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1. Ascorbic acid, diamide and N-ethylmaleimide inhibit Na+ + K+-ATPase activity in toad corneal epithelium. 2. Ascorbic acid, diamide and N-ethylmaleimide increase alpha-aminoisobutyric acid accumulation in this tissue. 3. The effects of these compounds on corneal amino acid and ion transport are not mediated through alterations in Na+ + K+-ATPase activity.
Coated vesicles were isolated from rat liver in about 80% fraction purity as determined from electron microscopy and analyses of marker enzymes and compared with Golgi apparatus and other membrane fractions isolated in parallel. The fractions were enriched in NADH-monodehydroascorbate reductase, ascorbate oxidase and ascorbic acid. The NADH-monodehydroascorbate reductase and ascorbate oxidase of the Golgi apparatus and coated vesicles differed from that of the endoplasmic reticulum in being inhibited by the sodium selective ionophore, monensin, at physiological concentrations while these activities were stimulated by ethylenediaminetetraacetic acid in coated vesicles but not in Golgi apparatus. Activities of both coated vesicles and Golgi apparatus fractions depleted in the coat protein, clathrin, were activated by the addition of clathrin-rich supernatant fractions. The results are discussed in the context of monodehydroascorbate as an acceptor for electron transport-mediated transfer of electrons from NADH by coated vesicles as part of a possible mechanism to drive membrane translocations or to acidify the interiors of vesicles.
Since activated macrophages generate potentially deleterious reactive oxygen species, we studied whether ascorbic acid might function as an antioxidant in these cells. Thioglycollate-elicited murine peritoneal macrophages contained about 3 mM ascorbate that was halved by culture in ascorbate-free medium. However, the cells took up added ascorbate to concentrations of 6-8 mM by a high-affinity sodium-dependent transport mechanism. This likely reflected the activity of the SVCT2 ascorbate transporter, since its message and protein were present in the cells. Activation of the cells by phagocytosis of latex particles depleted intracellular ascorbate, although not below the basal levels present in the cells in culture. Glutathione (GSH) was unaffected by phagocytosis, suggesting that ascorbate was more sensitive to the oxidant stress of phagocytosis than GSH. Phagocytosis induced a modest increase in reactive oxygen species as well as a progressive loss of alpha-tocopherol, both of which were prevented in cells loaded with ascorbate. These results suggest that activated macrophages can use ascorbate to lessen self-generated oxidant stress and spare alpha-tocopherol, which may protect these long-lived cells from necrosis or apoptosis.
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Combined radiotherapy and chemotherapy have represented a major advance in the therapeutic management of cancer therapy. However, the combination of doxorubicin (DXR) and cardiac irradiation (IRR) could precipitate the unexpected expression of congestive heart failure. Oxidative lesions induced by IRR and DXR could represent one of the pathogenic factors of myocardial dysfunction. Our investigations were performed to evaluate in the rat: 1) cardiac functional changes, 2) cardiac and plasma peroxidative damage and antioxidant defenses variations, that occur 24 h (acute effects) and 30 d (middle term effects) following DXR treatment 1 mg/kg(-1)/day(-1) IP for 10 d and a 1 x 20 Gy cardiac gamma-irradiation. Our results showed that DXR affected heart reactivity as early as the end of its administration, although irradiation exerted no detectable effect. Antioxidant defenses disturbances in hearts of DXR treated rats were characterized by vitamins C and E decreases, catalase activity induction and an increase in lipid peroxidation. Moreover, plasma vitamin C consumption and the lower level of plasma lipid peroxidation attested to the efficient solicitation of antioxidant defenses that probably contributed to the preservation of cardiac function at 24 h. After 30 d, cardiac dysfunction became symptomatic at rest, resulting from DXR cardiac toxicity. In spite of the persistent activation of cardiac catalase activity, antioxidant deficiency and increased plasma and cardiac lipid peroxidation highlighted defenses overtaken. Thus, different physiopathological mechanisms are involved in heart disturbance at acute and middle terms, IRR and DXR acting on distinct targets without disclosing synergistic effects. After 30 d, cardiac and plasma biochemical abnormalities were emphasized by the combined DXR+IRR therapy, pointing out the severity of the damage. Oxidative damage to the heart induced both by irradiation and DXR, may be one of the pathogenic factors of myocardial dysfunction. There is the possibility that the deleterious effects might be limited by the use of pharmacologic antioxidant agents.
Ascorbic acid is an essential reductant in biology but its reducing power is paradoxical. At physiological pH the predominant form of ascorbate (the monoanion) is a poor electron donor because it oxidizes to the energetically unfavorable neutral free radical. The ascorbate dianion forms the relatively stable semidehydroascorbate radical anion and is a powerful electron donor but its concentration at neutral pH is insufficient to produce the reaction rates observed. For example, ascorbate rapidly reduces cytochrome b561 from adrenal medullary chromaffin vesicles. This fast reaction rate may be rationalized by a mechanism involving concerted proton-electron transfer rather than electron transfer alone. This would permit reduction of the cytochrome by the abundant ascorbate monoanion but would circumvent formation of unfavorable intermediates. This may be a general mechanism of biological ascorbic acid utilization: enzymes using ascorbic acid may react with the ascorbate monoanion via concerted proton-electron transfer.
