Quercetin prevents glutathione depletion induced by dehydroascorbic acid in rabbit red blood cells

Exposure of rabbit red blood cells to dehydroascorbic acid (DHA) caused a significant decline in glutathione content which was largely prevented by quercetin, whereas it was insensitive to various antioxidants, iron chelators or scavengers of reactive oxygen species. This response was not mediated by chemical reduction of either extracellular DHA or intracellular glutathione disulfide. In addition, the flavonoid did not affect the uptake of DHA or its reduction to ascorbic acid. Rather, quercetin appeared to specifically stimulate downstream events promoting GSH formation.     

Quercetin prevents glutathione depletion induced by dehydroascorbic acid in rabbit red blood cells

Exposure of rabbit red blood cells to dehydroascorbic acid (DHA) caused a significant decline in glutathione content which was largely prevented by quercetin, whereas it was insensitive to various antioxidants, iron chelators or scavengers of reactive oxygen species. This response was not mediated by chemical reduction of either extracellular DHA or intracellular glutathione disulfide. In addition, the flavonoid did not affect the uptake of DHA or its reduction to ascorbic acid. Rather, quercetin appeared to specifically stimulate downstream events promoting GSH formation.     

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.     

Vitamin C recycling and function in human monocytic U-937 cells

The uptake, recycling, and function of ascorbic acid was evaluated in cultured U-937 monocytic cells. Dehydroascorbic acid, the two-electron oxidized form of the vitamin, was taken up on the glucose transporter and reduced to ascorbate to a much greater extent than ascorbate itself was accumulated by the cells. In contrast to dehydroascorbic acid, ascorbate entered the cells on a sodium- and energy-dependent transporter. Intracellular ascorbate enhanced the transfer of electrons across the cell membrane to extracellular ferricyanide. Rates of ascorbate-dependent ferricyanide reduction were saturable, fivefold greater than basal rates, and facilitated by intracellular recycling of ascorbate. Whereas reduction of dehydroascorbic acid concentrations above 400 microM consumed reduced glutathione (GSH), even severe GSH depletion by 1-chloro-2,4-dinitrobenzene was without effect on the ability of the cells to reduce concentrations of dehydroascorbic acid likely to be in the physiologic range (< 200 microM). Dialyzed cytosolic fractions from U-937 cells reduced dehydroascorbic acid to ascorbate in an NADPH-dependent manner that appeared due to thioredoxin reductase. However, thioredoxin reductase did not account for the bulk of dehydroascorbic acid reduction, since its activity was also decreased by treatment of intact cells with 1-chloro-2,4-dinitrobenzene. Thus, U-937 cells loaded with dehydroascorbic acid accumulate ascorbate against a concentration gradient via a mechanism that is not dependent on GSH or NADPH, and this ascorbate can serve as the major source of electrons for transfer across the plasma membrane to extracellular ferricyanide.     

N-terminal deletions and His-tag fusions dramatically affect expression of cytochrome p450 2C2 in bacteria

Mitochondria generate reactive oxygen species as by-products of oxidative metabolism. Since ascorbic acid can scavenge such destructive species, we studied the ability of mitochondria from rat liver and muscle to take up, recycle, and oxidize ascorbate. Freshly prepared mitochondria contain ascorbate, as do mitoplasts that lack the outer mitochondrial membrane. Both mitochondria and mitoplasts rapidly take up oxidized ascorbate as dehydroascorbic acid and reduce it to ascorbate. Ascorbate concentrations in mitochondria and mitoplasts rise into the low millimolar range during dehydroascorbic acid uptake, although uptake and reduction is opposed by ascorbate efflux. Mitochondrial dehydroascorbic acid reduction depends mainly on GSH, but mitochondrial thioredoxin reductase may also contribute. Reactive oxygen species generated within mitochondria oxidize ascorbate more readily than they do GSH and alpha-tocopherol. These results show that mitochondria can recycle ascorbate, which in turn might help to prevent deleterious effects of oxidant stress in the organelle.     

