Biomedical subjects

J M May

Publications and source records attributed to J M May.

At least 37 records · Page 2Linked to original sources

PubMed1999

Inhibition of aldose reductase in human erythrocytes by vitamin C.

Ascorbic acid, or vitamin C, has been reported to lower erythrocyte sorbitol concentrations, and present studies were performed to determine the mechanism of this effect. Incubation of erythrocytes with increasing concentrations of glucose (5-40 mM) progressively increased erythrocyte sorbitol contents, reflecting increased flux through aldose reductase. At extracellular concentrations of 90 microM, both ascorbic acid and its oxidized form, dehydroascorbate, decreased intracellular sorbitol by 25 and 45%, respectively. This inhibition was not dependent on the extracellular glucose concentration, or on erythrocyte contents of free NADPH or GSH. To test for a direct effect of ascorbate on aldose reductase, erythrocyte hemolysates were prepared and supplemented with 100 microM NADPH. Hemolysates reduced glucose to sorbitol in a dose-dependent manner that was inhibited with a Ki of 120 microM by the aldose reductase inhibitor tetramethylene glutaric acid. Above 100 microM, ascorbic acid also lowered hemolysate sorbitol generation by about 30%. Studies with ascorbic acid derivatives showed that the reducing capacity of ascorbic acid was not required for inhibition of sorbitol production from glucose in erythrocyte hemolysates. These results show that high, but physiologic, concentrations of ascorbic acid can directly inhibit erythrocyte aldose reductase, and provide a rationale for the use of oral vitamin C supplements in diabetes.

Aldehyde Reductase↗

PubMed1999

Ascorbate 6-palmitate protects human erythrocytes from oxidative damage.

Lipid-soluble antioxidants, such as alpha-tocopherol, protect cell membranes from oxidant damage. In this work we sought to determine whether the amphipathic derivative of ascorbate, ascorbate 6-palmitate, is retained in the cell membrane of intact erythrocytes, and whether it helps to protect the cells against peroxidative damage. We found that ascorbate 6-palmitate binding to erythrocytes was dose-dependent, and that the derivative was retained during the multiple wash steps required for preparation of ghost membranes. Ascorbate 6-palmitate remained on the extracellular surface of the cells, because it was susceptible to oxidation or removal by several cell-impermeant agents. When bound to the surface of erythrocytes, ascorbate 6-palmitate reduced ferricyanide, an effect that was associated with generation of an ascorbyl free radical signal on EPR spectroscopy. Erythrocyte-bound ascorbate 6-palmitate protected membrane alpha-tocopherol from oxidation by both ferricyanide and a water-soluble free radical initiator, suggesting that the derivative either reacted directly with the exogenously added oxidant, or that it was able to recycle the alpha-tocopheroxyl radical to alpha-tocopherol in the cell membrane. Ascorbate 6-palmitate also partially protected cis-parinaric acid from oxidation when this fluorescent fatty acid was intercalated into the membrane of intact cells. These results show that an amphipathic ascorbate derivative is retained on the exterior cell surface of human erythrocytes, where it helps to protect the membrane from oxidant damage originating outside the cells.

Antioxidants↗

PubMed1999

Vitamin C recycling and function in human monocytic U-937 cells.

