Preferential uptake and accumulation of oxidized vitamin C by THP-1 monocytic cells.

THP-1 cells preferentially accumulate vitamin C in its oxidized form. The uptake displays first-order kinetics and leads to a build-up of an outward concentration gradient which is stable in the absence of extracellular vitamin. The transport is faster than reduction by extracellular glutathione or by added cytosolic extract, and glutathione-depleted cells show the same uptake rates as control cells. In addition, energy depletion or oxidation of intracellular sulfhydryls does not inhibit accumulation of ascorbate. The accumulation, however, always occurs in the reduced form. The affinity for dehydroascorbate is lower (Km 450 microM vs 60 microM) than for reduced ascorbate, but the maximal rate is more than 30 times higher (581 compared to 19 pmol.min-1 per 106 cells), and it is independent of sodium, whereas the uptake of ascorbate is not. The sodium gradient also allows accumulation of reduced ascorbate. Inhibitors of glucose transport by the GLUT-1 transporter also inhibit uptake of dehydroascorbate (DHA), but there are some inconsistencies, because the Ki-values are higher than reported for the isolated transporter and one inhibitor (deoxyglucose) is noncompetitive. The preferential uptake of the dehydro-form of the vitamin may be useful for situations where this short-lived metabolite is formed by oxidation in the environment.     

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.     

Ascorbate-induced generation of 5-hydroxymethylcytosine is unaffected by varying levels of iron and 2-oxoglutarate

Tet (ten–eleven translocation) methylcytosine dioxygenases, which belong to the iron and 2-oxoglutarate (2OG)-dependent dioxygenase superfamily, convert 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC) in DNA. We recently reported that ascorbate (vitamin C) induces Tet-mediated generation of 5hmC. To initially delineate the role of ascorbate on 5hmC generation, we analyzed whether the effect of ascorbate is dependent upon the conditions of other components involved in the hydroxylation of 5mC catalyzed by Tet. We found that removing iron from the culture medium did not affect the induction of 5hmC by ascorbate (10 μM) in mouse embryonic fibroblasts (MEFs). The effect of ascorbate did not involve an increased expression of Tet1–3 or isocitrate dehydrogenases (IDH1–2), the enzymes responsible for producing 2OG. Interestingly, MEFs cultured with different concentrations of glucose, a major precursor of 2OG, exhibited nearly identical responses to ascorbate treatment. Further, blocking the uptake of the reduced form of vitamin C, ascorbic acid, through the sodium-dependent vitamin C transporters (SVCTs) inhibited the effect of ascorbate on 5hmC. However, inhibition of the facilitative glucose transporters (GLUTs), which mediate the incorporation of the oxidized form of vitamin C, dehydroascorbic acid (DHA), did not modify the ability of ascorbate to induce 5hmC generation. These results indicate that the effect of ascorbate on 5hmC is not dependent upon iron uptake, the expression of Tet and IDH, or the production of 2OG, suggesting that ascorbate may directly participate in the generation of 5hmC, most likely as a cofactor of Tet.     

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.     

Human neutrophils oxidize low-density lipoprotein by a hypochlorous acid-dependent mechanism: the role of vitamin C

Oxidatively modified low-density lipoprotein (LDL) has been strongly implicated in the pathogenesis of atherosclerosis. Peripheral blood leukocytes, such as neutrophils, can oxidize LDL by processes requiring superoxide and redox-active transition metal ions; however, it is uncertain whether such catalytic metal ions are available in the artery wall. Stimulated leukocytes also produce the reactive oxidant hypochlorous acid (HOCl) via the heme enzyme myeloperoxidase. Since myeloperoxidase-derived HOCl may be a physiologically relevant oxidant in atherogenesis, we investigated the mechanisms of neutrophil-mediated LDL modification and its possible prevention by the antioxidant ascorbate (vitamin C). As a sensitive marker of LDL oxidation, we measured LDL thiol groups. Stimulated human neutrophils (5x10(6) cells/ml) incubated with human LDL (0.25 mg protein/ml) time-dependently oxidized LDL thiols (33% and 79% oxidized after 10 and 30 min, respectively). Supernatants from stimulated neutrophils also oxidized LDL thiols (33% oxidized after 30 min), implicating long-lived oxidants such as N-chloramines. Experiments using specific enzyme inhibitors and oxidant scavengers showed that HOCl, but not hydrogen peroxide nor superoxide, plays a critical role in LDL thiol oxidation by neutrophils. Ascorbate (200 microM) protected against neutrophil-mediated LDL thiol oxidation for up to 15 min of incubation, after which LDL thiols became rapidly oxidized. Although stimulated neutrophils accumulated ascorbate during oxidation of LDL, pre-loading of neutrophils with ascorbate did not attenuate oxidant production by the cells. Thus, activated neutrophils oxidize LDL thiols by HOCl- and N-chloramine-dependent mechanisms and physiological concentrations of vitamin C delay this process, most likely due to scavenging of extracellular oxidants, rather than by attenuating neutrophil oxidant production.     

