Optimum marker selection of acute liver damage in rats in the experiment

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Abstract


Relevance. Assessment of liver damage and functional state is one of the leading tasks of clinical and laboratory diagnostics. Traditionally used methods for determining the activity of a number of indicator enzymes in blood with relative organ-specificity, such as aspartate aminotransferase, alanine aminotransferase, lactate dehydrogenase, sorbitol dehydrogenase, alkaline phosphatase, and γ-glutamyl transferase, have low specificity for liver diseases. In this regard, the determination of the optimal marker of acute liver injury is an urgent problem. Aim. The purpose of the study is to determine the dynamics of changes in liver damage markers in rats at different periods of reperfusion after 20 minutes of ischemia in order to select the indicators that most informatively characterize the state of test-animals under conditions of correction of ischemia-reperfusion syndrome. Materials and methods: the study was performed on 120 white nonlinear male rats weighing 200–250 grams. The animals were divided into 8 groups of 15 test-animals; all of them were simulated liver ischemia by clamping the analog of the hepatoduodenal ligament with a vascular clamp for 20 minutes. Then, blood was taken from different groups of rats at different reperfusion times – 5, 15, 30, 60, 120, 180 minutes, 8 hours and a day. In the blood plasma of laboratory animals, the activity of alanine aminotransferase (ALT), aspartate aminotransferase (AST), lactate dehydrogenase (LDH), glutathione transferase (GST), and lactate concentration were determined. Results: the results obtained allowed us to characterize two main peaks of indicators: a 5-minute period after restoration of blood flow – the maximum activity of glutathione transferase and lactate concentration, increased by 3.9–4.7 times; 60–180 minutes of reperfusion is the peak of aminotransferase activity, a significant increase in the activity of which begins 60 minutes after the restoration of blood flow and reaches its maximum by the 3rd hour of reperfusion, and LDH, the peak of which is recorded already by the 60th minute of revascularization. At the same time, after 8 hours of reperfusion, an obvious tendency for a decrease in all studied parameters was determined, which ends a day after modeling ischemia with a decrease to the level of control values. Conclusion: the assessment of organ damage in the ischemic period and the anti-ischemic effect of metabolic drugs can be carried out with the determination of an increase in lactate concentration and glutathione transferase activity almost immediately after restoration of blood flow. The development of injuries during the reperfusion period is more expedient to assess by determining AST, ALT and LDH after a 3-hour period of blood flow restoration, at which time the maximum values of markers are recorded under the condition of 20-minute total liver ischemia.


