The ability of O2 metabolites derived from the xanthine-xanthine oxidase system to inhibit mitochondrial function was examined using freshly isolated rat liver mitochondria. Under 2,4-dinitrophenol-uncoupled conditions, mitochondria exposed to free radicals exhibited a significant decrease in O2 consumption supported by NAD(+)-linked substrates, but showed almost no change in O2 consumption in the presence of succinate and ascorbate. Oxidative stress caused the loss of intramitochondrial nicotinamide nucleotides, and addition of NAD+ fully prevented any fall in O2 consumption with NAD(+)-linked substrates. The activity of electron-transfer complex I (NADH oxidase and NADH-cytochrome c oxidoreductase) and the energy-dependent reduction of NAD+ by succinate were unaltered by oxidative stress. Exposure to free radicals also had an uncoupling effect at all three coupling sites. The degree of mitochondrial swelling was closely correlated with the inhibition of State-3 oxidation of site-I substrates and with the increase in State-4 oxidation of succinate. The immunosuppressive agent cyclosporin A completely prevented the mitochondrial damage induced by oxygen free radicals (swelling, Ca2+ release, sucrose trapping, uncoupling and selective inhibition of the mitochondrial respiration of site-I substrates). The same protective effect was found when Ca2+ cycling was prevented, either by chelating Ca2+ with EGTA or by inhibiting Ca2+ reuptake with Ruthenium Red. These findings suggest that the deleterious effect of free radicals on mitochondria in the present experimental system was triggered by the cyclosporin A-sensitive and Ca(2+)-dependent membrane transition, and not by direct impairment of the mitochondrial inner-membrane enzymes.
A healthy, nonepileptic 16-month-old child ingested a massive overdose (approximately 4000 mg) of valproic acid (VPA). Upon admission to the hospital, he was in a deep coma and had generalized hypotonicity and no response to pain. His serum and urinary concentrations of VPA were 1316.2 and 3289.5 micrograms/mL, respectively. Urinary concentrations of the beta-oxidation metabolites of VPA were low, whereas concentrations of omega- and omega 1-oxidation metabolites were high. Moreover, 4-en-valproate (a potential hepatotoxin) was detected in the urine. Gastric lavage and general supportive measures were undertaken, including intravenous infusion to increase urine output and oral L-carnitine to correct hypocarnitinemia. Subsequently, the beta-oxidation metabolites increased, the omega- and omega 1-oxidation metabolites decreased, and 4-en-valproate was no longer detected. The patient recovered completely and was discharged on the eighth hospital day without any sequelae. This case suggests that enhanced drug excretion and L-carnitine supplementation may prevent potentially fatal hepatic dysfunction after VPA overdose.
Hepatic mitochondrial functions related to fatty acid metabolism, including the respiratory control ratio, fatty acid oxidative capacity and carnitine palmitoyltransferase I activity, were studied in vitro with mitochondria isolated from rats treated with thioacetamide for up to 12 wk. The levels of ketone bodies, carnitine, carnitine esters and malonyl-coenzyme A were also determined in liver extracts. Polarography of mitochondrial respiration from succinate or glutamate plus malate showed a lower respiratory control ratio in thioacetamide-treated rats, whereas uncoupled oxygen consumption was not altered. This suggests that the mitochondrial respiratory chain capacity remained intact in the thioacetamide-treated rats. The oxygen consumption associated with palmitoyl-coenzyme A and palmitoyl-L-carnitine oxidation by isolated liver mitochondria was increased by thioacetamide treatment on both a per-mitochondrial protein and a per-total liver basis. The carnitine palmitoyl-transferase I activity; the tissue levels of ketone bodies, carnitine and carnitine esters; and the beta-hydroxybutyrate/acetoacetate ratio were all higher in the livers of thioacetamide-treated animals than in control livers, whereas the hepatic malonyl-coenzyme A level was decreased by thioacetamide. These results indicate the increased diversion of cytosolic long-chain acyl-coenzyme As into the mitochondria for beta-oxidation rather than their esterification and use in lipogenesis. These intrahepatic metabolic changes induced by chronic thioacetamide administration may reflect the whole-body catabolic state and can be seen as adaptive for maintaining energy homeostasis under conditions of impaired glucose tolerance.
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