data on distribution of the patients according to age in the groups under consideration. table 1 shows that among the patients, school-age children prevailed – 69 (69%). the reason is the fact that it is the age when metabolic disorders are the most often manifested; it is associated with the transition of children to a general diet, violation of drinking regime, etc., and in the younger group – the diet is quite rational and metabolic changes are less pronounced. the distribution of patients in both groups depending on gender is presented in table 2. the data presented in table 2 shows that according to the gender distribution of patients, incidence of urolithiasis is higher among boys – 68 (68%) children than girls – 32 (32%). the most common complaints of patients with urolithiasis were pain in the lumbar region, fever, hematuria, turbidity of urine, increased or decreased urination. acute urinary retention and spontaneous discharge of concretions were noted in some cases. in dislocation of concretions in the ureter n/w the pain was present in the iliac region on the same side. the patients with renal insufficiency had charactable 1. age-related division of patients with urolithiasis and healthy children age of patients (years) control group (n=100) main group (n=100) total (n=200) n % n % n % 0-3 17 17 31 31 48 24 4-11 43 43 43 43 86 43 12-17 46 46 26 26 72 36 table 2. division of patients depending on gender gender control group (n=100) main group (n=100) total (n=200) n % n % n % girls 35 35 32 32 35 17.5 boys 65 65 68 68 165 82.5 sh.a. yusupov et al. 14 p e d ia t r ic s issn 2413-6077. ijmmr 2021 vol. 7 issue 1 teristic symptoms, i.e. headache, drowsiness, poor appetite. at admission pain syndrome was evidenced in 89 (89%) patients. also, a severe pain syndrome of renal colic was present in 15 (15%) patients. the nature and localization of the pain syndrome depended on the child’s age, presence of concomitant concretions in the urinary tract. in the patients of a younger age group, abdominal pain was typical. older patients complained for lumbar pain more often, sometimes complained of irradiation of pain along the ureter. in 3 (3%) children suffering from concretion in the lower third of the right ureter, pain syndrome led to unjustified appendectomy performed at the place of residence. in these patients ureterolithotomy was associated with some technical difficulties due to an adhesive process. dysuric manifestations were evidenced in 28 (28%) patients and mostly characterized by frequent and painful urination. acute urinary retention was present in 5 (5%) patients. dysuric manifestations were nearly 2 times more common in younger patients than other. acute urinary retention in all patients was managed by inserting an ad’mer catheter; in 3 (3%) patients, after the catheter was removed, spontaneous discharge of the concretion was evidenced. according to the localization of the concretions, the following were identified: renal calculi in 43 patients (left-13, right – 21, on both sides – 9); ureter stones in 12 patients (c/3 ureter – 1, b/3 ureter – 1, n/3 ureter – 10); bladder stones in 9 patients (recurrent bladder stone in 1 of them); stones of the proximal urethra in 4 patients; multiple urolithiasis in 10 patients. combined urolithiasis with urinary disorders were revealed in 22 children. genomic dna was separated from the whole blood of patients diagnosed for urolithiasis and almost healthy individuals of the control group. it was performed by means of reagents kit diatom dna prep 200 (isogen laboratory llc) according to a standard protocol. this kit comprised a lysing agent with guanidine thiocyanate, which was designed to destroy cells, solubilize cell debris and denature cell nucleases. with a lysing reagent the dna was absorbed on the nucleos™ sorbent, then washed from salts and proteins with an alcohol solution. the dna, which was eluted from the extra-genome sorbent, was used for further analysis. pcr was performed by means of special oligonucleotide primers and a reagents kit for pcr amplification of dna genepak™ pcr core (isogen laboratory llc). we used master mix tubes ready for amplification, which comprised a lyophilized state taq dna polymerase, deoxynucleose triphosphates, and magnesium chloride with final concentrations of 1 u, 200 microns, and 2.5 mm, respectively, and an optimized buffer system intended for pcr amplification. 5 µl of a primers mixture, a final concentration of 0.5 µm, 10 µl of diluent pcr, and 5 µl of the test dna were added to the master mix tubes. for pcr, the geneamp ® pcr system 9700 with a 96-cell block (applied biosystems) was used. the amplification program for the vdr gene involved 5 minutes of predenaturation at 95 °c, 34 cycles: 94 °c – 30 sec, 66 °c – 30 sec, 72 °c – 30 sec, and a final elongation for 7 minutes at 72 °c. the temperaturetime mode of amplification for the urokinase gene was: 5 minutes of preliminary denaturation at 95 °c, 40 cycles: 95 °c – 30 sec, 56 °c – 30 sec, 72 °c – 30 sec; as well as final elongation at 72 °c for 7 minutes. results bioinformatic search for the nucleotide sequences of these genes was carried out in the ensemble genome browser genomic database. after pcr amplification of the vdr gene fragment, the pcr-derived products were subjected to pcr analysis using fok i endonuclease (manufactured by npo sibenzyme) (fig. 1, table 3). the genotypes of the vdr gene polymorphisms were interpreted on the basis of different band patterns on the electrophoregram (fig. 2). after photodocumentation, the subsequent restriction fragments were genotyped according to the presence of corresponding fragments. table 3. chromosomal structure and localization of the studied gene gene, polymorphism primers nucleotide sequence restrictases vitamin d receptor fok-i polymorphism pr_vdr_f agctggccctggcactgactctgctct fok-i pr_vdr_r atggaaacaccttgcttcttctccctc sh.a. yusupov et al. 15 p e d ia t r ic s issn 2413-6077. ijmmr 2021 vol. 7 issue 1 genotyping analysis of vdr gene pdrf products (restriction products) was performed. one fragment weighing 278 bp indicated that this sample was a carrier of the homozygous genotype wt (wild type), and three fragments weighing 278 bp, 198 bp and 80 bp – a carrier of the heterozygous genotype wt/mut, the presence of two fragments weighing 198 bp and 80 bp – a carrier of the homozygous genotype mut (mutant) (fig.3). the frequency distribution of the vdr gene polymorphism fok1 genotypes in the control group corresponded to the hardy-weinberg distributions. a comparative analysis of the frequency distribution of the vdr gene polymorphism fig. 1. vdr gene fok-i polymorphism. fig. 2. fdrf analysis of the fok1 polymorphism of the vdr gene. sh.a. yusupov et al. 16 p e d ia t r ic s issn 2413-6077. ijmmr 2021 vol. 7 issue 1 fok1 genotypes showed a statistically significant association (p=0.02) of the f allele regarding the dominant inheritance model (ff +ff genotypes) in the group of urolithiasis patients compare to the corresponding indicators in the control group (fig. 4). as shown on the diagram, the genotype of heterozygotes f/f of the fok1 polymorphism of the vdr gene was the most often recorded in the control group (53%), and in the group of urolithiasis children, a decrease tendency was established, no confidence between the indicators (47%) (p>0.05) was evidenced. the f/f+f/f polymorphism fok1 of the vdr gene was the most informative genotype in urolithiasis, which occured almost 2 times more often compare to the control group (p<0.01). the frequency distribution of the heterozygotes f / f genotype was 44.4%; in the control group – 15%, respectively (χ2=0.47; p=0.24; or=1.35; 95% ci 0.57-3.17; df=1). f allele detection increased the risk of urolithiasis in children in 2.4 times compare to the f allele (95% ci=0.68-2.93, df=1). аllele determines the vitamin d receptor synthesis (427 amino acids); it is defined as *f, and the shorter form (424 amino acids) of the receptor is vdr*f. the study of the patient samples proved that the ff-normal genotype was revealed in 83 patients; a homozygous variant of the mutant ff genotype – in one case, a heterozygous ff genotype – in 127 cases by the vdr gene polymorphic marker foki (3663t>c). the allelic form of vdr*f*f was associated with a frequency of 74% and urolithiasis manifestations in the studied group of patients; the *f allele occurrence was 93 compare to the *f allele – 89. fig. 3. pdrf analysis of the fok1 polymorphism of the vdr gene. fig. 4. frequency distribution of fok1 polymorphism genotypes of the vdr gene in the control group and in patients with urolithiasis. 0 10 20 30 40 50 60 70 ff ff+ff 53% 37% 47% 63% % control group main group sh.a. yusupov et al. 17 p e d ia t r ic s issn 2413-6077. ijmmr 2021 vol. 7 issue 1 it is established that calcium urolithiasis is associated with the vdr*f*f genotype; in the individuals with this genotype the manifestations of urolithiasis at an early age are significantly more frequent; in the carriers of the vdr*f*f genotype the association with the development of urolithiasis is significantly lower; and in the individuals homozygous for the vdr*f*f genotype – intermediate. the analysis of the vdr gene genotype frequencies proved that the analysed genotypes distribution in our population corresponded to the hardy-weinberg distribution (rhb) (χ2=5.14; p=0.03) (table 4). thus, the attained results prove that the vdr gene is significant in revealing the disorders that contribute to urolithiasis development. table 4. statistical analysis of the genetic association of the vdr gene genotypes with urolithiasis according to the dominant inheritance model (test χ2, df=2) genotypes cases control χ2 р or n=100 n=94 case 95% ci genotype f/f 0.370 0.532 5.14 0.02 0.52 0.29-0.92 genotype f/f+f/f 0.630 0.468 1.93 1.09-3.43 discussion many scientists have proved that a violation of the metabolism of vitamin d leads to an imbalance in the phosphorus-calcium metabolism, which is a direct risk factor for the development of urolithiasis. a decrease in the level of vitamin d leads to a deterioration of health due to a range of physiological processes. however, many guidelines on urolithiasis provide recommendations on the limited administration of vitamin d due to the fear of development of increased lithogenesis. some researchers proved that 1.25 (oh)2d3 has a direct effect on calcium excretion [8]. currently, the relationship between the level of vitamin d and nephrolithiasis is debatable; the data obtained are contradictory, especially in paediatrics. schlingmann and co-authors stated that in children with urolithiasis, significant nephrocalcinosis and hypercalcemia had conditions of suppressed serum pth and a significant increase in the level of 1.25-dihydroxyvitamin d3 due to cyp24a1 mutations [13]. consequently, vdrs are encoded by the vdr gene with genetic polymorphism, i.e. the presence of various allelic variants of this gene in the population [5]. bsm i, folc i, taq i were the most significant polymorphisms of the vdr gene involved in disease development. the prevalence of vdr gene polymorphism has racial and ethnic differences. in one population, one genotype of a polymorphic marker prevails, in another population – another genotype of a polymorphic marker [11, 15, 17]. in this regard, it is relevant to study the distribution of genotypes of the polymorphic marker fok i of the vdr gene in the uzbek population in children with urolithiasis. the fok i locus is characterized by a t / c transition in the start codon (atg) of the second exon of the vdr gene, in which the length of the protein molecule of the receptor decreases due to the displacement of the start codon by three triplets. in the case of thymine (t) or the “f” allele, the m1 form of a molecule of 427 amino acids is synthesized; in the case of cytosine (c) or the “f” allele, a shorter and, accordingly, more active m4 form of the receptor with a length of 424 amino acids is synthesized [9]. according to the us researchers, in children with the ff genotype, bone mineral density was on average by 8.2% higher than in those with the recessive homozygous genotype (ff) and by 4.8% higher than in children with the ff genotype [16]. a group of scientists guided by s. azab conducted a study of the association of bsmi polymorphism of the vdr gene with the risk of development of nephropathy in patients with systemic lupus erythematosus; it showed that the bb (aa) genotype is a risk factor for this complication. however, there was no significant association of vdr gene variants with other clinical manifestations, laboratory profiles of systemic lupus erythematosus, the disease activity index or the level of 25-hydroxyvitamin d in the blood serum of patients [6]. according to the results of out study, it was established that in the uzbek population the genetic markers of predisposition to urolithiasis are: genotypes ff+ff of the vdr gene. a comparative analysis of the frequency distribution of the vdr gene polymorphism fok1 genotypes proved a statistically significant association (p=0.02) of the f allele regarding the dominant inheritance model (ff+ff genotypes) in the sh.a. yusupov et al. 18 p e d ia t r ic s issn 2413-6077. ijmmr 2021 vol. 7 issue 1 group of urolithiasis patients compare to the corresponding indicators in the control group. the genotype distribution frequencies of f/f heterozygotes were 44.4%, and 15% – in the control group, respectively (χ2 =0.47; p=0.24; or=1.35; 95% ci 0.57-3.17; df=1). revealing of the f allele increased the urolithiasis risk in children in 2.4 times, compare to the f allele (95%) (ci = 0.68-2.93, df=1). the heterozygosity of f/f mutations may predispose to formation of stones; therefore, screening should be carried out to determine the level of 25(oh)d in serum (which will be high) and levels of 24, 25(oh) vitamin d (which will be low), before prescribing vitamin d supplements to the patients with kidney stones to prevent exacerbation of calciuria. those to be screened and genetically tested are the patients with high or above average serum calcium levels and reduced pth levels. the molecular genetic method of prediction of urolithiasis occurrence allows identifying a disease predisposition at any age: just about from the birth of a person, since the genotype of an individual does not change throughout life. moreover, disease predisposition can be established by means of this method if no clinical or biochemical manifestations are present, specifically at the earliest preclinical stage of the pathology development. so, the earlier the genetic marker is detected, the more reliable and timely measures can be taken to prevent the disease. conclusions a comparative analysis of the frequency distribution of vdr gene polymorphism fok-1 genotypes showed a statistically significant association (p=0.02) of the f allele regarding the dominant inheritance model (ff +ff genotypes in total) in the group of urolithiasis patients compare to this indicator in the control group that was 63%. in the uzbek population, the genetic markers of predisposition to urolithiasis are: genotypes ff+ff of the vdr gene. it is appro priate to test this genotype as a comprehensive program of urolithiasis prevention in uzbekistan. conflict of interests authors declare no conflict of interest. author’s contributions yusupov shukhrat abdurasulovich, shamsiev azamat mukhitdinovich – conceptualization, methodology, formal analysis, writing – original draft, writing – reviewing and editing; shamsiev jamshed azamatovich, pulotov parviz amridinovich – data curation, writing – reviewing and editing, investigation, formal analysis. вітамін д і сечокам'яна хвороба в дитячому віці ш.а. юсупов1, а.м. шамсиев2, ж.а. шамсиев3, п.а. пулотов4 1, 2, 3, 4 – самаркандський державний медичний інститут, самарканд, республіка узбекистан вступ. сечокам'яна хвороба одна з найбільш актуальних проблем сучасної урології та медицини в цілому. це пов'язано в першу чергу з високою поширеністю сечокам'яної хвороби, яка, за даними декількох популяційних досліджень, становить від 3,5 до 9,6%. при цьому відзначається неухильне зростання захворюваності. мета. вивчити вміст поліморфного генетичного маркера гена рецептора вітаміну д асоційованого з розвитком і рецидивуванням сечокам'яної хвороби у дітей. методи. методом плр проведено генетичне дослідження поліморфного генетичного маркера гена рецептора вітаміну д асоційованого з розвитком і рецидивуванням сечокам'яної хвороби у 100 дітей. результати. результати, отримані в цьому дослідженні, вказують на те, що ген vdr сприяє визначенню порушень, що сприяють розвитку сечокам'яної хвороби висновки. в результаті проведеного порівняльного аналізу розподілу частот генотипів fok1 поліморфізму гена vdr встановлена статистично значуща асоціація (p=0.02) алеля f за домінантною моделлю спадкування (сумарно генотипи ff+ff) в групі хворих з уролітіазом порівняно з відповідним показником в групі контролю, який склав 63%. ключові слова: вітамін д, сечокам’яна хвороба, ген vdr, поліморфізм генів, дизурія sh.a. yusupov et al. 19 p e d ia t r ic s issn 2413-6077. ijmmr 2021 vol. 7 issue 1 information about the authors yusupov shukhrat abdurasulovich, phd, dcs, head of the department of pediatric surgery, samarkand state medical institute, samarkand, republic of uzbekistan. orcid 0000-0001-7259-028x, e-mail: shuchrat_66@mail.ru shamsiev azamat mukhitdinovich, phd, dcs, professor of the department of pediatric surgery, samarkand state medical institute, samarkand, republic of uzbekistan. orcid 0000-0001-8482-7037, e-mail: prof.ashamsiev@mail.ru shamsiev jamshed azamatovich, phd, dcs, professor of the department of pediatric surgery, samarkand state medical institute, samarkand, republic of uzbekistan. orcid 0000-0002-4193-7372, e-mail: jamshedshamsiev@mail.ru pulotov parviz amridinovich, assistant professor of the department of pediatric surgery, samarkand state medical institute, samarkand, republic of uzbekistan. orcid 0000-0003-1338-6873, e-mail: parviz8787@icloud.com references 1. apolikhin oi, sivkov av, konstantinova ov, slominsky pa, tupitsyna tv, kalinichenko dn. association of urolithiasis in patients with various family history of urolithiasis with polymorphisms of its candidate genes in the russian population. experimental and clinical urology 2014; (3): 50-52. 2. apolikhin oi, sivkov av, konstantinova ov, slominsky pa, tupitsyna tv, kalinichenko dn. the connection of unilateral and bilateral urolithiasis with genetic factors. experimental and clinical urology 2015;(2):68-70. 3. zhilenko mi, gusakova da, tyuzikov ia. prevalence of vitamin d deficiency/insufficiency in routine clinical practice. questions of dietology. 2017. vol. 7. no. 1. pp. 10-15. 4. kaprin ad, apolikhin oi, sivkov av, etc. analysis of uronephrological morbidity and mortality in the russian federation for 2003-2013. experimental and clinical urology. 2015. no. 2. pp. 3-7. 5. alagol f, shihadeh y, boztepe h, tanakol r, yarman s, azizlerli h, sandakci o. 2000. sunlight exposure and vitamin d deficiency in turkish wo men. j. endocrinol. invest. 23, 173-177. 6. azab sf, ali yf, farghaly ma, hamed me, allah ma, emam aa. vitamin d receptor gene bsmi polymorphisms in egyptian children and adolescents with systemic lupus erythematosus: a case-control study. medicine (baltimore) 2016; 95 (46): e5233. doi: 10.1097/md.0000000000005233 7. blomberg jm. vitamin d metabolism, sex hormones, and male reproductive function. reproduction. 2012. vol. 144. № 2. p. 135–152. 8. ferraro pm, taylor en, gambaro g, et al. vitamin d intake and the risk of incident kidney stones. 2017; 197(2):405-410. 9. genetics and biology of vitamin d receptor polymorphisms / a.g. uitterlinden [et al.] // gene. 2004. 338. 143–156. 10. girgis cm, baldock pa, downes m. vitamin d, muscle and bone: integrating effects in development, aging and injury. mol. cell. endocrinol. 2015. vol. 410. p. 10–13. 11. holick mf. vitamin d: photobiology, metabolism, etc., in “primer on the metabolic bone diseases and disorders of mineral metabolism. american society for bone and mineral research. 1996, philadelphia. 74-81. 12. holick mf, binkley nc, bischoff-ferrari ha et al. evaluation, treatment, and prevention of vitamin d deficiency: аn endocrine society clinical practice guideline. j. clin. endocrinol. metab. 2011. vol. 96. № 7. p. 1911–1930. 13. schlingmann kp, kaufmann m, weber s, et al. mutations in cyp24a1 and idiopathic infantile hypercalcemia. n engl j med 2011; 365(5):410-21. 14. skolarikos a, straub m, knoll t et al. meta bolic evaluation and recurrence prevention for urinary stone patients: eau guidelines // eur. urol. 2015. vol. 67. № 4. p. 750–763. 15. timms pm, mannan n, hitman ga et al: circulating mmp9, vitamin d and variation in the timp-1 response with vdr genotype: mechanisms for inflammatory damage in chronic disorders? qjm. 2002. 95. 787-796. 16. vitamin d receptor gene fok1 polymorphism predicts calcium absorption and bone mineral density in children / sk ames [et al.] // bone miner res. 1999. 14, n. 5. 740–746. 17. wang tj, pencina mj, booth sl, jacques pf et al. vitamin d deficiency and risk of cardiovascular disease. circulation. – 2008. №117. – с.503-511. 18. wehr e, pilz s, boehm bo et al. association of vitamin d status with serum androgen levels in men. clin. endocrinol. (oxf.). 2010. vol. 73. № 2. p. 243–248. 19. xu h, zisman al, coe fl, worcester em kidney stones: an update on current pharmacological management and future directions. expert opin. pharmacother. 2013. vol. 14. № 4. p. 435–447. received 25 may 2021; revised 19 jun 2021; accepted 20 jun 2021. this is open access article distributed under the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. sh.a. yusupov et al. 30 in t e r n a l m e d ic in e issn 2413-6077. ijmmr 2020 vol. 6 issue 2 *corresponding author: mariya marushchak, department of functional and laboratory diagnostics, i. horbachevsky ternopil national medical university, 1 maidan voli, 46001 ternopil, ukraine. e-mail: marushchak@tdmu.edu.ua international journal of medicine and medical research 2020, volume 6, issue 2, p. 30-36 copyright © 2020, tnmu, all rights reserved u.p. hevko et al. doi 10.11603/ijmmr.2413-6077.2020.2.11688 polymorphisms of insulin receptor substrate 1 as a risk factor for type 2 diabetes mellitus, obesity and chronic pancreatitis among population of ternopil region u.p. hevko, m.i. marushchak i. horbachevsky ternopil national medical university, ternopil, ukraine background. the course of type 2 diabetes mellitus (t2dm), obesity and chronic pancreatitis (cp) in most cases is not isolated, so it requires broadening the knowledge about the pathogenetic links at their combined course. despite significant advances in genome research, most of the genetic factors that cause development of t2dm are still unclear. objective. the aim of the study was to establish the prevalence of irs1 gene polymorphism in the patients with t2dm as well as obesity and cp. methods. the study involved 33 patients with t2dm who were hospitalized in the endocrinology department of ternopil university hospital in 2019-2020 and 10 apparently healthy patients. analysis of irs1 gene polymorphism (snp in the promoter region – rs2943640; gene localization 2q36.3) was performed on the basis of polymerase chain reaction data using specific primers. results. it was found that the frequencies of the genotype responsible for c/a polymorphism of irs1 gene in t2dm, t2dm with obesity and in the combined course of t2dm with obesity and cp did not deviate significantly from the hardy-weinberg equilibrium (p>0.05). the patients with combined course of t2dm, obesity and chronic pancreatitis experienced a probable influence of genotypes c/c and c/a of irs1 gene on the development of the studied comorbidity (p<0.05), which is confirmed by a probable difference in the dominant model of irs1 gene inheritance only when t2dm was combined with obesity and cp compared to the control group (p<0.001). conclusions. the presence of the c allele in both homozygous and heterozygous states may increase the risk of t2dm comorbidity, obesity and cp in the population of ternopil region. keywords: type 2 diabetes mellitus; obesity; chronic pancreatitis; insulin receptor substrate 1; genes polymorphism. introduction type 2 diabetes mellitus (t2dm) is a multifactorial genetic disease that causes significant morbidity and mortality worldwide [1]. multimorbidity, the presence of two or more chronic diseases [2], is typical for patients with t2dm, which makes multimorbidity in this population a significant clinical issue. nowakowska et al. found out that almost 75% of patients had at least one additional comorbidity at the time of t2dm diagnosis, and 44% – at least two comorbidities [3]. other studies indicate diabetic complications in 90% [4], 91.4% [5], 84.6% [6], 44% [7] of patients with t2dm, which probably depends on the age and gender coverage of the respondents in the study. the course of t2dm, obesity and chronic pancreatitis (cp) in most cases is not defined, so it requires broadening the knowledge about the pathogenetic links at their combined course [8-11]. it is obvious that t2dm is a poorly controlled epidemic that requires active research of the mechanisms of development, features of the course, methods of prevention, treatment and prevention of complications [12]. despite significant advances in genome research, most of the genetic factors that cause development of t2dm are still unclear [13, 14]. the insulin receptor substrate (irs) is a key central receptor in transmission of insulin signals. several irs polymorphisms have been identified, but the gly to arg 972 substitution of irs1 probably is pathogenetically crucial in the development of t2dm [15]. therefore, the aim of our study was to establish the prevalence of irs1 gene polymorphism in the patients with type 2 diabetes mellitus as well as obesity and chronic pancreatitis. methods the study involved 33 patients with t2dm who were hospitalized in the endocrinology 31 in t e r n a l m e d ic in e issn 2413-6077. ijmmr 2020 vol. 6 issue 2 department of ternopil university hospital in 2019-2020 and 10 apparently healthy patients. the distribution of groups is presented in table 1. there was no significant difference in age and gender between the patients of experimental groups. all patients were informed of the purpose of the clinical trial and gave written informed consent to participate in it. the privacy of the patient’s identity and health has been maintained. verification of t2dm was performed in accordance with the recommendations of the american diabetes association (2019) [16]. the criteria for t2dm diagnosing were based on the rate of glycated hemoglobin (hba1c) (≥6.5%), which was determined using the cobas 6000 automatic biochemical analyzer (roche hitachi, germany). verification of chronic pancreatitis (cp) was based on the unified clinical protocol of pri­ mary, secondary (specialized) medical care and medical rehabilitation “chronic pancreatitis” and the recommendations of the american pancreatic association [15,16]. bmi was calculated by the bmi formula = body weight (kg) / height (m2). the data were interpreted according to the who recommendations: normal weight in the range of 20.024.9 kg/m2; overweight (pre-obesity) – 25.029.9 kg/m2; class 1 obesity – 30.0-34.9 kg/m2; class 2 obesity – 35.0-39.9 kg/m2 and class 3 obesity >40 kg/m2 [17]. inclusion criteria were: clinical, laboratory and instrumental signs of t2dm, cp and obesity, no severe increase (not more than in 3 times from the upper of norm maximum) of alpha-amylase, lipase, alt, ast, alkaline phosphatase, gamma-glutamyltranspeptidase in the blood. exclusion criteria from the study were: signs of clinically significant neurological, mental, renal, hepatic, immunological, gastrointestinal, urogenital disorders, musculoskeletal lesions, skin, sense organs, endocrine (except t2dm) or hematological diseases that were uncontrolled, acute pancreatitis, unstable or lifethreatening heart diseases, malignant neoplasms not in complete remission for at least 5 years, medication (drug) dependence, alcohol dependence. analysis of irs1 gene polymorphism (snp in the promoter region – rs2943640; gene localization – 2q36.3) was performed on the basis of polymerase chain reaction data using specific primers. genomic dna was extracted from peripheral blood leukocytes using a commercially available dna isolation kit (qiaamp blood dna mini kit, qiagen, germany). the rs2943640 irs1 gene polymorphism was genotyped using the taqman real-time pcr method (applied biosystems, foster city, ca, usa). three genotypes of irs1 polymorphism (rs2943640) were defined (c/c, c/a and a/a). statistical analysis of the data was performed using the statistica 7.0 software. the hardyweinberg equilibrium was used to verify the conformity of the genotypes of the selected samples to the general population. comparison of the frequencies observed and expected, calculated according to the formula p2 + 2pq + q2 = 1 (the hardy-weinberg equilibrium), was performed using the pearson’s chi­square χ2. in assessment of the reliability coefficient p>0.05, the null hypothesis of the samples equality was taken into the account, i.e. the correspondence of the selected sample to the general population. comparative analysis of frequency tables was performed using the pearson’s chi-square χ2 and two-tailed p-value for fisher’s exact test (in cases of the expected frequencies of individual indicators not exceeding 5). to evaluate the influence of the factor (the presence of a certain genotype or allele of the gene) on the disease incidence, calculation of the odds ratio (or), its 95% confidence interval (ci) and the reliability coefficient p­value was used. results insulin receptor substrate (irs) molecules are key mediators in transmission of insulin signals. several polymorphisms have been defined in irs genes, but only the gly to arg table 1. characteristic features of the study groups (n=44) group № characteristics of the group n % 1 t2dm patients with normal body weight without chronic pancreatitis 9 22.7 2 t2dm patients with overweight / obesity without chronic pancreatitis 14 31.9 3 t2dm patients with overweight / obesity with concomitant chronic pancreatitis 10 22.7 4 apparently healthy patients (control) 10 22.7 u.p. hevko et al. 32 in t e r n a l m e d ic in e issn 2413-6077. ijmmr 2020 vol. 6 issue 2 972 substitution of irs-1 is pathogenically crucial in development of t2dm. however, other polymorphisms described in irs-1 gene are associated with insulin resistance (ir) in t2dm. the frequency distribution of polymorphic genotypes of irs1 gene (rs2943640) and verification of conformity with the hardy­ weinberg population equilibrium were performed in the experimental and control groups. it was found out that the frequencies of the genotype responsible for c/a poly morphism of irs1 gene in t2dm, t2dm with obesity and at the combined course of t2dm with obesity and cp did not deviate significantly from the hardy­ weinberg equilibrium (p>0.05), while in the control group the selected sample did not correspond to the general population (table 2). the corresponding frequencies for the genotypes of irs1 gene were as follows: 66.7% for c/a and 33.3% for a/a in the experimental group 1; 21.4% for c/a, 64.3% for c/a and 14.3% for a/a in the group 2; 70.0% for c/a, 10.0% for c/a and 20.0% for a/a in the group 3 and 100.0% for c/a in the control group (table 3). the frequencies of alleles of irs1 gene in the patients involved in the study are presented in table 2. in the patients with t2dm the allele c predominated, in the patients with t2dm + obesity + cp – allele a, while in the patients with t2dm + obesity and in the control group, the c allele and the a allele were almost equal. the obtained results, presented in table 4, indicate the absence of a statistically significant relationship between the factor (presence of alleles c or a) and the disease incidence (p>0.05). evaluation of the reliability coefficient in the analysis of odds ratio showed probable in flu­ ence of the genotypes c/c and c/a of the irs1 gene on development of t2dm combined with table 2. polymorphism of irs1 genes (rs2943640) according to the hardy-weinberg equilibrium in t2dm and its comorbidity genotypes t2dm (group 1) t2dm+obesity (group 2) t2dm+obesity+ср (group 3) control expected present expected present expected present expected present polymorphism of irs1 gene (rs2943640) homozygotes that are common с/с 1 0 4,0 3 6.4 7 2.5 0 heterozygotes с/а 4 6 7.0 9 3.2 1 5 10 homozygotes that are rare а/а 4 3 3.0 2 0.4 2 2.5 0 χ2, р χ2=2.25; р>0.05 χ2=1.20; р>0.05 χ2=2.45; р>0.05 χ2=10.00; р<0.01* table 3. frequency of the alleles of the irs1 genes (rs2943640) in t2dm and its comorbidity frequency of alleles t2dm (group 1) t2dm+obesity (group 2) t2dm+obesity+ср (group 3) control n % n % n % n % polymorphism of irs1 gene (rs2943640) allele c 6 33.33 15 53.57 16 80.00 10 50.00 allele a 12 66.67 13 46.43 4 20.00 10 50.00 pf (patients/control) рf=0.342 рf=0.999 рf=0.096 – table 4. the odds ratio for alleles in different study groups in t2dm and its comorbidity group irs1 gene (rs2943640) allele c allele a or 95 % ci p or 95 % ci p t2dm (group 1) 0.50 0.13-1.86 >0.05 2.00 0.54-7.45 >0.05 t2dm+obesity (group 2) 1.15 0.37-3.64 >0.05 0.87 0.27-2.73 >0.05 t2dm+obesity+ср (group 3) 4.00 0.98-16.27 >0.05 0.25 0.06-1.02 >0.05 u.p. hevko et al. 33 in t e r n a l m e d ic in e issn 2413-6077. ijmmr 2020 vol. 6 issue 2 obesity and cp (p<0.05) (table 5). this is con­ firmed by a probable difference in the dominant model of irs1 gene inheritance only in the group with combination of t2dm as well as obesity and cp compared with the control group (reliability coefficient for the chi­square p<0.001). thus, the presence of the c allele in both homozygous and heterozygous states may increase the risk of t2dm comorbidity, obesity and cp (table 6). it should be noted that in recessive models of irs1 gene inheritance in t2dm, t2dm + obesity and t2dm + obesity + cp no significant differences were established compared with the control group, but there was also a tendency to increase in the probability of development of these diseases in the presence of c allele (table 7). discussion it is established that genes are crucial in development of t2dm. researchers have suggested the interaction between many genetic factors and environmental factors that table 5. the odds ratio for genotypes in different study groups in t2dm and its comorbidity group genotype polymorphism of irs1 gene (rs2943640) cc cа аа or 95 % ci or 95 % ci or 95% ci t2dm (group 1) 1.11 0.02-61.38 0.09 0.01-2.00 11.31 0.50-256.21 t2dm+obesity (group 2) 6.39 0.29-138.94 1.73 0.03-99.96 4.20 0.18-97.55 t2dm+obesity+ср (group 3) 71.40* 3.00-1696.84 0.002* 0.001-0.13 6.18 0.26-146.79 note. * ‒ р<0.05. table 6. dominant model of irs1 gene inheritance (rs2943640) in t2dm and its comorbidity genotypes experimental group control pf or 95% ci p% % t2dm (group 1) с/с 0 0 ‒ 1.11 0.02‒61.38 >0.05 с/а+а/а 100.00 100.00 0.90 0.02‒50.25 >0.05 t2dm+obesity (group 2) с/с 21.43 0 0.239 6.39 0.29‒138.94 >0.05 с/а+а/а 78.57 100.00 0.16 0.01‒3.40 >0.05 t2dm+obesity+ср (group 3) с/с 80.00 0 <0.001* 71.40 3.00‒1696.84 0.008* с/а+а/а 20.00 100.00 0.01 0.001‒0.33 0.008* note. * – statistically significant results. table 7. recessive model of irs1 inheritance (rs2943640) in t2dm and its comorbidity genotypes experimental group control pf or 95 % ci p% % t2dm (group 1) с/с+с/а 66.67 100.00 0.087 0.09 0.01‒2.00 >0.05 а/а 33.33 0 11.31 0.50‒256.21 >0.05 t2dm+obesity (group 2) с/с+с/а 85.71 100.00 0.493 0.24 0.01‒5.53 >0.05 а/а 14.29 0 6.18 0.26‒146.79 >0.05 t2dm+obesity+ср (group 3) с/с+с/а 80.00 100.00 0.474 0.16 0.01‒3.85 >0.05 а/а 20.00 0 6.18 0.26‒146.79 >0.05 u.p. hevko et al. 34 in t e r n a l m e d ic in e issn 2413-6077. ijmmr 2020 vol. 6 issue 2 contribute to the disease development [20]. however, there are only isolated data in the literature on the role of irs1 polymorphism (rs2943640) in increased susceptibility to t2dm. thus, langenberg c et al. presented the results of the interact study, which proved the effect of c allele of irs1 gen e p olymorphism (rs2943640) on the risk of t2dm development [21]. in the study prepared by liu j and et al. 8 snps associated with t2dm were found, including rs2943640 variant of irs1 gene, which was also associated with body mass index [22]. a study of the physical activity genetics made by loos rj et al., which included irs1 (rs2943640) among the studied genes, showed an increased interaction of the gene with the risk of diabetes, in particular, a dependence on genetic susceptibility to insulin resistance [23]. another interesting result of this study is the established data on the higher genetic risk of t2dm in individuals with the highest level of physical activity that is consistent with the large-scale study of langenberg et al. (24), in which the predicted effect of t2dm genetic risk was the strongest among young, lean, and physically active individuals [21]. it should be noted that according to karaderi t. et al., rs2943640 variant of irs1 gene is associated with decreased bmi [24]. our results prove the effect of c allele of irs1 gene (rs2943640) on the increased susceptibility to the combination of t2dm+obesity+cp, but there is no probable effect of the factor (alleles c and a) on development of only t2dm and t2dm+obesity. there are no data in the literature on the role of irs1 gene (variant rs2943640) in increased susceptibility to cp. there are some limitations in this study that need to be considered when interpreting its results: the sample size is too small, so it is difficult to find significant relationships between the data; inclusion into the experimental groups only of patients with comorbidity t2dm+obesity and t2dm+obesity+cp; patients were not randomly selected generating a potential selection bias. therefore, we cannot exclude the hypothesis that the evaluated patients do not represent the whole population of the patients with comorbid t2dm, but these results are the first step in finding genes for increased susceptibility to the studied pathology, they reflect a more heterogeneous real­ world population characteristic in clinical practice. conclusions the patients with combined course of t2dm, obesity and chronic pancreatitis have a probable influence of genotypes c/c and c/a of irs1 gene on development of the studied comorbidity (p<0.05) that is confirmed by a probable difference in the dominant model of irs1 gene inheritance only when t2dm is combined with obesity and cp compare to the control group (p<0.001). thus, the presence of the c allele in both homozygous and heterozygous states may increase the risk of t2dm comorbidity, obesity and cp in the population of ternopil region. funding this research received no external funding. conflict of interest the authors declare no conflict of interest. author contributions hevko u.p. – methodology, software, validation, formal analysis, investigation, resources, data curation, visualization, writing – original draft, writing – reviewing and editing. marushchak m.i. – conceptualization, visualization, supervision, writing – reviewing and editing. поліморфізм субстрату інсулінових рецепторів 1 типу як фактор ризику розвитку цукрового діабету 2 типу, поєднаного з ожирінням та хронічним панкреатитом у населення тернопільської області у.п. гевко, м.і. марущак тернопільський національний медичний університет імені і.я. горбачевського моз україни, тернопіль, україна вступ. перебіг цукрового діабету 2 типу (t2dm), ожиріння та хронічного панкреатиту (cp) в більшості випадків не є ізольованим, тому потребує поглиблення знань стосовно патогенетичних u.p. hevko et al. 35 in t e r n a l m e d ic in e issn 2413-6077. ijmmr 2020 vol. 6 issue 2 ланок при їх поєднаному перебігу. незважаючи на значні успіхи у дослідженнях геному, більшість генетичних факторів, що спричинюють розвиток t2dm, залишаються невизначеними. мета дослідження – встановити поширення поліморфізму гена субстрату інсулінових рецепторів 1 типу (irs1) у хворих на t2dm у поєднанні з ожирінням та ср. методи дослідження. у дослідження було включено 33 хворих на t2dm, які перебували на стаціонарному лікуванні в ендокринологічному відділенні тернопільської університетської лікарні у 2019-2020 рр. та 10 практично здорових осіб. аналіз поліморфізму гена irs1 (snp у промоторному регіоні – rs2943640; генна локалізація 2q36.3) проведено на підставі даних полімерної ланцюгової реакції з використанням специфічних праймерів. результати. встановлено, що частоти генотипу, що відповідає за с/а поліморфізм гена irs1 при t2dm, t2dm з ожирінням та при поєднаному перебігу t2dm з ожирінням та ср суттєво не відхилялися від рівноваги харді–вайнберга (p>0,05). у хворих на поєднаний перебіг цукрового діабету 2 типу, ожиріння та хронічного панкреатиту виявляється вірогідний вплив генотипів с/с та с/а гена irs1 на розвиток досліджуваної коморбідності (р<0,05), що підтверджується вірогідною відмінністю у домінантній моделі успадкування irs1 гену тільки при поєднанні t2dm з ожирінням та ср порівняно з групою контролю (р<0,001). висновки. наявність алелі с як в гомозиготному, так і в гетерозиготному станах може підвищувати ризик виникнення коморбідності t2dm, ожиріння та ср. ключові слова: цукровий діабет 2 типу; ожиріння; хронічний панкреатит; субстрат інсулінових рецепторів 1 типу; поліморфізм гена. information about the authors hevko u.p. – phd student, department of functional and laboratory diagnostics, i. horbachevsky ternopil national medical university, ternopil, ukraine. orcid 0000-0001-5265-2842, e-mail: gevkoup@tdmu.edu.ua marushchak m.i. – professor, department of functional and laboratory diagnostics, i. horbachevsky ternopil national medical university, ternopil, ukraine. orcid 0000-0001-6754-0026, e-mail: marushchak@tdmu.edu.ua references 1. roy s, sherman a, monari-sparks mg, schweiker o, jain n, etty sims e, et al. association of comorbid and metabolic factors with optimal control of type 2 diabetes mellitus. n am j med sci. 2016; 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revised 20 dec 2020; accepted 23 dec 2020. this is open access article distributed under the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. u.p. hevko et al. 5 in t e r n a l m e d ic in e issn 2413-6077. ijmmr 2019 vol. 5 issue 2 doi 10.11603/ijmmr.2413-6077.2019.2.10334 stool antigen (hpsa) test in detection of helicobacter pylori infection among adult dyspeptic patients in tripoli, libya *g.m. huwiage1, a.a. nami2, a.h. akadh3 1 – high institute of medical sciences& technology, algarabolli, libya 2 – al-mergib university, al khums, libya 3 – mediterranean specialized clinics, tripoli, libya background. helicobacter pylori is the most common infection in the world. relationship between h. pylori and dyspepsia was confirmed by many studies, it has been strongly associated with peptic ulcer disease and gastric cancer. in that respect, several invasive and noninvasive methods for the diagnosis of h. pylori infection were utilized. objective. the aim of the study was to evaluate the association between dyspepsia and the positivity of h. pylori stool antigen test, to compare this test with serological igg test. methods. 125 adult patients were randomly selected from gastroenterology units of mediterranean and tajurah clinics in tripoli. stool samples were taken for detection of h. pylori antigen by enzyme immunoassay. blood samples for detection of anti-h. pylori igg antibodies were taken. data were statistically analyzed using spss. results. 125 dyspeptic patients: 47 male and 78 female, aged 18-83 years old were examined. 80 patients were infected by h. pylori that was proved by a positive stool test, 88 had a positive igg test. the prevalence was higher in the patients aged 28-47 years old. there was substantial relation to age, marital status and economic risk factors; there was no association between h. pylori and gender, sources of drinking water, living standards, smoking, family history of peptic ulcer, drug consumption, and blood groups. conclusions. relatively high rates of detection by hpsa prove that stool testing might be a reliable, simple, inexpensive, and non-invasive alternative test ащк detectшщт of h. pylori, diagnosing active infection and confirming cure. however igg test has a low sensitivity, specificity, and accuracy compare to the hpsa test. thus it can be used for screening purposes. key words: helicobacter pylori; dyspepsia; enzyme immunoassay; stool antigen test. *corresponding author: goma mohamed huwiage, ph.d., assistant professor, department of medical microbiology, high institute of medical sciences and technology, algarabolli, libya. e-mail: gomanagoma96@yahoo.com introduction the main definitive events of our knowledge of h. pylori came in 1979-1982 by the basic experiments of the pathologist robin warren, who identified the bacterium, and barry mar­ shall, who successfully cultured the bacterium. in their experiments, they emphasized the association of h. pylori with gastroduodenal diseases. in the end, the world health organization classified h. pylori is a class i carcinogen in 1994 due to its definite carcinogenic potential in humans [1]. h. pylori is a gram-negative spiral shaped, microaerophilic, and slowly growing bacterium. h. pylori-colonize the human stomach mainly in the mucus layer, rarely adhere to the mucosal cells, or intracellular. it is motile due to the presence of a tuft of polar flagella; moreover, it holds an important acid resistance mechanism through the generation of urease enzyme [2]. one of the principal structural parts of h. pylori is the flagella; it has unipolar flagella developed to move through the mucus barrier toward the gastric epithelial surface for colonization [3]. the other structural component is the outer membrane proteins (omps), these surface proteins have a specific important ad­ hesive virulence factor that can bind to receptors on the surface of the gastric mucosa. one of these factors is the baba (blood group antigen-binding adhesion) [3]. the other omp adhesion is the sialic acid binding adhesion (saba). bab a and sab a adhesions are usually linked to increased risk of gastro duodenal diseases [2]. lipopolysaccharide (lps) is another its structural element. it is significant in the integrity of the cell envelope of gram-negative bacteria; besides, it can affect the severity and chronicity of infection [4]. the most significant structure of h. pylori is the genome. there are international journal of medicine and medical research 2019, volume 5, issue 2, p. 5-14 copyright © 2019, tnmu, all rights reserved g.m. huwiage et al. 6 in t e r n a l m e d ic in e issn 2413-6077. ijmmr 2019 vol. 5 issue 2 two independent h. pylori genomes, that have been completely sequenced the h. pylori 26695 and h. pylori j99 genomes, although the 26695 genomes was 24 kb larger, both the j99 and 26695 genomes possessed a total (g+c)% of 39%. h. pylori have small genome (1,667,867 base pair for 26695 type while, it is 1.643.831 for j99 type). this genome accommodates about 1,500 genes [5]. virulence by definition is the ability of pathogens cause disease, and factors associated with this virulence are virulence factors. h. pylori virulence factors that are positively correlated with gastrointestinal diseases include structural, as well as secretory components. structural components include lps, omps, flagella, and the type iv secretion system (t4ss); however, its secretory compo­ nents involve ammonia by urease, cytotoxinassociated gene a (caga), vaculating cytotoxin (vaca), and other secretory enzymes [6]. the prevalence of h. pylori among adults is ~30% in the united states and other developed countries, while it is more than 80% in most de veloping countries h. pylori are usually acquired in childhood [2]. newly published two studies in african countries, reported that the prevalence of h. pylori infection is yet high in morocco and ethiopia (75.5% and 67.7% respectively), both surveys demonstrated a significant correlation with age. another survey in nigerian dyspeptic patients found that the prevalence was 93.6% by the serology test. these surveys showed that the prevalence was still high in this region; however, it looked lower in the western developed world in spite of higher rates among immigrants coming from countries with a high prevalence of h. pylori [7]. regarding important risk factors, it was accounted for that the prevalence of h. pylori was positively correlated with the living in rural areas, bad sanitary status, overcrowding, low educational level, and poor socioeconomic conditions [7]. transmission of the infection probably occurs in multiple pathways, which may differ in various societies and age groups, [8]. it has been established that there is still contention about the actual mode of transmission of h. pylori infection, although person-toperson transmission through fecal-oral or oraloral routes has been emphasized by many studies [9]. oral-oral route of transmission depends on the presence of h. pylori in the gastric juice, so could reach the oral cavity through reflux or vomitus. an early study reported that h. pylori can be cultivated from vomitus, as a result, h. pylori are potentially transmissible during gastrointestinal diseases, especially, during vomiting [9], and moreover, it has been shown that h. pylori could be transferred through contact between persons, particularly, during childhood. the primary path of transmission is regarded through oraloral, fecal-oral, or gastric-oral. nevertheless, the route of transmission appears to be different, between developing, and developed countries, because the age, race, and socioeconomic status are changeable [7]. dyspepsia could be separated into two types: ulcer dys­ pepsia (ud) related to organic cause and nonulcer dyspepsia (nud) also known as functional dyspepsia (fd), which is specified as chronic dyspepsia without organic disease [10]. for diagnosis, functional dyspepsia has been described that the patients higher than 55 years old, or those with alarm features such as git bleeding, weight loss, family history of gastric cancer, should undergo quick endoscopy to exclude organic diseases; nevertheless, in pa­ tients aged 55 years or younger with no alarm features, the clinician may consider testing and treat strategy using a non-invasive test for h.pylori diagnosis followed by the treatment of the h. pylori positive patients. it was recognized that the test and treatment option was preferable in populations with a moderate to high prevalence of h. pylori infection (≥10 %) [11]. the rapid hpsa test based on ica methods was more recently developed and had been used for the detection of h. pylori infection before and for follow up after the treatment; never­ theless, the accuracy of the rapid hpsa test is comparable, or a slight bit lower than the standard hpsa test [12]. the most distinct indications for treatment of h. pylori infection are those related to duodenal or gastric ulcers regardless of their activity status; other important in­ dications are patients with low-grade gastric b cell lymphoma and after gastric cancer re section, however, many guidelines now recommend h. pylori treatment in uninvestigated simple dyspepsia following noninvasive diagnosis, also recommend treatment in functional dyspepsia although, it was proved that only 5-10% of patients get benefit from such treatment [2]. combination regimens were developed to overcome the resistance to monotherapy. while, the best option is doing antibiotic sensitivities, but it is difficult and expensive and usually not indicated unless there is a failure of the second line of the treatment regimen. the recommended treatment regimen for h. pylori infections are the first line regimen involving g.m. huwiage et al. 7 in t e r n a l m e d ic in e issn 2413-6077. ijmmr 2019 vol. 5 issue 2 the usage of three drugs: omeprazole, clari­ thromycin, and amoxicillin for 7­14 days; or the use of omeprazole, clarithromycin, and metronidazole for 7-14 days. the second line suggests the use of four drugs: omeprazole, bismuth subsalicylate, tetracycline and metronidazole for 14 days [2]. the objectives of the study were to determine the prevalence of h. pylori among adult dyspeptic patients in tripoli region and to evaluate the performance of stool antigen test (hpsa) as a diagnostic method for the detection of h. pylori infection; to compare (hpsa) test with igg antibody test regarding sensitivity, specificity, accuracy and their useful applications; to determine the relationship of a proposed risk factor with h. pylori infection in the studied group. methods research design. the work was a crosssectional study conducted to find out the pre­ valence of h. pylori among the dyspeptic patients admitted to the gastroenterology units of the mediterranean and tajurah specialized clinics in tripoli from january to april 2017. labbased stool examination was carried out to collect data on the prevalence of h. pylori. in addition to this, data on risk factors were gathered from the study population using a questionnaire survey, which was focused on associated factors contributing to h. pylori infection. but the serological igg antibody test was applied to all the participants for comparison with the hpsa test. patients. in three months, (january to april 2017), one hundred and twenty­five different patients were selected randomly from those, who were attending gastroenterology units of the mediterranean and tajurah specialized clinics in tripoli city. all patients with complaints of dyspepsia were enrolled in this study. sample size. blood and stool samples were collected from all participants (125 randomly selected eligible patients) and all were interviewed to answer the questionnaire. sample collection. an volume of 5-gram stool samples was collected from each participant in a clean, dry, waterproof container containing no detergent, preservatives or transport media; the patients’ fecal samples were either frozen for long periods (60 days) or refrigerated for short periods (48 hours). sterile plastic stool container and elisa antibody kits (accudiag™, diagnostic automation, inc., california, usa) of 100% specificity and 0.5ng/ml sensitivity [see appendix b] were used for the study, as well as icebox for keeping the sample through transportation. a deep freezer for specimen storage was prepared as well as elisa micro plate reader (bio-tec) germany. assay procedure and principle of the test. h. pylori antigen in stool sample was determined by the elisa using a commercially available monoclonal antibody kit. stool samples were diluted, added (100μl) to antibody­coated micro wells and incubated for 30 minutes at room temperature. h. pylori­specific monoclonal antibodies were conjugated with the h. pylori antigens. all unbound materials were washed out (three time repeated washing process). then enzyme conjugate was added (red color solution) and incubated for 30 minutes; after adding the enzyme, it was bound to the antibody-antigen complex. the excess enzyme conjugate was washed out also, and the chromogenic substrate was added and incubated for 15 minutes. after the addition of the stop solution to stop the reaction, a visible yellow color reaction appeared indicating the presence of h. pylori. the intensity of the generated color was proportional to the amount of antigen in the sample. the results were read by a microwell reader compared in a parallel with a calibrator and controls. all the tests were performed according to the manufacturer’s instructions. the diagnostic elisa, hpsa test was used as a quantitative assay for detection of h. pylori antigens in a human stool specimen. the estimation of the sample concentration depended upon the construction of a standard curve, so the standard curve absorbance (o.d 450 nm) versus concentration of stool antigen in ng/ml for the quantitative detection of the stool antigen in each sample was applied, according to the elisa kit standard. serological tests for detection of igg antibodies. three ml venous blood was withdrawn from each participant at the time of delivering stool sample, then the blood was centrifuged at speed of 2200-2500 rpm (revolutions per minute) for 10 minutes to separate the serum that was analyzed immediately or was stored refrigerated at (4–8 °c) for up to 48 hours. for a longer storage, the serum was kept at -20 °c until analyzed for anti-h. pylori antibodies – igg detection using rapid anti-h. pylori kit (advanced quality™, intec products, inc, china). assay procedure and principle of rapid h. pylori igg test: we brought the device, sample diluent, and specimens to room temperature. g.m. huwiage et al. 8 in t e r n a l m e d ic in e issn 2413-6077. ijmmr 2019 vol. 5 issue 2 after we dispensed 1­2 drops (10 μl each drop) of serum to the circular sample well of the test card using the plastic dropper. we added two drops of sample diluent to the sample well immediately. we interpreted test results at 15 minutes. in positive results, we saw two lines, one of them referred to the control, while the other one referred to the infected individual. the principle of the test used for detecting igg antibodies in human serum or plasma is a visual, qualitative test. it was performed in a single-step procedure using one to two hanging drops of serum. the test was started with a sample applying to the sample kit and adding the provided sample diluent immediately. the h. pylori antigens-colloidal gold conjugate embedded in the sample pad reacted with the h. pylori antibody present in serum or plasma sample forming conjugate/ the h. pylori antibody complex. as the mixture was allowed to migrate along the test strip, the conjugate/ the h. pylori antibody complex was captured by an antibodybinding protein and immobilized on a membrane forming a colored test band in the test region. a negative sample did not pro duce a test line due to the absence of colloidal gold conjugate h. pylori antibody complex. a colored central band in the control region appeared at the end of test procedure regardless of test results. this control band was the result of colloidal gold conjugate binding to an anti-h. pylori antibody immobilized on the membrane. the control line indicated that the colloidal gold conjugate was functional. the absence of the control band evidenced that the test was invalid. hpsa should be used as the gold standard as the test offers excellent sen sitivity and specificity compare to the in vasive methods, such as gastric biopsy, culture, and the rapid urease test. [10]. statistical analysis. the data for prevalence and distribution of helicobacter pylori infection among sexes and different age groups generated from the study and the association between the prevalence of h. pylori infection and risk factors were determined and tabulated as microsoft excelled sheets and uploaded to the statistical package for social sciences (spss version 18) cross-tabulation of the variables were generated. chi-square was used to detect a statistically significant correlation between the variables. results study sample description. the survey was carried on during the period from january to april 2017. one hundred and twenty-five different dyspeptic patients were included in our study to investigate the prevalence of h. pylori infection and its related risk factors. during this period, all patients were interviewed, and they answered the questionnaire regarding personal information and their lifestyle. at the same time samples of stool and blood were collected for investigations. the participants were living in different geographical areas of tripoli; appro ximately 53% of them were from tajurah, 22% – tripoli central area and 17% – ain-zarra area. the following tables present a concise description of the study samples. the study population age ranged from 1883 years old. 37.6% were males with a mean age of 40.22 years old, and 62.4% were females with a mean age of39.10 years old. the male to female ratio was 1:1.66 (table 1). 2. detection of h. pylori infection by hpsa test table 2 presents the rate of positivity of h. pylori stool antigen test in adult dyspeptic table 1. division of patients with dyspepsia by age and sex age groups male no. % female no. % total no. % 18-27 5 20.83 19 79.17 24 19.2 28-37 11 34.38 21 65.62 32 25.6 38-47 16 55.17 13 44.83 29 23.2 48-57 6 28.57 15 71.43 21 16.8 >57 9 47.37 10 52.63 19 15.2 total 47 37.6 78 62.4 125 100% table 2. prevalence of h. pylori infection by hpsa test stool ag. detection the result of h. pylori stool ag. no. the result of h. pylori stool ag. % positive 80 64% negative 45 36% total no. 125 100% g.m. huwiage et al. 9 in t e r n a l m e d ic in e issn 2413-6077. ijmmr 2019 vol. 5 issue 2 pa tients among 125 adult patients, who underwent hpsa test. in (64%) 80 patients it was positive. 3. detection of h. pylori infection by igg test table 3 showed that the prevalence of h. pylori infection by igg was 70.4%. table 3. prevalence of h. pylori infection by igg test igg antibody detection the result of h. pylori igg test in no. the result of h. pylori igg test in % positive 88 70.4% negative 37 29.6% total no. 125 100% table 4.1. comparison of frequency hpsa test (gold standard) and igg test test hpsa positive no. hpsa negative no. total no. p value igg positive no. 64 24 88 0.002igg negative no. 16 21 37 total no. 80 45 125 table 4.2. igg test *hpsa test cross tabulation. hpsa test total positive negative igg test positive no. %within igg test %within hpsa test 64 72.7% 80.0% 24 27.3% 53.3% 88 100.0% 70.4% igg test negative no. %within igg test %within hpsa test 16 43.2% 20.0% 21 56.8% 46.7% 37 100.0% 29.6% total no. %within igg test %within hpsa test 80 64.0% 100.0% 45 36.0% 100.0% 125 100.0% 100.0% 4. stool antigen testing versus serology (igg) 4.1. comparison of frequency hpsa test (gold standard) and igg test. comparison of stool antigen detection test with serology (table 4.1) revealed that among 80 stool antigen positive cases, 64 (80%) were positive by igg. 24 (53%) were positive for igg out of 45 stool antigen-negative cases. this difference was statistically significant at p=0.002. 4.2. accuracy of igg test table 4.2 proved that sensitivity of the igg test was 80%, while the specificity was 46.7%, total agreement (accuracy) 68%, ppv=72.7%, and npv=56.8%. 4.3. correlations of hpsa and igg tests with the age, marital status, monthly income, and family number risk factors from the table above, the significant correlation of hpsa test with the age, marital status, and monthly income is evident, while igg was positively correlated with the marital status and family number. 5. socio-demographic characteristics of study participants 5.1. personal risk factors prevalence of h. pylori among some associated risk factors of the study participants (table 5.1). the correlation is significant at the level of 0.01 (2-tailed) regarding age (p=0.004) and 0.05 (2-tailed) regarding marital status risk factors (p=0.033). while there is no significant correlation between hpsa positivity and, gender (p=0.976), job (p=0.430) and educational level (p=0.819). 5.2. effects of socioeconomic status table 5.2 proved that the correlation was significant at the level of 0.05 for the monthly income (p=0.028); however, there is no corre­ lation between the number of people living in each household (p=0.086) and sources of water (p=0.716). 5.3. lifestyle variables, family history of gastroduodenal disease and drug consumption table 5.3 showed that there is no significant correlation between the rate of h. pylori infec tion g.m. huwiage et al. 10 in t e r n a l m e d ic in e issn 2413-6077. ijmmr 2019 vol. 5 issue 2 table 4.3. pearson correlations of the hpsa and igg tests with some of the risk factors correlations hpsa test igg test age of the tested group marital status monthly income hpsa test pearson correlation 1 0.280** -0.256** -0.191* -0.197* sig. (2-tailed) 0.002 0.004 0.033 0.028 n 125 125 125 125 125 igg test pearson correlation 0.280** 1 -0.116 -0.272** -0.090 sig. (2-tailed) 0.002 0.199 0.002 0.320 n 125 125 125 125 125 age of the tested group pearson correlation -0.256** -0.116 1 0.483** 0.077 sig. (2-tailed) 0.004 0.199 0.000 0.393 n 125 125 125 125 125 marital status pearson correlation -0.191* -0.272** 0.483** 1 -0.035 sig. (2-tailed) 0.033 0.002 0.000 0.694 n 125 125 125 125 125 monthly income pearson correlation -0.197* -0.090 0.077 -0.035 1 sig. (2-tailed) 0.028 0.320 0.393 0.694 n 125 125 125 125 125 family no. pearson correlation 0.154 0.224* 0.132 -0.253** -0.001 sig. (2-tailed) 0.086 0.012 0.143 0.004 0.988 n 125 125 125 125 125 notes. ** – correlation was significant at the 0.01 level (2-tailed); * – correlation was significant at the 0.05 level (2-tailed). table 5.1 personal risk factors variable negative no. hpsa % positive no. hpsa % p value age: 18-27 28-37 38-47 48-57 >57 15 12 9 4 5 62.5% 37.5% 31.0% 19.0% 26.3% 9 20 20 17 14 37.5% 62.5% 69.0% 81.0% 73.7% 0.004 sex: male female 17 28 36.2% 35.9% 30 50 63.8% 64.1% 0.976 marital status: single married 14 31 53.9% 31.3% 12 68 46.2% 68.7% 0.033 occupational status: employee housewife private student 15 15 7 8 34.1% 34.9% 28.0% 61.5% 29 28 18 5 65.9% 65.1% 72.0% 38.5% 0.430 education level: higher intermediate low 12 19 14 36.4% 33.9% 38.9% 21 37 22 63.6% 66.1% 61.1% 0.819 g.m. huwiage et al. 11 in t e r n a l m e d ic in e issn 2413-6077. ijmmr 2019 vol. 5 issue 2 and smoking (p=0.357), diseased family history (p=0.131) and drug consumption (p=0.363). h. pylori infection distribution according to the blood groups table 5.4 showed that there is no significant statistical correlation between the rate of h. pylori infection and blood groups despite higher prevalence rate among blood group o. table 5.2. effects of socioeconomic status variable hpsa negativeno. % hpsa positive no. % p value monthly income ld: <1000 1000-2000 >2000 23 20 2 42.6% 38.5% 10.5% 31 32 17 57.4% 61.5% 89.5% 0.028 number of people in household: 1-4 5-8 >8 10 22 13 31.3% 31.0% 59.1% 22 49 9 68.8% 69.0% 40.9% 0.086 sources of water for drinking: piped water and gallon well filtered water (tahlia) 9 16 20 27.3% 48.5% 33.9% 24 17 39 72.7% 51.5% 66.1% 0.716 table 5.3. lifestyle variables, family history of the gastroduodenal disease and drug consumption variable hpsa negativeno. % hpsa positive no. % p value smoking: no yes 37 8 38.14% 28.57% 60 20 61.86% 71.43% 0.357 family history of peptic ulcer or malignancy: no yes 38 7 39.58% 24.14% 58 22 60.42% 75.86% 0.131 consumed drugs: no yes 39 6 35.14% 42.86% 72 8 64.86% 57.14% 0.363 table 5.4. h. pylori infection distribution according to the blood groups blood group hpsa positive test no. hpsa negative test no. total no. p value a 24 17 41 0.761 b 20 8 28 0.819 ab 3 0 3 o 33 20 53 0.865 total 80 45 125 discussion the accurate diagnosis and management become a vital strategy for facing the h. pylori challenge because of its strong association with gastroduodenal diseases as well as its high rate of infectivity especially in the developing countries. although many diagnostic tests have been developed for detecting h. pylori, all have advantages as well as disadvantages. however, breath tests or fecal antigen tests are considered the best because of their excellent accuracy, as well as they are simple and non-invasive. in the study, hpsa was used as a proxy reference test for detection of h. pylori infection. as reported by many international studies, it was an accurate and a reliable test in comparison with the invasive tests and urea breath test for detection of active infection of h. pylori pa thogen [13­19]; moreover, to our best know ledge, there are no previous reports of assessing h. pylori infection g.m. huwiage et al. 12 in t e r n a l m e d ic in e issn 2413-6077. ijmmr 2019 vol. 5 issue 2 rates by stool antigen test in tripoli-libya. to avoid variations in polyclonal antibodies, we used a kit that included monoclonal antibodies specific for h. pylori antigens to assess the presence of h. pylori. monoclonal antibodies looked more accurate than polyclonal antibodies as it was documented by many studies [20]. our study focused mainly on determining the rate of h. pylori infection by hpsa test in adult dyspeptic patients of tripoli city, moreover, evaluating some of the potential risk factors associated with h. pylori infection, in addition, comparing hpsa test with the serological igg test. overall, 125 patients were involved in this study. the results proved that the age of the studied population ranged from 18-83 years old, with a mean age (40.22) (table 5.1). it was established that the majority of the examined patients were infected with h. pylori according to hpsa findings. the infection rates were increasing with age and married subjects (table 5.6). two non-invasive tests, hpsa test – the gold standard one, were used in this study to evaluate h. pylori infection as well as (igg) test for comparison. the study proved that the stool antigen assay (hpsa) has showed promising results for detection of h. pylori antigen in stool samples. conclusions the study proved that h. pylori infection was a serious health problem in the tripoli area. the rate of h. pylori infection among 125 adult dyspeptic subjects undergoing stool antigen test was relatively high (64%). most affected patients were at the age of 28-47 years old. it was more common in married subjects than single. the most significant risk factors for h. pylori infection was (age, marital status, and socio-economic state). sex, smoking, occupation, education level, number of persons in each home, water consumed, family history of peptic ulcer or gastric malignancy, drug consumption and blood group could not be considered as risk factors of h. pylori infection as evidenced by the results of our study in tripoli population. finally, hpsa test is comparatively inexpensive, noninvasive, simple and accurate test for detecting h. pylori in a stool sample. sats using monoclonal antibodies are useful for primary diagnosis as well as for the assessment of eradication therapy. recommendations according to the findings of the study the following recommendations can be shaped. there was a high prevalence rate of h. pylori infection in dyspeptic patients of this study area, so high attention should be paid by governments, local administration, and district health sector. they should contribute health education to increase the knowledge of society about h. pylori transmission, besides, the quality of water could be tested for h. pylori, and there is an urgent need to provide a wellprotected and treated drinking water to the community. creating good awareness on h. pylori infection and its risk factor is essential because the majority of respondents have no awareness about h. pylori and its risk factors, and they use any water source without care, they possess no information about the transmission pathway of h. pylori and its outcome. additionally, this study involved only stool test and questionnaire survey, so it is recommended that further studies must be carried out using different testing methods such as urea breath test or rapid urease test with a high number of patients. funding this research received no external funding. conflict of interests the authors declare no conflict of interest. author contributions goma m. huwiage – supervision, writing – review and editing, validation, data curation, abdurrazag a. nami – conceptualization, visualization, methodology, writing original draft, ali hussein akadh – project administration, resources, software, formal analysis, investigation, funding acquisition. g.m. huwiage et al. 13 in t e r n a l m e d ic in e issn 2413-6077. ijmmr 2019 vol. 5 issue 2 визначення helicobacter pylori за допомогою калового антигенного тесту (hpsa) у дорослих пацієнтів з диспепсичними розладами у тріполі, лівія g.m. huwiage1, a.a. nami2, a.h. akadh3 1 high institute of medical sciences& technology, algarabolli, libya 2 al-mergib university, al khums, libya 3 mediterranean specialized clinics, tripoli, libya вступ: тісний зв’язок helicobacter pylori з диспепсичними розладами, виразковою хворобою та раком шлунка підтверджено багатьма дослідженнями. мета роботи: встановити взаємозв’язок між диспепсичними розладами, позитивними результатами калового антигенного тесту та серологічної діагностики igg на h. pylori. методи: обстежено 125 дорослих пацієнтів з диспепсичними розладами (які лікувалися у гастроентерологічних відділень mediterranean та tajurah лікарень тріполі) за допомогою: іфа на визначення антигенів у калі та визначення у сироватці крові igg антитіл до h. pylori. статистичну обробку отриманих даних проводили за допомогою програмного забезпечення spss. результати: серед 125 пацієнтів з диспепсичними розладами (47 чоловіків та 78 жінок віком від 18 до 83 років) у 80 встановлено наявність h. pylori за результатами калового антигенного тесту, у 88 осіб – позитивний серологічний тест на igg. серед пацієнтів з позитивним результатами переважають особи віком 28-47 років. встановлено взаємозв’язок з такими факторами як вік, сімейний статус та економічне становище. не було достовірно значимих взаємозв’язків між h. pylori інфекцією та статтю, джерелами питної води, стандартами життя, палінням, сімейним анамнезом виразкової хвороби, вживанням лікарських засобів та групою крові. висновки: високий відсоток частоти визначення h. pylori за допомогою hpsa калового антигенного тесту вказує, що такий метод – надійний, недорогий та неінвазивний – може слугувати для діагностики активності інфекції та моніторингу динаміки лікування. в той час, як серологічна діагностика igg до h. pylori через нижчу чутливість, специфічність та точність може застосовуватися у якості скринінгу. ключові слова: helicobacter pylori; диспепсія; імуноферментний аналіз; каловий антигенний тест. information about authors goma mohamed huwiage – ph.d., assistant professor, department of medical microbiology, high institute of medical sciences& technology, algarabolli, libya. orcid 0000-0002-2727-148x, e­mail: gomanagoma96@yahoo.com abdurrazag altaher nami – ph.d., assistant professor, department of biology faculty of arts& sciences, al-mergib university, libya. orcid 0000­0002­1076­4368, e­mail: abdnami@gmail.com ali hussein akadh – msc in medical microbiology, mediterranean specialized clinics, tripoli, libya. orcid0000­0002­5439­7774, e­mail: alialhullali1955@gmail.com references 1. asaka m, sepulveda ar, sugiyama t, graham dy. gastric cancer. in: helicobacter pylori: physiology and genetics 2001. asm press. 2. longo d, fauci a. harrison's gastroenterology and hepatology, 2e. mcgraw­hill education; 2013. 3. marcus ea, scott dr. gastric colonization by h. pylori. in: helicobacter pylori. 2016 (pp. 23­34). springer, singapore. doi: 10.1007/978­981­287­706­2_2 4. moran ap. 10 lipopolysaccharides of helicobacter pylori: importance in gastric adaptation and pathogenesis. helicobacter pylori in the 21st century. 2010;17:190. doi: 10.1079/9781845935948.0190 5. alm ra, noonan b. the genome. in: mobley hlt, mendz gl, hazell sl, editors. helicobacter pylori: physiology and genetics. washington (dc): asm press; 2001. chapter 27 6. kim jm. h. pylori virulence factors: toxins (caga, vaca, dupa, oipa, icea). in: helicobacter pylori 2016 (pp. 77-88). springer, singapore. doi: 10.1007/978­981­287­706­2_5 g.m. huwiage et al. 14 in t e r n a l m e d ic in e issn 2413-6077. ijmmr 2019 vol. 5 issue 2 7. kim n. prevalence and transmission routes of h. pylori. in: helicobacter pylori 2016 (pp. 3­19). springer, singapore. doi: 10.1007/978­981­287­706­2_1 8. plonka m, targosz a, brzozowski t. can drinking water serve as a potential reservoir of helicobacter pylori? evidence for water contamination by helicobacter pylori. in: trends in helico­ bacter pylori infection 2014 apr 3. intechopen. doi: 10.5772/57568 9. sutton p, mitchell h, editors. helicobacter pylori in the 21st century. cabi; 2010. doi: 10.1079/9781845935948.0000 10. walker br, colledge nr. davidson's principles and practice of medicine e­book: with student consult online access. elsevier health sciences; 2013 dec 6. p. 872-8. 11. kim se. functional dyspepsia. in: helicobacter pylori, 2016 (pp. 207-218). springer, singapore. doi: 10.1007/978­981­287­706­2_18 12. yang hr. updates on the diagnosis of helicobacter pylori infection in children: what are the differences between adults and children? pediatric gastroenterology, hepatology & nutrition. 2016 jun 1;19(2):96­103. doi: 10.5223/pghn.2016.19.2.96 13. dore mp, pes gm, bassotti g, usai-satta p. dyspepsia: when and how to test for helicobacter pylori infection. gastroenterology research and practice. 2016;2016. doi: 10.1155/2016/8463614 14. el-shabrawi m, el-aziz na, el-adly tz, hassanin f, eskander a, abou-zekri m, mansour h, meshaal s. stool antigen detection versus 13c-urea breath test for non-invasive diagnosis of pediatric helicobacter pylori infection in a limited resource setting. archives of medical science: ams. 2018 jan;14(1):69­73. doi: 10.5114/aoms.2016.61031 15. iranikhah a, ghadir mr, sarkeshikian s, saneian h, heiari a, mahvari m. stool antigen tests for the detection of helicobacter pylori in children. iranian journal of pediatrics. 2013 apr;23(2):138­42. 16. calik z, karamese m, acar o, karamese sa, dicle y, albayrak f, can s, guvendi b, turgut a, cicek m, yazgi h. investigation of helicobacter pylori antigen in stool samples of patients with upper gastrointestinal complaints. brazilian journal of microbiology. 2016 mar;47(1):167­71. doi: 10.1016/j.bjm.2015.11.022 17. asaka m, kato m, takahashi si, fukuda y, sugiyama t, ota h, uemura n, murakami k, satoh k, sugano k. guidelines for the management of helicobacter pylori infection in japan: 2009 revised edition. helicobacter. 2010 feb;15(1):1­20. doi: 10.1111/j.1523­5378.2009.00738.x 18. kalach n, gosset p, dehecq e, decoster a, georgel af, spyckerelle c, papadopoulos s, dupont c, raymond j. a one‐step immune‐chromatographic helicobacter pylori stool antigen test for children was quick, consistent, reliable and specific. acta paediatrica. 2017 dec;106(12):2025­30. doi: 10.1111/apa.13968 19. tameshkel fs, niya mh, kheyri z, azizi d, roozafzai f, khorrami s. the evaluation of diagnostic and predictive values of helicobacter pylori stool antigen test in iranian patients with dyspepsia. iranian journal of pathology. 2018;13(1):3838­44. doi: 10.30699/ijp.13.1.39 20. zhou. x, su. j, xu. g& zhang, g. accuracy of stool antigen test for the diagnosis of helicobacter pylori infection in children: a meta­analysis. clinics and research in hepatology and gastroenterology, oct 2014; 38 (5), 629­38. doi: 10.1016/j.clinre.2014.02.001 received 07 august 2019; revised 20 october 2019; accepted 23 november 2019 this is open access article distributed under the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. g.m. huwiage et al. issn 2413-6077. ijmmr 2021 vol. 7 issue 266 d e n t is t r y doi 10.11603/ijmmr.2413-6077.2021.2.12426 bone tissue metabolism and changes in the oral cavity in reduced functional activity of the thyroid gland (literature review) *о.v. skochylo, s.i. boitsanyuk, n.o. tverdokhlib i. horbachevsky ternopil national medical university, ternopil, ukraine background. decreased functional activity of the thyroid gland leaves affects many organs and systems as well as bone tissue, pathological changes of which in the oral cavity are most often observed in periodontitis. however, the relationship between thyroid hypofunction and periodontitis or other inflammatory diseases of the oral cavity is still not confirmed. objective. the aim of the review was to study the published information and analyse bone metabolism and its relationships between autoimmune thyroiditis and oral diseases. methods. the articles in foreign periodicals on endocrinology, pathophysiology, dental surgery and therapy were the scientific sources for research. results. understanding the mechanisms of bone metabolism under the action of thyroid hormones is an important aspect of treatment and diagnostic process, as local treatment of dental pathology without reducing the impact on systemic factors ultimately does not have any positive result. decreased functional activity of the thyroid gland leads to homeostasis imbalance in the body. the thyroid hormones are important for bone metabolism, publications on periodontitis incidence in cases of autoimmune pathology of the thyroid gland are the most common. however, despite the number of studies, most authors agree that they are currently insufficient to clearly establish a causal relationship between autoimmune thyroid disease and maxillofacial disorders. conclusions. the study expands our knowledge, but there is still a need for further detailed studies that would clearly define the mechanisms of development of the disorders of the oral bone tissues and its relationships with autoimmune pathology of the thyroid gland. keywords: periodontitis; thyroid gland. *corresponding author: olha skochylo, department of dental surgery, i. horbachevsky ternopil national medical university, ternopil, ukraine. e-mail: skochyloov@tdmu.edu.ua international journal of medicine and medical research 2021, volume 7, issue 2, p. 66-75 copyright © 2021, tnmu, all rights reserved introduction increased attention to the diseases of the thyroid gland is caused by challenging statistics on the increase in its incidence according to the ministry of health of ukraine [1]. this trend is traced in the recent studies [2], their results show that the incidence of thyroid pathology has increased and is 46% of the total endocrinological morbidity. according to the author, during the last 5 years in the western region of ukraine the incidence of hypothyroidism increased by 28.4%, the increase of thyrotoxicosis – by 8%, and the prevalence of thyroiditis – by 12.7% [2]. these indicators are higher than the national average indicators and that of the north-eastern regions. according to official who data, about 1.5 billion people suffer from thyroid disease at present. however, despite the effective treatment of endocrine pathology, the tendency to its reduce in the world is not observed [3]. review hypothyroidism, as the most common pathology of the thyroid gland, surely affects the functions and morphology of all tissues and organs, the maxillofacial area as well. it is established that insufficient thyroid hormones adversely affect both tooth mineralization, bone mineral density and calcium-phosphorus metabolism that is clinically observed in the oral cavity as periodontitis, gingivitis or lesions of the tooth hard tissues [4,5]. understanding the mechanisms of bone metabolism under the action of thyroid hormones is an important aspect of treatment and diagnostic, as local treatment of dental pathology without reducing the impact on systemic factors does not have any positive result [6,7]. it is established that bone tissue is a constantly renewing tissue where remodeling processes take, place i.e. the processes of formation and destruction of osteotissue, provided by osteoblasts, osteoclasts and osteocytes, which functional activity depends on exogenous and endogenous factors, some of which are о.v. skochylo et al. issn 2413-6077. ijmmr 2021 vol. 7 issue 2 67 d e n t is t r y о.v. skochylo et al. thyroid hormones. although, according to allen m.r. et al. [8] there are 2 types of remodeling. the first type – targeted remodeling, in which a specific local signal directs the osteoclast to a specific location to begin remodeling (e.g., in the areas of microdamage); the second – chaotic remodeling, a random process in which osteoclasts begin remodeling without any signaling. this type of recovery is significant in calcium homeostasis. at the cellular level, both types of remodeling are equal [8]. according to present scientists, the remodeling cycle has 5 stages (fig. 1) [9]. in the first stage, the stage of activation, the stimulating signals by osteocytes and their transmission to cells of osteoclastic diferon are recognized. in response to this process, monocytic-macrophage cells are attracted to the bone surface, stimulated, proliferated and differentiated into osteoclasts. at the heart of the molecular understanding of regulation, rankl (receptor activator of nuclear factor kappa b ligand) is a transmembrane ligand of the nuclear factor activator receptor produced by osteoblasts that activates lymphocytes and macrophages. rankl molecules may remain attached to the surface of osteoblasts or stromal cells for some time. rank is a transmembrane receptor of nuclear factor activator. rankl interacts with rank, which is accompanied by the fusion of several osteoclast progenitor cells into one large structure and mature multinucleated osteoclasts are formed. thus, osteoblasts regulate formation of osteoclasts [11]. opg-osteoprotegerin is a protein synthesized by osteoblasts and bone marrow stromal cells. it is proved that at the stage of osteoclast formation, the process can be blocked by the protein opg-osteoprotegerin, which can bind to rankl, that prevents formation of the rankl/rank complex and thus stops resorption processes [12]. according to recent studies [13], not only in osteoblasts but also in osteocytes, most of the recently synthesized rankl form a protein complex with opg and is selectively directed to lysosomes. only a small fraction of newly synthesized rankl, which does not form a complex with opg, is transported to the cell surface. subsequently, transmembrane rankl is delivered to the surface of osteoclast precursors to stimulate rank, and induce activation of the subsequent signaling pathway. according to the authors, the ability of osteocytes to support formation of mature osteoclasts probably depends on the number of rankl molecules present on their cell surfaces. however, the way in which osteocytes embedded in the bone matrix deliver transmembrane rankl to the cell surfaces of osteoclast precursors that are localized in the bone marrow cavity should be elucidated. the second stage is the stage of resorption ‒ due to production of enzymes osteoclasts destroy the bone matrix. according to some scientists, at this stage, activated osteoclasts are phagocytes for bone. lysosomal collagenase is synthesized in large quantities that leads to disruption of the order in the structure of collagen. the products of hydrolysis enter the fig. 1. physiological bone remodeling, j.a. siddiqui and n.c. partridge [10]. issn 2413-6077. ijmmr 2021 vol. 7 issue 268 d e n t is t r y osteoblast from the “corrugated bristles” by endocytosis, and are released from the basement membrane, which is in contact with the blood vessel. the acidic environment in the area of resorption promotes leaching of calcium from apatites; the so-called “resorptive bone gaps” are formed; as a result, calcium and phosphates get into the blood. the third stage, the stage of reversion, osteoclasts undergo apoptosis, they are replaced by mesenchymal cells, which differentiate into osteoblasts [15]. active osteoclasts synthesize and secrete acid phosphatase, which dephosphorylates osteopontin, a sialoprotein that attaches to cells in the resorption zone. the connection with the bone surface becomes weak, so the resorption gradually decreases. a cementing line (a layer of secretory glycoproteins) is formed on the resorbed surface, which is able to hold colonies of osteoblasts, an additional prerequisite for this is the availability of local osteoprogenitors [16]. the fourth stage, the stage of bone for mation, osteoblasts synthesize the main organic substance of the bone matrix – collagen and substances that regulate mineralization (ostecalcin, osteonectin, etc.) [17]. the “matrix bubbles” of osteoblasts are significant in the process of mineralization. amorphous са3(ро4)2 is formed first in them, and later ‒ hydroxyapatite: “matrix bubbles”, which enter the extracellular space, contain high concentrations of calcium ions, according to vavilova t.p., in 25-50 times more, than in osteoblasts, as well as enzymes: alkaline phosphatase, pyrophosphatase. in the intercellular matrix, membrane vesicles are destroyed with the release of calcium ions. due to the influence of alkaline phosphatase, са2+ ions combine with ро43-, resulting in the formation of amorphous calcium phosphate. at the same time, са2+ and ро43ions bind to collagen and non-collagen proteins and matrix is formed, which is accompanied by the formation of nuclei. on the formed nucleus there are spiral structures, the growth of which takes place on the principle of adding new ions. the step of such a spiral is equal to the height of one structural unit of the crystal. crystal formation leads to the appearance of other crystals, this process is called epitaxis or epitaxic enucleation [18]. thus, mineralization occurs through the formation of calcium phosphate compounds that enter the bloodstream and their subsequent crystallization into hydroxyapatite followed by deposition of calcium hydroxyapatite along the collagen fibers [18]; osteoblasts play the main role in collagen synthesis [10]. the fifth stage, the final stage, is charac­ terized by differentiation of osteoblasts into osteocytes. after the bone formation stage is finished, the resting stage takes place, the osteoblasts are walled up in the matrix created in them, they lose activity and transform into osteocytes [19], which even recently were considered low metabolically active cells. but, as has been discovered, due to the mecha nosensory properties that occur through the indu ced flow of fluid through the lacuno­tu bu­ lar system, they regulate remodeling processes [20] and, as studied by cappuli [21], regulation is carried out by sclerostin protein (sost), which inhibits osteoblast differentiation. the process of remodeling is regulated by numerous hormonal and local factors, neuroendocrine and metabolic (fig. 2) [10]: gluco­ corticoids reduce synthesis of osteoblasts, which slows down formation of bone tissue; the influence of thyroid hormones (thyroxine and triiodothyronine) increases activity of osteoclasts, which contribute to catabolism of bone tissue. at the same time, sex hormones, especially estrogens, have antiresorptive properties. somatotropic hormone stimulates proliferation of osteoblasts and growth factors. it is established that with a decrease of the concentration of са2+ ions in the blood the secretion of parathyroid hormone (pth) increases, which is produced by cells of the parathyroid glands, and under its influence activates osteoclasts in bone tissue that increases bone resorption. as са2+ ions increase, the hormone calcitonin is secreted in the blood, which is produced by parafollicular thyroid cells and which bone mineralization increases and the number of osteoclasts reduces, i.e. resorption processes inhibits and, consequently, bone formation accelerates. vitamin d is important in regulation of concentration of са2+ ions in the blood, which are involved in the biosynthesis of са2+-binding proteins required for intestinal calcium absorption, renal reabsorption and mobilization of calcium from bones. recently, evidence has emerged that vitamin d is involved in deve lopment of many autoimmune diseases, including patients with autoimmune thyroid disease (aitd) [22]. it is established that thyroid hormones directly affect both remodeling processes, as they activate both osteoblasts and osteoclasts, and the process of calcium-phosphorus meо.v. skochylo et al. issn 2413-6077. ijmmr 2021 vol. 7 issue 2 69 d e n t is t r y о.v. skochylo et al. tabolism [24]. the thyroid gland secretes thyroxine (т4), triiodothyronine (т3) and calcitonin. the functional activity of the gland is regulated by thyroid-stimulating hormone (tsh), which is synthesized by the pituitary gland in res ponse to the secretion of thyroliberin (trg) by the hypothalamus. with a decrease in the con centration of thyroid hormones the secretion of tsh increases resulting in an increase in their formation in the thyroid gland, and, vice versa, with an increased concentration of thyroid hormones the formation of tsh decreases [17, 24]. thus, this cooperation of the main endocrine gland functions on the principle of “negative feedback” that ensures a constant level of hormones. violation of the concentration of one of the elements of the chain leads to changes in others that ultimately leads to dysfunction of both the endocrine glands and other organs and systems that depend on them. in the context of bone tissue, according to pankiv i.v. [4], and as seen at the scheme, thyroid hormones affect bone metabolism by increasing the activity of osteoclasts, which contribute to bone catabolism. there are studies [4, 25, 26], in which the authors indicate of osteoporosis of bone tissue with underlying hypothyroidism. according to [4], in persons with thyroid pathology, changes in bone mineral density were detected in 59 (39.9%) cases: osteopenia ‒ in 45 (30.4%) and osteoporosis ‒ in 14 (9.5%) cases. the incidence of osteopenia and osteoporosis was likely to increase in all groups of patients with thyroid functional disorders. the main factor that leads to decrease in bone strength in patients with thyroid disease is excessive or insufficient production fig. 2. systemic regulation and growth factor of bone remodeling, j. siddiqui, n. partridge [10]. issn 2413-6077. ijmmr 2021 vol. 7 issue 270 d e n t is t r y of thyroid hormones, as well as treatment with suppressive doses of levothyroxine. it is proved that the effect of thyroid hormones on the body cells is caused by the presence of receptors (tr) on their surface (fig. 3). it is established that there are several types of thyroid receptors: trα1, trα2, trβ1, trβ2 [10]. it was established that only trα and trβ receptors on the surface of osteoblasts and chondrocytes were detected in bone cells [28]. accordingly, т3 (triiodothyronine) induces osteogenesis by direct action on osteoblasts. however, recent studies [29] have proved that expression of thyroid receptor (tr) genes α1 and β1 is confirmed in osteoclasts, but it is still indefinite whether tri­ iodothyronine (т3) stimulates osteoclast activity directly or whether these processes are the result of т3 action in osteoblasts, osteocytes or other cells. as for the trβ2 receptor, there is evidence that it is associated with the hypothalamus and pituitary gland, where it inhibits the secretion of trg and tsh, so the hypothalamic-pituitarythyroid relationship is important in regulation of bone metabolism. to date, there are still debates on the key role in the functioning of bone tissue:by tsh or thyroid hormones [29]. the recommendations of the american thyroid association [30] state that serum tsh levels are one of the most informative indicators of thyroid function, and the association recommends that all patients have serum tsh levels determined from the age of 35 and monitored every 5 years, which is an important diagnostic aspect. however, confirmation is found in the lite­ rature: hypothyroidism causes general hypo­ metabolism [31], a decrease in osteoblast forma tion and resorption of osteoclasts, and leads to low bone metabolism or slowing down the remodeling process. according to the author, the processes of osteo formation are slowed down by 50%, the processes of resorption – by 40% [31]. calciuria decreases, serum concentrations of osteocalcin and alkaline phosphatase decrease, but the concentration of parathyroid hormone and vitamin d in the serum may increase [31]. analysing this data and drawing parallels with clinical symptoms, scientists patho genetically distinguish the following types of hypothyroidism: 1. primary hypothyroidism caused by primary pathology of the thyroid gland, which is divided into hypothyroidism due to a decrease in the amount of functionally active tissue of the gland and impaired biosynthesis of thyroid hormones. 2. secondary (pituitary) hypothyroidism caused by a decrease in tsh production. 3. tertiary (hypothalamic) hypothyroidism due to a decrease or production of thyroliberin. 4. peripheral (tissue) resistance to thyroid hormones [24]. autoimmune thyroiditis (ait) is the most common cause of primary hypothyroidism [32]. taking into account the complex mechanism of metabolic thyroid hormones metabolism, the question is whether thyroid dysfunction affects the course of oral diseases, the development of which is accompanied by destructive processes in bone tissue. fig. 3. the thyroid gland secretes the prohormone t4 and the active hormone t3, and circulating concentrations are regulated by the classical endocrine cycle of negative feedback, which maintains the physiological feedback between tsh and т4 and т3. pvn, paraventricular nucleus (bassett j.h, williams g.r.) [28] о.v. skochylo et al. issn 2413-6077. ijmmr 2021 vol. 7 issue 2 71 d e n t is t r y о.v. skochylo et al. periodontal diseases are foremost in the structure of dental pathology due to the sig nificant spread among the population of ukraine [33]. this pathology is characterized by inflam matory-destructive changes of the periodon tium, progressive nature of their course and leads to early tooth loss [34]. according to recent literature, the causes for this are both local and systemic factors [35], and sometimes a combi nation of both. local factors include microbial film, small orifice of the mouth, pathology of the bridles and the existing strands of the mucous membrane, crowding of the teeth, impaired occlusion. regarding the systemic factors that contribute to development of periodontal diseases, chronic cardiovascular, digestive and endocrine diseases are important. most scien tists today are inclined to the domi nant role of the influence of microorganisms and tissue in flam­ matory response as a con se quence of their activities [36]. the most com mon micro organisms of the dental micro bial film are gram­ negative anaerobic bacteria porphy romonas gingivalis, tannerella forsythia, treponema denticola, prevotellaintermedia [37, 44]. the mechanism of action of micro orga nisms is explained by their penetration through the connective tissue epithelium of the gingival sulcus that disrupts the integrity of the gingival junction and affects the pe riodontal tissues located deeper [37]. in cases of the inflammatory process in the periodontium microcirculation is violated that is accompanied by an increase in vascular permeability with insudation of blood plasma proteins into the walls of blood vessels and perivascular tissue. it has been proved that pathology in periodontal vessels is a trigger for progression of periodon titis [38]. in a com pa rative study of the micro circulation of healthy patients and in the exa mi nation of persons with periodontitis of va rying severity (1-3 degrees), (127 people), according to laser doppler flow metry, it was found that in pe­ riodontitis 1st degree severity, in hemody namics is decreased, compare to the healthy group of people, con gestion in the micro circulatory tract is present with further deve lopment of rheological disor ders. in generalized periodontitis of the 2nd degree, the main indicators of tissue blood flow are reduced, compare to the pre­ vious group, and in pe riodontitis of the 3rd degree microcir culatory disorders worsen with the involvement of all parts of compensatory regulation [38]. thus, violation of micro circulation in the presen ce of microbial mechanisms leads to slowing of blood flow, venous stasis, impaired vascular transport [40]. due to the possible systemic effects of thyroid dysfunction, periodontal tissue microcirculation in this aspect was studied by scardina g.a., messina p. (2008) [41], who assessed morphological microcirculation of interdental papillae in patients with hashimoto’s thyroiditis and possible associated periodontal disease. it was emphasized that the group of healthy patients deliberately did not involve the persons with conditions that disrupt microcirculation, such as diabetes or hypertension. all patients did not smoke. microcirculation was assessed by capillaroscopy. for each patient, visibility, course, tortuosity, average capillary loop size and number of visible capillary loops per square millimeter were investigated. in patients with hashimoto’s thyroiditis, a reduced capillary caliber, as well as a greater number and tor tuosity of capillary loops were evidenced. this study showed that changes in the capillaries in patients with hashimoto's thyroiditis occur red in cases of violation of gums microcirculation that is characteristic of periodontitis. the opinion that periodontitis is a multifactorial disease with a microbial initiator, the manifestation and progression of which is predisposed by a wide range of factors, one of which is hashimoto’s thyroiditis, is more and more popular [42, 43]. according to molaris a. et al. [42], after analyzing the data of 30 articles on the relationship between periodontitis and hashimoto’s thyroiditis regarding etiopathogenetic mechanisms, have established that it occurs because some of these mechanisms are accompanied by vascular endothelial dysfunction, microcirculation disorders, as well as due to the impact of hypothyroidism on alveolar protease metabolism, but a causal relationship between the two nosologies requires further research. according to patil b., patil s., gururaj, t. (2011) [7], their study was initiated due to the lack of effective local therapy of periodontitis in thyropatients. kothiwale s. et al. [5] presented a very interesting study regarding the impact of thyroid hormone dysfunction on the progression of periodontitis, systemic health of the patient, in which local treatment of periodontal tissues as a complex with endocrine compensation was proved. it was emphasized that the etiotropic phase of dental treatment lasted 8 weeks with the prescribed 150 mg of systemic thyroxine per day. during the dynamic obissn 2413-6077. ijmmr 2021 vol. 7 issue 272 d e n t is t r y servation, the hygienic index of the oral cavity improved, but the bleeding did not disappear. after 12 weeks of follow-up, after stabilization of thyroid hormones, a clear decrease in gingival bleeding was evidenced. the need for frequent professional evaluation, training of patients, motivating them to frequent systematic examinations was emphasized, as the treatment of such patients and achieving longterm remission would provide a positive result only in endocrinologist-dentist tandem. according to brankhar r.r. et al. (2017) [43] the endocrine system works together with the immune system. despite the fact that the bilateral effects of systemic diseases on the periodontium are proved, there are a few studies on the effects of periodontal therapy on hormone levels. in the study, the effect of non-surgical periodontal therapy (nspt) on serum stimulating hormone (tsh) levels in patients with hypothyroidism and periodontitis was assessed. clinical parameters and serum tsh levels were recorded at baseline in the experimental and control groups and compared with tsh data in 3 months after nspt in the patients with hypothyroidism. the results of the study showed that nspt was significant in improving the condition of the periodontium by reducing inflammatory markers and thus affecting thyroid hormone, a significant decrease in tsh in patients with hypothyroidism in 3 months after nspt. chingiz ragim ogly rakhimov, (2020) [44] presented data on the clinical efficacy of hyaluronic acid in the treatment of periodontal disease in patients with hypothyroidism. according to the evaluation of the main hygienic and periodontal indices, it was found that a decrease in the content of thyrohormones led to an increase in the frequency and inflam ma­ tory-destructive forms of periodontal disease. in such patients, high-frequency of porphyromonasgingivalis (25% and 15% in somatically healthy patients) and increased colonization of yeast-like fungi of the genus candida albicans were evidenced in the oral cavity. after the treatment, there was a positive clinical dynamic, but in patients with impaired gland function had worsening of the oral cavity in a month that confirmed the idea of ineffectiveness of only local dental treatment and the need for dynamic monitoring of hormonal status. there are studies on effectiveness of intraligamentous administration of vitamin d and calcium in the treatment of chronic periodontitis associated with hypothyroidism. in 3 months, there was a significant decrease in mobility, pocket depth and bleeding in the treatment of chronic periodontitis associated with hypothyroidism. the need for clinical trials with a large sample size and long-term observations was emphasized [45]. the significance of vita­ min d in the development of autoimmune thyroiditis was covered by bizzaro g. et al. [46, 47]. there is no doubt that patients with established hypothyroidism need a replacement therapy, and the study of periodontal status when taking thyroxine is the basis of further research [48]. after analyzing the plaque index, bleeding index, pocket probing depth [ppd], level of clinical attachment [cal] and radiological parameters, in the study group (52 patients) statistically significantly higher ppd and loss of clinical attachment compare to the control group was established. with the beginning of treatment of periodontitis and hypothyroidism improvement in oral hygiene and a decrease in bleeding gums was evidenced. regression analysis showed that hypothyroidism and thyroxine replacement therapy were important predictors of ppd and cal, but it still requires further study. the same opinion is traced in the study by hajer a. aldulaijan et al. (2020), who analyzed 847 publications and applied their inclusion and exclusion criteria; thus only 29 publications were selected, which were more critically analyzed. as a result, only four publications were used to further assess the hypothyroidism-periodontitis relationship, including one research note on association between hypothyroidism and periodontitis. hence, further well-controlled, clinical and immu nological studies are needed to confirm this relationship [6]. conclusions the results of the studies prove that there is a significant effect of the thyroid gland on the state of the oral cavity that can be manifested by periodontitis, which is accompanied by bone destruction and inflammatory processes in the gum tissue. local treatment of dental pathology without correction of thyrohormonal status does not provide effective treatment, so the correct diagnosis, selection of treatment and medical cooperation of a dentist and endocrinologist is necessary. such disorders are common, but there is still a lack of accurate and improved examinations of this problem, which will be the goal of our further research in the future. о.v. skochylo et al. issn 2413-6077. ijmmr 2021 vol. 7 issue 2 73 d e n t is t r y о.v. skochylo et al. conflict of interests authors declare no conflict of interest. authors’ contributions olha skochylo – investigation, formal analysis, writing – original draft. svitlana boitsanyuk – formal analysis, writing – reviewing and editing, data curation. nataliya tverdokhlib – conceptualization, methodology, writing – reviewing and editing. особливості метаболічного обміну у кістковій тканині та зміни зі сторони ротової порожнини на грунті зниження функціональної активності щитоподібної залози (огляд літератури) *о.в. скочило, с.і. бойцанюк, н.о. твердохліб тернопільський національний медичний університет імені і.я. горбачевського моз україни, тернопіль, україна вступ. зниження функціональної активності щитоподібної залози залишає свій слід на багатьох органах та системах. не виключенням є і кісткова тканина, патологічні зміни якої в ротовій порожнині найчастіше спостерігаємо при пародонтитах. проте, все ще залишається не підтвердженим взаємозв'язок між гіпофункцією тиреоїдної залози та пародонтитом чи іншими запальними захворюваннями ротової порожнини. метою нашого огляду власне, і було дослідження опублікованої інформації та її аналіз щодо метаболізму кісткової тканини та її взаємозв'язоку між автоімунним тиреоїдитом та захворюваннями ротової порожнини. методи. науковими джерелами були статті у зарубіжних періодичних виданнях з ендокринології, патофізіології, хірургічної, терапевтичної стоматологій. результати. розуміння механізмів кісткового метаболізму під дією гормонів щитовидної залози є важливим аспектом лікувально-діагностичного процесу, оскільки місцеве лікування стоматологічної патології без виключення впливу на системні чинники у підсумку не дає позитивного результату. зниження функціональної активності щитоподібної залози призводить до дисбалансу в гомеостазі організму. оскільки гормони щитоподібної залози відіграють важливу роль у обміні кісткової тканини, то найчастіше зустрічаються публікації стосовно виникнення пародонтиту на фоні автоімунної патології щитоподібної залози. проте, незважаючи на існуючі дослідження та зафіксовані зміни, більшість авторів згідні з думкою, що таких досліджень на даний час є недостатньо, щоб чітко встановити причинно-наслідковий взаємозв’язок між автоімунним процесом щитоподібної залози та патологією щелепно-лицевої ділянки. висновки. представлені дані розширюють існуючі знання, проте все ще існує потреба в подальших сучасних детальних дослідженнях, які б чітко дали відповідь на механізми виникнення та розвитку взаємозв'язку між автоімунною патологією щитоподібної залози та станом кісткової тканини ротової порожнини. ключові слова: пародонтит; щитоподібна залоза. information about the authors olha v. skochylo – phd, md, associate professor of the department of dental surgery, i. horbachevsky ternopil national medical university, ternopil, ukraine. orcid 0000­0002­8621­8275, e­mail: skochyloov@tdmu.edu.ua svitlana i. boitsanyuk – phd, md, associate professor of the department of dental therapy, i. horbachevsky ternopil national medical university, ternopil, ukraine. orcid 0000­0001­7742­1346, e­mail: boucanuk@tdmu.edu.ua nataliya o. tverdokhlib – phd, md, associate professor of the department of dental surgery, i. horbachevsky ternopil national medical university, ternopil, ukraine. orcid 0000­0002­1247­2430, e­mail: tverdohlibno@tdmu.edu.ua issn 2413-6077. ijmmr 2021 vol. 7 issue 274 d e n t is t r y references 1. kravchenko vi. iodine deficiency as a cause of high prevalence of thyroid pathology in the population of regions affected by the chernobyl accident. zhurnal namn ukrainy. 2016;22(2):222­9 [in ukrainian]. 2. chukur oo. dynamics of morbidity and prevalence of thyroid pathology among the adult population of ukraine. bulletin of social hygiene and health care organization of ukraine. 2018;4(78):19­25 [in ukrainian]. 3. vladymyrova im, heorhiiants va. farmakoterapevtychni napriamky zastosuvannia yodovmisnykh likarskykh roslyn pry riznykh hrupakh zakhvoriuvan shchytopodibnoi zalozy [pharmacotherapeutic directions of application of iodinecontaining medicinal plants in different groups of diseases of the thyroid gland]. scientific journal “sciencerise”. 2015;11/4 (16):46­54 [in ukrainian]. 4. pankiv iv. influence of the functional state of the thyroid gland on bone mineral density. trauma. 2015;6(16):33­41 [in ukrainian]. 5. kothiwale s, panjwani v. impact of thyroid hormone dysfunction on periodontal disease. j sci soc. 2016;43:34­47. 6. aldulaijan ha, cohen re, stellrecht e, levine mj. relationship between hypothyroidism and periodonti tis: a scoping review. clin exp dent res. 2020;6:147­57. https://doi.org/10.1002/cre2.247 7. patil b, patil s, gururaj t. probable autoimmune causal relationship between periodontitis and hashimotos thyroidits: a systemic review. nigerian journal of clinical practice. 2011;14:253­61. https://doi.org/10.4103/1119­3077.86763. 8. allen mr, burr db. bone modeling and remode ling. basic and applied bone biology 2014;chapter 4:75­90. 9. kenkre js., bassett j. the bone remodelling cycle. ann clin biochem. 2014;55(3):308­27. https://doi.org/10.1177/0004563218759371. 10. siddiqui ja, partridge nc. physiological bone remodeling: systemic regulation and growth factor involvement. physiology (bethesda), may, 31 (3). 2016;233-45. doi:10.1152/physiol.00061.2014. 11. martin tj, sims na. rankl/opg; 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revised 26 november 2021; accepted 7 december 2021. this is open access article distributed under the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. 84 b io m e d ic a l s c ie n c e s issn 2413-6077. ijmmr 2019 vol. 5 issue 1 doi 10.11603/ijmmr.2413-6077.2019.1.10308 antioxidant enzymes activity in experimental ischemia-reperfusion injury n.v. volotovska*, t.v. kashchak i. horbachevsky ternopil national medical university, ternopil, ukraine background. blood loss during civil and military limb trauma is the most common cause of preventable death. complications due to the use of a hemostatic tourniquet are widely investigated nowadays. therefore, the standards of the past have to be improved. objective. the aim of the research is to study the reaction of the enzyme chain of the liver antioxidant system in the presence of modifications of ischemia-reperfusion injury (iri). methods. 210 white male-rats, aged 5-5.5 months, were used in the research. the dynamics of antioxidant enzymes activity catalase (cat) and superoxide dismutase (sod) in liver tissue in cases of modifications of ischemiareperfusion injury (iri) were studied. the period of investigation was in 24 hours, 3, 7, 14 days after the injury. results. in cases of simulated iri the catalase level mainly decreased at each period of the experiment. the peak of sod activity was evidenced on the 1st, 3rd or 7th days after the experimental iri according to the degree of trauma severity. thus, iri combined with severe blood loss and mechanical trauma caused the severest affection of the antioxidant system. even a single application of hemostatic tourniquet caused similar wavelike reactions at different times. conclusions. the development of iri is accompanied by a significant depression of the liver antioxidant system. the most significant changes were evidenced in cases of iri combined with blood loss and mechanical trauma, but even a single application of a tourniquet caused active response of the antioxidant enzymes. key words: ischemia-reperfusion injury; trauma; blood loss; hemostatic tourniquet; catalase; superoxide dismutase. *corresponding author: nataliya v. volotovska, md, ph.d., department of physiology, bioethics and biosafety, i.horbachevsky ternopil national medical university, 1 maidan voli, ternopil, ukraine, 46001 e-mail: volotovskanv@tdmu.edu.ua introduction the damage to both skeletal muscles and internal organs caused by local ischemiareperfusion injury (iri) is mostly present as a result of intraoperative use of tourniquet for temporary stop of blood circulation in internal organs or for stop of blood loss from the limb [1-10]. primary damage leads to mechanical pressure, as for secondary mechanisms – reactive oxygen intermediates and lipid peroxidation are responsible for it [11]. nowadays, in state of affairs of ukrainian hostilities, gunshot wounds are widespread, and use of hemostatic tourniquet is one of efficient methods. but complications accompanying it are more serious than it was though previously. in the case of the hemostatic tourniquet use it is important to know about period of depression of antioxidant system in vitally important internal organs, for preventing exhaustion in further period consequences. oxidative stress is crucial in development of local and systemic damage and progress of ischemia-reperfusion injury in cases of oxygen insufficiency of the tissues [12]. overproduction of reactive oxygen species (ros) is combined with the violation of oxidative-reduction systems activity, damage of dna, membrane receptors, dysfunction of ion channels and changes in composition of membrane phospholipids, as well as activation of caspase mechanism of apoptosis [13, 14, 15]. all this is leading to activation of endogenous antioxidant defense [13, 16-22]. cellular systems of antioxidant protection are classified as enzymatic and non-enzymatic. the first one includes superoxide dismutase (sod), glutathione peroxidase (gp), and catalase (cat), which provide the first line of defense against the action of ros in that way, when the product of the first reaction becomes a substrate for the next one [23]. overexpression of sod1 prevents neuronal death in the area of hippocampus [24]. however, the extremely short period of half-life of sod1 in circulating blood makes it difficult to use enzyme therapy for brain damage. in international journal of medicine and medical research 2019, volume 5, issue 1, p. 84-90 copyright © 2019, tnmu, all rights reserved n.v. volotovska et al. 85 b io m e d ic a l s c ie n c e s issn 2413-6077. ijmmr 2019 vol. 5 issue 1 some studies gp provides even greater defense against oxidative stress than sod – its expression, as well as sod provides cytoprotective effect [16, 18]. cat is a very important element in maintaining the intracellular concentration of reduced glutathione and is crucial in neutralization of free radicals [24, 25]. various methods are used nowadays in therapeutic treatment: from lowering the temperature of the limb to the use of various drugs. thus, the protective effect of curcumin on the myocardium, kidneys, nervous tissue and lung has been proved in cases of this pathology [26-29]; montelucast reduces the level of local and systemic manifestations [29], legalon, thiotriazolin, emoxipine, silymarin have antioxidant effect [3-9, 25-30]. studying of changes in the activity of antioxidant enzymes is needed for prediction of the effect of a specific antioxidant corrector. this is essential to avoid exhaustion of the impaired body systems. objective. the aim of the research was to study the reaction of enzyme chain of the liver antioxidant system in the presence of modifications of iri. methods the experiments were performed on 210 non-linear white male rats, 250-270 g in weight and 5-5.5 months of age. they were divided into 5 groups, each of them contained 10 animals: the control group involved rats, which were administered thiopental-sodium anesthesia (40 mg/kg of body weight intramuscularly) only, the 1st experimental group (tourniquet was applied to the upper thigh 1/3 for 2 hours, reperfusion for 1 hour; the 2nd experimental group (blood loos in amount of 40% of circulating blood volume was simulated; the 3rd experimental group (a tourniquet on a thigh was combined with 40% blood loss from femoral vein of another lower limb), the 4th experimental group (a tourniquet on a thigh was combined with femoral bone fracture of another lower limb), the 5th experimental group (a tourniquet on a thigh was combined with 40% blood loss and femoral bone fracture of another lower limb). the experiments were performed in the vivarium of i. horbachevsky tnmu in the morning. special room had stable temperature (18-22 °c), relative humidity (40-60%) and illumination 250 lux. animals were sacrificed on the 1st, 3rd, 7th and 14th days after the trauma by thiopental-sodium anesthesia (40 mg/kg of body weight intraperitoneally by total bloodletting from the heart. an activity of catalase (mckat/kg) and superoxide dismutase (u/mg) of 10% liver homogenate samples were determined by means of koroliuk ma, et al. (1988) and chevari s, et al. (1985) methods respectively. all experimental stages of the research were performed following the european convention for the protection of vertebrate animals used for experimental and other scientific purposes (strasbourg, 1986), resolution of the first national congress on bioethics (kyiv, 2001) and the order of the ministry of health of ukraine no. 690, dated september 23, 2009. a statistical analysis of the attained data was performed by excel (microsoft, usa). statistically significant differences between the independent indices were estimated by the student t-test at normal distribution and by nonparametric methods in other cases. the results were presented as (m±m), where m means value, m – standard error. correlation analysis was performed for the attained data. linear correlation coefficient (r) and its significance (b) were evaluated as well. the link was considered to be lost if the r index was 0, the link evidenced of a week correlation when the range was 0-0.03. in the case of the range 0.3-0.7 – a medium link was established, the range of 0.7-1.0 proved a strong interaction correlation. the correlation coefficient was significant at p<0.05. results the total content of the investigated enzymes of the liver antioxidant system is presented in table 1. on the 1st day, compare to the control, an increase of sod in 3.1 times (p1<0.01), by 80% (p2<0.01), by 13% (p3<0.002), in 70% (p4<0.003), in 4.6 times (p5<0.006) was evidenced. as for cat, its level decreased the most. thus, on the 1st day its decrease in 24.1 times (p1<0.01), in 13.5 times (p2<0.01), in 17.8 times (p5<0.004) took place as well as an increase by 16% (p3<0.004) and 38.6% (p4<0.002). on the 3rd day, compare to the control, in all experimental groups there was an total increase of sod activity in 3 times (p1<0.003), in 5.4 times (p2<0.007), in 3.6 times (p3<0.03), in 2.48 times (p4<0.01), by 86% (p5<0.007). as for cat, its dynamic was variable. a decrease in its level in 24 times (p1<0.003), in 11.9 times (p2<0.005) was evidenced, but in cases of other traumas – an increase of its level in 1.8 times n.v. volotovska et al. 86 b io m e d ic a l s c ie n c e s issn 2413-6077. ijmmr 2019 vol. 5 issue 1 (p3<0.3), by 77.6% (p4<0.05), in 1.8 times (p5<0.08). on the 7th day, compare to the control, there was an evident increase if sod activity in all experimental groups: in 13 times (p1<0.02), in 3.1 times (p2<0.006), in 12.8 times (p3<0.02), in 4.2 times (p4<0.009), in 5.3 times (p5<0.003). in all groups, except for the 3rd one, a decrease of catalase activity was evidenced: in 3.5 times (p1<0.02), in 8.3 times (p2<0.005), in 15.2 times (p4<0.005), in 21.8% (p5<0.02). so, in the 3rd group an increase in 12.8 times (p3<0.02) took place. the similar dynamic of sod, compare to the control, was evidenced on the 14th day. an increase in its level in 2.2 times (p1<0.01), in 2.4 times (p2<0.004), in 5.8 times (p<0.006), in 2.36 times (p4<0.007), in 5.4 times (p5<0.01) took place. as for cat mostly depression was evidenced: a decrease of activity, compare to the control, in 10.3 times (p2<0.005), in 13.2 times (p3<0.004), in 1.67 times (p4<0.05), in 21.4% (p5<0.04), and a very slight increase of it in cases of isolated tourniquet – by 17% (p1<0.05). in cases of activation of lipid peroxidation and oxidative stress, according to the literature, different levels of activity of the antioxidant system in all organs of the affected organism take place. the attained results proved that in 1 day in liver homogenate, the sod activity increased in all experimental groups, but the most– in cases of a tourniquet alone and iri combined with blood loss and mechanical trauma, when its level, compare to the control, increased in 3.1 and 4.6 times respectively. in all groups, a wavelike reaction of enzyme activity was revealed. thus, in cases of isolated iri on the 1st and 3rd days it increased in 3-3.1 times; it suddenly increased (including the steady rates, compared with the previous periods) in 13 times, compare to the control, on the 7th day – in 13 times; it did not reached the norm on the 14th day. blood loss only, associated with primarily hypoxia of the ischemic genesis, triggered activation with peak of its activity on the 3rd day; it was still high. the combination of classic iri with blood loss contained elements of both abovementioned manifestations: a mild increase in the activity (apparently due to the fight against hypoxia) in the presence of blood loss only as well as in cases of iri only; the peak of increased sod activity on the 7th day reached 12.8-fold rate compare to the control. the activity of sod in cases of iri combined w it h mech a n ica l t ra u ma in crea sed an d decreased very smoothly with its peak on the 7th day, then it did not reached a normal level on the 14th day, being higher than the initial level in 2.36 times, and lower than the previous period index by 44%. in cases of iri combined with blood loss and mechanical trauma, two peaks of the sod activity increase were evidenced: on the 1st day (in 4.6 times, compare to the control) and in 5.3 times on the 7th day, compare to the control. on the 3rd day, which was obviously lifethreatening for this type of injury, the index table 1. changes in superoxide dismutase (u/mg) and catalase activity (mckat/kg) in 10% liver homogenates of the studied rats in cases of modifications of ischemic reperfusion injury and isolated blood loss, (m±m). group indices 1 2 3 4 5 post traumatic period control tourniquet t blood loss bl t+bl t+f t+bl+f in 24 hours after trauma sod 0.15±0.09 cat 4.44±0.36 sod 0.47±0.06* 0.27±0.04* 0.17±0.01 0.26±0.02* 0.70±0.02* cat 0.18±0.08* 0.33±0.07* 5.17±0.02* 6.15±0.01* 0.25±0.02* in 3 days after trauma sod 0.45±0.02 0.82±0.03*,** 0.54±0.16*,** 0.37±0.01*,** 0.28±0.03*,** cat 0.18±0.01* 0.37±0.02* 8.01±1.3*,** 7.89±0.3*,** 8.09±0.36*,** in 7 days after trauma sod 1.94±0.05*,** 0.47±0.03*,** 1.93±0.06*,** 0.29±0.02*,** 0.80±0.02*,** cat 1.28±0.16*,** 0.54±0.02*,** 6.63±0.27*,** 0.63±0.04*,** 3.47±0.05*,** in 14 days after trauma sod 0.32±0.06*,** 0.36±0.03*,** 0.88±0.02*,** 0.35±0.03*,** 0.83±0.06* cat 5.20±0.36*,** 0.43±0.03* 0.34±0.02*,** 2.65±0.20*,** 3.49±0.20* notes: * – statistical significance compare to the control, ** – statistical significance compare to the previous period of the study. n.v. volotovska et al. 87 b io m e d ic a l s c ie n c e s issn 2413-6077. ijmmr 2019 vol. 5 issue 1 slightly exceeded the initial control level, but was statistically significantly lower than on the 1st day (in 2.5 times). on the 14th day it was still increased and the same as on the 7th day. in cases of iri only, the dynamics of cat activity was depressed with a peak of decrease on the 3rd day (in 24 times). in the presence of blood loss only in all study periods, the activity of catalase was statistically significantly lower compare to the control. the peculiar features of the iri course combined with blood loss was an increase in the activity of this enzyme on the 3rd day in 1.8 times compare to the control; on the 14th day the index decreased in 13.2 times compare to the control, and in 23.6 times compare to that on the 3rd day. a somewhat similar dynamics of catalase activity was evidenced in cases of iri combined with mechanical trauma, when after a gradual increase till the 3rd day, it suddenly decreased in 15.2 times compare to the control on the 7th day. although, the index was still decreased on the 14th day being lower than the initial level in 1.67 times. the peculiar features of the cat dynamics in cases of iri combined with blood loss and mechanical trauma, was a sudden decrease of its activity on the 1st day compare to the control; then the cat activity suddenly increased on the 3rd day in 1.8 times compare to the control and was of the initial level up to the end of the experimental period, which obviously proved the reduction of enzymatic component on the 14th day. discussion the correlative analysis proved that, in development of ischemia-reperfusion syndrome caused by application of hemostatic tourniquet, liver failure is significant. this is the focus of the further research on morphological changes of liver tissue. hepatic active response is a predictable reaction, which belongs to the multiply organ failure syndrome. association of iri as the cause of such pathological effect in the organism was proved by a couple of researchers [32-36]. probably, the depression of sod is caused by overproduction of malonic dialdehyde and other derivates of peroxidation. detection of reactive oxygen intermediates accumulation, enzymes of cytolysis and oxidative modification of proteins might be studied in our future experiments, as these indexes are typical in such course of pathological external affection [37, 38]. moreover, the data on changes in the activity of serum and organ catalase in cases of trauma and blood loss vary. both decrease and increase of cat level were presented in different experimental studies [13, 21, 22]. and in our research such fluctuations of this enzyme activity were proved. in our case active response of the liver antioxidant system was evidenced by an increased activity of antioxidant enzymes. our studies coincide with those of other researches. e. orlova et al. proved that sod activity was the highest in liver and they advised correction of its insufficiency with vin-vita [23]. affection of liver tissue caused by application of tourniquet led to possible development of multiple organ failure due to the ischemic-reperfusion limb syndrome [37]. besides, we consider that iri combined with mechanical trauma may cause development of abrupt affecting of the liver antioxidant system that was proved by higher level of sod activity than in other groups and which was still increased till the end of experiment. such dynamics of sod and cat activity is a result of lipid peroxidation, i.e. increase in malonic dialdehyde level [2, 11]. it has been established that reperfusion syndrome affects the development of systemic changes in cases of combined trauma complicated by bleeding that is manifested by a significant activation of lipid peroxidation. conclusions even a single use of a hemostatic tourniquet leads to significant changes in the activity of enzymatic level of antioxidant defense. in present hostilities, blood loss is often combined with skeletal injuries, so it is advisable to limit the time of use of a hemostatic tourniquet or to take measures that counteract the development of lipid peroxidation caused by an injury in the need of bleeding stopping with a tourniquet. conflict of interest the authors declare no conflict of interest. n.v. volotovska et al. 88 b io m e d ic a l s c ie n c e s issn 2413-6077. ijmmr 2019 vol. 5 issue 1 зміна активності ферментів антиоксидного захисту при експериментальному ішемічно-реперфузійному синдромі н.в. волотовська, т.в. кащак тернопільський національний медичний університет імені і.я. горбачевського, тернопіль, україна вступ. крововтрата при цивільній та бойовій травмі кінцівок є найпоширенішою причиною т.з. «превентивних смертей». оскільки стандарти минулого щодо надання невідкладної медичної допомоги для зупинки кровотеч потребують поліпшення, сьогодні широко досліджуються ускладнення при накладанні гемостатичного джгута. мета дослідження. вивчити особливості реакції ферментативної ланки антиоксидантної системи печінки на тлі модифікацій ішемічно-реперфузійного синдрому (ірс). методи дослідження. у експерименті використано 210 білих щурів-самців віком 5-5,5 місяців. досліджено динаміку активності антиоксидантних ферментів каталази (кат) та супероксиддисмутази (сод) в тканині печінки при розвитку модифікацій ірс. забір зразків тканини здійснювали через 24 години, на 3, 7 та 14 доби після травми. результати. у наших моделях ірс рівень кат в основному зменшувався в кожному періоді експерименту. пік активності сод спостерігався на 1, 3 або 7 добу після експериментального ірс – згідно модифікацій ступенів тяжкості травми. таким чином, застосування кровоспинного джгута в поєднанні з втратою крові та механічною травмою, викликали найважчі ураження антиоксидної системи. при цьому, навіть одноразове застосування джгута викликало подібні хвилеподібні реакції. висновки. розвиток ірс супроводжується сильним пригніченням антиоксидної системи печінки. найбільш значні зміни спостерігалися на тлі застосування кровоспинного джгута, поєднаного з втратою крові та механічною травмою, але навіть ізольоване застосування джгута викликало суттєву зміну активності антиоксидних ферментів. ключові слова: ішемічно-реперфузійний синдром; травма; втрата крові; гемостатичний джгут; каталаза; супероксиддисмутаза. інформація про авторів волотовська наталія володимирівна – канд. мед. наук, асистент кафедри фізіології з основами біоетики та біобезпеки, тернопільський національний медичний університет імені і.я. горбачевського моз україни. кащак тетяна василівна – асистент кафедри фізіології з основами біоетики та біобезпеки, тернопільський національний медичний університет імені і.я. горбачевського моз україни. information about authors nataliya v. volotovska – md, ph.d., department of physiology, bioethics and biosafety, i. horbachevsky ternopil national medical university. orcid 0000-0003-4073-3148, е-mail: volotovskanv@tdmu.edu.ua tetiana v. kashchak – assistant professor, department of physiology, bioethics and biosafety, i. horbachevsky ternopil national medical university. e-mail: kachaktv@tdmu.edu.ua references 1. khanna a, cowled pa, fitridge ra. nitric oxide and skeletal muscle reperfusion injury: current controversies (research review). journal of surgical research. 2005 sep 1;128(1):98-107. doi: 10.1016/j.jss.2005.04.020 2. wang wz, baynosa rc, zamboni wa. update on ischemia-reperfusion injury for the plastic surgeon: 2011. plastic and reconstructive surgery. 2011 dec1;128(6):685e-92e. doi: 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article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. n.v. volotovska et al. 113 b io m e d ic a l s c ie n c e s issn 2413-6077. ijmmr 2019 vol. 5 issue 2 doi 10.11603/ijmmr.2413-6077.2019.2.10698 impact of nitric oxide synthesis modulators on the cytokines profile in experimental antiphospholipid syndrome *o.z. yaremchuk, k.a. posokhova, i.p. kuzmak, m.i. kulitska, o.o. shevchuk, a.s. volska, p.h. lykhatskyi i. horbachevsky ternopil national medical university, ternopil, ukraine background. antiphospholipid syndrome (aps) is an autoimmune disease characterized by the presence of specific antibodies. objective. the aim of the study was to investigate the effect of combined use of l-arginine and aminoguanidine on cytokine profile (il-1β, il-6, tnf-α, il-4, il-10) in experimental aps. methods. the study was performed on balb/c female mice. l-arginine (25 mg/kg) and aminoguanidine (10 mg/kg) were used for correction. serum cytokines concentrations were assessed using an elisa test. results. it was found that in aps the concentration of proinflammatory cytokines il-1β, il-6 and tnf-a increases in 3.2, 2.3 and 4.5 times respectively, compare to the control. at the same time a decrease of the il-4 and il-10 in 1.9 and 2.2 times was evidenced. aminoguanidine, a selective inos inhibitor, caused a significant decrease of tnf-α by 57% (p<0.001), but there were no changes in il-1β, il-6, il-4 and il-10 compare to the aps-group. l-arginine combined with aminoguanidine caused a significant decrease in the concentration of il-1β by 30% (p<0.01), il-6 – by 16% (p<0.05), tnf-a – by 59% (p<0.001) compare to the control. at the same time, the concentration of il-4 increased by 35% (p <0.01), il-10 – by 25% (p<0.005). conclusions. combined use of the precursor of the no synthesis l-arginine and aminoguanidine, a selective inos inhibitor, leads to a decrease in the concentrations of il-1β, il-6, tnf-a and an increase of il-4 and il-10 compare to the group of the balb/c mice with aps and the group of animals administered with aminoguanidine. key words: antiphospholipid syndrome; cytokines; nitric oxide; l-arginine; aminoguanidine. *corresponding author: yaremchuk olha, ph.d., associate professor of the department of medical biochemistry, i. horbachevsky ternopil national medical university, maidan voli, 1, ternopil, ukraine. e-mail: yaremchuk@tdmu.edu.ua introduction antiphospholipid antibody syndrome (aps) is an autoimmune condition characterized by the presence of antiphospholipid antibodies (apl) [1], encompassing primary aps, secondary aps, seronegative aps (snaps) and catastrophic aps (caps) [2]. secondary aps can be found in association with other autoimmune conditions such as systemic lupus erythematosus, rheumatoid arthritis, autoimmune thyroid disease, crohn’s disease, sjogren syndrome, systemic sclerosis, lymphoma or leukemia, malignancies of the ovary and cervix, drug induced as with oral contraceptive pills or in infectious disease such as hiv or syphilis [1]. in caps a systemic inflammatory response, systemic endothelial dysfunction and dic develop. these processes are the pathogenetic basis for development of multiple organ failure [3, 4]. snaps is negative for lupus anticoagulant and antiphospholipid antibodies [2]. the diagnostic aps criteria are anticardiolipin (acl), antiβ2­glycoprotein­i (aβ2gpi) and lupus anticoagulant (la) [1, 5]. aps can be classified only in the presence of thrombotic (non­inflam­ matory arterial, venous or small vessel thrombosis) obstetric complications (death of one or more morphologically normal fetus at or beyond the 10th week of gestation; one or more premature birth of normal fetus before the 34th week due to eclampsia, pre-eclampsia or placental insufficiency), or increased apl level [2]. the mechanisms of thrombosis in aps have not been fully studied yet [6]. aβ2gpi antibodies are central in pathogenic aps mechanisms and, although the full pathogenesis of aps is not clear yet, the binding of these apl antibodies to the antigens on the cell surface of platelets, monocytes, endothelial cells and trophoblasts triggers intracellular signaling with subsequent activation and alteration international journal of medicine and medical research 2019, volume 5, issue 2, p. 113-121 copyright © 2019, tnmu, all rights reserved o.z. yaremchuk et al. 114 b io m e d ic a l s c ie n c e s issn 2413-6077. ijmmr 2019 vol. 5 issue 2 of diverse cell functions. cellular activation starts after the binding of the complex aβ2gpi antibody/β2gpi [5, 7]. β2gpi is the most important antigenic target [2]. platelets activation and the subsequent release of thrombo xane favor their aggregation. thrombosis at the fine vasculature of the target organ is thought to be more dependent from antibodies against the anticoagulant anv. endothelial cells and monocytes activation determine a proaggregation status due to up-regulated expression of adhesion molecules, such as e-selectin, and release of tissue factor (tf) and proinflammatory cytokines [5]. many patients with apl antibodies remain asymptomatic [2]. an important factor in aps immuno pathogenesis is dysregulation of cytokine balance with increased synthesis of proinflammatory cytokines [8, 9]. cytokines are the most versatile system of regulation. cytokines, being synthesized at the inflammation site, affect virtually all cells invol­ ved in the inflammation development, as well as granulocytes, macrophages, fibroblasts, endothelial cells, epithelium cells, t and b lymphocytes [10].the inflammatory processes are controlled by the proinflammatory (il­1, il­2, il­6, il­8, il­12, tnf­α, ifn) and anti­ inflammatory (il­4, il­10, tgf) cytokines [11]. therefore, the study of pathobiochemical mechanisms of aps development, particularly establishment of the role of the cytokine system in development of this pathology, and search for effective methods of its treatment is an urgent and social issue [1, 6, 11, 12]. one of the links that are involved in the mechanisms of aps development is the nitric oxide (no) system. in obstetric aps, the synthesis and bioavailability of nitric oxide (no), which is involved in the regulation of vascular tone and blood coagulation properties, are impaired in the endothelium [4]. according to cella m [13], a decrease in no levels causes abortion and premature birth. on the other hand, no overproduction mediated with inducible no synthase (inos) increases uterine contractions and the risk of miscarriage [13]. contradictions of the existing information on the involvement of the no system in aps development as well as on the efficacy of no precursors in reducing the manifestations of this pathology necessitates further study of the role of this system in aps. the objective of research is to investigate the effect of combined use of l-arginine and aminoguanidine on cytokine profile (concent­ ration of il­1β, il­6, tnf­α, il­4, il­10) in experimental antiphospholipid syndrome. methods female balb/с mice, which were kept on a standard vivarium diet, were used in the research. the experiments were carried out following the principles of bioethics according to the “general ethical principles of animal experiments”, approved by the first national congress on bioethics (kyiv, 2001) and in accordance with the provisions of the “european convention for the protection of vertebrate animals used for experimental and other scientific purposes” (strasbourg, 1986) and eu directive 2010/10/63 eu for animal experiments. aps was modeled using cardiolipin (sigma, usa), which was injected intramuscularly four times (30 μg per 1 injection, the injection inter­ val was 14 days) [14]. to enhance the effectiveness of the immune response, cardiolipin was emulsified in 75 μl of complete freund’s adjuvant (first injection); subsequent injections were performed with incomplete freund’s adjuvant. aps was developing for 2 weeks after the last cardiolipin injection. the experimental animals were divided into 4 groups: the 1st – the intact; the 2nd – the balb/c mice with aps; the 3rd – the animals with aps administered with aminoguanidine, the 4th – the animals with aps administered with l-arginine in combination with aminoguanidine. l-arginine (sigma, usa, 25 mg/kg) and aminoguanidine (khimlaboratorreaktiv, ukraine, 10 mg/kg) were administered intraperitoneally once a day for 10 days after aps development. the animals of the control group were managed with the same volumes of the solvent intraperitoneally. in 10 days after confirmation of aps the animals were taken out of the experiment by thiopental sodium anesthesia (intraperitoneal admi nistration of 1% solution at a dose of 50 mg/kg of animal body weight). the concentration of cytokines il­1β, il­6, tnf­α, il­10, il­4 in the serum of balb/c mice was determined by enzyme immunoassay using standard kits adapted for mice of express biotech international, usa (mouse il­1β elisa assay, mouse il­6 elisa assay, mouse tnf­α elisa assay, mouse il-10 elisa assay, mouse il-4 elisa assay). the concentration of cytokines was expressed in pg/ml. statistical processing of digital data was performed by means of excel software (microsoft, usa) and statistica 6.0 (statsoft, usa) using non-parametric methods of estimation o.z. yaremchuk et al. 115 b io m e d ic a l s c ie n c e s issn 2413-6077. ijmmr 2019 vol. 5 issue 2 o.z. yaremchuk et al. for the attained data. the arithmetic mean (m), its variance and standard error of the mean (m) were assessed for all parameters. the significant difference between the independent quantitative values was determined using the mannwhitney test. the changes were statistically significant at p≤0.05. results according to the attained results, an increase of the concentration of il­1β in 3.2 times (p<0.001) was proved in the balb/c mice with aps compare to the control (fig. 1). an increase in the concentration of il-6 in 2.3 times (p<0.001) in the serum of the animals with aps was evidenced compare to the intact animals (fig. 2). tnf­α concentration increased in 4.5 times (p<0.001) in the serum of the balb/c mice with aps compare to the control (fig. 3). at the same time, the anti­inflammatory cytokine il-4 concentration reduced in 1.9 times (p<0.001) and il-10 – in 2.2 times (p<0.001) compare to the control (fig. 4-5). the administration of aminoguanidine, a selective inos inhibitor, did not cause significant changes concentrations of il­1β and il­6 in the serum of the balb/c mice with aps compare to the control (fig. 1-2). in the case of amino guafig. 1. il­1β content in the blood serum of the balb/c mice with aps in the case of administration of l-arginine and aminoguanidine (m±m, n=10). notes: herein, and figures 2-5. conventional name of animal groups: 1 – control; 2 – antiphospholipid syndrome (aps); 3 – aps + aminoguanidine; 4 – aps+l-arginine+aminoguanidine. * – р<0.05 compare to the control group; ** – р<0.05 compare to the group of animals with aps. fig. 2. il-6 content in the blood serum of the balb/c mice with aps in the case of administration of l-arginine and aminoguanidine (m±m, n=10). fig. 3. tnf­α content in the blood serum of the balb/c mice with aps in the case of administration of l-arginine and aminoguanidine (m±m, n=10). fig. 4. il-4 content in the blood serum of the balb/c mice with aps in the case of administration of l-arginine and aminoguanidine (m±m, n=10). fig. 5. il-10 content in the blood serum of the balb/c mice with aps in combined administration of l-arginine and aminoguanidine (m±m, n=10). statistical processing of digital data was performed by means of excel software (microsoft, usa) and statistica 6.0 (statsoft, usa) using non-parametric methods of estimation for the attained data. the arithmetic mean (m), its variance and standard error of the mean (m) were assessed for all parameters. the significant difference between the independent quantitative values was determined using the mann-whitney test. the changes were statistically significant at p≤0.05. results according to the attained results, an increase of the concentration of il-1β in 3.2 times (p <0.001) was proved in the balb/c mice with aps compare to the control (fig. 1). fig. 1. il-1β content in the blood serum of the balb/c mice with aps in the case of administration of l-arginine and aminoguanidine (m±m, n=10). notes: herein, and figures 2-5. conventional name of animal groups: 1 – control; 2 – antiphospholipid syndrome (aps); 3 – aps + aminoguanidine; 4 – aps+l-arginine+aminoguanidine. * – р<0.05 compare to the control group; ** – р<0.05 compare to the group of animals with aps. an increase in the concentration of il-6 in 2.3 times (p <0.001) in the serum of the animals with aps was evidenced compare to the intact animals (fig. 2). 0 10 20 30 40 50 60 1 2 3 4 il -1 β, p g/ m l * ** fig. 2. il-6 content in the blood serum of the balb/c mice with aps in the case of administration of l-arginine and aminoguanidine (m±m, n=10). tnf-α concentration increased in 4.5 times (p <0.001) in the serum of the balb/c mice with aps compare to the control (fig. 3). fig. 3. tnf-α content in the blood serum of the balb/c mice with aps in the case of administration of l-arginine and aminoguanidine (m±m, n=10). at the same time, the anti-inflammatory cytokine il-4 concentration reduced in 1.9 times (p <0.001) and il-10 – in 2.2 times (p <0.001) compare to the control (fig. 4-5). 0 5 10 15 20 25 1 2 3 4 il -6 , p g/ m l * ** 0 10 20 30 40 50 60 70 80 1 2 3 4 tn fα, p g/ m l * ** ** fig. 2. il-6 content in the blood serum of the balb/c mice with aps in the case of administration of l-arginine and aminoguanidine (m±m, n=10). tnf-α concentration increased in 4.5 times (p <0.001) in the serum of the balb/c mice with aps compare to the control (fig. 3). fig. 3. tnf-α content in the blood serum of the balb/c mice with aps in the case of administration of l-arginine and aminoguanidine (m±m, n=10). at the same time, the anti-inflammatory cytokine il-4 concentration reduced in 1.9 times (p <0.001) and il-10 – in 2.2 times (p <0.001) compare to the control (fig. 4-5). 0 5 10 15 20 25 1 2 3 4 il -6 , p g/ m l * ** 0 10 20 30 40 50 60 70 80 1 2 3 4 tn fα, p g/ m l * ** ** fig. 4. il-4 content in the blood serum of the balb/c mice with aps in the case of administration of l-arginine and aminoguanidine (m±m, n=10). fig. 5. il-10 content in the blood serum of the balb/c mice with aps in combined administration of l-arginine and aminoguanidine (m±m, n=10). the administration of aminoguanidine, a selective inos inhibitor, did not cause significant changes concentrations of il-1β and il-6 in the serum of the balb/c mice with aps compare to the control (fig. 1-2). in the case of aminoguanidine use, a significant decrease in the concentration of tnf-α by 57% (p <0.001) was evidenced compare to the intact animals (fig. 3). it was found out that, under the influence of aminoguanidine, the concentrations of il-4 and il-10 did not change significantly compare to the control group of animals (fig. 4-5). 0 5 10 15 20 25 30 35 1 2 3 4 il -4 , p g/ m l * ** 0 10 20 30 40 50 60 70 80 1 2 3 4 il -1 0, p g/ m l * ** fig. 4. il-4 content in the blood serum of the balb/c mice with aps in the case of administration of l-arginine and aminoguanidine (m±m, n=10). fig. 5. il-10 content in the blood serum of the balb/c mice with aps in combined administration of l-arginine and aminoguanidine (m±m, n=10). the administration of aminoguanidine, a selective inos inhibitor, did not cause significant changes concentrations of il-1β and il-6 in the serum of the balb/c mice with aps compare to the control (fig. 1-2). in the case of aminoguanidine use, a significant decrease in the concentration of tnf-α by 57% (p <0.001) was evidenced compare to the intact animals (fig. 3). it was found out that, under the influence of aminoguanidine, the concentrations of il-4 and il-10 did not change significantly compare to the control group of animals (fig. 4-5). 0 5 10 15 20 25 30 35 1 2 3 4 il -4 , p g/ m l * ** 0 10 20 30 40 50 60 70 80 1 2 3 4 il -1 0, p g/ m l * ** 116 b io m e d ic a l s c ie n c e s issn 2413-6077. ijmmr 2019 vol. 5 issue 2 nidine use, a significant decrease in the con­ centration of tnf­α by 57% (p<0.001) was evi­ denced compare to the intact animals (fig. 3). it was found out that, under the influence of aminoguanidine, the concentrations of il-4 and il­10 did not change significantly compare to the control group of animals (fig. 4-5). in the case of administration of the predecessor of the synthesis of no l-arginine in combination with aminoguanidine, a significant decrease in the concentration of il­1β by 30% (p<0.01), il-6 by 16% (p<0.05), tnf-a by 59% (p<0.001) was established compare to the control (fig. 1-3). at the same time, the concentration of anti­inflammatory cytokines il­4 increased by 35% (p <0.01) and il-10 – by 25% (p<0.005) compare to the control animals (fig. 4-5). the results of the study proved that a significant decrease in the concentration of il­1β by 22% (p<0.05), il­6 by 23% (p<0.005) was evidenced in the case of combined administration of l-arginine and aminoguanidine compare to the indicators of the 3rd group of animals, which were administered with aminoguanidine (fig. 1-2). an increase of the antiinflammatory cytokine il­4 concentration by 32% (p<0.05) and il-10 by 19% (p<0.05) was proved compare to the 3rd group of the balb/c mice administered with aminoguanidine (fig. 4-5). discussion besides the pathogenic role of the apl, proinflammatory cytokines and chemokines are significant in the pathogenesis of aps [12]. il­1, tnf-a and endotoxins induce tissue factor (tf) expression in endothelial cells, monocytes, macrophages promoting blood clotting [10, 15]. the inhibitors of il-1 production are il-4, il-10, il­12, tnf­α [16]. il­6 is involved in regulation of t and b cell interactions, macrophage, endotheliocytes activity. il-6 induces production of acute-phase proteins, stimulates hematopoiesis and platelet formation [16]. the attained results on increased concentrations of proinflammatory cytokines (il­1β, il­6, tnf­α) in the serum of the experimental animals with aps conform with the literature [6, 17, 18]. according to n.v. seredavkina [6], an increased concentration of il­6 and tnf­α in the patients with aps compare to the control group was established. it is not clear whether apl affect endothelial cells directly or through tnf­α. regardless of the mechanism, the pro­ thrombotic condition, typical of aps, is associated with both significantly increased apl levels as well as high tnf­α concentration [11]. according to j. swadzba et al. [15] tnf­α is one of the main proinflammatory cytokines in aps; its level is increased and reflects pathological processes in endothelial cells. according to the literature, apl and tnf-a can activate the endothelium and induce prothrombotic phenotype of endothelial cells, leading to increased thrombin production. activation of endothelial cells causes upregulation of tf, which has been suggested to be a major potential mechanism of aps-related thrombosis. once endothelial cells are activated, tf regulation can be more enhanced by a synergizing effect of tnf-a and factor xa, thus expression of adhesion molecules (icam-1, vcam-1, e and p selectins) and for mation of endothelial microparticles take place [15]. according to a. farzaneh-far et al. [17], who investigated the levels of crp il 6, isam-1, ptnf α­p2, ptnf α­p2 in the patients with sle, only increased ptnf α­p1 and ptnf α­p2 were asso­ ciated with apl positivity. according to nv seredavkin a negative correlation between crp and i g g β 2 g p 1 l e v e l s w a s e s t a b l i s h e d [ 6 ] . r.r. forastiero et al. [18] established that il 6 levels were greater in the patients with aps and apa carriers than in the control group. tnf concentration was the same in the patients with aps and apa carriers but higher than in the control group. in the patients with positive apa, a direct correlation between il 6 and tnf α was proved [18]. under the experimental conditions it has been established that tnf-a may manifest antiplatelet and antithrombotic activity [15] according to the literature, il­1β activates the synthesis of il­6, s100b, α1­antihymotrypsin, inducible nitric oxide synthase (inos) causing increased no synthesis [19, 20]. the inos is crucial in the primary proinflammatory response in macrophages [21]. ag is a nucleophilic hydrazine compound, structurally similar to l-arginine in that these compounds contain two chemically equivalent guanidino nitrogen groups and to l-arginine analogues that com petitively inhibit no synthase. ag completely prevents inflam­ matory stimuli induced for mation of no, and it is a potent inhibitor of the cytokine-inducible isoform nos [22]. the results of our studies proved that introduction of aminoguanidine, a selective inos inhibitor, did not cause significant changes in the concentration of proinflammatory cytokines (il­1β and il­6) and anti­inflammatory cytokines (il-4 and il-10) in the serum of the balb/c mice o.z. yaremchuk et al. 117 b io m e d ic a l s c ie n c e s issn 2413-6077. ijmmr 2019 vol. 5 issue 2 o.z. yaremchuk et al. with aps compare to the control group of animals. at the same time, in the case of aminoguanidine administration a significant decrease in tnf­α concentration was proved compare to the intact animals. according to ei ferreira et al. [23] aminoguanidine decreases tnfα levels, oxidative stress indicators, and no metabolites. it is established that increased concentrations of tnf­α are associated with pregnancy miscarriage in aps [6, 8], endotheliocytes activation, and chemokine amplification that leads to subendothelial leukocyte accumulation, endothelial dysfunction, microcirculation distur bances [16]. early endothelial dysfunction was observed in aps [24]. patients with aps displaying thrombosis exhibited low plasma levels of nitrites and nitrates, which are the stable metabolites of no breakdown. apl can act as antagonists of endothelial nitric oxide synthase (enos) through β2gpi, and this interaction may impair no synthesis. in particular, attenuation of enos activation by apl was mediated by reduced phosphorylation of enos serine. this inhibition of enos phosphorylation was shown to be dependent upon protein phosphatase 2a, β2gpi, and apolipoprotein e receptor 2. apl inhibition of enos activity contributes to thrombus formation, increased leukocyte adhesion, and alterations in vascular tone [4]. it is established that violation of the bioavailability of no may be one of the causes of endothelial dysfunction. this may be associated with both the lack of substrate for no l-arginine synthesis as well as formation of superoxide anion which rapidly binds and inactivates no [24]. no synthesis is not dependent on l-arginine concentration in physiological states. in pathological conditions, the availability of l-arginine may determine production of no. it is proved that l-arginine is necessary for adequate translation of inos. when inos is being activated, superoxide anion is produced, which forms a highly reactive peroxynitrite, which in turn produces nitrosylation of amino acid residues sensitive to it, especially tyrosine, that leads to conformational changes in the structure of pro tein molecules. with administration of l-arginine the functional characteristics of t-cells enhance, production of antibodies increases as well. no-dependent effect of l-arginine on the immune system may be hormone-mediated [25]. the next objective of our study was to investigate the effect of combined use of l-arginine and aminoguanidine on cytokine profile in aps. in the case of the use of the precursor of no l-arginine synthesis in combination with aminoguanidine, a selective inos inhibitor, a significant decrease in the concentration of proinflammatory cytokines (il­1β, il­6, tnf­a) and an increase in the concentration of antiinflammatory cytokines (il­4, il­10) was estab­ lished compare to the control group of animals. the attained results are consistent with the literature [25, 26]. these effects can be explained by the fact that glutamine formed from l-ar ginine is a conditionally essential amino acid and reduces the level of tnf­α soluble receptors [26]. according to vm sheibak et al. [25] ad ministration of l-arginine decreases the level of il-6. according to p. soltesz et al. [12], besides the conventional th1 pathway, th2 cytokines are crucial in the mechanisms of aps development, i.e. il-4 and il-10. various immunocompetent cells regulate the proinflammatory cascade that leads to cytokine imbalance and activated circulating lymphocytic pool in aps. this proinflammatory process leads to endo­ thelial dysfunction, development of arterial and venous thrombosis [12]. as a result of the research, a decrease in anti­inflammatory cyto­ kines (il­4, il­10) in aps was established; the results are consistent with the literature [11, 12]. according to p. soltesz et al. [12], the markers of endothelial dysfunction positively correlate with il-4 levels in aps. it allows suggesting that by activation of the humoral and cellular immune responses, il-4 is crucial in development of endothelial dysfunction, atherosclerosis, arterial and venous thrombosis. il-4 stimulates b and t cell proliferation as well as differentiation of cd4 + t cells into th2 cells [12]. according to a. menachem et al. [11] cytosolic and secreted il­10 and ifn­γ levels in eaps mice were lower at 6 and 15 weeks and higher at 24 weeks after immunization compared to adjuvant mice. il­10 is significant in autoimmune diseases. as a result of other studies, il-10 level was decreased in the serum of the patients with aps [12]. il-10 inhibits secretion of il-4, il-5 and ifn­γ, growth factors and chemokines, and therefore acts as a key counter-regulator of autoimmune processes [12]. one of the functions of il-10 is inhibition of the synthesis of proinflammatory cytokines: il­1, il­6, il­12 and tnfα via a stat3­dependent mechanism [27] and enhancement of il-1 receptor antagonist 118 b io m e d ic a l s c ie n c e s issn 2413-6077. ijmmr 2019 vol. 5 issue 2 expression [19]. decreased level of il-10 in the serum in cases of aps compare to the control confirms the fact that il10­mediated processes are impaired in aps that is why it leads to vascular damage [12]. low il­10 levels enable tnf­α unregulated production, resulting in procoagulant state. decreased il-10 levels can be associated with lymphocyte activation, which leads to the continuation of the autoimmune response. during the b-cell activation, il-10 delivers signals that promote the apoptosis of b cells [11]. conclusions thus, that in the serum of balb/c mice with aps, an increase in the concentrations of proinflammatory cytokines (il­1β, il­6, tnf­a) and a decrease in the concentrations of anti­inflam­ matory cytokines (il-4 and il-10) was established compare to the control parameters. with the introduction of aminoguanidine, a selective inos inhibitor, a decrease in the concentration of tnf­α was proved compare to that of the animals with aps. in the case of the use of the precursor of no synthesis l-arginine in combination with aminoguanidine, a significant decrease in concentrations of il­1β, il­6, tnf­α and an increase of il-4 and il-10 was evidenced compare to the group of balb/c mice with aps and the group of animals administered with aminoguanidine. funding this research received no external funding. conflict of interests the authors declare no conflict of interest. authors contributions yaremchuk o.z. – writing – original draft, conceptualization, project administration, methodology, investigation, formal analysis, posokho va k.a. – supervision, conceptualization, writing – review & editing, kuzmak i.p. – data curation, kulitska m.i. – investigation, shevchuk o.o. – investigation, writing – review & editing, volska a.s. – investigation, lykhatskyi p.h. – data curation. вплив модуляторів синтезу оксиду азоту на показники цитокінового профілю при експериментальному антифосфоліпідному синдромі о.з. яремчук, к.а. посохова, і.п. кузьмак, м.і. куліцька, о. о. шевчук, а.с. вольська, п.г. лихацький тернопільський національний медичний університет імені і. я. горбачевського, тернопіль, україна вступ. антифосфоліпідний синдром (афс) – це автоімунне захворювання, яке характеризується наявність антифосфоліпідних антитіл, артеріальними та венозними тромбозами, тромбоцитопенією, невиношування вагітності. мета дослідження. дослідити вплив комбінованого застосування l-аргініну та аміногуанідину на показники цитокінового профілю (концентрацію il-1β, il-6, tnf-α, il-4, il-10) при експериментальному антифосфоліпідному синдромі. методи дослідження. дослідження виконано на мишах-самках лінії balb/с, в яких моделювали афс. для корекції використовували l-аргінін (25 мг/кг) та аміногуанідин (10 мг/кг). визначення концентрації цитокінів il-1β, il-6, tnf-α, il-10, il-4 у сироватці крові мишей balb/c проводили методом імуноферментного аналізу з використанням стандартних наборів реактивів. результати й обговорення. отримані результати свідчать, що у сироватці крові мишей balb/с за умов афс відбувається зростання концентрації прозапальних цитокінів il-1β у 3,2 раза, il-6 у у 2,3 раза, tnf-а в 4,5 разів, відносно контролю. спостерігалось зниження концентрації протизапальних цитокінів il-4 в 1.9 раза та il-10 в 2,2 раза у групі тварин з афс, порівняно із показниками контролю. на фоні застосування селективного інгібітора inos аміногуанідину встановлено достовірне зниження концентрації tnf-а на 57 % (р<0.001), проте концентрація il-1β, il-6 il-4 та il-10 достовірно не змінювалася у сироватці крові мишей balb/c з афс, порівняно з показниками тварин з афс. на фоні застосування попередника синтезу no l-аргініну в комбінації з аміногуанідином встановлено достовірне зниження концентрації il-1β на 30 % (р<0.01), il-6 на 16 % (р<0.05), tnf-а на 59 % (р<0.001), відносно контролю. водночас зростала концентрація протизапальних цитокінів il-4 на 35 % (р<0.01) та il-10 на 25% (р<0.005), порівняно з показниками групи мишей balb/c з афс. o.z. yaremchuk et al. 119 b io m e d ic a l s c ie n c e s issn 2413-6077. ijmmr 2019 vol. 5 issue 2 o.z. yaremchuk et al. висновки. встановлено, що комбіноване застосування попередника синтезу no l-аргініну та селективного інгібітора іnos аміногуанідину призводить до зниження концентрації il-1β, il-6, tnf-а та зростання il-4 та il-10, порівняно з показниками групи мишей balb/c з афс та групи тварин, яким вводили аміногуанідин. ключові слова: антифосфоліпідний синдром; цитокіни; оксид азоту; l-аргінін; аміногуанідин відомості про авторів яремчук ольга зеновіївна – кандидат біологічних наук, доцент кафедри медичної біохімії тернопільського національного медичного університету імені і.я. горбачевського, тернопіль, україна. посохова катерина андріївна – доктор медичних наук, професор кафедри фармакології з клінічною фармакологією тернопільського національного медичного університету імені і.я. гор­ бачевського. кузьмак ірина петрівна – кандидат біологічних наук, доцент кафедри медичної біохімії тернопільського національного медичного університету імені і.я. горбачевського. куліцька марія іванівна – кандидат біологічних наук, доцент кафедри медичної біохімії тернопільського національного медичного університету імені і.я. горбачевського. шевчук оксана олегівна – кандидат медичних наук, доцент кафедри фармакології з клінічною фармакологією тернопільського національного медичного університету імені і.я. горбачевського. вольська аліна станіславівна – кандидат біологічних наук, доцент кафедри фармакології з клінічною фармакологією тернопільського національного медичного університету імені і.я. гор­ бачевського. лихацький петро григорович – доктор біологічних наук, професор кафедри медичної біохімії тернопільського національного медичного університету імені і.я. горбачевського. information about the authors yaremchuk o.z. – ph.d., associate professor of medical biochemistry department, i. horbachevsky ternopil national medical university, ternopil, ukraine. orcid 0000-0001-5951-1137, e­mail: yaremchuk@tdmu.edu.ua posokhova k.a. – md, ph.d., dsc., professor of pharmacology and clinical pharmacology department, i. horbachevsky ternopil national medical university, ternopil, ukraine. orcid 0000-0002-2696-5738, e­mail: posokhova@tdmu.edu.ua kuzmak i.p. – ph.d., associate professor of medical biochemistry department, i. horbachevsky ternopil national medical university, ternopil, ukraine. orcid 0000­0002­5035­8815, e­mail: kuzmak@tdmu.edu.ua kulitska m.i. – ph.d., associate professor of medical biochemistry department, i. horbachevsky ternopil national medical university, ternopil, ukraine. orcid 0000­0001­7116­6655, e­mail: kulitskam@tdmu.edu.ua shevchuk o.o. – md, ph.d., associate professor of pharmacology and clinical pharmacology department, i. horbachevsky ternopil national medical university, ternopil, ukraine. orcid 0000-0003-2473-6381, e­mail: shevchukoo@tdmu.edu.ua volska a.s. – ph.d., associate professor of pharmacology and clinical pharmacology department, i. horbachevsky ternopil national medical university, ternopil, ukraine. orcid 0000-0002-4985-9559, e­mail: volska@tdmu.edu.ua lykhatskyi p.h. – ph.d., dsc., professor of medical biochemistry department, i. horbachevsky ternopil national 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(in russian) 27. howes a, taubert c, blankley s, spink n, wu x, graham cm, zhao j, saraiva m, ricciardi-castagnoli p, bancroft gj, o’garra a. differential production of type i ifn determines the reciprocal levels of il-10 and proinflammatory cytokines produced by c57bl/6 and balb/c macrophages. the journal of immunology. 2016 oct 1;197(7):2838­53. doi: 10.4049/jimmunol.1501923 received 04 september 2019; revised 14 october 2019; accepted 25 october 2019. this is open access article distributed under the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. 98 p h a r m a c y issn 2413-6077. ijmmr 2019 vol. 5 issue 2 doi 10.11603/ijmmr.2413-6077.2019.2.10898 qsar-analysis of polysubstituted functionalized aminothiazoles with antihypertensive activity і.v. drapak danylo halytsky lviv national medical university, lviv, ukraine background. qsar analysis is an important tool for the identification of pharmacophore fragments in biologically active substances and helps optimize the search for new effective drugs. objective. the aim of the study was to determine the molecular descriptors for qsar analysis of polysubstituted functionalized aminothiazoles as a theoretical basis for purposeful search de novo of potential antihypertensive drugs among the investigated compounds. methods. calculation of molecular descriptors and qsar-models creation was carried out using the hyperchem 7.5 and buildqsar packages. results. the calculation of a number of molecular descriptors (electronic, steric, geometric, energy) was performed for 15 new polysubstituted functionalized aminothiazoles, with established in vivo antihypertensive activity. according to the calculated molecular descriptors and antihypertensive activity parameter, the qsar models were derived на = a + b ∙ x1 + c ∙ x2 + d ∙ x3 , where the activity parameter на is antihypertensive activity and x1, x2, x3 are molecular descriptors. conclusion. the study of ‘the structure antihypertensive activity’ relationship for polysubstituted functionalized aminothiazoles was carried out. qsar analysis revealed that volume, area, lipophilicity, dipole moment, refractivity, polarization of the molecule and energy of the lowest unoccupied molecular orbital have the most significant effect on antihypertensive activity. it was suggested that the attained qsar-models may have antihypertensive activity within abovementioned row of compounds and can be considered as theoretical basis for de novo design of new potential antihypertensive drugs. key words: polysubstituted functionalized aminothiazoles; antihypertensive activity; molecular descriptors; qsar-analysis. corresponding author: iryna drapak, phd, department of general, bioinorganic, physical and colloidal chemistry, danylo halytsky lviv national medical university, 69 pekarska, 79010, lviv, ukraine. email iradrapak@ukr.net introduction qsar analysis is an important tool for identification of pharmacophore fragments in biologically active substances and helps optimize the search for new effective drugs. to date, systematic correlation analysis is a necessary step for lead­compounds identifi cation in the search for a new drug. the directed search for new biologically active substances can be succeeded by the use of virtual methods of research, which are carried out by means of qsar/qsrr analysis (quantitative structureactivity/quantitative structure-property relationship), which allows establishing quantitative patterns of relation between the activity or properties of the investigated compounds and the parameters of their molecular structure. quantitative structure activity relationship is nowadays widely used as a method for predicting the biological activity of new compounds. [1-3]. thiazole derivatives are a promising class for the search for biologically active compounds, since the thiazole nucleus is a strong biophore fragment for the rational design of ‘drug-like molecules’, and thiazole derivatives exhibit various types of biological activity: anti­inflam­ matory [4], antihypertensive [5], cardioprotective [6], antioxidant [7]. the objectieve of the study was to determine the molecular descriptors for qsar analysis of the new polysubstituted functio nalized aminothiazoles as a theoretical basis for search for new antihypertensive drugs among abovementioned types of compounds. methods 15 polysubstituted functionalized aminothiazoles, which antihypertensive activity was determined, were used [6, 8-9]. calculation of molecular descriptors was carried out using hyper-chem 7.5 software [10] (license on hyperchem 7.5 software is available for danylo halytsky lviv national medical university); buildqsar software was used for qsar-model building [11]. international journal of medicine and medical research 2019, volume 5, issue 2, p. 98-104 copyright © 2019, tnmu, all rights reserved і.v. drapak 99 p h a r m a c y issn 2413-6077. ijmmr 2019 vol. 5 issue 2 і.v. drapak results target polysubstituted functionalized aminothiazoles were synthesized as described [6, 8-9]. the structures of the test compounds are presented in fig. 1. antihypertensive activity (на) studies were performed in vivo experiments on laboratory white rats [9]. the activity of the test compounds was determined by a decrease in the blood pressure in the tail artery of the animal in 60 minutes after intraperitoneal injection. for screening studies of antihypertensive activity, substances at a dose of 7 mg/kg were used. the antihypertensive activity of the test compounds are presented in table 1. the 2d structures of the molecules of the test compounds were converted into 3d using hyper-chem 7.5 software [10]. the optimization of the structures of the tested substances was carried out by the method of molecular mechanics mm+, and the final energy minimization was carried out by semi-empirical quantumchemical am1 method until a standard deviation of 0.001 kcal/mol was achieved. the calculation of number of descriptors (electronic, steric, geometric, energy) [12,13] was carried out for the tested compounds. the molecular descriptors were calculated using hyper-chem 7.5: the surface and grid area of the molecule 1. r= 4-ome, r1=2,5-dime, r2= (ch2)2oh, r 3=h 2. r= 4-ome, r1=4-ochf2, r 2= (ch2)3oh, r 3=me 3. r= 4-ochf2, r 1=4-ome, r2= (ch2)2oh, r 3=h 4. r1= h, r2 = n-piperydinyl(ch2)2, r 3=h, 5. r1= 4-ome, r2= n-morpholinyl(ch2)2-, r 3=oet, 6. r1= 2,3-dime, r2 = (ch2)2oh, r 3=oet, 7. r1= 4-me, r2= 3-pyridinyl-ch2, r 3=h, 8. r1= 4-me, r2= n-morpholinyl, r3=h, 9. r1= 4-oet, r2= n-morpholinyl, r3=h, 10. r1= 4-me, r2= 3,4-diome-c6h3(ch2)2, r 3=h, 11. r1= h, r2= 3,4-diome-c6h3(ch2)2, r 3=h, 12. r1= 2-me, r2= 3,4-diome-c6h3(ch2)2, r 3=h, 13. r1= 2,3-dime, r2= 3,4-diome-c6h3(ch2)2, r 3=h, fig. 1. structures of the tested compounds. (sa and sg), volume of the molecule (v), lipophilicity parameter (logp), refractivity (r), polarizability (p), molecular weight (m), dipole moment (d). the parameters of the calculated molecular descriptors of the test compounds are presented in table. 1. the following parameters were also calculated for oxygen (ch_o), sulfur (ch_s), nitrogen of thiazole cycle (ch_nt) and nitrogen of amino­group (ch_n); distances between atoms of sulfur and oxygen (d_s­o), sulfur and nitrogen nt (d_s­nt), nitrogen of the amino­ group and nitrogen of the thiazole cycle (d_nt­ n); angles between the atoms of the nitrogen of the thiazole cycle, sulfur and nitrogen of the amino group (a_n­s­nt), nitrogen of the amino group, oxygen and nitrogen of the thiazole cycle (a_n­o­nt). the attained parameters are presented in table 2. the energy parameters of the studied molecules were assessed: total energy (te), binding energy (be), isolated atomic energy (iae), electronic energy (ee), core-core interaction (cci), heat of formation (hf), energies of the highest occupied molecular orbital and lowest unoccupied molecular orbital (homo and lumo), hydration energy (eh). the attained results of energy parameters are presented in table 3. 100 p h a r m a c y issn 2413-6077. ijmmr 2019 vol. 5 issue 2 in the study, the optimal set of molecular descriptors was chosen by a sequential algorithm and the multiple linear regression (mlr) method to obtain qsar models by means of buildqsar [11]. statistically, the number of compounds tested (n) and independent variables (m) used in the model should correspond to a ratio of n / m ≥ 5. descriptors with high pairwise correlation were excluded from the multidimensional descriptor space. firstly, the descriptors from different groups were used to build individual models, and then descriptors that most fully describe the change in biological activity were used to obtain mixed models. this allowed us to choose single or multi-parameter models with the maximal correlation coefficient (r) and the minimal standard deviation (s). the models were further investigated for their adequacy by means of the fisher coefficient (f) and predictive ability using the q2 crossvalidation coefficient and sum of squares of prediction error (srress). based on the calculated molecular descriptors and the parameter of antihypertensive activity, the qsar models were derived ah=a+ b∙x1+c∙x2+d∙x3 , where the activity parameter ah is antihypertensive activity and x1, x2, x3 are molecular descriptors: table 1. аntihypertensive activity and molecular descriptors of polysubstituted functionalized aminothiazoles no. ah sa sg v logp r p m d 1 8 546.8 667.17 1142.6 1.3 122.22 43.77 435.38 1.99 2 11.9 705.14 731.37 1248.5 0.93 130.69 46.06 501.39 7.11 3 10.5 642.6 666.28 1141.6 0.85 120.04 42.39 473.33 4.49 4 6.2 551.29 605.91 1063.4 1.22 113.24 41.49 380.94 3.29 5 14,3 666.39 680.82 1224.9 1.7 124.63 45.78 470.42 7.44 6 5.5 514.36 560.73 969.72 0.68 99.07 36.3 334.43 4.21 7 8.3 511.78 577 981.94 0.68 107.71 38.47 337.44 2.2 8 6.6 570.15 579.33 1015.7 0.68 106.46 38.94 367.89 2.83 9 8.2 618.67 628.25 1094.2 -0.12 113.31 41.41 397.92 3.07 10 7.8 588.77 639.32 1147.8 0.42 128.78 45.96 410.53 2.04 11 12.2 549.76 614.72 1100.6 0.27 124.5 44.1 396.5 2.24 12 2.1 567.3 629.3 1137.4 0.42 106.94 42.56 410 2.49 13 11.1 659 672.16 1266 1.36 142.63 51.19 505.47 3.97 14 9.2 692.98 743.14 1354 -0.94 146.17 52.54 484.86 3.72 15 10.8 530.62 639.61 1153.6 4.88 123.15 46.06 455.88 2.28 table 2. charges, distances, angles between atoms of polysubstituted functionalized aminothiazoles no. ch_o ch_s ch_nt ch_n d_s­o d_s­nt d_s­n d_nt­n a_n­s­nt a_n­o­nt 1 0 0.382 -0.204 -0.243 0 2.59 2.79 2.37 52.27 0 2 0 0.285 -0.227 -0.205 0 2.60 2.79 2.38 52.41 0 3 0 0.352 -0.215 -0.229 0 2.59 2.79 2.38 52.31 0 4 -0.281 0.392 -0.204 -0.24 3.53 2.59 2.79 2.37 52.32 21.86 5 -0.363 0.449 -0.227 -0.207 2.6 2.61 2.63 2.49 56.86 22.08 6 -0.354 0.489 -0.243 -0.214 3.37 2.60 2.63 2.50 56.93 23.3 7 -0.271 0.384 -0.209 -0.223 3.34 2.59 2.79 2.38 52.42 22.16 8 -0.268 0.366 -0.117 -0.191 3.42 2.63 2.77 2.41 51.79 32.96 9 -0.261 0.374 -0.125 -0.193 3.27 2.62 2.77 2.41 53.1 23.14 10 -0.287 0.405 -0.203 -0.248 3.16 2.59 2.78 2.38 52.5 23 11 -0.286 0.406 -0.202 -0.25 3.17 2.59 2.78 2.38 52.53 23.04 12 -0.285 0.405 -0.203 -0.251 3.18 2.59 2.78 2.38 52.4 22.85 13 -0.286 0.361 -0.206 -0.238 3.13 2.59 2.78 2.38 52.37 23.13 14 -0.282 0.356 -0.211 -0.273 3.23 2.6 2.76 2.4 53 22.94 15 0 0.292 -0.183 -0.251 0 2.64 2.76 2.41 52.97 0 і.v. drapak 101 p h a r m a c y issn 2413-6077. ijmmr 2019 vol. 5 issue 2 і.v. drapak h a = 0 . 0 4 4 ( ± 0 . 0 1 9 ) s a + 0 . 0 4 1 ( ± 0 . 0 1 1 ) v -24.062(±7.933)lumo-21.062(±8.297) (1) (n=015; r=0.936; s=1.171; f=26.006; q2=0.799; spress=1.492) ha=+0.048(±0.015)v+0.004(±0.001)iae -27.025(±9.356)lumo-29.957(±10.172) (2) (n=015; r=0.930; s=1.227; f=23.353; q2=0.718; spress=1.767) ha=+0.019(±0.011)v+0.731(±0.866)logp13.142(±9.671)lumo-19.284(±13.242) (3) (n=015; r=0.825; s=1.879; f=7.844; q2=0.500; spress=2.354) ha=+0.051(±0.028)r-0.002(±0.001)be17.152(±10.844)lumo -16.857(±13.668) (4) (n=015; r=0.813; s=1.940; f=7.132; q2=0.086; spress=3.470) h a = + 0 . 0 3 3 ( ± 0 . 0 2 6 ) r + 0 . 4 2 3 ( ± 0 . 3 0 4 ) p-17.522(±10.543)lumo -20.150(±14.952) (5) (n=015; r=0.825; s=1.880; f=7.840; q2=0.295; spress=2.795) ha=+1.237(±0.685)p+0.002(±0.001)ee22.339(±12.913)lumo-36.725 (±21.841) (6) (n=015; r=0.817; s=1.920; f=7.359; q2=0.313; spress=2.760) ha=+1.165(±0.669)p-0.002(±0.001)cci20.886(±12.661)lumo-35.268(±21.945) (7) (n=015; r=0.807; s=1.968; f=6.835; q2=0.296; spress=2.793) h a = + 0 . 0 1 9 ( ± 0 . 0 1 2 ) v 1 1 . 7 7 7 ( ± 1 0 . 5 9 9 ) lumo+0.275(±0.771)a_n-s-nt-32.425(±42.963) (8) (n=015; r=0.778; s=2.092; f=5.624; q2=0.115; spress=3.132) h a = + 0 . 0 1 9 ( ± 0 . 0 1 2 ) v 1 1 . 1 9 7 ( ± 1 1 . 6 8 5 ) lumo+6.871(±57.283)ch_n-16.433(±20.007) (9) (n=015; r=0.766; s=2.142; f=5.196; q2=0.158; spress=3.055) h a = + 0 . 5 2 6 ( ± 0 . 3 7 3 ) p 1 2 . 8 0 7 ( ± 1 1 . 9 6 9 ) l u m o + 1 9 . 0 9 1 ( ± 3 3 . 9 3 4 ) d _ n n t 6 5 . 1 6 7 (±86.579) (10) (n=015; r=0.721; s=2.307; f=3.973; q2=0.028; spress=3.284) ha=+0.023(±0.012)v+0.233(±0.391)eh14.248(±10.989)lumo-21.927(±15.461) (11) (n=015; r=0.799; s=2.001; f=6.492; q2=0.351; spress=2.683) h a = + 0 . 0 1 2 ( ± 0 . 0 1 5 ) v + 0 . 5 3 5 ( ± 1 . 0 7 2 ) logp+0.528(±0.934)d-6.815(±14.912) (12) (n=015; r=0.705; s=2.362; f=3.622; q2=0.048; spress=3.409) discussion according to the analysis of the derived qsar models (1-12), it was established that the antihypertensive activity increases with the increase of the parameters of the following molecular descriptors: volume, lipophilicity, dipole moment, refractivity and polarization of the molecule and decrease of the surface area of the molecule. among the energy parameters, the most significant effect is the energy of the lowest unoccupied molecular orbital, with increasing antihypertensive activity and decrease in this parameter. antihypertensive activity also increases with increasing charge on the nitrogen atom of the amino group, with increasing distance between this atom and the nitrogen atom of the thiazole cycle and increase of the angle a_n­s­nt. the dependence of the observed and predicted antihypertensive activities for the qsar models (1-3) is presented in fig. 2. the search for quantitative regularities of the dependence of the antihypertensive activity table 3. energy parameters of polysubstituted functionalized aminothiazoles no. te be iae ee cci hf homo lumo eh 1 -103278 -5050 -98228 -780638 677360 4.33 -7.81 -0.27 -5.71 2 -136024 -5476 -130548 -989792 853768 -153.1 -8.12 -0.56 -12.2 3 -128838 -4913.8 -123924 -872337 743499 -141.4 -7.92 -0.59 -11.9 4 -100651 -4965.6 -95685 -785689 685038 19.91 -7.97 -0.34 -3.82 5 -117058 -5558.6 -111499 -960851 843794 -71.13 -8.34 -0.48 -2.35 6 -93372 -4600.7 -88771 -679708 586336 -78.24 -8.26 -0.42 -5.84 7 -88276 -4631.9 -83644 -648490 560214 69.86 -8.01 -0.41 -3.63 8 -99334 -4601.6 -94733 -733476 634141 3.26 -8.22 -0.45 -2.42 9 -110306 -4971.6 -105334 -827964 717658 -32.06 -8.05 -0.45 -4.36 10 -112313 -5773 -106539 -940854 828541 -17.24 -7.93 -0.48 -3.82 11 -108718 -5490 -103228 -889289 780571 -9.39 -8.03 -0.489 -5.25 12 -112312 -5772 -106539 -952370 840058 -16.28 -7.95 -0.46 -4.33 13 -124384 -6144 -118239 -1097703 973319 -34.41 -8.12 -0.65 -3.25 14 -137848 -6793 -131054 -1237984 1100137 -92.91 -8.19 -0.21 -6.53 15 -101712 -5379 -96332 -842684 740972 15.79 -8.21 -0.31 0.58 102 p h a r m a c y issn 2413-6077. ijmmr 2019 vol. 5 issue 2 of the studied compounds on the parameters of their molecular descriptors allowed deriving statistically qualitative qsar models (1-3) (r=0,825÷0,936) characterized by sufficient ade­ quacy (f=7.8÷26.006) and predictive ability (q2=0.5-0.799). conclusions thus, according to the study of the ‘structure-antihypertensive’ activity relationship for polysubstituted functionalized aminothiazoles, the qsar analysis revealed that the volume, area, lipophilicity, dipole moment, refractivity, polarization of the molecule and energy of the lowest unoccupied molecular orbital have the most significant effect on antihypertensive activity. the derived qsar-models are suggested for antihypertensive activity prediction within abovementioned row of compounds and can be a theoretical basis for de novo development of new potential antihypertensive drugs. funding this research received no external funding. conflict of interests the authors declare no conflict of interest. fig. 2. the dependence of the observed and predicted antihypertensive activities for the qsar models 1, 2, 3. 1 2 3 qsar-аналіз полізаміщених функціоналізованих амінотіазолів з гіпотензивною активністю і.в. драпак львівський національний медичний університет імені данила галицького, україна вступ. qsar-аналіз є важливим інструментом ідентифікації фармакофорних фрагментів в біологічно активних речовинах та дозволяє оптимізувати пошук нових ефективних ліків. і.v. drapak 103 p h a r m a c y issn 2413-6077. ijmmr 2019 vol. 5 issue 2 і.v. drapak мета дослідження. визначення молекулярних дескрипторів для qsar аналізу полізаміщених функціоналізованих амінотіазолів як теоретичної основи для цілеспрямованого пошуку нових антигіпертензивних засобів. методи дослідження. розрахунок молекулярних дескрипторів та побудова qsar-моделей проводились з використанням програмних пакетів: hyper-chem 7.5 та buildqsar. результати. для 15 нових полізаміщених функціоналізованих амінотіазолів із встановленою in vivo гіпотензивною активністю проведено розрахунок ряду молекулярних дескрипторів (електронних, стеричних, геометричних, енергетичних). на основі розрахованих молекулярних дескрипторів та параметра гіпотензивної активності були отримані qsar-моделі: ah = a + b ∙ x1 + c ∙ x2 + d ∙ x3, де ah гіпотензивна активність, а x1, x2, x3 молекулярні дескриптори. висновки. проведено дослідження зв'язку "структура – гіпотензивна активність" для ряду полізаміщених функціоналізованих амінотіазолів. за результатами qsar-аналізу встановлено, що об'єм, площа, ліпофільність, дипольний момент, рефрактивність, поляризованість молекули та енергія нижчої незайнятої молекулярної орбіталі мають найбільш значний вплив на гіпотензивну активність. одержані qsar-моделі будуть використані для прогнозування активності даного ряду сполук і можуть розглядатися як теоретична основа для de novo дизайну нових потенційних гіпотензивних препаратів. ключові слова: полізаміщені функціоналізовані амінотіазоли; гіпотензивна активність; молекулярні дескриптори; qsar-аналіз. інформація про автора драпак і.в. – кандидат фармацевтичних наук, доцент кафедри загальної, біонеорганічної та фізколоїдної хімії львівського національного медичного університету імені данила галицького information about the author drapak i.v. – phd, associate professor of the department of general, bioinorganic, physical and colloidal chemistry, danylo halytsky lviv national medical university, lviv, ukraine. orcid 0000­0002­6830­2908, e­mail: iradrapak@ukr.net references 1. cherkasov a, muratov en, fourches d, varnek a, baskin ii, cronin m, dearden j, gramatica p, martin yc, todeschini r, consonni v. qsar modeling: where have you been? where are you going to?. journal of medicinal chemistry. 2014 jan 6;57(12): 4977-5010. doi:10.1021/jm4004285 2. wang t, wu mb, lin jp, yang lr. quantitative structure – activity relationship: promising advances in drug discovery platforms. expert opinion on drug discovery. 2015 dec 2;10(12):1283­300. doi: 10.1517/17460441.2015.1083006 3. tetko iv, gasteiger j, todeschini r, mauri a, livingstone d, ertl p, palyulin va, radchenko ev, zefirov ns, makarenko as, tanchuk vy. virtual computational chemistry laboratory–design and description. journal of computer-aided molecular design. 2005 jun 1;19(6):453­63. doi: 10.1007/s10822­005­8694­y 4. giri rs, thaker hm, giordano t, williams j, rogers d, sudersanam v, vasu kk. design, synthesis and characterization of novel 2-(2, 4-disubstitutedthiazole-5-yl)-3-aryl-3h-quinazoline-4-one derivatives as inhibitors of nf­κb and ap­1 mediated transcription activation and as potential anti­inflammatory agents. european journal of medicinal chemistry. 2009 may 1;44(5):2184­9. doi: 10.1016/j.ejmech.2008.10.031 5. abdel-wahab bf, mohamed sf, amr ae, abdalla mm. synthesis and reactions of thiosemicarbazides, triazoles, and schiff bases as antihy pertensive α­blocking agents. monatshefte für chemie­ chemical monthly. 2008 sep 1;139(9):1083­90. doi: 10.1007/s00706­008­0896­2 6. drapak i, perekhoda l, demchenko n, suleiman m, rakhimova m, demchuk i, taran s, seredynska n, gerashchenko i. cardioprotective activity of some 2-arylimino-1, 3-thiazole derivatives. scientia pharmaceutica. 2019 mar;87(1):7­8. doi: 10.3390/scipharm87010007 7. perekhoda l, yeromina h, drapak i, kobzar n, smolskiy o, demchenko n. the antioxidant properties of 1-[2-(r-phenylimino)-4-methyl-3-(3-[morpholine4-yl] propyl)-2, 3-dihydro-1, 3-thiazol-5-yl] ethane-1one derivatives under conditions of artificial oxidative stress in vitro. saudi journal of medical and pharmaceutical sciences. 2017;3(1):55­9. doi: 10.21276/sjmps.2017.3.9 8. yeromina ho, drapak iv, perekhoda lo, yaremenko vd, demchenko am, perekhoda la. synthesis of 2­(4­aryl (adamantyl)­2­phenylі­ mіnothіazol­3­yl)­ethanol derivatives and prediction of their biological activity. der pharma chemica. 2016;8(3):64­70. 9. drapak iv, zimenkovsky bs, seredynska nm, demchenko am. 1-{3-[2-(3,4-dimethoxy-phenyl) ethyl]-4-methyl-1-2-phenylimino-2,3-dihydro-thiazol5-yl}-ethanone hydrochloride, which is antihypertensive action. no 19960 ua patent ua. 2019 august 27. 10. laxmi k. structural elucidation of drug aspirin by using various software tools like hyper104 p h a r m a c y issn 2413-6077. ijmmr 2019 vol. 5 issue 2 chem, argus lab, chemsketch, avogrado and chemeo database. j theor comput sci. 2019;5(163):2. 11. de oliveira db, gaudio ac. buildqsar: a new computer program for qsar analysis. quantitative structure­activity relationships: an international journal devoted to fundamental and practical aspects of electroanalysis. 2000 dec;19(6):599­601. doi: 10.1002/1521­3838(200012)19:6%3c599:: aid­qsar599%3e3.0.co;2­b 12. todeschini r, consonni v. handbook of molecular descriptors. 2000. ny, toronto: wiley­vch. doi: 10.1002/9783527613106 13. todeschini r, consonni v. molecular descriptors for chemoinformatics: volume i: a l p h a b e t i c a l l i s t i n g / v o l u m e i i : a p p e n d i c e s , references. john wiley & sons; 2009 oct 30. doi: 10.1002/9783527628766 received 05 september 2019; revised 29 october 2019; accepted 26 november 2019. this is open access article distributed under the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. і.v. drapak 58 b io m e d ic a l s c ie n c e s issn 2413-6077. ijmmr 2019 vol. 5 issue 1 doi 10.11603/ijmmr.2413-6077.2019.1.10306 pharmacokinetics of the new cerebropetector ferrum bis(citrato)germanate at the stage of its distribution to the organs in closed head injury v.d. lukianchuk1*, t.a. bukhtiarova1, i.i. seifullina2, e.m. polishchuk1, o.e. martsinko2, h.a. topolnytska1 1 – institute of pharmacology and toxicology of the national academy of medical sciences of ukraine, kyiv, ukraine 2 – odesa i.i. mechnikov national university, odesa, ukraine background. previous studies showed that new coordinate compound cerebrogerm (ferrum bis(citrato) germanate) is a promising cerebroprotector. objective of the study is a comparative analysis of the central stage of the pharmacokinetics of cerebrogerm as well as its distribution in vital organs in cases of closed head injury. methods. on the experimental original model of closed brain injury in rats the parameters of the central stage of cerebrogerm pharmacokinetics: the distribution in brain, liver, kidneys, were studied. results. it is established that, in cases of closed head injury cerebrogerm reaches maximum concentration first in the brain (in 3.75 h), then in the kidneys (in 3.92 h), and finally in the liver (in 4.17 h). in this case, the magnitude of the cmax of the coordinating compound of germanium that is being investigated in different biosubstrates of the rats with closed head injury may be presented in descending order as follows: brain (7.95 mg/l) > liver (6.22 mg/l) > kidneys (1.79 mg/l). conclusions. the compound cerebrogerm studied easily gets through the blood-brain barrier and meets the present requirements for cerebroprotectors and antihypoxants. the attained results allow noting that in the early post-traumatic period of closed head injury, the blood circulation in the kidneys does not change and cannot modify the absorption-elimination processes of xenobiotics. it has been also established that cerebrogerm is distributed faster in the examined organs in cases of closed head injury. the highest concentration of the drug is present in the brain and the smallest one – in the kidneys. key words: ferrum bis(citrato)germanat; closed head injury; pharmacokinetics; brain; liver; kidneys. *corresponding author: victor lukianchuk, md, ph.d., dsc, professor, institute of pharmacology and toxicology of nams of ukraine, 14 anton tsedik str., kyiv, 03680, ukraine e-mail: lvdlug@ukr.net phone number: +38(044)4569118 introduction closed head injury is characterized by a high and steadily increasing prevalence, which is especially marked during the period of hostilities. consequences of brain injury have significant medical and social components that are related to treatment of patients and their rehabilitation. the variety of clinical manifes_ tations and the severity of closed head injury conditioned the need to develop a strategy for scientific approaches to its pathogenetic treatment [1, 2, 3]. in the medical practice, a very large arsenal of pharmacological methods of correction of closed head injury effects is currently used. at the same time, not all of them meet the current *requirements for their effectiveness and s a f e t y . t h e r e f o r e , t h e s e a r c h f o r a n d development of new cerebroprotective drugs with multidimen sional pharmacodynamic effects and an accep table pharmacokinetic profile are the dilemma. in this regard, the original compounds of germanium with different bioligands based on coor dination bonds are particularly inte resting [4, 5, 6]. the creation of new drugs is impossible without determination of pharmacokinetic parameters, which is a mandatory step in their preclinical studies and a prerequisite for further study of a potential drug in clinical practice. it is established that the clarification of the pro cesses of getting drugs in the area of their phar macodynamic effects (including cerebro protective) has a great scientific and practical value in terms of developing a dosage regimen and optimizing rational pharmacotherapy in general. international journal of medicine and medical research 2019, volume 5, issue 1, p. 58-65 copyright © 2019, tnmu, all rights reserved v.d. lukianchuk et al. 59 b io m e d ic a l s c ie n c e s issn 2413-6077. ijmmr 2019 vol. 5 issue 1 according to the results of screening studies on the model of traumatic brain damage in the original coordinating compounds of germanium with citric acid, it has been proved that the most active one is the complex compound ferrum bis(citrato)germanate of a conventional name cerebrogerm [7]. the objective of the study was a comparative analysis of the central stage of the pharmacokinetics of ferrum bis(citrato)germanate, its distribution in vital organs in cases of normal as well as closed head injury. methods the experiments were performed on 62 white mature inbred 160-220 g rats reared in the animal facility of the institute of pharmacology and toxicology of the national academy of medical sciences of ukraine. all procedures were performed according to the ukrainian legislation, the european convention on the protection of vertebrate animals used in experimental research and for other scientific purposes (1986) and local ethics committee. the animals were on a standard diet and received food and water ad libitum. the experimental model of the closed head injury is an acute pathological process that developed in rats kept in a fixed modified chamber after a single application of a forcemetered and localization-oriented free-falling 45 g-load from the height of 80 cm onto the parietal skull [8]. pharmacokinetic studies were conducted in accordance with the guidelines [9, 10] by determining the concentrations of cerebrogerm by germanium in pathology-free tissues of rats and in those that received the studied coordinating compound for pharmacological correction of pathological changes occurring in the early post-traumatic period in cases of closed head injury. cerebrogerm was determined in blood serum and in vital organs: cerebral cortex, liver and kidneys by the method [11]. sampling of biomaterials in order to determine the pharmacokinetic profile of cerebogerm was performed in dynamics: in 0.5; 2; 6; 12 and 24 hours from the time of single intraperitoneal rats injection of ferrum bis(citrato)germanate according to the previously established dosage regimen (126 mg/kg 70 min after closed head injury) [12]. for a maximum correct evaluation of the pharmacokinetics of a potential cerebroprotector, a number of parameters was defined. these parameters characterizing the process of distribution of a potential cerebroprotector in the body of a two-chamber kinetic model with absorption in health animals and with closed head injury comprise: time of reaching the maximum concentration (tmax, hours), maximum concentration (cmax, mg/l), period of half-distribution to the organ (t1/2,a, hours), constant of the speed of direct mass transfer (k12, h -1), area under the pharmacokinetic curve (auc, mg/l·min), period of the reverse mass transfer (t1/2, hours), constant of the speed of reverse mass transfer (k21, h -1), average time in the organ (mrt, hours). the attained results were statistically processed on a personal computer using the student’s t-distribution criteria and mathematica v.5.0. differences were considered statistically significant at p<0.05. the statistical analysis was conducted at a 95% confidence level. results the results of the pharmacokinetic study which characterize the process of distribution in organs are presented in fig. 1 and 2 in the form of kinetic curves that indicate cerebrogerm distribution into the brain, liver and kidneys. pharmacokinetic parameters that characterize the process of distribution of cerebrogerm in vital organs of healthy animals (control group) and in those with closed head injury are presented in table 1. it is established that, in cases of closed head injury cerebrogerm reaches maximum concentration first in the brain (in 3.75 h), then in the kidneys (in 3.92 h), and finally in the liver (in 4.17 h). in this case, the magnitude of the cmax of the coordinating compound of germanium that is being investigated in different biosubstrates of the rats with closed head injury may be presented in descending order as follows: brain (7.95 mg/l)> liver (6.22 mg/l)> kidneys (1.79 mg/l). at the same time, cerebrogerm is identified in maximum concentration in the brain of healthy animals in 5.26 h, in the kidneys in 5.10 h and in the liver only in 6.20 h, with the presumable difference from the experimental group in relation to tmax in all biosubstrates. simultaneously, under normal conditions the organs of rats can be placed according to the size of the cmax of cerebrogerm in descending order as follows: brain (7.09 mg/l)> liver (5.71 mg/l)> kidneys (1.68 mg/l). the distribution (its intensity and com pleteness) of cerebrogerm to the peripheral v.d. lukianchuk et al. 60 b io m e d ic a l s c ie n c e s issn 2413-6077. ijmmr 2019 vol. 5 issue 1 fig. 1. pharmacokinetic curves showing the concentration of cerebrogerm in the brain of rats of the control group as well as in cases of closed head injury after a single intravenous injection at the dose of 126 mg/kg. fig. 2. pharmacokinetic curves of the concentration of cerebrogerm in the kidneys and liver of the control group rats as well as in those with closed head injury after a single intravenous injection at the dose of 126 mg/kg. v.d. lukianchuk et al. 61 b io m e d ic a l s c ie n c e s issn 2413-6077. ijmmr 2019 vol. 5 issue 1 ta bl e 1. p h ar m ac ok in et ic p ar am et er s of c er eb ro ge rm in t h e pr oc es s of it s di st ri bu ti on t o th e pe ri ph er al c h am be rs of t h e ki n et ic m od el o f h ea lt h y ra ts ( th e co n tr ol g ro u p) a n d th os e w it h c lo se d h ea d in ju ry a ft er a si n gl e in tr av en ou s in je ct io n a t th e do se o f 12 6 m g/ kg ( n = 6 ). ph ar m ac ok in et ic p ar am et er g ro u ps o f an im al s b ra in li ve r k id n ey s co n tr ol g ro u p cl os ed h ea d in ju ry co n tr ol g ro u p cl os ed h ea d in ju ry co n tr ol g ro u p cl os ed h ea d in ju ry ti m e of r ea ch in g m ax im um c on ce nt ra tio n (t m ax , y ea r) 5. 26 ±0 .3 2 3. 75 ±0 .2 7* 6. 20 ±0 .4 7 4. 17 ±0 .1 2* * 5. 10 ±0 .1 1 3. 92 ±1 .3 4* m ax im um c on ce nt ra tio n (c m ax , m g/ l) 7. 09 ±0 .1 5 7. 95 ±0 .1 0* * 5. 71 ±0 .1 7 6. 22 ±0 .1 4 1. 68 ±0 .0 5 1. 79 ±0 .0 2 pe ri od o f h al fdi st ri bu tio n to th e or ga n, (t 1/ 2, a , h ) 1. 99 ±0 .0 5 1. 08 ±0 .0 7* ** 1. 59 ±0 .0 6 1. 25 ±0 .0 2* * 4. 79 ±0 .0 1 2. 68 ±0 .0 4* ** co ns ta nt o f t he s pe ed o f d ir ec t m as s tr an sf er , ( k 1 2, h1 ) 0. 35 ±0 .0 1 0. 64 ±0 .0 01 ** * 0. 44 ±0 .0 01 0. 55 ±0 .0 01 ** * 0. 14 ±0 .0 01 0. 26 ±0 .0 01 ** * ar ea u nd er th e ph ar m ac ok in et ic cu rv e (a u c, m g/ l· m in ) 42 43 67 .3 0 ± 27 44 .0 2 51 12 45 .7 0 ± 16 82 .7 7* ** 32 21 45 .4 0 ± 15 67 .3 7 22 43 94 .9 0 ± 78 4. 12 ** * 13 70 29 .2 0 ± 76 8. 50 12 71 40 .0 ± 22 4. 17 ** * pe ri od o f r ev er se m as s tr an sf er , (t 1/ 2, h) 2. 05 ±0 .0 4 3. 07 ±0 .1 6* ** 1. 79 ±0 .0 9 1. 24 ±0 .0 7* * 2. 37 ±0 .2 2 2. 45 ±0 .0 8 co ns ta nt o f t he s pe ed o f r ev er se m as s tr an sf er , (k 21 , h -1 ) 0. 34 ±0 .0 1 0. 23 ** *± 0. 01 0. 39 ±0 .0 1 0. 56 ±0 .0 2* ** 0. 29 ±0 .0 1 0. 28 ±0 .0 2 av er ag e tim e in th e or ga n (m rt , h ) 24 .7 ±0 .7 3 24 .5 ±2 .1 7 30 .1 ±1 .9 7 21 .7 ±1 .4 6* 31 .9 ±2 .7 2 30 .7 ±1 .4 7 n ot es : * – p < 0. 05 c om pa re to th e he al th y an im al s; * * – p <0 .0 1 co m pa re to th e he al th y an im al s; * ** – p < 0. 00 1 co m pa re to th e he al th y an im al s. v.d. lukianchuk et al. 62 b io m e d ic a l s c ie n c e s issn 2413-6077. ijmmr 2019 vol. 5 issue 1 chamber is present in the half-distribution period (t1/2, a, h) from the central chamber to the studied organs and the constant of the speed of direct mass transfer (k12). it has been established that in cases of closed head injury (research), the coordinating compound of germanium reaches the brain the most fast, the liver slightly slower, and the kidneys the slowest, as evidenced by the parameters of t1/2, a, which denotes the time of reaching the levels of the compound which is equal to 50% of equilibrium concentrations between blood and other tissues. taking into account that the parameter k12 defines the relation k12 = 0.693/t1/2,a, that is the parameter is inversely proportional to the halfdistribution period in the organs, then the revealed changes of k12 also confirm the abovedescribed character of the distribution of the compound in the organs. it is established that cerebrogerm in both groups of animals equally enters the kidneys slowly, and the brain – the most intensively. such a parameter as the area under the pharmacokinetic curve (auc) is particularly informative. it is proportional to the amount of the compound present in blood. it was established that under normal conditions the highest concentration of the drug was revealed in the brain and the smallest – in the kidneys. the following sequence follows the auc indices: brain > liver > kidneys (see table 1). a comparative estimation of the auc of pathology-free animals and those of the experimental group indicates a significant difference between the concentrations of cerebrogerm in liver. it is established that the potential cerebroprotector is revealed in the liver under normal conditions by 43.6% more than in cases of closed head injury. it should be noted that cerebrogerm enters the brain (t1/2, a) very fast, but eliminates (t1/2) slowly in both compared groups of animals. it is significant that cerebrogerm is almost identically eliminated from the kidneys of the rats with or without traumatic brain damage (p> 0.05). similarly, the organs are classified according to the constant of the speed of reverse mass transfer. as presented in table 1, cerebrogerm eliminates from the liver of the animals of the experimental group the fastest and from the brain the slowest. among the pharmacokinetic parameters studied during the preclinical stage, the average time the compound stays in the body has high informational value since the implementation of the pharmacological effect of a drug directly depends on its time in the whole body as well as in a particular organ or tissue. the results presented in table 1 evidence that in the pathology-free animals the studied organs are classified according to the mrt decrease as follows: kidneys (31.9 h)> liver (30.1 h)> brain (24.7 h). in cases of traumatic brain damage, cerebrogerm is eliminated by 27.9% faster (21.7 hours) from the liver with no significant differences in mrt of the brain and kidneys (p <0.05). discussion the attained data, which characterizes the speed of cerebrogerm being delivered from blood to various animal organs (see table 1), indicates that coordination compound of germanium enters the peripheral chambers of the biokinetic model in different speed and amount both under normal and closed head injury conditions. it is worth noting that cerebrogerm passes through the histo-hematic barriers of the studied organs in different speed. the defined pharmacokinetic parameters of cerebrogerm, characterizing the process of getting from the peripheral chambers of the kinetic model into the blood under normal conditions as well as in cases of closed head injury, require more detailed analysis and discussion. particular attention should be paid to the period of reverse mass transfer (t1/2), that is the time the compound returns to the central circulation of the peripheral chamber (organ). according to this indicator, the pathology-free organs of the animals take the order of: liver > kidneys > brain. the revealed character of the distribution of cerebrogerm in cases of closed head injury indicates, in our opinion, the direction of its action in terms of cerebroprotective effect. under normal conditions in the animals cerebrogerm is first rewealed in liver, and then in the brain and kidneys. in this case, a 32.4% (p <0.001) decrease was defined in the rate of the reverse mass transfer constant of cerebrogerm from the brain into the blood in cases of closed head injury caused by post-traumatic exudation of the brain tissue which was subjected to mechanical damage. consequently, cerebral edema which occurs in cases of closed head injury makes the coordination of germanium stay in the brain longer that eventually causes a decrease in the v.d. lukianchuk et al. 63 b io m e d ic a l s c ie n c e s issn 2413-6077. ijmmr 2019 vol. 5 issue 1 speed of cerebrogerm transition from the brain into the bloodstream. this data reaffirms our hypothesis about cerebral edema, which impedes the transition of сerebrogerm from the brain into the blood, as evidenced by (p<0.001) increase of the auc indices by 20.5% in cases of closed head injury compare to normal conditions. this experimentally proved fact can indirectly show the detoxification function of the liver being saved in terms of its direct involvement in the processes of elimination of cerebrogerm as a xenobiotic. thus, it should be noted that in the early post-traumatic period of closed head injury, blood circulation in kidneys does not change to the extent that might substantially modify the absorption-elimination processes of xenobiotics. this is quite appropriate in view of the fact that kidneys have rich blood supply. thus, the results of the complex preclinical studies prove that the defined pharmacokinetic profile of the distribution of cerebrogerm in cases of closed mechanical brain trauma differs from that under the normal conditions. it has been experimentally proved that cerebrogerm enters the examined organs in cases of closed head injury faster than it does without it. it should be noted that the highest concentration of the drug, as evidenced by the area under the pharmacokinetic curve, occurs in the brain of the animals with closed head injury and the smallest – in the kidneys. this should be considered as a key pharmaco kinetic parameter that clearly proves the ability of cerebrogerm to get through the blood-brain barrier. the attained data also proves that the coordination compound of germanium corresponds precisely to the current requirements for cerebroprotectors and antihypoxants. this kind of drugs is capable to implement a number of pharmacodynamic effects through the accumulation in the brain, as in the central biophase. these effects aim at organ protection, mainly the protection of cerebral neurons. conclusions determination of the pharmacokinetic parameters of the potential сerebroprotector ferrum bis(citrato)germanate at the stage of its distribution in the body in cases of traumatic b r a i n d a m a g e i n d i c a t e s a s i g n i f i c a n t modification of the kinetic profile of the compound compared to the normal conditions. a number of parameters that characterize the process of cerebrogerm’s distribution from the central chamber to the peripheral (t1/2, a, k12, k21, auc) substantially (p<0.05 0.001) undergoes a ‘traumatic’ correction. according to the parameters characterizing the pharmacokinetic spectrum of the distribution of cerebrogerm into organs, it can be represented in the following order: brain > liver > kidneys. the studied compound cerebrogerm easily gets through the blood-brain barrier and meets the present requirements for cerebroprotectors and antihypoxants. the attained results allow noting that in the early post-traumatic period of closed head injury, the blood circulation in the kidneys does not change and cannot modify the absorption-elimination processes of xenobiotics. it has been also established that cerebrogerm is distributed faster in the examined organs in cases of closed head injury. the highest concentration of the drug is present in the brain and the smallest one – in the kidneys. conflict of interest the authors declare no conflict of interest. фармакокінетика нового церебропротектора ферум біс(цитрато) германату на етапі його розподілу в органи на тлі закритої черепно-мозкової травми в.д. лук’янчук1, т.а. бухтіарова1, і.й. сейфулліна2, є.м. поліщук1, о.е. марцинко2, г.а. топольницька1 1 – ду “інститут фармакології та токсикології намн україни”, київ, україна 2 – одеський національний університет імені і.і. мечникова, одеса, україна вступ. попередні скринінгові фармакота токсикометричні дослідження нової координаційної сполуки цереброгерм (ферум біс(цитрато)германат) показали, що це перспективний потенційний засіб церебропротекції. v.d. lukianchuk et al. 64 b io m e d ic a l s c ie n c e s issn 2413-6077. ijmmr 2019 vol. 5 issue 1 мета дослідження провести порівняльний аналіз центрального етапу фармакокінетики цереброгерму – розподілу у життєвоважливі органи: головний мозок, печінка, нирки, в нормі та при зчмт. матеріали. на оригінальній моделі зчмт визначали низку фармакокінетичних параметрів ферум біс(цитрато)германату що всебічно характеризують процес розподілу в периферичні камери моделі в нормі та за умов зчмт. результати. показано, що при зчмт максимум концентрації цереброгерму досягається спочатку у мозку (після 3,75 год), потім у нирках (після 3,92 год) та у печінці (після 4,17 год). в той же час, за величиною сmax цереброгерму органи щурів контрольної групи можна розташувати у порядку зменшення наступним чином: головний мозок (7,09 мг/л) > печінка (5,71 мг/л)> нирки (1,68 мг/л). величина сmax координаційної сполуки германію у щурів із зчмт виглядає так: головний мозок (7,95 мг/л) > печінка (6,22 мг/л)> нирки (1,79 мг/л). висновки. отримані дані дозволяють дійти висновку, що досліджувана сполука доволі легко проникає через гематоенцефалічний бар'єр і відповідає сучасним вимогам до церебропротекторів та антигіпоксантів. отримані результати дозволяють зазначити, що у ранньому постравматичному періоді зчмт стан кровообігу у нирках не змінюється у такій мірі, щоби суттєво модифікувати абсорбційно-елімінаційні процеси ксенобіотика. доведено також, що за умов зчмт цереброгерм надходить до органів, що досліджувались, більш швидко, ніж у здорових щурів. найбільша концентрація препарату, про яку судили за показником площі під фармакокінетичною кривою, знаходиться у головному мозку, а найменша в нирках травмованих тварин. ключові слова: ферум біс(цитрато)германат; закрита черепно-мозкова травма; фармакокінетика; головний мозок; печінка; нирки. інформація про авторів лук’янчук віктор дмитрович – д-р мед. наук, професор, головний науковий співробітник відділу медичної хімії, ду “інститут фармакології та токсикології намн україни”, м. київ, україна. бухтіарова тетяна анатоліївна – член-кореспондент намн україни, д-р мед. наук, директор ду “інститут фармакології та токсикології намн україни”, м. київ, україна. сейфулліна інна йосипівна – д-р хімічних наук, професор, завідувач кафедрою загальної хімії та полімерів одеського національного університету імені і.і. мечнікова, м. одеса, україна. поліщук євген миколайович – канд. мед. наук, пошукувач ду “інститут фармакології та токсикології намн україни”, м. київ, україна. марцинко олена едуардівна – д-р хімічних наук, професор кафедри загальної хімії та полімерів одеського національного університету імені і.і. мечнікова, м. одеса, україна. топольницька ганна андріївна – пошукувач ду “інститут фармакології та токсикології намн україни”, м. київ, україна. information about authors victor lukianchuk – md, ph.d., dsc, professor, institute of pharmacology and toxicology of nams of ukraine, kyiv, 03680, ukraine. orcid 0000-0002-7734-4739, e-mail: lvdlug@ukr.net tetiana a. bukhtiarova – md, ph.d., dsc, corresponding member of nams of ukraine, institute of pharmacology and toxicology of nams of ukraine, kyiv, 03680, ukraine. e-mail: bukhtiarova@yahoo.com inna i. seifullina – ph.d., dsc, professor, head of the department of general chemistry and polymers, odesa i.i. mechnikov national university, odesa, ukraine. evgen m. polishchuk – md, ph.d., institute of pharmacology and toxicology of nams of ukraine, kyiv, 03680, ukraine. e-mail: polishchyk@ukr.net olena e. martsinko – ph.d., dsc, professor, department of general chemistry and polymers, odesa i.i. mechnikov national university, odesa, ukraine. e-mail: lborn@ukr.net hanna a. topolnytska – institute of pharmacology and toxicology of nams of ukraine, kyiv, 03680, ukraine. v.d. lukianchuk et al. 65 b io m e d ic a l s c ie n c e s issn 2413-6077. ijmmr 2019 vol. 5 issue 1 references 1. shevchuk ov. experimental ground for α-lipoic acid use in closed head injury. [autoabstract]. kyiv: institute of pharmacology and toxicology of nams of ukraine; 2007. 21 p. [in ukrainian] 2. sadovnik ov. the efficacy of corvitin in closed head injury. [autoabstract]. kharkiv: national pharmaceutical university; 2011. 20 p. [in ukrainian] 3. fedorova vs. the efficacy of acetylcysteine in closed head injury. [autoabstract]. kharkiv: national pharmaceutical university; 2012. 18 p. [in ukrainian] 4. vysotska lv. the search of cerebroprotectors among the new compounds of germanium with bioligands. [autoabstract]. kharkiv: national pharmaceutical university; 2009. 20 p. [in ukrainian] 5. rysukhina nv. the search of means to correct endotoxicities of posttraumatic origin [autoabstract]. kyiv: institute of pharmacology and toxicology of nams of ukraine; 2010. 20 p. [in ukrainian] 6. lukianchuk vd, nizhenkovsky oi, fedorova vs, kravets ds, seifullina ii, martsinko oe, pesaroglo og. screening analysis of coordination compounds of germanium in model of closed head injury. pharmacology and drug toxicology. 2012;6:63–8 [in ukrainian]. 7. lukianchuk vd, polishchuk em, seifullina ii, rysukhina nv, martsinko oe, chebanenko oa. comparative estimation of cerebroprotective activity of newly synthesized coordination compounds of germanium in the model of closed head injury. pharmacology and drug toxicology. 2014;38(2):36-43 [in ukrainian]. 8. lukianchuk vd, shevchuk ov, badinov ov, assignee. the animal model of closed head injury; luhansk state medical university. ukraine patent ua 13678. 2006 april 17. 8 p. [in ukrainian] 9. kravets ds. optimization of calculation of parameters of chemobiokinetics using the eom software. ukrainian medical almanakh. 2000; 2:90-1 [in russian]. 10. golovenko nya, lukianchuk vd, zhuk ov, zinkovsky vg, kravets ds, zhuk ms. methodical recommendations in computer calculation of pharmacokinetics parameters of drugs. kyiv: state scientific-expertise center; 1999. 70 p. 11. kresiun vi, vidavska ag, shemonaeva kf. extraction-fotometric evaluation of micro amounts of germanium in tissues of experimental animals. odesa medical journal. 2000;62(6):7-11. 12. polishchuk en, kravets ds. experimental mathematical analysis of dose regimen of potential cerebroprotector cerebrogerm. medical journal of western kazakhstan. 2014; 44(4): 26-31 [in russian]. received 22 february 2019; revised 22 march 2019; accepted 25 april 2019. this is an open access article distributed under the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. v.d. lukianchuk et al. 35 in t e r n a l m e d ic in e issn 2413-6077. ijmmr 2021 vol. 7 issue 1 doi 10.11603/ijmmr.2413-6077.2021.1.12251 main indicators of the oxidant-antioxidant system and their relationship with the force of the respiratory muscles in adult patients with community-acquired pneumonia *v.i. bereznyakov kharkiv medical academy of postgraduate education, kharkiv, ukraine background. currently, pneumonia is the 4th-5th in the world in the structure of death causes after cardiovascular and cancer diseases, cerebrovascular pathology, injuries and poisonings. objective. the aim of the study was to evaluate the indicators of the oxidative-antioxidant system and their relationship with the strength of the respiratory muscles in adult patients with community-acquired pneumonia. methods. the study was carried out in the period 2017-2020 at the therapeutic department of the municipal non-profit enterprise “city clinical multidisciplinary hospital no. 25” of kharkiv city council. the study involved 52 adult patients with community-acquired pneumonia (cap) aged 18 to 80 years old. the control group consisted of 20 apparently healthy humans. the activity of malondialdehyde, catalase and superoxide dismutase, level of reduced glutathione, glutathione reductase and glutathione peroxidase were determined. the assessment of the rm strength was investigated by recording the maximum static pressures at the level of the mouth and nose with “closed” airways using a microrpm apparatus on the 1st and 10th days of illness. results. dysfunction of expiratory respiratory muscles prevailed in patients with non-severe cap, and inspiratory respiratory muscles – in the patients with severe cap. significant negative correlations of malondialdehyde with indicators of respiratory muscles strength and positive correlations with glutathione reductase, glutathione peroxidase, catalase, and superoxide dismutase were established. conclusions. the relations between prooxidant and antioxidant indicators and respiratory muscles strength complements the concept of the body systemic response on pulmonary inflammation – one of the markers of respiratory muscles dysfunction. keywords: community-acquired pneumonia; respiratory muscles; oxidative-antioxidant system. *corresponding author: vladyslav bereznyakov, phd, associate professor, kharkiv medical academy of postgraduate education, kharkiv, 61037 ukraine. email: vladyslavbereznyakov@gmail.com international journal of medicine and medical research 2021, volume 7, issue 1, p. 35-41 copyright © 2021, tnmu, all rights reserved v.i. bereznyakov introduction community-acquired pneumonia (cap) is s t i l l o n e o f t h e m o s t u r g e n t i s s u e s o f contemporary medicine. cap leads to a steadily high and growing morbidity. according to the who data in recent years, lower respiratory tract infections have become the third leading cause of death in the world, and over the past 30 years, the incidence rate has risen [1]. the mortality rate in hospitalized patients with a severe form of the disease varies from 14 to 40 % and significantly increases among patients over 60 years old [2]. according to the literature, the number of disease forms with severe or protracted course is increasing with frequent complications of pneumonia such as abscess formation, acute respiratory failure, infectious toxic shock, etc. [3, 4, 5]. in each 3-4 patients with cap, the disease has a protracted course [1, 6, 7]. that is why study continues for clarifying individual relations in the pathogenesis of cap, improving programs for its diagnosis and therapy. one of the leading factors in the pathogenesis of cap is the excessive production of reactive oxygen intermediates (roi), which is associated with bacterial or viral-bacterial aggression and insufficiency of the compensatory potential of antioxidant defence (aod). an imbalance in the oxidative-antioxidant system is one of the key factors in the development of oxidative stress (os), which plays an important role in the implementation of the molecular-cellular mechanisms of the pathogenesis of respiratory diseases [8, 9]. free radical processes are typical general biological protective reactions of the body, normally providing energy metabolism, proliferation, dif ferentiation of cells, gene expression, immune and adaptive responses, etc. [10]. at the 36 in t e r n a l m e d ic in e issn 2413-6077. ijmmr 2021 vol. 7 issue 1 same time, excessive roi initiates lipid peroxidation (lpo) with subsequent damage of cellular membranes, uncoupling oxidative phosphorylation, formation of energy in sufficiency, disturbance of enzymatic activity of the detoxification systems [11, 12, 13]. some studies have established that os is the most important component of endogenous intoxication – an obligate manifestation of cap, which has a significant effect on its clinical course. in the pathogenesis of cap the glutathione redox system (enzymes form thioredoxin and glutaredoxin-dependent complexes) play a special role that maintain intracellular homeostasis, which resists the destructive effects of oxidative stress factors [12]. it has been proven that low glutathione intracellular concentration contributes to imbalance between prooxidants and antioxidants in the lungs, aggravation of inflammatory reactions and complications development. also, one of the most important issues in the pathogenesis of cap, is dysfunction of the respiratory muscles (rm). formation of rm dysfunction at cap is characterized by the effect of local (alveolar inflammation) and systemic (endogenous intoxication) factors on muscle contraction [2, 12]. it is assumed that these changes are caused by the destabilization of cell membranes, decrease in the rate of myopotential conduction and violation of the potassium and calcium intracellular transport, which provide muscle contraction [11, 13]. at the same time, there is no data in the scientific literature about influence of free radical processes on the functional status of rm in patients with cap. the aim of the study was to evaluate the indicators of the oxidative-antioxidant system and their relationship with the strength of the respiratory muscles in adult patients with community-acquired pneumonia. methods the study involved 52 patients with cap aged 20-60 years (mean age 36.5±10.3), who were treated in the therapeutic department of the municipal non-profit enterprise “city clinical multidisciplinary hospital no. 25” of kharkiv city council in 2017-2020. diagnosis of cap was based on the results of clinical, radiological, microbiological and laboratory studies. non-severe cap (ncap) was diagnosed in 56 (72%) patients, severe cap (scap) – in 22 (28%). unilateral subsegmental inflammatory infiltration of the lung tissue was recorded in patients with ncap, and scap was characterized by the presence in one or both lungs of multisegmental, lobar, or bilobar infiltrates. the etiological structure of cap is mainly represented by streptococcus pneumonia – 57.4%, haemophilus influenza – 23.4%, myсoplasma pneumonia – 13.4%, chlamydophila pneumonia – 5.8%. the patients were treated according to the recommendations of the international society of pulmonologists and the f.h. yanovskyi national institute of phthisiology and pulmonology (kyiv, 2019). the average hospitalization period was 10.80±0.67 days. all the patients signed informed consent. the control group involved 45 apparently healthy humans (ahh) of the same age (mean age 39.5±12.5). the intensification of lpo processes was determined by the level of the final product – malondialdehyde (mda) in erythrocytes [14]. the total antioxidant activity (aoa) was assessed by the integral index in blood plasma. the intensity of the first line of aod was investigated by the activity of the enzymes catalase and superoxide dismutase (sod). catalase activity was determined by the rate of utilization of hydrogen peroxide in the reaction mixture [15], and sod in erythrocytes – by the ability to suppress the reduction of nitro blue tetrazolium. the state of the redox system was studied by the level of reduced glutathione (rg), the activity of glutathione reductase (gr), and glutathione peroxidase (gpo) in whole blood [16]. the assessment of the rm strength was carried out by recording the maximum static pressures at the level of the mouth and nose with “closed” airways using a microrpm apparatus (carefusion, great britain) on the 1st and 10th days of illness. the maximum inspiratory pressure (mip), maximum expiratory pressure (mep) and sniff nasal inspiratory pressure (snip) were evaluated. mip and snip characterized the strength of the inspiratory muscles, and mep – the expiratory muscles. the correlation of snip with the level of transhiatal pressure can refer it to the indicators of the functional activity of the diaphragm. the maximum rate of expiratory and inspiratory pressures rises in the oral cavity (maximal rate of pressure development – mrpdexp and mrpdinsp) was assessed using the additional software puma (micro medical, uk). the rm strength was assessed in the sitting position after 3-fold execution of respiratory movements with the fixation of maximum result. the v.i. bereznyakov 37 in t e r n a l m e d ic in e issn 2413-6077. ijmmr 2021 vol. 7 issue 1 required values (rv) for mep, mip, snip were calculated using a previously developed model [17]. to analyse the statistical significance of differences between the groups, statistical processing of the research results was carried out depending on the distribution origin of the data as follows: if the distribution was close to normal, the analysis was performed using the methods of variation statistics, the statistica 8.0 software package – the statistical two-way anova method (fisher lcd post-hoc test). if it was significantly different from normal, the differences between the groups were determined using the kruskal-wallis anova and median test method. correlation analysis was carried out in the same statistica 8.0 package by means of parametric and nonparametric methods depending on the type of distribution. the statistical significance of the differences between the indicators of the control and experimental groups was determined by the student’s and kruskal-wallis’s criteria using the excel program. the level of p<0.05 was statistically significant [18]. results group 1 involved 36 (69.2%) patients with ncap. their metabolic profile was characterized by minimal violations of the oxidative-antioxidant balance. the patients of this group showed slight decrease of rg and moderate increase of redox system rg-gpo and gr enzymes at normal values of sod and catalase (table 1). the analysed indicators showed adequate response to antioxidant protection, which ensured the neutralization of roi and lpo products, which was confirmed by normal mda values in erythrocytes. among these patients, the absolute values of the strength indices of expiratory rm were significantly lower in the group of ahh (mep – on 54.6%), and the limitation of their inspiratory function was less evident (mip – on 17%, snip – on 7%) (fig. 1, 2). in this case, the ratio of measured and rm was 78% for mep and 88% for mip and snip, which indicated a predominantly expiratory variant of rm dysfunction. group 2 involved 16 (30.8%) patients with scap, whose indicators were characterized by the most significant imbalance, as evidenced by significant increase of mda level (by 29%) against the background of decrease in aoa (by 18%) and catalase (by 59%). the inhibition of the rg redox system was expressed by decrease in the concentration of rg in the blood (by 48%) and decrease in gpo (in 1.7 times compare to the ahh). at the same time, compensatory increase in the content of gr (by 11%) and sod (by 45%) did not ensure the restoration of oxidative-antioxidant homeostasis. the study of the rm functional state in the patients of group 2 showed that with increase of the cap severity and imbalance in the oxidative-antioxidant system, the degree of their strength signs deviation from the level of ahh significantly increased and reached the maximum level. in this case, the indicators of mep and mrpdexp in relation to the ahh group decreased in 2.0 times, snip – in 1.5, and mip – in 1.75 times, which indicated about development of pronounced expiratory-inspiratory type rm dysfunction. the obtained results confirmed that the power of inspiratory rm was more evidence limited in scap with a significant weakening of functional activity of the diaphragm. the analysis of paired correlations showed that at the height of disease, there were multidirectional relations of varying intensity between the indicators of rm strength and table 1. oxidative-antioxidant indicators of patients with cap in various groups, ме (95% ci) indicators apparently healthy humans (n=45) patients with cap (n=52) group 1 group 2 mda, μmol/1ghb 7 [6,9; 71,0] 7,3 [7,1; 7,6] 9 [8,9; 9,2]* аоа, % 70,3 [68; 72,0] 68,6 [66,2; 70,1] 59,4 [57,5; 61,8]* мdа/аоа, c.u. 0,14 [0,13; 0,16] 0,13 [0,11; 0,15] 0,16 [0,15; 0,17]* rg, mol/1ghb 6,7 [6,4; 7,1] 5,8 [5,6; 6,0] 4,5 [4,2; 4,8]* gpo, μmol rg/1ghb/hour 128 [127; 133] 149 [142; 155]* 74 [71,5; 76]* gr, μmol nadph/1ghb/hour 154,3 [153,5; 156] 164 [158; 169]* 171,3 [160; 181]* catalase, % 80 [75; 84] 77 [73; 81] 51,2 [48,6; 54]* sod, % 54 [51; 58] 57,3 [56; 59] 78,3 [77; 80]* notes: * – р<0,05 statistically significant difference compare to the apparently healthy humans. v.i. bereznyakov 38 in t e r n a l m e d ic in e issn 2413-6077. ijmmr 2021 vol. 7 issue 1 some indicators of the oxidative-antioxidant status. thus, negative correlations of the average strength of mda with mep (r=-0.56), mda with mrpdexp (r=-0.53), mda with snip (r=-0.61) and mda with mip (r=-0.58) were evidenced. direct correlations were found between the indicators mip with gpo (r=0.76), snip with catalase (r=0.65), sod with snip (r=0.51), gr with mep (r=0.48). the obtained result indicates about the heterogeneity of the effect of prooxidant and antioxidant factors on the functional status of rm and confirm their role in the development of dm dysfunction. on the 10th day, the patients of the group 1 showed tendency to restoration of the strength indicators to the level of the ahh. at the same time, only mip, snip, and mrpdinsp indicators reached the values of ahh group, while mep and mrpdexp significantly differed, which indicated persisting signs of isolated expiratory rm dysfunction. in the patients of the group 2 on the 10th day of studying, the medians of all indicators of the rm strength were minimal compare to other groups. discussion analysis of the scientific literature showed that the role of dm in the pathogenesis of cap is not fully understood. it was noted, that decrease in the peak expiratory flow rate at the height of cap is associated not with increase of bronchial resistance to expiratory air flow, but with dm dysfunction. in the works of geltser b.i. et al. (2019) the pathophysiological significance of endogenous intoxication in the development of dm dysfunction in alveolar inflammation has been proven [12]. its development in this case is associated with the effect of systemic factors of acute inflammation on dm, including profig. 1. indicators of rm strength in patients with community-acquired pneumonia in various groups, ме 95% ci) notes: * – р<0.05 statistically significant difference compare to the apparently healthy humans. fig. 2. indicators of rm strength in patients with community-acquired pneumonia in various groups, ме (95% ci) notes: * – р<0.05 statistically significant difference compare to the apparently healthy humans. deviation from the level of ahh significantly increased and reached the maximum level. in this case, the indicators of mep and mrpdexp in relation to the ahh group decreased in 2.0 times, snip – in 1.5, and mip – in 1.75 times, which indicated about development of pronounced expiratory-inspiratory type rm dysfunction. the obtained results confirmed that the power of inspiratory rm was more evidence limited in scap with a significant weakening of functional activity of the diaphragm. fig. 1. indicators of rm strength in patients with community-acquired pneumonia in various groups, ме 95% ci) notes: * р<0.05 statistically significant difference compare to the apparently healthy humans. fig. 2. indicators of rm strength in patients with community-acquired pneumonia in various groups, ме (95% ci) notes: * р<0.05 statistically significant difference compare to the apparently healthy humans. the analysis of paired correlations showed that at the height of disease, there were multidirectional relations of varying intensity between the indicators of rm strength and some deviation from the level of ahh significantly increased and reached the maximum level. in this case, the indicators of mep and mrpdexp in relation to the ahh group decreased in 2.0 times, snip – in 1.5, and mip – in 1.75 times, which indicated about development of pronounced expiratory-inspiratory type rm dysfunction. the obtained results confirmed that the power of inspiratory rm was more evidence limited in scap with a significant weakening of functional activity of the diaphragm. fig. 1. indicators of rm strength in patients with community-acquired pneumonia in various groups, ме 95% ci) notes: * р<0.05 statistically significant difference compare to the apparently healthy humans. fig. 2. indicators of rm strength in patients with community-acquired pneumonia in various groups, ме (95% ci) notes: * р<0.05 statistically significant difference compare to the apparently healthy humans. the analysis of paired correlations showed that at the height of disease, there were multidirectional relations of varying intensity between the indicators of rm strength and some v.i. bereznyakov 39 in t e r n a l m e d ic in e issn 2413-6077. ijmmr 2021 vol. 7 issue 1 ducts of oxidative stress, excessive proteolysis, bacterial toxins, pro-inflammatory cytokines and other inflammatory mediators that impair the efficiency of respiratory myofibrils. in previous study of segizbaeva m. o, aleksandrova n. p. (2014) it was found that in cap, even endogenous intoxication could cause limitation of the contractile function of dm [13]. in resistive respiration, intensely contracting myocytes are able to transform into metabolic bridgehead producing spectrum of proinflammatory cytokines. at the same time, breathing resistance is considered as an “immune challenge” to the body with excessive synthesis of inflammatory mediators. it has been established that dm fatigue can also develop in healthy individuals with intense respiratory muscle stress [12, 13]. it is established, that the deficiency of glutathione enzymes contributes to damage of cell membranes and macromolecules – proteins, lipids, dna and can determine the development of extrapulmonary manifestations of alveolar inflammation, including through damage to the myofibrils of skeletal muscles [7]. the obtained results can identify patho physiological determinants that combine changes in the oxidative-antioxidant system and the rm functional activity in cap. this is an imbalance in the lpo-aod system, characterized by excess lipoperoxides and hypofunction of the first and second lines of aod. deficiency of the key intracellular antioxidant – glutathione, contributes to increase in os, which becomes as key factor in the initiation of the molecular-cellular mechanisms of alveolar inflammation and rm dysfunction. criteria for the severity of cap in most cases corresponded to the severity of changes in the prooxidant and antioxidant system. at the same time, the heterogeneity of prooxidant processes among patients with ncap, who were included in the group 1, may be due to individual typological characteristics of the organism, including genetic [19]. the important role of individual components of lpo-aod (mda, gpo, gr, catalase, sod) in the development of expiratory and inspiratory rm dysfunction was confirmed by the results of correlation analysis. the presence of interrelations between prooxidants and antioxidants and indicators of rm strength, complements the concept of the body systemic response on pulmonary inflammation – one of the markers of rm dysfunction. conclusions an imbalance in patients with communityacquired pneumonia in the system of lipid peroxidation and antioxidant defence, which is characterized by overproduction of lipoper oxidases and hypofunction of antioxidant defence has been established. increased oxidative stress in patients with community-acquired pneumonia causes deficiency of the key intracellular antioxidant – glutathione. glutathione is key factor in the initiation of the molecular and cellular mechanisms of alveolar inflammation and respiratory muscles dysfunction in patients with community-acquired pneumonia. the relations between prooxidant and antioxidant indicators and the respiratory muscles strength complements the concept of the body systemic response on pulmonary inflammation – one of the markers of respiratory muscles dysfunction. the obtained results will optimize the diagnosis of community-acquired pneumonia and its severity. conflicts of interest authors declare no conflict of interest. authors’ contributions vladyslav ber eznyakov – investigation, conceptualization, formal analysis, writing – original draft. основні показники оксидантно-антиоксидантної системи і їх взаємозв'язок з силою дихальних м'язів у дорослих хворих, що страждають на позалікарняну пневмонію в.і. березняков харківська медична академія післядипломної освіти, харків, україна вступ. в даний час пневмонії займають 4–5 місце в світі в структурі причин смерті після серцевосудинних і онкологічних захворювань, цереброваскулярної патології, травм і отруєнь. мета дослідження – оцінити показники оксидантно-антиоксидантної системи та їх взаємозв'язок з силою дихальних м'язів у дорослих хворих, що страждають на позалікарняну пневмонію. v.i. bereznyakov 40 in t e r n a l m e d ic in e issn 2413-6077. ijmmr 2021 vol. 7 issue 1 методи дослідження. дослідження проведено у період 2017-2020 рр. на базі терапевтичного відділення комунального некомерційного підприємства “міська клінічна багатопрофільна лікарня № 25“ харківської міської ради. у дослідженні брали участь 52 дорослих пацієнтів, що страждали на пп, віком 18 до 80 років. контрольну групу складали 20 практично здорових людей. визначено активність малонового диальдегіду, каталази та супероксиддисмутази, рівень відновленого глутатіону, глутатіонредуктази та глутатіонпероксидази. оцінку сили дихальних м’язів досліджували шляхом реєстрації максимальних статичних тисків на рівні порожнини рота та носа при “закритих” дихальних шляхах за допомогою апарату microrpm на 1-у та 10-у добу захворювання. результати. дисфункція експіраторних дихальних м’язів переважала у пацієнтів з нетяжкою формою пп, а інспіраторних дихальних м’язів – у пацієнтів з тяжкою формою пп. були встановлені негативні кореляції між рівнем малонового диальдегіду та показниками сили дихальної мускулатури, а також позитивні кореляції – з глутатіонредуктазою, глутатіонпероксидазою, каталазою та супероксиддисмутазою. висновки. виявлені взаємозв'язки між проі антиоксидантними показниками і силою дихальних м'язів доповнюють уявлення про системну реакції організму на легеневе запалення, одним з маркером якої є дисфункція дихальних м'язів. ключові слова: позалікарняна пневмонія; дихальні м’язи; оксидантно-антиоксидантна система. information about the authors vladyslav bereznyakov, phd, associate professor, department of general practice – family medicine, kharkiv medical academy of postgraduate education, kharkiv, ukraine. orcid 0000-0001-7818-4864, e-mail: vladyslavbereznyakov@gmail.com references 1. avdeev sn, chuchalin ag. 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(in russian). v.i. bereznyakov 41 in t e r n a l m e d ic in e issn 2413-6077. ijmmr 2021 vol. 7 issue 1 16. saydam n, kirb a, demir o, hazan e, oto o, saydam o, güner g. determination of glutathione, glutathione reductase, glutathione peroxidase and glutathione s-transferase levels in human lung cancer tissues. cancer lett. 1997; 119(1): 13-9. doi: 10.1016/s0304-3835(97)00245-0. 17. kabitz hj, walterspacher s, mellies u. et al. recommendations for respiratory muscle testing. pneumologie 2014; 68(5): 307-314. doi: 10.1055/s-0034-1365283 18. lapach sn, chubenko av, babich pn. [statistical methods in biomedical research using exel]. 2nd ed. k.: morion, 2001; 408 p. (in russian). 19. somov da, makarova mr, makarova in. [the value of muscle imbalance for the development of therapeutic exercises in patients with pneumonia]. questions of balneology, physiotherapy and physical therapy 2015; 3(92): 7-10. (in russian). received 27 may 2021; revised 20 jun 2021; accepted 21 jun 2021. this is open access article distributed under the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. v.i. bereznyakov 77 sh o r t c o m m u n ic a t io n issn 2413-6077. ijmmr 2020 vol. 6 issue 2 doi 10.11603/ijmmr.2413-6077.2020.2.11485 favipiravir and dexamethasone in management of sars-cov2 infection (pilot study) m. latief1, o. shafi2, z. hassan3, *f. abbas4 1 – gandhi medical college and hospital, hyderabad, india 2 – flushing hospital medical center new york, usa 3 – government medical college, baramulla, india 4 – pathology division government medical college, srinagar, india background. the clinical presentation of coronavirus disease 19 (covid-19) varies from mild symptoms to severe illness including multiorgan dysfunction. favipiravir is an antiviral agent which has been previously used for treatment of influenza and was recently approved for treatment of mild to moderate covid-19 in india. objective. the objective of this study was to assess the role of favipiravir and dexamethasone in patients with covid-19. methods. a total of 17 patients were included in this observational study. the included patients were rt-pcr for sars-cov-2 positive with increased inflammatory markers. all patients received antiviral therapy, anticoagulation (enoxaparin 0.4mg subcutaneous twice daily), steroids (dexamethasone 8mg daily for 5days and 4mg daily for 5 days). viral clearance (time to rt-pcr negative), time to defervescence after antiviral therapy, time to become independent of oxygen support was studied. results. fever, myalgias, dry cough and dyspnea were the commonest presentation of covid-19. all of our patients had lymphopenia. in our study 11 (64.7%) patients had bilateral ground glass opacities on ct chest while 6 had consolidation in addition to ground glass opacities. in two patients, who required non-invasive ventilation, favipiravir was stopped and these patients received remdesivir for a total of 5 days. in patients who received favipiravir only, the median time to rt-pcr negative, defervescence and oxygen independence was 8,3 and 6 days respectively. conclusion. our observational study demonstrated improvement in the majority of patients with covid-19 with use of favipiravir. additional studies are needed to compare the efficiency of favipiravir with remdesivir. keywords: sars-cov-2; favipiravir; remdesivir; covid-19. *corresponding author: farhat abbas, senior resident, pathology division government medical college, srinagar, 190011, india. e-mail: farahabbas.m@gmail.com international journal of medicine and medical research 2020, volume 6, issue 2, p. 77-81 copyright © 2020, tnmu, all rights reserved m. latief et al. introduction covid-19 or coronavirus disease 2019, is a novel illness caused by recently discovered severe acute respiratory syndrome coronavirus 2 (sars-cov-2). the virus was first identified in wuhan, a city in the hubei province of china in december 2019 and has since rapidly spread into a global pandemic causing considerable morbidity and mortality worldwide [1, 2]. the disease is classified as either mild to moderate di sease (with no or mild symptoms up to mild pneu monia), severe disease (with hypoxia, dysp nea or >50% lung involvement on imaging within 48 hours), or critical illness (with respiratory failu re, multiorgan dysfunction or shock) [3-6]. however, the vast majority (around 80 to 85%) of infections result in a mild to moderate illness [7]. the ideal therapies for management of covid-19 are still under investigation. favipiravir is an antiviral agent which has been previously used for treatment of influenza and was recently approved for treatment of mild to moderate covid-19 in india [8, 9]. it has shown promise in an early non-randomized open label clinical trial in patients with non-severe disease, where use of favipiravir was associated with faster rates of viral clearance (median time to clearance 4 versus 11 days) and more frequent radiographic improvement (in 91 versus 62 per cent by day 14) compared with lopinavirritonavir [10]. other robust studies are underway to assess the utility of favipiravir in the ma nagement of covid-19. we describe our early experience with the use of favipiravir in patients with covid-19. methods a total of 17 patients were included in this observational study. the included patients were rt-pcr positive for sars-cov-2 with increased inflammatory markers. clinical characteristics 78 sh o r t c o m m u n ic a t io n issn 2413-6077. ijmmr 2020 vol. 6 issue 2 of the patient population was studied. the inflammatory markers done in all patients were interlekin-6, ferritin, c-reactive protein, lactate dehydrogenase (ldh), d-dimer. the other investigations done in all the included patients were complete blood count, liver function and kidney function tests, urine examination, blood cultures, serum procalcitonin. ultrasound abdo men, xray chest and high-resolution computed tomography chest. all patients received antiviral therapy, anticoagulation (enoxaparin 0.4mg sc twice daily), steroids (dexamethasone 8mg daily for 5days and 4mg daily for 5 days). viral clearance (time to rt-pcr negative), time to defervescence after antiviral therapy, time to become independent of oxygen support and development of any complication during hospital stay was studied. patients requiring mecha nical ventilation at presentation to hospital were not included in this study. results out of 17 patients, there were 7 females and 10 male patients. the mean age of patients was 43.88±14.62 and mean body mass index (bmi) was 23.54±1.62 kg/m2. all had high grade fever and myalgias, while 9 (52.9%) patients had dry cough on presentation. 5 patients complained of sore throat and shortness of breath was seen in 9 patients (table 1). there were 4 patients with known diabetes mellitus and 3 of them were hypertensive as well. rest of the patients did not have any underlying comorbidity. however, in two patients, who developed new onset hyperglycemia during steroid course, were found to have hba1c in prediabetic range. out of 17 patients, 11 (64.7%) patients had bilateral ground glass opacities on ct chest while 6 had consolidation in addition to ground glass opacities. all of our patients had absolute lymphocyte count (alc) below 1500 while 4 patients had severe lymphopenia (alc<1000 cells). the inflammatory markers and biochemical parameters are shown in table 2. in our study, 10 patients had mild transaminitis before favipiravir and 3 developed self-limiting transaminitis after favipiravir, 4 had normal liver function test throughout. two patient had acute kidney injury (akin class 1) that resolved during hospital stay. blood culture was sterile in all the patients and serum procalcitonin was negative in all the patients. nine patients required oxygen support. among them 7 were on oxygen support via nasal canula and 2 patients required non-invasive ventilation (niv) during hospital stay. favipiravir was given at a dose of 1800 mg twice on day 1 and subsequently 800 mg twice daily from day 2-10. in two patients, who required niv favipiravir was stopped, and these patients received remdesivir for a total of 5 days. in patients who received favipiravir only, the median time to rt-pcr negative, defervescence and oxygen inde pendence was 8,3 and 6 days respectively. there was no thrombocytopenia, bleeding bacterial sepsis in our patient population. there was no mortality in our study population. discussion the optimal management of patients with covid-19 is rapidly evolving based on extensive ongoing research. initial studies have suggested a clinical benefit with remdesivir (antiviral agent) and a mortality benefit with the use of glucocorticoids. remdesivir, a nucleotide analogue, has in vitro activity against sars-cov-2 [11]. although more data from comparative, randomized trials are emerging [12, 13], available reports suggest there is likely some clinical benefit to remdesivir prompting emergency use authorization by the fda for severe covid-19 in hospitalized children and adults [14]. remdesivir needs to be administered intra venously and should be avoided in patients with transaminitis (alt ≥5 times normal) or in table 1. clinical features of patient population clinical feature n (%) fever 17 (100) myalgia 17 (100) cough 9 (52.9) sore throat 5 (29.4) vomiting 2 (11.7) dyspnea 9 (52.9) epigastric discomfort 1 (0.05) table 2. biochemical parameters and inflammatory markers parameter mean±sd hemoglobin (g/dl) 13.08±1.83 platelet (lac/ µl) 3.4±0.71 ferritin (µg/l) 615.41±307.03 alt (iu/l) 111.5±35.7 absolute lymphocyte count 1180±213.2 bilirubin (mg/dl) 0.92±0.23 triglyceride (mg/dl) 125.4±21.01 d-dimer (ng/ µl) 566.13±207.92 ldh (u/l) 615±187.06 il 6 (pg/µl) 80.58±98.06 m. latief et al. 79 sh o r t c o m m u n ic a t io n issn 2413-6077. ijmmr 2020 vol. 6 issue 2 patients with an estimated glomerular filtration rate (egfr) <30 ml/min per 1.73 m2. it should also not be coadministered with hydroxy chloroquine or chloroquine due to potential drug interactions. the use of dexamethasone (oral or intravenous) provided a mortality benefit at 28 days in hospitalised patients with covid-19 compared to usual care alone as per a preli minary report of the recovery trial, a large randomized open-label study in the united kingdom [15]. however, no benefit was reported among patients who did not require either oxygen or ventilatory support; with a statistically non-significant trend towards higher mortality (17.8 versus 14 percent, rr 1.19, 95% ci 0.911.55). also, there remain uncertainties in this preliminary report, as the baseline mortality rate in this report was higher than that from some other trials. thus, the absolute mortality benefit in other settings may not be as high as observed in this trial. addi tionally, adverse effects (including secondary infections) were not reported in the preliminary report. another antiviral agent which is being in vestigated for its use in patients with covid-19 is a purine nucleoside analogue, favipiravir. it selectively in hibits rna dependent rna polymerase (rdrp), an enzyme needed for rna viral replication within human cells, by getting incorporated instead of guanine and adenine [8]. the drug is converted into its active phosphorylated form intracellularly and subsequently is recognized as a substrate by the viral rdrp. the incorporation of a single molecule of the active form of favipiravir terminates the elongation of viral rna [8]. favipiravir is known to have a broad spectrum of activity towards rna viruses (like influenza, bunyavirus, arenavirus, flavivirus, and filoviruses causing hemorrhagic fever) including activity against oseltamivirand zanamivir-resistant influenza viruses [8, 16]. wang et al studied the in vitro antiviral efficiency of several drugs including favipiravir against sars-cov-2, reporting that favipiravir reduced the viral infection, albeit at higher concentrations half-maximal effective concentration (ec50) = 61.88 μm, half-cytotoxic concentration (cc50) > 400 μm, selectivity index (si) > 6.46 [11]. pertinently, in a previous study evaluating the efficiency of favipiravir against ebola virus, even with a high ec50 value in vero e6 cells of 67 μm, the antiviral agent demonstrated 100% in vivo effectiveness in protecting mice against ebola virus; suggesting that further clinical studies could better evaluate the in vivo response of this antiviral nucleoside [17]. early clinical studies of favipiravir for covid-19 have been promising. a non-randomized open-label study by cai et al. reported a significant reduction in time taken for viral clearance in covid-19 patients treated with favipiravir compared to historical controls who had received lopinavir/ritonavir [10]. this study from china involved administration of favipiravir (1600 mg orally twice daily on day 1 follo wed by 600 mg orally twice daily on days 2–14) in patients with mild to moderate covid-19. patients ≥75 years old, those having severe or critical covid-19, chronic liver disease or end-stage renal disease were excluded from the study. in addition to a significant reduction (p < 0.001) in median time to viral clearance in the favipiravir arm (4 days; iqr = 2.5–9) compared with the historical lopinavir/ritonavir arm (11 days; iqr = 8–13), the vast majority of patients (91.4%) in the former group had radiographic improvement versus 62.2% in the latter group at 14 days. there was a significantly lower rate of adverse events in patients receiving favipiravir (11.4% versus 55.6%; p < 0.01). in our study those patients who received favipiravir only, the median time to rt-pcr negative, defervescence and oxygen independence was 8,3 and 6 days respectively. chen et al conducted a prospective multicenter randomized open label study comparing outcomes in adult covid-19 patients after administration of umifenovir versus favipiravir in addition to conventional therapy [18]. in their preprint article, they report a statistically non-significant (p=0.1396, difference of recovery rate: 0.0954; 95% ci: -0.0305 to 0.2213) higher clinical recovery rate at day 7 in the favipiravir group (61.2%) compared to the umifenovir group (51.6%). also, the favipiravir group had significantly shorter latencies to relief in pyrexia (difference: 1.70 days, p<0.0001) and cough (difference: 1.75 days, p<0.0001). they reported only mild and manageable side effects from the use of favipiravir. although there is a paucity of highgrade evidence for the effectiveness of favipiravir in covid-19, the in vitro activity and benefits seen in early studies strongly suggest the potential for using favipiravir in sars-cov-2 infection. our observational study demonstrated improvement in the majority of patients with covid-19 with use of favipiravir. conclusions ease of oral administration and a profile of mild adverse effects are advantages of favipiravir. further evidence from well-designed m. latief et al. 80 sh o r t c o m m u n ic a t io n issn 2413-6077. ijmmr 2020 vol. 6 issue 2 randomized controlled trials should enable clinicians to better understand the role of favipiravir in the management of the ongoing coronavirus pandemic. additional studies are needed to compare the efficiency of favipiravir compared to remdesivir. conflicts of interest authors declare no conflict of interest. funding no funding was received for this study. acknowledgements we would love to thank dr. summyia farooq of pathology division gmc srinagar for helping us during this study. author contributions muzamil latief, obeid shafi – concep tualization, methodology, formal analysis, writing – original draft, writing – reviewing and editing; muzamil latief, zhahid hassan, farhat abbas – data curation, writing – reviewing and editing; muzamil latief, obeid shafi, farhat abbas – investigation, formal analysis. фавіпіравір та дексаметазон у лікуванні sars-cov2 інфекції (пілотне дослідження) m. latief1, o. shafi2, z. hassan3, *f. abbas4 1 – gandhi medical college and hospital, hyderabad, india 2 – flushing hospital medical center new york, usa 3 – government medical college, baramulla, india 4 – pathology division government medical college, srinagar, india вступ. клінічна картина коронавірусної хвороби (covid-19) варіює від легкого перебігу до тяжких проявів з поліорганною дисфункцією. противірусний засіб фавіпіравір, який раніше застосовувався для лікування грипу, нещодавно був схвалений для лікування covid-19 легкого та середнього ступеня тяжкості в індії. мета. завданням цього дослідження – оцінка ефективності фавіпіравіру та дексаметазону при covid-19. методи. у пілотне дослідження було включено 17 пацієнтів, у яких були позитивні плр тести до sars-cov-2 та підвищені маркери запалення. усі пацієнти отримували противірусну терапію, антикоагулянт (еноксапарин 0,4 мг підшкірно двічі на день), глюкокортикостероїди (дексаметазон 8 мг щодня протягом 5 днів та 4 мг щодня протягом 5 днів). досліджувалися такі показники: тривалість перебігу хвороби до негативних результатів плр тесту, час нормалізації температури на тлі противірусної терапії, швидкість відновлення самостійного дихання. результати. лихоманка, міалгія, сухий кашель та задишка були найпоширенішими симптомами covid-19. у всіх пацієнтів була лімфопенія. при проведенні комп’ютерної томографії грудної клітки, у 11 (64,7%) пацієнтів було знайдено симптом «матового скла», тоді як у 6 спостерігалися і симптом «матового скла» і ущільнення. у двох пацієнтів, яким була потрібна неінвазивна вентиляція, фавіпіравір було відмінено, і ці пацієнти отримували ремдесевір протягом 5 днів. у пацієнтів, які отримували лише фавіпіравір, медіана часу до негативного значення плр тесту, швидкість повернення температури до нормальної та швидкість відновлення самостійного дихання становила 8,3 та 6 днів відповідно. висновок. наше пілотне дослідження продемонструвало тенденцію до покращення у більшості пацієнтів із covid-19 при застосуванні фавіпіравіру. головні обмеження – мала кількість спостережень, та необхідність проведення додаткових досліджень для порівняння ефективності фавіпіравіру та ремдесевіру . ключові слова: sars-cov-2; фавіпіравір; ремдесевір; covid-19. information about the authors muzamil latief, dm resident, nephrology division gandhi medical college and hospital, secunderabad, india orcid 0000-0002-2267-6322, e-mail: muzamillatief.b@gmail.com obeid shafi, chief resident, flushing hospital medical center, new york, usa orcid 0000-0003-2669-199x, e-mail: obeidshafi@gmail.com zhahid hassan, consultant, government medical college, baramulla, india orcid 0000-0003-3724-8387, e-mail: xahid6676@yahoo.co.in farhat abbas, senior resident, pathology division government medical college, srinagar, india orcid 0000-0003-1922-4752, email: farahabbas.m@gmail.com m. latief et al. 81 sh o r t c o m m u n ic a t io n issn 2413-6077. ijmmr 2020 vol. 6 issue 2 references 1. world health organization. director-general’s remarks at the media briefing on 2019-ncov on 11 february 2020. available at: https://www.who.int/dg/ speeches/detail/who-director-general-s-remarks-atthe-media-briefing-on-2019-ncov-on-11-february 2020. accessed july 28, 2020. 2. wu f, zhao s, yu b, chen ym, wang w, song zg, et al. a new coronavirus associated with human respiratory disease in china. nature. 2020 mar;579 (7798):265-69. doi: https://doi.org/10.1038/s41586-020-2008-3 3. bajema kl, oster am, mcgovern ol, lindstrom s, stenger mr, anderson tc, et al. persons evaluated for 2019 novel coronavirus united states, january 2020. mmwr morb mortal wkly rep. 2020 feb 14;69(6):166-70. doi: https://doi.org/10.15585/mmwr.mm6906e1 4. wang d, hu b, hu c, zhu f, liu x, et al. clinical characteristics of 138 hospitalized patients with 2019 novel coronavirus-infected pneumonia in wuhan, china. jama. 2020 mar 17;323(11):1061-69. doi: https://doi.org/10.1001/jama.2020.1585 5. liu k, fang yy, deng y, liu w, wang mf, ma jp, et al. clinical characteristics of novel coronavirus cases in tertiary hospitals in hubei province. chin med j (engl). 2020 may 5;133(9):1025-31. d o i : h t t p s : / / d o i . o r g / 1 0 . 1 0 9 7 / c m 9 . 0000000000000744 6. yang x, yu y, xu j, shu h, xia j, liu h, et al. clinical course and outcomes of critically ill patients with sars-cov-2 pneumonia in wuhan, china: a single-centered, retrospective, observational study. lancet respir med. 2020 may;8(5):475-481. d o i : h t t p s : / / d o i . o r g / 1 0 . 1 0 1 6 / s 2 2 1 3 2600(20)30079-5 7. wu z, mcgoogan jm. characteristics of and important lessons from the coronavirus disease 2019 (covid-19) outbreak in china: summary of a report of 72 314 cases from the chinese center for disease control and prevention. jama. 2020 apr 7;323(13):1239-42. doi: https://doi.org/10.1001/jama.2020.2648 8. furuta y, komeno t, nakamura t. favipiravir (t-705), a broad spectrum inhibitor of viral rna polymerase. proc jpn acad ser b phys biol sci. 2017;93(7):449-63. doi: https://doi.org/10.2183/pjab.93.027 9. glenmark becomes the first pharmaceutical company in india to receive regulatory approval for oral antiviral favipiravir, for the treatment of mild to moderate covid-19. glenmark pharmaceuticals ltd. press release on 20 june 2020. available at: https:// www.glenmarkpharma.com/sites/default/files/ glen mark-becomes-the-first-pharmaceut-cal-company-in-india-to-receive.pdf. accessed july 28, 2020. 10. cai q, yang m, liu d, chen j, shu d, xia j, et al. experimental treatment with favipiravir for covid-19: an open-label control study. engineering (beijing). 2020 oct;6(10):1192-98. doi: https://doi.org/10.1016/j.eng.2020.03.007 11. wang m, cao r, zhang l, yang x, liu j, xu m, et al. remdesivir and chloroquine effectively inhibit the recently emerged novel coronavirus (2019-ncov) in vitro. cell res. 2020 mar;30(3):269-271 doi: https://doi.org/10.1038/s41422-020-0282-0 12. beigel jh, tomashek km, dodd le. remdesivir for the treatment of covid-19 preliminary report. reply. n engl j med. 2020 sep 3;383(10):994. doi: https://doi.org/10.1056/nejmoa2007764 13. wang y, zhang d, du g, du r, zhao j, jin y, et al. remdesivir in adults with severe covid-19: a randomised, double-blind, placebo-controlled, multicentre trial. lancet. 2020 may 16;395(10236):1569-78. doi: https://doi.org/10.1016/s0140-6736(20) 31022-9 14. remdesivir letter of eua. us fda. available at: https://www.fda.gov/media/137564/download. accessed on: july 22, 2020. 15. recovery collaborative group, horby p, lim ws, emberson jr, mafham m, bell jl, linsell l, et al. dexamethasone in hospitalized patients with covid-19. n engl j med. 2021 feb 25;384(8):693-704 doi: https://doi.org/10.1056/nejmoa2021436 16. shiraki k, daikoku t. favipiravir, an antiinfluenza drug against life-threatening rna virus infections. pharmacol ther. 2020;209:107512. doi: https://doi.org/10.1016/j.pharmthera. 2020.107512 17. oestereich l, lüdtke a, wurr s, rieger t, muñoz-fontela c, günther s. successful treatment of advanced ebola virus infection with t-705 (favipiravir) in a small animal model. antiviral res. 2014;105:17-21. doi: https://doi.org/10.1016/j.antivi ral.2014. 02.014 18. chen c, zhang y, huang j, et al. favipiravir versus arbidol for covid-19: a randomized clinical trial. medrxiv; 2020. doi: https://doi.org/10.1101/2020.03.17. 20037432 received 31 oct 2020; revised 17 dec 2020; accepted 24 dec 2020. this is open access article distributed under the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. m. latief et al. issn 2413-6077. ijmmr 2019 vol. 5 issue 276 d e n t is t r y doi 10.11603/ijmmr.2413-6077.2019.2.10448 diagnostic and prognostic significance of microbial flora imbalance in gingival biofilm g.a. loban, t.o. petrushanko, v.v. chereda, *m.o. faustova, m.m. ananieva, ya.o. basarab ukrainian medical stomatological academy, poltava, ukraine background. periodontal tissues inflammatory diseases are widespread among young people. objective. this study was aimed at elaborating the method to assess risks of periodontal inflammatory diseases and determining its efficacy depending on the state of dental tissues, gum tissues and sex. methods. the study included 182 students (93 men, 89 women) aged 19-29: 22 individuals had no lesions of hard dental tissues and no signs of periodontal disease; 51 individuals were found to have dmf index <6; 52 individuals – dmf index ≥6; 57 individuals were diagnosed with chronic catarrhal gingivitis. primary groups were formed in autumn; re-examination was carried in spring. the research participants were assessed for detection of risks of periodontal inflammatory disease by the method developes by the authors (patent ua 54041). results. the study revealed that the risk of development of preiodontitis increases in individuals with high caries and gingivitis intensity. in spring, more individuals suffer from microbial imbalance in in the composition of gingival sulcus fluid and decrease in the mean stability coefficient value that indicates an increased risk of inflammatory periodontal disease development. women were less likely to experience seasonal dysbiotic changes in the gingival sulcus fluid composition compared with men. conclusions. the method suggested for assessment of the risk of periodontal inflammatory diseases is of high informativeness. it allows clinicians detecting early pre-nosological signs of oral microbiocenosis imbalance that enhances the effectiveness of early diagnosis of inflammatory periodontal diseases. key words: biofilms; microflora; gingivitis; risk of morbidity. *corresponding author: maria faustova, ukrainian medical stomatological academy, 23 shevchenko street, poltava 36011, ukraine. e-mail: mashafaustova@ukr.net introduction according to the recent reports, oral and dental health has significantly improved in most countries, but the prevalence of inflammatory diseases of periodontal disease is still high [7, 9, 10]. inflammatory diseases of periodontal tissues are reported to be quite common among young population [7]. these diseases are in the focus of researchers and clinicians as they are a main cause of tooth loss as well as they increase risks of systemic pathologies even in young age. therefore, early diagnosis of inflammatory gum diseases and prognosis of their outcomes are one of topical issues of contemporary dentistry. the oral cavity is an ecological system harbouring various types of microorganisms forming a biofilm [1,8,11,13]. in the oral cavity, bacteria can be present in the planktonic state (e. g., in saliva) and can develop as colonies that adhere to organic structures and build up plaque, and are able to organize associations for joint survival. the colonies may develop their complex and unexpected distinct properties. according to the current interpretation, the bacterial plaque is called a biofilm, which is a specialized bacterial ecosystem that provides the viability and preservation of microbial species forming the biofilm and promotes their general population increase [14]. moreover, biofilm is an independent and self­regulating biological system, far from being an amorphous association of different bacteria. at present, early pre-nosological signs of the risk of inflammatory gum diseases are hardly diagnosed in dental practice. the relations between the early micro-ecological imbalance of the oral cavity and risk of gingival inflammation development are still unclear as well as the issues of gum disease prediction. the development of accessible and easy-to-use methods for early diagnosis and prediction of gum diseases and their progression in young age allows clinicians providing an evidencebased approach to choose the proper tactics in managing such patients. international journal of medicine and medical research 2019, volume 5, issue 2, p. 76-82 copyright © 2019, tnmu, all rights reserved g.a. loban et al. issn 2413-6077. ijmmr 2019 vol. 5 issue 2 77 d e n t is t r y g.a. loban et al. this study was aimed at elaborating the prognostic criteria to assess risks of periodontal inflammatory diseases and determining its efficacy depending on the state of dental tissues, gum tissues and sex. methods 182 students of a medical college aged 1929. 22 individuals (11 men and 11 women), who had no lesions of hard dental tissues and no signs of periodontal disease, made up the control group. 51 individuals (26 men, 25 women) were found to have dmf index <6; 52 individuals (27 men, 25 women) – dmf index ≥6; 57 individuals (29 men, 28 women) were diagnosed with chronic catarrhal gingivitis. primary groups were formed in autumn (october­november); re­examination of the test groups was carried out in 6 months in spring (april-may). the study was conducted in accordance with the helsinki declaration of the world medical association on the ethical principles for medical research involving human subjects [15]. signed written informed consents to participate in research study were given by research project participants that was an indispensable condition for the inclusion of students in the study. the research participants underwent standard dental clinical examination to determine caries intensity index (dmf index), oral hygiene index (greene-vermilion index) (ohi-s), pma gingival index modified by c. parma, muhlemann bleeding index, muhlemann-saxer index (pbi), interdental hygiene index (hyg), complex perio dontal index (cpi). all subjects were assessed for the risk of periodontal inflammatory disease by the method elaborated by the authors – the patent (utility model ua 54041, published information bulletin) [2]. the method of assessment of the risks of periodontal inflammatory diseases develop­ ment is: gingival fluid is obtained in 1­2 hours following tooth brushing with a sterile paper pin of 10 mm long, by inserting its end in the orifice of the gingival groove. when the paper pin gets soaked with the fluid of the gingival groove, it is placed in 0.1 ml of sterile saline and washed thoroughly. after that this saline suspension of microorganisms is put onto degreased sterile slide with following drying, fixing, staining by gram techniques. immersion microscopy is used to count the number of gram-positive cocci, gram-negative cocci, gram-positive rods, gram-negative rods, gram-negative spirilla as a percentage of total bacterial cells counted. the stability coefficient (sc) is calculated by the ratio of the sum of the number of grampositive cocci and gram-positive rod-shaped microorganisms as percentage to the sum of the number of gram-negative rods and gramnegative spirilla as percentage. when the sc value equals 2-4, this indicates the ecological balance between bacterial populations, prevalence of symbiotic stabilizing microbiota, and no risks of inflammatory periodontal disease. when the sc value is >4 (sc shift to the right), this points out an increase in the number of gram-positive bacteria residing on the gum tissues. these microorganisms are constituents of the dental plaque and contribute to development of inflammatory response characteristic of gingivitis, that is, the risk of inflammatory periodontal diseases increases. when the sc value is <2 (sc shift to the left), this evidenced an increase in obligatory anaerobic gram-negative rod-shaped bacteria (bacteroids) and spirilla that have periodontopathogenic effect, i.e., the risk of periodontitis development increases [2]. statistical analysis of the findings obtained was carried out using the spss 17.0 and microsoft excel 2003 programs. the obtained quantitative indicators were processed by the methods of mathematical statistics with definition of mean values (m) and errors of mean values (m) in the groups of individuals under the study. the statistically significance of differences between the investigated indicators was estimated by the student’s t-test criterion. for comparison of the particles in separate groups, χ2 criterion was used to determine the statistical significance of their differences. results the assessment of the risk of inflammatory periodontal diseases that was carried out in the autumn has revealed the following. in the control group, the incidence rate of sc within the range of 2-4 made up 95.5%, in 4.5% of individuals there was a sc shift to the right (fig. 1). the development of caries was accompanied by changes in the frequency structure of the sc gradations. in the individuals with dmf index <6, the number of people with sc= 2­4 decreased by 34.7%; the sc shift to the left was observed to be as more often as by 29.4%, and the frequency of the sc shift to the right increased by 5.3% (χ2=97042.761, p=0.0001). the increase in the caries intensity to dmf index ≥6 issn 2413-6077. ijmmr 2019 vol. 5 issue 278 d e n t is t r y was accompanied by a decrease in the number of individuals with sc=2­4 to 53.8%; 25.0% of the subjects were found to have sc<2, the number of individuals with sc shift to the right increased to 21.2% (χ2=71518240, p=0.0001). among the patients with gingivitis, subjects with sc<2 (73.7%) and sc>4 (26.3%) prevailed, while the patients with sc=2-4 were not detected (χ2=648866.373, p=0.0001). the mean sc value decreased in the sub jects with dmf index <6 by 18.2% (2.92±0.18 vs. 3.57±0.11 in control, p=0.002) and especially in patients with gingivitis, by 38.7 % (2.19±0.20, p=0.0005). the regularities of the frequency in detecting certain sc gradations and their absolute values did not depend on sex. in spring, the frequency of detecting sc gradations depended on the condition of teeth and gums (fig. 2). the number of individuals with dysbiotic shifts in the gingival fluid micro­ biota in comparison with the control group, increased in the individuals with dmf index <6 by 6.2% (χ 2=3876.859, p=0.0001), in the individuals with dmf index ≥6 by 16.8 % (χ2= 8653.959, p=0.0001), and in the patients with gingivitis there was an increase by 59,1% (χ2=13941.459, p=0.0001). the mean sc value in the individuals with caries did not differ from those in the control group (2.54±0.16 for dmf index <6; 2.82±0.19 for dmf index ≥6 vs. 2.62±0.19 for the control). the mean sc value in the individuals with catarrhal gingivitis was 1.6 times lower compared with the control (1.67±0.17, p=0.0004). in spring, compared to the fall, the frequency profile in all the study groups changed due to an increase in the number of individuals with sc shift to the left: in the control group, it incre­ ased by 40.9% (χ2=77307.546, p=0.0001), in the individuals with dmf ≥6 – by 15.4% (χ2=17.484, p=0.0001), and in the individuals with catarrhal gingivitis – by 15.8% (χ2=7.329, p=0.007). the mean sc value decreased in the control group fig. 1. rate of sc gradations in adolescents in autumn. fig. 2. rate of sc gradations in adolescents in spring. 4 estimated by the student’s t-test criterion. for comparison of the particles in separate groups, χ2 criterion was used to determine the statistical significance of their differences. results the assessment of the risk of inflammatory periodontal diseases that was carried out in the autumn has revealed the following. in the control group, the incidence rate of sc within the range of 2 – 4 made up 95.5%, in 4.5% of individuals there was a sc shift to the right (fig. 1). the development of caries was accompanied by changes in the frequency structure of the sc gradations. in the individuals with dmf index <6, the number of people with sc = 2 – 4 decreased by 34.7%; the sc shift to the left was observed to be as more often as by 29.4%, and the frequency of the sc shift to the right increased by 5.3% (χ2 = 97042.761, p = 0.0001). the increase in the caries intensity to dmf index ≥6 was accompanied by a decrease in the number of individuals with sc = 2 – 4 to 53.8%; 25.0% of the subjects were found to have sc <2, the number of individuals with sc shift to the right increased to 21.2% (χ2 = 71518240, p = 0.0001). among the patients with gingivitis, subjects with sc <2 (73.7%) and sc >4 (26.3%) prevailed, while the patients with sc = 2 – 4 were not detected (χ2 = 648866.373, p = 0.0001). 0,0% 53,8% 60,8% 95,5% 73,7% 25,0% 29,4% 0,0% 26,3% 21,2% 9,8% 4,5% 0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100% gingivitis dmf≥6 dmf<6 control 2-4 scores <2 scores >4 scores fig. 1. rate of sc gradations in adolescents in autumn. 5 the mean sc value decreased in the subjects with dmf index <6 by 18.2% (2.92 ± 0.18 vs. 3.57 ± 0.11 in control, p = 0.002) and especially in patients with gingivitis, by 38.7 % (2.19 ± 0.20, p = 0.0005). the regularities of the frequency in detecting certain sc gradations and their absolute values did not depend on sex. in spring, the frequency of detecting sc gradations depended on the condition of teeth and gums (fig. 2). the number of individuals with dysbiotic shifts in the gingival fluid microbiota in comparison with the control group, increased in the individuals with dmf index <6 by 6.2% (χ2 = 3876.859, p = 0.0001), in the individuals with dmf index ≥6 by 16.8 % (χ2 = 58653.959, p = 0.0001), and in the patients with gingivitis there was an increase by 59,1% (χ2 = 13941.459, p = 0.0001). the mean sc value in the individuals with caries did not differ from those in the control group (2.54 ± 0.16 for dmf index <6; 2.82 ± 0.19 for dmf index ≥6 vs. 2.62 ± 0.19 for the control). the mean sc value in the individuals with catarrhal gingivitis was 1.6 times lower compared with the control (1.67 ± 0.17, p = 0.0004). 0,0% 42,3% 52,9% 59,1% 89,5% 40,4% 41,2% 40,9% 10,5% 17,3% 5,9% 0,0% 0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100% gingivitis dmf≥6 dmf<6 control 2-4 scores <2 scores >4 scores fig. 2. rate of sc gradations in adolescents in spring. in spring, compared to the fall, the frequency profile in all the study groups changed due to an increase in the number of individuals with sc shift to the left: in the control group, it increased by 40.9% (χ2 = 77307.546, p = 0.0001), in the individuals with dmf ≥6 – by 15.4% (χ2 = 17.484, p = 0.0001), and in the g.a. loban et al. issn 2413-6077. ijmmr 2019 vol. 5 issue 2 79 d e n t is t r y g.a. loban et al. in 1.4 times (p=0.0001), and in the patients with gingivitis – in 1.3 times (p=0.047). in spring, in men and women, the sc frequency profile also depended on the dental status and was characterized by a significantly higher number of individuals with dysbiosis in the gingival sulcus biofilm in all the studied groups compared to the control. the mean sc value in men with gingivitis was 1.6 times lower than in the control group (1.61±0.24 versus 2.59±0.26 in control, p=0.019), while in women it did not differ significantly (1.77±0.24 vs. 2.64±0.27 in the control). in spring, in comparison with autumn, the number of persons with sc shift to the left increased in both men and women: in the control groups of men it increased by 45.5% (χ2=22721.819, p=0.0001), in women – by 34.6% (χ2=15994.909, p=0.0001); in men with dmf index ≥6, it increased by 22.3% (χ2=6.9661, p=0.031), in women with gingivitis – by 17.9% (χ2=4.375, p=0.036). the mean sc value in the control groups decreased in men in 1.4 times (2.59±0.26 in the spring compared with 3.71±0.12 in autumn, p=0.002), and in women, it – in 1.3 times (2.64±0.27 in spring compared to 3.43±0.18 in autumn, p=0.028). in spring, male subjects showed gradations of sc<2 and sc>4 more often than female subjects, and in particular, in the male patients with high caries intensity, it was observed to be more frequent (by 26.4%) (χ2=8.397, p=0.015). the attained results testify that women are less likely to experience seasonal dysbiotic changes in the gingival sulcus biofilm. in the individuals, whose gums were assessed as intact in autumn, the inflammation de­ velopment in spring was detected: in the control group, there were 3 individuals with signs of inflammation (1 woman, 2 men) con­ stituting 13.6% of the whole group, in the adolescents with dmf index <6, there were 16 individuals (9 men, 7 women) that made up 31.4% of the relevant group; and in the indivi­ duals with dff index ≥6, there were 21 patients (14 men, 7 women) that made up 40.4% of the individuals. the highest periodontal indexes were observed in the patients with catarrhal gingivitis, but in the patients with caries, their pma, muhlemann, and pbi indices were significantly higher than those of the control group. in the study group of patients with ca tarrhal gingivitis in spring, there was a tendency to increase in the periodontal indices, compared with the autumn. in particular, the pma was 27.7±0.89% (compared with 0.91±0.51% in the control group), the muhlemann index – 1.45±0.057 (in the control 0.02±0.013), the pbi index – 0.91±0.034 (0.01±0.006 in the control group). in the patients with caries, pma, muhlemann and pbi indexes were also significantly higher than the controls. thus, in persons with dmf <6 pma, this index was in 3.9 times higher in the control group (p<0.05), the muhlemann index – in 3.0 times (p<0.05), and the pbi index – in 6.0 times (p<0.05). in the patients with dmf ≥6, the periodontal indexes were even higher than the corresponding control group values, namely: pma – in 4.6 times (p<0.05), muhlemann index – in 4.0 times (p<0.05), the pbi index – in 7.0 times (p<0.05). in the study group of patients with catarrhal gingivitis in spring a tendency to increase in the periodontal indices was observed, compared with the autumn season. in men and women, in spring, the changes in indexes compare to the groups by dental status and by season had the same features as in the groups without regard to sex. discussion the study revealed an increase in the incidence of inflammatory processes in periodontal tissues of adolescents in spring. a number of reports have emphasized polyetiological nature of periodontal diseases. and in addition to that, inflammatory reactions provoked by gingival biofilm microflora are established to greatly contribute to development of periodontal diseases [5,6,12]. the suggested criteria have demonstrated an increase in the incidence of dysbiosis in the gingival biofilm in adolescents in spring that, in our opinion, determine an increase in the incidence of gingivitis in spring compared with autumn. the quantitative and specific composition of the oral microbial flora of each healthy indi­ vidual is relatively stable, since there are a number of factors that maintain its constancy. the most important factor in maintaining the stability of the oral microbial composition is the antagonism inherent in the resident microbial flora relative to pathogenic and opportunistic microorganisms, when a stable microbial community crowds out pathogenic agents from the oral cavity [13]. compensatory properties of symbiotic micro bial flora are far from being limitless and under the influence of various factors, the dynamic equilibrium between a normal and pathogenic flora may be disrupted [3,4]. for instance, the disorders of swallowing, chewing issn 2413-6077. ijmmr 2019 vol. 5 issue 280 d e n t is t r y and salivation always lead to an increase in the number of pathogenic microorganisms in the oral cavity. as a result, sharp suppression of normal microbial flora representatives occurs, i.e. dysbiosis develops, that means qualitative and/or quantitative changes in the resident microbial flora resulting from the impact of various exogenous or endogenous factors on the body [5]. conclusions the suggested prognostic criteria for assessing the risk of periodontal inflammatory di­ seases are of high informativeness, they allow clinicians detecting early pre-nosological signs of oral microbiocenosis imbalance that enhances the effectiveness of early diagnosis of inflammatory periodontal diseases, and can be used as a marker to evaluate the degree of body adaptation to the environment factors. the imbalance of indigenous and periodontopathogenic microbial flora has a significant impact on the oral status of adolescents regardless of sex. the risk of periodontitis development increases in individuals with caries severity and gingivitis intensity. in spring, more individuals were identified to have microbial imbalance in the composition of gingival sulcus fluid and a decrease in the mean stability coefficient that indicates an increase in the risk of inflammatory periodontal disease development. women were less likely to experience seasonal dysbiotic changes in the gingival sulcus fluid composition compared with men. in clinical dental practice early diagnosis of periodontal diseases and prediction of their development is significant for elaborating effective preventive measures. to predict the probable progression of the disease, taking into account the patterns of pathological processes and the course of the disease, the attained results require wider applying of mathematical analysis (correlation and regressive). detection of a wide range of relations not only improves the effectiveness of personalized prediction of microecological imbalance in the oral cavity but also allows choosing and prescribing appropriate preventive therapy or start treatment at the right time. funding this research received no external funding. conflict of interests the authors declare no conflict of interest. authors contributions loban’ g.a. – conceptualization, methodology, project administration, writing – review & editing; petrushanko t.o. – methodology, project administration; chereda v.v. – investigation, visualization; faustova m.o. – formal analysis, writing – review & editing; ananieva m.m. – visualization, writing – original draft; basarab ya.o. – writing – original draft. діагностичне і прогностичне значення дисбалансу мікрофлори ясенної біоплівки г.а. лобань, т.о. петрушанко, в.в. череда, м.о. фаустова, м.м. ананьєва, я.о. басараб українська медична стоматологічна академія, полтава, україна вступ. запальні захворювання тканин пародонта широко поширені серед молоді. мета. це дослідження було спрямоване на розробку методу оцінки ризику запальних захворювань пародонта та визначення його ефективності залежно від стану тканин зубів та ясен і гендерного фактору. методи. обстежено 182 студенти (93 чоловіків і 89 жінок) віком 19-29 років, з яких 22 особи не мали уражень твердих тканин зубів та пародонта, 51 особа з рівнем кпв<6, 52 особи з рівнем кпв≥6, 57 осіб з діагностованим хронічним катаральним гінгівітом. первинні групи були сформовані восени, повторне обстеження проводилося навесні. в усіх досліджуваних провели визначення стоматологічного статусу, виявлення ризику розвитку запальних захворювань пародонта здійснили за власною методикою (патент ua 54041). результати. проведені дослідження показали, що ризик розвитку пародонтиту підвищується в осіб з високою інтенсивністю карієсу та гінгівітом. у весняний період року виявили більшу кількість осіб з дисбалансом біоплівки ясенної борозни та зниження середньої величини коефіцієнту сталості порівняно з осіннім сезоном, що свідчило про збільшення ризику розвитку запальних захворювань g.a. loban et al. issn 2413-6077. ijmmr 2019 vol. 5 issue 2 81 d e n t is t r y g.a. loban et al. пародонта навесні. у жінок рідше спостерігали сезонні дисбіотичні зміни біоплівки ясенної борозни порівняно з чоловіками. висновки. запропонований метод оцінки ризику запальних захворювань пародонта має високу інформативність, дозволяє виявити ранні донозологічні порушення мікробіоценозу порожнини рота, що підвищує ефективність ранньої діагностики. запальних захворювань пародонта, і може бути використаний як маркер ступеня адаптації організму до факторів зовнішнього середовища. ключові слова: біоплівки; мікрофлора; гінгівіт; ризик захворювань. відомості про авторів лобань галина андріївна – доктор медичних наук, професор, завідувачка кафедри мікробіології, вірусології та імунології української медичної стоматологічної академії, полтава, україна. петрушанко тетяна олексіївна – доктор медичних наук, професор, завідувачка кафедри терапевтичної стоматології української медичної стоматологічної академії, полтава. череда вікторія володимирівна – кандидат медичних наук, асистент кафедри терапевтичної стоматології української медичної стоматологічної академії, полтава. фаустова марія олексіївна – кандидат медичних наук, викладач кафедри мікробіології, вірусології та імунології української медичної стоматологічної академії, полтава, україна. ананьєва майя миколаївна – кандидат медичних наук, доцент кафедри мікробіології, вірусології та імунології української медичної стоматологічної академії, полтава, україна. басараб ярослав олексійович – викладач кафедри мікробіології, вірусології та імунології української медичної стоматологічної академії, полтава, україна. information about the authors loban’ g. a. – md, ph.d., dsc, head of microbiology, virology and immunology department, ukrainian medical stomatological academy, poltava, ukraine. orcid 000­0003­0055­7696, e­mail: galina.loban@gmail.com petrushanko t.o. – md, ph.d., dsc, head of therapeutic dentistry department, ukrainian medical stomatological academy, poltava, ukraine. orcid 0000­0002­1001­5404, e­mail: petrusankotatana@gmail.com chereda v. v. – phd, lecturer of therapeutic dentistry department, ukrainian medical stomatological academy, poltava, ukraine. orcid 0000­0002­5823­9642, e­mail: viktoria.chereda@gmail.com faustova m. o. – phd, lecturer of microbiology, virology and immunology department, ukrainian medical stomatological academy, poltava, ukraine. orcid 0000­0001­5327­6324, e­mail: mashafaustova@ukr.net ananieva m. m. – md, phd, associate professor of microbiology, virology and immunology department, ukrainian medical stomatological academy, poltava, ukraine. orcid 0000­0001­9435­7622, e­mail: anfila@ukr.net basarab ya. o. – md, lecturer of microbiology, virology and immunology department, ukrainian medical stomatological academy, poltava, ukraine. orcid 0000­0001­7644­0713, e­mail: basarab.yaroslav.doc@gmail.com references 1. kassebaum nj, smith ag, bernabé e, fleming td, reynolds ae, vos t et al. gbd 2015 oral health collaborators. global, regional, and national preva lence, incidence, and disability-adjusted life years for oral conditions for 195 countries, 19902015: a syste matic analysis for the global burden of diseases, inju ries, and risk factors. j dent res. 2017; 96,4:380­7. doi: 10.1177/0022034517693566 2. machado v, botelho j, amaral a, proença l, alves r, rua j et al. prevalence and extent of chronic periodontitis and its risk factors in a portuguese subpopulation: a retrospective cross­sectional study and analysis of clinical 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resilience of the oral microbiota in health: mechanisms that prevent dysbiosis. j dent res. 2018;97,4:371­80. doi: 10.1177/0022034517742139 8. faustova mo, ananieva mm, basarab yo, dobrobolska ov, vovk im, loban' ga. bacterial factors of cariogenicity (literature review). wiad lek. 2018; 71,2: 378­82. 9. wma declaration of helsinki – ethical principles for medical research involving human subjects. https://www.wma.net/policies­post/wma­declara­ tion-of-helsinki-ethical-principles-for-medicalresearch-involving-human-subjects/ 10. chereda vv, petrushanko to, loban' ga. a method for assessing the risk of inflammatory periodontal disease. health newsletter. kyiv: ukrmedpatentinform ministry of health of ukraine. 2012;21,136:3. [in ukrainian]. 11. ebersole jl, dawson d, emecen-huja p, nagarajan r, howard k, grady me, et al. the periodontal war: microbes and immunity. periodontol 2000. 2017;75,1:52­115. doi: 10.1111/prd.12222 12. herrero er, fernandes s, verspecht t, ugarteberzal e, boon n, proost p et al. dysbiotic biofilms deregulate the periodontal inflammatory response. j dent res. 2018; 97,5:547­55. doi: 10.1177/0022034517752675 13. rafiei m, kiani f, sayehmiri k, sayehmiri f, tavirani m et al. prevalence of anaerobic bacteria (p.gingivalis) as major microbial agent in the incidence periodontal diseases by meta-analysis. j dent (shiraz). 2018; 19,3:232­42. 14. ananieva mm, faustova mo, basarab io, loban' ga. kocuria rosea, kocuria kristinae, leuconostoc mesenteroides as caries-causing representatives of oral microflora. wiad lek. 2017;70,2,2:296­8. 15. ananieva m, nazarchuk o, faustova m, basarab y, loban g. pathogenicity factors of kocuria kristinae contributing to the development of periimplant mucositis. mal j med health sci. 2018;14,3: 34-8. received 05 september 2019; revised 11 october 2019; accepted 21 october 2019. this is open access article distributed under the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. g.a. loban et al. 44 p h a r m a c y issn 2413-6077. ijmmr 2020 vol. 6 issue 2 doi 10.11603/ijmmr.2413-6077.2020.2.11388 elaeocarpus serratus l. exhibits potential analgesic and antidiarrheal activities in mice model a.a.h. pinkey1, *z.i. khan2, m.a. taher1, m.a. soma1 1 – department of pharmacy, state university of bangladesh, dhaka, bangladesh 2 – department of health technology and informatics, the hong kong polytechnic university, hong kong, china background. elaeocarpus serratus l. (family: elaeocarpaceae) is a tropical fruit tree, traditionally used in the treatments of poisoning, diarrhea, arthritis, and other diseases. objectives. the current study was performed to conduct the analgesic, antidiarrheal, and hypoglycemic activity of e. serratus in mice model using methanolic bark crude extract. methods. to assess the peripheral and central analgesic activity, the acetic acid-induced writhing and tail immersion methods were respectively used. the castor-oil mediated antidiarrheal method was used to assess the antidiarrheal activity whereas, the tail tipping technique was conducted to determine the hypoglycemic activity of the plant extract. results. in the peripheral analgesic assay, the methanolic bark crude extract of e. serratus significantly inhibits the number of writing 69.77% (200 mg/kg) and 73.26% (400 mg/kg) respectively (p<0.05) which was strongly comparable with standard nsaid drug diclofenac sodium 75.58% (p<0.05). similarly, it shown a significant tail flicking response for 30 minutes, 60 minutes and 90 minutes of central analgesic activity assay. in antidiarrheal activity assay, the e. serratus substantially reduced the number of diarrheal feces 64.26% (200 mg/kg, p<0.05) and 78.57% (400 mg/kg, p<0.05) which was also comparable with the positive control loperamide. the hypoglycemic activity of e. serratus extract was not convincing. conclusions. our investigation demonstrated the significant analgesic and antidiarrheal activities of methanolic bark extract of e. serratus (200 and 400 mg/kg) in mice model. keywords: elaeocarpus serratus; analgesic activity; antidiarrheal activity; hypoglycemic activity. *corresponding author: md zahirul islam khan, department of health technology and informatics, the hong kong polytechnic university, 100077, hong kong, china. e-mail: zahir.islamkhan@connect.polyu.hk international journal of medicine and medical research 2020, volume 6, issue 2, p. 44-51 copyright © 2020, tnmu, all rights reserved a.a.h. pinkey et al. introduction medicinal plants or natural drugs are traditionally and historically used around the globe by human beings for curing various ailments. the plant-derived natural drugs are widely accepted to all due to their diverse pharmacological activities, reduced toxicity, cost-effective, availability for drug discovery, and application to the chemical biology [1, 2]. although, the incessant investigation is being carried out to screen potential pharmacological activities of natural products but the numbers are very limited considering all medicinal plants distributed throughout the world [3]. so far, a considerable number of experimental research have been reported the use of natural products as an antioxidant agent, blood glucose-lowering agent, antimicrobial agent, central nervous system (cns) stimulating agent, anti-diarrheal agent, anti-helminthic agent, anti-inflammatory agent, and anti-cancer agent [4]. by considering the previous studies, we explored the pharmacological activities of elaeocarpus serratus l. (e. serratus) in a number of biological uses. e. serratus (english name: rosary nut, ceylon olive, bengali name: jalpai) belongs to the family elaeocarpaceae, a tropical fruit tree grown up to 18 meters tall, distributed in evergreen forests, and sometimes also cultivated for its edible fruit and medicinal applications [5, 6]. it is mostly found in the indian subcontinent regions including india, bangladesh, pakistan, sri lanka and nepal. however, it is also found in indo-china regions including, myanmar, indonesia, thailand and malaysia [5]. the e. serratus is a plant having both nutritional and medicinal values. for instance, the gc-ms analysis revealed that the plant contains numerous compounds including fatty acid, alcohol, aldehyde, hydrocarbons and alkenes which are biologically active [7]. in 45 p h a r m a c y issn 2413-6077. ijmmr 2020 vol. 6 issue 2 addition, the leaf of e. serratus contains alkaloids, flavonoids, and glycosides (eg. anthraqui none) [7]. moreover, a list of bioactive com pounds also contained in e. serratus such as myricitrin, mearnsetin 3-o-β-d-glucoside, mearn sitrin, and tamarixetin 3-o-α-l-rhamno pyranoside where, myricitrin is an established potential antioxidant [8]. historically, leaves of e. serratus extracts are used for the treatments of arthritis and various poisoning [9]. equally, appetite, diarrhea, dysentery and other neuro-motors related diseases are commonly treated with fruits or fruit extracts [6, 10]. moreover, the previous studies also reported that the leaf, bark and fruit of e. serratus have antimicrobial and antifungal activities [11, 12]. for all we know, there is no scientific report conducted on analgesic, hypoglycemic, and anti-diarrheal properties of e. serratus yet. therefore, our main objective was to assess the analgesic, hypoglycemic, and antidiarrheal activity of methanolic bark crude extract of e. serratus in mice model. methods collection and extraction of plant in february 2018, the bark of e. serratus was acquired from chandpur, bangladesh. the collection of bark samples was verified by bangladesh national herbarium (bdnh), dhaka, bangladesh. an herbarium specimen number (dacb-31155) was provided and preserved for their further reference. the barks were cleaned and cut into small pieces to accelerate the drying process. then the sundried fragments were crushed to a fine powder. about 400 g of powder was put in a flat bottom amber sterile glass container and socked with 1.5l methanol for two weeks. continuous shaking and stirring were maintained over time. afterward, the entire mixture was filtered with cotton and repeated second filtration with whatman filter paper (bibby re200, sterilin ltd., uk). the filtrate was then kept for a week allowed to concentrate with a rotary evaporator at 45°c and 50 rpm. finally, 28.0 g (yield 5.63%) of a black gummy substance was obtained referring to crude methanol extract of e. serratus bark. the extract was aliquot into 2 ml centrifuge tubes and stored at 4°c for further uses. experimental animals to conduct the experiments, the swiss albino mice (20-24gm) were brought from international centre for diarrheal disease research, bangladesh (icddr). the animals had a typical environmental condition (at 24.0 ± 1°c and 55 65% relative humidity), in cages with 12 hours of dark and light cycles. until starting of the experiments, the animals were housed to embrace the local laboratory conditions for a week. in each bioassay, the animals were selected randomly and sub-divided into four separated groups consist of positive control (pc) group, negative control (nc) group and two experimental groups receiving e. serratus crude extract at doses of 200 mg/kg body weight (b.w.) (es-i) and 400 mg/kg b.w. (es-ii). mice were remarkably observed for a week to monitor any suffering or distress and fasted overnight prior to the experiments. the animal experiments were conducted according to the ethics committee of state university of bangladesh (sub), dhaka, bangladesh. drug treatments and chemical reagents diclofenac sodium, glibenclamide, and loperamide hydrochloride were purchased from beximco pharmaceuticals ltd (bangladesh). phenobarbitone sodium and morphine sulphate were supplied by incepta pharmaceuticals ltd (bangladesh), and beacon pharmaceuticals ltd (bangladesh). tween 80, normal saline (0.9% nacl) and castor oil were kindly given by bdh chemicals ltd (united kingdom). the remaining chemicals and reagents were purchased from sigma-aldrich (munich, germany). peripheral analgesic activity the acetic acid-mediated writhing method by kaushik, d., et al. 2012 was copied to assess the peripheral analgesic activity of the e. serratus crude extract [13]. the intraperitoneal acetic acid injection was given to all mice with a view to exhort the abdominal pain followed by writhing in mice. the potentialities of the test samples were measured by calculating their competency in the reduction of writhing numbers. test group (es-i and es-ii) were orally administered, containing the doses of 200and 400 mg/kg of body weight (b.w.), respectively. whereas, the nc group orally received 1% tween 80 in saline and the pc group orally received diclofenac sodium at 50 mg/kg dose [14]. to induce writhing in mice, 1% v/v acetic acid was given intraperitoneally to all mice (10 ml/kg b.w.) followed by a resting period of 40 minutes after test samples administration. the writhing cases were carefully observed and documented for 10 minutes after giving 10 minutes resting period. the acetic acid-induced pain reduction was calculated by using the following equation: a.a.h. pinkey et al. 46 p h a r m a c y issn 2413-6077. ijmmr 2020 vol. 6 issue 2 central analgesic activity pizziketti et al., 1985 described the tail-flick method was implemented to assess the central analgesic activity of e. serratus crude extract in mice [15]. in this method, the mice were orally given a different dose of drugs and e. serratus, and the tips of the mice tails were submerged in a constant radiant heat source (hot water bath at 55±0.5 °c). the reaction time (mice tail deflects from the heating source) of each mouse was recorded using a stopwatch. to prevent the damage of tail, we maintained a cut off period of 15 seconds. similar to the peripheral analgesic study, the nc group orally received 1% tween-80 in saline, and the pc group was subcutaneously injected with morphine (2 mg/kg b.w.) [16]. the es-i and es-ii were prescribed orally to the test groups of mice. the tail-flick reaction was counted and recorded in 0 minutes, 30 minutes, 60 minutes, and 90 minutes after administration of the test samples. the following equation was used to measure the pain inhibition per centage (pip). % of writhing inhibition= (mean writhing of control–mean writhing of test)×100% mean writhing of control pip= (mean latency of treatment–mean latency of control)×100% mean latency of control percentage inhibition= mean defecation of control–mean defecation of test sample or standard mean writhing of control ×100% % of writhing inhibition= (mean writhing of control–mean writhing of test)×100% mean writhing of control pip= (mean latency of treatment–mean latency of control)×100% mean latency of control percentage inhibition= mean defecation of control–mean defecation of test sample or standard mean writhing of control ×100% hypoglycemic activity the tail tipping technique according to the method described by durschlag et al., 1996 was repeated to assess the hypoglycemic activity of test samples in mice model [17]. here, the nc group was treated with 1% tween-80 (0.1 ml/10 m g b . w . ) , a n d p c g r o u p t r e a t e d w i t h glibenclamide (5 mg/kg b.w.) whereas, groupiii and group-iv were treated with es-i and es-ii respectively. all treatments were applied orally [14]. to accelerate the blood sugar level of mice, a 10% glucose solution was orally given to all mice after an hour resting period at dose 2 g/kg b.w. blood is withdrawn from the tail tip and blood sugar was measured and recorded by using a glucometer (bioland g-423 s) in 0 minutes, 30 minutes, 60 minutes, 120 minutes, and 180 minutes respectively. antidiarrheal activity the antidiarrheal activity of e. serratus crude extract was determined by the method described by shaoba and thomas [18], where f o r c e f u l d i a r r h e a i s i n d u c e d b y o r a l l y administrating 1.0 ml of castor oil to all mice. similar to other studies, various oral treatments were applied to the mice such as nc group received 1% tween-80 in saline, pc group received loperamide hydrochloride (50 mg/kg b.w.) and the remaining two groups were given es-i and es-ii respectively [19]. all groups of mice were housed in individual cages with a blotting paper placed beforehand. the number of diarrheal feces were recorded for each mice over four hours of the experiment. the percentage of diarrheal prohibition was accounted for using the following formula: % of writhing inhibition= (mean writhing of control–mean writhing of test)×100% mean writhing of control pip= (mean latency of treatment–mean latency of control)×100% mean latency of control percentage inhibition= mean defecation of control–mean defecation of test sample or standard mean writhing of control ×100% statistical analysis the values are represented here are set of mean ± standard error of mean (m±sem). all the calculation was performed using student t-test or one way anova followed by dunnett’s test to determine the statistically significant differences between the groups. a p-value < 0.05 was considered statistically significant. results the peripheral analgesic activity of e. serratus crude extract is demonstrated in table 1. a significant reductions of abdominal muscle contractions caused by the administration of 0.1 ml acetic acid were exhibited in both experimental groups where es-ii showed higher writhing inhibition and was close to the pc group. our results indicated that the e. serratus bark crude extracts significantly inhibit the number of writhing 69.77% and 73.26% at dose 200 and 400 mg/kg b.w. gradually whilst diclofenac sodium displayed 75.58% writhing inhibition. values are represented here as mean of ±sem. nc=1% tween 80 in water, pc= diclofenac sodium, es-i: e. serratus crude extract-i, es-ii: e. serratus crude extract-ii. m1-4=mice 1 to 4 respectively. (n=4, *p<0.01) the result of the tail-flick method to assess the central analgesic activity of e. serratus are a.a.h. pinkey et al. 47 p h a r m a c y issn 2413-6077. ijmmr 2020 vol. 6 issue 2 shown in table 2. both experimental groups es-i and es-ii increased the response time by 37.32% and 53.72% respectively in the initial 30 minutes of the experiment, whereas pc morphine increased by 85.24%. in addition, 82.76% (200 mg/kg b.w.), 98.94% (400 mg/kg b.w.) tail flicking response were recorded in 60 minute, and 149.27% (200 mg/kg b.w.) and 179.46% (400 mg/kg b.w.) were recorded in 90 minutes of the experiments. the whole experiment followed a dose-dependent tail flicking response over the time. values are represented here as mean of ±sem. nc= 1% tween 80 in water, pc= morphine sulfate, es-i: e. serratus crude extract-i, and es-ii: e. serratus crude extract-ii. (n=4, *p<0.01) the bark crude extract of e. serratus not displayed any significant blood glucose-lowering activity at doses 200 and 400-mg/kg b.w. however, the percent of blood sugar reducing activity was found to be followed in a dosedependent manner. the results shown in table 3 indicated that the highest glucose lowering activity was displayed at dose 400 mg/kg b.w. relative to es-i groups. values are represented here as mean of ±sem. nc=1% tween 80 in water, pc=gli benclamide, es-i: e. serratus crude extract-i, and es-ii: e. serratus crude extract-ii. (n=4, *p<0.01) the remarkable antidiarrheal activities were displayed by es-i and es-ii in mice. the potential antidiarrheal activity of the e. serratus crude extract is shown in table 4. the es-i and es-ii substantially reduced the number of castor oilincited diarrheal feces by 64.29% and 78.57% compared to the nc. the highest diarrheal reduction was shown by pc group. values are represented here as mean of ±sem. nc=1% tween 80 in water, pc=loperamide hydrochloride, es-i: e. serratus crude extract-i, table 1. peripheral analgesic activity of e. serratus bark crude extract mice group writhing count (sec) number of writhing (mean±sem) % inhibition of writhingm-1 m-2 m-3 m-3 m-4 nc 20 21 23 22 21.50±0.65* – pc 5 6 5 5 5.25±0.25* 75.58 es-i 7 6 6 7 6.50±0.29* 69.77 es-ii 6 6 5 6 5.75±0.25* 73.26 table 2. central analgesic activity of e. serratus bark crude extract mice group 30 minutes of assay 60 minutes of assay 90 minutes of assay m±sem % of elongation m±sem % of elongation m±sem % of elongation nc 3.49±0.32* – 3.55±0.08* – 3.60±0.20* – pc 6.47±0.11* 85.24 9.57±0.25* 169.39 13.19±0.35* 266.07 es-i 4.79±0.34* 37.32 6.49±0.35* 82.76 8.98±0.25* 149.27 es-ii 5.37±0.32* 53.72 7.07±0.39* 98.94 10.07±0.42* 179.46 table 4. antidiarrhoeal activity of e. serratus bark crude extract mice group dose number of diarrheal feces (mean±sem) % reduction of diarrhea nc 10 ml/kg b.w. 3.5±0.58* – pc 50 mg/kg b.w. 0.5±0.48* 85.71 es-i 200 mg/kg b.w. 1.25±0.85* 64.29 es-ii 400 mg/kg b.w. 0.75±0.71* 78.57 table 3. hypoglycemic activity of e. serratus bark crude extract mice group 60 minutes of assay 120 minutes of assay 180 minutes of assay m±sem (mmol/l) % reduction m±sem (mmol/l) % reduction m±sem (mmol/l) % reduction nc 12.60±0.87 8.55±0.31 4.45±0.34 pc 5.63±0.39 55.36 4.23±0.57 50.58 2.6±0.17 41.57 es-i 10.18±0.61 19.25 7.05±0.39 17.54 3.95±0.23 11.24 es-ii 10.08±0.93 20.04 6.85±0.38 19.88 3.75±0.19 15.73 a.a.h. pinkey et al. 48 p h a r m a c y issn 2413-6077. ijmmr 2020 vol. 6 issue 2 and es-ii: e. serratus crude extract-ii. (n=4, *p<0.01) discussion acetic acid may trigger the writhing reflex in experimental animals where visceral pain is generated through activation of pain receptors on the visceral surface and extreme secretion of histamine, prostaglandins, bradykinin and serotonin [20]. in the experimental animals, acetic acid induces visceral pain which is commonly treated with nsaid drugs or chemicals, such as phenyl quine (prostaglandin e2 inhibitor). in addition, the level of analgesia is measured by calculating the percent reduction of abdominal contraction by drugs or crude extract after intraperitoneal administration of acetic acid to mice. in this study, e. serratus extracts significantly reduced the sum of abdominal contraction of 69.77% and 73.26% by es-i and es-ii compared to nc. importantly, the results of peripheral analgesic activity by es-i and es-ii were almost equal to the diclofenac treatment. therefore, by considering our results, we assumed that e. serratus extract may be inhibited the synthesis or release of endogenous substances in mice to act its potential peripheral analgesic activity. however, further research may need to explore the exact mechanisms. in the central analgesic assay, the relative promotion of tail-flicking response (in percent) was obtained from e. serratus extract in a dose and time-dependent manner. although, the responses from e. serratus crude extracts were a bit of lower than the pc-morphine however, higher dose might be shown an equals or higher potentiality like morphine. pizziketti, et al., 1985 demonstrated that the tail flicking response is mostly generated from spinal reflex caused by radiant heat source however it may involve higher neuronal complex signals. in general, the pain is centrally originated via a number of complex signaling such as opiate, dopaminergic, noradrenergic and serotonergic nervous systems [15]. our results described that e. serratus displayed a significantly higher level of pain threshold activity at 200 and 400 mg/kg b.w. respectively in mice model. the core mechanisms may be associated with the receptor-bind inhibition of pain-related nervous system or through peripheral mechanisms involved prohibited prostaglandins, leukotrienes, and other endogenous substances release and synthesis which are key mediators of pain [21]. our results might be followed the same mechanisms to exhibit the potential analgesic activity in mice model. our bark crude extract of e. serratus shown lack of blood glucose lowering activity. notwithstanding, a considerable number of studies have concluded that plant extracts exhibit potential anti-hyperglycemic activity by accelerating or regenerating β cells or promoting the secretion of insulin [22, 23]. the hypoglycemic activity by the natural product may also associated with excessive insulin secretion from β cells or trigger the peripheral glucose consumption, or promote insulin-mediated blood sugar absorbing mechanisms [22-24]. apart from this, the statistical evaluation revealed that both doses of e. serratus showed a significant dose-dependent anti-diarrheal activity in mice. the ricinoleic fatty acid or 12-hydroxy-9-cis-octadecenoic acid is an active metabolite of castor oil. this metabolic fatty acid enhanced peristaltic activity in the small intestine to trigger the permeability of mucosal electrolytes thus resulting diarrhea [25, 26]. furthermore, ricinoleic fatty acid enhanced mucosal irritation and inflammation which cont ribute to the excessive endogenous prostaglandin secretion. moreover, in castor oilinduced diarrheal mechanisms it involved a cascade of signaling including, intestinal na+/ k+-atpase inhibition, adenylate cyclase activation or promotion camp-mediated plateletactivating factor secretion [25, 27]. in summary, the plant e. serratus contained several flavonoids, anthraquinone glycosides, fatty acid, alcohol, aldehyde, hydrocarbons alkaloids, terpenoids, and steroids. [7, 8, 28]. the presence of glycosides, steroids, and flavonoids which exhibited potential analgesic, hypoglycemic and antidiarrheal activities in many plants [29-31]. in the present study, we concluded that e. serratus extract may contain a variety of bioactive phytochemicals. after successful isolation and characterization of phytochemicals, it might be used as an analgesic, and as an antidiarrheal agent. conclusion the bark extract of e. serratus exhibited potential peripheral and central analgesic activity, very mild hypoglycemic activity but effective antidiarrhoeal activity in mice model. therefore, further investigations are needed to isolate and characterization of bioactivite molecules present in this plant. further research may open a new therapeutic agents in the treatments of various diseases. a.a.h. pinkey et al. 49 p h a r m a c y issn 2413-6077. ijmmr 2020 vol. 6 issue 2 acknowledgement all the authors acknowledge that all the experiments were ethically approved by the department of pharmacy, state university of bangladesh. no additional fund was provided for this study. we thankful to dr. azam for assistance with the english improvements. conflict of interests authors declare no conflict of interest. funding no funding was received for this study. author׳s contributions asma aul husna pinkey, mohammad abdullah taher – conceptualization, methodology; asma aul husna pinkey, investigation, data curation, formal analysis, writing – original draft; zahirul islam khan – data curation, formal analysis, writing – reviewing and editing; mahfuza afroz soma – writing – reviewing and editing. преклінічні дослідження знеболюючої та протипроносної дії elaeocarpus serratus l. на мишах a.a.h. pinkey1, *z.i. khan2, m.a. taher1, m.a. soma1 1 department of pharmacy, state university of bangladesh, dhaka, bangladesh 2 department of health technology and informatics, the hong kong polytechnic university, hong kong, china вступ. elaeocarpus serratus l. (родина elaeocarpaceae) тропічне плодове дерево, фрукти, кора та інші частини якого традиційно використовуються при лікуванні отруєнь, діареї, артриту та інших захворювань. мета – дослідити фармакологічну активність (знеболювальну, протидіарейну та гіпоглікемічну дію) сухого метанольного екстракту кори e. serratus на мишах. методи. для експериментальної оцінки центрального та периферичного компонентів у механізмі знеболювальної дії екстракту використовували метод оцінки больової реакції, що викликається хімічним подразненням – метод «оцтовокислих корчів», та метод теплового подразнення, суть якого полягає в зануренні хвоста миші у гарячу воду (55±0.5°c). для оцінки протипроносної активності використовували модель діареї, викликаної введенням рициновою олією, для визначення гіпоглікемічної активності екстракту використали метод durschlag et al., 1996, забір крові проводили шляхом надрізів хвоста. результати. встановлено, що застосування сухого метанольного екстракту кори e. serratus достовірно зменшує частоту розвитку корчів на 69,77% (200 мг/кг) та 73,26% (400 мг/кг) відповідно (p<0,05), що досягає рівня активності стандартного нпзп диклофенаку натрію, який зменшує показник на 75,58% (p<0.05). такі ж результати щодо частоти реакції хвоста піддослідних тварин протягом 30, 60 та 90 хвилин – показника центральної знеболюючої активності екстракту. щодо протипроносної активності, то e. serratus зменшував частоту діареї на 64.26% (200 мг/кг, p<0,05) та 78,57% (400 мг/кг, p<0,05), що також досягало також ж ефективності, які і у групі позитивного контролю з лоперамідом. щодо гіпоглікемічної активності екстракту e. serratus – отримані нами результати були непереконливими. висновок. наше дослідження продемонструвало значну знеболювальну та протидіарейну активність сухого метанольного екстракту кори e. serratus (200 та 400 мг/кг) на мишах. ключові слова: elaeocarpus serratus; знеболювальна активність; протипроносна активність; гіпоглікемічна активність information about the authors asma aul husna pinkey, graduate student, state university of bangladesh, dhaka, bangladesh orcid 0000-0002-6851-269x, e-mail: pinkeykhanam88@gmail.com md zahirul islam khan, full-time phd student, the hong kong polytechnic university, hong kong, china orcid 0000-0001-7048-2613, e-mail: zahir.islamkhan@connect.polyu.hk mohammad abdullah taher, lecturer and coordinator, state university of bangladesh, dhaka, bangladesh orcid 0000-0002-0701-470x, e-mail: taher@sub.edu.bd mahfuza afroz soma, lecturer and student counselor, state university of bangladesh, dhaka, bangladesh orcid 0000-0003-2903-8822, e-mail: soma@sub.edu.bd a.a.h. pinkey et al. 50 p h a r m a c y issn 2413-6077. ijmmr 2020 vol. 6 issue 2 references 1. saleh-e-in mm, sultana n, hossain mn, hasan s, islam mr. pharmacological effects of the phytochemicals of anethum sowa l. root extracts. bmc complement altern med. 2016 dec;16(1):464. doi: https://doi.org/10.1186/s12906-016-1438-9 2. khan mf, khan zi, uddin mr, rahman ms, rashid ma. in vivo hypoglycemic and alloxan induced antidiabetic activity of xeromphis uliginosa retz. afr j pharm pharmacol. 2015;9(11):363-6. doi: https://doi.org/10.5897/ajpp2015.4293 3. al-snafi ae. encyclopedia of the constituents and pharmacological effects of iraqi medicinal plants. rigi publication; 2015. 4. van wyk b-e, wink m. medicinal plants of the world. cabi; 2018. doi: https://doi.org/10.1079/9781786393258. 0000 5. baruah ps, deka k, lahkar l, sarma b, borthakur sk, tanti b. habitat distribution modelling and reinforcement of elaeocarpus serratus l. a thre atened tree species of assam, india for improvement of its conservation status. acta ecologica sinica. 2019;39(1):42-9. doi: https://doi.org/10.1016/j.chnaes.2018. 06.002 6. de lima ff, breda ca, cardoso cal, duarte mct, sanjinez-argandoña ej. evaluation of nutritional composition, bioactive compounds and antimicrobial activity of elaeocarpus serratus fruit extract. afr j food sci. 2019;13(1):30-7. doi: https://doi.org/10.5897/ajfs2018.1760 7. geetha d, rajeswari m, jayashree i. chemical profiling of elaeocarpus serratus l. by gc-ms. asian pac j trop biomed. 2013;3(12):985-7. doi: https://doi.org/10.1016/s2221-1691(13) 60190-2 8. jayasinghe l, amarasinghe nr, arundathie bs, rupasinghe gk, jayatilake nan, fujimoto y. antioxidant flavonol glycosides from elaeocarpus serratus and filicium decipiens. nat prod res. 2012; 26(8):717-21. doi: https://doi.org/10.1080/14786419.2010.55 1514 9. geetha d, jayashree i, rajeswari m. in vitro anti-arthritic activity of elaeocarpus serratus linn. 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2020; revised 11 nov 2020; accepted 14 dec 2020. this is open access article distributed under the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. a.a.h. pinkey et al. 128 b io m e d ic a l s c ie n c e s issn 2413-6077. ijmmr 2019 vol. 5 issue 2 doi 10.11603/ijmmr.2413-6077.2019.2.10901 positive effect of enterosorption in doxorubicin-induced cardiohemodynamics alteration *o.o. shevchuk1, g.v. portnichenko2, t.y. lapikova-bryginska2, s.v. goncharov2, v.g. nikolaev3, v.e. dosenko2 1 – i. horbachevsky ternopil national medical university, ternopil, ukraine. 2 – o.o. bogomoletz institute of physiology of the national academy of science of ukraine, kyiv, ukraine. 3 – r.e. kavetsky institute of experimental pathology, oncology and radiobiology of the national academy of science of ukraine, kyiv, ukraine. background. anthracycline antibiotics are one of the most effective anti-cancer drugs, but their cardiotoxicity what limits its therapeutic use. objective. to analyze the efficiency of enterosorption in doxorubicin-induced cardiohemodynamics violation. methods. subchronic doxorubicin toxicity was modeled by injecting the anthracycline antibiotic intraperitoneally at a dose of 5 mg/kg once a week for 4 weeks, in total 20 mg/kg. male wistar rats were randomly distributed into 3 groups: control; dox-group and dox + enterosorbent c2 rats (γ=0.18 g/cm3, bet area 2162 m2/g). cardiohemodynamics was studied by the millar instruments, heart morphometry – by avtandilov’s method. results. mortality rate in dox-group was 25%. ejection fraction and stroke work indices were lower compared to the control group, preload adjusted maximal power decreased by 57.6%, minimum volume and end-systolic volume increased by 76,2 and 67.5% respectively. end-systolic stiffness of left ventricle (emax) as well as arterial elastance (ea) and end-systolic pressure had tended to decrease. indices of left ventricle (lv) volume at systole increased: v@dpdtmax – by 73.3%, v@dpdtmin – by 81.9%. end-diastolic volume increased by 54.6%. as for the dpdtmin, and tau constant we observed the slight tendency to its decline. endocardial surface of lv increased by 42.7%, planimetric index – by 40.4% compared to the control group of rats. in dox+c2 group mortality rate was 18.75%. we observed the strong tendency to normalization of the main indices compared to the dox group and shrinking of the lv. we want to underline the positive trends especially in ejection fraction (from 39.62±10.50% to 46.23±11.46%) and stroke work (from 6406.50±3345.83 to 10363.14±7329.55 mmhg×ul) as important indicators of the effectiveness of cardiac pump function. conclusions. enterosorption demonstrated positive impact on the doxorubicin-induced violated cardiohemodynamics and decreased the mortality rate. it is a ground for further investigations. key words: doxorubicin-induced subchronic toxicity; heart damage; enterosorption; cardiohemodynamics parameters. *corresponding author. oksana shevchuk, associate professor, pharmacology and clinical pharmacology department, i. horbachevsky ternopil national medical university, maidan voli, 1, ternopil 46001, ukraine. e-mail: shevchukoo@tdmu.edu.ua introduction anthracycline antibiotics are widely used to treat many types of malignancies because of their high efficacy. but, also, they are cardiotoxic, what limits their therapeutic use and cumulative dose [1,2]. doxorubicin (adriamycin, a derivative of rubomycin – 14-hydrorubomycin) is a part of chemotherapy schemes for treatment of breast and prostate cancer, solid tumors in children, sarcomas, and others. multiple mechanisms of heart damage by anthracyclines are recognized. oxidative stress and generation of reactive oxygen species (ros) by “anthracycline-iron” complex; cardiac muscle’s accumulation of highly reactive alcoholic metabolite doxorubicinol (doxol); cytokines disturbances; as a consequence of endogenic intoxication and bacterial translocation because of mucositis – are only a few possible ways of cardiomyocytes injury [3–7]. till today there are no definite and 100% efficient methods for prevention and treatment of anthracyclines-induced cardiotoxicity. ironchelating agent dexrazoxane was implemented into protocols based on its capability to prevent free radical release [8,9]. but there are some facts that this agent could decrease the efficacy of anti-cancer chemotherapy [10]. that is why the search of effective means to ameliorate the cardiotoxicity of anthracyclines, which do not international journal of medicine and medical research 2019, volume 5, issue 2, p. 128-136 copyright © 2019, tnmu, all rights reserved o.o. shevchuk et al. 129 b io m e d ic a l s c ie n c e s issn 2413-6077. ijmmr 2019 vol. 5 issue 2 o.o. shevchuk et al. attenuate the anti-tumor activity of drugs, remains actual. sorption detoxification is a well­ known method for cleaning of body fluids from toxic endogenous or exogenous compounds. the most widely used types of this method are the purification of blood or its components (hemosorption), oral administration of sorption materials (enterosorption), and applicationsorption therapy of wounds and burns [11]. our previous studies with enterosorbents carboline and carbon granular oral adsorbent c2 demonstrated promising results to alleviate the side effects caused by cytostatic agents melphalan and cisplatin (bone marrow suppression, gastrointestinal toxicity, testes damage, etc.) [12–15]. enterosorbent c2, which has optimized and shifted to mesopores porous structure, in combination with an officinal biosimilar of gra­ nulocyte colony­stimulating factor (filgrastim) ameliorated hematologic toxicity and oxidative stress indexes much better than each of these preparations alone [16]. the objective of this study is the assessment of the capability of carbon granular oral adsorbent c2 to diminish the doxorubicininduced heart damage. methods materials doxorubicin hydrochloride (doxorubicin teva 10 mg/5ml, concentrate for solution for infusion, teva pharmachemie, the netherlands) was used for experiments. carbon oral adsorbent c2 was specially designed at the department of means and methods of sorption therapy of r.e. kavetsky institute of experimental pathology, oncology and radiobiology (iepor). parameters of enterosorbent c2 are next: bulk density γ=0.18 g/cm3, granules with a diameter of 0.15–0.25 mm, the porous structure of c2 is well developed and shifted toward mesopores, which surface is 565 m2/g. bet (brunauer emmett-teller) surface area is 2162 m2/g. animal studies all experiments were carried out with male wistar rats, 180-220 g of primary weight, which were reared at tnmu animal facility (ternopil, ukraine). all procedures were done according to the local bioethical committee guidelines which conform to the rules and requirements of european convention for the protection of vertebrate animals used for experimental and other scientific purposes (1986) and eu direc­ tive on the use of animals for research directive 2010/63/eu. a common light-dark cycle was maintained for rats and fed on common rodent chow diet with tap water ad libitum, according to the guidelines for animal care. a well-documented regimen was used for the induction of heart damage by doxorubicin [17]. animals were randomly assigned to 3 groups: 1) control group (n=7); 2) rats treated with dox (dox­group) (n=16); 3) rats treated with both dox and carbon enterosorbent c2 (dox+c2) (n=16). subchronic doxorubicin toxicity was modeled by injecting the anthracycline antibiotic intraperitoneally at a dose of 5 mg/kg once a week for 4 weeks, in total 20 mg/kg [17]. the animals serving as control received the same volume of saline intraperitoneally once a week for a total of 4 weeks. newly designed carbon oral adsorbent c2 was given into the stomach via a custom rigid tube once a day at a dose of 5 ml per kg (or 1 ml for each 200 g of rat body weight; or 900 mg of the dry mass of the enterosorbent). we started enteral sorption therapy the next day after the first injection of doxorubicin. the sorbent was given as a suspension in an appropriate volume of distilled water. the rats of the control group received an equal volume of distill water. on the days of doxorubicin injection and one day before it, the enterosorbent was not given to avoid any pharmacokinetics disruption. cardiohemodynamics measurements for the direct cardiac function evaluation, we used millar pressure-volume (p-v) system (mpvs-300, millar instruments, houston, tx, usa). on the 29th day of the experiment counting from the first injection of doxorubicin, under urethane general anesthesia (1.5 g/kg) the right carotid artery was exposed and ligated distally, the artery was clamped and incised, and a 0.5 cm long 90 pe tube was inserted as a catheter guide. a 2-fr mikro-tip catheter (spr838, millar instruments, houston, tx, usa) was advanced through the guide into the lv under pressure control; a ligature was then tightened around the catheter to avoid blood loss [18]. after stabilization for 5 min, signals were continuously sampled at a sampling rate of 1000 samples/sec by the mpvs-300 system, recorded, and displayed on a personal computer by the powerlab system and charttm v.5.4.2 software (adinstruments, millar instruments) for 15-20 min. the relation of pressure and volume of the left ventricle was performed by software pvan 3.6 (ad instruments, millar instruments) with the conversion of relative volume units (rvu) into absolute one (equation slope 20,25×rvu – 130 b io m e d ic a l s c ie n c e s issn 2413-6077. ijmmr 2019 vol. 5 issue 2 intercept 29,05). the millar p-v system simultaneously and continuously measures left ventricle (lv) pressure (p) and volume (v) from the beating heart, producing characteristic pv loops readings of which a variety of cardiovascular parameters, such as heart rate (hr), cardiac output (co), stroke volume (sv), ejection fraction (ef), stroke work (sw), dp/dtmax, and dp/dtmin are derived. end-systolic pressure (esp), end-systolic volume (esv), end-diastolic pressure (edp), end-diastolic volume (edv), stroke volume (sv), stroke work (sw), maximum dp/dt (dpdtmax), minimum dp/dt (dpdtmin), tau, maximum dv/dt (dvdtmax), minimum dv/dt (dvdtmin), maximum pressure (pmax), minimum pressure (pmin), maximum volume (vmax), and minimum volume (vmin) were also analyzed. morphometrics of the heart to estimate chronic changes of the shape and size, the hearts of the rats were used for measuring and evaluating of the planimetric index. for indirect planimetry of the endocardial surface of the rats’ hearts, ventricles were taken accordingly to avtandilov g.g. method[19] in esypova i.k. et al. modification [20]. we measu­ red the endocardial surfaces of the left (elv) and right ventriculi’s wall area (erv). planimetric index (pi) was calculated as: pi=elv÷erv. where elv is the endocardial surfaces of the left ventricle wall area and erv is the endocardial surfaces of the right ventricle wall area. statistical analysis the normality of data distribution was tested using kolmogorov-smirnov test, homogeneity of variance – levene’s test. mannwhitney test and one-way anova was applied to test the differences between the groups. statistical analysis was performed using microsoft excel хр (usa) and statistica 10.0 (statsoft inc., usa). differences were considered significant if the probability of type i error was less than 0.05. p<0.05 was considered significant. results in dox-group the number of prematurely deceased rats was four, in dox+c2 group – three rats died before the end of the experiment. all abovementioned animals died after the 4th injection of doxorubicin during the last week of the experiment. among survived rats we observed typical clinical signs of heart failure: rats showed clear signs of dyspnea, from mild to severe ascites, different stages of hydrothorax and liver enlargement; their common activities were reduced compared to the rats of the control group. those sings had less intensity compared to the untreated group of animals. the pump function of the heart was analyzed by next parameters: ejection fraction, stroke volume and stroke work and cardiac output, as well as maximal power and preload adjusted maximal power (pamp) (table 1). these parameters are load-dependent and con sequently represent poor contractility indices. increased cardiac output, high heart rate as well as stroke volume are the typical signs of cardiac dysfunction and followed systemic hemodynamic changes and our results supposed it. it was a strong tendency for increasing of the all abovementioned indices, but enteral sorption therapy partly disrupts it. ejection fraction in dox-group rats was lower, but in the group dox+c2, we see the tendency to its normalization. the same tendency was for the stroke work: from 6406.50±3345.83 in rats, which received injections of doxorubicin, it increased to 10363.14±7329.55 mmhg×ul for rats which got oral adsorbent concomitantly. while the index in rats of the control group was 7036.43±5036.46 mmhg×ul. minimum volume increased by 76,2%, endsystolic volume – by 67.5%. for both indices, we observed the tendency for decreasing by oral adsorbent therapy, but they did not come close to the numbers of control group rats. in the rats of dox-group the index of maximal power did not change significantly, but the strong tendency to its decreasing we saw, while preload adjusted maximal power (pamp) was lower by 57.6% compared to the control group. enteral sorption therapy promoted the tendency to normalization of the indices. end-systolic stiffness of left ventricle (emax) had a strong tendency to decreasing from 4.55±2.93 to 2.74±2.02 (what means that left ventricle was dilated and lost end-systolic elastance), while carbon oral adsorbent c2 increased this index to 5.30±0.44. specific parameters as the volume at the point of maximal speed of pressure change (v@dpdtmax) and volume at the point of maximal speed of pressure decline (v@dpdtmin) are used for assessment of lv volume at systole. so, v@dpdtmax increased by 73.3%, while v@dpdt min – by 81.9%. during diastole, the myocardium stops shortening and generating force and relaxes. diastolic function was analyzed by changes of end-diastolic pressure and volume, the peak rate of pressure decline (dpdtmin) – isovolumic relaxation, constant tau by weiss method (τw). o.o. shevchuk et al. 131 b io m e d ic a l s c ie n c e s issn 2413-6077. ijmmr 2019 vol. 5 issue 2 o.o. shevchuk et al. end-diastolic volume increased by 54.6% in the group of rats, which received doxorubicin compared to the control rats. as for the dpdtmin, we observed a slight tendency to its decline, as well as for the tau constant. after 4 injections of doxorubicin on 29th day of the experiment, the endocardial surface of the left ventricular wall area increased by 42.7% (p<0.001) compared to control group of rats (table 2). at the same time, there were no changes in the right ventricular wall area. in rats which received enterosorption together with doxorubicin, the endocardial surface of the left ventricular wall area index decreased table 1. cardio-hemodynamics indices in rats, which received doxorubicin and enteral sorption therapy with oral carbon adsorbent c2. index control group dox group dox + с2 group hr (min−1) heart rate 321.0±43.89 356.64±48.82 378.38±33.86 maximum volume (ul) 146.79±13.08 232.76±76.58 236.68±102.07 minimum volume (ul) 78.27±15.17 137.93±46.96* 126.05±35.68 end-systolic volume (ul) 84.87±17.73 142.16±47.56* 130.76±36.68 end-diastolic volume (ul) 142.56±11.36 220.44±70.94* 223.75±85.04 maximum pressure (mmhg) 116.24±21.53 102.0±22.51 111.85±14.35 minimum pressure (mmhg) 4.97±3.89 4.15±3.39 2.10±1.65 end-systolic pressure (mmhg) 109.57±25.12 96.22±24.07 104.58±16.72 end-diastolic pressure (mmhg) 9.10±4.28 6.72±4.54 5.99±3.77 stroke volume (ul) 68.52±21.34 94.835±39.90 120.88±78.45 ejection fraction (%) 46.15±11.83 39.62±10.50 46.23±11.46 cardiac output (ul/min) 22222.85±8424.99 33103.55±13814.04 44721.45±28511.89 stroke work (mmhg×ul) 7036.43±5036.46 6406.50±3345.83 10363.14±7329.55 arterial elastance (ea), (mmhg/ul) 2.25±0.71 1.06±0.49 1.11±0.56 dpdt max (mmhg/sec) 11758.0±5232.28 9897.43±3142.76 12769.50±2861.17 dpdt min (mmhg/sec) -7312.86±2477.79 -7062.50±1742.62 -8928.12±3274.57 dvdt max (ul/sec) 2811.14±1048.07 4783.57±1703.76 4489.87±2985.50 dvdt min (ul/sec) -3345.57±1283.04 -3893.5±1345.52 -4751.13±2491.48 p@dvdt max (mmhg) 38.27±32.11 40.16±35.79 34.62±36.27 p@dpdt min (mmhg) 88.67±50.41 64.69±13.78 74.00±21.68 v@dpdt max (ul) 124.90±25.62 216.46±76.45* 228.02±102.85* v@dpdt min (ul) 79.91±15.49 145.33±52.64* 129.76±36.71 tau(w) (msec) 12.69±5.87 11.41±5.01 9.25±1.96 tau(g) (msec) 18.34±11.18 13.80±5.68 11.90±3.39 maximal power (mwatts) 43.89±34.61 38.94±18.71 57.61±32.30 preload adjusted maximal power, pamp (mwatts/µl^2) 20.81±15.88 8.82±4.78* 12.38±5.77 emax 4.55± 2.93 2.74±2.02 5.30±0.44 notes. the data are expressed as means (m) ± standard deviation (sd);* – p<0,05 comparing to control group; dpdtmax – peak rate of pressure rise; dpdtmin – peak rate of pressure decline; dvdtmax – peak rate of volume rise; dvdtmin – peak rate of volume decline; p@dvdtmax – pressure at dv/dt max; p@dpdtmin – pressure at dv/dtmin; v@dpdtmax – volume at dp/dtmax; v@dp/dtmin – volume at dp/dtmin; tau (g) – relaxation time constant calculated by glantz method (regression of dp/dt versus pressure); tau (w) – relaxation time constant calculated by weiss method (regression of log(pressure)); emax – end-systolic elastance. table 2. the influence of enterosorption on morphometric indexes of the heart ventricles in subchronic doxorubicin toxicity in rats. index control group dox-group dox+с2 group the endocardial surface of left ventricular wall area, mm2 118.0±4.45 168.4±7.63* 132.6±3.06*, ** the endocardial surface of right ventricular wall area, mm2 132.2±6.27 134.8±6.54 137.8±4.88 planimetric index (pi) 0.894±0.010 1.25±0.032* 0.965±0.022** notes: the data are expressed as means (m) ± standard error (se). p<0.05 compared to * – control group; ** – dox-group. 132 b io m e d ic a l s c ie n c e s issn 2413-6077. ijmmr 2019 vol. 5 issue 2 by 21.3% compared to the dox group, but it was still larger than in rats of the control group. doxorubicin injections increased the planimetric index (pi) the by 40.4% compared to the control group (from 0.89±0.01 to 1.25±0.03), while in dox+с2 group it decreased by 22.8% (0.96±0,02, p<0.001). discussion our study deals with the effect of enterosorption on doxorubicin-associated cardiac toxicity. doxorubicin’s use in patients is limited by its cardiac toxicity. today a new subspecialty appeared – cardio-oncology, which focuses on prevention, detection, monitoring, and treat_ ment of cardiovascular pathology during anticancer chemotherapy [21]. it is a marker of the high importance of this problem because long term survival of childhood cancers is more than 70% for now [6] and continued to increase [22]. strong links between cancer and heart disease are recognized, that is why a clinical need for optimized cardio-oncology patient management is growing. among anti-cancer agents, the most capable drugs to cause the dilative cardiomyopathy are anthracyclines and cyclophosphamide [16,23]. dose-dependent irreversible heart damage occurs in 1.7% of patients mostly via oxidative stress activation and by inhibition of transcriptions of genes, which are responsible for the synthesis of the contractile proteins [3,16]. up to 3% of heart transplantations were done for patients because of doxorubicin therapy [6]. it is known that the prognosis of patients who develop doxorubicin-induced congestive heart failure is poor: approximately ~50% mortality in 1 year [3]. monoclonal antibody trastuzumab and low molecular tyrosine kinase inhibitors as sunitinib and sorafenib may cause heart damage too [23]. they modulate mitochondrial integrity, deplete atp and lead to contractile dysfunction. but in this case, the contractile function of the left ventricle improves after drugs discontinuation [23]. we used typical widespread modeling to induce congestive heart failure in rats: four injections of dox at the dose of 5 mg/kg for cumulative dose 20 mg/kg and got the cardiohemodynamic disruption [17]. so, this model could be used for assessment of the capability of different substances and drugs to impact the heart systolic and diastolic function. one of the experimental morphometric methods to measure and estimate the type and deepness of heart injury is weighing and weight measurement of different parts of the organ namely left and right ventricle with the septum (ventricle index, fulton index, etc.). the planimetric method allows estimating changes of both ventricles by measuring the endocardial surfaces area [19]. and this method is validated to estimate the chronic changes of the heart morphology, while cardiohemodynamics violations measured by millar instruments are quite good for assessment of acute functional changes in heart work. a wide variety of indexes that can be quantified by analyzing pressure­volume (pv) loops have been proposed to characterize the left ventricle systolic and diastolic performance. in the present study, doxorubicin-associated cardiac dysfunction was manifested by a reduction in cardiac systolic and diastolic hemodynamic function. we have shown statistically significant differences between dox and cont­ rol groups in parameters of end-systolic and end-diastolic volumes as well as volumes at the point of maximal speed of pressure change and pressure decrease. also, we have shown a 57.6% decrease in preload adjusted maximal power. doxorubicin at the cumulative dose of 20 mg per kg promoted the heart dilation, which was confirmed by increased indices of the endocardial surface of the left ventricular wall area and planimetric index. our previous study demonstrated the decreased mass of the heart in subchronic doxorubicin toxicity. so, despite only the slight tendency of ejection fraction declining, these important changes already indicate the onset of the dilated cardiomyopathy. such results are supported by research on the male new zealand white rabbits with doxorubicin-induced heart damage [24]. so, we may conclude, that early myocardial effects of doxorubicin-induced cardiotoxicity are presented. we may talk about early stages of dilated cardiomyopathy with still preserved ejection fraction, but with clinical signs of congestion in survived rats – non-failing dilated left ventricle in survived animals. our results are confirmed by the study of lodi m. et al. [25]: a significantly reduced ejection fraction was seen on day 80 only. they modeled cardiomyopathy by 6 iv injection of dox at the dose of 1.5 mg/kg on the 8th, 11th, 14th, 17th, 20th and 23rd days of the experiment [25]. also, the results of our histological examination of heart tissues presented revealed loss of myofibrils and striations, as well as cytoplasmic edema. our previous study demonstrated that enterosorption with c2 ameliorates the morphological sings of heart damage [26]. also, we o.o. shevchuk et al. 133 b io m e d ic a l s c ie n c e s issn 2413-6077. ijmmr 2019 vol. 5 issue 2 o.o. shevchuk et al. observed improvements of hematological parameters, kidney’s function and decrease of endogenous intoxication markers. those data are in press. it is important to mention that the concomitant course of enterosorption during this experiment decreased the mortality rate. in rats which received doxorubicin, it was 25% (4 rats from 16), while at the dox+c2 group – 18.75% (3 rats from 16). dox+c2 rats’ group has shown a statistically significant difference compared to the control group in the parameter of volume at the point o f m a x i m a l s p e e d o f p re s s u re c h a n g e . v@dpdtmax increased by 82.6%. all 26 parameters of cardiohemodynamics were altered in rats which received doxorubicin at the total dose of 20 mg/kg. more than a half (14 parameters) among them demonstrated tendency to normalization under the influence of enteral sorption therapy. especially we want to notice the positive tendency in indices of preload adjusted maximal power, pamp (from 8.82±4.78 to 12.38±5.77 mwatts/µl^2), maximal power (from 38.94±18.71 to 57.61±32.30 mwatts, and it was even higher than in the control group – 43.89±34.61 mwatts), stroke work (from 6406.50±3345.83 to 10363.14±7329.55 mmhg×ul, while the control group index was 7036.43± 5036.46 mmhg×ul) and ejection fraction from 39.62±10.50% to 46.23±11.46%, when in the control group it was 46.15±11.83%). conclusions doxorubicin at the total dose of 20 mg/kg caused pronounced violation of cardio hemodynamics. systolic indices as ejection fraction, stroke work, end-systolic elastance (emax), endsystolic pressure – all these indices demonstrated a tendency to decline, preload adjusted maxi_ mal power (pamp) was lower by 57.6% compared to the control group. it is a marker of weaker pump function and poor contractility of the heart. morphometry showed dilation of the left ventricle and increased planimetric index. at the same time, the diastolic indices were disrupted too. end­diastolic volume significantly increased by 54.6%, the index of peak rate of pressure had a tendency for declining, as well as tau(w). the indices of volume at the point o f m a x i m a l s p e e d o f p r e s s u r e c h a n g e (v@dpdtmax) and volume at the point of maximal speed of pressure decline (v@dpdtmin) significantly increased in rats, which received doxorubicin. it confirms the diastolic dysfunction presence. enteral sorption therapy mostly normalized and improved violated indices and decreased the rate mortality of rats. we observed shrinking of the endocardial surface of the left ventricular wall area by 21.3% and decreasing of planimetric index. those results demonstrate that enterosorption could prevent remodeling of the heart chambers. our cardiohemodynamics investigations included more than 20 parameters and though mostly they are not statistically significant we want to underline the positive trends in dox+c2 rats comparing to dox-group, especially in ejection fraction and stroke work parameters as they are the important indicators of the effectiveness of cardiac pump function. such results could be explained by the fact that measurement of hemodynamics was done one week later after the last 4th injection of doxorubicin, and we observed the consequences of mechanisms of adaptation in survived rats. our research demonstrated promising results of the efficiency of carbon granular oral adsorbent c2 to ameliorate the doxorubicinassociated cardiohemodynamics changes and are the ground for further future investigation of different combinations of enterosorption and cardio-tropic drugs. funding this research received no external funding. conflict of interests the authors declare no conflict of interest. author contributions shevchuk o.o. – investigation, conceptualization, resourses, writing – original draft; portnichenko g.v. – formal analysis, visualization, investigation, writing – original draft, data curation, lapikova-bryginska t.y. – investigation, data curation, goncharov s.v. – investigation, data curation; nikolaev v.g. – conceptualization, project administration, writing (review and editing), supervision; dosenko v.e. – project administration, writing (review and editing), supervision. 134 b io m e d ic a l s c ie n c e s issn 2413-6077. ijmmr 2019 vol. 5 issue 2 позитивний вплив ентеросорбції на порушення кардіогемодинаміки, спричинені доксорубіцином о.о. шевчук1, г.в. портніченко2, т.є. лапікова-бригінська2, с.в. гончаров2, в.г. ніколаєв3, в.є. досенко2 1 – тернопільський національний медичні університет імені і.я. горбачевського, тернопіль, україна 2 – інститут фізіології імені о.о, богомольця нан україни, київ, україна 3 – інститут експериментальної патології, онкології і радіобіології імені р.є. кавецького нан україни, київ, україна вступ. кардіотоксичність протипухлинних лікарських засобів, і особливо антрациклінових антибіотиків, є одним з лімітуючих факторів ефективного лікування злоякісних новоутворів. мета. дослідити можливості ентеросорбції для пом’якшення кардіогемодинамічних змін, викликаних доксорубіцином в експерименті. методи. субхронічна доксорубіцинова токсичність моделювалася чотирьохкратним введенням доксорубіцину інраперитонеально в дозі 5 мг/кг один раз на тиждень протягом 4 тижнів у сумарній кумулятивній дозі 20 мг/кг. щури були рандомізовані у 3 групи: контроль, група тварин, що отримувала доксорубіцин (dox-група) та групу, котра окрім останнього отримувала ентеросорбент с2 (γ=0.18 г/см3, bet – 2162 м2/г). параметри кардіогемодинаміки вивчалися за допомогою millar instruments, морфомтричні зміни серця – за методом автанділова. результати. летальність у dox-групі склала 25%. показники фракції викиду та ударної роботи серця знижувалися порівняно з показниками контрольної групи. показник максимальної потужності, зрівноваженої на переднавантаження був достовірно нижчим на 57,6%, а мінімальний об’єм та кінцевосистолічний об’єм зросли на 76,2 та 67,5%, що свідчить про розвиток застійних явищ. показники v@dpdtmax зросли на 73.3%, v@dpdtmin – на 81.9%. кінцево-діастолічний об’єм був вищим на 54.6%. спостерігалася тенденція до зниження dpdtmin та tau константи. ендокардіальна поверхня лівого шлуночка зросла на 42,7%, а планіметричний індекс – на 40,4%. у групі dox+c2 летальність склала 18,75%. спостерігалася виражена тенденція до нормалізації усіх показників. особливо ми хочемо підкреслити позитивний ефект застосування вуглецевого ентеросорбента с2 на показники фракції викиду (з 39.62±10.50% до 46.23±11.46%) та ударної роботи (з 6406.50±3345.83 до 10363.14±7329.55 мм.рт.ст×мкл) як важливих показників насосної функції серця. висновки. в статті наведені дані, котрі демонструють здатність ентеральної сорбційної терапії зменшувати зрушення показників кардіогемодинаміки, спричинені введенням доксорубіцину. окрім цього, ентеросорбція сприяла зменшенню показника летальності піддослідних тварин. ключові слова: cубхронічна доксорубіцинова токсичність; пошкодження серця; ентеросорбція; параметри кардіогемодинаміки. відомості про авторів шевчук оксана – канд. мед. наук, доцент кафедри фармакології з клінічною фармакологією, тернопільський національний медичний університет імені і.я. горбачевського, тернопіль , україна портніченко георгій – канд. біол. наук, відділ загальної та молекулярної патофізіології, інститут фізіології імені о.о.богомольця нан україни, київ, україна. лапікова-бригінська тетяна – відділ загальної та молекулярної патофізіології, інститут фізіології імені о.о.богомольця нан україни, київ, україна. гончаров сергій – відділ загальної та молекулярної патофізіології, інститут фізіології імені о.о.богомольця нан україни, київ, україна. ніколаєв володимир григорович – чл.­кор. нан україни, професор, д­р мед. наук, завідувач відділу засобів та методів сорбційної терапії, інститут експериментальної патології, онкології і радіо­ біології імені р.є. кавецького нан україни, київ, україна. досенко віктор євгенович – професор, д­р мед. наук, завідувач відділу загальної та молекулярної патофізіології інституту фізіології імені о.о. богомольця нан україни, київ, україна. information about the authors oksana o. shevchuk – md, ph.d., pharmacology and clinical pharmacology department, i. horbachevsky ternopil national medical university, ternopil, ukraine. orcid 0000­0003­2473­6381, e­mail: shevchukoo@tdmu.edu.ua georgii v. portnichenko – ph.d., department of general and molecular pathophysiology, bogomoletz institute of physiology of the national academy of science of ukraine, kyiv, ukraine. orcid 0000­0001­8834­3013, e­mail: krissaegrim1@gmail.com o.o. shevchuk et al. 135 b io m e d ic a l s c ie n c e s issn 2413-6077. ijmmr 2019 vol. 5 issue 2 o.o. shevchuk et al. tetiana y. lapikova-bryhinska – department of general and molecular pathophysiology, bogomoletz institute of physiology of the national academy of science of ukraine, kyiv, ukraine. orcid 0000­0003­3405­6566, e­mail: tlapikovabr@gmail.com sergii v. goncharov – department of general and molecular pathophysiology, bogomoletz institute of physiology of the national academy of science of ukraine, kyiv, ukraine. e­mail: sergei_goncharov@bigmir.net volodymyr g. nikolaev – md, ph.d., dsc, professor, department of means and methods of sorption therapy, r.e. kavetsky institute of experimental pathology, oncology and radiobiology of the national academy of science of ukraine, kyiv, ukraine. e­mail: aosiepor2@gmail.com victor e. dosenko – md, ph.d., dsc, professor, department of general and molecular pathophysiology, bogomoletz institute of physiology of the national academy of science of ukraine, kyiv, ukraine. orcid 0000­0002­6919­7724, e­mail: dosenko@biph.kiev.ua references 1. jain d, russell rr, schwartz rg, panjrath gs, aronow w. cardiac complications of cancer therapy: pathophysiology, identification, prevention, treat ment, and future directions. curr cardiol rep 2017;19:36. doi:10.1007/s11886­017­0846­x 2. linnik o, drevytska ti, tarasova k, portnichenko g, dosenko ve, mankovskaya in. doxorubicininduced disturbances of cardiomyocyte contractile activity. int j physiol pathophysiol 2018;9:9–16. doi:10.1615/intjphyspathophys.v9.i1.20 3. chatterjee k, zhang j, honbo n, karliner js. doxorubicin cardiomyopathy. cardiology 2010;115: 155–62. doi:10.1159/000265166 4. curigliano g, cardinale d, dent s, criscitiello c, aseyev o, lenihan d, et al. 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advantages of prophylactic versus conventionally scheduled heart failure therapy in an experimental model of doxorubicin-induced cardiomyopathy. j transl med 2019;17:229. doi:10.1002/ejhf.1197 26. shevchuk oo, datsko t v., posokhova ka, nikolaev vg. histological findings at application of newly designed granular carbon enterosorbent under subchronic doxorubicin toxicity in rats. pharmacol drug toxicol 2019;13:119–30. received 04 september 2019; revised 09 october 2019; accepted 21 october 2019. this is open access article distributed under the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. o.o. shevchuk et al. 5 in t e r n a l m e d ic in e issn 2413-6077. ijmmr 2020 vol. 6 issue 1 *corresponding author: abdulraheem b. bawa-allah, department of physiology, college of medicine, university of lagos, ishaga road, idi-araba, 01234, lagos. e-mail: abbawaallah@gmail.com. international journal of medicine and medical research 2020, volume 6, issue 1, p. 5-13 copyright © 2020, tnmu, all rights reserved a.b. bawa-allah et al. doi 10.11603/ijmmr.2413-6077.2020.1.11014 serum lipid profile and arterial stiffness in non-dippers *a.b. bawa-allah1, m.m. mashao2, t.f. nyundu2, e.m. phukubje2, b.g. nkosi2, m.v. ngema2, b.w. mlambo2, m.j. maseko2 1 – college of medicine, university of lagos, lagos, nigeria 2 – university of the witwatersrand, johannesburg, south africa background. a non-dipping blood pressure profile (ndp) is associated with increased arterial stiffness and other cardiovascular target organ damage. serum lipid profiles have been shown to be important determinants of arterial stiffness. objective. the aim of the research was to assess serum lipid profiles and arterial stiffness in non-dippers. methods. this cross-sectional study was conducted involving 796 (288 males and 508 females) participants of black african origin. a twenty-four-hour ambulatory bp monitoring was done using a spacelabs 90207 (spacelabs inc., redmond, washington, usa) monitor. carotid-femoral pulse wave velocity measurements were performed using a high fidelity spc-301 micromanometer (millar instruments inc., houston, tx). results. of the 288 males, 140 were classified as non-dippers. of the 508 females, 273 were classified as non-dippers. in the general population, males had higher triglycerides when compared with females 1.46±0.96 vs 1.13±1.02, p<0.0001. additionally, dipper males had higher serum trgl when compared with dipper females 1.32±0.98 vs 1.06±0.58, p = 0.0012. non-dipper males also had higher serum trgl when compared with nondipper females 1.61±2.05 vs 1.19±1.14, p=0.0078. serum hdlc was lower in the non-dipper male group when compared to the non-dipper female group (p=0.008). in both male and female groups, non-dippers had higher cfpwv when compared with dippers; 7.53±3.60 vs 5.74±2.47, p<0.0001 and 6.64±2.52 vs 5.98±2.23, p=0.0021 respectively. however, non-dipper males had significantly higher cfpwv when compared with non-dipper females (7.53±3.60 vs 6.64±2.52, p=0.0031). conclusions. therapies targeting a reduction of serum triglycerides levels might be beneficial in improving arterial compliance with or without the presence of non-dipping. keywords: non-dipping; arterial stiffness; lipids. introduction a non-dipping blood pressure profile (ndp) is defined as failure of the night-time blood pressure (bp) values to drop by a minimum of 10% of the daytime values [1]. a non-dipping blood pressure profile (ndp) is associated with increased arterial stiffness and other cardiovascular target organ damage and is also a stronger predictive index of cardiovascular morbidity and mortality [2]. arterial stiffness, which is one of the complications of non-dipping, is an established marker of arteriosclerosis and subclinical atherosclerosis and is an independent predictor of future adverse cardiovascular events [3]. various techniques are available to assess arterial stiffness but some of these are inconvenient. carotid-femoral pulse wave velocity (cf-pwv) is a simpler, practical and more reproducible way of evaluating arterial stiffness. it is considered the ‘gold standard’ for the measurement of arterial stiffness [4]. it has been proved that increase in arterial stiffness is an independent predictor of all causes of cardiovascular morbidity and mortality [5]. cardiovascular risk factors such as age, diabetes and hypertension have been established to be positively associated with arterial stiffness [6]. however, the contribution of serum lipids to the development of arterial stiffness is not clear. studies on the relationship between serum lipids and arterial stiffness have been conflicting [7, 8]. additionally, there is no data investigating the possible role of serum lipid profile in non-dipping related increase in arterial stiffness. thus, the study was focused on describing the serum lipid profile in nondipping related increase in arterial stiffness measured by cf-pwv. methods study population this study was approved by the human research ethics committee hrec (medical) of the university of the witwatersrand johannesburg south africa (hrec approval number 6 in t e r n a l m e d ic in e issn 2413-6077. ijmmr 2020 vol. 6 issue 1 m170213). this study was carried out according to the helsinki declaration on ethical principles for medical research involving human subjects. the participants gave informed, written consents before the commencement of the study. standard questionnaire was given to the participants to record data on medical history, smoking habits, alcohol intake, gender and age. the participants were randomly recruited from the soweto area of johannesburg south africa and were of black african origin. of the 1,219 participants enrolled in this study only 796 had complete 24-hour ambulatory bp monitoring reports and their data was included in the statistical analysis. the minimum age of the participants was 18 years old. anthropometric measurements height, weight, waist and hip circumference were measured while the participants were in the standing position with no shoes on. height was expressed in meters (m) while waist and hip circumferences were expressed in centimetres (cm). body mass index (bmi) was expres sed as kg/m2. ambulatory blood pressure measurements a twenty-four-hour ambulatory bp monitoring was carried out during the participants’ typical working day or a day involving their usual activities using a spacelabs 90207 (spacelabs inc., redmond, washington, usa) monitor. the monitor was programmed to measure bp at 15-minute intervals from 06:00 to 22:00 and then 30-minute intervals from 22:01 to 05:59. participants were instructed to note the time they go to bed and the time they wake up in the morning in an activity diary which was used to determine ‘awake’ and ‘asleep’ periods. upon completion, data was transferred from the ambulatory bp monitors to a computer for analysis. a recording was considered as successful if at least 90% of valid recordings were obtained. participants were classified as dippers if their nocturnal drop in 24-hour systolic bp was >10% and as non-dippers if they had a nocturnal drop in 24-hour systolic bp of <10%. carotid-femoral pulse wave velocity (cfpwv) measurement carotid-femoral pwv measurements were conducted to determine the level of arterial stiffness. after resting for about 15 minutes in the supine position, cf-pwv was measured using a high fidelity spc-301 micromanometer (millar instruments inc., houston, tx) interfaced with a computer running sphygmocor software version 9.0 (atcor medical pty. ltd., west ryde, new south wales, australia). pulse pressure waveforms were recorded from the right common carotid artery and the right femoral artery. pulse wave velocity was calculated automatically by the software. blood sample collection venous blood samples to determine serum lipid profiles were taken for the participants by an experienced qualified nurse who is part of the research group. the participants were asked to relax, and blood sample was collected in the sitting position. for plasma analyses, blood samples were collected into 9 ml edta vacuette® (greiner bio one international gmbh) blood collection tubes. blood samples were collected into 5ml vacuette® serum gel tubes, gently inverted and allowed to clot for 30 minutes. the sample was then centrifuged at 2200 g for 15 minutes and the serum was carefully removed with a fine-bore pipette and then stored in properly labelled sterile vials and stored at 8 °c before pick-up by the laboratory. samples were then picked up by the staff of cls (clinical laboratory services) at the national health laboratory service (nhls), south africa, for analysis. all samples were taken to the laboratory within 7 days of blood collection. statistical analysis a l l d a t a w e re a n a l y s e d u s i n g s tata (statacorp llc texas usa) data analysis and statistical software version 13.0. data were expressed as mean ± standard deviation (sd) for continuous variables. categorical variables were expressed as absolute or relative frequencies or as percentages. a test for normality of continuous variables was assessed using shapiro-wilk’s statistic or χ2 test for categorical variables. comparisons between dipper and non-dipper groups as well as gender groups were done using independent student’s t-test or one-way analysis of variance (anova) (with post hoc bonferroni tests). simple regression was used to determine the relationship between carotid femoral pulse wave velocity and age in both genders. a p-value of <0.05 was considered as statistically significant. results demographic and clinical characteristics the study population consisted of 796 participants and was classified according to gender and bp dipping status. there were a total of 288 males and 508 females. of the 288 males, 148 were classified as dippers while 140 were classified as non-dippers. of the 508 females, 235 were classified as dippers while 273 were classified as being non-dippers. in a.b. bawa-allah et al. 7 in t e r n a l m e d ic in e issn 2413-6077. ijmmr 2020 vol. 6 issue 1 both gender groups, non-dippers were older than dippers; 49.79±18.84 vs 39.06±17.88, p<0.0001 in the male group and 46.61±17.45 vs 41.25±17.33, p=0.0003 in the female group. body mass index (bmi) was higher in females when compared to males in the general population 31.13±7.89 vs 24.96±5.14, p<0.0001. additionally, females had higher bmi than males irrespective of dipping status. waist circumference was higher in the female nondipper group when compared with the male dipper group (88.23±15.22 vs 83.42±13.31, p=0.02) and also higher in the female nondipper group when compared with the female dipper group (94.13±17.38 vs 89.50±15.35, p=0.0008). females had higher waist circu mference when compared with males irrespective of dipping status and gender group. there were more hypertensive participants in the nondipper groups when compared with the dipper groups irrespective of gender. there were also more diabetic participants in the male nondipper group when compared to the male dipper group. males in the general population had higher triglycerides (trgl) when compared with females in the general population 1.46±0.96 vs 1.13±1.02, p<0.0001. additionally, dipper males had higher serum trgl when compared with dipper females 1.32±0.98 vs 1.06±0.58 p=0.0012. non-dipper males also had higher serum trgl when compared with non-dipper females 1.61±2.05 vs 1.19±1.14, p=0.0078. within the same gender group, serum triglycerides were not different between dipper and nondippers. serum hdlc was lower in males when compared with females in the general population (p<0.0001). serum hdlc was also lower in the non-dipper male group when compared to the non-dipper female group (p=0.008). females had higher serum ldlc when compared to males in the general population 2.72±0.89 vs 2.48±0.62, p<0.0001. serum ldlc levels did not differ between dippers and non-dippers within gender groups; however, ldlc levels were higher in the female non-dipper group when compared with the male non-dipper group (p<0.05). there were no differences in other clinical and demographic characteristics. these results are presented in table 1. carotid femoral pulse wave velocity (cfpwv) by gender and dipping status in both male and female groups, nondippers had higher cfpwv when compared with dippers; 7.53±3.60 (n=130) vs 5.74±2.47 (n=134), p<0.0001 and 6.64±2.52 (n=232) vs 5.98±2.23 (n=203), p=0.0021 respectively. nondipper males however had significantly higher cfpwv when compared with non-dipper females (7.53±3.60 vs 6.64±2.52, p=0.0031). these results are presented in figure 1. relationship between age and carotid femoral pulse wave velocity (cfpwv) carotid femoral pulse wave velocity increased with age amongst males and females irrespective of dipping status. however, the age-related increase in cfpwv was steeper in males when compared to females (p<0.0001) irrespective of dipping status. the age-related increase in cfpwv was steeper in both nondipper males and females when compared to dipper males and dipper females (p<0.0001). these results are presented in figure 2. fig. 1. carotid femoral pulse wave velocity (cfpwv) of the participants according to blood pressure dipping status and gender. results are presented as mean±sd. male female 0 5 10 15 dippers non-dippers p<0.0001 p=0.0031 p=0.0021 c fp w v ( m /s ) a.b. bawa-allah et al. 8 in t e r n a l m e d ic in e issn 2413-6077. ijmmr 2020 vol. 6 issue 1 ta bl e 1. d em og ra ph ic a n d cl in ic al c h ar ac te ri st ic s of p ar ti ci pa n ts a cc or di n g to g en de r an d bl oo d pr es su re d ip pi n g st at u s m al es fe m al es a ll d ip pe rs n on -d ip pe rs p a ll d ip pe rs n on -d ip pe rs p n (% ) 28 8 (3 6) 14 8 (5 1. 