It has been suggested that oxidation of low-density lipoprotein (LDL) is a critical initiating step in human atherosclerosis, and that antioxidants can act as antiatherogens. Vitamin C (ascorbic acid, AA) is an antioxidant nutrient known to protect LDL against atherogenic modification in vitro more effectively than any other known natural or synthetic antioxidant. However, the mechanism of vitamin C's protective effect is unexplained, and it is not known whether vitamin C can act as antioxidant and antiatherogen in vivo. This proposal seeks to answer these questions. In the first part of the proposal, in vitro experiments will be conducted to identify the molecular mechanism of LDL protection by vitamin C. We have already shown that this mechanism is not explained by scavenging of free radicals by AA, but involves stable modification of LDL with decomposition product(s) of dehydroascorbic acid (DHA), the oxidation product of AA. Thus, the effects of AA, DHA, and selected DHA decomposition products on atherogenic modification of LDL will be investigated. LDL will be exposed to metal-ion-dependent (cupric ions or monocyte-macrophages) or metal-ion-independent (peroxyl radicals) oxidizing conditions. Progression of atherogenic modification of LDL will be assessed by measuring lipid peroxidation, electrophoretic mobility, and uptake of LDL by monocyte-macrophages. Possible mechanisms of action of vitamin C will be explored by investigating covalent modification of apolipoprotein B, chelation of metal ions, and peroxidase activity. We will also examine whether the reactions and mechanisms identified in vitro are operative in LDL in its natural plasma environment. Plasma will be incubated under oxidative stress to oxidize AA to DHA, or with DHA or its decomposition products. LDL will be isolated and examined for increased resistance to atherogenic modification. In the second part of the proposal, vitamin C's in vivo antioxidant and antiatherogenic effects will be investigated. Vitamin C-adequate and -marginally deficient guinea pigs on an atherogenic cholesterol diet will be exposed to hyperoxia, and compared to control animals on the same diets but without exposure to oxidative stress. In vivo lipid peroxidative damage will be assessed by measuring prostaglandin-like compounds formed by a free radical-catalyzed mechanism. LDL will be isolated from the animals and examined for antioxidant content and redox status as well as for susceptibility to atherogenic modification in vitro. Extent of atherosclerotic lesion development and tissue oxidative damage will be assessed at the conclusion of the study. In summary, in this proposal we will carry out a detailed analysis of vitamin C's antioxidant and antiatherogenic activities by studying its effects on three different levels, in vitro, in its natural plasma environment, and in vivo. The studies will elucidate fundamental mechanisms of antiatherogenesis and determine the importance of vitamin C as a dietary antioxidant and antiatherogen.
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