Shalu Mendiratta

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 K m of 2.5 mm for dehydroascorbate, and a k catof 90 min−1. Addition of 2.8 μm purified rat liver thioredoxin lowered the apparent K m to 0.7 mm, without affecting the turnover (k cat 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.

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.

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.

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.

Role of ascorbic acid in transferrin-independent reduction and uptake of iron by U-937 cells.

The role of ascorbic acid in transferrin-independent ferric iron reduction and uptake was evaluated in cultured U-937 monocytic cells. Uptake of 55Fe by U-937 cells was doubled by 100 microM extracellular ascorbate, and by pre-incubation of cells with 100 microM dehydroascorbic acid, the two-electron-oxidized form of ascorbate. Reduction of extracellular ferric citrate also was enhanced by loading the cells with dehydroascorbic acid. Dehydroascorbic acid was taken up rapidly by the cells and reduced to ascorbate, such that the latter reached intracellular concentrations as high as 6 mM. However, some ascorbate did escape the cells and could be detected at concentrations of up to 1 microM in the incubation medium. Further, addition of ascorbate oxidase almost reversed the effects of dehydroascorbic acid on both 55Fe uptake and ferric citrate reduction. Thus, it is likely that extracellular ascorbate reduced ferric to ferrous iron, which was then taken up by the cells. This hypothesis also was supported by the finding that during loading with ferric citrate, only extracellular ascorbate increased the pool of intracellular ferrous iron that could be chelated with cell-penetrant ferrous iron chelators. In contrast to its inhibition of ascorbate-dependent ferric iron reduction, ascorbate oxidase was without effect on ascorbate-dependent reduction of extracellular ferricyanide. This indicates that the cells use different mechanisms for reduction of ferric iron and ferricyanide. Therefore, extracellular ascorbate derived from cells can enhance transferrin-independent iron uptake by reducing ferric to ferrous iron, but intracellular ascorbate neither contributes to this reduction nor modifies the redox status of intracellular free iron.

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.

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.

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.

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.