The uptake, recycling, and function of ascorbic acid was evaluated in cultured U-937 monocytic cells. Dehydroascorbic acid, the two-electron oxidized form of the vitamin, was taken up on the glucose transporter and reduced to ascorbate to a much greater extent than ascorbate itself was accumulated by the cells. In contrast to dehydroascorbic acid, ascorbate entered the cells on a sodium- and energy-dependent transporter. Intracellular ascorbate enhanced the transfer of electrons across the cell membrane to extracellular ferricyanide. Rates of ascorbate-dependent ferricyanide reduction were saturable, fivefold greater than basal rates, and facilitated by intracellular recycling of ascorbate. Whereas reduction of dehydroascorbic acid concentrations above 400 microM consumed reduced glutathione (GSH), even severe GSH depletion by 1-chloro-2,4-dinitrobenzene was without effect on the ability of the cells to reduce concentrations of dehydroascorbic acid likely to be in the physiologic range (< 200 microM). Dialyzed cytosolic fractions from U-937 cells reduced dehydroascorbic acid to ascorbate in an NADPH-dependent manner that appeared due to thioredoxin reductase. However, thioredoxin reductase did not account for the bulk of dehydroascorbic acid reduction, since its activity was also decreased by treatment of intact cells with 1-chloro-2,4-dinitrobenzene. Thus, U-937 cells loaded with dehydroascorbic acid accumulate ascorbate against a concentration gradient via a mechanism that is not dependent on GSH or NADPH, and this ascorbate can serve as the major source of electrons for transfer across the plasma membrane to extracellular ferricyanide.

Ascorbic Acid↗

PubMed1999

Is ascorbic acid an antioxidant for the plasma membrane?

Ascorbic acid, or vitamin C, is a primary antioxidant in plasma and within cells, but it can also interact with the plasma membrane by donating electrons to the alpha-tocopheroxyl radical and a trans-plasma membrane oxidoreductase activity. Ascorbate-derived reducing capacity is thus transmitted both into and across the plasma membrane. Recycling of alpha-tocopherol by ascorbate helps to protect membrane lipids from peroxidation. However, neither the mechanism nor function of the ascorbate-dependent oxidoreductase activity is known. This activity has typically been studied using extracellular ferricyanide as an electron acceptor. Whereas an NADH:ferricyanide reductase activity is evident in open membranes, ascorbate is the preferred electron donor within cells. The oxidoreductase may be a single membrane-spanning protein or may only partially span the membrane as part of a trans-membrane electron transport chain composed of a cytochrome or even hydrophobic antioxidants such as alpha-tocopherol or ubiquinol-10. Further studies are needed to elucidate the structural components, mechanism, and physiological significance of this activity. Proposed functions for the oxidoreductase include stimulation of cell growth, reduction of the ascorbate free radical outside cells, recycling of alpha-tocopherol, reduction of lipid hydroperoxides, and reduction of ferric iron prior to iron uptake by a transferrin-independent pathway.

Antioxidants↗

PubMed1999

Uptake and antioxidant effects of ergothioneine in human erythrocytes.

Ergothioneine is a fungal metabolite that may have antioxidant functions in mammalian cells. Although it accumulates to low millimolar concentrations in liver and other tissues, it is not thought to be taken up by mature erythrocytes. During a study of the function of ergothioneine as an antioxidant in human erythrocytes, we found that these cells do take up ergothioneine from the surrounding medium. Ergothioneine concentrations in freshly prepared erythrocytes were 2-9-fold higher than in plasma from the same donor. Slow but progressive accumulation of ergothioneine to about 125% of basal levels was observed in erythrocytes over a 4 h incubation. After a 2 h incubation, intracellular ergothioneine concentrations rose on addition of increasing amounts of ergothioneine to the incubation medium, although saturation was not evident in cells from all donors. Both initial levels and rates of ergothioneine uptake varied in erythrocytes from different donors. Intracellular ergothioneine was stable to depletion of GSH by N-ethylmaleimide and to a more severe oxidant stress induced by hydrogen peroxide in the presence of catalase. These results show that human erythrocytes do take up ergothioneine; however, the GSH results do not support an antioxidant role for ergothioneine in erythrocytes.

Adult↗

PubMed1998

Reduction of the ascorbyl free radical to ascorbate by thioredoxin reductase.