Vitamin C homeostasis in skeletal muscle cells

In skeletal muscle, vitamin C not only enhances carnitine biosynthesis but also protects cells against ROS generation induced by physical exercise. The ability to take up both ascorbic and dehydroascorbic acid from the extracellular environment, together with the ability to recycle the intracellular vitamin, maintains high cellular stores of ascorbate. In this study, we examined vitamin C transport and recycling, by using the mouse C2C12 and rat L6C5 muscle cell lines, which exhibit different sensitivity to oxidative stress and GSH metabolism. We found that: (1) both cell lines express SVCT2, whereas SVCT1 is expressed at very low levels only in proliferating L6C5 cells; furthermore L6C5 myoblasts are more efficient in ascorbic acid transport than C2C12 myoblasts; (2) C2C12 cells are more efficient in dehydroascorbic acid transport and ascorbyl free radical/dehydroascorbic acid reduction; (3) differentiation is paralleled by decreased ascorbic acid and dehydroascorbic acid transport and reduction and increased ascorbyl free radical reduction; (4) differentiated cells are more responsive to oxidative stress induced by glutathione depletion; indeed, myotubes showed increased SVCT2 expression and thioredoxin reductase-mediated dehydroascorbic acid reduction. From our data, SVCT2 and NADPH-thioredoxin-dependent DHA reduction appears to belong to an inducible system activated in response to oxidative stress.     

Mammalian thioltransferase (glutaredoxin) and protein disulfide isomerase have dehydroascorbate reductase activity

Homogeneous native and recombinant porcine liver thioltransferase (glutaredoxin), bovine thymus and human placenta thioltransferase (glutaredoxin) were examined for dehydroascorbate reductase activity (EC 1.8.5.1) involving the direct catalytic reduction of dehydroascorbic acid (DHA) by glutathione. Each enzyme had substantial activity with apparent Km and Vmax for dehydroascorbate between 0.2 and 2.2 mM and 6-27 nmol min-1, respectively, and for gluathione between 1.6 and 8.7 mM and 11-30 nmol min-1, respectively. In the presence of purified bovine liver thioredoxin reductase, homogeneous bovine liver thioredoxin failed to reduce DHA to ascorbic acid as measured by NADPH oxidation. Highly purified bovine liver protein disulfide isomerase (PDI) reacted directly with DHA and GSH to catalyze the reduction of DHA to ascorbic acid. The apparent Km for DHA was 1.0 mM and the Vmax was 8 nmol min-1, and for GSH were 3.9 mM and 14 nmol min-1, respectively. These results suggest that thioltransferase and PDI contribute to the regeneration of oxidized ascorbic acid in mammalian cells, and based on their cellular location, thioltransferase is proposed to be the major cytoplasmic activity, whereas interaction of DHA with microsomal membrane PDI may catalyze regeneration of ascorbic acid and initiate oxidation of intralumenal protein thiols to disulfides.     

Absence of a Causal Relationship between Auxin-Induced Growth and Changes in the Content of Ascorbic and Dehydroascorbic Acids in Excised Plant Tissues

The data reported indicate that the oxidation-reduction balance of the ascorbic acid system is not causally related to the auxin-regulation of cell elongation. There was no shift in the ascorbic acid (AA) to dehydroascorbic acid (DHA) ratio with growth-promoting concentration of auxin in several plant tissues. The AA to DHA ratio was experimentally increased without altering the growth rate. Inhibition of growth by supra-optimal auxin was associated with a decrease in the AA to DHA ratio. Since the AA to DHA ratio was lowered by EDTA treatment without altering growth, it seems unlikely that the decrease in the AA to DHA ratio related to the inhibition of growth by high levels of auxin.     

Unequivocal evidence in support of the nonenzymatic redox coupling between glutathione/glutathione disulfide and ascorbic acid/dehydroascorbic acid.

