[Frontiers in Bioscience 3, d1-10, January 1, 1998]

	[Frontiers in Bioscience 3, d1-10, January 1, 1998] Reprints PubMed CAVEAT LECTOR
	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 5. ASCORBATE RECYCLING IN ERYTHROCYTES: MECHANISMS AND CAPACITY 5.1 Mechanisms of erythrocyte ascorbate recycling The mechanism by which erythrocytes reduce intracellular DHA to ascorbate was initially considered to be NADH-dependent (23). However, more recent studies have suggested that such recycling is primarily GSH- and NADPH-dependent (figure 2 ) (24). Excess GSH can chemically reduce DHA to ascorbate (7, 25). This reaction has been documented in dialyzed erythrocyte hemolysates following addition of GSH, and with a GSH-regenerating system (26). We found additional evidence for direct GSH-dependent recycling of DHA to ascorbate in resealed erythrocyte ghosts, in which the cytoplasm is diluted 40-fold. In ghosts that had been resealed to contain 4 mM GSH, recycling of DHA to ascorbate was several-fold more efficient than was recycling in ghosts resealed without additives, or than was recycling in ghosts resealed in the presence of 400 micromolar NADH (20). Erythrocytes also have enzymes that can facilitate GSH-dependent reduction of DHA to ascorbate. The thioltransferase glutaredoxin has been shown to reduce DHA using GSH as a cofactor (27), and an enzyme with similar properties has been purified from human erythrocytes (28). The most likely mechanism for either direct (29) or enzyme-mediated (27) GSH-dependent DHA reduction involves nucleophilic addition of the thiyl anion of GSH to carbon-3 of DHA, followed by reduction by another molecule of GSH to form the ascorbate double bond and GSSG (figure 3). It was first shown in lens epithelial cells that added DHA depletes GSH and causes a rise in GSSG and in the activity of the hexose monophosphate shunt (29, 30). In erythrocytes incubated without glucose, we also found that addition of DHA in low millimolar concentrations decreases GSH and increases GSSG proportionately (20). In the presence of glucose, DHA did not affect the erythrocyte content of GSH. This failure of GSH to fall in the presence of glucose probably reflects recycling of GSSG to GSH by glutathione reductase when adequate NADPH is available from the hexose monophosphate shunt (26). Two additional results also support a GSH-dependent mechanism of erythrocyte DHA reduction. First, depletion of cellular GSH by 50-70% with diamide decreased the ability of glucose-depleted cells to recycle DHA to ascorbate (20). Diamide selectively oxidizes GSH to GSSG (31), although the agent can react with pyridine nucleotides (32). Second, in electron paramagnetic resonance studies, we found no increase in the AFR signal during the first few minutes of uptake and reduction of 1 mM DHA by intact erythrocytes (20). If the AFR is an intermediate in the recycling of DHA to ascorbate, it should have been apparent in this experiment. DHA can also be reduced to ascorbate by the NADPH-dependent enzyme thioredoxin reductase (33). This reaction is facilitated by the presence of thioredoxin. Thioredoxin reductase has been shown to be present in erythrocytes by immunologic methods (34), and has been purified from this cell type (35). Additional support for enzyme-dependent DHA reduction, whether mediated by GSH or NADPH, is our observation that DHA loading of erythrocytes is rapid and saturable, with an apparent K_m of 200 micromolar (16). We have also found that erythrocyte hemolysates support NADPH-dependent reduction of DHA to ascorbate, which is enhanced by 5 micromolar thioredoxin from E. coli, and inhibited by 10 micromolar aurothioglucose (Mendiratta, S. and May, J.M., unpublished observations). At a concentration of 10 micromolar, aurothioglucose is selective for thioredoxin reductase (36). Whereas thioredoxin can recycle DHA to ascorbate in the erythrocyte, GSH-dependent mechanisms are likely to predominate. This may not be the case for other cell types, since HL-60 cells show no decrease in their ability to recycle DHA to ascorbate when their GSH is severely depleted (37). Figure 3. Proposed mechanism for direct reduction of DHA by GSH. 5.2 Capacity of erythrocytes for ascorbate recycling The ability of erythrocytes to recycle DHA to ascorbate is substantial. We have used DHA-stimulated reduction of ferricyanide by erythrocytes to estimate this capacity. Ferricyanide is a mild oxidant that does not penetrate the erythrocyte cell membrane (38, 39). Its extracellular reduction to ferrocyanide can be easily measured in a sensitive spectrophotometric assay (40). Assay of ferricyanide reduction is non-destructive to the cells, and DHA-induced increases in ferricyanide reduction provide an integrated measure of ascorbate recycling. Orringer and Roer (23) proposed that DHA-induced ferricyanide reduction by erythrocytes involves uptake of DHA by the cells, intracellular reduction of DHA to ascorbate by an NADH-dependent process, exit of ascorbate from the cells, and direct reaction with ferricyanide. Subsequent studies (41, 42) have shown that extracellular ferricyanide reduces intracellular, rather than extracellular ascorbate. The mechanism of this trans-membrane electron transfer from ascorbate to ferricyanide has not been established. It may be mediated by a trans-plasma membrane oxidoreductase, which has long been known to use NADH as an electron donor (43, 44). We found that ascorbate can also serve as an electron donor to this process. In ascorbate-loaded cells, the vitamin may be the major electron donor (45). Basal rates of ferricyanide reduction by erythrocytes are increased many-fold by providing the cells with DHA for conversion to ascorbate. Further, DHA-induced ferricyanide reduction is saturable with regard to the extracellular DHA concentration, and parallels the intracellular ascorbate content of the cells (42, 45). Using the initial rate of ferricyanide reduction at non-saturating DHA concentrations, which is about 40 nmol (ml erythrocytes)^-1 min^-1, we have calculated that erythrocytes in a milliliter of blood can regenerate a 40 micromolar concentration of ascorbate in this blood about once every three minutes (42). Similar estimates of ascorbate recycling capacity were determined from more recent studies in which initial rates of DHA reduction to ascorbate were measured directly in erythrocytes (16). The ascorbate recycling capacity of erythrocytes provides ascorbate to supplement the already substantial antioxidant defenses of the cells (42), and to serve as a source of ascorbate for export into plasma, as discussed in the next section.

