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PubMed1976

Regulation of glucose transport in chick fibroblasts: bicarbonate, lactate and ascorbic acid.

The rate at which chick embryo fibroblasts in primary or secondary culture transport glucose or 3-O-methyl glucose is strongly influenced by the presence of bicarbonate ion in the culture medium. Cells growing or maintained on glucose at physiologic concentration (5.5 mM) have an 8 to 10 fold higher rate of glucose uptake than their counterparts cultivated without bicarbonate. These cells also produce more lactate as a consequence of their more rapid intake of glucose. The hydrogen acceptors, methylene blue and dehydroascorbate added to the culture medium reduce the cell capacity to transport glucose and 3-O-methyl glucose to levels obtaining in the bicarbonate-free medium. There is a concomitant reduction in glucose utilized by cells during 24 hours and further reduction in lactate formed per molecule of glucose metabolized.

Animals↗
PubMed1998

Protection and recycling of alpha-tocopherol in human erythrocytes by intracellular ascorbic acid.

Ascorbic acid can recycle alpha-tocopherol from the tocopheroxyl free radical in lipid bilayers and in micelles, but such recycling has not been demonstrated to occur across cell membranes. In this work the ability of intracellular ascorbate to protect and to recycle alpha-tocopherol in intact human erythrocytes and erythrocyte ghosts was investigated. In erythrocytes that were 80% depleted of intracellular ascorbate by treatment with the nitroxide Tempol, both 2,2'-azobis(2-amidinopropane) dihydrochloride (AAPH) and ferricyanide oxidized alpha-tocopherol to a greater extent than in cells not depleted of ascorbate. In contrast, in erythrocytes in which the intracellular ascorbate concentration had been increased by loading with dehydroascorbate, loss of alpha-tocopherol was less with both oxidants than in control cells. Protection against AAPH-induced oxidation of alpha-tocopherol was not prevented by extracellular ascorbate oxidase, indicating that the protection was due to intracellular and not to extracellular ascorbate. Incubation of erythrocytes with lecithin liposomes also generated an oxidant stress, which caused lipid peroxidation in the liposomes and depleted erythrocyte alpha-tocopherol, leading to hemolysis. Ascorbate loading of the erythrocytes delayed liposome oxidation and decreased loss of alpha-tocopherol from both cells and from alpha-tocopherol-loaded liposomes. When erythrocyte ghosts were resealed to contain ascorbate and challenged with free radicals generated by AAPH outside the ghosts, intravesicular ascorbate was totally depleted over 1 h of incubation, whereas alpha-tocopherol decreased only after ascorbate was substantially oxidized. These results suggest that ascorbate within the erythrocyte protects alpha-tocopherol in the cell membrane by a direct recycling mechanism.

Amidines↗
PubMed1999

Ascorbic acid recycling in Nb2 lymphoma cells: implications for tumor progression.

Analysis of cultured rat "Nb2 lymphoma" cell lines, showing different degrees of malignant progression, can lead to identification of phenotypic changes associated with this phenomenon in T-cell cancers. In the present study we have compared the metastatic sublines, Nb2-11 and Nb2-SFJCD1, with regard to ascorbate and glutathione recycling, important processes in cellular protection from oxidative stresses. Whereas the Nb2-11 subline is prolactin (PRL)-dependent, the genetically related Nb2-SFJCD1 subline is growth factor-independent and shows more chromosomal alterations, indicative of more advanced progression. The Nb2-SFJCD1 cells, compared to the Nb2-11 cells, were less sensitive to toxic effects of dehydroascorbate, a potentially toxic oxidation product of ascorbate. Results were consistent with a significantly higher production of reducing equivalents (e.g., NADPH, GSH) and an accelerated reduction of dehydroascorbate by homogenates of Nb2-SFJCD1 cells. However, the increased resistance was apparently not directly related to the cellular uptake and reduction of dehydroascorbate by whole cells, which was similar in both cell lines. Observations indicate that Nb2 lymphoma cells, in their progression to malignancy, can acquire an enhanced capability to protect themselves from oxidative damage assisting them in withstanding the oxidative stress that anti-neoplastic drugs can cause. The adaptation may also be a mechanism that is utilized by tumor cells in suppressing apoptosis and other protective cellular functions facilitating, or potentiating, a tumor cell's ability to become more metastatic. However, the mechanism leading to this augmented capacity of Nb2 lymphoma cells to resist oxidative stress in not known and is the subject for further study.

