The Generation of Superoxide Radicals by Cardiac Mitochondria and the Antioxidant Effect of the Water-Soluble Form of Ubiquinol-10

Biophysics - We have studied the effect of the water-soluble form of ubiquinol-10 (CoQ10-H2) on the processes of electron transport, oxidative phosphorylation, and the formation of reactive oxygen...     

Interaction of Oxoferrylmyoglobin and Dinitrosyl-Iron Complexes

It is shown that dinitrosyl-iron complexes (DNIC) with glutathione can reduce oxoferrylmyoglobin forming on interaction of tert-butyl hydroperoxide and metmyoglobin. A rapid decrease in the DNIC concentration was observed under the conditions of production of tert-butyl free radicals; however, destruction of DNIC in the presence of oxoferrylmyoglobin alone was negligible. It is demonstrated that DNIC reduces oxoferrylmyoglobin more than an order more efficiently than S-nitrosoglutathione and glutathione. DNIC also inhibits formation of the thiyl radicals of glutathione in a medium containing metmyoglobin and tert-butyl hydroperoxide. A mechanism of the antioxidant action of DNIC based on regeneration of the nitrosyl complexes from the products of their interaction with oxoferrylheme is proposed.     

Interaction of reactive oxygen and nitrogen species with albumin- and methemoglobin-bound dinitrosyl-iron complexes.

Destructive effect of superoxide anions O2- derived from KO(2) or xanthine-xanthine oxidase system on dinitrosyl-iron complexes bound with bovine albumin or methemoglobin (DNIC-BSA or DNIC-MetHb) was demonstrated. The sensitivity of DNIC-BSA synthesized by the addition of DNIC with cysteine, thiosulfate or phosphate (DNIC-BSA-1, DNIC-BSA-2 or DNIC-BSA-3, respectively) to destructive action of O2- decreased in row: DNIC-BSA-1>DNIC-BSA-3>DNIC-BSA-2. The estimated rate constant for the reaction between O2- and DNIC-BSA-3 was equal to approximately 10(7)M(-1)s(-1). However, hydrogen peroxide and tert-butyl hydrogenperoxide (t-BOOH) did not induce any noticeable degradation of DNIC-BSA-3 even when used at concentrations exceeding by one order of magnitude those of the complex. As to their action on DNIC-MetHb both hydrogen peroxide and t-BOOH-induced rapid degradation of the complex. Both agents could induce the process due to the effect of alkylperoxyl or protein-derived free radicals formed at the interaction of the agents with ferri-heme groups of MetHb. Peroxynitrite (ONOO(-)) could also initiate protein-bound DNIC degradation more efficiently in the reaction with DNIC-BSA-3. Higher resistance of DNIC-MetHb to peroxynitrite was most probably due to the protective action of heme groups on ONOO(-). However, the analysis allows to suggest that the interaction of protein-bound DNICs with O2- is the only factor responsible for the degradation of the complexes in cells and tissues.     

Mechanisms of oxidative modification of low density lipoproteins under conditions of oxidative and carbonyl stress

Low-molecular-weight aldehydes (glyoxal, methylglyoxal, 3-deoxyglucosone) generated on autooxidation of glucose under conditions of carbonyl stress react much more actively with amino groups of L-lysine and epsilon-amino groups of lysine residues of apoprotein B-100 in human blood plasma low density lipoproteins (LDL) than their structural analogs (malonic dialdehyde (MDA), 4-hydroxynonenal) resulting on free radical oxidation of lipids under conditions of oxidative stress. Glyoxal-modified LDL aggregate in the incubation medium with a significantly higher rate than LDL modified by MDA, and MDA-modified LDL are markedly more poorly absorbed by cultured human macrophages and significantly more slowly eliminated from the rat bloodstream upon intravenous injection. Studies on kinetics of free radical oxidation of rat liver membrane phospholipids have shown that ubiquinol Q(10) is the most active lipid-soluble natural antioxidant, and suppression of ubiquinol Q(10) biosynthesis by beta-hydroxy-beta-methylglutaryl coenzyme A reductase inhibitors (statins) is accompanied by intensification of lipid peroxidation in rat liver biomembranes and in LDL of human blood plasma. Injection of ubiquinone Q(10) protects the human blood plasma LDL against oxidation and prevents oxidative stress-induced damages to rat myocardium. A unified molecular mechanism of atherogenic action of carbonyl-modified LDL in disorders of lipid and carbohydrate metabolism is discussed.     

