The study of biochemical mechanisms of mitochondrial dysfunction in rats’ hepatocytes during experimental hyperhomocysteinemia
AbstractMethionine is an essential proteinogenic amino acid found in many foods. During its metabolism homocysteine is formed. With elevated level of homocysteine in the blood – hyperhomocysteinemia – increased risk of developing certain diseases, such as non-alcoholic fatty liver disease, is associated. There is evidence that the homocysteine is able to reduce the effect of nitric oxide and induce mitochondrial dysfunction. The present study investigates the relationship of the functional state of the liver cells mitochondria and the level of nitric oxide metabolites in them in experimental hyperhomocysteinemia caused by excessive intake of methionine. The experiment was conducted on 17 male Wistar rats with an initial weight of 220–270 g, rats were divided into 2 groups. A 25% suspension of methionine was administered (in a dose of 1.5 g of methionine per kg body weight) two times a day for 21 days intragastrically (by gavage) to rats of the first group (n=9) while instead of drinking water animals received a 1% aqueous solution of methionine. Drinks daily volume of methionine solution was 17.2 [15.5; 18.1] ml. In the experiment 8 animals were used, in which severe hyperhomocysteinemia (> 100 mmol/l) was developed. The second group (n=8) served as a control. These rats were administered suspension base containing no methionine (10% Tween-80, 1% starch, 89% water). The total homocysteine concentration was measured in blood serum by ELISA. In the suspension of liver mitochondria total protein was measured by Lowry method; the concentration of NO metabolites by screening method; succinate dehydrogenase activity – under the reaction of hexacyanoferrate (III) potassium reduction; lactate dehydrogenase activity – by decrease of NADH concentration in the reaction of pyruvate’s reduction; activity of H+-ATPase – by measuring the inorganic phosphate; superoxide dismutase – by inhibition of quercetin auto-oxidation, the level of Ca2+ – by reaction with Arsenazo III. Oxidative modification of proteins was evaluated based on the reaction between carbonyl and imino groups of the amino acid residues oxidized with 2,4-dinitrophenylhydrazine to form 2,4-dinitrophenylhydrazone having a specific absorption spectrum in the ultraviolet and visible regions of the spectrum. During three weeks of the experiment the body weight of rats treated with methionine increased from 246 (229; 262) to 302 (283; 311) g, and of control animals – from 256 (231; 264) to 307 (275; 314) g. The difference in body weight gain was not statistically significant. In the study it was revealed that intragastric administration of the methionine for 3 weeks with the addition of this amino acid in the drinking water caused hyperhomocysteinemia. On the one hand it lead to an intensification of energy metabolism in rat liver mitochondria, resulted in increase of lactate dehydrogenase (by 63.0%), succinate dehydrogenase (by 76.1%) and H+-ATPase (by 62.5%) activities. On the other hand it lead to the disruption of the Ca2+ deposit (Ca2+ level in mitochondria was reduced by 68.2%) and to enhance of mitochondrial protein carbonylation (by 52.2%) with a predominance of the aggregation process and a reduction of the reserve-adaptive capacity, despite an increase in superoxide dismutase activity (by 87.7%). The reason for these changes in the mitochondria can be the decrease in production of nitric oxide (the level of its metabolites decreased by 21.3%).
Keywords:methionine, hyperhomocysteinemia, nitric oxide, oxidative stress, mitochondrial dysfunction