The lipid-soluble antioxidant alpha-tocopherol (vitamin E) is the principal cellular membrane protectant against injury cased by reactive free radical metabolites of toxic and carcinogenic chemicals. Vitamin E is thought to function in membranes via a redox cycle in which vitamin E transfers electrons to free radicals to yield a reversibly oxidized vitamin E product, which is then regenerated by other cellular reductants. Failure to complete the redox cycle results from irreversible reactions of vitamin E and leads to vitamin E turnover. The purpose of the proposed research is to develop techniques for quantifying the protectant function of vitamin E and to apply these techniques to examine differences in the capacity of vitamin E to protect different cellular membranes from different tissues. The initial objective of this work is to identify and develop assays for vitamin E oxidation products in a model membrane system (liposomes) and to test the utility of these procedures in a more complex biological membrane system (rat liver microsomes). The next objective is to identify vitamin E oxidation products that could be used as marker products for the passage of vitamin E through redox cycles and for vitamin E turnover. Estimation of vitamin E redox cycling and turnover will then be tested in liposomes and in microsomes. The third objective is to apply these techniques to examine the impact of endogenous glutathione-dependent antioxidant enzymes on vitamin E function in microsomes. Finally, techniques for quantifying vitamin E function will be applied to subcellular membrane fractions from tissues that in their rates of vitamin E turnover in vivo. Membrane fractions will be challenged with toxic chemicals known to induce free radical injury including paraquat, methyl ethyl ketone peroxide, doxorubicin, and tertbutyl hydroperoxide. This work will develop means of monitoring vitamin E function in living tissues and can be applied to future studies in vivo of the effects of toxic and carcinogenic chemicals that produce free radicals.
