Involvement of vitamin E and protein thiols in the inhibition of microsomal lipid peroxidation by glutathione

Abstract

Iron-ascorbate stimulated lipid peroxidation in rat liver microsomes can be inhibited by glutathione (GSH). The role of protein thiols and vitamin E in this process was studied in liver microsomes isolated from rats fed diets either sufficient or deficient in vitamin E and incubated at 37°C unde 100% O₂. Lipid peroxidation was induced by adding 400 μM adenosine 5′-triphosphate, 2.5 to 20 μM FeCl₃, and 450 μM ascorbic acid. One mL of the incubation mixture was removed at defined intervals for the measurement of thiobarbituric acid reactive substances (TBARS), protein thiols and vitamin E. In vitamin E sufficient microsomes, the addition of GSH enhanced the lag time prior to the onset of maximal TBARS accumulation and inhibited the loss of vitamin E. Treatment of these microsomes with the protein thiol oxidant diamide resulted in a 56% loss of protein thiols, but did not significantly change vitamin E levels. However, diamide treatment abolished the GSH-mediated protection against TBARS formation and loss of vitamin E during ascorbate-induced peroxidation. Liver microsomes isolated from rats fed a vitamin E deficient diet contained 40-fold less vitamin E and generated levels of TBARS similar to vitamin E sufficient microsomes at a 4-fold lower concentration of iron. GSH did not affect the lag time prior to the onset of maximal TBARS formation in vitamin E deficient microsomes although total TBARS accumulation was inhibited. Similar to what was previously found in vitamin E sufficient microsomes [Palamanda and Kehrer, (1992)Arch. Biochem. Biophys. 293, 103–109], GSH prevented the loss of protein thiols in vitamin E deficient microsomes. However, GSH did not protect efficiently against the loss of residual vitamin E in deficient microsomes. These data provide support for the concept that GSH protects against microsomal lipid peroxidation by maintaining protein thiols, and consequently vitamin E, in the reduced state. The lack of protection in vitamin E deficient microsomes may be related to the inability of such low levels of vitamin E to inhibit peroxidation.