Experiments were performed to evaluate the nonenzymatic reaction between glutathione (GSH) and dehydroascorbic acid (DHA). Though both ascorbic acid and glutathione disulfide (GSSG) are formed from this reaction, previous work has focused almost exclusively on measurements of ascorbic acid. In contrast, there is very little information about the formation of GSSG under the same conditions as those used to produce ascorbic acid. The emphasis on ascorbic acid stems from the fact that a spectrophotometric technique is available for its measurement, whereas 1H-NMR or an amino acid analyzer has been used to measure GSSG. The present experiments use a simple, rapid method for accurately and precisely measuring the concentrations of GSSG in a solution. The spectrophotometric (340 nm) procedure uses NADPH and glutathione reductase; analysis time is very short, many replicate samples can be tested and as little as 0.05-0.1 mM GSSG can be detected. Using this method, it is shown that there is an equimolar production of GSSG and ascorbic acid from GSH and DHA and that the decrease in GSH is stoichiometrically related to the increase in the concentration of GSSG. The present findings provide additional insight into the interaction between the GSH/GSSG redox couple and the ascorbic acid/DHA redox couple.     

Changes Induced by Abscisic Acid and Light in the Redox State of Ascorbate in the Apoplast of Epicotyls of Vigna angularis

Levels of ascorbic acid (AA) and dehydroascorbic acid (DHA)were examined in epicotyl segments and intact epicotyls undervarious conditions. It appears that not only the redox stateof an AA-DHA system but also the level of AA plus DHA in theapoplast might be affected by growth conditions. (Received March 26, 1994; Accepted June 4, 1994)     

Intracellular flavonoids as electron donors for extracellular ferricyanide reduction in human erythrocytes.

Reduction of extracellular ferricyanide [Fe(CN)(6)](-3) to ferrocyanide by intact cells reflects the activity of a trans-plasma membrane oxidoreductase that, in human red blood cells, utilizes ascorbic acid as an electron donor. We herein report that the flavonoids quercetin and myricetin, while inhibiting dehydroascorbic acid uptake-and thus the erythrocyte ascorbic acid content-effectively stimulate the extracellular reduction of ferricyanide. Other flavonoids such as rutin, acacetin, apigenin, and genistein do not show the same effect. The notion that quercetin or myricetin may serve as an intracellular donor for a trans-plasma membrane oxidoreductase is supported by the following lines of evidence: (i) they afford direct reduction of ferricyanide; (ii) extracellular reduction of ferricyanide was not mediated by direct effects of the flavonoids released by the cells and was abolished by the sulphydryl reagent parachloromercuribenzenesulfonic acid (pCMBS); (iii) the intracellular concentrations of quercetin or myricetin well correlate with increases in ferricyanide reduction; (iv) the intracellular concentration of the flavonoids dramatically declines after ferricyanide exposure. Taken together, the results presented in this study demonstrate that myricetin and quercetin, which accumulate in large amounts in red blood cells, act as intracellular substrates of a pCMBS-sensitive trans-plasma membrane oxidoreductase. This may represent a novel mechanism whereby these flavonoids exert beneficial effects under oxidative stress conditions.     

Determination of ascorbic and dehydroascorbic acids in blood plasma and serum by liquid chromatography

A liquid-chromatography (LC) method with ultraviolet detection for measuring ascorbic (AA) and dehydroascorbic acid (DHA) in human blood and serum was studied. The method used an ODS reversed-phase column and cetyltrimethylammonium bromide as an ion-pairing agent. AA was measured before and after the reduction of DHA with dithiothreitol. The absene of interferences resulting from hemolysis products was verified and also the stability of the ascorbic acid in metaphosphoric acid extracts. The analytical parameters, linearity (1–80 μg/ml), accuracy (recovery, 96.7–100.7%) and precision (C.V.=3.1%), show that the method is reliable and adequate for measuring the total vitamin C content in serum and plasma.     