Animals↗
PubMed2003

Dehydroascorbate uptake activity correlates with cell growth and cell division of tobacco bright yellow-2 cell cultures.

Recently, ascorbate (ASC) concentration and the activity of a number of enzymes from the ASC metabolism have been proven to correlate with differences in growth or cell cycle progression. Here, a possible correlation between growth and the activity of a plasma membrane dehydroascorbate (DHA) transporter was investigated. Protoplasts were isolated from a tobacco (Nicotiana tabacum) Bright Yellow-2 cell culture at different intervals after inoculation and the activity of DHA transport was tested with (14)C-labeled ASC. Ferricyanide (1 mM) or dithiothreitol (1 mM) was included in the test to keep the external (14)C-ASC in its oxidized respectively reduced form. Differential uptake activity was observed, correlating with growth phases of the cell culture. Uptake of DHA in cells showed a peak in exponential growth phase, whereas uptake in the presence of dithiothreitol did not. The enhanced DHA uptake was not due to higher endogenous ASC levels that are normally present in exponential phase because preloading of protoplasts of different ages did not affect DHA uptake. Preloading was achieved by incubating cells before protoplastation for 4 h in a medium supplemented with 1 mM DHA. In addition to testing cells at different growth phases, uptake of DHA into the cells was also followed during the cell cycle. An increase in uptake activity was observed during M phase and the M/G1 transition. These experiments are the first to show that DHA transport activity into plant cells differs with cell growth. The relevance of the data to the action of DHA and ASC in cell growth will be discussed.

Ascorbic Acid↗
PubMed1987

Effect of ascorbate and dehydroascorbate on tissue uptake of glucose.

In vitro studies have suggested that ascorbate or dehydroascorbate share with glucose the same tissue-transport carrier. To determine if ascorbic acid or its oxidized form can inhibit tissue uptake of glucose, the brain uptake index (BUI) and muscle uptake index of glucose were determined by single arterial injection tissue-sampling technique. The injectate was either buffered Ringer's solution with varying concentrations of ascorbate, dehydroascorbate (pH 7.4), or 70% serum from individuals on vitamin C supplements. Ascorbic acid over a wide range of concentrations (0-10,000 mg/L) did not reduce the BUI. Ascorbic acid reduced BUI from the control value of 33 +/- 3.2 to 20.1 +/- 2.2% (P less than .01) only at 100,000 mg/L; this effect was probably secondary to osmotic disruption of blood-brain barrier. In contrast, dehydroascorbate inhibited the BUI of glucose from baseline value of 32.8 +/- 1.1 to 10.7 +/- 0.67%, with an estimated Ki of 13.0 mM. Masseter muscle glucose uptake was not significantly altered over a wide range of ascorbate or dehydroascorbate concentrations in the injectate. Dehydroascorbate (7500 mg/L) did not significantly reduce the BUI of [14C]phenylalanine (55.2 +/- 4.4 vs. 62.1 +/- 4.2% in controls). When serum from six individuals on calcium ascorbate (3-5 g/day) was compared with that of nine controls, the BUI was not different (19.3 +/- 1.7 vs. 19.3 +/- 1.1%). Similarly, supplementing the diet of eight healthy volunteers with 1 g calcium ascorbate for 8 days did not alter the BUI of glucose.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1995

The uptake of ascorbic acid into human umbilical vein endothelial cells and its effect on oxidant insult.