40 ) 14 0 (4 8. 61 ) 50 8 (6 4) 23 5 (4 6. 25 ) 27 3 (5 3. 74 ) ag e (y ea rs ) 44 .2 ±1 9. 09 39 .0 6± 17 .8 8 49 .7 9± 18 .8 4# <0 .0 00 1* 44 .1 4± 17 .5 8 41 .2 5± 17 .3 3 46 .6 1± 17 .4 5 0. 00 03 * b m i ( kg m -2 ) 24 .9 6± 5. 14 £ 24 .3 2± 4. 92 ɸ 25 .6 3± 5. 30 # 0. 35 31 .1 3± 7. 89 30 .1 2± 7. 38 32 .0 0± 8. 22 0. 30 w ai st c ir c (c m ) 85 .7 6± 14 .4 5£ 83 .4 2± 13 .3 1ɸ 88 .2 3± 15 .2 2# 0. 00 2* 91 .9 8± 16 .6 2 89 .5 0± 15 .3 5 94 .1 3± 17 .3 8 0. 00 08 * sm ok er s (% ) 36 % 36 % 31 % 5% 6% 3% al co ho l u se (% ) 38 % 40 % 36 % 11 % 11 % 10 % h yp er te ns iv e (% ) 8% 25 % 50 % 40 % 33 % 46 % d ia be tic s (% ) 10 % 5% 14 % 8% 6% 5% tc h o l (m m ol /l ) 4. 47 ±1 .0 6 4. 45 ±1 .0 5 4. 48 ±0 .9 9 0. 40 1 4. 67 ±1 .0 2 4. 59 ±1 .0 3 4. 57 ±1 .3 1 0. 42 5 tr g l (m m ol /l ) 1. 46 ±0 .9 6£ 1. 32 ±0 .9 8ɸ 1. 61 ±2 .0 5# 0. 06 1. 13 ±1 .0 3 1. 06 ±0 .5 8 1. 19 ±1 .1 4 0. 06 h d lc (m m ol /l ) 1. 35 ±0 .4 2 1. 40 ±0 .5 3 1. 30 ±0 .4 3# 0. 08 1. 44 ±0 .2 1 1. 47 ±0 .4 4 1. 44 ±0 .3 8 0. 20 5 ld lc (m m ol /l ) 2. 48 ±0 .6 2£ 2. 45 ±0 .8 7 2. 50 ±0 .8 7# 0. 31 31 2. 72 ±0 .8 9 2. 63 ±0 .9 4 2. 79 ±1 .0 6 0. 09 1 bm i = bo dy m as s in de x; h ip c ir c, h ip c ir cu m fe re nc e; w ai st c ir c, w ai st c ir cu m fe re nc e; t ch o l = to ta l c ho le st er ol ; t rg l = tr ig ly ce ri de s; h d lc = h ig h de ns ity li po pr ot ei n ch ol es te ro l; ld lc = l ow de ns ity li po pr ot ei n ch ol es te ro l. d at a w er e pr es en te d as m ea n± sd o r nu m be r (% ). *a p v al ue ≤ 0. 05 d ep ic ts s ig ni fic an t di ff er en ce b et w ee n di pp er s an d no ndi pp er s of s am e ge nd er . # d ep ic ts si gn ifi ca nt d iff er en ce (p <0 .0 5) b et w ee n no ndi pp er m al es a nd n on -d ip pe r fe m al es . ɸ d ep ic ts s ig ni fic an t d iff er en ce (p <0 .0 5) b et w ee n di pp er m al es a nd d ip pe r fe m al es . £ d ep ic ts s ig ni fic an t d iff er en ce (p <0 .0 5) b et w ee n m al es a nd fe m al es in th e ge ne ra l p op ul at io n. a.b. bawa-allah et al. 9 in t e r n a l m e d ic in e issn 2413-6077. ijmmr 2020 vol. 6 issue 1 discussion in this cross-sectional study of black south africans, it was demonstrated that non-dippers have higher arterial stiffness as measured by cfpwv when compared with dippers. we also show that male non-dippers have greater arterial stiffness and greater age-related increase in arterial stiffness when compared with female non-dippers. males had significantly higher serum triglycerides when compared with females irrespective of blood pressure dipping status. to our knowledge, this is the first time that serum lipid profiles are considered in nondipping related arterial stiffness. the males in our cohort had greater levels of triglycerides irrespective of dipping status irrespective of dipping status. we believe that serum triglycerides might explain the higher arterial stiffness observed in males. this is consistent with the findings of wang and colleagues where they showed in a communitybased study involving 1, 447 participants, that lower levels of triglycerides were significantly associated with decreases in cfpvw even after adjusting for confounding variables [9]. zhao and colleagues also showed similar results [10]. they established that serum triglycerides had a positive relationship with brachial ankle pulse wave velocity (bapwv) in 1133 participants aged from 50 to 90. the present study shows a significantly lower serum hdlc in non-dipper males when compared with non-dipper females. this might also explain the gender differences in arterial stiffness between non-dipper males and females. the role of hdlc in arterial stiffness has been studied and it has been suggested that low levels of hdlc is a risk factor for cardiovascular and cerebrovascular diseases [11]. similarly, different studies have shown an inverse relationship between hdlc and pulse wave velocity, suggesting that higher levels of hdlc might be associated with an improved arterial distensibility [12]. wang and colleagues in a cross-sectional study showed that in 15, 302 participants, hdlc had an inverse relationship with cfpwv [13]. because of the large population of this study and similarity in technique used in the assessment of arterial stiffness, we are confident about the validity of our results. of interest is also the study by shen and colleagues where it is shown that high serum levels of hdlc are associated with a lower risk of arterial stiffness specifically in women older than 50 years of age but not in age matched men and not in individuals younger than 50 years old [14]. the exact mechanism behind the relationship between hdlc and cfpwv is not quite clear but it has been postulated that high hdlc levels are inversely proportional to advanced glycation end products levels (ages) [15] which stimulate inflammatory pathways and stress signaling and can damage elastin molecules in the vascular wall leading to arterial stiffness [16]. it has also been shown that hdlc has beneficial effects on the vascular wall via its anti-apoptotic, anti-inflammatory and antithrombotic properties [17]. additionally, we also show in this study that female non-dippers had higher ldlc concenfig. 2. age related increase in pulse wave velocity. panel a represents dippers population while panel b represents non-dippers. pwv = pulse wave velocity. male r2 = 0.476; p<0.0001 female r2 = 0.424; p<0.0001 male r2 = 0.498; p<0.0001 female r2 = 0.494; p<0.0001 a.b. bawa-allah et al. 10 in t e r n a l m e d ic in e issn 2413-6077. ijmmr 2020 vol. 6 issue 1 trations when compared with non-dipper males. although high serum levels of ldlc have been attributed to higher degrees of arterial stiffness [18], this might not explain the observed gender differences in arterial stiffness in this study. this suggests that ldlc might be less important at determining arterial stiffness when compared with hdlc and triglycerides in this population. this assumption may be supported by trials that have shown significant residual cardiovascular risk even when ldlc levels have reached treatment target levels. this residual cardiovascular risk has been attributed to high triglycerides levels despite normal ldlc levels [19]. it has also been shown that levels of hdlc have better prognostic capabilities when compared to levels of ldlc [20]. the significantly higher pulse wave velocity in non-dipper males could have also been accounted for by higher prevalence of diabetes in this group. alecu and colleagues showed in 207 participants a strong correlation between diabetes and arterial stiffness and a stronger relationship between pulse wave velocity and age in diabetic individuals when compared with non-diabetic individuals [21], although the nondipping component was not considered. a relatively larger study with 1,415 participants showed higher prevalence of arterial stiffness amongst diabetic participants. they also showed a higher risk of increased arterial stiffness amongst diabetic individuals in a 5-year follow-up study [22]. the present study shows an increase in cfpwv with age irrespective of gender, with males having a steeper increase in age related arterial stiffness when compared to females irrespective of dipper status. these observations are consistent with those of alghathrif and colleagues where they showed in a population of 777 individuals with 354 men and 423 women, that men had a steeper longitudinal increase in cfpwv with advancing age when compared with women, which makes the males to have higher cpfpwv values after their fifth decade [23]. our results are also in line with another study by wen and colleagues [24] in which arterial stiffness was assessed by cardio-ankle vascular index (cavi) in 18, 336 subjects and it was observed that aging led to a higher arterial stiffness in men when compared with women. magalhães and colleagues also showed that males above 50 years old had a greater pulse wave velocity when compared to age matched females [25]. however, in contrast to our findings there are observations by nethononda and colleagues where it was evidenced that females showed a steeper increase in aortic stiffness with age when compared with males [26]. this study consisted of 777 individuals with 408 females and 369 males. the pulse wave velocity was assessed using cardiovascular magnetic resonance. similarly, a study by tomiyama and colleagues reported that age induced arterial stiffness as measured by brachial ankle pulse wave velocity was higher in females when compared to males. this study involved 7,881 subjects with 4,488 males and 3,393 females [27]. the differences in observations between the latter mentioned studies are not quite clear, but despite their large sample sizes, they employed different methods to assess arterial stiffness. all the studies mentioned above have been conducted in general populations and the nocturnal bp dipping statuses of the participants were not taken into consideration. the reports that studied the non-dipping bp pattern alongside arterial stiffness did not consider age and gender diffe rences [28]. our results suggest that non-dipping related arterial stiffness is more severe in males when compared to females causing them to have a higher non-dipping related arterial stiffness and a steeper increase in age related arterial stiffness when compared with females. aging is associated with increased sympathetic activation [29] and may lead to inflammation. inflammation has been shown to induce arterial stiffness [30]. our study population was modest in size and consisted entirely of black africans living in africa, this might limit the generalizability of our findings. also, our study was crosssectional, therefore, cause-effect relationships cannot be established. conclusions the nocturnal non-dipping blood pressure profile is associated with higher arterial stiffness and a steeper age-related increase in arterial stiffness irrespective of gender. nondipper males have higher level of arterial stiffness and age-related increase in arterial stiffness when compared with non-dipper females. therapies targeting a reduction of serum triglycerides levels might be beneficial in improving arterial compliance with or without the presence of non-dipping. conflict of interest the authors report no conflict of interests in this study a.b. bawa-allah et al. 11 in t e r n a l m e d ic in e issn 2413-6077. ijmmr 2020 vol. 6 issue 1 funding this work was supported the university of the witwatersrand enablement grant, and the faculty of health sciences research grant, grant number: 001.251.8521101.5121105. 000000. 0000000000.4550. acknowledgements all participants formed an integral part of this study, without which the study would have been realizable. we appreciate their voluntary contributions. author’s contribution abdulraheem b. bawa-allah – concep tu alization, formal analysis, investigation, methodology, project administration, software, validation, visualization, writing-original draft, writing-review and editing; mercy m. mashao – investigation, methodology, project administration; thamsanqa f. nyundu – investigation, methodology, project administration; edgar m. phukubje – investigation, methodology, project administration; brian g. nkosi – investigation, methodology, project administration; mandisa v. ngema – investigation, methodology, project administration; bongubuhle w. mlambo – investigation, methodology, project administration; muzi j. maseko – conceptualization, investigation, methodology, project administration, validation, visualization, writingreview and editing, supervision. ліпідний спектр крові та артеріальна жорсткість у пацієнтів з non-dipper-добовим профілем артеріального тиску *a.b. bawa-allah1, m.m. mashao2, t.f. nyundu2, e.m. phukubje2, b.g. nkosi2, m.v. ngema2, b.w. mlambo2, m.j. maseko2 1 – college of medicine, university of lagos, lagos, nigeria 2 – university of the witwatersrand, johannesburg, south africa вступ. добовий профіль артеріального тиску (ат) non-dipper, який супроводжується недостатнім зниженням ат вночі, асоціюється з підвищеною жорсткістю артерій та ураженням органів-мішеней. ліпідний профіль – одна з важливих детермінант артеріальної жорсткості. мета роботи. мета дослідження – оцінити взаємозв’язки між ліпідним спектром крові та артеріальною жорсткістю у пацієнтів з добовим профілем ат non-dipper. методи. було проведене поперечне (одномоментне) дослідження 796 (288 чоловіків та 508 жінок) осіб-африканців. 24-годинний добовий моніторинг ат проводили з використанням обладнання spacelabs 90207 (spacelabs inc., redmond, washington, usa). швидкість поширення пульсової хвилі по артеріях еластичного типу (шппх) визначали за допомогою мікроманометра spc-301 (millar instruments inc., houston, tx). результати. серед 288 чоловіків у 140 добовий профіль ат був типу non-dipper. серед 508 жінок цей тип був у 273. у загальній когорті рівень тригліцеридів був вищим у чоловіків порівняно з жінками 1,46±0,96 vs 1,13±1,02, p<0,0001. окрім того, у чоловіків з добовим профілем ат dipper рівень тригліцеридів був вищим відносно жінок-dippers 1,32±0,98 vs 1,06±0,58 (p=0,0012) відповідно. non-dippers-чоловіки мали вищий рівень тригліцеридів також відносно і жінок-non-dippers: 1,61±2,05 vs 1,19±1,14 (p=0,0078) відповідно. рівень ліпопротеїдів високої щільності був достовірно нижчим у чоловіків-non-dippers порівняно з жінками з групи non-dippers (p=0.008). у обох статей і чоловіків і жінок non-dippers шппх була вищою у порівнянні з dippers; 7,53±3,60 vs 5,74±2,47, p<0,0001 та 6,64±2,52 vs 5,98±2,23 (p=0,0021) відповідно. однак у чоловіків non-dipper шппх була достовірно вищою порівняно з аналогічною групою жінок (7,53±3,60 vs 6,64±2,52, p=0,0031). висновки. враховуючи отримані нами дані, фармакотерапія спрямована на зниження рівня тригліцеридів у сироватці крові може впливати на перебіг артеріальної гіпертензії та її наслідки незалежно від типу добового профілю артеріального тиску. ключові слова: non-dipping профіль артеріального тиску; артеріальна жорсткість; ліпідний профіль крові. information about authors abdulraheem b. bawa-allah – department of physiology, college of medicine, university of lagos, lagos nigeria. orcid https://orcid.org/0000-0003-0829-3007, e-mail: abbawaallah@gmail.com a.b. bawa-allah et al. 12 in t e r n a l m e d ic in e issn 2413-6077. ijmmr 2020 vol. 6 issue 1 references 1. head ga, mihailidou as, duggan ka, beilin lj, berry n, brown ma, bune aj, cowley d, chalmers jp, howe pr, hodgson j. definition of ambulatory blood pressure targets for diagnosis and treatment of hypertension in relation to clinic blood pressure: prospective cohort study. bmj. 2010 apr 14;340:1104. doi: 10.1136/bmj.c1104 2. hansen tw, jeppesen j, rasmussen s, ibsen h, torp-pedersen c. ambulatory blood pressure monitoring and risk of cardiovascular disease: a population based study. american journal of hypertension. 2006 mar 1;19(3):243-50. doi: 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support of blood pressure in women. hypertension. 2014 feb;63(2):303-8. doi: 10.1161/hypertensionaha.113.02393 30. johnson cp, baugh r, wilson ca, burns j. age related changes in the tunica media of the vertebral artery: implications for the assessment of vessels injured by trauma. journal of clinical pathology. 2001 feb 1;54(2):139-45. doi: 10.1136/jcp.54.2.139 received 27 mar 2020; revised 19 may 2020; accepted 05 june 2020. this is open access article distributed under the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. a.b. bawa-allah et al. 19 in t e r n a l m e d ic in e issn 2413-6077. ijmmr 2020 vol. 6 issue 1 doi 10.11603/ijmmr.2413-6077.2020.1.11103 impact of molecular method for the diagnosis of acute bacterial meningitis in a tertiary health care centre in north india m.s. raza1, *b.k. das1, v. goyal1, r. lodha1, r. chaudhry1, s. sood1, v. sreenivas1, d. nair2, s. mohapatra1, h. gautam1, a. kapil1 1 – all india institute of medical sciences, new delhi, india 2 – vmc, safdarjung hospital, india background. acute bacterial meningitis is one of the significant infectious diseases that add an immense burden to the health system. proper management of meningitis is an invincible need to overcome the severe consequences. objectives. the aim of the study is detection of the etiological agents of acute bacterial meningitis by pcr. methods. total 267 csf samples collected from suspected bacterial meningitis cases were processed for the detection of s. pneumoniae, h. influenzae, n. meningitidis, e. coli and group b streptococci by conventional and molecular diagnosis method. csf was inoculated on blood, chocolate and macconkey agar plates and incubated at 37 °c for 24-48 hrs. bacteria grown were identified by matrix assisted laser desorption/ionization time of flight (maldi-tof). multiplex pcr of the enlisted bacteria was performed using dna extracted from csf by dna extraction kit (qiagen, usa). results. 5 (1.87%) out of the total 267 csf samples were culture positive (3 s. pneumoniae and 2 e. coli) and 28 (10.49%) had detectable dna by conventional pcr. out of these 28 samples, 20 (71.43%) were s. pneumoniae and 8 (28.57%) were e. coli. 15 (53.57%) out of total s. pneumoniae were present in children below 5 years of age. similarly, e. coli was predominant in neonates. sensitivity and specificity of the pcr was 100% and 95-98% respectively. conclusions. streptococcus pneumoniae is the commonest cause of community acquired bacterial meningitis in children below five years of age. hence, for the promising outcome, pcr should be implemented for the diagnosis of acute bacterial meningitis. key words: cerebrospinal fluid (csf); acute bacterial meningitis; pcr; maldi-tof. *corresponding author: dr. bimal kumar das, department of microbiology, all india institute of medical sciences, new delhi, india. email: tezpur.bimal@gmail.com m.s. raza et al. introduction bacterial meningitis is one of the dangerous infectious diseases of central nervous system (cns). meningitis is an inflammation of delicate membrane known as meninges covering brain and spinal cord and marked by intense headache, fever and nuchal rigidity. despite advances in treatment, acute bacterial meningitis (abm) is the major cause of mortality and morbidity in developed countries as well as in developing ones [1]. in india and other developing countries mortality rate varies 16-32% [2-5]. individuals with suspected meningitis require prompt diagnosis and treatment. delay in management can result in poor outcome of the disease [6]. the most common bacteria causing abm are streptococcus pneumoniae, haemophilus influenzae, neisseria meningitidis, listeria monocytogenes, group b streptococci and e. coli [7]. etiological agents of abm vary globally depending on age, geographical area and immune status [8, 9]. hence, there is need for regularly reviewing of bacterial meningitis. culture is considered as the ‘gold standard’ for the diagnosis of acute bacterial meningitis as well as equally important for antibiotic susceptibility testing of the causative agents that account for treatment [10]. however, it takes longer for identification of the pathogens and its outcome is influenced by intake of antibiotics prior to lumber puncture and number of bacteria (cfu/ml) present in the csf. to overcome this diagnostic glitch, polymerase chain reaction (pcr) is gaining momentum for a rapid diagnosis of abm [11-17]. hence, this experiment have been designed and performed to detect the etiological agents of community acquired bacterial meningitis by molecular method as pcr does not rely on viability of the organism. international journal of medicine and medical research 2020, volume 6, issue 1, p. 19-25 copyright © 2020, tnmu, all rights reserved 20 in t e r n a l m e d ic in e issn 2413-6077. ijmmr 2020 vol. 6 issue 1 methods a perspective study was designed and carried out at the department of microbiology, all india institute of medical sciences, new delhi, india. total 267 cerebrospinal fluids (csf) samples from suspected acute bacterial meningitis cases were collected in march, 2015 – april, 2018. all samples were processed for cytological, biochemical, microbiological (gram stain, latex agglutination and culture) and molecular examination. csf was centrifuged at 10,000 rpm for 10 minutes. sediment was used for gram stain and culture. blood, chocolate and mac-conkey agar plates were used for growth of the bacteria. after inoculation, the plates were incubated at 37 °c for 24-48 hrs with 5% co2. isolates grown on the culture plates were identified by maldi-tof (vitek ms biomerieux, france). the isolates were further processed for antibiotic susceptibility tests. nosocomial infection was defined as a positive bacterial infection that was not present at the time of hospital admission or clinical evidence of an infection no sooner than 48 hours after admission. remaining patients were considered to have community acquired infection. identification of isolates by maldi-tof. for identification of the bacteria, a single bacterial colony was taken, and the smear was made on the maldi-tof slide with the help of wooden stick and 0.5 μl matrix (α-cyano-4hydroxycinnamic acid) was added. the slide was kept in air at a room temperature for 5 mi nutes to dry and finally slide was kept in the maldi-tof machine (vitek ms biomerieux, france) for acquisition and identification. the isolates identified were further processed for antibiotic susceptibility test. antibiotics susceptibility test. mueller hinton agar was used to perform an antibiotic susceptibility test. the isolate was inoculated in 0.5% peptone water and incubated at 37 °c for 2 hrs. turbidity was compared with 0.5 mcfarland solutions so that bacterial count to be >105 cfu/ml. antibiotics used were amikacin (10 μg), co-trimoxazole (25 μg), cephotaxime (30 μg), ceftazidime (30 μg), ceftriaxone (30 μg), ciprofloxacin (30 μg), erythromycin (30 μg), penicillin g (10units), piperacillin + tazobactam (100/10 μg), imipenem (10 μg), netilmicin (30 μg), chloramphenicol (30 μg) and vanco mycin (30 μg). the antibiotic discs were obtained from hi media laboratories, mumbai, india. antibiotic susceptibility results were interpreted as per clsi guideline. the escherichia coli atcc25922 and staphylococcus aureus atcc25923 strains were used for quality control. l a t e x a g g l u t i n a t i o n t e s t ( l at ) . a supernatant of the centrifuged csf was used for detection of soluble bacterial antigen of streptococcus pneumoniae, neisseria meningitidis a, b, c, and w135, streptococcus agalactiae, escherichia coli, and haemophilus influenzae type b by latex agglutination test (lat) following manufacturer’s instructions. the directigentm meningitis combo kit (bd, usa) was used for the lat. extraction of dna. extraction of dna from csf samples was carried out using the mini dna extraction kit (qiagen, usa) following manufacturer’s instructions. dna was eluted in 100 μl elution buffer and stored at -20 °c for future use in the experiment. primer design and pcr conditions. for detection of s. pneumoniae, h. influenzae and n. meningitidis semi-nested multiplex pcr was designed. however, for e. coli & group b streptococcus the multiplex pcr was done. primers were designed accordingly that is presented in table 1. table1. sequence of primers used in this study organisms target gene primer amplicon length (bp) reference seminested multiplex pcr universal primer u3 (16s rrna) 5'-gtg cct gca gcc gcg gta at-3' 1000 chakrabarti p et al, 2009 [18] u8 (16s rrna) 5'-aag gag ggg tgt gtac-3' s. pneumoniae ply 5'-gta caa cga gtc gca agc-3' 293 h. influenzae bex 5'-cct aag aag aga tcg ag-3' 543 n. meningitidis ctr 5'-tgt tgc gca acc tga ttg-3' 710 multiplex pcr e. coli 16s rrna 5'-tgc ctg atg gag ggg gat aa-3' 776 this study 5'-ttt aac ctt gcg gcc gta ct-3' s. agalactiae sip 5'-aca acg gaa ggc gct act gtt c-3' 255 bergh k et al, 2004 [19]5'-acc tgg tgt ttg acc tga act a-3' m.s. raza et al. 21 in t e r n a l m e d ic in e issn 2413-6077. ijmmr 2020 vol. 6 issue 1 amplification of dna extracted from the csf. the pcr mixture (25 μl) contained 1×pcr buffer 2.5 μl with 2.5 mm mgcl2, 0.5 μl dntps (10mm each), 0.25 μl taq polymerase (promega, 500u) & primer 0.2 μl (10 μm each) for seminested multiplex pcr. however, for multiplex pcr, primer concentration used was 1 μl (10 μm each) and 2.5 μl of the target dna. finally, the volume of the pcr mixture was adjusted by adding milli q water. pcr was performed in the thermocycler (agilent technologies, surecycler 8800, usa) with the following cycling condition for multiplex pcr, i.e. initial denaturation at 95 °c for 5 min, denaturation at 95 °c for 1 min, annealing 55 °c for 30 sec, extension 72 °c for 30 sec and final extension at 72 °c for 10 min with 4 °c holding temperature, the number of cycles was 35. after amplification of the bacterial dna, gel electrophoresis of the pcr product was run for 30 minutes in 1.5% agarose with ethi dium bromide and visualize under uv fluores cence to check the product size. representative image of the gel electrophoresis is presented in fig. 1. statistical analysis. of the tests was performed by the wilcoxon singed-rank test. sensitivity, specificity, positive predictive value (ppv) and negative predictive value (npv) were evaluated. ethical approval. the study was approved by the institute ethics committee of all india institute of medical sciences, new delhi (no: iesc/t-20) results the finding of the study was interpreted and correlated with the etiological agents of abm in all age group (0 days-84 years) with conventional and molecular techniques. out of total 267 cases of suspected meningitis patients, 194 (72.60%) were male and 73 (27.34%) were female. the main clinical feature shown by the patients were fever 258 (96.63%), headache 210 (78.65%) and altered sensorium 181 (67.79%) details are given in table 2. the csf glucose and protein level of the majority of the patients were <45 mg/dl and >55 mg/dl respectively. out of 267 csf samples, 5 (1.87%) were proved to be positive by culture. among these 5 isolates, 3 (60%) were s. pneumoniae and 2 (40%) were e. coli. similarly, total 28 (10.49%) bacteria were detected by conventional pcr (cpcr) as the culture positive bacteria were also positive by pcr; out of 23 bacteria detected by pcr only, 6 (26.09%) were e. coli and 17 (73.91%) were s. pneumoniae. main cases of the organisms revealed were of the age group of 0-5 yearsold. likewise, 4 (50%) cases of e. coli were revealed in neonates. in case of s. pneumoniae, 15 (53.57%) isolates were revealed in children below 5 years of age. the details of distribution of bacteria revealed are presented in table 3. latex agglutination test (lat) was used to detect the bacterial soluble antigen of the common etiological agents of acute bacterial menin gitis: streptococcus pneumoniae, neisseria meningitidis a, b, c & w135, streptococcus aga lactiae, escherichia coli, and haemophilus influ enzae type b. however, only s. pneumoniae and e. coli was detected by the lat in this study. the lat has detected more organisms than the cul ture. total 15 isolates were detected by the lat. out of 15 lat positive isolates 10 (5 s. pneu moniae and 5 e. coli) were positive by the lat only. however, fig.1. a gel image of pcr product of s. pneumoniae in 1.5% agar. l1 = ladder, l2 = positive control, l3 = negative control & l4,5,6,7 = csf sample. table 2. demographic details of the meningitis patients clinical features number of patients fever 258 (96.63%) headache 210 (78.65%) nuchal rigidity 57 (21.34%) altered sensorium 181 (67.79%) nausea/vomiting 54 (20.24%) kernig’s and brudzinski's sign 51 (19.10%) bulging anterior fontanelle 32 (11.98%) m.s. raza et al. 22 in t e r n a l m e d ic in e issn 2413-6077. ijmmr 2020 vol. 6 issue 1 3 s. pneumoniae and 2 e. coli that were culture positive were also positive by the lat. taking culture as gold standard, the sensitivity, specificity, positive predictive value and negative predictive value were evaluated. sensitivity and specificity of cpcr for s. pneumoniae and e. coli were found in 100% and 95-98% cases, respectively, with 95% ci that is presented in table 4. the antibiotic susceptibility test was performed for e. coli and s. pneumoniae detected from csf by culture. most of the antibiotics tes ted were susceptible to s. pneumoniae (100%). one (33.36%) s. pneumoniae showed re sistance to co-trimoxazole. for e. coli, the most susceptible antibiotic was amikacin (100%) followed by colistin (100%) and imipenem (100%). table 3. age wise distribution of the isolates detected from csf isolates methods of detection age group totalneonates 1-3 months 3-24 months 2-5 yrs 5-50 yrs >50 yrs e. coli culture 01 – – – – 01 02 cpcr 03 02 – – – 01 06 s. pneumoniae culture – – 01 01 01 – 03 cpcr – 02 07 04 02 02 17 total 04 04 08 05 03 04 28 table 4. sensitivity, specificity, ppv, npv of cpcr comparing with culture statistics conventional pcr e. coli (n=6) s. pneumoniae (n=17) value with 95% ci value with 95% ci sensitivity 100% 15.81% – 100% 100% 28.24% – 100% specificity 98.28% 96.29% – 99.36% 95.26% 95.53% – 97.22% ppv 25% 13.10% – 42.42% 15% 9.99% – 21.92% npv 100% – – 95.30% accuracy 98.29% 96.31% – 99.37% 95.30% 92.59% – 97.24% discussion community acquired acute bacterial meningitis is an urgent health issue as most of the infection occurs in children below 5 years of age. in this study, total 267 csf samples were included irrespective of the age limit. of the total number of csf, only 5 (1.87%) were found po sitive by culture for different etiological agents of community acquired bacterial meningitis. a study conducted by zahra b. h et al. has shown culture positivity 2.5% [20]. positivity of culture decreases 60-70% in case of csf collected after administration of antibiotics [21]. in our study, low culture positivity may be due to intake of antibiotics prior to lumber puncture as our hospital is a referral center where majority of the patients admitted had already been ad ministered with various antibiotics before getting admitted. we detected 28 (10.14%) positive csf by cpcr which were almost 10% more than the culture positives. our finding is concurrent with the finding (11.76%) shown by nour m et al. [22] but less than the finding by zahra b. h. et al. and chokrabarty p. et al. who have reported 15% and 19.8% positivity by mpcr respectively [18, 20]. low positivity may be due to difference in sample size and organisms detected. sensitivity and specificity of convention pcr was found 100% and 95-98% respectively for e. coli and s. pneumoniae with 95% ci. finding was concurrent with the findings of wu et al. [23]. in children below 1-24 months of age s. pneu moniae 10 (50%) was the main organism. however, e. coli 4 (50%) were found in neonates that was also a significant finding as e. coli and s. agalactiae are the major pathogens of the neonates [24, 25]. we have detected total 20 s. pneumoniae, 15 (75%) of which were in those less than 5 years of age. likewise, out of these 15 cases, 8 (53.3%) were detected in those 3-24 months of age. more number of s. pneumoniae in children below 5 years of age was reported from india [26, 27]. however, 1 previous study conducted in 1999 by isib has reported h. influenzae as the main cause of bacterial meninm.s. raza et al. 23 in t e r n a l m e d ic in e issn 2413-6077. ijmmr 2020 vol. 6 issue 1 gitis prior to the introduction of pentavalent vaccines [28]. in our study, only e. coli and s. pneumoniae were detected among the etiological agents of community acquired acute bacterial meningitis that may be a result of implementation of the hib vaccination in national immunization program in india. h. influenzae detection rate has decreased drastically in recent times. similarly, in india, incidence of n. meningitidis increased greatly during the epidemics and the epidemic cycle of n. meningitidis is at an interval of 15-20 years. last episode of n. meningitidis epidemics was in 2005 [29]. antibiotic susceptibility test result showed that most of the antibiotics used were susceptible to s. pneumoniae. one (33.3%) s. pneumoniae out of 3 was resistance to co-trimoxazole. however, no penicillin resistance s. pneumoniae was found. similarly, amikacin, colistin and imipenem were the most susceptible (100%) antibiotics for e. coli though ciprofloxacin has shown (50%) resistance followed by cefo + salbactam (50%). conclusions acute bacterial meningitis is a severe medical emergency and s. pneumoniae is still the a major cause of community acquired bacterial meningitis in children. molecular diagnosis is gaining momentum for a rapid detection of etiological agents with high sensitivity and specificity since it is unaffected by viability of the organisms. hence, pcr should be implemen ted in the diagnosis to overcome the dependency on conventional method. conflict of interest the authors declare no conflict of interest. acknowledgements authors acknowledge the indian council of medical research (icmr), new delhi funding this work was supported by grant of institute research fund. institute grant no: 8-297/a-297/2014/rs authors contributions m.s. raza – conceptualization, investigation, writing (original draft), b.k. das – сconceptualization, project administration, resources, super vision (review and editing), v. goyal, r . l o d h a , r . c h a u d h r y , s . s o o d , d. n a i r , s. mohapatra, a. kapil – сconceptualization, supervision, writing (review and editing), h. gautam – conceptualization, writing (review and editing), v. sreenivas – formal analysis, visualization writing, data curation. молекулярні методи діагностики гострого бактеріального менінгіту на третинному рівні надання медичної допомоги у лікарнях північної індії m. s. raza1, b. k. das1*, v. goyal1, r. lodha1, r. chaudhry1, s. sood1, v. sreenivas1, d. nair2, s. mohapatra1, h. gautam1, a. kapil1 1 – all india institute of medical sciences, new delhi, india 2 – vmc, safdarjung hospital, india вступ. гострий бактеріальний менінгіт – одне з найбільш небезпечних інфекційних захворювань, що слугує додатковим тягарем для системи охорони здоров’я. відповідне лікування – необхідна умова для попередження та лікування тяжких наслідків хвороби. мета дослідження – встановлення етіологічних чинників гострого бактеріального менінгіту за допомогою плр. методи. загалом було відібрано 276 зразків спинномозкової рідини (смр) від пацієнтів з підозрою на бактеріальний менінгіт для визначення його основних збудників s. pneumoniae, h. influenzae, n. meningitidis, e. coli та стрептококів групи b типовим способом та за допомогою методів молекулярної діагностики. для проведення культурального дослідження зразків смр використовували живильні середовища: кров’яний агар, шоколадний агар, середовище макконкі. зразки інкубували при 37°c протягом 24-48 годин. ріст бактерій досліджували методом мас-спектрометрії з лазерною десорбцією/ іонізацією maldi-tof. мультиплексна плр зразків смр проводилася з використанням наборів для екстракції днк qiagen (usa). результати. із 276 досліджених зразків смр, п’ять (1.87%) були культуро-позитивними (три – s. pneumoniae та два зразки – e. coli). за допомогою типової плр днк збудника було знайдено у 28 зразках (10.49%). з цих 28 зразків у 20 (71.43%) – були ідентифіковані s. pneumoniae, а у 8 зразках (28.57%) – e. coli. m.s. raza et al. 24 in t e r n a l m e d ic in e issn 2413-6077. ijmmr 2020 vol. 6 issue 1 references 1. mcintyre pb, o’brien kl, greenwood b, van de beek d. effect of vaccines on bacterial meningitis worldwide. lancet. 2012; 380:1703–11. doi: 10.1016/s0140-6736(12)61187-8 2. kabra sk, praveen kumar, verma ic, mukherjee d, chowdhary bh, sengupta s, et al. bacterial meningitis in india: an ijp survey. indian j pediatr. 1991;58:505-11. doi: 10.1007/bf02750932 3. tang lm, chen st, hsu wc, lyu rk. acute bacterial meningitis in adults: a hospital-based epidemiological study. qjm. 1999;92:719-25. doi: 10.1093/qjmed/92.12.719 4. ayaz c, mehmet fg, hosoglu s, mustafa kc, akalin s, omer fk. characteristics of acute bacterial meningitis in southeast turkey. indian j med sci. 2004;58:327-33. 5. chinchankar n, mane m, bhave s, bapat s, bavdekar a, pandit a, niphadkar kb, dutta a, leboulleux d. diagnosis and outcome of acute bacterial meningitis in early childhood. indian pediatrics. 2002 oct 13;39(10):914-21. 6. van de beek d, de gans j, tunkel ar, wijdicks ef. community-acquired bacterial meningitis in adults. new england journal of medicine. 2006 jan 5;354(1):44-53. doi: 10.1056/nejmra052116 7. schuchat a, robinson k, wenger jd, harrison lh, farley m, reingold al, lefkowitz l, perkins ba. bacterial meningitis in the united states in 1995. new england journal of medicine. 1997 oct 2;337(14): 970-6. doi: 10.1056/nejm199710023371404 8. kasanmoentalib es, brouwer mc, van de beek d. update on bacterial meningitis: epidemiology, trials and genetic association studies. current opinion in neurology. 2013 jun 1;26(3):282-8. doi: 10.1097/wco.0b013e328360415c 9. liu l, oza s, hogan d, perin j, rudan i, lawn je, cousens s, mathers c, black re. global, regional, and national causes of child mortality in 2000–13, with pro jections to inform post-2015 priorities: an updated systematic analysis. the lancet. 2015 jan 31;385(9966):430-40. doi: 10.1016/s0140-6736(14)61698-6 серед зразків, де було виявлено s. pneumoniae, 15 (53.57%) належали дітям до 5 років. e. coli переважала у новонароджених. чутливість та специфічність плр була 100% та 95-98% відповідно. висновки. streptococcus pneumoniae – найбільш поширений збудник бактеріального менінгіту у дітей віком до 5 років. враховуючи результати наших досліджень, плр-діагностика повинна впроваджуватися у комплекс методів діагностики гострого бактеріального менінгіту. ключові слова: спинномозкова рідина (смр); гострий бактеріальний менінгіт; плр; maldi-tof. information about authors m. s. raza – ph.d, post-doctoral fellow, department of microbiology, all india institute of medical sciences, new delhi, india orcid https://orcid.org/0000-0002-5787-031x, e-mail sahidktm2000@gmail.com b. k. das – md, frcp, professor, department of microbiology, aiims, new delhi, india orcid https://orcid.org/0000-0003-1970-5046, email tezpur.bimal@gmail.com r. chaudhry – md, professor, department of microbiology, aiims, new delhi, india orcid https://orcid.org/0000-0002-7381-1504, email drramach@gmail.com s. sood – md, professor, department of microbiology, aiims, new delhi, india orcid https://orcid.org/0000-0002-8797-5286, e-mail: seemalsood@gmail.com a. kapil – md, professor, department of microbiology, all india institute of medical sciences, new delhi, india orcid https://orcid.org/0000-0001-7834-2754, e-mail: akapilmicro@gmail.com v. goyal – md. dm. professor, department of neurology, aiims, new delhi, india orcid https://orcid.org/0000-0002-7813-7117, e-mail: drvinaygoyal@gmail.com r. lodha –md, professor, department of pediatrics, aiims, new delhi, india orcid https://orcid.org/0000-0003-2608-1163, e-mail rlodha1661@gmail.com v. sreenivas –ph. d, professor, department of biostatistics, aiims, new delhi, india orcid https://orcid.org/0000-0001-9768-336x, e-mail vishnubhatla@gmail.com d. nair – md, professor, department of microbiology, vmc, safdarjung hospital, new delhi, india orcid https://orcid.org/0000-0002-0081-4034, e-mail deepthinair2@gmail.com h. gautam – md, associate professor, department of microbiology, aiims, new delhi, india orcid https://orcid.org/0000-0002-1409-1543, e-mail drhitender@gmail.com s. mohapatramd, associate professor, department of microbiology, aiims, new delhi, india orcid https://orcid.org/0000-0002-8311-4112, e-mail drsarita2005@gmail.com m.s. raza et al. 25 in t e r n a l m e d ic in e issn 2413-6077. ijmmr 2020 vol. 6 issue 1 10. brouwer mc, tunkel ar, van de beek d. epidemiology, diagnosis, and antimicrobial treatment of acute bacterial meningitis. clinical microbiology reviews. 2010 jul 1;23(3):467-92. doi: 10.1128/cmr.00070-09 11. greisen k, loeffelholz m, purohit a, leong d. pcr primers and probes for the 16s rrna gene of most species of pathogenic bacteria, including bacteria found in cerebrospinal fluid. journal of clinical microbiology. 1994 feb 1;32(2):335-51. doi: 10.1128/jcm.32.2.335-351.1994 12. lu jj, perng cl, lee sy, wan cc. use of pcr with universal primers and restriction endonuclease digestions for detection and identification of com mon bacterial pathogens in cerebrospinal fluid. journal of clinical microbiology. 2000 jun 1;38(6):2076-80. doi: 10.1128/.38.6.2076-2080.2000 13. rådström p, bäckman a, qian ny, kragsbjerg p, påhlson c, olcén p. detection of bacterial dna in cerebrospinal fluid by an assay for simul taneous detection of neisseria meningitidis, haemophilus influenzae, and streptococci using a seminested pcr strategy. journal of clinical microbiology. 1994 nov 1;32(11):2738-44. doi: 10.1128/jcm.32.11.2738-2744.1994 14. saravolatz ld, manzor o, vandervelde n, pawlak j, belian b. broad-range bacterial polymerase chain reaction for early detection of bacterial meningitis. clinical infectious diseases. 2003 jan 1;36(1):40-5. doi: 10.1086/345438 15. xu j, millar bc, moore je, murphy k, webb h, fox aj, cafferkey m, crowe mj. employment of broad‐ range 16s rrna pcr to detect aetiological agents of infection from clinical specimens in patients with acute meningitis–rapid separation of 16s rrna pcr amplicons without the need for cloning. journal of applied microbiology. 2003 feb;94(2):197-206. doi: 10.1046/j.1365-2672.2003.01839.x 16. schuurman t, de boer rf, kooistra-smid am, van zwet aa. prospective study of use of pcr amplification and sequencing of 16s ribosomal dna from cerebrospinal fluid for diagnosis of bacterial meningitis in a clinical setting. journal of clinical microbiology. 2004 feb 1;42(2):734-40. doi: 10.1128/jcm.42.2.734-740.2004 17. deutch s, pedersen ln, pødenphant l, olesen r, schmidt mb, møller jk, ostergaard l. broad-range real time pcr and dna sequencing for the diagnosis of bacterial meningitis. scandinavian journal of infectious diseases. 2006 jan 1;38(1):27-35. doi: 10.1080/00365540500372861 18. chakrabarti p, das bk, kapil a. application of 16s rdna based seminested pcr for diagnosis of acute bacterial meningitis. indian journal of medical research. 2009 feb 1;129(2):182‐8. 19. bergh k, stoelhaug a, loeseth k, bevanger l. detection of group b streptococci (gbs) in vaginal swabs using real-time pcr with taqman probe hybridization. indian journal of medical research. 2004 may 1;119:221-3. 20. bonab zh, farajnia s, ghotaslou r, nikkhah e. evaluation of nested pcr method for diagnosis of meningitis due to neisseria meningitidis and haemophilus influenzae. turkish journal of biology. 2012 nov 21;36(6):727-31. 21. nigrovic le, malley r, macias cg, kanegaye jt, moro-sutherland dm, schremmer rd, schwab sh, agrawal d, mansour km, bennett je, katsogridakis yl. effect of antibiotic pretreatment on cerebrospinal fluid profiles of children with bacterial meningitis. pediatrics. 2008 oct 1;122(4):726-30. 22. nour m, alaidarous a. simultaneous detection of bacterial meningitis in suspected cases of meningitis in children using pcr assay. arch clin microbiol. 2017; 8:1-6. 23. henry m wu, soraia m cordeiro, brian h harcourt. bmc infectious diseases. 2013, 13:26. 24. camacho-gonzalez a, spearman pw, stoll bj. neonatal infectious diseases: evaluation of neonatal sepsis. pediatric clinics of north america. 2013 apr;60(2):367–89. doi: 10.1016/j.pcl.2012.12.003 25. chang cj, chang wn, huang lt, huang sc, chang yc, hung pl, lu ch, chang cs, cheng bc, lee py, wang kw. bacterial meningitis in infants: the epidemiology, clinical features, and prognostic factors. brain and development. 2004 apr 1;26(3): 168-75. doi: 10.1016/s0387-7604(03)00122-0 26. manoharan a, manchanda v, balasubramanian s, lalwani s, modak m, bai s, vijayan a, shet a, nagaraj s, karande s, nataraj g. invasive pneumococcal disease in children aged younger than 5 years in india: a surveillance study. the lancet infectious diseases. 2017 mar 1;17(3):305-12. doi: 10.1016/s1473-3099(16)30466-2 27. jayaraman y, veeraraghavan b, chethrapilly pgk. burden of bacterial meningitis in india: preliminary data from a hospital-based sentinel surveillance network. plos one. 2018; 13(5): e0197198. doi: 10.1371/journal.pone.0197198 28. invasive bacterial infection surveillance (ibis) group, international clinical epidemiology network (inclen). prospective multicentre hospital sur veillan ce of streptococcus pneumoniae disease in india. lancet. 1999; 353: 1216-21. doi: 10.1016/s0140-6736(98)07228-6 29. sachdeva a, kukreja s, jain v, dutta ak. meningococcal disease-outbreak in delhi. indian pediatrics. 2005 jun 17;42(6):547-56. received 19 mar 2020; revised 10 apr 2020; accepted 30 may 2020. this is open access article distributed under the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. m.s. raza et al. 28 in t e r n a l m e d ic in e issn 2413-6077. ijmmr 2021 vol. 7 issue 1 doi 10.11603/ijmmr.2413-6077.2021.1.12258 radiation exposure in accessory pathway ablation procedures in cardiac electrophysiology: a retrospective analysis *m. ali1,2, b. banavalikar1, m.k. ghadei1, a. kottayan1, d. padmanabhan1, j. shenthar1 1 – department of cardiac electrophysiology, sri jayadeva institute of cardiovascular sciences and research, bangalore, india 2 – department of cardiology, sher-i-kashmir institute of medical sciences, srinagar, kashmir, india background. radiofrequency catheter ablation (ca) has been the treatment of choice in patients with accessory pathway (ap)-mediated tachycardias. most of these procedures are done under fluoroscopic guidance, leading to significant radiation exposure to the patient and the laboratory personnel. in this analysis, we have looked at the amount of radiation exposure in ap ca procedures performed without the support of a threedimensional electroanatomic mapping system. we have analyzed changes in exposure indices over the study period and the impact of change in fluoroscopy frame rate (ffr). objectives. the objectives of this study are to quantify radiation exposure in accessory pathway ablation procedures; to analyze the radiation exposure trend over time; and to evaluate the effect of fluoroscopy frame rate reduction on the radiation exposure indices in these procedures. methods. all the ap ablation procedures performed at our institute from january 2016 to december 2019 were retrospectively analyzed. the collected data were age, sex, location of aps based on successful site of ablation on fluoroscopy, procedure time, fluoroscopy time, and dose-area product (dap). effective dose (ed) was estimated from dap. the data of procedures performed before january 2018 (“pre” group) were compared with those of the procedures performed after that date (“post” group). pre-group procedures were performed at an ffr of 7.5 frames per second (fps), and post-group procedures – at an ffr of 3.75 fps. results. the total number of procedures included in the analysis was 635. the mean age of the patients was 39±14 years, and 401 of them (63%) were males. the most common location of the aps was left lateral (38%). procedure time and radiation indices showed a significant decrease over the study period (p < 0.001). post group procedures had significantly shorter procedure time and lower radiation exposure than pre group procedures. conclusions. a decrease in the ffr was associated with a significant reduction in radiation exposure in ap ablation procedures keywords: accessory pathway; catheter ablation; dose-area product; fluoroscopy time; radiation exposure. introduction international journal of medicine and medical research 2021, volume 7, issue 1, p. 28-34 copyright © 2021, tnmu, all rights reserved *corresponding author: dr. muzaffar ali, department of cardiology, skims, srinagar, kashmir, india. email: dralimuzaffar@gmail.com the ablation of accessory pathways (ap) using radiofrequency energy was first introduced by borggrefe and colleagues in 1987 when they ablated a right-sided accessory pathway for the first time in humans using radiofrequency energy [1]. since then, radiofrequency ablation has been the treatment of choice in patients with accessory pathwaymediated tachycardias [2,3]. although radiofrequency ablation for ap-mediated tachycardias is a highly efficacious and safe treatment option, some of the adverse effects of this procedure are not because of the procedure itself but because of the ionizing radiation used to visualize the catheters during the procedure. ionizing radiation exposure affects the patient and the laboratory personnel because of the fluoroscopic imaging used during cardiac electrophysiology (ep) procedures. the ionizing radiation exposure can cause long-term hazardous effects, including malignancy, cataract formation, thyroid dysfunction, dermatitis, germline mutations, etc. this is due to double-strand breaks induced in the dna backbone brought about by the free radicals m. ali et al. 29 in t e r n a l m e d ic in e issn 2413-6077. ijmmr 2021 vol. 7 issue 1 generated due to the exposure of ionizing radiations [4]. the hazard of radiation exposure increases with the higher radiation dose, which in turn increases with the longer duration of ep procedure for the patient and increase in the number of ep procedures for the laboratory staff. thus, it is essential to evaluate the benefit of the intended ep procedure over the risk of expected exposure to ionizing radiation and minimize the radiation exposure levels to minimum achievable levels for both the patients and the laboratory staff to have an overall beneficial outcome of the ep procedure [4]. one way to decrease radiation exposure in cardiac electrophysiology procedures is to use a lower fluoroscopy frame rate. we have previously reported of the impact of fluoroscopy frame rate reduction in complex catheter ablation procedures performed under a threedimensional electroanatomic mapping system [5]. in this study, we analyzed radiation exposure indices in our laboratory’s ap ablation procedures. we have also analyzed the impact of fluoroscopy frame rate reduction on radiation exposure indices during these procedures. the objectives of this study are: to quantify radiation exposure in accessory pathway ablation procedures; to analyze the radiation exposure trend over time; and to evaluate the effect of fluoroscopy frame rate reduction on the radiation exposure indices in these procedures. methods a retrospective analysis of accessory pathway ablation procedures carried out in a tertiary care referral institute in southern india was performed. the data were collected from january 2016 to december 2019. the data available were age, sex, location of successful ablation, fluoroscopy time, and dose area product (dap). procedures without such data were exclu_ ded. patients with multiple accessory pathways were also excluded from the analysis. the procedure time for catheter ablation procedures was defined as the time from the administration of a local anesthetic agent to removing catheters from the patient’s body. our institute has an ongoing cardiac electrophysiology fellowship program, and the fellows assist in all the catheter ablation procedures performed in our laboratory. four physicians performed the catheter ablation procedures with an experience of 21, 11, 6, and 4 years in interventional electrophysiology. all the catheter ablation procedures were performed without threedimensional electroanatomic mapping. the institute has an active ep fellowship program running since 2009, and the fellows assist in all the procedures performed in the laboratory. the laboratory is equipped with the philips allura xper fd 10 system (philips healthcare, the netherlands), and all the procedures were performed using the same system. the fluoroscopy frame rate used in the laboratory was 7.5 fps before february 2018. from the beginning of february 2018, fluoroscopy at 3.75 fps have been used. the location of the accessory pathways was determined by the location of successful ablation in the left anterior oblique (lao) projection, as shown in figure 1. the effective dose (ed, msv) was estimated from dap provided by the x-ray system by multiplying dap with the following conversion factors depending on the age of the patient: 5-10 years of age: 1.0; 10-15 years of age: 0.6; 15-20 years of age: 0.4; adult females: 0.28 and adult males: 0.2. lifetime attributable risk (lar) of cancer incidence and mortality was derived from the ed by multiplying it with 0.0001/msv (the standardized beir vii conversion factor) [5]. the data were summarized using standard descriptive statistics and presented as the arithmetic means with standard deviation (sd) or median with interquartile range, as appropriate. nonparametric statistical tests like the kruskal-wallis test and the mann-whitney u test were used to analyze the procedure time and radiation exposure parameters over the study period and the effect of fluoroscopy frafig. 1. location of accessory pathways as seen on a left anterior oblique (lao) projection. hb: his bundle; lal: left anterolateral; ll: left lateral; lpl: left posterolateral; lp: left posterior; lps: left posteroseptal; rps: right posteroseptal; rp: right posterior; rpl: right posterolateral; rl: right lateral; ral: right anterolateral; ra: right anterior; ras/ph: right anteroseptal/para-hisian; ms: midseptal. m. ali et al. 30 in t e r n a l m e d ic in e issn 2413-6077. ijmmr 2021 vol. 7 issue 1 me rate over various parameters, respectively. the level of significance was fixed at p<0.05. institutional ethical committee approval was not applied for this retrospective analysis. informed consent was obtained from all patients before the procedures, which included the clause that their data could be used for scientific purposes in the future. statistical package for the social sciences (spss), version 20, was used to perform statistical analysis. results six hundred and thirty-five pathway ablation procedures were included in the analysis. the mean age of the patients was 39±14 years. the mean age of male patients (n=401; 63% of the total) was 39±13 years, and female patients (n=234; 37% of the total) were 40±14 years. the location of the accessory pathways and the associated procedure time and radiation indices are presented in table 1. the number of the pathways ablated from the left side was 372 (58.6%), and that from the right were 263 (41.4%). the most common location of the ablated pathways was left lateral, which was 38.4 % of all the pathways, and the second most common location was right posteroseptal, which was 27.2% of all the pathways. the least common location of accessory pathways was mid septal (0.3%) followed by right anterolateral (0.5%) and right anterior (0.5%) locations. trends in various indices over the study period are shown in table 2. 15% (96) of all the procedures were performed in 2016. in the years 2017, 2018, and 2019 the proportion of the procedures performed was 23% (149), 31% (196), and 31% (194), respectively. trends in the indices before (pre group) and after (post group) the change in fluoroscopy frame rate are shown in table 3. 41% of the procedures (n=260) were performed under fluoroscopy frame rate of 7.5 fps, and 59% of the procedures (n=375) were performed under fluoroscopy frame rate of 3.75 fps. discussion table 1. procedure time and radiation indices based on the successful location of ablation on fluoroscopy in left anterior oblique projection pathways no. (%) proceduretime (min) fluoroscopy time (min) dap (cgy/cm2) ed (msv) lar, % lal 18 (2.8) 55 (45-98) 10 (8-14) 538 (313-706) 1.1 (0.8-1.5) 0.01 (0.008-0.02) ll 244 (38.4) 55 (45-75) 11 (8-17) 796 (480-1343) 1.8 (1.1-3.1) 0.02 (0.01-0.03) lpl 34 (5.4) 55 (44-81) 12 (7-17) 787 (483-1884) 1.9 (1.0-4.1) 0.02 (0.01-0.04) lp 22 (3.5) 58 (48-66) 13 (7-19) 1014 (456-1570) 2.3 (1.1-3.5) 0.02 (0.01-0.03) lps 54 (8.5) 80 (55-106) 20 (10-29) 1637 (795-2384) 3.6 (1.8-5.3) 0.04 (0.02-0.05) rps 173 (27.2) 60 (43-90) 13 (8-19) 988 (465-1609) 2.1 (1.1-3.6) 0.02 (0.01-0.04) rp 12 (1.9) 60 (36-94) 15 (7-26) 1372 (411-3420) 2.7 (0.8-8.2) 0.03 (0.01-0.08) rpl 24 (3.8) 69 (55-100) 19 (11-26) 1220 (738-2448) 3.5 (1.8-5.5) 0.04 (0.02-0.06) rl 27 (4.3) 90 (70-120) 27 (15-43) 1534 (848-2529) 3.6 (1.7-7.5) 0.04 (0.02-0.08) ral 3 (0.5) 105 (45-105) 17 (11-17) 701 (288-701) 2.8 (0.6-2.8) 0.03 (0.005-0.3) ra 3 (0.5) 65 (60-65) 21 (18-21) 1596 (723-1596) 4.4 (1.5-4.4) 0.04 (0.01-0.4) as/ph 19 (3.0) 65 (46-80) 15 (7-18) 555 (441-862) 1.2 (0.9-2.3) 0.01 (0.01-0.02) ms 2 (0.3) 100 (45-100) 21 (7-21) 1549 (377-1549) 3.1 (0.8-3.1) 0.01 (0.007-0.01 total 635 (100.0) 60 (45-90) 13 (8-19) 889 (488-1660) 2.1 (1.1-3.7) 0.02 (0.01-0.04) notes: lal: left anterolateral; ll: left lateral; lpl: left posterolateral; lp: left posterior; lps: left posteroseptal; rps: right posteroseptal; rp: right posterior; rpl: right posterolateral; rl: right lateral; ral: right anterolateral; ra: right anterior; ras/ph: right anteroseptal/para-hisian; hb: his bundle; ms: midseptal. table 2. change in procedure time and radiation indices over the study period year number procedure time (mins) fluoroscopy time (mins) dap (cgy/cm 2) ed (msv) lar, % 2016 96 75 (51-94) 19 (11-25) 1254 (751-2527) 3.1 (1.6-5.8) 0.03 (0.02-0.06) 2017 149 75 (60-110) 17 (12-30) 1604 (789-2713) 3.5 (1.8-5.7) 0.04 (0.02-0.06) 2018 196 60 (45-80) 12 (9-17) 798 (445-1284) 1.8 (1.0-3.1) 0.02 (0.01-0.03) 2019 194 50 (40-65) 9 (6-12) 596 (335-1079) 1.4 (0.8-2.4) 0.01 (0.008-0.02) p-value <0.001 <0.001 <0.001 <0.001 <0.001 m. ali et al. 31 in t e r n a l m e d ic in e issn 2413-6077. ijmmr 2021 vol. 7 issue 1 the main findings of our study are: a. there was a significant increase in the number of catheter ablation procedures performed in our laboratory over the study period. b. the procedure time and the radiation indices (fluoroscopy time, dap, ed, and lar) significantly decreased over the study period (p<0.001 for all the parameters). c. the trend of a significant decrease in procedure time and radiation exposure continued when the data were divided into two groups based on the fluoroscopy frame rate (p<0.001 for all the parameters). the radiation indices reported in different studies of catheter ablation procedures are presented in table 4. the radiation indices re ported are comparable to that of the published data. injury caused by exposure to ionizing radiation can be classified into two groups: a) the deterministic effects and b) the stochastic effects. deterministic effects are dose-de pendent (e.g., cataracts and skin injuries). a threshold radiation dose is a radiation dose below which the deterministic effects are not produced. on the other hand, even a tiny radiation dose involves an increased risk of stochastic effects (e.g., cancer), and the chances of suffering that effect are directly proportional to the radiation dose. as no radiation dose is safe, the policy of maintaining radiation exposure level is dependent on the “as low as reasonably achievable” (alara) principle. [4] some of the factors that influence the extent of ionizing radiation exposure in a cardiac ep laboratory are: 1) operator dependent: a) experience of the operators, b) training of operators with simulators, c) radiation awareness of the staff, d) c-arm projection used during the procedure, e) fluoroscopy frame rate, f) cine duration, g) cine substitution by stored fluoroscopy, h) fluoroscopy use during catheter removal from the body, i) collimation of the x-ray system, j) pelvic radiation, k) written report of the patient’s radiation exposure during the procedure. table 3. impact of fluoroscopy frame rate change on procedure time and radiation indices. pre-group: procedures performed at 7.5 fps; post-group: procedures performed at 3.75 fps number procedure time (mins) fluoroscopy time (mins) dap (cgy/cm2) ed (msv) lar, % pre 260 75 (55-104) 18 (12-27) 1432 (780-2564) 3.2 (1.8-5.7) 0.03 (0.02-0.06) post 375 55 (40-75) 10 (7-15) 698 (392-1176) 1.6 (0.9-2.7) 0.02 (0.01-0.03) p-value <0.001 <0.001 <0.001 <0.001 <0.001 table 4. reported radiation exposure in catheter ablation of supraventricular tachycardias (svt). adapted from [9] study type of study number of patients fluoroscopy time (mins) dap (cgy/cm 2) effective dose (msv) smith ir et al. [10] retrospective avnrt 270 avrt 135 2.1 (1.3-4.5) 23.8 (13.4-45.3) 260 (170-610) 2690 (1600-5410) – rogers dp et al. [7] observational pre drm 147 (avnrt/avrt) post drm 257 (avnrt/avrt) – 2040±2690 800±1030 3.3 1.24 heidbuchel h et al. [4] ehra practical guide – – – 4.4 (1.6-25) casella m et al. [6] multicentre randomized pre drm 128 post drm 134 14.32 (9.0822.43) 0 (0-0.2) 2036 (54–5297) 278 (80–791) 8.87 (3.67–22.01) 0 (0–0.08) see j et al. [11] observational pre drm avrt 55 post drm avrt 44 49.0 ± 36.3 14.1 ± 13.4 3292 ± 3282.7 654.4 ± 645.5 – casella m et al. [9] retrospective 979 (svt) 13(6-21) 1721(727-3884) 4.1(1.8-9.1) our data retrospective pre 260 post 375 18 (12-27) 10 (7-15) 1432 (780-2564) 698 (392-1176) 3.2 (1.8-5.7) 1.6 (0.9-2.7) m. ali et al. 32 in t e r n a l m e d ic in e issn 2413-6077. ijmmr 2021 vol. 7 issue 1 2) patient dependent: a) body habitus of the patient, b) arrhythmic lesion to be ablated. 3) technology dependent: a) x-ray system, b) combination with computed tomography (ct), c) three-dimensional electroanatomic mapping systems, d) shielding of the laboratory personnel. [4] the factors that lead to decreased radiation exposure in the cardiac ep laboratory are: 1) operator dependent: a) expert operators, b) operators who have been trained with simulators, c) radiation aware staff, d) predominant usage of right anterior oblique c-arm projection than an anteroposterior of left anterior oblique projection, e) low frame rate of fluoroscopy (<6 fps), f) short cine duration, g) frequent cine substitution by stored fluoroscopy, h) catheter withdrawal from the body without using fluoroscopy, i) optimized and adapted collimation, j) avoidance of pelvic radiation, k) a written report of the patient’s radiation exposure that includes air kerma or dose area product received during the procedure. 2) patient dependent: lean body habitus of the patients, supraventricular tachycardia ablation than atrial fibrillation ablation or ventricular tachycardia ablation, 3) technology dependent: cardiac ep tuned x-ray system which has been adequately maintained and inspected for quality control, no preprocedural or rotational computed tomography, the predominant use of threedimensional electroanatomic mapping systems, proper shielding which includes above and below the table or cabin shielding [4]. one of the most efficient ways to decrease radiation exposure during the ablation of accessory pathways is to perform these procedures with the help of three-dimensional electroanatomic mapping, which has been shown to decrease radiation exposure in such procedures significantly. no-party trial was the first multicentre, prospective, randomized trial that compared conventional fluoroscopy-guided catheter ablation procedures with procedures performed using the three-dimensional electroanatomic mapping system in the patients undergoing ep study for supraventricular tachycardias, 36% of whom were ap ablation procedures [6]. in this trial, three-dimensional electroanatomic mapping significantly reduced fluoroscopy time and ed (0 seconds and 0 msv) when compared to the procedures performed under fluoroscopic guidance (859 seconds and 8.87 msv) (p<0.00001). but the use of three-dimensional electroanatomic mapping systems is restricted due to various logistic reasons (availability, lack of expertise); we need to find alternative ways to decrease radiation exposure during catheter ablation procedures [4]. rogers a j et al. conducted a study to evaluate the effect of radiation dose-reduction maneuver on the radiation exposure in ep laboratory. atrioventricular nodal reentrant and ap-mediated tachycardia was 64% of catheter ablation procedures. they showed that with the simple maneuver of removing a secondary radiation grid to improve image quality and reducing the fluoroscopy pulse rate from 12.5 to 6.25 pulses/second, a radiation dose reduced from 20.4 to 8.0 gycm2. additionally, the risk of radiation-related fatal malignancy was reduced by 63 due to the implementation of the maneuver. this study showed the importance of selecting electrophysiology laboratory parameters in reducing radiation exposure during catheter ablation procedures [7]. voskoboinik a et al. conducted a prospective study to analyze the trends in radiation exposure during af ablation at a single center over 12 years. a significant and progressive decrease in the fluoroscopy time and ed was observed over time. the significant decrease in the patient and operator radiation exposure was attributed to increased operator experience, higher annual case volume, technology evolution over time, and recent use of contact force-sensing catheters. [8] more recently, casella m et al. have reported fluoroscopy data from their retrospective analysis of various ep and device implantation procedures at a large volume laboratory over 7 years. fluoroscopy time, dap, and ed showed a statistically significant reduction trend for all ep procedures. based on the obtained results, it was proposed that a combination of operator awareness about the fluoroscopy-associated risk and technological advancement can be used for optimizing the use of fluoroscopy in ep procedures [9]. one of the significant limitations of this study, other than being a retrospective analysis, is that one can argue that a significant decrease in the procedure duration can solely explain the changes in the radiation exposure in the procedure time, which is also reflected in the decrease in the fluoroscopy time. if there is a significant decrease in the procedure time, the radiation exposure will automatically decrease. m. ali et al. 33 in t e r n a l m e d ic in e issn 2413-6077. ijmmr 2021 vol. 7 issue 1 some of the other limitations of this study are that data regarding some of the other factors that influence radiation exposure during procedures (e.g., body mass index of the patients, cine exposure duration during the procedures) performed under fluoroscopy was not available. this study is based on retrospective data collected from a single center, and the results cannot be generalized to other centers. as already alluded to, radiation exposure also depends on the individual expertise of operators, but data regarding the radiation exposure of individual operators was not available. we propose that in our study, the decrease in the radiation indices is not solely because of the reduction in fluoroscopy frame rate but because of other factors, too, one being a more than 200% increase in the yearly number of ablation procedures performed over the study period. any modern electrophysiology laboratory will face the same scenario. conclusions a reduction in the fluoroscopy frame rate and improvement in the electrophysiology laboratory workflow leads to a significant decrease in the radiation exposure during catheter ablation procedures of accessory pathway-mediated tachycardias. conflict of interest the authors declare no conflict of interest. authors’ contributions muzaffar ali: formal analysis, writing – original draft, investigation, formal analysis. bharatraj banavalikar: writing – reviewing and editing, investigation, formal analysis. milan kumar ghadei: formal analysis, writing – original draft, investigation, formal analysis. anju kottayan: formal analysis, writing – original draft, investigation, formal analysis. deepak padmanabhan: writing – reviewing and editing, investigation, formal analysis. jayaprakash shenthar: conceptualization, methodology, writing – reviewing and editing іонізуюче опромінення при радіочастотній абляції додаткових шляхів проведення у електрофізіології серця: ретроспективний аналіз *m. ali1,2, b. banavalikar1, m.k. ghadei1, a. kottayan1, d. padmanabhan1, j. shenthar1 1 – department of cardiac electrophysiology, sri jayadeva institute of cardiovascular sciences and research, bangalore, india 2 – department of cardiology, sher-i-kashmir institute of medical sciences, srinagar, kashmir, india вступ. радіочастотна катетерна абляція (ка) метод вибору лікування у пацієнтів з тахікардією, додатковими шляхами (дш). більшість таких процедур проводиться під флюо роскопічним наглядом, що призводить до значного опромінення пацієнта та персоналу лабораторії. у цьому аналізі ми розглянули радіаційне опромінення в процедурах кадш, виконаних без підтримки тривимірної системи електроанатомічного відображення. ми проаналізували зміни показників експозиції за період дослідження та вплив зміни частоти кадрів флюороскопії (ffr). мета. метою цього дослідження є (1) кількісна оцінка радіаційного опромінення в процедурах абляції додаткових шляхів, (2) аналіз тенденції радіаційного опромінення з часом та (3) оцінка впливу зменшення частоти кадрів флюороскопії на показники радіаційного опромінення під час цієї процедури. методи. ми ретроспективно проаналізували всі процедури абляції дш, проведені в нашому інституті з січня 2016 року по грудень 2019 року. зібрані дані: вік, стать, місце розташування дш на основі успішного місця абляції на флюороскопії, час процедури, час флюороскопії та індекс доза опромінення відносно площі (dap). ефективну дозу (ед) оцінювали за dap. дані процедур, проведених до січня 2018 р. (група “до”), порівнювались із даними процедур, проведених після цієї дати (група “після”). процедури групи “до” виконувались із частотою ffr 7,5 кадрів в секунду (fps), а процедури групи “після” – з частотою ffr 3,75 fps. результати. загальна кількість процедур, включених до аналізу, становила 635. середній вік пацієнтів становив 39±14 років, і 401 з них (63%) були чоловіками. найчастіше зустрічалися ліволатеральні дш (38%). показники тривалості процедури та опромінення зменшилися протягом періоду дослідження (р<0,001). процедури групи “після” мали значно коротший час проведення ка та менший радіаційний вплив, ніж процедури групи “до”. висновки. зменшення ffr було пов'язане зі значним зменшенням радіаційного опромінення в процедурах абляції дш. ключові слова: додаткові шляхи проведення; катетерна абляція; дозування опромінення; час флюороскопії; радіаційне опромінення. m. ali et al. 34 in t e r n a l m e d ic in e issn 2413-6077. ijmmr 2021 vol. 7 issue 1 information about the authors muzaffar ali md, dm: department of cardiac electrophysiology, sri jayadeva institute of cardiovascular sciences and research, bangalore, india. orcid 0000-0002-3489-5864, email: dralmuzaffar@gmail.com bharatraj banavalikar md, dm: department of cardiac electrophysiology, sri jayadeva institute of cardiovascular sciences and research, bangalore, india. orcid 0000-0003-1116-3967, email: bharatrajnb@gmail.com milan kumar ghadei md, dm: department of cardiac electrophysiology, sri jayadeva institute of cardiovascular sciences and research, bangalore, india. dr.milan80@gmail.com anju kottayan bsc: department of cardiac electrophysiology, sri jayadeva institute of cardiovascular sciences and research, bangalore, india. anjukottayan@gmail.com deepak padmanabhan md, dm: department of cardiac electrophysiology, sri jayadeva institute of cardiovascular sciences and research, bangalore, india. orcid 0000-0002-9127-9275, email: deepak.padmanabhan@gmail.com jayaprakash shenthar md, dm, facc, frcp (lond): department of cardiac electrophysiology, sri jayadeva institute of cardiovascular sciences and research, bangalore, india. orcid 0000-0003-4509-7515, email: jpsbhat@gmail.com references 1. borggrefe m, budde t, podczeck a, breithardt g. high frequency alternating current ablation of an accessory pathway in humans. 10:7. doi: 10.1016/ s0735-1097(87)80200-0 2. page rl, jogler ja, caldwell ma. 2015 acc/aha/ hrs guideline for the management of adult patients with supraventricular tachycardia. heart rhythm. 2015;13(4):86. doi: 10.1016/j.hrthm.2015.09.019 3. katritsis dg, arbelo e, arribas f. the task force for the management of patients with supraventricular tachycardia of the european society of cardiology (esc). eur heart j. 41(2020):655–720. doi: 10.1093/ eurheartj/ehz467 4. heidbuchel h, wittkampf fhm, vano e, ernst s, schilling r, picano e, et al. practical ways to reduce radiation dose for patients and staff during device implantations and electrophysiological procedures. ep eur. 2014 jul 1;16(7):946–64. doi: 10.1093/ europace/eut409 5. ali m, padmanabhan d, kanjwal k, ghadei mk, kottayan a, banavalikar b, et al. effect of fluoroscopy frame rate on radiation exposure and in-hospital outcomes in three-dimensional electroanatomic m a p p i n g g u i d e d p ro c e d u re s . j a r r h y t h m i a . 2021;37:97–102. doi: 10.1002/joa3.12496 6. casella m, dello russo a, pelargonio g, del greco m, zingarini g, piacenti m, et al. near zero fluoroscopic exposure during catheter ablation of supraventricular arrhythmias: the no-party multicentre randomized trial. europace. 2016 oct;18(10): 1565–72. doi: 10.1093/europace/euv344 7. rogers dps, england f, lozhkin k, lowe md, lambiase pd, chow awc. improving safety in the electrophysiology laboratory using a simple radiation dose reduction strategy: a study of 1007 radiofrequency ablation procedures. heart. 2011 mar 1;97(5):366–70. doi: 10.1136/hrt.2010.204222 8. voskoboinik a, kalman es, savicky y, sparks pb, morton jb, lee g, et al. reduction in radiation dose for atrial fibrillation ablation over time: a 12-year single-center experience of 2344 patients. heart rhythm. 2017 jun;14(6):810–6. doi: 10.1016/j. hrthm.2017.02.014 9. casella m, dello russo a, russo e, catto v, pizzamiglio f, zucchetti m, et al. x-ray exposure in cardiac electrophysiology: a retrospective analysis in 8150 patients over 7 years of activity in a modern, large-volume laboratory. j am heart assoc. 2018 jun 5;7(11). doi: 10.1161/jaha.117.008233 10. smith ir, rivers jt, hayes j, stafford w, codd c. reassessment of radiation risks from electro physiology procedures compared to coronary angiography. heart lung circ. 2009 jun;18(3):191–9. doi: 10.1016/j.hlc.2008.10.006 11. see j, amora j, lee s, lim p, teo w, tan b, et al. non-fluoroscopic navigation systems for radiofrequency catheter ablation for supraventricular tachycardia reduce ionising radiation exposure. singapore med j. 2016 jul;57(07):390–5. doi: 10.11622/smedj.2016017 received 23 may 2021; revised 20 jun 2021; accepted 22 jun 2021. this is open access article distributed under the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. m. ali et al. 68 b io m e d ic a l s c ie n c e s issn 2413-6077. ijmmr 2020 vol. 6 issue 1 doi 10.11603/ijmmr.2413-6077.2020.1.11502 morphometric evaluation of spermatogenic epitheliocytes structure under the rubomycin influence in rats m.s. hnatiuk, *s.о. konovalenko, l.v. tatarchuk, о.b. yasinovsky i. horbachevsky ternopil national medical university, ternopil, ukraine background. over the last 20 years, the quality of men’s sperm around the world has deteriorated twice. the causes are poor environment, stress, sedentary lifestyle, prevalence of sexually transmitted infections, obesity, alcohol abuse and various stimulants. the structure and function of the testes, when exposed to various drugs and chemicals, attracts the attention of present researchers. objectives. the aim of the research was to study the structural changes of the spermatogenic epithelium in testicular damage caused by rubomycin hydrochloride. methods. the spermatogenic epithelium of the testes of 30 white adult white male rats, which were divided into 2 groups, was morphometrically examined. the 1st group comprised 15 experimental intact animals, the 2nd – 15 rats, in which testicular damage with rubomycin hydrochloride was simulated. euthanasia of rats was performed by bloodletting under thiopental anesthesia one month after the experiment beginning. quantitative morphological parameters were statistically processed. in spermatocytes of the 1st p-order, spermatogonia and spermatids of testes, their height, diameter of nuclei, nuclear-cytoplasmic ratio and relative volumes of damaged cells of spermatogenic epithelium were evaluated. results. it was established that spermatogenic epithelial cells were damaged in the simulated experimental conditions, which was morphometrically confirmed by changes in nuclear-cytoplasmic relations in the 1st, 2nd order spermatocytes, spermatogonia and spermatids. the relative volumes of damaged spermatogenic epithelial cells also increased significantly. the most significant morphometric parameters were altered in spermatids. thus, the nuclear-cytoplasmic ratio in the studied cells was statistically significantly (p <0.001) increased by 7.4% compared with the similar control morphometric parameter. the relative volume of damaged spermatids in these experimental conditions reached (32.50±0.18) %, which with a high statistically significant difference (p<0.001) exceeded the same control value in almost 15.8 times. optically, histological preparations of the testes showed severe vascular disorders, characterized by dilation and plethora mostly of venous vessels, which pointed to violation of venous drainage of the studied organs. conclusions. according to the attained research results it has been established that nuclear-cytoplasmic relations are an objective and valuable informative indicator of a functional condition of cells and their structural changes in pathological conditions. when rubomycin hydrochloride is exposed to experimental animals, the most significant changes in nuclear-cytoplasmic relations are found in spermatic spermatozoa. key words: rubomycin hydrochloride, testes, spermatogenic epithelium, white rats, morphometry. international journal of medicine and medical research 2020, volume 6, issue 1, p. 68-73 copyright © 2020, tnmu, all rights reserved *corresponding author: s.о. konovalenko, md, ph.d., associate professor, department of operative surgery and clinical anatomy, i. horbachevsky ternopil national medical university, ternopil, ukraine; e-mail: konovalenko @tdmu.edu.ua m.s. hnatiuk et al. introduction it is established that in the structure of infertile marriages, the male factor is 20% [2, 4, 8, 12, 17]. the reason for this is a decrease in spermatogenic and hormonal function of the testes. often the reason for this is the effect of various toxic factors on the body [2, 13, 14, 19]. in recent years, many researchers are interested in changes in the structure and function of the testes in different physiological and pathological conditions [10, 11, 15, 19]. rubomycin is an antitumor antibiotic of the anthracycline series with a significant cytostatic effect, which when administered to men can lead to azoospermia (absence of sperm in the ejaculate) [5]. the effect of rubomycin on the structure of the spermatogenic epithelium has been studied insufficiently. the aim of our study was to investigate the features of structural changes in the spermato genic epithelium in cases of testicular damage by rubomycin hydrochloride. methods the testicular structures of 30 laboratory adult white male rats, which were divided into 2 groups, were studied by morphometric methods. the 1st group comprised 15 exper mental intact animals, the 2nd – 15 rats with simulated 69 b io m e d ic a l s c ie n c e s issn 2413-6077. ijmmr 2020 vol. 6 issue 1 m.s. hnatiuk et al. testicular damage by rubomycin hydrochloride [18]. one month after the beginning of the experiment, euthanasia of experimental animals was performed by bloodletting under conditions of thiopental-sodium anesthesia. pieces were cut from the testes, which were fixed in 10 % neutral formalin solution, passed through ethyl alcohols of increasing concentration and placed in paraffin. microtome sections 5-6 mcr thick after dewaxing were stained with hematoxylin-eosin, according to van gizon, mallory, weigert, toluidine blue [7]. histological micropreparations determined the height of spermatogenic cells, the diameter of their nuclei, nuclear-cytoplasmic ratios, the relative volume of damaged epitheliocytes (vode) [1, 6, 16]. morphometry of these structures was performed using a light microscope “olimpus bx-2” with a digital video camera and application package “video test 5.0” and “video size 5.0”. quantitative values were processed statistically by statistika software package. the differences between the comparative values were determined by the mann-whitney and student’s tests [9]. the studies were performed according to national and international regulations on carrying out experimental tests (“european convention for the protection of vertebrate ani mals used for experimental and other scientific purposes” (strasbourg, 1986), “general ethical principles of animal experiments”, adop ted by the first national congress on bioethics (kiev, 2001)) [8], as well as the law of ukraine “on protection of animals from cruelty”, dated february 21, 2006. results the attained morphometric parameters are presented in table 1. a comprehensive analysis of the data showed that most of the studied morphometric parameters of the spermatogenic epithelium changed significantly. thus, the height of spermatocytes of the 1st order decreased slightly by only 0.65% (p<0.05). the diameters of the nuclei of these cells with a high statistically significant difference (p<0.001) increased from (5.25±0.03) µm to (5.76±0.04) µm, i.e. by 9.7%. uneven changes in the spatial characteristics of the nucleus and cytoplasm of spermatocytes of the 1st order led to violations of nuclear-cytoplasmic relations in them. this morphometric index increased by 3.2% (p<0.05), which pointed to violation of the studied elements of structural cellular homeostasis [1]. the relative volume of damaged first-order sper matocytes under these experimental conditions with a high statistically significant difference (p<0.001) increased almost in 9.7 times. the morphometric parameters of p-order spermatocytes changed almost similarly under the influence of rubomycin hydrochloride. the height of these cells was almost the same as in the control, the diameters of the nuclei were statistically significantly (p<0.05) increased by 2.4%, and the nuclear-cytoplasmic ratio – by 4.9% (p<0.01). the relative volume of damaged table 1. morphometric characteristics of cells of spermatogenic epithelium of testes of the experimental animals (m±m) cells morphometric parameter height, microns diameter of the core, microns nuclear-cytoplasmic relations vode, % control group spermatocytes of the first order 7.65±0.05 5.25±0.03 0.556±0.003 2.20±0.01 spermatocytes of the second order 5.88±0.04 3.76±0.03 0.410±0.002 2.26±0.02 spermatogonia 6.12±0.04 4.80±0.03 0.615±0.004 2.15±0.02 spermatids 4.98±0.03 3.07±0.03 0.389±0.002 2.06±0.02 experimental group spermatocytes of the first order 7.60±0.05 5.76±0.04*** 0.574±0.004* 21.30±0.15*** spermatocytes of the second order 5.85±0.04 3.85±0.03* 0.430±0.003** 25.60±0,15*** spermatogonia 6.05±0.04 4.79±0.03 0.628±0.004* 29.80±0.15*** spermatids 4.92±0.03 3.18±0.02* 0.418±0.002*** 32.50±0.18*** notes. * – р<0.05; ** – р<0.01; *** – р<0.001. vode – volume of damaged epitheliocytes. 70 b io m e d ic a l s c ie n c e s issn 2413-6077. ijmmr 2020 vol. 6 issue 1 p-order spermatocytes under these experimental conditions was equal to (25.60±0.15)%. this morphometric parameter with a high statistically significant (p<0.001) difference exceeded the similar quantitative morphological index in 11.3 times. the height of spermatogonia of the testes in the experimental animals affected by the studied drug medium decreased by only 1.1% (p<0.05), the diameters of their nuclei did not change significantly, and nuclear-cytoplasmic ratios increased by 2.1% (p<0.05). the relative volume of damaged spermatogonia in these experimental conditions statistically signi ficantly (p<0.001) increased in 13.8 times and reached (29.80±0.15)%. analysis of the obtained data revealed that under the influence of rubomycin hydrochloride the height of testicular spermatids decreased by only 1.2% (p<0.05), the diameters of their nuclei increased by 3.6% (p<0.05). nuclearcytoplasmic ratios in the studied cells statistically significantly (p<0.001) increased by 7.4% compared with the similar control morphometric parameter. the relative volume of damaged spermatids under these experimental conditions reached (32.50±0.18) %. this morphometric parameter with a high statistically significant difference (p<0.001) exceeded the similar control value in almost 15.8 times. discussion analysis of the above morphometric parameters proved that the introduction of rubomycin hydrochloride into the body of ex perimental animals led to structural rearrangement and damage to cells of spermatogenic epithelium of the testes. the most pronounced changes were found in spermatids in comparison with other studied cells. according to present researchers, nuclearcytoplasmic relations are a valuable morphometric indicator that adequately reflects the peculiarities of cell function and metabolism, degree of disturbances of cellular structural homeostasis [1, 6, 16]. the nucleus and cytoplasm of cells are separated from each other, but at the same time they are closely interconnected and integrated and form a single structural and functional system. it follows that the isolated study of quantitative morphological parameters of only the cytoplasm of the cell or only its nucleus allows getting a one-sided idea of these structural components of the cell. the study of nuclear-cytoplasmic relationships makes it possible to obtain more adequate, objective and in-depth information on the relationship between the nucleus and cytoplasm of cells and their changes in various physiological and pathological conditions. nuclear-cytoplasmic relations can change at hyperfunction of bodies as well as hypofunction, and also in cases of their damage. it is established that nuclear-cytoplasmic ratios depend on cell differentiation. the specified morphometric parameter decreases in case of maturation of cells and their differentiation that is caused to some extent by increase in cytoplasm and its hyperplasia of specific functioning organelles. the relationship between the spatial charac teristics of the nucleus and cytoplasm of the cell can also change with cell division, growth, diploidy [6, 16]. in the optical examination of testicular micro preparations, significant vascular disorders were evidenced, which were characterized by dilation and plethora of mainly venous vessels, which pointed to violation of venous drainage from the studied organs (fig. 1) [3]. a severe edema of the stroma, foci of dystrophically, necrobiotically altered spermatogenic epitheliocytes, stromal structures, local cellular infiltrates, sclerotic processes, desquamation and proliferation of vascular endothelial cells was present (pic. 2). the latter pointed to the presence of hypoxia. foci of vascular en dothelial cell edema, permeation of their membrane with plasma proteins were also present. foci of fibrinoid swelling and necrosis in some vessels were evidenced that indicated their severe damage. among spermatogenic epitheliocytes in the studied conditions of the experiment, spermatids were the most morpho logically altered, in which the degree of disorders fig. 1. stromal edema, partial reduction of layers and damage of spermatogenic epithelial cells, their desquamation, thickening of the membranes of the tortuous seminal tubules of the testis of a white rat under the influence of rubomycin hydrochloride. hematoxylin-eosin staining. ×140. m.s. hnatiuk et al. 71 b io m e d ic a l s c ie n c e s issn 2413-6077. ijmmr 2020 vol. 6 issue 1 of nuclear-cytoplasmic relations and the relative volume of damaged cells were the largest. it points to the fact that the studied morphometric parameter also reflects the severity and depth of cell damage. conclusions nuclear-cytoplasmic relations are an objective and significant informative indicator of functional state of the cells and their structural changes in pathological conditions. when rubomycin hydrochloride is exposed to experimental animals, the most significant changes in nuclear-cytoplasmic relations are found in spermatic spermatozoa. funding this research received no external funding. conflict of interests the authors declare no conflict of interest. author contributions hnatiuk m.s. – supervision, morphometry, morphological analysis of micropreparations, writing – review and editing; konovalenko s.о. – writing, generalization of results, project administration, tatarchuk l.v. – supervision, review and editing , yasinovsky о.b. – literature analysis, data curation. fig. 2. structural changes in the vessel wall, perivasal sclerosis, dystrophy, edema, desquamation of spermatogenic epitheliocytes in the tortuous seminal tubules of the testis of experimental animals under the influence of rubomycin hydrochloride. hematoxylin-eosin staining. ×140. морфометрична оцінка структурної перебудови клітин сперматогенних епітеліоцитів сім′яників експериментальних тварин під впливом рубоміцину гідрохлориду м.с. гнатюк, с.о. коноваленко, л.в. татарчук, о.б. ясіновський тернопільський національний медичний університет імені і.я.горбачевського, тернопіль, україна вступ. за останні 20 років якість сперми чоловіків у всьому світі погіршилась у 2 рази. причини: погана екологія, стреси, малорухливий спосіб життя, поширеність статевих інфекцій, ожиріння, зловживання алкоголем і різними стимуляторами. структура та функція сім′яників при дії на організм різних медикаментозних середників та хімічних речовин привертає увагу сучасних дослідників. мета. вивчити особливості структурних змін сперматогенного епітелію при ушкодженні сім’яників рубоміцином гідрохлориду. методи. морфометрично досліджено сперматогенний епітелій сім′яників 30 білих статевозрілих білих щурів-самців, які були були поділені на 2-і групи. 1-а група включала 15 дослідних інтактних тварин, 2-а – 15 щурів, у яких моделювали ушкодження сім′яників рубоміцином гідрохлоридом. евтаназію щурів здійснювали кровопусканням в умовах тіопенталового наркозу через місяць від початку досліду. кількісні морфологічні показники обробляли статистично. у сперматоцитах 1-го. п-го порядків, сперматогоніях та сперматидах сім′яників вимірювали їх висоту, діаметр ядер, ядерно-цитоплазматичні відношення та відносні об′єми пошкоджених клітин сперматогенного епітелію. результати. встановлено, що у змодельованих експериментальних умовах пошкоджувалися клітини сперматогенного епітелію, що морфометрично підтверджувалося змінами ядерно-цитоплазматичних відношень у сперматоцитах 1-го, 2-го порядків, сперматогоніях та сперматидах. виражено зростали також відносні об′єми пошкоджених вказаних клітин сперматогенного епітелію. найбільш виражено морфометричні параметри виявилися зміненими у сперматидах. так, ядерно-цитоплазматичні відношення у досліджуваних клітинах статистично достовірно (р<0,001) збільшилися на 7,4 % порівняно з аналогічним контрольним морфометричним параметром. відносний об′єм пошкоджених сперматид у даних умовах експерименту досягав (32,50±0,18) %, що з вираженою статистично достовірною різницею (р<0,001) перевищував аналогічну контрольну величину майже у 15,8 рази. світлооптично на гістологічних препаратах сім'яників cпостерігалися виражені судинні розлади, що характеризувалися m.s. hnatiuk et al. 72 b io m e d ic a l s c ie n c e s issn 2413-6077. ijmmr 2020 vol. 6 issue 1 розширенням та повнокров′ям переважно венозних судин, що свідчило про порушення венозного дренажу від досліджуваних органів. висновки. на основі проведених досліджень та отриманих результатів встановлено, що ядерноцитоплазматичні відношення є об′єктивним та цінним інформативним показником функціонального стану клітин та їх структурних змін в патологічних умовах. при дії на організм дослідних тварин рубоміцину гідрохлориду найвираженіші зміни ядерно-цитоплазматичних відношень виявлені у сперматидах сім′яників. ключові слова: рубоміцин гідрохлорид, сім′яники, сперматогенний епітелій, білі щурі, морфометрія. інформація про авторів гнатюк михайло степанович – доктор медичних наук, професор, завідувач кафедри оперативної хірургії та клінічної анатомії, тернопільський національний медичний університет імені і.я. горбачевського. orcid http://orcid.org/0000-0002-4110-5568, e-mail: hnatjuk@tdmu.edu.ua коноваленко сергій олександрович – канд. мед. наук, доцент кафедри оперативної хірургії та клінічної анатомії, тернопільський національний медичний університет імені і.я. горбачевського. orcid http://orcid.org/0000-0002-3478-462x, e-mail: konovalenko @tdmu.edu.ua татарчук людмила василівна – доктор медичних наук, асистент кафедри фізіології з основами біоетики та біобезпеки, тернопільський національний медичний університет імені і.я. горбачевського. orcid http://orcid.org/0000-0002-4678-4205, e-mail: tatarchyklv@tdmu.edu.ua ясіновський олег борисович – канд. мед. наук, асистент кафедри оперативної хірургії та клінічної анатомії, тернопільський національний медичний університет імені і.я. горбачевського. orcid http://orcid.org/0000-0002-5121-3140, e-mail: yasinovskyi@tdmu.edu.ua information about the authors hnatjuk m.s. – md, ph.d., dsc, professor, head of the department of operative surgery and clinical anatomy, i. horbachevsky ternopil national medical university, ternopil, ukraine. orcid http://orcid.org/0000-0002-4110-5568, e-mail: hnatjuk@tdmu.edu.ua konovaleno s.о. – md, ph.d., associate professor, department of operative surgery and clinical anatomy, i. horbachevsky ternopil national medical university, ternopil, ukraine. orcid http://orcid.org/0000-0002-3478-462x, e-mail: konovalenko @tdmu.edu.ua tatarchuk l.v. – md, ph.d., dsс, assistant professor, department of operative surgery and clinical anatomy, i. horbachevsky ternopil national medical university, ternopil, ukraine. orcid http://orcid.org/0000-0002-4678-4205, e-mail: tatarchyklv@tdmu.edu.ua yasinovsky о.b. – md, ph.d., assistant professor, department of operative surgery and clinical anatomy, i. horbachevsky ternopil national medical university, ternopil, ukraine. orcid http://orcid.org/0000-0002-5121-3140, e-mail: yasinovskyi@tdmu.edu.ua references 1. avtandilov gg. fundamentals of quantitative pathology. м.: meditsina.2002;240 p. (in russian). 2. bazalitskaya sv, gorpinenko ii, romanenko am. diagnostic methods and criteria for evaluating biopsy material in male infertility. men’s health. 2004;3:216-221. 3. baibakov vm. model of morphofunctional changes of testicular drainage systems in the experiment. pediatric surgery. 2016;1-2:43-45. 4. bedenyuk ad, tverdokhlib vv, mysak ai, nesteruk so. improving spermatogenesis in the complex treatment of male infertility. achievements of clinical and experimental medicine. 2019;2:83-87. 5. burbela at, shabrov av, denisenko pp. modern medicines. m.: olma-press, 2003;862 p. 6. hnatyuk ms, slabiy ob, tatarchuk lv. nuclearcytoplasmic relations in cardiomyocytes and ventricular endotheliocytes of the pulmonary heart. clinical anatomy and operative surgery. 2016;15(1):6770. 7. goralsky lp, khomych vt, kononsky oi. fundamentals of histological technique and morphofunctional research methods in normal and pathology. zhytomyr: polissya.2011;288 p. 8. gorpinchenko ii, romanyuk mg. male infertility: etiology, pathogenesis, diagnosis and modern methods of treatment. men's health. 2016;1:816. 9. grzhibovsky ai, ivanov ov, gorbatova ma. comparison of quantitative data of two paired samples with the implementation of statistica and m.s. hnatiuk et al. 73 b io m e d ic a l s c ie n c e s issn 2413-6077. ijmmr 2020 vol. 6 issue 1 spss software: parametric and nonparametric criteria. science and health. 2016;3:5-25. 10. gritsulyak bv, glodan oya. cytological changes in the testicle under conditions of blockade of blood flow from it in the experiment. bulletin of the precarpathian national university. v. stefanyk, series "biology", 201;xv.201-204. 11. hrytsulyak bv, spaska am, hrytsulyak vb. orchoepididymitis. ivano-frankivsk: precarpathian national university. 2010;188 p. 12. dariy os. optimization of tactics of assisted reproductive technologies in married couples with a burdened genetic history. women’s health. 2018;8:39-42. 13. dolinko np. influence of ginkgo biloba on morphometric and functional parameters of spermatozoa in conditions of chronic ethanol intoxication. bulletin of mogilev state university named after aa kuleshov. natural science series. 2015:2 (46):99 102. 14. dubinina aa. toxic substances and methods for their determination. kharkiv: khduht, 2016;106 p. 15. pastukhova va. porial analysis of the structural organization of the testicles of rats in the age aspect. taurian medical and biological bulletin. 2009.4(48):238-241. 16. weak ob, hnatyuk ms, tatarchuk lv. nuclearcytoplasmic relations in endotheliocytes of arterial and venous channels of the atria of the pulmonary heart. achievements of clinical and experimental medicine. 2018;2:253-254. 17. stus vp, polion ym, polion myu. restoration of spermatogenesis in patients with excretory-toxic infertility. men’s health. 2016;2:143-145. 18. konovalenko so., hnatiuk ms, yasinovskyi ob, tatarchuk lv. morphometric analysis of peculiarities of the testicular arteries remodeling under the influence of rubomycin hydrochloride. “science" poland (issue 11), warsaw. 2019;77-83. 19. mallidis c, agbaje і, mcclure n, kliesch s. the influence of diabetes mellitus on male reproductive function: a poorly investigated aspect of male infertility. urologe. 2011;50 (1):33–37. received 26 mar 2020; revised 07 apr 2020; accepted 29 may 2020. this is open access article distributed under the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. m.s. hnatiuk et al. 44 su r g ic a l d is e a se s issn 2413-6077. ijmmr 2022 vol. 8 issue 1 doi 10.11603/ijmmr.2413-6077.2022.1.13043 clinical outcomes of reversal of hartmann’s procedure i. ya. dzyubanovskyy, a. d. bedeniuk, *s. y. grytsenko i. horbachevsky ternopil national medical university, ternopil, ukraine. background. reversal of hartmann’s procedure (hp) is one of the most complex surgical interventions in abdominal surgery with high morbidity rates of up to 58% in the patients after hp and mortality of up to 3.6%. objective. this was a retrospective observational study to analyze the hartmann’s reversal (hr) outcomes. methods. the study involved 31 patients (16 males and 15 females), average age 59.6±10.31 (range 26-80), who underwent hr at ternopil regional hospital in 2010 2021. the reasons for the hp were: cancer in 20 (64.5%) cases, perforation of the diverticulum – 6 (19.3%), traumatic rupture – 3 (9.7%) and crohn’s disease 2 (6.4%) cases. results. median time from hartmann’s procedure to reversal was 11.13±9.24 months (interval 3-38). intraoperative time was 210.33±56.91 minutes (range 120-330). hr was performed in 30 (96.8 %) patients. dense pelvic adhesions of the stump of the rectum was diagnosed in 13 (41.9%) patients. hand-sewn anastomosis was performed for 22 (71%) patients, stapler anastomosis – 4 (12.9%), pull-through technique – 3 (9.7%) patients. al occurred in 3 (9.7%) patients on the 15th, 23rd and 35th postoperative days. pseudomembranous colitis was diagnosed in 2 (6.4%) patients with al. the mortality rate was 1 (3.3%) as a result of septic complications due to al; this case was not operated by a colorectal surgeon due to administrative issue in the hospital. conclusions. hartmann’s reversal is still one of the most difficult operations in colorectal surgery with high incidence of postoperative complications. sound selection of patients with low comorbidity and in suitable time period is crucial for successful hr. keywords: hartmann’s procedure; reversal; anastomotic leakage. *corresponding author: stepan grytsenko, assistant professor, i. horbachevsky ternopil national medical university, ternopil, 46000, ukraine. e-mail: grytsenko_s@tdmu.edu.ua international journal of medicine and medical research 2022, volume 8, issue 1, p. 44-47 copyright © 2022, tnmu, all rights reserved introduction a century has passed since henry hartmann firstly performed the widely known operation, but it has not lost its relevance and is frequently used around the world to pre vent anastomotic leakage (al), which remains an eternal problem in colorectal surgery [1]. reversal of hartmann’s procedure (hp) is one of the most complex surgical interventions in abdominal surgery with high morbidity rates of up to 58% in the patients after hp and mortality of up to 3.6% [6, 8]. multifactorial technical difficulties particular to reversal include dense pelvic adhesions, pelvic infection, adhesions, difficulty in identification of rectal stump and perfoming of colorectal anastomosis with short rectal stump [1, 7]. as a result, more than two-thirds of patients remain with a permanent stoma following hartmann’s procedure, either due to inability to perform reversal or due to anastomotic leak and stoma restoration [1, 6, 7]. the majority of ostomy patients become socially and psychologically maladapted need to manage the physical and psychological challenges associated with a stoma [7]. methods this was a retrospective observational study to analyze the hartmann’s reversal (hr) outcomes. the study involved thirty-one patients (16 males and 15 females) of average age 59.6±10.31 (range 26-80), who underwent hr at ternopil regional hospital in 2010-2021. the median bmi was 25.2 (range 18-39). most of the patients (80.6 %) had an asa score 3-4. median time from hartmann’s procedure to reversal was 11.13±9.24 months (interval 3-38). intraoperative time was 210.33±56.91 minutes (range 120-330). blood loss was 331.67±191.4 ml (range 150-1000). the reasons for the hp were: cancer in 20 (64.5%) cases, perforation of the diverticulum – 6 (19.3%), traumatic rupture – 3 (9.7%) and crohn’s disease – 2 cases (6.4%) (table 1). i. ya. dzyubanovskyy et al. 45 su r g ic a l d is e a se s issn 2413-6077. ijmmr 2022 vol. 8 issue 1 i. ya. dzyubanovskyy et al. quantitative variables were calculated with the median. all calculations were performed using the statistica 64 software. results hr was performed for 30 (96.8 %) patients. mobilization of the splenic flexure was per­ formed for 5 (16.1%) patients. dense pelvic adhesions of the stump of the rectum was diagnosed in 13 (41.9%) patients. hand-sewn anastomosis was performed for 22 (71%) patients, stapler anastomosis – 4 (12.9%), reduction – 3 (9.7%) patients. paracolostomy hernia was diagnosed in 7 (22.6%) patients. the mean hospital stay was 14.03±11.3 days (interval 7-62). al developed in 3 (9.7%) patients on the 15th, 23rd and 35th postoperative days (table 2). pseudomembranous colitis was diagnosed in 2 (6.4%) patients with al. 1 patient after hr was affected with covid-19 on the 12th postoperative day, probably it was one of the major favorable factors of al development which was diagnosed on the 35th postoperative day due to systemic infection and reducing the reactivity of the immune system. the mortality rate was 1 (3.3%) as a result of both sepsis and purulent peritonitis due to al; this case was not operated by a colorectal surgeon due to administrative issue in the hospital. discussion adequate patient selection and preoperative planning are extremely important for hr procedures to reduce the risk of postoperative complications [1]. patients with a benign disease, aged under 69 years old and with low comorbidity are more likely of undergoing hr [6, 8]. in our study al was diagnosed in 3 (9.7%) patients aged 63-80 years old and asa score 3-4. chronic infection (cured abscess and infected pelvis infiltratis) is one of the major factors influencing hr outcome and usually creates technical difficulties during surgery in the form of dense pelvic adhesions [4, 7]. these challenges made performing hr impossible in one patient in our study. one of the important factors is the optimal interval between hp and hr. according to the literature the best time to perform hr is at least 6 months after the surgery [5, 7]. in our study t h e i n t e r v a l b e t w e e n h p a n d h r w a s 11.13±9.24 months (interval 3-38), however, such results were obtained by inclusion of 3 patients, who underwent hr at 36-38 months after hp. other authors show that timing of surgery (more than 6 months) do not affect surgical complications rate or severity or the length of hospital stay [5]. in the study paracolostomy hernia was evidenced in 7 (22.6%) patients, nevertheless at the same time it did not increase the complication rate. laparoscopic hr has significant privilege under open hr with less short-term complications regarding the overall morbidity, less frequency of wound infection and postoperative ileus [2, 3]. table 1. indications for hartmann’s procedure indications for hartmann’s procedure n % colorectal cancer 20 64.5 perforation of the diverticulum 6 19.3 traumatic rupture 3 9.7 crohn’s disease 2 6.7 table 2. analasis of patients with al clinical data/patients 1 2 3 age 80 63 69 sex female female female indications for hp perforation colorectal cancer perforation months between hp and hr 4 14 13 relaparotomy + + + al on … postoperative day 15 23 37 hand-sewn anastomosis + + + pseudomembranous colitis – + + covid-19 – – + hospital discharge/death 19(death) 42 56 46 su r g ic a l d is e a se s issn 2413-6077. ijmmr 2022 vol. 8 issue 1 conclusions hartmann’s reversal is still one of the most difficult operations in colorectal surgery with a high incidence of postoperative complications. sound selection of patients with low comorbidity and in period of 6-12 months after hp is crucial for successful hr. conflict of interests authors declare no conflict of interest. authors’ contributions. ihor dzyubanovsky – conceptualization, writing – reviewing and editing; anatoliy bedeniuk – methodoly, formal analysis; stepan grytsenko – investigation, data curation, wri­ ting – original draft. клінічні результати закриття стоми після операції гартмана і. я. дзюбановський, а. д. беденюк, с. й. гриценко тернопільський національний медичний університет імені і. я. горбачевського моз україни, тернопіль, україна вступ. закриття стоми після операції гартмана є однією з найскладніших хірургічних втручань в абдомінальній хірургії з високим рівнем захворюваності до 58% і смертністю до 3,6%. мета. це ретроспективне обсерваційне дослідження з метою аналізу клінічних результатів закриття стоми після операції гартмана. методи. тридцять один пацієнт (16 чоловіків та 15 жінок) із середнім віком 59,6±10,31 років (діапазон 26-80), які проходили курс лікування у тернопільській обласній лікарні з 2010 по 2021 рік. причинами операції гармана були: рак у 20 (64, 5%) випадків, перфорація дивертикула – 6 (19,3%), травматичний розрив – 3 (9,7%) та хвороба крона – 2 (6,4%). результати. середній час між операцією гартмана та реконструктивно-відновною операцією становив 11,13±9,24 місяця (інтервал 3-38). інтраопераційний час становив 210,33±56,91 хв (діапазон 120-330). реконструктивно-віднову операцією виконано у 30 (96,8 %) пацієнтів. щільні тазові спайки кукси прямої кишки діагностовано у 13 (41,9%) хворих. 22 (71%) пацієнтам накладено ручний анастомоз, степлерний анастомоз у 4 (12,9%), низведення у 3 (9,7%). неспроможність анастомозу виникла у 3 (9,7%) пацієнтів на 15, 23 та 35 добу після операції. у 2 (6,4%) хворих з неспроможністю анастомозу діагностовано псевдомембранозний коліт. летальність у 1 (3,3%) випадку внаслідок септичних ускладнень зумовлених неспроможністю анастомозу, цей випадок неоперований колоректальним хірургом. висновки. закриття стоми після операції гартмана залишається однією з найскладніших операцій в колоректальної хірургії з високим відсотком післяопераційних ускладнень. правильний відбір пацієнтів з низьким рівнем супутньої патології та в правильний період часу є важливим ключем до успішного виконання реконструктивно-відновної операції. ключові слова: операція гартмана; закриття стоми; неспроможність анастомозу. information about the authors ihor dzyubanovsky, professor, i. horbachevsky ternopil national medical university, ternopil, ukraine. https://orcid.org/0000­0001­8852­3938, e­mail: dzubanovsky@tdmu.edu.ua anatoliy bedeniuk, professor, i. horbachevsky ternopil national medical university, ternopil, ukraine. https://orcid.org/0000­0003­1649­7886, e­mail: bedenyuk@tdmu.edu.ua stepan grytsenko, assistant professor, i. horbachevsky ternopil national medical university, ternopil, ukraine. https://orcid.org/0000­0002­6422­093x, e­mail: grytsenko_s@tdmu.edu.ua i. ya. dzyubanovskyy et al. 47 su r g ic a l d is e a se s issn 2413-6077. ijmmr 2022 vol. 8 issue 1 references 1. alberto bitran, roberto rasslan, fabio de oliveira ferreira, edivaldo massazo utiyama, samir rasslan. hartmann’s reversal as a safe procedure for selected patients: analysis of 199 patients at a high-volume center in sao paulo. surg today. 2020; 50(8):855­62. https://doi.org/10.1007/s00595­020­01957­0. 2. chen z, nair n, hanif u. outcomes of laparoscopic vs. open reversal of hartmann’s pro cedure: a single centre experience. cureus. 2021;13(8): e17242. https://doi.org/10.7759/cureus.17242. 3. chavrier d, alves a, menahem b. is laparoscopy a reliable alternative to laparotomy in hartmann’s reversal? an updated meta-analysis. tech coloproctol. 2022;26(4):239­52. https://doi.org/10.1007/s10151­021­02560­2. 4. hess gf, schäfer j, rosenthal r, kettelhack c, oertli d. reversal after hartmann’s procedure in patients with complicated sigmoid diverticulitis. colorectal dis. 2017;19(6):582­8. https://doi.org/10.1111/codi.13553. 5. horesh n, lessing y, rudnicki y, kent i, kammar h, ben-yaacov a et al. timing of colostomy reversal following hartmann’s procedure for perforated diverticulitis. j visc surg. 2020;157(5):395­ 400. https://doi.org/10.1016/j.jviscsurg.2020.01.005. 6. horesh n, rudnicki y, dreznik y, zbar ap, gutman m, zmora o et al. reversal of hartmann’s procedure: still a complicated operation. tech coloproctol. 2018;22(2):81­7. https://doi.org/10.1007/s10151­017­1735­4. 7. nir horesh, yonatan lessing, yaron rudnicki, ilan kent, haguy kammar, almog ben-yaacov et al. considerations for hartmann’s reversal and hartmann’s reversal outcomes-a multicenter study. int j colorectal dis. 2017;32(11):1577­82. https://doi.org/10.1007/s00384­017­2897­2. 8. hallam s, mothe bs, tirumulaju rmr. hartmann’s procedure, reversal and rate of stoma-free survival. ann r coll surg engl. 2018;100:301–7. https://doi.org/10.1308/rcsann.2018.0006. received 19 may 2022; revised 29 may 2022; accepted 14 june 2022. this is open access article distributed under the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. i. ya. dzyubanovskyy et al. issn 2413-6077. ijmmr 2021 vol. 7 issue 2 15 p u b l ic h e a lt h a n d e p id e m io l o g y l.b. romanyuk et al. doi 10.11603/ijmmr.2413-6077.2021.2.12675 retrospective analysis of gender-age structure and comorbid pathology of patients with viral and bacterial pneumonia associated with covid-19 *l.b. romanyuk, i.r. volch, n.y. kravets, t.i. pyatkovskyy, o.m. zahrychuk i. horbachevsky ternopil national medical university, ternopil, ukraine background. in december 2019 first case of covid-19 was first registered in wuhan, hubei province, china. this infectious disease primarily causes respiratory tract infection, but can also affect the other organs and systems. objective. in this study, the features of the gender-age structure and comorbid pathology of the patients with viral and bacterial pneumonia who were treated in inpatient department in the period from september to december 2020 are summarized. methods. the research was conducted in ternopil municipal hospital no. 3, ternopil, ukraine. clinical cases of 262 patients with viral and bacterial pneumonia associated with covid-19 were studied retrospectively. results. both men and women has been affected by this pathology in ternopil region with the same frequency in contrast with the established fact that men are less resistant to infections that women. most of the patients ranged in age from 51 to 70 years, however, a fair number of patients were over the age of 70 years. concomitant pathology was diagnosed in 65.5% of the patients with coronavirus infection. coronary heart disease (34.5%) is leading in the structure of comorbid conditions. conclusions. during the covid-19 pandemic, timely diagnosis and medical care of the patients over 50 years of age is of great importance. the analysis of concomitant pathology proves that the people with cardiovascular pathology are in the increased risk group. keywords: viral and bacterial pneumonia; covid-19; age; comorbidity; cardiovascular system. international journal of medicine and medical research 2021, volume 7, issue 2, p. 15-19 copyright © 2021, tnmu, all rights reserved *corresponding author: lidiya romanyuk, associate professor, i. horbachevsky ternopil national medical university, ternopil, 46000, ukraine. e-mail: romanyuk@tdmu.edu.ua introduction more than 238 million people have been infected and nearly 5 million have died worldwide since the beginning of the coronavirus pandemic [1]. this infectious pathology has become not only a medical but also an economic and social problem for many countries including ukraine [2, 3]. nowadays, ukraine, like the rest of the world, continues to fight covid­19. every day the officials of the ministry of health report the number of new cases of infection, the number of fatalities, of those who recovered, and of vaccinated people. although, coronavirus infection, which has spread to the whole world, has not ruled out the possibility of contracting the other respiratory viral infections the attention to latter has decreased somewhat. over the last decades, seasonal rises in the incidence of influenza and acute viral respiratory infections have been regularly registered in our country, the etiological structure of which included certain strains of coronaviruses along with parainfluenza viruses, rhinoviruses, rs viruses [4, 5]. back in 2017, domestic and world researchers noted that coronaviruses cause epidemic outbreaks of varying intensity, the peak of which occurs during the period of maximum circulation of the influenza virus (decem­ ber-january, march-april, sometimes octobernovember). the number of infected people among the susceptible individuals during the outbreaks can be quite significant which indi­ cates high contagiousness of this pathogen. the duration of outbreaks varies on average from 1 to 3 months depending on the coro navirus strain, the number of susceptible individuals and their age. however, this relates to the best studied strains of human coronavirus: hcov-229e, hcovoc43, hecov, and sars-cov [6]. the epidemiological analysis of the 2019– 2020 season confirmed the validity of these statements regarding the current coronavirus covid-19. however, the emergence of the issn 2413-6077. ijmmr 2021 vol. 7 issue 216 p u b l ic h e a lt h a n d e p id e m io l o g y second wave of the coronavirus pandemic, which began in august–september 2020, somewhat changed the epidemic stereotype. nevertheless, the increase in the number of inpatients and severe cases of coronavirus infection in the period from february to march 2021 fits into the general peculiarities of the epidemiology of coronavirus infection. according to many researchers, the increased detection of patients with coronavirus infection in the summer months may be due to greater availability and number of tests [4, 6]. methods the research was conducted in ternopil municipal hospital no.3, ternopil, ukraine. the clinical cases of 262 patients with viral and bacterial pneumonia associated with covid-19 were retrospectively studied. these patients were treated in a specialized department from september to december 2020. this period had been chosen due to the epidemic data within ukraine regarding the spread of covid-19 infection during the epidemic season 2019– 2020, which demonstrated the high contagiousness of the pathogen and the epidemic rise of the morbidity rate in the fall to spring seasons. data related to age, sex, and concomitant pathology were processed. data was collected and tabulated using ms excel 2013, qualitative data was presented as percentages and proportions. the diagnosis of covid-19 has been made by sars clinical signs detectable by computer tomography for pneumonia and confirmed by polymerase chain reaction (pcr) tests [6,10]. the collection of the samples for pcr was performed at home or in a clinic. results a total of 262 clinical cases of the patients with covid-19 were retrospectively studied. among patients of ternopil municipal hospital no. 3, men and women were almost equally affected (50.4% and 49.6%, respectively). in terms of age, all patients were divided into 4 age groups: patients under 30 years, only 0.4%; inpatients aged from 31 to 50 years – 9.9%; patients aged from 51 to 70 years – 60.3%; and the elderly over the age of 70 – 29.4%. their demographic details are presented in table 1. according to the results of processing of patients’ medical records in ternopil municipal hospital no. 3 concomitant pathology was diagnosed in 65.6 % of inpatients (172) with coronavirus infection. ischemic heart disease in various manifestations (cardiosclerosis, angina pectoris, etc.) (34.9%) was leading in the structure of comorbid conditions. hypertension of various severity (30.8%) was the second by frequency. overall, cardiovascular pathology accounted for 44.6% of patients with covid-19 associated viral and bacterial pneumonia. this pathology comorbid with type ii dm occurred in 29.1% of patients. as presented in figure 1, the other comorbid conditions were much less common: chronic obstructive pulmonary disease – 3.5%, others – 2.3%. discussion covid-19 is characterized not only by seasonal differences, but also pronounced age differences [7, 8]. according to the center for public health in ukraine, the majority of patients with coronavirus infection were the elderly: the majority of patients were ukrainians aged 30-59; 22% – people from 50 to 59 years; 21% – people from 30 to 39 years; 20% – people from 40 to 49 years. numerous studies of the characteristics of coronavirus infection indicate that it affects men more often than women [9]. this is due to a number of social, behavioral, physiological and genetic factors. according to the statistics, for example in china, where the new coronavirus began to spread around the world, mortality among men is more than twice as high as among women from this disease [10]. in the us two-thirds of the patients were male and this sex was associated with a higher risk of death [11]. among patients of ternopil municipal hospital no.3, men and women were affected table 1. demographics of participants demographic details n % gender males 132 50.4 females 130 49.6 age under 30 years 1 0.4 31-50 years 26 9.9 51-70 years 158 60.3 over 70 years 77 29.4 l.b. romanyuk et al. issn 2413-6077. ijmmr 2021 vol. 7 issue 2 17 p u b l ic h e a lt h a n d e p id e m io l o g y almost equally (50.4% and 49.6%, respectively), which does not align with the statistics of other countries [9, 10, 11]. according to ternopil regional state administration, the age structure of the population at the end of 2019 was as follows: children under the age of 17 – 6.5%, young people under the age of 30 – 12.4%, people aged from 30 to 50 years – 32.9%, residents of the region aged 51-70 years – 33.5%, over 70 – 14.7%. comparing the age structure of inpatients with viral and bacterial pneumonia associated with covid-19 with the age structure of the population of the region as a whole it was established that the number of young people under the age of 30 was the lowest although this age group in ternopil region is quite significant. this may be due to the features of the current covid-19 pandemic that indicates mild severity in young people and a correspondingly low rate of hospitalization. on the other hand, the maximum percentage of patients was observed in the age group of 51-70 years old while in the age structure of the region’s inhabitants such patients were about a third. analysis of clinical symptoms and severity of the disease proves a higher risk of disease in the elderly. in particular, people of age more than 50 years, especially with somatic chronic pathology, which usually complicates the course of coronavirus infection, have worse prognosis for recovery. for comparison, we used data from the analysis of medical records of 578 french patients with coronary heart disease (chd) [12]. according to the analysis of patients’ medical records, concomitant pathology was diagnosed in 34.6% of french and 65.5% of ukrainian patients. among the ukrainian patients, cardiovascular pathology dominated that cor responds with the data of french clinics. however, the frequency of these diseases in ternopil region was much higher. the incidence of dm in both countries is similar. chronic obstructive pulmonary disease (copd) was more common in france and in the structure of comorbid conditions of patients in ternopil region its incidence was almost the same. conclusions according to the results of the gender-age analysis of the morbidity rate of viral and bacterial pneumonia associated with covid-19, the following conclusions have been drawn: this pathology in ternopil region affects both men and women with the same frequency which does not align with the statistics of other countries; which claims that coronavirus infection affects men more often. by age, the maximum number of inpatients was between 51 and 70 years old. however, a fair amount of the patients, compare to the age structure of the region, was over 70 years old. this indicates a more severe course of covid-19 in these age groups. cardiovascular pathology: coronary heart disease and hypertension, dominated among the comorbid pathology in patients with pneumonia. dm was the second in frequency of comorbid pathologies. thus, the risk group for development of complicated pneumonia with underlying covid-19 comprise people over 50 years of age, equally men and women with chronic cardiovascular disease and dm. conflict of interests authors declare no conflict of interest. fig. 1. frequency of comorbid conditions in patients with covid-19 treated in ternopil municipal hospital no.3, ternopil, ukraine. 34,9% 30,8% 29,1% 3,5% 2,3% chd hypertension dm copd others 0,0% 10,0% 20,0% 30,0% 40,0% l.b. romanyuk et al. issn 2413-6077. ijmmr 2021 vol. 7 issue 218 p u b l ic h e a lt h a n d e p id e m io l o g y author’s contributions romanyuk lidiya b., volch iryna r. – conceptualization, methodology, formal analysis, writing – original draft, writing – reviewing and editing; romanyuk lidiya b., volch iryna r., kravets nataliia y., pyatkovskyy taras i., zahrychuk oksana m. – data curation, writing – reviewing and editing; romanyuk lidiya b., zahrychuk oksana m. – investigation, formal analysis. ретроспективний аналіз гендерно-вікової структури та коморбідної патології у стаціонарних пацієнтів, хворих на вірусно-бактеріальну пневмонію, асоційовану із covid-19 л.б. романюк, і.р. волч, н.я. кравець, т.і. п’ятковський, о.м. загричук тернопільський національний медичний університет імені і. я. горбачевського моз україни, тернопіль, україна вступ. у грудні 2019 року перший випадок covid-19 був вперше зареєстрований в ухані, провінція хубей, китай. це інфекційне захворювання в першу чергу викликає інфекцію дихальних шляхів, але може вражати й інші органи та системи. мета. у даному дослідженні узагальнено особливості гендерно-вікової структури пацієнтів та коморбідної патології з вірусною та бактеріальною пневмонією у стаціонарних хворих терапевтичного відділення, котрі перебували на лікуванні у період з вересня по грудень 2020 року. методи. дослідження проводили в міській комунальній лікарні № 3 м. тернополя. ретроспективно досліджено історії хвороби 262 хворих на вірусно-бактеріальну пневмонію, асоційовану з covid-19. результати. на тернопіллі ця патологія з однаковою частотою уражає як чоловіків, так і жінок, на відміну від відомого факту, що чоловіки менш стійкі до інфекцій, ніж жінки. більшість пацієнтів були у віці від 51 до 70 років, проте значна кількість пацієнтів була старше 70 років. у 65,6% хворих на коронавірусну інфекцію діагностовано супутню патологію. провідне місце в структурі коморбідних станів займала ішемічна хвороба серця (34,9%). висновки. у період пандемії covid-19 велике значення має своєчасна діагностика та надання кваліфікованої медичної допомоги пацієнтам у віці понад 50 років. аналіз супутньої патології, дозволяє стверджувати, що групою підвищеного ризику є особи із патологією серцево-судинної системи. ключові слова: вірусна та бактеріальна пневмонія; covid-19; вік; супутні захворювання; серцево-судинна система. information about the authors romanyuk lidiya b. – associate professor, i. horbachevsky ternopil national medical university, ternopil, ukraine. orcid 0000­0002­8844­8082, romanyuk@tdmu.edu.ua volch iryna r. – assistant professor, i. horbachevsky ternopil national medical university, ternopil, ukraine. orcid 0000­0002­7333­3354, volch@tdmu.edu.ua kravets nataliia y. – associate professor, i. horbachevsky ternopil national medical university, ternopil, ukraine. orcid 0000­0002­7593­1753, kravecnj@tdmu.edu.ua pyatkovskyy taras i. – associate professor, i. horbachevsky ternopil national medical university, ternopil, ukraine. orcid 0000­0003­1240­1680, pyatkovskyy@tdmu.edu.ua zahrychuk oksana m. – assistant professor, i. horbachevsky ternopil national medical university, ternopil, ukraine. orcid 0000­0003­0260­4740, zahrychuk.oks@tdmu.edu.ua l.b. romanyuk et al. issn 2413-6077. ijmmr 2021 vol. 7 issue 2 19 p u b l ic h e a lt h a n d e p id e m io l o g y references 1. li j, lai s, gao gf, shi w. the emergence, genomic diversity and global spread of sars-cov-2. nature. 2021 dec 8:1­1. https://doi.org/10.1038/s41586­021­04188­6 2. morato mm, bastos sb, cajueiro do, normeyrico je. an optimal predictive control strategy for covid-19 (sars-cov-2) social distancing policies in brazil. annual reviews in control. 2020 jan 1;50: 417-31. https://doi.org/10.1016/j.arcontrol.2020.07.001 3. hryhoruk p, khrushch n, grygoruk s, grygoruk s, prystupa l, gorbatiuk k. assessing the impact of covid-19 pandemic on the regions’ socioeconomic development: the case of ukraine. 4. wiersinga wj, rhodes a, cheng ac, peacock sj, prescott hc. pathophysiology, transmission, diagnosis, and treatment of coronavirus disease 2019 (covid­19): a review. jama. 2020 aug 25;324(8):782­93. https://doi.org/10.1001/jama.2020.12839 5. feshchenko yi, gavrisyuk vk, dziublyk iv, dziublyk oy, gumeniuk gl, gumeniuk mi, kapitan gb, yachnik va. infectious exacerbation of chronic obstructive pulmonary disease: place and role of respiratory viral pathogens. medicni perspektivi (medical perspectives). 2019;24(4):30­5. https://doi.org/10.26641/2307­0404.2019. 4.189191 6 . k o p c h a v s . f e at u r e s o f i m m u n o dependent manifestations at covid­19. іх [internet]. 2021 jun. 30 [cited 2021 dec. 26];(2):4­16. available from: https://ojs.tdmu.edu.ua/index.php/ inf-patol/article/view/12159 https://doi.org/10.11603/1681­2727.2021.2.12159 7. world health organization. clinical management of covid­19: interim guidance. may 27.2020. available online: https://www.who.int/ publications/i/item/clinical-management-ofcovid-19. 8. xia w, shao j, guo y, peng x, li z, hu d. clinical and ct features in pediatric patients with covid‐19 infection: different points from adults. pediatric pulmonology. 2020 may;55(5):1169­74. doi: 10.1002/ ppul.24718. https://doi.org/10.1002/ppul.24718 9. bwire gm. coronavirus: why men are more vulnerable to covid­19 than women?. sn comp­ rehensive clinical medicine. 2020 jul;2(7):874­6. https://doi.org/10.1007/s42399­020­00341­w 10. zeng f, dai c, cai p, wang j, xu l, li j, hu g, wang z, zheng f, wang l. a comparison study of sars‐cov‐2 igg antibody between male and female covid‐19 patients: a possible reason underlying different outcome between sex. journal of medical virology. 2020 oct; 92(10):2050­4. https://doi.org/10.1002/jmv.25989 11. gupta s, hayek ss, wang w, chan l, mathews ks, melamed ml, brenner sk, leonberg-yoo a, schenck ej, radbel j, reiser j. factors associated with death in critically ill patients with coronavirus disease 2019 in the us. jama internal medicine. 2020 nov 1; 180(11):1436­47. 12. jakhmola s, indari o, baral b, kashyap d, varshney n, das a, chatterjee s, jha hc. comorbidity assessment is essential during covid-19 treatment. frontiers in physiology. 2020 aug 4;11:984. https://doi.org/10.3389/fphys.2020.00984 received 25 november 2021; revised 30 november 2021; accepted 9 december 2021. this is open access article distributed under the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. l.b. romanyuk et al. 53 su r g ic a l d is e a se s issn 2413-6077. ijmmr 2022 vol. 8 issue 2 doi 10.11603/ijmmr.2413-6077.2022.2.13162 fetal bilateral adrenal hemorrhage (case report) j. s. randhawa1, *r. nagamahendran2, r. shankaran3 1 – indian naval hospital ship kalyani, visakhapatnam, india 2 – indian naval hospital ship sandhani, mumbai, india 3 – institute of naval medicine, mumbai, india background. fetal supra renal mass revealed incidentally by routine antenatal ultrasound is a great challenge for diagnosis and management by a surgeon. this is a matter of parental anxiety and diagnostic dilemma to a physician. indeed, such masses turn out to be complicated by an intra-tumor hemorrhage in neuroblastoma or antenatally diagnosed adrenal hemorrhage. the first one needs intensive management and the latter needs watchful observation. objective. a case of bilateral fetal adrenal mass revealed by routine fetal ultrasound examination at 28th week of gestation which turned out to be adrenal hemorrhage is presented. this is aimed to make awareness to ensure that clinicians always keep benign etiologies first and thoroughly investigate in case of incidentally detected fetal adrenal mass. methods. the study is a single case report of incidentally revealed supra renal mass. this case report encompasses differentiating features between the two and investigations that aid the surgeon to avoid unnecessary intervention in a benign hemorrhage. results. the baby was kept on follow up with serial ultrasound scans in the postnatal period and by the second scan in a month, the hemorrhage had resolved completely. conclusion. in cases of benign looking masses like adrenal hemorrhage or spontaneously resolving neuroblastoma, appropriate antenatal assessment and close monitoring with serial ultrasound scans can avoid surgery. keywords: bilateral fetal adrenal hemorrhage; neuroblastoma; adrenal mass. *corresponding author: dr. r. nagamahendran, assistant professor of the department of general surgery, indian naval hospital ship sandhani, mumbai, 400704, india. e-mail: nagaa.mahendran@gmail.com international journal of medicine and medical research 2022, volume 8, issue 2, p. 53-55 copyright © 2022, tnmu, all rights reserved j. s. randhawa et al. introduction the supra renal glands were discovered in 1522 by eustachius and described by him in 1563. the adrenal glands are responsible for essential physiologic functions including stress response, electrolyte balance, metabolism, immune function, blood pressure regulation and sexual development. supra renal masses have been a longstanding source of fascination for both physicians and surgeons especially in neonatal period. adrenal glands are relatively larger in size with high vascularity during fetal and neonatal period. this predisposes to its he morrhage in early life. neonatal adrenal hemorrhage is rare and is seen in 0.2-0.5% of ca ses [1]. with the recent advancement in imaging modalities, diagnosis and management of supra renal masses has become more effi­ cient. assessment of hemodynamic stability, adrenal insufficiency and exclusion of a hor­ monal active adrenal tumor proves to be a topical issue in management of such suprarenal masses [2]. association of adrenal hemorrhage with thrombosis of major blood vessels is also reported in the literature [3]. thus, a rare case of antenatally diagnosed bilateral adrenal mass which turned out to be benign adrenal hemorrhage is presented. this case report envisages the need for watchful observation and importance of short interval follow up imaging in management of benign disorders of adrenal gland before any surgical intervention. case report rare presentation of neonatal adrenal masses is a diagnostic dilemma in differentiating benign adrenal fetal hemorrhage from neuroblastoma. this case report encompasses differentiating features between the two and investigations that aid the surgeon to avoid unnecessary intervention in a benign he morrhage. following is the case report of a 27 years old gravida 3, para 2, live 2 woman, who was referred to our pediatric surgery center at 28 week of gestation for evaluation of bilateral supra renal cystic mass. her previous medical history was unremarkable. on evaluation with sonography, bilateral cystic mass was located 54 su r g ic a l d is e a se s issn 2413-6077. ijmmr 2022 vol. 8 issue 2 superior to both kidneys measuring 15×10 mm on the right side and 14×9 mm on the left side. fetus was evaluated further by doppler examination of umbilical cord, biophysical profile and fetal biometry and were in norm. evaluation with antenatal mri scan on 28 weeks of gestation was suggestive of bilateral fetal adrenal hemorrhage. the antenatal mother was kept on serial ultrasound follow up, scans at 32 weeks and 35 weeks revealed multicystic lesions of both adrenals. the right adrenal measured 21×12 mm and the left one – 20×13 mm without any abnormal vascularity. mother underwent elective lower segment caesarean section at 38 weeks of gestation. the surgery and postoperative period was uneventful. a 2.815 kg, healthy female child was delivered with normal peripartum period. discussion ultrasonography of abdomen on the 2nd day of life revealed mild resolution of bilateral suprarenal areas to 15×15 mm in the right and 13×14 mm in the left with hypo echoic foci within. further biochemical evaluation of serum aldosterone, renin, cortisol and plasma acth levels were in norm. the baby was kept on follow up with serial ultrasound scans and by the second scan in a month, the hemorrhage had resolved completely. spencer, in 1892 first described adrenal hemorrhage in stillborn infants. based on exten sive autopsy reports, the incidence of adrenal hemorrhage is estimated to be about 1.7 per thousand births and the incidence of antenatal detection is not established due to its rarity [2]. bilateral adrenal hemorrhage is found in 5-15% of cases with right side preponderance with right and left side ratio of 3:1 [4]. the increased use of perinatal ultrasound has led to early detection of an increasing number of neonatal suprarenal masses [5]. in ultrasound, the adrenal glands typical looks like wishbone appearance in the form of cap or an inverted v over the kidneys [6]. adrenal hemorrhage is rarely associated with adrenal insufficiency. even in cases with severe bilateral adrenal hemorrhage, it is hardly ever encountered as less than 10% availability of functional glandular component that is enough to prevent adrenal insufficiency [7]. adding to the existing knowledge of management of supra renal masses, which are detected antenatally, it is prudent from our study that serial radiological monitoring is crucial. masses with radiological reduction in size should be given full consideration for conservative management [8]. rare occurrence of fetal adrenal hemorrhage should be kept in mind in those cases where surgery can be avoided. conclusion differentiation between fetal adrenal hemorrhage and congenital neuroblastoma is difficult and urgent. in cases of radiologically benign looking supra renal masses, appropriate antenatal assessment and close monitoring with serial ultrasound scans can avoid unnecessary surgical intervention as they resolve spontaneously. limitations single case report in a tertiary care center. conflict of interests authors declare no conflict of interest. acknowledgements acknowledgements if any. author’s contributions dr. jaskiran singh randhawa – conceptualization, methodology, formal analysis, writing – reviewing and editing; dr. r. nagamahendran – investigation, writing – original draft, writing – reviewing and editing; dr. r. shan karan – investiga tion, formal analysis, data curation. двостороння кровотеча з надниркових залоз у плода (клінічний випадок) j. s. randhawa1, r. nagamahendran2, r. shankaran3 1 – indian naval hospital ship kalyani, visakhapatnam, india 2 – indian naval hospital ship sandhani, mumbai, india 3 – institute of naval medicine, mumbai, india вступ. утворення в наднирниках плода, випадково виявлені під час звичайного допологового ультразвукового дослідження, викликають занепокоєння батьків і створюють діагностичну проблему для лікуючого лікаря. такі утворення можуть виявитися ускладненим внутрішньопухлинним кровоj. s. randhawa et al. 55 su r g ic a l d is e a se s issn 2413-6077. ijmmr 2022 vol. 8 issue 2 references 1. izbizky g, elias d, gallo a, farias p, sod r. prenatal diagnosis of fetal adrenal carcinoma bilateral. ultrasound obstet gynecol. 2005; 26:669­71. https://doi.org/10.1002/uog.2623 2. alabsi sy, layland t. adrenal hemorrhage in neonates: unusual presentation. neonatal netw. 2015; 34(4):220­6. https://doi.org/10.1891/0730­0832.34.4.220 3. peruri g, suthar r, v, bhatia a. thrombosis of bilateral renal veins, inferior vena cava, and superior sagittal sinus with adrenal hemorrhage in a neonate. j postgrad med edu res 2019; 53(2):89­90. https://doi.org/10.5005/jp­journals­10028­ 1320 4. shin si, yoo jg, park iy, cheon jy. prenatal diagnosis – fetal adrenal hemorrhage and endo crinologic evaluation. obstet gynecol sci. 2016; 59(3):238­40. https://doi.org/10.5468/ogs.2016.59.3.238 5. maki e, oh k, rogers s, sohaey r. imaging and differential diagnosis of foetal suprarenal masses. j ultrasound med. 2014;33:895­904 https://doi.org/10.7863/ultra.33.5.895 6. spencer hr. on visceral hemorrhages in stillborn children. trans obstet soc london. 1892; 33:203. 7. glenn jf. neonatal adrenal hemorrhage. j urol. 1962 may;87:639­42 https://doi.org/10.1016/s0022­5347(17)65019­5 8. comline rs, silver m. catecholamine secretion by the adrenal medulla of the foetal and new-born foal. j physiol. 1971 aug; 216(3):659­82 https://doi.org/10.1113/jphysiol.1971.sp009546 received 5 september 2022; revised 20 october 2022; accepted 1 november 2022. this is open access article distributed under the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. виливом у нейробластому або антенатально діагностованим крововиливом у надниркові залози. перший потребує інтенсивного лікування, а другий – уважного спостереження. мета. представлено випадок двостороннього утворення в надниркових залозах плода, виявленого під час планового ультразвукового дослідження плода на 28-му тижні вагітності, які виявилися крововиливом у надниркові залози. випадок представлено з метою загострення уваги та настороженості клініцистів щодо доброякісної патології та проведення ретельного обстеження у випадку випадкового виявлення новоутворень надниркових залоз плода. методи. представлено випадок випадково виявлених утворень надниркових залоз. описано відмінності між ними та дослідження, які допомагають хірургу уникнути непотрібного втручання при доброякісному крововиливі. результати. у післяпологовому періоді дитину спостерігали за допомогою серійних ультразвукових досліджень. до другого узд через місяць крововилив повністю розсмоктався. висновки. у випадках доброякісних новоутворень, таких як крововилив у надниркові залози або нейробластома, що спонтанно розсмоктується, відповідна антенатальна оцінка та ретельний моніторинг за допомогою серійних ультразвукових досліджень можуть допомогти уникнути операції. ключові слова: двостороння фетальна надниркова кровотеча; нейробластома; утворення в наднирниках. information about the authors dr. jaskiran singh randhawa – associate professor and head, department of surgery, indian naval hospital ship kalyani, vishakapatnam, andhra pradesh, india https://orcid.org/0000­0002­0081­7382, e­mail: moshesingh2000@yahoo.co.in dr. r. nagamahendran – assistant professor, department of general surgery, indian naval hospital ship sandhani, mumbai, india https://orcid.org/0000­0002­9854­7236, e­mail: nagaa.mahendran@gmail.com dr. r. shankaran – dean and professor, department of general surgery, institute of naval medicine, mumbai, india https://orcid.org/0000­0002­0105­2270, e­mail: drshankaran@gmail.com j. s. randhawa et al. 62 p sy c h ia t r y issn 2413-6077. ijmmr 2022 vol. 8 issue 1 doi 10.11603/ijmmr.2413-6077.2022.1.13090 attitude towards psychiatry and psychiatrists among medical students in tertiary health care hospital r. jahagirdar zydus medical college and hospital, dahod, gujarat, india background. psychiatry is a medical subject; many medical students ignore its importance in medicine. regarding mental illness, many medical professionals and students are less aware as mental illness is very important in affecting the quality of care the patient receives and in choosing psychiatry as a career. this study specializes in psychiatry based on many factors, however undergraduate students’ attitude towards career choice is the most important. objective. this study aims to explore attitude towards psychiatry subject among undergraduate medical students. methods. this is a descriptive cross-sectional study done at vikhe patil medical college, ahmednagar. the total of 295 volunteer students from all phases of mbbs (bachelor of medicine and bachelor of surgery) were enrolled by purposive sampling, a 30-item self-administered questionnaire, atp-30 (attitude towards psychiatry) was used to measure the attitude of medical students. data collected was analysed using spss version 24. the results were reported as percentage and frequencies. results. in the study, 47.4% of the respondents were males and 52.6% were females. out of total study participants 52.3% of our study respondents strongly agreed that psychiatry was a respected branch of medicine; 56.7% strongly agreed that psychiatry should be an important part of medical curriculum; 19.4% of study respondents considered it in the list of the career choice while the majority did not think that it should be in their career list options. conclusions. in the study, the students from the later phases of mbbs showed positive attitude than that of the initial phases. many students found it as respected subject and essential field of medicine for their choice of profession. however, shortcoming knowledge and awareness was observed in the responses pertaining to present available treatment and recent trends in the field. keywords: attitude; undergraduate medical students; psychiatry; india. *corresponding author: dr. rohan jahagirdar, associate professor, zydus medical college and hospital, dahod, gujarat, india. email: drjahagirdarrohan@gmail.com international journal of medicine and medical research 2022, volume 8, issue 1, p. 62-66 copyright © 2022, tnmu, all rights reserved introduction according to the world health organization (who), mental disorders and mental illness are today the most significant part of the global burden of diseases [1]. psychiatric disorders affect 10% of adult population [2]. whereas mental disorders are either ignored by majority of people in their life or hesitated to share having very few psychiatrists. globally, psychiatry as a subject, psychiatrists as professionals, and patients with psychiatric disorders are subjected to cultural stereotypes and negative attitude by the general population. the alarming concern is that these prejudices exist within the medical community as well [3, 4]. this information has serious implications on psychiatry. most mental illness in people are undiagnosed or underdiagnosed as people with mental disorders hesitate and remain silent to pursue medical care due to presenting stigma and discrimination to them [5]. stigma exists even in the health care system and even students carry prejudices to choose and enter into psychiatry, which affects medicine. in this aspect, evaluation of undergraduate students for their attitude towards mental health issues is important as they are future of nation and going to influence directly or indirectly the health system [6]. psychiatric teaching in the undergraduate medical curriculum appears negligible and short due to insufficient number of allotted lectures, no r. jahagirdar 63 p sy c h ia t r y issn 2413-6077. ijmmr 2022 vol. 8 issue 1 r. jahagirdar available qualified teaching staff as well as lack of proper clinical positions, which results to inadequate exposure of medical students to psychiatry [7, 8, 9]. this study aims at evaluating the current attitudes towards psychiatry as a subject, which can further help in revising the curriculum, teaching and learning methods of undergraduates in order to improve the understanding of mental health issues in community and medical profession. methods this descriptive study was done after approval from the ethical committee, was conducted at vikhe patil medical college. ahmednagar for 6 months duration from july to december 2019. the study includes 295 medical students from all phases of mbbs (bachelor of medicine and bachelor of surgery) selected with purposive sampling. purpose of the present study was informed to participants about research and voluntary participation was invited. informed consent was taken and confidentiality was maintained and ensured for every participant though questionnaire did not included any information for identification. those not giving consent and not willing to participate were excluded from study. modified short atp-30 (attitude towards psychiatry developed in canada by burra et al (1982) a 30 item selfadministered questionnaire was used to measure the attitude of medical students in this study [10]. this scale measured the attitude of respondents such as towards psychiatry patients, psychiatry illness, psychiatry knowledge, psychiatry treatment including drugs, psy chiatrists, psychiatry career choice, and psychiatry training. the atp uses a 5-point likert type scale: 1; strongly agree, 2; agree, 3; neutral opinion, 4; strongly disagree, and 5; disagree. total score on the scale indicates positive or negative attitude, with a high total score indicating positive and low score indicating negative attitudes toward different aspects of psychiatry. data was analysed using spss version 24. results were entered in microsoft excel and were presented as frequencies and percentages. results all 295 students were analysed in order to assess their attitude towards psychiatry by a questionnaire. the distribution of the respondents according to gender and professional year is presented in table 1. among all 295 participants involved for the analysis, 53.98% (n=159) were males and 46.10% (n=136) were females. the highest number of participants were from final year of study (n=110, 37.2%), followed closely by the second professional phase (n=139, 34.4%) and the 1st year of study (n=119, 29.45%). apart from the 1st year students, all other participants (n=212, 71.5%) had been exposed to psychiatry postings and lectures as per the current mbbs curriculum. the majority of students (n=185, 63%) considered psychiatry to be a respected branch of medicine and 79.9% (n=230) thought that psychiatric illnesses deserve as much attention as physical illnesses. 240 respondents (81.1%) agreed that psychiatry should have been an important part of school curriculum (table 2). discussion the interlinking has always existed between mental and physical health in all individuals. undergraduate attitude towards mental health has significant influence in their future medical practice and the quality of life of patients. all mbbs undergraduates of all phases of study were involved into the study fairly. the formal students of pre and para clinical subjects did not receive any formal introduction to psychiatry while the other students studied psychiatry either in the forms of lectures or clinical postings. the majority of our study respondents are from the final phase of study (37.2%) among the respondents involved. the analysis of the study results shows that the majority of students has positive attitude towards psychiatry. 47.5% of our study respondents strongly agree that psychiatry is a respected branch of medicine; 40.7% believe most patients do not go table 1. demographic data of sample population year of study males n (%) females n (%) total n (%) first 48 (57.83) 35 (42.16) 83 (28.1) second 52 (50.98) 50 (49.01) 102 (34.5) final 59 (53.63) 51 (46.36) 110 (37.2) total 159 (53.89) 136 (46.10) 295(100) 64 p sy c h ia t r y issn 2413-6077. ijmmr 2022 vol. 8 issue 1 table 2. results of atp-30 questionnaire item strongly agree agree neutral strongly agree disagree n (%) n (%) n (%) n (%) n (%) psychiatry is a respected branch of medicine 115 39.0% 70 23.7% 45 15.3% 25 8.5% 41 13.6% people taking up psychiatry are running away from “real” medicine 27 9.2% 35 11.9% 42 14.2% 73 24.7% 118 40.0% psychiatric disorders deserve as much attention as physical illness 158 53.6% 75 25.4% 40 13.6% 17 5.8% 5 1.7% psychiatry is unscientific and imprecise 7 2.4% 18 6.1% 45 15.3% 55 18.6% 170 57.6% psychiatry is an important part of the medical school curriculum 164 55.6% 77 26.1% 28 9.5% 19 6.4% 7 2.4% there has been significant advancement in the understanding and treatment of psychiatric disorders in recent years 157 53.2% 71 24.1% 29 9.8% 20 6.8% 18 6.1% with the treatment available today, most psychiatric patients experience improvement 160 54.2% 74 25.1% 34 11.5% 22 7.5% 5 1.7% i find it hard to believe that patient’s symptoms can improve by “talk therapy” 100 33.9% 95 32.2% 54 18.3% 39 13.2% 7 2.4% genes and family history are important in the theology of mental illnesses 144 48.8% 86 29.2% 31 10.5% 24 8.1% 10 3.4% i believe that spiritual healer can help the mentally illnesses 4 1.4% 24 8.1% 45 15.3% 97 32.9% 125 42.4% i believe that ghosts (jinn) and magic (jadoo) can cause mental illnesses 5 1.7% 11 3.7% 42 14.2% 100 33.9% 137 46.4% mental illness can be a punishment for past sins 4 1.4% 12 4.1% 49 16.6% 95 32.2% 135 45.8% most psychiatric patients are violent and dangerous 15 5.1% 18 6.1% 52 17.6% 81 27.5% 129 43.7% psychiatric hospitals are like prisons 7 2.4% 9 3.1% 24 8.1% 98 33.2% 157 53.2% psychiatrists are mentally unstable compare to other doctors 2 0.7% 7 2.4% 21 7.1% 105 35.6% 160 54.2% if i was asked to name three most exciting medical specialties, psychiatry would be in the list 21 7.1% 26 8.8% 15 5.1% 94 31.9% 139 47.1% psychiatrists are less respected than other specialists 19 6.4% 29 9.8% 15 5.1% 92 31.2% 140 47.5% psychiatrists get less satisfaction from their work than other specialists 31 10.5% 32 10.8% 15 5.1% 103 34.9% 115 39.0% psychiatrists earn less than other doctors 76 25.8% 71 24.1% 36 12.2% 58 19.7% 54 18.3% most patients do not go to a psychiatrist even if they have mental problems 120 40.7% 70 23.7% 24 8.1% 44 14.9% 37 12.5% there is an acute shortage of psychiatrists in our country 88 29.8% 124 42.0% 18 6.1% 34 11.5% 31 10.5% r. jahagirdar 65 p sy c h ia t r y issn 2413-6077. ijmmr 2022 vol. 8 issue 1 to a psychiatrist even if they have mental health problems. many students strongly agree that psychiatry should be an important part of medical curriculum (81%). in certain aspects of the study, positive outcomes were noted regarding positive attitude toward psychiatrists. many of the students thought of psychiatry as an important stimulating discipline and psychiatric practice perceived to have promising prospects. our study findings were positively surprising compare to previous ones, which had reported same numbers with fewer respondents viewing psychiatry and psychiatrists in a positive light [11, 12]. however, a few studies in uk, australia and france shows opposing results [13, 14]. in fact, many students believe in magic, ghosts and family history [15]. thus, the study results are consistent with the research findings of the respondents, who consider psychiatry as exciting specialty and in the list of their future career choice, while a few has neutral opinion and some students consider it in the list of top specialties that they will likely to choose in the future [16]. conclusions even though lack of sufficient knowledge about the reasons behind mental problems students have positive attitude towards psychiatrists and psychiatry because of its importance in medicine. the study highlights the attitude of medical students towards psychiatry as their future preference of choice. this might be because a majority of students find psychiatry to be a respected and essential field of me­ dicine. the study also focuses on the need for further development of theoretical and clinical teaching of psychiatry at the undergraduate level to overcome these faults. conflicts of interest the author declares no conflict of interest. acknowledgements we thank all the participants for active participation of the study. the research did not have any financial support and sponsorship. ставлення до психіатрії та психіатрів серед студентів-медиків у закладах третинної медичної допомоги r. jahagirdar zydus medical college and hospital, dahod, gujarat, india вступ. психіатрія є медичним предметом, проте багато студентів-медиків ігнорують її важливість у галузі медицини. щодо психічних захворювань, багато медичних працівників і студентів-медиків є менш обізнаними, незважаючи на те, що психічні захворювання відіграють дуже важливу роль у впливі на якість лікування, яке отримує пацієнт, а також у виборі психіатрії як професії. мета. це дослідження мало на меті вивчити ставлення до предмету психіатрії серед студентівмедиків. методи. це описове перехресне дослідження, проведене медичним коледжем імені vikhe patil у місті ахмеднагар. в експерименті взяло участь 295 студентів-добровольців з усіх курсів mbbs (бакалавр медицини та бакалавр хірургії), для оцінювання ставлення студентів-медиків використовувався опитувальник із 30 пунктів самостійного заповнення, atp-30 (ставлення до психіатрії). зібрані дані були проаналізовані за допомогою spss версії 24. результати були представлені у відсотках і частотах. результати. у цьому дослідженні 47,4% респондентів були чоловіками та 52,6% жінками. із загальної кількості учасників дослідження 52,3% респондентів повністю згодні з тим, що психіатрія була шанованою галуззю медицини, а 56,7% повністю згодні з тим, що психіатрія також має бути важливою частиною медичної навчальної програми, ще 19,4% респондентів дослідження розглядають її як кар’єрний вибір, тоді як більшість не вважала, що психіатрія має бути в списку варіантів кар’єри. висновки. у цьому дослідженні студенти старших курсів mbbs демонструють більш позитивне ставлення до психіатрії ніж студенти молодших курсів. багато студентів вважали цей предмет шанованим і важливою галуззю медицини для свого вибору професії. однак у відповідях, що стосуються наявного лікування та останніх тенденцій у цій галузі, спостерігався недолік знань та обізнаності. ключові слова: ставлення; студенти бакалаврату медичного факультету; психіатрія; індія. r. jahagirdar 66 p sy c h ia t r y issn 2413-6077. ijmmr 2022 vol. 8 issue 1 references 1. who. investing in mental health. 2003 2. geneva: who; 2003. who. investing in mental health magnitude and burden of mental disorders; p. 8. 3. jiloha rc. image of psychiatry among medical community. indian j psychiatry. 1989;31:285­7. 4. minhas fa, mubbasher mh. attitude of medical students towards psychiatry in pakistan. j coll physicians surg pak. 2003;10:69­72. 5. pradhan sn, sharma sc, malla dp, et al. a study of help seeking behavior of psychiatric patients. journal of kathmandu medical college. 2014 may 31;2(1):21­4. https://doi.org/10.3126/jkmc.v2i1.10538 6. malhi gs, parker gb, parker k, carr vj, kirkby kc, yellowlees p, et al. attitudes toward psychiatry among students entering medical school. acta psychiatr scand. 2003;107:424­9. https://doi.org/10.1034/j.1600­0447.2003. 00050.x 7. chawla jm, balhara yp, sagar r, shivaprakash undergraduate 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https://doi.org/10.4103/0019­5545.174381 16. desai nd, chavda pd. attitudes of undergraduate medical students toward mental illnesses and psychiatry. journal of education and health promotion. 2018;7. https://doi.org/10.4103/jehp.jehp_87_17 received 15 may 2022; revised 11 june 2022; accepted 13 june 2022. this is open access article distributed under the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. r. jahagirdar 34 su r g ic a l d is e a se s issn 2413-6077. ijmmr 2022 vol. 8 issue 2 doi 10.11603/ijmmr.2413-6077.2022.2.13221 a rare tumor – adrenal angiosarcoma (case report) *a. kumar m., n. roy, h. jafri, v. popli, v. s. karthik, t. phuntsho armed forces medical college, pune, india background. primary adrenal angiosarcoma is an exceptionally rare tumor with an incidence of <1% of soft tissue sarcomas. less than 50 cases being described in literature; most of them were nonfunctional presenting with symptoms of abdominal lump, weight loss, fatigue or weakness. the treatment remains multi-modal with involvement of surgeon as well as medical oncologist. objective. the aim of this case report is to describe an entity which is rare in terms of incidence. it was functional as well as metastatic and managed with multimodality therapy. methods. the case is a 47-year-old man diagnosed with metastatic adrenal angiosarcoma. the patient was managed with chemotherapy followed by surgery. results. the final histopathological examination of the specimen and immunohistochemistry revealed angiosarcoma. at present the patient is under follow-up. conclusion. even though surgery forms the main stay of management, the role of multi-modality therapy forms the cornerstone for adrenal angiosarcomas. keywords: adrenal angiosarcoma; suprarenal; adrenalectomy; vasoformative tumor. *corresponding author: arun kumar m., assistant professor, armed forces medical college, pune, 411040, india. e-mail: ak3.doc@gmail.com international journal of medicine and medical research 2022, volume 8, issue 2, p. 34-37 copyright © 2022, tnmu, all rights reserved a. kumar m. et al. introduction a tumor with an incidence of <1% of soft tissue sarcomas, angiosarcoma is a rare lesion arising out of vascular endothelium [1, 2]. primary angiosarcomas of visceral organs and bones are exceedingly rare, though they account for metastatic sites. most of the primary lesions are either from the skin or subcutaneous tissue [3,4]. out of this meagre incidence of primary visceral angiosarcomas, adrenal angiosarcomas constitute an exceptionally uncommon variety with less than 50 cases reported in literature [5]. hence this case report becomes important and unique due to the rarity of the tumor. this case depicts the management of a 47­year­old man with meta_ static left adrenal angiosarcoma. case report a 47-year-old man with no previous comorbidities initially presented with blurring of vision of one-month duration. his initial clinical examination revealed a blood pressure of 225/150 mm of hg for which he was diagnosed as hyper tensive emergency with retinopathy and treated with antihypertensives. assessment of his hema tological and biochemical parameters revealed an abnormal serum urea 26.9 mg/dl, creatinine 2.14 mg/dl and uric acid 9.5 mg/dl. he was further evaluated for cause of the same and was found to have a left suprarenal mass measuring 15×18×22 cm extending on to the peripancreatic region on abdominal sonography. further imaging with mri of the abdomen revealed a large suprarenal mass lesion measuring 13×17×18 cm with fat, soft tissue areas and calcification with inferior displacement of kidney and loss of fat planes with left dome of diaphragm, anterior and posterior renal fascia. in addition, multiple soft tissue deposits were found along anterior abdominal wall, largest measuring 4.5 cm along left hypochondrium. multiple retroperitoneal lymph nodes were also enlarged. whole body pet ct revealed diffuse fdg uptake in left suprarenal lesion of similar size along with multiple nodules in both lungs with fdg uptake in paratracheal lymph nodes. fdg uptake was also noted along the anterior abdominal wall in the left hypochondrium. ultrasound guided fine needle aspiration cytology from left adrenal mass lesion as well as anterior abdominal wall deposits revealed a malignant vasoformative tumor with cells having large hyperchromatic nuclei and abundant vesicular cytoplasm. these cells stained positive for erg, cd34 and cd31 on ihc. patient was diagnosed as a case of metastatic adrenal angiosarcoma based on the imaging as well as tissue biopsy findings. 35 su r g ic a l d is e a se s issn 2413-6077. ijmmr 2022 vol. 8 issue 2 patient was started on chemotherapy based on paclitaxel and administered six cycles. follow up pet ct after 6 months of initial presentation revealed a relatively stable disease with no newer lesions. however, there was no regression in size of the lesions. in view of the same, patient was referred for left adrenalectomy. he underwent left open adrenalectomy along with splenectomy. intra-operatively 15×15 cm highly vascular tumor was found with dense adhesions between spleen, greater curvature of stomach and tail and body of pancreas with involvement of splenic artery by the tumor necessitating a splenectomy. left kidney was free of tumor. patient had an eventful post-operative course with surgical site infection requiring frequent dressings for a period of two weeks and secondary closure. he was administered requisite vaccines against opsi in the post-operative period. histopathological examination revealed tumor composed of sheets of highly bizarre plump endothelial cells forming anastomosing vascular channels with a high n:c ratio, marked nuclear pleomorphism, vesicular nuclei and prominent nucleoli. brisk mitosis and numerous multinucleated giant cells were also found along with areas of extensive necrosis and blood lakes. spleen was free of any atypia or ma lig nancy. cd31 & cd34 were positive in neoplastic cells on ihc. the findings were suggestive of angiosarcoma. the patient is presently on follow-up and his antihypertensive medications have been stopped in view of normalization of blood pressure post-surgery. he is being planned for further follow-up imaging to assess the status of metastatic lesions. discussion adrenal angiosarcoma, first case described in 1988 in a 54-year-old male by kareti et al., is a highly aggressive and rare malignancy [6]. the median age of presentation ranges from 34 to 85 years with a gender predilection for fig. 1. intraoperative findings of a bilobed left adrenal tumor adherent to pancreas and spleen. fig. 2. specimen of adrenal tumor along spleen. a. kumar m. et al. 36 su r g ic a l d is e a se s issn 2413-6077. ijmmr 2022 vol. 8 issue 2 males as reported by the countable number of cases reported in literature [7]. the symptomatology varies from typical abdominal or flank pain, palpable mass, weight loss, fatigue or weakness to being an incidental finding on imaging. patients with systemic symptoms tend to have a poor prognosis [5,7]. the etiology of adrenal angiosarcoma is usually unknown, with few cases showing association with arsenic and vinyl chloride [3]. hyperfunctioning tumors are yet to be reported in literature [8]. detection of adrenal masses are on a rise due to the advances in imaging, most of them being nonfunctional and benign in contrast to this case [9]. this adrenal lesion was an incidental finding, but functional as well as malignant. though rare, these tumors have a high malignant potential with local infiltration as well as distant metastasis being commonly found at diagnosis [10]. this usually warrants removal of adjacent organs during surgical resection of adrenals as was with the splenectomy in our case. being a tumor with very low incidence, there is no standardized treatment protocol described for these lesions [5]. surgical resection usually results in a good outcome [11]. adjuvant therapies are described for these lesions due to their aggressive nature [12]. the pathologists also have a dilemma in diagnosing primary adrenal angiosarcomas due to the non-specific histopathological characteristics with necrosis, hemorrhage and cystic components. most adrenal angiosarcomas have a solid epithelioid pattern in contrast to other angiosarcomas with a vasoformative pattern [10, 13]. a definitive pathological diag­ nosis requires a wider immunohistochemistry staining in the form of cd31, cd34, fli1 and factor viii in addition to cytokeratins for epithelial components [14]. the tumor in our case stained positive for cd31 and cd34, thereby confirming the diagnosis. conclusion this case of a rare tumor showcased the importance of multi-modality approach with medical oncology, surgery, radiology and pathology for the challenging diagnosis as well as treatment. a standard treatment protocol needs to be developed even though the incidence of this lesion is very low. surgical excision remains the mainstay of treatment along with adjuvant therapeutic options. conflict of interests authors declare no conflict of interest. acknowledgements no acknowledgements author’s contributions arun kumar m. – conceptualization, methodology, formal analysis, writing – original draft; nilanjan roy – conceptualization, methodology, formal analysis, writing – reviewing and editing, data curation, investigation; haris jafri – investigation, formal analysis, writing – reviewing and editing, data curation; vitesh popli – conceptualization, methodology, investigation, writing – reviewing and editing; thinley phuntsho roy – methodology, data curation, investigation; v. s. karthik – writing – reviewing and editing, data curation. рідкісна пухлина ангіосаркома наднирників (клінічний випадок) *a. kumar m., n. roy, h. jafri, v. popli, v. s. karthik, t. phuntsho armed forces medical college, pune, india вступ. первинна ангіосаркома надниркових залоз є виключно рідкісною пухлиною з частотою <1% сарком м'яких тканин. у літературі описано менше 50 випадків, більшість з них були нефункціональними і супроводжувалися симптомами припухлості в животі, втрати ваги, втоми або слабкості. лікування залишається мультимодальним із залученням хірургів та онкологів. мета. метою був опис цього рідкісного захворювання, яке було функціональним, метастатичним і лікувалося за допомогою мультимодальної терапії. методи. описаний випадок 47-річного чоловіка, у якого діагностували метастатичну ангіосаркому надниркових залоз, після чого було проведено хіміотерапію та згодом операцію. результати. остаточне патогістологічне дослідження препарату та імуногістохімія виявили ангіосаркому. на даний момент пацієнт перебуває під наглядом. a. kumar m. et al. 37 su r g ic a l d is e a se s issn 2413-6077. ijmmr 2022 vol. 8 issue 2 висновки. незважаючи на те, що хірургічне втручання є основним напрямком лікування, роль мультимодальної терапії є наріжним каменем для лікування ангіосаркоми надниркових залоз. ключові слова: ангіосаркома надниркових залоз; супраренальний; адреналектомія; судинна пухлина. information about the authors dr. arun kumar m. – assistant professor (surgery), armed forces medical college, pune, india https://orcid.org/0000­0002­6577­5937, e­mail: ak3.doc@gmail.com dr. nilanjan roy – professor (surgery), armed forces medical college, pune, india https://orcid.org/0000­0001­6233­8929, e­mail: nilanjanroyd604@rediffmail.com dr. haris jafri – assistant professor (surgery), armed forces medical college, pune, india https://orcid.org/0000­0002­9513­3345, e­mail: harick_14@yahoo.com dr. vitesh popli – assistant professor (surgery), armed forces medical college, pune, india maharashtra, india https://orcid.org/0000­0002­8473­0662, e­mail: vitesh03@gmail.com dr. v. s. karthik – sr (surgery), armed forces medical college, pune, india https://orcid.org/0000­0002­2975­3191, e­mail: vellalakarthik@gmail.com dr. thinley phuntsho – jr (surgery), armed forces medical college, pune, india https://orcid.org/0000­0002­2071­5041, e­mail: thinley806@gmail.com references 1. weiss sw, goldblum jr, folpe al. enzinger and weiss’s soft tissue tumors. elsevier health sciences; 2007. 4348 p. 2. li xm, yang h, reng j, zhou p, cheng zz, li z, et al. a case report of primary adrenal angiosarcoma as depicted on magnetic resonance imaging. medicine (baltimore). 2017 nov 10;96(45):e8551. https://doi.org/10.1097/md.0000000000008551 3. grajales-cruz a, baco-viera f, rivé-mora e, ramírez-tanchez c, tasso d, arroyo-portela n, et al. primary adrenal angiosarcoma: a rare and potentially misdiagnosed tumor. cancer control. 2017 apr;24(2):198­201. https://doi.org/10.1177/107327481702400213 4. stavridis s, mickovski a, filipovski v, sasho b, sasho d, ljupcho l. epithelioid angiosarcoma of the adrenal gland. report of a case and review of the literature. maced j med sci. 2010 dec 15;3. 5. antao n, ogawa m, ahmed z, piao j, poddar n. adrenal angiosarcoma: a diagnostic dilemma. cureus. 11(8):e5370. 6. kareti lr, katlein s, siew s, blauvelt a. angiosarcoma of the adrenal gland. arch pathol lab med. 1988 nov;112(11):1163­5. 7. fuletra jg, ristau bt, milestone b, cooper hs, browne a, movva s, et al. angiosarcoma of the adrenal gland treated using a multimodal approach. urol case rep. 2016 dec 1;10:38­41. https://doi.org/10.1016/j.eucr.2016.11.003 8. livaditou a, alexiou g, floros d, filippidis t, dosios t, bays d. epithelioid angiosarcoma of the adrenal gland associated with chronic arsenical intoxication? pathol ­ res pract. 1991 mar 1;187(2): 284-9. https://doi.org/10.1016/s0344­0338(11)80785­5 9. kumar a, popli v, roy n, jafri h, ponnam a. giant adrenal endothelial cyst: a case report. med sci int med j. 2022 aug 21;11. https://doi.org/10.5455/medscience.2022.04.102 10. sung jy, ahn s, kim sj, park ys, choi yl. angiosarcoma arising within a long-standing cystic lesion of the adrenal gland: a case report. j clin oncol off j am soc clin oncol. 2013 mar 20;31(9):e132­6. https://doi.org/10.1200/jco.2012.44.0800 11. rodriguez-pinilla sm, benito-berlinches ab, ballestin c, et al. angiosarcoma of adrenal gland: report of a case and review of the literature. rev esp patol. 2002;35(2):227­32. 12. criscuolo m, valerio j, gianicolo me, et al. a vinyl chloride-exposed worker with an adrenal gland angiosarcoma: a case report. ind health. 2014; 52(1):66­70. https://doi.org/10.2486/indhealth.2013­0044 13. hendry s, forrest c. epithelioid angiosarcoma arising in an adrenal cortical adenoma: a case report and review of the literature. int j surg pathol. 2014;22(8):744­8. https://doi.org/10.1177/1066896914532541 14. national institutes of health. nih state-ofthe-science statement on management of the clinically inapparent adrenal mass (“incidentaloma”) nih consens state sci statements. 2002;19(2):1­25. received 27 september 2022; revised 28 october 2022; accepted 18 november 2022. this is open access article distributed under the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. a. kumar m. et al. 81 b io m e d ic a l s c ie n c e s issn 2413-6077. ijmmr 2022 vol. 8 issue 1 doi 10.11603/ijmmr.2413-6077.2022.1.12903 structural organization of rat tissues in experimental combined trauma of the chest and both thighs *m. marushchak¹, o. mialiuk2, m. kaskiv2, m. demjanchuk2, i. krynytska1 1 – i. horbachevsky ternopil national medical university, ternopil, ukraine 2 – rivne medical academy, rivne, ukraine background. in cases of severe trauma, fractures of long bones are the most often combined with trauma of the chest, head, and abdomen, as well as development of hemorrhagic shock. therefore, it is reasonable to study the combined trauma of the chest and lower extremities in details, as well as the post-traumatic multiple organ dysfunction especially in early manifestation stage. objective. the aim of the study was to identify the features of structural organization of the lungs, heart and liver with underlying combined trauma of the chest and both thighs on the 7th day of the post-traumatic period. methods. the experimental study involved 22 adult nonlinear white male rats with body mass of 200-210 g, kept on a standard diet at animal facility with food and drinking regimes recommended by the standards for laboratory animals. all animals were divided into 2 groups: the control group (1st, n=10), the experimental group (the 2nd) chest trauma and both thighs trauma, observation for 7 days (n=12). the animals of the experimental group were simulated for right-sided closed pneumothorax with a rib fracture by a trocar under thiopental-sodium anesthesia (40 mg/kg of body weight of the rat, intraperitoneally); it was combined with a fracture of the left and right femurs. skeletal injury was modeled on each thigh that caused a closed fracture by a single dosed blow with a specially designed device. the blow energy was 0.375 j that corresponds to a severe injury. the associated injury was simulated by successive infliction of both injuries. results. examination of the liver of animals on the 7th day of the experiment revealed a rapid growth of lesions in their parenchyma. the central veins were poorly visible and contained single erythrocytes; the vessels of medium caliber of myocardial stroma were dilated and blood-filled, which was manifested by the development of perivascular oedema. in the lungs of experimental animals, a moderate decrease of interstitial tissue oedema of the interalveolar septa was present, while cellular infiltration of mast cells, macrophages and lymphocytes also decreased significantly. conclusions. multiple organ injuries, which are characterized by structural changes in the liver, heart and lungs in the combined trauma of the thorax and thighs on the 7th day of experiment were evidenced. keywords: combined trauma; lungs; heart; liver; morphology. *corresponding author: mariya marushchak, professor, i. horbachevsky ternopil national medical university, ternopil, 46002, ukraine. e-mail: marushchak@tdmu.edu.ua international journal of medicine and medical research 2022, volume 8, issue 1, p. 81-87 copyright © 2022, tnmu, all rights reserved m. marushchak et al. introduction at the present time, the significant increase of multiple and combined traumatic injuries is one of the major health problems in ukraine as well as in many countries around the world, [1] that leads to polysystemic and multiorgan lesions, which in turn increase mortality rate [2, 3]. the most common injuries and major causes of death are traumatic brain injury [4] and chest injury [5]. according to other research, limb fractures predominate in the structure of polytrauma [6, 7]. in the case of severe trauma, fractures of long bones of the extremities are most often combined with trauma of the chest, head, and abdominal organs, as well as development of hemorrhagic shock [8]. it is established that among patients with trauma, the frequency of polytrauma is 13.7%, and polytrauma with combined multiple fractures of long bones of the lower extremities is 0.5% [9]. the same authors note that more than 70% of chest injuries, traumatic brain injuries, and injuries of the musculoskeletal system (16.7% of patients have fractures of the lower extremities) dominate as for the localization. therefore, it is reasonable to study the combined trauma of the chest and lower extremities, as well as the post-traumatic multiple organ dysfunction especially in early manifestation stage (on the 7th day). 82 b io m e d ic a l s c ie n c e s issn 2413-6077. ijmmr 2022 vol. 8 issue 1 the aim of the study was to define the features of the structural organization of the lungs, heart and liver with underlying combined trauma of the chest and both thighs on the 7th day of the post-traumatic period. methods the experimental study involved 22 adult nonlinear white male rats weighing 200-210 g, which were kept on a standard diet of vivarium with food and drinking regimes at the level recommended by the standards for laboratory animals. all animals were divided into 2 groups: the control group (1st, 10 animals); the experimental group (2nd) – chest trauma and both thighs trauma (ct + 2t), observation during 7 days (12 animals). the animals of the experimental group were simulated right-sided closed pneumothorax with a rib fracture by a trocar under thiopental-sodium anesthesia (40 mg/kg of the body weight of the rat, intraperitoneally); it was combined with a fracture of the left and right femurs. skeletal injury was modelled on each thigh that caused a closed fracture by applying a single dosed blow by a specially designed device [10]. the blow energy was 0.375 j, which corresponds to a severe injury. the associated injury was simulated by successive infliction of both injuries. while working with animals, the rules of handling experimental animals were followed. mortality rate of animals was 16.7%. samples of heart, liver and lung tissues, under the conditions of simulated associated trauma and correction, were fixed with 1.5% solution of glutaraldehyde in 0.2 m cacodylate buffer (ph 7.2) at t=4 °c for 1 h. the samples were washed with cacodylate buffer and additionally fixed with a 2% solution of osmium tet ro xide in the same buffer for 1 h (t=4 °c). the preparations were washed from the fixatives and dehydrated in increasing concentrations of ethyl alcohol (50%, 70%, 90% and 100%). additionally, they were dehydrated in 2 changes of propylene oxide and placed in epoxy resin epon-812. the sections were obtained using an ultramicrotome umtp-6 and a diamond knife, they were contrasted with 2% uranyl acetate solution for 15 min and additionally with lead citrate according to reynolds. sections were observed and photographed using an electron transmission microscope pem-100. all animal studies have been approved by the appropriate bioethics committee of i. horbachevsky ternopil state medical university (protocol № 41 of june 1, 2017). results in the most severe period of the maximum probability of complications (from 3 to 10 days) development of respiratory hypoxia, cardio vascular disorders, metabolic acidosis, programmed and induced cell death is possible. during this period the syndrome of multiple organ dysfunction is diagnosed. therefore, liver, heart and lungs were investigated as target organs in multiple organ dysfunction during associated trauma in 7 days of the experiment. examination of the liver of animals in 7 days of the experiment revealed a rapid growth of lesions in its parenchyma. the central veins were poorly visible and contained single erythrocytes. the lumens of the sinusoids were practically not visualized, but erythrocytes were traced in their single preserved areas (fig. 1). the lobular organization of hepatocytes was disturbed, the vast majority of cells did not have a clear structure, their contours were not clear, the size was difficult to visualize. the cytoplasm of hepatocytes of all parts of the lobe was devastated, enlightened, or granular (fig. 1), which indicated development of severe protein hyaline-droplet and hydropic dystrophy with transition to focal necrosis (fig. 1). the vast majority of nuclei was crumpled, became smaller, or disappeared, but in some cells they were visualized with loose chromatin, multiple nucleoli. intercellular connections were largely lost. uneven blood supply and focal lymphoand histiocytic perivascular infiltration were ob­ served in the vessels of the portal tracts. in 7 days of the experiment, the vessels of medium calibre of myocardial stroma were still dilated and blood­filled, which was manifested by development of perivascular oedema. dilated capillaries and diapedesis of erythrocytes were found in the fibre thickness. cardio myo­ cytes were well visualized in the vast majority of visual fields, but focal intracellular myocytolysis and pyknotically altered nuclei were evidenced in some of them, indicating the presence of focal dystrophic-necrotic changes. the perivasal stroma was loosened due to oedema, which also spread to the intercellular spaces, disintegrating the muscle layer (fig. 2). in 7 days of the experiment in the lungs of the experimental animals a moderate decrease of interstitial tissue oedema of the interalveolar septa was evidenced, while cellular infiltration of mast cells, macrophages and lymphocytes a l s o d e c r e a s e d s i g n i f i c a n t l y . s i g n s o f dystelectasis (partial decline) were still present m. marushchak et al. 83 b io m e d ic a l s c ie n c e s issn 2413-6077. ijmmr 2022 vol. 8 issue 1 that led to a decrease in the airiness of the lung t i s s u e . s o m e p n e u m o c y t e s u n d e r w e n t dystrophic-necrotic changes, and a small amount of exfoliated epithelium and serous exudate appeared in the lumens of the alveoli (fig. 3). the blood supply to the vessels was still uneven, with a predominance of venous capillary ones. leukocytes focally accumulated in the perivascular spaces (fig. 3). discussion the severity of the patient’s condition with trauma is largely determined by development of various metabolic disorders in organs and tissues with underlying severe endotoxicosis [11]. these pathological phenomena cause development of deregulatory pathology, one of the manifestations of which is multiorgan fig. 1. structural changes in the liver in cases of simulation of combined trauma in 7 days of the experiment (acute hyaline-drip and hydropic dystrophy with transition to focal necrosis). hematoxylin and eosin stai ning. ×200. fig. 2. the structure of cardiomyocytes in the simulation of associated trauma in 7 days of the experiment (pericellular oedema combined with erythrodiapedesis, focal dystrophic changes in cardiomyocytes). hematoxylin and eosin staining. ×200. m. marushchak et al. 84 b io m e d ic a l s c ie n c e s issn 2413-6077. ijmmr 2022 vol. 8 issue 1 failure involving lungs, heart and liver into the pathological process. according to the pathogenesis shortly after the injury the systemic inflammatory response syndrome is established. a number of studies have shown that oxidative stress is crucial in activation of inflammation. from a genetic point of view, oxidative stress modulates inflammation by regulating the activity of certain transcription factors, such as nuclear kappa factor b (nf-kb), signal transducer and activator of transcription 3 signal transducer and activator of transcription 3 (stat 3), hypoxia induction factor 1 alpha (hif-1alpha), and protein-1 activator (ap-1) [12]. activation of peroxidation processes is one of the factors destabilizing the membranes. it is established that when hydroxyl radicals influence on diene conjugates of fatty acids, lipid hydroperoxides are formed, which cause conformational disturbances in the cell membranes. the data ob tained indicate that the intensity of peroxidation in different tissues of the body depends on the type of injury. analysis of the scientific fig. 3. structural changes of the lung in cases of simulation of combined trauma in 7 days of the experiment: a (perivascular accumulation of leukocytes), b (uneven vascular blood supply, dystrophic­necrotic changes of epitheliocytes, slight effusion in the lumen of the alveoli, dystelectasis, focal perivascular leukocyte infiltration). hematoxylin and eosin staining. ×200. а b m. marushchak et al. 85 b io m e d ic a l s c ie n c e s issn 2413-6077. ijmmr 2022 vol. 8 issue 1 sources shows that oxidative stress in the patients with various injuries develops almost immediately after the initial injury moving from the macroscopic level to the cellular and molecular one [13-15]. currently, scientists have proven that the maximum of lipoperoxidation coincides with the intensity of the body’s response to inflammation [16]. thus, kozak dv notes that the intensity of lipoperoxidation due to hip injury and blood loss is fluctuating with a period of development to the 3rd day, a period of temporary well-being in 14 days and a reincrease in 21 days [17]. the results of the study of oxidative processes in the case of combined trauma of the chest and both thighs show that the most intense free-radical processes occur in the lungs on the 7th and 14th days of observation. on the 3rd day of the experiment, the production of mitochondrial superoxide anion radical increases equally in the tissues of the heart and lungs, while in the tissues of the liver this rate increases to a maximum within 3-14 days [18-20]. it has been established that acute hyperproduction of reactive oxygen species by leukocytes in 1 day of the experiment causes disorganization of bioelectrical activity of mitochondrial membranes, which is characterized by a dynamic increase in the ratio of leukocyte cells suspension with reduced transmembrane potential and the ratio of leukocytes with signs of apoptosis with a maximum in 7 days [21]. in cases of extensive metabolic changes in the body tissues of rats with simulated combined trauma, structural changes in the liver, heart and lungs after 1-3 days of the experiment were evidenced. these changes were mani fested by severe circulatory disorders, erythrostasis and development of dystrophic-necrotic changes, especially in epithelial structures [22]. according to our study, these structural changes progressed in the lungs, heart and liver up to the 7th day of observation that is comparable to the severity of oxidative stress in the body tissues and apoptotic death of leukocytes. conclusions in cases of combined trauma of the thorax and thighs of the experimental animals, structural changes develop in the liver, heart and lungs on the 7th day of observation. these changes are characterized by severe hyalinedrip and hydropic dystrophy with transition to focal necrosis of liver tissue, pericellular oedema and dystrophic-necrotic changes of pneumocytes. conflict of interest authors declare no conflict of interest. author contributions mariya marushchak, inna krynytska – con­ ceptualization, methodology, formal analysis, writing – original draft, writing – reviewing and editing; mykhailo demjanchuk, oksana mialiuk – data curation, investigation, formal analysis; marjana kaskiv – writing, original draft. особливості структурної організації тканин щура за умов поєднаної травми грудної клітки та обох стегон м. марущак1, о. мялюк2, м. каськів2, м. демянчук2, і. криницька1 1 – тернопільський національний медичний університет імені і. я. горбачевського моз україни, тернопіль, україна 2 – комунальний заклад вищої освіти “рівненська медична академія” рівненської обласної ради, рівне, україна вступ. за умови тяжкої травми переломи довгих кісток кінцівок найчастіше поєднуються з травмою грудної клітки, голови, органів черевної порожнини, розвитком геморагічного шоку тому, обґрунтованим є дослідження поєднаної травми грудної клітки та нижніх кінцівок, а також поліорганність їх ураження, особливо у фазу маніфестації. мета. виявити морфологічні особливості структурної організації легень, серця і печінки на тлі поєднаної травми грудної клітки та обох стегон через 7 діб моделювання травми. методи. експериментальне дослідження виконано на 22 дорослих нелінійних білих щурах-самцях масою 200–210 г, яких утримували на стандартному раціоні віварію з підтриманням харчового і питного режимів на рівні, рекомендованому нормами утримання лабораторних тварин. усіх тварин m. marushchak et al. 86 b io m e d ic a l s c ie n c e s issn 2413-6077. ijmmr 2022 vol. 8 issue 1 було поділено на 2 групи: контрольну (1-а, 10 тварин), дослідну групу: 2-а – травма грудної клітки й обох стегон (тгк+2с), спостереження 7 діб (12 тварин). тваринам дослідної групи під тіопенталнатрієвим наркозом (40 мг/кг маси тіла щура внутрішньочеревно) за допомогою троакара моделювали правобічний закритий пневмоторакс із переломом ребра та поєднували їх з переломом лівої і правої стегнових кісток. скелетну травму моделювали шляхом нанесення однократного дозованого удару спеціально розробленим пристроєм по кожному стегну, який викликав закритий перелом (kozak, 2011). енергія удару становила 0,375 дж, що відповідало травмі важкого ступеня тяжкості. поєднану травму моделювали шляхом послідовного нанесення обох ушкоджень. результати. дослідження печінки тварин через 7 діб експерименту встановило різке зростання ураження її паренхіми. центральні вени проглядалися слабо, міс тили поодинокі еритроцити, судини середнього калібру строми міокарда залишалися розширеними та наповненими кров’ю, що проявилося розвитком перивакулярного набряку. у легенях піддослідних тварин спостерігалося помірне зменшення набряку інтерстиціальної тканини міжальвеолярних перегородок, а також значно зменшилась клітинна інфільтрація тучними клітинами, макрофагами та лімфоцитами. висновки. при поєднаній травмі грудної клітки та стегон експериментальних тварин структурні зміни в печінці, серці та легенях на 7 добу спостереження, що характеризуються вираженою гіаліновокрапельної та гідропічної дистрофії з переходом у вогнищеві некрози тканин печінки, перицелюлярним набряком, поєднаним з еритродіапедезом, вогнищевими дистрофічними змінами кардіоміоцитів та дистрофічно-некротичними змінами пневмоцитів. ключові слова: поєднана травма; легені; серце; печінка; морфологія. information about the authors mariya marushchak, professor, i. horbachevsky ternopil national medical university, ternopil, ukraine https://orcid.org/0000­0002­1177­412x, e­mail: marushchak@tdmu.edu.ua oksana mialiuk, assistant professor, rivne medical academy, rivne, ukraine https://orcid.org/0000­0002­5090­6607, e­mail: oksankamp@ukr.net marjana kaskiv, assistant professor, rivne medical academy, rivne, ukraine https://orcid.org/0000­0002­6914­0867, e­mail: maryana_kaskiv@ukr.net mykhailo demjanchuk, assistant professor, rivne medical academy, rivne, ukraine https://orcid.org/0000­0001­8729­5144, e­mail: dmr­rv@ukr.net inna krynytska, professor, i. horbachevsky ternopil national medical university, ternopil, ukraine https://orcid.org/0000­0002­0398­8937, e­mail: krynytska@tdmu.edu.ua 5. chrysou к, halat g. hoksch b, schmid rа, kocher gj. lessons from a large trauma center: impact of blunt chest trauma in polytrauma patients– still a relevant problem? scandinavian journal of trauma, resuscitation and emergency medicine. 2017;25(1):42. 6. probst c, pape hc, hildebrand f, regel g, mahlke l, giannoudis p, krettek c, grotz mr. 30 years of polytrauma care: an analysis of the change in strategies and results of 4849 cases treated at a single institution. injury. 2009;40(1):77­83. https://doi.org/10.1016/j. injury.2008.10.004 7. banerjee m, bouillon b, shafi zadeh s, paffrath t, lefering r, wafaisade a, german trauma registry group. epidemiology of extremity injuries in multiple trauma patients. injury. 2013;44(8):1015­ 1021. https://doi.org/10.1016/j.injury.2012.12.007 8. horst k, simon tp, pfeifer r, teuben m, almahmoud k, zhi q, et al. characterization of blunt references 1. huriev so, tanasiienko pv, satsyk sp. clinical and epidemiological characteristics of victims of infectious diseases caused by polytraumas of road accidents. medicine today and tomorrow. 2012;1:54. 2. world health organization (who) injuries and violence: the facts. 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international journal of medicine and medical research. 2018;2(2):52­55. 21. khudobiak мм, marushchak мі, holova­ tiuk lm, datsko tv. morphological changes in lungs, heart and liver caused by experimental associated chest and thigh trauma. international journal of medicine and medical research. 2017;3(1):79­83. 22. marushchak mi, khudobiak mm, habor hh, mialiuk op. mitochondrial mechanisms of apoptosis in combined trauma to the chest and thighs and justification for the use of antioxidants in the expe­ riment. bulletin of vinnytsia national medical university. 2017;1(2):204­210. received 12 may 2022; revised 26 may 2022; accepted 7 june 2022. this is open access article distributed under the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. m. marushchak et al. 67 b io m e d ic a l s c ie n c e s issn 2413-6077. ijmmr 2022 vol. 8 issue 1 doi 10.11603/ijmmr.2413-6077.2022.1.12924 antioxidant protection in white rats on the background of drinking water use with excessive nitrate concentration m. v. danchyshyn, o. v. lototska i. horbachevsky ternopil national medical university, ternopil, ukraine background. the problem of nitrate pollution in terms of its scale and consequences has reached a global level. approximately one-third of groundwater bodies for which information is currently available exceed the maximum allowable concentrations of nitrates in drinking water. objectives. the aim of the research is to study the features of antioxidant protection in the body of white rats on the background of drinking water with excessive concentrations of nitrates for 30 days regarding the age. methods. the experiment was performed on 2 groups of outbred white female rats of different age (mature and immature), 30 animals each, divided into 5 subgroups, which differed in the quality of drinking water they consumed independently. in a 30-day study, nitrates were given to rats with drinking water at a dose of 50, 150, 250, 500 mg of sodium nitrate per liter. the animals of the control group drank water from the city water supply. the effect of nitrates on superoxide dismutase (sod), catalase (cat) and ceruloplasmin (cp) in the serum of rats was evaluated. results. the research has shown increased sod, cat and cp in blood serum on the background of drinking water with a nitrate concentration of 150 to 500 mg/l in the experimental groups of animals of both ages. according to the research, the most significant changes in parameters are caused by drinking water with nitrates at a concentration of 500 mg/l. more significant changes were in immature animals then the mature ones. conclusions. excessive concentrations of nitrates in drinking water adversely affect the body of experimental animals causing activation of the studied parameters of the antioxidant system. keywords: drinking water; nitrates; superoxide dismutase; catalase; ceruloplasmin. *corresponding author: maryana danchyshyn, phd student, i. horbachevsky ternopil national medical university, ternopil, 46002, ukraine. e-mail: danchyshyn@tdmu.edu.ua international journal of medicine and medical research 2022, volume 8, issue 1, p. 67-73 copyright © 2022, tnmu, all rights reserved m. v. danchyshyn et al. introduction the problem of nitrate pollution in terms of its scale and consequences has reached the global level. it consists in total pollution of the biosphere, deterioration of plant agricultural products and drinking water, negative impact on the human body, especially young children and farm animals. approximately one third of the groundwater bodies, for which information is currently available, exceeds the maximum permissible concentrations (mpc) of nitrates in drinking water that is equal to 50 mg/dm3. monitoring of nitrate levels in groundwater in different districts of ternopil region showed that the maximum concentration ranges from 1.1 to 7.3 times or from 55 to 365 mg/l and even more [1, 2]. this is due to the widespread use of nitrogen-containing organic and inorganic fertilizers in agriculture. however, unfortunately, residents of houses have been using such water for drinking for many years, because nitrates, even their excessive concentrations are not felt or tasted by humans. nitrates are non-toxic, but entering the gastrointestinal tract they are reduced to nitrites, which due to their oxidizing properties are ten times more toxic than nitrates. nitrites are absorbed into the blood, and consequently reach tissues. the main mechanism of nitrite toxicity is the oxidation of fe2+ to fe3+, the conversion of hemoglobin into methemoglobin (methb) [3]. the use of nitrates with drinking water affects the nervous and cardiovascular systems, development of embryos; it can cause colorectal cancer, thyroid disease and neural tube defects [4, 1]. when systematically ingested, they can lead to disorders of the endocrine and central nervous system, blood circulation, etc. [5]. in addition, it is established that nitrites in the presence of amines can form n-nitrosamines, which have carcinogenic activity. nitrates can also inhibit the enzyme systems involved in tissue respiration. as a result, a large number of free radicals is formed. at the same time, at 68 b io m e d ic a l s c ie n c e s issn 2413-6077. ijmmr 2022 vol. 8 issue 1 the cellular level the processes of free radical oxidation of phospholipids of biological cell membranes take place that lead to development of oxidative stress [6]. this can be overcome by reducing the intensity of lipid peroxidation (lpo) in the body with antioxidant system (aos) which controls and inhibits all stages of reactions of free radicals capable of damaging the cell from their initiation to formation of hydroperoxides and malonic dial [7]. this protective system combines a number of substances of different nature; each of them acts in close contact with its other structural elements, harmoniously complements, and in many cases enhances the action of each other. the main antioxidants of blood plasma are superoxide dismutase (sod), catalase (cat) and ceruloplasmin (cp) [8, 9]. when an organism is in unfavorable living conditions, in particular ecological, or when it is exposed to adverse factors the activity of these antioxidants increases sharply [10]. therefore, the aim of the research was to study the features of antioxidant protection in the body of white rats on the background of drinking water with excessive concentrations of nitrates for 30 days regarding the age. methods the study was conducted on 60 outbred white female rats which were kept on the standard diet of the vivarium of i. horbachevsky ternopil national medical university. animals were divided into 2 age categories: the 1st category – 30 mature animals weighing 180­200 g and the 2nd – 30 immature animals weighing 60-80 g. the experimental animals of each age category were divided into 5 groups with 6 rats in each. the groups were selected by randomization. the animals of control group drank drinking water from the city water supply. the rats of the 1st group drank drinking water with nitrate content of 50 mg/l, the 2nd – 150 mg/l, the 3rd – 250 mg/l, the 4th – 500 mg/l. to create model aqueous solutions, drinking water from city water supply was used; taking into account background concentrations, sodium nitrate (nano3) was added into it to create aqueous solution with nitrates at a dose of 50, 150, 250, 500 mg per liter. the experiments were performed in accordance with the european convention for the protection of vertebrate animals used for research purposes (strasbourg, 1986) [12] and the norms of biomedical ethics and the “general ethical principles of animal experiments” adopted by the first national congress on bioethics (kyiv, 2001). the animals were taken out of the experiment by bloodletting under thiopental-sodium anesthesia in 30 days after the beginning of the experiment. the activity of catalase (cat), ceruloplasmin (cp) and superoxide dismutase (sod) was determined in the serum by means of a semiautomatic biochemical analyzer humalyzer 2000 using standard kits by human diagnostics (wiesbaden, germany). the kits used for evaluation of cat, cp and sod were adapted for their determination in animal serum. the results were statistically processed using computer programs statistica 10.0 and excel 2010. the choice of statistical research method was based on the correct distribution of the studied features. further pairwise comparison of groups was performed using the mann-whitney u­test, the statistical significance p<0.05. results the results of our studies showed that when drinking water with different concentrations of nitrates, the aos of the experimental animals of both ages underwent significant changes. the key enzyme of aos is sod. it was found that the level of sod, cat and cp in the 1st experimental group of both age categories did not differ significantly from the data in the control group. the increase in sod activity in other groups of experimental rats of both ages compare to the controls was directly proportional to the concentration of nitrates in water. the level of sod in the 2nd group of mature animals increased significantly by 43.7%, in the 3rd – by 56.2% and in the 4th – by 62.5% (fig. 1). in the immature animals, a more significant increase was evidenced. thus, in the 2nd group the activity of sod significantly increased by 61.1%, in the 3rd – by 94.4%. however, the activity of sod increased the most in the 4th group – by 116.6% (fig. 2). sod always works in conjunction with cat, which converts hydrogen peroxide to water and molecular oxygen. when determining the activity of cat in the serum of mature animals it increased by 16.0% in the 2nd group, in the 3rd – by 30.5% (p<0.05), and in the 4th group – by 97.2% (p<0.05) compare to the control (fig. 3). in the immature animals, insignificant increase in the activity of this parameter was found in the 2nd and 3rd groups (by 6.7% and 18.3%, respectively). however, in the animals m. v. danchyshyn et al. 69 b io m e d ic a l s c ie n c e s issn 2413-6077. ijmmr 2022 vol. 8 issue 1 fig. 1. the level of superoxide dismutase in the serum of mature experimental animals when drinking water with different concentrations of nitrates (unit/g). note: * – the differences are significant comparing to the control group (p<0.05). error bars indicate ± standard deviation of the mean after three replicates. fig. 2. the level of superoxide dismutase in the serum of immature experimental animals when drinking water with different concentrations of nitrates (unit/g). note: * – the differences are significant comparing to the control group (p<0.05). error bars indicate ± standard deviation of the mean after three replicates. * * * 0 0.05 0.1 0.15 0.2 0.25 0.3 0.35 control group 1 group 2 group 3 group 4 th e le ve l o f s up er ox id e di sm ut as e, un it/ g * * * 0 0.05 0.1 0.15 0.2 0.25 0.3 0.35 0.4 0.45 0.5 control group 1 group 2 group 3 group 4 th e le ve l o f s up er ox id e di sm ut as e, un it/ g of the 4th group the parameter increased by 28.8% (p<0.05) compare to the control group (fig. 4). the nonenzymatic part of aos was assessed for the presence of сp in the blood serum, which is also one of the main antioxidants in the human’s body. a main feature of this protein is its high stability to the toxic action of reactive oxygen species which allows it to maintain biological activity under conditions of their intensive generation [7, 8]. increased level of cp in both age categories was established; it differed not much. in the 2nd group of mature animals the parameter increased by 22.1%, in the 3rd group – by 23.4%, in the 4th group – by 53.3% (p<0.05) (fig.5). the number of sr in the immature animals of the 2nd group increased by 28.4%, in the 3rd – by 39.0% (p<0.05), in the 4th – by 61.0% (p<0.05) (fig. 6). discussion according to the literature the antioxidant substance in the cell is present at low con centrations and significantly reduces or prevents oxidation of the oxidizable substrate. the most m. v. danchyshyn et al. 70 b io m e d ic a l s c ie n c e s issn 2413-6077. ijmmr 2022 vol. 8 issue 1 efficient enzymatic antioxidants contain glu­ tathione peroxidase, catalase and superoxide dismutase. sod is one of the most potent intracellular enzymatic antioxidants, and it catalyzes the conversion of superoxide anions to dioxygen and hydrogen peroxide. the hydrogen peroxide is removed by catalase. this enzyme is present in the peroxisome of aerobic cells and is very efficient in promoting the conversion of hydrogen peroxide to water and molecular oxygen [13]. an increase in the activity of cat and sod is usually observed in cases of environmental pollutants since sod-cat system represents the first line of defense against oxidative stress [14, 15]. according to krishnamoorthy and sangeetha [16], sodium nitrite (300 mg/kg body weight) significantly increased the lipoperoxidation and the activities of liver marker enzymes such as alanine aminotransferase, aspartate aminotransferase, acid phosphatase and lactate dehydrogenase. analyzing the dynamics of changes in the activity of aos in the serum of rats, significantly higher sod against cat and cp were established. the sod interrupts the chains of oxygen-dependent free radical reactions, causing dismutation of the superoxide anion radical with the release of hydrogen peroxide, which may fig. 3. the level of catalase in the serum of mature animals when drinking water with different concentrations of nitrates (mcat/l). note: * – the differences are significant comparing to the control group (p<0.05). error bars indicate ± standard deviation of the mean after three replicates. fig. 4. the level of catalase in the serum of immature animals when drinking water with different concentrations of nitrates (mcat/l). note: * – the differences are significant comparing to the control group (p<0.05). error bars indicate ± standard deviation of the mean after three replicates. * * 0 1 2 3 4 5 6 7 8 9 10 control group 1 group 2 group 3 group 4 th e le ve l o f c at al as e, m ca t/ l * 0 1 2 3 4 5 6 7 8 9 control group 1 group 2 group 3 group 4 th e le ve l o f c at al as e, m ca t/ l m. v. danchyshyn et al. 71 b io m e d ic a l s c ie n c e s issn 2413-6077. ijmmr 2022 vol. 8 issue 1 precede formation of the most toxic hydroxyl radical (oh-) and triplet oxygen [10]. increased activity of sod is an additional damaging factor and may cause pathological processes as a result of increased cytotoxic action of hydrogen peroxide, formed as a result of dismutation of superoxide [8]. the increase of cat in serum does not exclude the presence of cytolysis on the background of drinking water with different concentrations of nitrates, because cat is an intracellular enzyme and its increased activity in the blood occurs in cases of increased permeability of cell membranes. cp neutralizes free radicals and protects cell membranes due to its ability to inhibit lipid peroxidase. it prevents activation of the floor, formation of toxic iron ions and damage to cell membranes. this enzyme acts as an “extracellular superoxide dismutase” neutralizing superoxide anions in the extracellular environment, blood, amniotic and cerebrospinal fluid, muscle, kidney and myocardium. cp increases with iron deficiency and excessive fig. 5. the level of ceruloplasmin in the serum of mature animals when drinking water with different concentrations of nitrates (mg/l). note: * – the differences are significant comparing to the control group (p<0.05). error bars indicate ± standard deviation of the mean after three replicates. fig. 6. the level of ceruloplasmin in the serum of immature animals when drinking water with different concentrations of nitrates (mg/l). note: * – the differences are significant comparing to the control group (p<0.05). error bars indicate ± standard deviation of the mean after three replicates. * 0 2 4 6 8 10 12 14 control group 1 group 2 group 3 group 4 th e le ve l o f c er ul op la sm in , m g/ l * * 0 2 4 6 8 10 12 control group 1 group 2 group 3 group 4 th e le ve l o f c er ul op la sm in , m g/ l m. v. danchyshyn et al. 72 b io m e d ic a l s c ie n c e s issn 2413-6077. ijmmr 2022 vol. 8 issue 1 copper, in response to hypoxia, insulin, thrombin, estradiol, proinflammatory cytokines [11]. increased oxidation processes are extremely crucial in the genesis of many pathological conditions, including inflammatory lesions of various tissues. in case of increased intake of xenobiotics, depletion of antioxidant depots and influence of negative factors, oxidative stress develops; it is characterized by prooxidant and antioxidant disbalance and development of oxidative damage [17]. conclusions the study allowed drawing the conclusion that excessive concentrations of nitrates in drinking water adversely affected the body of experimental animals causing activation of the studied parameters of the antioxidant system. as a result, the increased activity of sod, cat and cp in blood serum against consumption of drinking water with concentration of nitrates from 150 to 500 mg/l within 30 days in the experimental groups of both age categories has been revealed. the most pronounced changes in the parameters were regarding drinking water with nitrates at a concentration of 500 mg/l. the changes were more pronounced in immature animals then in the mature ones. conflict of interests the authors declare no conflict of interest. acknowledgements this research received no external funding. autho’s contributions maryana danchyshyn – investigation, data curation, formal analysis, writing – original draft, writing – reviewing and editing; olena lototska – conceptualization, methodology, writing – reviewing and editing. особливості антиоксидантного захисту в організмі білих щурів на тлі вживання питної води з понаднормативною концентрацією нітратів м. в. данчишин, о. в лотоцька тернопільський національний медичний університет імені і. я. горбачевського моз україни, тернопіль, україна вступ. проблема забруднення довкілля нітратами за своїми масштабами та наслідками вийшла на глобальний рівень. приблизно в одній третині підземних водних об’єктів, щодо яких наразі доступна інформація, перевищено гранично допустимі концентрації вмісту нітратів у питній воді. мeтою цієї роботи стало вивчення особливості антиоксидантного захисту в організмі білих щурів на тлі вживання питної води з понаднормативною концентрацією нітратів впродовж 30 днів в залежності від віку. матеріали. експеримент проводили на 2 групах безпородних білих самок-щурів різного віку (статевозрілих та статевонезрілих) по 30 особин у кожній, розділених на 5 підгруп, які відрізнялися за якістю питної води, яку вони споживали самостійно. у 30-денному дослідженні нітрати давали щурам у питній воді в дозі 50, 150, 250, 500 мг нітрату натрію на літр. тварини контрольної групи пили воду з міського водопроводу. оцінювали вплив нітратів на активність супероксиддисмутази (сод), каталази та церулоплазміну (ср) в сироватці крові щурів. результати. у результаті досліджень виявлено підвищену активність сод, каталази та ср у сироватці крові на тлі вживання питної води з концентрацією нітратів від 150 до 500 мг/л у піддослідних групах обох вікових категорій. встановлено, що найбільш виражені зміни показників викликає вживання питної води з нітратами у концентрації 500 мг/л. більш виражені зміни були у статево незрілих тварин. висновки. присутність нітратів у питній воді у понаднормативних концентраціях негативно впливає на організм піддослідних тварин, викликаючи активацію досліджуваних показників антиоксидантної системи. ключові слова: питна вода; нітрати; супероксиддисмутаза; каталаза; церулоплазмін. information about the authors maryana danchyshyn, phd student, i. horbachevsky ternopil national medical university, ternopil, ukraine https://orcid.org/0000­0002­7422­2883, e­mail: danchyshyn@tdmu.edu.ua olena lototska, professor, i. horbachevsky ternopil national medical university, ternopil, ukraine https://orcid.org/0000­0002­1393­7914, e­mail: lototska@tdmu.edu.ua m. v. danchyshyn et al. 73 b io m e d ic a l s c ie n c e s issn 2413-6077. ijmmr 2022 vol. 8 issue 1 references 1. ward mh, jones rr, brender jd, de kok tm, weyer pj, nolan bt, villanueva cm, van breda sg. drinking water nitrate and human health: an up­ dated review. int j environ res public health. 2018 jul 23;15(7):1557. https://doi.org/10.3390/ijerph15071557. 2. lototska ov, prokopov vo. risk assessment of drinking water consumption with high nitrate content for the health of the population of ternopil region. dovkillia i zdorovia (environment and health). 2018;4(89):21­5. https://doi.org/10.32402/dovkil2018.04.020 [in ukrainian] 3. fossen johnson s. methemoglobinemia: infants at risk. curr probl pediatr adolesc health care. 2019 mar;49(3):57­67. https://doi.org/10.1016/j.cppeds.2019.03.002. 4. brender jd, weyer pj. agricultural compounds in water and birth defects. curr environ health rep. 2016 jun;3(2):144­52. https://doi.org/10.1007/s40572­016­0085­0. 5. singh s, anil ag, kumar v, kapoor d, subramanian s, singh j, ramamurthy pc. nitrates in the environment: a critical review of their distribution, sensing techniques, ecological effects and remediation. chemosphere. 2022 jan;287(pt 1):131996. https://doi.org/10.1016/j.chemosphere.2021. 131996. 6. leskiv hya. the effect of nitrates on the state of the antioxidant system of piglets. naukovyi visnyk lvivskoho natsionalnoho universytetu veterynarnoi medytsyny ta biotekhnolohii im. gzhytskoho (scientific bulletin of the lviv national university of veterinary medicine and biotechnology named after gzhitskyi). 2011;13(4(1)):234­7 [in ukrainian]. available from: https://cyberleninka.ru/article/n/ vpliv-nitrativ-na-stan-antioksidantnoyi-sistemiporosyat 7. bielenichev if, levytskyi yel, hubskyi yui, kovalenko si, marchenko om antioxidant body protection system (review). modern problems of toxicology. 2002;3:24–31 [in ukrainian]. 8. lavryshyn yuyu, varholyak is, martyshuk tv, huta za, ivankiv lb, paladiychuk or, murska sd, gutiy bv, gufrii df. biological significance of the system of antioxidant protection of the animal organism. scientific bulletin of the lviv national uni­ versity of veterinary medicine and biotechnology named after s.z. gzhitskyi. naukovyi visnyk lvivskoho natsionalnoho universytetu veterynarnoi medytsyny ta biotekhnolohii imeni s.z. gzhytskoho (scientific bulletin of the s.z. gzhitskyi lviv national university of veterinary medicine and biotechnology). 2016;2 (66):100­11. 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[in ukrainian] available from: http://nbuv.gov.ua/ujrn/nvlnu_2011_13_4(2)__3 11. unguryan tm, zamorskii ii. changes in the content of ceruloplasmin in blood plasma under conditions of myoglobinuric form of acute kidney damage. ukrainian journal of medicine, biology and sport. 2018;3(6):67–72 [in ukrainian]. 12. european convention for the protection of vertebrate animals used for experimental and other scientific purposes. council of europe, strasbourg, 1986:53. 13. kurutas eb. the importance of antioxidants which play the role in cellular response against oxidative/nitrosative stress: current state. nutr j. 2016 jul 25;15(1):71. https://doi.org/10.1186/s12937­016­0186­5. 14. pavlović sz, borković mitić ss, radovanović tb, perendija br, despotović sg, gavrić jp, saicić zs. seasonal variations of the activity of antioxidant defense enzymes in the red mullet (mullus barbatus l.) from the adriatic sea. mar drugs. 2010 feb 26;8(3): 413-28. https://doi.org/10.3390/md8030413. 15. sierra-campos e, valdez-solana ma, camposalmazán mi, avitia-domínguez c, hernández-rivera jl, lira-sánchez ja de, garcia-arena g, téllezvalencia a. nitrate and nitrite in drinking water affect antioxidant enzymes in erythrocytes of rats. the ukrainian biochemical journal. 2018;4(90):90­101. https://doi.org/10.15407/ubj90.04.090. 16. krishnamoorthy p, sangeetha m. hepatoprotective effect of vitamin c on sodium nitriteinduced lipid peroxidation in albino rats. indian j biochem biophys. 2008;45(3):206­8. 17. marnett l. lipid peroxidation-dna damage by malondiaaldehyde. mutat. 1999. 424(1­2):83–95. received 18 may 2022; revised 29 may 2022; accepted 10 june 2022. this is open access article distributed under the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. m. v. danchyshyn et al. 5 in t e r n a l m e d ic in e issn 2413-6077. ijmmr 2022 vol. 8 issue 2 doi 10.11603/ijmmr.2413-6077.2022.2.13152 sickle cell thalassemia in an adult from central india showing massive splenic infarction and gamna-gandy bodies in splenic parenchyma with concomitant plasmodium falciparum infection (case report) *t. s. singh1, r. singh2, r. pandey3 1 – 155 base hospital, tezpur, india 2 – indian naval hospital ship asvini, mumbai, india 3 – command hospital, pune, india background. sickle cell thalassemia is a heterozygous state of hbs/β+ or hbs/β0 manifested clinically either as an asymptomatic carrier or have features akin to sickle cell anemia. objective. the aim of the study is to review the literature and discuss the varied clinical manifestations and diagnosis of a case report of haemoglobinopathy in an adult from central india. methods. a case of haemoglobinopathy from central part of india is being investigated. results. a case of haemoglobinopathy in an adult presenting to a tertiary hospital with chronic back ache was reported. the patient was found to have massive splenomegaly with evidence of splenic infarction. gross examination of spleen revealed multiple soft yellowish pasty areas, which on microscopic examination showed significant necrosis with presence of great amount pale amorphous yellow substance, gamna-gandy bodies (ggbs) and massive foreign body cell reaction in splenic parenchyma. post-splenectomy peripheral blood smear (pbs) showed thrombocytosis and plasmodium falciparum gametocytes. the hb electrophoresis revealed both elevation of hb s (49.7%), hb f (46.7%); hb a2 (3.0%) and (hb a 0.7%) consistent with as sickle cell thalassemia hb s/β+ thalassemia. conclusion. sickle cell thalassemia with long standing huge splenomegaly, splenic infarction with ggbs deposition and concomitant falciparum malaria in clinically stable patient is rare indeed. keywords: sickle-cell anemia; beta-thalassemia; malaria; plasmodium falciparum; gamna-gandy bodies. *corresponding author: thongam sachin singh, assistant professor (pathology), 155 base hospital, tezpur, 784001, india. e-mail: sachinthongam@gmail.com international journal of medicine and medical research 2022, volume 8, issue 2, p. 5-10 copyright © 2022, tnmu, all rights reserved t. s. singh et al. introduction hemoglobinopathies are the most common inherited rbc disorders, thalassemia and sickle cell disease – the most frequent. co-existent both sickle cell mutation and thalassemia mutation, commonly beta thalassemia, facilitating development to a compound heterozygous state known as sickle cell beta thalassemia, was first reported and described by silvestroni and bianco in 1944 [1]. the incidence of this condition is very common in mediterranean region. its prevalence in indian subcontinent is underreported, perhaps due to underdiagnosis as most of these patients are normal and asymptomatic. documented prevalence of sickle cell beta thalassemia in india is less than 1% in various studies [2]. inherited as autosomal recessive disorder it occurs when one abnormal gene for production of hemoglobin s is inherited from one parent and one abnormal gene for production of beta thalassemia is inherited from the other parent. the genes for both hemoglobin and beta thalassemia are both located on chromosome 11. manifestation of the condition was studied in 1973 by serjeant at el [3]. clinical manifestations vary depending upon heterozygosity from being completely asymptomatic (hb s/ β+) to signs and symptoms very much akin to sickle cell anemia (hb s/ β0) [4]. case report a 29-year-old serving soldier, the resident of central india, while being employed in high altitude area complained of chronic pain abdomen, which was found to be due to massive splenomegaly (23 cm in span) with sonographic evidence of splenic infarction. approximately 7 years earlier, he had similar episode of abdomen pain for which he was worked up to rule 6 in t e r n a l m e d ic in e issn 2413-6077. ijmmr 2022 vol. 8 issue 2 any hematological disorder. he had microcytic hypochromic anemia with normal tlc and dlc. thrombocytosis was noted. there was no documented evidence of any organomegaly in the past. past hb electrophoresis reports revealed hbs – 49.7%, hb a – 0.7%, hb a2 – 3.0% and hb f – 46.7%; consistent with as sickle cell thalassemia (hb s/β+ thalassemia). his general, systemic examinations were within norm during pre-anesthetic check-up. he was taken up for the splenectomy operation and specimen sent to dept of pathology for histopathological examination. lab received a grossly enlarged specimen of splenectomy weighing 1.2 kg and having apparently normal architecture externally. serial cut section of the fresh specimen showed multiple soft yellowish areas of splenic infarction. formalin-soaked cotton were placed in between these cut surfaces and whole specimen was put in 10 % formalin for 24 hours. multiple sections from these necrosed soft areas were submitted along with normal appearing areas of spleen. h&e stains slides were studied by microscopy. histopathological examination revealed significant necrosis in infarcted area and there were multiple areas showing deposition of yellowish granular substances in splenic tissue which were congo-red negative and biref ringence negative under polarized light. sickle cells in the sinusoids were also seen. post splenectomy period was uneventful with no fever, however, there was incidental findings of different forms of plasmodium fal­ ciparum including gametocytes in the peripheral blood. thrombocytosis was also noted as expected. discussion sickle cell thalassemia, described originally as micro-drepanocytic disease by silvertroni and bianco [1], is a clinical condition where mutations in both hbs gene and thalssemia gene (commonly beta gene) occur resulting in two heterozygous states of either hb s/ β+ or hb s/ β0. heterogenicity in the beta thalassemia mutations leads to different beta globin synthesis and hence different amounts of hba are synthesized which result in variable clinical manifestations ranging from nearly asymptomatic to a severe condition similar to sickle cell anemia (homozygous hbs) [4]. laboratory investigations reveal microcytic hypochromic red cells with target cells and occasionally sickled forms. hemoglobin elec trophoresis may reveal 60-90% hb s, 0-30% hb a, 1–20% hb f and an increased hba2 level. in hb s/β+thalassemia, as in our case, variable amounts of hb a dilute hb s and consequently inhibit polymerization-induced cellular damage. increased levels of hb a are usually associated with a milder phenotype [2, 5, 6, 7]. in our case, in the pre-splenectomy period, the patient was consistently having microcytic anemia as shown in table 1 with leukopenia and increased ldh indirectly suggesting hemolysis. platelet was adequate. no malarial parasite was seen in the peripheral smear. post splenectomy, anemia improved but mild leukocytosis and thrombocytosis was evidenced as expected. ldh was found decreasing. incidentally, peripheral smear showed numerous gametocytes of plasmodium falciparum. molecular tests for confirmation of sickle cell thalassemia were unavailable in the lab. hence diagnosis was purely based on tests for sickling and hb electrophoresis. role of pbs examination and relevant histopathological sections could not be undermined. individually, both thalassemia and sickle cell trait offer protection from malarial infection, however when both occurs in the same individual protection offered by thalassemia can be reversed by another additional genetic polymorphism such as hbs mutation. the two mutations essentially cancel each other and the individual becomes susceptible to malaria [8]. heterozygotes for the sickle gene (hb genotype as) are relatively protected against death due table 1. relevant present investigations sr no. pre splenectomy post splenectomy hb (gm/dl) 6.6 10.1 mcv (fl) 68 69 tlc 2450 11220 dlc p-69%, l-23%, m-07%, e-01% p-82%, l-11%, m-04%, e-03% platelets (105/μl) 1.1 5.56 ldh (u/l) >1000 634 pbs no malarial parasites malarial parasites evidenced t. s. singh et al. 7 in t e r n a l m e d ic in e issn 2413-6077. ijmmr 2022 vol. 8 issue 2 fig. 1. gross specimen before and after fixation showing areas of infarction. fig. 2. photomicrographs from h&e sections showing deposition of yellowish fine granular to amorphous (inset) substances in spleen (gamna-gandy bodies) and sickle cells. fig. 3. photomicrographs showing numerous gametocytes and trophozoites of plasmodium falciparum and thrombocytosis. fig. 4. photomicrographs showing positive sickling with malarial gametocytes. t. s. singh et al. 8 in t e r n a l m e d ic in e issn 2413-6077. ijmmr 2022 vol. 8 issue 2 to malaria, probably through accelerated clearance by macrophages of plasmodium falciparum-infected erythrocytes and by inhibiting growth of parasites in rbcs by increasing intracellular potassium, reduced ph and increasing endothelial adherence of infected cells [9]. on the other hand, homozygous (hb ss) sickle cell anemia patients are not protected from malaria or any other infections [10,11] and, hence, there is increased chance of dying from malaria which is additionally aggravated by having dysfunctional spleen [12,13] or in the post-splenectomy period as removal of ab normal rbcs and other intracytoplasmic inclusion bodies is the primary function of spleen [14,15]. in our case, the pre-splenectomy period blood did not show any malarial parasites due to effective hyperactive enlarged spleen but once patient’s spleen was removed, numerous forms of plasmodium falciparum were seen in the blood as the individual was devoid of major protection offered by spleen including removal of infected rbcs and other inclusion bodies. people living in the malaria endemic areas have some degree of acquired immunity against malaria which may weaken over absence of recurrent exposures or when individuals moved to non-endemic areas [16]. this partial acquired immunity against malaria develops during the first 5 years of life and is dependent on the intensity of transmission, on exposure frequency and so it decreases with time if re-exposure does not occur [17]. in sickle cell disease, auto-splenectomy or functional asplenia occurs in early adulthood in homozygous state, however, in sickle beta-thalassemia the spleen may persist and remain enlarged even in adults. this splenomegaly may cause massive pooling of blood in the spleen (splenic sequestration), which leads to its enlargement and resulting in severe anemia and hence a more compelling indication for splenectomy in sickle beta-thalassemia. this reduces substantially the rate of blood trans fusion [18]. also, multiple areas in splenic parenchyma with deposition of yellow­brownish fine gra­ nular to amorphous substance with associated foreign-body giant cell reaction were found [19]. they were gamna-gandy bodies. initially described by marini as siderotic nodules in 1904, french physician charles gandy in 1907 found it in biliary cirrhosis patient. in 1921, the etiology was better described by an italian pathologist carlos gamna, who observed deposits of amorphous material composed of iron and calcium sulfate in the spleen of a patient, who died of chronic hemolytic anemia; it was named splenogranulomatosisiderotica. the name “gamna-gandy bodies” has been widely used ever since [20]. it is formed in sickle cell disease (scd) due to chronic episodes of vasoocclusion and hemolysis in the central arteriole of the white pulp with periarteriolar hemorrhages followed by mineral elements of the blood. it can be seen as either fine granular deposits in red pulp or as perivascular deposits or subcapsular deposits. ggbs can vary in size, ranging from 10 to 49 microns [21]. the ggds is not pathognomonic of sickle cell disease as it can be found in many other diseases and in several other organs. conclusion splenomegaly in a case of sickle cell thalassemia is an established association, however to such extent that infarction with deposition of gamna-gandy bodies deposition is rare indeed. loss of protective mechanism due splenectomy also predispose the individual to manifest subclinical malarial infection. our case highlighted patient’s high endurance and acquired immunity against all the conditions mentioned by virtue of his birth with inherited mutations in an endemic area of malaria. diagnosis in the era of molecular tests still count on hb electrophoresis, sickling test and peripheral blood smear. conflict of interests authors declare no conflict of interest. acknowledgements sincere thanks to all the staff of the department of pathology, 155 base hospital, tezpur, india fig. 5. hb electrophoresis at alkaline ph showing a prominent band in sdg region, hb f and faint band in hba2 (hb s – 47.7%, hb f – 30.7%, hb a – 18.5 % and hb a2 – 3.1 %). t. s. singh et al. 9 in t e r n a l m e d ic in e issn 2413-6077. ijmmr 2022 vol. 8 issue 2 author’s contributions thongam sachin singh – conceptualization, methodology, formal analysis, writing – original draft, writing – re viewing and editing; raj singh – data curation, writing – reviewing and editing; rahul pandey – investigation, formal analysis. серповидно-клітинна таласемія з масивним інфарктом селезінки та вузлами гамна-ганді в паренхімі селезінки з супутньою малярійною інфекцією у дорослого з центральної індії (клінічний випадок) t. s. singh1, r. singh2, r. pandey3 1 – 155 base hospital, tezpur, india 2 – indian naval hospital ship asvini, mumbai, india 3 – command hospital, pune, india вступ. серпоподібноклітинна анемія – це спадкова гемоглобінопатія, яка характеризується гетерозиготним станом hb s/β+ або hb s/β0 і клінічно може не проявлятися у гетерозиготних носіїв мета. метою дослідження є огляд літератури та обговорення різноманітних клінічних проявів і діагностики клінічного випадку гемоглобінопатії у пацієнта із центральної індії. методи. описано випадок гемоглобінопатії у пацієнта із центральної індії. результати. ми повідомляємо про випадок гемоглобінопатії у дорослого, який звернувся до лікарні третього рівня з хронічним болем у спині. було виявлено масивну спленомегалію з ознаками інфаркту селезінки. при загальному дослідженні селезінки виявлені множинні м’які жовтуваті тістоподібні ділянки, які при мікроскопічному дослідженні показали виражений некроз з наявністю великої кількості блідо-аморфної жовтої субстанції, тілець гамна–ганді (ggbs) і виражену реакцію гігантських клітин на чужорідне тіло в паренхімі селезінки. мазок периферичної крові після спленектомії показав тромбоцитоз і гаметоцити plasmodium falciparum. електрофорез гемоглобіну виявив підвищення hb s (49,7%), hb f – 46,7%; hb a2 – 3,0% & hb a – 0,7%, що відповідає серповидноклітинній таласемії hb s/β+ таласемії. висновки. серповидноклітинна таласемія з тривалою спленомегалією, інфарктом селезінки з тільцями гамна-ганді і супутньою малярією у клінічно стабільних пацієнтів є справді рідкісним явищем. ключові слова: серповидно-клітинна анемія; бета-таласемія; малярія; малярійний плазмодій; тільця гамна–ганді. information about the authors thongam sachin singh – assistant professor (pathology), 155 base hospital, tezpur, india https://orcid.org/ 0000­0002­5029­8066, e­mail: sachinthongam@gmail.com raj singh – associate professor (pathology), indian naval hospital ship asvini, mumbai, india https://orcid.org/0000­0001­8423­6296, e­mail: rajsingh2u@yahoo.com rahul pandey – associate professor (surgery), command hospital, pune, india https://orcid.org/ 0000­0003­2624­5113, e­mail: rahuladviksimpy@gmail.com references 1. silvestroni e, bianco i. microdrepanocitoanemia in un soggetto di razza bianca. boll a accad med roma 1944; 70:347. 2. mohanty d, colah rb, gorakshakar ac, patel rz, master dc, mahanta j, sharma sk, chaudhari u, ghosh m, das s, britt rp, singh s, ross c, jagannathan l, kaul r, shukla dk, mu thuswamy v. prevalence of β­thalassemia and other haemo­ globinopathies in six cities in india: 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2413-6077. ijmmr 2022 vol. 8 issue 2 67 d e n t is t r y doi 10.11603/ijmmr.2413-6077.2022.2.13121 fluorides and oral health s. i. boitsaniuk, *m. o. levkiv i. horbachevsky ternopil national medical university, ternopil, ukraine background. the importance of fluorides in caries prevention is discussed in the article and practical recommendations for the use of fluoride containing products are presented. objectives. the primary objective of this systematic review is to assess the evidence on the comparative effectiveness of topical fluoride therapy (tft) in the form of toothpastes, mouth rinses, gels and varnishes in prevention of dental caries in children and adolescents. methods. a systematic electronic literature search was conducted in the pubmed and web of science databases using search terms. current reports in the literature concerning means and methods of use of fluoridecontaining medications are considered. results. a large number of scientific studies, reviews based on them and meta-analyses conclude that the central element of caries prevention, on which the decrease in caries prevalence and caries experience is based, is the regular topical use of fluoride medications for oral cavity. conclusions. topical use of fluorides is a key element in successful caries prevention. keywords: caries prevention; fluoride; mechanisms of action; fluoride side effects. *corresponding author: mariana levkiv – associate professor of the department of dental therapy, i. horbachevsky ternopil national medical university, ternopil, 46002, ukraine. e-mail: levkiv@tdmu.edu.ua international journal of medicine and medical research 2022, volume 8, issue 2, p. 67-73 copyright © 2022, tnmu, all rights reserved s. i. boitsaniuk et al. introduction dental caries is the most prevalent chronic disease afflicting a significant proportion of the world population, including around 60% to 90% of school-aged children and the vast majority of adults. untreated caries causes progressive destruction of the crowns of the teeth, often accompanied by severe pain and suffering, especially in children. thus, it can result in poorer quality of life and general health [1]. prevention of caries in children and adolescents is considered a priority for dental services and is more cost­efficient than treatment [2, 3]. dental caries develops because of demineralization of tooth structure by organic acids formed by oral bacteria present in dental plaque through the anaerobic metabolism of dietary sugars. the causal role of sugars in caries is well established. most caries lesions in children’s permanent teeth progress relatively slowly, with an average lesion taking three years to progress through tooth enamel to dentine [4, 5, 6]. the dental caries process is influenced by the susceptibility of the tooth surface, the bacterial profile, the quantity and quality of saliva and the presence of fluoride, which promotes remineralization and inhibits demineralization of the tooth structure. [7]. the caries process can be affected in several ways. one of the most effective methods to prevent caries is promoting remineralization and slowing down demineralization. this can be accomplished with fluoride therapy [8, 9]. however, it should be emphasized that successful caries prevention is based on the general concept of all caries prevention strategies that have been proven to be effective. in addition to topical fluoride use, this means implementation of adequate oral hygiene to reduce cariogenic biofilm and appropriate diet to reduce the intake of low molecular weight carbohydrates, primarily sucrose. review mechanisms of action of fluorides on teeth. three principal mechanisms by which fluoride is considered to inhibit dental caries have been identified. fluoride reduces the enamel solubility in acid by pre-eruptive incorporation into the hydroxyapatite crystal. it promotes remineralization and inhibits demineralization of early carious lesions. it inhibits glycolysis, the process by which cariogenic issn 2413-6077. ijmmr 2022 vol. 8 issue 268 d e n t is t r y bacteria metabolizes fermentable carbohydrates [10]. the hydroxyapatite of tooth enamel is primarily composed of phosphate ions (po43–) and calcium ions (ca2+). in norm, there is a stable equilibrium between the calcium and phosphate ions in saliva and the crystalline hydroxyapatite that comprises 96 % of tooth enamel. when the ph drops below a critical level (approximately 5.5 for enamel, and 6.2 for dentin), it causes dissolution of tooth mineral (hydroxyapatite) in a process called demineralization. when the natural buffer capacity of saliva elevates ph, the minerals are reincorporated into the tooth through remineralization [12, 13]. under cariogenic conditions, carbohydrates are converted to acids by bacteria in the plaque biofilm. when the ph drops below 5.5, the biofilm fluid becomes undersaturated with phosphate ion and enamel dissolves to restore balance. when fluoride (f–) is present, fluora­ pa tite is incorporated into demineralized enamel and subsequent demineralization is inhibited. when fluoride is present in oral fluids (i.e., saliva), fluorapatite, rather than hydroxyapatite, is formed during the remineralization process. (ca10(po4)6(oh)2→ca10(po4)6(f)2). fluoride ions (f-) replace hydroxyl groups (oh–) in formation of the apatite crystal lattice (fig. 1). in fact, the presence of fluoride increases the rate of re­ mineralization [11]. fluoride is available from many sources divided into 3 major categories: tap water (and foods and beverages processed with fluoridated water), home administered, and professionally applied. effects of topical fluorides on oral bacteria in addition to its direct mineralizing effect on enamel, fluoride may affect oral plaque bacteria. oral plaque bacteria secrete acids onto tooth surfaces (the byproducts of carbohydrate fermentation), which initiates tooth demineralization. the entry of fluoride into the bacterial cell interferes with acid production, thus re ducing, potential enamel destruction. [14, 15, 16]. in addition, fluoride can prevent the attach­ ment of microorganisms to the surface of the tooth. this effect is demonstrated, in particular, by fluorides of tin and amine. this can reduce formation of plaque that contributes to tooth decay. streptococci that cause caries bind glucan on tooth enamel surfaces by means of glucanbinding molecules called lectins. fluoride interferes with this specific binding and thus inhibits bio­film formation by the streptococci that demineralize enamel. the fluoride ion also fig. 1. fluorapatite formation. (a) fluoride ions (f–) replace hydroxyl groups (oh–) in hydroxyapatite to form fluorapatite in the tooth enamel. (b) a portion of the apatite crystal lattice shows replacement of hydroxide for fluoride. (adapted from: posner, 1985.20). s. i. boitsaniuk et al. issn 2413-6077. ijmmr 2022 vol. 8 issue 2 69 d e n t is t r y inhibits chain formation (growth) in streptococci and effects physiological capabilities of the micro-organism to metabolize sucrose. however, these effects are manifested only in high fluo­ ride concentrations, making the fluoride an expected process for caries reduction in remineralization [17]. in concentrations of fluoride administered orally via toothpastes or rinses, the clinical effect of inhibiting bacterial metabolism is insufficient to have an appreciable effect on caries development. systemic use of fluorides the correct way to use fluorides is that the body ingests fluorides through the digestive tract and passes them through the gastrointestinal tract. the fluorides are absorbed into the blood circulation and then transferred to the tissues such as teeth and saliva to prevent caries. there are four main methods in the systemic use of fluorides, which are water fluoridation, salt fluoridation, milk fluoridation, and fluoride tablets. water fluoridation. fluorides are present naturally in all water sources. community water fluoridation is the process of adjusting the fluoride content of fluoride­deficient water to the recommended level for optimal dental health, which is currently recommended at 0.7 parts fluoride per million parts water. water fluoridation is an effective and inexpensive means of obtaining the fluoride necessary to prevent tooth decay. water fluoridation continues to be effective in reducing tooth decay by 20-40 % in children and adults, even in the era of widespread availability of fluoride from other sources, such as fluoride toothpaste [18, 19, 20]. table 1 provides some of the fluoride salts that are commonly used for a ‘systemic method’. local treatment with fluorides toothpaste with fluorides tooth brushing with fluoride toothpaste is close to an ideal public health method that is convenient, inexpensive, culturally approved, widespread and available [21, 22]. it is scientifically proven and substantiated that fluoride toothpaste has preventive effect of caries for people of all ages. fig. 2. sources of fluoride in the environment. table 1. fluoride compounds and concentrations that are usually used in different ‘systemic’ fluoride methods (source: sampaio & levy, 2011) f-methods f-compounds f-concentrations water fluoridation hydrofluorosilicate (fsa), sodium fluorosilicate, sodium fluoride 0.7-1.2 mg/l salt fluoridation potassium fluoride, sodium fluoride 250-300 mg/kg milk fluoridation sodium fluoride or disodium monofluorophosphate 5 mg/l dietary f­supplements sodium fluoride, acidulated phosphate fluoride, potassium fluoride, calcium fluoride 0.25-1.0 mg/day s. i. boitsaniuk et al. issn 2413-6077. ijmmr 2022 vol. 8 issue 270 d e n t is t r y table 2. recommended fluoride toothpastes for children from guidelines on the use of fluoride for caries prevention in children: an updated eapd policy document age (years) (mg/kg f) frequency amount (g) size first tooth-up to 2 years 1000 twice daily 0.125 grain of rice 2-6 years 1000* twice daily 0.25 pea over 6 years 1450 twice daily 0.5–1.0 up to full length of brush note. *for children, 2-6 years old, 1000+ fluoride concentrations may be considered based on the individual risk of caries. mouthwashes with fluoride a new strategy developed for toothpastes can also be used to mouthwashes. a new generation of fluoride rinses is expected to contain soluble calcium salts that help retain fluoride in the mouth to release over time. the concept of including soluble calcium in the prerinse before a fluoride rinse is proved to increase the amount of fluoride in saliva almost fivefold 1 hour after the rinse compared to a rinse containing naf at the same fluoride concentration [24, 25, 26]. over­the­counter (otc) fluoride rinse pro­ vides a lower concentration of sodium fluoride than toothpaste or varnish. the concentration is most commonly 230 mg/kg (0.05% sodium fluoride). fluoride varnish is a concentrated topical fluoride applied to the teeth that sets on contact with saliva. advantages of this modality are that it is well tolerated by infants and young children, has a prolonged therapeutic effect, and can be applied by both dental and nondental health professionals in a variety of settings. the concent ration of fluoride in varnish is 22 600 mg/kg (2.26 % fluoride ion), and the active ingredient is sodium fluoride. there are cogent clinical data that prove anti-caries effectiveness of f-containing varnish. recent studies have shown that f-containing varnish has long-term effectiveness in preventing caries. [27, 28]. the primary cariostatic effect of f-varnish is caused by the effect of fluoride on the chemical stability of the tooth mineral that converts the enamel to fluorapatite, which is much less susceptible to acid attack than the enamel. for prevention of caries of permanent teeth, it is recommended to use f-containing varnish with an interval of 6 months. for children not at risk of caries, f-varnish may not be useful; however, the risks of therapy are low and the benefits outweigh the risks [29]. fluoride varnishes are not only effective from the point of view of primary prevention of caries development, but also have a positive effect in terms of secondary prevention in cases of already existing initial caries. at this point, regular use of fluoride varnish combined with regular oral hygiene can often stop caries (preventing further mineral loss) [30]. side effects of fluorides excessive exposure to fluorides is associated with a number of health issues: � dental fluorosis. � skeletal fluorosis. � thyroid problems. � neurological problems. � other health problems. � fluoride poisoning. there is a distinction between acute and chronic effects (fig. 3). acute toxic effect as a result of swallowing a large amount of fluorine­containing oral care products, as well as by children, may cause at least a temporary manifestation of dizziness and nausea. infants and preschool children may suffer from enamel fluorosis, the risk of which increases with increased constant fluoride intake. fluorosis can develop only at the stage of enamel formation; in other age groups, this side effect from the use of fluoride is nor provoked. the fluoride concentration of over-thecounter (otc) toothpaste ranges from 1000 to 1100 mg/kg. this translates into 1 mg of fluoride in a 1­inch (1 g) strip of paste. therefore, a pea-sized amount of toothpaste containing 1000 to 1100 mg/kg fluoride would have approximately 0.25 mg of fluoride. the use of fluoride toothpaste should begin with the eruption of the first tooth. for children younger than 3 years, the recommended amount is a smear or grain of rice size (approximately 0.1 mg of fluoride) (table 2) [23]. s. i. boitsaniuk et al. issn 2413-6077. ijmmr 2022 vol. 8 issue 2 71 d e n t is t r y conclusions fluorides are regarded as effective materials for control of dental caries which can benefit both prevention and treatment. topical application of fluorides is a key element in successful caries prevention. conflict of interests authors declare no conflict of interest. authors’ contributions svitlana boitsaniuk – investigation, formal analysis, writing – original draft; mariana levkiv – formal analysis, writing – reviewing and editing, data curation. fig. 3. effects of fluoride toxicity. фториди і здоров'я порожнини рота с. і. бойцанюк, *м. о. левків тернопільський національний медичний університет імені і. я. горбачевського моз україни, тернопіль, україна. вступ. у статті обговорюється важливість застосування фтору для профілактики карієсу та представлені практичні рекомендації щодо застосування препаратів фтору. мета. основною метою цього систематичного огляду є оцінка доказів щодо порівняльної ефективності місцевої фторотерапії (мфп) у вигляді зубних паст, ополіскувачів, гелів та лаків у профілактиці карієсу зубів у дітей та підлітків. методи. здійснено систематичний електронний пошук літератури в базах даних pubmed та web of science за пошуковими термінами. розглянуто поточні повідомлення в літературі, які стосуються засобів та методів застосування фторвмісних препаратів. результат. велика кількість наукових досліджень, оглядів на їх основі та метааналізів приходять до висновку, що центральним елементом профілактики карієсу, на якому ґрунтується це зниження поширеності карієсу та досвіду розвитку карієсу, є регулярне місцеве застосування препаратів фтору в порожнині рота. висновок. місцеве застосування фторидів є ключовим елементом успішної профілактики карієсу. ключові слова: профілактика карієсу; фтор; механізм дії; побічні ефекти фтору. information about the authors svitlana i. boitsaniuk – phd, md, associate professor of the department of dental therapy, i. horbachevsky ternopil national medical university, ternopil, ukraine. https://orcid.org/0000­0001­7742­1346, e­mail: boucanuk@tdmu.edu.ua mariana о. levkiv – phd, md, associate professor of the department of dental therapy, i. horbachevsky ternopil national medical university, ternopil, ukraine. https://orcid.org/0000–0001–7327–051x, e­mail: levkiv@tdmu.edu.ua s. i. boitsaniuk et al. issn 2413-6077. ijmmr 2022 vol. 8 issue 272 d e n t is t r y references 1. sheiham a, james, w. a new understanding of the relationship between sugars, dental caries and fluoride use: implications for limits on sugars con­ sum ption. public health nutrition. 2014;17(10): 2176-84. https://doi:10.1017/s136898001400113x 2. horst ja, tanzer jm, milgrom pm. fluorides and other preventive strategies for tooth decay. dent clin north am. 2018;62(2):207­34. https://doi:10.1016/j.cden.2017.11.003 3. toumba kj, twetman s, splieth c, parnell c, van loveren c, lygidakis nα. guidelines on the use of fluoride for caries prevention in children: an updated eapd policy document. eur arch paediatr dent. 2019;20(6):507­16. https://doi:10.1007/s40368­019­00464­2 4. fontana m, carrasco-labra a, spallek h, eckert g, katz b. improving caries risk prediction modeling: a call for action. j dent res. 2020;99(11): 1215-20. https://doi:10.1177/0022034520934808 5. featherstone jd. the caries balance: the basis for caries management by risk assessment. oral health prev dent. 2004;2 suppl 1:259­64. 6. benson pe, parkin n, dyer f, millett dt, furness s, germain p. fluorides for the prevention of early tooth decay (demineralised white lesions) during fixed brace treatment. cochrane database syst rev. 2013;(12). https://doi:10.1002/14651858.cd003809.pub 7. urquhart o, tampi mp, pilcher l, et al. nonrestorative treatments for caries: systematic review and network meta­analysis. j dent res. 2019;98(1): 14-26. https://doi:10.1177/0022034518800014 8. cury ja, tenuta lm. enamel remineralization: controlling the caries disease or treating early caries lesions? braz oral res. 2009;23 suppl 1:23­30. https://doi:10.1590/s1806­83242009000500005. 9. jullien s. prophylaxis of caries with fluoride for children under five years. bmc pediatr. 2021; 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https://doi:10.1002/14651858. 27. arruda ao, senthamarai kannan r, inglehart mr, rezende ct, sohn w. effect of 5% fluoride varnish application on caries among school children in rural brazil: a randomized controlled trial. community dent oral epidemiol. 2012;40:267–76. https://doi:10.1111/j.1600­0528.2011.00656.x s. i. boitsaniuk et al. issn 2413-6077. ijmmr 2022 vol. 8 issue 2 73 d e n t is t r y 28. american dental association council on scientific affairs. professionally applied topical fluoride, evidence-based clinical recommendation. j am dent assoc. 2006;137:1151–9. 29. carey cm. focus on fluorides: update on the use of fluoride for the prevention of dental caries. j evid based dent pract. 2014;14 suppl:95­102. https://doi:10.1016/j.jebdp.2014.02.004 30. autio-gold jt, courts f. assessing the effect of fluoride varnish on early enamel carious lesions in the primary dentition. j am dent assoc. 2001; 132(9):1247­318. https://doi:10.14219/jada.archive.2001.0367 31. ullah r, zafar ms, shahani n. potential fluoride toxicity from oral medications: a review. iran j basic med sci. 2017;20(8):841­8. https://doi:10.22038/ijbms.2017.9104 received 19 august 2022; revised 12 october 2022; accepted 1 november 2022. this is open access article distributed under the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. s. i. boitsaniuk et al. 11 in t e r n a l m e d ic in e issn 2413-6077. ijmmr 2022 vol. 8 issue 1 doi 10.11603/ijmmr.2413-6077.2022.1.13156 gender differences in the influence of comorbid conditions on the long-term outcomes of patients with acute coronary syndrome *t. v. lunova, i. m. klishch i. horbachevsky ternopil national medical university, ternopil, ukraine background. gender differences in the baseline characteristics of patients with the acute coronary syndrome (acs) have been widely acknowledged. women are known to be generally older with a higher prevalence of comorbidities. objectives. gender differences in the baseline characteristics of patients with the acute coronary syndrome (acs) have been widely acknowledged. women are known to be generally older with a higher prevalence of comorbidities. at the same time, it is now yet clear which comorbid conditions have the most significant impact on the long-term outcomes of patients with acs and if there are any gender differences in this respect. methods. we performed a retrospective cohort study of 167 patients (109 men and 58 women) admitted to the acute coronary unit of ternopil municipal hospital with acs in 2016-2017. all relevant clinical information has been recorded in the pre-designed data charts. the incidence of repeated major adverse cardiovascular events (maces) has been assessed over 36 months after the hospital discharge via an e-health electronic system. results. in this cohort, female patients with acs had a higher prevalence of comorbid conditions: 15.5% of women vs. 11% of men with acs had ≥5 comorbidities, 65.5% of women and 60% of men had 2-4 comorbid conditions, and 19% of women vs. 29% ≤1 concomitant disease. the comorbidity structure also differed between genders. women more often had concomitant cerebrovascular diseases (17.2% vs. 7.3%, p=0.05), dementia (15.5% vs. 5.5 %, p=0.03), connective tissue disorders (17.25 vs. 6.42%, p=0.03) and thyroid disease (12.1% vs. 2.8%, p=0.02). men more often suffered from peptic ulcer (13.7% vs. 3.4%, p=0.05). the incidence of maces during the follow-up period rose sharply with age and was higher in females (55% vs. 33%, p=0.003). in the multivariable model, pad (or 9.5, 95% ci: 1.7-52.3, p=0.01) and thyroid disease (or 7.2, 95% ci: 1.19-43.2, p=0.03) demonstrated the most significant impact on the long-term event-free survival of females in the cohort. in turn, a solid metastatic tumor was the most significant predictor of poor prognosis in men (or 6.3, 95% ci: 2.13-18.9, p=0.001). conclusions. we observed significant gender differences in the prevalence of comorbid conditions and their influence on the three-year event-free survival of patients with acs. the predictive value of comorbidities should be further investigated, preferably, with the involvement of larger cohorts. keywords: acute coronary syndrome; gender; outcomes. *corresponding author: tetiana lunova, ph.d student, i. horbachevsky ternopil national medical university, ternopil, 46002, ukraine, e-mail: lunovatetiana@gmail.com international journal of medicine and medical research 2022, volume 8, issue 1, p. 11-17 copyright © 2022, tnmu, all rights reserved t. v. lunova et al. introduction cardiovascular disease (cvd) remains the number one cause of death of males and females all around the world with coronary heart disease (chd) representing the major proportion [1]. the common misconception about chd being predominantly “male” disease has been confronted few decades ago, and gender differences in clinical presentation and treatment strategies have been widely acknowledged. significant body of evidence suggests that females presenting with acute coronary syndrome (acs) are usually older and more often have concomitant arterial hypertension (ah) and diabetes mellitus (dm) [7]. it is also known that female patients with acs have generally worse short-term and long-term outcomes as compared with males [3-6]. studies indicate that such disparities may be attributed to older age and higher prevalence of comorbidities in female patients [5, 6]. nevertheless, it is still not well-established which comorbid conditions have the most significant impact on the long-term outcomes of men and women with acs. in this study, we analyzed existing gender differences in the prevalence of comorbid conditions in patients with acs. we also aimed at identifying which comorbidities had the most significant impact on the long­term prognosis of men and women with acs. 12 in t e r n a l m e d ic in e issn 2413-6077. ijmmr 2022 vol. 8 issue 1 methods we performed a retrospective cohort study of 167 patients admitted to the acute coronary unit of ternopil municipal hospital with acs in 2016-2017. study sample was divided into two gender groups (109 males and 58 females). relevant clinical data has been extracted from medical records and indicated in pre-designed data charts. comorbidity structure in the following cohort has been assessed drawing on charlson comorbidity index. the 3-year followup of the patients has been conducted through e-health system and in rare cases patients have been contacted by phone. the incidence of repeated major adverse cardiovascular events (maces) throughout 36 months after hospitalization has been assessed which included all-cause death, acute myocardial infarction and unstable angina. acs included st-segment elevation myocardial infarction (stemi), non-st-segment elevation myocardial infarction (nstemi) and unstable angina (ua). comorbid conditions of patients were mentioned in their medical records or diagnosed during their hospital stay according to the guideline-based diagnostic criteria. such comorbidities from charlson comorbidity index were analyzed in the study: prior acute myocardial infarction (prior ami), congestive heart failure (hf), ah, peripheral artery disease (pad), chronic kidney disease (ckd), liver disease, chronic obstructive pulmonary disease (copd), leukemia, hemiplegia, me tastatic solid tumor, aids, lymphoma, connective tissue disorders, cerebrovascular disease, dementia, dm, peptic ulcer. additionally, we analyzed the prevalence of thyroid disease and anemia in selected patients. all statistical information has been pro cessed using ibm spss statistics 22.0 software. continuous variables were expressed as mean± standard deviation (sd). numbers and percentages were used for categorical data. differences between groups were analyzed using student’s t-test for continuous data and pearson’s chi-square test for categorical variables. a p­value of <0.1 was considered significant. the evaluation of prognostic impact of particular comorbidities was performed in multivariable analysis adjusting to various confounders, such as age, cardiogenic shock, elevated cardiac troponin levels. the predictors of mace were presented as adjusted odds ratios (or). the analysis was performed separately for two gender groups. results women in the following cohort were in general 10 years older than men (69.5±21.5 vs. 59.5±28.5, p<0.001). female patients with acs more often experienced nausea, vomiting, palpitations and fatigue instead of typical chest pain. they were also more likely to be hospitalized more than 12 hours after the symptom onset (64% vs. 41%, p=0.006). men were more often smokers while women were more often obese. female patients with acs more often than male had preserved ejection fraction (55.0% vs. 38.0%, р=0.04), but at the same time, they more often developed cardiogenic shock (12.0% vs. 3.7%, р=0.04). stemi was more often diagnosed in male patients (55% vs. 36%, p=0.02), while nstemi and ua more often occurred in females (64% vs. 45%, p=0.02). we observed significant gender differences in clinical profiles of patients in the following cohort. in general, female patients with acs had higher prevalence of comorbid conditions: 15.5% of women vs. 11% of men with acs had ≥5 comorbidities, 65.5% of women and 60% of men had 2-4 comorbid conditions and 19% of women vs. 29% ≤1 concomitant disease (table 1). in the following cohort of patients with acs, women more often had concomitant cerebrovascular diseases (17.2% vs. 7.3%, p=0.05), dementia (15.5% vs. 5.5 %, p=0.03) and connective tissue disorders (17.25 vs. 6.42%, p=0.03). thyroid disease was also more often diagnosed in female patients (12.1% vs. 2.8%, p=0.02). men more often suffered from peptic ulcer (13.7% vs. 3.4%, p=0.05). we did not obser ve statistically significant gender differences in the prevalence of arterial hypertension (ah), peripheral artery disease (pad), prior myocardial infarction (prior ami), chronic kidney disease (ckd), liver disease and copd in the selected cohort. diabetes mellitus was more often found in female patable 1. gender differences in the prevalence of comorbid conditions number of comorbidities 1 group (men) 2 group (women) ≤1 32 (29%) 11 (19%) 2-4 66 (60%) 38 (65.5%) ≥5 12 (11%) 9 (15.5%) t. v. lunova et al. 13 in t e r n a l m e d ic in e issn 2413-6077. ijmmr 2022 vol. 8 issue 1 tients (34.5% vs. 25%), but the difference was not considered significant (p=0.1). gender dif­ ferences in the comorbidity structure are demonstrated in the table 2. the incidence of maces during the followup period was significantly higher in females than in males (55% vs. 33%, p=0.003). the probability of adverse outcomes rose sharply with age. among female patients with acs aged 65 and older, the cumulative maces rate was significantly higher than in younger women (66% vs. 30%). similarly, 27% of men younger than 65 years old experienced recurrent maces within the following 3 years after hospital discharge and 44% of men, aged 65 and older. kaplan-meier analysis identified significant gender differences in the 3-year event-free survival of patients older than 65 years old and no sex differences in the prognosis of younger patients (figure 1, 2). table 2. gender differences in the comorbidity structure comorbid condition 1 group (men)n=109 2 group (women) n=58 p-value prior acute myocardial infarction n=32 (29%) n=18 (31%) p=0.8 congestive heart failure n=16 (15%) n=10 (17%) p=0.5 peripheral artery disease n=11 (10%) n=5 (9%) p=0.9 dementia n=6 (5.5%) n=9 (15.5%) p=0.03 diabetes mellitus n=27 (25%) n=20 (34.5%) p=0.1 cerebrovascular disease n=8 (7%) n=10 (17%) p=0.05 copd n=6 (5.5%) n=3 (5%) p=0.9 connective tissue disorders n=7 (6%) n=10 (17%) p=0.03 peptic ulcer n=15 (14%) n=2 (3%) p=0.05 chronic kidney disease n=17 (16%) n=8 (14%) p=0.7 hemiplegia n=0 (0%) n=0 (0%) – leukemia n=1 (1%) n=0 (0%) – lymphoma n=0 (0%) n=0 (0%) – metastatic solid tumor 6 (5.5%) n=1 (2%) p=0.2 liver disease n=26 (24%) n=14 (24%) p=0.9 aids n=0 (0%) n=0 (0%) – arterial hypertension n=89 (82%) n=53 (91%) p=0.1 other comorbidities thyroid disease n=3 (3%) n=7 (12%) p=0.02 anemia n=13 (12%) n=7 (12%) p=0.2 time (months) men women three-year event-free survival of patients with acs younger than 65 years old fig. 1. event-free survival (36 months) of patients older than 65 years. t. v. lunova et al. 14 in t e r n a l m e d ic in e issn 2413-6077. ijmmr 2022 vol. 8 issue 1 multivariable analysis identified 7 comorbid conditions that most significantly influenced the prognosis in men (table 3). according to obtained results, the greatest negative impact on the event-free survival of male patients in this cohort had metastatic solid tumor (or 6.3, 95% ci: 2.13­18.9, p=0.001). cerebrovascular disease, peptic ulcer, dementia and ckd were associated with more than twice higher chances of maces in the future while pad and prior mi with 1.5 times higher probability of maces. in the multivariable model, pad (or 9.5, 95% ci: 1.7­52.3, p=0.01) and thyroid disease (or 7.2, 95% ci: 1.19­43.2, p=0.03) demonstrated the most significant impact on the long­term event-free survival of females in this cohort. congestive hf (or 2.92, 95% ci: 0.6­14.2, p=0.06), prior mi (or 1.86, 95% ci: 0.6­5.7, p=0.075) and ah (or 2.3, 95% ci: 0.4­12.0, p=0.06) also negatively affected the prognosis of women with acs. higher incidence of recurrent maces was found in females with acs and gynecological diseases, such as endometriosis, polycystic ovary syndrome, dyshormonal states of the reproductive system, uterine extirpation, etc. (or 5.4, 95% ci: 0.9­10.3, p=0.04), (table 4). discussion this article provides an additional insight into gender-related differences in the prevalence of comorbidity in patients with acs as well as the prognostic impact of comorbid conditions men women time (months) three-year event-free survival of patients with acs older than 65 years old fig. 2. event-free survival (36 months) of patients younger than 65 years. table 3. multivariable analysis of the prognostic impact of comorbid conditions in men with acs variables 95% ci se p-value or lower upper age 0.017 0.510 1.011 0.979 1.045 elevated cardiac troponin levels* 0.380 0.849 1.075 0.511 2.264 ectopic rhythm 0.493 0.563 0.752 0.286 1.975 cardiogenic shock 0.045 0.501 1.054 0.453 1.843 prior mi 0.424 0.090 1.403 0.612 3.219 congestive hf 0.577 0.740 0.826 0.267 2.558 pad 0.580 0.084 1.901 0.610 5.925 dementia 0.948 0.096 2.591 0.404 16.606 dm 0.550 0.077 0.781 0.129 1.110 cerebrovascular disease 0.951 0.098 2.555 0.396 16.475 peptic ulcer 0.496 0.065 2.493 0.943 6.585 ckd 0.533 0.083 2.088 0.734 5.935 metastatic tumor 0.556 0.001 6.343 2.132 18.868 t. v. lunova et al. 15 in t e r n a l m e d ic in e issn 2413-6077. ijmmr 2022 vol. 8 issue 1 in male and female patients. according to the results obtained, female patients with acs more often experienced nausea, vomiting, palpitations and fatigue instead of typical chest pain. they were also more likely than men to be hospitalized >12 hours after the symptom onset. in the following cohort, stemi was more often diagnosed in men and nstemi or ua more often occurred in women. a substantial body of evidence suggests that female patients with acs are usually older and have more comorbid conditions, such as diabetes mellitus (dm) and arterial hypertension (ah) [2, 7-10]. at the same time, limited number of scientific articles analyzes the prevalence of other comorbidities. in accordance with previous studies, women in the following cohort were also older and had generally worse risk profiles. nevertheless, we did not observe sig­ nificant gender differences in the prevalence of dm and ah in our study. instead, women more often than men had concomitant cerebrovascular diseases, dementia, connective tissue disorders and thyroid disease. in turn, men more often suffered from peptic ulcer (13.7% vs. 3.4%, p=.05). female patients with acs showed significantly higher 3­year cumulative maces rates, as compared with their male counterparts. nevertheless, such gender difference was mainly attributed to the higher incidence of recurrent events in older patients (>65 years old). kaplan–meier analysis did not identify any significant gender differences in the prognosis of younger patients (< 65 years old). the above mentioned results reflect the general trend in the outcomes of women with acs. it is widely acknowledged that premenopausal women are generally protected with the anti-atherogenic and vasoprotective effects of estrogen [11, 12]. the situation changes dramatically when females reach the menopause. in such women, we observe not only a sharp increase in the in cidence of de novo cvd cases, but also a worse overall prognosis after acute ischemic events [13]. according to the results of numerous studies, female patients with acs have generally worse unadjusted short-term and long-term mortality rates after acs. nevertheless, after adjustment for various factors, the gender diffe rence was attenuated [3-6, 14]. scientists suggest that the mortality is most likely attributed to the older age and higher prevalence of comorbidity in female patients [5, 6]. in this study, we analyzed the prognostic impact of comorbid conditions, which were included to charlson comorbidity index. we also inves tigated the prognosis of patients with concomitant thyroid disease and the prognosis of females with gynecological diseases. the greatest impact on the 3-year eventfree survival of male patients with acs in this cohort demonstrated solid metastatic tumor. this comorbid condition was associated with six-fold risk of maces during the follow-up period. men with acs and concomitant cerebrovascular disease, peptic ulcer, dementia and ckd had more than twice higher chances of maces as compared with men with no such conditions. finally, pad and prior mi increased the probability of recurrent maces in 1.5 times in male patients with acs. the influence of other comorbidities on the long-term prognosis of men in this cohort was less significant. pad and thyroid disease demonstrated the most significant impact on the long­term event­ free survival of women with acs in this cohort. females with gynecological diseases also had significantly higher incidence of recurrent maces within 3 years after acs as compared with females with no history of gynecological issues. congestive hf, prior mi and ah also negatively affected the prognosis of female table 4. multivariable analysis of the prognostic impact of comorbid conditions in women with acs factors 95% ci se p-value or lower upper age 0.034 0.001 1.22 1.050 1.198 prior mi 0.574 0.075 1.860 0.604 5.724 congestive hf 0.805 0.060 2.925 0.604 14.166 pad 0.870 0.010 9.498 1.725 52.303 liver disease 0.634 0.880 0.909 0.262 3.149 ah 0.843 0.063 2.301 0.441 12.010 thyroid disease 0.916 0.031 7.188 1.194 43.253 gynecological diseases 0.675 0.044 5.412 0.895 10.342 t. v. lunova et al. 16 in t e r n a l m e d ic in e issn 2413-6077. ijmmr 2022 vol. 8 issue 1 patients with acs in this cohort. the prognostic impact of other comorbid conditions was not considered statistically significant. conclusions we observed significant gender differences in the clinical profiles and the prevalence of comorbidity in patients with acs. the cumulative incidence of 3-year maces was higher in women than in men. at the same time, statistically significant gender difference in the long­term outcomes has been observed exceptionally in the cohort of older patients. the influence of comorbid conditions on the event-free survival of patients with acs differed between genders. the predictive value of comorbidities should be further investigated, preferably, with an involvement of larger cohorts. limitations first of all, this is a retrospective study and the cohort of patients involved is relatively small. that is why we deem it reasonable to conduct further research on this subject with an involvement of larger patient samples. second, the selected patients never (or very rarely) developed leukemia, aids, hemiplegia and lymphoma; hence, it was impossible to investigate the influence of these conditions on the long-term prognosis in the following cohort. third, we did not have a chance to observe the 3-year event-free survival in female patients with diagnosed metastatic tumor due to a small number of cases. conflict of interests authors declare no conflict of interest. author’s contributions tetiana lunova – conceptualization, me­ thodology, formal analysis, writing – original draft, writing – reviewing and editing; ivan klishch – data curation, writing – reviewing and editing. гендерні відмінності впливу коморбідних станів на віддалені наслідки у пацієнтів з гострим коронарним синдромом *т. в. луньова, і. м. кліщ тернопільський національний медичний університет імені і. я. горбачевського моз україни, тернопіль, україна вступ. вплив статі на клінічний перебіг гострого коронарного синдрому (гкс) у пацієнтів є широко відомим. жінки з таким діагнозом зазвичай старші та мають більше супутніх захворювань. мета. у даній роботі ми мали на меті дослідити, які фактори впливають на довготривалий прогноз та відділені наслідки у пацієнтів з гкс. методи. ретроспективне когортне дослідження включало 167 пацієнтів (58 жінок та 109 чоловіків), які проходили лікування на базі тернопільської міської комунальної лікарні № 2 у 2016-2017 рр. з діагнозом гкс. уся інформація була записана у спеціально створені карти пацієнтів. частоту повторних серцевосудинних подій оцінювали протягом 36-ти місяців після виписки зі стаціонару. результати. жінки у цій когорті були старшими та мали вищу частоту супутніх захворювань: 15,5% жінок та 11% чоловіків з гкс мали ≥5 коморбідностей, 65,5% жінок і 60% чоловіків мали 2-4 коморбідності, і 19% жінок та 29% чоловіків ≤1 коморбідне захворювання. жінки частіше мали супутні цереброваскулярні захворювання (17,2% проти 7,3%, p=0,05), деменцію (15,5% проти 5,5 %, p=0,03), хвороби сполучної тканини (17,25% проти 6,42%, p=0,03) і захворювання щитоподібної залози (12,1% проти 2,8%, p=0,02). чоловіки частіше мали виразкову хворобу (13,7% проти 3,4%, p=0,05). частота повторних серцево-судинних подій була вищою у жінок (55% проти 33%, p=0,003). хвороба периферичних артерій (or 9,5, 95% ci: 1,7-52,3, p=0,01) хвороби щитоподібної залози (or 7,2, 95% ci: 1,19-43,2, p=0,03) продемонстрували найбільший вплив на прогноз у жінок. онкологічні захворювання мали найбільших вплив на виживання у чоловіків (or 6,3, 95% ci: 2,13-18,9, p=0,001). висновки. ми спостерігали суттєві гендерні відмінності у структурі коморбідності та вільному від подій виживанні пацієнтів з гкс. доцільно провести подальші дослідження із залученням більших когорт пацієнтів. ключові слова: гострий коронарний синдром; стать; 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https://doi.org/10.1007/bf00226405 13. lunova t, levytska l, kucher s, shatskyi v, habor h, klishch i. observation of serious adverse cardiovascular events over 3 years in patients with advanced atherosclerosis: is there a gender diffe­ rence? pol merkur lekarski. 2021;49(291):171­5. 14. stähli be, wischnewsky mb, jakob p, et al. gender and age differences in outcomes of patients with acute coronary syndromes referred for coronary angiography. catheter cardiovasc interv. 2019; 93(1):16­24. https://doi.org/10.1002/ccd.27712 received 10 june 2022; revised 13 june 2022; accepted 14 june 2022. this is open access article distributed under the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. t. v. lunova et al. koncor1_2014.pm6 54 b io m e d ic a l s c ie n c e s ijmmr 2015 vol. 1 no. 1 nitric oxide and allyl alcohol induced hepatotoxicity m. m. korda i. ya. horbachevsky ternopil state medical university, ternopil, ukraine background. nitric oxide (no) is an important mediator of hepatotoxicity. no in liver can be derived from two sources: (1) constitutive no synthase (enos) in endothelial cells, and (2) inducible no synthase (inos) in hepatocytes and kupffer cells. objectives. the present study was aimed to examine the effect of nonselective nos inhibitor (l-name) and selective inos inhibitor (1400w) on the development of allyl alcohol (aa) induced hepatitis in rats. methods. male wistar rats were treated with intraperitoneal injection of saline or aa and l-name or 1400w. no in liver was measured by electrochemical method after enos stimulation by calcium ionophore. total nos activity and nitrite/nitrate content were measured in liver and blood serum. the activity of free radical oxidation in liver was measured by chemiluminescent method. alanine aminotransferase (alt) and aspartate aminotransferase (ast) activities were assayed in blood serum results. aa increased the activity of free radical processes in liver and markers of cytolysis in serum, as well as decreased enos and increased inos activities. l-name considerably inhibited enos and augmented the necrosogenic properties of aa, whereas 1400w partially prevented liver damage. conclusion. it has been concluded that in aa intoxication no produced from enos is beneficial to the liver, but no derived from the upregulated inos has deleterious effect. key words: nitric oxide, toxic hepatitis, nos inhibitors. address for correspondence: mykhaylo korda, medical biochemistry department, i. ya. horbachevsky ternopil state medical university, m. voli, 1, ternopil, 46001, ukraine tel.: +380963952764; fax: +380352253998; e-mail: kordamm@yahoo.com introduction allyl alcohol (aa) is a well known hepatotoxin widely used in animal experiments to induce liver necrosis. aa in hepatocytes is oxidized to acrolein by alcohol dehydrogenase [1]. acrolein is a potent electrophile being able to attack nucleophilic molecules, e.g. sulfhydryl groups of reduced glutathione. the depletion of reduced glutathione affects the glutathione peroxidase antioxidant mechanisms and results in the activation of free radical oxidation. it has been suggested that the oxidative stress is the key event leading to liver necrosis in aa intoxication [2]. we used the aa model of hepatocyte cell damage in order to investigate the role of nitric oxide (no) in the pathogenesis of necrotic liver injury. nitric oxide (no) is an important mediator of hepatotoxicity, and changes in its generation are paradoxically implicated in both cytoprotection and cytotoxicity [3]. no in liver can be derived from two sources. hepatocytes and kupffer cells contain inducible no synthase (inos), activity of which is markedly increased in inflammation. endothelial cells contain constitutive no synthase (enos). no produced by enos plays important role in liver microcirculation. it has been shown that aminoguanidine, a relatively selective inhibitor of inos, protected against acetaminopheninduced hepatotoxicity [4], as well as thioacetamideinduced hepatotoxicity [5]. another study concluded that inos plays an important role in liver cirrhosis following ccl 4 intoxication to rats [6]. recently, we have demonstrated that n-(3-(aminomethyl)benzyl)acetamidine (1400w), a strongly selective inhibitor of inos, prevented the depletion of reduced glutathione level in liver injured by allyl alcohol (aa) [7]. at the same time, in acute hepatitis caused by thioacetamide, methyl ester of n-nitro-l-arginine (l-name), the nonselective inhibitor of nos, increased liver damage [8]. the present work was carried out to study the effects of nonselective (l-name) and selective inos (1400w) inhibitors on the formation of no in liver in aa intoxication, as well as to investigate the correlation between no production and severity of the liver damage. methods animals and treatment male wistar rats weighing 250-300 g were used in experiment. the animals were maintained under control conditions (23 °c, 12-hour light-dark cycle) with water and standard laboratory chow being available ad libitum. all rats were randomly assigned to four groups of ten animals each. rats from the first group were treated with intraperitoneal (i.p.) injection of saline and served as a control. aa (30 mg/kg) was diluted in saline and administered one time intraperitoneally into rats of the second, third and m. m. korda international journal of medicine and medical research 2015, volume 1, number 1, p. 54-57 copyright © 2015, tsmu, all rights reserved 55 b io m e d ic a l s c ie n c e s ijmmr 2015 vol. 1 no. 1 fourth groups. third and fourth groups animals received correspondingly l-name (10 mg/kg, i.p.) and 1400w (1.5 mg/kg, i.p.) 30 min prior to aa injection. on the next day of experiment rats were anesthetized with a mixture of 100 mg/kg ketamine and 10 mg/kg xylazine and liver was dissected to carry out the studies described below. blood was also taken for the analysis. all procedures were in compliance with national and international laws and guidelines for the use of animals in biomedical research [9]. measurement of no in liver the measurement of no was performed with highly sensitive electrochemical nanosensor which was prepared as previously described [10]. briefly, a carbon fiber (tip diameter about 500 nm) was covered with polymeric film of nickel (ii) tetrakis (3-methoxy-4-hydroxy-phenyl) porphyrin. measurements were performed using a three-electrode system that consisted of a nanosensor as the working electrode, a platinum counter electrode, and a silver-silver chloride reference electrode. liver tissue was cut into thin pieces of 5–6 mm in length using a razor blade and slices were placed on the bottom of thermostated organ chamber filled with hank’s solution. counter and reference electrodes were positioned in the chamber near the liver slice and the active tip of the nanosensor was lowered near (2–4 �m) the surface of liver tissue with the aid of micromanipulator. to stimulate the release of no, 10 �l of enos receptor independent agonist calcium ionophore a23187 was injected with a nanoinjector onto the surface of liver slice to reach a final concentration in the medium of 1 �mol/l. the changes in no concentration from the background level were monitored with time (amperometry) with a computer-based gamry vfp600 multichannel potentiostat (detection limit of 1 nmol/ l and resolution time <50 ms). no concentration was calculated by means of a calibration curve. measurement of the total nos activity in liver and nitrite/nitrate content in liver and blood serum the activity of no generating system was assayed in 40 mm tris hcl buffer, ph 8, containing liver homogenate (1 mg of protein/ml), 4 mm fad, 4 mm h 4 biopterin, 3 mm dithiothreitol, and 1 mm l-arginine. the reaction was initiated by adding nadph (2 mm) and run for 3 h at 37 °c [11]. the combined concentration of nitrite and nitrate, which accumulate quantitatively as the stable oxidation products of no, was determined as described below. measurement of the nitrite/nitrate concentration the combined nitrite/nitrate concentration in the no generating system as well as in liver homogenate and blood serum was assayed after the reduction of nitrite to nitrate by cadmium using improved griess method with the help of commercially available quantichromtm nitric oxide assay kit (bioassay systems). free radical oxidation assay the activity of free radical oxidation in liver homogenate was measured by chemiluminescent method [12]. evaluation of hepatotoxicity serum alanine aminotransferase (alt) and aspartate aminotransferase (ast) activities were assayed as the markers of hepatocellular death using commercially available kits (sigma-aldrich). statistical analysis values are expressed as mean±sem from 4 to 6 experiments. values p<0.05 are considered as statistically significant. statistical analysis was performed using anova followed by the student t test. results the release of no was measured ex-vivo using electrochemical nanosensors placed on the surface of the liver tissue. we stimulated no release by a calcium ionophore, as it causes a large increase in intracellular calcium, rapid enos activation and maximal receptor-independent no production. so far as, inos is not sensitive to ca2+, we can assume that the amount of no measured by this method directly reflects the functional state of the enos located in the hepatic sinusoidal endothelial cells. the maximum or peak concentration of released no from the liver of control rats was 160.0±12.2 nmol/l (figure 1). by contrast, stimulation of liver from the aa injured animals resulted in an approximately twofold reduction in the amount of no release. administration of the nonselective nos inhibitor � �� �� �� �� ��� ��� ��� ��� ��� � � � � ���������� ��� ��� �������� � � �� � � � � fig. 1. the effect of l-name and 1400w on calcium ionophore (a23187, 1 mmol/l) stimulated nitric oxide release from liver of aa-treated rats. rats were given aa (30 mg/kg, i.p.) with or without the administration of l-name (10 mg/kg, i.p.) or 1400w (1.5 mg/kg, i.p.) 30 min before aa. * — significantly different from control, p<0.05; # — significantly different from aa alone, p<0.05. values are mean±sem of 5–6 rats. m. m. korda 56 b io m e d ic a l s c ie n c e s ijmmr 2015 vol. 1 no. 1 fig. 2. the effect of l-name and 1400w on the total nos activity in liver of aa-treated rats. rats were given aa (30 mg/kg, i.p.) with or without the administration of l-name (10 mg/kg, i.p.) or 1400w (1.5 mg/kg, i.p.) 30 min before aa. * — significantly different from control, p<0.05; # — significantly different from aa alone, p<0.05. values are mean±sem of 6–8 rats. ��� ��� ��� ��� ��� ��� ��� ��� ��� � �� � ����������� ��� ���������� � � � �� � � � � � � � � � � � � �� � �� �� � �� �� �� �� �� �� � � fig. 3. the effect of l-name and 1400w on the combined nitrite/nitrate content in blood serum and liver of aa-treated rats. rats were given aa (30 mg/kg, i.p.) with or without the administration of l-name (10 mg/kg, i.p.) or 1400w (1.5 mg/kg, i.p.) 30 min before aa. * — significantly different from control, p<0.05; # — significantly different from aa alone, p<0.05. values are mean±sem of 6–8 rats. � � � � � � � �� �� �� �� �� � � � � � � � � � � � � � � � � � � � � � � � � � � � �� � � � � � � � � � � � � � � � � � � � � �� � ��� ����� �� �� ��� � � � � � � � �� � � �� � � � � � � � � �� � � �� � � � fig. 4. the effect of l-name and 1400w on the chemiluminescence intensity of aa-treated rats liver homogenate. rats were given aa (30 mg/kg, i.p.) with or without the administration of l-name (10 mg/kg, i.p.) or 1400w (1.5 mg/kg, i.p.) 30 min before aa. * — significantly different from control, p<0.05; # — significantly different from aa alone, p<0.05. values are mean±sem of 6–8 rats. � �� �� �� �� �� �� �� � � � � � � � � � � � � � � � � � � � �� � � � � � � � fig. 5. the effect of l-name and 1400w on alt and ast activities in blood serum of aa-treated rats. rats were given aa (30 mg/kg, i.p.) with or without the administration of l-name (10 mg/kg, i.p.) or 1400w (1.5 mg/kg, i.p.) 30 min before aa. * — significantly different from control, p<0.05; # — significantly different from aa alone, p<0.05. values are mean±sem of 6–8 rats. � ��� ��� ��� ��� ���� ���� � � � � � � � � � � �� � � � � � � � � � � � � � � � � � �� � � � � � � � � � � � � � � � � � � � � � � � �� � � � ��� ��� l-name markedly decreased the peak no release (more than in five times, as compared to control), whereas inos inhibitor 1400w did not change the no production significantly. in contrast to the calcium ionophore stimulated no yield, the activity of no generating system was about 1.5-fold increased after intoxication, thereby suggesting that aa liver damage is accompanied by the considerable inos induction (figure 2). both l-name and 1400w significantly diminished the total nos activity. serum and liver nitrite/nitrate concentrations correlated well with total nos activity and were significantly increased 24 hours after aa administration (figure 3). both inhibitors effectively prevented the increase in nitrite/nitrate level. the activity of free radical oxidation evaluated by the intensity of spontaneous chemiluminescence of the liver homogenate was almost three-fold higher in the poisoned animals in comparison with control (figure 4). it is worth noting that 1400w significantly inhibited the free radical processes in hepatocytes, whereas l-name administration resulted in the increase of the chemiluminescence intensity. m. m. korda 57 b io m e d ic a l s c ie n c e s ijmmr 2015 vol. 1 no. 1 the activities of serum alt and ast indicate the presence of hepatocyte cytolysis in animals treated with aa. these markers were especially elevated in the group administered with aa and l-name, which suggests significant damage of hepatocyte membranes in these rats (figure 5). inos inhibitor partially decreased alt and ast activity, as compared to the animals treated with aa alone. discussion it is generally accepted that no is an important regulator in the hepatotoxicity of many chemicals [4, 5, 6]. however, the role of no in the development of liver necrosis is still not fully known and demands further studies. no is reported to be produced in liver by both enos (endothelial cells) and inos (parenchymal and kupffer cells). previously we have shown that necrosogenic poison aa sharply decreases the reduced glutathione content in hepatocytes but inhibition of inos partially prevents this effect [7]. in this study we demonstrate that aa rapidly upregulates inos in liver. depletion of the glutathione may weaken cellular antioxidant defense to such a point that the no produced by inos may cause tissue injury. this suggestion is confirmed by our observation. the activity of inos as well as the intensity of free radical processes in liver tissue and the markers of cytolysis in serum (alt and ast), which were markedly increased after aa injection, went down significantly in animals treated with the strongly selective inos inhibitor 1400w. these results distinctly show that the upregulation of inos in macrophages and hepatocytes following by the activation of free radical reactions in liver cells may be at least one of the aa necrosogenic effect mechanisms. we also demonstrate here that in contrast to inos, hepatic enos is significantly inhibited in liver necrosis. this effect of aa, like the activation of inos, can also be the reason of the necrotic liver damage. after administration of the nonselective nos inhibitor l-name we observed the deep depression of enos activity simultaneously with the considerable raise of the chemiluminescence intensity in liver and cytolytic markers activity in serum. this phenomenon can be be explained by the role of no derived from enos in maintaining perfusion, and preventing hypoxia, platelet adhesion and thrombosis in liver capillaries. conclusion in the aa intoxication no produced from enos is clearly beneficial to the liver, whereas the inducible no production has an opposite effects. further research should provide a solid basis for therapeutic approaches to either supplement no to the liver for its protective effect or suppress inos to prevent liver damage. references 1. belinsky sa, bradford bu, forman dt, glassman eb, felder mr, thurman rg. hepatotoxicity due to allyl alcohol in deermice depends on alcohol dehydrogenase. hepatology 1985; 5: 1179–1182. 2. pompella a, romani a, benedetti a, comporti m. loss of membrane protein thiols and lipid peroxidation in allyl alcohol hepatotoxicity. biochem pharmacol 1991; 41: 1255–1259. 3. wang y, vodovotz y, kim pk, zamora r, billiar tr. mechanisms of hepatoprotection by nitric oxide. ann n y acad sci 2002; 962: 415–422. 4. gardner cr, heck de, yang cs, thomas pe, zhang xj, degeorge gl, laskin jd, laskin dl. role of nitric oxide in acetaminophen-induced hepatotoxicity in rat. hepatology 1998; 27: 748–754. 5. diez-fernandez c, sanz n, alvarez am, zaragoza a, cascales m. influence of aminoguanidine on parameters of liver injury and regenaration induced in rats by a necrogenic dose of thioacetamide. br j pharmacol 1998; 125: 102–108. 6. ortiz mc, fortepiani la, martinez c, atucha nm, gracia-estan j. renal and pressure effects of aminoguanidine in cirrhotic rats with ascites. j am soc nephrol 1996; 7: 2694–2699. 7. korda mm, yaroshenko tya. the effect of inos inhibitor n-(3-(aminomethyl)benzyl) acetamidine on the hepatotoxicity of allyl alcohol. med chem 2004; 6: 114–117. 8. chu cj, wang ss, lee fy, chang fy, lin hc, hou mc, chan cc. detrimental effects of nitric oxide inhibition on hepatic encephalopathy in rats with thioacetamide-induced hepatic failure. eur j clin invest 2001; 31: 156–163. 9. giles ar. guidelines for the use of animals in biomedical research. thromb res 1987; 58: 1078–1084. 10. malinski t, taha z. nitric oxide release from a single cell measured in situ by a porphyrinic based microsensor. nature 1992; 358: 676–678. 11. stuehr d, kwon ns, nathan c, griffiths o. nw-hydroxy-l-arginine is an intermediate in the biosynthesis of nitric oxide from l-arginine. j biol chem 1991; 266: 6259–6263. 12. korda mm. lipid peroxidation is a prerequisite for galactosamine-induced damage. curr. topics in biophys 2000; 24(2): 83–87. received: 2014.06.01 m. m. korda 23 p u b l ic h e a lt h a n d e p id e m io l o g y issn 2413-6077. ijmmr 2016 vol. 2 issue 1 k. barański, j. e. zejda doi 10.11603/ijmmr.2413-6077.2016.1.6375 supply of antihypertensive drugs and cardiovascular mortality in poland in 2000–2010 k. barański, j. e. zejda medical university of silesia, katowice, poland medical school in katowice, katowice, poland background and objective. in poland, the sale of antihypertensive drugs has significantly increased since 2000. according to that fact, the aim of our study was to determine if the increased use of antihypertensive drugs correlates with the decreasing mortality due to cardiovascular diseases (cvd) including hypertension (ht). methods. the analysis is based on data on annual national sales (million units) of four types of antihypertensive drugs in 2000–2010. for the same period standardized mortality rates were calculated based on the data available from the central statistical office in poland. data analysis involved correlation analysis between annual mortality rates due cvd and ht and country-wide annual sales of antihypertensive drugs (2000–2010). results. in the period 2000–2010, standardized mortality rates of cvd in the whole population followed a decreasing trend. analysis of correlation of cvd with specific drug provided the following findings: diuretics (r=-0.97; p<0.0001) beta-blockers (r=-1.0; p<0.0001) renin-angiotensin system (ras) inhibitors (r=-0.72 p=0.01) calcium-channel blockers (r=-0.82; p=0.001) standardized mortality rates for the ht showed fluctuating trend. correlations of that mortality with global sale of these drugs were no longer negative: r=0.54; p=0.08, r=0.56; p=0.08 r=0.55; p=0.07; r=0.63; p=0.03, respectively. conclusions. in poland, in 2000–2010, an improved access to pharmacological control of ht was associated with an apparent reduction in mortality from cvd but not from ht. the latter findings might reflect imprecise definition of ht as a cause of death or the fact that ht leads to other cardiologic events usually reported as a cause of death. key words: antihypertensive drug therapy, cardiovascular disease, hypertension. introduction standardized mortality rates caused by cardiovascular diseases (cvd) have been de creasing in developed countries since 1980 [1]. in poland, in the last 20 years, cvd mortality decreased twice and the dynamics was strong between 1990 and 2000 [2]. many hypotheses have addressed the issue, including changes of lifestyle and diet, improved access to modern medical technologies [3]. another potential factor could be related to better pharmacological control of hypertension (ht) [4]. such an explanation can be explored in ecological manner through analysis of correlation between the nation-wide sale of hypertensive drugs and cvd/ht mortality, over a period of time. the major classes of antihypertensive drugs include beta-blockers (bb), renin-angiotensin system inhibitors (res), diuretics (d) and calcium channel blockers (ccb). clinical studies confirm that those drugs used in the treatment of ht are effective in preventing heart attack and stroke [5]. less is known about populationbased impact of the increased supply of the above drugs on ultimate outcome of ht, such as cvd mortality. in poland, increased sales of and access to modern antihypertensive drugs has occurred over the two last decades and annual whole-country sales are available for a recent period (2000–2010), thus making such analysis possible. the principal aim of our study was to determine if the increased use of antihypertensive drugs correlates with the decreasing mortality due to cardiovascular diseases (cvd) including hypertension (ht), in general population of poland. the secondary aim of the study was to explore the hypothesized correlation in three age groups: 0–44 years, 45–64 years, above 64 years. materials and methods the analysis is based on data on annual sales (million units) of four types of antihy pertensive drugs in 2000–2010: beta-blockers (bb), renin-angiotensin system inhibitors (res), address for correspondence: kamil barański, department of epidemiology, medical school in katowice, medyków 18th street, katowice, 40-752 tel.: +48 32 208 85 38 e-mail: kbaranski@sum.edu.pl international journal of medicine and medical research 2016, volume 2, issue 1, p. 23–27 copyright © 2016, tsmu, all rights reserved 24 p u b l ic h e a lt h a n d e p id e m io l o g y issn 2413-6077. ijmmr 2016 vol. 2 issue 1 diuretics (d) and calcium channel blockers (ccb). annual standardized mortality rates due to cvd and ht were calculated from raw data provided by the reports of the central statistical office in poland, using direct method (reference population: who european standard population [6]. correlation analysis of cvd and hr with drug sales (spearman method) was performed for the entire population and for three age groups (0 to 44, 45 to 64 and over 64 years of age). statistical significance was assumed at p=0.05. all calculations were performed in sas software (version 9.20, sas institute, cary, n.c.). results standardized mortality rates due to cvd (n/100 000) in polish population between 20002010 were: 439, 428, 412, 412, 391, 377, 365, 358, 350, 350, 331, respectively. in the same period the annual sales of all antihypertensive drugs (mln units globally) were: 100, 97, 96, 100, 105, 117, 123, 134, 143, 154, 158. figure 1 shows annual antihypertensive drugs supply in each defined category and annual mortality rates due to cvd, over the same period. correlation analysis of cvd mortality with the defined classes of drugs showed negative and statistically significant coefficients: bb (r=-1.0 p<0.001), res (r=-0.72 p=0.01), d (r=-0.97; p<0.001), ccb (r=-0.82 p=0.001). additional analyses involving stratification for age showed larger coefficients of correlation in older segments of the population (table 1). standardized mortality rates due to ht (n/100 000) in polish population between 2000– 2010 were: 11,73; 10,82; 10,44; 11,30; 11,36; 12,53; 13,02; 12,60; 11,53; 12,38; 12,0; 10,24, respectively. figure 1 shows annual antihy pertensive drugs supply in each defined category and annual mortality rates due to ht, over the same period. trends of antihypertensive drugs supply with standardized mortality rate caused with ht are shown in figure 2. fig. 1 annual sales of antihypertensive drugs and cvd standardized mortality rate in 2000-2010 in polish population. legends: cvd – cardiovascular diseases, bb – beta-blockers, res – renin angiotensin system inhibitors, d – diuretics, ccb – calcium channel blockers. table 1. correlation of the supply of selected antihypertensive drugs (bb, res, d, ccb) with cvd standardized mortality rate in polish population between 2000–2010 type of antihypertensive drug specified group age bb res d ccb 0–44 years r=-0.82 p<0.05 r=-0.46 ns* r=-0.756 p<0.05 r=-0.52 ns* 45–64 years r=-0.99 p<0.05 r=-0.71 p<0.05 r=-0.96 p<0.05 r=-0.82 p<0.05 >64 years r=-0.98 p<0.05 r=-0.72 p<0.05 r=-0.96 p<0.05 r=-0.82 p<0.05 legends: ns – not significant, bb – beta-blockers, res – renin angiotensin system inhibitors, d – diuretics, ccb – calcium channel blockers. k. barański, j. e. zejda 25 p u b l ic h e a lt h a n d e p id e m io l o g y issn 2413-6077. ijmmr 2016 vol. 2 issue 1 correlation analysis of ht mortality with the defined classes of drugs showed negative and statistically significant coefficients: beta-blockers (r=0.56 p=0.07), res (r=0.55 p=0.07), diuretics (r=0.54; p=0.08), ccb (r= 0.63 p=0.03). additional analyses involving stratification for age showed larger coefficients of correlation in older segments of the population (table 2). discussion high mortality due to cardiovascular diseases, seen for decades in the xx century in poland, declined rapidly in 1991–1994. since then the pace of decline is slower, but apparent. for example, cvd standardized mortality rates significantly decreased from 431 in 2000 to 339 in 2010. the observed phenomenon is attributed to a number of factors, including change of lifestyle with particular focus on healthy diet. some estimates suggest that the reduction of dietary cholesterol intake in the analyzed period has explained a 39% reduction in general mortality due to coronary heart disease (chd), fig. 2 annual sales of antihypertensive drugs and ht standardized mortality rate in 2000-2010 in polish population. legends: ht – hypertension, bb – beta-blockers, res – renin angiotensin system inhibitors, d – diuretics, ccb – calcium channel blockers. table 2. correlation of the supply of selected antihypertensive drugs (bb, res, d, ccb) with ht standardized mortality rate in polish population between 2000–2010 type of antihypertensive drug specified group age bb res d ccb 0–44 years r=-0.58 ns* r=-0.29 ns* r=-0.52 ns* r=-0.31 ns* 45–64 years r=0.79 p<0.05 r=0.70 p<0.05 r=0.78 p<0.05 r=0.75 p<0.05 >64 years r=0.92 p<0.05 r=0.76 p<0.05 r=0.87 p<0.05 r=0.83 p<0.05 legends: ns – not significant, bb – beta-blockers, res – renin angiotensin system inhibitors, d – diuretics, ccb – calcium channel blockers. in poland [7]. other factors include reduced smoking and increased physical activity with the estimated contribution to decreased chd mortality at the levels of 11% and 10%, respectively [8]. other studies suggest an important role of tobacco smoking cessation (as most important cause), followed by blood pressure control (10%) and cholesterol (10%) [9]. the role of smoking habit together with high density lipoprotein cholesterol concentrations, triglyceride concentrations, diabetes, body mass index, height, alcohol intake, physical activity, and level of education have been also confirmed by ecological findings in the polish population [10]. interest in the impact of environmental factors on cvd mortality is justified by given socioeconomic changes in poland, in recent decades. however, the role of clinical control of cvd cannot be neglected. after 1990 the polish population has an increasing access to modern therapeutical measures, including effective medical technologies and effective k. barański, j. e. zejda 26 p u b l ic h e a lt h a n d e p id e m io l o g y issn 2413-6077. ijmmr 2016 vol. 2 issue 1 medicaments used in the treatment of cvd. in poland, the contribution of those factors to the diminished chd mortality could be as large as 37% [8]. the effect is likely to reflect not only a better access to modern pharmacological mana gements but also could be attributed to some changes in health care organization including physician-patient interactions. social aspects could play a role and result in improved compliance of patients thus leading to stronger preventive outcomes in patients with chronic cvd [11]. our findings showed a negative correlation between country-wide sale of antihypertensive drugs and chd mortality, particularly in older segment of the polish population. the result corresponds with a view concerning beneficial effect of pharmacological prevention of chd mortality. such effect was also seen in other study that addressed a risk of so called composite cvd events, stroke and all-cause mortality [12]. a large meta-analysis covering more than 40000 patients with hypertension provided interesting evidence concerning cardiovascular events and mortality: antihypertensive therapy was associated with the reduction of all-cause mortality rate by 13%, the risk of death from all-cardiac causes by 18%, cv events by 21, and stroke by 30%, including fatal stroke by 39% [13]. with regard to our findings it is of relevance that the gain is much larger in older patients than in young patients with hypertension [14]. it remains unknown to what extent the agerelated difference reflects age-related duration of disease and, consequently, age-related duration of the treatment. such explanation cannot be ignored when discussing no apparent effect of the treatment of hypertension on chd mortality over the period of 4-5 years [15]. our findings did not show major differences between correlations provided by different groups of antihypertensive drugs analyzed in the study. the study protocol hampers a more detailed discussion of that point. the ecological approach used in our analysis has well-known limitations but a general conclusion about a beneficial impact of increased access of the population to modern antihypertensive therapy on a risk of cvd mortality in that population seems to be convincing. we did not see a similar effect in relation to ht mortality. a lack of such effect could be explained by a relatively infrequent occurrence of ht as a reported cause of death in poland. death certificates usually include fatal complication of ht thus making this diagnosis an unreliable index of causespecific mortality, with a very limited pertinence to conclusive statistical analyses. conclusions in poland, an improved access of the population to pharmacological control of ht, as suggested by increased country-wide sale of modern antihypertensive drugs, is associated with an apparent reduction in mortality from cvd but not from ht. the latter finding might reflect imprecise definition of ht as a cause of death or the fact that ht leads to other cardiologic events usually repor ted as a cause of death. the findings seem to confirm an important contribution of pharma cological measures in prevention of cvd mor tality, in addition to the well explored role of socioeconomic and life-style factors, in po land. references: 1. danaei g, finucane mm, lin jk, singh gm, paciorek cj, cowan mj, et al. national, regional, and global trends in systolic blood pressure since 1980: systematic analysis of health examination surveys and epidemiological studies with 786 country-years and 5.4 million participants. lancet 2011; 377: 568–577. 2. barański k, zejda je.: nadciśnienie tętnicze jako główna przyczyna zgonów w polsce w latach 1990-2010. nadciśnienie tętnicze, 2014; 18(2): 74. 3. cheng a, braunstein jb, dennison c, nass c, blumenthal rs. reducing global risk for cardiovascular disease: using lifestyle changes and pharmacotherapy. clin cardiol. 2002; 25(5): 205–12. 4. gudmundsson ls, johannsson m, thorgeirsson g, sigfusson n, sigvaldason h, witteman jc. hypertension control as predictor of mortality in treated men and women, followed for up to 30 years. cardiovasc drugs ther. 2005; 19(3): 227–35. 5. white wb.: update on the drug treatment of hypertension in patients with cardiovascular disease. am j med. 2005; 118(7): 695–705. 6. who age standardization of rates: a new who standard: ahmad ob, boschi-pinto c, lopez ad, murray c, lozano r, inoue m.: gpe discussion paper series: no.31 eip/gpe/ebd world health organization 2001. k. barański, j. e. zejda 27 p u b l ic h e a lt h a n d e p id e m io l o g y issn 2413-6077. ijmmr 2016 vol. 2 issue 1 7. zatonski wa, willett w.: changes in dietary fat and declining coronary heart disease in poland: population based study bmj 2005; 331:187. 8. bandosz p, o’flaherty m, drygas w, koziarek j, wyrzykowski b, rutkowski m, et al.: explaining the decline in coronary heart disease mortality in poland between 1991 and 2005 esc congress, stockholm, sweden (2010 28 aug–01 sep) abstract 1119. 9. unal b, critchley ja, capewell s.: explaining the decline in coronary heart disease mortality in england and wales between 1981 and 2000. cir culation, 109 (2004): 1101–1107. 10. zatonski wa, mcmichael a j, powles jw, ecological study of reasons for sharp decline in mortality from ischaemic heart disease in poland since 1991 bmj. 1998 apr 4; 316(7137): 1047–105. 11. papp r, csaszar a, paulik e, balogh s.: correlations between prescription of anti-hypertensive medication and mortality due to stroke. bmc cardiovascular disorders 2012, 12: 1. 12. thompson am, hu t, eshelbrenner cl, reynolds k, he j, bazzano la.: antihypertensive treatment and secondary prevention of cardiovascular disease events among persons without hypertension: a meta-analysis. jama. 2011; 305(9): 913–22. 13. ostrowski m, zanchetti a, nikfar s, muntner p, aronow ws, howard vj, wong nd, howard g, abdollahi m, banach m.: the effect of hypertension pharmacotherapy in older adults. the results of a meta-analysis of 11 randomized control trials with 40325 patients. european heart journal (2014) 35 (abstract supplement), 1192. 14. perez mi, musini vm, wright jm.: effect of early treatment with anti-hypertensive drugs on short and long-term mortality in patients with an acute cardiovascular event. cochrane database syst rev. 2009 oct 7;(4):cd006743. 15. diao d, wright jm, cundiff dk, gueyffier f. pharmacotherapy for mild hypertension. cochrane database of systematic reviews 2012, issue 8. received: 2015-03-11 k. barański, j. e. zejda 5 in t e r n a l m e d ic in e issn 2413-6077. ijmmr 2016 vol. 2 issue 1 doi 10.11603/ijmmr.2413-6077.2015.2.6371 effectiveness of canephron® n in the complex management of subclinical gouty nephropathy s. i. smiyan, m. v. franchuk, r. r. komorovsky i. horbachevsky ternopil state medical university, ternopil, ukraine background. the risk of chronic kidney failure increases by 3–10 times with the steady increasing of uric acid level in the blood. it is known that the protein fractions is closely correlated with the level of uric acid. objective. microalbuminuria and microglobulinuria are predictors of kidney damage. the study involved 50 patients with gout who had never received preventive treatment of gouty nephropathy. we choosed canephron n (bionorica, neumarkt, germany) as a combined phytodrug with nephroprotective effect. all studied patients were men with obesity. results. according to standard examination kidney damage haven’t been found, but laboratory tests on microproteinuria showed that the vast majority of patients have signs of subclinical gouty nephropathy. conclusions. canephron n in complex gout treatment helps to decrease uric acid level in the blood and increase its excretion. key words: gout, chronic kidney disease, hyperuricemia, canephron n introduction the term “gouty nephropathy” (gn) com­ prises all renal pathology that may occur in patients with gout, including urate nephro li­ thiasis, tophi in the renal parenchyma, glome­ rulosclerosis, arteriosclerosis with subsequent nephrosclerosis, interstitial nephritis and chro­ nic renal failure. moreover, the use of non­ steroidal anti-inflammatory drugs (nsaids) for symptomatic treatment of patients with gout is associated with nephrotoxicity and may result in acute tubular necrosis, acute interstitial nephritis, proteinuria, hypertension, hyperka­ lemia [6, 13]. the prevalence of kidney damage in patients with gout ranges from 30 to 70%. hence it is essential that patients with chronic gout receive therapy for prevention of gn without any (or with minimal) side effects and contraindications. for this purpose we have chosen a herbal based medicine – canephron® n (bionorica, neumarkt, germany) which is an approved medicinal product containing a fixed combination of centaury herb (centaurium sp.), lovage root (levisticum officinale koch), and rosemary leaves (rosmarinus officinalis l.) [19]. it has been available on the european market for more than 40 years. the drug has diuretic [12, 33], spasmolytic [1, 32], anti-inflammatory [11, 23, 29], antimicrobial [7, 8, 17], nephroprotective [18] and hypouricemic [25] effects. some clinical studies show a therapeutic benefit in patients with urinary tract infections [9, 21, 26] and diabetic nephropathy [19]. materials and methods we examined 50 patients with gout (all men), who were hospitalized in the department of rheumatology of ternopil university hos pital. the patients were not previously diagno sedor tested for gn, nor were previously tested for gn. all the patients underwent main labora tory and instrumental methods of inves tigation and some additional tests for micro albumin and microglo­ bulin levels in morning urine performed by means of elisa method. micro proteinuria means any manifestation of microalbuminuria (ma), microglobulinuria (mg) or their combina tions. the patients were divi ded into 2 groups: the patients of group i (n=25), the study group, received standard urate lowering therapy (allo pu rinol), nsaids for pain control and cane phron® n; the patients of group ii (n=25), the control group, received only allopurinol and nsaids. body mass index (bmi), plasma and urine uric acid (ua) levels, blood creatinine, urea and glomerular filtration rate (gfr) were tested. kidney ultrasound and joint x­ray were also per for med. ca ne phron® n was prescribed, 2 tablets 3 times per day for 6 weeks. after 6 weeks pati ents’ microalbumin, microglobulin levels in urine and ua levels in plasma and urine were re­examined. s. i. smiyan et al. corresponding author: svitlana smiyan, department of inter�svitlana smiyan, department of inter� nal medicine no 2, i. horbachevsky ternopil state medical university, 1 maidan voli, ternopil, ukraine, 46001 tel. +380352273377 e�mail: smyjan@tdmu.edu.ua international journal of medicine and medical research 2016, volume 2, issue 1, p. 5–8 copyright © 2016, tsmu, all rights reserved 6 in t e r n a l m e d ic in e issn 2413-6077. ijmmr 2016 vol. 2 issue 1 continuous variables are expressed as mean±standard error of the mean. differences between the variables were determined by unpaired or paired t-test, as appropriate. a value of p<0,05 was considered to be statistically significant. results and discussion baseline indicators of the examinations are presented in table 1. due to the bmi, the majority of patients in both groups were diagnosed with obesity. blood creatinine level, urea and gfr were uninformative, because the findings were normal in both groups. also nephrolithiasis was found in both groups. laboratory and instrumental investigations showed that a significant proportion of the patients had subclinical gn (fig. 1). in the study group 48% patients had ma and 60% of them had mg. in the control group 44% patients had ma and 52% of them had mg. the patients from the study group received a combined treatment (allopurinol, nsaids + canephron n) and had better test results than the group which received standard treatment (allopurinol, nsaids) (table 2). the pathogenesis of gn is associated with hyperproduction of ua and imbalance between the processes of its tubular secretion and reabsorption. but currently there is no enough evidence that hyperuricemia (hu) is a marker of renal dysfunction or a risk factor for kidney disorders. the controversial results of the impact of hu on development of chronic kidney disease (ckd) are, partly, due to difficulties in diagnosis of gn at the early stage because of a long subclinical period [5, 10, 24, 27]. hyperproduction of ua and its excretion decrease leads to hu. the risk of chronic kidney table 1. baseline indicators markers study group(n=25) control group (n=25) p-value age, (yrs) 54,98±1,22 50,63±1,19 <0,05 disease duration, (yrs) 8,96±0,57 7,42±1,43 >0,05 bmi 28,64±0,61 30,62±1,37 >0,05 creatinine, (mmol/l) 72,12±0,39 78,31±1,21 >0,05 urea, (mmol/l) 5,05±0,65 4,96±0,27 >0,05 gfr, ml/min 130,88±1,22 129,64±0,31 >0,05 uric acid in plasma, (mcmol/l) 0,556±0,08 0,552±0,12 >0,05 x-ray stage, (%) i ii iii i ii iii – 11,4 79,5 9,1 11,8 76,5 11,7 nephrolithiasis, (%) 28 36 – p – significant differences between the baseline indicators of the study and control groups. 0 10 20 30 40 50 60 70 study group control group presence of microproteinuria, (p < 0,05) absence of microproteinuria, (p < 0,05) fig. 1. microproteinuria in patients suffering from gout. table 2. dynamics of changes at the beginning of the study and after 6 weeks of combined treatment with canephron n and standard therapy (the study group), and standard therapy only (the control group) study group (n = 25) control group (n = 25) markers baseline after treatment baseline after treatment p-value microalbuminuria, (mg/l) 131,81±3,45 62,38±2,99* 114,72±3,91 122,19±2,11 <0,05 microglobulinuria, (mg/l) 20,08±2,06 13,37±3,32* 22,36±3,12 21,19±2,94 <0,05 uric acid in plasma, (mcmol/l) 0,556±0,08 0,457±0,06* 0,562±0,12 0,498±0,23** <0,05 uric acid in urine, (mmol/l/day) 5,21±1,18 6,72±1,34* 5,02±1,09 5,08±1,02 <0,05 p – significant differences between the post-treatment markers of the study and control groups; * – after treatment in comparison with baseline markers of the study group (p<0,05); ** – after treatment in comparison with baseline markers of the control group (p<0,05). s. i. smiyan et al. 7 in t e r n a l m e d ic in e issn 2413-6077. ijmmr 2016 vol. 2 issue 1 failure (ckf) rises in 3–10 times caused by increase in ua in blood. every 4th patient has crf as a gout complication [3, 4, 5, 22]. several studies have shown relations of hu to the signs of kidneys damage [30, 31]. also ua can affect renal hemodynamics due to vasoconstriction in cortical layer and increase the expression of renin. an additional mechanism in kidneys damage is the ua impact on the formation of the endothelial dysfunction by increasing monocyte chemoattractant protein-1 (mcp-1) in vascular smooth muscle fibres and cells of the proximal renal tubules. mcp-1 is a main pathogenetic chemokine of ckd and atherosclerosis [16]. it is established that the protein fraction is closely correlated with the level of ua in blood; that is why hu causes endothelial dysfunction and ma [2]. if albumins and other highmolecular weight proteins are found in urine, glomerular injury is present. microglobulins (β2-, α1and retinol-binding protein), mg, are tubular disorders characterized by low-molecular weight proteins in urine. ma and mg are predictors of kidneys damage [14, 15, 19, 20, 28]. reference limits of ma and mg are presented in table 3. conclusions microalbuminuria and microglobulinuria are the main early symptoms of renal damage in patients with gout. the prevalence of kidney damage is significantly (p<0,05) higher than the incidence of gouty nephropathy in clinical practice. blood creatinine level, urea and gfr are uninformative at the stage of gn formation, which is asymptomatic. gn was diagnosed in 64% patients of the study group and in 56% ones of the control group according to the levels of ma and mg. uncontrolled hyperuricemia, which does not reach the target level, is a major risk factor for development of gn. the group of the patients who received canephron n as a standard gout treatment had better results after re-examination. this herbal based medicine has uricosuric effect because it decreases ua level in plasma. microproteinuria decreased in 2 times after the combined treatment with canephron n. also we did not detect any side effects during 6 weeks of the study. so, canephron n is recommended for treatment of subclinical gouty nephropathy. table 3. reference limits of microalbuminuria and microglobulinuria type indicators (mg/l) normoalbuminuria <20 microalbuminuria 20–200 macroalbuminuria >200 normoglobulinuria <12 microand macroglobulinuria >12 references 1. abdul-ghani as, el-lati sg, sacaan a, et al. anticonvulsant effects of some arab medicinal plants. int j crude drug res 1987; 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34: 16–20. 10. gerald d, nazia r, fang n. effect of uratelowering therapies on renal disease progression in patients with hyperuricemia. the journal of rheumatology, 2014. 955–962 11. gracza l, koch h, löffler e. isolierung von rosmarinsäure aus symphytum officinale und ihre antiinflammatorische wirksamkeit in einem in-vitro. modell. arch pharm 1985; 318:1090–1095. 12. haloui m, louedec l, michel b, lyoussi b. experimental diuretic effects of osmarinus officinalis and centaurium erythraea. j ethnopharmacol 2000; 71: 465–472. 13. hossamel z, brian f. managing gout: how is it different in patients with chronic kidney disease? s. i. smiyan et al. 8 in t e r n a l m e d ic in e issn 2413-6077. ijmmr 2016 vol. 2 issue 1 cleveland clinical journal of medicine volume 77 / number 12, 2010, 919–928. 14. hovind p. serum uric acid as a predictor for development of diabetic nephropathy in type 1 diabetes. an inception cohort study. diabetes. 2009. – v. 58. 1668–1671. 15. jalal d. serum uric acid levels predict the development of albuminuria over 6 years in patients with type 1 diabetes: findings from the coronary artery calcification in type 1 diabetes study. nephrol dial transplant. 2010. n25. 1865–1869. 16. kanbay m, solak y, dogan e et al. uric acid in hypertension and renal disease: the chicken or the egg? blood purif, 2010. – n 30. 288–295. 17. kumarasamy y, nahar l, cox pj, et al. bioactivity of secoiridoid glycosides from centaurium erythraea. phytomedicine 2003; 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inhibition by rosmarinic acid. biochem pharmacol 1986; 35: 1397–1400. 24. richard j, takahiko n, diana j, laura g et al. uric acid and chronic kidney disease: which is chasing which? nephrology dialysis transplantation. volume 28, issue 9. 2221–2228. 25. shuba n, voronov t, tkachenko n. influence of complex phytodrug canephron n on the level of uric acid in patients with hyperuricemia and arterial hypertension. ml №1 (77) 2011. 77–79. 26. sterner w, heisler e, popp ho, fischer h. studien über die canephron-wirkung bei chronischen nierenerkrankungen. physikalische medizin rehabilitation 1973; 14: 239–258. 27. testa a, mallamaci f, spoto b et al. association of a polymorphism in a gene encoding a urate transporter with ckd progression. clinical journal of the american society of nephrology, 2014. 1059–1065. 28. the study of renal function [e-resource]. – access mode: url: http://www.biochemmack.ru/ upload/uf/204/2042dda688fc61b5d5db08ad2350d6 dc.pdf. 29. valentao p, fernandes e, carvalho f, et al. hydroxyl radical and hypochlorous acid scavenging activity of small centaury (centaurium erythraea) infusion. a comparative study with green tea (camellia sinensis). phytomedicine 2003;10: 517–522. 30. viazzi f, leoncini g, ratto e et al. mild hyperuricemia and subclinical renal damage in untreated primary hypertension. ajh, 2007. v. 20; 1276–1282. 31. weiner d. uric acid and incident kidney disease in the community. j am soc nephrol, 2008. – v. 19, n 6. 1204–1211. 32. yamahara j, konoshima i, sawada i, fujimura h. biologically active principles of crude drugs: pharmacological actions of swertia japonica extracts, swertiamarine and gentianine. yakugaku zasshi 1978; 98: 1446–1451. 33. yarnell e. botanical medicines for the urinary tract. world j urol 2002; 20: 285–293. received: 2016-02-10 s. i. smiyan et al. issn 2413-6077. ijmmr 2017 vol. 3 issue 150 p u b l ic h e a lt h a n d e p id e m io l o g y doi 10.11603/ijmmr.2413-6077.2017.1.6955 influence of lactobacillus spp. on colonization and anti-infectious resistance of the mucous membranes of the upper respiratory tract s. v. kalinichenko1, o. o. korotkykh1, s. i. pokhil1, m. g. bakumenko2 1national academy of medical sciences of ukraine, 1mechnikov institute of microbiology and immunology, kharkiv, ukraine 2kharkiv city student hospital, kharkiv, ukraine background. lactobacilli are very important for the formation of colonization resistance and have pronounced antagonistic effect against a wide range of microorganisms. that is why the lactobacilli have extensive use as a component of classic probiotic agents that are widely used to prevent and treat dysbiotic conditions of digestive and genital systems of people. objective. the aim of the research was to study the effect of lactobacilli on anti-infectious resistance of mucous membranes of upper respiratory tract. methods. the colonization degree (lg cfu / g) of nasal mucosal membranes by lactobacillus spp. and s. aureus was determined in all carriers before the experiment. also, the level of lysozyme and secretory immunoglobulin a (siga) in nasal secretions cavities was identified. results. it was established a clear dysfunction of anti-infectious resistance in carriers of staphylococcus aureus a decrease of colonization resistance and local immunity of mucous membranes of upper respiratory tract. as for the anti-infectious resistance of nasal mucosal of s. aureus carriers, the level of lysozyme and secretory immunoglobulin a gradually increased after the application of probiotic strain l. rhamnosus gg, and in 21 days it reached rates of healthy individuals. conclusions. it was found out that probiotics for nasal passages sanitation in staphylococcus aureus carriers lead to gradual eradication of the pathogen (s. aureus) with restoration of colonization and anti-infectious resistance, mucous membranes and upper respiratory tract. keywords: lactobacillus spp.; lysozyme; siga; upper respiratory tract. corresponding author: svitlana kalinichenko, laboratory of viral infections, national academy of medical sciences of ukraine mechnikov institute of microbiology and immunology, 14 pushkinska street, kharkiv, ukraine, 61057. phone number: +380577313151 e-mail: imidir@ukr.net introduction lactobacilli are widely distributed in the environment and have a high biological activity. they are an important part of normal microflora of digestive and genital human tracts and they belong to the resident microflora of nasopharynx [1]. lactobacilli are actively involved in the formation of colonization resistance of mucous membranes and have significant antagonistic action against a wide range of bacteria [2-4]. that is why the lactobacilli are widely used as a component of classic probiotic agents [3-5]. probiotics based on lactobacilli are widely used to prevent and treat dysbiotic conditions of digestive and genital human system [6-8]. nowadays, research on the use of lactobacilli for prevention or treatment of infections and recovery of anti-infectious resistance of mucous membranes of upper respiratory tract (urt) are actual [9]. the aim of the research was to study the effect of lactobacillus spp. on colonization and anti-infectious resistance of mucosal membranes of upper respiratory tract in staphylococcus aureus carriers. the objects of the study were the carriers of staphylococcus aureus (n=29) among the medical staff of a hospital, kharkiv (ukraine). methods the colonization degree (lg cfu / g) of nasal mucosal membranes by lactobacillus spp. and s. aureus was determined in all the carriers before the experiment. also, the level of lysozyme and secretory immunoglobulin a s. v. kalinichenko et al. international journal of medicine and medical research 2017, volume 3, issue 1, p. 50–52 copyright © 2017, tsmu, all rights reserved issn 2413-6077. ijmmr 2017 vol. 3 issue 1 51 p u b l ic h e a lt h a n d e p id e m io l o g y s. v. kalinichenko et al. (siga) in nasal secretions cavities was identified. then the carriers were divided into groups: i (n=7, control) – the persons, who received saline as the sanitation agent (0.85% nacl, ph 7.2) during 30 days, 2–3 drops twice per day in each nostril; ii (n=11) – the persons, who received as the sanitation agent a suspension of probiotic strain l. rhamnosus gg (in saline) at a concentration of 5×109 cfu/ml (according to the group i scheme). the degree of colonization of nasal mucous membranes by lactobacillus spp. and s. aureus, lysozyme and siga levels in nasal cavities secretions were determined in the individuals taking part in the experiments repeatedly in 7, 14 and 21 days of sanitation. the material was selected as follows: in each nostril, alternatively, with special replicator sterile foam tape was injected and mucus impregnation was held for 5 minutes, after it the tape was immersed in a sterile tube containing 1.0 ml of phosphate-buffered saline (pbs, ph 7.2) for 30 minutes and then separated from the foam carrier mucus by insulin syringe, forcing it into the pbs. in the comparisons group there were persons, who did not have upper respiratory tract infection within 6 months (almost healthy, n=15). lysozyme levels were determined by means of nephelometric method [10]. the concentration of lysozyme was rated in micrograms/ml (μg/ml). to determine siga, ridascreen®siga elisa kits of r-biopharm ag company (germany) were used. the results of calculations were carried out on stat fax enzyme immunoassay analyser. siga concentration was rated in mg/ml. results it was established that in the healthy individuals (n=15) the degree of colonization of the na sal mucous membranes lactobacillus spp. was 6.64±0.9 lg cfu/g, the amount of lysozyme and siga in nasal secretions cavities were respec tively at 17.45±2.9 μg/ml and 129.3±21.7 mg/ml. the degree of colonization of nasal mucosa lactobacillus spp. was 2.2±0.7 lg cfu/g, the amount of lysozyme and siga in nasal secretions cavities were respectively 7.45±2.1 μg/ml and 63.9±19.8 mg/ml in the s. aureus carriers. discussion consequently, the degree of colonization of nasal mucosa lactobacillus spp. in the carriers of staphylococcus aureus was in 3.8–5.0 (p<0.01) times lower compared to the healthy individuals. the amount of lysozyme and siga in nasal secretions cavities were also lower in comparison with the healthy individuals, respec tively in 1.5–3.8 (p<0.05), and 1.2–3.4 (p<0.05) times. thus, it was established a clear dysfunction of anti-infectious resistance in the carriers of staphylococcus aureus: the decrease of colonization resistance and local immunity of mucous membranes of upper respiratory tract. according to the literature, application of probiotics or synbiotic treatment regimens in respiratory infections reduces relapse respiratory tract infections and bronchial obstruction in children [9]. but the authors did not conduct any studies to determine the number of lactobacilli in secrets of urt and their influence on anti-infectious resistance of mucous membranes. in connection with the foregoing, we conducted a study on the possibility of recovery colonization and anti-infectious resistance of mucous membranes of upper respiratory tract in the carriers of s. aureus using the probiotic strain l. rhamnosus gg. the data in the table 1 prove that the probiotic strain l. rhamnosus gg for sanitation the carriers decreased the degree of colonization by staphylococcus aureus mucosal in 7 and 14 days after the sanitation of lactobacilli suspension, and in 21 days there was a complete eradication of s. aureus from nasal mucous membranes. moreover, the decrease of s. aureus degree colonization on mucosal of the carriers occurred against the background of increase in the number of lactobacillus spp. on mucous membranes. as for the anti-infectious resistance of nasal mucosal of s. aureus carriers, the level of lysozyme and secretory immunoglobulin a after the application of the probiotic strain l. rhamtable 1. average indexes of mucosal colonization resistance after sanitation, (m±m) groups the degree of mucosal colonization urt, lg cfu / g lactobacillus spp. s. aureus in 7 days in 14 days in 21 days in 7 days in 14 days in 21 days i 2.3±0.7 2.2±0.6 2.1±0.7 4.7±0.7 4.9±0.6 4.8±0.6 ii 3.8±0.4* 4.5±0.4* 5.3±0.4* 1.8±0.6* 0.6±0.6* 0±0* note: * – significant difference between these indexes of i and ii groups (p<0.05). issn 2413-6077. ijmmr 2017 vol. 3 issue 152 p u b l ic h e a lt h a n d e p id e m io l o g y nosus gg significantly increased, and in 21 days it reached rates of healthy individuals (table 2). to be precise, after the sanitation of probiotic nasal of the carriers staphylococcus aureus, the level of lysozyme and siga rose: 7 days, an average of 1.6 (p <0.05) and 1.4 (p<0.05) times, respectively; at 14 days of 1.8 (p<0.05) and 1.6 (p<0.05) times, respectively; after 21 days – 2.1 (p<0.05) and 1.8 (p<0.05) times, respectively, compared with the group the carriers, that sanitation was conducted with saline. conclusions thus, it was found that the application of probiotics for nasal passages sanitation in the table 2. average indexes of mucosal anti-infectious resistance after sanitation, (m±m) group indicator of local immunity level of lysozyme, μg/ml level of siga, mg/ml in 7 days in 14 days in 21 days in 7 days in 14 days in 21 days i 7.3±2.4 7.4±2.2 7.4±2.3 61.3±21.3 62.1±18.8 61.9±19.3 ii 11.6±3.3* 13.3±2.9* 15.4±2.7* 84.8±19.8* 102.9±21.1* 112.2±19.1* note: * – significant difference between these indexes of i and ii groups (p<0.05). carriers of s. aureus leads to the gradual eradication of the pathogen (s. aureus) with colonization restoration (increased degree of mucosal lactobacilli settlement), and anti-infectious resistance (increased level of siga and lysozyme) in mucous membranes and upper respiratory tract. this study demonstrated the positive effect of lactobacillus spp. on the formation of colonization and anti-infectious resistance of upper respiratory tract mucous membranes and provided an opportunity to consider probiotics as a useful option for immune exposure to chronic infections of staphylococcal origin. references 1. ryzhkova ta. the peculiarities of antagonistic activity of lactobacillus strains (isolated from different econiches and probiotics) under cultivation conditions different by gas composition. annals of mechnikov institute. 2014;2:64–69. 2. bondarenko vm, rybalchenko ov. analysis of preventive and therapeutic action of probiotic drugs from the standpoint of new scientific technologies. microbiology journal. 2015;2:90–104. 3. sishel lm. immunomodulatory activity of probiotic strains laktoand bifidobacteria in vitro and in vivo. abstracts xiii congress of the society of microbiologists of ukraine. 2013;278. 4. sishel lm. imunobiotics – a group of new biotechnology drugs with antimicrobial and immunomodulatory properties abstracts xiii congress of the society of microbiologists of ukraine. 2013;279. 5. kalinichenko sv. microecological disorders and their modern correction. ukrainian biopharmaceutical journal. 2016;2(43):6–12. 6. babenko lp. effect of probiotic strains of lactobacilli and bifidobacteria in range of microbiota and immune reactivity urogenital tract with staphylococca infection. imv nasu. 2015:28. 7. bobyr vv. intestinal virus and human normal microflora: features of interaction. annals of mechnikov institute. 2015;2:25–29. 8. osolodchenko tp. role of distal intestinal tract microflora in support of oxalate homeostasis. annals of mechnikov institute. 2015;2:42–46. 9. stojkovic a, simovic a, bogdanovic z, bankovic d, poskurica m. clinical trial or experimental study (consort compliant): optimal time period to achieve the effects on synbiotic-controlled wheezing and respiratory infections in young children. srp arh celok lek. 2016;144(1–2):38–45. 10. dorofeychuk vg. determination of lysozyme activity by nephelometric method. laboratory work. 1968;1:28–30. received: 2016-11-18 s. v. kalinichenko et al. 51 in t e r n a l m e d ic in e issn 2413-6077. ijmmr 2021 vol. 7 issue 1 doi 10.11603/ijmmr.2413-6077.2021.1.12120 systematic review on the psychometric, reliability and validity properties of translated neuropathic pain screening tools (dn4, lanss and pdq) 1 january 2005 – 19 july 2019 t.r. fagbohun university of the witwatersrand, johannesburg, south africa background. different neuropathic pain screening tools (dn4, lanss and pdq) have been developed, translated into several local languages, and validated. to determine the reliability of these tools and their ability to differentiate between diagnosing neuropathic pain quality from nociceptive pain, a systematic review was conducted to synchronize properties and suggest the reliability of the translated version of these neuropathic pain-screening tools. objective. to conduct an evidence-based systematic review to assess the psychometric, reliability and validity of the translated version of dn4, lanss and pdq between january 2005 and 2019. methods. two independent reviewers adopted the use of online (internet) search machine (pubmed, scopus and web of science) to search for the relevant articles based on jbi (joanna briggs institute) inclusion criteria. data extracted from the articles were synthesis in tabular form. results. twenty-six articles were included from dn4 (n=11), lanss (n=8) and pdq (n=4) translated from english language to eight local languages. the sensitivity and specificity of the dn4 studies ranged from 75% to 98% and 37.3% to 96%, respectively. the internal reliability (α) of the translated version of the dn4 ranged from 0.55-0.862. the sensitivity and specificity of the lanss studies ranged from 75% to 98% and 37.3% to 96%, respectively. the internal reliability (α) of the translated version of the lanss ranged 0.67-0.96. the sensitivity and specificity of the pdq studies ranged from 75% to 98% and 37.3% to 96%, respectively. the internal reliability (α) of the translated version of the pdq ranged 0.81-0.86. conclusions. all the translated instruments reviewed showed good internal consistency of the items, high sensitivity and positive predictive value (ppv) but not to a suitable level compared with the original version. therefore, these screening tools are suggested to be used in conjunction with the clinical testing for appropriate diagnosis of patients with neuropathic pain quality. keywords: neuropathic pain; positive likelihood; negative likelihood; positive predictive value; negative predictive value. introduction neuropathic pain (np) is classified as one of the worse pains reported by chronic pain patients [1]. an estimated 1 out of 10 chronic pain patients develop neuropathic pain, depending on the population study [2]. the prevalence may be as high as 51.9 % in the patients being managed for chronic pain clinic [3]. evidence indicates that neuropathic pain affects both physical and emotional state of the patients [4], thereby. this type of pain decreases the quality of life of patients [4, 5] and results in a negative interaction with society in general [6]. neuropathic pain is associated with lesion or disease of the somatosensory pathway that leads to abnormality observed at the peripheral and central region of the system function (hyperalgesia or allodynia) [7]. the common symptoms associated with neuropathic pain are: sharp, burning, pins and needles, tingling, painful cold, numb and shooting [2]. diagnosing standards among pain physicians and researchers of neuropathic pain in chronic pain patients have been a challenge [8]. the five np screening tools are lanss [9], neuropathic pain questionnaire [10], douleur neuropathique 4 ‘dn4’ [11], id pain [12] and paindetect [13]. these instruments have been validated and adapted in different languages from different countries. among these instruments, dn4, lanss and paindetect are the most commonly used tools in the assessment of the quality of neuropathic *corresponding author: temitope richard fagbohun, research student, university of the witwatersrand, johannesburg, 2193, south africa. e-mail: temitopesms@aol.com international journal of medicine and medical research 2021, volume 7, issue 1, p. 51-67 copyright © 2021, tnmu, all rights reserved t.r. fagbohun 52 in t e r n a l m e d ic in e issn 2413-6077. ijmmr 2021 vol. 7 issue 1 pain in chronic pain patients due to their high sensitivity and specificity, short duration of the assessment, easy understanding of the terms and application by the pain experts [14, 15]. translation of these tools from the original language to local languages is essential for good communication and effective assessment of pain quality between the researcher or pain expert and the patients. critically appraising the data measurement of these instruments may be valuable for the clinician and researchers in decision making based on evidence from peer-reviewed articles that adopted these instruments in their studies. therefore, the aim of this study was to conduct a systematic review on the translated version of the douleur neuropathique en 4 ques tionnaire (dn4), leeds assessments of neuropathic symptoms and signs (lanss) and the paindetect questionnaire (pd-q) tools with the objective to evaluate their psychometric, reliability and validity properties. methods study design: systematic review of studies was conducted according to prisma guidelines [16]. the systematic review was conducted using a developed protocol registered on prospero (crd42015016752) by the authors. pico method was adopted to define our study question: p (patient or population): patients with chronic pain i (intervention): diagnostic screening tool c (comparator): none o (outcome): psychometric and diagnostic properties of neuropathic pain screening tools: dn4*, lanss**, and pd-q (* includes the dn4interview, ** includes the self-complete (s)lanss) study inclusion criteria the following article selection criteria were used: • language of publication: no restrictions; • geographic location: no restrictions; • publication date: 1 january 2005 to 31 july 2019; • publication type: original articles and abstracts; search strategy the search strategy was as follows: databases: pubmed, scopus, web of science. secondary search: reference lists of selected publications were checked. search terms: (“douleur neuropathique” or dn4 or dn-4 or “leeds assessment of neuropathic signs and symptoms” or lanss or paindetect or “pain detect” or pdq or pd-q) and pain and (neuropathy or neuropathic or neuralgia or neuritis or central or stroke or spinal) and (translation or adaptation or validation or reliability or validity). data management search results were transferred to mendeley desktop reference manager (elsevier), where all references retrieved were combined, and duplicates were removed. screening initial screening of the articles included was done by title and abstract and was performed by tf (temitope fagbohun) and checked by pk (peter kamerman). the excluded articles were removed, and the reason for their exclusion was recorded. the full text of all retained studies was then screened by tf and pk and a consensus list of studies was generated to include into the review. data extraction the following data were extracted: 1. bibliographic information; 2. study characteristics: a. name of the translated questionnaire; b. language of translation; c. setting (study population;) d. study methods; e. measures of reliability (reliability of the screening tools was determined by the following measures: test-retest reliability (intraclass correlation coefficient, pearson’s or spearman’s correlation coefficient), inter-rater reliability (cohen’s kappa lowest and highest score), and internal consistency (cronbach’s alpha)); f. diagnostic properties (measures of diagnostic performance: diagnostic performance was assessed by measures of sensitivity, specificity, positive likelihood, negative likelihood, positive predictive value, and negative predictive value. results a total of 1,493 articles were obtained from the initial electronic databases search and 27 articles were finally included in the final review. the details of the study identification and selection process in accordance with the preferred reporting items for systematic review and meta-analysis (prisma) statement [16] are described in fig. 1. one hundred and twenty-two articles were excluded due to duplications. the abstract and the title of 1,371 articles were screened, 1,337 articles were excluded due to not meeting the inclusion t.r. fagbohun 53 in t e r n a l m e d ic in e issn 2413-6077. ijmmr 2021 vol. 7 issue 1 criteria of this study. thirty-four (34) articles were further screened for full-text inclusion, and three (3) articles were excluded for not applying dn4, lanss or paindetect instrument neuropathic pain screening tools. thirty (30) articles were further screened for check of validity test; five (5) articles were further excluded due to no validity test. twenty-six (26) articles were included in this review for extraction. summary of the articles included twenty-six articles where included in this review – 11 dn4 articles [17-27], 8 lanss articles [20, 24, 28-33], 4 pd-q articles [15, 34-36] and 3 s-lanss [28, 37, 38]. the total sample size reported was 2,075. out of this, 1,056 were diagnosed with neuropathic pain, 874 – nociceptive pain and 55 were patients with mixed pain. eighty-two (82) participants had mixed pain included in the neuropathic pain participants [24] (table 1). dn4 description of the dn4 articles eleven (11) studies were included in the dn4 screening tool in this review (table 2). two (2) studies [17, 21] further evaluated the reliability and validity properties of the tools at different cut off. the dn4 was translated to eight different languages which includes: the arabic language (n=2) [17, 21], brazilian portuguese (n=1) [26], korean (n=2) [19, 20], spanish (n=2) [24, 25], farsi (n=1) [22], greek (n=1) [23], italian (n=1) [27] and japanese (n=1) [18]. the total sample size reported n=1,756. out of this, n=880 was diagnosed with neuropathic pain, n=731 – nociceptive pain and n=55 were patients with mixed pain. eighty-two (82) participants had mixed pain included in the neuropathic pain total articles obtained from electronic database search (n=1,493) sc re en in g in cl u d ed el ig ib ili ty id en ti fi ca ti o n articles screened for abstracts/tittles (n=1,371) articles records screened (n=34) articles records excluded due to none use of dn4, lanss or ppd-q (n=3) full-text articles assessed for methodology quality (n=31) full-text articles excluded due to no validity study (n=5) final articles included in the review (n=26) duplicate articles removed (n=122) articles excluded after evaluation of abstract/tittles. (n= 1,337 ) fig. 1. flow chart of the final selected articles. t.r. fagbohun 54 in t e r n a l m e d ic in e issn 2413-6077. ijmmr 2021 vol. 7 issue 1 ta bl e 1. d es cr ip ti on o f th e in cl u de d st u di es au th or n p to ol la ng ua ge sa m pl e si ze fo rw ar d tr an sl at io n n o of fo rw ar d tr an sl at io ns b ac kw ar d tr an sl atio n n o of ba ck w ar d tr an sl at io ns ex pe rt as se ss m en t pi lo t te st to ta l n eu ro pa th ic n oc ice pt iv e m ix ed s am pl e m at su ki e t a l. 18 d n 4 ja pa ne se 18 7 10 0 87 0 ye s 1 ye s 1 ye s n o ch at ila e t a l. 17 d n 4 ar ab ic 19 5 99 96 0 ye s 3 ye s n s ye s ye s te rk aw i e t a l. 21 d n 4 ar ab ic 12 4 77 47 0 ye s 5 ye s 2 ye s ye s ki m e t a l. 19 d n 4 ko re an 83 43 40 0 ye s ye s pa rk e t a l. 20 d n 4 ko re an 83 40 43 0 sy ki ot i e t a l. 23 d n 4 g re ek 23 7 12 3 59 55 ye s n s ye s n s ye s m ad an i e t a l. 22 d n 4 fa rs i 17 5 86 89 0 ye s 5 ye s 3 ye s ye s h am da n et a l. 24 d n 4 sp an is h 19 2 12 1 91 *s ee fo ot no te sp al lo ne e t a l. 27 d n 4 it al ia n 22 1 50 61 0 n s n s n s n s n s n s sa nt os e t a l. 26 d n 4 b ra zi lia n po rt ug ue se 10 1 42 59 0 ye s 2 ye s 2 ye s ye s pe re z et a l. 25 d n 4 sp an is h 15 8 99 59 0 ye s 2 ye s 2 ye s n s b at is ta ki e t a l. 28 la n ss g re ek 10 0 58 42 0 ye s 2 ye s 2 ye s ye s pa rk e t a l. 20 la n ss ko re an 21 3 11 3 10 0 0 ye s 2 ye s 2 ye s ye s sp an os e t a l. 30 la n ss g re ek 70 35 35 0 n s n s n s n s n s n s b ar bo sa e t a l. 29 la n ss po rt ug ue se 16 7 10 3 64 0 ye s 2 ye s 1 ye s ye s h am da n et a l. 24 la n ss sp an is h 19 2 12 1 91 *s ee fo ot no te tü rk el e t a l. 32 la n ss tu rk is h 14 8 99 49 0 ye s n s ye s 1 ye s ye s sc he st at sk y et a l. 31 la n ss b ra zi lia n po rt ug ue se 90 34 44 12 ye s 2 ye s 1 ye s n s yu ce l e t a l. 33 la n ss tu rk is h 10 1 49 52 0 ye s 1 ye s 1 ye s n o g ud al a et a l. 15 pa in d et ec t h in di 16 0 80 80 0 ye s 2 ye s 2 ye s ye s m at su ba ya sh i e t a l. 36 pa in d et ec t ja pa ne se 11 3 60 53 0 ye s 2 ye s 2 ye s n o al ka n et a l. 34 pa in d et ec t tu rk is h 24 0 80 80 80 ye s 2 ye s 2 ye s n o d e an dr és e t a l. 35 pa in d et ec t sp an is h 22 1 71 71 79 ye s 2 ye s 2 ye s n s ló pe zde -u ra ld evi lla nu ev a et a l. 38 sla n ss sp an is h 18 2 71 11 1 0 ye s 2 ye s 2 ye s ye s b at is ta ki e t a l. 28 sla n ss g re ek 10 0 54 46 0 ye s 2 ye s 2 ye s ye s ko c an d er de m og lu 37 sla n ss tu rk is h 15 4 13 7 10 7 0 ye s 2 ye s n s ye s ye s n ot e. * 82 (4 2% ) h ad m ix ed p ai n, b ut w er e in cl ud ed in th e ne ur op at hi c pa in g ro up n s – no t s pe ci fie d t.r. fagbohun 55 in t e r n a l m e d ic in e issn 2413-6077. ijmmr 2021 vol. 7 issue 1 ta bl e 2. m ea su re s of v al id it y fo r tr an sl at ed v er si on s of t h e d n 4 au th or as se ss ed in te rn al va lid ity cr on ba ch al ph a as se ss ed te st -r et es t re lia bi lit y ic c* co rr el at io n co effi ci en t as se ss ed in te rra te r re lia bi lit y co he n’ s ka pp a (lo w es t sc or e) co he n’ s ka pp a (h ig he st s co re ) co rr el at io n co effi ci en t ic c ch at ila e t a l. 17 ye s 0. 55 to 9 3 n o ye s 0. 92 (b ru sh in g) 1. 0 (h yp oe st he si a to br us hi ng , h yp oes th es ia to p in pr ic k) 0. 99 ch at ila e t a l. 17 ye s 0. 86 ** n o ye s 0. 92 (b ru sh in g) 1. 0 (h yp oa es th es ia fo r to uc h, h yp oa es th es ia fo r pi np ri ck ) 0. 99 te rk aw i e t a l. 21 ye s 0. 7 ye s te rk aw i e t a l. 21 ye s 0. 67 ye s 0. 81 0. 81 (s pe ar m an ) n o sa nt os e t a l. 26 ye s 0. 76 n o ye s 0. 92 (t ot al s co re ) 0. 92 (t ot al s co re ) m ad an i e t a l. 22 ye s 0. 86 2 ye s 0. 95 7 ye s 0. 41 6 (e le ct ri c sh oc ks ) 0. 82 6 (n um bn es s) 0. 95 7 sy ki ot i e t a l. 23 ye s 0. 65 ye s 0. 95 6 ye s 0. 81 8 (t ot al sc or e) 0. 81 8 (t ot al s co re ) sp al lo ne e t a l. 27 n o n o n o m at su ki e t a l. 18 ye s ye s 0. 82 7 pa rk e t a l. 20 ye s 0. 81 9 ye s 0. 81 3 ye s 0. 82 3 (it ch in g) 0. 94 6 (n um bn es s) ki m e t a l. 19 ye s 0. 81 9 ye s 0. 81 3 ye s 0. 82 3 (it ch in g) 0. 94 6 (n um bn es s) pe re z et a l. 25 ye s 0. 7 ye s 0. 94 9 ye s 0. 68 (n ot sp ec ifi ed ) 0. 79 (n ot s pe ci fie d) 0. 92 6 h am da n et a l. 24 n o n o * in tr acl as s co rr el at io n co effi ci en t;* * o nl y pr ov id ed in te rn al c on si st en cy fo r th e en tir e qu es tio nn ai re (b y ku de rri ch ds on fo rm ul a) . t.r. fagbohun 56 in t e r n a l m e d ic in e issn 2413-6077. ijmmr 2021 vol. 7 issue 1 participants [24]. all the included articles that used dn4 instruments were published between 2007 and 2018 (table 2). forward translation was reported in eight studies [17-19, 21-27] with the translation conducted in two studies in 5-times [21, 22], one study in 3-times [22], two studies in 2-times [25, 26], one study in 1-time [18]. similarly, backward translation was reported in eight studies [17-19, 21-23, 25, 26]. one study conducted in 3-times [22], three studies conducted in 2-times [21, 25, 26], three studies were not specific on the number of times backward translation was done, and three studies did not report on the backward translation (table 2). expert assessment was involved in seven (7) studies [17, 18, 21-23, 25, 26] and four (4) studies conducted a pilot test [17, 21, 22, 26] (table 2). validity and reliability of the dn4 instrument internal validity was reported in nine (9) studies [17-23, 25, 26] of the included eleven (11) studies (table 2). cronbach was reported in eight studies [17, 19-23, 25, 26]. findings on test-retest validity were reported in seven (7) studies [18-23, 25] with icc values between 0.81 and 0.96. one study reported the coefficients of correlation spearman value 0.81 [21]. the inter-rated reliability was conducted in seven (7) studies [17, 19, 20, 22, 23, 25, 26] in translated dn4, cohen’s kappa lowest scored values of 0.92 were reported for brushing in one (1) study [17], while total score values of 0.818 and 0.92 were reported in two (2) studies [23, 26], scored values of 0.823 was reported for itching in two studies [19, 20] (table 2). cohens kappa high values of 1.0, 0.8260.946 and 0.9 were reported for hypoesthesia to brushing and hypoesthesia to pinprick in two studies [17, 21] and numbness [19, 20, 22]. also, total cohens kappa highest score values of 0.818 and 0.92 (total score) were reported in two (2) studies [23, 26]. sensitivity, specificity, negative and positive likelihood different cut offs were adopted to differentiate the neuropathic pain from nociceptive pain in this instrument (table 3). studies included at cut off of 3 showed sensitivity between 93.3-100%, specificity between 3-100%, positive likelihood between 5.2-5.5, negative likelihood between 0-3, ppv ranges between 84.3-85.6% and npv of 72.1% – 97.5% in three studies. at cut off 4, sensitivity reported ranges between 80-96%, while the specificity was between 6.8-95%, positive likelihood 8.4-20.2 reported in three (3) studies, negative likelihood range between 0.1-0.2; ppv was between 63.995% and the npv was between 69-95.5% reported in eight (8) studies. at cut off of 5, sensitivity reported ranges between 75-91% in four (4) studies, specificity was between 51-99%, positive likelihood ranged between 5-150, negative likelihood was between 0.1-0.2, the ppv was between 84.3-93.7% and npv was between 53.2-92.9%. youden index values with cut off of three ranges between 0.46-0.92, cut off 4, was between 0.6-0.932 and cut off of 5 ranges between 0.6-0.89 (table 2). dn4-interview two studies were included in this review instrument [17, 27] conducted in the arabic and italian languages respectively (table 1) and reported between 2012 and 2017 were participants in arabic, and italian population with a total sample size of 611. patients with neuropathic pain (np) were 248. the number of nociceptive pain patients’ range was 253 patients, and none had mixed pain (mp). forward translation was conducted in two studies thrice (3-times), and out of the three (3) studies that adopted this dn4-interview, one (1) study was not specific on the conduct and the number of times it was conducted. similarly, backward translation was conducted in two (2) studies out of the three (3) studies adopted in dn4-interview, but the number of times conducted was not specific. expert assessment involved, and a pilot study was conducted in two studies included. measurement of the validity of dn4interview instrument internal validity was assessed in two studies with cronbach alpha value between 0.55-93 and 0.86 (using kuder-richardson formula to assess the internal consistency of the whole questionnaire). in two studies cohens kappa lowest score value of 0.92 (brushing) and cohen’s kappa highest score values of 0.9 (electric shocks) and 1.0 (hypoesthesia to brushing, hypoesthesia to pinprick). measurement of reliability of dn4interview roc was conducted in two studies included using this instrument (table 6). at cut off of 2, the sensitivity was 99%, specificity value of 58.3%, positive likelihood value 2.4, ppv of 71%, npv of 98.2%. two studies employ cut off 3 with sensitivity of 97%, specificity value of between 82-82.3, positive likelihood value of 5.5., negative likelihood of 0, ppv of 85% and npv of 96.3%. one study reported cut off value of 4 with sensitivity value of 84%, specificity value t.r. fagbohun 57 in t e r n a l m e d ic in e issn 2413-6077. ijmmr 2021 vol. 7 issue 1 of 90%. positive likelihood value of 8.1, and negative likelihood of 0.2, ppv 89.2% and npv of 84.3%. youden index value at cut off of 2 was 0.56, cut off 3 was 0.79 and cut off 4 was 0.37. lanss description of the lanss articles demographic characteristic of eight studies included using lanss instrument in assessment of neuropathic pain (table 1). eight (8) studies [20, 24, 28-33] were included in this review. two studies each were reported in the greek [28, 30] and turkish languages [32, 33]. each of the following languages reported one study – brazilian portuguese [31], korean [20], spanish [24] and portuguese [29]. total sample size reported was 1,081. out of this, 612 were diagnosed with neuropathic pain, while 477 were classified to have nociceptive pain. one study reported 42% of the neuropathic pain participants also had mixed pain [24]. forward translation was conducted in four studies twice and one study once. backward translation was conducted twice in two studies. four studies conducted backward translation once. six studies involved expert assessment while four studies conducted pilot studies. lanss measurement of validity internal validity assessment was conducted in six studies, with the cronbach alpha ranging between 0.65-0.96 and the test-retest reliability conducted in four studies (table 4). one study reported the intra class coefficient value of 0.77 [28] with pearson correlation coefficient reported in two studies (0.912-0.990 and 0.940). the inter-rated reliability was reported in two studies [28, 31] with pearson value of 0.87 in one study [28]. measure of reliability of lanss instrument five studies conducted roc at cut off of 12. six (6) cut off values were reported to different neuropathic pain from nociceptive pain. cut off 2, two (2) studies reported 80.2 and 89.8%, respectively. specificity was 100% and 94.2%. one (1) study reported ppv of 93.6% and npv of 90.74. while one (1) study reported youden index value of 0.8. two (2) studies reported cut off 2, sensitivity was 80.2 and 89.9, specificity was 94.2 and 100, npv was 93.6. ppv was 90.74 with youden index value 0.8. one (1) study reported cut off 7 with sensitivity of 91.2%, specificity value of 83%, positive likelihood of 5.4, negative likelihood value of 0.1, ppv of 86% and npv 89% with youden index value of 0.74. cut off of 10.5 was reported in one (1) study with sensitivity of 88%, specificity of 95%. one (1) study reported the use of cut off 11 with sensitivity of 100%, specificity of 95.9%, ppv of 93.6 and npv of 100. four studies used cut off 12 with sensitivity ranging 72.6-98%, specificity range between 74-98%. negative likelihood was reported in two (2) studies, npv, at cut of 7 with value 5.4 and cut off 12 with value 36.3, negative likelihood value 0.1 at cut off 7 and 0.3 at cut off 12, the sensitivity range between 72.6-98, specificity range between 74 98%. positive likelihood was reported in a study with value of 36.3 and negative likelihood 0.3. positive predictive value range between 85-99%, negative predictive value of 76-96%. one study applied cut off 13, with sensitivity of 95%, specificity of 98%, ppv of 99 and negative predictive v=value of 90.57. in addition, one (1) study reported the use of cut off 14 with sensitivity 84, specificity 82.8, ppv 88.7 and npv 76.8. self-lanss description three (3) included studies adopted lanssself between 2010-2016, one (1) study in the greek language [28], one study in the spanish language [38] and one study in the turkish language [37] (table 1). internal validity was reported in three (3) studies with cronbach alpha between 0.67-0.74. test-retest validity was reported in two (2) studies with r-coefficient 964-spearman, 0.97-pearson, respectively. one (1) study reported inter-rater reliability and second r-coefficient. roc was conducted in three (3) studies [28, 37, 38]. three (3) different cut offs were used in this study labelled cut off 1, cut off 2, cut off 3, to distinguish neuropathic pain from nociceptive pain. reliability roc was conducted in three (3) studies (table 7.3) with three different cuts off. one (1) study reported cut off 10 with sensitivity 78.8%, specificity 76.6%, ppv 81.2%, npv 73.9%. one (1) study adopted cut off 10.5, sensitivity 87%, specificity 88%, cut off 11, sensitivity 90.1%, specificity 72.1 %, positive likelihood value of 3.23, negative likelihood value of 0.2, ppv 67.4 and npv 91.0% with youden index value of 0.62. three (3) studies adopted cut off 12 with sensitivity ranging between 72.3-88.7%, specificity ranging between 78.8-95.2%, positive likelihood value of 3.8 and positive likelihood value 0.2 was recorded in one (1) study (lópez-de-uraldevillanueva et al., 2018); ppv ranging between 70.8-96.2%, npv between 69.4-91.4% with youden index value 0.61 reported in one study. cut off of 13 showed sensitivity of 81.7%, specificity 79.3%, positive likelihood value of 4, t.r. fagbohun 58 in t e r n a l m e d ic in e issn 2413-6077. ijmmr 2021 vol. 7 issue 1 ta bl e 3. m ea su re o f re lia bi lit y fo r th e d n 4 in st ru m en t tr an sl at ed au th or ro c* cu t o ff us ed se ns iti vi ty (% ) sp ec ifi ci ty (% ) po si tiv e li ke lih oo d n eg at iv e li ke lih oo d pp v* * (% ) n pv ** * (% ) yo ud en in de x ch at ila e t a l. 17 ye s 3 98 81 .3 5. 2 0 84 .3 97 .5 0. 79 4 96 89 8. 4 0. 1 89 .6 95 .5 0. 85 5 93 96 22 0. 1 85 .8 92 .8 0. 89 ch at ila e t a l. 17 ye s 5 93 95 .8 22 0. 1 95 .8 92 .9 0. 89 4 96 89 8. 4 0. 1 98 .5 95 .5 0. 85 te rk aw i e t a l. 21 te rk aw i e t a l. 21 ye s 4 88 .3 75 .4 sa nt os e t a l. 26 ye s 3 1 37 .3 85 80 0. 62 7 4 1 6. 8 0. 93 2 5 91 5. 1 0. 85 4 m ad an i e t a l. 22 ye s 3 95 88 8. 5 0 89 95 83 4 90 95 20 .2 0. 1 95 91 85 5 83 99 15 0 0. 2 99 86 82 sy ki ot i e t a l. 23 ye s 3 93 .3 52 .5 85 .6 72 .1 0. 46 4 89 78 92 .4 69 .7 0. 67 5 75 85 93 .7 53 .2 0. 6 sp al lo ne e t a l. 27 ye s 4 80 91 .7 9. 6 0. 2 81 .6 90 .8 m at su ki e t a l. 18 pa rk e t a l. 20 ye s 3 10 0 88 .2 0. 88 2 4 87 94 ki m e t a l. 19 ye s 3 10 0 88 .2 0. 88 2 4 87 94 0. 81 2 pe re z et a l. 25 ye s 4 79 .8 78 85 .9 69 0. 58 4 82 78 79 80 .7 0. 6 4 82 78 63 .9 90 .2 0. 6 h am da n et a l. 24 ye s 4 95 97 .2 0. 92 ye s 3 97 82 .3 5. 5 0 85 96 .3 0. 79 t.r. fagbohun 59 in t e r n a l m e d ic in e issn 2413-6077. ijmmr 2021 vol. 7 issue 1 ta bl e 4. m ea su re s of v al id it y fo r tr an sl at ed v er si on s of t h e la n ss au th or as se ss ed in te rn al va lid ity cr on ba ch al ph a as se ss ed t es tre te st re lia bi lit y ic c co rr el at io n co effi ci en t as se ss ed in te rra te r re lia bi lit y co rr el at io n co effi ci en t ic c sc he st at sk y et a l. 31 ye s 0. 67 n o ye s 0. 97 sp an os e t a l. 30 ye s 0. 89 5 ye s 0. 91 to 0 .9 9* (p ea rs on ) n o b at is ta ki e t a l. 28 ye s 0. 65 ye s 0. 94 0 (p ea rs on ) ye s 0. 87 (p ea rs on ) pa rk e t a l. 20 ye s 0. 82 n o n o b ar bo sa e t a l. 29 ye s 0. 77 ye s 0. 77 n o h am da n et a l. 24 n o n o yu ce l e t a l. 33 n o n o n o tü rk el e t a l. 32 ye s 0. 96 ye s 0. 95 (s pe ar m an ) # se e fo ot no te s *r an ge o f r c oe ffi ci en ts fo r ea ch it em : # d iffi cu lt to in te rp re t t es tre te st , a nd in te rra te r re lia bi lit y be ca us e of th e m an ne r of r ep or tin g ta bl e 5. m ea su re s of r el ia bi lit y fo r tr an sl at ed v er si on s of t h e la n ss au th or *r o c* cu t o ff u se d se ns iti vi ty (% ) sp ec ifi ci ty (% ) po si tiv e li ke lih oo d n eg at iv e li ke lih oo d pp v* * (% ) n pv ** (% ) yo ud en in de x sc he st at sk y et a l. 31 n o sp an os e t a l. 30 n o b at is ta ki e t a l. 28 ye s 12 82 .8 95 .2 96 80 10 .5 88 95 pa rk e t a l. 20 ye s 12 72 .6 98 36 .3 0. 3 98 76 n ot s pe ci fie d 7 91 .2 83 5. 4 0. 1 86 89 0. 74 b ar bo sa e t a l. 29 12 89 74 85 81 14 84 82 .8 88 .7 76 .8 h am da n et a l. 24 ye s 2 80 .2 10 0 0. 