Formation of superoxide radicals in isolated cardiac mitochondria: effect of adriamycin

The effect of adriamycin (doxorubicin) on superoxide radical formation in isolated rat heart mitochondria was studied by the spin trapping technique. The samples were placed into the cavity of EPR spectrometer in thin - wall gas - permeable capillary tubes, which allowed keeping the mitochondria of suspension in aerobic conditions. TIRON was used as a spin trap. We demonstrated that the rate of superoxide generation by isolated mitochondria depended radically on the presence of 1-150 microM adriamycin in incubation medium and was considerably higher than in control. The effect of adriamycin could be observed in the presence of both complex I (succinate) or complex II (glutamate and malate) substrates. The results obtained let to conclude that isolated cardiac mitochondria modified by adriamycin have a higher rate of production of superoxide radicals, which can react with spin traps not penetrating through the internal membrane.     

Influence of dicarbonyls on kinetic characteristics of glutathione peroxidase

Se-containing glutathione peroxidase (GSH-Px) is one of the key enzymes of the body’s antioxidant system. The kinetic characteristics of GSH-Px (substrate is tert-butyl hydroperoxide) after modification of the enzyme by various concentrations of natural dicarbonyls (glyoxal, methylglyoxal, malonic dialdehyde) were studied. It was shown that dicarbonyls affected both K _m and V _max for GSH-Px. It is suggested that the effect of various dicarbonyls on GSH-Px depends on the molecular mechanisms of their interaction with the amino acid residues of the enzyme.     

The generation of superoxide radicals by complex III in heart mitochondria and the antioxidant effect of dinitrosyl iron complexes at different partial pressures of oxygen

EPR spin trapping and EPR oximetry were used to study the superoxide radical generation in heart mitochondria from Wistar rats at various oxygen concentrations. Lithium phthalocyanine and TEMPONE-¹⁵N-D₁₆ were chosen to determine the oxygen content in a gas-permeable capillary containing mitochondria. TIRON was used as a spin trap. Several oxygen concentrations in the incubation mixture were tested; heart mitochondria were found to generate superoxide in complex III at various partial pressures of oxygen, including deep hypoxia (<5% O₂). Dinitrosyl iron complexes with glutathione (the drug Oxacom) exerted an antioxidant effect regardless of the partial pressure of oxygen; the magnitude and kinetic characteristics of the effect depended on the drug concentration.     

Antioxidants decreases the intensification of low density lipoprotein in vivo peroxidation during therapy with statins

The oxidative modification of low density lipoprotein (LDL) is thought to play an important role in atherogenesis. Drugs of -hydroxy--methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors (statins) family are usually used as a very effective lipid-lowering preparations but they simultaneously block biosynthesis of both cholesterol and ubiquinone Q₁₀ (coenzyme Q), which is an intermediate electron carrier in the mitochondrial respiratory chain. It is known that reduced form of ubiquinone Q₁₀ acts in the human LDL as very effective natural antioxidant. Daily per os administration of HMG-CoA reductase inhibitor simvastatin to rats for 30 day had no effect on high-energy phosphates (adenosin triphosphate, creatine phosphate) content in liver but decreased a level of these substances in myocardium. We study the Cu²⁺-mediated susceptibility of human LDL to oxidation and the levels of free radical products of LDL lipoperoxidation in LDL particles from patients with atherosclerosis after 3 months treatment with natural antioxidants vitamin E as well as during 6 months administration of HMG-CoA reductase inhibitors such as pravastatin and cerivastatin in monotherapy and in combination with natural antioxidant ubiquinone Q₁₀ or synthetic antioxidant probucol in a double-blind placebo-controlled trials. The 3 months of natural antioxidant vitamin E administration (400 mg daily) to patients did not increase the susceptibility of LDL to oxidation. On the other hand, synthetic antioxidant probucol during long-time period of treatment (3–6 months) in low-dose (250 mg daily) doesn't change the lipid metabolism parameters in the blood of patients but their high antioxidant activity was observed. Really, after oxidation of probucol-contained LDL by C-15 animal lipoxygenase in these particles we identified the electron spin resonance signal of probucol phenoxyl radical that suggests the interaction of LDL-associated probucol with lipid radicals in vivo. We observed that 6 months treatment of patients with pravastatine (40 mg daily) or cerivastatin (0.4 mg daily) was followed by sufficiently accumulation of LDL lipoperoxides in vivo. In contrast, the 6 months therapy with pravastatin in combination with ubiquinone Q₁₀ (60 mg daily) sharply decreased the LDL initial lipoperoxides level whereas during treatment with cerivastatin in combination with probucol (250 mg daily) the LDL lipoperoxides concentration was maintained on an invariable level. Therefore, antioxidants may be very effective in the prevention of atherogenic oxidative modification of LDL during HMG-CoA reductase inhibitors therapy.     

Dinitrosyl Iron Complexes with Glutathione Ligands Intercept Peroxynitrite and Protect Hemoglobin from Oxidative Modification

Applied Biochemistry and Microbiology - Dinitrosyl iron complexes with glutathione ligands (GS-DNICs) are a physiological form of nitric oxide that is stored and transported in the organism. In...