Intracellular reduced ascorbate (AA) levels in confluent cultures of human umbilical vein endothelial (HUVE) cells, grown under conventional conditions, were shown to be very low, ranging between undetectable, < 0.1 nmol/mg protein, and 0.3 nmol/mg protein. Reduced ascorbate was accumulated into the endothelial cells from M199 culture medium in time- and concentration-dependent manners, and was saturated at medium concentrations related to the normal plasma concentrations of the antioxidant (i.e. between 50 microM and 100 microM). Cells derived from different individuals demonstrated considerable inter-individual variation in these AA uptake parameters. The uptake of AA was sensitive to temperature and the presence of the structural analogue isoascorbate in the medium, indicating the involvement of an active transport mechanism. A role for the glucose transporter is, however, not indicated, as AA uptake was not sensitive to phloretin, an inhibitor of the cellular glucose transporter, nor greatly enhanced by depletion of glucose from the medium. Incubation of HUVE cells with dehydroascorbate (DHAA) caused a dose-dependent, but transient increase in intracellular AA. This indicates that HUVE cells are both competent in the uptake and intracellular reduction of oxidised ascorbate, and may resecrete AA into the medium. Indeed, reduced ascorbate in the medium was shown to be preferentially maintained in the presence of cells. The uptake of AA was not sensitive to the presence of DHAA in the medium, perhaps indicating different transporters for reduced and oxidised forms of ascorbate in these human cells. Pre-loading HUVE cells with AA was shown to protect control cells only weakly from the acute, sub-lethal toxicity of H2O2 generated by xanthine oxidase (1 U/mL or 10 U/mL). Protection was optimal at intracellular levels of 3-4 nmol AA/mg protein, with higher concentrations lacking a protective effect. Additionally, the presence of the iron chelator, desferoxamine, significantly protected GSH-depleted HUVE cells only in response to the peroxide, but did not potentiate the protective action of intracellular AA in either control or GSH-depleted cells. This indicates that ascorbate-driven redox-cycling of the Fe2+/Fe3+ does not hamper the intracellular protective function of ascorbate during hydrogen peroxide-derived oxidative stress. These results are discussed in terms of the central role of endothelial cells in the distribution of AA to the tissues of the body, the use of the HUVE cell system for model studies of the toxicity of oxidants in the human endothelium, and the balance between the antioxidant and pro-oxidant actions of AA.

Ascorbic Acid↗
PubMed1989

Electrogenic Na+-ascorbate cotransport in cultured bovine pigmented ciliary epithelial cells.

The high level of ascorbic acid (AA) in the aqueous humor of many mammals suggests an active transport of AA across the double-layered ciliary epithelium from blood to aqueous humor. We used [14C]AA to study AA uptake in bovine pigmented ciliary epithelial cells in tissue culture. We observed a 40-fold intracellular accumulation of AA, which was dependent on extracellular Na+. With labeled dehydroascorbate (DHA, the oxidized form of the vitamin) in the medium, there was a 20-fold intracellular accumulation of the label. However, the time course of DHA uptake was different compared with AA uptake and was not Na+ dependent, suggesting different transport systems for AA and DHA. AA uptake was inhibited by 1 mM phloretin and in the presence of isoascorbate. Furthermore, AA uptake was markedly reduced when intracellular Na+ was elevated by preincubation with ouabain or amphotericin B. With increasing AA concentration, Na+-dependent AA uptake exhibited first-order saturation kinetics with half-maximal uptake at 76 microM AA. Na+ dependence of AA uptake revealed a sigmoidal curve of Na+-dependent AA uptake vs. Na+ concentration with a half-maximal AA uptake at 45.4 mM Na+. The slope of the Hill plot from these data was 1.94, suggesting a transport system translocating two or more Na+ for one AA. This stoichiometry implies electrogenicity of the transporter. We, therefore, measured membrane potentials using conventional microelectrodes. Addition of 200 microM AA resulted in a depolarization of the membrane voltage by 4.9 +/- 0.5 mV (n = 22), which was absent in Na+ free medium and was markedly reduced by phloretin.(ABSTRACT TRUNCATED AT 250 WORDS)

Amphotericin B↗
PubMed2003

Vitamin C metabolomic mapping in the lens with 6-deoxy-6-fluoro-ascorbic acid and high-resolution 19F-NMR spectroscopy.

