N-terminal deletions and His-tag fusions dramatically affect expression of cytochrome p450 2C2 in bacteria

Mitochondria generate reactive oxygen species as by-products of oxidative metabolism. Since ascorbic acid can scavenge such destructive species, we studied the ability of mitochondria from rat liver and muscle to take up, recycle, and oxidize ascorbate. Freshly prepared mitochondria contain ascorbate, as do mitoplasts that lack the outer mitochondrial membrane. Both mitochondria and mitoplasts rapidly take up oxidized ascorbate as dehydroascorbic acid and reduce it to ascorbate. Ascorbate concentrations in mitochondria and mitoplasts rise into the low millimolar range during dehydroascorbic acid uptake, although uptake and reduction is opposed by ascorbate efflux. Mitochondrial dehydroascorbic acid reduction depends mainly on GSH, but mitochondrial thioredoxin reductase may also contribute. Reactive oxygen species generated within mitochondria oxidize ascorbate more readily than they do GSH and alpha-tocopherol. These results show that mitochondria can recycle ascorbate, which in turn might help to prevent deleterious effects of oxidant stress in the organelle.     

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.     

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.     

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     

Specificity of ascorbate analogs for ascorbate transport. Synthesis and detection of [(125)I]6-deoxy-6-iodo-L-ascorbic acid and characterization of its ascorbate-specific transport properties.

Cellular ascorbic acid accumulation occurs in vitro by two distinct mechanisms: transport of ascorbate itself or transport and subsequent intracellular reduction of its oxidized product, dehydroascorbic acid. It is unclear which mechanism predominates in vivo. An easily detectable compound resembling ascorbate but not dehydroascorbic acid could be a powerful tool to distinguish the two transport activities. To identify compounds, 21 ascorbate analogs were tested for inhibition of ascorbate or dehydroascorbic acid transport in human fibroblasts. The most effective analogs, competitive inhibitors of ascorbate transport with K(i) values of 3 microM, were 6-deoxy-6-bromo-, 6-deoxy-6-chloro-, and 6-deoxy-6-iodo-L-ascorbate. No analog inhibited dehydroascorbic acid transport. Using substitution chemistry, [(125)I]6-deoxy-6-iodo-L-ascorbate (1.4 x 10(4) mCi/mmol) was synthesized. HPLC detection methods were developed for radiolabeled and nonradiolabeled compounds, and transport kinetics of both compounds were characterized. Transport was sodium-dependent, inhibited by excess ascorbate, and similar to that of ascorbate. Transport of oxidized ascorbate and oxidized 6-deoxy-6-iodo-L-ascorbate was investigated using Xenopus laevis oocytes expressing glucose transporter isoform GLUT1 or GLUT3. Oxidation of ascorbate or its analog in media increased uptake of ascorbate in oocytes by 6-13-fold compared with control but not that of 6-deoxy-6-iodo-L-ascorbate. Therefore, 6-deoxy-6-iodo-L-ascorbate, although an effective inhibitor of ascorbate transport, either in its reduced or oxidized form was not a substrate for dehydroascorbic acid transport. Thus, radiolabeled and nonradiolabeled 6-deoxy-6-iodo-L-ascorbate provide a new means for discriminating dehydroascorbic acid and ascorbate transport in ascorbate recycling.     

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.     

Ascorbate biosynthesis in mitochondria is linked to the electron transport chain between complexes III and IV

Ascorbic acid is synthesized from galactono-gamma-lactone (GL) in plant tissues. An improved extraction procedure involving ammonium sulfate precipitation of membrane proteins from crude leaf homogenates yielded a simple, quick method for determining tissue activities of galactono-gamma-lactone dehydrogenase (GLDH). Total foliar ascorbate and GLDH activity decreased with leaf age. Subcellular fractionation experiments using marker enzymes demonstrated that 80% of the total GLDH activity was located on the inner mitochondrial membrane, and 20% in the microsomal fraction. Specific antibody raised against potato (Solanum tuberosum L.) tuber GLDH recognized a 56-kD polypeptide in extracts from the mitochondrial membranes but failed to detect the equivalent polypeptide in microsomes. We demonstrate that isolated intact mitochondria synthesize ascorbate in the presence of GL. GL stimulated mitochondrial electron transport rates. The respiration inhibitor antimycin A stimulated ascorbate biosynthesis, while cyanide inhibited both respiration and ascorbate production. GL-dependent oxygen uptake was observed in isolated intact mitochondria. This evidence suggests that GLDH delivers electrons to the mitochondrial electron transport chain between complexes III and IV.     