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In medical practice, the assessment of liver damage and functional state is one of the leading tasks of clinical and laboratory diagnostics. For this purpose, the determination of the activity of a number of indicator enzymes in the blood with relative organ-specificity is traditionally used, such as aspartate aminotransferase (AST), alanine aminotransferase (ALT), lactate dehydrogenase (LDH), alkaline phosphatase and γ-glutamyltransferase [1]. A change in the activity of aminotransferases often indicates cytolysis of liver cells, the activity of alkaline phosphatase increases with cholestasis [2, 3]. Taking into account the proteinsynthesizing function of the liver, in some cases it can be informative to determine the content of serum albumin in the blood plasma, especially in chronic diseases, and the prothrombin time, which reflects the activity of blood coagulation factors, also synthesized in the liver. Against the background of the detoxification function, the determination of bilirubin and its metabolites may be informative [4]. The main disadvantage of all the listed markers is their low specificity in relation to liver diseases. Meanwhile, by verification of liver damage by other methods, the assessment of the above indicators is justified for laboratory monitoring purposes [5, 6]. There are dynamic methods for assessing the functional state of the liver, based on determining the rate of metabolism in the liver of various substrates, such as bromsulphthalein, indocyanine green, lidocaine, etc. [7-9]. These methods are rarely used in clinical practice, since they are invasive, require complex equipment and a long study time. The principles and approaches to the assessment of experimental liver damage in rats are similar; however, there are features associated with the severity and diagnostic significance of certain markers, as well as with the dynamics of changes in the process of damage [10, 11]. The purpose of the present study was to determine the dynamics of changes in liver damage markers in rats at different periods of reperfusion after 20 minutes of ischemia in order to select the indicators that characterize the state of animals under conditions of correction of ischemia-reperfusion syndrome most informatively. Material and methods The study was carried out on 120 laboratory animals, white non-linear male rats (weighing 200- 250 grams). The animals were divided into 8 groups of 15 test-animals; all animals were simulated liver ischemia by clamping the analog of the hepatoduodenal ligament with a vascular clamp for 20 minutes. Then, blood was taken from different groups of rats at different reperfusion times - 5, 15, 30, 60, 120, 180 minutes, 8 hours and a day. Experimental work with laboratory animals was performed under general anesthesia using Zoletil 100 (Virbac, France) at a dosage of 10 mg/kg intramuscularly. The study was approved by the independent ethics committee at the FSBEI HE «KubSMU» of the Ministry of Health Care of Russia (Protocol No. 51 of May 23rd, 2017) and was carried out in accordance with the principles set out in the «European Convention for the Protection of Vertebrate Animals» (Strasbourg, 1986). Comparison of the data obtained in the experiment in the rats of the experimental groups was also carried out with the results of evaluating similar indicators in the group of intact animals (control group, n = 20) kept in similar conditions [2]. In the blood plasma of laboratory animals, the activity of alanine aminotransferase (ALT), aspartate aminotransferase (AST), lactate dehydrogenase (LDH), and lactate concentration were determined by kinetic methods using the Vital Development Corporation reagent kits (St. Petersburg, Russia). The activity of glutathione transferase (GST) was determined according to a method involving the registration of changes in optical density at 340 nm as a result of condensation of glutathione with dinitrochlorobenzene [12]. The data obtained were statistically processed by means of the AnalystSoft Inc. software and the statistical analysis program StatPlus, Version 7 (link: www.analystsoft.com/ru/). Taking into account the small sample size and abnormal distribution (assessed by the Shapiro-Wilk test), the nonparametric KruskalWallis test (for several independent groups) was used to determine the significance of differences between the parameters of the groups of animals with different reperfusion times, as well as the control group with the subsequent (if necessary) pairwise comparison by means of the Mann-Whitney test. The results are presented in the article as a median, error bars reflect the spread between the 25th and 75th percentiles. Differences between the group indicators were considered statistically significant at p <0.05. Results and discussion Analysis of changes in classical markers in the dynamics of the development of ischemia-reperfusion damage in rats showed different results for the activity of ALT, AST, LDH, GST and lactate concentration. The activity of all markers increased already at the 5th minute of reperfusion, but later on, the dynamics of indicators was different. ALT activity (Table 1) increased by the 5th minute of the reperfusion period by 2.1 times, a further statistically significant increase in this indicator was recorded after 60 minutes of reoxygenation; the ALT activity during this period was 3.8 times higher than the control indices. 