Oxidation of LDL induced by free radicals proceeds by a chain mechanism to give phosphatidylcholine hydroperoxides and cholesteryl ester hydroperoxides as the major primary products. In addition, apolipoprotein B100 is also oxidized. Various antioxidants suppress the oxidative modification of LDL. Water-soluble radical-scavenging antioxidants such as vitamin C and uric acid act as the first defense to suppress the chain initiation. Lipophilic radical-scavenging antioxidants in LDL such as vitamin E and ubiquinol scavenge radicals attacking from outside and also within the LDL. The overall importance and potency of antioxidants depend not only on chemical reactivity but also on the physical factors such as location and mobility at the microenvironment in LDL.

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We studied the location of α -tocopherol (α -Toc) in the liposome membranes, and the dynamics of its radical scavenging and recycling by ascorbic acid. The quenching efficiency of α -Toc fluorescence by acrylamide, a water soluble quencher with a very low capacity to penetrate through phospholipid bilayers, was very low in dimyristoyl-phosphatidylcholine (DMPC) liposomes with and without charges, but relatively high in sodium dodecylsulphate (SDS) or tetradecyl-trimethylammonium bromide (TTAB) micelles. These findings indicate the low exposure of the chromanol at the surface of the liposome membranes. α -Toc was oxidized by positively charged Fe³⁺ more slowly in DMPC liposomes negatively charged with dicetylphosphate (DCP) (1st order rate constant, 1.41×10^-3 sec^-1) than in negatively charged SDS micelles (7.14×10^-1 sec^-1). Assuming that 100% of the OH-groups of α -Toc are at the membrane surface of the SDS micelles, as the oxidation rate of α -Toc in liposomes is 0.32μM sec^-1, which is about 150 times slower than that in micelles (49.3μM sec^-1), only 0.65% of the OH-groups of α -Toc are probably present at the membrane surface of the liposomes. The fluorescence of α -Toc was most effectively quenched by interaction with the spin group of the probe 5-(N-oxyl-4, 4'-dimethyloxazolidin-2-yl) stearic acid (5-NS), indicating that its OH-group was located in a position corresponding to an inner 5-methylene carbon under the membrane surface. Ascorbic acid (AsA) was rapidly oxidized by 2, 2'-azobis (2, 4-dimethylvaleronitrile) (AMVN) when it was ionically trapped at the positively charged membrane surface of egg yolk phosphatidylcholine (egg PC) liposomes with stearylamine (SA), but was scarcely oxidized in negatively charged egg PC-DCP liposomes because it was present in the bulk water phase. These findings suggest that lipid peroxy-radicals move from the hydrophobic region to near the membrane surface, where they are trapped by α -Toc. The electron spin resonance (ESR) spectra of 5-NS and 16-NS labeled in DMPC or DMPC-DCP liposomes were not changed by the addition of AsA in the buffer solution of pH 7.0, indicating that negatively charged AsA could not penetrate into neutrally or negatively charged membranes. α -Toc inhibited AMVN-induced lipid peroxidation and AsA extended its inhibition period, but glutathione (GSH) did not affect this inhibition period. Oxidation of α -Toc in association with inhibition of lipid peroxidation was suppressed completely in the presence of negatively charged AsA and slightly in the presence of neutrally charged GSH, although AsA and GSH could not penetrate into these liposome membranes. These findings suggest that the resulting reversible oxidized product, probably the α -Toc cation, moves to the membrane surface, where α -Toc is regenerated by AsA.

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Endogenous oxidation reactions are essential for the normal biochemistry of life and are especially critical for leukocyte microbial killing mechanisms in host defense to infectious diseases. However, reactive oxidative intermediates can damage normal tissues unless kept under antioxidant control. Three selected examples of oxidant-antioxidant systems involved in infectious diseases are discussed, regulation of molecular iron catalyzed oxidations, superoxide scavengers and inhibitors of nitric oxide synthase in septic shock, and the use of glutathione replacement therapy in HIV infection and AIDS. The data suggest that antioxidants, and therapy based on increasing antioxidant potential, have a major impact on clinical infectious diseases.

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This paper reviewed the recent advance in the antioxidant therapy for digestive diseases. Many reports have supported that lipid peroxidation mediated by oxygen radicals is implicated in the pathogenesis of gastric mucosal injury, intestinal damage, acute pancreatitis, and liver injury, and several kinds of antioxidant, which are divided into the preventive antioxdant and chain-breaking antioxdant, are effective in the treament of these diseases. A new therapeutic approach using synthesized antioxidants, such as zinc-carnosinc chelate compound and ebselen, has been also proposed in these field.

