ACTIVE DYNAMICS OF THE ENZYMES OF THE ANTIRADICAL PROTECTION AND THE GENERAL ANTIOXIDATIVE ACTIVITY BY THE DEVELOPMENT OF THE EXPERIMENTAL ISCHEMIC REPERFUSION OF LIVER

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Abstract


The purpose of the study: to research the active changes in several enzymes of the antioxidative protection within the dynamical development of the liver reperfusion in rats. Materials and methods: the study has been performed on 95 white non-lineal male rats with the mass of 240-280 g divided into groups, the biological material of which has been sampled within 5-20 minutes of the ischemic period and 5-20 minutes of the reperfusion period with the interval of 5 minutes. To evaluate the changes of the antioxidative system the activity of the superoxide dismutase, the erythrocyte catalase and the liver homogenate as well as the general antioxidative activity have been determined. Results: the performed study has demonstrated the imbalance development for the relation between the activity of catalase and superoxide dismutase by the development of the ischemic reperfusion syndrome with the change of this relation to the prevalence of the catalase activity on the systemic level and the superoxide dismutase activity on the organic level after the circulatory restoration. Besides the increase in activity of the studied enzymes from the erythrocyte, meal by 2 times in comparison with the control group has been determined especially at the ischemic period as well as the tendencies to the progressive activity decrease of the same enzymes in the liver tissue. The general antioxidative activity in the period of ischemic reperfusion has been decreasing to the fifth minute of ischemia by 2 times and has lasted on the same level (0.04-0.05 mg/l of vitamin C in terms of 1 gram of homogenate protein) during the entire experiment. Conclusion: the received results have demonstrated the possibility for the evaluation of the functional state for the system of oxidative restorative homeostasis in an animal by ischemic reperfusion of the liver against the background of the lowered general antioxidative activity. It was marked also the necessity of the metabolic support of this link within the system of non-specific resistance for the correction of the developing disorders.


The hepatic ischemia-reperfusion syndrome is a complex of pathological and adaptive reactions of an organism including a variety of metabolic, immunological and morphological changes developing as a result of the reduced blood circulation within an organ with its further restoration [1-2]. The cessation of the oxygen supply leads to the metabolic inversion from aerobic oxidation to glycolysis, which is less energy efficient; the change is accompanied by the sufficient decrease of the ATP synthesis in tissues and the accumulation of the under-oxidized products. The further liver reperfusion is often a cause to the considerable damage of hepatocytes, capillary endothelium and epithelium of the bile ducts. The leading pathobiochemical development mechanism of the ischemia-reperfusion syndrome of liver is the oxidative stress. The imbalance between the tissue necessity in oxygen and its delivery as well as the rapid increase of its partial pressure in the liver tissue after the circulative restoration provides favorable conditions for the formation of free radicals (superoxide anion, hydrogen peroxide, and hydroxyl radical). In its turn that leads to the activation of processes concerning the peroxide oxidation of lipids which causes the alteration of cellular and subcellular membrane structures of hepatocytes [3-7]. The idea of processes accompanying the ischemic reperfusion of liver may become the basis for developing new scientifically justified methods, which allow improve quality of transplant and other general operations on the organs of the hepatobiliary system and also lowering the risk of postoperative complications. The purpose of the present research is to study the active changes in several enzymes of the antioxidative protection within the dynamical development of the liver reperfusion in rats. MATERIALS AND METHODS The study has been performed on 95 white non-lineal adult male rats with the mass of 240-280 g. The test animals have been captivated at the educational training-and-production department (vivarium) of the Kuban State Medical University of the Ministry of Health of Russia. All studies have been performed in accordance with “The Rules Accepted in the European Convention on the Protection of Vertebrate Animals” (Strasbourg, 1986) and has been approved by the local ethical committee of the Kuban State Medical University of the Ministry of Health of Russia. (protocol N 51 of 23.05.2017). All manipulations have been performed under the general anesthesia by means of Zoletil 100 remedy (“Virbac”, France) in proportion of 10 mg/kg intramuscular. The control group (Group 1) has been presented by animals (n = 12) that have been sampled blood in volume of 150 mcl from the caudal vena cava in 5, 10 and 15 minutes after the performed laparotomy. The second control group (Group 2) has been made up of animals (n = 10) that have been sampled blood in volume of 150 mcl from the caudal vena cava in 5, 10, 15 and 20 minutes after 15-minutes-period after the performed laparotomy. The animals of the 3rd group (n = 15) after the median laparotomy have been undergone the clamping of hepatoduodenal ligament (HDL) analogue for 15 minutes including the blood sampling in 5, 10 and 15 minutes after the ischemia beginning in volume of 150 mcl from the caudal vena cava. The animals of the 4th group (n = 14) have been undergone the clamping of HDL analogue for 15 minutes including the blood sampling in volume of 150 mcl from the caudal vena cava in 5, 10, 15 and 20 minutes of reperfusion. To evaluate the changes on the organic level 4 groups have been formed that have been sampled the liver tissue in mass of 0,15-0,20 g. The control group (Group 1p) has been presented by animals (n = 10), that has been sampled the liver tissue in 5, 10, 15 and 20 minutes after the performed laparotomy. The second control group (Group 2p) has been presented by animals (n=10), that has been sampled the liver tissue in 5, 10, 15 and 20 minutes after 20 minutes after the performed laparotomy. The animals of the 3rd group (n = 12) after the median laparotomy have been undergone the clamping of HDL analogue for 20 minutes including the blood sampling in 5, 10, 15 and 20 minutes of ischemia. The animals of the 4th group (n=12) have been undergone the clamping of HDL analogue for 20 minutes including the liver sampling in 5, 10, 15 and 20 minutes of reperfusion. Thus the biological material of the groups of animals have been formed has been sampled within the period of ischemia from 5 to 20 minutes and within the period of reperfusion from 5 to 20 minutes with the 5-minute interval. Since blood and liver have been sampled many times from the same animals, so for further evaluation of the influence of the blood loss and liver damage because of its partial resection the analogical control groups of rats have been formed. To evaluate the changes of antioxidative system the activity of superoxide dismutase (SOD), catalase (CAT), erythrocyte hemolysate and liver homogenate as well as the general antioxidative activity (AOA) in the liver homogenate have been determined. The CAT activity has been determined in accordance to the utilization of hydrogen peroxide that has been registered according to the absorption of light by 260 nm [8]. The SOD activity has been determined in accordance to the slowdown of the quercetin self-oxidation [9]. The general AOA has been determined by means of the FRAP method (Ferric Reducing/Antioxidant Power) including the excessive injections of Fe3+ ions and the photometric reagent, namely 2,2’-dipyridyl [10]. In the liver homogenate, the protein concentration has been determined in accordance with the Bradford protein assay including Coomassie dyes (Coomassie Brilliant Blue