Reduced glutathione effects on α-tocopherol concentration of rat liver microsomes undergoing NADPH-dependent lipid peroxidation

Abstract

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 α-tocopherol acetate for eight weeks produced a lag in peroxidation of >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 α-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 α-tocopherol. The results ofin vivo labeling of liver microsomes with [¹⁴C] α-tocopherol demonstrated that i) GSH addition to thein vitro peroxidizing medium reduced the disappearance of α-tocopherol, and ii) a compound that interfered with the determination of α-tocopherol was separated by HPLC and was not an oxidation product of α-tocopherol. A portion of the microsomal¹⁴C-labeled α-tocopherol was converted to an unidentified product with HPLC retention characteristics that was similar, but not identical, to α-tocopherol quinone.