Enzymatic recycling of ascorbic acid from dehydroascorbic acid by glutathione‐like peptides in the extracellular loops of aminergic G‐protein coupled receptors

The intracellular recycling of ascorbic acid from dehydroascorbic acid by the glutathione–glutathione reductase system has been well‐characterized. We propose that extracellular recycling of ascorbic acid is performed in a similar manner by cysteine‐rich, glutathione‐like regions of the first and second extracellular loops of some aminergic receptors including adrenergic, histaminergic, and dopaminergic receptors. Previous research in our laboratory demonstrated that ascorbic acid binds to these receptors at a site on their first or second extracellular loops, significantly enhancing ligand activity, and apparently recycling hundreds of times their own concentration of ascorbate in an enzymatic fashion. In this study, we have synthesized 25 peptides from the first and second extracellular loops of aminergic and insulin receptors and compared them directly to glutathione for their ability to prevent the oxidation of ascorbate and to regenerate ascorbate from dehydroascorbic acid. Peptide sequences that mimic glutathione in containing a cysteine and a glutamic acid‐like amino acid also mimic glutathione activity in effects and in kinetics. Some (but not all) peptide sequences that contain one or more methionines instead of cysteine can significantly retard the oxidation of ascorbic acid but do not recycle it from dehydroascorbate into ascorbate. Peptides lacking both cysteines and methionines uniformly failed to alter significantly ascorbate or dehydroascorbate oxidation or reduction. We believe that this is the first proof that receptors may carry out both ligand binding and enzymatic activity extracellularly. Our results suggest the existence of a previously unknown extracellular system for recycling ascorbate. Copyright © 2016 John Wiley & Sons, Ltd.     

Mitochondrial recycling of ascorbic acid as a mechanism for regenerating cellular ascorbate

Mitochondria are the major source of potentially damaging reactive oxygen species in most cells. Since ascorbic acid, or vitamin C, can protect against cellular oxidant stress, we studied the ability of mitochondria prepared from guinea pig skeletal muscle to recycle the vitamin from its oxidized forms. Although ascorbate concentrations in freshly prepared mitochondria were only about 0.2 mM, when provided with 6 mM succinate and 1 mM dehydroascorbate (the two-electron-oxidized form of the vitamin), mitochondria were able to generate and maintain concentrations as high as 4 mM, while releasing most of the ascorbate into the incubation medium. Mitochondrial reduction of dehydroascorbate was strongly inhibited by 1,3-bis(chloroethyl)-1-nitrosourea and by phenylarsine oxide. Despite existing evidence that mitochondrial ascorbate protects the organelle from oxidant damage, ascorbate failed to preserve mitochondrial alpha-tocopherol during prolonged incubation in oxygenated buffer. Nonetheless, the capacity for mitochondria to recycle ascorbate from its oxidized forms, measured as ascorbate-dependent ferricyanide reduction, was several-fold greater than total steady-state ascorbate concentrations. This, and the finding that more than half of the ascorbate recycled from dehydroascorbate escaped the mitochondrion, suggests that mitochondrial recycling of ascorbate might be an important mechanism for regenerating intracellular ascorbate.     

Quantitative data on the contribution of GSH and Complex II dependent ascorbate recycling in plant mitochondria

The reduction of dehydroascorbate, the oxidized form of ascorbate plays important role in the maintenance of sufficient level of ascorbate. In plant mitochondria two DHA reducing mechanisms, the GSH-dependent and the mitochondrial electron transfer chain dependent ascorbate recycling have been characterized. Although both pathways have been extensively studied quantitative information about the electron fluxes from one or another direction for the reduction of DHA is not known. The cellular, mitochondrial glutathione pools and mitochondrial DHA reducing capacity was measured in BSO treated and control tobacco cells. While BSO caused dramatic decrease of cellular GSH content the difference was much smoother at mitochondrial level. The difference in DHA reduction capacity was even smoother affirming the existence of alternative, non-GSH dependent DHA reducing mechanism(s) in plant mitochondria. On the base of the parallel determination of mitochondrial GSH content and ascorbate production upon DHA addition, GSH (consumption) is responsible for the ~ 20 % of ascorbate production. Almost 90 % enhancement of ascorbate production could be provoked by the addition of Complex II substrate succinate which could be almost totally prevented by the concomitant addition of malonate or TTFA. On the base of these results, the importance of mitochondrial Complex II compared to GSH-dependent mechanisms in mitochondrial ascorbate recycling has been underestimated so far.     

