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9THE CHANGES OF INDICES OF L-ARGININE/NITRIC OXIDE/ARGINASE SYSTEM AND OXIDATIVE PROCESSES IN BLOOD PLASMA IN PATIENTS WITH DIABETIC NEPHROPATHY BEFORE AND AFTER HEMODIALYSIS
Ivanochko R.B.
nephrolog, Lviv Regional Hospital, researcher, Department of Biochemitfry, Danylo Halytsky Lviv National Medical University
Sklyarov O.Ya.
MD, Professor, Head of Department of Biochemitfry, Danylo Halytsky Lviv National Medical University
Hutnyk I.N.
nephrolog, Lviv Regional Hospital
ABSTRACT
The aim of research was to determine blood plasma changes of indices of lipid peroxidation processes, flatus of antioxidant system and interrelationship between the content of L-arginine, nitric oxide and arginase activity in patients with diabetic nephropathy.
Materials and methods of invefligations. We carried out clinical supervision and biochemical invefligations of blood plasma of 21 patients (males - 8, females - 13) with diabetic nephropathy, who receive renal replacement therapy by the method of hemodialysis 3 times per week (12 hours) in bicarbonate regimen. Average age of patients was 52 years. Comparison group included blood of 20 donors with an average age of 48 years.
Results of fludies. In donors' blood creatinine and urea concentration were within normal range (0.09±0.02 and 3.24±0.6 mmol/l relevantly). In patients with chronic kidney insufficiency concentration of creatinine and urea increased 7.2- and 7.5-fold relevantly (p<0.01). After hemodialysis creatinine concentration in blood plasma decreased for 57% (p<0.05) and urea - for 68% (p<0.05).
In blood plasma of donors L-arginine concentration was on the level of 93.3±19 ^g/ml, concentration of nitrite-anion made 23.6±2.3 ^mol/l and arginase activity - 0.24±0.06 ^mol/minmg (table 1).
In patients, L-arginine concentration decreased for 29% (p<0.001), a tendency to decrease of the content of nitrite-anion was noted and arginase activity in blood plasma increased. After hemodialysis L-arginine concentration decreased for 27% (p<0.001), nitrite-anion content and arginase activity had tendency to decrease compared to relevant indices before hemodialysis.
Conclusions. Increase of the nitrosooxidative flress in lymphocytes compared to control group was shown. Hemodialysis caused acute decrease of the activity of iNOS, eNOS, content of TBA-active products content and L-arginine in lymphocytes lysate of patients with diabetic nephropathy. Hemodialysis in patients with diabetic nephropathy resulted in the decrease of nitrosooxidative flress in lymphocytes, at that eNOS activity acutely diminished. Decrease of eNOS and L-arginine in lymphocytes after hemodialysis may cause decline of the immunologic defense in patients with diabetic nephropathy and affect duration of life.
Keywords: diabetic nephropathy, NO-synthase, arginase, TBA-active products, nitrite-anion, L-arginine, blood plasma.
Introduction. Recently increasing attention has been paid to the evaluation of the relationship between the progression of chronic kidney disease (CKD) and development of different complications with oxidative flress, that is characterized by disbalance between pro- and antioxidant syflems [18, p. 8, 21, p. 1]. Such fludies were conducted in patients, receiving renal replacement therapy (RRT) by the method of hemodialysis [9, p. 9] and peritoneal dialysis [25, p. 7].
Actually it was revealed that CKD should be considered not only as local damage of renal flructures but also as general disease of the organism, in which all kinds of metabolism alter, including trace elements turnover [20, p. 4].
High toxicity of the products of lipid peroxidation together with decreased activity of antioxidant defense on the background of the trace elements disorders result in the diflurbance of the integrity of renal cells membranes, disintegrate regenerative processes [22, p. 3].
In patients with CKD, who receive RRT by the method of hemodialysis oxidative flress and insulin resiflance occur. High levels of urea, which are detected in patients with CKD, exacerbate oxidative flress. Alteration of the response to insulin and glucose transport are one of the consequences of this. These changes are directly interrelated with the increase of resiflance
to insulin and increase of the content of adipokinins, associated with insulin resiflance [8, p. 8, 28, p. 5].