Changes in ascorbic acid levels in apoplastic fluid during growth of pine hypocotyls. Effect on peroxidase activities associated with cell walls

The apoplastic fluid of pine ( Pinus pinaster Aiton) hypocotyls contains ascorbic acid (AA) and dehydroascorbic acid (DHA). The amounts of ascorbic and dehydroascorbic acids were in the nmol (g fresh weight)⁻¹ range and decreased with the hypocotyl age as well as along the hypocotyl axis. The ratio AA/(AA+DHA) also decreased with the hypocotyl age and along the hypocotyl. Both ascorbic oxidase and peroxidase activity against ascorbic acid showed very low activity not only in the apoplastic fluid but also in the fractions ionically and covalently bound to the cell walls. However, the peroxidase activity in the three abovementioned fractions was strongly increased in the presence of ferulic acid. That stimulation effect increased with the hypocotyl age and from the apical towards the basal region of the hypocotyls of 10-day-old seedlings. Furthermore, the oxidation of ferulic acid by apoplastic and ionically- and covalently-bound peroxidases was inhibited by ascorbic acid as long as ascorbate was available. A regulatory role of apoplastic ascorbic acid levels in the formation of dehydrodiferulic bridges between wall polysaccharides catalysed by cell wall peroxidases and thus in the cell wall stiffening during plant growth is proposed.     

Possible mechanisms responsible for the increased ascorbic acid content of Plasmodium vinckei-infected mouse erythrocytes

The possible mechanisms underlying the acquisition of an increased ascorbic acid content by mouse erythrocytes containing the malarial parasite Plasmodium vinckei were investigated. Ascorbic acid was taken up readily by parasitized red blood cells but not by controls, whilst its partly oxidized form, dehydroascorbic acid, entered both. The uptake of both ascorbic acid and dehydroascorbic acid into erythrocytes was increased as a result of malarial infection. Lysates prepared from parasitized red blood cells reduced exogenous dehydroascorbic acid to ascorbic acid at a higher rate than control red blood cell lysates; this difference was abolished following dialysis of the lysates, a process which removes endogenous reduced glutathione (GSH). The rates of chemical and enzymatic reduction of dehydroascorbic acid to ascorbic acid by GSH were of similar magnitude, thus calling into question the existence of a specific dehydroascorbate reductase in erythrocytes and parasites. These observations suggest that the increased uptake of dehydroascorbic acid into parasitized red blood cells may be a result of enhanced dehydroascorbate-reducing capacity, whilst the presence of the parasite induces a selective increase in the permeability of the erythrocyte plasma membrane to ascorbic acid. The endogenous ascorbic acid content of livers obtained from infected mice was 55% below the normal concentration and its relative rate of destruction during incubation in vitro was enhanced in comparison with that of control livers. Furthermore, the capacity of liver homogenates to synthesize ascorbic acid from glucuronic acid was greatly reduced in infected mice. Therefore it is unlikely that the increase in ascorbic acid content of parasitized red blood cells is a consequence of increased biosynthesis and release of ascorbic acid by the host liver. We have not been able to exclude the possibility that the malarial parasite itself may be capable of de novo synthesis of ascorbic acid.     

Recycling of vitamin C from its oxidized forms by human endothelial cells

Endothelial cells encounter oxidant stress due to their location in the vascular wall, and because they generate reactive nitrogen species. Because ascorbic acid is likely involved in the antioxidant defenses of these cells, we studied the mechanisms by which cultures of EA.hy926 endothelial cells recycle the vitamin from its oxidized forms. Cell lysates reduced the ascorbate free radical (AFR) by both NADH- and NADPH-dependent mechanisms. Most NADH-dependent AFR reduction occurred in the particulate fraction of the cells. NADPH-dependent reduction resembled that due to NADH in having a high affinity for the AFR, but was mediated largely by thioredoxin reductase. Reduction of dehydroascorbic acid (DHA) required GSH and was both direct and enzyme dependent. The latter was saturable, half-maximal at 100 microM DHA, and comparable to rates of AFR reduction. Loading cells to ascorbate concentrations of 0.3-1.6 mM generated intracellular DHA concentrations of 20-30 microM, indicative of oxidant stress in culture. Whereas high-affinity AFR reduction is the initial and likely the preferred mechanism of ascorbate recycling, any DHA that accumulates during oxidant stress will be reduced by GSH-dependent mechanisms.     