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Hypercholesterolemia attributable to increased plasma concentrations of low density lipoproteins is a well recognized risk factor for the premature development of coronary atherosclerosis in both experimental animals and humans. Recent studies have indicated that modifications to low density lipoprotein result in enhanced uptake of the modified lipoproteins by macrophages and lead to accelerated rates of lipid deposition and the creation of foam cells. Oxidation of low density lipoprotein has been shown to be one of the modifications which leads to uptake of this lipoprotein by scavenger receptors present on macrophages and results in intracellular lipid accumulation. Treatment of hypercholesterolemic animals with antioxidant drugs, including probucol, has been shown to reduce the development of atherosclerosis and xanthoma regression has been observed in patients with severe hypercholesterolemia treated with this drug. Epidemiologic studies support the view that low plasma concentrations of antioxidant vitamins, including vitamin E are associated with higher rates of coronary atherosclerosis in humans and that supplementation with vitamin E is associated with a decreased incidence of coronary artery disease. Prospective clinical trials to assess the potential benefit of antioxidant supplementation in high risk patients are currently in progress and these trials, when completed, should provide definitive information concerning the potential benefits to be derived from supplementation with antioxidant vitamins as an adjunctive therapy to prevent the premature development of atherosclerosis.

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This work was carried out to estimate the preventive effect of vitamin E on oxy radical-enhanced lung tumorigenesis in ddY mice. We have reported that oxy radicals could be an important factor contributing to the promotive effect of glycerol on 4-nitroquinoline 1-oxide (4NQO)-induced lung tumorigenesis (1). The glycerol-promoted lung tumorigenesis of mice treated with 4NQO was reduced in mice feeding on excessive vitamin E in this study. The levels of nuclear thiobarbituric acid reactive substances (TBARS) and oxidative damage of DNA estimated as DNA single strand breaks (DNA-SSB) were significantly higher in the lungs of mice treated with 4NQO + glycerol than in those treated with 4NQO at 4 weeks after 4NQO administration, This increase was suppressed by the feeding of excessive vitamin E for 4 weeks after 4NQO injection. At 23 weeks after 4NQO injection, the feeding of excessive vitamin E for 4 and 23 weeks after 4NQO injection could cancel the promotive effect of glycerol on lung tumorigenesis. Additionally, the α-tocopherol level in serum was related with the degree of lung tumorigenesis at 23 weeks after 4NQO injection. These findings suggest that vitamin E can act as a useful agent to protect mice from oxy radical-promoted lung tumorigenesis.

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Under selected conditions, vitamin A and carotenoids can both accept and donate electrons, and carotenoids can also quench singlet oxygen. Thus both sets of compounds can theoretically participate in a biological antioxidant network. Under physiological conditions, vitamin A esters are transported and stored in a lipid matrix that contains other antioxidants, and retinol and its active metabolites are largely bound in clefts of specific retinoid-binding proteins. Thus, vitamin A seems to be protected in vivo by other antioxidants and proteins rather than protecting other molecules. Carotenoids are largely distributed in lipoproteins, membranes, and the lipid phases of intracellular structures, usually together with vitamin E. Carotenoids can interact with other antioxidants in vitro, but whether they play similar significant roles in vivo is not clear. Nonetheless, some genetic conditions and precancerous lesions respond to carotenoids, and the dietary intake of carotenoids has been associated with a reduced risk of several chronic diseases. Carotenoids seem to act per se in such systems rather than by their conversion into vitamin A.

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The nutritional status with respect to vitamins A and E, and beta-carotene was examined in elderly Japanese subjects in two institutions at Osaka and Kyoto. Only the plasma vitamin E level has been determined in the majority of previous investigations. In this study, vitamin E levels were determined in red blood cells (RBCs), platelets (PLT), mononuclear cells (MN), polymorphonuclear cells (PMN), and buccal mucosal cells (BMC), using HPLC with electrochemical detection. Alpha-tocopherol levels in plasma and RBCs did not differ between elderly and young adults, while those in PLT, MN, and BMC were lower in the elderly. Thus, the vitamin E status of elderly Japanese individuals appears to be inadequate of the cellular levels. The daily vitamin E intake of the elderly subjects was below the recommended dietary allowance for the Japanese population. Plasma levels of retinol and beta-carotene were also assessed. The vitamin A status did not differ between elderly and young adults on the basis of the levels of retinol and retinol-binding protein (RBP). The daily intake of retinol (as retinol equivalent) by the elderly subjects was more than 2, 000 IU. With respect to beta-carotene, there was a large sex difference (female>male), which was more prominent in the young adults and became smaller in the elderly. This sex difference was partly attributable to a difference in plasma total lipids. No clear age-related trend was noted.

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Dietary restriction is widely recognized to be the most effective means of intervening in the aging process in laboratory animals. Recent studies have produced substantial evidence that indicates this nutritional manipulation can modulate many aspects of free radical metabolism, including free radical generation, lipid peroxidation, DNA damage and cytosolic defense systems. Some of our laboratory's recent work is summarized in this presentation.

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