G-250) [11], the calculation of enzyme activity and glutathione concentration have been performed to 1 gram of protein homogenate. The results of the study have been statistically processed by means of the system of statistical analysis Stat plus LE. The received data have been presented in form of a median (Ме), 25th and 75th percentile (р0,25/р0,75). Against the background of the Mann-Whitney U-test the index changes have been registered in animals of different groups while the Wilcoxon signed-rank test has been used for evaluation of the difference in indices received from animals of the same group, the difference by р < 0,05 has been considered to be reliable. RESULTS AND DISCUSS By the analysis of the received data, the most expressed increase of the catalase activity in the erythrocyte meal to the 10th minute of the ischemic period has been revealed by 2 times in comparison with the period of 5-minute-clamping of the HDL analogue. Besides, during the comparison with the control indices the catalase activity by the clamping of the HDL analogue for 5 minutes has been lowered by 14%. It is necessary to underline that the changes in activity of the observed enzyme in the control group of animals both during the ischemic and reperfusion period have been not detected. The further development of the ischemic and reperfusion period has been characterized by the high stable indices of the catalase activity of erythrocytes on the level of 10-12 mmol/(l x min). Only to the 20th minute of the restored circulation the small increase of the enzymatic activity by 10-20% has been observed in comparison with the same indices by the previous samplings. The activity of superoxide dismutase of erythrocytes has varied within the greater limits and its changes have not been that definite as by the changes of catalase activity. To the 5th minute of ischemia the SOD activity of the erythrocyte meal has decreased by 10%, then similarly to the changes in the catalase activity the increase by 1,9 times has been detected. In what follows the increase of the SOD activity by more 20% to the 15th minute of ischemia has been detected as well as the relatively low activity indices of this enzyme in the erythrocyte meal to the 5th minute of the restored blood circulation which has been higher than the control indices by 1,5 times. In general, the reperfusion period has been characterized by the lower values of SOD activity in comparison with the ischemic period and only to the 20th minute of reperfusion the increase in activity of this enzyme to the values of the 5-minute ischemia has manifested itself (Table 1). The evaluation of the activity proportion for CAT/SOD has revealed it’s maintaining on the level close to the one during the entire ischemia period which may characterize (including the data of the Table 1 The changes in the activity of enzymes of the antioxidative protection of erythrocytes by the ischemia-reperfusion of rat’s liver (Ме(р0,25/р0,75)) Period Time, min. C-CAT, mmol/(l×min) CAT, mmol/(l×min) C-SOD, rel.u. SOD, rel.u. Ischemia 5 5,29 (4,86/5,65) 4,56* (4,28/4,89) 30,2 (28,6/32,6) 27,8* (25,4/28,9) 10 5,08 (4,72/5,44) 10,26*^ (9,18/10,60) 33,0 (31,3/33,7) 51,8*^ (47,0/54,3) 15 6,02 (5,55/6,34) 10,30* (9,83/10,79) 35,7^ (32,2/36,8) 61,3*^ (55,5/63,9) Reperfusion 5 5,33 (4,95/5,70) 11,45* (10,57/11,80) 32,4 (30,4/34,0) 47,3*^ (45,6/50,1) 10 5,25 (4,85/5,5,77) 11,09* (10,78/11,80) 31,1 (30,2/33,5) 40,0*^ (37,4/45,0) 15 5,40 (5,02/5,82) 11,84* (11,48/12,35) 34,5 (31,6/36,8) 38,9* (37,3/43,5) 20 5,23 (4,90/5,56) 13,06*^ (12,40/13,85) 33,5 (30,8/34,8) 48,3*^ (44,5/50,4) Note: * - statistically significant differences (р < 0,05) in comparison with the indices of the control group; ^ - statistically significant differences (р < 0,05) between two indices determined with the 5-minute-interval of ischemia-reperfusion. Abbreviations: CAT, SOD - the enzyme activity in rats of the test groups; C-CAT, C-SOD - the enzyme activity of the control groups. activity increase in both enzymes by 2 times) the adaptive changes aimed at the preparation for the development of oxidative stress during the period of active restoration of the blood circulation. To a certain degree such effect may be caused by the increase in the activating influence of the superoxide anion-radical on SOD and the hydrogen peroxide - on CAT against the background of the general relative increase of the oxygen pressure in blood by the liver release out of the systemic blood stream. The reperfusion period is characterized by the increase of the CAT/SOD proportion to 1,5-1,9 units which indicates the primary generation of hydrogen peroxide and the possibly lowered resistance to the formation of superoxide anion-radical. The enzymes of the antioxidative protection in the liver tissue have undergone changes mainly to the progressive decrease of its activity. It’s interesting to underline that the CAT activity in control animals has lowered nearly to the indices by 1,5 time less than the initial. In rats that have undergone ischemia-reperfusion the catalase activity of the liver tissue has decreased by 20% to the 10th minute of the ischemic period and by more than 20% to the 20th minute of the same period in comparison with the previous value of the studied index. In general, the indices of the catalase activity at this period of the liver damage have corresponded with the control indices. The expressed lowering of CAT activity in average by 1,5-2 times in comparison with the control indices has been revealed in animals at the reperfusion period. The SOD activity in the liver tissue of rats from the control group has not changed in time by blood sampling without the modeling of ischemia-reperfusion. In animals of the test groups the