PURPOSE: Metabolomics, or metabolic profiling, is an emerging discipline geared to providing information on a large number of metabolites, as a complement to genomics and proteomics. In the current study, a fluorine-labeled derivative of ascorbic acid (F-ASA), a major antioxidant- and UV-trapping molecule in the aqueous humor and the lens, was used to investigate the extent to which the lens accumulates potentially toxic degradation products of vitamin C. METHODS: Human lens epithelial cells (HLE-B3) and rat lenses were exposed to hyperglycemic or oxidative stress in vitro or in vivo and probed for accumulation of F-ASA, fluoro-dehydroascorbate (F-DHA), fluoro-2,3-diketogulonate (F-DKG), and their degradation products in protein-free extracts, by proton-decoupled 750-MHz (19)F-nuclear magnetic resonance (NMR) spectroscopy. RESULTS: F-ASA and F-DHA were taken up into HLE B-3 cells by an Na(+)-dependent transporter. Their uptake was unexpectedly only slightly affected by hyperglycemia in vitro, unless glutathione was severely depleted. Glycemic stress catalyzed oxidation of F-ASA into a single novel F-compound at -212.4 ppm, whereas F-DHA and F-DKG were the major degradation products observed after GSH depletion. In contrast, F-ASA uptake was markedly suppressed in diabetic cataractous rat lenses, which accumulated both the F-DHA and the -212.4-ppm compound. In an unexpected finding, the latter formed only from F-ASA and not F-DHA or F-DKG, suggesting a novel pathway of in vivo F-ASA degradation. Both the cells and the intact rat and human lenses were permeable to several advanced F-ASA and F-DHA degradation products, except F-DKG. The unknown compound at -212.4 ppm was the only F-ASA degradation product that spontaneously formed in rabbit aqueous humor upon incubation with F-ASA. CONCLUSIONS: These studies suggest the existence of a novel ascorbic-acid-degradation pathway in the lens and aqueous humor that is influenced by the nature of the oxidant stress. Under similar culture conditions, intact lenses are more prone to hyperglycemia-mediated oxidant stress than are lens epithelial cells, but both are permeable to various F-ASA degradation products, the structure and biological roles of which remain to be established.

2,3-Diketogulonic Acid↗
PubMed2000

Plasma levels and redox status of ascorbic acid and levels of lipid peroxidation products in active and passive smokers.

Both active and passive smoking are regarded as risk factors for various diseases. To clarify the effects of active and passive smoking on plasma vitamin C levels and lipid peroxidation status, we examined the plasma levels of ascorbic acid (AA), its redox status [ratio of dehydroascorbate (DHAA) to total AA], the levels of thiobarbiturate reactive substance (TBARS), and the levels of lipid peroxides (LPO) in smokers, nonsmokers, and nonsmokers regularly exposed to environmental cigarette smoke (passive smokers). The study population consisted of 149 healthy males: 75 active smokers (consumption of > 15 cigarettes/day for more than 5 years), 36 passive smokers (more than 10 hr/week exposure to environmental cigarette smoke), and 38 nonsmokers (no cigarette smoke exposure). There were no significant differences in plasma TBARS and LPO levels among the three groups. Plasma levels of AA, the reduced form of vitamin C, were significantly lower in active smokers than in the combined nonsmoking groups (7.2 +/- 3.5 and 8.4 +/- 3.4 microg/mL, respectively; p < 0.05). Although no significant differences were found in plasma DHAA levels among the three groups, the ratios of DHAA to total AA were significantly higher in active and passive smokers than nonexposed nonsmokers (11.2, 10.3, and 7.1%, respectively; p < 0.05). These results indicate that passive smoking, as well as direct inhalation of cigarette smoke, affects the redox status of plasma AA. In passive smokers, the altered redox status of plasma AA suggests an oxidative stress.

Adult↗
PubMed1995

Phycobilin biosynthesis: reductant requirements and product identification for heme oxygenase from Cyanidium caldarium.

Algal heme oxygenase is a soluble enzyme from Cyanidium caldarium that catalyzes the first committed step of phycobilin biosynthesis by converting protoheme to biliverdin IX alpha. Although the physiological substrate (protoheme) of algal heme oxygenase is identical to that of microsomal heme oxygenase, which catalyzes heme catabolism in animals, the two enzyme systems differ in several respects including the nature of the required reductants and solubility of the enzymes. Addition of the strong Fe3+ ion chelators, desferrioxamine and Tiron (4,5-dihydroxy-1,3-benzenedisulfonic acid), greatly increased the yield of solvent-extracted bilin product. The effect of the Fe3+ chelators was approximately equal whether they were added during or after the enzyme incubation. Postincubation treatment of the enzyme reaction mixture with strong acid also greatly increased the product yield. Addition of desferrioxamine to the reaction mixture after the incubation was terminated caused the appearance of an absorption spectrum, indicating an increase in the concentration of free bilin product. Acid and Fe3+ chelators are known to cause dissociation of Fe(III)-bilin complexes. These results indicate that the in vitro enzymic reaction product of algal heme oxygenase is a nonenzyme-bound Fe(III)-biliverdin IX alpha complex that is poorly extracted and/or quantitated unless it is first dissociated. Algal heme oxygenase required the simultaneous presence of both reduced ferredoxin and a second reductant such as ascorbate for activity. The requirement for L-ascorbate could be substituted by Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid) or D-ascorbate, but not by dehydroascorbate or dithiothreitol. Heme oxygenase was purified over 200-fold from C. caldarium by differential (NH4)2SO4 precipitation and serial column chromatography over reactive blue 2-Sepharose, DEAE-cellulose, Sephadex G-75, and ferredoxin-Sepharose.

