Human erythrocyte recycling of ascorbic acid: relative contributions from the ascorbate free radical and dehydroascorbic acid

Recycling of ascorbic acid from its oxidized forms helps to maintain the vitamin in human erythrocytes. To determine the relative contributions of recycling from the ascorbate radical and dehydroascorbic acid, we studied erythrocytes exposed to a trans-membrane oxidant stress from ferricyanide. Ferricyanide was used both to induce oxidant stress across the cell membrane and to quantify ascorbate recycling. Erythrocytes reduced ferricyanide with generation of intracellular ascorbate radical, the concentrations of which saturated with increasing intracellular ascorbate and which were sustained over time in cells incubated with glucose. Ferricyanide also generated dehydroascorbic acid that accumulated in the cells and incubation medium to concentrations much higher than those of the radical, especially in the absence of glucose. Ferricyanide-stimulated ascorbate recycling from dehydroascorbic acid depended on intracellular GSH but was well maintained at the expense of intracellular ascorbate when GSH was severely depleted by diethylmaleate. This likely reflects continued radical reduction, which is not dependent on GSH. Erythrocyte hemolysates showed both NAD- and NADPH-dependent ascorbate radical reduction. The latter was partially due to thioredoxin reductase. GSH-dependent dehydroascorbate reduction in hemolysates, which was both direct and enzyme-dependent, was greater than that of the radical reductase activity but of lower apparent affinity. Together, these results suggest an efficient two-tiered system in which high affinity reduction of the ascorbate radical is sufficient to remove low concentrations of the radical that might be encountered by cells not under oxidant stress, with back-up by a high capacity system for reducing dehydroascorbate under conditions of more severe oxidant stress.     

Ascorbic acid spares alpha-tocopherol and decreases lipid peroxidation in neuronal cells

Ascorbic acid is considered an antioxidant in the central nervous system, but direct evidence that ascorbate protects neuronal cells from oxidant stress is lacking. Differentiated SH-SY5Y cells in culture took up ascorbic acid on the sodium-dependent vitamin C transporter Type 2 and retained it much more effectively than dehydroascorbic acid. Intracellular ascorbate spared alpha-tocopherol, both in cells loaded with alpha-tocopherol in culture and in cells under oxidant stress due to extracellular ferricyanide. Sparing of alpha-tocopherol in response to ferricyanide was associated with protection against lipid peroxidation in cell membranes. These results show that neuronal cells concentrate ascorbate, and that intracellular ascorbate, either directly or through sparing of alpha-tocopherol, protects them against oxidant stress.     

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.     

Vitamin C homeostasis in skeletal muscle cells

In skeletal muscle, vitamin C not only enhances carnitine biosynthesis but also protects cells against ROS generation induced by physical exercise. The ability to take up both ascorbic and dehydroascorbic acid from the extracellular environment, together with the ability to recycle the intracellular vitamin, maintains high cellular stores of ascorbate. In this study, we examined vitamin C transport and recycling, by using the mouse C2C12 and rat L6C5 muscle cell lines, which exhibit different sensitivity to oxidative stress and GSH metabolism. We found that: (1) both cell lines express SVCT2, whereas SVCT1 is expressed at very low levels only in proliferating L6C5 cells; furthermore L6C5 myoblasts are more efficient in ascorbic acid transport than C2C12 myoblasts; (2) C2C12 cells are more efficient in dehydroascorbic acid transport and ascorbyl free radical/dehydroascorbic acid reduction; (3) differentiation is paralleled by decreased ascorbic acid and dehydroascorbic acid transport and reduction and increased ascorbyl free radical reduction; (4) differentiated cells are more responsive to oxidative stress induced by glutathione depletion; indeed, myotubes showed increased SVCT2 expression and thioredoxin reductase-mediated dehydroascorbic acid reduction. From our data, SVCT2 and NADPH-thioredoxin-dependent DHA reduction appears to belong to an inducible system activated in response to oxidative stress.     