lowering of the SOD activity of the liver has been determined by 15% to the 5th minute of ischemic damage of the organ. The reperfusion period has been characterized by the indices of SOD activity within the limits of control indices in 5-15 minutes after the restoration of blood stream including the lowered enzyme activity to the 20th minute by 25% in comparison with the control indices (Table 2). The proportion of CAT/SOD activity of liver homogenate at the ischemic period has been characterized by the values close to one (1,2-1,6 units) including the unreliable prevalence of the catalase activity. It may be necessary to the partial return of active oxygen forms to the link of the electron transference in conditions of the lowered partial oxygen pressure in the ischemic tissue and provides the more effective running of energetic metabolism. At the reperfusion period, the CAT/SOD proportion has changed towards the prevalence of the SOD activity (0.5-0.8 units). At this period, the acute increase of the liver oxygenation has been accompanied by the hyperproduction of free radicals, first of all - of the product of monoelectronic oxygen restoration, namely the superoxide anion-radical. Table 2 The changes in the activity of enzymes of the antioxidative protection of rat’s liver by its ischemia-reperfusion (Ме(р0,25/р0,75)) Period Time, min C-CAT, mmol/(l×min×g of protein) CAT, mmol/(l×min×g of protein) C-SOD, rel.u./g of protein SOD, rel.u./g of protein Ischemia 5 2,71 (2,35/2,90) 2,52 (2,30/2,78) 12,5 (11,8/12,8) 10,7* (10,2/11,1) 10 2,17^ (2,02/2,42) 2,01^ (1,79/2,20) 11,6 (11,4/12,0) 9,0*^ (8,4/9,6) 15 1,88 (1,60/2,03) 2,39 (2,00/2,48) 12,0 (11,7/12,4) 9,7* (9,2/10,0) 20 1,88 (1,59/2,02) 1,72^ (1,54/1,95) 11,8 (11,5/12,3) 9,1* (8,8/9,4) Reperfusion 5 2,31 (2,01/2,56) 1,41*^ (1,23/1,59) 11,7 (11,3/12,1) 12,4^ (11,7/12,8) 10 2,17 (2,00/2,41) 1,61* (1,43/1,72) 11,8 (11,5/12,1) 11,8 (11,5/12,2) 15 1,83^ (1,60/1,97) 0,80*^ (0,73/1,05) 12,0 (11,6/12,3) 12,2 (11,8/12,5) 20 1,94 (1,64/2,10) 1,23* (0,95/1,36) 11,5 (11,3/11,9) 8,5*^ (8,0/9,0) Note: * - statistically significant differences (р < 0,05) in comparison with the indices of the control group; ^ - statistically significant differences (р < 0,05) between two indices determined with the 5-minute-interval of ischemia-reperfusion. Abbreviations: CAT, SOD - the enzyme activity in rats of the test-groups; C-CAT, C-SOD - the enzyme activity of the control groups. The research of the general antioxidative activity of the liver homogenate in control animals has revealed its restoration during the entire experiment on the level of 0.08-0.10 mg/l of vitamin C (taken as standard) for 1 gram of homogenate protein. At the period of ischemic reperfusion the general antioxidative activity has lowered already to the 5th minute of ischemia by two times and has been maintained on the level of 0.04-0.05 mg/l of vitamin C for 1 gram of homogenate during the entire experiment. At the moment of reperfusion the significant changes could be expected but it is possible that the increase of free-radical processes at that period is balanced by the supply of antioxidants from blood. CONCLUSION The received data describes the changes in the enzyme activity of the first lines of the antioxidant protection and the general antioxidative activity on systemic and organic level in details during 20 minutes of ischemic liver damage and at the early reperfusion period. 20-minute clamping of HDL to exclude the liver out of the systemic blood circulation by surgical interventions or the break of blood supply of the organ as a result of other reasons for 20-30 minutes are the most urgent time periods for studying the fundamental and clinical aspects of ischemic reperfusion of liver. At that time, the organ maintains its viability and it is still possible to preserve it by the circulatory restoration; by the metabolic influence on the most sensible pathogenic links the prevention of the developing liver insufficiency at late stages of reperfusion. The results of the present study have demonstrated the role of changes in the activity of catalase and superoxide dismutase of blood and liver during the observed pathological process. So the development of imbalance of CAT/ SOD activity proportion by the development of the ischemic reperfusion syndrome with the change of the proportion towards the prevalence of the catalase activity on the systemic level and of the superoxide dismutase on the organic level after the restoration of blood circulation. Besides the increase in activity of the studied enzymes in the erythrocyte meal especially at the ischemic period has been determined as well as the tendency to the progressive activity decrease of the same enzymes in the liver tissue. This proves the possible evaluation of the functional systemic state of the system of the oxidative-restorative homeostasis in animals by the ischemic reperfusion of liver; against the background of the lowered general antioxidative activity it presents the necessity of the metabolic support of this link in the system of the non-specific resistance.

K A Popov

Kuban State Medical University of the Ministry of Health of Russia

Author for correspondence.
Email: ilya.bh@mail.ru

I M Bykov

Kuban State Medical University of the Ministry of Health of Russia

Email: ilya.bh@mail.ru

PhD, MD, Head of the department of Fundamental and Clinical Biochemistry of The Kuban State Medical University of the Ministry of Health of Russia

G A Ermakova

Kuban State Medical University of the Ministry of Health of Russia

Email: ilya.bh@mail.ru

I Y Tsymbalyuk

Kuban State Medical University of the Ministry of Health of Russia

Email: ilya.bh@mail.ru

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