Unequivocal evidence in support of the nonenzymatic redox coupling between glutathione/glutathione disulfide and ascorbic acid/dehydroascorbic acid.

Experiments were performed to evaluate the nonenzymatic reaction between glutathione (GSH) and dehydroascorbic acid (DHA). Though both ascorbic acid and glutathione disulfide (GSSG) are formed from this reaction, previous work has focused almost exclusively on measurements of ascorbic acid. In contrast, there is very little information about the formation of GSSG under the same conditions as those used to produce ascorbic acid. The emphasis on ascorbic acid stems from the fact that a spectrophotometric technique is available for its measurement, whereas 1H-NMR or an amino acid analyzer has been used to measure GSSG. The present experiments use a simple, rapid method for accurately and precisely measuring the concentrations of GSSG in a solution. The spectrophotometric (340 nm) procedure uses NADPH and glutathione reductase; analysis time is very short, many replicate samples can be tested and as little as 0.05-0.1 mM GSSG can be detected. Using this method, it is shown that there is an equimolar production of GSSG and ascorbic acid from GSH and DHA and that the decrease in GSH is stoichiometrically related to the increase in the concentration of GSSG. The present findings provide additional insight into the interaction between the GSH/GSSG redox couple and the ascorbic acid/DHA redox couple.     

Stimulation of thiamine diphosphatase activity by ascorbic acid in rat brain microsomes.

The effect of ascorbic acid on microsomal thiamine diphosphatase activity in rat brain was examined. Ascorbic acid at 0.02--0.1 mM increased the thiamine diphosphatase activity by 20--600% and produced a significant amount of lipid peroxide, which was measured with thiobarbiturate under the same conditions as the enzyme. A lag period of about 10 min was observed in the process of stimulation of enzyme activity by ascorbic acid. The stimulation of enzyme activity and the lipid peroxidation induced by ascorbic acid were blocked by metal-binding compounds (EDTA, alpha,alpha'-dipyridyl, o-phenanthroline) and an antioxidant (N,N'-diphenyl p-phenylenediamine). GSH significantly enhanced the stimulation of enzyme activity and formation of lipid peroxide by 0.02--0.05 mM ascorbic acid. The effect of GSH was due in part to maintenance of the concentration of ascorbic acid in the medium, since GSH could convert dehydroascorbic acid, an oxidized form of ascorbic acid, to ascorbic acid.     

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     

Stimulation of thiamine diphosphate activity by ascorbic acid in rat brain microsomes

The effect of ascorbic acid on microsomal thiamine diphosphate activity in rat brain was examined. Ascorbic acid at 0.02–0.1 mM increased the thiamine diphosphate activity by 20–600% and produced a significant amount of lipid peroxide, which was measured with thiobarbiturate under the same conditions as the enzyme. A lag period of about 10 min was observed in the process of stimulation of enzyme activity by ascorbic acid. The stimulation of enzyme activity and the lipid peroxidation induced by ascorbic acid were blocked by metal-binding compounds (EDTA, α,α′-dipyridyl, o-phenanthroline) and an antioxidant (N,N′-diphenyl p-phenylenediamine). GSH significantly enhanced the stimulation of enzyme activity and formation of lipid peroxide by 0.02–0.05 mM ascorbic acid. The effect of GSH was due in part to maintenance of the concentration of ascorbic acid in the medium, since GSH could convert dehydroascorbic acid, an oxidized form of ascorbic acid, to ascorbic acid.     

The extent of N epsilon-(carboxymethyl)lysine formation in lens proteins and polylysine by the autoxidation products of ascorbic acid.

