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Oxidation of the biological antioxidant alpha-tocopherol (vitamin E; TH) by peroxyl radicals yields 8a-(alkyldioxy)tocopherones, which either may hydrolyze to alpha-tocopheryl quinone (TQ) or may be reduced by ascorbic acid to regenerate TH. To define the chemistry of this putative two-electron TH redox cycle, we studied the hydrolysis and reduction of 8a-[(2,4-dimethyl-1-nitrilopent-2-yl)dioxy]tocopherone (1) in acetonitrile/buffer mixtures and in phospholipid liposomes. TQ formation in acetonitrile/buffer mixtures, which was monitored spectrophotometrically, declined with increasing pH and could not be detected above pH 4. The rate of TQ formation from 1 first increased with time and then decreased in a first-order terminal phase. Rearrangement of 8a-hydroxy-alpha-tocopherone (2) to TQ displayed first-order kinetics identical with the terminal phase for TQ formation from 1. Both rate constants increased with decreasing pH. Hydrolysis of 1 in acetonitrile/H2(18)O yielded [18O]TQ. These observations suggest that 1 loses the 8a-(alkyldioxy) moiety to produce the tocopherone cation (T+), which hydrolyzes to 2, the TQ-forming intermediate. Incubation of either 1 or 2 with ascorbic acid in acetonitrile/buffer yielded TH. Reduction of both 1 and 2 decreased with increasing pH. In phosphatidylcholine liposomes at pH 7, approximately 10% of the T+ generated from 1 was reduced to TH by 5 mM ascorbic acid. The results collectively demonstrate that T+ is the ascorbic acid reducible intermediate in a two-electron TH redox cycle, a process that probably would require biocatalysis to proceed in biological membranes.

A phospholipid liposome system was employed to model peroxyl radical trapping reactions of alpha-tocopherol (1) in biological membranes. Peroxyl radicals generated by thermolysis of 2,2'-azobis(2,4-dimethylvaleronitrile) (AMVN) at 37 degrees C oxidized 1 to 8a-[(2,4-dimethyl-1-nitrilopent-2-yl)dioxy]tocopherone (3a), 8a-(hydroperoxy)tocopherone (3b), alpha-tocopherol quinone (4), 4a,5-epoxy-8a-hydroperoxytocopherone (6), 2,3-epoxy-alpha-tocopherol quinone (7), and 5,6-epoxy-alpha-tocopherol quinone (8). The products were purified by high-performance liquid chromatography and characterized by UV-vis spectroscopy, mass spectrometry, and cochromatography with authentic standards. Products accumulated in approximately constant proportion as 1 was consumed. Tocopherones 3a/3b decomposed in the bilayer primarily by hydrolyzing to produce 4. Tocopherone decomposition also produced small amounts of epoxides 6-8, apparently by unimolecular tocopherone decomposition rather than by peroxyl radical dependent oxidation, since neither AMVN nor 1 affected the rate of 3a loss or the distribution of products. Epoxides 6-8 appear to be formed primarily by autoxidation reactions that compete with the peroxyl radical trapping reactions that form tocopherone 3a. Epoxide products may thus serve as biochemical markers for irreversible oxidation of 1 by peroxyl radicals in membranes.

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