8 yu ce l e t a l. 33 ye s 2 89 .9 94 .2 93 .6 90 .7 4 tü rk el e t a l. 32 ye s 11 10 0 95 .9 98 10 0 12 98 98 99 96 13 95 98 99 90 .5 7 t.r. fagbohun 60 in t e r n a l m e d ic in e issn 2413-6077. ijmmr 2021 vol. 7 issue 1 negative likelihood value of 0.2, ppv of 71.6%, negative predictive value 87.1 with youden index value of 0.61. paindetect description four (4) studies were included in the translated original using this instrument between 2012 to 2017 in population with local language hindi [15], japanese [36], spanish [35] and turkish [34]. total sample size reported was 974, out of this, 371 participants had neuropathy, 364 and 239 participants were diagnosed with nociceptive and mixed pain respectively. five (5) studies included reported forward translation, the translation was reported twice in five (5) studies. backward translation was reported in five (5) studies conducted twice in five studies. expert assessment was conducted in five studies. pilot study was conducted in one (1) study [15]. paindetect validity characteristic internal validity of the terms was reported in the five (5) studies by cronbach alpha reported ranges from 0.78-0.86. test-retest validity was reported in five (5) studies and icc reported ranges between 0.934-0.98 in five (5) studies included. four (4) studies reported roc [15, 34-36]. paindetect reliability characteristic table 8.3: four cut offs were reported in the studies included using paindetect instrument. cut off 12, the sensitivity was between 84-93%, specificity 66-68%, two studies reported pl 2.7 and 2.9; nl 0.1 and 0.2; ppv was reported by four (4) studies ranging between 73-87%, npv 65-88% and youden index value of 0.575 and 0.519 reported in two studies (table 3). two (2) studies made use of cut off 17, sensitivity 81%, specificity 80 and 81%; positive likelihood 4.1 and 4.3; negative predictive value 65 and 81 with youden index values of 0.613 and 0.624. one (1) study reported cut off of 18, sensitivity 83%, specificity 91%, ppv 90% and npv 84%. three (3) studies adopted cut off of 19, specificity between 71-79%, specificity 83-93 %, positive likelihood reported in two (2) studies with values of 4 and 4.4, negative likelihood 0.3 and 0.4, ppv reported in three (3) studies ranging between 82-90%, npv between 55-79% with youden index reported in two (2) studies of 0.531 and 0.613 (table 3). discussion the aim of the study was to conduct a qualitative systematic review to determine the psychometric property of translated, validation and reliability of neuropathic pain screening tools (lanss, dn4 and pd-q). dn4 instrument the participants’ average sample size was above 30 in all the included studies, and this indicated that all the included studies had the sample size sufficient to represent the popu lation and achieve the aim of the study, and the sample mean on normal distribution. forward and backward translation were conducted in 90% of the included studies from local languages (arabic, brazillian-portuguies, farsi, greek, italian, japanese, korean and spanish) into the english language and from english into local languages in the included studies. tsang et al. [39] reported that this process is an important step in translation, the more times the trans lation the better chances of avoiding the error of bias. the reported cronbach alpha value was higher than 0.6 that indicated an acceptable internal consistency among the items and accu rate translation with exception of one study [17]. the involvement of expert assessment in over 80% of the included studies was in agreement with the set-out guidelines for the process of accurate translation [39]. further more, a pilot study was conducted in most of the studies in cluded which is an essential step in determination of the reliability of the items involved in the questionnaire and the validity of the test instru ment. the value of cohen’s kappa was low and high score reported by the pain expert was higher than normal values indicating a profound agreement among the pain experts. the average high sensitivity values and specificity values reported in the instrument pointed to a good validity of this instrument in differentiating neuropathic pain cohort from non-neuropathic pain groups. the high average value of positive likelihood decreased as the cut off increases. the same pattern of decrease was observed in positive likelihood, however no obvious change in the negative likelihood was evidenced. positive likelihood and negative likelihood were considered important factors in the measurement of sensitivity and specificity in test population. considering the reports from the included studies, an optimum value sensitivity, specificity, positive predictive value was reported at cut off 4 at average value, which could make it a better cut off in agreement with bouhassira et al. [11] in the original development of the dn4 instrument. dn4-interview the participants sample size in this review (n = 416) was greater than 30 in all the included t.r. fagbohun 61 in t e r n a l m e d ic in e issn 2413-6077. ijmmr 2021 vol. 7 issue 1 studies [17, 27] using dn4-interview instrument; this indicated that all the studies were statistically adequate, and the sample mean was on normal distribution. forward and backward translation were conducted in 67% from local languages (arabic and italian) to the english language and from english to local languages at least (three times) which were reported as important steps in translation process, as mentioned previously, the more times the translation – the better chances of avoiding the error of bias. tsang et al. [39] recommended a minimum of twice forward and backward translation for a good translation procedure. there was no specification on the number of times for backward translation that could lead to a possible limitation when using this instrument. the re_ viewed cronbach alpha value using this instrument (0.55-0.862) was averagely higher than 0.6 (the minimum cronbach alpha value for a good internal consistency) that indicated an acceptable internal consistence among the items, a general internal consistency measured (dichotomized measurement of reliability) by the kuder-richardson formula (0.86) which is close to 1 as recommended for a good reliability [40] with exception of one study [17]. moreover, expert assessment in over 80% of the included studies was also corroborating with the set-out guidelines for the process of adequate translation [39]. inter-rated reliability review showed a close point (0.9) to 1 in bru_ shing at low cohen kappa and 1 in hypoesthesia to brushing and pinprick. this indicates a high reliability in these two signs of measuring neuropathic pain and shows that this instrument is a good instrument and is consistent among the pain-expert. therefore, this instrument could be used to distinguish neuropathic pain from non-neuropathic pain. pilot study was conducted in most of the studies (67%) included studies, this is an essential step in determination of the reliability of the items involved in the questionnaire and hence the validity of the test instrument. the optimum sensitivity (84-99%) and specificity (58-90%) reported in the instrument pointed to the fact that this instrument (dn4-interview) was a highly sensitive and valid in distinguishing neuropathic pain quality from non-neuropathic pain. comparing the optimal test scores value of the translated dn4-interview instrument in the included studies with the original dn4interview test score values, the performance of the translated was not as good as the original version. lanss our review on the psychometric translation properties using lanss showed the sample size (n=90-213) indicating a good statistical sample. forward and backward translation were conducted (80%) for the review [20, 28, 29, 31, 33] of the reviewed studies as compared with the original version of lanss. test-retest reliability was conducted in 55% of the studies within the pain experts. only one study evaluated the intra-class correlation coefficient (0.77) [29]. this is contrary to the expectation from the original version, which showed that the included studies reported cronbach alpha (0.67-0.9612) higher than 0.6, indicating a good internal consistency among the items. forward and backward translation were conducted with the instrument good translation procedures in 75% of the included studies. fifty percent (50%) complied with minimum of twice forward translation while twenty percent (20%) complied with minimum of twice backward translation. this shows that there were gaps in translation procedures in 70% of the included studies [24, 29, 31-33]. inter-rated reliability was conducted in 20% of the included studies, which were considered as an important step in measurement of reliability in instrument testing and the validity of the instrument as compared with original lanss translation procedure. this is in contrary to the setout procedure for a good neuropathic pain instrument. the average sensitivity value (85%) and specificity values (92%) observed using this instrument indicated that lanss was a very sensitive instrument and specific to measure the neuropathic components of a pain patient across all languages. average positive predictive value (93.7%) showed that the instrument was an effective instrument in the determination of com ponents of lanss questionnaire that marked out neuropathic pain components. this agrees with the bennett study on the development of lanss neuropathic pain screening tool [9]. s-lanss the is a modification of the original lanss instrument without the clinical examinations that was developed by bennett et al. [41]. our review indicated that the internal consistence measured by cronbach alpha (0.67-0.74) showed high internal consistency among the items included in the instrument in a population sample size of average (n=145), which was statistically dependable sample size. forward and backward translations were conducted in t.r. fagbohun 62 in t e r n a l m e d ic in e issn 2413-6077. ijmmr 2021 vol. 7 issue 1 ta bl e 6: m ea su re s of r el ia bi lit y fo r tr an sl at ed v er si on s of t h e sla n ss au th or as se ss ed in te rn al va lid ity cr on ba ch al ph a as se ss ed te st -r et es t re lia bi lit y ic c co rr el at io n co effi ci en t as se ss ed in te r ra te r re lia bi lit y co he ns k ap pa (lo w es t s co re ) co he ns ka pp a (h ig he st sc or e) co rr el at io n co effi ci en t ic c b at is ta ki e t a l. 28 ye s 0. 67 ye s 0. 96 4 (s pe ar m an ) ye s 0. 54 (p ea rs on ) ló pe zde -u ra ld evi lla nu ev a et a l. 38 ye s 0. 71 n o n o -ko c an d er de m og lu 37 ye s 0. 74 ye s 0. 97 (p ea rs on ) n o ta bl e 7. m ea su re s of r el ia bi lit y fo r tr an sl at ed v er si on s of t h e sla n ss au th or ro c* cu t o ff us ed se ns iti vi ty (% ) sp ec ifi ci ty (% ) po si tiv e li ke lih oo d n eg at iv e li ke lih oo d pp v* *( % ) n pv ** *( % ) yo ud en b at is ta ki e t a l. 28 ye s 12 86 .2 95 .2 96 .2 83 .3 3 10 .5 87 88 ló pe zde -u ra ld e vi lla nu ev a et a l. 38 ye s 11 90 .1 72 .1 3. 23 0. 1 67 .4 91 .9 0. 62 12 88 .7 76 .6 3. 8 0. 2 70 .8 91 .4 0. 65 13 81 .7 79 .3 4 0. 2 71 .6 87 .1 0. 61 ko c an d er de m og lu 37 ye s 12 72 .3 80 .4 82 .5 69 .4 -10 78 .8 76 .6 81 .2 73 .9 re ce iv er o pe ra tin g ch ar ac te ri st ic , * * – po si tiv e pr ed ic tiv e va lu e, * ** – n eg at iv e pr ed ic tiv e va lu e. t.r. fagbohun 63 in t e r n a l m e d ic in e issn 2413-6077. ijmmr 2021 vol. 7 issue 1 ta bl e 8. m ea su re s of v al id it y fo r tr an sl at ed v er si on s of t h e pa in d et ec t au th or as se ss ed in te rn al va lid ity cr on ba ch al ph a as se ss ed te st -r et es t re lia bi lit y ic c co rr el at io n co effi ci en t as se ss ed in te rra te r re lia bi lit y co he ns ka pp a (l ow es t s co re ) co he n’ s ka pp a (h ig he st s co re ) co rr el at io n co effi ci en t g ud al a et a l. 15 ye s 0. 83 ye s 0. 94 n o m at su ba ya sh i e t a l. 36 ye s 0. 78 ye s 0. 94 n o d e an dr és e t a l. 35 ye s 0. 86 ye s 0. 93 4 n o al ka n et a l. 34 ye s 0. 81 ye s 0. 98 al ka n et a l. 34 ye s 0. 81 ye s 0. 98 ta bl e 9. m ea su re s of r el ia bi lit y fo r tr an sl at ed v er si on s of t h e pa in d et ec t au th or ro c* cu t o ff u se d se ns iti vi ty (% ) sp ec ifi ci ty (% ) po si tiv e li ke lih oo d n eg at iv e li ke lih oo d pp v* *( % ) n pv ** *( % ) yo ud en g ud al a et a l. 15 ye s 19 79 93 91 81 12 90 66 73 87 18 83 91 90 84 m at su ba ya sh i e t a l. 36 d e an dr és e t a l. 35 ye s 12 93 68 2. 9 0. 1 87 80 0. 60 9 19 75 84 4. 7 0. 3 92 60 0. 59 5 17 81 81 4. 3 0. 2 91 65 0. 62 4 al ka n et a l. 34 ye s 12 90 68 2. 8 0. 2 73 87 0. 57 5 19 78 83 4. 4 0. 3 82 79 0. 6 17 81 80 4. 1 0. 2 80 81 0. 61 3 al ka n et a l. 34 ye s 12 84 68 2. 7 0. 2 86 65 0. 51 9 19 71 83 4 0. 4 90 55 0. 53 1 t.r. fagbohun 64 in t e r n a l m e d ic in e issn 2413-6077. ijmmr 2021 vol. 7 issue 1 all the studies (two times) that was the minimum number of times it should be carried out; pilot study was conducted with involvement of expert assessment in all the included studies. this review proved cronbach alpha (0.67-0.74 range) of above 0.6 recommended for a good internal consistency in translation research [42]. however, the test – retest reliability was conducted (90%) of the included article, which is an important procedure to measure good translation procedure. our review on this instrument showed an average sensitivity value (83.02%) and specificity (80.3%); these values indicated that the instrument was sensitive in determination of the neuropathic component of pain patients. the value of the sensitivity deceased as the cut off value increases from 12 to 13 in a population sample size of 154 [38] in association with decrease in the value of npv as the cut off value deceased. this review showed an optimum value of sensitivity, spe cificity, and the positive predictive value at cut off 12, indicating a high performance of the instrument. this cut off 12 was also reported by the three studies, which could make it an acceptable cut off to distinguish neuropathic pain patients from non-neuropathic pain patients. pd-q all the included studies conducted forward and backward translation twice (in agreement with the recommended standard). most of the studies did not conduct any pilot study. this is contrary to the guideline for translation procedures, which is an important primary step. the internal consistency of the items measured by evaluating the cronbach alpha values (0.780.86) showed a high internal consistency among the items listed on the instrument. our review showed that test-retest reliability was conducted in all the included studies with the average value of interclass correlation coefficient (0.95), suggesting a high validity of the instrument in all the translated versions of pd-q reviewed in the diagnosis of neuropathic pain from non-neuropathic pain. the studies showed an average sensitivity value (82.3%) and specificity (80.5%) that indicated that pdq was a sensitive instrument in the determination of neuropathic pain component, and the items were very specific in the determination of the symptoms of neuropathic pain. conclusions the original dn4 and lanss had the most evidence for their psychometric, reliability and validity properties in peer-reviewed articles. these tools were designed to assess the neuropathic pain quality in a test population through differentiating signs and symptoms between neuropathic and non-neuropathic pain patients. furthermore, these screening questionnaires may provide an indication of the presence of neuropathic pain quality; however, they cannot replace a clinical assessment. it is clear from the studies that most of the instruments do not assess psychometric, reliability and validity properties effectively. for those that were assessed, not all of them were satisfactory, and most of the findings were supported by low or very low level of evidence. in conclusion, we recommend that both the clinical assessment and neuropathic painscreening tool are pivotal in the diagnosis of neuropathic pain component in pain patients in clinical settings. recommendations these three neuropathic pain screening tools (dn4, lanss and pdq) translated version as performed ultimately well in other local languages at their test population but none of these has been developed in the african language, it will be a valuable interest also to evaluate the performance of this tool by determining the reliability and validity properties. to increase the sensitivity, reliability and validity of these screening tools, efforts should be taken to carry out forward and backward translation more than twice from original version of the tools. additionally, the use of language translation experts in both the original version and the translated local version of the original screening tool should be used for transcription and translation process. moreover, translation into as many local languages as possible should be made to ensure consistency of the methodology and the properties should be measured by the tools. this would be especially valuable in the sub-saharan african region where most of the population might not be proficient in the lingua franca. we have just completed the translation of the dn4 screening tool into isizulu (a commonly spoken nguni african language in south africa) in our research group. in future studies, we will assess the sensitivity, reliability, and validity of this translated version and evaluate its feasibility in line with other previously translated versions. conflict of interests there are no conflicts of interest between the author and other researchers that contributed to data extraction. t.r. fagbohun 65 in t e r n a l m e d ic in e issn 2413-6077. ijmmr 2021 vol. 7 issue 1 acknowledgements my profound gratitude to professor peter kamerman and dr antonia wadley of pain laboratory, school of physiology, faculty of health sciences, university of witwatersrand medical school for their intellectual contributions in screening of the articles. i also appreciate the funding support (frc funding) from the faculty of health sciences and professor william daniels for funding this research through the nrf supervisor link funding, wits university, johannesburg, south africa. autho’s contributions temitope richar d fagbohun – conceptualization, methodology, formal data collection and analysis, writing – original draft, writing – reviewing and editing. систематичний огляд методів психометрії, надійності та валідності перекладених опитувальників для способів скринінгу нейропатичного болю (dn4, lanss і pdq) 1 січня 2005 – 19 липня 2019 t.r. fagbohun university of the witwatersrand, johannesburg, south africa вступ. для скринінгу нейропатичного болю використовують різні опитувальники (dn4, lanss та pdq), які були перекладені на декілька локальних мов та валідовані. щоб визначити надійність цих засобів та їх здатність відрізняти нейропатичний біль від ноцицептивного при діагностиці, було проведено систематичний огляд для синхронізації властивостей та припущення про надійність перекладеної версії цих засобів скринінгу нейропатичного болю. мета. провести обґрунтований систематичний огляд для оцінки психометрії, надійності та валідності dn4, lanss та pdq у період з січня 2005 по 2019 рік. методи. двоє незалежних рецензентів провели пошук відповідних статей у pubmed, scopus та web of science на основі критеріїв включення jbi (інститут джоани бріггс). дані, отримані зі статей, були синтезовані у вигляді таблиці. результати. в огляд були включені двадцять шість статей з dn4 (n=11), lanss (n=8) та pdq (n=4), перекладених з англійської мови на вісім місцевих мов. чутливість та специфічність шкали dn4 коливалися від 75% до 98% та 37,3% до 96% відповідно. внутрішня надійність (α) перекладеної версії dn4 коливалася в межах 0,55-0,862 чутливість та специфічність шкали lanss коливалися від 75% до 98% та 37,3% до 96% відповідно. внутрішня надійність (α) перекладеної версії lanss перебувала в межах 0,67-0,96 чутливість та специфічність шкали pdq коливалися від 75% до 98% та 37,3% до 96% відповідно. внутрішня надійність (α) перекладеної версії pdq знаходилася в межах 0,81-0,86. висновки. усі перекладені інструменти продемонстрували хорошу внутрішню узгодженість елементів, високу чутливість та позитивне прогностичне значення, однак не досягали рівня оригіналів. тому для належної діагностики пацієнтів з нейропатичним болем ці скринінгові інструменти пропонується використовувати разом з клінічним обстеженням. ключові слова: нейропатичний 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of test reliability. psychometrika. 1937 sep;2(3):151-60. doi: 10.1007/bf02288391 41. bennett mi, smith bh, torrance n, potter j. the s-lanss score for identifying pain of predominantly neuropathic origin: validation for use in clinical and postal research. the journal of pain. 2005 mar 1;6(3):149-58. doi: 10.1016/j.jpain.2004.11.007 42. taber ks. the use of cronbach’s alpha when developing and reporting research instruments in science education. research in science education. 2018 dec;48(6):1273-96. doi: 10.1007/s11165-016-9602-2 received 20 may 2021; revised 22 jun 2021; accepted 23 jun 2021. this is open access article distributed under the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. t.r. fagbohun 44 b io m e d ic a l s c ie n c e s issn 2413-6077. ijmmr 2016 vol. 2 issue 1 doi 10.11603/ijmmr.2413-6077.2016.1.6382 molecular apoptosis mechanisms with underlying experimental acute lung injury m. i. marushchak, i. m. klishch, yu. i. bondarenko, l. p. mazur i. horbachevsky ternopil state medical university, ternopil, ukraine background. current data suggest systemic autoimmune activation in the pathogenesis of bronchopulmonary diseases. the imbalance in the system of proand anti-inflammatory cytokines is very important in immunopathogenesis. objective. the aim of our research was to determine the caspase-3 rate in the dynamics of experimental acute lung injury and to study the relationship between their level and the number of cells carrying membrane binding tnf receptor type 1 to define the main mechanisms of cell death. results. the analysis of the results of caspase-3 rate in lung homogenate showed that this cysteine proteinase was uniformly increasing in all experimental groups during simulating of ali induced by administration of hydrochloric acid (p<0.001). when comparing the results of caspase course of apoptosis it was defined that, despite the progressive increase in caspase-3 rate in lung homogenate, cysteine proteinase rate in plasma did not change. the receptor mechanism of apoptosis was studied by establishing correlation relationships with the number of cells carrying membrane binding tnf type 1 (tnf-r1) receptor. a strong positive correlation relationship between the number of neutrophils with tnf-r1 and caspase-3 rate in lungs of all research groups was determined. conclusions. the implementation of neutrophils death by apoptosis is caused by change of activity of caspase cascade effector components, such as caspase-3, in cases of ali induced by intratracheal administration of hydrochloric acid. one of the potential mechanisms responsible for the activation of caspase course is excessive generation of active forms of oxygen and increase in the number of neutrophils carrying membrane binding tnf receptor type 1. key words: caspase-3, tumour necrosis factor alpha receptor 1, acute lung injury introduction current data suggest systemic autoimmune activation in the pathogenesis of broncho­ pulmonary diseases. the imbalance in the system of proand anti-inflammatory cytokines is very important in immunopathogenesis. acute lung injury is manifested by acute inflammatory response in the lung parenchyma that is associated with the severity of damage to the epithelial and endothelial barriers [1]. the latest researches have proved that cytokines, such as tumour necrosis factor alpha (tnf), are the signal molecules of the beginning, deve lopment and progression of inflammatory res ponse at local and systemic levels. tnf antagonists are soluble forms stnf-ri and stnf-rii, which are formed by separation of active receptor extracellular part from cell membrane [2, 3]. currently some rather contradictory evidence was published on the effect of cytokines on the programmed cell death. cytokine rate can be determined by its dose, type of target cells, their functional state and lesions [4]. two courses of apoptosis are: internal or mitochondrial by bcl-2 protein family, cyto­ chrome c and caspase­9; and external by cas­ pase-8 activation upon binding of specific fas cells receptor – and soluble receptors of tumour necrosis factor on the cell surface [5]. caspases, or cysteine asparagine­protease can be consi­ dered a critical effector molecules of prog ram­ med cell death, in this case caspase-3 is im portant for the implementation of both mito chon­ drial and receptor apoptosis activating [6]. the aim of our research was to determine the caspase-3 rate in the dynamics of experimental acute lung injury and to study the m. i. marushchak et al. corresponding author: marya marushchak, department of functional diagnostics and clinical pathophysiology, i. hor-i. horbachevsky ternopil state medical university, 1 maidan voli, ternopil, ukraine, 46001 tel.: +380979901202 e-mail: marushchak@tdmu.edu.ua international journal of medicine and medical research 2016, volume 2, issue 1, p. 44–48 copyright © 2016, tsmu, all rights reserved 45 b io m e d ic a l s c ie n c e s issn 2413-6077. ijmmr 2016 vol. 2 issue 1 relationship between their level and the number of cells carrying membrane binding tnf receptor type 1 to define the main mechanisms of cell death. materials and methods the experiments were performed on 54 white nonlinear mature male rats 200–220g in weight, which were kept on a standard diet at the vivarium of ternopil state medical university. the animals were kept and experiments were conducted in accordance with the “european convention for the protection of vertebrate ani mals used for experimental and other scientific purposes” [7]. the animals were divided into 5 groups: the 1st – control group (n=6), the 2nd – animals affected by hydrochloric acid for 2 hours (n=12), the 3rd – animals affected by hydrochloric acid for 6 hours (n=12), the 4th – animals affected by hydrochloric acid for 12 hours (n=12), the 5th – animals affected by hydrochloric acid for 24 hours (n=12). anaesthesia for the rats was administrated intraperitoneally with sodium thiopental, 40 mg/ kg of animal weight. the ventral side of the neck was treated with chlorhexidine and a 0.5 cm medisection was made to visualize the trachea. animals were placed in horizontal po sition at an angle of 45°, hcl, ph 1.2, 1.0 ml/kg was injected by insulin syringe into the trachea at inhale. physiologic saline, 1.0 ml/kg was administered to the animals of the control group. in 2, 6, 12 and 24 hours euthanasia was performed for rats by administration of sodium thiopental, 90 mg/kg of the animal weight, following the principles of humane treatment of animals. after their death chest was prosected and cardiopulmonary complex was separated. heparinized whole blood, lung homogenates and bronchoalveolar lavage (bal) was used for the research. the standard technique was performed to obtain bal from lungs [8]. to determine caspase rate in lung homogenate supernatant and leukocyte-lymphocyte blood fractions, 0.25 ml of buffer and 50 mcl of 2 mm devd-p-na was added to 0.7 ml of the test liquid and it was incubated for 2 hours at 37°c; the intensity of light absorbance was measured at 405 nm, which is directly proportional to the product of hydrolysis of acetyl-aspglu-val-asp n-nitroanilide caspase – 3-n-nitroanilide [9]. the number of bal neutrophils that keep membrane binding tnf receptor type 1 (tnf-r1) was evaluated by the method of flow laser cytometry by means on flow cytometer epics xl (beckman coulter, usa) using radio-labeled monoclonal antibodies to tnf-r1 (cd120a) (hycult biotech, netherlands) [10]. statistical analysis was conducted using the software statistica 6.0. to compare the differences between groups we used t-test in cases of a parametric distribution of alternatives, for calculating other data – one-way anova (fisher lcd post-hoc test), nonparametric analysis (mann-whitney test). the values are presented as mean±sd, where mean denotes the mean rate, sd – standard mean error. p<0.05 was considered statistically significant. correlation analysis was performed between all the studied rates. coefficient of linear corre lation (r) and its fidelity (p) was calculated that was accordingly denoted in the tables (correlation matrices). the correlation coefficient was significant at p<0.05. results caspase-3, which cleaves proteins important for maintaining of cellular homeostasis, is considered to be the main effector molecule of the ‘executive’ stage in many models of apoptosis. so it was reasonable to determine its rate during apoptosis induced by hydrochloric acid when simulating ali. our research on caspase-3 rate showed that the content of this proteinase in blood of the experimental animals suffering from ali did not change if compared with the data of the control and experimental groups (p>0.05) (table 1). the analysis of the results of caspase-3 rate in lung homogenate showed that this cysteine proteinase was uniformly increasing in all experimental groups during simulating of ali induced by administration of hydrochloric acid (p<0.001). so, in 2 hours after the beginning of the experiment the caspase-3 rate increased by 49.33% in comparison with the control, in 6 hours – by 26.94% if compared to the second experimental group, in 12 hours – by 23.67% if compared to the third experimental group and in 24 hours – by 28.66% if compared to the previous group (table 1). when comparing the results of caspase course of apoptosis it was defined that, despite the progressive increase in caspase-3 rate in lung homogenate, cysteine proteinase rate in plasma did not change. this evidenced the difference in the implementation of programmed cell death, which could be caused by: 1. the varying levels pro-apoptotic signals in blood and lungs; 2. different amount of cells bearing apoptogenic receptors. m. i. marushchak et al. 46 b io m e d ic a l s c ie n c e s issn 2413-6077. ijmmr 2016 vol. 2 issue 1 it was established that all populations of white blood cells, which are involved in the inflammatory process of ali such as neutrophils, secrete cytokines, and vascular endothelium is their main target [11]. the recent researches have proved that cytokines, such as tumour necrosis factor alpha (tnf), are signal molecules of the beginning, development and progression of inflammatory response at local and systemic levels. the bioactivity of tnf depends on the content of cytokine corresponding receptors on the surface of target cells and the number of circulating antagonists [12]. so, the receptor mechanism of apoptosis was studied by establishing correlation relationships with the num ber of cells carrying membrane binding tnf type 1 (tnf-r1) receptor. a strong positive correlation relationship between the number of neutrophils with tnf-r1 and caspase-3 rate in lungs of all research groups (table. 2) was determined. table 1. rates of caspase-3 in blood plasma and lung homogenate of rats with underlying experimental acute lung injury (m±m) rate control group n=6 experimental groups 2 n=6 3 n=6 4 n=6 5 n=6 caspase-3, pmol/mg of protein (blood) 19,43±0,88 18,50±1,45 16,65±1,64 15,98±1,41 16,23±1,36 р р1>0,05 р1,2>0,05 р1,2>0,05 р1,2>0,05 caspase-3, pmol/mg of protein (bal) 23,96±4,40 35,78±2,54 45,42±2,72 56,17±3,42 72,27±4,71 р р1<0,001 р1,2<0,001 р1,2<0,001 р1,2<0,001 legends: р1 – significant difference if compared to the control animals; р2 – significant difference if compared to the affected animals. table 2. possible relationships between caspase-3 rate and the number of cells carrying membrane binding tnf type 1 receptor in cases of acute lung injury rate experimental groups correlation coefficient, rxy correlation relationship probability, р tnf-r1 rate in ronchoalveolar lavage, % caspase-3 rate in lung homogenate, pmol/mg of protein 2 0,88 <0,01 3 0,90 <0,001 4 0,95 <0,001 5 0,81 <0,01 discussion a significant increase in caspase-3 rate could be caused by involvement of mitochondrial course of apoptosis, which was associated with the pro-apoptotic signals from inside the cells, such as active forms of oxygen. previously we proved that intensification of free radical peroxidation processes happened in cases of ali, and active forms of oxygen were the main cause of that [13]. the generation of oxygen radicals stimulated apoptosis by decrease in mitochondrial membrane potential that verified the mitochondria cell membrane poration and depolarization [14]. caspase-8, which is activated by the interaction of tumour necrosis factor-α and membrane binding receptor of this interleukin, con tribute to pores formation. as a result, mitochondrial matrix swelling developed; internal mitochondrial membrane ruptured; and cytochrome c, aif (apoptosis inducing factor), which stimulated caspase-3, secondary activator of caspases of mitochondrial origin and other pro-apoptotic proteins released from the intermembranous space into cytosol [15, 16] (figure 1). caspase-3 rate is regulated by both external and internal tnf receptor mediated mechanisms of apoptosis. currently, it is established that most of the cytotoxic effects of tnf are mediated by tnf-r1 due to its interaction with tradd (death domains caused by tnf-r1) [17]. our research also proved it. we evidenced a significant increase in caspase-3 rate with increase in percentage of neutrophils carrying tnf-r1 in cases of ali induced by intratracheal administration of hydrochloric acid. conclusions the implementation of neutrophils death by apoptosis is caused by change of activity of caspase cascade effector components, such as m. i. marushchak et al. 47 b io m e d ic a l s c ie n c e s issn 2413-6077. ijmmr 2016 vol. 2 issue 1 caspase-3, in cases of ali induced by intratracheal administration of hydrochloric acid. one of the potential mechanisms responsible for the activation of caspase course is excessive generation of active forms of oxygen and increase in the number of neutro phils carrying membrane binding tnf receptor type 1. acute lung injury induced by administration of hydrochloric acid neutrophils activation, move to lesion generation of active forms of oxygen decrease in mitochondrial membrane potential aif, apaf-1 release into cytoplasm caspase-3 activation apoptotic cell death figure 1. pathogenic justification of mitochondrial course of apoptosis in cases of acute lung injury. future prospects of the research in the further research, for pathogenetic study of programmed cell death course we plan to conduct a comparative analysis of the correlation relationships between the early apoptosis level and mitochondrial trans mem brane potential rates, active forms of oxygen and caspase rate in blood and bronchoalveolar lavage in rats to detect additional pathogenetic mechanisms of acute lung injury deve lopment. references 1. mitchell rs, martin tr. lung cytokines and ards. chest 1999; 116: 2–8. 2. schneider-brachert w, tchikov v, neumeyer j et al. compartmentalization of tnf receptor 1 signa ling; internalized tnf receptosomes as death vesicles. j. immunity 2004; 21 (3): 415–428. 3. macewan dj. tnf ligand and receptors – a matter of life and death. british jour. of pharm 2002; 135: 855–875. 4. roth z’graggen b, tornic j, müller-edenborn b et al. acute lung injury: apoptosis in effector and target cells of the upper and lower airway compartment. clinical and experimental immunology 2010; 161: 324–331. 5. kaminski m, kiebling m, suss d et al. novel role for mitochondria: protein kinase cθ-dependent oxidative signaling organelles in activation-induced t-cell death. mol cell biol 2007; 27 (10): 3625-3639. 6. глумчер фс, березняков иг, решедько гк. 1 украинский конгресс по вопросам антимикробной терапии: событие для отечественного здравоохранения. здоров’я україни 2007; 2 (1): 16–18. 7. european convention for the protection of vertebrate animals used for experimental and other scientific purposes. council of europe. strasbourg 1986; 123: 52. 8. гудима аа, марущак мі, габор гг, куліцька мі. патогенетична роль нейтрофільних гра нулоцитів у розвитку гострого ураження ле гень. буковинський медичний вісник 2011; 3: 82–86. 9. bonomini m, dottori s, amoroso l et al. increased platelet phosphatidylserine exposure and caspase activation in chronic uremia. j thromb haemost 2004; 2(8): 1275–1281. 10. часовских ню. роль протеинкиназ jnk и р38 в регуляции апоптоза мононуклеарных лейкоцитов крови при окислительном стрессе. бюллетень сибирской медицины 2008; 3: 38–43. 11. пасечник ав, фролов ва. апоптоз нейтрофилов как параметр воспалительной реакции m. i. marushchak et al. 48 b io m e d ic a l s c ie n c e s issn 2413-6077. ijmmr 2016 vol. 2 issue 1 при патологии. вестник рудн. серия медицина 2004; 25 (1): 103. 12. mann dl. recent insights into the role of tumor necrosis factor in the failing heart. heart fail rev 2001; 6(2): 71–80. 13. грищук ла, марущак мі. динаміка перекисного окиснення ліпідів та антиоксидантного захисту в щурів за умов гострого ураження легень. туберкульоз, легеневі хвороби, віл-інфекція 2011; 2 (05): 16–20. 14. мишуніна тм, тронько мд. oсновні молекулярні механізми апоптозу та їх порушення при канцерогенезі щитоподібної залози (огляд літератури). журн амн україни 2006; 12 (4): 611–633. 15. райхлин нт, райхлин ан. регуляция и проявление апоптоза в физиологических условиях и в опухолях. вопр онкол 2002; 48 (2): 159–171. 16. мишуніна тм, калініченко ов, тронько мд, зурнаджи лю. характеристика змін проник ності мембран мітохондрій з тканини папі лярних карцином щитоподібної залози та з її тканини за інвазії пухлинних клітин. журн амн україни 2010; 16 (1): 5–22. 17. chopra m, reuben js, sharma ac. acute lung injury:apoptosis and signaling mechanisms. experimental biology and medicine 2009; 234: 361–371. received: 2016-02-02 m. i. marushchak et al. 63 b io m e d ic a l s c ie n c e s issn 2413-6077. ijmmr 2016 vol. 2 issue 2 doi 10.11603/ijmmr.2413-6077.2016.2.7001 endogenous intoxication in animals of different age groups in case of polytrauma s. r. pidruchna, h. m. stepanova, b. o. pereviznyk, n. a. vasylyshyn, a. a. hudyma, l. s. donchenko i. horbachevsky ternopil state medical university, ternopil, ukraine background. associated injury is a worldwide social and economic problem. age related aspects of endogenous intoxication are not studied comprehensively. annually, from 44 000 to 65 000 citizens die because of traumatic injuries. as a result, this number increased by 32.6% for the last 10 years. the detoxification system, as a component of the functional systems of the organism, experiences significant changes in case of polytrauma. objective. the study was aimed to discover pathogenetic peculiarities of the multiple trauma in age aspect in different disease periods and to explore the level of endogenous intoxication in this condition. methods. the experiments were performed on 72 white male rats aged 3, 6 and 12 months, which underwent simulation of severe skeletal trauma and examination of the contents of middle mass molecules and endogenous intoxication index (markers of endogenous intoxication) in 1, 4 and 24 hours after the associated injury. results. the most significant increase of the middle mass molecules was fixed in 24 hours after modeling of severe skeletal injuries in all groups of animals, especially it was the most pronounced in 12-month-old animals. the erythrocyte intoxication index reached the highest level in 4 hours after the injury, its increase was most significant in sexually mature adult animals. conclusions. a significant increasing of endogenous intoxication markers in 12-month-old rats, if compared to 3and 6-month-old animals, can be caused by the decrease in compensatory protection mechanisms. key words: multiple trauma, endogenous intoxication, age. corresponding author: svitlana pidruchna, department of medical biochemistry, i. horbachevsky ternopil state medical university, 1 maidan voli, ternopil, ukraine, 46001 phone number: +380352254784 e-mail: pidruchna@tdmu.edu.ua introduction these days, the increased number of associated is one of the most important social and economic problems [1–7]. in ukraine the mortality rate, as a consequence of injury, is 91.8% of cases per 100 000 citizens. annually, from 44 000 to 65 000 citizens die because of traumatic injuries. as a result, this number increased by 32.6% for the last 10 years. the detoxification system, as a component of the functional systems of the organism, experiences significant changes in case of multiple trauma. the disturbance of functioning of this system leads to the development of endogenous intoxication syndrome (eis), which causes the primary diseases and the complications due to increased tissue decay, escalation of catabolic processes, insufficient functioning of internal organs [11, 12]. among the causes of death during terminal stage are multiple organ failure and consistent progressive failure of organ systems as a result of injury. nowadays the age-related mechanisms of eis development are not studied comprehensively. there are no complete pathogenetic approaches to the explanation and prognosis of early and late complications of multiple trauma. the study was aimed to discover pathogenetic peculiarities of the multiple trauma in age aspect in different disease periods and to explore the level of endogenous intoxication in this condition. material and methods in this experimental study we used 72 white rats of the age of 3, 6, and 12 months old. the experimental model of multiple trauma was implemented during the study [9]. the control group was comprised of intact animals, which international journal of medicine and medical research 2016, volume 2, issue 2, p. 63-66 copyright © 2016, tsmu, all rights reserved s. r. pidruchna et al. 64 b io m e d ic a l s c ie n c e s issn 2413-6077. ijmmr 2016 vol. 2 issue 2 were kept under the standard vivarium conditions. decapitation was accomplished under thiopental anaesthesia on 1st, 4th, and 24th hour of the experiment in accordance to the european convention for the protection of vertebrate animals used for experimental and other scientific purposes (strasbourg, 1986). we used blood serum for the research. the degree of toxic syndrome was evaluated by the level of middle mass molecules (mmm) [8] and erythrocyte intoxication index (eii) [10]. the received data was processed by method of statistical variation and student t-test. the results were considered as significant at the value р<0.05. results and discussion in our experiment we detected the significant changes of mmm in all 3 animal groups and compared the results with the intact ones. in the 3-month-old rats the increase of мmм254 was observed during the 1 st hour after multiple trauma. the level of these molecules increased by 39%, and after 4 hours it was 48% if compared to the intact animals. on the 1st day after the multiple trauma mmm254 was on 43% higher than normal rates. the similar changes were observed in the 6-month-old rats. during the 1st hour the мmм254 was increasing even by 73%, in 4 hours — by 83% and in 1 day of the experiment it was by 79% higher than in the intact group. the most significant increase in mmm254 was evidenced in the 12-month-old animals. after the 1st hour it increased by 93%, in 4 hours — by 96% if compared to the intact animals, in 1 day — more than in 2 times. the increase of mmm level in animals after multiple trauma proved the enforcement of catabolic processes. the rise of mmm levels, which can include oligopeptides, fragments of nucleic acids, fatty acids, and triglycerides, can prove the injury of the hepatocytes membranes and mmm254 that include purine bases, uric acid and aromatic amino acids — the suppression of the detoxifying function of liver [13]. the differences in the dynamics of changes of mmm280 between the 1 st, 2nd and 3rd groups were manifested (fig. 2). after the multiple trauma the level of mmm280 in the 3-month-old animal group was higher by 82% if compared to intact animals. in 4 hours it was exceeding the level of control group by 108%, in 1 day — by 98%. fig. 1. the level of мmм254 in rats of different age after multiple trauma (in percentage if compared to the level of the intact animals) in dynamics. fig. 2. the level of мmм280 in rats of different age after multiple trauma (in percentage if compared to the level in the intact animals) in dynamics. 142,73 179,09 148,18139,39 100 172,73 182,73 203,64192,73 196,36 0 50 100 150 200 250 intact 1 hour 4 hour 1 day 3 months 6 months 12 months 198218 208 182 100 204 236 448 370 434 0 50 100 150 200 250 300 350 400 450 500 intact 1 hour 4 hour 1 day 3 months 6 months 12 months s. r. pidruchna et al. 65 b io m e d ic a l s c ie n c e s issn 2413-6077. ijmmr 2016 vol. 2 issue 2 in the 6-month-old group mmm280 was higher by 104% if compared to the control group, in 4 and 24 hours — by 136% and 98% respectively. the highest level of mmm280 was observed in the 12-month-old animals in all periods of the experiment. in 1 hour after multiple trauma, it was exceeding the level of the intact animals by 270%, in 4 hours — by 344%, in 1 day — by 448%. we argue that the increase in mmm levels is a manifestation of catabolic processes enforcement in cells of vital tissues. the possible cause for these changes can be the activation of intracellular, particularly lysosomal proteases under the influence of these toxins [14]. the multiple trauma also influenced on the level of erythrocyte membranes impairment (fig. 3). 192,24 213,04 197,2 184,47 100 207,14 224,22 248,76227,33 244,72 0 50 100 150 200 250 300 intact 1 hour 4 hour 1 day 3 months 6 months 12 months fig. 3. the level of eii in rats of different age after multiple trauma (in percentage if compared to the level of the intact animals) in dynamics. eii significantly increased during the 1st hour after multiple trauma in the 3-month-old rats and exceeded the level of the intact animals by 85%. in 4 and 24 hours of the experiment it increased by 97% and 92% respectively. in the 6-month-old rats, in 1 hour the eii was higher than in the intact animals by 107%, in 4 hours — by 124%, in 1 day – by 213%. in the 12-month-old rats, in 1 hour the eii was higher by 127% if compared to the intact rats, in 4 hours — by 245%, and in 1 day — by 149%. in case of mmm the eii reached the highest level. the data obtained prove the action potentiation of endogenous toxins that are excreted to blood in case of multiple trauma; it can be accompanied by either high level of catabolic processes or the suppression of the detoxification system functional activity [15]. conclusions mmm and eii proved serious age-related differences between the animals with multiple trauma. in all animal groups the highest value was reached during the 1st hour after the injury and continued with concomitant slow decline, especially in the 3and 6-month-old animals in 4 hours after the injury. however, in the 12-month-old rats the increase of mmm and eii continued; it proved prolongation of endogenous intoxication. the further research on endogenous intoxication will broaden the view on the pathogenesis of traumatic diseases in people of different age and in various periods of the diseases that will provide the opportunity to make prognoses on further course of the disease. references 1. asehnoune k, еdouard a. inflammatory response and polytrauma: an update. reanimation 2006; 15: 568–575. 2. balogh z, varga e, tomka j, et al. the new injury severity score is a better predictor of extended hospitalization and intensive care unit admission than the injury severity score in patients with multiple orthopaedic injuries. j orth trauma 2003; 17: 508–512. 3. brenneman fd, boulonger br, mclellan ba, et al. measuring injury severity: time for a change? j trauma 1998; 44: 580–582. s. r. pidruchna et al. 66 b io m e d ic a l s c ie n c e s issn 2413-6077. ijmmr 2016 vol. 2 issue 2 4. dimar jr, carreon ly, riina j, et al. early versus late stabilization of the spine in the polytrauma patient. spine 2010; 35: 187–192. 5. hildebrand f, giannoudis p, kretteck c, pape h. damage control: extremities. injury 2004; 35: 678–689. 6. huber-lang m, wiegner r, lamp l, rolf e. brenner mesenchymal stem cells after polytrauma: actor and target stem. cells international 2016; 10. 7. pomeranz, jl, shaw lr, yu ns, moorhouse md. polytrauma and life care planning: managing the complex interaction of multiple injuries. work 2008; 31(3): 319–326. 8. andreichyn ma, bekh md, demyanenko vv. method of diagnosis of endogenous intoxication of organism: methodical recommendations 1998: 10– 13. (in ukrainian). 9. sekela tia, hudyma aa. patent for the utility model 30028 ukraine ipc 2006 g 09b 23/00. modeling method of polytrauma. ternopil medical university. u 2007 10471; stated. 21.09.2007; published.11.02.08; biul. 3: 4. (in ukrainian). 10. togaybaiev aa, kurguzkin av, rikun iv, karibzhanova rm. method of diagnosis of endogenous intoxication. lab. work 1988; 9: 22–24. (in russian). 11. valdmol bm, volchehorskyi yv, puzhevskyi as, et al. middle molecular peptides of blood as endogenous regulators of lipids peroxidation in normal and thermal burns. medical chemistry questions 1991; 37 (1): 23–26. (in russian). 12. erokhyn ya, havrylyn sv, nemchenko ns. endotoxicosis with severe concomitant injury. surgery journal2001; 160 (5): 120–124. (in russian). 13. kyshkun aa, kudynova as, ofytova ad, myshuryna rb. value of average molecules in appreciation of the endogenous intoxication level military medical journal 1990; 2: 41–44. (in russian). 14. pidruchna sr, vasylyshyn n a, kulyanda oo, kulyanda is. corrective effect of xenodermoplasticson the indexes of endogenous intoxication in severe injury. medical chemistry 2011; 13 (1) 58–62. (in ukrainian). 15. kulyanda oo, pidruchna sr, kulyanda is, zakharchuk um, ostrivka oi. the dynamics of endogenous intoxication in severe injury. pain. anesthesia and intensive care 2010; 2: 54–58. (in ukrainian). received: 2016-10-24 s. r. pidruchna et al. issn 2413-6077. ijmmr 2016 vol. 2 issue 2 37 p u b l ic h e a lt h a n d e p id e m io l o g y issn 2413-6077. ijmmr 2016 vol. 2 issue 2 doi 10.11603/ijmmr.2413-6077.2016.2.7032 physical activity levels in female students of ternopil state medical university d. v. popovych1, j. bergier2, o. m. sopel1, l. s. tsybulska1, m. m. korda1 i. horbachevsky ternopil state medical university, ternopil, ukraine 1 pope john paul ii state school of higher vocational education in biala podlaska 2 background. lifestyle is one of the major factors in the optimal development of systems and functions of human organism and health. the appropriate level of physical activity is the integral component of a healthy lifestyle. objective. the study was aimed to investigate the level of physical activity of the female students of medical university. methods. we applied the international physical activity questionnaire (ipaq) in the long version as a research method. results. the study was conducted among 333 female students of medical university of the first, second and third year of study. detailed international physical activity questionnaire evaluating physical activity, such as work, housework, sport, and physical activity during leisure time, was completed. conclusions. the examined female students of medical university are characterized by high level of total physical activity. place of residence during the studies does not differentiate significantly the level of physical activity of female students. key words: physical activity, the international physical activity questionnaire, female students, medical university. introduction lifestyle is one of the major factors in the optimal development of systems and functions of human organism and health. healthy lifestyle is a way of living aimed at preserving and improving state of health. the appropriate level of phy sical activity is the integral component of a healthy lifestyle (a natural biological need defi ned by the set of movements performed by a person) [1, 2]. many scientific studies proved the value of physical activity for the human body [3–8]. physical activity is an integral part of man's life which largely affects the cardiovascular and muscular-bone systems of the body, and has a significant impact on body metabolism. in current living conditions people focus less on physical activity due to well-developed transporting infrastructure, sedentary work and passi ve rest at home. such way of live may lead to the development of various health risks. at lack of physical activity and excessive neuroemotional stress the functional state of the central nervous system, as a mediator between the muscles and internal organs, is damaged. this causes the dysfunction of individual organs and systems of the body and contributes to the development of various diseases. the influence of physical activity on human health is widely recognized and studied among international researchers [9–13]. the issues of physical activity of students both in bio-physiological and cultural terms are more and more commonly discussed on different conti nents: south asian [14], south africa [15, 16], europe [17–19] and in other different countries [6, 7, 20–22], including medical universities students [23–26]. the research studies [6, 7] show the tendency of the decrease of physical activity among students of higher educational institutions. therefore, the subject of our research was to international journal of medicine and medical research 2016, volume 2, issue 2, p. 37-41 copyright © 2016, tsmu, all rights reserved corresponding author: dariya popovych, department of physical rehabilitation, human health and physical education, i. horbachevsky ternopil state medical university, 1 maidan voli, ternopil, ukraine, 46001 phone number: +380352253669 e-mail: kozak@tdmu.edu.ua d. v. popovych et al. issn 2413-6077. ijmmr 2016 vol. 2 issue 238 issn 2413-6077. ijmmr 2016 vol. 2 issue 2 investigate the level of physical activity of the female students of ternopil state medical university. material and methods the study was conducted among 333 female students of ternopil state medical university during october 2015. the participants were the first­, second­ and third­year­students aged 17-20 years old. the total of 333 female students included: 106 first­year­students (31.83%), 116 second-year-students (34.83%), and 111 third-year-students (33.33%). they were asked to complete questionnaires on demographic and general physical activity. we applied the international physical activity questionnaire (ipaq) in the long version [27] as a research method. detailed international physical activity questionnaire (ipaq) evaluating physical activity, such as work, housework, sport, and physical activity during leisure time was completed. dependence analyses were carried out using t-test and pearson correlation coefficient. the level of statistical significance was adopted at p<0.05. results the results of the research have shown significantly higher level of total physical activity of female students 5590.2 met min/week than in poland [28] and turkey [20]. on the basis of the international physical activity questionnaire (ipaq) it was evidenced that the activity associated with work amounted 1428.9 met min/ week. in case of the activity associated with movement it amounted 1443.7 met min/week. for physical activity associated with housework the results were recorded at 1162.2 met min/ week. рhysical activity in sport amounted 1555.4 met min/week. thus, the highest level of physical activity was identified in female students during sports workouts and the lowest during housework, but these results did not differentiate significantly (fig. 1). based on the data obtained from ipaq questionnaire on the course of study, we analysed the dynamics of physical activity during in the learning process. we established that the highest level of total physical activity of 6955.8 met prevails in sophomores, which is by 24.42% higher than the total-level of pa, while the lowest level of total physical activity of 3855.5 met was found in the third-year female stu_ dents, which is by 31.0% less than the total level of pa. such high rate of physical activity index in the second-year-students was observed due to high levels of physical activity at work/study, which was in 2812.7 met (40.43%), when the index of physical activity at home was 1212.2 met (17.42%), in the sport — 1519.4 met (21.84%), and the rest — 1411.6 met (20.29%). however, in the third-year female students compared to the students of the first and second years of study the lowest level of physical activity during work/training of 463.3 met (12.01%), with activity around the house of 939.1 met (24.35%), in the sport — 1380.7 met (35.81%), and the rest — 1072.4 met (27.81%) was evidenced. when comparing different types of activities of the female students, excluding physical activity during work / study, it was found that freshman have higher physical activity among students at home — 1341.3 met, in sport — 1777.7 met, and the rest — 1867.6 met, figure 1. the level and areas of physical activity of the female students m et -m in /w ee k d. v. popovych et al. issn 2413-6077. ijmmr 2016 vol. 2 issue 2 39 p u b l ic h e a lt h a n d e p id e m io l o g y issn 2413-6077. ijmmr 2016 vol. 2 issue 2 than the secondand third-year female students, which have the lowest physical activity in all types of physical activity (fig. 2, table 1). we found it interesting to discover whether the place of living of the recipients influence upon the level of their physical activity. three places of residence were used for the analysis: dormitory, family home and parents’ apartment. also this combination showed no significant differences in the total physical activity of the female students depending on the place of daily residence (fig. 3, table 2). discussion as a result of our research we have discovered a high level of overall physical activity in female students of medical university (55902 met min/week). after analysing the dynamics of physical activity of female students depending on the year of study we have found out that the highest overall physical activity (6955.8 met) prevailed in the second-year students. such high rate of physical activity in sophomores was due to high level of physical activity at work/ study (it was 40.43% of the total recipients of physical activity). this fact can be explained by the location of university departments and buildings, where classes for the second-yearstudents took place and by the lack of direct transportation between those locations. the average level of physical activity at the work/ school is 463.3 met in the third-year-students and it may explained by the increased duration of classes. in all other areas of physical activity (work, at home, leisure or sport) students showed high level of the researched activity, but it tended to slight decrease from the first to the third year of study. as for the residence of students, no direct connection between the place of residence (dormitory, apartment or parents’ house) and the physical activity was found [17]. thus, we can assume that regardless of the year of study and the place of residence the main factor of physical activity type among the female students of medical university is their correct (common sense) understanding of the role of this factor in a healthy lifestyle. however, is should be emphasised that a questionnaire is the cheapest method of collectable 1. the differentiation of the level and areas of physical activity of the female students with regard to the year of study kruskal-wallis test physical activity type h p differentiation total activity 29,47 0,0001* 1-3; 2-3** activity at work 38,21 0,0001* 1-2,3; 2-3** activity while commuting 14,30 0,0008* 1-3** activity at home 7,84 0,0198* 1-3** sport activity 8,47 0,0145* 1-3** note. * — significant differences at p<0,05; ** — years of study between which there is a statistically significant variation. figure 2. the level and areas of physical activity of the female students with regard to the year of study m et -m in /w ee k d. v. popovych et al. issn 2413-6077. ijmmr 2016 vol. 2 issue 240 issn 2413-6077. ijmmr 2016 vol. 2 issue 2 ting data on a large number of respondents (national and international research) regardless of their sex, age or health [29]. conclusions the female students of medical university are characterized by high level of total physical activity compared with the male students. figure 3. the level and areas of physical activity of the female students with regard to the place of residence table 2. the differentiation of the level and areas of physical activity of the female students with regard to the place of residence kruskal-wallis test physical activity type h p differentiation total activity 0,32 0,8505 activity at work 0,58 0,7477 activity while commuting 0,11 0,9484 activity at home 2,03 0,3616 sport activity 5,14 0,0767 m et -m in /w ee k the highest level of total physical activity prevails in the second-year female students. the place of residence during the studies (dormitory, family house or apartment) does not differentiate significantly the level of physical activity of the female students. references 1. krutsevych tyu, bezverhnya hv. recreation in physical culture at different groups population. kyiv: olympiyskaya literatura 2010; 248. 2. rybkovskyy ag, kanishevsky pm. system organization of human motor activity 2003; 436 p. 3. balsevich vk, zaporozhanov va. physical activity of a person. health 1987; 224. 4. dubogay a, yevtushok m. the nature and impact of school innovation in health-system technology. physical education, sport and culture of health in modern society 2008; 1: 36–40. 5. roviy as, rovno va, rovno aa. physiology of motor activity. kharkov 2014; 344. 6. tsos av, kasarda oz, shevchuk ab, samchuk an, khomich av. special physical activity of students of higher educational institutions. modern problems of health and healthy lifestyle athletic education 2015; 129: 330–334. 7. bergier b, tsos a, bergier j. factors determining physical activity of ukrainian students. annals of agricultural and environmental medicine 2014; 21: 613–616. d. v. popovych et al. issn 2413-6077. ijmmr 2016 vol. 2 issue 2 41 p u b l ic h e a lt h a n d e p id e m io l o g y issn 2413-6077. ijmmr 2016 vol. 2 issue 2 8. nowak ma, troczyńska n, forjasz j, nowak l. chosen aspects of lifestyles of physical activity women aged 45–59 and 60–74. health problems of civilization 2016; 10 (3): 30–37. 9. cavil n, biddle s, sallis jf. health enhancing physical activity for young people. statement of the united kingdom expert consensus conference. pediatric exercise science 2001; 13:12-25. 10. paffenbarger rs, lee im. a natural history of athleticism, health and longevity, fournal of sports sciences 1998; 16: 31–45. 11. powell ke, bricker sk, blair sn. treating inactivity. american journal of preventive medicine 2002; 23: 1–2. 12. warburton der, nicol c, bredin ssd. health benefits of physical activity: the evidence. canadian medical journal 2006; 174 (6): 801–809. 13. chastin sfm, dall pm, tigbe ww, grant mp, ryan cg, rafferty d, granat mh. compliance with physical activity guidelines in a group of uk-based postal workers using an objective monitoring technique. european journal of applied physiology 2009; 106: 893–899. 14. shani d, nimbalkar a, phatak a, nimbalkar s. training in dietary practices and physical activity to improve health among south asian medical students. advances in preventive medicine 2014; http:// dx.doiorg/10.1155/2014/610180 15. bioemhoff hj. gender and race-related physical activity levels of south african university students. african journal for physical, health education, recreation and dance ajpherd 2010; 16(41): 25–35. 16. pengpid s, peltzer k. physical inactivity and associated factors among university students in south africa. african journal for physical, health education, recreation and dance apherd 2013; 19 (l): 143–53. 17. bergier i, kapka skrzypczak l, bilinski p, paprzycki p, wojtyta a. physical activity of polish adolescents and young adults according to ipaq: a population based study. ann agric environ med 2012; 19: 109–115. 18. baar j, romppel m, igel d. associations between the residential environment and physical activity in german college students and the role of self-efficacy as a mediator. zeitschrift fur gesundheitspsychotogie 2014; 22(3): 118–128. 19. pedisic z, rakovac m, bennie j, jurakic d, bauman ae. levels and correlates of domain-specific physical activity in university students: crosssectional findings from croatia. kinesiology 2014; 46(1): 12–22. 20. soguksu k. physical activity level between polish and turkish university students (ipaq). in: bergier b, editor. physical activity in health and disease. biata podlaska: pope john paul ii state school of higher education in biata podlaska 2011; 19–27. 21. zhao y, sigmunt e, sigmundova d, lu y. comparison of physical activity between olomouc and beijing university students using an international physical activity questionare. acta univ palacki olomuc gymn 2007; 37(4): 107–114. 22. haase a, steptoe a, sailis f, wardle j. leisuretime physical activity in university students 23 countries: associations with health beliefs, risk awareness, and national economic development. prev med 2004; 39 (1): 182–90. 23. buchman bp, sallis jf, criqui mh, dimsdale je, kaplan rm. physical activity, physical fitness, and psychological characteristics of medical students. j. psychosom res 1991; 35: 197–208. 24. lobelo f, duperly j, frank e. physical activity habits of doctors and medical students influence their counselling practices. br. sports med 2009; 43: 89–92. 25. stanford fc, durkin mw, blair sn, powell ck, poston mb, stallworth jr. determining levels of physical activity in attending physicians, resident and fellow physicians and medical students in the usa. br j.sports med. 2012; 46: 360–364. 26. dabrowska-galas m, plinta r, dqbrowska j, skrzypulec-plinta v. physical activity in students of the medical university of silesia in poland. phys ther 2013; 93 (3): 384–392. 27. booth ml. assessment of physical activity: an international perspective. res. quart. exerc. sport 2000; 71: 114–120. 28. biernat e. aktywność fizyczna mieszkańców warszawy na przykładzie wybranych grup zawodowych. szkoła główna handlowa, urząd m. st. warszawy. biuro sportu i rekreacji, warszawa, 2011. 29. biernat e. international physical activity questionnaire. polish long version. polish journal of sports medicine 2013; 29 (1): 1–15. received: 2016-12-16 d. v. popovych et al. koncor1_2014.pm6 63 b io m e d ic a l s c ie n c e s ijmmr 2015 vol. 1 no. 1 information support system of medical system research v. p. martsenyuk1, i. ye. andrushchak2 1ternopil state medical university, ternopil, ukraine 2lutsk national technical university, lutsk, ukraine background. medical system research requires information support system of implementing data mining algorithms resulting in decision trees or if-then rules. besides that, this system should be object-oriented and web-integrated. objective. the aim of this study was to develop information support system based on data mining algorithms applied to system analysis method for medical system research. methods. system analysis methods are used for qualitative analysis of mathematical models diseases. algorithms such as decision tree induction and sequential covering algorithm are applied for data mining from learning data set. results. taking into consideration the complexity of mathematical equations (nonlinear systems with delays), scientific community requires the appearance of new powerfull methods of exact parameter identification and qualitative analysis. from the point of view of theoretical medicine, uncertainties arising in models of diseases require to develop treatment schemes that are effective, take into account toxicity constraints, enable better life quality, have cost benefit. multivariate method of qualitative analysis of mathematical models can be used for pathologic process forms of classification. conclusions. the complex qualitative behavior of diseases models depending on parameters and controllers was observed in our investigation even without considering probabilistic nature of the majority of quantities and parameters of information models. key words: data mining, system analysis, medical research, decision making introduction here, we would like to present our results in field of application of system analysis methods to problem of clinical medicine. we emphasize that effects of uncertainty should be taken into account in such complex systems. it will be shown that even considering deterministic models of such nonlinear systems, we observe different qualitative behavior closely dealt with parameters values. let’s start from the origin of this problem. nowadays, a lot of models describing physiological indices of human body at different diseases and treatment schemes are obtained. primarily, they are based on regression analysis. more complex ones use neural networks and evolutionary programming. the most significant attempts to construct mathematical models at different levels of hierarchy of human organism were made by john murray [3], keener and sneyd [2], g.i.marchuk [1], mackey and glass (they investigated nonlinear phenomena applying dynamic systems and introduced notion of dynamic diseases). without considering uncertainty all these models can be applied for patients from determined groups (primarily for given age and a lot of other restrictions). methods as for projects stimulating given research, we would like to note the following. during the last years medical informatics department performs investigations initiated by healthcare ministry of ukraine in order to develop and use general system analysis algorithm to study different diseases [4–9]. namely, in fields of oncology (melanoma, leukemia), infectious diseases (flu), therapy (bone tissue diseases). naturally, there arises a problem to develop a general model for disease. it is incorrect to state that we managed to offer unique universal algorithm to construct disease general model. more correctly is to say that this approach can be used for diseases of different nature. we believe this approach can be extended to processes in sociology and demography, as well as for economy and finance. a lot of them have the same nature as human diseases. let’s take into consideration special medical terminology (as little as possible). first of all, the most recognized definition of disease states that disease is a set of address for correspondence: vasil martsenyuk, i. ya. horbachevsky ternopil state medical university, m. voli, 1, ternopil, 46001, ukraine e-mail: marceniuk@yahoo.com v. p. martsenyuk et al. international journal of medicine and medical research 2015, volume 1, number 1, p. 63-67 copyright © 2015, tsmu, all rights reserved 64 b io m e d ic a l s c ie n c e s ijmmr 2015 vol. 1 no. 1 pathologic processes weakening vitality and activity of a human organism. here, pathologic process is a set of pathologic (that is abnormal) and protectoral reactions within human organism. the most significant is modeling pathologic process. results based on this reason we offered general model for pathologic process including three counterparts: (i) the reason or cause of disease (it may be some external factor (like bacteria, chemicals) or own modified cells (tumor cells); (ii) immune system supports organism with help of specific antibodies (sort of predators) and plasmatic cells (their ancestors); (iii) normal cells, tissues and organs (it is necessary to consider them to satisfy some constraints of toxicity). we used our own software for these researches: software environment for medical system researches (semsr). conceptual model of software environment of system medical investigations support is developed. model implementation of data structure for medical investigations in terms of xmltechnology is offered. interface which is webintegrated, user-oriented and adjustable is developed. mathematical methods of system analysis of pathologic processes in form of java-classes hierarchy are implemented. software tools to execute system medical investigations, to prepare results obtained for presentation in internet and visualization are developed. uncertainties in medical system research uncertainties in such models may be parametric. some of the parameters may be unknown functions. as for uncertainty in control, it is necessary to take into account all possible scenarios. note, the purpose of this article is not to present methods to identify these uncertainties. for these purpose we need to present powerful and deep mathematical apparatus of adjoint systems, sensitivity functions and minimax aposteriorial estimation. here, we would like to answer two questions: (i) why is it so important to take into account uncertainties? (ii) the basic uncertainties in models of diseases. to answer question № 1, we should say that the mathematical solutions of equations have different qualitative behavior. in practice we can observe different forms of disease (subclinical, acute, chronic, lethal). search of treatment scheme is dependent on such forms. in our research we investigated uncertainties in the following issues: maturation time for plasmatic cells �, influence of antigen on target-organ damage rate �, relation between target-organ damage rate and immune response ��(m), therapy scheme (polychemiotherapy, radiotherapy), surgery interventions. note, the three last ones are non-parametric. they depend on unknown function like controller. approach of compartmental systems problems of population dynamics, pharmacokinetics, mathematical epidemiology, and others are described by compartmental systems with time delay. even in the linear case, the solution of such equations leads to approximate computation procedures, which makes it impossible to find solutions of the following problems in explicit form: – determining the time instant at which the number of infected persons does not exceed some level i* (mathematical epidemiology); – estimating the time when no more than d* medical product units (pharmacokinetics) remain in the organism of a patient, etc. explicit solutions of such problems can be obtained on the basis of exponential type estimates. a number of works are devoted to the construction of exponential estimates for systems with delay. in [1], an estimate for a linear system is obtained on the basis of the cauchy formula. an approach based on lyapunov functions with conditions of the razumikhin type was developed in [2]. in [3], an estimate is found from the solution of a difference inequality for a lyapunov–krasovskii functional. in [4], a differential difference inequality is constructed for a lyapunov–krasovskii functional. for compartmental systems, a promising approach is proposed in [5] and the method of construction of a class of exponential estimates is based on the hale– lunel inequality. software development based on data mining technology the objective is to develop and implement an algorithms of diagnostic classification applying decision tree induction and sequential covering methods and to study problem of their computational complexity. the solved problem belongs to wide class of differential diagnostics problems. in medicine the notion of “differential diagnostics” means systemic approach based on evidence for determining causes of symptoms observed in case if there are few alternative explanations and also to reduce list of possible diagnoses. one of approaches expressing natural process of thinking for differential diagnostics is data mining method. we are interested in the problem of computational complexity of the algorithms for real clinical data such as, for a example, for biochemical data in case of polytraumas. v. p. martsenyuk et al. 65 b io m e d ic a l s c ie n c e s ijmmr 2015 vol. 1 no. 1 software implementation of decision tree induction the methods are implemented within netbeans developer system in java language. the database of learning tuples is deployed on mysql server. at fig.1 the conceptual model of informational system is presented. class decisiontree implements decision tree induction method. class datamanager is processing calls from decisiontree running queries to mysql database retrieving learning data. database mysql consists of two tables – table attribute for storage of information on attributes and table categorized_data – for learning tuples. the structure of tables in sql syntax is shown below: create table mysql.attribute ( id integer not null unique, attribute_name varchar(25), attribute_field_name varchar(25), primary key (id) ) engine=innodb; create table mysql.categorised_data ( id integer not null unique, a1 varchar(12), a2 varchar(8), a3 varchar(7), ………………….. a21 varchar(7), class varchar(28), primary key (id) ) engine=innodb; classes of this project are included in package decision_tree.model. there are beans-classes attribute, attribute_for_list and categoriseddata for processing data of corresponding tables. sqlqueries for retrieving corresponding data including calculations of information indices are implemented in class attributelistpeer. problem of computational complexity of decision tree induction algorithm as it was shown in the work [11], time of decision tree induction algorithm running is estimated with value ���������������� �� (1) our goal was to check this result experimentally. experiments were executed varying amount of attributes �. decision trees were constructed for each value of �. at fig. 2 and 3 there are shown estimates of decision tree induction times according to [4]. computational complexity of sequential covering algorithm due to analysis of sequential covering algorithm we conclude that computational complexity is determined by product of amount of possible values of class attribute � (quantity of external cycle iterations) and computational complexity of procedure mine_one_rule (d, att_vals, c) executed inside each cycle. procedure mine_one_rule (d, att_vals, c) includes execution of � iterations. for each iteration for a certain attribute � � we calculate the measure for each of � � values of attribute. that is internal body of cycle in procedure mine_one_rule (d, att_vals, c) is executed � � � �� �� times. the measure is executed as a result of 4 sql-queries with complexity ��������� (according with mysql 5.0 documentation). that is procedure mine_one_rule (d, att_vals, c) has computational complexity �� � � �� � � �� � �������� � �� � . summarizing we have sequential covering algorithm complexity of the order �� � � �� � � ��� � � �� � ��������� (2) conclusions so, even without considering probabilistic nature of the majority of quantities and parameters, we saw the complex qualitative behavior of diseases models depending on parameters and controllers. at different values of these quantities we observed subclinical, acute, chronic or lethal forms of pathologic processes. taking into consideration complexity of mathematical equations (nonlinear systems with delays), ��������������� ��� ������� � � � � � ������ � ����� ���������� �� ��������� ����� ��������� ������ fig. 1. conceptual model of informational system of decision tree induction v. p. martsenyuk et al. 66 b io m e d ic a l s c ie n c e s ijmmr 2015 vol. 1 no. 1 it requires appearance of new powerfull methods of exact parameter identification and qualitative analysis. from point of view of theoretical medicine, uncertainties arising in models of diseases require development of treatment schemes that are effective, take into account toxicity constraints, enable better life quality, have cost benefit. in future works our idea will be to compare behavior of pathologic processes using both deterministic and stochastic models and to extend such models to demographic processes. in the work here we considered the problem of development and implementation of decision tree induction and sequential covering methods based on information indices for construction of diagnostic classification algorithm. while investigating this example, the problem of computational complexity of decision tree induction algorithm was observed that: – decision tree induction time based on information indices is well approximated with estimate (1) at small number of attributes (in this case to 15–16); – when increasing number of attributes (in this example over 15–16), the time of decision tree induction begins to deviate essentially from estimate (1) independent on search of information measure; – at small number of attributes decision trees induced constructed based on either information gain or information gain are identical; i.e., information measure determining splitting attribute doesn’t affect on decision tree induced; �� ���� ����� ����� ����� ����� ����� ����� �� �� �� �� �� ��� ��� ��� ��� ��� ��� �������� � �������� ���� ������� ��� � �� ��������� ���� ��������� ���� ���� ������������ ���� ��������� ���� fig. 2. estimate of algorithm complexity based on information gain. � ���� ���� ���� ���� ����� ����� � � � �� �� �� � � �� �������� � �������� ��� ������� �� �� ������������������� ������������������ ���� ����������������� ����������� � fig. 3. estimate of complexity of sequential covering algorithm. �� ����� ����� ����� ����� ����� ����� �� �� �� �� � ��� ��� ��� ��� � � ��� �������� � �������� ���� ������� ��� �� ����� ������� �� ����������� ������� ����� ������� �� �������������� � fig. 4. estimate of complexity based on information gain ratio. v. p. martsenyuk et al. 67 b io m e d ic a l s c ie n c e s ijmmr 2015 vol. 1 no. 1 references 1. mathematical modelling in immunology and medicine. – proc. of the ifip tc-7 working conf., moscow, ussr, 5–11 july 1982, ed. by g.i.marchuk, l.n.belykh. amsterdam, new york, oxford: northholland; 1983: 246. 2. keener j, sneyd j. mathematical physiology. new york: springer verlag; 1998: 149. 3. murray jm. mathematical biology. new york: springer-verlag; 1989: 214. 4. martsenyuk vp. on the problem of chemotherapy scheme search based on control theory. j automation information sci 2003; 35 (4): 64–69. 5. martsenyuk vp. on hopf bifurcation and periodic solutions in g.i. marchuk model of immune protection. j automation information sci 2003; 35 (8): 154–157 6. marzeniuk vp. taking into account delay in the problem of immune protection of organism. nonlinear analysis: real world applications 2001; 2 (4): 483–496. 7. nakonechnyi ag, martsenyuk vp. controllability problems for differential gompertzian dynamic equations. cybernetics systems analysis 2004; 40 (2): 252–259. 8. martsenyuk vp. on stability of immune protection model with regard for damage of target organ: the degenerate liapunov functionals method. cybernetics systems analysis 2004; 40 (1): 126–136. 9. marzeniuk vp. qualitative analysis of human cells dynamics: stability, periodicity, bifurcations, control problems. adv math res 2003; 1 (5): 137–200. 10. khusainov dya, martsenyuk vp. double-ended estimates for solutions of linear systems with delay. dop. nan ukr 1996; 8: 8–13. 11. han j, kamber m. data mining: concepts and techniques, morgan kaufmann, san francisco, 1st edition; 2001: 312. 12. hastie t, tibshirani r, friedman jh. the elements of statistical learning, springer, new york, 1st edition; 2001: 125. 13. ordonez c., comparing association rules and decision trees for disease prediction. in proc. acm hikm workshop 2006: 17–24. 14. ordonez c. integrating k-means clustering with a relational dbms using sql, ieee transactions on knowledge and data engineering (tkde) 2006; 18(2): 188–201. 15. quinlan jr. induction of decision trees. machine learning 1986; 1: 81–106. 16. quinlan jr. c4.5: programs for machine learning. morgan kaufmann; 1993: 205. 17. breiman l, friedman j, olshen r, stone c. classification and regression trees. wadsworth international group; 1984: 124. 18. martsenyuk vp, semenets av. medical informatics. developer and expert systems, ternopil, ukrmedknyha, 2004: 222. received: 2014.05.07 – computational complexity of sequential algorithm is well approximated by (2). such estimate was checked changing an amount of attributes as well as number of learning tuples. the perspective of this investigation is comparative performance analysis depending on volume of set of learning tuples. v. p. martsenyuk et al. issn 2413-6077. ijmmr 2016 vol. 2 issue 226 d e n t is t r y issn 2413-6077. ijmmr 2016 vol. 2 issue 2 doi 10.11603/ijmmr.2413-6077.2016.2.7028 qualitative and quantitative changes in the cellular composition of gums in patiets with periodontal diseases n. v. нasiuk i. horbachevsky ternopil state medical university, ternopil, ukraine background. the results of a comprehensive study of cytological features of epithelial differentiation processes in gums in patients with generalized periodontitis are presented in the article. objective. the objects of clinical observations were patients with generalized periodontitis. the disease duration was 3–5 years. methods. material for the study was gums epithelium taken from a marginal area of gums in young adults, with generalized periodontitis. the epithelium was removed with a spatula, with subsequent transfer to a glass plate and drying in the open air for 3–5 minutes. the colouring of the material was done according to romanovskyyhimza, with a next microscopic and morphological analysis, taking into account the percentage of different forms of epithelial cells in normal and due to an age aspect. results. one of cytological signs of periodontal pathology in young people is epithelial cells with signs of irritation. it should be noted that these cellular elements belong to the intermediate layer of cells that meet histological sections and ribbed surface layers are characterized by homogenization and vacuolization of cytoplasm as a manifestation of prolonged irritation by bacterial cell aggression. conclusions. analysis of cytograms proved general periodontitis in the examined patients, in absence of adequate therapy as automotive continuous process. its components may regress under the influence of effective treatment and re-activate during exacerbation of the inflammatory process under the influence of pathogenic factors such as microbial, hygienic and immune. key words: gingiva, epithelium, cells, nucleus, cytoplasm. introduction as a part of the mucous membrane, buccal epithelium is active within irritating factors that influence it from external and internal environ­ ment. it allows to use it for studying the physiology and reactivity of mucous membranes, and in particular as an indicator of local and general disorders of homeostasis [1, 2]. each anatomical area of gums has a zonal type of keratinization and on histological sections is characterized by basal, ribbed, granular and stratum layers; keratinization occurs in it due to ortokeratoz — a phased process, and as well due to apoptotic changes in the upper layer of epithelium [3]. an only exception is gum sulcus epithelium that is not keratinized and reduces its barrier function and is a target organ at the stage of periodontal pathology development [4]. taken into account the spread of general periodontitis among young people [5] and the prevailing current points of view about the disease pathogenesis [6, 7, 8], the processes of differentiation of gums epithelial cells in the course of general periodontitis is an urgent matter, as well as diagnosis objectification, determination of severity degree and invol vement of adjacent connective tissue to predict course of the disease, exacerbation rate and relapse [9, 10, 11, 12, 13]. our study was aimed to determine the characteristic features of cellular structure of gums cytogram in patients with general periodontitis. material and methods material for the study was gums epithelium, taken from a marginal area of gums in young international journal of medicine and medical research 2016, volume 2, issue 2, p. 26-29 copyright © 2016, tsmu, all rights reserved corresponding author: natalya hasiuk, department of therapeutic dentistry, i. horbachevsky ternopil state medical university, 1 maidan voli, ternopil, ukraine, 46001 phone number: +380352259802 e-mail: gasyknv@tdmu.edu.ua n. v. нasiuk issn 2413-6077. ijmmr 2016 vol. 2 issue 2 27 d e n t is t r y issn 2413-6077. ijmmr 2016 vol. 2 issue 2 adults, with generalized periodontitis. the disease duration was 3–5 years. the epithelium was removed with a spatula, with subsequent transfer to a glass plate and drying in the open air access for 3–5 minutes. the colour of the material was done according to romanovskyy-himza, with a next microscopic and morphological analysis, taking into account the percentage of different forms of epithelial cells in norm and due to an age aspect. in order to standardize the epithelium layer and a deeper studying, in our research we used cytological classification; according to it basal, parabasal, intermediate, superficial cells were in the epithelium of gum in the area of keratinized epithelium of gums, horny scales. statistical analysis of the results was done at the department of statistical research of i. horbachevsky ternopil state medical university. parametric methods were used for the indi ca tors, their distribution met general requirements. to evaluate the nature of distribution the coefficients of skewness and kurtosis were determined. the test was performed with normal asymmetry test by shapiro-wilkie. probable differences of the results for different groups were determined using student t-test. the difference was considered likely in common in the medical and research error probability p<0.05. the probability of error was evaluated by the student t-test tables due to the size of the experimental groups. the rate of distribution was statistically significantly different from the expected normal nonpa rametric criterion (u) mann-whitney nonparametric analogue of a student t-test. results it is established that a major component of the cellular composition of the gums’ scraping c y t o g ra m i n t h e c o u r s e o f g e n e ra l i z e d periodontitis are the stratified squamous epithelial cells. they are present in cytograms under normal conditions and during pathology. it should be emphasised that the squamous cells are heterogeneous, reflecting the heterogeneity of epithelial cells gums’ cover. in the case of generalized periodontitis a c y t o l o g i c a l c h a ra c t e r o f s c ra p c h a n g e s significantly both in epithelial and connective tissue constituents. detailed statistical analysis of cytogram, made it possible to determine the percentage change in gingival epithelial cells of people examined due to the different severity of generalized periodontitis. this ratio is (2,20±0,10) : (9,30±0,16) : (41,6±0,36) : (37,0±0,31) : (9,9±0,16) (table 1). discussion the qualitative and quantitative cellular composition of cytogram in periodontitis depends on the clinical course and intensity of inflammatory and degenerative processes in periodontal tissues. the received data was significantly different from the stereotypical percentage of epithelial differentiation of stratified squamous epithelium of gums [3], and ratio that was determined for the examined patients and is characterized by violation of differentiation: disorder of keratinization as evidenced by the presence of all components of different epithelial cells. this makes it possible to assert that during generalised periodontitis the mechanisms of epithelial desquamation disruption take place causing barrier function of gums at generalized periodontitis; that is the outcome of different epithelial cells differentiation caused by inflammatory infiltrate of lamina propria. the presence of parabasal cells in cytograms was explained by activation of compensatory mechanisms of epithelium, taken into account high mitotic activity of gum epithelium and increased glycogen-containing epithelial cells. table 1. percentage change in different cells classes of stratified squamous gums epithelium in the course of generalized periodontitis indicator cells of smears basic parabasal intermediate superficial horny scales norm (v.l. bykov) 0 0 60 8 32 norm for young person 0 0 59,20±1,15* 7,60±0,34* 33,2±0,65* generalized periodontitis 2,20±0,10** 9,30±0,16** 41,60±0,36** 37,0±0,31** 9,90±0,16** note: * — p<0,05 compared with the control group; ** — p<0,05 if compared with the experimental group. n. v. нasiuk issn 2413-6077. ijmmr 2016 vol. 2 issue 228 d e n t is t r y issn 2413-6077. ijmmr 2016 vol. 2 issue 2 at the same time, reflecting the severity, course and intensity of general periodontitis and intensity of inflammatory and degenerative processes in periodontal tissues, cellular composition of gums in cytograms was divided into four types. the first type is characterized by presence of intermediate and parabasal cells stained by romanovskyy-himza, with smaller size compared to intermediate. these epithelial layers correspond to deep ribbed cells in histological sections. they are larger in size if compared to basic cells and have an elongated shape. the core is large, well-countered, vesicular, with small diffuse located chromatin inclusions and relatively wide rim of basophilic cytoplasm. the lack of hematogenous cells row is noted. it is obvious that this cellular composition obtained in cytograms proves evidence of proliferation, i.e. reproduction of parabasal cells and specific prognostic criterion of gene­ ralized periodontitis of initial stage. in the second type of cytogram there were individual representatives of rod flora with advantage of basophilic intermediate (azur positive) epithelial cells from cytological smears of gums. they have cubic, rectangular or polygonal form; cytoplasm contains azure-positive granules. the core is round, sometimes oval. the trend towards clusters of cells location of this class is noted. intermediate cells in histological sections correspond to ribbed parts of surface layer. these intermediate cells are found in normal and mostly without elements of cytopathology. we argue that this type of cytograms proves chronic generalized perio dontitis. in the third type of cytogram there were cytograms of intermediate cells staining by romanovskyy-himza. they have azure-positive cytoplasm nucleus centric located. the cytoplasm is elongated. the cells are usually located in clusters. the change in the microbial compo sition is determined that in this type of cytogram is represented mainly by cocci that adhere on the surface of epithelial cells. in addition to the above mentioned epithelial cells in cytogram of the third type of the examined people there are segmented leukocytes, which are split into 3–4 segments core also there are isolated mononuclear leukocytes or young white blood cells it suggests a redistribution pool of myeloid cells in response to the presence of foci of active inflammatory process that causes the process of differentiation of leukocytes available. present cytological pattern characterizes pointer of general periodontitis in the examined people. in the fourth type of cytograms there were single basal epithelial cells. during staining by romanovskyy-himza, if compared with basal cells of periodontal pockets they have relatively smaller and more rounded shape of cytoplasm. the core is round-shaped and has a narrow rim of cytoplasm. basal epithelial cells are surrounded by segmented leukocytes. the predominant cells in the fourth type of cytograms are the cells of intermediate layer, their cytological organization corresponds a lot to the class affiliation and level of differentiation, a pathogenic microflora is evidenced as well as the cells of inflammatory response. superficial cells are also verified. a powerful microbial composition initiates further necrobiotic processes in epitheliocytes and in segmented leukocytes. due to phagocytosis, the destruction of the cytoplasm of segmented leukocytes occur, it is ‘incomplete phagocytosis’. coccal microflora adheres not only on the surface of epithelial cells, but also on the surface of segmented leukocytes. along with haematogenous cells in cytograms predo minantly coccoid flora and single thread of pseudomycelia of candida fungi are evidenced. so cytograms of the fourth type present the increase of phagocytic reaction of segmented leukocytes, and as a result of incomplete phagocytosis formation of ‘pus cells’ occurs. during phagocytosis of leukocytes specific changes in the form of restructuring of its nuclear takes place, while in epitheliocytes of gums a necrobiotic process occurs primarily in the cytoplasm and in the nucleus. the described type of cytogram corresponds to the clinical manifestations of general periodontitis with abscess formation. the emergence of epithelial cells with signs of irritation in cytograms is one of cytological signs of periodontal pathology in young people. these cellular elements belong to the intermediate layer of cells that on histological sections correspond to ribbed surface layers and are characterized by homogenization and vacuolization of the cytoplasm as a manifestation of prolonged bacterial irritation. the vacuolization of epithelial cells cytoplasm in general periodontitis gingival complex proves disorders of water and electrolyte and protein metabolism, causing the changes in colloid-osmotic pressure in the cell. the violation of cell membrane permeability, accompanied by their collapse is also significant. it leads to lysosomal membranes labilization with activation of their hydrolytic n. v. нasiuk issn 2413-6077. ijmmr 2016 vol. 2 issue 2 29 d e n t is t r y issn 2413-6077. ijmmr 2016 vol. 2 issue 2 enzymes that break intramolecular connection with water. in fact, these changes are a manifestation of early cell focal necrosis [5]. at the same time the increase in the volume of epithelial cells was evidenced, the cytoplasm is full of vacuoles, containing a clear liquid. the core is moved to the periphery of the cell, sometimes are vacuoles or core shrinks. later disintegration of ultra-structural elements of cells occurs and it overflows with water. these changes are the features of the intermediate stage of cellular metabolism disorders caused by inflammation and preceding cell cytotransformation into balloons filled with fluid. such changes characterize balloon cell degeneration. analysing the cytogram we can reach the conclusion that unlike previous smear from epithelial gums it has the greater degree of destruction accompanied by cariopiknosis and kariorexis of nucleus and cytoplasm homogenization. horny scales in cytograms decreased in comparison to their quantitative composition in cytograms of people with health perio dontium. they are mainly eosinophilic, of polygonal shape, characterized by no clear contours. conclusions the changes in the gums in the course of generalized periodontitis are subject to rather wide limits, which depend on the clinical course and exogenous factors. analysis of cytograms gave the opportunity for consideration of general periodontitis in the examined patients due to the absence of adequate therapy as automotive continuous process. its components (inflammatory infiltrative and destructive) may decrease after the effective treatment and reactivate during the exacerbation of inflammatory process under the influence of pathogenic factors such as microbial, hygienic and immune. references 1. bainbridge bw. porphyromonas gingivalis lipopolysaccharide: an unusual pattern recognition receptor ligand for the innate host defense system. acta odontol scand 2001; 59: 131–138. 2. bourgeois d. epidemiology of periodontal status in dentate adults in france. j. periodontal. res 2007; 42, (№ 3): 219–227. 3. bykov vl. functional morphology of epithelial barrier of oral mucosa. dentistry, 1997; (№ 3): 12–17. 4. hasiuk nv. morphofunctional organization of gums in normal state and in case of inflammation. thesis of dis. for phd: specialty 14.03.09 histology, cytology, embryology, 2009; 19. 5. hasiuk nv. ultrastructural arrangement of gum furrow epithelial. bulletin of ukrainian medical dental academy: topical matters of contemporary medicine, 2013; 13 (42): 189–191. 6. grudianov ai. periodontal diseases. m : me­ dical news agency, 2009: 336. 7. kornman ks. the “innovator’s dilemma” for periodontists. j. periodontol 2010; 81 (№ 5): 646–649. 8. kononen e. population-based study of salivary carriage of periodontal pathogens in adults. j. clin. microbiol 2007; 45, (№ 8): 2446–2451. 9. schmalz g. release of prostaglandin e2, il-6 and il-8 from human oral epithelial culture models after exposure to compounds of dental materials. eur. j. oral. sci. 2000; 108: 442–448. 10. schnare m. toll­like receptors: sentinels of host defence against bacterial infection. j. allergy immunol 2006; (139): 75–85. 11. shi d. inflammatory bowel disease requires the interplay between innate and adaptive immune signals. cell res. 2006; (16): 70–74. 12. van dyke te. inflammation and factors that may regulate inflammatory response. j. periodontol. 2008; 79 (8): 1503–1507. 13. vitkov lv. bacterial internalization in periodontitis. oral microbiol. immunol. 2005; 20 (5): 317–321. received: 2016-12-01 n. v. нasiuk 54 b io m e d ic a l s c ie n c e s issn 2413-6077. ijmmr 2016 vol. 2 issue 1 doi 10.11603/ijmmr.2413-6077.2016.1.6387 патогенетичні механізми легеневих уражень м. і. марущак, і. я. криницька, г. г. габор, о. з. яремчук тернопільський державний медичний університет імені і. я. горбачевського вступ. на сучасному етапі розвитку медико-біологічної науки увага більшості дослідників прикута до ключових механізмів багатьох захворювань людини, які тісно пов’язані з порушенням клітинної смерті. однією з провідних причин дисрегуляції танатогенної програми клітин є активація біологічних ефектів фактора некрозу пухлин альфа. мета. узагальнити існуючі наукові дані щодо внеску метаболітів кисню і монооксиду нітрогену в механізми легеневого ураження. методи. аналіз літературних даних щодо механізму легеневого ураження. результати й висновки. у зв’язку з вищесказаним, актуальною є проблема розуміння сигналів та механізмів, відповідальних за регулювання процесів вільнорадикального окиснення і системи антиоксидантного захисту, роль прота протизапальних молекул, вплив активних метаболітів на процеси репарації і виживання клітин респіраторного тракту при гострих ураженнях легень. розуміння суті даних процесів дозволить поглибити існуючі й отримати нові знання при легеневій патології. ключові слова: легеневе ураження, патогенез. вступ на сучасному етапі розвитку медикобіологічної науки увага більшості дослідників прикута до ключових механізмів багатьох захворювань людини, які тісно пов’язані з порушенням клітинної смерті. однією з провідних причин дисрегуляції танатогенної про грами клітин є активація біологічних ефек тів фактора некрозу пухлин альфа (фнп-α) [1]. метою даної роботи було узагальнити існуючі наукові дані щодо внеску метаболітів кисню і монооксиду нітрогену в механізми легеневого ураження. проведений аналіз наукових джерел показав, що фнп-α, або кахектин, – плейотропний прозапальний цитокін з молекулярною масою 17 400 кда, переважно макрофагального походження, який у сироватці крові людей практично не визначається. фнп-α може діяти як незалежно, так і разом із широким спектром інших факторів, порушувати фенотип і метаболізм клітин у кожної тка нини організму. на даний час фнп-α визнають центральним медіатором серед широ кого спектра фізіологічних та імуно логічних функцій. ця молекула проявляє різні біологічні ефекти, в тому числі індукує цитотоксичні ефекти на ендотеліальні кліти ни, підсилює до них адгезію нейтрофілів шляхом підвищення вироблення хемокінів та адгезивних молекул, збільшує судинну проникність через активацію нейтрофілів, а також стимулює продукування інших прозапальних цитокінів (інтерлейкінів 1, 6, 8) [2, 3]. респіраторний відділ легень є важливим компонентом системного гомеостазу, який при багатьох патологічних станах, незважаючи на ряд деструктивних і дистрофічних змін у своєму складі, проявляє широкі ком пен саторні властивості. провідне значення в розвитку ускладнень і летальних наслідків належить тяжким прогресуючим респіра торним порушенням, що є одним із компо нен тів синдрому поліорганної недостатності [4]. основні чинники легеневого ураження поділяють на прямі та непрямі. до легеневих факторів належать ті, які безпосередньо уражають легеневий епітелій (віруси, бактерії, гриби, токсичний газ, аспірація шлункового вмісту) [5]. позалегеневі чинники є більш поширеними і зустрічаються при багатьох захворюваннях (цироз печінки, травма, сепсис, панкреатит) внаслідок циркуляції в крові corresponding author: marya marushchak, department of functional diagnostics and clinical pathophysiology, �. �or-�. �orbachevsky ternopil state medical university, 1 maidan voli, ternopil, ukraine, 46001 tel.: +380979901202 e-mail: marushchak@tdmu.edu.ua м. і. марущак та ін. international journal of medicine and medical research 2016, volume 2, issue 1, p. 54–58 copyright © 2016, tsmu, all rights reserved 55 b io m e d ic a l s c ie n c e s issn 2413-6077. ijmmr 2016 vol. 2 issue 1 системних запальних факторів, які мають пошкоджувальну дію на легені. так, локальна дія хімічно активного травмувального чинника на легеневу паренхіму зумовлює вибуховий викид біологічно активних речовин, зокрема фнп-α та інших цитокінів. за умов портальної гіпертензії порушується бар’єрна функція печінки, що зумовлює накопичення в крові ендотоксинів. ендотоксемія стимулює продукування макрофагами печінки і легень вазоактивних субстанцій (оксиду азоту, ендотеліну-1) та цитокіну макрофагального походження – фнп-α [6–8]. отже, в основі респіраторного порушення лежить безпосереднє або опосередковане пошкодження епітеліального й ендотеліального бар’єрів, що зумовлює надмірне виділення медіаторів запалення в кровотік, і, як наслідок, ініціюється, активується і поширюється системна запальна відповідь. сироваткові цитокіни, зокрема фнп-α, у свою чергу, є важливими гуморальними регуляторами апоптозу і можуть контролювати дані процеси на генетично детермінованому рівні, а порушення їх апоптотичної активності може призводити до прогресування патологічних станів [9]. фундаментальні та клінічні дослідження вказують на унікальну здатність фнп-α ініціювати і апоптотичну загибель клітин, і некроз клітин парен хіматозних органів за умов гіперпродукування фнп-α [10]. метаболізм клітини в більшості випадків залежить від характеру інформації, яку несуть первинні месенджери – цитокіни. фнп-α, як і il-1β, активує надфн-оксидази нейтрофільних гранулоцитів, які окиснюють надфн до надф+ за рахунок відновлення о2 до супероксидного аніон-радикала. ферментативна генерація супероксидного аніонрадикала в організмі людини здійснюється також ксантиноксидазою, цитохромом р450, альдегідоксидазою, ліпоксигеназою, циклооксигеназою нейтрофілів і моноцитів [10, 11]. у результаті виникає так званий кисневий спалах, зумовлений активними формами кисню, серед яких мієлопероксидаза каталізує реакцію утворення гіпохлориту з аніона хлору і пероксиду водню, а також відбувається утворення гідроксильного радикала з пероксиду водню і гіпохлориту за наявності іонів заліза (рис. 1) [12, 13]. дисмутація су пероксиданіон-радикалів під дією супероксиддисмутаз у біологічних тканинах спричиняє утворення пероксиду водню, який здатний легко проникати через мембрани клітин [14, 15]. активні форми кисню продукуються також при активації такого ферменту, як noсинтаза (nos). оксид азоту (no) відіграє важливу роль у регуляції функцій легень і в патофізіології захворювань системи дихання [16–19]. у легенях no виробляється під впливом конститутивної nos (cnos) в ендотеліальних клітинах легеневої артерії та вени. у ряді клітин, наявних у легенях і здатних виробляти no, включаючи макрофаги, нейтрофіли, гладкі клітини, ендотеліальні, гладком’язові, епітеліальні клітини та, можливо, клітини інших типів, представлена експресія індуцибельної nos (inos). дослідження, проведені пізніше, показали, що в дихальних шляхах cnos характеризується високою гомологічністю до inos та міститься в епітеліальних клітинах [17]. утворення ендогенного no можна розглядати як механізм, який підтримує сталість стану гладких міоцитів у легеневій циркуляції, і, відповідно, недостатність синтезу або ж вивільнення no зумовлює надмірну їх проліферацію. проведені наукові дослідження вказують на те, що в нормальному легеневому руслі no не лише сприяє адекватній вазодилатації, але й перешкоджає вазоконстрикторній дії гіпоксії та ендотеліну [17, 19]. втрата ендотелієзалежної релаксації судинами під час гіпоксії спричиняє підвищення їх тонусу, утворення тромбів, порушення мікро циркуляції, що призводить до проліферації гладких міоцитів [7, 8]. літературні дані останніх років свідчать про те, що при запальних захворюваннях органів дихання зростає утворення no в епітелії дихальних шляхів людини [16]. синтез no підвищується за рахунок активації inos під впливом макрофагальних цитокінів, що включають й інші клітинні структури в активне утворення медіаторів запалення, а також ендотоксемії. запальні медіатори, що виділяються мастоцитами, і гіперпродукування no сприяють збільшенню проникності судинної стінки з формуванням інтерстиційного та альвеолярного набряку [17]. метаболізм no відбувається таким чином (рис. 1). основний шлях – реакція з гемопротеїнами: клітинні ефекти no здій сню ються при зв’язуванні з гемовмісним ферментом гуанілатциклазою, no реагує з гемоглобіном еритроцитів з утворенням метгемоглобіну. внаслідок цього no перетворюється в іон нітриту (no2 -), а за наявності гемового заліза no2 переходить у стабільний іон нітрату (no3 -). також no при взаємодії з м. і. марущак та ін. 56 b io m e d ic a l s c ie n c e s issn 2413-6077. ijmmr 2016 vol. 2 issue 1 супероксид-аніоном утворює пероксинітрит і гідроксил-радикал. утворені сполуки належать до активних форм кисню і мають деструктивну дію відносно білків та ліпідів. метаболізм no теж відбувається шляхом утворення нітрозотіолів та динітрозольних комплексів негемового заліза, що є депом no [18, 19]. інактивація вільних радикалів здійснюється антиоксидантною системою, яка включає в себе активні антиокиснювачі та антиоксидантні ферменти, що розривають лан цю ги молекул під час реакції віль но радикального окиснення [16]. у надепітеліальній рідині респіраторного тракту є неферментні антиоксиданти: церулоплазмін, трансферин, вітаміни, амінокислоти, іони металів. до основних ферментів антиоксидантної системи, що містяться в надепітеліальній рідині дихальних шляхів, належить супер оксиддисмутаза, яка дисмутує супероксидний радикал, глутатіонпероксидазу, що інактивує пероксид ліпідів, каталазу, яка руйнує пероксид водню. зміни структури і функції субстратів, на які діють активні форми кисню та азоту, залежать загалом від співвідношення активності вільних радикалів та анти оксидантів. потрібно зауважити, що при гострих легеневих ураженнях антиоксидантні системи швидко виснажуються, а надмірне продукування активних метаболітів кисню може мати руйнівний вплив на епітеліоцити й інші клітини легеневої тканини, що є одним з основних факторів тяжкості захворювань органів дихання [6, 11, 21]. активні форми кисню та азоту впливають на прой антиапоптичні механізми клітин респіраторного тракту безпосередньо або через внутрішньоклітинні редоксзалежні сигнальні системи [20, 22]. так, альвеолоцити i і ii типів є чутливими до проапоптичної дії активних метаболітів кисню, які здатні активувати каспазу-3, посилювати експресію проапоптичного протеїну bax [23, 24]. монооксид азоту також є регулятором апоптозу, зокрема низька концентрація no пригнічує, а висока – індукує програмовану клітинну смерть. пошкодження днк активними радикалами азоту зумовлює накопичення р53, що вважають індикатором no-опосередкованого апоптозу [25, 26]. висновок у зв’язку з вищесказаним, актуальною є проблема розуміння сигналів та механізмів, відповідальних за регулювання процесів вільнорадикального окиснення і системи антиоксидантного захисту, роль прота протизапальних молекул, вплив активних метаболітів на процеси репарації і виживання клітин респіраторного тракту при гострих ураженнях легень. розуміння суті даних процесів дозволить поглибити існуючі й отримати нові знання, а також намітити подальші напрямки корекції патологічних змін при легеневій патології. рис. 1. шляхи цитотоксичної дії активних форм кисню та азоту. м. і. марущак та ін. 57 b io m e d ic a l s c ie n c e s issn 2413-6077. ijmmr 2016 vol. 2 issue 1 література 1. parsons pe, matthay ma, ware lb, eisner md. national heart, lung, blood institute acute respiratory distress syndrome clinical trials network. elevated plasma levels of soluble tnf receptors are associated with morbidity and mortality in patients with acute lung injury. am. j. physiol. lung cell. mol. physiol. 2005; 288: 426–431. 2. locksley rm, killeen n, lenardo mj. the tnf and tnf receptor superfamilies: integrating mammalian biology. cell 2001; 104: 487–501. 3. lundblad lk, thompson-figueroa j, leclair t. tnf-alpha over-expression in lung disease: a single cause behind a complex phenotype. am. j. respir. crit. care med. 2005; 171:1363–1371. 4. marshall jc. inflammation, coagulopathy, and the pathogenesis of multiple organ dysfunction syndrome. crit. care med. 2001; 29: s99-s106. 5. ware lb, matthay ma. the acute respiratory distress syndrome. n. engl. j. med. 2000; 342: 1334– 1349. 6. varghese j, ilias-basha h, dhanasekaran r et al. hepatopulmonary syndrome – past to present. ann hepatol. 2007; 6: 135–142. 7. huffmyer jl, nemergut ec. respiratory dysfunction and pulmonary disease in cirrhosis and other hepatic disorders. respir. care. 2007; 52: 1030–1036. 8. zhang hy, han dw, wang xc et al. experimental study on the role of endotoxin in the development of hepatopulmonary syndrome. world j. gastroentеrol. 2005; 11: 567–572. 9. maianski na, maianski an, kuijpers tw, roos d. apoptosis of neutrophils. acta haematol. 2004; 111: 56–66. 10. de dooy jj, mahieu lm, van bever hp. the role of inflammation in the development of chronic lung disease in neonates. eur. j. pediatr. 2001; 160: 457–463. 11. lee wl, downey gp. neutrophil activation and acute lung injury. curr. opin. crit. care. 2001; 7: 1–7. 12. asai t, ohno y, minatoguchi s. the specific free radical scavenger edaravone suppresses bleomycin-induced acute pulmonary injury in rabbits. clin. exp. pharmacol. physiol. 2007; 34: 22–26. 13.tamagawa k, taooka y, maeda a. inhibitory effects of a lecithinized superoxide dismutase on bleomycin-induced pulmonary fibrosis in mice. am. j. respir. crit. care med. 2000; 161: 1279–1284. 14. glosli h, tronstad kj, wergedal h. human tnf-alpha in transgenic mice induces differential changes in redox status and glutathione-regulating enzymes. faseb j. 2002; 16: 1450–1452. 15. ishii y, partridge ca, del vecchio pj, malik ab. tumor necrosis factor-alpha-mediated decrease in glutathione increases the sensitivity of pulmonary vascular endothelial cells to h2o2. j. clin. invest. 1992; 89: 794–802. 16. shiloh mu, macmicking jd, nicholson s. phe-shiloh mu, macmicking jd, nicholson s. phenotype of mice and macrophages deficient in both phagocyte oxidase and inducible nitric oxide synthase. immunity. 1999; 10: 29–38. 17. sittipunt c, steinberg kp, ruzinski jt. nitric oxide and nitrotyrosine in the lungs of patients with acute respiratory distress syndrome. am. j. respir. crit. care med. 2001; 163: 503–510. 18. tracey wr, xue c, klinghofer v. immunochemical detection of inducible no synthase in human lung. am. j. physiol. lung cell mol. physiol. 1994; 266: 722–727. 19. de andrade ja, crow jp, viera l. protein nitration, metabolites of reactive nitrogen species, and inflammation in lung allografism. am. j. respir. crit. care med. 2000; 161: 2035–2042. 20. hiwari bs, belenghi b, levine a. oxidative stress increased respiration and generation of reactive oxygen species, resulting in atp depletion, opening of mitochondrial permeability transition, and programmed cell death. plant physiol. 2002; 128: 1271–1281. 21. ware lb, matthay ma. alveolar fluid clearance is impaired in the majority of patients with acute lung injury and the acute respiratory distress syndrome. am. j. respir. crit. care med. 2001; 163: 1376–1383. 22.melley dd, evans tw, quinlan gj. redox regulation of neutrophil apoptosis and the systemic inflammatory response syndrome. clin. sci. 2005; 108: 413–424. 23. mikhailov v, mikhailova m, degenhardt k. association of bax and bak homo-oligomers in mitochondria. bax requirement for bak reorganization and cytochrome c release. j. biol. chem. 2003; 278: 5367–5376. 24.choi iw, sun-kim, kim ys. tnf-alpha induces the late-phase airway hyperresponsiveness and airway inflammation through cytosolic phospholipase a (2) activation. j. allergy clin. immunol. 2005; 116: 537–543. 25. степовая еа, жаворонок тв, стариков юв. регуляторная роль оксида азота в апоптозе нейтрофилов. бюл. эксперим. биологии и медицины. 2008; 146: 646–650. 26. choi bm, pae ho, jang si, chung ht. nitric oxide as a pro-apoptotic as well as anti-apoptotic modulator. j. biochem. mol. biol. 2002; 35: 116– 126. м. і. марущак та ін. 58 b io m e d ic a l s c ie n c e s issn 2413-6077. ijmmr 2016 vol. 2 issue 1 pathogenetic mechanisms of lung injury m. i. marushchak, i. ya. krynytska, g. g. gabor, o. z. yaremchuk �. �orbac�evsky ternop�l state med�cal un�vers�ty background. �n contemporary life science research development most attention is paid to mechanisms of many human diseases that are associated with the violation in cell death. one of the main causes of cell thanatologic disregulation program is the changes in their production and activation of the biological effects of tumor alpha necrosis factor. objective is to summarize current scientific data about role of activated oxygen and nitric metabolites in the system of lung pathogenetic injuries. methods: analysis of the research data on mechanisms of lung injury. results and conclusions. the topical issue of lung pathogenetic injury is to understand the signs and mechanisms responsible for regulation of free radical oxidation and antioxidant defense system, the role of pro and anti-inflammatory molecules, the influence of active metabolites on the process of restoration and survival of the respiratory tract cells in cases of acute lung injury. the studies of this processes will help to obtain more knowledge on lung pathology. key words: lung injury, pathogenesis. м. і. марущак та ін. received: 2016-03-10 koncor1_2014.pm6 58 b io m e d ic a l s c ie n c e s ijmmr 2015 vol. 1 no. 1 l�arginine, but not l�name protects against liver injury induced by experimental ischemia�reperfusion o. m. oleshchuk, k. a. posokhova, a. ye. mudra i. ya. horbachevsky ternopil state medical university, ternopil, ukraine background. hepatic ischemia-reperfusion (i/r) injury occurs upon restoration of hepatic blood flow after a period of ischemia. objective. the study establishes that stimulation or blockade of nitric oxide synthesis has a protective effect during ischemia-reperfusion. methods. male albino rats which were divided into four equal groups: sham-operated control, ischemia and reperfusion group (0.9 % saline i.p.) for 3 days, group pre-treated with l-arginine (25 mg/kg i.p.), group pre-treated with l-name (10 mg/kg i.p.) for 3 days before ischemia-reperfusion maneuver. complete ischemia of the median and left hepatic lobes was induced by clamping the left branches of the portal vein and the hepatic artery for 45 min. rats were sacrificed after 3-h reperfusion. nitric oxide synthase 3 (endothelial) and nitric oxide synthase 2 (inducible) expression, nitric oxide stabile metabolites (no 2 , no 3 ) content, ast and alt activities were determined. histological examination of liver tissue was performed. conclusions. relative no deficiency, due to enos inhibition, is central in the pathogenesis of hepatic ischemia reperfusion injury. replacing no content with either precursors or via donor drugs represents novel methods in ameliorating ischemia-reperfusion injury. key words: hepatic ischemia-reperfusion, nitric oxide, nos isoforms, l-arginine, l-name. introduction ischemia-reperfusion (i/r) of the liver is a complex of pathophysiological processes that occurs during surgery, trauma, liver transplantation and can lead to serious liver injury. basic mechanisms of liver ir are not completely understood at present time, but we claim that reactive oxygen and nitrogen are important in this process. overproduction of free radicals by activated kupffer cells and neutrophils causes a number of toxic effects, including changes in lipid and protein oxidation, excretion of proinflammatory mediators and microvascular dysfunction, which together lead to cell and tissue damage [1]. according to bahde h. (2010), the hepatic microcirculation violations and endothelial dysfunction also play main role in the pathophysiology of i/ r at the time of reperfusion [2]. the biologically active molecule of nitric oxide (no) may play a pivotal role in the process of free radicals formation. however, the data about the protective or damaging role of no during i/r are controversial. according to some studies the application of no precursors minimizes the adverse effects of reperfusion and improves hepatic microcirculation [3,4]. on the other hand, studies of some researchers suggest that inhibitors of no synthesis can prevent liver damage during i/r [5]. this study shows that stimulation or blockade of nitric oxide synthesis has a protective effect during ischemia-reperfusion. methods no precursor – l-arginine and non-selective nos blocker – l-name (n-nito-l-arginine methyl ester) were procured from sigma; alanine aminotransferase (alt) and aspartate aminotransferase (ast) kits were procured from “filisit–diagnostic”, while tnf-�, il-1�, il-6, enos, and inos elisa kits were procured uscn limited inc. animal model male white rats (ternopil state medical university vivarium, ukraine) 8 to 10 weeks old, were used for these experiments. all animals were fasted 12 h before experimentation and allowed water ad libitum. all animals received care in compliance with the “guide for the care and use of laboratory animals” (national institute of health publication № 85-23, revised 1985). the studies were carried out according to the national institute of health guide for the care and use of laboratory animals and were approved by the local animal protection committee. induction of ischemic and reperfusion injury the hepatic ischemia-reperfusion (i/r) protocols were performed as described in a previous study by oleshchuk, 2012 [6]. there was no mortality address for correspondence: oleshchuk o.m., m. voli, 1, ternopil, 46001, ukraine tel.: +380 352523987. o. m. oleshchuk et al. international journal of medicine and medical research 2015, volume 1, number 1, p. 58-62 copyright © 2015, tsmu, all rights reserved 59 b io m e d ic a l s c ie n c e s ijmmr 2015 vol. 1 no. 1 observed associated with this model. the liver of each rat was exposed through a midline laparotomy after the induction of anesthesia (thiopental sodium 20 mg/kg i.p.). complete ischemia of the median and left hepatic lobes was induced by clamping the left branches of the portal vein and the hepatic artery for 45 min. the right hepatic lobe was perfused to prevent intestinal congestion. after the time frame sufficient for ischemia development, the ligatures around the left branches of the portal vein and hepatic artery were removed. in order to accurately evaluate the blood flow in the median and left hepatic lobes after ischemia, the right branches of the portal vein and the hepatic artery were ligated to prevent shunting to the right lobe after reperfusion and perfused for 2 h. the wound was stitched with 3.0 silk suture. sham-operated animals were similarly prepared except that no ligature was placed to obstruct the blood flow to the left and median hepatic lobes. instead, it the blood flow to the right lobe of the liver was occluded. rats were sacrificed in all groups after 1-h ischemia followed by 3-h reperfusion. twenty four white rats were divided into four equal groups (n=6 each group). group 1 (shamoperated control group) and group 2 (ischemia and reperfusion group) were given 0.9 % saline (1 ml/kg, i.p.) for 3 days. group 3 was pre-treated with l-arginine (25 mg/kg i.p.), group 4 – with l-name (10 mg/kg i.p.) for 3 days before, (last time – 10 min before) induced ischemia-reperfusion maneuver. peripheral blood and hepatic tissue procurement blood sampling for determination of liver enzymes. blood samples were obtained from the right ventricle via left anterior thoracotomy at the time of sacrifice. blood was collected in a sterile syringe without anticoagulant and centrifuged at 2000 g to separate the serum. the serum samples were stored at -20 °c until use for ast and alt assays. blood sampling for cytokine and nos determination. serum was removed from blood samples by clotting for 2 hours on ice; serum was centrifuged at 2,500�g (4 °c), filtered, aliquoted, and frozen at -20 °c until assayed for enos and inos. small liver samples were collected from each rat, then frozen immediately and stored in liquid nitrogen until used for enos and inos assays. determination of liver enzymes function determination of ast (ec 2.6.1.1) and alt (ec 2.6.1.2) in plasma was performed by raytmanfrenkel method, using a standard kit “filisitdiagnostic” (ukraine) according to the manufacturer’s instructions. the activity of ast and alt in serum were expressed in mmol/( l�h). no 2 and no 3 assays. no 2 and no 3 in serum of blood and liver homogenates (1:10) were determined as described by several authors [7]. nos assays determination of enos activity was performed by elisa method using “enzyme-linked immunosorbent assay kit for rat nitric oxide synthase 3, endothelial (nos3)”, uscn, life science inc, e90868ra. determination of inos activity was done by elisa method using “enzyme-linked immunosorbent assay kit for rat nitric oxide synthase 2, inducible (nos2)”, uscn, life science inc, e90837ra. enos and inos expression was investigated in blood plasma and liver tissue. blood was collected using edta as an anticoagulant. samples were centrifuged for 15 min at 1000 g/min at 2–8 °c within 30 min after collection. determination was carried out immediately or frozen at -20 °c. the procedure of liver cells lysis was performed as follows: 1. preparing liver homogenates in isotonic nacl solution at a ratio of 1:10. 2. liver cells were centrifuged for 5 min at 300 g, and then the supernatant was removed. 3. cells were washed twice with isotonic nacl solution, after each wash the suspension was centrifuged at 300 g for 5 min. 4. buffer (pbs) was added to the normal liver tissue lysate (1 ml of buffer at 1�106 liver cells). it was centrifuged for 5 min at 300 g. 5. the supernatant was collected. determination of enzyme activity was carried out immediately or the supernatant was frozen at -20 °c. determination of enos and inos concentration were performed by elisa method and according to the manufacturer’s instruction. enos activity in serum and in hepatocytes (1 ml – 1�106 cells) was expressed as u/ml, and inos activity was expressed as ng/ml. histopathology study a portion of the tissue from the ischemic liver lobe was fixed in 10 % neutral-buffered formalin solution for 5 days, embedded in paraffin, and sectioned. the sections were stained with hematoxylin and eosin. statistical analysis statistical analysis was carried out by originpro program. results are expressed as mean±standard deviation. differences between experimental groups were analyzed with an unpaired 2-tailed student t test. all differences were considered statistically significant at a p<0.05. results the results indicate that after 45 min postischemia followed by 2 h reperfusion the activity of alt in blood increased from (0.44±0.10) in the sham control group compared to (2.35±0.09) in i/r group o. m. oleshchuk et al. 60 b io m e d ic a l s c ie n c e s ijmmr 2015 vol. 1 no. 1 animals, which is 5.3 times increase. accordingly, ast activity increased from (1.63±0.12) to (4.27± 0.37), which is 2.6 times increase. the described dynamics indicates that cytolysis process of hepatocytes is evolving under simulated ir injury (table 1). the significant increase in alt and ast activities, that occurred in i/r group, was significantly suppressed by pre-treatment with 25 mg/kg l-arginine (table 1). thus, the transaminases activity was significantly lower: alt (53.2 %) and ast (33.6 %). the activity of alt and ast increased by 38 % and 84 % with the introduction of l-name compared to those of animals with the i/r alone. the activity of these enzymes was different from the control values by 7.4 and 4.8 times, respectively. this highlights a further progression of cytolysis and output of enzymes from the damaged hepatic tissue as a result of l-name pre-treatment. therefore, it was found that stimulation of no synthesis in the animals with i/r improved function of the liver and blockage of no synthesis by nonselective nos inhibitor l-name aggravates liver injury. stimulated inos activity was registered in i/r treatment, content of which in the liver increased by 57.6 %, while in the serum – by 185.0 %. whereas, the concentration of enos in the liver was reduced by 38.5 % and in the blood by 18.2 % compared with the group 1 (table 2). these changes took place on the background of a decline in serum levels of stable metabolite of nitric oxide no 2 – by 51.7 %. this value has not changed significantly in the liver relative to the control group of similar animals. the level of no 3 – in the blood did not change significantly compared to the liver where it was reduced by 65.5 % (table 3). quantitative indicators can be evaluated as a manifestation of a decrease in the total content of final metabolites of no in both blood and liver during i/r trial. the intensity of metabolism of nitric oxide under conditions of prophylactic administration of its precursor was evaluated by the concentration of its stable metabolites (table 3) and the activity of nosynthase isoforms (table 2). the level of nitric oxide metabolites in the blood increased: no 2 – – by 3.7 times, and no 3 – – by 31.7 %, respectively, after administration of l-arginine, and in liver homogenates the content of nitrite anion was not changed (p>0.05), and the nitrate anion increased respectively by 3.1 times. we observed one-way influence of the substance on the expression of no-synthase isoforms, both in blood and in the liver. enzyme immunoassay results showed that the use of l-arginine in i/r treatment leads to increased enos content in hepatocytes (43.5 %) and the reduction of inos (19.2 %) (table 2). a similar pattern was observed table 1. effect of l-arginine on alt and ast activity in the liver of sham and experimental groups of rats results are expressed as mean±sd (n=6); significantly different: p – from sham, p 1 – from i/r injury group; activity of alt and ast are expressed in mmol/(l´h) ����� ����� �� �� ����� �������� ��� � �� ���� ��� ������������ �� � ��� ����� ����� � �� �!�� ��"� #$!!%#$�#� �$�&%#$#'� ()#$##�� �$�#%#$#'� ()#$##&� (�)#$##�� �$�&%#$��� ()#$##�� (�)#$##�� ��"� �$*�%#$��� !$�+%#$�+� ()#$##�� �$,�%#$�#� ()#$#�� (�)#$#&� +$,+%#$�&� ()#$##�� (�)#$##�� � table 2. enos and inos content in liver and blood of sham and experimental groups ������ ��� � � �� �� ���� ����� ����� ������� ���� ������ ��������������� ����� ����� ������� ����������������� ����� ������� �� ���� � �!"� �#� $% &'"� &#� # &'"� (�� $ (!"� �(� )�*��� �� �$�� $ (�"� �%� +,� �'� #� $$"% ��� +,� ���� % !("� $%� +,� ���� % $$"� �(� +,� ���� ��� ����� -)�*� �� �� ���� � '�"� $%� +.� �� +�,� �'� '� '%"� #%� +,� ���� +�,� ���� ( &!"� �#� +.� �� +�,� ���� � %�"� �%� +,� �'� +�,� ���� �/�012-)�*� �� �� �%�� � �%"� �$� +,� ���� +�,� ���� �� %�"� (�� +,� ���� +�,� ���� $ &&"� $�� +,� ���� +�,� ���� $ '("� �(� +.� �� +�,� ���� for this and for the following table the results are expressed as mean±sd (n=6); significantly different: p – from sham, p 1 – from i/r injury group o. m. oleshchuk et al. 61 b io m e d ic a l s c ie n c e s ijmmr 2015 vol. 1 no. 1 in blood. inducible no-synthase was reduced by 24.8 % after the use of l-arginine, but remained higher by more than 2.1 times, compared with sham group of animals. endothelial isoform of the enzyme increased by 34.5 % compared to its value in group 1. to summarize, we can say that prophylactic administration of no precursor l-arginine before i/r was followed by the activation of a constitutive nosynthase, with a simultaneous decrease of inducible nos activity and increase in nitric oxide metabolites level. it is logical that the expression of both isoforms of nitric oxide synthase, on the background of inhibition of l-name, decreased enos content was even lower than in the group of animals without correction by 56.5 and 38.5%, and the expression of inos decreased by 55.9 and 39.3 % in blood and hepatocytes. therefore, l-name reduced expression of all nos forms (table 2). it was also shown that the level of nitrite and nitrate anions after administration of non-selective blocker of no synthesis decreased by 45.0 and 12.6% in the serum and by 41.1 and 46.0 % in liver homogenates compared to ir. the levels were also significantly lower in the affected organ (by 1.7 and 1.8 times) compared to the group 1 of animals. we can hypothesize that reduction of end metabolites of nitric oxide synthesis correlates with inhibition of no synthase expression in both serum and liver (table 3). during histological examination of liver tissue of animals, which previously were administered larginine and subsequently simulated ischemiareperfusion, we found that the trabecular structure of the liver lobules was preserved. the central veins were dilated and well visualized. periportal areas had lymphocytic and histiocytic infiltration, particularly around the bile duct (fig.1). histological examination of liver tissue in ischemia-reperfusion model on the background of the previous l-name administration showed that the trabecular structure of the liver lobules was significantly changed (fig. 2). sinusoids were greatly expanded and they were free from red blood cells in central lobular areas. narrowed and saturated cell macrophages in the central and peripheral parts of the liver lobules were not present as well. portal tracts expanded mainly due to the plethora of vessels and severe perivascular lymphocytic and histiocytic infiltration. signs of small droplets of fat and protein degeneration were present. enhanced bile ducts and the presence of a table 3. no 2 – and no 3 – content in liver and blood of sham and experimental groups ������ ��� �� � �� �� ��� ��� ��� ������� ��� ��� ��� ������� ��� ��� ��� ��������� ��� ��� ��� ��������� ������� �� ���� ���� !�!"� �!��" !�#�� ��$! !���� %��% !�&%� '�(��� �� ���� !�$" !�!&� )*!�!!�� %�%! !�#�� )+!�!&� ���$ !���� )+!�!&� ,��! !�$$� )*!�!!�� �-� ��.�. /'�(� �� �� �,�� ��"# !�!$� )*!�!!�� )�*!�!!�� �,�!& !��,� )*!�!�� )�*!�!�� ��!$ !��!� )+!�!&� )�+!�!&� %�%# !��!� )+!�!&� )�*!�!!�� �0�123/'�(� �� �� �#�� !�#, !�!#� )*!�!!�� )�*!�!�� "��& !���� )*!�!&� )�*!�!&� !�%" !�!,� )*!�!!�� )�*!�!&� ��$, !�!$� )*!�!&� )�+!�!&� � fig. 1. histological structure of the liver after administration of l-arginine in ischemia-reperfusion injury. hematoxylineosin. magnification power 160�. fig. 2 histological structure of the liver during ischemiareperfusion and application of l-name. hematoxylin-eosin. magnification power 160�. o. m. oleshchuk et al. 62 b io m e d ic a l s c ie n c e s ijmmr 2015 vol. 1 no. 1 small amount of bile can be estimated as intrahepatic cholestasis. a large number of cells with pyknotic nuclei and necrotic cells were observed during histological observation in periportal zone of acinus. discussion i/r is a series of multifaceted cellular events which take place on the resumption of oxygen delivery to the affected organ after a period of hypoxia. ir occurs in the liver during procedures that are associated with vascular inflow obstruction followed by restoration of blood flow. i/r may result in major hepatocellular damage [8]. no plays a significant role in the acute phase of i/r, as this phase is associated with a rapid decrease in available no. this decrease occurs either by depressed production by enos in sinusoidal endothelial cells, increased degradation by reactive oxygen species, or both [9]. in this i/r study expression of liver inos increased and enos level decreased. it was determined during our previous i/r studies that the levels of il-1�, il-6 and tnf-� in i/r were significantly increased [6]. therefore, cytokines provoked stimulation of inos expression. to summarize, there is a lack of synthesis of no during the early periods of reperfusion, which may be due to inhibition of enos [9]. as for this study, we have found that after 2 hours of reperfusion levels of nitrates do not change significantly in the blood with decrease in the liver, and conversely decreased blood nitrite levels. we have shown that the application of l-arginine during i/r trial causes activation of nitric oxide synthesis. increased concentrations of no stable metabolites nitrite and nitrate anions in both blood and liver is consistent with our previous results and other scientific data [10, 11]. we observed oneway impact on the content of no-synthase isoform in blood and in the liver. the level of endothelial form increased and inducible – declined. results of our histological studies have shown that the precursor of nitric oxide synthesis has protective influence on the morphological structure of hepatocytes. blocking the enzymatic synthesis of no in liver i/r led to the aggravation of the pathological process, as evidenced by the results of our biochemical and histological studies. thus, the introduction of non-selective blocker of nos l-name increased activity of alt and ast, indicating a further increase cytolysis of hepatocytes and this is consistent with l. m. wang et al. (2003) [12]. our results on the liver degradation by complete blocking enzymatic no formation during i/r suggest no protective role during early reperfusion. conclusion. ischemia reperfusion injury is a well-defined threat to the liver during periods of interruption and restoration of oxygen delivery. it occurs in certain procedures such as hepatic resections and orthotopic liver transplantation. relative no deficiency, due to enos inhibition, is central in the pathogenesis of this injury. replacing no per se either precursors or via donor drugs represents a novel method for ameliorating hepatic ischemiareperfusion injury. references 1. selzner n, rudiger h, graf r, clavien p. protective strategies against ischemic injury of the liver. gastroenterol 2003; 125: 917–936. 2. bahde h, spiegel hu. hepatic ischemia-reperfusion injury from bench to bedside. bri j surg 2010; 97: 1461–1475. 3. ohmori h, dhar d, nakashima y et al. beneficial effects of fk409, a novel nitric oxide donor on reperfusion injury of rat liver. transplantation 1998; 66: 579–585. 4. shah v, kamath sp. nitric oxide in liver transplantation: pathobiology and clinical implication. liver transplantation 2003; 1 (9): 1–11. 5. lin hi, wang d, leu f-j. et al. ischemia and reperfusion of liver induces enos and inos expression: effects of no donor and nos inhibitor. chin j physiol 2004: 47 (3): 121–127. 6. oleshchuk om. experimental study of nitric oxide precursors in hepatic ischemia-reperfusion. hospital surgery 2012; 4(60): 42–47. (in ukrainian) 7. kiselyk io, lutsyk md, shevchenko lyu. features determination of nitrate and nitrite in the blood of patients with viral hepatitis and jaundice of different etiology. lab diagnostyka 2001; 3: 43–45. (in ukrainian) 8. phillips l, lopez–neblina f, toledo–pereyra lh. nitric oxide mechanism of protection in ischemia and reperfusion injury j invest sur 2009; 22: 46–55. 9. hines in, kawachi s, harada h et al. role of nitric oxide in liver ischemia and reperfusion injury. mol cell biochem 2002; 234/235: 229–237. 10. chattopadhyay p, shukla g, wahi ak. protective effect of l–arginine against necrosis and apoptosis induced by experimental ischemic and reperfusion in rat liver. saudi j gastroenterol 2009; 15 (3): 156–162. 11. chander v, chopra k. renal protective effect of molsidomine and l–arginine in ischemia–reperfusion induced injury in rats. j surg res 2005; 128: 132–139. 12. wang lm, tian xf, song qy et al. expression and role of inducible nitric oxide synthase in ischemia– reperfusion liver in rats. hepatobiliary pancreat dis int 2003; 2: 252–258. received: 2014.04.30 o. m. oleshchuk et al. 24 r a d ia t io n m e d ic in e a n d o n c o l o g y issn 2413-6077. ijmmr 2022 vol. 8 issue 2 *corresponding author: dr. yumkhaibam s. devi, associate professor, department of radiation oncology, regional institute of medical sciences, imphal, manipur, 795004, india. e-mail: sobitadeviy@gmail.com international journal of medicine and medical research 2022, volume 8, issue 2, p. 24-33 copyright © 2022, tnmu, all rights reserved k. baidya et al. doi 10.11603/ijmmr.2413-6077.2022.2.13118 hypofractionated radiotherapy with concurrent chemotherapy with weekly cisplatin in locally advanced relatively radioresistent subsites of head and neck cancers k. baidya1, *y. s. devi1, a. s. devi1, y. i. singh1, d. das2, r. mahawar1, n. n. devi1 1 – regional institute of medical sciences, imphal, manipur, india 2 – atal bihari vajpayee regional cancer institute, agartala, tripura, india background. locoregionally advanced head and neck cancers are more aggressive and locoregional failure rate after conventional radiotherapy is high. objective. the aim of the study is to assess the tumor response and toxicities of hypofractionated radiation therapy with concurrent chemotherapy in treatment of four relatively radioresistent tumor sites of head and neck. methods. a prospective randomised control trial was conducted in 27 head and neck cancer patients. all patients were treated with hypofractionated radiotherapy at 250cgy/fraction once daily to a maximum of 62.5gy in 25 fractions with concurrent cisplatin 30 mg/m2. data were evaluated with spss version 21.0 for windows with p-value <0.05. results. complete and partial responses were achieved in 15 (57.7%) and 8 (30.8%) patients respectively with an overall response rate of 88.5% and three patients having stable disease. grade 3 and 4 acute mucositis was experienced by 17 patients (65.4%) and seven patients (27%), respectively. grade 3 dysphagia was found in 21 patients (80.7%) and grade 3 and 4 skin reactions – in 11 and 2 patients, respectively. most patients had manageable acute toxicities. most of the late complications were of grade 2 and 3. the median time to locoregional recurrence was 12 months and one year progression-free survival attained by 61.5% patients. conclusion. treatment with hypofractionated radiotherapy with concurrent cisplatin appears feasible and safe and is associated with a good response rate. although grade 3 and 4 toxicities were comparatively high but it was manageable. late toxicities were within tolerable levels. keywords: head and neck cancer; hypofractionated; radioresistent; response; mucositis. introduction the term “head and neck cancers” usually refers to the variety of neoplasms arising from upper aero-digestive tract with majority (approximately 80%) of head and neck malignancies of squamous cell carcinomas [1]. oral cavity and laryngeal cancers are the most common head and neck cancers globally [2]. in india, age adjusted rate (aar) of incidence for head and neck squamous cell carcinoma (hnscc) is approximately 988.9 per 100000 populations with male to female ratio 3:1. in north­east of india, aar incidence of hnscc is 459.7 per 100000 populations, hypopharyngeal cancer being the commonest cancer [3]. hnscc have been a disease of older males with heavy lifelong tobacco use, high alcohol consumption, poorly preserved diet, and bad dentition usually presents with pain, dysphagia, neck mass etc. [4]. locoregionally advanced (la) hnscc is treated by multimodality approaches combining surgery, radiotherapy and chemotherapy; radio therapy being the main modality for unresctable tumors [5]. nevertheless, the outcome of conventional radiation was disappointing, especially in advanced stage (i.e., stage iii or iv) with a 5-year survival less than 60% only [6, 7]. one of the important causes of failure of conventional radiation is accelerated repopulation of tumor clones which usually starts around the 4th week of radiotherapy. to prevent this an altered fractionation scheme was introduced [8]. 25 r a d ia t io n m e d ic in e a n d o n c o l o g y issn 2413-6077. ijmmr 2022 vol. 8 issue 2 tumor hypoxia is a major concern in hnscc causing treatment failures with radiation therapy. whereas many sites of head and neck are radiosensitive like nasopharynx or larynx, few inherently radioresistant tumors of base of tongue, vallecula, pyriform sinus, retromolar trigone are associated with increased metastases and decreased sensitivity to ionizing radiation [9]. hence, hypofractionated schedules by using a small number of fractions with a larger dose per fraction and shortening of the overall treatment duration have been tried to counter the tumor hypoxia and repopulation [10]. a meta-analysis of 16 randomized clinical trials demonstrate that optimized hypofractionation can markedly improve tumor control probability by 35% to 49% for late-stage head and neck cancer by overcoming tumor repopulation in fast-growing tumors [11]. addition of concurrent chemotherapy to radiation would increase the efficacy of the treatment along with tumor control [12]. the aim of this study is to assess the tumor response and toxicities of hypofractionated radiation therapy with concurrent chemotherapy in treatment of four relatively radioresistent tumor sites of head and neck. after reviewing the literature and surveillance data, we have selected four subsites for this study retromolar trigone (rmt), base of tongue (bot) and vallecula and pyriform sinus (pfs) because of their rarity and aggressive behaviour along with increased incidence of treatment failure and less long-term survival. cancer in these sites is more hypoxic. methods a prospective non-randomized experimental study was undertaken in the department of radiation oncology, regional institute of medical sciences, imphal, manipur for two years starting from october 1, 2019 to september 30, 2021 among 27 patients. the permission of the research ethics board (reb), rims, imphal, manipur was obtained (no. a/206/rebcomm(sp)/rims/2015/597/75/2019) before initiating the study. inclusion criteria: 1. patients aged between 30-75 years of age. 2. patients with histopathologically confirmed squamous cell carcinoma of the selected head and neck subsites of base of tongue, vallecula, pyriform sinus, retromolar triangle (ajcc 8th edition stage iii, iva & ivb). 3. patients who were not eligible for curative resection due to advanced stage. 4. karnofsky performance status (kps) ≥60%. 5. normal complete blood count, liver and kidney function, blood glucose and normal audiometry. exclusion criteria: 1. subsites other than those included in inclusion criteria. 2. patient not willing to give consent. 3. patient with second malignancy, previously treated with radiation therapy / chemotherapy and / or surgery. 4. patients with uncontrolled co-morbid illness, psychotic disease, pregnant and lactating women. all patients were treated with external beam radiotherapy by theratron 780-c, telecobalt machine administered on a 5-week schedule by shrinking field technique and hypofractio­ nated radiotherapy at 250 gy/fraction once daily to a maximum of 62.5 gy in 25 fractions (bed=78.12). primary tumor and whole neck were irradiated up to a dose of 45gy in 18 fractions. doses equivalent to conventional fractionation were calculated for late reacting tissue and spinal cord shielding was performed after 40gy in 16 fractions (bed=73.33), which was biologically equivalent with 46 gy/23 fractions (bed=76.66) in conventional radiotherapy. clinically uninvolved areas were excluded after 45 gy. primary tumor with metastatic nodal regions was irradiated up to 62.5 gy. concurrent cisplatin was administered at a dose of 30 mg/m2 weekly for a total of 5 doses. the patients received standard hydration, mannitol infusion and prophylactic antiemetic medications for cisplatin therapy. laboratory requirements before the dose of cisplatin were granulocytes ≥1,500/μl, platelets ≥100,000/μl and serum creatinine ≤1.5 mg/dl and normal audio metry. during the whole period of treatment, the patients were reviewed every to note any adverse or untoward side effect, performance status, subjective response. tumor response was assessed in 6 weeks after the completion of radiotherapy using recist criteria (version 1.1) [13] with local examination and contrast enhance computed tomography (cect) imaging. acute radiation toxicities were assessed during each week of the radiation treatment and graded according to the rtog acute radiation toxicity grading. late radiation toxicities were assessed in 3 months after completion of treatment and thereafter every 3 months till 1 year (minimum 6 months) using the rtog late radiation morbidity grading [14]. k. baidya et al. 26 r a d ia t io n m e d ic in e a n d o n c o l o g y issn 2413-6077. ijmmr 2022 vol. 8 issue 2 statistical analysis: spss­version 21 for windows (ibm corp, armonk, ny, usa) was used for statistical analysis. variables like age, karnofsky performance scores were presented as descriptive analysis using mean, median, mode and standard deviation. categorical variables like gender, primary site, staging, histology, tumor response and toxicity profile were presented in terms of percentages and proportions as frequency tables or charts. test of significance was done with chi­square test using p-value 0.05 for assessing the association of multiple factors with treatment response and progression-free survival (pfs). pfs was analyzed using kaplan-meier survival curve and descriptive survival table. results total 27 cases of confirmed locoregionally advanced hnscc were studied during the study period. one patient stopped treatment in between and lost follow up was excluded from the study. hence data assessment was done for the 26 patients who completed their full coarse treatment. seventeen patients completed the treatment within the planned period of 35 days. the median time of delivery for these patients was 37 days (range, 33–65 days). delay in the treatment contributed by environmental factor (covid-19 pandemic), social factors (lockdown), patient factors (noncompliance), treatment factors (side effects) and mechanical factors (machine break down). characteristic features of the patients and tumor are presented in tables 1 and 2, respectively. 19 out of 26 patients had history of tobacco consumption whereas 16 patients had history of alcohol consumption and 9 patients had consumed both tobacco and alcohol. median follow up period was 11 months with a range of 18 months (6-24 months). there were 16 patients of pyriform sinus (5 stage iii, 7 stage iva and 4 stage ivb patients), 5 patients of retromolar trigone (3 stage iii and 2 stage iva patients), 3 patients of vallecula (2 stage iva and 1 stage ivb patients) and 2 patients of base of tongue (both stage iva). table 3 shows the overall treatment response at the end of 6 weeks after completion table 1. patients characteristics variables sub variables frequency(n=26) percentage (%) median age in years 58.5 sex male 20 77 female 6 23 kps 60 2 7.7 70 9 34.6 80 10 38.5 90 5 19.2 table 2. tumor characteristics variables sub variables frequency (n=26) percentage (%) t stage t1 2 7.7 t2 10 38.5 t3 10 38.5 t4 4 15.4 n stage n0 3 11.5 n1 4 15.4 n2 13 50.0 n3 6 23.1 stage grouping iii 8 30.8 iv a 13 50.0 iv b 5 19.2 grade well differentiated 7 26.9 mod differentiated 12 46.2 poor differentiated 6 23.1 un differentiated 1 3.8 k. baidya et al. 27 r a d ia t io n m e d ic in e a n d o n c o l o g y issn 2413-6077. ijmmr 2022 vol. 8 issue 2 of treatment in the study population. all the 26 patients were available for assessment at the end of the 1st month. the patients were evaluated with local examination and cect imaging. cr was disappearance of all target lesions in primary site as well as secondary nodes whereas pr was defined as at least 30% decrease in size of target lesions. complete and partial responses were achieved in 15 (57.7%) and 8 (30.8%) patients respectively with an overall response rate of 88.5% and 3 patients were having stable disease. one patient with t4 disease and two patients with n3 disease had radiological cr. all patients were assessed weekly during treatment. most of them had no hematological toxicity. grade 2 anemia occurred in four patients, grade 2 neutropenia was in three patients for which they received blood transfusion and granulocyte colony stimulating factors. grade 3 and 4 mucositis was experienced by 17 patients (65%) and seven patients (27%), respectively. grade 3 dysphagia was evidenced in 21 patients (80.7%). the patients suffering from grade 2 and 3 skin reactions were 9 and 11, respectively. more than 88 % experienced grade 2 hoarseness, and grade 2 and 3 acute salivary gland reaction was found in 11 and 15 patients, respectively. grade 3 and 4 toxicity developed after 3 weeks of radiation which corresponds with 3750 cgy in 15 fractions dose. most patients had manageable acute toxicity. late complications were assessed at the 3rd, 6th and 9th month after completion of radiation. grade 2 and 3 dysphagia was seen in 19 (73%) and 5 (19%) patients, respectively, and grade 2 mucositis was experienced by 38.5% of the study population. grade 2 xerostomia was present in 20 patients at the 3rd months and reduced on further follow up. grade 2 sub cutaneous reaction was evidenced in 12 patients at the 3rd month. almost all the toxicities were reduced on further follow up. the median time to locoregional recurrence was 12 months (ranging 2-24 months). in total 10 patients experienced recurrence. three patients had recurrence at primary as well as nodal site and six patients – at nodal site only. table 3. response rate in the patients according to response evaluation criteria in solid tumor (recist) criteria after 6 weeks of completion of treatment tumor response frequency (n=26) percentage (%) complete response (cr) 15 57.7 partial response (pr) 8 30.8 stable disease (sd) 3 11.5 overall response rate (orr) [cr + pr] 23 88.5 fig. 1. (a) axial ct image of neck showing extensive pyriform fossa mass; (b) response in the same patient in 6 weeks after completion of treatment. а в k. baidya et al. 28 r a d ia t io n m e d ic in e a n d o n c o l o g y issn 2413-6077. ijmmr 2022 vol. 8 issue 2 table 4. acute radiation toxicity in study population according to the rtog criteria, n=26 (%) toxicity grade week 2 week 3 week 4 week 5 anemia grade 2 0 2 (7.7) 3 (11.5) 2 (7.7) grade 3 0 0 1 (3.8) 0 grade 4 0 0 0 0 leucopenia grade 2 0 1 (3.8) 3 (11.5) 0 grade 3 0 0 1 (3.8) 1 (3.8) grade 4 0 0 0 0 mucositis grade 2 3 (11.5) 12(46.2) 8 (28.6) 7 (27.0) grade 3 0 12 (46.2) 13 (50.0) 17 (65.4) grade 4 0 0 7 (27.0) 2 (7.7) skin reaction grade 2 0 6 (23.1) 9 (34.5) 7 (27.0) grade 3 0 0 6 (23.1) 11 (42.3) grade 4 0 0 2 (7.7) 1 (3.8) dysphagia grade 2 3 (11.5) 13 (50.0) 4 (15.4) 7 (27.0) grade 3 0 12 (46.2) 21 (80.7) 20 (76.9) grade 4 0 0 0 0 hoarseness grade 2 0 12 (46.2) 23 (88.5) 18 (69.2) grade 3 0 0 1 (3.8) 4 (15.4) grade 4 0 0 0 0 salivary gland grade 2 0 17 (65.4) 11 (42.3) 11 (42.3) grade 3 0 3 (11.5) 15 (57.7) 14 (53.8) grade 4 0 0 0 0 table 5. late radiation toxicity in study population according to the rtog criteria, n=26 (%) toxicities grade 3rd month 6th month 9th month skin reaction grade 1 17 (65.4) 19 (73.1) 11 (42.3) grade 2 4 (15.4) 5 (19.2) 2 (7.7) grade 3 0 0 0 subcutaneous fibrosis grade1 13 (50.0) 9 (34.6) 4 (15.4) grade 2 12 (46.2) 13 (50.0) 9 (34.6) grade 3 0 0 0 mucositis grade 1 13 (50.0) 12 (46.2) 4 (15.4) grade 2 11 (42.3) 10 (38.5) 6 (23.1) grade 3 0 0 0 dysphagia grade 1 6 (23.1) 6 (23.1) 2 (7.7) grade 2 19 (73.1) 13 (50.0) 8 (30.7) grade 3 1 (3.8) 5 (19.2) 3 (11.5) hoarseness grade 1 13 (50.0) 16 (61.5) 10 (38.4) grade 2 13 (50.0) 9 (34.6) 1 (3.8) grade 3 0 0 0 xerostomia grade 1 5 (19.2) 7 (27.0) 5 (19.2) grade 2 20 (76.9) 16 (61.5) 7 (27.0) grade 3 1 (3.8) 2 (7.7) 1 (3.8) recurrence with distant metastasis was evi denced in one patient. the patients with re current disease were planned for further sal vage treatment. when we have evaluated the dependence of progression free survival on the different factors related to patient and disease, tobacco consumption in any form along with the stage at presentation were found to be the two most important factors affecting the pfs with significant p-value (p=0.038 and 0.004 respectively). k. baidya et al. 29 r a d ia t io n m e d ic in e a n d o n c o l o g y issn 2413-6077. ijmmr 2022 vol. 8 issue 2 discussion national comprehensive cancer network (nccn) recommends for surgical resection of primary tumor in t3-t4a, n0 tumor along with neck dissection in n+ patients followed by rt, but for patients with stage t4b, n0-3, unresectable nodal status and unfit of surgery patients should be treated with concurrent chemoradiotherapy [15]. prevalence of the head and neck malignancy in north eastern part of india is high due to their continuous use of tobacco in various forms and their lifestyle. most of them come with the advanced stage due to their negligence, unawareness, and poverty. achieving long-term local control (lc) in locally advanced squamous cell carcinoma of the head and neck remains a challenge [16]. from radiobiological, economic and logistical points of view, a hypofractionated schedule would be the most suitable option. first, the treatment is completed before accelerated repopulation becomes a significant radiobiologic factor. second, the reduction in the number of fractions also allows a more efficient use of resources, which can help avoid long waiting times for other patients; and lastly, considering that this group of patients are usually of elderly age and often have a poor performance status as well as significant co­morbidities, it is almost mandatory to keep the overall treatment time (ott) as short as possible [17]. moreover, during the recent first peak of the covid-19 pandemic, many radiotherapy departments had reviewed their protocols for chemoradiation to make the ott short by using hypofractionated schedules [18]. the patients characteristics were comparable with other studies [5, 19]. in this research by study design, pfs was the most common subsite followed by the rmt, vallecula and bot. comparison between these subsites are not available in the literature for references. other tumor characteristics were consistent with the previous studies [10, 20]. response: in this study, clinical complete response was observed in 15 (57.7%) patients and partial response in 8 (30.8%) patients with overall response rate of 88.5%. this report is consistent with the study by proceddu et al. where 56% patients had complete response to the primary site and overall objective response rate was 80% [10]. similarly, in the study by teckie et al. 79% patients showed response to hypofractionated rt [21]. our study has shown better result than the study by nguyen nta et al., where patients were treated with 8 gy per fraction on the 0,7 fig. 2. progression free survival (pfs) of the study population. k. baidya et al. 30 r a d ia t io n m e d ic in e a n d o n c o l o g y issn 2413-6077. ijmmr 2022 vol. 8 issue 2 and 21 st day and documented complete response in only 30.9% patients with ort of 81.8%, which means hypofractionated protocol of 62.5 gy in 25 fractions is superior [20]. koukourakis mi et al. studied conformal hypofractionated rt with amifostine combined with cisplatin and cetuximab in patients with lahnc, where the complete response rate was 68.57% [22]. the discrepancy is due to use of conformal hypofractionated rt, the use of amifostine, which reduced mucositis, that contributed to less interruption in treatment and some patients underwent nodal dissection. the study by valentina k et al. using conventional radiotherapy showed complete response in 72.3% patients which was superior than the present study findings. use of 3dcrt with linear accelerator in patients’ treatment could be a factor for such result [23]. rt at either preoperative or post-operative stage in advanced hnscc cases may influence the control rates, but many investigators in prospective studies have shown better response with concurrent chemoradiotherapy practice at any condition [24]. this suggests that patients undergoing accelerated rt with bulky lymph nodes sometimes may require neck dissection to gain benefit compare to conventional fractionation. toxicity: acute radiation reactions were high as expected, it contributed the major and minor deviation in the treatment plan for some patients and increase in overall treatment time. in this study, grade 3 and 4 acute toxicities especially mucositis, skin reaction and dysphagia were high and developed after 3 weeks of radiation which is almost concordance with the previous studies [25, 26]. study by paul sanghera et al. [27] using hypofractionted rt with carboplatin showed minimal grade 3 mucositis was experienced by 60 patients (74%), with 5 (5%) patients with grade 4 mucositis. grade 3 dysphagia was evidenced in 44 patients (54%). the incidence of acute toxicity was very low in the patients treated with amifostine as observed by koukourakis mi et al. [22]. the incidence of late side effects assessed at the 3rd, 6th and 9th months of treatment completion within a median of 11 months of followup was within acceptable levels, despite the use of hypofractionation. longer follow-up is certainly needed to better estimate late adverse events. reducing overall irradiation time by hypofractionation is an applicable alternative to conventional regimens with concerns that large fraction sizes may lead to greater toxicity in late-responding tissue supported by earlier studies. the reduction in number of fractions avoids long waiting times. there is no doubt that hypofractionation offers major potential advantages to patients and to the economy of health systems [19, 28, 29]. progression free survival: the median progression free survival in this study was 12 months. 38.5% patients had developed recurrence during the study period. pfs at the 6th and 12th month was 84.6% and 61.5%, respectively. this finding is almost similar with the study by agarwal jp et al. [5] where 1-year pfs was 55%. another study by teckie et al. [21] demonstrated a locoregional pfs rate of 70% at the 6th month, 43% at the 12th month, and 29% at the 2nd year with median follow up of 11.9 months. though overall response rate is good in this study, the sites chosen for the study are prognostically bad due to more hypoxia and less radiation responsiveness leading to poor progression free survival. of the various prognostic factors studied, tobacco consumption and higher stage had significantly affected the pfs. other prognostic factors like age, sex, kps, weight, site of tumor, t and n stage and the tumor grade did not show statistically significant difference on the pfs. all the patients who had loco-regional failure presented with n3 node at initial presentation. this denotes that patients with a large nodal burden are probably less likely to be benefited by hypofractionation. this out­ come is well-corroborated with march collaborative group meta-analysis, which showed that the effect of altered fractionation was significantly more pronounced on the primary tumor than on the nodal disease [30]. limitations of our study include a limited follow-up time, small sample size, treating with 2-d cobalt-60 teletherapy machine. in view of the non-randomized single arm nature of the study, it would not be correct to draw any definite conclusions regarding local recurrence and survival patterns from this study. the use of neck dissection and the ability to complete treatment within the prescribed overall treatment time would have contributed better results. conclusion treatment of locoregionally advanced head and neck cancer (lahnc) with hypofractionated rt with 62.5 gy in 25 fractions concurrently with k. baidya et al. 31 r a d ia t io n m e d ic in e a n d o n c o l o g y issn 2413-6077. ijmmr 2022 vol. 8 issue 2 cisplatin is feasible and safe and is associated with a good response rate. although grade 3 and 4 toxicities were comparatively high but it was manageable. late toxicities were within tolerable levels. these data highlight the potential usefulness of hypofractionation for lahnc, especially for low to medium income countries, where access to advanced techniques of rt is poor. long-term follow up with more sample size and comparative trials are needed for further recording of late response as well as toxicities to provide definitive conclusions about hypofractionated rt in the selected subsites lahnc. conflict of interests authors declare no conflict of interest. acknowledgements we would like to express our sincere gratitude to the head of the department of radiation oncology, rims, imphal, manipur, respected teachers and above all our patients for their kind cooperation and participation in the study. author’s contributions kishalay baidya, yumkhaibam sobita devi, yengkhom indibor singh – conceptualization, methodology, formal analysis, writing – original draft; kishalay baidya, akoijam sunita devi, deepsikha das – data curation, writing – reviewing and editing; rahul mahawar, nongmaithem 0ima devi – investigation, formal analysis. роль гіпофракціонованої променевої терапії з одночасною хіміотерапією цисплатином при локально поширеному відносно радіорезистентному раку голови та шиї k. baidya1, *y. s. devi1, a. s. devi1, y. i. singh1, d. das2, r. mahawar1, n. n. devi1 1 – regional institute of medical sciences, imphal, manipur, india 2 – atal bihari vajpayee regional cancer institute, agartala, tripura, india вступ. місцево-поширений рак голови та шиї є більш агресивним, і частота невдач терапії після звичайної променевої терапії є високою. мета. оцінити реакцію пухлини та токсичність гіпофракціонованої променевої терапії з одночасною хіміотерапією при лікуванні чотирьох відносно радіорезистентних пухлин голови та шиї. методи. проспективне рандомізоване контрольне дослідження було проведено за участю 27 пацієнтів з раком голови та шиї. усі пацієнти отримували гіпофракціоновану променеву терапію в дозі 250 сгр/фракція один раз на день до максимуму 62,5 гр у 25 фракціях з одночасним цисплатином 30 мг/м2. дані були оцінені за допомогою spss версії 21.0 для windows із значенням p<0,05. результати. повна та часткова відповіді були досягнуті у 15 (57,7%) та 8 (30,8%) пацієнтів відповідно із загальною частотою відповіді 88,5%, а у трьох пацієнтів захворювання було стабільним. гострий мукозит 3 і 4 ступеня мали 17 пацієнтів (65,4%) і 7 пацієнтів (27%) відповідно. дисфагію 3 ступеня спостерігали у 21 пацієнта (80,7%), а шкірні реакції 3 і 4 ступеня — у 11 і 2 пацієнтів відповідно. більшість пацієнтів мали керовану гостру токсичність. більшість пізніх ускладнень були 2-го та 3-го ступенів. середній час до локального рецидиву склав 12 місяців і однорічну виживаність без прогресування захворювання досягли 61,5% пацієнтів. висновки. лікування гіпофракціонованою променевою терапією з одночасним прийомом цисплатину видається можливим і безпечним і пов’язане з хорошим рівнем відповіді. хоча 3 і 4 ступені токсичності були порівняно високими, але це можна було контролювати. пізня токсичність була в межах допустимого рівня. ключові слова: рак голови та шиї; гіпофракціонована; радіостійкий; відповідь; мукозит. information about the authors kishalay baidya – senior resident, department of radiation oncology, regional institute of medical sciences, imphal, manipur, india, https://orcid.org/0000­0002­1178­0749, e­mail: amikishalay@gmail.com yumkhaibam sobita devi – associate professor, department of radiation oncology, regional institute of medical sciences, imphal, manipur, india, https://orcid.org/0000­0001­7151­5667, e­mail: sobitadeviy@gmail.com k. baidya et al. 32 r a d ia t io n m e d ic in e a n d o n c o l o g y issn 2413-6077. ijmmr 2022 vol. 8 issue 2 references 1. semple c, parahoo k, norman a, mccaughan e, humphris g, mills m. psychosocial interventions for patients with head and neck cancer. cochrane database syst rev 2013; 7: cd009441. doi: 10.1002/14651858.cd009441.pub2. 2. bray f, ferlay j, soerjomataram i, siegel rl, torre 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radiochemotherapy with cetuximab, cisplatin, and amifostine for locally advanced head and neck cancer. a feasibility study. int j radiat oncol 2010;77(1):9­15. doi: 10.1016/j.ijrobp.2009.04.060. 23. krstevska v, stojkovski i, zafirova­ivanovska b. concurrent radiochemotherapy in locally regionally advanced oropharyngeal squamous cell carcinoma. analysis of treatment results and prognostic factors. j radiat oncol 2012;7(6):78­82. doi: 10.1186/1748­717x­7­78 24. yi j, huang x, xu z, liu s, wang x, he x, et al. phase iii randomized trial of preoperative concurrent chemoradiotherapy versus preoperative radio_ therapy for patients with locally advanced head and neck squamous cell carcinoma. oncotarget 2017; 8(27):44842­50. doi: 10.18632/oncotarget.15107. 25. bonner ja, harari pm, giralt j, cohen rb, jones cu, sur rk, et al. radio therapy plus cetuximab for locoregionally advanced head and neck cancer: 5-year survival data from a phase 3 randomized, and relation between cetuximab induced rash and survival. lancet oncol 2010;11(1):21­8. doi: 10.1016/s1470­2045(09)70311­0. 26. goswami u, banerjee s, dutta s, bera a. treatment outcome and toxicity of hypofractionated radiotherapy with concomitant chemotherapy versus conventional fractionated concomitant chemoradiation in locally advanced head-and-neck carcinoma: a comparative study. asian j pharm clin res 2022;15(7):167­71. doi: 10.22159/ajpcr.2022v15i7.44857. 27. sanghera p, mcconkey c, ho kf, glaholm j, hartley a. hypofractionated accelerated radiotherapy with concurrent chemotherapy for locally advanced squamous cell carcinoma. int j radiat oncol biol phys 2007;67(5):1342­51. doi: 10.1016/j.ijrobp.2006.11.015. 28. spartacus rk, dana r, rastogi k, bhatnagar ar, daga d, gupta k. hypofractionated radiotherapy for palliation in locally advanced head and neck cancer. indian j palliat care. 2017;23(3):313­16. doi: 10.4103/ijpc.ijpc_9_17. 29. kodaira t, kagami y, shibata t, shikama n, nishimura y, ishikura s, et al. results of a multiinstitutional, randomized, non-inferiority, phase iii trial of accelerated fractionation versus standard fractionation in radiation therapy for t1-2n0m0 glottic cancer: japan clinical oncology group study (jcog0701). ann oncol. 2018;29(4):992­7. doi: 10.1093/annonc/mdy036. 30. lacas b, bourhis j, overgaard j, zhang q, grégoire v, nankivell m, et al. role of radiotherapy fractionation in head and neck cancers (march): an updated meta­analysis. lancet oncol 2017;18(9): 1221–37. doi:10.1016/s1470­2045(17)30458­8. received 15 august 2022; revised 5 october 2022; accepted 2 november 2022. this is open access article distributed under the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. k. baidya et al. koncor1_2014.pm6 39 r a d ia t io n m e d ic in e a n d o n c o l o g y ijmmr 2015 vol. 1 no. 1 prognostic significance of cd56 expression in acute leukemias b. m. ahmed1, n. a. kantoush2, m. a. ismail1, d. a. abd-el haleem2 1ain shams university, cairo, egypt 2national research center, cairo, egypt background. cd56 expression was extensively investigated in cases of acute leukemia. many studies associated it with short overall survival, unfavorable outcome, lower rates or short complete remission, however the results remain controversial. objectives. the aim of this study was to investigate the frequency and prognostic relevance of cd56 expression in patients with acute leukemia and to compare its value with other standard prognostic factors, such as age, gender, leukocytosis, morphologic subtypes, extramedullary invasion, cytogenetic abnormalities and performance status. methods. forty cases of acute leukemia treated at ain shmas university hospitals were investigated. they were classified by the french-american-british group (fab) criteria, flow cytometry, and cytogenetics data. they included twenty cases of acute myeloid leukemia (aml) and twenty cases of acute lymphoblastic leukemia (all). results. cd56 positive expression was detected in nine cases of aml (45 %), and only in two patients with all (10 %). the highest incidence of cd56 positivity was in fab subtypes m1 (35 %) and m2 (35 %).association studies between cd56 expression and other prognostic factors in aml cases showed no significant association with age, gender, clinical presentation, hematological data or cytogenetic risk groups. incidence of relapse was higher in aml patients expressing cd56 than those who did not (66.7 % vs 10 %, p=0.01). higher death rates were encountered in aml cases with cd56 expression than those without (55.6 % vs 10 %, p=0.032). conclusions. cd56 antigenic expression in aml cases represents an adverse prognostic factor. it should be regularly investigated in cases of aml for better prognostic stratification and assessment. key words: cd56; leukemia, myeloid; prognosis introduction acute leukemia is a heterogeneous group of disorders. they have various morphological, immunophenotypic and cytogenetic patterns. identifying these characteristics is useful for prediction of therapy responses and prognosis of the disease [1]. several phenotypic markers demonstrated to have clinical significance including detection of minimal residual disease [2, 3] rate and duration of complete remission (cr), disease-free survival, and overall survival (os) [3]. cd56, a neural cell adhesion molecule (ncam), is an early described natural killer cell-associated antigen. it mediates cell-to-cell interaction and is possibly involved in cell-mediated cytotoxicity. this antigen is also found in a subset of cd3+ cytotoxic t-cells and a small population of cd4+ t-cells and monocytes [4]. it is expressed in various hematopoietic neoplasms, including acute myeloid leukemia(aml) [5], acute lymphoblastic leukemia (all) [6], lymphoma [7], and myeloma cells [8]. being a cell adhesion molecule, cd56 expression on tumor cells is believed to play a role in their localization with involvement of extramedullary metastasis [9]. the prognostic value of cd56 expression in cases of acute leukemia has been extensively investigated but with few consistent results [3]. some investigators associated cd56 expression with short os [5, 10–12], and lower cr rates [10] in aml patient. although ferrara et al.[12] reported short os in aml cases expressing cd56; they could not detect association of this marker with the rate of cr in m3 aml cases. moreover, chang et al. studied 379 cases of aml including all subtypes except m3 and reported that the cr rate was not associated with cd56 expression, but with cd34 and hla-dr expression [13]. the aim of this study is to evaluate the prognostic role of cd56 expression in newly diagnosed acute address for correspondence: mona ahmed ismail, department of clinical and chemical pathology, faculty of medicine, ain shams university, cairo, egypt b. m. ahmed et al. international journal of medicine and medical research 2015, volume 1, number 1, p. 39-44 copyright © 2015, tsmu, all rights reserved 40 r a d ia t io n m e d ic in e a n d o n c o l o g y ijmmr 2015 vol. 1 no. 1 leukemia including aml and all cases and correlate the results with other prognostic factors. methods patients this study was carried out on 40 newly diagnosed cases of acute leukemia. it included 20 aml and 20 all patients presented at the hematology/ oncology clinics in the internal medicine and pediatric hospitals, ain shams university, cairo, egypt. diagnosis and classification of acute leukemia was based on who (2008) [14] and fab [15] criteria. bone marrow and peripheral blood samples obtained at presentation were examined for morphology, immunophenotyping and complementary cytogenetics. another bone marrow samples were taken on the 28th day to evaluate response to induction therapy. response to treatment was considered ineffective if more than 5 % blasts were detected in bm on the 28th day. immunophenotyping flow-cytometric analyses were performed as previously described [16]. the reactivity for the following markers was analyzed: a panel of fluorescein isothiocyanate (fitc)/phycoerythrin (pe) conjugated monoclonal antibodies to b-lineage markers (cd19, cd10, cd20, cd79a), t-lineage markers (cd2, cd3, cd5, cd7, cytcd3), myeloid markers (cd13, cd33, cd15, mpo, cd61, glycophorin, cd117), monocytic markers (cd14), and common progenitors markers (cd34, hla-dr), supplied by beckman coulter, france. in addition, moab ncam-pe (clone ncam) (miltenyibiotec, germany) for detection of cd56 was used. cells were considered positive for a certain marker when �20 % of blasts expressed it, except for cd56, cd34 and intracellular mpo, where expression of �10 % of blasts was reported as positive. cytogenetic analysis leukemic cells were cultured, and the chromosomes were banded. cytogenetic abnormalities were determined according to the international system for human cytogenetic nomenclature [17]. cytogenetic results were categorized into favorable, intermediate and unfavorable risk group. statistical analysis statistical analysis of the data was performed by using spss 15 software package and windows 7® operating system. categorical data parameters were presented in terms of frequency and percent age. comparisons and associations that involve categorical variables were done by chi square test or fisher exact test depending on the nature of the data. continuous data parameters were described as middle, standard deviation (sd), median and interquartile range (iqr). results forty acute leukemia patients (20 aml and 20 all) were enrolled in this study. table 1 summarizes age, gender, and clinical findings in the studied aml and all cases. according to fab criteria, aml patients were classified into: 7 (35 %) m1, 7 (35 %) m2, 4 (20 %) m3, one (5 %) m4 and one (5 %) m5 case. all were classified into 17 (85 %) b-all and 3 (15 %) t-all cases. successful mitosis was encountered in 18/20 (90 %) aml cases. ten of these cases (55 %) were categorized as favorable [t(8:21), inv(16),t(15:17)], 6 (33.3 %) were intermediate (+8, normal) and 2 (11 %) cases were poor risk group [t(9:22), 11q23]. successful mitosis was obtained in 12(60 %) of all cases. three all cases (25 %) were classified as favorable risk (del 6q, hyperdiploidy>50), 5(41 %) were intermediate risk (normal), and 4(33 %) were poor risk group ( t(9:22), hypodyploidy<45). our follow up of aml cases during induction period showed that 12 (63.2 %) aml cases showed good response to chemotherapy and achieved complete remission, while 7 patients (36.8 %) relapsed, and one case was missed. six of the relapsed patients died. meanwhile, 14 (70 %) all patients showed good response to chemotherapy and achieved complete remission, while five cases (25 %) relapsed, 3 of them died. cd56 expression and association studies: nine aml cases (45 %) were positive for cd56, while 11 (55 %) were negative. cd56 and cd34 coexpression was found in 2 cases (10 %). none of our aml cases co-expressed cd56 and cd7.only 2 all cases (10 %) expressed cd56. one all case co-expressed cd56 and cd34.there was a statistically significant positive association between cd56 positive expression and cd117 in aml (p<0.05). no significant association was found between cd56 positive expression and immunotable 1. demographic and clinical findings in aml and all cases � ���� ����� ����� ����� � ��� ���� � �� �� � � � � ����� ������ � ��� ������ � � ����������� �������� ������� � � � ���������� ������� ������� � ��� !�"� ��� �� ��� �� # $ !"� �������� �������� �������� %$� �"� �������� �����&�� �������� ���$' � �"$ !'������� �������� ��������� ( ��"������� ��������� �����)�� � * ������� �����)�� ��������� +� ��� ������� �������� �������� b. m. ahmed et al. 41 r a d ia t io n m e d ic in e a n d o n c o l o g y ijmmr 2015 vol. 1 no. 1 phenotype profile of all (p>0.05). because of the small all sample, expressing cd56, the analysis of results of these cases questions its statistical reliability. no significant association was detected between cd56 positive expression and age (p=0.806) (table 2), gender, clinical or hematological data in aml patients (table 3). similarly, we could not associate this marker expression with cytogenetics risk groups (p=0.118) (table 4). aml cases showed a statistically significant association between cd56 positivity and poor outcome. we missed one case and only 19 patients were evaluated including nine cd56 positive and table 2. association studies between cd56 and age in aml patients ��������� ��� ���� ��������� ��� ��� ��� �������� ��� ����� ��� � � ��� ������������� � !�� �"!##� "!"�� "!$"��� ��� ��������� ���� ������ ��������� ���� ����� � �� �� �� �� � ��� � �� ���� �� �� ������ �� ������ ������ ������� ������� �� �� ������ �� ������ � � � � ��� ������ !� !!�!"� ������ ������� ���#���$� %� �� ����� �� ������ � � � � �� "��"�� �� ������ &��&�� ������� ����'(���� %� �� �"��"� �� ������ � � � � "� !��!&� "� ""�"�� ��"�"� ������� ���#��� %� &� �!��!� �� ������ � � � � �� ������ &� &&�&&� ��&�&� ���!��� �����((�$� %� �� ������ �� ����!� � � � � !� �&���� �� ������ ���&�� ������� �������$� %� �� &��&�� �� ������ � � � � �� "��"�� �� ����!� ������ ����!�� ���)(��*���� %� �� �"��"� &� &&�&&� � � � +��� �� "��"�� "� ""�"�� ������ ������� ���,-� ���� �� �"��"� �� ������ � � � +��� "� !��!&� �� ������ ������ ��&��� ���.) � ���� &� �!��!� �� ������ � � � +���� �� "��"�� !� !!�!"� ������ ������� ����#/� ����� �� �"��"� �� ������ � � � +��� �� ������ �� ������ ������ ������� ����)�)(����� ���� �� ������ �� ������ � � � +!�� �� ������ �� ������ ������ ������� ���)��)(����� �!�� �� ������ �� ������ � � � table 3. association studies between cd56 expression and gender, clinical and hematological data in aml patients n: number of cases, hb: hemoglobin, wbc: white blood counts, plt: platelets count, pb: peripheral blood, bm: bone marrow sig: significant, ns: non-significant. ��������� ���� ������ ��������� ��� ����� ��� �� ���� � �� �� �� �� � � � ����� �� ������ �� ������ ���!"������� #� ������ �� ������ $��������%� &��'��!�()� ���!� �� *��*�� +� +� #����� +�**�,� ��� table 4.association between cytogenetics risk groups and cd56 expression in aml patients *2 cultures failed, n: number, sig: significant, ns: non-significant n = number of cases, ns: non-significant. b. m. ahmed et al. 42 r a d ia t io n m e d ic in e a n d o n c o l o g y ijmmr 2015 vol. 1 no. 1 10 negative cases. six patients out of the 9 positive cd56 cases (66.7 %) relapsed and only 3 (33.7 %) developed remission. five (55.6%) of the cd56 expressing patients died. on the other hand, the cd56 negative patients group showed one (10 %) case relapse and death, while nine (90 %) patients developed remission. statistical comparison between those patient with cd56 expression and those without showed significant increased incidence of relapse (p=0.01) (table 5, figure 1) and deaths (p=0.032) among patients expressing cd56 (table 5, figure 2). discussion acute leukemia comprises a heterogeneous group of diseases that differ in their etiology, pathogenesis, and prognosis. our study investigated the prognostic significance of cd56 expression in these cases to evaluate its association with other prognostic factors, and its influence on the outcome during induction therapy. cd56 positivity was observed in 45 % of aml cases, and 10 % all cases. these results are in concordance with fischer et al. [18] who reported that cd56 expression was not restricted to aml samples and could be detected in both b-cell and t-cell alls (14 %) as well. they concluded that it is not reliable for lineage distinction between aml and all. however, montero et al. [19] reported expression of this marker in only 4 patients (2 %) out of 200 all cases. these differences could be explained by lower number of cases in our study as compared to 200 cases studies, or due to methodology variation. the study of fischer et al. [18] investigated cd56 in 452 newly diagnosed t-all patients included in the gmall trial. the marker was expressed in 13.9 % of patients, predominating in the non-thymic subtypes, whereas thymic t-all was most common in the cd56 negative group. in addition, the authors reported that cd56 expression was associated with higher resistance to therapy. our statistical analysis showed that cd56 expression in aml cases was not associated with age (table 2), sex, or any laboratory variables including blasts counts, wbc, hemoglobin concentration or platelets counts (table 3). similarly yang et al. [20] could not associate cd56 expression with any laboratory variables in aml cases. however, other investigators [1] reported a higher proportion of cd56 expression in men. the presence of lymphadenopathy, hepatomegaly, and splenomegaly provide an indirect measurement of leukemic cell burden. we could not detect significant association of positive cd56 and presence of any of these clinical conditions in aml cases. also, no significant association was found with other clinical features as presence of fever, pallor or bleeding tendency (table 3). these findings are compatible with other published data (table 18, 21). our work detected only two cases of all expressing cd56, these were of b-all subtype and were not characterised by hepatomegaly or splenomegaly. although ravandiet al. [6] considered that table 5. association between treatment outcome, fate and cd56 expression in aml patients n = number of cases, *= one case was missed. fig. 1: association between cd56 expression and treatment outcome in aml patients. fig. 2. association between cd56 expression and mortalityin aml patients ��������� ��� ����� ��������� ��� ��� ��� �� ��� ����� ��� � � �� � �� �������� � �� ��� �� ����� � ������ ��!�"��� �� ��� �� ����� ������� ������� ��� ����� �� #!���� �� ��� $� $$�$� %���� ��&� �� ��� �� ����� $��$�'� ����(�� ��� ����� �� b. m. ahmed et al. 43 r a d ia t io n m e d ic in e a n d o n c o l o g y ijmmr 2015 vol. 1 no. 1 cd56 expression predicts occurrence of cns disease in all, none of our positive cd56 all cases had cns involvement. in addition, no association was found between cd56 expression and demographic, clinical presentation or laboratory tests (p>0.05). however, the low number of all cases expressing cd56 incapacitated our statistical and association analysis and doubts these results. cd56 expression was heterogeneous in different fab subtypes. the more frequent cd56 expression was observed in m1 (35 %) and m2 (35 %). allegrettiet al. [1] detected highest incidence of cd56 positivity among fab subtypes m4 and m5, while di bona et al. [22] found more cd56 expression in m5 patients. these differences could be attributed to different number of patients in sample. to elucidate the prognostic value of cd56, its expression was studied in relation to treatment response in our aml patients. cd56 expression was shown to be significantly related to response to chemotherapy with higher relapse (66.6 % vs 10 %, p=0.010) and death rate (55.5 % vs 10 %, p=0.32) among patients with positive cd56 expression as compared to patients without it. the investigation [1] in a single center in brazil that investigated cohort of 48 aml patients supported our results and documented the association of cd56 expression with worse prognosis. the authors observed higher death rate during induction in the cd56 positive cases. their study detected short os among cases with positive cd56 expression compared to cd56 negative patients. similarly, baer et al. stated that cd56 expression was significantly correlated with a short complete remission (cr) period and low os [21].on the contrary, ferrara et al. reported that there was no association between cd56 expression and the rate of cr in patients with aml [12]. moreover, di bona et al. failed to demonstrate the influence of cd56 positivity on cr duration or os in 171 studied cases of aml [22]. aml with t(8;21) is considered a group with favorable outcome, usually marked by high cr rate and prolonged disease-free survival. we encountered five cases with this subtype (25 %), three of these patients (60 %) expressed cd56 and they relapsed and died. although the small number of our cases (n=5) questions the significance of these results, the fatal outcome is too striking to be ignored. a previous large-scale studies investigated 144 aml patients with t(8;21) in the jalsgaml97 study. univariate analysis showed that increased white blood cell counts (wbc�20�109/l),cd19 negativity, and cd56 positivity were critical adverse factors for relapse after cr. multivariate analysis showed that wbc count and cd56 expression were independent adverse risk factors. the authors concluded that cd56 expression has a possible role in risks tratification for patients with aml with t(8;21) [23]. our study included only four cases of promyelocytic leukemia (apl), three of these patients expressed cd56 (75%). relapses were evident in two (50%) of those cases expressing this marker. the clinical significance of cd56 expression was studied in a large series of patients with apl, treated with all-trans retinoic acid and anthracycline-based protocols. the authors documented that expression of cd56 is an independent adverse prognostic factor for relapse in these patients and suggested implementing this marker as a risk-adapted therapeutic strategies in apl [24]. conclusions the association between cd56 expression and the poor response of aml to current treatments is not fully understood. raspadori et al. [25] hypothesized that cd56+ aml blasts might overexpress p-glycoprotein (pgp), a multidrug-resistance (mdr) related protein, and reduce responsiveness to chemotherapy. their data underlined the independent negative prognostic role of cd56 and pgp expression in cases of acute myeloid leukemia. in contrast, other investigators [26] showed that cd56 expression did not correlate with pgp expression, function, or with expression of the other mdr proteins. other authors [27] have shown that cd56 expression by aml cells is positively regulated by runx1 p48 and is negatively regulated by other splice variants. their findings suggest that p48 and runx1-driven nf-b and bcl2 pathways are new elements for targeted treatments in high-risk cd56amls. references 1. alegretti ap, bittar cm, bittencourt r, piccoli ak, schneider l, silla lm, bo sd, xavier rm. the expression of cd56 antigen is associated with poor prognosis in patients with acute myeloid leukemia. revistabrasileira de hematologia e hemoterapia 2011; 33: 202–206. 2. syrjala m, anttila vj, ruutu t, jansson se. flow cytometric detection of residual disease in acute leukemia by assaying blasts co-expressing myeloid and lymphatic antigens. leukemia 1994; 8: 1564–1570. 3. schabath r, ratei r, ludwig wd. the prognostic significance of antigen expression in leukaemia. best pract res clinhaematol 2003; 16(4): 613–628. 4. lanier ll, le am, civin ci, et al. the relationship of cd16 (leu-11) and leu-19 (nkh-1) antigen expression b. m. ahmed et al. 44 r a d ia t io n m e d ic in e a n d o n c o l o g y ijmmr 2015 vol. 1 no. 1 on human peripheral blood nk cells and cytotoxic t lymphocytes. j immunol 1986; 136: 4480–4486. 5. chang h, brandwein j, yi ql, chun k, patterson b, brien b.extramedullary infiltrates of aml are associated with cd56 expression, 11q23 abnormalities and inferior clinical outcome. leuk res 2004; 28 (10): 1007–1011. 6. ravandi f, cortes j, estrov z, thomas d, giles fj, huh yo, et al. cd56 expression predicts occurrence of cns disease in acute lymphoblastic leukemia. leuk res 2002; 26 (7): 643–649. 7. suzuki r, kagami y, takeuchi k, kami m, okamoto m, ichinohasama r, et al. prognostic significance of cd56 expression for alk-positive and alk-negative anaplastic large-cell lymphoma of t/null cell phenotype. blood 2000; 96 (9): 2993–3000. 8. cook g, dumbar m, franklin im. the role of adhesion molecules in multiple myeloma. actahaematol 1997; 97 (1–2): 81–89. 9. tiftik n, bolaman z, batun s, ayyildiz o, isikdogan a, kadikoylu g, muftuoglu e. the importance of cd7 and cd56 antigens in acute leukaemias 2004; 58(2): 149–152. 10. murray ck, estey e, paietta e, howard rs, edenfield wj, pierce s, et al. cd56 expression in acute promyelocytic leukemia: a possible indicator of poor treatment outcome? j clin oncol 1999; 17 (1): 293–297. 11. raspadori d, damiani d, lenoci m, rondelli d, testoni n, nardi g, et al. cd56 antigenic expression in acute myeloid leukemia identifies patients with poor clinical prognosis. leukemia 2001; 15 (8): 1161–1164. 12. ferrara f, morabito f, martino b, specchia g, liso v, nobile f, et al. cd56 expression is an indicator of poor clinical outcome in patients with acute promyelocytic leukemia treated withsimultaneous all-trans-retinoic acid and chemotherapy. j clin oncol 2000; 18 (6): 1295–1300. 13. chang h, salma f, yi ql, patterson b, brien b, minden md. prognostic relevance of immunophenotyping in 379 patients with acute myeloid leukemia. leuk res 2004; 28 (1): 43–48. 14. swerdlow sh, campo e, harris nl, et al. (eds.) who classification of tumours of haematopoietic and lymphoid tissues lyon: iarc; 2008. 15. bennet jm, catovsky d, daniel mt, flandrin g, galton dag, gralnick hr, sultan c. proposals for the classification of acutemyeloidleukemias. br j haematol 1976; 33: 451–458. 16. jennings cd, foon ka. recent advances in flow cytometry: application to the diagnosis of hematologic malignancy. blood 1997; 90: 2863–2892. 17. shaffer lg, tommerup n iscn 2005: an international system for human cytogenetic nomenclature. basal. karger publishers; 2005. 18. fischer l, gokbuget n, schwartz s, burmeister t, rieder h, bruggemann m, hoelzer d, and thiel e. cd56 expression in t-cell acute lymphoblastic leukemia is associated with non-thymic phenotype and resistance to induction therapy but no inferior survival after risk-adapted therapy. haematologica 2009; 94 (2): 224–229. 19. montero i, rios e, parody r, perez-hurtado jm, martin-noya a, rodriguez jm. cd56 in t-cell acute lymphoblastic leukaemia: a malignant transformation of an early myeloid-lymphoid progenitor? haematologica 2003; 88 (9): e127–e128. 20. yang dh, lee jj, mun yc, shin hj, kim yk, cho sh, chung ij, seong cm, kim hj. predictable prognostic factor of cd56 expression in patients with acute myeloid leukemia with t(8:21) after high dose cytarabine or allogeneic hematopoietic stem cell transplantation. am j hematol 2007; 82: 1–5 21. baer mr, stewart cc, lawrence d, arthur dc, byrd jc, davey fr, schiffer ca, bloomfield cd. expression of the neural cell adhesion molecule cd56 is associated with short remission duration and survival in acute myeloid leukemia with t(8;21)(q22;q22). blood 1997; 90: 1643–1648. 22. di bona e, sartori r, zambello r, guercini n, madeo d, rodeghiero f. prognostic significance of cd56 antigen expression in acute myeloid leukemia. haematologica 2002; 87: 250–256. 23. iriyama n, hatta y, takeuchi j, ogawa y, ohtake s, sakura t, mitani k, ishida f, takahashi m, maeda t, izumi t, sakamaki h, miyawaki s, honda s, miyazaki y, taki t, taniwaki m, naoe t. cd56 expression is an independent prognostic factor for relapse in acute myeloid leukemia with t(8;21). leuk res 2013; 37: 1021– 1026. 24. montesinos p, rayon c, vellenga e, brunet s, gonzalez j, gonzalez m, et al. clinical significance of cd56 expression in patients with acute promyelocytic leukemia treated with all-trans retinoic acid and anthracycline-based regimens. blood 2011; 117: 1799–1805. 25. raspadori d, lenoci m, rondelli d, testoni n, nardi g, sestigiani c. cd56 antigenic expression in acute myeloid leukemia identifies patients with poor clinical prognosis. leukemia 2001; 15: 1161–1164. 26. suvannasankha a, minderman h, o’loughlin kl, sait sn, stewart cc, greco wr, baer mr. expression of the neural cell adhesion molecule cd56 is not associated with p-glycoprotein overexpression in core-binding factor acute myeloid leukemia. leuk res 2004; 28: 449– 455. 27. gattenloehner s, chuvpilo s, langebrake c, dirk reinhardt d, muller-hermelink h, serfling e, vincent a, marx a. novel runx1 isoforms determine the fate of acute myeloid leukemia. blood 2007; 110: 2027–2033. received: 2014. 02.26 b. m. ahmed et al. issn 2413-6077. ijmmr 2022 vol. 8 issue 274 p u b l ic h e a lt h a n d e p id e m io l o g y doi 10.11603/ijmmr.2413-6077.2022.2.13594 communication of oncological patients with oncologists: main problems and problem-solving strategies *v. о. zub1, a. s. kotuza2, o. k. tolstanov1 1 – shupyk national healthcare university of ukraine, kyiv, ukraine 2 – feofaniya clinical hospital, state management of affairs of ukraine, kyiv, ukraine background. physician-patient сommunication is one of the main supportive care provided to oncological patients. it affects treatment outcomes, as well as the interaction of patients within the society, their family and colleagues. furthermore, covid-19 and military invasion of russia into ukraine makes the issue of improvement of physician-patient communication urgent. objective. the aim of the study is to develop recommendations based on the analysis of denfined problems in physician-patient communication in of oncological treatment. methods. the sociological research involved 419 patients during their inpatient treatment in the period from november 2021 to february 2022 using the eortc qlq-comu26 questionnaire. results. in the pre-war period, in ukrainian patients with oncological diseases the worst indicators were established for “active role of a patient” (82.00) and “correction of misunderstandings by a specialist” (89.19). the best indicators were established for “skills of the specialist (verbal/non-verbal)” with the score of 93.25 by a 100-point grading scale, as well as “satisfaction with communication” – 97.04 by a 100-point grading scale. conclusions. when developing programs for organization of oncological care, mechanisms for providing psychological care to patients through effective communication between patients and specialists regarding providing medical and psychological care must be taken into account. key words: communication; quality of life; cancer. *corresponding author: valeriy zub, shupyk national healthcare university of ukraine, kyiv, ukraine. е-mail: zub.valeriyoleksiyovych@gmail.com international journal of medicine and medical research 2022, volume 8, issue 2, p. 74-82 copyright © 2022, tnmu, all rights reserved v. о. zub et al. introduction physician-patient communication is one of the main aspects of care provided to oncological patients. a number of studies have shown that communication focused on a patient can influence patient’s satisfaction, decision­ma king, well-being, compliance with treatment of oncological diseases [1]. communication focused on a patient is a cornerstone of a high-quality care [2]. proper communication allows physician to respond better to the patients’ needs regarding information and support. for this purpose, recommen dations for improving the communication skills of medical workers are available in many countries [3]. the effective physician-patient communication may reduce the anxiety, keep hope [4], and increase satisfaction from communication in patients [5]. communication fo cused on a patient has also been developed in order to improve communication skills training of a physician [6]. these measures are aimed at involving a patient into counseling and thus increasing awareness of a physician and timely taking into account patients’ expec_ tations. the present circumstances should also be taken into account. the global crisis caused by covid-19 outbreak in 2019 has led to unexpected and difficult situations [7]. physical bar­ riers and non­visitation policy had a significant impact on communication in a hospital settings. physicians need new strategies to keep in touch with patients and their families/relatives [8, 9]. in the era of covid-19, in order to solve the challenges for effective physician-patient communication for oncological patients, when physical distancing is required, different solutions have been suggested, particularly, video calls were introduced by using smartphones and tablets [10]. thousands of oncological patients have suffered in connection with the war in ukraine, as they lost the opportunity to receive treatment and were left alone with their disease. the necessary medicines are difficult to get, chemo­ therapy and radiotherapy are carried out irregularly, physicians are often out of the telephone reach. moreover, cancer patients face psychoissn 2413-6077. ijmmr 2022 vol. 8 issue 2 75 p u b l ic h e a lt h a n d e p id e m io l o g y logical problems – uncertainty and the inability to plan and control their lives. most of them have complex depressive, anxiety-depressive and phobic symptoms. excessively expressed negative emotions (depression, anxiety, phobias) can cause suicidal thoughts in such patients. the oncologist-patient communication is especially difficult when it comes to information about prognosis and treatment outcomes, which is essential for achieving a shared understanding of disease state and treatment goal between patients and oncologists [11, 12]. the oncologists have frequent difficulties in assessing the patients’ information preferences [13]. in patients with advanced cancer, the prognosis information is often missing [14], and patients do not understand that the treatment provided will unlikely cure their oncological diseases [15]. the aim of the study is to develop recommendations based on the analysis of identified problems in physician-patient communication in patients with oncological diseases during treatment. methods the sociological research was perfomed by surveying 419 patients during their inpatient treatment. the required number of participants was calculated according to the glen’s method, and involved 398 individuals. taking into account the possibility of elimination (10%), 440 questionnaires were sent proportionally to different regions of ukraine (northern, southern, western, eastern, central); 419 questionnaires were received back. the inclusion criteria were the patients hospitalized with a histologically confirmed cancer diagnosis and received inpatient treatment. exclusion criteria were lack of written consent to participate in the study. the research was carried out at oncological institutions in nine regions of ukraine: chernihiv, zaporizhzhia, dnipropetrovsk, kyiv, poltava, khmelnytskyi, ivano-fran kivsk, zakarpattia and lviv regions. the primary data collection was performed from november 2021 till february 2022. all the patients gave written consents to participate in the trial. the average age of patients who participated in the trial was 59.62±10.33 years old. the research was conducted according to the unified protocol providing for the use of questionnaires which were designed by the european organisation for research and treatment of cancer (eortc). the questionnaire included 26 questions regarding communication of oncological patients with their health workers. the eortc­qlq­comu26 module was composed of six scales and four separate issues [16, 17, 20]. the following scales are involved in the eortc­qlq­comu26: the ability of the specialist to manage patient’s emotions (emot), the “specialist-patient” relationships (relat), the ability of the specialist to build relationships (qual), the active role of the patient (act), the skills of the specialist (verbal/non-verbal) (skill), the information skills of the specialist (info). the additional issues of eortc­qlq­comu26 are the following: correction of misunderstan­ dings by the specialist (misun), sufficient privacy (priva), taking into account patient’s preferences (pref), satisfaction with communication (satis). the permission to use this questionnaire from “eortc quality of life group” was re­ ceived in november 2021. the calculations were carried out according to the eortc qlq­comu26 methodology [16, 17]. the results were calculated according to unified scales or individual indicators. average score (raw score – rs) was presented as м±sd. besides, all scales and individual indicators of eortc qlq­comu26 questionnaire were calculated by a 100-point grading scale (ss) for easier interpretation according to the formula: ss = ((rs–1)/range)·100 where rs – raw score, range – scale range which is determined by the difference between the maximum and minimum values of the scale. the low raw score and high score by a 100-point grading scale testified to high quality of communication with a specialist. for scales that consist of 2 or more questions, cronbach’s alpha was calculated as an indicator of scale consistency. results according to the findings of qlq­comu26 questionnaire in the patients with oncological diseases, the worst indicators were established for the “active role of the patient” scale. the score was 82.00 by a 100-point grading scale (table 1), rs was 3.46±0.82 (fig. 1). this scale involved questions about sufficient opportu­ nities to communicate with a specialist (physician), opportunity to ask questions freely, opportunity to show emotions. v. о. zub et al. issn 2413-6077. ijmmr 2022 vol. 8 issue 276 p u b l ic h e a lt h a n d e p id e m io l o g y according to the findings of the qlq­ comu26 questionnaire, a better result was established for the scale of “information skills of the specialist”, where the score was 90.75 a 100-point grading scale. the following questions were included in this scale: whether the specialist was convinced that a patient understood all about the disease before providing any new information, whether the physician specified understanding by a patient the infor­ mation provided. it is quite natural that the patients with oncological diseases are often worried about their future. thus, in the scope of this scale, the patients were asked about the specialists’ exhaustive replies to difficult ques­ tions, as well as whether these answers satisfied the patients. generally, the sense of comp leteness of specialists’ answers, their explanations are very important, which is also discussed in this scale. fewer difficulties happened in the physician­ patient communication in the “ability of the specialist to manage the patient’s emotions” scale, where the score was 91.75 by a 100-point grading scale. a cancer patient badly needs support from their family, friends, relatives, but the effort of specialist to understand the current situation of a patient is certainly important as well. in this scale there were questions regarding the specialist’s help to master patient’s emotions, for example: sadness, anger, fear, anxiety, etc., and also the ability to listen when the patient demonstrated his/her emotions. the better result was obtained in the scale “specialist-patient relationships”, where the score was 91.94 by a 100-point grading scale. this scale involved questions whether the specialist had spent enough time to communicate, whether there was a mutual trust between the patient and specialist, whether the patient felt that they and the physician had the common understanding of the disease and its treatment. table 1. results of questionnaire of different qlq-comu26 scales in oncological patients scale issue directory code score by a 100-point scale cronbach’s alpha active role of the patient act 82.00 0.63 information skills of the specialist info 90.75 0.83 ability of the specialist to manage the patient’s emotions emot 91.75 0.64 specialist-patient relationships relat 91.94 0.66 ability of the specialist to build relationships qual 93.19 0.78 skills of the specialist (verbal/non-verbal) skill 93.25 0.67 fig. 1. raw score of different qlq­comu26 scales in oncological patients. v. о. zub et al. issn 2413-6077. ijmmr 2022 vol. 8 issue 2 77 p u b l ic h e a lt h a n d e p id e m io l o g y the highest scores characterized the ability of the specialist to establish the relatioships with the patient. this issue was 93.19 by a 100-point grading scale. this scale described the attitude of the specialist to the patient: whether the physician treated the patient with respect, as an equal, whether the physician showed sincerity, whether it was easy and open to discuss the troubling problems of the patient, whether the physician took them seriously. the best indicators of the qlq­comu26 questionnaire were established for the “skills of the specialist (verbal/non-verbal)” scale, where the score was 93.25 by a 100-point grading scale. this scale included the following questions: did the specialist use the language understandable to the patient (avoiding me dical terminology, using understandable terms), did the specialist openly answer the patient’s questions, did the specialist look at the patient during their conversation, did the specialist speak in a calm voice. it should be mentioned that in the qlq­ comu26 questionnaire in patients with oncological diseases, the respondents’ answers were of the same type in the “ability of the specialist to build relationships” and “information skills of the specialist” scales, which was shown by sufficient and high consistency, determined by cronbach’s alpha within 0.78–0.83. for the rest of the indicators, cronbach’s alpha was in the range of 0.63–0.67. according to the findings of qlq­comu26 questionnaire regarding the individual indicators, in the oncological patients the worst indica tors were in the “correction of misunderstandings by a specialist” scale. the score was 89.19 by a 100-point grading scale (table 2), the raw score was 3.68±0.54 (fig. 2). this indicator included questions regarding whether the specialist explained again in another way the information which the patient had not understand. this indicates the problem of the physician-patient communication in this scale. one of the probable causes of such misunderstandings may be lack of time allocated per patient, vulnerable emotional state of the patient with a potentially fatal disease, and, in table 2. results of the qlq-comu26 questionnaire regarding the individual indicators of oncological patients indicators directory code score by a 100-point scale correction of misunderstandings by a specialist misun 89.19 taking into account the wishes of the patient pref 89.62 sufficient privacy priva 93.08 satisfaction from communication satis 97.04 fig. 2. average score of individual indicators by qlq­comu26 in oncological patients. v. о. zub et al. issn 2413-6077. ijmmr 2022 vol. 8 issue 278 p u b l ic h e a lt h a n d e p id e m io l o g y the majority of cases, lack of medical literacy to comprehend information, which is difficult for understanding. the indicator “taking into account the wishes of the patient” was found to be practically comparable. the score was 89.62 by a 100-point grading scale. for this indicator, the patient was asked whether the specialist took into account the method of obtaining information preferred by the patient. slightly better results were established for the indicator “sufficient privacy”. the score was 93.08 by a 100-point grading scale. this indicator included the questions concerning sufficient privacy of the confidential conversation between the patient and specialist. the best qlq­comu26 questionnaire re­ sults regarding the individual indicators in oncological patients were established for the issue “satisfaction from communication”, which was 97.04 by a 100-point grading scale. discussion communication is one of the most important tasks in the professional practice of a clinician, especially for those who deal with the lifethreatening diseases. in this case, effective dialogue can be quite challenging, however, clinicians face this daily. in recent decades, clinical communication has been studied a lot, and, several structured approaches have been developed and suggested [8, 18, 19]. patients with advanced oncological diseases often forget their questions during consultation with a physician, they doubt the legality of the request, indirectly express concern, feel fear of a possibly humiliating answer and physicians do not often encourage them to ask questions [21-23]. patients may also have different needs and expectations, depending on time of the course of the disease [13, 24, 25]. discrepancy between the perceptions of the patient and oncologist regarding the goals of treatment, duration of disease treatment can lead to medical decisions that do not meet the life goals which are very important for the patients [26]. it leads to even greater psychological stress in patients [27]. according to the findings of the qlq­ comu26 questionnaire for the oncological patients, the worst indicators were established for the “active role of the patient” scale. the score was 82.00 by a 100-point grading scale which was less than the results attained by the german colleagues, where this issue was 85.0 [1]. a possible reason for a low activity of patients could be their self­absorbedness: pa­ tients are not able to talk about their problems freely, ask questions about everything concerning them or those they don’t completely understand. the least difficulties for the ukrainian patients were caused by the scales with nearly comparable indicators for the “ability of the specialist to build relationships” (93.19 points) and the “skills of the specialist (verbal/nonverbal)” (93.25 points). these results are higher than the results obtained by the german colleagues: the score of the “ability of the specialist to build relationships” scale was 89.6 points, while the score of the “skills of the specialist (verbal/non-verbal)” was 90.3 points [1]. generally, the european patients need more sincerity and ease, openness in discussing problems during the physician-patient communication. it should be taken into account that if before covid-19 the “physicians-patient” consultations were held in person at prearranged time, during the sars-cov-2 pandemic communication often took place usually over the phone. in this unprecedented situation often one-way communication cause a feeling of uncertainty and suffering in those who stay at home and have to wait for news [28]. it should be noted that relatives of the seriously ill patients already have the increased risk of anxiety, depression or post-traumatic stress disorder [29]. moreover, the loss of non-verbal signals, such as eye contact and gestures complicate communication and formation of empathy between the communication provider and the patient. thus, the uncertainty and experiencing psychological problems by patients make communica tion between cancer patients and physicians difficult. according to the findings of the qlq­ comu26 questionnaire regarding the individual indicators in oncological patients, the worst results were established for the “correction of misunderstandings by a specialist” indicators. the score was 89.19 by a 100-point grading scale that was less than the results attained by the german colleagues, where this issue was 84.8 [1]. the best indicators for the ukrainian patients with oncological diseases in the qlq­ comu26 questionnaire regarding the individual indicators were established for the “satisfaction with communication” issue, which was 97.04 that was significantly higher compared to the data of the german colleagues, where the indicator was 84.3 [1]. v. о. zub et al. issn 2413-6077. ijmmr 2022 vol. 8 issue 2 79 p u b l ic h e a lt h a n d e p id e m io l o g y an interesting and important fact should be taken into account for providing high-quality medical care is that communication with critically ill patients is often three-way, rather than two-way involving not only the specialist and the patient but also family members who are often the main support, an important part of the medical team and the component of communication for the health of their relatives. a high level of depression and burden on the caregiver was registered among the patients and relatives of the hospitalised oncological patients [8, 30]. therefore, targeted intervention in the field of physician­patient communication can improve the quality of life for both patients and their families. family and friends can be a great support for an oncological patient. the patient can rely on a family member or a trusted friend in a similar situation for help, for example asking to accompany them to visit a physician. the presence of a supportive person is a reminder that the cancer patient is not alone and it can help them take the initiative to communicate. generally, the inappropriate communication leads to unsatisfied patient needs for informa­ tion and support. understanding how to improve the physician-patient communication is important for all involved into patient care. conclusion the physician-patient communication is one of the main aspects of support provided to oncological patients. effective communication of the physician and patient may reduce patient’s anxiety, keep hope, increase satis faction from communication. in today’s covid-19 pandemic, physical barriers and no family visitation policy for patients have significantly affected the physician-patient communication for patients with oncological diseases in the hospital settings, and physicians need new strategies in order to keep contact with the patients. according to the findings of the qlq­ comu26 questionnaire for the ukrainian oncological patients, the worst indicators were established for the “active role of the patient” scale, its score was 82.00, which was less then the results obtained by the german colleagues (85.0). the best indicators in this questionnaire were established for the issue “skills of the specialist (verbal/non-verbal)”, the score was 93.25 by a 100-point grading scale. according to the findings of the qlq­comu26 ques­ tionnaire regarding the individual issues, the worst indicators were established for the “correc tion of misunderstandings by a specialist” scale. the score was 89.19 by a 100-point grading scale, which was higher the results obtained by the german colleagues (84.8 points). the best results of this questionnaire were established for the “satisfaction with communication” scale – 97.04 by a 100-point grading scale, which was significantly higher compared to the data of the german colleagues, where the indicator was 84.3. according to the findings of the qlq­ comu26 questionnaire, in ukrainian oncological patients in the pre-war period the worst indicators were established for the issues of “active role of the patient” and “correction of misunderstandings by a specialist”. thus, to provide a high-quality medical care it should be taken into account that communication with critically ill patients is often threeway rather than two-way, where not only the specialist and the patient are involved, but family members, who are often the main support, an important part of the medical team and a significant component of communication for their relatives. when developing programs for organization of oncological care, mechanisms for providing psychological care to these patients through effective communication between patients and specialists regarding providing medical and psychological care should be taken into account. during the russia’s military invasion of ukraine, many oncological patients had to go abroad in order to receive specialized treatment. thus, this information can be used for better communication of the foreign physicians with the ukrainian patients. conflict of interests authors declare no conflict of interest. author’s contributions valeriy zub – conceptualization, methodology, formal analysis, writing – original draft, writing – reviewing and editing; andrii kotuza – conceptualization, methodology, investigation, writing – reviewing and editing; oleksandr tolstanov – conceptualization, methodology, data curation, writing – reviewing and editing. v. о. zub et al. issn 2413-6077. ijmmr 2022 vol. 8 issue 280 p u b l ic h e a lt h a n d e p id e m io l o g y references 1. rogge aa, helmer sm, king r, et al. effects of training oncology physicians advising patients on complementary and integrative therapies on patientreported outcomes: a multicenter, cluster­rando­ mized trial. cancer. 2021;127(15):2683­2692. doi: https://doi.org/10.1002/cncr.33562 2. levinson w. patient­centred communication: a sophisticated procedure. bmj qual saf. 2011;20(10): 823-825. doi: https://doi.org/10.1136/bmjqs­2011­000323 3. stiefel f, kiss a, salmon p, peters s, razavi d, cervantes a, et al. training in communication of oncology clinicians: a position paper based on the third consensus meeting among european experts in 2018. ann oncol. 2018;29(10):2033­2036. doi: https://doi.org/10.1093/annonc/mdy343 4. clayton jm, hancock k, parker s, butow pn, walder s, carrick s, et al. sustaining hope when communicating with terminally ill patients and their families: a systematic review. 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суспільстві, з родиною та колегами. крім того, поширення коронавірусної хвороби covid-19 та військове вторгнення росії в україну зумовлюють необхідність покращення комунікації між лікарем та пацієнтом. мета. мета роботи – розробка рекомендацій на основі аналізу виявлених проблем в комунікації “лікар-пацієнт” у хворих на онкологічні захворювання під час лікування. методи. соціологічне дослідження проведено за участі 419 пацієнтів, на етапі їхнього стаціонарного лікування, в період з листопада 2021 року по лютий 2022 року з використанням опитувальника eortc qlq-comu26 результати. в українських пацієнтів з онкологічними захворюваннями, у довоєнний період, найгірші показники припали на шкали «активна роль пацієнта (82.00) та “корегування фахівцем непорозумінь” (89.19). найкращі дані встановлено для пункту “навички фахівця (вербальні-невербальні)”, на який припало 93.25 балів за 100-бальною шкалою та “задоволення від комунікації” – який склав 97.04 балів за 100бальною шкалою. висновки. при опрацюванні програм щодо організації онкологічної допомоги повинен бути врахований механізм щодо забезпечення надання психологічної допомоги пацієнтам шляхом ефективної комунікації між пацієнтами та спеціалістами з надання медичної та психологічної допомоги. ключові слова: комунікація; якість життя; онкологія. information about the authors valeriy zub – phd, doctoral student, shupyk national healthcare university of ukraine, kyiv, ukraine https://orcid.org/0000­0001­9823­4216, e­mail: zub.valeriyoleksiyovych@gmail.com andrii kotuza – professor, feofaniya clinical hospital, state management of affairs of ukraine, kyiv, ukraine https://orcid.org/0000­0002­1910­0197, e­mail: kotuza.andrii@gmail.com oleksandr tolstanov – professor, shupyk national healthcare university of ukraine, kyiv, ukraine https://orcid.org/0000­0002­7459­8629, e­mail: o.tolstanov@nuozu.edu.ua v. о. zub et al. issn 2413-6077. ijmmr 2022 vol. 8 issue 2 81 p u b l ic h e a lt h a n d e p id e m io l o g y velopment and 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https://doi.org/10.1016/j.pec.2021.02.003 29. kross ek, engelberg ra, gries cj, nielsen el, zatzick d, curtis jr. icu care associated with symptoms of depression and posttraumatic stress disorder among family members of patients who die in the icu. chest .2011;139(4):795–801. doi: https://doi.org/10.1378/chest.10­0652 30. seo yj, park h. factors influencing caregiver burden in families of hospitalised patients with lung cancer. j clin nurs. 2019;28(9­10):1979–1989. doi: https://doi.org/10.1111/jocn.14812 received 1 november 2022; revised 30 november 2022; accepted 16 december 2022. this is open access article distributed under the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. v. о. zub et al. 52 b io m e d ic a l s c ie n c e s issn 2413-6077. ijmmr 2016 vol. 2 issue 2 doi 10.11603/ijmmr.2413-6077.2016.2.7034 lipid peroxidation in multiple organ failure caused by associated chest and hip trauma m. i. marushchak, m. m. khudobіak, i. ya. krynytska, i. v. antonyshyn i. horbachevsky ternopil state medical university, ternopil, ukraine background. the injured with polytrauma are classified as extremely severe patients, as their emergency care and intensive care is followed by significant difficulties, frequent development of complications, high mortality and survivors disability. objective. this study is aimed to evaluate the effect of lipid peroxidation in heart, lungs, liver and blood of rats in early and late periods of combined traumatic injury of chest and hips and to establish their influence on the development of multiple organ failure. methods. the study was conducted on 70 adult male white nonlinear rats. it was determined the content of lipid hydroperoxides and concentration of tba-active products in erythrocyte mass, heart, lungs and liver. results. the data prove activation of free radical oxidation at the first day of post-traumatic period. analysis of the data evidences the increase in of lipid hydroperoxides (hpl) rate in liver homogenate in group e1 in 1.5 times, in all subsequent periods of the research the increase in rate fluctuated within 1.9-2.0 times. in blood and heart the hpl rate increased twice in group e1, reached maximum in group e2 and gradually decreased till the end of the experiment, data exceeded the control group. the highest level of hpl was determined in lung tissues (r≤0.01). in the post traumatic period the highest tba-ap was detected in lungs and liver. conclusions. in case of simulated trauma (injury of chest with fractures of both hips) hyperactivation processes of free radical oxidation is observed in 1 day, reaching a peak in 7–14 days of post-traumatic period in blood, tissues, liver, heart and lungs if compared to the control group causing multiple organ failure. key words: trauma, lipid peroxidation, lung, liver, heart. introduction nowadays one of the most urgent problems of medicine are multiple and combined injuries [1, 2]. this is due to the constant increase of the number of multisystem and multiorgan injuries on the background of some general manifesta­ tions of injuries stabilization, which leads to mortality increase [3, 4]. as a result of constant technological advances the increase in the number of man­made disasters has led to a significant increase in the number of patients with polytrauma. according to who trauma is one of the top five causes of death among people aged 15–44; mortality from injuries and accidents takes the first place [5]. the injurees with polytrauma are classified as extremely se vere patients, as emergency care and inten­ sive care is accompanied by significant diffi­ culties, frequent development of complications, high mortality and survivors’ disability [6]. in case of multiple trauma, wounded are primarily affected by phenomena of traumatic­haemor­ rhagic shock and tissue hypoxia. in the post­ traumatic period unnoticed injuries, infection, sirs, sepsis and multiple organ failure threaten their life and health [7]. therefore, our study was aimed to evaluate the effect of lipid peroxidation in heart, lungs, liver and blood of rats in early and late periods of combined traumatic injury of chest and hips and establish their influence on the development of multiple organ failure. material and methods the study was conducted on 70 adult male white nonlinear rats of 200–220 g body weight. rats were housed under standardized labo­ ratory conditions, with 12 h dark/light cycle and free access to food and tab water ad libi tum. all procedures were conducted according to the european convention for the protection of corresponding author: marya marushchak, department of functional diagnostics and clinical pathophysiology, i. horbachevsky ternopil state medical university, 1 maidan voli, ternopil, ukraine, 46001 phone number: +380979901202 e-mail: marushchak@tdmu.edu.ua international journal of medicine and medical research 2016, volume 2, issue 2, p. 52–55 copyright © 2016, tsmu, all rights reserved m. i. marushchak et al. 53 b io m e d ic a l s c ie n c e s issn 2413-6077. ijmmr 2016 vol. 2 issue 2 vertebrate animals used for experimental and other scientific purposes (strasbourg, 1986) and general ethical principles of experiments using animals (first national congress of bio­ ethics, kyiv, 2001). the animals were randomly divided into 8 groups: 1 control (c1) and 5 expe­ rimental (e1, e2, e3, e4, e5) groups, each comprising 12 animals. rats of experimental groups. the animals in the experimental group underwent thiopental sodium anaesthesia (40 mg/kg intraperitoneal rat) using a trocar modeled right­closed pneumothorax from frac­ tured ribs and combined them with a broken left and right femur. skeletal injury was mo­ deled by applying a single shock dosed by a specially designed device on each thigh, which caused a closed fracture [8]. impact energy was 0.375 j, which corresponded to the injury of mo derate severity. combined injury was mode­ led by sequential administration of these two injuries. mortality of animals in each group was e1(12/11), e2(12/9), e3(12/10), e4(12/10), e5(12/9). at the end of the experimental period, the rats were sacrificed by decapitation. the content of lipid hydroperoxides in erythrocyte mass, heart, lungs and liver was determined by [9]. this method is based on spectrophotometrical optical density measu­ rement of the products of ammonium thiocya­ nate, hydrochloric acid and mohr salt reaction. lipids from the samples were preliminarily extracted with ethanol. selection of tissue samples and preparation for extraction were performed at 4°c. ethanol (2.8 ml) and 0.05 ml of 50% trichloroacetic acid (tca) were added to 0.2 ml of hemolisate (dissolved in buffer solu­ tion with ph 7.4), and shaken for 5–6 min. obtained protein precipitate was separated by centrifugation at 700 g. ethanol (1.2 ml), 0.02 ml of concentrated hcl, and 0.03 ml of 1% mohr salt solution in 3% hcl were added to 1.5 ml of supernatant. the mixture was stirred. after 30 s, 0.2 ml of 20% ammonium thiocyanate was added, and then the absorbance of the solution was determined at λ=480 nm. in a control sample, the appropriate amount of bidistilled water was added instead of supernatant. the content of lipid hydroperoxides was calculated by the difference between experimental and control values, and expressed in arbitrary units of optical density for 1 mg of protein. the concentration of tba­active products, characterizing the lpo rate, was assessed by korobeinikova method based on the reaction between malondialdehyde (mda) together with other products of peroxidation and thiobar­ bituric acid (tba), occurring at high temperature and in acidic environment, and forming the colored complex of one mda and two tba mo­ lecules [10]. protein concentration was measu­ red by lowry [11]. all reagents used were obtained from sigmaaldrich and fluka (usa). the experimental data were processed by variation statistics methods by the originpro 8 program. student t­test was used to determine the likely differences between the means of the samples. in all cases, reliable differences were considered by p value under 5% (p<0.05). results and discussion the data prove activation of free radical oxidation at the first day of post­traumatic period (table 1 and 2). activation of peroxide oxidation is one of the factors destabilizing membrane [12]. it is established that the action of hydroxyl radicals in diene conjugates fatty acids, lipid hydroperoxide is formed, which cause conformational irregularities in cell mem­ table 1. the rates of lipid hydroperoxides in rat tissues (cu/mg protein) after combined trauma of chest and both hips (m±m) animal group blood heart lungs liver control 0.98±0.05 0.83±0.02 0.63±0.02 0.83±0.02 trauma of chest and hips, 1st day of observation 1.96±0.01* р≤0.01 1.64±0.02* р≤0.01 1.62±0.01* р≤0.01 1.26±0.01* р≤0.05 trauma of chest and hips, 3rd day of observation 2.36±0.08* р≤0.05 2.20±0.07* р≤0.01 1.79±0.01* p≥0.05 1.58±0.02* р≤0.01 trauma of chest and hips, 7th day of observation 2.03±0.06* р≤0.01 1.82±0.01* – 2.04±0.05* р≤0.01 1.80±0.01* р≤0.01 trauma of chest and hips, 14th day of observation 1.88±0.06* – 1.78±0.02* – 1.85±0.01* р≤0,01 1,65±0.01* р≤0,05 trauma of chest and hips, 28th day of observation 1.71±0.04* – 1.62±0.04* – 1.62±0.01* р≤0.01 1.57±0.02* р≤0,01 notes. tables 1 and 2: * — significant differences between the baseline indicators of the studied and control groups; p — significant differences between the experimental groups. m. i. marushchak et al. 54 b io m e d ic a l s c ie n c e s issn 2413-6077. ijmmr 2016 vol. 2 issue 2 branes. analysis of the data evidences the increase in hpl rate in liver homogenate in group e1 in 1.5 times, in all subsequent periods of observation the increased rate fluctuated within 1.9–2.0 times. in blood and heart the hpl rate increased twice in group e1, reached a maximum in group e2, and gradually decreased until the end of the experiment, data exceeded the control group (table 1). the highest level of hpl was determined in lung tissues, parti­ cularly in e1 it exceeded in 2.6 times the control rate, respectively e2 — in 2.9 times, e3 — in 3.2 times, e4 — in 3.0 times and e5 — in 2.5 times (r≤0.01). due to the fact that in case of trauma in lung tissues the activation of neutrophils is the main source of reactive oxygen species [13], and taking into account the administered chest injury, we believe that the pathological pro­ cesses that occur in lungs in case of simulated injuries have a significant contribution to the development of multiple organ failure. it is established that fatty acids in the ground connection break into fragments of aldehyde groups at the ends to form malonic dialdehyde. analysing the change of active products thiobarbituric acid in rat tissues, dif­ ferent activity of lipid peroxidation in the stud­ ied rat organs was revealed (table 2). the post traumatic period following combined chest trauma of both thighs on the 1st day surveillance the highest tba­ap was detected in lungs and liver, respectively, in 3 days — in blood and lungs, in 7 days and in subsequent periods of observation — in blood and liver (table 2). for a comparative analysis of the flow of free radical oxidation in various organs combi­ ned chest trauma of both thighs was adminis­ tered, the control group was regarded as 100%. it is established that the phenomenon of lipid peroxidation in heart was the most intense on the 3rd day after polytrauma simulation, respec­ tively, in lungs — from the 3rd to the 7th day, in blood — on the 7th day, in liver — on the 14th day (fig. 1). scientists have proved that the maxi mum intensity of lipid peroxidation coin­ cides with the body's response to inflammation table 2. the rate of active products of thiobarbituric acid in rat tissues (mmol/mg protein) after combined trauma of chest and both hips (m±m) animal group blood heart lungs liver control 0.22±0.01 0.36±0.01 0.20±0.08 0.40±0.02 trauma of chest and hips, 1st day of observation 0.40±0.01 p≥0.05 0.70±0.02* р≤0.01 0.42±0.02 p≥0.05 0.87±0.02* р≤0.01 trauma of chest and hips, 3rd day of observation 0.75±0.02* р≤0.05 0.97±0.03* р≤0.01 0.68±0.02* р≤0.01 1.23±0.02* р≤0.01 trauma of chest and hips, 7th day of observation 1.06±0.03* р≤0.01 0.80±0.02* p≥0.05 0.67±0.01* p≥0.05 1.76±0.03* р≤0.01 trauma of chest and hips, 14th day of observation 0.86±0.02* р≤0.01 0.75±0.02* p≥0.05 0.60±0.02* р≤0.01 2.08±0.04* р≤0.05 trauma of chest and hips, 28th day of observation 0.72±0.03* p≥0.05 0.68±0.03* p≥0.05 0.53±0.02* p≥0.05 1.87±0.02* p≥0.05 fig. 1. severity of the processes of lipid peroxidation (%) in rat tissues (as example, mda­ap) in post­traumatic period following combined trauma of chest and both hips. 0 100 200 300 400 500 600 1 day 3 day 7 day 14 day 28 day blood heart lung liver m. i. marushchak et al. 55 b io m e d ic a l s c ie n c e s issn 2413-6077. ijmmr 2016 vol. 2 issue 2 [14]. the development of hypoxemia caused by chest trauma affected lipid peroxidation sti­ mulation causing disruption of cell membranes structure and consequently cell death closing the ‘vicious’ circle, which in our and other authors opinion cause multiple organ failure with involvement of heart, lungs, liver in the pathological process [15]. the data indicate that the intensity of lipid peroxidation in different tissues of body de­ pends on injury type. thus, kozak d.v. in the research [16] notes that the intensity of lipid peroxidation due to blood loss and hip trauma has oscillatory nature of the duration increase up to the third day, the period of temporary welfare in 14 days and repeated increase in 21 days. our results prove that various tissues experience peroxidation activation and its maximum occurs at different time. conclusions in case of simulated trauma (chest injury with fractures of both hips) hyperactivation processes of free radical oxidation is observed in the 1st day, reaching a peak in 7–14th days of post­traumatic period in blood, tissues, liver, heart and lungs if compared to the control group that causes multiple organ failure. the future prospects of the research are investigation of oxidant system dynamics after traumatic injuries of chest and hips and estab­ lishment of antioxidant­prooxidant disbalance. references 1. guriev sa, tanasienko pv, satsyk sp. clinical and epidemiological characteristics of patients with infectious complications of trauma due to accidents. medicine today and tomorrow 2012; 1: 54. 2. gao jm, gao yh, zeng jb, wang jb. polytrauma with thoracic and / or abdominal injuries: experience in 1540 cases. j. traumatol 2006; 9: 108–114. 3. who. trauma care checklist [electronic resource]. – access: http://www.whp.int/patient­ safety/implementation/checlists/trauma/en/index. html. 4. aghajanian vv, ustyantseva im, pronskiy aa. polytrauma. emergency care and transportation. novosibirsk: nauka 2008; 320. 5. malysh ir, dvorskyy pd, zhrzheblovskaya lv, krylyuk va. the strategy of empirical antibiotic thera­ py in patients with severe polytrauma. materials of all­ukrainian scientific­practical conference with in ter national participation. zaporizhzhia 2010; 95–97. 6. tsybulyak gn. treatment of severe damage. st. petersburg: hyppokrat 1995; 424. 7. adams ha, trenz o. uter mitarbeit der iag schock der divi. anaesthesieologie und intensiv medizin 2007; 73–97. 8. prydruha sm, borys rm. a violation of the humoral component of immunity during the period of late manifestations of wound dystrophy and its correction by thiotriazoline. bukovyn. med. visn 2013; 17 (1): 96–101. 9. vlizlo vv, fedoruk rs, ratych ib. laboratory methods of research in biology, animal husbandry and veterinary medicine. lviv: spolom 2012; 764. (in ukrainian). 10. korobeynikova e. a modification of lipid per­ oxi dation products assessment in the reaction with thio barbituric acid. lab. delo 1989; (7): 8–9. (in russian). 11. lowry oh, rosebrough nj, farr al, randall rj. protein measurement with the folin vp rosalovsky, sv grabovska, yut salyha 132 issn 2409­4943. ukr. biochem. j., 2015, vol. 87, n 5 phenol reagent. j. biol. chem 1951; 193 (1): 265–275. 12. marushchak m, krynytska i, petrenko n, klishch i. the determination of correlation linkages between level of reactive oxygen species, contents of neutrophiles and blood gas composition in experimental acute lung injury. georgian medical news 2016; 253: 98–103. 13. dushianthan a, grocott mp, postle ad, cusack r. acute respiratory distress syndrome and acute lung injury. postgrad. med. j. 2011; 87(1031): 612–622. 14. henynh tp, kseyko da. indicator peroxide oxidation and antioxidant protection lipids in the system 'blood sera – erythrocytes "in acute circula­ tion hypoxi.a success of modern natural science 2004; 4: 17–20. 15. malysh ir, kozlov vk, zhrzheblovskaya lv. profile of cyrculating cytokines and their mononuclear production in dynamics in post traumatical period injuired with polytrauma. cytokines and inflammation 2007; l 6 (3): 49–56. 16. kozak d. features indices of lipid peroxidation in the dynamics of early and late periods of trauma. actual problems of emergency 2012; 3 (29): 103–106. received: 2016-12-10 m. i. marushchak et al. koncor1_2014.pm6 30 o b s t e t r ic s a n d g y n e c o l o g y ijmmr 2015 vol. 1 no. 1 instrumental and diagnostic criteria of hemodynamic disorders and endothelial dysfunction correction in pregnants with arterial hypertension s. m. heryak, i. ye. humenna i. ya. horbachevsky ternopil state medical university, ternopil, ukraine background. according to the who, hypertension is associated with 20-33 % of maternal death during pregnancy within extragenital pathology statistics. there are complications of the fetus and newborn associated with hypertension at 140/90 mm hg and higher. objective. a comparative analysis of antihypertensive therapy effectiveness in pregnants with arterial hypertension was performed using modern clinical and instrumental methods of endothelial function diagnostic, central and utero-placental hemodynamic estimation. methods. we examined 63 pregnant women with hypertension at 28 to 32 weeks of gestation. women were randomized into three groups:group i (control group – 20 women), that included pregnant women with hypertension treated with basic antihypertensive therapy as recommended by health ministry of ukraine standards (metildofa and nifedipin); group ii – 21 pregnant women receiving metildofa and metoprolol; group iii (22 pregnant women) receiving metildofa and nebivolol. conclusions. it was found that the brachial artery ultrasound measuring and occlusive plethysmography procedure by dietz is an early and safe method of endothelial dysfunction diagnostic in pregnants with hypertension. doppler ultrasound of blood flow in uterine, umbilical arteries, and middle cerebral arteries of the fetus allows timely diagnosis of the side effect of antihypertensive drugs on the fetus. the therapy of choice for pregnants with stage ii arterial hypertension should be based on methyldopa and calcium channel antagonists or selective beta-blockers combination. highly selective beta-blockers with vasodilative effect (nebivolol hydrochloride) and l-arginine (tivortin) allow to prevent perinatal adverse effects of antihypertensive therapy, to correct hemodynamic disorders and endothelial dysfunction in pregnants with arterial hypertension. key words: arterial hypertension, uterine-placental hemodynamics, endothelial dysfunction. introduction according to the who, hypertension is associated with 20-33 % of maternal death during pregnancy within extragenital pathology statistic. there are complications of the fetus and newborn associated with hypertension at 140/90 mm hg and higher [1, 5, 7]. mechanisms of increased blood pressure, leading to hypertension and/or strengthening of symptoms during pregnancy are very diverse. a major role of endothelial dysfunction (ed) in the development of hypertension is shown in the recent years. it is characterized by reduced synthesis of nitric oxide as one of the main vasodilators [3, 8, 9]. unfortunately, ongoing antihypertensive therapy does not improve pregnancy outcomes for the fetus, and even lead to higher frequency of low weight for gestational age newborns. overall, high blood pressure decreases due to drug therapy and may improve pregnancy outcomes for the mother but not for the fetus [4, 6, 11]. antihypertensive drug therapy choice during pregnancy is metyldophamine as it was proved to be safe for the fetus. the second-line drugs include calcium channel blockers. they are recommended for the treatment of high arterial blood pressure in pregnant females when refractory hypertension to methyldopa therapy occurs [2, 12]. the antihypertensive drugs of the second line for pregnant women also include �-blockers, but their use is limited due to the development of adverse effects to the fetus [1, 2, 10]. it is necessary to choose an optimal set of long-term antihypertensive treatment for pregnant women which will not have a negative effect on the fetus. that is why it is very important to perform detailed comparative analysis of differential approaches to the treatment of hypertension during pregnancy and perinatal complications. the aim is to investigate endothelial dysfunction in pregnants with hypertension using non-invasive instrumental methods and offer an optimal therapy program for pregnants with arterial hypertension and address for correspondence: s.m. heryak, medical biochemistry department, m. voli, 1, ternopil, 46001, ukraine tel.: +380 352254891 s. m. heryak et al. international journal of medicine and medical research 2015, volume 1, number 1, p. 30-34 copyright © 2015, tsmu, all rights reserved 31 o b s t e t r ic s a n d g y n e c o l o g y ijmmr 2015 vol. 1 no. 1 to minimize the negative impact of the antihypertensive drugs on fetus. methods we examined 63 pregnant women with hypertension at 28 to 32 weeks of gestation, which were hospitalized to the ternopil regional perinatal center “mother and child” of the department of extragenital pathology for pregnant. clinical research was performed for all pregnant women according to the ministry of health of ukraine standards: clinical analysis of blood and urine, biochemical blood analysis, assessment of urinary albumin excretion to detect microalbuminuria, as well as ecg, echocardiography, ultrasound investigation of renal and peripheral vessels. we used modified technique by dietz n. m. (1996) [6, 10], based on the measurement of the diameter of the vessel (by ultrasound) and peripheral vascular resistance (occlusal plethysmography), in order to assess endothelial function and its effect on uteroplacental hemodynamic. initially, the brachial artery diameter (distance between opposite walls of the artery) was measured in systole phase via linear 7 mhz sensor. then arterial blood pressure was measured with classic manual tonometer. the pressure, equal to systolic plus 50 mm hg, was injected into the cuff for 5 minutes. measuring of the artery diameter was carried out immediately and again after 10 minutes. cardiotocography (ctg) with a score on a fisher’ scale and biophysical profile were used to assess the functional status of the fetus. the development of the fetus was evaluated by comparing ultrasonic fetometry under percentile tables in dynamic survey of 4 weeks after the previous test. blood flow was assessed at uterine arteries, umbilical arteries, and middle cerebral arteries of the fetus with ultrasonic doppler. women were randomized into three groups: group i (control group – 20 women), which included pregnant women with hypertension treated with basic antihypertensive therapy as recommended by moh ukraine standards (metildofa and nifedipin); group ii – 21 pregnant women receiving metildofa and metoprolol;group iii (22 pregnant women) receiving metildofa and nebivolol. pregnants of all three groups were treated orally with low-dose aspirin, calcium supplements, and magnesium for the prevention of preeclampsia according to the ministry of health of ukraine standards. statistical analysis: data analysis was done and verified using statsoft statistica 10 at systemic statistical analysis department of ternopil state medical university. results basic antihypertensive therapy for all groups included dopegit four times a day. women of the group i were treated with nifedipin 20 mg per day in four divided doses to stabilize blood pressure. pregnants of the group ii were treated with metoprolol at a dose of 25 mg per day in two divided doses. pregnant women of the group iii were prescribed nebivolol hydrochloride 5 mg once a day as a highly selective third generation beta blocker with vasodilative properties and ability to modulate the synthesis of the nitric oxide by the blood vessels endothelium. the endothelial dysfunction treatment should be considered as a strategic line for effective prevention of cardiovascular complications of the arterial hypertension as pregnancy treatment. therefore, women of the second and the third groups were administered l-arginine aspartate (tivortin) 1 g (5 ml) orally six times per day for 14 days in addition to antihypertensive preparations. the gestational term of 28-32 weeks and arterial hypertension stage ii were the criterions for randomization. study groups were homogeneous for age, obstetric history, and the course of the underlying disease. exclusion criteria were defined as following: hypertension stage iii, severe concomitant extragenital pathology (diabetes, diseases of the thyroid gland, other cardiovascular pathology). we observed different hemodynamic normalization and stability of blood pressure after treatment in each group (table 1). table 1. blood pressure and heart rate in women of the surveyed groups during dynamic treatment (m±m) ������ ��� �� � ��� ��� ��� ����� ����� ����� ����� ���� �� � �� ��������� ���� ��������� � ��!�!����!� ������!�� ������"���#���� $ ��������� ���� ��%�����&�� ��&����&� ������ &� �� ��������� ���� ��&�!������ ����&���� � ������!�� ������""���#���� $ ��������� ���� ��!����� !� ������&�� �!�������� �� ��������� ���� ����!������ ��!�!��� �� ������!!� ������"""���#��� $ ��������� ���� ����������� !��������� !��������� note: p<0.01 – data accuracy. s. m. heryak et al. 32 o b s t e t r ic s a n d g y n e c o l o g y ijmmr 2015 vol. 1 no. 1 pregnant women of the group i showed poor tolerability to the therapy. particularly, we observed sudden changes in blood pressure (hypotension episodes below 100/60 mm hg or hypertensive crises (blood pressure above 160/110 mm hg) in three pregnant women; four pregnant women developed swelling and redness of the face, seven women complained about palpitations, dizziness and weakness. simultaneously, eight patients of the second group experienced bradycardia (less than 60 bpm), while another five complained about dyssomnia, dizziness, weakness, and increased edema. the best tolerability to the therapy was observed in the third group, where only two pregnant women noted weakness and headache while the heart rate slowed down below 60 bpm in two women. also, as it is shown in table 1, the clinically most effective scheme of arterial pressure normalization was the combination of nebivolol hydrochloride and metildofa on the background intake of l-arginine. forty four (70.9 %) of the surveyed women experienced ecg changes before treatment: the deviation of the electrical axis of the heart to the left, st segment depression, inversions or flattening of t wave in leads ii, iii and avf. in pregnants of the group i tachyarrhythmia was observed, including seven women (36.8 %) with arrhythmias. bradyarhythmia and conduction disturbances (different degrees of blockages) were observed in nine pregnants of the group ii (42.9 %). conduction disorders and slowing heart rate were observed in six women of the third group (27.2 %). as for results of the sonogram of the heart, ejection fraction in all three groups before treatment was 63.3 %±3.4 %, and it decreased insignificantly after the treatment only in the second group. all females in all three groups (38 females, 62.1 %) were equally affected by end-diastolic increase in the size of the right ventricle, and an increase of the index of left ventricular mass by 10–15 %. ten pregnant women (16.1 %) showed increase of the index of the left ventricular mass from 15.1 to 27.8 %, which may be classified as excessive. combined preeclampsia developed in seven females (two pregnants from the first group, three from the second group, and two from the third group). data for the indicators of the endothelial function are presented in table 2. in all three groups, the diameter of the brachial artery before treatment should be assessed as narrowed. therefore, an endothelial dysfunction is observed in pregnants with hypertension, which is manifested by spasm of the arteries. there was a significant dilatation of the brachial artery with slow normalization of arterial tonicity for the group i after the treatment (diameter 4.4 mm±0.2 mm and endothelium-dependent dilatation was more than 22 %, which is relatively higher than in healthy pregnant women). pregnants of the second group experienced spasm of the arteries, and this condition did not normalize. as for the third group, the diameter of the brachial artery was significantly restored to the values close to the healthy pregnant women and endothelium-dependent vasodilation (18.4 %) showed a normal tone of arteries and their ability to respond adequately to stimulation. therefore, the endothelial function was restored in the group iii of patients to the highest level. we have also established a relationship between fetometric performance relative to the gestational table 2. indicators of the endothelial function (m±m) note: p<0.01 – data accuracy. ������ ��� �� � � ���� � � ���������������������� ��� ������ � � �������� ����� �� �� ����������� ����� ������������ � �� ��� !"�"�� #�"!"�� � �������� $���������� � �� �� �!"�"�� %%��!"�& � ������������ � �� ����!"�"&� #�%!"��%� ���������� $���������� � �� ���#!"�"&� &�"!"��'� ������������ � �� ���'!"�"�� #� !"�%(� ����������� $���������� � �� ��#"!"�"�� #��!"��'� table 3. the main parameters of fetometry during treatment of pregnants with hypertension of 32–34 weeks term (m±m) ������ ��� �� � � ���� � �������������� ����� ������� � ���� � ������ ����� ������ � �� ����� ���� ������ � ������������ � �� �� !"#! $�� �� "%#! &�� %& '"#! %&� ������(�� )*!�� ����������� � �� &% %*#! �'� &� +"#$ $�� "* !'#! ��� ������������ � �� �& *�#! *$� �� $'#! +&� %& $"#! *&� ������((�� )*$�� ����������� � �� &* +!#! &"� &% *%#! �!� "! !+#$ *+� ������������ � �� �& !$#! **� �� $!#! $�� %& +!#! %'� ������(((�� )**�� ����������� � �� &" +!#! �%� �$ *!#! &%� "' *%#! ��� s. m. heryak et al. 33 o b s t e t r ic s a n d g y n e c o l o g y ijmmr 2015 vol. 1 no. 1 term and the option of antihypertensive therapy on the basis of surveys (table 3). the size and weight of the fetus is less than average in the general population according to the gestational age in women with hypertension, which is often due to the disruption of hemodynamics in the mother-placenta-fetus system. therefore, according to the ultrasound examination of the surveyed patients, the average biparietal diameter decreased for a given gestational age and evidence about disruption of uteroplacental hemodynamics; morevover, after the treatment, biparietal diameter was the biggest in the third group, followed by the first group and the second group. as for indicative parameters such as average diameter of the abdomen and femur length, best values were observed in the third group that explains normal fetal fatness and skeleton development, and therefore the optimal normalization of utero-placental hemodynamics, which correlated with indicators of endothelial function. thus, the maximum normalization of the biometric parameters was observed in the third group of pregnant women. doppler velocimetry is helpful to evaluate blood circulation in the placenta and detect early signs of poor circulation. to assess the blood circulation, the following indices are used: systolic-diastolic ratio (sdr), pulsation index (pi) and index of resistance (ir), all of those are shown in table 4. the degree of hemodynamic disorders in the mother-placenta-fetus system is evaluated according to the medvedev classification: 1a – isolated violations of the utero-placental blood flow; 1b – isolated violations of the feto-placental circulation; ii – violation of uteroplacental and feto-placental circulation does not reach the critical meanings; iii – decompensated disorders and circulatory centralization. based on our results, we can conclude that pregnant women of the group i experienced slight improvement of the parameters of the uterine artery blood flow, but no significant improvement of blood flow in the umbilical artery and middle cerebral artery of the fetus. pregnant women of the second group showed insignificant worsening of the blood flow in the uterine arteries, umbilical artery and middle cerebral artery. pregnant women of the third group were characterized by a slight improvement in blood flow of the uterine artery and nonsignificant improvement in umbilical artery and middle cerebral artery of the fetus. note: p<0.01 – statistical significance and data accuracy. discussion summing up the results of the abovementioned surveys, we can assume that in order to minimize side effects during gestation, the cardioselective �-blockers with vasodilation properties should be preferred. these drugs will prevent the increase in total peripheral vascular resistance and can smooth the negative effects of classical beta blockers (delayed intrauterine development, bradycardia, hypotension, hypoglycemia, respiratory depression of the fetus). conclusions 1. it was found that the brachial artery ultrasound measuring and occlusive plethysmography procedure by dietz is an early and safe method of endotable 4. doppler velocimetry in surveyed groups of pregnant women after 30 weeks of gestation � ������� �� �� �� �� � �� ����������� � �� � ��������� �� � ��� � � �� ���� ����������� ����������� ���������� �� ���!������� ���"������� �����������# $%��&��'��� ��� ���������"� ����������� ����������� ���� ����������� ����������� ��"�����!�� �� ���!������� ����������� ��"��������# $%���&��'��� ��� ����������� ����������� ����������� ���� ����������� ����������� ��"������"� �� ����������� ����������� ��""�������# $%����&��'��� ��� ���"������� ����������� ���"������� thelial dysfunction diagnostic in pregnants with hypertension. doppler ultrasound of blood flow in uterine arteries, umbilical arteries and middle cerebral arteries of the fetus allows timely to reveal the side effect of antihypertensive drugs on the fetus. 2. the therapy of choice for pregnants with stage ii arterial hypertension should be based on methyldopa and calcium channel antagonists or selective beta-blockers combination. 3. highly selective beta-blockers with vasodilative effect (nebivolol hydrochloride) and l-arginine (tivortin) let us to prevent perinatal adverse effects of antihypertensive therapy, to correct hemodynamic disorders and endothelial dysfunction in pregnants with arterial hypertension. s. m. heryak et al. 34 o b s t e t r ic s a n d g y n e c o l o g y ijmmr 2015 vol. 1 no. 1 references ? k���)� !�@� +���( �� .� lmln� �#"��" )�� � ��)�����#����� ���o�� � ��a�1? ?2 2::/ 2 k���)�!�@�+���( ��.�1>/n�7���)���"��-" � ����#�����-" ��,"����p�"* ���o�� �*�����," ����)� �� �����)�%�(� ,"��� �(� ��#�,�o�� #��� ���"����� �� o�#"��" )�(a�2/ :0 2:?2 1 !"��"��������'�� ���"�%�(�)�"� ���o" ����� �( #�����o�(���o�� �*a�8�(�a�2:?:n�?:q2/: / 7�� ���,� r!�� 7�%""�� ���� 7��,������ r!� ��,� � �s� !" ��� "'�������� �"��� '"��p���" �p '�������������"�" ���,�o����3�,"�" ��6����"�����p ���o�#"��" )�����'"�"," t* �!"��%� ��"� ��� �� 2:?1a�2n�m:qm0 0 r���," ���u���8���,���" ���u���v $������� ! @� �� ��� �� u������ �� �� ��,#�"� �,�� ������ � ��o�� �*� )� *�� �� �$� ���"����� �$� o�#"��" )�w$ @������"�-" &� t�2:?2a�/�3m:6n�l2ql0 l @�#���-� � �!�� ��%�"�� ���� 8� ����� � � ���� �x� �#� �'� "� ��)�� �(� ���o � ����� �� �%��p ��� �o�� �� �� " ���"��$� #��� ��o�� � �� � 7��" �� +���( � �k�,"��#��" ��n�mm;>/ �@����" �n�?; :l 2:?2 �#�'������ �n�?? :1 2:?1 7. henriques acpt, carvalho fhc, feitosa hn, macena rhm, mota rms, alencar jcg. endothelial dysfunction after pregnancy-induced hypertension. int j gyn ob 2014; 124 (3): 230–234. 8. mosca l. et al. effectiveness-based guidelines for the prevention of cardiovascular disease in women – 2011 update: a guideline from american heart association. circulation 2011; 123 (11): 1243–1262. 9. morton js, davidge st arterial endotheliumderived hyperpolarization: potential role in pregnancy adaptations and complications. journal cardiovasc. pharmacol 2013; 61(3): 197–203. 10. dietz nm, engelke ka, halliwill jr, proctor dn, joyner mj contribution of nitric oxide and prostaglandins to reactive hyperemia in human forearm. j. appl. physiol 1996; 81(4): 1807–1814. 11. yinon y, kingdom jc, odutayo a, moineddin r, drewlo s, lai v, cherney dz, hladunewich ma. vascular dysfunction in women with a history of preeclampsia and intrauterine growth restriction: insights into future vascular risk. circulation 2010; 122: 1846–1853. 12. romundstad pr, magnussen eb, smith gd, vatten lj. hypertension in pregnancy and later cardiovascular risk: common antecedents? circulation 2010; 122: 579–584. received: 2014.06.02 s. m. heryak et al. koncor1_2014.pm6 21 s u r g ic a l d is e a s e s ijmmr 2015 vol. 1 no. 1 comparison of short�term analgesic effects of extracorporeal shock wave therapy (eswt) and conservative treatment (ct) in men with chronic heel spur (hs) p. lizis holy cross college, kielce, poland background. the surgical removal of heel spur (hs) provokes many controversial opinions, but clinical studies show the risk of complication after operation. an alternative to the surgical removal of hs is extracorporeal shock wave therapy (eswt), because it is non-invasive method. objective. the aim of the study is to compare the short-term analgesic effects of eswt and conservative treatment (ct) in males with chronic hs. methods. sixty patients (mean age 54.9±4.3 years; range 42 to 59 years) were examined who had pain associated with hs. in the shockwave group (group 1) patients received 1000–2000 impulses of shocks to the affected heel in a single session. the patients in this group took a series of 5 eswt in 1 week intervals. patients in the control group (group 2) at the same time received ct consisting of nonsteroidal anti-inflammatory drugs (nsaids), orthotics and a local cortisone injection. the basic method of research was to evaluate pain according to visual analogue scale (vas) 0–10. the patients of the two groups were tested before the treatment, after the last treatment and 3 months and 6 months after the treatment. results. a significant decrease of vas (p=0.000) was seen in the shockwave group. in the control group no significant decrease of vas was seen. conclusions. eswt reduces pain more effectively than ct in men with chronic hs. key words: extracorporeal shock wave therapy, conservative treatment, visual analogue scale, heel spur, pain. address for correspondence: pawel lizis, phd, holy cross college, department of physiotherapy, 51, mielczarskiego str., 25-709 kielce, poland tel.: +48413623018; fax: +48413327451; e-mail: pawel_lizis@poczta.onet.pl introduction chronic inflammation of the plantar fascia, in the place of its connection to calcaneal tuberosity, can be a major cause of pain in the adult population. due to a visible x-ray imaging calcification – bone spurs near the heel – the condition is commonly called “heel spur” (hs). among other causes of the condition, there are: an inflammation of the achilles tendon associated with inflammation of the achilles tendon bursitis, inflammation of adipose tissue beneath calcaneal tuberosity by repeated micro damages that cause degeneration of the tissue, fatigue fractures of the calcaneal tuberosity, neuropathy caused by pressure on the plantar branch of the tibial nerve by an overgrown plantar aponeurosis. risk factors also include excessive body weight, running and jumping, work that entails standing or lifting heavy objects, flat feet, rheumatic changes [1– 5]. originally, the condition may be asymptomatic – pain rarely occurs in the early stages of the condition. most patients come to the doctor in a fairly advanced stage of the condition, complaining on severe pain in the heel that escalates after they walk on hard ground or carry heavy objects and that usually disappear after they rest. the treatment of hs and plantar fascia provokes many controversial opinions, many authors point to the need for surgical removal of calcaneal bursitis and resection of hs, but clinical studies show the risk of complication after operation [6–12]. an alternative to the surgical treatment of hs is ct that involves the use of physiotherapy treatments, such as ionophoresis, laser, ultrasound, and recently more and more often eswt is used [13–16]. eswt is a very effective method which effectiveness is comparable to surgery. the most important is the fact that it is a completely non-invasive method. it has not yet been fully explained how it works exactly, but it probably involves micro destructions – the application of eswt causes micro breaks in avascular or poorly-vascularized tissue thus stimulating appropriate revascularization and stem cell growth. patients may continue to work or train the next day. given the significant reduction of activity limitations and short duration of the treatment, p. lizis international journal of medicine and medical research 2015, volume 1, number 1, p. 21-26 copyright © 2015, tsmu, all rights reserved 22 s u r g ic a l d is e a s e s ijmmr 2015 vol. 1 no. 1 eswt is not only comparable to surgical treatment, but, in general, it is also less expensive than a few months of ct [17–18]. the aim of the study was to compare the shortterm analgesic effects of eswt and ct in males with chronic hs. methods continuous research was carried out in the period from january 2010 to july 2012, and involved sixty patients (middle age 54.9±4.3 years; range from 42 to 59 years) who had pain associated with hs. the average bmi was 24.29±0.78. none of the men was obese. the study was conducted in busko-zdroj spa. the patients had unilateral pain. the average duration of pain was 15.2±5.3 months. inclusion criteria were pain over the x-ray examined hs, unsuccessful ct (iontophoresis, cryotherapy, laser therapy, ultrasound and phonophoresis) during the six months before referral to spa in busko-zdroj. exclusion criteria were arthritis (rheumatoid arthritis, spondarthritis, crystal induced arthropathies) diabetes mellitus, neurological abnormalities, age under 18 years, infectious or tumorous diseases, skin ulcerations, and bursitis. all patients were informed about the principles of the treatment, and they signed a written permission for treatment and participation in the study. in the shockwave group (group 1) patients received – during the first treatment 1000 impulses, 1500 impulses in the second, and 2000 impulses in the third, fourth and fifth treatment (the pressure of 2.5 bar, the frequency of 8 hz, the energy density of 0.4 mj/mm2). the patients in the shockwave group took a series of 5 eswt in 1 week intervals. apparatus btl-5000 swt was used for the treatment. the active engagement between the head of the apparatus with a diameter of 15 mm and skin was gel used in ultrasound scan. the procedure was performed in the area of most intense pain – calcaneal tuberosity. treatment time did not exceed 10 minutes. patients in the control group (group 2) at the same time received ct consisting of nsaids, orthotics and a local cortisone injection with 0.5 ml of betamethasone (7 mg/ml). the basic method of research was to evaluate pain according to vas, where 0 – no pain; 10 – severe (maximum) pain at rest, after walking on awakening, and after normal daily activity. the patients of two groups were tested before the treatment, at the end of the last treatment. the patients were also examined for 3 months and 6 months after the end of treatment. the results observed were classified as excellent improvement (a vas reduction of over 50 %), good improvement (a vas reduction of over 30 % to 50 %), slight improvement (a vas reduction of over 15 % to 30 %). in order to evaluate the early results of the treatment, the obtained data was subjected to statistical analysis. the statistical distributions of the analyzed characteristics were examined by the use of kolmogorov-smirnov’s test. it was shown that the tested variables had normal distribution. basic descriptive statistics was used for the analysis. the mean arithmetic (�x) and standard deviation (sd) of the studied traits of men in both groups were calculated. in order to evaluate the statistical significance of differences in the intensity of pain before the treatment, after the last treatment and 3 and 6 months later, while at rest, while walking on awakening, and after normal daily activity, one-way analysis of variance (anova) was used in both studied groups. the value of the function f snedecor was observed in various combinations. the statistical significance of differences in the intensity of pain was assessed at the 0.05 level. calculations were performed at the department of computer science at holy cross university in kielce with the use of medcalc software – version 11.4.3.0, licensed to holy cross college. the research project was authorized by the bioethics committee at holy cross college in kielce, resolution 1/10/kb dated 29.01.2010. results a significant decrease of vas (p = 0.000) was seen in the shockwave group (group 1) just after the end of eswt and in the period after the end of eswt 3 months later and in the period from 3 months to 6 months after the end of eswt at three reference points – at rest, when walking on awakening, and after normal daily activity. no significant decrease of vas was seen in the control group (group 2) just after the end of ct at rest (p=0.202), when walking on awakening (p=0.367) and after normal daily activity (p=0.341). no significant decrease of vas was seen in the period after the end of ct 3 months later at rest (p=0.367), when walking on awakening (p=0.630) and after normal daily activity (p=0.633). also, no significant decrease of vas was seen in the period from 3 months to 6 months after the end of ct at rest (p=0.608), when walking on awakening (p=0.337) and after normal daily activity (p=0.393). studies have shown that eswt reduces pain more effectively than ct in males with chronic hs (table 1). the analgesic efficacy of eswt is confirmed by the detailed analysis of differences in the frequency of occurrence of pain intensity between groups. in the shockwave group (group 1) excellent improvement (a vas reduction of over 50 %) was seen in 22/30 (73.3 %) patients, good improvement (a vas reduction of over 30 % to 50 %) was seen in 6/30 (20 %) patients and slight improvement (a vas p. lizis 23 s u r g ic a l d is e a s e s ijmmr 2015 vol. 1 no. 1 table 1. the intensity of pain (vas) in the following terms of research at three reference points �������� �� ������ ������� �� �� � �� � ������ �� ����� �� �� ����������������� ����� ��� ��� ������������������� �� ���� � � !�"�� #$���� � �$���� � � !�"� #$���� � �$���� � � !�"� #$���� � �$���� � ����� % �� ��� �&�'��� ����� (�� ��� ��� �� ����&�'�� � � )*+!,*-� � � .*/!,*)� .-*,))0� ,*,,,� /*1!,*-� � � 2*�!,*/� �,,*�1/0� ,*,,,� /*.!,*-� � � 2*3!,*)� �33*,))0� ,*,,,� ����� (�� ��� ��� �� ����&�'�� ����� 1������ �� ��� �&�'�� � � .*/!,*)� � � 1*-!,*/� -.*/+�0� ,*,,,� 2*�!,*/� � � +*+!,*2� /2*),,0� ,*,,,� 2*3!,*)� � � +*2!,*)� )/*12)0� ,*,,,� ����� 1������ �� ��� �&�'�� ���� 2������ �� ��� �&�'�� � � 1*-!,*/� � � 3*.!,*)� .3*,1.0� ,*,,,� +*+!,*2� � � 1*,!,*)� 2-*�)20� ,*,,,� +*2!,*)� � � 1*.!,*2� +3*),20� ,*,,,� 4��� ���� ������ ����3�� ����� % �� � ��� ����� � � ��� ��� ����� (�� ��� ��� �� ��� ����� � � ��� ��� � � � /*+!,*-� � � � /*�!,*-� �*22)� ,*3,3� /*2!,*-� � � � /*+!,*/� ,*/3/� ,*12)� /*)!,*-� � � � /*.!,*)� ,*-31� ,*1+�� ����� (�� ��� ��� �� ��� ����� � � ��� ���� ����� 1������ ���� � ��� ����� � � ��� ���� � � � /*�!,*-� � � � /*1!,*/� ,*/3/� ,*12)� /*+!,*/� � � � /*.!,*/� ,*31+� ,*21,� /*.!,*)� � � � /*2!,*-� ,*31�� ,*211� ����� 1������ ���� � ��� ����� � � ��� ���� ����� 2������ ���� � ��� ����� � � ��� ���� � � � /*1!,*/� � � � /*+!,*)� ,*32.� ,*2,/� /*.!,*/� � � � /*)!,*/� ,*-1)� ,*11)� /*2!,*-� � � � /*/!,*-� ,*)+�� ,*1-1� � reduction of over 15 % to 30 %) was seen 2/30 (6,7 %) patients at three reference points – at rest, when walking on awakening, and after normal daily activity. there was no decreasing in pain intensity in the control group (group 2), as it was unchanged in 24/30 (80 %) patients and worsened in 6/30 (20 %) patients at these points (table 2). discussion hs is a condition that affects more and more people of all ages. it affects their quality of life, reduces socio-professional activity and becomes the cause of frequent sickness absence. the effectiveness of treatment for hs causes many controversial opinions. there is no consensus on how p. lizis 24 s u r g ic a l d is e a s e s ijmmr 2015 vol. 1 no. 1 to treat it, conservatively or surgically. endoscopy is used most often in surgical treatment. this method is cost-effective, non-invasive, with low risk of postsurgery complications and allows patients to return to socio-professional activity after surgical treatment earlier [8, 19–22]. an alternative to surgical treatment is to use eswt for treatment of chronic heel spur. there are different opinions on this subject, but the prevalent among them are views highlighting the analgesic efficacy of eswt in the treatment of chronic hs. the analgesic success of eswt probably depends on the total dose of energy, not on the density of stream of emitted energy [18, 23– 25]. review of subject literature shows that the analgesic efficacy of eswt with different physical characteristics and over a different period of time concerning was investigated. hammer, adam, kreutz, kohn, seil [14] assessed the analgesic efficacy of eswt in patients with painful chronic inflammation of the plantar fascia. patients treated with eswt were given 3000 impulses of shocks with energy density of 0.2 mj/mm2 at weekly intervals. two years after the end of treatment the level of pain on a vas scale in patients treated with eswt decreased 94 %. in subsequent studies, the previously mentioned authors used medical ultrasonography to assess the effects of eswt on changes in thickness of plantar fascia that had been affected by chronic inflammation. during the treatment, 3 sessions of 3000 shock wave impulses with the stream density of 0.2 mj/mm2 were applied at weekly intervals. the thickness of plantar fascia was measured 2 cm distally to calcaneal tuberosity. in addition, the level of pain was recorded in the vas scale. the ultrasound scan 6 months after the end of eswt showed a significant reduction in the thickness of plantar fascia of the affected foot and the level of patients pain was reduced 79 % in the vas scale [26]. metzner, dohnalek, aigner [18] also used eswt in patients with chronic plantar fascia. each patient received 1000 impulses of shock wave, the density of energy stream was 0.35 mj/mm2. the patients were examined 6 weeks, 16 months and 72 months after the end of eswt. it turned out that pain was reduced in 81% of patients after 6 weeks, in 88 % of patients – after 16 months, and in 96 % of patients in the last examination – 72 months after the end of eswt. based on these results, the authors concluded that the applied doses of eswt effectively reduced pain and the achieved therapeutic effects provide satisfactory long-term results. yalin, keskin akca, selcuk, kurtaran, akyuz [27] used eswt for analgesic purposes in patients with hs and treated them for 5 weeks with 2000 impulses of shock waves, ranging from 0.05 to 0.4 mj/mm2. clinical results demonstrated excellent results (no pain) in 66.7 % of the cases, good results (50 % of pain reduced) in 15.7 % of the cases, and unsatisfactory outcome (no reduction in pain) in 17.6 % of all cases. at the same time, the study showed no correlation between the reduction of pain and a significant modification of the hs in x-ray picture. moretti, garofalo, patella, sisti, corrado, mouhsine [28] evaluated the analgesic efficacy of low doses of eswt for foot plantar fascia inflammation accompanying hs in runners – athletes. the subjects received a weekly shock wave of 1000 impulses, 0.06 mj/mm2 energy density. eswt continued for four weeks. the patients were examined after the last session of eswt. clinical results were excellent in 59 % of cases, good in 12 % of cases, satisfactory in 21 % and clearly unsatisfactory in 8 %. ultrasound examination after the end of eswt showed the disappearance of inflammatory symptoms in 61 % of athletes. cosentino, falsetti, manca, de stefano, frati, frediani et al. [29] evaluated the analgesic efficacy eswt at patients with calcaneal enthesophytosis. patients were randomly assigned into two equal groups. the shockwave group (group 1) received six treatments (one every 7–10 days), each treatment consisting of 1200 shocks with a frequency of 120 shocks/min; the energy density used varied from 0.03 to 0.4 mj/mm2 and the control group (group 2) went through the identical process but energy density was simulated (0 mj/mm2). the results revealed significant reduction of pain in the shockwave group (group 1). in the control group (group 2) no significant decrease of vas was seen. wang, wang, yang, weng, ko [30] evaluated patients with an established diagnosis of chronic plantar fasciitis, including patients in the shockwave treatment group and patients in the control group. in the shockwave group, patients received 1500 table 2. the analgesic effects of eswt ��������� ��� ����������� ������������������������� �� �������� �� �������� ����������� ��!��� ��� "#$#��� %� %� ����#�&������� ��!��� '� ����� %� %� ������&#������ ��!��� �� '$"��� %� %� ������� ���� � %� %� �(� )���� ���� �� ��� � %� %� '� ����� p. lizis 25 s u r g ic a l d is e a s e s ijmmr 2015 vol. 1 no. 1 impulses of shockwaves at 16 kv to the affected heel in a single session. patients in the control group received ct consisting of orthotics, physical therapy, an exercise program, and/or a local cortisone injection. before treatment, groups showed no significant differences in the scores for pain. after treatment, the shockwave group showed significantly better pain scores in comparison with the control group. the overall results were 69.1 % excellent, 13.6 % good, 6.2 % fair, and 11.1 % poor for the shockwave group and 0 % excellent, 55 % good, 36 % fair, and 9 % poor for the control group. our study confirmed the results of other authors. the doses used in eswt gradually decreased pain intensity in the shockwave group (group 1) and this trend persisted up to 6 months from the last session of eswt. the observed differences in this period were statistically significant. in the control group (group 2) no significant decrease of vas was seen after the treatment and in the period from 3 months to 6 months after the end of ct and in the period from 3 months to 6 months after the end of ct in patients with hs. furthermore, statistically significant differences in pain were also found between the two groups. excellent improvement of pain intensity was seen in 73.3 % patients, good improvement was seen in 20 % patients and slight improvement was seen in 6.7 % patients in the shockwave group (group 1) at three reference points: at rest, when walking on awakening, and after normal daily activity. as for the control group (group 1), there was no reduction of pain intensity; it remained unchanged in 80 % of patients and worsened in 20 % of patients at these points. summing up, the results of shortterm studies show that eswt effectively reduces pain, which, in turn, reduces mental and physical discomfort in patients with chronic hs. conclusion the short-term studies have demonstrated the analgesic effectiveness of eswt. we achieved a significant reduction of pain, that persisted for 6 months in shockwaves group. eswt reduces pain more effectively than ct in males with chronic hs. the studies have shown that eswt is a repeatable and non-invasive treatment, and, therefore, is a valuable alternative therapeutic option for surgical treatment and other ct in patients suffering from pain due to chronic hs. references 1. agostinelly j, ross ja. infracalcaneal heel pain in the athlete. clin podiatr med surg 1997; 14: 503–509. 2. aquino w, payne c. function of the plantar fascia. foot 1999; 9: 73–78. 3. kim w, voloshin as. role of plantar fascia in the load besring capacity of the human foot. j biomech 1995; 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14: 1029–32. 29. cosentino r, falsetti p, manca s, de stefano r, frati e, frediani b et al. efficacy of extracorporeal shock wave treatment in calcaneal enthesophytosis. ann rheum dis 2001; 60: 1064–1067. 30. wang cj, wang fs, yang kd, weng lh, ko jy. long-term results of extracorporeal shockwave treatment for plantar fasciitis. am j sports med 2006; 34: 592–596. received: 2014.07.10 p. lizis koncor1_2014.pm6 5 in t e r n a l m e d ic in e ijmmr 2015 vol. 1 no. 1 oxidative stress in human thyroid gland under iodine deficiency nodular goiter: from harmlessness to hazard depending on copper and iodine subcellular distribution h. falfushynska1,2, l. gnatyshyna1,2, a. shulgai1, v. shidlovski1, o. stoliar2 1 i. ya. horbachevsky ternopil state medical university, ternopil, ukraine 2 v. hnatiuk ternopil national pedagogical university, ternopil, ukraine background. thyroid disorders are the second most common endocrinopathies found in humans and animals. determination of their key molecular markers presents a special interest. objective. we studied iodine and copper accumulation in nodular, paranodular and contralateral (not affected tissue by node) tissues of human thyroid gland in relation to the level of metal-binding proteins, potential antioxidants, and oxidative changes in tissue for this goal. lower level of organificated iodine and higher level and mass fraction of inorganic iodine and copper in the nodular and paranodular tissue versus contralateral part of thyroid gland was established. results. the level of both metal-binding and apo-form of metallothioneins was higher. content of reduced glutathione was lower in node-affected tissue compared to the contralateral part. signs of oxidative stress (higher activity of superoxide dismutase, catalase, glutathione-transferase and level of oxyradicals) and cytotoxicity (higher cathepsin d activity, higher level of dna strand breaks and glycolysis activation) in affected tissue were observed. the range of indice variability in paranodular tissue was smaller than in nodule compared to the parenchyma of contralateral part. conclusions. excess of copper unbound to metallothionein in goitrous-changed tissue and high level of inorganic iodine could be the reason for elevated dna fragmentation and increased lysosomal membrane permeability and activation of antioxidant defense. the main criterions of goiter formation were represented by low level of organificated iodine and high level of dna damage in thyroid gland. key words: iodine deficiency nodular colloidal goiter, iodine, copper, metallothioneins, oxidative stress, cytotoxicity address for correspondence: h. falfushynska, general chemistry department, i. ya. horbachevsky ternopil state medical university, m. voli, 1, ternopil, 46001, ukraine tel.: +380506782021; e-mail: halynka.f@gmail.com introduction thyroid disorders are the second most common endocrinopathies found in humans and animals [1]. determination of the key molecular markers is of considerable interest as they can be used to predict such pathologies. iodine deficiency in thyroid pathology occurrence rate is rapidly increasing [2]. it has miscellaneous origins as a result of complex interaction of endogenous and exogenous factors and arises in the setting of high level of “nonspecific” goitrogens, among them copper, in the environment. copper is an essential element for humans and animals, especially for antioxidant defense and the metabolism of the amino acid tyrosine, which is needed for the production of thyroid hormones [3]. besides these ones, excessive amounts of copper in the body can pose a risk. the mechanisms underlying the acute toxicity effects of copper in humans are not well understood. it is reasonable to speculate that they probably represent a combination of significant oxidative stress at different body areas together with marked perturbations in several components of the endocrine system. it was shown that the increase of copper level in the thyroid gland of patients with colloidal goiter was combined with the prooxidant changes in tissue [4]. it should be noted that ternopil region with combination of moderate iodine deficiency and high scale of copper water pollution [5] presents an interest to study the relationship between copper accumulation and progress of thyroid pathology. free radical-mediated oxidative damage has been implicated in pathogenesis of many diseases. however, the reasons of this phenomenon are disputable under endemic nodular thyroid goiter. it is believed that the prooxidant effect of copper depends on the specific accumulation by metal binding protein, such as metallothioneins [6]. metallothioneins (mts) are low-molecular weight proteins of 6–7 kda, with high content of cysteine (30 %) and complete absence of aromatic amino acids and histidine, capable of binding transition metals with high affinity. mts synthesis can be induced by a variety of metals, cytotoxic agents, stress-producing conditions, cytokines, and glucocorticoid hormones [7]. additionally, mts can function as efficient scavengers of reactive oxygen species to preserve homeostasis of cells. the latest study have indicated a possible role of mts as a tumor suppressor in papillary thyroid cancer [8] and h. falfushynska et al. international journal of medicine and medical research 2015, volume 1, number 1, p. 5-11 copyright © 2015, tsmu, all rights reserved 6 in t e r n a l m e d ic in e ijmmr 2015 vol. 1 no. 1 their part in the distribution of metals, thereby optimizing the function of thyroid gland [4]. thus, the aim of this study was to evaluate the relation between the function of mts and oxidative stress in node, paranodular and non-affected by node contralateral part tissue of human thyroid gland under iodine deficiency endemic nodular thyroid goiter. molecular markers of cytotoxicity were used to assess the severity of the pathological process. methods the target population in this study were the people with unilateral euthyroid iodine deficiency nodular thyroid goiter, who had lived in ternopil region for at least 22 years. small samples of nodule, paranodular and contralateral (not affected by node tissue) tissues from thyroid gland were dissected in 25 patients. they were operated at the general surgery department of the local ternopil emergency hospital. all experimental studies were conducted in accordance with the approval of the first national congress on bioethics (kyiv, 2000) and the approval of the bioethics commission of i. ya. horbachevsky ternopil state medical university. tissue samples were homogenized (1/10 w/v) in 0.1 m phosphate buffer, ph 7.4, containing 100 mm kcl and 1 mm edta, as well as 0.1 mm phenylmethylsulfonyl fluoride (pmsf) for proteolysis inhibition for enzymatic measurements. homogenates were centrifuged for 10 min at 6,000 g. protein concentration in the supernatant (soluble protein) was measured by the method of lowry et al. (1951), using bovine serum albumin as a protein standard. the absorbance values were measured on an uv/ vis spectrophotometer “lomo-56” (russia), and extinction/emission values were measured on the f-max fluorescence microplate reader [molecular device (usa)]. reduced glutathione (gsh), 5,5'-dithio-bis(2nitrobenzoic acid) (dtnb), glutathione reductase from baker’s yeast (s. cerevisiae), 2-vinylpyridine, dihydrorhodamine, salmon sperm dna, hoescht 33342, serum albumin, phenazinemethosulfate, phenylmethylsulfonylfluoride (pmsf), �-mercaptoethanol, nadh, �-nadph, and edta were purchased from sigma-aldrich. all chemicals were of the analytical grade or better. mts were determined in parts of thyroid gland tissue after ethanol/chloroform extraction by thiol measurement with dtnb according to the method of viarengoet al. (1997) [9]. the level of mt-related thiols (mt-sh) was calculated by using following relationship: 1 mol mt-sh = 20 mol gsh. the level of mts was defined as µg of mts per gram of fresh weight (fw) tissues. to assess metal concentration in the mts (mt-me), they were isolated as thermostable proteins by size-exclusion chromatography on sephadex g-50 with necessary adjustments needed to avoid their oxidation [10], as described previously [11]. a 5 % homogenate (w/v) was prepared in ice-cold 10 mm tris-hcl buffer, ph 8.0, containing 10 mm 2-mercaptoethanol for maintaining of the reduced conditions and 0.1 mm pmsf for the inhibition of proteolysis. fractions of the chromatographic peak with high absorbance at 254 nm and comparative high density ratio d 254 /d 280, identified as mt-containing peak [12], were pooled (total 10 ml) and applied to metal determination. to determine copper and zinc concentration, fresh tissues (500 mg) and pooled eluate of mts fraction after the size-exclusion chromatography (10 ml) were digested in 5 ml hno 3 (merck) for 3 h at 105 °c for metal analysis using hermetic acidcleaned teflon bomb. concentration of metals was analyzed by the atomic absorption spectrometer with flame detector (c-115, “lomo”, russian federation). the metal detection limits were 0.1 �g·g-1 fw. the analytical methods were validated by external intercalibrations. quality control was assessed by the quality control sample of trace metal and method of standard addition (www.dentalmercury.com/245_1.pdf). metal concentration in the tissues and mts was presented as �g·g-1 fw and nmol·g-1 fw. subcellular distribution of iodine in the thyroid gland was determined by previously developed method [13]. superoxide dismutase (sod, ec 1.15.1.1) activity was measured by the method of beauchamp and fridovich (1971) [14] based on the aerobic reduction of nbt at 535 nm by superoxide radicals and expressed as units·mg-1 soluble protein; 1 unit of sod activity is defined as the amount of protein causing 50 % inhibition of the rate of nbt reduction. catalase (ec 1.11.1.6) activity was measured by monitoring decomposition of 10 mm h 2 o 2 according to aebi (1974) [15] at 240 nm (�=40 m-1·cm-1.) in buffer containing 50 mm kh 2 po 4 (ph 7.0) and approximately 150 �g of proteins. the results were related to soluble protein. total glutathione (gsht) concentration was quantified by the glutathione reductase recycling assay [16]. to estimate the oxidized glutathione (gssg) level, the protein free sample was treated with 2-vinylpyridine for 60 min prior to assay at 2 % final concentration [17]. the rate of 5-thionitrobenzoic acid formation was monitored spectrophotometrically at 412 nm. standards were prepared from gsh, and concentrations were defined as nmol per g wet weight. the redox-index of glutathione (ri gsh) was calculated as the ratio of concentrations [gshr]/[gsh]. evaluation of oxyradical formation in thyroid gland tissue (1/10 w/v) homogenates was determined using the non-fluorescent derivative, dihydrorh. falfushynska et al. 7 in t e r n a l m e d ic in e ijmmr 2015 vol. 1 no. 1 hodamine, which is converted to the fluorescent dye, rhodamine-123, while reacting with reactive oxygen species [18]. the fluorescence signal was detected by using af-max fluorescence plate-reader [excitation = 485 nm, emission = 538 nm] immediately, and in 20 min. glutathione-s-transferase (gst, ec 2.5.1.18) activity was measured by cdnb as the substrate [19]. enzymatic activity was determined at 25 °c by monitoring changes in absorbance at 340 nm for 2 min. the gst activity was defined as nmol min-1·mg-1 soluble protein. the activity of lactate dehydrogenase (ldh, ec 1.1.1.27) was determined using the uv assay with pyruvate and nadh [20] by determining of the amount of nadh oxidation at 340 nm. phosphate/ pyruvate solution (3 ml) (50 mm phosphate, ph 7.5, 0.63 mm pyruvate) was pipetted into cuvettes and 50 ml nadh solution (11.3 mmb-nadh) was added. following this, 100 ml of sample was spiked and mixed. the extinction was checked after very minute interval for a period of 4 min. a molar extinction coefficient of 6.22·106 m-1·cm-1 was used. dna fragmentation was evaluated by the determination of the levels of protein-free dna strand breaks in the digestive gland by the alkaline dna precipitation assay [21] using hoescht 33342. the reduction of possible interference of traces of sds in the supernatant was measured in the presence of 0.4 m nacl, 4 mm sodium cholate, and 0.1 m tris (ph 9). probe fluorescence signal was detected by using af-max fluorescence plate-reader [excitation = 360 nm, emission = 450 nm]. quantitation was done using known amounts of salmon sperm dna in the same mixture without addition of sample and determined as ng of protein-free dna per mg tissue soluble proteins. cathepsin d (ec 3.4.23.5) activity was assayed spectrometrically as described previously [22] in incubation mixtures containing buffered haemoglobin solution [4% (w/v) in 0.25m-sodium acetate buffer, ph5.0]. all measurements were carried out using samples from 25 patients. the results were defined as means ± standard deviation (sd). data were tested for normality and homogeneity of variance using kolmogorov-smirnoff and levene’s tests. since data were not normally distributed (lilliefors’ test), non-parametric tests (kruskall–wallis anova and mann–whitney u-test) were performed (significant at p<0.05). for detection of correlation, the pearson’s correlation test was also performed at a 0.05 level of significance. data were subjected to multiple regression analysis and principal component analysis (pca) with nipals algorithm to differentiate the group by the set of their indices and select distinguished criterions. classification trees were built using classification and regression tree (cart) software on the basis of all determined biological characteristics. all statistical calculations were performed by means of statistica v 8.0 and excel for windows 2000. results the results showed the coherent activation of superoxide dismutase (by 81 %), catalase (up to two times) and glutathione-transferase (by 212 %), decrease of gsh level (by 33 %) and the increase of metallothioneins level (both mt-sh and mt-me) in affected part of thyroid gland (fig. 1, 2). higher level of oxyradicals (by 21 %) and gssg (up to 41%) has been also detected in these samples. a relation between mt-sh and oxyradical level in thyroid gland was proved. signs of cytotoxicity, higher free cathepsin d activity (up to 84.6 % and 134.4 % in paranodular tissue and node respectively), and higher level of dna strand breaks in node (up to 22.6 %), were observed (fig. 3). also, activation of glycolysis in the affected part of thyroid gland was observed. the accumulation of reactive oxygen species (r = 0.72, p<0.01) and initiation of oxidative stress in the cell could be the reason for shifting of a b fig. 1. subcellular distribution of iodine (a) and copper (b) in the human thyroid gland under iodine deficiency euthyroid nodular goiter, µg×g-1 fw, mean ± sd (n=25). in fig. 1 – 4, the significant differences were regarded as (p < 0.05); a, differences compare to contralateral part; b, differences between paranodular tissue and nodule. h. falfushynska et al. 8 in t e r n a l m e d ic in e ijmmr 2015 vol. 1 no. 1 energy balance to anaerobiosis [23]. range of indices variability in paranodular tissue compared to parenchyma of contralateral part was lesser than in the nodular tissue, but had generally the same trend. the most important finding was lower level of organificated iodine (by 23 and 15 %, respectively) and higher level (by 46 and 32 %, respectively) and mass fraction (by 42% and 82% respectively) of inorganic iodine in the nodular and paranodular tissue versus contralateral part of thyroid gland (fig. 4). the disruption of iodine organification in hyperplastic thyrocytes could be caused by elevated copper level (more than twice) in thyroid gland. copper excess in the affected tissue partly accumulated in mts. the binding ability of mts with copper in the nodule was lesser than in the paranodular tissue. according to pca test with nipals algorithm (fig. 5, a), two sets were the most distinguished: nodule was characterized by oxidative stress indices, mts characteristics, and copper distribution, whereas non-affected contralateral part was located jointly with organificated iodine and gsh level. there was no clear characteristic related to the paranollar tissue. furthermore, it was important to select the main distinguished criterion for pathological process progress at the nodule formation. we used cart analysis (fig. 5, b) to achieve this. followed by analysis of all biochemical parameters of every group, the contralateral part of thyroid gland was singled out by its ability to iodine organification. paranodular tissue and nodule in affected part of thyroid gland were distinguished by level of dna fragmentation. discussion it is generally known that nodules develop in pathologically affected thyroid tissue [2]. we established that hyperplastic thyroid epithelium under iodine deficiency nodular colloidal goiter sacrificed its ability for iodine organification and therefore increased of level of inorganic iodine. this pattern designated with higher copper level (r = 0.69, p<0.01), manifestation of oxidative damage (increased of sod, catalase and gst activity (r = 0.73, r = 0.59 and r = 0.64 correspondingly, p<0.01 and mt-sh level (r = 0.97, p<0.001)) and cytotoxicity (increased dna fragmentation, r = 0.51, p<0.01) in affected by nodule part of thyroid gland. therefore, it can be assumed that under deprivation of iodine organification surplus of it has been created in human thyroid gland and determined further formation of iodine toxic intermediates in hyperplastic thyrocytes after its oxidation by thyroperoxidase [24]. this scenario of a stimulating effect on the antioxidant defense system was proved. similar results, such as increased lipofuscin level, lipid peroxidation, necrosis of epithelial cells, and destabilization of mitochondrial membranes and development of autoimmune processes in the tissue of the thyroid gland were obtained after iodine application in micromolar range into human body [25]. it was proved that apoptosis was among the major determinants of pathological conditions progression [24]. cathepsin d is a lysosomal a b c d fig. 2. oxidative stress parameters in the human thyroid gland under iodine deficiency euthyroid nodular goiter. data for a: superoxide dismutase, b: catalase, c: glutathione transferase, d: oxyradical level are present as means ± sd (n=25). h. falfushynska et al. 9 in t e r n a l m e d ic in e ijmmr 2015 vol. 1 no. 1 fig. 3. characteristics of metallothioneins (a-c) and glutathione (d-f) in the human thyroid gland under iodine deficiency euthyroid nodular goiter. concentration of mt-sh (a); concentration of mt-me (b); mt-sh/mt-me concentration ratio (c); levels of reduced glutathione (d), oxidized glutathione (e) and redox index of glutathione (ri gsh) (f). the values are expressed as the mean ± sd (n=25). a b c d e f endopeptidase. it involves in the processing of thyroglobulin, and belongs to mediators of ifn-� and tnf-�-induced lysosomal programmed cell death pathway [26]. we were observing its increased level in patients with endemic nodular goiter. it was related to the increase of copper level in the thyroid tissue (r=0.63, p <0.01), and particularly, in potentially toxic, unbound with mts form (r=0.67, p<0.01). we can speculate that copper accumulated in lysosomes and consistently caused their swelling, activation of calcium-dependent phospholipase a2 [27], and determined destabilization of lysosomal membranes. the in vivo release of the enzymes from the organelles, which happened consequently, has been suggested to play a fundamental role in mediating caspase activation, dna fragmentation and apoptosis [28]. recently, it has been shown in vitro that mts and gsh form a part of the pool of cellular thiols and their function in the cell is cohered [29]. we can conclude the same regulation in human thyroid tissue (r=-0.79, p<0.001) based on our observations. mts might act as an effective scavenger against reactive oxygen species due to high level of sh-group under weakness of glutathione functional ability, which could indicate a compensatory mechanism. despite the fact that the mts level in the cell is lower than gsh, they have exhibited 50x higher antioxidant activity by the molar ratio of thiols[30]. we detected a reverse relation for the mt-sh level and oxyradicals level, which indicated a leading role of mts in detoxification of oxyradicals: oxyradicals=-268.4+88.2�gssg–4.7�gsh+405.5�ri gsh+6.4�gst–2.1�mt-sh*; r2=0.45, f(5,12)=4.7, p<0.03 (* – indicated significant contribution into the mathematical model). similar results were obtained by comparing of goitrous-changed and intact human thyroid gland tissue [4]. conclusions to summarize, the combination of endemic iodine deficiency with a high environmental copper level increases the risk of node formation and progress of pathological changes. at low level of h. falfushynska et al. 10 in t e r n a l m e d ic in e ijmmr 2015 vol. 1 no. 1 fig. 4. markers of cytotoxicity in the human thyroid gland under iodine deficiency euthyroid nodular goiter. data for a: dna strand breaks, b: cathepsin d; c: lactate dehydrogenase are present as means ± sd (n=25). a b c ��� ���� ��� ��� ��� ��� �� �� ���� ��� ��� ���������� �������� ����������������� ������ ������� �� �� ��� ���� ���� ���� ���� ���� ���� ��� ��� ��� ��� ��� ��� ��� � ���� ���� ���� ���� ���� ���� ��� ��� ��� ��� ��� ��� ��� � � ��� ���� ��� ��� ��� ��� �� �� ���� ��� ��� ���������� �������� ����������������� ������ ������� �� �� ��� fig. 5. principal component analysis with nipals algorithm (a) and classification tree models using cart algorithm (b) of total data from human thyroid gland under iodine deficiency euthyroid nodular goiter depending on deepness of pathology processes. data for b showing node types, split variables, and associated split values of all attributes of tissues. �������� � �� � �������������� ����� �������� ���� � � � � � �������� � �� ���� � �� �� �� �� �� �� �� �� � �� �� � a b h. falfushynska et al. 11 in t e r n a l m e d ic in e ijmmr 2015 vol. 1 no. 1 iodine organification and high copper level in goitrous tissue of thyroid gland, metallothioneins may provide a partial compensatory effect on prooxidative processes. the effects on the secretion of the cathepsin d can depend on the accumulation of copper inside thyrocyte lysosomes. nodule formation in human thyroid gland has been followed by disruption of iodine organification by thyrocytes (general characteristic of affected by nodule tissue) and increased level of dna fragmentation (specific character of the nodule). acknowledgements this work has been granted 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10.11603/ijmmr.2413-6077.2016.1.6379 enzyme markers activity and bile formation function of liver in cases of tuberculostatics and hexavalent chromium compounds affection in rats n. i. burmas, l. s. fira, p. h. lyhackyy i. horbachevsky ternopil state medical university, ternopil, ukraine background. currently, the growing incidence of toxic lesions of the liver is associated with industrial chemicalization and uncontrolled use of hepatotoxic drugs in everyday life. there are about one thousand drugs with high or low hepatotoxicity, such as anti-tb drugs. objective. in this research we studied the intracellular enzymes activity and bile formation function of the liver in rats of different ages in cases of tuberculostatic (isoniazid and rifampicin) affection and chromium (potassium dichromate) intoxication. methods. the experimental affection of rats of different ages was performed by combined injection of hexavalent chromium compounds (a solution of potassium dichromate, 3 mg/kg), isoniazid (0.05 g/kg) and rifampicin (0.25 g/kg). on the 7th and 14th days the rats were injected with enterosorbent sorbex (150 mg/kg). enzyme markers activity of the liver was evaluated due to alanine and aspartate aminotransferases (alt and ast) and alkaline phosphatase (alp) rates. bile formation function of the liver was evaluated by total bilirubin and bile acids content in blood. results. the disorders in hepatocytes plasma membranes permeability were defined by the increased rates of alt, ast and alkaline phosphatase in blood serum which were decreased in the liver. it was determined that total bilirubin and bile acids content in blood serum of the affected animals increased. it influenced hepatocytes excretion in bile capillaries and caused cholestasis and revenues decrease in bile. conclusions. the most significant metabolic disorders in cases of chrome-isoniazid-rifampicin affection were defined in immature and senior animals in comparison with mature animals. key words: isoniazid, rifampicin, hexavalent chromium compounds, liver enzymes, bile formation. n. i. burmas et al. address for correspondence: nataliya burmas, department of general chemistry, i. horbachevsky ternopil state medical university, 1 maidan voli, ternopil, ukraine, 46001 tel.: +380976123434 e-mail: burmas@tdmu.edu.ua introduction considering the role of liver in the chemical compounds metabolism we can assert that there are no drugs that in certain conditions would not cause impairment. more and more information about their hepatotoxic effect indicates that medical affection of the liver is one of the major problems of hepatology [1–3]. antituberculosis drugs are of great interest [4–6]. according to the centre of monitoring of adverse reactions of drugs (2007), isoniazid – 29.2%, rifampicin – 26.7%, capreomycin – 17.1%, ethambutol – 10.2% dominate among monopreparations in high incidence of the adverse reactions in world [5]. thus, the risk of the development of hepatitis increases in patients who take rifampicin together with isoniazid. in this case hepatitis incidence is 5-8%. during the isoniazid monotherapy, the incidence of hepatitis is 1.2%, but during the rifampin monotherapy – 0.3% [4]. according to the researches [7, 8], the hepatotoxicity of isoniazid may be developed in two ways: 1. the accumulation of free radicals with activation of lipid peroxidation and the formation of reactive metabolites: acetylisoniazid, hydrazine, monoacetylhydrazine; 2. the increased activity of n-acetylisoniazid by n-hydroxylation and formation of acetyl radical and acetyl carbonium ion. the metabolism of acetyl hydrazine and microsomal monooxygenases cause hepatotoxic effect as a result of the covalent addition of international journal of medicine and medical research 2016, volume 2, issue 1, p. 32–38 copyright © 2016, tsmu, all rights reserved 33 b io m e d ic a l s c ie n c e s issn 2413-6077. ijmmr 2016 vol. 2 issue 1 acetyl groups to the liver proteins [7], which manifest as temporary asymptomatic increase of transaminases activity. the hepatotoxicity of rifampin is also due to the formation of toxic metabolites as a result of its deacetylation in the liver that leads to hepatocytes dystrophy [9, 10]. however, increasing saturation of the environment by the compounds of heavy metals leads to the accumulation of these compounds in human bodies, of those who live on the contaminated areas from early childhood [11–13]. according to some studies, in the future the compounds of heavy metals as the threat for the environment can become the urgent matter, leaving nuclear stations wastes and organic anthropogenic pollution behind [13]. today chromium (vi) is recognized by the international agency of the cancer researches of the european union to be one of six chemical elements (arsenic, beryllium, cadmium, cobalt, nickel and chromium) that reveal the carci nogenic effect on human body [14, 15]. the influence of the compounds of chromium (vi) is accompanied by toxic effects and serious internal organs (kidneys, lungs, liver) da mage. but, the recent studies do not provide full concept of the of liver enzymes activity and its influence on bile formation function in cases of combined effect of hexavalent chromium compounds on age-dependent isoniazid-rifampicin destruction of the body, which is presented in this research. materials and methods the experiments were conducted on outbred white male rats of three age groups: the 1st group – immature (3-month-old animals, 90–110 g in weight); 2nd group – mature (6-month-old animals, 150–170 g in weight); and the 3rd group – senile (18-months-old animals, 280–300 g in weight). the rats were kept on a standard diet at the vivarium of ter nopil state medical university. the manipulations on the animals were carried out according to the article 26 of the law of ukraine “on protection of cruelty to ani mals” from 21.02.2006. no. 3447-iv, “european convention on protection of vertebrate animals used for experimental and other scientific purposes”, “general ethical principles of the experiments on animals", approved 20.09.2001 during the 1st ukrainian national con gress on bioethics considering regulations of nih guide on care and use of laboratory animals [16]. the experimental toxic affection of the animals was simulated by combined effect of hexavalent chromium compounds, isoniazid and rifampicin. hexavalent chromium compounds were administered intragastrically to animals every day for one group by the solution of potassium dichromate, 3 mg/kg. for another group on the 7th and 14th days isoniazid and rifampicin by metallic probe in aqueous solution, 0.05 g/kg and 0.25 g/kg accordingly, were administered intragastrically. for combined action, the xenobiotics mentioned above were administered in the same doses. euthanasia was performed by means of thiopental sodium on the 7th and 14th days from the first day of xenobiotics administration. the study of liver homogenate and blood serum was performed. the blood was taken from the heart of animals by centrifugation at 3000 rev/ min during 30 min. the obtained blood serum, sedimentary liquid, was used for researches. liver (250 mg) was put into 10% homogenate and different homogenisation methods were used after previous perfusion in physiological solution. the activity of liver enzyme markers was determined by the rate of aminotransferases (alt and ast) and an alkaline phosphatase (alp) (the reagents of ooo npp filisitdiagnostics, ukraine) in blood serum and liver homogenate. the evaluation of alt rate was conducted by compound of 2-oxoglutaric acid and l-alanine, which under the influence of alanine aminotransferase formed l-glutamic and pyruvic acids. the interaction of pyruvic acid and 2.4-dynitrophenylhydrazine in alkaline medium formed 2.4-dynitrophenylhydrazones that had high coefficient of the molar extinction, so its optical density registered on the fec was directly proportional to the activity of the enzyme. the enzyme activity was estimated by the calibration graph due to the content of pyruvic acid in mkmol/(l∙h) [17]. ast rate was evaluated by optical density measuring of 2.4 nitro phenylhydrazones of 2-oxoglutaric and pyruvic acids in alkaline medium. hydrazone of pyruvic acid has a higher coefficient of the molar extinction, so there is a directly proportional relationship of optical density of the reaction solution to the enzyme activity. the enzyme activity was evaluated by the calibration graph due to the content of pyruvic acid, mkmol/(l∙h) [17]. estimation of the alkaline phosphatase rate was based on the property of the enzyme to hydrolyse the etheric bond in β-glyceron. i. burmas et al. 34 b io m e d ic a l s c ie n c e s issn 2413-6077. ijmmr 2016 vol. 2 issue 1 phosphate and eliminate the phosphoric acid. phosphorus was determined by colorimetric method due to the reaction with molybdenum reagent in the presence of a reducing eikonogen or ascorbic acid. the product of reaction was molybdenum blue; its colour intensity was directly proportional to the amount of phosphorus in the simple evaluation of the enzyme activity [18]. the bile formation function of the liver in the animals was defined by the content of total bilirubin and bile acids in blood serum. the content of total bilirubin was determined by caffeine reagent, which together with diazotized sulphanilic acid formed azobilirubin of pinkpurple colour. the colour intensity of this solution was directly proportional to the concentration of total bilirubin in the sample. evaluation of total bilirubin in blood serum was performed by the calibration graph, mmol/l [18]. determination of bile acids content was based on the reaction of colour products formation by condensation, which interacted with bile acids and oxymethyl furfural. these solutions were obtained from fructose. they are the products of hydrolysis by adding concentrated sulfuric acid to sucrose. bile acids content was evaluated by the calibration graph due to the tauroholic acid content, g/l [18]. the statistical processing of the results was performed on a pc by means of programs "microsoft excel" and "statistica 6.0" on the basis of arithmetic middling and errors according to student's t-test [19]. the changes were considered to be reliable at p≤0.05. results we noted the increased alt rate (table 1) in the blood serum of animal groups of all ages. it was the highest in rats affected by tuberculostatics and potassium dichromate. on the 14th day of xenobiotics administration the rate increased in 2.9 times in immature animals in comparison with the control group, in 2.5 times – in mature animals and in 3.2 times – in senior animals. the mature animals, which were affected by hexavalent chromium compounds, proved to be the least sensitive. their affection rate exceeded the normal range in 2.3 times. in the immature rats the alt rate increased by 120% on the 7th day of the research and by 145% – on the 14th day of the affection by isoniazid and rifampicin, in the mature animals this rate exceeded the level of the intact control by 30% on the 7th day and by 128% till the end of the experiment. the senior animals were more sensitive to anti-tb drugs. alt rate in blood serum increased by 181% on the 14th day after the affection. the most significant changes were observed in the liver of the senior animals which underwent the aforementioned xenobiotics table 1. alanine aminotransferase rate in blood serum (mkmol/l·h) and liver (mkmol/kg·h) of rats affected by isoniazid, rifampicin and hexavalent chromium compounds, (m±m) research material group of animals age group of animals immature mature senior research duration, days 7th 14th 7th 14th 7th 14th blood serum intact control, n=6 0.83±0.05 2.96±0.18 2.34±0.15 affected by potassium dichromate, n=6 1.51± 0.10* 1.93± 0.16* 4.03± 0.32* 6.66± 0.17* 3.75± 0.36* 6.16± 0.37* affected by isoniazid and rifampicin, n=6 1.83± 0.11* 2.03± 0.20* 3.84± 0.24* 6.74± 0.26* 4.44± 0.32* 6.58± 0.30* affected by potassium dichromate, isoniazid and rifampicin, n=6 2.21± 0.12* 2.38± 0.14* 4.69± 0.19* 7.46± 0.42* 5.87± 0.30* 7.57± 0.26* liver intact control, n=6 5.40±0.12 8.30±0.21 6.08±0.33 affected by potassium dichromate, n=6 3.96± 0.26* 2.97± 0.16* 6.55± 0.23* 3.96± 0.26* 2.97± 0.16* 6.55± 0.23* affected by isoniazid and rifampicin, n=6 3.68± 0.20* 2.83± 0.11* 6.48± 0.27* 3.68± 0.20* 2.83± 0.11* 6.48± 0.27* affected by potassium dichromate, isoniazid and rifampicin, n=6 3.18± 0.22* 2.42± 0.12* 5.78± 0.31* 3.18± 0.22* 2.42± 0.12* 5.78± 0.31* note: here and in the following tables * – significant differences between the animals of intact controls and the affected animals, р≤0.05. n. i. burmas et al. 35 b io m e d ic a l s c ie n c e s issn 2413-6077. ijmmr 2016 vol. 2 issue 1 compounds. alt rate decreased till the end of the experiment by 58% (p≤0.05) in this group, when in the immature group – by 55%, in the mature one – by 45% in comparison with animals of the intact control. in blood serum of the experimental animals, affected by k2cr2o7, the ast rate was gradually increasing and reached the highest rate at the end of the experiment by 251% in the immature animals, by 181% – in the mature animals and 267% – in the senior animals if compared to the control group of animals (table 2). in the immature and mature rats ast rate increased in 3.0 and 2.1 times accordingly in comparison with the control group. during the research the decrease in ast rate in liver of the affected animals of all age groups was determined (table 2). in the immature animals the activity of this enzyme decreased by 17% on the 7th day of the research after the administration of k2cr2o7, in the ma ture animals – by 33% and in the senior ani mals – by 39%. it was proved that the accumu lation of heavy metals, which got into the body of animals from the environment, took place in hepatocytes [11, 12]. we studied alkaline phosphatase rate in blood serum and liver of the affected rats (table 3). it was established that this enzyme is a marker of liver disorder and indicates the inflammation in it. the administration of potassium dichromate into the body of the immature animals caused the increase of alkaline phosphatase rate in blood serum in 1.3 times on the 7th day of the experiment, in the mature animals – in 1.5 times and in the senior animals – in 1.3 times, that caused toxic affection of liver. these changes were significant (р≤0.05). we evidenced the highest rate of alkaline phosphatase in blood serum of the immature animals after combined effect of potassium dichromate, isoniazid and rifampicin, which was 100% on the 7th day after affection and 127% – on the 14th day in comparison with the animals of the intact control. in liver of the affected animals the alp rate decreased during the experiment in all experimental groups (table 3). the lowest rate of alkaline phosphatase was on the 14th day of the research in the mature animals after the combined effect of xenobiotics (772.86±29.62) nmol/(s·g) that is in 1.7 times lower than in the control group (1338.23±54.21 nmol/(s·g). we evidenced a significant increase (р≤0.05) in total bilirubin content in blood serum of the animals of all age groups in comparison with control rats (table 4). on the 14th day of the experiment the total bilirubin content increased by 53% in the immature animals, by 40% in the mature ones and by 28% in the senior rats after the affection with anti-tb drugs in comparison with the animals of the intact control. we evidenced the highest content of total bilirubin at the end of the research in the mature animals after combined administration of k2cr2o7, isoniazid and rifampicin, which was 206% in comparison with intact animals that is table 2. aspartate aminotransferase rate in blood serum (mkmol/l·h) and liver (mkmol/kg·h) of rats affected by isoniazid, rifampicin and hexavalent chromium compounds, (m±m) research material group of animals age group of animals immature mature senior research duration, days 7th 14th 7th 14th 7th 14th blood serum intact control, n=6 0.70±0.06 0.64±0.04 1.05±0.20 affected by potassium dichromate, n=6 0.94± 0.04* 1.76± 0.08* 0.93± 0.05* 1.16± 0.04* 2.04± 0.14* 2.80± 0.09* affected by isoniazid and rifampicin, n=6 1.05± 0.05* 1.84± 0.10* 1.10± 0.05* 1.21± 0.06* 2.61± 0.09* 3.40± 0.14* affected by potassium dichromate, isoniazid and rifampicin, n=6 1.21± 0.07* 2.07± 0.14* 1.19± 0.06* 1.34± 0.08* 3.22± 0.14* 3.85± 0.18* liver intact control, n=6 1.21±0.11 2.66±0.06 2.48±0.12 affected by potassium dichromate, n=6 1.01± 0.05 0.91± 0.02* 1.79± 0.06* 1.44± 0.05* 1.52± 0.04* 1.25± 0.04* affected by isoniazid and rifampicin, n=6 1.02± 0.04 0.86± 0.03* 1.66± 0.10* 1.34± 0.07* 1.45± 0.04* 1.18± 0.03* affected by potassium dichromate, isoniazid and rifampicin, n=6 0.87± 0.03* 0.75± 0.04* 1.45± 0.10* 1.25± 0.06* 1.34± 0.04* 1.13± 0.04* n. i. burmas et al. 36 b io m e d ic a l s c ie n c e s issn 2413-6077. ijmmr 2016 vol. 2 issue 1 by 13% and 20% higher than in the immature and senior animals, respectively. the results of the research on the bile acids content in blood serum of the rats of all age groups are presented in table 5. in animals, which potassium dichromate was administrated to, the content of bile acids increased in all age groups (р≤0.05). on the 7th day of the research this rate increased by 35% in the immature animals, by 53% – in the mature animals and by 57% – in the senior animals if compared with the animals of the intact control. during the investigation of blood serum of the animals, which anti-tb drugs were administrated to, we evidenced a significant increase in bile acids content in the immature animals in 1.42 and 1.71 times on the 7th and 14th days of the research, respectively. on the 7th day in the mature and the senior animals the bile acids content increased in 1.54 and 1.76 times; on the 14th day – in 1.50 and 1.80 times, respectively, in comparison with the animals of the intact control. discussion it is established, that aminotransferases catalyze the reaction of transamination between the aminoand α-keto acids, take part in synthesis and forming of body proteins. the increasing activity of blood plasma enzymes, such as alt and ast, demonstrates the level of hepatocytes damage and indicates liver disorders. due to this thesis, we studied aminotransferase rate in blood serum and liver of the rats of different age groups affected by xenobiotics. in the liver of the rats after combined administable 3. alkaline phosphatase rate in blood serum (nmol/s·l) and liver (nmol/s·g) of rats affected by isoniazid, rifampicin and hexavalent chromium compounds, (m±m) research material group of animals age group of animals immature mature senior research duration, days 7th 14th 7th 14th 7th 14th blood serum intact control, n=6 1924.64±113.32 2405.80±159.13 3007.25±240.58 affected by potassium dichromate, n=6 2766.67± 159.13* 3518.48± 227.04* 3488.41± 206.60* 3849.28± 184.40* 3939.49± 205.29* 4360.51± 225.44* affected by isoniazid and rifampicin, n=6 3247.83± 192.09* 3849.28± 206.60* 4210.15± 159.13* 4540.94± 142.96* 4330.43± 208.35* 4781.52± 137.81* affected by potassium dichromate, isoniazid and rifampicin, n=6 3849.28± 159.13* 4360.51± 182.92* 4691.30± 104.17* 4751.45± 144.85* 4961.96± 137.81* 5352.90± 159.13* liver intact control, n=6 739.78±30.90 1338.23±54.21 1705.11±74.21 affected by potassium dichromate, n=6 634.53± 20.53* 610.47± 14.30* 1004.42± 50.54* 878.12± 33.16* 1377.32± 41.84* 1305.14± 19.02* affected by isoniazid and rifampicin, n=6 562.35± 19.44* 496.20± 20.17* 941.27± 38.82* 836.01± 35.68* 1341.23± 30.79* 1235.14± 16.64* affected by potassium dichromate, isoniazid and rifampicin, n=6 514.24± 21.22* 457.10± 13.72* 878.12± 20.66* 772.86± 29.62* 1205.91± 26.93* 1106.67± 22.18* table 4. total bilirubin content in blood serum (mkmol/l) of rats affected by isoniazid, rifampicin and hexavalent chromium compounds, (m±m) group of animals age group of animals immature mature senior research duration, days 7th 14th 7th 14th 7th 14th intact control, n=6 12.19±0.55 12.49±0.47 15.09±0.78 affected by potassium dichromate, n=6 14.82± 0.98 17.97± 1.13* 14.10± 0.91 16.53± 1.17* 19.25± 1.27* 20.97± 1.61* affected by isoniazid and rifampicin, n=6 13.38± 0.69 18.68± 1.50* 16.53± 1.17* 17.53± 1.61* 18.97± 1.12* 19.25± 1.27* affected by potassium dichromate, isoniazid and rifampicin, n=6 16.97± 1.62* 23.53± 1.61* 23.10± 1.55* 25.67± 1.28* 23.82± 1.09* 28.08± 1.44* n. i. burmas et al. 37 b io m e d ic a l s c ie n c e s issn 2413-6077. ijmmr 2016 vol. 2 issue 1 tration of toxicants we evidenced decrease in this enzyme rate, which indicated the cytolysis of hepatocytes and liver protein synthesis dysfunction in cases of affection by tuberculostatics and hexavalent chromium compounds. it was determined that the increase in alt rate in blood serum was higher than in liver. it could be caused by toxic influence of potassium dichromate with underlying isoniazidrifampicin affection of liver. the damaged liver cannot synthesize this enzyme because of hepatocytes damage and their release in blood serum; so we evidenced increased rate of this enzyme. the determination of aminotransferase rate in blood serum is a sound indicator of the level of pathological process in liver. so we investigated ast rate. ast rate in blood serum increased and was the highest on the 14th day of the research (in 3.7 times higher than the normal range) in the senior animals affected by chrome-isoniazid-rifampicin. the decrease in ast rate in liver proved the transamination of aspartate according to breaking of the citric acid cycle and negligible release of protein enzyme from tissue cells into blood. it was determined that increase in alkaline phosphatase rate in blood serum of the rats of all age groups after the administration of the investigated toxins into their bodies cause the release of alp out of the damaged hepatocytes as well as the restoration of its synthesis in bile tubules. we consider that this dynamic activity of alkaline phosphatase may evidence the development of hepatocytes destruction and intrahepatic cholestasis caused by liver architectonics damage and possible development of cirrhosis. it was established that hepatotoxicity of the metabolites of isoniazid and rifampicin caused the lipid peroxidation of hepatocyte biomembranes and bile formation dysfunction. rifampicin can also inhibit the glucuronil-trans aminases and cause bilirubin metabolism disorders and jaundice [10]. so, we had to examine total bilirubin content in blood serum of the animals affected by isoniazid, rifampicin and hexavalent chromium compounds. increase in total bilirubin content under the influence of the toxicants evidenced the damage of cell membranes and erythrocytes predominantly and decrease in haemolysis as well as liver excretory dysfunction. it was established, that in case of druginduced hepatitis the intestines and liver suffer from the affection, which was caused by the disorders of biosynthesis and hepatoenteral circulation of bile acids. after the combined administration of toxins (potassium dichromate, isoniazid and rifampicin) into the animals’ bodies we evidenced that the highest level of bile acids content in immature rats was at the end of the experiment, 239%, in comparison with the animals of the intact control. the increase in bile acids content in blood serum of the affected animals may have the toxic effect on hepatocyte mitochondria that caused increase in ions permeability to internal membrane of mitochondria, ions swelling and release of cytochrome c into cytosol as well as cells apoptosis. the immature animals were the most sensitive to bile formation after administration of tuberculostatics and k2cr2o7, although the increase in bile acids content was evidenced in the mature and senior animals. conclusions we determined the increase in aminotransaminases and alkaline phosphatase rate in blood serum and their decrease in liver. it proved the toxic effect of hexavalent chromium compounds and tuberculostatics on the body of animals of all ages. it caused the disorder of table 5. bile acids content in blood serum (g/l) of rats affected by isoniazid, rifampicin and hexavalent chromium compounds, (m±m) group of animals age group of animals immature mature senior research duration, days 7th 14th 7th 14th 7th 14th intact control, n=6 6.95±0.43 9.48±0.58 12.02±0.64 affected by potassium dichromate, n=6 9.36± 0.68* 11.16± 0.62* 14.50± 0.50* 16.37± 0.67* 18.33± 0.98* 20.90± 0.95* affected by isoniazid and rifampicin, n=6 9.90± 0.49* 11.89± 0.55* 14.60± 0.81* 16.69± 0.65* 18.09± 0.87* 21.59± 0.84* affected by potassium dichromate, isoniazid and rifampicin, n=6 14.26± 0.55* 16.61± 0.43* 18.23± 0.75* 21.37± 0.87* 22.42± 0.81* 24.16± 0.95* n. i. burmas et al. 38 b io m e d ic a l s c ie n c e s issn 2413-6077. ijmmr 2016 vol. 2 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інфекційні хвороби 2002; 2: 104–106. 11. трахтенберг ім, короленко тк, коршун мм. експериментальне вивчення впливу важких металів на організм тварин різних вікових груп. гигиена труда 2004; 35: 158–170. 12. duffus jh. heavy metals – a meaningless term? pure and applied chemistry 2002; 74: 793– 807. 13. recommended health-based limits in occupational exposure to heavy metals. geneva: who, 1980: 205. 14. hantson р, caenegem о, decordier і. hexavalent chromium ingestion: biological markers of nephrotoxicity and genotoxicity. clin. toxicol. (phila) 2005; 43: 111–112. 15. maeng sh, chung hw, kim kj. chromosome aberration and lipid peroxidation in chromiumexposed workers. biomarkers 2004; 9: 418–434. 16. guide for the care and use of laboratory animals: eighth edition. the national academies press, washington, dc. – 2011. 17. reitman s, frankel s. definition of biochemical indicators of the toxicity of liver. amer j clin. path 1957; 28: 56–60. 18. гонський яі, саяк нп, рубіна лм. біологічна хімія. лабораторний практикум. тернопіль. укрмедкнига; 2001: 287. 19. лапач сн, чубенко ав, бабич пн. статистические методы в биологических исследованиях с использованием excel. київ. морион; 2000: 320. received: 2015-12-28 n. i. burmas et al. 56 b io m e d ic a l s c ie n c e s issn 2413-6077. ijmmr 2016 vol. 2 issue 2 doi 10.11603/ijmmr.2413-6077.2016.2.7030 damaging effect of immobilizing stress on hypoxia high and low-resistant rats of both sexes iu. m. ordynskyi, o. v. denefil i. horbachevsky ternopil state medical university, ternopil, ukraine background. cardiovascular morbidity is a topical issue; stress is an essential contributing factor. pathogenic links in damaging stress impact on the animal units of different reactivity is promising in disease prevention and development of individual correction methods. objective. stress-induced development of cardiovascular pathology is undeniable, the stress impact depending on individual systemic response, age and sex. methods. 96 hypoxia highand low-resistant (hr and lr, respectively) wistar rats aged 5.5-6 months were used in experiments. changes of lipid peroxidation processes, as well as protein oxidative modification, nitrite anion content and the indices of antioxidant defence under immobilizing stress were studied in the research. results. immobilizing stress causes the development of oxidative and carbonyl stress in hr and lr rats that is more pronounced in lr group; and the activation of antioxidant defence system. in males, stress development is concomitant with increased catalase activity as well as that of blood peroxidase, ceruloplasmin and reduced glutathione content, whereas increased catalase and ceruloplasmin activity has been found in hr females, and that of superoxide dismutase and ceruloplasmin — in lr group. as compared with the females, more intensive oxidative and nitroxidative stress, protein oxidative modification, and stress-related accumulation of circulating immune complexes have been found in the males. conclusions. the most intensive oxidative and nitroxidative stress, protein oxidative modification, and stress-related accumulation of circulating immune complexes have been found in the hypoxia low-resistant males. key words: immobilizing stress, resistance to hypoxia, heart, damage. introduction stresses are common in daily life and work [1], the long-lasting ones being the most dangerous. the same stressor impacts the people of various sex and age differently [2], the aftermath depends on a person’s reactivity, nervous and endocrine regulation, immune system, resistance to surroundings, etc. cardiovascular morbidity is a topical issue; stress is an essential contributing factor [3]. pathogenic links in damaging stress impact on the animal units of different reactivity is promising in disease prevention and development of individual correction methods. therefore, the research was aimed to determine the lipid peroxidation changes, nitrite anion and protein oxidative modification products content as well as of the antioxidant defence in heart of stress-exposed hr and lr rats of both sexes. material and methods 96 hypoxia highand low-resistant (hr and lr, respectively) wistar rats aged 5.5–6 months were used in the research. the animals were divided into groups: control and experimental (exposed to chronic stress), each group comprising 12 males and 12 females. animal units with different resistance to hypoxia were separated in accordance with v. ya. berezovskiy method (1978) [4]. stress was simulated by immobilization of the rats in supine position (1 hour/4 times, stress episodes at 72-hour interval). 24 hours after the 4th fixation the ani­ mals were withdrawn from the procedure [5]. experiments were performed in a special room within the morning at 18–22 °с, relative humidity 40–60 % and illumination250 lux. the regulations of the european convention for the protection of vertebrate animals used for experimental and other scientific purposes (strasbourg, 18.03.1986), resolution of the i national congress on bioethics (kyiv, 2001) and the order of the ministry of health of ukraine № 690 of 23.09.2009, were strictly followed. corresponding author: olha denefil, department of pathological physiology, i. horbachevsky ternopil state medical university, 1 maidan voli, ternopil, ukraine, 46001 phone number: +380352431262 e-mail: denefil@tdmu.edu.ua international journal of medicine and medical research 2016, volume 2, issue 2, p. 56–62 copyright © 2016, tsmu, all rights reserved iu. m. ordynskyi et al. 57 b io m e d ic a l s c ie n c e s issn 2413-6077. ijmmr 2016 vol. 2 issue 2 the rats were euthanized by total heart bloodletting and previous thiopental sodium anaesthesia (60 mg.kg-1 b.w. intraperitoneally). blood and heart samples were taken for the research. concentration of diene conjugates (dc), triene conjugates (tc), schiff bases [6], tba-active products [7]; indices of protein oxidative modification (pom 370 and pom 430) [8], nitrite anion [9], circulating immune complexes (cic) [10], superoxide dismutase (sda) [11], catalase [12] activity, and ceruloplasmin (cp) content [13] were determined in heart homogenate, whereas peroxidase blood activity (pba) [14], reduced glutathione (rgl) [15], as well as glutathione peroxidase and glutathione reductase activity[16] were determined in blood serum. statistical analyses of digital data was performed by department of systemic statistical research of the university by means of excel (microsoft, usa) and statistica 6.0 (statsoft, usa). reliability of the value difference between independent quantities was determined by student t-test at normal distribution and by nonparametric methods in other cases. results and discussion lower lipid peroxidation activity was revealed in the control hr males as compared with the lr animals, that was confirmed by 6.78% lower (p<0.001) indices of tba-active products (table 1). under chronic stress, the significant increase of primary and intermediate lipid peroxidation products: dc — in 2.26 times (p<0.001), tc — in 3.56 times (p<0.001), tbaactive products — in 4.45 times (p<0.001), whereas schiff bases decreased by 32.32% (p<0.001) was revealed the hr group. in the lr group the dc value increased in 5.99 times (p<0.001), tc — in 6.80 times (p<0.001), tbaactive products — in 5.81 times (p<0.001), and schiff bases decreased by 22.84% (p<0.001). as compared with the lr group, the hr group presented lower lipid peroxidation values: dc — in 2.73 times (p<0.001), tc — in 1.99 times (p<0.001), tba-active products — in 1.39 times (p<0.001), and schiff bases — by 9.35% (p<0.001). this data evidenced progressing oxidative and carbonyl stress in the hr and lr male rats that was more pronounced in the last group. as compared with lr group, higher activity of primary and final lipid peroxidation products (dc — by 14.38%, p<0.001, schiff bases — by 31.12%, p<0.001) and lower concentration of tba-active products (by 8.01%, p<0.001) were re vea led in the control hr females. the data pro ved more intensive lipid peroxidation pro cess in the intact hr females, probably due to more potent activity of antioxidant and endo crine system. the former is confirmed by the fact that in hr females tc values were by 14.67% (p<0.001) higher and schiff bases values — by 22.12% (p<0.001) lower as compared with the hr males. table 1. stress-related changes of lipid peroxidation indices in hr and lr animals of both sexes м±m (n=12) group index diene conjugates, standard unit. g.-1 triene conjugates standard unit. g.-1 тba­а active products mcmol/kg schiff bases standard unit males hr control 0.975±0.021 0.994±0.013 0.906±0.012 1.413±0.100 stress 2.209±0.057* 3.537±0.103* 3.537±0.103* 0.956±0.009* lr control 1.005±0.010 0.994±0.013 0.967±0.006** 1.354±0.055 stress 6.025±0.794*,** 7.039±0.701*,** 5.625±0.163*,** 1.044±0.019*,** females hr control 0.952±0.024 1.140±0.032# 0.899±0.002 1.100±0.016# stress 1.023±0.001*,# 0.972±0.005*,# 1.319±0.008*,# 1.043±0.006*,# lr control 0.861±0.006**,# 0.966±0.017# 0.971±0.005** 0.932±0.016**,# stress 1.027±0.013*,# 1.074±0.006*,**,# 1.865±0.014*,**,# 1.038±0.003* notes: * — reliable indices as compared with the controls; ** — reliable indices as compared with the hr; # — reliable indices as compared with the males of corresponding group. iu. m. ordynskyi et al. 58 b io m e d ic a l s c ie n c e s issn 2413-6077. ijmmr 2016 vol. 2 issue 2 as compared with the hr females, tc indices lower by 14.67% (p<0.001) and schiff bases values higher by 22.12% (p<0.001) were revealed in the control hr males. in comparison with the lr females, dc indices higher by 14.38% (p<0.001) were revealed in the lr males, whereas tc and schiff bases values were by 6.63% (p<0.002) and 31.12% (p<0.001) higher, respectively. these findings indicated more intensive lipid peroxidation process in females alongside with potent activity of antioxidant system that provided neutralization of lipid peroxidation products through decreased schiff bases’ production. more intensive lipid peroxidation course under stress was revealed in the males. as compared with hr females, the increase in dc in 2.16 times (p<0.001) was revealed in the hr males, whereas tc increased in 3.64 times (p<0.001), tba-active products — in 3.06 times (p<0.001), and schiff bases — by 9.06% (p<0.001). in comparison with the lr fema les the increase in dc in 5.86 times (p<0.001) was evidenced in the lr males, whereas tc increased in 6.56 times (p<0.001), and tba-active pro ducts — in 3.01 times (p<0.001). thus, stress impact is responsible for more intensive lipid peroxidation course in males that can be attri bu ted to activated sympathetic division of auto nomic nervous system, as well as to the less potent or more intensive antioxidant discharge, and to the decrease of sex hormones protective effect. the study of protein oxidative modification revealed reduced pom 370 and pom 430 values in the control hr males as compared with the lr group (by 7.23%, p<0.001 and by 10.11%, p<0.001, respectively, table 2). under stress impact, pom 370 decreased by 11.64%, p<0.001 in the hr group, while pom 430 increased by 65.69%, p<0.001. the pom 370 index decreased in the lr group by 24.32%, p<0.001; pom 370 and pom 430 values were higher in the hr group (by 8.17%, p<0.001 and by 26.63%, p<0.001, respectively). as against the lr group, higher pom 370 (by 13.57%, p<0.001) was revealed in the control hr females. under stress impact, the decreased pom 370 (by 13.07%, p<0.001) and increased pom 430 (by 69.19%, p<0.001) were revealed in the hr group, whereas in the lr group pom 370 decreased by 23.59%, p<0.001, but pom 430 decreased by 46.39%, p<0.001. pom 370 and pom 430 were higher in the hr group (by 24.02%, p<0.001 and 16.14%, p<0.001, respectively). as compared with the females, higher pom 370 and pom 430 indices (in the hr – by 11.57%, p<0.001 and by 15.41%, p<0.001; in the lr – by 28.72%, p<0.001 and by 25.59%, p<0.001, respectively) were revealed in the control males. under stress impact, higher pom 370 and pom 430 indices were revealed in the hr males in comparison with the females (by table 2. stress-related changes in the indices of protein oxidative modification nitric oxide anion-radical, and circulating immune complexes (cic) in hr and lr rats of both sexes, м±m (n=12) group index pom 370, mmol/g of protein pom 430, mmol/g of protein nitrite anion, х10-3, mkmol’/l cic, standard unit males hr control 1175.08±3.71 712.69±11.09 1.044±0.075 57.66±1.67 stress 1038.32±7.84* 1180.86±4.74* 9.564±0.301* 118.00±3.85* lr control 1260.00±3.51** 784.74±3.80** 1.189±0.083 56.25±0.84 stress 953.53±38.05*,** 866.38±53.34** 5.410±0.124*,** 103.25±1.63*,** females hr control 1039.11±17.20## 602.83±13.05## 0.988±0.059 54.00±1.41 stress 903.26±8.45*,## 1019.42±20.91*,## 1.792±0.048*,## 86.00±1.33*,## lr control 898.14±35.92**,## 583.93±11.77## 1.156±0.039** 50.00±0.77**,## stress 686.31±13.02*,**,## 854.83±14.20*,** 1.876±0.038*,## 83.50±1.66*,## notes: * –reliable indices as compared with the controls; ** – reliable indices as compared with the hr; ## – reliable indices as compared with the males of corresponding group. iu. m. ordynskyi et al. 59 b io m e d ic a l s c ie n c e s issn 2413-6077. ijmmr 2016 vol. 2 issue 2 13.01%, p<0.001 and by 13.67%, p<0.001, respectively), whereas in the lr males pom 370 index was higher by 28.025%, p<0.001. thus, decreasing pom 370 index was revealed in all groups due to the stress effect, whereas pom 430 increased in all hr rats and lr females, indicating the development of oxidative stress, and is likely to be related to free-radical oxidation of lipids which act on the proteins as potent oxidizers. no reliable difference in nitrite anion values between the control hr and lr males was found. under stress impact, the index increased in the hr group in 9.2 times, p<0.001, and in the lr group — in 4.5 times, p<0.001 that was higher by 43.43%, p<0.001 in the hr group. the values of nitrite anion of the control hr females were by 16.95%, p<0.005 lower as compared with the lr group. under stress, the value increased in 43.84 times, p<0.001 in the hr group and in 32.66 times, p<0.001 — in the lr group. no significant difference in nitrite anion values as against the females was revealed in the control males. under stress, the values of the females increased as compared to the males (in the hr group — by 81.26%, p<0.001, in the lr group — by 65.32%, p<0.001). thus, in comparison with the males, more intensive nitrite anion accumulation has been found in the females that may evidence the development of active oxygen forms in the females. as against the lr group, cic values in the control hr males were similar. under stress, they increased in the hr group in 2.0 times (p<0.001) and in the lr group — in 1.8 times (p<0.001) that was by 12.50% (p<0.001) lower in the lr group. in the control hr females the cic value was 7.41% (p<0.001) higher as compared with the lr group. under stress, the value increased in 2.90 times (p<0.001) in the hr group and in 3.29 times (p<0.001) in the lr group that was higher in the latter by 5.16% (p<0.001). as compared with the females, the cic value was by 11.11% (p<0.001) higher in the control lr males. under stress, the cic values were by 27.12% (p<0.001) higher in the hr males as compared with the females, and in the lr group — by 19.13% (p<0.001) higher. the research findings proved more signi­ ficant cic increase in the males as compared with the females, and in the hr males against the lr males. the cic increase is fraught with the development of autoimmune diseases. the study of antioxidant defence enzymatic link (table 3) revealed that sod activity in the control hr males was by 37.33% (p<0.001) higher as compared with the lr group, whereas ceruloplasmin content — by 15.90% (p<0.001) higher, and blood peroxidase activity (bpa) — by18.13% (p<0.001). these findings evidence the minor lipid peroxidation products content in the lr males as compared with the hr group. table 3. stress-related changes in antioxidant state in heart of hr and lr rats of both sexes, м±m, n=12 group index sod standard unit, mg-1 catalase, mcat/kg ceruloplasmin, mg/l bpa, mg/l males hr control 0.98±0.02 1.61±0.01 2.35±0.09 342.90±1.21 stress 0.19±0.01* 2.21±0.04* 14.85±0.44* 788.28±7.99* lr control 0.71±0.01** 1.26±0.06 2.03±0.05** 322.48±3.38** stress 0.08±0.02*,** 2.58±0.04*,** 13.89±0.14*,** 599.71±6.99*,** females hr control 0.81±0.01# 0.35±0.03# 3.63±0.17# 283.71±1.97# stress 0.79±0.02# 0.88±0.01*,# 6.50±0.07*,# 97.43±2.79*,# lr control 0.65±0.01**,# 1.09±0.01**,# 2.43±0.06**,# 270.38±3.76**,# stress 0.81±0.02*,# 0.24±0.01*,**,# 5.95±0.05*,**,# 122.57±5.42*,**,# notes: * — reliable indices as compared with the controls; ** — reliable indices as compared with the hr; # — reliable indices as compared with the males of corresponding group. iu. m. ordynskyi et al. 60 b io m e d ic a l s c ie n c e s issn 2413-6077. ijmmr 2016 vol. 2 issue 2 in the males, stress impact caused the decrease in sod activity (in the hr group — by 80.99%, p<0.001; in the lr group — by 89.22%, p<0.001), increase in catalase activity (in the hr group — in 1.38 times, p<0.001, in the lr group — in 2.04 times, p<0.001), ceruloplasmin content (in the hr group — in 6.32 times, p<0.001, in the lr group — in 6.85 times, p<0.001), and bpa (in the hr group — in 2.30 times, p<0.001, in the lr group — in 1.86 times, p<0.001). in the hr group, sod activity was by 58.72% (p<0.001) higher, as well as ceruloplasmin content (by 6.48%, p<0.05) and bpa (by 23.92%, p<0.001). however, catalase activity was by 16.62% (p<0.001) lower. the data prove that minor lipid peroxidation products content was provided by higher activity of antioxidant systemic defence in the hr rats. as against the lr group, the increase in ceruloplasmin content by 25.65% (p<0.001), the increase in sod activity by 33.13% (p<0.001), increase in bpa by 4.70% (p<0.01) in the control hr females were revealed, whereas catalase activity decreased in 3.07 times (p<0.001). these findings evidenced the intensive lipid peroxidation process and adequate antioxidant defence in the hr rats. as compared with the females, higher sod activity (by 16.79%, p<0001 in the hr group and 9.32%, p<0.001 — in the lr group), catalase activity (by 77.95%, p<0.001 in the hr group, by 13.72%, p<0.01 — in the lr group), bpa (by 17.26%, p<0.001 in the hr group and by 16.15%, p<0.001 in the lr group) were found in the intact males. meanwhile, higher ceruloplasmin content (by 54.56%, p<0.001 in the hr group and by 19.78%, p<0.001 — in the lr group) was evidenced in the females. in the hr females under stress, catalase activity increased in 2.48 times (p<0.001), ceruloplasmin content — in 1.79 times (p<0.001), whereas bpa decreased by 65.66% (p<0.001). in the lr females, catalase activity decreased by 78.13% (p<0.001) and bpa decreased by 54.67% (p<0.001), while sod activity increased by 25.38% (p<0.001) and ceruloplasmin content increased in 2.45 times (p<0.001). as compared with the lr females, higher catalase activity by 72.92% (p<0.001) and higher ceruloplasmin content by 8.42% (p<0.001) were revealed in the hr group, while bpa was by 25.81% (p<0.001) lower. this data indicated that minor lipid peroxidation products content in the hr group was provided by more potent activity of antioxidant system. in comparison with the males, sod activity was higher in the females (in 4.27 times, p<0,001 and in 10.55 times, p<0,001 in the hr and lr groups, respectively). however, higher catalase indices were in the males (by 60.22%, p<0.001 in the hr group and by 90.76%, p<0.001 in the lr group), ceruloplasmin (by 56.26%, p<0.001 in the hr rats and 57.16%, p<0.001 in the lr animals), and bpa (87.64 %, p<0.001 in the hr group and 79.56%, p<0.001 in the lr group. this table 4. stress-related changes in glutathione system in heart of the hr and lr rats of both sexes, м±m, n=12 group index reduced glutathione, mcmol/g glutathione peroxidase, mcmol/min.kg glutathione reductase mcmol/min.kg males hr control 776.32±22.04 0.441±0.002 0.621±0.004 stress 1393.27±40.89* 0.199±0.004* 0.319±0.001* lr control 625.73±25.31** 0.226±0.004** 0.616±0.004 stress 1057.02±12.80*,** 0.290±0.001*,** 0.254±0.001*,** females hr control 820.17±13.68 0.512±0.001## 0.303±0.012## stress 486.84±24.63*,## 0.132±0.002*,## 0.155±0.011*,## lr control 644.74±13.68** 0.228±0.003** 0.281±0.004## stress 451.75±19.92*,## 0.094±0.002*,**,## 0.094±0.002*,**,## notes: * — reliable indices as compared with the controls; ** — reliable indices as compared with the hr; ## — reliable indices as compared with the males of corresponding group. iu. m. ordynskyi et al. 61 b io m e d ic a l s c ie n c e s issn 2413-6077. ijmmr 2016 vol. 2 issue 2 data evidenced minor lipid peroxidation activation due to sod effect that provided minor accumulation of secondary and final lipid peroxidation products in the females. during the study of glutathione system indices (table 4), the reduced glutathione (rg) was revealed by 24.06% (p<0.001) higher in the hr males as compared with the lr group, glutathione peroxidase values higher by 95.57% (p<0.001). in the hr females the reduced glutathione values were by 21.39% (p<0.001) higher and glutathione peroxidase values were by 55.46% (p<0.001) higher. enzyme activity was lower in the hr females than in the males (glutathione peroxidase by 16.17%, p<0.001, glutathione reductase by 51.27%, p<0.001), and glutathione reductase in the lr females (by 54.41%, p<0.001). in the males, stress impact caused the increase in reduced glutathione values (the hr group — in 1.79 times, p<0.001, the lr group — in 1.69 times, p<0.001) and decrease in glutathione reductase activity (the hr group — by 48.62%, p<0.001, the lr group — by 58.80%, p<0.001), while glutathione peroxidase values decreased in the hr group by 54.89%, p<0.001 and increased in the lr group by 28.67%, p<0.001. stress caused the decrease of reduced glutathione indices (in the hr group — by 40.64%, p<0.001, in the lr group — by 29.93%, p<0.001), while glutathione reductase activity decreased in the hr group (by 80.26%, p<0.001) and increased in the lr group (by 88.39%, p<0.001). in the hr group glutathione peroxidase activity remained higher (by 28.79%, p<0.001), as compared with the lr group, but glutathione reductase activity was higher (by 39.26%, p<0.001). all the studied indices in the females under stress impact were lower in comparison with the females (reduced glutathione index in hr group — by 65.06% (p<0.001), glutathione peroxidase — by 66.33% (p<0.001), and glutathione reductase — by 51.53% (p<0.001). in the lr group, the indices were 57.26% (p<0.001), 67.61% (p<0.001) and 62.96 % (p<0.001), respectively. reduced glutathione increase has been noted in the males only; it evidenced of increasing hydroxyl radical which is crucial for the modification of amino acid residues. besides, in the males the decrease and in the females — increase of sod-enzyme were revealed that inactivates superoxide-anion radical. apparently, the processes of free radical oxidation progress differed in the hr and lr rats of both sexes. conclusions as compared with the lr animals of the same age, minor activity of lipid peroxidation processes owing to tba-active products were revealed in the intact adult hr male rats, whereas increased activity due to diene conjugates and schiff bases were evidenced in the female rats. immobilizing stress causes the development of oxidative and carbonyl stress in the hr and lr rats, more pronounced in the lr group, and the activation of antioxidant defence system. in the males, stress development was concomitant with the increased catalase activity as well as blood peroxidase, ceruloplasmin and reduced glutathione content, whereas the increased catalase and ceruloplasmin activity was evidenced in the hr females, and that of superoxide dismutase and ceruloplasmin — in the lr group, thus failing to provide neutralization of lipid peroxidation products in the latter group. as compared with the females, more intensive oxidative and nitroxidative stress, protein oxidative modifi­ cation, and stress-related accumulation of circulating immune complexes has been found in the males. references 1. rodin r, bonanno ga, rahman n, et al. expressive flexibility in combat veterans with posttraumatic stress disorder and depressionю j. affect. disord 2016; 207: 236–241. 2. crea f, battipaglia i, andreotti f. sex differences in mechanisms, presentation and management of ischaemic heart disease. atherosclerosis 2015; 241 (1): 157–168. 3. pimple p, shah aj, rooks c, et al. angina and mental stress-induced myocardial ischemia. j. psychosom. res 2015; 78 (5): 433–437. 4. berezovsky va. hypoxia and individual features of reactivity. k.: naukova dumka 1978; 216. (in russian). 5. kulinskiy vi, olkhovskiy ia. two adaptation strategies in adverse conditions: resistant and toleriu. m. ordynskyi et al. 62 b io m e d ic a l s c ie n c e s issn 2413-6077. ijmmr 2016 vol. 2 issue 2 ant. the role of hormones and receptors. successes of modern biology 1992; 112: 697–711. (in russian). 6. khyshiktuyev bs, khyshiktuyeva na, ivanov vn. methods of determination of lipid peroxidation products in exhaled breath condensate and their clinical significance. clinical laboratory diagnostics 1996; 3: 13–15. (in russian). 7. preclinical studies of medication: guidelines; ed. by corr. members of ams of ukraine a.v. stefanov. k., avicenna 2001; 528. (in ukrainian). 8. archakov ai, mikhosoev im. modification of proteins by active oxygen and their decomposition. biochemistry 1998; 54 (2): 179–185. (in russian). 9. green ic, davie aw, golawski j et al. analisis of nitrate, nitrite and [15n] nitrate in biological fluids. anal. biochem 1982; 126 (1): 131–138. 10. hayevska myu. circulating immune complexes in the conditions of norm and pathology. journal of scientific research 2000; 4: 37–40. (in ukrainian). 11. chevari c, chaba i, sokey i.the role of superoxide dismutase in cell oxidation process and the method of its determination in biological materials. lab. bus 1985; 11: 678–681. (in russian). 12. koroljuk ma, ivanova li, mayorova ig, tokarev ve. the method of determining the activity of catalase. lab. bus 1988; 1: 16–19. (in russian). 13. clinical and laboratory diagnosis. normative directive legal documents. k: medinform 2003; 856. (in ukrainian). 14. popov t, neykovska l. method of determining the peroxidase activity of blood. hygiene and sanitation 1971; 10: 89–93. (in russian). 15. moffat ja, armstrong pw, marks gs. investigations into the role of sulfhydryl groups in the mechanism of action of the nitrates. canadian journal of physiology and pharmacology 1982; 60 (10): 1261–1266. 16. kruhlіkova ho, shtutman im. glutathione peroxidase and glutathione reductase activity of the liver of rats after introduction of sodium selinate. ukrainian biochemical journal 1976; 2: 227–233. (in ukrainian). received: 2016-12-09 iu. m. ordynskyi et al. 13 r a d ia t io n m e d ic in e a n d o n c o l o g y issn 2413-6077. ijmmr 2016 vol. 2 issue 1 h. i. falfushynska international journal of medicine and medical research 2016, volume 2, issue 1, p. 13-18 copyright © 2016, tsmu, all rights reserved doi 10.11603/ijmmr.2413-6077.2016.1.6373 detection of oxidative stress, apoptosis and molecular lesions in human ovarian cancer cells h. i. falfushynska1,2,3 1i. horbachevsky ternopil state medical university, ternopil, ukraine 2volodymyr hnatiuk ternopil national pedagogical university, ternopil, ukraine 3university of north carolina at charlotte, charlotte, usa background. ovarian cancer has the highest mortality rate of gynaecological cancers. this is partly due to the lack of effective screening markers. indices of oxidative stress are well-recognized prognostic criteria for tumorous transformation of tissue, but their value depends on the type of tumor and the stage of its development. objective. the aim of this study is to clarify the relationship between antioxidant/pro-oxidant ratio and the signs of molecular lesions and apoptosis rate in blood of ovarian cancer patients and non-cancer ones. results. the ovarian cancer group is marked by antioxidant/prooxidant balance shifting to oxidative damage in blood as the consequence of overexpression of oxyradicals (by 300%). higher level of glutathione (by 366%), lower level of metallothioneins (by 65%) as well as higher level of lipid peroxidation (by 174%) and protein carbonyls (by 186%) in blood of ovarian cancer patients compared to the normal ovarian group have been observed. the signs of cytotoxicity are determined in blood of ovarian cancer patients: an increased (compared to control) level of dna fragmentation (by 160%), choline esterase (up to twice), higher rate of both caspase dependent and caspase independent lysosomal mediated apoptosis. conclusions. cathepsin d activity both total and free, choline esterase activity, tba-reactive substance and protein carbonyls level in blood could be used as the predictive markers of worse prognosis and the signs of human ovarian cancer. key words: ovarian cancer, oxidative stress, apoptosis, caspase-3, cathepsin d, choline esterase, metallothionein. corresponding author: halina falfushynska, department of general chemistry, i. horbachevsky ternopil state medical university, 1 maidan voli, ternopil, ukraine, 46001 research laboratory of comparative biochemistry and molecular biology, volodymyr hnatiuk ternopil national pedagogical university, 2 maksym. kryvonis street, ternopil, ukraine, 46027 tel.: +380673506531 e-mail: halynka.f@gmail.com introduction ovarian cancer dominates among the death causes of malignant tumours. in particular, according to the international agency for cancer research, more than 165 thousand of newly diagnosed cases of ovarian cancer are registered each year over the world. despite intensive studies, every year, more than 100 000 women die from this disease worldwide. due to minimal and non-specific early stage symptoms, ovarian cancer diagnosis is late and prognosis is usually poor [1]. currently there are no screening programs for precancerous and malignant ovarian pathology diagnostic, with the help of which specialists could have reduced the incidence and fatalities of this disease [2]. therefore, exploration of new and/or low-cost early-warning molecular signatures of disease is a key goal of ovarian cancer research. one of the most common mechanisms of diseases development and aggressiveness of pathology which is on the focus of biomedical research has been linked to overexpression of free radicals and oxidative stress initiation [3, 4, 5, 6, 7]. the damage that reactive oxygen species (ros) can cause to the cell not only depends on their internal concentration but also on the equilibrium between the ros and the endogenous antioxidants normally protect cells against oxidative stress [4, 6]. when the antioxidant/prooxidant system becomes unbalanced, oxidative stress is generated, altering and damaging many biomolecules, including dna, lipids and proteins, in turn, decreasing cell viability [5, 6, 8, 9]. we have shown before that higher content of the products of oxidative damage of proteins, lipids, dna fragmentation and cathepsin d activity, particularly free one are the main characteristic signs of malignant ovarian tissue [10]. however, it can be difficult 14 r a d ia t io n m e d ic in e a n d o n c o l o g y issn 2413-6077. ijmmr 2016 vol. 2 issue 1h. i. falfushynska to obtain a biopsy from ovarian tissue for routine biochemical measurements with pro gnostic mission of diseases development. instead of them correspondent blood test would be highly recommended. thus, here we aim to clarify the relationship between anti oxi dant/ pro-oxidant ratio and the signs of mo le cular lesions and apoptosis rate in blood of ova rian cancer patients and normal ovarian ones. materials and methods for this research we used the venous blood samples taken from the 15 newly diagnosed patients of reproductive age who had been operated for epithelial ovarian cancer at the department of gynaecology of ternopil regional oncological hospital. cancer pathology was verified histologically. according to figo classification, all diagnosed patients had stage iii disease. none of the operated oncologic female patients had been previously treated with platinum-based drugs (cisplatin/carbo platin/ cycloplatam) or cyclophosphamide. venous blood of 15 females not affected by the relevant pathology were taken as controls. all experimental studies were conducted in accordance with the rules of the national congress on bioethics (kyiv, 2000) and the decision of the commission on bioethics of the ternopil state medical university (№ 3, 2013). all the procedures on bloods were carried out at 4o c. all the reagents, except those specified below, were produced by “synbias”, ‘chemically pure’ grade. the determination was conducted in the soluble fraction of blood, which had been recei ved to determine superoxide dismutase (sod) as a result of centrifugation of blood within 10 min at 6000 g (s6), to determine the level of oxygen radicals – as a result of its centrifugation within 45 min at 12 000 g (s12) and to determine the concentration of metallo thioneins – within 45 min at 16 000 g (s16). superoxide dismutase activity (sod) (ec 1.15.1.1) was measured by a decrease in the rate of the reduction of nitrotetrazolium blue in the presence of phenazine methosulfate and nadh [11]. enzymatic activity was expressed in conventional units (cu). enzyme activity, which was able to cause a decrease in optical density in the process of the reduction of nitrotetrazolium blue in 50% test sample per 1 mg of protein from the homogenate in soluble form, was taken as 1 cu. the content of total glutathione was determined in blood after complete reduction of glutathione through the use of glutathione reductase (sigma, usa) and with the help of ellman’s reagent [12]. the level of 5-trinitrobenzoic acid was monitored with a spectrophotometer at 412 nm. the determination of protein carbonyls (pc) was conducted due to their ability to form 2,4dinitrophenylhydrazones under the blood plasma incubation in the presence of 0.1 m 2,4-dinitrophenylhydrazine in hcl, 2 m. the light absorbance was registered at 370 nm against the control, and the content of phenylhydrazone was calculated using a molar extinction coeffi cient of 2.1×104 m-1cm-1[13]. lipid peroxidation was characterized by the products of interaction between deproteinized blood after precipitation of proteins with trichloroacetic acid (with final concentration of 5%) from blood samples and 2-thiobarbituric acid (tba). the formation of tba-reactive substance (tbars) was calculated by the intensity of the absorption of a pinkcoloured complex at 532 nm by the molar extinction coefficient of the complex equal to ε=1.56×105 м–1cm–1 [14]. the content of oxyradicals (or) in s12 fraction of the blood plasma was evaluated using the non-fluorescent derivative, dihydror hodamine, which is converted to the fluorescent dye, rhodamine-123, after a reaction with re active oxygen species. the fluorescence sig nal was detected by using an f-max fluorescence platereader [excitation=485 nm, emission=538 nm] immediately and after 20 min and used to deter mine the rate of ros formation [10, 15] and expressed in relative fluorescence units (rfu) per 1 mg of protein. the content of metallothioneins (mts) in s16 fraction of blood was evaluated by the content of thiol groups (mt-sh) with slight modification [8, 9, 10, 16] with 5,5'-dithio-bis-2-nitrobenzoic acid (dtnb, sigma, usa) after the chloroform-ethanol extraction of mt, and calculated assuming that 1 mol of mt contains the same amount of sh-groups as 20 moles of gsh. choline esterase (che, ec 3.1.1.7) activity was determined in blood as the acetylthiocholinecleaving che activity according to the colorimetric method of ellman et al. (1961) at 25oc [17]. enzyme activity was calculated using a molar extinction coefficient of 13.6×103 м–1сm–1 and standardized to the soluble protein content. dna damage was determined by the content of the fragmented deproteinized dna in the total dna by the method of alkaline pre15 r a d ia t io n m e d ic in e a n d o n c o l o g y issn 2413-6077. ijmmr 2016 vol. 2 issue 1 h. i. falfushynska cipitation of 10% blood plasma in 50 mm trisedta buffer, ph 8.0, containing 0.5% sodium dodecyl sulfate (sigma, usa). the supernatant contains damaged dna molecules when the pellet contains protein and a whole dna. dna content in the supernatant and in the pellet was determined by the hoescht dye in the presence of 0.4 m nacl, 4 mm of sodium cholate and 0.1 m tris (ph 9.0) at the excitation wave (ex.)=360 nm and emission (em.)=450 nm [18]. the content of fragmented dna was expressed as a percentage to the total dna in the sample. caspase-3 activity in blood samples was measured using a colorimetric assay based on the hydrolysis of peptide acetyl-asp-glu-val-asp p-nitroanilide (ac-devd-pna) by caspase-3, resulting in a release of the p-nitroaniline (pna) moiety. p-nitroaniline was detected at 405 nm (εm=10,500 m -1cm -1) [19, 20]. the enzymatic activity of cathepsin d (ec 3.4.23.5) was determined spectrophotometrically by the formation of acid-soluble products of hemoglobin enzymatic hydrolysis [10, 21]. the reaction mixture contained 50% blood serum in 0.25 m of sucrose solution and, as a substrate, 1% solution of bovine hemoglobin (sigma, usa) in 0.1 m acetate buffer (ph 5.0). the enzymatic reaction was stopped by adding 10% solution of trichloracetic acid up to a final concentration of 2%. to determine the total activity of cathepsin d, the blood serum sample was previously treated with 1% solution of triton x-100 (sigma, usa) for 10 min at +37ос. the control sample was incubated at +4ос for 30 min before adding 10% trichloracetic acid solution. the activity of cathepsin d was calculated by the difference in the optical density of experimental and control samples at 280 nm wave length and expressed as nmol of tyrosine/(min×ml). the results of the measurements were presented as means ± standard deviation (sd) m±sd for 15 samples of the blood of the ovarian cancer patients and the reference group. if the data was not normally distributed according to the lilieford test, it had been transformed using the box-cox method. for the data that were not normally distributed even after the transformation, non-parametric tests (kruskall–wallis anova and mann–whitney u-test) were performed. differences were considered significant if the probability of type i error was less than 0.05. for evaluation of the antioxidative-pro oxidative equilibrium we proposed the integrated oxidative stress (ios) index as the ratio of antioxidant factors (sod, gsh and mt-sh level) and prooxidant manifestations (oxyradicals (or), tbars and pc) after the standardization of data [8, 9]. the main classification criterion and relationship between biochemical parameters of the ovarian tissue samples was evaluated using the discriminant analysis and correlation analysis (pearson’s correlation coefficient r under the probability of the value p<0.05). all statistical calculations were performed by means of statistica v10.0 and excel for windows 2010. results and discussion the results of an evaluation of antioxidant defense system parameters (table 1) show glutathione level (by 366%) in blood of the cancer patients is higher than in the control ones and sod activity just commensurate in both groups. the mt-sh level (by 65%) is lower under the ovarian cancer. at the same time, the intensity of the oxyradicals formation (by 300%), the concentration of tbars (by 174%) and protein carbonyls (by 186%) are higher in the blood of the cancer patients compared to the correspondent control ones. the signs of cytotoxicity are determined in blood of the ovarian cancer patients: an increased (compared to the control ones) level of dna fragmentation (by 160%), caspase-3 activity (by 43%) and cathepsin d activity, both of its total (by 546%) and, particularly, free forms (by 952%). the activity of choline esterase is twice as high as in the ovarian cancer group compared to the intact one. the application of discriminant functional analysis (a subtype of the multivariate data analysis) to the set of studied biochemical parameters has shown that the characteristics of cathepsin d activity both total and free, choline estrase activity, tbars and protein carbonyls level are the main criteria for differentiation of the studied groups (f(11,18)=2353,6, p<0,0000). for an appreciation of the quantitative equilibrium between antioxidant defence and oxidative destruction, we calculated the ios index after standardizing the data. it was lower in blood of the cancer patients (ios=-39%) indicating the balance shifting to oxidative damage as the consequence of tolerance limits of antioxidant defence system increased in ovarian cancer patient. recent studies have shown an important role for reactive oxygen species (ros) in cancer development [5]. ros can be produced in different endogenous sources, such as mito chondria, peroxisomes etc. [3] and are accu mulated 16 r a d ia t io n m e d ic in e a n d o n c o l o g y issn 2413-6077. ijmmr 2016 vol. 2 issue 1h. i. falfushynska under an imbalance between their production and elimination by protective mechanisms, referred to as antioxidants. ros are overproduced a long time, then, may cause dna, protein, and/or lipid damage [7]. in our case the ovary cancer is also said to be under oxidative stress (ios less than zero) associates with the most prominent upraise of oxidative lesions (triple of them, tbars, protein carbonyls and oxyradicals). we speculate that oxidative stress causes injury to cells, among them initiation of dna strand breaks (dnasb=1.65–0.02×tbars+ 1.93×pc*+0.65×or*, r2=0.98, f(6,23)=212,69, p<0.001; * – parameter makes a presumable con tri bution to the mathematical model) and liberation of cathepsin d from lysosomes (cathepsin d(free) = –3.69+0.19×tbars*+0.78× pc+0.48×or, r2=0.96, f(6,23)=210,05, p<0.001). it is established that cathepsins, released from lysosomes in course of oxidative injury of membranes [22], cleave bid, which activates the mitochondrial apoptosis pathway [23]. thus, human ovary cancer progression is linked to oxidative stress directly by increasing dna fragmentation and caspase-independent lysosomal-mediated apoptosis pathway. a failure to undergo apoptosis is considered to be a key event in cancer formation and progression [24]. using the key mediators of mitochondrial events of apoptosis, caspase 3, we have shown that ovarian cancer exhibits higher rates of apoptosis than either samples of the control group. data about caspase 3 role in cancer progress and patients survival are controversial; and it has been demonstrated that expression of caspase-3 does not correlate with the extent of apoptosis in primary carcinomas [25]. some investigators have also reported increased rates of apoptosis in dif ferent types of cancer, breast carcinomas among them. but, on the other hand, it has been shown that loss of caspase-3 expression may decrease breast cancer cells resistant to chemotherapy and radiation therapy [26]. many studies have found that the expression level of ache and/or bche increases during apoptosis in various cell types and alter human breast, lung cancer, leukemia etc progress [27]. many tumours, including ovarian carcinoma, express ches, indicating that the enzymes may be functionally important in neoplastic cell transformation, and can be considered a marker of early differentiation [28]. in breast cancer ache activity is as in twice as high as in normal breast. the difference in lectin reactivity between erythrocyte and breast ache, the lack of ache in blood plasma suggest that breast epithelial cells produce ache for membrane attachment [27]. it has also been demonstrated that ache can hydrolyse lipid peroxides, which may support the possibility that a reduction in enzyme activity augments oxidative stress and cellular damage of bronchopulmonary epithelial cells [29]. we have also shown in our study the concordant changes of choline esterase and caspase-3 activity (r=0.83, p<0.001) strongly suggest that che is an important component in the common pathway leading to apoptosis in human ovarian cancer and future studies of such mechanisms would be of great interest. several in vitro studies have demonstrated the importance of mts expression for cancer cell growth and survival. on the one hand over expression of mts has been reported for different type of cancers and mt levels have been correlated with increasing tumour grade in different ones [30, 31]. but, on the other hand, the downregulation of mt protein pro duction table 1. biochemical parameters in blood of ovarian cancer and normal ovarian patients, m±sd, n=15 parameter groups normal ovarian ovarian cancer mt-sh content, µg⋅ml-1 of blood 16.2±1.9 5.6±0.7* total glutathione content, mmol⋅l-1 of blood 0.9±0.1 4.2±0.8* sod activity, cu⋅mg-1 of protein 1.8±0.2 1.6±0.2 tbars content, nmol⋅ml-1 of blood 19.4±2.5 53.1±6.2* protein carbonyls level, nmol⋅mg-1 of plasma proteins 0.7±0.2 2.0±0.3* cholin esterase, nmol⋅min-1⋅mg-1 proteins 0.38±0.04 1.25±0.18* oxygen radicals content, rfu⋅mg-1 of protein 0.94±0.08 3.76±0.42* caspase-3, pmol⋅min-1⋅mg-1 proteins 7.7±0.8 11.0±1.3* total activity of cathepsin d, nmol⋅min-1⋅ml-1 of blood serum 2.2±0.3 14.2±1.2* free cathepsin d activity, nmol⋅min-1⋅ml-1 of blood serum 0.96±0.07 10.10±0.89* content of fragmented dna in total dna, % 3.0±0.4 7.8±0.5* note. * – differences compared to the blood samples of the reference group, р<0.05. 