Ascorbic acid is known to circulate free in the plasma of several species and is therefore filtered in the kidney; reabsorption subsequently takes place and prevents urinary loss. However, no specific mechanism of renal ascorbic acid transport has previously been presented. In the present study, rat and guinea pig kidney were incubated as slices or as isolated tubules in vitro in the presence of low concentrations of [14C]ascorbic acid. The kidneys of both species handle ascorbic acid similarly. Ascorbic acid accumulates in the renal tissue to a concentration three to four times that present in the bathing media. Recently absorbed ascorbic acid diffuses freely from the kidney and is predominantly nonmetabolized during absorption. Uptake is reduced following replacement of bathing solution sodium by lithium or cesium, or when incubation is performed in the presence of metabolic inhibitors or at low temperatures. The results indicate that ascorbic acid is reabsorbed in the kidney by a sodium-dependent active transport mechanism that operates by concentrating ascorbic acid in the cellular fluid. Renal slices and tubules both appear to transport ascorbic acid and galactose across the brush-border membrane; this indicates that the tubular lumens in these preparations are not collapsed or sealed off.
The tissue distribution of L-ascorbic acid (AsA) and dehydro-L-ascorbic acid (DAsA) in guinea pigs injected with DAsA intravenously was examined using high-performance liquid chromatography. DAsA injected into guinea pigs fed normal diets containing AsA (control group) was readily taken into erythrocytes, and AsA contents of plasma and other tissues rapidly increased after DAsA injection. In animals fed vitamin C-deficient diets, DAsA was also detected in erythrocytes; however, the increase of AsA in their tissues was considerably less than that of control group. From these results, it was suggested that utilization of DAsA as AsA in vitamin C-deficient guinea pigs was less than that of control animals, and the reduction mechanism of DAsA to AsA in vitamin C-deficient guinea pigs may have differed from that of control groups.
We describe two independent HPLC procedures for the rapid, accurate analysis for ascorbic acid in human plasma. No sample extraction or phase separation is required. We also describe a procedure for preparing a human plasma reference material for use in clinical laboratory analysis for ascorbic acid. The ascorbic acid in plasma can be determined in 15 min, with as little as 50 microL of sample. Analytical recoveries are near 100% with direct injection of deproteinized plasma. Extensive stability data under several conditions, with dithiothreitol as a preservative (antioxidant), indicate that ascorbic acid remains stable in stored plasma for as long as 57 weeks. CVs for round-robin analysis of 11 normal human blood samples by two independent methods were between 0.1% and 5.3%. These clinical samples appear to be stable for at least 50 days under the described conditions of stabilization and sample treatment. Finally, because ascorbic acid prepared by the described procedures is stable at room temperature for at least 18 h, these methods can be readily adapted to clinical laboratory automation at room temperature.
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Both reduced and oxidized ascorbates were measured in aqueous, neural retina, and pigment epithelium-choroid complex (PE-C) of pigmented guinea pigs. Normal values for total ascorbate of 16 mg/dl in aqueous, 22 mg/dl in neural retina, and 7 mg/dl in PE-C were found. After mild photic damage caused by varying lengths of exposure of 10,000 to 20,000 lux of fluorescent lighting, reduced ascorbate concentrations generally decreased in the neural retina, while oxidized ascorbate generally increased in PE-C. In both normal and light-exposed retinas, reduced ascorbate was predominant in the neural retina, and oxidized ascorbate was predominant in the PE-C. Histochemical localization of reduced ascorbate occurred in the Müller cell fibers and at the apices of the retinal pigment epithelium.
Purified human serum albumin and immunoglobulin G (IgG) were investigated for their metal-chelating activity, using ESR spectroscopy. Both copper (Cu2+) and iron ions (Fe3+) enhanced the radical intensity of both sodium ascorbate and sodium 5,6-benzylidene-L-ascorbate (SBA). Albumin significantly reduced the stimulation effect of copper, but not that of iron. On the other hand, IgG effectively reduced the radical intensity of iron, without affecting that of copper. The present study demonstrates the specific chelating action of these serum proteins, suggesting their possible preventive effects on metal-catalyzed pathogenic diseases.
Ascorbate peroxidase (APX) is a hydrogen peroxide-scavenging peroxidase which uses ascorbate (AsA) as the specific electron donor. APX has not been isolated in mammals. Ocular tissue contains AsA at high concentrations, and we detected APX activity in bovine retinal pigment epithelium (RPE) and choroid. We purified APX from bovine RPE and choroid by four chromatographic steps. The purified APX was a monomeric hemoprotein with a molecular mass of 43 kDa. The amino acid sequence of the amino-terminal region of the purified APX showed a high degree of homology to that of plants. The primary product of the APX reaction was identified as the monodehydroascorbate radical. The APX showed high specificity for AsA as an electron donor. This is the first isolation and characterization of APX from mammals, and its role in the protection against active species of oxygen in ocular tissue is discussed.