1,2-Dihydroxybenzene-3,5-Disulfonic Acid Disodium ↗
PubMed2003

Up-regulation of the 31 kDa dehydroascorbate reductase in the modified skeletal muscle cell (nurse cell) during Trichinella spp. infection.

Ascorbic acid (AA) is an important factor of defence against oxidative stress. AA is maintained in the reduced functional form by glutathione (GSH)-dependent dehydroascorbate (DHA) reducing enzymes, including the cytosolic glutaredoxin, the microsomal protein disulphide isomerase, and a DHA reductase of 31 kDa, hereafter referred to as DHAR, purified from rat liver cytosol and human red cells. As these mechanisms have rarely been studied in parasites, we looked for the possible presence of this 31 kDa protein in Trichinella spiralis L(1) larvae. Biochemical data, immunoblot analysis and immunohistochemical studies suggested the absence of this protein within parasites at this stage. However, they possess a low DHA reducing ability, which is probably due to the presence of glutaredoxin. On the other hand, immunohistochemical studies performed in histological sections of muscle tissue from Trichinella-infected animals showed an increase in DHAR in the nurse cell (NC) of T. spiralis- and Trichinella britovi-infected animals, compared with the surrounding muscle fibres. This result was confirmed by immunoblot analysis, whereas no such increase was observed in Trichinella pseudospiralis-infected animals. In the modified skeletal muscle cell also haeme oxygenase 1 increased, as well as lipoperoxidised proteins. Both findings suggest an oxidative stress of the NC, which might be related to the intense inflammatory reaction which surrounds the NC-parasite complex. Another possibility to explain the increase in DHAR could be that the NC needs to recycle a substantial amount of AA to synthesise the collagen capsule.

Animals↗
PubMed2003

Increasing vitamin C content of plants through enhanced ascorbate recycling.

Vitamin C (ascorbic acid) is essential to prevent disease associated with connective tissue (e.g., scurvy), improves cardiovascular and immune cell functions, and is used to regenerate alpha-tocopherol (vitamin E). In contrast to most animals, humans lack the ability to synthesize ascorbic acid as a result of a mutation in the last enzyme required for ascorbate biosynthesis. Vitamin C, therefore, must be obtained from dietary sources and, because it cannot be stored in the body, it must be obtained regularly. Once used, ascorbic acid can be regenerated from its oxidized form in a reaction catalyzed by dehydroascorbate reductase (DHAR). To examine whether overexpression of DHAR in plants would increase the level of ascorbic acid through improved ascorbate recycling, a DHAR cDNA from wheat was isolated and expressed in tobacco and maize, where DHAR expression was increased up to 32- and 100-fold, respectively. The increase in DHAR expression increased foliar and kernel ascorbic acid levels 2- to 4-fold and significantly increased the ascorbate redox state in both tobacco and maize. In addition, the level of glutathione, the reductant used by DHAR, also increased, as did its redox state. These results demonstrate that the vitamin C content of plants can be elevated by increasing expression of the enzyme responsible for recycling ascorbate.

Amino Acid Sequence↗
PubMed1997

Local and Systemic Responses of Antioxidants to Tobacco Mosaic Virus Infection and to Salicylic Acid in Tobacco (Role in Systemic Acquired Resistance).

Changes in ascorbate and glutathione levels and in activities of ascorbate peroxidase, catalase, dehydroascorbate reductase (DHAR), glutathione reductase (GR), glutathione S-transferase (GST), and superoxide dismutase (SOD) were investigated in tobacco mosaic virus (TMV)-inoculated lower leaves and in non-inoculated upper leaves of Nicotiana tabacum L. cv Xanthi-nc. In separate experiments the effects of exogenous salicylic acid (SA) were also studied. Symptom appearance after TMV inoculation was preceded by a slight, transient decline of ascorbate peroxidase, GR, GST, and SOD activities in the inoculated lower leaves, but after the onset of necrosis these activities and the glutathione level substantially increased. Ascorbic acid level and DHAR activity declined and dehydroascorbate accumulated in the inoculated leaves. In upper leaves, the glutathione level and the activities of GR, GST, and SOD increased 10 to 14 d after TMV inoculation of the lower leaves, concomitantly with the development of systemic acquired resistance. From the six distinct SOD isoenzymes found in tobacco leaves, only the activities of Cu,Zn-SOD isoenzymes were affected by TMV. SA injection induced DHAR, GR, GST, and SOD activities. Catalase activities were not modified by TMV infection or SA treatment. It is supposed that stimulated antioxidative processes contribute to the suppression of necrotic symptom development in leaves with systemic acquired resistance.

