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.     

Ascorbic acid: metabolism and functions of a multi-facetted molecule

Ascorbic acid (vitamin C) is the most abundant antioxidant in plants. Its biosynthetic pathway via GDP-D-mannose and L-galactose, which was proposed only recently, is now supported by molecular genetic evidence from Arabidopsis thaliana and transgenic potato plants. Except for the last step (which is located on the inner mitochondrial membrane) the pathway is cytosolic, sharing GDP-sugar intermediates with cell-wall polysaccharide and glycoprotein synthesis. Ascorbate peroxidase is emerging as a key enzyme in the fine control of H(2)O(2) concentration; its expression being controlled by redox signals and H(2)O(2). Convincing evidence of the involvement of ascorbate in cell division and growth is also accumulating. Its role as a cofactor in the synthesis of cell wall hydroxyproline-rich glycoproteins is one mechanism for this function.     

Escherichia coli xthA mutant is not hypersensitive to ascorbic acid/copper treatment--an H2O2 generating reaction

Ascorbate (vitamin C) in the presence of copper yields H2O2, which seems to be responsible for its toxic effects in bacteria. However, we found that the Escherichia coli xthA mutant strain, which is hypersensitive to H2O2, has almost the same sensitivity as the wild-type strain to ascorbate and copper treatment. Our results suggest that the DNA damage induced in E. coli by H2O2 generated in oxidized ascorbate solutions is different from that induced by direct H2O2 treatment.     

Ascorbate uptake is decreased in the hippocampus of ageing rats

Ascorbate, an intracellular antioxidant, has been considered critical for neuronal protection against oxidant stress, which is supported especially by in vitro studies. Besides, it has been demonstrated an age-related decrease in brain ascorbate levels. The aims of the present study were to investigate ascorbate uptake in hippocampal slices from old Wistar rats, as well as its neuroprotective effects in in vitro and in vivo assays. Hippocampal slices from male Wistar rats aged 4, 11 and 24 months were incubated with radiolabeled ascorbate and incorporated radioactivity was measured. Hippocampal slices from rats were incubated with different concentrations of ascorbate and submitted to H(2)O(2)-induced injury, cellular damage and S100B protein levels were evaluated. The effect of chronic administration of ascorbate on cellular oxidative state and astrocyte biochemical parameters in the hippocampus from 18-months-old Wistar rats was also studied. The ascorbate uptake was decreased in hippocampal slices from old-aged rats, while supplementation with ascorbate (2 weeks) did not modify any tested oxidative status in the hippocampus and the incubation was unable to protect hippocampal slices submitted to oxidative damage (H(2)O(2)) from old rats. Our data suggest that the decline of ascorbate uptake might be involved in the brain greater susceptibility to oxidative damage with advancing age and both in vitro and vivo assays suggest that ascorbate supplementation did not protect hippocampal cells.     

Progress in manipulating ascorbic acid biosynthesis and accumulation in plants

l -Ascorbic acid (vitamin C) is synthesized from hexose sugars. It is an antioxidant and redox buffer, as well as an enzyme cofactor, so it has multiple roles in metabolism and in plant responses to abiotic stresses and pathogens. Plant-derived ascorbate also provides the major source of vitamin C in the human diet. An understanding of how ascorbate metabolism is controlled should provide a basis for engineering or otherwise manipulating its accumulation. Biochemical and molecular genetic evidence supports synthesis from GDP- d -mannose via l -galactose ( d -Man/ l -Gal pathway) as a significant source of ascorbate. More recently, evidence for pathways via uronic acids has been obtained: overexpression of myo -inositol oxygenase, d -galacturonate reductase and l -gulono-1,4-lactone oxidase all increase leaf ascorbate concentration. Interestingly, this has proved more effective in pathway engineering than overexpressing various d -Man/ l -Gal pathway genes. Ascorbate oxidation generates the potentially unstable dehydroascorbate, and the overexpression of glutathione-dependent dehydroascorbate reductase has resulted in increased ascorbate. Ascorbate is catabolized to products such as oxalate, l -threonate and l -tartrate. The enzymes involved have not been identified, so catabolism is not yet amenable to manipulation. In the examples of pathway engineering so far, the increase in ascorbate has been modest on an absolute or proportional basis. Therefore, a deeper understanding of ascorbate metabolism is needed to achieve larger increases. Identifying genes that control ascorbate accumulation by techniques such as analysis of quantitative trait loci (QTL) or activation tagging may hold promise, particularly if regulatory genes can be identified.     

Redox cycling of myoglobin and ascorbate: a potential protective mechanism against oxidative reperfusion injury in muscle

Metmyoglobin catalyzes the decomposition of H2O2 as well as other hydroperoxides by using ascorbic acid as a substrate. The ratio of H2O2 reduced to ascorbate oxidized is close to one, whereas the rate of oxidation is directly proportional to both H2O2 and metmyoglobin concentrations. Ascorbate also prevents the protein modifications and the O2 evolution that accompany the reaction of metmyoglobin with hydroperoxides. In the absence of ascorbate, myoglobin and H2O2 promote the peroxidation of unsaturated fatty acids and, thus, may cause damage to cellular constituents. However, lipid peroxidation is inhibited in the presence of ascorbate and, for this reason, it is suggested that this heme protein functions in the opposite manner. The redox cycling of myoglobin by ascorbate may act as an important electron "sink" and defense mechanism against peroxides during oxidative challenge to muscle.     

Effectiveness of ascorbate and ascorbate peroxidase in promoting nitrogen fixation in model systems

Ascorbate and ascorbate peroxidase are important antioxidants that are abundant in N2-fixing legume root nodules. Antioxidants are especially critical in root nodules because leghemoglobin, which is present at high concentrations in nodules, is prone to autoxidation and production of activated oxygen species such as O2.- and H2O2. The merits of ascorbate and ascorbate peroxidase for maintaining conditions favorable for N2 fixation were examined in two model systems containing oxygen-binding proteins (purified myoglobin or leghemoglobin) and N2-fixing microorganisms (free-living Azorhizobium or bacteroids of Bradyrhizobium japonicum) in sealed vials. The inclusion of ascorbate alone to these systems led to enhanced oxygenation of hemeproteins, as well as to increases in nitrogenase (acetylene reduction) activity. The inclusion of both ascorbate and ascorbate peroxidase resulted in even greater positive responses, including increases of up to 4.5-fold in nitrogenase activity. In contrast, superoxide dismutase did not provide beneficial antioxidant action and catalase alone provided only very marginal benefit. Optimal concentrations were 2 mM for ascorbate and 200 micrograms/ml for ascorbate peroxidase. These concentrations are similar to those found in intact soybean nodules. These results support the conclusion that ascorbate and ascorbate peroxidase are beneficial for maintaining conditions favorable for N2 fixation in nodules.     

Ascorbate does not act as a pro-oxidant towards lipids and proteins in human plasma exposed to redox-active transition metal ions and hydrogen peroxide

The combination of ascorbate, transition metal ions, and hydrogen peroxide (H(2)O(2)) is an efficient hydroxyl radical generating system called "the Udenfriend system." Although the pro-oxidant role of ascorbate in this system has been well characterized in vitro, it is uncertain whether ascorbate also acts as a pro-oxidant under physiological conditions. To address this question, human plasma, used as a representative biological fluid, was either depleted of endogenous ascorbate with ascorbate oxidase, left untreated, or supplemented with 25 microM-1 mM ascorbate. Subsequently, the plasma samples were incubated at 37 degrees C with 50 microM-1 mM iron (from ferrous ammonium sulfate), 60 or 100 microM copper (from cupric sulfate), and/or 200 microM or 1 mM H(2)O(2). Although endogenous and added ascorbate was depleted rapidly in the presence of transition metal ions and H(2)O(2), no cholesterol ester hydroperoxides or malondialdehyde were formed, i.e., ascorbate protected against, rather than promoted, lipid peroxidation. Conversely, depletion of endogenous ascorbate was sufficient to cause lipid peroxidation, the rate and extent of which were enhanced by the addition of metal ions but not H(2)O(2). Ascorbate also did not enhance protein oxidation in plasma exposed to metal ions and H(2)O(2), as assessed by protein carbonyl formation and depletion of reduced thiols. Interestingly, neither the rate nor the extent of endogenous alpha-tocopherol oxidation in plasma was affected by any of the treatments. Our data show that even in the presence of redox-active iron or copper and H(2)O(2), ascorbate acts as an antioxidant that prevents lipid peroxidation and does not promote protein oxidation in human plasma in vitro.     

Ascorbate enhances the toxicity of the photodynamic action of Verteporfin in HL-60 cells

As a reducing agent, ascorbate serves as an antioxidant. However, its reducing function can in some settings initiate an oxidation cascade, i.e., seem to be a "pro-oxidant." This dichotomy also seems to hold when ascorbate is present during photosensitization. Ascorbate can react with singlet oxygen, producing hydrogen peroxide. Thus, if ascorbate is present during photosensitization the formation of highly diffusible hydrogen peroxide could enhance the toxicity of the photodynamic action. On the other hand, ascorbate could decrease toxicity by converting highly reactive singlet oxygen to less reactive hydrogen peroxide, which can be removed via peroxide-removing systems such as glutathione and catalase. To test the influence of ascorbate on photodynamic treatment we incubated leukemia cells (HL-60 and U937) with ascorbate and a photosensitizer (Verteporfin; VP) and examined ascorbic acid monoanion uptake, levels of glutathione, changes in membrane permeability, cell growth, and toxicity. Accumulation of VP was similar in each cell line. Under our experimental conditions, HL-60 cells were found to accumulate less ascorbate and have lower levels of intracellular GSH compared to U937 cells. Without added ascorbate, HL-60 cells were more sensitive to VP and light treatment than U937 cells. When cells were exposed to VP and light, ascorbate acted as an antioxidant in U937 cells, whereas it was a pro-oxidant for HL-60 cells. One possible mechanism to explain these observations is that HL-60 cells express myeloperoxidase activity, whereas in U937 cells it is below the detection limit. Inhibition of myeloperoxidase activity with 4-aminobenzoic acid hydrazide (4-ABAH) had minimal influence on the phototoxicity of VP in HL-60 cells in the absence of ascorbate. However, 4-ABAH decreased the toxicity of ascorbate on HL-60 cells during VP photosensitization, but had no affect on ascorbate toxicity in U937 cells. These data demonstrate that ascorbate increases hydrogen peroxide production by VP and light. This hydrogen peroxide activates myeloperoxidase, producing toxic oxidants. These observations suggest that in some settings, ascorbate may enhance the toxicity of photodynamic action.     

Involvement of Protein Phosphorylation in Water Stress-induced Antioxidant Defense in Maize Leaves

Using pharmacological and biochemical approaches, the role of protein phosphorylation and the interrelationship between water stress-enhanced kinase activity, antioxidant enzyme activity, hydrogen peroxide (H2O2) accumulation and endogenous abscisic acid in maize (Zea mays L.) leaves were investigated. Water-stress upregulated the activities of total protein phosphorylation and Ca2+ -dependent protein kinase, and the upregulation was blocked in abscisic acid-deficient vp5 mutant. Furthermore, pretreatments with a nicotinamide adenine dinucleotide phosphate oxidase inhibitor and a scavenger of H2O2 significantly reduced the increased activities of total protein kinase and Ca2+-dependent protein kinase in maize leaves exposed to water stress. Pretreatments with different protein kinase inhibitors also reduced the water stress-induced H2O2 production and the water stress-enhanced activities of antioxidant enzymes such as superoxide dismutase, catalase, ascorbate peroxidase and glutathione reductase. The data suggest that protein phosphorylation and H2O2 generation are required for water stress-induced antioxidant defense in maize leaves and that crosstalk between protein phosphorylation and H2O2 generation may occur.     

Involvement of Polyamine Oxidase in Abscisic Acid induced Cytosolic Antioxidant Defense in Leaves of Maize

Using pharmacological and biochemical approaches, the role of maize polyamine oxidase (MPAO) in abscisic acid (ABA)induced antioxidant defense in leaves of maize (Zea mays L.) plants was investigated. Exogenous ABA treatment enhanced the expression of the MPAO gene and the activities of apoplastic MPAO. Pretreatment with two different inhibitors for apoplastic MPAO partly reduced hydrogen peroxide (H2O2) accumulation induced by ABA and blocked the ABA-induced expression of the antioxidant genes superoxide dismutase 4 and cytosolic ascorbate peroxidase and the activities of the cytosolic antioxidant enzymes. Treatment with spermidine, the optimum substrate of MPAO, also induced the expression and the activities of the antioxidant enzymes, and the upregulation of the antioxidant enzymes was prevented by two inhibitors of MPAO and two scavengers of H2O2. These results suggest that MPAO contributes to ABA-induced cytosolic antioxidant defense through H2O2, a Spd catabolic product.     

Oxygen reduction in chloroplasts and the ascorbate cycle

Current views concerning the generation of superoxide radicals and hydrogen peroxide in chloroplasts as well as their toxic influences on photosynthesis are presented. Systems of H2O2 detoxification including the ascorbate peroxidase reactions and the ascorbate regenerating reactions are described. Data concerning mechanisms of monodehydroascorbate reduction by the photosynthetic electron transport chain are reviewed. The participation of the Mehler-peroxidase reaction in building of a proton gradient across the thylakoid membrane and its possible input in ATP synthesis and in protection from photoinhibition are analyzed. Ascorbate functions in chloroplasts and the need to consider the high concentration of ascorbate in chloroplasts when photosynthetic reactions in vivo are discussed are briefly reviewed.     

The Function of Ascorbic Acid in Photosynthetic Phosphorylation

Ascorbate is oxidized to the free radical monodehydroascorbate by O2.- and by H2O2 (through the action of ascorbate peroxidase) formed in the Mehler reaction by isolated spinach (Spinacia oleracea) thylakoids. Light-dependent electron transport from water to monodehydroascorbate is shown to be coupled to ATP formation with a ratio ATP:O2 of 2. In the presence of ascorbate the net O2 exchange balance of the Mehler reaction is close to zero, and the synthesis of ATP is increased 2 to 3 times due to the extra electron transport to the monodehydroascorbate free radical. A scheme of the electron transport in the presence of ascorbate is discussed.     

Ascorbate interacts with reduced glutathione to scavenge phenoxyl radicals in HL60 cells

We have compared the abilities of ascorbate and reduced glutathione (GSH) to act as intracellular free radical scavengers and protect cells against radical-mediated lipid peroxidation. Phenoxyl radicals were generated in HL60 cells, through the action of their myeloperoxidase, by adding H2O2 and phenol. Normally cultured cells, which contain no ascorbate; cells that had been preloaded with ascorbate; and those that had been depleted of GSH with buthionine sulfoximine were investigated. Generation of phenoxyl radicals resulted in the oxidation of ascorbate and GSH. Ascorbate loss was much greater in the absence of GSH, and adding glucose gave GSH-dependent protection against ascorbate loss. Ascorbate, or glucose metabolism, had little effect on the GSH loss. Glutathionyl radical formation was detected by spin trapping with DMPO in cells lacking ascorbate, and the signal was suppressed by ascorbate loading. Addition of phenol plus H2O2 to the cells caused lipid peroxidation, as measured with C11-BODIPY. Peroxidation was greatest in cells that lacked both ascorbate and GSH. Either scavenger alone gave substantial inhibition but optimal protection was seen with both present. These results indicate that GSH and ascorbate can each act as an intracellular radical scavenger and protect against lipid peroxidation. With both present, ascorbate is preferred and acts as the ultimate radical sink for phenoxyl or glutathionyl radicals. However, GSH is still consumed by metabolically recycling dehydroascorbate. Thus, recycling scavenging by ascorbate does not spare GSH, but it does enable the two antioxidants to provide more protection against lipid peroxidation than either alone.     

"Antioxidant" (reducing) efficiency of ascorbate in plasma is not affected by concentration

Ascorbate (vitamin C), an important dietary derived antioxidant, reportedly shows decreasing "antioxidant efficiency" with increasing concentrations in indirect radical trapping methods of antioxidant capacity. This study investigated the effect of concentration on antioxidant efficiency of ascorbate using a direct test of antioxidant capacity, the ferric reducing/antioxidant power test (FRAP assay). Results showed that the antioxidant efficiency factor of ascorbate was 2 and was constant over a wide concentration range in both plasma and pure aqueous solution. However, the absolute amount of ascorbate lost per unit of time increased with concentration. Furthermore, ascorbate was less stable in plasma than in aqueous solutions of similar pH and less stable in ethylenediamine tetraacetic acid (EDTA) than in heparinized plasma. Results indicate that previously reported concentration-dependent changes in antioxidant efficiency of ascorbate may have been caused by loss of ascorbate prior to and during testing, and by methodologic characteristics of indirect peroxyl radical trapping tests of antioxidant capacity. Therefore, it is suggested that the premise that the antioxidant efficiency of ascorbate is concentration-dependent is largely methodologically derived and does not reflect the antioxidant behavior of ascorbate per se.     

Dithiothreitol induces the sacrificial antioxidant property of human serum albumin in a metal-catalyzed oxidation and gamma-irradiation system

The species *OH or H2O2 are produced by both metal-catalyzed oxidation (MCO) of reducing equivalents and gamma-irradiation. Intact or Cys-34-modified human serum albumin (HSA) was significantly degraded in the MCO system containing dithiothreitol (DTT) as electron donor, but as long as it lasted, HSA prohibited *OH or H2O2 from initiating molecular damage of DNA. However, in the GSH and ascorbate (nonthiol) MCO system, HSA was not sacrificially degraded, and indeed accelerated the formation of DNA strand breaks. In the y-irradiation system producing *OH from H2O, only DTT attenuated the generation of DNA strand breaks by HSA. It did not degrade more H2O2 in the presence of reduced GSH (thiol-linked peroxidase) than in its absence. Therefore it would seem that in an MCO system, the antioxidant activity of HSA depends on the effectiveness of reducing equivalents to induce exposure of a functional group scavenging the *OH or H2O2 species, by reduction of its disulfide-bonds. In the presence of DTT, disulfide bonds in HSA were quantitatively reduced to cysteinyl residues but not significantly reduced by ascorbate or GSH. In conclusion, the antioxidant activity of HSA in the D     

Effect of ascorbate and its oxidation products on H2O2 production in cell-suspension cultures of Picea abies and in the absence of cells

Dehydroascorbate and traces of ascorbate were present apoplastically in living spruce (Picea abies) twigs. Since the proposed apoplastic ascorbate degradation pathway contains several steps that possibly generate H(2)O(2), the effects of ascorbate and some of its degradation products were tested on apoplastic H(2)O(2) concentrations in a cell culture of P. abies as a model and on non-enzymic H(2)O(2) production in vitro. Ascorbate scavenged H(2)O(2) in the culture medium of lignin-producing Picea cells and in spent and boiled spent medium; in the presence of Cu(2+) or fresh medium, ascorbate led to the non-enzymic generation of H(2)O(2). Preparations of dehydroascorbate (the initial oxidation-product of ascorbate), and diketogulonate (the hydrolysis-product of dehydroascorbate) induced H(2)O(2) accumulation both non-enzymically and enzymically in Picea cell-suspensions. Paper electrophoresis showed that the dehydroascorbate and diketogulonate preparations contained several degradation products; some of these probably contributed to H(2)O(2) production and/or scavenging in these experiments, and would also do so in vivo. These results indicate a complex ability of apoplastic ascorbate, dehydroascorbate, diketogulonate, and further products to modulate H(2)O(2) concentrations, with potential consequences for the control of growth, development and lignification.     