17 r a d ia t io n m e d ic in e a n d o n c o l o g y issn 2413-6077. ijmmr 2016 vol. 2 issue 1 h. i. falfushynska resulted in dramatic growth inhibition of several human cancer cell lines. similarly, when mt expression was abolished in human pc-3 prostate and skov-3 ovarian cancer cell lines using a specific ribozyme, there is a marked increase in spontaneous apoptosis [32]. we have also found that lower level of mts in ovarian cancer group potentially caused the caspasedependent apoptosis pathway activa tion (r=-0.86, p<0.001). we speculate that apo ptotic activation is dependent on mts levels via zinc, because it is a potent inhibitor of caspase-3 [33]. it is recognised that depletion of zinc by chelation has been shown to promote apoptosis in vitro [34]. therefore, zinc bound to mts may play a crucial role in the control of apoptosis. apoptosis rate would be as higher as less zinc is liberated in cells containing less mts. our present findings are in good correlation with our previous work that demonstrated decreased zinc concentration in the samples of ovarian cancer tissue compared with normal ovarian [10]. conclusions using the multi-marker analysis of stressrelated processes in the ovarian cancer we determined a number of blood characteristics, which compound the pathological changes in the malignant tissue; the imbalance between antioxidants and oxidative lesions, appearance of dna strand breaks and activation of both caspase dependent and caspase independent lysosomal mediated apoptosis pathway are among them. cathepsin d activity, both total and free, choline esterase activity, tbars and protein carbonyls level can be used as the predictive markers of worse prognosis and the signs of human ovarian cancer. metallothioneins down-regulation and choline esterase activation in blood are ultimately resulting in intrinsic apoptosis pathway in ovarian cancer. the further studies are needed to elucidate the mechanism by which choline esterase regulates caspase-3 dependent apoptosis in human ovarian cancer. acknowledgements the research was supported by the westukrainian biomedical center, the 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biochem 1998; 124: 300–303. 33. chimienti f, seve m, richard s, mathieu j, favier a. role of cellular zinc in programmed cell death: temporal relationship between zinc depletion, activation of caspases, and cleavage of sp family transcription factors. biochem pharmacol 2001; 62: 51–62. 38 su r g ic a l d is e a se s issn 2413-6077. ijmmr 2022 vol. 8 issue 2 doi 10.11603/ijmmr.2413-6077.2022.2.13197 intraoperative state of the hemocoagulation system in patients with open and endovascular revascularization of infraingual arterial segment in the presence of stenotic-occlusive process of tibial arteries i. k. venger, s. ya. kostiv, b. p. selskyy, m. p. orlov, *n. i. tsiupryk i. horbachevsky ternopil national medical university, ternopil, ukraine background. among all postoperative complications associated with revascularization of atherosclerotic steno-occlusive process of the femoral-distal artery, thrombosis of the reconstruction segment is 6-32%. objective. prevention of thrombotic complications through vascular revascularization of the lower extremity by using a pathogenetically reasonable system of postoperative thromboprophylaxis. methods. the study involved 97 patients with atherosclerotic stenotic-occlusive process of the infrainguinal artery in cases of stenotic-occlusive lesions of the tibial arteries. in order to study the state of the hemocoagulation system on patients, the methods for studying the indicators of coagulation, fibrinolytic and aggregation systems were used. results. development of hypercoagulable disorders in the patients who underwent endovascular revascularization of the arterial segment is more intense than in those who underwent open vascular revascularization methods. hypercoagulation of blood at the intraoperative stage of revascularization takes place primarily due to the activity of factor iia of the hemocoagulation cascade. taking into account these circumstances, already at the surgery stage for thromboprophylaxis non-fractionated heparin (nfh) should be prescribed. at the same time for prevention of thromboembolic complications double anti-thrombocyte therapy: clopidogrel, acetylsalicylic acid should be prescribed. conclusion. hypercoagulant ability of the blood system, which develops after arterial reconstructive interventions, takes place against the background of low activity of the fibrinolytic blood system, nevertheless a gradual increase in the activity of the aggregation capacity of the blood. development of hypercoagulable disorders at the intraoperative stage of surgery in the patients with endovascular methods of arterial reconstructions is more intense than in those treated with open methods of arterial revascularization. taking into account these circumstances, already at the surgery stage for thromboprophylaxis an anticoagulant should be prescribed that has a targeted effect on factor iia of the hemocoagulant cascade with simultaneous prevention of thromboembolic complications – double anti-thrombocyte therapy. keywords: blood coagulation system; aggregative assay; revascularization; thrombosis. *corresponding author: n. i. tsiupryk, phd student, i. horbachevsky ternopil national medical university, ternopil, 46001, ukraine. e-mail: tsyupryk_cnad@tdmu.edu.ua international journal of medicine and medical research 2022, volume 8, issue 2, p. 38-43 copyright © 2022, tnmu, all rights reserved i. k. venger et al. introduction treatment of patients with multilevel atherosclerotic lesions of the arterial bed of the lower extremities is still challenging and, in many cases, unresolved problem. this is especially true of the choice of surgical tactics for atherosclerotic lesions of several anatomical zones of the main arteries [1]. open reconstructive interventions are leading in the revascularization of multilevel atherosclerotic lesions of the arterial bed of the lower extremity. this is important in revascularization of the arterial bed below the knee in cases of chronic critical ischemia [3]. one of the ways to solve the problems of surgical treatment of multilevel lesions of the arterial bed of the lower extremities is use of hybrid revascularization technology such as endovascular angioplasty of one arterial segment in combination with bypass surgery of another [2]. according to some researchers, endovascular angioplasty of the femoral-distal artery and especially the tibial arteries in chronic critical ischemia is the only chance to delay or avoid amputation of the lower limb [4]. at the same time, the use of the suggested methods of revascularization (open, hybrid, endovascular) of atherosclerotic stenotic-occlusive process of the femoral-distal artery causes development of a number of compli ca tions of 39 su r g ic a l d is e a se s issn 2413-6077. ijmmr 2022 vol. 8 issue 2 6-32% of cases diagnosed with throm bosis of the reconstruction segment [6, 8]. thus, the main goal of the study was to find a way for prevention of development of thrombotic complications of revascularization of the arterial bed of the lower extremity by using a pathogenetically reasonable system of postoperative thromboprophylaxis. methods the study involved 97 patients with atherosclerotic stenotic-occlusive lesions of the in frainguinal arterial bed in cases of stenoticocclusive lesions of the tibial arteries. according to the classification of fontaine (1954) the following degrees of chronic arterial insuffi­ ciency were established: 2b degree in 47 pa­ tients, 3 degree in 21 patients and 4 degree in 29 patients. 53 (54.2%) patients were treated with open methods of revascularization, 44 (45.7%) – endovascular methods of revascu larization of the arterial bed of the lower extremity. the level of indicators of coagulative, fib­ rinolytic and aggregate systems was carried out as follows: fibrinogen (fg) – gravimetric method according to rutberg r.a. (1964); fibrinostabilizing factor activity (fsf) – with the “kit for determining factor xiii” by research and production company simko ltd (lviv); thromboplastic activity of blood (tpa) – by the method of kudryashov b.a. and ulitina p.d.; plasma recalcification time (prt) – according to the method of bergerhof and roca; plasmin (pl), plasminogen (pg), total fibrinolytic activity (tfa) – by the method of monastyrsky v.a. et al. (1988); euglobulin clots lysis time (eclt) – using the “kit for determining the fibrinolytic activity (fla) of blood plasma” by research and p ro d u c t i o n c o m p a n y s i m ko l t d ( l v i v ) ; antithrombin iii (at iii) activity was determined by the method of morbet and wiltertein in the modification of katsadze y.l., kotovshchikova m.a. (1982); platelet aggregation properties were studied using adp at a con centration of 2.5 μmol/l with recording aggregatogramms on the analyzer ar 2110 solar; d-dimer was studied by immunoanalytical method using a coagulometer; determination of soluble fibrin monomer complexes (sfmcs) was performed by the tablet method; von willebrand factor (vwf) was studied by the method of barkagan z.s. (1988). statistical analysis was performed using the statistical software package statistica 6.0 for windows (statsoft, inc. tulsa ok, usa) and microsoft excel (microsoft office 2013, usa) with data in the form of mean and standard deviation (m±sd). comparisons between several groups were performed by one-way analysis of variance (anova) and tukey’s post-hoc analysis to analyse mean differences. a statistical p value of <0.05 was considered statistically significant. results at the preoperative stage, an increased level of blood coagulation was observed in the patients of both study groups. thus, when analysing the indicators of the coagulation system of patients’ blood it was found that the level of the blood fibrinogen was significantly higher than the same indicator in the control group. the same was observed regarding the content of sfmcs, tfa, fibrin degradation products (fdp), fibrinopeptide a (fpa): these indicators significantly exceeded the level of similar indicators in the control group. along with the results of the study, the level of at iii and prt in the patients at the preoperative stage was within the control level, 88.56±7.49% and 91.17±5.23 sec. respectively, which differed from that of patients of the control group (table 1). revascularization of the femoral / popliteal arterial bed of the lower extremity stimulated development of changes in the hemocoagulative system – contributed to maintenance of increasing hypercoagulant properties of blood during surgery. similar changes in the hemocoagulable blood system occured with varying degrees of activity in both groups of patients and depended on the method of revascularization of the infrainguinal arterial arterial bed of the lower extremity. thus, with the preoperative fg blood level of 5.56±0.48 g/l, in the period of 1.5-2.0 hours and 3.0-4.0 hours of the surgery with the use of the open revascularization methods it reached the level of 5.61±0.49 g/l and 5.76±0.53 g/l, respectively. the endovascular method of revascularization led to fg level of 5.98±0.53 g/l and 6.39±0.52 g/l at the same time points (table 2). more noticeable changes in the patients of both study groups were observed for the sfmcs, fpa and fdp levels. the level of these indicators at 3.0-4.0 hours of the surgery with the open method of revascularization was higher than in the preoperative period in 1.2, 1.9 (p<0.05) and 1.7 (p<0.05) times respectively, and with the use of the endovascular revascularization method – in 1.8 (<0.05), 2.1 (p<0.05) and 2.1 (p<0.05), respectively. at the same time, i. k. venger et al. 40 su r g ic a l d is e a se s issn 2413-6077. ijmmr 2022 vol. 8 issue 2 there was a significant difference between the indicators of the above components of the hemocoagulation in the patients treated with different methods of revascularization. thus, the sfmcs, fpa and fdp levels in the patients treated with endovascular method were significantly higher than those treated with open methods, in 1.5 (p<0.05), 1.3 (p<0.05) and 1.3 (p<0.05) times, respectively (table 2). along with the above, fla and at iii content in the blood of patients during the surgery decreased gradually. the decrease in the fla and at iii blood level in the patients with endovascular revascularization was more significant than in those treated with open revascularization methods. crp with a significant difference between its indicators in the patients of both study groups was not evidenced (table 2). all these changes in the coagulation system occurred in the background of a gradual shortening of the prt. however, no significant difference between its levels in the patients of both study groups was observed (table 2). lower extremity arterial bed revascularization, regardless of the method of restoring blood flow, contributed to activation of the anticoagulant system. thus, in the 3.0-4.0 h of the surgery, pl serum level increased in 1.4 times (p<0.05), and eclt was prolonged in 1.3 times (p<0.05) compared with the pre­ operative period. this took place due to a 1.2 time (p<0.05) reduction of the pg serum level, which contributed to a 1.2-time reduction in the tfa of the serum. it should be noted that the results of the anticoagulant blood system study indicated its inability to resist the increase in hemocoagulable capacity of the blood at the final stage of revascularization. the results of the blood aggregation study at the preoperative stage did not reveal any visible difference between the indicators in the patients and the control group individuals. revascularization of the femoral-distal arterial bed helped to activate the blood aggregation system. thus, at 1.5-2 h of the surgery thrombocyte aggregation velocity (tav) increased in table 1. preoperative state of hemocoagulation in patients with stenotic-occlusive atherosclerotic process of infrainguinal arterial bed in cases of stenotic-occlusive process of tibial arteries indicators control preoperative period fg, g/l 3.68±0.41 5.56±0.48* sfmcs, ext. units 0.48±0.06 0.71±0.15* fpa, ng/ml 1.97±0.38 2.69±0.35* fdp, μmol/ml 4.78±1.45 7.75±1.67* fla, % 53.18±4.52 56.23±4.57 ат ііі, % 81.41±6.71 88.56±7.49 prt, sec. 103.61±4.97 91.17±5.23 note. * р<0.05 compare to the control group indicators. table 2. intraoperative state of the hemocoagulative system in the conditions of open and endovascular revascularization of the infrainguinal arterial bed indicator before surgery intraoperative period of revascularization 1.5-2.0 h of the surgery 3.0-4.0 h of the surgery 3.0 h after the surgery open endovasc. open endovasc. open endovasc. fg, g/l 5.56±0.48 5.61±0.49 5.98±0.51 5.76±0.53 6.39±0.52 5.87±0.51 6.89±0.54 sfmcs, ext.units 0.71±0.15 0.74±0.12 0.89±0.12 0.87±0.15 1.27±0.17* 0.94±0.16 1.33±0.19* fpa, ng/ml 2,69±0.35 3.68±0.31 4.47±0.31* 5.13±0.31 5.71±0.32* 5.45±0.31 6.67±0.35* fdp, μmol/ml 7.75±1.67 9.56±2.31 12.74±2.41* 13.14±2.37 16.61±2.46* 14.28±3.21 17.69±3.27* fla. % 56.23±4.57 52.19±4.37 49.58±4.41 50.47±4.21 46.53±4.37 47.15±4.34 43.57±4.52 ат ііі. % 88.56±7.49 85.14±7.39 84.17±6.28 83.78±6.31 82.13±6.35 84.34±6.41 83.47±6.45 prt. sec. 91.17±5.23 85.15±5.39 82.25±5.38 82.37±5.26 79.14±5.19 80.24±5.03 78.21±5.53 note. * р<0.05 – significant difference between the indicators in patients treated with open revascularization and those who underwent endovascular revascularization. i. k. venger et al. 41 su r g ic a l d is e a se s issn 2413-6077. ijmmr 2022 vol. 8 issue 2 1.3 times (p<0.05) and thrombocyte aggregation rate (tar) slightly increased with a simultaneous acceleration of thrombocyte aggregation time (tat) by 12.42%. during the surgery blood platelets level increased. thus, in 3.0-4.0h of the surgery its level increased almost in 1.3 times compared to the preoperative period. as for the tat, it was further reduced, reaching the level of 7.68±0.75 min, which was in 1.3 times (p<0.05) less than the preoperative tat level. when analysing the results of the blood aggregation system state it was found that there was a difference in its performance at the different stages of surgical treatment by different methods of revascularization. thus, the patients treated with endovascular methods had a significantly higher tav and tar with a significant reduction in tat than the patients treated with open revascularization (table 3). discussion the study has shown that revascularization of the femoral / popliteal arterial bed at the intraoperative stage of surgery is accompanied by an increase in the hypercoagulant ability of the blood system. it takes place in the background of low blood fibrinolytic system activity, nevertheless a gradual increase in the blood aggregation activity. revascularization of the infrainguinal arterial bed of the lower extremity contributes to increase of hypercoagulable properties of blood during surgery. similar changes in the hemocoagulable blood system occur with varying degrees of activity in both groups of patients and depend on the method of revascularization of the infrainguinal arterial artery of the lower extremity. thus, the fg blood content in the period of 1.5-2.0 h and 3.0-4.0 h of the surgery with open revascularization method reached the level of 5.61±0.49 g/l and 5.76±0.53 g/l, respectively, and with the endovascular method of revascularization, 5.98±0.53 g/l and 6.39±0.52 g/l, respectively. the level of sfmcs, fpa, fdp at 3.0-4.0 h of the surgery using the open method of revascularization was in 1.2, 1.9 (p<0.05) and 1.7 (p<0.05) times higher, respectively, and with the endovascular revascularization method – in 1.8 (p<0.05), 2.1 (p<0.05) and 2.1 (p<0.05) times higher, respectively, than in the preoperative period. at the same time, a significant difference was found between the blood levels of sfmcs, fpa, fdp in the patients who underwent surgeries by different methods of revascularization. thus, in those operated by the endovascular method, the levels of sfmcs, fpa, fdp in the blood were significantly higher, in 1.5 (p<0.05), 1.3 (p<0.05) and 1.3 (p<0.05) times, respectively, than in those treated with open methods of revascularization. the differences in the results of the coagulation system study in the patients of both groups can be explained by trauma to the inner surface of the arterial bed during endovascular manipulations and, accordingly, the activation of blood coagulation ability [1]. development of hypercoagulable disorders (syndrome) at the intraoperative stage of revascularization: increased fg content, increased sfmcs levels, increased thrombin levels, high fpa content and increased fdp, indicate the activity of factor iia hemocoagulant cascade. taking into account these circumstances, already at the surgery stage an anticoagulant that would have a targeted effect on factor iia of the hemocoagulant cascade should be prescribed. unfractionated heparin (ufh) has table 3. state of the blood aggregation system at the intraoperative stage of the infrainguinal arterial bed revascularization indicator platelet number, ×109/l tav, min. tar, % tat, min. open endo-vasc. open endovasc. open endovasc. open endovasc. before surgery 301.56±14.06 14.17±1.58 74.46±0.62 10.15±0.68 1.5-2.0 h of the surgery 321.26± 13.68 352.68± 15.42 13.26± 1.21 11.34± 1.17* 77.16± 0.87 82.37± 0.89* 9.52±0.81 8.33±0.77 3.0-4.0 h of the surgery 329.46± 12.78 383.45± 14.31 12.19± 1.13 9.68± 1.19* 89.41± 1.05 93.45± 1.01* 8.41±0.81 7.75±0.85 3.0 h after surgery 331.19± 13.83 378.75± 15.24 11.71± 1.11 9.57± 1.18* 85.82± 1.11 90.26± 1.07* 8.02±0.83 7.56±0.86 note. * р<0.05 – significant difference between the indicators rates in patients treated with open revascularization and those who underwent endovascular revascularization. i. k. venger et al. 42 su r g ic a l d is e a se s issn 2413-6077. ijmmr 2022 vol. 8 issue 2 a targeted effect on factor iia, which activates markers of hypercoagulation at the intraoperative stage of surgery [8]. thus, according to the study results, the thromboprophylaxis method for reconstructive interventions on the main arteries of the lower extremity should be as follows: immediately after the surgery, the first dose of ufg should be prescribed with its continuation to the next 7-9 days (aptt control) of the early postoperative period. another scheme of thromboprophylaxis is as follows: ufg administration immediately after surgery with the continuation of its use until 12-24 hours of the early postoperative period. also, after 12-24 hours, the thromboprophylaxis with low-molecular-weight heparin (lmwh) continues [9]. at the same time, for prevention of thromboembolic complications due to activation of the aggregate blood system at the intraoperative stage, following the recommendations of the european society of vascular surgeons (esvs), 2017, and the european society of vascular medicine (esvm), 2019, dual antiplatelet therapy should be prescribed: clopidogrel, acetyl­ salicylic acid for a month or longer. conclusion revascularization of the arterial bed of the lower extremity is accompanied by development of hypercoagulable syndrome at the intraoperative stage of surgery. development of hypercoagulable syndrome at the intraoperative stage of surgery in the patients who underwent endovascular revascularization is more intense than in those treated with open methods of revascularization of the arterial bed. conflict of interests. authors declare no conflict of interest. authors contributions i. k. venger, s. ya. kostiv – conceptualization, methodology, formal analysis, writing – original draft, writing – reviewing and editing; s. ya. kostiv, b. p. selskyy – data curation, writing – reviewing and editing; m. p. orlov, n. i. tsiupryk – investigation, formal analysis. інтраопераційний стан системи гемокоагуляції у хворих з відкритою та ендоваскулярною реваскуляризацією інфраінгвального артеріального сегменту за наявності стенотико-оклюзійного процесу артерій гомілки і. к. венгер, с. я. костів, б. п. сельський, м. п. орлов, н. і. цюприк тернопільський національний медичний університет імені і.горбачевського моз україни вступ. серед усіх післяопераційних ускладнень, пов'язаних з реваскуляризацією атеросклеротичного стено-оклюзійного процесу стегново-дистальної артерії, тромбоз реконструкційного сегмента становить 6-32%. мета. профілактика тромботичних ускладнень шляхом реваскуляризації судин нижньої кінцівки з використанням патогенетично обґрунтованої системи післяопераційної тромбопрофілактики. методи. у дослідження включено 97 пацієнтів з атеросклеротичним стенозо-оклюзійним процесом підпахвинної артерії на тлі стенозо-оклюзійного ураження великогомілкових артерій. для вивчення стану системи гемокоагуляції у хворих застосовували методи дослідження показників коагуляційної, фібринолітичної та агрегаційної систем. результати. формування гіперкоагуляційних розладів у пацієнтів, які застосовували ендоваскулярні методи реваскуляризації артеріального сегмента, відбувається більш інтенсивно, ніж у пацієнтів, яким проводили відкриті методи реваскуляризації судин. гіперкоагуляція крові на інтраопераційному етапі реваскуляризації відбувається в першу чергу за рахунок активності фактора ііа каскаду гемокоагуляції. враховуючи вищезазначені обставини, вже на етапі завершення оперативного втручання з метою тромбопрофілактики слід призначити – нефракціонований гепарин (нфг). одночасно для профілактики тромбоемболічних ускладнень слід призначати подвійну антитромбоцитарну терапію: клопідогрель, ацетилсаліцилову кислоту. висновки. гіперкоагулянтна здатність системи крові, яка виникає після реконструктивних артеріальних втручань, виникає на тлі низької активності фібринолітичної системи крові, але з поступовим підвищенням активності агрегаційної здатності крові. формування гіперкоагуляційних розладів на інтраопераційному етапі хірургічного втручання у пацієнтів з ендоваскулярними методами артеріальних реконструкцій відбувається більш інтенсивно, ніж у пацієнтів з 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endovascular revascularization only in patients with critical limb ischemia complicated with wound infection and diabetes mellitus. european journal of vascular and endovascular surgery. 2013 nov;46(5):575–82. doi: 10.1016/j.ejvs.2013.08.002 8. venher ik, kostiv sya, zarudna oi, kostiv oi. ways of prevention a postoperative venous thrombo embolic complications in patients with plan surgical pathology. hospital surgery. journal named by l.ya. kovalchuk. 2017 nov 8;(3): 49–53. doi: 10.11603/2414­4533.2017.3.8122 9. rosenberg af, zumberg m, taylor l, leclaire a, harris n. the use of anti­xa assay to monitor intra­ venous unfractionated heparin therapy. journal of pharmacy practice. 2010 may 6;23(3):210–6. doi: 10.1177/0897190010362172 received 21 september 2022; revised 2 november 2022; accepted 30 november 2022. this is open access article distributed under the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. артеріальної реваскуляризації. враховуючи ці обставини, вже на етапі завершення оперативного втручання з метою тромбопрофілактики слід призначити антикоагулянт, що має цілеспрямовану дію на фактор ііа гемокоагулянтного каскаду з одночасною профілактикою тромбоемболічних ускладнень – подвійну антитромбоцитарну терапію. ключові слова: система згортання крові; сукупний аналіз; реваскуляризація; тромбоз. information about the authors k. venger – professor of the department of surgery no. 2, i. horbachevsky ternopil national medical university, ternopil, ukraine. https://orcid.org/0000­0003­0170­1995, e­mail: vengerik@tdmu.edu.ua s. ya. kostiv – professor of the department of surgery no. 2, i. horbachevsky ternopil national medical university, ternopil, ukraine https://orcid.org/0000­0002­7963­5425, e­mail: kostivsj@tdmu.edu.ua b. p. selskyy – assistant of the department of surgery no. 2, i. horbachevsky ternopil national medical university, ternopil, ukraine https://orcid.org/0000­0001­6787­4843, e­mail: selskyi_bp@tdmu.edu.ua m. p. orlov – phd student of the department of surgery no. 2, i. horbachevsky ternopil national medical university, ternopil, ukraine https://orcid.org/0000­0002­3400­5243, e­mail: orlov_mypa@tdmu.edu.ua n. i. tsiupryk – phd student of the department of surgery no. 2, i. horbachevsky ternopil national medical university, ternopil, ukraine https://orcid.org/0000­0002­3989­1563, e­mail: tsyupryk_cnad@tdmu.edu.ua i. k. venger et al. 37 su r g ic a l d is e a se s issn 2413-6077. ijmmr 2022 vol. 8 issue 1 *corresponding author: thokchom bishwajit singh, assistant professor (general surgery), department of surgery, military hospital, haryana, 133001, india. e-mail: bishwajitsingh6068@gmail.com. international journal of medicine and medical research 2022, volume 8, issue 1, p. 37-43 copyright © 2022, tnmu, all rights reserved t. b. singh et al. doi 10.11603/ijmmr.2413-6077.2022.1.13083 an observational study to find relationship of acute appendicitis to menstruation cycle in northern and northeastern part of india *t. b. singh1, r. kumar2, a. nigam3, t. r. devi4 1 – military hospital, haryana, india 2 – military hospital, tezpur, assam, india 3 – public health department gangtok, sikkim, india 4 – jawaharlal nehru institute of medical sciences, imphal east, india background. diagnosing acute appendicitis correctly in a female patient is a challenge for a practicing surgeon. rate of misdiagnosis of acute appendicitis is very high among female patients. there are many studies carried out to find incidence of acute appendicitis as per various phases of menstruation cycle but the results were conflicting. objective. the study was conducted to find the relationship of acute appendicitis with the different phases of the menstruation cycle. methods. this research was an observational study carried out in two regional hospitals in northern and north-eastern part of india. duration of study was 24 months between 01 june 2019 and 31 may 2021. inclusion criteria were for all female patients, non-pregnant and menstruating, who were histopathologically confirmed as acute appendicitis. all pathologically proven negative appendectomy patients were excluded from this study. all female patients taking oral contraceptive pills (ocp) were excluded from the study. results. a total of 96 females were hospitalized in both hospitals during the study period; 12 of them did not attain menarche (12.5%), 6 had menopause (6.3%) and 78 were menstruating (81.25%). of those 78 patients, who had physiological menstrual cycles, 6 were in menstrual phase (7.6%), 18 were in proliferative phase (23%), and 54 in luteal phase (69.2%). in our study, there were only 6 cases of acute appendicitis during menstruation. therefore, the expected ratio of cases was 6/14=0.42. the corresponding expected rate for the proliferative phase was 2.78×9=25 cases, whereas for the luteal phase it was 2.78×14=38.92. the expected ratio was 18/25=0.72 for the proliferative phase and 54/39=1.38 for the luteal phase. there was a significant increase in number of cases of acute appendicitis among the menstruating women in luteal phase with p value <0.05. mean age of the study participants was 28.31±9.56. conclusions. according to the result of the study, the incidence of acute appendicitis significantly differs in different phases of menstruation cycle with highest incidence in luteal phase. hence, female hormones (estrogen and progesterone) are significant in causing acute appendicitis. keywords: acute appendicitis; menstruation phase; proliferative phase; luteal phase. introduction diagnosing an acute appendicitis in female patients is a challenge for a practicing surgeon. the rate of misdiagnosis is comparatively high in female patients especially in reproductive age group. the lifetime risk of appendicitis is 6.7% for females [1]. diseases of the female i n t e r n a l re p ro d u c t i v e o r g a n s t h a t m a y erroneously be diagnosed as appendicitis in approximately descending order of frequency include pelvic inflammatory disease, ruptured graffian follicle, twisted ovarian cyst or tumor, endometriosis and ruptured ectopic pregnancy [1]. timely diagnosis and early treatment can prevent complications associated with the disease. the sonographic diagnosis of acute appendicitis has reported sensitivity of 55% to 96% and a specificity of 85% to 98% [1]. ap­ pendicitis is also associated with gastrointestinal symptoms like nausea (sensitivity, 58%; specificity, 36%), vomiting (sensitivity, 51%; speci­ ficity, 45%), and anorexia (sensitivity, 68%; specificity, 36%) [1]. gastrointestinal symptoms that develop before the onset of pain suggest a different etiology such as gastroenteritis [1]. 38 su r g ic a l d is e a se s issn 2413-6077. ijmmr 2022 vol. 8 issue 1 the ratio of cases of appendicitis to cases of pelvic inflammatory disease is low in females in the early phase of the menstrual cycle and high during the luteal phase [1]. many studies showed different results. arnbjo rnsson et al. showed that appendicitis is more common during the luteal phase of the cycle [2] whereas robinson et. al. showed that acute appendicitis is equally common in all phases of the cycle [3]. the aim of the current study was to find out the relationship of acute appendicitis to menstruation cycle. methods this study was an observational study carried out in two regional hospitals in northern and north­eastern parts of india. the study was aimed to assess the relationship of phases of menstruation cycle to acute appendicitis. duration of study was 24 months between 01 june 2019 and 31 may 2021. inclusion criteria: all female patients, nonpregnant and menstruating, who were his­ topathologically confirmed as acute appen­ dicitis. exclusion criteria: all pathologically proven negative appendectomy patients were excluded from this study. all female patients, who were con suming in ocp, were excluded from the study. menstrual cycle was divided into 3 phases: 1. menstruation phase; 2. proliferative phase; 3. luteal phase. duration of two phases was considered as constant (a) menstruation phase: 05 days of menstruation in physiological cycle from the beginning of bleed; (b) luteal phase: last 14 days of cycle (calculated retrospectively 14 days from the first day of menstruation bleed of next cycle excluding the day 1 of bleed). hence, the period of proliferative phase was variable. it was also considered that ovulation occurred at the 14th day of the cycle. results a total of 96 females were hospitalized in both hospitals during the study period (table 1); 12 of them did not attained menarche (12.5%), 06 had menopause (6.3%), and 78 were mens­ truating (81.25%). of those seventy­eight pa­ tients, who had physiological menstrual cycles, 6 were in menstrual phase (7.6%), 18 were in proliferative phase (23%), and 54 – in luteal phase (69.2%). fig. 1. phases of menstruation cycle. menstruation phase: 5 days proliferative phase: [n-(5+14) days] luteal phase: 14 days total days in a cycle: n days table 1. acute appendicitis cases (total number and percentage) as per phases of menstruation number of females percent not attained menarche 12 12.5 menstrual phase 6 6.3 proliferative phase 18 18.8 luteal phase 54 56.3 post menopause 6 6.3 total 96 100.0 t. b. singh et al. 39 su r g ic a l d is e a se s issn 2413-6077. ijmmr 2022 vol. 8 issue 1 t. b. singh et al. four out of seventy-eight acute appendicitis cases (table 2) in menstruating females were complicated type, whereas none in non­menst­ ruating female group had any complications. the expected daily occurrence of acute appendicitis for patients with physiological menstrual cycles (for a 28­day cycle) would have been 78/28 = 2.78 cases/day. the expected rate for cases of acute appendicitis during menstruation (accepting that menstruation lasts for 5 days), would have been 2.78×5=14 cases. in current study there were only 06 cases of acute appendicitis during menstruation. the observed: expected ratio of cases therefore was 06/14=0.42. the corresponding expected rate for the proliferative phase was 2.78×9=25 ca­ ses, whereas for the luteal phase it was 2.78×14=38.92. the observed: expected ratio therefore was 18/25=0.72 for the proliferative phase and 54/39=1.38 for the luteal phase. there was a significant increase in number of cases of acute appendicitis among the menstruating women in luteal phase with p value <0.05. the mean age of the study participants was 28.31±9.56. table 2. total complicated and non-complicated cases menstruating non menstruating total complicated 4 0 4 non complicated 74 18 92 total 78 18 96 table 3. observed frequency, expected frequency and p value with respect to different phases of menstruation cycle phase of menstrual cycle observed frequency expected frequency p value menstrual 6 14 p value 0.034066proliferative 18 25 luteal 54 39 chi-square statistic 6.7589 table 4. mean, median, std deviation and ranges statistics n valid 78 mean 28.31 median 28.00 std. deviation 9.558 range 39 minimum 13 maximum 52 discussion the previous studies by arnbjornsson [2] and eldar et. al. [4] reported that incidence of histologically confirmed acute appendicitis was less common during menstruation phase [1] but the difference were not statistically sig­ nificant. on literature search before these cur­ rent studies, there were conflicting observations. some studies [1, 4] showed higher incidence of acute appendicitis in luteal phase whereas some other [2] – in proliferative and menstrua­ tion phase. another study by robinson et. al. failed to show any difference [3]. the study had established that there was a significant increase in number of cases of acute appendicitis among the menstruating women in luteal phase with p value <0.05. similar result was shown in the studies by eldar et. al. the cause of this significant raise in incidence of acute appendicitis may be because of low estrogen level and high progesterone level modulating immune response and increasing susceptibility for gut infections. souza et. al. [5] reported increased nk activity in follicular phase as compared with luteal phase. progesterone receptors and progesterone induced apoptosis of nk cells and suppresses il-12-induced ifn-g production of killer ig­like receptor (kir)+ nk cells [6]. whereas estrogen has been reported in many stu dies to prevent b cell apoptosis, enhance sur vival and activation of autoreactive cells [7], and increase expression of cd4+ t cell che­ mokine receptors [8]. estrogen exerts immune regu lation via estrogen receptors (ers) on the lymphocytes, and receptors for estrogen, progesterone, androgen, and glucocorticoid are found in lymphoid organs and/or lymphocytes [9, 10]. 40 su r g ic a l d is e a se s issn 2413-6077. ijmmr 2022 vol. 8 issue 1 hall et. al described that concentrations of progesterone fluctuated over the life course in females, with increased concentrations at puberty, cyclical changes during the menstrual cycle, and a steady rise during pregnancy followed by a sharp decline post­partum [11]. at menopause, concentrations steadily declined to levels that are similar to those prior to puberty [11]. progesterone hormone level started rising after ovulation on the 14th day of cycle and reached its highest level during mid-luteal phase (fig. 2). speroff et al. in his study mentioned that serum levels of progesterone fluctuate during the menstrual cycle with a peak of 20 ng ml-1 during the luteal phase and the nadir (<1ng ml-1) during the follicular phase [12]. progesterone generally inhibits inflammatory innate immune responses [11]. in vitro studies by hardy et. al. and jones et. al. showed that progesterone can suppress activation of macrophages and dendritic cells [13, 14]. when progesterone is bound to its receptor, it directly interferes with the transcription factor nuclear factor­kappa b (nf­kb) through transrepression and inhibits gene transcription downstream of the nf­kb pathway, including cyclooxygenase­2 to decrease inflammation [13, 15]. progesterone can also decrease inflammation by inhibiting the production of proinflammatory cytokines (e.g., tnf­a, ifn­g, and il­12) and increasing production of anti-inflammatory cytokines, including il-10 [14, 16]. raised level of progesterone also suppresses th1 response and enhances il-10 producing th2 cells. miyaura et. al. in his study mentioned that progesterone was established to suppress th1 response and enhance il-10 producing th2 cells [17]. ehring et. at. in his study had showed that progesterone suppressed immune functions of t cells by a non-genomic mechanism, which was the blocking of k+ channel, ca2+ signaling and nf of activated t cells driven gene expression [18]. siiteri et. al. had demonstrated in vivo the immunosuppressive effects of progesterone in his study by prolonged survival of xenografts near silastic implants containing progesterone at concentrations typically found in the placenta [19]. hence, such high pro­ gesterone level in luteal phase may have caused unfavorable immunity changes leading to more susceptibility to gut infection and causing highest incidence of acute appendicitis in luteal phase. estrogen hormone increases its level from menstruation phase and reaches its peak at late proliferative phase. estrogen then decline its level and reaches minimum level at late luteal phase. hence, maximum proliferation of both lymphocytes and macrophages occurs during proliferative phase whereas minimum proliferation of both lymphocytes and macrophages occurs during luteal phase. fig. 2. relationship between different level of estrogen and progesterone with percentage of acute appendicitis in different phases of menstruation cycle. t. b. singh et al. 41 su r g ic a l d is e a se s issn 2413-6077. ijmmr 2022 vol. 8 issue 1 t. b. singh et al. whitacre et. al. stated in his study that the effects of estrogen were pleotropic on immune cell function and estrogen had been shown to significantly stimulate the proliferation of both lymphocytes and macrophages [20]. in many studies, estrogen has been shown to regulate immune response by impairing negative selection of high affinity auto­reactive b cells, modulating b cell function and leading to th2 response [21, 22]. estrogen also influences phy­ siological functions via ers which are expressed in brain, gut epithelial cells, lymphoid tissue cells as well as immune cells [23, 24]. r. mo et. al has stated that estrogen also induces t cell homing by enhancing the expression of c-c chemokine receptor type 5 (ccr5), a homing marker [8]. many studies has also shown that estrogen regulates immune res ponse via modulation of endosomal tlrs and tlr8 expression thus hormonal balance determines the overall response to infection in females [25, 26, 27]. hence, such low estrogen level with high progesterone level in luteal phase may cause unfavorable immunity changes leading to more susceptibility to gut infection and causing highest incidence of acute appendicitis in luteal phase and vice versa in proliferative phase. conclusions according to the results of the study, it can be concluded that the incidence of acute appendicitis significantly differs in different phases of menstruation cycle with highest incidence in luteal phase. hence, female hormones (estrogen and progesterone) are significant in causing acute appendicitis. this relationship can be used as additional guiding tool or information when there is diagnostic dilemma for acute appendicitis in females. limitations small sample size. conflict of interests authors declare no conflict of interest. acknowledgements i express thanks to all my interns mr. rameez, mr. praveen, mr. suryakant, mr. ashok, mr. sryakant and mr. agyastha for helping me in collection of data, tabulation and statistical analysis. author’s contributions thokchom bishwajit singh, ranjan kumar – conceptualization, methodology, formal ana­ lysis, writing – original draft, writing – reviewing and editing; ankur nigam – investigation, formal analysis; tekcham roshini devi – data curation, writing – reviewing and editing. взаємозв'язок гострого апендициту та та фази менструального циклу у жителів північної та північно-східної частині індії *t. b. singh1, r. kumar2, a. nigam3, t. r. devi4 1 – military hospital, haryana, india 2 – military hospital, tezpur, assam, india 3 – public health department gangtok, sikkim, india. 4 – jawaharlal nehru institute of medical sciences, imphal east, india вступ. діагностування гострого апендициту у пацієнта може бути проблемною для практикуючого хірурга. частота неправильної діагностики гострого апендициту дуже висока серед пацієнтів. існує багато досліджень, проведених для виявлення частоти гострого апендициту відповідно до різних фаз циклу менструації, але їх результати є суперечливими. мета. дане дослідження проводилося для встановлення та оцінки взаємозв'язку гострого апендициту з різними фазами менструального циклу. методи. дослідження було спостережним, проведеним у двох регіональних лікарнях у північній та північно-східній частині індії. тривалість дослідження становила 24 місяці з 01 червня 2019 року по 31 травня 2021 року. критерії включення: усі пацієнти, які не порушень менструального циклу та які мали гістопатологічне підтвердження діагнозу гострий апендицит. критерії виключення: усі пацієнти яким не проводилася апендектомія чи які приймали оральні контрацептиви, були виключені з дослідження. результати. всього 96 жінок були госпіталізовані в обох лікарнях протягом періоду дослідження. з них 12 не досягли менархе (12,5%), 6 мали менопаузу (6,3%), а 78 – мали менструальні цикли (81,25%). з тих 78 пацієнтів, які мали фізіологічні менструальні цикли; 6 були в менструальній фазі (7,6%), 18 у 42 su r g ic a l d is e a se s issn 2413-6077. ijmmr 2022 vol. 8 issue 1 проліферативній фазі (23%) та 54 у лютеїновій фазі (69,2%). у нашому дослідженні під час менструації було лише 6 випадків гострого апендициту. таким чином, спостережуване: очікуване співвідношення випадків становило 6/14 = 0,42. відповідна очікувана цифра для проліферативної фази становила 2,78×9 = 25 випадків, тоді як для лютеїнової фази було 2,78×14 = 38,92. таким чином, спостережуване/очікуване співвідношення становило 18/25 = 0,72 для проліферативної фази та 54/39 = 1,38 для лютеїнової фази. спостерігалось значне збільшення кількості випадків гострого апендициту серед менструальних жінок у лютеїновій фазі (р<0,05). середній вік учасників дослідження становив 28,31 ± 9,56. висновки. за результатами нашого дослідження можна зробити висновок, що захворюваність на гострий апендицит значно відрізняється різними фазами циклу менструації з найвищою частотою в лютеїновій фазі. отже, жіночі гормони (естроген та прогестерон) відіграють важливу роль у виникненні гострого апендициту. ключові слова: гострий апендицит; фаза менструації; проліферативна фаза; лютеїнова фаза. information about the authors thokchom bishwajit singh, assistant professor (general surgery), department of surgery, 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[internet]. 2014; 151(1):66–77. available from: http:// dx.doi.org/10.1016/j.clim.2014.01.006 and https:// w w w . s c i e n c e d i r e c t . c o m / s c i e n c e / a r t i c l e / p i i / s1521661614000114. received 19 may 2022; revised 27 may 2022; accepted 14 june 2022. this is open access article distributed under the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. 5 in t e r n a l m e d ic in e issn 2413-6077. ijmmr 2022 vol. 8 issue 1 *corresponding author: larysa levytska, professor, i. horbachevsky ternopil national medical university, ternopil, 46002, ukraine. e-mail: levytskal@tdmu.edu.ua international journal of medicine and medical research 2022, volume 8, issue 1, p. 5-10 copyright © 2022, tnmu, all rights reserved l. v. levytska et al. doi 10.11603/ijmmr.2413-6077.2022.1.12910 predictors of low functional reserves in rehabilitation of patients with myocardial infarction complicated by comorbid pathology *l. v. levytska, v. v. yurkiv, m. m. korda i. horbachevsky ternopil national medical university, ternopil, ukraine background. myocardial infarction (mi) is one of the leading causes of death in working age population; the risk of cardiovascular complications for survivors of acute mi complicated by comorbid pathology (cp) is very high. objective. the study is aimed to search for reliable prognostic markers for risk of reducing the functional reserves of the cardiovascular system in myocardial infarction with comorbid pathology. methods. the prospective study involved 371 patients with mi, who received non-invasive therapy and were observed for a 90-day period after admission to the hospital. all patients were examined and treated according to current protocols. results. it was found that 6-minute walk test (6mwt) is a specific and highly sensitive prognostic marker of functional reserves for patients with mi with cp (specificity – 100%, sensitivity – 63%, prognostic value of a positive result – 100%) with the charlson comorbidity index (cci) ≥2. the correlation of 6mwt performed on the 10th, 30th and 90th day of rehabilitation was revealed with the age of patients, spo2, respiratory rate, systolic blood pressure, heart rate, left ventricular ejection fraction, levels of troponin t, creatinine, the number of lymphocytes in the peripheral blood, cci (p<0.05). conclusions. during the 90-day rehabilitation period of a patient with mi complicated by cp, the markers of reduced exercise tolerance to be monitored are: blood pressure levels, respiratory rate, troponin t, creatinine, cholesterol, low-density lipoprotein, spo2, the number of lymphocytes in the peripheral blood. to improve control over the process of rehabilitation in patients with mi complicated by cp the 6mwt and cci should be used. keywords: myocardial infarction; comorbid pathology, charlson comorbidity index, 6-minute walk test, predictors of functional reserves. introduction myocardial infarction (mi) is the main cause of cardiovascular mortality in the human population [1, 2]. to date, there is no single strategy for the use of clinical, laboratory, imaging parameters to predict risks in the treatment and rehabilitation of patients with mi. the task of risk prevention in patients with mi with comorbid pathology (cp) is particularly difficult [3, 4]. the objectives of this study were to identify and evaluate the predictors of severe mi with cp in the stages of rehabilitation. methods the prospective study involved 371 patients with mi, who received non-invasive therapy and were observed for a 90-day period from admission to the hospital in 2014-2019. all patients were examined and treated according to the current protocols. clinical, laboratory and instrumental examinations were performed on the 1st, 10th, 30th and 90th days. cardiovascular functional reserves were determined by the degree of heart failure (killip and nyha), echocardiography and 6-minute walk test (6mwt) on the 10th, 30th and 90th days of mi. risk assessment was performed according to the grace scale and the aacpr cardiovascular risk scale. the charlson comorbidity index (cci) was used to estimate the degree of comorbidity [5, 6, 7]. statistical analysis was performed using software products msexcel 2000, eviews 5.1. and spss. results there were 67.4% of men and 32.6% of women aged (66.2±10.4) years involved in the study. comorbid pathology was detected in 6 in t e r n a l m e d ic in e issn 2413-6077. ijmmr 2022 vol. 8 issue 1 93.8 % of the patients: arterial hypertension – in 84.9%; diabetes mellitus – in 25.1%; vascular pathology – in 35.6%. analysis of exercise tolerance and rehabilitation potential in the study cohort showed that inpatients had predominantly high heart failure and reduced cardiac reserves. thus, the six-minute walk test performed in patients on the 10th day after admission was (76.1±35.2) m; on the 30th and 90th days – (133.8±49.6) m and (207.6±74.1) m, respectively. their nyha functional class was high (2.9±0.7), and the risk on the grace scale and the aacpr risk scale was high and very high (2.9±0.3) and (2.7±0.6), respectively. rehabilitation and hemodynamic potentials, reflected by the functional class according to the rehabilitation classification and parameters of left ventricular systolic function, also indicated a significant depletion of functional reserves of the cardiovascular system. thus, the functional class according to echocardioscopy was (2.9±0.8) points, and the class according to the rehabilitation classification was (3.3±0.7). the possible correlations between the degrees of cardiovascular risk according to the grace, aacpr scales, functional classes according to the rehabilitation classification, func­ tional classes of heart failure (killip, nyha) and the presence and severity of comorbid pathology were assessed. high reliability (p<0.0001) relationships were found between the presence of cp and the categorical indicators of functional class of heart failure according to killip, nyha and the degree of risk according to aacpr (table 1). the proportion of patients at maximum risk for grace was significantly higher in patients with mi with cp than in those without cp (z-test =-4.399; p<0.0001). risk levels for grace in patients with mi with cp directly correlated with age (r=0.267; p<0.0001), respiratory rate (r=0.248; p=0.001), glucose levels (r=0,2; p=0.007) and cci (r=0.275; p<0.0001). high specificity (92.5%) and sensitivity (87.1%) of the grace calculator and the aacpr risk scale (94.2% and 71.0%, respectively) with a high prognostic value of a positive result (97% for both risk scales) and cci ≥2 was determined. the obtained data allow using the grace calculator and the aacpr risk scale to predict early risks and possible negative consequences of the standard rehabilitation program at the hospital stage, as well as stratification of co­ morbid patients into individualized rehabilitation programs in the acute phase of rehabilitation. the correlation of high reliability of cci with risk levels according to grace (r=0.542 according to the association coefficient), functional class according to nyha and 6mwt on the 10th, 30th and 90th days of rehabilitation was revealed; (r6mwt10=-0.318; r6mwt30=-0.397; r6mwt90=-0.425) (p<0.0001). it was established that the six-minute walk test is a specific and highly sensitive prognostic marker of functional reserves for patients with mi and cp (specificity – 100%, sensitivity – 63 %, prognostic value of a positive result – 100%), cci≥2. the correlation of 6mwt performed on the 10th, 30th and 90th days of rehabilitation (6mwt10, 6mwt30 and 6mwt90) was revealed regarding the age of patients (r6mwt10=-0.199; r 6mwt30=-0.287; r 6mwt90=-0.410 respectively; p<0.05), spo2 (r6mwt10=0.399; r6mwt30=0.265; r 6 m w t 9 0= 0 . 2 4 8 ; р < 0 . 0 5 ) , r e s p i r a t o r y r a t e (r6mwt10=-0.318; r6mwt30=-0.357; r6mwt90=-0.298; р<0.0001), systolic blood pressure (r6mwt10=0.179; r6mwt90=0.205; p<0.05), heart rate (r6mwt30=-0.259; r6mwt90=-0.179; p<0.05), left ventricular ejection fraction (r6mwt10=0.706; r6mwt30=0.706; 0.670; r6mwt90=0.583; p<0.0001), levels of troponin t (r6mwt10=-0.210; r6mwt30=-0.312; p<0.05), creatinine (r6mwt10=-0.148; p<0.05), lymphocyte count in peripheral blood (r6mwt10=-0.303; r6mwt30=-0.278; r6mwt90=-0.294; p<0.0001), cci (r6mwt10=-0.323; r6mwt30=-0.398; r6mwt30=-0.398; r6mwt90=-0.427; p<0.0001). table 1. dependence of functional categorical indicators in patients with myocardial infarction on the presence of comorbid pathology index fc according to killip fc according to nyha risk degree according to ааcpr fc according to rehabilitation classification pearson’s coefficient of mutual conjugation 0.258 0.237 0.370 0.286 chuprov coefficient of mutual conjugation 0.189 0.206 0.334 0.251 cramer’s coefficient of mutual conjugation 0.267 0.244 0.398 0.299 χ2 (p-value) <0.0001 <0.0001 <0.0001 <0.0001 l. v. levytska et al. 7 in t e r n a l m e d ic in e issn 2413-6077. ijmmr 2022 vol. 8 issue 1 according to the results obtained, most of these dependence factors are repeated at all three stages of rehabilitation; therefore, their significant impact on exercise tolerance during the 90-day period of the cardiorehabilitation program is obvious. in general, the patients with mi with cp and high age indices, rr, heart rate, creatinine, troponin t and charlsson comorbidity index, as well as low sbp in the acute period of mi, spo2 and lvef have significantly lower tolerance to physical activity at all stages of rehabilitation. therefore, the above parameters can be considered to be the main functional clinical, hemodynamic and laboratory markers of reduced tolerance to exercise; they should be used to monitor the response to increased physical activity and predict the tolerability of rehabilitation measures in patients with mi associated with comorbid pathology. discussion profound disturbances of functional re serves in this category of patients were associated with late treatment (20.34±15.11 hours), and, accordingly, the lack of timely revascularization of the infarct-related artery. the age category of patients (mean age (66.16±10.41) years) and the presence of comorbid pathology in most patients (93.8%) were also significant. this profile of infarct patients, which is characterized by late treatment, and, consequently, the loss of timely revascularization of infarct-related artery, the presence of comorbid pathology and old age of patients, is still common in ukraine and requires special approaches unprescribed in the current protocols of rehabilitation. most studies [8­13] confirm the significant negative impact of comorbid pathology on the functional state and reserve capacity of the cardiovascular system in patients with mi with a high efficiency of adequate rehabilitation programs. the study confirms the possibility of using the charlson index to quantify the degree of comorbidity in patients with mi and various comorbid pathologies and predict their reaction to exercise tolerance in the rehabilitation stages. so, the interdependence of the degree of charlson comorbidity and the six-minute walk test in the stages of rehabilitation was analyzed [14, 15]. it was established that 6mwt10, 6mwt30 and 6mwt90, which reflect exercise tolerance, in the acute (inpatient) and subacute (early and late outpatient) phases of rehabilitation process in the patients with a higher degree of comorbidity were significantly lower (p<0.0001), r6mwt10=-0.318; r6mwt30=-0.397; r6mwt90=-0.425, respectively. analysis of the specificity and sensitivity of the six-minute walk test on the 10th day of rehabilitation of postinfarction patients with the parameters of the charlson comorbidity index ≥2 showed high specificity and prognostic value of a positive result of this marker for comorbid patients (table 2, fig. 1). according to the results of the roc analysis, it was established that the best quality of the model for 6mwt10 (auc=0.75, 95% ci (0.672–0.828) provides a threshold value (distribution point) <83.5 m with the specificity of 100%, sensitivity – 63%, and the prognostic value of a positive result – 100%. the corresponding values for 6mwt performed on the 30th and 90th days after the beginning of mi were also with the maximum specificity (100%) and prognostic value of a positive result (100 %) for 6mwt30 values in the range from 147 to 166.5 m (95% ci (0.766-0.904), p=0.005; area under the roc curve 0.835) and 6mwt90 in the range of 199.5-227.5 m (95% ci (0.741-0.933), p=0.005; area under roc curve 0.837). thus, the analysis of the specificity and sensitivity of the six-minute walk test on the 10th, 30th and 90th days of rehabilitation of postinfarction patients with cci values ≥2 showed maximum specificity (100%) and prognostic value of a positive result (100 %) of this marker in comorbid patients. accordingly, the 6mwt targets by the end of the first decade of the rehabilitation period in patients with mi with the charlsson comorbidity index ≥2 were in the range of 83.5­98 m, by the end of the first month of rehabilitation – 147.0­166.5 m and by the end of the first trimester after the index date – 199.5­227.5 m. the results suggest that the charlson comorbidity index and the sixtable 2. matrix for sensitivity and specificity of 6mwt10 at cci values ≥2 6mwt10 value sensitivity specificity prognostic value of a positive result 83.5 0.63 1 1 98.0 0.80 0.33 0.97 100.5 0.81 0.33 0.97 note. 95% ci (0.672–0.828), p=0.038. l. v. levytska et al. 8 in t e r n a l m e d ic in e issn 2413-6077. ijmmr 2022 vol. 8 issue 1 minute walk test are highly specific and sen­ sitive prognostic markers of exercise tolerance in patients with mi with comorbid pathology at all stages of rehabilitation. conclusions during the 90-day rehabilitation period of a patient with miocardial infarction and cp, the markers of reduced exercise tolerance to be monitored are blood pressure levels, heart rate, respiratory rate, spo2, troponin t, creatinine, the number of lymphocytes in the peripheral blood. the basic test for monitoring functional reserves in patients with mi and cp during the 90-day period is 6mwt. to improve control over the process of rehabilitation in patients with mi and cp, the charlson comorbidity index should be used in addition to grace and aacpr cardiovascular risk scales. conflict of interests authors declare no conflict of interest. author’s contributions larysa v. levytska – conceptualization, methodology, formal analysis, writing – original draft, writing – reviewing and editing; viktoriia v. yurkiv – data curation, writing – reviewing and editing; mykhaylo m. korda – investigation, formal analysis, writing – reviewing and editing. fig. 1. roc­curve of sensitivity and specificity of 6mwt10 at values of ci >2 (area under the curve 0.75, standard error 0.040; p=0.038). l. v. levytska et al. 9 in t e r n a l m e d ic in e issn 2413-6077. ijmmr 2022 vol. 8 issue 1 предиктори низьких функціональних резервів у хворих на інфаркт міокарда, ускладнений коморбідною патологією *л. в. левицька, в. в. юрків, м. м. корда тернопільський національний медичний університет імені і. я. горбачевського моз україни, тернопіль, україна вступ. інфаркт міокарда (ім) є однією з головних причин смертності у працездатному віці, а ризик серцево-судинних ускладнень для тих, хто виживає в гострий період ім з коморбідною патологією (кп), є дуже високим. мета. пошук надійних прогностичних маркерів ризику зниження функціональних резервів серцевосудинної системи при інфаркті міокарда з коморбідною патологією. методи. у проспективне дослідженні було включено 371 хворого на інфаркт міокарда, які отримували неінвазивне лікування та спостерігалися протягом 90 днів з моменту надходження до стаціонару. усі пацієнти були обстежені та проліковані згідно з діючими протоколами. результати. встановлено, що тест шестихвилинної ходьби (тшх) є специфічним і високочутливим прогностичним маркером функціональних резервів у хворих на ім з кп (специфічність – 100%, чутливість – 63%, прогностичне значення позитивного результату – 100% ) з ікч ≥2. виявлено кореляцію тшх, проведеного на 10, 30 і 90 день реабілітації, з віком пацієнтів, частотою дихання (чд), spo2, систолічним артеріальним тиском, частотою серцевих скорочень (чсс), фракцією викиду лівого шлуночка (фв), рівнями тропоніну т, креатиніну, кількістю лімфоцити в периферичній крові та індексом коморбідності чарльсон ікч (р<0,05). висновки. протягом 90-денного періоду реабілітації хворих на інфаркт міокарда з коморбідною патологією маркерами зниженої толерантності до фізичних навантажень, які потребують моніторингу, є рівні артеріального тиску, чсс, чд, spo2, тропоніну т, креатиніну, кількість лімфоцитів в периферичній крові. для покращення контролю за процесом реабілітації у хворих на ім з кп доцільно використовувати тшх та ікч. ключові слова: інфаркт міокарда; коморбідна патологія, індекс коморбідності чарльсон, тест 6-хвилинної ходьби, предиктори функціональних резервів. information about the authors larysa v. levytska, professor, i. horbachevsky ternopil national medical university, ternopil, ukraine https://orcid.org/0000­0002­1327­441x, e­mail: levytskal@tdmu.edu.ua viktoriia v. yurkiv, student, i. horbachevsky ternopil national medical university, ternopil, ukraine https://orcid.org/ 0000­0002­8712­6807, e­mail: yurkiv_vikvit@tdmu.edu.ua mykhaylo m. korda, professor, i. horbachevsky ternopil national medical university, ternopil, ukraine https://orcid.org/0000­0002­6066­5165, e­mail: kordamm@yahoo.com references 1. heart disease and stroke statistics-2021 update: a report from the american heart asso­ ciation. circulation. 2021 feb 23;143(8):254­743. https://www.ahajournals.org/doi/10.1161/cir. 0000000000000950 2. moroz dm. [problems of health and medical care and a model of improvement in modern conditions: a manual for cardiologists, rheumatologists, therapists, health organizers and general practitioners]. in: kovalenko vm, kornatsky vm., editors. institute of cardiology of m.d. strazhesko of the ams of ukraine. kyiv: hordon printing house; 2016 (in ukrainian). 3. caughey ge, ramsay en, vitry ai, gilbert al, luszcz ma, ryan p, et al. comorbid chronic diseases, discordant impact on mortality in older people: a 14-year longitudinal population study. j. epidemiol. community health. 2010;64(12):1036­1042. https://doi.org/10.1136/jech.2009.088260 4. simić­panić d, bošković k, milićević m, rabi žikić t, cvjetković bošnjak m, tomašević­todorović s, et al. the impact of comorbidity on rehabilitation outcome after ischemic stroke. acta clin croat. 2018; 57(1):5–15. https://doi.org/10.20471/acc.2018.57.01.01 5. charlson me, pompei p, ales kl, mackenzie cr. a new method of classifying prognostic comorbidity in longitudinal studies: development and validation. journal chronic disease. 1987;40(5):373­383. https://doi.org/10.1016/0021­9681(87)90171­8 l. v. levytska et al. 10 in t e r n a l m e d ic in e issn 2413-6077. ijmmr 2022 vol. 8 issue 1 6. bellet rn, francis rl, jacob js, healy km, bartlett hj, adams h, et al. repeated six-minute walk tests for outcome measurement and exercise prescription in outpatient cardiac rehabilitation: a longitudinal study. arch phys med rehabil. 2011; 92(9):1388­1394. https://doi.org/10.1016/j.apmr.2011.04.014 7. shved mi, levytska lv. [modern strategies of treating patients with acute coronary syndrome]. kyiv: publishing house medknyha; 2018. p. 176 (in ukrainian). 8. deyo ra, cherkin dc, ciol ma. adapting a clinical comorbidity index for use with icd-9-cm administrative databases. j clin epidemiol. 1992; 45(6):613­619. https://doi.org/10.1016/0895­4356(92)90133­8 9. listerman j, bittner v, sanderson b, brown t. cardiac rehabilitation outcomes: impact of comor­ bidities and age. j cardiopulm rehabil prev. 2011; 31(6):342–348. https://doi.org/10.1097/hcr.0b013e31822f189c 10. rashid m, kwok cs, gale cp, doherty p, olier i, sperrin m, et al. impact of co-morbid burden on mortality in patients with coronary heart disease, heart failure and cerebrovascular accident: a sys­ tematic review and meta-analysis. eur heart j qual care clin outcomes. 2017;3(1):20–36. https://doi.org/10.1093/ehjqcco/qcw025 11. shved mi, levytska lv. [approaches to the assessment of risks and functional reserves of the cardiovascular system in patients with myocardial infarction with comorbid pathology, who are in the acute period of cardio-rehabilitation]. world of medicine and biology. 2018;4(66):124­130 (in ukrainian). https://doi.org/10.26724/2079­8334­2018­4­66­ 124-130 12. levytska l, shved m, korda m. estimation of functional reserves of the body and risk of car diovascular events in patients with myocardial infarction with comorbid pathology undergoing rehabilitation health problems of civilization 2019;13(3):178­186. https://doi.org/10.5114/hpc.2019.86209 13. levytska lv. [constellation of biochemical and physical markers of the functional state of the body in patients with myocardial infarction with of sistolic and diastological arterial pressure levels and the possibility of their using in the rehabilitation pro cess]. mcch. 2019;1:92­102 (in ukrainian). https://doi.org/10.11603/mcch.2410­681x.2019. v0.i1.10013 14. grolla dl, tob t, bombardierc c, wright jg. the development of a comorbidity index with physical function as the outcome. j clin epidemiol. 2005;58(6):595­602. 15. bellet rn, francis r, jacob js, healy km, bartlett hj, adams l, et al. repeated six-minute walk tests for outcome measurement and exercise prescription in outpatient cardiac rehabilitation: a longitudinal study. arch phys med rehabil. 2011; 92(9):1388–1394. https://doi.org/10.1016/j.apmr.2011.04.014 received 15 may 2022; revised 29 may 2022; accepted 14 june 2022. this is open access article distributed under the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. l. v. levytska et al. 18 su r g ic a l d is e a se s issn 2413-6077. ijmmr 2022 vol. 8 issue 1 doi 10.11603/ijmmr.2413-6077.2022.1.13098 antibacterial therapy for patients with burn injuries s. y. zaporozhan, *d. b. fira, o. v. pokryshko i. horbachevsky ternopil national medical university, ternopil, ukraine background. treatment of burn wound infection is an urgent issue of contemporary medicine, including surgery, combustiology and microbiology. it is established that infectious complications are a challenge for burn patients. in the course of wound reparation, infectious complications may worsen. along with surgical treatment, mechanical removal of pathogens from burn wounds is also important as well as antimicrobials for patients with severe burns. objective. the aim of the study was to define the most common pathogens of purulent-inflammatory complications of burn wounds and their susceptibility to antibiotics. methods. the study involved patients treated at the center of thermal trauma and plastic surgery of lviv i-territorial medical association, the unit of st. luke hospital of lviv. collection of material from wound secretions of burn wounds was performed with sterile swab. the study was performed before prescription of antibiotics, at the end of the first and second weeks of the disease. the pathogens were isolated and identified. antibiotic susceptibility was studied using standard research methods. the obtained results were analyzed by means of the software package of the microbiological monitoring system whonet 5.2 (who collaborating centre for surveillance of antimicrobial resistance) and the program microsoft office excel 2007. results. the study of smears from burn wounds proved that 240 strains of gram-positive and gram-negative microorganisms that caused purulent-inflammatory processes were isolated. among the selected causative agents of a burn wound complicated by a purulent-inflammatory process, gram-negative bacteria predominated (60.8% of all detected microorganisms). gram-positive flora of s. epidermidis and s. aureus were more common in the wound surface during the first week of the disease. in most patients with severe burns, bacterial associations were isolated from the wound surface (66.3%) in two and three weeks, and in three weeks candida spp. were isolated. non-fermenting rods a. baumannii and p. aeruginosa dominated among the gram-negative flora isolated from the wound surface of burns. the analysis of susceptibility of microorganisms isolated from patients with burns to antibiotics showed that almost all of the cultures were polyresistant. conclusions. gram-negative microorganisms, strains of non-fermenting bacteria predominated among the pathogens isolated from burn wounds complicated by purulent inflammation; staphylococcus aureus prevailed among the gram-positive ones. the most significant clinical strains were highly polyresistant to antibiotics. keywords: smears from burn wounds; strains of microorganisms; antibiotics; resistance. *corresponding author: dmytro fira, associate professor, i. horbachevsky ternopil national medical university, ternopil, 46002, ukraine. e-mail: firadb@tdmu.edu.ua international journal of medicine and medical research 2022, volume 8, issue 1, p. 18-24 copyright © 2022, tnmu, all rights reserved introduction according to the who, injuries, burns, poisonings, etc. are the third in the structure of human mortality. every year about 840 million people suffer from burns and about 180 thousand people die in the world. in ukraine, more than 100,000 cases of burns are registered annually, and 60-80% of those burned have superficial burns of the skin of ii­iii a degree, which do not require surgical intervention [1, 2, 3, 4]. treatment of burn wound infection is an urgent issue of contemporary medicine, in particular, surgery and combustiology. according to literature [5, 6], despite the constant improvement of wound healing methods, the frequency of its infectious complications in surgery is 30%. traditional remedies and treatments for infected burns are often ineffective. this necessitates further search for new and improvement of existing medications and treatment that stimulate reparative processes in infected wounds, as well as in-depth study of the mechanisms of action of antibiotics [7]. today, there is a wide range of medications for conservative treatment of burns, but none of them is sufficiently effective. it is established that infectious complications are a challenge for patients with burns. according to the literature, their frequency correlates with the depth and area of burns. complications s. y. zaporozhan et al. 19 su r g ic a l d is e a se s issn 2413-6077. ijmmr 2022 vol. 8 issue 1 s. y. zaporozhan et al. in patients with burns are caused by disturbance of barrier function of the skin, reduction of its protective properties due to the action of traumatic factors and suppression of the immune system of these patients [8, 9, 10]. the most common cause of patient mortality is infection, which accounts for about 76.3% of burn mortality. in cases of thermal damage, coagulation necrosis of the epidermis, of various layers of the dermis and adjacent tissues develops that creates favorable conditions for massive microbial invasion. infectious complications worsen the course of reparative processes in the wound. in patients with severe burns antimicrobial therapy is important together with surgical treatment aimed at mechanical removal of pathogens from burn wounds [11, 12, 13]. the aim of the study was to define the most common pathogens of purulent­inflammatory complications of burn wounds and their susceptibility to antibiotics. methods the study involved patients treated at the center of thermal trauma and plastic surgery of lviv i-territorial medical association, the unit of st. luke’s hospital of lviv. collection of material from the wound secretions of burn wounds was performed with sterile swab. burn wounds in all patients were studied before prescription of antibiotics, at the end of the first and second weeks of the disease, which included isolation of pathogens, their identification by morphological, cultural and biochemical properties. antibiotic susceptibility was studied using standard research methods according to the order of the ministry of health of ukraine no. 167 “on approval of guidelines for determination of susceptibility of microorganisms to antibac terials”, dated april 05, 2007 and the recommendations of the international committee of clinical standards (nccls, 2002). statistical processing and analysis of the results was performed using the software package of the microbiological monitoring system whonet 5.2 (who collaborating centre for surveillance of antimicrobial resistance) and microsoft office excel 2007 [14]. results the results of the studies showed that in patients with burn trauma during the first week of the disease gram­positive flora of s. epidermidis and s. aureus were more common according to microbiological examination of smears from wound surfaces. candida spp. were isolated in patients with severe burns on the third week of the disease, which might have been associated with immunosuppression due to thermal trauma and development of antibacterial resistance [15]. depending on this, susceptibility of the main pathogens to antibacterials was evidenced. the study found that s. aureus showed high resistance to ceftriaxone (78-80%) and carbapenems (70-73%), high susceptibility to fluoroquinolones, including ciprofloxacin (71.5%) and levofloxacin (67.5%). p. aeruginosa strains were susceptible to carbapenems, in particular to meropenem (80 %) and imipenem (95%). on day 18-20 from the moment of the injury, pseudomonas aeruginosa was isolated from the wound in 65-70% of patients. strains of pseudomonas aeruginosa were moderately resistant, retaining susceptibility to carbapenems. as a result of microbiological examination of smears from burn wounds, etiologically signi ficant pathogens of infectious complications in patients with dermal burns were isolated. a total of 240 strains of gram-positive and gramnegative microorganisms were isolated from burn wound surfaces, which led to development of purulent­inflammatory processes. on the seventh day after burns in 62.3% of cases, microorganisms were isolated from patients in monoculture, and only 37.7% – in associations. in two and three weeks of the disease, most patients with severe burns had bacterial associations isolated from the wound surface (66.3%), and on the third week of the disease candida spp. were isolated due to possible immunosuppression on the background of thermal trauma. acinetobacter baumannii (30.0%), pseudomonas aeruginosa (20.4%) and staphylococcus aureus (15%) are leading in the spectrum of isolated clinical strains (fig. 1). most often, they formed associations from the second week of the disease. among the isolated pathogens of purulentinflammatory complications of burn wounds, gram-negative bacteria predominated (60.8 % of all isolated microorganisms) (fig. 2). non-fermenting rods a. baumannii and p. aeruginosa (48.3% and 34.9%, respectively) dominated in the gram­negative flora isolated from the burn wound surface; they were most often isolated after the first week of the disease. in contrast, enterobacteria accounted for only 17.1 % (fig. 3); escherichia coli (8.7 % of isolated 20 su r g ic a l d is e a se s issn 2413-6077. ijmmr 2022 vol. 8 issue 1 fig. 3. species of gram-negative bacteria isolated from burn surfaces,% fig. 1. the range of microorganisms isolated from burn surfaces,%. fig. 2. the range of microorganisms isolated from purulent­inflammatory surfaces of burn wounds,%. 0 5 10 15 20 25 30 15,0 11,6 4,6 2,1 2,5 1,3 5,4 1,7 1,3 0,4 1,6 30,0 20,4 2,1 60,8 % 37,1 % 2,1 % gram-negative bacteria gram-positive bacteria yeast fungi 8,7 % 2,7 % 2,0 % 0,7 % 2,7 % 48,3 % 34,9 % e. coli k. pneumoniae p. mirabilis p. vulgaris e. cloacae a. baumannii p. aeruginosa s. y. zaporozhan et al. 21 su r g ic a l d is e a se s issn 2413-6077. ijmmr 2022 vol. 8 issue 1 s. y. zaporozhan et al. gram-negative rods), enterobacter cloacae (2.7 %), klebsiella pneumoniae (2.7 %), proteus mirabilis (2.0%), proteus vulgaris (0.7%) were isolated among them. gram-positive cocci were isolated 1.6 times less (37.1%) (fig. 4). staphylococci were predominant among them. they accounted for 75 % of isolated strains of cocci, and cultures of s. aureus (40.4% of all identified strains of cocci) were most often isolated among them. coagulase-negative cocci were represented by cultures of s. epidermidis and s. haemolyticus (31.5 % and 12.4%, respectively). enterococci were represented by strains of enterococcus faecalis and enterococcus faecium, which accounted for 10.1 % of the coccal flora, respectively. streptococcus pyogenes was isolated only in 5.6%. enterococci were more often isolated in the first week of the disease, staphylococci in the following weeks. candida spp. accounted for only 2.1 % of all isolated microorganisms. the analysis of the susceptibility of microorganisms isolated from patients with burn disease to antibiotics showed that these clinical strains had high resistance to antibacterials, especially those that most often infect the wound surfaces. almost all isolated strains were polyresistant. the identified strains of p. aeruginosa were low susceptible to cephalosporins, in particular: cefepime (92.3%), ceftriaxone (86.5%), ceftazidime (80.8%), cefotaxime (69.2%). almost half of the isolated cultures of pseudomonas aeruginosa were resistant to aminoglycosides: to gentamicin – in 46.2% cases, to amikacin – 42.3 %. meropenem and imipenem showed also low effectiveness, although they were reserve antibiotics. clinical strains of p. aeruginosa showed resistance in 51.9 % and 82.7 % cases, respectively. the most effective antimicrobial against pseudomonas aeruginosa was doxycycline. resistance to it was only 19.2 %. like p. aeruginosa strains, isolated cultures of a. baumannii had a high level of antibiotic resistance. cephalosporin antibiotics, in particular ceftazidime (97.2 %), ceftriaxone (95.8 %), cefepime (91.7 %), cefotaxime (86.1 %), were ineffective against acinetobacteria. strains of a. baumannii to gentamicin and amikacin (73.6 % and 79.2 %, respectively) were highly resistant. they were also low susceptible to fluoroquinolones, i.e.: levofloxacin (76.4 %), ciprofloxacin (68.1%), gatifloxacin (63.9%). meropenem and imipenem were more effective than other groups of antibacterials against acinetobacteria, only in 31.9% and 40.2% of resistant cultures of a. baumannii. isolated cultures of s. aureus were resistant to oxacillin (63.9%) that indicated methicillin resistance of these strains, as well as cephalosporin antibiotics, in particular: cefepime, ceftazidime, ceftriaxone (from 19.4 % to 33.3 %, respectively). staphylococcus aureus was highly resistant to azithromycin (66.7 %) and lincosamides such as clindamycin (72.2 %) and doxycycline (52.8 %). fluoroquinolones were also low effective, i.e.: ciprofloxacin (58.3 %) and fig. 4. species of gram-positive bacteria isolated from burn surfaces,% 40,4 % 31,5 % 12,4 % 5,6 % 6,7 % 3,4 % s. aureus s. epidermidis s. haemolyticus s. pyogenes e. faecalis e. faecium 22 su r g ic a l d is e a se s issn 2413-6077. ijmmr 2022 vol. 8 issue 1 levofloxacin (25.0­44.4 %), as well as aminogly­ cosamides: gentamicin (30.6 %) and amikacin (38.9 %). in contrast to non-fermenting bacteria, clinical strains of s. aureus were the most susceptible to carbapenems: meropenem and imipenem resistant cultures of staphylococcus aureus were only 8.3% and 5.6% of strains. discussion burns destroy the first barrier of human innate immunity that protect tissue from the colonised external world, and microorganisms can easily spread and infiltrate necrotic tissue [15, 16]. our data confirmed many published studies, which have reported gram-negative bacteria as the commonest microorganisms that colonize burn wounds [17]. initial burn wounds are sterile. however, within a few days, gram-positive strains, such as staphylococcus aureus, coagulase-negative staphylococcus, and streptococcus spp., start to colonize the wounds from deeper structures (hair follicles and glands). in the second phase, a gram-negative shift takes place, where p. aeruginosa, e. coli, and proteus are the predominant isolates [18, 19, 20]. if left untreated, this colonization can lead to infection [20, 21]. our data confirmed the steps of infectious process developing on burn wounds. the study had established that s. aureus among gram-positive microbes and p. aeruginosa among gram-negative microbes were the most frequent microbial isolates in our patients(40.4% and 34.9% respectively). similar observation was seen in the study by tsolakidis s. et. al. [18]. in our study, we found a variable percentage of antibiotic resistance among the cultured bacteria. the analysis of the studied isolates susceptibility to antibiotics showed that most often infecting the wound surfaces clinical strains had high resistance to antibacterials. almost all isolated bacteria were multiresistant. 63.9% of identified s. aureus belong to mrsa staphylococci. this had less incidence with other studies on mrsa in burn patients by mandal [21]. isolates of s. aureus (more than 70 % of all of them) were found as highly resistant to cephalosporins, including cefepime, ceftazidime, ceftriaxone, to lincosamides, such as clindamycin (72.2 %) and doxycycline (52.8 %). s. aureus was highly resistant to fluo­ roquinolones, i.e.: ciprofloxacin (58.3 %) and levofloxacin (25.0­44.4 %), as well as aminogly­ cosamides: gentamicin (30.6 %) and amikacin (38.9 %). however, isolates of s. aureus were susceptible to fluoroquinolones, including cipro floxacin (71.5 %) and levofloxacin (67.5%). in contrast to non-fermenting bacteria, clinical strains of s. aureus were the most susceptible to carbapenems: meropenem and imipenem; only 8.3 % and 5.6 % of strains of s. aureus were resistant to these antibiotics. p. aeruginosa strains as well as s. aureus were resistant to cephalosporins, in particular: cefepime (92.3 %), ceftriaxone (86.5 %), ceftazi dime (80.8 %), cefotaxime (69.2 %); to aminoglycosides: gentamicin (46.2 %), amikacin (42.3 %); to meropenem and imipenem (almost half of the isolated cultures of p. aeruginosa). and moderate resistant to carbapenems, in particular: to meropenem (80 %) and imipenem (95 %). the most resistant antibiotics found in most of studies were cephalosporins and quinolones [7, 8, 23, 24, 25]. in contrast, some authors reported no isolated bacteria found resistant to gentamicin [24], or susceptible to aminoglycosides [25]. conclusions it was established that among the pathogens isolated from complicated purulent­inflam ma­ tory burn wounds, gram-negative micro organisms of non-fermenting bacteria predominated and staphylococcus aureus among gram-positive ones. the most significant clinical strains were highly multiresistant to antibiotics. funding this research received no external funding. conflict of interest the authors declare no conflict of interests in this study. author’s contributions stepan zaporozhan – conceptualization, writing – original draft; dmytro fira – formal analysis, writing – original draft, writing – reviewing and editing; olena pokryshko – data curation, writing – reviewing and editing. s. y. zaporozhan et al. 23 su r g ic a l d is e a se s issn 2413-6077. ijmmr 2022 vol. 8 issue 1 s. y. zaporozhan et al. антибактеріальна терапія у хворих із опіковою травмою с. й. запорожан, д. б. фіра, о. в. покришко тернопільський національний медичний університет імені і. я. горбачевського, тернопіль, україна вступ. лікування опікової ранової інфекції є актуальною проблемою сучасної медицини, зокрема хірургії, комбустіології й мікробіології. відомо, що у хворих з опіками інфекційні ускладнення становлять серйозну проблему. інфекційні ускладнення погіршують перебіг репаративних процесів у рані. водночас із хірургічними методами лікування, спрямованими на механічне видалення збудників з опікових ран, важливе значення має застосування антимікробних лікарських засобів у хворих із тяжкими опіками. мета. визначити найбільш поширені збудники гнійно-запальних ускладнень опікових ран у хворих та їх чутливість до антибіотиків. методи. дослідження проводилось у пацієнтів, що перебували на стаціонарному лікуванні у центрі термічної травми і пластичної хірургії кнп «і-е територіальне медичне об'єднання м. львова» відокремленого підрозділу «лікарня святого луки» м. львів. забір матеріалу із ранових виділень опікових ран здійснювали за допомогою стерильного тампону. дослідження проводили у хворих до початку застосування антибіотиків, наприкінці першого і другого тижня захворювання. виділяли збудники, ідентифікували їх. чутливість до антибіотиків вивчали за допомогою стандартних методів дослідження. отримані результати піддавали аналізу, який включав пакет програм системи мікробіологічного моніторингу "whonet 5.2" (who collaborating centre for surveillance of antimicrobial resistance) та програму «microsoft office exel 2007». результати. у результаті проведеного дослідження мазків з опікових ран виділено 240 штамів грампозитивних та грамнегативних мікроорганізмів, які спричиняли гнійно-запальні процеси. серед виділених збудників гнійно-запальних ускладнень опікових ран переважали грамнегативні бактерії (60,8% усіх виділених мікроорганізмів). впродовж першого тижня захворювання у рановій поверхні частіше зустрічалися грампозитивна флора s. epidermidis та s. aureus. після двох і трьох тижнів захворювання у більшості пацієнтів із важкою опіковою травмою з ранової поверхні висівали асоціації бактерій (66,3%) та на третьому тижні захворювання виділяли ще й гриби роду candida spp. у грамнегативній флорі, висіяній із ранової поверхні опіків, домінували неферментуючі палички a. baumannii та p. aeruginosa. результати аналізу чутливості мікроорганізмів, виділених від хворих із опіковою хворобою, до антибіотиків показали, що практично всі висіяні культури були полірезистентними. висновки. серед виділених збудників гнійно-запальних ускладнень опікових ран переважали грамнегативні мікррорганізми, штами неферментуючих бактерій, серед грампозитивних – золотисті стафілококи. найбільш значущі клінічні штами були високо полірезистентними до антибіотиків. ключові слова: мазки з опікових ран; штами мікроорганізмів; антибіотики; резистентність. information about the authors stepan zaporozhan, professor, i. horbachevsky ternopil national medical university, ternopil, ukraine https://orcid.org/0000­0002­4038­2010, e­mail: zaporozhan@tdmu.edu.ua dmytro fira, associate professor, i. horbachevsky ternopil national medical university, ternopil, ukraine https://orcid.org/0000­0002­0590­8910, e­mail: firadb@tdmu.edu.ua olena pokryshko, associate professor, i. horbachevsky ternopil national medical university, ternopil, ukraine https://orcid.org/0000­0001­9640­0786, e­mail: pokryshko@tdmu.edu.ua references 1. world health organization media center. burns. available online: http://www.who.int/media­ centre/ factsheets/fs365/en/ (accessed on 1 march 2017). 2. vikas singh, bharat mishra, rahul pandey. burn wound infection and antibiotic susceptibility patterns in a tertiary care teaching centre in western india. international journal of scientific research. 2019 sep; 8(9):1­2. https://doi.org/10.36106/ijsr 3. rafla k, tredget ee. infection control in the burn unit. burns. 2011;37:5–15. https://doi.org/10.1016/j.burns.2009.06.198. 24 su r g ic a l d is e a se s issn 2413-6077. ijmmr 2022 vol. 8 issue 1 4. greenhalgh dg, saffle jr, holmes jh. american burn association consensus conference to define sepsis and infection in burns. j. burn care res. 2007; 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bacteriological profile and antimicrobial resistance patterns of burn wound infections in a tertiary care hospital. heliyon. 2019;5(12):e02956. https://doi.org/10.1016/j.heliyon.2019.e02956. received 20 may 2022; revised 29 may 2022; accepted 14 june 2022. this is open access article distributed under the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. s. y. zaporozhan et al. 55 su r g ic a l d is e a se s issn 2413-6077. ijmmr 2022 vol. 8 issue 1 doi 10.11603/ijmmr.2413-6077.2022.1.12934 short term study on clinical relevance and outcomes of foetal hydronephrosis j. s. randhawa1, *n. rajendran2, j. husain2, r. shankaran1, v. v. nair2, s. dorai b.3 1 – institute of naval medicine, inhs asvini, mumbai, maharashtra, india 2 – armed forces medical college, pune, maharashtra, india 3 – tata memorial hospital and research centre, mumbai, maharashtra, india background. hydronephrosis is the most common urinary tract pathology detected on antenatal screening by ultrasound. the detection of fetal hydronephrosis by ultrasound pres ents a treatment dilemma to the treating surgeon and parental anxiety. objective. this study aims to examine the role of serial ultrasounds in antenatally detected fetal hydronephrosis to know the disease progression, and to assess indications and timing of surgery in these patients to preserve renal function. methods. this is an observational study of 30 cases of foetal hydronephrosis conducted at a tertiary care paediatric surgery centre. a foetal renal pelvic antero-posterior diameter (apd) >7 mm at 32 weeks of gestation is considered to indicate foetal hydronephrosis. these patients are followed up for a period of two years from 2018 to 2020. progression of disease and need for surgical intervention is noted. results. the study comprised 30 cases of antenatal foetal hydronephrosis (42 renal units). of 26 renal units with mild hydronephrosis, all are resolved in the post-natal period. of 10 renal units with moderate hydronephrosis, 3 (30%) resolved and 7 (70%) worsened and required surgical intervention. of 6 renal units with severe hydronephrosis, all required surgical intervention and underwent surgery between 12 to 18 months of age. conclusions. these results suggest a grading system with antero-posterior diameter of renal pelvis distinguishes those cases with moderate and severe degrees of hydronephrosis that are at higher risk of surgery. keywords: antero-posterior renal pelvic diameter; antenatal fetal hydronephrosis; pyeloplasty. *corresponding author: nagamahendran rajendran, clinical tutor, department of surgery, armed forces medical college, pune, maharashtra, 411040, india. e-mail: nagaa.mahendran@gmail.com international journal of medicine and medical research 2022, volume 8, issue 1, p. 55-61 copyright © 2022, tnmu, all rights reserved j. s. randhawa et al. introduction antenatal ultrasound surveillance detects significant fetal anomaly in 1% of pregnancies; 20-30% of them are genitourinary cases, and 50% – manifest as hydronephrosis [1]. much of the controversy arises from diagnostic dilemmas and difficulties in distinguishing lesions which are obstructive and potentially harmful to the developing fetal kidney. controversy remains on the efficacy of therapeutic inter­ vention due to limited knowledge of the underlying natural history and determining appropriate outcome measures. furthermore, early diagnosis of hydronephrosis may cause anxiety to parents in rest of the pregnancy. early onset of fetal hydronephrosis is directly related to prognosis [2]. antenatally detected hydronephrosis is diagnosed if the renal pelvic ap diameter equals or exceeds 4 mm before 28 weeks or 7 m thereafter [3]. the study took place at the tertiary care paediatric surgery centre to assess the progression and disease course of antenatally detected fetal hydronephrosis. the issue which is still ambiguous at present and is the area of much study is how to differentiate a dilated but non obstructed system from a dilated and obstructed system [4]. to alleviate this pitfall, an integrated approach right from antenatal ultrasonogram to the latest available investigation techniques including dmsa, schintigraphy scan was performed to deepen the understanding of progression of disease in patients with suspected puj obstruction [5]. follow up of antenatally detected fetal hydronephrosis with serial ultrasounds was carried out to identify the disease progression in terms of antero-posterior diameter of renal pelvis. clinical and imaging parameters were 56 su r g ic a l d is e a se s issn 2413-6077. ijmmr 2022 vol. 8 issue 1 analyzed in antenatal and postnatal period to assess the functional status of urinary system. the indication and right time for surgical intervention was assessed to preserve renal function. this understanding of the functional status of the urinary system also guides in timely surgical intervention. methods this observational study was done at the tertiary care centre in the department of paediatric surgery after due permission from the institutional ethics committee and after taking written informed consent from the patients. once the patients were enrolled for the study, a thorough history and physical examination was done as per proforma (table 1). the complete profile with clinical, radio­ logical imaging and renal scan details were taken as per study proforma and were followed up and outcomes were assessed for complete resolution, partial resolution and persisting cases requiring surgical intervention (fig. 1). the antenatal usg at around 32 weeks was performed to diagnose anh, and renal pelvic anteroposterior diameter (apd) between <7mm was taken as mild, 7-10 mm was taken as moderate and >10mm was taken as severe hydronephrosis (table 2, 3). imaging with ultrasound: all new-borns with history of anh underwent postnatal usg between days 3-7 of life (table 4). neonates with suspected posterior urethral valves, oligohydramnios or severe bilateral hydro nephrosis underwent ultrasonography within 24-48 hours of birth [6]. subsequent ultrasounds were done at 4-6 weeks, 3 months, 6 months, and 6 monthlies thereafter till resolution/ cor rection. two consecutive normal ultra sounds were considered as the criteria for resolution. voiding cystourethrogram: vcug was performed for cases with moderate to severe hydronephrosis, if hydronephrosis worsens on follow up usg, presence of ureteral dilatation, features of lower urinary tract obstruction and in urinary tract infection. fig 1. consort flow diagram. assessed for eligibility (n=30) excluded (n=0) underwent surgery (n=0) resolved spontaneously (n=15) analysis follow-up usg apd at second trimester enrollment moderate (apd 7-10 mm) (n=9) severe (apd >10 mm) (n=6) allocation resolved spontaneously (n=2) resolved spontaneously (n=0) underwent surgery (n=7) underwent surgery (n=6) mild (apd 4-7 mm) (n=15) j. s. randhawa et al. 57 su r g ic a l d is e a se s issn 2413-6077. ijmmr 2022 vol. 8 issue 1 radioisotope studies: diuretic renography was done at 6-8 weeks of life. radioisotope studies were done in moderate to severe hydronephrosis, worsening hydronephrosis and dilated ureter on usg. antibiotic prophylaxis: the postnatally confirmed anh of moderate grade, severe grade or with dilated ureters were put on antibiotic prophylaxis with syp. cephalexin 10 mg/kg/ day as per our institution protocol till evaluation were complete (table 5). table 1. demographic data parameter no. of patients no. of kidneys gender female 8 (26.67 %) 10 male 22 (73.33 %) 32 affected side left 12 (40 %) 12 right 6 (20 %) 6 bilateral 12 (40 %) 24 second trimester usg mild 15 (50 %) 26 moderate 9 (30 %) 10 severe 6 (20 %) 6 third trimester usg resolved 13 (43.33 %) 24 mild 4 (13.3 %) 4 moderate 5 (16.66 %) 6 severe 8 (26.66) 8 table 4. severity of anh, post-natal (kidneys=42) u/l vs b/l resolved (n=26) mild 4-7 mm (n=0) moderate 7-10 mm (n=4) severe >10 mm (n=10) no of kidneys no of kidneys no of kidneys no of kidneys unilateral 4 (15.38%) 0 (0%) 2 (50%) 10 (100%) bilateral 22 (84.62%) 0 (0%) 2 (50%) 0 (0%) table 2. severity of anh, second trimester (kidneys=42) u/l vs b/l mild 4-7 mm (n=26) moderate 7-10 mm (n=10) severe >10 mm (n=6) no of kidneys no of kidneys no of kidneys unilateral 4 (15.38%) 8 (80%) 6 (100%) bilateral 22 (84.62%) 2 (20%) 0 (0%) table 3. severity of anh, third trimester (kidneys=42) u/l vs b/l resolved (n=24) mild 4-7 mm (n=4) moderate 7-10 mm (n=6) severe >10 mm (n=8) no of kidneys no of kidneys no of kidneys no of kidneys unilateral 2 (8.33%) 4 (100%) 4 (66.67%) 8 (100%) bilateral 22 (91.67%) 0 (0%) 2 (33.33%) 0 (0%) table 5. descriptive analysis of uti in study population (n=30) urine c/s frequency percentages no growth 23 76.67% growth 7 23.33% e. coli 4 57.14% kelbsiella pneumoniae 1 14.29% mixed growth 1 14.29% staph hominis 1 14.29% j. s. randhawa et al. 58 su r g ic a l d is e a se s issn 2413-6077. ijmmr 2022 vol. 8 issue 1 results there was significant male preponderance in the study population; 73% of the study population was comprised by male gender. while comparing right and left apd in cases of unilateral hydronephrosis, left apd was significantly more than the right. post­natal apd mean difference was also the highest with left sided hydronephrosis. as the trimester increases, distension of urinary bladder was on rise and it was directly proportional to the degree of anh in the early trimester. oligohydramnios was an indirect marker for severe congenital anh with almost 50% of the antenatal mothers had oligohydramnios during their pregnancy. in our study, out of 30 patients, 17 cases underwent spontaneous resolution. those cases with mild hydronephrosis, all were resolved postnatally. in the moderate hydronephrosis cases, some of them were resolved, and in the severe hydronephrosis group, further evaluation and surgical intervention was done in all cases. it was further observed that, for those cases, where outlet obstruction was suspected, radio nuclide studies were completed to assess the excretory function of kidney. 13 patients with persistent severe hydronephrosis in post-natal period underwent further evaluation with mcu and mru. in those patients, e. coli remained the table 6. post-natal analysis of operated vs non-operated cases severity operated non-operated mild (n=15) 0 (0%) 15 (100%) moderate (n=9) 7 (77.78%) 2 (22.22%) severe (n=6) 6 (100%) 0 (0%) table 7. operated post-natal pathology (n=13) pathology operated cases percentage puj obstruction 9 69.23% vesico ureteric reflux 2 15.38% posterior urethral valve 1 7.69% vuj obstruction 1 7.69% table 8. descriptive analysis of time of surgery in the study population (n=13) time of surgery frequency percentages within 10 days 1 7.69% 10 days to 6 months 3 23.07% 6 months to 12 months 3 23.07% 12 months to 18 months 6 46.15% table 9. descriptive analysis of surgical procedure done in the study population (n=30) post-surgical outcome frequency percentages underwent surgery 13 43.33% underwent mah 9 69.23% ureterostomy with ureter reimplantation 3 23.07% fulguration 1 7.69% surgery: the progression of disease was screened by postnatal ultrasound. in cases of persistent and severe hydronephrosis, after assessing the functional status of kidneys, surgery was planned according to the etiology (table 6, 7). the type of surgery performed and the ideal timing of surgery were assessed (table 8, 9). quantitative data was presented by the mean and standard deviation. comparison among the study groups was performed by the unpaired t test as per results of normality test. qualitative data was presented via the frequency and percentage table. association among the study groups was assessed by the fisher test, student’ t-test and chi-square test. p value less than 0.05 was taken as significant. j. s. randhawa et al. 59 su r g ic a l d is e a se s issn 2413-6077. ijmmr 2022 vol. 8 issue 1 most common infectious organism in urine amounting to 57% of cases in patients with symptomatic uti. pelvi ureteric junction obstruction was the commonest indication for surgery amounting to 61%. in the study, most of the infants born with persistent and severe hydronephrosis underwent corrective surgery in 12 months. this complies with the institution protocol of more conservative and follow up approach for definitive period before a surgical decision was taken. among the operated 13 cases, left sided modified anderson hyne’s pyeloplasty was the commonest procedure amounting to 62.5% of the cases operated. in the surgical patients, left sided poor functioning kidney was maximum with less than 50 % split function in more than 80 % of the surgical group. there was significant male preponderance in our study population. 73% of the study population was comprised by male gender. while comparing right and left apd in cases of unilateral hydronephrosis, left apd was significantly more than the right. post­ natal apd mean difference was also the highest with left sided hydronephrosis. as the trimester increases, distension of urinary bladder was on rise and it was directly proportional to the degree of anh in the early trimester. oligohydramnios was an indirect marker for severe congenital anh with almost 50% of the antenatal mothers suffering from oligohydramnios during their pregnancy. in the study, out of 30 patients. 17 cases underwent spontaneous resolution. all cases with mild hydronephrosis were resolved postnatally. in the moderate hydronephrosis cases, some of them resolved and in the severe hydronephrosis group, further evaluation and surgical intervention was done for all cases. it was further observed that, those cases where outlet obstruction was suspected, radio nuclide studies were performed to assess the excretory function of kidney. 13 patients with persistent severe hydronephrosis in postnatal period underwent further evaluation with mcu and mru. in those patients, e. coli was the most common infectious organism in urine amounting to 57% of cases. pelvi ureteric junction obstruction was the commonest indication for surgery amounting to 61%. in the study, the most of the infants born with persistent and severe hydronephrosis underwent corrective surgery in 12 months. this complies with the institution protocol of more conservative and follow up approach for definitive period before a surgical decision was taken. among the operated 13 cases, left sided modified anderson hyne’s pyeloplasty was the commonest procedure amounting to 62.5% of the cases operated. in the surgical patients, left sided poor functioning kidney was maximum with less than 50 % split function in more than 80% of the surgical group. discussion the study reports on the independent association of grading of anteroposterior diameter of renal pelvis and its direct association with the risk for surgical intervention. the results support the use of prenatal and postnatal ultrasonograms in assessment of disease progression. while the renal pelvic apd varies with gestation; bladder distension was also an objective parameter in assessing severity of hydronephrosis. if anh is detected, a followed up with ultrasound including evaluation for lower urinary tract, renal dysplasia, and extra renal structural malformations is carried out [7]. two follow up ultrasounds were performed, one in third trimester and the other in postnatal period irrespective of severity of hydro nephrosis. in the study 100% of severe hydro nephrosis and 77.78% of moderate hydronephro sis needed surgical management. hence, frequent ultrasound evaluation is suggested in cases with moderate and severe hydronephrosis in 6-8 weekly interval to assess disease progression. we recommend that all newborns with history of anh irrespective of any trimester should undergo postnatal ultrasound exa mination within the first week of life. in neonates with moderate or severe hydronephrosis, ultrasonography should be performed within first or second day of birth. we suggest that a micturating cystourethrogram (mcu) being performed within 4 days of life in patients with unilateral or bilateral, moderate and severe hydronephrosis [8]. our institution performs diuretic renography after 10 weeks of age in persistent severe and progressing moderate hydronephrosis and repeats the same in the 6th month of post-natal life. our indication for surgical intervention is persistent severe hydronephrosis with obstruction and with evidence of reduced differential renal function on repeat evaluation [6]. as per our institution protocol, in 12th month of birth, we repeat the diuretic renograpy and watch for disease progression and worsening of split renal function to decide on surgical correction [9]. the patients were started on oral antibiotics j. s. randhawa et al. 60 su r g ic a l d is e a se s issn 2413-6077. ijmmr 2022 vol. 8 issue 1 for all cases of moderate and severe hydronephrosis with syp cephalexin as the incidence of uti in these groups of patients was maximum and e coli was the most commonly isolated organism in urine [10]. conclusions the prenatal diagnosis of uropathies is today the main contributor to the evolution of individuals suffering from this disease. when faced with a diagnosis of pyelocalyceal dilation, postnatal observation should be planned in order to minimize parental anxiety and to monitor progression of nephrourinary pathology. because of the extremely elastic nature of the fetal renal system, it was established that an evaluation of antenatal hydronephrosis via ultrasound should be conducted on more than one occasion via a standardized methodology [11]. the apd grade may be used as reliable predictor of the outcome of fetal hydronephrosis, and may be an independent indicator for postpartum surgery. limitations single centre observational study conflict of interests the authors declare no conflict of interest. author’s contributions j. s. randhawa, nagamahendran r., jafar husain – conceptualization, methodology, for­ mal analysis, writing – original draft, writing – reviewing and editing; vipin v. nair, shankaran r. – data curation, writing – reviewing and editing; shakthi dorai b. – investigation, formal analysis. короткострокове дослідження клінічної значущості та наслідків гідронефрозу плода j. s. randhawa1, *n. rajendran 2, j. husain2, r. shankaran1, v. v. nair2, s. dorai b.3 1 – institute of naval medicine, inhs asvini, mumbai, maharashtra, india 2 – armed forces medical college, pune, maharashtra, india 3 – tata memorial hospital and research centre, parel, mumbai, maharashtra, india вступ. гідронефроз найпоширеніша патологія сечовивідних шляхів, яка виявляється при допологовому обстеженні за допомогою узд. виявлення гідронефрозу плода за допомогою узд ставить перед лікуючим хірургом дилему лікування та викликає занепокоєння у батьків. мета. це дослідження мало на меті вивчити роль серійних ультразвукових досліджень при антенатально виявленому гідронефрозі плода, щоб визначити прогресування захворювання, а також оцінити показання та терміни хірургічного втручання у цих пацієнтів для збереження функції нирок. методи. спостережне дослідження 30 випадків гідронефрозу плода, проведене в центрі дитячої хірургії третинної медичної допомоги. передньо-задній діаметр ниркової миски плода розміром понад 7 мм на 32 тижні вагітності вважається ознакою гідронефрозу плода. пацієнти перебували під спостереженням протягом двох років з 2018 по 2020 рік. відзначалоться прогресування захворювання та необхідність оперативного втручання. результати. дослідження охопило 30 випадків антенатального гідронефрозу плода (42 нирки). з 26 нирок з легким гідронефрозом всі одужали в післяпологовому періоді. з 10 нирок із помірним гідронефрозом 3 (30%) одужали, а у 7 (70%) стан погіршився, що вимагало хірургічного втручання. з 6 нирок з важким гідронефрозом всі вимагали хірургічного втручання і були прооперовані у віці від 12 до 18 місяців. висновки. результати свідчать про те, що система класифікації за передньо-заднім діаметром ниркової миски дозволяє розрізняти випадки з помірним і важким ступенями гідронефрозу, які мають вищий ризик хірургічного втручання. ключові слова: передньо-задній діаметр ниркової миски; антенатальний гідронефроз плода; пієлопластика. j. s. randhawa et al. 61 su r g ic a l d is e a se s issn 2413-6077. ijmmr 2022 vol. 8 issue 1 information about the authors j. s. randhawa, assistant professor general surgery & pediatric surgeon, institute of naval medicine, inhs asvini, mumbai, maharashtra, india https://orcid.org/0000­0002­0081­7382, moshesingh2000@yahoo.co.in nagamahendran rajendran, clinical tutor, armed forces medical college, pune, maharashtra, india https://orcid.org/0000­0002­9854­7236, nagaa.mahendran@gmail.com jafar husain, assistant professor general surgery, armed forces medical college, pune, maharashtra, india https://orcid.org/0000­0003­2400­1473, jafarhusain01@gmail.com r. shankaran, professor and head, department of surgery, institute of naval medicine, inhs asvini, colaba, mumbai, maharashtra, india https://orcid.org/0000­0002­0105­2270, drshankaran@gmail.com vipin v. nair, associate professor, armed forces medical college, pune, maharashtra, india https://orcid.org/0000­0001­6903­6368, vipinvenugopalnair@gmail.com shakthi dorai b., research fellow, oral & maxillofacial pathologist, tata memorial hospital and research centre, mumbai, maharashtra, india https://orcid.org/0000­0002­1819­7951, drshakthidorai@gmail.com references 1. sinha a et al. revised guidelines on management of antenatal hydronephrosis. indian journal of nephrology 2013;23,2:83­97. 2. chiodini b, ghassemi m, khelif k, ismaili k. clinical outcome of children with antenatally diagnosed hydronephrosis. front pediatr. 2019; 7:103. 3. has, recep, and tugba sarac sivrikoz. “prenatal diagnosis and findings in ureteropelvic junction type hydronephrosis”. frontiers in pediatrics 2020;8 492.4. https://doi.org/10.3389/fped.2020.00492 4. orabi m, abozaid s, sallout b, abu shaheen a, heena h, al matary a. outcomes of isolated antenatal hydronephrosis at first year of life. oman med j. 2018 mar;33(2):126­32. https://doi.org/10.5001/omj.2018.24. 5. miyakita h, hayashi y, mitsui t, okawada m, kinoshita y, kimata t, koikawa y, sakai k, satoh h, tokunaga m, naitoh y, niimura f, matsuoka h, mizuno k, kaneko k, kubota m. guidelines for the medical management of pediatric vesicoureteral reflux. int j urol. 2020 jun;27(6):480­90. https://doi.org/10.1111/iju.14223. 6. zhang d, sun x, chen x, yu b, li t, cheng y, ye m, lin l, ma l, zhao y, li p. ultrasound evaluation for prediction of outcomes and surgical decision in fetal hydronephrosis. exp ther med. 2019 aug;18(2): 1399-406. https://doi.org/10.3892/etm.2019.7683. 7. li b, mcgrath m, farrokhyar f, braga lh. ultrasound-based scoring system for indication of pyeloplasty in patients with upjo-like hydro nephrosis. front pediatr. 2020 jul 2;8:353. https://doi.org/10.3389/fped.2020.00353. 8. cho hy, jung i, kim yh, kwon jy. reliability of society of fetal urology and onen grading system in fetal hydronephrosis. obstet gynecol sci. 2019 mar; 62(2):87­92. https://doi.org/10.5468/ogs.2019.62.2.87. 9. asl as, maleknejad s. clinical outcome and follow-up of prenatal hydronephrosis. saudi j kidney dis transpl. 2012 may;23(3):526­31. 10. rianthavorn p, phithaklimnuwong s. the role of antibiotic prophylaxis in mild to moderate isolated hydronephrosis detected in antenatal screening. investig clin urol. 2020 mar;61(2):200­6. https://doi.org/10.4111/icu.2020.61.2.200. 11. madden-fuentes rj, mcnamara er, nseyo u, wiener js, routh jc, ross ss. resolution rate of isolated low-grade hydronephrosis diagnosed within the first year of life. j pediatr urol. 2014 aug;10(4): 639-44. https://doi.org/10.1016/j.jpurol.2014.07.004. received 20 may 2022; revised 29 may 2022; accepted 14 june 2022. this is open access article distributed under the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. j. s. randhawa et al. 11 r a d ia t io n m e d ic in e a n d o n c o l o g y issn 2413-6077. ijmmr 2022 vol. 8 issue 2 doi 10.11603/ijmmr.2413-6077.2022.2.13282 impact of covid-19 pandemic on the radiotherapy and cancer patients in a tertiary health care center of assam, northeast india: a retrospective single institution study *c. baruah, k. deka, t. paul, b. sarma, p. patowary, s. sreeni, b. mahanta assam medical college and hospital, dibrugarh, india background. the covid-19 pandemic has affected every sector of health care system including the cancer care. objective. the purpose of the study is to analyse the impact of the current covid-19 pandemic on routine radiotherapy practice at a tertiary hospital of northeast india. methods. this is a retrospective study of all patients planned for radiotherapy at assam medical college, a tertiary care centre, in assam, india from january 1, 2020 to may 31, 2020. the study samples are divided into two groups: group a – patients treated from january 1, 2020, to march 20, 2020 (pre-lockdown,) and group b – patients treated from march 21, 2020, to may 31, 2020 (lockdown). the department registration numbers of all the patients were collected from the entry register, and the respective paper files were then accessed to obtain the required data. results. a total of 153 patients were planned for radiotherapy during the study period, of these 113 patients receiving radiotherapy in the pre-lockdown period and 40 patients receiving it in the lockdown period. the number of female patients decreased from 66 (58.40%) in the pre-lockdown period to 20 (50%) during the lockdown. the number of male patients also decreased from 47 to 20 but the proportion increased from 41.5% to 50%. the proportion of patients who completed radiotherapy was 90.8% in group b compare to 88.3% in group a. conclusion. there was a decrease in number of cancer patients, especially female during the lockdown. the pandemic has severely disrupted every aspect of cancer care. keywords: covid-19; radiotherapy; pandemic; lockdown; oncology. *corresponding author: chiranjiv baruah, post-graduate trainee, department of radiation oncology, assam medical college and hospital, dibrugarh, 786002, india. e-mail: chiranjivbaruah92@gmail.com international journal of medicine and medical research 2022, volume 8, issue 2, p. 11-17 copyright © 2022, tnmu, all rights reserved c. baruah et al. introduction the coronavirus outbreak caused by the severe acute respiratory syndrome started in wuhan in december 2019. since then it has spread across every corner of the world [1]. with the spread of covid-19 disease in 2019, countries have instituted lockdowns for the benefit of public health at the expense of national economy. during the lockdown, in the fear of getting infected with the virus in the public health care, people avoid screening, diagnosis and treatment of other non-covid diseases like cancer, which have impacted proper treatment of the cancer patients. the prognosis of cancer like complex set of diseases depends largely on the timing of diagnosis and immediate interventions [2]. the government of india witnessed a sudden and complete lockdown, in phased wise manner to combat the covid-19 pandemic in late march 2020. this lockdown affected the entire country [3]. due to the restrictions during the lockdown every sector of health care delivery system has been affected along with the cancer care system. cancer patients remain very much susceptible to different kind of infections due their immunosuppressed status. they are also at risk of severe complications if they get infected with the coronavirus compare to the general populations [4]. radiation therapy is one of the primary and useful modality of cancer treatment and it remained a valid treatment option during coronavirus pandemic as well. radiation therapy in generally does not need any intensive care unit bed or respirators which are in demand resources during the pandemic. moreover, majority of radiotherapy regimens are comparatively less immunosuppressive than to chemotherapy regimens. lasting by following proper personal protection equipment and practicing social distancing proper cancer care and treatment can be given to the patients through radiation 12 r a d ia t io n m e d ic in e a n d o n c o l o g y issn 2413-6077. ijmmr 2022 vol. 8 issue 2 therapy according to the schedule of the respective patient [5]. although, in the treatment of cancer patients, radiotherapy is one of the primary options but the high cost of machines and asso ciated infrastructure along with the re quirement of trained personnel have restricted its availability to only a few urban centres of india. theref ore, the travel restrictions imposed during the lockdown are likely to have affected the access to this treatment for patients coming from the distant places to radiotherapy centres [6]. considering this, we have planned this study to assess the impact of the current covid-19 pandemic on the patients receiving radiotherapy at a tertiary care hospital of upper assam region of northeast india. methods this is a retrospective study completed in the department of radiation oncology at assam medical college, a tertiary cancer centre in assam, india, from january 1, 2020, to may 31, 2020. the inclusion criterion was all the patients who were planned for and who received radiotherapy at the centre during the study period, and the patients who have defaulted after planning radiotherapy were excluded from the study sample. in our analysis, the patients were divided into two groups: group a included the patients treated from january 1, 2020, to march 20, 2020, the period before the lockdown; and group b included all the patients treated from march 21, 2020, to may 31, 2020, the period of the lockdown. the study is planned with a primary objective to assess whether the impact of the covid-19 pandemic varied in patients with cancer depending on their age, gender, site of disease, and intent of radiotherapy. the department registration numbers of all the patients receiving radiation therapy during the study period were collected from the entry register, and the respective paper files were then accessed to obtain the required data. the patients’ characteristics and results between the two groups are compared by the pearson chi-square test. results are considered to be of significant difference if p­value is ≤0.05. screening for covid-19 was done for every patient before starting the radiation therapy and thereafter weekly during radiation therapy. patients who were asymptomatic were not tested prior to starting radiotherapy. there was no patient tested positive for covid-19 while on radiotherapy during the study period in the hospital. results in the study a total of 153 patients were planned for radiotherapy during the study period from january 1, 2020, to may 31, 2020. out of these 153 patients, 113 patients are placed in group a (pre-lockdown period); and 40 patients – in group b (lockdown period). none of patients has defaulted from treatment in both groups. there is decrease in number of new patients receiving radiotherapy during the lockdown compare to the pre-lockdown period but it is not statistically significant. age distri­ bution is almost similar in both groups; about 95% of the patients were aged between 30 and 70 years old in both groups. the gender-wise analysis showed that there were 66 (58.40%) female patients in the pre-lockdown period and the number decreased to 20 (50%) in the lockdown period. likewise, the number of male pa tients decreased from 47 to 20 but the proportion increased from 41.5% to 50%. in our study in both groups, three the most common sites of occurrence of cancer were the head and neck cancers, gynaecological cancers and breast cancers. the number of patients with head and neck, gynaecological, and breast cancers were 43 (38.05%), 21 (18.58%), and 17 (15.04%), respectively, in the pre-lockdown period, and 17 (42.50%), 8 (20%), and 7 (17.5%), respectively, in the lockdown period. this indicates there was an increase in the proportion of patients with head and neck cancer and a decrease in the proportion of patients with gynaecological and breast cancers from the pre-lockdown to lockdown period. our study shows that 102 (90.3%) patients were treated with a radical intent in the group a, and 11 (9.7%) patients were treated with palliative intent. while in the group b 34 (85%) patients were treated with radical intent and 6 (15%) patients were treated with palliative intent. the patients treated with a palliative intent increased from 11 (9.7%) in the group a to 6 (15%) in the group b. there was no modification in the radiation fractionation used for the patients treated with a curative intent or palliative intent in both groups. the compliance to the planned treatment protocol was similar in both groups. the proportion of patients who completed radiotherapy was 90% in the group b as compared to 87.61% in the group a. the changes in data reported, either increase or decrease in the lockdown period compared c. baruah et al. 13 r a d ia t io n m e d ic in e a n d o n c o l o g y issn 2413-6077. ijmmr 2022 vol. 8 issue 2 to the pre-lockdown period, with respect to any variable is found to be statistically insignificant. discussion our study showed a decrease in the number of patients planned for radiotherapy and the number of new patients starting radiotherapy at their scheduled time during the lockdown period compared to the pre-lockdown period. however, once the treatment was started, almost the same number of patients completed the planned radiation therapy in the prelockdown and lockdown periods. the age distribution of patients was similar in both groups. there was a decrease of female patients coming for radiotherapy during the lockdown period. both during the pre-lockdown and the lockdown periods three the most common sites for occurrence of cancer were head and neck followed by gynaecological and breast cancers. curative intent radiotherapy was delivered to 90.3% of patients in the pre lockdown period as compared to only 85% during the lockdown period. the proportion of patients receiving palliative intent radiotherapy have increased during the lockdown. table 1. distribution of patients in the lockdown and pre-lockdown periods group a pre-lockdown group b lockdown time period jan 1, 2020 to mar 22, 2020(n=113) mar 23, 2020 to may 31, 2020: (n=40) number of patients defaulted 0 0 number of patients, who completed radiotherapy as per schedule 113 40 number of patients, who completed radiotherapy as per schedule 99 (87.61%) 36 (90%) total number of patients 113 40 table 2. distribution of gender, intent of treatment, age and radiotherapy completion in the cohort of patients receiving radiotherapy during the study period group a pre-lockdown period (n=113), n (%) group b lockdown period (n=40), n (%) absolute difference (%) p gender male 47 (41.6%) 20 (50%) 8.4 0.459 female 66 (58.4%) 20 (50%) 8.4 0.459 intent of treatment radical 102 (90.3%) 34 (85%) 5.3 0.386 palliative 11 (9.7%) 6 (15%) 5.3 0.386 age below 30 years 1 (1%) 1 (2.5%) 1.5 1 30-70 year 108 (95.5%) 38 (95%) 0.5 1 above 70 years 4 (3.5%) 1 (2.5%) 1 1 concurrent chemotherapy 38 (33.62%) 13 (32.5%) 1.12 1 table 3. site-wise distribution of patients receiving radiotherapy during the study period site group a pre-lockdown period (n=113), n (%) group b lockdown period (n=40), n (%) absolute difference (%) p head and neck 43 (38.05) 17 (42.5) 4.45 0.707 gynaecological 21 (18.58) 8 (20) 1.42 1 breast 17 (15.04) 7 (17.5) 2.46 0.801 gastrointestinal 13 (11.50) 6 (15) 3.50 0.582 others 19 (16.81) 2 (5) 11.81 0.105 c. baruah et al. 14 r a d ia t io n m e d ic in e a n d o n c o l o g y issn 2413-6077. ijmmr 2022 vol. 8 issue 2 there was no modification in the radiation fractionation or the use of concurrent chemotherapy in the patients treated with radical intent or palliative intent in either of the two groups. a study done by chauhan et al. [6], in a charitable hospital of eastern india have also reported decrease in number of patients during the lockdown period compared to the prelockdown period. they also showed a significant decrease in the number of female patients in the lockdown period. the decrease in the number of cancer patients in the lockdown was more drastic during the first week of lockdown, then the numbers improved gradually but were still low compared to the pre-lockdown period. restriction on public transport was the main reason for the increase of patients. the study was conducted at a government medical hospital of northeast india. here the most of the patients came from the distant rural areas for their treatment and most of them also belonged to the low socioeconomic stratum. they came from remote villages and were mainly depended on the public transport for their arrival to the hospital. moreover, many patients as well as the attendants feared of getting infected with coronavirus on exposure to the health care setup. therefore, many asymptomatic patients requiring postoperative adjuvant radiotherapy delayed their treatment. the covid-19 pandemic has affected every sector of health care system along with the cancer care. due to the restrictions during the lockdown there is a halt in national cancer scree ning program [7]. in developing countries like india there is no easy access to affordable screening and diagnosing setup for cancer along with a lack of awareness among people about the suspected signs and symptoms of cancer that leads to delayed diagnosis of cancer. the strict lockdown also leads to closing of hotels and guest houses nearby the hospital, local people also avoid renting theirs houses to any patients in fear of coronavirus pandemic; thus, there is hardly any place for accommodation near the cancer treatment centre for the patients. another reason for the increase of patients during the lockdown is the continuously affected poor financial status of the patients and their family. as majority of the patients belong to lower socioeconomic status, many of them are daily wedge earner or run small business. restrictions imposed on their workplaces due to the lockdown have affected their financial condition badly. our study also shows a decrease of female patients during the lockdown compared to the pre-lockdown period. a similar decrease in number of female patients in lockdown is also reported by chauhan et al. [6]. financial and social dependency on male is one of the reasons for this decrease of female patients in the lockdown. moreover, the female patients might be reluctant to spend the limited resources of the family on themselves during the pandemic. in our study we have reported that there is an increased number of patients coming for palliative intent radiotherapy during the lockdown period compared to the pre-lockdown period. the studies by chauhan et al. [6] and ghosh et al. [8] have also reported similar increase of number of patients coming for palliative intent radiotherapy in lockdown period. in most of the parts of india including the northeast india there is a lack of social and personal awareness in between the people regarding the cancer related early sign and symptoms, lack of communication and knowledge regar ding importance of screening for common cancers, eligible age groups for screening and different risk factors for different cancers. there is also a lack of affordable cancer care centres easily accessible for the public [9]. all of this leads to diagnosis of cancer in an advance stage in many people for whom palliative radiation therapy would be an effective option. these patients suffer unbearable pain and other symptoms which can be relieved by palliative therapy. our study shows that the majority of the patients in both the lockdown and pre-lockdown periods are in between 30-70 years of age. similar findings were seen in the study by chauhan et al [6]. our study also shows that the most common type of cancer in both the lockdown and pre-lockdown periods is head and neck cancer followed by gynaecological cancer and breast cancer. the major cause of the head and neck cancers primarily of the gingivobuccal complex in the developing countries like india are smokeless tobacco by men [10]. although smoking of bidis, chewing tobacco, betel nut quid and areca nut as well as alcohol consumption are main risk factors for head and neck squamous cell carcinoma; nevertheless a strong association of human papillomavirus (hpv) with head and neck squamous cell carcinoma in non-smoking cases is also reported [11]. head and neck cancer are followed by cervical and other gynaecological cancer and c. baruah et al. 15 r a d ia t io n m e d ic in e a n d o n c o l o g y issn 2413-6077. ijmmr 2022 vol. 8 issue 2 breast cancer. in northeast india and many other rural areas of india the cases of cervical cancer are reported more than breast cancer although breast cancer is more common overall. this can be explained by the lack of knowledge and awareness regarding the screening age group, sign and symptoms of cervical cancer, in practicular no vaccination and risk factors like low socioeconomic status, early marriage, multiparty etc. [9,12]. our study was performed in the department of radiation oncology, assam medical college and hospital, dibrugarh. assam medical college and hospital, dibrugarh is a tertiary cancer care centre in the state of assam of northeast india. the department of radiation oncology is equipped with one cobalt teletherapy machine. more than 30 patients are treated in the department of radiation oncology daily. the institute caters are mainly the patients from districts of upper assam and arunachal pradesh. during the covid-19 pandemic some changes were taken in treatment protocol following various guidelines published in high impact journals, government advisories, and hospital policies [13]. regular opd work was minimized to bare minimum and follow up and non-emergency visits are, avoided if possible, by giving telephone consultation during the pandemic for the safety of patients and health care workers [14]. this is the analysis of the few studies regarding the impact of covid-19 pandemic in radiation therapy practice and cancer patients as a whole in upper assam area of the north east india. as our study is a retrospective and observational in design, the study has many limitations. the generalised stage data of the cancer patients for the study during the study period could not be assessed. another main limitation of our study is that no follow up is included in the study for analysis. conclusion a decrease in number of cancer patients planned for radiation therapy during the lockdown compared to the pre-lockdown period has been established in our study. covid-19 pandemic is one of the deadly natural disaster of modern era. the covid-19 pandemic has disrupted every aspect of health care system, including screening, diagnosis and management of the cancer patients. being a deadly immunosuppressive disease, cancer patients suffer a lot during the pandemic that affects them physically, financially as well as psychologically. conflict of interests authors declare no conflict of interest. author’s contributions chiranjiv baruah – conceptualization, methodology, formal analysis, writing – original draft, writing – reviewing and editing, kankan jyoti deka – writing – reviewing and editing, trishan paul – writing – reviewing and editing, biswajit sarmak – writing – reviewing and editing, partha pratim patowary – writing – reviewing and editing, sharath sreeni – writing – reviewing and editing, badurjya mahanta – formal analysis. вплив пандемії covid-19 на доступ до променевої терапії та онкологічних пацієнтів у центрі третинної медичної допомоги в ассамі, північно-східна індія: ретроспективне дослідження *c. baruah, k. deka, t. paul, b. sarma, p. patowary, s. sreeni, b. mahanta assam medical college and hospital, dibrugarh, india вступ. пандемія covid-19 вплинула на всі сектори системи надання медичної допомоги, включаючи систему надання онкологічної допомоги. мета. метою нашого дослідження був аналіз впливу поточної пандемії covid-19 на рутинну практику променевої терапії у закладі третинної медичної допомоги на північному сході індії. методи. проводилося ретроспективне дослідження із залученням пацієнтів, яким запланована променева терапія, в медичному коледжі ассама, центрі третинної медичної допомоги, в ассамі, індія, з 1 січня 2020 р. по 31 травня 2020 р. вибірку дослідження поділено на дві групи, група а включає пацієнтів, які лікувалися з 1 січня 2020 р. по 20 березня 2020 р. (прелокдаун), а група б включає пацієнтів, які проходили лікування з 21 березня 2020 р. по 31 травня 2020 р. (локдаун). c. baruah et al. 16 r a d ia t io n m e d ic in e a n d o n c o l o g y issn 2413-6077. ijmmr 2022 vol. 8 issue 2 результати. загалом 153 пацієнтам було заплановано проведення променевої терапії протягом періоду дослідження, з них 113 пацієнтів отримували променеву терапію в період до локдауну, а 40 пацієнтів отримували її у період карантину. кількість пацієнток різко зменшилася з 66 (58,40%) у прелокдауні до 20 (50%) під час локдауну. пацієнтів чоловічої статі також зменшилося з 47 до 20, але частка зросла з 41,5% до 50%. частка пацієнтів, які завершили променеву терапію, становила 90,8% у групі б порівняно з 88,3% у групі a. висновки. під час карантину кількість онкологічних пацієнтів, яким надавалася медчна допомога, особливо жінок, зменшилася. пандемія серйозно порушила всі аспекти системи надання медичної допомоги онкологічним захворюванням. ключові слова: covid-19; радіотерапія; пандемія; локдаун; онкологія information about the authors chiranjiv baruah – post graduate trainee, department of radiation oncology, assam medical college and hospital, dibrugarh, india https://orcid.org/0000­0002­3154­150x, e­mail: chiranjivbaruah16@gmail.com kankan jyoti deka – assistant professor, radiation oncology, assam medical college, dibrugarh, india https://orcid.org/0000­0001­5811­8691, e­mail: email­knkn.dk@gmail.com trishan paul – assistant professor, radiation oncology, assam medical college, dibrugarh, india https://orcid.org/0000­0002­5807­0461, e­mail: trishanpaul2422@gmail.com biswajit sarma – registrar, department of radiation oncology, assam medical college, dibrugarh, india https://orcid.org/0000­0003­1270­2504, e­mail: drsarmabiswajit@gmail.com partha pratim patowary – post graduate trainee, department of radiation oncology, assam medical college, dibrugarh, india https://orcid.org/0000­0003­2595­249x, e­mail: partha7patowary@gmail.com sharath sreeni – post graduate trainee, department of radiation oncology, assam medical college and hospital, dibrugarh, india https://orcid.org/0000­0001­7066­2390, e­mail: sharathsreeni@gmail.com badurjya mahanta – post graduate trainee, department of community medicine, assam medical college and hospital, dibrugarh, india. https://orcid.org/0000­0002­2533­4598, e­mail: baidurjya15@gmail.com references 1. singh ak, misra a. impact of covid-19 and comorbidities on health and economics: focus on developing countries and india. diabetes metab syndr clin res rev. 2020 nov 1;14(6):1625­30. https://doi.org/10.1016/j.dsx.2020.08.032 2. sharpless ne. covid-19 and cancer. science (80­). 2020;368(6497):1290. https://doi.org/10.1126/science.abd3377 3. jiwnani s, ranganathan p, tiwari v, ashok a, niyogi d, karimundackal g, et al. covid-19 pandemic and its gendered impact on indian physicians. jco glob oncol. 2021;(7):1093­100. https://doi.org/10.1200/go.21.00020 4. al­quteimat om, amer am. the impact of the covid-19 pandemic on cancer patients. 2020; available from: www.amjclinicaloncology.com 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30 in t e r n a l m e d ic in e issn 2413-6077. ijmmr 2021 vol. 7 issue 2 doi 10.11603/ijmmr.2413-6077.2021.2.12682 drug therapy for protein composition changes of blood in hypertension and in cases of comorbidity yu.r. dzordzo, s.m. andreychyn i. horbachevsky ternopil national medical university, ternopil, ukraine background. the binding function of serum albumin (bfsa) and its changes in various diseases in recent years are of interest to researchers. hypertension (ht) in combination with comorbidities, including non-alcoholic steatohepatitis (nash) and type 2 diabetes mellitus (dm) can contribute to bfsa. objective. the aim of this study is to evaluate the relationship between quantitative changes in bfsa, protein fractions and indicators of endogenous intoxication (ei) in ht in combination with nash and type 2 diabetes and to suggest drug therapy of the disorders revealed. methods. 123 patients with stage 2 ht and degree 2-3 arterial hypertension were examined; they were divided into three groups: group 1 included 28 patients without concomitant diseases, 2 – 48 patients with concomitant nash, 3 – 47 patients with nash and type 2 diabetes. groups 3 and 4 were divided into two subgroups (a and b): patients of the subgroup a received basic ht therapy and additionally antral® 200 mg 3 times a day for 60 days, b – only basic ht therapy. all patients underwent a standard clinical examination, as well as for bfsa, total protein, albumin, globulins and albumin-globulin ratio, medium mass molecules (mmm) at 280 and 254 nm and erythrocyte intoxication index (eii). the comparison group consisted of 25 healthy individuals. results. it was found out that antral® in patients with ht in combination with nash and with nash and type 2 diabetes with a statistically significant decrease in bfsa, total protein and albumin, as well as with increased indicators of ei (msm254, msm280 and eii) caused significant improvement in bfsa, increase of total protein, serum albumin, reduce of msm254, msm280, eii and strengthening of all correlations. conclusions. antral® therapy in patients with ht in combination with nash as well as nash and type 2 diabetes causes significant increase in bfsa, serum protein fractions and decreases ei. keywords: hypertension; non-alcoholic steatohepatitis; type 2 diabetes mellitus; binding function of serum albumin; antral®. yu.r. dzordzo et al. international journal of medicine and medical research 2021, volume 7, issue 2, p. 30-36 copyright © 2021, tnmu, all rights reserved *corresponding author: yurii dzordzo, postgraduate student of the department of internal medicine propedeutics and phthisiology, i. horbachevsky ternopil national medical university, ternopil, ukraine, 46000. e-mail: dzordzo@tdmu.edu.ua introduction one of the important factors in the normal functioning of all organs and systems of the human body is the stability of protein content and their role in biological fluids. albumin as the most common protein in the body is of particular interest of all the protein fractions. normally, its content is about 55% of all proteins, so the importance of changing its content and function is not overestimated [1, 2]. the structure of the albumin molecule de termines a number of its properties; the binding function of serum albumin (bfsa) is among them; bfsa is the ability to bind and transport a significant amount of biological substances of endogenous and exogenous nature: fatty acids, nitric oxide, chlorine and calcium ions, toxins, synthetic drugs and others. violation of bfsa can have a negative impact on the body directly and reduce the effectiveness of drug treatment in various pathological conditions [3]. endogenous intoxication (ei) is an important indicator of homeostasis. development of ei indicates the presence of a pathological process in the body. dysfunction of serum albumin, bfsa in particular, may directly affect the increase of ei. in diseases that are accompanied by endotoxicosis, a significant number of meta­ bolites accumulate in the blood; most of them belong to the medium mass molecules (mmm) [4]. the accumulation of mmm as a marker of endotoxemia is a sign of intensification of the pathological process. in addition to mmm, another important indicator of ei is the erythrocyte intoxication index (eii), which evidences the permeability of erythrocyte membranes and may indicate signs of endotoxic damage to organs and tissues [5]. 31 in t e r n a l m e d ic in e issn 2413-6077. ijmmr 2021 vol. 7 issue 2 disorders of protein metabolism with the development of ei can be caused by many diseases. however, little is known about the possibility of such changes in cases of hypertension (ht) and related comorbid conditions, in particular when combined with non-alcoholic steatohepatitis (nash) and type 2 diabetes mellitus (dm) – a very common disease that causes systemic adverse effects on the whole body [6, 7]. in addition, they are associated with long-term use of various drugs that are mainly metabolized in the liver and can cause damage to this organ, which is the main source of protein synthesis and in particular albumin [8, 9]. methods the study included 123 patients with stage 2 ht and degree 2-3 hypertension in combination with diastolic heart failure fc 1–3 according to the nyha. they were divided into three groups. group 1 – 28 patients with ht without concomitant pathology (12 men and 16 women) aged 45-76, mean age (60.71±1.95) years. group 2 involved 48 patients (21 men and 27 women) diagnosed with ht and concomitant nash, aged 46-78, mean age (64.68±1.07) years. it was divided into two subgroups: 2a (27 patients) – undergoing basic ht therapy and additionally antral® 200 mg 3 times a day for 60 days, 2b (21 patients) – only basic ht therapy. group 3 included 47 patients (21 men and 26 women) with ht combined with concomitant nash and type 2 diabetes in the stage of subcompensation, aged 58-82, the average age was (68.72±0.86) years. it was also divided into two subgroups: 3a (27 patients) – in addition to basic treatment of ht and type 2 diabetes taking antral® 200 mg 3 times a day for 60 days, 3b (20 patients) – only basic ht therapy. the comparison group consisted of 25 healthy individuals, comparable in age and sex (control group). the duration of ht in patients ranged from 6 to 25 years. the study did not include patients with symptomatic hypertension, people who drink alcohol (more than 40 ml of ethanol per week for men and 20 ml for women), as well as those who had at the time of examination or history of acute coronary syndrome, acute disorders of cerebral circulation, cancer, viral, drug and autoimmune hepatitis, mental disorders. the diagnosis of nash was established according to the recommendations of the unified clinical protocol of primary, secondary (specialized) medical care “non-alcoholic steatohepatitis” (order of the ministry of health of ukraine no. 826, dated november 6, 2014) and the recommendations of the european association for the study of the liver (easl). the functional state of the liver was examined by sonoelastography on the ultima sm-30 device by the swei method to determine the stiffness of the liver parenchyma, which averaged 8.42 kpa in the patients with nash. all patients were determined bfsa by the method of s. i. chager, the content of mmm at wavelengths of 280 and 254 nm and eii by the method of n. i. gabrielyan [10]. the content of total protein, albumin, globulins in the blood serum was studied by bochemical methods and the albumin­globulin coefficient was calculated. all patients were treated according to the criteria of the unified protocol of medical care for patients with hypertension (order of the ministry of health of ukraine no. 384, dated may 24, 2012) and the recommendations of the european society of cardiologists (esc). patients with type 2 diabetes were treated according to the unified clinical protocol of primary, secondary (specialized) medical care “diabetes mellitus, type 2” (order of the ministry of health of ukraine no. 1118, dated december 21, 2012). analysis of the obtained digital data was performed by the method of parametric pearson correlation. the correlation dependence was considered strong at r=0.7-0.99, medium at r=0.3-0.69, and weak at r=0.01-0.29. statistica 10 and microsoft excel software were used. results in the control group (almost healthy individuals) indicators of bfsa, protein fractions and ei were within norm. the analysis of the correlation of bfsa with serum proteins showed a positive relationship of medium strength with the level of albumin (r=0.68, p<0.05), total protein (r=0.52, p<0.05) and albumin-globulin ratio (r=0.69, p<0.05), as well as a negative relationship of medium strength with the content of globulins (r=­0.68, p<0.05). no significant correlations were observed between bfsa and endotoxicosis indicators (p>0.05) (table 1). in patients with ht without concomitant pathology (group 1), the values of bfsa and protein fractions were close to those in almost healthy individuals. at the same time, there was a significant increase in eii and mmm280 concentrations (p<0.05). the analysis showed a po sitive relationship between the average yu.r. dzordzo et al. 32 in t e r n a l m e d ic in e issn 2413-6077. ijmmr 2021 vol. 7 issue 2 strength of bfsa with the level of albumin (r=0.45, p<0.05), total protein (r=0.38, p<0.05) and albumin-globulin coefficient (r=0,49, p<0,05) and a negative relationship of medium strength with the content of globulins (r=-0,45, p<0,05). no significant correlations of ei indi­ cators with the content of bfsa were observed. in patients with ht in combination with nash (subgroup 2a) before drug therapy, a significant decrease in bfsa, total protein and serum albumin was found. in addition, there was an increase in all studied indicators of ei (p<0.05). the presence of positive relationships of medium strength of bfsa with the content of total protein (r=0.69, p<0.05), albumin (r=0.63, p<0.05) and with albumin-globulin ratio (r=0.62, p<0.05), as well as a negative relationship of medium strength with the level of globulins (r=-0.6, p<0.05). at the same time, there were significant negative correlations between the mean strength of bfsa and eii (r=-0.46, p<0.05), the concentration of mmm254 (r=-0.48, p<0.05) and mmm280 (r=-0.41, p<0.05). similar changes were observed in the subgroup 2b, but the strength of the relationship between bfsa and albumin, globulins and albumin-globulin ratio were slightly higher than in the previous subgroup. regarding the level of total protein, the correlation was almost the same. ei relationships in this subgroup were also similar, but the strength of the correlation of bfsa with mmm254 was slightly higher, and with eii, on the contrary, lower. in patients with ht combined with nash and type 2 diabetes (subgroup 3a) before correction there was even more pronounced decrease in bfsa, total protein, albumin, and albumin-globulin ratio. at the same time, there was a significant increase in all studied indi­ cators of ei (p<0.05). the presence of a positive correlation between the average strength of bfsa and the level of total protein (r=0.63, p<0.05), albumin (r=0.54, p<0.05) and albuminglobulin coefficient (r=0.56, p<0.05), as well as a negative relationship of medium strength with the level of globulins (r=-0.56, p<0.05). there was also a negative correlation of the average strength of bfsa with eii (r=-0.63, p<0.05) and mmm254 (r=-0.60, p<0.05) and strong with mmm280 (r=-0,70, p<0.05). similar changes were observed in the subgroup 3b, but there was an increase in the negative correlation with mmm254 compare to the previous subgroup. normalization of bfsa, total protein, albumin and albumin-globulin ratio was proved to be associated with antral® in patients of the subgroup 2a. also, a normalization of the concentrations of mmm254 and mmm280 and a significant decrease in the level of eii compare to subgroup 2b (p<0.05) were evidenced. a strong positive relationship of bfsa with the level of total protein (r=0.84, p<0.05) was noted table 1. correlation coefficients between bfsa and indicators of protein composition of blood and ei in patients with ht combined with nash and their correction by antral® indicator control group (n=25) group і (n=28) group іі (n=48) subgroup 2a (n=27) subgroup 2b (n=21) before treatment after treatment before treatment after treatment total protein 0.52 р<0.05 0.38 р<0.05 0.69 р<0.05 0.84 р<0.05 0.72 р<0.05 0.68 р<0.05 albumin concentration 0.68 р<0.05 0.45 р<0.05 0.63 р<0.05 0.70 р<0.05 0.73 р<0.05 0.70 р<0.05 concentration of globulins -0.68 р<0.05 -0.44 р<0.05 -0.60 р<0.05 -0.66 р<0.05 -0.73 р<0.05 -0.74 р<0.05 albumin-globulin ratio 0.69 р<0.05 0.49 р<0.05 0.62 р<0.05 0.67 р<0.05 0,71 р<0,05 0.72 р<0.05 eii -0.07 р>0.05 -0.33 р>0.05 -0.46 р<0.05 -0.62 р<0.05 -0,31 р>0,05 -0.19 р>0.05 mmm254 -0.14 р>0.05 0.01 р>0.05 -0.48 р<0.05 -0.76 р<0.05 -0,62 р<0,05 -0.65 р<0.05 mmm280 -0.29 р>0.05 -0.07 р>0.05 -0.41 р<0.05 -0.64 р<0.05 -0,38 р>0,05 -0.25 р>0.05 note. p – statistical significance of the correlation coefficient (p<0.05). yu.r. dzordzo et al. 33 in t e r n a l m e d ic in e issn 2413-6077. ijmmr 2021 vol. 7 issue 2 in association with antral®, as well as a positive relationship of medium strength with albumin content (r=0.70, p<0.05) and negative medium strength with globulin content (r=-0.66, p<0.05). compare to the subgroup without antral® correction, the correlation of bfsa with the level of total protein was stronger, and with the level of globulins, on the contrary, slightly lower. in addition, negative correlations were found with eii (r=-0.62, p<0.05) and mmm280 (r=-0.64, p<0.05); compare to the patients undergoing no treatment it changed from negative weak to medium strength, as well as a negative relationship with the content of mmm254 (r=-0.76, p<0.05), which changed from medium strength to strong. in subgroup 2b, all indicators did not change significantly. the patients in subgroup 3a had a statistically significant increase in bfsa, total protein and serum albumin during antral® treatment. there was also a significant reduction in all indicators of endotoxicosis. an analysis of bfsa correlations revealed that statistically significant strong correlations with all protein fractions occurred in cases of antral® treatment. thus, there was a positive relationship with the level of total protein (r=0.76, p<0.05), albumin content (r=0.81, p<0.05) and albumin-globulin ratio (r=0.78, p<0.05), as well as negative – with the content of globulins (r=-0.81, p<0.05). analysis of the relationship of bfsa with ei showed a strong negative correlation with the level of eii (r=-0.74, p<0.05), which was stronger than in the subgroup without correction, where it was moderate. in addition, medium-strength negative correlations with the content of mmm254 (r=-0.69, p<0.05) and mmm280 (r=-0.69, p<0.05) were revealed, which compare to the subgroup without treatment were also stronger, but not significantly. the correlations in the subgroup 3b did not change significantly. discussion therefore, in patients with ht and nash and with ht combined with nash and type 2 diabetes, there is a statistically significant correlation of bfsa with eii, mmm254 and mmm280, which may indicate development of endotoxicosis, not excluded due to liver cell damage and systemic pathological effects of comorbid diseases [12, 13]. since the liver is the main organ that metabolizes many toxemia products, most of which belong to medium molecular weight peptides, damage to this organ leads to an increase in mmm. in addition, in cases of development of liver disease the accumulation of free radical forms of oxygen, which in addition to promoting the increase of ei products, cause damage to cell membranes that is manifested by an increase in eii. as we can see, the changes in protein composition and endotoxicosis and table 2. correlation coefficients between bfsa and indicators of protein composition of blood and ei in the patients with ht combined with nash and type 2 diabetes and their correction by antral® indicator control group (n=25) group ііі (n=47) subgroup 3a (n=27) subgroup 3b (n=20) before treatment after treatment before treatment after treatment total protein 0.52 р<0.05 0.63 р<0.05 0.76 р<0.05 0.69 р<0.05 0.62 р<0.05 albumin concentration 0.68 р<0.05 0.54 р<0.05 0.81 р<0.05 0.50 р<0.05 0.55 р<0.05 concentration of globulins -0.68 р<0.05 -0.56 р<0.05 -0.81 р<0.05 -0.51 р<0.05 -0.54 р<0.05 albumin-globulin ratio 0.69 р<0.05 0.56 р<0.05 0.78 р<0.05 0.53 р<0.05 0.58 р<0.05 eii -0.07 р>0.05 -0.63 р<0.05 -0.74 р<0.05 -0.65 р<0.05 -0.65 р<0.05 mmm254 -0.14 р>0.05 -0.60 р<0.05 -0.69 р<0.05 -0.68 р<0.05 -0.63 р<0.05 mmm280 -0.29 р>0.05 -0.70 р<0.05 -0.69 р<0.05 -0.64 р<0.05 -0.60 р<0.05 note. p – statistical significance of the correlation coefficient (p<0.05). yu.r. dzordzo et al. 34 in t e r n a l m e d ic in e issn 2413-6077. ijmmr 2021 vol. 7 issue 2 their relationship with bfsa are more pronounced in patients with ht combined with nash and type 2 diabetes, which can also be explained by the processes of glycolysis of proteins, and in particular albumin, which has a direct effect on bfsa. the severity of ei syndrome in these cases may be a marker of the severity of the pathological process [14]. antral® treatment improves bfsa and serum protein composition, as well as strengthens significant correlations with all studied indi­ cators of protein fractions and ei. these positive changes can be explained by the anti­inflam­ matory and detoxifying effects of antral® on the whole body and in particular on the function of the liver as the main protein-synthesizing organ [15, 16]. the drug has a positive effect on hepatocytes by stabilizing cell membranes and lysosomal membranes and increasing the synthesis of phospholipids. by improving the energy supply and functioning of monooxygenase systems, antral® restores the antitoxic effect of the liver. due to its angioprotective action by improving capillary hemoperfusion, this drug can improve microcirculation in the liver and other organs, which has a positive effect on the whole body [17]. conclusions in ht without concomitant diseases an increase in eii and mmm280 and a decrease in the strength of correlations of bfsa with the concentration of protein fractions in serum take place. in the conditions of ht combined with nash as well as ht with nash and type 2 diabetes a decrease in bfsa, total protein and albumin content and increase in ei, as well as a decrease in the correlation of bfsa with albumin, globulin and albumin levels, globulin coefficient and strength increase – with the content of total protein and ei, are evidenced. treatment of the patients with ht with concomitant nash with antral® normalizes bfsa and the content of total protein and albumin, as well as mmm254 and mmm280 and significantly decreases eii. in cases of ht with nash and type 2 diabetes a significant increase in bfsa, the content of total protein and albumin in the serum and a decrease in ei take place with the drug. all studied correlations of bfsa are enhanced under the action of the drug in both comorbid states. prospects for further research. in the future, it is planned to identify other effects on the changes of bfsa and development of ei in comorbid diseases associated with ht, as well as to implement the data into clinical practice. conflict of interests authors declare no conflict of interest. author’s contributions yurii r. dzordzo – conceptualization, methodology, formal analysis, investigation, writing – original draft, writing – reviewing and editing; serhiy m. andreychyn – data curation, writing – reviewing and editing. медикаментозна корекція змін білкового складу крові при гіпертонічній хворобі в умовах коморбідності ю.р. дзьордзьо, с.м. андрейчин тернопільський національний медичний університет імені і. я. горбачевського моз україни, тернопіль, україна вступ. зв’язувальна функція сироваткового альбуміну (зфса) та її зміни при різних захворюваннях в останні роки викликають інтерес у дослідників. гіпертонічна хвороба (гх) при поєднанні з супутніми захворюваннями, зокрема неалкогольним стеатогепатитом (насг) і цукровим діабетом (цд) 2-го типу може сприяти порушенню зфса. мета дослідження – дати оцінку взаємозв’язків кількісних змін зфса, білкових фракцій та показників ендогенної інтоксикації (еі) при гх у поєднанні з насг і цд 2-го типу та запропонувати медикаментозну корекцію виявлених порушень. yu.r. dzordzo et al. 35 in t e r n a l m e d ic in e issn 2413-6077. ijmmr 2021 vol. 7 issue 2 методи дослідження. обстежено 123 пацієнтів з гх іі стадії зі ступенем артеріальної гіпертензії 2-3, які були розділені на три групи. до і увійшли 28 осіб без супутніх захворювань, до іі – 48 пацієнтів із супутнім насг, до ііі – 47 осіб із насг і цд 2-го типу. іі та ііі групи, своєю чергою були поділені на дві підгрупи (а та б): хворі підгруп а – отримували базову терапію гх та додатково препарат антраль по 200 мг 3 рази на добу протягом 60 днів, б – лише базову терапію гх. усі хворі пройшли стандартне клінічне обстеження, а також у них досліджували зфса, вміст загального білка, альбуміну, глобулінів та альбуміно-глобуліновий коефіцієнт, молекули середньої маси (мсм) при довжині хвилі 280 та 254 нм та еритроцитарний індекс інтоксикації (еіі). групу порівняння склали 25 практично здорових осіб. результати. встановлено, що застосування антралю у хворих з гх у поєднанні з насг та з насг і цд 2-го типу на тлі статистично достовірного зниження зфса, вмісту загального білка та альбуміну, а також збільшення показників еі (мсм254, мсм280 та еіі) супроводжується істотним покращенням зфса, зростанням вмісту загального білка, альбуміну, та зниженням – мсм254, мсм280, еіі й посиленням усіх кореляційних зв’язків. висновки. лікування антралем у хворих на гх в поєднанні з насг та з насг і цд 2-го типу супроводжується істотним підвищенням зфса, вмісту фракцій білка сироватки крові та зниженням показників еі. ключові слова: гіпертонічна хвороба, неалкогольний стеатогепатит, цукровий діабет 2-го типу, зв’язувальна функція сироваткового альбуміну, антраль. information about the authors yurii r. dzordzo – postgraduate student of the department of internal medicine propedeutics and phthisiology of i. horbachevsky ternopil national medical university, ternopil, ukraine. orcid 0000­0002­8871­8257, e­mail: dzordzo@tdmu.edu.ua serhiy m. andreychyn – professor, head of the department of internal medicine propedeutics and phthisiology of i. horbachevsky ternopil national medical university, ternopil, ukraine. orcid 0000­0002­8770­7353, e mail: andreychynsm@tdmu.edu.ua references 1. kligunenko en, zozulya oa. human serum albumin (past and future). meditsina neotlojnyih sostoyaniy. 2017;5(84):26­30. 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[in russian]. received 30 november 2021; revised 3 december 2021; accepted 10 december 2021. this is open access article distributed under the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. yu.r. dzordzo et al. 10 in t e r n a l m e d ic in e issn 2413-6077. ijmmr 2016 vol. 2 issue 2 doi 10.11603/ijmmr.2413-6077.2016.2.7021 vitamin d and diabetes mellitus type 2 n. v. pasyechko, v. m. kulchinska, l. v. radetska, l. v. naumova, i. v. smachylo, a. o. bob, i. p. savchenko, m. ye. havryliuk,n. i. yarema, a. i. balaban, o. o. bob, m. a. dzhula, t. i. krytskyi i. horbachevsky ternopil state medical university, ternopil, ukraine background. as it is established that diabetes mellitus causes metabolic disturbances of all types (first of all because of carbohydrate metabolism), affection of blood vessels, nerves, different organs and tissues. however, the influence of dm on the level of microelements is still not investigated enough. despite the sufficient variety of medicaments, compensation of diabetes mellitus is achieved in less than 30% of patients. for this reason, the search of new ways of treatment which are aimed at the improvement of carbohydrate metabolism is an urgent issue. objective. the research was aimed to reveal the deficiency of 25­hydroxyvitamin d3 [25(oh)d3] and its correlation with carbohydrate metabolism. methods. thirty five patients with diabetes mellitus type 2 aged 55–74 with illness duration 2–4 years were examined. the control group included 35 healthy people of the same age and sex. levels of 25-hydroxyvitamin d3 [25(oh)d3] were tested by means of radioimmunoassay. the level of glycated haemoglobin was tested by means of liquid chromatography. results. correlation of the level of vitamin d with the degree of diabetes mellitus type 2 compensation has been detected. conclusions. a clear tendency to the improvement of diabetes mellitus type 2 compensation after medication correction with vitamin d was evidenced. key words: diabetes mellitus, vitamin d, glycated haemoglobin, glycemic control. introduction diabetes mellitus (dm) — is a non-infectious epidemic of xxi century. according to world statistics data, the amount of people with dm doubles every 13-15 years. today there are 415 million patients with dm in the world, and their amount will increase to 642 million in 2040. new drugs do not contribute to the disease suppression, and the intensification of diagnostic criteria causes rapid increase in the number of patients. mainly the increase of patients mainly with dm type 2. today, in ukraine the dm compensation is unsatisfactory because the average rate of glycated haemoglobin (hba1c) within dm type 1 is about 9.0%, and within dm type 2 – about 8.5% [2]. in our day, it is ultimately important to search for pathogenetic links for efficiency of patients’ with dm treatment improvement. so, much attention is paid to the study of vitamin d influence on different organs and systems in case of different diseases, dm as well. recently scientists began to study the influence of vitamin d on the course of dm type 2 [4,5,6,7]. s o m e a u t h o r s s u g g e s t , t h a t v i t a m i n d insufficiency is a risk factor of dm type 2 [6], other — that vitamin d supports glucose ho meo stasis and is inversely proportional to the level of glycated haemoglobin in dm type 2 [8]. also there is a suggestion, that an adequate vitamin d intake may be connected with lower risk of a gestational diabetes mellitus develop ment [9]. physiological effects of vitamin d is polyhedral, it is difficult to select some less signi­ ficant factors. vitamin d influences the phos­ phorus­calcium exchange: calcium and other microelements cannot be absorbed in its defi­ ciency. activity of vitamin d is directly connected with many physiological processes in organism, in particular with vitamin e admission, liver function and function of intestine, lipid metabolism. vitamin d is necessary for providing international journal of medicine and medical research 2016, volume 2, issue 2, p. 10-13 copyright © 2016, tsmu, all rights reserved corresponding author: veronika kulchinska, department of internal medicine № 1, i. horba chevsky ternopil state medical university, 1 klinichna, ternopil, ukraine, 46000 phone number: +380978484206 e-mail: v.m.kulchinska@gmail.com n. v. pasyechko et al. 11 in t e r n a l m e d ic in e issn 2413-6077. ijmmr 2016 vol. 2 issue 2 normal functioning of immune system, reproductive and sexual health, and hematopoietic system. that is why, cholecalciferol and its active metabolites is applied for treatment of immunodeficiency, anaemia, various patholo­ gical conditions of liver, gastrointestinal tract, diabetes, cardiovascular system, tuberculosis, cancers of breast and bowel, prostate, locally for the treatment of psoriasis. according to statistical data [3], among all residents of ukraine, only 4.6% of the examined people had normal level of vitamin d, 13.6% of the population was diagnosed with its failure, and in 81.8% of residents the deficit was pronounced. however, how the level of vitamin d can change, and whether its change can influence on indicators of compensation for diabetes remains poorly examined. according to the literature [10], objective criterion of vitamin d security, is the determination of 25-hydroxy vitamin d3 [25 (oh) d3] contents. the study is aimed to reveal the deficiency of 25-hydroxy vitamin d3 and its cross-correlation with glycated haemoglobin of patients with diabetes type 2. material and methods 35 patients with diabetes mellitus type 2 were examined, the individuals were aged from 55 to 74, illness duration was 2–4 years. the control group included 35 healthy people of the same age and sex. the patients, who suffered from dm type 2, took oral hypoglycaemic drugs of biguanides in therapeutic doses (1000-2500 mg daily). additionally patients of both group received the drug alpha d3 teva, dose 1 mkg daily during 3 month. levels of 25-hydroxyvitamin d3 [25(oh)d3] were tested by means of radioimmunoassay. the level of glycated haemoglobin (hba1c) was tested by means of liquid chromatography. the results of 25(oh)d3 and hba1c were determined before the start of vitamin d taking and after 3 months of treatment with it. statistical evaluation of results was realized by methods of variation statistics with calculation of frequency performance indicators (p) and averages (the arithmetic mean x). statistical significance of the result if compared the pa­ tient groups with the control one was evaluated using student t-test. regression analysis was used to determine the relationship between hba1c levels and 25(oh)d3. results after examination of patients with dm type 2, all of them were identified either with the deficiency or insufficiency of vitamin d. in accordance to classification (holick mf et al) at the level of 25(oh)d3 50 nmol/l (20 ng/ml) to 75 nmol/l (30 ng/ml) d-vitamin failure is diagnosed, and decreased level of 25(oh)d3 below 50 nmol/l (20 ng/ml) — vitamin d3 deficiency. we have found out that among 35 patients with dm type 2 nobody has normal level of vitamin d. 62.9% of patients were diagnosed with the deficiency of vitamin d (p<0.01), moreover in 22.7% of them good compensation was observed (p<0.01), in 31.8 % of the patients compensation was satisfactory (p<0.01), and in 45.5% of the patients — unsatisfactory (p<0.01). inverse correlation connection between the decrease level of vitamin d and increase level of glycated haemoglobin was detected (r=-0.40, p<0.05). we considered that compensation of dm was good when the level of glycated haemoglobin was <7.5%, satisfactory — at the level of glycated haemoglobin 7.5-8.5%, unsatisfactory — when the level of glycated haemoglobin was >8.5%. we justified the increased rate of glycated haemoglobin compensation because we examined patients of older age who were recommended to maintain higher indicators of hba1c to avoid hypoglycaemia and brain hypoxia. 37,1% of the patients with dm type 2 (p<0,01) suffered from vitamin d failure, moreover 61.5% of them had satisfactory compensation of diabetes (the level of glycated hemoglobin was 7.5-8.5%) (p<0,01), and 38.5% – unsatisfactory (the level of glycated haemoglobin > 8.5%) (p<0,01). inverse correlation between the decrease of vitamin d and increase of glycated haemoglobin was detected (r=-0.39, p<0.05). in the control group (without diabetes mellitus) there were only 25.7% of patients with a good level of vitamin d (p<0.01), 54.2% of people in the same group suffered from vitamin d insufficiency (p<0.01), and 20.1% of people were diagnosed with vitamin d deficiency (p<0.01). the low implementation of vitamin d for the older patients without diabetes may be associated with alimentary factor and age features of the body. in the control groups the level of glycated haemoglobin was 5.1±0.05% (p<0.01). n. v. pasyechko et al. 12 in t e r n a l m e d ic in e issn 2413-6077. ijmmr 2016 vol. 2 issue 2 the patients of both groups (except those with no deficiency) were provided with a drug alpha d3 teva – 1 mkg daily for three month to correct vitamin deficiency. after that, the levels of 25(oh)d3 and hba1c were tested again. the improvement of diabetes mellitus compensation is clearly stated for patients: decrease the number of patients with unsatisfactory compensation and increase of number of patients with good and satisfactory compensation. 51.4% (p<0.01) of the patients were diagnosed with optimal levels of vitamin d and 11.5% (p<0.01) had its failure. inverse correlation connection between the increased level of vitamin d and decrease level of glycated haemoglobin was detected (r=-0.38, p<0.05). we have established that a number of the patients with unsatisfactory compensation decreased, a number of the patients with satisfactory compensation increased, and patients with high compensation of diabetes was in evidence. after treatment with alpha d3 teva, 34.2% out of 37.1% of the patients with vitamin d failure had normal vitamin d level (p<0.01), and 2.9% still had its insufficiency (p<0.01). inverse correlation between the increased level of vitamin d and decreased level of glycated haemoglobin has been detected (r=-0.39, p<0.05). in the control group, after three months of treatment with alpha d3 teva, 74.2% of the patients had normal vitamin d level (p<0.01), 20% had its failure (p<0.01), 5.8% were diagnosed with the of vitamin d deficiency (p<0.01). discussion many authors consider the matter of vitamin d deficiency, which is a predictor of various diseases and also promotes decom pensation of chronic diseases [11]. it is a topical issue in whole world and in ukraine as well [12]. so, its deficiency correction should not seem less important. the drugs of cholecalciferoli are applied the most for its treatment [13], which, according to research literature, are first­line drugs that include ergocalciferol and structural analogue of vitamin d3-dyhidrotahisterol. the second-line drugs are calcitriol and alfacalcidol. as first­line drugs are exposed to 25­hydroxy­ lation in liver, afterwards are converted into active metabolites in kidneys, therefore, the metabolism of these drugs is decreased in elderly patients with different types of primary and secondary osteoporosis and then don’t have positive effect on bone system. these defects are absent in the second-line drugs [14]. we suggest the drug alphacalcidol for vitamin d deficiency correction, because in our research we examined elderly patients. also, many patients suffer from osteoporosis; in such case this drug is more efficient. the efficiency of alphacalcidol (1 mkg per day) compared to the combination of vitamin d (880 mo per day) and calcium carbonate (1000 mg per day) for patients with postmenopausal osteoporosis and absence of vitamin d deficiency in blood plasma is presented in research literature. in 12 months in the females, who took alfacalcidol, mineral density of lumbar bone was increased by 2.33% (from base level) and in 18 months by 2.87% (p<0.001), and in the group of patients, who received vitamin d and ca — only by 0.7% [15]. there are various conclusions on calcitriol efficacy. some researches proved significant figure 1. the levels of glycated haemoglobin of the patients with diabetes mellitus type 2, who were diagnosed with vitamin d deficiency (before and after treatment with alpha d3 teva). figure 2. the levels of glycated haemoglobin of the patients with diabetes mellitus type 2, who were diagnosed with vitamin d failure (before and after treatment with alpha d3 teva). 0 2 4 6 8 10 <7,5% 7,5-8,5% >8,5% before treatment after treatment 0 1 2 3 4 5 6 7 8 9 7,50% 7,5-8,5% >8,5% before treatment after treatment n. v. pasyechko et al. 13 in t e r n a l m e d ic in e issn 2413-6077. ijmmr 2016 vol. 2 issue 2 effect of vitamin d on mineral density of bone for patients with postmenopausal osteoporosis [16, 17], others stated that such outcome was not obtained that may be associated with low doses [18]. thus, we can argue that the drug alphacalcidol can be recommended for patients with vitamin d deficiency, diabetes type 2 and age­ related osteoporosis. conclusions a clear tendency to the improvement of diabetes mellitus type 2 compensation after medication correction with vitamin d was evidenced. so, by correcting vitamin d deficiency the compensation of diabetes is improved. references 1. pankiv vi. symposium № 156. diabetes mellitus: definition, classification, epidemiology, risk factors. international journal of endocrinology 2013; № 7: 95–104. 2. pankiv vi. the aim of modern treatment of patients with diabetes mellitus: add not only years to life, but life to years. practical angiology 2011; 8 (1). 3. povorozniuk vv,balatska ni, muts via, vdovina oa. deficiency and insufficiency of vitamin d in citizens of ukraine .pain.joints.spine 2011; 4 (04): 5–13. 4. yefimov as,mykhalchuk lm. deficiency of vitamin d and vascular injures at diabetes mellitus type 2 . international journal of endocrinology 2013; 5: 10–13. 5. komisarenko yui. deficiency of vitamin d and its role in development of breach metabolism at diabetes mellitus . clinical endocrinology and endocrine surgery 2013; (3): 69–74. 6. pittas a, lau j, hu f, dawson-hughes b the role of vitamin d and calcium in type 2 diabetes. a systematic review and meta-analysis. j clin endocrinol metab 2007; 92: 2017–2029. 7. pittas a, nelson j, mitri j, hillmann w, gar ganta c, nathan d et al. plasma 25-hyd roxy vitamin d and progression to diabetes in patients at risk for diabetes: an ancillary analysis in the diabetes pre­ vention program. diabetes care 2012; 35: 565–573. 8. lau s, gunton j, athayde n, byth k, cheung n serum 25-hydroxyvitamin d and glycated haemoglobin levels in women with gestational diabetes mellitus. med j aust 2011; 194: 334–337. 9. alzaim m, wood r vitamin d and gestational diabetes mellitus. nutr rev 2013; 71: 158–167. 10. bodnar pm, mykhalchyshyn hp, komisarenko yui. endocrinology. vinnytsia: new book 2013. 11. kienreich k, tomaschitz a, verheyen n, et al. vitamin d and cardiovascular disease. nutrients 2013; 5(8): 3005–3021. 12. povorozniuk vv, balatska ni. deficiency of vitamin d in population of ukraine and risk factors of its development. pain.joints.spine 2012; 4(8): 5–11. 13. komisarenko yui. correction of the breach of metabolism in patients with diabetes mellitus type 1 and type 2 by vitamin d3. ukrainian biochemical journal 2014; 1: 111–116. 14. shvarts hia. deficiency of vitamin d and its pharmacological correction. russian journal of medicine 2009; 7: 477. 15. shupyna my, nechaeva hy, lohynova en, shupyn dv. alfacalcidol in the treatment of osteoporosis: influence on bone density, risk of falls and fractures. treating doctor 2016; 04. received: 2016-12-05 n. v. pasyechko et al. issn 2413-6077. ijmmr 2016 vol. 2 issue 234 issn 2413-6077. ijmmr 2016 vol. 2 issue 2 doi 10.11603/ijmmr.2413-6077.2016.2.7029 medical errors in clinical practice of physicians in ternopil region (ukraine) v. v. franchuk i. horbachevsky ternopil state medical university, ternopil, ukraine background. the professional occupation of a doctor quite often meets different imperfections, which have negative outcome for patients. objective. the study was aimed to investigate the expert characteristics of improper performance of the professional duties by medical staff on the example of a particular region of ukraine. methods. in the study the archival materials (commission forensic medical examinations) held in ternopil regional bureau of forensic medical examination in 2007–2014 years were analysed. the research results are summarized and processed with the use of general statistical methods. results. it is defined that during this period 112 examinations concerning medical malpractice were implemented (9.05% of all commission examinations). conclusions. medical errors were combined, specifically during the diagnostics, treatment and in medical records. the majority of cases (82.1%) of medical malpractice were caused by the objective reasons. key words: medical malpractice, medical errors, forensic medical examinations, dereliction of duty by medical personnel, negligence. introduction the professional occupation of a doctor quite often meets different imperfections, which have negative outcome for patients. after the implementation of criminal code of ukraine in 2001, especially article 140 “unjustifiable dereliction of duty by the medical or pharmaceutical practitioner”, the number of criminal proceedings on the so-called “medical cases” significantly increased. this article became very important for forensic medical examination and for law in general, because of unintended events during medical care delivery and their legal correct qualification were foreseen in ukrainian law for the first time. the experts of the world health organization consider that near 6–7 patients die every day in ukraine as a result of medical errors. the journal of patient safety released a new study in 2013 that estimated number of preventable medical errors above 440,000 annually [1]. every case of inadequate medical care becomes an object of investigation which can’t be implemented without the conclusion of the commission on forensic medical examination. this problem is not st udied enough in contemporary ukrainian forensic medicine. that is why the study of structure, occurrence and peculiarities of medical malpractice is the aim of the research. material and methods in the study archival materials (commission forensic medical examinations) handled in ternopil regional bureau of forensic medical examination (trbfme) in 2007–2014 years were analysed. the research results are summarized and processed by general statistical methods. results and discussion during the studied period 112 examinations were conducted in trbfme for “medical matters” (9.05% of all commission examinations). improper medical care was revealed in majority of the cases (92 (82.1%)) when dereliction of duty by medical personnel occurred. the notion improper medical care or medical malpractice covers all unintended and wilful failures, omissions and defects of medical care delivery, treatment and intervention. due to anglosaxon system of law all failures in the doctors international journal of medicine and medical research 2016, volume 2, issue 2, p. 34–36 copyright © 2016, tsmu, all rights reserved corresponding author: valentyn franchuk, department of pathological anatomy with forensic medicine course, і. horbachevsky ternopil state medical university, 12 ruska street, ternopil, ukraine, 46001 phone number: +380679835870 e-mail: franchukv@tdmu.edu.ua v. v. franchuk issn 2413-6077. ijmmr 2016 vol. 2 issue 2 35 p u b l ic h e a lt h a n d e p id e m io l o g y issn 2413-6077. ijmmr 2016 vol. 2 issue 2 conduct are divided into two types: medical negligence and professional misconduct [2]. from the point of ukrainian legislation all cases of unintentional medical intervention or lack of such can by classified into three main groups: medical maloccurrence, professional crimes of medical practitioners and medical errors. the term maloccurrence is the same both anglosaxon and slavonic law systems: inspite of good medical attention and care, an individual fails to respond properly or may suffer from adverse reactions of the drug [3]. the definition “professional crimes of medical staff” means inadequate medical care or medical intervention that foresees criminal liability of defendant. gross breach of the standards of medical care occurs in those cases the severe consequences develop for a patient. this is similar to the term “criminal negligence” in anglo-saxon law system. other cases of unsuccessful medical care are usually qualified in ukrainian law as “medical errors” (similar to “civil negligence”). totally, 19 cases (20.6%) met unjustifiable medical care and were determined as crimes in medical sphere. medical errors (civil negligence) were recognized by the forensic medical expert commissions in 73 cases (79.4% of all). the majority of such deviations were committed by hospital doctors and were performed during emergency medical care delivery [4]. the proportion of physicians facing an unintentional medical intervention or lack of such [5] ranged from 39.3% in anaesthesiology, 21.4% in internal medicine, 18.7% in obstetrics and gynaecology, 17.8% in paediatrics, 14.3% in general surgery and 4.5% in family medicine, ophthalmology, neurosurgery, 3.6% in neurology, 2.7% in oncology and 1.8% in psychiatry (table 1). the following mistakes were diagnosed by forensic medical examination: misjudgement of diagnostics (61.1% of cases), mistakes in medical records (46.4%); mistakes during invasive procedures or medical treatment including medication errors (40.2%), institutional defects (27.7%), failures in the doctors conduct which impinge upon their professional relationships (deontological) were determined in 21.4% of cases. as a rule, forensic medical expert com mis sions established the presence of two different types of the mistakes (23.2%) simultaneously, if for example misjudgement of clinical symptoms was combined with medication errors. the presence of 4 different types of mistakes were ascertained in 16.7% of “medical cases”; 3 defects (14.3%); all kinds of defects (3.6%). the following failures in the diagnostics were normally revealed: insufficient or incom­ plete general-clinical, laboratory and instrumental examinations; misjudgement of clinical symptoms; inadequacy of the clinical diagnosis; unskilful implementation of conventional special diagnostic methods and procedures. mistakes during medical treatment including medication errors took place: the excess doses of medication, insufficient or excess amount of infusion, unjustified prescription of many the­ table. 1. the prevalence of malpractice among medical practitioners specialty of a physician the number of cases in which deficiencies of paramedics of certain specialties were established (n=112) the percentage on total amount of research cases (%) anaesthesiologists 44 39,3% therapists 24 21,4% obstetricians 21 18,7% paediatricians 20 17,8% surgeons 16 14,3% traumatologists 15 13,4% іnfectiologists 14 12,2% emergency doctors 10 8,9% family doctors 5 4,5% ophthalmologists 5 4,5% neurosurgeons 5 4,5% radiologists and clinicians ultrasound 5 4,5% neurologists 4 3,6% oncologists 3 2,7% psychiatrists 2 1,8% note: digital values exceed the total number of cases due to (in one case) deficiency assumptions on medical professionals of different specialties. v. v. franchuk issn 2413-6077. ijmmr 2016 vol. 2 issue 236 issn 2413-6077. ijmmr 2016 vol. 2 issue 2 rapeutic agents concurrently. besides of these, the breach of care delivery standards, lack of monitoring data about a patient, omission of patient’s condition, unforeseen complications etc. were detected too. the following institutional defects seemed more frequent: equip­ ment malfunctions, absence of consultations; incorrect admission of a patient; inadequate monitoring of hospital administration about medical care. numerous mistakes in medical records were found: the absence of hemo­ dynamic parameters; superficial and incorrect description of objective state, lack of informed consent of a patient, inadequate clinical diagnosis, incomplete epicrisis etc. all deontological errors were commonly conducted with a poor qualification of medical staff, negative rela­ tionships between a patient and doctor, doctor and patient’s relatives [6]. approximately 30% of all studied cases faced the breach of medical care standards. forensic medical examination confirmed the objective reasons for medical malpractice in almost 80% of cases. they were: the severity of patient’s condition or the presence of comorbidity (32.1% of cases); rapid duration of pathological process (21.4%); difficulties in diag­ nostics or atypical manifestations of disease (13.4%). the subjective reasons (i.e., due to poor qualification of medical personnel) were detected in 9.8% of cases. all these cases were qualified as criminal negligence. dereliction of duty by medical staff as a rule was accompanied by severe consequences for patients (fatality, grievous degree of severity, prolongation of illness, moderate degree of severity). conclusions dereliction of duty by medical personnel is confirmed more than 80% of “medical cases” in the conclusions of the commission forensic medical examinations. the occurrence of unintentional medical intervention or lack of such in anaesthesiology, therapeutic, obstetrical/gynaecological, paediatric and surgical cases was more frequent. unintended medical incidents were associated with and occurred during diagnostics, treatment, medical care, medication and in medical records. considered inadequate medical care was usually insufficient and combined with breach of care standards. the majority of medical errors happened due to different objective reasons. criminal negligence was identified in about 20% of medical malpractice cases. references 1. james, john t. a new, evidence-based estimate of patient harms associated with hospital care. journal of patient safety 2013; 9(3): 122–128. 2. shepherd r. medical malpractice (in). simpson’s forensic medicine. london. arnold publi sher; 2003: 15–26. 3. narayan reddy k.s. professional negligence (malpraxis) (in). the essentials of forensic medicine and toxicology. hyderabad. k. suguna devi publisher; 2006: 30–45. 4. sorensen b, lynnerup n. fatal outcome after unintended events following medical intervention: a forensic pathological case survey. rrfms 2014; 4: 11–17. 5. anupam b. jena, seth seabury, darius lakdawalla, amitabh chandra. malpractice risk according to physician specialty. n engl j med 2011; 365 : 629–636. 6. meng you, xu wang, di lu, haidong zhang, shengli di, fengqin zhang et al. analyses of medical malpractice in judicial appraisal: 505 cases. forensic sci med 2015; 1: 21–25. received: 2016-12-09 v. v. franchuk 18 r a d ia t io n m e d ic in e a n d o n c o l o g y issn 2413-6077. ijmmr 2022 vol. 8 issue 2 doi 10.11603/ijmmr.2413-6077.2022.2.13154 anthropometric variables in breast lesions of women of reproductive age in university of port harcourt teaching hospital *c. d. orupabo, c. g. odoya rivers state university, port harcourt, nigeria background. benign breast diseases are a potential health concern to a large number of women. the incidence of cancer is increasing worldwide. a steady increase in incidence has been observed in most developed and developing counties. objective. thus, our study seeks to investigate the anthropometric variables implicated in breast lesions of women of reproductive age. methods. this is a descriptive and prospective study with a random sampling method; the data was obtained from the department of surgery, radiotherapy and oncology unit of university of port harcourt teaching hospital. a total number of 146 subjects (18-49 years) were involved into this study. a number of 90 were breast lump patients and 56 were breast cancer patients. results. data obtained was analysed with microsoft excel. our results show no statistically significant difference (p> 0.05) in the body mass index (bmi) and waist-hip-ratio (whr) of both the breast lump and breast cancer patients. this study supports the fact that high bmi is a predictive marker for breast lumps and cancer in women. conclusion. the study has also identified that high bmi favours the incidence of breast lumps and breast cancers in women of reproductive age. keywords: anthropometric variables; bmi; waist-hip-ratio; breast cancer; breast lump. *corresponding author: dr. clinton david orupabo, lecturer 1, faculty examination officer, faculty of basic medical sciences, college of medical sciences, rivers state university, port harcourt, pmb 5080, nigeria. e-mail: clinton.orupabo@ust.edu.ng international journal of medicine and medical research 2022, volume 8, issue 2, p. 18-23 copyright © 2022, tnmu, all rights reserved c. d. orupabo et al. introduction benign breast diseases are common throughout a woman’s lifetime, from early reproductive life to the postmenopausal part of life, making it a potential health concern to a large number of women. malignant lesions on the other hand are cancerous in nature and a r e c h a r a c t e r i z e d b y p r o g r e s s i v e a n d uncontrolled growth. breast cancer is one of the most common types of malignant diseases, affecting millions of women around the world, with a high rate of morbidity and mortality. the prevalence rate of breast cancer, particularly in nigeria, accounts for 20% to 25% of tumours in women with an annual incidence of about 800 to 1000 cases [1]. african breast cancer patients tend to present at a younger age, with a lesion characterized by large and palpable tumour, multiple nodal involvements, and have poorer clinical and pathological prognostic factors compared with caucasian patients [2]. these documented characteristics are somewhat similar to that of african-americans but are in contrast with those of non-hispanic whites in t h e u s a , w h i c h t h e re f o re c a l l s f o r t h e consideration of genetic factors in the aetiology of breast cancer in general, and in people of african origin in particular [3, 4]. in 2017, an estimated 252,710 new cases of invasive breast cancer were diagnosed among women and 2,470 cases were diagnosed in men and thus approximately 40,610 women and 460 men were expected to die from breast cancer in 2017 [5]. the incidence of cancer is increasing worldwide. a steady increase in incidence has b e e n o b s e r v e d i n m o s t d ev e l o p e d a n d developing counties. apart from incidence, cancer related deaths are also increasing. there were over 230,000 new cases of breast cancer each year in the united states as at 2015. some studies had affirmed that the risk of breast cancer increase with age, and in the united states approximately one out of eight women will get breast cancer at some point in their lives [6, 7, 8]. thus with 1.7 million new cases diagnosed in 2012, constituting 12% of all new cancer cases and 25% of all cancers in women, breast cancer remains the commonest site19 r a d ia t io n m e d ic in e a n d o n c o l o g y issn 2413-6077. ijmmr 2022 vol. 8 issue 2 specific malignancy affecting women and the most common cause of cancer death in women. in nigeria, female breast cancer is recognized as major cause of morbidity and mortality with incidence rate ranging from 36.3 to 50.2/100,000 live birth [9]. more so, late presentation of patients at advanced stages when little or no benefit can be derived from any form of therapy is the hall mark of breast cancer. reasons given include poverty, under-education, lack of knowledge and poor access to care [10]. indeed, the ratio of mortality to incidence in africa is 0.73, compared to below 0.23 in north america and 0.354 in latin america and the caribbean [11]. risk factors associated with breast cancer development include non­modifiable ones such as female sex, increasing age, a history of breast cancer in close relatives especially in mothers and siblings. non­modifiable ones include early menarche before the age of 14 years or menopause later than the age of 55 years. other risks include overweight and obesity, prolonged use of hormone replacement therapy, oral contraceptives, tobacco use, alcohol intake, nulliparity or having first child after age 35. thus, our study seeks to investigate the anthropometric variables implicated in breast lesions of women of reproductive age. high body weight (measured in terms of body mass index, bmi) has been recognized as an important risk factor for breast cancer among postmenopausal women in many previous epidemiological studies [12, 13, 14, 15]. after menopause, epidemiological evidence found a substantial positive association between bmi and breast cancer risk [16]. a meta-analysis showed that the substantial positive association between post-menopausal bmi and breast cancer risk was confined to hormonal factors [17]. some studies have shown a decreased risk for premenopausal women [18, 19], while others have shown no association [20, 16]. breast cancer has been documented in other studies as the most frequently occurring cancer in women of reproductive age [21]. methods this is a descriptive and prospective study with a random sampling method; the data was obtained from the department of surgery, radiotherapy and oncology unit university of port harcourt teaching hospital. demographic information such as age, body weight, height, waist circumference, hip circumference was obtained from the patients. a consent form was given to the patients prior to data collection. the population of this study includes breast lumps and breast cancer patients, the age range of 18 49 years old, of university of port harcourt teaching hospital, rivers state, nigeria. the participants voluntarily consented to the study which was conducted in compliance with the ethical standards. a total number of 146 subjects were involved into the study; 90 of them were breast lump patients and 56 were breast cancer patients. data obtained was analysed with microsoft excel. the following anthropometric variables were measured. i. weight (kg): weight is measured to the nearest 0.1 kg when the subject is standing and putting on light indoor clothes excluding shoes, belt and sweater using a weighing scale. heavy jewellery if any present was removed and pockets emptied. ii. height (cm): standing height is the measurement of the maximum distance from the floor to the vertex, when the subject is facing forward. iii. the bmi: it was calculated as the ratio of weight in kilogram by square of height in meters (m2). i.e.: bmi (kg/m2) = weight (kg)/ height (m2). it is a measure of body mass corrected for height which is used to assess the extent of weight deficit or excess. bmi also provides a practical indicator of adiposity and hence overweight or obesity. iv. waist circumference (cm): the tape is used to circle the waist (like a belt would circle the waist) at the natural waistline, which is midpoint between the lowest rib and the iliac crest. the subject is asked to stand erect while measurements are taken. the measurement is taken at the midpoint between the lowest rib and the iliac crest. the measuring tape is placed perpendicular to the long axis of the body and horizontal to the floor, with sufficient tension to avoid slipping off but without compressing the skin. the measurement is made at the end of a normal expiration to the nearest 0.1 cm v. hip circumference (cm): the subject stands erect, the weight is evenly distributed on both feet. the tape is placed at the maximum extension of the buttocks, horizontal to the floor, with sufficient tension to avoid slipping off. the tape is held a bit tighter but without compressing the buttocks. the zero end of the tape is held under the measurement value recorded to the nearest 0.1 cm. c. d. orupabo et al. 20 r a d ia t io n m e d ic in e a n d o n c o l o g y issn 2413-6077. ijmmr 2022 vol. 8 issue 2 vi. w a i s t h i p r a t i o ( w h r ) : t h i s i s calculated by dividing the waist measurement by the hip measurement, since the hips are the widest part of the buttocks. the formula is: whr = waist circumference/hip circumference. ethical approval ethical approval was obtained from the research ethics committee of university of port harcourt teaching hospital with the number of upth/adm/90/s.ii//vol.xi/1270. informed consent was obtained from all respondents prior to data collection. results the data in table 1 shows that the mean bmi for age group (18-25) is 23.71, which is normal according to world health organization (who) classification. table 2 shows the anthropometric data for women with breast cancer who had the mean bmi of 26.89, 27.92, 26.85, and 28.62 for the various age groups studied. they therefore just like their counterparts with breast lumps are within the overweight category. a t value of -0.88 means that there is some similarity between the bmi of those with breast lump and those with breast cancer; however, the t value shows some difference does exist. the negative t value shows that bmi values affect both the occurrence of lumps and that of cancer. the data shows that both patients with breast lumps and those with breast cancers all have high bmi: overweight. a p value of 0.38 (>0.05) shows that there is no statistical difference in the bmi of patients with breast lump and breast cancer. thus, high bmi favours the occurrence of breast lumps and cancers in women of reproductive age (table 3). a p value of 0.44 (> 0.05) shows that there is no statistical difference in the whr of the patients with breast lump and breast cancer. however, those with breast cancer have a higher mean bmi and a higher mean whr. thus, bmi and whr affects the incidence of breast cancer (table 4). discussion the results of this study showed that many of the participants involved were overweight. also, for age groups (26-33), (34-41) and (41-42), their mean bmi were 25.69, 27.54 and 29.93, table 1. average anthropometric values of reproductive age women with breast lump age group (years) n weight (kg) height (m) bmi x wc (cm) hc (cm) whr x 18-25 15 63.93 1.64 23.71 normal 69.09 84.16 0.80 normal 26-33 26 67.42 1.62 25.69 overweight 74.25 89.66 0.83 normal 34-41 29 73.97 1.63 27.54 overweight 79.18 97.91 0.81 normal 42-49 20 78.85 1.63 29.93 overweight 83.95 101.48 0.83 normal notes. n – number of subjects, bmi – body mass index, wc – waist circumference, hc – height circumference, whr – waist-hip ratio. whr (women): <0.85 = normal, >0.85 = risk, x – classification. table 2. average anthropometric values of reproductive age women with breast cancer age group (years) n weight (kg) height (m) bmi x wc (cm) hc (cm) whr x 18-25 2 68 1.59 26.89 overweight 76.20 88.9 0.85 risk 26-33 9 75.78 1.65 27.92 overweight 83.82 101.04 0.84 normal 34-41 25 72.24 1.64 26.85 overweight 82.30 98.14 0.84 normal 42-49 20 73.90 1.61 28.62 overweight 84.20 105.54 0.80 normal notes. n – number of subjects; bmi – body mass index; wc – waist circumference; hc – height circumference; whr – waist-hip ratio. whr (women): < 0.85 = normal, > 0.85 = risk, x – classification. table 3. relationship between bmi of breast lump and breast cancer patients irrespective of age group x n percentage, % mean±sd t value p value inference breast lump 90 61.64 26.90±4.71 -0.88 0.38 not significant breast cancer 56 38.35 37.66±5.28 notes. x – classification; bmi – body mass index; sd – standard deviation. c. d. orupabo et al. 21 r a d ia t io n m e d ic in e a n d o n c o l o g y issn 2413-6077. ijmmr 2022 vol. 8 issue 2 respectively, indicating that they were all in the overweight category. the mean waist hip ratio (whr) was within the norm for all the women within this age group and classification. our data shows the anthropometric data for women with breast cancer, who had the mean bmi of 26.89, 27.92, 26.85, and 28.62 for the various age groups studied. they therefore just like their counterparts with breast lumps are within the overweight category. their whr were also within norm except for women within the age group 18-25 (table 2). again, both tables 3 and 4 shows that there is no statistically significant difference (p> 0.05) in the bmi and whr of both the breast lump and breast cancer patients. thus, the same variables implicated in the incidence of breast lumps also affected those of breast cancers. our study therefore shows that the same anthropometric variables present in reproductive age women with breast lump were also found in those women with breast cancer. high bmi thus influences the incidence of breast lump and breast cancers proportionately, as more than seventy percent of the subjects are within the overweight category. the whr in the study was not a major factor to have influenced the lumps and cancers. about 62% of the patients studied had breast lumps and about 38% had breast cancers. the findings were consistent with several other authors who reported that high bmi favours the occurrence of breast cancer in women, especially postmenopausal women [16, 22], although our study covered women within the reproductive age. it is therefore apparent to affirm that high bmi favours the occurrence of both breast lumps and cancers in women. one most classical biological explanation is as a r e s u l t o f a f e m a l e h o r m o n e r e l a t e d mechanism. this is for the fact that adipose tissue may be a major source of oestrogens [14], which are critical mitogens for mammary epithelial cells [12, 15]. despite the fact that our findings are associated high bmi with cancer among women of reproductive age, some other studies among premenopausal women are inconsistent and unclear [23]. some studies have shown a decreased risk for breast cancer among premenopausal women [18, 19] while others have shown no association [16, 20]. our study did not compare breast cancer risk between healthy women and cancer patients though, as we only focused on the prevalent anthropometric variables among women with breast lesions. most probably this comparison would have shown the level of risk association between the two groups as argued by previous researchers. nonetheless our study has identified the association of high bmi with breast lumps and cancers in women of reproductive age. conclusion this study supports the fact that high bmi can be used as a tool to predict breast cancer as well as breast lumps for women. more so the study has also identified that high bmi favours the incidence of breast lumps and breast cancers in women of reproductive age. the findings did not show any statistically significant correlation between high whr and breast lumps and/or breast cancers as most of the subjects in this category were within the norm despite being diagnosed of a lump and/or cancer. conflict of interest authors declare no conflict of interest. funding the research was funded by the authors. acknowledgements we the most sincerely appreciate the staff and matrons of the surgical and oncology outpatient clinic of the university of port harcourt teaching hospital for the support and cooperation which enabled us to obtain the data needed. again, we thank the hospital management and ethics review committee for granting the approval for the study. author’s contributions clinton david orupabo – conceptualization, methodology, formal analysis, writing – reviewing and editing; chelsea odoya george – investigation, data curation, writing – original draft. table 4. relationship between the waist-hip ratio of breast lump and breast cancer patients irrespective of age group x n percentage, % mean±sd t value p value inference breast lump 90 61.64 0.82±0.06 -0.77 0.44 not significant breast cancer 56 38.35 0.83±0.07 notes. x – classification, whr – waist-hip ratio, sd – standard deviation. c. d. orupabo et al. 22 r a d ia t io n m e d ic in e a n d o n c o l o g y issn 2413-6077. ijmmr 2022 vol. 8 issue 2 антропометричні параметри уражень молочної залози у жінок репродуктивного віку в університетській лікарні порт-харкорт c. d. orupabo, c. g. odoya rivers state university, port harcourt, nigeria. вступ. зростання частоти та поширеності доброякісних захворювань молочної залози становить потенційну загрозу для значної кількості жінок. ця тенденція спостерігається в більшості розвинутих країн і країнах, що розвиваються. мета. наше дослідження було спрямоване на дослідження антропометричних змінних уражень молочних залоз у жінок репродуктивного віку. методи. проведене описове проспективне дослідження методом випадкової вибірки, дані були отримані з відділення хірургії, променевої терапії та онкології університетської лікарні порт-харкорта. усього в цьому дослідженні було залучено 146 пацієнток (18-49 років). з них 90 були пацієнтами з ущільненням грудей і 56 – з раком молочної залози. результати. статистична обробка даних зроблена за допомогою microsoft excel. аналіз даних не виявив статистично значущої різниці (p> 0,05) між індексом маси тіла (імт) та показниками співвідношення талії і стегон (whr) як у пацієнтів із доброякісними новоутворами молочної залози, так і у пацієнтів з раком молочної залози. однак, отримані нами дані вказують на те, що високий імт є прогностичним маркером утворення і розвитку доброякісних і злоякісних захворювань молочної залози. висновки. високий індекс маси тіла є сприятливим фактором виникнення доброякісних та злоякісних захворювань молочної залози у жінок репродуктивного віку. ключові слова: антропометричні змінні; індекс маси тілі (імт); співвідношення талії та стегон; рак молочної залози; доброякісні захворювання молочної залози. information about the authors clinton david orupabo – lecturer 1, faculty examination officer, faculty of basic medical sciences, college of medical sciences, rivers state university, port harcourt, nigeria. https://orcid.org/0000 0002 4784 4273, e­mail: clinton.orupabo@ust.edu.ng chelsea odoya george – graduate assistant, department of human anatomy, faculty of basic medical sciences, rivers state university, port harcourt, nigeria. e­mail: odoya.chelsea.george@gmail.com references 1. adebamowo ca and ajayi, oo. breast cancer in nigeria. west african journal of medicine. 2000; 19, 179-91. 2. amin jt, isyak mk and mimham sd. breast cancer in sub-saharan africa. african journal of medical science. 1993; 1:5­10. 3. ajayi oo. breast cancer in nigeria. west african journal of medicine. 2001; 20:211­9. 4. ganiy oj and ganiyu ar. epidemiology of breast cancer in europe and africa. 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7 ­ 0215(19980529)76:5<647::aid­ijc6>3.0.co;2­q 21. mccray dk, simpson ab, flyckt r, liu y, o’rourke c, crowe jp. fertility in women of reproductive age after breast cancer treatment: practice, patterns and outcomes. ann surg oncol. 2016; 23(10):3175­81. https://doi.org/10.1245/s10434­016­5308­y 22. cold s. effect of pregnancy as a risk factor for breast cancer in brca1/brca2 mutation carriers. international journal of cancer. 2002; 117:988­91. 23. morimoto lm, white e, chen, z, chlebowski, rt, hays, j, kuller, l et al. obesity, body size, and risk of postmenopausal breast cancer: the women’s health initiative (united states). cancer causes control. 2002; 13(8):741­51 https://doi.org/10.1023/a:1020239211145 received 30 august 2022; revised 28 october 2022; accepted 17 november 2022.. this is open access article distributed under the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. c. d. orupabo et al. 9 o b st e t r ic s a n d g y n e c o l o g y issn 2413-6077. ijmmr 2016 vol. 2 issue 1 doi 10.11603/ijmmr.2413-6077.2016.1.6372 complex approach to treatment of subchorionic hematoma in early threatened abortion s. n. heryak, n. v. petrenko, i. ya. kuziv, o. y. stelmakh, n. i. bagniy, i. v. korda, v. yu. dobryanska, l. v. bagniy i. horbachevsky ternopil state medical university, ternopil, ukraine background. currently, miscarriage is considered to be a multietiological disorder with trombofilic violations and hormone deficiency as the leading factors. despite the achievements in treatment of miscarriage, the frequency of preterm termination of the wanted pregnancies is still high and the number of perinatal losses is significant. therefore, pathogenetically based therapy, safe for the foetus, is very important in management of pregnancy interruption in the first trimester. a proper drugs administration provides optimal concentration of active ingredients and fast action. the aim is to improve effectiveness of the early threatened abortion treatment in cases of subchorionic hematoma (sch) by combination of sublingual natural micronized progesterone and tranexamic acid objective. we examined 50 pregnant women with early threatened abortion with sch. we studied system of haemostasis, basic hormonal markers and ultrasound criteria of threatened abortion. we compared efficacy of treatment between traditional (supportive) therapy (sedation, spasmolytic, haemostatic drug) and combination of supportive therapy in combination with tranexamic acid and natural micronized progesterone. results. the result of lab tests showed minimal signs of hypercoagulation, hyperfibrinogenemia and platelet hyperactivity, a significant β-hcg level decrease and approximate decrease in progesterone and free estriol production. sonographic examination showed presents of local myometrial hypertonus, deformation of fertilized egg, hypoplasia of chorion, low location of fertilized ovum, retarded growth of crl. the research proved that combined administration of sublingual micronized progesterone and tranexamic acid for the treatment of threatened abortion with sch has more significant positive effect for pregnancy maintenance due to clinical, biochemical, hormonal and ultrasound results if compared with the group which underwent supportive therapy. conclusions. complex application of natural micronized progesterone 100 mg three times a day sublingually and 500 mg of tranexamic acid dissolved in 200 ml normal saline solution improves the dynamics of the main hormonal, haemostatic and ultrasound markers of abortion and significantly reduces reproductive losses. tranexamic acid treatment proved a rapid and effective action on hematoma and absence of embryotoxical and сoagulopathyc influence. tranexamic acid does not cause any significant disorders of hemostatic system. this is very important at the early gestation because of intravascular coagulation, physiological hypercoagulable condition during pregnancy that can cause microthrombosis and disrupt placentation. on the other hand, it is dangerous for the mother’s health because of the increased risk of thrombosis. key words: threatened miscarriage, subhorial hematoma, micronized progesterone, tranexamic acid. introduction currently, miscarriage is considered to be a multietiological disorder with trombofilic violations and hormone deficiency as the leading factors. the problem of early pregnancy loss remains urgent because these factors are the most common complications without any downward tendency [1–4]. according to the latest concepts, 80% of all pregnancy losses occur at the early stages. one of the first signs that threatens the pregnancy is subchorionic hematoma (sch) [5, 6]. it is the most unfortunate sign of threatened abortion. it is established that the disruption of trophoblast invasion in the 1st trimester of pregnancy leads to the advanced gestational complications: threatened abortion, preeclampsia, preterm delivery, pla cental abruption, which increase perinatal, in fant and s. n. heryak et al. corresponding author: svitlana heryak, department of obstetrics and gynecology no 2, i. horbachevsky ternopil state medical university, 1 maidan voli, ternopil, ukraine, 46001 tel.: +380352254891 e-mail: heryak@tdmu.edu.ua international journal of medicine and medical research 2016, volume 2, issue 1, p. 9–12 copyright © 2016, tsmu, all rights reserved 10 o b st e t r ic s a n d g y n e c o l o g y issn 2413-6077. ijmmr 2016 vol. 2 issue 1 maternal mortality. the protective func tion of the immune system at the early stages of gestation such as the suppression of natural killer cells in the endometrium, mother’s blocking fac tors production is provided by diffe rent mostly hormonal dependent mechanisms; their disorders lead to defects of implantation, sch and can cause early pregnancy termination [3, 7]. the endometrium and decidua contain a huge number of immune system cells, all of them are able to secrete cytokines. main cyto kines that are secreted in the body inhibit the embryo development, proliferation and deve lop ment of normal trophoblast. they affect the embryo, both directly and indirectly, depending on the intensity of secretion and differentiation of target tissues [6]. cytokine cascade can be triggered by infectious agent and by endogenous factors (hypoxia, hormones, etc.) as well. per sis tent viral bacterial infection, neuroen docrine disorders, chromosomal ab nor malities, the toxic effects, stress and envi ronment are also etio logical factors of early abortion [8, 9]. despite the achievements in treatment of mis carriage, the frequency of preterm termination of the wan ted pregnancies is still high and the number of perinatal losses is significant. therefore, pathogenetically based therapy, safe for the foetus, is very important in management of pre gnancy interruption in the first trimester. a proper drugs administration provides optimal concentration of active ingredients and fast action. the aim is to improve effectiveness of the early threatened abortion treatment in cases of subchorionic hematoma by combination of sublingual natural micronized progesterone and tranexamic acid. materials and methods the examination of 50 pregnant women was conducted at the department of gynaecology of ternopil regional perinatal centre “mother and child”. they received treatment for threatened abortion in gestational age from 8 to 12 weeks. all women were diagnosed with sch by means of ultrasound. to exclude infection as an etiological factor of abortion, bacteriological research of torch and sexually transmitted infections were conducted. all patients had negative results of the tests for these infections. system of haemostasis was evaluated by determination of platelet and aggregation time, prothrombin index (pi), thrombin clotting time, activated partial thromboplastin time (aptt), fibrinogen concentration. basic hormonal markers of abortion were also studied: the concentration of free estriol and progesterone by radioimmunoassay method, sets made by company “sorin” (france), and radioisotope sets amerlayt made by international company amersham. β-subunit of human chorionic gonadotropin (β-hcg) concen tration was determined by immune fluorescence analysis with time resolution (test system delfiya, wallac, perkinelmer) (according to the manufacture standard protocol of test systems). to determine efficacy of the proposed complex of therapeutic measures, all the pregnant were tested with transvaginal ultrasound on the 14th day of the treatment. the pregnant of the 1st group (n=25; the comparison group) received supportive therapy – sedation, spasmolytic, haemostatic drug. the pregnant of 2nd group (n=25), together with supportive therapy, received tranexamic acid 2 ml (500 mg) dissolved in 200 ml normal saline solution intravenously till the arrest of bleeding and natural micronized progesterone 100 mg three times a day sublingually. the control group consisted of 20 healthy pregnant women without any symptoms of threatened abor tion. results and discussion the evaluation of plasma-coagulation and vascular-platelet hemostasis showed minimal signs of hypercoagulation, hyperfibrinogenemia and platelet hyperactivity if compared with the women with physiological pregnancy. this suggests a thrombophilia as an etiopathogenetic factor of early abortion with sch in the examined women. the evaluation of hormonal homeostasis showed, that at the early gestational age a significant β-hcg level decrease and approximate decrease in progesterone and free estriol production were the diagnostic markers of threatened abortion with sch the results of ultrasound examination showed, that sch was located in the area of the lower chorion in 32 (64%) of the pregnant women, in the central part – in 18 (36%) pati ents. also sonographic examination showed other markers of abortion. so, local myometrial hyper tonus was diagnosed in 43 (86%) pregnant women, deformation of fertilized egg – in 32 (64%), hypoplasia of chorion – in 12 (24%), low location of fertilized ovum – 8 (16%), retarded growth of crl – in 2 (4%). the research proved that combined administration of sublingual micronized progesterone s. n. heryak et al. 11 o b st e t r ic s a n d g y n e c o l o g y issn 2413-6077. ijmmr 2016 vol. 2 issue 1 and tranexamic acid for the treatment of threatened abortion with sch has more sig­ nificant positive effect for pregnancy main­ tenance due to clinical, biochemical, hormonal and ultrasound results if compared with the group which underwent supportive therapy. after the symptomatic treatment a spon­ taneous abortion happened in 6 (24%) women, missed abortion in – 6 (24%), gestational pro­ cess complicated by hyperemesis gravidarum which required additional therapy – in 4 (16%). among 25 women of the 2nd group spontaneous abortion occurred in 3 (12%) patients, missed abortion – in 1 (4%) woman. the results of our research have shown that in women of the 2nd group the level of platelets increased and did not significantly differ from the control group findings. in patients of the 1st group it was significantly lower than in women of the 2nd and control groups. the similar chan­ ges were evidenced in the evaluation of pi. in 2 weeks of the proposed therapy, the level of β­hcg increased in both groups of women, but in the 2nd group it did not sig­ nificantly differ from the control group. after the treatment the level of free estriol and pro­ gesterone increased and did not significantly differ from the control group with the similar results. symptomatic and complex treatment sig­ nificantly reduced the sonographic signs of abortion, but in the 2nd group these changes pronounced more positive trend than in the 1st group. so, in 18 (72%) pregnant women of the 2nd group and in 4 (16%) – of the i group (p<0,05) there was a complete resumption of sch, hematoma decreased in size in 3 (12%) women of each group. thus, the prescription of micronized proges­ terone sublingually provides an inhibitory effect on the contractile activity of myofibrils, prevents the further chorine detachment that leads to pregnancy maintenance and prevents repro­ ductive losses. our research demonstrates the necessity of the inclusion of micronized progesterone in 100 mg 3 times daily sublingually and tranexamic acid 500 mg intravenously to the treatment protocol of early threatened abortion with sch. also this treatment is more effective if compared with the traditional supportive therapy (seda­ tion, spasmolytic, haemostatic drug). conclusions 1. complex application of natural micronized progesterone 100 mg three times a day sub­ lingually and 500 mg of tranexamic acid dissolved in 200 ml normal saline solution improves the dynamics of the main hormonal, haemostatic and ultrasound markers of abor­ tion and significantly reduces reproductive losses. 2. tranexamic acid treatment proved a rapid and effective action on hematoma and absence of embryotoxical and сoagulopathyc influence. tranexamic acid does not cause any significant disorders of hemostatic system. this is very important at the early gestation because of intravascular coagulation, physiological hyper­ coagulable condition during pregnancy that can cause microthrombosis and disrupt pla­ centation. on the other hand, it is dangerous for the mother’s health because of the in­ creased risk of thrombosis. references 1. венцківський бм. стан імунного та гормо­ нального статусусфетоплацентаного комплексу при недоношуванні вагітності. педіатрія, акушер­ ство та гінекологія 2012; 3: 40­43. 2. резніченко гі. профілактика невиношуван­ ня вагітності і передчасних пологів. жіночий лікар 2013; 3: 10­12. 3. soldo v, cutura n, zamurovic m. threatened miscarriage in the first trimester and retrochorial hematomas: sonographic evaluation and signifi­ cance. clin exp obstet gynecol 2013; 40(4): 548­50. 4. şukur ye, goç g, kose o, acmaz g, ozmen b, atabekoglu cs, koc a, soylemez f. the effects of subchorionic hematoma on pregnancy outcome in patients with threatened abortion. j turk ger gyne­ col assoc 2014; 15(4): 239­242. 5. dongol a, mool s, tiwari р. outcome of preg­ nancy complicated by threatened abortion. kath­ mandu univ med j 2011; 9(33): 41­44. 6. odeh m, ophir е, grinin v, tendler r, kais м, bornstein j. prediction of abortion using three­ dimensional ultrasound volumetry of the gestational sac and the amniotic sac in threatened abortion. j clin ultrasound 2012; 40(7): 389­393. 7. biesiada l, krekora m, krasomski g. subcho­ rionic hematomaas a risk factor of pregnancy and s. n. heryak et al. 12 o b st e t r ic s a n d g y n e c o l o g y issn 2413-6077. ijmmr 2016 vol. 2 issue 1 delivery in women with threatening abortion. ginekol pol 2010; 81(12): 902-906. 8. hodgson dt, lotfipour s, fox jc. vaginal bleeding before 20 weeks gestation due to placental abruption leading to disseminated intravascular coagulation and fetal loss after appearing to satisfy criteria for routine threatened abortion: a case report and brief review of the literature. j emerg med 2009; 32(4): 387-392. 9. stamatopoulos n, lu c, infante f, menakaya u, casikar i, reid s, mongelli m, condous g. does the presence of subchorionic haematoma increase the risk of miscarriage? ultrasound in obstetrics & gynecology 2013; 42 (1): 54. received: 2015-12-01 s. n. heryak et al. koncor1_2014.pm6 35 o b s t e t r ic s a n d g y n e c o l o g y ijmmr 2015 vol. 1 no. 1 ultrastructural research of the endometrium receptivity in the conditions of pre�conceptional preparation in refractory pregnancy loss i. o. marinkin, d. l. nepomnyashikh, v. m. kuleshov, n. a. ilizarova, t. o. semchenko, s. v. aidagulova novosibirsk state medical university, novosibirsk, russia background. refractory pregnancy loss (rpl) is a multifactoral pathology in women of reproductive age which treatment represents great difficulties. great role in rpl pathogenesis is stipulated by chronic endometritis resulting from disturbances of implantation followed by gestational sac malfunction, which is not less significant than progesterone deficiency. objective. the aim of research was to study the dynamics of ultrastructural changes of endometric epithelial cells in the conditions of pre-conception preparation in refractory pregnancy loss. methods. 63 samples of endometrium bioptates obtained from female patients of reproductive age with insufficient middle luteal phase of the cycle were studied. light and electronic microscopy was used to study the morphological changes in endometric epithelial cells. results. pre-conceptional cyclic hormone therapy (1 mg 17�-estradiol and 20 mg of didrogesteron) in comparison with monotherapy of 20 mg of didrogesteron during 3 months contributed to intracellular regeneration and restoration of secretor phenotype of endometrium epithelial cells corresponding to the status of receptivity with “opened window of implantation”. conclusions. cyclic hormonotherapy in combination with complex metabolic therapy as a pre-conceptional preparation contributing to pregnancy illustrates much higher effectiveness of pathogenetically proved preconceptional hormonotherapy key words: refractory pregnancy loss, endometrium receptivity, hormone therapy, ultrastructure. introduction refractory pregnancy loss (rpl) is a multifactoral pathology in women of reproductive age, which treatment represents great difficulties [1, 3, 4]. in the majority of cases the termination of pregnancy in prl is due to inadequate reaction to endogenic progesterone and disturbance of secretory transformation of endometrium that results in the disturbance of blastocyst receptivity. great role in rpl pathogenesis is stipulated by chronic endometritis resulting from disturbances of implantation followed by gestational sac malfunction, which is not less significant than progesterone deficiency [11]. weak receptive sensitivity of the endometrium in maintaining normal level of hormones is connected with a wide prevalence of chronic infectious and inflammatory processes accompanied by high content of cytotoxic cells cd16+ and cd56+ and their products such as anti-inflammatory cytokines that, in their turn, contribute to activation of prothrombinase with following thrombosis and placental infarctions [5, 8]. besides of all mentioned causes of rpl and chromosomal fetus pathologies, there are idiopathic misbirths (approximately 15– 25 %), pathogenesis of which is not clear and empirically progesterone is used [4]. hormone therapy, as well as drugs possesing direct or indirect action on the steroid metabolism, are used to recover endometrium receptivity in case of correction of luteal insufficiency in rpl [12]. however, a great percentage of misfortune in the recovery of endometrium receptivity and achievement of fertility in rpl needs development of new approaches to the therapy and improvement of schemes of pre-conceptional preparation [9]. aim of the research: the research was aimed to study the dynamics of ultrastructural changes of epithelial cells of the endometrium in the conditions of pre-conceptional hormone therapy in rpl. methods morphological studying of endometrium sampling bioptates in 63 women of reproductive age with rpl was performed. the patients were empirically divided into 2 groups (31 and 32 female patients 27.2±1.2 and 28.3±0.9 years old, respectively) depending on the scheme of pre-conceptional address for correspondence: igor marinkin, state budget educational institution of higher professional education “novosibirsk state medical university” of the ministry of public health, krasny prospect, 52, novosibirsk, 630091, russia i. o. marinkin et al. international journal of medicine and medical research 2015, volume 1, number 1, p. 35-38 copyright © 2015, tsmu, all rights reserved 36 o b s t e t r ic s a n d g y n e c o l o g y ijmmr 2015 vol. 1 no. 1 hormone therapy performed during 3 menstrual cycles. the first scheme is a cyclic hormone therapy, in which femostin was administered 1/10 (1 mg 17�-estradiola and 10 mg didrogesterone) from the 1st to the 28th day of the cycle with additional supplementation of dodrogesterone (dufastone) in the dosage 10 mg per 24-hours from the 16th till 25th day. it’s important to note that in comparison with recommended schemes [4], the 17�-estradiol dosage was decreased twice to stimulate insignificantly proliferative and differentiated activity of cellular endometrium populations. the first scheme included administration of 20 mg of didrogesterone (dufastone) per 24-hours from the 16th to 25th days of cycle. in both groups hormone therapy was used on the background of cyclic metabolic therapy including 100 mg of vitamin e in combination with 0.012 g of lipoic acid and 0.1 g of calcium pantothenate from the 5th to 15th days of the cycle, and with 1 mg of folic acid and 0.5 mg potassium orotate from the 16th to 25th days, respectively. sampling bioptates of endometrium were obtained according to the indications on the 22-24th day of the menstrual cycle till pre-conceptional preparation. bioptates were fixed in 4 % solution of paraform. paraffin sections colored with haematoxylin and eosin, as well as semithin sections colored with schiff’s reagent and toluydine blue, were studied using microscope axio scope.a1 with camera axiocam mrc5 and software zen blue (c.zeiss). ultrathin sections of epithelial cells of endometrium contrasted with uranyl acetate and lead citrate were studied with electronic microscope jem100s. written agreement on performing examination and treatment of rpl was taken in female patients. results before pre-conceptional hormone therapy the complex of structural changes of endometrium in female patients with rpl in the middle secretory stage of menstrual cycle in light-optical studying of paraffin and semithin sections corresponded to general pathological agreement about severe dystrophy and atrophy. endometrial glands were reduced in the amount, being shortened and polymorphic according to the maturity: secretory glands did not prevail and were desynchronized with the phase of menstrual cycle, with dystrophy and atrophy of epithelium. subepithelial stroma of endometrium was characterized by weak signs of decidualization of cellular elements and the presence of simple subepithelial lymphatic cells (fig. 1), that didn’t correspond to the severe inflammatory changes in chronic endometritis. electronic microscopic examination of epithelial cells of endometrium revealed ultrastructural signs of a considerable reduction of the level of biosynthetic processes. the decreasing of height of epithelial cells, apical membrane of which didn’t form pinopods, associated with severely decreased amount of nucleolus and components of canalicular system in nucleus and was accompanied with the reduction of elements of granular cytoplasmatic net in the absence of mega mitochondria. the decrease of synthesized protein and energetic potentials of epithelial cells was due to the accumulation of only singular glycogen grains. thus, ultrastructural equivalents of the disturbance of endometrium receptivity in rpl were insufficiently expressed by elements of protein synthesized complex of the nucleus and cytoplasm that in combination with little content of glycogen and absence of mega mitochondria confirmed insufficiency of plastic and energetic material for providing of secretory transformation. disbalance of hormonedependent cellular differentiation of epithelial cells of endometrium in cyclic stage in female patients with rpl is possible to be interpreted as systemic manifestation of universal syndrome of regenerative and plastic insufficiency [7], which essence consists of the disturbance of processes of intracellular regeneration and forming of plastic deficiency in different organs and tissues undergoing chronic unfavorable influences. the use of the 1st scheme of hormone therapy during 3 menstrual cycles has stimulated intracellular regenerative reactions of epithelial cells since an adequate stage of the cycle by means of differentiation and expression of secretor phenotype of endometrium (fig. 2) more than proper level of reproductive health with “opened window of implantation” in comparison with the 2nd group (fig. 3). gigantic mega mitochondria contributing to hormone-induced cytological differentiation were revealed in the epithelial cells of endometrium. there were large karyosomes with clasters of regular tubular structures, those membranes were appropriate to the tubules of granular cytoplasmatic net with numerous polysomes in the nucleus of the majority of cells (fig. 4); gross deposits of glycogen were localized in the cytoplasm diffusively and like super nuclear aggregates. high polymorphic protrusions of apical plasmolemma as pinopods were formed in 47-63 % of epithelial cells (fig. 5), other cells had polymorphic microvillus and cilia on their apexes. discussion as a result of using the 2nd scheme of preconceptional preparation during three cycles, it was noted the growth of proliferative activity of epithelial cells which corresponds to the luteal phase of the menstrual cycle – the glands of stellar and serrated forms with wide lumens, and folded counters lined i. o. marinkin et al. 37 o b s t e t r ic s a n d g y n e c o l o g y ijmmr 2015 vol. 1 no. 1 by prismatic epithelium with ultrastructural signs of adequate cytodifferentiation. however, taking into account the complex of clinical data we can observe that the given scheme in most cases did not provide the stimulation expression level of plastic and energy potential of endometrial epithelial cells necessary to “open a window to implantation”. normally the epithelium and the endometrium stroma are rapidly renewing tissues under the influence of a particular combination of reproductive hormones and should be exposed to remodeling for the normal receptivity of blastocytes. the basis of luteal phase deficiency is a reduced level of progesterone receptor expression as a consequence of inadequate estrogen content [6]. however, the morphological “immaturity” of endometrium can be observed even during the insufficient progesterone production as a result. what is important is not only the level of steroids in the organism, but also the adequate realization of hormonal effect [8, 10]. fig 1. refactory pregnancy loss. endometrial biopsy sample on the 23rd day of menstrual cycle. dystrophy of epithelial cells, absence of pinopods; isolated stromal cells. semifine section, coloring with schiffs reagent and toluidine blue. magnification 1050. fig. 2. refactory pregnancy loss after cyclic hormonotherapy during 3 months. endometrium biopsy sample on the 23d day of menstrual cycle. numerous pinopods on the apical pole of epithelial cells. semifine section, coloring with schiffs reagent and toluidine blue. magnification 1050. fig. 3. refactory pregnancy loss after dydrogesterone hormonotherapy during 3 months. endometrium biopsy sample on the 24th day of menstrual cycle. isolated pinopods on the epical poles of epithelial cells. semifine section, coloring with schiffs reagent and toluidine blue. magnification 1050. fig. 4. refactory pregnancy loss after cyclic hormonotherapy during 3 months. fragments of endometrium of granulocytes on the 23rd day of menstrual cycle: the nucleolus in the nucleus is with the clusters of granular tubular structures; the cytoplasm contains free and attached polysoms. electrondiffraction pattern. magnifying power 10 000. fig. 5. refactory pregnancy loss after cyclic hormonotherapy during 3 months. high pinopods on the apical poles of endometrium epithelial cells on the 23rd day of menstrual cycle: numerous exosomes and vesicles. electron-diffraction pattern. magnifying power 15 000. i. o. marinkin et al. 38 o b s t e t r ic s a n d g y n e c o l o g y ijmmr 2015 vol. 1 no. 1 in our trial the low doses of estrogens at the stage of pre-conceptional preparation of patients with refectory pregnancy loss contributed both to proliferation and hormone-mediated epithelium differentiation with receptors expression both to estrogens and to progesterone with the increased dosages to the 16th day of a cycle inducing secretory transformation. in other words, the cyclic hormonotherapy, providing the adequate supply of reproductive steroid hormones, consequently induced the intracellular regeneration of endometrium epithelial cells with the restoration of the necessary level of the receptor apparatus to estrogens and progesterone. the result of this process was the realization of status of endometrium receptivity corresponding to the indexes of reproductive health. mucous membranes including endometrium play a leading role in the adaptation processes; complex morphological studies of mucous membrane based on endoscopic biopsies demonstrate the permanence in their reaction that allows to formulate the conception of “reaction unity of mucous membranes” frequently realized by the syndrome of regenerative and plastic insufficiency based on the reducing of the processes of intracellular regeneration [2]. this syndrome can become a fundamental basis to create principally new approaches to the therapy of chronic diseases especially of endometrium by means of remedies inducing regenerative reactions in opposition to aggressive antibiotic therapy [3]. conclusions thus, the obtained results allow us to distinguish a new aspect in morphogenesis of endometrium pathology in case of refractory pregnancy loss. in the complex of interrelated pathologic phenomena, there are the endocrine disorders, thrombophilia, and chronic endometritis. one of the important positions is concerned with endometrium dystrophy, which is evident in the reduction of intracellular regenerative epithelium reactions. the ultrastructural equivalents of reducing the endometrium receptivity is the reduction of protein synthesizing nuclear and cytoplasmic organelles in epithelial cells that underlie the discrepancy of the structure of luteal phase cycle with a reduction of secretory activity and «closed door to implantation». the strategy basis of refractory pregnancy loss treatment is the intensification of regenerative reactions in cellular endometrium populations that contributed to the induction of biosynthetic reactions with endometrium receptivity and fertility. cyclic hormonotherapy in combination with the complex metabolic therapy as a pre-conceptional preparation contributed to pregnancy in 16 (51.6 %) women of the 1st group and 13 (40.6 %) women in the 2nd group; full-term children with apgar scores 8-9 were born in the 1st group – in 10 women (32.3 %), in the 2nd group – in 8 women (25.0 %) demonstrating higher effectiveness of pathogenetically proved preconceptional hormonotherapy. references 1. milovanov ap, gerasimova op. clinical and morphological control of the efficiency of pregravid treatment in women after non-developing pregnancies. obstetrics and gynecology 2013; 5: 87–90. 2. непомнящих г. и. биопсия бронхов: морфо& генез общепатологических процессов в легких. м. : изд&во рамн; 2005: 384. 3. radzinskiy ve, dimitrova v.i, mayskova iu. nondeveloping pregnancy. manual. m.; 2009: 200. 4. sidelnikova vm. habitual pregnancy loss. m.; 2005: 303. 5. sukhikh gt, vanko lv. immunology of pregnancy. m.; 2003: 400. 6. khmelnitskiy ok. pathomorphological diagnostics of gynecological diseases. s-petersburg; 1994: 480. 7. tsellarius ug, semenova la, nepomnyashchikh lm et al. favorites fundamental problems of myocardial histopathology bul. of sib. div. of ramn 1982; 3: 22–29. 8. badawy am, el-gharib me, zalata k. should endometrial biopsy be a routine investigation for recurrent early-pregnancy loss? gynaecol. endosc 2002; 11 (6): 397–399. 9. ford hb, schust dj. recurrent pregnancy loss: etiology, diagnosis, and therapy. rev. obstet. gynecol. 2009; 2 (2): 76–83. 10. lea r.g., oliver s.s. immunoendocrine aspects of endometrial function and implantation. reproduction. 2007; 134 (3): 389–404. 11. prado-drayer a, teppa j, sanchez p, camejo mi. immunophenotype of peripheral t-lymphocytes, nk cells and expression of cd69 activation marker in patients with recurrent spontaneous abortions during the mid-luteal phase amer. j. reprod. immunol 2008; 60 (1): 66–74. 12. szekeres-bartho j, balasch j progestagen therapy for recurrent miscarriage. hum. reprod. update 2008; 14 (1): 27–35. received: 2014.06.09 i. o. marinkin et al. issn 2413-6077. ijmmr 2017 vol. 3 issue 164 p u b l ic h e a lt h a n d e p id e m io l o g y doi 10.11603/ijmmr.2413-6077.2017.1.7000 research in sensitivity to antibiotics, antiseptics in pseudomonas aeruginosa strains isolated from patients with infectious complications o. a. nazarchuk, d. v. paliy, n. i. osadchuk vinnytsia national pirogov memorial medical university, vinnytsia, ukraine background. infections caused by pseudomonas are one of the topical issues of medicine. objective. the aim of the research was to study sensityvity to antibiotics, antiseptics of p. aeruginosa clinical strains that cause infectious complications in patients with burns. methods. microbiological study of biological material, received from 435 patients with burns of the 3rd stages (2011-2015 years). in early terms of burn disease 127 clinical strains of p. aeruginosa were isolated from patients. standard methods were used to identify clinical isolates of p. aeruginosa by their morphological, tinctirial, culture and biochemical properties. the research of antimicrobial action of antiseptics, antibiotics against pseudomonas were carried out by means of standard methods according to the directive of the ministry of health of ukraine (no. 167 from 05.04.2007 р.) and guidelines of national committee of clinical and laboratory study (nccls, 2002). results. it was established that p. aeruginosa caused infectious complications in 23.9% of patients among other pathogens. clinical isolates of p. aeruginosa were found to be low sensitive to amoxicillin/clavulanate (30.76%), ceftazidime (25.92%), cefoperazonum/sulbactam (46.15%), aztreonam (51.85%), tobramycin (38.46%), amicacin (70.34%), doxiciclini (26.92%), fluoroquinolones (59.26%). the analitical progistic criteria of decrease of sensitivity to ceftazidime, cefepim, meropenem and gatifloxacin were found in p. aeruginosa. this pathogen was determined to be sensitive to decasan ®, antimicrobial composition of decamethoxine ®, iodine pvidone. conclusions. clinical strains of pseudomonas aeruginosa, being highly resistant to antibiotics, are also very sensitive to antiseptics decasan ®, antimicrobial of decamethoxine®, povidone iodine. key words: antibiotics; antiseptics; infectious complications; resistance. introduction infection is a determinative factor of recovery of patients with different diseases in critical state. it is the main reason of severe complications and mortality among seriously ill patients. infectious complications, caused by such non-fermenting gram-negative bacilli as pseudomonas, are one of the topical matter in contem porary medicine. the incidence of infectious complications, caused by p. aeruginosa has no tendency to decrease, despite the use of up-todate methods of diagnostics and treatment of diseases with antimicrobial agents. management of p. aeruginosa related infection is of great significance among burn patients [1, 2]. it is established that unsatisfactory results of prevention, treatment of infectious complications of burns are due to distribution of p. aeruginosa polyantibioticresistant strains in surgical clinics. environmental factors active antimicrobial prevention and management by environmental factors provide selective effect on bacteria, which obtaines new qualities under this action (resistance to environmental factors, high virulence and resistance to antibiotics). such qualities are determined by genome of microorganisms and can be caused by mutations that prove high adaptation to bacteria. in these cases, infectious complications caused by p. aeruginosa are still a current issue [3, 4]. current principles of prevention and treatment of infectious complications in patients with burns include antimicrobials straight after clinical sympthoms are evidenced. so, wide spectrum antimicrobials are empirically used. nevertheless in conditions of high resistance of microorganisms to antibiotics the absolute therapeutic effect cannot be achieved. complex approach is one of the most perspective tendency in fight against pseudomonas infection, international journal of medicine and medical research 2017, volume 3, issue 1, p. 64–69 copyright © 2017, tsmu, all rights reserved corresponding author: oleksandr nazarchuk, department of microbiology, vinnytsia national pirogov memorial medical university, 56 pirogov street, vinnytsia, 21018 phone number: +380432570561 e-mail: nazarchukoa@gmail.com o. a. nazarchuk et al. issn 2413-6077. ijmmr 2017 vol. 3 issue 1 65 p u b l ic h e a lt h a n d e p id e m io l o g y it include combined use of antibiotics and antiseptics. that is why, the research in the effectiveness of antimicrobials against strains of p. aeruginosa that cause infectious complications is essential [5]. the aim of the research was to study sensitivity of p. aeruginosa clinical strains, isolated from patients with infectious complications, to antibiotics and antiseptics methods microbiological research of wound exudate samples taken from burn surfaces of 435 patients with deep burns of the 3rd stages took place in 2011–2015. all patients enrolled into the research had infectious complications. they were treated in burns centre of n. i. pirogov vinnitsya regional clinical hospital. microbiological research was carried out in bacteriological laboratory of the department of microbiology of vinnytsya national pirogov memorial medical university of the ministry of health of ukraine. the research consisted of isolation of pure culture of pathogens identified by their morphological, tinctirial, culture and biochemical properties. the research samples were separated by sterile plug before antibacterial therapy. 645 strains of opportunistic bacteria were isolated from patients with burn trauma. during the first 7 days of patients’ treatment in the burn centre p. aeruginosa caused infectious complications in 23.9% of cases in comparison with other etiological pathogens. so the sensitivity of p. aeruginosa isolates to antibiotics and antiseptics was studied. biochemical activity of clinical strains of p. aeruginosa was defined by means of test-system (nefermtest-24 (pliva – lachema a. s., chech republic), which consisted of biochemical tests (indol, arginin, urease, lysine, glucose, fructose, inositol, saccharose, phosphatase, beta-galactosidase, beta-glucuronidase, n-acetyl-beta-dglucosaminidase, mannitol, xylose, cellobiose, galactose, nitrates, nitrites, eskulin, gamma glutamyltransferase, lactose, maltose, tregalose, citrate of simonce). cytocromeoxidase activity was additionally studied (oxy-test). the study of bacteria sensitivity to antibiotics and antiseptics (decasan® (ds), miramistine (mr), chlorhexidine digluconate (chd), povidon iodine (pi), antimicrobial composition of decamethoxine® (amc) was conducted by standard methods according to the directive of the ministry of health of ukraine (no. 167 from 05.04.2007 р.) and guidelines of national committee of clinical and laboratory study (nccls, 2002). sensitivity rate of p.aeruginosa clinical strains to antiseptics was evaluated by the value of minimal bactericidal concentration (mbcc, in mkg/ml) that was obtained by the serial broth dilution method [7]. in the research mathematical and statistical analysis of sensitivity of p. aeruginosa clinical strains to antibiotics, antiseptics were conducted. the methods of statistical analysis, used in the research, enabled definition regular relationship between numeric values of variable indications and probability of realization of these values in array observations. to find out the effectiveness of antibiotics current methods of mathematical prediction were used. so, accurate sensitivity of p. aeruginosa and its extrapolation on the studied system by building a series of standard normative mathematical models of predicted sensitivity of the pathogen to antibiotics was foreseen. thus, analytical methods with concretization of absolute and relative optimum meanings were used. predictive models of p. aeruginosa sensitivity to antibiotics of different groups were investigated as summation of mathematical formulas, which were used to determine these qualities of microorganisms accordingly to their meanings, extrinsic and initial conditions and time. the significance of the results, possible sensitivity rate to antimicrobials was assessed by the coefficient of determination (r2). the obtained data underwent computer processing by means of original programs “statistica 7”; “matlab 7.11”. for each group of microorganisms strains for each year of study arithmetic mean (м), error arithmetic mean (m), standard deviation (σ) were defined. using the methods of applied mathematics the approximation and interpolation of the data were conducted. due to this analytic dependence the possible dynamic of sensitivity rate of p. aeruginosa strains, isolated from the patients, was estimated [8]. results as the result of the research, 154 clinical strains of p. aeruginosa were isolated and identified that looked like typical bacteria of this specimen due to their tinctirial, morphological, and cultural properties. strains of p. aeruginosa, isolated from the patients, had typical biochemical features (table 1). all clinical isolates of p. aeruginosa (100%) produced haemolysins, oxidase and decom posed d-mannitol. as the results of the research, it was proved that p. aeruginosa was sensitive to aminopenicillin o. a. nazarchuk et al. issn 2413-6077. ijmmr 2017 vol. 3 issue 166 p u b l ic h e a lt h a n d e p id e m io l o g y combined with amoxicillin/clavulanate only in 12.30–30.76% of cases (fig. 1). the study of cephalosporins effectiveness showed that sensitivity of p. aeruginosa clinical isolated to ceftazidime had not exceeded 25.92% for five years of our study. the efficacy of ceftazidime was decreased by 8.5% in 2014– 2015. by means of mathematical analysis the table 1. enzymatic properties of isolated strains of p. aeruginosa (n 154) test p. aeruginosa oxidase + catalyse + dcellobiose glucose + γglutamyltransferase + d-mannitol + l₁-prolinarylamidase + lipase urease +/malonate + phosphatase +/llactate n-acetyl-beta-d-glucosaminidase dmannose lhistidine fig. 1. sensitivity of p. aeruginosa isolates, received from seriously ill patients with burns (%). o. a. nazarchuk et al. issn 2413-6077. ijmmr 2017 vol. 3 issue 1 67 p u b l ic h e a lt h a n d e p id e m io l o g y predicted decrease of p. aeruginosa sensitivity to ceftazidime has been proven (formula 1; fig. 2). 2 1 ceftazidim ln ln /a bx x c x x = + + , (1) a=-55358.47; b=2.2527981; c=1.1111315е+10 according to the results of the study of cepha losporins activity, the efficacy of cefepime was not proved (20.55–30.76%). prognostic curve of sensitivity of p. aeruginosa clinical strains to cefepime could refer to parabolic function, presenting future decrease of the efficacy of this antibiotic. it was found out that p. aeruginosa colonized at burn surfaces was resistant to cefalosprins combined with sulbactam. only 20.55–44.44% of p. aeruginosa clinical strains were sensitive to cefoperazone/sulbactam. in 2011–2015, the tendency of decrease of p. aeruginosa clinical strains sensitivity to meropenem (28.75%), imipenem (24.34%) was registered. predictive analysis of p. aeruginosa sensitivity to meropenem was revealed by its linear decreasing in 2011-2015 (formula 2; fig. 3): meropenem=a+bx3+cx/lnx, (2) a=1719659.7; b=8.4437694е-05; c=-9101.5317 similar decrease of pseudomonas sensitivity to imipenem was defined in the research. the estimation of p. aeruginosa sensitivity to aminoglycosides proved low efficacy of tobramicin (16.23–38.46%). the decrease of p. aeruginosa sensitivity to amikacin was also evidenced in the research (formula 3; fig. 4). amikacin 2,5 1,5 d y a bx cx x = + + + , (3) a=-5.8244079e+9; b=2892515.8; c=-8.0030078; d=1.3157016e+14; х – year thus, p. aeruginosa clinical strains were found to be significantly sensitive to amikacin in 2011 (70.37%). but in 2015 the number of p. aeruginosa sensitive strains was significantly lower (16.23%). analytical prediction revealed linear decrease of p. aeruginosa sensitivity to amikacin. it was found out, that isolates of p. aeruginosa were of low sensitivity to aztreonam (34.61– 51.85%). five-year-study of p. aeruginosa sensitivity to aztreonam presented its depression (21.73%) (formula 4; fig. 5). aztreonam y=a+bx2+cx4+dx6 (4) a=-6.5573499e+8; b=485.57507; c=-0.000119856; d=9.861468e-12; х – years fig. 2. predictive sensitivity to ceftazidime of p. aeruginosa clinical strains, received from the patients with burn wounds (%). fig. 3. predictive sensitivity of p. aeruginosa strains to meropenem (%). o. a. nazarchuk et al. issn 2413-6077. ijmmr 2017 vol. 3 issue 168 p u b l ic h e a lt h a n d e p id e m io l o g y in p. aeruginosa clinical strains of low antibiotic sensitivity, different sensitivity rates to antiseptic remedies were found (table 2). in the research, clinical strains of p. aeruginosa were found to be sensitive to amc (mbcc 53.15±1.27 mkg/ml). inhibitory effect of dc® (46.65±0.76 mkg/ml), mr (47.83±2.04 mkg/ml) did not significantly differ from amc (р>0.05). clinical strains of p. aeruginosa were resistant to chd. thus, mbcc of chd versus p. aeruginosa exceeded 165.85±10.9 mkg/ml that proved its low efficacy against these bacteria. dc® and mr had in 1.8 times higher antimicrobial qualities versus p. aeruginosa in comparison with chd (р<0.001). antimicrobial activity of amc was 3 times higher than in chd (р<0.001). antibiotic resistant clinical strains of p. aeruginosa were studied to obtain high sensitivity to bactericidal concentrations of povidone iodine that is contained in the official pharmaceutical form of this remedy. discussion however, in the research the obtained data proved that the amount of isolates of p. aeruginosa in general population was high and reached 23.9% of all microorganisms, isolated from burn wounds in patients, who suffered from infectious complications. such tendency did not differ from established trend of the increasing importance of p.aeruginosa as a prominent pathogen of infectious complications [2, 5]. the results of the research proved low sensitivity of p. aeruginosa clinical strains to antibifig. 4. predictive sensitivity of p. aeruginosa strains to amikacin (%). fig. 5. predictive sensitivity of p. aeruginosa strains to aztreonam. table 2. antimicrobial activity of antiseptics against p. aeruginosa isolates antiseptic minimal inhibitory concentrations minimal bactericidal concentrations (м±m), mkg/ml antimicrobial composition of decamethoxine® (amc) 31.0±0.17 53.15±1.27 decasan® (dc) 46.65±0.76† 91.34±1.69* miramistin (mr) 47.83±2.04† 96.85±3.25* clorhexidine digluconate (chd) 106.3±4.76*** 165.85±10.9*** povidon iodine 3858.27±192.13*** 6742.13±397.27*** notes. † – р>0.05, * – р<0.05 ** – р<0.01, *** – р<0.001 in comparison with amc. o. a. nazarchuk et al. issn 2413-6077. ijmmr 2017 vol. 3 issue 1 69 p u b l ic h e a lt h a n d e p id e m io l o g y otics of different groups. the low sensitivity of pseudomonas to antibiotics, containing betalactam ring in their molecule, was defined. however, in some clinics these antibiotics are widely used as agents of choice for prevention and treatment of infectious complications in patients with burns. low efficacy of cephalosporins against p. aeruginosa according to their sensitivity rate was also determined. higher sensitivity rate to carbapenems was proved in p. aeruginosa (74.07–77.77%). the estimation of the antibiotic sensitivity of p. aeruginosa to aminoglycosides showed their low effectiveness (16.23-38.46%). it was proved, that p. aeruginosa isolates, received from the patients with deep burns, were of low sensitivity to aztreonam (34.61–51.85%) [3, 4]. bactericidal qualities of antiseptics were established that proved high efficacy of decamethoxine, decasan, povidone iodine against clinical strains of p. aeruginosa, as pharmaceutical forms contained in adequate doses of these drugs. in the majority of cases bactericidal concentrations of miramistin exceeded the dose in the remedy officinal form [2, 6]. conclusions clinical strains of p. аeruginosa, which cause infectious complications in seriously ill patients with burn disease, have moderate sensitivity to beta-lactam antibiotics (amoxicillin/clavulanate – 30.76%; ceftazidime – 25.92%; cefoperazone/sulbactam – 46.15%; meropenem – 77.77%; imipenem – 74.07%; aztreonam – 51.85%); aminoglycosides (tobramicin – 38.46%; amikacin – 70.34%). analytical models predict parabolic decrease of sensitivity of p. aeruginosa to ceftazidime, cefepime; lineal tendency of decrease of their sensitivity to meropenem, imipenem. clinical strains of pseudomonas aeruginosa, being highly resistant to antibiotics, save their high sensitivity to antiseptics decasan®, antimicrobial composition of decamethoxine®, povidone iodine. the use of antiseptics is very promising in prevention and treatment of infectious complications caused by p. aeruginosa. references 1. church d, elsayed s, reid o, et. al. burn wound infection. clin microb rew. 2006;19:403–434. 2. nahajchuk vi, nazarchuk oa, paliy ig, et al. in addition to characteristics of modern infectious complications in patients with burns. ukr med j. 2014;5(103):123–126. 3. bhandari s, banjara mr, lekhak b, et al. multidrug and pan-drug resistant pseudomonas aeruginosa: a challenge in post-antibiotic. nep j of sc tech. 2012;13(2):197–202. 4. chander a, raza ms. antimicrobial susceptibility patterns of pseudomonas aeruginosa clinical isolates at a tertiary care hospital in kathmandu, nepal. ains j of pharm and clin res. 2013;6(3):235–238. 5. frieden t. antibiotic resistance threats in the united states. u.s. department of health and human services: centers for disease control and prevention. 2013:114. 6. paliy gk, nazarchuk oa, gonchar oo, et al. the research of physical and chemical, antimicrobial properties of decamethoxin® remedy. med and clin chem. 2016;18(1):36–44. 7. nakrasova ls, svyta vm, glushkevych tg, et al. the determination of sensitivity of microorganisms to antibacterial drugs: methodological recommendations mr 9.9.5. кyiv. 2007:74. 8. yunkerov vi, grigoryev sg, rezvantsev mv. mathematical and statistical processing of medical research data. saint-petersburg: vmeeda. 2011:318. o. a. nazarchuk et al. received: 2016-11-23 28 p u b l ic h e a lt h a n d e p id e m io l o g y issn 2413-6077. ijmmr 2016 vol. 2 issue 1 doi 10.11603/ijmmr.2413-6077.2016.1.6376 epidemiological and epizootic aspect of leptospirosis evolution in ternopil region n. a. vasylieva, yu. a. kravchuk i. horbachevsky ternopil state medical university, ternopil, ukraine ternopil regional laboratory centre of the state sanitation and epidemiological service of ukraine, ternopil, ukraine background. ternopil region is endemic on leptospirosis. its natural conditions (slightly alkaline or alkaline soils, air temperature, sufficient rainfall) contribute to the existence of major natural reservoir of the pathogen – mouse-like rodents. in the region, different serovariants of leptospira are exuded by rodents and farm animals. objective. the materials of the department of highly infectious diseases of ternopil regional laboratory centre of the state sanitation and epidemiological service of ukraine, ternopil regional laboratory of veterinary medicine, clinic of infectious diseases of tsmu were studied. leptospiras were detected by dark ground microscopy (dfm) of blood of patients, trapped rodents and examined farm animals. results. the circulating of pathogens between different sources (rodents, animals) and annual disease in-disease incidence evidences that new leptospira serovar are carried onto endemic area mostly by farm animals; humans are infected from them through the environment sometimes in 3-5 years intervals; the further diffusion to the new areas of this pathogen serovars in all kinds of the examined mouse-like rodents is noticed. it is established that farm animals and rodents are competing reservoirs. to predict the future epidemiological situation of leptospirosis among the humans and to improve its diagnosis the constant monitoring of the population, infection and leptospira carriage among mouse-like rodent and farm animals and expanding of the panel of diagnostic leptospira strains including new pathogen variants in animals is necessary. conclusions. the development of additional reservoirs in animals, with circulating of other pathogen serovars among them, such as mouse-like rodents, which were previously absent in the main natural reservoir, cause the change of etiological structure in human leptospirosis at the endemic areas. the range of human leptospirosis pathogens and its further spreading among all kinds of rodents increased during our research. the results of detection of leptospirosis pathogens among the various contingents which were studied evidence that the farm animals and rodents are competing reservoirs that cause human infection through environment. key words: leptospirosis, disease incidence, source of infection, rodents, farm animals. introduction leptospirosis is a �o��o� i��e�tio�s �is��o��o� i��e�tio�s �is� i��e�tio�s �is� ease a�o�g h��a�s a�� a�i�als. the �eat�res o� this �isease are: �ostly severe �o�rse, high �ortality, a�� great so�ial a�� e�o�o�i� losses. every year �ore tha� 1.03 �illio� �ases a�� 58.900 �eaths happe� ��e to leptospirosis i� the worl�. the highest i��i�e��e a�� �ortality rate is i� so�th a�� so�theast asia, o�ea�ia, lati� a�eri�a, east a�ri�a [1]. this �isease was re�or�e� i� al�ost all regio�s o� ukrai�e, i� ter�opil regio� as well. the �o�se�like ro�e�ts a�� �at�ral �o��i� tions (slightly alkaline and alkaline soils, suffi­ �ie�t rai��all, appropriate air te�perat�re [2, 3]) as main natural pathogen reservoir define the e��e�i�ity o� leptosirosis area a�� �o�tri� b�te to preservatio� a�� �isse�i�atio� o� this �isease. the aim o� the resear�h was to i�vestigate reg�larities o� epi�e�i� a�� epizooti� pro�esses o� leptospirosis a�� st��y it’s pathoge� �ir��� latio� betwee� �i��ere�t �i�i (ro�e�ts, a�i�als) a�� perso�. materials and methods the �aterials o� the depart�e�t o� highly i��e�tio�s diseases o� ter�opil regio�al labo� ratory ce�tre o� the state sa�itatio� a�� epi� �e�iologi�al servi�e o� ukrai�e, ter�opil re� n. a. vasylieva, yu. a. kravchuk corresponding author: nataliya vasylieva, department of infectious diseases and epidemiology, dermatology and venereology, i. horbachevsky ternopil state medical university, 1 maidan voli, ternopil, ukraine, 46001 tel.: +380352524725 e-mail: vasylyeva@tdmu.edu.ua i�ter�atio�al jo�r�al o� me�i�i�e a�� me�i�al resear�h 2016, vol��e 2, iss�e 1, p. 28–31 �opyright © 2016, tsmu, all rights reserve� 29 p u b l ic h e a lt h a n d e p id e m io l o g y issn 2413-6077. ijmmr 2016 vol. 2 issue 1 gional laboratory of veterinary medicine, clinic of infectious diseases of tsmu were studied. leptospiras were detected by dark ground microscopy (dfm) of blood of patients, trapped rodents and examined farm animals. pathogen serovar was determined according to serological investigation in microhemagglutination test of live leptospira cultures (rmal); standard diagnostic set consisted of 11 leptospira serogroups that consisted of both diagnostic strains proposed by the who and strains registered in ukraine and identified in accordance with international reference cultures. the leptospirosis incidence among the humans in ternopil region in 1980-2014, leptospira contamination of animals at private and collective farms and also rodents in the wild were studied. results and discussion in ternopil region there were two rather large “bath” leptospirosis outbreaks in children (laboratory deciphered l. grippotyphosa) in july 1963 (18 patients) and in august 1972 (22 patients). it happened after swimming in the river, livestock farms were situated upstream of it. the rodents trapped near the river contaminated with leptospira too. since 1972 regular monitoring of leptospirosis among the humans took place. the leptospirosis incidence among humans in the region differed: 1,05–12,17 per 100 thousand of population (2–149 cases per year); the highest rate was in 1992–2001, the maximum incidence was in 1994, in 2014 − 3,26, which exceeded the average rate in the country all the time and was the highest in ukraine for many years. till 1981 l. grippotyphosa was the main leptospirosis pathogen in humans (90% cases etiologically deciphered). the percentage of this leptospira in the structure of leptospirosis decreased by 41,1–51,4% in 1988-1989, by 3.4% in 1994. from 1999–2000 there were no incidence of leptospirosis caused by this pathogen. l. icterohaemorrhagiae was the main etiological factor of this disease in the 90s (in 1991 − 93,2%, in 2000 − 100,0%). the disease was rarely caused by other leptospira serogroups. the disease caused by l. hebdomadis (5,9–45,4%), l. canicola (14,2– 60,0%), l. pomona (3,1–21,4%) was registered in 2002; the rate of l. icterohaemorrhagiae decreased by 37.5%, there were only some rare cases of l. grippotyphosa (fig. 1). the rate of combined leptospirosis significantly increased in recent years (34.3% of all registered in 2014). the etiologic spectrum of leptospirosis in humans and in mouse-like rodents in the first stage of observation did not coincide (1981– 1993). in the natural habitats the antibodies to leptospira of grippotyphosa serogroup were found in mostly different kinds of voles, field and house mice; only to icterohaemorrhagiae – in domestic grey rats. hebdomadis segroup was also found in voles and mice and at home foci as well. during the same period the disease caused by the above mentioned pathogens as well as canicola and pomona (1982) (fig. 1), further kabura (2005) polonica (2007), was registered in humans, but in rodents these pathogens had not been found before that figure 1. etiological structure of human leptospirosis incidence in ternopil region (annual percentage fraction of pathogens, 1980-2015, %). 1981 1983 1985 1987 1989 1991 1993 1995 1997 1999 2001 2003 2005 2007 2009 2011 2013 l. grippotyphosa l. icterohaemorrhagiae l. hebdomadis l. canicola l. pomona n. a. vasylieva, yu. a. kravchuk 30 p u b l ic h e a lt h a n d e p id e m io l o g y issn 2413-6077. ijmmr 2016 vol. 2 issue 1 time. in 2010 one case of leptospirosis in humans caused by l. australis was registered. according to the epidemiological anamnesis the source of infection has not been established. the contamination possibly happened at other area. the animals can be the source of leptospirosis infection too. the vaccination process, natural infection or animal disease might cause their serological positivity. the vaccine do not contain l. kabura, (l. hebdomadis), l. bratislava, which are often detected in animals. every year the carriers of leptospirosis and disease incidence in farm animals were registered at private and collective farms of ternopil region which ended up with animal death and abortion; from 1983 epizootic diseases were rare, clinically apparent forms of leptospirosis were not registered, however, sufficiently high titers (1: 400) of antibodies were defined, which evidenced about carrying or subclinical form of the disease in animals and we should consider them as potential sources of leptospirosis for humans. it is possible that the animals’ infection happened after restocking from other farms and areas. according to the recent researches, the duration of leptospirosis carrying in cattle is up to 6 months: in pigs − up to1 year; 10% of cattle and 30% of pigs are carriers for life, as well as rats and mice [4]. however, some authors believe that at most areas the domestic and farm animals are not very important for the epidemiology of leptospirosis because rats usually are revealed at these loci [5]. etiological structure of animal leptospirosis is diverse and includes l. icterohaemorrhagiae, pomona, grippotyphosa, hebdomadis, polonica, canicola, kabura, tarassovi; l. bratislava was registered in 2007 (87.5% in pigs in 2008), often diagnostically significant titers of antibodies to two different serovars are defined at the same time (71,4–78,0% in cattle in 2003–2006). till 1983 only leptospira of grippotyphosa and icterohaemorrhagiae serogroups, sometimes of hebdomadis, was detected; later antibo dies to other serovars were identified: canicola in rats (2004), kabura (2006) in different kinds of voles, except grippotyphosa, also icterohae morrhagiae, pomona, canicola, hebdomadis; the expanding of pathogens and their further distribution in all kinds of examined mouse-like rodents took place. in 2015 leptospira polonica was identified in rodents for the first time. the range of serologically identified pathogens was listed during the monitoring of humans, farm animals and mouse-like rodents (table 1). due to this study and our previous researches [6–8] we verify that leptospira serovars in farm animals cause the human diseases because these pathogens were not exuded by rodents in this region before. new leptospira serovars, which are carried mostly by farm animals and humans are infected through environment from them, in humans are usually registered in 3–5 years after they are exuded from animals. we also evidence the further spread of the new pathogen serovars at this area in all kinds of examined mouse-like rodents. so, it was proved that farm animals and rodents are competing reservoirs of leptospirosis pathogens. to predict future epidemiological situation of leptospirosis in humans and to improve its diagnosis the monitoring of the population, infection and leptospira carriage among mouse-like rodents and farm animals and expanding of the panel of diagnostic leptospira strains including new pathogen variants in animals is necessary. table 1. leptospirosis pathogens serologically identified in humans, farm animals and rodents (1972-2015). rodents farm animals humans l. icterohaemorrhagiae (1972) l. icterohaemorrhagiae (1972) l. icterohaemorrhagiae (1972) l. grippotyphosa (1972) l. grippotyphosa (1972) l. grippotyphosa (1972) l. hebdomadis (1983) l. hebdomadis (1981) l. hebdomadis (1981) l. canicola (1982, 2004) l. canicola (1979) l. canicola (1982) l. pomona (2008) l. pomona (1979) l. pomona (1982) l. kаburа (2006) l. kаburа (2002) l. kаburа (2005) l. polonica (2015) l. polonica (2002) l. polonica (2007) l. tarassovi (2002) l. bratislava (2007) l. australis (2010) n. a. vasylieva, yu. a. kravchuk 31 p u b l ic h e a lt h a n d e p id e m io l o g y issn 2413-6077. ijmmr 2016 vol. 2 issue 1 thus, the factors that contribute to the evolution of the epidemic process in leptospirosis are: – biological changes of the natural reservoir – species composition of rodents, their number and contamination; – expanding of new leptospira strains into endemic areas of farm animals, which cause human infection through environment and further spread among mouse-like rodents and pathogen establishment there. prevention: annual examination for leptospirosis of breeding stock and animals purchased for sale in other farms; sterilization of animal carriers; vaccination; diratisation; draining of wetlands could decrease (possibly avoid) the expanding of new leptospira strains into endemic areas and prevent the disease incidence in humans. conclusions 1. the development of additional reservoirs in animals, with circulating of other pathogen serovars among them, such as mouse-like rodents, which were previously absent in the main natural reservoir, cause the change of etiological structure in human leptospirosis at the endemic areas. 2. the range of human leptospirosis pathogens and its further spreading among all kinds of rodents increased during our research. 3. the results of detection of leptospirosis pathogens among the various contingents which were studied evidence that the farm animals and rodents are competing reservoirs that cause human infection through environment. references 1. costa f, hagan je, calcagno j et al. global morbidity and mortality of leptospirosis: a systematic review. plos negl trop dis 2015; 9 (9): 3898. 2. виноград но, кіріяк оп, мурзова лі та ін. еколого-епідеміологічні особливості лептоспірозу на івано-франківщині. сучасні інфекції 2004; 1: 60-65. 3. кравчук юа, васильєва на. епізоотологоепідеміологічні особливості лептоспірозу в тернопільській області. анали мечниківського інституту 2015; 2: 165-171. 4. задорожна ві, протас св, гопко нв та ін. епізоотологічні та епідеміологічні аспекти лептоспірозу в україні. к; 2014: 46. 5. бернасовська єп, кондратенко вм, мельницька ов. проблема лептоспiрозу в українi. iнфекцiйнi хвороби 1996; 2: 37-39. 6. васильєва на, буртняк тв, блажкевич бв, грузина ло. захворюваність людей на лептоспіроз та інфікованість патогенними лептоспірами гризунів у тернопільській області. інфекційні хвороби 1995; 2: 22-25. 7. васильєва на, поліщук юа, івахів ол та ін. епідеміологічні особливості лептоспірозу в західному регіоні україни. інфекційні хвороби 2008; 2: 14-18. 8. васильєва на, луцук ос, павлів ов. еволюція епідемічного процесу лептоспірозу (за матеріалами тернопільської області). профілактична медицина 2011; 2: 69-73. received: 2016-02-19 n. a. vasylieva, yu. a. kravchuk 14 p e d ia t r ic s issn 2413-6077. ijmmr 2016 vol. 2 issue 2 international journal of medicine and medical research 2016, volume 2, issue 2, p. 14-17 copyright © 2016, tsmu, all rights reserved doi 10.11603/ijmmr.2413-6077.2016.2.7033 nail changes in case of enteroviral infections in western ukraine: a report of 34 cases o. r. boyarchuk1, l. a. volyanska1, o. m. dyvonyak2 1 i. horbachevsky ternopil state medical university, ternopil, ukraine 2 municipal children hospital, ternopil, ukraine background. enteroviral infections are common infections, mostly affect children. nail changes of enteroviral infection including transverse ridging (beau’s lines) and nail shedding (onychomadesis) were described in many countries worldwide. objective. the aim of the research was to investigate the clinical features of the infection that was followed by nail changes during its outbreak in summer–autumn 2016 in ternopil region, ukraine. methods. a case report of 34 children with nail changes that were observed in october 2016 in ternopil region, ukraine is presented. all patients were from one kindergarten. nail trauma, periungual dermatitis, significant medication intake history, systemic diseases were excluded. the survey and clinical examination of cases was performed. faecal samples were obtained from 13 children with onychomadesis. results. the median age of the exemined children was 3.97±0.78 years. interval between onsets of enteroviral infection to nail changes varied from 4 to 12 weeks. clinical signs that preceded nail changes are fever (64.7%), herpangina (32.6%), pharyngitis (17.6%), gastrointestinal symptoms (44.1%), cutaneous lesions (82.4%) as maculopapular, vesicular rash (44.2%) or/and skin desquamation (41.2%). these clinical data indicate preceded enterovirus infection. in 21 (61.8%) patients hand-foot-mouth disease was suggested. nail changes were presented by beau's lines and onychomadesis (nail shedding). the number of affected nails varied from 1 to 16, the median number was 4.88±4.09. conclusions. our study proved association between the outbreak of onychomadesis (nail shedding) and enteroviral infection, mainly hand-foot-mouth disease, during summer–autumn 2016 in western ukraine (ternopil region). enteroviral infection was followed by onychomadesis in 4–12 weeks. clinical features of enteroviral infection were very variable, with prevalence of cutaneous lesions. more studies are necessary to determine the serotype of the virus that causes onychomadesis. key words: enteroviral infections, hand-foot-mouth disease, onychomadesis, children, ukraine. introduction enteroviral infections are commonly encountered infections, caused by group of rna viruses, especially affect infants and children. enteroviruses cause more than half of all infectious diseases in children, are accompanied by fever [1–2]. clinical features of enterovirus infection are very variable; from mild benign fever to life-threatening encephalitis, myocarditis, or sepsis [1]. the enteroviral group includes coxsackievirus, echovirus, and poliovirus. enteroviral infections may be caused by 23 coxsackievirus a, 6 coxsackievirus b, 28 echovirus, and 5 unclassified enteroviruses. hand-foot-mouth disease (hfmd) is one of the forms of enteroviral infections. hfmd is characterized by sores in mouth (vesicular, erosive stomatitis) or on mouth and on hands, feet and sometimes buttocks and legs (maculopapular, vesicular lesions). the most common cause of hfmd is coxsackievirus a6, a10, a16 and enterovirus 71 [2, 3]. first cases of nail changes after hfmd were reported in 2000 in 5 children in chicago, usa [4]. in europe first four cases of nail changes following hfmd were described in 2001 [5]. nail changes of enteroviral infection are described as nail matrix arrest, including transverse ridging (beau’s lines) and nail shedding (onychomadesis) [4, 6]. short-term slowing down of nail formation leads to beau’s lines, while long-term stop of nail growth causes onychomadesis [7]. corresponding author: oksana boyarchuk, department of childrens diseases and pediatric surgery, i. horbachevsky ternopil state medical university, 2 acad. sakharova street, ternopil, ukraine, 46023 phone number: +380352269061 e-mail: boyarchuk@tdmu.edu.ua o. r. boyarchuk et al. 15 p e d ia t r ic s issn 2413-6077. ijmmr 2016 vol. 2 issue 2 the aim of the research was to investigate the clinical features of the infection that was associated with nail changes during an outbreak in summer-autumn 2016 in ternopil region, ukraine. material and methods we present a case report of 34 children with nail changes that were observed in october 2016 in ternopil region, ukraine. all patients were from one kindergarten. nail trauma, periungual dermatitis, significant medication intake history, systemic diseases were excluded. the study of cases was performed. it included a questionnaire containing the following parameters: age, gender, personal and family history of the children. a questionnaire was completed by parents. the patients were clinically examined, and their paediatric and dermatological records were studied to confirm precedent enteroviral infection. direct microscopic examination and cultures for fungi were performed in the first 3 cases. faecal samples were obtained from 16 children with onychomadesis for enterovirus testing. parents of all patients provided written informed consent before any study-related procedure. the study conformed to the principles outlined in the declaration of helsinki. the results have been analysed using standard procedures with statistica statsoft 6.0 software package. values are expressed as mean ± standard deviation (sd) for continuous variables and n (percentages) for categorical variables. results the median age of the exemined children was 3.97±0.78 years (range, 2–5 years). 19 (55.9%) were male. all patients were presented with nail changes. due to the history of present illnesses it was revealed that interval between onsets of enteroviral infection to nail changes varied from 4 to 12 weeks. clinical features of enteroviral infection were very variable (table 1). table 1. clinical features of patients with nail changes after enteroviral infection clinical features n % fever 22 64.7 respiratory symptoms 16 47.1 herpangina 11 32.6 pharyngitis 6 17.6 gastrointestinal symptoms 15 44.1 cutaneous lesions 28 82.4 maculopapular, vesicular rash 15 44.1 skin desquamation 14 41.2 conjunctivitis 2 5.9 the average duration of fever was 1.87±0.92 days. maximum temperature varied from 37.6 to 40.0 °c. gastrointestinal symptoms were presented by nausea, vomiting, diarrhea without blood and mucus. in 7 (20.6%) cases maculopapular, vesicular rash was on hands, feet and around mouth. skin rash was followed by skin desquamation in 3 (8.8%) patients in 2-6 weeks. in other cases skin desquamation occurred in children without history of skin rash. skin desquamation was both on the palms and soles in 6 (17.6%) cases, only on palms in 3 (8.8%) and only on soles of feet in 5 (14.7%) cases. the first manifestation of the disease occurred in the middle of summer (july — 8/23.5% cases), in august (10/29.4% cases) and in autumn (september — 13/38.2% cases). in 3 cases parents did not indicate previous problems or any symptoms of enterovirus infection, so we can suggest asymptomatic duration of the infection. faecal samples from onychomadesis patients were positive for enterovirus in one case. nail changes were presented by beau's lines and onychomadesis (nail shedding) (figure 1–4). the number of the affected nails varied from 1 to 16. the mean number of the affected nails was 4.88±4.09. fingernails were involved more often than toenails (3.79±2.61, range 1 to 10; 3.31±2.20, range 1 to 9, respectively). in two children we observed changes of toenails colour (figure 5). o. r. boyarchuk et al. 16 p e d ia t r ic s issn 2413-6077. ijmmr 2016 vol. 2 issue 2 discussion since 2000 a lot of enteroviral infection outbreaks associated with nail matrix arrest have been reported throughout the world [8– 12]. hand-foot-mouth disease was mostly followed by nail changes. however, in ukraine such cases have been not yet described. formerly nail changes were associated with nail trauma, periungual dermatitis, significant medication intake history, autoimmune disorders, etc. now onychomadesis are described as late complication of enteroviral infection [6, 8]. onychomadesis are caused by a temporary arrest in nail plate formation [2, 8], but the enteroviruses influence on this process remains unclear today. frequently onychomadesis was associated with coxsackievirus (c) a6 serotype of enterovirus [10, 11]. however identification of other enteroviral strains (ca10, ca16 and ente rovirus 71) was reported too [9, 10, 12]. ente rovirus serotypes ca10 and coxsackievirus b1 were mainly detected as a monoinfection or coinfection in valencia, spain, where an onychomadesis outbreak occurred [12]. osterback r. et al. from finland [9] detected cva6 also in a fragment of shed nail. they suggest that virus replication damages nail matrix and results in temporary nail dystrophy. fig. 5. the deformation of toenail bed, toenail plate thickening and change of its colour. fig. 1. beau’s lines on the right fingernails after enteroviral infection. fig. 2. different stages of fingernail shedding (onycho­ madesis) in a 4-year-old child after enteroviral infection. fig. 3. onychomadesis on both great fingernails after enteroviral infection. fig. 4. beau’s lines and onychomadesis on both great toenails after enteroviral infection. o. r. boyarchuk et al. 17 p e d ia t r ic s issn 2413-6077. ijmmr 2016 vol. 2 issue 2 the study from taiwan proves that in 51% ca6 patients desquamation of palms and soles occurred after the infection episode and in 37% ca6 patients onychomadesis developed, which only occurred in 7 (5%) of 145 cases with nonca6 enterovirus infection [11]. the authors con firmed association of ca6 infection with deve loping nail abnormalities (p<0.001). unfortunately, we were unable to determine the serotype of the virus in our cases. enterivirus was identified only in one case (6.3%), probably because of a long period from the first symp­ toms of the disease. study in spain [12] reported about 59% positive samples collected from 29 onychomadesis case-patients (23 with hfmd) and 9 exposed persons (1 with hfmd). in our study typical preceding clinical signs indicated enteroviral infection. in the most cases (21/61.8%) cutaneous lesions (maculopapular, vesicular rash and skin desquamation) were located on hands, feet and around the mouth, which indicated hfmd. enteroviral diseases in countries with temperate climate are characterized by summerautumn seasonality. unlike, in tropical countries, where the virus is circulating throughout the year seasonal outbreaks are not typical. the first manifestation of the disease in our cases began in the middle of summer (july) and lasted till autumn (september). an outbreak of hfmd with onychomadesis was reported in finland in 2008 starting in august and continuing at least until the end of the year [10]. in valencia, spain hfmd, followed by onychomadesis occurred during summer and autumn 2008 [12]. but in northern greece, in the region of thessaloniki an onychomadesis outbreak related to the outbreak of hfmd occurred during autumnwinter 2012–2013 [6]. conclusions our study proved association between onychomadesis (nail shedding) outbreak and outbreak of enteroviral infection, mainly handfoot-mouth disease, during summer-autumn 2016 in western ukraine (ternopil region). enteroviral infection was followed by onychomadesis in 4–12 weeks. clinical features of enteroviral infection were very variable, with prevalence of cutaneous lesions. more studies are necessary to determine the serotype of the virus causing onychomadesis. references 1. de crom scm. rossen, jwa, van furth am, obihara cc. enterovirus and parechovirus infection in children: a brief overview. eur j pediatr 2016; 175: 1023–1029. 2. shin jy, cho bk, park hj. a clinical study of nail changes occurring secondary to hand-foot-mouth disease: onychomadesis and beau's lines. ann der­ matol 2014; apr; 26(2): 280–283. 3. stock i. hand, foot and mouth disease — more than a harmless "childhood disease". med monatsschr pharm 2014; jan; 37(1): 4–10; quiz 11–2. 4. clementz gc, mancini aj. nail matrix arrest following hand­foot­mouth disease: a report of five children. pediatr dermatol 2000;17: 7–11. 5. bernier v, labrèze c, bury f, taïeb a. nail matrix arrest in the course of hand, foot and mouth disease. eur j pediatr 2001; 160: 649–651. 6. apalla z, sotiriou e, pikou o, lefaki i, lallas a, lazaridou e, ioannides d. onychomadesis after hand-foot-and-mouth disease outbreak in northern greece: case series and brief review of the literature. int j dermatol 2015; 54: 1039–1044. 7. haneke e. onychomadesis and hand, foot and mouth disease — is there a connection? euro surveill 2010; 15: pil: 19664. 8. bettoli v, zauli s, toni g, virgili a. onychomadesis following hand, foot, and mouth disease: a case report from italy and review of the literature. int j dermatol 2013; 52: 728–730. 9. davia jl, bel ph, ninet vz, bracho ma, gonzález-candelas f, salazar a, et al. onychomadesis outbreak in valencia, spain associated with hand, foot, and mouth disease caused by enteroviruses. pediatr dermatol 2011; 28: 1–5. 10. osterback r, vuorinen t, linna m, susi p, hyypiä t, waris m. coxsackievirus a6 and hand, foot, and mouth disease, finland. emerg infect dis 2009; 15: 1485–1488. 11. wei sh, huang yp, liu mc, tsou tp, lin hc, lin tl, et al. an outbreak of coxsackievirus a6 hand, foot, and mouth disease associated with onychomadesis in taiwan, 2010. bmc infect dis 2011; 11: 346. 12. bracho ma, gonzález-candelas f, valero a, córdoba j, salazar a. enterovirus co-infections and onychomadesis after hand, foot, and mouth disease, spain, 2008. emerg infect dis 2011; 17: 2223–2231 received: 2016-12-03 o. r. boyarchuk et al. 11 in t e r n a l m e d ic in e issn 2413-6077. ijmmr 2017 vol. 3 issue 1 doi 10.11603/ijmmr.2413-6077.2017.1.6879 screening for dyslipidemia and expedience of statin therapy for the citizens of transcarpathia valley regions with overweight and obesity a. v. kedyk, m. v. rishko, o. o. kutsyn uzhhorod national university, uzhhorod, ukraine background. the lipid profiles of patients with overweight, obesity and healthy individuals, the citizens of transcarpathia valley regions were analysed. objective. the study was aimed at evaluation of dyslipidaemia frequency in patients with overweight and obesity, determination of expedience of statins prescription. methods. all patients were divided into 2 groups: group 1 – patients with overweight; group 2 – patients with obesity of i and ii degree. estimation of lipid profile parameters was conducted by means of spectrophotometric device siemens dimension rxl max. statistical analysis of the data was conducted using microsoft excel 2007. results. the patients with obesity had higher level of total cholesterol (6.03±0.53 mmol/l), lower hdl-c (1.15±0.07 mmol/l) and higher level of ldl-c (4.19±0.46 mmol/l) compare with overweight patients. in 46% of patients with overweight, dyslipidaemia was evidenced and required correction, 27% of them had high cvr and needed statin therapy, 19% of people with obesity had moderate cvr and didn’t need statins. 77% of obese patients needed lipid correction, 54% of them with very high and averagely high level of cvr required statin therapy; 23% of people with obesity had moderate cvr and did not need statins. conclusions. in the studied overweight and obese patients, atherogenic dyslipidaemia was established in 46% and 77% of cases respectively. correction of dyslipidaemia with statin was compulsory for 27% of patients with overweight and for 54% with obesity. key words: body mass index; overweight; obesity; lipid profile; statins. corresponding author: antonina kedyk, department of hospital therapy, uzhhorod national university, 15a timiryazeva street, uzhhorod, 88000 phone number: +380968844247 e-mail: kedyk.tonja@gmail.com introduction in 2000, the who first suggested using the term ‘epidemic’ to distinguish the situation with obesity prevailing in the world; it was recognized that the number of people with overweight and obesity is progressively increasing. over the last 35 years the number of obese patients has increased twice, and over the last 10 years it increased by 75%. according to who data in 2003 there were 1.7 billion of the adult population suffering from overweight (bmi≥25 kg/m2), the statistics in 2014 announced a new data: there were more than 1.9 billion people with overweight and obesity [1]. in 2016 the results of a meta-analysis on the dynamics of bmi in adults of over 18 years old bet ween 1975 and 2014 were published. 1698 data on population studies was analysed, which inclu ded 19.2 million people from 186 countries. the increase of average bmi from 21.7±0.4 kg/m2 to 24.2±0.2 kg/m2 for men and from 22.1±0.4 kg/m2 to 24.4±0.2 kg/m2 for women was proved [2]. the negative effect of obesity on population is established: this pathology correlates with increased morbidity, mortality, quality of life, and is a significant socioeconomic burden for the world [3]. several studies prove that obesity increases the cost of medical care and worsens the quality of life [4]. to reduce mortality from cardiovascular diseases in people aged under 65, the council of europe in 2002 declared a plan “heart of europe”, according to which it is necessary to reduce cholesterol level in the population by less than 5.0 mmol/l, lower blood pressure in the population to a level of <140/90 mm hg., reduce the number of smokers by 1% per 1 year. thus, by 2020 the mortality rate should dea. v. kedyk et al. international journal of medicine and medical research 2017, volume 3, issue 1, p. 11–14 copyright © 2017, tsmu, all rights reserved 12 in t e r n a l m e d ic in e issn 2413-6077. ijmmr 2017 vol. 3 issue 1 crease by 40% (2.4% per year). to achieve this goal, primary care physicians must reorient to early detection of lipid metabolism disorders, their adequate correction and prevention of atherosclerosis associated with cardiovascular diseases and their complications [5]. in most patients with obesity such lipid disorders are observed: rising of small particles of low density lipoprotein (ldl) in blood plasma [5, 6, 7], which is often associated with increased levels of triglycerides (tg) and decreased level of high-density lipoprotein cholesterol (hdl cholesterol) [5]. algorithm development for selection of patients for statins therapy, adequate drug choice and its efficacy control, evaluation of target level of cholesterol are the topical matter for a family doctor today. however, the questions ‘how often sub clinical dyslipidaemia in obese patients is revealed’, and ‘how reasonable the prescription of statins for these patients is’ are still open, because of the absence of supply for routine lipid profile determination in the practice of family doctors in ukraine. the study was aimed at evaluation of dyslipidaemia frequency in patients with overweight and obesity, determination of the expedience of statins prescription. methods 40 individuals who attended clinics of family medicine in uzhhorod from october to december 2015 were examined. we analysed the following data in the examined persons: age, sex, level of total cardiovascular risk (cvr), body mass index (bmi), total cholesterol (tc), high-density lipoprotein cholesterol (hdl-c), low density lipoprotein cholesterol (ldl-c), triglycerides (tg), atherogenic coefficient (ac). estimation of lipid profile parameters was conducted by means of spectro photometric device siemens dimension rxl max. the study was approved by the ethics committee of the transcarpathian regional clinical cardiology dispensary. the patient had to sign the informed consent to involvement in the research. for diagnosis of the overweight or obese we used a body mass index, which was calculated as the ratio of body weight to height squared (kg/m2). the persons, whose bmi was from 25 to 29.99 kg/m2 composed the group of patients with overweight. the persons with bmi from 30 to 34.99 kg/m2 were diagnosed with obesity i degree, in the persons with bmi from 35 to 39.99 kg/m2 obesity ii degree was established. the results of lipid profile were interpreted according to guidelines of the esc and target lipid levels of patients with different cvr. all patients were divided into 2 groups: group 1 – patients with overweight; group 2 – patients with obesity of i and ii degree (hereinafter patients with obesity). the share of patients with overweight was 55% (22 people), with obesity – 45% (18 persons). due to the terms and conditions of the research, only those patients, who did not take statins for at least one month before the date of screening, took part in the study. patients’ data was normally distributed. statistical analysis of the data: the t-test and pearson rank correlation coefficient, was conducted using microsoft excel 2007. results the average age of group 1 patients was 57.2±1.2 years old, bmi 27.06±0.42 kg/m2. the average age of group 2patients was 59.7±1.1 years old, bmi 35.9±1.17 kg/m2. patients with obesity had the following differences in lipid spectrum compared with those who were overweight: higher level of total cholesterol (6.03±0.53 mmol/l vs 4.76±0.23 mmol/l, p=0.048), lower hdl-c level (1.15±0.07 mmol/l and 1.51±0.12 mmol/l, p=0.02) and higher level of ldl-c (4.19±0.46 mmol/l and 2.89±0.21 mmol/l, p=0.026). in the group 1, among all, who required correction of lipid spectrum, 19% of patients had moderate cvr and didn't need statin therapy because the average level of ldl-c was 2.5±0.13 mmol/l; respectively, 27% had increased cvr and average level of ldl-c was 3.4±0.21 mmol/l and they needed statin therapy. among the obese 77% of patients required lipid correction. 21% of group 2 patients had a very high average level of cvr because ldl-c level was 4.4±0.32 mmol/l and they needed correction of lipid profile with statins; 33% of patients had high cvr with an average level of ldl cholesterol 3.7±0.51 mmol/l and also needed statin therapy; 23% of patients with obesity had moderate cvr and did not need statins (fig.1). in addition to determination of significant differences between different groups, we also conducted correlation analysis of the groups of patients. in group 1 a direct average relationship was revealed between: the level of total a. v. kedyk et al. 13 in t e r n a l m e d ic in e issn 2413-6077. ijmmr 2017 vol. 3 issue 1 cholesterol and bmi (r=0.5), bmi and ldl cholesterol (r =0.4), bmi and tg (r=0.56), bmi and atherogenic ratio (r=0.34), inverse middle relationship between age and the level of hdl cholesterol (r=-0.52). similar correlations were found in the group 2: between bmi and total cholesterol (r=0.42), bmi and ldl cholesterol (r=0.37), bmi and tg (r=0.46). discussion thus, with increase of bmi, lipid profile got worse: the levels of total cholesterol and ldl cholesterol increased and of hdl cholesterol decreased. according to the literature [8,9, 10], in patients with overweight and obesity most often such lipid disorders are found: rising of small particles of ldl in blood plasma, which is often associated with increased level of tg and decreased level of hdl-c [6]. according to the framingham heart study about 10% of patients with overweight have increased blood concentrations of cholesterol by 0.3 mmol/l. in young patients with overweight the increased level of cholesterol was twice more than in people of the same age with normal weight. according to the results of population survey conducted in the national scientific center “m.d. strazhesko institute of cardiology” mas of ukraine; the occurrence of hypercholesterolemia in patients with overweight is 65%, 70% in the obese, and only 40% in people with normal weight [10]. the method of dyslipidaemia correction was chosen according to the recommendations of esc/aha and association of cardiologists of ukraine. 46% of patients with overweight had dyslipidaemia, which required correction: lifestyle modification in 19% and/or statin therapy in 27%. thus, with bmi levels increase the levels of total cholesterol, ldl-c and tg significantly increase, in addition in patients of the group 1 hdl-c level decreased with age. conclusions in the examined overweight and obesity patients, atherogenic dyslipidaemia was established in 46% and 77% of cases respectively. correction of dyslipidaemia by statin was compulsory for 27% of patients with overweight and 54% with obesity. regardless of gender and age, in the patients with increased bmi level, the increased levels of total cholesterol, ldl cholesterol and atherogenic factor were observed. fig. 1. percentage of the obese with different cardio-vascular risks and lipid correction is compulsory. references 1. obesity: world statistic. 2015. https://medaboutme.ru/zdorove/publikacii/stati/sovety_vracha/ ozhirenie_mirovaya_sta tistika/?utm_source=copypaste&utm_me dium=referral&utm_campaign=copypaste? utm_source=copypaste&utm_me dium= referral&utm_campaign=copypaste. accessed 31 june 2015. 2. ncd risk factor collaboration (mariachiara di cesare, james bentham, gretchen a stevens and other more than 150 authors). trends in adult bodya. v. kedyk et al. 14 in t e r n a l m e d ic in e issn 2413-6077. ijmmr 2017 vol. 3 issue 1 mass index in 200 countries from 1975 to 2014: a pooled analysis of 1698 population-based measurement studies with 19·2 million participants. lancet. 2016;387:1377–96. 3. frühbeck g, toplak h, woodward e, yumuk v, maislos m, oppert jm. obesity: the gateway to ill health – an easo position statement on a rising public health, clinical and scientific challenge in europe. obes facts. 2013;6:117–120. 4. zhang j, shi xm, liang xf. economic costs of both overweight and obesity among chinese urban and rural residents, in 2010. zhonghua liu xing bing xuezazhi. 2013;34(6):598–600. 5. magkos f, mohammed bs, mittendorfer b. effect of obesity on the plasma lipoprotein subclass profile in normoglycemic and normolipidemic men and women. int j obes. 2008;32(11):1655–1664. 6. rosolova h, nussbaumerova b. cardio-metabolic risk prediction should be superior to cardiovascular risk assessment in primary prevention of cardiovascular diseases. epma j. 2011;2(1):15–26. 7. rizzo m, mikhailidis dp. there is more to predicting vascular disease than just established risk factors. curr pharm des. 2011;17(33):3608–3610. 8. tvorogova m.g. reverse cholesterol transport. cardiology 2011;2:66–70. 9. rizzo m, kotur-stevuljevic j, berneis k, spinas g, rini gb, jelic-ivanovic z, et al. atherogenic dyslipidaemia and oxidative stress: a new look transl res. 2009;153(5):217–223. 10. sokolova ei. decrease of the importance of antiatherogenic high-density lipoproteins in obese. cardilogy. 2010;2:45–50. received: 2016-11-14 a. v. kedyk et al. koncor1_2015.pm6 75 p u b l ic h e a l t h a n d e p id e m io l o g y issn 2413�6077. ijmmr 2015 vol. 1 no. 1 удк 616�002.5:614.254.3 doi туберкульоз у практичній діяльності сімейного лікаря і. т. п’ятночка, с. і. корнага, т. в. корнага тернопільський державний медичний університет імені і. я. горбачевського вступ. у статті дано характеристику сучасному сімейному лікарю як універсалу, який стоїть на сторожі збереження здоров’я людини, і показано його роль у боротьбі з туберкульозом. надзвичайної гостроти набула проблема хіміорезистентного туберкульозу, зокрема мультирезистентного, від якого в україні страждають 23,4 % хворих на вперше діагностований туберкульоз та 58,6 % із повторними випадками захворювання. мета роботи – дати характеристику сімейному лікарю як універсалу, а також показати його роль у боротьбі з туберкульозом на сучасному етапі. результати. у тернопільській області в останні роки спостерігають позитивну динаміку епідеміологічного процесу, зменшення захворюваності й смертності від туберкульозу. в 2014 р. ефективність лікування хворих на вперше діагностований туберкульоз легень з бактеріовиділенням становила 87,6 %. однак кількість хворих, які померли від туберкульозу протягом одного року спостереження, за останнє десятиріччя зросла з 15,6 до 22,2 % (р<0,05). це свідчить про несвоєчасне виявлення туберкульозу на різних рівнях медичної служби, зокрема і первинної ланки. летальність серед хворих на мультирезистентний туберкульоз становила 17,2 %, з них до одного місяця – 1/3 осіб. висновки. сімейний лікар надає першу медичну допомогу та ліквідовує вогнища інфекційних захворювань, проводить серед населення пропаганду здорового способу життя, відповідального ставлення до свого здоров’я як найвищої особистої і суспільної цінності. ключові слова: туберкульоз, сімейний лікар. вступ туберкульоз відомий у світі, відколи існує людство. це одна з найдавніших хвороб, які можуть уражати всі органи людини. періоди епідемії туберкульозу змінювалися періодами стабілізації. останній спалах туберкульозу розпочався з 90-х років минулого століття. у 1995 р. в україні проголосили епідемію туберкульозу, захворюваність перевищила епідемічний поріг – 50 випадків на 100 тис. населення. у 2005 р. цей показник був найвищим – 84,1 на 100 тис. населення. упродовж 2006–2015 рр. спостерігали покращення епідеміологічної ситуації, і вже у 2014 р. захворюваність на туберкульоз, поширеність і смертність від нього становили, відповідно 53,2, 90,2 та 12,2 випадку на 100 тис. населення. водночас зросла захворюваність на ко-інфекцію туберкульоз/віл/ снід – 10,4 на 100 тис. населення. надзвичайної гостроти набула проблема хіміорезистентного туберкульозу, зокрема мультирезистентного (стійкість до найефективніших протитуберкульозних препаратів – ізоніазиду і рифампіцину), від якого в україні страждають 23,4 % хворих на вперше діагностований туберкульоз та 58,6 % із повторними випадками захворювання. у результаті проведення основного курсу хіміотерапії вилікування нових випадків бацилярного туберкульозу легень спостерігають у 55 % пацієнтів від тих, які захворіли [1]. такий низький відсоток видужання зумовлений несвоєчасним виявленням хворих, помилками діагностики, низькою прихильністю пацієнтів до тривалої хіміотерапії, як і самими вадами її проведення. усе це зобов’язує невідкладно спрямувати значні зусилля на покращення профілактики і, передусім, своєчасну діагностику туберкульозу на рівні первинної ланки (сімейної медицини), де помилки сягають 80,2–96,4 % [2]. причини помилок щодо діагностики туберкульозу, в кінцевому результаті, зводяться і до зниженої фтизіaddress for correspondence: s. i. kornaha, department of internal medicine propedeutics and phthisiology, i. horbachevsky ternopil state medical university, 1 maidan voli, ternopil, ukraine, 46001 tel. +380352525929 e-mail: kornaha@tdmu.edu.ua і. т. п’ятночка та ін. international journal of medicine and medical research 2015, volume 1, number 1, p. 75-79 copyright © 2015, tsmu, all rights reserved 76 p u b l ic h e a l t h a n d e p id e m io l o g y issn 2413�6077. ijmmr 2015 vol. 1 no. 1ijmmr 2015 vol. 1 no. 1 атричної настороженості, що неприпустимо в період значного поширення мультирезистентного туберкульозу. лікування такого туберкульозу надзвичайно складне, тривале (≥2 роки), дороге (іноді в 100 разів дорожче, ніж терапія чутливого до ліків туберкульозу), малоефективне і часто супроводжується побічними токсичними реакціями [3]. мультирезистентний туберкульоз – надзвичайно небезпечна форма туберкульозу, яка нещадно карає людей за їх байдужість до свого здоров’я, слабку обізнаність у питаннях боротьби із цією недугою, що є наслідком вкрай недостатнього проведення санітарно-просвітницької роботи серед населення і хворих на різних рівнях медичної служби та прорахунків в організації боротьби з туберкульозом взагалі. виникла нагальна потреба перебудови санітарно-просвітницької діяльності на зразок економічно розвинутих європейських країн. суть її полягає у виробленні навичок ведення здорового способу життя в дитячому та юнацькому віці; пропагування здорового способу життя в соціальних популяціях людей, об’єднаних спільним устроєм життя чи специфікою праці; зміні пасивного ставлення людини до свого здоров’я шляхом підтримування її матеріальної зацікавленості у збереженні здоров’я в доброму стані, тобто людині не вигідно буде хворіти; розробці для кожної людини індивідуальної програми профілактики захворювань з усуненням впливу вже добре відомих негативних чинників (алкоголь, куріння, гіподинамія тощо), а також наданні людині рекомендацій щодо вибору професії з урахуванням показників психофізіологічних і професійних тестів [4]. втілення в життя такої індивідуальної програми профілактики захворювань є можливим лише за присутності сімейного лікаря, який найбільш повно ознайомлений із характерологічними особливостями конкретного пацієнта і станом його здоров’я. великий досвід у цьому напрямку мають деякі країни європи [5]. адже і поняття “лікар загальної практики” склалося еволюційним шляхом у хііі– хіх ст. в окремих європейських країнах, коли лікарську допомогу надавали в індивідуальному комерційному порядку. в результаті цього з’явився сталий контингент пацієнтів, які вважали доцільним протягом усього життя звертатися виключно до “свого” лікаря, якому довіряли завдяки його професіоналізмові та індивідуальним якостям. за таких умов лікар був одночасно сімейним і лікарем загальної практики. мета роботи – дати характеристику сімейному лікарю як універсалу, а також показати його роль у боротьбі з туберкульозом на сучасному етапі. матеріали і методи використано літературні дані про сімейного лікаря як універсала, наведено основні епідеміологічні показники щодо туберкульозу в україні й по тернопільській області за останні 10 років і сформульовано головні завдання, які стоять перед сімейним лікарем відповідно до сучасного уніфікованого клінічного протоколу первинної, вторинної (спеціалізованої) та третинної (високоспеціалізованої) медичної допомоги дорослим “туберкульоз” (2014) [6]. результати та їх обговорення сімейний лікар – якісно нова форма лікарської діяльності, що володіє великим об’ємом знань і вмінь. це лікар-універсал, який має ґрунтовні знання з головних розділів медицини – профілактики та діагностики. він надає першу медичну допомогу, попереджує та ліквідовує вогнища інфекційних захворювань, проводить серед населення пропаганду здорового способу життя. за даними світової статистики, кваліфікований сімейний лікар може самостійно вирішити до 80 % проблемних завдань на етапі первинної допомоги, включаючи і лікування в домашньому або денному стаціонарі. поряд із цим, однією з основних вимог щодо високої якості роботи сімейного лікаря є доброзичливе ставлення до пацієнта і високий професіоналізм та, безперечно, відсутність грубості й байдужості до хворої людини. ще у хіі ст. англійський лікар sydenham сказав, що лікар повинен так поводитися із хворим, як би він хотів, щоб поводилися з ним – увічливо і з турботою. до того ж, сімейний лікар зобов’язаний очищувати свою мову від нетактовних і брутальних слів. не слід думати, що слово – дрібниця, не в словах справа. адже слово виражає думку, а думка породжує дії, які можуть закінчуватися фатально. слід уникати і такої крайності, коли діагноз має таємничий характер. це особливо лякає хворого, який не повинен знати про своє захворювання. у противному разі, залежно від психіки пацієнта і стану його здоров’я, рідні повинні бути інформовані про діагноз, методи лікування, прогноз. інформацію про захворювання слід надати хворому в спокійній, простій, доступній формі з надією на видужання чи, хоча б, на покращення його стану. дуже важливими в діяльності сімейного лікаря є його індивідуально-психологічні особливості. надто важливо встановити розумний, щиросердечний, безкорисливий контакт із хворим та завоювати його довіру. отож, якщо люди шукають у тебе допомоги – ти хороший лікар. цього можна досягнути, поряд із високою і. т. п’ятночка та ін. 77 p u b l ic h e a l t h a n d e p id e m io l o g y issn 2413�6077. ijmmr 2015 vol. 1 no. 1 професійною майстерністю, індивідуальним підходом сімейного лікаря до хворого, манерою звертання та, зрештою, зовнішнім виглядом. до того ж, незайвим є нагадати слова гіппократа: “лікар повинен бути скромним і стриманим, добрим та ввічливим, постійно збагачувати свої знання і прислухатися до думки колег, бачити свою мету не у здобуванні слави та грошей, а в полегшенні страждань і зціленні хворих, у самовідданому служінні людям, які звертаються до нього за допомогою та за порадою”. отже, сімейна медицина – це медицина сьогодення і майбутнього у вітчизняній охороні здоров’я. особливий, довірливий характер стосунків з родиною, тривале спостереження за кожним її членом дають змогу постійно і ретельно займатися лікувально-профілактичною роботою, спрямованою на відновлення та збереження здоров’я людини. дуже важлива роль сімейних лікарів у масових заходах, спрямованих на раннє виявлення і профілактику туберкульозу. до того ж, більшість пацієнтів із симптомами респіраторних захворювань звертається в лікувальні заклади загальної медичної мережі, де і виявляють туберкульоз та навіть завершують лікування хворого. отже, ефективне проведення протитуберкульозних заходів не можливе без широкої участі в цій роботі лікарів загальної практики – сімейної медицини. переваги сімейної медицини полягають у тому, що сімейному лікарю відомі конкретні умови праці, побуту, бюджет і харчування сім’ї, наявність хворих, зокрема і на туберкульоз, а також осіб з підвищеним ризиком виникнення цього захворювання, яких щороку направляють на профілактичні обстеження. до слова, в останні роки ризик інфікування продовжує зростати, оскільки сучасні заходи щодо розриву ланцюга передачі туберкульозної інфекції від хворої людини до здорової недостатньо ефективні: несвоєчасне виявлення хворих, незадовільна їх ізоляція та малоефективне лікування, зокрема мультирезистентного туберкульозу, сприяють поширенню туберкульозної інфекції. отож пацієнтів із такими симптомами, як кашель, що триває понад 2–3 тижні, нічне потовиділення, загальна слабість, безпричинне схуднення, тривале, понад тиждень, підвищення температури тіла більше 37 °с, необхідно направляти на рентгенологічне обстеження, дворазове дослідження мокротиння на мікобактерії туберкульозу. сімейний лікар проводить лікування хворих на туберкульоз в амбулаторних умовах, здійснює контрольовану хіміопрофілактику в групах підвищеного ризику за рекомендацією фтизіатра. туберкульоз у групах ризику активно виявляють шляхом призначення скринінгового флюорографічного обстеження 1 раз на рік. крім цього, в лабораторних умовах і вдома сімейний лікар діагностує та надає екстрену допомогу при таких невідкладних станах, як спонтанний пневмоторакс, гостра дихальна недостатність, кровохаркання і легеневі кровотечі, гострі алергічні реакції на протитуберкульозні препарати. випадки туберкульозу та латентної туберкульозної інфекції за умов епідемії туберкульозу виявляють шляхом щорічної туберкулінодіагностики (проба манту з 2 то), яку проводять здоровим дітям від 4 до 14 років. дітям до 4 років та дітям підліткового віку туберкулінодіагностику проводять за бажанням батьків, у групах ризику – за епідпоказаннями, зокрема в туберкульозних вогнищах. профілактичну променеву діагностику призначають дітям віком 15 і 17 років. у разі виявлення на рентгенограмі порожнин розпаду, дрібновогнищевої дисемінації пацієнта направляють до фтизіатра на дообстеження з результатами дворазової бактеріоскопії мазка мокротиння. у випадку негативних мазків мокротиння і за наявності вогнищевих або інфільтративних змін на рентгенограмі легень хворому призначають антибіотики широкого спектра дії (за винятком рифампіцину, аміноглікозидів та фторхінолонів) до 2 тижнів, після чого повторюють рентгенограму легень. за відсутності позитивної динаміки пацієнта негайно скеровують до фтизіатра. до фтизіатра направляють осіб при підозрі на позалегеневі форми туберкульозу (кістковосуглобового, периферичних лімфатичних вузлів, сечостатевих органів, очей, нервової системи та інших локалізацій). до дитячого фтизіатра на консультацію направляють дітей: які контактували з хворим на туберкульоз; із гіперергічними реакціями на пробу манту з 2 то (інфільтрат 17 мм і більше або реакція з наявністю везикул, некрозу чи лімфангоїту незалежно від розміру інфільтрату); при зростанні розміру інфільтрату на пробу манту на 6 мм і більше; за наявності ускладнень вакцинації бцж (холодні підшкірні абсцеси, периферичні лімфаденіти, виразки розміром понад 10 мм, келоїдні рубці, персистувальна та дисемінована бцж-інфекція з ураженням різних органів); при інших ситуаціях, що пов’язані з ризиком захворювання на туберкульоз. сімейний лікар організовує щеплення вакциною бцж дітей, які не були вакциновані в пологовому будинку, контролює розвиток післявакцинних реакцій. лікування хворих на туберкульоз проводять за стандартними режимами під безпосереднім наглядом медперсоналу відповідно до уніфікованого клінічного протоколу первинної, вторині. т. п’ятночка та ін. 78 p u b l ic h e a l t h a n d e p id e m io l o g y issn 2413�6077. ijmmr 2015 vol. 1 no. 1 ної (спеціалізованої) та третинної (високоспеціалізованої) медичної допомоги дорослим “туберкульоз” (2014) [6]. хіміопрофілактику дітям та дорослим проводять на основі призначених фтизіатром препаратів. найпоширенішою схемою хіміопрофілактики є шестимісячне приймання ізоніазиду (5–8 мг/кг). кількість курсів превентивної хіміотерапії призначають індивідуально. сімейний лікар повинен проводиьти санітарно-просвітницьку роботу в сім’ї, з хворим, як і з населенням загалом. санітарно-просвітницька робота – це безперервний процес, який полягає у висвітленні властивостей збудника туберкульозу, шляхів його передачі, проявів захворювання, лікування та профілактики. без чіткого розуміння хворим, що таке туберкульоз, без належних гігієнічних навичок, правильної поведінки в сім’ї, колективі, розумної прихильності до лікування боротьба з туберкульозом, зокрема хіміорезистентним, приречена на невдачу. однак не варто впадати в “закодований” песимізм, потрібно нівелювати негативні явища стосовно навіть такої небезпечної форми, як мультирезистентний туберкульоз. у зв’язку з цим, сімейний лікар повинен бути проінформований про позитивні зрушення в подоланні туберкульозу, недоліки в роботі як загальномедичної, фтизіатричної служби, так і первинної ланки, тобто сімейного лікаря. у тернопільській області в останні роки спостерігають позитивну динаміку епідеміологічного процесу, зменшення захворюваності й смертності від туберкульозу. в 2014 р. ефективність лікування хворих на вперше діагностований туберкульоз легень з бактеріовиділенням становила 87,6 %. однак, поряд з цим, спостерігали низку недоліків. зокрема, кількість хворих, які померли від туберкульозу протягом одного року спостереження за останнє десятиріччя зросла з 15,6 до 22,2 % (р<0,05). це свідчить про несвоєчасне виявлення туберкульозу на різних рівнях медичної служби, зокрема і первинної ланки. виявлено недоліки і при роботі у вогнищах туберкульозної інфекції, оскільки за останні 10 років захворюваність контактних осіб у вогнищах туберкульозної інфекції (на 1000 контактних) зросла з 5,2 до 9,9 (р<0,05). це вже явний недолік у роботі фтизіатрів і сімейних лікарів. за 2010–2014 рр. на тлі зниження захворюваності на туберкульоз легень (з 506 до 444 осіб) значно збільшилась кількість рецидивів туберкульозу – з 23,8 до 61,2 % відповідно (р<0,05). до слова, із загальної кількості хворих, які перебували на стаціонарному лікуванні в обласному протитуберкульозному диспансері, мультирезистентність констатовано у 8,5 % осіб, зокрема з 2005 до 2014 р. вона зросла з 4,2 до 17,4 % (р<0,05). летальність серед хворих на мультирезистентний туберкульоз становила 17,2 %, з них до одного місяця – 1/3 осіб. найбільшу поширеність мультирезистентного туберкульозу спостерігають у м. тернопіль, тернопільському, теребовлянському та підволочиському районах (53,7 %). це надзвичайно тривожна ситуація в роботі медичної служби, передусім фтизіатричної, у цих районах. висновки на завершення, акцентуємо увагу на тому, що сімейна медицина – це медицина сьогодення і майбутнього, а сімейний лікар – універсал, який маючи ґрунтовні знання з основних розділів медицини, зокрема і фтизіатрії, надає первинну профілактичну допомогу прикріпленому за сімейно-територіальним принципом населенню в умовах амбулаторно-поліклінічного закладу чи вдома. сімейний лікар надає першу медичну допомогу та ліквідовує вогнища інфекційних захворювань, проводить серед населення пропаганду здорового способу життя, відповідального ставлення до свого здоров’я як найвищої особистої і суспільної цінності. адже “здоров’я – це не все, але все без здоров’я – ніщо” (сократ). список літератури 1. туберкульоз в україні : аналіт.-стат. довід. к. 2015; 142. 2. мельник в. туберкульоз – проблема соціальна й економічна. ваше здоров’я 2012; 40–41: 8. 3. фещенко юі, мельник bm. контроль за туберкульозом в умовах адаптаційної дотс-стратегії. к.: медицина 2007; 480. 4. саєнко ю. соціальний портрет україни і моделі майбутнього. універсум 2004: 10–12: 4–10. 5. свінціцький ас. здоров’я населення як важливий чинник державотворення та національної безпеки. практ. лікар 2013; 2: 7–13. 6. уніфікований клінічний протокол первинної, вторинної (спеціалізованої) та третинної (високоспеціалізованої) медичної допомоги дорослим “туберкульоз” 2014; 179. і. т. п’ятночка та ін. 79 p u b l ic h e a l t h a n d e p id e m io l o g y issn 2413�6077. ijmmr 2015 vol. 1 no. 1 received: 2015.06.10 udc 616�002.5:614.254.3 tuberculosis in family practice i. t. piatnochka, s. i. kornaha, t. v. kornaha i. horbachevsky ternopil state medical university background. in this article we describe a modern family doctor as a comprehensive paediatrician, who preserves human health, and their importance in fight with tuberculosis. drug-resistant tuberculosis has become an urgent matter, multidrug-resistant tb in particular. in ukraine 23.4% of patients suffer from tb diagnosed for a first time and 58.6% from recurrent cases of the disease. objective. the aim of our research is to present a family doctor as a comprehensive paediatrician and demonstrate their importance in fight with tuberculosis in modern times. results. in recent years in ternopil region the positive dynamics of epidemiological process, decrease in incidence and mortality from tuberculosis is evidenced. in 2014, the efficiency of treatment of patients who were diagnosed with bacterial pulmonary tuberculosis for a first time, was 87.6%. however, the number of patients, who died of tb within one year of observation over the last decade, increased from 15.6% to 22.2%, (p <0.05). this proves an untimely tuberculosis diagnosis at different levels of medical services, at primary one in particular. mortality rate in patients with multidrug-resistant tuberculosis is 17.2%, 1/3 persons died up to one month. conclusions. family doctor provides first medical aid, eliminates foci of infectious diseases, promotes healthy lifestyle among population, and is responsible for their health as the highest personal and social value. key words: tuberculosis, family doctor, healthcare. і. т. п’ятночка та ін. 47 b io m e d ic a l s c ie n c e s issn 2413-6077. ijmmr 2016 vol. 2 issue 2 doi 10.11603/ijmmr.2413-6077.2016.2.7036 age related oxidative processes and endogenous intoxication dynamics of rats arter tobacco smoke affection p. g. lyhatskyy, o. b. rytsyk, l. s. fira, o. z. yaremchuk i. horbachevsky ternopil state medical university, ternopil, ukraine background. in an experiment on rats in the content of reactive oxygen species and lipid peroxidation products in blood and liver of rats of different age groups after 45-day affection with tobacco smoke was studied. objective. the study was aimed to investigate the rate of reactive oxygen species formation, especially the processes of lipid peroxidation and degree of endogenous intoxication in rats of different age groups in terms of 45 day affection with tobacco smoke. methods. the content of ros was determined in blood neutrophils method gradient centrifugation, the activity of free radical processes in rats evaluated the content of tba active products (tba ap), the degree of endogenous intoxication the content average molecular weight (amw) of the two factions – mw1 (dominated by chain amino acids) and mw2 (dominated by aromatic amino acids). blood, blood serum and liver of the experimental rats were used for the investigation. it was prepared 10% homogenate in saline from liver tissue. results. we have noticed that the destruction of rats by tobacco smoke for 45 days caused the increase of ros in blood content, which is the result of toxic effect on the body. the immature rats were the most sensitive to the affection, which ros contents in neutrophils increased in 2.75 times till the end of the experiment, in the mature animals it was in 1.65 times higher than in the intacts, in the senile it was higher in 2.43 times than normal rate. it was found that the level of oxygen metabolites and tba-active products increased during the experiment (on the 15th, 30th and 45th day of toxicity). conclusions. the most pronounced changes were inherent for the immature rats. accumulation of active toxic metabolites was conducted with endogenous intoxication intensifying that was proved by the high content of catabolism products in the body – middle mass molecules that are likely to grow in serum of rats of all age groups. key words: oxidative processes, lipid peroxidation, reactive oxygen species, endogenous intoxication, tobacco smoke, rats. introduction at present tobacco smoke is one of the most common anthropogenic agents that has a wide range of effects on morphofunctional status of various systems of the body [8, 10, 15, 16]. the share of tobacco smoke in the overall air pollution is quite considerable and each year continues to rise, so it is one of powerful polluters of the environment. it is established that the basis of the pathogenic action of contaminated pollutants or cigarette smoke air is the oxidant aggression on the mucosa of respiratory tract reactive oxygen species, nitrogen dioxide and sulfur and other free radicals, which cause the activation of lipid peroxidation and damage of biological membranes [3, 11]. smoking generates the reactive oxygen species (ros: o2 •, o2 1, oh, h2o2, etc.), which are important for many physiological and biochemical processes: the regulation of vascular tone, cell proliferation, prostaglandins synthesis, signals transmission from intercellular signalling molecules of regulatory system that control the expression of phagocytes genes antimicrobial action [6, 9, 14]. under the influence of extreme factors of various origin (chemical contamination, ionizing radiation, hyperand hypoxia, toxic substances, inflammatory processes) formation of ros in organisms is enhanced [12, corresponding author: petro lyhatskyy, department of medical biochemistry, i. horbachevsky ternopil state medical university, 1 maidan voli, ternopil, ukraine, 46001 phone number: +380352254784 e-mail: luhatsky@tdmu.edu.ua international journal of medicine and medical research 2016, volume 2, issue 2, p. 47-51 copyright © 2016, tsmu, all rights reserved p. g. lyhatskyy et al. 48 b io m e d ic a l s c ie n c e s issn 2413-6077. ijmmr 2016 vol. 2 issue 2 13, 17, 18]. the last causes free radical oxidation activation that leads to increased lipid peroxidation (lpo), oxidative modification of proteins (omp), degradation of nucleic acids, carbohydrates, increasing of endogenous intoxication in the organism. however, in literature there is no definitive data on the rate of formation of ros and processes of lipid peroxidation activation in rats of different ages after prolonged exposure with tobacco smoke. accordingly, the study was aimed to investigate the rate of reactive oxygen species forma tion, especially the processes of lipid peroxidation and degree of endogenous intoxication in rats of different age groups in terms of 45-day affection with tobacco smoke. material and methods the experiments were conducted on white outbred male rats, which were kept on a vivarium standard diet of ternopil state medical university. the rats were divided into three age groups: the first — immature, weight 60–80 g, the second — mature, weight 180–200 g and the third — senile, weight 300–320 g. each age group consisted of two subgroups: an intact control (c) and an experimental group (e). the rats of the experimental groups were affected with tobacco smoke during 45 days. the model of the chronic smoke was created by means of airtight chamber volume of 30 litres that allowed animals to fumigate free behaviour. tobacco smoke was formed by smoking of 6 cigarettes prima sribna (synia) (0.6 mg of nicotine and 8mg of tar), was served into it through openings in the chamber. six animals were simultaneously in the chamber during 6 minutes. the animals of the control group were also 6 minutes in a sealed chamber, but were not subjected to smoke. in 15, 30 and 45 days after the beginning of the affecting the animals with tobacco smoke, they were taken out of the experiment by euthanasia, which was performed with thiopental anaesthesia. blood, blood serum and liver of the experimental rats were used for investigation. 10% homogenate in saline of liver tissue was prepared. the content of ros was determined by blood neutrophils method [6], the activity of free radical processes in rats evaluated the content of tba-active products (tba-ap) [5], the degree of endogenous intoxication — the average molecular weight (amw) content of two factions [2, 7]: mw1 (dominated by chain amino acids) and mw2 (dominated by aromatic amino acids). we followed general principles of animal experiments in the research that were approved at the national congress on bioethics (kyiv, ukraine, 2001) and consistent with the provisions of the european convention for the protection of vertebrate animals used for experimental and other scientific purposes (strasbourg, france, 1985) [4]. parametric (according to student) and non-parametric (according to wilcoxon) methods for data statistical analysis were used. changes were considered as significant at p≤0.05. results and discussion any stress reaction normally is accompanied by a brief increase in the number of ros [6]. this is due to adaptation to extreme conditions in which ros play a role of a secondary messenger participating in signal transduction and activation of transcription factors and related genes, including those encoding enzymes antioxidants. we noticed that the affection of the rats by tobacco smoke during 45 days caused the increase of ros in blood content (table 1), which is the result of toxic factor on the body. the immature rats were the most sensitive to the affection, the ros contents in neutrophils increased in 2.75 times at the end of the experiment, in the mature animals it was in 1.65 times higher than in the intacts, in the senile it was in 2.43 times higher than normal rate. it is established that under the influence of various extreme factors (chemical contamitable 1. the contents of ros (%) in blood neutrophils of the rats affected with tobacco smoke of all ages (m±m; n=72) research time, days groups of the experimental animals immature rats mature rats senile rats intact rats 15.06±0.71 18.47±0.22 19.87±0.86 15th day of the affection 17.19±0.83 28.58±2.53* 25.38±1.95 30th day of the affection 39.25±1.29* 29.54±0.50* 41.89±0.78* 45th day of the affection 41.52±2.37* 30.63±0.66* 48.32±0.57* note: * — differences between the intact rats and the rats affected with tobacco smoke (p≤0.05). p. g. lyhatskyy et al. 49 b io m e d ic a l s c ie n c e s issn 2413-6077. ijmmr 2016 vol. 2 issue 2 nation, ionizing radiation, hyperand hypoxia, toxic substances, inflammatory processes) the formation of ros in organisms is enhanced. among the reasons that causes of the increased production of ros are: violation of electron transport in the respiratory chain of mitochondria and electron transport chain microsomes, the intensification of synthesis and oxidation of catecholamines, the increased degradation adenylate nucleotides and activation of xanthine oxidase, the emergence of a pool of catalytically active metal ions of variable valence (especially fe2+), the synthesis of prostaglandins from arachidonic acid (reactions catalysed cyclooxygenase and lipoxygenase), the activation of inducible form of nitric oxide synthase, the increased activity of phagocytes [14]. the intensification of free radical oxidation reactions is one of the manifestations of oxygen metabolites toxic effects. free radical oxidation is a universal mechanism, which controls the most important homeostatic physical and chemi cal parameters of cells: strength, integrity and selective permeability of cell membranes [3]. a significant increase of ros, which was evidenced after poisoning the animals with tobacco smoke, caused the intensification of free radical oxidation including lipid peroxi dation. as one of the indicators of lipid peroxidation, the contents of tba­ap significantly increased in serum and liver of the rats after tobacco smoke intoxication (table 2). in serum of the im mature animals on the 15th day of the intoxication, this indicator increased by 30% (changes were not significant). the other age groups in this period were more sensitive: the tba­ap content in serum increased by 87% in the mature animals and by 58% in the senile (p≤0.05). the intoxication with tobacco smoke during 45 days caused the significant activation of lipid peroxidation, which was evidenced by considerable increase of tba-ap content in serum of the animals of all experimental groups (in the immature rats the indicator increased in 2 times, in the immature — in 2.1 times, in the senile — in 1.8 times). in liver of the experimental animals a similar increase of investigational product lipid peroxidation was observed during the experiment. by the end of tobacco smoke toxicity (on the 45th day of the research), the increase of tba-ap content in liver of the mature and senile animals in 1.8 times respectively was evidenced. the immature animals were more sensitive to this indicator and the content of intermediate peroxidation increased in 3.2 times in the test organ. perhaps this is due to insufficient decontaminating of liver, including oxidation processes and microsomal enzymes that took part in it. in pathological processes the average molecular weight (amw) is quite important, some its fraction exhibit high biological activity [1]. the spectrum of pathological action of these compounds has a different character: they break the physical and chemical properties of cell membranes, inhibit tissue respiration and oxidative phosphorylation, inhibit atp activity and dna synthesis, activate lipid peroxidation and reduce the activity of antioxidant defence cells, stimulate cellular proliferation and inhibiting the process of apoptosis [2]. amw accumulation is a consequence of the activation of catabolic processes and reduction of liver detoxification function. metabolic syndrome intoxication is developing, which leads to severe functional impairment and morphological damage to various organs and systems. activation of ros-formation processes and lipid peroxidation causes increased proteolysis reactions in the body affected with tobacco smoke, accompanied by accumulation of both factions amw — amw1 (dominated by chain amino acids) and amw2 (dominated by aromatic amino acids). the results of the table 2. the content of tba-ap in blood serum (mmol/l) and liver (mmol/kg) of the rats in dynamics of tobacco smoke affection (m±m; n=72) research time, days groups of the experimental animals immature rats mature rats senile rats blood serum intact rats 3.28±0.23 1.85±0.14 2.35±0.14 15th day of the affection 4.28±0.31 3.47±0.29* 3.71±0.28* 30th day of the affection 4.85±0.36* 4.28±0.31* 4.42±0.28* 45th day of the affection 6.62±0.08* 3.94±0.27* 4.23±0.17* liver intact rats 15.49±1.28 14.42±0.71 16.55±0.98 15th day of the affection 18.69±1.28 23.50±1.35* 25.12±2.09* 30th day of the affection 30.86±1.55* 28.73±0.78* 29.06±1.46* 45th day of the affection 49.66±3.38* 26.49±1.51* 29.69±2.39* p. g. lyhatskyy et al. 50 b io m e d ic a l s c ie n c e s issn 2413-6077. ijmmr 2016 vol. 2 issue 2 research of this indicator in blood serum are presented in table 3. after 15-day toxicity with tobacco smoke in blood serum of the immature rats the amw1 content increased by 35%, amw2 – by 38%. by the end of the experiment, these indices increased in 3 and 2.2 times respectively in this age group. at the end of the research in the mature rats the amw1-content in blood serum was in 3 times higher than in the intact animals, amw2-content was in 1.7 times higher than normal. similarly, the increased content of msm in the senile rats in 45 days after the tobacco intoxication in blood serum of this age group the amw1-content was in 2.8 times higher than the level of the intact control, the amw2-content exceeded the level of the healthy animals in 1,9 times. analysing the processes of catabolism in the affected organism we can notice that they were activated in all age groups of rats similarly. obviously, tissue hypoxia is the primary in the development of destructive processes in the organism after tobacco smoke toxicity due to the breach of oxygen transport. conclusions due to the chronic affection with tobacco smoke on the rats of different ages the massive formation of reactive oxygen species in neutrophils causes activation of lipid peroxidation and permanent changes of endogenous intoxication (the increase of tba-active products in blood serum and liver of the effected rats, the accumulation of average molecular weight). immature rats were the most sensitive to tobacco smoke; metabolic disorders were the most pronounced as well. table 3. amw content in blood serum (cu/l) of the rats in dynamics of tobacco smoke affection (m±m; n=72) research time, days groups of the experimental animals immature rats mature rats senile rats amw1 intact rats 14.00±1.15 11.00±0.85 13.66±0.61 15th day of the affection 19.00±1.52 17.66±1.20* 18.00±1.55 30th day of the affection 37.00±1.12* 31.00±1.12* 35.66±0.61* 45th day of the affection 41.66±0.95* 33.00±1.52* 38.00±1.03* amw2 intact rats 16.66±0.84 12.67±0.84 15.33±0.67 15th day of the affection 23.00±2.29 15.33±0.66 20.33±1.82 30th day of the affection 28.67±0.67* 18.66±0.67* 23.33±0.99* 45th day of 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m e d ic a l s c ie n c e s issn 2413-6077. ijmmr 2016 vol. 2 issue 1 address for correspondence: hanna saturska, department of social medicine, health care management, economy and medical statistics, i. horbachevsky ternopil state medical university, 1 maidan voli, ternopil, ukraine, 46001 tel. +380352527233 e-mail: saturska@tdmu.edu.ua doi 10.11603/ijmmr.2413-6077.2016.1.6383 influence of trimetazidine metabolic therapy on connective tissue metabolism in experimental diffuse ischemic necrotic cardiosclerosis in rats with different rates of hypoxia resistance h. s. saturska, yu. і. bondarenko, u. v. saturska i. horbachevsky ternopil state medical university, ternopil, ukraine background. the change in metabolism of the connective tissue elements of heart is the central chain in pathogenesis of diffuse ischemic necrotic cardiosclerosis (dinc), which occurs after repeated epinephrine injury of myocardial tissues. objective. this study proves that trimetazidine (tm) metabolic therapy has a protective effect on the development of dinc in rats with different rates of hypoxia resistance. methods. male white rats were divided into three groups due to the different rates of hypoxia resistance by means of the method of hypobaric hypoxia: rats with low, middle and high rates of hypoxia resistance. each group was divided into equal subgroups: a control group, a dinc group (injections of epinephrine hydrotartrate (0,5 mg/kg of body weight) and calcium gluconate (5 mg/kg of body weight) two times), a control group administrated with trimetazidine dihydrochloride (10 mg/kg of body weight), a dinc group treated with tm every day (10 mg/kg of body weight) for all period of observation. concentration of protein-bound oxyproline in blood serum was evaluated on the 7th, 14th and 30th days after the pathology simulation. histological examination of masson trichrome staining of myocardium was performed on the 30th days after the pathology simulation. results. dinc increased the concentration of protein-bound oxyproline in blood serum on the 7th, 14th and 30th days after the pathology simulation, and followed by metabolic imbalances in diffuse connective tissue elements, which are rich in collagens. dinc+tm increased the concentration of protein-bound oxyproline in blood serum less intensively. conclusions. the intensity of metabolic imbalances in diffuse connective tissue elements is the highest in the low resistant animals to hypoxia. those results are confirmed by histological examination of the myocardium of rats with different resistance to hypoxia. fibrotic regions in myocardium are rich in collagens. it has been revealed that the most pronounced therapeutic effect of tm is observed in animals with low resistance to hypoxia, slight – in animals with medium resistance to hypoxia, and the lowest – in animals with high resistance to hypoxia. key words: hypoxia, heart, diffuse cardiosclerosis, trimetazidine, oxyproline. introduction the pathology of the cardiovascular system is the major medical and social problem, because it takes the main cause of morbidity and mortality [1-2]. the special attention is paid to the research on diagnostic markers of degradation and reparation of myocardial tissue [3-5], which would reflect the dynamic changes in myocardium and were predictors of diffuse cardiosclerosis [2-3]. the purpose of this investigation was to determine the changes in the content of protein-bound oxyproline in blood as a diagnostic marker of metabolic activity of collagen at the experimental diffuse ischemic necrotic cardiosclerosis in rats with different rate of hypoxia resistance. recently, we demonstrated that the use of trimetazidine as an endogenous cardioprotection inducer in the development of diffuse ischemic necrotic cardiosclerosis is manifested by decrease in manifestations of oxidative and nitrooxidative stress, optimization of immune and cytokinе response, stabilization of humoral immune responsiveness [6]. the research was carried out to study the effects of tm on the improvement of connective tissue elements metabolism, indicating inhibition of cardiosclerotic process. h. s. saturska et al. international journal of medicine and medical research 2016, volume 2, issue 1, p. 49–53 copyright © 2016, tsmu, all rights reserved 50 b io m e d ic a l s c ie n c e s issn 2413-6077. ijmmr 2016 vol. 2 issue 1 materials and methods animals and treatment experiments were done on 192 male white rats (190-250 g) of the vivarium of ternopil state medical university, ukraine. all animals received care in compliance with the “guide for care and use of laboratory animals” (national institute of health publication № 85-23, revised 1985). the studies were carried out according to the national institute of health guide for care and use of laboratory animals and were approved by the local animal protection committee. the experimental animals were divided into three groups according to different rates of hypoxia resistance by means of the method of hypobaric hypoxia [6] [berezovskyi, 1975; markova, 1998]: rats with low, middle and high rates of hypoxia resistance. each group was divided into equal subgroups: a control group, a diffuse ischemic necrotic cardiosclerosis group (injections of epinephrine hydrotartrate (0,5 mg/kg body weight) and calcium gluconate (5 mg/kg of body weight) two times), a control group administered with trimetazidine dihydrochloride (10 mg/kg of body weight) every day [6], diffuse ischemic necrotic cardiosclerosis group treated with trimetazidine dihydrochloride (10 mg/kg of body weight) every day for all period of observation (n=8 of each group). evaluation of protein-bound oxyproline in blood serum concentration of protein-bound oxyproline in blood serum was determined biochemically [7] on the 7th, 14th and 30th days after the pathology simulating. histopathology study the tissue from myocardium ventricles was taken on the 30th day after pathology simulation, then put in 10 % neutral-buffered formalin solution for 5 days, embedded in paraffin, and sectioned. histological examination of masson trichrome staining of myocardium was performed [8]. statistical analysis statistical analysis was carried out by originpro program. the results were presented as mean±standard deviation. differences between experimental groups were analyzed with an unpaired two-tailed student t-test [9]. values were considered to be statistically significant at p<0.05. results before the dinc simulation the proteinbound oxyproline concentration in blood serum of rats with low hypoxia resistance was 17.8% (p<0.05) higher than in blood serum of rats with middle hypoxia resistance (table 1), oxyproline concentration in blood serum of high hypoxia resistant animals was 21.9% lower (p<0.05) than in blood serum of rats with middle hypoxia resistance. after the dinc simulation proteinbound oxyproline concentration in blood serum of rats gradually increased at all groups. under the influence of trimetazidine metabolic therapy the changes in protein-bound oxyproline concentrations in blood serum of the animals with low hypoxia resistance were less pronounced (table 2). concentration of this metabolite collagen in 7 days after pathology simulation with tm was by 11.0% (p<0.05) (figure 1) lower than in the group of untreated animals at this stage of observation. on the 14th day after dinc simulation with tm, proteinbound oxyproline concentration in blood serum of the rats with low hypoxia resistance was by 25.3% lower (p<0.001) than in the untreated animals, and on the 30th day of observation it was by 33.9% (p<0.001) lower than in the table 1. protein-bound oxyproline concentration in blood serum in cases of experimental diffuse ischemic necrotic cardiosclerosis (dinc) with innate hypoxia resistance in rats hypoxia resistance rate in animals control group (n=8) stages of dinc observation 7 days (n=8) 14 days (n=8) 30 days (n=8) low 49,55±0,59 р<0,05 57,45±1,78 р<0,01 р*<0,01 79,15±2,66 р<0,01 р*<0,01 104,84±3,42 р<0,01 р*<0,01 middle 42,07±1,10 47,92±0,62 р*<0,01 58,24±1,00 р*<0,01 73,38±3,30 р*<0,01 high 34,52±0,92 р<0,05 38,53±0,55 р<0,01 р*<0,05 42,65±1,19 р<0,01 р*<0,01 56,43±2,84 р<0,01 р*<0,01 notes: p<0.05 – significantly different from middle hypoxia resistant animals at all stages of observation; *р<0.05 – significantly different from the control group at all stages of observation. h. s. saturska et al. 51 b io m e d ic a l s c ie n c e s issn 2413-6077. ijmmr 2016 vol. 2 issue 1 untreated animals with low hypoxia resistance at the similar stage of cardiosclerotic process development without any correction. in blood serum of the rats with middle hypoxia resistance, protein-bound oxyproline concentration on the 7th day after dinc simulation and trimetazidine correction was by 8.3% (p<0.05) (fig. 1) lower than in the group of untreated animals at this stage of observation. at the next stage of observation, on the 14th day of dinc simulation, protein-bound oxyproline concentration in blood serum of these animals was by 18.6% lower (p<0.001) than in the untreated ones, and on the 30th day of dinc observation – by 28.2% (p<0.001) lower than in the untreated rats. there was no significantly difference between the treated and untreated animals with high hypoxia resistance on the 7th day of dinc observation. on the 14th day after the pathology simulation, protein-bound oxyproline concentration was lower by 11.6% (p<0.05) than in the group of untreated animals at this stage of observation; and on the 30th day of dinc observation and correction with metabolic therapy, concentration of protein-bound oxyproline in blood serum was 28.4% (p<0.001) lower than in the untreated animals with high hypoxia resistance without any correction. histological examination of the myocardium on the 30th day of dinc observation showed that at heart micropreparations of the healthy animals with different rates of hypoxia resistance, connective tissue was observed slightly in the form of thin collagen fibers (figure 2), but in heart micropreparations of the animals with dinc (figure 3) focal cardiosclerosis, perivascular sclerosis hyperelasticity of vessels table 2. influence of trimetazidine on protein-bound oxyproline concentration in blood serum in cases of experimental diffuse ischemic necrotic cardiosclerosis (dinc) with innate hypoxia resistance in rats hypoxia resistance rate in animals control tm group (n=8) stages of dinc+tm observation 7 days (n=8) 14 days (n=8) 30 days (n=8) low 45,58±1,51 51,15±1,36 р*<0,05 р<0,001 59,09±1,85 р*<0,001 р<0,001 69,32±1,86 р*<0,001 р<0,001 middle 40,58±1,83 43,97±1,34 40,58±1,83 р*<0,05 52,72±3,15 р*<0,05 high 34,90±0,96 р<0,05 36,33±0,91 р<0,001 37,72±1,52 р<0,001 34,90±0,96 р*<0,05 р<0,01 notes: p<0.05 – significantly different from the middle hypoxia resistant of animals at all stages of observation; *р<0.05 – significantly different from the control group at all stages of observation. fig. 1. influence of trimetazidine on protein-bound oxyproline concentration in blood serum in cases of experimental diffuse ischemic necrotic cardiosclerosis (dinc) with innate hypoxia resistance in rats. notes: the indices of the control groups were presented in 100%; * – significantly different from the control group at all stages of observation, p<0.05; # – significantly different from the untreated rats at all stages of observation, p<0.05. 0 50 100 150 200 250 control dinc 7 days dinc 14 days dinc 30 days lrh lrh+tm mrh mrh+tm hrh hrh+tm % * * * * * * * ** # # # # # # ### h. s. saturska et al. 52 b io m e d ic a l s c ie n c e s issn 2413-6077. ijmmr 2016 vol. 2 issue 1 discussion evaluation of protein-bound oxyproline concentrations in blood serum in dinc simulation with and without trimetazidine correction proved that increased collagen production and products of its metabolism [4-5] can be used as a biological marker of the intensity of collagen synthesis in tissue infarction. so we can make the following conclusion: the intensity of metabolic imbalance of connective elements in cases of diffuse ischemic necrotic cardiosclerosis and trimetazidine correction depends on hypoxia resistance of animals. in the low hypoxia resistance animals, maximum effect of trimetazidine correction was manifested; however, more pronounced changes in oxyproline concentration were in dinc simulation without any correction. this effect was not enough for denoting differences between animals with different rates of hypoxia resistance. this matter is characteristic feature of animals with middle hypoxia resistance, but the changes were less pronounced. animals with high hypoxia resistance were characterized by lower oxyproline concentration, which changed after dinc simulation, so the effect was manifested less, but in general, they are characterized by minimal metabolic disorders of connective tissue elements in the development of dinc and correction with trimetazidine [10-16]. the activity of connective tissue metabolism was studied in experimental diffuse ischemic ne crotic cardiosclerosis with different rates hy poxia resistance of a body. the investigations were based on the changes in concentration of protein-bound oxyproline in blood serum that pro ved adequate metabolic changes in collagen [4–5]. conclusions the development of the experimental diffuse ischemic necrotic cardiosclerosis at all stages of observation was accompanied by metabolic imbalance in connective tissue of heart, and was proved by the increase in oxyproline level in blood serum of animals with different rates of hypoxia resistance. the intensity of metabolic imbalances in diffuse connective tissue elements was the highest in low hypoxia resistant animals. those results were confirmed by histological examination of myocardium of rats with different rates of hypoxia resistance. fibrotic regions in myocardium are rich in collagens. it has been revealed that the most pronounced therapeutic effect of tm is observed in animals with low hypoxia resistance, fig. 2. myocardium of the control rat. masson trichrome staining of myocardium. х 400. fig. 3. myocardium of the low hypoxia resistant rat with dinc. fibrotic regions in myocardium are rich in collagens and therefore appear in blue upon masson trichrome staining. in addition, centralized nuclei as well as shape and size distribution of myofibers were detected that was the evidence of pronounced cardiomyocytes hypertrophy. 30-day dinc. masson trichrome staining of myocardium. х400. inner membrane, cardiomyocyte hypertrophy, diffuse proliferation of connective tissue were presented. fibrotic regions in myocardium are rich in collagens and therefore appear in blue upon masson trichrome staining. in addition, centralized nuclei as well as shape and size distribution of myofibers were detected that was the evidence of pronounced cardiomyocytes hypertrophy. all the above-mentioned symptoms are the highest in the low hypoxia resistant animals, indicating the intense development of diffuse cardiosclerosis in animals with low hypoxia resistance and confirm the results obtained during evaluation of the concentration of protein-bound oxyproline serum of rats with different rates of hypoxia resistance. h. s. saturska et al. 53 b io m e d ic a l s c ie n c e s issn 2413-6077. ijmmr 2016 vol. 2 issue 1 slightly less – in animals with medium hypoxia resistance, and the lowest – in animals with high hypoxia resistance. this matter was evidenced at all stages of observation, but it was the most pronounced in the early period of cardiosclerotic process, indicating the feasibility of early use of metabolic therapy. it explains the absence of cardioprotective effect of trimetazidine in the later stages of cardiosclerosis, when the myocardial fibrosis is already formed. references 1. lopez ad, mathers cd. measuring the global burden of disease and epidemiological transitions: 2002–2030. ann trop med parasitol. 2006; 100(5–6): 481–499. 2. salemi vm, leite jj, picard mh et al. echocardiographic predictors of functional capacity in endomyocardial fibrosis patients. eur j echocardiogr 2009; 10(3): 400–405. 3. iglezias sd, benvenuti la, calabrese f. et al. endomyocardial fibrosis: pathological and molecular findings of surgically resected ventricular endomyocardium. virchows arch 2008; 453(3); 233–241. 4. ito a, yamagiwa h, sasaki rj. effects of aging on hydroxyproline in human heart muscle. am geriatr soc. 1980; 28(9): 398–404. 5. hoerstrup sp, zünd g, ye q, et al. tissue engineering of a bioprosthetic heart valve: stimulation of extracellular matrix assessed byhydroxy proline assay. asaio j. 1999; 45(5): 397–402. 6. saturska hs. peculiarities of cardioprotective effect of trimetazidine at experimental cardiosclerosis in rats with different sensitivity to hypoxia. vestnik of vitebsk state medical university 2015; 14(1): 34–40. (in russian). 7. sharaev pn. method for determination of free and bound hydroxyproline in serum. lab business 1981; 5: 283–285. (in russian). 8. merkulov ga. course of histological techniques. – l . : medicine, 1969; 422 p. (in russian). 9. orlov аi. mathematics cases: probability and statistics – the basic facts: a tutorial. m. : m-press, 2004; 100 p. (in russian). 10. detry jm, sellier p, pennaforte s, et al. trimetazidine: a new concept in the treatment of angina: comparison with propranolol in patients with stable angina. trimetazidine european multicenter stu dy group. br j clin pharmacol 1994; 37: 279–288. 11. gupta r, sawhney jp, narain vs. treatment of stable angina pectoris with trimetazidine modified release in indian primary-care practice. am j cardiovasc drugs. 2005; 5(5): 325–329. 12. marzilli m, klein ww. efficacy and tolerability of trimetazidine in stable angina: a metaanalysis of randomized, double-blind, controlled trials. coron artery dis 2003; 14: 171–179. 13. sellier p, broustet jp. assessment of antiische mic and antianginal effect at trough plasma concentration and safety of trimetazidine mr 35mg in patients with stable angina pectoris: a multicenter, double-blind, placebo-controlled study. am j cardiovasc drugs 2003; 3: 361–369. 14. szwed h, sadowski z, pachocki r, et al. antiischaemic efficacy and tolerability of trimetazidine in elderly patients with angina. clin drug invest 2000; 19: 1–8. 15. szwed h, sadowski z, pachocki r, et al. combination treatment in stable effort angina using trimetazidine and metoprolol: results of a randomized, double-blind, multicentre study (trimpol ii). trimetazidine in poland. eur heart j 2001; 22: 2267–2274. 16. szwed h, sadowski z, pachocki r, et al. the antiischemic effects and tolerability of trimetazidine in coronary diabetic patients: a substudy from trimpol 1. cardiovasc drugs ther 1999; 13: 217–222. received: 2016-03-04 h. s. saturska et al. 48 su r g ic a l d is e a se s issn 2413-6077. ijmmr 2022 vol. 8 issue 1 doi 10.11603/ijmmr.2413-6077.2022.1.13157 endovascular angioplasty for multi-level stenoticocclusive lesions of the femoral-distal arterial bed in cases of stenotic-occlusive process of the tibial arteries i. k. venher, s. ya. kostiv, *b. p. selskyi, d. v. khvalyboha, m. yu. orlov, i. v. faryna, n. i. tsiupryk i. horbachevsky ternopil national medical university, ternopil, ukraine background. occlusive-stenotic lesions of the lower extremity arteries are the second in the structure of cardiovascular diseases, after only coronary heart disease. surgical treatment of patients with infrainguinal atherosclerotic lesions of the arterial segment is challenging in a number of cases. at the same time, revascularizing surgical interventions on the femoral-distal artery and especially on the tibial arteries in chronic critical ischemia is the only chance to avoid amputation of the lower limb. in this regard, the search and study of optimal methods of revascularizing surgery on the infrainguinal artery for stenotic-occlusive lesions of the arteries of the tibial segment is urgent. objective. this study is aimed at promotion of endovascular and hybrid technology in cases of stenoticocclusive process of the tibial arteries and choosing the optimal surgical treatment tactics in this case. methods. endovascular interventions of the femoral-distal arterial bed in cases of stenotic-occlusive process of the tibial arteries performed for 135 patients at the department of vascular and cardiac surgery of ternopil regional clinical hospital have been analysed. results. in most cases, endovascular angioplasty of two tibial arteries in hybrid and endovascular methods of revascularization of multilevel atherosclerotic process of the femoral-distal arterial segment of the lower extremity prevents development of thrombosis in tibial segment and allows maintaining the patency of the reconstruction segment and preservation of the lower extremity in 97.57% and 93.44% of cases, respectively. conclusions. endovascular angioplasty of two tibial arteries provides a better result than angioplasty of one isolated tibial artery and allows maintaining the patency of the reconstructed segment for more than 90 percent compare to one isolated angioplasty. keywords: vascular surgery; endovascular angioplasty; stenotic-occlusive process; atherosclerosis; revascularization. *corresponding author: boryslav selskyi, assistant professor, i. horbachevsky ternopil national medical university, ternopil, 46000, ukraine. e-mail: selskyi_bp@tdmu.edu.ua international journal of medicine and medical research 2022, volume 8, issue 1, p. 48-54 copyright © 2022, tnmu, all rights reserved introduction occlusive-stenotic lesions of the lower extremity arteries are the second in the structure of cardiovascular diseases, only after coronary heart disease [1]. more than 55% of atherosclerotic lesions of the femoral-distal arterial segments were detected [2]. occlusivestenotic lesions of the infrainguinal artery in 35-65% of cases lead to chronic critical ischemia [3]. surgical treatment of patients with infrainguinal atherosclerotic lesions of the arterial bed is challenging in a number of cases [4]. the complexity of surgical interventions in the femoral-distal arterial area is due to multilevel atherosclerotic lesions of the arterial bed, the spread of lesions to adjacent arterial basins, collateral arterial basins and the state of outflow pathways – tibial arteries. at the same time, revascularizing surgical interventions on the femoral-distal artery and especially on the tibial arteries in chronic critical ischemia is the only chance to avoid amputation of the lower limb [13]. in this regard, the search and study of optimal methods of revascularizing surgery on the infrainguinal artery for stenotic-occlusive lesions of the tibial arteries is urgent. methods endovascular angioplasty of the femoraldistal arterial bed in cases of stenotic-occlusive process of the tibial arteries was performed for 135 patients. according to fontaine’s classification, 50 patients were diagnosed with stage i. k. venher et al. 49 su r g ic a l d is e a se s issn 2413-6077. ijmmr 2022 vol. 8 issue 1 i. k. venher et al. iib of chronic arterial insufficiency (cai), 63 patients – stage iii of cai, 22 patients – stage iv. the method of hybrid revascularization was applied for 74 patients, and 61 patients underwent endovascular angioplasty of the infrainguinal arterial bed of the lower extremity. to diagnose occlusive-stenotic lesions of the aorta and main arteries of the lower extremities, computed tomography with angio-ampli fication was performed by philips brilliance ct64 (philips medical systems, the netherlands) and ultrasound doppler sonoscape s8 exp (italy) for examination of the arterial bed of lower extremities, angiography in the settings of the endovascular x-ray operating room, by angiograph siemens axiom artis (germany). before endovascular surgery double disaggregating therapy was prescribed to all patients wre: acetylsalicylic acid drugs one day (160-300 mg/d) and thieperidine drugs (clopidogrel 75 mg/d). one hour before the operation, premedication was prescribed. promedol 2%, 1.0 ml was most often administered intramuscularly or 30-40 minutes prior intramuscular injection of sol. relanii or sol. seduxeni 2.0. endovascular interventions were performed under local anaesthesia: 20.0­40.0 ml of 0.5% solution of novocaine. before the operation, after access, heparin solution was administered at a dose of 70-80 u/kg of the patient body weight (5000-7500 u). to prevent arterial spasm of the tibial arteries, it was effective to prescribe nifedipine 10 mg under the tongue for 15-20 minutes in combination with intravenous administration of 2.0 ml of 2% solution of papaverine under control of blood pressure. check-flo performer by cook incorporated (usa) and balton (eu), size 4-7 fr were used to ensure the change of the balloon catheter and the free introduction of contrast. during the endovascular stage of hybrid surgery, intraluminal angioplasty of the tibial arteries was performed, and in 14 – subintimal angioplasty and subsequent stenting. in all cases, long cylinders (80-150 mm) armada 35 ll by abbott vascular (usa) and amphirion deep (medtronic) coyote (boston scientific), stents completese (medtronic), smart (cordis) were used. in all cases, long cylinders (80-150 mm) armada 35 ll by abbott vascular (usa) and amphirion deep (medtronic) coyote (boston scientific), stents completese (medtronic), smart (cordis) were used. retrograde transfemoral access was applied for endovascular manipulations on the femoral-popliteal-tibial arterial bed. the puncture site of the cfa was placed 2-3 cm below the inguinal fold. subsequently, endo vascular manipulations on the arteries of the femoralpopliteal segment were performed through the 6f introducer. endovascular angio plasty of the stenotic-occlusive process of the femoral segment was performed with balloon catheters pan medical (pekico), opta pro (cordis). after hybrid and endovascular angioplasty, unfractionated heparin was administered at a dose of 70-80 u/kg of body weight immediately after surgery with continued use until 12-24 hours of the early postoperative period. in 12 to 24 hours, thromboprophylaxis was continued with low molecular weight heparin. disaggregating therapy with acetylsalicylic acid 100 mg and clopidogrel 75 mg was prescribed. the criteria for inclusion of patients in the group for surgery was the presence of a multilevel atherosclerotic stenotic-occlusive lesions of the infrainguinal artery. the atherosclerotic process in the tibial arteries was extensive, parietal with segmental stenoticocclusive lesions of the vascular lumen, the length of which was within 2.8-4.3 cm. thus, endovascular angioplasty of two tibial arteries was performed to form a functionally suitable outflow pathway in the tibial arteries in cases of stenotic-occlusive process. statistical analysis was performed using the statistical software package statistica 6.0 for windows (statsoft, inc. tulsa ok, usa) and microsoft excel (microsoft office 2013, usa) with data in the form of mean and standard deviation (m±sd). spearman correlation analyses were conducted to evaluate correlations between resistance index and type of angioplasty. results hybrid revascularization of the infrainguinal artery in cases of stenotic-occlusive lesions of the tibial arteries was performed for 74 patients. according to fontaine’s classification of stages of chronic arterial insufficiency, stage iib of cai was detected in 26 patients, in 34 patients – stage iii; in 14 patients with stage iv of cai trophic changes on the toes were found. atherosclerotic lesions of the arterial bed of the lower extremity was multilevel. it was established that occlusive process in one tibial artery with simultaneous development of stenotic lesions of the other two was detected in 6 (9.83%) cases. the occlusive process of two tibial arteries was established in 59 (79.73%) 50 su r g ic a l d is e a se s issn 2413-6077. ijmmr 2022 vol. 8 issue 1 cases. in 9 (14.76%) cases, segmental occlusive process was detected simultaneously in all tibial arteries. in 17 (22.97%) cases, atherosclerotic occlusion spread from the level of bifurcation of the common femoral artery (cfa) to the superficial femoral artery; in 38 (51.35%) cases the superficial femoral artery (sfa) was affec­ ted; in 7 (9.46%) cases the occlusive process spread from the initial segment of the middle third of the sfa to the level of the first section of the popliteal artery (pa), in 12 (16.22%) – to the ii-iii section of pa. ultrasound duplex examination in 17 (22.97%) cases revealed stenosis at the level of 54-68% of the mouth (first section) of the deep femoral artery (dfa). hybrid surgery included open reconstruction of the femoral-popliteal segment and endovascular angioplasty of the tibial arteries and was staged one-step. surgical treatment began with isolation of the femoral and popliteal arteries at typical access sites. in all cases, the distal anastomosis of the autovenous conduit was formed at the level of pa, and in 51 (68.92%) cases it was formed by the end-to-end type from autovein to pa. subsequently, the autovenous conduit was moved anatomically through the canal along the vascular-nerve bundle to the selected level of the femoral artery. the proximal anastomosis of the autovenous bypass in 17 (22.97%) cases was formed at the level of cfa bifurcation. in these cases, the formation of the proximal anastomosis preceded the profundoplasty. in 57 (77.03%) cases, the proximal anastomosis was formed at the level of sfa: in 38 (66.67%) cases – at the level of the upper third of sfa, in 19 (33.33%) – at the level of the initial section of the middle third of sfa. prior to the completion of the first stage of the hybrid operation, a venous stump was formed at the level of the proximal anastomosis of the autovenous conduit to fix the introducer. this was achieved as follows: in the formation of the proximal anastomosis autovein was taken with an excess length of 3-4 cm, the stump was formed from it. an introducer was inserted into it, which was fixed with a tourniquet and was passed through a puncture at a distance of 5-6 cm from the edge of the wound to the skin surface. the venous stump to insert an introducer was used in 65 cases. in 9 cases, the introducer was performed at the level of the proximal anastomosis through the venous branch of the autovenous bypass. thus, 122 endovascular angioplasties of the tibial arteries were performed: angioplasty of only one of the tibial arteries was performed in 26 (35.13%) cases, and angioplasty of two vessels simultaneously – in 48 (64.87%) cases. in 14 patients with stage iv of cai, trophic changes were found on the toes: in 10 cases superficial trophic changes of the skin of the toes were detected; in 3 cases – deep ulcer of the toes, which involved subcutaneous adipose tissue, tendons and muscles; in one case – a deep ulcer involving subcutaneous adipose tissue, tendons, muscles with bone lesions, 1-3 fingers with spreading to their base. at the end of revascularization on the infrainguinal arterial segment, surgical treatment of trophic changes of the distal segments of the feet was performed in 4 cases. in cases of deep ulcer, which involved subcutaneous adipose tissue, tendons and muscles, exarticulation of the second and third toes was performed, and in one case of deep ulcer involving subcutaneous adipose tissue, tendons, muscles with bone lesions – metatarsal amputation of the distal segment of the foot. so, endovascular revascularization interventions on the infrainguinal artery in stenotic-occlusive lesions of the tibial arteries were performed for 61 patients. instrumental examination of a vascular bed of the lower extremity showed multilevel stenotic-occlusive atherosclerotic lesion of the infrainguinal arterial bed. it was established that atherosclerotic lesions of the arteries of the tibial segment were parietal with the spread of the process along the entire length of the artery, which narrowed its lumen and led to segmental occlusion. the length of occluded segments in the tibial arteries was 2.7-3.5 cm. the occlusive process in one vessel was found only in 6 (9.83%) cases with stenotic lesions of the other two tibial arteries. in the vast majority of cases, 42 (68.85%), the occlusive process of two tibial arteries with stenotic lesions of the third of the tibial arteries was revealed. in 9 (14.75%) cases the occlusive process was established simultaneously in all tibial arteries. tibioperoneal trunk occlusion was revealed in 4 (6.56%) cases. the results of computed tomography and ultrasound doppler of the arterial bed of the lower extremities were taken as the basis for the choice of endovascular method of revascularization of the stenotic-occlusive process of the infrainguinal arterial bed of the lower extremity. after balloon angioplasty, self-opening stents smart control (cordis) were used in 8 cases, carbostent (flype) in 6 cases, and vascui. k. venher et al. 51 su r g ic a l d is e a se s issn 2413-6077. ijmmr 2022 vol. 8 issue 1 i. k. venher et al. lar stent (bard) in 11 cases. that is, in 40.98% of cases, self-opening stents were used to ensure the effectiveness of endovascular angioplasty of the femoral segment. the second stage of endovascular intervention on the femoral-popliteal-tibial artery was angioplasty of the tibial arteries. intraluminal angioplasty of the tibial arteries was performed in 48 cases, and subintimal angioplasty – in 13 cases, followed by stenting. thus, in total 108 endovascular angioplasties of the tibial arteries were performed: angioplasty of only one of the tibial arteries was performed in 6 (5.56%) cases, and two tibial vessels at the same time – in 51 (83.61%) cases. endovascular angioplasty of the tibioperineal trunk was perfor med in 4 cases. among 61 patients who underwent endovascular revascularization methods, trophic changes of the toes were revealed in 8 (13.15%) patients with stage iv of cai; superficial ulcer was found in 5 of them, in 2 cases – a deep ulcer in which subcutaneous adipose tissue, tendons and muscles were involved in the process; and in one case – a deep ulcer involving subcutaneous adipose tissue, tendons, muscles with bone damage. at the end of revascularization manipulations on the infrainguinal arterial bed, surgical treatment of trophic changes of the toes was performed; in 3 cases the toes (2-3 toes) were amputated. complications were diagnosed during hybrid revascularization of the infrainguinal artery in cases of stenotic-occlusive lesion of the tibial arteries in 74 patients; 6 of them (8.11) were diagnosed with complications in the early postoperative period. in 5 (6.76%) cases, thrombosis of the reconstruction segment was revealed, and in one (1.35%) – ineffectiveness of surgery. after endovascular revascularization of the femoral-distal artery in cases of stenotic-occlusive lesions of the tibial arteries in 61 patients, complications were diagnosed in 5 (8.17%) cases. in 4 (6.56%) cases, thrombosis of the reconstruction segment was revealed and in one (1.64%) – ineffectiveness of surgery. after hybrid and endovascular revascu larization of multilevel atherosclerotic lesions of the femoral-distal arterial bed of the lower extre mity thrombosis of the revascularization segment developed in 9 (6.67%) patients: in 8 cases – thrombosis of the popliteal segment, in 1 case – thrombosis of the femoral­popliteal segment. in 8 patients with thrombosis of the po pliteal segment, a minimally invasive thrombectomy method was used [14]: rheological throm­ boextration with the angiojet rheological thromboextraction device (possies, usa). in two cases rethrombosis of the reconstruction segment developed in the early postoperative period of the minimally invasive method of thrombectomy. thrombolytic and active conservative therapies were ineffective that led to amputation of the lower extremity. in 1 case thrombosis of the femoral-popliteal segment developed on the 6th day after two-level endovascular revascularization of atherosclerotic lesions of the femoral-distal arterial segment. in cases of acute arterial insufficiency of the stage iib, according to savelyev’s classification, open femoral­popliteal autovenous bypass and thrombectomy from the tibial segment were performed. the postoperative period was without complications. in two (1.48%) cases, both hybrid and endovascular methods of revascularization of the infrainguinal artery were not successful: after surgery, the preservation and progression of lower extremity ischemia was evidenced. intensive conservative therapy for 7-9 days did not stop development of ischemia that led to amputation of the lower limb. discussion at the moment, there are various opinions regarding the choice of method of revascularization of the infrainguinal arterial bed of the lower extremity: open, endovascular or hybrid [7]. the main purpose of revascularizing sur gical interventions is to use the optimal method of revascularization in multilevel atherosclerotic lesions of the femoral-distal arterial bed of the lower extremity and prevent development of a number of postoperative complications [8]. at the same time, many researchers point to development of postoperative complications, including thrombosis of the revascularization segment, the lack of effectiveness of surgery, which are more common when using endo vascular revascularization methods [9, 10]. in a number of studies on revascularization of the multilevel stenotic-occlusive process of the femoral-distal arterial bed in cases of stenotic-occlusive lesions of the tibial arteries, endovascular angioplasty of only one tibial artery was used [11, 12]. it is possible that in these circumstances revascularization of only one tibial artery cannot fully perform the tract outflow, which leads to development of throm­ bosis of the reconstruction segment. 