Roles of catalase and cytochrome C in hydroperoxide-dependent lipid peroxidation and chemiluminescence in rat heart and kidney mitochondria

A recent report (Radi et al., J. Biol. Chem. 266:22028–22034, 1991) showed that rat heart mitochondria contain catalase. The protective role of mitochondrial catalase was tested by exposing heart or kidney mitochondria and mitoplasts to two oxidants (H₂O₂) or tert-butyl hydroperoxide, t-BOOH), estimating lipid peroxidation (as thiobarbituric acid-reactive substances, TBARS) and overall oxidative stress (as chemiluminescence). Additional controls included heart and kidney preparations from aminotriazole-treated (catalase-depleted) rats. Both oxidants increased TBARS in catalase-free preparations to similar extents over their respective controls (between 200 to 350%). In catalase-containing preparations, H₂O₂ lipid peroxidation increased by only 40 to 96% over controls. Similar qualitative results were obtained when measuring chemiluminescence. The catalytic role of cytochrome c in mitochondrial lipid peroxidation was investigated by exposing either control or cytochrome-c-depleted kidney mitoplasts (catalase free) to either H₂O₂ or t-BOOH. Hydrogen-peroxide-dependent mitochondrial lipid peroxidation varied with cytochrome c concentrations, remaining close to controls when cytochrome c concentration decreased by 66%, even though there was no catalase present. Tert-butyl hydroperoxide-dependent lipid peroxidation was less affected by cytochrome c remaining 2.3-fold above controls under the same conditions, suggesting that organic peroxides are more likely to remain in the less polar membrane environment being decomposed by heme or nonheme iron imbedded in the inner mitochondrial membrane. Chemiluminescence was less affected by cytochrome c depletion. Comparing control and cytochrome-c-deficient mitochondria, chemiluminescence was 1.7-fold and 2.8-fold higher when control preparations were challenged with t-BOOH or H₂O₂, respectively.     

Mitochondrial superoxide production and respiratory activity: Biphasic response to ischemic duration

Long bouts of ischemia are associated with electron transport chain deficits and increases in free radical production. In contrast, little is known regarding the effect of brief ischemia on mitochondrial function and free radical production. This study was undertaken to examine the relationship between the duration of ischemia, effects upon electron transport chain activities, and the mitochondrial production of free radicals. Rat hearts were subjected to increasing ischemic durations, mitochondria were isolated, and superoxide production and electron transport chain activities were measured. Results indicate that even brief ischemic durations induced a significant increase in superoxide production. This rate was maintained with ischemic durations less than 15 min, and then increased further with longer ischemic times. Mechanistically, brief ischemia was accompanied by an increase in NADH oxidase activity, reflected by a specific increase in complex IV activity. In contrast, longer ischemic durations were accompanied by a decrease in NADH oxidase activity, reflected by deficits in complexes I and IV activities.     

Role of vitamin E as a lipid-soluble peroxyl radical scavenger: in vitro and in vivo evidence

Multiple reactive oxygen/nitrogen species induce oxidative stress. Mammals have evolved with an elaborate defense network against oxidative stress, in which multiple antioxidant compounds and enzymes with different functions exert their respective roles. Radical scavenging is one of the essential roles of antioxidants and vitamin E is the most abundant and important lipophilic radical-scavenging antioxidant in vivo. The kinetic data and physiological molar ratio of vitamin E to substrates show that the peroxyl radicals are the only radicals that vitamin E can scavenge to break chain propagation efficiently and that vitamin E is unable to act as a potent scavenger of hydroxyl, alkoxyl, nitrogen dioxide, and thiyl radicals in vivo. The preventive effect of vitamin E against the oxidation mediated by nonradical oxidants such as hypochlorite, singlet oxygen, ozone, and enzymes may be limited in vivo. The synergistic interaction of vitamin E and vitamin C is effective for enhancing the antioxidant capacity of vitamin E. The in vitro and in vivo evidence of the function of vitamin E as a peroxyl radical-scavenging antioxidant and inhibitor of lipid peroxidation is presented.     

Sensitivity of mitochondria isolated from liver and kidney of rat and bovine to lipid peroxidation: A comparative study of light emission and fatty acid profiles

Much work has been carried out on non-enzymatic–induced lipid peroxidation of mitochondria obtained from different tissues of monogastric animals, but little information is available about this process in poligastric animals. Studies were carried out to determine the sensitivity of mitochondria isolated from liver and kidney of rat and bovine to lipid peroxidation (ascorbate-Fe²⁺ dependent) by comparison of light emission and fatty acid profiles. Mitochondria from both species were susceptible to lipid peroxidation. Measurements of chemiluminescence indicate that the lipid peroxidation process was more effective in mitochondria from rat liver than in the organelle obtained from bovine, whereas changes were not observed in mitochondria from rat and bovine kidney. The fatty acid composition of total lipids isolated from liver and kidney mitochondria of both species was substantially modified when subjected to non-enzymatic lipid peroxidation with a decrease of arachidonic and docosahexaenoic acids. The polyunsaturated fatty acid (PUFA) composition was higher in mitochondria obtained from rat liver (43.11± 4.16) than in bovine (15.78 ± 0.76). As a consequence, the unsaturation index (UI), was higher in mitochondria of rat liver than in bovine. Nevertheless, the PUFA composition of kidney mitochondria from both species was similar; therefore, statistically significant differences in the UI were not observed. The results suggest that mainly the PUFAs present in hepatic and kidney mitochondria were sensitive to oxidative damage. The lipid peroxidation process was more effective in rat liver mitochondria than in bovine. (Mol Cell Biochem xxx: 77–82, 2005) Member of Carrera del Investigador Científico, Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET)