Human erythrocyte recycling of ascorbic acid: relative contributions from the ascorbate free radical and dehydroascorbic acid

Recycling of ascorbic acid from its oxidized forms helps to maintain the vitamin in human erythrocytes. To determine the relative contributions of recycling from the ascorbate radical and dehydroascorbic acid, we studied erythrocytes exposed to a trans-membrane oxidant stress from ferricyanide. Ferricyanide was used both to induce oxidant stress across the cell membrane and to quantify ascorbate recycling. Erythrocytes reduced ferricyanide with generation of intracellular ascorbate radical, the concentrations of which saturated with increasing intracellular ascorbate and which were sustained over time in cells incubated with glucose. Ferricyanide also generated dehydroascorbic acid that accumulated in the cells and incubation medium to concentrations much higher than those of the radical, especially in the absence of glucose. Ferricyanide-stimulated ascorbate recycling from dehydroascorbic acid depended on intracellular GSH but was well maintained at the expense of intracellular ascorbate when GSH was severely depleted by diethylmaleate. This likely reflects continued radical reduction, which is not dependent on GSH. Erythrocyte hemolysates showed both NAD- and NADPH-dependent ascorbate radical reduction. The latter was partially due to thioredoxin reductase. GSH-dependent dehydroascorbate reduction in hemolysates, which was both direct and enzyme-dependent, was greater than that of the radical reductase activity but of lower apparent affinity. Together, these results suggest an efficient two-tiered system in which high affinity reduction of the ascorbate radical is sufficient to remove low concentrations of the radical that might be encountered by cells not under oxidant stress, with back-up by a high capacity system for reducing dehydroascorbate under conditions of more severe oxidant stress.     

Mitochondrial ascorbic acid transport is mediated by a low-affinity form of the sodium-coupled ascorbic acid transporter-2

Despite the fundamental importance of the redox metabolism of mitochondria under normal and pathological conditions, our knowledge regarding the transport of vitamin C across mitochondrial membranes remains far from complete. We report here that human HEK-293 cells express a mitochondrial low-affinity ascorbic acid transporter that molecularly corresponds to SVCT2, a member of the sodium-coupled ascorbic acid transporter family 2. The transporter SVCT1 is absent from HEK-293 cells. Confocal colocalization experiments with anti-SVCT2 and anti-organelle protein markers revealed that most of the SVCT2 immunoreactivity was associated with mitochondria, with minor colocalization at the endoplasmic reticulum and very low immunoreactivity at the plasma membrane. Immunoblotting of proteins extracted from highly purified mitochondrial fractions confirmed that SVCT2 protein was associated with mitochondria, and transport analysis revealed a sigmoidal ascorbic acid concentration curve with an apparent ascorbic acid transport K_m of 0.6 mM. Use of SVCT2 siRNA for silencing SVCT2 expression produced a major decrease in mitochondrial SVCT2 immunoreactivity, and immunoblotting revealed decreased SVCT2 protein expression by approximately 75%. Most importantly, the decreased protein expression was accompanied by a concomitant decrease in the mitochondrial ascorbic acid transport rate. Further studies using HEK-293 cells overexpressing SVCT2 at the plasma membrane revealed that the altered kinetic properties of mitochondrial SVCT2 are due to the ionic intracellular microenvironment (low in sodium and high in potassium), with potassium acting as a concentration-dependent inhibitor of SVCT2. We discarded the participation of two glucose transporters previously described as mitochondrial dehydroascorbic acid transporters; GLUT1 is absent from mitochondria and GLUT10 is not expressed in HEK-293 cells. Overall, our data indicate that intracellular SVCT2 is localized in mitochondria, is sensitive to an intracellular microenvironment low in sodium and high in potassium, and functions as a low-affinity ascorbic acid transporter. We propose that the mitochondrial localization of SVCT2 is a property shared across cells, tissues, and species.     

Determination of ascorbic acid with immobilized green zucchini ascorbate oxidase

Ascorbate oxidase from zucchini squash was immobilized onto CH-Sepharose via carbodiimide. The properties of the immobilized enzyme were found to be similar to those of the free ascorbate oxidase. The immobilized enzyme was utilized in a flow-through system equipped with a polarographic detector which monitors the oxygen depletion due to the reaction ascorbic acid + 1/2 O2----dehydroascorbic acid + H2O. This method, the response of which is linear between 3 X 10(-7) and 5 X 10(-4) M ascorbate, was utilized to measure the ascorbic acid in biological samples such as human plasma and fruit juices at a rate of about 60 determinations every hour with a standard deviation lower than 5%.     