Superoxide dictates the mode of U937 cell ascorbic acid uptake and prevents the enhancing effects of the vitamin to otherwise nontoxic levels of reactive oxygen/nitrogen species

Exposure of U937 cells to low micromolar levels of ascorbic acid or dehydroascorbic acid, while resulting in identical ascorbic acid accumulation, is unexpectedly associated with remarkably different responses to exogenous oxidants. We observed that otherwise nontoxic levels of hydrogen peroxide, tert-butylhydroperoxide or peroxynitrite promote toxicity in cells preloaded with ascorbic acid, whereas hardly any effect was detected in cells pretreated with dehydroascorbic acid. Further experiments performed with peroxynitrite in cells preloaded with ascorbic acid provided evidence for a very rapid nonapoptotic death, preceded by early Bax mitochondrial translocation and by mitochondrial permeability transition. The notion that conversion of extracellular ascorbic acid to dehydroascorbic acid prevents the enhancing effects on oxidant toxicity and nevertheless preserves the net amount of vitamin C accumulated was also established using ascorbate oxidase as well as various sources of superoxide, namely, xanthine/xanthine oxidase or ATP-driven NADPH oxidase activation. These findings suggest that superoxide-dependent conversion of extracellular ascorbic acid to dehydroascorbic acid represents an important component of the overall survival strategy of some cell types to reactive oxygen/nitrogen species.     

Cellular vitamin C accumulation in the presence of copper

Under the cell-free condition, copper is known to oxidize ascorbic acid (the active form of vitamin C) and the event leads to the loss of vitamin C. However, the biological consequence of this interaction was never examined in the presence of cells. We demonstrated in intestinal epithelial cells that dehydroascorbic acid (the oxidized form of ascorbic acid), when generated from ascorbic acid in the presence of copper, can be efficiently transported into the cells and reduced back to ascorbic acid. We also observed in other types of cells the transport and intracellular reduction of dehydroascorbic acid in the presence of copper. In the presence of iron, a metal that also oxidizes ascorbic acid, we observed similar oxidation-related accumulation in intestinal cells. Other metals that do not interact with ascorbic acid had little effect on vitamin C transport. A nonmetal pro-oxidant, hydrogen peroxide, is known to oxidize ascorbic acid and we observed that the oxidation is also accompanied by an increased intracellular accumulation of vitamin C. The efficient coupling between dehydroascorbic acid transport and intracellular reduction could help to preserve the important nutrient when facing oxidative metals in the intestine.     

Recycling of vitamin C by a bystander effect

Human cells transport dehydroascorbic acid through facilitative glucose transporters, in apparent contradiction with evidence indicating that vitamin C is present in human blood only as ascorbic acid. On the other hand, activated host defense cells undergoing the oxidative burst show increased vitamin C accumulation. We analyzed the role of the oxidative burst and the glucose transporters on vitamin C recycling in an in vitro system consisting of activated host-defense cells co-cultured with human cell lines and primary cells. We asked whether human cells can acquire vitamin C by a "bystander effect" by taking up dehydroascorbic acid generated from extracellular ascorbic acid by neighboring cells undergoing the oxidative burst. As activated cells, we used HL-60 neutrophils and normal human neutrophils activated with phorbol 12 myristate 13-acetate. As bystander cells, we used immortalized cell lines and primary cultures of human epithelial and endothelial cells. Activated cells produced superoxide anions that oxidized extracellular ascorbic acid to dehydroascorbic acid. At the same time, there was a marked increase in vitamin C uptake by the bystander cells that was blocked by superoxide dismutase but not by catalase and was inhibited by the glucose transporter inhibitor cytochalasin B. Only ascorbic acid was accumulated intracellularly by the bystander cells. Glucose partially blocked vitamin C uptake by the bystander cells, although it increased superoxide production by the activated cells. We conclude that the local production of superoxide anions by activated cells causes the oxidation of extracellular ascorbic acid to dehydroascorbic acid, which is then transported by neighboring cells through the glucose transporters and immediately reduced to ascorbic acid intracellularly. In addition to causing increased intracellular concentrations of ascorbic acid with likely associated enhanced antioxidant defense mechanisms, the bystander effect may allow the recycling of vitamin C in vivo, which may contribute to the low daily requirements of the vitamin in humans.     

Proteasome-mediated degradation of tau proteins occurs independently of the chymotrypsin-like activity by a nonprocessive pathway

Mitochondria can regenerate ascorbic acid from its oxidized forms, which may help to maintain the vitamin both in mitochondria and in the cytoplasm. In this work, we sought to determine the site and mechanism of mitochondrial ascorbate recycling from dehydroascorbic acid. Rat skeletal muscle mitochondria incubated for 3 h at 37 degrees C with 500 microM dehydroascorbic acid and energy substrates maintained ascorbate concentrations more than twice those observed in the absence of substrate. Succinate-dependent mitochondrial reduction of dehydroascorbic acid was blocked by inhibitors of mitochondrial Complexes II and III. Neither cytochrome c nor the outer mitochondrial membrane were necessary for the effect. The ascorbate radical was generated by mitochondria during treatment with dehydroascorbic acid and was abolished by ferricyanide, which does not penetrate the mitochondrial inner membrane. Together, these results show that energy substrate-dependent ascorbate recycling from dehydroascorbic acid involves an externally exposed portion of mitochondrial complex III.     