Erythrocytes reduce extracellular ascorbate free radicals using intracellular ascorbate as an electron donor

Ascorbate is readily oxidized in aqueous solution by ascorbate oxidase. Ascorbate radicals are formed, which disproportionate to ascorbate and dehydroascorbic acid. Addition of erythrocytes with increasing intracellular ascorbate concentrations decreased the oxidation of ascorbate in a concentration-dependent manner. Concurrently, it was found, utilizing electron spin resonance spectroscopy, that extracellular ascorbate radical levels were decreased. Control experiments showed that these results could not be explained by leakage of ascorbate from the cells, inactivation of ascorbate oxidase, or oxygen depletion. Thus, this means that intracellular ascorbate is directly responsible for the decreased oxidation of extracellular ascorbate. Exposure of ascorbate-loaded erythrocytes to higher levels of extracellular ascorbate radicals resulted in the detection of intracellular ascorbate radicals. Moreover, efflux of dehydroascorbic acid was observed under these conditions. These data confirm the view that intracellular ascorbate donates electrons to extracellular ascorbate free radical via a plasma membrane redox system. Such a redox system enables the cells to effectively counteract oxidative processes and thereby prevent depletion of extracellular ascorbate.     

Modulation of sulfide oxidation and toxicity in rat mitochondria by dehydroascorbic acid

Hydrogen sulfide is enzymatically produced in mammalian tissues and functions as a gaseous transmitter. However, H(2)S is also highly toxic as it inhibits mitochondrial respiration at the level of cytochrome c oxidase, which additionally is involved in sulfide oxidation. The accumulation of toxic sulfide levels contributes to the pathology of some diseases. This paper demonstrates that sulfide toxicity can be modified, and dehydroascorbic acid functions as an effector in this process. It significantly reduces the inhibitory effect of sulfide on cytochrome c oxidase, resulting in higher rates of respiration and sulfide oxidation in rat mitochondria. After the addition of dehydroascorbic acid mitochondria maintained more than 50% of the oxygen consumption and ATP production rates with different substrates in the presence of high concentrations of sulfide that would normally lead to complete inhibition. Dehydroascorbic acid significantly increased the sulfide concentration necessary to cause half maximal inhibition of mitochondrial respiration and thus completely prevented inhibition at low, physiological sulfide concentrations. In addition, sulfide oxidation was stimulated and led to ATP production even at high concentrations. The decrease in sulfide toxicity was more pronounced when analyzing supermolecular functional units of the respiratory chain than in isolated cytochrome c oxidase activity. Furthermore, the protective effect of dehydroascorbic acid at high sulfide concentrations was completely abolished by quantitative solubilization of mitochondrial membrane proteins with dodeclymaltoside. These results suggest that binding of cytochrome c oxidase to other proteins probably within respiratory chain supercomplexes is involved in the modulation of sulfide oxidation and toxicity by dehydroascorbic acid.     

Non-transferrin iron reduction and uptake are regulated by transmembrane ascorbate cycling in K562 cells

K562 erythroleukemia cells import non-transferrin-bound iron (NTBI) by an incompletely understood process that requires initial iron reduction. The mechanism of NTBI ferrireduction remains unknown but probably involves transplasma membrane electron transport. We here provide evidence for a novel mechanism of NTBI reduction and uptake by K562 cells that utilizes transplasma membrane ascorbate cycling. Incubation of cells with dehydroascorbic acid, but not ascorbate, resulted in (i) accumulation of intracellular ascorbate that was blocked by the glucose transporter inhibitor, cytochalasin B, and (ii) subsequent release of micromolar concentrations of ascorbate into the external medium via a route that was sensitive to the anion channel inhibitor, 4,4'-diisothiocyanatostilbene-2,2'-disulfonate. Ascorbate-deficient control cells demonstrated low levels of ferric citrate reduction. However, incubation of the cells with dehydroascorbic acid resulted in a dose-dependent stimulation of both iron reduction and uptake from radiolabeled [(55)Fe]ferric citrate. This stimulation was abrogated by ascorbate oxidase treatment, suggesting dependence on direct chemical reduction by ascorbate. These results support a novel model of NTBI reduction and uptake by K562 cells in which uptake is preceded by reduction of iron by extracellular ascorbate, the latter of which is subsequently regenerated by transplasma membrane ascorbate cycling.     