In vitro oxidation of ascorbic acid and its prevention by GSH

The interaction of glutathione (GSH) with ascorbic acid and dehydroascorbic acid was examined in in-vitro experiments in order to examine the role of GSH in protecting against the autoxidation of ascorbic acid and in regenerating ascorbic acid by reaction with dehydroascorbic acid. If a buffered solution (pH 7.4) containing 1.0 mM ascorbic acid was incubated at 37 degrees C, there was a rapid loss of ascorbic acid in the presence of oxygen. When GSH was added to this solution, ascorbic acid did not disappear. Maximum protection against ascorbic acid autoxidation was achieved with as little as 0.1 mM GSH. Cupric ions (0.01 mM) greatly accelerated the rate of autoxidation of ascorbic acid, an effect that was inhibited by 0.1 mM GSH. Other experiments showed that GSH complexes with cupric ions, resulting in in a lowering of the amount of GSH in solution as measured in GSH standard curves. These results suggest that the inhibition of ascorbic acid autoxidation by GSH involves complexation with cupric ions that catalyze the reaction. When ascorbic acid was allowed to autoxidize at 37 degrees C the subsequent addition of GSH (up to 10 mM) did not lead to the regeneration of ascorbic acid. This failure to detect a direct reaction between GSH and the dehydroascorbic acid formed by oxidation of ascorbic acid under this condition was presumably due to the rapid hydrolysis of dehydroascorbic acid. When conditions were chosen, i.e., low temperature, that promote stability of dehydroascorbic acid, the direct reaction between GSH and dehydroascorbic acid to form ascorbic acid was readily detected. The marked instability of dehydroascorbic acid at 37 degrees C raises questions regarding the efficiency of the redox couple between GSH and dehydroascorbic acid in maintaining the concentration of ascorbic acid in mammalian cells exposed to an oxidative challenge.     

Estimation of dehydroascorbic acid in blood of diabetic patients.

A modified method has been described for the estimation of ascorbic acid (AA) and dehydroascorbic acid (DHA) in blood and plasma. DHA is practically absent in the blood of normal human beings. On the other hand, diabetic patients have a persistently high blood DHA level. The DHA from diabetic blood has been isolated as the 2,4-dinitrophenylhydrazone derivative and identified by thin-layer chromatography and spectrophotometry.     

Differential Implication of Glutathione, Glutathione-Metabolizing Enzymes and Ascorbate in Tomato Resistance to Pseudomonas syringae

We studied the response of glutathione‐ and ascorbate‐related antioxidant systems of the two tomato cultivars to Pseudomonas syringae pv. tomato infection. In the inoculated susceptible A 100 cultivar a substantial decrease in reduced glutathione (GSH) content, oxidised glutathione accumulation and GSH redox ratio decline as well as glutathione peroxidase activity increase were found. The enhanced glutathione reductase activity was insufficient to keep the glutathione pool reduced. A transiently increased dehydroascorbic acid (DHA) content and ascorbic acid (AA) redox ratio decrease together with ascorbate peroxidase activity suppression were observed. Adversely to the progressive reduction in GSH pool size, AA content tended to increase but the changes were more modest than those of GSH. By contrast, in interaction with the resistant Ontario cultivar the glutathione pool homeostasis was maintained throughout P. syringae attack and no significant effect on the ascorbate pool was observed. Moreover, in the resistant interaction there was a significantly higher constitutive and pathogen‐induced glutathione‐S‐transferase (GST) activity. The relationship between GST activity and DHA content found in this study indicates that this enzyme could also act as dehydroascorbate reductase. These results reflect the differential involvement of GSH and AA in tomato‐P. syringae interaction and, in favour of the former, they clearly indicate the role of GSH and GSH‐utilizing enzymes in resistance to P. syringae. The maintenance of glutathione pool homeostasis and GST induction appear to contribute to tissue inaccessibility to bacterial attack.     