Comparison of protein oxidation and aldehyde formation during oxidative stress in isolated mitochondria

Oxidative stress is known to cause oxidative protein modification and the generation of reactive aldehydes derived from lipid peroxidation. Extent and kinetics of both processes were investigated during oxidative damage of isolated rat liver mitochondria treated with iron/ascorbate. The monofunctional aldehydes 4-hydroxynonenal (4-HNE), n-hexanal, n-pentanal, n-nonanal, n-heptanal, 2-octenal, 4-hydroxydecenal as well as thiobarbituric acid reactive substances (TBARS) were detected. The kinetics of aldehyde generation showed a lag-phase preceding an exponential increase. In contrast, oxidative protein modification, assessed as 2,4-dinitrophenylhydrazine (DNPH) reactive protein-bound carbonyls, continuously increased without detectable lag-phase. Western blot analysis confirmed these findings but did not allow the identification of individual proteins preferentially oxidized. Protein modification by 4-HNE, determined by immunoblotting, was in parallel to the formation of this aldehyde determined by HPLC. These results suggest that protein oxidation occurs during the time of functional decline of mitochondria, i.e. in the lagphase of lipid peroxidation. This protein modification seems not to be caused by 4-HNE.     

Inhibition of human leukocyte 3-hydroxy-3-methylglutaryl coenzyme A reductase activity by ascorbic acid. An effect mediated by the free radical monodehydroascorbate

3-Hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase activity in microsomes isolated from cultured lymphoid (IM-9) cells or freshly isolated human leukocytes was markedly decreased by either ascorbic acid or its oxidized derivative, dehydroascorbate. Inhibition of IM-9 leukocyte HMG-CoA reductase activity was log linear between 0.01 and 10 mM ascorbic acid (25 and 81% inhibition, respectively) and 0.1 and 10 mM dehydroascorbate (5 and 75% inhibition, respectively). Inhibition was noncompetitive with respect to HMG-CoA (Km = 10.2 microM (RS); ascorbic acid, Ki = 6.4 mM; dehydroascorbate, Ki = 15 mM) and competitive with respect to NADPH (Km = 16.3 microM; acetic acid, Ki = 6.3 mM; dehydroascorbate, Ki = 3.1 mM). Ascorbic acid and dehydroascorbate are interconverted through the free radical intermediate monodehydroascorbate. Reducing agents are required to convert dehydroascorbate to monodehydroascorbate, but prevent formation of the free radical from ascorbate. In microsomes from IM-9 cells, the reducing agent, dithiothreitol, abolished HMG-CoA reductase inhibition by ascorbate but enhanced inhibition by dehydroascorbate. In addition, the concentration of monodehydroascorbate present in ascorbate solutions was directly proportional to the degree of HMG-CoA reductase inhibition by 1.0 mM ascorbate. Fifty per cent inhibition of enzyme activity occurred at a monodehydroascorbate concentration of 14 microM. These data indicate that monodehydroascorbate mediates inhibition of HMG-CoA reductase by both ascorbate and dehydroascorbate. This effect does not appear to be due to free radical-induced membrane lipid modification, however, since both ascorbate and dehydroascorbate inhibited the protease-solubilized, partially purified human liver enzyme. Since inhibition of HMG-CoA reductase occurs at physiological concentrations of ascorbic acid in the human leukocyte (0.2-1.72 mM), this vitamin may be important in the regulation of endogenous cholesterol synthesis in man.     