52 su r g ic a l d is e a se s issn 2413-6077. ijmmr 2022 vol. 8 issue 1 the choice of the extent of surgery was based on the recommendations of the international classification tasc­ii (2007) and the european recommendation for the management of patients with the lesions of aortic/ iliac-femoral-popliteal arterial bed (2014). according to the european recommendation, the following types were singled out: type a – single stenosis or single occlusion of the fe moral segment up to 10 cm – 38 cases; type b – multiple lesions of the femoral segment, each of which <5 cm, single lesion <5 cm above the slit of the knee joint, any lesions in the absence of patency of the tibial arteries <5 cm, single stenosis of the popliteal artery – 17 cases; type c – multiple lesions of the femoral segment with a length of >15 cm, without or with calcification, restenosis or reocclusion after two endovascular interventions – 5 cases; type d – chronic ste­ notic-occlusive process of the femoral segment >20 cm with spread to the popliteal artery – 1 case. the criterion for inclusion in the study was the presence of multilevel atherosclerotic stenotic-occlusive lesion of the infrainguinal artery with underlying stenotic-occlusive lesion of the tibial arteries. atherosclerotic process in the tibial arteries had extensive, parietal character with segmental stenotic-occlusive lesions of the vascular lumen at the level of 2.8 to 4.3 cm. so, there was a need to form a functionally suitable outflow pathway in the tibial segment. this can be achieved by endovascular angioplasty of the two arteries of the tibia. in the study of the level of blood flow at the level of the popliteal artery the patients underwent endovascular revascularization of multilevel atherosclerotic stenotic-occlusive lesions of femoral-distal arterial bed with endovascular angioplasty of one or two tibial arteries. resistance index (ri) in the patients with endovascular angioplasty of one tibial artery was at the level of 0.88±0.09 – 0.86±0.10 unit, while ri in patients with endovascular angioplasty of two tibial arteries was 0.67±0.13 unit, which is 1.3 in times lower (p<0.05) than ri in endovascular angioplasty of one tibial artery. the results of the study have proved that endovascular angioplasty of the two tibial arteries produces satisfactory functional conditions of the outflow tract in the tibial segment. a similar level of incidence of postoperative complications was achieved due to the fact that in revascularization of multilevel atherosclerotic lesions of the femoral-distal arterial bed of the lower extremity by both hybrid and endovascular methods, mainly endovascular angioplasty of two tibial arteries was performed in 64.87% and 83.61% of cases, respectively. complications of the early postoperative period developed in those cases when endovascular angioplasty of only one of the tibial arteries was performed during revascularization of the infrainguinal artery. the analysis of the results of revascularization surgeries during the first month of the early postoperative period has proved that a high clinical level of effectiveness is achieved by hybrid and endovascular methods of revascularization of the stenotic-occlusive process of infrainguinal arterial bed in cases of atherosclerotic lesions of the tibial arteries, endovascular angioplasty of the two tibial arteries in 64.87% and 83.61% of cases, respectively. the preservation of the patency of the reconstruction segment as well as saving the lower extremity was obtained by hybrid methods and endovascular revascularization in 97.57% and 93.44% of cases, respectively. conclusions in most cases, endovascular angioplasty of two tibial arteries in hybrid and endovascular methods of revascularization of multilevel atherosclerotic process of the femoral-distal arterial segment of the lower extremity prevents development of thrombosis in tibial segment and allows maintaining the patency of the reconstruction segment and preservation of the lower extremity in 97.57% and 93.44% of cases, respectively. conflict of interests. authors declare no conflict of interests. acknowledgments this paper and the research behind it would not have been possible without the everyday great dedication and hard team work of the department of surgery of the municipal noncommercial enterprise “ternopil regional clinical hospital” of ternopil regional council. author’s contributions. ihor venher, sviatoslav kostiv, boryslav selskyi – concep tua­ lization, methodology, formal analysis, writing – original draft, writing – reviewing and editing; dymytriy hvalyboha, mykola orlov, ihor faryna, nadiia tsiupryk – data curation, writing – reviewing and editing, investigation, formal analysis. i. k. venher et al. 53 su r g ic a l d is e a se s issn 2413-6077. ijmmr 2022 vol. 8 issue 1 i. k. venher et al. ендоваскулярна ангіопластика при багаторівневому стенотично-оклюзивному процесі стегно-дистального артеріального русла в поєднанні з стенотично-оклюзивним процесом гомілкових артерій і. к. венгер, с. я. костів, б. п. сельський, д. в. хвалибога, м. п. орлов, і. в. фарина, н. і. цюприк тернопільський національний медичний університет імені і. я. горбачевського моз україни, тернопіль, україна вступ. у структурі серцево-судинних захворювань стенотично-оклюзійні ураження артерій нижніх кінцівок посідають друге місце, поступаючись лише ішемічній хворобі серця. хірургічне лікування хворих з атеросклеротичним ураженням інфраінгвінального артеріального сегмента у ряді випадків є складним завданням. у той же час реваскуляризуючі оперативні втручання на стегново-дистальному артеріальному руслі, особливо на гомілкових артеріях при хронічній критичній ішемії залишаються єдиним шансом уникнути ампутації нижньої кінцівки. у зв’язку з цим пошук і апробація оптимальних методів реваскуляризуючого хірургічного втручання на гомілкових артеріях спрямовані на ліквідацію стенотично-оклюзивного ураження артерій гомілки. мета. це дослідження спрямоване на популяризацію ендоваскулярних та гібридних хірургічних втручань при стенозно-оклюзійному процесі гомілкових артерій та вибір оптимальної тактики та об’єму лікування в цьому випадку. методи. проведено та проаналізовано результати 135 пацієнтів яким були проведені ендоваскулярні втручання на стегно-дистальному руслі в умовах стенотично-оклюзивного процесу гомілкових артерій на базі відділенні серцево-судинної хірургії комунального некомерційного під приємства «тернопільська обласна клінічна лікарня» тернопільської обласної ради. результати. у більшості випадків проведення ендоваскулярної ангіопластики двох гомілкових артерій при гібридних та ендоваскулярних методах реваскуляризації багаторівневих атеросклеротичних уражень стегно-дистального сегменту нижньої кінцівки, запобігає розвитку тромбозу гомілкового сегмента та дозволяє підтримувати прохідність сегменту реконструкції та збереження нижньої кінцівки у 97,57% та 93,44% випадків відповідно. висновки. проведення ендоваскулярної ангіопластики двох гомілкових артерій дає достовірно кращий результат, ніж ангіопластика однієї ізольованої гомілкової артерії і дає можливість зберегти прохідність сегменту реконструкції більше ніж на 90 відсотків при порівнянні з однією ізольованою ангіопластикою. ключові слова: судинна хірургія; ендоваскулярна ангіопластика; стенотично-оклюзивний процес; атеросклероз; реваскуляризація. information about the authors ihor k. venher, professor of the department of surgery no. 2, i. horbachevsky ternopil national medical university, ternopil, ukraine. https://orcid.org/0000­0003­0170­1995, e­mail: vengerik@tdmu.edu.ua sviatoslav ya. kostiv, professor of the department of surgery no. 2, i. horbachevsky ternopil national medical university, ternopil, ukraine. https://orcid.org/0000­0002­7963­5425, e­mail: kostivsj@tdmu.edu.ua boryslav p. selskyi, assistant professor of the department of surgery no. 2, i. horbachevsky ternopil national medical university, ternopil, ukraine. https://orcid.org/0000­0001­6787­4843, e­mail: selskyi_bp@tdmu.edu.ua dymytriy v. hvalyboha, assistant professor of the department of surgery no. 2, i. horbachevsky ternopil national medical university, ternopil, ukraine. https://orcid.org/0000­0003­0168­0848, e­mail: hvalyboga@tdmu.edu.ua mykola yu. orlov, phd student of the department of surgery no. 2, i. horbachevsky ternopil national medical university, ternopil, ukraine. https://orcid.org/0000­0002­3400­5243, e­mail: orlov_mypa@tdmu.edu.ua ihor v. faryna, phd student of the department of surgery no. 2, i. horbachevsky ternopil national medical university, ternopil, ukraine. https://orcid.org/0000­0002­1767­9721, e­mail: faryna_ivol@tdmu.edu.ua nadiia i. tsiupryk, phd student of the department of surgery no. 2, i. horbachevsky ternopil national medical university, ternopil, ukraine. https://orcid.org/0000­0002­3989­1563, e­mail: tsyupryk_cnad@tdmu.edu.ua 54 su r g ic a l d is e a se s issn 2413-6077. ijmmr 2022 vol. 8 issue 1 references 1. rollins k, jackson d, coughlin p. meta-analysis of contemporary shortand long-term mortality rates in patients diagnosed with critical leg ischemia. journal of vascular surgery. 2014;59:9. https://doi.org/10.1016/j.jvs.2013.11.010 2. neville r, lidsky m, capone a, babrowicz j, rahbar r, sidawy a. an expanded series of distal bypass using the distal vein patch technique to improve prosthetic graft performance in critical limb ischemia. european journal of vascular and endovascular surgery. 2012;44:177­2. https://doi.org/10.1016/j.ejvs.2012.04.014 3. raju r, mathur k, ayyappan m, vijayakumar j, venkatesan v, venkatachalapathy b. composite sequential bypass using profunda vein hitchhike. journal of vascular surgery. 2016;64:526­9. https://doi.org/10.1016/j.jvs.2016.04.050 4. nolan b, de martino r, stone d, schanzer a, goodney p, walsh d, cronenwett j. prior failed ipsilateral percutaneous endovascular intervention in patients with critical limb ischemia predicts poor outcome after lower extremity bypass. journal of vascular surgery 2011;54:730­6. https://doi.org/10.1016/j.jvs.2011.03.236 5. maurel b, lancelevee j, jacobi d, bleuet f, martinez r, lermusiaux p. endovascular treatment of external iliac artery stenoses for claudication with systematic stenting. annals of vascular surgery. 2009;23:722­8. https://doi.org/10.1016/j.avsg.2008.05.019 6. nakayama m, sakamoto f. proximal direct endarterectomy combined with simultaneous distal endovascular therapy for chronic full-length occlusion of the superficial femoral artery in elderly patients. asian journal of surgery. 2013;136:104­10. https://doi.org/10.1016/j.asjsur.2012.11.006 7. menard m, farber a. the best­cli trial: a multidisciplinary effort to assess whether surgical or endovascular therapy is better for patients with critical limb ischemia. seminars in vascular surgery. 2014;27:82­4. https://doi.org/10.1053/j.semvascsurg.2015. 01.003 8. motaganahalli r, menard m, koopman m, farber a. best endovascular versus best surgical therapy in patients with critical limb ischemia (best-cli) trial. vascular and endovascular review. 2020. https://doi.org/10.15420/ver.2019.12 9. hicks c, najafian a, farber a, menard m, malas m, black j, abularrage c below-knee endo vascular interventions have better outcomes compared to open bypass for patients with critical limb ischemia. vascular medicine. 2016;22:28­4. https://doi.org/10.1177/1358863x16676901 10. gentile f, lundberg g, hultgren r. outcome for endovascular and open procedures in infrapopliteal lesions for critical limb ischemia: registry based single center study. european journal of vascular and endovascular surgery. 2016;52:643­9. https://doi.org/10.1016/j.ejvs.2016.07.013 11. tan h, zhang l, guo q, yao y, sun s, wang t, li y, xiong k. “one-stop hybrid procedure” in the treatment of vascular injury of lower extremity. indian journal of surgery. 2013;77:75­8. https://doi.org/10.1007/s12262­013­0897­1 12. borgia f, di serafino l, sannino a et al. angiojet® rheolytic thrombectomy for acute superficial femoral artery stent or femoropopliteal by-pass thrombosis. monaldi archives for chest disease. 2015. https://doi.org/10.4081/monaldi.2010.271 13. kobza i, yarema y, zhuk r, fedoriv d. reconstructive operations on pedal arteries in the treat ment of critical limb ischemia. umj heart &vessels. 2018;1:37­9. [in ukrainian] https://doi.org/10.30978/hv2018137 received 29 may 2022; revised 10 june 2022; accepted 13 june 2022. this is open access article distributed under the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. i. k. venher et al. koncor1_2014.pm6 45 r a d ia t io n m e d ic in e a n d o n c o l o g y ijmmr 2015 vol. 1 no. 1 method of oncoplastic breast resection i. y. galaychuk i. ya. horbachevsky ternopil state medical university, ternopil, ukraine background. in women with breast cancer the significant postop lateral deviation of the nipple-areola complex may occur if the partial breast resection with lymphadenectomy was performed through one-line straight incision from the middle axilla to the edge of areola. objective. to describe radical oncoplastic breast resection with avoiding of nipple-areola deviation and preservation of the symmetry of breasts. methods. the study included 16 women, age 32 to 67 years. breast cancer at the stage i was found in 5 patients, stage iia – in 4, stage iib – in 2, and stage iiia – in 5 patients. we proposed to use curved (broken line) incision with 120° angle instead of linear incision. oncoplastic breast resection began with incision of skin and soft tissues in axilla, along the pectoral muscle downward to lateral contour of breast, and then direction was changed at an angle of 120° towards areola. the tumor projection was encircled by two semi-oval incisions, and then partial breast resection with axillary lymphadenectomy was done. after morphological confirmation of “clear” surgical margins the wound was sutured along the trajectory of the incision. results. all women rated the postop cosmetic result as “good” and “satisfactory”. the selected configuration of tissue incisions provided physiological position of the operated breast. conclusions. method of “broken line” incision of skin and soft tissue at an angle of 120° allows performing the radical breast resection with axillary lymph node dissection and prevents postoperative lateral deviation of nipple-areola, and maintains symmetry of the breasts. key words: breast cancer, oncoplastic resection. address for correspondence: igor y. galaychuk, medical department of oncology and radiology, i. ya. horbachevskyternopil state medical university, ternopil, ukraine m. voli, 1, ternopil, 46001, ukraine tel.: +380506975723; fax: 380352268648 e-mail: halaychuk@gmail.com introduction in ukraine, breast cancer of i-ii stages is diagnosed in 76.9 % of patients according to the national cancer registry [1]. thus we can expect increasing the likelihood of organ-sparing operations in patients with breast malignancies. in the world, breast-conserving surgery (bcs) with postoperative whole breast irradiation is generally accepted as a routine method for most patients with early-stage breast cancer [2]. the proportion of patients treated with bcs has increased from 37 % between 1990 and 1992 to 62 % between 2002 and 2004 [3]. nowadays 70 % of breast cancer patients are treated with bcs, 27 % with mastectomy alone, and 3 % – with mastectomy plus radiation [4]. bcs of early-stage cancer has been proven to be as equally effective as mastectomy in term of local control, distant disease, breast-cancer-specific and overall survival [3, 5]. bcs depends on the quadrant and distance from the nipple-areolar complex (nac). they may require an oncoplastic approach to avoid the nipple or cutaneoglandular retraction with aesthetic sequelae [6]. objective: to present a method of oncoplastic breast resection with avoiding of nipple-areola deviation and preservation of symmetry of breasts. methods the study included 16 women, age 32 to 67 years (mean 51.7±2.5 yr.). breast cancer at the first stage (t1n0m0) was found in 5 patients, stage iia (t2n0m0, t1n1m0) – in 4, stage iib (t2n1m0) – in 2, and stage iiia (t1n2m0) – in 5 patients. category t1 tumor was diagnosed in 12, and t2 – in 4 patients. in 11 patients the tumor was located in the upper outer quadrant, and in 5 cases – in the inferolateral quadrant. staging procedures included: clinical examination of breasts and regional lymph nodes, ultrasound of breasts and ways of lymphatic drainage, mammography, lung x-ray or ct scan, liver ultrasound, fine needle aspiration or core biopsy of tumor, blood analyses and ca-15.3. histological and biological evaluation results: tumor size, histologic type, grading, margin histology, receptors (er, pr, her2neu) and ki-67 status. the general scheme of treatment consisted of neoadjuvant chemotherapy, 2–3 cycles (6 patients) or chemoradiation therapy (10 patients). all patients underwent sectoral (or partial) resection of breast that included 2–3 cm of normal tissue surrounding i. y. galaychuk international journal of medicine and medical research 2015, volume 1, number 1, p. 45-48 copyright © 2015, tsmu, all rights reserved 46 r a d ia t io n m e d ic in e a n d o n c o l o g y ijmmr 2015 vol. 1 no. 1 the malignant tumor; also the overlying skin and underlying fascia had to be removed. these radical sectoral resections were performed with axillary lymph nodes dissection by using the suggested oncoplastic technique [7]. in postoperative period the correction of anticancer treatment was done with regard to molecular subtypes of breast cancer. patients’ quality of life was assessed by eortc scale qlq-c30 br23. oncoplastic breast resection. technical consideration. radical sectoral resection in breast cancer patients consists of removing of the outer breast sector with simultaneous axillary lymphadenectomy. cosmetic effects of such operations are not always satisfactory and mostly because resection of the breast and axillary lymph node dissection are performed on one-line tissue incision from the middle axilla to areola (fig. 1 – ac ). postoperative linear scar causes significant lateral deviation of the areola and nipple (fig. 1 – d). to avoid deviation and preserve symmetry, surgeon must perform the extra step operation – the centralization of nipple-areola complex (nac), i.e., medial transposition of the nac (fig. 1 – c1). we propose, instead of linear incision (ac), to apply the curved (broken line) incision with 120° angle between two lines ab and bc (fig. 1). because of this incisional geometry, when abc>ac, the postoperative sutures (or scars) have discordant power vectors that provide physiological position of the operated breast. clinical case. in patient n., 41 years old, the 10 mm tumor in upper quadrant of right breast was found by ultrasound and mammographic examination. fine needle aspiration biopsy confirmed moderately differentiated cancer. axillary lymph nodes were 18 mm with margin hyperplasia according to the ultrasound findings. diagnosis: cancer of the right breast ct1nxm0. treatment scheme: neoadjuvant chemotherapy (doxorubicin–cyclophosphamide, �2 cycles) and preoperative co60 external radiation therapy on breast and axilla (4.5-5.0 gy per fraction, 5 days, doses on axilla 34.2, on breast 38.2 gy equivalent); surgery – radical partial breast resection with axillary dissection; postoperative radiation therapy on the breast only (2.2 gy�10 days, total dose on breast 60.2 gy) and chemotherapy (cisplatin – paclitaxel). surgery. the operative designs made in upright position of patient marking tumor’s projection on skin with help of breast ultrasound. oncoplastic breast resection was started with incision of the skin and soft tissues in axilla along the pectoral muscle downward to the lateral contour of breast, and then changed direction at an angle of 120° towards areola (fig. 2). the tumor projection was embraced by two semi-oval incisions, and then partial breast resection with axillary lymphadenectomy was done (fig. 3). after morphological confirmation of “clear” margins in surgical specimen the wound was sewn along the trajectory of the incision (fig. 4). surgical specimen: a tumor size of 5�7 mm at a distance of 2.0 cm to the margins of resection, with adequate volume ratio “tumor – to – breast tissue”. the 11 lymph nodes ranging in size from 5 to 15 mm were present in axillary fat (fig. 5). histology: invasive ductal carcinoma, grade g2; lymph nodes with hyperplasia and angiomatosis; margin tissues histologically negative. immunohistochemistry: er–, pr–, her2fig. 1. geometry of incisions: a – axilla, c – nipple-areola complex (nac), b – lateral border of breast, d – direction of deviation, c1 – transposition of nac. fig. 2. clinical case: trajectory of “broken line” incision on axilla and breast. fig. 3. on-table result of radical breast’s resection with axillary lymphadenectomy. i. y. galaychuk 47 r a d ia t io n m e d ic in e a n d o n c o l o g y ijmmr 2015 vol. 1 no. 1 neu++. according to the clinical and morphological data, final diagnosis was determined as: cancer of the right breast pt1bn0m0g2, stage i, er/pr-, her2-neu++. in postoperative period wound healing was without any complication. patient completed treatment by radiation therapy on operated breast and systemic chemotherapy. two years follow-up with clinical examination, breast ultrasound (every 3 months) and annual x-ray mammography revealed no signs of relapse. outcome of oncoplastic surgery is satisfactory; breasts are located symmetrically with good cosmetic effect (fig. 6). results complications. swelling of the operated breast was observed in 11 (69.0 %) women, and it lasted up to 6 months; lymphedema of upper extremity was noted in 4 (25.0 %); seroma around postoperative suture was found in 7 (44.0 %) of patients; pain and stiffness in the shoulder joint occurred in 6 (38.0 %) women. the postoperative cosmetic result all patients rated as “good” and “satisfactory”. the selected configuration of tissue incisions provided a physiological position of the operated breast. during the 1-4 years of follow-up, 14 patients (88.0 %) were without evidence of recurrence; one patient died due to metastases into meningeal membranes; juxtaregional lymph node metastases developed in one patient after two years . the organ-sparing surgery was performed in cases of tumor placing at a short distance from the regional lymph nodes in the outer quadrants of the sufficient developed breasts, when the proportion “tumor-to-breast volume” was more than 1:75-100. it was noted that neoadjuvant anticancer chemoand radiation therapy reduced the volume of primary breast tumor and lymph nodes and by these enabled to do breast-conserving surgery. on the other side the organ-sparing surgery can be safely performed after neoadjuvant chemoand radiation therapy in patients with breast cancer. discussion breast-conservative surgery is considered today as equivalent, if not superior, to mastectomy in terms of breast-cancer-specific survival. as shown in agarwal et al. analysis of 132,149 women with earlystage invasive breast cancer (tumor �4 cm; �3 positive lymph nodes) that underwent bcs have a higher rate of disease-specific survival than those who undergo mastectomy. the 5-year breast cancer-specific survival rates of patients who underwent bcs, a mastectomy alone, or a mastectomy with radiation were 97 %, 94 %, and 90 %, respectively (p<0.001) [4]. fig. 4. completed of oncoplastic breast’s resection. fig. 5. surgical specimen (1 – cancer, 2 – breast tissue, 3 – axillary fat, 4 – axillary lymph nodes). fig. 6. cosmetic results 2-years after oncoplastic resection of right breast. i. y. galaychuk 48 r a d ia t io n m e d ic in e a n d o n c o l o g y ijmmr 2015 vol. 1 no. 1 the comparative study of 112,154 eligible women (t1/t2, stages i or ii disease) included 61,771 who received lumpectomy (bcs) and radiation, and 50,383 who had mastectomy without radiation was published recently [3]. the 51 % of women under the age of 40 underwent bcs compared with 59 % of women age 50 to 59 years and 51 % of women age 70 to 80 years. the group achieving greatest benefit in overall survival with bcs relative to mastectomy was women at the age of 50 and older who were diagnosed with hr-positive tumors. the smallest benefit was seen among women who were under age of 50 diagnosed with hr-positive tumors. kaplan-meier survival estimation showed significantly greater overall and breast cancer-specific survival favoring bcs over mastectomy [3]. in another prospective, randomized study of 749 elderly patients with tumor less than 25 mm and pn0, er+, g1-2, ki-67<20 %, concluded that whole breast irradiation after bcs can be omitted in selected patient. after 9 years of median follow-up no significant statistical difference was found regarding overall survival and distant disease free survival between both groups with wide resection of the breast tumor (bcs) and bcs plus 50 gy breast radiotherapy [5]. thus, these data and our findings provide confidence that bct remains an effective alternative to mastectomy for early stage disease regardless of age or hr status. conclusions method of “broken line” incision of skin and soft tissue at an angle of 120° allows performing radical breast resection with axillary lymph node dissection and prevents postoperative scar’s lateral deviation of nipple-areola, and maintains the symmetry of the breasts. suggested method of oncoplastic resection is advisable to apply in the case of lateral localization of tumor in the breast. organ-sparing surgery can be safely performed after neoadjuvant chemoand radiation therapy in patients with breast cancer. received: 2014.10.09. references 1. national cancer registry of ukraine. bulletin no. 14. cancer in ukraine, 2011-2012. kyiv; 2013. 2. veronesi u, stafyla v, luini a, veronesi p. breast cancer: from “maximum tolerable” to “minimum effective” treatment. front oncol 2012; 2: 125–130. 3. hwang es, lichtensztajn dy, gomez sl, fowble b, clarke ca. survival after lumpectomy and mastectomy for early stage invasive breast cancer. the effect of age and hormone receptor status. cancer 2013; 119: 1402–1411. 4. agarwal s, pappas l, neumayer l, kokeny k, agarwal j. effect of breast conservation therapy vs mastectomy on disease-specific survival for early-stage breast cancer. jama surg. available at http://archsurg. jamanetwork.com/article.aspx?articleid=1813803 accessed january 15, 2014. 5. tinterri c, gatzemeier w, costa a, gentilini ma, zanini v, regolo l. et al. breast-conservative surgery with and without radiotherapy in patients aged 55–75 years with early-stage breast cancer: a prospective, randomized, multicenter trial analysis after 108 months of median follow-up. ann surg oncol 2014; 21: 408–415. 6. fitoussi a, berry mg, couturaud b, salmon rj. oncoplastic and reconstructive surgery for breast cancer. the institute curie experience. – springer-verlag berlin heidelberg; 2009. 7. patent 86401u ua, rl?� 17/00. method of oncoplastic resection of mammary gland. galaychuk i.y. – . 2013 08870; appl. 15.07.13; publ. 25.12.13. bulletin . 24. i. y. galaychuk koncor1_2014.pm6 27 s u r g ic a l d is e a s e s ijmmr 2015 vol. 1 no. 1 application of transurethral microwave thermotherapy for patients with acute urinary retention and severe combined comorbidity from benign prostatic hyperplasia a. mysak, w. lamptey ternopil university hospital, i. ya. horbachevsky ternopil state medical university, ternopil, ukraine background. the incidence of benign prostatic hyperplasia (bph) has grown by almost 35 % over the last five years with an incidence rate of 4438.2 per 100 000 of the respective population (147 677 total patients) in ukrainian men beyond working age in 2007 [3]. objective. the aim of the study was to assess the efficacy of tumt in men where bph was complicated by aur and severe comorbidities. method. tumt was performed using the domestically produced equipment ‘almgp-01’ at the frequency of 1300 hz, rectal temperature of 42.5 °c, and urethral temperature of 44.5 °c. the average session duration is 46-55 minutes. results and discussion. subjective self-assessment: no effect of the procedure was detected (urination not restored) in 9 patients (11.11 %); poor current status with prior temporary improvement (6-9 months of independent urination with repeated aur) in 14 patients (17.28 %); satisfactory current status with occasional dysuric episodes (however better than pre-tumt status) in 40 patients (49.38 %). eight patients (22.22 %) have assessed their status as ‘good’ and ‘excellent’; one patient noted substantial improvement compared to pre-procedure status. the nine patients with lack of success of restoring physiological urination had the following peculiarities: intra-vesical growth of bph and prostatic volume over 85 cm3 in majority of these patients. conclusions. taking into consideration the minimally invasive nature, favourable tolerability and absence of adverse effects, tumt can be considered as a method of choice in patients with bph-triggered aur and contraindications to major surgical treatments and general anaesthesia. remote outcomes of tumt may be evaluated as satisfactory, with good effects in 71.62 % patients. however, in prostatic volumes exceeding 85 cm3 and pronounced intravesical pattern of bph growth the efficacy of tumt is arguable. key words: benign prostatic hyperplasia, acute urinary retention, transurethral microwave thermotherapy. address for correspondence: mysak andriy, ternopil university hospital, department of endoscopy, miniinvasive surgery, urology, orthopaedics and traumatology, str. chornovola 2/12, 46001, ternopil, ukraine, tel. +380672558899; e-mail: mysak.andriy@gmail.com introduction benign prostatic hyperplasia is a very common condition, found in middle-aged, senior and elderly males, according to epidemiological studies [1]. as for the age, the incidence of this disease is up to 85 % [2]. the incidence of benign prostatic hyperplasia (bph) has grown by almost 35 % over the last five years which is 4438,2 per 100000 of the respective population (147 677 patients) in 2007 in ukrainian men beyond working age [3]. the painand stress-free urination is known to be an essential component of optimal quality of life. however, there are moments when the perception of the quality of life undergoes a radical change. one of such moments is acute urinary retention (aur). occasionally, acute urinary retention occurs due to certain triggering factors, such a surgery under general anaesthesia, excessive fluid intake and medications with sympathomimetic or anticholinergic action [4]. however, the main or principal group consists of over 90% males in whom aur is a result of natural development of benign prostatic hyperplasia (bph) [5, 6]. european urological community identified the following risk factors of aur: age >70 years, prostatic volume >30 cm3, uroflowmetric results <12 ml/sec, ipss score >7 points, residual urine >50 ml and psa levels >1.5 ng/ml [7, 8]. it is generally accepted that the principal methods of aur relief in patients with bph are short-term bladder catheterisation, trocar-assisted or suprapubicepicystostomy, transurethral resection or open prostatectomy. however, there are situations when despite a disappointing effect of medical management, it is difficult to decide in favour of radical elimination of aur due to severe comorbidities. then the physician faces a difficult question how to solve this problem and which treatment modality is best to spare hold the patient harmless and restore spontaneous micturition. a. mysak et al. international journal of medicine and medical research 2015, volume 1, number 1, p. 27-29 copyright © 2015, tsmu, all rights reserved 28 s u r g ic a l d is e a s e s ijmmr 2015 vol. 1 no. 1 throughout the last decades, there has been a search for new therapeutic approaches in symptomatic bph [9]. for a technique to be recognized as effective it is essential for it to be less invasive and nevertheless effective, to require no general anaesthesia, to have an outpatient option and to have as few complications as possible. one of the urological technology achievements to meet the above criteria is transurethral microwave thermotherapy (tumt). the aim of the study was to assess the efficacy of tumt in men where bph was complicated by aur and severe comorbidities. methods the ternopil university hospital has purchased and is currently using (since 2002) a local microwave prostatic hyperthermia device (almgp-01), manufactured by the jsc radmir state enterprise company ndirv (kharkov). 516 tumt procedures have been performed in bph patients with high surgical risk at the urology department between years of 2002 and 2013. clinical analysis was performed in medical records of 81 (15.69 %) patients with aur-complicated bph. patient complaints were assessed using the ipss symptom scale, developed by the american urological association, including the quality of life question in dysuric patients (qol).the volume of the urinary bladder, the thickness of its walls, prostatic size and volume, as well as residual urine were assessed with transabdominal ultrasound. upper urinary tract function was assessed using radiological and radionuclide method; the levels of prostate specific antigen (psa) in all patients were within normal limits (up to 4 ng/ml). radical surgical techniques for bph could not be used due to severe concurrent comorbities in the patients. in the given investigation, twenty-nine patients were diagnosed with coronary artery disease (cad), diffuse and post-mi cardiosclerosis and class iia-iib heart failure; 34 patients had cad with complicated arrhythmias; 13 patients had residual post-cva findings; 32 patients had stage iii hypertension; 17 patients had chronic bronchitis, emphysema and stage ii respiratory failure; 8 patients had severe type 2 diabetes; 21 patients had varicose leg veins with stage ii–iii chronic venous failure; 9 patients had stage ii chronic kidney disease; 2 patients had ankylosing spondylitis (bechterew’s disease) and 3 patients had bilateral coxarthrosis. each of the tumt patients had three to five concurrent comorbidities. tumt was performed using equipment ‘almgp-01’ (kharkov, ukraine) at the frequency of 1300 hz, rectal temperature of 42.5 °c and urethral temperature of 44.5 °c. the average session duration was 46-55 minutes. according to ultrasound examination, the prostatic volume was within the range of 46 cm3 to 102 cm3, with a size of 74.5 cm3 on average. the patients had a foley catheter inserted repeatedly and were administered oral antibacterial drugs, usually fluoroquinolones or cephalosporins after completion of the tumt session. postoperative hospital stay was 4 to 7 days, 4.92 days on the average. three days prior to urethral catheter removal, �1a-adrenoblocker tamsulosin (omnic 0.4 mg/day) was used. the catheter was removed on week 4 post-tumt, after completed resorption of cellular necrosis. results most patients tolerated the tumt session favourably. only 5 patients (6.17 %) had a shortterm urethrorrhagia; 2 patients (2.46 %) had an acute pyelonephritis and 2 patients (2.46 %) had a single day urethral fever in post operative period. the patients had the following baseline preoperative findings: ipss 21.62±1.14 and qol: 4.42±0.36. the following results were obtained in analysis of these findings 9–12 months post-tumt: the ipss score decreased to 18.42±1.63 and the quality of life index (qol) decreased to 3.07±0.24. physiological urination was restored in 72 patients (88.89 %) after removal of the foley catheter. residual urine in the patients where urination was restored was between 15 and 145 ml, 48.25±18.36 ml on the average. psa levels were subsequently within normal limits. discussion subjective self-assessment of tumt efficacy by the aur patients was the following: no effect of the procedure was detected (urination not restored) in 9 patients (11.11 %); poor current status with prior temporary improvement (6–9 months of independent urination with repeated aur) in 14 patients (17.28 %); satisfactory current status with occasional dysuric episodes (however better than pre-tumt status) in 40 patients (49.38 %). eight patients (22.22 %) have assessed their status as ‘good’ and ‘excellent’; one patient note substantial improvement compared to pre-procedure status. the nine patients with lack of success restoring physiological urination had the following peculiarities: intra-vesical growth of bph and prostatic volume over 85 cm3 in majority of these patients. within a year’s span, post-tumt surgical treatment was undertaken in 8 patients with lack of independent urination (2 patients had transvesical prostatectomy, 4 patients had turp and 2 more patients had suprapubic cystostomy placed). one patient refused surgical treatment and was on an indwelling catheter for 3 years already. concerning the temporary improvement population, 11 patients a. mysak et al. 29 s u r g ic a l d is e a s e s ijmmr 2015 vol. 1 no. 1 had surgeries (4 patients had transvesical prostatectomy, 6 patients had turp and 1 more patient had suprapubic trocar cystostomy placed). three patients of this group had repeated tum session after recurrent aur with independent urination successfully restored. it is noteworthy that open posttumt prostatectomies were associated with difficulty enucleating hyperplastic nodules due to proliferative changes in the nodes and the adjacent tissues. another peculiar observation was that post-tumt open prostatectomies, as well as post-tumt turp procedures, were associated with less pronounced and storter haemorrhage. conclusions taking into consideration the minimally invasive nature, favourable tolerability and absence of adverse effects, tumt can be considered a method of choice in patients with bph-triggered aur and contraindications to major surgical treatments and general anaesthesia. remote outcomes of tumt may be evaluated as satisfactory, with good effects in 71.62 % patients. however, the efficacy of tumt is arguable in prostatic volumes exceeding 85 cm3 and pronounced intravesical pattern of bph growth. references 1. возіанов оф, пасєчніков сп, сайдакова но прогнозування результатів застосування тамсулозину для відновлення сечовипускання у хворих на доброякісну гіперплазію prostate, ускладнену гострою затримкою сечі. здоровье мужчини 2008; 3: 118–123. 2. иремашвили вв острая задержка моче& испускания у мужчин. здоровье мужчины 2007; 4(23): 126–129. 3. переверзев ас острая задержка мочи – неотложное клиническое состояние матеріали все& української науково&практичної конференції. чернівці 2007; 127–129. 4. сайдакова но, старцева лм основні показники урологічної допомоги в україні за 2006–2007 рік (відомче видання) к.; 2008. received: 2014.04.06 5. aua practice guidelines committee. aua guideline on management of benign prostatic hyperplasia (2003). chapter 1: diagnosis and treatment recommendations. j urol 2003; 30–47. 6. elhilali m, vallancien g, emberton m. et al. management of acute urinary retention (aur) in patients with bph. a worldwide comparison. j urol 2004; 171: 407–444. 7. emberton m, anson k. acute urinary retention in men: an age old problem. bmj 1999; 921–925. 8. fitzpatrick jm, kirby rs management of acute urinary retention. bju int 2006; 97 (suppl 2): 16–20. a. mysak et al. 18 su r g ic a l d is e a se s issn 2413-6077. ijmmr 2016 vol. 2 issue 2 doi 10.11603/ijmmr.2413-6077.2016.2.7035 embossed autodermotransplants: theoretical substantiation and practical usage v. v. koptiukh, i. y. galaychuk i. horbachevsky ternopil state medical university background. it is established that granulation wound has uneven bumpy surface and modern electric dermatomes cut skin with a smooth internal surface. therefore there is no tightly contact between the smooth surface of a graft and granulation tissue. objective. the method of harvesting by split-skin grafts with variable thickness (embossed) is proposed. methods. an embossed skin graft with sinusoidal surface of inner side was obtained by modified dermatome with special technological plate. the embossed autografting of skin was applied in 16 patients (21–60 years old) with deep burns of 7–9% of body. for clinical comparison the part of granulating wounds in each patient was covered with embossed transplants and the other part with smooth split-thickness grafts. results. during ultrasound doppler examination of grafts on the fifth postop day the active blood flow in embossed transplants with 4–6 blood outbreaks and only 2–3 flashes in smooth graft were found. clinically the embossed transplants were engraftment on 6–7th day. on 18–21st day the roughness of grafts surface disappeared. in two months the graft together with subcutaneous fatty tissue was taken in crease. donor wound: on 14–15th day its surface was like “slate” but on 25–30th day the undulation of the re-epithelisation skin disappeared. conclusions. the improvement of dermatome design with technological plate allows cutting scalloped split-skin grafts of varied thickness (embossed). split-thickness embossed grafts combine anatomic and physiological features of skin cover granulation wound to adhere tightly because of the enlargement of area of their internal surface without retraction and/or secondary shrinkage. donor wounds, after the harvesting of embossed skin grafts, contain the morphological elements of dermis which contribute to their complete spontaneous healing. key words: embossed split-thickness skin graft, granulating wound. international journal of medicine and medical research 2016, volume 2, issue 2, p. 18-21 copyright © 2016, tsmu, all rights reserved introduction skin transplantation is a method of choice in the treatment of patients with deep burns. in order to do this procedure, split-thickness skin grafts are harvested from donor areas. the donor wound regrows epithelium within two weeks [1–3, 5]. sometimes there are complications with transplant skin engraftment to a granulating wound, sometimes areas of donor wounds suppurate [4, 6, 7, 11]. important aspect of skin graft healing is wound contraction that may present serious functional and cosmetic concerns, depending on the location and severity. myofibroblasts may cause contraction. the ability of a skin graft to resist contraction is related to the thickness of deep dermal component included in the graft, not just the absolute thickness of the graft. this deep dermal component is able to suppress myofibroblast function. the severity of wound contraction from the least to the most is as follows: full­thickness skin graft cause least contraction, then thick and thin split-thickness skin graft, and finally the open wound has the most contraction [14]. modern electric dermatomes cut skin with a smooth internal surface, in the meantime granulation wound has uneven bumpy surface [1, 12]. in certain cases there is no close contact between the smooth surface of a skin graft and granulation tissue; there are gaps and voids, where discharge from the wound collects, colonies of microorganisms form, which may cause hematomas or seromas and local inflam­ matory complications [4, 8, 9, 11]. thus, for successful healing by a skin graft without contraction of the wound, it is necessary to have the surface adapted to the granulating corresponding author: igor galaychuk, department of oncology, radiology diagnostics and therapy and radiation medicine, i. horbachevsky ternopil state medical university, 1 maidan voli, ternopil, ukraine, 46001 phone number: +380506975723 e-mail: halaychuk@gmail.com v. v. koptiukh et al. 19 su r g ic a l d is e a se s issn 2413-6077. ijmmr 2016 vol. 2 issue 2 wound surface and contained with the elements of dermis. the question we are aiming to address is to find out if it is possible to cut skin graft with an uneven embossed surface, similar to the relief of granulations. the study is aimed to introduce a method of harvesting the split-skin grafts of variable thickness. material and methods theoretical consideration. the upper boundary of granulation tissue on the tentative vertical cut of the wound is a sinusoid with smaller or larger range, depending on which granulations (small-, medium-, or largegrained) fill the wound. theoretically these granulations should be covered with analogic sinusoidal surface (fig. 1). obviously, the larger is the surface of granulations contact with the graft, the faster graft retention will be. optimal configuration of internal surface of skin graft with spacing between ledges of π/2, π/4, π/8 mm (equals to 1.57 mm, 0.78 mm and 0.39 mm) is proved mathematically. technical solution. to obtain split-skin grafts with variable thickness (embossed) it is sug gested to add a so called technological plate with embossed surface [10]. a technological plate is made of plastic mass or metal and consists of a base and shaped elements on it which have ledges of rectangular or other geometric figure shapes (fig. 2). method of embossed skin graft cutting. instruments and donor area do not differ from regular grafting. the technological plate is fixed to the base of the dermatome. the embossed surface of the plate should adjoin the skin of donor area tightly. technological plate should be chosen according to the size of granulation on the wound: small­, medium­ and large­ grained. fig. 3 shows embossed skin graft harvest of the variable thickness and donor wound with sinusoidal surface. an embossed skin graft has sinusoidal surface from the inner side, which considerably enlarges area of its contact with granulations on the wound (fig. 4). e.g., the inner surface is embossed skin graft with 0.78 mm space between ledges is larger by 29.3% if compared to a smooth graft. full-scale adhesion to wound surface contributes to a quick engraftment of dermal transplant and minimizes the local postoperative comp lications. the epithelisation of donor wound is not affected because all skin appendages are preserved. clinical application. the embossed autografting of skin was applied for 16 patients with deep burns of 7–9% of the body. the patients aged 21–60 years, 13 mail (81.0%), and 3 female (19.0%). part of granulating wounds in each patient was covered with embossed transplants, and part of them with smooth splitthickness grafts. thus it was possible to conduct a clinical comparison of survival of two types of grafts. next day after the surgery the surface of the embossed skin grafts was of different colours — pale and cyanotic lines along the grafts. on the second or third day cyanosis fig. 1. scheme of sinusoidal surface of granulation wound with the sinusoid length of π/2 mm (medium grained granulation tissue). fig. 2. the scheme of cutting edge of the dermatome: 1 — blade, 2 — protective plate of the dermatome, 3 — splitthickness embossed skin graft, 4 — technological plate. fig. 3. scheme of split-thickness embossed skin graft cutting: а — embossed skin graft, в — donor wound. fig. 4. scheme of covering the granulation wound with embossed skin graft (а — skin graft, в — granulation wound). v. v. koptiukh et al. 20 su r g ic a l d is e a se s issn 2413-6077. ijmmr 2016 vol. 2 issue 2 decreased, and on 4–5th days the colour of the embossed grafts became even. clinically the embossed transplants were engrafted on 6–7th day. on 18–21st day the roughness of grafts surface disappeared. in two months the graft together with subcutaneous fatty tissue was taken in the crease. donor wound: on 14–15th day its surface was like “slate”, but on 25–30th day the undulation of the re-epithelized skin disappeared. ultrasound doppler (siemens g60s) examination of grafts was performed on the 5th day. active blood flow was found in some parts of the embossed transplants, there were on average 4–6 blood outbreaks on the length of the linear sensor (l10-5; 7.5–10.0 mhz), but in smooth graft, which was still swelling, there were only 2–3 flashes. therefore, variable thickness skin graft integrated with granulation tissue faster. thus, our clinical study proved the use of embossed skin grafts in closing the granulating wounds. results and discussion the embossed epidermo-dermal transplants with variable thickness contain morphological elements of dermis which the thin smooth split-thickness skin grafts do not have. the thin part of the sinusoidal graft is represented by the epidermis; the thick part contains papillary and reticular dermis. so there are all structures of the skin: loose and dense connective tissue containing blood and lymphatic capillaries and vessels, elastic and collagen fibres, reticular layer with fibroblasts, nerve endings, and epidermal appendages in the gel-like ground substance. the same components are also in donor sites and play a crucial role in its re-epithelialization. that is why after placement of sinusoidal graft, its initial adherence to the wound bed via a fibrin binding is favourable if compared to the straight skin graft. it is established that the smooth split skin grafts shrink along fibres on cleavage lines and in transverse directions after cutting. moreover, shrinkage along the cleavage lines is almost twice as much as transverse shrinkage. convolution of split-thickness skin grafts to internal surfaces is its characteristic feature [1, 6]. the shrinkage of skin grafts increases with the increase of its thickness. thin transplants of 0.1–0.4 mm shrink minimally, transplants of medium thickness (0.5–0.6 mm) shrink by 15– 25%, thick (0.7–1.0 mm) — up to 50%, full-layer (>1.1 mm) — even more. moreover, longitudinal and lateral components of shrinkage differ in 1.3–1.6 times [1, 3, 4, 12, 14]. because of the embossed surface the shrinkage of epidermo-dermal transplant decreases considerably and the preserved morphological structure of dermis contributes to fast healing of burn wound as well as regeneration of functions of the transplanted skin. the choice of skin grafts thickness is very important for successful autodermoplastic surgeries. autodermoplastics with thin grafts have several advantages: the transplant survives for an average of 3–4 days, and the donor wound epithelizes for 12–14 days. however, this method has its deficiencies as well. secondary shrinkage of the transplant occurs with usage of thin split grafts for wound covering; besides fatty tissue does not grow under this type of transplant [6, 8, 13]. usage of full-thickness skin grafts has distinct advantages. they withstand mechanical loads, full fatty tissue grows under them, thanks to the renewed skin integument, which receives the functional and dynamic features. long term retraction and retention of full-layer grafts are significant disadvantages. they suppurate and are rejected more often. the usage of such grafts creates problems for the treatment of a do nor wound, which has to be covered with local tissues or autodermotransplants [1, 7, 9, 12, 14]. fig. 5. embossed split-thickness skin graft (clinical example). fig. 6. fragment of sinusoidal surface of donor wound. v. v. koptiukh et al. 21 su r g ic a l d is e a se s issn 2413-6077. ijmmr 2016 vol. 2 issue 2 epidermis of various topographical anatomical body areas is different in terms of relief, color and thickness (0.5–0.2 mm). human skin thickness varies from 0.4 up to 4.0 mm. thickness of the skin’s dermal layer varies from 0.35 up to 4.0 mm and is a significant resource for harvesting split flaps [1, 14]. opposite to the smooth split grafts, embossed dermotransplants with variable thickness retain anatomical and physiological features of skin in monofunctional structural integrity; herewith there is no substantial trans plant shrinkage. the area of contact of transplant and wound increases because of the positioning of surface granulations, which hasten the process of its engraftment. conclusions the improvement of dermatome design with technological plate allows cutting scalloped split-skin grafts of varied thickness (embossed). split-thickness embossed grafts combine anatomic and physiological features of skin cover granulation wound to adhere tightly because of the enlargement of area of their internal surface without retraction and/or secondary shrinkage. donor wounds, after the harvesting with the embossed skin grafts, contain the morphological elements of dermis which contributes to their complete spontaneous healing. references 1. ratner d. skin grafting. from here to there. dermatol clin 1998; 16(1): 75–90. 2. white n, hettiaratchy s, papini rp. the choice of split­thickness skin graft donor site: patients’ and surgeons’ preferences. plast reconstr surg 2003; 112 (3): 933–934. 3. mimoun m, chaouat m, picovski d, et al. the scalp is an advantageous donor site for thin-skin grafts: a report on 945 harvested samples. plast reconstr surg 2006; 118 (2): 369–373. 4. unal s, ersoz g, demirkan f, et al. analysis of skin­graft loss due to infection: infection­related graft loss. ann plast surg 2005; 55 (1): 102–106. 5. demirtas y, yagmur c, soylemez f, ozturk n, demir a. management of split-thickness skin graft donor site: a prospective clinical trial for comparison of five different dressing materials. burns 2010; 36 (7): 999–1005. 6. rudolph r. the effect of skin graft preparation on wound contraction. surg gynecol obstet 1976; 142 (1): 49–56. 7. zuidam jm, coert jh, hofer so. closure of the donor site of the free radial forearm flap: a comparison of full-thickness graft and split-thickness skin graft. ann plast surg 2005; 55 (6): 612–616. 8. terrill pj, goh rc, bailey mj. split-thickness skin graft donor sites: a comparative study of two absorbent dressings. j wound care 2007; 16 (10): 433–438. 9. liu j, li y, rong x, lin w, zhang t, wang b, et al. application of crystalline cellulose membrane (veloderm) on split-thickness skin graft donor sites in burn or reconstructive plastic surgery patients. j burn care res 2013; 34 (3): e176–e182. 10. patent ua 69020. ipc а61в 17 /322 (2006.01). dermatome for cutting quasithick autodermograft / v.v. koptiukh (ua). no. u201108642; appl. 11.07.2011. publ. 24.04.2012. bulletin № 8. 11. world union wound healing societies (wuwhs). wound infection in clinical practice – an international consensus. international wound journal 2008; 5 (3): 1–4. 12. northern sydney central coast health (nscch.) skin graft management guidelines [internet]. sydney: nscch; sep, 2008. [cited 2013 dec 12]. available from: http://web.archive.org/web/ 20130420112308/http:/www.nscchealth.nsw.gov.au/ services/wound.care/final-skingraftma na gementdraft.pdf. 13. brolmann fe, eskes am, goslings jc, niessen fb, de bree r, vahl ac, et al. randomized clinical trial of donor-site wound dressings after split-skin grafting. br j surg 2013; 100 (5): 619–627. 14. wax mk, meyers ad. split-thickness skin grafts. [updated: mar 18, 2015]. available from: http://emedicine.medscape.com/article/876290. received: 2016-10-08 v. v. koptiukh et al. koncor1_2014.pm6 17 p e d ia t r ic s ijmmr 2015 vol. 1 no. 1 clinical and laboratory diagnostic criteria of immune resistance of healthy and sick with pneumonia children of different age o. fedortsiv, n. haliyash, s. nykytyuk i. ya. horbachevsky ternopil state medical university, ternopil, ukraine background. the universality of the general adaptation syndrome and the need to study the impact of general adaptive responses on the occurrence and consequences of pathological processes, as well as detection of early signs of disadaptation, have been proven by numerous works. objective. the aim of study was to explore the peculiarities of general non-specific adaptive reactions in pediatric population and to discuss the diagnostics effectiveness of general nonspecific adaptive reactions in early detection of health disorders in children of different age. methods. the paper presents the results of laboratory and clinical tests, conducted in 185 healthy children age 4-12 years and 42 children of the same age with community-acquired pneumonia. the characteristics and prevalence of general nonspecific adaptative reactions in healthy children of different age were given. determination of adverse adaptative reactions (stress, training and overactivation) based on analysis of the general blood analysis is an early diagnostic criterion of individual health reduction of children. results. the features of clinical course, the nature and direction of immune disorders of children with community-acquired pneumonia, depending on their adaptive systems, were revealed. the influence of conventional treatment regimens of children with pneumonia on the state of adaptation processes was determined. clinical diagnostic criteria for prognosis and treatment efficacy based on diagnostics of nonspecific adaptive responses in the dynamic of pneumonia were established. the proportion of morphometrically different blood lymphocytes in healthy children was determined. it was revealed that the blood of preschool age children dominated with type i cells (61.3±1.7) %. the relative content of lymphocytes (morphological type ii) (28.7±1.6) %, type iii – (6.1±0.6)%, type iv – (3.9±0.6)% was observed. conclusions. general nonspecific adaptative reactions of mild and increased activation, as the most conducive to children’s age, demonstrate a balance of the relative content of different types of lymphocytes according to morphometrical data. key words: children, cell-mediated immunity, adaptive reactions, community-acquired pneumonia. introduction currently, it is not possible to assess the health of the child without taking into account the processes of adaptation to the external environmental factors, indicators of physical and mental health of an individual [1]. the universality of the general adaptation syndrome and the need to study the impact of general adaptive responses on the occurrence and consequences of pathological processes, as well as detection of early signs of disadaptation have been proven by numerous works [2, 3, 4]. according to the previous studies, poor adaptation processes are the background for the implementation of specific pathogenic factors [5, 6]. resistance and adaptation are provided with blood components, which serve as a clinical indicator of the organism status, provide immune supervision and act as effectors at various adaptive-trophic effects [7]. lymphocytes represented by a diverse in form and functions cell population are recognized as central cells of the immune system. damaging factor may cause activation or depression of the immune system that necessarily is reflected in the number and structure of lymphocytes. the objective of the study is to explore the peculiarities of general non-specific adaptive reactions in pediatric population and to discuss the diagnostics effectiveness of general nonspecific adaptive reactions in early detection of health disorders in children of different age and to improve the treatment control and prognosis depending on the immune resistance in case of communityacquired pneumonia. methods 185 healthy children age four to twelve years were examined. the sample was formed on the basis of pre-schools and secondary schools in ternopil and ternopil region. only children without congenital malformations or chronic disease, during the physical well-being and not less than three weeks after the last acute illness or vaccination were involved into the research. the general totality was formed by probabilistic method (during the general dispensary address for correspondence: nataliya haliyash, department of pediatrics � 2, i. ya. horbachevsky ternopil state medical university, m. voli, 1, ternopil, 46001, ukraine tel.: +380979215292; fax: +380352524183; e-mail: nhaliyash@tdmu.edu.ua, nhaliyash@yahoo.com o. fedortsiv et al. international journal of medicine and medical research 2015, volume 1, number 1, p. 17-20 copyright © 2015, tsmu, all rights reserved 18 p e d ia t r ic s ijmmr 2015 vol. 1 no. 1 examinations), thus excluding the element of subjectivity. distribution of surveyed children by age and sex is shown in fig. 1. type of the general nonspecific adaptive reactions (gnar) was determined by the indexes of lymphocytes as white blood count (wbc). also, the study involved 42 children of the relevant age with community-acquired pneumonia who were treated in the pulmonological department of ternopil regional children’s hospital (trchh). all patients in the hospital underwent clinical, laboratory and instrumental examination in accordance with approved protocols of treatment in the specialty “children’s pulmonology” (ministry of health of ukraine � 18 from 13.01.2005). in all cases the diagnosis of pneumonia was confirmed radiographically. the determination of gnar was done by analyzing the correlation of different types of leukocytes in wbc [2]. the character of gnar (harmonious or strained) was determined on the basis of adaptation index, calculated as the ratio of relative values of lymphocytes to segmented neutrophils. the level of gnar reactivity (low, medium, high) was determined by the presence and severity of strain signs at wbc. these strain signs include an increase or decrease in the number of white blood cells, the relative levels of band neutrophils, eosinophils, monocytes, and the emergence of young and immature forms of leukocytes. as a marker of the state of perfect health in children, we accepted gnar of increased activation at a high level of reactivity. studies of the cellular immunity was conducted by determining the number of lymphocytes and their subpopulations (cd3+, cd4+, cd8+, cd16+, cd22+) in venous blood by indirect immunofluorescence using monoclonal antibodies (“granum”, kharkiv). indicators of endogenous intoxication were determined by colorimetric method over the serum content of molecules of medium mass (mmm) using the technique of gabrielian, n.i. and levitsky, e.r. (1984). statistical processing of the research results was performed by parametric analysis with the calculation of student’s t-test using the software package “microsoft excel 5.0”. results we have found that among children of preschools and secondary schools of the ternopil region and ternopil only 41 children were healthy (22.2 %) (1st group). in 144 children (77.8 %), who were considered healthy as well (2nd group), some insignificant deviations were found, including functional minor pathology of the respiratory system (61.6 %) and digestive system (42.7 %) at leading positions (fig. 2). the gnar values of increased activation (51.3 %) and over-activation (25.2 %) in preschool children were detected. we have revealed reactions of mild activation (11.3 %) and training (12.2 %) with somewhat less frequency in the same group of children. in children of the 1st group, the reaction of high activation predominated (64.7 %) (fig. 3). in the group of younger pupils the gnar of mild and increased activation (29.4 % and 38.2 % respectively) were predominant. simultaneoulsy, gnar of mild activation (50.2 %) was registered in the majority of the 1st group children. in the age group 10-12 years, the same gnar (high activation (39.0 %), mild activation (25.0 %), and training (25.0 %)) were identified; in the 1st group the prevailing finding was gnar of training (45.4 %) (fig. 3). it was found that general nonspecific adaptive reactions differ in the parameters of the immune response based on the subpopulations of lymphocytes results. thus, the gnar of stress and over-activation are characterized by a low content of cd3+ ((37.5±0.3) % and (42.0±1.3) %, respectively) and cd8+ lymphocytes ((11.7±0.2) % and (12.8±0.5) %, respectively), showing a pronounced tendency to immunosuppression. the reactions of mild and increased activation and training are characterized by minor changes of immune cells quantitative indicators that testify to active functioning of all components of the immune system. among the clinical peculiarities of children hospitalized in the pulmonary department of trchh, we detected prevailing focal (91.3 %) forms of community-acquired pneumonia that coincides with the literature data. the prevalence of bilateral forms of community-acquired pneumonia (in 69.0 %) was determined that differs from the reference data fig. 1. distribution of surveyed children by age and gender. � ����������� �� � �� �������� � �� � �� ���������� � �� ���� �������� � ���� � �� ��� �� � ����������� �� � � �� �������� �� ��� ������ �� ���� o. fedortsiv et al. 19 p e d ia t r ic s ijmmr 2015 vol. 1 no. 1 �� ��� ��� ��� ��� �� �� ��� ��� �� �� �� �� ��� �� ��� ���� ���� ��� ��� ����� �������� � ������� � � �������� � ���������������� ������ �������������� ���������������� ��������� ���� � fig. 3. types of general nonspecific adaptive reactions in surveyed children from child care institutions of ternopil region. testifying the predominance of unilateral pulmonary inflammatory processes in childhood. among all analyzed cases, uncomplicated and complicated forms of community-acquired pneumonia were recorded (52.6 % and 58.3 %, respectively). the most commonly observed disorder that complicated the disease was obstructive syndrome (recorded in 26.2 % of cases), with a tendency to increasing the frequency of this complication with age. allergic history of these patients was not burdened. the most common symptoms of community-acquired pneumonia were cough (95 %), mostly wet and unproductive (50.0 %), respiratory failure events (73,9-78,9 %); objectively dull sound on the lungs percussion in the projection of the inflammatory focus (85.7 %), fine rales (31.0 %) and small-vesicles rhonchi (38.1 %) on auscultation. detection of general nonspecific adaptive reactions in patients with community-acquired pneumonia at baseline showed that the gnar of stress (42.9±1.2) % and training (35.7±1.1) % were recorded more often (table 1). consecutive determination of gnar was conducted on the 710th day of hospitalization. mean values of endogenous intoxication indices in a group of healthy children were within reference values. the increase of mmm1 and mmm2 was stated at gnar of stress, training and overactivation; they were significantly lower at activation reaction. in children with community-acquired pneumonia the average values of mmm1 and mmm2 increased by 1.5 and 1.3 times, respectively, in comparison to healthy subjects. discussion it was found that general nonspecific adaptive reactions differ regarding parameters of the immune response according to the subpopulations of lymphocytes. thus, the gnar of stress and overactivation are characterized by a low content of cd3+ (37.5±0.3 % and 42.0±1.3 %, respectively) and cd8+ lymphocytes (11.7±0.2 % and 12.8±0.5 %, respectively), showing a pronounced tendency to immunosuppression. the reactions of mild and increased activation and training are characterized by minor changes of immune cells quantitative indicators that testify to active functioning of all components of the immune system. in children with community-acquired pneumonia gnar of stress and training prevailed, that was accompanied by insufficiency of t-cell immunity and increased levels of endogenous intoxication. the fig. 2. minor deviations found in apparently healthy surveyed children ����� ����� ������ ������ �� �� ������� ���� ������ ������ ����� ��� �� ��� �� ����� ����� ��� �� ������ ��� �� ������ ������ ����� ������ ������ ���� ���� �� � �� �� � �� � �� � �� ��������������� ��������� ��������� � ���������� ��������� � ���������� � ����� �� ������ ������ �������������� � ����� ����� ��� ���������� ������� ��� ������ ��� ��� ��� ��� ������ �� ���� ���������� �� � ��� ������������ ���� � ������ �� � ������ � o. fedortsiv et al. 20 p e d ia t r ic s ijmmr 2015 vol. 1 no. 1 reactions of stress and training differ from other adaptive reactions by low levels of all subpopulations of lymphocytes. these changes can be interpreted as immunosuppression. we have identified the peculiarities of gnar types changing in children with community-acquired pneumonia under the influence of treatment. in particular, there was a reduction in the number of patients with reactions of stress to 7.1 % and training to 23.8 % and increased the frequency of gnar of mild and increased activation (in 38.1 % and 23.8 % of children, respectively) to the 10th day of hospitalization, which indicates an improvement of adaptive processes and satisfactory mobilization of reserve capacity of the organism. however, we observed the prolonged retention of reactions unfavorable to recovery in 7 patients (16.7 %). devolution on a scale of adaptation reactions was revealed in 2 children (4.8 %) indicating a reduction in the activity of adaptive processes. the detection of gnar of stress and training by 7–10 day of treatment, even against the normalization of clinical data, may be regarded as criteria of the bronchopulmonary disease treatment inefficiency. recovery of these patients should not be considered complete. prolonged retention of the reaction of training, according to the theory of adaptation reactions, creates conditions for chronic pathology. table 1. the frequency of gnar in children with community-acquired pneumonia �������� ��� � �� ������������� ��� � �� ������������� �������� � � � ������� � ������� �������� ��� �� ������ !� "���#�$�%� ��� &�� �'� !'�"������ �#� �!�����#�� ( ������ ��� � � '� ��� �#������ �$� !��������%� ) ����������� ��� � � !� "���#�$���� �#� �!�����#���%� *���+��� ��� � � �� �� �#� ���� !� "���#�$���� notes: 1 – statistically significant difference (p<0.05) if compared with stress; 2 – statistically significant difference (p<0.05) if compared with reaction of training; 4 – statistically significant difference (p<0.05) if compared with increased activation; 5 – statistically significant difference (p<0.05) if compared with over-activation; * – statistically significant difference (p<0.05) in the treatment process. conclusions 1. considering the level of adaptability and resistance of the organism among children surveyed, the condition of perfect health was diagnosed in 13.5 %, the average health – in 58.4 %, the state of preexisting diseases – in 27.6 %, and the state of the vulnerability to the pathological process – in 0.5 %. 2. it was proved that the most common symptoms of community-acquired pneumonia in children age 4–12 years were cough, mostly wet and unproductive, dyspnea, dull sound on percussion in the projection of the inflammatory focus in the lungs, and fine rales on auscultation. majority of patients had progressing pathological process occurred despite the reduction of immune resistance that was evidenced by gnar of stress and training, insufficiency of t-cell immunity and increased parameters of endogenous intoxication. 3. the process of recovery with effective treatment occurs against the transition of gnar of stress into anti-stress reactions of mild and increased activation of a high level of reactivity through reaction of training. patients with adverse gnar types (stress, over-activation) are of high risk of a protracted disease course. detection of the gnar type allows to identify the risk group of unfavorable community acquired pneumonia course by identifying adaptive reactions of stress and training or over-activation development. references 1. danylenko gm, podrihalo lv, shashkevych sa effect of social hygiene and psychosocial factors on the internal school environment and the functional state of elementary school students. bulletin of hygiene and epidemiology 2008; 12(1): 114–118. 2. garkavi lh, kvakina eb, kuz’menko ts. antistress reactions and activation therapy. moscow: publishing house “b,"�� ”; 1998: 654. 3. maikova tv. stress as a factor of the development of combined pathology of the digestive system. contemporary gastroenterology 2004; 4 (18): 49–53. 4. reshchikov va. features of the 4–6 years children health in industrial region today. ukrainian morphological almanac, 2007; 3: 123–124. 5. podgorna nv. description of the adaptive processes and their correction in children with gastroduodenal pathology. modern pediatrics, 2008; 5: 117–121. 6. lychkovska ol, kryvko yuya. the features of nonspecific adaptive reactions in children with gastroduodenal pathology. experimental and clinical physiology and biochemistry 2010; 4: 54–59. 7. andrushchuk ao, barzylovich ad. assessment of children’s health on the basis of adaptation indicators. the medicine of transport of ukraine 2005; 1: 74–77. received: 2014.01.30 o. fedortsiv et al. 19 p h a r m a c y issn 2413-6077. ijmmr 2016 vol. 2 issue 1 doi 10.11603/ijmmr.2413-6077.2015.2.6374 qualitative composition and organic acids content in the aboveground part of plants from families lamiaceae, asteraceae, apiaceae and chenopodiaceae s. m. marchyshyn, m. i. shanayda, i. z. kernychna, o. l. demydiak, i. s. dahym, t. s. berdey, i. m. potishnyj i. horbachevsky ternopil state medical university, ternopil, ukraine background. organic acids are the compounds of aliphatic or aromatic orders, which are widespread in flora and have a wide range of biological activity. we studied the qualitative composition and quantitative contents of organic acids in the aboveground part of some unofficial medicinal plants from families lamiaceae, asteraceae, apiaceae and chenopodiaceae is relevant. objective. the objects of the research are the aboveground part of unofficial medicinal plants from families lamiaceae, asteraceae, apiaceae and chenopodiaceae. methods. identification of organic acids was performed by means of thin-layer and paper chromatography, their content was determined by means of gas chromatography, the quantitative amount of organic acids was defined by titrimetric analysis. results. in the studied raw plants the quality of organic acids and their total contents were determined (in terms of malic acid). it is established that the maximum content of organic acids is accumulated in the grass hyssopus officinalis l. (lamiaceae), and the minimal is in the leaves of chrysánthemum xhortorum l. variety apro (asteraceae). in all studied raw plants the dominance of aliphatic acids (citric, malic, oxalic and malonic) was determined by means of gas chromatography. benzoic is predominant among the aromatic acids. conclusions. in the studied raw plants the quality of organic acids and their total content were determined. the following results can be used in developing the methods of quality control of the studied raw plants and during the study of new bioactive substances. key words: organic acids, lamiaceae, asteraceae, apiaceae, сhenopodiaceae, grass, leaves, thinlayer chromatography, gas chromatography, paper chromatography. introduction organic acids are the biologically active substances which are in plants in the free state, in the form of salts, esters, dimers and compounds with other substances. they are intermediate products of plants’ metabolism: involved in the oxidation of carbohydrates, fats, amino acids and proteins; used in the synthesis of amino acids, alkaloids, steroids, etc. [1, 2]. organic acids have a wide range of biological effects. they enhance the secretory and motor activity of the digestive tract, improving digestion; help to reduce nitration processes in the organism and to reduce chemical carcinogenesis; raise the protective strength and vitality of the organism. the antioxidant, antiallergic, antiinflammatory, antiseptic properties of these compounds are established [2]. according to the information above, we consider that the study of the qualitative composition and quantitative contents of organic acids in the aboveground part of some unofficial medicinal plants from families lamiaceae, asteraceae, apiaceae and chenopodiaceae is relevant and is of significant scientific and practical interest [3–5]. this will make it possible to justify the use of these plants in the future pharmaceutical researches. the aim of our research is to define the qua litative composition and quantitative con tents of free organic acids in the aboveground part of medicinal plants from families lamiaceae (hyssopus officinalis l., lophanthus anisatus l.), asteraceae (bellis perennis l. — cultivated, ta getes tenuifolia cav., chrysánthemum xhor torum l. variety apro), apiaceae (angelica syl vestris l.) and chenopodiaceae (chenopodium album l.). the leaves of chrysánthemum xhortorum and angelica sylvestris and the grass of the rest species were used for phytochemical analysis. raw plants for research were harvested during their mass flowering. s. m. marchyshyn et al. corresponding author: svitlana marchyshyn, department of pharmacognosy and medical botany, i, horbachevsky ternopil state medical university, 1 maidan voli, ternopil, ukraine, 46001 tel.: +3800352520518 e-mail: marchyshyn@tdmu.edu.ua international journal of medicine and medical research 2016, volume 2, issue 1, p. 19–22 copyright © 2016, tsmu, all rights reserved 20 p h a r m a c y issn 2413-6077. ijmmr 2016 vol. 2 issue 1 materials and methods identification of free organic acids in raw materials was performed out by means of thinlayer chromatography (tlc), paper chromatography (pc) and gas chromatography (gc) according to [6, 7]. for tlc and pc the aqueous extracts of raw plants were prepared. as standard samples for pc and gc benzoic, oxalic, malic, tartaric, succinic, salicylic, citric acids and the following solvent systems: n-butanol-formic acid-water (10:1:4), 95% ethanol-chloroformconcentrated solution of ammonia-purified water (70:40:20:2); 95% ethanol-concentrated solution of ammonia (16:4.5) and ethyl acetateformic acid-water (3:1:1) were used. the chromatograms were developed after drying in 0.05% alcohol solution bromothymol blue and 0.1% solution of 2,6-dichlorophe nolindophenol sodium salt hydrate. the action of ammonia vapours on chromatograms after a few seconds improved the contrast of spots. the quantitative contents of organic acids in aqueous extracts of raw plants were determined according to [7] by titrimetric method. the contents of free organic acids (x) in terms of malic acid in absolutely dry raw materials in percentage were calculated by the formula: , )w100(10m 1001002500067,0v x −×× ×××× = where: v — volume of 0.1 m sodium hydroxide solution consumed on titration, ml; 0.0067 — the amount of malic acid corresponding to 1 ml of 0.1 m sodium hydr oxide solution; m — mass of raw material, g; w — loss in weight because of drying, %. the quantitative contents of individual organic acids were defined by modified methods for determining the fatty acids in raw plants with further detection of organic acids. it is based on getting acid methyl esters (fatty, organic, phenolic) by methylating agent and their removal for further chromatographing by means of the gas chromatograph agilent technologies 6890 n with mass spectrometric detector 5973 n. methyl esters of organic acids were obtained by a modified method of a. carrapiso [8]. results 3–5 organic acids were identified in the raw plants of every studied species by tlc and pc methods (table 1). according to table 1, all studied species contain citric, oxalic and malic organic acids; tartaric, salicylic, benzoic and succinic acids were found only in some representatives. the results of quantitative contents of organic acids determination (in terms of malic acid) are shown in table 2. according to table 2, the maximum contents of organic acids accumulate in the grass hyssopus officinalis, the lowest is in the leaves of chrysánthemum xhortorum. the component composition and quantitative contents of individual organic acids in the aboveground organs of some studied species was defined by means of gs method (table 3). 13 organic acids in raw plants of hyssopus officinalis, 19 — in lophanthus anisatus, 5 — in bellis perennis, 5 — in chrysánthemum xhortorum, 18 — in chenopodium album, 15 — in angelica sylvestris were determined. discussion according to the results, aliphatic organic acids (citric, oxalic, malonic and malic) were defined in all studied raw plants by means of the methods of thin-layer, gas and paper chromatable 1. results of organic acids identification in raw plants from families lamiaceae, asteraceae, apiaceae and chenopodiaceae acid types of plants hyssopus officinalis (grass) lophanthus anisatus (grass) bellis perennis (grass) tagetes tenuifolia (grass) chrysánthemum xhortorum (leaves) chenopodium album (grass) angelica sylvestris (leaves) succinic + + – + – + traces tartaric – – traces – – – – citric + + + + + + + salicylic – traces + – – – traces oxalic + + + + + + + malic + + traces + + + + benzoic + + – – – + – note: “+” — labels identified compounds, “–” — labels unidentified compounds. s. m. marchyshyn et al. 21 p h a r m a c y issn 2413-6077. ijmmr 2016 vol. 2 issue 1 tography. the detection of organic acids is a topical issue of phytochemical researches. the study on composition and contents of these compounds in raw plants was not the urgent matter before [2, 9, 10]. citric acid is very widespread in nature and is used in medicine as a part of drugs to improve energy metabolism; malic and oxalic acids are used in food industry [2, 9]. we consider that among all investigated objects the grass chenopodium album is the most promising source of oxalic acid, and the grass hyssopus officinalis — of citric and malic. malonic acid is the predominant organic acid in all investigated raw plants except the grass chenopodium album. this dicarboxylic acid is an important component of biochemical reactions in a plant organism and a precursor in the synthesis of cinnamic acids and flavonoids; its significant contents indicates the level of metabolism in plants during the preparation of raw plants (in the period of their flowering). its accumulation in plants depends on the intensity of photosynthetic activity, enzymic reactions, temperature, etc. studying the role of this acid in plants and its effect on the biological activity of phytosubstances of plants is a promising area for scientific investigations [11]. benzoic acid is quantitatively dominant among the aromatic acids in the grass family lamiaceae (table 3), which is rather useful in pharmacy because it has anti-inflammatory, antibacterial and immunotropic properties [2]. the grass hyssopus officinalis is the most promising for benzoic acid among the studied species. conclusions the quantitative content of organic acids was studies and determined in the aboveground parts (grass or leaves) of the plants from families lamiaceae, asteraceae, apiaceae and chenopodiaceae. the component composition of free organic acids in raw plants from families lamiaceae (hyssopus officinalis, lophanthus anisatus), asteraceae (bellis perennis — cultivated, tagetes tenuifolia, chrysánthemum xhortorum variety apro), apiaceae (angelica sylvestris) and chenopodiaceae (chenopodium album) were analysed by means of gas chromatography for the first time. the dominance of aliphatic acids (citric, malic, oxalic and malonic) was determined. the following results can be used to develop the methods of quality control of the studied raw plants and during the study of new bioactive sub stances. table 2. quantitative contents of organic acids in raw plants from families lamiaceae, asteraceae, apiaceae and chenopodiaceae types of plants hyssopus officinalis (grass) lophanthus anisatus (grass) bellis perennis (grass) tagetes tenuifolia (grass) chrysánthemum xhortorum (leaves) chenopodium album (grass) angelica sylvestris (leaves) contents of acids, % 3,26±0,03 2,49±0,02 0,69±0,01 2,78±0,16 0,34±0,05 2,37±0,03 0,69±0,29 table 3. contents of main organic acids in raw plants from families lamiaceae, asteraceae, apiaceae and chenopodiaceae acid component content, mg/kg hyssopus officinalis (grass) lophanthus anisatus (grass) bellis perennis (grass) chrysánthemum xhortorum (leaves) chenopodium album (grass) angelica sylvestris (leaves) oxalic 84 370 70 310 20257 0,69±0,29 malonic 3731 3578 1069 557 963 1066 succinic 82 214 – – 727 37 benzoic 466 101 – – 81 – malic 946 590 – 2797 297 1101 citric 3063 1500 438 1562 792 1122 s. m. marchyshyn et al. 22 p h a r m a c y issn 2413-6077. ijmmr 2016 vol. 2 issue 1 references 1. dibner j, butin р. use of organic acids as a model to study the impact of gut microflora on nutrition and metabolism. j. appl. poultry research 2002; 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(in russian) 3. venkateshappa sm, sreenath kp. potential medicinal plants of lamiaceae. american int. j. of research in formal, applied & natural sciences 2013; 3(1): 82-87. 4. kokanova-nedialkova z, nedialkov p, nikolov s. the genus chenopodium: phytochemistry, ethnopharmacology and pharmacology. pharmacognosy review 2009; (3): 280-306. 5. ramya r, mahna s, bhamunathi s, bhat s. analysis of phytochemical composition and bacteriostatic activity of tagetes sp. int. research j. of pharmacy 2012; 3(11): 114-115. 6. benzel’ il, darmohrai rl, benzel’ lv. investigation of content of ascorbic acid and free organic acids in herbal substances of bergenia crassifolia. pharm. journal 2010; 1: 98–101. (in ukrainian) 7. emelyanova iv, kovaliov vs, kovalev sv, zuravel io. investigation of qualitative composition and dynamics of the accumulation of free organic acids in vegetative and generative organs of grindelia squarrosa. pharm. journal 2009; 1: 80–84. (in ukrainian) 8. carrapiso a, garcía c. development in lipid analysis: some new extraction techniques and in situ transesterification. lipids 2000; 35 (11): 1167–1177. 9. brul s, coote p. preservative agents in foods, mode of action and microbial resistance mechnismes. intl. j. food microbiology 1999; 50 (1–2): 1–17. 10. chirikova nk., olennikov dn., rokhin av. organic acids from medicinal plants: scutellaria baicalensis (lamiaceae). chem. of nat. compounds 2008; 44 (1): 84-86. 11. kenneth ek. kurt wf., schatz fp. a one-step synthesis of cinnamic acids using malonic acid. journal of chem. educ. 1990; 67 (12): 304-308. received: 2015-11-12 s. m. marchyshyn et al. issn 2413-6077. ijmmr 2016 vol. 2 issue 222 a n e st h e si o l o g y a n d c r it it a l c a r e issn 2413-6077. ijmmr 2016 vol. 2 issue 2o. v. oliynyk et al. international journal of medicine and medical research 2016, volume 2, issue 2, p. 22-25 copyright © 2016, tsmu, all rights reserved doi 10.11603/ijmmr.2413-6077.2016.2.7031 the influence of different ventilation patterns on treatment of patients with severe traumatic brain injury o. v. oliynyk1,2, b. o. pereviznyk1, a. shlifirchyk2 1i. horbachevsky ternopil state medical university, ternopil, ukraine 2pope john paul ii state school of higher education, biala podlaska, poland background. respiratory support is a vital method for temporary compensation of external breathing function in patients with severe traumatic brain injury. however, it is not always possible to deal with severe respiratory dysfunction even with the usage of up-to-date respiratory technologies. this work is aimed to find an answer how different pattern of mechanical ventilation influence on a treatment of patients with severe traumatic brain injury. objective.the influence of respiratory support, as a main method for temporary compensation of external breathing function, on treatment result for patients with severe traumatic brain injury. methods. treatment results of 253 patients with severe traumatic brain injury of ternopil university hospital were evaluated due to the type of respiratory support used. the results were separately evaluated in alive and dead patients. results. mortality rate of patients depended on the type of mechanical ventilation that was used. the highest mortality (58.69 %) was in the group, when a patient was transferred to forced ventilation a volume control. the mortality rate was decreasing by 51.78% in case of adding peep. the strategy of using accessory lung ventilation patterns cpap and bipap caused significant (in 1.48 times) decrease of mortality in this group of patients. conclusions. the survival of patients with severe traumatic brain injury, who were ventilated by the method of consistent combination of forced ventilation with pressure control (cрv) and 2 patterns of accessory lung ventilation: constant positive airway pressure (cpap) and biphasic positive airway pressure (bipap), is reliably higher than in the case of forced ventilation with volume control with positive end-expiratory pressure. key words: severe traumatic brain injury, lung ventilation, treatment, intensive care. introduction the process of treating the patients with severe traumatic brain injury (stbi) is a topical issue of contemporary medicine [1]. it is established that the mortality rate in case of stbi is 80%, it is caused by the development of multiple organ failure [2]. stbi is one of the causes of mortality and disability all over the world. it is also one of the main reasons of human mortality until the age of 40 [3]. in great britain stbi frequency is 1 500 people per every 100 000 population. 9 patients out of 100 000 population die because of traumatic brain injury [1, 2]. the problem of stbi treatment is today not only medical, but social as well [2-5]. among the complications of stbi the prominent role is given to lung affection which develops in 70–80% of patients and becomes one of the contributory factors for the worsening of the patient state with the further negative treatment results [4]. morphological changes of lungs with underlying stbi are detected in 95–97.7% of cases [4]. the most frequent cause of patients mortality on the 3–5th day of stbi is acute respiratory distress syndrome (ards) [2]. it happens in almost every stbi patient with multiple organ failure [6, 7]. respiratory support is a main method for the temporary compensation of external breathing function [8]. however, it is not always possible to deal with severe respiratory dysfunction even using up-to-date respiratory technologies. the question is: how will it influence the treatment result? this study is aimed to find an answer. material and methods treatment results of 253 patients with stbi of ternopil university hospital were evaluated due to the type of respiratory support used. corresponding author: oleksandr oliynyk, department of anaesthesiology and critical care medicine, i. horbachevsky ternopil state medical university, 1 maidan voli, ternopil, ukraine, 46001 phone number: +380352273341 e-mail: oliynyko@tdmu.edu.ua issn 2413-6077. ijmmr 2016 vol. 2 issue 2 23 a n e st h e si o l o g y a n d c r it it a l c a r e issn 2413-6077. ijmmr 2016 vol. 2 issue 2 the results were separately evaluated in alive and dead patients. as the criteria of treatment effectiveness we used the period of patients stay on mechanical ventilation. 4 different patterns of breathing were used: independent or spontaneous breathing, forced ventilation with volume control (cmv) and forced ventilation with pressure control (cрv). we also used 2 patterns of accessory lung ventilation: constant positive airway pressure (cpap) and biphasic positive airway pressure (bipap), a type of ventilation with 2 phases of positive pressure in airway. for ventilation with volume control we used a mechanical ventilation device “bryz”. for ventilation in cpap, bipap and cрv modes we used a mechanical ventilator carina drager. in 2012–2015 years in ternopil university hospital there were 253 patients with stbi. mechanical ventilation was delivered for all these patients. they were divided into 3 groups. in the 1st group patients were ventilated in cmv (5–6 ml/kg). the strategy in 2nd group was the same except during cmv mechanical ventilation we used positive pressure in the end of expiration (peep), which didn’t exceed 6–9 mbar. in the 3rd group we used the accessory ventilation. if the saturation of these patients in case of independent breathing decreased more than by 92%, the accessory ventilation through endotracheal tube in cpap pattern or alternatively tracheostomy was implemented. if saturation was lower than 90%, patients were transferred to bipap pattern. in case of full absence of independent breathing, the patient of this group was ventilated in cpv pattern. at this strategy peep didn’t exceed 6–9 mbar, and inspiratory pressure — 20–22 mbar. patients usually died because of multiple organ failure, which was followed by ards. ards was the most frequent cause of patients’ deaths during the first 5 days after the surgery. we used guidelines for the management of severe traumatic brain injury of american asso ciation of neurosurgeons (2007) as the treatment strategy which was modified accor­ ding to our conditions [4]. in all groups of patients during spontaneous breathing and mechanical ventilation as well, we tried not only to keep the partial oxygen pressure but also to prevent hypocapnia, so the hyperventilation didn’t occur. infusion therapy was implemented in normovolemic mode in order to avoid hypotension episodes. additionally, we tried to keep central venous pressure on the level of 8–10 mm hg and hemodynamic support with the usage of vasopressors was also performed. we also tried to prevent the decrease of average blood pressure (bp) lower than 80 mm hg. dopamine and adrenaline were the most frequently used among the vasopressors. the highest point of systolic bp was 160 mm hg. as for the infusion therapy we preferred normal saline solution (nss) or ringer’s solution. analgosedation was used for eradicating hyperventilation and synchronization with ventilator. the ventilation was implemented with paco2 35–40 mm hg. peep was 5–8 mm hg (not higher than 15 mm hg). according to the treatment strategy we also cured hyperthermia and anemia, prevented convulsions with carbamazepine and gabantin, used low molecular weight heparins at the absence of hemorrhage. for prevention of stress ulcers was conducted and nutrition therapy was applied. we preferred the fastest administration of enteric nutrition by means of standard food mixes and obligatory glycemia control. corticosteroids (cs) were used only in case of ards development. if intracranial pressure was rising we also used barbiturates. antibiotic therapy was chosen due to the type of microflora. the drugs of choice were meropenem, colomycin, amikacin. the mortality rate and ventilation period in each studied group were counted. the ventilation period was counted separately: for those who stayed alive and died. the permission for research implementation was issued by the commission on bioethics of i. horbachevsky ternopil state medical university (protocol №29 from may 20, 2015). results mortality rate of patients depended on the type of mechanical ventilation that was used. the highest mortality (58.69%) was in the 1st group, when the patient was transferred to cmv pattern in terms of insufficient independent breathing. the mortality rate decreased by 51.78% when peep was added. the strategy of using accessory lung ventilation patterns cpap and bipap caused significant (in 1.48 times) mortality decrease in this group of patients. the usage of different ventilation patterns caused the changes in the period of patients’ stay on mechanical ventilation. in the 1st group (cmv), dead patients were on accessory ventilation for very short period and died quickly. patients, who stayed alive, adversely, stayed on mechanical ventilation for longer period. the accession of peep improved ventilation results: o. v. oliynyk et al. issn 2413-6077. ijmmr 2016 vol. 2 issue 224 a n e st h e si o l o g y a n d c r it it a l c a r e issn 2413-6077. ijmmr 2016 vol. 2 issue 2 dead patients lived longer and those, who stayed alive, were taken of ventilator earlier. the best results were in groups with accessory ventilation patterns cpap and bipap. mortality in this group was lower than in 1st and 2nd groups. among the alive patients with such mechanical ventilation support, the process of excluding patients from mechanical ventilation was much easier, though the mortality in this group was in average for a few days longer if compared to 1st and 2nd group. discussion to our mind, the obtained results depend on peculiarities of ventilation pattern and analgosedation. anesthesiologists always see that a patient feels pain because vital signs, above all pulse, bp and respiratory rate increase. however, we tried not to use the excessive dosages of analgesics. if there are no signs of pain, we could give no analgesics to a patient at all. in case of even small signs of pain we prescribed narcotic analgesics. frequently, even not high dosage of analgesics caused the impairment of patients’ state. patient started breathing worse and mechanical ventilation was necessary. the same situation was on in case of hyperventilation, which promoted hypercapnia development. even small dosages of analgesics caused the decrease in patients’ state according to gcs level, partial oxygen pressure in arterial blood less than 60 mm hg and promoted the necessity of mechanical ventilation. the main differences and advantages of accessory ventilation are: in cpap pattern the ventilator helps the patient to inhale even if there are 40 inhales per 1 minute. the ventilator will support the positive pressure in the end of exhale on the determined level, which equals 6-9 cm of water. also, it helps to make every effort of inhale according to determined measurement of inspiratory support (20 mbar). in bipap pattern the respiratory rate is maintained by a ventilator. inhalation starts by the patients’ effort to inhale, and then the inspiratory support level is achieved. the important thing is that patient can breathe independently and breathe between respiratory cycles, which are provided by mechanical ventilation. in order to achieve the synchronization with it, we need smaller dosages of analgesics. we observed the smaller interruption of consciousness and breathing due to gcs during the last year we succeed in mortality decrease almost in 4 times: before the rate was 9 to 100 000 and now it is 9 up to 200 000. the main causes of this are 2 main factors: the earlier described strategy of respiratory support and the cs in treatment of ards. the most significant problem of intensive care unit is increase of bacteria resistance to carbapenem. in terms of insufficient financial support it is hard to solve. table 1. treatment results of patients with stbi followed by severe sepsis and ards depending on type of respiratory support and cs usage ventilation mode amount of patients average age ventilation period in days survival, % sv+ іmv alive 19 37.52 19.47 58.69 dead 27 52.96 5.22 sv+іmv+peep alive 27 41.0 17.14 51.78 dead 29 52.1 6.76 cpap+bipap+cpv alive 91 40.85 13.93 39.73 dead 60 50.0 14.1 dead patients sv+іmv sv+іmv+peep cpap+bipap+cpv sv+іmv – 0.0734 0.000050 sv+іmv+peep 0.0734 – 0.000099 cpap+bipap+cpv 0.000050 0.000099 – alive patients sv+іmv sv+іmv+peep cpap+bipap+cpv sv+іmv – 0.8194 0.1769 sv+іmv+peep 0.8194 – 0.4688 cpap+bipap+cpv 0.1769 0.4688 – o. v. oliynyk et al. issn 2413-6077. ijmmr 2016 vol. 2 issue 2 25 a n e st h e si o l o g y a n d c r it it a l c a r e issn 2413-6077. ijmmr 2016 vol. 2 issue 2 conclusions the survival of patients with stbi which were ventilated by consistent combination of forced ventilation with the pressure control (cрv) and 2 patterns of accessory lung venti­ lation: constant positive airway pressure (cpap) and biphasic positive airway pressure (bipap) is significantly higher than in the case of forced ventilation with the volume control with positive end-expiratory pressure. references 1. bullock r. neurotrauma. guidelines for the management of severe traumatic brain injury 2007; 24: 101–106. 2. churlyaev а. acute respiratory distress syndrome with severe traumatic brain injury. common resuscitation 2009; 2: 21–27. 3. shekhovtseva k. epidemiology of traumatic brain injury and the organization of assistance to victims of stavropol territory. dissertation for the candidate of medical sciences: russian research neurosurgical institute — st. petersburg, 2006. www. dissercat.com. 4. saidov s. violation of hypo coagulating lung function in severe traumatic brain injury. dissertation for the candidate of medical sciences: moscow, 2004. www.dissercat.com. 5. arabi y, haddad s, tamim h. mortality reduction after implementing a clinical practice guidelines-based management protocol for severe traumatic brain injury. j critcare 2010; 25: 190–195. 6. lewandowski k, lewandowski m: epidemiology of ards. minerva anestesiol 2006; 72: 473–477. 7. eloise m harman. acute respiratory distress syndrome treatment & management. emedicinehealth 2014, 2, www// emedicine.medscape.com. 8. atul malhotra. benefits of low tidal volume ventilation in ards. crit care clin 2008; 18: 79–89. 9. luce j. corticosteroids in ards. an evidencebased review. crit care clin 2002; 1: 79–89. 10. meduri g. effect of prolonged methylprednisolone therapy in unresolving acute respiratory distress syndrome. a randomized controlled trial. jama 2008; 280: 159–165. 11. meduri g. methylprednisolone infusion in early severe ards: results of a randomized controlled trial. chest 2007; 131: 954–963. 12. steinberg k. efficacy and safety of corticosteroids for persistent acute respiratory distress syndrome. n engl j med 2007; 354: 1671–1684. 13. meduri g. steroid treatment in ards: a critical appraisal of the ards network trial and the recent literature. intensive care medicine 2008; 34: 61–69. 14. izaguirre-anariba d. acute respiratory distress syndrome: a complex clinical condition, medscape 2015. www. reference.medscape.com 15. roberts i, yates d, sandercock p. crash trial collaborators. effect of intravenous corticosteroids on death within 14 days in 10008 adults with clinically significant head injury (mrc crash trial): randomised placebo­controlled trial. lancet 2008; 364: 1321–1328. 16. martin-loeches i, lisboa t, rhodes a. use of early corticosteroid therapy on icu admission in patients affected ey severe pandemic (h1n1) v influenza a infection. intensive care med. feb 2011; 37(2): 272–283. 17. maclaren r, jung r. stress-dose corticosteroid therapy for sepsis and acute lung injury or acute respiratory distress syndrome in critically ill adults. pharmacotherapy 2002; 22: 1140–1156. 18. agarwal r, aggarwal a etiology and outcomes of pulmonary and extrapulmonary acute lung injury/ards in a respiratory icu in north india. chest 2006; 130: 724–729. 19. brun-buisson c, richard j, mercat a. early corticosteroids in severe influenza a/h1n1 pneumonia and acute respiratory distress syndrome. am j respir crit care med 2011; 183: 1200–1206. received: 2016-11-29 o. v. oliynyk et al. 56 p sy c h ia t r y issn 2413-6077. ijmmr 2022 vol. 8 issue 2 doi 10.11603/ijmmr.2413-6077.2022.2.13123 comparative characteristics of symptoms in neurotic and affective patients after covid-19 o. venger, a. m. koval, m. shved, *t. ivanitska i. horbachevsky ternopil national university, ternopil, ukraine background. covid-19 pandemic has negatively affected people’s mental health and created new barriers for those who have already suffered from mental illnesses. objective. the aim of the study was to compare the symptoms of mental disorders in neurotic and affective patients, who recovered from covid-19. methods. the features of disorders during the pandemic in 35 neurotic and 31 affective patients were analysed. the hamilton anxiety and depression scales (ham-a, ham-d), insomnia severity index (isi), schulte tables, jacobson short-term memory study were used. results. moderate and severe anxiety was more likely to develop in neurotic patients (34.29% and 37.14%, respectively), mild and moderate – in affective ones (38.71% and 32.26%). neurotic patients had a higher level of depression (moderate – by 31.42%, severe – by 48.57%) than affective patients (mild – by 32.26%, severe – by 35.48%). the degree of memory loss was higher in affective patients (51.62% – low, 35.48% – very low) than in neurotic ones (41.94% – low, 20.00% – very low). most of patients had reduced attention. insomnia severity was higher in patients with neurotic disorders (25.72% – subthreshold, 45.71% – moderate) than in affective patients (38.71% – subthreshold, 22.58% – moderate). a higher level of anxiety and depression severity was observed in patients who had covid-19 in 2021 than in those who had it in 2020, while the severity of insomnia and memory impairment – vice versa. conclusion. anxiety, depression, insomnia, memory and attention loss were detected. anxiety and depression were more severe in neurotic patients but the degree of memory loss was higher in affective patients. keywords: pandemic; mental health; depression; anxiety; insomnia. *corresponding author: tetiana ivanitska, assistant professor of the department of psychiatry, narcology and medical psychology, i. horbachevsky ternopil national medical university, ternopil, 46027, ukraine. e-mail: ivanitska_te@tdmu.edu.ua international journal of medicine and medical research 2022, volume 8, issue 2, p. 56-66 copyright © 2022, tnmu, all rights reserved o. venger et al. introduction severe acute respiratory syndrome coronavirus 2 (sars­cov­2), first detected in wuhan, china, in december 2019, rapidly spread around the world in 2020. the outbreak of coronavirus disease (covid-19) is still having a global impact on people today. the association between sars-cov-2 infection and mental illness is a topical issue, as the covid-19 pandemic has negatively affected mental health of many people and created new challenges for those already suffering from a mental illness [1]. with an increase in the number of infected people, there is a simultaneous increase in the number of recovered patients with preserved clinical manifestations of coronavirus infection after complete elimination of the virus from the organism, which indicates the post-covid syndrome. a lot of people report persistent fatigue, joint and bone pain, rapid heartbeat, headache, dizziness, insomnia, anxiety, and depression [2, 3]. residual phenomena of the disease are manifested by damage to many internal organs, including the nervous system, causing neuropsychiatric syndromes that affect cognitive, behavioural, affective and perceptual disorders [4]. the aetiology of psychiatric consequences of coronavirus infection is multifactorial and may include direct effect of the virus, cerebrovascular diseases, hypoxia, immune response, social isolation, psychological impact of a new potentially fatal disease, anxiety about infecting others and stigma or discrimination [4]. sarscov-2 is a neurotropic virus that can enter the brain in various ways, including the olfactory neural pathway, and trigger inflammatory res­ ponse affecting both the peripheral and central nervous systems (cns) [3]. at least seven mechanisms of development of affective and neurotic disorders and cognitive dysfunction are established, i.e.: viral replication in innate immune cells, dysregulation of the 57 p sy c h ia t r y issn 2413-6077. ijmmr 2022 vol. 8 issue 2 immune response, cytokine storm, antibodymediated response, hypoxemia, ischemia due to hypercoagulation, neuronal damage [1, 2]. lung damage and consequently hypoxemia caused by covid-19 is likely to contribute to neuronal damage and further cognitive decline. sars-cov-2 affects the vascular endothelium causing endothelial dysfunction followed by coagulopathy and thrombotic complications. the virus also causes systemic vasculitis and cytokine storm, which can damage brain structures. the hypercoagulatory and hyperinflammatory states evidenced in covid­19 may contribute to delusions and future cog nitive decline. in addition, peripheral pro­inflam­ matory cytokines such as tumour necrosis factor alpha (tnf­α), interleukin­1 (il­1), il­6 effect the permeability of the blood-brain barrier through cyclooxygenase­2 (cox­2), upregulation and activation of matrix metalloprotease and enter the cns, causing microglial activation and oxidative stress which induce apoptosis and eventually alterations in neurotransmitter signalling leading to synergistic cognitive impairment, psychiatric disorder development and progression. induced neuroinflammation can contribute to short-term delusions and severe long-term cognitive deficits [1, 2]. the aim of our study is to compare the symptoms of mental disorders in patients with neurotic and affective mental disorders, who have recovered from covid-19. methods in compliance with the principles of biomedical ethics, on the basis of informed consents, 61 respondents (15 men and 46 women) with no previous psychiatric history were interviewed; 30 respondents among them were not previously infected with covid-19 and 31 respondents who had suffered from this disease before; and 66 recovered patients (26 men and 40 women) who got previously sick with coronavirus infection and at the time of the study were undergoing inpatient treatment in the psychiatric departments of the ternopil regional clinical psychoneurological hospital (the psychiatric illness of the cohort was diagnosed before coronavirus disease). the average age of respondents of the control group was (39±7) years, ranging from 19 to 47 years old. the average age of patients was (50±13) years, ranging from 20 to 63 years old. among respondents who were infected with covid-19, 24 respondents had a mild course (77.42%), 7 respondents – moderate severity (22.58%). among the patients, 35 respondents had a mild course (53.03%), 21 – moderate course (31.82%) and 10 – severe course (15.15%). the study was conducted from december 2021 to january 2022 at i. horbachevsky ternopil national medical university and the ternopil regional clinical psychoneurological hospital. the printed questionnaires were distributed among the volunteer respondents who agreed to participate after being invited on private social media pages. to collect data and optimize the obtained results the following tools were used: 1. general questionnaire with sociodemographic data (age, sex), type of activity, hobbies and support of relatives, previous history of mental disorders. 2. information about the date of covid-19 onset and the course of the disease. 3. hamilton anxiety rating scale (ham-a). 4. hamilton depression rating scale (ham-a). 5. insomnia severity index (isi). 6. schulte table test. 7. short-term memory study. results the respondents were divided into four groups: two control groups (respondents who were not previously infected with covid-19 and recovered respondents with no mental dis order), neurotic patients (35 patients: 10 patients with f40, 16 patients with f41, 9 patients with f43), affective patients (31 patients: 9 patients with f31, 12 patients with f32, 10 patients with f33), and six subgroups according to the date of covid­19 onset: 1. group 1 – 30 respondents who were not previously infected with covid-19. 2. group 2 – 31 respondents who had covid­19: subgroup 2.1 – 15 people who were ill in 2020 (48.39%); subgroup 2.2 – 16 people who were ill in 2021 (51.61%). 3. group 3 – 35 neurotic patients who recovered from covid­19: subgroup 3.1 – 13 people who were ill in 2020 (37.14%); subgroup 3.2 – 22 people who were ill in 2021 (62.86%). 4. group 4 – 31 affective patients who recovered from covid­19: subgroup 4.1 – 13 people who were ill in 2020 (41.94%); o. venger et al. 58 p sy c h ia t r y issn 2413-6077. ijmmr 2022 vol. 8 issue 2 subgroup 4.2 – 18 people who were ill in 2021 (58.06%). based on the questionnaire which included the hamilton anxiety rating scale (ham-a), the level of anxiety was assessed in the respondents, who were not previously infected with covid-19, and the recovered respondents with no mental disorder, neurotic patients and affective patients, who recovered from covid-19. during our study it was established that among the respondents-volunteers, who were not previously infected with covid-19 (group 1), 30.00% did not experience anxiety, 70.00% had mild anxiety. among the respondents with covid-19 (group 2), only 6.45% had no anxiety, mild anxiety was evidenced in 54.84% of people, moderate and severe anxiety were found in 25.81% and 12.90%, respectively. among the patients with neurotic disorders (group 3), all of them suffered from anxiety, 28.57% of patients had mild anxiety. in this group moderate and severe anxiety were more common in 34.29% and 37.14% of patients, respectively. among the patients with affective disorders (group 4,) 6.45% had no anxiety, mild and moderate anxiety prevailed in 38.71% and 32.26% of them, respectively, and the other 22.58% of patients had severe anxiety (fig. 1). therefore, an increase in the frequency and severity of anxiety in the recovered respondents and patients was evidenced compared to the respondents who were not previously infected with covid-19. the patients in groups 3 and 4 were more likely to experience moderate and severe anxiety, while the respondents in group 2 were more likely to have mild anxiety. comparing the anxiety severity in the respondents of groups 3 and 4, moderate and severe anxiety was more common among the neurotic patients while mild and moderate anxiety – among the affective patients. assessing the anxiety severity in the patients by the date of covid onset, mild anxiety was evidenced more in those patients, who were ill in 2020 (53.85% of neurotic and 53.85% of affective patients), and the patients, who were ill in 2021, had moderate anxiety (50.00% of neurotic and 50.00% of affective patients) (table 1). based on the survey, which involved the hamilton depression rating scale (ham-d), the level of depression was assessed in the respondents-volunteers, who were not previously infected with covid-19, and the recovered ones, neurotic and affective patients, who recovered from covid-19. during our study it was established that among the respondents of group 1, 80.00% had no depression, 16.67% had mild depression, and only 3.33% of people had moderate depression. among the respondents, who recovered (group 2), only 25.81% of them had no depression, 41.93% had mild depression, and 16.13% – moderate and severe depression each. among the patients with neurotic disorders (group 3), no depression was observed only in 5.71% of them, 14.29% had mild depression. most often, the patients in this group suffered from moderate or severe depression, in 31.43% and 48.57% of people, respectively. among the patients with affective disorders (group 4), 9.68% had no depression, 32.26% had mild depression, 22.58% had moderate depression, and 35.48% had severe depression (fig. 2). fig. 1. anxiety severity according to ham-a. group 1 (respondents who were not ill) group 2 (respondents who were ill) group 3 (neurotic patients) group 4 (affective patients) absent 30,00% 6,45% 0,00% 6,45% mild 70,00% 54,84% 28,57% 38,71% moderate 0,00% 25,81% 34,29% 32,26% severe 0,00% 12,90% 37,14% 22,58% 30 ,0 0% 6, 45 % 0, 00 % 6, 45 % 70 ,0 0% 54 ,8 4% 28 ,5 7% 38 ,7 1% 0, 00 % 25 ,8 1% 34 ,2 9% 32 ,2 6% 0, 00 % 12 ,9 0% 37 ,1 4% 22 ,5 8% 0,00% 10,00% 20,00% 30,00% 40,00% 50,00% 60,00% 70,00% 80,00% o. venger et al. 59 p sy c h ia t r y issn 2413-6077. ijmmr 2022 vol. 8 issue 2 there was an increase in the severity of depression in the respondents and patients, who had covid-19, compared to the respondents, who were not previously infected. people of the groups 3 and 4 were more likely to experience moderate to severe depression (more severe in the neurotic patients), while the respondents of group 2 were more likely to experience mild depression. comparing the level of depression severity in the patients of groups 3 and 4, it was noticed that the neurotic patients were more likely to have moderate and severe depression, while the affective patients– mild and severe. assessing depression severity in the patients by the date of covid-19 onset, it was established that the patients, who were ill in 2020, usually experienced mild depression (30.77% of neurotic patients and 46.16% of affective patients), and the patients, who were ill in 2021 – severe depression (54.54% of neurotic patients and 50.00% of affective patients) (table 2). the respondents’ memory was evaluated using the “short-term memory study”. it was found out that among the respondents of group 1, 6.67% of them had very high memory, 30.00% had high memory, 43.33% had average memory, and 20.00% had low memory. among the recovered respondents (group 2), only 6.45% of them had high memory, 19.35% – ave rage memory, and low memory was evidenced the most often in 70.97% of people. among the patients with neurotic disorders (group 3), 48.86% of them had average memory, low memory in 41.94% of patients, and very low memory – in 20.00%. among the patients with affective disorders (group 4), average memory was found in 12.90% of respondents, low memory – in 51.62%, and very low memory – in 35.48% (fig. 3). there was a decrease in memory of the respondents and patients, who recovered from table 1. anxiety severity according to ham-a severity of anxiety group 3 (neurotic patients) 35 people group 4 (affective patients) 31 people group 3.1 were ill in 2020 13 respondents group 3.2 were ill in 2021 22 respondents group 4.1 were ill in 2020 13 respondents group 4.2 were ill in 2021 18 respondents no % no % no % no % assessment of anxiety absent 0 0 0 0 1 7.69 0 0 mild 7 53.85 3 13.64 7 53.85 4 22.22 moderate 1 7.69 11 50.00 2 15.38 9 50.00 severe 5 38.46 8 36.36 3 23.08 5 27.78 fig. 2. depression severity according to ham-d. group 1 (respondents who were not ill) group 2 (respondents who were ill) group 3 (neurotic patients) group 4 (affective patients) absent 80,00% 25,81% 5,71% 9,68% mild 16,67% 41,93% 14,29% 32,26% moderate 3,33% 16,13% 31,43% 22,58% severe 0,00% 16,13% 48,57% 35,48% [з н а че н н я] 25 ,8 1% 5, 71 % 9, 68 % 16 ,6 7% 41 ,9 3% 14 ,2 9% 3 2, 26 % 3, 33 % 16 ,1 3% 31 ,4 3% 22 ,5 8% 0, 00 % 16 ,1 3% 48 ,5 7% 35 ,4 8% 0,00% 10,00% 20,00% 30,00% 40,00% 50,00% 60,00% 70,00% 80,00% 90,00% o. venger et al. 60 p sy c h ia t r y issn 2413-6077. ijmmr 2022 vol. 8 issue 2 covid, compared to the respondents, who were not infected. the people of groups 3 and 4 were more likely to have low and very low memory (more pronounced in the affective patients), while the respondents of group 2 were found to have low memory. moreover, the level of attention of respondents according to the “schulte table” test was evaluated. it was established that 100% of the respondents of group 1 had normal attention, among the people of group 2, 93.50% had normal attention and only 6.45% had reduced atten tion. among the neurotic patients, only 17.14% of people had normal attention, while 82.86% had reduced attention. among the affective patients, only 19.35% of people had normal attention, while 80.65% had reduced attention (fig.4). therefore, the majority of neurotic and affective patients, who were previously infected with covid-19, had reduced attention, 82.86% and 80.65%, respectively, compared to the recovered respondents without mental illnesses. evaluating memory and attention in patients by the date of covid-19 onset, there was no significant difference in attention changes in the patients, who were ill in 2020 and in 2021. as for memory, the neurotic patients, who were ill in 2021, generally had better memory (45.46% – normal memory, 36.36% – low memory, 18.18% – very low memory) than those, who were ill in 2020 (38.46% – average memory, 38.46% – low memory, 23.08% – very low memory). worse results were found in the affective patients, who had covid­19 in 2021: 5.55% of patients had normal memory, 61.11% – low memory, 33.34% – very low memory, while among the patients, who had covid-19 in 2020, 15.38% had average memory, 38.46% – low memory and 46.16% – very low memory (table 3). fig. 3. assessment of memory according to short-term memory study. table 2. depression severity according to ham-d depression severity group 3 (neurotic patients) 35 people group 4 (affective patients) 31 people group 3.1 were ill in 2020 13 respondents group 3.2 were ill in 2021 22 respondents group 4.1 were ill in 2020 13 respondents group 4.2 were ill in 2021 18 respondents no % no % no % no % assessment of depression absent 1 7.69 1 4.55 3 23.08 0 0 mild 4 30.77 1 4.55 6 46.16 4 22.22 moderate 3 23.08 8 36.36 2 15.38 5 27.78 severity 5 38.46 12 54.54 2 15.38 9 50.00 group 1 (respondents who were not ill) group 2 (respondents who were ill) group 3 (neurotic patients) group 4 (affective patients) very high 6,67% 0,00% 0,00% 0,00% high 30,00% 6,45% 0,00% 0,00% avarage 43,33% 19,35% 48,86% 12,90% low 20,00% 70,97% 41,94% 51,62% very low 0,00% 3,23% 20,00% 35,48% 6, 67 % 0, 00 % 0, 00 % 0, 00 % 30 ,0 0% 6, 45 % 0, 00 % 0, 00 % 43 ,3 3% 19 ,3 5% 48 ,8 6% 12 ,9 0% 20 ,0 0% 70 ,9 7% 41 ,9 4% 51 ,6 2% 0, 00 % 3, 23 % 20 ,0 0% 35 ,4 8% 0,00% 10,00% 20,00% 30,00% 40,00% 50,00% 60,00% 70,00% 80,00% o. venger et al. 61 p sy c h ia t r y issn 2413-6077. ijmmr 2022 vol. 8 issue 2 based on the questionnaire, the insomnia severity index was evaluated. it was found out that 90.00% of the respondents of group 1 had no insomnia, and 10.00% had subthreshold insomnia. among the respondents, who had covid-19 (group 2), only 45.16% of respondents had no insomnia, 38.71% had subthreshold insomnia, 12.90% had moderate insomnia and only 3.23% had severe insomnia. among the patients with neurotic disorders (group 3), only 8.57% of patients had no insomnia, 25.72% had subthreshold insomnia, 45.71% had moderate insomnia, and 20.00% – severe insomnia. among the patients with affective disorders (group 4), 22.58% had no insomnia, 38.71% had a subthreshold insomnia, 22.58% had moderate insomnia, and 16.13% had severe insomnia (fig. 5). therefore, a higher degree of insomnia was found in the respondents and patients, who were previously infected with covid-19, compa re to the respondents, who did not suffer from the disease. the neurotic patients had moderate insomnia, while the affective patients had subthreshold insomnia. moderate to severe insomnia was more common in the patients than in the respondents without previous psychiatric history. assessing the level of insomnia severity in the patients by the date of covid-19 onset, it was established that in neurotic patients, who were ill in 2020, subthreshold and moderate insom nia prevailed (30.77% and 53.85%, respectively), and in the patients of this group, who were ill in 2021 – moderate and severe insomnia (40.91% and 27.27%, respectively). severe in somfig. 4. assessment of attention according to schulte table test. group 1 (respondents who were not ill) group 2 (respondents who were ill) group 3 (neurotic students) group 4 (affective patients) normal 100,00% 93,50% 17,14% 19,35% reduced 0,00% 6,45% 82,86% 80,65% 10 0, 00 % 93 ,5 0% 17 ,1 4% 19 ,3 5% 0, 00 % 6, 45 % 82 ,8 6% 80 ,6 5% 0,00% 20,00% 40,00% 60,00% 80,00% 100,00% 120,00% table 3. assessment of memory and attention severity group 3 (neurotic patients) 35 people group 4 (affective patients) 31 people group 3.1 were ill in 2020 13 respondents group 3.2 were ill in 2021 22 respondents group 4.1 were ill in 2020 13 respondents group 4.2 were ill in 2021 18 respondents no % no % no % no % assessment of attention normal 2 15.38 4 18.18 3 23.08 3 16.67 reduced 11 84.62 18 81.82 10 76.92 15 83.33 assessment of memory very high 0 0 0 0 0 0 0 0 high 0 0 0 0 0 0 0 0 average 5 38.46 10 45.46 2 15.38 1 5.55 low 5 38.46 8 36.36 5 38.46 11 61.11 very low 3 23.08 4 18.18 6 46.16 6 33.34 o. venger et al. 62 p sy c h ia t r y issn 2413-6077. ijmmr 2022 vol. 8 issue 2 nia prevailed in the affective patients, who were ill in 2020 (38.46% of people), and sub threshold insomnia prevailed in the patients of this group, who were ill in 2021 (50.00 %) (table 4). according to the survey, the severity of mental and cognitive changes in the patients were evaluated depending on the date of covid-19 onset (2020 or 2021 year). thus, it was established that 96.15% of patients, who were ill in 2020, and 100% of patients, who were ill in 2021, suffered from anxiety. therefore, the anxiety severity in the patients, who had covid-19 in 2021, was higher than in those who had it in 2020. the respondents, who were ill in 2021, were more likely to have moderate and severe anxiety in 50.00% and 32.50%, respectively. as for the patients, who were ill in 2020, mild and severe anxiety was more common in 53.85% and 30.76% of them, respectively. depressive disorders were observed in 84.62% of patients, who were ill in 2020, and in 97.50% of patients, who were ill in 2021. the severity of depression was also higher in the patients, who were ill in 2021. in this group of patients, moderate depression was most often observed in 32.50% of patients and severe depression – in 52.50%. among the patients, who were ill in 2020, severe depression was observed in 26,92% of them and mild depression – in 38,46% of people. attention was reduced in the majority of patients, who were ill in 2020 and 2021, 80.77% and 82.50%, respectively. reduced memory was evidenced in 72.50% of patients, who were ill in 2021, and in 73.08% of patients, who were ill in 2020, but the degree of memory loss was higher in those who were ill in 2020 (low memory – in 38.46%, very low memory – in 34.62%). among the patients, who fig. 5. assessment of insomnia severity according to isi. group 1 (respondents who were not ill) group 2 (respondents who were ill) group 3 (neurotic patients) group 4 (affective patients) absent 90,00% 45,16% 8,57% 22,58% subthreshold 10,00% 38,71% 25,72% 38,71% moderate 0,00% 12,90% 45,71% 22,58% severe 0,00% 3,23% 20,00% 16,13% 90 ,0 0% 45 ,1 6% 8, 57 % 22 ,5 8% 10 ,0 0% 38 ,7 1% 25 ,7 2% 38 ,7 1% 0, 00 % 12 ,9 0% 45 ,7 1% 22 ,5 8% 0, 00 % 3, 23 % 2 0, 00 % 16 ,1 3% 0,00% 10,00% 20,00% 30,00% 40,00% 50,00% 60,00% 70,00% 80,00% 90,00% 100,00% table 4. assessment of insomnia severity according to isi insomnia severity group 3 (neurotic patients) 35 people group 4 (affective patients) 31 people group 3.1 were ill in 2020 13 respondents group 3.2 were ill in 2021 22 respondents group 4.1 were ill in 2020 13 respondents group 4.2 were ill in 2021 18 respondents no % no % no % no % assessment of insomnia severity absent 1 7.69 2 9.09 1 7.69 5 27.78 subthreshold 4 30.77 5 22.73 4 30.77 9 50.00 moderate 7 53.85 9 40.91 3 23.08 4 22.22 severe 1 7.69 6 27.27 5 38.46 0 0 o. venger et al. 63 p sy c h ia t r y issn 2413-6077. ijmmr 2022 vol. 8 issue 2 were ill in 2021, low memory was evidenced in 47.50%, very low – only in 25.00%. sleep disorders affected 82.50% of patients, who were ill in 2021, and 92.31% of those, who were ill in 2020. the severity of insomnia was higher in the patients, who were ill in 2020: subthreshold insomnia was observed in 30.77% of people, moderate insomnia – in 38.46%, severe insomnia – in 23.08%. among the patients, who were ill in 2021, subthreshold insomnia was evidenced in 35.00% of people, moderate insomnia – in 32.50%, severe insomnia – only in 15.00% (table 5). discussion it is established that covid-19 negatively affects mental health of the population around the world. fear of coronavirus infection associated with the possible risk of infection, unpredictable course of the disease, lack of pathogenetic treatment and total uncertainty causes negative psychological reactions, such as distress and maladaptation [5]. as for patients with a primary psychiatric history, usually after covid-19, the symptoms of mental disorders were more significant [6]. according to the survey it was found out that 100% of neurotic and 92.31% of affective patients experienced anxiety after covid-19. moderate and severe anxiety was more common in the patients with neurotic disorders (in 34.29% and 37.14%, respectively), and mild and moderate anxiety was evidenced in the a f f e c t i v e p a t i e n t s ( 3 8 . 7 1 % a n d 3 2 . 2 6 % , respectively). it was established that 94.29% of neurotic patients and 90.32% of affective patients experienced depression after recovering from covid-19. the neurotic patients had a higher degree of depression (moderate – in 31.42%, severe – in 48.57%) than the affective patients (mild depression – in 32.26% and severe – in 35.48%) table 5. comparison of the severity of mental and cognitive disorders depending on the date of covid-19 onset (year 2020 and 2021) severity all patients 65 patients were ill in 2020 26 patients were ill in 2021 40 patients no % no % assessment of anxiety absent 1 3.85 0 0 mild 14 53.85 7 17.50 moderate 3 11.54 20 50.00 severe 8 30.76 13 32.50 assessment of depression absent 4 15.38 1 2.50 mild 10 38.46 5 12.50 moderate 5 19.23 13 32.50 severe 7 26.92 21 52.50 assessment of attention normal 5 19.23 7 17.50 reduced 21 80.77 33 82.50 assessment of memory very high 0 0 0 0 high 0 0 0 0 average 7 26.92 11 27.50 low 10 38.46 19 47.50 very low 9 34.62 10 25.00 assessment of insomnia absent 2 7.69 7 17.50 subthreshold 8 30.77 14 35.00 moderate 10 38.46 13 32.50 severe 6 23.08 6 15.00 o. venger et al. 64 p sy c h ia t r y issn 2413-6077. ijmmr 2022 vol. 8 issue 2 additionally, impaired cognitive functions were found in the respondents with coronavirus infection, i.e. memory and attention. the memory loss was diagnosed in 61.94% of patients with neurotic disorders and 87.10% of patients with affective disorders. the degree of memory loss was higher in the affective patients (51.62% – low memory, 35.48% – very low memory) than in the neurotic patients (41.94% – low memory, 20.00% – very low memory). reduced attention was observed in the majority of the examined patients: 82.86% of neurotic patients and 80.65% of affective patients. sleep disorders were evidenced in 91.43% of neurotic patients and 77.42% of affective patients after covid-19. subthreshold insomnia and moderate insomnia were more common in both groups of patients. the frequency of their occurrence was higher in the patients with neurotic disorders (25.72% of patients – subthreshold insomnia, 45.71% – moderate insomnia) than in those with affective disorders (38.71% – subthreshold insomnia, 22.58% – moderate insomnia). we have identified features of mental and cognitive disorders among the patients with a previous psychiatric history, who recovered from covid-19, depending on the date of covid­19 onset (2020 and 2021 year): – depressive disorders and anxiety in the neurotic and affective patients, who were ill in 2020, were mostly mild, and in those, who were ill in 2021 – moderate; – memory was more worsened in the neurotic patients, who were ill in 2020, than in those, who were ill in 2021. the affective patients had the opposite result – the better memory was in those, who had covid-19 in 2020; – sleep disorders also correlated with the date of covid-19 onset. the neurotic patients, who were ill in 2020, had subthreshold and moderate insomnia, and those, who were ill in 2021, had moderate and severe insomnia. in the affective patients, the results were opposite: those, who were ill in 2020, had severe insomnia, and those, who were ill in 2021, had subthreshold insomnia. numerous studies also show that people with psychiatric illnesses are at higher risk of covid-19 infection and related complications due to a variety of factors [6]. comorbidities, altered immune functions, sleep problems and various socioeconomic risk factors contribute to an increase in covid-19 infection rates [6]. studies [7, 8] from around the world have established a significant association between existing psychiatric disorder and an increased risk of covid-19 infection and covid-19-related hospitalization, morbidity and mortality. the prognosis was found to be worse among patients with affective mental illnesses than for patients with neurotic ones [7, 8]. threats of coronavirus disease, violation of the usual life stereotype, restriction of leisure activity, harmful interest in news about the pandemic, usage of tobacco are important in the me chanisms of distress development. these conditions increase the risk of stress, anxiety and depression [9]. therefore, patients with mental disorders should be considered a vulnerable group for covid-19 infection, and the results of this study are of particular significance and value in predicting development of mental disorders and optimizing treatment after an infectious disease in people suffering from neurotic and affective disorders during this pandemic or subsequent ones. in the perspective of this study, after the survey and interpreting the attained results, a conversation with the respondents took place and a course of cognitive-behavioural and biosuggestive psychotherapy was offered to correct the symptoms of mental disorders and improve resocialization of the patients. the data will be published in the following publications. conclusion anxiety, depression, insomnia, memory and attention loss were found in examined patients. anxiety and depression were more severe in the patients with neurotic disorders while the degree of memory loss was higher in the patients with affective disorders. reduced attention was observed in most of the examined patients with affective and neurotic disorders. our survey found that 100% of neurotic and 92.31% of affective patients experienced anxiety after covid-19. moderate and severe anxiety was more common in the patients with neurotic disorders and mild and moderate anxiety was evidenced in the affective patients. it was established that 94.29% of neurotic patients and 90.32% of affective patients experienced depression after recovering from covid-19. the neurotic patients had more severe depression than the affective ones. additionally, impaired cognitive functions in the respondents with coronavirus infection, i.e. memory and attention, were found. memory loss was diagnosed in 61.94% of patients with neurotic o. venger et al. 65 p sy c h ia t r y issn 2413-6077. ijmmr 2022 vol. 8 issue 2 disorders and 87.10% of patients with affective disorders. memory loss was more significant in the affective patients than in neurotic ones. reduced attention was observed in the majority of the examined patients: 82.86% of neurotic patients and 80.65% of affective patients. sleep disorders were evidenced in 91.43% of neurotic patients and 77.42% of affective patients after covid-19. subthreshold insomnia and moderate insomnia were more common in both groups of patients. their frequency was higher in the patients with neurotic disorders than in those with affective ones. more severe anxiety and depression was observed in the patients, who had covid-19 in 2021, than in those, who had it in 2020, while severity of insomnia and memory impairment vice versa. conflict of interests authors declare no conflict of interest. author’s contributions o l e n a v e n g e r – c o n c e p t u a l i z a t i o n , methodology, formal analysis, writing – reviewing and editing; anna-maria koval, mykola shved, tetiana ivanitska – data curation, investigation, writing – original draft. порівняльна характеристика симптомів у невротичних та афективних пацієнтів після covid-19 о. венгер, а. м. коваль, м. швед, т. іваніцька тернопільський національний медичний університет імені і. я. горбачевського, тернопіль, україна вступ. пандемія covid-19 негативно вплинула на психічне здоров’я людей і створила нові бар’єри для тих, хто вже страждає на наявні психічні захворювання. мета. порівняти симптоми психічних порушень у пацієнтів з невротичними та афективними психічними розладами, які перенесли covid-19. методи. проаналізовано особливості порушень під час пандемії у 35 невротичних і 31 афективних пацієнтів. було використано: шкалу тривоги та депресії гамільтона (ham-a, ham-d), індекс вираження безсоння (isi), «таблиці шульте», «дослідження короткотривалої пам’яті за джекобсоном». результати. встановлено, що тривога помірного та тяжкого ступеня частіше розвивається у пацієнтів з невротичними розладами (34,29 % та 37,14% відповідно), а легкого і помірного ступеня – у афективних (38,71% та 32,26% відповідно). більший рівень вираженості депресії був у невротичних пацієнтів (помірна – у 31,42% осіб, тяжка – у 48,57%), ніж у афективних (переважала легка– у 32,26% та тяжка – у 35,48%). ступінь зниження пам’яті вищий у афективних пацієнтів (у 51,62% – низька, у 35,48% – дуже низька), ніж у невротичних (у 41,94% – низька, у 20,00% – дуже низька). у більшості обстежених виявлено порушення уваги та безсоння. частота безсоння більша у пацієнтів із невротичними розладами (у 25,72% осіб – субклінічне, у 45,71% – помірне), ніж в афективних (у 38,71% осіб – субклінічне, у 22,58% – помірне). вищий рівень тривоги та депресії спостерігався у пацієнтів, які хворіли у 2021, а тяжкість безсоння та порушення пам’яті – у тих, які хворіли у 2020. висновки. виявлено тривогу, депресію, безсоння, зниження пам’яті та уваги. рівень тривоги та депресії більше виражений у невротичних пацієнтів, натомість ступінь зниження пам’яті вищий у афективних пацієнтів. знижена увага спостерігалася у більшості обстежених пацієнтів із афективними та невротичними психічними розладами. ключові слова: пандемія; психічне здоров’я; депресія; тривога; безсоння. information about the authors olena venger – prof., dsc, phd, md, head of the department of psychiatry, narcology and medical psychology, i. horbachevsky ternopil national medical university, ternopil, ukraine https://orcid.org/0000­0002­5823­9415, e­mail: venger_ol@tdmu.edu.ua anna-maria koval – student, i. horbachevsky ternopil national medical university, ternopil, ukraine https://orcid.org/0000­0002­4825­4806, e­mail: koval_annvol@tdmu.edu.ua mykola shved – student, i. horbachevsky ternopil national medical university, ternopil, ukraine https://orcid.org/0000­0003­4014­7704, e­mail: shved_mykand@tdmu.edu.ua tetiana ivanitska – assistant professor of the department of psychiatry, narcology and medical psychology, i. horbachevsky ternopil national medical university, ternopil, ukraine https://orcid.org/0000­0001­7289­0514, e­mail: ivanitska_te@tdmu.edu.ua o. venger et al. 66 p sy c h ia t r y issn 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lxxiv 9(1):2175­82. doi: 10.36740/wlek202109126 received 19 august 2022; revised 4 october 2022; accepted 1 november 2022. this is open access article distributed under the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. o. venger et al. koncor1_2014.pm6 49 s p o r t s m e d ic in e ijmmr 2015 vol. 1 no. 1 anthropometric profile, body composition and vertical jump score in boxers and swimmers a. s. roy1, r. dalui1, m. kalinski2, a. bandyopadhyay1 1 university of calcutta, university colleges of science and technology, kolkata , india 2 murray state university, murray, usa background. vertical jump test (vjt) is used in some sport disciplines to evaluate an individual’s lower extremity power. however, vjt data is unavailable in young indian boxers and swimmers. objective. the given study was aimed to evaluate the vjt, anthropometric profile and body composition in young male indian swimmers and boxers and compare the data with sedentary control group. the study also explored the relationship of vjt with anthropometric parameters and different components of body composition in the studied groups. methods. male boxers (n=40), swimmers (n=40) and sedentary subjects (n=40) with similar socio-economic background with age ranging between 21 and 25 years were sampled for the study from kolkata, india. body composition was determined by skinfold measurements and vjt was evaluated by sargent jump test. results. one way anova shows significant (p<0.001) difference in body mass, %fat, total body fat (tf), lean body mass (lbm) and vjt score in boxers, swimmers and sedentary groups. significant positive correlation of lbm with vjt score was associated with a greater jumping height in swimmers. on the other hand, vjt had significant negative (p<0.05) correlation with sum of all the skinfolds, individual skinfold and total fat in both athlete group as well as in the sedentary group. conclusion. significant intergroup difference was found in the studied parameters. vjt scores obtained in all the groups were well comparable. higher value of body %fat imposed the unfavourable effect towards achieving higher jumping height mainly in sedentary group. key words: vjt, boxers, swimmers, %fat, lbm. introduction vertical jump test (vjt) is used to evaluate the leg power or leg strength which is an important component of fitness testing in athletes as well as in sedentary population [1, 2]. muscular strength is also assessed by vertical jump testing procedure [3]. plyometric exercise training improves vertical jump performance and leg strength [4]. vertical jumping and lower extremity power are significant in achieving success in volleyball players [5]. higher value of vjt of international male volleyball players showed a better performance of explosive strength and a better use of arms during jumping activities [6]. low fat percentage (%fat) appeared to coincide a better performance in vjt score also in elite male handball players [7]. to the opposite, higher %fat and bmi values exhibited poor physical fitness and lower value of vjt score in volleyball players [8]. swimming performance depends on muscular power [9], muscular endurance [10], anthropometric characteristics, as well as on body composition in relation to vjt score. swimmers need high muscle power functioning in lower limbs to achieve a sound performance [11]. the swimming start can be seen as an explosive event (jump), which requires high force production over a short period of time. there are, however, a few citations in the literature regarding the jump performance of swimmers [12]. previous study showed that amateur boxers had lower vjt score, compared to wrestlers [13]. there is no data concerning jump performance (vjt) in indian boxers and swimmers. moreover, the relationship of vjt with anthropometric parameters and body composition in swimmers and boxers has not yet been investigated in indian context. therefore, the purpose of the present study was to: evaluate the vjt, anthropometric profile and body composition in young male indian swimmers and boxers; compare the vjt, anthropometric profile and body composition between the boxers, swimmers and with sedentary control group; explore the address for correspondence: dr. amit bandyopadhyay, sports and exercise physiology laboratory, department of physiology, university of calcutta, university colleges of science and technology, 92, a. p. c. road, kolkata: 700009, india. tel.: +91 33 23508386, fax: +91 33 23519755 e-mail: bamit74@yahoo.co.in a. s. roy et al. international journal of medicine and medical research 2015, volume 1, number 1, p. 49-53 copyright © 2015, tsmu, all rights reserved 50 s p o r t s m e d ic in e ijmmr 2015 vol. 1 no. 1 relationship of vjt with anthropometric parameters and different components of body composition in the studied population. methods selection of subjects young male swimmers (n=40) and boxers (n=40) belonging to 20–24 years age group with an average of five years training experience were sampled for the study from different sports academies in kolkata, india. the sedentary control group (n=32) was randomly selected from the postgraduate section of the university of calcutta, kolkata, india. all participants had similar socio-economic background, and were disease-free, took no medication during the study period, and had no history of previous bone fracture or heavy injury. written informed consent was obtained from each participant in accordance with the policy of the university of calcutta ethics board. the study was approved by the human ethics committee of the department of physiology, university of calcutta. the study was conducted at a room temperature ranging between 20–23 °c and relative humidity ranging between 40–45 %. after coming in the laboratory, subjects took rest for half an hour. body height was measured with the subject standing barefoot with an accuracy of ±0.50 cm whereas the body mass was measured to an accuracy of ±0.1 kg by using the weight measuring instrument fitted with height measuring rod (avery india ltd., india) with the subject wearing minimum clothing. determination of body composition [14] body composition was determined by skinfold measurement using the following formulae: body density (bd; gm·cc-1) was determined by means of the following equations: bd = 1.10938 – 0.0008267x 1 + 0.0000016x 1 2 – 0.0002574x 2 (x 1 = sum of chest, abdominal, thigh skinfolds, x 2 = age in nearest yrs) %fat = 495/bd – 450 percentage of lean body mass (%lbm), total body fat, and total lbm were calculated using the following equations: • % lean body mass (%lbm) = 100 – %fat • total fat or tf (kg) = %fat/100 � body mass (kg) • lbm (kg) = body mass (kg) – total fat (kg) determination of vjt score the vjt score of each subject was measured according to the protocol elaborated in earlier reports [15]. statistical analyses data have been presented as mean±sd. one way anova was conducted to determine the significance of difference in the undertaken parameters among the studied groups. pearson’s product moment correlation was conducted to calculate the relationship between physical parameters and vjt score. regression analysis was adopted to calculate the prediction norms for predicting vjt score from different physical parameters. results values of physical and anthropometric parameters and vertical jump score in boxers and swimmers and their sedentary counterparts are shown in table 1. table 2 represents the values of correlation coefficient of vjt scores with different anthropometric parameters and body composition in boxers, swimmers and sedentary group. simple and multiple regression norms for the prediction of vjt score in indian boxers and swimmers and sedentary groups are shown in table 3 and table 4, respectively. discussion mean values of age, body weight, and body height, bmi, %fat, tf, lbm and vjt score are displayed in table 1. among the three groups, one way anova shows significant inter-group difference (p<0.05) in the parameters (body height, bmi, %fat, tf, lbm and vjt score), except the body height (table 1). bonferroni correction was performed in the cases when it was reasonable. according to the classification of arkinstall vjt scores, obtained in boxers and swimmers, were below average level [16]. table 1. values of physical and anthropometric parameters and vertical jump score in boxers and swimmers values are mean±sd, *p<0.05, #p<0.05 when compared to control, # when compared to boxer and swimmer. � ����� ���� � ���� � ��� � ����� � ��� � ���� ������� � ��� � �� � �� ����� � ��� � ���� ��� � � ��!"���� �"� � !�#�$ ���� �$%&' � ''()&*� +()� +,-(./*� &('� ,,(.+*� -(-� '&(,/*� '(),� +/()0*� &('1� 1(0*� &(.,� ),('*� )('1� &)()*� '(-� ��2��� �$%/. � ''(+'*� +(++� +,,(+,*� &(&&3!� )&(&'*� '(0+4� +1(&&*� +()+4� ,(')*� +(/-4� &(&-*� .(1)4� /(1)*� '(.,4� &-(&+*� /('0� !5������ �$%/. � ''(1)*� +(&-� +,)(''*� /()-� )0(&'*� -())4�� '+('0*� +(1.4�� +.(11*� +(0+�� ,(&1*� +()/4�� )+(1'*� -(.04� /.(0.*� /(+)4�� a. s. roy et al. 51 s p o r t s m e d ic in e ijmmr 2015 vol. 1 no. 1 in the present study vjt score of the swimmers was significantly (p<0.001) higher than the boxers, and sedentary group. papadopoulos et al. [12] reported lower values of vertical jumps performance in much younger (14.5±1.3 years old) swimmers, compared to an average of 22 years old swimmers in present study. similarly, the vjt score in boxers in present study (age of an average of 22) was higher, compared to the jumping performance of the younger subjects (male kick boxers with the age 18.5±1.85 years), reported by ouergui [13]. the variances of the results in jumping performance can be explained by the age of subjects in different studies. it is worth mentioning that in the given study vjt score of the swimmers was lower, compared to data reported for the vertical jump performance in american football players of similar age [17]. the variances of the results in jumping performance in these two studies can be attributed possibly to the special type of movements with higher specific requirements for power performance of the american football players, compared to swimmers. in the given investigation vjt score was significantly (p<0.001) higher in boxers and swimmers than their sedentary counterparts. this finding was similar to the earlier report in male elite volleyball players who also demonstrated significant positive correlation between height and vjt score [5]. malaysian male taekwondo players had significantly higher vjt score than their female counterparts [18]. � � ���� ���� � �� � ������� ��� � �� � ������� ��� � ��� ������� � ������� ���������� ��� � ����� �� � �� ��� � ! �� ��� � "# � ��� � $� � %&%%'� (%&%)*� %&%+)� (%&'%,� (%&%,%� (%&%*-� %&%++� (%&''� � � (%&.+� %&%'*� (%&..� (%&.%� (%&'-� (%&'*� (%&.*� %&.%� ���� �� � %&',� %&%)� %&.'� %&%+� %&%/� %&'/� %&'+� (%&'*� $� � � %&+/0� %&)*000� %&,)000� %&1%000� %&/,000� %&),000� (%&,.000� � � � %&')� %&1,000� %&/%000� %&1)000� %&/*000� %&)1000� (%&-.0� 2� �� � � %&/-000� %&)*000� (%&..� (%&.%� %&-100� %&)/000� %&+'0� $� � � � %&%1� %&%,-� %&%,.� %&')� %&-*0� (%&'/� � � � � (%&-)� %&''� %&''� %&'+� %&.%� (%&.,� 3� �� � � � %&,+000� (%&%+� (%&%.� %&-/0� %&/.000� %&+*0� $� � � � � %&1.000� %&1+000� %&/,000� %&//000� (%&,%000� � � � � � %&,-000� %&,+000� %&,/000� %&1+000� (%&'/� ��� �� � � � � (%&.,� (%&.-� %&-+0� %&)-000� %&.'� $� � � � � � %&))000� %&)*000� %&-,00� (%&,,000� � � � � � � %&)1000� %&))00� %&,+00� (%&-'0� ������� ���������� �� � � � � � %&))000� %&1-000� (%&+*0� (%&-+0� $� � � � � � � %&),000� %&-100� (%&,,000� � � � � � � � %&))000� %&,.000� (%&-%0� ����� �� � � � � � � %&1*000� (%&+-0� (%&-*0� $� � � � � � � � %&,/000� (%&1%000� � � � � � � � � %&1%000� (%&-.0� �� �� � � � � � � � %&+-0� (%&.%� $� � � � � � � � � (%&*%00� � � � � � � � � � (%&+/0� ! �� �� � � � � � � � � %&+,0� � � � � � � � � � �"# � �� � � � � � � � � � table 2. values of correlation coefficient of vjt scores with different anthropometric parameters and body composition in boxers, swimmers and sedentary group ***p<0.001, **p<0.01,*p<0.05, b = boxer group, s = swimmer group, c= sedentary group. table 3. simple regression norms for the prediction of vjt score in indian boxers and swimmers ������ �� � ���� �������� ��������� ������ �� ���������� ��������� !�"#�$%��� &�!'� (�)��� �������*� +�+'�$%��� ,��&� �-� ��� ����*&�!' +�!&�$%��� ,�"+� ���������� �����*�&' !�&,��� &�!�� (�)��� �����'�&, !�,"��� ,��!� �-� ��� ����*&�*� !�,!��� ,�"*� ���������� ����*!�*# !�&'�.(/� &�,�� (�)��� ����"'�,& !�"#�.(/� ,��'� �-� ��� ����&��'�0!�&+�.(/� ,�#"� s = sum of skinfolds, lbm = lean body mass a. s. roy et al. 52 s p o r t s m e d ic in e ijmmr 2015 vol. 1 no. 1 earlier studies reported significant positive correlation of height with vjt score in male elite volleyball players [5]. in the given study significant positive correlation was found between height and vjt score in swimmers. this finding corroborated with the earlier study in malaysian martial art players [19]. vertical jump score of athletes could be predicted by %fat which is related to the work performed during vertical jump [20]. previous research suggests that a reduction of body fat by proper dietary planning would help to develop leg power [21]. in the present study the vjt score had significant (p<0.001) negative correlation with the individual skinfold, sum of skinfolds, %fat and tf. however, lbm showed significant (p<0.05) negative correlation with vjt score in boxers only. analogous findings were also reported in malaysian martial art players [19]. similarly, poor vjt score in professional rugby league players was attributed to the higher value of %fat [22]. generally, sedentary individuals have a larger body %fat. hence, athletes with lower body %fat have advantages in vertical jumps. body %fat is the best predictor of vertical jump for recreational male athletes. in the present study, sum of skinfold thicknesses has significant negative correlation with vjt score [1,20]. therefore, an individual who has lower body fat percentage would be able to make more jumping height [18,23]. simple regression equations have been calculated to find out norms for the prediction of vjt score from %fat, sum of skin folds and total fat (tf). multiple regression equations were also calculated to predict vjt score in both the athlete groups from sum of skinfolds and total fat (tf). in the multiple regression equations %fat was not considered as a predictor variable since it was calculated from the sum of skinfolds. standard errors of estimate (see) of the computed multiple regression equations were smaller than the simple regression equations and the values of these see were substantially small enough to recommend the multiple regression norms for practical use in epidemiological studies and also in clinical settings. conclusion one way anova showed significant (p<0.001) difference in body mass, %fat, total body fat (tf), lean body mass (lbm) and vjt score in boxers, swimmers and sedentary groups. the vjt scores, obtained in the athlete groups, were below average, compared to data from other studies. higher value of body %fat imposed the unfavourable effect on achieving higher jumping height mainly in sedentary group. significant positive correlation of lbm with vjt score was associated with a greater jumping height in swimmers. on the other hand, vjt had significant negative (p<0.05) correlation with sum of all the skinfolds, individual skinfold and total fat in both athlete group as well as in the sedentary group. table 4. multiple regression norms for the prediction of vjt score in indian boxers and swimmers lbm = lean body mass references 1. roschel h, batista m, monteiro r, bertuzzi rc, barroso r, loturco i. association between neuromuscular tests and kumite performance on the brazilian karate national team. j sports sci med 2008; 8: 20–24. 2. sargent d. the physical test of a man. am phys edu rev 1921; 26: 188–194. 3. ostojic sm, stojanovic m, ahmetovic z. vertical jump as a tool in assessment of muscular power and anaerobic performance. med pregl 2010; 63(5-6): 371– 375. 4. ioannis g, athanasios z, leontsini 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!�%"�&'(� !�)!��*+!�!!%�� !���!%� %��)� '�,��� ��������% %�""�#$�� !����&'(� !��-��*+!�!"�� !�%.��� -�.�� �/������ ����-)��! !�.)�#$�� !�"��&'(� !�0%��*+!�!%�� !�"�!%� -�0)� a. s. roy et al. 53 s p o r t s m e d ic in e ijmmr 2015 vol. 1 no. 1 9. hawley ja, williams mm, vickovic mm, handcock pj. muscle power predicts freestyle swimming performance. br j sports med 1992; 26: 151–155. 10. wakayoshi k, lj d’acquisto, cappaert jm, troup jp. relationship between oxygen uptake, stroke rate and swimming velocity in competitive swimming. int. j sports med 1995; 16: 19–23. 11. mameletzi d, siatras t. sex differences in isokinetic strength and power of knee muscles in 10–12 year old swimmers. isokinet exerc sci 2003; 11: 231–237. 12. papadopoulos c, sambanis m, gissis i, noussios g, gandiraga e, manolopoulos e, papadimitriou id. evaluation of force and vertical jump performance in young swimmers with different force-time curve characteristics. biol sport 2009; 26: 301–307. 13. ouergui i, 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briscope da, markowski ct, saville se, taylor cj. physical characteristics that predict vertical jump performance in recreational male athletes. phys ther sport 2003; 4: 167–174. 21. shin n, hyun w, lee h, ro m, song k. a study on dietary habits, health related lifestyle, blood cadmium and lead levels of college students. nutr res pract 2012; 6(4): 340–348. 22. gabbett tj. physiological and anthropometric characteristics of amateur rugby league players. br j sports med 2000; 34: 303–307. 23. dizon jmr, somers kg. making filipino taekwondo athletes internationally competitive: an international comparison of anthropometric and physiologic characteristics. j sport med doping studies 2012; 2: 1–7. received: 2014.06.12 a. s. roy et al. 75 b io m e d ic a l s c ie n c e s issn 2413-6077. ijmmr 2017 vol. 3 issue 1 v. v. ilika doi 10.11603/ijmmr.2413-6077.2017.1.7051 morphological research on free-residue oxidation processes in cases of decidual cells of placenta in chorioamnionotis and basal deciduitis combined with iron-deficiency anemia in the pregnant v. v. ilika bukovinian state medical university, chernivtsi, ukraine background. the oxidative modification of proteins is lately pivotal to pathologists and it is a new way of research on different pathological conditions, as well as the diagnostics of inflammation processes in placenta. objective. the study was aimed at the research of nitro peroxides and establishing the specific features of oxidative modification of proteins in inflammation of placenta with iron deficient anaemia in the pregnant. methods. сhemiluminescent and histochemical technique (with bromphenol blue on ‘acidic’ and ‘basic’ proteins according to mikel calvo) was applied. results. the intensity of nitro peroxides glow in chorioamnionitis and basal deciduitis increased in comparison with the samples of physiological and iron deficient anaemia gestation. at the same time in chorioamnionitis the glow intensity is higher than in basal deciduitis. due to the results of immune histochemical technique held while analysing the samples, together with chorioamnionitis and basal deciduitis the r/b increases and in basal deciduitis the rate, is probably, higher, than in chorioamnionitis. at the same time, the extent of oxidative modification of proteins in cases of inflammation with iron deficient anaemia in the pregnant is on the average higher than with no iron deficient anaemia in the pregnant. conclusions. high level of nitro peroxides in placentae basal plate in secundines inflammation, the increase in r/b rate, in other words the prevalence of ‘acidic’ proteins over ‘basic’ ones, is evidenced due to the increase of the intensity of oxidative modification processes of proteins in cases of deciduitis. key words: inflammation; placenta; anaemia; iron-deficiency. introduction the formation of active forms of oxygen and oxidative modification of macromolecules is usual and important biological processes. but the creation of oxygenous residues excess could damage cells and facilitate the development of many illnesses [1], and in certain cases, a molecular pathology of the organism [2]. the oxidative modification of proteins is lately pivotal to pathologists [3], and it is a new way of research on different pathological conditions [4], as well as the diagnostics of inflammation processes in placenta [5]. in this area the scholars used a range of techniques aimed at detection of nitro peroxides [6], as the most responsive and long lasting oxidants among free residues, and at estimation of the degree of protein oxidative modification in various structures of placenta [7]. however, this aspect in combination with iron deficient anaemia in the pregnant has not been explored yet [8] and has been being studied that is the objective of our study. objective chemiluminescent technique with luminol was applied in the research of nitro peroxides in cases of inflammation. the aim of the study was to establish the specific features of oxidative modification of proteins in cases of deceduitis of basal plate in long lasting basal deciduitis and chorioamnionitis in combination with iron deficient anaemia and without it in the pregnant by means of immune histochemical and micro spectral photometric procedure. corresponding author: vitaliy ilika, department of pathologic anatomy, bukovinian state medical university, 15 chelyuskintsev street, chernivtsi, 58000. phone number: +380951477073 e-mail: ilika.vitaliy@mail.ru international journal of medicine and medical research 2017, volume 3, issue 1, p. 75–78 copyright © 2017, tsmu, all rights reserved 76 b io m e d ic a l s c ie n c e s issn 2413-6077. ijmmr 2017 vol. 3 issue 1 methods the control group consisted of placenta (n=20) received at the urgent delivery of clinically healthy women in childbirth and in cases of iron-deficiency anaemia in gravidas (n=21). experimental group consisted of the placenta (n=123) received from women in childbirth at gestation period of 37-40 weeks with chorioamnionitis and basal deciduitis combined with iron deficiency anemia in gravidas and without it. the first procedure (a chemiluminescent technique) was performed on the frozen sections of placenta. the luminol originated chemiluminescence was studied under the luminescent microscope lumam p8. on digital photomicrographies, obtained by means of computer estimation of glow intensity in a 256 gradations scale: from 0 (glow absence indicator) to 255 (glow top intensity), the quantitative measuring of luminescence was carried out [8]. for validity estimation of averages contrast student’s t-test was used, after positive verification of the samples for normal distribution in it was determined by the shapiro-wilk test (computer program past 3. 14, free license, o. hammer, 2016) [9]. for approval or rejection of statistic hypothesis the significant difference p≤0.05 was used. to evaluate the extent of oxidative modi fication of proteins, histochemical technique was carried out with bromphenol blue on ‘acidic’ and ‘basic’ proteins according to mikel calvo [10]. optic images were converted into digital using the digital camera and were analysed with licensed copy of the imagej computer program (1.48, w. rasband, national institute of health, usa) using probe computer spectrometry in the system of colour analysis rgb. according to the designated system of colour analysis the intensity of red and blue colours in coloration was assessed. according to the mikel calvo technique, red coloration matches carbonyl groups, whereas blue coloration matches protein amines; therefore establishment of mathematical relation between coloration intensity in two colours (spectrum areas) the r/b was received and the extent of oxidative modification of proteins was evaluated (relation of carboxylic and amines) [11,12]. results chemiluminescent glow of nitro peroxides was evaluated and the level of physiological gestation of placentae was estimated in cases of iron deficient anaemia in the pregnant. the averages are introduced in table 1. the results of quantitative indicators of chemiluminescent glow of nitro peroxides and the r/b rate in cases of deciduitis of placenta basal plate in acute and long lasting chorioamnionitis, and basal deciduitis in combination with iron deficient anemia in the pregnant are presented in table 2. discussion according to the data above, we should emphasise that in physiological gestation of pregnancy in cases of iron deficient anaemia, significant deviations in any conducted technique was not evidenced. the data in table 2 proves that in chemiluminescent research technique the intensity of nitro peroxides glow in cases of chorioamnionitis and basal deciduitis increases in comparison with the samples of physiological and iron deficient anaemia gestation. the fact that, in cases of placentitis with underlying iron deficient anaemia in the pregnant, the quantitative indicators are higher than with no anaemia. at the same time, in cases of chorioamnionitis, the glow intensity is higher than in basal deciduitis. table 1. the quantitative indicators of chemiluminescent nitro peroxide glow and r/b rate (histochemical technique on ‘acidic’ and ‘basic’ proteins with bromphenol blue according to mikel calvo) in cases of deciduitis of placenta basal plate in physiological gestation and iron deficient anaemia in the pregnant basal pate structures research groups observation of physiological gestation (n=20) observation of iron deficient anemia in the pregnant without inflammation of secundines (n=21) decidual cells of basal plate chemilumi nescent glow of nitro peroxides with luminol histochemical technique on ‘acidic’ and ‘basic’ proteins with bromphenol blue on mikel calvo chemilumi nescent glow of nitro peroxides with luminol histochemical technique on ‘acidic’ and ‘basic’ proteins with bromphenol blue on mikel calvo 34±3.8 1.04±0.008 38±4.2 p>0.05 1.06±0.009 p>0.05 v. v. ilika 77 b io m e d ic a l s c ie n c e s issn 2413-6077. ijmmr 2017 vol. 3 issue 1 v. v. ilika the photomicrographs of histological images give an idea of how some structures of placental basal plate look like, decidual cells in particular, when the immunohistochemically technique for ‘acidіc’ and ‘basic’ proteins according to mikel calvo is applied (fig.1). when assessing visual histochemical preparations, decidua cells are clearly stained that is applicable for quantitative research, cell boundaries are defined by clear cell membrane colouring and contrasting colour around decidua cells fibrinoid. nuclei and nucleoli were visualized fairly well. ‘basic’ proteins prevailed in nucleoplasm, while ‘sour’ in the nucleolus. the decidua cells cytoplasm specific colour was mostly granular in nature and spectral characteristics and optical density of colour varied greatly. relatively invariable data in assessing the degree of oxidative modification of proteins in placentas of physiological gravidity and gravidas are observed, which can be interpreted as the manifestation of involutory changes in placenta. the r/b factor is only slightly higher than what can be evaluated as a slight predominance of ‘sour’ proteins over ‘basic’ ones. these figures are important in terms of the r/b factor assessing, as an indicator of oxidative modification of proteins. however while analysing the samples with chorioamnionitis and basal deciduitis the r/b increases and in basal deciduitis the rate is probably higher than in chorioamnionitis. at the same time, the extent of oxidational modification of proteins in cases of inflammation in combination with iron deficient anaemia in the pregnant is on the average higher than with no iron deficient anaemia in these patients. taking into account our findings of high level of nitro peroxide in basal lamina of placentable 2. quantitative indicators of chemiluminescent glow of nitro peroxides and the r/b rate (histochemical technique on ‘acidic’ and ‘basic’ proteins with bromphenol blue on mikel calvo) in cases of deciduitis of placenta basal plate in acute and long lasting chorioamnionitis and basal deciduitis in combination with iron deficient anemia in the pregnant research groups chemiluminescent glow of nitro peroxides with luminol histochemical technique on ‘acidic’ and ‘basic’ proteins with bromphenol blue on mikel calvo r/b rate (m=m) secundines inflammation secundines inflammation in cases of iron deficient anaemia in the pregnant secundines inflammation secundines inflammation in cases of iron deficient anaemia in the pregnant acute chorioamnionitis 154±4.9 (n=20) 186±5.1 (n=21) p=0.003 1.24±0.011 (n=20) 1.64±0.016 (n=21) p<0.001 acute basal deciduitis 130±4.4 (n=21) 164±4.5 (n=20) p=0.002 1.89±0.015 (n=21) 2.14±0.018 (n=20) p<0.001 fig. 1. photomicrograph of placental basal plate. нistochemical technique for ‘acidic’ and ‘basic’ proteins with bromphenol blue according to mikel calvo. on the right – observation during physiological pregnancy. on the left – in cases of chronic deciduitis in placental basal plate. | 50 micrometers | | 50 micrometers | 78 b io m e d ic a l s c ie n c e s issn 2413-6077. ijmmr 2017 vol. 3 issue 1 tas which are characterized by secundines inflammation, increase in, i.e. the prevalence of ‘sour’ proteins over ‘basic’ ones; correlation between the increasing intensity of oxidative modification of proteins in cytoplasm of deciduitis and secundines inflammation would be reasonable. conclusions due to the chemiluminescent technique of nitro peroxides evaluation and immune histochemical technique of ‘acidic’ and ‘basic’ proteins assessment in cases of deciduitis of placenta basal plate, the impetuous increase of indicators is observed. the inflammation of secundines in combination with iron deficient anaemia in the pregnant was evidenced by higher average rates than with no anaemia. considering the received data that proves high level of nitro peroxides in placentae basal plate in secundines inflammation, the increase in r/b rate, in other words prevalence of ‘acidic’ proteins over ‘basic’ ones, is evidenced due to the increase of intensity of oxidative modification processes of proteins in cases of deciduitis. references 1. reactive oxygen species and oxidative modification of macromolecules: the benefits, harm and protection. http://www.pereplet.ru/obrazo vanie/ stsoros/696.html 2. gubskyi yi. cell death: free radicals, necrosis, apoptosis: the monograph. vinnytsia new book. 2015;67–101. 3. ben messaoud ershyd, davydenko is. protein oxidative modification in the cytoplasm of epithelial cells of the endometrium in its various states of nonneoplastic and neoplastic nature. clinical anatomy and operative surgery. 2008;1:25–29. 4. muravleva le, molotov-luchanskyi vb, kliuiev da, bakenova ra, kultanov bj, tankibaeva na, koikov vv and oth. protein oxidative modification: problems and research perspectives. fundamental research. 2010;1:74–78. 5. benirschke k, burton gj, baergen rn. pathology of the human placenta. new york springer. 2012;6:974. 6. davydenko is, davydenko om. nitro peroxide chemiluminescent determination in the cytoplasm of placental plasmodium chorionic villi in the placenta with purulent chorioamnionitis. proceedings of viii international congress of pathologists of ukraine "modern problems of pathological anatomy". poltava. 2008;26–27. 7. shenderiuk op, davydenko is. method of measuring protein oxidative modification in the structure of placenta. patent of ukraine for invention №13712 u.–17.04.2006. bull. №4. 2c. (appl 14.10.2005, №u200509673, bsmu, shenderiuk op, davydenko is. protein oxidative modification in the cytoplasm of placental plasmodium chorionic villi in the pla centa with purulent chorioamnionitis (histochemical data). the world of medicine and biology. 2008;2(3):88–90. 8. іlіka vv, davydenko іs. nitro peroxide chemiluminescent research in the nidus of inflammation in chorioamnionitis and basal deciduitis in gravidas with iron deficiency anemia. prospective directions of modern perinatology. proceedings of the scientific-practical conference with international participation on the occasion of the 100th anniversary of professor boryma tv birthday. chernivtsi medical university. 2014;108–111. 9. hammer o. past: paleontological statistics, version 3.14. reference manual. oslo natural history museum university of oslo. 2016;243. 10. davydenko is. standardization measures of histochemical methods in terms of protein oxidative modification. ukrainian medical almanac. 2013;3: 180–181. 11. vorobiova li, peresunko op, zelinska nv, davydenko oi. computer microspectrophotometry of the system "epithelium-connective tissue" in cervical cancer diagnosis. http://dspace.bsmu.edu. ua:8080/xmlui/handle/123456789/3890. accessed 2012. 12. davydenko is, davydenko om. histochemical peculiarities in protein oxidative modification in the cells of the renal glomeruli in glomerulonephritis with acute postinfectious glomerulonephritis. bukovyna medical journal. 2012;16(3):106–107. received: 2017-01-17 v. v. ilika 39 b io m e d ic a l s c ie n c e s issn 2413-6077. ijmmr 2016 vol. 2 issue 1 doi 10.11603/ijmmr.2413-6077.2016.1.6380 levels of nitric oxide metabolites in rats with hepatopulmonary syndrome i. ya. krynytska i. horbachevsky ternopil state medical university, ternopil, ukraine background. system of nitric oxide (no), which consists of no, and its metabolites, is very important for various biological processes. no is signalling molecules and mediators of intracellular and intercellular interaction that causes relaxation of smooth muscles of blood vessel walls, inhibits platelet aggregation and their adherence, is involved in the transmission of nerve impulses, cell proliferation. objective. the aim of our research was to study the content of nitric oxide metabolites in blood serum and bronchoalveolar lavage, to substantiate their role in pathogenesis of hepatopulmonary syndrome in experiment. methods. the experiments were performed on 56 outbread male rats, 180-220 g in weight. the first experimental model of hepatopulmonary syndrome (hps) was made by imposition of double ligature on common bile duct and its further dissection with a scalpel. the second experimental hps model was made by 8-week intragastric administration of oil solution ccl4 (400 g per 1 l), 0.5 ml per 100 g of body weight on the first day of the experiment, 0.3 ml per 100 g on the third day of the experiment and then every third day until the end of the experiment 0.3 ml per 100 g. a mixture of corn flour, lard and cholesterol and alcohol solution was added to the standard diet of the rats. results. the total content of nitric oxide metabolites in blood serum of the rats of the experimental group no.1 (on the 31st day after the common bile duct ligation) was significantly increased in 3.9 times (p1<0,001) if compared with the control group №1. in the rats of the 2nd experimental group (with carbon tetrachloride induced cirrhosis) the total content of nitric oxide metabolites in blood serum also significantly increased in 3.1 times (p1<0,001). comparison of nitric oxide metabolites content in blood serum and bronchoalveolar lavage, which directly indicated about the processes in lung tissue, was great importance. conclusions. so, in rats with experimental hepatopulmonary syndrome activation of nitroxydergic process by significant increase in nitric oxide metabolites in blood serum and bronchoalveolar lavage took place. keywords: hepatopulmonary syndrome, nitric oxide metabolites. introduction system of nitric oxide (no), which consists of no, and its metabolites, is very important for various biological processes [2]. no is signalling molecules and mediators of intracellular and intercellular interaction that causes relaxation of smooth muscles of blood vessel walls, inhi­ bits platelet aggregation and their adherence, is involved in the transmission of nerve im­ pulses, cell proliferation. cytostatic activity is also presented in no. formation of this agent by immunocompetent cells provides protection of body from being infected by bacteria and cancer cells. the researches on participation of no in the process of apoptosis are very inte­ resting [1, 5, 10]. contemporary studies on pulmonary disorders are also associated with impaired nitroxidergic dysfunction [3, 7]. no is a molecule of high reactivity with an effective half­life from 2 to 30 sec, which is formed by the enzymatic oxidation of l­arginine under the influence of cytochrome p-450-like hemoproteins – no­synthase (nos). there are 3 isoforms of this enzyme, endothelial (enos), neuronal (nnos) or brain and inducible (inos) or macrophagal [4, 6]. as a lipophilic molecule, no easily diffuses through cell membranes into the neighbouring cells (e.g. from endothelial to myocytes of vessels) where the formed cyclic guanosine monophosphate decreases the level of free calcium and activates the kinase of myosin light chain causing dilatation of vessel [4]. i. ya. krynytska corresponding author: inna krynytska, department of clinical and laboratory diagnostics, i. horbachevsky ternopil state medical university, 1 maidan voli, ternopil, ukraine, 46001 tel.: +3800352254577 e-mail: krynytska@tdmu.edu.ua international journal of medicine and medical research 2016, volume 2, issue 1, p. 39–43 copyright © 2016, tsmu, all rights reserved 40 b io m e d ic a l s c ie n c e s issn 2413-6077. ijmmr 2016 vol. 2 issue 1 most cytotoxic effects of no belong to onoo that is formed in reaction with superoxide. peroxynitrite is much more active, nitrosates proteins intensively and can be a source of a highly toxic hydroxyl radical in reaction with superoxide anion radical. onooirreversibly inhibits enzymes of respiratory chain nitrosating them and taking iron away. inhibition of mitochondrial respiration can cause apoptosis [9]. production of no by alveoli can influence the hemodynamic and gas exchange in patients with liver cirrhosis. thus, a direct relationship between alveolar products of no and hyperdynamic type of circulation was established [12]. moreover, in experimental liver cirrhosis in rats, hyper-expression of both inducible and constitutional isoforms of no were observed – synthase in alveolar macrophages and lung endothelial cells [22]. the average life span of nitric oxide in the body is a few seconds. nitric oxide, which did not participate in chemical reactions, is rapidly oxidized to inactive compounds: nitrites and nitrates. these are nitric oxide stable metabolites, which are the method of this compound synthesis intensity evaluation [18]. so, the aim of our research was to study the content of nitric oxide metabolites in blood serum and bronchoalveolar lavage, to substantiate their role in pathogenesis of hepatopulmonary syndrome in experiment. material and methods the experiments were performed on 56 outbread male rats, 180–220 g in weight. during the simulation of the pathology 8 animals died. the first experimental model of hepatopulmonary syndrome (hps) was made by imposition of double ligature on common bile duct and its further dissection with a scalpel. [15] in the control group of animals № 1, common bile duct was separated from the tissue, but not dissected. postoperative wound was sewed up completely in layers. in the 31st day after the surgery the animals were taken out of experiment under thiopental anaesthesia. the second experimental hps model was made by 8-week intragastric administration of oil solution ccl4 (400 g per 1 l), 0.5 ml per 100 g of body weight on the first day of the experiment, 0.3 ml per 100 g on the third day of the experiment and then every third day until the end of the experiment 0.3 ml per 100 g. a mixture of corn flour, lard and cholesterol and alcohol solution was added to the standard diet of the rats. the control group of animals № 2 was on a standard diet of the vivarium and was administered intragastrically the equivalent amount of olive oil. [21]. animal care and experiments were performed in accordance with the european convention for the protection of animals used for experimental and other scientific purposes [14]. blood serum and bronchoalveolar lavage (bal) were the subjects of the research. quantitative assessment of no metabolites content was performed by evaluation of their amount, which included nitrite ions that were previously presented in the sample (no2 –) and also nitrate ions restored to nitrites (no3 –) [2]. recovery was performed using zinc dust in acidic environment. nitrites with sulphanilic acid underwent a reaction of diazotization, obtained diazotization solution of n-1 – naftyletylendiamin formed azo dye. optical density of the obtained colour solution was evaluated by spectrophotometry at absorption maximum and wavelength 536 nm. according to the evaluation results of calibration solutions optical density (y), calibration straight line was built and regressor was estimated: y=a+bx, y is optical density of calibration solutions; x – concentration of calibration solutions, mmol/l; b – regression coefficient; a – intercept. the concentration of no metabolites in the studied sample was estimated by the equation: x1=(y1–a)/b, y1 is optical density of the studied sample. statistical analysis of the data received was conducted by standard methods of variation statistics using statistical software package. results are presented as (m±m), m is mean value, m – standard error. statistical significance of the studied rates was determined by means of paired t-test. correlation analysis was performed between the data studied. linear correlation coefficient (r) and its significance (b) appropriately denoted in the tables (correlation matrices) were evaluated. if index r=0, link was considered to be lost, range 0–0,3 evidenced about weak correlation, index interval 0.3–0.7 demon-–0.7 demon-0.7 demonstrated medium link, and interval 0,7–1,0 proved a significant correlation interaction. the correlation coefficient was significant at p<0.05. results and discussion the total content of nitric oxide metabolites (no2 –+no3 –) are presented in table 1. the total content of nitric oxide metabolites in blood serum of the rats of the experimental i. ya. krynytska 41 b io m e d ic a l s c ie n c e s issn 2413-6077. ijmmr 2016 vol. 2 issue 1 group № 1 (on the 31st day after the common bile duct ligation) was significantly increased in 3.9 times (p1<0,001) if compared with the control group № 1. in the rats of the 2nd experimental group (with carbon tetrachloride induced cirrhosis) the total content of nitric oxide metabolites in blood serum also significantly increased in 3.1 times (p1<0,001). comparison of nitric oxide metabolites content in blood serum and bronchoalveolar lavage, which directly indicated about the processes in lung tissue, was great importance. it was determined that no production disorders took place unidirectionally towards the oxidative stress flare. thus, the total content of nitric oxide metabolites in bal (table 1) in the rats of the experimental group № 1 also significantly increased in 5,8 times (p1<0,001), and in the rats of the experimental group № 2 – in 4.5 times (p1<0,001). the correlative analysis showed that, in simulation of hepatopulmonary syndrome by common bile duct ligation, total content of nitric oxide metabolites in blood serum had strong positive correlative link with the content of no2 –+no3 – in bal (r=0,87) (p<0,01). in carbon tetrachloride induced cirrhosis (experimental mo del № 2) the total content of nitric oxide metabolites in blood serum also had a strong positive correlative relationship with the content of no2 –+no3 – in bal (r=0,84) (p<0.01). this evidenced the unidirectionality of changes in nitroxydergic processes in blood and lungs in cases of hepatopulmonary syndrome of the applied models. probably, the synthesis of nitric oxide in cases of experimental hepatopulmonary syndrome increased due to the activation of inducible no-synthase under the influence of proinflammatory cytokines and endotoxins, which caused increase in production of no by liver kupffer’s cells and alveolar macrophages. our results coincide with the studies of other authors. m. b. fallon et al. defined and emphasized the role of no in experimental model of liver cirrhosis, where overexpression of enos by pulmonary vessels caused increase in production of endothelin-1 (et-1) by cholangiocytes, whereby expression of endothelin receptors type b to et-1 at pulmonary vessels and table 1. nitric oxide metabolites content in blood serum and bronchoalveolar lavage in rats with experimental hepatopulmonary syndrome (m±m) experimental group control group № 1 (n=12) experimental group № 1 (n=12) control group № 2 (n=12) experimental group № 2 (n=12) blood serum no2 –+no3 –, mcmol/l 36,7±6,0 143,4±14,8 p1<0,001 33,4±4,4 104,2±9,3 p1<0,001 p2<0,05 bal no2 –+no3 –, mcmol/l 14,1±3,2 81,7±7,6 p1<0,001 12,0±3,2 54,7±6,9 p1<0,001 p2<0,05 legends: p1– significant difference if compared to the control animals; р2 – significant difference if compared to the affected animals. fig. 1. comparison of nitric oxide metabolites content in blood serum and bronchoalveolar lavage (* – significant difference if compared to the control animals (p<0,001); # – significant difference if compared to the affected animals (p<0,05). 0 20 40 60 80 100 120 140 blood serum bal control group № 1 experimental group № 1 control group № 2 experimental group № 2 i. ya. krynytska 42 b io m e d ic a l s c ie n c e s issn 2413-6077. ijmmr 2016 vol. 2 issue 1 synthesis of nitric oxide increased [16]. the level of no in expired air increased in patients with hps, and turned to normal in 3–12 months after liver transplantation. [19] degano b. et al. in a similar study found out that concentration of no in expired air in patients with liver cirrhosis was 3 times higher than that in the noncancer [12] by means of the method of flow cytofluorimetery that allows to differentiate alveolar and bronchial origin of no, the main alveolar increase in formation of no was determined [13]. it was revealed that no production by alveoli can influence hemodynamic disturbances and changes in gas exchange in patients with liver cirrhosis. thus, a close relationship between alveolar production of no and hyperdynamic circulation type was defined [20]. moreover, in experimental liver cirrhosis in rats, overexpression of both inducible and constitutional isoforms of no-synthase in alveolar macrophages and lung endothelial cells was observed [22]. the further studies of no showed that despite all mentioned above, relationship of no with portal hypertension, hyperdynamic circulation type and degree of liver damage is unclear. [17] in addition, other molecular mechanisms of vasodilation –nitric oxide independent: enzymatic formation of co by increase in expression of heme-oxygenase-1, enzymatic formation of h2s and stimulation of calcium-activated potassium channels through endothelial derivative – hyperpolarization factor are described in the literature [8, 11]. conclusions and further research 1. so, in rats with experimental hepatopulmonary syndrome activation of nitroxydergic process by significant increase in nitric oxide metabolites in blood serum and bronchoalveolar lavage took place. 2. after studying the results of nitric oxide metabolites content in blood serum and bronchoalveolar lavage, synchronous development of nitroxydergic processes on systemic and local levels and predominance of nitric oxide synthesis in lungs was determined. in the future, pro-inflammatory cytokines rate in rats with experimental hepatopulmonary syndrome should be studied for more profound pathogenetic substantiation of nitroxydergic processes intensification. fig. 2. comparison of increase in intensity of nitric oxide metabolites content in blood serum and bronchoalveolar lavage (no2 –+no3 – content in both control groups was equated to 100 %). 0 100 200 300 400 500 600 blood serum bal control group № 1 experimental group № 1 control group № 2 experimental group № 2 references 1. боярчук ор. вміст метаболітів оксиду азоту та прозапальних цитокінів у хворих із гострою ревматичною лихоманкою та хронічною ревматичною хворобою серця. український ревматологічний журнал 2010; 3 (41): 9-13. 2. козар вв, кудря мя, устенко нв, нікішина лe, кравченко св. визначення концентрації метаболітів оксиду азоту в сироватці крові. лабораторна діагностика 2010; 3 (53): 14–16. 3. марущак мі. нітроксидергічні аспекти патогенезу гострого ураження легень в експерименті. туберкульоз, легеневі хвороби, віл-інфекція 2011; 3 (6): 69-73. 4. сапатий ал, купновицька іг. метаболічні особливості оксиду азоту у формуванні 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krynytska issn 2413-6077. ijmmr 2017 vol. 3 issue 1 47 d e n t is t r y doi 10.11603/ijmmr.2413-6077.2017.1.7105 the effectivness of chronic gingivites treatment in patients with non-removable orthodontic apparatus according to the results of periodontal tissues index assessment ye. ya. kostenko, v. s. melnyk, l. f. horzov uzhhorod national university, uzhhorod, ukraine background. the risk of chronic gingivitis is increased in patients who undergo orthodontic treatment. it is known that the gingivitis is closely correlated with the level of hygiene. objective. the study involved 123 orthodontic patients with chronic catarrhal and hypertrophic gingivitis that developed during the first two months of active orthodontic treatment. we chose vitis ortнodontiс (dentaid international, spain) that contains active ingredients we need to support healthy state of oral cavity. all studied patients were 12–15 years old. methods. for dental examination we used health indices ohi-s according greene-vermillion (1964). gums bleeding was determined according to a modified sbi index by muhlemann (1971), inflammation of the gingival margin was assessed by pma index parma (1960). results. therapeutic treatment consisted of the following: all patients underwent correction of oral hygiene, removal of dental plaque. vitis orthodontic was prescribed due to the manufacturer's recommendations: rinse 15 ml for 30 seconds after normal procedures of oral hygiene. eating or drinking is not recommended during the next 30 minutes after using this product. the results proved a high anti-inflammatory efficacy of the treatment schemes. conclusions. the complex therapy of early manifestations of inflammation in periodontal had a positive effect on the subjective feelings of patients and health performance rate, gum inflammation and bleeding. key words: orthodontic patients; chronic gingivitis; dental examination; oral hygiene. corresponding author: lyudmyla horzov, children dentistry department, uzhhorod national university, 16a universytetska street, uzhhorod, ukraine, 88000 phone number: +380507626129 e-mail: lyudmila_buley@list.ru introduction dentition abnormalities impair the hygienic condition of the mouth, cariogenic effect exacerbate the situation and increase the risk factors of periodontal diseases. several researchers indicate high percentage of periodontal abnormalities with dentoalveolar lesions [4, 6, 9, 10]. thus, the prevalence of periodontal diseases in patients requiring orthodontic treatment was 81.4%. due to the anne-marie bollen symptoms of periodontal they were determined in 89.3% patients. periodontal tissues were affected in all kinds of bite anomalies [1]. thus gingivitis is in deep (46.3%) and an open (43.7%) occlusion, mesial (37.0%) and oblique (33.3%) bite [2]. the clinical picture may correspond to varying degrees of severity of periodontal tissues diseases. some authors [3, 5] diagnosed chronic periodontitis in 70%, gingivitis – in 30%, including chronic catarrhal gingivitis – in 15%, medium gravity of hypertrophic gingivitis – in 15% of people. the aim of the study was to improve the efficiency of treatment of catarrhal and hypertrophic gingivitis in patients with fixed orthodontic apparatus in mouth when using rinse aid. methods the examination and treatment of 123 orthodontic patients in permanent dentition period with symptoms of chronic catarrhal and hypertrophic gingivitis that were determined two months after the beginning of active orthodontic treatment by the technique of permanent braces version ‘the direct arc’. the patients' age was 12–15 years: 59 (47.9%) boys, 64 (52.1%) girls. all patients were almost healthy. two weeks before the beginning of orthodontic treatment, all patients were subjected to professional teeth cleaning procedure to remove all deposits and external staining, and ye. ya. kostenko et al. international journal of medicine and medical research 2017, volume 3, issue 1, p. 47–49 copyright © 2017, tsmu, all rights reserved issn 2413-6077. ijmmr 2017 vol. 3 issue 148 d e n t is t r y then tooth surface was polished. before the beginning of the study instructions on oral hygiene were provided for the patients and a standard method of teeth cleaning was recommended. in a study patients health indices ohi-s according to greene-vermillion (1964) were used. gums bleeding was determined by a modified sbi index by muhlemann (1971), inflammation of the gingival margin was assessed by pma index parma (1960) [7]. the methods of therapeutic treatment consisted of the following: all patients underwent correction of oral hygiene, removal of dental plaque. vitis orthodontic was prescribed due to the manufacturer’s recommendations: rinse 15 ml for 30 seconds after normal procedures of oral hygiene. eating or drinking is not recommended during the next 30 minutes after using this product. in two weeks of using vitis orthodontic twice daily, patients were examined again. when assessing the results of the drug, the views of patients about the taste of the drug, convenience of application was surveyed, we also evaluated the dynamics of the major indexes. statistical data processing was carried out by probability student t-test [8]. results initial data of periodontal state in the patients before orthodontic phase of treatment are presented in table 1. periodontium was clinically healthy. hygiene was satisfactory. within two months from the beginning of orthodontic treatment, due to the lack of oral hygiene, the availability of items for retention of dental plaque around the brackets, most patients complained of bleeding gums when brushing their teeth, swelling, halitosis. during objective examination, in 70 patients swelling, cyanosis gum, thickening in the area of gingival papilla were evidenced that pointed to a mild severity of catarrhal gingivitis. mechanical irritation was accompanied by bleeding. in the teeth there was the increased content of soft plaque (patients avoided brushing due to pain and bleeding gums). according to the results of objective examination, in 53 children hypertrophic gingivitis was determined that proved the visual assessment of size of crown and vertical sensing. all patients had mild severity of hypertrophic gingivitis that was manifested by proliferation of gum to 1/3 crown. to the touch gingival papillae dense produced false periodontal pockets, bleeding was absent, which evidences of the fibrous form of hypertrophic gingivitis. for gingivitis diagnosis we used classification of periodontal tissues diseases by mf danilevsky (1994). evaluation of patients’ performance in two months after the beginning of orthodontic treatment is presented in table 2. all the indicators increased in both groups of patients, oral hygiene index increased to 2.36 points, proving the deterioration in 3 times. the average plaque index increased to 2.6 points that verified unsatisfactory oral hygiene, gingivitis index increased on average by 52%, bleeding index – by 45%. after the application of hygiene measures and the vitis orthodontic solution the patients in 2–3-day reported about reduction of bleeding, gums swelling, disappearance of discomfort events. on examination, the detable 1. initial data evaluation of the studied patients (m±m) indicators girls (n=64) boys (n=59) ohi-s (points) 1.00±0.04; p<0.01 1.10±0.03; p<0.01 рма (%) 18.3±2.8; p<0.05 9.9±1.2; p<0.05 sbi (%) 7.70±0.02; p<0.01 8.50±0.12; p<0.01 note. statistical significance of differences between the relevant groups of girls and boys. table 2. evaluation of the patients examination in two months after the beginning of orthodontic treatment (m±m) indicators chronic catarrhal gingivitis, mild severity (n=70) chronic hypertrophic gingivitis, mild severity, fibrous form (n=53) ohi-s (points) 2.21±0.04; p1<0.05 2.51±0.03; p1<0.05 рма (%) 69.35±4.30; p1<0.05 71.67±3.70; p1<0.05 sbi (%) 47.17±0.14; p1<0.05 57.16±0.10; p1<0.01 note. p1 – difference reliability index compared with the initial data. ye. ya. kostenko et al. issn 2413-6077. ijmmr 2017 vol. 3 issue 1 49 d e n t is t r y ye. ya. kostenko et al. crease of gingival hyperemia was determined. in 8–10 days of the treatment, hyperemia, gingival swelling disappeared, gingival papillae compacted and acquired normal form. by the 14th day of rinses the state of oral health improved significantly. also with a significant improvement of subjective sensations the patable 3. evaluation of patients examination in two weeks after the treatment (м±m) indicators chronic catarrhal gingivitis (n=70) chronic hypertrophic gingivitis (n=53) ohi-s (points) 0.80±0.04; p2<0.05 0.90±0.03; p2<0.01 рма (%) 16.3±2.8; p2<0.05 19.9±1.7; p2<0.05 sbi (%) 8.20±0.12; p2<0.05 10.50±0.04; p2<0.05 note. p2 – differences reliability index compared with the initial data in two months after the beginning of orthodontic treatment. tients noted positive dynamics of the indices, and there was no oral mucosa irritant. none of them had any allergic reaction or any adverse side effects. all patients had good rinse results. teeth sensitivity was not changed. teeth were not stained. the results are presented in table 3. discussion the results of epidemiological studies of dental status of children in some areas of ukraine prove great variability and frequency of teeth anomalies, which varies in different regions from 30.8% to 85.4% and tends to increase. the treatment of anomalies of jaw apparatus teeth using fixed orthodontic structures is a priority in contemporary orthodontics, because it is highly effective and provides reliable retention of the obtained results. however, in the literature it is mentioned that periodontal tissue react to the treatment with braces, whereby the share of gingivitis according to petrushanko ta (2013) is 33.3% [3,4,5]. among the causes of inflammatory diseases of periodontal tissues in children this categories are defined: worsening of hygienic condition of oral cavity, microbial factors, hormonal changes and the effect of orthodontic forces. however, the development of complex therapeutic measures for chronic catarrhal and hypertrophic gingivitis in children with non-removable orthodontic apparatus has its own significance and importance that should be considered. thus, a comparative analysis of clinical trials revealed that the developed complex of therapeutic measures for patients with braces help to improve its clinical course that made it possible to achieve stable remission. conclusions inclusion in the complex therapy of early manifestations of inflammation in periodontium had a positive effect on the subjective feelings of patients and health performance rate, gum inflammation and bleeding. it is necessary to emphasize that the use of vitis orthodontic must be preceded by correction of oral hygiene and improvement of hygiene practices in patients. references 1. anne-marie bollen. effects of malocclusions and orthodontics on periodontal health: evidence from a systematic review. journal of dental education. 2008;8:912–918. 2. benkovskyy vv. clinical assessment of oral hygie ne in patients who use orthodontic equipment. 2011. 3. kudratova dm. effect of orthodontic treatment on the condition of periodontal tissue. journal of dentistry. 2008;1:66. 4. da silva pde l. gingivitis, psychological factors and quality of life in children. oral health prev dent. 2015;3:227–235. 5. lara-carrillo e. effect of orthodontic treatment on saliva, plaque and the levels of streptococcus mutans and lactobacillus. med oral patol oral cir bucal. 2010;15(6):924–929. 6. nesterenko om. estimated restructuring of jaw bone in adult patients in orthodontic treatment retention period: poltava; 2008. 7. who bulletin dental examination, basic methods. geneva. 1989;3:21. 8. zajcev vm. applied medical statistics. foliant. 2006;432. 9. ostrovska ly. the incidence of periodontal pregnant and mechanisms of development (literature review). bukovynskiy medical journal. 2016;20(77): 215–219. 10. denga ov. gingivitis in young people with decreased functional activity of salivary glands. ukrainian dental almanac. 2014;2:26–28. received: 2017-01-30 44 su r g ic a l d is e a se s issn 2413-6077. ijmmr 2022 vol. 8 issue 2 doi 10.11603/ijmmr.2413-6077.2022.2.13120 prospective descriptive study of non-thyroidal neck swellings in western indian population j. husain1, s. rajagopalan2, v. v. nair1, *r. nagamahendran1, p. sharma1, n. roy1, p. p. rao1 1 – armed forces medical college, pune, india 2 – dr chandramma dayananda sagar institute of medical education and research, harohalli, india background. nonthyroidal neck swellings are common and cause diagnostic challenges. this study highlights various diagnosis associated with non-thyroidal neck swellings and their management challenges. objective. this pilot study highlights the various diagnosis associated with nonthyroidal neck swellings and their management challenges. the clinical spectrum of two most common aetiology of nonthyroidal neck swelling: tubercular cervical lymphadenopathy and lymphomas, are also compared. methods. this prospective study was conducted in a tertiary care teaching hospital from 01 jun 2018 to 31 dec 2019. the first 100 nonthyroidal neck swellings presented to the surgical outpatient department were included and assessed clinically, radiologically and histopathologically. comparison was carried out between the first two common diagnosis of tubercular cervical lymphadenitis and lymphoma. continuous variables were analysed using student’s t-test and categorical data were analysed using the chi square test. a statistical p value <0.05 was taken as significant. results. there were 73 lymph node swellings and 27 non-lymph node swellings in the study. the most common diagnosis was tubercular cervical lymphadenitis (n=38) and lymphoma (n=17). the mean volume of lymph nodes in the lymphoma group (38.72±22.12 cm3) was significantly bigger than in the tuberculosis group (9.44±5.99 cm3) p=0.00001. the mean age (33.81±11.8 years) of tubercular patients was significantly less than the lymphoma (52.38±25.3 years) with p=0.000167. the clinical diagnosis was nearly accurate in 85% of cases. however, in 15 cases clinical diagnosis was changed after fine needle aspiration cytology. conclusions. ultrasonography and fine needle aspiration cytology are very useful adjunct in arriving at a definite diagnosis of a lymph node swelling in neck. tubercular cervical lymphadenopathy and lymphoma were two major diagnosis. tubercular cervical lymphadenopathy is significantly different from lymphomas in terms of early age of presentation and smaller size at the time of presentation. keywords: neck mass; cervical; lymphadenopathy; tubercular; lymphoma. *corresponding author: dr. r. nagamahendran, assistant professor of the department of general surgery, armed forces medical college, pune, 411040, india. e-mail: nagaa.mahendran@gmail.com international journal of medicine and medical research 2022, volume 8, issue 2, p. 44-52 copyright © 2022, tnmu, all rights reserved j. husain et al. introduction neck swellings are commonly seen in the general surgery outpatient departments. cervical lymphadenopathy constitutes around half of these swellings. thyroid swellings are the next common neck swelling which account for 32% of all neck mass [1]. non thyroidal neck swellings are varied and sometimes cause diagnostic dilemma. there are many more diagnostic variants, emerging diseases and diagnostic surprises when managing nonthyroidal neck mass. this pilot study highlights various diagnosis associated with nonthyroidal neck swellings and their management challenges. this study also compares the clinical spectrum of two most common aetiology of nonthyroidal neck swelling: tubercular cervical lymphadenopathy and lymphomas. methods this prospective study was conducted in a tertiary care teaching hospital in western india from 01 jun 2018 to 31 dec 2019. the first 100 nonthyroidal neck swellings presented to the surgical outpatient department were included in the study. the exclusion criteria were symptom that lasted less than one week, thyroid swelling, known primary with lymph node metastasis and recurrent swelling of neck. all patients were scrutinised with detailed history and clinical examination in a well-lit room. all swellings were classified into a lymphnodal swelling and non-lymphnodal swelling. after 45 su r g ic a l d is e a se s issn 2413-6077. ijmmr 2022 vol. 8 issue 2 clinical examination all patients with lymph node swelling underwent haematological examination for complete hemogram and base line biochemistry tests. three sputum samples, two early morning and one routine was sent for acid fast bacilli as per recommen dations. further tests and management were based on the hospital protocol as summarised in the consort diagram. radiological examination included ultrasonography for neck and chest radiogram posteroanterior view. all ultrasonography was performed in the dept of radiology by consultant radiologists in a siemens 4d ultrasound machine using multi frequency linear array probe. chest x­ray was performed according to the standard protocol for posteroanterior view. all chest x­rays were studied for presence of tuberculosis and malignancies. all patients underwent fine needle aspiration cytology (fnac) either free hand or usg guided for histopathological diagnosis. fnac was performed under aseptic condition using a 23-gauge needle fitted to 10 ml disposable syringe attached to a plunger. if the fnac findings were inconclusive, an excision biopsy was contemplated. all non-lymph node swellings also underwent surgical excision and specimen were sent for histopathology. a final diagnosis was made with histopathological examination of the excision biopsy or of the specimen in all 100 cases. no immunohistochemical studies were carried out. comparison was also carried out between the clinical and final histopathological diagnosis. computed tomography and magnetic resonance imaging were done in selected cases, in our study only for 16 cases, where the ultrasound and fnac were inconclusive or additional information was required. benign cases were managed medically or surgically depending upon aetiology but for malignant lesions, if primary – detected defi­ nitive treatment was carried out with radical excision and appropriate neck dissection. however, in cases of unknown primary, options of chemotherapy/radiotherapy/surgery were explored based on a case to case basis and existing protocols of the hospital. statistical analysis was completed using ibm spss version 26. for data with normal distribution the levene’s test of equity of variance was performed. means of continuous variables were analysed using the student’s t­test for statistical significance. categorical data were analysed using the chi square test. a statistical p value < 0.05 was taken as significant. j. husain et al. consort diagram enrollment classification exclusion criteria • symptom less than one week • thyroid swelling • known primary with lymph node metastasis and reccurent swelling of neck lymph node swelling (n=73)non lymphnodal swelling (n=27) tubercular cervical lymphadenopathy (n=38) non-tubercular (n=35) • lymphoma (n=17) • non­lymphoma (n=18) follow-up lost to follow-up (n=0) analysis lost to follow-up (n=0) analysed (n=38) • excluded from analysis (n=0) analysed (n=17) • excluded from analysis (n=18) all patients presenting with neck swelling to the general surgery outpatient department assessed for eligibility (n=100) 46 su r g ic a l d is e a se s issn 2413-6077. ijmmr 2022 vol. 8 issue 2 results a total of first 100 cases of nonthyroidal neck swellings were included in this study. there were 61 men (61%) and 39 women (39%). the mean age was 39.14±16.9 (range 1-76 years). the majority of the patients were in the third and fourth decade of life (47%). duration of the swelling was variable with 68% cases having a short duration of one to six weeks and 32% cases with swelling more than 6 weeks. most of the patients (42%) presented in first two weeks of swelling onset and had reactive lymphadenopathy managed with antibiotics. the commonest associated symptom was fever of low grade (42%), non-productive cough (18%) and weight loss (14%). a typical evening rise of temperature was evidenced in 26% of cases. brief spell of fever at the onset was reported in 14% cases. only five cases presented with specific complaint of loss of appetite, in rest of the cases it was either associated with fatigue, night sweats or dysphagia. the rest 46% of patients had no associated symptoms. the majority of swellings were unilateral 56%, and 44% were bilateral. only 22% cases presented with a solitary swelling, and 78% cases – with multiple swellings. the majority of swellings (47%) were in range of 1-3 cm (60%). there were 24 cases of swelling size of 3-5 cm and 29 cases with swelling of more than 5 cm. in our study, 73% were lymph node swelling and 27% were non-lymph node swelling. the majority of the lymphnodal swelling were clinically and histopathologically diagnosed to be benign. there were more cases of cervical tubercular lymphadenitis (n=38) compared to non-tubercular cervical lymphadenitis (n=35). it is pertinent to mention that the incidence of tuberculosis infection in this part of the country is 210 per one lakh population. the commonest non-tubercular cervical lymphadenitis were lymphoma (n=17) followed by reactive lymphadenopathy (n=8), carcinoma of unknown primary (n=4), hiv associated lymphadenopathy (n=4) and one patient each of kikuchi fujimoto disease and cat scratch disease (fig. 1-3). the incidence of lymphoma in this part of the country is 2.5 per 100000 population. the mean volume of lymph nodes in the lymphoma group (38.72±22.12cm3) was significantly bigger in the tuberculosis group (9.44±5.99cm3) with p=0.00001. it was also seen that the mean age (33.81±11.8years) of tubercular patients was significantly lower than the lymphoma (52.38±25.3years) with p=0.000167. there was no statistical significance in any other parameter among these groups (table 1). the clinical diagnosis was nearly accurate in 85% of cases. however, in 15 cases the clinical diagnosis was changed after fnac and a new pathology was discovered that changed the complete line of management. they were six cases of tubercular lymphadenitis, three cases of metastatic lymphadenopathy, and one case each of lymphangioma, neurofibroma, carotid body tumour, adenoid cystic carcinoma and inflamed branchial cyst. there were 27 non-lymph node swellings and they were lipoma (n=6), sebaceous cyst (n=6), salivary gland tumour (n=6), branchial fig. 1. a – cervical tubercular lymphadenopathy level 5; b – cystic hygroma; c – metastatic carcinoma mass in neck; d – dermoid neck; e – cold abscess; f – kikuchi fujimoto disease. j. husain et al. 47 su r g ic a l d is e a se s issn 2413-6077. ijmmr 2022 vol. 8 issue 2 fig. 2. investigation and operative pictures: a – vascular doppler neck mass – carotid body tumour; b – intra-operative image of carotid body tumour; c – ultrasonography neck showing cystic hygroma; d – intraoperative image of neck nymphangioma; e – mri right sided carotid body tumour. fig. 3. histopathological findings: a – follicular lymphoma (40×); b – hodgkin’s lymphoma (400×); c – reactive lymphadenitis (40×); d – reactive lymphadenitis (400×); e – necrotising granuloma (40×); f – necrotising granuloma (400×). table 1. comparison of tubercular cervical lymphadenopathy and lymphoma presenting as a neck mass variable tubercular lymphadenitis(n=38) lymphoma (n=17) p-value sex male 22 12 female 16 5 age (years) mean (sd) 33.81 (11.18) 52.38 (25.28) 0.000167 duration of symptoms (weeks) mean (sd) 4.28 (3.69) 3.83 (2.45) 0.318085 mean size (cm3) mean 9.44 (5.99) 38.72 (22.12) <0.00001 associated symptoms fever 19 9 0.9654 weight loss 11 5 0.972046 j. husain et al. 48 su r g ic a l d is e a se s issn 2413-6077. ijmmr 2022 vol. 8 issue 2 cyst (n=2), cystic hygroma (n=3), carotid body tumour (n=1), parathyroid adenoma (n=1), adenocystic carcinoma (n=1), lymphangioma (n=1). discussion thyromegaly is a major diagnosis in neck swelling and accounts for only 20-30 % of all neck swellings. the other neck masses are nonthyroidal in origin. more than 50% of the neck swellings revealed in the general po pulation originate from the cervical lymph nodes. commonly tuberculosis and malig nancies present as cervical lymphadenopathy. other considerations are infections, lymphoma, leukaemia, and metastatic swellings. neck swelling in younger age group tend to be more benign and are congenital, infectious or benign neoplasms. however, the chances of malignant swelling in neck increases to a greater extent after 40 years of age [2]. this correlates with our findings. von haller in 1743 was the first to describe a non-thyroidal swelling of the neck which later was known as carotid body tumour [3]. von mikulicz-radecki, in 1888, reported a patient who experienced bilateral, painless, symmetrical swelling involving lacrimal, submandibular and parotid gland. this disease was later termed as mikulicz disease [4]. martin in 1957 published his classic work “surgery of head and neck tumors” which first gave a varied and detailed description of neck masses [5]. there are other rare cases of non-thyroidal neck swellings described in medical literature. recently kikuchi fujimoto disease is an emerging cause of necrotizing cervical lymph node mass revealed particularly in the asians. the disease is very much amenable to management with steroids [6]. kawasaki disease is another uncommon cause of cervical lymphadenopathy but can deteriorate to multiorgan failure in a short span of time [7]. in 2021, kesavan et al. described two unusual cases of neck swelling. first was a case of achala sia cardia reported as neck swelling in a 75-year-old man [8]. abuzayad et al. described a swelling in the region of carotid in a 15-yearold girl, which was later diagnosed as castleman disease [9]. sometimes the neck masses may not be of any pathological significance. these lesions called pseudo lesions may be due to normal variant anatomy, atypical location of organ of other location and foreign bodies due to iatrogenic causes or self-introduction [10]. cervical ectopic thymus is one of the common such pseudo lesion and is widely reported [11]. many studies were conducted in regards to evaluation of neck swellings but the diagnostic accuracy of clinical examination was purely discussed. in our study we were accurate by 85% in correctly diagnosing the cause for nonthyroidal neck swellings. however, the physical examination frequently requires to be supplemented by proper investigation to improve the diagnostic accuracy. the two important investigations that help to diagnose neck swellings are ultrasound and fnac. ultrasound has been found to be a reliable and sensitive diagnostic tool in diagnosis of soft tissue cysts, vascular neoplasms, deep neck abscesses, palatal tumours, salivary gland diseases, fractures of facial bone, solid and cystic lesions of the jaws, temporomandibular joint pathology, head and neck cancer and nodal metastases. the recent indian study conducted in 2011 by chandak et al. pointed out that the diagnostic accuracy of clinical examination of 85.7% in diagnosis of neck swellings, while ultrasonography of neck had a sensitivity and accuracy of 98.5% [12]. in another study regarding the ultrasonographic evaluation of cervical lymphadenopathy, the most significant distinguishing feature is strong internal echoes seen in 84% of tubercular lymph nodes. this finding is found in only 11% of metastatic nodes and absent in lymphomatous nodes [13]. the other findings such as irregular margins, hypoechoic centre, fusion tendency, peripheral halo and absent hilus are helpful in differentiating reactive from diseased nodes but show considerable overlap in the 3 groups of tubercular, metastatic and lymphoma lymph nodes. however, ultrasound for neck masses is a valuable diagnostic tool, its accuracy depends on the characteristics of the ultrasound probe and the experience of the radiologist [14]. this vulnerability was overcome by higher modes of radiological diagnostic procedures. the capabilities of ct scan to distinguish between soft tissue structures coupled with the added advantage of utilizing intravascular contrast, particularly suits the examination of neck disorders. with improved equipment permitting thinner sections and shorter exposure times, applications in head and neck diagnosis are increased. ct is used in the evaluation of salivary gland enlargements, staging of known tumours and evaluation of a variety of neck swellings. in some cases, ct alone may be the only imaging procedure necessary, while in other situations, ct may comprise an important portion of comprehensive imaging evaluaj. husain et al. 49 su r g ic a l d is e a se s issn 2413-6077. ijmmr 2022 vol. 8 issue 2 tion [15]. important anatomic variants are pointed out so the surgeon can avoid potential intraoperative complications. secondly, nodal staging can be assessed in an effort to increase the number of abnormal nodes detected by physical examination and more importantly to precisely define their location by a standard classification system that can be understood and consistently applied by the radiologist, surgeon, radiation oncologist and pathologist. although secondary to the previously described tasks, ct scan imaging frequently enables saving the patient unnecessary risk and shortening the time to diagnosis and ultimate treatment [16]. fine needle aspiration cytology (fnac) is the most important step in the management of neck lumps. it is easy to perform and also relatively cheap. fnac has high sensitivity ( 9 4 . 6 % ) , s p e c i f i c i t y ( 9 7 . 9 % ) , a c c u r a c y (96.7%), and both positive predictive (95.9%) and negative predictive (97.2%) value in the diagnosis of neck masses [17]. fnac also serves as an excellent preliminary screening procedure. fnac can be performed free-hand or ultrasound­guided to increase confidence of diag­ nosis if the lesion is impalpable. fnac has excellent patient compliance, is simple and quick to perform in the outpatient department and can be readily repeated. studies reveal the burden of tubercular lymphadenitis to be significant in most developing countries [18]. fnac is deemed as frontline investigation with further investigations based on fnac result. however, histopathological examination is still the most dependable diagnostic tool. surgical intervention is definitely required in many cases, though most of the cases are medically curable [19]. out of 100 cases 36 (36%) had tubercular lymphadenitis. the high percentage of tubercular lymphadenopathy in the indian subcontinent setting is mirrored by similar findings by fatima et al in pakistan where the incidence of tubercular lymphadenopathy of 52.7% [20]. fnac was a prime diagnostic tool in establishing the diagnosis in this study. a total of 95 patients were subjected to fnac excluding cases of cystic hygroma, lymphangioma and carotid body tumour. of the 95 neck swellings, fnac was accurate in 81 cases, inconclusive in 11 cases and disparate with the final diagnosis in 3 cases. thus, in 85.26% the fnac diagnosis correlated with the final histo­ pathological diagnosis. this is similar to the findings by pandey et al., who reviewed the role of fnac in 395 patients [21]. in our study fnac has been accurate in 94% for diagnosis of tuberculosis. das et al. has shown in their series of 180 cases an accuracy of 84.4% for detection of tuberculosis [22]. in our study, a total of 72% of neck swellings were found to be of lymph nodal in origin. this is similar to the findings by jasani et al., which found the prevalence of lymph nodal swellings to be 69% in a series of 450 patients, who underwent fnac [23]. in our study, 4 patients were finally diag­ nosed with metastatic lymphadenopathy labelled as carcinoma of unknown primary source (cups). the success rate of diagnosis of metastatic carcinoma by fnac was 100 percent in this study. these findings are similar to the study published by narang et al., where the success rate of diagnosing metastatic carcinoma by fnac was 100% [24]. there were total of 17 cases of lymphoma in this series, comprising of 14 non-hodgkin’s (14%) and 3 hodgkin’s lymphoma (3%). this is similar to the findings in the british study by smith et al., where 11 cases of lymphoma were reported in a series of 100 cases [25]. there were 10 cases of nonspecific adenitis in our series, constituting 19.6% of lymph node swellings that was similar to the 2007 study by song et al., showing the prevalence of non­specific adenitis to be 22.4% in 147 patients with lymph node swellings [26]. in our series 8% of the neck swellings were diagnosed to be lipomas. similar incidence was seen in a series of 109 patients where four fifth of lipomas are 1.0-5.0 cm in maximum dimension [27]. in our series 4 lipomas were approximately 3 cm in size and 2 were 4-5 cm in size. there were two cases of cystic hygroma in our study, constituting 1.8% of benign tumour of the neck. both were located in the posterior triangle. this is similar to the finding in the older study which showed one case of cystic hygroma in 100 consecutive cases. there were two cases of branchial cyst in the same study. both swellings were in right lateral position. this conforms to previous published series by liston et al., where it is mainly either on right or on left side. it rarely occupies the midline position [28]. our study reported a total of 6 cases of salivary gland tumour. in our study there was one case of chronic sialadenitis of sub man dibular gland. this is consistent with the findings of hag et al., where less than 5% cases of sialadenitis were detected in 225 neck swellings [29]. conclusion non thyroidal neck swellings are commoner diagnosis than goitre. most of the time diagnoj. husain et al. 50 su r g ic a l d is e a se s issn 2413-6077. ijmmr 2022 vol. 8 issue 2 sis can be correctly established after a thorough history and clinical examination. the ultrasonographic and fine needle aspiration cytology are very useful adjunct in arriving at a definite diagnosis. tubercular cervical lymphadenopathy and lymphoma were two major diagnosis constituting more than 50% of nonthyroidal neck swellings. tubercular cervical lymphadenopathy was found to be significantly different from lymphomas in terms of mean age and the mean size of swelling. limitations the limitations of this study were: a) single centre observation b) small duration of study c) hospital-based study. the advantages of this study were the prospective data collection and adequate sample size. as this is a pilot study a multicentre prospective study with more participants is recommended based on this study design. conflict of interests authors declare no conflict of interest. author's contributions jafar husain, s. rajagopalan, vipin venugopal nair, r. nagamahendran, pawan sharma, pankaj p. rao – conceptualization, methodology; jafar husain, s. rajagopalan, vipin venugopal nair, r. nagamahendran, pawan sharma, nilanjan roy – investigation, data curation, formal analysis; jafar husain, vipin venugopal pawan sharma, pankaj p. rao – writing – original draft; rajagopalan, vipin venugopal nair, r. nagamahendran, pawan sharma, nilanjan roy, pankaj p. rao – writing – reviewing and editing. проспективне описове дослідження нетиреоїдних набряків шиї у населення західної індії j. husain1, s. rajagopalan2, v. v. nair1, r. nagamahendran1, p. sharma1, n. roy1, p. p. rao1 1 – armed forces medical college, pune, india 2 – dr chandramma dayananda sagar institute of medical education and research, harohalli, india вступ. нетиреоїдні набряки шиї є поширеним явищем і викликають труднощі у діагностиці. у цьому дослідженні висвітлюються різні діагнози, пов’язані з нетиреоїдними набряками шиї, і проблеми з їх лікуванням. мета. це пілотне дослідження висвітлює різні діагнози, пов’язані з нетиреоїдними набряками шиї, і проблеми з їх лікуванням. у цьому дослідженні також порівнюється клінічний спектр двох найпоширеніших етіологій нетиреоїдного набряку шиї – туберкульозної шийної лімфаденопатії та лімфом. методи. дане проспективне дослідження проводилося у навчальній лікарні третинного рівня з 01 червня 2018 р. по 31 грудня 2019 р. перші 100 нетиреоїдних набряків шиї, представлених до хірургічного амбулаторного відділення, були оцінені клінічно, радіологічно та гістопатологічно. проведено порівняння перших двох поширених діагнозів туберкульозного шийного лімфаденіту та лімфоми. безперервні змінні аналізувалися за допомогою t-критерію стьюдента, а категоричні дані аналізувалися за допомогою критерію хі-квадрат. статистичне значення p < 0,05 вважалося значущим. результати. у дослідженні було виявлено 73 випадків збільшень лімфатичних вузлів і 27 набряків не пов’язаних із лімфатичними вузлами. найбільш частим діагнозом був туберкульозний шийний лімфаденіт (n=38) та лімфома (n=17). середній об’єм лімфатичних вузлів у групі лімфоми (38,72±22,12 см3) був статистично більшим у порівнянні з об’ємом лімфовузлів у групі туберкульозу (9,44±5,99 см3) p=0,00001. середній вік хворих на туберкульоз був 33,81±11,8 років, порівняно з віком пацієнтів з лімфомою 52,38±25,3 років (p=0,000167). клінічний діагноз був точним у 85% випадків. однак у 15 випадках клінічний діагноз був змінений після тонкоголкової аспіраційної цитології. висновки. ультразвукове дослідження та тонкоголкова аспіраційна цитологія є дуже корисним допоміжним засобом для встановлення точного діагнозу збільшень лімфовузлів на шиї. туберкульозна шийна лімфаденопатія та лімфома були двома основними діагнозами. туберкульозна шийна лімфаденопатія суттєво відрізняється від лімфом раннім віком появи та меншими розмірами на момент появи. ключові слова: пухлини шиї; шийний; лімфаденопатія; туберкульозний; лімфома. j. husain et al. 51 su r g ic a l d is e a se s issn 2413-6077. ijmmr 2022 vol. 8 issue 2 references 1. sakorafas gh. historical evolution of thyroid surgery: from the ancient times to the dawn of the 21st century. world j surg. 2010;34(8):1793­804. https://doi.org/10.1007/s00268­010­0580­7 2. larsen cg, channir hi, kiss k, charabi b, lajer c, von buchwald c. udredning af knude på halsen hos voksne [diagnosing neck mass in adults]. ugeskr laeger. 2015 sep 21;177(39):v02150131. 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postcovid-19 kawasaki-like syndrome. ear nose throat j. 2021 mar 26:1455613211006011. https://doi.org/10.1177/01455613211006011 8. kesavan p, joshi s, gercek y. intermittent neck swelling: an unusual presentation of achalasia cardia. bmj case rep. 2021 sep 21;14(9):e243229. https://doi.org/10.1136/bcr­2021­243229 9. abuzayed b, said a, jamous o, al-abadi h, alashqar o, alawneh k. castleman disease: an unu­ sual cause of an isolated neck mass. j craniofac surg. 2021 jan­feb 01;32(1):e37­e38. https://doi.org/10.1097/scs.0000000000006853 10. gopal n, bhatt aa. ten must know pseudolesions of the head and neck. emerg radiol. 2021 feb;28(1):119­26. https://doi.org/10.1007/s10140­020­01807­z 11. rousslang l, meldrum j, veronica r, poutre a, biega t. ectopic cervical thymus: a common, yet rarely symptomatic pediatric neck mass. curr med imaging. 2021;17(4):544­48. https://doi.org/10.2174/1573405616999201027 214101 12. chandak r, degwekar s, bhowte r, motwani m, banode p, chandak m, et al. an evaluation of efficacy of ultrasonography in the diagnosis of head and neck swellings. dentomaxillofacial radiology. 2011;40(4):213­21. https://doi.org/10.1259/dmfr/68658286 13. khanna r, sharma ad, khanna s, kumar m, shukla rc. usefulness of ultrasonography for the evaluation of cervical lymphadenopathy. world j surg oncol. 2011;9:29. https://doi.org/10.1186/1477­7819­9­29 14. akinbami bo, ugboko vi, owotade e j, obiechina ae, adetiloye vo, ayoola o. applications of ultrasonography in the diagnosis of soft tissue swellings of the cervicofacial region. west afr j med. 2006 apr­jun;25(2):110­8. https://doi.org/10.4314/wajm.v25i2.28259 information about the authors jafar husain – associate professor of the department of surgery, armed forces medical college, pune, india https://orcid.org/0000­0003­2400­1473, e­mail: jafarhusain01@gmail.com s rajagopalan – medical director and vice principal, dr chandramma dayananda sagar institute of medical education and research, harohalli, india formerly professor & hod, department of surgery, armed forces medical college, pune, india https://orcid.org/0000­0003­3921­0171, e­mail: rajdel17@gmail.com vipin venugopal nair – associate professor of the department of surgery, armed forces medical college, pune, india https://orcid.org/0000­0001­6903­6368, e­mail: vipinvenugopalnair@gmail.com r. nagamahendran – assistant professor of the department of general surgery, armed forces medical college, pune, india https://orcid.org/0000­0002­9854­7236, e­mail: nagaa.mahendran@gmail.com pawan sharma – professor of the department of general surgery, armed forces medical college, pune, india https://orcid.org/0000­0003­4984­0708, e­mail: drpawansharma55@gmail.com nilanjan roy – professor of the department of general surgery, armed forces medical college, pune, india https://orcid.org/0000­0001­6233­8929, e­mail: nilanjanroyd604@rediffmail.com pankaj p. rao – professor & hod, department of surgery, armed forces medical college, pune, india https://orcid.org/0000­0002­1420­7622, e­mail: pankajrao@rediffmail.com j. husain et al. 52 su r g ic a l d is e a se s issn 2413-6077. ijmmr 2022 vol. 8 issue 2 15. gayler b, kashima h, martinez c. computed tomography of the neck. crit rev diagn imaging. 1985;23(4):319. 16. alberico ra, husain sh, sirotkin i. imaging in head and neck oncology. surg oncol clin n am. 2004 jan; 13(1):13­35. https://doi.org/10.1016/s1055­3207(03)00124­8 17. paker io, kulaçoğlu s, eruyar t, ergül g. fine needle aspiration cytology of head and neck masses: a cytohistopathological correlation study with emphasis on false positives and false negatives. kulak burun bogaz ihtis derg. 2013 may­jun;23(3): 163-72. https://doi.org/10.5606/kbbihtisas.2013.27048 18. göret cc, göret ne, özdemir zt, özkan ea, doğan m, yanık s, gümrükçü g, aker fv. diagnostic value of fine needle aspiration biopsy in non­thy­ roidal head and neck lesions: a retrospective study of 866 aspiration materials. int j clin exp pathol. 2015 aug 1;8(8):8709­16. 19. sreenidhi gm, nandeeshkumar gn. “clinicopathological study of cervical tubercular lymphadenopathy at kims hospital bangalore”. journal of evolution of medical and dental sciences 2013;2,(44), 04;8655-66. 20. fatima s, arshad s, ahmed z, hasan sh. spectrum of cytological findings in patients with neck lymphadenopathy-experience in a tertiary care hospital in pakistan. asian pac j cancer prev. 1873;12. 21. pandey p, dixit a, mahajan n. the diagnostic value of fnac in assessment of superficial palpable lymph nodes: a study of 395 cases. al ameen journal of medical sciences. 2013;6(4). 22. das dk. fine-needle aspiration cytology in the diagnosis of tuberculous lesions. lab medicine. 2000;31(11):625­32. https://doi.org/10.1309/uj0b­vdwv­u0le­e0qq 23. jasani jh, vaishnani hv, vekaria pn, patel d, shah yd, patel d, et al. retrospective study of fine needle aspiration cytology of head and neck lesion in tertiary care hospital. international journal of biomedical and advance research. 2013;4(4):253­7. https://doi.org/10.7439/ijbar.v4i4.337 24. narang r, pradhan s, singh r, chaturvedi s. place of fine needle aspiration cytology in the diag­ nosis of lymphadenopathy. ind j tub. 1990;37(1):29­ 31. 25. smith o, ellis p, bearcroft p, berman l, grant j, jani p. management of neck lumps--a triage model. ann r coll surg engl. 2000;82(4):223. 26. song jy, cheong hj, kee sy, lee j, sohn jw, kim mj, et al. disease spectrum of cervical lym phadenitis: analysis based on ultrasound­guided core­ needle gun biopsy. j infect. 2007;55(4):310­6. https://doi.org/10.1016/j.jinf.2007.06.004 27. rydholm a, akerman m, idvall i, persson bm. aspiration cytology of soft tissue tumours. a prospective study of its influence on choice of surgical procedure. int orthop. 1982;6(4):209­14. https://doi.org/10.1007/bf00267141 28. liston sl, siegel lg. branchial cysts, sinuses, and fistulas. ear nose throat j. 1979 dec;58(12):504­9. 29. el hag ia, chiedozi lc, al reyees fa, kollur sm. fine needle aspiration cytology of head and neck masses. acta cytol. 2011;47(3):387­92. https://doi.org/10.1159/000326538 received 18 august 2022; revised 6 october 2022; accepted 1 november 2022. this is open access article distributed under the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. j. husain et al. 5 in t e r n a l m e d ic in e issn 2413-6077. ijmmr 2016 vol. 2 issue 2 doi 10.11603/ijmmr.2413-6077.2016.2.7038 the remodeling of left atrium and vasodilation factors changes in ischemic heart failure v. i. denesyuk, о. v. denesyuk, n. o. muzyka national pirogov memorial medical university, vinnytsіa, ukraine background. according to the national registries of european countries and epidemiological studies, the prevalence of chronic heart failure (chf) among adults is 2,0–5,0%, and increases due to age, in people aged over 70 years old it is 10,0–20,0%. objective. to find out the specific features of remodeling of the left atrium and change of vasodilation factors in ischemic heart failure with reduced and preserved left ventricular ejection fraction and to establish correlation relationships. methods. a full clinical examination of 153 patients with chf (105 men and 48 women) was conducted to achieve this objective. the surveyed patients underwent clinical examinations; spectrophotometric parameters: quantification of markers of vasodilation, metabolites of monoxide nitrogen — nitrates and nitrites with gris reagent; content of endothelial nitric oxide synthase (enos) in serum — elisa for the set of nitric oxide synthase 3, endothelial (nos3) human elisa kit (cloud-clone corp, usa). electrocardiographic (ecg) examination was conducted in 12 standard conventional leads on electrocardiograph by the hungarian production heart screen 112 d. results. the 1st group of the examined patients with reduced lv ef prevails iii (significant) degree la dilatation in 33 (70.21%) cases, ii (moderate) degree of la dilatation was determined in 14 (29.78%), and i (initial) degree was not defined at all. in the 2nd group of the patients with preserved lv ef mainly the ii degree of la dilatation was determined in 44 (44.51%) cases, and decreased la dilation in 39 (36.79%) cases (p<0.01), and iii degree of la dilation was defined in 23 (21.69%) cases (p<0.01). in patients with stable coronary heart disease, complicated by heart failure with reduced lv ef and ii degree of la dilatation, enos levels in the serum was 449.00±39.91 pg/ml, whereas in patients with stable coronary heart disease, complicated by heart failure with preserved lv ef and ii stage of la dilatation — 673.56±50.98 pg/ml (p<0.01). at iii stage of la dilatation in patients of the 1st group level enos was 344.20±51.98 pg/ml in the patients of the 2nd group — 616.90±36.49 pg/ml (p<0.01). at the same degree and with la dilation in the patients of the 2nd group enos was 750.27±99.85 pg/ml. conclusions. the structural and functional changes of the left atrium and changing factors of vasodilation in patients with stable coronary artery disease of ii-iii functional classes complicated by heart failure of i–iii functional classes are studied. it is established that in the examined patients with stable coronary heart disease complicated by heart failure with reduced left ventricular ejection fraction mainly iii (significant) degree of dilatation of the left atrium was determined, while in patients with stable coronary heart disease, complicated by heart failure with preserved left ventricular ejection fraction mainly ii (moderate) degree of dilation of the left atrium was determined. in comparison with the results of research among the patients with stable coronary heart disease, complicated by heart failure with reduced left ventricular ejection fraction, and a group of patients with preserved left ventricular ejection fraction, it was determined a significant decrease in enos, nitrites, total amount of nitrites and nitrates. key words: heart remodeling factors of vasodilation, heart failure. introduction according to the national registries of european countries and epidemiological studies, prevalence of chronic heart failure (chf) among adults is 2.0–5.0%, and increases in proportion to age, people aged over 70 years it is from 10.0 to 20.0% [16]. considering demographic trends, in ukraine as well, to increase the number of population of older age groups, the provision of medical care to patients with chf is becoming a topical issue [2, 14]. distinguish systolic and diastolic options of chf. in version of systolic chf the presence of clinical signs in patients with ejection fraction (ef) of lv≤45.0%, at diastolic version with lv ef>45.0% is observed [4]. it is known that left ventricular hypertrophy (lvh) precedes the development of chf and is formed together international journal of medicine and medical research 2016, volume 2, issue 2, p. 5–9 copyright © 2016, tsmu, all rights reserved corresponding author: nadia muzyka, department of general medicine № 3, national pirogov memorial medical university, 96, khmelnytske chaussee street, vinnytsia, ukraine, 21000 phone number: +380673757410 e-mail: myzuchechka@gmail.com v. i. denesyuk et al. 6 in t e r n a l m e d ic in e issn 2413-6077. ijmmr 2016 vol. 2 issue 2 with or even is caused by this disease. analysis of prognostic value of lvh in patients with chf with preserved ejection fraction proves that the high risk of lethal outcome is observed in patients with left ventricular myocardial mass that exceeds 400 g [3]. it is established that at the progression of diastolic dysfunction an increased risk of sudden cardiac death in 80.0% occurs [4, 15]. endothelial dysfunction is very important in development of chf. the cause of nitric oxide (no) synthesis inhibition by endothelium as a major component of its dysfunction at chf is free radical stress [11]. the main cause of chf is negative impact of free radical factors that leads to lower ventricular myocardial contractile function followed by entire body hypoxia, endothelial dysfunction with the appropriate activation of cellular factors [17]. these processes can lead to increasing of blood coagulation capacity, which is one of the causes of thrombotic complications in patients with chf [12]. biosynthesis of no from l-arginine occurs in three major isoforms of no-synthase, two constitutive: neuronal (nnos) and endo­ thelial (enos), and inducible one (inos) [6]. the objective of the study is to find out the specific features of left atrium remodeling and vasodilation factors changes in case of ischemic heart failure with reduced and preserved left ventricular ejection fraction and to establish correlation relationships. material and methods to achieve this objective a full clinical examination of 153 patients with chf (105 men and 48 women) was conducted; they were treated in the department of cardiology for patients with arrhythmias of vinnitsa regional centre of cardiovascular disorders (ukraine). all patients were divided into 2 groups: group 1 consisted of patients with stable coronary heart disease complicated by heart failure with reduced lv ef (n=47); group 2 — patients with stable coronary heart disease complicated by heart failure with preserved lv ef (n=106). the control group consisted of 30 healthy individuals. the diagnoses of ch and fc was made due to clinical, laboratory and instrumental criteria, as recommended by the european society of cardiology (2012) and the association of cardiologists of ukraine (2012) [16]. diagnosis and treatment of clinical forms of coronary artery disease were made by order of the ministry of health of ukraine № 152 from 03.02.2016 [9]; functional class (fc) ch — by nyha classification. the criteria for inclusion. patients with stable coronary artery disease ii-iii fc followed by chf ii-iii fc on informed consent to participate in the study. exclusion criteria: chf fc iv by the nyha; period up to 3 months from the beginning of acute myocardial infarction, or stroke; implanted artificial pacemaker or need of im­ plantation; severe diseases of respiratory system, kidneys, liver with hepatic insufficiency, anaemic state of haemoglobin 90,0 g/dl and below; the formation of malignant disorders and severe neuro-psychiatric disorders. among the patients who participated in the study mail prevailed. the average age of patients was 68.80±0.90 years. hypertension was defined in 132 (86.27%) patients. systolic blood pressure (bp) was 142.40±5.01 mm hg, diastolic blood pressure — 88.30±2.95 mmhg the examined patients underwent clinical (complaints, case history, physical examination); laboratory tests (general clinical blood and urine tests, blood glucose, bilirubin, cholesterol, β­lipoprotein, creatinine, urea, coagulation para meters); spectrophotometric parameters: quantification of vasodilation markers meta bo­ lites of monoxide nitrogen: nitrates and nitrites with gris reagent; content of endothelial nitric oxide synthase (enos) in serum — elisa for the set of nitric oxide synthase 3, endothelial (nos3) human elisa kit (cloud-clone corp, usa). electrocardiographic (ecg) examination was conducted in 12 standard conventional leads on electrocardiograph heart screen 112 d made in hungary. statistical analysis was performed with standard statistical package statistica 6.0. for primary analysis of tables and intermediate calculations microsoft excel package was used. results and discussion the decrease in left atrium (la) dilation degree in patients with stable coronary heart disease complicated by heart failure with reduced and preserved lv ef is presented in table 1. in table 1 the 1st group of the examined pa tients with reduced lv ef prevails iii (significant) degree of la dilatation in 33 (70.21%) cases, ii (moderate) degree of la dilatation in 14 (29.78%), i (initial) degree is not defined at all. in the 2nd group of patients with preserved lv ef the de gree of dilatation ii la was determined in 44 (44.51%) cases, and slightly decreased degree of la dilation in 39 (36.79%) cases (p<0,01), iii de gree of la dilation in 23 (21,69%) cases (p<0,01). v. i. denesyuk et al. 7 in t e r n a l m e d ic in e issn 2413-6077. ijmmr 2016 vol. 2 issue 2 remodeling of myocardium, including hypertrophy and dilation of left ventricle with a changes of its geometry and a contraction violation precedes clinical skim chf manifestations [7]. thus, remodeling is a harbinger of heard activity decompensation [7, 13]. the features of myocardial remodeling are studied the most in patients after myocardial infarction with heart failure development [7, 8, 14]. these results are consistent with the data that myocardial ischemia may be accompanied by its dysfunction [7]. the study of factors of vasodilation levels and degrees of la dilatation in patients with stable coronary heart disease complicated by heart failure with reduced and preserved lv ef is presented in table 2. in patients with stable coronary heart disease complicated by heart failure with reduced lv ef and ii degree of la dilatation, enos levels in the serum is 449.00±39.91 pg/ml, whereas in patients with stable coronary heart disease complicated by heart failure with preserved lv ef and ii stage of la dilatation — 673.56±50.98 pg/ml (p<0.01). at iii stage of la dilatation in patients of group 1 the enos level is 344.20±51.98 pg/ml in patients of groups 2 — 616.90±36.49 pg/ml (p<0,01). at the same degree and with la dilation in patients of group 2 enos is 750.27±99.85 pg/ml. thus, in the examined patients with the progression of heart failure the endothelial factors decrease, e.g. enos. in the study of levels of metabolites of nitrogen monoxide — nitrites and nitrates in blood serum the following data were obtained: in patients with stable coronary heart disease, complicated by heart failure with reduced lv ef and ii and iii stage of la dilatation, the total level of nitrates and nitrites in serum was significantly lower than in patients with stable coronary heart disease, complicated by heart failure with preserved lvef and ii and iii stage of la dilatation (p<0.01). among the patients of group 1with reduced lv ef with the progression of la dilation degree from ii (moderate) stage to iii (large) one, enos levels in blood serum decreased by 23.34%. table 1. remodeling of left atrium in case of stable coronary heart disease complicated by heart failure with reduced and preserved left ventricular ejection fraction the degree o f dilation la patients with heart failure with reduced lv ef group 1, n=47 patients with heart failure with preserved lv ef group 2, n=106 p i (initial) la from 40.0 to 45.0 mm 0 39 (36.79 %) р<0,01 ii (moderate) la from 45.1 mm to 50.0 mm 14 (29.78%) 44 (41.51%)* р<0,01 iii (significant) la from 50.1 mm and more 33 (70.21%)* 23 (21.69%) р<0,01 notes: la — the size of the left atrium; * p<0.01 — reliable change index. table 2. vazodilating factors at different stages of left atrium dilation in case of stable coronary heart disease complicated by heart failure with reduced and preserved left ventricular ejection fraction (m±m) indices patients with heart failure with reduced lv ef group 1, n=47 patients with heart failure with preserved lv ef group 2, n=106 ii degree of la dilatation n=14 iii degree of la dilatation n=33 i degree of la dilation n=39 ii degree of la dilatation n=44 iii degree of la dilatation n=23 enos, pg/ml 449.00±39.91 344.20±51.98 750.27±99.85 673.56±50.98* 616.90±36.49# nitrite+nitrate, mmol/l 16.75±0.75 16.05±0.94 19.27±0.67 19.22±0.77* 19.05±1.10# nitrite, mmol/l 4.41±0.21 3.95±0.32 5.15±0.20 5.06±0.16* 5.30±0.37# nitrate, mmol/l 12.34±0.59 12.09±1.13 14.11±0.61 14.16±0.71* 13.74±0.85 notes: * — comparison of patients with stable coronary heart disease complicated by heart failure with reduced lv ef and preserved lv ef and ii degree of left atrium dilatation (p<0,01); # — comparison of patients with stable coronary heart disease complicated by heart failure with reduced lv ef and preserved lv ef and iii degree of left atrium dilatation (p<0,01). v. i. denesyuk et al. 8 in t e r n a l m e d ic in e issn 2413-6077. ijmmr 2016 vol. 2 issue 2 among the patients of group 2 with preserved lv ef with the progression of la dilatation from i (initial) stage to ii one, the level of enos in blood serum decreased by 12.89%, and with the progression of the ii stage to iii one la dilation reduced by 8.41%. in the examined patients with stable coronary artery disease complicated by heart failure with reduced left ventricular ejection fraction, the correlation analysis between indicators of linear dimensions of la and enos levels in the blood serum a direct correlation of medium strength (r=0.38) (p<0,01) was esta blished. carrying out a similar correlation ana lysis for patients with stable coronary heart disease complicated by heart failure with preserved lv ef also allowed to establish a direct correlation of weak force (r=0.21) (p<0.01). progressive hypertrophy and dilatation of heart was followed by subsequent first diastolic disruption and then systolic ventricular dysfunction, increased myocardial oxygen demand, changes in subendocardial blood flow, myo­ cardial bioenergy disorders and increased risk of life-threatening ventricular arrhythmias [1]. recent research proved instrumental and morphological parallels, so we expand on the remodeling of heart as a single clinical and morphological syndrome that is important for making prognosis in patients with coronary heart disease [18]. the processes of la remodeling are defined by the degree of new myocardial damage and the presence of viable myocardium. conclusions in patients with stable coronary heart disease complicated by heart failure with reduced left ventricular ejection fraction iii (significant) degree of left atrium dilatation was mainly determined, while in patients with stable coronary heart disease complicated by heart failure with preserved left ventricular ejection fraction ii (moderate) degree of left atrium dilation was mainly determined. in the patients with stable coronary heart disease complicated by heart failure with reduced left ventricular ejection fraction in comparison with the group of patients with preserved left ventricular ejection fraction, the significant reduction of enos, nitrites, total of nitrites and nitrates was defined during the study. the research allowed to establish the direct correlation of medium strength (r=0.38) between parameters of linear left atrial size and enos levels in blood serum of the patients with stable coronary artery disease complicated by heart failure with reduced left ventricular ejection fraction (p<0.01) and the direct correlation of weak force (r=0.21) in the examined patients with stable coronary artery disease complicated by heart failure with preserved left ventricular ejection fraction (p<0,01). references 1. amosowa k. clinical cardiology 1999: 710. 2. voronkov l. primary prevention of heart failure — one of the priorities of modern cardiology. ukrainian kardіology magazine 2004; 4: 42–47. 3. glaser m. left ventricular hypertrophy and prognosis in patients with chronic heart failure with preserved ejection fraction. heart failure 2012; 13: 19–25. 4. denesyuk o, denesyuk v. dynamics degrees of remodeling and systolic-diastolic left ventricular dysfunction in unstable angina influenced by the optimal long-term treatment. herald of problems of biology and medicine 2015; 2: 121–126. 5. dyuzhykov a, terentyev v. features of morpho-functional state of the myocardium in patients with ischemic and no ischemic cardiomyopathy. modern scientific technology 2007; 12: 23–28. 6. zharynova v. modern opportunities of opti mization of endothelium treading therapy in patients with chf (focus on enos). heart failure 2013; 3: 46–53. 7. zakirova an, oganov rg, zakirova ne, klochkova gr, musina fs. myocardial remodeling in ischemic heart disease. rational pharmacotherapy in carv. i. denesyuk et al. 9 in t e r n a l m e d ic in e issn 2413-6077. ijmmr 2016 vol. 2 issue 2 dio logy 2009; 5(1): 42–45. (in russ.) doi:10.20996/1819­ 6446-2009-5-1-42-45. 8. lang r, biering m, devereux rb et al. recommendations of chambers quantification. eur j echo­ cardiogr 2006; 7(2): 79–108. 9. mandate of the ministry of health of ukraine from 02.03.2016 №152 on approval of the introduction of medical and technological documents for standardization of care in stable coronary heart disease. news of medicine and pharmacology in ukraine 2016; 572: 26–60. 10. muhorlyamov n. cardiomyopathy. medicine 1999: 228. 11. oxidative stress in chronic heart failure. the possibilities of pharmacological correction. y belenkov, e privalov, y danilogorskaya et al. cardiology. cardiovascular surgery 2009; 1: 4–9. 12. patalakh i. participation of protein c in the regulation of the fibrinolytic capacity of plasma in patients with chronic heart failure. circulation of blood and hemostasis 2009; 3/4: 95–100. 13. pfeffer ma, braunwald e. ventricular remodeling after myocardial infarction. experimental observations and clinical implication. circulation 1990; 81(4):1161–1172. 14. recommendations of association of cardiologists of ukraine for the treatment of chronic heart failure in adults (revision 2011). heart failure 2011; 1: 101–116. 15. aljaroudi w. inpact of progression of dia stolic dysfunction on mortality in patients with nor mal ejection fraction. circulation 2012; 14: 782–788. 16. esc guidelines for the diagnosis and treatment of acute and chronic heart failure 2012: the task force for the diagnosis and treatment of acute and chronic heart failure 2012 of the european society of cardiology. developed in collaboration with the heart failure association (hfa) of the esc. european heart journal 2012; 33: 1718–1847. 17. ramani gv. chronic heart failure: contem­ porary diagnosis and management. mayo clin. proc. 2010; 85: 180–195. 18. ziuraitiene r, pangonyte d, stalioraityte e et al. cardiomyocyte remodeling in ischemic heart disease. medicina (kaunas) 2008; 44: 848–854. received: 2016-10-17 v. i. denesyuk et al. koncor1_2014.pm6 68 b io m e d ic a l s c ie n c e s ijmmr 2015 vol. 1 no. 1 the influence of antiretroviral and antituberculosis agents on the biochemical and histopathological indices of liver function in rats o. o. shevchuk, k. a. posokhova, o. m. oleshchuk, t. v. datsko i. ya. horbachevskyternopil state medical university, ternopil, ukraine background. standard antituberculosis treatment and highly active antiretroviral therapy are frequently associated with hepatotoxicity leading to drugs discontinuation. objective. this study aimed to assess the signs of hepatotoxicity in albino rats in case of simultaneous usage of tuberculostatics (tbs) and antiretroviral agents (art). methods. healthy rats were divided in 4 groups: 1st control group; 2nd group was given tuberculostatics (isoniazid – 50 mg/kg, rifampicin – 50 mg/kg and pyrazinamide – 1500 mg/kg); 3rd group was given art (efavirenz – 150 mg/kg and stavudine – 5 mg/kg); 4th group was given tbs and art. the animals were sacrificed painlessly on the 29th day; blood and liver samples were obtained. the main biochemical and histopathological indices were determined. conclusions. comparing with control group, repeated usage of tbs caused the prominent liver injury with cytolysis and cholestasis signs, decreasing of cyp3a and cyp2e1 isozymes activity and dysfunction of protein synthesis by the liver. art (efavirenz and stavudine) caused the elevation of transaminases activity with the increase of serum bilirubin level at the background of increase in cytochrome 450 isoforms 3a and 2e1 activities and total serum protein. the antiretroviral agents in case of simultaneous administration with the antituberculosis drugs diminished the hepatotoxic effects of first-line drugs for tuberculosis treatment which was confirmed by the study of liver histopathology. such results of our experimental study give encouragement for further detailed clinical research of drug-drug interaction of both pharmacological groups due to the rising cases of hiv-associated tuberculosis in the whole world. key words: isoniazid, rifampicin, pyrazinamide, efavirenz, stavudine, liver, cytochrome p450. introduction the global burden of tuberculosis (tb) is enormous today (fig. 1) [1]. the overlapping epidemiology of hiv and tb as simultaneous infections has had catastrophic consequences. the interaction between these diseases is bidirectional. hiv infection increases the risk of both primary and reactivated tb [2], and this risk increases markedly with advancing of hiv disease. the case fatality rates of hivassociated tb are high; the estimated aggregate case fatality rate of hiv-infected tb is about 40 %, and may be over 50 % in many developing countries [3]. when choosing therapy for patients with hiv and tb, drug–drug interactions should be carefully considered. rifampicin is an upregulator of cyp450 enzymes that catalyzes the metabolism of a number of other drugs, including the nnrti (non-nucleoside reverse-transcriptase inhibitors) efavirenz (efv). concomitant highly active antiretroviral therapy (haart) during tb therapy is complicated by high pill burden, concerns about drug-drug interactions, paradoxical immune reconstitution reactions and the main problem is overlapping drug toxicities (table 1) [4, 5, 6]. the treatment of tuberculosis is complicated by drug-induced hepatotoxicity, with reported rates ranging widely, from approximately 3 to 25 %, depending on the hepatotoxicity definitions, the regimens, the methodologies, and the study populations [7]. hepatotoxicity is a relatively common adverse drug reaction leading to treatment interruptions in hiv patients, observed with different drug combinations [8]. currently, the top priority for world health organization (who) is to increase coverage of art for hiv-positive tb patients towards the 100 % target [1]. isonazid (isonicotinylhydrazine, inh) in treatment of all types of tb is associated with mild to moderate elevation of liver enzymes and sometimes with severe hepatotoxicity. rifampicin (rfp), which is commonly used in combination with inh, was address for correspondence: shevchuk oo, medical biochemistry department, m. voli, 1, ternopil, 46001, ukraine tel.: +380352-52-39-87; e-mail: shevchukoksana777@gmail.com o. o. shevchuk et al. international journal of medicine and medical research 2015, volume 1, number 1, p. 68-74 copyright © 2015, tsmu, all rights reserved 69 b io m e d ic a l s c ie n c e s ijmmr 2015 vol. 1 no. 1 reported to result in higher rate of liver injury, especially with the simultaneous administration of pyrazinamide (pza). thus in this study we investigate the influence of first-line antituberculosis and antiretroviral drugs alone and in combination on main functions of the liver. methods inh, rfp and pza were secured from “borshchahivskiy cpp” (ukraine). antiretroviral agents – efv and d4t (aurobindo pharma ltd., india) were kindly provided by state institution “ukrainian center for aids prevention of the ministry of health of ukraine”. animals and study design the white inbred rats (200±20 g, n=24) were taken from tsmu vivarium. animals were randomly distributed into 4 groups (n=6): 1 – intact group; 2 – rats which were treated with combination of antituberculosis drugs isoniazid (50 mg/kg), pyrazinamide (1500 mg/kg) and rifampicin (50 mg/ kg); 3 – rats which were treated with the components of haart: efavirenz (150 mg/kg) and stavudine (d4t, 5 mg/kg); and 4 – rats for which both antiretroviral and antituberculosis drugs in the abovementioned doses were used. a suspension of granulated tablets was introduced via the tube into the rat stomach once a day during 28 days. rats of intact group were given equivalent quantity of distilled water. the rats were weighted and sacrificed under ketamine hydrochloride general anesthesia on the 29th day. the blood and liver samples were taken for investigation. all animals’ procedures were performed according to the rules and requirements of european convention for the protection of vertebrate animals used for experimental and other scientific purposes and local ethic committee. biochemical analysis serum activities of aspartate aminotransferase (ast) and alanine aminotransferase (alt), alkaline phosphatase, concentration of total bilirubin, serum total protein, creatinine and urea were determined table 1. overlapping or additive adverse drugs effects of antiretroviral and first-line antituberculosis agents ��������� ������� �������� ��� ������������������ ��� ���������� ������ �� � ����� �� ���� ��� � ��� ����� � � ����� �� ��� ��� �� � �� � ����� �� ��� ��� ���� � ����� � �������� �� ������� �� ��� ��������� ������� �� ����� ��� � ���� ��� � � ��� ���� � ���� ��� ��� ������ ��������� � ������ ����� ����� ����� � � ��! "������ � ��� ����� �#� ���� ��������� ���� ����� ������� �� ������� � ��� ������ ��������� � ������ ����� fig. 1. estimated tb incidence rates, 2012. reproduced from world health organization. global tuberculosis report 2013. o. o. shevchuk et al. 70 b io m e d ic a l s c ie n c e s ijmmr 2015 vol. 1 no. 1 using standard test kits «lachema». thiobarbituric acid-reactive substances (tbars), catalase activity were determined as described earlier [9, 10]. plasma ceruloplasmin level was determined as well [11]. the liver tissue was homogenized by using the homogenizer silent crusher s (heidolph, germany). the concentrations of lipid hydroperoxides [12], tbars, superoxide dismutase (sod) [13], catalase activity and reduced glutathione level (g-sh) [14] were analyzed in liver homogenates. p-hydroxylase activity as a marker of cyp2e1 and n-demethylase activity as a marker for cytochrome p450 – cyp3a activity in rat hepatic microsomes were determined; liver microsomes were prepared by differential ultracentrifugation [15]. we determined the content of the active form of inh in serum of experimental animals also [16]. histopathological studies slices of the liver (from six animals of each group) were fixed in 10 % formalin, and then lillyfixator was applied. samples were embedded in paraffin; 5–6 �m sections were routinely stained with haematoxylin and eosin and observed using microscope lomo biolam. statistical analysis results were presented as the mean ± standard error of the mean (m±m) and analyzed by the mannwhitney-test and anova-test using statsoft statistica 10 (at systemic statistical analysis department of tsmu). a probability level of less than 0.05 was considered as significant. the distribution of indices was estimated using shapiro-wilk normality test. results combination of the most effective and common antituberculosis agents has the most prominent negative effect on liver function. we observed the rising of liver transaminases levels and cholestatic syndrome: increasing of alt and ast activity by 163 and 118 %, respectively, increasing of alkaline phosphatase and total bilirubin level – by 77 and 104 %, respectively, in comparison with intact control (table 2). also, there are some problems with liver functions due to decreasing of total protein serum level by 40 %. the influence of antiretroviral agent on indices of cytolysis and cholestasis was milder. we noted the increase in alt and ast activities by 42 and 45 % comparing with the control group rats (table 2). the main positive findings of our study are that the concomitant administration of efv and d4t with inh, rmp and pza decreased the hepatotoxicity of antituberculosis drugs (figure 2). the group of rats which received the treatment of all five drugs had decreased levels of alt, ast and alkaline phosphatase activity by 34, 36 and 27 %, respectively, total bilirubin level was lower by 23 %, and total serum protein level was higher by 95 % comparing with rats which were treated with antituberculosis agents alone. however, there was no full restoration of all indices. it was noted that the alt, ast and alkaline phosphatase activities and total bilirubin levels were still higher in rats treated with antituberculosis agents table 2. the changes of biochemical indices of serum in case of antiretroviral and antituberculosis agents usage (m±m, n=6) index the group of animals alt, mccat/l ast, mccat/l alkaline phosphatase, mm�l/(l·hour) total bilirubin, mcmol/l total protein, g/l control rats 0.85±0.03 1.15±0.07 1.86±0.08 2.38±0.10 60.82±1.88 inh, rfp, pza 2.24±0.08* 2.51±0.06* 2.38±0.17* 4.86±0.29* 36.88±0.76* efv, d4t 1.21±0.04* 1.43±0.06* 2.07±0.04 3.23±0.05* 74.01±0.12* inh, rfp, pza+efv, d4t 1.48±0.04*#� 1.61±0.06*# 2.40±0.20*# 3.74±0.14*#� 71.87±4.54* �notes. statistical significance level is p<0.05 for this and for the following tables comparing with: 1. * – intact rats; 2. # – group of rats which received antituberculosis agents inh, rfp, pza; 3. • – group of rats which received antiretroviral agents efv, d4t. � �� � �� � �� � �� � �� ��� ��� ��� ��� ��� � � �� �� ��� ����������� ���� �� ������ ����������� �� �� !"��#$� �� �� !"��#$��%�&'(��)*� fig. 2. the cytolysis and cholestasis indices in case of antituberculosis and antiretroviral agents’ administration. notes: p<0.05 comparatively with * – intact rats, # – rats which were treated with combination of antituberculosis drugs isoniazid (inh, 50 mg/kg), pyrazinamide (pza, 1500 mg/kg) and rifampicin (rmp, 50 mg/kg). o. o. shevchuk et al. 71 b io m e d ic a l s c ie n c e s ijmmr 2015 vol. 1 no. 1 alone than in control rats by 73, 40, 29 and 57 %, respectively. the biochemical markers of intensive oxidative stress were detected after administration of combination of antituberculosis agents (table 3). it was observed that the levels of hpl and tbars in liver tissue increased by 86 and 144 % respectively; tbars in blood serum – by 99 % in comparison with intact group on the 29th day. the decrease in sod activity (by 39 %) and catalase activity in liver homogenates and blood serum – by 55% and 32.5%, respectively, was observed. simultaneously, the levels of g-sh and cp were lower by 29 %, the level of ceruloplasmin was higher by 19 %, respectively, compared to control group. antiretroviral agents caused some lipoperoxidation of cells membranes: hpl level in the liver increased by 21 %, tbars in the liver tissue and serum – by 50 and 23 %, respectively, comparing with control rats (table 3). as for indices of endogenic antioxidant defense, sod and catalase activity in the liver were lower by 46 and 40 %, in the serum – by 31 and 32 %, respectively, comparing with control group. the g–sh was lower by 22 %, ceruloplasmin level increased by 16 %. the levels of primary and secondary products of lipids membranes peroxidation were lower in the group of rats which received combination of antiretroviral and antituberculosis agents comparing with the rats which were treated with antituberculosis combination inh, rmp, pza alone. for example, hpl level was lower by 24 %, tbars in liver tissue and serum – by 32 and 30 %. as for activity of sod and catalase in the liver, they were higher by 20.8 and 21 %, respectively. the antiretroviral agents efv and d4t caused the increase of n-demethylase and p-hydroxylase activity by 34 and 26 %, respectively. similarly, antituberculosis drugs inh, rmp and pza caused n-demethylase and p-hydroxylase activity decrease by 54 and 33 %, respectively, all results comparing with intact rats (figure 3). the level of serum free inh was lower by 18 % in rats which received combined treatment of efv, d4t and inh, rmp, pza, comparing with the group of animals which were treated with antituberculosis agents alone (table 4). histologic findings were done as described below. we observed the lobular disarray, dilation of central veins, and signs of fatty and hyaline-drops protein dystrophy in case of treatment with antituberculosis drugs. all cells had different structure and sizes. we found increased number of cells with kariopicnosis, fragmentation and karyolysis (fig. 4 (1)). art had no the influence on lobular structure of the liver (fig. 4 (2)). however, we noticed the hyperplasia of hepatocytes due to the enlargement of homogenous cytoplasm and hyperplasia of granulated nuclei. cholestasis signs were not fig. 3. the influence of art and antituberculosis agents on n-demethylase and p-hydroxylase activity of liver microsomes. � � � � �� � � �� �� �� �� ��� ��� ��� ��� �� ������������������� � ��������������������� � ������������ �� !���" #$%!�& '!�()# #$%!�& '!�()#�*��� !���" �������� �� ��� ����� ������� �������������� �������������� ������ �� ������!�������"������ �� ��#��$%&�� '()*+(,-� .( /*+(,,0�� -().*+())0�� /(,+*+(,)0��� �1��2�3� 4��5���6���%&�� -(/7*+(,7� )-( '*+(7-0� )+(+8*+(,80�� ))(,,*+(-)0�� �1��2�3�����5���6���%�� +(8)*+(+ � )(-,*+(+,0�� )(+)*+(+70�� )() *+(+)0��� 29:�3� 4��5��$%&�� '(+/*+(+.� 7(+.*+(+.0�� ,(/-*+(+8�0�� 7(/7*+()70�� 29:�3�����5��$%�� ( 7*+(+/� 7(,7*+()'0� 7(+'*+(),�0�� 7(78*+()+0�� ���������3� 4��5��6��%&�� /(..*+(+/� 7('.*+(,70� (8,*+()/0�� (7'*+() 0�� ���������3�����5��6��%�� 8(-)*+(,'� '(8)*+(7,0� '(8,*+(7'0� -( *+( /� ;<2�������%&�� ,(7/*+(+ � )(-/*+(+70� )(8'*+(+80� )(/*+(+-� ������ ���� �����%�� 7,.(-*)+(+� 7.+(8*)7('0� 787('*-(.� 7/)(.*)+(8� � table 3. the influence of antiretroviral and antituberculosis agents on prooxidant and antioxidant homeostasis (m±m, n=6) notes. in this and all the following tables: statistical significance p<0.05 comparatively with: 1. * – control rats; 2. # – group of rats which received antituberculosis agents inh, rfp, pza; 3. • – group of rats which received antiretroviral agents efv, d4t. o. o. shevchuk et al. 72 b io m e d ic a l s c ie n c e s ijmmr 2015 vol. 1 no. 1 observed. in case of drug administration from both pharmacological groups, we noticed the saved lobular structure of the liver (fig. 4 (3)). the structure of hepatic cells was normal; cytoplasm was saturated and homogeneous in all cells of the liver lobule. the histological structure of the liver was improved comparing to the liver structure of the rats which were treated with combination of antituberculosis drugs. discussion at least one-third of the 34 million people living with hiv worldwide are infected with latent tb. persons co-infected with tb and hiv are 21–34 times more likely to develop active tb disease than persons without hiv. coinfection with hiv leads to difficulties in both the diagnosis and treatment of tuberculosis. the problem of simultaneous administration of antituberculosis and antiretroviral drugs in patients with hiv-associated tuberculosis is ambiguous due to overlapping side effect profiles of drugs both pharmacological groups, the adherence challenges of polypharmacy and their drug-drug interaction especially on the stage of metabolism by liver microsome enzymes. today state-of-art in combination of such drugs is described below. the treatment of patients with tb and hiv should include the first line drugs for tb treatment (common combination of isoniazid, rifampicin, and pyrazinamide) and nnrti based antiretroviral therapy regimen, if possible; efavirenz and rifampin are the preferred components [18]. it is known that evr is metabolized by isoenzyme of cyp450 – cyp3a preferably and by cyp2b and 2a also [6]. this nnrti causes the induction of cyp3a in the liver, but not in the intestine [19]. nucleoside reverse-transcriptase inhibitors (nrtis), like d4t, are metabolized by glucuronidation pathway and don’t have any influence on cyp450 [20]. concerning the antituberculosis drugs, rmp is a potent inducer of cyp3a isoenzyme, as well as of other p450 isoforms, more potent than rifapentine and rifabutin [21]. pza can induce cyp2e1 [22]. we also know that hepatotoxicity of inh depends on activity of this isoenzyme [23]. the sapit trial – an open-label randomized controlled trial in durban, south africa to determine optimal timing of art initiation in relation to tb treatment – showed that initiation of art during tuberculosis treatment in patients with sputum smear positive for tuberculosis and hiv-infection, reduced mortality by 56 % and was associated with improved outcomes [17]. our study reasserts the results of this trial. we can see the improving of all liver functions due to the repeated administration of components of art, especially total serum protein level. such results are explained because of induction of enzymes of the liver endoplasmic reticulum by efv [19]. the influence of antiretroviral agent on indices of cytolysis and cholestasis was mild. the increase in total bilirubin level was noted. we can explain this by the fact that one of the mechanisms of efvinduced cholestasis is inhibition of bile acids transport [24]. our results are supported by the clinical data of the similar studies which showed the absence of risk of isoniazid–associated hepatitis in case of concomitant haart [25]. �������� �� �� ��������� �������� ��������� ������ �������� ��������� ����� �� ��� ���� ������ ���������������� ��������� ��� ���� !�"� #�$ !�%&� notes. * – the significance level p<0.05 comparing with the rats which received antituberculosis drugs. table 4. the influence of antiretroviral and antituberculosis agents on serum isoniazid level in rats (m±m) fig. 4. histopathological structure of rat liver in case of administration of antiretroviral and antituberculosis agents alone and in combination: 1. liver structure of rats which were treated with combination of pza, inh and rmp. haematoxylin and eosin stain. �160; 2. liver structure of rats which were treated with antiretroviral agents for 28 days. haematoxylin and eosin stain. �160; 3. liver structure of rats which received combination of antituberculosis and antiretroviral agents. haematoxylin and eosin stain. �160. 1 2 3 o. o. shevchuk et al. 73 b io m e d ic a l s c ie n c e s ijmmr 2015 vol. 1 no. 1 liver biopsy is the traditional gold standard to ascertain the degree of liver injury. our biochemical results are confirmed by histological observations. functionally, the liver can be divided into three zones, based upon oxygen supply. zone 1 encircles the portal tracts where the oxygenated blood from hepatic arteries enters. zone 3 is located around central veins, where oxygenation is poor. zone 2 is located between the first two zones. the main mass of cyp450 enzymes is located in zone 3 of hepatic acinus [29]. some studies showed increased activity of cyp3a and cyp2e1 in case of isoniazid and rifampicin administration [30]. that’s why the decreasing of activity of these isozymes can be explained by higher hepatotoxicity of such dose of pza which was added to the combination of inh and rmp. we can observe the necrosis of hepatic cells in centrolobular part of liver lobule which is the main cause of depression of such liver function as detoxification. we can suggest that the decrease in concentration of toxic metabolite of inh due to the combination of rmp and evf is the key factor in decreasing of isoniazid hepatotoxicity, and the absence of cases of liver toxicity during preventive inh therapy with the haart background [25, 31]. possible mechanisms in decreasing the negative influence of antituberculosis agents in case of their combination with antiretroviral agents can be explained by the fact that inducers of cytochrome p450 enzymes stimulate synthetic and antitoxic liver functions and can decrease the quantity of necrotic hepatocytes [26]. due to this we can see improved metabolism and detoxification processes due to the stimulation of cytochrome p450 isozymes in case of simultaneous usage of antituberculosis and antiretroviral agents. hepatotoxicity of inh is linked to its metabolites monoacetyl-hydrazine, hydrazine, acetyl-isoniazid and others [27]. isoniazid is metabolized by genetically polymorphic arylamine nacetyltransferase type 2 (nat2), and recent studies showed that isoniazid concentrations may be more toxic for slow acetylators after a standard therapeutic dosing [28], because of relative overdosing and higher concentration of inh toxic metabolites in the blood. that’s why the increase in isonicotinyl hydrazine metabolism can accompany the mitigation of its hepatotoxic effects. conclusion the present study showed that the combination of first-line antituberculosis drugs (isoniazid, rifampicin and pyrazinamide) in case of their intragastric administration during 28 days caused the prominent liver injury with cytolysis and cholestasis signs, decreasing of cyp3a and cyp2e1 isozymes activity and dysfunction of protein synthesis by the liver. antiretroviral agents (efavirenz and stavudine), after their administration into the stomach for 28 days, caused the elevation of transaminases activity with the increase of serum bilirubin level at the background of cytochrome 450 isoforms 3a and 2e1 activity rising and the increase of total serum protein. the antiretroviral agents in case of simultaneous administration with the antituberculosis drugs diminished the hepatotoxic effects of the first-line drugs for tuberculosis treatment. such results of our experimental study may facilitate the detailed clinical research of drug-drug interaction from both pharmacological groups due to the rising cases of hivassociated tuberculosis in the whole world. references 1. world health organization. global tuberculosis report 2014. available at http://www.who.int/tb/publications/global_report/en/. aceessed october 3, 2014. 2. kwara a, flanigan tp, carter ej highly active antiretroviral therapy (haart) in adults with tuberculosis: current status. int j tuberc lung dis 2005; 9(3): 248–257. 3. lawn sd, kranzer k, wood r. antiretroviral therapy for control of the hiv-associated tuberculosis epidemic in resource-limited settings. clin chest med 2009; 30(4): 685–699. 4. cattamanchi a, smith r, steingart kr et al. interferon-gamma 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characteristic of its oxidizing systems (in) orechovich vn (ed) the modern methods in biochemistry. moscow. medicina; 1977: 49– 62 (in russian). 16. hrebennik li. about the determination of isonicotinic acid hydrazide derivatives and their transformation products in the body. probl tuberc 1961; 4: 69–74 (in russian). 17. karim sa, naidoo k, grobler a, et al. timing of initiation of antiretroviral drugs during tuberculosis therapy. n engl j med 2010; 362: 697–706. 18. kaplan je, benson c, holmes kh, et al. guidelines for prevention and treatment of opportunistic infections in hiv-infected adults and adolescents: recommendations from cdc, the national institutes of health, and the hiv medicine association of the infectious diseases society of america. mmwr recomm rep 2009; 58(rr-4): 1–207. 19. berruet n, sentenac s, auchere d, et al. effect of efavirenz on intestinal p–glycoprotein and hepatic p450 function in rats. j. pharm. pharmaceut. sci 2005; 8 (2): 226–234. 20. mouly s, rizzo–padoin n, simoneau g. effect of widely used combinations of antiretroviral therapy on liver cyp3a4 activity in hiv–infected patients. br. j. clin. pharmacol. 2006; 62: 200–209. 21. di perri g, marucco ad, mondo a, et al. drug– drug interactions and tolerance in combining antituberculosis and antiretroviral therapy expert opin drug saf 2005; 4 (5): 821-–836. 22. kovalenko vm, shayakhmetova am, bondarenko lb, et al. the influence of cytochrome p-450 2e1 on biochemical and histomorphological indices of rats liver. suchasni problemy toksikologii. 2006;3. available at http://www.medved.kiev.ua/arhiv_mg/st_2006/ 06_3_5.htm (in ukrainian). accessed october 4, 2014. 23. yue j, peng ren-xiu, yang j, et al. cyp2e1 mediated isoniazid-induced hepatotoxicity in rats. acta pharmacol sin 2004 may; 25 (5): 699–704. 24. mcrae mp, lowe cm, tian x. ritonavir, saquinavir, and efavirenz, but not nevirapine, inhibit bile acid transport in human and rat hepatocytes. j pharmacol exp ther 2006; 318 (3):1068–1075. 25. tedla z, nyirenda s, peeler c, et al. isoniazid– associated hepatitis and antiretroviral drugs during tuberculosis prophylaxis in hiv–infected adults in botswana. am. j. respir. crit. care med. 2010. available at http://ajrccm.atsjournals.org/cgi/reprint/200911– 1783ocv1. accessed october 4, 2014. 26. vengerovsky ai. pharmacological approaches to regulation of liver function. bull sib med 2002; 1: 25– 29 (in russian). 27. de rosa hj, baldan hm, brunetti il, ximenes vf, machado rg. the effect of pyrazinamide and rifampicin on isoniazid metabolism in rats. biopharm drug dispos 2007; 28(6): 291–296. 28. possuelo lg, castelan ja, debrito tc, et al. association of slow n–acetyltransferase 2 profile and anti– tb drug–induced hepatotoxicity in patients from southern brazil. eur j clin pharmacol 2008; 64: 673–681. 29. nepomnyashchikh gi, dyubanova ga, nepomnyashchikh dl, et al. universal markers of hepatotoxic influence of medical agents. bulletin so ramn 2008; 6(134): 86–92 (in russian). 30. chen j, raymond k. roles of rifampicin in drug– drug interactions: underlying molecular mechanisms involving the nuclear pregnane x receptor. ann clin microbiol antimicrob 2006; 5:3. available at http:// www.ann–clinmicrob.com/content/5/1/3. accessed october 4, 2014. 31. huang y–s, chern h–d, su w–j, et al. cytochrome p450 2e1 genotype and the susceptibility to antituberculosis drug–induced hepatitis. hepatology 2003; 37(4): 924–930. received: 2014.05.10 o. o. shevchuk et al. 22 in t e r n a l m e d ic in e issn 2413-6077. ijmmr 2017 vol. 3 issue 1 doi 10.11603/ijmmr.2413-6077.2017.1.7867 the effect of l-arginine on oxidative stress and microalbuminuria in patients with type 2 diabetes mellitus and chronic kidney disease l. p. martynyuk, l. z. vons, o. o. ruzhytska i. horbachevsky ternopil state medical university, ternopil, ukraine background. diabetic kidney disease (dkd) is one of the severest complications of diabetes. microalbuminuria (mau) is one of the first signals of dkd and an important pathogenetic mechanism of disease progression. with diabetes antioxidant properties worsen significantly. objective. the aim was to investigate the effect of l-arginine on oxidative stress parameters and microalbuminuria in type 2 diabetes mellitus and chronic kidney disease patients. methods. in total, 57 patients with type 2 diabetes mellitus and chronic kidney disease and 30 healthy individuals (control group) took part in the study. the patients were divided into 2 congruent groups. the 1-st group of patients (n=33), in addition to standard therapy, received l-arginine 4.2 g intravenously for 5 days, after that they took it 1.0 g orally three times a day during meals for 1 month. the second group of patients (n=24) received a standard therapy. the concentration of lipid peroxidation products was measured by a spectrophotometric method. the determination of mau was carried out in morning portion of urine immunological semiquantitative using test strips. results. significant improvement in indexes of lipid peroxidation was observed in both groups after therapy (p˂0.01), but in patients treated with l-arginine it was more expressed (p˂0.01). the standard therapy did not significantly affect the level of mau (p˃0.05). the patients treated with l-arginine, showed a significant reduction in mau (p˂0.01). conclusions. l-arginine facilitates the correction of lipid peroxidation processes and reduces the severity of microalbuminuria in patients with diabetic kidney disease that slows down its progression. key words: diabetes mellitus, chronic kidney disease, lipid peroxidation, microalbuminuria, l-arginine. corresponding author: liudmula vons, department of internal medicine №3, i. horbachevsky ternopil state medical university, 1 clinichna street, ternopil, ukraine, 46002 phone number: +380965406768 e-mail: vonsluyda@gmail.com introduction in recent decades a number of patients with diabetes mellitus (dm) is increased, the patients with type 2 diabetes mellitus are a significant share of them [1, 2, 3]. diabetic kidney disease (dkd) is one of the severest complications of diabetes; it is registered in 20–30% of patients with diabetes. among the patients, who receive renal replacement therapy, the share of people with dkd is about 40–50% [2, 4]. it is established that 1020% of patients with diabetes die from chronic renal failure [4, 5]. microalbuminuria (mau) is one of the first signals of dkd. it is not only the symptoms but also an important pathogenetic mechanism of progression [6, 7]. according to recent studies, mau is considered to be an independent predictor of progressive renal disease [6, 7]. despite the fact that mau is regarded as a risk factor, in the timely diagnosis and adequate treatment it can achieve the level of recourse normoalbuminuric [6, 8]. but in 30–45% of patients over 10 years old the disease progresses to proteinuria. on the other hand, in 20% of patients over 20 years old a terminal kidney failure is developing either [6, 7]. with diabetes antioxidant properties worsen significantly. the activity of antioxidant defence in the tissues is reduced; a lipid peroxidation is disordered that has repeatedly been proven in experimental diabetes [9, 10]. it is important to reveal as soon as possible the disorders of proand antioxidant system for diagnosis and monitoring of the health state. the timely correction of changes can prevent the progression of vascular complications of diabetes [9]. international journal of medicine and medical research 2017, volume 3, issue 1, p. 22–25 copyright © 2017, tsmu, all rights reserved l. p. martynyuk et al. 23 in t e r n a l m e d ic in e issn 2413-6077. ijmmr 2017 vol. 3 issue 1 the aim of our research was to investigate the effect of l-arginine on oxidative stress parameters and microalbuminuria in type 2 diabetes mellitus and chronic kidney disease patients. methods a total of 57 patients with type 2 diabetes mellitus and i-v stages of chronic kidney disease took part in the study. there were 27 (47%) men and 30 (53%) women among them. the average age of the patients was (58.7±1.2) years old. the average duration of diabetes is (11.4±0.9) years. the average illness period of ckd is (2.5±0.3) years. the average weight of patients was (81.7±1.4) kg. 30 healthy individuals composed the control group. the diagnosis was established by the second national congress which was adopted as nephrology classification of diseases of the urinary system in 2005. the stage of ckd was determined by glomerular filtration rate (gfr), defined by the formula ckd-epi [11]. the concentration of thiobarbituric acid reactive substances (tbars) was determined in the blood serum by a spectrophotometric method [12]. we also evaluated the concentrations of glutathione (sh-group) [13], catalase [14], superoxide dismutase (sod) [15] in the blood plasma. the determination of mau was carried out in morning portion of urine immunological semiquantitative using test strips clinitek® microalbumin 2 strips (germany) by means of the unit clinitek status® + analyser (germany). mau was diagnosed at the level of urinary albumin excretion within 30–300 mg/day, normoalbuminuria – less than 30 mg/day. the patients with dkd were divided into 2 congruent groups: the 1-st group of patients (n=33), who, in addition to standard therapy received l-arginine 4.2 g intravenously for 5 days, after that they took it 1.0 g orally three times a day during meals for 1 month. the second group of patients (n=24) received a standard therapy. the results were compared with those of the healthy controls (n 30). the data were subjected to statistical research processing. for this purpose the application package statistica (startsoft usa, v.12) was used: the method of parametric and nonparametric statistics, spearman rank correlation to determine the connection between the studied parameters. statistically significant differences in p˂0.05 are considered. results analyzing the results, it was found outthat 100% of patients with dkd have lipid peroxidation disorders (fig. 1). the average level of mau in the dkd patients before treatment is in 25.6 times higher than in the control group (fig. 2). we have analysed the findings of mau, depending on the method of treatment in dkd patients groups before and after treatment (table 2). discussion the average levels of tbars and sh-groups in the patients with dkd before treatment were significantly increased compared with the corresponding values in the control group (p˂0.01). significantly reduced sod and catalase activity were detected in the observed dkd patients before treatment compared with the control group (p˂0.01). no significant difference in lipid peroxidation indexes was noted between two dkd patients groups before treatment (р˃0.05). significant improvement in lipid peroxidation indexes was observed in both dkd patients groups after therapy (p˂0.01), but in the pafig. 1. the level of lipid peroxidation indexes in dkd patients before treatment and control groups. * – the difference between the control group and dkd patients group is significant (р˂0.01). fig. 2. the level of mau in the dkd patients before treatment and in the control groups. * – the difference between the control group and the dkd patients group is significant (р˂0.01) l. p. martynyuk et al. 24 in t e r n a l m e d ic in e issn 2413-6077. ijmmr 2017 vol. 3 issue 1 tients treated with l-arginine it was more expressed (p˂0.01). the standard therapy did not significantly affect the level of mau (p˃0.05). a significant reduction in mau (p˂0.01) was proved in the patients treated with l-arginine. the role of oxidative stress in the pathogenesis of diabetes has been examined repeatedly in the experimental models. renal cortical superoxide production is increased in the early stage of experimental diabetes that leads to vasoconstriction of afferent arteriole [16]. recent studies in experimental models of diabetic nephropathy indicate that vascular synthesis of no protects from progression of renal lesions in diabetes [17]. l-arginine is a semiessential amino acid and also the main source for the generation of no via nitric oxide synthase [18]. recent study demonstrated that l-arginine supplementation in type ii diabetic rats was beneficial by preserving glomerular filtration rates, presumably via increased renal endothelial nitric oxide synthase levels, that leads to renal vasodilation [19]. in our present research l-arginine supplementation, which stimulates nitric oxide synthesis, causes a pronounced improvement in lipid peroxidation indexes and a significant re duction in mau in the patients with dkd. conclusions in patients with dkd, the decrease in activity of sod and catalase and the increase of tbars the effects of treatment on lipid peroxidation indexes are presented in table 1. table 1. the dynamics of lipid peroxidation indexes in dkd patients before and after treatment depending on different treatment regimens, m±m indexes groups p group і (n=33) group іі (n=24) p1 p2 p3 tbars, mkmol/l before treatment 6.26±0.20 6.20±0.13 р˃0.05 after treatment 3.81±0.27 5.27±0.46 р˂0.01 р˂0.01 р˂0.05 sod, % before treatment 41.76±0.68 41.45±0.66 р˃0.05 after treatment 55.61±1.42 43.21±1.18 р˂0.01 р˂0.01 р˃0.05 sh-groups, mlmol/l before treatment 42.59±1.23 43.71±2.08 р˃0.05 after treatment 57.05±1.98 51.14±1.65 р˂0.05 р˂0.01 р˂0.05 catalase, % before treatment 53.74±2.16 51.46±1.18 р˃0.05 after treatment 31.39±2.80 47.25±2.88 р˂0.01 р˂0.01 р˃0.05 notes: 1. p1 – significant difference in performance between groups before and after treatment. 2. p2 – significant difference in performance in the first group before and after treatment. 3. p3 – significant difference in performance in the second group before and after treatment. table 2. the dynamics of mau levels, depending on different treatment regimens, m±m маu mg/day groups p group і (n=23) group іі (n=24) p1 p2 p3 before treatment 180.7±19.9 188.6±15.6 р˃0.05 after treatment 51.4±15.9 149.2±15.5 р˂0.01 р˂0.01 р˃0.05 notes: 1. p1 – significant difference in performance between groups before and after treatment. 2. p2 – significant difference in performance in the first group before and after treatment. 3. p3 – significant difference in performance in the second group before and after treatment. l. p. martynyuk et al. 25 in t e r n a l m e d ic in e issn 2413-6077. ijmmr 2017 vol. 3 issue 1 and sh-group parameters is observed that proved a lipid peroxidation disorder. l-arginine in the standard therapy facilitates the correction of lipid peroxidation processes and reduces the severity of microalbuminuria in patients with diabetic kidney disease that slows down its progression. references 1. schieppati a. chronic renal disease as a public health problem: epidemiology, social and economic implications. kidney int. 2005;98:7–10. 2. dudar i, loboda o, korol l, alieksieieva v. progression of chronic kidney disease and the role of oxidative stress in different stages of ckd. ukrainian journal of nephrology and dialysis. 2012; 2(34):18–24. 3. gaidaiev yu. endocrinological state service of ukraine and prospects of medical care to patients with endocrine disorders. international journal of endocrinology. 2006;2(4):9–14. 4. kamynsky a. diabetes: new insights and old misconceptions. part 2. prevention of type 2 diabetes. international journal of endocrinology. 2012;4:42–45. 5. kolesnyk m. endocrinology: science and practice. pharmacist. 2008;3:4–6. 6. loboda om, krasyuk іv, alexeeva vv, korol lv, drіyanska vy, dudar іo. the relationship of lipid peroxidation and chronic inflammation of atherosclerotic changes in vessels and poeaznykamy endothelial dysfunction in patients with ckd stages ii–iv. ukrainian journal of nephrology and dialysis. 2015;1(45):13–20. 7. sirenko yu. new opportunities determination of microalbuminuria in clinical practice. hypertension. 2010;3(11):55–58. 8. caramori m. the need for early predictors of diabetic nephropathy risk: is albumin excretion rate sufficient. diabetes. 2000;9:1399–1408. 9. radzyshevsky n. violation of homeostasis and oxidative carbohydrate metabolism and their effects on the heart and blood vessels in patients with comorbid diseases. therapeutic ukrainian magazine. 2016;1:30–35. 10. reheda m, bayda m. role of disorders of lipid peroxidation and activity of antioxidant enzymes in the trachea of guinea pigs in the pathogenesis of allergic alveolitis ekspyrymentalnoho and their correction thiotriazolin. experimental and clinical physiology. 2013;1:47–52. 11. levey as, stevens la, schmid ch, zhang yl, castro af, feldman hi, et al. a new equation to estimate glomerular filtration rate. ann intern med. 2009;150:604–612. 12. gavrilov v, mishkorudnaya m. spectrophotometric determination of lipid hyperoxides in blood plasma. laboratornoe delo. 1983;29:33–35. 13. ellman gl. tissue sulfhydryl groups. arch biochem. 1959;82:70–72. 14. koroluy m, ivanova l, majorova i. metod opredeleniya aktivnosti katalazi. laboratornoe delo. 1988;34:16–19. 15. chevari s, andyal t, shtrenger ya. determination of antioxidant parameters of blood and their significance in old age. laboratornoe delo. 1991;37:9–13. 16. ishii n, patel kp, lane ph, taylor t, bian k, murad f, et al. nitric oxide synthesis and oxidative stress in the renal cortex of rats with diabetes mellitus. j am soc nephrol. 2001;12:1630–1639. 17. kanetsunay, takahashi k, nagata m, gannon ma, breyer md, harris rc, et al. deficiency of endothelial nitric-oxide synthase confers susceptibility to diabetic nephropathy in nephropathyresistant inbred mice. am j pathol. 2007;170(5): 1473–84. 18. cherla g, jaimes ea. role of l-arginine in the pathogenesis and treatment of renal disease. j nutr. 2004;134(10):2801–2806. 19. claybaugh t, decker s, mccall k, slyvka y, steimle j, et al. l-arginine supplementation in type ii diabetic rats preserves renal function and improves insulin sensitivity by altering the nitric oxide pathway. int j endocrinol. 2014:171546. doi: 10.1155/2014/171546. received: 2016-12-07 l. p. martynyuk et al. 16 in t e r n a l m e d ic in e issn 2413-6077. ijmmr 2017 vol. 3 issue 2 doi 10.11603/ijmmr.2413-6077.2017.2.8242 left ventricular diastolic dysfunction and oxygen supply of lower extremities in patients with stable ischemic heart disease and concomitant type 2 diabetes mellitus n. i. yarema, n. v. pasechko, a. i. khomitska, i. p. savchenko, i. v. smachylo, l. v. naumova, l. v. radetska, a. o. bob, m. e. havrylyuk, o. о. bob, n. m. havrylyuk, o. i. kotsyuba i. horbachevsky ternopil state medical university, ternopil, ukraine background. the peculiarities of diastolic heart failure and indices of arterial and venous blood oxygenation in patients with stable ischemic heart disease and concomitant type 2 diabetes mellitus are presented in the article. obvious left ventricular diastolic dysfunction with the increased levels of natriuretic peptide, uric acid and decreased indices of arterial and venous blood oxygenation in the presence of comorbid type 2 diabetes mellitus have been revealed. objective. the research was aimed to study the peculiarities of left ventricular diastolic function disorders, levels of nt-probnp, uric acid and indices of arterial and venous blood oxygenation in patients suffering from stable exertional angina with underlying comorbid type 2 diabetes mellitus. methods. 70 patients with ihd: stable exertional angina of the ііі functional class, were examined. the first group comprised 39 patients with stable exertional angina of the ііі functional class with left ventricular diastolic dysfunction; the second group – 31 patients with stable exertional angina of the ііі functional class with left ventricular diastolic dysfunction and concomitant type 2 diabetes mellitus. all the examined patients underwent bdechocardioscopy, with the detailed evaluation of left ventricular diastolic function, nt-probnp and uric acid levels in venous blood were determined by immunoenzyme method, indices of arterial and venous blood oxygenation were evaluated too. results. the correlation between left ventricle diastolic function and oxygen volume consumed by the tissues of lower extremities in patients with stable ischemic heart disease and concomitant type 2 diabetes mellitus was determined. conclusions. in patients with stable ihd, left ventricular diastolic dysfunction and concomitant type 2 diabetes mellitus the levels of nt-probnp, uric acid and oxygen supply of lower extremities are significantly higher as compared to patients with ihd without type 2 diabetes mellitus. key words: stable ischemic heart disease; left ventricular diastolic dysfunction; diabetes mellitus; natriuretic peptide; blood oxygenation. corresponding author: alla khomitska, department of internal medicine №1, i. horbachevsky ternopil state medical university, 1 clinichna street, ternopil, ukraine, 282004 phone number: +0976860500 e-mail: balabanai@tdmu.edu.ua introduction stable ischemic heart disease (ihd) in patients with type 2 diabetes mellitus is diagnosed in 2–4 times more often than in people of the same age without diabetes [1]. according to some authors, diabetes mellitus negatively influences on left ventricular diastolic function, and the increase of diabetes mellitus duration is accompanied by chronic heart failure development [4]. there are some findings that left ventricular diastolic dysfunction may be caused by diabetes mellitus development regardless of the presence of ihd or arterial hypertension [2]. the results of some researches show that the prevalence of asymptomatic left ventricular diastolic dysfunction in patients with type 2 diabetes mellitus is 63.2 % and increases with age [4]. one of the main markers of chronic heart failure is n-terminal brain natriuretic propeptide (nt-probnp), its levels increase as chronic heart failure develops [3]. an asymptomatic hyperuricemia is also an important risk factor in the development of cardiovascular complications. therefore early diagnostic of this pathology is important for proper drug-induced correction in patients with ihd, left ventricular diastolic dysfunction and concomitant type 2 diabetes mellitus [5]. international journal of medicine and medical research 2017, volume 3, issue 2, p. 15–19 copyright © 2017, tsmu, all rights reserved n. i. yarema et al. 17 in t e r n a l m e d ic in e issn 2413-6077. ijmmr 2017 vol. 3 issue 2 n. i. yarema et al. the research was aimed to study the peculiarities of left ventricular diastolic dysfunction, the levels of nt-probnp, uric acid and indices of arterial and venous blood oxygenation in patients with stable exertional angina with comorbid type 2 diabetes mellitus. methods 70 patients with ihd: stable exertional angina of the ііі functional class, were examined. the average age of the examined patients was (58.38±0.64) years old. the first group comprised 39 patients suffering from stable exertional angina of the ііі functional class with left ventricular diastolic dysfunction; the second group counted in 31 patients with stable exertional angina of the ііі functional class with left ventricular diastolic dysfunction and concomitant type 2 diabetes mellitus. all patients were diagnosed with relaxation type of left ventricular diastolic dysfunction, and left ventricular ejection fraction in all examined patients was higher than 45%, which means that systolic function of the left ventricle was preserved. the control group comprised 20 healthy individuals of the same age and sex. all the examined patients underwent bd-echocardioscopy with the detailed evaluation of left ventricular diastolic function, nt-probnp and uric acid levels in venous blood were determined by immunoenzyme method, indices of arterial and venous blood oxygenation were evaluated too (saturation of arterial (sa.o2) and venous (sv.o2)) blood – with pulse oximeter; arterial (ca.o2) and venous (cv.o2) blood oxygenation in vitro including the assessment of oxygen volume consumed by the tissues of lower extremities da.o2-dv.o2) – with oximeter unistat apparatus (usa). all statistical analyses were performed with statistica 6.0 and microsoft excel. data are expressed as means±standard deviation or as number (%). continuous variables were analyzed by the student’s t­test and the mann–whitney test. p<0.05 was considered statistically significant. the authors had full access to information and take full responsibility for the integrity of the data. all authors have read and agreed to the manuscript as written. institutional review committee approval and informed consents were obtained. results in patients with ihd and concomitant type 2 diabetes mellitus correlation of е/а was by 34.8% (p<0.01) lower than in the control group and by 15.1% (p<0.05) higher than in patients with ihd without concomitant type 2 diabetes mellitus (table 1). ivrt and dt values also differed considerably in patients with ihd, left ventricular diastolic dysfunction and concomitant type 2 diabetes mellitus, specifically: dt value was by 30.5% (p˂0.01) higher than in the control group and by 24.1% (p<0.05) higher than in patients with ihd without type 2 diabetes mellitus. е' value was by 50.5% (p<0.01) lower in the 2nd group of patients as compared to the control group, and by 26.8% (p<0.05) lower than in patients with ihd without type 2 diabetes mellitus. in patients with stable ihd correlation of е/e' was increasing to (9.83±0.14) because of significant decrease in е' and was in 1.8 times higher than in the control group and by 25.9% (p<0.01) higher as compared to the patients with ihd without type 2 diabetes mellitus that proves an increased inflexibility of myocardium in patients with comorbidity of ihd and type 2 diabetes mellitus. nt-probnp value in patients with stable ihd and concomitant type 2 diabetes mellitus was (566.07±22.01) pg/ml and was by 35.6% (р˂0.01) higher as compared to the first group of the examined patients and by 78.7% (р˂0.01) higher as compared to the control group that evidences more significant chronic heart failure development in patients with stable ihd and concomitant type 2 diabetes mellitus (fig. 1). in patients with ihd, left ventricular diastolic dysfunction and concomitant type 2 diabetes mellitus the level of uric acid was by 53.3% (р˂0.01) higher as compared to the control group and by 21.3% (р˂0.05) higher as compared to the examined patients without config. 1. level of nt-probnp in the examined patients 18 in t e r n a l m e d ic in e issn 2413-6077. ijmmr 2017 vol. 3 issue 2n. i. yarema et al. comitant type 2 diabetes mellitus. the data obtained in our research confirm the results of some authors that hyperuricemia is an independent risk factor in the development of cardiovascular complications in patients with ihd and has negative effect on the clinical course of chronic heart failure. in patients with stable exertional angina and concomitant type 2 diabetes mellitus the decrease in arterial and venous blood oxygenation indices as compared to the patients with stable ihd without type 2 diabetes mellitus was determined (table 2). sa.o2 was slightly lower in the 2nd group than in the 1st group. sv.o2 value in the 2 group of patients with comorbid type 2 diabetes mellitus was by 21.8% (p<0.05) lower as compared to the patients of the 1st group. са.о2 and cv.o2 values were considerably lower in patients with left ventricular diastolic dysfunction and type 2 diabetes mellitus as compared to the patients without diabetes mellitus, respectively by 21.2% (p<0.01) and 18.5% (p<0.01), which indicates the deficiency of oxygen saturation of lower extremities tissues as the result of transvenous and transcapillary blood flow difficulties. the volume of oxygen consumed by tissues of lower extremities da.o2–dv.o2 in patients with stable ihd and type 2 diabetes mellitus was (44.85±0.94) ml·l-¹ and was by 9.6% (р<0.05) lower as compared to the patients without type 2 diabetes mellitus. so, in patients with stable ihd and concomitant type 2 diabetes mellitus the decrease in oxygen volume, which is transported to the tissues of lower extremities, was accompanied by the decrease in oxygen volume consumed by the tissues of lower extremities. the direct correlation between the decrease in value of e' decline and decrease oxygen volume consumed by the tissues of lower extremities in patients with stable ischemic heart disease and concomitant type 2 diabetes mellitus was revealed. discussion according to the recent literature [6,8], violation of oxygen transporting processes of in the organism is predefined by insufficiency of pumping function of myocardium, by deceleration of blood stream in the vessels of greater circulation, by uneven distribution of blood in the system of microcirculation [7]. таble 1. rates of left ventricular diastolic function in patients with ihd and concomitant type 2 diabetes mellitus index control group,n=20 group 1, n=39 group 2, n=31 р1-2 е, sm/sec 0.72±0.01 0.62±0.02* 0.56±0.02* р1-2<0.05 а, sm/sec 0.54±0.03 0.65±0.04* 0.70±0.03* р1-2˃0.05 е/а 1.21±0.04 0.93±0.05* 0.79±0.04* р1-2<0.05 е', sm/sec 0.121±0.010 0.082±0.007* 0.060±0.006* р1-2<0.05 е/е' 5.34±0.18 7.81±0.12* 9.83±0.14* р1-2<0.01 dt, msec 172.12±3.02 181.26±5.23* 224.76±13.62* р1-2<0.05 ivrt, msec 80.14±1.32 98,04±4,25* 112.61±3.62* р1-2<0.05 notes: 1. * – significant differences of indices in comparison with the control; 2. р1–2 – significant differences of indices between two groups of patients. таble 2. rates of arterial and venous blood oxygenation in patients with stable ihd, left ventricular diastolic dysfunction and type 2 diabetes mellitus index control group,n=20 1 group, n=39 2 group, n=31 р1-2 sa.o2, % 99.10±0.41 96.12±0.74* 91.53±0.51* р1-2<0.05 sv.о2, % 70.31±0.33 67.26±0.41* 52.64±0.58* р1-2<0.01 са.о2, ml·l -¹ 149.20±0.62 142.10±1.53* 112.57±2.65* р1-2<0.01 сv.o2, ml·l -¹ 98.33±0.62 94.14±1.41* 76.75±2.57* р1-2<0.01 da.о2-dv.o2, ml·l -¹ 50.96±0.66 47.91±0.62* 43.85±0.94* р1-2<0.05 notes: 1. * – significant differences of indices in comparison with the control, 2. р1-2 – significant differences of indices between two groups of patients. 19 in t e r n a l m e d ic in e issn 2413-6077. ijmmr 2017 vol. 3 issue 2 for patients with stable іhd the circulatory hypoxia is of systemic nature, develops in the presence of chronic heart failure and develops in comorbid states [4, 7], type 2 diabetes mellitus in particular. it was established that in efficiency of oxygen transporting to the tissues of the organism, a functional ability of cardiovascular system is crucial [8], and that is why a search for effective methods of comorbid states treatment is a topical issue. conclusions in the examined patients with stable ihd and concomitant type 2 diabetes mellitus more severe impairment of left ventricular diastolic function and the increase of its rigidity, caused by type 2 diabetes mellitus, were revealed. in patients with stable ihd, left ventricular diastolic dysfunction and concomitant type 2 diabetes mellitus the levels of nt-probnp and uric acid were significantly higher as compared to the patients with ihd without type 2 diabetes mellitus, which means that comorbidity with diabetes mellitus was the reason for more severe diastolic heart failure. in patients with stable exertional angina and concomitant type 2 diabetes mellitus there was obvious oxygen deficiency in peripheral tissues with the decrease in oxygen volume consumed by the tissues of lower extremities, which worsens the course of chronic heart failure. references 1. arques s. current clinical applications of spectral tissue doppler echocardiography as a noninvasive surrogate for left ventricular diastolic pressures is the diagnosis of heart failure with preserved left ventricular systolic function. cardiovascular ultrasound. 2007;5:28–16. 2. danzmann lc. left atrioventricular remodeling in the assessment of the left ventricle diastolic function in patients with heart failure: a review of the currently studied echocardiographic variables. cardiovascular ultrasound. 2008;6:69–56. 3. exiara t. left ventricular diastolic dysfunction in diabetes mellitus type 2. hypertension. 2010;28: 294–289. 4. galderisi m. diastolic dysfunction and diabetic cardiomyopathy: evaluation by doppler echocardiography. journal american college cardiology. 2006;48:1551–1548. 5. koh m. management of stable coronary artery disease. european heart journal. 2013;38:2949– 2300. 6. kovakenko vm. stable ischemic heart disease. reccomendations of association of cardiologists of ukraine. 2016;1:176–1. 7. roe mt. patterns and prognostic implications of blood oxigention in patients with ischemic heart disease. european heart journal. 2014;29:2488– 2480. 8. owan te. trends in prevalence and outcome of heart failure with preserved ejection fraction. national england journal of medicine. 2012;355: 254–251. received: 2017-09-08 n. i. yarema et al. 84 b io m e d ic a l s c ie n c e s issn 2413-6077. ijmmr 2017 vol. 3 issue 1 doi 10.11603/ijmmr.2413-6077.2017.1.7923 free radicals and inflammation in rats of different age in cases of sodium nitrites and tobacco smoke poisoning p. h. lykhatskyi, l. s. fira i. horbachevsky ternopil state medical university, ternopil, ukraine background. due to the wide use of nitrate fertilizers in agriculture and their migration into groundwater and food, the spreading of nitrate poisoning has become epidemic. however, people in the process of life get into bad habits: smoking, alcohol, drugs abuse. all these factors affect health and can cause fatal outcome. in real life, people are often exposed to more toxic factors that lead to general poisoning of the body and damage of many organs. objective. the research was aimed to study the activity of free radicals and inflammation in rats of different age in cases of sodium nitrite affection with underlying 45-day tobacco intoxication. methods. the content of nitrite ion (no2 -) was evaluated by gris reaction. the level of pro-inflammatory (interleukin 6 (il-6) and anti-inflammatory (interleukin 4 (il-4) cytokines was determined in serum by elisa method using test kits. results. it was proved that in rats of different age affected by sodium nitrite with underlying 45-day tobacco smoke intoxication, the content of nitrite ion in serum, liver, lungs and myocardium is increased. after poisoning the animals with the studied toxicants, inflammation was activated in the body that was evidenced by the increased pro-inflammatory cytokine il-6 and decreased inflammatory cytokine il-4 in serum. conclusions. the nitrite ion content in organs was the most significant and inflammation was manifested in the immature rats. in these animals the content of anti-inflammatory cytokines was the lowest. key words: tobacco smoke; sodium nitrite; rats of different age; nitrite ions; cytokines. corresponding author: petro lykhatskyi, department of medical biochemistry, i. horbachevsky ternopil state medical university, 1 maidan voli, ternopil, ukraine, 46001 phone number: +380974433614 e-mail: luhatsky@tdmu.edu.ua introduction the wide spread smoking is a global problem of humanity; many scientists and experts make efforts to solve it. smoking has a negative impact on the health of the smokers, but also on the health state of people, who do not smoke but are exposed to harmful effects of pollutants entering the atmosphere with tobacco smoke [1]. numerous scientific studies clearly highlighted the negative influence of smoking on the development of many diseases of different organs and systems of the human body [2, 3]. in recent years, the influence of smoking on free radical processes on the body has been extensively studied. it is established that smoking causes depletion of vitamins c and a, decreases serum levels of other antioxidants, which leads to tissue damage by free radicals [4, 5]. study of the oxidation-antioxidant status in passive smokers revealed similarity of such changes in active smokers [6]. due to the increasing technogenic and anthropogenic pollution, the study of combined effects on the body of the most common xenobiotics: heavy metals, nitrates and nitrites, tobacco smoke, medications, is urgent. considerable attention is attracted to intoxication mechanism of nitrite and nitrate. this happens because of the intensive application of chemicals in the industry, lack of efficient methods for purifying drinking water, high levels of pollution caused by nitrates and nitrites number in foods, especially early fruits and vegetables. hemic hypoxia, caused by nitrates that penetrate into the body, cause functional and morphological changes in many organs, including kidneys and liver, which can lead to further development of comorbidity [7, 8]. because of the summation of environmental risk factors, chronic inflammation may develop, which involves all organs and tissues. recently, the attention of researchers has been attracted to mediators of immune reinternational journal of medicine and medical research 2017, volume 3, issue 1, p. 84–88 copyright © 2017, tsmu, all rights reserved p. h. lyhatskyi, l. s. fira 85 b io m e d ic a l s c ie n c e s issn 2413-6077. ijmmr 2017 vol. 3 issue 1 p. h. lyhatskyi, l. s. fira sponses – cytokines. cytokines are produced by all body cells for cell-to-cell interaction and regulation of biochemical processes in cell [9]. imbalance of cytokines is important for the pathogenesis of toxic affection of the body. the aim of this research was to study the activity of free radicals and inflammation in rats of different age in cases of sodium nitrite affection with underlying 45-day tobacco intoxication. methods the experiments were conducted on white outbred male rats that were kept on a standard diet at the vivarium of ternopil state medical university. the rats were divided into three age groups: the first – immature, weighing 60-80 g, the second – mature, weighing 180-200 g, the third – senile rats, weighing 300-320 g; each age group consisted of two subgroups: an intact control and experimental group. rats study groups were affected with tobacco smoke for 45 days. the test animals were divided into 3 groups. one of them was administered with sodium nitrite, dose 45 mg/kg 24 hours before the end of the experiment; the second – with sodium nitrite 72 hours before euthanasia. the third group of rats was subjected to toxic affection with tobacco smoke only. depending on the chronic smoke effect, the model was simulated using a sealed chamber 30 litres in volume, allowing fumigating the animals in free behaviour. tobacco smoke, formed by combustion of 6 cigarettes “prima sribna (blue)”, containing 0.6 mg of nicotine and 8 mg of tar, was served into it through openings in the chamber. the camera was located 6 animals both within 6 minutes. the animals in the control group were well for 6 minutes in a sealed chamber, but were not subject to smoke. in 45 days after the beginning of the affection of the animals with tobacco smoke they were taken out of the experiment by euthanasia under thiopental anaesthesia. blood, serum, liver, lungs and myocardium of the animals were used for the study. the experimental tissues were used to prepare 10% homogenates using saline. the content of nitrite ion (no-2) was determined by gris reaction [10]. pro-inflammatory (interleukin 6 (il-6) and anti-inflammatory (interleukin 4 (il-4) cytokines levels in blood serum were determined by elisa methods using test kits. quantitative assessment of serum concentration in peripheral blood of these cytokines was performed by chemiluminescent enzyme-linked immunosorbent assay using elisa analyser rt-2100c. test systems and control serum il-4, il-6, russia, were used according to the test systems protocols. the results of the reaction were determined by a spectrophotometer ulab108ua, wavelength 450 nm. these cytokines concentration was evaluated using a calibration curve in picogram per 1 ml (pg/ml) [11]. the research was performed according to the general principles of animal experiments approved by the national congress on bioethics (2001, kyiv, ukraine) and is consistent with the regulations of the european convention for the protection of vertebrate animals used for experimental and other scientific purposes (strasbourg, france, 1985) [12]. statistical analysis of the data was performed using statistica 6.0, parametric student's t-test and nonparametric wilcoxon criterion for related samples. changes considered significant at p≤0.05 [13]. results after rats poisoning with sodium nitrite (sn) during a 45-day tobacco smoke (ts) toxicity we have noticed the increase in content of nitrite ion in serum and organs (table 1). in immature rats nitrite ion content in serum increased by 18% till 45 day of ts toxicity. after the poisoning with sodium nitrite (24 hours before the end of the experiment on the 45th day) the content of this indicator increased by 69%, and after the application of toxicants in 72 hours before the study deadline the content of nitrite ion increased by 132%. a similar increase in nitrite ion content was evidenced in serum of mature and old rats. using of both toxicants led to increase in its content in 2 times (72 hours after 45-day sn poisoning of ts animals). in liver of rats of all age, nitrite ion content increased after the affection with tobacco smoke. the use of sodium nitrite as an additional toxicant contributed to the formation of nitrite ion. in liver of the immature rats the rate increased in 3 times, in mature – in 3.5 times. the least sensitive liver was evidenced in the senile animals which nitrite ion content increased in 2.2 times in the final period of the study. similar dynamics was observed in lungs of rats of different age. the highest content of nitrite ion was in the groups of rats with underlying 45 day tobacco intoxication which 72 hours before the end of the experiment were administered with sodium nitrite. this rate 86 b io m e d ic a l s c ie n c e s issn 2413-6077. ijmmr 2017 vol. 3 issue 1 increased in the immature rats in 3.7 times, in the mature – in 3.2 times, and in the senile – in 12.7 times compared with the intact control group. during the study of myocardium of the smoke intoxicated rats almost the same increase in the content of nitrite ion in all age groups (in 1.7-1.9 times higher than normal) was noted. in the rats poisoned with both toxicants this rate also increased: in the immature and old rats it exceeded the intact control in 2.2-2.3 times respectively. in the mature rats the rate increase in myocardium exceeded 2 times. thus, the poisoning of rats for 45 days with tobacco smoke caused a moderate increase in nitrite ion content in blood serum and organs of rats of different age. after an additional affection of rats with sodium nitrite, this rate significantly increased (r≤0.05) and was considerably higher than in the intact animals. this increase in the content of nitrite ion may be caused by activation of free radical processes in the affected organism. we determined the content of proand anti-inflammatory cytokines in blood serum of rats after simultaneous affection with sodium nitrite and tobacco smoke. the results of the study of pro-inflammatory cytokine (il-6) content in blood of rats of different age groups are presented in table 2. in old rats inflammation development was less significant, as evidenced by the increase of il-6 content in serum after the affection with tobacco smoke in 2.4 times, and after the application of both toxicants in 2.6 times (at the end of the experiment). most inflammation was activated in the immature rats. poisoning of the young animals with tobacco smoke resulted in pro-inflammatory cytokine increase in 3.4 times. in the smoke affected animals, which 72 hours before table 1. the content of nitrite ion in serum (nmol/l) and organs (nmol/kg) of rats of different age affected by sodium nitrite with underlying 45 day tobacco smoke toxicity (m±m; n=72) research time, days groups of experimental animals immature rats mature rats senile rats blood serum intact rats 10.00±0.46 8.20±0.74 8.80±0.56 45 day affection with tobacco smoke 11.80±0.44* 10.20±0.18 10.70±0.46 45 day affection with tobacco smoke + 24 hours sodium nitrite poisoning 16.90±0.87* 15.30±0.22* 15.80±0.29* 45 day affection with tobacco smoke + 72 hours sodium nitrite poisoning 23.20±0.37* 16.40±0.25* 17.90±0.18* liver intact rats 7.60±0.22 3.20±0,16 9.90±0.39 45 day affection with tobacco smoke 10.20±0.61* 5.40±0.51* 11.40±0.43* 45 day affection with tobacco smoke + 24 hours sodium nitrite poisoning 15.60±0.21* 11.00±0.29* 16.10±0.79* 45 day affection with tobacco smoke + 72 hours sodium nitrite poisoning 23.00±0.59* 11.30±0.48* 21.50±0.73* lungs intact rats 1.30±0.14 1.00±0.11 1.70±0.19 45 day affection with tobacco smoke 2.90±0.28* 1.60±0.14* 2.60±0.23* 45 day affection with tobacco smoke + 24 hours sodium nitrite poisoning 3.40±0.31* 1.90±0.11* 3.10±0.17* 45 day affection with tobacco smoke + 72 hours sodium nitrite poisoning 4.80±0.21* 3.20±0.13* 4.60±0.32* lungs intact rats 2.20±0.14 1.60±0.15 1.80±0.15 45 day affection with tobacco smoke 4.00±0.26* 2.70±0.14* 3.40±0.14* 45 day affection with tobacco smoke + 24 hours sodium nitrite poisoning 4.40±0.21* 2.90±0.17* 3.70±0.20* 45 day affection with tobacco smoke + 72 hours sodium nitrite poisoning 4.90±0.16* 3.20±0.13* 4.10±0.13* note: here and in the following tables * – significant changes between intact rats and rats affected with tobacco smoke (p≤0.05). p. h. lyhatskyi, l. s. fira 87 b io m e d ic a l s c ie n c e s issn 2413-6077. ijmmr 2017 vol. 3 issue 1 p. h. lyhatskyi, l. s. fira the end of the experiment end (45th day of tobacco affection) were administered with sodium nitrite, the content of il-6 increased in 4.6 times. thus, the immature rats were the most sensitive to smoke and both toxins, inflammation in them was developed the fastest and most actively. the research of anti-inflammatory cytokines (il-4) content in the studied rats affected with xenobiotics was useful. the results are presented in figure 1. the rate of inflammatory cytokines activity is the lowest in the immature rats, in the simul_ taneous use of sodium nitrite and smoke the content of il-4 reaches 45% compared to the norm (on 45th day of smoke intoxication and 72nd hour of sodium nitrite poisoning), as presented in figure 1. in the immature and senile rats the content of this indicator after the affection with tobacco smoke was on the same level, 68% of the intact animals’ rate. the use of sodium nitrite as an additional toxicant contributed to the disorders in the system of anti-inflammatory cytokines formation. in the old rats after sodium nitrite application, the il-4 content in tabaco smoke intoxicated rats decreased by 45% and was 55% of norm, in the mature rats in this period it was 59%. therefore, sodium nitrite poisoning of rats with underlying tobacco intoxication leads to the imbalance in the system of pro-/anti-inflammatory cytokines, pro-inflammatory indicators are predominant. discussion it is established that after getting into the human body nitrates are restored to nitrites, which lead to the activation of methaemoglobin formation that causes tissue hypoxia. according to literature [7], sodium nitrite in contact with oxyhaemoglobin is a powerful generator of active radicals: ho2, o2 oh, no2. these metabolites damage biological systems, have a significant cytotoxic action, initiate the process of lipid peroxidation, can reveal a strong oxidizing action interacting with sh-groups of proteins, reduced forms of nucleotides, physiologically active compounds [14]. in the pathogenesis of various diseases, inflammation caused by immune mechanisms is of great importance that is confirmed by the numerous experimental studies [15]. inflammation is developed in response to damage and penetration into tissues of pathogens with table 2. the content of pro-inflammatory cytokines (il-6) in serum (ng/l) of rats affected with sodium nitrite with underlying 45-day tobacco smoke toxicity (m±m; n=72) research time, days groups of experimental animals immature rats mature rats senile rats intact rats 1.91±0.28 3.00±0.30 4.14±0.17 45 day affection with tobacco smoke 6.43±0.21* 8.31±0.19* 9.97±0.29* 45 day affection with tobacco smoke + 24 hours sodium nitrite poisoning 7.74±0.35* 9.35±0.32* 10.94±0.21* 45 day affection with tobacco smoke + 72 hours sodium nitrite poisoning 8.87±0.21* 10.50±0.26* 10.90±0.31* note: here and in the following tables * – significant changes between the intact rats and the rats affected with tobacco smoke (p≤0.05). fig. 1. the content of anti-inflammatory cytokines (il-4) in blood serum of the rats of different age groups affected by sodium nitrite with underlying 45-day tobacco intoxication, %. 88 b io m e d ic a l s c ie n c e s issn 2413-6077. ijmmr 2017 vol. 3 issue 1 participation of pro-inflammatory cytokines: il-1, tnf-α, il-6, il-8 chemokines. in case of local reactions violation, protective inflammatory response is intensified, synthesis of cytokines is increased; they get into the bloodstream and effect at a system level. in this case pro-inflammatory cytokines affect almost all organs and body systems. [15] the cytokine imbalance is important for the initiation and progression of inflammation in the body, but the role of cytokines during chronic inflammatory reactions still remains to be determined. conclusions the affection of rats of different age with sodium nitrite with underlying tobacco intoxication leads to nitrite ion accumulation in serum and organs of animals that may be caused by activation of free radical processes in the body. the violation in ratio proand anti-inflammatory cytokines was determined. the most significant increase of il-6 in the immature rats with underlying decrease of anti-inflammatory il-4 was proved; that is one of the causes of decreased activity of protective systems after injury. references 1. bakharev vs, kortsova ol, zharikova mo, gushchin av. bad habits as a factor in the formation of impact of environmental risks. environmental safety. 2009;1(5):68–72. 2. behera s, xian h, balasubramanian r. human health risk associated with exposure to toxic elements in mainstream and sidestream cigarette smoke. science of the total environment. 2014;472(15): 947–56. 3. hagstad s, bjerg a, ekerljung l, backman h, lindberg a, ronmark e, lundback b. passive smoking exposure is associated with increased risk of copd in never smokers. chest. 2014;145(6):1298–304. 4. goraca a, scibska b. plasma antioxidant status in healthy smoking and non-smoking men. bratisl lec listy. 2005;106(10):301–306. 5. kiral f, ulutas pa, fidanci ur. lipid peroxidation and antioxidant enzymes in rats affected with cigarette smoke. ancara univ vet fac derg. 2008; 117(6):145–148. 6. yokus b, mete n, cakir ud, toprak g. effects of active and passive smoking on antioxidant enzymes and antioxidant micronutrients. biotechnol eq. 2005;19:117–123. 7. el-sheikh n, khalil f. l-arginine and l-glutamine as immunonutrients and modulating agents for oxidative stress and toxicity induced by sodium nitrite in rats. food and chemical toxicology. 2011; 49(4):758–762. 8. green c, david a, golawski j. analysis of nitrate, nitrite and (15n) nitrate in biological fluids. anal biochеm. 1982;126(1):131–138. 9. symbyrtsev as. cytokines – a new system of regulation of protective reactions of the organism. cytokines and inflammation. 2002;1(1):9–17. 10. yrhashev ta, karimov ai. nitrates effect on the body of humans and animals (review). dushanbe: nodyr; 2009;58. 11. peremot sd, smilianska mv, wolanska a. the activity of cytokines in patients with acute coronary syndrome. annals of mechnikov institute. 2010;3: 33–37. 12. medical ethics and human rights, regulations on use of animals in biomedical experiments. experimental and clinical physiology and biochemistry. 2003;2(22):108–109. 13. lapach sn. statistical methods in medical and biology research using excel in experimental study. clinical test. analysis of the pharmaceutical market. kyiv: moryon; 2000;320. 14. нolovko ll. protective status of the organism under conditions combined effects of cadmium and lead salts and sodium nitrite. medical chemistry. 2004;6(3):176. 15. rahyno yi, chernyavskyy am, polonskaya y. inflammatory and destructive changes in the process of vulnerable atherosclerosis plaque modification. cardiology. 2007;9:62–66. received: 2017-03-06 p. h. lyhatskyi, l. s. fira koncor1_2014.pm6 12 p e d ia t r ic s ijmmr 2015 vol. 1 no. 1 association of familial and environmental factors with asthma and allergic diseases in ukrainian children population o. ye. fedortsiv1, j. e. zejda2, n. yu. luchyshyn1, g. m. brozek2 1 i. ya. horbachevsky ternopil state medical university, ternopil, ukraine 2 medical university of silesia, katowice, poland background. asthma and other allergic diseases as influenced by environmental and familial factors might be targeted using preventive measures. these diseases are a matter of some urgency in ukraine because of the clinical, social and economic importance in childhood. objective. the aim of this study was to investigate the relationship between prevalence of asthma, spastic bronchitis, allergic rhinitis, atopic eczema, unspecified hypersensitization and some selected environmental and familial factors in a population sample of 6 to14-yrs old western ukraine schoolchildren. methods. the data set of the study was collected using a questionnaire-based survey, containing the data of 4871 urban and rural children age 6 to 14 years. correlation of asthma and allergic disease with familial and environmental factors was examined by means of multivariate logistic regression. results. increased risk of asthma (1.7 %) was associated with the urban residence (or=1.8; p=0.04) and high parental education (or=1.8; p=0.02); spastic bronchitis (6.2%) – with parental allergy (or=1.3; p=0.03); atopic eczema (6.2 %) – with younger age (or=1.3; p=0.03), high parental education (or=1.3; p=0.03), parental allergy (or=1.4; p=0.02), tobacco smoke at home (or=0.7; p=0.01) and household density (or=0.6; p=0.001); diagnosis of unspecified allergic sensitization (11.8 %) was related to high parental education (or=1.2; p=0.03), parental employment (or=0.8; p=0.02) and pets at home (or=1.2; p=0.06). conclusions. this study identifies that lifeslyle and building factors are associated with an altered prevalence of common childhood allergic diseases. prevention may need to address the minimization of potential riskfactors. key words: asthma, environmental factors, children. introduction prevalence of asthma and allergies among children has become an increasing problem during the last few decades. asthma has become the most common chronic disease among children and is one of the major causes of hospitalization among those younger than 15 years of age. as more people become sensitized to allergens, allergic diseases may increase in europe in the coming years [1]. previous isaac studies revealed a relatively low prevalence of allergic diseases specifically in eastern europe. while the results of the studies from different areas in eastern europe have been reported, less is known about the current epidemiology of childhood allergic diseases in different areas of ukraine [2, 3]. with the prevalence of childhood asthma and allergic diseases increasing worldwide over the past decades, it is widely accepted that environmental factors, in addition to genetics, are closely related to allergic diseases [4, 5]. the role of environmental factors in relation to asthma and allergy increased through the 1990s. there has been widespread public concern that changing patterns of outdoor air pollution underlie the rising burden of asthma, but the professionals are not so sure about such correlations. the indoor environment, in which people spend most of their time, has received less attention [6, 7]. the aims of this study was firstly, to quantify the prevalence of asthma, spastic bronchitis, allergic rhinitis, atopic eczema, unspecified hypersensitization in schoolchildren in ternopil and ternopil region, and secondly, to evaluate associations between children's asthma and allergic diseases from one side and housing and familial characteristics from another. methods we used questionnaire which was based on the isaac symptom questionnaire in ukrainian language. the questionnaires were answered by parents or guardians of children, aged 6 to 14 years old from ternopil and surrounding rural area. issues and factors affecting the prevalence of physiciandiagnosed asthma, spastic bronchitis, allergic rhinitis, atopic eczema, and unspecified hypersensitization address for correspondence: n. yu. luchyshyn, medical department of paediatrics � 2, i. ya. horbachevsky ternopil state medical university, m. voli, 1, ternopil, 46001, ukraine tel.: +380660471120; e-mail: luna.ternopil@gmail.com o. ye. fedortsiv et al. international journal of medicine and medical research 2015, volume 1, number 1, p. 12-16 copyright © 2015, tsmu, all rights reserved 13 p e d ia t r ic s ijmmr 2015 vol. 1 no. 1 were included in the list of allergic diseases. the study protocol was approved by the local ethics committee at the ternopil state medical university (� 1 (b) from 07.04.2010). independent variables of interest were based on questionnaire self-report. subgroups of subjects defined by place of residence (urban/rural), gender (male/female), age group (<10 years old / >10 years old), parental education (less than high school/high school or above), parental employment (both unemployed/otherwise), any diagnosed parental allergy (at least one parent: yes/ no), tobacco exposure at home – smoking adult at home (yes/no), housing (multi-storey house/ otherwise), heating of the house (coal or wood stove/ otherwise), household density – defined as the ratio of persons living in the house to rooms in the house (�1 person per room/otherwise), dampness in house defined as moisture stains or signs of mold on the inner surfaces in the house (yes/no), contact with pets inside the house (yes/no). statistical analysis was performed by using statistica 7.1 software. descriptive analyses were used to examine the prevalence of each outcome with the personal and environmental characteristics of the study population. statistical significance was assessed using chi2 test. multivariable logistic regression analyses with each outcome were used to examine the association with the personal and environmental characteristics and to account for potential confounding. for these analyses, outcomes were the diseases or symptoms of interest. the strength of association was based on the odds ratio (or) and 95 % confidence intervals (ci). results analysis of 4871 completed questionnaires has found the following results: study population was 50.8 % females and 49.2 % males. 54.7 % of children lived in an urban area, 47.3 % – in rural. groups of children living in urban and rural areas were similar (p<0.05) in terms of age (10.4±2.0 years and 10.5±2.0 years, respectively) and gender (51.0 % and 50.5 %, respectively, were females). descriptive characteristics of the study population by urban and rural status are presented in table 1. the prevalence of respiratory and allergic diseases were: childhood asthma – 1.70 %, spastic bronchitis – 6.26 %, allergic rhinitis – 5.79 %, atopic dermatitis – 12.40 %, and current chest wheezing – 11.56 %. table 2 shows the prevalence of respiratory and allergic outcomes by characteristics of the study group. bronchial asthma prevailed in children from urban areas or those with a higher parental education. a higher prevalence of spastic bronchitis was found among those with a parental history of allergy. a higher prevalence of allergic rhinitis was also found among those in an urban area compared to a rural area. there were no statistically significant differences between categories of the characteristics considered for atopic eczema. as results from logistic regression analysis, we found that an increased risk of asthma was associated with urban residence (p=0.04) and high parental education (p=0.02). spastic bronchitis was associated with parental allergy (p=0.03). atopic eczema had statistically significant associations with younger age (p=0.03), high parental education (p=0.03), parental allergy (p=0.02), tobacco smoke at home (p=0.02) and household crowding (p=0.0007). diagnosis of unspecified sensitization was related to high parental education (p=0.03) and parental employment (p=0.02) (table 3). discussion this study was designed to assess the relationship between physician-diagnosed asthma, spastic bronchitis, allergic rhinitis, atopic eczema, table 1. descriptive characteristics of the study population in the ternopil region. ���������� ���� ���� ������ ���� ����� ������ ���� ����� ������ ���� !"� ����# �� $��� ��%� �&%& ��%� �%�� '��� "��(�� �&%� ��%& �&%� �%� )�"��� ������� �(������� ����� ����� �� ��%&� �&%�� ��%�� �%����� *������� ���+ ��$+��� ����� ����� �� �,%�� ��%�� ��%�� �%����� !�$ �����"�� (� �� � �� +����� �� -������ ��%�� �,%�� ��%�� �%����� ������ �. � ��� �� ��+� / +�0��" �(��� � ��+�� ��%,� ��%�� ��%�� �%�� �� �(���� �� � +����1 ����$ ��� � ��%� ��%� ��%� �%���� ���� ��2� �������"3���0��"� ,%, �%, �%& �%���� )�� ����( (�� ��$ 4� �� �� �� ���+ ��%� ��%� ��%� �%� 5�+ �� �� ��� � ,�%� ,�%& ,�%� �%���� ������� #��� �� �� �(� ��� ��� � ��%� ��%� ��%� �%���� o. ye. fedortsiv et al. 14 p e d ia t r ic s ijmmr 2015 vol. 1 no. 1 table 2. frequencies of questionnaire-based variables during the examinatoin of population ���������� ��� � ���� ��� ��� ��� ��������� ��� �������� ������� ��� ������ ������ ��� ������� ���� �������� ��� ����� �� �� !"� #�" �� $�� $" �% �%� #%" �" #&� %!! �## �"� '(��� �$ �# �&� #)* �" &�� $% �% &"� #&! �" %$� �&) �#� )�� '� ������ + ,��(� ! !% ! # ! % ! & ! � .��� %& �# �)� #�# �" �*� $% �% )"� #"! �" "&� �*% �#� �#� /����� )" �# $"� #�) �" �%� $" �% )$� #)" �� *#� �$# �## %&� 0����� + ,��(� ! % ! * ! * ! � ! % 1��� �& �� !*� #$! �" "#� #!$ �% *&� #*% �& !*� %�# �#� $*� 2 1��� �" �# �#� #�� �� $�� "# �� $)� ##% �� �"� ��% �#! %$� +������� ��(������ + ,��(� ! !# ! � ! !% ! !!$ ! !" 3� "� �# "&� �%& �" %*� #%* �% &�� �%" �" %"� )") �#� �#� � �# �# $#� "$ �� $�� %! �� �$� &! �" !�� ##! �* )&� +������� ���������� + ,��(� ! & ! � ! !� ! " ! !!� 3� #* �� #�� "* �& $�� %) �% $�� &� �$ #"� ##! �#� )&� � ") �# "!� �%" �� *�� #%� �% %$� �%) �� $&� )") �## "%� +������� ������� 4 ��� ������ �� + ,��(� ! � ! !% ! ) ! !# ! ) 3� %" �# �"� #)# �" ##� &$ �% %$� #�% �� %%� �&� �## *%� � )& �# $%� #") �" %*� *# �% ��� #$% �& #%� �** �## ""� 5������ �6�� (�� �� + ,��(� ! ) ! " ! & ! !!* ! & .(���7 ����� ��( � )* �# &&� #"� �� $�� *# �% �*� #&$ �" )%� %## �## �)� 8���� %) �# "�� #)% �" $!� &$ �% &#� #�$ �" !$� �"% �#� �!� 1�( ��� + ,��(� ! " ! # ! ) ! " ! # ����9:��� � �# �%� * �� ��� % �# $)� ## �" &�� #� �* �!� ������� $# �# &�� �*" �" �*� #"" �% �%� �*� �" �&� ��* �## $&� 1������ �� ��( � + ,��(� ! " ! " ! � ! $ ! � #;��� �� 9���� "& �# &�� ��* �" !!� #%$ �% "#� �#% �� �$� )�! �## &*� 2#��� �� 9���� #" �# ��� &" �& ��� %# �� *)� *% �$ $%� #�) �## &$� 1�( ����� ��� ��� + ,��(� ! " ! # ! � ! !!!# ! * 3� �" �# ��� #!� �" #)� �* �% )�� ** �� &*� #** �## ")� � �& �# $!� �!! �" %%� ##! �% )$� �!& �" ��� %&� �## $"� <����� �� ��( � + ,��(� ! ) ! $ ! * ! % ! $ 3� )# �# )*� #&� �" %�� #!) �% &&� #"� �� **� %)� �#� ��� � )� �# *$� #%! �" #)� "� �% !&� #)# �" ""� ��* �#! $�� ������� :��� ��� �� ��� ��� ��( � + ,��(� ! # ! & ! # ! % ! !" � unspecified hypersensitization and various family risk and environmental factors while using representative sample of schoolchildren in the 6 to 14 yrs-old age group from a defined geographical area. it was found that 1.7 % had physician diagnosed asthma, 6.26 % – spastic bronchitis, 5.79 % – allergic rhinitis and 12.40 % – atopic dermatitis. the prevalence of childhood asthma in the ternopil region was relatively low comparing to other studies from eastern europe and much lower than in the recent findings in children of kiev (8.1 % in 6–7 years old children and 6.1 % in the 13–14 year old children) [akopian a. z. – unpublished report]. there is an evidence to support an underdiagnosis of asthma in this study population. low prevalence of childhood asthma in the ternopil region and frequent occurrence of allergic diseases at the same time, within the range of the published data, suggests an asthma underdiagnosis. our study revealed a relatively frequent occurrence of spastic bronchitis (6.2 %), which is suggestive of an asthmalike response. however, it is lower then the previous report for central and eastern european children [8]. using multivariable logistic regression analysis, it was established that urban residence was positively associated with asthma, while parental allergy with spastic bronchitis in children. high parental education was related to asthma, atopic eczema and unspecified sensitization of children. parental allergy, tobacco smoke at home, household crowding were positively associated with children’s atopic eczema. keeping pets at home was related with unspecified sensitization of children. o. ye. fedortsiv et al. 15 p e d ia t r ic s ijmmr 2015 vol. 1 no. 1 conclusions this cross-sectional examination suggests that environmental and family risk factors are associated with prevalence of asthma, spastic bronchitis, atopic eczema and unspecified sensitization in 6 to 14-yrsold schoolchildren in ternopil. the determination of potentially preventable environmental and family factors affecting risk of allergic disease is important, considering the apparent world-wide increases in the prevalence of childhood allergic disease. this study identifies lifestyle, building factors which are associated with an altered prevalence of common childhood allergic disease. prevention may need to address the minimization of potential risk-factors. acknowledgments the authors would like to thank those individuals who participated in this data collection. the ministry of foreign affairs (poland) – polish aid 2011: “polish assistance to the prevention of large public impact lung diseases in belarus and ukraine” provided financial support for the project under the grant [249/ pr/2011]. the authors thank the children and parents who participated in this study. ��������� � �� ����� ������� �� ������� ���������� �������� ��������� ��������� �� ����������� �������� � �� ����� ����� !"!#� $"%&'!"%(�� !"!!� $")('!"#*�� !"&!� $"�('!"%��� !"*�� !"$*'!"(#�� !"$#� $")%'!"*#�� �����!$� �+$"�� �+$"$$%� �+$"$�� �+$"$&� �+$"(� !"%�� !"$*'&"!#�� $")�� $"(%'!"!��� $")(� $"��'!"*(�� $"��� $"%!'!"*%�� $"�*� $"%#'!"!#�� ,�-������������� �+$"$#� �+$"#� �+$"#� �+$"%� �+$"#� $")*� $"�&'!"*%�� !"$$� $"%�'!"*%�� !"$#� $"%�'!"&��� !"!$� $")%'!"&�� !"!!� $"�&'!"&*�� .������/�0���� �+$"&� �+$"�� �+$")� �+$"#� �+$"*� !"%)� !"$)'*"�*�� !"!!� $")('!"#*�� !"&&� $"��'!")(�� !"&!� !"$*'!"(%�� !"**� !"$!'!"#)�� 1����2�������� ��3���� �� �+$"$*� �+$"#� �+$"$�� �+$"$&� �+$"$&� !"&)� $")!'*"&&�� $"�*� $"(�'!"*#�� $"%�� $"#�'!"!#�� !"$$� $"%�'!"&#�� $"%%� $"(*'$"�%�� 2������������ ������ �+$"*� �+$"�� �+$"!� �+$"�� �+$"$*� !"*!� $"%!'*"$(�� !"&�� !"$!'!")$�� !"!$� $"%#'!"(*�� !"&%� !"$&'!")*�� !"$(� $")#'!"&&�� 2����������������/����� ����� ���� �+$"#� �+$"$&� �+$"(� �+$"$*� �+$"(� $"�$� $"�)'!"#$�� $"�%� $"%('!"**�� $"��� $"%$'!"&$�� $"%�� $"��'$"���� !"$&� $")%'!"*&�� 4 -��� ��5� �3������ � ��� �+$"(� �+$"%� �+$"%� �+$"$!� �+$"%� $"%$� $"#!'!"*$�� $"))� $"(('!"!%�� $"�*� $"(&'!"&(�� !"$%� $")$'!"#&�� $"�!� $"%&'!"!&�� 1 3����/�3���'�� ���� � 3��� �+$"!� �+$"&� �+$"(� �+$"(� �+$"&� $"�!� $"**'&"#��� $")*� $"#!'!"(#�� $"#)� $"!�'!"�*�� !"*$� $"(&'*"*)�� $"%!� $"#!'!"*&�� 1������� �� 3���/� � ��� ��6 ����� 7�� �+$"�� �+$"�� �+$"*� �+$"�� �+$"*� !"*(� $"%*'*"*!�� $")(� $"(�'!"!&�� !"*�� $")#'!")(�� $"(#� $"#�'$")&�� $"��� $")$'!"*&�� 1 3��� ����������� �!8���� �9� �� �+$"#� �+$"*� �+$"*� �+$"$$$%� �+$"�� $")�� $"�&'!"&%�� $"�&� $"%*'!"!��� $"�&� $"(%'!"&$�� $")�� $"(%'!"!#�� $"�#� $"%)'!"!#�� :������������ � 3��� �+$"#� �+$"�� �+$"(� �+$"�� �+$"�� $")!� $"�!'!"*%�� !"$#� $")!'!"&&�� !"*#� $"�$'!"%*�� $"��� $"%%'!"*%�� !"!%� $"�%'!"#!�� 2������� 3��� �+$"&� �+$"%� �+$"*� �+$"�� �+$"$(� table 3. associations of allergic diseases with environmental and familiar factors (multivariate logistic regressions) o. ye. fedortsiv et al. 16 p e d ia t r ic s ijmmr 2015 vol. 1 no. 1 references 1. the international study of asthma and allergies in childhood steering committee: worldwide variation in prevalence of symptoms of asthma, allergic rhinoconjunctivitis, and atopic eczema: isaac. lancet 1998; 351(9111): 1225–1232. 2. brozek m, zejda je, kowalska m, gebus m, kepa k, igielski m. opposite trends of allergic disorders and respiratory symptoms in children over a period of large-scale ambient air pollution decline. pol j environ stud 2010; 19(6): 1133–1138. 3. the prevalence rates of symptoms of asthma and allergic rhinoconjunctivitis in children aged 6–7 years and 13–14 years fact sheet no. 3.1 may 2007, code: rpg3 air e1. 4. wang tt, zhao zh, yao h et al. housing characteristics and indoor environment in relation to children’s asthma, allergic diseases and pneumonia in urumqi, china. chinese science bulletin 2013; 58(34): 4237–4244. 5. balo bb, makra l, matyasovszky i, csepe z. association of sociodemographic and environmental factors with allergic rhinitis and asthma. acta climatologica et chorologica 2012; 46: 33–49. 6. asher mi, stewart aw, mallol j, montefort s, lai ckw, at-khaled n, odhiambo j, the isaac phase one study group. which population level environmental factors are associated with asthma, rhinoconjunctivitis and eczema? review of the ecological analyses of isaac phase one. respir res 2010, 11:8. available at http:// respiratory-research.com/content/11/1/8. acessed may, 25, 2014. 7. strachan dp. the role of environmental factors in asthma. british medical bulletin 2000; 56(4): 865–882. 8. leonardi gs, houthuijs d, nikiforov b, volf j, rudnai p, zejda j, gurzau e, fabianova e, fletcher t, brunekreef b. respiratory symptoms, bronchitis and asthma in children of central and eastern europe. eur respir j 2002; 20(4): 890–898. 9. fedortsiv o, brozek gm, luchyshyn n, kubey i, lawson ja, rennie dc, zejda je. prevalence of childhood asthma, rhinitis, and eczema in the ternopil region of ukraine – results of bupas study. adv med sci 2012; 57(2): 282-289. received: 2014.05.26 o. fedortsiv et al. 5 e d it o r ia l issn 2413-6077. ijmmr 2018 vol. 4 issue 1 international journal of medicine and medical research 2018, volume 4, issue 1, p. 5-7 copyright © 2018, tsmu, all rights reserved corresponding author: janindra warusavitarne, b med fracs ph.d., department of surgery, st. mark’s hospital, london, united kingdom e-mail: ccolorectal@btinternet.com phone number: +07753061849 doi 10.11603/ijmmr.2413-6077.2018.1.9198 the evolution of anorectal manometry d. wickramasinghe, j. warusavitarne department of surgery, st. mark’s hospital, london, united kingdom the history of physiology testing the history of anorectal research can be dated back to 1200 bc to the chester beatty medical papyrus [1] which described anal diseases and contained scanty descriptions of the anal canal. this knowledge was further enhanced by the descriptions of galen and illustrations of versalius and john calcar. it was many centuries later that the physiology of the anal sphincter control was elucidated. in 1867, masius identified the center of the lower spinal cord as being responsible for anal sphincter tone and reflex contractions in the dog and rabbit. during the same period, manometry studies were first carried out to study anorectal function. the first phenomenon to be described was the recto-anal inhibitory reflex reported by gowers in 1877. this was followed by the observations of langley and anderson where they demonstrated that in the cat, the stimulation of lumbar sympathetic nerves causes relaxation of the rectum and the contraction of the internal anal sphincter. anorectal manometry joltrain et al first described a method for measuring colorectal pressures in 1919. he used a rectal tube after rectal infusion to measure pressures of the lower gastro intestinal tract. this method was refined in 1940 by white et al [2] who developed the colonometrogram which was based on cystometry. this primitive device contained of a vertical glass tube manometer connected in one side to an intravenous drip line and at the other end to a rectal tube (figure 1). he used this to assess the colonic tone in patients with injuries in the brain, spinal cord, cauda equina or sacral nerves and noted that the compliance of the colon and rectum depended on the level of the lesion. based on these initial maneuvers, anorectal manometry (arm) was developed. the procedure involves insertion of a catheter into the anorectum and connecting it to a pressure recorder to measure the intraluminal pressure. it had first been used in assessing patients in the 1980s , although more complex procedures had been attempted several decades previously [3]. transducers have often been developed first for oesophageal manometry and subsequently the same technology used to create devices for anorectal manometry. the initial devices had an intraluminal balloon. subsequently water perfused and solid-state manometers had been used. conventional manometry probes contained a few sensors that were spaced at 3-5 cm and incapable of acquiring the pressures the entire anal canal simultaneously. therefore, they required pullthrough manoeuvres or rotation to sample the entire area of interest. this prevented a continuous measurement of pressures throughout the entire anal canal. moreover, radial sensors required a pull through procedure that introduced motion artefacts. with the advancement of electronics and miniaturisation of sensors, more and more sensors could be fitted into the probes, and this resulted in the development of high-resolution anorectal manometry (hrarm) in 2007 [4]. in hrarm, the space between 2 adjacent sensors is less than 1 cm. most systems have circumferential sensors, each with 12 pressure sensitive segments arranged radially. ten of these sensors are fitted within 6 cm on the probe. the 12 sector pressures are averaged to obtain a single mean pressure value for each level. three dimensional (3d) high definition anorectal manometry (3dhdm) was introduced in 2010 [5]. this uses 16 sensors, each with 16 radial pressure sensitive sectors, arranged over a space of 6.4 cm. this sensor arrangement for the first time provided sufficient radial re solution to allow accurate, simultaneous circumferential assessment of the anal asc. it is also a static test and therefore minimises motion artefacts and other confounders. both these modern techniques are heavily dependent computer hardware and software for recording and interpolation of the data. there are several advantages and disadvantages in hrarm / 3dhdm when compared to conventional manometry [6] (see table 1). d. wickramasinghe et al. 6 e d it o r ia l issn 2413-6077. ijmmr 2018 vol. 4 issue 1 fig. 1. colonometrogram, developed by white et al [2]. table 1. comparison of conventional manometry and hrarm / 3dhdm, from lee et al [6] hrm / 3dhdm conventional manometry more sensors at close intervals (continuum in space and time) e-sleeve for high-pressure zone few sensors at wider intervals dent sleeve for high-pressure zone stationary examination, less discomfort pull through, can be uncomfortable color topographic display, better resolution allowing easier interpretation with less time lines display, poor anatomical resolution, less easy to interpret and time-consuming high resolution allows radial bedside pressure measurement only circular pressure measurement more fragile, shorter life span, greater maintenance required less susceptible to wear and tear, little maintenance and seldom malfunctions arm provides information about the resting pressure (rp), squeeze pressure (sp) and length of the anal canal (anal high pressure zone length – hpzl) by direct measurement. a balloon attached to the tip of the catheter allows additional measurements such as rectal sensory thresholds and rectoanal inhibitory reflex to be elicited. the normal pressure values for a given age and gender varies significantly, depending on the technique and the type of catheter used and thus it is recommended that every laboratory establish its own normal values for every technique. presently, there is a classification system for anal incontinence based on anorectal manometry findings [7]. d. wickramasinghe et al. 7 e d it o r ia l issn 2413-6077. ijmmr 2018 vol. 4 issue 1 a group of patients who are incontinent will demonstrate normal anorectal manometry findings under static conditions. they require monitoring of anorectal motor events over a prolonged period and in the fully ambulatory state. the methods used utilise micro pressure transducers with or without simultaneous emg recordings of the eas. ambulatory arm was first used in patient evaluation in the last decade of the 20th century [8]. the study by kumar et al [8] identified that spontaneous transient relaxations of the ias were more frequent and of longer duration in patients with idiopathic anal incontinence. furthermore, the motility index of the rectum and colon were lower in patients with slow transit constipation. despite the promising results, the clinical role of ambulatory arm has not yet been established. traditional manometry assesses the pressure in the anal canal at each level as a single value and ignores the possibility of radial asymmetry. vector manometry assesses the radial and longitudinal pressure profile along the entire length of the anal sphincter. radial asymmetry, which is expressed as a percentage calculates the degree to which the integrated cross sections deviate from a perfect circle. the vector symmetry index (vsi) on the other hand, is expressed as a value from 0 to 1 [9] with values closer to 0 indicate greater asymmetry. despite being shown to have an accuracy comparable to endoanal ultrasound and needle emg in some studies, yang and wexner found that the localisation of sphincter injuries with vector manometry is poor. the diagnostic utility of anorectal manometry anorectal manometry is useful in objectively evaluating a multitude of disorders. in patients with chronic constipation, arm helps identify patients with defaecatory disorders [10]. however, there can be significant overlap between the subtypes. manometry can help in distinguishing weaknesses in the internal and external anal sphincters in patients with anal incontinence [10]. the response to treatment can also be serially monitored using arm. arm is also useful in excluding dyssynergic defae cation in patients with proctalgia. references 1. banov jl. the chester beatty medical papyrus: the earliest known treatise completely devoted to anorectal diseases. surgery. 1965 dec;58(6):1037-43. 2. white jc, verlot mg, ehrentheil o. neurogenic disturbances of the colon and their investigation by the colonmetrogram: a preliminary report. ann surg. 1940 dec;112(6):1042-57. 3. duthie hl, watts jm. contribution of the external anal sphincter to the pressure zone in the anal canal. gut. 1965 feb;6(1):64-8. 4. jones mp, post j, crowell md. high-resolution m a n o m e t r y i n t h e e v a l u a t i o n o f a n o r e c t a l disorders: a simultaneous comparison with waterperfused ma nometry. am j gastroenterol. 2007 apr;102(4):850-5. 5. rao ss. advances in diagnostic assessment of fecal incontinence and dyssynergic defecation. clinical gastroenterology and hepatology. 2010 nov 1;8(11):910-919.e2. 6. lee yy, erdogan a, rao ss. high resolution and high definition anorectal manometry and pressure topography: diagnostic advance or a new kid on the block? curr gastroenterol rep. 2013 dec 1;15(12):360. 7. thekkinkattil dk, lim m, stojkovic sg, finan pj, sagar pm, burke d. a classification system for faecal incontinence based on anorectal investigations. br j surg. 2008 feb;95(2):222-8. 8. kumar d, waldron d, williams ns, browning c, hutton mr, wingate dl. prolonged anorectal manometry and external anal sphincter electromyography in ambulant human subjects. digestive diseases and sciences. 1990 may 1;35(5):641-8. 9. perry re, blatchford gj, christensen ma, thorson ag, attwood se. manometric diagnosis of anal sphincter injuries. the american journal of surgery. 1990 jan 1;159(1):112-7; discussion 116-7. 10. staller k. role of anorectal manometry in clinical practice. current treatment options in gastroenterology. 2015 dec 1;13(4):418-31. received: 2018-05-18 d. wickramasinghe et al. еволюція аноректальної манометрії d. wickramasinghe, j. warusavitarne st. mark’s hospital, london, united kingdom у статті розглянуто основні віхи розвитку методу аноректальної манометрії, який вико ристо вується для об’єктивної оцінки тонусу аноректальних м’язів і скоординованості скорочень прямої кишки та сфінктерів ануса шляхом прямого вимірювання. issn 2413-6077. ijmmr 2017 vol. 3 issue 234 d e n t is t r y doi 10.11603/ijmmr.2413-6077.2017.2.8228 biometric method of age estimation: development and efficiency, in cases of pathologies of teeth hard tissues 1m. yu. goncharuk-khomyn, 2kh. v. pohoretska, 2l. o. patskan 1uzhhorod national university, uzhhorod, ukraine 2i. horbachevsky ternopil state medical university, ternopil, ukraine background. the physiological changes of tooth are the criteria used in evaluation of regressive formula by kvaal et al. age estimation technique. but in cases of abnormal occlusion, abnormal chewing habits, bruxism, abrasive factors or structural defects of teeth the intensity of tooth aging accelerates. objective. the aim of the research was to define the options of age estimation according to dental state of individuals with pathological attrition. methods. 