Sepsis inhibits recycling and glutamate-stimulated export of ascorbate by astrocytes

Sepsis causes brain dysfunction. Because neurotransmission requires high ascorbate and low dehydroascorbic acid (DHAA) concentrations in brain extracellular fluid, the effect of septic insult on ascorbate recycling (i.e., uptake and reduction of DHAA) and export was investigated in primary rat and mouse astrocytes. DHAA raised intracellular ascorbate to physiological levels but extracellular ascorbate only slightly. Septic insult by lipopolysaccharide and interferon-gamma increased ascorbate recycling in astrocytes permeabilized with saponin but decreased it in those with intact plasma membrane. The decrease was due to inhibition of the glucose transporter (GLUT1) that translocates DHAA because septic insult slowed uptake of the nonmetabolizable GLUT1 substrate 3-O-methylglucose. Septic insult also abolished stimulation by glutamate of ascorbate export. Specific nitric oxide synthase (NOS) inhibitors and nNOS and iNOS deficiency failed to alter the effects of septic insult. Inhibitors of NADPH oxidase generally did not protect against septic insult, because only one of those tested (diphenylene iodonium) increased GLUT1 activity and ascorbate recycling. We conclude that astrocytes take up DHAA and use it to synthesize ascorbate that is exported in response to glutamate. This mechanism may provide the antioxidant on demand to neurons under normal conditions, but it is attenuated after septic insult.     

Mitochondrial ascorbic acid transport is mediated by a low-affinity form of the sodium-coupled ascorbic acid transporter-2

Despite the fundamental importance of the redox metabolism of mitochondria under normal and pathological conditions, our knowledge regarding the transport of vitamin C across mitochondrial membranes remains far from complete. We report here that human HEK-293 cells express a mitochondrial low-affinity ascorbic acid transporter that molecularly corresponds to SVCT2, a member of the sodium-coupled ascorbic acid transporter family 2. The transporter SVCT1 is absent from HEK-293 cells. Confocal colocalization experiments with anti-SVCT2 and anti-organelle protein markers revealed that most of the SVCT2 immunoreactivity was associated with mitochondria, with minor colocalization at the endoplasmic reticulum and very low immunoreactivity at the plasma membrane. Immunoblotting of proteins extracted from highly purified mitochondrial fractions confirmed that SVCT2 protein was associated with mitochondria, and transport analysis revealed a sigmoidal ascorbic acid concentration curve with an apparent ascorbic acid transport K_m of 0.6 mM. Use of SVCT2 siRNA for silencing SVCT2 expression produced a major decrease in mitochondrial SVCT2 immunoreactivity, and immunoblotting revealed decreased SVCT2 protein expression by approximately 75%. Most importantly, the decreased protein expression was accompanied by a concomitant decrease in the mitochondrial ascorbic acid transport rate. Further studies using HEK-293 cells overexpressing SVCT2 at the plasma membrane revealed that the altered kinetic properties of mitochondrial SVCT2 are due to the ionic intracellular microenvironment (low in sodium and high in potassium), with potassium acting as a concentration-dependent inhibitor of SVCT2. We discarded the participation of two glucose transporters previously described as mitochondrial dehydroascorbic acid transporters; GLUT1 is absent from mitochondria and GLUT10 is not expressed in HEK-293 cells. Overall, our data indicate that intracellular SVCT2 is localized in mitochondria, is sensitive to an intracellular microenvironment low in sodium and high in potassium, and functions as a low-affinity ascorbic acid transporter. We propose that the mitochondrial localization of SVCT2 is a property shared across cells, tissues, and species.     

