Oxidation of vitamin E and vitamin C and inhibition of brain mitochondrial oxidative phosphorylation by peroxynitrite

The effects of peroxynitrite (PN; product of the reaction between nitric oxide and superoxide) on mitochondrial respiration as well as oxidation of alpha-tocopherol and ascorbic acid were studied. Mitochondria were isolated from brain hemispheres of 4-month-old male Fisher rats by standard centrifugation procedures utilizing Ficoll gradients. Treatment of brain mitochondria with PN caused a concentration-dependent impairment of oxidative phosphorylation and depletion of the endogenous antioxidants alpha-tocopherol and ascorbic acid. PN-induced mitochondrial dysfunction was characterized by 1) decreases in state 3 respiration and oxidative phosphorylation, 2) loss of respiratory control [ratio of ADP-stimulated (state 3) to basal (state 4) respiration], and 3) uncoupling of oxidative phosphorylation. PN did not function as a pure uncoupler, insofar as the increase in state 4 respiration was accompanied by a larger decrease in state 3 respiration. This contrasts with the uncoupling action of the protonophore carbonyl cyanide m-chlorophenylhydrozone, which increases both state 3 and state 4 respiration. PN-induced reduction in respiratory control and oxidative phosphorylation closely paralleled the oxidation of membrane tocopherol and were preceded by loss of ascorbate. alpha-Tocopherol (the most potent biological lipid antioxidant) may have a unique role in protecting mitochondrial membranes from oxidative stress. The two antioxidant nutrients alpha-tocopherol and ascorbate (which interact with each other and glutathione) may be intimately involved in protecting mitochondria in situations in which excessive release of superoxide and nitric oxide occurs under normal and/or pathological conditions. (C) 2004 Wiley-Liss, Inc.