Vitamin C: poison,prophylactic or panacea?

Ascorbate is an essential enzyme cofactor but is often also regarded as an important antioxidant in vivo, protecting against cancer by scavenging DNA-damaging reactive oxygen species. Recent studies suggest that ascorbate sometimes increases DNA damage in humans. Although there is no evidence that any of these effects are deleterious to humans, we might need to change our thinking about the mechanisms of the antioxidant action of ascorbate in vivo.     

Nitric oxide modulates sodium vitamin C transporter 2 (SVCT-2) protein expression via protein kinase G (PKG) and nuclear factor-κB (NF-κB)

Ascorbate is an important antioxidant, which also displays important functions in neuronal tissues, including the retina. The retina is responsible for the initial steps of visual processing, which is further refined in cerebral high-order centers. The retina is also a prototypical model for studying physiologic aspects of cells that comprise the nervous system. Of major importance also is the cellular messenger nitric oxide (NO). Previous studies have demonstrated the significance of NO for both survival and proliferation of cultured embryonic retinal cells. Cultured retinal cells express a high-affinity ascorbate transporter, and the release of ascorbate is delicately regulated by ionotropic glutamate receptors. Therefore, we proposed whether there is interplay between the ascorbate transport system and NO signaling pathway in retinal cells. Here we show compelling evidence that ascorbate uptake is tightly controlled by NO and its downstream signaling pathway in culture. NO also modulates the expression of SVCT-2, an effect mediated by cGMP and PKG. Kinetic studies suggest that NO increases the transport capacity for ascorbate, but not the affinity of SVCT-2 for its substrate. Interestingly, NO utilizes the NF-κB pathway, in a PKG-dependent manner, to modulate both SVCT-2 expression and ascorbate uptake. These results demonstrate that NO exerts a fine-tuned control of the availability of ascorbate to cultured retinal cells and strongly reinforces ascorbate as an important bioactive molecule in neuronal tissues.     

Stimulation of the pentose phosphate pathway and glutathione levels by dehydroascorbate, the oxidized form of vitamin C.

Ascorbic acid, or vitamin C, generally functions as an antioxidant by directly reacting with reactive oxygen intermediates and has a vital role in defenses against oxidative stress. However, ascorbic acid also has pro-oxidant properties and may cause apoptosis of lymphoid and myeloid cells. The present study shows that dehydroascorbate, the oxidized form of vitamin C, stimulates the antioxidant defenses of cells, preferentially importing dehydroascorbate over ascorbate. While 200-800 microM vitamin C caused apoptosis of Jurkat and H9 human T lymphocytes, pretreatment with 200-1000 microM dehydroascorbate stimulated activity of pentose phosphate pathway enzymes glucose 6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, and transaldolase, elevated intracellular glutathione levels, and inhibited H(2)O(2)-induced changes in mitochondrial transmembrane potential and cell death. A 3. 3-fold maximal glutathione elevation was observed after 48 h stimulation with 800 microM dehydroascorbate. In itself, dehydroascorbate did not affect cytosolic or mitochondrial reactive oxygen intermediate levels as monitored by flow cytometry using oxidation-sensitive fluorescent probes. The data reveal a novel mechanism for increasing glutathione levels through stimulation of the pentose phosphate pathway and identify dehydroascorbate as an antioxidant for cells susceptible to the pro-oxidant and proapoptotic properties of vitamin C.