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PubMed1986

Distribution of ascorbate in the retina, subretinal fluid and pigment epithelium.

The posterior segment of the eye was divided into four compartments: retinal cytosol (R), subretinal fluid on the retinal surface (S/R), retinal pigment epithelial (RPE) cytosol, and subretinal fluid on the RPE surface (S/RPE). The volume of each compartment was estimated from the dilution of creatinine (in the extraction buffer) by the endogenous tissue fluid. The ascorbate concentrations in R, S/R, S/RPE, and RPE were 20.6, 12.3, 3.7, and 5.8 mg/dl respectively. Dehydroascorbate was observed only in the RPE and S/RPE. The decreasing ascorbate concentration from the retina to RPE, and the distribution of dehydroascorbate suggest a movement of ascorbate from the vitreous cavity into the subretinal space. The permeability of retinal cell layers to ascorbate was confirmed by the high radioactivity observed in the subretinal space after an intravitreal injection of C14-ascorbate. The occurrence of dehydroascorbate in the RPE and the S/RPE indicates the presence of oxidative reaction of ascorbate in these compartments, where light induced free radicals are located.

Animals↗
PubMed1980

Inhibition of protein and DNA synthesis in tissue culture cells by a derivative of methyl glyoxal and ascorbate.

The inhibitory effect of a methyl glyoxal-ascorbate (MGA) adduct (NFCR 278021) on protein and DNA synthesis in monolayer cultures of GPK epithelial cells has been compared with the inhibitory action of methyl glyoxal (MG). GPK cells exhibited an ID50 of 0.98 microM MG for both protein and DNA synthesis compared with an ID50 of 0.92 mM for the adduct. Hill plots demonstrate that the characteristics of the receptor saturation are the same for MG and MGA, suggesting that the action of the two agents is mediated through the MG moiety which is modified by the presence of the ascorbate portion of the molecule in MGA. It is shown that MGA undergoes spontaneous oxidation in solution and is a substrate for ascorbate oxidase, but that no additional MG activity is released by total enzymic oxidation of MGA, and oxidised MGA possesses the same inhibitory characteristics as MGA. Inhibition of protein synthesis by ascorbate or dehydroascorbate were not demonstrated in the dose range employed for MGA. The inhibitory effect of the adduct on protein synthesis was found to be diminished in the presence of glutathione and glyoxalase I (Glo I) and II( Glo II).

Aldehydes↗
PubMed1992

Selective inactivation of the hydroxylase activity of bifunctional rat peptidylglycine alpha-amidating enzyme.

Conversion of dansyl-Tyr-Val-Gly to dansyl-Tyr-Val-NH2 by recombinant type A rat 75-kDa peptidylglycine alpha-amidating enzyme (alpha-AE) is inactivated by ascorbate, dehydroascorbate, and hydrogen peroxide in a time- and concentration-dependent manner. Both ascorbate- and dehydroascorbate-mediated inactivation are saturable with apparent kinact/Kinact values of 1.7 and 0.23 s-1 M-1, respectively. Hydrogen peroxide-mediated inactivation is not saturable with a second-order rate constant of 50 s-1 M-1. Peptidyl-Gly substrates, EDTA, and H2O2 scavengers protect against ascorbate-mediated inactivation while EDTA and semidehydroascorbate scavengers protect against dehydroascorbate-mediated inactivation. Under similar conditions, ascorbate, dehydroascorbate, and H2O2 have no effect on the alpha-AE-catalyzed conversion of dansyl-Tyr-Val-alpha-hydroxyglycine to dansyl-Tyr-Val-NH2 which is consistent with the hypothesis that the 75-kDa enzyme consists of distinct peptidyl-Gly hydroxylase and peptidyl-alpha-hydroxyglycine lyase active sites.

Amino Acid Sequence↗
PubMed1985

Depletion of extracellular cysteine with hydroxocobalamin and ascorbate in experimental murine cancer chemotherapy.

