Long-term objectives: To clarify the significance of regeneration of vitamin E from its radical form (recycling) by studying the interaction of enzymatic and non-enzymatic recycling mechanisms involving water-and lipid- soluble antioxidants with various forms of vitamin E. This will prove useful in establishing an optimal (as opposed to minimal) daily intake of vitamin E, clarify the relative efficacy of various forms of vitamin E in a more physiological manner, and help in the design of strategies aimed at preventing or ameliorating diseases, such as cancer, thought to be induced at least in part by oxidants.
Specific aims : To investigate: (1) The mechanisms of vitamin E radical recycling in mitochondria. This will clarify enzymatic recycling of vitamin E in an intracellular compartment. Liver mitochondrial membrane preparations (submitochondrial particles), liver outer membrane preparations (whose antioxidant recycling potential has not been investigated), electron transport complexes isolated from bovine heart and incorporated into liposomes, and intact mitochondria from alpha-tocopherol- deficient, -sufficient, and -supplemented animals will be studied. (2) Non- enzymatic recycling of vitamin E in membranes and in LDL. Studies of the vitamin E radical in liposomes will establish what compounds (such as glutathione, uric acid, bilirubin, dihydrolipoic acid) serve to non- enzymatically recycle the vitamin E radical. This is aimed especially at elucidating which compounds directly reduce vitamin E radicals, and which act indirectly (by recycling ascorbate to its reduced form, which can then recycle vitamin E). An important extracellular pool of vitamin E is in low density lipoprotein (LDL), and we will examine the ability of plasma antioxidants to recycle LDL vitamin E. (3) The contribution of recycling to vitamin E's overall antioxidant effectiveness. In physiological membranes such as microsomes and submitochondrial particles, quantitate the contribution of recycling to vitamin E's overall antioxidant effectiveness, and determine the extent to which enzymatic and non-enzymatic recycling contribute to total recycling. (4) The relationship between the relative antioxidant efficiencies of isomers of vitamin E and their relative recycling abilities. Vitamin E has 8 isomers, four tocoperols and four tocotrienols. alpha-tocotrienol has 40- to 60-fold greater antioxidant activity in vitro then alpha-tocopherol, and it also is more efficiently recycled. The relationship between antioxidant effectiveness and recycling efficiency will be investigated for other isomers tocopherol and tocotrienol. It is predicted that the two properties will be closely related. Tools: Membranes from animals on vitamin E-sufficient, -deficient, and - supplemented diets, as well as electron spin resonance for the detection of radicals, HPLC for the detection of intact antioxidant molecules, and NMR to evaluate mobility and membrane organization of isomers of vitamin E in model membranes. Vitamin E-deficient, compared to normal membranes, will allow investigation of a potential adaptive enzymatic response to vitamin E deficiency that improves the vitamin E recycling to prevent vitamin loss.
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