[Frontiers in Bioscience 3, d1-10, January 1, 1998]
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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

5. ASCORBATE RECYCLING IN ERYTHROCYTES: MECHANISMS AND CAPACITY

5.1 Mechanisms of erythrocyte ascorbate recycling

The mechanism by which erythrocytes reduce intracellular DHA to ascorbate was initially considered to be NADH-dependent (23). However, more recent studies have suggested that such recycling is primarily GSH- and NADPH-dependent (figure 2 ) (24). Excess GSH can chemically reduce DHA to ascorbate (7, 25). This reaction has been documented in dialyzed erythrocyte hemolysates following addition of GSH, and with a GSH-regenerating system (26). We found additional evidence for direct GSH-dependent recycling of DHA to ascorbate in resealed erythrocyte ghosts, in which the cytoplasm is diluted 40-fold. In ghosts that had been resealed to contain 4 mM GSH, recycling of DHA to ascorbate was several-fold more efficient than was recycling in ghosts resealed without additives, or than was recycling in ghosts resealed in the presence of 400 micromolar NADH (20).

Erythrocytes also have enzymes that can facilitate GSH-dependent reduction of DHA to ascorbate. The thioltransferase glutaredoxin has been shown to reduce DHA using GSH as a cofactor (27), and an enzyme with similar properties has been purified from human erythrocytes (28). The most likely mechanism for either direct (29) or enzyme-mediated (27) GSH-dependent DHA reduction involves nucleophilic addition of the thiyl anion of GSH to carbon-3 of DHA, followed by reduction by another molecule of GSH to form the ascorbate double bond and GSSG (figure 3). It was first shown in lens epithelial cells that added DHA depletes GSH and causes a rise in GSSG and in the activity of the hexose monophosphate shunt (29, 30). In erythrocytes incubated without glucose, we also found that addition of DHA in low millimolar concentrations decreases GSH and increases GSSG proportionately (20). In the presence of glucose, DHA did not affect the erythrocyte content of GSH. This failure of GSH to fall in the presence of glucose probably reflects recycling of GSSG to GSH by glutathione reductase when adequate NADPH is available from the hexose monophosphate shunt (26). Two additional results also support a GSH-dependent mechanism of erythrocyte DHA reduction. First, depletion of cellular GSH by 50-70% with diamide decreased the ability of glucose-depleted cells to recycle DHA to ascorbate (20). Diamide selectively oxidizes GSH to GSSG (31), although the agent can react with pyridine nucleotides (32). Second, in electron paramagnetic resonance studies, we found no increase in the AFR signal during the first few minutes of uptake and reduction of 1 mM DHA by intact erythrocytes (20). If the AFR is an intermediate in the recycling of DHA to ascorbate, it should have been apparent in this experiment.

