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Tocopherols and tocotrienols (vitamin E) and ascorbic acid (vitamin C) as well as the carotenoids react with free radicals, notably peroxyl radicals, and with singlet molecular oxygen (1O2), this being the basis of their function as antioxidants. RRR-alpha-tocopherol is the major peroxyl radical scavenger in biological lipid phases such as membranes or low-density lipoproteins (LDL). L-Ascorbate is present in aqueous compartments (e.g. cytosol, plasma, and other body fluids) and can reduce the tocopheroxyl radical; it also has a number of metabolically important cofactor functions in enzyme reactions, notably hydroxylations. Upon oxidation, these micronutrients need to be regenerated in the biological setting, hence the need for further coupling to nonradical reducing systems such as glutathione/glutathione disulfide, dihydrolipoate/lipoate, or NADPH/NADP+ and NADH/NAD+. Carotenoids, notably beta-carotene and lycopene as well as oxycarotenoids (e.g. zeaxanthin and lutein), exert antioxidant functions in lipid phases by free-radical or 1O2 quenching. There are pronounced differences in tissue carotenoid patterns, extending also to the distribution between the all-trans and various cis isomers of the respective carotenoids. Antioxidant functions are associated with lowering DNA damage, malignant transformation, and other parameters of cell damage in vitro as well as epidemiologically with lowered incidence of certain types of cancer and degenerative diseases, such as ischemic heart disease and cataract. They are of importance in the process of aging. Reactive oxygen species occur in tissues and cells and can damage DNA, proteins, carbohydrates, and lipids. These potentially deleterious reactions are controlled in part by antioxidants that eliminate prooxidants and scavenge free radicals. Their ability as antioxidants to quench radicals and 1O2 may explain some anticancer properties of the carotenoids independent of their provitamin A activity, but other functions may play a role as well. Tocopherols are the most abundant and efficient scavengers of peroxyl radicals in biological membranes. The water-soluble antioxidant vitamin C can reduce tocopheroxyl radicals directly or indirectly and thus support the antioxidant activity of vitamin E; such functions can be performed also by other appropriate reducing compounds such as glutathione (GSH) or dihydrolipoate. The biological efficacy of the antioxidants is also determined by their biokinetics.

Nitrosothiols are powerful vasodilators. They act by releasing nitric oxide, which activates the heme protein guanylate cyclase. We have studied the kinetics of nitrosothiol formation of glutathione, cysteine, N-acetylcysteine, human serum albumin, and bovine serum albumin upon reaction with nitric oxide (NO) in the presence of oxygen. These studies have been made at low pH as well as at physiological pH. At pH 7.0, contrary to published reports, nitric oxide by itself does not react with thiols to yield nitrosothiol. However, formation of nitrosothiols is observed in the presence of oxygen. For all thiols studied, the rates of nitrosothiol formation were first order in O2 concentration and second order in NO concentration and at lower concentrations (< 5 mM thiol) also depended on thiol concentrations. Analysis of the kinetic data indicated that the rate-limiting step was the reaction of NO with oxygen. Analysis of the reaction products suggest that the main nitrosating species is N2O3: RSH+N2O3-->RSNO+NO2- + H+. Rate constants for this reaction for glutathione and several other low molecular weight thiols are in the range of 3-1.5 x 10(5) M-1 s-1, and for human and bovine serum albumins 0.3 x 10(5) M-1 s-1 and 0.06 x 10(5) M-1 s-1, respectively. The data further indicate that the reaction rate of the nitrosating species N2O3 with thiols is competitive with its rate of hydrolysis. At physiological concentrations nitrosoglutathione formation represents a significant metabolic fate of N2O3, and at glutathione concentrations of 5 mM or higher almost all of N2O3 formed is consumed in nitrosation of glutathione. Implications of these results for in vivo nitrosation of thiols are discussed.

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The analysis of beta-carotene and lycopene, the two predominant carotenoids in human serum and tissues, was extended to the level of geometrical (cis-trans) isomers by using an improved reversed-phase HPLC methodology. We separated five geometrical isomers of beta-carotene and seven of lycopene in human serum and tissues. 13-cis-beta-Carotene was identified as the predominant cisisomer in human serum, contributing about 5% to total beta-carotene. In tissue, however, considerable amounts of 9-cis- and traces of 15-cis-beta-carotene were also detected. In contrast to beta-carotene, the lycopene isomer patterns in human serum and tissues are quite similar.