The autoxidation of ascorbic acid (ASA) leads to the formation of compounds which are capable of glycating and crosslinking proteins in vitro. When the soluble crystallins from bovine lens were incubated with ASA in the presence of sodium cyanoborohydride, a single major adduct was observed, whose appearance correlated with the loss of lysine. When polylysine was reacted with equivalent amounts of ASA under the same conditions, this product represented half of the total lysine content after four weeks of incubation at 37 degrees C. This adduct was isolated and identified as N epsilon-(carboxymethyl)lysine (CML) by TLC, GC/MS and amino acid analysis. Several oxidation products of ASA were each reacted with polylysine in the presence of sodium cyanoborohydride to identify the reactive species. CML was the major adduct formed with either ASA and dehydroascorbic acid (DHA). Markedly diminished amounts were seen with L-2,3-diketogulonic acid (DKG), and L-threose, while no CML was formed with L-threo-pentos-2-ulose (L-xylosone). In the absence of sodium cyanoborohydride the yield of CML was similar with each of the ASA autoxidation products and required oxygen. Reactions with [1-14C]ASA gave rise to [14C]CML, but only with NaCNBH3 present. At least two routes of CML formation appear to be operating depending upon whether NaCNBH3 is present to reduce the putative Schiff base formed between lysine and DHA.     

Oxidative Decomposition of Vitamin C in Drinking Water

We have previously shown that vitamin C (ascorbic acid) can initiate hydroxyl radical formation in copper contaminated household drinking water. In the present study, we have examined the stability of vitamin C in copper and bicarbonate containing household drinking water. In drinking water samples, contaminated with copper from the pipes and buffered with bicarbonate, 35% of the added vitamin C was oxidized to dehydroascorbic acid within 15?min. After 3?h incubation at room temperature, 93% of the added (2?mM) ascorbic acid had been oxidized. The dehydroascorbic acid formed was further decomposed to oxalic acid and threonic acid by the hydrogen peroxide generated from the copper (I) autooxidation in the presence of oxygen. A very modest oxidation of vitamin C occurred in Milli-Q water and in household water samples not contaminated by copper ions. Moreover, addition of vitamin C to commercially sold domestic bottled water samples did not result in vitamin C oxidation. Our results demonstrate that ascorbic acid is rapidly oxidized to dehydroascorbic acid and further decomposed to oxalic- and threonic acid in copper contaminated household tap water that is buffered with bicarbonate. The impact of consuming ascorbic acid together with copper and bicarbonate containing drinking water on human health is discussed.     

Oxidative decomposition of vitamin C in drinking water

We have previously shown that vitamin C (ascorbic acid) can initiate hydroxyl radical formation in copper contaminated household drinking water. In the present study, we have examined the stability of vitamin C in copper and bicarbonate containing household drinking water. In drinking water samples, contaminated with copper from the pipes and buffered with bicarbonate, 35% of the added vitamin C was oxidized to dehydroascorbic acid within 15 min. After 3 h incubation at room temperature, 93% of the added (2 mM) ascorbic acid had been oxidized. The dehydroascorbic acid formed was further decomposed to oxalic acid and threonic acid by the hydrogen peroxide generated from the copper (I) autooxidation in the presence of oxygen. A very modest oxidation of vitamin C occurred in Milli-Q water and in household water samples not contaminated by copper ions. Moreover, addition of vitamin C to commercially sold domestic bottled water samples did not result in vitamin C oxidation. Our results demonstrate that ascorbic acid is rapidly oxidized to dehydroascorbic acid and further decomposed to oxalic- and threonic acid in copper contaminated household tap water that is buffered with bicarbonate. The impact of consuming ascorbic acid together with copper and bicarbonate containing drinking water on human health is discussed.     