Recycling of ascorbic acid from its oxidized forms is required to maintain intracellular stores of the vitamin in most cells. Since the ubiquitous selenoenzyme thioredoxin reductase can recycle dehydroascorbic acid to ascorbate, we investigated the possibility that the enzyme can also reduce the one-electron-oxidized ascorbyl free radical to ascorbate. Purified rat liver thioredoxin reductase catalyzed the disappearance of NADPH in the presence of low micromolar concentrations of the ascorbyl free radical that were generated from ascorbate by ascorbate oxidase, and this effect was markedly stimulated by selenocystine. Dehydroascorbic acid is generated by dismutation of the ascorbyl free radical, and thioredoxin reductase can reduce dehydroascorbic acid to ascorbate. However, control studies showed that the amounts of dehydroascorbic acid generated under the assay conditions used were too low to account for the observed loss of NADPH. Electron paramagnetic resonance spectroscopy directly confirmed that the reductase decreased steady-state ascorbyl free radical concentrations, as expected if thioredoxin reductase reduces the ascorbyl free radical. Dialyzed cytosol from rat liver homogenates also catalyzed NADPH-dependent reduction of the ascorbyl free radical. Specificity for thioredoxin reductase was indicated by loss of activity in dialyzed cytosol prepared from livers of selenium-deficient rats, by inhibition with aurothioglucose at concentrations selective for thioredoxin reductase, and by stimulation with selenocystine. Microsomal fractions prepared from rat liver showed substantial NADH-dependent ascorbyl free radical reduction that was not sensitive to selenium depletion. These results suggest that thioredoxin reductase can function as a cytosolic ascorbyl free radical reductase that may complement cellular ascorbate recycling by membrane-bound NADH-dependent reductases.

Animals↗

PubMed1998

Enzyme-dependent ascorbate recycling in human erythrocytes: role of thioredoxin reductase.

Human erythrocytes efficiently reduce dehydroascorbic acid (DHA) to ascorbate, which helps to maintain the ascorbate content of blood. Whereas erythrocyte DHA reduction is thought to occur primarily through a direct chemical reaction with GSH, this work addresses the role of enzyme-mediated DHA reduction by these cells. The ability of intact erythrocytes to recycle DHA to ascorbate, estimated as DHA-dependent ferricyanide reduction, was decreased in parallel with GSH depletion by glutathione-S-transferase substrates. In contrast, the sulfhydryl reagent phenylarsine oxide inhibited DHA reduction to a much greater extent than it decreased GSH in intact cells. DHA reduction in excess of that due to a direct chemical reaction with GSH was also observed in freshly prepared hemolysates. Hemolysates likewise showed NADPH-dependent reduction of DHA that appeared due to thioredoxin reductase, because this activity was inhibited 68% by 10 microM aurothioglucose, doubled by 5 microM E. coli thioredoxin, and had an apparent Km for DHA (1.5 mM) similar to that of purified thioredoxin reductase. Additionally, aurothioglucose-sensitive, NADPH-dependent DHA reductase activity was decreased 80% in hemolysates prepared from phenylarsine oxide-treated cells. GSH-dependent DHA reduction in hemolysates was more than 10-fold that of NADPH-dependent reduction. Nonetheless, the ability of phenylarsine oxide to decrease DHA reduction in intact cells with little effect on GSH suggests that enzymes, such as thioredoxin reductase, may contribute more to this activity than previously considered.

Arsenicals↗

PubMed1998

Erythrocyte defenses against hydrogen peroxide: the role of ascorbic acid.

Ascorbate has been reported to increase intracellular hydrogen peroxide (H2O2) generation in human erythrocytes. In the present work, the basis for this prooxidant effect of the vitamin was investigated in the context of erythrocyte defenses against H2O2. Ascorbate added to erythrocytes caused a dose-dependent increase in intracellular H2O2, which was measured as inactivation of endogenous catalase in the presence of 3-amino-1,2,4-triazole (aminotriazole). Ascorbate-induced catalase inactivation was not observed when only the intracellular ascorbate concentration was increased, when cells were incubated with ascorbate in plasma, or when extracellular Fe3+ was chelated. Together, these results suggest that the observed ascorbate-induced H2O2 generation is due to Fe3+-catalyzed oxidation of extracellular, as opposed to intracellular, ascorbate by molecular oxygen. Rather than generate an oxidant stress in erythrocytes, ascorbate was one of the most sensitive intracellular antioxidants to H2O2 coming from outside the cells. On the other hand, intracellular ascorbate contributed little to the detoxification of H2O2, which was found to be mediated by both catalase and by the GSH system.