Nitric oxide-induced oxidant stress in endothelial cells: amelioration by ascorbic acid

Nitric oxide has multiple beneficial effects in the blood vessel wall. However, high concentrations of nitric oxide in the presence of hydroperoxides have been shown to damage cultured cells. In this work, the effect of relatively high concentrations of nitric oxide alone on the function and antioxidant status of a human endothelial cell line (EA.hy926) was tested. Nitric oxide generated from 0.1 to 0.5mM spermine NONOate generated reactive species in the cells detected by triazole formation from diaminofluorescein and by oxidation of dihydrofluorescein. Intracellular ascorbic acid decreased this oxidant stress. Spermine NONOate also decreased intracellular ascorbate concentrations, although reduced glutathione was not affected unless cells had also been caused to reduce dehydroascorbic acid to ascorbate. Nitric oxide predictably inhibited both endothelial nitric oxide synthase and glyceraldehyde 3-phosphate dehydrogenase, and ascorbate partially prevented inhibition of the latter enzyme. These results suggest that relatively high concentrations of nitric oxide can cause oxidant stress in endothelial cells that is ameliorated by ascorbic acid.     

Apoptosis-inducing activity of vitamin C and vitamin K.

Apoptosis-inducing activity of vitamins C and K and of their analogs are reviewed. Vitamin C shows both reducing and oxidizing activities, depending on the environment in which this vitamin is present. Higher concentrations of vitamin C induce apoptotic cell death in various tumor cell lines including oral squamous cell carcinoma and salivary gland tumor cell lines, possibly via its prooxidant action. The apoptosis-inducing activity of ascorbate is stimulated by Cu2+, lignin and ion chelator, and inhibited by catalase, Fe3+, Co2+ and saliva. On the other hand, at lower concentrations, ascorbic acid displays an antioxidant property, preventing the spontaneous and stress or antitumor agent-induced apoptosis. Sodium 5,6-benzylidene-L-ascorbate, intravenous administration of which induces degeneration of human inoperable tumors and rat hepatocellular carcinoma in vivo, induces apoptotic or non-apoptotic cell death, depending on the types of target cells. On the other hand, elevation of intracellular concentration of ascorbic acid by treatment with ascorbate 2-phosphate or dehydroascorbic acid makes the cells resistant to the oxidative stress-induced apoptosis. Vitamin K2, which has a geranylgeranyl group as a side chain,and vitamin K3 induces apoptosis of various cultured cells including osteoclasts and osteoblasts, by elevating peroxide and superoxide radicals. Synergistic apoptosis-inducing actions have been found between vitamins C and K, and between these vitamins and antiproliferative agents. The possible therapeutic application of these vitamins is discussed.     

Electron spin resonance study on the formation of ascorbate free radical from ascorbate: The effect of dehydroascorbic acid and ferricyanide

Summary Ascorbate free radical is considered to be a substrate for a plasma membrane redox system in eukaryotic cells. Moreover, it might be involved in stimulation of cell proliferation. Ascorbate free radical can be generated by autoxidation of the ascorbate dianion, by transition metal-dependent oxidation of ascorbate, or by an equilibrium reaction of ascorbate with dehydroascorbic acid. In this study, we investigated the formation of ascorbate free radical, at physiological pH, in mixtures of ascorbate and dehydroascorbic acid by electron spin resonance spectroscopy. It was found that at ascorbate concentrations lower than 2.5 mM, ascorbate-free radical formation was not dependent on the presence of dehydroascorbic acid. Removal of metal ions by treatment with Chelex 100 showed that autoxidation under these conditions was less than 20%. Therefore, it is concluded that at low ascorbate concentrations generation of ascorbate free radical mainly proceeds through metal-ion-dependent reactions. When ascorbate was present at concentrations higher than 2.5 mM, the presence of dehydroascorbic acid increased the ascorbate free-radical signal intensity. This indicates that under these conditions ascorbate free radical is formed by a disproportionation reaction between ascorbate and dehydroascorbic acid, having aK _equil of 6 × 10^–17 M. Finally, it was found that the presence of excess ferricyanide completely abolished ascorbate free-radical signals, and that the reaction between ascorbate and ferricyanide yields dehydroascorbic acid. We conclude that, for studies under physiological conditions, ascorbate free-radical concentrations cannot be calculated from the disproportionation reaction, but should be determined experimentally.Abbreviations AFR ascorbate free radical - DHA dehydroascorbic acid - EDTA ethylenediaminetetraacetic acid - DTPA diethylenetri-aminepentaacetic acid - TEMPO 2,2,6,6-tetramethylpiperidinoxy     

Improved method for simultaneous determination of ascorbic acid and dehydroascorbic acid,isoascorbic acid and dehydroisoascorbic acid in food and biological samples

The total vitamin C amount in different food and plasma samples was determined by a dual detection system, after HPLC separation, with direct detection of ascorbic acid and indirect fluorimetric detection of dehydroascorbic acid after a post-column O-phenyldiamine derivatisation. The two active forms of vitamin C and their d-isomers were separated within 10 min. The repeatability was determined by measurement of several fruits and vegetables and ranged from 0.3 to 1.9% (relative standard deviation) for vitamin C. The reproducibility, based on double determinations, ranged from 1.9 to 3.6% for vitamin C, depending on the matrix. The reproducibility, based on several determinations of reference materials, ranged from 2.4 to 3.7% for ascorbic acid and from 4.3 to 5.8% for dehydroascorbic acid, again depending on the matrix.     

Vitamin C - vitamin E interaction in juvenile lake sturgeon (Acipenser fulvescens R.), a fish able to synthesize ascorbic acid

The hypothesis that vitamin C interacts with vitamin E in vivo was investigated in juvenile lake sturgeon. Ten-month old lake sturgeon were fed diets supplemented with either 0 or 1250 mg ascorbic acid/kg diet concomitantly with either 0 or 200 mg α tocopherol/kg diet for 7 weeks at 17°C. Dietary vitamin C supplement resulted in significant increases of ascorbate concentrations in the posterior kidney and liver of sturgeon. Dietary vitamin E omission affected liver concentrations of α-tocopherol (10.0 ± 4.5 μg/g) in comparison to sturgeon fed a diet supplemented with vitamin E and vitamin C (99.5 ± 22.9 μg/g). Dietary vitamin C supplement decreased liver α-tocopherol concentration in vitamin E-deprived sturgeon. Also, vitamin E supplement lowered posterior kidney and liver ascorbic acid concentrations in vitamin C-deprived sturgeon. Gulonolactone oxidase and dehydroascorbic acid reductase activities were stimulated in groups fed vitamin C. Thiobarbituric acid-reactive substances concentrations (an indicator of lipid peroxidation) were higher in sturgeon fed either of vitamins as compared to sturgeon deprived of both vitamins. The results suggested that large doses of vitamins C and E may be prooxidant in vivo.     

Quantitative analysis of ascorbic acid and dehydroascorbic acid by high-performance liquid chromatography

High-performance liquid chromatography with spectrophotometric detection has been used to separate and quantitate ascorbic acid and dehydroascorbic acid. These components of vitamin C are resolved on a Lichrosorb-NH₂ column. The technique is capable of quantitatively following oxidation of ascorbic acid to dehydroascorbic acid and the reverse reduction. The technique is demonstrated to be suitable for assay of vitamin C in biological samples, foods, and pharmaceutical vitamin preparations.     

Mechanisms of ascorbic acid recycling in human erythrocytes.

Vitamin C, or ascorbic acid, is efficiently recycled from its oxidized forms by human erythrocytes. In this work the dependence of this recycling on reduced glutathione (GSH) was evaluated with regard to activation of the pentose cycle and to changes in pyridine nucleotide concentrations. The two-electron-oxidized form of ascorbic acid, dehydroascorbic acid (DHA) was rapidly taken up by erythrocytes and reduced to ascorbate, which reached intracellular concentrations as high as 2 mM. In the absence of D-glucose, DHA caused dose-dependent decreases in erythrocyte GSH, NADPH, and NADH concentrations. In the presence of 5 mM D-glucose, GSH and NADH concentrations were maintained, but those of NADPH decreased. Reduction of extracellular ferricyanide by erythrocytes, which reflects intracellular ascorbate recycling, was also enhanced by D-glucose, and ferricyanide activated the pentose cycle. Diethylmaleate at concentrations up to 1 mM was found to specifically deplete erythrocyte GSH by 75-90% without causing oxidant stress in the cells. Such GSH-depleted erythrocytes showed parallel decreases in their ability to take up and reduce DHA to ascorbate, and to reduce extracellular ferricyanide. These results show that DHA reduction involves GSH-dependent activation of D-glucose metabolism in the pentose cycle, but that in the absence of D-glucose DHA reduction can also utilize NADH.     