The effect of ascorbic acid oxidation on the incorporation of sulfate by slices of calf costal cartilage

A marked inhibition of the incorporation of S³⁵-sulfate by normal calf costal cartilage was produced by potassium ascorbate in the presence of catalytic amounts of cupric ions. The effect of the various components of the ascorbic acid oxidizing system (potassium ascorbate, cupric ions, cuprous ions, hydrogen peroxide, dehydroascorbic acid) was investigated. The results of experiments in which hydrogen peroxide, catalase, or sodium azide were used singly or in combination suggest that the inhibition produced by the ascorbic acid oxidizing system is due, to a considerable extent, to the production of hydrogen peroxide. Dehydroascorbic acid was also found to inhibit the incorporation of S³⁵-sulfate by cartilage slices. However, the gradual fall in pH which resulted from the addition of dehydroascorbic acid could account to a large extent for the inhibitory effect observed because the incorporation of S³⁵-sulfate by cartilage slices decreases sharply as the pH is lowered. The incorporation of S³⁵-sulfate by cartilage slices is inhibited also by increasing the concentration of phosphate.     

Acid proteases. II. Fluorescence study of the interaction of Cladosporium acid protease with glycyl-DL-norleucine methyl ester in the presence of cupric ions.

Glycyl-DL-norleucine methyl ester (GN), a diazoacetyl-DL-norleucine methyl ester (DAN) analog, in the presence of cupric ions was found to partially quench the protein fluorescence of acid protease from Cladosporium sp. No. 45-2, and cupric ions were also found to quench the fluorescence. These quenchings were pH-dependent. GN alone did not quench the fluorescence of the enzyme. The interaction between the enzyme and GN in the presence of cupric ions was studied statically at pH 5.4 in terms of fluorescence change. The dissociation constant, Kd, of the enzyme-GN complex in the presence of a 20-fold molar excess of cupric ions (0.08 mM) determined by fluorescence titration at 30 degrees C (Kd = 1.86 mM) was in good agreement with that obtained for GN from kinetics of inhibition of DAN-induced inactivation in the presence of a 20-fold molar excess of cupric ions at 30 degrees C (KA = 1.94 mM) (Kanazawa, H. (1977) J. Biochem. 81, 1739-1744). At various concentrations of cupric ions, no change of Kd was found. These results suggest that cupric ions are attracted to a negatively charged carboxyl group responsible for the formation of the enzyme-GN complex.     

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.     

Ascorbic acid and copper in linoleate oxidation. II. Ascorbic acid and copper as oxidation catalysts

Both ascorbic acid and copper were strong prooxidants in the oxidation of linoleate in a buffered (pH 7.0) aqueous dispersion at 37 degrees C. Minimum concentrations at which catalytic activity was detected were 1.3 x 10(-7) m for copper and 1.8 x 10(-6) m for ascorbic acid. For concentrations up to 10(-3) m, the increase in rate of oxidation with increase in concentration of catalyst was greater for ascorbic acid than for copper. Ascorbic acid had maximum catalytic activity at 2.0 x 10(-3) m, but was still prooxidant at the highest concentration tested (5.0 x 10(-2) m). Dehydroascorbic acid was a weaker prooxidant than ascorbic acid. Further degradation products of ascorbic acid were not prooxidant. In early stages of the oxidation autocatalytic behavior was observed with copper, but not with ascorbic acid. Ascorbic acid functioned as a true catalyst, i.e., it accelerated the reaction but it was not oxidized simultaneously with the linoleate. It is proposed that the dehydroascorbic acid radical initiates the linoleate oxidation reaction.     

Inhibition of glutathione reductase by isoproterenol oxidation products

Oxidative stress induced by catecholamines is a well recognized toxic event. This effect has been extensively observed in the heart, where high levels of catecholamines cause enzyme inhibition, lipid peroxidation, energy depletion and myocardial necrosis. Catecholamines can be converted into o-quinones and undergo cyclization into aminochromes. This process can occur enzymatically or through autoxidation and involves the formation of free radicals. Aminochromes are highly reactive molecules that can cause oxidation of protein sulfhydryl groups and deamination catalysis, among other deleterious effects; in addition, inhibition of some enzymes has been also reported. We have studied the effects of isoproterenol oxidation products (IOP) on glutathione reductase (GR) activity in vitro. Isoproterenol (ISO) autoxidation was conducted at 37 degrees C in the dark, for 4 h at pH 7.0 and this process was monitored by UV spectrophotometry at both 340 and 490nm. Addition of the autoxidized solution to GR in the presence of oxidized glutathione (GSSG) and NADPH showed that IOP inhibits GR in a competitive mode and that this effect increases during the 4 h incubation period. This inhibitory effect of IOP was partially prevented by the addition of reduced glutathione (GSH), L-cysteine and ascorbic acid to the reaction mixtures.     

Inhibition of Glutathione Reductase by Isoproterenol Oxidation Products

Oxidative stress induced by catecholamines is a well recognized toxic event. This effect has been extensively observed in the heart, where high levels of catecholamines cause enzyme inhibition, lipid peroxidation, energy depletion and myocardial necrosis. Catecholamines can be converted into o-quinones and undergo cyclization into aminochromes. This process can occur enzymatically or through autoxidation and involves the formation of free radicals. Aminochromes are highly reactive molecules that can cause oxidation of protein sulfhydryl groups and deamination catalysis, among other deleterious effects; in addition, inhibition of some enzymes has been also reported. We have studied the effects of isoproterenol oxidation products (IOP) on glutathione reductase (GR) activity in vitro. Isoproterenol (ISO) autoxidation was conducted at 37 degrees C in the dark, for 4 h at pH 7.0 and this process was monitored by UV spectrophotometry at both 340 and 490 nm. Addition of the autoxidized solution to GR in the presence of oxidized glutathione (GSSG) and NADPH showed that IOP inhibits GR in a competitive mode and that this effect increases during the 4 h incubation period. This inhibitory effect of IOP was partially prevented by the addition of reduced glutathione (GSH), L-cysteine and ascorbic acid to the reaction mixtures.     

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.     

Kinetics of the reaction between nitric oxide and glutathione: implications for thiol depletion in cells

Nitric oxide in the absence of oxygen was suggested to react with 5-50 mM glutathione (GSH) over many minutes when [NO*] < [GSH] (N. Hogg et al., FEBS Lett. 382:223-228; 1996). However, Aravindakumar et al. (J. Chem. Soc. Perkin Trans. 2:663-669; 2002) provided data suggesting approximately 200-fold higher reactivity under conditions of [NO*] > [GSH]. To help resolve these differences, the rate of loss of NO* ( approximately 9 microM) in aqueous solutions of GSH (2.5-20 mM) was measured by chemiluminescence. An apparent second-order rate constant of 0.080 +/- 0.008 M(-1) s(-1) at pH 7.4, 37 degrees C, was calculated based on the total [GSH] and "pseudo-first-order" kinetics; thiolate anion was much more reactive than undissociated thiol. These data imply a half-life of approximately 30 min for low concentrations of NO* with 5 mM GSH, 37 degrees C, pH 7.4, in the absence of oxygen. Possible kinetic schemes that can partially explain the divergent literature reports are discussed, notably an equilibrium in the reaction between NO* and GSH. Human breast carcinoma MCF-7 cells were exposed to NO* (initially approximately 18 microM) in alidded six well plate in an anaerobic chamber in vitro; intracellular GSH levels decreased by half in approximately 60 min. Aerobic exposure depletes GSH in cells in vitro much faster because of autoxidation of NO* to NO2*, >10(8) times more reactive toward GSH.     

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.     

Possible mechanisms responsible for the increased ascorbic acid content of Plasmodium vinckei-infected mouse erythrocytes

