Vitamin E oxidation in rat liver mitochondria.

Antioxidant reactions of alpha-tocopherol (vitamin E, alpha-TH) were studied by examining the fate of alpha-TH during oxidative challenge to mitochondrial membranes. Rat liver mitochondria were exposed to increasing concentrations of the water-soluble radical initiator 2,2'-azobis(2-amidinopropane) dihydrochoride (ABAP), and damage was assessed by monitoring mitochondrial respiration, alpha-TH oxidation, and lipid peroxidation. Significant lipid peroxidation was observed after 50% of the initial alpha-TH was depleted. Oxidative damage produced by ABAP-generated peroxyl radicals inhibited mitochondrial use of O2, as indicated by decreases in the respiratory control ratio and in state 3 and state 4 respiration. Rat liver mitochondria were supplemented with [14C]-alpha-TH by incubation of liver homogenate with [14C]-alpha-TH for 30 min at room temperature, followed by isolation of mitochondria by differential centrifugation. This supplementation resulted in a distribution of 83.7% and 14.3% of the added alpha-TH to the inner and outer mitochondrial membranes, respectively, which is similar to the distribution of endogenous alpha-TH. [14C]-alpha-TH-supplemented mitochondria then were treated with ABAP, and alpha-TH oxidation products were identified by radiochromatographic analysis of mitochondrial extracts. Products observed included alpha-tocopherolquinone, alpha-tocopherolquinone-2,3-oxide, and alpha-tocopherolquinone-5,6-oxide, which were identified by comparing HPLC retention and UV spectra to those of authentic standards. Product identities were verified by GC-MS of product O-trimethylsilyl derivatives. Another product, which was identified by HPLC, UV, and mass spectral analysis as 8a-(ethyldioxy)tocopherone, was found to be an artifact of sample workup and was shown to be derived from 8a-hydroperoxytocopherone, which was formed by alpha-TH oxidation in the mitochondria. These results indicate that alpha-TH antioxidant reactions in mitochondria are similar to those identified in homogeneous solutions and model liposomal systems.