As it was eflablished that resiflance to insulin is an independent cardiovascular risk factor, therapeutic approaches directed on free radicals of oxygen and insulin resiflance induced by urea, potentially may be beneficial in decrease of the high morbidity and mortality, associated with the lafl flages of CKD [8, p. 8, 17, p. 4, 19, p. 6].
Peroxide oxidation of lipids (POL) is an assembly of important processes such as conflant renew of the lipid conflitution of cell membranes, support of the activity of lipiddependent receptors, synthesis of proflaglandins, oxidative phosphorylation in mithochondrias, phagocytosis etc. Under the conditions of the normal functioning of the organism, intensiveness of oxidative processes is relevantly low, what is mediated by the low level of the generation of POL products and balanced effect of the antioxidant defense syflem (ADS). However, accessive production of peroxide radicals, which interact with different cellular components - proteins, nucleic acids, lipids, produces high toxic hydroperoxides and new free radicals, that lead to the rapid damage of cell flructures and become the basis for pathogenesis of the chronic renal failure (CRF) [21]. Development of the inflammatory reaction causes activation of leukocytes and production of oxygen radicals by
leukocytes and resident cells. Produced active forms of oxygen initiate oxidative processes, damaging cells of kidney tissues. This results in the degradation of target cells with the production of Sable products of oxidative processes [26, p. 37].
CRF belongs to prevalent kidney diseases, that has unfavourable prognosis and high mortality rate. This pathology is characterized by disfunction of endotheliocytes, hypertension, increased content of cytokins, circulating in blood, changes of functions of blood cells, that are associated with uremia, oxidative flress and alteration of the syflem of L-arginine/NO-synthase/arginase [10, p. 16, 11, p. 1].
In patients with diabetic nephropathy, who receive RRT by the method of hemodialysis decreased concentration of amino acid L-arginine is noted, which is the subflrate for NO-synthases and arginase [3, p. 9, 29, p. 5]. The important role in the functioning of endothelial syflem and form elements of blood of patients with CRF belongs to the increase of oxidative processes and decreased activity of the antioxidant defense line [2, p. 4, 6, p. 6]. In addition to that in CRF patients decreased content of vitamin C is reported, which is an important antioxidant in plasma and cell cytoplasm [16, p. 6, 25, p. 7].
Aim of research was to evaluate changes in blood plasma indices of the processes of lipid peroxidation, flatus of antioxidant defense syflem and interrelationship between the content of L-arginine, nitric oxide and arginase activity in patients with CRF on the background of diabetes mellitus before and after dialysis.
Material and methods of invefligations. We carried out clinical supervision and biochemical invefligations of blood plasma of 21 patients (males - 8, females - 13) with diabetic nephropathy who receive RRT by the method of hemodialysis 3 times per week (12 h) in bicarbonate regimen. Average age of the patient was 52 years. Comparison group consifled of blood samples of 20 donors with an average age 48 years. Examination was conducted on the base of the Department of Chronic Hemodialysis of the Lviv Regional Clinical Hospital. RRT by the method of hemodialysis was conducted 3 times per week
Blood plasma content of L-arginine, nitrite-anion and argina hemodialysis
for 4 hours using synthetic dialisators and bicarbonate buffer. Blood for invefligations in every patient was collected from the formed vascular access of the arterio-venous fiflula before and after hemodialysis. The fludies were conducted due to bioethical norms.
For the evaluation of the level of lipid peroxidation processes in blood plasma before and after hemodialysis we detected the content of thiobarbituric acid products (TBA-active products) [30, p. 3], superoxide dismutase activity (SOD) [7, p. 5] and concentration of antioxidant vitamin C (total and oxidized form) [27, p. 3], for the evaluation of the syflem L-arginine/nitric oxide/arginase we determined the content of L-arginine [1, p. 239], nitrite-anion [13, p. 8] and arginase activity [12, p. 6]. We also determined blood plasma changes of creatinine and urea concentration using generally accepted methods.
Statiflical procession of experimental results we performed using applied program ANOVA "Statiflica". The difference at p < 0.05 was supposed to be flatiflically significant.
Results of fludies and their discussion. In the blood of donors concentration of creatinine and urea were within normal range (0.09±0.02 and 3.24±0.6 mmol/l relevantly). In patients with CRF concentration of creatinine and urea increased 7.2- and 7.5-fold relevantly (p<0.01). After hemodialysis creatinine concentration in blood plasma decreased for 57% (p<0.05), and urea - for 68% (p<0.05).