Treatment of mice bearing P388 lymphocytic leukemia with combined hydroxocobalamin (0.1 mmol/kg) and sodium ascorbate (1.0 mmol/kg) for 10 consecutive days resulted in 70% increase in survival. Treatment with dehydroascorbate (1.0 mmol/kg) similarly increased survival. The extended treatment for 15 days posttumor inoculation failed to improve survival as did lowering the tumor inocula from 10(6) to 10(4) cells in similarly treated mice. P388 cells in primary culture with cystine as a source of cysteine require exogenous thiols or disulfides for growth and also grow with cysteine but only when trace copper is sequestered with bathocuproine sulfonate. This is due to the copper catalyzed oxidation of cysteine to cystine, which these cells cannot utilize. Cells grew in medium preincubated for 8 h with cysteine and bathocuproine sulfonate, but lysed when the preincubated medium included hydroxocobalamin (10 microM) and sodium ascorbate (100 microM). This defect was corrected by resupplementation with cysteine or cystine plus bishydroxyethyl disulfide after addition of the cells. Further support for cysteine depletion as a factor in therapy is obtained by therapeutic interference with 4-thiamethionine (25 mumol/kg). This mixed disulfide of cysteine and methyl mercaptan enters cells as a methionine analogue, is reduced to supply cysteine, and thus withstands the oxidative environment that limits the utilization of cysteine as a nutrient. These results suggest that dehydroascorbate is generated from combined hydroxocobalamin and ascorbate and accounts for the thiol-prive activity of combined hydroxocobalamin and ascorbate therapy. The ultimate failure of therapy by thiol oxidation may be due to the increased availability of cysteine produced by host tissues, possibly infiltrating macrophages, that occurs independently of extended treatment and the size of the tumor inoculum.

Animals↗
PubMed1991

Effects of smoking and vitamin E on blood antioxidant status.

Despite similar vitamin E contents, erythrocytes of smokers have an increased tendency (P less than 0.01) to peroxidize in vitro compared with those of nonsmokers. This difference is abolished by vitamin E supplementation (1000 mg alpha-tocopherol acetate/d for 14 d). The increased susceptibility to erythrocyte peroxidation in the smokers may reflect lower glucose-6-phosphate dehydrogenase (P less than 0.02) and glutathione peroxidase (P less than 0.05) activities. Smokers seem to be under a sustained oxidant stress with increased plasma-conjugated dienes (P less than 0.01) and dehydroascorbate (P less than 0.05) and decreased ascorbate (P less than 0.06) concentrations. Additionally, plasma ceruloplasmin in smokers is elevated (P less than 0.01), consistent with an acute-stress response. Plasma total cholesterol is similar in smokers and nonsmokers and is unaffected by vitamin E supplementation. Indices of sustained oxidant stress in smokers are partially ameliorated by vitamin E supplementation.

Adult↗
PubMed2003

Role of ascorbate in oxidative protein folding.

Both in prokaryotic and eukaryotic cells, disulfide bond formation (oxidation and isomerization steps) are catalyzed exclusively in extracytoplasmic compartments. In eukaryotes, protein folding and disulfide bond formation are coupled processes that occur both co- and posttranslationally in the endoplasmic reticulum (ER), which is the main site of the synthesis and posttranslational modification of secretory and membrane proteins. The formation of a disulfide bond from the thiol groups of two cysteine residues requires the removal of two electrons, consequently, these bonds cannot form spontaneously; an oxidant is needed to accept the electrons. In aerobic conditions the ultimate electron acceptor is usually oxygen; however, oxygen itself is not effective in protein thiol oxidation. Therefore, a small molecular weight membrane permeable compound should be supposed for the transfer of electrons from the ER lumen. The aim of the present study was the investigation of the role of ascorbate/dehydroascorbate redox couple in oxidative folding of proteins. We demonstrated that ascorbate addition or its in situ synthesis from gulonolactone results in protein thiol (and/or glutathione; GSH) oxidation in rat liver microsomes. Since microsomal membrane is hardly permeable to ascorbate, the existence of a transport metabolon was hypothesized. Three components of the system have been described and partially characterized: (i) A microsomal metalloenzyme is responsible for ascorbate oxidation on the outer surface of the ER. Ascorbate oxidation results in ascorbate free radical and dehydroascorbate production. (ii) Facilitated diffusion of dehydroascorbate is present in microsomal vesicles. The transport is presumably mediated by a GLUT-type transporter. On the contrary, the previously hypothesized glutathione disulfide (GSSG) transport is practically absent, while GSH is transported with a moderate velocity. (iii) Protein disulfide isomerase catalyzes the reduction of dehydroascorbate in the ER lumen. Both GSH and protein thiols can be electron donors in the process. Intraluminal dehydroascorbate reduction and the consequent ascorbate accumulation strictly correlate with protein disulfide isomerase activity and protein thiol concentration. The concerted action of the three components of the system results in the intraluminal accumulation of ascorbate, protein disulfide and GSSG. In fact, intraluminal ascorbate and GSSG accumulation could be observed upon dehydroascorbate and GSH uptake. In conclusion, ascorbate is able to promote protein disulfide formation in an in vitro system. Further work is needed to justify its role in intact cellular and in vivo systems, as well as to explore the participation of other antioxidants (e.g. tocopherol, ubiquinone, and vitamin K) in the electron transfer chain responsible for oxidative protein folding in the ER.