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.     

Role of Ascorbate in Detoxifying Ozone in the Apoplast of Spinach (Spinacia oleracea L.) Leaves

Both reduced and oxidized ascorbate (AA and DHA) are present in the aqueous phase of the extracellular space, the apoplast, of spinach (Spinacia oleracea L.) leaves. Fumigation with 0.3 [mu]L L-1 of ozone resulted in ozone uptake by the leaves close to 0.9 pmol cm-2 of leaf surface area s-1. Apoplastic AA was slowly oxidized by ozone. The initial decrease of apoplastic AA was <0.1 pmol cm-2 s-1. The apoplastic ratio of AA to (AA + DHA) decreased within 6 h of fumigation from 0.9 to 0.1. Initially, the concentration of (AA + DHA) did not change in the apoplast, but when fumigation was continued, DHA increased and AA remained at a very low constant level. After fumigation was discontinued, DHA decreased very slowly in the apoplast, reaching control level after 70 h. The data show that insufficient AA reached the apoplast from the cytosol to detoxify ozone in the apoplast when the ozone flux into the leaves was 0.9 pmol cm-2 s-1. The transport of DHA back into the cytosol was slower than AA transport into the apoplast. No dehydroascorbate reductase activity could be detected in the apoplast of spinach leaves. In contrast to its extracellular redox state, the intracellular redox state of AA did not change appreciably during a 24-h fumigation period. However, intracellular glutathi-one became slowly oxidized. At the beginning of fumigation, 90% of the total glutathione was reduced. Only 10% was reduced after 24-h exposure of the leaves to 0.3 [mu]L L-1 of ozone. Necrotic leaf damage started to become visible when fumigation was extended beyond a 24-h period. A close correlation between the extent of damage, on the one hand, and the AA content and the ascorbate redox state of whole leaves, on the other, was observed after 48 h of fumigation. Only the youngest leaves that contained high ascorbate concentrations did not exhibit necrotic leaf damage after 48 h.     

Some vertebrates go with the GLO

Most vertebrates synthesize vitamin C (ascorbate) de novo from glucose, but humans and certain other mammals cannot. In this issue, Montel-Hagen et al. (2008) demonstrate that erythrocytes from these ascorbate auxotrophs switch the preference of their glucose transporter Glut1 from glucose to dehydroascorbate (DHA), the oxidized form of vitamin C. This substrate preference switch is mediated by the membrane protein stomatin and is an evolutionary adaptation to vitamin C deficiency.     

Stimulation of onion root elongation by ascorbate and ascorbate free radical inAllium cepa L.

Summary We report that ascorbate free radical stimulates onion root growth at 15 °C and 25 °C. The fully reduced form, ascorbate, also stimulates root elongation if culture conditions allow its oxidation. When ascorbate oxidation was inhibited, no stimulation of root growth was found. The effect of the fully oxidized form, dehydroascorbate, was inhibitory. We show also that ascorbate free radical generated by ascorbate oxidation, is reduced back probably by a transplasmalemma reductase. These results are discussed on the basis of an activation of a transplasma membrane redox system likely involved in processes related to cell growth.Abbreviations AFR ascorbate free radical - ASC ascorbate - DHA dehydroascorbate     

Metabolic control by dehydroascorbic acid: Questions and controversies in cancer cells

For a long time, the effect of vitamin C on cancer cells has been a controversial concept. From Linus Pauling's studies in 1976, it was proposed that ascorbic acid (AA) could selectively kill tumor cells. However, further research suggested that vitamin C has no effect on tumor survival. In the last decade, new and emerging functions for vitamin C have been discovered using the reduced form, AA, and the oxidized form, dehydroascorbic acid (DHA), independently. In this review, we summarized the latest findings related to the effects of DHA on the survival and metabolism of tumor cells. At the same time, we put special emphasis on the bystander effect and the recycling capacity of vitamin C in various cellular models, and how these concepts can affect the experimentation with vitamin C and its therapeutic application in the treatment against cancer.     