DHA can also be reduced to ascorbate by the NADPH-dependent enzyme thioredoxin reductase (33). This reaction is facilitated by the presence of thioredoxin. Thioredoxin reductase has been shown to be present in erythrocytes by immunologic methods (34), and has been purified from this cell type (35). Additional support for enzyme-dependent DHA reduction, whether mediated by GSH or NADPH, is our observation that DHA loading of erythrocytes is rapid and saturable, with an apparent K_m of 200 micromolar (16). We have also found that erythrocyte hemolysates support NADPH-dependent reduction of DHA to ascorbate, which is enhanced by 5 micromolar thioredoxin from E. coli, and inhibited by 10 micromolar aurothioglucose (Mendiratta, S. and May, J.M., unpublished observations). At a concentration of 10 micromolar, aurothioglucose is selective for thioredoxin reductase (36). Whereas thioredoxin can recycle DHA to ascorbate in the erythrocyte, GSH-dependent mechanisms are likely to predominate. This may not be the case for other cell types, since HL-60 cells show no decrease in their ability to recycle DHA to ascorbate when their GSH is severely depleted (37).

Figure 3. Proposed mechanism for direct reduction of DHA by GSH.

5.2 Capacity of erythrocytes for ascorbate recycling

The ability of erythrocytes to recycle DHA to ascorbate is substantial. We have used DHA-stimulated reduction of ferricyanide by erythrocytes to estimate this capacity. Ferricyanide is a mild oxidant that does not penetrate the erythrocyte cell membrane (38, 39). Its extracellular reduction to ferrocyanide can be easily measured in a sensitive spectrophotometric assay (40). Assay of ferricyanide reduction is non-destructive to the cells, and DHA-induced increases in ferricyanide reduction provide an integrated measure of ascorbate recycling. Orringer and Roer (23) proposed that DHA-induced ferricyanide reduction by erythrocytes involves uptake of DHA by the cells, intracellular reduction of DHA to ascorbate by an NADH-dependent process, exit of ascorbate from the cells, and direct reaction with ferricyanide. Subsequent studies (41, 42) have shown that extracellular ferricyanide reduces intracellular, rather than extracellular ascorbate. The mechanism of this trans-membrane electron transfer from ascorbate to ferricyanide has not been established. It may be mediated by a trans-plasma membrane oxidoreductase, which has long been known to use NADH as an electron donor (43, 44). We found that ascorbate can also serve as an electron donor to this process. In ascorbate-loaded cells, the vitamin may be the major electron donor (45). Basal rates of ferricyanide reduction by erythrocytes are increased many-fold by providing the cells with DHA for conversion to ascorbate. Further, DHA-induced ferricyanide reduction is saturable with regard to the extracellular DHA concentration, and parallels the intracellular ascorbate content of the cells (42, 45). Using the initial rate of ferricyanide reduction at non-saturating DHA concentrations, which is about 40 nmol (ml erythrocytes)^-1 min^-1, we have calculated that erythrocytes in a milliliter of blood can regenerate a 40 micromolar concentration of ascorbate in this blood about once every three minutes (42). Similar estimates of ascorbate recycling capacity were determined from more recent studies in which initial rates of DHA reduction to ascorbate were measured directly in erythrocytes (16).

The ascorbate recycling capacity of erythrocytes provides ascorbate to supplement the already substantial antioxidant defenses of the cells (42), and to serve as a source of ascorbate for export into plasma, as discussed in the next section.