Animals↗
PubMed2004

The ascorbic acid redox state controls guard cell signaling and stomatal movement.

H(2)O(2) serves an important stress signaling function and promotes stomatal closure, whereas ascorbic acid (Asc) is the major antioxidant that scavenges H(2)O(2). Dehydroascorbate reductase (DHAR) catalyzes the reduction of dehydroascorbate (oxidized ascorbate) to Asc and thus contributes to the regulation of the Asc redox state. In this study, we observed that the level of H(2)O(2) and the Asc redox state in guard cells and whole leaves are diurnally regulated such that the former increases during the afternoon, whereas the latter decreases. Plants with an increased guard cell Asc redox state were generated by increasing DHAR expression, and these exhibited a reduction in the level of guard cell H(2)O(2). In addition, a higher percentage of open stomata, an increase in total open stomatal area, increased stomatal conductance, and increased transpiration were observed. Guard cells with an increase in Asc redox state were less responsive to H(2)O(2) or abscisic acid signaling, and the plants exhibited greater water loss under drought conditions, whereas suppressing DHAR expression conferred increased drought tolerance. Our analyses suggest that DHAR serves to maintain a basal level of Asc recycling in guard cells that is insufficient to scavenge the high rate of H(2)O(2) produced in the afternoon, thus resulting in stomatal closure.

Ascorbic Acid↗
PubMed1994

A physiological level of ascorbate inhibits galactose cataract in guinea pigs by decreasing polyol accumulation in the lens epithelium: a dehydroascorbate-linked mechanism.

It was reported previously that dietary ascorbate (ASC) delays the development of galactose-induced cataract in guinea pigs compared to the rate which is observed in ASC-deficient animals. Experiments were conducted to explore the possible mechanism of this phenomenon. Guinea pigs were fed for a period of up to 4 weeks either a normal diet (1 g ASC/kg diet) or a scorbutic diet (< 0.04 g ASC/kg diet) combined with 10% galactose in the drinking water. After 2 weeks, levels of ASC in animals on the scorbutic diet decreased by 95% in the aqueous humor and by 78% in the lens. Slit lamp examination showed that galactose-induced vacuoles in the lens equator formed at a significantly faster rate in the scorbutic animals. However, examination of biochemical parameters in whole lenses of the two groups of animals after 2 weeks showed no significant differences with regard to accumulation of galactose and galactitol, decreases in the levels of myoinositol, taurine and GSH or changes in cation concentrations. In order to examine possible regional changes in the lenses, various parameters were studied in the lens capsule-epithelium. On day 4, the capsule epithelia of scorbutic animals on a galactose diet had a content of galactitol two-and-a-half times higher than that of normal galactose-fed animals. Scorbutic conditions also intensified the loss of Na(+)-K+ ATPase activity in the lens capsule-epithelium caused by galactose feeding. Oxidized glutathione was not detectable in the lens capsule epithelia of any of the animals studied. Hexose monophosphate shunt activity was elevated in lenses of normal galactose-fed animals during the first hour of culture after death whereas lenses of scorbutic galactose-fed animals were not. Consistent with the in vivo findings, galactitol accumulation in dog lens epithelial cells exposed to 30 mM galactose was significantly inhibited by the presence of either ASC or dehydroascorbate (DHA) in the medium. Hexose monophosphate shunt activity in the cells was stimulated to two-and-a-half times its initial level by either 1 mM DHA or 30 mM galactose and slightly more than three-fold by a combination of the two challenges. The results suggest that decreased polyol accumulation in the lens epithelium of the normal galactose-fed guinea pig, which has a high level of ASC in the aqueous humor, accounts for the delay in onset of cataract compared to that for the ASC-deficient animal.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed2004

Ascorbate modulation of H(2)O(2) and camptothecin-induced cell death in Jurkat cells.