Enzymes of the ascorbate biosynthesis and ascorbate-glutathione cycle in cultured cells of tobacco Bright Yellow 2

The ascorbate (ASC) and glutathione (GSH) metabolisms were studied in cultured Nicotiana tabacum cv. Bright Yellow 2 (TBY-2) cells. TBY-2 cells were found to be endowed with L-galactono-γ-lactone dehydrogenase (GLDH) (EC 1.3.2.3), an enzyme that converts L-galactono-γ-lactone into ASC. Cellular fractionation of TBY-2 protoplasts indicated that this enzyme is exclusively localised in mitochondria and associated to the membrane fractions. During the growth cycle of TBY-2 cell culture, GLDH transiently increased, reaching the maximum value on the third day of culture, at the beginning of the exponential phase, when the cell proliferative activity was also higher. Similar behaviour has been observed for ASC and GSH contents. The activities of ascorbate peroxidase (APX) (EC 1.11.1.11), ascorbate-free radical reductase (AFRR) (EC 1.6.5.4), dehydroascorbic acid reductase (DHAR) (EC 1.8.5.1) and glutathione reductase (GR) (EC 1.6.4.2) also transiently raised. However, the scale of the increases varied being about 4-fold for APX and AFRR, 2-fold for DHAR and more than 11-fold for GR. The behaviour of the ASC and GSH recycling enzymes allowed TBY-2 cells to maintain both dehydroascorbic acid and glutathione disulphide at low levels, even under conditions of high ASC and GSH utilisation. The relationship between the ASC and GSH metabolisms during the growth cycle of TBY-2 cell suspension cultures is also discussed.     

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.     

Ascorbate degradation in tomato leads to accumulation of oxalate,threonate and oxalyl threonate

Ascorbate content in plants is controlled by its synthesis from carbohydrates, recycling of the oxidized forms and degradation. Of these pathways, ascorbate degradation is the least studied and represents a lack of knowledge that could impair improvement of ascorbate content in fruits and vegetables as degradation is non‐reversible and leads to a depletion of the ascorbate pool. The present study revealed the nature of degradation products using [¹⁴C]ascorbate labelling in tomato, a model plant for fleshy fruits; oxalate and threonate are accumulated in leaves, as is oxalyl threonate. Carboxypentonates coming from diketogulonate degradation were detected in relatively insoluble (cell wall‐rich) leaf material. No [¹⁴C]tartaric acid was found in tomato leaves. Ascorbate degradation was stimulated by darkness, and the degradation rate was evaluated at 63% of the ascorbate pool per day, a percentage that was constant and independent of the initial ascorbate or dehydroascorbic acid concentration over periods of 24 h or more. Furthermore, degradation could be partially affected by the ascorbate recycling pathway, as lines under‐expressing monodehydroascorbate reductase showed a slight decrease in degradation product accumulation.     

Selenium spares ascorbate and alpha-tocopherol in cultured liver cell lines under oxidant stress.

The selenoenzyme thioredoxin reductase (TR) can recycle ascorbic acid, which in turn can recycle alpha-tocopherol. Therefore, we evaluated the role of selenium in ascorbic acid recycling and in protection against oxidant-induced loss of alpha-tocopherol in cultured liver cells. Treatment of HepG2 or H4IIE cultured liver cells for 48 h with sodium selenite (0-116 nmol/l) tripled the activity of the selenoenzyme TR, measured as aurothioglucose-sensitive dehydroascorbic acid (DHA) reduction. However, selenium did not increase the ability of H4IIE cells to take up and reduce 2 mM DHA, despite a 25% increase in ascorbate-dependent ferricyanide reduction (which reflects cellular ascorbate recycling). Nonetheless, selenium supplements both spared ascorbate in overnight cultures of H4IIE cells, and prevented loss of cellular alpha-tocopherol in response to an oxidant stress induced by either ferricyanide or diazobenzene sulfonate. Whereas TR contributes little to ascorbate recycling in H4IIE cells, selenium spares ascorbate in culture and alpha-tocopherol in response to an oxidant stress.     

Comparison of Two Methods for Preparing Mitochondria from Tomato Leaves to Study the Ascorbate-Glutathione Cycle Activity

Mitochondria were purified from tomato leaves by a differential centrifugation method and a Percoll density-gradient method. A comparative study on the ascorbate-glutathione cycle activity revealed that there was no difference in the specific activity of the antioxidative enzymes and ascorbate and glutathione concentrations between mitochondria prepared by these two methods. However, the contents of dehydroascorbic acid and oxidised glutathione in Percoll-separated organelles were 260 and 210 %, respectively, of those found in mitochondria purified by the differential centrifugation method.