108 panoramic x-ray photos of patients with pathological attrition of teeth were chosen by a randomized selection (49 males and 59 females). all photos were made by means of planmeca promax orthopantomograph. nine measurements were made for each tooth: the tooth length, pulp length, root length, root width and pulp width at three different levels: cement-enamel junction (level a, beginning of root), one-quarter of root length from a cement-enamel junction (level b), and mid-root (level c). due to these measurements, a number of ratios were calculated in accordance with kvaal et al. method. results. the errors that reached 27±8.4 years were found when evaluating the dental age using primary coefficients of equations suggested by the authors of the method used. by means of mathematical analyses, principal component regression method as well, the correlation coefficient of pearson and method of combining linear regression due to the tooth changes in cases of pathological attrition (lowering level of occlusal surface, dystrophy of pulp structures and deposition of tertiary reparative dentine) by regression analysis, the modified formulas for age estimation using radiographic technique were found. modified coefficients decreased the error to 13±0.8 years, which was relative to the real age upto nearly 42–48% compared to the primary coefficients of equations for pathological attrition. conclusions. age estimation technique can be improved taking into account morphological changes in pathological attrition and the calculated coefficients make it possible to expand the circle of person’s age which needs to be found. key words: age estimation; kvaal method; pathological attrition; regression analyses; reparative dentine deposition. corresponding author: myroslav goncharuk-khomyn, department of prosthetic dentistry, uzhhorod national university, 16/a universytetska str., uzhhorod, ukraine, 88000 phone number: +380991212813 e-mail: myroslav.goncharuk-khomyn@uzhnu.edu.ua introduction estimation of biological age of a person is significant in forensic science, especially for comparative and reconstructive identification antemortem and postmortem as recommended by interpol/icpo (international criminal police organization) and fbi (federal bureau of investigation). justice bodies in an ethnically heterogeneous society use the results of age estimation by dental status where age indicators affect the need of socio-vulnerable persons, illegal immigrants and children and allow benefiting from the state budget; it also influences on level of criminal responsibility of persons with regard to age limit [1]. age is the least variable and probably the most accurate determining parameter, since the aging process is the most independently reflected in changes of pulp and hard tissues of teeth than any other functional system of the body that is more vulnerable to the effects of pathologal features, constitution and physiological defects. practical determination of the age of adults is possible using morphological techniques of gustafson g. (“age determination on teeth”), bang g., ramm e. (“determination of age in humans from root dentine transparency”) johanson g. (“age determination from teeth”), maples w. r. (“an improved technique using dental histology for estimation of adult age”), international journal of medicine and medical research 2017, volume 3, issue 2, p. 34–38 copyright © 2017, tsmu, all rights reserved m. yu. goncharuk-khomyn et al. issn 2413-6077. ijmmr 2017 vol. 3 issue 2 35 d e n t is t r y m. yu. goncharuk-khomyn et al. and morphologically-radiographic techniques of solheim t. (“a new method for dental age estimation in adults”), kvaal s. i. et al (“age esti mation of adults from dental radiographs”) [2–10]. the most rational method which excludes extraction of teeth and subjective grading of morphological indicators is kvaal et al. technique, which involves calculating the ratio of length of crown and root to the length of pulp, width of root to the width of pulp in specifically designated areas, searches averages and uses standardized coefficients for the final result. however, this technique does not provide the effective use in cases of the presence of hard tissue lesions of teeth, pathological attrition is the most common. attrition is a constant form of retrogressive changes in teeth, which involves lowering the level of occlusal surface in the amount related to the normal process of aging. the physiological loss of hard tissue caused by tooth-to-tooth contact in occlusion and mastification depends on diet, dentition, force of mastificatory muscles and chewing habits. physiological attrition is proportional to the age of an individual as deposition of secondary dentine or pulp changes during lifetime [11–13]. the physiological changes of tooth are the criteria used in calculation of regressive formula by kvaal et al. age estimation technique. but in cases of abnormal occlusion (crowding of teeth, malposed teeth, lesions of prosthetics treatment), abnormal chewing habits, bruxism, abrasive factors or structural defects in teeth take place the intensity of tooth aging increases. this process is pathological attrition. we have found out that in pathological attrition the occlusal level of teeth lowers in a few times faster in dependence of forces that influence. formation of reparative tertiary dentine, closing volume of pulp chamber and dystrophy processes take place in pulp structure that is unusual for physiological attrition [14–20]. due to all these factors and principles we have approbated primary method of kvaal et al. age estimation technique and found modified regression formulas that approximate the calculation results with the real age. methods 108 panoramic x-rays photo of patients with pathological attrition of teeth were chosen by a randomized selection (49 males and 59 females). all photos were made by means of planmeca promax orthopantomograph [14– 15]. using graphical redactor adobe photoshop cs3 some teeth were cut from each panoramic photo: maxillary central incisor, lateral incisor and second premolar, mandibular later incisor, canine and first premolar, and all of them were positioned strongly on vertical axis. nine measurements of each tooth were made: tooth length, pulp length, root length, root width and pulp width at three different levels: cement­ enamel junction – (level a, beginning of root), one-quarter of the root length from the cement-enamel junction (level b), and mid-root (level c) [10]. all measurements were performed by means of measurement tool in adobe photoshop cs3 mainly in pixels and then converted into millimeters. for each value 7 measurements were made and for further calculation the average means were estimated (table 1). from the measurements, a number of ratios were calculated in accordance with kvaal et al.: p – the ratio of pulp length to root length; t – the ratio of tooth length to root length; r – the ratio of pulp length to tooth length; a – the ratio of width of pulp to root at level a; b – the ratio of width of pulp to root at level b; c – the ratio of width of pulp to root at level c; m – the mean values of all ratios; w – the mean value of width ratios from levels b and c; l – the mean value of length ratios p and r; w-l – the difference between w and l [10]. all ratios were calculated using standard microsoft office program package microsoft office excel. the mean of all ratios (m) was used as the first predictor, while the difference between the mean of the 2 width ratios and the mean of the 2 length ratios (w-l) was used as the second predictor. the results of estimated age differ significantly from real age and reach the error up to nearly 48%. using correlation coefficient and principal component regression method it was established that the strongest correlation between age results of patients with pathological attrition and calcula ted ratios was evidenced between r and further l, w, and also a (as the level closest to the centers of reparative dentine formation), which occurred because of significantly different level of tooth loss and closing volume of pulp chamber due to attrition (table 2). the statistical information was analyzed by means of statystics pro software and linear regression analysis, thus new modified coefficients for kvaal et al. primary formulas were estimated (table 3). as a result, the error reached 13±0.9 years and did not increase more. issn 2413-6077. ijmmr 2017 vol. 3 issue 236 d e n t is t r y results the most significant correlation results between tooth and age were established in upper and lower incisors, and lower premolar. the lowest correlation was found at lower canine in patients with pathological attrition because of the level of influence of pathological attrition on different types of tooth. the pearson correlation coefficients between chronological age and different ratios (p, t, r, a, b, c) calculated due to the length and width measurements on the orthopantomographs are displayed in table 2. the differences compared to primary correlation are significant at r, l, w table 1. example of measurement of tooth specific indicators of patient with pathological attrition specific parameters base/repeat measurements measurements and calculations number of measurements mean (mm) difference (mm) tooth length main measurement 7 22.07 0.03repeated measurement 7 22.10 pulp length main measurement 7 17.02 0.47repeated measurement 7 16.55 root length main measurement 7 15.00 0.29repeated measurement 7 14.71 pulp width a main measurement 7 0.82 0.01repeated measurement 7 0.81 pulp width b main measurement 60 0.72 0.02repeated measurement 60 0.70 pulp width c main measurement 60 0.39 0.01repeated measurement 60 0.40 root width a main measurement 60 5.14 0.04repeated measurement 60 5.10 root width b main measurement 60 4.36 0.07repeated measurement 60 4.29 root width c main measurement 60 3.82 0.07repeated measurement 60 3.75 table 2. correlation between age of patients with pathological attrition and the ratios of measurements upper central incisor upper lateral incisor upper second premolar lower lateral incisor lover canine lower first premolar p -0.11 -0.08 -0.16 -0.15 -0.07 -0.49 t -0.34 -0.07 -0.11 -0.12 -0.16 -0.44 r 0.24 -0.14 -0.16 -0.12 -0.04 -0.28 a -0.19 -0.30 -0.16 -0.22 -0.90 -0.10 b -0.30 -0.20 -0.16 -0.32 -0.14 -0.20 c -0.32 -0.30 -0.27 -0.31 -0.15 -0.20 m -0.31 -0.26 -0.21 -0.34 -0.17 -0.39 l -0.08 -0.11 -0.17 -0.27 -0.14 -0.23 w-l -0.39 -0.14 -0.08 -0.30 -0.02 0.21 table 3. modified regression equations of patients with pathological attrition equation r2 (coefficient of determination) significant predictors all six teeth age=45.1+5.42 m+3.76 w-l 0.21 none lower canine age=77.1–84.1 m–51.09 w-l 0.012 none lower lateral incisor age=24.6+4.06 m–19.01 w-l 0.04 none lower first premolar age=–21.4+16.5 m–36.1 w-l 0.356 m and w-l upper second premolar age=125.6–84.02 m+42.4 w-l 0.211 w-l upper lateral incisor age=35.11–16.5 m–38.1 w-l 0.214 none upper central incisor age=35.6–76.8 m–56.3 w-l 0.27 w-l upper three teeth age=30.14+14.7 m+2.10 w-l 0.045 m lower three teeth age=19.2+5.7 m–12.18 w-l 0.051 w-l m. yu. goncharuk-khomyn et al. issn 2413-6077. ijmmr 2017 vol. 3 issue 2 37 d e n t is t r y m. yu. goncharuk-khomyn et al. and a ratios because of specific processes in teeth in cases of pathological attrition. by regression analysis new formulas were developed (table 3) and the levels of absolute and relative errors were compared (table 4). discussion the results depend on the stage of pathological attrition. in the research we have found out that the attrition is caused by bruxism, abnormal occlusion due to dispositioned tooth, and inadequate prosthetic treatment may cause proportional constant intense deposition of tertiary reparative dentine and lowering of occlusal surface depending on pathology stage [12]. however, due to abnormal tooth structures or abrasion factors pathological attrition is not a progressive process during which pulp structures and hard tissues changes depend on time and stage of changes and enhanced process development may take place any time. also, the better result were established when the mean levels of all six teeth were cal culated, and the most distant result were reached when single measurements of mandible canine were included. improved regressive formulas were checked by new randomized samples of 50 x-ray photos of patients with pathological attri tion, no information about age was available. the results were ranged by the error not higher than 14±0.8 years old. the further research should be focused on verifying value of tertiary reparative dentine using computer cone beam tomography to determine the dynamics of pulp changes intensity in different stages of disorders that cause pathological attrition. it allows making retro spective analysis, which provides information about average changes of pulp chamber and hard tissues of tooth, so identification of phy siolo­ gical secondary dentine formation before the pathology come about and tertiary repa rative dentine formation in pathology develop ment gives a chance to create new regression analysis by two systems coefficients ‘before patho­ logy’ and ‘during pathology development’. thus all minimal errors occur in techniques of age de ter mination in cases of dental health disorders. conclusions during the study we used kvaal et al. age estimation technique for patients with pathological attrition and defined the level of errors which reaches about 47-49%. using component regression analysis and pearson’s coefficients we determined the correlation between age results and level of tooth surface attrition and deposition of tertiary reparative dentine due to the kind of pathology, which cause pathological attrition, and the time of pathology. the most significant correlation was found between changes in incisors and lower first premolar. the changes in canine in cases of pathology attrition do not affect the result significantly. individually calculated modified coefficients in equations by kvaal et al. age estimation technique in cases of pathological attrition showed the result more adjacent to real age, e.g. the level of absolute error in years was improved from 27±8.4 to 13±0.8 years. age estimation technique can be improved taking into account morphological changes in cases of pathological attrition, and the calculated coefficients allow expanding the circle of person’s age that should be defined. also the examination of two regression systems which stand for attrition of occlusal surface and tertiary reparative dentine deposition in cases of pathology (system 1) and lowering of physiological lever of tooth high as well as deposition of secondary dentine (system 2) exclude the number of errors for accurate age estimation with cone beam tomography. table 4. example of differences of age estimated by means of primary technique of kvaal et al. and modified technique for patient a with pathological attrition teeth/tooth groups age primary technique of kvaal et al. for patients with pathological attrition modified technique of kvaal et al. for patients with pathological attrition mean difference mean difference single tooth actual age 35 21 35 13estimated age 56 48 all six teeth actual age 35 16 35 10estimated age 51 45 three maxillary teeth actual age 35 18 35 12estimated age 53 47 three mandibular teeth actual age 35 17 35 14estimated age 52 39 issn 2413-6077. ijmmr 2017 vol. 3 issue 238 d e n t is t r y references 1. amandeep s. age estimation from physiological changes of teeth. j indian forensic sci. 2004;6(2): 113–121. 2. willems g. a review of commonly used dental age estimation techniques. j. forensic odontostomatol. 2001;19(1):9–17. 3. solheim t, sundnes pk. dental age estimation of norwegian adults: a comparison of different me­ thods. forensic sci int. 1980;16(1):7–17. 4. kostenko y, goncharuk-khomyn m. possibility of improving method of age determination during pathological attrition. the journal of forensic odontostomatology. 2013;3:67–68. 5. gustafson g. age determination on teeth. j am dent assoc. 1950;41(1):45–54. 6. bang g, ramm e. determination of age in humans from root dentine transparency. acta odontol scand. 1970;28:3–35. 7. johanson g. age determination from teeth. odontol revy. 1971;22(21 suppl.):1–126. 8. maples wr. an improved technique using dental histology for estimation of adult age. j. forensic sci. 1978;23(4):764–70. 9. solheim t. a new method for dental age estimation in adults. forensic sci int. 1993;59:137–47. 10. kvaal si, kolltveit km, thompsen io, solheim t. age estimation of adults from dental radiographs. forensic sci int. 1995;74:175–85. 11. bida vi. pathological attrition of hard tissue of teeth and main principles of it’s treatment. кyiv: kyiv truth, ukraine. 2002. 12. addy m, shellis rp. interaction between attrition,abrasion and erosion in tooth wear. monogr oral sci. 2006;20:17–31. 13. purkait swapan kumar. essentials of oral pathology. isbn 9789350252147. 3rd edition. 2011;648. 14. bosmans n, ann p, aly m, willems g. the application of kvaal’s dental age calculation tech­ nique on panoramic dental radiographs. forensic sci int. 2005;153:208–12. 15. landa mi, garamendi pm, botella mc, aleman i. application of the method of kvaal et al. to digital orthopantomograms. int j legal med. 2009; 123:123–128. 16. paewinsky e, pfeiffer h, brinkmann b. quantification of secondary dentine formation from ortho­ pantomograms – a contribution to forensic age esti mation methods in adults. int j legal med. 2005; 119:27–30. 17. sumit s, upender k, atul m, sharma gk. determination of age from teeth using index value of attrition. j forensic med toxicol. 2013;1:0973–1970. 18. solheim t. dental attrition as an indicator of age. gerodontics. 1988;4:299–304. 19. solheim t. amount of secondary dentin as an indicator of age. scand j dent res. 1992;100: 193–9. 20. morse dr. age-related changes of the dental pulp complex and their relationship to systemic aging. oral surg oral med oral pathol. 1991;72: 721–45. received: 2017-10-04 m. yu. goncharuk-khomyn et al. issn 2413-6077. ijmmr 2016 vol. 2 issue 230 d e n t is t r y issn 2413-6077. ijmmr 2016 vol. 2 issue 2 doi 10.11603/ijmmr.2413-6077.2016.2.7037 clinical effectiveness of treatment the patients with chronic apical periodontitis n. g. gadzhula national pirogov memorial medical university, vinnytsia, ukraine background. the success of endodontic treatment is provided by a thorough instrumental and antiseptic treatment of infected root canals, and it depends on the composition of filling material, the degree of adhesion to dentin, hermetic obturation of apical foramen, solubility of sealer. objective. the study was aimed to evaluate the effectiveness of root canal obturation with bioroottm rcs sealer in the treatment of patients with chronic apical periodontitis. methods. endodontic treatment of 23 teeth in 20 patients with chronic apical periodontitis by method of lateral compaction of gutta-percha was carried out. in the main group root canals were obturated with bioroottm rcs, in the control group the canals were filled with apexit plus. the percentage of efficient or non-efficient cases was evaluated on the basis of radiographic comparison of treated chronic apical periodontitis immediately after obturation, in three, six months and one year. radiographic conditions were defined as existing state, improvement and worsening. results. in a year of dynamic evaluation the final results were: in the main group — 54.55% of the patients had complete bone healing, in 27.27% of cases the focus of bone destruction was decreased by 1/2 or more of the initial sizes, 18.18% — resorption lesion was decreased by less than1/2; in the control group — 33.33% of improvement, 25.0% of existing state and 41.66% of worsening. conclusions. bioroottm rcs using for root canals obturation in the treatment of chronic apical periodontitis we proved the high effectiveness of the treatment undertaken: complete healing or improvement of radiographic conditions of periapical bone destruction with x-ray signs of bone regeneration. key words: apical periodontitis, endodontics, sealers, filling, root canals. corresponding author: nataliia gadzhula therapeutic dentistry department, national pirogov memorial medical university, pirogov street 52, vinnytsia, 21001 phone number: (0432) 61-85-79; 097-701-19-18 e-mail: natalidentist1@gmail.com introduction treatment of patients with chronic apical periodontitis is complex and is an important task of modern dentistry due to their high prevalence, frequent complications after traditional treatment and absence of consistent results [1, 2]. according to numerous researches, the basic principles of effective treatment of destructive forms of apical periodontitis are the thorough instrumental and antiseptic treatment of infected root canals with their subsequent filling with pastes based on calcium hydroxide [3, 4]. it prevents microbial contamination and its influence on surrounding tissues, accele­ ration the processes of apexification and bone regeneration [5, 6]. the success of endodontic treatment is also impossible without quality hermetic obturation of root-canal system for forming of reliable barrier between tooth cavity and periodontal ligament [1, 2, 6]. among all modern endodontic sealers a mineral-based root canal sealer bioroottm rcs “septodont” has the above-mentioned features. this material is bioactive by stimulating bone physiological process and mineralization of dentinal structure [7]. it creates a favourable environment for periapical healing and bioactive features including biocompatibility, hydroxyapatite formation, mineralization of dentinal structure, alkaline ph and sealing qualities. due to the use of active biosilicate technology which is monomer free, there is no shrinkage of bioroottm rcs during setting for a tight seal of root canal [8, 9]. therefore, this study was aimed to evaluate the effectiveness of root canal obturation with bioroottm rcs sealer in the treatment of patients with chronic apical periodontitis. international journal of medicine and medical research 2016, volume 2, issue 2, p. 30-33 copyright © 2016, tsmu, all rights reserved n. g. gadzhula issn 2413-6077. ijmmr 2016 vol. 2 issue 2 31 d e n t is t r y issn 2413-6077. ijmmr 2016 vol. 2 issue 2 material and methods endodontic treatment of 23 teeth from frontal and distal groups with chronic apical periodontitis (by an equal distribution between chronic granulating and granulomatous periodontitis) in 20 patients aged 25–45 years without accompanying pathology was carried out. disinfection and irrigation procedures of root canals were made by standard methods: the canals were shaped and underwent an appropriate tapered preparation, disinfected with a 3% sodium hypochlorite solution activated with mechanical agitation and rinsed with 17% edta. permanent obturation of root canals was carried out by method of lateral compaction of gutta-percha. in the main group the root canals of 11 teeth were obturated with bioroottm rcs “septodont” as a sealer, in the control group the root canals of 12 teeth were filled with apexit plus “ivoclar vivadent”. the percentage of efficient or non­efficient cases was evaluated by radiographic comparison of treated chronic apical periodontitis immediately after obturation, and in three, six and twelve months. radiographic conditions were defined as existing state, improvement and wor sening. dynamics of periapical tissue healing were also controlled by a periapical index pai [10]. descriptive statistics was compiled by microsoft excel and student’s t-test. mann-whitney u-test (nonparametric criteria) was used to evaluate differences between groups at baseline (immediate postoperative) and at the 12-month follow-up evaluation. changes in pai score for each group from baseline to 12-month follow-up evaluation were tested with wilcoxon signed rank test. the secondary outcome measures, proportion of teeth in each group that could be considered as improved (pai score decreased) or healed (pai<2) were assessed with chi-square test [3]. clinical symptoms and abnormal findings at the follow­up examination were recorded but not subjected to statistical analysis. results by using bioroottm rcs for nonsurgical treatment of destructive forms of chronic apical periodontitis in the patients of the main group we did not observe any complications after contact with this material. in three months after the treatment was completed the dynamics of clinical and radiographic changes were practically identical in both groups of patients, although complete healing of periapical bone area was not evidenced in any clinical cases. the average values of pai before and after 3 months of the treatment did not differ significantly in the study groups of the patients (p>0.05). in 6 months the patients of the main group were absolutely asymptomatic. clinical symptoms or abnormal findings (spontaneous pain, swelling, mobility, or sensitivity to percussion or palpation) in the main group of patients were absent. in 2 patients of the control group the exacerbation of chronic apical periodontitis was diagnosed and the teeth were re-treated. intergroup significant differences of pai were deter­ mined after 6 months of dynamic observation: in the main group — 2.09±0.26 points, in the control group — 2.92±0.31 points (p<0.05). in six months on the x-ray images of treated teeth in the main group of patients the absence of periapical zone destruction was determined in 36.36% of cases (4 teeth) and reduction of focus of apical bone resorption by ½ or more was established in 45.45% (5),the existing state was in 18.18% (2). in the control group in six months after root canal obturation the widening of periapical destruction zone was revealed in 25.0% (3) of cases, the focus of bone destruction was not changed in 33.33% (4), it was decreased by less than 1/2 in 25.0% (3), and it was decreased by 1/2 or more from the initial size in 16.67% (2). 12-month follow-up examinations proved that the patients of the main group did not have any complaints. during intraoral examination the gingiva in the area of tooth apexes was of normal colour without visible pathological changes, except scar formation from gingival fistula after the treatment of chronic granulating periodontitis. percussion reaction was painless, regional lymph nodes were not palpable. the teeth performed full functional load. mean pai score in the main group was 3.27±0.43 at baseline and decreased up to 1.55±0.35 in 1 year of observation, in the control group it was 3.25±0.39 and 2.75±0.38, respectively. the decrease in pai in the main group was up to 1.72, in the control group — up to 0.5. the decrease in pai was statistically significant in both groups of the patients (p<0.001). in a year of dynamic assessment of the main group the improvement of radiographic state was evidenced: 54.55% (6) of the patients had complete bone healing, 27.27% (3) of cases the focus of bone destruction was decreased by 1/2 or more of the initial sizes, 18.18% (2) — the resorption lesion was decreased by less than n. g. gadzhula issn 2413-6077. ijmmr 2016 vol. 2 issue 232 d e n t is t r y issn 2413-6077. ijmmr 2016 vol. 2 issue 2 1/2. final results in the control group of patients were: 33.33% (4) of improvement, 25.0% (3) of existing state and 41.66% (5) of worsening. the absence of positive dynamics during 12 months of observation of the main group patients was in 18.18% (2) of cases; the percentage of non­effi­ cient cases in the control group was 41.66% (5). discussion repair of periradicular tissues con sisted of a complex regeneration invol ving bone, periodontal ligament, and cementum. although clinical as well as radiographic data were used to monitor cases, the relative absence of clinical symptoms in chronic apical periodontitis made the evaluation primarily a radio graphic one [10]. due to the use of bioroottm rcs in the main group of pa tients the positive dynamics of clinical manifestations was confirmed radio graphically during periodic follow-up examinations. three months after the treatment was completed we revealed the lesions size reduction, increasing of the number of bone trabecules and decreasing of intertrabecular spaces. in six months of bioroottm rcs exposition in root canals the expansion of area destruction in periapical tissues and the appearance of new rarefaction or the increase in initial rarefaction were not determined in any clinical case. on x-ray images bone thickening was observed, periodontal contours were not widened. moreover, in six months the radiographic evidence of trabecular pattern of bone was deter mi ned at the area the periradicular lesion was present before. in a year of dynamic assessment a significant thickening of bone and regeneration of periapical tissues were evidenced. the contour, width, and structure of perio dontal margin were normal. the area of mineral loss gradually was filled with bone and the radiographic density was increased in most clinical cases. composition of bioroottm rcs (bio active tricalcium silicate setting system with the absence of any aluminate and calcium sulphate), high degree of adhesion to the dentin and gutta-per cha points, zero shrinkage as monomer-free sealer and great flowability created a favourable en­ vironment for periapical healing, the accele ration of apexification and regeneration processes of the bone, and provided a hermetic obtu ration of apical foramen [9]. there were no complications after the conducted treatment. comparing the clinical and radiographic results of both groups it should be noted that in the patients of the main group bioroottm rcs usage resulted in the improvement of clinical symptoms of the disease. repair of periapical bone structures and periodontal tissues, in creasing of mineral saturation of bone were accelerated by this sealer, especially it was a characteristic feature of pathological processes with size of destruction up to 3 mm in diameter. regeneration in these clinical cases occurred faster than in the cases of larger sizes of destructive lesions. although the action of bioroottm rcs was also effective in the patients with the large periapical lesions, but in the control group the healing of such lesions was not evidenced. conclusions bioroottm rcs using for root canal obturation in the treatment of chronic apical periodontitis we proved the effectiveness of the treatment undertaken: complete healing or improvement of radiographic states of periapical bone destruction with x-ray signs of bone regeneration. these changes were confirmed by the higher indicators of radiographic dynamics of periapical index in the main group of patients than in the other. references 1. siqueira jf, rocas in, ricucci d, hulsmann m. causes and management of post-treatment apical periodontitis. british dental j 2014; 216: 305–312. 2. yu vs, khin lw, hsu cs, yee r, messer hh. risk score algoritm for treatment of persistent apical periodontitis. j dent res 2014; 93 (11): 1076–1082. 3. gill gs, bhuyan ac, kalita c, das l, kataki r, bhuyan d. single versus multi-visit endodontic treatment of teeth with apical periodontitis: an in vivo study with 1-year evalution. annals of medical & health sciences res 2016; 6: 19–26. 4. tyagi s, mishra p, tyagy p. evolution of root canal sealers: an insight story. european j of den­ tistry 2013; 2 (3): 199. 5. murugesan gawthaman, selvarai vinodh, veerabadhran mahesh mathian, rangasamy vijayaraghavan, ramachandran karunakaran. apexification with calcium hydroxide and mineral trioxide aggren. g. gadzhula issn 2413-6077. ijmmr 2016 vol. 2 issue 2 33 d e n t is t r y issn 2413-6077. ijmmr 2016 vol. 2 issue 2 gate: report of two cases. j pharm bioallied sci 2013; 5 (2): 131–134. 6. nair pnr. on the causes of persistent apical periodontitis: a review. international endod j 2006; 39: 249–281. 7. camps j, jeanneau c, ei avachi i, laurent p. bioactivity of a calcium silicate-based endodontic cement (bioroot rcs): interactions with human periodontal ligament cells in vitro. j endod 2015; 41 (9): 1469–1473. 8. zhang w, li z, peng b. ex vivo cytotoxicity of a new calcium silicate based canal filling material. international endod j 2010; 42 (9): 769–774. 9. simon s, flouriot ac. bioroottm rcs a new biomaterial for root canal filling. j case studies collection 2016; 13: 4–11. 10. orstavik d, kerekes k, eriksen hm. the periapical index: a scoring system for radiographic assessment of apical periodontitis. endod dent traumatol 1986; 2 (1): 20–34. received: 2016-11-10 n. g. gadzhula 10 in t e r n a l m e d ic in e issn 2413-6077. ijmmr 2017 vol. 3 issue 2 doi 10.11603/ijmmr.2413-6077.2017.2.8009 serum soluble cd25 in hepatocellular carcinoma, shall we be able to change the natural history? 1e. a. sameea, 1t. zakareya, 1k. metwaly, 2a. a.-r. youssef, 2h. m. kamal, 2w. m. abdalla 1hepatology department, national liver institute, menoufiva university, egypt 2clinical and chemical pathology departmrnt, banha faculty of medicine, egypt background. although hepatocellular carcinoma (hcc) is one of the most common malignancy related mortality worldwide, it can be curable if detected in early stages. emergence of a new marker that can early detect hcc could help in early treatment and therefore ameliorate the outcome. objective. the aim of the research is to evaluate the performance of serum soluble cd25 (scd25) in the prediction of early hcc and compare it to α-fetoprotein (afp). methods. serum levels of scd25 and afp were measured in three groups of population; hcc group (40 patients), cirrhosis without hcc control group (20 patients) and healthy control group (20 patients). hcc group contained 20 early and 20 late stage patients according to barcelona clinic liver cancer (bclc) staging system (stage 0/a and b-d respectively). levels of both biomarkers were compared in all groups. predictive yield of both biomarkers for early hcc was evaluated using roc curve analysis. results. level of scd25 was significantly higher in patients with hcc than in both cirrhotic controls and healthy controls (p<0.0001and 0.013 respectively). for prediction of early hcc in patients with cirrhosis, the optimal scd25 cut-off level was 7.15 ng/ml with sensitivity and specificity of 90% and 60% respectively (auc=0.717; p=0.019) while sensitivity and specificity of afp were 70% and 85% respectively at a cut-off value of 9.85 ng/ml (auc=0.781; p=0.002) in the same settings. conclusion. scd25 seems to be a reliable biomarker for early detection of hcc and therefore could enhance the outcome. key words: hepatocellular carcinoma; soluble cd25; alfa fetoprotein. corresponding author: talaat zakareya, department of hepatology, national liver institute, menoufiya university, shebeen el-kom, menoufiya, egypt, 32511 phone number: +201111815877 e-mail: talaatzakareya@gmail.com introduction hepatocellular carcinoma is one of the most serious and life threatening complications of chronic liver disease. it represents the 5th most common malignancy in men, the 7th in women and the 3rd malignancy related mortality worldwide. curative treatment strategy can be achieved if detected in early stages [1–4]. the role of serum α­fetoprotein (afp), the widely used classical biomarker for hcc, has been stepped down in the recent european and american surveillance guidelines because of low sensitivity and specificity. this is based on the knowledge that almost 80% of small hccs do not show increased levels of afp, and the sensitivity decreases to 25% in tumors smaller than 3 cm [5–8]. looking for a new marker with a better diagnostic accuracy became an inevitable requi rement. this eventually would optimize the hcc surveillance program and improve the outcome through prompt application of the proper treatment strategy early in the course of the disease. serum soluble cd25 (scd25) has been recently investigated as a new marker for hepatocellular carcinoma. it quantitatively reflects the immunological activity against the tumor [9–11]. it represents the α­chain of inter­ leukin 2 receptor (il­2rα) which is composed of three polypeptide chains: α, β and γ. it is not found on the surface of resting t cells, but rapidly expressed on their surface after being activated. chronic t-cell stimulation, as in some malignancies, leads to shedding of il­2rα (cd25) into plasma with subsequent elevation of its level [11–16]. cabrena and colleagues reported that serum level of scd25 was correlating with tumor burden and poor survival in hcc patients and believed that measuring international journal of medicine and medical research 2017, volume 3, issue 2, p. 10–15 copyright © 2017, tsmu, all rights reserved e. a. sameea et al. 11 in t e r n a l m e d ic in e issn 2413-6077. ijmmr 2017 vol. 3 issue 2 serum level of scd25 might provide a clue for early diagnosis of hcc [12]. when we designed the current study, we hypothesized that scd25 could have an impressive diagnostic value and a potential ability for detection of early hcc. we assessed the performance of scd25 in the prediction of early hcc and its correlation with the tumor stage and compare it with afp. methods the study was conducted in national liver institute, menoufiya, egypt. after obtaining an informed consent, eighty persons in 3 groups were included; hcc on a background of cirrhosis (40 patients), liver cirrhosis with no evidence of hcc (20 patients) and healthy control group (20 patients). hcc group comprised 20 early and 20 late stage hcc patients, according to barcelona clinic liver cancer (bclc) staging system, (stage a and b-d respectively) (fig. 1). cirrhotic and healthy controls had matched age and sex with hcc patients. all included cases of hcc was diagnosed on the basis of the presence of typical vascular enhancement pattern of liver lesion (s) in contrast enhanced dynamic ct scan or mri [18]. diagnosis of cirrhosis was based on combined historical, clinical, laboratory and radiological findings. severity of cirrhosis was assessed by child pugh classification [19]. all patients had complete laboratory profile including cbc, liver panel, creatinin as well as serum level of scd25 and afp. elisa kit (elecsys e411, switzerland) was used to quantify blood level of afp while elisa kit (bender medsystems, vienna, austaria) was used to measure serum level of scd25. statistical methods spss, version 21 for windows (inc, chicago, il, usa) was used for all statistical analyses. qualitative data were presented as frequency and percentage. chi square and fisher’s exact tests were used to compare groups. quantitative data were presented as mean and standard deviation. for non-parametric data, student t-test and mann–whitney u test were used to compare level difference of scd25 between two groups while anova and kruskal wallis were used to compare level difference of scd25 between more than two groups. receiver-operator characteristic (roc) curve analysis was used to generate sensitivity and specificity at different cut-offs. the best cut-off was set at the value where sensitivity and specificity were maximal. correlation between serum level of scd25 and laboratory parameters was assessed by spearman's correlation coefficient. the sta­ tistical significance was set at p­value of less than 0.05 for all tests. results the studied populations were mostly males representing 77.5, 75 and 60% in hcc, cirrhotic and healthy control groups respectively. the mean age was 56.38±5.934 years in hcc group while was 53.75±7.383 and 54.20±5.863 years in cirrhotic and healthy controls respectively. hepatitis c virus (hcv) was the underlying etiology of cirrhosis in all patients in both hcc and cirrhotic control groups. the mean scd25 level was 13.07±6.645, 13.15±6.967, 8.938±6.487 and 4.97±3.031 ng/ml in early hcc, late hcc, cirrhotic and healthy control groups respectively. level of scd25 was significantly higher in patients with hcc than in both cirrhotic and healthy controls (p<0.0001 and 0.013 respec tively) and significantly higher in cirrhotic pa­ tients than healthy controls (p=0.042). scd25 level was significantly and positively correlated with the severity of liver disease as assessed by child-pugh classification (r=0.56, p<0.001). there was no statistical difference between scd25 in early and late hcc (p=0.968). the mean afp level was 17.66±12.092, 244±302.041, 8.01±6.965 and 2.95±2.175 ng/ml in early hcc, fig. 1. barcelona clinic liver cancer (bclc) staging and treatment strategy (adapted from llovet jm, et al. lancet 2003) [17]. ps – performance status; pei – percutaneous ethanol injection; rf – radiofrequency fig. 1. barcelona clinic liver cancer (bclc) staging and treatment strategy (adapted from llovet jm et al., lancet 2003) [17]. ps – performance status; pei – percutaneous ethanol injection; rf – radiofrequency e. a. sameea et al. 12 in t e r n a l m e d ic in e issn 2413-6077. ijmmr 2017 vol. 3 issue 2 table 1. statistical difference of demographic and laboratory data among the studied groups afp – α­fetoprotein; hb – hemoglobin; hcc – hepatocellular carcinoma; inr – international normalized ratio; lc – liver cirrhosis; na – not applicable; p – significance between hcc and healthy controls; p* – significance between liver cirrhosis and healthy controls; p^ – significance between hcc and liver cirrhosis; p# – significance between early and late hcc; scd25 – soluble cd25; ♂s – males; ♀s – females. table 2. correlation between scd25 and laboratory parameters among the studied groups total hcc (n=40) early hcc (n=20) late hcc (n=20) lc (n=20) control (n=20) r p r p r p r p r p hb (g/dl) -0.060 0.714 -0.038 0.875 0.040 0.866 0.304 0.193 -0.3710.118 wbcs (×103/dl) -0.228 0.157 -0.478 0.033 -0.063 0.792 -0.081 0.736 0.179 0.462 platelets (×103/dl) 0.128 0.431 0.068 0.777 0.290 0.215 -0.136 0.567 -0.269 0.265 inr 0.151 0.352 0.250 0.287 0.039 0.869 -0.224 0.343 0.035 0.887 albumin (g/dl) 0.002 0.991 0.205 0.387 -0.220 0.352 0.142 0.550 0.064 0.794 bilirubin (mg/dl) -0.038 0.816 -0.102 0.668 -0.021 0.928 -0.442 0.051 0.266 0.270 alt (u/ml) 0.093 0.570 0.078 0.745 0.049 0.838 -0.014 0.955 0.348 0.144 ast (u/ml) 0.124 0.445 0.179 0.450 0.078 0.744 -0.078 0.744 0.390 0.099 creatinin (mg / dl) 0.062 0.706 0.136 0.569 -0.043 0.856 -0.217 0.359 -0.249 0.303 afp (ng/ml) 0.023 0.890 0.196 0.407 -0.093 0.697 -0.254 0.279 0.503 0.028 total hcc (n=40) early hcc (n=20) late hcc (n=20) lc (n=20) healthy control (n=20) p p* p^ p# sex n (%) ♂s 31 (77.5) 15 (75) 16 (80) 15 (75) 12 (60) 0.156 0.311 0.829 0.705♀s 9 (22.5) 5 (25) 4 (20) 5 (25) 8 (40) age (years) mean±sd 0.212 0.822 0.133 0.53956.38± 5.934 58.40± 5.576 55.35± 5.706 53.75± 7.383 54.20± 5.863 hb (g/dl) 11.07± 1.097 11.14± 1.268 11.01± 0.925 10.52± 0.928 12.71± 1.091 <0.001 <0.001 0.058 0.724 wbcs (×103/dl) 4.88± 1.717 5.18± 2.247 4.59± 0.903 4.87± 1.242 7.00± 1.693 <0.001 <0.001 0.977 0.282 platelets (×103/dl) 119.65± 35.246 122.55± 34.264 116.75± 36.854 169.05± 31.749 217.80± 47.522 <0.001 <0.001 <0.001 0.609 inr 1.37± 0.196 1.43± 0.197 1.32± 0.185 1.31± 0.236 1.07± 0.081 <0.001 <0.001 0.225 0.091 albumin (g/dl) 3.19± 0.371 3.334± 0.382 3.04± 0.299 3.55± 0.445 4.34± 0.463 <0.001 <0.001 0.002 0.009 bilirubin (mg/dl) 1.64± 0.833 1.19± 0.415 2.09± 0.907 1.73± 0.692 0.84± 0.154 <0.001 <0.001 0.626 <0.001 alt (u/ml) 65.15± 15.184 61.75± 17.278 68.55± 12.262 57.05± 10.655 24.45± 5.276 <0.001 <0.001 <0.001 0.159 ast (u/ml) 89.48± 24.724 76.50± 17.021 102.45± 24.708 67.85± 10.069 27.25± 4.962 <0.001 <0.001 0.019 <0.001 creatinin (mg/dl) 0.93± 0.159 0.93± 0.180 0.94± 0.139 0.95± 0.161 1.04± 0.193 0.025 0.114 0.665 0.845 scd25 (ng/ml) 13.11± 6.719 13.07± 6.645 13.15± 6.967 8.938± 6.487 4.97± 3.031 <0.001 0.042 0.013 0.968 afp (ng/ml) 130.83± 240.106 17.66± 12.092 244± 302.041 8.01± 6.965 2.95± 2.175 0.008 0.926 0.010 0.003 child-pugh score n (%) a 6 (15) 6 (30) 0 (0) 12 (60) na na na 0.001 0.004b 29 (72.5) 14 (70) 15 (75) 8 (40) c 5 (12.5) 0 (0) 5 (25) 0 (0) afp – α­fetoprotein; hb – hemoglobin; hcc – hepatocellular carcinoma; inr – international normalized ratio; lc – liver cirrhosis; r – spearman’s correlation coefficient. e. a. sameea et al. 13 in t e r n a l m e d ic in e issn 2413-6077. ijmmr 2017 vol. 3 issue 2 late hcc, cirrhotic and healthy control groups respectively with statistical difference between hcc versus cirrhotics and early versus late hcc as well (p=0.010 and 0.003 respectively). the rest of demographic and laboratory data as well as their statistical differences between the studied groups are presented in table 1. cor relation analyses between scd25 and laboratory parameters among the studied groups are presented in table 2. there was no significant correlation with all laboratory parameters apart from a negative correlation with wbcs in early hcc group (r=-0.478, p=0.033) and a positive correlation with afp in healthy control group (r=-0.503, p=0.028). scd25 performed well in predicting hcc presence among patients with cirrhosis; sensitivity and specificity were 90% and 84.2% respectively at a cut-off value of 7 ng/ml (auc=0.969; p<0.0001). for prediction of early hcc in patients with cirrhosis, the optimal scd25 cutoff level was 7.15 ng/ml with sensitivity and specificity of 90% and 60% respectively (auc=0.717; p=0.019) while, sensitivity and specificity of afp were 70% and 85% respectively at a cut-off value of 9.85 ng/ml (auc=0.781; p=0.002) in the same settings (fig. 2). discussion hcc represents the most serious and lethal complication of cirrhosis. fortunately, early stages of hcc could be curative. axiomatically, detection of hcc in early stages would be helpful in changing the poor outcome of late stages by offering the proper treatment early in the course of the disease with subsequent amelioration of the outcome [20–22]. in the current study, we evaluated the performance of scd25 in predicting early hcc stages among patients with cirrhosis and compare it to afp. serum scd25 level was significantly higher in hcc patients than cirrhotics (p<0.0001) and healthy controls (p=0.013). in the same stream, it was significantly higher in cirrhosis than healthy controls (p=0.042). additionally, there was a significant positive correlation between serum scd25 and severity of cirrhosis (childpugh class) (r=0.56, p<0.001). the optimal scd25 cut-off level in detecting early hcc among cirrhotic patients was 7.15 ng/ml with sensitivity and specificity of 90% and 60% respectively (auc=0.717; p=0.019). on the other hand, sensitivity and specificity of afp were 70% and 85% respectively at a cut-off value of 9.85 ng/ml (auc=0.781; p=0.002) in the same settings .this higher sensitivity of scd25 highlights its substantial role as a screening marker for hcc. similar findings were reported by cabrena and his group. they reported scd25 cut-off level of 2899 pg/ml as the best cut-off with a sensitivity of 89.6% and a specificity of 39.3% (auc=0.630, p<0.0001). by comparison, fig. 2. receiver operator curve (roc) of scd25 and afp levels for the prediction of early hcc among patients with cirrhosis fig. 2. receiver operator curve (roc) of scd25 and afp levels for the prediction of early hcc among patients with cirrhosis e. a. sameea et al. 14 in t e r n a l m e d ic in e issn 2413-6077. ijmmr 2017 vol. 3 issue 2 at a cut-off value of 20 ng/ml, afp had a sensitivity of 41.7% and a specificity of 82.6% (auc=0.630, p=0.0257) [12] the difference between the optimal cuto-ff between the current study (7150 pg/ml) and that of cabrena et al. (2899 pg/ml) might be referred to the variability in the sample size, underlying etiology as well as dissimilarity in racial, ethnic, ge netic and environmental factors. it is noteworthy that, the main underlying etiology of liver disease was hcv representing 92.5 and 90% in hcc and cirrhosis groups respectively while 7.5 and 10% were referred to combined hcv and hbv etiology in the same groups respectively. in the study of cabrena et al., 60% were hcv, 13% were cryptogenic, 9% were alcoholic cirrhosis and 9% were non-alcoholic fatty liver disease (nafld) in hcc group while 72% were hcv, 5% alcoholic cirrhosis and 5% nafld and 3% were cryptogenic in cirrhosis group. in spite of the presence of a significant positive correlation between serum levels of scd25 and severity of liver cirrhosis, there was no significant difference in its level in early and late hcc which disclaims findings of cabrena et al., who reported a significant positive correlation between serum levels of scd25 and tumor stage [12]. we could not eventually find a reasonable explanation for these conflicting results however difference in underlying etiology, tumor differentiation/biology, interracial and inter-ethnic variations between both studies might be accused. a notable finding that should be considered the correlation between scd25 and afp in hcc and cirrhosis groups was absent denoting that measuring both markers in serum can improve the reciprocally holistic diagnostic value of hcc. conclusions serum scd25 sounds to be a good marker for predicting early hcc. there was some discrepancy between the optimal cut-off in the current and previous studies. this calls for a large scale study for further integration and unification of the current results and previous ones and to standardize the optimal cut-off taking into consideration addressing the relationship between scd25 level and tumor biology rather than tumor size and number. references 1. el-serag hb. epidemiology of viral hepatitis and hepatocellular carcinoma. gastroenterology. 2012;142:1264–1273. e1. pmid: 22537432 doi: 10.1053/j.gastro.2011.12.061. 2. el-serag hb. hepatocellular carcinoma. n engl j med. 2011;365:1118–1127. 3. parkin dm, bray f, ferlay j, pisani p. estimating the world cancerburden: globocan 2000. int j cancer. 2001;94:153–6. 4. llovet jm, burroughs a, bruix j. hepatocellular carcinoma. lancet. 2003;362:1907–1917. 5. el-serag hb, kramer jr, chen gj, duan z, richardson pa and davila ja. effectiveness of afp and ultrasound tests on hepa tocellular carcinoma mortality in hcv-infected patients in the usa. gut. 2011;60:992–997. 6. benowitz s. liver cancer biomarkers struggling to succeed. j natl cancer inst. 2007;99:590–591. 7. sherman m. current status of α­fetoprotein testing. gastroenterolhepatol (ny). 2011;7:113–114. 8. bruix j, sherman m. management of hepatocellular carcinoma: an update. hepatology 2011;53: 1020–1022. pmid: 21374666 doi: 10.1002/hep.24199. 9. cao m, cabrera r, xu y, et al. hepatocellular carcinoma cell supernatantsincrease expansion and function of cd4(+)cd25(+) regulatoryt cells. lab invest. 2007;87:582–90. 10. foss fm. immunologic mechanisms of antitumor activity. seminoncol. 2002;29:5–11. 11. cabrera r, ararat m, cao m, et al. hepatocellular carcinoma immunopathogenesis:clinical evidence for global t cell defects and an immunomodulatory role for soluble cd25 (scd25). dig dis sci. 2010;55:484–95. 12. cabrena r, fitian a, ararat m, et al. serum levels of soluble cd25 as a marker for hepatocellular carcinoma. oncology letters. 2012;4:840–846. 13. nakamoto y, guidotti lg, kuhlen cv, fowler p, chisari fv. immune pathogenesis of hepatocellular carcinoma. j exp med. 1998;188:341–350. 14. cacalano na and johnston ja: interleukin­2 signaling and inherited immunodeficiency. am j hum genet . 1999;65:287–293. 15. hoechst b, ormandy la, ballmaier m, et al. a new population of myeloid-derived suppressor cells in hepatocellular carcinoma patients induces e. a. sameea et al. 15 in t e r n a l m e d ic in e issn 2413-6077. ijmmr 2017 vol. 3 issue 2 cd4(+)cd25(+)foxp3(+) t cells. gastroenterology. 2008;135:234–243. 16. arun b, curti bd, longo dl, et al. elevations in serum soluble interleukin-2 receptor levels predict relapse in patients with hairy cell leukemia. cancer j sci am 6. 2000;21–24. 17. forner a, reig me, de lope cr, et al. current strategy for staging and treatment: the bclc update and future prospects. semin. liver dis. 2010;30 (01):61–74. 18. choi jy, lee jm, sirlin cb. ct and mr imaging diagnosis and staging of hepatocellular carcinoma: part i. development, growth, and spread: key pathologic and imaging aspects. radiology. 2014;272(3):635–54. 19. pugh rn, murray-lyon im, dawson jl, pietroni mc, williams r. transection of the oesophagus for bleeding oesophageal varices. br j surg. 1973; 60(8):646–9. 20. fattovich g, stroffolini t, zagni i, donato f. hepatocellular carcinoma in cirrhosis: incidence and risk factors. gastroenterology. 2004;127 suppl 1: s35-s50. 21. forner a, llovet jm, bruix j. hepatocellular carcinoma. lancet. 2012;379:1245–1255. pmid: 22353262 doi: 10.1016/s0140­6736(11)61347­0. 22. marrero ja. current treatment approaches in hcc. clin adv hematol oncol. 2013;11 suppl 5:15–18. received: 2017-07-25 e. a. sameea et al. 54 b io m e d ic a l s c ie n c e s issn 2413-6077. ijmmr 2017 vol. 3 issue 2 international journal of medicine and medical research 2017, volume 3, issue 2, p. 54–58 copyright © 2017, tsmu, all rights reserved n. ya. letniak et al. doi 10.11603/ijmmr.2413-6077.2017.2.8416 nanotubs increase tetrachloromethane induced oxidative stress n. ya. letniak, i. p. kuzmak, m. m. korda i. horbachevsky ternopil state medical university background. the unique physical and chemical properties of carbon nanotubes determine wide-ranging prospects for their use in biology and medicine. the capability of nanotubes to transport medicines and chemicals inside a cell makes the possibility of classical toxicants toxicity increase in case of their intake to the body with nanotubes, an urgent issue. objective. the aim of the research was to study the effect of carbon nanotubes on the capability of the chemical toxicant tetrachloromethane (tcm) to induce oxidative stress in serum and liver of rats. methods. the experiments were performed on outbred male rats, which were administered intraperitoneally with 0.5 ml of suspension of single-walled, multi-walled or multi-walled functionalized cooh nanotubes (60 mg/ kg) only or together with tcm (2 ml/kg). the animals were taken out of the experiment in 3, 6 and 48 hours after the administration of the nanotubes and tcm. the activity of catalase, superoxide dismutase, the content of thiobarbituric acid reactive substances (tars), reduced glutathione, ceruloplasmin and total antioxidant activity of serum were determined in serum and liver. results. it was established that under the influence of multi-walled carbon nanotubes the studied parameters changed significantly. the administration of tetrachloromethane to rats caused significant changes in all indicators. maximal changes in the rates were recorded in the group of animals that were administered with carbon nanotubes and tetrachloromethane togeather. in this case, a number of the studied parameters of blood and liver significantly changed compare to the similar indicators of the group of animals, which were administered with the chemical toxicant only. conclusions. carbon nanotubes increase the capability of the chemical toxicant tetrachloride to cause oxidative stress in liver and serum. key words: carbon nanotubes; tetrachloromethane; oxidative stress; rats. corresponding author: nataliia letniak, department of biochemistry, i. horbachevsky ternopil state medical university, 1 maidan voli, ternopil, ukraine, 46001 phone number: +380352254784 e-mail: letnyak@tdmu.edu.ua introduction nanotechnology today is the most promising direction in the development of world science. nanomaterials have caused a step forward in many industries and are used in our overall life. carbon nanotubes (cnt) are one of the priority types of nanomaterials. they are multifunctional materials that are actively studied due to their unique physical and chemical properties [2, 6]. they exist in various forms and can be chemically modified by functional groups of biomolecules. cnt have unique mechanical, electrical and thermal properties and are widely used in various industries. nanotubes are a promising nanomaterial for medical use due to their really high biocompatibility with blood, bones, cartilages and soft tissues [7, 9]. they can be used to create artificial heart valves, for the diagnosis and treatment of cancer, as well as for the transport of proteins, anti gens, genes, vaccines and medicinal substances into a cell. due to everyday increase of nanomaterial use, less attention is paid to the possible negative effects of nanoparticles on environment and on people’s health as a whole [14]. small size, specific structure, large surface area, and chemical composition alert of possible toxic effects on the human body. apart from the direct influence of carbon nanotubes on cells, they may interact with classical toxicants, e.g. tetrachloromethane (tcm). currently, the issue of biological effects of nanoparticles in case of their intake to the body together with traditional toxicants is urgent. thus it is necessary to study the toxicological properties of carbon nanotubes alone as well as in case of their intake to the body together with a toxicant. 55 b io m e d ic a l s c ie n c e s issn 2413-6077. ijmmr 2017 vol. 3 issue 2 the aim of the research was to study the effect of carbon nanotubes on the capability of the chemical toxicant tetrachloromethane (tcm) to induce oxidative stress in serum and liver of experimental rats. methods the experiments were performed on outbred male rats, 160 g in weight, which were kept on a standard vivarium diet. single-walled (swnt), multi-waledl (mwnt) and multi-walled functionalized (mwnt-cooh) nanotubes were administered to the animals in suspension (0.5 ml) intraperitoneally at a dose of 60 mg/kg. tcm was administered intraperitoneally in 50% oily solution at a dose of 2 ml/kg just the once. dispersion of nanoparticles in distilled water or tcm solution was carried out by means of the ultrasonic disperser uzdn-m750t (20–25 khz, 750 w) for 5 minutes. the experimental animals were divided into 8 groups: the 1st – the control (intact rats), administered with physical solution (0.5 ml/kg); the 2nd – the rats administered with swnt, the 3rd – the animals administered with mwnt, the 4th – the rats administered with mwnt-cooh, the 5th – the animals administered with tcm, the 6th – the rats administered with swnt suspension together with tcm, the 7th – the rats administered with thesuspension of mwnt+tcm, the 8th – the animals administered with the suspension of mwnt-cooh+tcm. the animals were taken out of the experiment under thiopental anesthesia in 3, 6 and 48 hours after the injection. liver homogenate and blood serum were the objects of the study. the animals were kept and the experiments were conducted in accordance with the guidelines of european convention for the protection of vertebrate animals used for experimental and other scientific purposes. the state of antioxidant system was evaluated by the activity of enzymes of superoxide dismutase (sod) [8], catalase (ct) [4], the content of ceruloplasmin (cp) [10] and reduced glu ta thione (gsh) [3]. the development of oxidative processes in the body was evidenced by the content of products that react with thio barbituric acid (tbars) [1]. the total antioxidant acti vity (taa) of plasma was also determined [13]. the nanopowder of single-walled carbon nanotubes (swcn, 90%, 1–2 nm), multi-walled nano tubes (mwcn, 99%, 13–18 nm) and carboxyfunctionalized nanotubes (mwcn-cooh, 95%, 30-50 nm)) produced by usresearch nano materials, inc. (usa) were used in the experiment. tetrachloromethane produced by makrokhim (ukraine) was used as a model toxicant. statistical processing of the results was performed at the department of system statistical study of i. horbachevsky ternopil state medical university using the software package statsoft statistica. the obtained indexes were compared using the mannwhitney non-parametric test. the changes were statistically significant at p<0.05. results in 3 and 6 hours after the administration of mwnt, the activity of sod significantly decreased in serum and liver compared to the control. after the administration of mwntcooh, the changes in the sod content in both tissues were significant only by the 6th hour of the experiment. at the same time, swnt did not caused significant changes of this para­ meter. another antioxidant defense enzyme that functions in blood and intercepts reactive oxygen intermediates is the cp. the content of cp in the blood of the animals administered with mwnt significantly exceeded the control indices in 1.3 times by the 6th hour of the experiment. after administration of nanotubes to the experimental animals, the processes of lipoperoxidation increased that was evidenced by the increase in the content of tbars in serum and liver. thus, in cases of mwnt administration, the tbars content in serum was significantly higher in 1.3 and 1.4 times compare to the control group of animals, respectively by the 3rd and 6th hours of the experiment. in cases of swnt and mwnt-cooh administration, the significant increase of this indicator was evidenced only by the 6th hour after injection. a significant increase in ct activity was observed in cases of the administration of swnt and mwnt-cooh by the 6th hour of the experiment (in 1.2 and 1.4 times respectively), as well as in 1.3 and 1.5 times by the 3rd and 6th hours after the administration of mwnt. the reduced glutathione is one of the main antioxidants of non-enzymatic nature, its deficiency in tissues or blood causes significant oxidative stress [12]. as presented in table 1, the administration of mwnt to animals caused a significant decrease in the content of reduced glutathione in 3 and 6 hours after injection, respectively in 1.3 and 1.5 times compared to the control, as well as in 1.4 times in 6 hours after the mwnt-cooh administration. the plasma taa varied equally to the gsh. it should be noted that all the indices changed wavelike, n. ya. letniak et al. 56 b io m e d ic a l s c ie n c e s issn 2413-6077. ijmmr 2017 vol. 3 issue 2 but in 48 hours after the administration of nanoparticles were normal again. thus, it was proved that multi-walled functionalized cooh nanotubes had the most significant toxic effect. the administration of tcm to the animals caused significant disorders of antioxidant system (table 2). above all, the content of tbars in serum and liver increased significantly in all periods of the study. significant changes of sod activity were evidenced (p<0.05 in all cases) with the maximum decrease by the 6th hour of the experiment (in 1.7 times in serum and in 1.6 times in liver). consecutively, ct activity and cp content, quite the opposite, were significantly increased in all periods of the study. the maximum increase of the catalase activity (in 1.9 times compare to the intact animals) was evidenced by the 6th hour of the experiment. the concentration of another important antioxidant – gsh, under the chemical toxicant influence, decreased in 1.5, 1.7 and 1.4 times compare to the control group of animals (p<0.05 in all cases). taa decreased significantly in 1.4, 1.5 and 1.3 times in the corresponding study periods. table 1. the influence of carbon nanotubes on the indices of oxidative stress intensity in blood serum and liver of rats (m±m, n=8) index groups of animals intact swnt mwnt mwnt-cooh time after the administration (hours) 3 6 48 3 6 48 3 6 48 blood plasma tbars, µmol/l 7.81 ±0.43 8.05 ±0.51 9.37* ±0.49 7.35 ±0.38 10.05* ±0.56 11.09* ±0.61 8.18 ±0.39 8.95 ±0.41 10.11* ±0.43 7.95 ±0.41 gsh, mmol/l 2.73 ±0.19 2.55 ±0.16 2.17 ±0.13 2.61 ±0.14 2.14* ±0.15 1.72* ±0.14 2.65 ±0.16 2.41 ±0.15 1.98* ±0.14 2.52 ±0.18 cт, mab/l 0.67 ±0.04 0.79 ±0.05 0.81* ±0.04 0.63 ±0.06 0.89* ±0.03 1.00* ±0.07 0.78 ±0.03 0.82 ±0.04 0.93* ±0.06 0.60 ±0.04 cp, mg/l 238.4 ±9.20 247.9 ±9.85 257.3 ±8.01 245.7 ±10.04 261.7 ±10.65 285.5* ±11.45 251.3 ±8.95 258.1 ±8.40 269.6* ±10.25 255.4 ±8.83 taa, % 61.49 ±4.10 60.72 ±3.52 58.05 ±2.70 61.1 ±4.26 53.31 ±3.02 45.57* ±2.98 55.13 ±4.05 57.45 ±3.80 50.23* ±2.75 59.01 ±5.12 sod, units/ ml 8.33 ±0.54 7.62 ±0.60 7.02 ±0.48 8.16 ±0.53 6.65* ±0.51 6.30* ±0.43 8.41 ±0.60 7.08 ±0.64 6.47* ±0.53 7.99 ±0.52 liver tbars, µmol/kg 62.53 ±2.04 64.11 ±1.98 72.06 ±1.64 59.32 ±2.12 69.33 ±1.94 87.54 ±2.42 64.02 ±1.85 65.84 ±1.78 81.23 ±1.66 59.83 ±2.03 sod, units/g 0.60 ±0.02 0.62 ±0.03 0,53* ±0.02 0.59 ±0.05 0.51* ±0.03 0.43* ±0.05 0.61 ±0.04 0.52 ±0.03 0.49* ±0.04 0.64 ±0.05 note: * – significant differences compared to the control (p<0.05). table 2. the influence of tetrachloromethane on the indices of oxidative stress in blood serum and liver of rats (m±m, n=8) index groups of animals ccl4 intact time after the administration (hours)3 6 48 blood plasma tbars, µmol/l 7.81±0.43 10.73*±0.48 12.91*±0.54 9.03±0.45 gsh, mmol/l 2.73±0.19 1.83*±0.14 1.65*±0.15 1.98*±0.15 cт, mab/l 0.67±0.04 1.08*±0.06 1.27*±0.07 0.93*±0.03 cp, mg/l 238.4±9.20 291.8*±8.47 322.6*±9.02 283.1*±9.11 taa, % 61.49±4.10 44.51*±2.41 41.29*±2.14 47.21*±2.95 sod, units/ml 8.33±0.54 6.12*±0.38 5.11*±0.42 6.57*±0.39 liver tbars, µmol/kg 62.53±2.54 79.85*±3.77 101.04*±3.25 84.49*±2.98 sod, units/g 0.60±0.02 0.43*±0.03 0.38*±0.02 0.41*±0.04 note: * – significant differences compared to the control (p<0.05). n. ya. letniak et al. 57 b io m e d ic a l s c ie n c e s issn 2413-6077. ijmmr 2017 vol. 3 issue 2 the most significant changes in the func­ tioning of antioxidant system were evidenced in the animals administered with total tetrahloromethane and carbon nanotubes (table 3). in this group of animals, significant changes were evidenced in all the studied parameters compare to the intact animals in all periods of the study. it should be noted that the most of indices of the animals administered with the nanotubes+tetrachloromethane combined were significantly lower than those in the corresponding periods in the animals administered with tetrachloromethane and no nanotubes. table 3. the influence of combined administration of carbon nanotubes and tetrachloromethane on the indices of oxidative stress in blood serum and liver of rats (m±m, n=8) index intact swnt+ccl4 mwnt +ccl4 mwnt-cooh+ccl4 time after the administration (hours) 3 6 48 3 6 48 3 6 48 blood plasma tbars, µmol/l 7.81 ±0.43 10.83* ±0.59 13.27* ±0.63 9.92* ±0.57 14.12*# ±0.61 16.43*# ±0.60 10.18* ±0.51 12.53*# ±0.57 14.83*# ±0.59 9.98* ±0.56 gsh, mmol/l 2.73 ±0.19 1.68* ±0.10 1.56* ±0.12 1.92* ±0.14 1.38*# ±0.12 1.16*# ±0.16 1.65* ±0.13 1.44* ±0.10 1.21*# ±0.14 1.73* ±0.16 cт, mab/l 0.67 ±0.04 1.18* ±0.07 1.30* ±0.06 0.99* ±0.05 1.33*# ±0.06 1.58*# ±0.07 1.08* ±0.06 1.21* ±0.09 1.51* ±0.08 1.02* ±0.06 cp, mg/l 238.4 ±9.20 311.2*# ±7.02 326.9* ±9.61 308.7* ±7.82 321.8*# ±8.63 345.3*# ±9.25 298.1* ±8.08 319.7* ±9.40 331.6* ±8.25 289* ±8.22 taa, % 61.49 ±4.10 42.31* ±2.62 37.91* ±3.18 44.77* ±2.91 38.31* ±2.65 30.44*# ±2.73 41.63* ±2.22 40.82* ±2.18 34.61*# ±2.09 43.65 ±3.58 sod, units/ml 8.33 ±0.54 5.98* ±0.38 5.05* ±0.32 6.10* ±0.41 5.27* ±0.38 4.65* ±0.34 5.49* ±0.39 5.18* ±0.44 4.82* ±0.39 5.73* ±0.42 liver tbars, mol/kg 62.53 ±2.04 87.18* ±2.68 103.7* ±2.64 79.5* ±2.22 98.68* ±3.04 118.0*# ±2.40 88.66* ±2.13 91.02* ±2.78 106.4* ±2.51 78.3* ±2.13 sod, units/g 0.60 ±0.02 0.43* ±0.04 0.34* ±0.02 0.48 ±0.04 0.36* ±0.03 0.28*# ±0.02 0.41* ±0.03 0.38* ±0.03 0.33* ±0.01 0.45* ±0.04 notes: * – significant differences compared to the control (p<0.05). # – significant differences compared to the group of animals administered with tetrachloromethane (p<0.05). discussion the study results brought us to the conclusion that the capability of the chemical toxicant tetrachloromethane to cause oxidative stress in serum and liver was significantly increased in case of its combined administration with carbon nanotubes. the effect of increased bioavailability of tetrachloromethane due to the capability of carbon nanotubes to absorb the toxin on its surface and to contribute to its transport to tissues and cells is the most likely explanation for the toxicity synergy of the investigated factors. according to the results of our research, as well as to the literature, nanotubes, especially mwnt, are able to induce the oxidative processes in tissues. it was established that the toxicity of nanotubes depended on their structure, size and surface area, as well as on the environment they are found in. the toxicity increased when the size of the particles decreased [2, 9]. conclusions carbon nanotubes are able to activate the oxidative processes in the tissues of the body. the carbon nanotubes are placed in the following order by the degree of toxicity: mwnt>mwnt-cooh>swnt. carbon nanotubes increase the capability of the chemical toxicant tetrachloride to cause oxidative stress in liver and serum. n. ya. letniak et al. 58 b io m e d ic a l s c ie n c e s issn 2413-6077. ijmmr 2017 vol. 3 issue 2 references 1. andrieyeva li, kozhemiakin la, kishkun aa. modification of the method of lipid peroxides determination in the test using thiobarbituric acid. laboratory science. 1988;11:41–43. 2. balabanov vi. nanotechnologies. science of the future. moscow: eksmo; 2009. p. 220. 3. kolb vg, kamyshnikov vs. guide to clinical chemistry. minsk: belarus; 1982. p. 311. 4. koroliuk ma, ivanova li, mayorova ig. method of catalase activity determination. laboratory science. 1988;1:16–18. 5. lapach sn, chubenko av, babich pn. statistical methods in biomedical research using excel. kyiv: morion; 2000. p. 320. 6. 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ve. mechanisms of carbon nanotube-induced toxicity: focus on oxidative stress. toxicol appl pharmacol. 2012 jun 1;261(2):121–33. 13. stock j, gutteridge jm, sharp rj, et al. assay using brain homogenate for measuring the antioxidant activity of biological fluids. clinical science and molecular medicine. 1974;47:215–222. 14. tsuda h, xu j, sakai y, et al. toxicology of engineered nanomaterials – a review of carcinogenic potential. asian pacific journal of cancer prevention. 2009;10:975–980. received: 2017-10-11 n. ya. letniak et al. 59 b io m e d ic a l s c ie n c e s issn 2413-6077. ijmmr 2017 vol. 3 issue 2 international journal of medicine and medical research 2017, volume 3, issue 2, p. 59–63 copyright © 2017, tsmu, all rights reserved doi 10.11603/ijmmr.2413-6077.2017.2.8035 chronic enterocolitis combined with streptozotocininduced diabetes in rats: mechanism of oxidative stress development 1n. v. lisnianska, 2м. і. marushchak, 2і. v. antonyshyn, 3о. p. mialiuk 1chernivtsi medical college of bukovinian state medical university, chernivtsi, ukraine; 2i. horbachevsky ternopil state medical university, ternopil, ukraine; 3rivne state basic medical college, rivne, ukraine background. despite numerous studies on chronic inflammatory processes in colon, the complex problem of chronic enterocolitis (cec) remains relevant. objective. the aim of the research is to evaluate the lipid peroxide oxidation and antioxidant defence system in rats suffering from chronic enterocolitis development with underlying streptozocine-induced diabetes mellitus. methods. the study involved 106 white non-linear male rats. diabetes mellitus (dm) was modeled by a single intraperitoneal administration of streptozotocin to animals (sigma aldrich, usa, at a dose of 60 mg/kg of body weight). cec was induced by a free access of animals to 1.0% solution of carrageenan in drinking water for 1 month. results. in the wall of small intestine of dm rats, lipid peroxide oxidation increases and the activity of enzyme link of antioxidant defence decreases reliably. the course of experimental cec in rats is accompanied by the increase in free radical oxidation in the wall of small intestine and simultaneous increase of enzyme activity of antioxidant defence system, compared to the control. in cases of cec with underlying dm in rats, the development of oxidative stress in the wall of small intestine is caused by the statistically significant increase in levels of diene conjugates and thiobarbituric acid reactive substances, (p<0.01) and the decrease in activity of sod and catalase. conclusions. the activation of free radical reactions is an important non-specific mechanism of inflammation development in tissues of small and large intestine in cases of diabetes mellitus. key words: chronic enterocolitis; diabetes mellitus; lipid peroxidation; antioxidant defence system; experiment. corresponding author: maria marushchak, department of functional diagnostics and clinical pathophysiology, i. horbachevsky ternopil state medical university, 1 maidan voli, ternopil, ukraine, 46001 phone number: +380979901202 e-mail: marushchak@tdmu.edu.ua introduction a difficult social and economic situation, deterioration of living conditions, aggressiveness of the environment and other negative stress factors contribute to an increase in incidence of digestive diseases in the population. digestive diseases are one of the most common among chronic diseases in developed countries. according to a number of studies, their share is 8–10% [1–5]. chronic enterocolitis (cec) is one of the diseases, which are based on the combination of elements of inflammation and dystrophy of mucous membrane with functional disorders in small and large intestines. this disease takes account of about 10% of the total chronic pathology of digestive system organs, and its prevalence is 5–12 cases per 1000 people [6]. despite numerous studies on chronic inflammatory processes in colon, the complex problem of chronic enterocolitis remains relevant [7, 8]. for a long time this term defined a variety of pathological conditions of intestine, which was due to the lack of sufficiently clear notions about the nature of the disease. from the standpoint of modern concepts, chronic enterocolitis should be considered as a clinical and morphological phenomenon characterized by pain and dyspeptic syndromes typical for intestinal diseases with mor phologically determined signs of epithelium dystrophy, the decrease in crypts depth and development of various severity lymphoplasmatic infiltration [9]. according to the offi­ cial data, the incidence of cec in the world is 50–230 cases per 100,000 people [10]. about n. v. lisnianska et al. 60 b io m e d ic a l s c ie n c e s issn 2413-6077. ijmmr 2017 vol. 3 issue 2 15–20% of the world’s population suffer from cec. in the united states, over 20 million adults have symptoms of this disease. [11]. the annual increase in number of enterocolitis patients in the world is 5–20 cases per 100,000 people. epidemiological calculations in the us proved that in white population, cec is found in 3–5 times more often than in african americans, while jews are threatened in 3.5 times more than non-jewish people. the disease occurs in all age groups, but the highest incidence is in 20–40-year-old individuals [12]. in the overall structure of gastroenterological pathology in america, cec ranks first and accounts for 28% of all cases of treatment by gastroenterologists. about 12% of the patients seek medical advice from a general practitioner with complaints specific to cec. cec is diagnosed the most commonly in young people: 13.5% of people are aged 15-34, 13% of them are 35–44 years old and 9% are aged 45 and older. therefore, the disease causes great economic harm to the society both the cost of medical care and by indirect factors, which include a compensation for temporary disability. [13]. however, it should be noted that in modern literature there is controversial information on cec as an independent nosological form, and the presence of functional intestinal disease – an irritable bowel syndrome, is mentioned more often, while the changes in the intestinal mucosa are interpreted by the authors as morphofunctional ones, which causes the complexity of clinical and laboratory diagnostics and different understanding of cec [14, 15]. from the pathogenetic point of view, the diseases related to the class of free radical pathology are widespread, starting with birth (bronchopulmonary dysplasia, retinopathy in preterm infants, enterocolitis, etc.). [16, 17]. the weakening of antioxidant defence and uncontrolled enhancement of lipid peroxidation processes is one of the important links in the pathogenesis of autonomic dysfunction, atopic dermatitis, dental pathology, diabetes mellitus, as well as pathology of gastrointestinal tract, enterocolitis as well [18, 19]. in this case peroxide lipid oxidation products, including malonic dialdehyde, which destabilize cellular membranes, reach high concentrations in blood and tissues [20, 21]. to date, a large amount of data has been accumulated proving the participation of free radical processes in the pathogenesis of cec [16]. therefore, the aim of our study was to evaluate the lipid peroxide oxidation and antioxidant defence system in rats suffering from chronic enterocolitis development with underlying streptozocine-induced diabetes mellitus. methods the study involved 106 white non-linear male rats, which were kept on a standard vivarium diet of i. horbachevsky ternopil state medical university. during the experiment the principles of the european convention for the protection of animals used for experimental and other scientific purposes were followed. the experimental rats were divided into four groups: the 1st – control (intact animals), the 2nd – animals with diabetes mellitus, the 3rd – animals with chronic enterocolitis, the 4th – animals with diabetes mellitus and chronic enterocolitis. diabetes mellitus (dm) was modeled by a single intraperitoneal administration of streptozotocin to the animals aged 2 months, (sigma aldrich, usa, at a dose of 60 mg/kg of body weight) [22]. right before its injection, streptozotocin was dissolved in 0.1 molar citrate buffer (ph 4.5); the control group received the appropriate amount of citrate buffer. the study involved the animals with the glucose rate at least 10.8 mmol/l 2 weeks after streptozotocin administration. chronic enterocolitis was induced by a free access of animals to 1.0% solution of carrageenan in drinking water for 1 month [23, 24]. euthanasia of animals was performed by heart puncture under anaesthesia, in accordance with the requirements of the animal care committee [25]. the state of lipid peroxide oxidation (lpo) was evaluated by the concentration of diene conjugates (dc) and thiobarbituric acid reactive substances (tbars). the content of dc was determined by direct spectrophotometry, the principle of which is to isolate native fatty acids by extraction with a mixture of equal volumes of heptane and isopropanol, followed by measuring the optical density of heptane phase of lipid extract. absorption at a wavelength of 232 nm evidences the content of dc [26]. to determine tbars, we used the method of m. mihara (1980), which consists in the formation of a coloured complex by the interaction of lipid peroxide oxidation products with thiobarbituric acid, by means of a standard set. the activity of antioxidant enzymes: catalase, was recorded simultaneously with the lpo processes [27], superoxide dismutase [28]. the obtained data were subjected to statistical processing [29, 30]. to verify the conformity of the data samples with the normal disn. v. lisnianska et al. 61 b io m e d ic a l s c ie n c e s issn 2413-6077. ijmmr 2017 vol. 3 issue 2 n. v. lisnianska et al. tribution law, the calculation by shapiro-wilk test was applied. due to the lack of data matching to the normal distribution at the significance rate p<0.05, the median characteristics were estimated: median (me), first and third quartiles (q25­q75). the level of statistical significance of sample differences was evaluated using nonparametric mann-whitney u test. differences were considered statistically significant at the achieved rate of p<0.05. results the increase of free radical oxidation processes in small intestine wall in the presence of the studied pathologies has been established (p<0.05). for instance, in cases of streptozotocininduced diabetes, the content of dc increased statistically significantly by 66.93% and of tbars by 71.22%, compared to the control values (table 1). as the lipid peroxide oxidation increased, the activity of enzyme system of antioxidant defence system decreased, in particular, the activity of sod decreased by 38.02% and that of catalase by 58.82%. experimental cec was accompanied by the increase of lipid peroxide oxidation in the wall of small intestine (dc by 35.16% and tbars by 27.65%, p<0.01) with simultaneous increase of enzyme activity of antioxidant defence system (sod by 29.90% and catalase by 21.65%, p<0.01) compared to the control (table 1). the statistically significant activation of free radical oxidation processes in the rats with cec combined with dm (the level of dc increased by 104.84% and of tbars by 115.02% respectively, p<0.01) was evidenced. a significant decrease in activity of sod by 131.09% and of catalase by 21.65% (p<0.001), compared to the control, was found. discussion thus, in cases of experimental diabetes, the prooxidant-antioxidant disbalance in the wall of small intestine was characterized by the development of oxidative stress. according to the literature, the increase in blood glucose levels with its auto-oxidation is one of the causes of intensification of free radical oxidation [31]. in current studies there are no reliable data on the effect of systematic consumption of carrageenan on the body of an adult, child, foetus and when this supplement comprises the diet of pregnant women. studying this problem in the clinic is very problematic, so there is an urgent need of studying the influence of ca rra geena n on meta bolic parameters in an experimental model. the obtained data of experimental cec prove that under the conditions of normal digestion due to acid hydrolysis, carrageenan splits into low and high molecular particles, which trigger free radical processes. previous s t u d i e s e v i d e n c e t h e i n v o l v e m e n t o f macrophages in the absorption of carrageenan with the formation of heterolysosomes, which leads to the implementation of harmful effects of lysosomal enzymes. on the other hand, macrophages are established to be the source of free radicals [32]. in order to ensure antioxidant equilibrium, an antioxidant defence system is activated simultaneously against the action of the external factor, proving the mobilization of protective and adaptive mechanisms associated with the excessive table 1. rates of free radical oxidation in the wall of small intestine of the rats with chronic enterocolitis combined with streptozotocin-induced diabetes, me (q25-q75) rate control dm(group 2) cec (group 3) dm+cec (group 4) dc, st.un./g of tissue 2.89 (2.50; 3.25) 4.83* (4.25; 5.29) 3.91* (3.71; 4.09) 5.92* (5.76; 6.15) р1,2<0.01 tbars, mmol/mg 3.33 (2.78; 3.80) 5.71* (5.53; 5.95) 4.26* (4.00; 4.57) 7.16* (6.98; 7.31)* р1,2<0.01 sod activity, st. un/mg of proteins 17.17 (16.28; 18.18) 10.64* (8.95; 12.02) 22.30* (21.43; 23.35) 7.43* (6.38; 8.08) р1,2<0.001 catalase activity, mmol·min/mg of proteins 41.64 (40.78; 42.55) 17.15* (16.16; 17.65) 50.66* (51.88; 54.55) 11.46* (10.85; 12.07) р1,2<0.001 notes: * – the difference between the control and experimental groups is statistically significant (р<0.05–0.001); р1 – the reliability value between the groups 2 and 4; р2 – the reliability value between the groups 3 and 4. 62 b io m e d ic a l s c ie n c e s issn 2413-6077. ijmmr 2017 vol. 3 issue 2 production of superoxide anion radical. darren n. seril et al. note that oxidative stress and free radical damage to mucous membranes of small and large intestines are one of the signs of peptic ulcer and, probably, one of the factors of carcinogenesis under these conditions [33]. the inflow of toxin (carrageenan) into the systemic circulation largely depends on the state of cytoplasmic membranes of organs and tissues that perform barrier functions, where the intestine is very important [34, 35]. one of the main pathogenetic factors regulating the permeability of membranes is the activation of free radical oxidation processes, which are crucial mechanism that provides the availability of lipid-protein complexes of membrane for phospholipase and proteases respectively [36]. this fact justifies the results obtained by us regarding the statistically significant activation of free radical oxidation processes in the rats with cec combined with dm. reducing sod activity may be due to the damaging effects of free radicals on the me talloprotein complex of enzyme containing copper, zinc or manganese. low activity of catalase may be associated with the increase in concentration of hydrogen ions, which leads to the development of a proton form of an enzyme having an altered catalytic activity. it should be noted that in the experimental group 4 the rates of lpo were the highest and those of the antioxidant system were the lowest, compared to the other experimental groups. therefore, the activation of free radical reac tions is an important nonspecific mechanism of inflammation develop­ ment in tissues of small and large intestine in cases of diabetes mellitus. conclusions in the wall of small intestine of streptozotocin-induced diabetic rats, lipid peroxide oxidation increases (dc content is higher than in the control by 66.93% and tbars by 71.22%, respectively p<0.001) and the activity of enzyme link of antioxidant defence decreases reliably (sod by 38.02% and catalase by 58.82%). the course of experimental chronic enterocolitis in rats is accompanied by the increase in free radical oxidation in the wall of small intestine (dc by 35.16% and tbars by 27.65%, p<0.01) with simultaneous increase of enzyme activity of antioxidant defence system (sod by 29.90% and catalase by 21.65%, p<0.01), compared to the control. in cases of chronic enterocolitis with underlying diabetes mellitus in rats, the development of oxidative stress in the wall of small intestine is associated with the statistically significant increase in levels of dc (by 104.84%) and tbars (by 115.02%), p<0.01 and the decrease in activity of sod (by 131.09%) and catalase (by 21.65%). references 1. dmitriieva tv. analysis of the patterns of the formation of morbidity and disability and the scientific justification of modern approaches to medical and social expertise in diseases of the digestive organs: the author's abstract of the diss. of doctor of med. sciences. moscow: littera; 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pantoea agglomerans; human health. international journal of medicine and medical research 2018, volume 4, issue 1, p. 78-82 copyright © 2018, tsmu, all rights reserved corresponding author: liudmyla butsenko, department of phytopathogenic bacteria, zabolotny institute of microbiology and virology of the national academy of sciences of ukraine, 154, akademika zabolotnoho str., kyiv, 03680, ukraine e-mail: plant_pathol@ukr.net introduction recently, the number of diseases caused by opportunistic bacteria has been increasing all over the world. opportunistic microorganisms are characterized by ubiquitous proliferation, flexibility in adaptation to the conditions of the environment, lack of specific relationship with the macroorganism. such bacteria are characterized by the ability to cause nonspecific toxic infections in weakened people and animals. it has been established that strains of certain species of microorganisms can cause damage to plants, insects, animals and humans [1]. this phenomenon is known as polybiotrophy [2], which is particularly spread among opportunistic microorganisms. for example, conditionally pathogenic bacteria for humans pseudomonas aeruginosa cause an internal of putrefaction onion during storage [3]. strains p. aeruginosa, isolated from sick people, under experimental conditions affect plants, nematodes and insects [4]. phytopathogenic bacteria are also able to infect both plants and animals [1, 2]. bacteria of the genus erwinia, which are well known exclusively as pathogenic to plants, are often isolated in pathological processes in humans and animals [5]. in this case isolates isolated from humans and animals are pathogenic to plants [2]. the causative agent of vascular bacteriosis of erwinia toxica cucumbers, in intraperitoneal administration to mice, leads to sepsis in animals. infected with these bacteria fruits of cucumbers cause poisoning in people [6]. a high degree of similarity established by dna hybridization analyses and phenotypic data between strains of erwinia herbicola, enterobacter agglomerans and erwinia milletiae led gavini et al. [7] to unite them as a single species, namely pantoea agglomerans (beijerinck 1888) comb. nov. p. agglomerans is widespread in numerous diverse natural habitats and is particularly associated with many different plants as a common epiphyte and endophyte [8]. additionally, it has been also isolated from seeds, water, humans (e.g., wounds, blood, l. m. butsenko et al. 79 b io m e d ic a l s c ie n c e s issn 2413-6077. ijmmr 2018 vol. 4 issue 1 urine and internal organs) as well as from animals [7, 9-12]. pantoea species are ubiquitous in nature and occasionally are associated with infections caused by contaminated clinical material. hence, pantoea agglomerans is considered as an opportunistic pathogen of humans [13]. the pseudomonas fluorescens bacteria, under conditions favorable to their development, can cause diseases of agricultural crops [14]. for some human diseases, antibodies to p. fluorescens lipopolysaccharide have been identified in the patients’ blood. therefore, the purpose of the research was to detect the pantoea agglomerans and pseudomonas fluorescens bacteria in various ecological niches and establish their virulence. methods materials for research were the plants with symptoms of damage of rye, wheat and weeds that grew in wheat agrocenoses. bacteriological analysis and isolation of bacteria were carried out using generally accepted methods [15]. virulent properties were investigated on the host: plant, aggressiveness of the pathogen was determined by a 4-point scale. the biological properties were investigated by the methods described by klement et al [15]. the bacteria were identified by comparing their properties with the characteristics of strains collection, and according to the bergey’s manual of systematic bacteriology [16]. results we have found out that yellow-pigmented bacteria, flat or with a conical center, are opaque, wavy edges; and the oxidase-positive gray-colored isolates with wavy edges were isolated from all investigated plant materials. it was established that a part of the investigated isolates of bacteria was avirulent for plants. in all other cases, the isolated bacteria caused diseases of rye, wheat and weeds. the yellow pigmented bacteria are polymorphic, short rods, single arranged, sometimes in pairs in the form of short chains. bacteria are mobile, gram negative, spores are not formed, oxidase negative. facultative anaerobes (table 1). on the meat-peptone broth they grow with the formation of uniform turbidity, ring, film and sediment. the bacteria are utilized glucose (anaerobic), reduce nitrates, acidify litmus serum. strains differ in the use of rafinose, sorbitol and inulin. all investigated strains do not use dulcitol, cause coagulation or peptonization of milk. the isolates obtained from the affected plants according to the physiological and table 1. physiological and biochemical properties of isolates test yellow-pigmented isolates p.аgglomerans [16] unpigmented isolates p.fluorescens [16] gram’s staining – – – – motility + + + + nitrate reduction + + – – oxidase – n/i + + formation of h2s –/+ n/i – n/i formation of indole – – – n/i gelatinase +/+ + n/i of-test facultative anaerob facultative anaerob aerob aerob utilization: d-glucose, l-arabinose, d-mannitol, dxylose + + + + fructose, galactose + + + n/i salicin + + – – lactose + d – – inositol + + +/– + raphinose +/– d +/– n/i dulcitol – – +/– n/i sorbitol +/– – +/– + inulin +/– n/i note: n/i – not investigated. l. m. butsenko et al. 80 b io m e d ic a l s c ie n c e s issn 2413-6077. ijmmr 2018 vol. 4 issue 1 biochemical properties are similar to each other and do not differ from the characteristics of the described p. agglomerans species [16]. according to this they were identified as p. agglomerans. gray colored oxidase-negative bacteria grow on the meat-peptone broth, form a film, ring and precipitate, and use glucose (aerobic), mannitol, xylose, fructose, arabinose, and do not utilize lactose, inulin, salicin, as a single source of carbon. most of the isolates utilize raffinose, maltose and dulcitol. all isolates hydrolyze gelatin, alkalinize the litmus serum, and do not reduce nitrates (table 1).the isolates of bacteria obtained from the affected cereals and weeds on the morphological and cultural-biochemical properties did not differ from the described strains of p. fluorescens and from the p. fluorescens characteristic given in bergey’s manual of systematic bacteriology [16]. all bacterial strains tested were virulent for wheat, rye and weed plants (table 2). it was established that the strains of the isolated bacteria are more aggressive on weeds than on agricultural crops. consequently, pathogenic bacteria for plants of the species pantoea agglomerans and pseudomonas fluorescens are able to come into contact with humans and animals and cause their diseases. this is evidenced by the fact that we detected antigens to the strains pantoea agglomerans and pseudomonas syringae pv. syringae in the serum of non-immunized rabbits (true phytopathogen) (table 3). thus, the study of phytopathogenic and opportunistic bacteria is important not only for the development of plant protection methods, but also from the point of view of studying their effects on human health, since these bacteria are widespread in nature and can be ingested by humans. discussion our research has found that rye, wheat and weeds that grow in the agroecosystem of wheat are affected by opportunistic bacteria pantoea agglomerans and pseudomonas fluorescens. our previous studies proved that strains of p. agglomerans and p. fluorescens were more commonly found in agricultural crops as epiphytes and did not cause plant diseases [17]. with the change of environmental factors and the influence of agronomic techniques, these opportunistic bacteria in recent years increasingly acquire virulent properties. for example, the bacteria of p. agglomerans were isolated from the affected locales of cotton bolls collec_ ted in a field in the usa and were able to cause comparable disease symptoms in greenhouse grown cotton fruit [18]. p. agglomerans, gram negative bacteria of enterobacteriaceae family, were isolated from feculent material, plants and soil. soft tissue and bone-joint infections due to p. agglomerans following penetrating trauma by vegetation and bacteraemia in association with intravenous fluid, total parenteral nutrition, blood products and anesthetic agent contamination were reported [19]. some authors on the basis of their studies suggested that, independent of their origin, all p. agglomerans strains might possess indistinguishable virulence potential [13]. p. agglomerans was also proved to be an antibiotics producer [20]. some researchers reported on isolation of p. agglomerans in two cases of septic mono arthritis after plant thorn and wood sliver injuries [21]. this indicated the transfer of p. agglomerans from the infected plant material to humans. table 2. virulence properties species aggressiveness (marks) on plant: rye wheat weed pantoea agglomerans 1–2 1–3 2–3 pseudomonas fluorescens 1–2 2 2–3 table 3. results of agglutination reaction species, strains titres of agglutination reaction with sera of non-immunized rabbits serum 1 serum 2 pantoea agglomerans, 116 50 100 pseudomonas syringae pv. syringae, ncppb 281 200 200 erwinia amylovora, 2024 0 0 l. m. butsenko et al. 81 b io m e d ic a l s c ie n c e s issn 2413-6077. ijmmr 2018 vol. 4 issue 1 when studying the effect of bacteria of the genus klebsiella (k. pneumoniae, k. hinoscleromatis, k. ozaenae), which, like the erwinia phytopathogenic genus, belonged to the entero bacteriaceae family, on leaves of potatoes, horse beans, beans, cabbage, cucumber, pumpkin and apple fruits jonathan found out that four strains of k. pneumoniae were capable to affect horse beans and potatoes. the phytopathogenic properties of klebsiella species did not correlate with their ability to produce pectinases [22]. at the same time, phytopathogenic properties can be presented by bacteria, which are traditionally pathogens of animals and humans. it is proved that 15% of the strains of bacteria of genera escherichia, citrobacter, enterobacter, proteus, pseudomonas, isolated from urological patients, have phytopathogenic properties. the most pronounced these properties were on fruits of tomatoes [23]. among the species of the genus pseudomonas, which produce pigments (p. aeruginosa, p. fluorescens, p. aureofaciens), it is proved that the strains have phytotoxic and entomocidal properties. the greatest number of such strains is found among the bacteria of p.fluorescens species. the cultural fluid of these strains suppresses the germination of seeds of radish, lettuce and, to a lesser extent, wheat, and also causes the death of 100% of mosquito larvae. entomopathogenic strains of p. fluorescens have antagonistic effects on some saprophytic bacteria (bacillus subtilis, b. megaterium, sarcina lutea, escherichia coli, mycobacterium sp.). thus, the toxins of pigmented strains of the genus pseudomonas are not narrowly specific and affect a wide range of organisms [24]. in some human diseases antibodies to the lipopolysaccharide of p. fluorescens 7769, which were isolated from affected rye tissues, were identified [25]. the adaptation of p. agglomerans to diverse microenvironments might suggest that this species maintain high genetic plasticity. p. agglomerans appears to be readily accessible to horizontal gene transfer driven by plasmids and other mobile elements [26], a trait that may explain its flexibility in adapting to different life styles. some researchers noted that pathogenic microorganisms had a lot in common in mechanisms of pathogenicity, regardless of which macroorganism they were infected with [1, 27, 28]. conclusions thus, in the agrophytocenosis of wheat, one of the most widespread agricultural crops in ukraine, there are virulent strains of opportunistic bacteria of p. agglomerans and p. fluorescens species. present agroecosystems, which are overloaded with chemical pollutants, create conditions for increasing the aggressiveness of opportunistic bacteria. it has been established that in the blood of healthy rabbits antibodies to opportunistic bacteria, which are spread in agrophytocenoses, are present. it proves the intake by animals of opportunistic bacteria together with plant food. since opportunistic bacteria can cause the infections processes in humans, animals, insects and plants, the control of their spread and investigation of peculiarities of virulent strains circulation is necessary. l. m. butsenko et al. опортуністичні бактерії в агроекосистемах україни л. м.буценко, л. а. пасічник інститут мікробіології і вірусології імені д. к. заболотного нан україни, київ, україна вступ. останнім часом кількість захворювань, спричинених опортуністичними бактеріями, зростає в усьому світі. опортуністичні мікроорганізми характеризуються значними темпами проліферації, гнучкістю в адаптації до умов навколишнього середовища, відсутністю специфічних зв’язків з макроорганізмом. фітопатогенні бактерії також здатні інфікувати як рослини, так і тварини. метою дослідження було виявлення бактерій pantoea agglomerans і pseudomonas fluorescens в різних екологічних нішах і встановлення їх вірулентності. методи дослідження. у дослідженні використовувалися класичні мікробіологічні, біохімічні та серологічні методи. ідентифікацію бактерій було здійснено за їх фенотиповими властивостями. результати дослідження. встановлено, що в агрофітоценозах пшениці присутні опортуністичні бактерії виду p. agglomerans і p. fluorescens. виділені бактерії вірулентні для рослин пшениці, жита та бур’янів. антитіла до опортуністичних бактерій, які поширюються в агрофітоценозах зернових культур, виявлені в крові здорових кроликів. висновки. встановлено, що вірулентні штами опортуністичних бактерій p. agglomerans та p. fluorescens поширюються в агрофітоценозах зернових культур. наявність антитіл до цих бактерій у крові здорових кроликів доводить, що опортуністичні бактерії з рослинного матеріалу потрапляють в організми тварин і людини. знання біології та поширення опортуністичних патогенів в агрофітоценозах необхідні для профілактики інфекцій, що викликаються цими бактеріями в організмі людини. ключові слова: опортуністичні бактерії; pantoea agglomerans; здоров’я людини. 82 b io m e d ic a l s c ie n c e s issn 2413-6077. ijmmr 2018 vol. 4 issue 1 9. cruz at, cazacu ac, allen ch. pantoea agglomerans, a plant pathogen causing human disease. j clinical microbiol. 2007;45:1989-1992. 10. williams aj, scott rj, lightfoot nf. erwinia herbicola as a cause of bacterial endocarditis. j infect. 1986;12(1):71-3. 11. milanowski j, dutkiewicz j, fafrowicz b. pathogenic action of erwinia herbicola. ii. in vivo investigations. pneumonol alergol pol. 1993;61(1112):598-605. 12. silva mc, attademo ff, freire ac, sousa gp, luna fo, lima dc et al. identification of bacteria in blood cultures from clinically ill captive antillean manatees (trichechus manatus manatus). j zoo wildl med. 2017;48(1):13-17. doi: 10.1638/2015-0094.1. 13. völksch b, thon s, jacobsen id, gube m. polyphasic study of plantand clinic-associated pantoea agglomerans strains reveals indistinguishable virulence potential. infect genet evol. 2009;9(6):138191. doi: 10.1016/j.meegid.2009.09.016. 14. gvozdyak ri, pasichnyk la, yakovleva lm, moroz sm, lytvynchuk oo, zhytkevych nv, et al. fitopatohenni bakterii. bakterialni khvoroby roslyn. kyiv: tov ’nvp interservis’; 2011.v.1:444 p. 15. кlement z, rudollf k, sands d. methods in phytobacteriology. budapest, academia kiado; 1990: 568 p. 16. bergey’s manual of systematic bacteriology. boore dr, castenholz rw editors, vol. 1: garrity gm, editor-in-chief. 2nd ed. new york, berlin, heidelberg: springer, 2005; 2, part b: 1106 p. 17. pasichnyk la, gvozdyak ri, khodos sf. epiphytic and endophytic microflora of healthy seeds and wheat plants. bulletin of the su, series biology. 2005; 2(15):141-148. 18. medrano eg, bell aa. role of pantoea agglomerans in opportunistic bacterial seed and boll rot of cotton (gossypium hirsutum) grown in the field. j a p p l m i c r o b i o l . 2 0 0 7 ; 1 0 2 : 1 3 4 1 4 3 . d o i : 10.1111/j.1365-2672.2006.03055.x 19. izzo i, lania d, castro a, lanzini f, bella d, pagani a, colombini p. seven cases of port-a-cath contamination caused by pantoea agglomerans in the oncological service of iseo hospital, brescia (italy). infez med. 2014;22(2):152-5. 20. wright sa, zumoff ch, schneider l, beer sv. pantoea agglomerans strain eh318 produces two antibiotics that inhibit erwinia amylovora in vitro. appl environ microbiol. 2001;67:284-292. 21. de champs c, le seaux s, dubost jj, boisgard s, sauvezie b, sirot j. isolation of pantoea agglomerans in two cases of septic monoarthritis after plant thorn and wood sliver injuries. j clin microbiol. 2000; 38(1):460-1. 22. turyanitsa ai, korobko ap. phytopathogenic properties of bacteria of the genus klebsiella. microbiol z. 1983;45(6):88-89. 23. gvozdyak ri, khodos sf, chaikovskaya vl. phytopathogenic properties of bacteria isolated from urological patient. microbiol z. 1986;48(2):30-33. 24. mikhnovskaya nd, shevtsova ni, ruban em, lysenko ln, vasilevskaya ia. problem on nonspecific toxigenicity of certain representatives of the genus pseudomonas. microbiol z. 1988;50(5):83-86. 25. zdorovenko gm, gvozdyak ri, gubanova nya, afonina gb, zdorovenko ei. characterization of the lipopolysaccharide from f pseudomonas fluorescens (biovar i). мikrobiology. 1999;68(3):330-339. 26. barash i, manulis-sasson s. virulence mechanisms and host specificity of gall-forming pantoea agglomerans. trends in microbiol. 2007;15: 538-545. 27. kempf vaj, hitziger n, riess t, autenrieth ib. do plant and human pathogens have a common pathogenicity strategy? trends in microbiol. 2002; 10(6):269-275. 28. rahme lg, stevens ej, wolfort sf, shao j, tompkins rg, ausubel fm. common virulence factors for bacterial pathogenicity in plants and animals. science. 1995;268(5219):1899-1902. received: 2018-04-27 l. m. butsenko et al. 6. gvozdyak ri. korobko ap, lemeshchenko gp. zoopathogenic properties of the causative agent of vascular bacteriosis of e. toxica cucumbers. in phytopathogenic bacteria. kyiv: nauk. dumka, 1975: 88-91. 7.gavini f, mergaert j, beji a, mielcarek c, izard d, kerster k, de ley j. transfer of enterobacter agglomerans (beijerinck 1988) ewing and file 1972 to pantoea gen. nov. as pantoea agglomerans comb. nov. and description of pantoea dispersa sp. nov. int j syst bacteriol.1989;39:337-345. 8. kobayashi dy, palumbo jd. bacterial endophytes and their effects on plants and uses in agriculture. in microbial endophytes. bacon cw, et al. (eds). basel, switzerland: marcel dekker inc. 2000;199-233. references 1. cao h, baldini rl, rahme lg. common mechanisms for pathogens of plants and animals. ann rev phytopathol. 2001;39:259-284. 2. gvozdyak ri. polybiotrophy of bacteria. microbiol z.1981;43(2):256-262. 3. gvozdyak ri, yakovleva lm. on the peculiar features of the pathogenicity of pseudomonas aeruginosa. j microbiol epidemiol immunol. 1987;3: 3-6. 4. mahajan–miklos s, rahme lg, ausubel fm. elucidating the molecular mechanisms of bacterial virulence using non–mammalian hosts. mol microbiol. 2000;37(5):981-988. 5. starr mp, chatteriee ak. the genus erwinia: enterobacteria pathogenic to plant and animals. ann rev microbiol. 1972;26:389-426. 20 su r g ic a l d is e a se s issn 2413-6077. ijmmr 2017 vol. 3 issue 2 doi 10.11603/ijmmr.2413-6077.2017.2.8381 choice of treatment options for cystic lymphstic malformations in the head and neck region: treatment experience of 81 children i. m. benzar bogomolets national medical university, kyiv, ukraine background. surgery has previously been the only treatment for lymphatic malformations (lms), but in the head and neck region is challenging due to the risk of scarring, nerve damage, recurrence. sclerotherapy may be a perfect alternative. objective. the aim of the study is to determine the efficacy and safety of the ok-432 sclerotherapy in the children with craniofacial lms. methods. 81 children with head and neck lm between december 2010 and march 2017were involved into the study. the follow-up period was from 6 to 79 months. according to the size of cysts, lms were classified into macrocystic, microcystic, and mixed. the result of the treatment of lms was determined by the percentage of reduction in size as excellent (decrease by more than 90%), good (by 50%–89%), satisfactory (by 20%–49%) and none (by less than 20%). results. the macrocystic lms diagnosed in 41.97% of patients, microcystic – in 12.35%, and mixed – in 45.68% of children. ok-432 sclerotherapy only was performed for 83.9% of patients and in 12.3% in combination with surgery. the range of sclerotherapy sessions was from 1 to 11. an excellent result in 96.97% of cases was evidenced in the patients with macrocystic lm. poor result was proved in the patients with microcystic lms; the most of them (55.56%) had satisfactory result. in the patients with mixed lm, an excellent and good result was evidenced in 83.33%. after 198 sessions of ok-432 sclerotherapy, complications associated with the treatment occurred in 5 (2.52%) cases. conclusions. ok-432 sclerotherapy is a safe and effective treatment of head and neck lms in children. macrocystic lms proved the best response to ok-432 treatment. key words: lymphatic malformations; sclerotherapy; ok-432; children. corresponding author: iryna benzar, department of pediatric surgery, bogomolets national medical university, 28/1 v. chornovola, kyiv, ukraine, 01135 phone number: +380951295882 e-mail: iryna.benzar@nmu.ua, ira_benzar@yahoo.com introduction cystic lymphatic malformations are benign vascular lesions, which develop as the result of embryological disturbances of lymphatic system. unfortunately, the terms often used to describe lms are imprecise. ‘cystic hygroma’ and ‘lymphangioma’ are often used incorrectly to describe malformations. both of these terms should be abandoned, as the suffix -oma connotes a neoplasm [1]. in 1982, a landmark article by mulliken and glowacki suggested a classification system designating vascular ano­ malies as either tumours or malformations accor ding to their biologic and pathologic features [2]. this system was adopted by the international society for the study of vascular anomalies (issva) and was last updated in 2014 [3]. vascular malformations are classified as capillary, venous, lymphatic, arterial, and mixed lesions, depending on their vascular tissue of origin. these lesions are present at birth and have a progressive clinical course. the incidence of lms is evidenced in about 1 to 6000 to 1 to 16,000 live births, with a frequency of hospitalization – 3 cases per 100,000 [4, 5]. lms near the principal lymphatic chains in the neck region are formed when a primordial lymph sac loses or fails to re-establish communication with the central veins (jugular and subclavian) from which it arises [1]. for many years, surgery has been the only treatment for lms of any anatomical localization. however, the results of the operation were often disappointing, especially in cases of lm of cheek, tongue, oral cavity, neck lateral triangle. the surgical treatment of lm in the region of head and neck is accompanied by complications in 12–33% and recurrences in 15–53% of cases. in addition, international journal of medicine and medical research 2017, volume 3, issue 2, p. 20–24 copyright © 2017, tsmu, all rights reserved i. m. benzar 21 su r g ic a l d is e a se s issn 2413-6077. ijmmr 2017 vol. 3 issue 2 re section of lm does not inhibit bones overgrowth in the region of lms [6, 7, 8]. recurrence and complications are usually the result of incomplete preoperative examination and incorrectly planned treatment. incomplete resection provokes rapid growth of malformation, causes postoperative lymphorrhea, and recurrent infections [9]. the role of surgical treatment in order to achieve an excellent cosmetic result in patients with lms is questionable. when planning an intervention, it is necessary to clearly understand that lm can sprout fascia, infiltrate different tissues and change normal anatomy. the damage of important nervous and vascular structures is unacceptable in the treatment of benign pathology [10]. lms usually infiltrate skin and subcutaneous tissue [1], therefore after the operation the asymmetry progresses, and later lymph nodes can sprout post-operative scars. currently, there is no sufficient evidence in the literature to create treatment algorithms and help determine the best choice of initial therapy, and in recent decades sclerotherapy has become the most common treatment option [5, 11, 12]. according to the results of the literature analysis in the pubmed, medline, cochrane databases, three therapeutic agents are widely used for cystic lm local treatment: bleomycin, doxycycline and ok-432 (picibanil) [13]. the features of endothelial cells lining the lms cysts determine the effectiveness of the ok-432 for the treatment of lm. the mechanism of action of the drug is based on the induction of inflam­ mation with subsequent activation of cytokines and apoptosis of cells that form the inner layer of the cysts [14]. in the cyst fluid the level of inflammatory mediators increases that confirms the inflammation in response to the injection of the drug [15]. the effectiveness of the ok-432 sclerotherapy in the treatment of cystic lms has been described in small series of some medical centres [16, 17]. since 2011 ok-432 is successfully used in ukraine [18]. objective of the study is to analyse the treatment experience of 81 child patients with lymphatic malformations (lm) in the head and neck region, and to determine the efficacy and safety of the ok-432 sclerotherapy in this cohort of patients. methods during the period from december 2010 to march 2017, 126 patients with isolated lms or lms in combination with other congenital vascular malformations were treated in the okhmatdyt national specialized children hospital. the study includes 81 clinical cases of lms that are localized in the head and neck region, representing 64.29% of all patients with lms during the established period of time. the follow up period was from 6 to 79 months. there were 44 (53.7%) male and 37 (45.1%) female patients. the following visualization methods were used to confirm the diagnosis of cystic lms: ultrasound scan in grey-scale and colour doppler was performed at the primary patient's examination and in dynamics, mri was per formed before treatment and at the stages of treatment, ct in urgent situation. according to the results of mri, the size and structure of lms and also their topographical relationship with neighbouring organs and tissues were deter mined as particularly mediastinum with retro pharyngeal space involvement associated with lms to the level of the hyoid bone. lms were classified as macrocystic, microcystic, or mixed (macrocysticmicrocystic), which has been proven useful in other sclerotherapy studies [19]. the classification was determined radio graphically by mri and ultrasound examination according to the cysts size: macrocystic (formed by cysts with a volume of more than 2 cubic centimetres (cc)), microcystic (cysts volume – less than 2 cc), and mixed, in which the microcystic component exceeds 50%. the size of lms was determined using the ellipse square formula. the size of lms up to 100 cm2 was evaluated as small, 100–199 cm2 – middle, 200–299 cm2 – large and more than 300 cm2 – gigantic. the result of the treatment of lm was determined by the percentage of reduction in the lesion size [3, 10] as excellent (decrease by more than 90%), good (50–89%), satisfactory (20–49%) and unsatisfactory (less than 20% reduction in size). the statistical analysis was performed using ibm spss statistics, version 23. the data is presented as an average of 95 percent confidence interval or standard deviation. the qualitative data (age, sex, localization, size, etс.) were analysed in a univariate analysis with the pearson’s correlation coefficient χ2 test. the logistic regression model was used for variants that significantly predict the outcome of the treat­ ment and/or the course of the disease. statistical significance was defined as p<0.05. results diagnosis of lms were established prenatally in 12 (14.81%) patients, at birth in 43 (53.08%), i. m. benzar 22 su r g ic a l d is e a se s issn 2413-6077. ijmmr 2017 vol. 3 issue 2 during the first year of life in 12 children (14.81%), at the age of one to three years in 4 (4, 94%) and in 10 (12.35%) children, the clinical signs of the disease were manifested at the age above three years. clinical signs of cystic lm in the head and neck region were: the asymmetry of face and neck due to mass in all children, compression of upper airway requiring tra cheostomy before sclerotherapy (n=4, 3.6%), transient stridor during the first year of life (n=8, 7,1%), macroglossia, disarticulation (n=5, 4,5%), disturbance of occlusion (n=6, 5,4%), recurrence lymphorrhoea in the children with mucosal and skin lesions (n=4, 3.6%), excessive salivation (n=3, 2.7%), visual impairment (n=1, 0.9%), lymphedema of upper limb (n=1, 0.9%). 68 patients were treated primary, 13 children underwent surgical interventions previously. there pa tients were treated conservatively with propranolol within 6–12 months without any clinical result. according to the visualization of lms using ultrasound and mri, the topographic features of lm and relationship to adjacent organs were established. the lesions were localized in suprahyoid region in 37 (45.68%) children, and in infrahyoid region – in 44 (54.32%) cases. bilateral mass were observed in 23 (28.39%) children, in other patients lms were localized on the one side of middle line. 34 (41.97%) patients had macro cystic lms, in 10 (12.35%) children microcytic lms were diagnosed, and in 37 (45.68%) children lms were mixed. the size of lms ranged from 23 to 517 сm2, in average 135.87 см2. in 37 (45.68%) patients lms were small; in 29 (35.80%) they were middle, in 7 (8.64%) lms were large and in 8 (9.88%) lms were gigantic. in 22 (2716%) children mediastinal involvement was diagnosed. in children with head and neck lms sclero therapy, surgical and combined treatments were performed. three patients (3.7%) underwent surgical resection of lms for the following reasons: recurrent inflammation requiring re­hospita­ lization and antibiotic therapy (n=2), difficulty in diagnostic due to sudden onset and atypical course of the disease (n=1). recurrence after surgery happened in one case. one patient had postoperative complication: transient paresis of facial nerve branch. giant lms, which occupy several anatomical sites and infiltrate muscles, bones, cellular spaces deserved a particular attention. bilateral lms narrowed airway and tracheostomy was evidenced in 5 patients. in this series, decannulation was eventually possible in 4 patients after multiple sessions of ok-432 sclerotherapy. one patient with bilateral lms, mediastinum, retropharyngeal space and lung involvement died due to septic complications. combined treatment was performed for 10 (12.3%) children, which consisted of the following procedures: resection of lm, tracheostomy prior to treatment, injection of ok-432 into residual cyst ca vity during and/or after surgery (n=5, 6.17%), thoracocentesis, pleural and mediastinal drainage in the patient with purulent mediastinitis with subsequent sclerotherapy after the elimination of complications (n=1), sclerotherapy and correction of scarring and tissue deformities (n=3), resection of tongue in the patient with pronounced macroglossia, disturbance of occlusion (n=1), resection of orbital part of lms due to visual impairment and subse quent sclerotherapy (n=1). the most patients (n=68, 83.90%) underwent the ok-432 sclerotherapy as a single method of head and neck lms. patients received from 1 to 11 sessions of sclerotherapy, in average 2.44±2.21 sessions per patient. after 1 session of the ok-432 sclerotherapy, an excellent result was achieved in 24 (29.63%) patients, according to localization and structure, these were unilateral macro cystic lms of neck. involvement of the mediastinum and retropharyngeal space was not a contraindication to the sclerotherapy of lms. i n 1 8 c h i l d r e n w i t h m e d i a s t i n a l a n d retropharyngeal part of lms an excellent result was evidenced in 5 (27.78%), good in 9 (50.0%), and satisfactory in 2 (11.11%) cases. however, in 1 case, the result was poor and in 1 case the result of the treatment was unsatisfactory. after 198 sessions of the ok-432 sclerotherapy, complications associated with the treatment occurred in 5 (2.52%) cases: signi­ ficant oedema with the need for hospitalization, puncture, and decompression of the cyst (n=4; 2.02%), and skin allergic reaction (n=1; 0.51%). response data for macro cystic lms were higher than in other types of lms with an excellent result in 96.97% and good result in 3.03% of patients. in the patients with microcystic lms, no positive result was evidenced, 33.33% of cases had a good treatment result, 55.56% were satisfactory and 11.11% had no results. in the patients with mixed lm, an excellent result was evidenced in 33.33% of cases, 50.0% of patients had good response to treatment, 11.11% – a satis factory one, and 2.77% of patients had no result. primary efficacy endpoints were evaluated using fisher’s exact test. covariates used in a i. m. benzar 23 su r g ic a l d is e a se s issn 2413-6077. ijmmr 2017 vol. 3 issue 2 univariate analysis included: lm type (macro­ cystic, microcystic, mixed), laterality (unilateral versus bilateral), mediastinal and retropharyngeal involvement, prior treatment, age during the first treatment, gender. it was establi­ shed, that the result of treatment was determined primarily by the type of lm and previous intervention: the best results were evidenced in the patients with macrocystic lms (p<0.005), previous intervention reduced the treatment efficacy (p<0.005). covariates that proved a significant association with the treatment result were used for logistic regression analysis, the probability of an excellent and good outcome for the patients with macrocystic lm in cases of no prior treatment was 86.6%. discussion this article presents a relatively large experience of the treatment of cystic lms in the head and neck region for a relatively short period of time (5.5 years). a peculiarity of this study is that for the first time in ukraine a new approach to the treatment of lms of challenging localization in children is presented. treatment of head and neck lms is accompanied by significant risks, therefore for children it is necessary to choose the most effective and safe way, until stronger evidence is present, the difference in complication rates is potentially the deciding factor in the choice the treatment options [13]. for decades, resection has been the mainstay for management of lms. more recently, sclerotherapy has become the first­line treat­ ment for head and neck lesions. experienced surgeons welcome the ascendancy of sclerosant management. although some lms can be successfully resected, complications are expected and results are often disappointing. one of the most safe and effective ways of cystic lms treatment in children is the ok-432 sclerotherapy [5, 16]. the ok-432 (chugai pharmaceuticals, tokyo, japan) is a lyophilized powder of streptococcus pyogenes (group a, type 3, su strain) incubated with benzylpenicillin. the drug was developed in japan in the late 1960s as an antitumor agent. although therapy of ok-432 did not increase the survival of the patients with cancer, its efficacy was proved in pleurodesis in cases of malignant pleural involvement. according to these studies, in the late 1980s, japanese authors [17] published the first results of using ok-432 as a safe and effective treatment for cystic lms; in 1994, the authors published the results of treatment of 94 patients with lms [19]. since then, the method has become widely used far beyond the borders of the country [20, 21]. the ok-432 is not exactly a sclerosing agent, because it doesn’t destroy vascular endothelium. the ok­ 432 induces apoptosis of lymphatic endothelium and local cellular inflammatory reaction [7, 16]. recent studies have proved that the pathway of the ok-432 action within lymphangiomas is probably cellular and cytokine-mediated [7, 16]. complications due to ok-432 management are temporary and predictable [22]. according to the results of the literature analysis, no systemic hematological, renal, hepatic or cardiac side effects were detected [23], which was confirmed in our study. the typical side effect of ok­432 sclerotherapy is an inflammatory response that is accompanied by local oedema and may be potentially dangerous in cases of an airway compromised by cervical lms. in our series, in the patients with impaired mechanics of breathing, tracheostomy was performed prior to treatment. all of those children had signs of respiratory failure, tracheostomy as a preventive procedure was not performed. unfortunately, none of the suggested methods for cystic lm treatment in children guarantees the complete recovery for all patients [24]; however, the introduction of a minimally invasive treatment of lm significantly reduced the percentage of open surgical interventions and made it possible to differentiate treatment in each individual clinical case. conclusions the ok-432 sclerotherapy is safe and effective treatment option of head and neck lms in children. macrocystic lms prove the best response to the ok-432 treatment, previous intervention and increasing the part of microcystic component reduces the efficacy of treat­ ment. complications associated with the ok432 treatment occurred in 2.52% cases. references 1. elluru r, balakrishnan k, padua h. lymphatic malformations: diagnosis and management. semin pediatr surg. 2014;23(4):178–85. doi: 10.1053/j. sempedsurg.2014.07.002. i. m. benzar 24 su r g ic a l d is e a se s issn 2413-6077. ijmmr 2017 vol. 3 issue 2 2. mulliken jb, glowacki j. hemangiomasand vascular malformations in infants and children: a classification based on endothelial characteristics. plast reconstr surg. 1982;69(3):412–22. 3. wassef m, blei f, adams d, alomari a, baselga e, berenstein a, et al. vascular anomalies classification: recommendations from the interna­ tional society for the study of vascular anomalies. pediatrics. 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18. benzar i. treatment of lymphatic malformations with ok­432: the first experience of a single hospital. internat j of biomed. 2014;4(4):237–41. 19. ogita s, tsuto t, nakamura k, deguchi e, iwai n. ok-432 therapy in 64 patients with lymphangioma. j pediatr surg. 1994;29(6):784–5. 20. poldervaart mt, breugem cc, speleman l, pasmans s. treatment of lymphatic malformations with ok-432 (picibanil). j craniofac surg. 2009; 20(4):1159–62. doi: 10.1097/scs.0b013e3181abb249. 21. tu jh, do hm, patel v, yeom kw, teng jmc. sclerotherapy for lymphatic malformations of the head and neck in the pediatric population. j neurointerv surg. 2017;9(10):1023–1026. doi: 10.1136/ neurintsurg-2016-012660. 22. smith mc, zimmerman mb, burke dk, bauman nm, sato y, smith rj. efficacy and safety of ok-432 immunotherapy of lymphatic malformations. laryngoscope. 2009;119(1):107–15. doi: 10.1002/ lary.20041. 23. kim dw. ok-432 sclerotherapy of lymphatic malformation in the head and neck: factors related to outcome. pediatr radiol. 2014;44(7):857–62. doi: 10.1007/s00247-014-2889-0. 24. trenor cc 3rd, chaudry g. complex lymphatic anomalies. semin pediatr surg. 2014;23(4):186–90. doi: 10.1053/j.sempedsurg.2014.07.006. received: 2017-10-13 i. m. benzar 67 b io m e d ic a l s c ie n c e s issn 2413-6077. ijmmr 2018 vol. 4 issue 1 doi 10.11603/ijmmr.2413-6077.2018.1.8979 carrageenan induces cell death in rats blood o. m. kopanytsia, m. i. marushchak, i. ya. krynytska i. horbachevsky ternopil state medical university, ternopil, ukraine background. because of its physical and chemical properties, carrageenan is fairly widely used. about 70 % of the carrageenan produced in the world is used in the food industry. previous studies point to the development of oxidative stress in rats, by means of which carrageenan chronic enterocolitis was modeled. objective. the aim of our study was to investigate the level of apoptosis and necrosis in the suspension of leukocytes in rats using 0.5 % and 1.0 % solutions of carrageenan. methods. annexin v (v) binding assays were performed using annexin v apoptosis kit (sigma aldrich, usa), caspase rate in leukocyte-lymphocyte blood fractions was determined by spectrofotometry. results. it was established that in the experimental application of carrageenan, the percentage of leukocytes with signs of apoptosis in both experimental groups statistically significantly increased. it was detected by the increased activity of effector caspase-3 in 1 month after the experiment in 1.5 times in the 2nd group and in 2.8 times in the 3rd group vs control data that point to caspase-dependent apoptotic pathway in case of carrageenan usage in rats. conclusions. oral use of carrageenan in rats was accompanied by the increase in the number of leukocytes with signs of apoptosis. the animals that consumed 1.0 % solution of carrageenan had more obvious increase in the activity of caspase-3 in serum relative to a group of rats consuming 0.5 % of carrageenan, proving the increase in the severity of apoptotic processes in intestine with the increase of the dose of carrageenan. key words: carrageenan, apoptosis, caspase-3, rat. introduction in world practice it is allowed to use about 500 different substances as nutritional supplements, not taking into account a huge number of different types of flavors and some varieties of combined supplements [1]. in the usa, the amount of nutritional supplements (ns) exceeds 1500, in the eu it reaches 1200, in russia it is 415, in the federal republic of germany – 350 [2]. by the beginning of 90s of the 20th century the use of nutritional s u p p l e m e n t s i n u k ra i n e w a s l i m i t e d i n comparison with other countries of europe and the usa [3], only 194 ns were permitted until 1994, and according to the cabinet of ministers resolution in 2000, 221 ns were permitted. in addition, in the eu countries it is allowed to use more than 400 flavors in food production. for food and in medical practice, there is a nutritional supplement called carrageen that is emitted by water extraction from marine algae [4]. this ns (e407) constitutes a family of polysaccharides (also known as irish moss) contained in the red seaweed chondrus crispus, eucheuma species, gigartina species, and others. by chemical nature carrageenan is close to agarose and represents unbranched sulfated heteroglycans which molecules are made of residues of derivatives of d-galactopyranose with a strict alternation of α(1,3) and β(1,4) b o n d s b e t w e e n t h e m , i . e . o f re p e t i t i v e disaccharide units including residues of β-dg a l a c t o p y r a n o s e a n d 3 , 6 a n h y d r o α d galactopyranose. depending on the peculiarities of the structure of disaccharide repeating units, there are three main types of carrageenans, for which the letters of the greek alphabet are used to define [1, 5, 6]. because of its physical and chemical properties, carrageenan is a fairly widely used. about 70 % of the carrageenan produced in the world is used in the food industry: in the production of dairy products (chocolate milk, sherbets, farmer cheese, cheese paste, baby food), meat and fish products (canned food, sausage casings, jelly coatings), seasonings, nonalcoholic beverages, bakery (bread dough, fruit muffins, sugar icing) and confectionery [7]. previous studies point to the development of oxidative stress in rats, by which carrageenan chronic enterocolitis was modeled [8]. the international journal of medicine and medical research 2018, volume 4, issue 1, p. 67-70 copyright © 2018, tsmu, all rights reserved corresponding author: mariya marushchak, professor, department of functional and laboratory diagnostics, i. horbachevsky ternopil state medical university, 1 maydan voli, ternopil, 46000, ukraine e-mail: marushchak@tdmu.edu.ua phone number: +380979981202 o. m. kopanytsia et al. 68 b io m e d ic a l s c ie n c e s issn 2413-6077. ijmmr 2018 vol. 4 issue 1 activation of lipid peroxidation is caused by direct stimulation of generation of active forms of oxygen by carrageenan, or indirectly, via the tumor-alpha necrosis factor [9]. the fundamental of carrageenan influence on the body is the development of oxidative stress as one of the mechanisms of damage of intestine as well as the major multiple organ lesions in heart, lungs and liver. therefore, the aim of our study was to investigate the level of apoptosis and necrosis in the suspension of leukocytes in rats using 0.5 % and 1.0 % solutions of carrageenan. methods the study was conducted on 36 mature white nonlinear male rats, which were kept on a standard diet at the vivarium of i. horbachevsky ternopil state medical university. during the study we followed the principles of the european convention for the protection of vertebrate animals used for experimental and other scientific purposes (strasbourg, 1986). the rats were divided into 1 control and 2 experimental groups: the 1st group – control (intact animals); the 2nd group comprised the animals that consumed 0.5 % solution of carrageenan, the 3rd group consisted of the animals that consumed 1.0 % solution of carrageenan. the 2nd and the 3rd groups of animals were provided with free access to 0.5 % solution of carrageenan and 1.0 % solution of carrageenan in drinking water for 1 month [10, 11]. a n n e x i n v ( v ) b i n d i n g a s s a y s w e r e performed using annexin v apoptosis kit (sigma aldrich, usa). apoptotic cells of blood leukocyte suspension were identified by flow cytometry using flow cytometer epics xl (beckman coulter, usa). to distinguish cells that had lost membrane integrity, propidium iodide (pi) was added to a final concentration of 10 mg/ml before the analysis. the results were presented as a percentage of the total number of cells as follows: live cells – not stained (v -/pi -), cells with early signs of apoptosis – stained with annexin (v+/pi-), cells with late signs of apoptosis – positive double fluorescence staining, cells with signs of necrosis – stained with propidium iodide (v-/ pi+). to determine caspase rate in leukocytelymphocyte blood fractions, 0.25 ml of buffer and 50 mcl of 2 mm devd-p-na was added to 0.7 ml of the test liquid. it was incubated for 2 hours at 37 °c; the intensity of light absorbance was measured at 405 nm, which was directly proportional to the product of hydrolysis of acetyl-asp-glu-val-asp n-nitroanilide caspase – 3-n-nitroanilide [12]. statistical analysis the results were analyzed using statistica 7.0 software and presented as mean with standard error of mean. the differences between all groups were determined using one-way anova, followed by post hoc the least significant difference test. a p-value <0.05 was considered statistically significant. results it was established that with the experimental application of carrageenan, the percentage of leukocytes with signs of apoptosis in both experimental groups increased significantly (table 1). thus, the percentage of v+/pi--cells in the 2nd group increased in 1.9 times, and in group 3 – in 2.2 times vs the control indexes (p<0.001). the percentage of leukocytes with later signs of apoptosis increased significantly, with respect to control: in group 2 – in 8.9 times, in group 3 – in 22.3 times (p<0.001). it should be noted that the level of necrotic cells when introducing 0.5 % ca rra geena n did not significantly differ from the normal indices, while the use of 1.0 % solution of carrageenan in drinking water caused the increase of v-/pi+ -cell in 1.7 times (p<0.001). с a s p a s e s i n g e n e r a l a r e i m p o r t a n t mediators in apoptosis, especially caspase-3, which is the main caspase effector that cleaves cell substrates. it was established that the activity of caspase-3 effector in 1 month of the experiment increased in 1.5 times in the 2nd group and in 2.8 times in the 3rd group vs control data that proved the caspase-dependent apoptotic pathway in case of carrageenan use for rats (fig. 1). discussion caspase-3 is probably the best understood of the mammalian caspases in terms of its specificity and roles in apoptosis. overall, recent progress has generally confirmed the notion of multiple, complex death pathways (some of which require caspase-3 in specific cell types) that converge on common events including cell shrinkage, blebbing, chromatin condensation and dna. two established ways of apoptosis include internal or mitochondrial, involving protein family bcl–2, cytochrome с and caspase – 9 and external with the activation of caspase–8 linking a specific cell receptor fas– and soluble tumor necrosis factor receptors on the cell o. m. kopanytsia et al. 69 b io m e d ic a l s c ie n c e s issn 2413-6077. ijmmr 2018 vol. 4 issue 1 o. m. kopanytsia et al. table 1. indicators of cell death in serum of the rats in experimental use of carrageenan me (q25-q75) index control 2nd group 3rd група alive leukocytes, % 95.76 (95.28; 96.77) 90.05* (89.26; 90.43) 83.87*# (82.62; 86.11) leukocytes with early signs of apoptosis, % 3.05 (2.20; 3.68) 5.89* (5.55; 6.20) 6.73*# (5.83; 7.55) leukocytes with late signs of apoptosis, % 0.36 (0.10; 0.60) 3.19* (2.78; 3.65) 8.02*# (6.75; 8.73) leukocytes with signs of necrosis, % 0.83 (0.65; 0.98) 0.86 (0.73; 0.98) 1.39*# (1.20; 1.61) notes: * – the difference between the control and the experimental group is statistically significant (р<0.05-0.001) # – the difference between the 2nd and the 3rd study groups is statistically significant (р<0.05) fig. 1. caspase-3 level in case of carrageenan intoxication (* – significant difference compared with the control group, # – significant difference compared with the experimental groups). 0 50 100 150 200 250 300 control group 2 group 3 * *# % surface [13]. caspase-3 is the most involved pathway which should be generated from its inactive protein (procaspase-3), caspase 3 is required for some apoptosis features (chromatin condensation, dna damage and apoptotic body formation) and its part may take place before cell viability suppression starts [14]. hridneva sv notes that in chronic enterocolitis endothelial functions are impaired, which manifests itself in the activation of free radical oxidation processes with underlying decrease in the activity of the antioxidant system that explains the excessive production of ros [15] and cell death. the activity of caspase-3 increases with increased carrageenan concentration, that proves a more obvious enterocyte apoptosis with the increase in daily intake of carrageenan. at the same time, animals of the 3rd group have a more obvious increase in the activity of caspase-3 in serum, indicating the increase in the severity of apoptotic processes in intestine. the obtained data contradicts the results of some studies that prove that exposure of human intestinal epithelial cells to carrageenan in vitro does not lead to the activation of caspase-3, caspase-7 or increased percentage of fragmented dna, suggesting no apoptotic alterations following carrageenan exposure [16]. otherwise, the results of other studies evidence that carrageenan induced chronic gastroenterocolitis is accompanied by the decrease in the activity of parp and elevation of mmp-2, mmp-9 and caspase-3 in blood serum of animals [17]. the results are controversial and require detailed consideration. therefore, further research is warranted to elucidate the role of carrageenan in intestinal caspase-depending cell death that may help define novel nutritional strategies for hindering the development of gut diseases. conclusions oral use of carrageenan in rats was accompanied by the increase of the number of leukocytes with signs of apoptosis: v+/pi-cells in the 2nd group increased in 1.9 times, and in the 3rd group – in 2.2 times, v+/pi+-cells increased in 8.9 and 22.3 times, compared with the control (p<0.001); the percentage of leukocytes with later signs of apoptosis was significantly increased too (p<0.001). animals that consumed 1.0 % solution of carrageenan had more obvious increase in the activity of caspase-3 in serum relative to the group of rats consuming 0.5 % carrageenan, proving the increase in the severity of apoptotic processes in intestine with the increase in the dose of carrageenan. 70 b io m e d ic a l s c ie n c e s issn 2413-6077. ijmmr 2018 vol. 4 issue 1 references 1. nechaev ap, kochetkova aa, zaytsev an. nutritional supplements. m.: kolosm; 2001. [in russian]. 2. flavoring substances and natural sources of flavourings. strasbourg; 1992. 3. smoliar vi. problems of using nutritional supplements in ukraine. abstracts of the international scientific and technical conference / nukht. k.: nauka; 1998. [in ukrainian]. 4. correc g, barabanova a, tuvikene r, truus k, yermak i, helbert w. comparison of the structures of hybrid κ-/β-carrageenans extracted from furcellaria lumbricalis and tichocarpus crinitus. carbohydrate polym. 2012;88:31-6. doi:10.1016/j. carbpol.2011.11.052 5. buldakov as. nutritional supplements. m.: deliprint, 2001. [in russian]. 6. nechaev ap, traubenberg se, kochetkova aa. food chemistry. spb.: giord, 2003. [in russian]. 7. osmanyan rg. prospects of using carrageenans in confectionery production (for the production of jelly sweets with functional properties. food and processing industry. abstract journal. 2008;2:432. 8. lisnianska nv, маrushchak mi, antony shyn iv, mialiuk op. chronic enterocolitis combined with streptozotocin-induced diabetes in rats: mechanism of oxidative stress development. international journal of medicine and medical research. 2017; 3 ( 2 ) : 5 9 6 3 . h t t p s : / / d o i . o r g / 1 0 . 1 1 6 0 3 / ijmmr.2413-6077.2017.2.8035. [in ukrainian]. 9. tkachenko as, gopkalov vg, martyinova sn. the method of correction of chronic carrageenaninduced gastroenterocolitis with antioxidant vitamins. bulletin of biology and medicine. 2015; 3(1):315-7. [in ukrainian]. 10. gubina-vakulyk gi, kolousova ng, ivanenko то, gorbach tv, korobchanskyy vo. method of modeling chronic gastroenterocolitis. patent of ukraine. № a201014510, 2012. [in ukrainian]. 11. moyana tn, lalonde jm. carrageenaninduced intestinal injury in the rat – a model for inflammatory bowel disease. ann clin lab sci. 1990; 20(6):420-6. 12. hug h, los m, hirt w, debatin k-m. rhodamine 110-linked amino acids and peptides as substrates to measure caspase activity upon apoptosis induction in intact cells. biochemistry. 1999; 38:13906-11. 13. illinska if. apoptosis, apocytosis and their role in the immune response (analytical review). lab diahnostyka. 2002;3:66-72. [in russian]. 14. mcilwain dr, berger t, mak tw. caspase functions in cell death and disease. cold spring harb perspect biol. 2013;5:a008656. 15. hridneva sv. the role of nitric oxide and lipoperoxidation processes in the development of chronic non-ulcerative colitis. modern gastroenterology. 2003;2:43-6. [in russian]. 16. gubina-vakyulyk gi, gorbach tv, tkachenko as. damage and regeneration of small intestinal enterocytes under the influence of carrageenan induces chronic enteritis. comp clin pathol. 2015; 24:1473. https://doi.org/10.1007/s00580-015-2102-3 17. tkachenko as, nakonechna oa, zhukov vi, gorbach tv, tkachenko mo. proteolytic degradation of poly (adp-ribose) polymerase in rats with carrageenan-induced gastroenterocolitis. journal of v. n. karazin’ khnu. series ’medicine’. 2017;34:41-46. [in ukrainian]. received: 2018-05-11 o.m. kopanytsia et al. карагінан спричиняє загибель клітин крові щурів о. м. копаниця, м. і. марущак, і. я. криницька тернопільський державний медичний університет імені і. я. горбачевського, тернопіль, україна вступ. завдяки своїм фізико-хімічним властивостям карагінан досить широко застосовується, а близько 70% усього виробленого в світі продукту використовується в харчовій промисловості. попередні дослідження вказують на те, що введення карагінану при моделюванні хронічного ентероколіту викликає розвиток оксидативного стресу. метою дослідження стало вивчення рівня апоптозу та некрозу в суспензії лейкоцитів у щурів при застосуванні 0,5% і 1,0% розчинів карагенану. методи. аналіз зв’язування анексину v (v) проводили з використанням набору sigma aldrich, сша; рівень каспази у суспензії лейкоцитів та лімфоцитів крові щурів визначали спектрофотометрично. результати. встановлено, що при застосуванні карагінану відсоток лейкоцитів з ознаками апоптозу в обох експериментальних групах статистично достовірно збільшувався. активність каспази-3 зростала в 1,5 рази у 2-й групі і в 2,8 рази в 3-й групі у порівнянні з контрольними даними, які вказують на каспазо-залежний шлях апоптозу клітин при введенні карагінанy. висновки. пероральне застосування карагінану в щурів супроводжувалося збільшенням кількості лейкоцитів з ознаками апоптозу. тварини, які споживали 1,0% розчину карагінану, мали більш значуще збільшення активності каспази-3 в сироватці відповідно до групи щурів, які споживали 0,5% каррагінану, що доводить дозо-залежний ефект апоптотичних процесів у кишечнику. ключові слова: карагінан; апоптоз; каспаза-3; щури. 34 su r g ic a l d is e a se s issn 2413-6077. ijmmr 2017 vol. 3 issue 1 doi 10.11603/ijmmr.2413-6077.2017.1.7291 chronic mycotic-associated surgical nail pathology complicated with ingrown nail (nail incarnation): the analyses of clinical cases and complex treatment a. r. vergun, b. m. parashchuk, m. r. krasnyy, z. m. kit, o. m. vergun danylo halytsky lviv national medical university, lviv, ukraine background. conservative treatment of secondary recurrent unguis incarnatus are not very effective and dupuytren’s method, emmert-schmiden surgeries etc. are very traumatic, disfigure nail bone, distort anatomic and functional unity of a nail. objective. the aim of our research was the optimal sequence of holiatry, surgical moving away of nails, local and system therapy after excision of the staggered nails in case of destructive onychomycosis complicated by secondary ingrown nail for some patients with complicated mycotic defeat of nails. clinical options of surgical treatment were clarified; morphologic changes were studied; causes of unsatisfactory outcomes of chronic pathology complex treatment were analyzed for prospective approaches to preventing of relapses. methods. the analysis justifies the feasibility of establishing of predictive relationships between clinical variants of chronic purulent necrotic infections and combined comorbidity. system therapy by itraconazole before operative treatment (basic onychial defeats sanation) and in a postoperative period was carried out. types of operative treatment applied may be divided into five statistic groups. results. over a five-year period (2010-2015) 436 unguis incarnates diagnosis (325 cases of incarnated onychomycosis) in 259 men and 177 women aged 12-67 were performed, 38 of them with incarnated onychogryphosis had diabetes mellitus and 24 had metabolic syndrome. analysis of subonychial scraping allowed stating the prevalence of red trichophytia in 31% of cases with the bacterial flora. patients with combined pathology got 4-5 five-day system ‘pulses’ of 400 mg/day itraconazole therapy. in patients with primary advantage of providing low-impact methods of nail excision with access via onycholisation structure a nail extirpation and marginal matricectomy was carried out; dermatophytoms and stratification on a nail bed were deleted. conclusions. type of onychectomy wound cytograms in the study group on the 10th day was defined as regenerative-inflammatory in 24.81%, regeneratory in 75.19% (p=0.031). the methods of surgical treatment of mycotical recurrent nail incarnation have been improved considering pathoand morphogenetic properties of destructive onychomycosis. key words: destructive onychomycosis; secondary recurrent ingrown nail; antimycotic therapy; surgical nail removal. introduction dermatophytes, infecting a nail matrix, were determined as the dystrophic changes of nail and a subnail hyperkeratinization and dermatophytoms [2, 3], surgical nail pathology, that compress a nail that is the driving nosotropic member of the secondary ingrowing and has an influence on curative tactics [4], in particular on the necessity of the surgical moving away [8, 21]. the pathogenetic links leading to increase of the probability of occurrence and development of feet mycosis and onychomycosis for diabetic patients include the pathology of cardiovascular and nervous systems [10], disruption of glycolysis, resulting in lower energy supply of skin cells and changes in metabolism, skin dysfunction, determining rapid progression and chronic mycosis [18, 20]. the objective was to study certain peculiarities of mycosis-associated pathology and treatment, including surgical removal of nails in patients with onychogryphosis and recurrent ingrown with underlying diabetes mellitus [20]. the results of determining of vitamins в1, в2, рр, в6 and с in blood and their derivatives are analysed [22]. some 50-year-old patients, who have high mycotic surgical nail pathology, have corresponding author: andriy vergun, department of outpatient care, family medicine and dermatology, venerology, danylo halytsky lviv national medical university, 1 konovaltsya street, lviv, ukraine, 79013 phone number: +380322757632 e-mail: plagiamail@meta.ua a. r. vergun et al. international journal of medicine and medical research 2017, volume 3, issue 1, p. 34–40 copyright © 2017, tsmu, all rights reserved 35 su r g ic a l d is e a se s issn 2413-6077. ijmmr 2017 vol. 3 issue 1 a. r. vergun et al. the endothelial dysfunctions and polyvitamin disturbances that are some factors from clinical corrections [10, 12, 22]. the study involves peculiarities of treatment, some indicators of lipid exchange of patients with diabetes mellitus [9, 10, 18, 20], metabolic syndrome accompanied by destructive onychomycosis and secondary nail ingrowth. clinical options of in-patient and out-patient surgical treatment for nail ingrowth (mono-lesions; complicated and combined mycotic-associated processes and relapses) were clarified; morphologic changes were studied; causes of unsatisfactory outcomes of chronic pathology complex treatment were analysed for prospective approaches to preventing relapses. if the infection involves the eponychium as well as the lateral fold, it is called eponychia [15, 17]. its extension to the opposite side of the fingernail, which is uncommon, is called runaround abscess. in these cases, the paronychia is compressed along the nail edge, trapping the abscess. all procedures that successfully treat paronychia separate it from the hard nail [23, 24]. if the infection is limited to less than onehalf of the eponychium, a single incision placed to drain the paronychium and to elevate the eponychial fold for excision of the proximal onethird of the nail is satisfactory. if the entire eponychium is involved, two incisions are required. the relevance of the problem of the ingrown nail (onychocryptosis, unguis incarnatus) in an urgent outpatient purulent surgery is caused by the increased frequency of its occurrence, chronicity, complications, not uncommon early and late postoperative relapses [8, 15, 17]. conservative and orthopedic treatment of incarnatus surgical nail pathology are not very effective while dupuytren’s method, emmert-schmiden surgeries etc. are very traumatic, disfigure nail bone, [4, 5, 21, 24] distort anatomic and functional unity of a finger and in 2–20% of cases (depending on absence or presence of onychocryptosis and fungal agents) cause a relapse. the clinical features and ways to optimize the treatment of patients with onychomycosis and destructive secondary ingrown nails are clarified in the article. the results of complex treatment of the patients with nail trichophytosis, associated with ingrown toenail, submitted according to dependence on a nail plate and eponycheal changes are presented in the publication. the aim of our research is the optimal sequence of holiatry, surgical moving away of nails, local and system therapy after moving away the staggered nails in cases of destructive onychomycosis complicated by the secondary ingrown nail for some patients with the complicated mycotic defeat of nails. methods certain peculiar features of the clinical course and comprehensive treatment were studied, including surgical removal of the nails in patients with onychogryphosis associated with an ingrown nail (onychocryptosis, nail incarnation). results of ingrown nail surgical correction were studied to improve the results of complex treatment. over a five-year period (2010–2015) 436 unguis incarnates diagnosis (325 cases of incarnated onychomycosis) in 259 men and 177 women aged 12–67 were performed. in 182 patients late relapses of onychocryptosis were confirmed after previous surgeries at other clinics. conservative treatment was recommended only at early stages of ingrowth. removal of the affected nails was performed for patients with mycotic lesions (local and systemic fungicide therapies were used). investigation of the morphogenesis of destructive aspect of the mycotic lesions was carried out. a variety of factors, aetiology, and pathogenesis of chronic purulent necrotic lesions of the foot, contributing to the occurrence, progression and recurrent course of these diseases, creates objective difficulties of diagnosis [14, 16, 19]. the analysis justifies the feasibility of establishing predictive relationships between clinical variants of chronic purulent necrotic infections and combined comorbidity [1, 25]. system therapy by itraconazole in operative treatment (basic onychial defeats sanation) and in a postoperative period was carried out [12, 25]. the applied types of operative treatment of surgical nail pathology may be divided into five main groups: 1 – emmertschmiden type surgeries (marginal excision of nail plate and eponychia with marginal removal of the growing part via partial matricectomy); 2 – dupuytren’s type surgeries (onychectomy – complete removal of nail plate); 3 – bartlett type surgeries (local tissue plastic re construction); 4 – marginal resection of marginal section of nail plate; 5 – meleshevych surgery; 6 – our modifications (with previous block-type eponychectomy). results over the five-year period we examined and treated 98 patients of 52–86 years old with in36 su r g ic a l d is e a se s issn 2413-6077. ijmmr 2017 vol. 3 issue 1 carnated onychogryphosis (fig. 1): 67 men (68.37%) and 31 women (31.63%) of the examined patients, 38 of them had diabetes mellitus and 24 had metabolic syndrome. three variants of dermatophytoma are differentiated: front centre, with up to 25% of eroded nail – 45 cases, subtotal, from 25 to 70% (without capturing the growth plate) – 38 cases, total from 70 to 90% (with affected growth plate of the nail) – the other 15 cases. in all cases, dermatophytoma affected distal and central part of the nail bed. analysis of subonychial scraping allowed stating the prevalence of red trichophytia [3, 4]: in 74% of cases it was associated with mold, in 26% of cases – with yeast fungi; in 31% of cases – with bacterial flora [1]. conglomerate of nail plate and subungual hyperkeratosis and trichophytosis calcinated completely, forming onychogryphosis with deformation [6, 7] (fig. 2) and secondary recurrent ingrown nail [15, 16, 22, 23]. in patients with onychomycosis, especially in severe destructive forms of subungual hyperkeratosis, large deterioration of microcirculation was noted. rheographic prevailed spastic type curves (p<0.01) [9, 10]. index of open capillaries was reduced by 31%. patients in both groups mainly were affected with hallucis on left foot – in 188 people, the other – on right foot (133 patients), the presence of pathological ingrown nail plates of hallucis of both feet [12, 15, 17] and otherfingers were examined in other patients. mycotic associated hyponychial panaritium was diagnosed in 16 patients with onychogryphosis (16.33%), purulent paronychia was diagnosed in 11 patients (11.22%), the other 5 patients of this group suffered from eponychial abscess (5.10%). patients of the main group underwent a three-day adjuvant systemic fungicide therapy: 400 mg itraconazole daily, during 4 days (the first 2 postoperative days) as pulse therapy. a similar dose at weekly intervals was carried out following five-day 2–3 cycle pulse [23, 25]. removal of the affected nails for patients with polyonychomycosis was performed in successive stages at add-back of certain systemic ‘pulses’ with itraconazole. patients with such combined pathology got 4–5 five-day system ‘pulses’ of 400 mg/day itraconazole therapy together with a simultaneous use of hepatoprotectors and correction of comorbid pathology [21–23]. provided adequate surgical treatment, in addition to standard decompression stage (complete removal of the nail plate), contained antirecurrent component to prevent from repeated ingrowth. along with antimycotic therapy and correction of comorbid pathology the following procedures were carried out: cutting pathologic eponychial tissues, hypergranulations and necroses [22, 24] (fig. 3); removing nail plate with partial marginal mafig. 1. trichophytial polyonychomycosis, onychogryphosis. clinical case. 81-year-old woman. fig. 2. total nail dermatophytosis (tr. rubrum), polyonychom ycosis. big polyonychial gryphosis. clinical case. 82-year-old man. fig. 3. surgical nail pathology, subnail subungual hyperkeratosis and dermatophytosis, big dermatophytoma, hypergranulations and local necroses. intraoperation photo. trichophytial polyonychomycosis, incarnated onychogryphosis. clinical case. 81-year-old woman. a. r. vergun et al. 37 su r g ic a l d is e a se s issn 2413-6077. ijmmr 2017 vol. 3 issue 1 a. r. vergun et al. trixectomy in the ingrowth area [2, 3, 20]. noninvasive methods of nail excision and marginal nail resection were preferred for patients with diabetes mellitus. during the five-year period we examined and treated 38 patients with onychogryphosis and diabetes mellitus type 2. we provide the research on the characteristic features of the pathological process to create the optimal scheme of complex treatment for patients with abnormal ingrowth of nail plate with underlying diabetes mellitus type 2 [9, 18]. 23 patients with ingrown onychogryphosis and underlying diabetes mellitus, diabetic micro and macroangiopathy (prospective material, the treatment group), and onychogryphosis and recurrent incarnation of toenail (pathology being clinically dominant and manifesting through pain syndrome) and multiple destructive mycotic lesions of other nail plates of both feet: 14 men and 9 women, 55–80 years old were involved in the surgery. the other 15 people with onychogryphosis of the first toe (hallux) and fungus of other nails constituted the control group. the duration of clinicallymanifesting nail mycotic process in all studied cases exceeded 5 years. mycosis-associated acute purulent pathology [19, 21, 23] was confirmed in 13 patients (34.21% of the examined, 52% cases) of the treatment group and in 5 patients of the control group (13.16% of the examined, 30% cases) correspondingly. systemic adjuvant pulse therapy with 400 mg itraconazole was applied during two days before the initial surgical treatment and during the first three days of postoperative period [19]. remediation of other affected nails in order to prevent from mycosis reinfection was carried out with antimycotic amorolfine 5% lacquer and ciclopirox 8% nail lacquer solution [2, 11–13, 22]. removal of other nails affected by hyperkeratosis with trichophytosis was performed through onycholysis by separate successive stages, where certain ‘pulses’ of therapy were supported with itraconazole [19, 21]. we prospectively examined 93 patients with metabolic syndrome [9, 10], associated with surgical nail pathology, destructive onychomycosis and secondary ingrown toenails. all patients underwent a comprehensive treatment of comorbid pathology, corrected using the systemic antimycotic therapy and terbinafine antifungal liniments, ciclopirox nail lacquer [11, 13] and surgical methods for removing nail plates [8, 9, 12, 22]. we studied lipid metabolism that manifested biochemical change indicators in the lipid profile, as well as their relations, which were significantly higher in both groups of patients (the main and control group), p<0.01 for both groups; we evidenced the positive correlation between the level of total cholesterol and leptin (p<0.01). the concentration of highdensity lipoprotein cholesterol in patients of the main group was 1.12±0.06 mmol/l compared with patients in the control group – 1.07±0.06 mmol/l. the average content of nitrogen oxide in the study group (metabolic syndrome) was higher than in the healthy patients – 15.06±0.97 mcmol/l, p<0.05. significant decrease of homa-index of β-cell function and increase of homa-index of insulin resistance [18, 20] (10.21±1.9 in the treatment group and 4.12±1.12 in the control group, p<0.01) was detected in patients with underlying diabetes mellitus type 2 (the treatment group) with polyonychomycosis and trichophytosis onychogryphosis, and recurrent incarnation of the nail edge. high total cholesterol over 5.18 mmol/l was detected for all patients of the main group, i.e., 7.28±0.07 mmol/l, and 5.45±0.12 mmol/l in the half of the control group; the deviation of laboratory parameters of low and high density lipoprotein cholesterol was also confirmed. polyonychomycosis and big trichophytic subungual hyperkeratosis with secondary nail ingrowth [6-9] were diagnosed in the main group comprising 62 patients with type 2 diabetes mellitus (48 males and 14 females, aged 42-65). metabolic syndrome was confirmed in the comparison group comprising 53 patients with arterial hypertension and complicated mycotic nail damage. the rest 276 patients constituted the control group. we studied biochemical findings, lipid blood spectrum: total cholesterol, high-density lipoprotein cholesterol, low-density lipoprotein cholesterol; nitric oxide of blood plasma; leptin [9]. lipid blood spectrum and nitric oxide level [10] were determined before and after pharmacotherapy along with continuous antihypertensive therapy (correction of intercurrent and comorbid pathology). the objective criterion of insulin resistance was the homa-ir index (the homeostasis model assessment) involving glucose and insulin levels on an empty stomach divided by 22.5 coefficient. patients of the main and the comparison groups with polyonychomycosis and trichophytic subungual hyperkeratosis with secondary ingrown nail experienced considerable decrease of homa-index of β-cells function and increasing homa-index of insulin resist38 su r g ic a l d is e a se s issn 2413-6077. ijmmr 2017 vol. 3 issue 1 ance (8.11±1.1 in the main group, 5.89±2.1 in the comparison group and 2.23±1.18 in the control group, p1=0,01; p2=0.01). considerable positive correlation between all indicators of carbohydrate exchange was identified in groups comprising patients with type 2 diabetes mellitus: glucose and insulin (r=0.51; p=0.01), and the homa-index (r=0.70; p=0.01), and glycolyzed haemoglobin (r=0.75; p=0.001); insulin and the homa-index (r=0.73; p=0.01) and glycolyzed haemoglobin (r=0.65; p=0.01); the homa-index and glycolyzed haemoglobin (r=0.67; p=0.01). considerable increase of circulating insulin was identified directly after surgical treatment (in the main group and the comparison group correspondingly 15.33±0.23 and 8.24±1.18 mkmo/l, p=0.01), the homa-index of insulin resistance (p=0.05) and the homa-index of β-cells function (p=0.05) in comparison with the control group with further tendency to some decrease in the process of treatment. we determined disturbances in lipid exchange, insulin resistance, lipid blood spectrum changes that were considerably higher in both groups of patients (the main and comparison groups), p=0.01 for both groups in comparison with the control group. discussion on one hand the subungual hyperkeratosis and dermatophytosis caused compression of the central part of the nail, epionychium edges ‘ingrew’ to periungual walls thus recurrent ingrown nail was formed; on the other hand, constant compression caused destruction of central part of nail bed; this process was typical for 32 (84.32%) cases. methods of surgical treatment of uncomplicated onychogryphosis [6, 7] and onychogryphosis complicated with recurrent nail incarnation [8, 15] were improved considering pathoand morphogenetic properties of destructive onychomycosis [16, 17]; removal of the mycotic affected nails of these patients should reasonably be conducted through onycholized structures with simultaneous removal of dermatophytoma, hyperkeratosis, and ingrowth areas with hypergranulation. surgical treatment was performed according to the standard algorithm due to patholo gical eponychial changes in the patients with primary advantage of providing low-impact methods of nail excision with access via onycholisation structure and wedge resection of the nail [2, 20]. when combined incarnation of onychomycosis and acute eponychial abscess comply with disclosure abscess, excision of abnormal tissue eponychial hypergranulation and focal necrosis, and removal of the nail plate, enlarged partial marginal matrixectomy in the ingrowth area are performed. when combined with onychomycosis and nail incarnation acute eponychial abscess, an autopsy ulcer, excision of abnormal tissue eponychial hypergranulation and focal necrosis, and removal of the nail plate, enlarged partial marginal matrixectomy in the ingrowth area are made. other patients (the control group) underwent a typical nail removal – operations such as dupuytren's: complete removal of the nail plate under the guise of ‘classical’ pulse therapy by itraconazole and terbinafine [13, 21]; in 45 cases this intervention combined with simultaneous excision of the modified cuticle and plastic – operations such as bartlett (plastic local tissue) and meleshevich. the processes of destruction of the nail in patients with trichophyton onychomycosis and type 2 diabetes mellitus / metabolic syndrome is much faster and is characterized by a more pronounced morphological variants of mycotic nail destruction that determine the occurrence of secondary ingrowth and attachment with intercurrent flora with the emergence of dermatophythoma with centres of decay and necrotic foci in the nail bed. a nail extirpation and partial marginal matricectomy mechanical carving and diathermocoagulation with the further scraping off by the folkman’s spoon were carried out; dermatophytoms and stratification on a nail bed were deleted. sanation of other nails for prevention of mycotic reinfection was carried out by ciclopirox nail lacquer [11]. application of system enzymic protheolitical therapy allows to considerably improve the primary results of holiatry of the complicated subnail hyperkeratinization, diminish a perifocal edema and inflammation; stimulate the necrolitical and reparative processes in an operating wound that clinically shows up to the accelerations of its granulation and epithelization; accelerate cicatrization of surface, abbreviate the terms of temporal non-operability. for patients with type 2 diabetes mellitus (the main and comparison groups) with polyonychomycosis and dermatophytoms as well as secondary ingrown nail, subnail hyperkeratinization were performed for certain significant [20] reduction of the homaindex of function and the increase of the homaindex of insuline resistance. the chart of holiatry applied proved the effectiveness of treatment of bad resistance cases of destructive a. r. vergun et al. 39 su r g ic a l d is e a se s issn 2413-6077. ijmmr 2017 vol. 3 issue 1 a. r. vergun et al. poly onychomycosis by a subnail hyperkeratinization and secondary ingrown nail, in particular for patients with the type 2 diabetes mellitus [9]. arguing that the removal of the nail plate in the patients with destructive onychomycosis with secondary incarnation nail advantageously carried out through onycholisation structure with simultaneous correction of pathological bed changes and cuticle, which in combination with antifungal treatment provides positive dynamics of regenerative type cytologic picture and shorter healing onychectomy wounds in 18 – 27 days to 12-25 days, with good early and long-term results. the type of transaction cytograms of onychectomy wounds in the study group on the 10th day of post-operative period is defined as the regenerative-inflammatory in 24.81%, as regenerative in 75.19% (p=0.031). in these embodiments, in the control group the indicators were respectively 53.12% and 46.88%. the advantage of regenerative option of cytological picture proves the correct choice of treatment strategy and accelerates wound healing after removing nail in patients of the main group. the factors that influence the occurrence and progression of incarnations, and disease recurrence after surgical treatment require further study [3, 4]. we have studied the results of onychocryptosis surgeries of surgical type 1 correction in 84 patients, type 2 – in 66 patients, type 3 – in the other 50 cases, type 4 – in 42 persons, type 5 – in another 27 patients, type 6 – in 56 patients (with the use of our modification of surgical treatment). three types of trichophytosis have been differentiated: frontal central – with erosion of up to 25% of nail area, subtotal – from 25 to 70% (without touching upon growth area), total – from 70 to 90% (with affected growth area). in 65 mycotic trichophitis patients with secondary nail incarnation a standard itraconazole pulse therapy was applied. the presence of onycholytic focuses and degradation of hyperkeratotic areas which result in lamination of a part of nail plate proves the feasibility of performing low-trauma onychectomy for patients with trichophyton onychomycosis with secondary incarnation via onycholized structure with the single-stage sequential removal of dermatophyte and ingrowth areas with changed eponychial folds. relapse causes after meleshevych, emmert-schmiden, bartlett surgeries were technical faults of surgical tools, intraoperative nail bed trauma, faults of postoperative anti-relapse treatments, surgical area trauma, wearing tight shoes, non-compliance with doctor’s recommendations for correction of orthopaedic pathology, onychomycosis. we believe that the less traumatic removal of nails through onycholysis should be prefered, particularly after such treatment, the patients with diabetes mellitus experienced healing time of operative wound (crust formation) during 16-23 days (average healing duration is 19 days) and had the indices tend to the control group; indices of the patients with diabetes and ‘classical’ nail removal (onicectomia) were normal in 24-30 days (average healing duration is 26 days), indices of the control group – in 14-22 days (average healing duration is 18 days). conclusions in all cases of mycotic onychocryptosis (secondary ingrown toenail) the patients underwent a comprehensive treatment of comorbid pathology corrected by the systemic antimycotic therapy and terbinafine antifungal liniments, ciclopirox nail lacquer and surgical methods for removing nail plates, supplemented the eponychial resections and partial marginal matrіxectomy. system therapy of itraconazole before operative treatment (basic onychial defeats sanation) and in a postoperative period was carried out. patients with combined pathology got 4-5 five-day system ‘pulses’ of 400 mg/day itraconazole therapy. in patients with primary advantage of providing low-impact methods of nail excision with access via onycholisation structure a nail extirpation and partial marginal matricectomy mechanical carving and diathermocoagulation with the further scraping off by the folkman’s spoon were carried out; dermatophytoms and stratification on a nail bed were deleted. sanation of other nails for prevention of mycotic reinfection was carried out by ciclopirox nail lacquer. methods of surgical treatment of uncomplicated onychogryphosis and onychogryphosis complicated with recurrent nail incarnation have been improved considering pathoand morphogenetic properties of destructive onychomycosis; removal of the mycotic affected nails of these patients should reasonably be conducted through onycholized structures with simultaneous removal of dermatophytoma, hyperkeratosis, and ingrowth areas with hypergranulation. type of onychectomy wounds cytograms in the study group on the 10th day of the post-operative period was 40 su r g ic a l d is e a se s issn 2413-6077. ijmmr 2017 vol. 3 issue 1 proved as the regenerative-inflammatory in 24.81%, regeneratory in 75.19% (p=0.031). the chart of holiatry applied proved the efficiency of treatment of bad resistance cases of destructive polyonychomycosis by a subnail hyperkeratinization and secondary ingrowing toenail; the less traumatic removal of nails through onycholysis should be preferred, particularly after such treatment the patients with the type 2 diabetes mellitus experienced healing time of operative wound (crust formation) during 16-23 days and the indices tend to the control group. references 1. adamski z, kaszuba a. dermatology for cosmetologists. wroclaw: elsevier urban & partner; 2011;186. 2. baran r, feuithade m, datry a. a randomized trial of amorolfine 5% nail lacquer combined with oral terbinafine compared with terbinafine alone in the treatment of dermatophytic toenail onychomycoses affecting the matrix region. br j dermatol. 200;142:1177–83. 3. blaszczyk-kostanecka m, wolska h. dermatology in practice. warsaw: medical publishing house pzwl; 2009;230. 4. brillowska-dabrowska a, saunte dm, arendrup mc. five hour diagnosis of dermatophytenail infections with specific detection of trichophyton rubrum. j clin microbiol. 2007;45(4):1200–1204. 5. brotzman sb, wilk ec. orthopedic rehabilitation. wroclaw: elsevier urban & partner; 2007;2(1):120. 6. chang р. onychogryphosis. our dermatol online. 2011;2(4):227–28. 7. chang p, meaux t. onychogryphosis: a report of ten cases. skinmed. 2015;13(5):355–59. 8. chapeskie h, kovac jr. case series: soft-tissue nail-fold excision: a 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ck. toenail onychomycosis: an important global disease burden. journal of clinical pharmacy and therapeutics. 2010;35:497–519. 22. vergun ar. multivitamin failure in patients with complicated destructive nail mycosis; analysis of clinical observations. 7 th lviv-lublin conference of experimental and clinical biochemistry. abstract book. lviv. 2013. p. 176–77. 23. welsh o. vera-cabrera l, welsh e. onychomycosis. clinics in dermatology. 2010;28:151–59. 24. yang kc, li yt. treatment of recurrent ingrown great toenail associated with granulation tissue by partial nail avulsion followed by matricectomy with sharpulse carbon dioxide laser. dermatol surg. 2002;28(5):419–21. 25. yin z, xu j, luo d. a metaanalysis comparing long term recurrences of toenail onychomycosis after successful treatment with terbinafine versus itraconazole. j dermatol treat. 2012;23(6):449–52. received: 2017-02-21 a. r. vergun et al. 89 m e d ic a l e d u c a t io n issn 2413-6077. ijmmr 2018 vol. 4 issue 1 doi 10.11603/ijmmr.2413-6077.2018.1.8710 conventional minimum in copyright protection (the berne convention) j. barg axiom, wroclaw, poland background. intellectual property rights are present in our everyday lives to a huge extent. law of intellectual property is generally governed by national law, with general principles set out in international treaties. copyrights strictly protect only the expression of ideas, not the underlying ideas, procedures, methods of operation, or mathematical concepts themselves. berne convention was first signed in 1886 and to this day is one of the most important international treaties concerning copyrights and moral rights. objective. this paper aims to shortly explain the basic rights and privileges provided to the authors by the berne convention in its present version, i.e. paris act of july 24, 1971, amended on september 28, 1979. results. berne convention provides a „conventional minimum”, meaning that all members must provide at least the rights granted by the berne convention to the authors. however, each member can grant more rights to the authors. in article 7 berne convention regulates the term of protection of copyrights, which is the life of the author and fifty years after her death. moral rights, provided in article 6bis, were added in 1928 and grant the author a right to claim authorship of the work and the right of respect. article 10 of the berne convention provides “certain free uses of works”. conclusions. the freedoms granted include possibilities of making quotations and of using the work of someone else to illustrate for teaching purposes. however, in both cases, an indication of the source of the work is required. key words: copyrights; moral rights; the berne convention; fair use. international journal of medicine and medical research 2018, volume 4, issue 1, p. 89-93 copyright © 2018, tsmu, all rights reserved introduction intellectual property rights are present in our everyday lives to a great extent. law of intellectual property is generally governed by national law, with general principles set out in international treaties, and by international and regional organizations such as world trade organization, world intellectual property organization (wipo), or european union. the basic rights of authors are set out in the berne convention for the protection of literary and artistic works (the berne convention), which will be the baseline regarding the rights of an author, the protected works, as well as the general understanding of copyrights and moral rights of authors. one of the most important issues regarding copyrights is that they strictly protect only the expression of ideas, not the underlying ideas, procedures, methods of operation, or mathematical concepts themselves [1]. this basically means that when one describes a method used 1 copyright. official website of wipo, accessed on 24th october 2017: http://www.wipo.int/copyright/en/ . in her experiment, only the published description is copyrighted, not the method used to obtain the results (the method could be patented, if it fulfills the patent requirements). similarly, only the way in which the results are presented in a paper are protected by copyrights. second important issue concerning copyrights is the fact that generally an author does not have to apply to be granted copyrights and moral rights (according to the berne convention, as well as in the majority of jurisdictions). nevertheless, most countries provide a registration system in one form or another. such systems are usually voluntary and help with licensing and transferring copyrights and in solving various disputes [2]. the berne convention was first signed in 1886 and to this day is one of the most important international treaties concerning copyrights and moral rights. not only was the berne convention the first international treaty relating to copyrights, but as of april 13, 2017, it has 173 members and hence applies almost all around the world. none of the international treaties provide a formal definition of copyrights, but generally such rights can be said to be ‘a set of exclusive rights granted by a sovereign state to 2 ibidem. corresponding author: julia barg, contract associate i., axiom, 3, kazimierza wielkiego, wroclaw, 50-077, poland e-mail: julka.barg@gmail.com phone number: +48600896343 j. barg 90 m e d ic a l e d u c a t io n issn 2413-6077. ijmmr 2018 vol. 4 issue 1 an author of an original work, for a limited period of time, and within a limited area’. the berne convention leaves the issue of fixing the work into a tangible medium up to the national legislation of member countries [3]. this paper will shortly explain the basic rights and privileges provided to the authors by the berne convention in its present version, i.e. paris act of july 24, 1971, amended on september 28, 1979. author’s exclusive rights – conventional minimum as stated above, intellectual property rights are granted at national level, rather than at the international one. therefore, the berne convention provides only a so called ‘conventional minimum’. all the members must provide at least the rights granted by the berne convention to authors. however, each member can grant more rights to the authors who seek to protect their works within that member’s territory. from the rights granted to the authors by the conventional minimum, several are of greater importance with relation to publishing research and scholar papers. provided that the author is a citizen of a member to the berne convention, they must be granted as a minimum the following copyrights: right of translation [4]; right of reproduction [5]; rights of adaptation, arrangement and alteration [6]; as well as moral rights [7]. there are several other copyrights set out in the berne convention that relate to public recitation of literary works, concerning dramatic and musical works, as well as broadcasting [8]. the first of analyzed copyrights, the right of translation, gives the author an exclusive right to translate their original work or to give permission for someone else to translate it. the right of reproduction allows the author to grant the right to copy, reproduce, and distribute such copies of their work. moreover, the scope of this right granted by the berne convention is very broad as it contains not only currently available means of reproduction and distribution but also those not yet invented [9]. finally, the right of adaptation gives the author an exclusive 3 berne convention for the protection of literary and artistic works, article 2, paragraph 2, official website of wipo, accessed on 24th october 2017: http://www.wipo.int/treaties/ en/text.jsp?file_id=283698 . 4 ibidem article 8. 5 ibidem, article 9. 6 ibidem, article 12. 7 ibidem, article 6bis. 8 ibidem, articles: 11, 11bis, 11ter, 14, 14bis, 14ter. 9 guide to the berne convention for the protection of literary and artistic works (paris act, 1971), wipo, geneva, 1978, p. 54. right to change the form of their work, i.e. make a derivative work, which can be both an infringement of the primary work (if done by someone other than the author and without their consent) and an original work itself [10]. in article 7 of the berne convention the term of protection of copyrights is regulated. generally, copyrights are protected during the life of the author and fifty years after their death. as stated above, the members of the berne convention may provide more protection to the authors than is guaranteed in the convention but cannot breach the conventional minimum. hence, in many member countries the term of copyright protection is longer than the conventional fifty years, i.e. in poland, ukraine, usa and many other countries the copyright term was elongated to the life of the author and seventy years after their death [11]. in cases of joint authorship, the copyright is protected during the lives of all authors and fifty years after their deaths. here however, the fifty years starts to run after the death of the last surviving co-author [12]. copyrights and moral rights in common law, copyrights tend to cover only the economic rights of the author. on the other hand, in continental law the term ‘copyright’ includes both economic and moral rights alike. hence, the need to unify the protection of the authors and their rights was urgent. this was achieved by including the moral rights of authors into the conventional minimum of the berne convention. moral rights, provided in article 6bis, were added in 1928 and grant the author (i) a right to claim authorship of the work and (ii) the right of respect. moral rights are granted to the author “independently of the author’s economic rights” and exist even after the author transfers their economic rights [13]. moreover, moral rights are maintained at least until the expiration of author’s economic rights. they can also be exercised after the death of the author by “persons or institutions authorized by the legislation of the country where protection is claimed” [14]. the first of the moral rights granted by the berne convention, the right to claim authorship, can be exercised in many various ways. firstly, 10 ibidem, p. 76. 11 for more information on copyright term in various countries visit: https://en.wikipedia.org/wiki/list_of_countries%27_copyright_lengths (accessed on 25th october 2017) 12 berne convention, article 7bis. 13 guide to the berne convention, p. 42. 14 berne convention…, article 6bis, paragraph 2. j. barg 91 m e d ic a l e d u c a t io n issn 2413-6077. ijmmr 2018 vol. 4 issue 1 j. barg the author may place their name on their work and its copies, hence claiming the authorship of the work. secondly, the author may publish their work under a pseudonym or even anonymously. moreover, the author can change their mind and change their pseudonym or quit their secrecy. finally, the author can use their right in a negative way by refusing their name to be put on a work that is not theirs. the right to claim authorship is a significant issue when considering fair use and publishing the work. the second moral right is the right of respect or, alternatively, the right to integrity of the work. according to article 6bis of the berne convention, the author can “object to any distortion, mutilation or other modification of, or other derogatory action in relation to, the said work, which would be prejudicial to their honor or reputation” [15]. this right prevents anyone from changing the work in any way without the consent of the author, even if the author transferred their economic rights. therefore, a publisher or an editor may not delete any part from the work of the author, unless they have the author’s informed and explicit consent. even though a work is usually proofread before publication, the changes should be sent to the author for approval. depending on the jurisdiction in which copyright protection is sought, the courts may allow some changes to be made when the work is being adapted into a different medium [16]. one of the most important differences between the economic and moral rights of the author is the issue of transferability. economic rights are freely transferable, while in many countries moral rights are inalienable – they stay with the author, even after licensing or transferring copyrights, and after the death of the author. this means that the author can license or sell their rights of translation, reproduction, or alteration (among others) to someone else, but they may not be able to do such a thing with their moral rights [17]. with respect to their moral rights, the author can agree not to exercise them. in many jurisdictions such a waiver must be in writing to be binding. certain free uses of works of others on one hand, as authors we would like to be protected for our works, on the other – as 15 ibidem, article 6bis, paragraph 1. 16 cotter, thomas f., pragmatism, economics, and the droit moral, 76 n.c. l. rev. 1, 1997, https://cyber.harvard.edu/metaschool/fisher/integrity/links/articles/cotter.html (accessed on 26th october 2017). 17 guide to the berne convention…, p. 42. scholars we are aware of the necessity of accessing the works of others as well as for ours to be accessed by other researchers and authors. hence, in article 10 of the berne convention provides ‘certain free uses of works’. the freedoms granted include possibilities of making quotations and of using the work of someone elseto illustrate for teaching purposes. however, in both cases indication of the source of the work is absolutely required. the berne convention permits quotations of someone else’s work under certain limitations. firstly, the work must have been made lawfully available to the general public, i.e. published by the author, with the author’s consent, or by means of compulsory license before it can be quoted [18]. secondly, the quotation must be “compatible with fair practice” [19]. as this concept is not defined in the berne convention, what is or is not fair is left for the national courts to decide. generally, the courts will consider, among other factors, the proportion between the size of the quote and the work in which it is used [20]. thirdly, the purpose of the quotation is also analyzed. the berne convention does not specify the purposes for which the quote may be used, stating only that the extent of the quote should “not exceed that justified by the purpose” [21]. most courts assume that the purpose of the quote should be educational, scientific, analytical, or parodist. when utilizing this ‘free use’, the author must remember that their own work cannot be overwhelmed by the quotes. in practice it means that quotes should be put into an original work to support the ideas of the author. the resulting original work of the author cannot be a compilation of quotes, with few commentaries in between them. rather, it should be an original work of the publishing author supported by quotes of more established researchers. “use of works by way of illustration for teaching” is present in copyright laws of most countries. plainly put, the teacher may reproduce a work of someone else during her class to compare, contrast, and analyze this work. nevertheless, the teacher must still abide by the limitations provided for quotations, i.e. the extent of the reproduction during the class must be “justified by the purpose” and the use must be “compatible with fair practice” [22]. it is worth mentioning, that the word ‘teaching’ includes all levels of education, i.e. “educational 18 ibidem, p.58. 19 berne convention…, article 10, paragraph 1. 20 guide to the berne convention…, p.59. 21 berne convention…, article 10, paragraph 1. 22 berne convention…, article 10, paragraph 2. 92 m e d ic a l e d u c a t io n issn 2413-6077. ijmmr 2018 vol. 4 issue 1 institutes, municipal and state schools and pri vate schools” [23]. generally speaking, a professor may reproduce the work of someone else during their lecture, for example by showing a reproduction of a picture or a painting, or a fragment of a movie. moreover, the teacher may make copies of such works (photocopies of reproductions of pictures, articles, or a page from a textbook) to be analyzed during the class, provided it is ‘justified by the purpose’. note, that you absolutely cannot photocopy and distribute more of someone else’s work than is necessary to teach the class. the professor must clearly indicate the author and the source of each and every work used by the way of illustration for teaching, be it a graph, table, photo, or a sentence from someone else’s work. note, that even if the professor modifies another’s work, they still must provide the source of the original work and indicate the implemented changes. from the practical point of view, right to quote is very close in nature to the ‘use of works by way of illustration for teaching’ – both allow us to use the work of someone else to some extent. however, the most important purpose of copyrights is to protect economic interests of the author. hence, in a commercial lecture we use the right to quote with the purpose being scientific or analytical rather than edu cational. as indicated above, with that come certain restrictions which are stricter than in case of the second fair use – in most cases the author may quote someone else’s work in their commercial lecture (presen tation), but most likely will be prohibited from distributing photocopies of the part of the quoted work used in the lecture. no matter under which paragraph of article 10 one uses someone else’s work, the source and the name of the author must absolutely be mentioned [24]. hence, article 10 paragraph 3 of the berne convention restates one of the moral rights of authors. the requirement to in dicate the name of the author (when possible) supports the notion of the right to claim author ship. one should also note that the indication of the source may infringe the cited author’s second moral right. misrepresentation of the cited author’s ideas or putting their words out of context can breach their right to integrity of their work. conclusions as stated in the beginning of this article, the protection of copyrights, both economic and 23 guide to the berne convention…, p. 60. 24 berne convention…, article 10, paragraph 3. moral rights of the authors, is within the national law. therefore, the protections afforded to authors in public international law should be considered a set of minimum rights and privileges. the berne convention does not provide for an enforcement body or a court to solve the disputes that arise under copyright laws. on the contrary, in many articles the convention leaves the means of redress for copyright infringement to national legislation and courts of the country where protection is sought. for scholars and researchers there are some practical points that should be kept in mind. firstly, always properly indicate the source of your quote or even paraphrase, customarily including (at least) the name of the author, the title of their work, the publisher or the title of the journal, and the year of publication. there are several styles of citation – which one to use generally depends on the publisher or the university. one important tip regarding the style of citation: it should be uniform across the paper. secondly, no matter the situation, always, always read what you agree to. when scholars submit their papers for publishing, there usually are terms of use, privacy policy, disclaimers, waivers or alike to sign or mark with annotation “i have read and agree to the following (…)”. in fine print there can be hidden various waivers of copyrights, for example stating that the author licenses (or transfers) their copyrights to the publisher, or that they will not submit the paper to be published in another journal while the publication is pending in the current one or simply ever. moreover, there can also be statements indicating that the author will not enforce their moral rights regarding this paper. as mentioned above, in many jurisdictions moral rights are inalienable – no one can demand that the author transfers or disclaims their moral rights. in such a case, only the enforcement of moral rights (in a court of law) can be disclaimed when the moral rights are infringed. infringement can happen for example by not putting the name of the author on the work or changing the work in any way without the author’s consent. to s u m m a r i z e , w h a t ev e r w a i v e r s o r statements might be included in terms of use or publishing contract be sure to read it carefully before agreeing or signing. to be honest, it is a good idea to read a document thoroughly before signing it, whatever the circumstances. j. barg 93 m e d ic a l e d u c a t io n issn 2413-6077. ijmmr 2018 vol. 4 issue 1 j. barg references 1. copyright. official website of wipo, accessed on 24th october 2017: http://www.wipo.int/ copyright/en/ . 2. berne convention for the protection of literary and artistic works. september 9, 1886, as amended on september 28, 1979. official website of wipo. 2017: http://www.wipo.int/treaties/en/text.jsp?file_ id=283698. 3. guide to the berne convention for the protection of literary and artistic works (paris act, 1971), wipo. geneva. 1978. 4. cotter, thomas f, pragmatism, economics, and the droit moral, 76 n.c. l. rev. 1, 1997, https:// cyber.harvard.edu/metaschool/fisher/integrity/ links/articles/cotter.html. accessed on 26th october 2017. received: 2018-03-15 конвенційний мінімум захисту авторського права (конвенція берне) j. barg axiom, wroclaw, poland вступ. в нашому повсякденному житті, значною мірою, використовуються права інтелектуальної власності. як правило, закон інтелектуальної власності регулюється національним законодавством, а загальні принципи викладені в міжнародних договорах. авторські права суворо захищають лише вираження ідей, а не основні ідеї, процедури, методи роботи або самі математичні поняття. бернська конвенція була вперше підписана в 1886 році і до цього дня є одним з найважливіших міжнародних договорів про авторські та моральні права. мета дослідження полягає у короткому поясненні основних прав і привілеїв, наданих авторам бернською конвенцією у її нинішній редакції, паризький акт від 24 липня 1971 року, змінений 28 вересня 1979 року. результати. бернська конвенція передбачає «гарантований мінімум», що означає, що авторам повинні гарантуватися щонайменше права, надані бернською конвенцією. однак, їм також може надаватися більше прав. у статті 7 бернської конвенції регулюється термін захисту авторських прав, який є життям автора і п’ятдесят років після його смерті. моральні права, передбачені статтею 6bis, були додані в 1928 році, і надають авторові право вимагати авторські права на власний твір/наукову роботу та право на повагу. стаття 10 бернської конвенції передбачає “безсумнівне вільне використання творів”. висновки. надані свободи включають в себе можливості цитування і використання наукових доробків інших авторів в навчальних цілях. проте в обох випадках обов’язково повинно вказуватися першоджерело. ключові слова: авторські права; моральні права; бернськa конвенція; використання прав. issn 2413-6077. ijmmr 2017 vol. 3 issue 1 53 p u b l ic h e a lt h a n d e p id e m io l o g y doi 10.11603/ijmmr.2413-6077.2017.1.7804 operations throughput as a determinant of golden-hour in mass-gathering medicine i. d. khan1, b. asima2, s. a. khan2 army college of medical sciences and base hospital, new delhi, india1 resident nuclear medicine, army hospital research and referral, new delhi, india2 background. golden-hour, a time-tested concept for trauma-care, involves a systems approach encompassing healthcare, logistics, geographical, environmental and temporal variables. golden-hour paradigm in massgathering-medicine such as the hajj-pilgrimage entwines along healthcare availability, accessibility, efficiency and interoperability; expanding from the patient-centric to public-health centric approach. the realm of massgathering-medicine invokes an opportunity for incorporating operations-throughput as a determinant of goldenhour for overall capacity-building and interoperability. methods. golden-hour was evaluated during the indian-medical-mission operations for hajj-2016; which established, operated and coordinated a strategic network of round-the-clock medical operations. throughput was evaluated as deliverables/time, against established standard-operating-procedures for various clinical, investigation, drug-dispensing and patient-transfer algorithms. patient encounter-time, waiting-time, turnaroundtime were assessed throughout echeloned healthcare under a patient-centric healthcare-delivery model. dynamic evaluation was carried out to cater for variation and heterogeneity. results. massive surge of 394 013 patients comprising 225 103 males (57.1%) and 168 910 females (42.9%) overwhelmed the throughput capacities of outpatient attendance, pharmacy, laboratory, imaging, ambulance, referrals and documentation. there was a delay in attendance, suspicion, diagnosis and isolation of patients with communicable infections. the situational-analysis of operations-throughput highlights wasted turnaround-time due to mobilization of medical-team, diverting critical healthcare resources away from emergency situations. conclusions. time being a crucial factor in the complexity of medical-care, operations-throughput remains an important determinant towards interoperability of bottlenecks, thereby being a determinant of golden-hour in mass-gathering-medicine. early transportation of a patient to definitive-care reduces treatment initiation-time, notwithstanding logistics of communication, evacuation, terrain and weather being deterministic in outcome. golden-hour needs to be emphasized under a population-based approach targeting the clientele towards administering first-aid and reaching out to hospital within the golden-hour. key words: golden-hour; operations throughput; mass-gathering medicine; turnaround-time; definitive-care; population-based approach. corresponding author: inam danish khan clinical microbiology and infectious diseases, army college of medical sciences and base hospital, new delhi 110010, india phone number: +91 9836569777 e-mail: titan_afmc@yahoo.com introduction golden-hour, a hitherto time-tested concept for trauma-care, has been found useful across the entire ambit of emergency health-systems. golden-hour involves a systems approach encompassing healthcare, logistics, geographical, environmental and temporal variables. mass-gathering-medicine applies to situations where a mass-gathering overwhelms accessibility, interoperability and public-safety response to medical-emergencies. mass-gathering-medicine involves higher rates of morbidity and mortality attributable to infections, trauma, environment, occupation, lifestyle, substance-abuse and disasters [1, 2]. hajj pilgrimage is a 5-day outdoor unbounded peaceful mass-gathering involving a moving assemblage of over 3.5 million pilgrims from 200 countries, engaged in prayers, supplications and strenuous rituals in densities of 9 people/ m2 or more, in harsh desert climate of saudiarabia. mass-gathering-medicine at hajj is challenged by issues of healthcare availability, accessibility, infection control, rapid-diagnosis, on-site treatment, referral, evacuation, and response to disasters and public-health emergencies [3, 4]. the realm of mass-gathering-medicine invokes an opportunity for incorporating operations-throughput as a determinant of goldeninternational journal of medicine and medical research 2017, volume 3, issue 1, p. 53–59 copyright © 2017, tsmu, all rights reserved i. d. khan et al. issn 2413-6077. ijmmr 2017 vol. 3 issue 154 p u b l ic h e a lt h a n d e p id e m io l o g y hour for overall capacity-building and interoperability. this prospective study explored the perspective of golden-hour across the spectrum of mass-gathering-medicine. methods the perspective of golden-hour as a determinant of operations-throughput was evaluated during the indian medical mission operations for hajj-2016, which established, operated and coordinated a strategic network of roundthe-clock medical operations in mecca, medina and jeddah during hajj-2016 pilgrimage from 01 aug to 30 oct 2016. the mission framework comprised of (a) mobile medical task-forces detailed for strategic mass-gathering locations such as mosques, religious places, bus-stops, train-stations and along the pilgrimage assemblage during the five critical days of hajj, to cater for 5000-100000 pilgrims per congregation. (b) 18 static-clinics having basic first-aid capabilities catered for 6000-9000 pilgrims/ clinic residing in over 400 buildings in mecca and medina (c) one mobile referral tent-clinic with 35 tent-clinics having medical attendance facilities only, catered for 3000-4000 pilgrims in over 5000 tents in mina and arafat, as well as 1,40,000 unsheltered pilgrims in muzdalifah. (d) two strategically sited 40-bedded secondarycare referral facilities for critical-care, internalmedicine, general and orthopaedic surgery, gynaecology, paediatrics, psychiatry, dermatology, isolation, lab-medicine and radiology (e) tertiary-care patient transfers coordinated with 28 saudi arabian hospitals. golden-hour as a determinant of operations throughput was evaluated across the spectrum of mass-gathering-medicine, through evaluation of throughput in terms of deliverables/per unit time, against established standard-operating-procedures for various clinical, investigation, drug-dispensing, patient-transfer, administrative and patient-safety algorithms. the average range of patient encountertime, waiting-time, turnaround-time were assessed throughout various echelons of healthcare under a patient-centric healthcare delivery model. even low-acuity patients were attended round-the-clock. subjective assessment of communication-time, visit-time and doctorpatient interaction on-site was done. encounter-time was defined as time taken by the patient for a healthcare operation such as outpatient attendance, investigation, drug-dispensing, minor-procedures and ambulance transfers. waiting-time was defined as pre-operation waiting time due to resource limitations attributable to health-system, while excluding waiting-time attributable to patient or otherwise such as delayed reporting, traffic-conditions and post-attendance time for decisions or other personal reasons. turnaround-time was defined as time taken to complete a task incorporating both operation and waiting-time, as applicable. communication-time/reportingtime/accessibility-time or patient-rescue time was defined as time taken for health-intelligence to reach the doctor. visit-time was defined as time taken for visit by medical team to reach the patient through ambulance/foot. doctor-patient interaction on-site was defined as time taken for elicitation of relevant history and administration of first-aid, if any. the turnaround-time for healthcare personnel was compared between site visits and that with patient being brought to healthcare facility. health emergency was defined as any condition threatening the life or limb of a patient. initiation of definitive-care was the end/start point of defining upstream (pre-hospital) and downstream (hospital-care) processes, being prior and after respectively. dynamic evaluation was carried out to cater for variation and heterogeneity. results a total of 394 013 patients comprising 225 103 males (57.1%), and 168 910 females (42.9%) were attended by a team of 144 doctors including 50 specialists, 146 paramedics and 74 ancillary staff. the patient distribution in mobile medical task-forces, static-clinics and tent-clinics was 13473, 374475 and 5135 patients. out of 930 secondary-care and 523 tertiary-care referrals, 585 and 495 patients were institutionalized respectively. total secondary-care bed days were 4626, average bed occupancy being 77.78% for one month around hajj and 32% otherwise. pooled unadjusted average length of stay of all patients was three days. 1505 minor surgical and 770 orthopaedic procedures were performed. 7850 laboratory, 2074 imaging and 1159 electrocardiograms were carried out. massive surge of patients overwhelmed the throughput capacities of outpatient attendance, pharmacy, laboratory, imaging, ambulance, referrals and documentation. there was delay in attendance, suspicion, diagnosis and isolation of patients with communicable infections. average encounter-time, waiting-time and operation turnaround-time for patients, i. d. khan et al. issn 2413-6077. ijmmr 2017 vol. 3 issue 1 55 p u b l ic h e a lt h a n d e p id e m io l o g y i. d. khan et al. procedures, investigations and ambulance operations is depicted in table 1. the turnaround-time for healthcare personnel has been compared in table 2. discussion golden-hour golden-hour conceptualizes early arrival of patient to definitive-care enabling early recogtable 1. indian medical mission for hajj–2016: encounter, waiting and operation turnaround time for patients, procedures, investigations and ambulance operations no. patient–care variables encounter time (minutes) waiting time (minutes) operation turnaround time (minutes) pre–hospital patient care (on-site primary-care through mobile-medical-task-force during mass–gathering congregations) 1 rescue/reporting/accessibility time to healthcare professional 1–30 0–5 20–60 2 immediate first-aid 5–20 0–5 20–60 3 ambulance arrival at site of injury/first-aid 0–15 0–15 30–150 4 stretcher/wheelchair transfer from site of first-aid to ambulance/primary-care/secondary-care 0–20 0–2 30–150 5 ambulance transfer from site of first-aid to primary/secondary-care 10–40 10–20 30–150 pre-hospital patient care (static-clinic based primary-care) 1 low acuity patients 2–10 0–60 2–60 2 high acuity patients 20–40 0–5 10–60 3 checking of vitals 3–5 0–30 3–30 4 systemic examination 2–15 0–15 2–15 5 blood glucose by glucometer 2–3 0–40 2–40 6 collection of medicines from pharmacy 2–10 0–60 2–60 7 ambulance transfer from static-clinic to secondary/tertiary-care 30–90 0–60 30–150 pre-hospital patient care (tent-clinic based primary-care during five critical days of hajj) 1 low acuity patients 2–10 0–20 2–20 2 high acuity patients 20–40 0–2 20–40 3 checking of vitals 3–5 0–10 3–10 4 systemic examination 2–15 0–10 2–25 5 blood glucose by glucometer 2–3 0–10 2–10 6 collection of medicines from pharmacy 2–10 0–20 2–20 7 ambulance transfer from tent-clinic to secondary/tertiary-care 10–20 0–30 10–60 hospital-based patient care (secondary-care) 1 low acuity patients 2–5 0–60 2–60 2 high acuity patients 20–40 0–2 20–40 3 electrocardiogram 5 0–30 5–30 4 urine routine and microscopy 10 0–20 10–20 5 blood glucose by glucometer 2–3 0–40 2–40 6 malaria/ dengue/hcv by rapid kits 30 0–30 30–60 7 haematology tests 30 0–30 30–60 8 clinical chemistry tests 30 0–30 30–60 9 manual x-ray 20 0–30 30–60 10 ultrasonography 5 0–20 5–20 11 minor surgical procedures 30 0–60 30–60 12 orthopaedic procedures 30 0–60 30–60 13 collection of medicines from pharmacy 2–10 0–60 2–60 14 ambulance transfer from secondary-care to tertiary-care 30–60 0–60 60–180 issn 2413-6077. ijmmr 2017 vol. 3 issue 156 p u b l ic h e a lt h a n d e p id e m io l o g y nition of disease-condition, triage, initiation of resuscitation, control of bleeding, intravenous fluids, antimicrobials, analgesics, investigations and treatment/surgery, thereby positioning the patient on the path to recovery [5]. the model has evolved with expansion in scope from trauma to head-injury, sepsis, shock, stroke, neonates, antimicrobial therapy, pressure-ulcer; weaning of mechanical resuscitation, icu admissions, ischemia, heat-stroke, laboratory, imaging, referral and evacuation etc. as well as expansion in time to evolve the “platinum halfhour”, “golden 10 minutes” and “silver-day” [6-9]. golden-hour is an essential pragmatic tool in pre-hospital care in both clinical and logistic fronts. golden-hour subsumes time to reach definitive-care, thereby including communication/accessibility/reporting time, doctor-patient interaction time and stabilization-time allowing critical clinical interventions. significant associations with deterioration of patient’s condition have been found with each incremental minute of patient arrival to definitive-care [6-9]. it is important to note that doctor-patient interaction time and stabilization-time may be as long as 27.55 minutes, which may nudge into the golden-hour [10]. logistics of communication, evacuation, terrain and weather considerations are deterministic in outcome [11-15]. there are three axes to golden-hour. firstly, golden-hour in a patient-centric scenario incorporates resource maximization towards protocolized treatment. secondly, golden-hour in an incident-accident centric scenario involves triage and evacuation under the ‘best for most’ approach. thirdly, golden-hour in mass-gathering-medicine insinuates a resource-limited scenario with surge of both high-acuity and low-acuity patient-crowds from diverse sociocultural backgrounds, expectations and needs [16]. the plot of golden-hour in mass-gatheringmedicine is shown in fig. 1. while patients’ arrival within first 60 minutes has been associated with better prognosis and early discharge, increased out-of-hospital time is associated with the contrary. pre-hospital life-saving procedures such as intubation may not reduce morbidity [17-18]. communication of information is critical for preparedness. even when encounter-time and waiting-time may be few minutes, together they lead to a turnaround-time spilling out of the goldenhour. ambulance turnaround-time are affected by confusing addresses of camps/sites, distance, overwhelming vehicle and pedestrian traffic as well as reorganized routes, which may lead to unusual delay. huge surge of patients lead to crowding of healthcare facilities reduces access to more deserving patients. little understanding of health emergencies by patients lead to increased waiting-time for patients deserving early attention [6, 19]. throughput in healthcare human system is an extremely complex system where existing knowledge is complemented by deductive algorithm and modelling to design protocolized goal-directed diagnosis and treatment modules. the throughput involving human lives is dependent on doctor-patient interactions, which incorporates real-life human dependence at both ends; and poses limitations in simulation-based real-time preparedness. despite advances in biomedical engineering, the throughput in health-systems table 2. comparative analysis of average time of initiation of definitive-care no. healthcare personnel turnaround time site/home visit by doctor patient brought to healthcare facility 1 mobile-medical-task-force during mass-gathering congregations 20–60 5–30 2 tent-clinic based primary-care 10–30 5–15 3 static-clinic based primary-care 10–60 5–15 4 hospital-based secondary-care 10–60 5–30 5 tertiary-care fig. 1. illustration of golden-hour in mass-gathering medicine. i. d. khan et al. issn 2413-6077. ijmmr 2017 vol. 3 issue 1 57 p u b l ic h e a lt h a n d e p id e m io l o g y i. d. khan et al. is not comparable with inanimate entities such as data packets, industrial processes and products; which function in systems invented, designed, standardized and calibrated through calculations and simulations. the throughput in medicine and health systems has had no defined maximum since antiquity, even in the absence of stochastic and deterministic deductions and quantifiable indicators. disasters, wars and pandemics have exemplified the doctor-patient equation in situations of overwhelming throughput. the concept of maximum theoretical throughput may at best be applied to quality healthcare including documentation which is restricted in scope in an overwhelming throughput scenario. the maximum achievable throughput is defined by resource-limitations [20, 21]. golden-hour in mass-gathering medicine golden-hour paradigm in mass-gathering medicine entwines along healthcare availability, accessibility, efficiency and interoperability; expanding from the patient-centric to publichealth centric approach. operational stringencies exist parallel to infrastructural and provisioning stringencies in makeshift healthcare facilities with intricate operational, procurement, distribution and disposal logistics. overwhelming number of patients may lead to downgrading of medical centres to resourcelimited setups, compromising standards of healthcare, clientele satisfaction and resource security. 22 it also leads to physical, mental and compassion fatigue amongst healthcare-personnel. healthcare availability involves dynamic planning of human, auxiliary and ancillary resources such as infrastructure, medicines, equipment and consumables. human resources are a critical component of goldenhour mandating deployment discretion. healthcare professionals are often required to multitask in resource-limited settings which hamper the technical efficiency towards focused and dedicated clinical work. accessibility can be improved by resource planning, information technology, communications, surveillance systems and outreach activities. internal communication for healthcare professionals should include information on local diseases, transmission, antimicrobial resistance, availability of medicines through acquired health intelligence [20, 21]. population-based approach to golden-hour the role of population is often not discussed within the golden-hour concept. golden-hour pre-hospital care is ought to be provided by people such as family, friends and the first people noticing the need; much before the arrival of first responders, paramedics or doctors. while significant expertise in first-aid may exist in progressive western communities, negligible help is available to victims in most developing communities leading to increased morbidity and mortality. the problem of spectator-inertia around accidents mandates behavioural change in attitude of the masses towards philanthropic efforts. community level expertise development programs can enable rapid firstaid availability. calling for a paramedic/doctor to reach the site is time consuming and doesn’t lead to definitive-treatment as most cases need to be shifted to hospitals for simple measures such as fluid resuscitation, application of plaster-casts or administration of analgesics, antiallergics etc. instead of sending a representative from the community to call for medical help, the time and effort should be utilized towards providing initial care and transportation support to the victim towards the hospital for definitive-care. patient being brought to definitive-care reduces treatment initiation-time by 50% as seen in this study. while the patient is brought early to a resourcerich environment compared to pre-hospital care, it also furthers simultaneous domiciliary care and monitoring of multiple patients. a multifold availability of doctor and auxiliary health professionals at medical facility facilitates adherence to protocols and optimization of processes, thereby improving outcome and streamlining throughput [19, 23, 24]. the situational-analysis of operations throughput highlights wastage of considerable time due to mobilization of medical team from the hospital to reach the patient on-site due to calls for low/moderate-acuity complaints resulting from inability of the patients to judge medical emergencies. it has been proven that trauma team activation doesn’t guarantee better survival [19]. visits consume the turnaround time for healthcare professionals, thereby reducing their availability to attend to deserving patients in need, and diverted critical healthcare resources away from emergency situations. sometimes, visit come at the cost of leaving the medical facility without a doctor. in any resource limited scenario, most patients who deserve time, access definitive-care beyond the golden-hour [7, 8, 25, 26]. there is an evolving paradigm of healthcare which is as explicit as right-to-heath or implicit under human/social rights/security within the issn 2413-6077. ijmmr 2017 vol. 3 issue 158 p u b l ic h e a lt h a n d e p id e m io l o g y boundaries defined by a nation/state for its citizens, or by an employer/insurer for beneficiaries. the kingdom of saudi arabia has been magnanimous to extend comprehensive quality healthcare to hajj pilgrims from all over the world, through a robust and efficient statesponsored single-tier health system, a vanguard equity healthcare-model difficult to emulate. nevertheless, operations throughput gets overwhelmed during hajj at all healthcare facilities [1, 2, 25]. the golden-hour is not a blind concept around buying time; rather it is a dynamic concept requiring reasonable discretion and prudence; failing which there exist limitations to its applicability. overwhelming fluid resuscitation and extensive procedures may lead to procedural errors, transmission of infections and antimicrobial overuse [17, 27-30]. since the study catered to 4.5 lakh patients across a wide network of medical facilities in a setting of mass-gathering medicine, limitations exist due to variability in patients, sites and medical facilities. elements of bias and confounding at patient and doctors’ may not be fully accounted. the variability of operations as well as that of observers may limit the exhaustive collation of data. conclusions time being a crucial factor in the complexity of medical-care, operations throughput remains an important determinant towards availability, accessibility, efficiency and interoperability of bottlenecks, thereby being a determinant of golden-hour in mass-gatheringmedicine. early transportation of patient to definitive-care reduces treatment initiationtime by 50% thereby improving outcome, and consequentially emphasizing patient-evacuations personal visits and interventions by medical/trauma team. evidence-based guidelines, standards, alert and response systems need to be developed with international cooperation and networking for enhancing capability and core competency. golden-hour needs to be emphasized under a population-based approach targeting the clientele towards administering first-aid and reaching out to hospital within the golden-hour. references 1. kollek d. an intro to mass gatherings. centre for excellence in emergency preparedness. ontario. canada. 2014:1–15. www.ceep.ca/publications/ mass_gatherings.pdf. accessed 2014. 2. who. epidemic and pandemic alert and response. communicable disease alert and response for mass gatherings. who/hse/epr/2008/8 technical workshop. geneva-switzerland, 29-30 apr 2008:1–29. http://www.who.int/csr/mass_gathering/ en/. accessed 2015. 3. memish za, stephens gm, steffen r, ahmed qa. emergence of medicine for mass gatherings: lessons from the hajj. lancet infect dis. 2012;12(1):56– 65. http://www.ncbi.nlm.nih.gov/pubmed/22192130. accessed 2012. 4. shujaa a, alhamid s. health response to hajj mass gathering from emergency perspective, narrative review. turk j emerg med. 2015;15(4):172–176. h t t p : / / w w w . n c b i . n l m . n i h . g o v / p m c / a r t i c l e s / pmc4882208. accessed 2015. 5. muck f, wirth k, muggenthaler m, et al. pretreatment mass casualty incident workflow analysis : comparison of two level 1 trauma centers. unfallchirurg. 2016;119(8):632–41. http://www.ncbi. nlm.nih.gov/pubmed/27351989. accessed 2016. 6. dinh mm, bein k, roncal s. redefining the golden hour for severe head injury in an urban setting: the effect of prehospital arrival times on patient outcomes. injury. 2013 may;44(5):606–10. doi: 10.1016/j.injury.2012.01.011. 7. van zanten ar. the golden hour of antibiotic administration in severe sepsis: avoid a false start striving for gold. crit care med. 2014;42(8):1931– 1932. 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(pmid – 24532901) 14. khan id. comorbid cerebral and pulmonary edema at 7010 m/23000 ft: an extreme altitude perspective. j medicine. 2013;14(2):153–155. 15. khan id. cerebral venous sinus thrombosis (cvst) masquerading as high altitude cerebral edema (hace) at extreme altitude (6700 m/22000 ft). int j travel med glob health. 2016;4(3):65–67. 16. kotwal rs, howard jt, orman ja, et al. the effect of a golden hour policy on the morbidity and mortality of combat casualties. jama surg. 2016;151(1):15–24. doi:10.1001/jamasurg.2015.3104. 17. lerner eb, moscati rm. the golden hour: scientific fact or medical “urban legend”? acad emerg med. 200l;8(7):758-60. http://www.ncbi.nlm. nih.gov/pubmed/11435197. accessed 2001. 18. platts-mills tf, evans cs, brice jh. prehospital triage of injured older adults: thinking slow inside the golden hour. j am geriatr soc. 2016. http://www. ncbi.nlm.nih.gov/pubmed/27556573. accessed 24 aug 2015. 19. samplais js, lavoie a, williams ji, et al. impact of on-site 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accessed 2015. 27. taghavi s, vora hp, jayarajan sn. prehospital intubation does not decrease complications in the penetrating trauma patient. am surg. 2014. 80(1):9– 14. http://www.ncbi.nlm.nih.gov/pubmed/24401498. accessed 2014. 28. jacob m, kumar p. the challenge in management of hemorrhagic shock in trauma. med j armed forces india. 2014 apr;70(2):163–9. http://www.ncbi. nlm.nih.gov/pmc/articles/pmc4017195 29. jindal ak, pandya k, khan id. antimicrobial resistance: a public health challenge. med j armed forces. india. 2014;71(2):178–181. http://www.ncbi. nlm.nih.gov/pubmed/25859082. accessed 2014. 30. khan id, sahni ak, bharadwaj r, lall m, jindal ak, sashindran vk. emerging organisms in a tertiary healthcare set up. med j armed forces. india. 2014;70(2):120–128. http://www.ncbi.nlm.nih.gov/ pubmed/24843199. accessed 2014. received: 2017-05-15 35 r a d ia t io n m e d ic in e a n d o n c o l o g y issn 2413-6077. ijmmr 2018 vol. 4 issue 1 international journal of medicine and medical research 2018, volume 4, issue 1, p. 35-40 copyright © 2018, tsmu, all rights reserved corresponding author: piotr młynarz, ph.d., d.sc., bioorganic chemistry group, faculty of chemistry, wrocław university of science and technology, wroclaw, poland e-mail: piotr.mlynarz@pwr.edu.pl phone number: +48713203616 doi 10.11603/ijmmr.2413-6077.2018.1.8921 is metabolomics the diagnostic tool for medical diagnostics of cancer? an example based on lung and breast cancer a. lamasz1, w. barg2, p. mlynarz1 1bioorganic chemistry group, faculty of chemistry, wrocław university of science and technology, wrocław, poland 2department of physiology, wroclaw medical university, wroclaw, poland background. metabolomics is a relatively new diagnostic tool that allows a deep insight into the body metabolism at a cellular level. objective. this paper provides a comprehensive view into the metabolomics methodology and shows usefulness of this approach in diagnosing and stratifying lung and breast cancers. methods. literature review of metabolomics studies and its clinical application in the diagnosis of cancerselected studies. results. in general, the metabolomic approach comprises three steps: 1) sampling and preparing biofluids or tissue homogenates, 2) identification of low-molecular weight compounds up to 1.0 kda using nuclear magnetic resonance, mostly 1h-nmr and/or mass spectrometry, and finally 3) data processing and analysing. it is possible to identify a set of metabolites, which is specific for a certain metabolic status (the metabolic fingerprint). furthermore, this set of metabolites provides information of possible pathomechanisms involved in the disease process i.e. information about the disease etiology. it has been proven that the change in metabolome precedes; not only clinical symptoms but other laboratory findings as well. consequently, this approach, if sufficiently validated, seems to be very promising especially in screening and early diagnosing. conclusions. it was demonstrated that metabolomic approach allows to discriminate patients with cancer from healthy persons, as well as to differentiate between clinical stages of the cancer. key words: metabolomics; metabolome; breast cancer; lung cancer. introduction to understand the complex processes occurring in the living systems the holistic approach should be used within employing all established multilevel approaches, currently is called ‘omics science’. in general, there are more than 100 types of omics subjects, i n c l u d i n g s c i e n c e s s u c h a s : g e n o m i c s , transcriptomic, proteomics, and metabolomics. this approach includes all-important live processes from information storage trans cription processes, protein production, up to e n z y m e s a c t i o n s t r a n s f o r m i n g c e r t a i n substrates to products maintaining the cell l i v i n g p r o c e s s e s . w h i l e g e n o m i c s a n d proteomics have been successfully introduced to routine diagnostics, metabolomics is still on the runway despite its assessed potential [1]. metabolomics refers to the analysis and interpretation of ongoing processes in living organisms at the end of life chain consisting of genomics-transcriptomics-proteomics-metabolo mics science. metabolomics might be strongly influenced by external factors such as environment, life style, diet, medications, as well as by additional metabolite input from the body’s microbiomes. metabolites are delineated as lowmolecular weight compounds (lmwc) up to 1.0 kda. those compounds are carbohydrates, fatty acids, lipids, amino acids, nucleosides, or other organic molecules which are involved in biochemical reactions as substrates, intermediates, and/or final products. the set of body’s metabolites ultimately form its characteristic metabolome. metabolome is a dynamic system, which is largely dependent on internal biochemical reaction and external factors mentioned above [1, 2]. metabolomics is a comparative science, e.g. the obtained information should be compared to the referenced one or vice versa. with this approach metabolomics can be a. lamasz et al. 36 r a d ia t io n m e d ic in e a n d o n c o l o g y issn 2413-6077. ijmmr 2018 vol. 4 issue 1 regarded as a diagnostic tool, which in principle enables to describe current metabolic status of cells, tissues, and organs. simply put, it can define the general health of a living organism. this is done by analysing body fluids, either secreted like urine, saliva, mother milk, stool, or obtained during standard medical sampling procedures such as serum, plasma, cereb rospinal fluid, tissue samples etc.. the samples collection for metabolomics studies is relatively simple, however certain procedures must be followed [3]. the patients must follow standard requirements: they should be fasting and reports all designated therapy, addictions, and medical history. the samples taken should be stored at -80 °c or preserved in liquid nitrogen [2, 4]. this low storage temperature ensures the maintaining of metabolic composition, i.e. minimalizing possible changes in the metabolic profile after sampling, and protects against potential influence of the donor’s microbiome [5, 6]. comprehensive or even partial determination of the metabolome is not possible with a single analytical method due to different levels of detection limits and different group determination. there are many analytical methods employed for metabolomics, each of them having both advantages and dis ad vantages. however, there are two main methods, which are routinely used in metabolomics studies: nmr (nuclear magnetic resonance, mostly 1h-nmr) and ms (mass spectrometry) hyphenated with separations techniques [2]. studies based on both nmr and ms have a common pathway: raw sample preparation and/or extraction of metabolites data collection nmr/gc or lc-ms data processing and data analysis one of the most important advantages of the ms method is the sensitivity to femtomole concentration, which allows analysing up to several thousand molecules in a certain sample, resulting in precise molecular description in the tested specimen. on the other hand, reproducibility with ms is usually worse as compared to nmr spectroscopy. unlike the ms, the nmr method provides high reproducibility with the ability to combine structure analysis and detection of the range of 30-90 molecules without sample destruction (biofluids). however, the detection limit of individual compounds is shifted several rows and strongly d e p e n d o n t h e t i m e o f m e a s u re m e n t s (acquisition). the advantage of this method is the ability to measure different compounds such as amino acids, nucleosides, amines, acids, etc. all at the same time. the data obtained with both methods are both quantitative and qualitative among the set of detected metabolites [7]. the results obtained are further elaborated using chemometric multidimensional discriminatory methods. for this type of studies, the methods most commonly used are: unsu pervised pca, supervised pls (partial least squares fig. 1. typical workflow for metabolomics studies [2]. discriminant analysis), and opls-da (orthogonal version of pls-da). these approaches allow describing each subject not with 2 or 3 variables (chemical compounds or characteristic parameters) as in routine statistical analysis, but even hundreds of variables (chemical compounds metabolites) can be analysed with selection of the most specific ones. those compounds or more often sets of compounds can be employed as biomarkers for discriminating patients from healthy individuals or for staging of the disease [8]. application of metabolomics in the diagnosis of cancer – selected studies lung cancer in europe, lung cancer accounts for approximately 20 % of all cancer deaths, with 376 000 deaths in 2008. unfortunately, the early s t a g e s o f t h i s c a n c e r a r e u s u a l l y l o w symptomatic and final diagnosis is made late in the natural history of the disease. consequently, effective treatment is not possible up to 90 % of cases, and the overall 5-year survival rate is 11.2 % for men and 13.9 % for women [9]. the metabolomic studies of lung cancer are based on various body fluids, predominantly blood serum, urine, and saliva. the most a. lamasz et al. 37 r a d ia t io n m e d ic in e a n d o n c o l o g y issn 2413-6077. ijmmr 2018 vol. 4 issue 1 common type of lung cancer is non-small cell lung cancer (nsclc). the main recognized risk f a c t o r f o r n s c lc i s c i g a re t t e s m o k i n g . additionally, copd is considered an independent risk factor of lung cancer [10]. our team has demonstrated a possibility to differentiate between patients with copd and with two stages of nsclc: an early nsclc (e-nsclc) and an advanced nsclc (a-nsclc). using 1h nmr method we were able to identify 45 metabolites in patients’ serum. con centrations of acetate, citrate, and methanol were significantly reduced in lung cancer subjects. in contrast, concentrations of n-acetylated glycoproteins, leucine, lysine, mannose, choline, lipids (l3 + l4), and two other unknown compounds were increased in for patients suffering cancer in all three performed comparisons: nsclc vs. copd, e nsclc vs. copd and ansclc vs. copd [10]. similar results were obtained by musharraf et al [11]. using gc-ms method for tracing metabolites in plasma, they were able to discriminate lung cancer patients from copd patients, as well as healthy non-smokers and healthy smokers with high sensitivity (96.2 %) and specificity (92.05 %). another res earch grou p focu s ed on differentiating early lung cancer patients from healthy controls. they used 1hmr and rapid resolution liquid chromatography (rrlc) methods to investigate metabolites in serum and identified 25 metabolites, which were up or down regulated. those findings proved disorders in glycolysis, lipid metabolism, choline phospholipid metabolism, one-carbon metabolism, and amino acid metabolism. the use of both methods enabled diagnosing early stage of lung cancer with a very high accuracy [12]. mass spectrometry hyphenated with gas chromatography (gc-ms) was used by horia and co-workers [13]. they demonstrated that the levels of 23 of 58 serum metabolites and 48 of 71 sampled from tissue were significantly changed in patients with lung cancer with i-iv stage as compared, with healthy volunteers. an early cancer stage was also investigated by other group using lc-ms method. in this study, the achieved auc value based on 12 metabolites was 0.836 [14]. urine also has a strong discriminative potential in lung cancer diagnosing. this was proven in a study performed using 1h nmr method by carrola and colleagues [15]. the main metabolites differentiating between healthy controls and lung cancer patients were: hippurate, trigonelline, β-hydroxyisovalerate, α-hydroxyisobutyrate, n-acetylglutamine, and creatinine. they were able to develop a classification model, which confirmed 93 % sensitivity and 94 % specificity with overall classification rate of 93.5 %. [15]. the studies performed by lc-ms method on urine samples from cohort of 469 patients with lung cancer and 536 controls revealed two biomarkers: creatine riboside and n-acetylneuraminic acid, which were significantly increased in non-small cell lung cancer and were associated with worse prognosis [16]. exhaled breath condensate (ebc) seems to be naturally associated with lung cancer. indeed, studies on ebc performed with gc-ms [ 1 7 ] a n d lc m s [ 1 8 ] p rov e d d i a g n o s t i c usefulness of ebc differentiating lung cancer patients, copd patients, smokers, ex-smokers, and healthy controls. in addition to the above mentioned biofluids, also sweat should be included, which was successfully used to differentiate the lung cancer patients from control individuals with risk factors and without them by its analysis by lc-ms system [19]. recently, a very promising study has been published by shen et al.. using two-stage study design and advanced metabolon platform they were able to identify four metabolites, which may be useful biomarker candidates for identifying patients, who may benefit from platinum-based chemotherapy in advanced nsclc [20]. the comprehensive review on lung cancer biomarkers and metabolomics methods has been published recently [21]. breast cancer breast cancer (bc) is the most common cancer in women worldwide and the morbidity keeps rising. incidence vary from 19.3 per 100,000 women in eastern africa to 89.7 per 100,000 in western europe [22, 23]. some decrease in the number of death caused by bc is observed mainly due to the improvement in early diagnostics [24]. nevertheless, routine methods currently used to detect bc usually are not effective, especially in the very early stages [25]. a new approach in cancer diagnostics involves the use of metabolomics tools for blood serum and urine analysis [25, 26, 27]. metabolomic-based diagnosing not only seems to allow discriminating healthy controls from bc subjects, but also differentiating metastatic bc from early stages of bc as well [28, 29]. previous studies proved that bc can be diagnosed by nmr spectroscopy and by ms, both by use of serum and urine [27, 30, 31]. a. lamasz et al. 38 r a d ia t io n m e d ic in e a n d o n c o l o g y issn 2413-6077. ijmmr 2018 vol. 4 issue 1 however, those metabolomic studies involved relatively small groups and, utilized various protocols and methods [32]. therefore, a more integrated and coherent methodology should be used [33]. the gc-ms method can be used for metabolic profiling of serum. using this method, it was possible to differentiate patients with bc from patients with non-malignant tumours, and from healthy controls. sets of amino acids, fatty acids, and lysolipids allowed differentiating these three groups [31]. there is also evidence that polar compounds analysis has been successfully applied for metabolomicbased bc diagnosing in tissue samples. chae and co-workers used hr-mas 1h nmr method for retrospective analysis in the patients with ductal carcinoma in situ (dcis) diagnosed on preoperative biopsy. the univariate analysis proved that choline-containing compounds did not differ between the groups, while gpc/pc ratio, myo-inositol, and succinate were higher in the ‘pure’ dcis group as compared to the invasive carcinoma subjects. multivariate analysis opls-da could discriminate to some degree between these two groups [34]. comparable results were obtained in other groups. in a study based on add space 1h nmr method the authors were able to distinguish between the non-invasive intraductal carcinoma and invasive ductal carcinoma patients. histidine con centration was significantly lower in the patients with invasive ductal carcinoma. in contrast, those patients presented higher concentrations of glucose, lactate, tyrosine, and lipids in plasma samples, as compared to the non-invasive carcinoma group [35]. it is also possible to differentiate metastatic bc patients from the ones with a localized, early-stage disease. serum analysis with nmr spectroscopy identified 9 metabolites: histidine, acetoacetate, glycerol, pyruvate, glycoproteins (n-acetyl), mannose, glutamate, and phenylalanine concentrations, all of which were significantly different, than those in the other studied groups [26]. volatile organic compounds (voc) from urine samples can also be used for bc diagnosis. in the study of silva et al., gas chromatographymass spectrometry was used to obtain metabolomic patterns of 26 bc patients and 21 healthy individuals. of the 79 volatile identified metabolites six compounds were of diagnostic power: (−)-4-carene, 3-heptanone, 1,2,4-trimethylbenzene, 2-methoxythiophene, phenol, and dimethylsufide. all of them were able to successfully discriminate between the groups [30]. more holistic approach was suggested by bro et al. in a retrospective study they combined 1h nmr data with other relevant biological and phenotypic information to construct a patient’s biocountour. with this approach the authors could predict an increased risk of bc, a few years before its occurrence with sensitivity and specificity well above 80 % [36]. a very interesting paper aiming on possible associations of dietrelated metabolites with bc risk was published recently by pleydon et al.. using ce-ms method they identified a bunch of metabolites, which were moderately correlated with increased risk of estrogen receptor related to bc development [37]. the annually increasing literature data proves the usefulness of metabolomics in bc [33, 38]. conclusions data from the literature clearly demonstrate the usefulness of metabolomic approach in diagnostics of lung cancer, breast cancer, and many other diseases. despite this, the metabolomic approach is still not routinely implemented into medical protocols. this results from many factors. analytical methods used for metabolomic research are neither standardized nor validated. consequently, it is difficult to: compare results obtained by various research groups, and determine the definitive clinical recommen dations. threfore we need more and more research to overcome these difficulties. however, in the future the metabolomic approach with fingerprinting and profilingbased methods, combined together with predictive-discrimination statistical models should be the method of choice for preventive, screening, and treatment research. a. lamasz et al. 39 r a d ia t io n m e d ic in e a n d o n c o l o g y issn 2413-6077. ijmmr 2018 vol. 4 issue 1 references 1. barding ga, salditos r, larive ck. quantitative nmr for bioanalysis and metabolomics. analytical and bioanalytical chemistry. 2012 sep 1;404(4):1165-79. 2. kettaneh n, berglund a, wold s. pca and pls with very large data sets. computational statistics & data analysis. 2005 jan 1;48(1):69–85. 3. https://www.erswhitebook.org/chapters/ lung-cancer/ 4. deja s, porebska i, kowal a, zabek a, barg w, pawelczyk k, stanimirova i, daszykowski m, kor zeniewska a, jankowska r, 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вступ. метаболоміка це відносно новий діагностичний підхід, який дозволяє аналізувати метаболічні процеси на рівні клітини. мета дослідження та методи – огляд технології аналізу метаболоміки та її використання у клінічній практиці для діагностики певних злоякісних захворювань. результати. загалом, метаболомічний підхід включає три етапи: 1) відбір проб біоологічних матеріалів, 2) ідентифікація низькомолекулярних сполук масою до 1,0 кда з використанням ядерного магнітного резонансу та/ або мас-спектрометрії, 3) обробка та аналіз даних. сьогодні ми можемо визначити набір метаболітів, що є специфічним для певного метаболічного статусу (так званий «метаболічний відбиток»). цей набір метаболітів дасть можливість оцінити певні патогенетичні ланки та визначитися з етіологією захворювання. доведено, що зміни в метаболомі передують, не лише появі перших клінічних ознак та симптомів, але й будь-яким змінам у клініко-лабораторних аналізах. отже, метаболомічний підхід може бути перспективним для скринінгу та ранньої діагностики. висновки. доведено, що метаболоміка дозволяє первинно діагностувати та провести диференціальний діагноз стадії злоякісного процесу. ключові слова: метаболоміка; метаболіти; рак молочної залози; рак легень. a. lamasz et al. 40 r a d ia t io n m e d ic in e a n d o n c o l o g y issn 2413-6077. ijmmr 2018 vol. 4 issue 1 15. yu l, li k, zhang x. next-generation metabo lomics in lung cancer diagnosis, treatment and precision medicine: mini review. oncotarget. 2017 dec 29;8(70):115774-86. 16. http://www.who.int/cancer/detection/ breastcancer/en/index1.html 17. zhang ah, sun h, qiu s, wang xj. metabolomics in noninvasive breast cancer. clinica chimica acta. 2013 sep 23;424:3-7. 18. senkus e, kyriakides s, ohno s, penault-llorca f, poortmans p, rutgers e, zackrisson s, cardoso f. primary breast cancer: esmo clinical practice guidelines for diagnosis, treatment and follow-up. annals of oncology. 2015 sep 1;26(suppl_5):v8-30. 19. asiago vm, alvarado lz, 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cancer risk in a prospective study. the american journal of clinical nutrition. 2017 jun 28;106(2):637-49. 33. yu l, jiang c, huang s, gong x, wang s, shen p. analysis of urinary metabolites for breast cancer patients receiving chemotherapy by ce-ms coupled with on-line concentration. clinical biochemistry. 2013 aug 1;46(12):1065-73. 34. menezes gl, van den bosch ma, postma el, el sharouni ma, verkooijen hm, van diest pj, pijnap pel rm. invasive ductolobular carcinoma of the breast: spectrum of mammographic, ultrasound and magnetic resonance imaging fi ings correlated w i t h p r o p o r t i o n of the l o b u l a r c o m p o n e n t . sprin ger plus. 2013 dec;2(1):621-33. 35. nam h, chung bc, kim y, lee k, lee d. combining tissue transcriptomics and urine meta bolomics for breast cancer biomarker identification. bioinformatics. 2009 sep 25;25(23):3151-7. 36. bro r, kamstrup-nielsen mh, engelsen sb et al. forecasting individual breast cancer risk using plasma metabolomics and biocontours. metabo lomics. 2015 mar 10; 11:1376-1380. 37. playdon mc, ziegler rg, sampson jn, et al. nutritional metabolomics and breast cancer risk in a prospective study. the american journal of clinical nutrition. 2017 jun 28; 106(2): 637-649. 38. menezes gl, van den bosch ma, postma el, et al. invasive ductolobular carcinoma of the breast: spectrum of mammographic, ultrasound and magnetic resonance imaging finding correlated with proportion of the lobular component. springer plus 2013 nov 20; 2:621-633. received: 2018-05-01 a. lamasz et al. пустая страница issn 2413-6077. ijmmr 2018 vol. 4 issue 1 45 d e n t is t r y doi 10.11603/ijmmr.2413-6077.2018.1.9255 reactive oxygen and nitrogen species role in experimental periodontitis development a. ye. demkovych i. horbachevsky ternopil state medical university, ternopil, ukraine introduction. activation of lipid peroxidation is one of the trigger mechanisms of periodontium injury, which is primary caused by cellular damage. reactive oxygen and nitrogen species (rons) are able to cause damage to a cell as well as final products of lipid peroxidation, including unsaturated aldehydes and other metabolites. objective. the aim of the research was to determine the role of rons and accumulation of lipid peroxidation derivatives in initial development and formation of chronical inflammatory process in periodontium. methods. experimental periodontitis was modeled in animals by injection of complex mixtures of microorganisms diluted in egg protein into periodontal tissues. the results of biochemical studies of free radical processes activity in blood serum were evaluated by content of diene, triene conjugates, tba-active products and total quantity of metabolites of nitric oxide (no2 –+no3 –), which were determined on the 7th, 14th and 30th days of the experiment. results. generation of active forms of oxygen is more influential, providing longevity of inflammatory process. this pays attention to typical dynamics of changes in active processes of lipid peroxidation in the development and course of experimental periodontitis. the study of inflammatory process with a bacterial-immune component in the rats’ periodontal complex proved accumulation of lipid peroxidation and nitric oxide metabolites in blood serum. conclusions. the preservation of increased lipid peroxidation and nitric oxide metabolites in blood serum of the experimental animals with acute periodontitis conduce enhance of alteration and delayed healing that result in its sequel into chronical periodontitis. key wo rd s: periodontitis; nitric oxide metabolites; tba-active products; diene conjugates; triene conjugates. introduction improvement of the established and creation of new methods of generalized periodontitis treatment is one of the urgent matters of contemporary dentistry [1, 2]. i nflammatory processes in the complex of periodontal tissues are the most common a m o n g i n f l a m m a t o r y c o m p l i c a t i o n s o f maxillofacial area [3, 4]. i t is indisputable that pathogenic infection is crucial in this case, as well as their combinations, in particular staphylococcus and streptococcus, which develop in cases of reduced resistance of oral tissues to infectious agents. [5, 6]. the etiology and pathogenesis of periodontal diseases are complicated and studied insufficiently however, the infectious factor and ability of immune protective mechanisms (local cellular unspecifi and general adaptive) are essential; the features of pathological process development, its subsequent treatment and prophylaxis affects depend on them [7]. it is unclear as well as the mechanisms, due to which different by nature and character of action local and general factors lead to inflammatory and destructive lesions of periodontal tissues [8]. the investigation of inflammatory process mechanisms in the tissues of periodontal complex is one of the current issues of contemporary dentistry due to a relatively wide spread and unfavorable prognosis, because the frequency of periodontal disease in world is within 5–20 % and increases with age up to 75 % [9, 10]. t he development of inflammatory-destru tive changes in periodontal tissues is caused by disturbances of microcirculation and transcapillary exchange with underlying severe hypoxia. a ctivation of reactive oxygen and nitrogen species (ron s) and exhaustion of antioxidant defense in biological tissues is the most serious of all effects and outcomes of international journal of medicine and medical research 2018, v olume 4, issue 1, p. 45-51 copyright © 2018, tsmu, all rights reserved corresponding author: andriy demkovych, department of orthopedic dentistry, i. horbachevsky ternopil state medical university, 1 maydan voli, ternopil, 46001, ukraine e-mail: demkovushae@tdmu.edu.ua phone number: +380979342501 a. ye. demkovych issn 2413-6077. ijmmr 2018 vol. 4 issue 146 d e n t is t r y hypoxia. activation of lipid peroxidation (l po ) is a trigger mechanism for oxidative stress with cellular metabolism disorders, which are primary caused by damage of cellular and subcellular membranes [11]. activation of l po and decrease of anti oxidant protection contribute to accumulation of deleterious free cholesterol, lysophosphatides, phosphatidylcholine, that changes the dynamic stability of cellular membranes due to pathological process development in periodontal complex [13]. all these facts about the influence of oxidative stress on the pathogenesis of periodontitis are present in the activity of lipid peroxidation as potential predictors of escalation of inflammatory lesions in periodontal disease. t he disturbance of antioxidant protection in the patients with hypertension, which was proved by changes in the activity of catalase, ceruloplasmin and saturation of transferrin by iron, and the increase in the level of diene conjugates and tba-active products in serum, which leads to the development of endogenous intoxication syndrome in the patients with general periodontitis. one of the parameters that allow estimating the state of free radical processes is the content of lipids hydroperoxides and t ba -active products formed by oxidation of unsaturated fatty acids, and aldehyde and ketone derivatives, which are developed by the action of active radicals on the amino acid residues in protein molecules [14]. t he components of bacterial toxins (especially lipopolysaccharide) and proin fla matory cytokines (mainly tnf -α, il -1 and interferon gamma-ifn -γ) produced by the affected tissues stimulate the production of nitric oxide (no ) by the inducible nitric oxide synthase (ino s) in different cell types [15]. it is proved that parodonotopathogenic bacteria are capable of inducing no formation by inducible no synthase. excessive n o formation, which occurs when ino s is stimulated by proinflammatory cytokines and endotoxins of pathogenic microfl ra of oral cavity, leads to nitrooxidative stress which, together with the activation of lipoperoxidation and oxidative modification of proteins, can cause increased disintegration of connective tissue components and progressing of periodontitis [16]. the aim of this investigation was to determinate the pathogenic influence of ron s and accumulation of lipid peroxidation derivatives in regard to initial development and formation of chronical inflammatory process in periodontal complex. methods t he experiments were carried out using white clinical healthy male rats, 150–200 g in weight, in environments of vivarium, on a standard diet balanced for the basic elements. the research related to animals’ use has been complied with all the relevant national regulations and institutional policies for the care and use of animals. the investigations was conducted following the general rules and regulations of the european convention for the protection of v ertebrate animals used for experimental and o ther scientific purposes (strasbourg, 1986), the general ethics on a nimal experimentation (kyiv, 2001). t he experimental animals were randomly selected and divided into 4 groups: the 1st – intact animal, controls (n=10); the 2nd – animals with experimental periodontitis on the 7th day of study (n=8); the 3rd – animals with experimental periodontitis on the 14th day of study (n=8); the 4th – animals with experimental periodontitis on the 30th day of study (n=8). experimental periodontitis (ep) was caused by introduction of complex mixtures of microorganisms diluted in egg protein into periodontal tissues [17]. simultaneously with the injections of the pathogen a complete freund’s adjuvant was injected in the rat paw to enhance the immune response. when conducting studies with animals of group 4, on the 14th day, repeated administration of the pathogen and injection of adjuvant was carried out. at the 7th and 14th days the experimental animals were euthanized by total heart bloodletting and previous thiopental anesthesia. serum samples were taken for further research. in blood serum the level of diene (dc) and triene conjugates (tc), tba-active products and total quantity of metabolites of nitrogen (іі) oxide were determined. t he concentration of diene conjugates (dc) and triene conjugates (tc) was evaluated by the method based on the fact that the extracted heptane-isopropyl hydroperoxide mixture had an appropriate absorption maximum: d c at a wavelength of 232 nm; tk at a wavelength of 275 nm [18]. the total nitric oxide metabolites in blood plasma: nitrite anion (no2¯) and nitrate anion (no3¯), were determined by photometry using a gray reagent (sulfanilamide solution and n-naphthyl ethylenediamine dihydrochloride in 30 % glacial acetic acid), which was used as a color reagent a. ye. demkovych issn 2413-6077. ijmmr 2018 vol. 4 issue 1 47 d e n t is t r y giving raspberry coloring in the presence of nitrogen oxide metabolites in a liquid [19]. t he method of determining the concentration of tba-active products consisted in the ability of malonic dialdehyde to interact with tiobarbituric acid in an acidic medium to form a colored complex which intensity is adequate to the content of tba-active products [20]. the results were statistically analyzed by means of nonparametric indexes [21]. t he data were presented in the arithmetic mean (m) ± standard deviation of the mean value (m) for a specific number of the animals (n). changes were considered statistically significan at p<0.05. excel 2010 (microsoft corporation) and statistica 10.0 (statsoft, usa) software were used. results these studies were performed in accordance with the suggested and patented patterns for experimental periodontitis [22], which presented the influence of bacterial and immune disorders on the mechanisms of inflammation development in periodontal complex. t he study of experimental periodontitis is associated with the fact that this type and values of bacterialimmune infl mmation has not investigated before. the results of the research proved that in the early period of inflammation development in periodontal complex, which included the period from the 1st to the 7th day of the experiment, there was an excessive accumulation of lipid peroxidation products in serum, as evidenced by increased concentration of dc (in 2.20 times, p<0.01) and tc (in 1.93 times, p<0.01) respectively, compared with the control group of experimental animals (t able 1, f ig. 1). o n the 14th day of experimental periodontitis model, there was a significant decrease of d c (in 1.53 times, p<0.01) and tc (in 1.52 times; p<0.01) in serum compared to the group of animals studied on the 7th day of the experiment, but these indices were higher than those of the intact animal group (in 1.44 times, p<0.01 and by 1.26 times, p<0.01, respectively). in the further observation, on the 30th day of inflammatory process development in the tissues of periodontal complex, the content of dc in blood serum slightly increased in comparison with the indices on the 7th day, but the data were statistically insignificant (p>0.05), but on the 14th day this index increased in 1.53 times (p<0.01). when comparing it with the indices of the control group, it was found out that the content of this metabolite in serum was significantly higher (in 2.21 times, p<0.01). the content of triene conjugates changed the same during the period of monitoring, however, the increase in their concentration in blood serum was less significa t – in 1.53 times (p<0.01), compared with the indices on the 14th day, and in 1.94 times (p<0.01), compared with the control group. when comparing them with the results of the group of animals with experimental periodontitis on the 7th day of the experiment, the changes were found to be statistically insignificant (p>0.05) when determining the ratio of dk/tc content (table 2) in blood serum, it was proved that that index significantly increased on the 7th day of the study (in 1.15 times; p<0.01) table 1. concentration of diene and triene conjugates in serum of the rats in different periods of experimental periodontitis (m±m) f orm of experiment d uration of experiment (days) number of animals dc, conditioned, units/ml tc, conditioned, units/ml control, intact animals 10 2.383±0.071 2.756±0.022 animals with periodontitis 7 8 5.250±0.242 p1<0,01 5.310±0.187 p1<0,01 14 8 3.431±0.089 p1<0.01, p2<0.01 3.485±0.107 p1<0.01, p2<0.01 30 8 5.266±0.141 p1<0.01, p2>0.05, p3<0.01 5.338±0.140 p1<0.01, p2>0.05, p3<0.01 notes: p1 – statistical significance of differences relative to the intact animals; p2 – statistical significance of differences relative to the animals with experimental periodontitis on the 7th day of the research; p3 – statistical significance of differences relative to the animals with experimental periodontitis on the 14th day of the research. a. ye. demkovych issn 2413-6077. ijmmr 2018 vol. 4 issue 148 d e n t is t r y * * * # * # * ● º* ● º 0 50 100 150 200 250 periodontitis 7th day periodontitis 14th day periodontitis 30th day control dc tc f ig. 1. changes in the indices of lipid peroxidation in rats’ serum in the experimental periodontitis followup (% of the control). notes: * – statistically significant differences relative to the intact animals (p<0.01); # – statistically significant differences relative to the animals with periodontitis on the 7th day of the experiment (p<0.01); ● – statistically significant differences relative to the animals with periodontitis on the 7th day of the experiment (p>0.05); º – statistically significant differences relative to the animals with periodontitis on the 14th day of the experiment (p<0.01). compared to the control group and remained on the same level throughout the duration of the experiment: it was higher on the 14th (in 1.16 times, p<0.01) and on the 30th day (in 1.15 times, p<0.01) of the indices of the intact animals. when comparing the same ratios in the rats at different periods of the experiment, in particular on the 7th, 14th, 30th days, the differences were statistically insignificant (p<0.05). as a result of the study of the main indices of lipid peroxidation – the content of t ba-active products, significant changes were also evidenced (table 3). in particular, it was found out that on the 7 th day of experimental periodontitis development in the rats, this serum level was higher in 4.22 times (p<0.01) compared to the control group. on the 14 th day of the experimental periodontitis model, a gradual decrease in the level of tba-active products (in 1.34 times, p<0.01) was evidenced in blood serum in comparison with the group of animals with in�ammatory process in periodontal tissues on the 7th day of the experiment, but these indices were still increased compared to the intact group of animals (in 3.16 times, p<0.01), that proved a signi�cant activation of free radical lipid oxidation processes during the entire period of inflammation development. the studies on the 30th day of the experiment proved that the content of tba-active products in serum gradually decreased (in 1.49 times, p<0.01 and in 1.11 times, p<0.01) respectively, compared to the groups of animals with experimental periodontitis on the 7th and 14th days of the experiment. at the same time, it was higher (in 2.84 times, p<0.01) than in the intact group of white rats (fig. 2). a t the early stage of experimental periodontitis development, that is on the 7th day, there was a signi�cant increase in the content of nitric oxide metabolites (no 2 -+no 3 -), which were classi�ed as unstable products of free radical oxidation in serum (in 6.86 times, p<0.01), but on the 14th day this index changed table 2. correlation of diene and triene conjugates in serum of the rats in different periods of experimental periodontitis development (м±m) f orm of experiment control, intact animals animals with periodontitis d uration of experiment (days) 7 14 30 number of animals 10 8 8 8 dc / tc 0.86±0.03 0.99±0.02 p1<0.01 1.00±0.04 p1<0.01, p2>0.05 0.99±0.01 p1<0.01, p2>0.05, p3>0.05 notes: p1 – statistically significant differences relative to the intact animals; p2 – statistically significant differences relative to the animals with experimental periodontitis on the 7th day of the research; p3 – statistically significant differences relative to the animals with experimental periodontitis on the 14th day of the research. a. ye. demkovych issn 2413-6077. ijmmr 2018 vol. 4 issue 1 49 d e n t is t r y table 3. the content of tba-active products and metabolites of nitrogen (іі) oxide (no2–+no3–) in serum of the rats in different periods of experimental periodontitis development (м±m) f orm of experiment d uration of experiment (days) number of animal tba-active products, mcmol/l no 2–+no 3–, mcmol/l control, intact animals 10 2.555±0.092 0.028±0.001 animals with periodontitis 7 8 10.774±0.122 p1<0.01 0.192±0.006 p1<0.01 14 8 8.066±0.143 p1<0.01, p2<0.01 0.147±0.003 p1<0.01, p2<0.01 30 8 7.255±0.103 p1<0.01, p2<0.01, p3<0.01 0.102±0.002 p1<0.01, p2<0.01, p3<0.01 notes: p1 – statistically significant differences relative to the intact animals; p2 – statistically significant differences relative to the animals with experimental periodontitis on the 7th day of the research; p3 – statistically significant differences relative to the animals with experimental periodontitis on the 14th day of the research. reversely, that is it decreased (in 1.31 times, p<0.01) compared with the animals on the 7th day of the experiment, but was increased compared to the intact group of animals (in 5.25 times, p<0.01) (table 3, fig. 3). characterizing the changes in the content of products of metabolism of nitric oxide in blood serum of the experimental animals with periodontitis, it should be noted that this active form of oxygen on the 30th day of the experiment along with the lipoperoxidation indices of previous studies also signi�cantly increased (in 3.64 times, p<0.01) compared to the results of the animals of control group. however, the data were lower than those in rats on the 7th (in 1.88 times, p<0.01) and 14th days (in 1.44 times, p<0.01) respectively. discussion i ntroduction of complex mixtures of microorganisms diluted in egg protein into periodontal tissues caused hyperergic in�am matory process with signi�cant changes in soft tissue of lower jaw accompanied by edema and hyperemia of mucous membrane and the manifestations were the same as the changes in humans [23]. inflammatory process in periodontal tissues was accompanied by cellular in�ltr ation of surrounding tissues and destructive changes in periodontal complex [24, 25]. the obtained data proved that generation of active forms of oxygen at a su�ciently high level, activation of free radical lipid oxidation were present during the entire period of * * # * # º 0 50 100 150 200 250 300 350 400 450 periodontitis 7th day periodontitis 14th day periodontitis 30th day control tba-active products f ig. 2. changes in the indices of t ba-active products in rats’ serum in the experimental periodontitis followup (% of the control). notes: * – statistically significant differences relative to the intact animals (p<0.01); # – statistically significant differences relative to the animals with periodontitis on the 7th day of the experiment (p<0.01); º – statistically significant differences relative to the animals with periodontitis on the 14th day of the experiment (p<0.01). a. ye. demkovych issn 2413-6077. ijmmr 2018 vol. 4 issue 150 d e n t is t r y * * # * # º 0 100 200 300 400 500 600 700 periodontitis 7th day periodontitis 14th day periodontitis 30th day control nitrogen (іі) oxide f ig. 3. changes in the indices of metabolites of nitrogen (іі) oxide in rats’ serum in the experimental periodontitis follow-up (% of the control). notes: * – statistically significant differences relative to the intact animals (p<0.01); # – statistically significant differences relative to the animals with periodontitis on the 7th day of the experiment (p<0.01); º – statistically significant differences relative to the animals with periodontitis on the 14th day of the experiment (p<0.01). in�ammatory reaction development, but the highest degree was during the peak of the in�ammatory process that corresponded to a more severe clinical picture in this group of animals. in a later period of periodontitis, despite a slight decrease in the intensity of l po, a complete reduction of the in�amed process in periodontal tissues did not take place, which may point to its chronicity. t he indices of lipid peroxidation activity: the content of tba-active products in serum proved that irrespective of the period of their study, during the development of bacterial-immune experimental periodontitis, the formation and accumulation of intermediate toxic products of lipid peroxidation in serum took place at diffe rent stages of its chain branching. also, the in�am matory reaction in periodontal complex in the acute period of development became a source of formation of reactive oxygen species, which were capable of triggering a cascade of free radical processes involving no-radical metabolites. active form of oxygen on the 30th day of the experiment proved the continuation of no generation, the enhancement of free radical processes activity and the disturbance of dynamic equilibrium with the antioxidant defense system. conclusions t he in�ammatory process with bacterialimmune component in periodontal complex is accompanied by increase of lipid peroxidation and nitric oxide metabolites in the blood serum that affects the course and completion of the in�ammatory pr ocess. a signi�cant increase of diene and triene conjugates level and tba-active products in blood serum in the acute period (on the 7th day of the experiment) and a temporary decrease on the 14th day, as well as further increase on the 30th day of the experiment evidence the increased generation of reactive oxygen species and their derivates for the entire period of in�ammation de velopment. t he preservation of increased lipid peroxida tion and nitric oxide metabolites in blood serum of the experimental animals with acute periodontitis conduce to enhance of altera tion and delayed healing that result in its sequel into chronical periodontitis. a. ye. demkovych роль активних форм кисню та нітрогену у розвитку експериментального пародонтиту а. є. демкович тернопільський державний медичний університет імені і. я. горбачевського, тернопіль, україна вступ. розвито оксидативного стресу є одним з пускових механізмів патогенезу ушкодженння пародонту. активні форми кисню та нітрогену здатні викликати пошкодження клітини, так само як і кінцеві продукти перекисного окислення ліпідів, включаючи ненасичені альдегіди та інші метаболіти. мета дослідження полягала у визначенні ролі активних форм кисню та нітрогену та накопичення продуктів перекисного окислення ліпідів у формуванні хронічного запального процесу в пародонті. методи. експериментальний пародонтит моделювали у тварин шляхом введення складних сумішей мікроорганізмів, розведених в яєчному білку. активність вільнорадикальних процесів у сироватці крові оцінювали за вмістом дієнових та трієнових кон’югатів, тбк-активних продуктів та метаболітів оксиду азоту (no2– та no3-) на 7-у, 14-у та 30-у доби експерименту. результати. генерація активних форм кисню забезпечує значну тривалість запального процесу. тому типова динаміка процесів перекисного окислення ліпідів у розвитку та перебізі експериментального періодонтиту викликає значний інтерес. результпти нашого дослідженння запального процесу з бактеріально-імунною складовою в періодонтальному комплексі щурів довело важливу роль накопичення продуктів перекисного окислення ліпідів і метаболітів оксиду азоту в сироватці крові. issn 2413-6077. ijmmr 2018 vol. 4 issue 1 51 d e n t is t r y a. ye. demkovych висновки. підвищений рівень продуктів перекисного окислення ліпідів та метаболітів оксиду азоту в сироватці крові експериментальних тварин з гострим парродонтитом сприяює поглибленню патологічного процесу та уповільнює загоєння, що призводить до розвитку хронічного перебігу захворювання. ключові слова: пародонтит; метаболіти оксиду азоту; тбк­активні продукти; дієнові кон’югати; трієнові кон’югати. references 1. borisenko a. periodontal disease. kyiv: medicine; 2013. 2.. dimi ova a, kolenko y. evaluating the effectiveness of various immunomodulators in complex treatment generalized periodontitis in young adults (18-25 years). j modern dentistry. 2013;2:38-9. 3 .. zabo lo tniy , markov a , shilіvsky i . generalіzed periodontitis. lviv: galdent; 2011. 4. tamaki n, takaki a, tomofuji t, endo y, kasuyama k, ekuni d. stage of hepatocellular carcinoma is associated with periodontitis. j clin periodontol. 2 0 1 1 ; 3 8 : 1 0 1 5 2 0 . d o i : 1 0 . 1 1 1 1 / j . 1 6 0 0 0 5 1 x . 2011.01777.x. 5.. erkaik mj, busscher hj, rustema-abbing m, slomp am, abbas f, van der mei hc. oral biofilm models for mechanical plaque removal. clin oral invest. 2010;14:403-9. doi: 10.1007/s00784-0090309-x. 6. kaplan jb. biofilm dispersal: mechanisms, clinical implications and potential therapeutic uses. j d e n t r e s . 2 0 1 0 ; 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1997. 19. sklyarov o, fedorovych ip, korobov vm. changes in no2 concentration in biological fluids in diseases of stomach cancer. med chemistry. 2004;6(3):55-7. 20. andreeva li, kozhemyakin la, kishkun aa. modification of the method for determination of lipid peroxides in the test with thiobarbituric acid. lab case. 1988;11:41-3. 21. berger rl, casella g. statistical inference 2nd ed. florida: duxbury press; 2001. 22. demkovych ay, bondarenko yi, inventor; i. horbachevsky ternopil state medical univ., assignee. pathogenetic basis periodontitis modeling in rats. ukraine patent 82388 u201303000. 2013 jul 25. 23.3. d ovych a. the necrotic-apoptotic changes in blood mononuclear phagocytes in the experimental bacterial-immune periodontitis development. world of medicine and biology. 2018;no.1(63):120-122. doi: 10.26.724/2079-83342018-1-63-120-122. 24. bullon p, cordero md, quiles jl, ramireztortosa mdel c. autophagy in periodontitis patients and gingival fibroblasts: unraveling the link between chronic diseases and inflammation. bmc med. 2012;17:10-122. doi: 10.1186/1741-7015-10-122. 25. jin cq, dong hx, cheng pp, zhou jw, zheng by, liu f. antioxidant status and oxidative stress in patients with chronic itp. scand j immunol. 2013;77:482-7. doi: 10.1111/sji.12048. received: 2018-04-06 без имени 71 b io m e d ic a l s c ie n c e s issn 2413-6077. ijmmr 2018 vol. 4 issue 1 international journal of medicine and medical research 2018, volume 4, issue 1, p. 71-77 copyright © 2018, tsmu, all rights reserved doi 10.11603/ijmmr.2413-6077.2018.1.8733 cytokines profile in experimental contact allergic dermatitis and use of nanoencapsulated agents i. i. khudan-tsilo, o. o. shevchuk, m. m. korda i. horbachevsky ternopil state medical university, ternopil, ukraine background. inflammation, oxidative and nitro-oxidative stress are the essentials of the pathogenesis of contact allergic dermatitis as well as cytokines imbalance. objective. the concentration of tnf-α, il-1β, il-4 and il-10 in blood serum of rats with nickel-induced contact allergic dermatitis was evaluated to determine whether it correlated with the use of free and nanoencapsulated preparations of betamethasone, superoxide dismutase (sod) and potent highly selective inhibitor of inos (1400w). methods. to induce contact dermatitis (cd), 5 % nickel sulfate was used for 12 days. experiments were performed on white inbred male rats, 180–220 g of body mass. all rats were divided into 10 groups (n=10). group i – the control one; ii – the animals with cd; iii – the rats with cd treated with empty polymeric chitosan nanoparticles; groups iv–vi – the rats with cd treated with free sod, 1400w and betamethasone; groups vii-ix – the rats administered with nanoencapsulated sod, 1400w and betamethasone; x – cd + nano-composition of all agents. results. the statistically higher serum concentrations of tnf-α, il-1β and decrease of il-4 and il-10 in experimental contact dermatitis is proved in comparison with the healthy rats. mono-treatment with betamethasone, sod and 1400w is efficient, but the use of nanoparticles loaded with these preparations surpasses its effects. the use of the combination of all nanoencapsulated medicines is the most effective. conclusions. chitosan nanoparticles loaded with topical anti-inflammatory glucocorticoid, and inhibitors of oxidative and nitro-oxidative stress is a promising method for treatment of allergic contact dermatitis and can be recommended for further research and use in clinics. key words: contact nickel dermatitis; cytokines; nanoparticles; betamethasone; sod; 1400w. introduction contact dermatitis (cd) frequency has been rising recently [1, 2]. its two types are differentiated: triggered by antigens as allergic contact dermatitis and by obligatory cutaneous irritants – irritant contact dermatitis (nonimmunologic driven reaction) [3]. all types of dermatitis (atopic, psoriatic, allergic, etc.) are challenge for health care system because of chronic and relapsing character. it is forth reason for doctor’s visiting among all skin disorders [4]. today the first choice and the mainstay of dermatitis drug therapy are topical glucocorticoids (tgs): betamethasone, dexamethasone, fluocinolone, triamcinolone, etc. in spite of their high efficacy a lot of side effects (systemic and local) are common: skin atrophy, acneiform eruption, striae, and telangiectasia, as well as hypothalamic–pituitary-adrenal axis suppression, glaucoma development, high risks of thrombosis and others [5, 6]. one more serious challenge for dermatitis treatment is transdermal delivery of active components. skin is a prime barrier against a lot of environmental physical, chemical, and biological stressors (ultraviolet irradiation, bacteria, viruses, allergens, etc.). stratum corneum, claudins and occludins of tight junctions are crucial in defense, but also impede the absorption of topical medicines [7]. nanoencapsulated medications have been developed as a vehicle into the deeper skin layers and demonstrated its benefits [8–10]. nanotechnology through the reduced particle size improves the absorption and concentration of the drug in the target tissue, its pharmacokinetic parameters and long-term release of the medication at the target site [11]. it is established at present that even regular use of tgs does not prevent the relapse of cd and does not provide the appropriate control of its severity [12]. cytokines are crucial in pathogenesis of different types of dermatitis (allergic, atopic, etc.). contact allergic dermatitis corresponding author: oksana shevchuk, associate professor, department of pharmacology and clinical pharmacology i. horbachevsky ternopil state medical university, 1 maydan voli, ternopil, 46000, ukraine e-mail: shevchukoo@tdmu.edu.ua phone number: +38(0352)52-44-92 i. i. khudan-tsilo et al. 72 b io m e d ic a l s c ie n c e s issn 2413-6077. ijmmr 2018 vol. 4 issue 1 is mediated via t lymphocytes. contacting the a l l e r g e n s e p i t h e l i a l c e l l s m a y p ro d u c e mediators, which intensify the cascade of immune response [13]. the combination of sensitized t cell plus antigen releases cytokines cascade and lead to inflammation [14]. the objective of the research is to study the cytokines profile in experimental allergic contact dermatitis and its correction with nanoencapsulated steroid anti-inflammatory agent, antioxidant and inos inhibitor. methods a n i m a l s a n d e x p e r i m e n t a l d e s i g n . experiments were performed on white inbred male rats 180–220 g of body mass. all rats were divided into 10 groups, 10 animals in the each one. group i – the control one involved the animals sensitized only with solid lanoline base; group ii – the animals with contact dermatitis (cd) induced by sensitization with 5 % niso4 dissolved in the base; group iii – the rats with cd treated with empty polymeric chitosan nanoparticles; groups iv–vi involved the rats with cd treated with free sod, 1400w and betamethasone accordingly; groups vii–ix – the rats, which were administered with nanoencapsulated sod, 1400w and betamethasone; group x – cd + nano-composition of sod, betamethasone and 1400w. to induce the contact dermatitis the hair on the dorsal area, 4x4 cm in size, was removed and the cleaned skin area was administered with 4 g of solid lanoline composition containing 5 % nickel sulfate for 12 days. all correctors were used for one week after the development of cd twice a day. we used sigma-aldrich (usa) beta methasone 17,21-dipropionate, pegylated superoxide dismutase (sod) and n-([3-(aminomethyl) phenyl]methyl)ethanimidamide dihydrochloride (1400w)-potent highly selective inhibitor of inos. chitosan nanoparticles loaded with above-mentioned preparations were prepared according to the method described by hussain z., et al. [15]. chitosan solution (25 ml, 0.2 % w/v, prepared in 1 % v/v acetic acid, ph 5.0) was incubated with betamethasone solution (1 mg/ml in a 30:70 mixture of ethanol/water) or 1400w (1 mg/ml) or sod solution (10 mg/ml) stirring for 30 minutes. loaded nanoparticles were spont a n e o u s l y f o r m e d by a d d i n g 1 0 m l o f pentasodium tripolyphosphate solution (0.1 % w/v, in distilled water) dropwise with constant stirring at 700 rpm. the nanoencapsulated preparations were harvested by ultra centrifugation (28,000 rpm) for 30 minutes and subsequently resuspended. the size of nanoparticles was determined using the morphometric program video-test–5.0, кappa imagebase, and it was 40–100 nm. all procedures for the animals were p e r f o r m e d a c c o rd i n g t o t h e r u l e s a n d requirements of european convention for the protection of vertebrate animals used for experimental and other scientific purposes, directive 2010/63/eu and were approved by local tsmu ethic committee. the rats were euthanized under the ketamine anesthesia on the 20th day of examination. amounts of cytokines in blood serum were evaluated by elisa test (raybiotech inc., norcross, ga, usa). the concentrations of tnf-α, il-1β, the th2specific cytokines il-4 and il-10 were analyzed. statistical analysis. the data are presented as mean±se standard error. statistical analysis was performed by the statistica 10.0 (statsoft inc., usa) program. the distribution of indices was estimated using shapiro-wilk normality test. the significance of the results was determined by the mann-whitney u-test and anova-test. a probability level of less than 0.05 was considered as statistically significant. results it has been established that in cd, induced by nickel sulfate, blood serum concentration of fig. 1. schematic diagram of experimental design. necropsy, blood samples day 20 correction of contact allergic dermatitis day 13–19 development of nickel-induced contact allergic dermatitis day 1–12 5 % niso4 free and nanoencapsulated betamethasone, sod, 1400w and its combination blood serum concentration of tnf-α, il-1β, il-4 and il-10 i. i. khudan-tsilo et al. 73 b io m e d ic a l s c ie n c e s issn 2413-6077. ijmmr 2018 vol. 4 issue 1 pro-inflammatory cytokines tnf-α and il-1β significantly increased in 5.5 and 3.7 times compared to the intact rats (table 1). antiinflammatory th2-specific cytokines il-10 and il-4 concentration was significantly lower, in 2.3 times and 2.4 times respectively. table 1. blood serum concentration of cytokines in experimental contact nickel dermatitis, m±m, n=10 groups interleu kins intact rats cd cd + e m pt y na no pa rt ic le s cd + b et am et ha so ne cd + n an obe ta m et ha so ne cd + s o d cd + n an oso d cd + 1 40 0w cd + n an o14 00 w cd + n an oco m bi na tio n of b et am et ha so ne , s o d an d 14 00 w tnf-α, pg/ml 7.48 ±0.56 41.16 ±4.10* 40.54 ±3.98* 26.98 ±2.30 *,** 20.94 ±1.16 *,**, #1 35.48 ±1.95 *,** 30.22 ±2.02 *,**, #2 36.22 ±1.78 *,** 30.12 ±1.56 *,**, #3 12.88±1.28 *,**,γ123 il-1β, pg/ml 10.46 ±0.88 37.98 ±2.42* 35.88 ±3.36* 24.77 ±1.75 *,** 19.35 ±1.28 *,**,#1 31,32 ±1.54*, ** 26.45 ±1.54 *,**, #2 32.16 ±1.98 * 26.66 ±1.38 *,**, #3 14.48±1.34 *,**,γ123 il-10, pg/ml 12.05 ±1.08 5.24 ±0.42* 5.15 ±0.42* 7.54 ±0.52 *,** 8.96 ±0.32 *,**,#1 6.88 ±0.50 *,** 7.88 ± 0.64 *,** 6.32 ±0.44* 8.48 ±0.46 *,**, #3 10.33±0.48 **,γ123 il-4, pg/ml 19.26 ±1.88 8.02 ±0.78* 7.68 ±0.66* 11.43 ±0.45 *,** 13.16 ±0.58 *,** 10.04 ±1.00* 11.25 ±0.78 *,** 11.14 ±0.32 *,** 13.26 ±0.44 *,**,#3 15.68±0.77 **,γ123 notes: statistical significance р<0.05 compared to: *intact rats; **cd rats; #1 – rats with cd treated with free form of betamethasone, #2 – free form of sod, #3 – free form of 1400w, γ1 – nanoencapsulated betamethasone, γ2 – nanoencapsulated sod, γ3 – nanoencapsulated 1400w. the use of empty nanoparticles did not cause any significant changes in the indices. use of each nanoencapsulated agent was more efficient to restore the cytokines imbalance in comparison with free form usage. but the most prominent effect was caused by the combination of all three nanoencapsulated correctors. betamethasone is a topical corticosteroid of high potency. its free form caused positive changes in cytokines profile: the decrease in pro-inflammatory tnf-α and il-1β concentration by 34.5 and 34.8 %; and increase of antiinflammatory il-10 and il-4 – by 43.9 and 42.5 % was evidenced. but nanoencapsulated tg was more effective and surpassed its free form efficacy. tnf-α and il-1β rates were statistically significantly lower in this group in comparison with cd group by 49.1 and 49.05 %; in comparison with its free form by 22.4 and 21.9 %. also the increase in anti-inflammatory interleukins il-10 and il-4 concentration was evidenced in group cd + nano-betamethasone: by 71.0 and 64.1 % in comparison with pathology; and by 18.8 % for il-10 in comparison with free form of tg. the same tendency was for free and nanoencapsulated forms of sod. the use of free sod caused decreased concertation of tnf-α and il-1β by 11.4 and 17.5 %; and increased rates of il-10 and il-4 by 31.3 and 25.2 % accordingly. nanoencapsulated sod surpassed the activity of its free form in decreasing of concentration of pro-inflam matory cytokines. the rate of tnf-α was statistically significantly lower by 17.2 %, il-1β – by 15.5 %. but there was a positive tendency only in correction of anti-inflammatory inter leukins indices. highly selective inhibitor of inos was also quite efficient. the concentration of tnf-α decreased by 9.6 % in use of its free form; and by 26.8 and 29.8 % – of nanoencapsulated 1400w accordingly. the last rates were statistically significantly improved by 19.1 % for tnf-α and 17.0 % – for il-1β in comparison with cd + 1400w group. il-4 concentration increased by 38.9 % in use of free 1400w; and by 65.3 % – of nanoencapsulated one in comparison with control pathology (the last index was higher by 19.0 % compared to cd + 1400w group). value of il-10 was statistically significantly different only in case of use of nano-form of 1400w by i. i. khudan-tsilo et al. 74 b io m e d ic a l s c ie n c e s issn 2413-6077. ijmmr 2018 vol. 4 issue 1 61.8 % in the control group and by 34.2 % in cd + 1400w group. the best results were evidenced in the group cd + nano-combination of beta methasone, sod and 1400w. the combination of all nanoencapsulated agents was the most efficient in restoring of cytokines balance in comparison with mono-use of each corrector (table 1). discussion among a lot of allergens, nickel is one of the most common metals responsible for allergic cd. its regular contacting for hypersensitive persons leads to a delayed-type hypersensitivity reaction and up to 20 % of the general population may be affected in europe [16–18]. high frequency of positive nickel patch tests and high serum nickel concertation are also observed in people with intrinsic atopic dermatitis [19]. pathogenesis of allergic contact dermatitis includes sensitization (or induction) and elicitation (or efferent) phases, which are innate (opposite to contact irritant dermatitis) [20]. it is established at present that cytokines are crucial in inflammation, including immuneinvolved dermatitis [21–23]. a lot of skin cells are able to produce cytokines [24] (table 2). dermal dendritic cells are crucial in the process of activating naive t cells; after contacting the allergen langerhans cells start to synthesize il-1β mrna and to release the protein; then, keratinocytes are activated and release tfn-α and gm-csf [25]. table 2. cytokines developed by epidermal cells epidermal cells cytokine (constitutive or inducible expression) keratinocytes i l 1 α , i l 1 β , i l 1 r a , i l 3 (mouse), il-6, il-7, il-8, il-10, il-12, il-15, il-18, tnf-α, g-csf, gm-csf, mcsf, gro, mip-2 (mouse), ip-10, rantes, mcp-1, tgf-α, tgf-β langerhans cells il-1α, il-1β, il-6, il-15, il-18, tnf-α, gro, mip-2, mip-1α, tgf-β melanocytes il-1α, il-1β, il-6, il-7, il-8, il10, il-12, tnf-α, g-csf, gmc s f , m c s f , g r o , m i p 2 (mouse), rantes, mcp-1, tgf-α, tgf-β skin hypersensitivity reactions are the result of imbalance between a variety of types of t-cell responses and inflammatory mediators, including t-helper (th) 2 cytokines and also t-helper 1 cells [26]. a th1/th2 hypothesis suggests that imbalance among these cells toward th2 is the key to allergic reaction intensifying [22, 27]. the th1 cells release of pro-inflammatory cytokines includes il-1α, il1β, il-8, il-13, tnf-α, and granulocyte-macrophage colony-stimulating factor (gm-csf). they increase the vascular permeability and cause swelling and redness associated with inflammation and immunologic reactions in skin exposed to irritants, also affect the proliferation and differentiation of keratinocytes and mediators of cellular infiltration [3, 21, 22]. the key regulators are tfn-α and il-1β. these pleiotropic cytokines are responsible for inflammation, apoptosis and necrosis of cells, phagocytic and cytotoxic activities [28]. il-10 is an anti-inflammatory interleukin produced mainly by monocytes, t cells, macrophages, and dendritic cells. mast cells can also produce il-10, which limits the rate of leukocyte infiltration, inflammation, and skin disorders such as contact dermatitis [21]. il-4 and il-10 suppress th1 immune response. il-10 deficiency is crucial in maintaining of cd clinical signs [29]. the pro-inflammatory cytokines tnf-α and il-1β and the th1/th2 cytokines ifnγ and il-4 are involved in both the induction and elicitation of cutaneous immune response and are modulated by glucocorticoids, that is also proved by the results of our study. gluco corticoids decrease tnf-α and il-1β production and shift the th1/th2 ratio in a th2 direction by augmenting production of il-4 [16, 30]. mechanisms of such imbalance are asso ciated with activation of oxidative stress, which is realized by generation of reactive oxygen and nitric species (ros and rns) and depletion of antioxidant defense. ‘vicious circle’ is running here: low antioxidant indices intensify the release of cytokines and inflammation processes that promotes the generation of ros and rns again. use of nano-encapsulated medications is emerging as potential therapeutics for a wide variety of diseases [22]. they have been successfully used for delivery of hydrophobic and hydrophilic small molecule drugs and biomacromolecules, nucleic acids of various sizes and structures. it is very important for skin diseases treatment to go through the stratum corneum to target tissues and subcellular compartments and nanoparticles are quite i. i. khudan-tsilo et al. 75 b io m e d ic a l s c ie n c e s issn 2413-6077. ijmmr 2018 vol. 4 issue 1 successive for that. nano-form of preparations, especially its combination was the most powerful to alleviate experimental contact dermatitis clinical sings and intensity of oxidative and nitro-oxidative stress [31, 32]. our previous findings proved the importance of oxidative and nitro-oxidative stress activation in pathogenesis of cd. the last one is aggravated by cytokines. nitric oxide synthase (nos) is an enzyme that catalyzes the synthesis of no and l-citrulline using oxygen and l-arginine as substrates. and its isoform inos is induced by the cytokines tnf-a, il-1 [33]. so, modulation of no-synthase (nos) activity, use of antioxidants and free radical scavengers are important and prospect part of complex treatment for different types of cd (plus common recommendations for allergens avoiding, emollients and others) [2, 33, 35]. the perspective results of combined treatment with betamethasone-, sod (superoxide dismutase) and 1400w (highly selective inhibitor of inducible nitric-oxide synthase, inos)-loaded nanoparticles were obtained. such combination of the potent anti-inflammatory steroid agent, the powerful antioxidant and inos-inhibitor in nanoencapsulated form was more effective in experimental contact dermatitis than use of their free forms [31, 36]. conclusions experimental contact dermatitis induced by 5 % nickel sulfate is accompanied by pathological cytokines imbalance. mono-treatment with betamethasone, sod (superoxide dismutase) and 1400w (highly selective inhibitor of inducible nitric-oxide synthase, inos) was efficient, but the use of nanoparticles loaded with these agents surpassed its effects. the use of combination of all nanoencapsulated medicines was the most effective. chitosan nanoparticles loaded with topical anti-inflammatory glucocorticoid, and inhibitors of oxidative and nitro-oxidative stress is a promising method for treatment of allergic contact dermatitis and can be recommended for further research and use in clinics. acknowledgments the research was conducted as a part of s c i e n t i f i c i n v e s t i g a t i o n a n d r e s e a r c h 0112u000542 “biochemical mechanisms of toxicity of nanoparticles of different origin and other anthropogenic and biogenic toxicants and biological systems” and 0116u003353 “biochemical mechanisms of metabolism disorders in case of body intake of toxicants of different genesis”. i. i. khudan-tsilo et al. цитокіновий профіль при експериментальному контактному дерматитi та використанні наноінкапсульованих препаратів і. і. худан-цільо, о. о. шевчук, м. м. корда тернопільський державний медичний університет імені і. я. горбачевського, тернопіль, україна вступ. запалення, оксидативний та нітрооксидативний стрес є основою патогенезу контактного алергічного дерматиту та розвитку дисбалансу цитокінів. мета дослідження полягала у вивченні змін цитокінів tnf-α, il-1β, il-4 і il-10 в сироватці крові щурів з контактним нікель-індукованим дерматитом при використанні вільних і наноінкапсульованих препаратів бетаметазону, супероксиддисмутази (сoд) і потужного високоселективного інгібітора inos (1400w). методи. для моделювання контактного дерматиту (кд) 5% сульфат нікелю наносили на шкіру щурам протягом 12 днів. білі щури-самці масою тіла 180–220 г було рандомізовано на 10 груп (n = 10): i група контрольна; ii тварини з кд; iii щури з кд, які отримували полімерні хітозанові наночастинки; iv – vi групи щури з кд, які отримували вільні форми сод, 1400w та бетаметазону відповідно; vii-ix групи щури, яким вводили наносод, нано-1400w і нано-бетаметазон; x група кд + нанокомпозиція всіх препартів корекції. результати дослідження. доведено, що при експериментальному контактному дерматиті достовірно зростає рівень прозапальних цитокінів tnf-α, il-1β у сироватці та спостерігається зниження il-4 і il-10 у порівнянні зі здоровими щурами. монотерапія препаратами бетаметазону, сод і 1400w є ефективною, однак використання нанаінкапсульованих форм перевищує результати монозастосування кожного препарату. найбільш ефективним є використання поєднання всіх наноінкапсульованих лікарських засобів. висновки. хітозанові наночастинки з інкапсульованими препаратами топічного протизапального глюкокортикоїда та інгібіторів оксидативного і нітрооксидативного стресу є перспективним методом лікування контактного дерматиту і можуть бути рекомендовані для подальшого вивчення з впровадженням у клінічну практику. ключові слова: контактний нікелевий дерматит; цитокіни; наночастинки; бетаметазон; сод; 1400w. 76 b io m e d ic a l s c ie n c e s issn 2413-6077. ijmmr 2018 vol. 4 issue 1 references 1. peiser m, tralau t, heidler j, api am, arts jhe, basketter da, et al. allergic contact dermatitis: epidemiology, molecular mechanisms, in vitro methods and regulatory aspects. cell mol life sci 2012;69:763–81. doi:10.1007/s00018-011-0846-8. 2. brasch j, becker d, aberer w, bircher a, kränke b, jung k, et al. guideline contact dermatitis. 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3 : 1 4 4 – 8 . d o i : 1 0 . 1 1 6 0 3 / 2 4 1 5 8 7 9 8 . 2017.3.8135 [in ukrainian]. received: 2018-03-29 i. i. khudan-tsilo et al. 10 in t e r n a l m e d ic in e issn 2413-6077. ijmmr 2019 vol. 5 issue 1 doi 10.11603/ijmmr.2413-6077.2019.1.9693 evaluation of serum γ-glutamyl transferase and its association with high sensitivity c-reactive protein and insulin levels in the patients with metabolic syndrome r. dharuni1*, b.v. maruthi prasad2, h.l. vishwanth2 1 – sapthagiri institute of medical sciences and research centre, bangalore, india 2 – bangalore medical college and research institute, bangalore, india background. metabolic syndrome (ms), a collection of cardiovascular risk factors, is a major worldwide public health problem. the gathered data prove that serum gamma-glutamyl transferase (γgt) activity is a true marker of atherosclerotic cardiovascular disease (cvd) and is of a prognostic importance as well as the highsensitivity c-reactive protein (hs-crp). objectives. in the study, we sought to evaluate serum γgt activity, hs-crp and insulin resistance in patients with ms. methods. the study involved 50 persons with metabolic syndrome and 50 healthy age and sex matched controls. fasting serum samples of all participants were investigated for γgt, hs-crp, insulin, blood glucose, lipid profile and liver function tests. anthropometric measurements and bmi were also calculated results. in that case 50% showed significantly high γgt compared to the controls, 30% proved increased hs-crp levels above >0.5 mmol/l, whereas 94% of the controls were within the reference range. 74% of cases revealed the presence of insulin resistance while 32% of the controls showed insulin resistance. high γgt levels were also observed in that case with deranged lipids levels and high bmi. conclusions. the study suggests that the patients with ms have a higher serum γgt activity. this study also proves that hs-crp and homa-ir, which are independent risk factors of cvd, are also associated with ms. the correlation between γgt and the components of ms are also found significant compared to hs-crp. thus, γgt can be considered as an inexpensive and authentic predictor of ms, which can be a manifestation of cvd in near future. key words: metabolic syndrome; gamma-glutamyl transferase; high sensitivity c-reactive protein; homa-ir. *corresponding author: dr. r. dharuni, department of biochemistry, sapthagiri institute of medical sciences & research centre, hesarghatta main road, bangalore, karnataka, india phone no.: +91-9535101939 email: dharunii@gmail.com introduction metabolic syndrome (ms) is defined by a constellation of risk factors of cardiovascular disease (cvd), that include abdominal obesity, dyslipidemia, hypertension, and impaired glucose tolerance, which increase the risk of cvd and diabetes mellitus [1]. ms has been considered as one of the threatening non communicable public-health problem globally [2]. serum gamma-glutamyl transferase (γgt) has long been considered a harbinger of hepatic dysfunction and alcohol intake [3]. recently, accumulating epidemiology studies have revealed that γgt contributes in several pathophysiological processes, including oxidative stress and lipid peroxidation, which are important for pathogenesis and develop ment of insulin resistance as well as ms [4, 5, 6]. in addition, when compared with other hepatic markers, γgt was the major predictor of type 2 diabetes [7,8,9]. γgt is a possible risk factor and a prognostic indicator of cvd. further information is needed regarding the magnitude of the risk associated with γgt activity and individual cardiometabolic disorders. such a relationship could help to decipher a high prevalence of ms. perhaps excessive energy consumption, which leads to obesity, is a more serious and frequent nutritional problem, but there can be a gradual and fairly predictable transition from simple obesity with no observable metabolic changes through insulin resistance. insulin resistance arises from the inability of insulin to act normally in regulating nutrient metabolism in peripheral tissues. increasing evidences of human population studies and animal research have established correlative as well as causative relations between chronic inflammation and insulin resistance [10]. chronic, systemic, subclinical inflammation has also been identified as a driving force for insulin resistance. since international journal of medicine and medical research 2019, volume 5, issue 1, p. 10-16 copyright © 2019, tnmu, all rights reserved r. dharuni et al. 11 in t e r n a l m e d ic in e issn 2413-6077. ijmmr 2019 vol. 5 issue 1 hs-crp is a marker of systemic inflammation, it might explain the prevalence of insulin resistance in ms. nevertheless, the relationship remains uncertain and has not been well researched yet. therefore, the aim of this study was to examine the associations of serum γgt, hs-crp and insulin resistance in the individuals with ms as well as its components. methods source of data this study was a hospital based cross sectional study, which comprised metabolic syndrome patients attending the outpatient and inpatient departments of medicine. the study was approved by the local ethical committee of the institute and the informed consents were obtained from all subjects, who took part in the study. selection of subjects all subjects were diagnosed according to national cholesterol education program, adult treatment panel iii criteria and it required the presence of 3 or more of the following [2]: a) fasting blood glucose ≥ 6.105 mmol/l; b) serum triglyceride ≥1.71 mmol/l or being on lipid lowering therapy; c) serum hdl < 2.220 mmol/l in men and < 2.775 mmol/l in women or being on antilipidemic therapy; d) blood pressure ≥130 mmhg systolic and/or ≥85 mmhg diastolic or being on antihypertensive therapy; and e) waist circumference >102 cm in men and >88 cm in women. the subjects with following history were excluded. alcohol intake more than 30 g/day (≈38 ml of 100% alcohol) and the patients with smoking history, hepatitis b or c infection or other known liver diseases, liver enzymes exceeding the upper reference range in three times, use of hepatotoxic drugs, acute infectious/inflammatory conditions, familial hyperlipidemia, new york heart association class 3-4 heart failure. sample size after consulting a statistician, sample size was estimated to be 100, with 50 cases and 50 age and sex matched healthy controls. type of study: a cross sectional observational study. method of sample collection the informed consents were taken from the patients and control subjects. the selected subject’s blood samples were collected with all aseptic precautions. 5 ml of blood was collected from median cubital vein. the collected blood was allowed to clot for 30 minutes in a clean dry test tube and was subjected to centrifugation to separate the serum. the serum samples were stored in a deep freezer at -800c till they were studied. the following parameters were considered appropriate for the study: 1. serum insulin levels defined by chemiluminescence method and insulin resistance by homeostasis of model assessment of insulin resistance (homa-ir). 2. serum γgt by colorimetric method. 3. hs-crp by chemiluminescence method. 4. renal and liver function tests by colorimetric method. 5. lipid profile by enzymatic, colorimetric method. 6. fasting blood sugar by hexokinase method. 7. measurement of body mass index. 8. to measure waist circumference, top of right iliac crest was located. a measuring tape was placed in a horizontal plane around abdomen at level of iliac crest. before reading the measurements, it was estimated that the tape was snug but did not compress the skin and was parallel to floor. the assessment was performed at the end of normal expiration. statistical analysis descriptive and inferential statistical analysis has been carried out during the study. the results on continuous measurements are presented on mean±sd (min-max) and the results on categorical measurements are presented in number (%). statistical processing of the research results was performed by parametric analysis with the calculation of student’s t-test using the software package microsoft excel 5.0. chi-square test was used to find the significance of study parameters on categorical scale between two or more groups. pearson correlation between γgt and homa-ir and hscrp were performed to measure the strength between variables and relationships. results the clinical characteristics of the study population are presented in table 1. the current study is a case control study, in which the serum γgt, hs-crp and insulin levels were determined in 50 metabolic syndrome subjects and were compared with 50 healthy age and sex matched controls. the results were tabulated and statistically analyzed. the metabolic syndrome patients were diagnosed according to the national cholesterol education program’s adult treatment panel iii criteria (ncep atp iii criteria). the study r. dharuni et al. 12 in t e r n a l m e d ic in e issn 2413-6077. ijmmr 2019 vol. 5 issue 1 population belonged to age group ranging 4070 years old, which was similar in the controls as well. the mean±sd of the cases and controls were 51.4±9. 7 years old and 50.2±9 years old respectively, which suggested that metabolic syndrome was prevalent in late middle ages. waist circumference (wc) and body mass index (bmi) are the two important anthropometric measurements among the various definitions of metabolic syndrome. the study proved the mean±sd for wc in that case as 104±9.5 cm and in the controls as 82.5±10.3 cm. and the mean bmi in that case was 29.58±3.96 kg/m2 and in the controls – 23.14±2.52 kg/m2. both these parameters were significantly higher in the cases with p≤0.001. the biochemical characteristics of the study population are presented in table 2. the mean concentration of fasting blood glucose in the controls was 4.1±0.93 mmol/l; in that case it was 6.5±2.1 mmol/l, which was significantly increased in the subjects with ms. increased triacylglycerols and decreased hdl-cholesterol were potential markers of cvd. in this study, mean triglycerides in metabolic syndrome cases was 1.86±0.96 mmol/l and in the controls, it was 1.41±0.8 mmol/l, which was significantly higher. hdl-cholesterol levels in cases were found to be 0.73±0.2mmol/l and 0.96±0.3mmol/l in the controls. the lower hdlcholesterol levels in that case was found to be significant with p<0.005. the mean±sd of γgt in that case was 6 0 . 9 6 ± 4 5 . 6 4 u / l a n d i n t h e c o n t r o l s 29.78±18.01u/l with a p value <0.001**. the mean±sd of serum insulin in that case was 29.34±26.94 μiu/ml and in the controls 11.97±5.98 μiu/ml with p value ≤0.01**. the mean±sd of hs-crp in that case was 76.2±47.6 mmol/l and in the controls 27.6± 11.4 mmol/l with p value ≤0.001**. the mean±sd of homair in that case was 9.44±4.39 and in the controls 2.32±1.48 with p value ≤0.001**. the comparison of γgt, insulin, hs-crp, homa-ir is presented in table 3. pearson correlation was completed to analyse the relationship between γgt, hs-crp and homa-ir in ms cases are as presented in table 4. γgt table 1. clinical characteristics of the study population parameters controls cases p value none of the subjects 50 50 sex (male/female) 18/32 20/30 0.68 age 50.2±9 51.4±9.7 <0.05* bmi (kg/m2) 21.5±3.5 29.6±3.9 <0.001** waist circumference (cm) 82.5±10.3 104±9.5 <0.001** note: the values expressed as mean ± sd. t-test was used for groups’ comparison. the differences in proportions were assessed by means of chi-square test. * suggestive significance (p value p<0.05); ** strongly significant (p value p≤0.001). table 2. biochemical characteristics of the study population variables controls cases p value glucose (mmol/l) 4.1±0.93 6.5±2.1 <0.01* total cholesterol (mmol/l) 3.8±1.16 4.3±1.38 <0.05* triglycerides (mmol/l) 1.41±0.8 1.86±0.96 <0.05* hdl cholesterol (mmol/l) 0.96±0.3 0.73±0.2 <0.05* serum albumin (mmol/l) 36.7±8.2 31.8±7.4 <0.01** aspartate aminotransferase (u/l) 19.8±7.9 24.26±15 0.06 alanine aminotransferase (u/l) 17±9.7 22.38±12.1 <0.01** alkaline phosphatase (u/l) 71.4±25.6 83.5±33.9 <0.05* serum phosphate (mmol/l) 1.1±0.2 0.9±0.2 <0.01** serum creatinine (μmol/l) 61.9±26.5 53.5±0.3 <0.01** notes: the values expressed as mean sd. t-test was used for groups comparison. * suggestive significance (p value <0.05); * moderately significant (p value <0.01); ** strongly significant (p value ≤0.001). r. dharuni et al. 13 in t e r n a l m e d ic in e issn 2413-6077. ijmmr 2019 vol. 5 issue 1 showed a positive correlation with homa ir and hs-crp which was of suggestive significance. discussion ms comprises a group of atherogenic factors [11]. besides, the gathered data have reported of many biochemical and anthropometric parameters associated with ms, together with parameters of obesity and products released by adipose tissue, plasma insulin levels, liver enzymes, and crp [12, 13]. many epidemiology studies have proved that circulating serum γgt levels may be associated with the evolvement and clinical progression of cvd, even after adjusting for confounding factor like alcohol consumption [14, 15]. although high levels of γgt have been speculated to be directly atherogenic [16], just like several other biomarkers for ms, a direct causation of atherosclerosis remains to be elucidated. as presented in table 3, a higher γgt along with insulin resistance levels in ms involves a potentially greater risk for subsequent development of type 2 diabetes. the increasing evidences have proved that the circulating γgt, which is primarily synthesized from liver, is a key target organ for development of ms. a number of studies have also shown that the serum level of γgt directly correlates with an increased risk of ms [17]. this was evidenced by significant correlations between γgt levels and all mets components, independently of age and gender, except for blood pressure values [18]. hardly any studies have proved increased γgt activity in hypertensives, which could be associated with the relation between γgt and ms [19, 20]. the association between the serum γgt and hs-crp (table 2), which is, as put forward by ortega et al. [21], the low-grade inflammation in liver caused by hepatic steatosis in ms, could have caused increase in γgt levels. hs-crp, an acute-phase reactant of hepatic origin and a sensitive marker for systemic inflammation, predicts the occurrence of diabetes, metabolic syndrome and atherosclerotic diseases in healthy subjects [23]. it has been hypothesized that increased γgt levels might occur before elevation in crp, and the related oxidative stress would give rise to a subsequent inflam_ matory response [24]. also, fatty infiltration in liver might have enhanced oxidative stress, leading to glutathione metabolism with com_ pensatory increase in γgt secretion. as γgt activity reflects oxidative stress and inflammation, the increased levels can actively predict the incidence of ms [17]. many studies have proved the association between the increased γgt and insulin resistance, as well as the subsequent development of type 2 dm [14, 19]. the increase of γgt levels in serum might be as a result of secondary hepatic inflammation [22]. conclusions this study suggests that increased gammag l u t a m y l t r a n s f e r a s e a c t i v i t y c o u l d b e considered as harbinger of low-grade systematic table 3. comparison of γ-gt, insulin, hs-crp, homa-ir in both study groups parameters controls cases p value γ-gt (u/l) 29.78±18.01 60.96±45.64 <0.001** insulin (μiu/ml) 11.97±5.98 29.34±26.94 <0.01* hs-crp (mmol/l) 27.6±11.4 76.2±47.6 <0.001** homa-ir 2.32±1.48 9.44±4.39 <0.01* notes: values expressed as mean±sd. z-test was used for groups comparison *. suggestive significance (p value <0.05). * moderately significant (p value <0.01); ** strongly significant (p value ≤0.001). table 4. pearson correlation of γ-gt, homa-ir, hs-crp in metabolic syndrome parameters metabolic syndrome (n=50) r value p value γgt vs homa-ir 0.26 0.060+ γgt vs hs-crp 0.252 0.078+ hs-crp vs homa-ir 0.207 0.15 notes: + suggestive significance. r. dharuni et al. 14 in t e r n a l m e d ic in e issn 2413-6077. ijmmr 2019 vol. 5 issue 1 inflammation and oxidative stress through mediation of glutathione transport. current study contributes to the increasing number of evidences that gamma-glutamyl transferase estimation in metabolic syndrome, which is simple and inexpensive, could be considered among the strongest serum predictors of insulin resistance, imminent type 2 diabetes and cardiovascular events. conflict of interest the authors declare no conflict of interest. взаємозв'язок між показниками гамма-глютамілтрасферази, високочутливого с-реактивного білка та рівня інсуліну у пацієнтів з метаболічним синдромом r. dharuni1, b.v. maruthi prasad2, h.l. vishwanth2 1 – sapthagiri institute of medical sciences and research centre, bangalore, india 2 – bangalore medical college and research institute, bangalore, india вступ. метаболічний синдром (мс), як сукупність факторів ризику розвитку серцево-судинних захворювань (ссз), є важливою проблемою охорони здоров'я. отримані дані свідчать про те, що активність сироваткової гамма-глютамілтрансферази (ггт), у якості маркера атеросклеротичного процесу при ссз, має прогностичне значення, як і показники високочутливого с-реактивного білка (вчсрб). мета. дослідити активність сироваткової ггт, вч-срб та інсулінорезистентність у пацієнтів з метаболічним синдромом. методи. дослідження включало 50 пацієнтів з мс та 50 здорових осіб. зразки сироватки були взяті у всіх учасників натщесерце для дослідження активності ггт, рівня вч-срб, інсуліну, глюкози в крові, оцінки ліпідного профілю та проведення печінкових проб. також були розраховані антропометричні показники та індекс маси тіла (імт). результати. у 50% досліджуваних з метаболічним синдромом активність ггт була достовірно вищою відносно контрольної групи, у 30% діагностовано підвищений рівень вч-срб (вище >0,5 ммоль/л), тоді як показники 94% осіб контрольної групи знаходилися в діапазоні норми. у 74% випадків виявлено наявність інсулінорезистентності у пацієнтів з метаболічним синдромом, тоді як у контрольній групі цей показник склав 32%. висока активність ггт також спостерігалася при порушеннях ліпідного профілю та високому імт. висновок. отримані нами дані свідчать, що у пацієнтів з метаболічним синдромом активність сироваткової гамма-глютамілтрансферази достовірно вища, порівняно з контрольною групою. також нами встановлено, що показники високочутливого с-реактивного білка та рівень інсулінорезистентності, які є незалежними факторами ризику серцево-судинних захворювань, також асоціюються з метаболічним синдромом. встановлено кореляційні зв’язки між активністю гамма-глютамілтрансферази та високочутливого с-реактивного білка. таким чином, показники сироваткової гамма-глютамілтрансферази можна розглядати як економічно доступний та достовірний предиктор метаболічного синдрому, що може передувати появі серцево-судинних захворювань у найближчому майбутньому. ключові слова: метаболічний синдром; гамма-глютамілтрансфераза; високочутливий с-реактивний білок; індекс нома-ir (homeostasis model assessment of insulin resistance). information about authors r. dharuni – assistant professor, department of biochemistry, sapthagiri institute of medical sciences and research centre, bangalore, karnataka, india. orcid 0000-0002-3543-2810, e-mail: dharunii@gmail.com b.v. maruthi prasad – professor, bangalore medical college and research institute, bangalore, karnataka, india. orcid 0000-0002-2951-9151, e-mail: maruthiprasadbv@gmail.com h.l. vishwanth – professor and head, bangalore medical college and research institute, bangalore, karnataka, india. orcid 0000-0001-7331-4308, e-mail: drvishwanathhl@gmail.com r. dharuni et al. 15 in t e r n a l m e d ic in e issn 2413-6077. ijmmr 2019 vol. 5 issue 1 references 1. alberti kg, eckel rh, grundy sm, zimmet pz, cleeman ji, donato ka, et al. harmonizing the metabolic syndrome: a joint interim statement of the international diabetes federation task force on epidemiology and prevention; 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109: 2818-2825. doi: 10.1161/01.cir.0000132467.45278.59 24. lee dh, jacobs dr jr. association between serum gammaglutamyltransferase and c-reactive protein. atherosclerosis 2005; 178: 327-330. doi: 10.1016/j.atherosclerosis.2004.08.027 received 16 december 2018; revised 04 january 2019; rerevised 14 march 2019; accepted 25 april 2019. this is an open access article distributed under the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. r. dharuni et al. 42 b io m e d ic a l s c ie n c e s issn 2413-6077. ijmmr 2016 vol. 2 issue 2 corresponding author: iryna bekus, department of medical biochemistry, i. horbachevsky ternopil state medical university, 1 maidan voli, ternopil, ukraine, 46001 phone number: +38035252433 e-mail: bekus@tdmu.edu.ua international journal of medicine and medical research 2016, volume 2, issue 2, p. 42-46 copyright © 2016, tsmu, all rights reserved doi 10.11603/ijmmr.2413-6077.2016.2.7107 biochemical parameters of lipid metabolism in animals affected by heavy metal salts and treated with carnitine chloride and sodium alginate i. r. bekus, m. v. kyryliv, i. b. ivanusa, o. b. furka, i. y. krynytska, m. i. marushchak i. horbachevsky ternopil state medical university, ternopil, ukraine background. lipid metabolism disorders in the organism affected by environmental pollutants, including poisoning with cadmium and lead salts are of topical matter nowadays. objective. the study was aimed to examine biochemical features of lipid metabolism in rats subjected to toxic damage by lead and cadmium salts and treated with carnitine chloride and algigel. methods. experiments were carried out on white mature outbred male rats weighing 180-200 g. to cause the toxic damage the animals were administered with aqueous solution of cadmium chloride and lead acetate daily for the period of 30 days using intra-gastric lavage. the indices of lipid metabolism were detected by biochemical methods. results. in animals treated with cadmium chloride and lead acetate the following changes were observed: hdl-cholesterol concentrations significantly decreased, resulting in 87% of the levels in the intact animals on the third day, 84% on the fifth and 80% on the seventh day. conversely, concentrations of hdl-cholesterol and vldlcholesterol significantly increased during the experiment. respectively, the ratios for hdl-cholesterol are 240%, 352%, and 388%; and for vldl-cholesterol 108%, 116%, and 132%. conclusions. lipids profile of the rats displayed changes in the levels of cholesterol, triglycerides and lipoproteins of low, high and very low density. key words: blood lipid profile, heavy metal toxicity. introduction the lipid metabolism disorders in the organism affected by environmental pollutants including poisoning with cadmium and lead salts were extensively studied. salts of these metals are assigned class ii of toxicity in the european union's classification system, which is regulated by directive 67/548/eec. they have a large migration ability (in the soil to plant, and animal to person systems), and also high cumulativeness [4, 14]. this creates a threat of their increased uptake if an organism is exposed to several contaminated sources at once: inhaling the air (car exhaust), soil particles along the roadways, industrial dust, and ingesting fertilizer residue with vegetables and produce [8, 9]. due to their association with industrial waste, these compounds often occur in combination. however, although there is a large amount of the literature describing mechanisms of action of either lead or cadmium salts on the body, the number of studies of the combined effect of these two toxins is limited [16, 17]. this study demonstrates that in rats lipid metabolism disorders caused by the combined toxic effect of cadmium (cd) and plumbum (pb) ions were effectively corrected after carnitine chloride and sodium alginate administration. this conclusion is proved by changes in the concentrations of lipid metabolism biomarkers in blood plasma. material and methods experiments were carried out on white mature outbred male rats weighing 180-200 g, which were kept on a standard diet and housed in the animal facility of ternopil state medical university. the animal maintenance, treatment and euthanasia were conducted in accordance with the internal safety and ethical regulations, as well as european convention for the proteci. r. bekus et al. 43 b io m e d ic a l s c ie n c e s issn 2413-6077. ijmmr 2016 vol. 2 issue 2 tion of vertebrate animals used for experimental and other scientific purposes [15], and applied recommendations for the maintenance and work with laboratory animals [10]. to cause the toxic damage, animals were administered with aqueous solution of cadmium chloride, dose 3.3 mg/kg (0.05 ld50), and lead acetate, dose 11 mg/kg (0.05 ld50), daily for the period of 30 days using intra-gastric lavage [2]. the treatment used for toxic damage correction consisted of 2% solution of carnitine, dose 50 mg/kg, and enterosorbent (chelator) algigel, dose 400 mg/kg [5, 12]. both of these substances were administered daily using intragastric lavage. all experimental animals were divided into the following groups: group 1, intact rats; group 2 (control), animals treated with cadmium chloride and lead acetate; group 3, animals treated with cadmium chloride, lead acetate as well as carnitine and algigel enterosorbent. after finishing the heavy metals and corrective agents administration, we tested the lipid metabolism rates on the 3rd, 5th and 7th days. to determine lipid metabolism the following methods were used: triacylglycerols (tg) were extracted from blood serum using isopropanol with simultaneous removal of phospholipids mix from the reaction by precipitation with aluminum oxide [3]; the presence of cholesterol was determined using a colour reaction whereby in the presence of acetate anhydride and a mixture of acetic and sulphuric acid a green compound was formed [1]. levels of cholesterol were evaluated by the intensity of the colour using colorimetry. free cholesterol was detected using digitonin precipitation [3]. cholesterol-digitonin complex was subsequently dissolved in chloroform, evaluated by liebermann–burchard test. the level of highdensity lipoprotein (hdl) cholesterol was determined in soluble fraction after low density (ldl) and very low-density (vldl) cholesterols in blood plasma were precipitated using heparin in the presence of manganese ions [4]. levels of ldl and vldl cholesterols were evaluated by mathematical models. the results of the study were analyzed by the department of statistical research of ternopil state medical university using statistica software (statsoft). arithmetic mean of the sample (m), its variance and mean squared error (mse) were calculated for all biochemical indices. significance of the difference between independent quantitative values was determined using student's t-test. results and discussion cadmium and lead belong to a group of heavy metals; their ions have the ability to accumulate in the body. since in this study these substances were administered for 30 consecutive days in subthreshold doses, we looked for general toxic effect in addition to the parameters of lipid metabolism. thus, for the duration of the experiment, we observed the influence of cadmium chloride and lead acetate on the overall condition of the animals, change in body mass, peripheral blood composition and certain other hematological parameters [7, 8, 11, 13]. the early symptoms of poisoning with heavy metal are: rejection of food by the animal, general depression, and weight loss. our observations indicate that on the 5th day of the experiment, the control group of animals sometimes refused their food or ate smaller quantities compared to the intact animals. we also observed lethargy, reduced movements, and changes in fur cover. to prove these observations, we measured body mass of the intact and control animals, which allowed us to determine its changes. we determined the levels of total cholesterol, triacylglycerols (tg), hdl, ldl and vldl cholesterol in plasma of the experimental and control animals [6]. the data presented in table 1 proves that in plasma of the animals injected with heavy metals, the concentrations of tg significantly increased compared to intact animals, for instance on the third day the it was 186%, on the fifth — 179%, and on the seventh — 166% of the levels of the intact animals. we consider that the increase of triacylglycerol levels in response to prolonged cadmium chloride and lead acetate intoxication occurs according to a specific mechanism. the intoxication by salts of lead and cadmium causes disruption of energy processes at the mitochondria. this evidences the activation of tissue lipases and mobilizing of fatty acids from fat depots, which are rapidly delivered to the liver synthesizing the surplus amounts of triacylglycerols. cholesterol is an important indicator allowing lipid metabolism evaluation. under physiological conditions, the levels of cholesterol in the body are in a state of equilibrium between hdl and ldl cholesterols: the amount of cholesterol incoming with food and synthesized de novo corresponds to what is excreted as bile i. r. bekus et al. 44 b io m e d ic a l s c ie n c e s issn 2413-6077. ijmmr 2016 vol. 2 issue 2 acids and free cholesterol. in this study, the concentration of total cholesterol in blood of the intact animals was 2.18±0.09 mmol/l (table 1), but it was different in the group of the animals that received heavy metal salts. the maximal differences between the two groups were recorded on the seventh day after the beginning of toxicant administration, when cholesterol concentration increased by 16% in the treated animals compared to the intact ones. cholesterol is present in blood as its free form and its esters of fatty acids. each of these forms has specific physiological functions and diagnostic value for understanding of lipid metabolism disorders. free cholesterol is exchanged between tissues, so its concentration in serum reflects the total content of cholesterol in the body. the results of this study prove that there are significant differences in the rates of different form of cholesterol between the intact and control groups. thus, toxic action of heavy metal ions disrupts esterification of cholesterol decreasing concentrations of its esters in blood serum. lipids are transported in blood as complex supramolecular complexes, lipoproteins. therefore, in this study we determined concentrations of different lipoprotein classes. in the animals treated with cadmium chloride and lead acetate the following changes were observed: hdl­cholesterol concentrations significantly decreased, resulting 87% of the levels in the intact animals on the third day, 84% on the fifth day and 80% on the seventh day. conversely, the concentrations of hdl-cholesterol and vldl-cholesterol significantly increased during the experiment. respectively, the rates for hdl-cholesterol were 240%, 352%, and 388%; and for vldl-cholesterol — 108%, 116%, and 132% (table 1). treatment of exogenous intoxication involves the use of antioxidants, metabolic factors, 90 95 100 105 110 115 120 3 day 5 day 7 day % intact treated w ith heavy metal salts * * figure 1. total cholesterol content in blood plasma of the animals. notes: * — significant difference if compared to the control animals. table 1. concentrations of total cholesterol, triacylglycerols, hdl, ldl and vldl cholesterol in rats’ blood serum during 30-day administration of cadmium chloride and lead acetate (n=6) intact animals, group 1 animals treated with cadmium chloride and lead acetate, group 2 3rd day 5th day 7th day 0.86±0.06 1.60±0.09 р1<0.01 1.54±0.08 р1<0.01 1.43±0.08 р1>0.05 total cholesterol, blood plasma, mmol/l 2.18±0.09 2.21 ±0.17 р1>0.05 2.46±0.18 р1>0.05 2.54±0.18 р1>0.05 esterified cholesterol, blood plasma, mmol/l 1.52±0.07 0.91±0.07 р1<0.05 0.96±0.09 р1<0.05 1.27±0.08 р1<0.05 free cholesterol, blood plasma, mmol/l 0.66±0.04 1.30±0.11 р1<0.05 1.50±0.11 р1<0.05 1.27±0.08 р1<0.05 hdl-cholesterol, blood plasma, mmol/l 1.54±0.02 1.34±0.11 р1>0.05 1.29±0.04 р1<0.01 1.24±0.04 р1<0.001 ldl-cholesterol, blood plasma, mmol/l 0.25±0.07 0.60±0,03 р1<0.01 0.88±0.13 р1<0.01 0.97±0.10 р1<0.01 vldl-cholesterol, blood plasma, mmol/l 0.39±0.01 0.27±0.03 р1<0.02 0.29±0.01 р1<0.001 0.33±0.04 р1>0.05 notes: р1 — significant difference if compared to the control animals. i. r. bekus et al. 45 b io m e d ic a l s c ie n c e s issn 2413-6077. ijmmr 2016 vol. 2 issue 2 cell membrane protectors and also reducing systemic concentrations of toxic components in the body. the use of so-called efferent therapies, including enterosorbtion is one of the approaches to removing foreign substances and metabolic products. in this study, we used the combination of carnitine and enterosorbent sodium alginate. the changes of total cholesterol in blood plasma of the experimental animals were as follows. during the whole experiment we observed significant and linear decrease of cholesterol contents in the experimental animals compared to the control group. on the third day cholesterol levels were by 75% lower in experimental group, on the fifth — by 62%, and on the seventh — by 56%, which was lower than in the intact animals (table 1). this decrease was in the free cholesterol, while the concentration of esterified forms significantly increased for the period of the experiment. in this study, we evidenced the positive effect of combined use of carnitine and sodium on the concentrations of hdl, ldl and vldl cholesterol. the combined effect of toxicants caused a marked reduction in the concentration of hdl cholesterol at all stages of the experiment compared to the intact animals (figure 2). subsequent treatment with carnitine and algigel caused a significant increase in these rates. thus, on the 3rd day it increased by 12%; on the 5th by 22%; and on the 7th by 26% compared to the control animals. these rates, however, were significantly different from the corresponding rates in the intact group. when treated with the corrective factors, carnitine and algigel, the animals demonstrated significant decrease of ldlc concentration. in particular, on the 3rd day this rate decreased in 3.6 times in comparison with the control animals. on the 5th day the concentration decreased more and was in 5.8 times lower than in the control animals, and on the 7th day it was in 8 times lower. in the experimental group, the concentration of vldl cholesterol on the 3rd day increased by 79% compared to the animals of the control group, and on the 5th and 7th days by 50% and 36% respectively. conclusions this study proved that carnitine in combination with sodium alginate had a clear positive effect on some parameters of lipid metabolism in blood plasma of rats. the proposed means of correcting biochemical changes caused by poisoning with lead and cadmium salts in subthreshold doses demonstrated the need for further studies to develop possible use in clinical settings for correction of lipid metabolism and reducing toxic effects of xenobiotics. hdl cholesterol 0 0,5 1 1,5 2 3 day 5 day 7 day m m ol /l * * * *# *# *# ldl cholesterol 0 0,5 1 1,5 2 2,5 3 day 5 day 7 day m m ol /l * * * # # # vldl cholesterol 0 0,1 0,2 0,3 0,4 0,5 3 day 5 day 7 day m m ol /l intact treated w ith heavy metal salts treated w ith heavy metal salts, plus carnitine and algigel * * * * # * figure 2. changes in the concentrations of hdl, ldl and vldl cholesterol in blood plasma of the animals. notes: * — significant difference if compared to the control animals; # — significant difference if compared to the affected animals. i. r. bekus et al. 46 b io m e d ic a l s c ie n c e s issn 2413-6077. ijmmr 2016 vol. 2 issue 2 references 1. harbarets bo, wysocki ij, kachanova a. workshop on biological chemistry. sumy 1997; 28. 2. gerasimenko ti, domnin sg, roslyi of, fedoruk aa. the evaluation of combined effects of leadcopper and lead-zinc binary mixtures. med tr prom ekol 2000; 8: 36–39. 3. horyachkovskyy a. clinical biochemistry in the laboratory diagnostics. odessa: ecology 2005: 607. 4. demkiv i, klisch i, bekus i, krynytska i condition of antioxidant system with acute intoxication by ethyl alcohol against a background of protracted intoxication by salts of lead and cadmium. second international conference of students and graduate “youth and progress of biology”, march 21–24. 2006: theses. lviv 2006; 46–47. 5. dmytrukha nm. assessment of calcium alginate influence on nonspecific resistance of white rat organism with lead intoxication. modern problems of toxicology 2004; 4: 15–17. 6. kamyshnikov vs. handbook of clinical and biochemical studies, and laboratory diagnosis. minsk: belarus 2000; 1–2. (in russian) 7. melnyk mk. exposure to ionizing radiation and lead for neuroendocrine system performance in rats and their correction: thesis abstract on competition sciences degree candidate. biol. sciences specials. 03.00.01. “biological chemistry”. kyiv 2003: 15. 8. mikhaleva lm. human pathology by cadmium dependent. archives of pathology 1988; 50 (9): 81–85. 9. mikhaleva lm. pathological anatomy expe rimental intoxication caused by cadmium chloride: ab­ stract of the thesis for the candidate scientific degree in medical sciences: 14.00.15. moscow 1990; 31. 10. kozhemyakin ym, khromov os, filonenko ma, sayfetdinova ga. scientific­practical advice on keeping laboratory animals and work with them. k.: avicenna 2002; 156. 11. rice rh, gulyaeva lf. biological effects of toxic compounds: lectures. novosibirsk state. univ. novosibrsk 2003; 208. 12. sidoryak ng, volgin dv. effect of l-carnitine on lipid peroxidation and lipid composition of blood serum at hemic hypoxia. ukr. biochem. mag 1996; 68 (5): 54–58. 13. yanchuk vv, vlasyk li. features combined effect of sodium nitrate and lead acetate in subacute experiment on rats of different ages. bukovina’s medical journal 2003; 7 (4): 163–167. 14. brind a, hurlstone a, edrisinghe d. the role of polymorphisms of glutathione s–transferases gstm 1, m3, p 1,t 1, and a 1 in susceptibility to alcoholic liver disease. alcohol 2004; 39 (6): 478–483. 15. european convention for the protection of vertebrate animals used for experimental and other scientific purposes. council of europe. strasbourg 1986; 123: 52. 16. rana s, verma s. protective effects of gsh, vitamin e and selenium on lipid peroxidation in cadmium fed rats. boil. trace element res 1998; 2: 161–168. 17. sies h. oxidative stress: oxidants and antioxidants. exp. physiol 1997; 82 (2): 291–295. received: 2016-12-16 i. r. bekus et al. 25 su r g ic a l d is e a se s issn 2413-6077. ijmmr 2017 vol. 3 issue 2 doi 10.11603/ijmmr.2413-6077.2017.2.7917 characteristic features of multiple organ failure in cases of peritoneal sepsis l. yu. ivashchuk, i. b. pizhitsky i. horbachevsky ternopil state medical university, ternopil, ukraine background. the study of hystomorphology of liver and small intestine in experimental peritonitis is presented. due to this information the criterion of pathogenetic moment transition sirs for peritoneal sepsis was determined. objective. the aim of the research was to study the morphology of terminal part of small intestine and liver in cases of experimental peritonitis. methods. for histological and electron microscope study the biopsy of liver, small intestine were taken; the samples were stabilized in a neutral formalin, dried in alcohol of increasing concentration and placed in paraffin. paraffin sections were painted with hematoxylin and eosin and studied under the light-optical microscope. results. apoptosis caused damage to enterocytes and hepatocytes of first bacterial translocation. mechanism of vasodilatation effect of no and its effect on apoptosis were determined. septic shock was accompaniment of two main levels of body cells damage: apoptosis and membranes destruction. peritoneal sepsis is a grave condition caused by progressive peritonitis and polyorgan insufficiency syndrome. the phases of peritonealis sepsis pathogenesis were defined. conclusions. the presented morphological criteria prove the initiation of apoptosis within 24 hours after the development of peritonitis in enterocytes and hepatocytes. key words: peritonealis sepsis; apoptosis; polyorgan insufficiency syndrome. corresponding author: larysa ivashchuk, department of general surgery, i. horbachevsky ternopil state medical university, 2 shpytalna street, ternopil, ukraine, 46008 phone number: +38679401031 e-mail: ivashchuk_lu@tdmu.edu.ua introduction twenty­five years ago at the chicago consensus conference on sepsis some traditional approaches and postulates as well as critical approach to the suggested concept has been changed [1, 2]. the accumulation of experimental and clinical data on the significance of cytokines in the pathogenesis of sepsis allows adequate formulation of clinical diagnosis and formation of accurate subject [5, 7]. the realization of systemic inflammatory response syndrome (sirs), as a part of diagnosis that characterizes the septic state of patients, allowed building algorithm due to patho genic effects. at the same time, much criticism by the supporters of the traditional concept of ‘sepsis as a bacteremia accompanied by appropriate clinical disruption of homeostasis’ that are inherent to sirs substantiate that clinical signs of the syndrome are a common reaction to surgical infection. in our clinic, the researchers believe that the determination of septic states is not reasonable enough: sepsis, severe sepsis, septic shock, hypotension syndrome and multiple organ failure. the concept peritoneal (abdominal) sepsis is even more terminologically confusing. our aim was to define the morphological criteria, which determine pathogenic moment of sirs transition that causes peritoneal sepsis at peritonitis. methods during the research the morphology of terminal part of small intestine and liver in experimental peritonitis was studied. the research took place in spring. 20 mature nonlinear white male rats, weighing 200–250 g were used. the animals were kept on a standard vivarium diet with free access to water in accordance with the requirements of the “european convention for the protection of vertebrate animals used for experimental and other scientific purposes” [6, 9]. international journal of medicine and medical research 2017, volume 3, issue 2, p. 25–29 copyright © 2017, tsmu, all rights reserved l. yu. ivashchuk et al. 26 su r g ic a l d is e a se s issn 2413-6077. ijmmr 2017 vol. 3 issue 2 acute peritonitis was modeled by administration of 10% fecal mixture into abdominal cavity by the method of lazarenko v. a., et al (2008) [8] that is comparable to etiological factors, clinical manifestations and phase transition of similar process in humans and allows the death of animals, which is acceptable for conducting a dynamic study during 10 days. this was performed by the administration of 0.5 ml of 10% fecal suspension filtered into peritoneal cavity of the studied rats. the suspension was obtained by mixing isotonic solutions and feces from the intestine of two or three intact animals and then it was filtered twice through a double layer of gauze. the resulting suspension no later than in 20 minutes after preparation was administered to the intact animals by puncture method. to avoid damage to internal organs when the fecal suspension was introduced into abdominal cavity, the animals were held upright, caudal end up. by the method of puncture of ventral wall in the middle of the central line of abdomen, the end of the needle was rotated in turn into the right and left hypochondria, the right and left ilium sections, and the same amount of fecal suspension was injected. in 24 hours the animals were killed by decapitation under general anaesthesia. for histological and electron microscope study biopsies of liver, small intestine were taken. the samples were stabilized in neutral formalin, dried in alcohols of increasing concentration and placed in paraffin. paraffin sections were painted with hematoxylin and eosin and studied under the light-optical microscope. electron microscope examination of biopsy samples was processed by standard methods and placed in epoxy resin. in addition, the method was used to identify membrane permeability due to ca++-atp. thick ultrathin sections 600 ǻ were managed in ultra­microtome lkb and reyhert. to improve the contrast the sections were painted by reynholds and studied through the electron microscope em-400, philips. results histological examination of small intestine wall in all animals, which were simulated with diffuse peritonitis, proved dystrophic and destructive changes of all its layers, mainly affecting submucosal tissues: oedema on the back­ ground of disturbed intra­organ blood flow was the most pronounced that was manifested with plethora expansion and main mass of microvessels, perivascular and interstitial haemorrhage. associated with oedema, histo-architectonic connective tissue stroma was damaged. mucous membrane was sometimes flaky in intestine and a large mass of microvilli was swollen and deformed. epithelial vessels microvilli were swollen with a clarified cytoplasm and basophilic nucleus was eccentrically placed. muscle shell was also swollen with more extended and filled with blood microvessels, as a result focal violations moved muscle fibres with expansion to intramuscular spaces. the electron microscope study of columnar epithelium intestinal mucosa proved pronounced dystrophic and destructive changes of nucleus and cytoplasm. nuclei fracture, chromatin aggregation and redistribution of its overwhelming pre-shell condensation were established; normal chromatin in the nuclei of epithelial cells of intestine was placed uniformly in plasma nuclei. in the cytoplasm of enterocytes intracellular accumulation of detritus was evidenced, proving fairly pronounced destructive changes in these cells. changes occur in the microvilli system that protrude the intestine in a gleam. histochemical reaction on membrane permeability using the ca++-atp-ase proved that compare to normal, reaction products fell in the sediment on the surface membranes of microvilli and determined in their gleam penetrating into the cytoplasm of cells. this evidenced membrane permeability microvilli violation associated with their swelling. external intestinal serosa studied on lightoptical level was slightly thickened and swollen. violations of connective tissue skeleton swelling and disorientation of collagen and elastic fibers and desquamation mesothelial cells were also noticed. numerous cavities extended microvascular events with accumulation of red blood cells and endothelial swelling. in the parietal layer of peritoneum the changes were less pronounced and comprised extended microvessels cavity and perivascular connective tissue swelling (fig. 1.) this resulted in pathological permeability of intestine inner lining and was accompanied by increased penetration of endotoxin, bacterial contamination from intestine cavity into mesenteric lymph nodes and portal system. microscopic examination of liver in all cases proved more or less pronounced swelling of parenchyma on the background of disturbed intra­organ blood flow, which was manifested by plethora expansion and main vessels massl. yu. ivashchuk et al. 27 su r g ic a l d is e a se s issn 2413-6077. ijmmr 2017 vol. 3 issue 2 es and focal perivascular haemorrhage. in areas of disturbed intra­organ blood flow quite pronounced swelling of liver cells with impaired liver histological structure and expanding intercellular spaces was evidenced. the investigation of hepatocytes structure in a significant increase in a series of semi-thin slices proved pronounced degenerative changes of liver cells with sharp cytoplasm enlightenment, significant decrease in the size of the nucleus, associated with swelling, decrease in the number and pre-shell condensation of chromatin. in addition, significantly increased sinusoids with focal desquamation of endothelial cells that route to the cavity of blood vessels was observed. electron microscopic examination of hepatocytes proved that some of these cells had pronounced degenerative changes associated with swelling of cytoplasm, manifested by quite significant expanding and vacuolization of endoplasmic reticulum, mitochondria and destruction of the significant decrease in the number of free and fixed ribonucleic granules. ultrastructural changes were detected in endothelial cells of hepatic sinusoid. these changes were of diverse nature: from dystro­ phic-atrophic to hyperplastic, proving the focal reparative effects. in hepatocytes that were adjacent to hyperplastic endothelial cells the effects reparative regeneration were established that comprised strengthening their energy-producing and protein synthesis function by hypertrophy of mitochondria and increase in the number of free ribosomes and polysomes (fig. 2.). thus, the presented morphological criteria proved the initiation of apoptosis in enterocytes and hepatocytes within 24 hours after the development of peritonitis. discussion kerr [2] described these effects in 1972 as ‘apoptosis’ cause, we believe that further destruction of cellular organelles took place. thus, in most enterocytes the damaged mitochondria, swollen and infected reticulum cisterns with a reduced number of ribonucleic granules fixed in their membrane were evidenced. the number of ribosomes and polysomes was reduces. according to the literature, the mentioned processes cause local activation of immune cells, cytokines and other inflammatory mediators (tnf, il-1,6,8), which are able to stimulate the production of prostaglandins, free radicals, nitric oxide, which in turn is a powerful factor in adaptive processes at a cellular level [10, 11]. the destruction of liver cells during peritonitis occurs also due to apoptosis. it is likely that this process is initiated by nitric oxide, which in living organisms is formed by the action of specific enzymes united under the title of nitric oxide synthase. the last were found in endothelial cells of arteries and veins of intestine, and were initiated by the generation of nitric oxide in all cells of liver [3, 4]. these changes may be enough to form multiple organ failure, especially such as bacterial translocation. in our opinion, the appearance of morphological, biochemical and clinical signs of organ failure should be the criteria for diagnosis ‘peritoneal sepsis’ (fig. 3.). further development of the pathological process is determined by two­way cell destruction: by apoptosis and degradation of membranes, and can lead to septic shock. if at this moment intensive multicomponent therapy does not help stabilize homeostasis, the irreversible changes fig. 1. modeling of acute peritonitis. oedema and violation of intracellular organelles integrity of intestine epithelial cells. electron micrographs ×13000 fig. 2. modeling of acute peritonitis. oedema of hepatocyte with violation of mitochondria integrity and reduction of endoplasmic reticulum. electron micrographs ×13000 l. yu. ivashchuk et al. 28 su r g ic a l d is e a se s issn 2413-6077. ijmmr 2017 vol. 3 issue 2 in vital organs and systems will take place that may lead to fatal consequences. we understand that the above concept is hypothetical and disputable to some extent, but morphological changes prove the need for further study of the problem, especially the determination of nitric oxide, its synthase and apoptosis. conclusions the affection of hepatocytes and enterocytes in experimental peritonitis begins with apoptosis. apoptosis, probably, can be morphological criteria of multiple organ failure initiation. peritoneal sepsis is a severe pathology resulting in progression of peritonitis and mul tiple organ failure. fig. 3. pathogenetic variants of peritoneal sepsis acute surgical diseases of abdominal cavity endotoxins, cytokines, leukotrienes increased permeability of cell membrane sirs unlimited peritonitis apoptosis limited peritonitis organ failure organ failure organ failure abdominal sepsis apoptosis and destruction of membrane no synthesis initiation fall vascular tone hypoperfusion tissues organ failure septic shock organ failure irreversible changes lethal consequence fig.3. pathogenetic variants of peritoneal sepsis. conclusions the affection of hepatocytes and enterocytes in experimental peritonitis begins with apoptosis. apoptosis, probably, can be morphological criteria of multiple organ failure initiation. peritoneal sepsis is a severe pathology resulting in progression of peritonitis and multiple 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2413-6077. ijmmr 2017 vol. 3 issue 2 8. lazarenko va, lipatov va, blinkov yuyu, skorikov dv. experimental model of widespread fecal peritonitis. man and his health. 2008;4:128–132. 9. kozhemiakin yum, khromov os, filonen ko ma, saifetdinova ha. scientific and practical advice on keeping laboratory animals and work with them. kyiv: avitsena. 2002;156. 10. titov vn. the role of macrophages in the formation of inflammation, the effect of interleukin-1, interleukin-6 and the activity of the hypothalamic-pituitary system. clinical laboratory diagnostics. 2003;12:3–10. 11. boelen a, kwakkel j, platvoetter schiphorst m. interleukin­18, a proinflammatory cytokine, contributes to the pathogenesis of non-thyroid al illness mainly via the central part of the hypothalamus pituitary-thyroid axis. eur j endocrinol. 2004; 151(4):497–502. l. yu. ivashchuk et al. received: 2017-06-22 13 in t e r n a l m e d ic in e issn 2413-6077. ijmmr 2018 vol. 4 issue 2 international journal of medicine and medical research 2018, volume 4, issue 2, p. 13-19 copyright © 2018, tsmu, all rights reserved corresponding author: dr inam danish khan, mbbs, md, dnb, dhcm, mipha, miscd, associate professor (clinical microbiology and infectious diseases), army college of medical sciences and base hospital, delhi cantt 110010 india e-mail: titan_afmc@yahoo.com, mobile: +91 8076324060, fax: +91 11 25693490 s. khan et al. doi 10.11603/ijmmr.2413-6077.2018.2.9317 prevalence of gestational diabetes mellitus in an urban indian cohort using diabetes in pregnancy study group in india (dipsi) criteria – validating one-step approach s. khan1, h. bal2, i. d. khan3, d. paul3 1 – inhs kalyani, visakhapatnam, india 2 – d. y. patil medical college, pune, india 3 – army college of medical sciences and base hospital, new delhi india background. india is the “world’s diabetes capital”, with half the diabetic population being women. early detection of glucose intolerance during pregnancy offers a timely opportunity for screening, management and prevention of gestational diabetes mellitus (gdm) and prevents fetal complications. objective. the study assessed the prevalence of gdm in an indian cohort using the diabetes in pregnancy study group of india (dipsi) criteria. methods. 200 pregnant women underwent two-phase testing with non-fasting 75-gram glucose challenge under diabetes in pregnancy study group of india (dipsi) criteria at <20 weeks and between 24-28 weeks period of gestation. a 3-hour 100-gm oral glucose tolerance test (ogtt) was used for confirmation. repeat testing was done for women negative during the first-phase. results. mean age was 24.26±3.75 years with 52.5% multigravidas. mean body mass index (bmi) was 20.7±3.07 kg/m2. the prevalence of gdm in study cohort was found to be 15.5% using the dipsi criteria while the prevalence of gdm after 100 g ogtt was 13.0%. gdm was mostly seen to occur in women of 26-30-year age group. statistically significant associations for age and gdm, and bmi and gdm were evidenced. conclusions. maternal age of ≥25 years should be adopted as a risk factor for the development of gdm. the dipsi criteria offer a cost-effective and an evidence-based protocol for a single-step definitive glucose test for both screening and diagnosis of pregnant patients belonging to any socio-economic strata; furthering its implementation for public health obstetrics. key words: gestational diabetes mellitus; dipsi criteria; screening; pregnancy; glucose tolerance test. introduction gestational diabetes mellitus (gdm) is established to be carbohydrate intolerance with onset or first diagnosis during pregnancy [1]. the who defines gdm as plasma glucose concentration of >140 mg/dl 2-hours by 75-gm oral glucose tolerance test (ogtt) similar to that of impaired glucose tolerance (igt) test in a non-pregnant state [2]. with advancement of pregnancy, insulin resistance and diabetogenic stress caused by placental hormones necessitates compensatory increase in insulin secretion, the inadequacy of which leads to the development of gdm. the patients with gdm are at a risk group of future diabetes mellitus (dm) development, predominantly type-2 dm, as well as their children are [3]. in addition, untreated gdm may possibly lead to increased risk of large for gestational age fetus, plunge in blood sugar and jaundice in the offspring. the prevalence of dm is increasing worldwide. developing countries sustain a major proportion of world population translating to the epidemic proportions of dm being encountered by the healthcare fraternity in limited resource public health infrastructure. india is projected as the “world’s diabetes capital”, with half the diabetic population being women. india is expected to contain the highest population of diabetics by 2025. the syndemic (synergistic epidemic) of dm and obesity compounding the problem of gdm exists under socio-epidemiological and anthropological perspectives of health disparity factored by poverty, living conditions, socio-economic status and dietary habits. gdm is the most common metabolic disease of pregnancy worldwide. the prevalence of gdm reaches up to 14% of all pregnancies, resulting in approximately 200,000 cases annually in the united states. asian and indian 14 in t e r n a l m e d ic in e issn 2413-6077. ijmmr 2018 vol. 4 issue 2 lifestyles are starkly different from western lifestyles translating into 11.3 times higher relative-risk of gdm in indian women compared to their western counterparts [4]. w i t h t h e p o p u l a t i o n ex p e r i e n c i n g a changing lifestyle and epidemiology of dm, it is pertinent to offer screening of gdm during the antenatal work-up. gdm holds out a significant opportunity for testing, development and implementation of clinical strategies for diabetes prevention in people [5]. timely screening of the pregnant women for glucose intolerance, succeeding euglycemia and adequate nutrition may prevent presumably the pathological cycle of vertical transmition of glucose intolerance. this necessitates the universal mandatory screening for gdm during pregnancy, which is a resource intensive concept in the developing country perspective. presently most institutions catering to women with adequate affordability are following the 2-phase procedure for screening gdm. the criteria by diabetes in pregnancy study group of india (dipsi) recommend a simplified onestep approach for the screening and diagnosis of gdm irrespective of fasting state of expectant mothers, which is a promising protocol for underprivileged communities having limited healthcare accessibility during pregnancy. timely revealing of glucose intolerance in pregnancy offers an opportunity for screening, management and prevention of gdm on time and prevents fetal complications thus improves neonatal outcomes [6, 7]. this necessitates the general mandatory screening for gdm during pregnancy, which is a resource intensive testing modality. this study was carried out to assess the incidence of gdm in an indian cohort using the dipsi criteria [8]. methods the triple-blind study was conducted amongst 200 patients admitted to the antenatal outpatient department (opd) of a tertiary-care hospital, containing 1600 beds, and medical teaching institute in western india; the study was approved by the ethics committee of these medical facilities as well as the written informed consents were attained from the patients. all pregnant females at 20 weeks or less period of gestation (pog) were involved in the study that lasted for two years: from may 2012 to apr 2014. the patients with gdm/impaired glucose tolerance (igt) in previous pregnancy, established morbidity of dm, dm in a first-degree relative or with a history of unexplained stillbirth, large for gestational age offspring, congenital anomalies or previous birth injuries, were excluded. relevant history, general examination for calculating body-mass index and evidence of insulin resistance along with obstetric/gynecological examination were carried out. triple-blinding of a patient, gyne cologist and laboratory medicine specialist was ensured to eliminate bias and confounding. the entire cohort of 200 patients was subjected to a two-phase testing at the pog of <20 weeks and for a second time at the pog of 24-28 weeks, a temporal separation was at least four weeks. in the first phase, all patients were given 75-gm anhydrous oral glucose at their first visit, irrespective of their fasting state, according to the dipsi criteria. the levels of plasma venous blood glucose were evaluated by glucose oxidase-peroxidase method in 2 hours. the indices of ≥140 mg/dl were positive by the dipsi criteria. a 3-hour 100-gm oral glucose tolerance test (ogtt) was used for confirmation. any indices of ≥95 mg/dl fasting, ≥180 mg/dl in 1 hour, ≥155 mg/dl in 2 hours, ≥140 mg/dl in 3 hours were considered to be positive. only one positive value in ogtt was considered as igt while two positive values were considered for gdm. in the second phase, women who were negative initially by dipsi criteria were made to undergo a repeat test with non-fasting 75-gm at 24-28 weeks as per the dipsi criteria. a 100gm ogtt was used for confirmation. data was analyzed using spss (version 21; ibm corporation) with χ2 test or fisher’s exact test for categorical variables and student’s t-test for continuous variables. all statistical tests were two-tailed and p values <0.05 were considered significant. clinicodemographic and diagnostic profiles were correlated for de scrip tive statistics and included frequency, per cen tages and 95% confidence intervals (95% ci). results the study cohort comprised of young patients with mean age was 24.26±3.75 years ranging from 20 to 28 years. most patients were between 21 to 25 years of age (102/200, 51%, 95% ci 43.87% – 58.09%), followed by 49/200, 24.5%, 95% ci 18.83% – 31.17%, between 26 to 30 years (24.5%) (table 1). 95/200, 47.5%, 95% ci 40.45% – 54.65% were primigravida while 105/200, 52.5%, 95% ci 45.35% – 59.55% were multigravida. mean body mass index (bmi) was 20.7 ± 3.07 kg/m2, range s. khan et al. 15 in t e r n a l m e d ic in e issn 2413-6077. ijmmr 2018 vol. 4 issue 2 between 14.33 to 30.81 kg/m2. most of the pregnant females (108/200, 54%, 95% ci 46.83% – 61.01%) were having bmi between 21-25 followed by 92/200 (46%, 95% ci 38.99% – 53.17%), who had bmi ≤20 kg/m2. there were no overweight or obese women in the cohort (table 2). the study was carried out with a 100% follow up with no drop outs. out of the 200 pregnant females in the cohort, in the first phase, 31/200 (15.5%, 95% ci 10.93% – 21.44%) were tested positive by the dipsi criteria prior to 20 weeks pog; 21/200 (10.5%, 95% ci 6.77% – 15.81%) of them were tested positive by 100-gm ogtt. in the second phase, the remaining 10 women tested positive by the dipsi criteria and negative by 100-gm ogtt were again subjected to 100-gm ogtt at a 24-28-week pog, resulting in five more being found positive by 100-gm ogtt. out of the 169 women tested negative by the dipsi criteria at less than 20 weeks pog, in the first phase, one aborted at 14 weeks pog and was excluded from the study. the remaining 168 women were again subjected to dipsi and then validated by 100-gm ogtt at a 24-28-week pog. none tested positive with either dipsi or 100-gm ogtt. the prevalence of gdm in study cohort was found to be 15.5% using dipsi criteria while the prevalence of gdm after 100-gm ogtt was 13% (table 1). gdm was mostly seen to occur in women of 26-30-year age group (12/26, 46.15%, 95% ci 27.14% – 66.25%) followed by 9/26 (34.62%, 95% ci 17.95% – 55.64%) in the 21-25year age group. statistically significant association for age and gdm (p=0.003) was seen by fisher’s exact test. almost all (25/26, 96.15%, 95% ci 78.41% – 99.8%) gdm was seen with bmi >20 kg/m2, with statistically significant (p=0.003) difference seen by fisher’s exact test. however, the association of gravidity was not significant (p=0.207) using chi square test. discussion disorders of maternal glucose metabolism during pregnancy are two-pronged. firstly, pre-existing type-2 dm accounts for 8% of dm in pregnancy. there is an increasing trend of type-2 dm in women of childbearing age group, attributable to sedentary lifestyles, dietary changes and the virtual epidemic of adolescent and childhood obesity. gdm accounts for 90% of diabetes in pregnancy. gdm represents the “tip of an iceberg” for the overall prevalence of dm in the population, thus being representative screening target for timely intervention. the prevalence of gdm varies from 1-20% depending upon population sample and diagnostic criteria. risk factors of gdm include a high bmi (a measure of body fat), gaining weight or low physical activity in pregnancy, excessive dietary eating of polyunsaturated fats, glucose intolerance (a sign of diabetes) or delivery of a large baby in previous pregnancies, as well as a family history of diabetes. excessive intake of saturated fat, low eating of polyunsaturated fat, and high gestational weight gaining may possibly increase the risk of gdm. a decreased risk of gdm is also associated with physical activity. obesity is one of the most significant risk factors for gdm, its prevalence has been increasing much over the last decades [9, 10, 11]. the study revealed gdm among young pregnant females up to 30 years of age in contrast to the development of dm in later age. the risk of gdm increases significantly from 25 years onwards [12]. the most predictive factor of gdm is maternal age ≥25 years, according to the recommendations of the american diabetes association (ada) on the age criteria of ≥25 years as a cut-off for screening for gdm. in population with lower diabetes prevalence, timing of screening depends on the risk profile. women at high risk are offered screening at table 1. age profile of pregnant patients (n=200) age (years) number of patients percentage (%) 95% confidence intervals ≤20 35 17.5 12.64-23.64% 21-25 102 51.0 43.87-58.09% 26-30 49 24.5 18.83-31.17% >30 14 7.0 4.03-11.71% table 2. body-mass index (bmi) profile of pregnant patients (n=200) bmi (kg/m2) number of patients percentage (%) 95% confidence intervals ≤20 92 46 38.99-53.17% 21-25 108 54 46.83-61.01% >26 0 0 – s. khan et al. 16 in t e r n a l m e d ic in e issn 2413-6077. ijmmr 2018 vol. 4 issue 2 first antenatal visit, moderate risk at 24-28 weeks as per ada guidelines. in general, screening and diagnostic tests are performed between 24 and 28 weeks, because at this point in gestation the diabetogenic effect of pregnancy is manifested. the study of kaiser permanente of colorado (kpco) proved a strong cohort influence on the prevalence of gdm. regardless of the age and ethnicity, the women, who were born more recently, were at an increased risk for gdm diagnosis compare to those born earlier. this finding most likely reveals an increased exposure to risk factors taking place before childbearing age [13]. in clinical practice, maternal age of ≥25 years should be adopted instead of ≥35 years or ≥40 years as a risk factor for the development of gdm [14, 15]. the present study also proved that the increased prevalence of gdm was evidenced together with increasing bmi. although the incidence of gdm in the pregnant females with normal weight (bmi 18.5-24.9) is 2.3%, it increased more than five-fold to reach 11.5% in extremely obese pregnant women with bmi 35-64.9 [16, 17, 18]. a systematic review of observational studies published over last 30 years, which elected maternal bmi as the only measure of obesity and where all diagnostic criteria for gdm were accepted; it revealed that for every 1 kg/m2 increase in bmi, the prevalence of gdm increased by 0.92% (95% ci 0.73% to 1.10%) [19]. indian women with gdm experience a higher risk of metabolic syndrome and diabetes [20]. the 15.5% prevalence of gdm by dipsi criteria found in this study compares well to other indian studies showing prevalence between 16.55% and 22%. in india the prevalence of gdm has been estimated at 16.55% by the who criteria of a 2-hour blood glucose level of 140 mg/dl. however, the prevalence for kashmiri women was 3.8% [21, 22, 23, 24, 25]. gdm was proved to be more widespread in urban areas than in rural. for this population and ethnicity, the incidence of gdm corresponds to the incidence of igt in non-pregnant adults within that population [26]. in indian context the prevalence of gdm is steadily increasing from 2% in 1982 to 12% in 1991 to 16.55% in 2002. variations in prevalence of gdm due to geography and ethnicity have similarly been reported in mexico [27]. certain ethnically diverse subpopulations have a much higher rate of gdm which renders them the susceptibility of a greater predisposition to dm in later age. the incidence of gdm differs in direct share to the incidence of type2 dm for tis ethnic group or population. in asian population, gdm reflects the prevalence of igt in the population. therefore, the general screening for gdm is necessary for asian and indian population [28]. in comparison to the selective screening, the general one for gdm distinguishes more patients and improves neonatal and maternal prognosis. currently, and after extensive deliberation, universal screening of all pregnant women is recommended by some professional associations. nevertheless, there exist challenges in quality control of laboratory testing in developing countries catering to mass-screening in resource limited laboratories, which needs to be taken into account for clinical decision making [29, 30]. in pregnancy, the choice to carry out a placebo-controlled trial involves clinical equi poise [31]. hence, there was no control group of unmanaged pregnant women in this study, as there are some publications confirming that management of gdm women, as defined by the who criteria, was associated with a decreased risk of pregnancy outcome. as the routine screening for glucose intolerance during pregnancy was not done initially, probably the undiagnosed glucose intolerance that was occurring in the past has resulted in the increased prevalence of diabetes in india. dipsi criteria are a major advance as they cater diagnosis and screening of all pregnant women regardless of the fasting state by a single-step approach with a 75-gm of a 2-hour glucose test and a cut-off of >140mg/dl for diagnostics. the study revealed 31 patients through dipsi criteria at ≤20 weeks pog, 21 of which were detected by 100-gm ogtt at ≤20 weeks pog and five were detected between 24-28 weeks pog. if the 75-gm criterion was reapplied at 32-34 weeks pog as recommended by dipsi, it is likely that even the five women, who tested negative, when validated with 100gm ogtt, could have tested positive for gdm. dipsi is very economical, practical, convenient and feasible for patients and obstetric healthcare practitioners [32, 33]. conclusions the incidence of gestational diabetes mellitus in the study cohort using dipsi criteria was significantly high (15.5%) and is comparable with other indian studies. in clinical practice, the maternal age of ≥25 years instead of 35 s. khan et al. 17 in t e r n a l m e d ic in e issn 2413-6077. ijmmr 2018 vol. 4 issue 2 years or ≥40 years should be adopted as a risk factor for gdm development. the dipsi criteria offer a cost-effective and an evidence-based protocol for a single-stage complete glucose test for both screening and diagnosis of pregnant patients of any socio-economic strata; furthering its implementation for public health obstetrics. references 1. metzger be, coustan dr. summary and recommendations of the fourth international workshopconference on gestational diabetes mellitus: the organizing committee. diabetes care. 1998;21:b161-7. 2. dirar am, doupis j. gestational diabetes from a to z. world journal of diabetes. 2017 dec 15;8(12):489. doi:10.4239/wjd.v8.i12.489. 3. varma k, surwade vm, sinha v, kachhawa p. prevalence of gestational diabetes and risk factors among women visiting antenatal clinic of a tertiary health care hospital. iosr journal of dental and medical sciences. 2017; 16(9):1-4. 4. rosenblatt b. prevalence, health behaviors, and preventive health practices among adult поширеність гестаційного цукрового діабету серед когорти міського індійского населення за критеріями dipsi – апробування одноетапного підходу s. khan1, h. bal2, i.d. khan3, d. paul3 1 – inhs kalyani, visakhapatnam, india 2 – d. y. patil medical college, pune, india 3 – army college of medical sciences and base hospital, new delhi india вступ. індія є «столицею діабету у світі», при чому половину населення, хворого на діабет, складають жінки. раннє виявлення порушення толерантності до глюкози під час вагітності дає можливість своєчасно проводити скринінг, лікування та профілактику гестаційного цукрового діабету (гцд) та запобігати розвиткові ускладнень вагітності. мета дослідження – встановити частоту розвитку гцд серед жінок з використанням критеріїв dipsi. методи дослідження. обстежено 200 вагітних жінок, яким проводили двофазне тестування навантаженням глюкозою (75 г глюкози натще серце) відповідно до критеріїв dipsi на термінах <20 тижнів та між 24-28 тижнями вагітності. тригодинний пероральний глюкозотолерантний тест (пгтт) (з навантаження 100г глюкози) використовували для повторного дослідження, яке проводили в тому числі і жінкам з негативними результатами, отриманими під час першої фази обстеження. результати. середній вік обстежуваних вагітних жінок склав (24,26±3,75) років, з них з 52,5 % – мали кілька вагітностей. середній індекс маси тіла (імт) становив (20,7±3,07) кг/м2. встановлено, що поширеність гцд у досліджуваній когорті становила 15,5 % відповідно до критеріїв dipsi, тоді як поширеність гцд після 100 г пгтт становила 13 %. гцд в основному спостерігався у жінок вікової групи 26-30 років. встановлено статистично достовірні кореляції між показниками віку та гцд, індексу маси тіла та гцд. висновки. вік майбутньої матері більше 25 років повинен розглядатися як фактор ризику розвитку гцд. критерії dipsi – це економічно ефективний і обґрунтований протокол для викоримтання глюкозотолерантного тесту для скринінгу та діагностики вагітних пацієнток, що належать до будь-яких соціально-економічних верств, який може бути рекомендований для подальшої імплементації у клінічну практику. ключові слова: гестаційний цукровий діабет; критерії dipsi; скринінг; вагітність; глюкозотолерантний тест. s. khan et al. 18 in t e r n a l m e d ic in e issn 2413-6077. ijmmr 2018 vol. 4 issue 2 coloradans with diagnosed diabetes: results from the behavioral risk factor surveillance system, 1997–2000. denver, colorado department of public health. 