Reduced glutathione effects on alpha-tocopherol concentration of rat liver microsomes undergoing NADPH-dependent...

Factors involved in reduced glutathione (GSH) and vitamin E-mediated inhibition of NADPH-dependent rat liver microsomal lipid peroxidation were examined. Lipid peroxidation was monitored over a time-course of 180 min by thiobarbituric acid reactive product formation. The addition of 5 mM GSH to the reaction system containing microsomes from rats fed a diet supplemented with 150 IU/kg of alpha-tocopherol acetate for eight weeks produced a lag in peroxidation of greater than 30 min. This effect was not observed for microsomes prepared from rats fed a diet deficient in vitamin E. Indeed, a prooxidant effect of 5 mM GSH was observed in assays containing microsomes from rats fed a diet deficient in vitamin E. The inhibition by GSH of lipid peroxidation in microsomes prepared from livers of vitamin E supplemented rats was not restricted by its availability, for it was found that approximately 92% of the GSH remained in the reduced form after 60 min. Additional experiments revealed that the alpha-tocopherol content of peroxidizing microsomes decreased rapidly in the absence of GSH. The addition of 5 mM GSH to the assay system markedly depressed the loss of microsomal alpha-tocopherol. The results of in vivo labeling of liver microsomes with [14C]alpha-tocopherol demonstrated that i) GSH addition to the in vitro peroxidizing medium reduced the disappearance of alpha-tocopherol, and ii) a compound that interfered with the determination of alpha-tocopherol was separated by HPLC and was not an oxidation product of alpha-tocopherol. A portion of the microsomal 14C-labeled alpha-tocopherol was converted to an unidentified product with HPLC retention characteristics that was similar, but not identical, to alpha-tocopherol quinone.