In blood plasma of donors L-arginine concentration was on the level of 93.3±19 ^g/ml, concentration of nitrite-anion made 23.6±2.3 ^mol/l and arginase activity - 0.24±0.06 ^mol/minmg (table 1).
In patients with CRF L-arginine concentration decreased for 29% (p<0.001), tendency to decrease of the nitrite-anion content was noted and arginase activity in blood plasma increased. After hemodialysis L-arginine concentration decreased for 27% (p<0.001), nitrite-anion content and arginase activity had tendency to decrease compared to relevant indices before dialysis.
Table 1
activity in patients with diabetic nephropathy before and after
Examined groups L-arginine^g/ml Nitrite-anion ^mol/l Arginase^mol/minmg
Control 94.3±16.7 23.6±2.3 0.24±0.06
Plasma of patients with CRF (before hemodialysis) 66.4±6.8*** 0.64±0.04** 0.34±0.07***
Plasma of patients with CRF (after hemodialysis) 48.6±11*** 0.57±0.04*** 0.24±0.07
*** - significance of changes compared to primary indices (p<0.001); ** significance of changes compared to indices before hemodialysis (p<0.01)
of SOD, bounding superoxide anion was 27.0±4.2 ^mol/NST/ minmg of protein, total level of vitamin C made 93.6±19.9 ^mol/l and oxidated form of vitamin C - 44.28±11.5 ^mol/l (table 2).
Table 2
Blood plasma concentration of TBA-active products, activity of superoxide dismutase and arginase in patients with diabetic nephropathy before and after hemodialysis
In healthy individuals in the course of metabolic processes certain amount of lipid peroxidation products is produced, which get to blood. In blood plasma of donors TBA-active products concentration made 82.8±6 ^mol/gHissue. Blood plasma activity
Examined groups TBA-active products ^mol/g •tissue SOD^mol NST/ minmg of protein Vitamin C (total form) ^mol/l Vitamin C (oxidized form)^mol/l
Control 82.8±6 27.0±4.2 91.5±13.8 44.3±11.5
Before dialysis 108±9*** 23.5±4.1* 57.5±11.3*** 38.9±8.5
After dialysis 94.2±9.2** 33.9±25.06* 41.4±10.41*** 26.15±6.7***
* - significance of changes compared to primary data (p<0.05); **- significance of changes compared to indices before hemodialysis (p<0,01 *** - significance of changes compared to indices after hemodialysis (p<0,001).
In blood plasma of patients with CRF increase of TBA-active products content for 30% (p<0.001) was revealed. Simultaneously we noted a decrease of vitamin C concentration - total form for 39% (p>0.001), oxidized form - for 12%. SOD activity did not change significantly. After dialysis TBA-active products content decreased for 13% (p<0.01). Concentration of vitamin C also decreased - total form for 28% (p<0,001), oxidized form - for 33% (p<0,001) compared to indices before dialysis.
Obtained results revealed decrease ofL-arginine concentration in blood plasma of patients with diabetic nephropathy, who receive RRT by the method of hemodialysis (for 29%, p<0.001), after hemodialysis decrease of the content of this amino acid was even more significant (for 27%, p<0.001) and for 51% compared to its content in the blood of healthy individuals.
Our fludies showed the decrease of the concentration of the total and oxidized form of vitamin C in patients with diabetic nephropathy, after hemodialysis the decrease of the level of this vitamin in blood is even more significant. Hemodialysis results in the decrease of the concentration of vitamin C in blood plasma for 28-33% in patients with CRF, being one of the factors of the decrease of the antioxidant defense [16, p. 6, 23, p. 6].
Literary data on the changes of nitric oxide and its derivatives in patients with CRF is disputable. Thus, some fludies showed an increase of endogenous nitric oxide, asymmetric dimethylarginine and methylated arginine in blood plasma of uremic patients [5, p. 6]. On the other hand, decreased production of nitric oxide was noted in CKD, being one of the causes of endothelial disfunction. Hemodialysis caused the decrease of NO concentration in blood plasma, but after one day its level increased to preliminary indices [4, p. 12, 14, p. 4].