Enhanced prostaglandin synthesis as a mechanism for inhibition of melanoma cell growth by ascorbic acid

Both ascorbic acid and the 1-series prostaglandins have been reported to be important regulators of cell growth and since ascorbic acid also increases the synthesis of the 1-series prostaglandins, it is possible that the effects of ascorbic acid on cell growth might be mediated by changes in 1-series prostaglandin synthesis induced by ascorbic acid. This study attempted to examine this possible relationship. The effects of ascorbic acid, prostaglandin E1 and the essential fatty acid precursors of the prostaglandins, linoleic acid and gamma-linolenic acid on the in vitro growth of transformed BL6 murine melanoma cells and untransformed monkey kidney (LLCMK) cells was determined. The effects of ascorbic acid addition on the growth inhibitory effect of the essential fatty acids and on the activity of delta-6-desaturase, a key enzyme in 1-series prostaglandin synthesis were also examined. Addition of ascorbic acid, prostaglandin E1 and both essential fatty acids was found to reduce BL6 growth while PGE1 and to a lesser extent the essential fatty acids reduced LLCMK cell growth. The growth inhibitory effect of the essential fatty acids was enhanced by ascorbic acid which was also found to stimulate delta-6-desaturase activity in BL6 cells. The growth inhibitory effect of ascorbic acid on BL6 cells may thus be mediated by changes in prostaglandin synthesis through an association with the metabolism of the essential fatty acid precursors of the prostaglandins.     

Mechanisms of ascorbic acid recycling in human erythrocytes.

Vitamin C, or ascorbic acid, is efficiently recycled from its oxidized forms by human erythrocytes. In this work the dependence of this recycling on reduced glutathione (GSH) was evaluated with regard to activation of the pentose cycle and to changes in pyridine nucleotide concentrations. The two-electron-oxidized form of ascorbic acid, dehydroascorbic acid (DHA) was rapidly taken up by erythrocytes and reduced to ascorbate, which reached intracellular concentrations as high as 2 mM. In the absence of D-glucose, DHA caused dose-dependent decreases in erythrocyte GSH, NADPH, and NADH concentrations. In the presence of 5 mM D-glucose, GSH and NADH concentrations were maintained, but those of NADPH decreased. Reduction of extracellular ferricyanide by erythrocytes, which reflects intracellular ascorbate recycling, was also enhanced by D-glucose, and ferricyanide activated the pentose cycle. Diethylmaleate at concentrations up to 1 mM was found to specifically deplete erythrocyte GSH by 75-90% without causing oxidant stress in the cells. Such GSH-depleted erythrocytes showed parallel decreases in their ability to take up and reduce DHA to ascorbate, and to reduce extracellular ferricyanide. These results show that DHA reduction involves GSH-dependent activation of D-glucose metabolism in the pentose cycle, but that in the absence of D-glucose DHA reduction can also utilize NADH.     

Symbiophagy as a cellular mechanism for coral bleaching

Coral bleaching is a major contributor to the global declines of coral reefs. This phenomenon is characterized by the loss of symbiotic algae, their pigments or both. Despite wide scientific interest, the mechanisms by which bleaching occurs is still poorly understood. Here we report that the removal of the symbiont during light and temperature stress is achieved using the host's cellular autophagic-associated machinery. Host cellular and sub-cellular morphologies showed increased vacuolization and appearance of autophagic membranes surrounding a variety of organelles and surrounding the symbiotic algae. Markers of autophagy (Rab 7 and LAS) corroborate these observations. Results showed that during stress the symbiont vacuolar membrane is transformed from a conduit of nutrient exchange to a digestive organelle resulting in the consumption of the symbiont, a process we term symbiophagy. We posit that during a stress event, the mechanism maintaining symbiosis is destabilized and symbiophagy is activated, ultimately resulting in the phenomenon of bleaching. Symbiophagy may have evolved from a more general primordial innate intracellular protective pathway termed xenophagy.     

Absorption of ascorbic acid and ascorbic sulfate and ascorbate metabolism in common carp (Cyprinus carpio L.)

Summary Ascorbate metabolism was analyzed in fasted common carp and carp offered diets lacking ascorbic acid or supplemented with ascorbic acid (AA) or ascorbic sulfate (AS). Ascorbic acid and ascorbic sulfate were analyzed in the contents collected from various parts of the digestive tract. The major site of the dietary ascorbate absorption was located in the first 20% of the anterior intestine region (58.7±10.2%), whereas absorption increased to 94.3±1.9% (in the whole gut). Considerable secretion of ascorbate into the initial part of the intestine was found (71 g AA · g^-1 dry food) in fish offered the diet lacking ascorbate, but this amount was completely reabsorbed in the following portions of the intestine. AS was concentrated in the contents of the digestive tract and the external marker method revealed no absorption of AS from the intestine. In fish fed the AA-supplemented diet, the concentration of ascorbate in plasma, hepatopancreas, kidney, intestine, spleen, and brain was significantly (P<0.01) higher than in similar tissues from the other groups, suggesting that ascorbic sulfate hydrolysis was ineffective. Small amounts of AS were found in the intestine and spleen of fish fed a diet supplemented with AS. Ascorbate analysis in the whole fish allowed the estimate of the catabolic rate of fasting and scorbutic-diet-fed fish, which amounted to 0.7% and 1.46% daily of the ascorbate body pool, respectively. There was no indication that ascorbic sulfate sulfohydrolase activity was induced in hepatic, kidney, or intestinal tissue of fish offered a diet with AS in comparison to other groups. It seems unlikely that cyprinid fish are able to utilize ascorbic sulfate as a vitamin C source, and thus resemble scurvy-prone mammals in this respect.Abbreviations AA ascorbic acid - AS ascorbic sulfate - TCA trichloroacetic acid - PCA perchloric acid - EDTA ethylenediaminetetraacetate dihydrate - DNPH dinitrophenyl hydrazine - ASS-ase ascorbic sulfate sulfohydrolase - AR-ase arylsulfatase - NCS p-nitrocatechol sulfate - DHA-rase dehydroascorbate reductase - DHA dehydroascorbic acid - GSH glutathione - G-6-Pase glucose-6-phosphate dehydrogenase - G-6-P glucose-6-phosphate     

Endothelial dysfunction as a cellular mechanism for vascular failure

The regulation of vascular tone, vascular permeability, and thromboresistance is essential to maintain blood circulation and therefore tissue environments under physiological conditions. Atherogenic stimuli, including diabetes, dyslipidemia, and oxidative stress, induce vascular dysfunction, leading to atherosclerosis, which is a key pathological basis for cardiovascular diseases such as ischemic heart disease and stroke. We have proposed a novel concept termed "vascular failure" to comprehensively recognize the vascular dysfunction that contributes to the development of cardiovascular diseases. Vascular endothelial cells form the vascular endothelium as a monolayer that covers the vascular lumen and serves as an interface between circulating blood and immune cells. Endothelial cells regulate vascular function in collaboration with smooth muscle cells. Endothelial dysfunction under pathophysiological conditions contributes to the development of vascular dysfunction. Here, we address the barrier function and microtubule function of endothelial cells. Endothelial barrier function, mediated by cell-to-cell junctions between endothelial cells, is regulated by small GTPases and kinases. Microtubule function, regulated by the acetylation of tubulin, a component of the microtubules, is a target of atherogenic stimuli. The elucidation of the molecular mechanisms of endothelial dysfunction as a cellular mechanism for vascular failure could provide novel therapeutic targets of cardiovascular diseases.     

Nitric oxide stimulates the erythrocyte for ascorbate recycling.

S-Nitrosothiols act as carrier and reservoir of nitric oxide (NO), and release NO under stimulation of ascorbate (Asc). Erythrocyte can regenerate Asc from its oxidised products, thus saving this powerful antioxidant. In this paper the effect of donors of NO, superoxide, and peroxynitrite (SpNONOate, KO(2), and SIN-1, respectively) on the erythrocyte production of Asc was investigated. We report here that NO stimulated, while superoxide and peroxynitrite decreased, the Asc recycling. The NO-stimulating effect on the erythrocyte production of Asc was confirmed by using GSNO, a natural occurring S-nitrosothiol, as NO donor. These data highlight a new property of NO, that is the stimulation of erythrocytes for their Asc recycling. Such a property might contribute to regenerate Asc from its oxidised forms, thus preventing its depletion in the circulation. Temperature and pH significantly affected, both in absence and presence of NO, the recycling of Asc by erythrocytes. We propose that a positive feedback, involving the reciprocal stimulation between Asc and S-nitrosothiols, might enhance productions of Asc by erythrocytes and NO release by circulating S-nitrosothiols.     