2002. 5. moon jh, kwak sh, jang hc. prevention of type 2 diabetes mellitus in women with previous gestational diabetes mellitus. the korean journal of internal medicine. 2017;32(1):26-41. doi:10.3904/ kjim.2016.203. 6. khan s, bal 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equipoise and the uncertainty principles both require further scrutiny. bmj 2001; 322:795. 32. khan s, khan id. the pregnant indian pilgrim at hajj and associated obstetric and neonatal outcomes: a 3-year experience of indian hajj medical mission. sultan qaboos univ med journal. epub ahead of print. 2018, eissn 2075-0528, issn 2075051x. doi: 10.18295/squmj 33. vij p, jha s, gupta sk, aneja a, mathur r, waghdhare s, panda m. comparison of dipsi and iadpsg criteria for diagnosis of gdm: a study in a north indian tertiary care center. international journal of diabetes in developing countries. 2015 sep 1;35(3):285-8. s. khan et al. 5 p sy c h ia t r y issn 2413-6077. ijmmr 2017 vol. 3 issue 1 international journal of medicine and medical research 2017, volume 3, issue 1, p. 5–10 copyright © 2017, tsmu, all rights reserved corresponding author: olena venger, department of neurology, psychiatry, narcology and medical psychology, i. horbachevsky ternopil state medical university, 1 maidan voli, ternopil, ukraine, 46001 phone number: +380352435774 e-mail: venger_ol@tdmu.edu.ua o. p. venger et al. doi 10.11603/ijmmr.2413-6077.2017.1.7039 improvement in the approaches to the treatment of emigrants and re-emigrants with depressive disorders o. p. venger, y. i. mysula, l. v. shkrobot i. horbachevsky ternopil state medical university, ternopil, ukraine background. social and clinical relevance of emigration and re-emigration is determined by a significant amount of ukrainians that emigrate and re-emigrate. clinical features of depressive disorders have not been studied;as well as risk factors and antiriscs of various forms of depression have not been established yet. development of comprehensive approaches to the treatment and rehabilitation of these patients is an effective way of resolving of this problem and is of significant scientific, practical, medical and social importance. objective. the development of measures for treatment of depressive disorders in emigrants and re-emigrants is based on investigation and analysis of clinical-psychopathological phenomenology of depression. methods. we examined 196 emigrants, 191 re-emigrants and 198 not emmigrants with final clinical diagnosis of depressive disorder according to icd-10 criteria. examination was conducted by means of depression rate according to m. hamilton, the montgomery-asberg and symptom checklist-90-revised and the scale of life quality by who qol-26. statistical analysis of the results was performed using nonparametric methods: mann-whitney test, wilcoxon criterion of signs. results. it was found that the maximum content of organic acids is accumulated in grass hyssopus officinalis l. (lamiaceae), and the minimal is in the leaves of chrysánthemum xhortorum l. variety apro (asteraceae). the dominance of aliphatic acids was determined by means of gas chromatography. benzoic is predominant among aromatic acids. conclusions. the suggested therapy schemes proved higher effectiveness compared with the traditional one, and also allowed to improve the life quality of emigrants and re-emigrants. key words: depressive disorders; emigrants; re-emigrants; clinical-psychopathological features. introduction emigration and re-emigration is one of the most important and actual public and social matter. the number of ukrainian emmigrants is estimated up to 6.5–7 million people, what is about 15% of the population, and tends to increa se [7]. re-emigration (coming back of emigrants) is independent medical and social problem; its amounts are comparable with migration. migration is a traumatic factor, provoking manifestation and exacerbation of endogenous mental disorders [1]. in the structure of personality changes in emigrants, emotional instability, anxiety, social introversion, behavioral conformity, suspicion, poor integration of perso nality traits as emotivity and frustration, low level of frustrational tolerance are detected. among mental disorders associated with emigra tion, psyhodisadaptative states, posttraumatic stress disorder, neurasthenia, pro longed depressive reaction and moderate depressive episode are the most common. however, despite the significance of the problem of emigration and re-emigration important issues related to the course of depressive disorders in emigrants and re-emigrants are have not been studied, treatmentrehabilitation programs have not been developed and implemented for emigrants and re-emigrants with depressive disorders. the aim of the study is the development and improvement of measures for treatment of depres sive disorders in emigrants and re-emigrants due to investigation and analysis of clinical-psychopathological phenomenology of depression. methods we examined 196 persons who for at least one last year lived outside ukraine and planned to return abroad soon (emigrants); and 191 persons who at least a year lived outside ukrai6 p sy c h ia t r y issn 2413-6077. ijmmr 2017 vol. 3 issue 1o. p. venger et al. ne and over the last year have returned for permanent residence in ukraine (re-emigrants). also we included 198 persons which were permanently living in the ukraine, and never traveled abroad for long stay (not emmigrants). all of the patients were treated in the ternopil regional municipal clinical psychoneurological hospital for the period from 2010–2014 years, with established final clinical diagnosis of depressive disorder according to icd-10 cri teria. psychogenic depressive disorder (icd-10 codes f43.21 and f43.22) was established in 69 of not emigrants, 68 of emigrants and 67 re-emigrants, endogenous (icd-10 codes f31.3, f31.4, f32.1, f32. 2, f33.1 and f33.2) – in 65, 66 and 63 persons accordingly, organic (icd-10 code f06.3) – in 64, 62 and 61 people accordingly. examination was conducted by means of depression rate according to m. hamilton [8], the montgomeryasberg [9] and questionnaire of psychopathological symptoms severity symptom checklist90-revised [10] and the scale of life quality by who qol-26. statistical analysis of the results was performed by non parametric methods: mann-whitney test, wilco xon a criterion of signs. results the determined in our study clinical-psy chopathological and patopersonal changes in emigrants and re-emigrants prove the necessity of improvement of psychiatric help for this group of patients. today in ukraine the absence of targeted treatment and rehabilitation programs for re-emigrants and emigrants determines the necessity of development of funda_ mental principles, aims and approaches to the treatment of depressive disorders in these patients. considering the clinical-psychopathological and patopersonological features evidenced during the examination of the emigrants and re-emigrants with depressive disorders of different genesis, we recommended the following schemes of medicational therapy and psychotherapy. the suggested scheme of treatment and rehabilitation due to the principles of an integrated, individual and differentiated approach, ensure the staging and continuity of treatment and rehabilitation. its objectives defined as more rapid elimination of depressive disorders (depression, anxiety-depressive, asthe nic-depressive, apatho-depressive syndromes), maximal recovery and social functioning of patients, prevention of recurrence of depressive disorders, providing early socia lization and social adaptation considering the features found in the emigrants and re-emi grants, engaging families and public organi zations. clinical features of depressive disorders (depressed mood, anhedonia, fatigue, anxiety, agitation, etc.), social desadaptation and disor ders of microsocial interaction, abnormal beha vioral patterns associated with the presence of depres sive disorders, as well as factors of emigration and re-emigration were defined as therapy goals. the suggested scheme was realized in four stages that consisted of succession and continuity of treatment and diagnostic measures. the first stage – diagnostical – included clinical psychopathological assessment of disorders that were evidenced in the patient’s psychoemotional sphere, analysis of anamnesis, clinical symptoms, dynamics and prognosis of the disease, the relationship of clinical and psychosocial factors. the second stage – complex treatment – included a compound of biological therapy and psychotherapy aimed to relieve depressive disorder, normalization of emotional state, social adaptation and readap tation. antidepressant therapy at this stage included prescription of agomelatine in a daily dose of 25 mg for the emigrants suffering from psychogenic depressive disorders, for the patients with endogenous depressive disorders – quetiapin in a daily dose of 200 mg, for the emigrants with depressive disorders of organic genesis – fluoxe tinein a daily dose of 20 mg, for the re-emigrants with psychogenic depressive disorders – mirtazapine in a daily dose of 30 mg, for the re-emigrants with endogenous depressive disorders – combination of fluoxetine and risperidone in a daily dose of 20 mg and 2 mg accordingly, for the re-emigrants with depressive disorders of organic genesis – fluoxetine in daily dose of 20 mg. differentiated psychotherapeutic correction for the emigrants included rational use of psychotherapy, cog nitive behavioral therapy and group psycho therapy, and for the emigrants suffering from psychogenic de pressive disorder – also psyho educational therapy. psychotherapeutic inter ven tions for the reemigrants consisted of rational, family, cognitive-behavioral therapy; the focus was on working with the automatic thoughts and dysfunctional beliefs. the crite rion of treatment efficacy was stable (at least two weeks) normalization of emotional state, disappearance of clinical signs of depressive disorder, working out adequate emotional response to real-life circumstances, including emigration (re-emi7 p sy c h ia t r y issn 2413-6077. ijmmr 2017 vol. 3 issue 1 o. p. venger et al. gra tion), disactualization of destructive and formation of constructive pat terns of behavior. the third stage – rehabilita tion – aimed at forming stable adequate emo tional-behavioral pattern, maximum adaptation and readaptation of a patient, preventing of depressive disorder relapse, while medicamental therapy was similar to that used during the treatment with the appropriate dosage correc tion due to the actual condition of the patient. differentiated psychotherapeutic work included the use of cognitive-behavioral therapy for the emigrants, and a combination of cognitive-behavioral therapy, family therapy and auto genous training for the re-emigrants. at this stage, also the measures for rehabilitation and social reintegration of the patients were also useful. the fourth stage – preventive – aimed at maintaining of normal emotional state, effective stress resistance and prevention of depressive disorder recurrence. medical thera py included treatment of primary disease in cases of depressive disorders of organic gene sis and seasonal prevention of depressions of endogenous character and psychotherapy – techniques of selfregulation, and measures for social adaptation. comparison analysis of effectiveness was conducted in three main directions: 1. dynamics of mental state (recovery, significant improvement of mental state, improvement of mental state, lack of dynamics of mental state, worsening of mental state). 2. dynamics of pathological manifestations severity according to psychometric scales. 3. dynamics of life quality rate during the treatment. reassessment performed 6 months after the beginning of the treatment. to evaluate the effictiveness of the suggested treatment, regimens were divided into 6 major groups due to the emigrants and reemigrants suffering from psychogenic, endogenous and organic depressive disorders, receiving the suggested treatment and 6 similar in all clinical and social characteristics comparison groups. comparison of the results was conducted in pairs of each clinical group due to the clinical variant and genesis of depressive disorder. comparing analyzes of effectiveness was conducted in three main directions: dynamics of mental state, dynamics of pathological manifestations severity according to psychometric rate and dynamics of life quality during treatment. assessment of mental state dynamics as a result of the conducted therapy proved significantly higher efficiency of the offered scheme of treatment compared to the traditional one (fig. 1). in the main group higher quality of treatment was achieved: in the group of the emigrants suffering from psychogenic depressive disorders in 81.2% of the patients clinical recovery, in 9.4% – a significant improvement, in 6.3% – improvement, no dynamics was detected in one patient (3.1%), cases of reverse dynamics of depressive disorders under the influence of treatment were not found, while in the control group of patients clinical recovery was achieved in 42.4% of cases, significant improvement – in 33.3%, improvement – in 15.2%, no changes – in 6.1%, and in one case (3.0%) in verse dynamics (p<0.05) was evidenced. in the emigrants with endogenous depressive disorder, who received the suggested scheme of treatment, clinical recovery was achieved in 59.3% of cases, significant improvement – in 21.9%, improvement – in 12.5%, lack of dynamics – in 6.3%, in the group of patients receiving traditional therapy clinical recovery was achieved in 24.2%, a significant improvement and improvement – in 27.3%, no dynamics was detected in 18.2%, worsening of state – in one case (3.0%) (p<0.05). in the group of emmigrants with depressive disorders of organic genesis, clinical recovery was achieved in 36.6%, a signifig. 1. comparison of the results of traditional and suggested treatment schemes for the emmigrants, patients with depressive disorders. 8 p sy c h ia t r y issn 2413-6077. ijmmr 2017 vol. 3 issue 1o. p. venger et al. ficant improvement – in 40.0%, improvement – in 16.7%, lack of dynamics – in 6.7%, in the comparison group clinical recovery was found in 19.3%, a significant improvement – in 19.3%, improvement – in 32.3%, no explicit dynamics – in 22.6%, worsening of state – in 6.5% (p<0.05). in the re-emigrants with depressive disorders higher effectiveness of the suggested scheme was also proved (fig. 2). in the re-emigrants suffering from psychogenic depressive disorder treated by the sugges ted scheme, clinical recovery was achieved in 71.0% of cases, significant improvement – in 16.1%, improvement – in 9.7%, the lack of dynamics occurred in one case (3.2%), the inver se dynamics was not detected, and there-emigrants with psychogenic depressive disorders who received conventional therapy achieved clinical recovery in 32.2%, a significant improvement – in 35.5%, improvement – in 22.6%, no dynamics was detected in 6.5%, inverse dynamics – in 3.2% of the patients (p<0.05). in the re-emigrants with endogenous depressive disor ders, treated by traditional therapy, clinical recovery was achieved in 51.5% of cases, significant improvement – in 22.6%, impro vement – in 19.4%, no dynamics was detected in 6.5% of the patients. after the treatment by the suggested clinical therapy, recovery and significant improvement occurred in 19.3%, improvement – in 42.0%, lack of dynamics – in 12.9%, inverse dynamics – in 6.5% of the patients (p<0.05). in the re-emigrants with depressive disorders of organic genesis, the suggested scheme has lead to clinical recovery in 33.3%, a significant improvement – in 36.7%, improvement – in 20.0%, no dynamics was detected in 10.0%, and in the comparison group of clinical recovery occurred in 13.3%, a significant improvement – 20.0%, improvement – in 40.0%, lack of dynamics – in 16.7%, state worsening – in 10.0% of the patients (p<0.05). discussion due to the the intended aim and objectives of our study the analisis of the received results made it possible to justify the treatment and rehabilitation programs for the emigrant and re-emigrant patients with depressive disorders. considering the features of depressive disorders in these groups of patients and lack of information on the developed approaches to treatment and rehabilitation, we suggested new therapeutic schemes. therefore, we invite you to the discussion of scientific research that proved positive results. analysis of quantitative indices by scale of depression according to m. hamilton also confirmed significantly higher efficiency of the suggested scheme for both the emigrant and re-emigrant patients with psychogenic, endogenous and organic depressive disorders. so, the suggested scheme of therapy for the emigrants suffering from psychogenic depressive disorder was significantly more effective against depressed mood, guilt, suici dal intentions, early, middle and late insomnia, working capacity and activity, retardation, agitation, mental anxiety, somatic symptoms, genital symptoms and hypochondria. the suggested scheme also promoted to significant decrease of the overall rate of depression and indicators of adynamic, agitated depression, depression with fear and undifferentiated depression. the suggested scheme of treatment enabled achievement of significantly improved indexes of main subrates of m. hamilton depression scale in the emigrants with endogenous depressive disorders. significant differences were detected by comparison of the indexes after reasonable treatment of depressed mood, guilt, early, middle and late insomnia, working capacity and activity, retardation, agitation, men tal anxiety, somatic symptoms and hypochondria, as well as general indicators of defig. 2. comparison of the results of traditional and suggested treatment schemes for the re-emigrants with depressive disorders. 9 p sy c h ia t r y issn 2413-6077. ijmmr 2017 vol. 3 issue 1 o. p. venger et al. pression, indexes of adynamic, agitated depression, depression with fear and undifferentiated depression. for the emigrants suffering from depressive disorders of organic genesis, the suggested scheme of treatment was significantly more effective against depressed mood, guilt, su icidal intentions, early, middle and late insomnia, working capacity and activity, retardation, agitation, mental anxiety, somatic symptoms, genital symptoms and hypochondria, as well as general indicators of depression, indexes of adynamic, agitated depression, depression with fear and undifferentiated depression. the suggested scheme of treatment also allowed decreasing anxiety in the re-emigrants and the emigrants with depressive disorders of different genesis. for the emigrants suffering from psycho genic depressive disorders, the suggested scheme was significantly more effective against an xious mood, tension, anxiety, insomnia, de pres sed mood, muscle somatic symptoms, somatic sensory symptoms, cardiovascular symptoms, respiratory symptoms, hastro intestional symptoms, autonomic symptoms, as well as general severity of psychic and somatic anxiety. for the emigrants with endogenous depressive disorder, the suggested scheme was significantly more effective against anxious mood, tension, insomnia, depressed mood, muscle somatic symptoms, somatic sensory symptoms, cardiovascular symptoms, respiratory symptoms, vegetative symptoms, as well as general severity of psychic and somatic anxiety. for the emigrants suffering from depressive disorders of organic nature, the suggested scheme was significantly more effective against anxious mood, tension, anxiety, insomnia, depressed mood, muscle somatic symptoms, cardiovascular symptoms, respiratory symptoms, vegetative symptoms, as well as general severity of psychic and somatic anxiety. in the re-emigrants the positive influence of the suggested scheme was evidenced. thus, for the re-emigrants suffering from psychogenic depressive disorder, the suggested scheme was more effective against anxious mood, tension, anxiety, insomnia, cognitive disorders, depressed mood, muscle somatic symptoms, somatic sensory symptoms, cardiovascular symptoms, respiratory symptoms, gastrointestinal symptoms, vegetative symptoms, as well as general severity of psychic and somatic anxiety. the suggested scheme of treatment for the re-emigrants with endogenous depressive disorder, was more effective against anxious mood, tension, anxiety, insomnia, cognitive disorders, depressed mood, muscle somatic symptoms, somatic sensory symptoms, cardiovascular symptoms, respiratory symptoms, gastrointestinal symptoms, vegetative symptoms, as well as the general severity of psychic and somatic anxiety. for the re-emigrants with depressive disorders of organic genesis, the suffested scheme significantly improved the rate of anxious mood, anxiety, insomnia, muscle somatic symp toms, somatic sensory symptoms, cardiovascular symptoms, respiratory symptoms, gastrointestinal symptoms and severity of psychic and somatic anxiety. the influence of the suggested therapy on the severity of psychopathological symptoms was also more effective compared to the traditional one. for the emigrants suffering from psychogenic depressive disorders, the suggested scheme proved significantly better results in the rate of somatization, obsessive-compulsive disorders, interpersonal sensitivity, depression, anxiety, hostility, phobic anxiety and general indicator (index gsi), the index of symptoms detecting (psi) and index of distress severity (pdsi). for the emigrants suffering from depressive disorders of organic genesis, the suggested scheme was proved effectiveness in the rate of of somatization, obsessive-compulsive disorders, interpersonal sensitivity, depression, anxiety, hostility, phobic anxiety and general indicator (index gsi), the index of symptoms detecting (psi) and index of distress severity (pdsi). in the patients with depressive disorders of different genesis significantly better rates of psychopathological symptoms severity influenced by the suggested scheme of therapy were achieved. thus, the re-emigrants suffering from psychogenic depressive disorders, treated by the suggested scheme achieved more pronounced decrease in the rates of somatization, obsessive-compulsive disorders, interpersonal sensitivity, depression, anxiety, hostility, phobic anxiety and general indicator (index gsi), the index of symptoms detecting (psi) and index of distress severity (pdsi). for the re-emigrants with endogenous depressive disorders, the suggested therapy scheme promoted to more effective decrease in the rates of somatization, obsessive-compul10 p sy c h ia t r y issn 2413-6077. ijmmr 2017 vol. 3 issue 1 sive disorders, interpersonal sensitivity, depression, anxiety, hostility, phobic anxiety and general indicator (index gsi), the index of symptoms detecting (psi) and index of distress severity (pdsi). for the re-emigrants with depressive disorders of organic genesis, the suggested treatment scheme was more effective for decrease in rates of somatization, obsessive-compulsive disorders, interpersonal sensitivity, anxiety, hostility, phobic anxiety and general indicator (index gsi), the index of symptoms detecting (psi) and index of distress severity (pdsi). significant improvement in quality of life during the treatment is an important factor that positively assesses the perspectives of implemen tation of the suggested treatment regimens. thus, in the group of emigrants suffering from psychogenic depressive disorders, the suggested scheme promoted to achievement of significant improvement of physical health and psychological qol. for the emigrants with endogenous depressive disorders, the use of the suggested scheme of therapy promoted to improvement of qol in physical health, psychological qol and environment. for the emigrants suffering from depressive disorders of organic genesis, the suggested scheme was more effective in improvement of qol in physical health and psychological qol. the improvement of qol during the treatment by the suggested scheme was also achieved in the re-emigrants. thus, in the re-emigrants with psychogenic depressive disor ders, significantly higher qol of physical health and psychological qol were defined, in the reemigrants with endogenous depressive disorders – of qol in physical health, psycho logical qol and environment, in the re-emigrants with depressive disorders of organic origins – of qol in physical health, psychological qol and environment. thus, the comparative analysis of the suggested treatment schemes for the emigrants and re-emigrants suffering from psychogenic, endogenous and organic depressive disorder proved significantly higher effectiveness comparing with traditional one. conclusions the analysis of the influence of the suggeste therapy schemes on the severity of psychopathological symptoms also proved higher effectiveness compared with the traditional one; it was evidenced by significant decrease in the rate of somatization, obsessive-compul sive disorders, interpersonal sensitivity, de pression, anxiety, hostility, phobic anxiety, as well as general index, the index of symptoms detection and the index of distress severity. the suggested therapy scheme of also promoted to improvement of life quality of the emigrants and re-emigrants with depressive disorders, significant increase in the rates of their physical health, psychological quality of life and environment compared to the comparison group. the results allow recommending the developed treatment scheme for implemen tation in the complex treatment of emigrants and reemigrants suffering from psychogenic, endogenous and organic depressive disorders. references 1. de wit m, tuinebreijer w, dekker j, et al. depressive and anxiety disorders in different ethnic groups m. social psychiatry and psychiatric epidemiology. 2008;43(11):905–912. 2. hamilton m. rating scale for depression by max hamilton. journal of neurology, neurosurgery, psychiatry. 1960;23:56–62. 3. hynie m, crooks va, barragan j. immigrant and refugee social networks: determinants and consequences of social support among women newcomers to canada. canadian journal of nursing research. 2011;43:26–46. 4. gavin be, kelly bd, lane a, et al. mental health of emigrants. irish medical journal. 2001;94:229–230. 5. guruge s, thomson ms, george u, chaze f. social support, social conflict, and immigrant women’s mental health in a canadian context: a scoping review. journal of psychiatric and mental health nursing. 2015;22:655–667. 6. lauber c, lay b, rossler w. patterns of psychiatric inpatient care in migrants: results from switzerland. swiss medical weekly. 2006;135:50–56. 7. migration in ukraine: facts and numbers. international organization of migration. kyiv: representation iom in ukraine; 2011;7. 8. montgomery sa, asberg м. a new depression scale designed to be sensitive to change. british journal of psychiatry. 1979;134:382–389. 9. selten jp, veen n, feller w, et al. incidence of psychotic disorders in immigrant groups to the netherlands. british journal of psychiatry. 2001; 178:367–372. received: 2016-12-29 o. p. venger et al. 20 in t e r n a l m e d ic in e issn 2413-6077. ijmmr 2018 vol. 4 issue 2 doi 10.11603/ijmmr.2413-6077.2018.2.9248 bullous pemphigoid a rare autoimmune disease: a case report s. m. biradar1, s. dhanavidya1, p. kavya1, t. keerthi1, n. sunanda1, s. c. marapur1, v. warad2, n. v. kalyane1 1 – bldea’s ssm college of pharmacy and research center, vijaypur, india 2 – shri b. m. patil medical college hospital and research centre, vijaypur, india background. bullous pemphigoid (bp) is a rare autoimmune blistering skin disease in the elderly and it is manifested by cutaneous blisters on the skin lesions. the objective was to emphasize the rare case of bp. methods. a case report of bp in a 58-year-old male patient admitted to a dermatology ward is presented. results. a 58-year-old male patient with complaints of fluid-filled skin lesions, was examined initially over the trunk, gradually progressed involving b/l upper and lower extremities. even though the patient was treated with the recommended therapy of corticosteroid (dexamethasone) along with adjuvant drugs, new skin lesions continued to develop, and the patient’s condition worsened. the prednisolone was started in place of dexamethasone on the fifth day of treatment at its higher dose (50mg/day), the prednisolone proved its efficacy to combat the extensive condition of bp. conclusions. bullous pemphigoid is a distressing blistering skin disease. untreated disease is often fatal because of the susceptibility to infection and fluid-electrolyte disturbances. the mortality of patients with bullous pemphigoid has been significantly reduced with the advent of new therapies and treatment modalities. the treatment with systemic and topical corticosteroids forms the mainstay of treatment along with other adjuvant drugs. in the present case study, the use of prednisolone has proven its efficacy in the extensive disease state of bp and improved the patient’s quality of life. key words: bullous pemphigoid; rare autoimmune disease; dexamethasone; prednisolone. introduction bullous pemphigoid (bp) is a rare autoimmune blistering disease and affects the elderly mostly. however, on rare occasions, it may affect children and young adults too. the annual incidence of bp has been estimated to range from 2 to 14 new cases per one million people. its incidence is expected to rise as a consequence of population aging. a recent study in france has established a 3-time increase in the annual incidence of bp over the last 15 years with 21.7 new cases per million inhabitants [1]. although several clinical variants have been recognized, bp usually presents with tense blisters arising on healthy or erythematous skin, typically involving the flexor surfaces. autoantibodies against well-characterized autoantigens, bp180 and bp230, are believed to play a crucial role in the pathogenesis of bp. the prognosis of bp has been studied by several research groups, but the results of the studies have been inconsistent [2]. the body’s immune system is confused and makes an antibody (a type of protein used to fight infection) that targets a part of the skin that normally holds it together. the attack on the skin causes blisters (firm, fluid-filled bubbles on the skin) to form. this disease most often involves only the skin, but sometimes it may affect to eyes, mouth, and genital organs. the following clinical variants are described: classic (described above), localized, nodular, vegetating, erosive, erythrodermic, juvenile and drug-induced [3]. the clinical presentation of bp is tense blisters, which are often seen on erythematous or normal-looking skin of limbs and trunk and may be widespread or localized. bullae and ⁄or erosions may be present in the oral and genital mucosa. pruritus alone or associated with erythema and/or urticated plaques may precede the formation of bullae by weeks or months; in some cases, bullae may not become clinically apparent [4]. a recent study conducted in the international journal of medicine and medical research 2018, volume 4, issue 2, p. 20-23 copyright © 2018, tsmu, all rights reserved corresponding author: dr. s m. biradar, dept. of pharm. d programmee, bldeas’ ssm college of pharmacy and research centre, vijauypur-586103, india. e-mail: smbiradar@rediffmail.com s. m. biradar et al. 21 in t e r n a l m e d ic in e issn 2413-6077. ijmmr 2018 vol. 4 issue 2 united kingdom found an incidence of 4.3 (95% confidence interval (95% ci), 4.0–4.6) per 100,000 person-years [5]. the bp patients have complex co-morbidity profiles, most notably neurological disorder, as well as autoimmune and are prone to infection. various studies have confirmed a strong association between bp and neurological disorders [6]. case study a 58-year-old male patient admitted to a dermatology ward with chief complaints of fluid-filled skin lesions (fig. 1), initially over the trunk, gradually progressed involving bilateral upper and lower extremities. the lesions did not rupture spontaneously and were associated with mild itching. upon admission, general physical examination was performed in which the patient was moderately built and nourished, conscious and co-operative. blood pressure was 80/60 mm hg. local examination of the skin was performed. it showed that multiple tense vesicles and bullae over the trunk, on both sides upper and lower extremities, few erosions, nikolsky’s sign, and bulla signs had spread. the initial blood investigation revealed total count (tc) 19970 cells/mm3, neutrophils 57.4%, lymphocytes 15.5%, eosinophils 24.5%, monocytes 2.3%, basophils 0.3%, rbc 5.05 ×1012/l, hb 15.0 g/dl, packed cell volume 45.3%, mcv 89.7 fl, mch 29.7 pg, mchc 33.1 %, platelet count 240000 cells/mm3, esr 10 mm/hour. initially, dexamethasone iv and oral were prescribed along with other drugs: ranitidine, calcimax (calcium+ vitamin d3+ magnesium + zinc) and teczine (levocetirizine). in two days, a few new lesions occurred, then tablet cefixl (cefixime+cloxacillin) 200 mg was prescribed. in 24 hours, multiple new lesions occurred again, then omnacortil (prednisolone) (higher dose) – a corticosteroid, was prescribed. gv lotion (gentian violet) and ointment liquid paraffin were prescribed twice a day (bid), as well as tablet dapsone (dapsone) once daily (od) and capsule nicoglow (nicotinic acid) bid. treatment was continued up to the time of discharge and the patient condition improved. discussion bullous pemphigoid is a rare autoimmune blistering disease; it typically affects the elderly and is followed by significant morbidity and mortality [7]. the clinical symptoms of the disease are development of oral lesions in about one-third of the patients; lesions may occur on the trunk, extremities, and intertriginous areas. in most of the cases, no clear precipitating factors are identified; some precipitating factors are exposure to ultraviolet light, radiation therapy and exposed to certain drugs like furosemide, penicillin, sulfasalazine, and captopril [7]. the disease is characterized by the formation of igg auto antibodies targeting dystonin (bullous pemphigoid antigen 1 (bpag1), and /or type xvii collagen also called bullous pemphigoid antigen 2 (bpag2), which is a component of hemides mosomes [2]. in the present case fluid-filled lesions were seen initially over the trunk and gradually progressed involving lower and upper extremities. the recommended treatment for bp is as follows [8]: initial therapy. initial therapy is determined by the extent and rate of progression of the lesions. the priority is to control lesions usually in a slowly progressive form of the disease; initial treatment includes intralesional injections of corticosteroids or topical applications of corticosteroids. maintenance therapy. once most lesions are healed, the dose and type of medication are gradually reduced to limit the risk of side effects. understanding the rate of dose reduction is determined by clinical response and overall disease activity. it is important to monitor this balance and limit use of unnecessary medication as many fatalities are related to complications associated with the therapy. the available treatments work via different mechanisms. some aim to suppress the inflammatory process e.g. corticosteroids, antibiotics, anti-inflammatory mediators. immune modulating treatments include intravenous immunoglobulins. intravenous immunoglobulin has been widely tried as an immunomodulatory agent in various auto-antibody mediated blistering diseases. fig. 1. skin lesions of bullous pemphigoid. s. m. biradar et al. 22 in t e r n a l m e d ic in e issn 2413-6077. ijmmr 2018 vol. 4 issue 2 systemic corticosteroids are most commonly used: dexamethasone and prednisolone. typical recommendations for wide spread disease are for a starting daily dose of about 1 mg/kg continued until cessation of new blister formation then gradually decreased. the starting dose ranges between 40 and 80 mg daily, usually 60 mg daily. lower starting doses of 20 to 40 mg daily have been recommended. antibiotics should be considered as the first line of treatment for both localized and mild to moderate disease. antibiotic treatment is provided for at least 2 weeks. azathioprine and methotrexate are also recommended [8]. in the present case study, the patient was prescribed with dexamethasone initially for four days, even though a few new lesions developed. in the next consecutive days the dexamethasone was replaced with prednisolone at its higher dose, and then the patients’ condition improved. similar kind of results was noticed with prednisolone prescription in the extensive disease state of bp [7]. dapsone was given in one week after patient’s admission as it was an immunosuppressant to reduce the action of auto-antibodies and nicotinamide was given on the last two days as it had some effects in reducing itching and redness. conclusions bullous pemphigoid is a distressing blistering skin disease. untreated, bullous pemphigoid is often fatal because of the susceptibility to infection and fluid-electrolyte disturbances, hence utmost importance is given for bp treatment. the mortality of patients with bullous pemphigoid has been significantly reduced with the advent of new therapies and treatment modalities. the treatment with systemic and topical corticosteroids forms the mainstay of treatment of bp along with other adjuvant drugs. in the present case study, the use of prednisolone has proven its efficacy in the extensive disease state of bp and improved the patient’s quality of life. бульозний пемфігоїд – рідкісне автоімунне захворювання (клінічний випадок) s. m. biradar1, s. dhanavidya1, p. kavya1, t. keerthi1, n. sunanda 1, s. c. marapur1, vijaykumar warad2, n. v. kalyane1 1 – bldea’s ssm college of pharmacy and research center1, vijaypur, india 2 – shri b. m. patil medical college hospital and research centre2, vijaypur, india вступ. бульозний пемфігоїд (бп) – це рідкісне автоімунне захворювання шкіри у літніх людей, яка проявляється епідермальними пухирями на пошкоджених шкірних покривах. мета – акцентувати увагу на рідкісному випадку бп. методи дослідження. представлено клінічний випадок бп у 58-річного пацієнта-чоловіка, який поступив у дерматологічне відділення. результати дослідження. обстежено 58-річного пацієнта із скаргами на заповнені рідиною утвори на шкіри, які з’явилися спочатку на тулубі, і поступово прогресували, поширюючись білатерально на верхні та нижні кінцівки. незважаючи на те, що пацієнт отримував рекомендовану терапію на базі кортикостероїду (дексаметазон) разом з ад’ювантними препаратами, шкірні елементи та пошкодження шкіри прогресували, і стан хворого погіршився. було застосовано преднізолон замість дексаметазону на п’ятий день лікування у більш високій дозі (50 мг/добу). останній довів свою ефективність для боротьби з прогресуючим бп. висновки. бульозний пемфігоїд – хронічне, набуте аутоімунне захворювання. нелікована хвороба часто смертельна через схильність до інфекцій та порушення водно-електролітного обміну. смертність пацієнтів з бульозним пемфігоїдом значно зменшилася з появою нових методів терапії. базисним є лікування системними і топічними кортикостероїдами у цьому дослідженні застосування преднізолону довело свою ефективність при генералізованій формі захворювання та дозволило поліпшити якість життя пацієнта. ключові слова: бульозний пемфігоїд; рідкісні аутоімунні захворювання; дексаметазон; преднізолон. s. m. biradar et al. 23 in t e r n a l m e d ic in e issn 2413-6077. ijmmr 2018 vol. 4 issue 2 references 1. teixeira vb, cabral r, brites mm, vieira r, figueiredo a. bullous pemphigoid and comorbidities: a case-control study in portuguese patients. anais brasileiros de dermatologia. 2014 apr;89(2):274-8. 2. kjellman p, eriksson h, berg p. a retrospective analysis of patients with bullous pemphigoid treated with methotrexate. archives of dermatology. 2008 may 1;144(5):612-6. 3. zanella rr, xavier ta, tebcherani aj, aoki v, sanchez ap. bullous pemphigoid in younger adults: three case reports. anais brasileiros de dermatologia. 2011 apr;86(2):355-8. 4. venning va, taghipour k, mustapa mm, highet as, kirtschig g. british association of dermatologists’ guidelines for the management of bullous pemphigoid 2012. british journal of dermatology. 2012 dec 1;167(6):1200-14. 5. bastuji-garin s, joly p, lemordant p, sparsa a, bedane c, delaporte e, roujeau jc, bernard p, guillaume jc, ingen-housz-oro s, maillard h. risk factors for bullous pemphigoid in the elderly: a prospective case–control study. journal of investigative dermatology. 2011 mar 1;131(3):637-43. 6. liu yd, wang yh, ye yc, zhao wl, li l. prognostic factors for mortality in patients with bullous pemphigoid: a meta-analysis. archives of dermatological research. 2017 jul 1;309(5):335-47. 7. varytimiadis d, sotiriou e, trigoni a, ioannides d. successful full clinical remission of bullous pemphigoid with intravenous pulse methylprednisolone and cyclophosphamide: a case report. aristotle university medical journal. 2012 apr 28;39(1):35-8. 8. bower c. bullous pemphigoid: guide to diagnosis and treatment. prescriber. 2006 jun 19;17(12): 44-50. received: 2018-06-26 s. m. biradar et al. 79 b io m e d ic a l s c ie n c e s issn 2413-6077. ijmmr 2018 vol. 4 issue 2 doi 10.11603/ijmmr.2413-6077.2018.2.9810 gender and age aspects of bioenergetics processes in experimental passive tobacco smoking and monosodium glutamate administration a. v. rutska, i. ya. krynytska i. horbachevsky ternopil state medical university, ternopil, ukraine background. active smoking and exposure to passive smoke are responsible for more than 5 million deaths each year. at the same time, a characteristic feature of present food technologies is the use of food additives that are not always safe for human health, such as monosodium glutamate (msg). objective. the aim of the research was to determine the changes in mitochondrial enzymes activity in rats in case of passive tobacco smoke combined with prolonged administration of msg in the sex and age aspects. methods. the evaluation of bioenergetics processes in the mitochondria of circulating neutrophils was carried out using succinate dehydrogenase (sdg) and cytochrome oxidase (co) activity. results. passive tobacco smoke combined with the msg administration in mature male-rats is accompanied by a significant inhibition of bioenergetics processes, as evidenced by a decrease in succinate dehydrogenase activity by 47.1% (p<0.001) compared to the intact animals, which is by 27.9% (p<0.001) below this index in case of the isolated effect of tobacco smoke and reduction of cytochrome oxidase activity by 27.5% (p<0.001) compared to the control group. conclusions. thus, the findings suggest that low dose intake of monosodium glutamate enhances the ability of tobacco smoke to disrupt the cell's bioenergetics processes by affecting the respiratory chain function and generation of atp. therefore, it is advisable to investigate the established toxic doses of e621, as well as to study the molecular mechanisms of the ‘safe’ (allowed) doses of msg effect on a living organism. key words: passive tobacco smoking; monosodium glutamate; cytochrome oxidase; succinate dehydrogenase. international journal of medicine and medical research 2018, volume 4, issue 2, p. 79-86 copyright © 2018, tsmu, all rights reserved corresponding author: inna krynytska, d.med.sci., professor, dept. of functional and laboratory diagnostics, i. horbachevsky ternopil state medical university, 1 majdan voli, ternopil 46001, ukraine. tel.: +380964790616. e-mail: krynytska@tdmu.edu.ua a. v. rutska et al. introduction the who estimates that in 2015 there was about 1.1 billion adult smokers worldwide, representing nearly a quarter (22%) of the global adult population. number of women of reproductive age, who smoke, is also increasing. it is expected that by 2025 more than 500 million women will be smokers, accounting for about 20% of the global female population [1-2]. according to solomenchuk t. m., in ukraine the prevalence of smoking among women has tripled over the past 30 years [3]. over 6 million people die from tobacco each year. more than 5 million of those deaths are the result of direct tobacco use while more than 600,000 are the result of nonsmokers b e i n g e x p o s e d t o s e c o n d h a n d s m o k e . secondhand smoke is a major health hazard, especially for infants and children. in the united states, it has been estimated that 43% of children aged from 2 months to 11 years live in a home with at least one person that smokes. the prevalence of passive infant smoking was reported to be around 40% in europe as well [4]. at the same time, the characteristic feature of present food technologies is the use of food additives. one of the most common food additives in ukraine as well as in europe is monosodium glutamate (msg). encoded e621, it is a food additive from a group of flavor enhancers, used in a wide range of foods, such as soups, sauces, mixed condiments, chips, meat products, and puddings. despite its taste stimulation and improved appetite enhancement, reports indicate that msg is toxic to human and experimental animals [5]. an important role in the implementation of toxic action of xenobiotics is the violation of the energy supply in a cell. the most toxic metabolites, as well as products of their initiated lipoperoxidation disturb oxidation of substrates with dehydrogenases, transport electrons 80 b io m e d ic a l s c ie n c e s issn 2413-6077. ijmmr 2018 vol. 4 issue 2 along the respiratory chain, causing the uncoupling of mitochondrial oxidative phosphorylation. irreversible disorders in the structure and functioning of mitochondria, caused by the action of excessive amounts of reactive oxygen species (ros), cause displacement of energy metabolism towards increasing the intensity of glycolysis and inhibition of oxidative phos phorylation [6]. the aim of this investigation was to determine the changes of mitochondrial enzymes activity in rats in case of secondhand tobacco smoke combined with prolonged administration of monosodium glutamate in the sex and age aspects. methods experimental studies were conducted on 32 inbred mature white male rats weighing 180200 g, 32 mature nonlinear white female rats weighing 180-200 g and 32 nonlinear immature white male rats weighing 60-80 g. each group of animals was divided into four subgroups: the 1st – the intact rats (n=8); the 2nd – the rats with modeled passive tobacco smoking (n=8); the 3rd – the rats, which were injected with monosodium glutamate (n=8); the 4th – the rats with modeled passive tobacco smoking combined with monosodium glutamate injection (n=8). the investigations were conducted following the general rules and regulations of the european convention for the protection of vertebrate animals used for experimental and other scientific purposes (strasbourg, 1986), the general ethics on animal experimentation (kyiv, 2001). the rats of the second experimental group were exposed to tobacco smoke for 30 days. the model of the passive tobacco smoking was created by means of airtight chamber volume of 30 liters that allows fumigating animals, which could move freely [7]. tobacco smoke, formed by smoking of 2 cigarettes prima sribna (chervona) (containing 0.8 mg of nicotine and 10 mg of tar), was delivered into it through openings in the chamber. four animals were simultaneously in the chamber for 30 minutes. the third group was administered msg diluted in a distilled water at a dose of 30 mg/kg body weight (corresponds dose 2 g per day in humans) for 30 days via intragastrical tube [8]. msg was produced by sigma-aldrich (usa). rats of the fourth group were exposed to tobacco smoke and msg administration in combination for 30 days. on the 31-st day the experimental animals were euthanized under thiopental anesthesia and the whole blood was used for further investigation. the population of neutrophils was obtained by whole blood centrifugation at double density gradient 1.077 and 1.093 of ficoll-urografin. after 40 minutes of centrifugation at 4 °c and the speed of 1500 rpm, two interphases were formed. the upper interphase (on the border of plasma – ficoll-urografin density 1.077) consisted of mononuclear cells: 80% lymphocytes, 15-18% monocytes and 2-3% granulocytes. lower interphase (on the border of solutions gradient density 1.077-1.092) was the population of neutrophils. the number of viable cells presented in a cell sus pen sion was 98-99% (trypan blue exclusion test). the analysis of cell samples to determine neutrophils with ros (hydrogen peroxide) overproduction was evaluated by the flow laser cytometry method on flow cytometer epics xl (beckman coulter, usa), using 2.7-dichlorodihydrofluorescein diacetate. the value of the studied parameter was expressed as a percentage (ratio of cells with ros overproduction to general cell count×100 %). the evaluation of bioenergetics processes in the mitochondria of neutrophils was carried out using succinate dehydrogenase activity (sdg), which was studied by the reaction of the reduction of potassium ferricyanide, which solution had a yellow color, to colorless potassium ferrocyanide by succinate under the influence of sdg and cytochrome oxidase activity (co) by the oxidation reaction of dimethyl-n-phenylenediamine. all spectrophotometric measurements were made on a sf-46 spectrophotometer. statistical processing of digital data was carried out using the software excel (microsoft, usa) and statistica 6.0 (statsoft, usa). the distribution of data was analyzed according to assessment of normality by kolmogorov-smirnov criterion. the obtained values had a normal distribution, so the difference between the groups was analyzed using the student’s t-criterion. all data were presented as m (mean)±m (standard error). a probability level (p value) of less than 0.05 was considered to be statistically significant. the influence of factors on the indices was determined using the one-factor dispersion analysis (anova). the linkage between the studied indices was established on the basis of the results of the correlation analysis using the pearson correlation coefficient. a. v. rutska et al. 81 b io m e d ic a l s c ie n c e s issn 2413-6077. ijmmr 2018 vol. 4 issue 2 results our studies have proved that succinate dehydrogenase activity in leukocytes mitochondria in the mature male-rats under passive tobacco smoking significantly decreases by 26.6% compare to the control group (table 1). passive tobacco smoking combined with the monosodium glutamate injection is accompanied by an even greater decrease in succinate dehydrogenase activity (by 47.1%, p<0.001) vs. the control group, which is 27.9% (p<0.001) below this indicator, providing isolated effect of tobacco smoke. in this case, the prolonged administration of glutamate monosodium results in a less significant decrease in succinate dehydrogenase activity (by 17.2%, p<0.02) compared to the control rats. in the mature female rats, passive tobacco smoking is accompanied with the decrease in succinate dehydrogenase activity in blood mitochondria of leukocytes by 39.8% (p<0.001) compared to the control group. passive tobacco smoking combined with the monosodium glutamate injection is accompanied by an even greater decrease in succinate dehydrogenase activity (by 62.1%, p<0.001) compared to the control group, which is 37.1% (p<0.001) below this indicator, provided isolated effect of tobacco smoke. at the same time, prolonged administration of monosodium glutamate results in a decrease in succinate dehydrogenase activity by only 9.6% (p<0.05) compared to the control rats. in the gender aspect, the intensity of changes in succinate dehydrogenase activity exceeded the rates of the mature male-rats in case of passive tobacco smoking by 13.2%, in case of its combination with the monosodium table 1. influence of passive tobacco smoking and monosodium glutamate on the energy processes in neutrophils mitochondria of rats (m±m, n=8) indicator groups of experimental animals intact passive tobacco smoking monosodium glutamate passive tobacco smoking+ monosodium glutamate mature male-rats sdg, nmol/(mg×min) 2.44±0.09 1.79±0.09 p1<0.001 2.02±0.10 p1<0.02 1.29±0.07 p1<0.001 p2<0.001 p3<0.001 cо, nmol/(mg×min) 2.00±0.07 1.69±0.09 p1<0.05 1.79±0.05 p1<0.05 1.45±0.07 p1<0.001 p2>0.05 p3<0.01 mature female-rats sdg, nmol/(mg×min) 2.51±0.08 1.51±0.07 p1<0.001 2.27±0.06 p1<0.05 0.95±0.05 p1<0.001 p2<0.001 p3<0.001 cо, nmol/(mg×min) 2.07±0.09 1.57±0,06 p1<0.001 2.03±0.08 p1>0.05 1.29±0.04 p1<0.001 p2<0.05 p3<0.001 immature male-rats sdg, nmol/(mg×min) 2.91±0.08 1.66±0.11 p1<0.001 2.12±0.08 p1<0.001 0.85±0.04 p1<0.001 p2<0.001 p3<0.001 cо, nmol/(mg×min) 2.19±0.07 1.49±0.04 p1<0.001 1.79±0.04 p1<0,002 1.01±0.05 p1<0.001 p2<0.001 p3<0.001 notes: р1 – statistical significance of the differences compared to the intact animals; р2 – statistical significance of the differences compared to the animals with experimental passive tobacco smoking; p3 – statistical significance of the differences compared to the rats affected by monosodium glutamate. a. v. rutska et al. 82 b io m e d ic a l s c ie n c e s issn 2413-6077. ijmmr 2018 vol. 4 issue 2 glutamate injection – by 15.0%, in cases of prolonged administration of monosodium glutamate – by 7.6%; the intensity of changes in succinate dehydrogenase activity was lower with respect to those in the mature male rats. in the immature male rats, passive tobacco smoking is accompanied by a decrease succinate dehydrogenase (sdg) activity in mitochondria of blood leukocytes by 42.9% (p<0.001) compare to the control group. passive tobacco smoking combined with the monosodium glutamate injection is accompanied by a more significant decrease in succinate dehydrogenase activity (in 3.4 times, p<0.001) compare to the control group, which is 48.8% (p<0.001) below this indicator, subject to the isolated effect of tobacco smoke. in this case, prolonged administration of monosodium glutamate results in a less significant decrease in succinate dehydrogenase activity (by 27.1%, p<0.001) compared to the control rats. in the immature male rats, the changes of succinate dehydrogenase activity exceed the indicators of the mature male rats of all experimental groups: in cases of modeled passive tobacco smoking – by 16.3 %, with the introduction of monosodium glutamate – by 9.9 %, in cases of modeled passive tobacco smoking combined with the monosodium glutamate injection – by 23.7 %. unidirectional changes are evidenced relation to the end-stage enzyme of mitochondrial respiratory chain – cytochrome oxidase (co). in the mature male rats with modeled passive tobacco smoking, the activity of co in mitochondria of blood leukocytes in the mature male rats decreased by 15.5% (p<0.05) compare to the control group. in the animals with modeled passive tobacco smoking combined with the monosodium glutamate it is also accompanied by a decrease in cytochrome oxidase activity (by 27.5% (p<0.001) compare to the control group, which does not significantly differ from this indicator, provided isolated effect of tobacco smoke. at the same time, prolonged administration of monosodium glutamate leads to a decrease in the activity of this enzyme (by 10.5 % (p<0.05) compared with the control rats. in the mature female-rats, passive tobacco smoking is accompanied by a decrease in co activity in mitochondria of blood leukocytes by 24.1 % (p <0.001) compare to the control group. in the animals with modeled passive tobacco smoking combined with the monosodium glutamate it is accompanied by an even greater decrease in co activity (by 37.7%, p<0.001) compare to the control group, which is 17.8% (p<0.05) below this indicator, provided isolated effect of tobacco smoke. in this case, prolonged administration of monosodium glutamate do not lead to a significant reduction in co activity compared with the control rats. in the sex aspect, in the mature male rats with modeled passive tobacco smoking, the changes of cytochrome oxidase activity exceed by 8.6%, in cases of passive tobacco smoking combined with the monosodium glutamate – by 10.2%. in the immature male rats, passive tobacco smoking is accompanied by a decrease in the activity of co in mitochondria of blood leukocytes by 32.0% (p<0.001) compare to the control group. in the animals with modeled passive tobacco smoking combined with the monosodium glutamate it is accompanied by a more significant decrease in the activity of co (by 53.9%, p<0.001) compare to the control group, which is 32.2% (p<0.001) below this indicator, provided isolated effect of tobacco smoke. in this case, prolonged administration of monosodium glutamate results in a signi ficant decrease in co activity (by 18.3% (p<0.002) compared with the control rats. in the age aspect, in immature male-rats, the intensity of changes in co activity was higher than those of the mature male-rats of all experimental groups: with passive tobacco smoking – by 16.5%, with monosodium glutamate administration – by 7.8%, in group passive tobacco smoking combined with the monosodium glutamate – by 26.4%. our studies have showed that the percentage of neutrophils with ros overproduction in the mature male-rats under passive tobacco smoking significantly increases by 2.2 times (p<0.001) vs the control group (table 2). passive tobacco smoking combined with the monosodium glutamate administration is accompanied by an even greater increase in the percentage of neutrophils with ros over production (by 3.1 times, p<0.001) vs. the control group. the prolonged administration of monosodium glutamate results in a less significant decrease in the percentage of neutrophils with ros overproduction (by 40.3%, p<0.001) compared to the control rats. in the mature female rats, passive tobacco smoking is accompanied with the increase in the percentage of neutrophils with ros overproduction by 3.0 times (p<0.001) compared to the control group. passive tobacco smoking a. v. rutska et al. 83 b io m e d ic a l s c ie n c e s issn 2413-6077. ijmmr 2018 vol. 4 issue 2 combined with the msg administration is accompanied by an greater increase in the percentage of neutrophils with ros overproduction (by 3.7 times, p<0.001) compared to the control group. at the same time, prolonged administration of msg results in an increase in the percentage of neutrophils with ros overproduction by only 25.1% (p<0.02) compared to the control rats. the percentage of neutrophils with ros overproduction in the immature male-rats under passive tobacco smoking significantly increases by 2.8 times (p<0.001) vs the control group. passive tobacco smoking combined with the msg administration is accompanied by the greater increase in the percentage of neutrophils with ros overproduction (by 4.0 times, p<0.001) vs. the control group. the prolonged administration of msg results in a less significant increase in the percentage of neutrophils with ros overproduction (by 69.4%, p<0.001) compared to the control rats. discussion the most important functions of mitochondria are oxidation of intermediate carbohydrate, lipid and protein metabolites such as pyruvate, fatty acids, acetate, and use of energy released upon decomposition of these compounds for the biosynthesis of atp. mitochondrial dysfunctions, associated with oxidative phosphorylation processes, structural integrity of mitochondria and information identity of their genetic apparatus, intensify in cases of oxidative stress, in diseases caused by metabolic disorders, and carcinogenesis [9]. mitochondrial respiratory chain is the main intracellular source for generation of reactive oxygen species (ros), and the activity of succinate dehydrogenase as a component of the 2nd complex of the respiratory chain largely determines the rate of use of oxygen and synthesis of atp in mitochondria [10]. both succinate dehydrogenase and cytochrome oxidase determine the functioning of the chain of transformations of energy substrates [11]. consequently, the intensity of energy processes in cases of passive tobacco smoking combined with the monosodium glutamate is significantly reduced in the animals of all experimental groups, which ultimately leads to ‘energy starvation’. decrease in cellular respiration and cellular energy abnormalities may table 2. influence of passive tobacco smoking and monosodium glutamate on the percentage of neutrophils with ros overproduction of rats (m±m, n=8) indicator groups of experimental animals intact passive tobacco smoking monosodium glutamate passive tobacco smoking+ monosodium glutamate mature male-rats ros+-cells, % 17.98±0.86 39.44±2.56 р1<0.001 25.23±1.19 р1<0.001 56.39±2.82 р1<0.001 p2<0.01 p3<0.001 mature female-rats ros+-cells, % 14.95±0.98 45.21±1.70 р1<0.001 18.71±0.78 р1<0.02 60.95±3.07 р1<0.001 p2<0.02 p3<0.001 immature male-rats ros+-cells, % 12.90±0.77 36.89±1.62 р1<0.001 21.85±0.87 р1<0.001 51.39±2.60 р1<0.001 p2<0.002 p3<0.001 notes: р1 – statistical significance of the differences compared to the intact animals; р2 – statistical significance of the differences compared to the animals with experimental passive tobacco smoking; p3 – statistical significance of the differences compared to the animals affected by monosodium glutamate. a. v. rutska et al. 84 b io m e d ic a l s c ie n c e s issn 2413-6077. ijmmr 2018 vol. 4 issue 2 be caused by endogenic intoxication and oxidative stress. mechanisms of tobacco-induced oxidative stress are primarily due to the fact that tobacco smoke is a substance that is directly a source of ros as superoxide anion radical, hydrogen peroxide, and hydroxyl radical. in general, according to yanbaeva d.g. and coauthors, tobacco smoke contains 1017 molecules of oxidants per one breath [12]. in addition, activation of inflammatory cells induced by tobacco smoke promotes the production of oxidants in tissues. many components of tobacco smoke can accumulate in mitochondria and affect the function of the respiratory chain, thereby affecting the cellular generation of adenosine triphosphate (atp). in particular, carbon monoxide can interact with the components of the mitochondrial respiratory chain and suppress cytochrome oxidase [13]. lykhatskyi p. h. et al. in the experiment on rats of different ages exposed with tobacco smoke for 45 days had established the inhibition of mitochondrial enzymes activity, indicating violations of bioenergetics processes in the body [14]. the most pronounced changes they had observed were in immature and senile rats. in cases of administration of msg, the mitochondrial respiratory chain is the main source of ros. in addition, the increase in extracellular level of glutamate increases production of hydroxyl radicals. studies by sharma a. proved increased activity of α-ketoglutarate dehydrogenase (α-kgdh) in cases of the use of msg, which could activate the oxygen and form anion superoxide and hydrogen peroxide [15]. most researches interweave oxidative stress and tissue damage through glutamate receptors. glutamate receptors include three families of ionotropic receptors (nmda – n-methyl-d-aspartate, ampa – α-amino-3 hydroxy-5-methyl-4-isoxazolepropionic acid and kainate) and three groups of metabotropic receptors (mglur) [5]. activation of mglur produces oscillatory increases in intracellular ca2+. increased ca2+ influx leads to excessive uptake of ca2+ into mitochondria, which can increase generation of ros. generation of ros by ca2+-loaded polarized mitochondria depletes the antioxidant potential of a cell, which causes final disruption of cytoplasmic homeostasis of calcium that can trigger numerous cellular reactions, including activation of nitric oxide synthase, protein kinase c and inhibition of mitochondria enzymes activity [16]. studies by s. wu have proved that oxidative stress can even lead to fragmentation of mitochondria [17]. a study by s. kumari et al. [18] showed that the glutamate induced cytotoxicity was associated with mitochondrial hyperpolarization, increased ros production and en hanced oxygen consumption, glutamatecaused mitochondrial dynamic imbalance and reduced the number of cells with fragmented mitochondria, up to the splitting and activated autophagy. analyzing the correlation interactions between generation of ros by blood neutrophils and their bioenergetics processes, without taking sex and age features into account, in the experimental groups of rats a negative moderate correlation (r =-0.60; p<0.05) between the generation of ros and sdg activity and a similar interaction (r =-0.57; p<0.001) between the generation of ros and co activity, provided isolated effect of tobacco smoke, has been established. considering the division of rats by age and sex, in the mature male rats a significant negative correlation (r =-0.85; p<0.05) between the ros generation and succinate dehydrogenase activity and a similar interaction (r=-0.85; p<0.05) between the generation of ros and cytochrome oxidase activity, provided isolated effect of tobacco smoke. in case of passive tobacco smoking combined with the monosodium glutamate, installed an significant negative correlation (r=-0.88; p<0.05) between the generation of ros and cytochrome oxidase activity has been evidenced. in the mature female rats with modeled passive tobacco smoking, a significant negative correlation (r=-0.72; p<0.05) between the ros generation and activity of cytochrome oxidase has been established. in the immature male rats, a significant negative correlation between the generation of ros and cytochrome oxidase activity has been established, provided isolated effect of tobacco smoke (r=-0.78; p<0.05) as well as in cases of passive tobacco smoking combined with the monosodium glutamate (r=-0.72; p<0.05). using anova test, the influence of sex and age on succinate dehydrogenase and cytochrome oxidase activity of neutrophils mitochondria in the rats of the experimental groups has been determined. in cases of passive tobacco smoking combined with the monosodium a. v. rutska et al. 85 b io m e d ic a l s c ie n c e s issn 2413-6077. ijmmr 2018 vol. 4 issue 2 glutamate, an age differences in the activity of mitochondrial enzymes (p<0.05) have been proved. conclusions passive tobacco smoking is accompanied by an significant inhibition of bioenergetics processes in the mitochondria of circulating neutrophils in rats. the combination of passive tobacco smoking with the monosodium glutamate administration is accompanied by more pronounced changes. in the sex aspect, the bioenergetics processes under the condition of passive tobacco smoking combined with the monosodium glutamate administration are more declining in mature female-rats, and with the age-old comparison of the changes in the activity of succinate dehydrogenase and cytochrome oxidase, was established their more intense reduction in immature male-rats. thus, the findings suggest that low dose intake of monosodium glutamate enhances the ability of tobacco smoke to disrupt the cell's bioenergetics processes by affecting the respiratory chain function and generation of atp. therefore, it is advisable to investigate the established toxic doses of monosodium glutamate, as well as to study the molecular mechanisms of ‘safe’ (allowed) doses of monosodium glutamate effect on a living organism. статеві та вікові аспекти біоенергетичних процесів при пасивному тютюнопалінні та застосуванні глутамату натрію в експерименті а. в. руцка, і. я. криницька тернопільський державний медичний університет імені і. я. горбачевського, тернопіль, україна вступ. активне та пасивне паління щороку є причиною понад 5 мільйонів смертей. водночас технічний прогрес у харчовій та переробній областях сприяє використанню харчових добавок, які не завжди безпечні для здоров’я людини, наприклад, глутамату натрію. мета дослідження – визначити зміни активності мітохондріальних ферментів у щурів при пасивному палінні у поєднанні з тривалим введенням глутамату натрію в статевому та віковому аспектах. методи дослідження. оцінку біоенергетичних процесів проводили за допомогою визначення активності сукцинатдегідрогенази та цитохромоксидази в мітохондріях циркулюючих нейтрофілів. результати. пасивне паління у поєднанні з введенням глутамату натрію у зрілих щурів-самців супроводжується значним гальмуванням біоенергетичних процесів, про що свідчить зниження активності сукцинатдегідрогенази на 47,1 % (p<0,001) порівняно з інтактними тваринами, що на 27,9 % (р<0,001) нижче цього показника при ізольованому ефекті тютюнового диму; і зниженні активності цитохромоксидази на 27,5 % (р<0,001) порівняно з контрольною групою. висновки. таким чином, отримані дані свідчать про те, що глутамат натрію підвищує здатність тютюнового диму порушувати процеси біоенергетики клітини, впливаючи на функцію дихального ланцюга і генерацію атф. тому доцільно досліджувати ефекти встановлених токсичних доз e621, а також вивчати молекулярні механізми впливу його “безпечних” (дозволених) рівнів на організм. ключові слова: пасивне тютюнопаління; глутамат натрію; цитохромоксидаза; сукцинатдегідрогеназа. references 1. reitsma mb. smoking prevalence and attributable disease burden in 195 countries and territories, 1990-2015: a systematic analysis from the global burden of disease study 2015. the lancet, 2017;389(10082),1885-1906. 2. xu x, bishop ee, kennedy sm, simpson sa, pechacek tf. annual healthcare spending attributable to cigarette smoking: an update. american journal of preventive medicine; 2015;48(3): 326-33. a. v. rutska et al. 86 b io m e d ic a l s c ie n c e s issn 2413-6077. ijmmr 2018 vol. 4 issue 2 received: 2018-09-15 3. solomenchuk tm, badzai ao, protsko vv. metabolic disorders in women with unstable angina, depending on the smoking habit. bukovinianmedical bulletin; 2017;21,2(1):85-88 [in ukrainian]. 4. peirson l, muhammad ua, meghan k, parminder r, sherifali d. interventions for prevention and treatment of tobacco smoking in school-aged children and adolescents: a systematic review and meta-analysis. preventive medicine; 2016;85:20-31. 5. husarova v, ostatnikova d. monosodium glutamate toxic effects and their implications for human intake: a review. journal of medical internet research; 2013;2013:1-12. 6. likhatsky pg, fira ls, gonsky yai. dynamics of changes of markers of bioenergetic processes and cytolysis in rats after damage by sodium nitrite against the background of tobacco intoxication. bulletin of biology and medicine problems; 2017; 2(136):147-152 [in ukrainian]. 7. lizurchik lv, shade ev. effect of tobacco smoke on the content of toxic elements in the rat. bull. ogu; 2014;6(167):71-74 [in russian]. 8. falaleeva tm, samonina ge, beregovaya tv, dzyubenko nv, andreev la. the effect of glyprolines on the structural and functional state of the gastric mucosa and body weight of rats under conditions of prolonged administration of sodium glutamate. physics of the living; 2010;18,1:154-159 [in russian]. 9. bilyk a, negelya a, garmanchuk l. activity of cytochrome oxidase and succinate dehydrogenase in primary culture of transfected carcinoma of lewis lungs at different stages of tumor growth. bulletin of taras shevchenko national university of kyiv; 2016;2(21):81-85 [in ukrainian]. 10. voloshchuk on, marchenko mm. enzymatic activity of components of the system of energy supply of mitochondria of blood leukocytes in the growth dynamics of guerin's carcinoma. siberian cancer journal; 2013;6(60):36-39 [in russian]. 11. vasilenko ov, bodnar oi, vinyarskaya gb, sinyuk yuv., grubinko vv. energy and nitrogen metabolism of uchlorella vulgaris beij(chlorophyta) under the influence of sodium selenite. algology; 2014;24(3):297-301 [in russian]. 12. yanbaeva dg, dentener ma, creutzberg ec, wesseling g, wouters ef. systemic effects of smoking. chest; 2007;131(5):1557-66. 13. heijink ih, pouwels sd, leijendekker c, de bruin hg, zijlstra gj, van der vaart h, ten hacken nh, van oosterhout aj, nawijn mc, van der toorn m. cigarette smoke–induced damage-associated molecular pattern release from necrotic neutrophils triggers proinflammatory mediator release. american journal of respiratory cell and molecular biology. 2015 may;52(5):554-62. 14. lykhatskyi ph., fira ls., fedorovich um. proteins oxidative modification and mitochondrial enzymes activity in rats of different ages under affection by sodium nitrites and tobacco smoke. ukrainian biopharmaceutical journal; 2017;3(50): 38-46. 15. sharma a. monosodium glutamate-induced oxidative kidney damage and possible mechanisms: a mini-review. journal of biomedical science; 2015; 22:93. 16. ward mw, rego ac, frenguelli bg, nicholls dg. mitochondrial membrane potential and glutamate excitotoxicity in cultured cerebellar granule cells j neurosci. 2000;20(19):7208-19. 17. wu s, zhou f, zhang z, xing d. mitochondrial oxidative stress causes mitochondrial fragmentation via differential modulation of mitochondrial fissionfusion proteins. febs j; 2011;278:941-954. 18. kumari s, mehta sl, li pa. glutamate induces mitochondrial dynamic imbalance and autophagy activation: preventive effects of selenium. plos one; 2012;7(6):e39382. a. v. rutska et al. 5 in t e r n a l m e d ic in e issn 2413-6077. ijmmr 2017 vol. 3 issue 2 doi 10.11603/ijmmr.2413-6077.2017.2.7935 endothelial dysfunction and cardiovascular risk in patients with ankylosing spondylitis s. i. smiyan, b. o. koshak, i. v. gnatko i. horbachevsky ternopil state medical university, ternopil, ukraine background. ankylosing spondylitis is a disease that induces damage to the musculoskeletal system. mortality rate among patients with as is in 1.5 times higher than the population level. it is caused by cardiovascular disease and chronic renal failure. objective. the research was aimed to study the prevalence of endothelial dysfunction and to establish its dependence on the factors of cardiovascular risk in patients with as. methods. 104 patients with ankylosing spondylitis (as) were examined using standard diagnostic methods, such as disease activity, lipidogram, ultrasound of the carotid artery intima media, and endothelium vasodilatation in response to reactive hyperaemia was evaluated. clinical activity of the disease was determined using the disease activity index basdai, basfi functional index, index basmi metrology, asqol quality of life. to estimate the 10year risk of cardiovascular disease, the qrisk scale was used. results. endothelial dysfunction (ed) was found in 47% cases. it was established that in the patients with ed<10% the incidence of lpl>1.7 mg/l, hdl-c<1.0 mmol/l, tim thickening>0.9 mm was higher than in the patients with ed>10%. in this group of patients, significant duration of the disease and essential differences in their progress in terms of vas, crp, esr, index activity and functional disorders were revealed. conclusions. the problem of cvd in patients with as may be caused by systemic inflammatory disease associated with the development of endothelial dysfunction and increased levels of atherogenic lipids. key words: ankylosing spondylitis; endothelial dysfunction; cardiovascular risk. corresponding author: bohdan koshak, department of internal medicine № 2, i. horbachevsky ternopil state medical university, 1 clinichna street, ternopil, ukraine, 460002 phone number: +380987123099 e-mail: koshak_bohdan@yahoo.com introduction ankylosing spondylitis (as) is a chronic systemic inflammatory disease primarily involving the axial skeleton (sacroiliac, intervertebral joints) [1; 12] and belongs to the group of seronegative spondylitis (sna). mortality among patients with as is in 1.5 times higher than the population levels. [8] it is caused by cardiovascular disease and chronic renal failure [3]. it is established that the presence of chronic systemic inflammation is an important predictor of cardiovascular (cv) disease [5; 7; 12] due to the development of endothelial dysfunction, and further – atherosclerosis and atherothrombosis, remodelling of vascular wall and myocardium [10] and, therefore, the main cause of numerous life-threatening adverse conditions. specifics of endothelial dysfunction deve­ lopment have been well studied in patients with rheumatoid arthritis, systemic lupus erythematosus. at the same time, despite widespread prevalence of as, this aspect has not been discussed and analyzed in a cohort of these patients so far. there is scanty information of the impact of systemic inflammation activity on the functional state of endothelium in cases of as, and of the possibility of endothelial dysfunction correction against the background of anti­inflammatory therapy. the research was aimed to study the prevalence of endothelial dysfunction and to establish its dependence on the factors of cardiovascular risk in patients with as. methods 104 patients (90 males and 14 females) with verified diagnosis of as were examined. they were hospitalized into the department of rheumatology of ternopil university hospital within 2015-2017. the study inclusion criteria were: the diagnosis of as according to the modified new york criteria and the informative patient’s consent to participate in the study. exclusion criteria were age over 60 years old, presence of psoriasis, crohn’s disease, ulce­ rative colitis, coronary heart disease, mani fesinternational journal of medicine and medical research 2017, volume 3, issue 2, p. 5-9 copyright © 2017, tsmu, all rights reserved s. i. smiyan et al. 6 in t e r n a l m e d ic in e issn 2413-6077. ijmmr 2017 vol. 3 issue 2 tations of peripheral atherosclerosis, clinically significant heart disease, circulatory insuffi­ ciency of any origin, diabetes, severe liver disease, kidney diseases and other chronic diseases in their acute phases. all patients who agreed to participate in the study underwent general clinical examination (common blood test, urinalysis, ecg, x-ray of sacroiliac joints), biochemical test of blood with determination of lipidograma and acute phase indicators (c-reactive protein, rheumatoid factor and etc.) and detection of hla b-27. clinical activity of the disease was determined using the disease activity index basdai, basfi functional index and basmi metrology index, asqol quality of life [2, 4]. endothelium vasodilatation was evaluated in response to reactive hyperaemia in 57 patients by the method, which was first described by d. celermajer [9], using acuson 128 xp/10 ultrasonic complex equipped with a 7 mhz linear transducer. the study was conducted in a duplex mode (ultrasound scanning in b-mode and doppler spectral analysis of the signal). tests with reactive hyperaemia (endothelium-dependent vasodilators) and nitro-glycerine (endothelium-independent vasodilators) were performed [10]. endothelium-dependent vasodilatation ( e dv d ) w a s c a l c u l a t e d by t h e f o r m u l a : edvd=(d60–d0)×100%/d0, where d60 is the brachial artery diameter 60 seconds after the restoration of blood flow, d0 – the initial dia­ meter of brachial artery. endothelium-independent vasodilatation (eivd) was calculated by the formula: eivd=(d5–d0)×100%/d0, where d5 – the diameter of brachial artery after 5 minutes of taking nitro-glycerine, d0 – the initial diameter of brachial artery. to determine the correlation between eivd and edvd, the reactivity index (ir) of brachial artery was calculated using the formula: ir=eivd/edvd. to assess the 10-year risk of cardiovascular disease, the scale qrisk was used (q-research cardiovascular risk algorithm). the advantage of this scale is the use of such risk factors: age, sex, smoking, systolic blood pressure, the ratio of total cholesterol and hdl cholesterol, body mass index, family history of coronary artery disease, socio eco nomic status, treatment with antihypertensive drugs and the presence of comorbidity, systemic inflammatory disease in an individual case. statistical analysis of the results was carried out using traditional methods of anova, spss 22 (© spss inc.). results the main part of patients was of working age (mean age – 35.9±17.1 years old, duration of the disease averaged 15.9±8.2 years (fig. 1)). peripheral form of the disease was found in 35 patients (33.7%), central form – in 69 patients (66.3%). evaluation of the disease proved the presence of high and moderate activity in the absence of disease in patients who had inactive or active course. fig. 1. classification of patients according to age and disease duration the research of endothelium functions in the patients with as and in the control group proved that remodulation of vessels occurred due to endothelium dilation of brachial artery in response to reactive hyperaemia and that they differ slightly in the quantitative value of the indicator in the control group. besides, edvd reduced (less than 10%), that is a sign of endothelial dysfunction, more common among patients with as as compared to the control group (45.7% vs. 11.8%, respectively). eivd is significantly higher than edvd in patients with as and healthy individuals. however, eivd in the patients with as was found to exceed significantly the edvd in the control group (table 1). in what follows, we evaluated clinical and laboratory characteristics of as in patients with reduced eivd (table 2). the data proved that in the patients with impaired endothelial vasoregulation function, a significantly longer duration of disease was revealed, the onset of the illness symptoms was evidenced in younger s. i. smiyan et al. 7 in t e r n a l m e d ic in e issn 2413-6077. ijmmr 2017 vol. 3 issue 2 age. in addition, the patients with impaired edvd had significantly higher indices of vas and activity (basdai) with significantly higher than quantitative indicators of crp, esr, functional disorders and dfi. among the patients with eivd<10% (26 patients), 14 (53.8%) are treated with nsaids, only 4 (28.5%) of them – continuously. among the patients with edvd>10% (31 patients), 28 persons underwent nsaids therapy, 19 of them – continuously (fig. 2) classical cardiovascular risk factors and arterial hypertension are crucial for the development of endothelial dysfunction, so their prevalence in the group of the examined patients was analysed (table 3). about every third patient suffered from ii degree hypertension, and the frequency of its registration was found to be similar to the prevalence among the general population (in the city area – 29.3%, in rural – the prevalence of hypertension – 36.3%) [13]. it should be noted that adequate antihypertensive therapy at the time of inclusion into the study was provided for 14 patients (39%). thickening of intima-media common carotid artery >1 mm was found in 19.2% patients with as. high incidence of smoking among patients with as can be due to the predominance of males. many patients had reduced levels of hdl and high ldl levels that can be attributed to scanty availability of patients with hypercholesterolemia. mean bmi in the studied patients was 20.2±4.9 kg/m2; 72 (69.2%) patients had normal weight (bmi 18.5-24.9 kg/m2), 18 (17.3%) pa ti ents had a deficit of body weight (bmi<18.5 kg/m2), table 1. results of endothelia vasoregulation function indices аs (n=57) the control group (n=20) p the initial diameter of brachial artery, mm 4.32±0.29 3.78±0.18 p<0.05 the wall thickness of brachial artery, mm 0.58±0.06 0.41±0.03 p<0.05 the output speed of blood flow, m/s 0.72±0.09 0.68±0.07 p<0.05 edvd, % 9.8±1.23 13.3±1.1 p<0.05 eivd, % 25.4±2.14 19.7±5.4 p<0.05 brachial artery reactivity index 2.35±0.12 1.37±0.14 p<0.05 note: p – significant differences between the baselines indicators of the study and control groups. table 2. clinical and laboratory characteristics of patients with as indices edvd>10%(n=31) edvd<10% (n=26) p disease duration, (yrs) 13.7±5.2 16.1±4.8 p>0.05 age of disease onset, (yrs) 25.8±3.2 23.2±2.1 p>0.05 the presence of peripheral arthritis n % n % p>0.05 10 32.3 9 34.6 basdai, sm 4.64±0.42 6.80±0.27 p<0.05 basfi, sm 4.34±0.28 5.74±0.18 p<0.05 dfi, points 15.8±1.1 17.1±0.89 p<0.05 vas, mm 61.9±14.5 80.3±12.4 p<0.05 с­reactive, mg/l 8.3±1.4 15.2±2.7 p<0.05 esr, mm/h 18.3±3.2 28.5±4.1 p<0.05 note: р – significant differences between еd<10% and ed>10%. fig. 2. dependence of edvd on the duration of nsaid treatment s. i. smiyan et al. 8 in t e r n a l m e d ic in e issn 2413-6077. ijmmr 2017 vol. 3 issue 2 and 10 (9.6%) patients had excessive body weight (bmi>25 kg/m2), including 4 (3.8%) with obesity (bmi>30 kg/m2). the next stage of our research was aimed at analysing the quantitative characteristics of cardiovascular risk factors in patients with impaired eivd (table 4). the data proved that the level of atherogenic lipids (ldl and hdl) was the sole indicator of significant differences between patients with normal and impaired eivd. on the other hand, it was found that patients in groups with normal and low edvd were comparable regarding most cardiovascular risk factors. thus, traditional factors are unlikely to play a major role in the processes of endothelial wall remodullation and edvd occurrence. further cardiovascular risk for the examined patients was established, the value for the study group (3.4 % [1.8; 5.4]) being about in three times higher than the risk value for the general population. it should be noted that the average cardiovascular risk in the patients with impaired edvd was 2.8% as compared to the index in normal eivd – 3.9%. discussion brachial artery active response to exogenous nitrate in patients with as proves more comparable index of reactivity of brachial artery, that is indicative of the ratio between eivd and edvd. a recent research suggests that excessive response of brachial artery to exogenous nitrate in patients with as is associated with deficient endogenous production of nitric oxide and can be regarded as a sign of endothelial dysfunction [5, 6]. the obtained findings prove table 3. the prevalence of traditional cardiovascular risk factors sign n % arterial hypertension 23 22.1 тім of common carotid artery >1 mm 49 47.1 smoking 34 32.7 hypercholesterolemia (tch>5.2 mmol/l) 22 21.2 low level of hdl (<1 mmol/l for male and <1.2 mmol/l for female) 63 60.6 high level of ldl (>3.0 mmol/l) 41 39.4 hypertriglyceridemia (тг >1.7 mmol/l) 14 13.5 overweight (bmi>25 kg/m2) 12 11.5 family history of early heart disease 16 15.4 table 4. summary of cardiovascular risk factors and hypertension in patients with normal and reduced endothelium vasodilation markers edvd >10% (n=31) edvd <10% (n=26) р average age, years 36.8±8.2 38.8±8.8 p>0.05 bmi, kg/m 22.8±4.3 23.9±4.1 p>0.05 smoking n % n % 12 39 10 38 p>0.05 history of smoking, years 10.2±2.4 9.2±1.9 p>0.05 family history of early heart disease n % n % 6 19 4 15 p>0.05 tch, mmol/l 4.52±0.94 4.28±0.81 p>0.05 hdl, mmol/l 1.16±0.31 0.71±0.21 p<0.05 ldl, mmol/l 2.61±0.18 3.22±0.29 p<0.05 тg, mmol/l 1.15±0.44 1.32±0.58 p>0.05 atherogenic index 3.4±1.21 3.9±1.26 p>0.05 тім of common carotid artery, mm 0.89±0.12 1.32±0.18 p<0.05 arterial hypertension n % n % 7 22.5 6 23.1 p>0.05 systolic arterial pressure, mm.hg. 124.5±18.3 122.5±22.5 p>0.05 diastolic arterial pressure, mm.hg. 78.5±14.5 76.5±18.5 p>0.05 note: р – significant differences between еd<10% and ed>10%. s. i. smiyan et al. 9 in t e r n a l m e d ic in e issn 2413-6077. ijmmr 2017 vol. 3 issue 2 that output diameter and wall thickness of brachial artery in the patients with as were significantly higher than similar indices in the control group, and the rate of blood flow through the artery was virtually identical. this may evidence the signs of brachial artery remodelling in patients with as, endothelium is crucial [5, 6]. the detected changes of lipid profile are typical for the patients with systemic inflam­ matory diseases, and are consistent with the literature [11]. it should be emphasized that a high cardiovascular risk in patients with as cannot be explained from the standpoint of the analysis of classical risk factors, as they do not explain high incidence of patients with endothelium dependent vasodilatation, which is a significant predictor of cardiovascular problems in patients with inflammatory diseases of joints. conclusions our findings suggest high prevalence of ed in patients with as (47% cases). ed <10% caused significant increase in the number of patients with ldl >1.7 mmol/dl, hdl-c <1.0 mmol/l, tim thickening >0.9 mm. at the same time, in this group of patients a significantly longer duration of disease and substantial differences in the course in vas, crp and esr indices, as well as activity index and functional disorders were revealed. with the qrisk score scale, ss risk was established as 3.4% that is about three times higher than the risk among the general population. among the traditional factors, increased hdl and decreased ldl are most often reported. thus, the problem of cvd is one of the most important aftermaths of systemic inflammatory disease that is associated with the development of endothelial dysfunction and increased levels of atherogenic lipids. references 1. braun j, sieper j. ankylosing spondylitis. lancet. 2007;369:1379–1390. 2. braun j, van den berg r, baraliakos x, et al. 2010 update of the asas/eular recommendations for the management of ankylosing spondy litis. ann rheum dis. 2011;70(6):896–904. 3. brown ma, kenna t, wordsworth bp (2016) genetics of ankylosing spondylitis-insights into patho genesis. nat rev rheumatol.12:81–91. 4. chen ha, chen сh, liao ht, et al. clinical, func­ tional, and radiographic differences among juvenileonset, adult-onset, and late-onset ankylosing spondylitis. j rheumatol. 2012;39(5):1013–8. 5. erre gl, sanna p, zinellu a, ponchietti a, fenu p, sotgia s, carru c, et al. plasma asymmetric di me thylarginine (adma) levels and atherosclerotic disease in ankylosing spondylitis: a cross­sectional study. clin rheumatol. 2001;30:21–27. 6. heeneman s. cardiovascular risks in spondyloarthritides. curr opin rheumatol. 2007. 19:358– 362. 7. kumar a, falodia sk, shankar s, grover r, marwaha v, aneja r, srivastava k, das n. assessment of serum nitrite as biomarker of disease activity in ankylosing spondylitis. indian j rheumatology. 2009;4:47–50. 8. mccarey d, sturrock rd. comparison of cardiovascular risk in ankylosing spondylitis and rheumatoid arthritis. clin exp rheumatol. 2009;27:s124– s126. 9. celermajer ds, sorensen ke, gooch vm, et al. non-invasive detection of endothelial dysfunction in children and adults at risk of atherosclerosis. lancet. 1992;340:1111–1115. 10. poddubnyi da, rebrov ap. endothelial dysfunction in patients with bechterew’s disease (anky­ losing spondylitis). klin med (mosk). 2007;85:66–69. 11. syngle a, vohra k, sharma a, kaur l. endothelial dysfunction in ankylosing spondylitis improves after tumor necrosis factor­α blockade. clin rheu­ matol. 2010;29:763–770. 12. taurog jd, chhabbra a, colbert ra. axial spondyloarthritis and ankylosing spondylitis. new engl j med. 2016;26:2563–2574. 13. unified clinical protocols of primary, emer­ gency and secondary (specialized) medical help: arterial hypertension. http://www.moz.gov.ua/ua/ portal/allresources/. accessed 2016. s. i. smiyan et al. received: 2017-06-26 45 b io m e d ic a l s c ie n c e s issn 2413-6077. ijmmr 2017 vol. 3 issue 2 doi 10.11603/ijmmr.2413-6077.2017.2.8372 acetaminophen effect on free radical oxidation indices in rats with type 2 diabetes mellitus о. b. furka, і. b. ivanusa, м. м. mykhalkiv, і. м. klishch i. horbachevsky ternopil state medical university, ternopil, ukraine background. acetaminophen is a drug used to relieve pain syndrome. it is used both independently and in composition of combined drugs. type 2 diabetes is an age-related disease that is associated with a violation of insulin synthesis by pancreas. objective. the aim of the research was to study the effect of acetaminophen on major free radical oxidation indices of rats with type 2 diabetes mellitus in time dynamics. methods. we conducted two series of experiments. the first series comprised rats with type 2 diabetes mellitus and acute acetaminophen toxic lesions. the second series involved rats with type 2 diabetes mellitus and acetaminophen administration at a dose of 55 mg/kg for the period of 7 days. results. administration of acetaminophen for rats with type 2 diabetes mellitus caused the increase in the content of malondialdehyde, diene and triene conjugates and schiff bases in blood plasma and malondialdehyde, diene and triene conjugates in liver homogenate. the maximum increase in these indices was observed on the first day of the experiment. gradually these indices decreased on the 3rd, 5th and 7th days of the experiment. conclusions. free radical oxidation increased in both series of the experiment. this process developed in rats with type 2 diabetes mellitus and acute acetaminophen toxic lesions more intensively, than in rats with type 2 diabetes mellitus and administration of acetaminophen at the highest therapeutic dose during 7 days. key words: acetaminophen; malondialdehyde; diene and triene conjugates; schiff bases; diabetes mellitus. corresponding author: olha furka, department of medical biology, i. horbachevsky ternopil state medical university, 2 yu. slovatskoho, ternopil, ukraine, 46001 phone number: +380352252584 e-mail: furkaob@tdmu.edu.ua introduction acetaminophen is a drug used to relieve pain syndrome. it is used both independently and in composition of combined drugs. a long time it was considered to be the safest drug among the group of analgesics/antipyretics [1, 2]. acetaminophen has a relatively low toxicity in therapeutic doses. however, a conscious and often uncontrolled administration of high doses of the drug causes complications that sometimes can lead to death due to hepatic insuffi ­ ciency [3, 4, 5]. type 2 diabetes is one of the most common diseases, every year its frequency is steadily increasing. the prevalence of diabetes is associated with changes in environmental factors, especially the populations (genetic, demographic), the concentration of risk factors in the populations (increased body weight, arterial hypertension, cardiovascular diseases, lipid metabolism disorders, etc.). considering all above, the aim of our research was to study free radical oxidation activity in rats with type 2 diabetes mellitus and acetaminophen toxic lesions. methods the experiments were carried out on white rats weighing 180–220 g on a standard diet and free access to water in vivarium. we conducted two series of experiments. in the first series toxic lesion was caused by a single intragastric administration of acetami nophen suspension in 2% starch solution for the animals at a dose of 1250 mg/kg (1/2 ld50). in the second series the suspension of acetami nophen in a 2% starch solution at a dose of 55 mg/kg was managed, which corresponds to the highest therapeutic dose during 7 days. non-genetic form of experimental type 2 dia be tes mellitus was modeled by islam s., choi h. method [7, 8], that is, a single intraperitoneal administration of streptozotocin solution at a dose of 65 mg/kg (sigma, usa) to the rats, which was diluted by citrate buffer (ph 4.5) with the previous (15 minutes ahead) intraperitoneal nicotinamide international journal of medicine and medical research 2017, volume 3, issue 2, p. 45–50 copyright © 2017, tsmu, all rights reserved о. b. furka et al. 46 b io m e d ic a l s c ie n c e s issn 2413-6077. ijmmr 2017 vol. 3 issue 2 administration at a dose of 230 mg/kg. the rats with the same body weight, which were given the same amount of solvent (citrate buffer ph 4.5), were used as the control group. in the first series of the experiment, the rats were divided into 4 groups: the 1st group was the intact (control); the 2nd group involved a single acetaminophen administration; the 3rd group comprised the animals with type 2 diabetes mellitus caused by streptozotocin administration; and the 4th group contained the rats with a single administration of acetaminophen after streptozotocin administration. in the second series of the experiment, the rats were divided into 4 groups: the 1st group was the intact (control); the 2nd group was with acetaminophen administration during 7 days; the 3rd group were the animals with type 2 diabetes mellitus caused by streptozotocin administration; the 4th group were the rats with administration of acetaminophen during 7 days after streptozotocin administration. the animals were removed from the experiment on the 5th, 3rd, 5th and 7th days after last acetaminophen administration by euthanasia under thiopental anaesthesia. all experiments on rats were carried out according to the guideline principles for the care and use of laboratory animals [10]. evaluation of the content of diene and triene conjugates was carried out by the method [6]. determination of malondialdehyde content was carried out by the method [9]. determination of the contents of the schiff bases was carried table 1. dynamics of content of malondialdehyde, diene and triene conjugates and schiff bases in blood plasma of rats with type 2 diabetes mellitus and acute acetaminophen toxic lesions (m±m; n=10) group of animals time after acetaminophen administration (days) content of 1st day 3rd day 5th day 7th day control n=10 mda, μmol/l 7.48±0.47 dc, u/l 1.12±0.03 тc, u/l 0.58±0.01 schiff bases, u/l 2.53±0.33 acetaminophen (single) n=10 mda, μmol/l 24.62±1.42 p1<0.001 22.63±1.11 p1<0.001 21.79±1.12 p1<0.001 21.47±1.13 p1<0.001 dc, u/l 4.62±0.49 p1<0.001 4.47±0.41 p1<0.001 3.42±0.48 p1<0.001 3.11±0.47 p1<0.001 тc, u/l 1.97±0.37 p1<0.001 1.80±0.24 p1<0.001 1.76±0.32 p1<0.001 1.65±0.31 p1<0.001 schiff bases, u/l 8.61±0.77 p1<0.001 8.04±0.71 p1<0.001 7.79±0.65 p1<0.001 7.60±0.62 p1<0.001 type 2 dm n=10 mda, μmol/l 14.62±0.92 p1<0.001 14.22±1.02 p1<0.001 14.03±0.69 p1<0.001 13.56±0.65 p1<0.001 dc, u/l 1.831±0.24 p1<0.001 1.79±0.31 p1<0.001 1.72±0.28 p1<0.001 1.66±0.31 p1<0.001 тc, u/l 1.20±0.18 p1<0.001 1.17±0.23 p1<0.001 1.08±0.16 p1<0.001 0.95±0.18 p1<0.001 schiff bases, u/l 5.10±0.47 p1<0.001 5.01±0.59 p1<0.001 4.87±0.44 p1<0.001 4.63±0.52 p1<0.001 acetaminophen (rats with type 2 dm) n=10 mda, μmol/l 39.01±1.12 p1<0.001 p2<0.001 38.72±0.91 p1<0.001 p2<0.001 38.13±1.19 p1<0.001 p2<0.001 37.58±1.22 p1<0.001 p2<0.001 dc, u/l 3.86±0.28 p1<0.001 p2<0.001 3.70±0.50 p1<0.001 p2<0.001 3.38±0.51 p1<0.001 p2<0.001 3.14±0.52 p1<0.001 p2<0.001 тc, u/l 4.00±0.39 p1<0.001 p2<0.001 3.91±0.49 p1<0.001 p2<0.001 3.77±0.54 p1<0.001 p2<0.001 3.53±0.48 p1<0.001 p2<0.001 schiff bases, u/l 13.33±1.15 p1<0.001 p2<0.001 13.20±0.93 p1<0.001 p2<0.001 12.98±0.97 p1<0.001 p2<0.001 12.81±0.61 p1<0.001 p2<0.001 notes: here and in the following tables p1 – significant difference compare with control animals; p2 – significant difference compare with the animals, which were administered with acetaminophen. о. b. furka et al. 47 b io m e d ic a l s c ie n c e s issn 2413-6077. ijmmr 2017 vol. 3 issue 2 о. b. furka et al. out by the method [12, 13]. quantitative indices were processed statistically. the results of the experiment were processed by means of statistical program statistica [11] using parametric student's t test and wilcoxon signed-rank test for non-parametric statistical hypothesis test. changes were considered significant at р≤0.05. results the content of malondialdehyde, diene and triene conjugates and schiff bases increased in animals with lesions caused by acetaminophen and type 2 diabetes mellitus. as presented in table 1, the content of malondialdehyde in blood plasma increased by 229.2% on the 1st day of the experiment in the 2nd group of experimental animals, it increased by 95.5% in the 3rd group of animals. the maximum of this index increase (in 5.21 times) was observed in the animals with type 2 diabetes mellitus and acute acetaminophen toxic lesions (the 4th group). this index decreased on the 3rd and 5th days of the experiment. the maximal decrease of malondialdehyde content was observed on the 7th day of the experiment in all groups of animals. the content of diene and triene conjugates in blood plasma of the animals with single acetaminophen administration (the 2nd group) increased in 4.11 and 3.36 times on the 1st day of the experiment to compare with the control animals. these indices increased by 62.8% and 104.8% in the 3rd group of animals with streptozotocin action. the content of diene conjugates increased in 3.43 times, and triene conjugates in 6.81 times in the animals with type 2 diabetes mellitus and acute acetaminophen toxic lesions (the 4th group). these indices decreased on the 3rd, 5th and 7th days of the experiment. the content of schiff bases increased in 3.4 times on the 1st day of the experiment in the 2nd group of experimental animals after acetaminophen administration. this index increased by 101.6% in the 3rd group of animals with streptozotocin administration. the maximal increase of schiff bases in 5.26 times was observed in the 4th group of animals with type 2 diabetes mellitus and single acetaminophen administration. according to the results (table 2), the content of malondialdehyde in liver homogenate increased in 4.76 times on the 1st day of the experiment in the 2nd group of experimental animals to compare with the control animals, and by 60.8% in the 3rd group of animals. the maximum of this index increase (in 5.7 times) was observed in the 4th group of animals. table 2. dynamics of content of malondialdehyde, diene and triene conjugates in liver homogenate of rats with type 2 diabetes mellitus and acute acetaminophen toxic lesions (m±m; n=10) group of animals time after acetaminophen administration (days) content of 1st day 3rd day 5th day 7th day control n=10 mda, μmol/l 13.67±1.16 dc, u/l 7.83±0.26 тc, u/l 3.69±0.16 acetaminophen (single) n=10 mda, μmol/l 65.10±3.57 p1<0.001 64.51±3.50 p1<0.001 63.67±2.41 p1<0.001 62.63±2.11 p1<0.001 dc, u/l 46.09±1.43 p1<0.001 44.21±2.07 p1<0.001 39.47±1.72 p1<0.001 38.94±2.06 p1<0.001 тc, u/l 17.93±0.78 p1<0.001 17.18±1.56 p1<0.001 16.14±1.69 p1<0.001 15.38±1.28 p1<0.001 type 2 dm n=10 mda, μmol/l 21.98±1.80 p1<0.001 21.55±2.04 p1<0.001 20.79±1.92 p1<0.001 20.19±2.08 p1<0.001 dc, u/l 23.44±1.00 p1<0.001 21.66±1.50 p1<0.001 20.47±1.13 p1<0.001 20.19±1.70 p1<0.001 тc, u/l 13.43±0.58 p1<0.001 12.78±1.37 p1<0.001 12.02±1.04 p1<0.001 11.47±1.11 p1<0.001 acetaminophen (rats with type 2 dm) n=10 mda, μmol/l 77.94±2.43 p1<0.001 p2<0.001 77.31±4.14 p1<0.001 p2<0.001 76.53±2.00 p1<0.001 p2<0.001 75.70±2.09 p1<0.001 p2<0.001 dc, u/l 52.98±1.03 p1<0.001 p2<0.001 51.25±1.89 p1<0.001 p2<0.001 51.03±2.27 p1<0.001 p2<0.001 47.34±2.34 p1<0.001 p2<0.001 тc, u/l 24.24±1.49 p1<0.001 p2<0.001 23.56±1.45 p1<0.001 p2<0.001 22.94±1.67 p1<0.001 p2<0.001 22.01±1.64 p1<0.001 p2<0.001 48 b io m e d ic a l s c ie n c e s issn 2413-6077. ijmmr 2017 vol. 3 issue 2 the content of diene and triene conjugates in the experimental animals increased the most in the 4th group of animals on the 1st day of the experiment in 6.76 and 6.56 times respectively. these indices increased on the 1st day of the experiment in 5.88 and 4.85 times in the 2nd group, and in 2.99 and 3.63 times in the 3rd group respectively. the content of malondialdehyde, diene and triene conjugates, schiff bases increased on the 1st day of the experiment in blood plasma (table 3) and liver homogenates (table 4). these indices decreased on the 3rd, 5th, 7th days in all series of the experiment. these changes were less pronounced than in the 1st series of the experiment. in the 2nd group of experimental animals with acetaminophen administration during 7 days, the content of malondialdehyde in blood plasma on the 1st day of the experiment increased by 126.5%, in liver homogenate – by 138.6%; diene and triene conjugates in blood plasma increased by 105.9% and 142.3%, in liver homogenate increased in 3.0 and 3.2 times; schiff bases increased by 134.3% in blood plasma to compare with control animals. in animals with type 2 diabetes and acetaminophen administration during 7 days (the 4th group), malondialdehyde in blood plasma increased the most in 4.13 times on the 1st day of the experiment, it increased in 4.9 times in lever homogenate; the content of diene and triene conjugates increased in blood plasma in 3.1 and 3.2 times, in 5.7 and 4.2 times in liver homogenate; schiff bases increased in 4.1 times in blood plasma. discussion the activation of free radical processes is universal mechanism in case of toxic action of the vast majority of toxic agents. the mechanism of cell damage by free radical metabolites, table 3. dynamics of content of malondialdehyde, diene and triene conjugates and schiff bases in blood plasma of rats with type 2 diabetes mellitus and acetaminophen administration at a dose of 55 mg/kg during 7 days (m±m; n=10) group of animals time after acetaminophen administration (days) content of 1st day 3rd day 5th day 7th day control n=10 mda, μmol/l 7.48±0.47 dc, u/l 1.12±0.03 тc, u/l 0.58±0.01 schiff bases, u/l 2.53±0.33 acetaminophen (7 days) n=10 mda, μmol/l 16.94±0.94 p1<0.001 16.54±0.99 p1<0.001 16.12±1.04 p1<0.001 15.77±0.96 p1<0.001 dc, u/l 2.31±0.29 p1<0.001 2.24±0.20 p1<0.001 2.00±0.21 p1<0.001 1.83±0.21 p1<0.001 тc, u/l 1.42±0.07 p1<0.001 1.24±0.21 p1<0.001 1.19±0.20 p1<0.001 1.17±0.28 p1<0.001 schiff bases, u/l 5.93±0.64 p1<0.001 5.75±0.71 p1<0.001 5.58±0.77 p1<0.001 5.43±0.65 p1<0.001 type 2 dm n=10 mda, μmol/l 14.62±0.92 p1<0.001 14.22±1.02 p1<0.001 14.03±0.69 p1<0.001 13.56±0.65 p1<0.001 dc, u/l 1.831±0.24 p1<0.001 1.79±0.31 p1<0.001 1.72±0.28 p1<0.001 1.66±0.31 p1<0.001 тc, u/l 1.20±0.18 p1<0.001 1.17±0.23 p1<0.001 1.08±0.16 p1<0.001 0.95±0.18 p1<0.001 schiff bases, u/l 5.10 ± 0.47 p1<0.001 5.01 ± 0.59 p1<0.001 4.87 ± 0.44 p1<0.001 4.63 ± 0.52 p1<0.001 acetaminophen (rats with type 2 dm) n=10 mda, μmol/l 30.90±0.55 p1<0.001 p2<0.001 30.54±0.73 p1<0.001 p2<0.001 29.91±0.87 p1<0.001 p2<0.001 29.40±1.31 p1<0.001 p2<0.001 dc, u/l 3.52±0.21 p1<0.001 p2<0.001 3.25±0.45 p1<0.001 p2<0.001 2.85±0.37 p1<0.001 p2<0.001 2.46±0.39 p1<0.001 p2<0.001 тc, u/l 1.88±0.33 p1<0.001 p2<0.001 1.83±0.33 p1<0.001 p2<0.001 1.76±0.36 p1<0.001 p2<0.001 1.63±0.30 p1<0.001 p2<0.001 schiff bases, u/l 10.60±0.77 p1<0.001 p2<0.001 10.41±0.82 p1<0.001 p2<0.001 10.22±0.82 p1<0.001 p2<0.001 10.03±0.68 p1<0.001 p2<0.001 о. b. furka et al. 49 b io m e d ic a l s c ie n c e s issn 2413-6077. ijmmr 2017 vol. 3 issue 2 о. b. furka et al. which are formed as a result of large number of biocidal xenobiotics biotransformation, including acetaminophen, involves their ability to initiate processes of lipid peroxidation and oxidation modification of proteins, covalently bind with bio-macromolecules (proteins, nucleic acids, lipids) and generate reactive oxygen intermediates (roi), which are highly toxic and capable to initiate new chains of free radical reactions. the severity of damage effect of free radicals depends on the intensity of their production and functional ability of antioxidant system. as the result of metabolic transformations of acetaminophen, as proved by a number of researchers [1, 2], free radical metabolites, as well as reactive oxygen intermediates are formed; the damage by acetaminophen is accompanied by the intensification of free radical processes. detoxification system is exhausted due to hyperglycaemia induced by streptozotocin injections, and reactive metabolites of acetaminophen prove even more toxic damage, the intensification of reactive oxygen intermediates formation and activation of lipid peroxidation processes in particular. table 4. dynamics of content of malondialdehyde, diene and triene conjugates and schiff bases in liver homogenate of rats with type 2 diabetes mellitus and acetaminophen administration at a dose of 55 mg/kg during 7 days (m±m; n=10) group of animals time after acetaminophen administration (days) content of 1st day 3rd day 5th day 7th day control n=10 mda, μmol/l 13.67±1.16 dc, u/l 7.83±0.26 тc, u/l 3.69±0.16 acetaminophen (7 days) n=10 mda, μmol/l 32.62±1.75 p1<0.001 31.83±2.78 p1<0.001 31.34±1.63 p1<0.001 29.79±1.84 p1<0.001 dc, u/l 23.63±1.60 p1<0.001 23.45±1.42 p1<0.001 21.43±1.40 p1<0.001 20.82±1.74 p1<0.001 тc, u/l 12.02±0.79 p1<0.001 11.22±0.90 p1<0.001 10.91±0.99 p1<0.001 10.03±0.90 p1<0.001 type 2 dm n=10 mda, μmol/l 21.98±1.80 p1<0.001 21.55±2.04 p1<0.001 20.79±1.92 p1<0.001 20.19±2.08 p1<0.001 dc, u/l 23.44±1.00 p1<0.001 21.66±1.50 p1<0.001 20.47±1.13 p1<0.001 20.19±1.70 p1<0.001 тc, u/l 13.43±0.58 p1<0.001 12.78±1.37 p1<0.001 12.02±1.04 p1<0.001 11.47±1.11 p1<0.001 acetaminophen (rats with type 2 dm) n=10 mda, μmol/l 67.00±1.78 p1<0.001 p2<0.001 66.54±2.63 p1<0.001 p2<0.001 65.66±2.20 p1<0.001 p2<0.001 65.43±2.18 p1<0.001 p2<0.001 dc, u/l 44.75±1.14 p1<0.001 p2<0.001 42.88±1.55 p1<0.001 p2<0.001 41.51±1.67 p1<0.001 p2<0.001 39.14±1.70 p1<0.001 p2<0.001 тc, u/l 15.62±1.12 p1<0.001 p2<0.001 15.19±1.26 p1<0.001 p2<0.001 14.92±1.25 p1<0.001 p2<0.001 14.42±1.25 p1<0.001 p2<0.001 consequently, we can assert that the radical oxidation processes in the rats with type 2 diabetes and acetaminophen action increases significantly that may cause excessive formation of free radicals and violation of their neutralization. conclusions acute lesions by acetaminophen (1/2 ld50) of animals with type 2 diabetes causes significant increase of lipids peroxidation processes in comparison with the animals without the simulated pathological process, and nondiabetic animals, which were modeled by acetaminophen poisoning, as indicated by the increase in the concentration of diene and triene conjugates, tba-active products, and schiff bases. the administration of acetaminophen at a higher therapeutic dose for the rats with hyperglycemia during 7 days was also accompanied by a significant increase in the concentration of lipid peroxidation products, but less pronounced than in cases of acute lesions. 50 b io m e d ic a l s c ie n c e s issn 2413-6077. ijmmr 2017 vol. 3 issue 2 references 1. stepanov yum, filippova ayu, kononov in. medicinal lesions of liver: pathogenesis, classification, diagnosis, treatment. pharmacist. 2006;5. 2. ushkalova yea. medicinal lesions of liver. gastroenterology. 2003;10(73):72–75. 3. buyeverov ao. fundamental understanding of medicinal lesions of liver. clinical prospects of gastroenterology, hepatology. 2002;4:7–11. 4. minushkin on. some hepatoprotectors in the treatment of hepatic diseases. therapist. 2002;6:55– 58. 5. sheen cl, dillon jf, bateman dn, et al. paracetamol toxicity: epidemiology, prevention and costs to the 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studying the stress and adaptive reactions of the body according to blood parameters. nizhniy novgorod: publishing house of nizhny novgorod state university; 2010. p. 25. 13. khyshiktuyev bs, khyshiktuyeva na, ivanov vn. methods of determining the products of lipid peroxidation in an exhaled breath condensate and their clinical significance. clinical laboratory diagnostics. 1996;3:13–15. received: 2017-10-11 о. b. furka et al. 72 b io m e d ic a l s c ie n c e s issn 2413-6077. ijmmr 2018 vol. 4 issue 2 doi 10.11603/ijmmr.2413-6077.2018.2.9836 melphalan-induced cytotoxicity in the bone marrow of rats by flow cytometry measurements b. i. gerashchenko1, i. m. todor1, o. o. shevchuk2, v. g. nikolaev1 1 – r. e. kavetsky institute of experimental pathology, oncology and radiobiology, national academy of sciences of ukraine, kyiv, ukraine 2 – i. horbachevsky ternopil state medical university, ternopil, ukraine background. bone marrow (bm) that contains hematopoietic cells of various lineages is a sensitive target for a number of cytotoxic agents including chemotherapy drugs. objective. flow cytometry (fcm) was chosen to test cytotoxicity in bm of rats, that received melphalan either intravenously (i.v.) or intraperitoneally (i.p.). methods. one group of rats received melphalan i.v. (3 mg/kg) followed by the bm examination on the 3rd and 7th day after drug administration, whereas another group of animals received this drug i.p. in total doses of 9 and 15 mg/kg followed by the bm examination on the next day after the 3rd and 5th injection of the drug. bm cells were stained with acridine orange and analyzed by fcm. cytotoxicity was assessed by determining the percentage of total nucleated cells (tnc%) among the whole bm cell population and by determining the percentage of polychromatic erythrocytes (pce%) among the whole population of enucleated erythrocytes. results. regardless of the dose and regimen of melphalan administration, either i.v. or i.p. administered drug caused a significant reduction of tnc%. on the average, the i.p. administered drug resulted in about 2.0fold decrease of tnc% (p<0.05), while the i.v. administered drug resulted in about 1.3-fold decrease of tnc% (p<0.05). as for enucleated erythrocytes, the i.p. administered drug resulted in about 1.4-fold decrease of pce% (p<0.05), whereas the i.v. administered drug did not cause any changes in the pce%. conclusions. under these experimental conditions, i.p. administrated melphalan is considerably more cytotoxic than i.v. administered melphalan. this cytotoxic effect is preferentially due to impaired erythropoiesis. key words: bone marrow; melphalan; cytotoxicity; flow cytometry; polychromatic erythrocytes; total nucleated cells. introduction the anti-tumor effect of alkylating chemotherapeutics primarily attributes to their ability to covalently bind dna via alkyl groups causing intraand inter-strand crosslinks [1]. any alkylating drug by induction of dna lesions can affect the replication of actively proliferating cells [2]. moreover, an impaired replication or repair of crosslinked dna is likely to lead to cell death [3]. although alkylating drugs can specifically target proliferating cells, they are not cell cycle phase-specific, and for this reason, cell death is believed to directly correlate with the dose of the drug [4]. an alkylating agent melphalan (known as interstrand dna-cros slinker [1]), which is mainly used for treatment of multiple myeloma, ovarian carcinoma, breast cancer, childhood neuroblastoma, and polycythaemia vera, however, may cause complications, particularly acute myeloid leukemia in the decade after therapy [5]. melphalan-treated individuals with an increased level of chromosomal aberrations in the peripheral blood lymphocytes are at risk of developing cancer later in life [5]. in the experimental animals, melphalan induces cancer of various localizations [5], and regardless of the route of administration, it is apparently genotoxic [6-10]. in the present work, flow cytometry (fcm) has been chosen to examine cytotoxicity in the bone marrow (bm) of rats that received melphalan either intravenously (i.v.) or intraperitoneally (i.p.). bm that contains hematopoietic cells of various lineages is a sensitive object of cytotoxic studies. as for the fcm, this technique is indispensable in many areas of biology and medicine not only because of its high-speed analysis, but also because of its ability to accurately discriminate cells of various types. the fcm usually discriminates cells based on their size, intracellular granularity and selective/ specific fluorescence labeling [11]. this unique international journal of medicine and medical research 2018, volume 4, issue 2, p. 72-78 copyright © 2018, tsmu, all rights reserved corresponding author: bogdan i. gerashchenko, m.d., ph.d. r. e. kavetsky institute of experimental pathology, oncology and radiobiology, national academy of sciences of ukraine, vasylkivska 45, kyiv 03022, ukraine phone: +380 44 2571177 fax: +380 44 2581656 e-mail: biger63@yahoo.com b. i. gerashchenko et al. 73 b io m e d ic a l s c ie n c e s issn 2413-6077. ijmmr 2018 vol. 4 issue 2 advantage of fcm can be applied for the study of cytotoxic effects in bm cells of different lineages and maturation stages. here we use a simple and reliable fcm approach for the analysis of bm cells stained with acridine orange (ao), a metachromatic dye that simultaneously interacts with dna and rna producing at λ=488 nm the dual emission spectra with peaks at 530 nm and 640 nm, respectively [12]. this proposed approach by criswell et al. [13] allows assessing cytotoxicity particularly in erythropoietic cells based on detection of differences in ao uptake between polychromatic erythrocytes (pce) and normo chromatic erythrocytes (nce). redistribution of erythrocytes towards nce is indicative of cytotoxic effect. suzuki et al. [14] suggested that the reduced pce/nce ratio is most likely caused by elevation of nce population as a result of mutageninduced rapid differentiation and multiplication or enucleation of erythroblasts which remained in the bm instead of entering the peripheral blood steam. on the other hand, von lebedur and shcmid [15] claimed that as a result of mutagen-induced partial depletion of the marrow cavities of nucleated blood cell precursors the newly formed erythrocytes can be retained along with inundation with peripheral blood. methods experimental animals and administration of melphalan adult outbred female rats (140-160 g) were taken from the animal house of r. e. kavetsky institute of experimental pathology, oncology and radiobiology (iepor) of the national academy of sciences of ukraine (kyiv, ukraine). immediately before the i.v. or i.p. injections, 0.6 mg/ml solution of melphalan (alkeran® produced by glaxosmithkline, uk) was prepared by diluting 20 mg/ml solution of this drug dissolved in acidified ethanol (96% ethanol and 12 n hcl mixed in the ratio of 150:1) with saline. one group of animals (n=3) was subjected to i.v. injection (via tail vein) of melphalan of a single dose of 3 mg/kg followed by the bm examination on the 3rd and 7th day after drug administration. another group of animals (n=3) was subjected to i.p. injections of melphalan every other day with single doses of 3 mg/kg followed by the bm examination on the next day after 3rd and 5th injection of the drug (total doses were 9 and 15 mg/kg, respectively). since melphalan after i.p. administration is assumed to be less readily delivered to the target tissue than after i.v. administration due to its gradual absorption into blood, this prompted us to increase the total dose of melphalan for delivery via i.p. route. there was also a group of animals that did not receive the drug at all (the intact control, n=3). the study with animals was performed according to the regulations of the ethics committee. bm isolation, specimen processing and fixation the femur removal and bm isolation procedures were in general performed as proposed [13]. the bm cells were thoroughly flushed from the femur with 4 ml of rpmi-1640 (pharm biotek, ukraine) and immediately placed in a refrigerator (+4-6 °c). at this temperature, the bm cells were kept no longer than 1.5 hour before they were resuspended by vortexing and centrifuged at 300× g for 5 min. specimen processing and fixation procedures were mainly performed according to the protocol [13]. the supernatant was discarded with further washing the cells in 5 ml of pbs using centrifugation at 300× g for 5 min. the supernatant was discarded with further resuspension of cells in 2 ml of pbs by vortexing. cell aggregates were dissociated by gentle syringing of the suspension through a 21-gauge needle. while vigorous vortexing, 0.2 ml of cell suspension was added to 5 ml of fixative solution: 1% glutaraldehyde (v/v) in pbs with 30 µg/ml of sds (merck, germany). the cells were fixed for 5 min and then centrifuged at 300× g for 5 min. the supernatant was removed with further resuspension of cells in 0.5 ml of pbs. fluorescence staining this procedure with slight modification was performed in accordance with the protocol [13]. solution a was prepared by dissolving of the following components in 100 ml (final volume) of distilled h2o: 0.1 ml triton x-100 (loba chemie, austria), 8 ml 1.0 n hcl, and 0.877 g nacl. solution b was prepared by mixing of 37 ml 0.1 m citric acid with 63 ml 0.2 m na2hpo4 (ph 6.0) and adding 0.877 g nacl, 34 mg edta disodium salt (sigma, usa), and 0.6 ml of acridine orange (ao; sigma) stock solution (1 mg/ml). the fixed cells (0.2 ml of cell suspension) were added to the mixture of solutions a and b (0.4 and 1.2 ml, respectively) that was chilled on ice in a 12×75 mm centrifuge tubes. while shaking, the cells were stained on ice for 30 min in the dark. then they were centrifuged at 300× g for 5 min. after the supernatant was carefully removed, 1 ml of pbs was added to resuspend the cells. before fcm, cell suspension b. i. gerashchenko et al. 74 b io m e d ic a l s c ie n c e s issn 2413-6077. ijmmr 2018 vol. 4 issue 2 was gently syringed through a 21-gauge needle to obtain a suspension of single cells. fcm analysis the samples were analyzed using an epics xl flow cytometer (beckman coulter, usa) equipped with a 15-mw argon-ion laser (488 nm). the forward light scatter (fsc; related to cell size) and side (90°) light scatter (ssc; related to intracellular granularity) signals were collected in a linear mode. the fluorescence signals of dnaand rna-bound ao were collected respectively in the green fluorescence channel (fl1) through a 525/10-nm band-pass filter and in the far-red fluorescence channel (fl4) through a 675/10-nm band-pass filter using a logarithmic amplification [13]. the acquisition rate was 500-1000 cells per second. at least 1.0× 105 events were collected for each sample. the analysis of the data was performed by publicly available software “winmdi” developed by dr. j. trotter (http://www.cyto. purdue.edu/flowcyt/software/winmdi.htm). the cells were gated on fsc-height vs. sscheight histograms to eliminate debris and aggregates from analysis (not presented here), although microscopic observation showed that their numbers were extremely low. the parameters that were examined are as follows: 1) per centage of total nucleated cells (tnc) of all bm cells, including enucleated cells such as pce and nce (this parameter is further denoted as tnc%); 2) percentage of pce of all enucleated erythrocytes (denoted as pce%). the reason why tnc% was also examined is based on the fact that the nucleated erythroid cells are most numerous in the bm, and accordingly, suppressed erythropoiesis may affect tnc%. thus, the decreased tnc% and pce% (particularly pce%) can be indicative of inhibited division and maturation of nucleated erythroid cells, the fact that has been previously reported [13-16]. the populations of tnc, pce and nce that demonstrate significant differences in ao uptake were determined on a fl1-height vs. fl4height histogram (fig. 1a). statistical analysis probability values with p<0.05 were considered statistically significant. the distribution of indices was esti mated by using the shapirowilk normality test. the statistical significance of the diffe rences between the means was assessed by the mann-whitney-test and anova-test using origin 7.5 software (originlab corporation, usa). results regardless of the dose and regimen of melphalan administration, i.v. delivered melpha lan did not cause any significant changes in the pce%, whereas i.p. delivered drug on the average resulted in about 1.4-fold decrease of pce% (p<0.05, compared with the control; fig. 2). as for tnc, melphalan administered either i.v. or i.p. resulted in a significant decre ase of fig. 1a. example of fcm determination of tnc, pce, nce populations in ao-stained unfractionated bm cells (bm cells were isolated from the femur of the control intact rat). fig. 1b. population of tnc gated on a fl1-height vs. fl4-height histogram (framed by the rectangular window; panel a) is shown on a fsc-height vs. ssc-height histogram to analyze the population ‘m’ comprised of the vast majority of myeloid cells. b. i. gerashchenko et al. 75 b io m e d ic a l s c ie n c e s issn 2413-6077. ijmmr 2018 vol. 4 issue 2 tnc% (p<0.05, compared with the control fig. 3). on the average, after i.p. and i.v. drug de livery there was a 2.0-fold and 1.3-fold decrease of tnc%, respectively (fig. 3). obviously, melphalan after several i.p. administrations (3×3 mg/kg or 5×3 mg/kg) was more cytotoxic than after a single i.v. administration (3 mg/kg). however, increasing the dose of i.p. injected melphalan up to 5×3 mg/kg did not result in more significant cytotoxic effect. perhaps, at lower dose of this drug (3×3 mg/kg) the maximal effect could be reached. as for the i.v. delivery of melphalan, we did expect that this route of drug administration would be more efficient in terms of causing cytotoxicity in the bm. that is why a single minimal dose of melphalan (3 mg/kg) was chosen for this route of delivery. although the aforementioned findings seem to be indicative of suppressed proliferation of erythroid cells, particularly in case of i.p. administered melphalan, it cannot be certainly claimed that proliferation of myeloid cells remains unaffected. since myeloid cells as well as erythroid cells are numerous in the bm [17], o n e c a n ex p e c t t h a t t h e d r u g i n d u c e d suppression in proliferation of these cells may also contribute to significant fluctuations of tnc%. to address this issue, we were able to identify within tnc the population of cells (population ‘m’; fig. 1b), the vast majority of which are likely to be myeloid. this assumption is simply based on the evidence that they are generally large with a specific intracellular granularity [17-19]. in fcm, cell size usually correlates with the fsc, while intracellular granularity correlates with the ssc [11]. the percentage of population ‘m’ of the whole tnc population (denoted as population ‘m’%) was similar to that revealed microscopically by the classic morphology-based evaluation (date not shown). notably, i.p. administered melphalan on the average caused a 1.3-fold increase of the portion of po pulation “m” (fig. 4) with a concordant decrease of pce% (fig. 2). however, together with the fact that the i.p. delivered drug resulted in a 2-fold decrease of tnc% (fig. 3) one can assume that granulopoiesis is likely be affected but certainly to a lesser extent then erythropoiesis. as for the i.v. delivered fig. 2. effect of variously administered melphalan on the pce%. the data presented are the mean ± standard error of the mean. each group consists of three animals. asterisks (*) show significant differences if compared with the control (p<0.05). fig. 3. effect of variously administered melphalan on the tnc%. the data presented are the mean ± standard error of the mean. each group consists of three animals. asterisk (*) shows significant difference if compared with the control (p<0.05). b. i. gerashchenko et al. 76 b io m e d ic a l s c ie n c e s issn 2413-6077. ijmmr 2018 vol. 4 issue 2 drug, there was a 1.3-fold decrease of population “m”%, but only on the 7th day of its injection (p<0.05, compared with the control; fig. 4). this decrease of population ‘m’% was accompanied by a slight increase of pce% on the same day after i.v. drug ad ministration (fig. 2). perhaps, on this day (the 7th day) after i.v. drug administration the erythroid cells recovered faster than myeloid cells. discussion regrettably, there is lack of reports concerning cytotoxic or genotoxic effects in the bm of rats caused by melphalan delivered either i.v. or i.p. instead, it was reported that the rats that intramuscularly received this alkylating agent of a single dose of 1 mg/kg developed a transient but significant increase of chromosomal aberrations in bm cells peaking on the next day after the drug administration (but there were almost no aberrations on the second day after the drug administration, similar to the control), while increasing the dose up to 10 mg/ kg led to the absence of mitotic figures, which is indicative of significant bm suppression [6]. thus based on the results of our research, one can assume that erythropoiesis is more readily affected by melphalan than granu lopoiesis was, but erythropoiesis seemed to be recovered faster than granulopoiesis. apparent erythropoietic cytotoxicity in the bm of rats was shown to be caused by another alkylating drug cyclophosphamide that at the range of doses 5-40 mg/kg resulted in a significant increase of myeloid/erythroid ratios on the second day after i.p. drug delivery [20, 21]. erythroid cells were shown to be very sensitive with respect to ionizing radiation (ir), dna damaging agent of a physical origin [22, 23]. for example, as a result of a whole-body irradiation of rats with the dose of x-rays about 7.0 gy (ld50) the erythroid cells appeared to be significantly more sensitive than the myeloid cells; however, erythropoiesis began recovering much earlier than granulopoiesis (obvious regeneration was first clearly observed on the 12th day after irradiation as evidenced by areas of erythropoiesis) [21]. notably, the cytotoxic effect of any alkylating agent is referred to as ‘radiomimetic’ be cause ir and alkylating agents are similar in terms of inducing cell death mechanisms (both of them induce the mitotic catastrophe) [24, 25]. interestingly, as for melphalan, its dose-response relationship resembles that for ir as evidenced by the shape of the survival curve [24]. a series of studies has been initiated towards tackling melphalaninduced bm suppression [26, 27], and, in this regard, monitoring of bm recovery by fcm could be helpful as well. conclusions under the present experimental conditions, i.p. administrated melphalan is considerably more cytotoxic than i.v. administered melphalan, and this effect is preferentially due to impaired erythropoiesis. granulopoiesis is less readily affected by the melphalan than erythropoiesis, but on the other hand, granulopoiesis, if affected, is slower recovering than erythropoiesis. it is expected that the fcm findings of this study could be helpful for experimental oncologists, who design experiments on antitumor effects of melphalan with less side effects. fig. 4. effect of variously administered melphalan on population ‘m’%. the data presented are the mean ± standard error of the mean. each group consists of three animals. asterisks (*) show significant differences if compared with the control (p<0.05). b. i. gerashchenko et al. 77 b io m e d ic a l s c ie n c e s issn 2413-6077. ijmmr 2018 vol. 4 issue 2 оцінка мелфалан-індукованої цитотоксичності на клітини кісткового мозку щурів за даними проточної цитометрії б. і. геращенко1, і. м. тодор1, о. о. шевчук2, в. г. ніколаєв1 1 – інститут експериментальної патології, онкології і радіобіології імені р. є. кавецького нан україни, київ, україна 2 – тернопільський державний медичний університет імені і. я. горбачевського, тернопіль, україна вступ. кістковий мозок, як основний орган гемопоезу, особливо чутливий до впливу цілого ряду цитотоксичних протипухлинних лікарських засобів. мета роботи: за допомогою проточної цитометрії встановити прояви цитотоксичності на клітини кісткового мозку щурів за умов довенного та інтраперитонеального введення мелфалану. методи. кістковий мозок щурів досліджували на 3-ю та 7-у доби після довенного введення мелфалану в дозі 3 мг/кг; а також на наступний день після 3-ї та 5-ї ін’єкцій препарату при його інтраперитонеальному застосуванні (при досягненні кумулятивної дози 9 та 15 мг/кг). клітини кісткового мозку забарвлювали акридиновим помаранчевим та аналізували за допомогою проточної цитометрії. цитотоксичність оцінювали за відсотком загальної кількості ядерних клітин (як%), а також за відсотком поліхроматофільних еритроцитів (пхе%) у складі всіх без’ядерних еритроцитів. результати. незважаючи на шлях введення та обрану дозу, мелфалан викликав достовірне зниження як%. в середньому, показник як% знижувався у 2 рази при інтраперитонеальному введенні (p<0.05) та в 1.3 рази – при довенному (p<0.05). стосовно без’ядерних еритроцитів, пхе% знижувався в 1.4 рази при інтраперитонеальнему введенні (p<0.05), тоді як при довенному введенні цей показник залишався без змін. висновки. в даних експериментальних умовах, на відміну від довенного застосування, інтраперитонеально введений мелфалан виявився більш цитотоксичним, переважно за рахунок порушень еритропоезу. ключові слова: кістковий мозок; мелфалан; цитотоксичність; проточна цитометрія; поліхроматофільні еритроцити; ядерні клітини. references 1. hurley lh. dna and its associated processes as targets for cancer therapy. nat rev cancer 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philadelphia: lippincott williams & wilkins; 2000, 588 p. 26. shevchuk oo, posokhova ka, sidorenko as, bardakhivska ki, maslenny vm, yushko la, et al. the influence of enterosorption on some haematological and biochemical indices of the normal rats after single injection of melphalan. exp oncol 2014; 36: 94-100. 27. shevchuk oo, posokhova ka, todor in, lukianova ny, nikolaev vg, chekhun vf. prevention of myelosuppression by combined treatment with enterosorbent and granulocyte colony-stimulating factor. exp oncol 2015; 37: 135-138. b. i. gerashchenko et al. 51 b io m e d ic a l s c ie n c e s issn 2413-6077. ijmmr 2017 vol. 3 issue 2 doi 10.11603/ijmmr.2413-6077.2017.2.8077 ecological features of microbiocenosis of the skin of mammary glands and vagina in pregnant women with threat of preterm labor v. ya. ivankiv, i. m. malanchyn, n. i. tkachuk i. horbachevsky ternopil state medical university, ternopil, ukraine background. the threat of preterm birth is one of the most topical issues in the world medicine. according to the statistics, from 12–13 to 25–35 % of all pregnancies end prematurely. one of the causes of preterm labour is chronic inflammatory processes of female genital organs and disorder of microbiocenosis. timely diagnosis and adequate treatment will reduce the risk of premature labour and avoid perinatal loss. objective. we examined and analysed the microflora of the skin of mammary glands and mucous membrane of vagina in healthy pregnant women and patients with threat of preterm labour. methods. the examination of the pregnant was conducted at the trmpc “mother and child” in several stages. first of all, we rinsed the skin of mammary glands and smeared from mucous membrane the posterior vault of vagina with sterile swabs pre-moistened in physiological solution. after that, the tampons were placed in sterile tubes and delivered to laboratory. sowing was carried out on petri dishes with sterile medium: zhsa, bloods mpa, endo, saburo, thioglycolic medium. results. as a result of the research we found saprophytic gram-positive and gram-negative microorganisms (in women with a physiological course of pregnancy). in pregnant women with preterm labor, there is an increase in the number of st. haemolyticus from 13% to 87%, appearance of representatives of the pathogenic flora – st. aureus (in 20%). conclusions. changes in the microbiocenosis of the mammary glands and mucous membranes of the vagina of pregnant women with preterm labor may indicate the presence of opportunistic microflora, or personal hygiene or the presence of associated bacterial infections. it requires the further investigation of possible links among the preterm birth and microbiota. key words: microbiocenosis of the skin of mammary glands; mucous membrane of vagina; preterm labour. corresponding author: natalia tkachuk, department of microbiology, virology and immunology, i. horbachevsky ternopil state medical university, 1 maidan voli, ternopil, ukraine, 46001 phone number: +380352250539 e-mail: tkachuk@tdmu.edu.ua introduction currently, the threat of preterm birth is one of the most pressing issues in the world. according to literary statistics, from 12–13 to 25–35% of all pregnancies ended prematurely [1, 3]. one of the causes of preterm labour is chronic inflammatory processes of female genital organs and disorder (dysbiosis) of microbiocenosis [2]. timely diagnosis and adequate treatment reduce the risk of premature labour and avoid perinatal loss. in normal conditions, women are dominated by lactic acid bacteria in vagina, which prefer to live in an acidified me­ dium of healthy vagina [4, 6]. they protect mucous membrane and may show slight scaling of mucous membranes. if this lactobacillus becomes little, conditionally pathogenic microbes begin to multiply instead of them. they are also normally in a womb of a healthy woman, but in very small quantities. if there are many of them the imbalance presents and bacterial vaginosis develops [5]. according to the literature, the main causes of the imbalance of microorganisms are: – changes of hormonal state in pregnancy, when progestogen hormones shift the vaginal medium to the paralysis; – dysbiosis in intestine, which promotes changes in the balance of microbes in vagina; – the use of drugs during pregnancy that affect microflora; – chronic infectious processes in the body of a pregnant woman with chronic inflammation in the urinary tract that is most often relevant; – chronic sex infections are not detected in time and aggravated during pregnancy (ford h. b, schust d. j, 2009). methods the examination of the pregnant was conducted at ternopil regional municipal perinatal international journal of medicine and medical research 2017, volume 3, issue 2, p. 51–53 copyright © 2017, tsmu, all rights reserved v. ya. ivankiv et al. 52 b io m e d ic a l s c ie n c e s issn 2413-6077. ijmmr 2017 vol. 3 issue 2 centre “mother and child” in several stages. first of all, we rinsed the skin of mammary glands and smeared from mucous membrane the posterior vault of vagina with sterile swabs pre-moistened in physiological solution. we took the material by scrolling all sides of cotton swab. after that, the tampons were placed in sterile tubes and delivered to laboratory. the time was 20-30 minutes from the stage of taking the research material to the crop. sowing was carried out on petri dishes in sterile medium: zhsa, bloods mpa (for detection of coccious microorganisms), endo (enterobacteriaceae), saburo (mushrooms of genus candida), thiogly colic medium (anaerobic microorganisms). each of the cups was marked with the indication accordingly. sowing on the medium was carried out with tampons: first touching one edge of petri cup, then – scrolling with all sides. then we continued sowing the sterilized bacteriological loop perpendicular to the sowing with a swab. the media was placed in a thermostat for 18-48 hours at an optimum temperature. we evaluated the growth of microorganisms on the media after incubation in the thermostat (their shape, colour, size of colonies, nature of surface and edges). next, we made smears from certain types of colonies, stained with gram method and microscopically. results as a result of microscopic examination, in 15 women from the control group (pregnant women with physiological pregnancy) were found: – on the skin of mammary gland e. soli, fuso bacterium, gram-positive non-spore baillus, m. roseus, streptococcus spp., lactosenegative enterobacteria in 7%; lactobacillus, st. haemolyticus – in 13%; st. saprophyticus – in 20%; corynebacterium – in 27%; clostridium, tetracoccus, bacillus spp. – in 33%; m. luteus – in 40%, peptostreptococcus, st. epidermidis, m. lylae – in 47%; bacteroideus – in 60% of the examined patients were present; – in the vaginal smears st. hominis, streptococcus, gram-positive non-spore bacillus, st. haemolyticus in 7%; streptobacillus, streptococcus spp., m. lylae – in 13%; tetracoccus, candida, e. soli, st. saprophyticus, lactosenegative enterobacteria – in 20%; m. luteus – in 33%; doderlein sticks, corynebacterium, st. epidermis, bacillus spp. – in 40%; clostridium, bacteroideus, enterococcus – in 47%; lactobacillus – in 73% of the examined women were evidenced. in the patients with a threat of preterm labour, it was found: – on the skin of mammary glands: m. sedentarius, st. hominis, m. roseus, lactobacillus, gamma-hemolyticus strepto coccus – in 7%; m. varians, bacillus spp. – in 13%; cofig. 1. cultures of microorganisms on petri dishes (endo medium) fig. 2. cultures of microorganisms on petri dishes (bloods mpa medium) fig. 3. cultures of microorganisms on petri dishes (zhsa medium) v. ya. ivankiv et al. 53 b io m e d ic a l s c ie n c e s issn 2413-6077. ijmmr 2017 vol. 3 issue 2 v. ya. ivankiv et al. rynebakterium spp., st. aureus, m. lylae – in 20%; m. luteus, st. epidermidis – in 33%; st. haemolyticus – in 87% of the exemined; – in the vagina smears: st. epidermidis, m. luteus, streptobacillus in – 7%; st. aureus, lactose-negative enterococcus – in 13%; alphahemolyticus streptococcus, beta-hemolyticus streptococcus in – 20%; bacillus spp., candida, e. coli – in 27%; enterococcus – in 53%; st. haemolyticus in 67% of the examined pregnant women. discussion according to foreign statistics, approximately 25% of all cases of preterm labour were caused by mother or foetus indications, 30% – due to premature rupture of membranes of foetus [8]. many scholars argue that most of the premature labour occurs on the background of uncomplicated pregnancy [7, 9]. but, despite this, and summing up the results of our research, we can assume that a violation of microflora of vagina and skin of ma mmary glands is one of the causes of preterm labour. that is why women with violations of microflora of vagina and skin of mammary glands should be under the obstetrician supervision of obstetrician-gynaecologist. each pregnant woman should be thoroughly examined to prevent complications during labour and avoid perinatal loss. in the detection of any pathology of female genital organs, it is necessary to conduct timely treatment. conclusions representatives of normal microflora: sap­ rophytic gram-positive and gram-negative microorganisms, found on the skin of mammary glands of the control group of women, coincide with the data of the literature. in the pregnant women with preterm labour, there is the increase in the number of st. haemolyticus from 13% to 87%, the presence of representatives of pathogenic flora – st. aureus (in 20% of the examined). in the study of microbiocenoses of mucous membrane of vagina in the examined women with threat of preterm labour compared with the control group, a sharp decrease in the number of lactic acid bacteria (doderlein sticks, lactobacillus) and the increase in the incidence of coccal flora (st. haemolyticus, st. aureus, alpha-hemolytic streptococci, beta-hemolytic streptococci). changes in the microbiocenosis of mammary glands and mucous membranes of vagina of pregnant women with preterm labour may indicate the presence of opportunistic micro flora or personal hygiene or the presence of associated bacterial infections. perhaps this is one of the causes of preterm birth in pregnant women. the carriage of opportunistic and pathogenic coccalmicroflora can contribute to inflam­ matory purulent-septic processes in infants and new-borns. references 1. order of the ministry of health of ukraine № 624: premature childbirth. accessed 03 nov 2008. 2. medved vi. selected lectures on extragenital pathology of pregnant women. kyiv; 2010:10–240. 3. zhuk si, kalinkaya, sidelnikova vм. missing pregnancy: a new look at the old problem. health of ukraine. 2007;5:1–35. 4. shchurevskaya od. stress of mother during pregnancy: implications for the fetus and the newborn. taking care of a woman. 2015;9:54–57. 5. golyanovsky v. screening and treatment of bacterial vaginosis during pregnancy. taking care of a woman. 2015;5:88–92. 6. reznichenko hi. prevention of miscarriage and premature delivery. zhinochyi likar. 2013;3:10–12. 7. khmil sv, kuchma zm, romanchuk li. obstetrics.ternopil. tutorials and manuals. 2010:313– 345. 8. ford hb, schust dj. recurrent pregnancy loss: etiology, diagnosis, and therapy. rev obstet gynecol. 2009;2(2):76–83. 9. christiansen ob, steffensen r, nielsen hs, varming k. multifactorial etiology of recurrent miscarriage and its scientific and clinical implications. gynecol obstet invest. 2008;66(4):257–267. received: 2017-09-20 60 b io m e d ic a l s c ie n c e s issn 2413-6077. ijmmr 2018 vol. 4 issue 1 corresponding author: victor lukianchuk, professor, institute of pharmacology and toxicology of nams of ukraine, 14 anton tsedik str., kyiv, 03680, ukraine e-mail: lvdlug@ukr.net phone number: +38(044)4569118 international journal of medicine and medical research 2018, volume 4, issue 1, p. 60-66 copyright © 2018, tsmu, all rights reserved doi 10.11603/ijmmr.2413-6077.2018.1.9253 cerebroprotection by germanium coordination compounds in experimental acute global brain ischemia v. d. lukianchuk1, i. i. seifullina2, o. e. martsinko2, o. o. shevchuk3 background. stroke is among the leading causes of death and disability worldwide with rising incidence among young people today. this is the third most common cause of disability-adjusted life-years worldwide objective. the present study evaluated the cerebroprotective action of coordination compounds of germanium with underlying global cerebral ischemia in rats. methods. global cerebral infarction was induced by bilateral common carotid artery occlusion. for primary screening we used numerous bis(citrate) germanates (stannates) compounds, which contained different metals: ol1-8, and vitagerm-1,2,3 and 4. all germanium complexes used were injected intraperitoneally (1 % aqueous solution at a dose of 50 and 100 mg/kg in 35 min after bilateral common carotid artery occlusion). piracetam was used as a reference drug. criteria of cerebroprotection efficacy survival of rats (%), et50 (median effective time), observational irwin’s test. results. almost all bis(citrate) germanates (stannates), which contained different metals, possessed antiischemic activity of different intensity. the exceptions were cobalt-containing ol-6 and ol-2 compounds. the most significant efficac of all investigated indices (which exceeded even reference drug) was evidenced for vitagerm-1 – a coordination compound of germanium, diethylenetriaminepentaacetic acid and lithium. conclusions. results of our experiments are the substitution for further more profound pharmacological investigation of vitagerm-1 for stroke cerebroprotection and its implementation into clinics. key words: global brain ischemia; germanium coordination compounds; screening. introduction today stroke is in the top 10 causes of death in the world. it maintains the position as the leading killer, and accounts for more than 6 million deaths annually [1]. in ukraine, it is near 100 000 cases each year, and almost 40 000 of them are fatal [2]. risk of stroke is rising in direct proportion to the patient’s age. however, it is observed its rising incidence among young people up to 40 years old in age [2, 3]. stroke affects men and women equally and causes major social and economic burdens to society: 80 % of survivors are disabled until the end of life [4]. this is the third most common cause of disability-adjusted life-years worldwide [4, 5]. more than 80 % of all strokes are ischemic and characterized by occlusion of brain vessel of thrombi or embolus. the aim of the study is to find the effective cerebroprotector among original synthesized coordination compounds of germanium in the experimental model of acute global cerebral ischemia. methods experiments were performed on white inbred rats, males and females, 180–220 g of body weight. acute brain ischemia was modeled by bilateral occlusion of both common carotid arteries before its bifurcation to external and internal branches under thiopental general anesthesia. all animals’ procedures were carried out according to the rules and requirements of european convention for the protection of vertebrate animals used for experimental and other scientific purposes as well as eu directives 2010/63/eu. for primary screening we used numerous bis(citrate)germanates (stannates) compounds, which contained different metals: magnesium bis(citrate) germanate (germacit), mangan bis(citrate) germanate (ol1), cobalt bis(citrate) germanate (ol2), nickel bis(citrate) germanate (ol3), zinc bis(citrate) germanate (ol4), magv. d. lukianchuk et al. 1 – institute of pharmacology and toxicology of the national academy of sciences of ukraine, kyiv, ukraine 2 – odesa i. i. mechnikov national university, odesa, ukraine 3 – i. horbachevsky ternopil state medical university, ternopil, ukraine 61 b io m e d ic a l s c ie n c e s issn 2413-6077. ijmmr 2018 vol. 4 issue 1 v. d. lukianchuk et al. nesium bis(citrate) stannate (ol5), cobalt bis(citrate) stannate (ol6), nickel bis(citrate) stannate (ol7), zinc bis(citrate) stannate (ol8), vitagerm-1 (coordination compound of germanium, diethylenetriaminepentaacetic acid and lithium), vitagerm-2 (coordination compound of germanium, di ethyl enetri aminepentaacetic acid and sodium), vitagerm-3 (coordination compound of germanium, diethylenetriaminepentaacetic acid and potassium), vitagerm-4 (coordination compound of germanium, diethyl ene tri aminepentaacetic acid and magnesium). all the compounds were primarily synthesized in the laboratory of department of general chemistry and polymers, i. mechnikov odesa national university, headed by prof. i. seifullina. all used germanium complexes were administered to rats at the doses according to the results of our previous studies [6, 7]. we injected them intraperitoneally in the form of 1 % aqueous solution at a dose of 50 and 100 mg/kg in 35 min after bilateral common carotid artery occlusion. as a comparator (reference) drug, piracetam medication was used (farmak, ukraine, 20 % solution in 5 ml ampules). it was injected intraperitoneally at a dose of 350 mg/kg in 30 min after ischemia. the rats of control group (ischemia without any correction) got equivalent dose of saline. criteria of cerebroprotector activity of the studied compounds were: survival of rats (%) each hour during the first 12 hours, then on the 24th, 48th and 72nd hours of ischemia; monitoring the clinical signs for et50 (median effective time – the time required for a half as organisms in a toxicity test to reveal the nonlethal response). it was evaluated to be et50=f(n/2) by interpolation of data of lethality time to mathematical function t=f(n), where t is the time of animals death, n – the number of animals. neurological status was assessed by irwin’s test in the model of acute brain ischemia and administration of g e r m a n i u m c o m p o u n d s . t h e p r i m a r y observation test was used for rodents to assess acute toxicity of a test agent and its effects on behavior and physiological function [8, 9]. statistical analysis. statistical analysis of animals’ survival have been carried out using the nonparametric criterion fisher’s exact test and average time of lifespan by use of t-criterion of students’ test. all data were presented as m (mean) ± m (standard error). a probability level (p value) of less than 0.05 was considered to be statistically significant. results the results obtained in the screening model of global brain ischemia are presented in the table 1. almost all bis(citrate) germanates (stannates), which contained different metals, possessed anti-ischemic activity of different intensity. the exception was the ol-6 compound, which comprised cobalt. even at the dose of 50 mg per kg in this group during the first hour of occlusion of aa. carotica communes the rate of rats survival was only 28.5 % opposite to 100 % in the control group. in addition, the other cobalt-containing compound ol-2 proved very low efficacy for cerebroprotection. as for the results in table 1, the most significant efficacy on the 48th hour of the experiment was evidenced for vitagerm-1 – a coordination compound of germanium, diethylenetriaminepentaacetic acid and lithium. the animals’ survival in this group was 57.1 % compared to 100 % lethality in the control group. it is important that the anti-ischemic activity of vitagerm-1 exceeded even the effect of reference drug piracetam. to estimate the obtained screening results more profoundly and detail, we suggested and used in our experiment the index of median effective time et50. according to the results presented in table 1, et50 for group of rats administered with vitagerm-1 was 53.31±2.77. it was in 7.5, 3.0 times better compared to the control and reference group respectively. at the same time to make an all-round comparison analysis of the results of screening series we assessed clinical signs of acute ischemia: neurological status of rats, which got the different types of germanium compounds. all data are presented in table 2. the rats with stroke, on the model we chose, experienced the severe clinical signs of global ischemia. the animals of control group after occlusion of both carotid arteries became less alert and more passive. it was proved by the indices of emotional condition: grooming decreased up to 0 points, vocalization increased up to 1.4. the tonus of limbs also diminished, ataxia signs increased up to 2.9 points. the character of these neurological changes testified quite a deep injury of brain cortex because of global ischemia. it resulted in the disruption of motor activity in the control group rats: suppression of reactions to touch and knock up to 1.5 and 1.9 points respectively. brain ischemia also strengthened the tremor up to 2.3 points. the suppression of ptosis reaction in 2.1 times as well as increased 62 b io m e d ic a l s c ie n c e s issn 2413-6077. ijmmr 2018 vol. 4 issue 1 ta bl e 1. t h e su rv iv al d yn am ic s (% ) an d th e av er ag e ti m e of d ea th ( et 50 ) of r at s w it h b ra in is ch em ia u n de r th e in fl u en ce of b is (c it ra te ) ge rm an at es ( st an n at es ) co m po u n ds w it h d if fe re n t m et al s (n =1 0) g ro up o f a ni m al (n =1 0) th e do se , m g/ kg r at s’ s u rv iv al ( % ) in t h e ce rt ai n p er io d of t im e (h ou rs ) p 1 p 2 et 50 (h ou rs ) p 3 p 4 1 2 3 4 5 6 7 8 9 10 11 12 24 48 72 in ta ct r at s 10 0 10 0 10 0 85 .7 57 .1 57 .1 57 .1 42 .8 42 .8 42 .8 42 .8 42 .8 14 .2 6. 96 ±0 .2 6 re fe re nc e gr ou p (p ir ac et am ) 35 0 10 0 10 0 10 0 10 0 85 .7 71 .4 71 .4 71 .4 71 .4 57 .1 57 .1 57 .1 42 .8 42 .8 42 .8 <0 .0 5 16 .1 2 ±0 .7 9 <0 .0 01 v it a g er m -1 10 0 71 .4 71 .4 71 .4 71 .4 71 .4 71 .4 71 .4 71 .4 71 .4 71 .4 71 .4 71 .4 57 .1 57 .1 42 .8 <0 .0 5 <0 .0 5 53 .3 1 ±2 .7 7 <0 .0 01 <0 .0 01 m ag ne si um b is (c itr at e) ge rm an at e (g er m ac it) 10 0 10 0 10 0 85 .7 85 .7 85 .7 85 .7 85 .7 85 .7 85 .7 85 .7 85 .7 71 .4 71 .4 28 .5 28 .5 >0 .0 5 >0 .0 5 43 .7 9 ±2 .3 6 <0 .0 01 <0 .0 01 m an ga n bi s (c itr at e) ge rm an at e (o l1 ) 10 0 10 0 85 .7 85 .7 85 .7 85 .7 85 .7 85 .7 85 .7 85 .7 71 .4 71 .4 57 .1 42 .8 42 .8 42 .8 <0 .0 5 <0 .0 5 33 .7 1 ±4 .0 5 <0 .0 01 <0 .0 1 co ba lt bi s( ci tr at e) ge rm an at e (o l2 ) 50 42 .8 42 .8 42 .8 42 .8 42 .8 42 .8 42 .8 42 .8 42 .8 42 .8 42 .8 42 .8 14 .2 14 .2 14 .2 >0 .0 5 >0 .0 5 9. 13 ±0 .8 2 <0 .0 5 <0 .0 01 ni ck el b is (c itr at e) ge rm an at e (o l3 ) 50 10 0 28 .5 28 .5 28 .5 28 .5 28 .5 28 .5 28 .5 28 .5 28 .5 28 .5 28 .5 >0 .0 5 >0 .0 5 5. 92 ±0 .4 7 <0 .0 5 <0 .0 01 zi nc b is (c itr at e) ge rm an at e (o l4 ) 50 10 0 10 0 10 0 10 0 10 0 10 0 10 0 71 .4 71 .4 42 .8 42 .8 42 .8 28 .5 >0 .0 5 >0 .0 5 11 .0 3 ±0 .8 2 <0 .0 1 <0 .0 1 m ag ne si um b is (c itr at e) st an na te (o l5 ) 10 0 10 0 10 0 10 0 10 0 10 0 57 .1 57 .1 57 .1 57 .1 57 .1 57 .1 28 .5 28 .5 >0 .0 5 >0 .0 5 10 .6 8 ±1 .4 9 <0 .0 5 <0 .0 5 co ba lt bi s (c itr at e) st an na te (o l6 ) 50 28 .5 28 .5 28 .5 28 .5 28 .5 28 .5 >0 .0 5 <0 .0 5 1. 09 ±0 .0 9 <0 .0 01 <0 .0 01 ni ck el b is (c itr at e) st an na te (o l7 ) 50 10 0 10 0 85 .7 14 .2 14 .2 14 .2 14 .2 14 .2 14 .2 14 .2 14 .2 14 .2 >0 .0 5 <0 .0 5 4. 31 ±0 .3 1 <0 .0 01 <0 .0 01 zi nc b is (c itr at e) st an na te (o l8 ) 10 0 10 0 10 0 10 0 10 0 57 .1 57 .1 57 .1 57 .1 57 .1 57 .1 28 .5 28 .5 28 .5 >0 .0 5 >0 .0 5 12 .2 6 ±0 .7 6 <0 .0 01 <0 .0 5 vi ta ge rm -2 10 0 10 0 10 0 10 0 57 .1 57 .1 28 .5 28 .5 14 .2 14 .2 14 .2 14 .2 14 .2 14 .2 14 .2 14 .2 >0 .0 5 >0 .0 5 5. 19 ±0 .1 7 <0 .0 01 <0 .0 01 vi ta ge rm -3 10 0 10 0 10 0 71 .4 71 .4 71 .4 57 .1 57 .1 57 .1 57 .1 57 .1 42 .8 42 .8 28 .5 28 .5 28 .5 >0 .0 5 >0 .0 5 10 .6 9 ±0 .4 9 <0 .0 01 <0 .0 01 vi ta ge rm -4 10 0 10 0 71 .4 71 .4 57 .1 57 .1 42 .8 28 .5 28 .5 28 .5 28 .5 14 .2 14 .2 14 .2 >0 .0 5 >0 .0 5 5. 14 ±0 .3 2 <0 .0 1 <0 .0 01 n ot es : s ta tis tic al s ig ni fic an ce w as e va lu at ed b y fi sh er e xa ct te st in 4 8 ho ur s af te r ar te ri es o cc lu si on ; p 1, p 3 – co m pa ed to th e co nt ro l g ro up ; p 2, p 4 – co m pa re d to p ir ac et am v. d. lukianchuk et al. 63 b io m e d ic a l s c ie n c e s issn 2413-6077. ijmmr 2018 vol. 4 issue 1 v. d. lukianchuk et al. ta bl e 2. 1. n eu ro ph ar m ac ol og ic al p ro fi le ( ir w in ’s o bs er va ti on t es t) o f bi s( ci tr at e) ge rm an at es ( st an n at es ) co m po u n ds , w h ic h c on ta in ed d if fe re n t m et al s, in a cu te b ra in is ch em ia n eu ro ph ar m a co lo gi ca l p ro fil e basal score statistical data co nt ro l pi ra ce ta m b is (c itr at e) g er m an at es (s ta nn at es ) c om po un ds w ith d iff er en t m et al s vitagerm-1 germacit ol-1 ol-2 ol-4 ol-5 ol-8 vitagerm-2 vitagerm-3 vitagerm-4 b eh av io ra l pr ofi le al er tn es s 4 m ±m p1 p2 0. 0± 0. 0 2. 9± 0. 3 <0 .0 01 3. 0± 0. 3 <0 .0 01 <0 .0 5 2. 6± 0. 2 <0 .0 01 >0 .0 5 2. 7± 0. 4 <0 .0 01 >0 .0 5 1. 7± 0. 5 <0 .0 1 >0 .0 5 2. 1± 0. 2 <0 .0 01 >0 .0 5 2. 2± 0. 2 <0 .0 01 >0 .0 5 2. 3± 0. 5 <0 .0 1 >0 .0 5 1. 5± 0. 3 <0 .0 1 >0 .0 5 2. 6± 0. 4 <0 .0 01 <0 .0 5 1. 6± 0. 3 <0 .0 01 >0 .0 5 pa ss iv ity 0 m ±m p1 p2 2. 9± 0. 4 2. 6± 0. 1 >0 .0 5 1. 2± 0. 1 <0 .0 1 <0 .0 01 1. 5± 0. 3 >0 .0 5 <0 .0 5 1. 4± 0. 3 <0 .0 5 <0 .0 1 2. 0± 0. 3 >0 .0 5 >0 .0 5 2. 3± 0. 3 >0 .0 5 >0 .0 5 2. 4± 0. 3 >0 .0 5 >0 .0 5 1. 9± 0. 3 >0 .0 5 <0 .0 1 1. 9± 0. 4 >0 .0 5 >0 .0 5 1. 6± 0. 3 >0 .0 5 <0 .0 5 1. 1± 0. 3 <0 .0 5 <0 .0 1 m oo d g ro om in g 4 m ±m p1 p2 0. 0± 0. 0 1. 5± 0. 2 <0 .0 01 2. 8± 0. 5 <0 .0 1 >0 .0 5 1. 0± 0. 2 <0 .0 1 >0 .0 5 0. 9± 0. 5 >0 .0 5 >0 .0 5 1. 8± 0. 2 <0 .0 01 >0 .0 5 1. 0± 0. 1 <0 .0 01 >0 .0 5 1. 1± 0. 1 <0 .0 01 >0 .0 5 2. 1± 0. 3 <0 .0 01 >0 .0 5 1. 4± 0. 4 <0 .0 1 >0 .0 5 1. 0± 0. 5 >0 .0 5 >0 .0 5 1. 7± 0. 2 <0 .0 01 >0 .0 5 vo ca liz atio n 0 m ±m p1 p2 1. 4± 0. 3 0. 0± 0. 0 <0 .0 01 0. 0± 0. 0 <0 .0 01 0. 0± 0. 0 <0 .0 01 0. 0± 0. 0 <0 .0 01 0. 0± 0. 0 <0 .0 01 0. 0± 0. 0 <0 .0 01 0. 0± 0. 0 <0 .0 01 0. 0± 0. 0 <0 .0 01 0. 0± 0. 0 <0 .0 01 0. 0± 0. 0 <0 .0 01 0. 0± 0. 0 <0 .0 01 m ot or ac tiv ity to uc h r e sp on se 4 m ±m p1 p2 1. 5± 0. 3 2. 1± 0. 2 >0 .0 5 3. 6± 0. 3 >0 .0 5 <0 .0 5 1. 5± 0. 3 >0 .0 5 2. 0± 0. 4 >0 .0 5 >0 .0 5 2. 0± 0. 3 >0 .0 5 >0 .0 5 2. 5± 0. 4 >0 .0 5 >0 .0 5 2. 0± 0. 2 >0 .0 5 >0 .0 5 2. 3± 0. 3 >0 .0 5 >0 .0 5 1. 9± 0. 4 >0 .0 5 >0 .0 5 2. 3± 0. 4 >0 .0 5 >0 .0 5 2. 0± 0. 2 >0 .0 5 >0 .0 5 kn oc k re sp on se 4 m ±m p1 p2 1. 9± 0. 2 2. 9± 0. 3 <0 .0 1 3. 5± 0. 3 <0 .0 1 >0 .0 5 1. 7± 0. 3 <0 .0 5 <0 .0 5 2. 0± 0. 4 >0 .0 5 >0 .0 5 1. 8± 0. 3 >0 .0 5 >0 .0 5 2. 3± 0. 4 >0 .0 5 >0 .0 5 2. 0± 0. 1 >0 .0 5 >0 .0 5 2. 6± 0. 5 >0 .0 5 >0 .0 5 1. 9± 0. 3 >0 .0 5 >0 .0 5 2. 4± 0. 3 >0 .0 5 >0 .0 5 1. 7± 0. 2 >0 .0 5 <0 .0 1 n ot es : i n ta bl es 2 .1 a nd 2 .2 : p 1 – st at is tic al s ig ni fic an ce c om pa ed to th e co nt ro l, p2 – c om pa re d to th e co m pa ra to r (r ef er en ce ) d ru g. 64 b io m e d ic a l s c ie n c e s issn 2413-6077. ijmmr 2018 vol. 4 issue 1 ta bl e 2. 2. n eu ro ph ar m ac ol og ic al p ro fi le ( ir w in ’s o bs er va ti on t es t) o f bi s( ci tr at e) ge rm an at es ( st an n at es ) co m po u n ds , w h ic h c on ta in ed d if fe re n t m et al s, in c as es o f ac u te b ra in is ch em ia n eu ro ph ar m a co lo gi ca l p ro fil e basal score statistical data control piracetam b is (c itr at e) g er m an at es (s ta nn at es ) c om po un ds w ith d iff er en t m et al s vitagerm-1 germacit ol-1 ol-2 ol-4 ol-5 ol-8 vitagerm-2 vitagerm-3 vitagerm-4 n eu ro lo gi ca l pr ofi le m ot or co or di na tio n li m b to ne 4 m ±m p1 p2 2. 5± 0. 2 2. 2± 0. 3 <0 .0 5 3. 3± 0. 3 <0 .0 5 >0 .0 5 0. 8± 0. 3 <0 .0 01 <0 .0 5 1. 3± 0. 3 <0 .0 1 >0 .0 5 1. 2± 0. 1 <0 .0 01 >0 .0 5 1. 2± 0. 2 >0 .0 5 1. 6± 0. 2 >0 .0 5 2. 2± 0. 3 >0 .0 5 >0 .0 5 1. 5± 0. 4 <0 .0 5 >0 .0 5 1. 4± 0. 4 <0 .0 5 >0 .0 5 1. 6± 0. 3 <0 .0 1 >0 .0 5 at ax ia 0 m ±m p1 p2 2. 9± 0. 3 1. 9± 0. 3 <0 .0 1 0. 0± 0. 0 <0 .0 01 <0 .0 01 0. 0± 0. 0 <0 .0 01 <0 .0 01 0. 0± 0. 0 <0 .0 01 <0 .0 01 0. 0± 0. 0 <0 .0 01 <0 .0 01 2. 0± 0. 3 <0 .0 5 <0 .0 5 2. 4± 0. 3 >0 .0 5 >0 .0 5 0. 0± 0. 0 <0 .0 01 <0 .0 01 0. 0± 0. 0 <0 .0 01 <0 .0 01 0. 0± 0. 0 <0 .0 01 <0 .0 01 0. 0± 0. 0 <0 .0 01 <0 .0 01 cn s st at us tr em or 0 m ±m p1 p2 2. 3± 0. 3 0. 0± 0. 0 <0 .0 01 0. 0± 0. 0 <0 .0 01 2. 1± 0. 3 >0 .0 5 <0 .0 01 0. 0± 0. 0 <0 .0 01 0. 0± 0. 0 <0 .0 01 0. 0± 0. 0 <0 .0 01 0. 0± 0. 0 <0 .0 01 0. 0± 0. 0 <0 .0 01 0. 0± 0. 0 <0 .0 01 0. 0± 0. 0 <0 .0 01 0. 0± 0. 0 <0 .0 01 au to no m ic pr ofi le pt os is 4 m ±m p1 p2 1. 9± 0. 2 0. 9± 0. 2 <0 .0 1 2. 2± 0. 3 <0 .0 5 <0 .0 01 1. 6± 0. 2 >0 .0 5 >0 .0 5 0. 8± 0. 3 <0 .0 5 >0 .0 5 1. 4± 0. 2 >0 .0 5 >0 .0 5 1. 5± 0. 2 >0 .0 5 <0 .0 5 0. 7± 0. 3 >0 .0 5 >0 .0 5 1. 5± 0. 4 >0 .0 5 >0 .0 5 1. 0± 0. 3 <0 .0 5 0. 9± 0. 3 <0 .0 5 >0 .0 5 1. 1± 0. 2 >0 .0 5 >0 .0 5 u ri na tio n 0 m ±m p1 p2 1. 4± 0. 1 0. 0± 0. 0 <0 .0 01 0. 0± 0. 0 <0 .0 01 0. 0± 0. 0 <0 .0 01 0. 0± 0. 0 <0 .0 01 0. 0± 0. 0 <0 .0 01 0. 0± 0. 0 <0 .0 01 0. 0± 0. 0 <0 .0 01 0. 0± 0. 0 <0 .0 01 0. 0± 0. 0 <0 .0 01 0. 0± 0. 0 <0 .0 01 0. 0± 0. 0 <0 .0 01 d ef eca tio n 0 m ±m p1 p2 0. 9± 0. 3 0. 0± 0. 0 <0 .0 5 0. 0± 0. 0 >0 .0 5 1. 9± 0. 3 >0 .0 5 <0 .0 5 0. 7± 0. 6 >0 .0 5 1. 1± 0. 2 >0 .0 5 <0 .0 01 1. 5± 0. 4 >0 .0 5 <0 .0 1 0. 0± 0. 0 >0 .0 5 1. 6± 0. 2 >0 .0 5 <0 .0 01 0. 8± 0. 3 >0 .0 5 >0 .0 5 0. 9± 0. 3 >0 .0 5 0. 8± 0. 2 >0 .0 5 >0 .0 5 v. d. lukianchuk et al. 65 b io m e d ic a l s c ie n c e s issn 2413-6077. ijmmr 2018 vol. 4 issue 1 v. d. lukianchuk et al. frequency of urination and defecation was evidenced in comparison to the indices of healthy group. the next stage of our experiment was to estimate the neurological and behavioral profile of rats under the influence of germanium compounds in global brain ischemia model. the most efficient compound was vitagerm-1 again (tables 2.1 and 2.2). the significant increase of alertness up to 3.0 points and decreasing of expressiveness of passivity by 41.0 % was fixed in this group compared to the control rats. this investigated compound increased the grooming and normalized the vocalization. administration of vitagerm-1 led to absence of ataxia and increase of extremities tonus up to 3.3 points that was nearly the same as the indices of the intact rats. responses to touch and knock intensified also up to 3.6 and 3.5 points respectively in comparison to the control. we noticed total positive effects of this germanium compounds in prevention of tremor, urination and defecation; the ptosis rate increased up to 2.2 points. discussion the pathophysiology of stroke is complex, and involves inflammatory pathways, oxidative damage, apoptosis, energy deficiency etc. the main goal of treatment is to preserve tissues in ischemic penumbra, where perfusion is decreased but sufficient to stave off infarction, and save the neurons (neuroprotection) [4, 5]. ischemic stroke and its consequences is a major public health problem. in spite of huge number of medications to treat it, we still have no perfect one according to the present requirements of safety and efficacy. that is why the search and investigation of newly synthesized organic and other compounds with capability for stroke protection is an actual challenge and target for chemists, pharmacologists, physicians and health professionals. numerous bis(citrate) germanates (stannates) compounds with wide spectrum of therapeutic activity are synthesized and investigated today. our previous experiments proved its low toxicity to warm-blooded species [10, 11, 12]. use of the rodent models is recommended and approved by different researches for screening and investigating of promising compounds to prevent and treat ischemic brain lesions [13]. in the screening series in the model of global brain ischemia (via occlusion of both common carotid arteries before its bifurcation), it was determined that almost all compounds had potency for stroke protection. such criteria as survival and median effective time plus irwin’s test results were adequate to evaluate it [14, 15]. piracetam was used as a reference drug [16]. a m o n g a l l i n v e s t i g a t e d b i s ( c i t r a t e ) germanate (stannates) compounds, which contained different metals, the most potent anti-ischemic activity was possessed by coordination compound of germanium, diethylenetriaminepentaacetic acid and lithium coded as vitagerm-1. it was a leader among the germanium compounds according to the analyses of the indices of survival and lifespan of rats from the moment of occlusion of coronary arteries. such positive effects were manifested by the highest level of survival and the most favorable clinical course of cerebrovascular blood circulation. median effective time et50 in this group exceeded in 7.5 and 3.0 times the index of control and reference group respectively. we noticed total positive effects of this germanium compounds in prevention of disorders of behavioral, neurological and autonomic profiles of rats. therefore, we concluded the prominent improvement of clinical course of acute brain ischemia under the vitagerm-1 impact. we suppose that the presence of the cobalt in structure of ol-2 and ol-5 molecules is responsible for animals lethality during the first hour after the disorder development. low efficacy of these germanium compounds could be explained by the fact that this metal is a cytochrome p450 inhibitor. the cytochromes p450 (cyps) enzyme family are crucial in processes of detoxification of the organism, and suppression of its activity is one of the most possible causes of high lethality in cases of experimental global brain ischemia [17]. conclusions almost all bis(citrate) germanates (stannates), which contained different metals, possessed anti-ischemic activity of different intensity. the exceptions were cobalt-containing ol-6 and ol-2 compounds. the most expressive efficacy for all investigated indices (which exceeded even the reference drug) proved for vitagerm-1 – a coordination compound of germanium, diethylenetriaminepentaacetic acid and lithium. the results of our experiments are suggested for the substitution for further deeper pharmacological investigation of vitagerm-1 for stroke cerebroprotection, and its implementation into clinical practice. 66 b io m e d ic a l s c ie n c e s issn 2413-6077. ijmmr 2018 vol. 4 issue 1 references 1. world health organization who updates fact sheet on top 10 causes of death. 27 jan 2017. 2. zozulia is. epidemiology of cerebrovascular diseases in ukraine. ukrainian medical journal. 2011;85(5):38–41 [in ukrainian]. 3 . s m a j l o v i c d s t r o k e s i n y o u n g a d u l t s epidemiology and prevention. vascular health and risk management. 2015;11:157–164. doi.org/10.2147/ vhrm.s53203. feb 2015. 4. musuka td wilton sb traboulsi m hill md diagnosis and management of acute ischemic stroke: speed is critical. cmaj : canadian medical association journal. 2015;187(12):887–893. doi. org/10.1503/cmaj.140355. aug 2015. 5. sas a, horvath l, olah c, valikovics a. a. review of neuroinflammatory mechanisms in ischemic stroke: background and therapeutic approaches. inmechanisms of neuroinflammation. 2017. intech. 6. lukianchuk vd, zhytina io, seifullina ii, martsinko oe, pesaroglo og. screening and com parative characteristic of anti ischemic e cacy of coordination compounds of germanium in acute cerebrovascular insuficiency pharmacology and drug toxicology [in ukrainian]. 7. lukianchuk vd, krylova ev, seifullina ii, tkachenko vn. screening of potential cerebro protectors among newly synthesized coordination compounds of germanium with complexions of hydroxycarboxylic acids in the model of global cerebral ischemia. go mozhaev journal of emergency medicine. 2008;9(4):123–126 [in russian]. 8. gad sc. drug safety evaluation. john wiley & sons. accessed 7 nov 2016. 9. stefanov ov. preclinical study of drugs: methodical instructions. k: avicenna. 2001:292–306. 10. chadova lv, seifulina ii, tkachenko vm. screening and comparative characteristic of antiischemic drugs among coordination compounds of germanium with bioligands in acute cerebrovascular insufficiency. odesa medical journal. 2005;6:19–22 [in ukrainian]. 11. lukianchuk vd, seifullina ii, litvinenko df, martsinko oe. pharmacodynamics of organic and coordination compounds of germanium – modern state of art pharmacology and drug toxicology. 2016;1:3–13 [in ukrainian]. 12. shutka ao, kravets ds, lukianchuk vd. mathematical-pharmacological analysis of dose regimen of new antihypoxant ok ukrainian journal o f c l i n i a c l l a n d l a b o r a t o r y m e d i c i n e . 2010;5(4):115–9. 13. canazza a, minati l, boffano c, parati e, binks s. experimental models of brain ischemia a review of techniques, magnetic resonance imaging, and investigational cell-based therapies. front neurol. frontiers media sa. 2014;5:19. 14. durukan a, tatlisumak t. acute ischemic stroke overview of major experimental rodent models, pathophysiology, and therapy of focal cerebral ischemia. pharmacology biochemistry and behavior. 2007;87(1):179–97. 15. fluri f, schuhmann mk, kleinschnitz c. animal models of ischemic stroke and their application in clinical research. drug design, development and therapy. 2015;9:3445. 16. winblad b piracetam a review of phar macological properties and clinical uses. cns drug rev. 2005;11(2):169–82. 17. navarro-mabarak c, camacho-carranza r, espinosa-aguirre jj. cytochrome p450 in the central n e r v o u s s y s t e m a s a t h e ra p e u t i c t a r g e t i n neurodegenerative diseases. drug metabolism reviews. 2018;50(2):95–108. received: 2018-03-06 v. d. lukianchuk et al. пошук церебропроекторів серед координаційних сполук германію на моделі ішемічного інсульту в. д. лук’янчук1, і. й. сейфулліна2, о. е. марцинко2, о. о. шевчук3 1 – ду «інститут фармакології та токсикології намн україни», київ, україна 2 – одеський національний університет імені і. і. мечникова, одеса, україна 3 – тернопільський державний медичний університет імені і. я. горбачевського, тернопіль, україна вступ. інсульт залишається однією з провідних причин смертності та інвалідності в усьому світі, однак спостерігається зростання захворюваності серед молоді. мета дослідження – скринінгове дослідженння церебропротекторної дії координаційних сполук германію на моделі гострої ішемії у щурів. методи. гостру ішемію моделювали двосторонньою оклюзією загальної сонної артерії. для первинного скринінгу використовувалися сполуки біс(цитрато)германати (станати), що містили різні метали: ol1-8, vitagerm-1,2,3 та 4. усі використовувані комплекси германію вводили внутрішньочеревно (1% водного розчину при дозі 50 і 100 мг/кг через 35 хв після двосторонньої загальної оклюзії сонної артерії). пірацетам використовували в якості референс препарату. критерії ефективності церебропротекції: виживання щурів (%), ет50 (середній час загибелі 50% тварин), тест ірвіна. результати. результати, отримані на скринінговій моделі тотальної ішемії головного мозку, показали, що практично всі різнометальні біс(цитрато) германати (станати) володіють антиішемічною активністю різної інтенсивності. виняток становили сполуки, що містять кобальт ol-6 і ol-2. найбільш значима ефективність всіх досліджених показників (які перевищували навіть еталонний препарат) була підтверджена для vitagerm-1 – координаційного з’єднання германію, діетилентриамінпентаоцтової кислоти і літію. висновки. результати наших експериментів слугуют підгрунтям для подальшого вивчення сполуки vitagerm-1 у якості церебропротектора та її впровадження в клінічну практику. ключові слова: тотальна ішемія головного мозку; координаційні сполуки германію; скринінг. issn 2413-6077. ijmmr 2018 vol. 4 issue 1 41 a n e st h e si o l o g y a n d c r it ic a l c a r e doi 10.11603/ijmmr.2413-6077.2018.1.8514 hemophagocytosis secondary to pharyngeal abscess in an immunocompetent patient (case report) i. d. khan, m. malik, k. s. rajmohan, p. banerjee, s. khan, p. s. panda, m. brijwal, s. gupta, kahkasha, s. gazala, e. sawarat, m. s. aguinaga army college of medical sciences and base hospital, new delhi, india background. hemophagocytosis is a rare, potentially fatal disorder, comprising pancytopenia, liver dysfunction, hepatosplenomegaly, hypertriglyceridemia, and hyperferritinemia presenting as fever, lymphadenopathy and skin rashes. objective. to attract the clinicians’ attention to a problem of hemophagocytosis in critical care management. methods. hemophagocytosis secondary to pharyngeal abscess in a 58 year old male is being reported. results. a 58-year-old immunocompetent patient presenting with hemophagocytosis secondary to pharyngeal abscess, was managed on ventilator and inotropic support, when he developed heathcare-associated urinary tract infection by escherichia coli and ventilator-associated pneumonia by acinetobacter baumanii. he developed neutropenic septic shock and multi-organ dysfunction and went through a downhill course leading to demise. conclusions. hemophagocytosis remains a sinister entity in modern intensive care despite astute clinical management. secondary superinfections with opportunistic multidrug resistant pathogens are difficult to treat. a high index of clinical suspicion, aggressive diagnosis and prompt treatment for hemophagocytosis and polymicrobial opportunistic superinfections with multidrug-resistant healthcare-associated pathogens needs to be addressed upfront. key words: hemophagocytosis; pharyngeal abscess; acinetobacter baumanii; pancytopenia; ventilator-associated pneumonia; sepsis. international journal of medicine and medical research 2018, volume 4, issue 1, p. 41-44 copyright © 2018, tsmu, all rights reserved introduction hemophagocytosis is a rare, potentially fatal disorder, comprising pancytopenia, liver dysfunction, hepatosplenomegaly, hypertriglyceridemia, and hyperferritinemia pre senting as fever, lymphadenopathy and skin rashes. primary hemophagocytosis may be genetic (x-linked lymphoproliferative syndrome a n d c h e d i a k h i g a s h i s y n d ro m e ) , w h i l e secondary hemophagocytosis may occur due to infections, malignancies (lymphomas) and autoimmune diseases (sarcoidosis). bacterial infections causing hemophagocytosis are t u b e r c u l o s i s , t y p h o i d , b r u c e l l o s i s a n d ehrlichiosis. many viruses such as swine influenza h1n1, avian influenza, measles, epstein-barr virus, human immunodeficiency virus, parvovirus, hepatitis viruses, herpes viruses and varicella zoster virus, parasitic diseases such as leishmaniasis, systemic mycosis and various emerging pathogens can also cause hemophagocytosis [1, 2, 3, 4, 5]. antigenic stimuli cause a progressive immune reaction with a cytokine storm, activate macrophages phagocytose erythrocytes and leucocytes in bone marrow and lymphoid tissues. hemophagocytosis secondary to pharyngeal abscess in a 58 year old male is being reported. case report a 58 year old male with mild odynophagia and nasal congestion of three year duration along with high grade continuous fever of one month duration, was referred to a tertiary-care facility. on examination at arrival, the patient had an intoxicated appearance with tachycardia, tachypnea, fever 105.3 °f, pallor and weight 66 kg. he was nursed in intensive care after tracheostomy with ventilator support in synchronized intermittent mandatory ventilation (simv) mode along with inotropic support for worsening hypotension. ventilator was readjusted to continuous positive airway pressure (cpap) mode. central venous pressure was 8 mm hg. hot potato voice and nasal twang were evidenced along with oral mucositis and coated tongue. oedematous and erythematous soft palate with superficial slough was present. corresponding author: dr inam danish khan, associate professor, clinical microbiology and infectious diseases, army college of medical sciences and base hospital, new delhi 110010, india e-mail: titan_afmc@yahoo.com mobile phone: +91 8076324060 i. d. khan et al. issn 2413-6077. ijmmr 2018 vol. 4 issue 142 a n e st h e si o l o g y a n d c r it ic a l c a r e ulcer on posterior pharyngeal wall extending from soft palate to floor of vallecula and multiple aphthous ulcers along the lateral border of tongue were evidenced. bilateral congested palpebral conjunctiva and arcus senilis were also present. ultrasonography revealed subcutaneous emphysema chest, hepatomegaly, distended gall bladder and grade ii prostatomegaly. cect chest revealed bilateral ground glass opacities in lung bases and early interstitial lung disease in posterobasal segment of right lung. repeat ncct neck revealed mass in right pyriform fossa which was confirmed by spiral ct neck, obliteration of right pyriform sinus, thickening of right aryepiglottic folds with restriction of right true vocal cord movement suggestive of mass lesion. mri showed diffuse oedema and thickening of posterior naso-oro-hypo pha ryn geal walls with effacement of right pyriform sinus and significant compromise of lumen. acute pansinusitis, bilateral mastoiditis, bilate ral pleural effusion and fibrotic opacity right lung were evidenced. subcutaneous emphysema of right subclavian region was present. history of low-grade continuous fever two months prior to highgrade fever was elicited for which he was treated with injectable antimicrobials with little improvement. there was history of loose stools 10–12 times and weight loss 2.5 kg over past three months. he underwent septoplasty three years earlier. during the course of his illness, haemoglobin dropped from 10 gm/dl to 7.5 gm/dl with leukopenia reducing from 3600/cu mm to 900/cu mm with neutrophil count 600/cu mm. erythrocyte sedimentation rate increased from 45 to 96 in one hour. anaemia workup revealed mean corpuscular volume 69 fl, serum iron 23 µg/dl, total iron binding capacity 936 µg/dl, ferritin 280 µg/dl. liver function tests included total bilirubin 3.4 mg/dl, direct bilirubin 0.6 mg/dl, alanine and aspartate amino transferases 125 and 55 mg/dl, alkaline phosphatase 78 mg/dl and gamma glutamyl transferase 180 mg/dl. coagulation profile was deranged with inr 2.6, d-dimers 3.17 µg/l and features of early disseminated intravascular coagulation. dyselectrolytemia and hypocalcaemia persisted. serum iga and igm were raised. serum protein electrophoresis revealed polyclonal bands in gamma regions. microbiological work up including mantoux test, tuberculosis polymerase chain reaction, widal test, malarial serology, tests for hiv, syphilis, hepatitis b and clostridium difficile toxin were negative. blood cultures were negative, urine cultures revealed escherichia coli. throat swab revealed streptococcus viridans and budding yeast cells. tracheal aspirates revealed acinetobacter baumanii susceptible to colistin and cryptococcus laurentii which was susceptible to all antifungals. histopathological work up involving punch biopsy from pharyngeal wall and nasopharynx revealed chronic inflammatory posterior pharyngeal wall abscess. repeat biopsy revealed acute ulceration with granulomatous tissue without malignant cells. bone marrow biopsy revealed reactive marrow with brisk haemophagocytosis. vasculitis work up was negative. no blasts or atypical cells were seen. rheumatologic workup for both anti-neutrophil cytoplasmic antibodies and myeloperoxidase were not contributory, however c-reactive proteins and gamma glutamyl transferase were raised. whole body dual-time positron emission tomography (pet)fludeoxyglucose f 18 (fdg) scan revealed avid inflammatory lesions in oropharynx and nonfdg avid mass in left pharyngeal spaces along with bony erosions in cervical vertebral bodies with adjacent soft tissue attenuation. a host of other investigations such as thyroid profile, vitamin b-12 assay, upper gastrointestinal endoscopy were not contributory. neutropenic septic shock and multi-organ dysfunction developed and the patient went through a downhill course leading to demise. discussion deep seated abscesses in pharyngeal spaces can be present insidiously with prolonged fever which may be difficult to localize and treat. apart from features of infection such as fever, they can cause pressure effects leading to variable degrees of dysphagia, odynophagia and respiratory obstruction and/or compromise. the abscess can spread to contiguous areas or the inflammation can affect surrounding tissues. pre-existing comorbidities can lead to rapid worsening of general condition despite the infection being localized, which may also preclude surgical intervention [6, 7]. conditions leading to hemophagocytosis can also cause granulomatous hepatitis. hemophagocytosis in this patient appears to be due to multiple etiology causing immunological stimulation in parallel. hemophagocytosis is known to occur in patients under intensive-care leading to sepsis and multiple organ failure as evidenced in this patient [8]. unexplained hypotension requiring inotropic support in this patient is likely to be due to pre-existing disease conditions i. d. khan et al. issn 2413-6077. ijmmr 2018 vol. 4 issue 1 43 a n e st h e si o l o g y a n d c r it ic a l c a r e such as pulmonary emphysema and liver disorder. pancytopenia in hemophagocytosis can lead to immunocompromised state facilitating opportunistic infections such as healthcareassociated urinary tract infection and ventilatorassociated pneumonia. acinetobacter baumanii was multidrug resistant and was susceptible only to colistin. secondary superinfections with opportunistic multidrug resistant pathogens were difficult to treat [9, 10, 11, 12]. ct, mri and histopathology localized the same lesion without detection of any other foci [13]. whole body dual-time pet-fdg scan revealed avid inflammatory lesions in oropharynx which could suggest active disease and non-fdg avid mass in left pharyngeal spaces hints towards a latent tuberculosis infection. also the bony erosions in cervical vertebral bodies with adjacent soft tissue attenuation point towards involvement of cervical spine. retropharyngeal space abscesses are a disease of infancy, usually resulting from abscess of lymph nodes draining infection of ear, nose or throat [14,15]. chronic inflammatory posterior pharyngeal wall abscess is usually evidenced in adults or slightly elder children. retropharyngeal abscess in adults is often pyogenic and usually secondary to pharyngeal or oesophageal perforation, sepsis in the throat or sinuses, penetrating injury, oral endotracheal intubation or trauma to pharynx. retro pharyngeal tuberculous abscess can rarely be present with odynophagia, neck pain, stridor, mediastinitis and life-threatening respiratory obstruction. diagnostic limitations preclude diagnostic outcome despite clinical intuition, especially in emerging and rare pathogens [16, 17, 18, 19, 20, 21]. conclusions hemophagocytosis remains a sinister entity in contemporary intensive care despite astute clinical management. a high index of clinical suspicion, aggressive diagnosis and prompt treatment for hemophagocytosis and polymicrobial opportunistic superinfections with multidrug-resistant healthcare-associated pathogens needs to be addressed upfront. i. d. khan et al. гемофагоцитоз внаслідок глоткового абсцесу в імунокомпетентного пацієнта (клінічний випадок) і. d. khan, m. malik, k. s. rajmohan, p. banerjee, s. khan, p. s. panda, m. brijwal, s. gupta, kahkasha, s. gazala, e. sawarat, m. s. aguinaga army college of medical sciences and base hospital, new delhi, india вступ. гемофагоцитоз – це рідкісний, потенційно небезпечний для життя розлад, який включає панцитопенію, порушення функції печінки, гепатоспленомегалію, гіпертригліцеридемію і гіперферритинемію, та проявляється у вигляді лихоманки, лімфадентопатії та шкірних висипань. мета дослідження привернути увагу лікарів до проблеми гемофагоцитозу у відділеннях невідкладної допомоги. методи. описано клінічний випадок гемофагоцитозу, який розвинувся на фоні глоткового абсцесу в чоловіка 58 років. результати. 58-річний імунокомпетентний пацієнт з гемофагоцитозом на фоні глоткового абсцесу перебував на штучній вентиляції легень та отримував інотропну фармакотерапію (кардіотоніки), коли у нього розвинулась інфекція сечовивідних шляхів, спричинена escherichia coli та вентиляційна пневмонія, спричинену acinetobacter baumanii. у пацієнта розвинувся нейтропенічний септичний шок і мультиорганна недостатність, які призвели до його смерті. висновки. незважаючи на інтенсивну терапію та сучасні засоби і методи лікування, гемофагоцитоз залишається небезпечним для життя станом. вторинні суперінфекції, спричинені опортуністичними мультирезистентними патогенами, важко піддаються лікуванню. клінічна настороженість, агресивна діагностика, раннє лікування гемофагоцитозу та мультирезистентних опортуністичних суперінфекцій є необхідністю у відділеннях інтенсивної терапії. ключові слова: гемофагоцитоз; глотковий абсцес; acinetobacter baumanii; панцитопенія; венти­ ляційна пневмонія; сепсис. issn 2413-6077. ijmmr 2018 vol. 4 issue 144 a n e st h e si o l o g y a n d c r it ic a l c a r e 13. silva-herzog e, detweiler cs. intracellular m i c ro b e s a n d h a e m o p h a g o c y t o s i s . c e l l u l a r microbiology. 2008;10(11):2151–2158. 14. mohamad i, jaafar r. an elderly man with acute anterior neck pain and odynophagia after a meal. malaysian family physician: the official journal of the academy of family physicians of malaysia. 2013;8(3):37–39. 15. poorey vk, karkare a. retropharyngeal abscess following accidental throttling. indian journal of otolaryngology and head & neck surgery. 1998;50(3):275-276. 16. khan id, gupta n, rangan nm, singh r, sharma ak, khurana a, rudra p, krushnarao ms. evaluation of pre and post analytical variables in clinical microbiology services in multidisciplinary icu of a medical college and tertiary care hospital. j basic & clin med 2016,5(1):2–4. 17. khan id, lall m, sen s, ninawe sm, chandola p. multiresistant elizabethkingia meningoseptica infections in tertiary care. mjafi 2014;71(3):66–67. 18. khan id, mukherjee t, gupta s, haleem s, sahni ak, banerjee s, konar j. ochrobactrum anthropi sepsis in intensive tertiary care. j basic & clin med 2014,3(1):18–20. 19. khan id, sahni ak, bharadwaj r, anwar i, jain v, khan s, chowdhury a, dwivedi ak, gupta ak, s h a m s u z z a m a n , a l a m s , k o n a r j . c e r e b r a l toxoplasmosis diagnosed by stereotactic brain biopsy leading to detection of hiv infection. j basic & clin med 2016,5(1):5–7. 20. khan id. outbreak of prototheca wickerhamii algaemia and sepsis in a tertiary care chemotherapy oncology unit. international journal of infectious diseases. 2016 apr 30;45:195. 21. khan id, rajmohan ks, gupta rm, sen s, hashmi a, ninawe sm, mukherjee b, kishore k, mishra m, yadav a, choubey a, shaikh s. invasive cerebral and pulmonary mucormycosis in an immunocompromised patient. j basic & clin med 2017,6(2):9–11. received: 2018-02-03 i. d. khan et al. references 1.. takahashi n, chubachi a, kume m, et al. a clinical analysis of 52 adult patients with h e m o p h a g o c y t i c s y n d ro m e : t h e p ro g n o s t i c significance of the underlying diseases. int j hematol. 2001;74:209–13. 2.. brastianos pk, swanson jw, torbenson m, sperati j, karakousis pc. tuberculosis-associated haemophagocytic syndrome. lancet infect dis. 2006 jul;6(7):447-54. 3. kumar n, gadpayle ak, singh r, tonk rs, dembla g. epstein-barr virus-induced haemophagocytic syndrome. natl med j india. 2012 sepoct;25(5):271-3. 4. khan id, sahni ak, bharadwaj r, lall m, jindal ak, sashindran vk. emerging organisms in a tertiary healthcare set up. med j armed forces india 2014; 70(2):120–128. 5.. johnson tm, brown ms, rabbat m, slim j. hemophagocytic lymphohistiocytosis associated with anaplasmosis. journal of global infectious diseases. 2017;9(2):76–78. 6. gaglani mj, edwards ms. clinical indicators of childhood retropharyngeal abscess. am j emerg med. 1995 may;13(3):333–336. 7. hashmi hrt, mishra r, niazi m, venkatram s, diaz-fuentes g. an unusual triad of hemophagocytic syndrome, lymphoma and tuberculosis in a non-hiv patient. the american journal of case reports. 2017;18:739–745. 8. rekik r, morazin f, lumbroso a, stirnemann j, montravers p, gauzit r. reactive haemophagocytic syndrome and multiple organ failure in intensive care unit patients. ann fr anesth reanim. 2004 dec;23(12):1189–1191. 9.. khan id, sahni ak. bacterial infections and emerging resistance in renal transplant recipients. bang j med sci. 2015;14(1):14–21. 10. khan id, basu a, kiran s, trivedi s, pandit p, chattoraj a. device-associated healthcare associated infections (da-hai) and the caveat of multiresistance in a multidisciplinary intensive care unit. med j armed forces india 2017;73(3):222–231. 11. khan id, gupta rm, sen s, rajmohan ks, jindal ak, makkar a, et al. emerging antimicrobial resistance and evolving healthcare: dangerous crossroads for the community and the military. journal of archives in military medicine. in press: e12097. 12.2 khan id, dogra pm, ramphal sk, khan s, konar j, palit a, srivastava n, agrawal p, haleem s, alam s. polymicrobial infections in a teenaged renal trans plantrecipient. j basic & clin med 2015,4(1): 37–39. 26 p e d ia t r ic s issn 2413-6077. ijmmr 2017 vol. 3 issue 1 corresponding author: iryna sarapuk, department of pediatrics №2, i. horbachevsky ternopil state medical university, 2 sakharova street, ternopil, ukraine, 46400 phone number: +380502081147 e-mail: prostoirusya@ukr.net i. m. sarapuk et al. international journal of medicine and medical research 2017, volume 3, issue 1, p. 26–33 copyright © 2017, tsmu, all rights reserved doi 10.11603/ijmmr.2413-6077.2017.1.7063 neurodevelopmental care of preterm infants and its key elements i. m. sarapuk1, h. a. pavlyshyn1, l. lacina2, b. królak-olejnik3 і. horbachevsky ternopil state medical university, ternopil, ukraine1 nidcap cincinnati training center, cincinnati children’s hospital medical center, cincinnati, ohio, usa2 wroclaw medical university, wroclaw, poland3 during the past few decades, advancements in the perinatal and neonatal intensive care have led to a significant survival of premature infants. however neurodevelopmental outcomes remain the major issues of concern. developmental care is an approach that aims to reduce the mismatches between the extraand intrauterine environment, decrease the stress of preterm newborns in neonatal intensive care units, and thus promote optimal neurobehavioral development of the infant. the newborn individualized developmental care and assessment program (nidcap) model was developed as a clinical framework for the implementation of developmental care. the model focuses on detailed reading of each individual infant’s behavior. by observing the infant during a routine assessment (before, during and after) and a detailed description of his/her behavioral responses, a professional can assess the ability of the infant’s immature nervous system to tolerate the environment and care manipulations. such evaluation will enable one to determine the adequacy of the environment and care to meet the infant’s opportunities and needs, with subsequent corrections and adaptation. nidcap’s aim is to support the infant in his/her interaction with the environment as an active participant in caregiving within a family. with the help of nidcap approaches in neonatal care medical staff will study how to read and interpret infants’ behavior, thus hearing their voices. key words: developmental care, nidcap, preterm infants. introduction today the number of premature births is increasing. according to the world health organization (2012), every year about 15 million babies are born prematurely – more than one in 10 of all babies born around the world, affecting the families around the world. in developed countries of europe and usa the incidence varies from 5 to 12% [1, 2, 3], and may reach 40% in less developed regions [2]. during the past few decades, advancements in the perinatal and neonatal intensive care (the use of antenatal corticosteroid therapy, surfactant therapy, and improved ventilatory techniques) have led to a significant survival of premature infants with gestational age less than 32 weeks [4]. the percentage of newborns with gestational age less than 32 weeks (extremely preterm and very preterm ones) is 1–2% in developed countries [1]. however, despite the positive results of survival, morbidity and complications associated with premature birth are growing rapidly [2, 3]. preterm babies meet with a large number of negative factors, since the beginning of their extra uterine life passes in the nicu (neonatal intensive care unit) environment, where such factors as light, noise, highly advanced noninvasive and invasive manipulation and pain dramatically affect the whole immature organism, resulting in early short-term and long-term complications. neurodevelopmental outcome has become a benchmark in determining the effectiveness of medical neonatal care [5]. the incidence of disability and neurodevelopmental problems among survivors of neonatal intensive care remains high and problematic [2, 6, 7]. severe neurological complications (cerebral palsy, moderate to severe mental retardation, epilepsy, poor motor skills, sensorineural hearing loss, blindness) still remain consistently high. moreover, by the age of 8 years, over 50% of very low birth weight preterm children require special 27 p e d ia t r ic s issn 2413-6077. ijmmr 2017 vol. 3 issue 1 i. m. sarapuk et al. educational services and 15% have repeated at least one grade in school [3, 8, 9]. however, these are not the only neurological problems. the implementation of extended follow-up monitoring, using modern methods and scales of neurological evaluation allow us to identify moвшіre and more neuro-developmental disorders in children. these disorders include learning disabilities, low average iq scores, attention deficit, hyperactivity disorders, neuropsychological deficits, visual motor disintegration, executive function difficulty, varying temperament difficulties, language delays, emotional problems and regulatory disorders [5, 8]. 50–70% of very low birth weight premature infants suffer from these dysfunctions [3, 10]. in order to provide not only survival, but to optimize long-term health and development, it is necessary to know the features of brain development of the fetus in the intra-uterine environment and of the premature baby in the extrauterine space. the development of the brain of the full-term infant takes place in utero where the infant resides in a temperature-stable environment, receives all the nutrients, undergoes circadian rhythm, and most importantly, feels maternal presence and permanent protection [11, 12]. prematurely born infants are deprived of all these favorable conditions in the most critical period when their brain is growing and developing most intensively [13]. at the same time, they are exposed to excessive manipulation in a high-technology medical nicu environment. the mismatch between the preterm infants’ need that are necessary for brain development, and the realities of intensive care units may cause serious problems of neuro-physiological, psycho-emotional and psychosocial development. developmental care is an approach that uses a range of medical and nursing interventions that aim to reduce the mismatches between extraand intra-uterine environment, decrease the stress of preterm neonates in nicus, and thus promote optimal neurobehavioral development of the infant. review the framework of developmental care views preterm infants as fetuses who find themselves too early and unexpectedly in a technologic hospital environment instead of the evolutionarily promised mother’s womb [3, 14]. behavioral individuality of each infant is the basis of neuro-developmental care. each infant is seen as an active participant in all elements and at all stages of care. an infant's behavior provides the best information to determines the care management [14, 15]. the newborn individualized developmental care and assessment program (nidcap) model was developed as a clinical frame work for the implementation of developmental care. nidcap is a comprehensive program which includes a behavioral observation methodology and creation of individual developmental caregiving support of the infant's own developmental goals [12]. the model focuses on detailed reading of each individual infant’s behavior [2]. by observing the child during a routine assessment (before, during and after) and a detailed description of his/her behavioral responses, medical staff can assess the ability of the infant’s immature nervous system to tolerate the environment and care manipulations. such evaluation will enable one to determine the adequacy of the environment and care to infant’s opportunities and needs, with subsequent corrections and adaptation [2, 15]. the infant’s ability to regulate and control the behavior occurs within continuous interaction with the environment (whether womb, nicu, home conditions) and is expressed through the infant's five systems: autonomic (physiological), motor, state, attention/ interaction and self-regulation. these systems are continuously interacting with one another. each system influences and supports the other, and interacts with the environment. the autonomic system functioning is manifested in an infant’s breathing quality and rate, color fluctuation, and visceral stability or instability. the functional state of motor system is estimated by the position, muscle tone and movement patterns of the face, trunk and extremities. state organization is observable in an infant’s range of available levels of consciousness, their robustness and modulation of the available states, and the patterns of transition from state to state. the acti vity of attention/interaction system is manifested by infant’s look, facial expressions and the ability to interact [3, 12, 15]. the self-regula tion system is manifested in infant’s ability to re-balance and bring into harmony the other sys tems when they have moved out of balance [2]. nidcap methodology was founded by heidelise als (psychologist from the harvard university) in 1986 and is based on the synactive theory of development. heidelise als has been a pioneer in helping medical staff and parents to understand how to “read” preterm infant's 28 p e d ia t r ic s issn 2413-6077. ijmmr 2017 vol. 3 issue 1i. m. sarapuk et al. behavior, especially those who need intensive care [16]. the synactive theory of newborn behavioral organiza tion and development (synaction n., or synactive adj. (from the greek syn “together” and the latin actio “action,” resulting in “together in action”). the synactive theory of newborn behavioral organization and development suggests that development of the human fetus, and later newborn, proceeds through the constant balancing of approach and avoidance behaviors, leading to a continuous interaction of the above mentioned subsystems and their increasingly defined delineations within the organism, and the organism’s interaction with the environment at large [12, 17]. the synactive theory of infant development provides that there is no possibility for development without stimulation, but there is also no harmonious development if the stimuli lead to disorganization and stress [15]. sensory inputs are the important and necessary parameters in fostering central nervous system development [3]. inappropriate stimuli penetrate into all systems and destroy them, while adequate and timely stimuli promote and improve the infant’s growth and development [11, 15]. the infant’s response to any stimulus manifests in signs of adaptation of the foregoing systems when he or she tolerates it, or in maladaptive signs if the infant is unable to cope. the infant is able to tolerate stimuli when they are appropriate in timing, complexity and intensity in relation to the infant’s thresholds of functioning. if the stimuli are too intense, complex or inappropriately in time, infant either has strategies to move away from inputs, avoid them, or suffer from them. such behavior is considered a stressful situation for an infant [2, 12]. the aim of the nidcap is to support the infant in his/her growing tolerance to stimuli, and to minimize stressful events and manipulations that require energy consumption, use of calories and sometimes break the physiological homeostasis [3]. each caregiver must recognize that for the unstable or fragile infant the best form of sensory input may necessitate a plan of reduction of surrounding stimuli [18]. signs of autonomic system adaptation in the infant are: stable color, regular breathing and heart rate within physiological ranges and stable digestion. self-regulatory balance of motor system is reflected by the appropriate muscle tone, harmonious and smooth movements, softly flexed posture with flexed arms and legs; hands near the face, around the mouth or inside it [12, 15]. sleep and waking cycles develop in time to become differentiated [15, 19]. the infant actively looks for a source of stimulation and visual contact with a person who cares for him/ her, the face is open, and the infant has attentive look. this stability of the systems manifests in smooth function and reflects intact organization and central nervous system control [3]. signs of autonomic system stress in the infant are: skin discoloration (pale, marble, red, cyanotic), breathing disorders (apnea, tachypnea) with or without desaturation, unstable heartbeat (bradycardia or tachycardia), functional gastrointestinal disorders (hiccups, regur_ gitation, vomiting) and neurological disorders (tremors, convulsions, yawning, sighing). motor signs of stress include: muscle hypotonia, extension of the trunk (arching), ourward movements of limbs (splaying, airplanning, saluting, sitting on air) and tongue extensions. sleep and waking cycles are not proper differentiated, with the infant avoiding stimulation and visual contact, there is lack of visual concentration. in general, extension behaviors are thought to reflect stress, and flexion behaviors are thought to reflect self-regulatory competence. diffuse behaviors are thought to reflect stress, and well-defined behaviors are thought to reflect regulatory balance [12, 15]. such stress reactions may indicate that environmental stimuli of light or noise or even caregiving events such as turning the infant or changing the diaper, may be overwhelming [3]. to overcome these stressful situations infant spends the energy that he needs to maintain his homeostasis [3, 15]. another behavioral scenario is possible when the infant cannot respond to environment or handling stimuli. the infant may lie in the bed, limp and flaccid, unable to put any energy into responding to caregiver; or just develop minimal ranges of responsiveness [15, 17]. the ability to regulate or control the infant’s autonomic, motor and state organization is a requirement of early development and it is called self-regulation [20]. early self-regulation is accomplished in the mother's body as a fetus, and continues over the course of the first three years of life after birth. the first tasks of selfregulation involve the regulation of physiological function as breathing, heart rate regulation, maintenance of visceral control, body temperature and homeostasis and day–night cycles regulation. later the infant learns to calm himself and relax even after mild stress. as an infant matures in the context of daily interactive care from consistent primary caregivers, self regula29 p e d ia t r ic s issn 2413-6077. ijmmr 2017 vol. 3 issue 1 i. m. sarapuk et al. tion becomes the task of learning to control motor behaviors, sleep–wake cycles, attention and interaction [3, 15, 20, 21]. eventually, these capacities merge into emotional regulation. the premature baby often needs external assistance in self-regulation. parents and caregivers become the infant's “co-regulators” and support infants by accurately reading and interpreting the infant's behavioral signals [3]. how assess and interpret the child's behavior according to nidcap? developmental specialists observe an infant’s behavior weekly or every other week throughout the hospitalization, starting with the phase of the infant’s initial stabilization. during each observation, the developmental specialist systematically records an infant’s behavior for approximately 20 minutes before a planned medical or nursing caregiving interaction and continues to observe throughout the duration of the interaction and for approximately 20 minutes after it. ninetyone behaviors, including autonomic (breathing, heart rate, color changes and visceral signs), motor (postures, muscle tone fluctuations and movements) and state organization behaviors (levels of arousal, patterns of transitions between states, and clarity and robustness of sleep and awake states) are monitored every 2 minutes. behaviors are conceptualized as regulatory and as stress according the foregoing signs of adaptation and disadaptation and are interpreted as indices of the infants’ current strength and vulnerabilities respectively [14]. after the observation the developmental specialists write descriptive neurobehavioral reports and suggestions, which describe the infant’s strengths, current sensitivities and apparent goals and thresholds to stress, and the infant’s self-regulatory efforts at rest and during care manipulations. it helps to structure caregiving procedures to the infant’s sleep/ wake cycle, adapt or modify the environment and care according to the child’s needs with the aim to maintain the infant’s well-regulated behavioral balance in an effort to promote the infant’s strengths and simultaneously to reduce the infant’s self-regulatory vulnerability [2, 14]. as the infant matures, these recommendations are modified appropriately [22]. key elements for implementation of individualized developmental family-centered care nidcap : 1) provision of sensitive care and handling for baby, 2) creation of a calm nicu environment, 3) parental involvement in infant care, their support, 4) presence of infant developmental specialist [3]. provision of sensitive care and handling for the infant based on an accurate reading of infant behavioral signals and respect for the message that the infant communicates, along with recognition that the infant actively participates in all aspects of neonatal care [3]. а) structure the infant’s 24-hour day in accordance with the individual infant’s sleep-wake cycles, state of alertness, medical needs and feeding competence. it may be necessary to divide or to combine procedures (eg. medical examination and care manipulations), to offer pauses according to the child’s behavioral responses or to perform care with two persons. this approach provides infant’s tranquility and thus promotes his/her growth [15]. b) non-nutritive sucking suppors infant’s physiological stability, improves sleep/waking cycle and reduces time of hospitalization [23]. c) infant’s flexed position reduces stress reactions, improves self-regulation, physiological stability and sensory stimuli tolerance and prevents the skeletal deformities [24, 25]. d) positioning in the “nests” provides flexed position of the whole body with pelvic support (physiological position of the fetus), which prevents the formation of pathological extension position and hypertonus [24, 25]. e) swaddling reduces physiological distress, maintains temperature homeostasis, improves motor development and optimizes the sleep/ waking cycle [26]. creation of a calm nicu. it is necessary to create a supportive, gentle environment for the most fragile infants (pic. 1, 2) а) noise reduction (reduction of alarm volume, decreased ringtone volume of telephones, modification of individual and collective behaviors – quiet conversation, avoiding the noisy gestures, individual rooms, installation of a sound measurement device. sound levels shall pic. 1. incubators in the nicu are covered to reduce the light and noise, to simulate day-night cycle. 30 p e d ia t r ic s issn 2413-6077. ijmmr 2017 vol. 3 issue 1i. m. sarapuk et al. not exceed 45 db and hourly l10 of 55 db; transient sounds should not exceed 70 db; equipment in nicu should have noise levels of 40 db [27, 28]. b) reduction of the light level (avoiding direct illumination into the infants’eyes, except during examination, incubator cover adapted to the infant's awakening, individual illumination lamps at each incubator, providing circadian rhythm with decreasing light intensity at night, access to daylight) [3, 27]. parental involvement in infant care, their support. а) early parental involvement in caregiving and participation in all care with day-and-night access in the nicu [15]. b) skin-to-skin contact with mother or father, including children who need invasive and non-invasive respiratory support. such contact has significant advantages for both the infant (temperature homeostasis, respiratory stability with a decrease in apnea and bradycardia, calm and lasting sleep, feeling of safety, positive dynamics of body weight, decreased risk of nosocomial infections) and mother (improving of lactation, stress reducing, sense of paternity) [15, 29, 30], (pic. 3, 4, 5, 6, 7). pic. 2. the device for monitoring the volume of sounds in hospital wards (green – normal, yellow – loud, red – noice). pic. 3. kangaroo mother care (“skin-to-skin”contact) of the preterm infant with ncpap. pic. 4. kangaroo mother care of the elbw infant with ncpap. pic. 5. kangaroo mother care of the twins. 31 p e d ia t r ic s issn 2413-6077. ijmmr 2017 vol. 3 issue 1 i. m. sarapuk et al. developmental care supports the relationship between the child and the family [12]. for the human species, parents are the familiar, constant and steady connection in an infant’s life. they assure their infant’s safety, and assure their child’s development of trust and as a whole person [2]. the core principle of nidcap is the collaboration with each infant and the infant’s parents as the primary caregivers and co-regulators; and the main goal of developmental care in the nicu and throughout the hospital stay is to improve child and family comfort and outcomes. the presence of infant developmental specialist. the developmental specialist conducts regular nidcap observations and staff/parent teaching, provides consistency of developmental support to medical staff, family and the infant, articulates and adjusts the nicu in the accordance with the developmental needs of the infant [3]. positive aspects of developmental care. the studies have documented the beneficial effect of nidcap in terms of shorter intensive care and overall hospital stay, decreased time on the ventilator, decreased oxygen days, shorter duration of parenteral feeding, shorter transition periods to full enteral feeding, better average daily weight gain, lower total hospital charges and effect in enhancing neurodevelopmental outcome [11, 314]. some research has also documented that the nidcap approach enhances brain structure and function when measured by sophisticated medical techniques such as eeg and mri [35]. nidcap also has significant benefits for parents. the studies of h. als and other developmental specialists show the reduction in parenting stress and enhanced parent perception of the infant [31, 36, 37]. except for shortterm positive effects, neonatal care according the nidcap has also long-term effects on the infant’s neurological development, behavior and mother–child interaction [34, 36]. conclusions as survival of high risk infants has continued to improve, greater emphasis has now been placed on improving neurological outcomes and quality of life. the implementation of individualized developmental care encourages the shift from a protocol-based task and schedule oriented framework of nicu care to an individualized and relationship-based framework of care. the introduction of nidcap in the neonatal departments will reduce the mismatch between intra-uterine environment and nicu by taking into account the individual infant’s current thresholds of behavioral organization, diminishing stress, and supporting each infant’s strengths and competencies. with the help of nidcap approaches in neonatal care all staff will study how to read infants’ behavior, to hear their voices and understand them. our smallest and most fragile patients should feel comfort and a sense of security that are so important for their healthy development. 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structure in preterm infants with severe intrauterine growth restriction journal of perinatology. 2012;32:797–803. 36. kleberg a, westrup b, stjernqvist k. developmental outcome, child behaviour and mother–child interaction at 3 years of age following newborn individualized developmental care and intervention program (nidcap) intervention. early human development. 2000;60:123–135. 37. kleberg a, hellstrom-westas l, widstrom a. mothers’ perception of newborn individualized developmental care and assessment program (nidcap) as compared to conventional care. early human development. 2007;83:403–411. i. m. sarapuk et al. 5 in t e r n a l m e d ic in e issn 2413-6077. ijmmr 2018 vol. 4 issue 2 doi 10.11603/ijmmr.2413-6077.2018.2.9870 kidney lesions in hiv-infected patients м. о. andrushchak bukovinian state medical university, chernivtsi, ukraine introduction. hiv prevalence is one of the most important issues of contemporary medicine. over a 30-year history of this disease more than 75 million people have been infected with hiv, nearly 30 million adults and children of died. in the future decades, its significance in world premature mortality rates continues to rise. the objective of the study was to establish clinical and laboratory features of kidney lesions in hiv infection. methods. the study involved 292 hiv-infected patients, who were managed outpatiently at the chernivtsi regional aids center. taking into account the main markers of kidney lesions: persistent proteinuria and glomerular filtration rate <60 ml/min/1.73 m2, 48 persons were diagnosed with chronic kidney disease (ckd), which was very frequently accompanied by dysfunction of these organs. results. increasing proteinuria rate is accompanied by a significant renal dysfunction and more frequently is combined with arterial hypertension as well as hematuria without significant differences in the incidence of opportunistic diseases. the mean reciprocal correlation between the levels of proteinuria and glomerular filtration rate (r=-0.562, p<0.01), as well as between the levels of proteinuria and hemoglobin (r=-0.596, p<0.01) have been established as well. conclusions. kidney lesions in hiv-infected are most often characterized by tubulointerstitial lesions. at the same time, glomerular kidney lesion, which is much less common, is accompanied by a significantly higher level of hiv rna. key words: hiv-infection; chronic kidney disease; tubulo-interstitial lesion; glomerular lesion of kidneys. introduction hiv prevalence is one of the most important issues of contemporary medicine. over a 30year history of this disease more than 75 million people have been infected with hiv, nearly 30 million adults and children died [1, 2]. in the future decades, its significance in world premature mortality rates continues to rise. the kidneys lesion, which is often characterized by severe clinical manifestations, can significantly affect the life expectancy in hivinfected patients [3, 4]. considering the increasing number of hiv-infected people in the world and a rise in the life expectancy of such patients, an increase in the number of hivinfected people in need of expensive substitution renal therapy as well as kidney transplantation is expected. the world scientific literature points out the factors associated with renal impairment in hiv infection: history of kidney disease, uncontrolled hiv infection, time spent on haart, older age, female sex, african origin (apol1 genetic variant), cd4+ lymphocytes <200 cells/ml, as well as the use of nephrotoxic drugs [5]. however, despite a large number of foreign publications concerning this topic, the issue of the kidney lesion in hiv infection is studied insufficiently in ukraine. the objective of the study was to establish clinical and laboratory features of kidney lesions in hiv infection. methods the study involved 292 hiv-infected patients, who were managed outpatiently at the chernivtsi regional aids center (chief physician v. m. mochulskyi). in establishing the diagnosis, clinical and epidemiological data as well as findings of the laboratory examination methods: serological and immunological (including determination of cd4+-lymphocyte contents), were taken into account. the initial screening of hiv-infected people was carried out, when they were registered for monitoring in accordance with the clinical protocol no. 551, dated july 12, 2010. the average age of all patients was (29.3±8.2) years (ranged from 19 to 55 years). there were 188 (64.4%) men and 104 (35.6%) international journal of medicine and medical research 2018, volume 4, issue 2, p. 5-12 copyright © 2018, tsmu, all rights reserved corresponding author: margaryta andrushchak, bukovinian state medical university, chernivtsi, ukraine phone number +380996019597 e-mail: margaritaassistent@gmail.com м. о. andrushchak 6 in t e r n a l m e d ic in e issn 2413-6077. ijmmr 2018 vol. 4 issue 2 women among the patients. the study mostly involved young patients (25-44 years old). among the patients, who were included in the study, 26 (8.9%) were diagnosed with the first stage of hiv infection, 40 (13.7%) individuals – with the second one, 108 (37.0%) patients – with the third, and 118 (40.4 %) were diagnosed with the fourth clinical stage of the disease. the screening of kidney lesion markers with albuminuria/proteinuria test systems by means of urinary strips (aution sticks-2ea) was performed. with the presence of proteinuria ≥1+ in the screening test, corresponding to a gradation of 30 mg/l, repeated urinalysis was performed with a quantitative protein deter mination by means of mikrolab-600 spec trophotometer using uni-test-bm reagents separated in the period from 3 days to one week. the functional status of the kidneys was evaluated by the integral index, which characterized the degree of active nephrons mass maintenance/loss. a decrease in glomerular filtration rate (gfr) <60 ml/min per 1,73 м2 was a cri terion of renal dysfunction [6, 7]. chronic kidney disease was diagnosed when proteinuria or that in combination with a decrease in gfr for 3 months or more was revealed. a screening study to identify the kidney lesion markers (permanent proteinuria, reduction in gfr that was detected for 3 or more months) in hiv-infected patients was conducted in accordance with the recommendations of the kidney disease outcome quality initiative, k / doqi, 2002, and infectious diseases society of america, idsa, 2005 [4, 7]. among the surveyed patients there were 105 (36.0%) people with markers of kidney lesion: albuminuria/ proteinuria. based on the main markers of kidney lesion: persistent proteinuria (pu) and gfr <60 ml/min/1.73 m2, in 16.4% of cases chronic kidney disease (ckd) was diag nosed, which was very frequently accompanied with renal impairment. hiv-associated nephropathy was revealed in 48 out of 292 (16.4%) patients (31 men and 17 women), in whom the markers of kidney lesion: persistent proteinuria or proteinuria combined with a decrease in gfr, were iden tified and confirmed in the course of the examination. statistical processing of the received data was carried out using the package of applications statistica 6.1 (statsoft, usa) and microsoft excel 2007 programs. the normal dissemination of the signs was determined by the graphical method, the lilliefors criterion and the w-criterion of shapiro-wildlife. dispersion of attributes was evaluated using the f-criterion in the anova dispersion analysis procedure. to describe the selective normal distribution of quantitative attributes, the arithmetic mean (m) and standard deviation (m) were calculated. if the dissemination of the sign differed from normal, for its description the median (me) and the interquartile scale with the boundaries of the segment [25%; 75%] was developed. when comparing several independent groups, the crackel-wallis dispersion analysis was used (to avoid multiple comparisons). nonparametric methods were used to compare two independent groups: the mann-whitney u-test and the kolmogorov-smirnov test, and the two dependent groups were for the wilcoxon criterion. the correlation analysis of two quantitative attributes was carried out using spierman’s rank method: the relationship between the indicators was considered weak in case r<0.3, moderate – at 0.3<r<0.7, strong – at r>0.7. the comparison of groups by qualitative features was carried out by nonparametric method through analyzing 2×2 conjugation tables using a two-sided exact fisher or χ2 for unrelated groups. multivariate logistic regression analysis was used to identify the predictors of kidney impairment. statistical differences were significant at p<0.05, very significant at p<0.01, the most significant at p<0.001 and insignificant at p>0.05. when describing qualitative signs, the percentage of patients with the presence or absence of the analyzed sign from the total number of patients in the group is presented. the results of studies, processed statistically and presented in tables or diagrams, allow establishing the dynamics of the parameter, reliability, as well as the relationship with the changes in other parameters in accordance with existing requirements. results 48 hiv-infected patients with kidney lesion had the following distinctive clinical symptoms and syndromes of ckd: ȃ urinary syndrome characterized by isolated proteinuria of varying degrees, by proteinuria in combination with hematuria/leukocyturia; ȃ arterial hypertension (ah); ȃ acute nephritic syndrome; ȃ nephrotic syndrome; м. о. andrushchak 7 in t e r n a l m e d ic in e issn 2413-6077. ijmmr 2018 vol. 4 issue 2 ȃ chronic renal insufficiency. it was established that in every fourth hivinfected person with ckd the urinary syndrome was characterized by isolated pu (27.1%). pu was most often combined with changes in the urine sediment: erythrocyturia and leukocyturia (17 persons – 35.4%) or hematuria (14 patients – 29.2%), with the latter most often accompanied by pu>1.0 g/day compared with the group of patients with a lower level of protein in the urine (90.5 and 51.9% respectively, p<0.01). in 4 patients (8.3%) pu was combined with leukocyturia. it should be noted that in more than half of patients transient non-bacterial leukocyturia was evidenced – more often at pu≤1.0 g/day. ah was diagnosed in 15 patients (31.3%) in the presence of proteinuria compared with 2.5% in its absence (p<0.001). acute nephritic syndrome was revealed in 5 patients (10.4%), nephrotic syndrome – in 7 (14.6%), reduction of gfr<60 ml/min/1.73 m2 – in 23 individuals (47.9%). according to the analysis of complaints, anamnestic information and clinical symptoms of kidney lesion, the patients were divided into 2 groups. the first group consisted of 31 (64.6%) out of 48 persons with tubulointerstitial and the second one – of 17 (35.4%) patients with glomerular diseases (table 1). the presented data confirm that hiv-infected kidney lesions are most often characterized by tubulointerstitial lesion. chronic tubulointerstitial diseases of kidneys were characterized by a minimal or insignificant pu (0.4 [0.3; 0.8] g/day) and only in 4 (12.9±6.0)% of patients it exceeded 1 g/day. pu only was evidenced in 9 – (29.0±8.1)% of cases, but in most people pu was combined with changes in urine sedimentation. for instance, pu was accompanied by hematuria, manifested by isomorphous erythrocytes and leukocyturia in 8 (25.8±7.9)% of patients, hematuria – in 2 (6.5±4.4)% and leukocyturia – in 4 (12.9±6.0)% of cases. in tubulointerstitial diseases, in comparison with the glomerular pathology of kidneys, the renal function impairment was diagnosed much less frequently (32.3±8.4) against (76.5±10.3) (p<0.01), as well as ah – (9.7±5.3) and (70.6±11.0)% respectively (p<0.001). glomerular kidney lesion was characterized by a significantly lower glomerular filtration rate – 48.7 [30.2; 78.9] vs. 84.5 [52.6; 107.2] ml/ table 1. clinical characteristics of patients with different variants of kidney damage criterion damage to the kidneys all patients (n=48) tubulointerstitial diseases (n=31) glomerular diseases (n=17) gfr, ml/min/1.73 m2, median [25%; 75%] 84.5 [52.6; 107.2] 48.7 [30.2; 78.9]* 60.2 [34.4; 80.3] ≥90, n (m%±m%) 15 (48.4±9.0) % 1 (5.9±5.7) %* 15 (31.3±6.7) % 60-89, n (m%±m%) 8 (25.8±7.9) % 3 (17.6±9.2) % 10 (20.8±5.9) % 30-59, n (m%±m%) 7 (22.6±7.5) % 8 (47.1±12.1) % 16 (33.3±6.8) % 15-29, n (m%±m%) 1 (3.2±3.2) % 2 (17.6±9.2) % 3 (6.3±3.5) % <15, n (m%±m%) 0 (0.0±0.0) % 3 (17.6±9.2) % 4 (8.3±4.0) % renal impairment, n (m%±m%) 10 (32.3±8.4) % 13 (76.5±10.3) %* 22 (45.8±7.2) % proteinuria, g/day, median [25%; 75%] 0.4 [0.3; 0.8] 1.3 [1.4; 3.0]* 0.8 [0.34; 1.42] ≤1 g / day, n (m%±m%) 27 (87.1±6.0) % 0 (0.0±0.0) %* 27 (56.3±7.2) % >1 g / day, n (m%±m%) 4 (12.9±6.0) % 17 (100.0±0.0) %* 21 (43.8±7.2) % isolated proteinuria, n (m%±m%) 9 (29.0±8.1) % 3 (17.6±9.2) % 12 (25.0±6.3) % proteinuria and hematuria, n (m%±m%) 2 (6.5±4.4) % 11 (64.7±11.6) %* 14 (29.2±6.6) % proteinuria, hematuria, leukocyturia, n (m%±m%) 8 (25.8±7.9) % 9 (52.9±12.1) %* 17 (35.4±6.9) % proteinuria and leukocyturia, n (m%±m%) 4 (12.9±6.0) % 0 (0.0±0.0) %* 4 (8.3±4.0) % acute nephritis syndrome, n (m%±m%) 0 (0.0±0.0) % 5 (29.4±11.0) %* 5 (10.4±4.4) % nephrotic syndrome, n (m%±m%) 0 (0.0±0.0) % 7 (41.2±11.9) %* 7 (14.6±5.1) % arterial hypertension, n (m%±m%) 3 (9.7±5.3) % 12 (70.6±11.0) %* 15 (31.3±6.7) % hemoglobin, g/l, median [25%; 75%] 124.0 [112.5; 133.0] 99.1 [83.0; 123.6]* 111.5 [85.5; 131.0] notes: * – significant difference between the groups of patients with glomerular and tubulointerstitial diseases (p<0.05-0.001). м. о. andrushchak 8 in t e r n a l m e d ic in e issn 2413-6077. ijmmr 2018 vol. 4 issue 2 min/1.73 m2 (p<0.05). accordingly, only 1 person with glomerular lesion had gfr higher than 90 ml/min/1.73 m2, which was significantly lower than the corresponding frequency of this feature in tubulointerstitial pathology – (48.4±9.0)% (p<0.001). at the same time, the final stage of ckd was in 3 (17.6±9,2)% of patients, 2 of whom were recommended substitution renal therapy by hemodialysis program. in cases of glomerular kidney lesion there was also a significantly higher level of pu – 1.3 [1.4; 3.0] vs. 0.4 [0.3; 0.8] g/day (p<0.05). for instance, it exceeded 3.0 g/day in 8 patients and reached 8.0 and 9.0 g/day in 2 of them. the combination of pu with hematuria (64.7±11.6) and (6.5±4.4)%, respectively (p<0.001), with hematuria and aseptic leukocyturia (52.9±12.1) and 25.8±7.9)%, respectively (p<0.05) were present much more frequently than in the patients with tubulointerstitial diseases. thus, in the majority of cases there was micro hematuria, manifested by dysmorphic erythrocytes, whereas episodic macrohematuria was evidenced in 2 patients. 7 (41.2±11.9)% patients were diagnosed with nephrotic and 5 (29.4±11.0)% people suffered from acute nephritic syndromes. it is noteworthy, that these syndromes were not revealed in any representative of the group with tubulointerstitial disease. expectedly, the level of hemoglobin in glomerular kidney lesion was reliably lower: 99.1 [83.0; 123.6] vs. 124.0 [112.5; 133.0] g/l (p<0.05). table 2. number of rnas of hiv, cd4+ lymphocytes and the ratio of cd4+/cd8+ in the patients with different variants of clinical kidney damage criterion damage to the kidneys all patients (n=48) tubulointerstitial diseases (n=31) glomerular diseases (n=17) viral load (rna of hiv), copies/ml 22 000 [5 125; 308 000] 250 000 [35 225; 690 500]* 135 000 [14 027; 460 000] hiv rna was not detected, n, (m±m, %) 1 (3.2±3.2) % 2 (11.8±7.8) % 4 (8.3±4.0) % ≤100 000, n (m±m, %) 11 (35.5±8.6) % 8 (48.1±12.1) % 18 (37.5±7.0) % >100 000, n (m±m, %) 19 (61.3±8.7) % 7 (41.2±11.9) % 26 (54.2±7.2) % cd4+ (median [25 %; 75 %]) 220.4 [34.6; 280.5] 197.5 [54.3; 309.0] 185.5 [60.9; 318.0] ≤200, n (m±m, %) 16 (51.6±9.0) % 8 (47.1±12.1) % 25 (52.1±7.2) % 201-350, n (m±m, %) 8 (25.8±7.9) % 5 (29.4±11.0) % 13 (27.1±6.4) % >350, n (m±m, %) 7 (22.6±7.5) % 4 (23.5±10.3) % 10 (20.8±5.9) % correlation cd4+/cd8+ (mediana [25 %; 75 %]) 0.2 [0.1; 0.5] 0.2 [0.1; 0.4] 0.2 [0.1; 0.4] duration of hiv infection, years, (mediana [25 %; 75 %]) 5.0 [3.5; 8.0] 6.0 [2.5; 7.5] 5.5 [3.0; 8.0] notes: * – significant difference between the groups of patients with glomerular and tubulointerstitial diseases (p<0.05-0.001). this may point to the direct effect of hiv on the glomerular apparatus, whereas tubulointerstitial kidney lesions are most likely due to the influence of opportunistic infections and drugs with nephrotoxic potential, as well as the use of psychotropic drugs and the uncontrolled administration of nonsteroidal anti-inflam matory drugs, which these patients often abuse of. the mean number of cd4+ lymphocytes in serum of the patients with proteinuria is much lower than in the hiv-infected individuals without markers of kidney lesion: 185.5 [2560.9; 75% – 318.0] vs. 312.0 [25% – 175.5; 75% – 469.0] cl/μl respectively (p<0.05). a decrease in cd4+ lymphocytes level ≤200 cl/μl was found in 52.1±7.2% of patients with proteinuria and in 30.0±7.2% of those without it (p<0.05). the difference between the ratios of cd4+/cd8+lymphocytes in the studied groups was also quite significant: 0.2 [0.1; 0.4] and 0.4 [0.2; 0.6] respectively (p<0.05) (table 2). the data concerning the hiv rna level and type of ckd are presented at fig. 1. depending on the level of proteinuria, the patients were divided into two groups. the first group consisted of 27 out of 48 (56.3%) patients with pu less than 1.0 g/day, the second group – 21 (43.7%) patients with pu more than 1.0 g/day, in 7 of them it reached the nephrotic level – more than 3.0 g/day. there were more males in both groups (70.4±8.4) and (57.1±10.9)% respectively, and people aged 25-44 (66.7±9.1) and (61.9±10,6)% м. о. andrushchak 9 in t e r n a l m e d ic in e issn 2413-6077. ijmmr 2018 vol. 4 issue 2 fig. 1. hiv rna level in the hiv-infected patients with different variants of kidney damage. fig. 1. hiv rna level in the hiv-infected patients with different variants of kidney damage. depending on the level of proteinuria, the patients were divided into two groups. the first group consisted of 27 out of 48 (56.3%) patients with pu less than 1.0 g/day, the second group – 21 (43.7%) patients with pu more than 1.0 g/day, in 7 of them it reached the nephrotic level – more than 3.0 g/day. there were more males in both groups (70.4 ± 8.4) and (57.1 ± 10.9)% respectively, and people aged 25-44 (66.7 ± 9.1) and (61.9 ± 10,6)% respectively, the same as in the total number of hiv-infected people. table 3. frequency of opportunistic diseases in the hiv-infected patients with different levels of proteinuria opportunistic infections proteinuria level р ≤1 g/day (n=27) >1 g/day (n=21) n m±m, % n m±m, % bacterial 3 11.1±6.0 2 9.5±6.4 >0.05 viral 5 18.5±7.5 7 33.3±10.3 >0.05 fungal 14 51.9±9.6 11 52.4±10.9 >0.05 parasitic 3 11.1±6.0 2 9.5±6.4 >0.05 tuberculosis 4 14.8±6.8 6 28.6±9.9 >0.05 250000 680000 -100000 400000 900000 1400000 h iv r n a , c op ie s/ m l tubulointerstitial nephropathies glomerular disease respectively, the same as in the total number of hiv-infected people. according to table 3, in both groups of patients, fungal diseases were the most common (51.9±9.6) and (52.4±10.9)% respectively, the same as the diseases of viral etiology – (18.5±7.5) and (33.3±10.3)%, respectively, without significant differences in their incidence (p>0.05). clinical description of the hiv-infected patients with different levels of proteinuria is presented in table 4. according to table 4, the hiv-infected patients with a level of pu more than 1 g/day were much more frequently diagnosed with arterial hypertension (52.4±10.9) versus (14.8±6.8)% in the patients with proteinuria not exceeding the indicated level (p<0.01). thus, the relationship between the level of pu and the presence of arterial hypertension was established. it should be noted that according to the level of blood pressure, the patients in the groups were distributed as follows: the first degree ah was diagnosed in 3 (11.1%) patients of the first group and in 3 (14.3%) patients of the second group; the second degree ah was revealed in 2 (7.4%) and 4 (19.0%) patients respectively, and the third degree ah was only found in one (3.7%) patient of the first group and in 3 (14.3%) persons with proteinuria >1 g/day. the incidence of hypercholesterolemia and hypoalbuminemia in the comparable groups was approximately the same (p>0.05). at the same time, the level of hemoglobin (99.0 [78.0, 123.0]) (median [interquartile scale]) was expectedly much lower in the patients with a higher level of pu vs. (124.0 [93.0; 139.0]) g/l (p<0.05), the incidence of hematuria was much higher as well (90.5±6.4) vs. (51.9±9.6)% (p<0.01). it is noteworthy that with the increase in pu the gfr levels decreased significantly from (72.0 [38.3; 99.6]) to (48.3 [30.5; 61.8]) ml/ min/1.73 m2 (p<0.01), and only in the group of patients with pu less than 1 g/day the gfr did not drop off below 30 ml/min/1.73 m2 (fig. 2). there were statistically significant intergroup differences in the severity of kidney lesion, depending on the level of proteinuria. for instance, the preserved renal function (gfr≥90 ml/min/1.73 m2) was more frequently evidenced table 3. frequency of opportunistic diseases in the hiv-infected patients with different levels of proteinuria opportunistic infections proteinuria level р≤1 g/day (n=27) >1 g/day (n=21) n m±m, % n m±m, % bacterial 3 11.1±6.0 2 9.5±6.4 >0.05 viral 5 18.5±7.5 7 33.3±10.3 >0.05 fungal 14 51.9±9.6 11 52.4±10.9 >0.05 parasitic 3 11.1±6.0 2 9.5±6.4 >0.05 tuberculosis 4 14.8±6.8 6 28.6±9.9 >0.05 fig. 2. correlation among the levels of proteinuria and glomerular filtration rate. the incidence of hypercholesterolemia and hypoalbuminemia in the comparable groups was approximately the same (p>0.05). at the same time, the level of hemoglobin (99.0 [78.0, 123.0]) (median [interquartile scale]) was expectedly much lower in the patients with a higher level of pu vs. (124.0 [93.0; 139.0]) g/l (p<0.05), the incidence of hematuria was much higher as well (90.5±6.4) vs. (51.9±9.6)% (p<0.01). it is noteworthy that with the increase in pu the gfr levels decreased significantly from (72.0 [38.3; 99.6]) to (48.3 [30.5; 61.8]) ml/min/1.73 m2 (p<0.01), and only in the group of patients with pu less than 1 g/day the gfr did not drop off below 30 ml/min/1.73 m2 (fig. 2). fig. 2. correlation among the levels of proteinuria and glomerular filtration rate. there were statistically significant intergroup differences in the severity of kidney lesion, depending on the level of proteinuria. for instance, the preserved renal function (gfr≥90 ml/min/1.73 m2) was more frequently evidenced in the patients of the 1st group (48.1±9.6)% and much less frequently in those with proteinuria >1 g/day – (9.5±6.4)% (p<0.01). on the contrary, the gfr, which corresponded to the 3rd stage of ckd, was evidenced in half of patients with proteinuria >1g/day (47.6±10.9)% and only in 18.5±7.5% of patients with pu ≤1 g/day (fig. 3). accordingly, the terminal 0 20 40 60 80 100 120 140 proteinuria ≤1 g/day proteinuria >1 g/day g fr , m l/ m in p er 1 .7 3 m 2 м. о. andrushchak 10 in t e r n a l m e d ic in e issn 2413-6077. ijmmr 2018 vol. 4 issue 2 renal insufficiency (gfr<15 ml/min/1.73 m2) was revealed only in the patients of the 2nd group, in 2 of them proteinuria exceeded 3.0 g/day. fig. 4. the morbidity with ckd in the hiv-positive patients depending on the level of proteinuria and glomerular filtration rate. the mean reciprocal correlation between the levels of proteinuria and the glomerular filtration rate (r=-0.562, p<0.01), as well as between the levels of proteinuria and hemoglobin (r=-0.596, p<0.01) have been also established. discussion thus the clinical manifestations of kidney lesion in the studied patients coincide with the typical ones for various pathologies in the general number of nephrology patients. there are numerous experiments on the study of a number of renal diseases in the hiv-infected individuals worldwide [4, 8, 9]. for instance, the studies conducted in the united states have revealed that, according to the renal biopsy, 52.7% of the patients with nephrotic pu were diagnosed with hivassociated nephropathy. they were all african americans. the high incidence of this pathology is associated with racial affiliation, as well as with a specific variant of the antigen/receptor to duffy 0 5 10 15 20 25 30 35 40 45 50 >90 60-89 30-59 15-29 <15 48,1 33,3 18,5 0 0 9,5 14,3 47,6 14,3 14,3 % ml/min per 1,73 м2 proteinuria ≤1 g/day proteinuria >1 g/day fig. 3. the morbidity with ckd in the hiv-positive patients depending on the level of proteinuria and glomerular filtration rate. table 4. clinical and laboratory characteristics of the hiv-infected patients with different levels of proteinuria indicator proteinuria level р ≤1 g/day (n=27) >1 g/day (n=21) arterial hypertension, n (m±m, %) 4 (14.8±6.8) 11 (52.4±10.9) <0.01 at systolic, mm hg (median [25%; 75%]) 135 [100; 170] 145 [110; 180] >0.05 diastolic blood pressure, mm hg (median [25%; 75%]) 90 [85; 100] 95 [90; 110] >0.05 cholesterol, mmol/l (median [25%; 75%]) 4.1 [3.2; 5.4] 4.3 [3.4; 5.8] >0.05 hypercholesterolemia, n (m±m, %) 5 (18.5±7.5) 4 (19.0±8.6) >0.05 hypoalbuminemia, n (m±m, %) 7 (25.9±8.4) 7 (33.3±10.3) >0.05 albumin, g/l (median [25%; 75%]) 36.9 [30.3; 42.8] 34.1 [28.7; 38.5] >0.05 hemoglobin, g/l (median [25%; 75%]) 124.0 [93.0; 139.0] 99.0 [78.0; 123.0] <0.05 hematuria, n (m±m, %) 14 (51.9±9.6) 19 (90.5±6.4) <0.01 gfr, ml/min /1.73 m2 (median [25%; 75%]) 72.0 [38.3; 99.6] 48.3 [30.5; 61.8] <0.01 ≥90, n (m±m, %) 13 (48.1±9.6) 2 (9.5±6.4) <0.01 60-89, n (m±m, %) 9 (33.3±9.1) 3 (14.3±7.6) >0.05 30-59, n (m±m, %) 5 (18.5±7.5) 10 (47.6±10.9) <0.05 15-29, n (m±m, %) 0 (0.0±0.0) 3 (14.3±7.6) <0.05 <15, n (m±m, %) 0 (0.0±0.0) 3 (14.3±7.6) <0.05 in the patients of the 1st group (48.1±9.6)% and much less frequently in those with proteinuria >1 g/day – (9.5±6.4)% (p<0.01). on the contrary, the gfr, which corresponded to the 3rd stage of ckd, was evidenced in half of patients with proteinuria >1g/day (47.6±10.9)% and only in 18.5±7.5% of patients with pu ≤1 g/day (fig. 3). accordingly, the terminal renal insufficiency (gfr<15 ml/min/1.73 m2) was revealed only in the patients of the 2nd group, in 2 of them proteinuria exceeded 3.0 g/day. the mean reciprocal correlation between the levels of proteinuria and the glomerular filtration rate (r=-0.562, p<0.01), as well as between the levels of proteinuria and hemo globin (r=-0.596, p<0.01) have been also established. м. о. andrushchak 11 in t e r n a l m e d ic in e issn 2413-6077. ijmmr 2018 vol. 4 issue 2 discussion thus the clinical manifestations of kidney lesion in the studied patients coincide with the typical ones for various pathologies in the general number of nephrology patients. there are numerous experiments on the study of a number of renal diseases in the hivinfected individuals worldwide [4, 8, 9]. for instance, the studies conducted in the united sta tes have revealed that, according to the re nal biopsy, 52.7% of the patients with ne phrotic pu were diagnosed with hiv-associated ne ph ropathy. they were all african americans. the high incidence of this pathology is associated with racial affiliation, as well as with a specific variant of the antigen/receptor to duffy chemokines, which are found in the renal tissue [10]. according to the results of multi center studies in france and italy, where most patients were of the caucasian race, among morpho logically verified diagnoses, immune deposit diseases were prevalent in the hiv-infected patients with kidney pathology [11, 12]. proteinuria is established to be one of the major laboratory criteria for ckd. therefore, the next stage of the work was the establishment of clinical and laboratory features of renal impairment depending on the level of protein in urine. thus, the analysis proved that the increase in pu levels was accompanied by a significant renal dysfunction and a more frequent combination with arterial hypertension and hematuria without significant differences in the frequency of opportunistic diseases. the inverse correlation between the level of proteinuria, gfr and hemoglobin value has been established. according to other indicators characterizing the course of hiv infection in people with different clinical variants of chronic kidney lesion, there were no reliable differences. conclusions kidney lesions in hiv-infected are most often characterized by tubulointerstitial lesions. at the same time, glomerular kidney lesion, which is much less common, is accompanied by a significantly higher level of hiv rna. an increase in proteinuria level is accompanied by a significant renal dysfunction and a more frequent combination with arterial hypertension and hematuria without significant differences in the incidence of opportunistic diseases. the mean reciprocal correlation between the levels of proteinuria and glomerular filtration rate (r=-0.562, p<0.01), as well as between the levels of proteinuria and hemoglobin (r=-0.596, p<0.01) have been established as well. ураження нирок у віл-інфікованих м. о. андрущак буковинський державний медичний університет, чернівці, україна вступ. однією з найважливіших проблем сучасності є епідемія віл-інфекції. за 30-річну історію цієї хвороби віл уразив понад 75 мільйонів людей, з них майже 30 мільйонів дорослих і дітей померли. у найближчі десятиліття, як і раніше, вони відіграватимуть істотну роль у світових показниках передчасної смертності. мета роботи – встановити клінічні та лабораторні особливості ураження нирок при віл-інфекції. методи. обстежено 292 хворих на віл-інфекцією, які перебували на амбулаторному спостереженні в чернівецькому обласному центрі з профілактики та боротьби зі снідом. на підставі основних маркерів пошкодження нирок (персистентна протеїнурія та швидкість клубочкової фільтрації <60 мл/хв/1,73 м2) у 48 осіб діагностовано хронічну хворобу нирок, яка з великою частотою супроводжувалася порушенням функції цих органів. результати встановили, що у віл-інфікованих ураження нирок найчастіше характеризується їх тубулоінтерстиційним ураженням. водночас гломерулярне ураження нирок, що буває значно рідше, супроводжується достовірно вищим рівнем рнк віл. підвищення рівня протеїнурії супроводжувалося достовірно значущим порушенням функції нирок і частішим поєднанням з артеріальною гіпертензією і гематурією за відсутності достовірних відмінностей у частоті опортуністичних захворювань. встановлено зворотну середньої сили кореляцію між рівнями протеїнурії і швидкістю клубочкової фільтрації – (r=-0,562, р<0,01), а також між рівнями протеїнурії та гемоглобіну (r=-0,596, р<0,01). м. о. andrushchak 12 in t e r n a l m e d ic in e issn 2413-6077. ijmmr 2018 vol. 4 issue 2 висновки у віл-інфікованих ураження нирок найчастіше характеризується їх тубулоінтерстиційним ураженням. водночас гломерулярне ураження нирок, що буває значно рідше, супроводжується достовірно вищим рівнем рнк віл. ключові слова: віл-інфекція; хронічна хвороба нирок; тубулоінтерстиційне ураження; гломерулярне ураження нирок. kidney disease. kidney international. 2011 sep 1;80(5):553-4. https://doi.org/10.1038/ki.2011.202 7. fine dm, perazella ma, lucas gm, atta mg. renal disease in patients with hiv infection. drugs. 2 0 0 8 m a y 1 ; 6 8 ( 7 ) : 9 6 3 8 0 . h t t p : / / d x . d o i . org/10.2165/00003495-200868070-00006 8. winston j, deray g, hawkins t, szczech l, wyatt c, young b, mayer kh. kidney disease in patients with hiv infection and aids. clinical infectious diseases. 2008 dec 1;47(11):1449-57. doi: 10.1086/593099 9. tada m, masumoto s, hinoshita f. clinical remission of iga nephropathy in an hiv-positive patient after combined treatment with tonsillectomy and steroid pulse therapy. cen case reports. 2015 nov 1;4(2):157-61. http://dx.doi.org/10.1007/s13730014-0158-6 10. wearne n, swanepoel cr, boulle a, duffield ms, rayner bl. the spectrum of renal histologies seen in hiv with outcomes, prognostic indicators and clinical correlations. nephrology dialysis transplantation. 2011 dec 26;27(11):4109-18. https:// doi.org/10.1093/ndt/gfr702. received: 2018-11-07 references 1. alfvén t, erkkola t, ghys pd, padayachy j, warner-smith m, rugg d, de lay p. global aids reporting-2001 to 2015: lessons for monitoring the sustainable development goals. aids and behavior. 2017 jul 1;21(1):5-14. 2. piot p, karim ss, hecht r, legido-quigley h, buse k, stover j, resch s, ryckman t, møgedal s, dybul m, goosby e. defeating aids-advancing global health. the lancet. 2015 jul 11;386(9989):171-218. 3. pokrovsky vv, yurin og, kravchenko av, belyaeva vv, ermak tn, canestri vg, shahgildyan vi, kozyrin nv, buravtsova vv, narsia rs, pokrovskaya av. national recommendations for follow-up and treatment of patients with hiv infection. clinical protocol. epidemiology and infectious diseases. 2015 (6): 14. [in russian]. 4. campos p, ortiz a, soto k. hiv and kidney diseases: 35 years of history and consequences. clinical kidney journal. 2016 oct 25:772-81. https:// doi.org/10.1093/ckj/sfw104 5. shylov em, editor. nephrology: guideline for postgraduate education. moscow: geotar media; 2008, 696 p. [in russian]. 6. woo kt, chan cm. kdigo clinical practice guidelines for bisphosphonate treatment in chronic м. о. andrushchak 50 r a d ia t io n m e d ic in e a n d o n c o l o g y issn 2413-6077. ijmmr 2018 vol. 4 issue 2 doi 10.11603/ijmmr.2413-6077.2018.2.9410 obesity: a causative risk factor of breast cancer a. s. roy, s. basu, a. bandyopadhyay university of calcutta, university college of science and technology, kolkata, west bengal, india background. obesity, a global health burden and one of the most deleterious diseases have substantially increased prevalence attributable to changing lifestyle of modern times. persuasive evidence indicates obesity as an independent prognostic factor for developing malignancy in the form of breast cancer particularly in postmenopausal women. objective. this review aims to focus in comprehensive manner on the biochemical, hormonal and immunological pathways governing the obesity linked breast cancer so that potential treatments may be improvised consequently to provide a cure to this menace, threatening the lives of many. methods. literature review of published materials that provide examination of recent or current literature on problem of obesity. results. increased body fatness, mainly visceral adiposity may account for predisposing an obese individual to the risk of encountering cancer although the mechanisms for such cancers may vary depending upon the organ affected. metabolic and biochemical alterations influencing obesity related carcinogenesis, consisting of heightened oxidative stress and bodily inflammation levels with the concomitant rise in pro-inflammatory cytokines are discussed. pertinent references about elevated levels of serum insulin, insulin-like growth factor, sex steroids and the imbalance in adipokines (adiponectin and leptin) are included as well. conclusions. persuasive evidence indicates obesity as an independent prognostic factor for developing malignancy in the form of breast cancer particularly in post-menopausal women. generation of novel and effective therapeutic interventions for combating the ailment along with positive lifestyle modifications may be improvised consequently to provide a cure to this menace, threatening the lives of many. key words: obesity; breast cancer; lipotoxicity; adiponectins. introduction obesity is a major health problem of this century, characterized by excess accumulation of fat due to positive energy balance, resulting from energy intake that exceeds the energy expenditure [1]. a 15-20% of body fat for men and 25% of body fat for women are generally accepted as ‘normal’, but these are not essentially the optimal values, as a 10% to 20% of excess body fat over the usual values is generally considered to be “obesity” [2]. according to the world health organization (who) criteria, a bmi greater than or equal to 25 kg/m2 is overweight, while obesity is defined as having a bmi equal to or higher than 30 kg/m2. obesity has been recognized, as a major risk factor for many cancers and, following tobacco use, may be the greatest modifiable cancer risk factor [3, 4, 5]. the incidences of overweight and obesity is dramatically rising in most parts of the world, and is generally higher in women than in men [6]. convincing data associate being overweight to the risk for various types of cancer as well as other chronic ailments, including cardiovascular disease, stroke and diabetes that are accountable to a large percentage of premature mortality [7, 8]. the international agency for research on cancer reviewed the literature on the involvement between excess body weight and cancer risk. they evaluated the available data as sufficient for a plausible connection with cancers of colon, female breast (postmenopausal), endometrium, kidney (renal cell), and oesophagus (adenocarcinoma). preliminary information also exists to indicate a relationship with ancillary cancer [9, 10]. specifically, obesity is related with a twofold increase in the risk of developing breast cancer in case of postmenopausal women while among premenopausal women it is associated with a reduced incidence [11]. numerous corresponding author: dr. amit bandyopadhyay, b.sc., m.sc., ph.d., assistant professor, sports and exercise physiology laboratory, department of physiology, university of calcutta, university colleges of science and technology, 92, a. p. c. road, kolkata: 700009, india. e-mail: bamit74@yahoo.co.in a. s. roy et al. international journal of medicine and medical research 2018, volume 4, issue 2, p. 50-58 copyright © 2018, tsmu, all rights reserved 51 r a d ia t io n m e d ic in e a n d o n c o l o g y issn 2413-6077. ijmmr 2018 vol. 4 issue 2 interacting hormonal and metabolic pathways seem to underlie the link between being overweight and cancer, with insulin-resistance harbouring a major role. since evidence is swelling that surplus body weight can also unfavourably influence cancer prognosis, obesity is a prime target for cancer management programs. this review explores the epidemiological and biological evidences concerning the linkage between excess body weight/obesity and particularly cancer in the breast in females, available from several accessible and thorough systematic literature surveys, along with a brief insight into the probable therapeutic interventions in vogue. obesity related health disorder now a day’s obesity and overweight are considered as main causative factors for several chronic diseases, most notably hypertension, type 2 diabetes, dyslipidaemia and coronary heart diseases, osteoarthritis and musculoskeletal disorders, fatty liver, gall stones, psychological disorders and psychosocial problems [12, 13]. direct relationship of obesity with mortality has also been documented [14]. among its many health consequences, obesity is increasingly recognized as a risk factor for numerous malignancies, and the obesitycancer link has recently received much attention [15,16]. sufficient evidences exist to link obesity with increased risk of colon cancer, postmenopausal breast cancer, endometrial cancer, renal cell cancer and adenocarcinoma of the oesophagus [17]. obesity and cancer world cancer research fund (wcrf) and american institute for cancer research (aicr) concluded that obesity is an established risk factor for several cancers [18]. according to the reports of the last 25 years, obesity was found as a reason of approximately 14% of cancer deaths in men and up to 20% of deaths due to cancer in women [19]. over this time-period, the commonness of overweight and obesity has gone up from 15% in 1980 to 35% in 2005 [20]. recent investigations count on the fact that the total health onus of overweight and obesity may surpass that for cigarette smoking [20]. a major review of weight, physical activity, and cancer incidence by the international agency for research on cancer (iarc) concluded in 2002, that obesity was the aetiology of 11% of colon cancer cases, 9% of postmenopausal breast cancer cases, 39% of endometrial cancer incidences, 25% of kidney cancer cases, and 37% of oesophageal cancer incidences [17]. additionally, data from the american cancer society indicated, that overweight and obesity are connected to mortality from liver cancer, pancreatic cancer, non-hodgkin’s lymphoma, and myeloma [19]. obesity and cancer – general mechanism general mechanisms of obesity and cancer the cause effect relationship of obesity and cancer are not well known. however, it is well established that it acts through obesity-related hormones, growth factors, multiple signalling pathways of calorie restriction and modulation of energy balance and inflammatory processes. these factors affect the promotion and progres sion of the cancer cells [21, 22, 23, 24, 25, 26]. obesity and breast cancer obesity has been marked as a noteworthy risk factor for breast cancer and the association varies depending upon the menopausal status in females. breast cancer, as evident from the recent estimates is the most frequent type of cancer in women (28.9% of all female incident cancers) of european population and is the second most common cancer overall [27, 28]. obesity is found to consistently rise in postmenopausal women by 30%-50% [29, 30, 31, 32]. breast cancer incidence varies considerably between developed and developing countries which may be attributed to nutritional factors and lifestyle behaviours due to different socioeconomic conditions and variation in ethnicity [33]. literature clearly indicated the intimate association between obesity and breast cancer that might provide insight in exploring and identifying the various mechanisms involved in this process. obesity linked breast cancer is multifactorial and involves a network of hormo nal and metabolic pathways. hence understanding the molecular and cellular mechanisms of the obesity-cancer link is imperative for developing potential therapeutics. mechanisms underlying obesity related breast cancer in females bio-energetic homeostasis and cancer metabolic parameters associated with body fatness might influence the bioenergetic balance of the cells and favour the expansion of cells with high anaerobic glycolytic capacity which is a characteristic feature regarding the bioenergetics adaptation of the cancer cells. this effect is termed as “warburg effect” described by intense lipogenesis and glycolysis and low mitochondrial oxidative phosphorylation capacity even in the presence of adequate oxygen [34, 35]. high blood glucose a. s. roy et al. 52 r a d ia t io n m e d ic in e a n d o n c o l o g y issn 2413-6077. ijmmr 2018 vol. 4 issue 2 levels and hyperinsulinaemia, which is frequent in obese individuals, are thought to pose a selective advantage for the growth of such cells [36]. increased risk of breast cancer attributed to higher energy intake has been reported in some research studies [37]. adenosine 5′-mono phosphate activated pro tein kinase (ampk) is a master sensor of cellular energy status that plays a key role in the regulation of whole-body energy ho meostasis [38]. recently, studies were con ducted to examine targets such as amp activated protein kinase (ampk), mammalian target of rapamycin (mtor), fatty acid syn thase, deacetylase sirt1 (sirtuin1) and epi genetic modulators as in nutrient sensing pathways coupled to insulin signalling have been hypo thesized to participate in carcino genesis [19]. insulin, igfs, igfbps and insulin resistance – the interplay insulin resistance is a metabolic state characterized by a reduced response capacity to insulin by the muscle and liver cells [10]. insulin resistance has been correlated to a sub sequent compensatory excess production of pancreatic insulin leading to hyperin suli naemia. chronic hyperinsulinaemia in turn is related to carcinogenesis and linked to breast cancer [39, 40]. this can be explained in the light of the postulates of insulin-igf hypothesis, which states that chronic hyperinsulinaemia decreases the production of insulin like growth factor binding proteins (igfbp1 and igfbp2) that results in the subsequent rise of plasma levels of free igf-1 with concomitant alterations in the cellular environment. both insulin and igf-1 are anabolic molecules that are capable of tumorogenesis by inhibiting apoptosis, stimulating cell proliferation and motility and being pro-angiogenic [41, 42, 43, 44]. high concentrations of circulating total igf-1, a major determinant of free igf-1 concentrations have been associated to an increased risk of premenopausal breast cancer [45]. however, the insulin-igf hypothesis has two shortcomings. first, levels of total igf-1 increases linearly with increased bmi but only up to a certain extent around 27 kg/m2 and thereafter it reduces with further increase in weight [44]. secondly, in overweight and/or obese individuals, who purposely lose weight (a presumed cancerprotective action), the total igf-1 concentration tends to escalate the insulin-signalling pathway. this is very much relevant in case of cancer progression because both extracellular signal regulated kinase (erk) and phosphatidyl inositol-3 kinase (pi3k) pathways are triggered by activation of the insulin receptor (ir). contrarily, over expression of the ir is evident in breast cancer patients [10, 46, 47, 48, 49]. insulin and igf-1 signal by mean of the akt/pi3k/mtor cascade for promotion of cell growth and proliferation, thereby inhibiting cell survival [50, 51]. this akt/pi3k/mtor cascade has emerged as a target of the obesity and cancer linkage and is activated by both insulin and igf-1 that are detected frequently at higher concentrations in the serum of the overweight and obese individuals, culminating [52, 53, 54]. alterations in sex hormones steroid hormones including oestrogen, progesterone, androgens and adrenal steroids are related with energy homeostasis and obesity related progression of different types of male and female cancers [55]. obesity increases the risk of developing breast cancer after menopause and it has been indicated that up to 50% of postmenopausal breast cancers are linked to obesity [37]. predisposing risk factors familiar in developing breast cancers are related to oestrogen e.g., early menarche, late menopause and hormone replacement therapy (hrt) [56, 57, 58, 59]. obesity and age has been ascertained as factors that may negatively influence the survival of patients with breast cancer [60,61]. increased adiposity may influence sterol synthesis and metabolism of oestrogens. obesity has been associated with increasing levels of oestrogen because of accelerated peripheral aromatization of adrenal androgens in adipose tissue among postmenopausal women, that can promote cell proliferation, have anti-apoptotic and pro-angiogenic effects [62,63]. in postmenopausal women, plasma levels of free oestradiol and testosterone are positively associated to breast cancer occurrence [64]. studies revealed that the relationship between obesity and breast cancer risk in postmenopausal women might be justified by heightened levels of oestrogens, particularly bioavailable oestradiol [65, 66]. further, in case of postmenopausal women the link between body mass index (bmi) and risk of breast cancer has been strongly evident among women, who do not use hormone replacement therapy (hrt), compared to women, who have undergone hrt [67]. some studies showed an inverse relationship between bmi and pre menopausal breast cancer and this may be supported by the fact that for pre menopausal women obesity is linked with a higher frequency of anovulatory cycles and with reduced levels a. s. roy et al. 53 r a d ia t io n m e d ic in e a n d o n c o l o g y issn 2413-6077. ijmmr 2018 vol. 4 issue 2 of circulating sex steroids [67]. another dimension to the association between bmi and breast cancer is mammographic density, the latter being negatively correlated with bmi. for adjust ment of mammographic density, estimates for bmi, cancer risk rise [68]. lipotoxicity cancer cells exhibit accentuated de novo lipogenesis by means of elevated fatty acid synthase (fasn), an enzyme responsible for synthesizing endogenous fatty acids, that may be modified and packaged into structural lipids required for cell division [69]. both obesity and cancer cell-derived lipolytic enzymes produce free fatty acids for the tumour to supply structural as well as oncogenic lipid signalling molecules such as platelet activating factor (paf), sphingosine 1-phosphate (s1p), lysophosphatidic acid (lpa) and prostaglandins [70]. elevated fasn enzyme, mrna, and enzymatic activity have been documented in human breast cancer cell lines and the rise in fasn is thought to be essential for evoking the malignant effects of proliferation and survival although this alone is not the reason for malignancy [71]. thus elevated basal lipolysis followed by increased plasma levels of free fatty acids (ffas) leads to enhanced intracellular accumulation which can impair non-adipose cells in their normal role as well as insulin signalling and the phenomenon is known as “lipotoxicity” [72]. obesity induced immunosuppression obesity induces chronic, low-grade inflammation leading to increased levels of local and systemic proinflammatory cytokines including prostaglandin e2 (pg e2), tumour necrosis factor-alpha (tnf-α), interleukin (il-2, il-8, il-10), c-reactive protein (crp) and monocyte chemoattractant protein (mcp-1). in this context activation of nf-κb complex may be cited as a possible mechanism by which inflammation may stimulate cancer progression [24,25]. thus, the proinflammatory state evident in the metabolic cells of adipocyte and the recruitment of immune cells along with the consequent release of inflammatory cytokines (tnf-α, il-6, adiponectin etc.) is the outcome of obesity. tumour necrosis factor–α or tnf–α a pro-inflammatory cytokine by nature tnf-α exerts several effects in adipose tissue encompassing lipid metabolism and insulin signalling in which the circulating levels are elevated with obesity and levels off with weight loss. a rise in tnf–α stimulates the secretion of other pro-inflammatory cytokines like il-6 while decreasing the levels of anti-inflammatory cytokines like adiponectin [73]. research findings indicated that tnf-α promoted adipocytes apoptosis and induced insulin resistance by means of inhibiting the insulin receptor substrate 1 signalling pathway [74,75]. interleukin–6 or il–6 macrophage is the preliminary source of circulating il-6 that plays a pivotal role in the whole-body energy homeostasis, as well as inflammation. the fact that il-6 has the potential to suppress the activity of lipoprotein lipase has been deduced from both in vitro and in vivo studies. expression of il-6 receptor is evident in certain brain regions and hypothalamus being one of them is responsible for controlling appetite and energy intake [76]. adiponectin contrary to the reduced levels of adiponectin as seen in cases of animal models of obesity and insulin resistance, weight loss has been found to elevate the adiponectin levels. regulation of lipid and glucose metabolism, increased sensitivity towards insulin, body weight and food intake regulation and protection against chronic inflammation are some of the vital roles of adiponectin [77]. intracellular pathways of inflammation overfeeding has been hypothesized to be the starting signal of inflammation in obesity and the pathway has its inception in the metabolic cells like the adipocyte, hepatocyte or myocyte. acute evocation of inflammatory responses due to consumption of nutrients has been suggested from studies in mice and humans [78, 79]. adipose tissue and liver in obese men and women, when compared to lean controls, exhibit hyperactivation of three kinases, namely: the c-jun n-terminal kinase (jnk), the inhibitor of k kinase (ikk) and the protein kinase r (pkr) capable of inducing inflammatory cytokines’ expression [80, 81]. the inflammasome and the toll-like receptors (tlrs) of the innate immune system are activated as well in those same metabolic tissues [82, 83, 84]. inflammatory signals or nutrients may trigger off the tlrs pathways and downstream jnk, ikk and pkr. these kinases control downstream transcriptional programs by means of the transcription factors activator pro tein-1 (ap-1), nf-κb and interferon regu latory factor (irf) inducing upregulation of inflammatory mediator gene expression. the rise in cytokines aggravates receptor activation through a positive feedback loop of inflam mation and the inhibitory signalling of metabolic pathways [85]. a. s. roy et al. 54 r a d ia t io n m e d ic in e a n d o n c o l o g y issn 2413-6077. ijmmr 2018 vol. 4 issue 2 dysregulation in adipokines the adipose tissue, known primarily as energy storage organ, by virtue of recent studies has also been established as an endocrine organ, producing and secreting polypeptide hormones, adipokines, among which leptin and adiponectin are most common and involved in cancer development [86]. adipokines (leptin, adiponectin and hepatocyte growth factor (hgf) are recognized for their participation in the mechanisms by which obesity and related metabolic disorders affect breast cancer risk [87]. the physiological and pathological commu nications of leptin and adiponectin are mostly antagonistic, as are their biological consequences on breast cancer cells [88]. leptin, a hormone essentially exclusive to adipose tissue acts centrally in the hypothalamus for regulation of body weight and peripheral energy expenditure [87, 89]. circulating leptin levels are strongly correlated to the body fat content and are prominent in obese subjects to normal individuals [90, 91, 92]. thus leptin, a potential mediator of obesity-related cancer influences cancer progression by activating pi3k, mapk and stat3 pathways, while the stimulatory effects of leptin on breast cancer growth were noted to occur primarily via oestrogen receptor activation [21, 26, 88, 93, 94]. further, evidences through extensive research suggest that adiponectin, the most abundant adipokine, affect the proliferation and insulin sensitivity of various types of cells [95]. unlike leptin, adiponectin is inversely related with adiposity, hyperinsulinaemia and inflammation [22]. moreover, adiponectin may incur anticancer effects by diminishing insulin/ insulin like growth factor (igf-1) and mtor signalling via activation of 5' amp-activated protein kinase (ampk) and providing antiinflammatory action by the inhibition of nuclear factor kappa-light chain enhancer of activated b cells (nf-κb) [22]. current findings indicate that the low serum adiponectin levels are significantly associated with an increased risk for breast cancer and that tumours arising in women with the low serum adiponectin levels have greater likelihood of expressing a biologically aggressive phenotype [95]. another adipokine, hepatocyte growth factor (hgf) or ‘scatter factor’ may exert a positive influence on tumorigenesis as a consequence of its antiangiogenic properties but is mainly known for its ability to promote cell invasion [88]. numerous investigations revealed that the serum concentration of hgf are often elevated in patients with breast cancer and particularly so in those suffering from the advanced disease stage [96, 97, 98]. conclusions the striking association between obesity and incidence of breast cancer has been established through several investigations and experiments until date. the various metabolic and endocrine mechanisms that account for the pathogenesis of obesity linked breast cancer have been discussed here to further probe into the nodal points of control in these cascades that may be beneficial to the researchers for generation of novel and effective therapeutic interventions for combating the ailment along with positive lifestyle modifications. currently hormonal therapy with se lective estrogen receptor modulators (serms) (such as tamoxifen and raloxifene) as well as aromatase inhibitors (such as exemastane, anastrozole, and letrozole) has been approved as standard mode of treatment of women with estrogen receptor-positive breast cancer. this therapy alongside adjuvant therapy acts in curing of advanced disease form though issues relating to their side effects are also a major concern [99, 100]. the efficacy of another drug which acts as an insulin lowering agent named metformin, in reducing breast cancer recurrence is presently being studied extensively [101, 102, 103]. simultaneously in the recent years yoga based lifestyle interventions that is a form of physical activity facilitating in accomplishing recommended levels of physical fitness have gained much attention and are found to effectively thwart and hinder the progression of cardiovascular and metabolic syndromes like that of obesity [104, 105]. the method of action of such benefit may be credited to a reduction in weight and stress, networking at mind and body levels, thereby leading to a decline in inflammation, and causation and progression of the disease [106]. thus, any further information about the drugs and other treatment modalities that can ameliorate the adverse effects of breast cancer by altering the markers of obesity may also be useful in this regard. acknowledgements the authors would like to express their heartfelt gratitude towards the works of researchers whose findings and reports have played integral roles in preparing this review. a. s. roy et al. 55 r a d ia t io n m e d ic in e a n d o n c o l o g y issn 2413-6077. ijmmr 2018 vol. 4 issue 2 ожиріння – фактор ризику раку молочної залози a. s. roy, s. basu, a. bandyopadhyay university of calcutta, university college of science and technology, kolkata, west bengal, india вступ. ожиріння – це глобальна проблема здоров’я, що набула значної поширеності зі зміною сучасного способу життя. також це незалежний прогностичний фактор розвитку раку молочної залози, особливо у жінок в період постменопаузи. мета огляду – комплексний аналіз біохімічних, гормональних та імунологічних чинників, які пов’язують ожиріння з раком молочної залози, та пошук потенційних методів лікування. методи дослідження. аналіз даних літератури для оцінки поточного стану проблеми. результати. надмірна вага, та головним чином накопичення вісцерального жиру, пов’язані з підвищеним ризиком розвитку злоякісних захворювань, однак механізми їх розвитку значно варіюють залежно від ураженого органу. обговорюються метаболічні та біохімічні показники, що впливають на канцерогенез, пов’язаний з ожирінням; включно з розвитком оксидативного стресу та ознак запального процесу з одночасним підвищенням рівня прозапальних цитокінів. а також такі фактори як підвищений рівень сироваткового інсуліну, інсуліноподібного фактора росту, статевих стероїдів та дисбалансу адипокінів (адипонектину і лептину). висновки. переконливі докази вказують на ожиріння як незалежний 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stewart st, cutler dm, rosen ab. forecasting the effects of obesity and smoking on us life expectancy. new england journal of medicine. 2009 dec 3;361(23):2252-60. received: 2018-08-16 a. s. roy et al. 64 b io m e d ic a l s c ie n c e s issn 2413-6077. ijmmr 2017 vol. 3 issue 2 doi 10.11603/ijmmr.2413-6077.2017.2.8237 age-related morphometric characteristics of remodeling of arterial bed of hind limbs in white rats with experimental hypercholesterolemia i. i. yuryk, ya. ya. bodnar, v. d. voloshyn, ya. i. yuryk i. horbachevsky ternopil state medical university, ternopil, ukraine background. the human organism is constantly affected by metabolic risk factors. all of them for a long time are asymptomatic and often form in early childhood long before clinical manifestations. however, morphogenesis and morphofunctional features of age-related structural changes in blood vessels of different sites are poorly studied. objective. to evaluate the morphometric characteristics of arterial bed remodeling of hind limbs in white rats of pra and ra with experimentally induced hypercholesterolemia. methods. the tissues biopsies from femoral, knee and shin areas were studied by means of histological and morphometric methods. results. experimental hypercholesterolemia caused remodeling of vessels with increasing thickness of medial and endothelial layer of vessels in the investigated parts of the rats’ body. in this case, the external diameter of vessels increased only in femoral area in the reproductive group of rats. in the group of pre-productive animals, the external diameter of the vessels studied was more or less unaffected. thus, increased thickness of intima and media led to a decrease in arteries capacity. the depth of vascular lesions and the decrease in functional capacity of blood vessels were directly proportional to the duration of the experiment. in the group of reproductive rats, the changes in vascular bed were more evident. conclusions. objective study of the processes allows providing a scientific basis for further research and understanding of the pathomorphism of vascular diseases in humans. key words: hypercholesterolemia; arteries; remodelling; morphometria. corresponding author: ihor yuryk, department of pathoanatomy with forensic medicine and sectional course, i. horbachevsky ternopil state medical university, 12 ruska str., ternopil, ukraine, 46008 phone number: +380352525844 e-mail: juryk@tdmu.edu.ua introduction cardiovascular diseases take the first place in the population mortality rate of the most countries of the world and are characterized by the increase in their prevalence rates, including those at young able-bodied age [1, 2]. according to state statistics [3], in ukraine over the past 10 years the prevalence of obesity among adolescents (15-17 years) per 1000 children population has increased in 2.5 times [3]. the human and animal organism is constantly affected by metabolic risk factors: insulin resistance, hyperinsulinemia, dyslipidemia, arterial hypertension, abdominal obesity, hyperuricemia are the most significant. all of them are asymptomatic for a long time and often form in early childhood long before clinical manifestations [4]. one of the first reactions to the toxic effects of metabolites are structural changes of vessels. however, morphogenesis and morphofunctional features of age-related structural changes in blood vessels of different sites are poorly studied. in the literature the established data is interpreted ambiguously. so, defining the morphometric rearrangement of arteries of femoral, knee and shin area of hind limbs in rats of pre-reproductive (pra) and reproductive age (ra) with hypercholesterolemia (hh) is very important. so, the aim of the research was to evaluate the features of morphometric characteristics of remodeling of arterial bed of hind limbs in white rats of pra and ra with experimentally induced hypercholesterolemia. methods studies were performed by means of 64 white rats. the experimental group consisted of 48 animals with biochemically confirmed hypercholesterolemia. they were divided into international journal of medicine and medical research 2017, volume 3, issue 2, p. 64–69 copyright © 2017, tsmu, all rights reserved i. i. yuryk et al. 65 b io m e d ic a l s c ie n c e s issn 2413-6077. ijmmr 2017 vol. 3 issue 2 i. i. yuryk et al. 2 groups: the first – 24 rats, 2–3 months­old in age, weighing 150–170 grams; and the second – 24 rats aged 10–11 months, weighing 230– 250 grams. the control group consisted of rats aged 2–3 and 10–11 months comprising 8 animals in each subgroup. hypercholesterolemia was modeled by feeding cholesterol at a dose of 0.5 g/kg with warmed vegetable oil. to suppress thyroid function, mercazolil was used at a dose of 10 mg/kg [5, 6]. this mixture was administered by means of an intragastric probe. the level of cholesterolemia was determined by a biochemical method by means of a semiautomatic biochemical analyzer humalyzer 2000 (germany) using a standard set of reagents and instructions for their use, human (germany). the biopsies of the tissues of femoral, knee and shin areas were fixed in a 10% neutral formalin solution and, according to a standard procedure, were sealed with paraffin. the dewaxed microtome sections were stained with hematoxylin and eosin, resorcin-fuchsin by weigert and van gieson, fuchsin hart, iron hematoxylin by heidenhain and alcian blue. during histological preparations for morphometry, the microscopes seoscan, mbi-15 were used. the image from the microscopes was displayed on a computer monitor using the programs vision color ccd camera and intervideowindvr. to determine the size of tissue structures of vascular wall, the images of histological sections were analysed using the computer programs videotest-5.0, kaare image base. the external diameter (ed) of arteries, the internal diameter (id) of arteries, the thickness of medial (tm), the thickness of intima (ti), the resistance index of bandwidth capacity of arteries (ribca) were determined. the latter was calculated as the ratio of wall area of vessel to the area of its lumen in percent [7]. laboratory rats were taken out from the experiment by bleeding after intraperitoneal injection of sodium thiopental at a dose of 50 mg/kg of body weight in 15, 30 and 45 days. the rats were kept and all experiments were carried out in compliance with the european convention for the protection of vertebrate animals used for experimental and other scientific purposes (strasbourg, 1986), the general ethical principles of experiments on animals adopted by the first national congress on bioethics (kiev, 2001). ), the helsinki declaration of the world medical association (2000), the order of the ministry of health of ukraine no. 281 from january 11, 2000. results the level of cholesterolemia in the rats of pre-reproductive age with experimental h y p e r c h o l e s t e r o l e m i a i n 1 5 d a y s w a s (2.15±0.03) mmol/l, and in the rats of reproductive age – (2.20±0.03) mmol/l. in 30 days the level of cholesterolemia in the pra rats was (2.68±0.04) mmol/l and in the ra rats – (2.82±0.03) mmol/l. in 45 days the cholesterolemia level in the pra rats was (2.83±0.05) mmol/l and in the ra rats – (3.03±0.03) mmol/l. in the intact rats group, the level of cholesterolemia was (1.80±0.04) mmol/l in the pra animals and (1.37±0.04) mmol/l in the ra animals. analysis of the obtained morphometric indices in the animals of pra and ra with experimental hh proved that they had a tendency to increase in comparison with the same parameters in the animals of intact group. in the rats of reproductive age, the structural changes are presented in table 1. thus, the external diameter of femoral artery of the intact animals of reproductive age i n c r e a s e d f r o m ( 5 2 5 . 4 1 ± 2 . 7 0 ) μ m t o (534.84±1.98) μm, which was 1.8% at p<0.01 in 45 days of the experiment. the internal diameter of femoral artery decreased by 4.65% in 30 days and by 6.32% in 45 days of the research (p<0.001). тtм of femoral artery i n c r e a s e d f r o m ( 1 0 0 . 0 4 ± 0 . 6 9 ) μ m t o (114.44±0.76) μm, which was 14.39% in 45 days of the experiment (p<0.001). the index of resistance to the femoral artery capacity gradually increased from (161.04±1.50) to (195.86±2.85)% in 30 days of the experiment and to (205.88±2,21)% in 45 days (p<0.001) and was higher by 21,62% and 27,84% respectively (fig. 1). the external diameter of popliteal artery and tibia artery in the rats of reproductive age was more or less unchanged till the end of the experiment. the internal diameter of popliteal artery decreased in 30 days of the experiment by 4.33% and in 45 days – by 5.55% (p<0.01). the ttm of popliteal artery increased from (81.29±0.37) μm to (83.35±0.74) μm in 15 days (2.6% (p<0.05)), to (89.11±1.12) μm in 30 days (9.6% (p<0.001)), and up to (90.69±0.94) μm (11.56% (p<0.001)) in 45 days of the experiment. bandwidth resistance index of popliteal artery increased by 5.56% in 15 days (p<0.01), in 30 days – by 20.55% (p<0.001), and in 45 days – by 24.53% (p<0.001). the internal diameter of artery of tibial area in the reproductive age animals in 15 days decreased by 2.75% (p<0.05), in 30 days – by 66 b io m e d ic a l s c ie n c e s issn 2413-6077. ijmmr 2017 vol. 3 issue 2 table 1. morphometric indices of arteries of hind limbs of the rats of reproductive age with experimental hypercholesterolemia time of the experiment area studied parameters ed, μm id, μm тtм, μm intact animals femoral artery 525.41±2.70 325.32±1.98 100.04±0.69 popliteal artery 416.69±1.50 254.12±0.97 81.29±0.37 tibia artery 139.17±0.70 83.18±0.37 28.00±0.19 within 15 days femoral artery 533.38±1.70* 321.17±2.52 106.11±1.01*** popliteal artery 416.91±1.90 250.21±1.71 83.35±0.74* tibia artery 139.45±1.21 80.90±1.06* 29.27±0.32** within 30 days femoral artery 533.15±2.75* 310.23±1.97*** 111.46±1.02*** popliteal artery 419.65±2.26 241.44±2.13*** 89.11±1.12*** tibia artery 140.26±1.32 79.71±1.06** 30.27±0.43*** within 45 days femoral artery 534.84±1.98** 305.96±1.41*** 114.44±0.76*** popliteal artery 419.95±2.37 238.57±1.43*** 90.69±0.94*** tibia artery 140.73±1.37 79.57±1.13** 30.58±0.66*** notes: * – р<0.05; ** – р<0.01; *** – р<0.001 ed – external diameter; id – internal diameter; ttm – thickness of tunica media. 4.2% (p<0.001) and in 45 days of the study – by 4.4% (p<0.001). statistical significance (p<0.01) of ttm arteries of tibial area increased in 15 days of the experiment, and in 45 days of the experiment it was by 9.2% higher (p<0.001). the ari in tibial area increased from (179.93±0.85)% to (216.80±7.92)% in 45 days (p<0.001) and was higher by 20.49%. the tunica intima (ti) in the rats of reproductive age (ra) in 15 days of the experiment was significant only in the knee area (+4.8% (p<0.05)). morphometric indices of other regions during this period were unreliable. after 30 days, the ti of femoral artery in the reproductive animals increased by 7.78% (p<0.001), and in 45 days – by 8.83% (p<0.001). in the popliteal region the ti increased by 8.00% (in 30 days) and by 10.45% (p<0.001) in 45 days of the experiment. the values of ti of tibial area increased by 8.62% in 30 days and by 8.95% (p<0.001) in 45 days. in the animals of pre-reproductive age (pra), the morphometric parameters were similar, although somewhat reduced (table 2). thus, the external diameter of femoral artery was more or less unchanged during the study. the internal diameter of femoral artery decreased by 2% in 15 days (p<0.05), by 5.44% in 30 days and by 670% in 45 days of the study (p<0.001). the parameters of tm of femoral artery i n c r e a s e d i n 3 0 d a y s b y 8 . 9 3 % f r o m (69.53±0.85) μm to (75.74±1.15) μm (p<0.001); i n 4 5 d a y s i t i n c r e a s e d b y 1 1 . 1 1 % t o (77.26±0.74) μm (p<0.001). the ihss of femoral artery gradually increased in 15 days by 6.4% from (164.23±2.58) to (174.72±2.61)% (p<0.01), in 30 days of the experiment – by 19.79% to table 2. morphometric parameters of arteries of hind limbs of the pre-reproductive age rats with experimental hypercholesterolemia, (m±m) time of the experiment area studied parameters ed, μm id, μm тtм, μm intact animals femoral artery 361.66±2.18 222.60±1.10 69.53±0.85 popliteal artery 295.09±1.66 179.79±1.43 57.65±0.79 tibia artery 94.83±0.87 56.54±0.47 19.15±0.29 within 15 days femoral artery 361.50±2.48 218.25±1.54* 71.62±0.81 popliteal artery 295.69±1.66 174.59±1.41* 60.55±1.41* tibia artery 95.01±0.63 55.24±0.64 19.88±0.23 within 30 days femoral artery 362.00±2.44 210.51±1.53*** 75.74±1.15*** popliteal artery 296.03±1.75 170.02±1.32*** 63.00±0.84*** tibia artery 95.28±0.96 54.21±0.53** 20.53±0.32** within 45 days femoral artery 362.10±2.25 207.70±1.57*** 77.26±0.74*** popliteal artery 296.83±1.84 169.98±1.46*** 63.43±0.68*** tibia artery 95.77±0.83 54.04±0.39*** 20.87±0.29*** notes: * – р<0.05; ** – р<0.01; *** – р<0.001. i. i. yuryk et al. 67 b io m e d ic a l s c ie n c e s issn 2413-6077. ijmmr 2017 vol. 3 issue 2 i. i. yuryk et al. (196.74±4.48)% (p <0.001) and in 45 days – by 24,67% to (204.76±3,10)% (p<0.001) (fig. 2). the external diameter of popliteal artery and tibial artery in the pra rats, (and in the ra animals) didn't change just about till the end of the experiment. the internal diameter of popliteal artery decreased in 15 days by 2.9% (p<0.05), in 30 days – by 5.44%, in 45 days – by 5.46% (p<0.001). the tm of popliteal artery increased in 15 days by 5% (p<0.05), in 30 days – by 9.28% (p<0.001) and in 45 days of the experiment – by 10.02%. the ribca of popliteal artery increased by 10.33% in 15 days (p<0.01), in 30 days – by 19.95%, and in 45 days – by 20.81% (p<0.001). the internal diameter of artery of tibial area decreased in 15 days of the study by 2.30%, in 30 days – by 4.11% (p<0.01), and in 45 days – by 4.43% (p<0.001). the tm of artery of tibial region increased by 7.26% (p<0.01) in 30 days of the experiment, and in 45 days of the experiment it increased by 8.98% (p<0.001). the ribca of tibial region arteries increased in 45 days of the experiment from (181.82±3.45) to (214.43±3.33)% (p<0.001) and was by 17.93% higher than the control. the thickness of intima of arteries in the pra rats with experimental hypercholesterolemia increased a bit less than in the rats of ra. in 15 days of the experiment, the thickening of the intima was not reliable at all levels of the study. in 30 days, the ti of femoral artery increased by 6.90% (p<0.01), and in 45 days – by 7.63% (p<0.001); the ti of popliteal artery increased in 30 days by 7.69% (p<0.01) and in 45 days – by 9.61% (p<0.001); the increase of ti of tibial artery was 6.58% (p<0.01) in 30 days and 8.40% (p<0.001) in 45 days of the experiment. discussion arteries remodeling is initiated as an adaptive process in response to altered circulatory conditions or to the influence of any humoral agents. the arterial vessels are shaped throughout life: the walls of vessels thicken, their rigidity increases. the endothelium dysfunction and its increased permeability are indicators of arteries aging. these negative processes result in an enhanced synthesis of vasoconstrictor substances: angiotensin ii and endothelin. at the same time, vasodilation development factors: nitric oxide, prostacyclin decrease, are manifested [8, 9]. in conditions of insufficient amount of nitric oxide, the endothelium of vessels cannot fulfil its inherent functions, and the decrease in the amount of no in the vessels is the cause of atherosclerosis, other cardiovascular diseases and one of the first signs of aging of vascular bed. intima diffusely thickens due to the aging of arteries that is caused by the accumulation of extracellular matrix proteins, smooth myocytes, collagen, glycosaminoglycans in it. these factors contribute to the enhanced adhesion of monocytes to the luminal surface of endothelium and are one of the stages of atherosclerotic plaque formation in arteries. there is evidence that vessel intima fig. 1. the resistance index of the bandwidth capacity of arteries (ribca) of hind limbs in the rats of reproductive age with experimental hypercholesterolemia 161,04 176,9 195,86 205,88 168,93 178,28 203,66 210,37 179,93 198,96 211,75 216,80 100 120 140 160 180 200 220 240 control through 15 days through 30 days through 45 days r ib c a , % femoral area knee area tibial area 164,23 174,72 196,74 204,76 170,44 188,05 204,45 213,93 181,82 196,63 209,58 214,43 100 120 140 160 180 200 220 240 control through 15 days through 30 days through 45 days r ib c a , % femoral area knee area tibial area 68 b io m e d ic a l s c ie n c e s issn 2413-6077. ijmmr 2017 vol. 3 issue 2 thickens in 2–3 times for the period of from 20 to 90 years old [10, 11]. the proteins of extracellular matrix of the middle shell of arteries undergo significant structural and functional changes in aging. in the vessels there is an excessive deposition of calcium, transition of smooth myocytes from media into intima. elastin of arteries is thinned that is characterized by fragmentation phenomena under the influence of matrix metalloproteinases and transforming growth factor­tgf­β [12, 13]. in contrast, the collagen protein synthesis is promoted with the assistance of angiotensin ii, which increases the rigidity of collagen itself and vascular wall [14, 15]. smooth myocytes in the intima of vessels are characterized by increased proliferation. this promotes the adhesion of blood cells to the endothelium of arterial vessels, thickening of fig. 2. the resistance index of bandwidth capacity of arteries (ribca) of hind limbs in the rats of pre-production age with experimental hypercholesterolemia intima, increasing its stiffness and development of atherosclerosis [16]. morphometric indices in animals with hypercholesterolemia prove that hypercholesterolemia was accompanied by remodeling of predominantly femoral and popliteal arteries that was severe in the animals of reproductive age. conclusions experimental hypercholesterolemia in the rats of reproductive age was accompanied by remodeling of predominantly femoral and popliteal arteries. it should be noted that the intensity of these changes in the arteries of large caliber in the animals of pre-reproductive age was lower than in the animals of reproductive group, and remodeling of lower leg arteries was more significant in the animals of reproductive age. references 1. go as, mozaffarian d, roger vl, benjamin ej, berry jd, borden wb, et al. executive summary: heart disease and stroke statistics – 2013 update. a report from the american heart association. circulation. 2013;127:143–52. 2. roger vl, go as, lloyd-jones dm, benjamin ej, berry jd, borden wb, et al. executive summary: heart disease and stroke statistics – 2012 update. circulation. 2012 jun 5;125(22):188–97. 3. tokarchuk ni, timchuk ev. overweight in young children is a factor of risk of illness in the future (review of literature). modern pediatrics. 2009; 6(28):54–6. 161,04 176,9 195,86 205,88 168,93 178,28 203,66 210,37 179,93 198,96 211,75 216,80 100 120 140 160 180 200 220 240 control through 15 days through 30 days through 45 days r ib c a , % femoral area knee area tibial area 164,23 174,72 196,74 204,76 170,44 188,05 204,45 213,93 181,82 196,63 209,58 214,43 100 120 140 160 180 200 220 240 control through 15 days through 30 days through 45 days r ib c a , % femoral area knee area tibial area i. i. yuryk et al. 69 b io m e d ic a l s c ie n c e s issn 2413-6077. ijmmr 2017 vol. 3 issue 2 i. i. yuryk et al. 4. halpern al, mancini mc, magalhaes me, fisberg m, radominski r, bertolami mc, et al. metabolic syndrome, dyslipidemia, hypertension and type 2 diabetes in youth: from diagnosis to treatment. diabetol. metab. syndr. 2010;2:55–75. https://doi. org/10.1186/1758-5996-2-55. 5. polyakov lm, lushnykova el, nepomnyashchykh lm, russkykh hs, byushkyna nh, klynnykova mh, et al. indicators of lipid metabolism and protein composition of blood plasma lipoproteins of hypothyroid rats at experimental hypercholesterolemia. fundamental research. 2014;(10):342–5. 6. mikhail gs, alshammari sm, alenezi my, mansour m, khalil na. increased atherogenic low-density lipoprotein cholesterol in untreated subclinical hypothyroidism. endocr. pract. 2008;14(5):570–5. 7. avtandilov hh. medical morphometry. moscow: medicina; 1990. 8. klinkova еv, otteva en, harbuzova оh, isakova vn, bandurko ev. assessment of arterial rigidity in patients with gout and arterial hypertension.. scientific and practical rheumatology. 2010;(6):40–5. 9. safar m, wang m, lakatta eg. central arterial aging: humans to molecules. handbook of hypertension: arterial stiffness in hypertension. amsterdam: elsevier. 2006. 10. palluy o, morliere l, gris jc, bonne c, modat g. hypoxia/reoxygenation stimulates endothelium to promote neutrophil adhesion. free radic. biol. med. 1992;13(1):21–30. https://doi: 10.1016/ 0891-5849(92)90161-9. 11. orlandi a, marcellini m, spagnoli lg. aging influences development and progression of early aortic atherosclerotic lesions in cholesterol-fed rabbits. arterioscler thromb vasc biol. 2000;20(4): 1123–36. 12. li z, froehlich j, galis zs, lakatta eg. increased expression of matrix metalloproteinase-2 in the thickened intima of aged rats. hypertension. 1999;33(1):116–23. 13. risinger gm jr, updike dl, bullen ec, tomasek jj, howard ew. tgfsuppresses the upregulation of mmp-2 by vascular smooth muscle cells in response to pdgf-bb. am j physiol cell physiol. 2010 jan; 2010;298(1):191–201. https://doi:10.1152/ajpcell. 00417.2008. 14. konova e, baydanoff s, atanasova m, velkova a. age-related changes in the glycation of human aortic elastin. exp gerontol. 2004;39(2):249–54. https://doi: 10.1016/j.exger.2003.10.003. 15. zieman sj. mechanisms, pathophysiology and therapy of arterial stiffness. arterioscler thromb vasc biol. 2005;25(5):932–43. https://doi: 10.1161/01. atv.0000160548.78317.29 16. strazhesko id, akasheva du, dudinskaya yen, tkacheva on. aging of blood vessels: basic signs and mechanisms. cardiovascular therapy and prevention. 2012;11(4):93–100. received: 2017-07-07 issn 2413-6077. ijmmr 2017 vol. 3 issue 160 p u b l ic h e a lt h a n d e p id e m io l o g y doi 10.11603/ijmmr.2413-6077.2017.1.7810 awareness of lyme borreliosis of the patients of ternopil regional tb dispensary l. p. melnyk1, l. a. hryshchuk2, m. koziol-montewka3, p. o. tabas2, r. o. klos2 1ternopil regional tb dispensary, ternopil, ukraine 2i. horbachevsky ternopil state medical university, ternopil, ukraine 3medical university of lublin, lublin, poland background. lyme disease has many clinical features similar tothose in sarcoidosis and tuberculosis. epidemiological data in the world, in particular in ukraine, proves the increase in lyme borreliosis incidence. ternopil region is endemic with lyme borreliosis. objective. the research was aimed to investigate the prevalence of infection with borrelia burgdorferi and epidemiology features of borreliosis among the patients of ternopil regional tb dispensary. methods. in total, 29 patients were admitted to departments of differential diagnostic, tb therapy and tb surgery of ternopil regional tb dispensary in october 2016-january 2017. all the surveyed answered the questions of an integrated international questionnaire, where they noted the area and a number of tick bites, described the removal method, noted the survey for borreliosis pathogen and complaints after tick bites. results. it was established that 5 respondents had a history of tick bites episodes, but only in one case the patient was examined of borreliosis. tick bites were noticed in 3 patients with sarcoidosis and 1 with tuberculosis (tb) and exudative pleurisy, respectively. conclusions. the absence of appeals for medical care, lack of sufficient information on lyme borreliosis and disuse of preventive measures for tick bites by the interviewed patients of ternopil regional tb dispensary departments proves the need of improvement of health education on lyme borreliosis (lb) among this category of population. 24 (82.7%) of 29 respondents did not remember the tick bite. the symptoms of (lb) are similar to those in sarcoidosis and tuberculosis (pleural lesions, heart, joints, nervous system, skin), and the presence of tick bites gives the reasons to examine these patients of borrelia burgdorferi senso lato. key words: lyme borreliosis; survey; sarcoidosis; tuberculosis; granulomatous diseases; lyme disease; borrelia burgdorferi. corresponding author: leonid hryshchuk, department of prope deutics internal medicine and phthisiology, i. horbachevsky ternopil state medical university, 14 kupchynskyi street, ternopil, ukraine, 46000 phone number: +3800352525929 e-mail: gryshchuk@tdmu.edu.ua intoduction lyme disease (systemic tick borreliosis, lyme borreliosis, chronic migrating erythema, clip erythema) is defined as the natural cell transmissible disease that is caused by borrelia burgdorferi and is manifested with erythema migrans, fever, lesions of the central and peripheral nervous systems, heart and large joints [1, 3] spirochetes are carried by ixodes tick. the epidemiological features of lb in the world, in particular in ukraine, proved the annual incidence increase [5, 12, 15, 17]. due to the location in the zone of moderate continental climate with warm humid summers and mild winters, chernozem, broadleaf and mixed forests, the territory of ternopil region is endemic with lb. spring-summer seasonality is usual for tick bites. but in recent years for ixodes ticks winter diapause takes place earlier, and seasonal activity continues till the third week of november, which leads to an increase in the number of ticks and, consequently, a large number of attacks on people. in 2016 the first tick bite in ternopil happened on the third week of february. during 10 months of 2016 1,211 cases of tick bites (in all districts of ternopil region) were compared to 1289 in the same period of 2015. according to the primary urgent messages in 2016 135 cases of lyme disease were registered compared to 100 in the same period in 2015 [11]. according to the literature, borrelia burgdorferi can act as a potential cause of systemic granulomatous disease such as sarcoidosis [2, international journal of medicine and medical research 2017, volume 3, issue 1, p. 60–63 copyright © 2017, tsmu, all rights reserved l. p. melnyk et al. issn 2413-6077. ijmmr 2017 vol. 3 issue 1 61 p u b l ic h e a lt h a n d e p id e m io l o g y l. p. melnyk et al. 4, 13, 14]. however, sarcoidosis can be caused by other etiological factors: infections (viruses: herpes, epstein-barr, retrovirus, coxsackie b virus, cytomegalovirus, bacteria: propionibacterium acnes, mycobacterium tuberculosis and other mycobacteria, mycoplasma), inorganic (beryllium, zirconium and aluminum) and organic (pollen, clay) agents [2, 4, 7, 10, 16]. lyme disease has many symptoms similar to those in sarcoidosis and tuberculosis (lesions of pleura, heart, joints, nervous system, skin) [6, 8, 9, 18]. assumptions about of borreliosis and sarcoidosis connection are based on the geographical spread of both diseases and family manifestations of these diseases [2, 4, 13]. therefore, the accuracy of sarcoidosis diagnosis depends on the thorough surveys of the patients with potential contact to organic and inorganic antigens [2, 10]. the objective of the study was to investigate the prevalence of borrelia burgdorferi infection and epidemiological features of borreliosis among the patients of ternopil regional tb dis pensary. the survey is a part of a joint ukrainian-polish project “research on the epidemiology, pathogenesis, clinical manifestations and prophylaxis of borreliosis” together with the pope john paul ii state school of higher education in biala podlaska (poland) within the research projects of the european union. methods in total, 29 patients were admitted to the departments of differential diagnostic, tb therapy and tb surgery of ternopil regional tb dispensary in october 2016-january 2017. the age of the respondents ranged from 20 to 65 years old. there were 17 (58.6%) men, 12 (41.4%) women. all the surveyed answered the questions of an integrated international questionnaire, where they noted the area and a number of tick bites, described the removal method, noted the survey for borreliosis pathogen and complaints after tick bites. the patients of ternopil regional tb dispensary departments also informed about the use of repellent at the entrance to the forest zone and the checking the skin after leaving it. the analysis of patients' awareness and media data about borreliosis, according to the questionnaires, proved the need of enhancement of their knowledge of lyme disease or other diseases associated with tick bites. results among the respondents city residents prevailed: 18 men (62.1%), 11 (37.9%) rural residents. due to the nosology there were 17 (58.6%) of the patients submitted with sarcoidosis, 5 (17.2%) with pulmonary tuberculosis and 7 (24.1%) with pulmonary tuberculosis complicated with exudative pleurisy. 23 (79.3%) of the respondents suffered from chronic diseases. among the surveyed 29 patients 5 (17.3%) of the respondents noted the tick bites, 24 (82.7%) did not remember the episode of bite. one respondent noticed the appearance of the erythema on the site of the bite within 12 hours. 5 respondents described the localization of tick bites. it should be noted that the most common tick bites the patients noticed on the trunk (2) and lower limbs (2). only one person no ticed tick bites on the abdomen. most of the bites took place in the forest – 4, in rural areas – 1. all the patients removed the tick be themselves (with fingers) within to 12 hours after the bite. the patients with erythema migrans or other symptoms did not ask for medical aid. according to the survey, only one respondent was examined to borreliosis. the diagnosis was confirmed by western blot and the patient was treated with macrolide antibiotics. according to the results of the survey the awareness of borreliosis in patients of ternopil regional tb dispensary is insufficient. table 1. characteristic features of the patients nosology number of patients number of patients % information about episode of tick bite information about the mites (patients) surveyed on lyme borreliosis sarcoidosis 17 58.6 3 1 pulmonary tuberculosis 5 17.2 1 – pulmonary tuberculosis complicated with exudative pleurisy 7 24.2 1 – total 29 100 5 1 issn 2413-6077. ijmmr 2017 vol. 3 issue 162 p u b l ic h e a lt h a n d e p id e m io l o g y quality medical history of illness and examination of patients is important for differential diagnosis of granulomatosis and lb for timely adequate therapy. incorrect or late diagnosis of lb may cause the development of complications in patients [3, 5, 15]. discussion according to the literature, tuberculosis and sarcoidosis histologically belongs to granulomatous diseases, which are based on caseous and non caseous granuloma. while tuberculosis agent is verified, sarcoidosis is a systemic disease which aetiology is still unknown [2]. however, according to the literature, the probable causes of sarcoidosis may be mycobacterium tuberculosis, borrelia burgdorferi, propiono bacterium acnes and others [13]. epidemiological data in the world, in particular in ukraine, prove the increase in lyme borreliosis incidence. the situation concerning tb also remains difficult, especially due to the spread of drug resistant tb. ternopil region is endemic with lyme borreliosis, due to the location in the zone of broad leaf and mixed forests. [3, 5, 15]. however, ukraine has no data determining borrelia burgdorferi infection in patients with granulomatous respiratory diseases, including sarcoidosis and tuberculosis. thus, it is an urgent matter to examine the prevalence of borrelia burgdorferi infection in patients with sarcoidosis and tuberculosis, because lyme disease has many clinical features similar to sarcoidosis and tuberculosis (pleural lesions, heart, joints, nervous system, skin) [7, 8, 14]. conclusions the absence of appeals for medical care, lack of sufficient information on lyme borreliosis and disuse of preventive measures for tick bite by the interviewed patients of ternopil regional tb dispensary departments, proves the need of improvement of health education on lyme borreliosis among this category of population. 24 (82.7%) of 29 respondents did not remember the tick bite. lyme borreliosis symptoms are similar to those of sarcoidosis and tuberculosis (pleural lesions, heart, joints, nervous system, skin), and tick bites give the reasons to examine these patients of borrelia burgdorferi senso lato. table 2. multiplicity of tick bites multiplicity of tick bites number of tick bites (n=5) number of tick bites, % 1 time 2 40 2 times 1 20 many (3 and more times) 2 40 table 3. localization of tick bites localization of bite number of bites (n=5) number of bites,% hands – – legs 2 40 trunk front back 2 40 abdomen 1 20 neck – references 1. andreychyn ma. infectious diseases in general practice and family medicine. ternopil: ukrmedknuha; 2007. 2. baughman rp, lower ee, du bois r. sarcoidosis. lancet 2003;361:111–1118. 3. vasylyeva na, ivakhiv ol, kachor vo, et al. lyme disease in ternopil region. infectious diseases. 2011;2:50–53. 4. nunes h, bouvry d, soler p, valeyre d. sarcoidosis. orphanet journal of rare diseases 2007. doi:10.1186/1750-1172-2-46. 5. shkilna mi. lyme borreliosis in forest workers of ternopil region. infectious diseases. 2016;83(1): 36–40. 6. antonova tv, zhevnerova ns. viral myocarditis: etiology and pathogenesis, problems of diagnostics. journal infectology. 2013;5(2):13–21. doi:10.22625/ 2072-6732-2013-5-2-13-21. 7. feshchenko yui, protsyk lm, cherednyk yuo. respiratory sarcoidosis: current state of problem. ukrainian journal pulmonological. 2006;3:5–10. l. p. melnyk et al. issn 2413-6077. ijmmr 2017 vol. 3 issue 1 63 p u b l ic h e a lt h a n d e p id e m io l o g y l. p. melnyk et al. 8. boyko lt. modern methods of laboratory diagnosis of serous meningitis. ukrainian journal psychoneurology. 2014;22(79):25–27. 9. duzhy id, oleshchenko hp, hresko iya, yurchenko av. pleural effusion syndrome and lyme disease. tuberculosis, pulmonary diseases, hiv infec tion. 2016;2(25):79–84. 10. gavrisiuk vk. respiratory sarcoidosis. kyiv: veles; 2015. 11. activity of ticks continues. 2016. http://www. terses.gov.ua. accessed 03 nov 2016. 12. solovey nv. lymeborreliosis. minsk. 2015. 13. vizel aa, guryleva me. potential infectious triggers in sarcoidosis. clinical microbiology and antimicrobial chemotherapy. 2002;4(4):313–324. 14. stinco giuseppe. lyme disease, atypical skin manifestations. acta dermatovenerologica apa. 1994;3(93)49–52. 15. popovych oo. lyme borreliosis: current problem of infectiology (clinical lecture). actual infectology. 2016;3(12):114–122. doi:10.22141/2312413x.3.12.2016.81725. 16. esteves t, aparicio g, garcia-patos v. is there any association between sarcoidosis and infectious agents?: a systematic review and meta-analysis. bmc pulmonary medicine. 2016;16(165):1–13. doi:10.1186/ s12890-016-0332-z. 17. mayne pj. clinical determinants of lyme borreliosis, babesiosis, bartonellosis, anaplasmosis, and ehrlichiosis in an australian cohort. international journal of general medicine. 2015;8:15–26. 18. allen hb, shaver cm, etzler ca, joshi sg. autoimmune diseases of the innate and adaptive immune system including atopic dermatitis, psoriasis, chronic arthritis, lyme disease, and alzheimer’s disease. immunochem immunopathol. 2015;1(112): 1–4. doi:10.4172/2469-9756.1000112. received: 2017-03-17 issn 2413-6077. ijmmr 2017 vol. 3 issue 2 39 p u b l ic h e a lt h a n d e p id e m io l o g y doi 10.11603/ijmmr.2413-6077.2017.2.8012 panresistant superbugs: are we at the edge of a ‘microbial holocaust’ 1i. d. khan, 1k. s. rajmohan, 2a. k. jindal, 3r. m. gupta, 4s. khan, 5m. shukla, 6s. singh, 7sh. mustafa, 8а. tejus, 8s. narayanan 1army college of medical sciences and base hospital, new delhi, india 2armed forces medical college, pune, india 3army hospital research and referral, new delhi, india 4inhs kalyani, vishakhapatnam, india 5esi hospital, rohini, new delhi, india 6army hospital research and referral, delhi cantt, india 7college of medicine, imam mohammad bin saud univesity, riyadh, saudi arabia 8army college of medical sciences and base hospital, delhi cantt, india contemporary healthcare has progressed towards world health security through advancements in medicationbased and surgical interventions, supported by the success of antimicrobial therapy. the emergence of panresistant infectious diseases is becoming a public health problem worldwide. panresistance is attributable to a complex interplay of antimicrobial overuse in healthcare facilities due to lack of regulatory commitment in the backdrop of natural mutations in pathogens and rise in immunocompromised hosts. developing countries are facing the brunt in epidemic proportions due to strained public health infrastructure and limited resource allocation to healthcare. panresistance is a biological, behavioural, technical, economic, regulatory and educational problem of global concern and combating it will require concerted efforts to preserve the efficacy of the available antimicrobials. an intensified commitment needs to be taken up on a war footing to increase awareness in the society, increase laboratory capacity, facilitate antimicrobial research, foster emphasis on infection control and antimicrobial stewardship, and legislation on manufacturing, marketing and dispensing of antimicrobials. key words: panresistance; antimicrobial resistance; totally drug resistant tuberculosis; infection control; antimicrobial stewardship. corresponding author: inam danish khan, clinical microbiology and infectious diseases, army college of medical sciences and base hospital, new delhi, india, 110010 phone number: +919836569777 e-mail: titan_afmc@yahoo.com introduction infectious diseases of the antiquity such as plague, cholera, influenza, smallpox, measles and malaria, which have been responsible for claiming billions of lives, have either been eradicated, eliminated or controlled in various parts of the globe due to advanced antimicrobial therapeutics and vaccines. the discovery of penicillin in 1940s and consequent success at wound healing and survival of soldiers in world war ii was a major breakthrough in the history of mankind. the subsequent discovery of a series of antimicrobials, some 22 of them credited to selman waksman, brought the menace of infectious diseases and ensuing sepsis under control. antimicrobials conferred safety and reliability upon a wide variety of diagnostic and therapeutic procedures including advanced surgeries, organ transplantation and immunotherapy, being heavily dependent on antimicrobial support. while the era of infectious diseases was being considered over, backed upon the success of antimicrobial therapy, there was reemergence of infectious diseases due to rise in immunocompromised populace. the resurgence of infectious diseases consequent to human immunodeficiency virus (hiv)­acquired immune deficiency syndrome (aids) pandemic resulted in 1.5 fold increase in infectious diseases mortality between 1980 and 1992. hivaids became the leading cause of mortality amongst infectious diseases in the following two decades [1]. antimicrobial resistance, being unanticipated in its entirety, evolved manifolds to reach dangerous connotations towards panresistance. world health organization (who) theme for world health day 2011 was international journal of medicine and medical research 2017, volume 3, issue 2, p. 39–44 copyright © 2017, tsmu, all rights reserved i. d. khan et al. issn 2413-6077. ijmmr 2017 vol. 3 issue 240 p u b l ic h e a lt h a n d e p id e m io l o g y “antimicrobial resistance: no action today, no cure tomorrow” [2]. since then, despite progressive steps towards concept development, the magnitude of panresistance overshadows control efforts [3, 4]. panresistance is a complete roadblock to years of progress made towards advanced healthcare. with infectious diseases being the second leading cause of mortality worldwide as per global health estimates, the day may not be far when it will be the leading cause of death due to emergence of panresistance emanating the realization of a ‘microbial holocaust’. evolution of panresistance the rise of panresistance is multifactorial. microbial factors include natural evolution of microorganisms conferring increase in virulence, infectivity, pathogenicity and antimicrobial resistance. opportunistic pathogens are crossing host barriers and are now being encountered as emerging pathogens [1, 5, 6, 7]. established pathogens are evolving into panresistant potentially untreatable mutants such as glycopeptide resistant gram negative bacteria, which are resistant to all available antimicrobials including tigecycline and colistin. in addition, totally drug resistant tuberculosis, multidrug resistant malaria and dual oseltamivir­adamantane resistant influenza viruses are emerging [8]. tuberculosis has emerged as the leading cause of mortality amongst infectious diseases overtaking hiv-aids due to the development of resistance. tuberculosis related deaths in 2014 were 1.5 million, surpassing 1.2 million hiv-aids related deaths. host factors include steep rise in immunocompromised populace owing to increased organ transplants, immunodeficiency disorders, neoplasms, old age as well as patients under intensive-care. prescription trend factors include aggressive exposure of multiple antimicrobials to patients harbouring multiresistant microorganisms. rising empiricism in antimicrobial therapy overshadows susceptibility guided therapy, facilitating development of resistance due to selection pressure [9, 10]. panresistant microorganisms can spread resistance-conferring mobile genetic elements to susceptible microorganisms and commensal flora, contributing to the development of a reservoir of antimicrobial resistance in human body. panresistant microorganisms can also colonize inanimate surfaces and create reservoirs from which they can get transmitted in healthcare facilities thereby rendering all patients and healthcare professionals at-risk. human factors involved include a complex interplay of antimicrobial misuse in healthcare facilities due to lack of regulatory commitment in the backdrop of natural mutations which has contributed to the development of panresistance [1, 3]. panresistance is increasingly being reported in gram negative microbes [6, 11]. the south and south-east asia region (sear) has one of the highest prevalence of tuberculosis with one death every few minutes [12]. all forms of resistant tuberculosis viz. multi drug resistant tuberculosis (mdr tb), extremely drug resistant tuberculosis (xdr tb) and totally drug resistant tuberculosis (tdr tb) have been reported. the recent reports of tdr tb from iran, india and italy represent the tip of an iceberg as antitubercular susceptibility testing occurs in only 5% patients worldwide [12-15]. the dots (directly observed treatment short course) program for developing countries has been challenged by the emergence of xdr tb and tdr tb not only due to resistance but also due to limitations of antitubercular susceptibility testing, which is offered only at highly specialized centres. a seven year study on dots plus reported 61% cure, 19% deaths, 18% defaulters, 3% failed treatments and an average delay of 5 months in initiation of therapy [16]. antimalarial resistance to artemisinin and quinine has been re ported in sear and africa [17, 18]. antiviral resis tance to almost all antivirals has been reported particularly in hepatitis b, herpes virus, cytomegalovirus, varicella zoster, influenza and hiv [19–23]. impact of panresistance the emergence of panresistant infectious diseases is becoming a public health problem worldwide. developing countries are facing the brunt in epidemic proportions due to strained public health infrastructure and limited resource allocation to healthcare. the rise of panresistance is discouraging the development of newer antimicrobials under private equity. any new antimicrobial loses economic value in a few years due to emergence of resistance compared to medicines for lifestyle diseases which remain economically rewarding for many years [24, 25]. inadvertent or intended release of panresistant bioweapons against humans, fauna and flora can wreak havoc leading to widespread disruption [26]. the future panresistance is a biological, behavioural, technical, economic, regulatory and educational problem of global concern and combating it will require concerted efforts to preserve the i. d. khan et al. issn 2413-6077. ijmmr 2017 vol. 3 issue 2 41 p u b l ic h e a lt h a n d e p id e m io l o g y i. d. khan et al. efficacy of available antimicrobials. an inten­ sified commitment needs to be taken up on a war footing. knowledge, attitude and practices of general public examples from successful programs such as “antibiotics are not automatic” in france, “get smart” in the us and “do bugs need drugs?” in canada need to be followed in developing countries [27]. health educators, public health specialists, government officials and community leaders should be sensitized about the hazards of using antimicrobials. there should be active community participation in the cause of positive health. citizens must foster sound belief and inculcate positive attitude and responsible behaviour in societal healthcare system. there is long standing need for increasing awareness about approach, operation, decision making and scope of healthcare amongst general population. attitude and expectations need a paradigm shift from ‘instant cure’ and ‘magic pill’ to ‘rational drug therapy’ and ‘evidence based healthcare’. patients should not engage into unjustified requests or arguments or frequent change of doctor’s advice. self ­medication, quack remedies, underdosing and uncompleted regimens should be stopped. left over drugs from the last prescription should not be taken again for a similarly perceived symptom. the society should ensure availability of trained pharmacists through legislation to ensure adherence to prescription safety. hospital infection control nosocomial pathogens evolve under continuous selection pressure to become panresistant. hospital infection control involves monitoring of hospital safety measures such as patient isolation, visitor control, contact precautions, barrier nursing, universal prophylaxis, hand hygiene, environmental surveillance and equipment sterilization in operation theatre, labour room, intensive care, oncology, burns, dialysis and transplant centres. carriers are identified, quarantined and organisms are eradicated from hospital environment. hand hygiene is considered to be the single most important step in controlling spread of panresistant pathogens. compliance is limited due to overbearing pressures of patient volume, time, undue multitasking and paucity of washing infrastructure. hand washing with soap followed by antiseptic handrub should be strongly encouraged as the standard of care for all healthcare practitioners and patients. a broad based policy and standards for infection control in healthcare facilities needs to be implemented. the jaipur declaration on amr-2011 for sear and the chennai de claration for india are efforts to this end [25, 28]. laboratory surveillance laboratory based surveillance of infectious diseases, pathogens, susceptibility patterns, resistance phenotyping, outbreak investigation, hospital environmental surveillance and epidemiological typing is mandated to keep a track of panresistance development. a number of pathogens such as viruses, parasites, certain bacteria and fungi surpass identification under the constraints of resources available in routine labs [1, 5–7]. antimicrobial susceptibility testing for tuberculosis, parasites, viruses and fungi are only available in reference labs which are far and few. unavailability of testing facilities promotes empirical antimicrobial therapy to save the patient, thereby contributing to the development of panresistance. enhancing laboratory capacity with automated phenotypic identification systems, molecular microbiology techniques and biostatistical softwares, precise organism identification to species level, anti­ microbial susceptibility patterns, resistance phenotypes, typing and data analysis has been facilitated. the resistogram generated can be used to guide infection control strategies with other collaborating centres through a worldwide free web repository. the potential of the microbiology lab is largely underutilized in developing countries due to deficiencies in lab equipment and specialized staff. antimicrobial stewardship antimicrobial stewardship including antimicrobial rotation and holiday, combination therapy and standard treatment guidelines have proven to be beneficial [29, 30]. a dynamic antimicrobial policy should specify as to when escalation and de-escalation to reserve antimicrobials such as carbapenems, colistin, tigecycline, vancomycin, teicoplanin and daptomycin, needs to be undertaken. spiralling empiricism, prophylactic antimicrobial usage and attitude to use the best antimicrobial should be discouraged and susceptibility guided therapy be promulgated [1, 5–7]. regular availability of required antimicrobials, prescription audits, formulary restriction, pre-authorization and stop orders should be advocated to ensure policy implementation which in turn should be linked to grant of accreditation to hospitals. a multidisciplinary approach would include building of consensus across issn 2413-6077. ijmmr 2017 vol. 3 issue 242 p u b l ic h e a lt h a n d e p id e m io l o g y clinicians and arbitration of disagreements. the who classification of antimicrobials into key, watch and reserve groups in jun 2017 can form a guideline towards the successful implementation of antimicrobial authorization and prescription prudence [31]. health resource allocation the present situation demands an increase in resource allocation in the health sector to boost healthcare infrastructure, public awareness and accessibility. this would entail accommodative policy for establishment of specialized medical varsities, superspeciality hospitals, specialized laboratories, biocontainment facilities, promotion of antimicrobial research through grants, medical journals, medical societies, involvement of private sector through public private partnership and mass health campaigns. comprehensive standards for surveillance and control should be established in association with international health regulations [32]. national surveillance systems similar to the national nosocomial infection surveillance (nnis) in the us and sentry antimicrobial surveillance program can be instituted [32]. antimicrobial research research on the development of newer antimicrobials has multipronged implications. one, effective antimicrobials would foster prompt treatment of infections caused by resistant pathogens and prevent progression to disseminated infection and sepsis. two, successful therapy will reduce transmission of resistant pathogens. three, chemoprophylaxis can be directed for prevention of infections in susceptible host population. four, behavioural research regarding non-adherence to prescribed drug schedules and self-medication are social issues in which there has been limited research. research needs to be undertaken on these behavioural factors, so that targeted intervention can be planned for bringing about changes at the societal level. public health measures robust public health infrastructure and human resource with strengthened vector control programs, immunization coverage, screening programs, national health programs, rapid outbreak investigation, quarantine and control measures are required. panresistant infectious diseases and resistant pathogens should be made notifiable. effective public health will reduce reliance on antimicrobials and break chain of transmission of resistant microbes [25]. legislation on manufacturing, marketing and dispensing of antimicrobials three important areas of intervention exist at manufacturing, marketing and dispensing of antimicrobials. quality assurance in antimicrobial dosage and efficacy from manufac­ turers and ethical marketing can have profound downstream effects. while regulation regarding prohibition of sale of antimicrobials without proper prescriptions is in place, it is not being implemented. regulatory mechanisms for ensuring good manufacturing practices, responsible marketing and dispensation by pharmacists need to be instituted and strengthened through industrial and marketing audit, and enhanced vigil on pharmacies. antimicrobial and infection control advisory bodies need to be actively involved to integrate surveillance and legislation. role of who who has issued a call for action to halt the spread of amr by introducing a six-point policy package for all countries to combat amr. this includes commitment to a comprehensive, financed national plan with accountability and civil society engagement; strengthening of surveillance and laboratory capacity; ensuring uninterrupted access to essential medicines of assured quality; regulation and promotion of rational use of medicines, including in animal husbandry, and ensuring proper patient care; reduction of antimicrobials usage in food-producing animals; enhancing infection prevention and control; and fosterage of innovations and research and development for new tools [2]. who has also laid down the procedure to establish national laboratory based surveillance including identification of pathogens and diseases of public health importance, creation of network of antimicrobial susceptibility testing (ast) and standardization of involved methodologies. world bodies such as association for prudent use of antimicrobials (apua) and world alliance against antibiotic resistance (waar) are efforts to this end [33]. who has advocated a priority pathogens list in 2017 to highlight a list of bacteria for which newer antimicrobials are urgently required [34]. who has classified antimicrobials into key access, watch group and reserve group to optimize usage guidelines worldwide [35]. conclusions panresistance is emerging in alarming proportions worldwide, thereby threatening the advances made towards public health i. d. khan et al. issn 2413-6077. ijmmr 2017 vol. 3 issue 2 43 p u b l ic h e a lt h a n d e p id e m io l o g y i. d. khan et al. security of the world. there is a dire need to i d e n t i f y t h i s t h re a t , d ev e l o p c o n c e r t e d multipronged strategy, develop infrastructure, foster expertise and take coordinated and urgent steps to tackle the serious public health challenge. it is time for action else we face the consequences of microbial genocide of mankind. resolute conviction towards astute measures with a sustained momentum will hold a promise for safeguarding health of future generations. references 1. khan id, sahni ak, bharadwaj r, lall m, jindal ak, sashindran vk. emerging organisms in a tertiary healthcare set up. med j armed forces india. 2014;70(2):120–128. 2. world health organization. world health day 2011: policy briefs. geneva, who, 2011. http://www. who.int/world-health-day/2011/policybriefs/en/ index.html. accessed 20 jun 2017. 3. jindal ak, pandya k, khan id. antimicrobial resistance: a public health challenge. med j armed forces india. 2014;71(2):178–181. doi:10.1016/j. mjafi.2014.04.011. 4. vijayvergia v, sahni ak, lal m, vijay k, khan id. phenotypic detection of esbl and amp c beta-lactamases in a tertiary care hospital. bang j med sci. 2013;12(4):378–384. 5. khan id, mukherjee t, gupta s, haleem s, sahni ak, banerjee s, et al. ochrobactrum anthropi sepsis in intensive tertiary care. j basic & clin med. 2014;3(1):18–20. 6. khan id, lall m, sen s, ninawe sm, chando la p. multiresistant elizabethkingia meningoseptica infections in tertiary care. med j armed forces india. 2014;71(3):66–67. doi:10.1016/j.mjafi.2014.02.002. 7. khan id, sati a, arif s, mehdi i, bhatt p, jain v, et al. streptococcus mitis/oralis corneal ulcer after corneal transplantation. j basic & clin med. 2016; 5(1):8–10. 8. sheu tg, fry am, garten rj, deyde vm, et al. dual resistance to adamantanes and oseltamivir among seasonal influenza a (h1n1) viruses: 2008­ 2010. j infect dis. 2010;203:13–7. 9. fraser gl, stogsdill p, dickens jd, et al. antibiotic optimization: an evaluation of patient safety and economic outcomes. arch intern med. 1997;157: 1689–94. 10. pelletier ll. hospital usage of parenteral antimicrobial agents: a gradated utilization review and cost containment program. infect control. 1985;6(6):226–30. 11. falagas me, bliziotis ia, kasiakou sk, samonis g, athanassopoulou p, michalopoulos a. outcome of infections due to pandrug-resistant (pdr) gram­negative bacteria. bmc infect dis. 2005;5: 24–8. 12. udwadia zf, amale ra, ajbani kk, rodrigues c. totally drug resistant tuberculosis in india. clin infec dis. 2011. doi:10.1093/cid/cir8898. 13. mahadev b, kumar p, agarwal sp, chauhan ls, srikantaramu n. surveillance of drug resistance to anti-tuberculosis drugs in districts of hoogli in west bengal and mayurbhanj in orissa. indian j 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jun 2017. 32. nnis system. national nosocomial infections surveillance (nnis) system report, data summary from january 1992 through june 2004, issued october 2004. atlanta, ga: centers for disease control and prevention, department of health and human services; 2004. http://www.cdc.gov. accessed 20 jun 2017. 33. carlet j, rambaud c, pulcini c. waar (world alliance against antibiotic resistance): safeguarding antibiotics. antimicrob resist infect control. 2012;1:25. 34. world health organization. global priority list of antibiotic-resistant bacteria to guide research, discovery and development of new antibiotics. http://www.who.int/mediacentre/news/releases/2017/bacteria-antibiotics-needed/en/. accessed 01 sep 2017. 35. world health organization. who model list of essential medicines. 20th list. mar 2017. http:// www.who.int/medicines/publications/essentialmedicines/en/. accessed 01 sep 2017. received: 2017-07-30 i. d. khan et al. 30 o b st e t r ic s a n d g y n e c o l o g y issn 2413-6077. ijmmr 2017 vol. 3 issue 2 international journal of medicine and medical research 2017, volume 3, issue 2, p. 30-33 copyright © 2017, tsmu, all rights reserved n. p. bondarenko et al. doi 10.11603/ijmmr.2413-6077.2017.2.8396 antenatal investigation of ductus venosus velocity as a method of detecting the fetal heart failure, caused by parvovirus b19 infection n. p. bondarenko, a. v. aksonova bogomolets national medical university, kyiv, ukraine background. the article describes the methods and results of investigation of blood flow velocity waveforms in fetal ductus venosus (dv). these studies are used to visualize the degree of fetal heart failure and determine its further clinical course. objective. the study was aimed to predict the development of heart failure in the fetuses that were infected with parvovirus b19 infection during 11-14 gestation weeks by measuring the doppler parameters of blood flow velocity in the dv. methods. our investigation involved 20 pregnant women aged from 18 to 30 years old who were infected with parvovirus b19 infection during the period from 11 to 14 weeks of gestation. the dv was determined by means of color doppler. fetal echocardiography (echocg) was performed by means of the ultrasound scanner philips hd iixe device (usa) using a transabdominal convex probe with the frequency of 3.5 mhz, operating in a cdc mode and the frequency filter at 100 hz. the a-wave directivity evaluation in the dv was investigated according to the guideline principles of the fetal medicine foundation (www.fetalmedicine.com). statistical processing of data was carried out using the package of applied programs microsoft office excel 2016 and statistica 6, stata 12. results. in 16 of 20 (80%) fetuses we did not observe any absent or reversed a-wave flow in the dv during atrial contraction as well as any fetal echocardiographic pathological signs. in 2 (10%) cases a reversed a-wave flow in the dv in a combination with echocg-signs of overload of left side of heart, resulting in enlargement (dilatation) of left atrium and left ventricle were detected. in 2 (10%) cases the presence of a reversed a-wave flow in the dv and echocg-signs of fetal heart failure (reduction of cardiac output, significant dilatation of left ventricle) were evidenced. the results of the study confirm that with the expansion of fetal nuchal translucency thickness, the systolic blood flow velocity in the dv increases with the correlation coefficient r=0.594, which proves a linear dependence between these two ultrasonography parameters. conclusions. the linear correlation between the presence of a reversed a-wave blood flow velocity in the dv and the overload of left side of fetal heart, development of heart failure (20% of the total number of examined women) were proved. key words: ductus venosus (dv); heart failure; parvovirus b19 infection; peak systolic blood flow velocity; fetal echocardiography (echocg). corresponding author: anastasiia aksonova, department of obstetrics and gynaecology №1, national bogomolets national medical university, 13 t. shevchenko, kyiv, ukraine, 03150 phone number: + 380673056011 e-mail:aks.anastasiia@gmail.com introduction parvovirus b19 infection is a potentially lifethreatening disease [1] for a fetus, especially during the first two trimesters of pregnancy that can cause a variety of signs of its damage (severe anemia, ventriculomegaly, hypertrophic myocardiopathy and pericardial effusion, nonimmune fetal hydrops, hydropic or nonhydropic intrauterine fetal death, intrauterine growth retardation, thrombocytopenia, meconium peri tonitis, hepatic calcifications, abnormal long­ term neurodevelopment, ascites, placentomegaly, etc.) [2]. the risk of fetal death depends on gestational age in cases of infection. according to gestational age, maternal infection in the first trimester leads to fetal death in 19%, in 13–20 weeks – 15% and after 20 weeks – 6% of cases [3]. it is quite widespread. approximately 1–5% of women is susceptible to parvovirus and develops serologic evidences during pregnancy period; the frequency of morbidity is rising to 3-34% in epidemic periods; about 50% of women have an asymptomatic course of the disease [4]. infection with parvovirus b19 affects many organs and systems, mostly cardiovascular system. the transmission rate of maternal parvovirus b19 infection to fetus is 17% to 33% 31 o b st e t r ic s a n d g y n e c o l o g y issn 2413-6077. ijmmr 2017 vol. 3 issue 2 [5]. transplacental viral transmission is probably explained by the presence in villous trophoblastic cells of placental tissues the maximum level of neutral glycolipid known as blood group antigen p (globoside), which also serves as a receptor for b19, during the first two trimesters of pregnancy. cardiomyocytes are the most important target cells for parvovirus. p-antigen expressed on fetal cardiac myocytes enables the virus to infect myocardial cells and induce myocarditis that aggravates cardiac failure. fetal heart failure may be caured by severe ane mia but may often be associated with myocarditis, which can cause arrhythmias or even cardiac arrest without evidence of anemia, cardiac failure or hydrops. infection within myocardium often results in acute inflammation, but may also lead to non­inflammatory damage of cells, and as a consequence, to infection-related cardiomyopathy. virus b19 enters the endothelium cells utilizing a globoside and its coreceptors a5 pi-integrin and ku80 and after connection with a receptor pass to the state of persistent infection in the endothelium of various organs, including heart. in consequence, numerous copies of the viral genome of b19 are determined in the endothelium of intramyocardial arterioles, capillaries and post-capillary venules, initiating inflammation process indu­ ced by the continuous expression of pro-inflammatory cytokines (tnf­a, interleukins (il): il-6, il-8, il-2)), igg, soluble interleukin 2 receptor, leukotrienes and prostaglandins [6]. the direct cytopathic effect of parvovirus, apoptosis, activation of innate and adaptive immune response lead to endothelial dysfunction, followed by ventricular remodeling and development of dilated cardiomyopathy. the asymptomatic course and late detection of parvovirus b19 infection during antenatal period are two real problems which may lead to the development of cardiac fetal insufficiency. the advanced ultrasound allows characterizing many complex conditions which are necessary to evaluate and understanding va rious pathologies that contribute to heart failure in fetus. doppler ultrasound tools have improved our understanding of fetal circulation and patho-physiological mechanism that controls fetal circulation. according to the literature, ductus venosus (dv) doppler measurements can give information on pregnancy courses, its prognosis and can therefore be recommended as a part of the routine workup of pregnancies, complicated by viral infections, such as parvovirus b19. the article describes the methods and results of investigation of blood flow velocity waveforms in fetal dv. these studies are used to visualize the degree of fetal heart failure and determine its further clinical course. the aim of our research was to predict the development of heart failure in the fetuses that were infected with parvovirus b19 infection during 11–14 gestation weeks by measuring the doppler parameters of blood flow velocity in the dv. methods 20 pregnant women, who were infected with parvovirus b19 during the period from 11 to 14 weeks of gestation, were involved into the study. the inclusion criteria were: age from 18 to 30 years old; singleton pregnancy; absence of extragenital pathology events in the suband decompensation stages; gestation period from 11 to 14 weeks; no episode of a threatened abortion during pregnancy; absence of fetal chromosomal abnormalities. the dv was identified by means of color doppler. given that its localization at the cephalic end of intraabdominal portion of umbilical vein we used two approaches in an oblique transverse and sagittal imaging planes through the fetal abdomen to visualize the communication between the umbilical vein and the inferior vena cava. the presence of characteristic high-velocity signals in the color doppler b-mode proved the identification. our record was taken in the inlet of dv with an insonation ange as near to the long axis of the vessel as possible. blood flow pat­ terns merely proved changes in the pressure between the vessel and the right atrium throughout the cardiac cycle. after activating the color doppler using a low velocity setting (<0.24 m/s), three vessels were detected in cross­section: the abdominal aorta, the inferior vena, and the dv. unlike the superior and inferior vena cavae in which there may be a re verse flow during atrial systole, the flow through the dv proves continuous forward flow towards the heart. normal flow in the dv was low velocity and triphasic consisting of maximal velocities during ventricular systole (s -wave) in relation with a rapid filling of the atria; the second peak (d-wave) corresponded to the early ventricular diastole, and the flow velocity was minimal during atrial contraction (a-wave). the a-wave directivity evaluation in the dv was investigated according to the guideline principles of the fetal medicine foundation (www. fetalmedicine.com). the waveforms were clasn. p. bondarenko et al. 32 o b st e t r ic s a n d g y n e c o l o g y issn 2413-6077. ijmmr 2017 vol. 3 issue 2 sified as normal or abnormal depending on whether the a-wave lowest forward velocity during atrial contraction in late diastole was positive or absent/reversed, respectively. absent or reverse a-wave in the dv proved fetal cardiac failure. fetal echocardiography (echocg) included assessment of the position of heart, the four­chamber view, the outflow tracts and the venous return to the heart. echocg was performed by the ultrasound scanner philips hd ii xe device (usa) using a transabdominal convex probe with the frequency of 3.5 mhz, operating in a cdc mode and the frequency filter at 100 hz. statistical processing of data was carried out using the package of applied programs microsoft office excel 2016 and statistica 6, stata 12. statistically significant differences at p<0.05 were considered. results analyzing the results of investigation, in 16 of 20 (80%) fetuses we didn’t observe any absent or reversed a­wave flow in the dv during atrial contraction as well as any fetal echocardiographic pathological signs of cardiac damage (fig. 1). in 2 (10%) cases the reversed a­wave flow in the dv in a combination with echocg-signs of overload of left side of heart, resulting in enlargement (dilatation) of left atrium and left ventricle were detected. in 2 (10%) cases the presence of reversed a-wave flow in the dv and echocg­signs of fetal heart failure (reduction of cardiac output, significant dila tation of left ventricle) were evidenced (fig. 2). in 20% of the examined women (4 cases) with the presence of reversed a-wave blood flow velocity in the dv we also made a ma­ thematical correlation between the peak systolic blood flow velocity in the dv and the fetal nuchal translucency thickness, as one of the echocg markers of fetal heart failure. the findings confirm that with the expansion of fetal nuchal translucency thickness there is an increased peak systolic blood flow velocity in the dv with the correlation coefficient r=0.594 (the coefficient of determination is r2=0.3534 with normal range r=0.161 (r2=0.0258)) that proves a linear dependence between these two ultrasonography parameters (fig. 3): the coefficient of determination is the percentage of response variable variation that is explained by a linear model (dependence on the changes of one parameter from another in percentages) – in our case 0,3534 (35,34%) which means, that the peak systolic blood flow velocity changes in the dv 35.34% depends on changes in the fetal nuchal translucency thickness and could predict the future fetal cardiac compromise (p<0.05). discussion dynamic observation of gestational process allows relate some features of ultrasound pattern evidenced in 11–14-week fetuses with an increased risk of heart failure development. parvovirus b19 infects primarily the erythroid cell line and may therefore cause fetal anemia and consequently cardiac failure with hydrops fetalis in some cases. doppler measurement technics today can modify treatment so that the number of fetuses infected with virus b19 and its attendant risks can be minimized, without missing the opportunity for timely, lifesaving intervention when it is warranted. fig. 1 (leftward). normal blood flow velocity measured in the ductus venosus during 12 gestation weeks in the woman infected with parvovirus b19 fig. 2 (rightward). reversed a­wave flow waveform in the ductus venosus during 12-13 gestation weeks measured in the woman infected with parvovirus b19 n. p. bondarenko et al. 33 o b st e t r ic s a n d g y n e c o l o g y issn 2413-6077. ijmmr 2017 vol. 3 issue 2 increased fetal nuchal translucency thickness was established to be a myocardial dysfunction and/or fetal anemia [7]. the correlation between the increased nuchal translucency and the presence of abnormal blood flow profile in the dv was proved. the measu­ rement of peak systolic blood flow velocity in the dv is the latest tool of non-invasive monitoring of fetuses at risk for anemia and cardiac insufficiency due to parvovirus b19. in the article, the increased fetal nuchal translucency thickness was found with reversed flow in the dv during atrial contraction, this findings were helpful indicators of the presence of fetal heart failure. in summary, the linear correlation between the presence of a reversed a­wave blood flow velocity in the dv and the overload of left fetal heart, the development of heart failure (20% of the total number of examined women) was proved. conclusions parvovirus b19 infection has a direct pathological effect on the endothelium of intra myocardial arterioles, capillaries and post-capillary venules. the development of dilated cardiomyopathy and heart failure are two pathological consequences of b19 cardiac damage. the most reliable way to diagnose fetal heart failure during antenatal period is the doppler and echocg investigation of blood flow velocity waveform in the dv and the measurement of nuchal translucency thickness. in case of left-sided heart insufficiency occur the volume overload of right side of fetal heart with increased pressure in the dv, the presence of a reversed a­wave flow velocity in the dv and increased peak systolic blood flow, the expansion of fetal nuchal translucency thickness and decreasing left ventricular isovolumic relaxation time. a linear relationship between the increased fetal nuchal translucency thickness and the peak systolic blood flow velocity in the dv was determined. according to the results of the study, we can conclude that there is a direct correlation between the appearance of a reversed a-wave blood flow in the dv and the development of fetal heart failure. fig. 3. correlation curve between the peak systolic blood flow velocity in the ductus venosus (cm/s) and fetal nuchal translucency thickness (mm) gestation weeks in the woman infected with parvovirus b19 figure 2 (rightward). reversed a-wave flow waveform in the ductus venosus during 1213 gestation weeks measured in the woman infected with parvovirus b19 in 20% of the examined women (4 cases) with the presence of reversed a-wave blood flow velocity in the dv we also made a mathematical correlation between the peak systolic blood flow velocity in the dv and the fetal nuchal translucency thickness, as one of the echocg markers of fetal heart failure. the findings confirm that with the expansion of fetal nuchal translucency thickness there is an increased peak systolic blood flow velocity in the dv with the correlation coefficient r=0.594 (the coefficient of determination is r2=0.3534 with normal range r=0.161 (r2=0.0258)) that proves a linear dependence between these two ultrasonography parameters (fig. 2): the coefficient of determination is the percentage of response variable variation that is explained by a linear model (dependence on the changes of one parameter from another in percentages) – in our case 0,3534 (35,34%) which means, that the peak systolic blood flow velocity changes in the dv 35.34% depends on changes in the fetal nuchal translucency thickness and could predict the future fetal cardiac compromise (p<0.05). fetal nuchal translucency thickness (mm)  normal psv ■ patology psv references 1. arabzadeh s. human parvovirus b19 in patients with beta thalassemia major from tehran, iran. blood res. 2017;52(1):50–54. 2. zavattoni m, paolucci s, sarasini a. diagnostic and prognostic value of molecular and serological investigation of human parvovirus b19 infection during pregnancy. new microbiol. 2016;39:181–185. 3. lamont rf. parvovirus b19 infection in human pregnancy. bjog. 2011;18(2):175–186. 4. kerr jr. the role of parvovirus b19 in the pathogenesis of autoimmunity and autoimmune disease. j clin pathol. 2016;69(4):279–91. 5. crane j. parvovirus b19 infection in pregnancy. j obstet gynaecol. 2014;36(12):1107–1116. 6. verdonschot j. relevance of cardiac parvovirus b19 in myocarditis and dilated cardiomyopathy: review of the literature. european journal of heart failure. 2016;18,1430–1441. 7. hichijo a, morine m. a case of fetal parvovirus b19 myocarditis that caused terminal heart failure. case reports in obstet and gynecol. 2014:1–4. received: 2017-10-19 n. p. bondarenko et al. 26 su r g ic a l d is e a se s issn 2413-6077. ijmmr 2018 vol. 4 issue 1 doi 10.11603/ijmmr.2413-6077.2018.1.9112 pathogenetic approaches and ways of prevention of thromboembolic complications in trauma patients l. yu. ivashchuk i. horbachevsky ternopil state medical university background. the study of thromboembolic complications prevention in trauma patients, who underwent surgery, is presented in the research. objective. patients were examined in the department of traumatology of ternopil municipal hospital. the first group, 263 people, (18.6 %) consisted of patients with polytrauma and unfavorable prognosis and significant disease severity. the second group comprised patients with combined trauma, 462 people (32.8 %) – a doubtful prognosis for life. the third group, 685 people (48.6 %) involved patients with isolated trauma and positive treatment outcome. methods. all patients, besides general clinical examination, underwent evaluation of the number of platelets, clotting time, duration of bleeding and study of coagulation (prothrombin index, prothrombin activity thrombotest, total fibrinogen, fibrinogen a, activated recalcification time). the venous system of lower limbs was examined using distal ascending phlebography, color doppler and duplex ultrasonography simens acusson x 300. results. a comprehensive prophylaxis of thromboembolic complications was carried out using low-molecular weight heparin as well as essential complex kinetic treatment. bemiparin in an appropriate dose was administered once a day for 10-14 days of postoperative stay in the hospital. for the patients with moderate risk and high surgery risk (major surgery, over 40 years old in age, obesity, and serious comorbidities) bemiparin was administered at a dose of 5000-7500 iu per day during patients’ stay in the hospital. in individuals with sub-acute and chronic thrombophlebitis of subcutaneous veins the surgical prophylaxis of thromboembolic complications was performed. conclusions. the combination of physical, drug and surgical prophylaxis prevented the thromboembolic complications in trauma patients. key words: thromboembolism; trauma; polytrauma; prevention; bemiparin. international journal of medicine and medical research 2018, volume 4, issue 1, p. 26-30 copyright © 2018, tsmu, all rights reserved corresponding author: larisa ivashchuk, department of general and minimally invasive surgery, i. horbachevsky ternopil state medical university, 1 maydan voli, ternopil, 46001, ukraine e-mail: ivashchuk_lu@tdmu.edu.ua phone number: +380679401031 introduction thromboembolism is one of the most formidable complications after surgical interventions that cause high mortality. the frequency of thromboembolism depends on the duration of traumatic procedures and increases with the larger its dimension and expansion of indications for them, especially in the elderly patients [1]. the situation in the departments of traumatology and orthopedics, where thromboembolic complications are present in more than a half of patients, is especially threatening [5]. diagnosis, treatment and prediction of the consequences of thromboembolism in this category of patients are accompanied by considerable difficulties. it is noteworthy that the largest number of fatal cases (up to 75.4 %) was registered not at the day of injury, but in 3-5 days after it or later [2, 4]. the etiology of thromboembolism has a multifactorial nature: varicose veins of lower extremities, phlebothrombosis of small pelvis veins, venous stasis of lower extremities, postoperative violation of blood rheology are the most important. significant cause of thromboembolic complications is the body's response to surgical trauma, which is accompanied by spasm of peripheral vessels, hypercoagulation and vascular wall injury, especially in cases of surgeries on lower extremities, hip joint and pelvic bones [6, 7, 8]. the deep veins thrombosis of lower extremities and pelvis complicates the postoperative period by 10-30% in the elder age group [3]. l. yu. ivashchuk 27 su r g ic a l d is e a se s issn 2413-6077. ijmmr 2018 vol. 4 issue 1 in many patients, the first clinical manifestation of deep vein thromboembolism may be thromboembolism of pulmonary artery [11]. with age in the body there are a number of hemodynamic (decreased shock, respiratory minute volume and blood circulation, increased peripheral vascular resistance) and hematologic changes (increase of aggregation and adhesion ability of platelets, activity of plasma coagulation factors, and concentration of fibrinogen), which contribute to the presence of deep vein thrombosis [9, 10]. methods the patients of the department of traumatology of the ternopil city communal emergency hospital were examined. over the last year, 1410 patients of different age groups were hospitalized: 588 (41.7 %) men, 822 (58.3 %) women. during the study the patients were divided into 3 groups depending on the severity of the injury. the first group, 263 men (18.6 %), consisted of patients with polytrauma and unfavorable prognosis due to the significant disease severity. the second group consisted of patients 462 people (32.8 %), with a combined injury and doubtful prognosis for life. the third group, 685 people (48,6 %), involved patients with isolated trauma and positive treatment outcome (table 1). the patients of the first group were in a state of severe combined shock. they were given resuscitation aimed at correction of hemodynamics and function of external respiration. the comprehensive examination of those patients included clinical, laboratory and radiological methods by means of laparocentesis, thoracentesis, sonography, computed tomography scan. in diagnostics of prolonged bleeding without delay, against the background of antishock therapy, surgical interventions aimed at stopping it were performed. on the damaged segments of musculoskeletal system, with satisfactory standing of fragments, plaster casts and skeletal extracts were used in the presence of displacements. in the second group of patients, treatment also began with actions for vital signs support (fight against bleeding, respiratory failure). all measures were carried out against the back_ ground of intensive anti-shock therapy and elimination of dominant internal organs damage after appropriate preoperative training. later, with satisfactory hemodynamic parameters, urgent surgeries were carried out on injuries of the musculoskeletal system. in cases of surgical interventions in the patients of the third group with severe predominant trauma of musculoskeletal system, among which there were fractures of thigh, leg bones, shoulder, forearm, foot, brush, shoulder girdle, metal osteosynthesis was carried out. exceptions were the cases of severe placement and tear of limbs. such patients underwent a primary surgical treatment of the wound or limb amputation without delay. all patients, besides general clinical examination, underwent evaluation of the number of platelets, clotting time, duration of bleeding and study of coagulation: prothrombin index, prothrombin activity thrombotest, total fibrinogen, fibrinogen, fibrinogen a, activated recalcification time. the venous system of lower extremities was studied by means of distal ascension phlebography, color dopplerography and duplex ultrasonography simens acusson x 300. accompanying pathology of cardiovascular, respiratory and endocrine systems was found in 1167 (82.8 %) persons. in patients with decompensation of accompanying pathology (coronary heart disease, hypertension, non-specific chronic diseases of lungs, diabetes mellitus), preoperative preparation was conducted in a hospital setting. the number of surgical interventions during the period of research in the department is 865 surgeries, including 410 urgent. the other patients were treated conservatively (table 2).fig. 1. the number of trauma patients, who were hospitalized. table 1. the number of trauma patients, who were hospitalized group number of patients % of patients і 263 18,6 іі 462 32,8 ііі 685 48,6 l. yu. ivashchuk 28 su r g ic a l d is e a se s issn 2413-6077. ijmmr 2018 vol. 4 issue 1 results in the examined patients, a number of factors of postoperative thrombotic complications were defined: thrombotic complications in history, coagulation disorders, cardiovascular and malignant diseases, overweight, traumatic complications, and traumatic nature. malignant diseases, breast cancer, prostate and rectum cancer were most commonly associated with pathological fractures of femur and shoulder bones. chronic cardiac insufficiency of the і-іі degree was diagnosed in 651 (46.2 %) patients, varicose enlargement under skin of lower limbs veins – in 624 (42.2 %) patients. obesity was evidenced in 237 (16.8 %) patients. hypercoagulation was revealed in 270 (18.2 %) patients. changes in the coagulogram did not always correspond to the severity and features of phlebothrombosis. only in 6 out of 33 pa_ tients fibrinogen b was present within 8 days after the surgery. a slight increase in fibrinogen a was observed in 22.3 % and significant one (up to 9.9 g/l) in 24.2 % of patients. in all other patients, its content was decreased up to 1.45 g/l. the changes in other indicators were not significant that did not cause the need for additional instrumental survey methods. diseases of thrombophlebitis of subcutaneous or deep veins of lower extremities were revealed in 72 (15.3 %) persons. the choice of anesthetic method also affected the number of thrombotic complications. one of the main causes of thromboembolic complications in trauma patients in the postoperative period is spasm of peripheral vessels caused by operational stress, which leads to tissue ischemia and thrombosis. the stress response of the body in general with anxiety is the result of insufficiency of blockade of nerve impulses that are transmitted through the sympathetic nervous system. the number of phlebothrombosis in patients who underwent surgery under general anesthesia with artificial ventilation was high – 22 cases (68.3 %). the decrease in the number of complications took place due to significant reduction of stress pulses in cases of spinal anesthesia (9 patients (27.2 %)) and conductive anesthesia in combination with intravenous anesthesia (2 patients (4.5 %)). discussion prevention of thrombotic complications was aimed at elimination of pathogenetic parts of phlebothrombosis, such as decreased blood flow, increased blood coagulation, damage of vascular wall and dependence on the risk factors presence. the mechanism of action was physical, drug and surgical methods of prevention of thrombophlebitis of lower extremities. over time, table 2. types of surgical interventions carried out in the department of traumatology type of surgery quantity number of thrombo-embolic complications 1. surgery of the segments of musculoskeletal system: 17.2 % 1.1. open reposition and metal osteosynthesis 376 1.1.1. intramedullary blocking and metal osteosynthesis 44 1.1.2. external fixing devices 25 1.1.3. repositioning 12 1.1.4. sketch plates 295 1.2. primary surgical treatment and metal osteosynthesis 74 1.3. primary surgical treatment without metal osteosynthesis 191 10.0 % 2. joints surgery 93 44.2 % 2.1 open reposition and metal osteosynthesis 38 2.2 removal of metal fixators 3 2.3 arthroscopy 3 2.4 endoprothesis 49 3. reconstructive surgery 31 24.1 %3.1. of soft tissues 27 3.2. of bones 4 4. removal of metal sections 56 5. minimally invasive intervention 19 6. palliative treatment of metastatic bone lesion 12 4.5 % 7. other 10 l. yu. ivashchuk 29 su r g ic a l d is e a se s issn 2413-6077. ijmmr 2018 vol. 4 issue 1 these methods were applied preoperatively, sub-operatively and postoperatively. preventive measures were taken on the 1st–2nd day of hospital stay. all patients fully underwent a prophylaxis of thromboembolic complications by means of low molecular weight anticoagulants and compulsory complex of kinetic therapy. bemiparin (low molecular weight heparin (lmwh), zibor 2500, berlin-chemie ag) at an appropriate therapeutical dose was prescribed from the 1st day of patients’ stay in the hospital for 10-14 days of the postoperative period once a day subcutaneously in the stomach area. for the patients with moderate risk and high surgery risk (major interventions, over 40 years old in age, obesity, and serious co-morbidities) bemiparin was prescribed at a dose of 50007500 iu per day while patients stay in the hospital [12, 13]. in case of changes of venous tract of legs, in the patients with a great anamnesis, elastic stockings and elastic bandings were worn. suboperative procedures into ankle joint, early arousal of the patient after surgery, medical physical exercises were performed for all patients. venotonics (detralex, phlebodia-600, etc.) were used in the complex treatment of the patients with chronic venous insufficiency [14]. in persons with subacute and chronic thrombophlebitis of subcutaneous veins the surgical prophylaxis of thromboembolic complications was performed. a crossectomy was carried out in 9 (1.9 %) patients, as the first stage before surgical intervention. not taking into account the complex of preventive measures, in 4-5 days after surgery, in 10 patients (1.1 %) thrombophlebitis of shin varicose veins, in 12 (1.3 %) – iliofemoral thrombosis developed, in 8 (0,9 %) patients deep thrombosis of lower extremities veins was diagnosed. drug therapy was effective in 16 people. 4 patients had ligation of thighs large subcutaneous vein. in 1 case, small intestinal tract was observed. there were no cases of lethal death associated with thromboembolic complications. no hemorrhagic complications were present. thus, the development of pathogenetic mechanisms of thromboembolic complications in the complex measures for their prevention allowed decreasing the number of postoperative complications and mortality in trauma patients. conclusions in trauma patients, who undergo surgical intervention, a high risk of thromboembolic complications is established that is a prerequisite for the preventive measures. prevention of thromboembolic compli cations should be complex and individual, taking into account the nature of the injury, the dimension of surgical intervention, and the associated risk factors. the combination of physical, drug and surgical prophylaxis prevent thromboembolic complications in trauma patients. l. yu. ivashchuk патогенетичні підходи та шляхи профілактики тромбоемболічних ускладнень у травматологічних хворих л. ю. іващук тернопільський державний медичний університет імені і. я. горбачевського, тернопіль, україна вступ. розлянуто питання профілактики тромбоемболічних ускладнень у травматологічних хворих. мета. пацієнтів відділення травматології тернопільської міської лікарні було поділено на кілька груп групи. перша група налічувала 263 особи (18,6%) з політравмою, несприятливим прогнозом і у тяжкому стані. другу групу (462 хворих або 32,8% вибірки) склали пацієнти з комбінованою травмою та сумнівним прогнозом для життя. третя група налічувала 685 осіб (48,6% вибірки) пацієнти із ізольованою травмою та сприятливим прогнозом. методи. усім пацієнтам, окрім загального клінічного обстеження, визначали наступні показники: протромбіновий індекс, протромбінову активність, тромботест, загальний фібриноген, фібриноген в, фібриноген а, активований час рекальцифікації. венозну систему нижніх кінцівок було досліджено за допомогою дистальної висхідної флебографії, кольорового допплерівського та дуплексного узд із simens acusson x 300. результати. усім хворим у повному об’ємі було проведено профілактику тромбоемболічних ускладнень шляхом застосування низькомолекулярних гепаринів і обов’язковим комплексом кінетерапії. беміпарин у відповідній дозі вводили один раз на день протягом 10-14 днів під час післяопераційного перебування в стаціонарі. для пацієнтів з помірним і високим ризиком (обширне оперативне втручання, вік понад 40 років, ожиріння і серйозні супутні захворювання) беміпарин вводили у дозі 5000-7500 мо на добу під час перебування пацієнтів у стаціонарі. у осіб з помірно гострим і хронічним тромбофлебітом підшкірних вен проводили хірургічну профілактику тромбоемболічних ускладнень. висновки. поєднання фізичної, фармакологічної та хірургічної профілактики запобігало тромбоемболічним ускладненням у травматологічних хворих. ключові слова: тромбоемболія; травма; політравма; профілактика; беміпарин. 30 su r g ic a l d is e a se s issn 2413-6077. ijmmr 2018 vol. 4 issue 1 boyko vv. assessment of the risk of venous thrombosis and embolism in surgical patients. clinical surgery. 2003;8:5-8 [in russian]. 9. kakkar ak, williamson rcn. prevention of venous thromboembolism in cancer patient. semin. thromb. hemost. 2009;25:239-243. 10. geerts wh, pineo gf, heit ja. prevention of venous thromboembolism. the seventh accp conference on antithrombotic and thrombolytic therapy. chest. 2004;126:338-400. 11. suchkov ia, martinez-gonzalez j, schellong sm, garbade t, falciani m. bemiparin dvt study group. comparison of once-daily bemiparin with twice-daily enoxaparin for acute deep vein thrombosis: a multicenter, open-label, randomized controlled trial. clinical drug investigation. 2018 feb 1;38(2):181-9. 12. ciccone mm, cortese f, corbo f, corrales ne, al-momen ak, silva a, zito a, pinto m, gesualdo m, scicchitano p. bemiparin, an effective and safe low molecular weight heparin: a review. vascular pharmacology. 2014 jul 1;62(1):32-7. 13. muñoa l, gonzález ab, de rada pd, valenti a, valenti jr. rivaroxaban is as efficient and safe as bemiparin as thromboprophylaxis in knee arthroscopy. musculoskeletal surgery. 2014 jun 1;98(1): 21-5. 14. kuznetsov mr, sapelkin sv, boldin bv, leont'ev sg, neskhodimov la. recanalization of lower-limb deep veins as an index of efficacy of treatment for acute venous thrombosis. angiology and vascular surgery. 2016; 22(3):82-8 [in russian]. received: 2018-05-14 l. yu. ivashchuk references 1. vinnik yua, volos yub, shmanko yuv. prevention of thromboembolic complications in cancer patients. x congress of oncologists of ukraine: materials of congress. kyiv. 2001;140-145 [in russian]. 2.. grint v ga, tantsyura vp, peschanyi rye, vysokiy ag. the role of hemodynamic disorders in the choice of tactics of surgical treatment of victims with combined trauma of the chest and limbs. journal of emergency and restorative medicine. 