The activity of glutathione regeneration enzymes in rats’hepatocytes under the conditions of

Abstract

In the present study, the investigation of the redox status of the glutathione system and the activity of glutathione regeneration enzymes in rats’ hepatocytes under the conditions of different protein and sucrose supplementation: the content of reduced and oxidized forms of glutathione, glutathione redox ratio, glutathione reductase and glucose-6-phosphate dehydrogenase activity are presented. During the experiment, the experimental animals consumed a semi-synthetic diet AIN-93 in accordance with the recommen-dations of the American Institute of Nutrition. Isolation of hepatocytes was performed by non-enzymatic method with Hanks' solution with the addition of 2 mM EDTA. The concentration of the reduced glutathione was investigated using Elman's reagent after the deproteinization of the samples on a CARY 60 spectrophotometer (USA) at a wavelength of 412 nm. To determine the content of oxidized glutathione, zinc dust was added to the reaction mixture in order to convert the oxidized form to the reduced one. Glutathione reductase activity in hepatocytes was determined by the oxidation rate of NADPH⁺•H⁺. Glucose-6-phosphate dehydrogenase activity was investigated by NADP⁺ reduction rate at 340 nm. A maximum decrease in GSH content (at 4.5 times compared to control) with a simultaneous increase in GSSG levels (at 12 times compared with control) in rats’ hepatocytes was found under the conditions of consumption of excessive sucrose content on the background of deficiency of dietary protein. These results are characterized by a significant decrease in the glutathione redox ratio and indicates a disruption of the redox balance in the conjugate conversion system GSH ↔ GSSG. Deficiency of dietary protein leads to the activation of glutathione regeneration enzymes - glutathione reductase and glucose-6-phosphate dehydrogenase in rats’ hepatocytes, which is probably aimed at maintaining a stable level of GSH under the conditions of disruption of its synthesis in γ-glutamylcysteine synthetase reaction. Excessive consumption of sucrose is a key factor in reducing the activity of the studied enzymes in rats’ liver cells, which leads to the blocking of the first stage of the pentose phosphate cycle. Therefore, the decrease in the amount of NADPH, as a consequence of the established changes, can be considered as one of the reasons for the decrease in the content of reduced glutathione in animals’ liver cells under the experimental conditions.