Ascorbic Acid↗

PubMed1998

Similarities in the metabolism of alloxan and dehydroascorbate in human erythrocytes.

The beta-cell toxin alloxan is reduced within cells to dialuric acid, which may then decompose to release damaging reactive oxygen species. We tested whether such redox cycling of alloxan occurs in the human erythrocyte, a cell with stronger antioxidant defenses than beta-cells. Erythrocytes incubated with increasing concentrations of alloxan progressively accumulated dialuric acid, as measured directly by HPLC with electrochemical detection. At concentrations up to 2 mM, alloxan decreased cellular GSH slightly, but did not affect erythrocyte contents of ascorbate or alpha-tocopherol. Intracellular H2O2 generation, measured as inhibition of endogenous catalase activity in the presence of 3-amino-1,2,4-triazole (aminotriazole), was decreased by alloxan. Despite its failure to induce significant oxidant stress in erythrocytes, 2 mM of alloxan doubled the activity of the hexose monophosphate pathway (HMP). This likely reflected consumption of reducing equivalents during reduction of alloxan to dialuric acid. Alloxan pretreatment enhanced the ability of erythrocytes to reduce extracellular ferricyanide while protecting alpha-tocopherol in the cell membrane from oxidation by ferricyanide. Ninhydrin, a hydrophobic derivative of alloxan, showed similar effects, but caused progressive GSH depletion and cell lysis at concentrations above 50 microM. The ability of alloxan to enhance ferricyanide reduction and to spare alpha-tocopherol suggests that dialuric acid or other reducing species within the cells can protect or recycle alpha-tocopherol and donate electrons to a transmembrane transfer process. This behavior resembles that observed for the dehydroascorbate (DHA)/ascorbate pair, and leads to the unexpected conclusion that alloxan increases the reducing capacity of the erythrocyte.

Alloxan↗

PubMed1998

Erythrocyte ascorbate recycling: antioxidant effects in blood.

Ascorbic acid is an important antioxidant in human plasma, but requires efficient recycling from its oxidized forms to avoid irreversible loss. Human erythrocytes prevented oxidation of ascorbate in autologous plasma, an effect that required recycling of ascorbate within the cells. Erythrocytes had a high capacity to take up dehydroascorbate, the two-electron oxidized product of ascorbate, and to reduce it to ascorbate. Uptake and conversion of dehydroascorbate to ascorbate was saturable, was half-maximal at 400 microM dehydroascorbate, and achieved a maximal intracellular ascorbate concentration of 1.5 mM. In the presence of 100 microM dehydroascorbate, erythrocytes had the capacity to regenerate a 35 microM ascorbate concentration in blood every 3 min. Ascorbate recycling from DHA required intracellular GSH. Depletion of erythrocyte GSH by more than 50% with diamide did not acutely affect the cellular ascorbate content, but did impair the subsequent ability of GSH-depleted cells to recycle dehydroascorbate to ascorbate. Whereas erythrocyte ascorbate recycling was coupled to GSH, an overwhelming extracellular oxidant stress depleted both ascorbate and alpha-tocopherol before the GSH content of cells fell appreciably. Recycled ascorbate was released from cells into plasma, but at a rate less than one tenth that of dehydroascorbate uptake and conversion to ascorbate. Nonetheless, ascorbate released from cells protected endogenous alpha-tocopherol in human LDL from oxidation by a water soluble free radical initiator. These results suggests that recycling of ascorbate in erythrocytes helps to maintain the antioxidant reserve of whole blood.

Antioxidants↗

PubMed1998

Protection and recycling of alpha-tocopherol in human erythrocytes by intracellular ascorbic acid.