Ascorbic acid depletion enhances expression of the sodium-dependent vitamin C transporters, SVCT1 and SVCT2, and uptake of ascorbic acid in livers of SMP30/GNL knockout mice

In this study, we examined whether ascorbic acid (AA) and dehydroascorbic acid (DHA), the oxidized form of AA, levels in tissues regulate the AA transporters, sodium-dependent vitamin C transporters (SVCT) 1 and SVCT2 and DHA transporters, glucose transporter (GLUT) 1, GLUT3, GLUT4 mRNA by using senescence marker protein-30 (SMP30)/gluconolactonase (GNL) knockout (KO) mice. These mice are incapable of synthesizing AA in vivo. AA depletion enhanced SVCT1 and SVCT2 mRNA expression in the liver and SVCT1 and GLUT4 mRNA expression in the small intestine, but not in the cerebrum or kidney. Next, we examined the actual impact of AA uptake by using primary cultured hepatocytes from SMP30/GNL KO mice. In the AA-depleted hepatocytes from SMP30/GNL KO mice, AA uptake was significantly greater than in matched cultures from wild-type mice. These results strongly affirm that intracellular AA is an important regulator of SVCT1 and SVCT2 expression in the liver.     

Regulation of Water Deficit-Induced Abscisic Acid Accumulation by Apoplastic Ascorbic Acid in Maize Seedlings

Water deficit-induced abscisic acid （ABA） accumulation is one of the most important stress signaling pathways in plant cells. Redox regulation of cellular signaling has currently attracted particular attention, but much less is known about its roles and mechanisms in plant signaling. Herein, we report that water deficit-induced ABA accumulation could be regulated by ascorbic acid （AA）-controlled redox status in leave apoplast. The AA content in non-stressed leaves was approximately 3 umol/g FW, corresponding to a mean concentration of 3 mmol/L in a whole cell. Because AA is mainly localized in the cytosol and chloroplasts, the volume of which is much smaller than that of the whole cell, AA content in cytosolic and chloroplast compartments should be much higher than 3 mmol/L. Water deficit-induced ABA accumulation in both leaf and root tissues of maize seedlings was significantly inhibited by AA and reduced glutathione （GSH） at concentrations of 500 umol/L and was completely blocked by 50 mmol/L AA and GSH. These results suggest that the AA-induced inhibition of ABA accumulation should not occur at sites where AA exists in high concentrations. Although water deficit led to a small increase in the dehydroascorbic acid （DHA） content, no significant changes in AA content were observed in either leaf or root tissues. When compared with the whole leaf cell, the AA content in the apoplastic compartment was much lower （i.e. approximately 70 nmol/g FW, corresponding to 0.7 mmol/L）. Water deficit induced a significant decrease （approximately 2.5-fold） in the AA content and an increase （approximately 3.4-fold） in the DHA content in the apoplastic compartment, thus leading to a considerably decreased redox status there, which may have contributed to the relief of AA-induced inhibition of ABA accumulation, alternatively, promoting water deficit-induced ABA accumulation. Reactive oxygen species （ROS） could not mimic water deficit in inducing ABA accumulation, suggesting that the inhibition of ABA accumulation by AA or GSH was not related to their ROS-scavenging ability. The results of the present study suggest that the redox status in the apoplastic compartment, as determined by AA and DHA, may play a vital role in the regulation of the signaling process for water deficit-induced ABA accumulation.