Mechanism of vitamin C inhibition of cell death induced by oxidative stress in glutathione-depleted HL-60 cells

Vitamin C is a well known antioxidant whose precise role in protecting cells from oxidative challenge is uncertain. In vitro results have been confounded by pro-oxidant effects of ascorbic acid and an overlapping role of glutathione. We used HL-60 cells as a model to determine the precise and independent role of vitamin C in cellular protection against cell death induced by oxidative stress. HL-60 cells do not depend on glutathione to transport or reduce dehydroascorbic acid. Depletion of glutathione rendered the HL-60 cells highly sensitive to cell death induced by H2O2, an effect that was not mediated by changes in the activities of glutathione reductase, glutathione peroxidase, catalase, or superoxide dismutase. The increased sensitivity to oxidative stress was largely reversed when glutathione-depleted cells were preloaded with ascorbic acid by exposure to dehydroascorbic acid. Resistance to H2O2 treatment in cells loaded with vitamin C was accompanied by intracellular consumption of ascorbic acid, generation of dehydroascorbic acid, and a decrease in the cellular content of reactive oxygen species. Some of the dehydroascorbic acid generated was exported out of the cells via the glucose transporters. Our data indicate that vitamin C is an important independent antioxidant in protecting cells against death from oxidative stress.     

Liquid chromatographic behavior of ascorbate on amine columns

Quantitation of ascorbate at concentrations normally found in biological samples and foods has previously been shown to be possible by HPLC analysis. Prefilled amine columns from three manufacturers were presently used to evaluate their potential for separating low concentrations of [14C]ascorbic acid from its degradation products, [14C]dehydroascorbic acid and [14C]diketogulonic acid. A successful separation was achieved on some columns with as little as 200 cpm (30 pmol) of total ascorbate injected. On other columns, injection of 30-500 pmol of ascorbate resulted in as much as 80% of [14C]ascorbic acid eluting with an unpredictable retention time. In these instances the inclusion of nonlabeled ascorbic acid (0.5 mg/ml) to the sample resulted in most of the [14C]ascorbic acid activity eluting at the expected retention time of ascorbic acid. The inclusion of ascorbic acid in samples injected onto the column also resulted in a more discrete peak in the elution of dehydroascorbic acid, and more complete recovery of the total [14C]activity (ascorbic acid, dehydroascorbic acid, and diketogulonic acid) injected onto the column.     

Mechanisms of vitamin C stabilization by K562 erythroleukemic cells

Summary K562 cells display several possibilities to keep ascorbic acid in the surrounding medium in the reduced state and prevent its loss by degradation of the oxidized form, dehydroascorbic acid: (1) A semidehydroascorbic acid reductase with high affinity for the ascorbate radical scavenges this before it disproportionates into the two parent forms of vitamin C (ascorbate and dehydroascorbic acid). (2) Dehydroascorbic acid in the extracellular medium is slowly converted to ascorbate by a different mechanism with low affinity which may or may not involve uptake of the oxidized and release of the reduced form. (3) Ascorbate remains relatively stable in the cell culture medium in presence, but also in absence of the cells after their removal, This is most probably due to the presence of released peptides in the cell-conditioned medium which can chelate transition metal ions and thus prevent catalytic autoxidation of ascorbate.     

Ascorbic acid recycling by cultured beta cells: effects of increased glucose metabolism

Ascorbic acid is necessary for optimal insulin secretion from pancreatic islets. We evaluated ascorbate recycling and whether it is impaired by increased glucose metabolism in the rat beta-cell line INS-1. INS-1 cells, engineered with the potential for overexpression of glucokinase under the control of a tetracycline-inducible gene expression system, took up and reduced dehydroascorbic acid to ascorbate in a concentration-dependent manner that was optimal in the presence of physiologic D-glucose concentrations. Ascorbate uptake did not affect intracellular GSH concentrations. Whereas depletion of GSH in culture to levels about 25% of normal also did not affect the ability of the cells to reduce dehydroascorbic acid, more severe acute GSH depletion to less than 10% of normal levels did impair dehydroascorbic acid reduction. Culture of inducible cells in 11.8 mM D-glucose and doxycycline for 48 h enhanced glucokinase activity, increased glucose utilization, abolished D-glucose-dependent insulin secretion, and increased generation of reactive oxygen species. The latter may have contributed to subsequent decreases in the ability of the cells both to maintain intracellular ascorbate and to recycle it from dehydroascorbic acid. Cultured beta cells have a high capacity to recycle ascorbate, but this is sensitive to oxidant stress generated by increased glucose metabolism due to culture in high glucose concentrations and increased glucokinase expression. Impaired ascorbate recycling as a result of increased glucose metabolism may have implications for the role of ascorbate in insulin secretion in diabetes mellitus and may partially explain glucose toxicity in beta cells.