Thus, acute decrease of the concentration of vitamin C and L-arginine on the background of uremia flate about the necessity of regular introduction of vitamin C and L-arginine aimed at improvement of oxidative processes and increase of the immunologic flatus of the patients with 5th flage CRF, who are treated with RRT by the method of hemodialysis [8, p. 8, 16, p. 6, 23, p. 6, 25, p. 7, 28, p. 5].
Conclusions. 1. In blood plasma of patients with CRF on the background of the significant increase of the concentration of creatinine and urea (7.2- and 7.5-fold relevantly) increased content of TBA-active products and arginase activity is noted, whereas L-arginine and vitamin C decreased. 2. After hemodialysis compared to indices before dialysis, blood plasma concentration of creatinine decreased for 57% (p<0.05) and urea for 68% (p<0.05), what was accompanied by decreased concentration of L-arginine, vitamin C and tendency to decrease of the content of TBA-active products, nitrite anion, arginase activity.
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ВПЛИВ 1МУНОМОДУЛЯТОР1В НА ПЕРЕБ1Г ВАГ1ТНОСТ1 ПРИ ЕКСПЕРИМЕНТАЛЬНИХ ШФЕКЦШНО-ЗАПАЛЬНИХ УРАЖЕННЯХ
МАТЕР1 ТА ПЛОДА
Па^ешвШ Н.М.
канд.мед.наук, головний лiкар, Харювський ^тчний перинатальний центр
Карпенко В.Г.
д.мед.н., професор, Харювська медична академiя пiслядипломноï освти
Прокопюк В.Ю.
канд.мед.наук, 1нститут проблем ^обюлогИ' та крюмедицини НАН Украши
Фалько О.В.
канд.мед.наук, 1нститут проблем ^обюлогИ' та крюмедицини НАН Украши THE INFLUENCE OF IMMUNOMODULATORY ON PREGNANCY IN EXPERIMENTAL INFECTIOUSAND INFLAMMATORY LESIONS OF THE MOTHER AND FETUS
Pasieshvili N.M., Ph.D., Head of Kharkiv regional clinical perinatal center; Karpenko V.G., MD, professor, Kharkiv Medical Academy of Postgraduate Education; Prokopyuk VU., Ph.D, Institute of Cryobiology and Cryomedicine Ukraine; Falco O.V, Ph.D, Institute of Cryobiology and Cryomedicine Ukraine АНОТАЦ1Я
У статп представлен результата впливу ÎMyHOMO^raTopy азоксимеру бромщу на перебн- шфекцшно-запальних ура-жень матерi та плоду в експерименп. Визначено його ефектившсть в комплексному лiкyваннi материнсько-плодово1' шфек-цп бактерiальноï та кандидозно1' етюлогп. ABSTRACT
The results of the impact of immunomodulators azoksymeru bromide in the course of infectious and inflammatory lesions of the mother and fetus experiment. Determined its effectiveness in the treatment of maternal-fetal infection and candida bacterial etiology. Ключовi слова: азоксимеру бромщ шфекцшно-запальш ураження матерi та плоду, материнсько-плодова шфекщя. Keywords: azoksymeru bromide, infectious and inflammatory lesions of the mother and fetus, maternal-fetal infections.
Постановка проблеми. Материнськоьплодова шфекщя (МП1) займае одне з провщних мюць в стрyктyрi перина-тальноï захворюваносп i смертносп новонароджених [1, 2151-2161]. В той же час при ваптносп з одного боку ю-нуе необхвдшсть швидкого подолання шфекцп для поперед-ження ïï шюдливого впливу на материнський та плодовий оргашзми, з шшого боку - призначення лшарських засобiв обмежено через 1'х негативну дш на стан вагiтноï та ново-
народженого [2, 206-215]. Останшм часом в акушерськш практищ поширено застосування iмyномодyляторiв з метою скорочення антибютикотерапп та зменшення впливу шфекцп на оргашзм матерi та плода. Одним з дозволених засобiв для акушерського застосування е iмyномодyлятор азоксимеру бромщ (полюкадонш) [3, 16-22], який впливае на фаго-цитарну систему, мае детоксикацшну та гепатопротекторну дш [4, 47-50]. Вплив препарату на систему мати-плацента-
