ASCORBATE FUNCTION AND METABOLISM IN THE HUMAN ERYTHROCYTE

James M. May

Departments of Medicine and Molecular Physiology and Biophysics, 736 Medical Research Building II, Vanderbilt University School of Medicine, Nashville, TN 37232-6303 USA

Received 11/5/97 Accepted 11/9/97

6. ANTIOXIDANT FUNCTION OF ASCORBATE IN PLASMA AND IN ERYTHROCYTES

6.1 Ascorbate as an antioxidant in plasma

Ascorbate has been shown to be the primary antioxidant in plasma, since its presence is required to prevent lipid hydroperoxide formation in plasma lipoproteins (46-48). More specifically, ascorbate directly protects against peroxide-mediated oxidation of plasma low- density lipoprotein (LDL) (49, 50). LDL is the primary atherogenic lipoprotein in human plasma, and is cleared by LDL receptors and by a scavenger pathway (50). If the surface lipids and protein sulfhydryls of LDL become oxidized, its receptor-mediated clearance is markedly impaired, and its uptake occurs largely by the scavenger pathway in monocyte-derived macrophages (50, 51). The latter in turn become the lipid-laden foam cells found in atherosclerotic plaques (52). Ascorbate probably acts synergistically with the tocopherols in protecting against LDL oxidation (49, 53). Ascorbate can both consume oxygen free radicals before they can oxidize alpha-tocopherol, and can reduce alpha-tocopherol in LDL in the face of an oxidant stress (49, 54). These antioxidant effects of ascorbate in plasma may explain the early observations of Willis that acute and chronic scurvy in guinea pigs produced lesions in the arterial intima that are indistinguishable from those of human atherosclerosis (55).

Erythrocytes are likely to be an important source of ascorbate in plasma, if nothing else because of their abundance. As noted earlier, erythrocytes take up ascorbate (as opposed to DHA) very slowly, and ascorbate efflux is also on the order of hours (16). Nonetheless, it appears that ascorbate efflux from erythrocytes can maintain the steady-state plasma ascorbate concentration during prolonged incubations in oxygenated serum (16, 19, 56), and even in the presence of an oxidant stress generated by AAPH (42). The latter is a free radical initiator that releases water-soluble radicals at a constant rate as a function of the temperature. These radicals are efficiently scavenged by ascorbate in plasma (47).

We also found that erythrocytes and ascorbate generated by the cells protect alpha-tocopherol in LDL against oxidation by AAPH, as diagramed in figure 4 (16). This protection was due in part to consumption by erythrocytes of oxygen free radical species generated from AAPH. Nonetheless, loading erythrocytes with ascorbate by incubating them with DHA provided even greater protection against loss of alpha-tocopherol in LDL than did erythrocytes alone. The protective effect of DHA-loading on LDL alpha-tocopherol was reversed when ascorbate oxidase was present outside the cells, which suggests that it was due to ascorbate that had left the cells.

Figure 4. Tocopherol recycling by ascorbate. Abbreviations: RBC, human erythrocyte; ASC, ascorbate; T, alpha-tocopherol; X, free radical species.

In addition to release of ascorbate from erythrocytes, the presence of an AFR reductase on the outer surface of the cells could also contribute to ascorbate recycling. One-electron oxidation of ascorbate outside erythrocytes would provide AFR to this enzyme for recycling back to ascorbate using intracellular reducing equivalents. Evidence for this activity has been presented for leukemic cells in culture (57, 58) and AFR reductase activity has been demonstrated in isolated erythrocyte membranes (59). The source of reducing equivalents for this process is presumably intracellular, but has not been identified.

By helping to maintain plasma ascorbate concentrations, erythrocyte ascorbate recycling may preserve alpha-tocopherol in LDL, prevent LDL oxidation, and scavenge oxidants released by monocytes and leukocytes. The latter effect will in turn prevent damage to the vascular endothelium.

6.2 Role of ascorbate in protecting erythrocytes from oxidative stress

A question related to whether ascorbate recycled by erythrocytes protects plasma lipoproteins from oxidative damage is whether ascorbate recycling protects the erythrocyte itself from oxidant stress, especially since this cell is equipped with so many different types of defenses against oxygen free radicals (60). In the cytoplasm, enzymatic defenses against both endogenous and exogenous oxidants include catalase (61), glutathione peroxidase/reductase, and superoxide dismutase (62). The primary non-enzymatic antioxidant defenses in the erythrocyte cytoplasm are GSH and ascorbate (63), although less well studied thiols such as ergothioneine (64, 65) may also contribute. In unpublished observations, we have found that ascorbate is more sensitive to oxidation by exogenous H₂O₂ generated by the glucose oxidase system than are GSH and alpha-tocopherol, but that it contributes little to defense of the cells against overwhelming oxidant stress.

Defenses against oxidant damage in the erythrocyte plasma membrane involve prevention and reversal of peroxidation of unsaturated fatty acids in the lipid bilayer (66). In the face of a profound external oxidant stress to the erythrocyte, the plasma membrane is often the initial site of damage; the resulting peroxidation of membrane lipids then causes hemolysis (67). Quantitatively, alpha-tocopherol is the most important antioxidant in the cell membrane (66). This vitamin scavenges peroxide free radicals and converts them to less toxic lipid hydroperoxides (figure 5) (3). In so doing, it protects the cell membrane and decreases hemolysis (66, 68). Several studies have shown that the erythrocyte content of alpha-tocopherol correlates directly with the resistance of the cell to oxidant-induced hemolysis (69-71). Indeed, prior to the availability of more specific assays, H₂O₂-induced hemolysis was used as an indirect measure of the membrane content of this vitamin (72, 73). Further, addition of alpha-tocopherol to erythrocytes either in vivo or in vitro protects the cells against hemolysis induced by gamma irradiation (70) and by aqueous free radicals (74, 75). To a lesser extent, ubiquinol-10 (76, 77) and membrane protein sulfhydryls (78) may also contribute to protection of the membrane. The GSH-dependent phospholipid hydroperoxidase can reduce membrane lipid hydroperoxides (79, 80), and can also spare alpha-tocopherol in cellular membranes (81). This enzyme has not been shown to directly reduce alpha-tocopherol, however.