3 hours after the restoration of blood flow in the ischemic liver, the activity of the analyzed marker reached its maximum values in blood plasma - 8.8 times higher than that of intact animals. After that, the ALT activity gradually decreased during the day, so 8 hours after the start of reperfusion, the activity exceeded the control figures by 5.2 times, and after a day - only by 1.6 times. One of the interesting features is that a wide scatter of indicators in animals at the stages of 1 and 2 hours of reperfusion should be highlighted, which can be regarded as an increase in ALT activity in animals, manifested to varying degrees depending on the individual characteristics of the animals, different initial states of the organism. At the same time, a statistically significant increase in the indicator was recorded only in the group of animals after a 3-hour period of blood flow restoration. This makes it possible to recommend the use of this period in experimental studies of ischemia-reperfusion syndrome, including its correction, more reasonably. To use a 1-2 hour reperfusion period for the same purposes, it is necessary to consider the possibility of either increasing the sample or preliminary assessment of the resistance of rats to hypoxia-reoxygenation. Table 1 Changes in hepatocyte cytolysis markers at different periods of reperfusion after 20-minute ischemia of rat liver (Ме(р0,25/р0,75)) Studied groups/ Reperfusion time Studied indices ALT, U/l AST, U/l LDH, U/l Control 23,5 (18,4/25,5) 44,6 (42,9/51,9) 152,3 (99,2/167,5) 5 min. 49,8 (45,1/60,0)* 62,2 (55,1/67,0)* 224,5 (203,8/313,5)* 15 min. 78,6 (51,8/93,7)* 65,3 (47,8/100,3)* 325,0 (227,0/521,5)* 30 min. 47,4 (44,5/78,2)* 85,2 (80,0/90,7)* 498,0 (479,0/544,0)* 60 min. 89,6 (82,1/138,5)*^ 156,6 (130,8/199,6)*^ 1519,0 (971,4/2585,0)*^ 120 min. 84,4 (77,4/143,8)* 85,6 (82,1/159,6)* 1113,4 (823,7/1545,9)* 180 min. 207,9 (149,1/241,5)*^ 240,9 (156,6/268,9)*^ 1112,8 (764,3/1320,3)* 8 h. 121,8 (93,8/170,1)* 118,55 (86,8/176,7)*^ 459,5 (308,2/783,8)*^ 1 day 38,3 (33,9/48,2)*^ 79,8 (68,7/82,4)*^ 390,7 (330,8/450,7)* Note:* - statistically significant differences (р<0,05) from the same index of the control group; ^ - statistically significant differences (р<0,05) from the index of the previous group. The dynamics of AST activity during the development of reperfusion syndrome practically coincided with that for ALT (Table 1). A feature was the initially higher values of the enzyme activity in animals of the control group and in animals of the experimental groups accordingly against the background of a pathological process. AST activity, as well as ALT activity, increased by 1.4 times already after 5 minutes of reperfusion. 60 minutes after the restoration of blood flow in the vascular bundle of the hepatic hilus, the AST activity exceeded the control values by 3.5 times. This period (60-120 minutes of reperfusion) was also characterized by amplitude changes in the activity of the enzyme with a significant increase by the 3rd hour of reoxygenation - up to values 5.4 times higher than the level of a similar parameter in intact rats. Determination of AST activity after 8 hours and a day showed a gradual decrease in the indicator, similar to the dynamics of changes in ALT activity. In general, the increase in ALT activity was characterized by high relative values in comparison with the group of intact animals. Table 2 Changes in hepatocyte cytolysis markers at different periods of reperfusion after 20-minute ischemia of rat liver (Ме(р0,25/р0,75)) Studied groups/ Reperfusion time Studied indices GSТ, U/l Lactate, mmol/l Control 0,023 (0,020/0,026) 2,8 (2,6/3,0) 5 min. 0,108 (0,094/0,120)* 11,0 (10,6/11,4)* 15 min. 0,095 (0,067/0,127)* 8,0 (7,0/8,3)*^ 30 min. 0,087 (0,085/0,101)* 8,1 (6,8/10,5)* 60 min. 0,123 (0,112/0,192)*^ 7,0 (6,8/9,2)* 120 min. 0,052 (0,050/0,056)*^ 3,9 (3,8/4,9)*^ 180 min. 0,050 (0,048/0,058)* 5,9 (4,3/6,1)* 8 h. 0,030 (0,025/0,037)^ 5,1 (4,3/6,7)* 1 day 0,027 (0,025/0,031) 2,5 (2,3/2,7)^ Note:* - statistically significant differences (р<0,05) from the same index of the control group; ^ - statistically significant differences (р<0,05) from the index of the previous group. The dynamics of changes in LDH activity in animals of the experimental groups was significantly different (Table 1). There was also a slight increase in the parameter already 5 minutes after the restoration of blood flow - by 1.5 times. Subsequently, the average values of the enzyme activity and the scatter of the index within the group gradually increased, but a statistically significant