The effect of ascorbate on cell death was examined in Jurkat cells (human T-cell leukemia) by incubation with dehydroascorbate (DHA), which is rapidly taken up by cells and efficiently reduced to ascorbate. Apoptosis was evaluated by caspase-3 activity in cell extracts and flow cytometry of annexin V-labeled cells. In parallel, necrosis was estimated by the release of lactate dehydrogenase. Minor effects on cell death were observed when Jurkat cells were incubated with either DHA alone (100-1,000 microM) or a single dose of 10 microM H(2)O(2). However, pre-incubation with DHA followed by exposure to H(2)O(2) clearly stimulated both apoptosis and necrosis. In complete contrast, pre-incubation of cells with DHA significantly inhibited apoptosis, but did not affect necrosis, induced by the topoisomerase I inhibitor camptothecin. Our results indicate that intracellular ascorbate can modulate cell death in a manner which depends upon the nature of the apoptotic stimulus, which in turn has critical implications regarding the mechanism and potential application of ascorbate in cancer therapy.

Ascorbic Acid↗
PubMed1992

Dihydrolipoic acid--a universal antioxidant both in the membrane and in the aqueous phase. Reduction of peroxyl, ascorbyl and chromanoxyl radicals.

Thioctic (lipoic) acid is used as a therapeutic agent in a variety of diseases in which enhanced free radical peroxidation of membrane phospholipids has been shown to be a characteristic feature. It was suggested that the antioxidant properties of thioctic acid and its reduced form, dihydrolipoic acid, are at least in part responsible for the therapeutic potential. The reported results on the antioxidant efficiency of thioctic and dihydrolipoic acids obtained in oxidation models with complex multicomponent initiation systems are controversial. In the present work we used relatively simple oxidation systems to study the antioxidant effects of dihydrolipoic and thioctic acids based on their interactions with: (1) peroxyl radicals which are essential for the initiation of lipid peroxidation, (2) chromanoxyl radicals of vitamin E, and (3) ascorbyl radicals of vitamin C, the two major lipid- and water-soluble antioxidants, respectively. We demonstrated that: (1) dihydrolipoic acid (but not thioctic acid) was an efficient direct scavenger of peroxyl radicals generated in the aqueous phase by the water-soluble azoinitiator 2,2'-azobis(2-amidinopropane)-dihydrochloride, and in liposomes or in microsomal membranes by the lipid-soluble azoinitiator 2,2'-azobis(2,4-dimethylvaleronitrile); (2) both dihydrolipoic acid and thioctic acid did not interact directly with chromanoxyl radicals of vitamin E (or its synthetic homologues) generated in liposomes or in the membranes by three different ways: UV-irradiation, peroxyl radicals of 2,2'-azobis(2,4-dimethylvaleronitrile), or peroxyl radicals of linolenic acid formed by the lipoxygenase-catalyzed oxidation; and (3) dihydrolipoic acid (but not thioctic acid) reduced ascorbyl radicals (and dehydroascorbate) generated in the course of ascorbate oxidation by chromanoxyl radicals. This interaction resulted in ascorbate-mediated dihydrolipoic acid-dependent reduction of the vitamin E chromanoxyl radicals, i.e. vitamin E recycling. We conclude that dihydrolipoic acid may act as a strong direct chain-breaking antioxidant and may enhance the antioxidant potency of other antioxidants (ascorbate and vitamin E) in both the aqueous and the hydrophobic membraneous phases.

Animals↗
PubMed2001

Effect of peroxynitrite on glutaredoxin.

Glutaredoxin is an important enzyme in thiol homeostasis. As a thioltransferase, it reduces oxidized thiols. It also has dehydroascorbate reductase (DHAR) activity to reduce dehydroascorbate (DHA) to ascorbic acid. Peroxynitrite (ONOO-) is one of the most active elements of oxidative stress that can be formed wherever nitric oxide and superoxide are produced simultaneously. ONOO- is known to react with free thiols easily. To observe the effect of ONOO on glutaredoxin, rat liver cytosolic fractions were incubated with 0-250 microM ONOO-. Thioltransferase activity was found to be decreased as ONOO concentration increased. The inhibition was not reversible with dithiothreitol (DTT). In cytosol besides glutaredoxin, another enzyme with DHAR activity is also present. In our study, the cytosolic DHAR activity which consisted both enzymes, was also inhibited by ONOO-, but DTT was able to return the activity almost completely.

Animals↗
PubMed1997

Measurement of ascorbate and dehydroascorbate contents in biological fluids.