Although smooth muscle and endothelial cells in pig coronary artery are morphologically and functionally distinct, ascorbate uptake has been characterized only in smooth muscle cells. Ascorbate transporters in kidney and intestinal epithelial cells differ from those in smooth muscle. We examined ascorbate transport and mRNA expression of sodium-dependent vitamin C transporters (SVCT) by RT-PCR in the pig coronary artery endothelial cell cultures. When 14C-ascorbate uptake in endothelial cells was examined as 14C or by HPLC, the two values did not differ from each other. 14C-ascorbate uptake was Na(+)-dependent, stereoselective for L-ascorbate and inhibited by sulfinpyrazone. The kinetic characteristics of the uptake were: Km = 27 +/- 3 microM (Hill coefficient = 1) for ascorbate and Km = 73 +/- 14 mM (Hill coefficient = 2) for Na+. Surprisingly, endothelial cells had similar kinetic parameters as smooth muscle cells, except for a slightly lower uptake velocity in endothelial cells. Comparison with the smooth muscle showed that both tissue types expressed mRNA for SVCT2. Endothelial cells differ from epithelial cells which express mainly SVCT1 but resemble smooth muscle cells in this respect.
A membrane preparation enriched in the brush-border component of the plasma membrane was isolated from rat renal superficial cortex by a divalent cation precipitation procedure. Uptake of dehydro-L-ascorbic acid, the oxidized form of L-ascorbic acid, by the brush-border membrane vesicles was studied. The uptake mechanism was found to be sodium-independent and insensitive to the trans-membrane electrical potential difference. Uptake was saturable and subject to cis-inhibition. Concentrative uptake was demonstrated only under conditions of trans-stimulation by structural analogs. The results suggest a mechanism of facilitated diffusion for the uptake of dehydro-L-ascorbic acid in renal brush-border membranes.
This study investigated whether the age-related decline in hepatic ascorbic acid (AA) levels in rats was due to altered AA uptake. AA concentrations were 68% lower in freshly isolated hepatocytes from old (24-26 months) versus young (3-5 months; p<0.0005) Fischer 344 rats. When incubated with 100 microM AA, cells from old as compared to young rats showed a 66% decline in both the rate of AA transport and the steady state intracellular levels. Sodium-free media significantly reduced AA uptake, suggesting that the sodium-dependent vitamin C transporter (SVCT) was largely responsible for declines in AA transport. Analysis of SVCT messenger RNA (mRNA) levels shows that one isoform of this transport protein, SVCT1, declines 45% with age, with no significant changes in SVCT2 mRNA levels. These results show for the first time that sodium-dependent AA transport declines during the aging process, which may account for much of the loss in tissue AA content.
Endothelial cells are exposed to potentially damaging reactive oxygen species generated both within the cells and in the bloodstream and underlying vessel wall. In this work, we studied the ability of ascorbic acid to protect cultured human-derived endothelial cells (EA.hy926) from oxidant stress generated by the redox cycling agent menadione. Menadione caused intracellular oxidation of dihydrofluorescein, which required the presence of D-glucose in the incubation medium, and was inhibited by intracellular ascorbate and desferrioxamine. At concentrations of 100 microM and higher, menadione depleted the cells of both GSH and ascorbate, and ascorbate loading partially prevented the decrease in GSH due to menadione. Menadione increased L-arginine uptake by the cells, but inhibited endothelial nitric oxide synthase, an effect that was prevented by acute loading with ascorbate. Ascorbate blunts menadione-induced oxidant stress in EA.hy926 cells, which may help to preserve nitric oxide synthase activity under conditions of excessive oxidant stress.
Ascorbyl free radical (AFR), can be considered as an atoxic and endogenous indicator of oxidative stress. The purpose of our experiments was to investigate the influence of the severity and length of ischemia on the extent of AFR release during myocardial ischemia and reperfusion. For that purpose, isolated perfused rat hearts were submitted to a global ischemia, either total (residual flow 0%) or low flow (residual flow 5%), of 20 or 60 min length. Coronary effluents were collected at different times of experimentation and analyzed with Electron Spin Resonance (ESR) spectroscopy. AFR ESR doublet (g = 2.0054, aH = 0.188 MT) was not detected in coronary effluents collected during control perfusion periods. Nevertheless, during low-flow ischemia, a weak AFR release was noted. Moreover, a sudden and massive AFR liberation was observed at the time of reperfusion: this AFR release was weaker after low-flow ischemia than after total ischemia and was enhanced when the duration of ischemia increased from 20 min to 60 min. The large liberation of AFR noticed during global total ischemia was associated with a greater depression in myocardial contractile function and a lower recovery in coronary flow. In conclusion, our study demonstrates that AFR production at the time of reperfusion depends on the duration and strength of the ischemia, and is related to free radical injury. According to previously described ascorbate/AFR properties, we can conclude that AFR liberation in coronary effluents could represent a marker of oxidative stress during ischemia and/or reperfusion of hearts. This AFR release could be considered a sign of the severity of the ischemic episode, and could be related to the functional impairment during reperfusion.