Vitamin C function in the brain: vital role of the ascorbate transporter SVCT2

Ascorbate (vitamin C) is a vital antioxidant molecule in the brain. However, it also has a number of other important functions, participating as a cofactor in several enzyme reactions, including catecholamine synthesis, collagen production, and regulation of HIF-1α. Ascorbate is transported into the brain and neurons via the sodium-dependent vitamin C transporter 2 (SVCT2), which causes accumulation of ascorbate within cells against a concentration gradient. Dehydroascorbic acid, the oxidized form of ascorbate, is transported via glucose transporters of the GLUT family. Once in cells, it is rapidly reduced to ascorbate. The highest concentrations of ascorbate in the body are found in the brain and in neuroendocrine tissues such as adrenal, although the brain is the most difficult organ to deplete of ascorbate. Combined with regional asymmetry in ascorbate distribution within different brain areas, these facts suggest an important role for ascorbate in the brain. Ascorbate is proposed as a neuromodulator of glutamatergic, dopaminergic, cholinergic, and GABAergic transmission and related behaviors. Neurodegenerative diseases typically involve high levels of oxidative stress and thus ascorbate has been posited to have potential therapeutic roles against ischemic stroke, Alzheimer's disease, Parkinson's disease, and Huntington's disease.     

Differential rapid analysis of ascorbic acid and ascorbic acid 2-sulfate by dinitrophenylhydrazine method

Ascorbic acid 2-sulfate (AAS) has recently been detected in animals (1,2), and the suggestion has been made that it is involved in some physiological functions (3–5).AAS has been determined by spectrophotometer measurements at 254 nm, ? = 17,000, after isolation from animal tissues (1,6,7). This procedure, however, is complicated and time consuming. Baker et al. (8) reported a rapid assay for AAS from biological samples based on a dinitrophenylhydrazine (DNPH) method which is a standard method for ascorbic acid (AA) determination in biological materials. In this method, AA is oxidized to the osazone by incubation with DNPH in a dilute sulfric acid solution. According to Baker et al. (8), AAS reacts with DNPH during a 3-hr incubation at 60°C but not during a 1-hr incubation at 37°C, and the difference in the reading at 540 nm between the two temperatures corresponds to AAS.This report is concerned with a more rapid and specific method with DNPH for differential measurement of AA and AAS, that is, the differential oxidation of AA and AAS with 2,6-dinitrophenolindophenol (2,6-Dye) and KBrO₃ and the determination of the osazone produced with the original method by Roe and Kuether (9).     

Ozone detoxification in the apoplasm and symplasm of spinach,broad bean and beech leaves at ambient and elevated concentrations of ozone in air

Spinach (Spinacia oleracea L.), broad bean (Vicia faba L.) and beech (Fagus sylvatica L.) plants were exposed to ozone at concentrations often measured in air during the summer months (120–300 g·m^–3) and antioxidants were determined in the leaf tissue and in the aqueous phase of the cell wall, the apoplasm. Concentrations of both reduced ascorbate (AA) and its oxidized form, dehydroascorbate (DHA), showed the tendency to increase transiently in the apoplasm of spinach leaves 6–24 h after starting fumigation with ozone. In beech leaves, apoplasmic AA and DHA increased 3–7 d after beginning of treatment. At the very high concentration of 1600 g O₃·m^–3, an increase of apoplasmic AA was already measured after 1 d in beech leaves. Apparently, spinach and beech leaves respond to oxidative stress by increasing AA transport into the apoplasm and by accelerating DHA export. In contrast to these observations, DHA accumulated during 3 d of fumigation with only 120 g O₃·m^–3 in the apoplasm of broad bean leaves, while AA contents did not increase. After termination of fumigation, the extracellular redox state of ascorbate normalized within 1 d. Glutathione could not be detected in the apoplasm of any of the three leaf species. Intracellular AA changed its redox state in response to exposure to elevated concentrations of ozone. After 4–6 weeks of fumigation with 200–300 g O₃·m^–3 an increase of intracellular DHA was measured in beech leaves. At the same time, chlorophyll contents decreased and characteristic symptoms of ozone damage could be observed. However, no significant change in the redox state of apoplasmic ascorbate could be detected in beech leaves. Evidently, detoxification of ozone by apoplasmic AA was insufficient to protect the leaf tissue. Fumigation with a high ozone concentration (1600 g·m^–3) caused an appreciable increase in the cellular contents of the oxidized forms of ascorbate and glutathione in beech leaves. Whereas in spinach leaves intracellular antioxidant contents and redox states were not altered during fumigation with 120–240 g O₃·m^–3, in broad bean leaves the intracellular DHA concentration increased and intracellular ascorbate became more oxidized after fumigation of the plants with 120 g O₃·m^–3. Apparently, broad bean leaves are more sensitive to ozone than beech and spinach leaves.Abbreviations AA ascorbate, reduced form - DHA ascorbate, oxidized form (dehydroascorbate) - FW fresh weight - GSH glutathione, reduced form - GSSG glutathione, oxidized form - IWF intercellular washing fluid - V_air intercellular air space volume of leaves - V_apo apoplasmic water volume of leaves This work was supported within the Sonderforschungsbereich 251 of the University of Würzburg.     