Ascorbic acid can recycle alpha-tocopherol from the tocopheroxyl free radical in lipid bilayers and in micelles, but such recycling has not been demonstrated to occur across cell membranes. In this work the ability of intracellular ascorbate to protect and to recycle alpha-tocopherol in intact human erythrocytes and erythrocyte ghosts was investigated. In erythrocytes that were 80% depleted of intracellular ascorbate by treatment with the nitroxide Tempol, both 2,2'-azobis(2-amidinopropane) dihydrochloride (AAPH) and ferricyanide oxidized alpha-tocopherol to a greater extent than in cells not depleted of ascorbate. In contrast, in erythrocytes in which the intracellular ascorbate concentration had been increased by loading with dehydroascorbate, loss of alpha-tocopherol was less with both oxidants than in control cells. Protection against AAPH-induced oxidation of alpha-tocopherol was not prevented by extracellular ascorbate oxidase, indicating that the protection was due to intracellular and not to extracellular ascorbate. Incubation of erythrocytes with lecithin liposomes also generated an oxidant stress, which caused lipid peroxidation in the liposomes and depleted erythrocyte alpha-tocopherol, leading to hemolysis. Ascorbate loading of the erythrocytes delayed liposome oxidation and decreased loss of alpha-tocopherol from both cells and from alpha-tocopherol-loaded liposomes. When erythrocyte ghosts were resealed to contain ascorbate and challenged with free radicals generated by AAPH outside the ghosts, intravesicular ascorbate was totally depleted over 1 h of incubation, whereas alpha-tocopherol decreased only after ascorbate was substantially oxidized. These results suggest that ascorbate within the erythrocyte protects alpha-tocopherol in the cell membrane by a direct recycling mechanism.

Amidines↗

PubMed1998

Ascorbate function and metabolism in the human erythrocyte.

Ascorbic acid, or vitamin C, is an important antioxidant in plasma, where it consumes oxygen free radicals and helps to preserve alpha-tocopherol (vitamin E) in lipoproteins. Erythrocytes, as the most plentiful cell in blood, help to preserve ascorbate in the blood plasma. In contrast to nucleated cells, which avidly concentrate ascorbate, the erythrocyte ascorbate concentration is the same as that in plasma. Erythrocytes nonetheless have a high capacity to regenerate the vitamin from its two electron-oxidized form, dehydroascorbic acid (DHA). DHA is rapidly taken up by these cells on the abundant glucose transport protein, GLUT1. Intracellular DHA is rapidly reduced to ascorbate by GSH in a direct chemical reaction, although enzyme-dependent mechanisms involving both glutaredoxin and thioredoxin reductase have also been demonstrated. Ascorbate, which carries a negative charge at physiologic pH, enters and leaves the cells slowly. Nonetheless, this slow release of ascorbate from erythrocytes can preserve both the plasma concentration of the vitamin, and prevent oxidation of alpha-tocopherol in low-density lipoprotein. In addition, intracellular ascorbate can spare and possibly recycle alpha-tocopherol in the erythrocyte membrane. In turn, alpha-tocopherol protects the cell membrane from lipid peroxidation. The ability of erythrocytes to recycle ascorbate, coupled with the ability of ascorbate to protect alpha-tocopherol in the cell membrane and in lipoproteins, provides a potentially important mechanism for preventing lipid peroxidative damage in areas of inflammation in the vascular bed, such as those involved with atherosclerosis.

Antioxidants↗

PubMed1997

Reduction of dehydroascorbate to ascorbate by the selenoenzyme thioredoxin reductase.