Instabilities of ascorbate and dehydroascorbate throughout sample processing are clearly a significant aspect of quantifying of them. Contents of ascorbate in biological fluids decrease with measurable oxidation occurring within minutes to hours. Similarly, dehydroascorbate disappears with chemical or enzymatic degradation within minutes. The half-life of dehydroascorbate in human heparinized plasma was approximately 2 min. These results indicated that the amount of dehydroascorbate present in sample solutions is a function of both the oxidation of ascorbate and the degradation of dehydroascorbate during the processing of biological fluids. To quantify ascorbate and dehydroascorbate concentrations in biological fluids including circulating blood plasma and urine, we established a high-performance liquid chromatographic method, which requires no pretreatment of sample solutions.

Ascorbic Acid↗
PubMed1980

Studies on the lysyl hydroxylase reaction. II. Inhibition kinetics and the reaction mechanism.

Product inhibition of lysyl hydroxylase (peptidyllysine, 2-oxoglutarate:oxygen 5-oxidoreductase, EC 1.14.11.4) was studied with succinate, CO2, dehydroascorbate and hydroxylysine-rich polypeptide chains. The product inhibition patterns and addition data are consistent with a reaction mechanism involving an ordered binding of Fe2+, alpha-ketoglutarate, O2 and the peptide substrate to the enzyme in this order, and an ordered release of the hydroxylated peptide, CO2, succinate and Fe2+, in which Fe2+ need not leave the enzyme during each catalytic cycle and in which the order of release of the hydroxylated peptide and CO2 is uncertain. Ascorbate probably reacts by a substitution mechanism, either after the release of the hydroxylated peptide, CO2 and succinate or after the release of all products, including Fe2+, and dehydroascorbate is released before the binding of Fe2+. It is suggested that the ascorbate reaction is required to reduce either the enzyme-iron complex or the free enzyme, which may be oxidized by a side-reaction during some catalytic cycles, but not the majority. The mechanisms of the prolyl 4-hydroxylase and lysyl hydroxylase reactions are suggested to be identical. Zn2+, several citric acid cycle intermediates, nitroblue tetrazolium and homogentisic acid inhibited lysyl hydroxylase competitively with regard to Fe2+, alpha-ketoglutarate, O2 and ascorbate respectively, and epinephrine non-competitively with regard to all cosubstrates. Apparent Ki values are given for the product and other inhibitors.

Animals↗
PubMed1993

Cigarette smoke exposure of school children: effect of passive smoking and vitamin E supplementation on blood antioxidant status.

Despite similar vitamin E concentrations, erythrocytes of 25 children of smoking parents had an increased tendency (p < 0.01) to peroxidize in vitro as compared with those of 28 children of nonsmoking parents. This difference was abolished by vitamin E supplementation (100 mg alpha-tocopherol acetate/day for 14 days). The increased susceptibility to erythrocyte peroxidation in the smokers may reflect lower erythrocyte glucose-6-phosphate dehydrogenase, glutathione peroxidase and superoxide dismutase activities (p < 0.001, p < 0.005, and p < 0.02, respectively) in children of smoking parents. Children of smoking parents seem to be under sustained oxidant stress with increased plasma-conjugated dienes (p < 0.01) and dehydroascorbate (p < 0.002) concentrations. Total plasma cholesterol was similar in children of smoking and nonsmoking parents, and was unaffected by vitamin E supplementation. Indices of sustained oxidant stress in children of smoking parents were partially ameliorated by vitamin E supplementation.

Antioxidants↗
PubMed1975

Ascorbate and phagocyte function.

Scorbutic guinea pig neutrophils (PMN) were found to produce H2O2 and kill Staphylococcus aureus as well as control PMN, suggesting that ascorbate does not contribute significantly to phagocyte H2O2 production or bacterial killing. Total and reduced ascorbate contents of human PMN was observed to fall upon phagocytosis, whereas dehydroascorbate increased to a lesser extent. These observations are consistent with the view that ascorbate constitutes a functional part of the PMN's redox-active components and may thus function to protect cell constituents from denaturation by the oxidants produced during phagocytosis.

2,3-Diketogulonic Acid↗
PubMed1983

Interaction between glucose and dehydroascorbate transport in human neutrophils and fibroblasts.

Dehydroascorbate (DHA) competitively inhibits the uptake of 2-deoxy-D-glucose and of 3-O-methyl-D-glucose by human blood neutrophils; 2-deoxyglucose and 3-O-methylglucose competitively inhibit dehydroascorbate uptake. Ratios of Km to Kl are near unity for each of these ligands. Exposure of cultured human fibroblasts to insulin enhances cytochalasin B-inhibitable uptake of DHA and of 2-deoxyglucose, both to the same degree. These observations suggest that DHA transport is mediated by glucose transport systems in human neutrophils and fibroblasts.

3-O-Methylglucose↗