The possible mechanisms underlying the acquisition of an increased ascorbic acid content by mouse erythrocytes containing the malarial parasite Plasmodium vinckei were investigated. Ascorbic acid was taken up readily by parasitized red blood cells but not by controls, whilst its partly oxidized form, dehydroascorbic acid, entered both. The uptake of both ascorbic acid and dehydroascorbic acid into erythrocytes was increased as a result of malarial infection. Lysates prepared from parasitized red blood cells reduced exogenous dehydroascorbic acid to ascorbic acid at a higher rate than control red blood cell lysates; this difference was abolished following dialysis of the lysates, a process which removes endogenous reduced glutathione (GSH). The rates of chemical and enzymatic reduction of dehydroascorbic acid to ascorbic acid by GSH were of similar magnitude, thus calling into question the existence of a specific dehydroascorbate reductase in erythrocytes and parasites. These observations suggest that the increased uptake of dehydroascorbic acid into parasitized red blood cells may be a result of enhanced dehydroascorbate-reducing capacity, whilst the presence of the parasite induces a selective increase in the permeability of the erythrocyte plasma membrane to ascorbic acid. The endogenous ascorbic acid content of livers obtained from infected mice was 55% below the normal concentration and its relative rate of destruction during incubation in vitro was enhanced in comparison with that of control livers. Furthermore, the capacity of liver homogenates to synthesize ascorbic acid from glucuronic acid was greatly reduced in infected mice. Therefore it is unlikely that the increase in ascorbic acid content of parasitized red blood cells is a consequence of increased biosynthesis and release of ascorbic acid by the host liver. We have not been able to exclude the possibility that the malarial parasite itself may be capable of de novo synthesis of ascorbic acid.     

Changes in ascorbate, glutathione, and related enzyme activities during thidiazuron-induced bud break of apple

The changes of ascorbic acid, dehydroascorbic acid, and glutathione content and related enzyme activities were studied in apple buds during dormancy and thidiazuron-induced bud break. An increase in ascorbic acid, reduced form of glutathione (GSH), total glutathione, total non-protein thiol (NPSH) and non-glutathione thiol (RSH) occurred as a result of induction by thidiazuron during bud break, whereas dehydroascorbic acid and oxidized glutathione (GSSG) decreased during the same period. Thidiazuron also enhanced the ratio of GSH/GSSG, and activities of ascorbate free radical reductase (AFR; EC 1.6.5.4), ascorbate peroxidase (EC 1.11.1.11). dehydroascorbate reductase (DHAR; EC 1.8.5.1) and glutathione reductase (GR; EC 1.6.4.2). The ascorbic acid content and the activities of AFR, ascorbate peroxidase, and DHAR peaked when buds were in the side green or green tip stage just prior to the start of rapid expansion, and declined thereafter. The GSH, NPSH, RSH, ratio of GSH/GSSG, and activities of GR increased steadily during bud development.     

Mammalian thioltransferase (glutaredoxin) and protein disulfide isomerase have dehydroascorbate reductase activity

Homogeneous native and recombinant porcine liver thioltransferase (glutaredoxin), bovine thymus and human placenta thioltransferase (glutaredoxin) were examined for dehydroascorbate reductase activity (EC 1.8.5.1) involving the direct catalytic reduction of dehydroascorbic acid (DHA) by glutathione. Each enzyme had substantial activity with apparent Km and Vmax for dehydroascorbate between 0.2 and 2.2 mM and 6-27 nmol min-1, respectively, and for gluathione between 1.6 and 8.7 mM and 11-30 nmol min-1, respectively. In the presence of purified bovine liver thioredoxin reductase, homogeneous bovine liver thioredoxin failed to reduce DHA to ascorbic acid as measured by NADPH oxidation. Highly purified bovine liver protein disulfide isomerase (PDI) reacted directly with DHA and GSH to catalyze the reduction of DHA to ascorbic acid. The apparent Km for DHA was 1.0 mM and the Vmax was 8 nmol min-1, and for GSH were 3.9 mM and 14 nmol min-1, respectively. These results suggest that thioltransferase and PDI contribute to the regeneration of oxidized ascorbic acid in mammalian cells, and based on their cellular location, thioltransferase is proposed to be the major cytoplasmic activity, whereas interaction of DHA with microsomal membrane PDI may catalyze regeneration of ascorbic acid and initiate oxidation of intralumenal protein thiols to disulfides.     

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.