Recycling of ascorbate from its oxidized forms is essential to maintain stores of the vitamin in human cells. Whereas reduction of dehydroascorbate to ascorbate is thought to be largely GSH-dependent, we reconsidered the possibility that the selenium-dependent thioredoxin system might contribute to ascorbate regeneration. We found that purified rat liver thioredoxin reductase functions as an NADPH-dependent dehydroascorbate reductase, with an apparent Km of 2. 5 mM for dehydroascorbate, and a kcat of 90 min-1. Addition of 2.8 microM purified rat liver thioredoxin lowered the apparent Km to 0.7 mM, without affecting the turnover (kcat of 71 min-1). Since thioredoxin reductase requires selenium, we tested the physiologic importance of this enzyme for dehydroascorbate reduction in livers from control and selenium-deficient rats. Selenium deficiency lowered liver thioredoxin reductase activity by 88%, glutathione peroxidase activity by 99%, and ascorbate content by 33%, but did not affect GSH content. NADPH-dependent dehydroascorbate reductase activity due to thioredoxin reductase, on the basis of inhibition by aurothioglucose, was decreased 88% in dialyzed liver cytosolic fractions from selenium-deficient rats. GSH-dependent dehydroascorbate reductase activity in liver cytosol was variable, but typically 2-3-fold that of NADPH-dependent activity. These results show that the thioredoxin system can reduce dehydroascorbate, and that this function is required for maintenance of liver ascorbate content.

Animals↗

PubMed1997

Liposomes containing alpha-tocopherol and ascorbate are protected from an external oxidant stress.

The interaction between alpha-tocopherol and ascorbate in protecting membrane lipids from peroxidation was studied in unilamellar liposomes in which alpha-tocopherol was incorporated into the liposomal membrane, and ascorbate was trapped within the vesicles. Extravesicular ferricyanide was reduced by ascorbate-derived electrons, and this was enhanced by the presence of alpha-tocopherol in the lipid bilayer. When a water-soluble free radical initiator was added to the outside of liposomes, intravesicular ascorbate prevented oxidation of alpha-tocopherol, and this effect was associated with complete protection against peroxidation of membrane lipids. These results suggest that ascorbate-dependent recycling of alpha-tocopherol can protect biological membranes from peroxidation by oxidants originating across the membrane bilayer from ascorbate.

Antioxidants↗

PubMed1996

Interaction of ascorbate and alpha-tocopherol in resealed human erythrocyte ghosts. Transmembrane electron transfer and protection from lipid peroxidation.

A role for ascorbate-derived electrons in protection against oxidative damage to membrane lipids was investigated in resealed human erythrocyte ghosts. Incubation of resealed ghosts with the membrane-impermeant oxidant ferricyanide doubled the ghost membrane concentration of F2-isoprostanes, a sensitive marker of lipid peroxidation. Incorporation of ascorbate into ghosts during resealing largely prevented F2-isoprostane formation due to extravesicular ferricyanide. This protection was associated with a rapid transmembrane oxidation of intravesicular ascorbate by extravesicular ferricyanide. Transmembrane electron transfer, which was measured indirectly as ascorbate-dependent ferricyanide reduction, correlated with the content of alpha-tocopherol in the ghost membrane in several respects. First, ascorbate resealed within ghosts protected against ferricyanide-induced oxidation of endogenous alpha-tocopherol in the ghost membrane. Second, when exogenous alpha-tocopherol was incorporated into the ghost membrane during the resealing step, subsequent ferricyanide reduction was enhanced. Last, incubation of intact erythrocytes with soybean phospholipid liposomes, followed by resealed ghost preparation, caused a proportional decrease in both the membrane content of alpha-tocopherol and in ferricyanide reduction. Incorporation of exogenous alpha-tocopherol during resealing of ghosts prepared from liposome-treated cells completely restored the ferricyanide-reducing capacity of the ghosts. These results suggest that the transmembrane transfer of ascorbate-derived electrons in erythrocyte ghosts is dependent in part on alpha-tocopherol and that such transfer may help to protect the erythrocyte membrane against oxidant stress originating outside the cell.

Ascorbic Acid↗

PubMed1996

Functional organization of mammalian hexokinase II. Retention of catalytic and regulatory functions in both the NH2- and COOH-terminal halves.