Journal Article↗
PubMed2006

Cellular disulfide-reducing capacity: an integrated measure of cell redox capacity.

To assess the disulfide reduction capacity of intact cells, EA.hy926 endothelial cells were incubated with alpha-lipoic acid in the presence of 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB). Alpha-lipoic acid was reduced within cells to dihydrolipoic acid, which could be quantified upon efflux from the cells as reduction of DTNB. Uptake of both alpha-lipoic acid and alpha-lipoamide occurred at least in part via a medium chain fatty acid transporter, based on inhibition by octanoate. Alpha-lipoic acid was reduced within cells by pyridine nucleotide-disulfide oxidoreductases, since it is not reduced by GSH and since its reduction was inhibited by carmustine. Nonetheless, reduction was also dependent on the cellular redox environment, since it was inhibited by the redox cycling of menadione, by decreasing intracellular GSH, and by reduction of dehydroascorbate. Together, these results show that alpha-lipoic acid-dependent DTNB reduction provides a simple method to assess the disulfide-reducing capacity of intact cells, especially as determined by pyridine nucleotide-disulfide oxidoreductases.

Cell Line↗
PubMed2001

Antioxidants attenuate early up regulation of retinal vascular endothelial growth factor in streptozotocin-diabetic rats.

AIMS/HYPOTHESIS: A strong positive correlation has been found between lipid peroxidation product and vascular endothelial growth factor concentrations in the vitreous of patients with proliferative diabetic retinopathy. To establish a causal relation between diabetes-associated enhanced oxidative stress and vascular endothelial growth factor production, we evaluated two antioxidants, DL-alpha-lipoic acid and taurine, on retinal vascular endothelial growth factor protein and mRNA expression and on parameters of oxidative stress in streptozotocin-diabetic rats. METHODS: Our experiments were on control rats and streptozotocin-diabetic rats with a 6-week duration of diabetes, treated with or without DL-alpha-lipoic acid (100 mg x kg(-1) x d(-1), i.p.) or taurine (1% in the diet) starting from induction of diabetes. Vascular endothelial growth factor protein in retinal homogenates was assessed by sandwich ELISA with an affinity-purified polyclonal antibody and vascular endothelial growth factor mRNA by ribonuclease protection assay. Retinal lipid peroxidation products i.e. malondialdehyde plus 4-hydroxyalkenals were quantified with N-methyl-2-phenylindole. Retinal reduced and oxidized glutathione, ascorbate, dehydroascorbate, and sorbitol pathway intermediates were measured spectrofluorometrically, and taurine by reverse-phase HPLC. RESULTS: Vascular endothelial growth factor protein concentration (means +/- SD) was increased in diabetic rats compared with control rats (33+/-7 vs 19+/-5 pg/mg total protein, p < 0.01) This increase was attenuated by taurine (26+/-8, p < 0.05) and prevented by DL-alpha-lipoic acid (21+/-4, p < 0.01). Vascular endothelial growth factor mRNA abundance was reduced by 1.4-fold in diabetic rats compared with control rats and this decrease was attenuated but not completely prevented by both antioxidants. Malondialdehyde plus 4-hydroxyalkenal concentration was increased in diabetic rats compared with control rats, and both antioxidants arrested accumulation of lipid peroxidation products. Taurine, reduced glutathione, oxidized glutathione, ascorbate, dehydroascorbate and sorbitol pathway intermediate concentrations as well as oxidized glutathione/reduced glutathione and dehydroascorbate/ascorbate ratios were similar in control and diabetic rats treated with or without taurine. CONCLUSION/INTERPRETATION: Oxidative stress is directly involved in up regulation of vascular endothelial growth factor protein in the retina during early diabetes.

Alternative Splicing↗
PubMed1985

Reductive metabolism of ascorbic acid in the central nervous system.

