Vitamin C Is an Essential Antioxidant That Enhances Survival of Oxidatively Stressed Human Vascular Endothelial Cells in the Presence of a Vast Molar Excess of Glutathione

Abstract

Cellular glutathione levels may exceed vitamin C levels by 10-fold, generating the question about the real antioxidant role that low intracellular concentrations of vitamin C can play in the presence of a vast molar excess of glutathione. We characterized the metabolism of vitamin C and its relationship with glutathione in primary cultures of human endothelial cells oxidatively challenged by treatment with hydrogen peroxide or with activated cells undergoing the respiratory burst, and analyzed the manner in which vitamin C interacts with glutathione to increase the antioxidant capacity of cells. Our data indicate that: (i) endothelial cells express transporters for reduced and oxidized vitamin C and accumulate ascorbic acid with participation of glutathione-dependent dehydroascorbic acid reductases, (ii) although increased intracellular levels of vitamin C or glutathione caused augmented resistance to oxidative stress, 10-times more glutathione than vitamin C was required, (iii) full antioxidant protection required the simultaneous presence of intracellular and extracellular vitamin C at concentrations normally found in vivo, and (iv) intracellular vitamin C cooperated in enhancing glutathione recovery after oxidative challenge thus providing cells with enhanced survival potential, while extracellular vitamin C was recycled through a mechanism involving the simultaneous neutralization of oxidant species. Therefore, in endothelial cells under oxidative challenge, vitamin C functions as an essential cellular antioxidant even in the presence of a vast molar excess of glutathione.Human cells contain two important water soluble antioxidants, vitamin C and the tripeptide glutathione (L-␥-glutamyl-L-cysteinyl-glycine). Vitamin C plays an important physiological role in cells as a reducing agent and antioxidant, free radical scavenger, and enzyme cofactor (1, 2). Glutathione is the most abundant non-protein thiol in mammalian cells and participates in multiple functions central to the physiology of cells, acting as a reducing agent, antioxidant, and free-radical scavenger and is involved in the metabolism and detoxification of xenobiotics, and alterations in GSH levels and metabolism have been associated with different human diseases (3, 4). Glutathione and vitamin C show a strong functional interdependence in vivo. Disruption of glutathione metabolism in vivo in rats and guinea pigs by treatment with buthionine-(SR)-sulfoximine (BSO), 5 a potent and specific glutathione synthesis inhibitor, revealed that the dysfunction and mortality associated with glutathione deficiency can be ameliorated by vitamin C supplementation (3, 5). Inversely, glutathione ester supplementation can protect or delay the effects of a vitamin C-free diet in newborn rats and guinea pigs unable to synthesize vitamin C (3, 6).Although a functional relationship between glutathione and vitamin C has been clearly established in rats and guinea pigs, we know little about how they cooperate in providing human cells with potent antioxidant defense mechan...

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