Glutathione-dependent ascorbate recycling activity of rat serum albumin

An efficient regeneration of vitamin C (ascorbate) from its oxidized byproduct, dehydroascorbate (DHAA), is necessary to maintain sufficient tissue levels of the reduced form of the vitamin. Additionally, the recycling may be more significant in mammals, such as guinea pigs and humans, who have lost the ability to synthesize ascorbate de novo, than it is in most other mammals who have retained the ability to synthesize the vitamin from glucose. Both a chemical and an enzymatic reduction of DHAA to ascorbate have been proposed. Several reports have appeared in which proteins, including thioltransferase, protein disulfide isomerase, and 3-alpha-hydroxysteroid dehydrogenase, characterized for other activities have been identified as having DHAA reductase activity in vitro. Whether these previously characterized proteins catalyze the reduction of DHAA in vivo is unclear. In the present study, a 66 kD protein was purified strictly on the basis of its DHAA-reductase activity and was identified as rat serum albumin. The protein was further characterized and results support the suggestion that serum albumin acts as an antioxidant and exerts a significant glutathione-dependent DHAA-reductase activity that may be important in the physiologic recycling of ascorbic acid.     

Ascorbate uptake in normal and diabetic rat retina and retinal pigment epithelium

Oxidative stress is an important causative factor in the pathogenesis of diabetic retinopathy. Therefore, it becomes important to understand the mechanisms that help maintain appropriate levels of a small molecule antioxidant such as ascorbate in the retina. The outer blood-barrier which results from the tight junctions between the retinal pigment epithelial cells (RPE) restricts the flow of nutrients reaching the retina. In this study, we characterized the transport properties of carboxyl-(14)C ascorbate (AA) in normal rat retina and RPE, and compared them with those in streptozotocin-diabetic rats. Retina and RPE accumulated AA by a temperature-sensitive and energy-dependent kinetic mechanism with an apparent K(M) of 380 and 420 microM, respectively. Accumulation of AA was significantly reduced in a sodium-free medium. Although high glucose concentrations reduced AA uptake by 40%, this was not affected by cytochalasin B. The RPE and retina of diabetic rats presented lower levels of AA accumulation. These findings suggest the presence of the specific vitamin C transporter SVCT in retina and RPE, which may be involved in the manifestation of diabetic retinopathy.     

Use of a redox-sensing GFP (c-roGFP1) for real-time monitoring of cytosol redox status in Arabidopsis thaliana water-stressed plants

Using Arabidopsis plants transformed with a redox-sensing green fluorescent protein targeted to the cytosol (c-roGFP1), we have demonstrated, in real time, measurements of reversible changes of redox status in the cytosol of plants subjected to a gradual water-stress, followed by re-watering. Plants sensed water stress, and changed the redox potential of their cytosol to a more oxidized value after a gradually-imposed water stress. Small variations in the cytosol redox potential and ascorbate (AA) values suggest that this parameter was tightly regulated. The re-watering was paralleled by a return of water stress, redox potential and ascorbate to initial values, showing the reversibility of water stress and its consequences.