Rat and feline brain and feline spinal cord were examined for the presence of semidehydroascorbate reductase (EC 1.6.5.4) and dehydroascorbate reductase (EC 1.8.5.1). Semidehydroascorbate reductase (SDAR), as monitored by both ascorbyl radical-dependent nicotinamide adenine dinucleotide (NADH) oxidase activity and NADH-dependent ascorbyl radical quenching, was present in all tissues studied. Rat cerebrum exhibited the highest levels and feline spinal cord the lowest. SDAR activity was about twice as high in feline cerebral cortex as in underlying white matter, and paralleled ascorbic acid levels. Subcellular fractionation of rat cerebrum localized most SDAR in a large granular fraction. In contrast, dehydroascorbate reductase was not detectable in any of the tissues examined. The results suggest that semidehydroascorbate reductase is the major enzyme catalyzing the regeneration of reduced ascorbic acid in the central nervous system.

Animals↗
PubMed1995

Intracellular ascorbic acid inhibits the Na(+)-Ca2+ exchanger in cultured rat astrocytes.

The effect of ascorbic acid on Ca2+ uptake in cultured rat astrocytes was examined in the presence of ouabain and monensin, which are considered to drive the Na(+)-Ca2+ exchanger in the reverse mode. Ascorbic acid at 0.1-1 mM inhibited Na(+)-dependent Ca2+ uptake significantly but not Na(+)-dependent glutamate uptake in the cells, although the inhibition required pretreatment for more than 30 min. The effect of ascorbic acid on the Ca2+ uptake was blocked by simultaneous addition of ascorbate oxidase (10 U/ml). Na(+)-dependent Ca2+ uptake was also inhibited by isoascorbate at 1 mM but not by ascorbate 2-sulfate, dehydroascorbate, and sulfhydryl-reducing reagents such as glutathione and 2-mercaptoethanol. The inhibitory effect of ascorbic acid was observed even in the presence of an inhibitor of lipid peroxidation, o-phenanthroline, or a radical scavenger, mannitol, and the degrading enzymes such as catalase and superoxide dismutase. On the other hand, the inhibitory effect was not observed under the Na(+)-free conditions that inhibited the uptake of ascorbic acid in astrocytes. When astrocytes were cultured for 2 weeks in a medium containing ascorbic acid, the content of ascorbic acid in the cells was increased and conversely Na(+)-dependent Ca2+ uptake was decreased. These results suggest that an increase in intracellular ascorbic acid results in a decrease of Na(+)-Ca2+ exchange activity in cultured astrocytes and the mechanism is not related to lipid peroxidation.

Animals↗
PubMed1995

Purification from placenta, amino acid sequence, structure comparisons and cDNA cloning of human glutaredoxin.

Glutaredoxin is generally a glutathione-dependent hydrogen donor for ribonucleotide reductase and also catalyses general glutathione (GSH)-disulfide-oxidoreduction reactions in the presence of NADPH and glutathione reductase. A Glutaredoxin from human placenta was purified to homogeneity, as judged by SDS/PAGE and IEF (12 kDa). Purification was monitored by the activity with hydroxyethyl disulfide as a substrate. Values of pI for glutaredoxin were obtained by IEF; the pI of the protein shifted from 7.3 in its fully reduced state to 9.0 in the oxidized state after treatment with excess hydroxyethyl disulfide. The glutaredoxin preparation showed GSH-dependent hydrogen-donor activity with recombinant mouse ribonucleotide reductase, it exhibited dehydroascorbate reductase activity as well as hydroxyethyl-disulfide-reducing activity. The amino acid sequence (residues 3-104) of glutaredoxin was determined by peptide sequencing and residues 1, 2 and 105 by cDNA sequence analysis. The glutaredoxin sequence comprised the classical active site for glutaredoxins -Cys22-Pro-Tyr-Cys25- and three additional half-cystine residues; two of these in positions 78 and 82. The sequence was similar to other known mammalian glutaredoxins (about 80% identities), with important differences such as one additional Cys residue (Cys7) and no Met residue. The sequence of human glutaredoxin was compared to that of Escherichia coli glutaredoxin with known three-dimensional structure in solution to identify conserved residues and predict a structure from alignment. In particular the GSH-binding site of glutaredoxin was conserved between all molecules. A cDNA that encodes the entire glutaredoxin gene (grx) and flanking sequences was isolated from a human spleen cDNA library. The nucleotide sequence of this cDNA (0.8 kb) was determined, including the complete grx gene.

Amino Acid Sequence↗