2011;5(2): 230-232 [in russian]. 3. yefetova ts. prevention of microcirculatory disorders caused by operational trauma and associated complications in radical surgery for stomach cancer. clinical surgery. 2012;8:37-41 [in russian]. 4.. maz enko ov, kuzmin vyu. analysis of mortality victims with isolated and combined trauma. clinical surgery. 2008;12:21-23 [in ukrainian]. 5.. st odubtseva mb, demyanenko en, shpakov ei. intensive therapy of pulmonary embolism in cancer patients using fractiparin. x congress of oncologists of ukraine: materials of congress. kyiv. 2001; 56-68 [in russian]. 6. protsyk ai. the fundamental approach to providing medical care to victims of prehospital polytrauma. clinical surgery. 2015;4:28-29 [in ukrainian]. 7.. savel v vs. prevention of postoperative venous thromboembolic disorders. the russian consensus. 2010; 20 [in russian]. 8. sayenko vf, mazur ap, grubnik vv, fillipenko va, kobza i i, popik mp, kalinin og, krivoruch ko ia, 47 b io m e d ic a l s c ie n c e s issn 2413-6077. ijmmr 2019 vol. 5 issue 1 doi 10.11603/ijmmr.2413-6077.2019.1.9737 probing breast cancer therapeutic responses by dna content profiling b.i. gerashchenko1*, k. salmina2, j. eglitis3, j. erenpreisa 2 1 – r.e. kavetsky institute of experimental pathology, oncology and radiobiology of the national academy of sciences of ukraine, kyiv, ukraine 2 – latvian biomedical research and study centre, riga, latvia 3 – university of latvia, riga, latvia background. discrepancies in the interpretation of breast cancer therapeutic responses still exist mainly because of lack of standardized assessment criteria and methods. objective. dna content profiling of cells in the affected (cancerous) tissue before and after neoadjuvant chemotherapy (nac) was applied to facilitate interpretation of therapeutic responses. methods. both diagnostic biopsy and operation materials representing the tissue of primary tumors surgically removed after nac were subjected to dna image cytometry. polyploidy and aneuploidy in dna histograms were evaluated with a prognostic auer typing. stemline dna index (di) values and percentages of cells that polyploidize (>4.5c) were also determined. immunofluorescence staining was applied to evaluate proliferation (ki-67), invasiveness (cd44), and self-renewal factors characteristic for stem cells (sox2 and nanog). results. dna content profiles of 12 breast cancer cases, of which 7 were triple-negative, revealed the features of tumor non-responsiveness to nac in 7 cases, of which 5 were triple-negative. among non-responsive cases there were 3 cases that showed enhanced polyploidization, suggesting the negative nac effect. near-triploid (di=1.26-1.74) triple-negative cases were determined as most resistant to nac. cycling near-triploid cells may contribute to the excessive numbers of >4.5c cells. polyploid cells were positive for ki-67, cd44, sox2, and nanog. conclusions. dna content profiling data provide additional helpful information for interpreting therapeutic responses in nac-treated breast cancers. polyploid tumor cells possessing stem cell features can be induced by nac. because nac effects in some cases may be unfavorable, the use of the further treatment strategy should be carefully considered. key words: breast cancer; polyploid cells; near-triploidy; dna content profiling; therapeutic response; cancer stem cells. corresponding author*: bogdan i. gerashchenko, md, ph.d. r.e. kavetsky institute of experimental pathology, oncology and radiobiology of nasu, 45 vasylkivska str., kyiv 03022, ukraine phone: +380 44 2571177, fax: +380 44 2581656 e-mail: biger63@yahoo.com introduction neoadjuvant therapy that was initially designed for the preoperative treatment of patients with locally advanced breast cancer [1] presently provides a good opportunity to evaluate therapeutic response of primary tumors and customize subsequent conventional non-surgical treatment [2−5]. therapeutic response is usually assessed by means of histological examination of the affected breast tissue stained with hematoxylin and eosin. to date, most histological criteria consider the absence of invasive cancer cells in the breast and regional lymph nodes as a pathologic complete response (pcr) [3, 4]. contrary to the patients, who have partial or no response, those patients, who achieve pcr, largely have favorable prognosis (improved long-term, diseasefree, and overall survival) [5]. although the histological assessment of therapeutic responses significantly helps to determine prognosis in breast cancer, the problem caused by discrepancies in the interpretation of results obtained from different pathologists remains unresolved mainly because of lack of standardized assessment criteria and methods [4, 5]. moreover, the term pcr still needs to be applied in a consistent, standardized manner [6]. breast cancer as many other solid tumors is predisposed to high degrees of aneuploidy (abnormal chromosome number) [7, 8] that correlates with the resistance to anti-cancer treatment and poor prognosis [9]. to minimize misinterpretations in histopathologic scoring of therapeutic responses, dna content profiling of cells in the affected breast tissue before and after neoadjuvant therapy seems to be a good option since this technique is capable of detecting tumor cell dna aneuploidy that may international journal of medicine and medical research 2019, volume 5, issue 1, p. 47-57 copyright © 2019, tnmu, all rights reserved b.i. gerashchenko et al. 48 b io m e d ic a l s c ie n c e s issn 2413-6077. ijmmr 2019 vol. 5 issue 1 often be unseen by histopathologists. in other words, the advantage of the technique is in detection of very small numbers of aneuploid tumor cells in a large cohort of analyzed cells. in this regard, the intra-tumor genetic diversity that can potentially be manifested in clonal heterogeneity is worth mentioning [10, 11]. there are situations when in a tumor the drugsensitive dominant clones can be eliminated, while the minor resistant clones or sub-clones can be hiding in a dormant state followed by accelerated cell proliferation. also, to date, the issue of detecting and quantifying the resistant tumor cells that polyploidize in response to genotoxic therapies is of pivotal importance [12, 13]. cytometric determination of dna content in tissue specimens for detecting malignancies, monitoring responses to therapy, and prognosing disease outcome is very important and demanding [12, 14]. notably, cell nuclei isolated from aneuploid-gated distribu tions by means of fluorescence activated cell sorting can be subjected to single-cell dna and rna sequen cing to study breast cancer chemoresistance evolution [15]. in this study, the image cytometry-based dna content profiling of cells of the affected breast tissues of women predominantly with locally advanced stage iii disease before and after neoadjuvant chemotherapy (nac) with paclitaxel and doxorubicin was performed. dna histograms were categorized into 4 types (i−iv) according to auer [16] to estimate the grade of malignancy for diagnostic and prognostic purposes. positions of stemline dna aneuploidy peaks as well as the number of polyploidizing cells (>4.5c) were taken into account while analyzing dna content profiles. the dna content profiling data were compared with miller-payne histopathologic grades specifically designed for evaluation of therapeutic responses [17]. one of the therapy-resistant cases that showed extensive polyploidization was chosen for immunocytochemical staining to evaluate cell proliferation (ki-67), invasiveness (cd44), and self-renewal factors typical for stem cells (sox2 and nanog). methods patients and clinico-pathologic information the study involved 12 breast cancer patients (age range: 33–75), who underwent diagnostic procedures at the latvian oncology centre of the riga east university hospital in 2014 and 2015. the tissue specimens were collected after the patients’ informed consent was obtained in accordance with the regulations of the committee of medical ethics of latvia. the clinico-pathologic information about these patients, such as staging (ranged from i to iv), anatomic extent of disease according to tnm classification, overall grading (ranged from 1 to 3), proliferation status (based on scoring of ki-67-positive cells in biopsies before treatment) and the status of er, pr and her2 receptors, was obtained from the aforementioned clinics. in the study group, 11 patients suffered from locally advanced breast cancer (predominantly stage iii disease). a case was diagnosed as triple-negative breast cancer (tnbc) if lack of er and pr expression was accompanied by lack of her2 expression as confirmed by commercial herceptest (dako, glostrup, denmark) showing her2 levels at 0 or 1+. there were 7 cases diagnosed as tnbc. such an excessive number of tnbc cases were collected intentionally because they were expected to be resistant to nac. after completion of diagnostic procedures, the patients underwent 3–9 courses of nac with standard doses of paclitaxel and doxorubicin followed by surgical removal of the affected tissue. nac effects (therapeutic responses) were evaluated by miller-payne histopathologic grading system [17]. in brief, this grading system based on the comparison of tumor cellularity of the core biopsy (before nac) with that of the resected tumor (after nac) is presented as follows: grade 1 – no reduction in overall cellularity; grade 2 – a minor loss of tumor cells (<30%); grade 3 – moderate loss (30–90%); grade 4 – a significant loss (>90%); and grade 5 – no residual invasive cancer. dna image cytometry the samples of diagnostic core biopsy or operation material (resected breast tissue specimens) were prepared on poly-l-lysinecoated microscope slides (thermo fisher scientific, waltham, ma, usa) and stained with a stoichiometric dna dye toluidine blue (tb; thermo fisher scientific) according to the published procedures [18, 19]. in brief, once samples had been fully air-dried on slides, they were fixed in acetone/ethanol mixture (1:1) for at least 30 min at 4 °c and dried again. samples were treated with 5n hci for 20 min at room temperature, washed in a distilled water (5×1 min), then stained with 0.05% tb in 50% mcilvain citrate-phosphate buffer (ph 4.0). immediately after staining, samples were rapidly washed in distilled water (3 times by dipping) and promptly blotted with absorbing sheets of paper followed by dehydration in b.i. gerashchenko et al. 49 b io m e d ic a l s c ie n c e s issn 2413-6077. ijmmr 2019 vol. 5 issue 1 butanol (2×3 min at 37°c). samples were then i m m e r s e d i n x y l e n e ( 2 × 3 m i n a t ro o m temperature) and embedded in dpx mounting medium (sigma-aldrich, st. louis, mo, usa). digital images of at least 200 randomly selected and undistorted cell nuclei were collected with 100× objective magnification using ergolux l03-10 microscope (leitz, germany) equipped with dxc-390p color video camera (sony, tokyo, japan) calibrated in the green channel. dna content per cell nucleus was measured as integral optical density using image-pro plus 4.1 software (media cybernetics, rockville, md, usa). dna content histograms were analyzed in accordance with the basic performance standards of diagnostic image cytometry [20]. dna peaks were identified as aneuploid, if they deviated more than 10% from the normal diploid (2c in g1) or tetraploid (4c in g2) region (10% is the estimated integral error of the method). as a reference for locating normal 2c peaks, the nuclei of leukocytes persisting in a sample were analyzed as well. to classify breast cancer dna content his tograms, the approach proposed by auer et al. [16] was used. for example, the histograms characterized by a single peak in 1.5–2.5c region (diploid and near-diploid region) were classified as type i, whereas the histograms characterized by a single peak in 3.5–4.5c region (tetraploid and near-tetraploid region) but sometimes supplemented with an additional peak in 1.5–2.5c region were classified as type ii. in the type ii histograms, the number of cells with ploidy of >4.5c together with the cells of ploidy ranged from 2.5c to 3.5c (predominantly near-triploid range) was less than 10%. the type iii histograms represented highly proliferating cells (>5%) that scattered between normal or near-normal 2c and 4c peak positions. less than 5% of cells had >4.5c. the type iv histograms were characterized by a large fraction of aneuploid cells (>5%) with increased and scattered dna content significantly exceeding the normal 4c peak position (>4.5c). dna histograms of types iii and iv are indicative of the worst prognosis [16]. ploidy-related parameters such as dna index (di) that characterizes aneuploidy and the percentages of cells exceeding 4.5c that characterize polyploidization were also determined. di introduced by barlogie et al. [21] was defined as the ratio of the modal dna value of the abnormal cells in g1 to the modal dna value of the normal diploid cells in g1. immunofluorescence staining the imprints of surgically removed fresh tissue specimens were prepared on poly-llysine-coated microscopy slides (thermo fisher scientific) followed by fixation and staining according to the published procedures [19]. samples were fixed in methanol at −20°c for 7 min and dipped 10 times in ice-cold pure acetone. after fixation, samples were rinsed with tris-buffered saline (tbs; ph 7.4) supplemented with 0.01% tween 20 (washing solution abbreviated as tbst) 3×5 min and blocked in tbs supplemented with 0.05% tween 20 and 1% bsa at room temperature. after blocking, samples were covered with tbs, 0.025% tween 20, and 1% bsa, containing primary antibodies to ki-67 (1:50, rabbit polyclonal, pa5-16785, pierce), sox2 (1:50, mouse monoclonal, ma1014, pierce), nanog (1:50, mouse monoclonal, n3038, sigma), and incubated in a humidified chamber overnight at 4°c. then samples were washed with tbst 3×5 min at room temperature and incubated for 40 min at room temperature in the dark with the appropriate secondary antibodies diluted in tbst: goat anti-mouse igg alexa fluor 488 (1:300, a31619, invitrogen) or goat anti-rabbit igg alexa fluor 594 (1:300, a31631, invitrogen). finally, samples were rinsed with tbst 3×5 min followed by rinsing with phosphate buffered saline (pbs; ph 7.3) 1×2 min. to clearly visualize cell nuclei, samples were counterstained with dapi (sigma-aldrich) at concentration of 0.25 µg/ml for 2 min, rinsed with pbs, and embedded in the anti-fade reagent prolong gold (invitrogen). as for staining of cd44, samples were fixed in 4% paraformaldehyde in pbs for 15 min at room temperature followed by rinsing them with pbs containing 0.1% glycine 3×5 min. all subsequent steps were identical to those described above. dilution of the primary antibody to cd44 (rabbit polyclonal, hpa005785, sigma) was 1:50. results table 1 presents the clinico-pathologic information for each of 12 breast cancer cases supplemented by corresponding tumor dna content analysis data that were obtained before and after nac. the cases categorized in this study as near-triploid (~3c), whose di=1.26−1.74 (n=7), were those that had at least one ~3c clone in spite of persisting the clones of other ploidy. the rest of the cases were defined as near-euploid, whose di values fall into the category of <1.26 and/or >1.74 (n=5). the reason why we focused on the ‘triploid group’ b.i. gerashchenko et al. 50 b io m e d ic a l s c ie n c e s issn 2413-6077. ijmmr 2019 vol. 5 issue 1 ta bl e 1. c lin ic opa th ol og ic al v ar ia bl es a n d im ag e cy to m et ry -b as ed t u m or d n a c on te n t an al ys is d at a of 1 2 br ea st c an ce r ca se s. pa ti en t n o. a ge st ag e g ra de er % pr % h er ce p te st (h er 2) k i67 % a u er & d i be fo re n a c a u er & d i af te r n a c > 4. 5c % be fo re n a c > 4. 5c % af te r n a c m ill er pa yn e 3* 57 ii ib (t 2n 3m 0) 3 0 0 0 86 iv (1 .5 0) iv (1 .5 9) 22 .5 23 .8 1 5* 33 ii a (t 2n 0m 0) 3 0 0 0 67 iv (1 .0 0 /1 .6 4) ii i (1 .0 0) 9. 7 2. 9 2 11 50 ii ia (t 2n 2m 0) 3 0 0 3+ 62 ii i (1 .0 0) ii i (1 .0 0/ 1. 73 ) 4. 7 5. 0 3 13 59 ii ia (t 2n 2m 0) 2 65 0 3+ 46 iv (1 .4 8/ 1. 70 ) ii (1 .0 7) 6. 6 0 2 15 75 ii ic (t 2n 3m 0) 3 70 65 3+ 72 iv (1 .2 7/ 1. 47 ) iv (1 .0 0/ 1. 53 ) 9. 1 13 .6 2– 3 17 * 46 ii ic (t 2n 3m 0) 3 0 0 0 78 iv (1 .0 0/ 1. 53 ) iv (1 .0 0/ 1. 47 ) 11 .8 19 .5 1 26 57 ii ic (t 4c n 3m 0) 2 0 0 2+ 78 iv (1 .1 3) i (1 .0 0) 4. 9 0 4 27 * 56 ii ia (t 2n 2m 0) 3 0 0 0 92 ii i (1 .0 0) ii (1 .0 0) 1. 3 0. 9 2 29 * 65 ii ib (t 3n 2m 0) 3 0 0 1+ 87 ii i (1 .1 8) ii i (1 .1 4) 1. 9 2. 7 1 30 * 38 ii ic (t 2n 3m 0) 3 0 0 0 84 iv (1 .0 7/ 1. 67 ) iv (1 .0 0/ 1. 64 ) 8. 8 14 .2 1– 2 33 * 34 ii ib (t 3n 2m 0) 2 0 0 1+ 24 iv (1 .1 3/ 2. 00 /2 .2 7) ii (1 .2 0) 5. 0 0. 3 3 34 68 ii ic (t 4c n 3m 0) 3 0 0 3+ 62 iv (1 .0 0/ 1. 53 /2 .0 0) ii (1 .4 1) 19 .0 2. 7 3 d en ot at io ns : pa tie nt n um be r is g iv en a s re gi st er ed f or t hi s st ud y; a st er is k (* ) – tr ip le -n eg at iv e br ea st c an ce r; n ac – n eo ad ju va nt c he m ot he ra py ; au er – d n a hi st og ra m ty pi ng th at c ha ra ct er iz es th e ex te nt o f a bn or m al ity (t yp es i− iv ) a cc or di ng to a ue r e t a l. [1 6] ; d i – d n a in de x th at s ho w s th e ex te nt o f a ne up lo id y; tr ip lo id or n ea rtr ip lo id d i v al ue s ar e un de rl in ed ; m ill er -p ay ne (g ra de s 1– 5) – tu m or r es po ns e to n ac a cc or di ng to o gs to n et a l. [1 7] . b.i. gerashchenko et al. 51 b io m e d ic a l s c ie n c e s issn 2413-6077. ijmmr 2019 vol. 5 issue 1 is that this category of patients compared with others was found to predominantly have highly malignant tumors leading to poor prognosis [14, 22−24]. in addition, the near-triploid cases can correlate with significant genomic gains (>5c) occurring presumably due to active proliferation of ~3c clones [22]. although by examining diagnostic biopsies we did find an apparent increase in the number of >4.5c cells in near-triploid tumors compared with neareuploid ones (fig. 1a), this finding was not clearly attributed to the enhanced proliferation of tumor cells, as evidenced by the results of counting of ki-67-positive cells in both groups (fig. 1b). perhaps, much larger cohort of data is needed to delineate the role of tumor cell proliferation (particularly proliferation of ~3c clones) in gaining the numbers of >4.5c cells. because an increase in the number of >4.5c cells assuming polyploidization is indicative of tumor aggressiveness and poor disease outcome, it seems reasonable to clarify whether nac can eliminate them or considerably reduce their presence. figure 2a shows that nac completely eliminated >4.5c cells in 2 cases (13 a n d 2 6 ) a n d c o n s i d e ra b l y re d u c e d t h e percentage of such cells in 3 cases (5*, 33*, and 34), suggesting the apparent positive effect of nac in 5 of 12 cases regardless of the ploidy group they belong to. however, among other 7 non-responsive cases (3*, 11, 15, 17*, 27*, 29*, and 30*), there were 3 cases (15, 17*, and 3 0 * ) t h a t d i s t i n c t l y s h o w e d e n h a n c e d polyploidization, as eviden ced by a significant i n c re a s e i n p e rc e n t a g e s o f > 4 . 5 c c e l l s , suggesting the negative (opposite) nac effect. all those 5 aforementioned cases that were responsive to nac showed the improved dna content profiles evaluated according to auer (fig. 2b). notably, the only one case (27*) of all non-responsive cases showed an improvement in dna histogram from type iii to type ii. to demonstrate whether auer dna histogram typing data can correlate with miller-payne histopathologic scoring data, figure 3 shows both of these parameters well interrelated. a distinct group of tnbc (3*, 5*, 17*, 29*, and 30*) is likely to be the most resistant to nac, and all cases in this group except the case 29* belong to the near-triploid class. although the case 27* showed an improved dna histo gram (fig. 2b), this one had the low grade 2 (millerpayne scale, fig. 3) confirming its resis tance to nac. as for the case 13 that was well responsive to nac (figs. 2a and 2b), this one, however, like the case 27*, had the low grade 2 (miller-payne scale, fig. 3). of all 12 cases, the best therapeutic effects were achieved with regard to the neareuploid case 26, which was not tnbc (auer type i vs. miller-payne grade 4, fig. 3). regrettably, pcr was not achieved in all these cases because none of them had the highest miller-payne grade 5. discussion thus, the auer types of dna content profiles of post-nac breast cancer cases were generally in agreement with corresponding miller-payne histopathologic grades, but quantification of >4.5c cells seems superior in terms of identifig. 1. percentages of >4.5c cells (a) and ki-67-positive cells (b) in near-triploid (di values are ranged from 1.26 to 1.74) or near-euploid (di values are <1.26 and/or >1.74) breast cancer cases before nac. asterisks (*) denote triple-negative breast cancer. b.i. gerashchenko et al. 52 b io m e d ic a l s c ie n c e s issn 2413-6077. ijmmr 2019 vol. 5 issue 1 fig. 2. effects of nac on percentages of >4.5c cells (a) and dna content profiles (typed according to auer) (b) in each of breast cancer cases. asterisks (*) denote triple-negative breast cancer. fig. 3. mapping of 12 breast cancer cases based on auer typing of tumor dna content histograms (after nac) vs. miller-payne histopathologic scoring. auer scale presents the types of histograms (i−iv), while miller-payne scale presents the histopathologic grades (1−5). most resistant cases are encircled. asterisks (*) denote triple-negative breast cancer. fication of those cases that show the negative nac effect (in this study those cases were 15, 17*, and 30*). polyploidization that in some non-responsive cases was gained by nac is likely to be attributed to ~3c clones. the most of non-responsive cases examined here are near-triploid tnbc, of which one case should especially be pointed out. this is the case 17*, which substantially differs from the rest of nonresponsive cases in terms of spectacular postnac polyploidization of predominant 3c tumor cells resulting in the growth of the fraction of b.i. gerashchenko et al. 53 b io m e d ic a l s c ie n c e s issn 2413-6077. ijmmr 2019 vol. 5 issue 1 6c cells accompanied by the appearance of additional fractions of 12c and 24c cells generated due to ploidy doublings (fig. 4a vs. fig. 4b). a significant increase of the fraction of 2c cells (fig. 4b) may take place due to depolyploidization [25]. microscopic examination of the operation material obtained after nac revealed the existence of multinucleated cells, although those ones were rarely seen (fig. 4b). in this particular case (17*), like in some other non-responsive cases, cells with large nuclei were often observed as ki-67-positive (fig. 5a), supporting the assumption that these cells are capable of cycling. among cd44-positive cells, cells with large nuclei were widely present as well (fig. 5b), suggesting their invasive potential. moreover, ~80% of 2c cells that may represent descendants of de-polyploidizing cells were cd44-positive. in basal-like breast cancers, of which 77% are tnbc [26], cd44-positive tumor cells that can potentially be associated with cancer stem cell phenotype are characteristic of highly aggressive tumors with enhanced invasiveness [27, 28]. generation of drugresistant polyploid tumor cells gaining mesenchymal characteristics with elevated expression of cancer stem cell markers (cd44 and cd133) has been reported [29]. in addition to the expression of ki-67 and cd44, many cells with large or small nuclei (up to 75%) can express self-renewal factors characteristic for stem cells, such as sox2 and nanog (figs. 5c-d and figs. 5e-f, respectively). notably, stress-induced polyploidization of tumor cells (including breast cancer cells) expressing self-renewal factors was also demonstrated in vitro in other studies by several groups of researchers, prompting us to suppose that the polyploid cells and their descendants released by de-polyploidization can possess stem cell features [30-33]. this type of stem cell-like gene expression signature is characteristic of poorly differentiated aggressive tumors including breast cancer often of basallike subtype [34]. interestingly, ionizing radiation may induce a breast cancer stem-cell phenotype cd44+/cd24−/low in differentiated breast cancer cells with the involvement of polyploid cells that express oct4, sox2, nanog, and klf4, in a dose-dependent manner [32]. the number of examples demon strating radioor chemotherapy-induced either stem-like and/or therapy-resistant state in fig. 4. dna content histograms of a triple-negative case (patient 17*) before nac (a) and after nac accompanied with microphotographs of tb-stained nuclei (scale bars = 10 μm) (b). b.i. gerashchenko et al. 54 b io m e d ic a l s c ie n c e s issn 2413-6077. ijmmr 2019 vol. 5 issue 1 fig. 5. microphotographs of immunofluorescence-stained imprints of the surgical post-nac material of patient 17* depicting cells positive for ki-67, cd44, sox2 and nanog. (a) a group of large nuclei expressing ki-67 in 15% of all ki-67-positively stained nuclei. (b) among cd44-positive cells (45%) there are many cells with large nuclei. (c, d) among sox2-positive cells (75%) there are many cells with large nuclei. (e, f) among nanog-positive cells (75%) there are many cells with large nuclei (in this image, two largest nuclei may have 24c). all immunofluorescence preparations were counterstained with dapi for dna. scale bars = 10 μm. tumor cells is growing [15, 35, 36]. a recent study of tnbc has shown that pre-existing subclones can be adaptively selected by nac (adaptive resistance), followed by transcriptional reprogramming to evolve the resistant phenotypes [15]. conclusions dna content profiling provides a facile, prompt, and accurate means of interpreting therapeutic responses in nac-treated breast cancers. because nac effects for some breast cancers may be negative, the use of the further treatment strategy should be carefully considered. therapy-resistant polyploid tumor cells possessing stem cell features can be induced in vivo as well, assuming that this process is not autonomous, but rather stipulated by the tumor microenvironment and intra-tumor heterogeneity. these polyploid cells are not quiescent and they manifest the invasiveness phenotype that together with stemness are also seen in descendants after de-polyploidization. acknowledgments this work was performed within the frame of the project supported by the european social fund (grant number: 2013/0023/1dp/1.1.1.2.0/ 13/apia/viaa/037). conflict of interest the authors declare no conflict of interest. b.i. gerashchenko et al. 55 b io m e d ic a l s c ie n c e s issn 2413-6077. ijmmr 2019 vol. 5 issue 1 оцінка відповіді раку молочної залози на протипухлинну терапію за даними днк-цитометрії б.і. геращенко1, k. salmina2, j. eglītis3, j. ērenpreisa 2 1 – інститут експериментальної патології, онкології і радіобіології імені р.є. кавецького нан україни, київ, україна 2 – latvian biomedical research and study centre, riga, latvia 3 – university of latvia, riga, latvia вступ. розбіжності в інтерпретації відповіді раку молочної залози (рмз) на протипухлинну терапію все ще існують переважно через відсутність стандартизованих критеріїв оцінки та методів. мета роботи. оцінка ефективності застосування профілювання вмісту днк у клітинах рмз до і після неоад'ювантної хіміотерапії (нхт) для спрощення інтерпретації терапевтичного ефекту. методи. препарати діагностичної біопсії та операційного матеріалу (тканини первинних пухлин, хірургічно видалених після нхт), досліджували за допомогою днк-цитометрії. поліплоїдію та анеуплоїдію у днк гістограмах оцінювали застосовуючи прогностичну типізацію ауера. на тих же гістограмах визначали днк індекси (di) клітинних клонів і відсотки поліплоїдизуючих клітин (>4.5c). імунофлуоресцентне забарвлення застосовували для оцінки проліферації (ki-67), інвазивності (cd44) та факторів самооновлення, притаманних стовбуровим клітинам (sox2 та nanog). результати. профілювання вмісту днк 12 випадків рмз, з яких 7 були тричі-негативними, виявило ознаки нечутливості пухлини до нхт у 7 випадках, з яких 5 були тричі-негативними. серед нечутливих до нхт випадків були 3 випадки, які показали посилену поліплоїдизацію, що свідчить про негативний ефект нхт. пара-триплоїдні (di = 1.26-1.74) тричі-негативні випадки визначені як найбільш резистентні до нхт. проліферуючі пара-триплоїдні клітини можуть призводити до зростання кількості поліплоїдних клітин позитивних на ki-67, cd44, sox2 і nanog. висновки. дані днк-цитометрії забезпечують додатковою корисною інформацією для інтерпретації відповіді раку молочної залози рмз на протипухлинну терапію. поліплоїдні пухлинні клітини з особливостями стовбурових клітин можуть бути індуковані нхт. оскільки в деяких випадках нхт може викликати несприятливу відповідь пухлин, стратегію подальшої протипухлинної терапії необхідно ретельно переглянути. ключові слова: рак молочної залози; поліплоїдні клітини; пара-триплоїдія; днкцитометрія; терапевтичний ефект; стовбурові клітини раку. інформація про авторів геращенко богдан іванович – канд. біол. наук, відділ засобів та методів сорбційної терапії, інститут експериментальної патології, онкології і радіобіології ім. р.є. кавецького нану, вул. васильківська 45, київ 03022, україна. information about authors bogdan i. gerashchenko – md, ph.d., department of means and methods of sorption therapy, r.e. kavetsky institute of experimental pathology, oncology and radiobiology of nasu, 45 vasylkivska str., kyiv 03022, ukraine. orcid 0000-0001-6404-2320, e-mail: biger63@yahoo.com kristine salmina – ph.d., cancer research laboratory, latvian biomedical research and study centre, 1 ratsupites str., riga, lv-1067, latvia. orcid 0000-0002-8994-773x, e-mail: salmina.kristine@gmail.com janis eglitis – md, ph.d., faculty of medicine, university of latvia, 3 jelgavas str., riga, lv-1004, latvia. orcid 0000-0002-8784-1544, e-mail: dreglitis@gmail.com jekaterina erenpreisa – dr. habil. med., cancer research laboratory, latvian biomedical research and study centre, 1 ratsupites str., riga, lv-1067, latvia. orcid 0000-0002-2870-7775, e-mail: katrina@biomed.lu.lv b.i. 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international journal of medicine and medical research 2018, volume 4, issue 1, p. 8-12 copyright © 2018, tsmu, all rights reserved corresponding author: nadiya makarchuk, department of first emergency medical aid and emergency medical treatment, 1 maydan voly, ternopil, 46001, ukraine e-mail: nadiya_rm@ukr.net phone number: +380971322995 doi 10.11603/ijmmr.2413-6077.2018.1.8717 dn4 questionnaire in family practice for evaluation of clinical manifestations of neuropathic pain in type 2 diabetes patients treated by light therapy n. r. makarchuk i. horbachevsky ternopil state medical university, ternopil, ukraine background. peripheral diabetic polyneuropathy (dpn) is one of the most frequent neurological complications of diabetes mellitus (dm). despite the large number of pharmacological agents, its treatment is not sufficiently effective, which necessitates the search for new therapies. objective. the aim of the study was to increase the effectiveness of treatment of neuropathic pain in the patients with diabetic polyneuropathy by incorporating procedures using polarizing polychromatic non-coherent light (bioptron light therapy) into the complex therapy of this disease. methods. we examined 67 patients with type 2 diabetes complicated with diabetic polyneuropathy. patients were divided into two groups: group 1 consisted of 32 patients, who received standard treatment; group 2 comprised 35 patients, who additionally underwent 12 light therapy treatments by means of the bioptron physiotherapy unit. the evaluation of neuropathic pain intensity was performed using a modified questionnaire dn4. results. a positive clinical effect of treatment was evidenced in both groups in 12 days of treatment. in 3 months, the intensity of complaints was significantly lower (p<0.05) only in the group with additional use of polarizing light. in 6 months, the positive effect of the therapy was leveled in the patients of both groups. conclusions. the use of the dn4 questionnaire with a modified scale for assessing the parameters of neuropathic pain can optimize its diagnosis. the light therapy procedures together with the standard complex therapy of diabetic polyneuropathy increase the clinical efficacy of neuropathic pain treatment and help to preserve the therapeutic effect within 3 months. key words: diabetic polyneuropathy; neuropathic pain; dn4 questionnaire; polarized polychromatic noncoherent light (bioptron). introduction peripheral diabetic polyneuropathy (dpn) is a typical early and most frequent complication of diabetes mellitus (dm) [1]. it develops due to affection of nerve fibers, caused by diabetes and occurs in more than 50 % of patients with this illness. polyneuropathy is revealed in both young and elderly patients with type 1 and type 2 diabetes mellitus [2]. there is no single classification of peripheral diabetic neuropathy. some authors recommend defining hypohyper­ glycemic, generalized, focal and multifocal types (thomas p.k. 1997); whereas others insist on singling outing asymptomatic, symptomatic and marked symptoms (dyck p. j. 1999) or mono, polyneuropathy and autonomic polyneuropathy (i. i. dedov et al., 2002). according to the protocol of medical care [11], peripheral polyneuropathy is divided into somatic (motor, sensory and sensory-motor), vegetative and mononeuropathy. to diagnose pathological process in the nervous tissue boulton et al (2005) suggested allocating three clinical forms of peripheral dpn (silent, acute pain and chronic pain). chronic pain form of dpn is the most commonly reveled in the patients with diabetes [1]. the severity of this complication depends on its clinical consequences, in particular, trophic disorders and neuropathic pain, which adversely affect patients’ quality of life. a number of questionnaires are used to diagnose neuropathic pain [3]. dn4 questionnaire, being one of the most practical, where a positive response to four or more questions out of ten substantiates ‘neuropathic pain’ diagnosis, is used as a screening to detect neuropathic pain syndromes [4]. despite the large number of pharmacological agents, treatment of patients with diabetic polyneuropathy is not sufficiently effective [5, 6, 7], which necessitates the search for new methods of treatment. since the mid-80s, there have been physiotherapeutic devices that emit visible (wavelength 80-3400 nm) linearly polarized (95 %) incoherent (desynchronized in time and space) low-energy (non-destructive, n. r. makarchuk 9 in t e r n a l m e d ic in e issn 2413-6077. ijmmr 2018 vol. 4 issue 1 with the energy flux intensity of 40 mw/cm2) (pyler) light, and the light stream, transformed by polarization, that lacks both ultraviolet and a significant part of the infrared rays. studies conducted at the end of the last century proved a positive effect of physiotherapeutic procedures using this light for treatment of diseases with lesions of peripheral nerves [8, 9, 10]. the aim of the study is to increase the effectiveness of treatment of neuropathic pain in patients with diabetic polyneuropathy by including light therapy procedures into the complex treatment of this disease. methods 67 patients were examined (36 males (53.7 %) and 31 females (46.3 %)) with type 2 diabetes and dpn. the diagnosis of dpn was based on anamnesis and clinical examination data. the age of the examined persons ranged from 45 to 65 years old (mean age 57.0±5.2 years old). duration of diabetes mellitus was from 4 to 19 years (average duration 9.4±3.7 years), and of dpn was from 1 to 12 years (which averaged 5.5±2.9 years). for the convenience of systematization and objectification of data comparing, all examined patients were divided into two groups: the 1st – control group comprised 32 patients with type 2 dm and dpn, who received standard treatment according to a unified clinical protocol of primary and secondary (specialized) medical care (no.1118, dated december 21, 2012) [11]. the 2nd group involved patients undergoing standard treatment together with 12 light therapy treatments by means of the bioptron physiotherapy unit [12]. the duration of the procedure was 10 minutes with a directed flow of light on the lower limbs. general characteristic features of patients with diabetes are presented in table 1. clinical examination of patients was perfor­ med before the treatment and on the 12th day after the beginning of the treatment. neuropathic pain was diagnosed using the modifi questionnaire dn4 (2005) [13, 14]. the questionnaire structure included two blocks of questions: 7 questions of the fi block re­ vealed sensory symptoms, including sponta neous pain (burning, painful cold, electric shocks), paresthesia and dysesthesia (tingling, pins and needles, numbness, itching); three conclusions of the physician, based on the clinical examination, which comprised the second block, give the physician the opportunity to identify the allodynia and negative sensory symptoms. neuropathic pain was set at a score of 4 or more points. for the details of each question, we modi fied the dn4 questionnaire by ranging the intensity (scale 1 to 10) of the sensations listed in the first question block. evaluation of the results was carried out at the admission of patients to the hospital and in 12 days after the start of diabetic polyneu ropathy treatment. long-term results of the therapy were administered in 3 and 6 months by performing the call-in poll among the patients using the first question block of the questionnaire. the analysis and processing of statistical data of clinical examinations results were carried out on a personal computer using statistica 10 and ms excel xp application packages. all data are presented as mean value and standard deviation (m±σ). relations between continuous variables were examined by the pearson correlation coefficient χ². comparison of the rates between the groups was carried out using the student t-test, and those within the group were compared using wilcoxon matched paired test. the difference in rates was considered statistically significant at p<0.05. results according to the dn4 questionnaire, 49 (73.1 %) of the surveyed patients suffered neuropathic pain before treatment, which is consistent with the literature [15]. no signifi differences between the groups were noticed before treatment (p>0.05) (table 2). subjective symptoms in the general group of patients were presented as follows: 67.2 % of patients suffered burning sensation, while 31.34 % experienced painful cold. 74.6 % of people were disturbed by tingling. 40.29 % of patients with diabetes sensed electric shocks. pins and needles sensation and that of numbness troubled 58.2 % and 59.7 % of patients respectively. 34.3 % of respondents had complains of itching. the table 1. general characteristic features of the patients (m±σ) characteristic features of the groups of patients 1st group, n=32 2nd group, n=35 p age, years 58.7±5.2 55.6±4.9 р˃0.05 duration of dm, years 8.6±3.3 10.1±4.0 р˃0.05 duration of dpn, years 4.9±2.8 6.0±3.1 р˃0.05 10 in t e r n a l m e d ic in e issn 2413-6077. ijmmr 2018 vol. 4 issue 1 table 2. follow-up of neuropathic pain severity in the examined groups of patients with diabetes mellitus according to the dn4 questionnaire before and after treatment (m±σ) 1st group 2nd group p before treatment 4.7±1.4 4.8±1.6 p˃0.05 after treatment 3.4±1.5 2.9±1.2 p˃0.05 notes: * – significant difference before and after treatment (p<0.05). objective examination of the patients’ lower extremities proved that the pain was localized in the area with a reduced sensitivity to touching (in 70.2 %), pricking (in 37.3 %) and in the area of irritation with a brush (in 19.4 %), indicating a tactile and sensory sensitivity disturbance. the intensity of each of the following complaints before treatment in the examined groups of patients is presented in fig. 1. after the course of treatment, a decrease in the level of neuropathic pain was evidenced, together with a positive dynamics of the in tensity of subjective complaints of the patients. the analysis of data of the dn4 questionnaire proved a decrease in the signs of neuropathic pain by 41.7 % (χ²=2.5; p>0.05) in the patients of control group, and by 64.0 % (χ²=27.6; p˂0.05) in the group with additional light therapy procedures. the rate of neuropathic pain presence after the course of treatment was much lower in the 2nd group. the study of individual rates of neuropathic pain in each of the groups on the 12th day after the beginning of treatment proved that the patients of the 1st and the 2nd groups experienced burning sensation decrease by 15.6 % (χ²=1.6; p>0.05) and 34.4 % (χ²=6.9; p˂0.05) respectively. painful cold sensation insignificantly decreased by 6.3 % (χ²=0.291; p>0.05) in group 1 and by 5.7 % (χ2=0.3; p>0.05) in group 2. the sensation of electric shocks was reduced by 28.13 % (χ²=6.5; p˂0.05) in the patients of the 1st group and by 28.13 % (χ²=5.9; p˂0.05) in those of group 2. tingling worried the patients with diabetes less by 15.6 % (χ²=1.6; p>0.05) of group 1 and by 22.9 % (χ²=4.2; p˂0.05) of group 2. pins and needles as well as numbness decreased by 21.88 % (χ²=3.1; p˂0.05) and 3.1 % (χ²=3.1; p>0.05) in the patients of the 1st group, and by 25.7 % (χ2=4.629; p˂0.05) and 28.6 % (χ²=5.7; p<0.05) in those of the 2nd group. itching began to bother less the respondents of the 1st group by 3.1 % (χ²=0.1; p>0.05) and by 14.3 % (χ²=1.7; p>0.05) those in the 2nd group. the pain, which was localized in the area of reduced sensitivity to touching, decreased by 15.6 % (χ²=1.9; 1st group 2nd group 1st group 2nd group * * before treatment in 12 days after treatment * 1st group 2nd group * * 1st group 2nd group * * in 3 months after treatment in 6 months after treatment fig. 1. comparison of the intensity of complaints between the patient groups undergoing treatment. notes: sensation of 1 – burning; 2 – painful cold; 3 – electric shocks; 4 – tingling; 5 – pins and needles; 6 – numbness; 7 – itching; * – р˂0.05. 10 n. r. makarchuk 11 in t e r n a l m e d ic in e issn 2413-6077. ijmmr 2018 vol. 4 issue 1 p>0.05) and 14.3 % (χ2=1.4; p>0.05), to pricking by 25 % (χ²=4.4; p˂0.05) and 22,9 % (χ²=4.6; p˂0.05), and to irritation with a brush by 6,5 % (χ²=0.3; p>0.05) and 5.71 % (χ²=0.7; p˂0.05) in the examined groups 1 and 2, respectively (table 3). the survey of the patients in 3 months after the treatment, proved that in the 1st group such sensations as burning, painful cold, electric shocks, tingling, pinsand needles, numbness and itching were experienced by 17 (53.1 %), 10 (31.3 %), 12 (37.5 %), 20 (62.5 %), 10 (31.3 %), 18 (56.3 %) and 9 (28.1 %) patients, whereas in the 2nd group – by 7 (20 %), 8 (22.9 %), 6 (17.1 %), 23 (65.7 %), 14 (40 %), 11 (31.4 %) and 7 (20 %) patients respectively. the analysis of survey of the patients with diabetes mellitus, conducted in 6 months after the treatment, proved that the patients with diabetes had sensations of burning, painful cold, electric shocks, tingling, pins and needles, numbness and itching in 22 (68.5 %), 12 (37.5 %), 14 (43.8 %), 22 (68.5 %), 15 (46.9 %), 21 (65.6 %) and 9 (28.1 %) cases in group 1, and in 23 (65.7 %), 10 (28.6 %), 13 (37.1 %), 26 (74.3 %), 20 (57.1 %), 14 (40 %) and 12 (34.3 %) cases in group 2 respectively. the comparison of com plaints intensity between the patient groups is presented in fig. 1. discussion the results of our study are consistent with the recent literature [1]. the standard therapy (α­lipoic acid, actovegin and complex of vitamins of the group b) decreases the intensity of pain and neuropathic disorders [11]. the adding of light therapy procedures allows not only the achievement of this effect, but also its long-term preservation [10, 12]. the use of the questionnaire dn4 has long been practiced for the diagnosis of neuropathic pain [13, 14], but only its modification [9] allows evaluating the therapy effec­ tiveness. the obtained results prove that signifi­ cant improvement in the dn4 questionnaire’s quantitative indicators occurred in 12 days after the beginning of treatment and persisted for three months after the treatment in both groups. at the same time, the intensity of the indicators of block 1 of the questionnaire in three months after the treatment was considerably less signifi in the patients who received additional light therapy. six months later, quantitative and quali­ tative indicators of the presence and inten sity of neuropathic pain resumed to the initial level. conclusions using a modified dn4 questionnaire in the patients with type 2 diabetes can improve the diagnosis of neuropathic pain. table 3. follow-up of neuropathic pain intensity in the examined groups of patients under the influence of therapy (m±σ) in de x before treatment after treatment in р1 р2 р3 р4 р512 days 3 months 6 months 1st group 1 4.7±3.7 1.7±1.9 1.7±1.9 4.6±3.5 <0.05 ˃0.05 <0.05 <0.05 ˃0.05 2 2.3±3.3 0.7±1.3 0.8±1.3 2.4±3.4 <0.05 ˃0.05 <0.05 <0.05 ˃0.05 3 2.3±3.4 0.5±1.5 0.6±0.8 2.0±2.6 <0.05 ˃0.05 <0.05 <0.05 ˃0.05 4 3.6±3.5 2.0±2.6 2.2±2.1 3.8±3.2 <0.05 ˃0.05 <0.05 <0.05 ˃0.05 5 2.2±3.7 1.3±2.3 1.4±2.2 2.3±3.1 <0.05 ˃0.05 <0.05 <0.05 ˃0.05 6 3.0±3.3 1.3±2.2 1.5±1.7 3.1±2.9 <0.05 ˃0.05 <0.05 <0.05 ˃0.05 7 1.750±2.828 0.9±1.9 1.7±2.9 1.8±3.1 <0.05 ˃0.05 <0.05 <0.05 ˃0.05 2nd group 1 5.1±3.8 0.2±0.4 0.3±0.6 4.7±3.9 <0.05 ˃0.05 <0.05 <0.05 ˃0.05 2 2.1±3.5 0.5±1.0 0.5±1.1 1.8±3.1 <0.05 ˃0.05 <0.05 <0.05 ˃0.05 3 2.4±3.2 0.4±1.0 0.5±1.3 1.8±2.6 <0.05 ˃0.05 <0.05 <0.05 ˃0.05 4 4.3±3.1 2.0±2.0 2.0±2.6 3.9±3.1 <0.05 ˃0.05 <0.05 <0.05 ˃0.05 5 2.5±2.5 1.1±1.8 1.3±1.8 2.4±2.4 <0.05 ˃0.05 <0.05 <0.05 ˃0.05 6 3.6±3.3 0.4±0.5 0.5±0.9 2.9±3.7 <0.05 ˃0.05 <0.05 <0.05 ˃0.05 7 1.7±2.8 0.4±1.0 0.5±1.3 1.1±2.3 <0.05 ˃0.05 <0.05 <0.05 ˃0.05 notes: sensation of 1 – burning; 2 – painful cold; 3 – electric shocks; 4 – tingling; 5 – pinsand needles; 6 – numbness; 7 – itching; р1 – significant differences of indexes before and in 12 days after treatment; р2 – significant differences of indexes in 12 days and in 3 months after treatment; р3 – significant differences of indexes before and in 3 months after treatment; р4 – significant differences of indexes in 12 days and in 6 months after treatment; р5 – significant differences of indexes before and in 6 months after treatment. n. r. makarchuk 12 in t e r n a l m e d ic in e issn 2413-6077. ijmmr 2018 vol. 4 issue 1 the presence of phototherapeutic procedures by the bioptron together with the standard complex therapy of diabetic polyneuropathy has a pronounced clinical effect and contributes to a 3-month-long reduction of quantitative and qualitative indicators of neuropathic pain. the temporary clinical effect of the use of polarizing light in the treatment of diabetic polyneuropathy proves the feasibility of studying new therapies that would influence the pathogenesis of neuropathic pain. references 1. pankiv iv. efficiency of alpha lipoic acid therapy at diabetic foot syndrome. pain. 2014;3(59):108-114. 2. bansal d, gudala k, muthyala h, et al. prevalence and risk factors of development of peripheral diabetic neuropathy in type 2 diabetes mellitus in a tertiary care setting. diabetes investig. 2014; 5:714– 721. 3. mardzvik vm. efficacy of neogabyn in treat­ ment of postherpatic neuralgia. ukrainian journal of psychoneurology. 2015;1(82):137-140 [in ukrainian]. 4. borenstayn d. epidemiology, etiology, diag nostic evaluation and treatment of lumbar pain. international medical journal. 2000;1:36-43 [in russian]. 5. dedov ii, shestakova mv, mayorov ay. standards of specialized diabetes care. 8th edition. мoscow.: print publishing company; 2017 60. available at http://webmed.irkutsk.ru/doc/pdf/ algosd.pdf [in russian]. 6. yang w, dall tm, halder p, gallo p, kowal sl, hogan pf. economic costs of diabetes in the u.s. in 2012. diabetes care. 2013;36(4):1033-1046. 7. alleman cj, westerhout ky, hensen m, et аl. humanistic and economic burden of painful diabetic peripheral neuropathy in europe: a review of the literature. diabetes res. clin. pract. 2015;2:215-225. 8. gulyar sa. anthology of lightt herapy. medical bioptron technology (theory, practice, prospects). the book of scientific works. kiev. ifb of nas of ukraine. 2009;1024 [in russian]. 9. martynyuk lp, shved, mi, makarchuk nr, chernetskyi vi. east european scientific journal. 2018;1(29):39-41. 10. limanskiyyu.p. the phenomenon of analgesia under the action of piler-light apparatus bioptron on point acupuncture. anthology of light therapy. the book of scientific works. kiev. ifb of nas of ukraine. 2009:184-189 [in russian]. 11. unified clinical protocols of primary, emergency and secondary (specialized) medicalhelp: diabetes mellitus type 2. available at http://old.moz. g ov . u a / u a / p o r t a l / d n _ 2 0 0 9 0 8 0 5 _ 5 7 4 . h t m l [ i n ukrainian]. 12. gulyar sa, limansky yuр, tamarova za. pain and bio ptron : treatment of pain syndromes by polarized light. zepter. 2000 [in russian]. 13. povoroznyuk vv. neuropatic component of pain in skeletal-muscle in diseases. pain. joints. spine part i. 2014;4(16):5-13 [in ukrainian]. 14. mathieson s, maher cg, terwee cb, follyde campos t, lin cw. j clin epidemiol. neuropathic pain screening questionnaires have limited measurement properties. a systematic review. 2015;68(8):957-66. 15. bregovskiy vb. painful diabetic polyneuropathy of the lower extremities: current knowledge and treatment options (review). pain. 2008;1(18);2934 [in russian]. gov.ua/ua/portal/dn_20090805_574. html [in ukrainian]. received: 2018-05-03 опитувальник dn4 у практиці сімейного лікаря для оцінки нейропатичного болю у хворих з цукровим діабетом 2-го типу при лікування світлотерапією н. р. макарчук тернопільський державний медичний університет імені і. я. горбачевського, тернопіль, україна вступ. периферична діабетична полінейропатія є одним з найбільш частих неврологічних ускладнень цукрового діабету. незважаючи на широкий спектр існуючих лікарських засобів, її лікування є недостатньо ефективним, що зумовлює пошук нових статегій та засобів. мета дослідження – підвищити ефективність лікування нейропатичного болю у хворих на цукровий діабет шляхом включення процедур iз використанням поляризаційного поліхромного некогерентного світла (біоптронна світлотерапія). методи. обстежено 67 хворих на діабет 2 типу, який ускладнений діабетичною поліневропатією. пацієнтів було розділено на дві групи: 1 група – 32 пацієнтів, які отримали стандартне лікування; 2 група 35 хворих, які додатково пройшли 12 процедур світлотерапії за допомогою світлотерапевтичного пристрою біоптрон. оцінку інтенсивності невропатичного болю проводили за допомогою модифікованої анкети dn4. результати. через 12 днів лікування позитивний клінічний ефект лікування спостерігався в обох групах. однак, через 3 місяці інтенсивність скарг була значно меншою (p<0,05) в 2-ій групі пацієнтів, які отримували додатково лікування поляризаційним світлом. через 6 місяців ефект терапії був однаковим у пацієнтів обох груп. висновки. використання опитувальника dn4 з модифікованою шкалою для оцінки параметрів нейропатичного болю може оптимізувати його діагностику та оцінку. світлотерапевтичні процедури у поєднанні зі стандартною комплексною терапією діабетичної поліневропатії підвищують клінічну ефективність лікування невропатичного болю і допомагають зберегти терапевтичний ефект протягом 3 місяців. ключові слова: діабетична поліневропатія; невропатичний біль; опитувальник dn4; поляризоване поліхроматичне некогерентне світло (біоптрон). n. r. makarchuk 21 in t e r n a l m e d ic in e issn 2413-6077. ijmmr 2018 vol. 4 issue 1 international journal of medicine and medical research 2018, volume 4, issue 1, p. 21-25 copyright © 2018, tsmu, all rights reserved doi 10.11603/ijmmr.2413-6077.2018.1.8701 bone mineral status and metabolism disorders in patients with chronic spinal cord injury m. a. bystrytska d. f. chebotarev institute of gerontology of the national academy of sciences of ukraine, kyiv, ukraine ukrainian scientific medical center of osteoporosis, kyiv, ukraine background. the study of late complications, including osteoporosis, is urgent due to the increasing life expectancy of patients with complete spinal cord injury (sci). objective. the aim of the study is to evaluate bone mineral status and bone turnover markers in the patients with chronic sci. methods. 73 sci patients and 57 healthy persons were examined. bone status was determined by ultrasound (us) densitometry. bone turnover markers and vitamin d were evaluated by electrochemiluminescence method. results. in the sci patients bone mineral status was significantly lower compared with the individuals of control group. the stiffness index (si) was 51.4±11.8% vs. 98.5±16.6%, p<0.05 in men and 50.1±9.8% vs. 92.9±11.1%, p<0.05 in women. in the sci patients the levels of bone turnover markers were significantly higher than the reference values. the bone mineral status of patients with sci, which occurred before peak bone mass development, is significantly worse, compared with the individuals with the already developed peak bone mass, that makes them a high risk group for fracture. conclusions. sci leads to increased bone resorption with development of secondary osteoporosis (according to the ultrasound densitometry of calcaneal bone). in chronic sci, bone resorption is higher than in the individuals with combined low levels of vitamin d, and the absence of axial load results in continued loss of bone mass. key words: spinal cord injury; osteoporosis; tetraplegia; paraplegia; bone turnover markers; ultrasound densitometry. introduction improved social adaptation and increased life expectancy of persons with complete spinal cord injury pay attention to late complications of spinal cord injuries (sci) including osteo porosis and fragility fractures. sci-induced osteoporosis has been studied since the first half of the 20th century, but no consistent approach to its diagnosis and treatment has been developed. currently, diagnostic criteria for osteoporosis in postmenopausal women and men over 50 years are established, but the vast majority of sci patients are young men. most of studies are devoted to bone changes in the first days, weeks and months after the injury, when intense bone resorption starts. during the fi years after complete spinal cord injury, rapid decrease in bone mass takes place, which reaches 20-50% of bone mineral density (bmd) of paralyzed limbs [1]. demineralization of spongy bone occurs faster than that of cortical bone, due to its higher metabolic corresponding author: maryna bystrytska, d. f. chebotarev institute of gerontology of the national academy of medical sciences of ukraine, ukrainian scientific medical center of osteoporosis, 67, vyshgorodska str., kyiv, 04114, ukraine e-mail: miroslava_br@ukr.net phone number: +380674185573 activity [1]. also there is no consensus for increased resorption duration after sci. some studies have proved that bone loss slows down 3-5 years after the injury [2]. other study findings evidenced continued bone loss in later stages of the post-traumatic period [3, 4]. the extent and duration of bone loss are caused by many factors, which depend on the charac teristics of the injury as well as on many pa rameters of various systems of the patient. the significance of a degree of spinal cord injury and opportunities for verticalization and mo vement of bone loss has been proved, but the role of factors affecting bone tissue in the patients with complete spinal cord injury has not been defined so far. objective of the study is to evaluate the bone mineral status and parameters of bone metabolism in the patients with chronic spinal cord injuries. methods 73 people who suffered a spinal trauma with complete spinal cord injury with tetraor para plegia were examined: 38 men, average age 40.1±12.2 years old; 35 women, average age 41.3±11.3 years old; duration of the post-trau matic period was 15.7±10.6 years. patient m. a. bystrytska 22 in t e r n a l m e d ic in e issn 2413-6077. ijmmr 2018 vol. 4 issue 1 examination was conducted in cooperation with the association of disabled persons with spinal cord injuries. the examined patients lead an active lifestyle that excluded the impact of prolonged bed rest. volunteers accompanying people with sci were also examined as a comparison group. volunteers were usually represented by patients’ relatives: parents, children, spouses, which could partially reduce the impact of other factors affecting bone tissue, including eating habits, living envi ronment, and genetic factors. bone status was determined by ultrasound (us) densitometry of calcaneal bone using quantitative ultrasonometer sahara (hologic inc., model 04874, 2008). ultrasound densi tometry evaluated: speed of sound (sos, m/s); broadband ultrasound attenuation (bua, db/ mhz); stiffness index (si, %), calculated by computer on the basis of sos and bua para meters; extrapolated mineral density of cal caneal bone; t-score (patient’s si difference from the index of conventionally healthy adults aged 20 years old was presented in standard deviations); z-index (patient’s si difference from the index of conventionally healthy adults of the same age was presented in standard deviations). bone remodelling markers in peripheral blood (osteocalcin, procollagen type 1 propeptide (p1np), collagen type 1 cross-linked c-telopeptide (β-ctx)) were defined by electrochemilu minescence method on the analyser elessys 2010 (roche diagnostics, germany) using cobas testsystems. the statistical analysis was performed using statistica 10 software. results were presented as mean values and standard deviation (m±m). the difference in parameters between the groups was defined using one-way analysis of variance anova. intergroup differences were evaluated by scheffe’s test. the difference in parameters was statistically significant at p<0.05. results the results of ultrasound densitometry of the individuals of the main group proved low bone mineral density (z-score≤-2.0 sd) in all women of reproductive age (n=21) and all men younger than 50 years old (n=25). osteoporosis (t-score≤-2.5 sd) was evidenced in 12 postmeno pausal women and 13 men older than 50 years. none of the patients with spinal cord injuries had normal parameters of bone tissue structural-functional state. in the patients of control group, osteoporosis was not diagnosed, low bone mineral density was revealed in 10 in dividuals (3 men and 7 women), in all postmenopausal women, and 2 men older than 50 years. the results of ultrasound densitometry in the patients with spinal cord injuries proved significantly lower parameters of bone density compared with those of the control group (tabl. 2). it should be noted that the analysis of data in tabl. 2 proved no impact of gender on the parameters of bone mineral status (p>0.05). there was also no significant dependence of bone parameters on age, height, injury level and duration. significant moderate correlation between si and body weight was obtained in men of the main group. relationship was described by the linear regression equation: si (%)=8.01+0.55* weight (kg); r=0.41; p=0.017; r2=0.17. in the women no influence of anthropometric data on bone mineral status was revealed. bone mineral density was found to depend on the trauma age in men (r=0.41; p=0.019; r2=0.16) and women of reproductive age (r=0.40; p=0.048; r2=0.16). it is established that complete spinal cord injury leads to intensive bone loss in the first years after the injury [5]. furthermore, the rate of loss slows down and the studied bone parameters are less dependent on the duration table 1. characteristic features of the examined sci patients and persons of control group parameter main, n=73 control, n=57 р gender men, n=38 women, n=35 men, n=21 women, n=36 men women age, years 40.10±12.20 41.30±11.30 39.70±13.80 46.80±12.20 0.91 0.06 height, cm 178.60±7.20 164.10±5.60 177.30±5.60 163.20±5.80 0.48 0.52 weight, kg 72.90±9.10 59.20 ± 5.70 71.20±10.60 62.30±9.60 0.51 0.11 bmi, kg/cm2 22.90±2.90 22.10±2.20 22.70±3.30 23.50±4.00 0.77 0.01 trauma age, years 23.60±7.50 26.50±8.30 trauma duration, years 16.50±11.40 14.90±6.60 trauma level c4-6=9 th2-12=19 l1=11 c6=1 th4-12=22 l1=12 m. a. bystrytska 23 in t e r n a l m e d ic in e issn 2413-6077. ijmmr 2018 vol. 4 issue 1 23 in t e r n a l m e d ic in e issn 2413-6077. ijmmr 2018 vol. 4 issue 1 women no influence of anthropometric data on bone mineral status was revealed. bone mineral density was found to depend on the trauma age in men (r=0.41; p=0.019; r2=0.16) and women of reproductive age (r=0.40; p=0.048; r2=0.16). it is established that complete spinal cord injury leads to intensive bone loss in the first years after the injury [5]. furthermore, the rate of loss slows down and the studied bone parameters are less dependent on the duration of post-traumatic period, but there is a proven relationship of osteoporosis risk and bone characteristic features at the time of the injury [6]. the results of the study performed by professor vv povoroznyuk in 1998 prove that in the ukrainian population women reach peak bone mass at the age of 23-24 years old and men – at the age of 25-26 [7]. in present study, patients were divided into 2 subgroups according to their trauma age. a cutting-off point for men was 25 years and for women – 23 years of age. in the patients with the injury that occurred before reaching peak bone mass, the bone mineral status according to ultrasound densitometry was significantly worse than that of the patients of corresponding age, anthropometric parameters and duration of the posttraumatic period with the onset of injury at the age over 23 and 25 years, respectively (fig. 1, 2). no difference was proved in the bone mineral status of the women depending on their age at the injury onset, but after excluding postmenopausal women from both study groups, it was found out that bone mineral status was significantly worse in the women with trauma at the age of 23 years old (fig. 2). a significant difference in stiffness index in the women of reproductive age and postmenopausal women was not evidenced due to small size and heterogeneity of sample. in addition, the impact of menopause should be considered in certain groups of women, depending on the age at the injury onset before or after reaching peak bone mass. among the examined individuals, 18 patients had fractures of hip (11 patients) or tibia fractures (7 patients) due to a low-energy trauma in the period after a spinal cord injury. 66% of them had a spinal cord injury at the age table 2. bone mineral status in the patients with chronic spinal cord injury and persons of control group parameter men women main group control group main group control group т-score -3.20±0.80* -0.40±0.80 -3.10±0.50* -0.60±0.60 z-index -2.70±0.70* 0.40±0.20 -2.90±0.50* -0.40±0.60 si 51.40±11.80* 98.50±16.60 50.10±9.80* 92.90±11.10 extrapolated bmd 0.25±0.08* 0.55±0.10 0.24±0.06* 0.51±0.07 bua 40.50±13.80* 78.70±15.70 37.40±12.70* 76.90±11.30 sup 1477.00±19.00* 1554.00±26.00 1473.00±25.00* 1542.00±18.00 note:* – p<0.05, differences in parameters of the main and control groups. mean mean±se mean±1,96*se under 25 more than 25 36 38 40 42 44 46 48 50 52 54 56 58 60 s tif fn es in de x, % р <0.05 mean mean±se mean±1,96*se under 23 more than 23 38 40 42 44 46 48 50 52 54 56 58 60 62 s tif fn es in de x, % р <0.05 m. a. bystrytska of post-traumatic period, but there is a proven relationship of osteoporosis risk and bone characteristic features at the time of the injury [6]. the results of the study performed by professor vv povoroznyuk in 1998 prove that in the ukrainian population women reach peak bone mass at the age of 23-24 years old and men – at the age of 25-26 [7]. in present study, patients were divided into 2 subgroups accor ding to their trauma age. a cutting-off point for men was 25 years and for women – 23 years of age. in the patients with the injury that occurred before reaching peak bone mass, the bone mineral status according to ultrasound densitometry was significantly worse than that of the patients of corresponding age, anthro pometric parameters and duration of the posttraumatic period with the onset of injury at the age over 23 and 25 years, respectively (fig. 1, 2). no difference was proved in the bone mineral status of the women depending on their age at the injury onset, but after excluding postmenopausal women from both study groups, it was found out that bone mineral status was significantly worse in the women with trauma at the age of 23 years old (fig. 2). a significant difference in stiffness index in the women of reproductive age and postmenopausal women was not evidenced due to small size and heterogeneity of sample. in addition, the impact of menopause should be considered in certain groups of women, depending on the age at the injury onset before or after reaching peak bone mass. among the examined individuals, 18 pa tients had fractures of hip (11 patients) or tibia fractures (7 patients) due to a low-energy trauma in the period after a spinal cord injury. 66% of them had a spinal cord injury at the age of 25 years old (p=0.001). other differences between the group of patients with fractures and without fractures were not detected. bone metabolism according to remodelling markers was analysed separately for women table 2. bone mineral status in the patients with chronic spinal cord injury and persons of control group parameter men women main group control group main group control group т-score -3.20±0.80* -0.40±0.80 -3.10±0.50* -0.60±0.60 z-index -2.70±0.70* 0.40±0.20 -2.90±0.50* -0.40±0.60 si 51.40±11.80* 98.50±16.60 50.10±9.80* 92.90±11.10 extrapolated bmd 0.25±0.08* 0.55±0.10 0.24±0.06* 0.51±0.07 bua 40.50±13.80* 78.70±15.70 37.40±12.70* 76.90±11.30 sup 1477.00±19.00* 1554.00±26.00 1473.00±25.00* 1542.00±18.00 note:* – p<0.05, differences in parameters of the main and control groups. and men, and compared with the reference data for the ukrainian population (table 3) [8]. according to the results of previous studies, bone metabolism is much increased after sci, and then it gradually decreases, but remains high in the first years after the injury [4]. this paper proves that bone remodelling markers remain elevated in the later period of the injury as well (traumatic disease duration from 5 to 38 years, average duration – 14.7±1.2). in both women and men with sci, tp1np levels are significantly higher than the reference values. men are also characterized by higher levels of bone resorption marker (β-ctx). the level of fig. 1. bone mineral status of men depending on the spinal cord injury age. fig. 2. bone mineral status of reproductive age women with sci depending on the age at trauma incidence. m. a. bystrytska 24 in t e r n a l m e d ic in e issn 2413-6077. ijmmr 2018 vol. 4 issue 1 vitamin d3 is significantly lower in the sci pa tients than the reference values. no significant relationships of the levels of bone remodelling markers and sci duration were evidenced, but upon division of patients into 2 groups depen ding on the post-traumatic period duration: subgroup a<14.7 and subgroup b>14.7 years, the β-ctх level was significantly higher in subgroup a, 0.55±0.07 pg/ml, in subgroup b – 0.39±0.04 pg/ml, t=2.4, p<0.05, proving gradual slowing of the destruction process in the late posttraumatic period. discussion increased bone resorption in the sci pa tients occurs very quickly after the injury and affects bones below the spinal cord injury. according to the results of previous studies, the patients with signs of complete spinal cord injury, who had a fragility fracture, were diagnosed with a significant loss of bone mineral density in the distal femur (54%) and distal tibia bone (73%), which took place for the first 5-7 years after the injury [5]. interestingly that bone loss is greater in the distal section. the above trend suggests that at the level of calcaneal bone, the loss will be even greater, also due to a higher content of spongy bone in the calcaneal bone. the use of ultrasound densitometry of calcaneal bone as a screening method in the diagnosis of osteoporosis is based on the close relationship of parameters of vertebral bone and calcaneal bone. in the sci patients, this relationship is missing. after the complete spi nal cord injury, bone changes differently depend on the location. in parts of the skeleton, located above the injury level, bone mineral density decreases immediately after the injury, but gradually returns to normal parameters and even increases with increased load, such as bone mineral density of upper limbs in the patients with paraplegia. in the sections below the injury, with impaired innervation and reduced static load, such as lower limbs, bone mineral density decreases progressively [4, 9]. thus, regardless of the injury level, the lumbar vertebrae and calcaneal bone is in different conditions and changes in them are opposite. distal femur and proximal tibia are the most frequent localization of fractures in these patients and therefore an area of interest. these areas consist mainly of spongy bone and are closer in composition to calcaneal bone, therefore lose bone tissue in a similar way in the absence of axial load and adequate trophic effects. the results of ultrasound densitometry of calcaneal bone can also diagnose osteoporosis, but according to the literature the capabilities of this method in the follow up of sci patients are limited [8-10]. the loss of bone tissue after a severe spinal injury develops rapidly, resulting in the development of osteoporosis and significantly increased fracture risk, but after 3-5-10 years, the rate of bone resorption slows down. further, the post-traumatic period duration has less effect on the bone mineral status [4]. inverse correlation was proved between the post-traumatic period duration and the number of proximal tibia trabecules that confirmed the continued predominance of resorption processes in spongy bone. simultaneously, the thinning of cortical layer of long bones of lower extremities took place, but the loss of cortical layer occurred much more slowly than that of trabecular bone. slow cortical layer thinning may explain the fact that the average period until the first fracture in sci patients is 9 years after the injury [3]. thus, correction of structural-functional state of bone as fracture prevention has quite a large therapeutic window – 9 years. among the leading risk factors for osteoporosis in the sci patients along with severity of the injury and absence of axial load in the post-traumatic period, the low baseline values of mineral density and high levels of markers of bone remodeling are present [11]. this coincides with the results of our study. the patients, who had injury before bone mass peak, have lower parameters of structural and functional state of bone and increased risk of fractures. all patients in our study had low bone mineral density (according to z-criterion or t-score, depending on the age). according to the 2014 national osteoporosis foundation guidelines, sci is one of the diseases and conditions that contribute to the development of osteoporosis and fractures, and therefore the patients with sci and low bone mineral table 3. bone turnover markers and vitamin d level in the sci patients bone metabolism parameters bone metabolism reference values bone metabolism parame ters in the sci patients р for men for women in men in women men women tp1np, ng/ml 34.50±5.80 44.70±4.40 43.80±4.30 57.40±4.50 <0.05 <0.05 oc, ng/ml 24.30±1.70 23.40±1.50 24.50±1.90 19.60±1.50 >0.05 <0.05 β-ctx, ng/ml 0.37±0.06 0.41 ±0.07 0.58±0.05 0.36±0.04 <0.05 >0.05 25(oh)d3, nmol/ml 44.60±5.20 40.40±5.60 26.80±7.80 20.30±4.50 <0.05 <0.05 m. a. bystrytska 25 in t e r n a l m e d ic in e issn 2413-6077. ijmmr 2018 vol. 4 issue 1 density can be diagnosed with secondary osteoporosis. the prevalence of this condition among the sci patients encourages further researches. conclusions sci leads to increased bone resorption with development of secondary osteoporosis (accor ding to the ultrasound densitometry of calcaneal bone). in chronic sci, bone resorption is higher than in the individuals with combined low levels of vitamin d, and the absence of axial load results in continued loss of bone mass. acknowledgments i appreciate the help of the ukrainian asso ciation of osteoporosis (the president – pro fessor povoroznyuk v. v.) and association of disabled persons with spinal cord injuries (the president – marusevych i. m.) in conducting research. references 1. dolbow jd, dolbow dr, gorgey as, adler ra, gater dr. the effects of aging and electrical stimu lation exercise on bone after spinal cord injury. aging dis. 2013;4(3):141–53. 2. frotzler a, berger m, knecht h, eser p. bone steady-state is established at reduced bone strength after spinal cord injury: a longitudinal study using peri pheral quantitative computed tomography (pqct). bone.2008;43(3):549–55. doi: 10.1016/j. bone.2008.05.006. 3. battaglino ra, lazzari aa, garshick e, morse lr. spinal cord injury-induced osteoporosis: pathogenesis and emerging therapies. curr osteoporos rep. 2012;10(4):278–85. doi: 10.1007/s11914-0120117-0. 4. bauman wa, cardozo cp. osteoporosis in individuals with spinal cord injury. pm&r. 2015 feb 1;7(2):188-201. doi: 10.1016/j.pmrj.2014.08.948. 5. bryson je, gourlay ml. bisphosphonate use in acute and chronic spinal cord injury: a systematic review. j spinal cord med. 2009;32(3):215-25. 6. gifre l, vidal j, carrasco jl, muxi a, portell e, monegal a et all. risk factors for the development of osteoporosis after spinal cord injury. a 12-month follow-up study osteoporos int. 2015;26(9):2273-80. doi: 10.1007/s00198-015-3150-x. 7. povoroznyuk vv, orlyk tv, kreslov yeo. a modern view of the problem of osteoporosis in men in ukraine. pain. joints. spine. 2012: 2(6). http://www. m i f u a . c o m / a rc h i v e / a r t i c l e _ p r i n t / 3 4 3 3 5 . [ i n ukrainian]. 8. vaida vm. (donetsk national medical university). gender and age features of osteoporosis and its complications. donetsk; 2011. 310 p. [in ukrainian]. 9. bittar c, cliquet a. utility of quantitative ultrasound of the calcaneus in diagnosing osteo porosis in spinal cord injury patients. american journal of physical medicine & rehabilitation. 2011 jun;90(6):477-81. pmid: 21765270. doi: 10.1097/ phm.0b013e31821a7386. 10. chow yw, inman c, pollintine p, sharp ca, et al. ultrasound bone densitometry and dual energy x-ray absorptiometry in patients with spinal cord injury: a cross-sectional study. spinal cord.1996; 34:736-41. doi: 10.1038/sc.1996.134. 11. cosman f, de beur sj, leboff ms, et al. clinician’s guide to prevention and treatment of osteoporosis. osteoporos int. 2014 oct;25(10):2359 81. pmid: 25182228. pmcid: pmc4176573. doi: 10.1007/s00198-014-2794-2. received: 2018-05-11 зміни мінеральної щільності та метаболізму кісткової тканини в пацієнтів зі спінальною травмою м. а. бистрицька ду «інститут геронтології ім. д. ф. чеботарьова намн україни», київ, україна український центр лікування остеопорозу, київ, україна вступ. вивчення пізніх ускладнень при травмах спинного мозку, у тому числі остеопорозу, є актуальним через збільшення тривалості життя таких пацієнтів. мета дослідження визначити мінеральну щільність та особливості метаболізму кісткової тканини у хворих із травматичною хворобою спинного мозку. методи. обстежено 73 хворих із травмами спинного мозку та 57 здорових осіб. за допомогою ультразвукової денситометрії визначили мінеральну щільність кісток; маркери ремоделювання кісткової тканини та вміст вітаміну д стан – методом електрохімілюмінесценції. результати. у хворих із травмами спинного мозку стан мінерального компонента кісткової тканини був значно нижчим порівняно з хворими контрольної групи. індекс міцності кісткової тканини становив 51,4±11,8% у порівнянні з 98,5±16,6 % (p<0,05) у чоловіків і 50,1±9,8 % у порівнянні з 92,9±11,1 % (p<0,05) у жінок. у хворих із травмами спинного мозку рівень маркерів ремоделювання кісткової тканини був значно вищим, ніж у контрольній групі. стан мінерального компонента кісткової тканини у хворих із травмами спинного мозку значно гірший, порівняно з особами з вже розвинутою піковою кістковою масою, що дає змогу їх віднести до групи підвищеного ризику виникнення переломів. висновки. травми спинного мозку зумовлюють посилення резорбції кістки з розвитком вторинного остеопорозу (за даними ультразвукової денситометрії п’яткової кістки). процеси резорбції є значно інтенсивнішими при травмах спинного мозку у хворих із низьким рівнем вітаміну d, а також при відсутності осьового навантаження. ключові слова: травма спинного мозку; остеопороз; тетраплегія; параплегія; маркери ремоделювання кісткової тканини; ультразвукова денситометрія m. a. bystrytska issn 2413-6077. ijmmr 2018 vol. 4 issue 2 67 p u b l ic h e a lt h a n d e p id e m io l o g y doi 10.11603/ijmmr.2413-6077.2018.2.9286 infrastructure, resources, services evaluation and gap analysis of integrated maternal and child development services in india s. kaur, r. gupta, i. d. khan, a. k. jindal, s. prajapati, a. makkar, k. s. rajmohan army college of medical sciences and base hospital, new delhi, india background. integrated child development services (icds) is an indian community-centric government program organized under anganwadi centres catering to supplementary nutrition, health and preschool education, primary healthcare, growth monitoring and counselling the children under six years old along with their mothers. it is the world’s largest outreach program in a developing country covering a population of 1.35 billion; the variations in service delivery were analysed involving cross-sectional rural and urban anganwadi centers in new delhi. methods. data were collected by assessment of children and mothers, interview of anganwadi workers and observation of service delivery parameters and conduction of activities. infrastructural, beneficiaries, services and content were evaluated by a suitable pre-tested questionnaire based on the national institute of public cooperation and child development (nipccd) evaluation proforma. the data was analysed by a descriptive statistics. results. gaps were found in respect of infrastructure, resources, health and nutrition facilities especially at rural anganwadi centre which was inadequate in terms of implementation of nutrition and health program, supplementary nutrition, preschool education and nutrition rehabilitation centre for existing beneficiaries. both anganwadi centres were not catering for new who growth standards and adolescent health. conclusions. gaps found in respect of infrastructure, resources, health and nutrition facilities can affect performance of icds program and the services delivered by anganwadi centres, which need a boost. both urban and rural centres have a direct opportunity towards delivering adolescent health program focusing on nutrition and education of girls prior to their pregnancy, and adoption of new who growth standards. key words: icds, anganwadi centre, nutrition, maternal health, growth charts. international journal of medicine and medical research 2018, volume 4, issue 2, p. 67-71 copyright © 2018, tsmu, all rights reserved corresponding author: dr inam danish khan, mbbs, md, dnb, dhcm, mipha, miscd, associate professor (clinical microbiology and infectious diseases), army college of medical sciences and base hospital, delhi cantt 110010 india e-mail: titan_afmc@yahoo.com, mobile: +91 8076324060, fax: +91 11 25693490 s. kaur et al. introduction integrated child development services (icds) is a medico-social, gender-neutral, community-centric philanthropic program organized under rural courtyard shelters known as anganwadi centres, catering to supplementary nutrition, nutrition and health education, preschool non-formal education, primary healthcare, immunization and growth monitoring of the children under six years old along with their mothers [1]. icds was launched in 1975 in accordance with the national policy for children in 33 places throughout india complemented by 13.3 lakh anganwadi centres across the country. organization of icds is implemented at five different levels, i.e. central level, state/union territory level, district level, block level and village level covering rural areas, tribal areas and urban slums to serve the extremely underprivileged communities of backward and remote areas of the country. each anganwadi centre covers 40-42 children from 0-6 years old and 20-25 pregnant and lactating mothers delivering services right at the doorsteps of the beneficiaries to ensure their maximum participation [2]. the icds team comprises child development project officers, district program officers, and auxiliary staff. being the world’s largest outreach program in targeting infants and children below six years old, expectant and nursing mothers, the icds has generated interest worldwide among academicians, planners, policy makers, admi nistrators and those responsible for its im plementation [3]. given its effectiveness over the last few decades, the government of india is committed towards ensuring universal availaissn 2413-6077. ijmmr 2018 vol. 4 issue 268 p u b l ic h e a lt h a n d e p id e m io l o g y bility of icds. the icds program at anganwadi centres that are meant to adhere to certain laid-down standards given by the government for various infrastructural, resource and service parameters, for optimal functioning of the unit. the resources/infrastructure available on ground and services delivered vary widely. these variations account for the difference in standards of service-delivery, which in-turn might make a difference in overall effectiveness of icds. thus, the availability of infrastructure, human resources and delivery of services should be studied and compared against the laid-down standards, for ascertaining gap analysis. it is obvious that bridging the gaps will help improve the program and offset malnutrition. various evaluations of icds have revealed outcomes requiring improvement in the nutritional status of children which were falling short of expectations. this crosssectional study has evaluated and analysed the gap, with respect to infrastructure, resources and icds services vis a vis the laid-down standards, at rural and urban anganwadi centres in new delhi. methods a cross-sectional evaluation study was carried out among two anganwadi centres in new delhi. the data were collected by assessment of children and mothers, interview of anganwadi workers and observation of service delivery parameters and conduction of activities. infrastructural assessment of icds program via anganwadi centres was done by observing building infrastructure, floor, roof, electricsupply, availability of kitchen or washroom, kitchen-utilities, drinking-water, preschool education material, guidebook, growth charts, proportion of malnourished children, supplementary nutrition to pregnant females, counselling activities towards nutrition, health, immunization, maintenance of records about beneficiaries and services availed. suitable pretested and adequately modified questionnaire based on the national institute of public cooperation and child development (nipccd) evaluation proforma was designed to interview the anganwadi worker and the beneficiaries (mothers/children). data was analysed under descriptive statistics. results both anganwadi centres rented cemented building with adequate indoor space, childfriendly unisex toilets, electricity supply and water. both anganwadi centres were provided with hot cooked food containing a varied combination of pulses, cereals, oil, vegetables and sugar as well as take-home rations. the urban and rural anganwadi centres involved 11 and 12 pregnant women, seven lactating women each, 35 and 36 children aged from 6 months to 3 years, as well as 30 and 18 children aged 3-6 years respectively. no children were found to be underweight. no adolescent girls were beneficiaries at either anganwadi centres. pregnant women were counselled towards utilisation of key services i.e. antenatal care, iron and folic-acid supplementation, adequate extra-care from family and rest during pregnancy. there was no interruption in supplementary nutrition in past six months. supplementary nutrition program in urban anganwadi centre was more effectively used by beneficiaries as compared to rural anganwadi centre, where quality was neither satisfactory nor accepted by beneficiaries, while it was adequately accepted in the urban centre. in rural anganwadi centre, nutrition and health education program was not followed. as for infrastructural facilities, both anganwqadi centres had good electricity supply with electric points above 5 feet from ground that was safe for children. direct tapwater was stored in covered containers as per standards however there was no provision of either water-filters or reverse osmosis plants. new who growth standards were not implemented in both anganwadi centres, but weighing, plotting, interpretation and counselling of mothers were accurately performed and they were provided with who growth chart. early childhood care and education program was conducted in both anganwadi centres every month as per laid-down standards. urban anganwadi centre was adequately provided with preschool education materials such as time-table, guide-book for story-telling, counting numbers, free conversation sessions to enable speaking and organized smallactivities related to fine muscle coordination and development such as painting, drawing, threading and matching colour, reading simple words, writing alphabets words, distinguish objects, recognise pictures etc. the rural anganwadi centre was also provided with preschool education materials but not adequate ones as per laid-down standards. both anganwadi centres were undertaking routine health check-up. it was noted that in the urban anganwadi centre, the frequency of health check-up was monthly for children aged s. kaur et al. issn 2413-6077. ijmmr 2018 vol. 4 issue 2 69 p u b l ic h e a lt h a n d e p id e m io l o g y 1-3 years, quarterly for children aged 3-6 years, and four check-ups were provided to pregnant females starting in first trimester. in rural anganwadi centre, pregnant females were seen only twice starting in second-trimester. both anganwadi centres didn’t include adolescent girls for health check-up. in both anganwadi centres, immunization program was running effectively. iron and vitamin a supplementation were provided to children and pregnant women under the icds program. discussion recognizing the need for early intervention to ensure the development of a young child’s body, mind, and intellect to its maximum potential, the government of india started the icds, a centrally sponsored scheme, which is a step toward responding to the child’s needs in a comprehensive and holistic perspective. the national goals of icds program are reduction of infant mortality rate to <60/1000, reduction in child mortality rate to <10/1000, and reduction in maternal mortality rate by at least 50%, through a focused health check-up, immunization, deworming, basic treatment of minor illnesses like fever, diarrhoea etc., referral services for severe illness, supplementary feeding, growth monitoring and early childhood day care [4, 5]. infrastructural evaluation was satisfactory in this study whereas other studies found toilet facilities present only in 57.1% anganwadi centres [6,7]. the rural anganwadi centre was lagging the laid-down standards towards nutrition and health program, supplementary nutrition, preschool education and adoption of nutrition rehabilitation centre, which was also found in other rural anganwadi centres in india by various studies [8, 9, 10, 11]. these services are meant for effective transmission of certain basic health and nutrition messages to enhance the level of awareness of mothers about the child's needs and their capacity for care, protection, and development of the child. supplementary nutrition is strategic as it not only improves the nutritional level of children and reduces malnutrition, but it also works as an incentive for promoting attendance of children and mothers to participate in the activities of anganwadi centres, and as such plays a vital role in icds program. inadequacy on these fronts bears a direct connotation towards inadequate service delivery and fetomaternal outcomes as india is mostly a rural country and requires strengthening of rural anganwadi centres [12, 13, 14, 15]. in current study it was also observed that the new who growth standards were not implemented in both anganwadi centres, but weighing, plotting, interpretation and counselling of mothers were accurately performed, and they were provided with the who growth chart. similarly, adolescent health is a completely neglected area under the icds program which needs to be intensified to ensure nutrition and health of girls prior to pregnancy to enable them to make better choices towards family planning, contraception and perinatal care of themselves and their children [16]. in turn, this will universalize and maximise the concept development of icds program in context and content; and further capacity building initiatives amongst beneficiaries for better families, better management of anganwadi centres and a sustainably better future in a tropical developing country withholding a population of 1.35 billion [17, 18, 19]. conclusions gaps found in respect of infrastructure, resources, health and nutrition facilities can directly affect the performance of icds program and the services delivered by the anganwadi centres. there is a felt opportunity to boost the service delivery ecosystem of rural anganwadi centre in term of implementation of nutrition and health program, supplementary nutrition, preschool education and nutrition rehabilitation centre for existing beneficiaries. both urban and rural centres have a direct opportunity towards delivering adolescent health program focusing on nutrition and education of girls prior to their pregnancy, in order to maximise the potential of icds program and universalise the acceptance and availability of icds. there is a scope of improving upon adoption of new who growth standards. s. kaur et al. issn 2413-6077. ijmmr 2018 vol. 4 issue 270 p u b l ic h e a lt h a n d e p id e m io l o g y references 1. integrated child development services (icds) scheme, child development. new delhi: ministry of women and child development, government of india. rapid facility survey of infrastructure at anganwadicenters (rfs-awcs) by ncaer. available from: http://wcd.nic.in/icds.htm. 2. kapil u, sachdev hp. recommendations: national consultation to review the existing guidelines in icds scheme in the field of health and nutrition. indian pediatr. 2002;38:721-31. 3. patil sb, doibale mk. study of profile, knowledge and problems of anganwadi workers in icds blocks: a cross sectional study. online j health a l l i e d s c i 2 0 1 3 ; 1 2 : 1 . h t t p : / / w w w . o j h a s . o r g / issue46/2013-2-1.html. 4. three decades of icds an appraisal. national institute of public cooperation and child development (nipccd), 2006.http://www.nipccd.nic.in/reports/ icdsvol3.pdf. 5. thakare mm, kurll bm, doibale mk, goel nk. knowledge of anganwadi workers and their problems in an urban icds block. j med coll chandigarh. 2011;1(1):15-9. 6. chudasama rk, patel uv, verma pb, vala m, rangoonwala m, sheth a, viramgami a. evaluation of anganwadi centres performance under integrated child development services (icds) program in gujarat state, india during year 2012-13. journal of mahatma gandhi institute of medical sciences. 2015 jan 1;20(1):60. 7. datta ss, boratne av, cherian j, joice ys, vignesh jt, singh z. performance of anganwadi centres in urban and rural area: a facility survey in coastal south india. indian j matern child health 2010;12:1-9. 8. icds iv project (ida assisted). new delhi: ministry of women and child development, government of india. guidelines and processes to be followed for the preparation of state project implementation plans and district annual plans, central project management unit. february 2008. 9. dhingra r, shrama i. assessment of preschool education component of icds scheme in jammu district. global j human social sci. 2011;11(6):13-8. 10. kapil u, saxena n, nayar d, gnanasekaran n. status of growth monitoring activities in selected icds projects in rajasthan. indian pediatr 1996; 33:949-52. 11. gurukartick j, ghorpade ag, thamizharasi a, dongre ar. status of growth monitoring in anganwadi centres of a primary health centre, thirubhuvanai, puducherry. online j health allied scs. 2013; інтегровані центри матері і дитини в індії: інфраструктура, ресурси, оцінка діяльності та прогалини у роботі s. kaur, r. gupta, i. d. khan, a. k. jindal, s. prajapati, a. makkar, k. s. rajmohan army college of medical sciences and base hospital, new delhi, india вступ. об’єднані центри дитячого розвитку (icds) – це державна соціально-орієнтована програма у індії під егідою анганваді центрів, яка забезпечує додаткове харчування, санітарно-гігієнічну просвітницьку діяльність та дошкільну освіту, надання первинної медичної допомоги, моніторинг та консультування дітей віком до шести років разом з їхніми матерями. це найбільша у світі за охопленням (коло 1,35 мільйонів населення) програма у країнах, які розвиваються. мета роботи – проаналізувати діяльність анганваді центрів у міській та сільській місцевостях нью делі. методи. дані збиралися шляхом опитування матерів та дітей і працівників анганваді центрів, а також оцінки діяльності центрів. опитувальники щодо інфраструктури, переваг та наданих центрами послуг базувалися на формі, запропонованій національним інститутом громадського співробітництва та розвитку дітей. дані аналізувалися за допомогою описової статистики. результати. були знайдені недосконалі моменти у роботі анганваді центрів, особливо у сільській місцевості, а саме щодо термінів впровадження програм здорового харчування та дошкільної освіти та програм реабілітації. у обох центрах не було запроваджено нові стандарти вооз. висновки. виявлені прогалини можуть вплинути на ефективність роботи об’єднаних центрів дитячого розвитку та якість послуг, які надаються у анганваді центрах. однак, як міські, так і сільські центри мають можливість здійснювати програми охорони здоров'я підлітків, зосереджуючись на харчуванні та вихованні дівчат до їх вагітності, а також прийняття нових стандартів вооз. ключові слова: icds програма; анганваді центр; харчування; репродуктивне здоров’я жінок; розвиток дітей. s. kaur et al. issn 2413-6077. ijmmr 2018 vol. 4 issue 2 71 p u b l ic h e a lt h a n d e p id e m io l o g y received: 2018-08-02 12(2):2. available from: http://www.ojhas.org/ issue46/2013-2-2.html 12. sood ak, sood vp. the evaluation of supplementary nutrition component of icds – a study at block beri, rohtak, haryana. health & population – perspectives and issues. 1987;10:207-212. 13. sharma m, soni gp, sharma n. assessment of coverage of services among beneficiaries residing in area covered by selected anganwadi in urban project i and ii of raipur city. j community med health educ. 2013;3:195. 14. saiyed f, seshadri s. impact of the integrated package of nutrition and health services. indian j pediatr. 2000;67:322-8. 15. khan s, bal h, khan id, paul d. feto-maternal outcome in women diagnosed for gestational diabetes mellitus in western india: a two-year prospective study. iranian journal of diabetes and obesity.epub ahead of print.2018, eissn 2345-2250, issn 2008-6792. 16. khan id. kapb study on contraceptives among married armed forces personnel. indian journal for the practicing doctor. 2008;5:2. 17. surwade jb, mantri sb, wadagale av. utilization of icds scheme in urban and rural area of latur district with special reference to pediatric beneficiaries. international j recent trends sci technology. 2013;5:107-10. 18. avachat s, phalke vd, phalke db. epidemiological study of malnutrition (under nutrition) among under five children in a section of rural area. pravara med rev. 2009;1:20-2. 19. dixit s, sakalle s, patel gs, taneja g, chourasiya s. evaluation of functioning of icds project areas under indore and ujjain divisions of the state of madhya pradesh. online j health allied scs. 2010;9(1):2. s. kaur et al. 15 in t e r n a l m e d ic in e issn 2413-6077. ijmmr 2017 vol. 3 issue 1 o. m. oleshchuk, h. ya. loi doi 10.11603/ijmmr.2413-6077.2017.1.7563 leptin resistance and type 2 diabetes o. m. oleshchuk, h. ya. loi i. horbachevsky ternopil state medical university, ternopil, ukraine leptin is one of adipocyte-secreted hormones. it signals to the brain and other tissues about the status of body energy reserves. circulating leptin levels are directly proportional to the amount of the body fat. leptin concentration increases when surfeit and decreases during fasting. obese patients are hyperleptinemic compared with thin persons and they are tolerant to the central hypothalamic effects of leptin. the reduced sensitivity toward exogenous and endogenous leptin is commonly referred to as leptin resistance. alterations in the signaling of the long isoform of the leptin receptor play the crucial role in leptin resistance. surfeit may induce leptin resistance and other metabolic sequelae of obesity. leptin insensitivity and insulin resistance play a major role in the development of type 2 diabetes. metformin remains the preferred first-line pharmacologic agent for the treatment of type 2 diabetes. it reduces hepatic glucose production, increases glucose uptake in peripheral tissue and can lead to weight loss. metformin decreases both insulin and leptin concentration, restores the sensitivity to these hormones. but some studies have shown poor relationship between metformin action and leptin level. and the mechanism of metformin action on leptin resistance remains unclear. thus, these issues should be studied as well as polymorphisms in genes encoding metformin action. key words: leptine resistance; type 2 diabetes; metformin. corresponding author: halyna loi, department of pharmacology with clinical pharmacolology, i. horbachevsky ternopil state medical university, 1 maydan voli, ternopil, ukraine, 46001 phone number: +380 978975239 e-mail: loy@tdmu.edu.ua leptin role in endocrine system adipose tissue is a complex, essential, and highly active metabolic and endocrine organ. it responds to afferent signals from traditional hormone systems and central nervous system but also expresses and secretes factors with important endocrine functions [1]. the discovery of leptin in 1994 has changed the understanding of hormonal regulation of energy homeostasis and the role of adipose tissue from that of a depot storage organ to that of an active endocrine organ producing several bioactive peptides [2]. leptin, a 167–amino acid peptide [fig. 1], is one of the adipocyte-secreted hormones [1]. it is also expressed in other tissues, including placenta, mammary gland, testes, ovary, endometrium, stomach, hypothalamus, pituitary, and others [3]. leptin signals to brain and other tissues about the status of body energy reserves. thus, that appropriate changes in food intake, energy expenditure, and nutrient partitioning can occur to maintain whole-body energy balance [4]. circulating leptin levels are directly proportional to the amount of the body fat [5] and fluctuate with acute changes in caloric intake [6]. leptin concentration increases when surfeit and decreases during fasting [7]. circulating leptin crosses blood brain barrier and mediates its action through the janus kinase (jak)–signal transducer and activator of transcription (stat) pathway (jak–stat3) [8]. leptin exerts pleiotropic effects by binding and activating specific leptin receptors in the hypothalamus and other organs, has direct and indirect effects in metabolically active tissues, and regulates some neuroendocrine axes. several isoforms of the leptin receptor, resulting from alternative splicing, convey biological activity and are involved in mediating leptin's actions in brain and peripheral organs [2]. the long leptin receptor isoform (obrb) is expressed particularly in hypothalamus, where it regulates energy homeostasis and neuroendocrine function [9]. in ventromedial hypothalamus leptin stimulates expression of anorexigenic brain-derived neurotrophic factor [10]. in addition, leptin influences neurons that directly or indirectly regulate levels of other circulating hormones (e.g. thyroid hormone, sex steroids, and gh) [9, 11]. obrb is also expressed in multiple peripheral tissues, including international journal of medicine and medical research 2017, volume 3, issue 1, p. 15–21 copyright © 2017, tsmu, all rights reserved 16 in t e r n a l m e d ic in e issn 2413-6077. ijmmr 2017 vol. 3 issue 1 pancreatic islets, adipose tissue, skeletal muscle, liver, and immune cells [12, 13]. in the pancreatic islets, leptin directly inhibits insulin expression and secretion [14]. in liver and white adipose tissue, leptin inhibits lipogenesis and stimulates lipolysis [12, 13]. leptin directly promotes fatty acid oxidation in isolated adipocytes and skeletal muscle [10, 15] and decreases lipid levels in isolated livers [16]. leptin takes part in the regulation of energy homeostasis, insulin action and lipid metabolism [1] by improving insulin sensitivity and reducing intracellular lipids [17–19]. the ability of leptin to inhibit feeding is related to activation of pi3 kinase in the hypothalamus. blockade of pi3 kinase activity prevents the anorectic action of leptin [20]. congenital leptin deficiency is associated with hyperphagia, impaired thermogenesis, insulin resistance, hyperlipidemia, and central hypogonadism, which can be reversed by leptin treatment [20, 21]. women have higher leptin concentrations than men. but after menopause a significant reduction in the amount of circulating leptin occurs [22]. such sexual differences can be explained by the difference in fat mass, body fat distribution, and sex hormones. leptin level is also independent of body mass index. subcutaneous fat expresses more leptin mrna than omental (visceral) fat, and this may partially contribute to increased leptin levels in women compared with men [23]. deficient leptin signaling results in hyperphagia and decreased energy expenditure [24]. the result is not only an increasing degree of obesity associated with increased lipid storage in muscle, liver, and other tissues but also dysfunction of several neuroendocrine axes, including the reproductive, thyroid, and adrenal axes, as well as abnormal function of the immune and autonomic system (i.e. thermoregulation, energy expenditure, and others) [17,24]. normally leptin reduces along with insulin during fasting and mediates the suppression of thyroid hormone, growth hormone and reproductive hormones. leptin causes stimulation of appetite and inhibition of sympathetic nerve activity [25, 26]. chronic weight loss in females and obese patients on dietary restriction decreases leptin, resulting in suppression of reproductive hormones, disruption of menstrual cycles, and energy use by muscle [27, 28]. leptin may control glucose homeostasis independently of its effects on adiposity [29]. it regulates glycaemia via the cns, but it may also directly regulate the physiology of pancreatic β-cells [30, 31] and peripheral insulin-sensitive tissues [32]. it was also proved that leptin acting through lrb regulates insulin receptor substrate-1 and 2, mitogen-activated protein kinase, extracellular signal-regulated kinase, akt, and pi3 kinase, raising the possibility of interaction between leptin and insulin [33]. antidiabetic properties of leptin as a suppressor of glucagon are shown in some literature [34–36]. leptin also limits accumulation of triglycerides in liver and skeletal muscle through a combination of direct activation of ampk and indirect actions mediated through central neural pathways, thereby improving insulin sensitivity [37]. attenuation of leptin sensitivity in brain leads to excess triglyceride accumulation in adipose tissue, as well as muscle, liver, and pancreas, resulting in impaired insulin sensitivity and secretion [38]. although obese subjects are hyperleptinemic compared with thin persons [39], they appear either to be tolerant or resistant to the central hypothalamic effects of leptin. the reduced sensitivity toward exogenous and endogenous leptin is commonly referred to as leptin resistance [40]. however, leptin is not widely used in the clinical field because obesity is accompanied by increased serum leptin and responds poorly to the pharmacological administration of exogenous leptin, which ordinarily potently promotes fat mass loss and body weight reduction in thin subjects [41, 42]. fig. 1. leptin structure [81]. o. m. oleshchuk, h. ya. loi 17 in t e r n a l m e d ic in e issn 2413-6077. ijmmr 2017 vol. 3 issue 1 alterations in the signaling of the long isoform of the leptin receptor, especially in the hypothalamic arcuate nucleus, seem to play a crucial role in leptin resistance. additional mechanisms that were proposed to induce resistance toward the effects of leptin include alterations in the transport of leptin across the blood-brain barrier. the protein tyrosine phosphatase 1b is another potential mechanism that was proposed to interfere with leptin signaling by inhibiting signaling of the long isoform of the leptin receptor [24, 43]. leptin insensitivity and insulin resistance play a major role in the development of type 2 diabetes [44]. it can be explained by high levels of insulin and leptin as well as with hyperphagia in patients with obesity [45]. leptin resistance as a development factor of obesity and type 2 diabetes the who has declared obesity as the lar gest global chronic health problem in adults, and it is considered the 5th leading risk for life globally [46]. obesity is a ‘gateway’ for diabetes [47]. worldwide, 415 million people have diabetes, and this number will reach 642 million by 2040 [48]. type 2 diabetes accounts for 90-95% of all diabetes. this form encompasses individuals who have relative (rather than absolute) insulin deficiency and have peripheral insulin resistance [49]. nearly 90% of individuals with type 2 diabetes are either overweight or obese [50]. excess weight itself causes some degree of insulin resistance. patients with type 2 diabetes may have insulin levels that appear normal or increased; the increased blood glucose levels in these patients would be expected to result in even higher insulin values and to have their b-cell function in norm. thus, insulin secretion is defective in these patients and insufficient to compensate for insulin resistance [49]. leptin resistance was introduced in an apparent analogy with that of insulin resistance to explain why hyperleptinemia associated with obesity fails to correct the defect in energy balance and feeding behaviour [51]. in obesity, the transport of leptin across the blood brain barrier is decreased and levels of suppressors of cytokine signaling-3 (socs-3), an inhibitor of leptin signaling is increased in hypothalamus, which leads to leptin resistance [52]. therefore, the concept of ‘leptin resistance’ has been challenged recently by an alternate concept of ‘hypothalamic leptin insufficiency’ [53]. it is assumed that leptin resistance contributes to hyperphagia [54, 55]. it is also possible that surfeit may induce leptin resistance and other metabolic sequelae of obesity [45]. this rapid adaptation to increased energy availability may be designed to curtail the leptin system to facilitate storage of nutrients into lipid stores [54, 56]. this may be accomplished by restraining leptin biosynthesis [57–59] and/or by inducing leptin resistance [54, 56]. these mechanisms would be particularly well developed in individuals predisposed to weight gain and diabetes [54, 60]. consistent with the ‘thrifty genotype’ hypothesis, this sequence of events would be tightly coupled to the onset of insulin resistance [45, 54]. inadequate early increase in leptin secretion and biosynthesis (table 1) in response to overeating may also play a role in the development of obesity and glucose intolerance [61]. table 1. the scheme of metabolic disorders in the human body initiated by leptin resistance (based on the fig. 2 by kalra s.p., 2008) diminished leptin transport across blood–brain barrier ↓ leptin resistance ↓ hypothalamic leptin insufficiency with defections in hypothalamic regulation of energy homeostasis ↓ decreased restraint on pancreatic insulin secretion decreased glucose metabolism in peripheral organs decreased energy expenditure ↓ ↓ ↓ hyperinsulinemia with insulin resistance → hyperglycemia with increased energy stores → adipogenesis and obesity ↓ hyperleptinemia metabolic syndrome type 2 diabetes o. m. oleshchuk, h. ya. loi 18 in t e r n a l m e d ic in e issn 2413-6077. ijmmr 2017 vol. 3 issue 1 metformin action in patients with leptin resistance metformin remains the preferred first-line pharmacologic agent for the treatment of type 2 diabetes [62]. the use of metformin as a firstline therapy was supported by the findings of a large meta-analysis with selection of secondline therapies based on patient-specific considerations [63]. metformin acts mainly by reducing hepatic glucose production via inhibition of gluconeogenesis [64, 65] and increases glucose uptake in peripheral tissue [65]. metformin is associated with a low risk of hypoglycemia and is weight neutral or can lead to weight loss [66]. moreover, metformin decreases leptin concentration in morbidly obese subjects [67, 68] and in normal-weight healthy men [69]. although leptin concentration is closely related to body fat mass, the leptin-reducing effect of metformin cannot be fully explained by body weight reduction because metformin reduces leptin level even without changing body weight in normal-weight healthy men [69]. it was proved that weight loss achieved by metformin was correlated with pre-treatment plasma leptin levels. this effect of metformin was paralleled by a stimulation of the expression of the leptin receptor gene (obrb) in the arcuate nucleus. thus, identifying the hypothalamic obrb as a gene was modulated after metformin treatment [70]. it has been recently reported that metformin targets the amp-activated protein kinase (ampk), which is also activated by leptin [71–73]. several researches have been performed to analyse the molecular mechanism behind the effect of metformin on leptin levels. an invitro study reports that metformin inhibits leptin secretion by inhibiting mapk signaling pathway in adipocytes [74]. the other studies has proved that metformin restores leptin sensitivity in obese rats with leptin resistance and metformin treatment increases cerebrospinal fluid leptin concentrations in both standard chow and high fat-fed obese rats compared with the untreated rats. the authors have also reported that metformin increases hypothalamic pomc (an anorexigenic peptide) expression by leptin treatment in high fat-fed obese rats, whereas this is not observed in untreated high fat-fed obese rats [75]. as the effect of leptin is associated with the activation of pomc, failure to activate pomc expression by leptin is an evidence of leptin resistance [76]. distinct reduction in serum leptin level was observed in non-obese healthy individual on metformin, without any reduction in body weight [77]. but some studies have shown poor relationship between metformin action and leptin level [78, 79]. although many researches have been conducted, the mechanism of metformin action on leptin resistance remains unclear. moreover, similar investigations have contrary outcomes. therefore, this issue requires further research. conclusions leptin, the adipocyte-secreted hormone, has direct and indirect effects on metabolically active tissues, and regulates several neuroendocrine axes. it takes part in the regulation of energy homeostasis, insulin action and lipid metabolism, and signals primarily on the status of the body energy reserves in fat to brain and other tissues. circulating leptin levels are directly proportional to the amount of body fat. leptin concentration are increased in obesity and decreased during fasting. in obesity, the transport of leptin across the blood brain barrier is diminished, which leads to leptin resistance. leptin insensitivity and insulin resistance play a major role in the development of type 2 diabetes. metformin decreases both insulin and leptin concentration, restores the sensitivity to these hormones. but the mechanism of metformin action on leptin resistance remains unclear. the leptinreducing effect of metformin cannot be fully explained only by body weight reduction. furthermore, some studies have proved poor relationship between metformin action and leptin level. all these aspects require an in-depth research. considering the topical matter of exploring metformin action due to polymorphisms in genes encoding drug receptors, transporters, and metabolizing enzymes [80], the study of the relationship between genetic variants in solute carrier transporters and changes in leptin levels under metformin therapy would be reasonable. o. m. oleshchuk, h. ya. loi 19 in t e r n a l m e d ic in e issn 2413-6077. ijmmr 2017 vol. 3 issue 1 references 1. kershaw e, flier j. adipose tissue as an endocrine organ. the journal of clinical endocrinology & metabolism. 2004;89(6):2548–2556. 2. bluher s, mantzoros c. leptin in humans: lessons from translational research. the american journal of clinical nutrition. 2009;89(3):991–997. 3. leibel rl. molecular physiology of weight regulation in mice and humans. international journal of obesity. 2008;32:98–108. 4. 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aral y. leptin concentrations are related to glycaemic control, but do not change with short‐term oral antidiabetic therapy in female patients with type 2 diabetes mellitus. diabetes, obesity and metabolism. 2000; 2(5):313–6. 79. adamia n, virsaladze d, charkviani n, skhirtladze m, khutsishvili m. effect of metformin therapy on plasma adiponectin and leptin levels in obese and insulin resistant postmenopausal females with type 2 diabetes. georgian med news. 2007; 145:52. 80. pasechko nv, oleshchuk om, loi gy. рharmacogenetic aspects of metformin action in patients with type 2 diabetes mellitus. international journal of endocrinology. 2016;8(80):66-72. 81. leptin (150-167) (human). 2016. http://www. chemicalbook.com/productchemicalpropertiescb3304602_en.htm. accessed 2016. received: 2017-03-25 o. m. oleshchuk, h. ya. loi 13 in t e r n a l m e d ic in e issn 2413-6077. ijmmr 2018 vol. 4 issue 1 o. v. yermishev doi 10.11603/ijmmr.2413-6077.2018.1.8902 the levels of functional-vegetative homeostasis as criteria for magnetotherapy efficacy o. v. yermishev vasyl stus donetsk national university, vinnytsia, ukraine background. disorders of autonomic nervous system caused up to 80% of functional disorders. there is no information about the influence of magnetotherapy (mt) on the indicators of vegetative homeostasis, which disturbance is a cause of functional pathology. objectives. the aim of the study is to investigate vegetative rehabilitation trend of mt in various initial conditions of functional-vegetative disorders. methods. functional-vegetative diagnostics method by v.g. makats was chosen as a method of control of mt impact. the diagnostic complex biotest-12m was a technical tool. 38 children of different age and gender treated in the department of physiotherapy of vinnytsia regional children clinical hospital in 2016-2017 were involved in the research. the patients were divided into 7 groups according to the levels of functional vegetative homeostasis. results. mt had a different effect on vegetative activity as well as systemic and functional dependence, according to the coefficient of functional vegetative homeostasis. the most positive effect was evidenced in a group with a significant parasympathicotonia (group 1). in the group with severe sympathicotonia (group 6) it had negative effect. there were no gender and age-related characteristic features of the influence of mt on the dispersion of vegetative levels. conclusions. rehabilitation expediency requires maintenance of functional vegetative homeostasis at the level of fcp-vb-fcs in conjunction with functional-vegetative diagnostics using the method of v.g. makats. magnetotherapy can be recommended to be used only for patients with significant and expressed parasympathicotonia. key words: magnetotherapy; functional-vegetative diagnostics; vegetative homeostasis; vegetative level; vegetative coefficient. international journal of medicine and medical research 2018, volume 4, issue 1, p. 13-20 copyright © 2018, tsmu, all rights reserved corresponding author: oleh yermishev, phd, associate professor of department of plants physiology and biochemistry, biological faculty, vasyl stus donetsk national university, 21 600-richchya str., vinnytsia, 21000, ukraine e-mail: o.yermishev@donnu.edu.ua phone number: +380676504433 introduction the disorders of autonomic nervous system (ans) caused 25-80 % of functional disorders of reserves and mechanisms of adaptation to the changing environment, physical and psychosomatic efforts [1, 2]. being of integrative importance at the central level, the ans ensures regulation of vegetative homeostasis, systemic disorders of which determine the pathogenetic basis of functional and somatic diseases [1, 3, 4]. their fundamental evaluation goes beyond certain parameters of vegetative regulation, have need of a systematic approach and study of a holistic organism as a multilevel interdependent system [5, 6]. a significant support for the problem of vegetative pathogenesis is the normalization of vegetative indices in the rehabilitation period. in this case, special attention should be paid to the use of traditional faz (functionally active skin zones) [7-9]. nowadays, according to the who recommendations, ’electro acupuncture diagnostics and reflexotherapy’ should become the basis for rehabilitation medicine (the who international council, yerevan, 19.09.03). its general assembly (2014) herewith officially advises the states (the who participants) to include these areas into national health programs and draws attention to the development of biophysical methods of controlling the rehabilitation effectiveness of physiotherapeutic and preformed factors [5, 10, 11]. therefore, much attention should be paid to the recently discovered ’functional-vegetative system of a human’ (fvs) [12], which has proved the biophysical reality of traditional ‘acupuncture channels’ as well as their direct relation to the vegetative homeostasis and, in addition, requires defining of its place in the classical physiological system [13, 14]. 14 in t e r n a l m e d ic in e issn 2413-6077. ijmmr 2018 vol. 4 issue 1 low-frequency magnetotherapy (mt) is widely used in modern physiotherapeutic practice using the magnetic component of lowfrequency electromagnetic fields [15-17]. mt has an impressively wide range of indications (anti-inflammatory, anti-oedematous, trophic, hypocoagulant, vasoactive, anaesthetic, stimulating reparative processes and immuno-modelling) [18-20]. the official list of absolute and relative official contraindications is, concurrently, also impressive. the attention is drawn to complete absence of information on the influence of mt on the indicators of vegetative homeostasis, the disturbance of which is conditioned by the development of any functional pathology. the aim of the study is to investigate autonomic rehabilitation of mt at different initial states of functional vegetative disorders. methods the research is a fragment of the program ’two-stage system of rehabilitation of vegetative disorders in children living in the zone of ecological (radiation) control of ukraine’ (is being carried out according to the assignment of the cabinet of ministers of ukraine no. 12010/87). ’functional-vegetative diagnostics’ (fvd) by the method of v.g. makats has been chosen as a method of control of functional and vegetative efficiency of mt [1214]. this method has been admitted to be used in medical practice by the academic council of the ministry of health of ukraine and the joint session of the republican problem commissions (rpc) of paediatrics, obstetrics and gynaecology, quantum medicine, haematology and transfusiology, new medical technology and new diagnostic tools (minutes no. 1, 08-01 dated september 11, 1994). the diagnostic complex biotest-12m is a technical tool of fvd. the latter does not use traditional external power sources and the rpc ’new medical technology and new methods of diagnostics, prevention and rehabilitation’ by the ministry of health of ukraine (minutes no. 5 dated december 25, 1991) has been approved for practical use. statistical significance of the obtained data was estimated by means of parametric and nonparametric statistics. the analysis of the results was carried out by means of the computer programs ‘search’ (developed by the european centre for postgraduate education of the unap). functional autonomic systems (‘acupun cture channels’) are based on the international ‘acupuncture nomenclature’ (ian) suggested by the who (table 1). the following zones (levels) of functional-vegetative homeostasis are scientifically based on the coefficients of functional-vegetative homeostasis (k-v) [3]: pa-sig (k-v to 0.75 – a zone of significant parasympathetic activity); pa-exp (k 0,76-0,86 – a zone of expressed parasympathetic activity); fcp (k-v 0,87-0,94 – a functional compensation zone of parasympathicotonia); vb (k-v 0,951,05 – a zone of vegetative balance); fcs (k-v 1,06-1,14 – a zone of functional compensation of sympathicotonia); sa-exp (k-v 1,14-1,26 – a zone of clear sympathetic activity); sa-sig. (k-v≥1,26 – a zone of significant sympathetic activity). the planned research activities were conducted under the guidance of a high-level expert of the national academy of sciences of ukraine, doctor habilitatus in medicine, professor v.g. makats. 38 children (7-16 years old, 13 females and 25 males) were involved in the study; they underwent treatment at the department of physiotherapy of the vinnytsia regional children clinical hospital in 2016-2017. the children were divided into 7 groups according to the levels of functional vegetative homeostasis: the 1st group – with significant parasympathetic activity (pa-sig); the 2nd – with expressed parasympathetic activity (pa-exp); the 3rd – with the zone of admissible functional vegetative norm (zan); the 4th – with expressed sympathetic activity (sa-exp); the 5th – with a significant sympathetic activity (sa-sig). they were also divided into 2 groups according to gender: a female group (fg) – 13 children and table 1. functional autonomic systems ‘acupuncture channels’ (based on the international ‘acupuncture nomenclature’ (ian) suggested by the who) traditional channel ман traditional channel ман lungs lu bladder bl large intestine li kidneys ki stomach st pericardium pc spleen, pancreas sp triple heater te heart ht gallbladder gb small intestine si liver lr o. v. yermishev 15 in t e r n a l m e d ic in e issn 2413-6077. ijmmr 2018 vol. 4 issue 1 a male one (mg) – 25 children. fvd was twice held in the first half of the day (10:00-12:00) before and after the mt session. the bioelectric activity of 12 symmetrical pairs of functionally active skin zones (24 fazs) was studied, 2016 tests were carried out. the attention was paid to the direction of dispersion of integral vegetative homeostasis levels. the following areas of influence were chosen for mt: parasternal, epigastric and anterior abdominal wall, intrascapular, lumbar-sacral regions and the region of pelvic (femoral), knee and ankle joints. results the study of the influence of mt on vegetative activity as well as systemic and functional dependence in the initial significant parasympathicotonia (group 1) proved that, regardless of the topography of the effect, mt positively affects the vegetative homeostasis transforming its vegetative coefficients to a higher level of functional activity at the functional compensation zone of parasympathicotonia (fcp), as evidenced by the increase of functional-vegetative homeostasis coefficient (k-v) from 0.66 to 0.88 (fig. 1, table 2). table 2. the effect of mt on systemic-functional dependence мт k-v lu pc ht si te li sp lr ki bl gb st initial significant parasympathicotonia (group 1) 1* 0.66 8.7 7 7 3.8 2.8 3.8 12 12 13 11.1 8.4 9.8 2* 0.88 8.7 7.7 5.1 8.2 2.6 5.1 8.2 12 12 14.1 7.2 10.2 initial expressed parasympathicotonia (group 2) 1 0.85 11.2 9.4 10.2 12.6 2.7 8.3 8.8 8.6 6.7 9.4 5.3 7.8 2 0.90 8.0 8.4 10.5 8.8 6.3 4.2 8.0 11.3 6.3 9.2 10.5 8.4 initial functional compensation of parasympathicotonia (group 3) 1 0.90 7.4 9.7 9.5 11.4 5.9 6.7 8.6 9.0 8.0 9.9 7.6 5.9 2 1.25 6.8 6.5 6.8 13.5 9.5 7.2 9.0 7.2 9.0 11.3 7.9 7.2 initial autonomic balance of vns (group 4) 1 1.06 11.0 9.3 8.2 11.4 3.0 7.6 11.0 4.0 4.9 18.3 5.3 5.5 2 0.86 6.7 8.3 8.8 10.2 5.4 10.0 14.0 8.0 7.9 10.6 4.8 5.4 initial functional compensation of sympathicotonia (group 5) 1 1.07 7.5 7.2 8.7 13.7 5.0 8.0 8.7 7.5 8.6 10.1 7.2 7.7 2 0.86 7.5 9.2 9.6 11.7 4.7 4.9 10.0 8.2 9.6 9.2 7.0 8.8 initial significant and expressed sympathicotonia (groups 6,7) 1 1.26 8.8 6.3 5.7 9.1 10.0 11.0 7.1 8.5 8.0 8.8 6.0 11.1 2 2.28 4.6 6.8 3.0 6.1 10.6 11.0 9.5 2.7 3.8 31.6 3.8 6.1 notes: 1 * – here and further. before the exposure of magnetic radiation. 2 * – here and further. after the exposure of magnetic radiation. fig. 1. the influence of mt at pa-sig. o. v. yermishev 16 in t e r n a l m e d ic in e issn 2413-6077. ijmmr 2018 vol. 4 issue 1 the study of the influence of mt on vegetative activity and systemic-functional dependence in the initial expressed parasympathicotonia (group 2) revealed that, regardless of the topography of the effect, mt affects the vegetative homeostasis relatively positively, translating its vegetative coefficients to higher levels of functional activity, as proved by the increase of the coefficient of functional vegetative homeostasis (kv) from 0.85 to 0.9 (fig. 2, table 2). what is more, the dispersion of the levels of vegetative balance (lvb) under the influence of mt was present, which was accompanied by the transition of 28.57 % of children to the fcp zone and 14.28 % to the zone of vb and fcs (table 3). the system correlation of ‘acupuncture channels lu-pc-ht, si-te-li, st-gb-ki-lr) was in a state of interrelated dynamic functional compensation (fig. 2). the study of the influence of mt on vegetative activity and systemic an functional dependence taking into account the initial functional compensation of parasym pa thicotonia (group 3), the initial autonomic balance of vns (group 4) and the initial functional compensation of sympathicotonia (group 5) proved that the effect of magnetotherapy was relatively neutral concerning functional vegetative homeostasis, which in approximately 70 % of cases fluctuated within the functional compensation of sympathetic and parasympathetic activities (table 3). the value of vetable 3. the influence of mt on the dispersion of the levels of vegetative balance (lvb), % мт pa-sig pa-exp fcp vb fcs sа-exp sа-sig initial significant parasympathicotonia (group 1) 1 100 2 33.33 33.33 33.33 initial expressed parasympathicotonia (group 2) 1 100 2 14.28 28.57 28.57 14.28 14.28 initial functional compensation of parasympathicotonia (group 3) 1 100 2 37.50 25.00 25.00 12.50 initial autonomic balance of vns (group 4) 1 100 2 20.00 20.00 30.00 20.00 10.00 initial functional compensation of sympathicotonia (group 5) 1 100 2 12.50 12.50 50.00 12.50 12.50 initial significant and expressed sympathicotonia (groups 6,7) 1 100 2 25.00 50.00 25.00 fig. 2. the influence of mt at pa-exp. o. v. yermishev 17 in t e r n a l m e d ic in e issn 2413-6077. ijmmr 2018 vol. 4 issue 1 getative coefficients (kv) pointed to the tendency to ‘maintain stability’ and the systemic ratio of acupuncture channels lu-pcht, si-te-li, st-gb-ki-lr, in its turn, was staying in a state of interdependent dynamically functional compensation (table 2). the study of the influence of mt on vegetative activity and systemic and functional dependence in the initial expressed and significant sympathicotonia (groups 6-7) revealed that mt, regardless of the topography of the effect, negatively affected the vegetative homeostasis increasing its level of significant neo-rewards (fig. 3, table 2), which was accompanied by a significant increase in the value of kv from 1.26 to 2.28. in case of mt only 25 % were transformed to the poor level of functional activity at the zone of functional compensation of sympathicotonia (fcs) (table 3). the systemic ratio of acupuncture channels lu-pc-ht, si-te-li, st-gb-ki-lr), in this case, was in a state of interdependent dynamic functional compensation (fig. 3). investigating the gender and age characteristic features of the influence of mt on the dispersion of the vegetative levels, the same type of dispersion of vegetative levels in the female and male study groups was revealed (table 3). the analysis proved that there was no gender and age-related influence of mt on the variance of vegetative levels. under all conditions, this factor positively affected only the initial levels of the benefits of parasympathetic activity. its usage in other vegetative disorders caused development of a higher sympathetic orientation. the conclusion was drawn according to the directed dispersion of vegetative levels at various states of initial vegetative disorders (table 4). discussion the data on the effect of magnetic radiation on the vegetative status of animals and humans can be found in the literature. the effect of weak (up to 3.5 mt) and low-frequency (up to 100 hz) impulse magnetic field on the state of vegetative nervous system of animals has been studied by analyzing the variability of heart rate. the effect of magnetic field was evaluated by a individually designed complex for recording cardiac signals of animals. several specially selected regimes of impulse magnetic fields were studied. it was proved that the impulse magnetic field was of a high biological activity at all regimes used, and the indices of vegetative nervous system after the exposure to impulse magnetic field were about to the values typical for normotonic animals. this made it possible to use magnetic fields at those regimes in magnetotherapy [21]. the study of the effect of transcranial magnetotherapy on 63 people with the 1st degree ag at the age of 38-50 years old proved that after the course of therapy the number of patients with hyper-sympathicotonia decreased from 24 (69 %) to 14 (40 %) and the number of patients with normal and asympathicotonic vegetative regulation were 39 % and 21 % respectively [22]. the effect of electromagnetic field of ultrahighfrequency range results in the optimization of autonomic balance of the body in the patients with autonomic nervous system disorders was also evidenced. the electro magnetic field (emf) had sympatholytic, parasympatolytic and tonic action on a person [23]. we still believe that the data obtained from these studies cannot fully characterize the effect of magnetic and wave effect on the body due to the selected methods of vegetative state fig. 3. the influence of mt at initial sa-exp (sig). o. v. yermishev 18 in t e r n a l m e d ic in e issn 2413-6077. ijmmr 2018 vol. 4 issue 1 diagnosing. in the first two cases rhythmocardiography was used. diagnostic tables were used in the third case. these research methods of vns record the functional state of individual vns subsystems and separate mechanisms of vegetative regulation. the method of functionalvegetative diagnostics by v.g. makats provides the results more complete, stable, and comparable in time periods. due to the fact that the organism is a multilevel hierarchical system the effect of magnetic fields manifests itself at different levels of organization and depends on many external and internal factors. the reactions of the organism to the action of magnetic fields are characterized by the diversity, instability and phase flow, during which an opposite change is often observed. it is determined by the differences in individual sensitivity of the organism, its systems, and their initial state as well as by the nonspecific character of the action of magnetic fields. the effect of magnetic fields in many cases is normalizing or corrective. to start with, it should be noted that the central parasympatolytic effect during the action of low-frequency magnetotherapy in all groups of observations was revealed. that happens possibly due to the improvement of the parameters of peripheral hemodynamics and sanogenesis processes. this effect, along with the sedative effect of low-frequency magnetotherapy, is realized by affecting the subcortical centres and the pituitary-hypothalamic system. it should also be noted that low-frequency magnetotherapy is capable of exerting an activating influence on the processes of sanogenesis which contributes to restoration of disturbed self-regulation of many functional systems and the organism as a whole. it forms effective protective reactions as well as compensatory and adaptive processes and, moreover, it expands the range of homeostatic response of the organism in conditions of disturbed mechanisms of self-regulation. the obtained data do not coincide with the literature, where the corrective effect of the magnetotherapeutic influence on the parasympathetic and sympathetic units of vegetative ns or pronounced sympatholytic activity is described. this is primarily due to the age characteristic features of the child’s organism and its vegetative status. the second important aspect is the method of functional and vegetative diagnosis according to v.g. makats that was chosen to determine the vegetative status of children, as more universal and complete one. table 4. the effect of magnetotherapy (mt) on the vegetative dispersion at various stages of initial vegetative disorders in female and male groups № female group (in %) male group (in %) pa -s ig pa -e xp fc p vb fc s sa -e xp sa -s ig pa -s ig pa -e xp fc p vb fc s sa -e xp sa -s ig initial significant parasympathicotonia 1 100 100 2 100 50 50 initial expressed parasympathicotonia (group 2) 1 100 100 2 100 20 20 20 20 20 initial functional compensation of parasympathicotonia 1 100 100 2 100 42,8 42,8 14,3 initial autonomic balance of vns (group 4) 1 100 100 2 50 25 25 40 60 initial functional compensation of sympathicotonia (group 5) 1 100 100 2 66,6 16,6 16,6 50 50 initial significant and expressed sympathicotonia 1 100 100 2 10 60 30 25 50 25 o. v. yermishev 19 in t e r n a l m e d ic in e issn 2413-6077. ijmmr 2018 vol. 4 issue 1 функціонально-вегетативні рівні як критерій ефективності магнітотерапії о. в. єрмішев донецький національний університет імені василя стуса, вінниця, україна вступ. близько 80 % функціональних порушень зумовлено розладами вегетативної нервової системи. на сьогодні немає достатньо даних щодо впливу магнітотерапії (мт) на показники вегетативного гомеостазу при функціональній патології. мета дослідження – дослідити тенденції реабілітації при мт на початкових етапах різних функціональновеге тативних розладів. методи. як метод контролю впливу мт була обрана «функціонально-вегетативна діагностика» (фвд) за в. г. макацом. технічним засобом фвд виступав діагностично-реабілітаційний комплекс біотест-12м. у дослідженні взяли участь 38 дітей різного віку та статі, які проходили лікування у відділенні фізіотерапії вінницької обласної дитячої клінічної лікарні у 2016-2017 роках. пацієнтів поділили на 7 груп відповідно до рівнів функціонального вегетативного гомеостазу. результати. за коефіцієнтом функціонального вегетативного гомеостазу мт мала різний вплив як на вегетативну активність, так і на системну, і функціональну залежність. у групі зі значною парасимпатікотонією (група 1) виявлено найбільш позитивний ефект. у групі з вираженою симпатикотонією (група 6) – негативний ефект. не виявлено гендерних та вікових характерних особливостей впливу мт на вегетативні рівні. висновки. доцільність здійснення реабілітаційних заходів вимагає підтримки функціонального вегетативного гомеостазу на рівні fcp-vb-fcs у поєднанні з функціонально-вегетативною діагностикою з використанням методу в. г. макац. можна рекомендувати використовувати магнітотерапію лише для хворих зі значно вираженою парасимпатокотонією. ключові слова: магнітотерапія; функціонально-вегетативна діагностика; вегетативний гомеостаз; вегетативний рівень; вегетативний коефіцієнт. conclusions the results of the studies have proved that mt has a different effect on the vegetative activity as well as systemic and functional dependence in the study groups that depends on the coefficient of functional vegetative homeo-stasis. that is, in a group with a significant parasympathicotonia (group 1), mt has a positive effect; in the group with the expressed parasympathicotonia (group 2) it has a relatively positive effect; in the groups with functional compensation of parasympathicotonia (group 3), with the autonomic balance of vns (group 4) and in the group with functional compensation of sympathicotonia (group 5) mt has a relatively neutral effect; in a group with severe sympathicotonia (group 6) it has negative effect and in a group with significant parasympathicotonia (group 7) it has negative effect as well. according to the results of our research, the absence of gender and age-related influence of mt on the dispersion of vegetative levels has b e e n rev e a l e d . s y m p a t h e t i c v e g e t a t i v e orientation is typical for a low-frequency magnetotherapy in all study groups. therefore, it can only be used to patients with significant and severe parasympathicotonia. the rehabilitation expediency requires the maintenance of functional vegetative homeostasis at the level of fcp-vb-fcs in conjunction with functional-vegetative diagnostics using the method of v.g. makats. the method of functional-vegetative diagnosis is easy to use; it provides repeated comparable results and can be applied in hospital and out-patient environment. references 1. veyn ам. vegetative disorders: clinic, treatment, diagnosis. moscow: мiа; 2000. 752 p. 2. messina a, monda m, valenzano a, messina g, villano i, moscatelli f, nigro e. functional changes induced by orexin a and adiponectin on the sympathetic/parasympathetic balance. frontiers in physiology. 2018;9.259:1-6. 3. chang wh, lee ih, chi mh, lin s-h, chen kc, chen ps, yang yk. prefrontal cortex modulates the correlations between brain-derived neurotrophic factor level, serotonin, and the autonomic nervous system. scientific reports. 2018;8.2558:1-9. 4. guan l, collet j-p, mazowita g, claydon ve. autonomic nervous system and stress to predict o. v. yermishev 20 in t e r n a l m e d ic in e issn 2413-6077. ijmmr 2018 vol. 4 issue 1 secondary ischemic events after transient ischemic attack or minor stroke: possible implications of heart rate variability. frontiers in neurology. 2018;9.90: 1-16. 5. makats vg, makats еv. unknown chinese acupuncture (reality, errors, problems). vol. і. vinnytsia: nilan-ltd ; 2016. 276 p. 6. makats vg, makats еv. unknown chinese acupuncture (biophysical atlas of systemic de pendency). vol. ii . vinnytsia: nilan-ltd ; 2016. 204 p. 7. muzhikov v, vershinina e, belenky v, muzhikov r. comparative assessment of the heart’s functioning by using the akabane test and classical methods of instrumental examination. journal of acupuncture and meridian studies. 2017; 10(3): 171179. 8. vargas-lunaa fm, perez-aldaya ea, huertafrancob mr, delgadillo-holtforta i. electric characterization of skin near biological active points and meridians. international journal of bioelectromagnetism. 2010; 12(2): 76-80. 9. hong y, shang h, yang h, kong q, wang m, zhang q. a 3d recognition and projection system for meridians and acupoints, 2017 ieee international conference on bioinformatics and biomedicine (bibm), kansas city, mo, usa. 2017:1357-1363. 10. hegyi g, hamvas s, havasi m, petrovics g. dry needling stimulation (acupuncture) in evidence based medicine. austin journal of nuclear medicine and radiotherapy. 2016;3(2):1–9. 11. muzhikov v, vershinina e, belenky v, muzhi kov r. comparative assessment of the heart’s func tioning by using the akabane test and classical me thods of instrumental examination. journal of acu puncture and meridian studies. 2017;10(3):171-179. 12. makats vg, nagaychuk vi, makats еv, yermishev ov. unknown chinese acupuncture (problems of vegetative pathogenesis). vol. iv. vinnytsia: nilan-ltd ; 2017. 286 p. 13. makats vg, nagaychuk vi, makats еv. unknown chinese acupuncture (problems of functional vegetales). vol. iii. vinnytsia: nilan-ltd; 2017. 204 р. 14. makats vg, nagaychuk vi, yermishev ov. fundamentals of functional vegetology and physiotherapy. vinnytsia: nilan-ltd ; 2017. 254 p. 15. pesqueira t, costa-almeida r, gomes me. uncovering the effect of low-frequency static magnetic field on tendon-derived cells: from mechanosensing to tenogenesis. scientific reports. 2017;10948. 16. cichoń n, czarny p, bijak m, miller e, śliwiński t, szemraj j, at al. benign effect of extremely low-frequency electromagnetic field on brain plasticity assessed by nitric oxide metabolism during poststroke rehabilitation. oxidative medicine and cellular longevity. 2017;2181942,9. 17. cichoń n, bijak m, miller e, saluk j. extremely low frequency electromagnetic field (elf-emf) reduces oxidative stress and improves functional and psychological status in ischemic stroke patients. bioelectromagnetics. 2017;38(5):386–396. 18. yermishev ov, petruk rv, ovchynnykova yy, kostiuk vv. functional health of children as an ecological bioindicator of ukraine. vinnytsia: nilan ltd; 2017. 226 p. 19. ciejka e, skibska b, gorąca a. influence of low frequency magnetic field used in magnetotherapy on interleukin 6 (il-6) contents in rat heart and brain. medycyna pracy. 2017;68(4):517–523. 20. pasi f, sanna s, paolini a, alquati m, lascialfari a, corti me, et al. effects of extremely lowfrequency magnetotherapy on proliferation of human dermal fibroblasts. electromagnetic biology and medicine. 2016;35(4):343-352. 21. kraiukhina k, lobkaeva ep, deviatkova ns. effect of the low-frequency impulse magnetic field on the autonomic nervous system in animals. biofizika. 2010;55(4): 20-6. 22. nagornev sn, frolkov vk, kulish av, gurevich kg, puzyreva ga, samsonova os. the mechanism of realization of hypotensive action of transcranial magnetotherapy in complex treatment of patients with arterial hypertension. man and his health. 2017;1:5-11. 23. sayakhov rf, kildebekova rn, mingazova lr, nizamov ak. the effectiveness of physiotherapeutic effects in the treatment of autonomic nervous s y s t e m d i s o r d e r s . f u n d a m e n t a l r e s e a r c h . 2010;11:112-114. received: 2018-05-08 o. v. yermishev 44 o b st e t r ic s a n d g y n e c o l o g y issn 2413-6077. ijmmr 2018 vol. 4 issue 2 doi 10.11603/ijmmr.2413-6077.2018.2.9347 microbiota of vagina and mammary glands skin in the pregnant women with preeclampsia v. ya. ivankiv, i. m. malanchyn, n. i. tkachuk i. horbachevsky ternopil state medical university, ternopil, ukraine background. timely prediction, diagnosis and prevention of complications during the gestational period leading to perinatal loss and maternal mortality are the main tasks of contemporary obstetrics. about 50,000 women die from preeclampsia and eclampsia every year and perinatal mortality ranges from 15 to 25%. objective. the aim of the research was to study and analyze the microecology of vagina and mammary glands skin in the pregnant women with underlying preeclampsia. methods. the pregnancy examination was conducted at the ternopil regional clinical perinatal center “mother and child”. the research involved 25 pregnant women with preeclampsia (the main group) and 15 healthy women with a physiological course of pregnancy (the control group). material from the pregnant women was taken out of the skin of mammary glands and mucous membrane of vagina, then it was plated out in the nutrient medium for the cultivation and the microorganisms were defined. results. the quantitative composition of a normal microflora of vagina and mammary glands skin in the control group was within the normal range. the quantitative composition of a normal microflora of vagina and mammary glands skin in the pregnant women of the main group decreased, the representatives of opportunistic and pathogenic flora were found. conclusions. in the pregnant women with preeclampsia, abnormal microbiocenosis of vagina and breast skin was revealed, the degree of abnormality correlated with the severity of the disease. our results may provide useful clinical knowledge to a broader understanding of microbiota role in pregnancy complications. key words: preeclampsia; microbiocenosis of mammary glands skin; perinatal mortality. introduction in the structure of maternal and perinatal morbidity and mortality, preeclampsia is one of the most leading causes. the incidence of preeclampsia in ukraine ranges from 6 to 16%. perinatal mortality in preeclampsia is high and, according to various authors ranges from 15 to 25%. today, preeclampsia is established to be an inability of the adaptive mechanisms of a parent organism to adequately provide the needs of fetal development, manifested by perfusion-diffuse insufficiency in the motherplacenta-fetus of varying degrees of severity [1, 2, 3, 4, 5]. preeclampsia is not an independent disease; it is just a manifestation of a systemic inflammatory response syndrome to oxidative stress, endothelial dysfunction, thrombophilia, metabolic homeostasis, and normal microbiota. in the pregnant women with preeclampsia, often a violation of the qualitative and/or quantitative composition of the representatives of normal microflora of the body takes place. this leads to adaptive or irreversible changes in the appropriate microbiological link, which is termed ‘dysbiosis’ [6, 7, 8, 9]. physiological colonization is important because normal flora supports immunological homeostasis, suppresses pathogenic microorganisms. the process of formation of microflora of a newborn and the development of its immune system may be disturbed if the expectant mother suffers from pathology of gastrointestinal tract, dysbiosis, has sources of chronic infection, or suffered from preeclampsia during pregnancy. identifying the beneficial and detrimental microbial components and their roles during pregnancy may have extremely important implications. according to the literature, there are rare reports on the study of microbiota of mammary gland skin [15, 17], so, the aim of our research was to study the microbiota of vagina and international journal of medicine and medical research 2018, volume 4, issue 2, p. 44-49 copyright © 2018, tsmu, all rights reserved corresponding author: viktoria ivankiv, i. horbachevsky ternopil state medial university 1 maydan voli, ternopil 46001, ukraine phone number +380972340892 e-mail vivankiv@meta.ua v. ya. ivankiv et al. 45 o b st e t r ic s a n d g y n e c o l o g y issn 2413-6077. ijmmr 2018 vol. 4 issue 2 mammary glands skin in the pregnant women with preeclampsia. methods the study involved 25 pregnant women with preeclampsia (the main group) and 15 healthy women with a physiological course of pregnancy (the control group). mild preeclampsia was diagnosed in 15 (60%) of the examined patients, and the severe one was in 10 (40%). classification of preeclampsia in the pregnant women was conducted in accordance with the amendment 10 of the icd (1995), order of the ministry of health of ukraine dated december 31, 2004 no. 676. material from the pregnant women was taken out of the skin of mammary glands and the mucous membrane of vagina, then it was plated out in the nutrient medium and the microorganisms were defined. the examination of the pregnant women was conducted at the ternopil regional municipal perinatal center “mother and child” in several stages. first, the skin of the mammary glands was rinsed, and the posterior vault of the vagina was smeared from the mucous membrane with sterile swabs pre-moistened in physiological solution. the material was taken by scrolling all the sides of cotton swab. after that, the tampons were placed in sterile tubes and delivered to the laboratory. the time was 20-30 minutes from the time of taking the research material to the crop. the samples were carried out on petri dishes with sterile media: yolk-salt agar, bloods mpa (for detection of cocci microorganisms), endo (enterobacteriaceae), sabouraud’s dextrose agar (fungus of the genus candida), thioglycollate broth (anaerobic microorganisms). the media was placed in an incubator for 18-48 hours at an optimal temperature. we evaluated the growth of microorganisms in the media after incubation in the incubator (their shape, color, size of the colonies, nature of the surface and edges). next, smears of certain types of colonies were made, stained with gram method and examine microscopically. results as a result of microscopic examination, in 15 women of the control group (pregnant women with a physiological pregnancy course) the following was revealed: 1) on the skin of mammary glands: e. coli, fusobacterium spp., aerobic non–spore-forming gram-positive bacilli, m. roseus, streptococcus spp., lactose-negative gram negative rods in 6,7% of the examined patients; lactobacillus spp., s. haemolyticus in 13,3%; s. saprophyticus in 20,0%; corynebacterium spp. in 26,77%; clostridium spp., bacillus spp. in 33,0%; m. luteus in 40,0%, peptostreptococcus spp., s. epidermidis, m. lylae in 47,0%; bacteroides spp. in 60,0% (fig. 1). 2) in the vaginal smears: s. hominis, aerobic non spore-forming gram-positive bacillia, s. hae molyticus at 6,7%; streptobacillus spp., fig. 1. colonization of microorganisms on the skin of the mammary glands (control group), %. fig.1 colonization of microorganisms on the skin of the mammary glands (control group), % 2) in the vaginal smears: s. hominis, aerobic non spore-forming gram-positive bacillia, s. haemolyticus at 6,7%; streptobacillus spp., streptococcus spp., m. lylae in 13,3%; candida spp., e. coli, s. saprophyticus, lactose-negative gram negative rods in 20,0%; m. luteus in 33,0%; lactobacillus delbrueckii, corynebacterium spp., s. epidermidis, bacillus spp. in 40,0%; clostridium spp., bacteroides spp., enterococcus spp. in 47,0%; lactobacillus spp. in 73,3% of the examined women (fig. 2) 6,7 6,7 6,7 6,7 6,7 6,7 13,3 13,3 20 26,7 33 33 40 47 47 47 60 0 10 20 30 40 50 60 70 e. coli fusobacterium spp aerobic nonspore-forming gram-positive bacillia m. roseus streptococcus spp. lactose-negative gram negative rods lactobacillus spp. s.haemolyticus s. saprophyticus corynebacterium spp clostridium spp. bacillus spp. m. luteus peptostreptococcus spp s.epidermidis m. lylae bacteroides spp. v. ya. ivankiv et al. 46 o b st e t r ic s a n d g y n e c o l o g y issn 2413-6077. ijmmr 2018 vol. 4 issue 2 streptococcus spp., m. lylae in 13,3%; candida spp., e. coli, s. saprophyticus, lactose-negative gram negative rods in 20,0%; m. luteus in 33,0%; lactobacillus delbrueckii, corynebacterium spp., s. epidermidis, bacillus spp. in 40,0%; clostridium spp., bacteroides spp., enterococcus spp. in 47,0%; lactobacillus spp. in 73,3% of the examined women (fig. 2). in 15 pregnant women of the main group with mild preeclampsia: 1) on the skin of mammary glands: саndida spp. in 6,7%; m. lylae, βhemolytic streptococcus in 13,3%; corynebacterium spp., s. saprophyticus in 20,0%; s. aureus in 26,7%; bacillus spp., s. epidermidis in 33,3%; m. luteus, s. haemolyticus in 87,0% of the examined women (fig. 3). 2) in the vagina smears: s. epidermidis, m. lylae in 6,7%; streptococcus spp., m. luteus, lactose-negative gram-negative rods in 13,6%, e. сoli, s. aureus in 26,6%; s. haemolyticus, enterococcus spp. in 33,3%; candida spp. in 46,6%; corynebacterium spp. in 53,3%; bacillus spp. in 60,0% (fig. 4). in 10 pregnant women of the main group with severe preeclampsia: 1) on the skin of mammary glands: streptococcus spp., bacillus spp. in 20,0%; m. catarrhalis in 26,6%; s. epidermidis in 33,3%; s. aureus, corynebacterium spp. in 40%; s. haemolyticus in 66,7%, m. luteus in 73,3% (fig. 5). 2) in the vagina smears: corynebacterium spp., p. vulgaris in 20,0%; lactose-negative gram fig.2 colonization by microorganisms of the vaginal mucosa (control group), % in 15 pregnant women of the main group with mild preeclampsia: 1) on the skin of mammary glands: саndida spp. in 6,7%; m. lylae, βhemolytic streptococcus in 13,3%; corynebacterium spp., s. saprophyticus in 20,0%; s. aureus in 26,7%; bacillus spp., s. epidermidis in 33,3%; m. luteus, s. haemolyticus in 87,0% of the examined women (fig. 3). 6,7 6,7 6,7 6,7 6,7 6,7 13,3 13,3 20 26,7 33 33 40 47 47 47 60 0 10 20 30 40 50 60 70 e. coli fusobacterium spp aerobic nonspore-forming gram-positive bacillia m. roseus streptococcus spp. lactose-negative gram negative rods lactobacillus spp. s.haemolyticus s. saprophyticus corynebacterium spp clostridium spp. bacillus spp. m. luteus peptostreptococcus spp s.epidermidis m. lylae bacteroides spp. 6,7 13,3 13,3 20 20 26,6 33,3 33,3 87 87 0 10 20 30 40 50 60 70 80 90 100 саndida spp. m. lylae βhemolytic streptococcus corynebakterium spp s. saprophyticus s.aureus bacillus spp. s.epidermidis m. luteus s. haemolyticus fig. 2. colonization by microorganisms of the vaginal mucosa (control group), %. fig.2 colonization by microorganisms of the vaginal mucosa (control group), % in 15 pregnant women of the main group with mild preeclampsia: 1) on the skin of mammary glands: саndida spp. in 6,7%; m. lylae, βhemolytic streptococcus in 13,3%; corynebacterium spp., s. saprophyticus in 20,0%; s. aureus in 26,7%; bacillus spp., s. epidermidis in 33,3%; m. luteus, s. haemolyticus in 87,0% of the examined women (fig. 3). 6,7 6,7 6,7 6,7 6,7 6,7 13,3 13,3 20 26,7 33 33 40 47 47 47 60 0 10 20 30 40 50 60 70 e. coli fusobacterium spp aerobic nonspore-forming gram-positive bacillia m. roseus streptococcus spp. lactose-negative gram negative rods lactobacillus spp. s.haemolyticus s. saprophyticus corynebacterium spp clostridium spp. bacillus spp. m. luteus peptostreptococcus spp s.epidermidis m. lylae bacteroides spp. 6,7 13,3 13,3 20 20 26,6 33,3 33,3 87 87 0 10 20 30 40 50 60 70 80 90 100 саndida spp. m. lylae βhemolytic streptococcus corynebakterium spp s. saprophyticus s.aureus bacillus spp. s.epidermidis m. luteus s. haemolyticus fig. 3. colonization of microorganisms on the skin of the mammary glands (the main group, a mild preeclampsia), %. v. ya. ivankiv et al. 47 o b st e t r ic s a n d g y n e c o l o g y issn 2413-6077. ijmmr 2018 vol. 4 issue 2 negative rods, e. сoli in 26,6%; саndida spp., s. aureus in 33,3%; enterococcus spp., bacillus spp. in 46,6%, s. haemolyticus in 73,3% (fig. 6). discussion since the pathogenesis of preeclampsia is not fully studied, it is practically impossible to prevent its development. however, it is established that in the presence of some risk factors, development of preeclampsia is more frequent. at present, there is no ‘gold standard’ in the diagnosis of preeclampsia, and maternal and infant mortality from this pathology is still high, therefore, there is a need for further search for new pathogenesis as well as development of screening diagnostic methods. fig. 3 colonization of microorganisms on the skin of the mammary glands (the main group, a mild preeclampsia), %. 2) in the vagina smears: s. epidermidis, m. lylae in 6,7%; streptococcus spp., m. luteus, lactose-negative gram-negative rods in 13,6%, e. сoli, s. aureus in 26,6%; s. haemolyticus, enterococcus spp. in 33,3%; candida spp. in 46,6%; corynebacterium spp. in 53,3%; bacillus spp. in 60,0% (fig. 4). fig. 4 colonization by microorganisms of the vaginal mucosa (the main group, a mild preeclampsia), %. in 10 pregnant women of the main group with severe preeclampsia: 1) on the skin of mammary glands: streptococcus spp., bacillus spp. in 20,0%; m. catarrhalis in 26,6%; s. epidermidis in 33,3%; s. aureus, corynebacterium spp. in 40%; s. haemolyticus in 66,7%, m. luteus in 73,3% (fig. 5). 6,7 6,7 13,3 13,3 13,3 26,6 26,6 33,3 33,3 46,6 53,3 60 0 10 20 30 40 50 60 70 m. lylae s.epidermidis streptococcus spp. m.luteus lactose-negative gram negative rods e. coli s.aureus s.haemolyticus enterococcus spp. candida spp corynebakterium spp. bacillus spp. fig. 4. colonization by microorganisms of the vaginal mucosa (the main group, a mild preeclampsia), %. fig. 5. colonization of microorganisms on the skin of the mammary glands (the main group, severe preeclampsia), % 2) in the vagina smears: corynebacterium spp., p. vulgaris in 20,0%; lactose-negative gram-negative rods, e. сoli in 26,6%; саndida spp., s. aureus in 33,3%; enterococcus spp., bacillus spp. in 46,6%, s. haemolyticus in 73,3% (fig. 6). fig. 6. colonization by microorganisms of the vaginal mucosa (the main group, severe preeclampsia), %. discussion 20 20 26,6 33,3 40 40 66,6 73,3 0 10 20 30 40 50 60 70 80 streptococcus spp. bacillus spp. m.catarrhalis s.epidermidis s.aureus corynebakterium spp s.haemolyticus m.luteus 20 20 26,6 26,6 33,3 33,3 46,6 46,6 73,3 0 10 20 30 40 50 60 70 80 p.vulgaris corynebakterium spp lactose-negative gram negative rods e.coli s.aureus саndida spp enterococcus spp bacillus spp. s. haemolyticus fig. 5. colonization of microorganisms on the skin of the mammary glands (the main group, severe preeclampsia), %. fig. 6. colonization by microorganisms of the vaginal mucosa (the main group, severe preeclampsia), %. fig. 5. colonization of microorganisms on the skin of the mammary glands (the main group, severe preeclampsia), % 2) in the vagina smears: corynebacterium spp., p. vulgaris in 20,0%; lactose-negative gram-negative rods, e. сoli in 26,6%; саndida spp., s. aureus in 33,3%; enterococcus spp., bacillus spp. in 46,6%, s. haemolyticus in 73,3% (fig. 6). fig. 6. colonization by microorganisms of the vaginal mucosa (the main group, severe preeclampsia), %. discussion 20 20 26,6 33,3 40 40 66,6 73,3 0 10 20 30 40 50 60 70 80 streptococcus spp. bacillus spp. m.catarrhalis s.epidermidis s.aureus corynebakterium spp s.haemolyticus m.luteus 20 20 26,6 26,6 33,3 33,3 46,6 46,6 73,3 0 10 20 30 40 50 60 70 80 p.vulgaris corynebakterium spp lactose-negative gram negative rods e.coli s.aureus саndida spp enterococcus spp bacillus spp. s. haemolyticus v. ya. ivankiv et al. 48 o b st e t r ic s a n d g y n e c o l o g y issn 2413-6077. ijmmr 2018 vol. 4 issue 2 the further health care of a woman with preeclampsia is a topical issue. previously, it was established that the problem of preeclampsia disappears after all. however, according to current literature, this condition has a significant impact on the woman’s health and is manifested by the increased risk of development of arterial hypertension, cardiovascular diseases and cerebral circulation disorders in the future [10, 11, 12]. recent scandinavian studies have argued that pregnant women, who suffered from preeclampsia before the 37th week of gestation, have an 8-time increase of a risk of cardiovascular death over the next 13 years. therefore, it is advisable to recommend to women, who suffered from preeclampsia, an annual examination to evaluate cardiovascular risk as well as keeping a healthy lifestyle in order to avoid long-term consequences of late gestosis [13, 16]. the search for new pathogenesis of preeclampsia would reduce the level of maternal and perinatal mortality and improve the quality of life of pregnant women and childbirth. conclusions the representatives of normal microflora: saprophytic gram-positive and gram-negative microorganisms, found on the skin of mammary glands of the control group of women, coincide with the literature data. in the pregnant women with mild preeclampsia, there is an increase in the number of s. haemolyticus from 13% to 87%, the presence of the representatives of pathogenic flora: s. aureus (in 27% of the examined). in the patients with moderate preeclampsia, the number of s. aureus (40%). depending on the composition of the microflora of a pregnant woman (normocenosis or dysbiosis) and the functional state of mother-placenta-fetus system the microflora of newborns is developed, and their immune system is established. in the study of microbiocenoses of vaginal mucosa in the examined pregnant women with preeclampsia, in comparison with the control group, a significant decrease in lactic acid bacteria (lactobacillus delbrueckii, lactobacillus spp.) was evidenced as well as the increase in the incidence of coccal flora (s. haemolyticus, s. aureus, β-hemolytic streptococcus). all this evidence the presence of one of the main signs of dysbiosis – a decrease in the frequency of lactic acid bacteria. dysbiosis of the skin of mammary glands is found in 87% of the examined of the main group, mucous membrane of vagina – in 73%. in the pregnant women with preeclampsia, abnormal microbiocenosis of vagina and breast skin was revealed; the degree of changes correlated with the severity of preeclampsia. the analysis of individual variants of microbiota in the examined main group proved that destabilization of a microbial ecosystem takes place before childbirth, which, in our opinion, is associated with changes in the immune system during pregnancy and with certain obstetric pathologies such as preeclampsia. the most unfavorable combination is preeclampsia together with dysbiosis, where significant dysbiotic disorders of both vagina and mammary glands skin with the phenomena of colonization of conditionally pathogenic and transient flora in high diagnostic concentrations are present. it is necessary to make correction of dysbiotic disorders in the pregnant women, and in the future, to maintain normal microflora of the newborns that reduces the number of chronic diseases and slows down the aging process. мікрофлора піхви та шкіри молочних залоз у вагітних жінок з прееклампсією в. я. іванків, і. м. маланчин, н. і. ткачук тернопільський державний медичний університет імені і. я. горбачевського, тернопіль, україна вступ. своєчасне прогнозування, діагностика та профілактика ускладнень вагітності, які призводять до перинатальних втрат і материнської смертності є важливим завданням сучасного акушерства. близько 50 тисяч жінок щорічно помирають від прееклампсії та еклампсії, а перинатальна смертність становить від 15 до 25%. мета дослідження полягала у вивченні та аналізі мікробіоценозу піхви і шкіри молочних залоз у вагітних з прееклампсією. v. ya. ivankiv et al. 49 o b st e t r ic s a n d g y n e c o l o g y issn 2413-6077. ijmmr 2018 vol. 4 issue 2 references 1. order of the ministry of health of ukraine № 676: about the approval of clinical protocols for obstetric and gynecological care; 31-12-2004. available at http://old.moz.gov.ua/ua/portal/ dn_20041231_676.html 2. acker lv, warshavskyi bya, elganinova sa. indicators of oxidative and antioxidant stress in pregnant women with gestosis. obstetrics and gynecology 2013;4:17-20. 3. mellina in. a combination of preeclampsia in pregnant women with hypertension: warning possible and necessary. medical aspects of women's health 2011;4(44):5-13. 4. saveleva gm, shalina ri. gestosis in modern ob stetrics. international medical journal 2015;1:50-53. 5. shyphman em. preeclampsia, eclampsia, nellp syndrome. petrozavodsk. intel-tech;2012: 38-129. 6. magee la, hellewa m, moutquin jm, von dadelszen p. diagnosis, evaluation, and management of the hypertensive disorders of pregnancy. journal of obstetrics and gynaecology canada 2016;3:1-48. 7. national clinical guideline center. hypertension in pregnancy. the management of hypertensive disorders during pregnancy. national institute for health and clinical excellence. nice clinical guideline; 2010:288. 8. brown ma. preeclampsia: a lifelong disorder. medical journal 2013;4(179):181-182. 9. definitions of preeclampsia updated 2014. available at http://www.obgyn.net/. 10. odent m. hypothesis: preeclampsia as a maternal-fetal conflict. medgenmed 2011; 5:20-21. 11. preventing pre-eclampsia – are dietary factors the key? – updated 2014. available at http:// www.ncbi.nlm.nih.gov/pubmed 12. the pre-eclampsia community guideline (precog): how to screen for and detect onset of pre-eclampsia in the community – updated 2015. a v a i l a b l e a t h t t p s : / / w w w . n c b i . n l m . n i h . g ov / pubmed/15760998. 13. roberts et al. preventing and treating eclamptic seizures. bmj 2012; 325:609-610. 14. international classification of diseases 10 views – updated 1995. available at http://mkh10.com. ua/ 15. malanchyn im. condition of microbiocenosis of the skin of the mammary glands, intestines and vagina in pregnant women with preeclampsia. actual problems of pediatrics, obstetrics and gynecology 2010;1:140-2. 16. malanchyn im, koptyukh vi, franchuk oa. remote consequences postponed preeclampsia. actual questions of pediatrics, obstetrics and gynecology 2014;2:160-162. 17. ivankiv vya, malanchyn im, tkachuk ni. ecological features of microbiocenosis of the skin of mammary glands and vagina in pregnant women with threat of preterm labor. international journal of medicine and medical research 2017;2:51-53. received: 2018-06-26 методи дослідження. у дослідження включено 25 вагітних з прееклампсією (основна група) та 15 здорових жінок з фізіологічним перебігом вагітності (контрольна група) (тернопільський обласний клінічний перинатальний центр “мати і дитина”). у обстежуваних жінок брали мазки зі шкіри молочних залоз і слизової оболонки піхви, матеріал висівали в живильні середовища для культивування. результати. у вагітних основної групи кількісний склад нормальної мікрофлори піхви і шкіри молочних залоз був порушений, виявлено представників опортуністичних бактерій і патогенної флори. ступінь аномалій мікробіоценозу корелює з тяжкістю клінічних проявів. висновки. наші результати дослідження дозволять розширити розуміння ролі і зв’язку змін мікрофлори та ускладнень вагітності. ключові слова: прееклампсія; мікробіоценоз шкіри молочних залоз; перинатальна смертність. v. ya. ivankiv et al. issn 2413-6077. ijmmr 2018 vol. 4 issue 152 p u b l ic h e a lt h a n d e p id e m io l o g y international journal of medicine and medical research 2018, volume 4, issue 1, p. 52-59 copyright © 2018, tsmu, all rights reserved doi 10.11603/ijmmr.2413-6077.2018.1.8971 epidemiologic evaluation of thyroid diseases morbidity of ukrainian adult population from 2000 to 2013 а. м. antonenko, м. м. korshun, o. p. vavrinevych, s. t. omelchuk, v.h. bardov o. o. bogomolets national medical university, kyiv, ukraine background. the diseases of thyroid gland have been attracting considerable attention in recent decades. this is partly due to the fact that thyroid gland reacts actively to geochemical state and pollution of the environment with industrial and agricultural waste products with the subsequent incidence of certain pathological processes. the objective of the research was to analyze the morbidity of adult population of ukraine for thyroid gland diseases in the period from 2000 to 2013. methods. the methods of empirical and theoretical research of scientific information: analysis, synthesis, induction, deduction and systematization, as well as epidemiological and statistical methods were used. using the microsoft office excel (2007) and ibm spss statisticsbase v.22 program the correlation and regression analyzes were conducted. results. from 2000 to 2013, high levels of adult population endocrinopathies, thyroid in general and diffuse goiter of varying degrees, general and primary morbidity were registered in the western and northern regions of ukraine, low – in the central, eastern and southern regions. statistically significant (p<0.001) positive correlation between the level of prevalence and the level of incidence of endocrine pathology, diseases of thyroid gland as a whole, as well as individual nosology was detected. conclusions. regional peculiarities of the levels and dynamics of changes in the incidence rates of thyroid morbidity among adult population of ukraine can be related to the urgent environmental factors for each region. this factor requires further study to develop effective methods of prophylaxis and defense. key words: morbidity; thyroid gland; adult population. corresponding author: аnna antonenko, hygiene and ecology department № 1, o. o. bogomolets national medical university, 34, peremogy avenue, kyiv, 03680, ukraine e-mail: antonenko1985@ukr.net phone number: +380991466331 introduction the diseases of thyroid gland have been attracting considerable attention in recent decades. in the structure of endocrine pathology prevalence in ukraine they are the first: on average 44 %, and in the endemic with iodine deficiency western regions – up to 70 % [1]. this is partly due to the fact that thyroid gland reacts actively to geochemical state and pollution of the environment with industrial and agricultural waste products with the subsequent occurrence of certain pathological processes [2]. in the structure of endocrinological pathology, different types of goiter are the largest share [3, 4]. minimizing thyroid dysfunction rates is an important task for most countries of the world, since, according to the who publications, nearly 2 billion planet’s inhabitants are at risk for thyroid diseases, including iodine-dependent diseases [4, 5]. the objective of the research was to analyze the morbidity of adult population of ukraine for thyroid gland diseases in the period from 2000 to 2013. methods the object of our research was the level and the dynamics of endocrine system (class iv, e00-e90 according to the international diseases classification of the tenth revision) and thyroid gland (e00-e07) general morbidity (prevalence1) and primary morbidity (incidence2) of adult population in 24 regions of ukraine, the crimea as well as kyiv and sevastopil cities, in the period from 2000 to 2013. the sources of information were the reports of the endocrinology service of ukraine ̒the main indices of the activity of the endocrinology service of ukraine...̓ for the period of 8 years: 2000, 2004, 2005, 2006, 2009, 2010, 2011 and 2013 [7–14], according to which the evaluation of general endocrinology morbidity, incidence of diffuse nontoxic goiter degree i, diffuse non1 the term recommended by who. 2 the term recommended by who. а. м. antonenko et al. issn 2413-6077. ijmmr 2018 vol. 4 issue 1 53 p u b l ic h e a lt h a n d e p id e m io l o g y toxic goiter degree ii–iii, nodule goiter, hypothyroidism, thyrotoxicosis, thyroiditis and thyroid cancer (ii class of neoplasm, c73) was carried out. the spreadsheet was developed in microsoft office excel (2007). the methods of empirical and theoretical research of scientific information: analysis, synthesis, induction, deduction and systematization, as well as epidemiological and statistical methods were used. for the analysis of changes in the level of general and primary thyroid disease, the average absolute increase (ai), which characterizes the average annual increase rate, increase rate (air,%) and increase rate (ir,%), were evaluated. the estimation of the basic (relative to the level of the initial year) and the chain (relative to the level of the previous year) indices were carried out. using the microsoft office excel (2007) and ibm spss statisticsbase v.22 program corre_ lation (with pearson correlation coefficient – r) and regression analyzes to estimate the dynamics of population morbidity over a fourteen-year period, the ranking of investigated administrative territories (at) of ukraine according to the levels of prevalence and incidence for 2000–2013 years and the rate of their increase, the identification of the re lationship between the incidence rate of individual thyroid diseases and the relationship between the levels and the rate of morbidity index increase according to the calculation of the spearman rank correlation coefficient (rs), were conducted results the current who statistics prove that the pathology of endocrine system takes the third place after cardiovascular and cancerous diseases in the structure of the overall morbidity and causes of mortality in most countries of the world and it continues to increase [1]. a similar situation is evidenced in ukraine also, where the increase in the number of patients with various endocrinopathies in the period from 2005 to 2010 amounted to 9.85 % [14]. it has been established that in the period from 2000 to 2013, the overall endocrinology incidence (prevalence of endocrinopathies) of adult population in 16 regions of ukraine, the crimea, kyiv and sevastopil increased, as evidenced by the statistically significant coefficients of the pearson correlation pair (r>rtabl; rtabl=0.707 at n=8, p=0.05). regarding the primary incidence of endocrine system diseases, in the period of monitoring, a positive correlation was established only in 6 regions, another one (cherkasy) proved a tendency to increase (r>rtabl; rtabl=0.629 at n=8, p=0.1). at the same time, the largest air and ir of both general and primary morbidity were observed in zaporizhia region (ir 137.1 and 118.6 % respectively), kyiv (113.3 and 33.8 %), mykolaiv (102.7 and 55.8 %), kharkiv (100.7 and 64.8 %), and poltava (100.1 and 57.4 %) regions (respectively, 1, 2, 3, 4 and 5 rank positions for ir of general morbidity and 1, 5 , 4, 2 and 3 – ir of primary morbidity). significant (p<0.05) decrease of the total endocrinological morbidity that correlated with the year was observed only in three regions: volyn (administrative center is lutsk), sumy and chernihiv. in the other (five) regions the statistically significant correlation between the preva lence of endocrine pathology and the year was not proved. regarding the primary morbidity, a statistically significant inverse correlation relationship was established in 8 regions (including volyn, sumy and chernihiv); in ano ther one (kirovohrad, administrative center kropyvnytskyi) and crimea regions there was a tendency to index decrease (0.05<p<0.1). in the other (10) investigated at, the level of primary morbidity did not significantly change during 14 study years (p>0.1). in general, between the ir of the general and ir of the primary incidence of endocrine system diseases in the period from 2000 to 2013 there was a significant positive correlation (rs=0.916; rs tabl=0.597 at n=27, p=0.001). the same correlation was found between the levels of prevalence and incidence of endocrine pathology (rs=0.841). in general, in the period from 2000 to 2013, the highest levels of both indices of endocrine disease were reported in vinnytsia region and the regions of western ukraine: zakarpattia (administrative center is uzhgorod), rivne, volyn (table. 1). primary morbidity was high also in ivano-frankivsk, ternopil and khmelnitsky regions, and prevalence – in the northern regions (kyiv, chernihiv and sumy regions). it should be noted that these areas have the highest incidence of diffuse goiter of varying degrees; in the northern regions of nodular goiter and thyroid cancer as well. the seven last ranked places according to both indices of endocrine system diseases incidence were occupied by the eastern (lu hansk), southern (zaporizhia, kherson), central (poltava) regions and sevastopil. primary morbidity was also low in donetsk and odesa regions, and prevalence – in kirovohrad and mykolaiv. the а. м. antonenko et al. issn 2413-6077. ijmmr 2018 vol. 4 issue 154 p u b l ic h e a lt h a n d e p id e m io l o g y ta bl e 1. r an ki n g of a dm in is tr at iv e te rr it or ie s ac co rd in g to t h e le ve ls o f th yr oi d ge n er al ( g m ) an d pr im ar y (p m ) m or bi di ty o f ad u lt p op u la ti on d u ri n g th e pe ri od o f 20 00 –2 01 3 re gi on s ad m in is tr at iv e te rr ito ry ra nk s en do cr in e sy st em d is ea ses (e sd ) d iff us e go ite r of i de gr ee (d g -і ) d iff us e go ite r of ii -i ii d eg re es (d g -і і+ іі і) n od ul ar g oi te r (n g ) th yr oi d ca nc er (t c) h yp ot hy ro id is m (h t) th yr ot ox ic os is (т t) th yr oi di tis (t d ) зз п з зз п з зз п з зз п з зз п з зз п з зз п з зз п з 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 w es te rn vo ly n 2* * 5* * 1* * 2* * 4* * 4* * 12 16 27 ’’ 25 ’’ 10 24 ’’ 17 18 25 ’’ 26 ’’ za ka rp at tia 3* * 1* * 2* * 3* * 1* * 1* * 20 27 ’’ 25 ’’ 27 ’’ 14 13 2* * 3 ** 24 ’’ 24 ’’ iv an ofr an ki vs k 8 4* * 7* * 4* * 17 17 18 23 ’’ 26 ’’ 26 ’’ 1* * 8 4* * 4 ** 22 ’’ 20 lv iv 11 12 6* * 6* * 14 16 24 ’’ 22 ’’ 17 15 16 18 8 21 ’’ 14 14 ri vn e 5* * 2* * 4* * 1* * 2* * 2* * 14 14 23 ’’ 21 ’’ 22 ’’ 19 16 10 23 ’’ 22 ’’ te rn op il 12 6* * 13 5* * 10 13 19 25 ’’ 22 ’’ 22 ’’ 19 21 ’’ 15 19 27 ’’ 27 ’’ kh m el ni ts ky 9 7* * 14 10 11 8 8 12 18 20 6* * 4 * 1* * 1 ** 17 12 ch er ni vt si 10 8 12 12 5* * 6* * 22 ’’ 19 11 24 ’’ 25 ’’ 23 ’’ 10 26 ’’ 12 18 n or th er n zh yt om yr 15 16 11 13 9 9 7* * 13 15 12 24 ’’ 26 ’’ 13 16 19 23 ’’ ky iv 4* * 15 3* * 11 3* * 3* * 2* * 3* * 2* * 2* * 8 10 21 ’’ 17 6* * 4* * su m y 7* * 9 10 9 7* * 5* * 4* * 5* * 8 4* * 17 15 6* * 12 7* * 9 ch er ni hi v 6* * 19 5* * 14 6* * 15 3* * 4* * 6* * 6* * 21 ’’ 20 7* * 13 9 10 th e ci ty o f k yi v 16 17 21 ’’ 20 15 12 1* * 1* * 1* * 1* * 2* * 1* * 24 ’’ 7 ** 1* * 1* * ce nt ra l vi nn yt si a 1* * 3* * 8 7* * 8 11 5* * 2* * 4* * 7* * 5* * 5* * 5* * 15 8 7, 5* * d ni pr op et ro vs k 17 18 16 16 12 10 13 8 7* * 9 4* * 2* * 9 6* * 5* * 6* * ki ro vo hr ad 21 ’’ 13 15 15 24 ’’ 19 21 ’’ 20 19 16 26 ’’ 27 ’’ 20 27 ’’ 13 15 po lta va 22 ’’ 24 ’’ 18 21 ’’ 19 24 ’’ 9 6* * 13 11 18 14 14 8 15 13 ch er ka sy 13 10 20 18 13 7* * 10 10 12 10 11 12 12 2* * 21 ’’ 19 ea st er n d on et sk 19 21 ’’ 22 ’’ 23 ’’ 21 ’’ 22 ’’ 11 9 9 13 3* * 3* * 3* * 5* * 2* * 3* * lu ha ns k 27 ’’ 26 ’’ 23 ’’ 22 ’’ 20 18 27 ’’ 21 ’’ 24 ’’ 23 ’’ 27 ’’ 25 ’’ 25 ’’ 14 26 ’’ 25 ’’ kh ar ki v 18 14 17 17 16 14 25 ’’ 15 16 19 13 6* * 27 ’’ 24 ’’ 3* * 2* * so ut he rn za po ri zh ia 26 ’’ 26 ’’ 24 ’’ 24 ’’ 26 ’’ 25 ’’ 16 11 10 88 23 ’’ 17 19 11 11 5* * m yk ol ai v 24 ’’ 20 19 19 22 ’’ 21 ’’ 26 ’’ 18 21 ’’ 7* * 9 26 ’’ 23 ’’ 10 11 а. м. antonenko et al. issn 2413-6077. ijmmr 2018 vol. 4 issue 1 55 p u b l ic h e a lt h a n d e p id e m io l o g y ta bl e 2. c or re la ti on b et w ee n t h e le ve ls o f th yr oi d ge n er al ( g m ) an d pr im ar y (p m ) m or bi di ty o f ad u lt p op u la ti on o f u kr ai n e du ri n g th e pe ri od of 2 00 0– 20 13 . sp ea rm an ’s r an k co rr el at io n co effi ci en ts es d g m es d p m d g -і g m d g -і pm d g іі +і іі g m d g іі +і іі pm n g -g m n g -p m tc g m tc -p m h tg m h t p m тt -g m тt -p m тd -g m es d -g m 1 es d -p m 0. 84 1* 1 d g -і -g m 0. 88 2* 0. 76 4* 1 d g -і -p m 0. 84 4* 0. 90 6* 0. 91 6* 1 d g -і і+ іі іg m 0. 85 8* 0. 69 2* 0. 78 3* 0. 71 4* 1 d g -і і+ іі іpm 0. 79 7* 0. 75 0* 0. 71 7* 0. 71 6* 0. 92 0* 1 n g -g m 0. 36 0^ 0. 03 7 0. 16 0 0. 02 3 0. 35 0^ 0. 26 5 1 n g p m 0. 16 0 -0 .1 24 0. 02 3 -0 .1 51 0. 23 5 0. 17 6 0. 82 2* 1 tc g m -0 .1 14 -0 .4 22 ’’ -0 .3 35 ^ -0 .4 99 * -0 .0 56 -0 .1 44 0. 64 0* 0. 70 2* 1 tc p m -0 .1 84 -0 .4 88 * -0 .3 28 ^ -0 .4 93 * -0 .1 59 -0 .2 19 0. 69 1* 0. 71 7* 0. 91 1* 1 h t g m 0. 23 4 0. 18 4 0. 04 1 0. 06 3 0. 03 4 0. 07 3 0. 32 3^ 0. 33 2^ 0. 19 4 0. 15 6 1 h t p m 0. 07 9 0. 06 3 -0 .1 04 -0 .0 83 -0 .0 60 -0 .0 01 0. 29 1 0. 44 2’ ’ 0. 32 5^ 0. 25 3 0. 86 8* 1 tt g m 0. 55 9* 0. 46 9’ ’ 0. 39 6’ ’ 0. 40 9’ ’ 0. 39 2’ ’ 0. 30 8 0. 21 1 0. 02 1 -0 .0 53 -0 .1 69 0. 22 3 0. 18 3 1 tт p m 0. 24 5 0. 21 7 0. 13 2 0. 18 1 0. 19 0. 25 9 0. 41 8’ ’ 0. 31 3 -0 .0 33 0. 00 9 0. 43 0’ ’ 0. 50 7* 0. 49 4* 1 тd g m -0 .1 63 -0 .3 38 ^ -0 .2 46 -0 .4 08 ’’ -0 .1 22 -0 .1 58 0. 38 8’ ’ 0. 67 2* 0. 77 2* 0. 67 3* 0. 37 6^ 0. 54 5* -0 .1 18 -0 .0 92 1 тd p m -0 .1 53 -0 .3 22 -0 .2 48 -0 .3 97 ’’ -0 .1 75 -0 .1 84 0. 37 9^ 0. 69 8* 0. 70 1* 0. 64 2* 0. 45 6’ ’ 0. 67 2* -0 .0 83 0. 06 5 0. 94 0* n ot e. s pe ar m an ’s ra nk c or re la tio n co effi ci en ts (п = 27 ): * – r s ta bl =0 .4 87 a t р =0 .0 1; ’’ – r s ta bl л =0 .3 81 a t р =0 .0 5; ^ – r s ta bl = 0. 32 3 at р =0 .1 . co nt in ua tio n of ta bl e 1 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 so ut he rn о de sa 20 23 ’’ 26 ’’ 27 ’’ 25 ’’ 26 ’’ 23 ’’ 26 ’’ 14 14 15 16 11 25 ’’ 20 17 kh er so n 23 ’’ 22 ’’ 25 ’’ 26 ’’ 18 23 ’’ 6* * 7* * 3* * 3* * 9 11 22 ’’ 20 4* * 7, 5* * cr im ea 14 11 9 8 23 ’’ 20 15 17 20 17 12 7* * 18 9 18 16 se va st op il 25 ’’ 27 ’’ 27 ’’ 25 ’’ 27 ’’ 27 ’’ 14 24 ’’ 5* * 5* * 20 22 ’’ 23 ’’ 21 ’’ 16 21 ’’ n ot e. ** – th e hi gh es t l ev el s of m or bi di ty , ’ ’ – th e lo w es t l ev el s of m or bi di ty . а. м. antonenko et al. issn 2413-6077. ijmmr 2018 vol. 4 issue 156 p u b l ic h e a lt h a n d e p id e m io l o g y lowest rates of diffuse goiter of different degrees are presented in table 1. however, it should be noted that in the regions that occupy the last ranked positions according to the levels of both morbidity indices, air and ir were high. on the contrary, in regions with a high morbidity, air and ir were low or even negative. this was confirmed by the spearman’s rank correlation coefficients, which proved a statistically significant backward correlation between the level and the ir of the total (rs=-0.576; rs tabl=0.478 at n=27, p=0.01) and the primary (rs=-0.509, p<0.01) incidence of endocrinopathies among the population of 27 investigated at of ukraine. in general, the incidence rates of adult population for endocrine diseases correlate with the prevalence and morbidity rates of diffuse goiter of various degrees (table 2), which is explained by a high specific gravity of the latter in the structure of endocrine diseases. thus, in ukraine in 2010, in the structure of endocrinopathy, diffuse goiter of degrees i-iii was the second (29.99 %) after diabetes mellitus (31.88 %), and the share of all thyroid gland diseases was 46.67 % [14]. in the analysis of both indices of thyroid morbidity, it was found out that during the period of investigation, the highest levels were registered in the western and northern regions of ukraine, and the lowest ones were in the central, eastern and southern (except in the autonomous republic of crimea) regions (fig. 1). at the same time, in most of the western and northern regions during 14 years there was a decrease in both (volyn, lviv, zakarpattia, ivano-frankivsk, sumy oblasts), or one (ternopil, zhytomyr, kyiv, chernihiv) of the morbidity indices, while the changes in the other were insignificant (fig. 2). kyiv was the exception: both indices, especially the level of general morbidity, increased; in rivne and chernivtsi regions the general morbidity significantly increased without significant changes in the primary one, and in khmelnitsky region significant changes in both indices did not take place. the situation in the central, eastern and southern regions was the opposite, where in the majority of regions there was a significant increase in both (cherkasy, poltava, kharkiv, donetsk, zaporizhia, mykolaiv, odesa) or one (vinnytsia, dnipropetrovsk, kherson, luhansk regions and sevastopil) of indices with minor changes of the other. kirovograd region and crimea were the exceptions: the general morbidity did not fig. 1. thyroid morbidity. а. м. antonenko et al. issn 2413-6077. ijmmr 2018 vol. 4 issue 1 57 p u b l ic h e a lt h a n d e p id e m io l o g y change significantly, and the primary one – decreased (fig. 2). thus, in relation to thyroid pathology, as well as in case of endocrine diseases in general, a statistically significant inverse relationship between the level and ir of the general (rs= -0.687, p<0.001) and primary (rs=-0.735, p<0.001) morbidity of adult population in 27 investigated at of ukraine was revealed. concerning certain nosological forms of thyroid pathology, during the period of monitoring, significant regional features were revealed both for the levels of morbidity indices and the direction of their changes in the period of study. thus, the levels of primary and general morbidity of diffuse goiter (dg) were the highest in the western (zakarpattia, rivne and volyn regions – dg of degrees i and ii–iii, chernivtsi – dg of degrees ii–iii, lviv and ivanofrankivsk – dg of degree i) and northern (kyiv, sumy, chernihiv) regions, and the lowest ones – in the eastern (donetsk) and southern (odesa, zaporizhia, kherson regions and sevastopil) regions (table 1). during 14 years of the study, the general morbidity rate for diffuse goiter of degrees i (dg-i) and іі–ііі (dg-іі–ііі) in 11 and 6 regions fig. 2. dynamics of changes in the thyroid general and primary morbidity. respectively decreased, in 8 and 13 at – increased, which was confirmed by statistically significant correlation coefficients. in 3 and 2 at respectively, there was a tendency to increase (0.707>r>0.622, 0.05<p<0.1); in the rest of the at there were no significant changes. regarding the primary morbidity of diffuse goiter of different degrees, its level decreased in 16 at (p<0.05), in one region – a tendency to decrease was evidenced; in the other 10 at there were no significant changes. it should be noted that between the level and gr of both general and primary morbidity with diffuse goiter of degrees i and ii–iii there was a reliable inverse relationship (from rs=-0.390 to rs=-0.582; rs tabl=0.381 at n=27 and p=0.05): the lower the level of incidence in the region, the greater the pace of its growth. the highest levels of nodular goiter primary and general morbidity were registered in the northern regions (kyiv city, kyiv, chernihiv and sumy regions), vinnytsia and kherson regions (table 1). at the same time, in those regions, as well as in sevastopil, the highest levels of thyroid cancer morbidity incidence and prevalence were proved. the lowest levels of morbidity rates for both nodular goiter and thyroid cancers were registered in western а. м. antonenko et al. issn 2413-6077. ijmmr 2018 vol. 4 issue 158 p u b l ic h e a lt h a n d e p id e m io l o g y (zakarpattia, ivano-frankivsk, ternopil) and luhansk regions; only nodular goiter – in lviv and odesa, only cancer – in volyn and rivne regions. in general, between the levels of nodular goiter primary and general morbidity, on the one hand, and cancer, on the other hand, there was a significant positive correlation (table 2). regarding the dynamics, in the study period, the nodular goiter general morbidity in 25 of 27 at increased, which was confirmed by a statistically significant positive correlation coefficient; in zhytomyr region there was a tendency to increase (0.707>r>0.622, 0.05<p<0.1) and only in volyn region the index decreased (r=-0.902; p<0.01). nodular goiter primary morbidity increased in 22 at (r>0.707, p<0.05); in three – it did not change significantly, remaining steadily high in kyiv and sumy regions; and in volyn and zhytomyr – decreased ( r = 0 . 9 0 8 , p < 0 . 0 1 a n d 0 . 7 4 8 , p < 0 . 0 5 , respectively). regarding the overall incidence of thyroid cancer, in general in the period of 2000–2013 there was no statistically significant relationship in the period of monitoring between 25 out of 27 at (p>0.05), only in kyiv region the increase (r=0.784 , p<0.05) took place, while in chernivtsi – the decrease (r=-0.716, p<0.05) of the index. the primary incidence of thyroid cancer in the period of investigation increased in 15 regions and the crimea autonomous republic (r>0.707, p<0.05), while in 4 regions (rivne, zhytomyr, kropyvnytskyi, and odesa), there was a tendency to increase (0.707>r>0.622, 0.05<p<0.1), in the rest of the at the relationship between the level of the indices and the year of monitoring was not detected (p>0.05). the highest levels of both indices of the incidence of adult thyroiditis in ukraine were registered in kyiv city, kharkiv, donetsk, kyiv, dnipropetrovsk and kherson regions, and the lowest – in ternopil, volyn, luhansk, zakarpattia and rivne regions (table 1). in general, both thyroiditis morbidity indices correlated with the incidence of thyroid cancer, nodular goiter and hypothyroidism (table 2). during the study period, the overall incidence of thyroiditis, hypothyroidism and thyrotoxicosis increased in most (24, 20 and 26, respectively) of the studied at, which was confirmed by a statistically significant positive coefficient of correlation. regarding the primary morbidity of thyroiditis, hypothyroidism and thyrotoxicosis, the indices increased in 13, 17 and 11 respectively. discussion the regional peculiarities of the levels and dynamics of changes in the incidence rates of the thyroid morbidity among adult population of ukraine can be related to the priority environmental factors for each region. it is esta blished that the western and northern regions of ukraine are different from the rest of the low natural content in the soil of iodine. the northern (kyiv, chernihiv, zhytomyr, rivne) and cherkasy regions are significantly contaminated by radioactive substances, including radioactive iodine isotopes (predominantly i-131), as a result of the chernobyl accident. in the eastern region, dnipropetrovsk and zaporizhia regions, powerful industrial centers are concentrated, in which relatively high levels of environmental pollution by industrial toxicants, including heavy metals, are registered. the central and southern regions have developed agricultural production, which today widely uses chemical protection products for plants. the study of the environmental factors influence on the ukrainian adult population thyroid morbidity will be a topical issue of our further research. conclusions in the period from 2000 to 2013, high levels of adult population endocrinopathies, thyroid glands in general and diffuse goiter of varying degrees, general and primary morbidity have been recorded in the western and northern regions of ukraine, low – in the central, eastern and southern regions. statistically significant (p<0.001) positive correlation between the level of prevalence and the level of incidence of endocrine pathology, diseases of thyroid gland as a whole, as well as individual nosology has been detected. it has been established that endocrine di sea ses incidence rates of adult population correlate (p<0.01) with the prevalence and morbi dity rates of diffuse goiter of various degrees, which is explained by a high specific gravity of the latter in the structure of endocrine diseases in general and thyroid pathology, in particular. the factors that cause development of various diseases of thyroid gland as well as regional peculiarities of their development require further study to develop effective methods of protection and prevention. * kropyvnytskyi is a city in central ukraine, and is the administrative center of the kirovohrad oblast. between 1939 and 2016 it was called kirovohrad. а. м. antonenko et al. issn 2413-6077. ijmmr 2018 vol. 4 issue 1 59 p u b l ic h e a lt h a n d e p id e m io l o g y references 1. karachentsev yui, tymchenko am, kozakov ov. current trends of endocrine morbidity and prospects for endocrinology service development. health of ukraine. available at http://health-ua.com/ article/110.html. feb 2018. 2. kravchenko vi, postol sv. the dynamics of morbidity in pathology of thyroid gland in ukraine. intern journal of endocrinology. 2011;3(35):12–16. 3. kiptenko li, budko hiu, vasko lv, piddubna ti. statistical analysis of diseases of the thyroid gland in the sumy region population. the world of medicine and biology. 2009;2–3:17–21. 4. pyrohova vh, kravchenko vi. dynamics of diseases of the thyroid gland caused by iodine de ciency in the population of the transcarpathian region scienti c bulletin of the uzhgorod university 2011;3:132–139. 5. who. unisef. iccidd. global database on iodine deficiency iodine status worldwide geneva 2004. 48 p. 6. report of endocrinology service of ukraine. main indicators of activity of the endocrinology service of ukraine for ams and мes of ukraine, se v.p. komisarenko institute of endocrinology and metabolism named after of nams of ukraine. 2000:31. 7. report of endocrinology service of ukraine. main indicators of activity of the endocrinology service of ukraine for ams and мes of ukraine, se v.p. komisarenko institute of endocrinology and metabolism named after of nams of ukraine. 2005:31. 8. report of endocrinology service of ukraine. main indicators of activity of the endocrinology service of ukraine for ams and мes of ukraine, se v.p. komisarenko institute of endocrinology and metabolism named after of nams of ukraine. 2006:33. 9. report of endocrinology service of ukraine. main indicators of activity of the endocrinology service of ukraine for ams and мes of ukraine, se v.p. komisarenko institute of endocrinology and metabolism named after of nams of ukraine. 2007:33. 10. report of endocrinology service of ukraine. main indicators of activity of the endocrinology service of ukraine for ams and мes of ukraine, se v.p. komisarenko institute of endocrinology and metabolism named after of nams of ukraine. 2010:31. 11. report of endocrinology service of ukraine. main indicators of activity of the endocrinology service of ukraine for ams and мes of ukraine, se v.p. komisarenko institute of endocrinology and metabolism named after of nams of ukraine. 2011:33 12. report of endocrinology service of ukraine. main indicators of activity of the endocrinology service of ukraine for ams and мes of ukraine, se v.p. komisarenko institute of endocrinology and metabolism named after of nams of ukraine. 2012:36. 13. report of endocrinology service of ukrainе. main indicators of activity of the endocrinology service of ukraine for ams and меs of ukraine, se v.p. komisarenko institute of endocrinology and metabolism named after of nams of ukraine. 2014:40. 14. larin os, pankiv vi, selivanenko mi, hrachova ap. analysis of the activity of the endocrinology service of ukraine in 2010 and prospects for the development of medical care for patients with endocrine pathology. intern journal of endocrinology 2011;3 (35):1–7. received: 2018-05-10 а. м. antonenko et al. аналіз захворюваності дорослого населення україни на патологію щитоподібної залози з 2000 по 2013 роки а. м. антоненко, м. м. коршун, о. п. вавріневич, с. т. омельчук, в. г. бардов національний медичний університет імені о. о. богомольця, київ, україна вступ. патологія щитоподібної залози останнім часом привертає значну увагу фахівців клінічної та профілактичної медицини. частково це пов’язано з тим, що щитоподібна залоза активно реагує на геохімічний стан і забруднення навколишнього середовища промисловими і сільськогосподарськими відходами з наступним поширенням певних патологічних процесів. метою дослідження було проаналізувати захворюваність дорослого населення україни на захворювання щитовидної залози в період з 2000 по 2013 роки. методи. використовувалися методи емпіричного та теоретичного дослідження наукової інформації: аналіз, синтез, індукція, дедукція та систематизація, а також епідеміологічні та статистичні методи. за допомогою програми microsoft office excel (2007) та програми ibm spss statisticsbase v.22 були проведені кореляційний та регресійний аналіз. результати. з 2000 по 2013 рр. зареєстровано високий рівень загальної та первинної захворюваності на ендокринопатії, дифузний токсичний та ендемічний зобу у дорослого населення західних і північних областей україни, низький рівень – у центральних, східних і південних регіонах. виявлено статистично значущу (p <0,001) позитивну кореляцію між рівнем поширеності та рівнем захворюваності ендокринною патологією, захворюваннями щитовидної залози, а також окремою нозологією. висновки. регіональні особливості рівню та динаміки частоти захворюваності щитовидної залози серед дорослого населення україни можуть бути пов’язані з екологічними факторами у кожному регіоні. цей фактор потребує подальшого вивчення для розробки ефективних методів профілактики та захисту. ключові слова: захворюваність; щитоподібна залоза; дорослe населення, україна. 24 p e d ia t r ic s issn 2413-6077. ijmmr 2018 vol. 4 issue 2 doi 10.11603/ijmmr.2413-6077.2018.2.9694 prevalence and risk factors for vitamin d deficiency in overweight and obese adolescents in ukraine a-m. a. shulhai, h. a. pavlyshyn i. horbachevsky ternopil state medical university, ternopil, ukraine background. vitamin d affects the function of many organs and systems. lipid metabolism disorder is established to be one of the risk factors for vitamin d deficiency, and the amount of adipose tissue is crucial. objective. the aim of the study was to determine the prevalence and risk factors for vitamin d deficiency in overweight and obese adolescents. methods. 146 children with excessive weight and obesity as well as 63 healthy children with normal body weight were examined. in the study groups, there were no children taking vitamin d. vitamin d status was evaluated by the level of 25(oh)d in blood serum. vitamin d deficiency was diagnosed at the level of 25(oh)d between 20 and 29 ng/ml, and significant deficiency – below 20 ng/ml, normal calcidiol content was 30-100 ng/ml. results. the average level of 25(oh)d in the adolescents with normal body weight was 19.76±4.28 ng/ml, in the adolescents with excessive body weight – 15.24±3.47 ng/ml, and in the obese children – 13.87±2.71 ng/ml. the prevalence of vitamin d deficiency in the overweight adolescents was 70.62%, and in the adolescents with obesity – 77.19%. conclusions. vitamin d deficiency is prevalent in the adolescents with overweight and obesity. to prevent the development of hypovitaminosis and vitamin d deficiency, it is necessary to carry out educational activities with adolescents for promotion of healthy lifestyle and healthy food, as well as to develop an optimal program for improving vitamin d status in the obese children. key words: vitamin d; children; calcidiol; prevalence; obesity. corresponding author: anna-maria shulhai, department of pediatrics no. 2, i. horbachevsky ternopil state medical university, 1 maydan voli, ternopil, 46001, ukraine e-mail: shulhai_aa@tdmu.edu.ua phone number: +380972171870 a-m. a. shulhai et al. introduction vitamin d, due to the biological properties of its derivatives, affects the function of many organs and systems. vitamin d deficiency leads to a decrease in calcium concentration in blood, impairment of calcium and phosphorus absorption in intestines and kidneys because of its active metabolite 1.25-dihydroxyvitamin d [1]. it has been proved that vitamin-d endocrine system affects electrolytes concentration, cell proliferation, angiogenesis, stimulation of insulin synthesis, inhibition of renin secretion [2, 3, 4]. the presence of interconnections between calcidiol level as well as lipid and carbohydrate metabolism in children [5, 6] has been established. moreover, special attention is paid by the researchers to the development of cardiometabolic risk factors and their relations with the concentration of calcium in blood and levels of parathyroid hormone in cases of vitamin d deficiency [7, 8]. the period of puberty is characterized by a rapid, peak increase in bone and muscle mass, and requires higher calcium and phosphorus intake, and, therefore, maintenance of proper levels of vitamin d metabolites in blood plasma [9]. however, adolescents frequently suffer from hypovitaminosis d and are characterized by increased tendency towards a sedentary lifestyle, spending much time at a computer or in front of the tv [10]. leading a sedentary lifestyle in such children reduces the time spent in sunlight and outdoors, which is a direct risk factor for obesity and vitamin d deficiency [1]. investigating metabolic abnormalities in children of different ages, researchers identified inverse relationship between vitamin d levels and metabolic factors, in particular, insulin resistance, body mass index, triglyceride levels and total testosterone, and direct relationship with insulin sensitivity [11]. it is established that lipid metabolism disorder is one of the risk factors for vitamin d deficiency, and the amount of adipose tissue is crucial in its metabolism and biological significance [12]. numerous clinical studies have proved that for those suffering from obesity, international journal of medicine and medical research 2018, volume 4, issue 2, p. 24-30 copyright © 2018, tsmu, all rights reserved 25 p e d ia t r ic s issn 2413-6077. ijmmr 2018 vol. 4 issue 2 vitamin d intake should be 2-3 times higher than for those with normal body weight. there is a pathogenetic connection between obesity and vitamin d deficiency, since vitamin d is a fat-soluble substance, distributed in the adipose tissue, which leads to decrease in its concentration in plasma [13]. moreover, attention is drawn to the fact that with the increase in the amount of adipose tissue there is a limitation of the bioavailability of vitamin d, which is associated with its engulfment by adipocytes and deposition in the adipose tissue. thus, spanish researchers have established existing relations between low-level serum concentration of 25(oh)d with high triglyceride levels regardless of age, sex, body mass index and physical activity [14]. childhood obesity is an important public health problem. in ukraine, 12% of children aged 7 to 17 years old suffer from excessive weight [15], among which about 10% are diagnosed with obesity by body mass index. mo reover, the number of obese children has a positive annual increasing rate. taking into consideration the increase in the number of overweight and obese adolescents in ukraine, it has become necessary to determine the prevalence of vitamin d deficiency among the overweight and obese adolescents and to identify the main factors affecting the vitamin d status of such children. the aim of the research is to determine the prevalence and risk factors for vitamin d deficiency in the adolescents with excessive weight and obesity. methods the research was conducted in the period of 2016-2018 at the communal institution of the ternopil regional council “ternopil re gional children clinical hospital”. the patient safety rules and the ethical standards and procedures for research involving human beings (2000) have been followed in carrying out the study. in all cases, informed consent has been obtained from the patients and/or their parents. the research involved on 146 adolescents (78 boys and 68 girls) aged 12 to 17 years old, which, depending on the body mass index (bmi), were divided into two groups: the overweight children and the obese children. the adolescent age of each child was determined according to the tanner scale (2-5 stages) [5, 12]. the control group consisted of 63 healthy children aged 12-17 years old, who lived in the city of ternopil and sought medical consultations for various reasons and chronic diseases. none of the causes of seeking medical help and disease affected their growth, body structure, nature of nutrition, physical activity. the experimental groups did not include children, whose obesity was due to endocrine diseases (hypothyroidism, hypercorticism, hypopituitarism, traumas of hypothalamic-pituitary area), taking antiepileptic drugs or glucocorticoids. all children were ukrainian (caucasians) and lived in ternopil region, ukraine. in anthropometric studies, body height and weight were determined, and bmi was calculated according to the formula (mass (kg)/height2 (m2)). anthropometric examinations: body weight (within the accuracy of 0.1 kg), height (within the accuracy of 0.1 cm), were carried out by the established methods by means of floor weight, height meter and flexible centimeter tape. bmis were evaluated according to standard percentile tables [5, 14]. thus, children with bmi from 15 to 85 percentiles were assigned to have normal body mass, the excessive body mass corresponded to 85-95 percentiles and over 95 percentiles – to obesity. to determine the factors affecting vitamin d status, the children were asked to fill in a questionnaire, which included data that ascertained the age of the child, sex, place of residence (city or village), the season of the questionnaire (november-march, april-october), income per family member (above or below the average living wage), daily milk consumption (up to 1 cup per day, from 1 to 3 cups and more), the use of vitamin d supplements, fish oil, the state of physical activity, which was determined by the number of active hours per week (up to 2 hours, from 2 to 5 hours, more than 5 hours), the duration of the daily stay in the open air (up to 30 minutes, more than 30 minutes), passive rest in front of the computer or tv (up to 2 hours per day, 2-4 hours per day, more than 4 hours per day). vitamin d status was determined according to the level of 25(oh)d in blood serum. for this, fasting blood test from the vein was taken. by centrifugation, serum was isolated, frozen and stored at -80 °c. the level of calcidiol was determined by the immunoassay method using 25-oh vitamin d elisa test kit (euroimmun, germany), with an intra-assay cv 3.2-4.9% and an inter-assay cv 4.0-7.8%. an assessment of the results of 25(oh)d level was conducted according to the recommendations of the international society of endocrinology (2011) [12]. vitamin d insufficiency was established at a a-m. a. shulhai et al. 26 p e d ia t r ic s issn 2413-6077. ijmmr 2018 vol. 4 issue 2 level of calcidiol ranging 20-29 ng/ml (50 75 nmol/l), vitamin d deficiency was established at 25(oh)d below 20 ng/ml (less 50 nmol/l), the normal calcidiol level was at 25(oh)d 30 100 ng/ml (76-250 nmol/l). the content of 25(oh)d above 100 ng/ml (250 nmol/l) was considered to be excessive. the attained results of the research were subjected to statistical processing. descriptive statistics was used to evaluate the concentration of calcidiol in serum and to determine the weight-height ratios of bmi. the level of calcidiol in serum was presented in the form of mean values and their standard errors. the comparison of frequency indices in the study groups was carried out using the wilcoxon signed-rank test for continuous variables and the chi-square test, or the fisher’s exact test for categorical variables. the comparison of mean values and their standard errors in different study groups with their accurate distribution was performed by the student’s t-test for independent samples, and if distribution of the values is not normal the nonparametric mann–whitney u test was used. the multiple logistic regression was used to determine the effect of each independent variable of the probable risk factor in the development of a 25(oh)d deficiency in the adolescents with obesity. all statistical studies were conducted using spss (statistical package for social sciences) for windows software 21.0 version. the differences between the values were statistically significant at p<0.05. results the research has established low levels of 25(oh)d in serum. in the adolescents with normal body weight, the mean values of 25(oh)d were 19.76±4.28 ng/ml, in the adoles cents with overweight – 15.24±3.47 ng/ml, and in the children with obesity – 13.87±2.71 ng/ml. the results of the study of 25 (oh) d levels, depending on the body mass index, are presented in table 1. vitamin d status in the adolescent children of 25(oh)d in most cases was manifested by its deficiency. in the adolescents with normal body weight, in blood serum of 14.32% of the children the level of 25(oh)d remained within the normal levels and in 29.46% was deficient. the highest deficiency rate of vitamin d was determined in the adolescents with obesity, which prevailed with a significant difference in comparison with the incidence of vitamin d deficiency (p=0.022) in the control group of adolescents with normal body weight. it has been confirmed that with the increase in bmi, a simultaneous increase in the proportion of vitamin d deficiency and a decrease in the proportion of individuals with normal levels and insufficiency of calcidiol was ob served. according to the results of statistical processing of the children’s answers in the questionnaire, the frequency of manifestations of the main risk factors with underlying vitamin d deficiency in the adolescents with normal body weight, overweight and obesity has been established. the predicted risk factors for vitamin d deficiency development among the study groups, depending on the body mass index, are presented in table 2. actual data have established that sex and place of residence do not have a significant impact on the prevalence of vitamin d deficiency in the adolescents with overweight and obesity. the frequency of diagnosis of vitamin d deficiency is more common in the adolescent boys with obesity, which was 42.2% (p=0.193). other factors that strongly influenced the significantly greater prevalence of vitamin d were: the season of blood serum collection from november to march, low income per family member, daily milk consumption, failing to take vitamin d supplements or fish oil, low physical activity, spending much time at the computer or in front of the tv. the time spent in the open air, both with overweight (p=0.448) and obesity (p=0.417), had no effect on the incidence of vitamin d deficiency in the adoles cents. for the adolescents with overweight, the duration of physical activity during the week did not influence a reliable dependence on low levels of calcidiol (p=0.450). table 1. level of 25(oh)d in adolescents depending on the body mass index (%) level 25(oh)d, ng/ml normal body weight, (%) p=63 excessive body weight, (%) n=68 obesity, (%) n=78 30-100 14.32 6.75 3.83 20-29 29.46 22.61 19.17 <20 57.35 70.72 77.19* notes. * – significant difference between the values compare to the group with normal body weight (p<0.05). a-m. a. shulhai et al. 27 p e d ia t r ic s issn 2413-6077. ijmmr 2018 vol. 4 issue 2 according to the results of the multiple logistic regression analysis, it has been found out that factors affecting the development of vitamin d deficiency include excessive body weight and obesity (table 3). moreover, in the presence of this factor, the likelihood of vitamin d deficiency increases in 1.54 times. in addition, a significant effect on the development of vitamin d deficiency is due to winter-spring season of the study (p=0.002), low income per family member (p=0.015), low daily milk consumption (p=0.032), physical activity up to 2 hours per week (p=0.042) and more than 4 hours a day spent at the computer table 2. frequency of manifestations of risk factors in the adolescents with deficiency of 25(oh)d depending on bmi % (95% ci) characteristics specific proportion of the children with deficiency 25(oh)d n the study group, % (95% ci) normal body weight, n=63 p excess body weight, n=68 p obesity,n=78 p sex 0.184 0.481 0.193 men 36.1 (25.4-50.8) 40.2 (31.4-49.3) 42.2 (31.2-53.1) women 25.0 (17.5-37.2) 31.3 (22.5-42.1) 35.9 (26.9-43.6) place of residence 0.569 0.725 0.515 rural areas 27.2 (18.3-36.5) 34.8 (24.1-46.2) 35.5 (24.3-46.4) city 31.9 (22.7-46.3) 39.7 (27.9-48.1) 43.6 (34.9-51.7) season 0.026 0.035 0.002 april-october 18.6 (9.1-27.7) 29.4 (19.1-41.3) 26.2 (20.5-35.9) november-march 41.3 (32.5-53.1) 44,1 (36.8-54.2) 52.8 (44.661.1) income per family member 0.019 0.032 0.006 above the average 15.7 (10.5-25.6) 20.5 (10.2-31.8) 28.8 (22.1-38.5) below the average 41.0 (31.6-45.9) 48.5 (36.8-61.3) 50.6 (43.4-59.5) milk consumption 0.035 0.003 0.001 up to 1 cup per day 37.7 (26.1-44.6) 50.6 (40.2-62.5) 60.5 (51.3-69.8) from 1 to 3 cups a day and more 20.8 (11.4-29.7) 20.1 (10.8-31.4) 19.8 (14.1-29.5) use of vitamin d (fish oil) supplements 0.178 0.002 0.001 yes 23.4 (12.9-32.3) 20.3 (11.8-30.9) 14.2 (7.8-23.9) no 32.5 (23.8-44.2) 52.4 (42.6-67.6) 62.1 (53.8-70.2) physical activity 0.198 0.450 0.001 up to 2 hours/week 21.2 (12.7-30.4) 30.9 (20.6-41.2) 48.7 (39.2-57.8) from 2 to 5 hours per week 22.8 (14.3-31.5) 21.5 (14.7-33.8) 17,9 (11.5-25.6) more than 5 hours per week 12.3 (6.3-22.8) 19.1 (11.8-32.4) 12.8 (6.4-20.5) daily stay in the open air 0.251 0.484 0.417 up to 30 min/day 34.5 (23.8-45.3) 29.3 (20.8-38.2) 41.9 (30.6-50.1) more than 30 min/day 23.0 (17.5-34.9) 41.2 (32.9-50.4) 35.5 (28.2-44.3) time spent at the computer or in front of the tv 0.059 0.034 0.001 up to 2 hours/day 9.3 (4.8-19.2) 12.2 (6.8-22.1) 14.4 (9.3-21.8) from 2 to 4 hours/day 21.3 (12.7-33.2) 25.9 (17.5-38.4) 28.8 (23.1-36.5) more than 4 hours/day 28.2 (19.4-39.3) 35,3 (23.5-46.2) 45.4 (36.2-56.4) a-m. a. shulhai et al. 28 p e d ia t r ic s issn 2413-6077. ijmmr 2018 vol. 4 issue 2 or tv (p=0.027). along with this, it has been found out that sex (p = 0.869), place of residence (p=0.156), taking of vitamin d supplements, fish oil (p = 0.698), daily outdoor exposure (p=0.062) have no significant effect on the development of vitamin d deficiency in the children with overweight and obesity. discussion the results of the study have proved that the prevalence of vitamin d deficiency in the adolescents is significant as in many other countries [2, 3, 12]. it has been established that there is an inverse relationship between the level of 25(oh)d in blood serum and the body mass index in the adolescents. in cases of excessive body weight, the frequency of diagnosing vitamin d deficiency increased in 1.23 times, and with obesity – by 1.35 times. the mean serum calcidiol content in blood serum of the adolescents with obesity was 1.43 times lower than that of the children with normal body weight. the data attained during the study showed a similar trend of change in the status of vitamin d in the children of different ages according to the results of epidemiological studies in ukraine but were lower compared with the data of the studies in the usa, spain, and italy [5, 9]. researchers explain the low levels of 25 (oh) d in blood serum by depositing calcidiol in the adipose tissue, reducing bioavailability, and reducing its synthesis under the influence of ultraviolet rays [14]. according to the results of the conducted studies, it has been established that the prevalence of vitamin d deficiency in the adolescents with obesity and overweight is unrelated to sex and place of residence. the latter were also not recognized as probable risk factors for vitamin d deficiency. however, according to spanish pediatric school [5], vitamin d deficiency was more often reported during puberty in obese girls. via the multiple logistic regression analysis, it has been established that the degree of influence of independent predictors do affect development of vitamin d deficiency in the adolescents with obesity and overweight. it has been proved that the greatest influence is exerted by the season of blood collection in the period of november-march, in which the probability of development of vitamin d deficiency increases in 2.74 times compared with the april-october season. the amount of time spent at the computer and watching tv more than 4 hours a day increases the chances of vitamin d deficiency development in 1.91 times and, together with low physical activity, belongs to the three main independent variables in the development of vitamin d deficiency in the adolescents with obesity and overweight. research results also indicate that the daily milk consumption of up to 3 cups or more reduces development of vitamin d deficiency in 1.49 times compared with the adolescents, who do not consume or consume table 3. logistic regression analysis of probable risk factors for vitamin 25(oh)d deficiencies risk factor b (se) or ci 95% p sex (men versus women) -0.14 (1,05) 0.87 0.11-6.82 0.869 place of residence (city versus rural areas) 0.16 (0.48) 1.07 0.39-2.18 0.156 season (november-march versus april-october) 1.29 (0.55) 2.74 1.05-7.38 0.002 income per family member (below the average versus above the average) 2.08 (1.17) 1.31 0.52-6,14 0.015 milk consumption (up to 3 cups or more versus up to 1 cup) -1.54 (0.95) 0.67 0.24-0.93 0.032 the use of vitamin d supplements (fish oil) (no versus yes) 0.91 (1.07) 1.46 0.31-5.79 0.698 physical activity up to 2 hours/week versus more than 5 hours/week 1.36 (0.42) 1.61 0.83-3.45 0.042 2 to 5 hours/week versus more than 5 hours/week 0.48 (0.76) 1.01 0.45-2.15 0.253 daily stay outdoors up to 30 minutes/day versus more than 30 minutes/day -0.72 (0.93) 0.89 0.24-2.09 0.062 time spent at the computer or in front of the tv 2 to 4 hours/day versus 2 hours/day 0.32 (0.83) 1.27 0.28-7.03 0.720 more than 4 hours/day versus 2 hours/day 0.27 (0.69) 1.91 0.35-8.46 0.027 excessive weight, obesity 0.43 (0.85) 1.54 0.37-3.02 0.012 a-m. a. shulhai et al. 29 p e d ia t r ic s issn 2413-6077. ijmmr 2018 vol. 4 issue 2 up to 1 cup of milk per day. our data support the results of studies conducted by the scientists from other countries [8, 10] concerning the degree of insufficiency or deficiency of vitamin d caused by the above-mentioned risk factors. for a comparative assessment of the impact of poverty and the level of income per family members on vitamin d status, we included in the questionnaire the information about the income of the adolescent’s family. it has been established that the level of low income per family member increases in 1.31 times the likelihood of vitamin deficiency in adolescents (p = 0.015). the findings confirm the results of other studies conducted in different countries, but in that case, the risk ratio was 1.36, while in the usa it was 1.6 [13], and in canada – 3.14 [14]. on the other hand, we have not confirmed the significance of vitamin d supplements and fish oil as a factor for vitamin d deficiency. in our opinion, it is mainly due to the low amount of food and milk products enriched with vitamin d or their use in insufficient quantities, as well as irregular use of fish oil. consequently, according to the results of the conducted studies, the prevalence of vitamin d deficiency and factors of its development in the children with overweight and obesity have been defined as well as the main probable factors of its development. conclusions vitamin d deficiency is prevalent in adolescents with overweight and obesity. the main risk factors for vitamin d deficiency development include winter and spring seasons, spending more than 4 hours per day at the computer, low physical activity up to 2 hours per week, taking small portions of milk less than 1 cup per day and low income per family member. to prevent development of hypovitaminosis and vitamin d deficiency, it is necessary to carry out educational activities with adolescents aimed at healthy lifestyle and healthy eating, and to develop an optimal program for improving vitamin d status in obese children. поширеність та фактори ризику розвитку дефіциту вітаміну д у підлітків з надмірною масою тіла та ожирінням a-m. а. шульгай, г. а. павлишин тернопільський державний медичний університет імені і. я. горбачевського, тернопіль, україна вступ. вітамін д бере участь у функціонуванні багатьох органів і систем організму. одним з факторів ризику розвитку гіповітамінозу д є порушення обміну ліпідів, і кількість жирової тканини відіграє вирішальну роль за даних обставин. метою дослідження стало вивчення поширеності та факторів ризику розвитку недостатності вітаміну д серед підлітків з надмірною масою тіла та ожирінням. методи дослідження. обстежено 146 дітей з надмірною масою тіла та різним ступенем ожиріння та 63 здорових дітей з нормальною вагою. усі включені у дослідження підлітки не вживали препарати вітаміну д. для оцінки стану забезпеченості організму визначали рівень кальцидіолу 25(oh)d у сироватці крові. недостатність вітаміну д діагностували при значеннях показника 20-29 нг/мл, а його дефіцит – при рівні менше 20 нг/мл. нормальний вміст кальцидіолу коливається в межах 30-100 нг/мл. результати. середній рівень 25(oh)d у підлітків з нормальною масою тіла склав (19,76±4,28) нг/мл, з надмірною масою тіла – (15,24±3,47) нг/мл, з ожирінням – (13,87±2,71) нг/мл. поширеність дефіциту вітаміну д у дітей з надмірною масою тіла склала 70,62 %, з ожирінням – 77,19 %. висновки. дефіцит вітаміну д переважає серед підлітків з надмірною масою тіла та ожирінням. для профілактики розвитку його недостатності необхідно пропагувати здоровий спосіб життя, заохочувати фізичну активність та здоровий спосіб харчування та розробити оптимальні програми для покращення ситуації серед дітей з ожирінням. ключові слова: вітамін д; діти; кальцидіол; поширеність; ожиріння. a-m. a. shulhai et al. 30 p e d ia t r ic s issn 2413-6077. ijmmr 2018 vol. 4 issue 2 received: 2018-09-05 references 1. barja-fernández s, aguilera cm, martínezsilva i, vazquez r, gil-campos m, olza j et al. hydroxyvitamin d levels of children are inversely related to adiposity assessed by body mass index. j physiol biochem. 2018;74(1):111-118. pmid: 28744831. doi: 10.1007/s13105-017-0581-1. 2. antonucci r, locci c, clemente m, chicconi e, antonucci l. vitamin d deficiency in childhood: old lessons and current 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[in ukrainian]. a-m. a. shulhai et al. issn 2413-6077. ijmmr 2019 vol. 5 issue 140 p u b l ic h e a lt h a n d e p id e m io l o g y doi 10.11603/ijmmr.2413-6077.2019.1.9857 mortality analysis of the patients with alcoholic liver cirrhosis n.r. matkovska ivano-frankivsk national medical university, ivano-frankivsk, ukraine background. alcohol is considered to be the main risk factor for adverse event deaths around the world. in ukraine, mortality due to alcoholic liver disease (ald) has taken the second place in the structure of death causes from diseases of the digestive system. objective. the aim of the research was to study the peculiarities of the causes of death in the patients with alcoholic liver disease at the stage of liver cirrhosis (lc) based on the analysis of protocols of pathoanatomical research. methods. the analysis of 149 protocols of the pathoanatomical study of the patients, who died from alcoholic lc, has been carried out at the premises of the pathoanatomical department of the ivano-frankivsk regional clinical hospital in 2006-2018. results. most people were young and middle aged. fatal cases were caused by decompensation of the lc with the development of hepatic, hepatic-renal, cardio-pulmonary insufficiency, pancreatic necrosis, gastrointestinal bleeding (gib), sepsis, hepatocellular carcinoma (hcc). in 37.6 % of the patients the concomitant illness was coronary heart disease (chd), 10.7 % of the people had hypertension. in 6 % of the patients, ischemic stroke of the brain was diagnosed. in most people atherosclerotic vascular changes were revealed. conclusions. excessive consumption of alcohol and, consequently, the development of lc, can be considered as an adverse factor in the reducing social standard of living. in the majority of people, who died from the decompensation of alcoholic lc, atherosclerotic vascular lesions have been detected. this indicates a significant prevalence of lipid metabolism disturbance in the people with alcoholic lc. key words: liver cirrhosis, alcoholic; cause of death; coronary disease; atherosclerosis. corresponding author: natalia r. matkovska, ivano-frankivsk national medical university, 16 kropyvnytskyi str., ivano-frankivsk 76018 e-mail: nmail4you@gmail.com introduction demographic processes in ukraine have signs of a protracted demographic crisis, which is closely related to the historical and socioeconomic peculiarities of the country's development. it is well-known that ukraine belongs to countries with a progressive decline in demographic and reproductive potential, which leads to depopulation and population decline. at the beginning of 2016, the number of children in the population of ukraine was 16.1 %, those of working age – 61.8 %, older than ablebodied – 22.1 %. the prevalence of women in the elderly as a result of higher mortality among males is the peculiarity of gender imbalance. demographic aging, that is, the steady increase in the proportion of economically and socially inactive elderly people, combined with a decrease in the proportion of able-bodied people, has a direct impact on various spheres of life in the ukrainian society [1, 2]. along with the aging of the population, the increase in the mortality rate, which is associated with endogenous diseases (circulatory and neoplastic diseases) and exogenous (respiratory diseases, digestive disorders, infectious and parasitic diseases, external) causes, is a topical issue. in recent years, much attention has been paid to the increase in the incidence of so-called non-infectious diseases, which is becoming a sign of not only an epidemic, but also a possible future pandemic [3, 4, 5]. according to the who, 41 million people die from these diseases every year, accounting for 71% of all deaths in the world. annually 15 million people between 30 and 69 years of age die from them. cardio vascular diseases (stroke, heart attack) and malignant neoplasms are leading in prevalence and mortality among non-infectious diseases [6, 7]. non-communicable diseases are the result of the combination of genetic, physiological, environmental and behavioural factors. behavioural factors belong to the modified factors, and they can be changed. these include the use of tobacco, lack of physical activity, inappropriate nutrition, and alcohol abuse. the other not less international journal of medicine and medical research 2019, volume 5, issue 1, p. 40-46 copyright © 2019, tnmu, all rights reserved n.r. matkovska issn 2413-6077. ijmmr 2019 vol. 5 issue 1 41 p u b l ic h e a lt h a n d e p id e m io l o g y important factors include metabolic factors: increased blood pressure, overweight/obesity, hyperglycemia, hyperlipidemia [8, 9, 10]. among non-infectious diseases caused by these risk factors, the digestive diseases together with the increase of the proportion of deaths are significant. fibrosis and lc, alcohol and non-alcoholic liver diseases, which accounts for more than 50 % of those who died of digestive diseases, contributed much to mortality from this class of deaths causes. the reasons for this are the continuous increase of quantitative and qualitative types of such patients, frequent chronic diseases, prolonged and severe courses, unfavourable consequences of a disease, prevailing affection of people of working age that is associated with medical and socio-economic factors [11, 12, 13]. the main causes of liver damage are alcohol, viruses, non-alcoholic fatty liver disease. alcohol is considered to be the main risk factor for adverse lethal outcomes around the world. alcohol abuse is third among the causes of mortality among young people after tobacco and arterial hypertension and secondarily among the causes of liver transplantation in europe. in ukraine, mortality due to ald takes the second place in the structure of causes of death from digestive diseases [14, 15, 16]. the aim of the research was to study the peculiar features of the death causes in the patients with ald at the stage of lc based on the analysis of protocols of pathoanatomical investigation. methods the analysis of 149 protocols of pathoanatomical research of the patients, who died from alcoholic lc at the premicies of the pathoanatomical department of the ivanofrankivsk regional clinical hospital in 20062018, was conducted. the average age of patients was (56±12.1) years old (y. o.): women – (44.6±9.2) y. o., men – (57.6±11.8) y. o., the average duration of the disease was (6.6±1.8) years. the patients were divided into groups by age as follows: 44 young people (31 males and 13 females), 76 middle-aged persons (64 males, 12 females), 29 elderly patients (19 males, 10 females). results according to the child-pugh criteria, among the patients with the lc, who died, there are 9 (6%) persons with stage a of the disease, 26 (17.5%) – with stage b, 114 (76.5%) persons – with stage c. the causes of death in the patients with the lc of alcoholic etiology were pancreatic necrosis (4.7 % of patients with stage a, 8.8 % of patients with stage b and 1.3 % of patients with stage c), gastrointestinal bleeding (gib) – in 4 % of patients with stage b and in 36.3 % of patients with stage c, liver failure – in 17.4 % of patients with stage c, liver and kidney failure – in 1.3 % of people with stage b and in 10.7 % of persons with stage c, sepsis – in 1.3 % of patients with stage b and in 6.7 % of patients with stage c, cardio-pulmonary insufficiency – in 1.3 % of patients with stage a and 2 % of patients with stage b, hcc – in 4 % of people with stage c (table 1). according to the medical records, in all patients who died, the signs of portal hypertension, hepatosplenomegaly, cytolytic, hepatodepressive, mesenchymal-inflammatory syndrome were revealed. among the manifestations of hepatic hyperazotemia there were hepatic encephalopathy and hepatopulmonary syndrome in all patients and hepatorenal syndrome – in 87.9 % (3.4 %, 10.7 %, 4.7 % of cases in young, middle aged and elderly patients), respectively. the signs of cholestasis syndrome were detected in 88.6 % of patients. excessive subcutaneous fat was in 34.9 % of cases, satisfactory – in 42.3 %, insufficient – in 22.8 %. 134 (89.9 %) patients had ascites; 18.1 table 1. death causes in the patients with liver cirrhosis death causes stage of child-pugh criteria а (n=9) в (n=26) с (n=114) n % n % n % pancreatic necrosis 7 4.7 13 8.8 2 1.3 gastrointestinal bleeding 6 4 54 36.3 liver failure 26 17.4 liver and kidney failure 2 1.3 16 10.7 sepsis 2 1.3 10 6.7 cardio-pulmonary insufficiency 2 1.3 3 2 hepatocellular carcinoma 6 4 n.r. matkovska issn 2413-6077. ijmmr 2019 vol. 5 issue 142 p u b l ic h e a lt h a n d e p id e m io l o g y % of them suffered from ascites-peritonitis. septicemic condition was revealed in 8.7 % of cases and steatonecrosis of the omentum – in 16.1 % of people. hydrotorax was diagnosed in 83.2 % of cases, hydropericardium – in all cases. varicose of the oesophagus veins was diagnosed in all the lethal outcomes: of the 1st stage – in 9.4 % (14 persons), 2nd stage – in 20.8 % (31 persons), 3rd stage – in 69.8 % (104 persons) of cases, varicose veins of the stomach were present in 40.9 % of cases. the bleeding from varicose veins of the oesophagus was in 57.7 %, a combination of bleeding from the veins of the oesophagus and the stomach – in 30.2 % of cases. depending on the age, the gib was in 10.7 %, 18.8 %, 6.7 % of the patients of young, middle and old age, respectively. among men, gib was revealed in 35.5 % of young people, in 35.9 % of middle-aged patients, in 15.4 % of the elderly. among women, gib was revealed in 38.5 % of young people, in 41.7 % of middleaged patients and 30 % of the elderly (table 2). erosive gastroduodenitis was revealed in 66.4 % (99 persons) of cases, acute ulcers of the duodenal bulb (db) – in 10.7 % (16 persons), acute ulcers of the stomach – in 2.7 % (4 persons). the umbilical hernia was present in 12.7 % of cases. chronic hemorrhoids were diagnosed in 10.7 % of people (with bleeding in 4.7 % of cases). mild anemia was evidenced in 14.1 % of patients, moderate – in 39.6 %, severe – in 49.7 %. in 86.6 % of cases, the signs of chronic pancreatitis were revealed, 55 % of patients had pancreatic lipodystrophy, 14.8 % suffered from pancreatic necrosis (young and middle-aged persons), 7.4 % had cholelithiasis, 61.7 % – the signs of chronic cholecystitis, in 2 % of people the signs of cholangitis were present. hypotonia of the gall bladder was revealed in 12.1 % of all cases. type 1 diabetes mellitus was diagnosed in 2 % of young people, type 2 – in 4.7 % of middleaged and elderly people. chronic pyelonephritis table 2. the revealed pathoanatomical changes of internal organs depending on the liver cirrhosis decompensation stage pathological changes of internal organs stage by child-pugh criteria а в с n % n % n % 9 6 26 17.5 114 76.5 cardiovascular system: hydropericardium hypertension ihd atrial fibrillation ischemic stroke of the brain in anamnesis chronic venous insufficiency of the vessels of the lower extremities atherosclerosis of the coronary arteries -stage of the lipid stain -stage of atherosclerotic plaque -complicated plaque with ulceration -narrowing of lca by 10% -narrowing of lca by 30% -narrowing of lca by 50% atherosclerosis of the iliac arteries -stage of the lipid stain -stage of atherosclerotic plaque atherosclerotic changes in the aorta -stage of the lipid stain -stage of atherosclerotic plaque -complicated plaque with ulceration 9 6 3 3 9 2 5 3 3 4 4 9 2 4 3 6 4 2 2 6 1.3 3.4 2 2 2.7 2.7 6 1.3 2.7 2 26 8 22 3 6 24 10 10 4 3 2 3 8 6 2 26 9 12 5 17.5 5.4 14.8 2 4 16.1 6.7 6.7 2.7 2 1.3 2 5.3 4 1.3 17.5 6 8 3.4 114 2 31 2 7 22 5 17 2 2 5 5 84 36 42 6 76.5 1.3 20.8 1.3 4.7 14.8 3.4 11.5 1.3 1.3 3.4 3.4 56.4 24.2 28.2 4 respiratory system: hydrothorax pulmonary edema pulmonary emphysema pneumonia pleurisy corp 9 6 10 26 2 7 6 3 6.7 17.5 1.3 4.7 4 2 114 114 4 98 35 8 76.5 76.5 2.7 65.8 23,5 5.4 n.r. matkovska issn 2413-6077. ijmmr 2019 vol. 5 issue 1 43 p u b l ic h e a lt h a n d e p id e m io l o g y pathological changes of internal organs stage by child-pugh criteria а в с n % n % n % 9 6 26 17.5 114 76.5 digestive system: portal hypertension cytolytic syndrome hepatodepressive syndrome mesenchymal-inflammatory syndrome cholestasis syndrome ascites ascites-peritonitis steatonecrosis of the omentum splenomegaly umbilical hernia varicose of the esophagus veins 1-st degree 2-nd degree 3-d degree -with bleeding varicose veins of the stomach -with bleeding erosive gastroduodenitis acute ulcers of the duodenal bulb acute ulcers of the stomach chronic hemorrhoids hemorrhoidal bleeding chronic pancreatitis pancreatic lipodystrophy pancreatic necrosis cholelithiasis chronic cholecystitis cholangitis hypotonia of the gall bladder type 1 diabetes mellitus type 2 diabetes mellitus 9 9 9 9 9 9 9 10 2 6 6 6 6 6 6 6 6.7 1.3 26 26 26 26 18 20 26 26 5 21 8 3 15 6 8 5 16 2 17.5 17.5 17.5 17.5 12.1 13.4 17.5 17.5 3,4 14.1 5.3 2 10.1 4 5.4 3.4 10.7 1.3 114 114 114 114 114 114 27 24 114 19 114 10 104 86 61 45 91 16 4 13 7 114 76 4 4 76 3 18 3 5 76.5 76.5 76.5 76.5 87.9 86.6 18.1 16.1 76.5 12.7 76.5 6.7 69.8 57.7 40.9 30.2 61.1 10.7 2.7 8.7 4.7 76.5 51 2.7 2.7 51 2 12.1 2 3.4 urinary system: chronic pyelonephritis kidney cyst 2 1.3 12 3 8.1 2 nervous system: encephalopathy alcoholic delirium in anamnesis 9 7 6 4.7 26 4 17.5 2.7 114 76.5 was present in 9.4 % of cases, kidney cyst – in 2 %. pneumonia was diagnosed in 70.5 %, pleurisy – in 27.5 %, chronic obstructive pulmonary disease (copd) – in 7.4 %, pulmonary emphysema – in 4 %. in 4.7 %, chronic venous insufficiency of the vessels of the lower extremities was revealed. among the concomitant diseases, hypertension was present in 10.7 % of middle aged and elderly patients, ischemic heart disease (ihd) – in 37.6 % of cases, 3.4 % of these patients suffered from atrial fibrillation. 6 % of patients had ischemic stroke of the brain in anamnesis, and 7.4 % – alcoholic delirium. the signs of atherosclerosis were also evidenced. particularly, the biochemical parameters of the lipid spectrum were characterized by an increase in the content of total cholesterol in the blood, low and very low-density lipoprotein cholesterol. moreover, the degree of increase of these indicators was directly proportional to the degree of decompensation of lc. atherosclerotic changes in the aorta at the stage of lipid stain were present in 31.5 % of cases, at the stage of atherosclerotic plaque – in 38.9 %, at the stage of complicated plaque with ulceration – in 9.4 % of people. atherosclerosis of the left coronary artery (lca) was revealed in 11.4 % at the lipid staining stage, at the stage of atherosclerotic plaque – in 21.5 %, ulcerated atherosclerotic plaque was present in 2.7 %. n.r. matkovska issn 2413-6077. ijmmr 2019 vol. 5 issue 144 p u b l ic h e a lt h a n d e p id e m io l o g y narrowing of lca by 10% was revealed in 5.3 % of cases, by 30 % – in 4.7 %, by 50 % – in 2 %. atherosclerosis of iliac arteries was present in 11.4 % of patients. discussion consequently, analyzing clinical records of the pathoanatomical study of the patients with alcoholic lc, it was established that most patients were young and middle aged and died from decompensated lc with the development of multiple organ failure. in all cases, there was a lesion of the pancreas, in half of them lipodystrophy was revealed. bleeding from varicose veins of the esophagus and stomach was the cause of death in 57.5 % of people, 14.8 % of patients died from pancreatic necrosis, 4 % – from hcc. ihd was the concomitant illness in 37.6 % of the patients. more than 70 % of the dead had atherosclerotic lesions of aorta, and one third of the persons had injured coronary vessels. excessive consumption of alcohol and, consequently, the development of lc, can be considered as an adverse factor in the reducing social standard of living. fatal cases were caused by decompensation of lc, development of multiple organ failure, pancreatic necrosis, bleeding from the esophagus and stomach veins, hcc. special attention should be paid to the combination of ald with atherosclerotic vascular changes revealed in 79.8 % of people, indicating a significant prevalence of lipid metabolism disorders among people with decompensated alcoholic lc. consequently, analyzing clinical records of the pathoanatomical study of the patients with alcoholic lc, it was established that most patients were young and middle aged and died from decompensated lc with the development of multiple organ failure. in all cases, a lesion of the pancreas was present, in half of them pancreatic lipodystrophy was revealed. the cause of death in 40.3 % of people was bleeding from varicose veins of the esophagus and stomach with subcompensation and decompensation of lc, 17,4 % of patients died from hepatic failure with decompensation of lc, 13.5 % of patients died from pancreatic necrosis with compensation and subcompensation of lc. hepatic-renal failure was cause of death of the patients with subcompensation and decompensation of lc. sepsis caused death of 8 % of people with subcompensation and decompensation of lc, 4 % of patients died from hcc. cardio-pulmonary failure caused death in the patients with compensation and subcompensation of lc. the cytolytic, hepato-depressive, mesenchymal-inflammatory, hepatic encephalopathy, cholestatic, hepatorenal, anemic, hepa topulmonary syndromes and portal hyper tension were especially significant in the patient with subcompensation and decompensation of lc. more than a third of people had overdeveloped subcutaneous fat. ihd was the concomitant illness in 37.6 % of the patients. more than 70 % of the dead had atherosclerotic lesions of aorta, and one third of the persons had injured coronary vessels. excessive consumption of alcohol and, consequently, the development of lc, can be considered as an adverse factor in the reducing social standard of living. fatal cases were caused by decompensation of lc, development of multiple organ failure, pancreatic necrosis, bleeding from the esophagus and stomach veins, hcc. special attention should be paid to the combination of ald with atherosclerotic vascular changes revealed in 79.8 % of people, indicating a significant prevalence of lipid metabolism disorders among people with decompensated alcoholic lc. conclusions analyzing clinical records of the pathoanatomical study of the patients with alcoholic liver cirrhosis, it was established that most patients were young and middle aged and died from decompensated lc with the development of multiple organ failure. bleeding from varicose veins of the esophagus and stomach were the causes of death in 57.5 % of people, 14.8 % of patients died from pancreatic necrosis, 4 %– from hcc. ihd was the concomitant illness in 37.6 % of the patients. more than 70% of the dead had atherosclerotic lesions of aorta, and one third of the persons had injured coronary vessels. pancreatic necrosis and cardio-pulmonary failure were the causes of death in most people with stages a and b. gib, hepatic and hepatic-renal failure, sepsis, and development of hcc were the causes of death in most people with stage c. ihd was the concomitant illness in 37.6 % of the patients. conflict of interest the author declares no conflict of interest. n.r. matkovska issn 2413-6077. ijmmr 2019 vol. 5 issue 1 45 p u b l ic h e a lt h a n d e p id e m io l o g y аналіз смертності хворих на алкогольний цироз печінки н.р. матковська двнз “івано-франківський національний медичний університет”, івано-франківськ, україна вступ. алкоголь визнають основним фактором ризику несприятливих летальних завершень у всьому світі. в україні смертність внаслідок алкогольної хвороби печінки (ахп) посіла друге місце в структурі причин смерті від хвороб органів травлення. мета. метою було вивчити особливості причин смерті у осіб з ахп на стадії цирозу печінки (цп) на основі аналізу протоколів патологоанатомічного дослідження. методи. проведено аналіз 149 протоколів патологоанатомічного дослідження померлих на алкогольний цп на базі патологоанатомічного відділення івано-франківської обласної клінічної лікарні за період 2006-2018 рр. результати. більшість осіб були молодого та середнього віку. летальні випадки були зумовлені декомпенсацією цп з розвитком печінкової, печінково-ниркової, серцево-легеневої недостатності, панкреонекрозу, шлунково-кишкових кровотеч, сепсису, гепатоцелюлярної карциноми. у 37,6% померлих супутньою хворобою була ішемічна хвороба серця, у 10,7% осіб – гіпертонічна хвороба. у 6% осіб в анамнезі був перенесений ішемічний інсульт головного мозку. у більшості осіб виявлено атеросклеротичні зміни судин. висновки. надмірне вживання алкоголю і, як наслідок, розвиток цирозу печінки можна вважати несприятливим чинником зниження рівня здоров’я населення. у більшості осіб, що померли внаслідок декомпенсації алкогольного виявлено атеросклеротичні ураження судин. це вказує на значну поширеність порушення ліпідного обміну у осіб з алкогольним цирозом печінки. ключові слова: алкогольний цироз печінки; причини смерті; ішемічна хвороба серця; атеросклероз. інформація про автора матковська наталія романівна – доцент кафедри терапії і сімейної медицини післядипломної освіти, канд. мед. наук, двнз “івано-франківський національний медичний університет”, вул. кропивницького, 16, м. івано-франківськ, 76018. information about the author natalia r. matkovska – md, ph.d, assosiate professor of the department of therapy and family practice of postgraduate study faculty, ivano-frankivsk national medical university, 16 kropyvnytskyi str., ivano-frankivsk, 76018. orcid 0000-0002-9924-2127, e-mail: nmail4you@gmail.com references 1. melnyk ps, slabkyi ho, dziuba om, chepelevska la, kudrenko mv. annual report on the health status of the population, the sanitary and 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therapy for portal hypertension in liver cirrhosis: current progress and perspectives. world j gastroenterol. 2018;24(33):3738-3748. doi: 10.3748/wjg.v24.i33.3738 received 19 february 2019; revised 18 march 2019; accepted 12 april 2019 this is an open access article distributed under the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. n.r. matkovska 26 r a d ia t io n m e d ic in e a n d o n c o l o g y issn 2413-6077. ijmmr 2019 vol. 5 issue 1 doi 10.11603/ijmmr.2413-6077.2019.1.9819 adipose tissue and its role in microenvironment of the colorectal adenocarcinoma cancer cell a.a. burlaka national cancer institute, kyiv, ukraine introduction. the mechanisms of adipose-tissue’s influence on tumor progression has been studied a lot, but the way of interaction of adipocytes with tumor cells have not been well defined until now. objective. the aim of this study was to evaluate the mechanisms of adipocytes and tumor cells interaction under the influence of radiation and chemo-radiation therapy in locally advanced rectal cancer (larc) patients. material and methods. a prospective randomized single-center study was conducted. it involved 110 patients with larc and pre-obesity. the patients were randomized into a main group a (radiation therapy and oxaliplatin-based chemotherapy) and a comparison group b (radiation therapy and fluoropyrimidine-based mono-chemotherapy). superoxide free radicals and no levels generated by mitochondria of adipocytes were evaluated in both groups’. also, there was estimated the indices of mmp-2, mmp-9, 8-oxog, and free fatty acids (ffa) level. results and discussion. level of superoxide radicals in tumor-adjacent adipose tissue was 0.58±0.15 (main group) and 0.70±0.12 nmol/g·min (comparison group) (p<0.001). blood levels of ffa increased in group a up to 2.05±0.15, and in group b up to 2.48±0.20 mmol/l (while in it was 0.57±0.11 mmol/l). 8-oxog levels in tumoradjacent adipose tissue had no statistically significant differences. conclusions. the tumor-adjacent adipose tissue is an energy depot that can act as a promoter of tumor progression supplying the locally advanced rectal cancer with an energy substrate ffa. it has been established that the level mmp-2 activity significantly reduces the degree of intercellular matrix remodeling by the xelox chemotherapy. key words: locally advanced rectal cancer; adipose tissue; tumor progression; chemoradiotherapy. corresponding author: burlaka anton, md, ph.d., national cancer institute, 33/43 lomonosov str., kyiv, 03022, ukraine e-mail nir.burlaka@gmail.com introduction a number of clinical studies have proved a direct effect of obesity on the epidemiology of colorectal cancer (crc), disease progression, and a direct effect of adipose tissue (at) on malignant neoplasms development, as well as the ‘survival’ of the latter. adipocyte is the main structural unit of at that provides a significant resource of lipids, cytokines and adipokines. it has been established that the lipids, cytokines and adipokines provide regulation of signaling metabolic cascades and spread of malignant cells, including crc adenocarcinoma cells. adipocytes can modify a tumor microenvironment that in its turn stimulates/accelerates the tumor metabolism and leads to formation of an aggressive phenotype of crc tumor due to paracrine secretion and the presence of significant levels of free fatty acids (ffa). the cells of malignant neoplasms can accelerate the rate of proliferation, which is impossible without the modification of energy metabolism and the initiation of de novo-lipogenesis [1]. ffa are used for the accumulation of adipose tissue and the membranes construction, which is the basis for the malignant cells’ survival. most studies have proved that lipids provide survival of adenocarcinoma cells while their own de novo-lipogenesis is in a state of inhibition [2]. thus, it becomes clear that adenocarcinoma cells carry out proliferation without performing the synthesis of their own energy but using energy rich fatty acids of microenvironment [3]. ffa as a powerful source of energy becomes a key element in an aggressive rectal cancer (rc) phenotype formation. it worth remembering that lipids can enhance the warburg effect in tumor cells [4]. over the past decade, the awareness of the mechanisms of the at influence on rc tumor progression has improved considerably. however, the mechanisms of adipocytes and tumor cells interaction under the influence of radiation and chemo-radiation therapy have not been revealed until now. international journal of medicine and medical research 2019, volume 5, issue 1, p. 26-32 copyright © 2019, tnmu, all rights reserved a.a. burlaka 27 r a d ia t io n m e d ic in e a n d o n c o l o g y issn 2413-6077. ijmmr 2019 vol. 5 issue 1 material and methods a prospective randomized single-center study was conducted. the study involved 110 patients with locally advanced rectal cancer (larc) (ymrt3-4an0-2m0-1, crm-positive) with overweight (pre-obesity), who were treated in national cancer institute in january 2016 – december 2018. the patients were randomized into a main group (a) n=57 (radiation therapy with a total focal dose of 50.4 gy (1.8 gy×28) and oxaliplatin-based polychemotherapy) and comparison group (b), n=53 (radiation therapy with a total focal dose of 50.4 gy (1.8 gy×28) and mono-chemotherapy based on fluoropyrimidine). the patients’ division into groups was 1:1. the characteristic features of the groups of patients are provided in table 1. in each clinical case, multidisciplinary approach was used, where surgeons, oncologists, chemotherapists and radiologists took part. in all cases, the diagnosis was confirmed cytologically/ histologically after the primary tumor biopsy taken during the colonoscopy in order to assess the resectability of the primary tumor, to detect metastatic lesions in regional lymph nodes and to identify possible distant metastases. a common method of magnetic resonance imaging (mri) of the small pelvis and abdominal cavity with intravenous contrast and computer tomography (ct) of the chest cavity, abdominal cavity and small pelvis with intravenous contrast, according to international protocols was carried out [5]. positron-emission tomography was conducted only for planned multivisceral resections and in clinically com plicated cases as well as for differential diagnosis for the presence of metastatic lesions of other organs/sites. surgical techniques comprised implementation of standardized approaches for rc removing (total mesorectal excision and d3 lymphatic dissection); most of the interventions were performed laparoscopically. sampling of at was performed for 20 minutes since the removal of the tissue sample at a distance of 1-5 cm from the tumor; a surgeon and a morphologist took part in it. the samples were shaped by a special mold, frozen in liquid nitrogen, with subsequent electron paramagnetic resonance spectra (epr), they were recorded in a paramagnetic pure quartz devuar. the levels of superoxide free radicals (sr) generated by mitochondria of adipocytes, as well as the levels of no content were evaluated by means of epr method and spin traps technology. the state of intracellular matrix was assessed by the activity of matrix metallo proteinases (mmp-2 and mmp-9) in polyacrylamide gel using zymography technique. after the washing the active forms of mmp-2 and mmp-9 in gel visualized as a discolored stripe on a blue background; their localization was determined according to the molecular weight standards (sigma) and corresponded to the molecular weight of each of the enzymes (72 and 92 kda, respectively). the proteolytic activity was evaluated by measuring the area of the lysis zone using a standard set of mmp-2 and mmp9 (sigma) for comparison. one a.u. of the enzyme activity assumed as 1 mcg of enzyme in 1 g of control sample. the results were protable 1. characteristic features of the groups indices group а (n=57) group b (n=53) p value age 61.4±2.3 64.2±1.9 0.16 body mass index (bmi) 31.5±2.3 29.2±3.8 0.84 sex (male/female), (n) 37/21 24/28 0.45 presence of distant metastases, (n) 14 5 0.19 crm positive 20 12 0.32 emvi positive 9 3 0.44 trg ≥3 10 4 0.25 pn+ 28 14 0.21 cea level 27.4±5.6 12.2±3.1 0.18 patient’s condition (asa scale): і-іі 47 39 0.65 ііі 10 14 0.67 notes: crm – ‘circumferential resection margin’; emvi – ‘extramural venous invasion’; trg – ‘tumor regression grade’. asa – american society of anesthesiologists scale. a.a. burlaka 28 r a d ia t io n m e d ic in e a n d o n c o l o g y issn 2413-6077. ijmmr 2019 vol. 5 issue 1 cessed using the standard totallab 1.01 program. the levels of dna guanine oxidation (marker 8-oxog) in adipose tissue and blood ffa level were determined spectrophotometrically. the content of free (un-esterified) fatty acids (hf) in the blood was determined by spectrophotometric method at λ=546 nm (the intensity of the red dye formed by the interaction of hydrogen peroxide and tronder compounds is directly proportional to the concentration of non-esterified fatty acids in the knowledge). the attained results were statistically analyzed by means of spss 20.0 statistics (ibm, armonk, new york, usa). the differences were statistically different at p<0.05. results in at superoxide radicals (sr) and their derivatives are produced by mitochondria and nox immunocompetent cells. in phy siological conditions there is a balance between the sr generation and its elimination by antioxidants. in cases of pathological conditions, in particular, in malignant tumors, there is an increase in sr levels both in tumor and in tissues surrounding it that affects signaling regulatory pathways of tumor cells. the results of the at studying are presented in table. 2. the rate of sr generation in at stripped at a distance of 1 cm from the macroscopically visible edge of the larc tumors was determined. in the physiological conditions, the rate of sr generation was 0.18±0.03 nmol/g of tissue·min [6]. the rate of sr generation in tumor-adjacent at was 0.58±0.15 (group a) and 0.70±0.12 nmol/g tissue·min (group b) (p<0.001). the increased blood level of ffa was evidenced in group a: 2.05±0.15 mmol/l, and in group b – 2.48±0.20 mmol/l, the control value was 0.57±0.11 mmol/l. thus, the damaging effect on the surrounding tissues, in particular in cases of the radiation therapy and administration of platinum, is lower compare to the scheme comprising radiation therapy and fluoropyrimidines. the growth of sr and ros, res pectively, can cause lipids oxidation, 4-hydroxynonenal and malonic dialdehyde formation. the malonic dialdehyde can stimulate the activity of cyclooxygenase-2 (cox-2), which in turn catalyzes synthesis of prostaglandins and induces angiogenesis in rs tumors [7]. fatty acids, the level of which increases in tumor d u r i n g t h e i r ow n m e t a b o l i s m , p ro d u c e electrons; their energy in mitochondrial etc of tumor cells transform into atp and sr due to fact that adipocytes mitochondria are dysfunctional (table 2). oxidative dna damage is also a result of unregulated increase of sr and ros levels and their effects on human cells. the most common product of the pathological effect of sr on a dna molecule is 8-oxoguanine (8-oxog) formation; the level of it starts to exceed the normal values already in the early stages of the disease [8]. 8-oxog can be accumulated both in nuclear and mitochondrial dna. that is why they are considered to be a highly informative marker for the development of malignant neoplasms and their progression. according to our previous studies, 8-oxog levels in tumor-adjacent at proved to have no statistically significant differences: 0.80±0.08 and 0.84±0.13, respectively for groups a and b, p=0.052 (table 1 that may be explained by the fact that medicines used for hemotherapy lead to genotoxic effects). platinum containing medicines can in addition initiate sr generation. nitric oxide (no) is a pleiotropic, regulatory signaling molecule that is crucial for a variety of biological processes, including vasodilation, neurotransmission, and immune response [9]. no is synthesized out of l-arginine by no synthase (nos) [10]. the increased expression of inducible nos (inos) in tumor cells proves direct proportional correlation with survival of patients with rc and other malignant neoplasms of epithelial origin [11]. the activity of inos in tumor-adjacent adipose tissue is determined in accordance with the procedure described above. normal value of the no content is 1.16±0.20 nmol/g of tissue. direct evaluation of no level is 0.51±0.11 and 0.38±0.08 nmol/g of table 2. comparison of the studied molecular markers between groups of patients. values norm values(mean±se) group а (mean±se) group b (mean±se) t-value р-value speed of sr generation, nmol/g tissue•min 0.18±0.03 0.58±0.15 0.70±0.12 4.43 <0.001 no content level, nmol/g tissue 1.16±0.20 0.51±0.11 0.38±0.08 -7.24 <0.001 8‐oxo‐g level, nmol/g tissue 0.21±0.03 0.8±0.08 0.84±0.13 1.96 0.052 ffa levels, mmol/l 0.57±0.11 2.05±0.15 2.48±0.20 2.36 0.045 a.a. burlaka 29 r a d ia t io n m e d ic in e a n d o n c o l o g y issn 2413-6077. ijmmr 2019 vol. 5 issue 1 tissue in group a and b, respectively (p<0.001) (table 2) that indicates a disturbance of no bioavailability in tumor-adjacent at. the changes of at cells mitochondrial etc in the patients with rc in cases of different treatment regimens were revealed. in epr spectra of at intensive epr signals with g=2.03 were recorded that proved formation and increase of no-fes proteins complexes in complex i (etc) of adipocytes mitochondria in the group a (fig. 1, epr 3 spectrum, g=2.03). the level of no-fes proteins complexes was 0.27±0.14 a.u. this value in norm was 0.08±0.05 a.u. in the group b of patients, who received fluoropyrimidines instead of oxaliplatin, the level of no-fes proteins complexes in etc were significantly higher (0.36±0.09 a.u.) that indicated that fluoropyrimidines-based at formed hypoxic microenvironment, which was more favorable for the tumor. the level of nofes proteins complexes in etc of adipocytes mitochondria correlates with the degree of rc differentiation (r=0.79, p<0.05). it should also be noted that the level of mmp-2 activity in at was significantly lower in the group a, indicating that oxaliplatin had less significant effect on redox state of at and subsequently the intracellular matrix remodeling. tissue intracellular matrix remodeling, including degradation and reorganization of its structures developed with the participation of specific enzymes: matrix metalloproteinases (mmp). mmps are a family of zn2+ and cu2+dependent endopeptidases with a common functional domain and activation mechanism. at present, more than 20 types of mmps different by their cellular localization, substrate specificity, functional activity, have been established: gelatinase, collagenase, stromelysines, membrane bound mmp as well as a group of insufficiently studied mmp [12]. the increased level of gelatinase subgroup members (mmp-2 and mmp-9) proves the presence of tissue matrix damage. the factors activating the inactive forms of mmp (pro-mmp) a re i n t e n s i f i c a t i o n o f s r g e n e ra t i o n i n endothelial cells mitochondria and nox immunocompetent cells of at; sr reacts with no forming onooas a result that leads to disorder of no bioavailability in at [11, 13]. it was already noted that in the patients, involved into the research, independently from the treatment method a decrease in the no content in at adjusted to tumor was evidenced (table 2). in fig.2 the results of the gelatinases activity (mmp-2 and mmp-9) in the patients with rc is presented. it was proved that mmp-2 activity rates were ( 3 . 5 1 ± 1 . 0 6 ) a n d ( 5 . 7 2 ± 1 . 1 3 ) a t p < 0 . 0 0 1 , respectively in groups a and b. mmp-9 activity fig.1. graphic representation of epr spectra in studied groups of patients. i – epr spectra of at in patients with rc: 1 – at of the patients with bmi 25.0–30.0. 2 – group b patients with bmi ≥25. 3 – group a patients with bmi ≥25. ii – zymograms showing mmp-2 activity in at of the corresponding groups. a.a. burlaka 30 r a d ia t io n m e d ic in e a n d o n c o l o g y issn 2413-6077. ijmmr 2019 vol. 5 issue 1 rate was (6.82±1.03) and (8.06±1.36) in the main and control groups, respectively (p<0.001). these results indicate that in the group a p a t i e n t s , w h o r e c e i v e d o x a l i p l a t i n o r capecitabine, the level of intercellular matrix degradation was lower compare to the group b patients administered with fluoropyrimidines. discussion redox molecules are capable of inducing lipolysis in adenocarcinoma cells that results in glycerol production, which can be incorporated into the glycolytic metabolic pathway [14]. such pathological changes in normal metabolism c a n a c t i v a t e m o r e r a p i d g r o w t h a n d subsequently much significant malignant metastatic tumor phenotype, including rc [15]. such changes are accompanied by changes in the microen viron men t of h ealt hy cells, infiltration of tumor-associated immune cells that trigger the process of chronic inflammation and enhance oxidative stress. the analysis proves that activation of de novo-lipogenesis in most of the patients with crc, is caused by high levels of cr and, consequently leads to mmp activation. the mmp activation, in its turn, proves that the adipocytes of tumor-adjacent at in the patients with rc are an ‘additional’ source of energy involved in metabolic processes of the rc adenocarcinoma that contributes to their faster growth and formation of metastases. in addition, a significant difference in the studied molecular markers proved a promising use of a modified scheme of preoperative course of chemotherapy-based oxaliplatin and capecitabine (xelox). we believe that such approach can help reduce the percentage of distant metastasis and local relapses. conclusions tumor-adjacent at is an energy depot that can act as a promoter of tumor progression, supplying the rc tumor with the energy substrate – ffa. in addition, the oxaliplatin based chemotherapy reduces the level of dna oxidative-induced point mutations in adipocytes mitochondria. it has been established that the level mmp-2 activity significantly reduces the degree of intercellular matrix remodeling by the xelox chemotherapy. conflict of interest the author declares no conflict of interest. fig. 2. mmp-2 and mmp-9 activity in at of the patients. mmp-2 activity in at was significantly lower in the group a, indicating that oxaliplatin had less significant effect on redox state of at and subsequently the intracellular matrix remodeling. tissue intracellular matrix remodeling, including degradation and reorganization of its structures developed with the participation of specific enzymes: matrix metalloproteinases (mmp). mmps are a family of zn2+ and cu2+-dependent endopeptidases with a common functional domain and activation mechanism. at present, more than 20 types of mmps different by their cellular localization, substrate specificity, functional activity, have been established: gelatinase, collagenase, stromelysines, membrane bound mmp as well as a group of insufficiently studied mmp [12]. the increased level of gelatinase subgroup members (mmp-2 and mmp-9) proves the presence of tissue matrix damage. the factors activating the inactive forms of mmp (pro-mmp) are intensification of sr generation in endothelial cells mitochondria and nox immunocompetent cells of at; sr reacts with no forming onooas a result that leads to disorder of no bioavailability in at [11, 13]. it was already noted that in the patients, involved into the research, independently from the treatment method a decrease in the no content in at adjusted to tumor was evidenced (table 2). in fig.2 the results of the gelatinases activity (mmp-2 and mmp-9) in the patients with rc is presented. fig. 2. mmp-2 and mmp-9 activity in at of the patients. 0 2 4 6 8 10 12 mmp-2 activity mmp-9 activity re la tiv e un its group а group в control p < 0.001 p < 0.001 a.a. burlaka 31 r a d ia t io n m e d ic in e a n d o n c o l o g y issn 2413-6077. ijmmr 2019 vol. 5 issue 1 жирова тканина та її роль в мікрооточенні клітин аденокарциноми колоректального раку а.а. бурлака національний інститут раку моз україни, м. київ, україна вступ. за останні десять років наше розуміння механізмів впливу жирової тканини (жт) на пухлинне прогресування значно покращилось, однак до цього часу не розкрито механізми взаємодії адипоцитів з клітинами пухлин раку прямої кишки (рпк) за умов проведення променевої та хіміотерапії мета. дослідити механізми взаємодії адипоцитів та клітин аденокарциноми за умов променевого та хіміопроменевого лікування у хворих на місцево поширені форми раку прямої кишки. методи. було проведено проспективне рандомізоване одноцентрове дослідження. у дослідженні брали участь 110 хворих на місцево-поширений рак прямої кишки (мпрпк), (ymrt3-4an0-2m0-1, crm – positive) з надлишковою вагою в період з січня 2016 р. до грудня 2018 р. пацієнтів рандомізували у співвідношенні 1:1 на основну групу (а) n=57 (променева терапія сумарною осередковою дозою 50,4 гр (1,8 гр х 28) та поліхімотерапія на основі оксаліплатини), та на групу порівняння (б), n=53 (променева терапія сумарною осередковою дозою 50,4 гр (1,8 гр х 28) та монохіміотерапія на основі фторпіримідинів). у обох групах вивчали рівні мітохондріальних супероксидиних радикалів та no в адипоцитах прилеглої до пухлини жирової тканини. також досліджували рівень матриксних металопротеїназ (ммп-2 та ммп-9), маркери окисного пошкодження днк (8-oxog) та рівень жирних кислот. результати. зареєстровані рівні швидкості генерування супероксидних радикалів (ср) в жт, яка контактувала з пухлиною складали 0,58±0,15 (група а) та 0,70±0,12 нмоль/г тканини•хв (група б) (р<0,001). в крові цих хворих виявлено зростання рівня вільних жирних кислот (вжк): у групі а до значень 2,05±0,15 ммоль/л, а в групі б цей показник склав 2,48±0,20 ммоль/л при значеннях норми 0,57±0,11 ммоль/л. рівні 8-oxog в прилеглій до пухлини жт хворих досліджуваних груп не мали статистично значущої відмінності – 0,80±0,08 та 0,84±0,13 відповідно для груп а та б, при р = 0,052. висновки. прилегла до пухлини жт представляє собою енергетичне депо, здатне виступати в ролі промотора пухлинного прогресування, забезпечуючи пухлину мпрпк енергетичним субстратом – вжк. було показано, що хіміпроменева терапія (xelox) знижує рівень ремоделювання міжклітинного матриксу прилеглої жирової тканини. ключові слова: місцево-поширений рак прямої кишки; жирова тканина; пухлинне прогресування; хіміопроменева терапія. інформація про автора антон анатолійович бурлака – канд. мед. наук, старший науковий 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attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. a.a. burlaka 5 in t e r n a l m e d ic in e issn 2413-6077. ijmmr 2019 vol. 5 issue 1 doi 10.11603/ijmmr.2413-6077.2019.1.9768 proximal brachial monomelic amyotrophy or hirayama disease: no longer an alias? (case report) a. rao ramaiah medical college, bangalore, karnataka, india background. brachial monomelic amyotrophy (bmma) has been called as hirayama disease (hd) when it is characterized by unilateral distal upper limb weakness and atrophy that shows progression for a limited period and is associated with typical features on mri of cervical spine in flexion. objective was to explore the differences when bmma affects the proximal upper limb muscles with the help of case report. methods. a case report of bmma in an adult indian male is represented. results. a 30-year-old man presented to us with a history of weakness in the proximal aspect of his left upper limb that began four years ago. the weakness was progressive up until 6 months prior to his presentation since when the weakness had neither worsened nor improved. cervical spine contrast enhanced mri revealed mild loss of cervical lordosis, but no features of hd like localized cord atrophy, loss of attachment of dura from subjacent lamina on neutral position axial t2wi mri, nor any presence of posterior epidural crescentic enhancing mass on flexion contrast sagittal t1wi mri. the patient was managed with supportive therapy and has been under regular follow up ever since. his clinical status has been stable. conclusions. we support the suggestion to consider proximal brachial monomelic amyotrophy to be a separate entity and to be distinguished from hirayama disease that should be reserved for patients with distal upper limb involvement with cervical mri findings on flexion studies. key words: brachial monomelic amyotrophy; hirayama disease; cervical spine flexion mri. introduction traditionally hirayama disease (hd), also known as brachial monomelic amyotrophy (bmma), has been described as a condition characterized by insidious onset unilateral upper limb weakness and wasting [1]. hd is considered to be a focal degenerative motor neuron disease (mnd) affecting the cervical spinal cord. though it is considered as a variant of motor neuron disease, it differs from other forms of mnd, since it clinically involves only one limb and, after a brief period of progression, the neurological deficit becomes static [2]. recently there have been arguments to recognize hd and bmma as separate diseases with different clinical and neuroimaging features and therefore possibly different etiopathologies. hassan k.m. et al. suggested that “hirayama disease” should be used for cases with clinically unilateral upper limb distal muscle weakness and atrophy with cervical spine magnetic resonance imaging (mri) demonstrating and the typical findings highlighted below, restricting the term “brachial monomelic amyotrophy” for cases of proximal upper limb atrophy without typical mri features of hd [3]. typical mri findings in the cervical spine in hirayama disease include [3]: in neutral position • abnormal cervical curvature (loss of cervical lordosis). • localized lower cervical cord atrophy with asymmetric cord flattening. • intramedullary hyperintensity in lower cervical cord. flexion position • loss of attachment between posterior dural sac and subjacent lamina. • anterior shifting of posterior cervical dural wall on flexion with prominent epidural flow voids. • enhancing epidural mass in lower cervical region. so, we report a case with isolated weakness and atrophy of proximal left upper limb muscles, without the typical mri findings of hd. case study a 30-year-old man, who was a farmer by occupation, presented to us in july 2015, with international journal of medicine and medical research 2019, volume 5, issue 1, p. 5-9 copyright © 2019, tnmu, all rights reserved corresponding author: akshay rao, assistant professor, department of general medicine, ramaiah medical college, bangalore, karnataka, india. e-mail: akshayrao19@yahoo.com a. rao 6 in t e r n a l m e d ic in e issn 2413-6077. ijmmr 2019 vol. 5 issue 1 history of weakness in the proximal aspect of his left upper limb that began four years ago. the weakness was progressive up until 6 months prior to his presentation since when the weakness had neither worsened nor improved. there was no trauma or fever preceding the onset of weakness, no family history of similar complaints nor any exposure to toxins or heavy metals. he denied pain, numbness, diplopia, dysphagia, ptosis, muscle cramps, fasciculations, and headache or neck pain. examination revealed marked atrophy and weakness in the proximal aspect of his left upper limb (fig. 1). fasciculations were noted in the left deltoid. the left biceps and supinator reflexes were diminished. the rest of the motor system examination was unremarkable. sensory and cranial nerve examinations were normal. electroneuromyography (enmg) showed denervation pattern with reduced motor action potentials and fasciculations in the left deltoid (fig. 2). motor nerve conduction study showed reduced compound motor action potentials (cmap) amplitudes in left axillary, musculocutaneous and infraspinatus, but the sensory nerve conduction was normal. cervical spine contrast enhanced mri revealed mild loss of cervical lordosis, but no features of hd like localized cord atrophy, loss of attachment of dura from subjacent lamina on neutral position axial t2wi mri, nor any presence of posterior epidural crescentic enhancing mass on flexion contrast sagittal t1wi mri (fig. 3). the patient was managed with supportive therapy and has been under regular follow up ever since. his clinical status has been stable. fig. 1. photographs displaying atrophy of the proximal aspect of the left upper limb with loss of contour of the left shoulder. fig. 2. electroneurogram comparison of the nerves supplying upper limb muscles revealed reduced compound motor action potential (cmap) on the left musculocutaneous nerve. a. rao 7 in t e r n a l m e d ic in e issn 2413-6077. ijmmr 2019 vol. 5 issue 1 discussion hirayama disease is a sporadic disease characterized by wasting and weakness confined to only one upper limb, initially progressing for 2-4 years which is followed by a stationary course, without clinical involvement of other limbs. there is no cranial nerve, cerebral, pyramidal tract or sensory involvement. in upper limb involvement, atrophy of the medial forearm muscles and the small muscles of hand are common [4]. emg is characterized by fibrillations, positive sharp waves, and fasciculations consistent with the underlying pathophysiologic process of denervation [5]. mri abnormalities in hd have been consistently reported in studies by hassan et al., sonwalkar et al. and raval et al. [3, 6, 7]. the debate continues whether hd represents a focal primary lower mnd or a local consequence of anatomical variation in the cervical spine [8]. it has been suggested that an imbalanced growth causes disproportional length between the patient’s vertebral column and spinal canal contents, leading to tightening of dural sac and anterior displacement of posterior dural wall on neck flexion [9], thereby compressing the cord against the posterior margin of adjacent vertebrae producing microcirculatory disturbances in the lower cervical cord resulting in necrosis of anterior horn cells, gliosis and localized lower cervical cord atrophy, consistent with pathologic findings on autopsy [10]. as with any case presenting with unilateral upper limb weakness and atrophy, there are other differential diagnoses to be considered for this patient [11, 12]: multifocal motor neuropathy with conduction blocks involves upper limbs in asymmetrical manner with predominant distal muscle involvement, atrophy isn’t a prominent feature and enmg shows demyelination with conduction blocks and has high serum titers of anti-gm1 ganglioside antibodies. in spinal muscular atrophy and motor neuro pathy, such as that due to lead toxicity, upper limbs involvement is bilateral and symmetric. a chronic focal myositis can be differentiated by an elevated serum creatine phosphokinase, and the emg, and the muscle histologic features. neuralgic amyotrophy or brachial neuritis is present with characteristic deep shoulder aching dysesthesia. there is good prognosis for full recovery. an entity called “late-progression of poliomyelitis” merits a comment. in this disease there are: 1) a definite history of poliomyelitis; 2) residual but stable neurologic deficits; 3) a quiescent period of ten years or more. our patient did not have any history of poliomyelitis in his past. cases of proximal brachial mma reported in the literature are very limited, and no such cases have been shown to have dynamic compression of cervical cord on mri. in 2000, de freitas m.r.g. et al. published a case series of brachial mma patients including one patient with proximal disease, in whom cervical mri was normal [11]. in 2008, orsini m. et al. reported two young men with proximal right upper limb mma, and both had a normal cervical mri [12]. in 2013, hassan k.m. et al. also described two young males with proximal left upper limb mma with only mild loss of cervical lordosis on mri [3]. in 2015, yoo s.d. et al. reported a patient with proximal upper limb atrophy with flexion cervical mri showing typical features of hd, but this patient had certain atypical features such as left deltoid tenderness and upper motor neuron signs in ipsilateral upper and lower limbs [13]. in 2017, aundhakar s.c. et al. reported a case of right upper limb wasting and weakness with typical mri findings of hd but were involved of proximal as well as distal upper limb muscles [14]. fig. 3. mri studies of the cervical spine in flexion failed to demonstrate the typical changes associated with hirayama disease. a. rao 8 in t e r n a l m e d ic in e issn 2413-6077. ijmmr 2019 vol. 5 issue 1 hence, it appears that proximal brachial bmma may thus have a pathogenesis different from the dynamic ischemia of lower cervical cord postulated for distal upper limb wasting of hd. perhaps such patients need not be subjected to cervical spine flexion contrast mri; an mri of the cervical spine in neutral position may suffice to exclude compressive myelopathy due to other causes. conclusions we support the suggestion of hassan et al. to consider proximal brachial monomelic amyotrophy to be a separate entity and to be distinguished from hirayama disease that should be reserved for patients with distal upper limb involvement with cervical mri findings on flexion studies. thus, there is a need to explore a different pathological mechanism for brachial monomelic amyotrophy. we also suggest that performing flexion contrast mri of the cervical spine is not necessary in proximal brachial monomelic amyotrophy patients. conflict of interest the author declares no conflict of interest. проксимальна плечова мономелічна аміотрофія або хвороба хіраяма: більше не одне і те ж? (клінічний випадок) a. rao ramaiah medical college, bangalore, karnataka, india вступ. брахіальну мономелічну аміотрофію (бмма) ще називають хворобою хіраяма, якщо вона характеризується однобічною дистальною слабкістю верхньої кінцівки і атрофією, яка прогресує протягом невеликого періоду часу, а мрт виявляє типові ознаки у шийному відділі хребта у положенні його згинання. мета роботи дослідити особливості та відмінності перебігу хвороби при ізольованому ураженні проксимального відділу верхньої кінцівки на прикладі клінічного випадку. методи. описано та проаналізовано клінічний випадок брахіальної мономелічної аміоатрофії у дорослого чоловіка, жителя індії. результати. 30-річний чоловік звернувся зі скаргами на слабкість у проксимальному відділі лівої верхньої кінцівки, яка триває близько чотирьох років. слабкість прогресувала приблизно півроку після її появи, з того часу – без змін, не спостерігалося ні погіршення ні покращення стану руки. мрт шийного відділу з контрастом виявила невелике сплощення шийного лордозу, однак не було виявлено типових ознак хвороби хіраяма: локалізованої вогнищевої атрофії спинного мозку, відшарування задньої стінки дурального мішка від стінок хребцевого каналу в нейтральному положенні на сагітальних t2ві зображеннях, чи наявність додаткових мас-утворів на задній поверхні твердої мозкової оболонки на сагітальних t2ві зображеннях мрт при згинанні шийного відділу хребта. пацієнтові було призначено підтримуючу терапію, він знаходиться під регулярним спостереженням лікаря. клінічний стан стабільний. висновки. на основі аналізу клінічного випадку, ми підтримуємо пропозицію розглядати брахіальну мономелічну атрофію як окрему нозологічну одиницю, відмінну від хвороби хіраяма, у пацієнтів з ураженням верхньої кінцівки на підставі результатів обстеження мрт шийного відділу хребта у положенні згинання. ключові слова: брахіальна мономелічна аміотрофія; хвороба хіраяма; мрт шийного відділу хребта у положенні згинання. information about the author akshay rao – assistant professor, department of general medicine, ramaiah medical college, msrit post, msr nagar, mathikere, bangalore, karnataka, india, pin 560054 orcid 0000-0002-4111-5424, e-mail: akshayrao19@yahoo.com a. rao 9 in t e r n a l m e d ic in e issn 2413-6077. ijmmr 2019 vol. 5 issue 1 references 1. hirayama k, toyukura y, tsubaki t. juvenile muscular atrophy of unilateral upper extremity: a new clinical entity. psychiatr neurol jpn.1959;61:2190-7 2. uncini a, servidei s, delli pizzi c, cutarella r, di muzio a, gambi d, et al. benign monomelic amyotrophy of lower limb: report of three cases. acta neurol scand. 1992;85:397-400. doi: 10.1111/j.1600-0404.1992.tb06035.x 3. hassan km, sahni h. nosology of juvenile muscular atrophy of distal upper extremity: from monomelic amyotrophy to hirayama disease--indian perspective nosology of juvenile muscular atrophy of distal upper extremity: from monomelic amyotrophy to hirayama disease – indian perspective. biomed res int. 2013;2013:478516. doi: 10.1155/2013/478516 4. gourie-devi m, suresh t.,shankar s. monomelic amyotrophy. arch neurol. 1984; 41:388-394. doi: 10.1001/archneur.1984. 04050160050015 5. de frietas marcos r. g., nascimento osvaldo j.m., benign monomelic amyotrophy: a study of 21 cases. arq neuropsiquiatr.2000; 58(3-b):808-813. https://pdfs.semanticscholar.org/4da3/405f8bf181 55c0b988f1dea56d2fe26d8b8e.pdf 6. sonwalkar ha, shah rs, khan fk, gupta ak, bodhey nk, vottath s, et al. imaging features in hirayama disease. neurol india 2008;56:22-6. doi: 10.4103/0028-3886.39307 7. raval m, kumari r, dung aa, guglani b, gupta n, gupta r. mri findings in hirayama disease. indian j radiol imaging. 2010;20(4):245-9. doi: 10.4103/0971-3026.73528 8. turner mr, talbot k mimics and chameleons in motor neurone disease. practical neurology. 2013;13:153-164. doi: 10.1136/practneu rol-2013000557 9. hirayama k. juvenile muscular atrophy of distal upper extremity (hirayama disease). intern med. 2000;39:283-90. doi: 10.2169/internalmedicine.39.283 10. hirayama k., tomonaga m., kitano k., yamada t., kojima s., arai k. the first autopsy case of 'juvenile muscular atrophy of unilateral upper extremity'. shinkei naika. 1985;22(2):85-86. 11. de freitas mrg, nascimento ojm. benign monomelic amyotrophya study of twenty-one cases. arq neuropsiquiatr. 2000;58(3-b):808-813. doi: 10.1590/s0004-282x2000000500003 12. orsini m, freitas mrg, catharino a, mello mp, nascimento ojm. upper limb proximal form of benign monomelic amyotrophy: on purpose of 2 cases. rev bras neurol.2008; 44 (3): 13-17. https://pdfs.semanticscholar.org/4da3/405f8bf 18155c0b988f1dea56d2fe26d8b8e.pdf 13. yoo sd, kim hs, yun d h, kim dh, chon j, lee sa, et al. monomelic amyotrophy (hirayama disease) with upper motor neuron signs: a case report. ann rehabil med. 2015; 39(1):122-7. doi: 10.5535/arm.2015.39.1.122 14. aundhakar sc, mahajan sk, chhapra da. hirayama's disease: a rare clinical variant of amyotrophic lateral sclerosis. adv biomed res. 2017;6:95. published 2017 jul 28. doi: 10.4103/2277-9175.211797. received 16 february 2019; revised 20 february 2019; accepted 18 march 2019. this is an open access article distributed under the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. a. rao 83 b io m e d ic a l s c ie n c e s issn 2413-6077. ijmmr 2018 vol. 4 issue 1 doi 10.11603/ijmmr.2413-6077.2018.1.9254 the alteration of proand anti-inflammatory cytokines in adrenaline-calcium induced myocardial damage and its correction with quercetin in rats о. v. denefil, а. m. мusiienko i. horbachevsky ternopil state medical university, ternopil, ukraine background. cardiac insufficiency is one of the main causes of morbidity and mortality in the world. objective. the aim of the study was to determine the content of interleukins in male rats with adrenalinecalcium model of myocardial injury (acm) and under correction by quercetin. methods. 143 wistar male-rats aged 5-6 months were used in the experiments. the changes of interleukins (il-1beta, il-2, il-4, il-6, il-10, tnf-alpha) content in blood serum in the development of heart injury by adrenaline and calcium (ac) and use of quercetin for protection were studied. the period of investigation was in 1, 2, 24 hours, 3, 7, 14, 21, 28 days. results. in acm model of myocardial injury tnf-alpha increased, il-2, il-4 decreased at each period of investigation. il-1beta increased in 2 and 3 days after the injury, at all other periods (except in 1 hour and 21 days) those indices were decreased. also we observed similar wave changes of il-6 and il-10 content. injections of quercetin for 7 days caused a significant decrease of il-2, il-4, il-10, and increase of il-6. in 7 days of quercetin administration after ac-injury, decreased tnf-alpha was observed as well as increased il-4, il-6, and il-1beta rates. in 14 days, the increase of tnf-alpha, normalization of il-10 to control was evidenced. in 28 days, increased activation of il-1beta, decreased content of tnf-alpha was fixed. conclusions. the development of adrenaline-calcium heart damage is accompanied by bright cytokine wavelike reaction at different time points. quercetin causes normalization of interleukins level. key words: heart damage; rats; adrenaline-calcium model; quercetin; interleukins. international journal of medicine and medical research 2018, volume 4, issue 1, p. 83-88 copyright © 2018, tsmu, all rights reserved corresponding author: olha denefil, professor of department of pathophysiology, i. horbachevsky ternopil state medical university, 1 maydan voli, ternopil, 46001, ukraine e-mail: denefil@tdmu.edu.ua phone number: +380978536918 о. v. denefil et al. introduction cardiac insufficiency is one of the main causes of morbidity and mortality in the world. one of the causes of its development is myocardial infarction, when cardiomyocytes die due to prolonged ischemia. myocardial infarction is a dynamic process, which is accompanied by transition of reversible changes to irreversible ischemic damage and completes in replacement of the diminished part of myocardium by a fibrous scar. development of fibrosis is a dysfunctional process in which myofibroblasts, the main fibrous cellular elements, are metabolically active and are able to produce and upregulate cytokines; they also have contractile properties [1]. myocardial infarction leads to rapid necrosis of cardiac myocytes. to achieve tissue integrity and function, inflammatory cells are activated, including monocytes/macrophages. impaired monocyte/ macrophage function is a not established new pathophysiological mechanism for left ventricular thrombus development after myocardial infarction [2]. myocardial damage in myocardial infarction takes place mainly due to ischemic necrosis and inflammatory mechanisms while apoptosis is the main mechanism of cell death in ‘reperfusion injury’ in addition to limited ischemic necrosis [3]. in the diseased heart, cardiomyocytes are lost to necrotic cell death, and phenotypically transformed fibroblast-like cells, termed ‘myofibroblasts’, are activated to initiate a ‘reparative’ fibrosis. the structural integrity of myocardium is preserved by this scar tissue, although the expense of its remodeled structure, which increases tissue stiffness and propensity to arrhythmias. a persisting population of activated myofibroblasts turns this fibrous tissue into a living ‘secretome’ that generates angio_ tensin ii and its type 1 receptor, fibrogenic growth factors, all of which collectively act as a signal-transducer-effector signaling pathway to type i collagen synthesis and, therefore, fibrosis. persistent myofibroblasts, and the resultant fibrous tissue they produce, cause progressive adverse myocardial remodeling, a 84 b io m e d ic a l s c ie n c e s issn 2413-6077. ijmmr 2018 vol. 4 issue 1о. v. denefil et al. table 1. division of animals by groups group quantity of animals total mortality control 10 – in 1 hour after аcм 10 – in 2 hours after аcм 10 – in 24 hours after аcм 10 – in 3 days after аcм 10 – in 7 days after аcм 10 – in 14 days after аcм 10 – in 21 days after аcм 10 – in 28 days after аcм 12 16.67 % quercetin 10 – in 7 days after аcм + quercetin 10 – in 14 days after аcм + quercetin 10 – in 21 days after аcм + quercetin 10 – in 28 days after аcм + quercetin 11 8.33 % pathological hallmark of the failing heart irrespective of its etiologic origin [4]. cardiomyocyte necrosis activates an inflam­ matory response that serves to clear the injured myocardium from dead cells, and stimulates repair, but may also extend injury. recently the studies in the literature have identified inter­ leukin­1a and rna released by necrotic cardio­ myocytes as key danger signals that trigger the inflammatory response following infarction. interleukin­1 promotes activation of a proinflam­ matory phenotype in leukocytes and fibroblasts, and delays myofibroblast transdifferentiation. inhibitory lymphocytes are crucial in negative regulation of postinfarction inflammatory res­ ponse by modulating macrophage and fibroblast phenotype. cardiac macrophages exhibit signifi­ cant heterogeneity and phenotypic plasticity and may manage the reparative response following infarction. in adult mice­ animals replacement of resident macrophage populations with mono­ cyte­derived macrophages may induce inflam­ mation while inhibiting cardiac regeneration [5]. taking into account that the main pathogenic links are the development of inflammation, oxidative and nitric explosion, spasm of coronary vessels, necrosis, fibrosis, the study of the effec­ tiveness of cardioprotective activities for the treatment of the basic pathogenesis is necessary. these medications include quercetin, which has antioxidant, anti­spasm, anti­inflammatory, anti­ sclerotic properties [6]. therefore, the aim of our study was to deter­ mine the content of interleukins in male rats with adrenalin­calcium model of myocardial injury (acm) and to correct the pathological process by quercetin. methods the experiments were performed on 143 albino wistar male rats 5­6 months of age. the animals were divided into 14 groups (table 1). each group comprised 10 animals. the rats were once intramuscularly administered with a 0.18% solution of adrenaline hydrotartrate at a dose of 0.5 mg/kg of body weight (pharma­ ceutical firm “darnitsa”, ukraine) and intrape­ ritoneally with 5% solution of calcium gluconate (“dniprofarm”, ukraine) at a dose of 1 ml/100 g of body weight. for the correction the solution of quercetin at a dose of 200 mg/kg of body weight was injected intraperitoneally once a day for 7 days after adrenaline and calcium injections. experiments were performed in a special room in the morning at 18­22 °с, relative humidity 40­60% and illumination 250 lux. the regula tions of the european convention for the pro tec tion of vertebrate animals used for expe­ ri mental and other scientific purposes (stras­ bourg, 1986), resolution of the first national congress on bioethics (kyiv, 2001) and the order of the ministry of health of ukraine № 690 dated september 23, 2009, were strictly followed. the rats were euthanized by total heart bloodletting and previous thiopental sodium anesthesia (60 mg·kg­1 of body weight intra­ peritoneally). blood samples were taken for the research. concentration of interleukins il­1beta, il­2, il­4, il­6, il­10, tumor necrosis factor alpha (tnf­alpha) was determined in blood serum by the method of immunoenzyme analysis using a set of reagents produced by zao vector­best (russia). 85 b io m e d ic a l s c ie n c e s issn 2413-6077. ijmmr 2018 vol. 4 issue 1 о. v. denefil et al. a statistical analysis of digital data was performed by means of excel (microsoft, usa) and statistica 6.0 (statsoft, usa). statistical significance of the differences between inde­ pendent indices was estimated by student t­test at normal distribution and by nonparametric methods in other cases. results in 1 hour after adrenaline and calcium injection, the increase of tnf­alpha in 7.56 times (p<0.001), decrease of il­4 by 35.63% (p<0.001) and of h­10 by 46.08% (p<0.001) were determined (table 2). in 2 hours il-1 beta was increased by 45.69 % (p<0.001) and tnf­alpha – in 5.9 times (p<0.001); il­2 was decreased by 18.24% (p<0.002) and il­6 – by 48.57% (p<0.001). the increase of il­1 beta by 45.82% (p<0.01), of il­10 in 2.1 times (p<0.001), the decrease of il­6 by 47.55% (p<0.001) were determined compared with the previous study. in 1 day, compared to the control, there was a decrease of il­1beta by 38.58% (p<0.001), il­2 – by 30.40% (p<0.001), il­4 – by 23.45% (p<0.001), and increase of il­6 in 46.68% (p<0.01), tnf­alpha – in 5.21 times (p<0.001), and compared to the previous study, there was a decrease of il­1beta by 57.84% (p<0.001), il­2 – by 14.88% (p<0.01), il­10 – by 23.37% (p<0.01), increase of il­6 in 2.85 times (p<0.001). in 3 days, compared to the control, the level of il­1beta was higher by 21.94% (p<0.001), tnf­alpha – in 7.06 times (p<0.01), il­10 – by 37.16% (p<0.001), and il­2 was lower by 56.08% (р<0.001), il­4 – by 39.66% (p<0.001), il­6 – by 34.67% (p<0.01), and compared with the previous period, il­1beta was increased by 22.06% (р<0.001), il­10 – in 2.547 times (p<0.001), il­2 was decreased by 83.20% (p<0.02), il­6 – by 33.38% (p<0.001), il­4 – by 6.26% (p<0.001). in 7 days compared to the control the increase of tnf­alpha in 17.88 times (p<0.001) was observed, as well as the decrease of il­1 beta by 45.81% (p<0.001), il­2 – by 47.97% (p<0.001), il­4 – by 20.54% (p<0.001), and compared with the previous period of study, il­1beta was decreased by 55.57% (p<0.001), il­10 – by 16.57% (p<0.001), il­6 was increased by 70.81% (p<0.01), tnf­alpha – in 2.53 times (p<0.001), il­4 – by 31.69% (p<0.001). in 14 days compared to the control the in­ crease of tnf­alpha in 19.69 times (p<0.001) was evidenced, as well as the decrease of il­1 beta by 40.45% (p<0.001), il­2 – by 37.84% (p<0.001), il­4 – by 21.08% (p<0.001), il­10 – by 39.67% (p<0.002), and compared with the previous study period – the decrease of il­10 by 47.28% (p<0.001). in 21 days compared with the control, the increase of tnf­alpha in 2.1 times (p<0.001), il­6 – by 72.91% (p<0.001), the decrease of il­4 by 28.06% (p<0.001), il­2 – by 22.30% (p<0.02), and compared with a previous period of study – the decrease of tnf­alpha by 89.87% (p<0.001), increase of il­6 by 70.73% (p<0.001) and il­10 – by 78.85% (p<0.001) were present. in 28 days the increase of tnf­alpha in 25.89 times (p<0.001), il­6 – by 36.94% (p<0.02), the decrease of il­1 beta by 45.38% (p<0.001), il­2 – by 35.13% (p<0.001), il­4 – by 19.52% (p<0.001), and compared to the previous period of study, the increase in tnf­alpha in 11.87 times (p<0.001), and the decrease of il­1beta by 39.43% (p<0.05) were evidenced. injections of quercetin for 7 days caused a significant decrease of il­2 by 62.84% (p<0.001), il­4 – by 32.32% (p<0.001), il­10 – by 51.92% (p<0.001), and increase of il­6 in 2.7 times (p<0.001). in 7 days after the injury, quercetin correction proved increased level of il­1beta in 2.61 times (p<0.001), il­4 – by 35% (p<0.001), il­6 – in 2.27 times (p<0.001), and decrease of tnf­alpha in 32.99 times (p<0.001). in this period, il­2, il­4, il­10 levels did not significantly differ from the control ones, and il­1beta was increased by 41.6% (p<0.001), il­2 – by 29.73% (p<0.001), il­6 – in 2.54 times (p<0.001). il­1 beta level was also higher by 60% (p<0.01), tnf­ alpha – in 9.26 times (p<0.001), il­4 – by 58.5% (p<0.002), il­10 – in 2.69 times (p<0.001) compared to the quercetin level. in 14 days after the injury, the quercetin correction proved higher level of tnf­alpha by 55.14% (p<0.01) and il­10 by 84.76% (p<0.001). in this period, only il­10 level did not significantly differ from the control ones, il­6 was increased by 49.59% (p<0.05), tnf­alpha – in 32.1 times (p<0.001), il­1beta was decreased by 28.5% (p<0.01), il­2 – by 28.38% (p<0.001), il­4 – by 23.7% (p<0.001), and compared to a previous period of study il­1 beta was decreased by 49.5% (p<0.001), il­6 – by 40.97% (p<0.002), and il­4 – by 28.87% (p<0.05), tnf­alpha was increased in 2.68 times (p<0.001). il­2 was also higher in 1.93 times (p<0.001), tnf­alpha – in 24.8 times (p<0.001), il­10 – in 2.32 times (p<0.001) and il­6 was lower by 44.7% (p<0.01) compared to the quercetin level. in 21 days after the injury, the quercetin correction proved higher level of h­6 by 51.42% (p<0.002), tnf­alpha in 9.59 times (p<0.001), lower level of h­2 by 50.43% (p<0.001). in this period, only il­1 beta level did not significantly differ from the control, h­6 was higher in 2.62 times (p<0.001), tnf­alpha – in 20.1 times (p<0.001), il­10 – by 20.07% (p<0.001), il­2 was 86 b io m e d ic a l s c ie n c e s issn 2413-6077. ijmmr 2018 vol. 4 issue 1 ta bl e 2. c h an ge s in in te rl eu ki n in di ce s in r at s’ b lo od w it h a dr en al in -c al ci u m m yo ca rd ia l d am ag e an d it s co rr ec ti on b y qu er ce ti n , n =1 0, м ±m g ro up in de x іl -1 be ta , p g/ m l іl -2 , х 10 -3 , p g/ m l іl -6 , p g/ m l tn fal ph a, p g/ m l іl -4 , p g/ m l іl -1 0, p g/ m l co nt ro l 66 .3 0± 2. 34 1. 48 ±0 .0 7 0. 78 ±0 .0 4 0. 05 4± 0. 00 2 2. 16 ±0 .0 2 10 .2 5± 0. 81 in 1 h ou r af te r аc м 66 .2 4± 5. 09 3. 87 ±1 .5 6 0. 77 ±0 .0 1 0. 40 8± 0. 08 7* 1. 39 ±0 .1 3* 5. 53 ±0 .4 2* in 2 h ou rs a ft er а cм 96 .5 9± 7. 91 *, # 1. 21 ±0 .0 3* 0. 40 ±0 .0 4* ,# 0. 31 8± 0. 07 8* 1. 83 ±0 .1 7 11 .6 3± 0. 87 # in 2 4 ho ur s af te r аc м 40 .7 2± 5. 50 *, # 1. 03 ±0 .0 6* ,# 1. 15 ±0 .1 2* ,# 0. 28 1± 0. 03 9* 1. 65 ±0 .0 7* 8. 91 ±0 .4 3# in 3 d ay s af te r аc м 80 .8 5± 0. 14 *, # 0. 65 ±0 .1 3* ,# 0. 51 ±0 .0 9* ,# 0. 38 1± 0. 12 0* 1. 30 ±0 .0 4* ,# 14 .0 6± 0. 54 *, # in 7 d ay s af te r аc м 35 .9 3± 5. 54 *, # 0. 77 ±0 .0 6* 0. 87 ±0 .0 8# 0. 96 6± 0. 08 1* ,# 1. 71 ±0 .0 3* ,# 11 .7 3± 0. 41 # in 1 4 da ys a ft er а cм 39 .4 8± 4. 82 * 0. 92 ±0 .1 2* 0. 79 ±0 .0 9 1. 11 7± 0. 19 3* 1. 70 ±0 .1 1* 6. 18 ±0 .9 2* ,# in 2 1 da ys a ft er а cм 59 .7 9± 9. 76 1. 15 ±0 .1 2* 1. 35 ±0 .1 2* ,# 0. 11 3± 0. 01 6* ,# 1. 55 ±0 .0 6* 11 .0 6± 1. 03 # in 2 8 da ys a ft er а cм 36 .2 1± 3. 07 *, # 0. 96 ±0 .0 4* 1. 07 ±0 .1 0* 1. 34 1± 0. 22 6* ,# 1. 74 ±0 .1 1* 10 .9 6± 0. 55 q ue rc et in 58 .6 7± 11 .1 6 0. 55 ±0 .0 4* 2. 12 ±0 .3 3* 0. 07 0± 0. 01 1 1. 46 ±0 .0 3* 4. 93 ±0 .2 6* in 7 d ay s af te r аc м + q ue rc et in 93 .8 8± 3. 03 *, ** ,& 1. 04 ±0 .2 4 1. 99 ±0 .1 8* ,& 0. 64 7± 0. 10 7* ,* *, & 2. 31 ±0 .2 6* *, & 13 .2 7± 2. 12 ** in 1 4 da ys a ft er а cм + q ue rc et in 47 .4 0± 5. 63 *, # 1. 06 ±0 .0 7* ,* * 1. 17 ±0 .1 7* ,* *, # 1. 73 4± 0. 09 9* ,* *, # ,& 1. 65 ±0 .0 9* ,# 11 .4 3± 1. 04 ** ,& in 2 1 da ys a ft er а cм + q ue rc et in 60 .9 8± 5. 73 0. 57 ±0 .0 4* ,# ,& 2. 05 ±0 .0 8* ,# ,& 1. 08 5± 0. 15 7* ,* *, # ,& 1. 37 ±0 .0 7* ,# 12 .3 1± 0. 11 *, ** in 2 8 da ys a ft er а cм + q ue rc et in 81 .7 3± 2. 56 *, # ,& 0. 86 ±0 .0 6* ,* *, # 1. 30 ±0 .0 6* ,* *, # 0. 47 9± 0. 16 3* ,* *, # ,& 1. 56 ±0 .0 6* ,# 9. 89 ±0 .3 3* *, # n ot es : * – st at is tic al ly s ig ni fic an t i nd ic es a s co m pa re d w ith th e co nt ro ls ; ** – s ta tis tic al ly s ig ni fic an t i nd ic es a s co m pa re d w ith th e qu er ce tin ; # – s ta tis tic al ly s ig ni fic an t i nd ic es a s co m pa re d w ith th e pr ev io us p er io d of in ve st ig at io n; & – s ta tis tic al ly s ig ni fic an t i nd ic es a s co m pa re d w ith th e re su lts w ith ou t c or re ct io n. о. v. denefil et al. 87 b io m e d ic a l s c ie n c e s issn 2413-6077. ijmmr 2018 vol. 4 issue 1 о. v. denefil et al. lower by 61.49% (p<0.001), il­4 – by 36.5% (p<0.001), and compared with a previous period of study the decrease of il­2 by 46.23% (p<0.001), tnf­alpha – by 37.4% (p<0.001), il­4 – by 16.77% (p<0.05), the increase of il­6 by 74.91% (p<0.002) were present. tnf­alpha was also higher in 15.53 times (p<0.001) and il­10 – in 2.5 times (p<0.001) compared to the quercetin level. in 28 days after the injury, the quercetin correction proved higher level of il­1 beta in 2.26 times (p<0.001), lower level of tnf­alpha by 64.38% (p<0.01). in this period, only il­10 did not significantly differ from the control level, il­1 beta was higher by 23.28% (p<0.001), il­6 – by 65.62% (p<0.001), tnf­alpha – in 8.87 times (p<0.01), il­2 was lower by 41.89% (p<0.001), il­4 – by 27.48% (p<0.001), and compared with a previous period of study the decrease of il­6 by 36.74% (p<0.002), tnf­alpha by 55.88% (p<0.001), il­10 by 19.68% (p<0.001), the increase of il­1 beta by 34.04% (p<0.001), il­2 by 50.88% (p<0.001), il­4 by 14.19% (p<0.05) were present. il­2 level was also higher by 56.36% (p<0.001), tnf­alpha – in 6.85 times (p<0.05) il­10 – in 2 times (p<0.001), and il­6 was lower by 38.82% (p<0.05) compared to the quercetin level. discussion in first hour a direct damage of heart muscle by adrenaline was present, since the рroinflam­ matory cytokine tnf­alpha was a mediator of cardiac pathology. tnf­alpha was synthesized by activated macrophages, and also stimulated them, promoted cytotoxic, immunomodulating, anti­inflammatory action. decreases in il­10 and il­4 may be associated with the early period of damaging effects of adrenaline and calcium when the increase of one anti­inflammatory cytokine was sufficient with protection. in two hours a high tnf­alpha content still was present. it could cause damage to endothelial cells, increase of microvascular permeability, activate the hemostasis system with dic synd­ rome development. normalizing the concen tra­ tion of il­4 and il­10 may evidence development of immune response with involvement of t­ and b­lymphocytes. that was evidenced by the increase of il­1beta. the decrease of il­2 and il­6 proved immunosuppressive effect. in day 1 high values of tnf­alpha and il­6, in the presence of decreased il­4, may evidence the activation of coagulation hemostasis with the development of blood clots. the increase of il­6 also evidenced development of hypoxia, which may take place due to necrosis of cardio­ myocytes. the increase of il­1beta and tnf­alpha in day 3 could be caused by more severe hypoxia and ischemia, products of fibrin degradation. the increase of il­10 proved its protective influence. in day 7, compared to the previous period, il­6 normalization may evidence the worsening of hypoxia. tnf­alpha increase and decrease of h­10 only confirmed this assertion. that was observed in 14 days also. in 21 and 28 days the same changes as in a 24 hour period were present: high level of tnf­ alpha and il­6 in the presence of decreased il­4. it may prove the secondary activation of coagulation hemostasis by means of alteration. in the infarcted myocardium, necrotic cardiomyocytes release danger signals activating an intense inflammatory reaction that serves to clear the wound from dead cells and matrix debris, but may also extend injury. dead cardiomyocytes release il­1α that may function as a crucial alarm in triggering the post­in­ farction inflammatory reaction. il­1β is markedly up­regulated in the infarcted myocardium; activation of inflammasome in both cardio­ myocytes and interstitial cells results in release of bioactive il­ip in the infarcted area. binding of il­1 to the type 1 receptor triggers an in­ flammatory cascade, inducing recruitment of proinflammatory leukocytes and stimulating a matrix­degrading program in fibroblasts, while delaying myofibroblast conversion. il­1 mediates dilative remodeling following infarction and may influence on the pathogenesis of postinfarction heart failure [7]. injection of quercetin in the experiments increased contents of il­6, suppressing the formation of il­2, il­4 and il­10. adrenaline and calcium lesion of heart in which quercetin was used had a favorable course. thus, in 7 days, the decreased activation of tnf­alpha was observed, and increased – of il­4, il­6, and il­1 beta. in 14 days, the increased activation of tnf­ alpha, normalization of h­10 to the control level was evidenced. the increased content of il­6, decreased – of il­1 beta, il­2, il­4 were present. in 21 days, the increased content of tnf­alpha, il­6 was evidenced. il­10 level did not differ from the control, il­2 content was decreased. in 28 days, the increased activation of il­1beta, decreased content of tnf­alpha was observed. il­10 improved myocardial function after acute global i/r and suppressed inflammation of stat3 pathway [8]. also the profibrotic effect of il­10 autocrine loop promoted macrophages to secret osteopontin and tgfp. these media­ tors activated cardiac fibroblasts to produce collagen that caused cardiac fibrosis and in crea­ sed cardiac stiffness [9]. increased il­6 con tent was associated with a cardiovascular disease and protected cells against apoptosis whilst being important for normal metabolism [10]. 88 b io m e d ic a l s c ie n c e s issn 2413-6077. ijmmr 2018 vol. 4 issue 1о. v. denefil et al. references 1.. seg a am, frazier oh, buja lm. fibrosis and heart failure. heart fail rev. 2014;19(2):173–85. doi: 10.1007/s10741-012-9365-4. 2. frantz s, hofmann u, fraccarollo d, schäfer a, kranepuhl s, hagedorn i, nieswandt b, nahrendorf m, wagner h, bayer b, pachel c, schön mp, kneitz s, bobinger t, weidemann f, ertl g, bauersachs j. monocytes/macrophages prevent healing defects and left ventricular thrombus formation after myocardial infarction. faseb j. 2013;27(3):871–81. doi: 10.1096/fj.12-214049. 3.. ashmi s, al-salam s. acute myocardial infarction and myocardial ischemia-reperfusion injury: a comparison. int j clin exp pathol. 2015;8(8): 8786–96. 4. weber kt, sun y, bhattacharya sk, ahokas ra, gerling ic. myofibroblast-mediated mechanisms of pathological remodelling of the heart. nat rev cardiol. 2013;10(1):15–26. doi: 10.1038/nrcar dio. 2012.158. 5. frangogiannis ng. inflammation in cardiac injury, repair and regeneration. curr opin cardiol. 2015;30(3): 240–5. doi: 0.1097/hco.0000000000000158. 6. sheremeta l. antiulcerous activity of liposomal quercetin at experimental subacute gastric ulcer. herald ssu. the series ’medicine’. 2008;1:43–7. [in ukrainian]. 7.. angogiannis ng. interleukin-1 in cardiac injury, repair, and remodeling: pathophysiologic and translational concepts. discoveries (craiova). 2015; 3(1): e41. doi: 10.15190/d.2015.33 8. manukyan mc, alvernaz ch, poynter ja, wang y, brewster bd, weil br, abarbanell am, herrmann jl, crowe bj, keck ac, meldrum dr. interleukin-10 protects the ischemic heart from reperfusion injury via the stat3 pathway. surgery. 2011;150(2):231–9. doi.org/10.1016/j.surg.2011.05.017 9.. cih ova d. interleukin-10 stiffens the heart. journal of experimental medicine. 2018;215(5): doi: 10.1084/jem.20180049 10.0. ywardena my, leifert wr, warnes ke, varghese jn, head rj. cardiovascular biology of interleukin-6. current pharmaceutical design. 2009; 15:1809–21. received: 2018-05-03 the obtained data has proved the inclusion of pro­ and anti­inflammatory mechanisms of protection against damage, when used with the protective effect of quercetin. quercetin in cases of more severe fibrosis with underlying injury of acm of rats prevents cardiac necrosis. conclusions the development of adrenaline­calcium heart damage is accompanied by bright cytokine wavelike reaction at different time points. in early time a direct damage of heart muscle by adrenaline and calcium is present. then can be development of dic syndrome, immune res­ ponse, hypoxia, and ischemia. at day 3 increases the protective influence – anti­inflammatory interleukins level. then evidence the worsening of hypoxia, secondary activation of coagulation hemostasis by means of alteration. quercetin causes normalization of interleukins level. зміни прозапальних та протизапальних цитокинів на моделі адреналіново-кальцієвого ураження міокарда у щурів та корекції кверцетином о. в. денефіль, а. м. мусієнко тернопільський державний медичний університет імені і. я. горбачевського, тернопіль, україна вступ. серцева недостатність є однією з основних причин захворюваності та смертності у світі. метою дослідження було визначення коливань інтерлейкінів на моделі адреналіново-кальцієвого ураження міокарда та його корекції кверцетином. методи. досліди проведені на 143 щурах-самцях лінії вістар віком 5-6 місяців. було досліджено зміни вмісту інтерлейкінів il-1beta, il-2, il-4, il-6, il-10, tnf-альфа у сироватці крові при розвитку ураження серця та застосування кверцетину. дослідження проводили у 1-у, 2-у, 24-у години, на 3-ю, 7-у, 14-у, 21-у та 28-у доби. результати. адреналіно-кальцієва модель ураження міокарда характеризувалася підвищенням рівня tnf-альфа; а вміст il-2 та il-4 поступово достовірно знижувався. рівень il-1бета зростав на 2-у і 3-ю доби після формування ураження серцевого м’яза, у інших періодах часу досліджень (за винятком 1 години і 21-ї доби) ці показники знижувалися. спостерігалися також хвилеподібні коливання вмісту il-6 і il-10. ін’єкції кверцетину протягом 7 днів зумовили достовірне зниження il-2, il-4, il-10 та підвищення вмісту il-6. також спостерігалося зниження рівня tnf-альфа, та підвищення вмісту il-4, il-6 та il-1. через 14 днів все ще спостерігалося підвищення рівня tnf-альфа та нормалізація показників il-10. на 28-у добу зафіксовано підвищений вміст il-1beta та зниження рівня tnf-альфа. висновки. розвиток адреналіно-кальцієвого ураження серця супроводжується яскравою хвилеподібною реакцією цитокінів у різні досліджувані проміжки часу час. кверцетин викликає нормалізацію рівня інтерлейкінів. ключові слова: адреналіново-кальцієва модель ураження міокарду; щури; кверцетин; інтерлейкіни. 75 b io m e d ic a l s c ie n c e s issn 2413-6077. ijmmr 2019 vol. 5 issue 1 doi 10.11603/ijmmr.2413-6077.2019.1.9897 anthropometry of the external ear among adult ijaws in bayelsa state of nigeria e.i. edibamode1, k. mordi1, l.k. david1, a.m. eghoi2* 1 – university of port harcourt, port harcourt, nigeria 2 – abia state university, uturu, nigeria background. external ear measurement is of utmost importance in reconstructive surgeries. objectives. the present study is aimed at ascertaining sexual dimorphism in external ear anthropometry and ear lobe attachments among adults ijaws in bayelsa, nigeria. methods. a total of 112 adults within the age range of 18-50 yrs, who met the inclusion criteria, were involved in the study. four linear dimensions of the ear, which are ear length (el), ear width (ew), lobular length (ll) and lobular width (lw), were measured for both genders. the lobular attachment for both ears for males and females were also examined and results recorded. these data were subjected to student t-test, chi-square test, and pearson’s correlation using spss version 20.0. results. the mean values for el, ew, ll, and lw for the left auricle in the males and females were 58.14±0.60, 27.41±0.37, 14.47±0.27, 13.50±0.34 and 57.90±0.16, 27.45±0.65, 15.41±0.31, 13.43±0.38 respectively. however, for the right auricle in the males and females, the values were 58.40±0.45, 28.21±0.68, 14.32±0.31, 13.04±0.32, and 56.66±1.10, 27.51±0.65, 15.58±0.29, 13.28±0.34 respectively. the left and right lobular length were the only parameters that proved statistical significance (p<0.05) in females compare to males. pearson’s correlations between right and left sides for each of the parameters were positive and significant. chi-square analysis revealed no significant relationship (p>0.05) between earlobe attachments and gender. conclusions. sexual dimorphism was thus established in the ijaw population as regards lobular length dimensions. it is believed that the results of this study would be very useful for ear morphology and reconstructive surgeries. key words: external ear (auricle); bayelsa ijaws; anthropometry; sexual dimorphism; lobular attachment. corresponding author: azibaediya m. eghoi, m.sc.,department of human anatomy, faculty of basic medical sciences, abia state university, uturu, abia state, nigeria e-mail azibsmil32@yahoo.com introduction anthropometry refers to the branch of anthropology concerned with comparative measurements of the human body for the purpose of understanding of physical variations in humans [1]. these body measurements have been shown to vary according to sex, age and race, thus the study of these physical variations is of utmost importance in plastic surgery, prosthetics, clothing designs, and ergonomics [2-3]. it is also important for development of personal individuality [2]. the human external ear is an important organ contributing to the esthetics of human face. its size, shape, position, and projection influence the facial appearance of the individual [4]. it consists of external, middle and internal parts. the external ear is made up of a shell-like auricle (pinna), which collects sound, and the external acoustic meatus, which conducts s o u n d f ro m t h e p i n n a t o t h e t y m p a n i c membrane [5]. the auricle, helical in shape, is made up of an elastic cartilage covered by skin and has several depressions and elevations. the lobule (earlobe) of the auricle is a tag of skin containing fibrofatty tissue and blood vessels and is easily pierced for taking blood samples and inserting earrings [5,6]. it is the last part of the auricle to develop [7]. earlobe attachments were mainly classified into attached and free (unattached) earlobes [8-12]. many authors, however, have pointed out that there are many people with intermediate (or tapering) earlobes [13-14]. individuals having earlobes that hang freely are known to have free (unattached) earlobes, whereas those having earlobes fused with the sides of proximal head are termed as attached (or adherent) earlobes [15-16]. several anthropometric studies on ear measurements have been carried out on different populations. according to the studies international journal of medicine and medical research 2019, volume 5, issue 1, p. 75-83 copyright © 2019, tnmu, all rights reserved e.i. edibamode et al. 76 b io m e d ic a l s c ie n c e s issn 2413-6077. ijmmr 2019 vol. 5 issue 1 carried out in india and urhobo people of southern nigeria, females were found to have a statistically higher lobular length and width compare to males [3,17]. the studies in american, italian, indian, israeli and turkish populations suggested that boys and men had larger ears than girls and women [8, 19-26]. similar studies have shown that auricular dimensions in northern nigeria are higher in males compare to females [27]. the reports by sharma [3], however, documented that ear dimensions were higher in males than females [3,17]. the studies by wang, et al [18] of a chinese population, however suggested that lobular dimensions were not significantly different in both genders. despite a large number of world publications on the anthropometric measurement of the external ear, the studies correlating the dimensions of the external ear and sexual dimorphism in adult nigerian population show paucity of information. thus, the purpose of this study is to determine sexual dimorphism in mean ear dimensions and earlobe attachments among adult ijaws in bayelsa state of nigeria. previous studies on the morphometric analysis of the external ear have been carried out previously in several countries including nigeria, however, the studies correlating the dimensions of the external ear and sexual dimorphism in adult bayelsa ijaws show paucity of information and have necessitated the need for this study. hence the present study aims to ascertain the anthropometry of the external ear among adults ijaws in bayelsa state of nigeria. the objectives of this study are: to determine the mean dimensions of ear length (el), ear width (ew), lobular length (ll) and lobular width (lw) among bayelsa ijaws. to ascertain if there may exist a sexual dimorphism among the parameters mentioned above. to ascertain if there is any correlation in the measured parameters of the external ear in sexual dimorphism. to determine the variation in attachment of earlobe among ijaws. methods the study involved the subjects from various local governments in bayelsa state: yenagoa, sagbama, southern ijaw, nembe, k o l o k u m a , e k e r e m o r a n d o g b i a l o c a l government area. the study was conducted on adult ijaws, whose parents were indigenes of bayelsa state in nigeria up to their second generation. the sampling method used for the selection of subjects was a simple random sampling method. a total of 112 subjects were involved: 59 males and 53 females. only the subjects within the age range of 18-50 years of the ijaw ethnic group in bayelsa state without anomalies to their external ears comprised this study. the subjects with ear defects and those with previous history of ear surgery were excluded from the study. also the subjects, who had not given their consent, were also excluded from the study. the procedure for ear measurements was explained to the subject in order to get maximum cooperation. with the subject’s head in frankfort horizontal plane, direct measurements were taken using a digital vernier caliper with resolution of 0.01 mm as follows [3]. the anthropometric parameters of the external ear measured were (fig. 1): total length of ear (tle) – measured as the distance between the superior projection of the helix and inferior projection of the external ear (ab). total width of ear (twe) – measured as the distance between the anterior and superior points of the external ear (cd). total lobular width (tll) – measured from the midpoint of the intertragic notch to the lowest point of the lobule (gb). fig.1. photograph of the lateral surface of the auricle displaying the measured parameters. notes: ab – total length of ear; cd – total width of ear; bg – total lobular length; ef – total lobular width. e.i. edibamode et al. 77 b io m e d ic a l s c ie n c e s issn 2413-6077. ijmmr 2019 vol. 5 issue 1 total lobular width (tlw) – measured as the transverse distance of the ear lobule passing through the center of the length of the lobule (ef). data analysis was performed using statistical package for social sciences (spss) version 20.0 with results expressed as mean and standard error of mean (mean ± se). the student independent sample t-test with a twotail distribution was applied to compare the ear measurements between males and females in the study population. p-values, less than 0.05 (p<0.05) were considered to be significant. chisquare test was used to determine the relationship between gender and left lobular attachment of the population. pearson’s correlation coefficient test was conducted to determine the symmetry between the right and left side ear measurements of each gender and for the study population. correlation coefficient (r value) was interpreted as follows: a. <0.2 – a slight correlation b. 0.2–0.4 – a weak correlation c. 0.4–0.7 – a moderate correlation d. 0.7–0.9 – a high correlation e. >0.9 – an almost perfect correlation results the distribution of the subjects involved in the study according to the various local government areas of bayelsa state is presented in table 1. table 1. detailed analysis of the subjects according to gender and local government areas (lga) in bayelsa state. s/n lga gender female male 1 ekeremor 11 6 2 kolokuma 4 6 3 nembe 9 2 4 ogbia 2 4 5 sagbama 5 11 6 southern ijaw 15 17 7 yenagoa 8 12 total 54 58 seventeen of the subjects hailed from ekeremor, 10 from kolokuma, 11 from nembe, 6 from ogbia, 17 from sagbama, 20 from yenagoa, 27 from southern ijaw local government area of bayelsa state. the result prove that out of the total number of the subjects, who consented and participated in the study, table 2. general characteristics of the study population (n=112) variables mean sem sd range min max variance age 26.69 0.71 7.56 32.00 18.00 50.00 57.08 lel 58.02 0.38 4.02 30.34 39.77 70.11 16.12 lew 27.43 0.36 3.86 36.33 19.97 56.30 14.91 lll 14.93 0.21 2.18 11.12 9.12 20.24 4.76 llw 13.47 0.25 2.69 16.85 4.40 21.25 7.24 rel 57.56 0.58 6.16 53.20 16.20 69.40 37.96 rew 27.87 0.47 4.98 39.78 19.98 59.76 24.77 rll 14.92 0.22 2.34 13.28 9.56 22.84 5.47 rlw 13.15 0.23 2.45 10.68 7.60 18.28 6.02 notes: rel – right ear length; lel – left ear length; rew – right ear width; lew – left ear width; rll – right lobular length; lll – left lobular length; rlw – right lobular width; llw – left lobular width; sem – standard error of mean; sd – standard deviation. table 3. comparison of the measurements of the study population (n=112). variable n t p inference left ear length-right ear length 112 0.788 0.433 ns left ear width-right ear width 112 -0.813 0.418 ns left lobular length-right lobular length 112 0.004 0.997 ns left lobular width-right lobular width 112 2.011 0.047 ns notes: p – the statistical significance was determined using paired sample t-test; p<0.05 – significance between mean values; n – sample size; ns – not significant; s – significant. e.i. edibamode et al. 78 b io m e d ic a l s c ie n c e s issn 2413-6077. ijmmr 2019 vol. 5 issue 1 54 are females and 58 – males (table1). the mean values, standard deviation, standard error and variance for each variable of each ear measured in the study population (n=112) are enlisted in table 2. the results also proved that values for the right ear measurements did not significantly differ (p>0.05) from that on the left (tables 2 & 3) for the study population. the results of the population data subjected to chi-square analysis at 95% and 99% confidence interval is presented in table 4. the results attained proved that there was a weak correlation (r=0.3) in the left and right ear length of the study population (n=112). the p value for the correlations was found to be <0.05 (p=0.001), hence the correlation was statistically significant. left and right ear width however showed a slight correlation, which was not significant (p=0.073; r=0.2). significant correlations were found in the study population between the right and left side of lobular length (p=0.000; r=0.7) and that of lobular width (p=0.000; r=0.7). in tables 5, 6, and 7, four important indices (ear width, ear length, lobular length and lobular width) were assessed and compared in both and within the sexes. it was evidenced that females had a significantly larger dimensions (p<0.05) of left lobular length and right lobular length compare to that of males (table 5). the dimensions for left lobular length and right lobular length were 15.41±0.31 and 15.58±0.29 in females, whereas in males – 14.47±0.27 and 14.32±0.31 respectively. apart from the left and right lobular length that proved higher mean values in females, the mean values of other parameters were not consistently higher (p>0.05) in one gender with respect to a side (table 5). within the male population, the right ear measurements did not significantly statistically differ (p>0.05) from that on the left (table 6). table 4. pearson correlation of right and left side measurements of the study population (n=112). variable n r r2 p left ear length-right ear length 112 0.307 0.094 0.001** left ear width-right ear width 112 0.170 0.029 0.073 left lobular length-right lobular length 112 0.654 0.428 0.000** left lobular width-right lobular width 112 0.799 0.638 0.000** notes: n – sample size; r – coefficient of correlation; p – statistical significance; superscript * – correlation at 0.05 level (p˂0.05); ** – correlation at 0.01 level (p<0.01). table 5. comparison of ear measurements between the male and female study population. variable sex n mean±se t value p value inference left ear length male 58 58.14±0.60 0.318 0.751 ns female 54 57.90±0.46 left ear width male 58 27.41±0.37 -0.055 0.956 ns female 54 27.45±0.65 left lobular length male 58 14.47±0.27 -2.327 0.022 s female 54 15.41±0.31 left lobular width male 58 13.50±0.34 0.125 0.901 ns female 54 13.43±0.38 right ear length male 58 58.40±0.45 1.505 0.135 ns female 54 56.66±1.10 right ear width male 58 28.21±0.68 0.747 0.457 ns female 54 27.51±0.65 right lobular length male 58 14.32±0.31 -2.954 0.004 s female 54 15.58±0.29 right lobular width male 58 13.04±0.32 -0.508 0.613 ns female 54 13.28±0.34 notes: values are presented as mean±se; p – statistical significance was determined using independent sample t-test; p<0.05 – statistical significance between mean values; se – standard error; n – sample size; ns – not significant; s – significant. e.i. edibamode et al. 79 b io m e d ic a l s c ie n c e s issn 2413-6077. ijmmr 2019 vol. 5 issue 1 similarly, the right ear indices (right ear length, right ear width, right lobular length, right lobular width) did not statistically signifi cantly differ (p>0.05) from that of the left (left ear length, left ear width, left lobular length, left lobular width) in female population (table 7). tables 8 and 9 present the relationship between the lobular attachments of the ear in males and females subjects of ijaw ethnicity. the proportion of the male subjects, whose left lobular attachment is either attached, free (unattached) or intermediate, were 22 (19.6%), 17 (15.2%) and 19 (17.0%) respectively (table 8). for female subjects, the proportions were 27 (24.1%), 8 (7.1%) and 19 (17.0%) respectively. results proved no significant relationship between the left lobular attachment and gender (x2=3.612; p=0.164). the proportion of the male subjects, whose right lobular attachment is either attached, free or intermediate, were 21 (18.8%), 11(9.8%), and 26 (23.2%) respectively (table 9). on the other hand, the proportion of the female subjects, whose right lobular attachment was either attached, free or intermediate, were 14 (12.5%), 6 (5.4%), and 34 (30.4%) respectively. there was no significant table 6. comparison of measurements in males (n=58). variable n t p inference left ear length-right ear length 58 -0.443 0.660 ns left ear width-right ear width 58 -1.180 0.243 ns left lobular length-right lobular length 58 0.543 0.589 ns left lobular width-right lobular width 58 1.920 0.060 ns notes: p – statistical significance was determined using paired sample t-test; p<0.05 – significance between mean values; n – sample size; ns – not significant; s – significant. table 7. comparison of the left and right ear measurements in females (n=54). variable n t p inference left ear length-right ear length 54 1.189 0.240 ns left ear width-right ear width 54 -0.660 0.948 ns left lobular length-right lobular length 54 -0.813 0.420 ns left lobular width-right lobular width 54 0.800 0.427 ns notes: p – statistical significance was determined using paired sample t-test; p<0.05 – statistical significance between mean values; n – sample size; ns – not significant; s – significant. table 8. relationship between gender (sex) and left lobular attachment of the population (n=112). sex left lobular attachment total chi-square analysis attached free intermediate df x2 p-value male 22 (19.6%) 17 (15.2%) 19 (17.0%) 58 (51.8%) 2 3.612 0.164 female 27 (24.1%) 8 (7.1%) 19 (17.0%) 54 (48.2%) total 49 (43.8%) 25 (22.3%) 38 (33.9%) 112 (100.0%) notes: the values are presented as frequencies and percentages; p<0.05 – the association (or relationship) between variables. table 9. relationship between gender (sex) and right lobular attachment of the population (n=112). sex left lobular attachment total chi-square analysis attached free intermediate df x2 p-value male 21 (18.8%) 11(9.8%) 26 (23.2%) 58 (51.8%) 2 3.799 0.150 female 14 (12.5%) 6 (5.4%) 34 (30.4%) 54 (48.2%) total 35 (31.3%) 17(15.2%) 60 (53.6%) 112 (100.0%) notes: the values are presented as frequencies and percentages. p<0.05 means there is an association (or relationship) between variables. e.i. edibamode et al. 80 b io m e d ic a l s c ie n c e s issn 2413-6077. ijmmr 2019 vol. 5 issue 1 relationship between gender and right lobular attachments (x2=3.799; p= 0.150). discussion the current study presents anthropometric data regarding the external ear measurements for individuals of bayelsa state, nigeria. gender (sexual) dimorphism was presented in the study as regards the measurement of lobular length. the results revealed that lobular length (left and right) were significantly higher (p<0.05) in female subjects compared to that in males. these findings are supported by other studies carried out involving india and urhobo people of southern nigeria, where females were also found to have a statistically higher lobular length than males [3,17]. our findings however contradict with reports in northern nigeria which showed that lobular dimensions were higher in males compare to females [27]. when the mean value for lobular length, measured on both sides with respect to gender, of our study was compared with other anthropometric studies in nigeria, it was evidenced that urhobo people of southern nigeria had similar mean values, while the residents of maiduguri in northern nigeria had lower values. this implies that within nigeria, people of different ethnicity and geopolitical zones have different dimensions of lobular length. the findings by wang et al. [18], in a chinese population, however suggested that lobular dimensions were not significantly different in the two genders. the disparity in mean ear dimensions, across subjects in nigeria as well as other countries of the world, could be attributed to differences in social and ethnic background. the data of the present study was analyzed using pearson correlation and it was observed that all parameters on both sides of the ear proved to have a positive linear relationship, which was highly significant (p<0.05). this was similar to the findings of ekanem et al. [4] and eboh [17]. analyzing the data with regards to sex of the subjects, it was observed that ear length and width did not vary significantly (p>0.05) between males and females. our findings however contradict with the reports in northern nigeria, which showed that auricular dimensions were higher in males compare to females [4]. similarly, our findings also contradicts with reports of azaria et al. [8], sharma et al. [9], brucker et al. [19], bozkir et al. [22] purkait and singh [27], ito et al. [28], and meijerman et al. [29], who suggested that males have longer ears compare to females. the disparity in mean ear dimensions across the subjects in different countries of the world could be associated with difference in social and ethnic background. various authors have studied the attachment of ear lobules in different populations and its significance in population genetics [11, 15]. the results of the present study, from data analyzed using chi-square test, revealed no association ( p > 0 . 0 5 ) b e t w e e n g e n d e r a n d l o b u l a r attachment for both ears. the frequency of attached, free and intermediate ear lobule in ijaw population of bayelsa state, nigeria, were 43.8%, 22.3% and 33.9% respectively for the left ear and 31.3%, 15.2% and 53.6% respectively for the right ear. in other words, attached and intermediate ear lobules were found to be the predominant (p>0.05) type of ear lobule in the ijaw population of bayelsa state, nigeria. our study corroborates with findings by munir et al. [15], shah et al. [30], kim et al. [31], who revealed a higher frequency of attached and intermediate ear lobules in japanese/chinese and indian population respectively. in a similar research carried out by munier et al. [15], it was proved that no association (p>0.05) existed between ear lobular attachment and gender. the present study however differs from the findings by nwaopara et al. [32] and singh and sengupta [33], who have reported a higher frequency for free (unattached) ear lobule (63.4%) amongst the population of ekpoma, nigeria and assamese, india respectively. conclusions the present anthropometric study on ear measurements revealed a significantly higher lobular length in females compare to males, thus sexual dimorphism was established as regards the measurement of lobular length in ijaws population. other parameters such as ear length, ear width, lobular width, and lobular attachment did not exhibit significant gender differences in the study population. there was a high correlation in the dimensions of ear length, lobular length, and lobular width of the pinna between the right and left sides in the study population. it is believed that the results of this study would be very useful in ear morphology and reconstructive surgeries. acknowledgements authors are thankful to the management and staff of bmh for assisting them at different stages of the study. special thanks to the entire e.i. edibamode et al. 81 b io m e d ic a l s c ie n c e s issn 2413-6077. ijmmr 2019 vol. 5 issue 1 members of staff of the department of anatomy for their technical input and assistance. conflict of interest the authors declare no conflict of interest. антропометричні дослідження зовнішнього вуха серед дорослого населення іджо штату байелса у нігерії e.i. edibamode1, k. mordi1, l.k. david1, a.m. eghoi2 1 – university of port harcourt, port harcourt, nigeria 2 – abia state university, uturu, nigeria вступ. вимірювання зовнішнього вуха є надзвичайно важливим у морфології вуха та реконструктивній хірургії. мета роботи – дослідити статевий диморфізм у антропометрії зовнішнього вуха (довжина вуха, ширина вуха, висота вушної сережки (мочки), ширина мочки) та прикріплення мочки серед дорослого населення етнічної групи іджо у штаті байелса, нігерія. методи. обстежено 112 дорослих віком від 18 до 50 років, які відповідали критеріям дослідження. чотири лінійних виміри зовнішнього вуха, а саме довжина вуха (дв), ширина вуха (шв), довжина мочки (дм) та ширина мочки (шм) були проведені у досліджуваних осіб обох статей. прикріплення мочки у чоловіків та жінок також фіксувалося. отримані дані обробляли з використанням t-критерію стьюдента, критерію хі-квадрата кореляції пірсона за допомогою програмного забезпечення spss версії 20.0. результати. середні величини для досліджуваних показників дв, шв, дм,шм склали у чоловіків та жінок 58,14±0,60, 27,41±0,37, 14,47±0,27, 13,50±0,34 та 57,90±0,16, 27,45±0,65, 15,41±0,31, 13,43±0,38 відповідно. однак для правого вуха у чоловіків і жінок ці показники були 58,40±0,45, 28,21±0,68, 14,32±0,31, 13,04±0,32 та 56,66±1,10, 27,51±0,65, 15,58±0,29, 13,28±0,34 відповідно. статистично достовірні відмінності у розмірах були лише у показника довжини лівої і правої мочки у жінок відносно до чоловіків (p<0.05). коефіцієнт кореляції пірсона між параметрами правого та лівого вуха був достовірним та позитивним. за критерієм xі-квадрата не було знайдено достовірних взаємозв’язків (p>0.05) у прикріпленні мочки вуха у різних статей. висновки. проведене нами дослідження дозволило встановити статеві відмінності у довжині мочки вуха у популяції іджо. ці дані будуть корисними при вивченні морфології вуха та при проведенні реконструктивних втручань на вусі. ключові слова: зовнішнє вухо(auriclа); іджо штату байелс; антропометрія; статевий диморфізм; прикріплення мочки вуха. information about authors ezon-ebidor i. edibamode – phd, department of human anatomy, faculty of basic medical sciences, university of port harcourt, choba,port harcourt, rivers state, nigeria. e-mail: innocent.edibamode@uniport.edu.ng kurotimi mordi – b.sc, department of human anatomy, faculty of basic medical sciences, university of port harcourt, choba, port harcourt, rivers state, nigeria. e-mail: morditimi@gmail.com lekpa k. david – phd, department of human anatomy, faculty of basic medical sciences, university of port harcourt, choba, port harcourt, rivers state, nigeria. e-mail: lekpa.david@uniport.edu.ng azibaediya m. eghoi – m.sc, department of human anatomy, faculty of basic medical sciences, abia state university, uturu, abia state, nigeria. e-mail: azibsmil32@yahoo.com e.i. edibamode et al. 82 b io m e d ic a l s c ie n c e s issn 2413-6077. ijmmr 2019 vol. 5 issue 1 references 1. stedman tl. stedman's medical dictionary, 27th ed. baltimore: lippincott williams and wilkins; 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m, sueno k, arikuni t, kida a, et al. a morphological study of age changes in adult human auricular cartilage with special emphasis on elastic fibers. laryngoscope 2001;111:881-6. doi: 10.1097/00005537-200105000-00023 e.i. edibamode et al. 83 b io m e d ic a l s c ie n c e s issn 2413-6077. ijmmr 2019 vol. 5 issue 1 29. meijerman l, van der lugt c, maat gj. crosssectional anthropometric study of the external ear. j forensic sci. 2007;52:286-93. doi: 10.1111/j.1556-4029.2006.00376.x 30. shah a, fareed m, hussain m, afzal m. phylogenetic relationships of muslim populations of manipur based on morphogenetic markers. physical anthropology. 2012;8:463-480. 31. kim ke, song wj, kim dk. reevaluation of the earlobe types in koreans. pubmed.2018; 69(6): 377380. doi: 10.1016/j.jchb.2018.10.003 32. nwaopara ao, anibeze cip, akpuaka fc, agbotaen of. morphogenetic traits combination pattern amongst the population of ekpoma, nigeria: focus on tongue rolling, ear lobe attachment, blood groups and genotypes. african journal of biotechnology. 2008;7:3593-3598. 33. singh j, sengupta s. some morphogenetic and behavioural traits among the assamese sikhs. anthropologist. 2004;6(4):253-255. doi: 10.1080/09720073.2004.11890862 received 03 april 2019; revised 24 april 2019; accepted 15 may 2019. this is an open access article distributed under the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. e.i. edibamode et al. issn 2413-6077. ijmmr 2018 vol. 4 issue 2 59 d e n t is t r y doi 10.11603/ijmmr.2413-6077.2018.2.9650 dynamics of periodontal tissues microbiocenosis under the complex treatment of catarrhal gingivitis and chronic gastroduodenitis in the adolescents i. s. lisetska, m. m. rozhko, r. v. kutsyk ivano-frankivsk national medical university, ivano-frankivsk, ukraine background. the key links in the etiology and pathogenesis of periodontal tissue diseases are the quantitative and qualitative changes in the composition of the microflora of oral cavity, with the simultaneous deterioration of oral hygiene, reduction of local and general immunity, which occurs more often in the presence of somatic diseases. objective. the aim of the research was to determine the clinical and microbiological efficacy of the developed treatment-prophylactic complex in the adolescents with catarrhal gingivitis and chronic gastroduodenitis before and after treatment. methods. changes were made to and before the treatment of clinical parameters, gingival microbiocenosis of 38 adolescents with generalized catarrhal gingivitis and chronic gastroduodenitis aged 12-18 years old, who comprised the main group. in the comparison group 25 adolescents of similar age diagnosed with generalized catarrhal gingivitis without any somatic diseases were involved. results. it has been established that used combination (drug of plant origin with antimicrobial properties + dental gel with metronidazole benzoate and chlorhexidine digluconate + capsules of probiotics) yields the conventional treatment as well as exceeds it for examined clinical indicators and indexes. the treatment and prophylaxis with suggested complex have proved a significant positive effect on the gums microbiocenosis in adolescents with generalized catarrhal gingivitis and underlying concomitant gastroduodenitis. conclusions. the suggested therapeutic and prophylactic complex provides a reduction in the massiveness and colonization frequency of the gum mucosa by pathogenic aerobic microflora (β-hemolytic streptococcus, golden staphylococcus, and yeast-like candida fungi). key words: catarrhal gingivitis; chronic gastroduodenitis; adolescents; microbiocenosis; complex treatment. introduction recent epidemiological studies prove a high intensity and prevalence of periodontal tissue diseases in childhood. according to many authors, children are mainly diagnosed with chronic catarrhal gingivitis, which prevalence reaches 90% [6, 13, 19, 20]. according to the contemporary concept, the development of periodontal tissue diseases is closely related to the microflora of the oral cavity, that is the reduction of the number of normal flora, the increase of conditionally pathogenic microorganisms, excessive indigestion and infection with periodontopathogens with simultaneous deterioration of oral hygiene, reduction of local and general immunity in the presence of somatic diseases are key links of etiology and pathogenesis of the disease [1, 4, 11, 12, 16]. at the present time, the normophyll of the human body is considered to be a combination of microbiocenosis, which is part of a holistic system that performs the most important functions in the body: it is a supplier of biologically active substances, a powerful metabolic and detoxification body, it determines the formation of the overall immunological status of the organism and local immunity, and most importantly creates an anterior line of nonspecific microorganism protection [3, 17, 22, 23]. the oral microbiome is one of the most complex and diverse microbial communities in the human body. in the development of dysbiosis disorders of the organism, conditionally pathogenic microbes prevail, among which are clones with medicinal resistance and genetic determinants that determine the virulence and pathogenicity of bacteria, but it is established that streptococci (representatives of normal flora) in the stage of primary inflammation of international journal of medicine and medical research 2018, volume 4, issue 2, p. 59-66 copyright © 2018, tsmu, all rights reserved corresponding author: irina s. lisetska, ivano-frankivsk national medical university, 2 galitska st., ivano-frankivsk 76000, ukraine phone 0679275100 e-mail: lisecka9@gmail.com i. s. lisetska et al. issn 2413-6077. ijmmr 2018 vol. 4 issue 260 d e n t is t r y the gum are significant in the development of a pathological process, for example, the fixation of p. gingivalis and p. intermedia on the gum surface occurs after the appearance on these areas of streptococcus mitis and streptococcus sanguis, which contribute to attachment of parodontal microflora forming an intermediate layer between them and the outer membrane of epithelial cells [8, 14, 18]. the issue of creation of effective integrated treatment schemes is urgent, due to defeats in treatment, lack of a stable clinical effect, presence of relapses, resulting in a one-way approach to treatment without considering the features of the existing microflora, characteristics of local resistance and general condition of the organism [2, 10, 21]. the aim of the research was to determine the clinical and microbiological efficacy of the treatment and prophylactic complex aimed at correction of microbiocenosis of periodontal tissues in the adolescents with catarrhal gingivitis and chronic gastroduodenitis by clinical and microbiological monitoring before and after treatment. methods a clinical dental examination of 38 adolescents with generalized catarrhal gingivitis and chronic gastroduodenitis aged 12-18 years old, who comprised the main group, was conducted (group 1). in the comparison group, 25 adolescents of the same age diagnosed with generalized catarrhal gingivitis, who, at the time of the survey, had no complaints of violations of somatic health and were not on the dispensary records of related specialists, were involved (group2). as a control, similar studies were performed in 20 adolescents of the same age without any signs of gum inflammation and somatic diseases. clinical examination of the adolescents was carried out according to the generally accepted technique using subjective and objective methods. the received data of each patient was entered in an outpatient documents of a dental patient and the record of our examination developed by us. at an objective dental examination of the patients, the depth of the pride of oral cavity and the features of the attachment of bridles, bite, tooth row and its integrity, the presence of seals and their condition were studied. particular attention was paid to the condition of the gums: color (pale pink, hyperemia, cyanosis), relief of the gingival margin (exacerbation of the peaks of gingival papillae, conical incision, swollen papillae) and consistency (normal tone, edema, pastiness), a kind of gingivitis, prevalence. the evaluation of the condition of gums around the tooth was carried out by sounding a periodontal probe. the index score was used to determine the initial state of periodontal tissues in the pre-existing groups and during monitoring after treatment. in order to assess the oral hygiene status, all patients were given the simplified oral hygiene index (green-vermillion, 1964), which allowed them to detect not an only plaque but also a tooth_brush. to evaluate the inflammatory process in gums, the pma index (papillary-marginal-alveolar index by c. parma, 1960) was used. to establish the diagnosis and prognosis of the treatment of periodontal tissue diseases pa pillary bleeding index (saxer and muhlemann, 1975) was used. simultaneously, microbiological studies were carried out on the contents of the toothasparagus furrow. a material for bacteriological examination for revealing of aerobic and extraanaerobic microflora from the tooth-asparagus was conducted on tooth brushing, using a calibrated bacteriological loop no.1 on blood agar, endo medium, and a potassium-iodinestarch indicative medium system (for identification of producers of hydrogen peroxide) and it was delivered to a microbiological laboratory within an hour. plating was per formed by the gold method [9]. the seeds were incubated for 1 day at 37 °c under aerobic and anaerobic conditions (in a hermetically sealed desiccator) in an atmosphere enriched with co2. the bacteriological examination was carried out in order to isolate pure cultures of microorganisms and their identification according to generally accepted microbiological methods for bacteria identification [5]. identification of the isolated pure cultures was carried out by a complex of morphological, cultural and biochemical methods (streptotest 16, staphytest 16, erba lachema, czech republic). quantitative records of colonies were conducted according to their species (or generic) affiliation. the results of the quantitative study of microflora were registered in colony-forming units, converted to 1.0 ml – cfu/ml, taking into account only those microorganisms which concentration in the specimen was not less than 1×103 cfu/ml. based on the analysis of crop yields for each group, the population level pl (lg cfu/ml) and the continuous index (ci) [5] were determined. integrated therapy of gcg was carried out in accordance with the protocols approved by i. s. lisetska et al. issn 2413-6077. ijmmr 2018 vol. 4 issue 2 61 d e n t is t r y the order of the ministry of health of ukraine no.566 dated 23.11.2004 “on approval of protocols for the provision of medical aid to children for the specialty pediatric therapeutic dentistry”. the patients of the main group and the comparison group were divided into a and b subgroups according to treatment schemes. patients of 1a and 2a subgroups were prescribed a combined herbal antimicrobial medicine in the form of paddling with 15% aqueous solution (about 10 ml of the preparation dissolved in ¼ cups of water) of the oral cavity 3-4 times a day, application to the gum mucosa and introduction into the interdental dentately gaps 2 times a day. combined herbal antimicrobial medicine is a mixture of a mixture of chamomile flowers, oak bark, sage leaves, arnica herbs, ayer rhizomes, peppermint herbs, and thyme grass. for general treatment, probiotics (capsules of yogurt) were prescribed 1-2 capsules 3 times a day with a meal. for local treatment of the patients of 1b and 2в subgroups, irrigation of the gums was used with 0.05% chlorhexidine digluconate solution, herbs (chamomile, calendula) 3-4 times a day for 7 days, applications on the gum mucosa and insertion into the interdental gaps’ ointment with mefenamic acid 2 times a day. the course lasted 10 days. the data were expressed as the mean±standard error of the mean (m±m). probability values with p<0.05 were considered statistically significant. the distribution of indices was estimated by using the shapiro-wilk normality test. the statistical significance of the differences between means was assessed by student’s t-criterion using statistica 5.0 (statsoft, usa). the research was carried out in accordance with the principles of the helsinki declaration. the protocol of the study was approved by the local ethics committee (lec) of all institutions mentioned in the work. in accordance with the requirements of bioethics “on conducting laboratory research of biological material”, written consent was received from the parents (guardians) of each child and the adolescents for the study of biomaterials. results according to the results of the clinical examination, the prevalence of catarrhal gingivitis in the adolescents of the main group was higher than in the comparison group, 69.8% versus 52.7%, respectively. the course of gingivitis in the patients of the main group in most cases was chronic or in the stage of aggravation, of moderate severity, with the main complaint of bleeding gums. in the comparison group, mild chronic catarrhal gingivitis was predominantly diagnosed. the pma index evidenced that the degree of severity of gingivitis was higher in the adolescents of the main group – 36.8±1.21%, which corresponds to the average severity of gingivitis, 19.2±1.07% in the adolescents of the comparison group, which corresponds to mild gingivitis. the mean value of bleeding index was 1.23±0.01 points in the main group and 0.8±0.01 points in the comparison group. the correlation between the level of oral hygiene and the prevalence of inflammatory events in periodontal tissues was defined. analyzing the results of oral hygiene state, it was found that the average value of hygiene index in the adolescents of the main group and the comparison group was a satisfactory and unsatisfactory condition of the oral cavity. thus, in the adolescents of the main group, the average index was 1.76±0.03 points, in the comparison group – 1.32±0.03 points. after the treatment, oral hygiene improved by 0.31±0.04 points and 0.17±0.02 points in the main and in the comparison group respectively, which corresponded to a good oral hygiene condition. at the end of the course of complex treatment and elimination of clinical manifestations of the disease, the complaints in all adolescents were absent. gums were pale pink, of a denseelastic consistency, did not bleed when probing in the area of the tooth-spatula furrow. however, the adolescents of subgroups 1a and 2a, who received the suggested improved treatment, were more likely to have shortened treatment terms than the adolescent of subgroups 1b and 2b receiving traditional treatment. during the treatment, all patients of the main group, as well as the comparison group, proved a positive dynamic of the studied parameters. thus, the dynamics of the pma index tended to reduce the signs of inflammation: the value of the pma index after the end of treatment in the adolescents of the main group, subgroup 1a was 3.7±0.12% and the subgroup 1b – 6.8±0.14%. in the adolescents of the comparison group, subgroup 2a it was 1.6±0.08% and the subgroup 2b – 2.9±0.13%. a similar trend was evidenced in the study of the dynamics of the index of bleeding: the index after the end of the treatment course in the adolescents of the main group, subgroup 1a was 0.11±0.02 points and the subgroup 1b – i. s. lisetska et al. issn 2413-6077. ijmmr 2018 vol. 4 issue 262 d e n t is t r y 0.17±0.03 points. in the adolescents of the comparison group, subgroup 2a it was 0.07±0.01 points and the subgroup 2b – 0.17±0.02 points. however, in 6 months of monitoring, a slight worsening in clinical performance was evidenced in all groups, but in the adolescents of groups 1a and 2a, the increase in clinical indices was less significant than in the groups 1в and 2в. thus, the pma index for the adolescents of groups 1a and 2а in 6 months was 4.5±0.11% and 2.4±0.12% respectively, which was less than in the adolescents of groups 1в and 2в – 7.4±0.25% and 4.5±0.14% (p<0.01), respectively. the abnormal pattern was evidenced in the study of the iodine of gums bleeding: in the adolescents of groups 1a and 2а in 6 months it was 0.23±0.01 points and 0.18±0.01 points, respectively, which was less than in the adolescents of groups 1в and 2в – 0.31±0.01 points and 0.27±0.01 points (p<0.01), respectively. the initial microbiological examination proved that, compared to the control group, in the adolescents with generalized catarrhal gingivitis, both in the context of gastroduodenitis and without concomitant pathology, a higher level of colonization of gum mucus by the representatives of resident α-hemolytic streptococci (p<0.01) and transient microflora of the oral cavity: epidermal staphylococcus (p<0.05), stomatococcus (p<0.05), and corynebacterium (diphtheroids) (p<0.05), was evidenced. in addition, the presence of active inflammatory process on the gum mucosa is accompanied by a significantly higher level of colonization of the affected areas by staphylococcus aur eus (p<0.05), β-hemolytic streptococci (p<0.05), and yeast-like fungi of the genus candida. in this regard, a therapeutic and prophylactic complex aimed at correction of microbiocenosis of periodontal tissues (patients of subgroups 1a and 2a) was developed. the effectiveness of the suggested complex was evaluated by a comparison of the dynamics of microbiological parameters with patients of subgroups 1b and 2b, who received protocol traditional treatment. by quantitative indicators of microbiocenosis of gum mucosa (population level and index of the constancy of different microorganisms), obtained during the initial examination before treatment, the comparable subgroups of patients (1a and 2а, 1b and 2b) practically did not differ. the main representatives of the resident microflora of the oral cavity: α-hemolytic streptococci were plated in all patients without exception in all periods of the monitoring. however, both therapeutic complexes demonstrated significant reducing the massiveness of colonization of gum mucus by α-hemolytic streptococci (fig. 1). the most significant dynamics was evidenced after the treatment in the patients of subgroups 1a and 1b: population-level decreased by 6.43±0.15 lg cfu/ml to 4.72±0.13 lg cfu/ml (p<0.01) and from 6.27±0.15 lg cfu/ml to 5.7±0.15 lg cfu/ml (p<0.01), which corresponds to normal age indices in the control group without dental pathology. significant positive dynamics was also evidenced in the patients of subgroup 2b (population level decreased from 5.05±0.26 lg cfu/ml to 4.23±0.13 lg cfu/ml, p<0.05). however, in the latter case, pl α-hemolytic streptococci dipped below the age standard (4.74±0.31 lg cfu/ml). during a long-term after treatment period (in 6 months), the level of colonization of mucosal α-hemolytic streptococci in subgroups 1b and 2a increased again (p<0.05). the patients of the subgroup 1b did not experience any fig. 1. dynamics of population-level changes (pl) of α-hemolytic streptococci on gingival mucosa of the adolescents with gkg in the use of various therapeutic complexes. i. s. lisetska et al. issn 2413-6077. ijmmr 2018 vol. 4 issue 2 63 d e n t is t r y improvement; however, the α-hemolytic streptococcal fraction was below the age standard. in the patients of the subgroup 1a (patients with ketal gingivitis and underlying gastroduodenitis), the suggested therapeutic complex allowed achieving a stable normalization of this index (pl α-hemolytic streptococci 4.84±0.12 lg cfu/ml, p<0.01 compared to the pre-treatment stage). it should be noted that the suggested therapeutic complex also contributed to normalization of the species composition of α-hemolytic streptococci on gingival mucosa of the adolescents followed up. thus, before treatment of catarrhal gingivitis 68.4±3.32% of the patients of the main and 64.0±3.43% of the comparison group, streptococcus gordonii, strept ococcus sanguis, streptococcus constellatus, streptococcus anginosus were isolated (which, in comparison with other α-hemolytic streptococci, have wider sets of factors of virulence). the overwhelming majority (96.4±2.3%) of α-hemolytic streptococci cultures from dermato logically healthy individuals were defined as streptococcus salivarius and streptococcus mitis. after the treatment, in the patients with scar tissue of subgroups 1a and 2a, the latter in creased to 84.6±3.2% and 76.5±3.9% respectively. in the patients treated for gcg in the tra ditional way (subgroups of 1b and 2b), there was a significant decrease in the massiveness of colonization of gum mucosa by transient representatives of normal microflora of oral cavity: stomatococcus, non-series, and diphtheroids (table 1). in the long term after treatment period (in 6 months), low population levels of these representatives of normophyll in the patients of the subgroups 1b and 2b were still present. at the same time, they were lower than the age norm, which may evidence a stable deficit of minor representatives of normal microbiocenosis of the oral cavity. the dynamics of changes in the index of constancy (plating frequency) of these microorganisms in the subgroup 2b was similar (table 2). on the contrary, the massive colonization of gum mucus by epidermal staphylococci in the treatment of the patients of subgroups 1b and 1b, on the contrary, proved a tendency to increase (table 1). nevertheless, the used therapeutic measures allowed achieving a short-term decrease in the frequency of plating s. epi der midis from the gum in these groups (table 2). the suggested new therapeutic complex, which included milder local action of antiseptic agents in combination with probiotics, proved a gentler normalizing effect on germ microbiocenosis. in the patients of subgroup 1a (gcg with underlying gastroduodenitis) immediately after the course of treatment and in 6 months, the rate of pr and ie representatives of transient microflora of oral cavity was close to normal age values (tables 1 and 2). table 1. the massiveness of gingival mucosa colonization with transient representatives of the oral cavity normal microflora (population level, lg cfu/ml) during treatment of the adolescents with gcg patients subgroups s. epidermidis stomatococcus mucilaginosus neisseria sp. corynebacterium sp. control 3.78±0.21 3.39±0.08 3.57±0.22 3.00±0.05 gcg+gastroduodenitis 1 а before treatment 4.89±0.24 † 4.83±0.38 † 3.93±0.16 † 4.20±0.16 † after treatment 3.68±0.09 * 3.57±0.14 * 3.57±0.12 * 3.00±0.05 * in 6 months 3.89±0.24 * 3.68±0.15 * 3.50±0.13 * 3.47±0.09 */† 1 b before treatment 4.18±0.30 4.78±0.30 † 3.74±0.27 4.00±0.19 † after treatment 4.05±0.24 3.45±0.12 * 4.35±0.11 */† 3.00±0.05 * in 6 months 4.38±0.17 † 3.69±0.07 * 3.14±0.07 */† 3.50±0.16 */† gcg 2 а before treatment 3.43±0.15 4.03±0.13 † 4.13±0.27 † 3.85±0.06 † after treatment 3.43±0.24 3.73±0.11 */† 3.35±0.11 * 3.57±0.14 † in 6 months 3.60±0.17 3.83±0.04 † 3.35±0.14 * 3.60±0.12 † 2 b before treatment 3.94±0.27 4.76±0.27 † 3.85±0.35 3.80±0.25 † after treatment 3.90±0.25 3.57±0.15 * 3.00±0.05 */† 3.00±0.05 * in 6 months 4.02±0.12 3.73±0.11 */† 3.35±0.14 * 3.18±0.10 * notes: * – p<0.05 compared to the initial level in the corresponding subgroup (before treatment); † – compare to the control (dental-healthy adolescents without git comorbidity). i. s. lisetska et al. issn 2413-6077. ijmmr 2018 vol. 4 issue 264 d e n t is t r y in the patients of subgroups 1a and 2a, the new therapeutic complex allowed achieving a steady decrease in the massiveness and colonization frequency of gum mucosa by pathogenic aerobic microflora: β-hemolytic streptococcus s. pyogenes, s. aureus, golden staphylococcus, and candida genus yeast fungi. in the adolescents with gcg and underlying gastroduodenitis (subgroup 1a) immediately after treatment, the fact of a full disappearance of golden staphylococci from germ microbiocenosis was established. in six months after the treatment, it was revealed in only one patient (ci 5.3±1.2% with a minimum pl of 3.0 lg cfu/ ml). at the same time, a significant decrease in β-hemolytic streptococcus gum (ci 10.5±1.6%, p<0.05) was also evidenced compared to the initial levels as well as in yeast-like fungi of genus candida (ci 5.3±1.2%, p<0.05). in the patients with gcg without concomitant gastroduodenal disease (subgroup 2a), the suggested therapeutic complex promoted to the disappearance of yeast fungus from gum mucus, but this event was not longlasting. in 6 months in 2 patients (5.4±2.3%) the colonization of gums by candidiasis was present again with an average pr 3.35±0.14 lg cfu/ml. in the subgroup 1b (gcg in combination with gastroduodenitis), the traditional therapeutic complex did not allow achieving a significant decrease in gonadal colonization rates by pathogenic microbiota. discussion the attained results of clinical examination of the adolescents of the main group, who underwent the suggested comprehensive treatment of catarrhal gingivitis, proved a more significant positive dynamics of the indices compared to the adolescent of the comparison group. thus, the developed new therapeutic complex for treatment of adolescents with gcg and underlying concomitant gastroduodenitis proved a significant corrective effect on the nature of microbiocenosis gums. this allowed achieving a stable normalizing effect on the resident and transient normoflora and ensured a decrease in the proportion of pathogenic aerobic microorganisms in oral microbiocenoses of the examined adolescents. the analysis of the conducted microbiological study prove that the microbiocenosis of gum mucus is caused by increased colonization by representatives of resident α-hemolytic streptococci (p<0.01) and transient microflora of oral cavity: epidermal staphylococcus (p<0.05), stomatococcus (p<0.05) and corynebacteria (diphtheroids) (p<0.05), and also is accompanied by a higher level of colonization of the affected areas by staphylococcus aur eus (p<0.05), β-hemolytic streptococci (p<0.05) and yeast-like fungi of the genus candida, which confirms the established present concept of inflammatory diseases with underlying periodontal disease, which deals with the microbial factor triggering table 2. the frequency of plating of normal microflora transient representatives (continuous index, %) out of gingival mucosa of the adolescents with gcg during treatment patients subgroups s. epidermidis stomatococcus mucilaginosus neisseria sp. corynebacterium sp. control 30.0±3.3 45.0±3.6 15.0±2.6 10.0±2.1 gcg + gastroduodenitis 1 а before treatment 42.1±2.6 † 47.4±2.6 21.1±2.2 † 10.5±1.6 after treatment 26.3±2.3 * 52.6±2.6 */† 15.8±1.9 * 5.3±1.2 */† in 6 months 42.1±2.6 † 47.4±2.6 21.1±2.2 † 15.8±1.9 1 b before treatment 63.2±2.5 † 57.9±2.6 † 26.3±2.3 † 21.1±2.2 † after treatment 42.1±2.6 */† 31.6±2.5 */† 10.5±1.6 * 10.5±1.6 * in 6 months 52.6±2.6 */† 42.1±2.6 26.3±2.3 † 10.5±1.6 * gcg 2 а before treatment 53.9±3.8 † 53.9±3.8 † 23.1±3.2 † 15.4±2.8 after treatment 30.8±3.6 * 46.2±3.8 * 30.8±3.6 */† 23.1±3.2 */† in 6 months 61.5±3.7 */† 53.9±3.8 † 15.4±2.8 * 30.8±3.6 */† 2 b before treatment 41.7±4.1 † 50.0±4.2 † 16.7±3.1 25.0±3.6 † after treatment 25.0±3.6 * 25.0±3.6 */† 8.3±2.3 */† 8.3±2.3 * in 6 months 41.7±4.1 † 58.3±4.1 † 8.3±2.3 */† 33.3±3.9 */† notes: * – p<0.05 compared to the initial level in the corresponding subgroup (before treatment); † – compare to the control (dental-healthy adolescents without git comorbidity). i. s. lisetska et al. issn 2413-6077. ijmmr 2018 vol. 4 issue 2 65 d e n t is t r y inflammation in cases of periodontal disease, in children and adolescents [8,14]. therefore, the detail study of the changes of microbiocenosis in adolescents, to be precise at the initial stage of inflammatory development, is urgent; much attention should be paid to the constant factors that may contribute to a longterm development of the disease with its transition to a more severe degree as well as occurrence of relapses. in addition, the results of microbiological examination can be a diagnostic criterion for the effectiveness of treatment and prognostication of the subsequent course of the inflammatory process in gums. the dynamics of clinical parameters and their changes in microbial associations in the course of treatment confirms the necessity of a repeated course of treatment for the adolescents with concomitant somatic pathology and underlying chronic gastroduodenitis, as well as without any somatic pathology, in order to obtain stable results and prevent recurrence. the attained results are important for dental practice as well as for general pediatric practice, since the oral cavity is the initial part of gastrointestinal tract, periodontal tissues can be a reservoir for opportunistic and pathogenic microflora, and therefore cause not only periodontal tissue diseases but reinsert the lower sections of gastrointestinal tract and affect the course and results of treatment of common somatic diseases. consequently, the results prove the need for the development of the scheme of treatment and prophylactic complex of professional oral hygiene, hygiene training, monitoring the stable motivation to comply with individual oral hygiene in addition to drugs aimed at various pathogenesis links of the disease, as is also evidenced by other researchers [18]. conclusions the attained results allow us drawing a conclusion that a high clinical efficacy of the suggested complex, which contributes to a prolonged positive dynamics and stable changes in periodontal tissues at the early period of treatment, which is confirmed by positive changes in the indexes. the therapeutic and prophylactic complex for treatment of the adolescents with gcg has a steady corrective effect on normal gum microflora (the composition of α-hemolytic streptococci, their quantitative characteristics of colonization, population level (pl) and index of constancy (continuous index, ci) stomatococci, non-toxic, and diphteroids). the suggested treatment and prophylaxis complex reduce the massivity and colonization frequency of gum mucosa by pathogenic aerobic microflora (β-hemolytic streptococci, staphylococcus aureus, and yeast-like fungi of genus candida). динаміка змін мікробіоценозу тканин пародонта за умов комплексного лікування у підлітків з катаральним гінгівітом та хронічним гастродуоденітом і. с. лісецька, м. м. рожко, р. в. куцик івано-франківський національний медичний університет, івано-франківськ, україна вступ. якісні та кількісні зміни мікрофлори, нехтування гігієною ротової порожнини та ослаблення місцевого і системного імунітету, які частіше зустрічаються на фоні соматичної патології, відіграють ключову роль у етіології та патогенезі захворювань тканин пародонту. мета дослідження встановити клінічну та мікробіологічну ефективність застосування запропонованого лікувально-профілактичного комплексу у підлітків з катаральним гінгівітом та хронічним гастродуоденітом. методи. обстежено 38 підлітків віком 12-18 років з катаральним гінгівітом та хронічним гастродуоденітом (основна група). у групу порівняння включено 25 підлітків з катаральним гінгівітом без наявності супутньої соматичної патології. результати. встановлено, що застосування запропонованої нами для лікування комбінації (засіб рослинного походження з антимікробними властивостями, застосування дентального гелю з метронідазолом та хлоргексидином разом з капсулами пробіотика) значно перевищує за своєю i. s. lisetska et al. issn 2413-6077. ijmmr 2018 vol. 4 issue 266 d e n t is t r y received: 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