Effects of ascorbic acid and sodium ascorbate on cyclic nucleotide metabolism in human lymphocytes.

L-ascorbic acid (LAA) augmented cGMP many-fold in highly purified human peripheral blood lymphocytes. The cGMP response occurred within 10 sec and persisted for at least 60 min. D-ascorbic acid (DAA) and dehydroascorbic acid (DHAA) were also equally active in enhancing cGMP concentrations but metabolic precursors of ascorbic acid and other inorganic acids did not increase cGMP levels. Determination of the amount of DHAA contaminating the LAA precluded the possibility that it was solely responsible for the enhanced cGMP levels. The sodium or calcium salts of ascorbic acid did not increase cGMP concentrations. If these neutralized preparations were acidified, increased cGMP concentrations were then noted. In broken cell preparations, LAA, DAA, and DHAA and to a lesser extent sodium ascorbate (NaA) enhanced guanylate cyclase activity while neither inhibited cAMP or cGMP phosphodiesterase (PDE) activity. The possible role of H2O2, fatty acid liberation, prostaglandin production, oxidizing-reducing agents, and free radical formation in mediating the effects of ascorbic acid on cGMP levels were evaluated, but none of these potential mechanisms were definitively proven to be a required intermediary for the cGMP enhancing activity of ascorbic acid. LAA, DHAA or NaA did not induce lymphocyte transformation or modulate lectin-induced mitogenesis.     

Macrophage uptake and recycling of ascorbic acid: response to activation by lipopolysaccharide

To test whether ascorbic acid might be involved in the antioxidant defenses of inflammatory cells, we studied ascorbate uptake and recycling by quiescent and lipopolysaccharide-activated RAW264.7 murine macrophages. These cells concentrated ascorbate 100-fold in overnight culture, achieving steady-state concentrations of more than 10 mM at extracellular concentrations of 20-100 muM. This steep gradient was generated by high-affinity sodium-dependent ascorbate transport. The latter likely reflects function of the SVCT2 (SLC23A2), since this protein was detected on immunoblots. Dehydroascorbate, the two-electron oxidized form of ascorbate, was also taken up and reduced to ascorbate by the cells. Dehydroascorbate reduction required rapid recycling of GSH from GSSG by glutathione reductase. Activation of ascorbate-containing macrophages with lipopolysaccharide transiently depleted intracellular ascorbate without affecting GSH. Recovery of intracellular ascorbate required function of the SVCT2 transporter, the activity of which was modestly enhanced by lipopolysaccharide. Lipopolysaccharide treatment nearly doubled intracellular GSH concentrations over 2 h. Despite lipopolysaccharide-induced oxidant stress, this GSH increase was associated with a comparable increase in reduction of dehydroascorbate to ascorbate. These results show that macrophages maintain millimolar concentrations of ascorbate through function of the SVCT2 and that activated cells have an enhanced ability to transport and recycle ascorbate, possibly reflecting its role as an intracellular antioxidant.     

Changes in ascorbic acid content and ascorbate peroxidase activity during the development of Acetabularia mediterranea

Abstract. The level of peroxidase activity utilizing ascorbic acid changes during the development of the green alga, Acetabularia mediterranea. During development almost parallel levels of peroxidase activity and ascorbic acid content are detectable: both steadily decrease as algae progress from very young, slowly growing cells to the rapid growth stage and then to cells exhibiting differentiation into primordium and cap. Changes in the levels of the enzyme and its substrate in the cytoplasm and periplasm were demonstrated using biochemical and cytochemical procedures. Concomitant with these developmental changes, we also observed changes in the stage-specific patterns of ascorbic acid concentration: growing algae exhibit a pronounced negative apicobasal gradient of ascorbic acid. Acetabularia cultivated at 1,200 lux (the normal intensity in a 12-h-light/12-h-dark cycle) and at 700 lux (intensity at which growth is reduced, and cap formation is delayed) were also compared. The higher light intensity induced a moderate decrease in the ascorbic acid content without noticeable changes in the compartmental distribution in the cytoplasm and periplasm, and an increase in the level of periplasmic peroxidase activity with little change in the total peroxidase activity. Catalase was found to be present at very low levels and is unlikely to play a role in H₂O₂ catabolism. Possible roles for ascorbic acid and peroxidase in the development of Acetabularia are discussed.     

Changes in ascorbic acid content and ascorbate peroxidase activity during the development of Acetabularia mediterranea

We cloned ras-related sequences from goldfish genomic libraries constructed as recombinants using the lambda phage. Restriction enzyme mapping of the clones obtained revealed three kinds of ras-related sequences among approximately 350,000 genomic clones. One of these clones was partially sequenced. Comparison with the nucleotide sequences of mammalian ras genes showed that the determined sequences covered the predicted amino acid coding regions and parts of the intervening regions. The predicted amino acid sequences of the cloned ras-related goldfish gene suggested that the coding region is localized separately in DNA, and that its exon-intron boundaries are exactly the same as those of corresponding mammalian genes. The nucleotide and amino acid sequences of the goldfish ras-related gene may have extensive homologies to mammalian p 21 protein. Among the three mammalian ras proteins, the predicted amino acid sequence of the sequenced ras-related goldfish clone is most closely homologous (96%) to the Kirsten ras protein. Differences in the predicted amino acid sequence were greatest in the sequence predicted from the fourth exon; fewer differences were found in the sequence from the third exon, and only slight or no differences were found in the sequence predicted for the first and second exons. The 12th and 61st amino acids from the N-terminal of the protein, which are thought to be critical positions for GTP binding and catalysis, are both conserved in the goldfish protein.(ABSTRACT TRUNCATED AT 250 WORDS)     

Glutathione-dependent ascorbate recycling activity of rat serum albumin

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

Glucose inhibits cellular ascorbic acid uptake by fibroblasts in vitro

It has been suggested earlier that the local deficiency of ascorbic acid in tissues could be responsible for development of various angiopathies in diabetes. Hyperglycemia is one of the factors which could contribute considerably to the development of local ascorbic acid deficiency. Therefore, the effect of glucose on uptake of L-[1-14C] ascorbic acid by fibroblasts was studied in vitro. The data clearly show that ascorbic acid uptake is inhibited instantly by glucose in a concentration dependent fashion. The results support the contention that local ascorbic acid deficiency in tissues could be a natural consequence of hyperglycemia of whatever cause. The rate of ascorbic acid uptake under various conditions suggests that additional supplements of ascorbic acid might be helpful to individuals in averting deleterious effects of hyperglycemia on tissue ascorbic acid supply.     

DNA-decorated nanoparticles as nanosensors for rapid detection of ascorbic acid

We designed an assay for rapid detection of ascorbic acid (AA) with a DNAzyme cleaving its DNA substrate in the presence of Cu(2+) and AA. The sensor consists of two DNA strands that form a complex between each other. The 5'-end of the DNAzyme binds the substrate DNA via Watson-Crick bonding and the 3'-end binds through formation of a DNA-triplex via Hoogsteen hydrogen bonding. The substrate DNA was prepared by two different methods. In the first case the nucleic acid was modified with fluorescein/dabcyl FRET pair across the cleavage site. In the second case the nucleic acid modified with fluorescein was immobilised on gold nanoparticles. DNAzyme contains a loop forming a complex with Cu(2+) ions. The oxidation of ascorbic acid (AA) with oxygen yields hydrogen peroxide. The latter interacts with Cu(2+) to give hydroxyl radicals. They break substrate DNA in close vicinity to the copper/DNA complex to separate fluorescein from gold nanoparticles leading to the increase in fluorescence intensity. Use of substrate DNA modified with the fluorescein/dabcyl couple allowed to measure AA concentration within 3 min with the detection limit of 2.5 μM. Employment of gold nanoparticles decorated with fluorescein-modified DNA allowed to improve the detection limit of AA quantification by two orders of magnitude due to enhanced cleavage of DNA catalysed by Au clusters. Fructose, sucrose, glucose, urea, and citric acid did not interfere with our assay even at concentration of 1mM. Good selectivity allowed us to apply our rapid and sensitive assays to detection of AA in vitamin C tablets, urine and orange juice.