The mammalian hexokinase (HK) family includes three closely related 100-kDa isoforms (HKI-III) that are thought to have arisen from a common 50-kDa precursor by gene duplication and tandem ligation. Previous studies of HKI indicated that a glucose 6-phosphate (Glu-6-P)-regulated catalytic site resides in the COOH-terminal half of the molecule and that the NH2-terminal half contains only a Glu-6-P binding site. In contrast, we now show that proteins representing both halves of human and rat HKII have catalytic activity and that each is inhibited by Glu-6-P. The intact enzyme and the NH2- and COOH-terminal halves of the enzyme each increase glucose utilization when expressed in Xenopus oocytes. Mutations corresponding to either Asp-209 or Asp-657 in the intact enzyme completely inactivate the NH2- and COOH-terminal half enzymes, respectively. Mutation of either of these sites results in a 50% reduction of activity in the 100-kDa enzyme. Mutation of both sites results in a complete loss of activity. This suggests that each half of the HKII molecule retains catalytic activity within the 100-kDa protein. These observations indicate that HKI and HKII are functionally distinct and have evolved differently.

Animals↗

PubMed1996

Ascorbate recycling in human erythrocytes: role of GSH in reducing dehydroascorbate.

Human erythrocytes regenerate ascorbate from its oxidized product, dehydroascorbate. The extent to which such ascorbate recycling occurs by a GSH-dependent mechanism was investigated. In the presence of glucose, erythrocytes took up over 90% of extracellular [14C]dehydroascorbate and rapidly converted it to [14C]ascorbate, which was trapped within the cells. Dehydroascorbate uptake and reduction was not associated with generation of a monoascorbyl free radical intermediate. Uptake and reduction of dehydroascorbate by glucose-depleted erythrocytes coordinately decreased GSH and raised GSSG concentrations in erythrocytes. This effect was reversed by D-glucose, but not by L-lactate. Conversely, depletion of cellular GSH decreased the ability of cells to recycle dehydroascorbate to ascorbate, as reflected in the extent to which cells were able to reduce extracellular ferricyanide. Monoascorbyl free radical was formed during the reduction of extracellular ferricyanide, indicating that one electron transfer steps were involved in this process. In GSH-depleted cells, addition of L-lactate as an energy source for glycolysis-dependent NADH regeneration did cause a partial recovery of the ability of cells to reduce ferricyanide. However, in resealed erythrocyte ghosts containing either 4 mM GSH or 400 mu M NADH, only the GSH-containing ghosts supported regeneration of ascorbate from added dehydroascorbate. These results suggest that in human erythrocytes ascorbate regeneration from dehydroascorbate is largely GSH dependent, and that it occurs through either enzymatic or nonenzymatic reactions not involving the monoascorbyl free radical.

Ascorbic Acid↗

PubMed1996

Accessibility and reactivity of ascorbate 6-palmitate bound to erythrocyte membranes.

Lipophilic derivatives of ascorbic acid may protect lipid bilayers and micelles against lipid peroxidation. In this work the binding, accessibility, and reducing capacity of ascorbate 6-palmitate (A6P) were studied in human erythrocyte membranes. In contrast to less lipophilic carbon-6-modified ascorbate derivatives, A6P bound to erythrocyte membranes in a concentration-dependent manner. This binding was preserved following centrifugation washes, but was largely reversed by extraction with bovine serum albumin. Most of the ascorbyl groups of membrane-bound A6P were readily accessible to oxidation by water-soluble oxidants. Ferricyanide quantitatively oxidized membrane-bound A6P, but the latter spared endogenous tocopherols from destruction. In EPR studies, A6P was much more effective than ascorbate in reducing nitroxide spin labels positioned at either carbon-5 or carbon-16 of membrane-bound stearic acid in both intact cells and in membranes. A6P, thus, appears to intercalate into the erythrocyte membrane with the ascorbyl group located superficially, but with access to the hydrophobic membrane interior, and with the ability to recycle endogenous alpha-tocopherol during oxidant stress.

Antimutagenic Agents↗