increase in the marker was recorded by the 60th minute of reperfusion, while the values of LDH activity increased by 7.3-10.0 times in animals after 2and 3-hour reoxygenation. Later, as well as for aminotransferases, a decrease in the activity of the analyzed enzyme was recorded, which did not reach the control values. Thus, the main feature of the dynamics of LDH activity was the achievement of the maximum value already by 60 minutes of reperfusion and the absence of a peak in the indicator after 180 minutes of the experiment. The activity of glutathione transferase changed in a different way (Table 2), in contrast to the activity of transaminases and LDH. The most significant increase in the activity of the analyzed enzyme was observed already at the 1st stage of the experiment after 5 minutes of reperfusion. The activity of glutathione transferase in this period increased by 4.7 times. Subsequently, a slight increase in activity was determined after 60 minutes of restoration of blood flow - the value of the considered indicator exceeded the control figures by 5.3 times, which was especially distinguished against the background of a slight trend towards a decrease in enzymatic activity in animals after 15-30 minutes of reperfusion. Against the background of 2-hour reperfusion, the activity of glutathione transferase decreased to a level 2.3 times higher than the control values, and after 8 hours it did not differ from the value of the corresponding parameter in the group of intact rats. The concentration of lactate is not a marker of hepatocyte cytolysis, but it was interesting to follow its changes against the background of its expected growth in the presence of hypoxia. In fact, the concentration of the analyzed metabolite was maximal immediately, i. e. 5 minutes after the end of the ischemic period. At this stage, the concentration of lactate exceeded the control values by 3.9 times. During further observation, the content of lactic acid in blood plasma progressively decreased. The most significant points of decrease in lactate concentration were 15 minutes and 120 minutes, in addition, the normal level of lactic acid was determined one day after the restoration of blood flow. Thus, in our opinion, two main peaks of indicators can be distinguished: a 5-minute period after the restoration of blood flow - the maximum activity of glutathione transferase and the concentration of lactic acid; 60-180 minutes of reperfusion is the peak of aminotransferase activity, a significant increase in the activity of which began from 60 minutes after the restoration of blood flow and reached a maximum by the 3rd hour of reperfusion and LDH, the peak of which was recorded already by the 60th minute of revascularization. An increase in the first two indicators by the 5th minute of the experiment is associated with changes in the ischemic period, an increase in the activity of AST, ALT and LDH to a greater extent reflects an increase in liver damage during the reperfusion period, the time limits of the main damaging effect of which are also indicated by an increase in glutathione transferase activity and a delay in the decrease in concentration lactate. Conclusion Depending on the goals and objectives, a reasonable choice of one or another marker of liver damage in an acute experiment is required. Assessment of organ damage in the ischemic period and the anti-ischemic effect of metabolic drugs can be carried out by determining the increase in lactate concentration and glutathione transferase activity almost immediately after the restoration of blood flow. It is more expedient to evaluate the development of injuries during the reperfusion period using the classic markers of hepatocyte cytolysis - AST, ALT, and LDH. More homogeneous and easily comparable results can be obtained when determining these indicators after a 3-hour period of blood flow restoration, at the same time the maximum values of markers are recorded under the condition of 20-minute total liver ischemia.

About the authors

K. A. Popov

Kuban State Medical University

Author for correspondence.
Email: naftalin444@mail.ru
SPIN-code: 9456-9710
Krasnodar, Russian Federation

I. Y. Tsymbalyuk

Kuban State Medical University

Email: naftalin444@mail.ru
SPIN-code: 4493-0738
Krasnodar, Russian Federation

R. I. Sepiashvili

Peoples’ Friendship University of Russia (RUDN University)

Email: naftalin444@mail.ru
SPIN-code: 6921-7356
Moscow, Russian Federation

I. M. Bykov

Kuban State Medical University

Email: naftalin444@mail.ru
SPIN-code: 9977-6613
Krasnodar, Russian Federation

E. S. Ustinova

Kuban State Medical University

Email: naftalin444@mail.ru
Krasnodar, Russian Federation

M. I. Bykov

Kuban State Medical University

Email: naftalin444@mail.ru
SPIN-code: 2909-3520
Krasnodar, Russian Federation

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Copyright (c) 2020 Popov K.A., Tsymbalyuk I.Y., Sepiashvili R.I., Bykov I.M., Ustinova E.S., Bykov M.I.

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