Figure 5. Recycling of lipid peroxyl radicals by alpha-tocopherol (Toc), and of alpha-tocopherol by ascorbate (Asc).

Given the primacy of alpha-tocopherol in protecting the erythrocyte membrane, it is crucial that it either be replaced or recycled when oxidized. Transfer of the vitamin from plasma lipoproteins likely provides the major source of new alpha-tocopherol to the cells, but such transfer is relatively slow, with a half-time of several hours (82, 83). Thus it is likely that alpha-tocopherol is recycled in the cell membrane from the alpha-tocopheroxyl free radical (figure 5). This process appears to be efficient, given the observation that erythrocyte membranes contain low concentrations of alpha-tocopherolquinone, the major two-electron oxidation product of the vitamin (69). The most plausible mechanism for alpha-tocopherol recycling involves ascorbate.

Ascorbate does not directly affect membrane lipid peroxidation (84), but it may perform this function indirectly by reducing the tocopheroxyl free radical in the lipid bilayer (85, 86). The mechanism of such recycling presumably involves reduction of the alpha-tocopheroxyl free radical at the aqueous-lipid interface of the membrane bilayer (figure 5) (3). Ascorbate has been shown to recycle the alpha-tocopheroxyl radical in variety of in vitro systems: in solution (85, 87), in lipid or detergent micelles (85, 87-89), in liposomes (84, 90, 91), in microsomes and mitochondria (92, 93), and in isolated erythrocyte membranes (94). However, there is controversy regarding whether ascorbate can recycle alpha-tocopherol in cells (95, 96).

If ascorbate recycles alpha-tocopherol in intact cells, the resistance to oxidation of alpha-tocopherol in the cell membrane should parallel changes in the intracellular ascorbate content. This was first demonstrated by Stocker, et al. (97), who reported that increased intracellular ascorbate protects membrane alpha-tocopherol in Plasmodium vinckei-infected erythrocytes. We recently found that increasing the erythrocyte ascorbate content by loading with DHA protects against ferricyanide- and AAPH-induced loss of erythrocyte alpha-tocopherol (98). Conversely, selective ascorbate depletion with the nitroxide Tempol (99) increases loss of alpha-tocopherol in response to both oxidants. The rationale for these experiments is that both oxidants are restricted to the extracellular space because of their size and charge, and thus should attack alpha-tocopherol in the membrane before interacting with intracellular ascorbate. The observed preservation of alpha-tocopherol in proportion to the cellular ascorbate content is in line with a recycling mechanism. A role for ascorbate recycling of alpha-tocopherol would also be supported by demonstrating that the vitamin is depleted with or before alpha-tocopherol and GSH in the face of an oxidant stress (100). This is complicated in intact cells by the recycling of DHA to ascorbate, since such recycling will tend to maintain the ascorbate concentration. In intact erythrocytes, we were able to show that loss of alpha-tocopherol lags behind that of ascorbate (100). On the other hand, Glascott, et al. (95, 96) found in cultured rat liver cells that ascorbate and alpha-tocopherol behaved independently during an exogenous oxidant stress. However, both erythrocytes and hepatocytes can recycle ascorbate, and rat hepatocytes can synthesize it. These capacities are likely to confound detection of any relationship between ascorbate and alpha-tocopherol. To overcome this problem, we used resealed erythrocyte ghosts, in which the capacity to recycle ascorbate is greatly diminished by a 40-fold dilution of the cytoplasmic contents. These ghosts can be tightly sealed in the presence of ascorbate, and followed for their ability to preserve alpha-tocopherol in the membrane in response to an oxidant stress. Intravesicular ascorbate protected against loss of alpha-tocopherol in the ghost membranes, whether induced by ferricyanide (101) or by AAPH (98). In the latter studies, alpha-tocopherol began to decrease only after intravesicular ascorbate was almost depleted. These results in both cells and ghosts show that ascorbate can spare, and perhaps recycle alpha-tocopherol in the cell membrane.

Despite the above in vitro evidence in support of a role for ascorbate in recycling alpha-tocopherol, there remain caveats in the reported in vivo experiments in the guinea pig model. Guinea pigs, like humans, cannot synthesize ascorbate, and so can be used to obtain different stages of ascorbate deficiency. Burton, et al. (102) studied the kinetics of alpha-tocopherol turnover at various levels of ascorbate depletion or repletion in these animals, and found that the turnover kinetics were not affected by the ascorbate status of the animal. On the other hand, Bendich et al. (103) found that ascorbate supplementation in ascorbate-deficient guinea pigs increased the alpha-tocopherol content in plasma at one level of alpha-tocopherol intake, and increased the alpha-tocopherol content in lung at all levels of alpha-tocopherol intake. As noted by Liebler (100), use of oxidatively stressed animals in such studies might provide a better test of whether ascorbate can "spare" alpha-tocopherol in vivo. Despite the conflicting in vivo results, the findings outlined above at the cell level do favor a sparing or recycling role for ascorbate in the preservation of alpha-tocopherol, both in plasma lipoproteins, and in the cell membrane.