Age-related decline of sodium-dependent ascorbic acid transport in isolated rat hepatocytes

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.     

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.     

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.     

Effect of oxygen on ascorbic acid uptake and concentration in embryonic chick brain

The effects of oxygen on ascorbic acid concentration and transport were studied in chick embryo (Gallus gallus domesticus). During normoxic incubations, plasma ascorbic acid concentration peaked on fetal day 12 and then fell, before increasing again on day 20 when pulmonary respiration began. In contrast, cerebral ascorbic acid concentration rose after day 6, was maintained at a relatively high level during days 8–18, and then fell significantly by day 20. Exposure of day 16 embryos for 48 h to 42% ambient O₂ concentration decreased ascorbic acid concentration by four-fifths in plasma and by one-half in brain, compared to values in normoxic (21% O₂) or hypoxic (15% O₂) controls. Hyperoxic preincubation of embryos also inhibited ascorbic acid transport, as evidenced by decreased initial rates of saturable and Na⁺-dependent [¹⁴C]ascorbic acid uptake into isolated brain cells. It may be concluded that changes in ascorbic acid concentration occur in response to oxidative stress, consistent with a role for the vitamin in the detoxification of oxygen radicals in fetal tissues. However, changing O₂ levels have less effect on ascorbic acid concentration in brain than in plasma, indicating regulation of the vitamin by brain cells. Furthermore, the effect of hyperoxia on cerebral vitamin C may result, in part, from inhibition of cellular ascorbic acid transport.     

Detection of Sendai virus fusion with human erythrocytes by fluorescence photobleaching recovery

The subunit stoichiometry of the ATP synthetase (CF₁-CF₀) immunoprecipitated from Triton X-100 extracts of chloroplast thylakoid membranes was determined to be α₃, β₃, γ, δ, ? (CF₁) and I_0.3, II_0.6–0.9, III₄₍₆₎ (CF₀). Antibodies against the polypeptides α, β, γ, δ, I, II and ? combined specifically with the isolated subunits as analysed by the protein blotting method. Applying this technique, antibodies against the CF₁ subunits were found to form complexes with the corresponding polypeptides of thylakoids, whereas those against I (M_r 20 000) and II (M_r 17 000) combined with M_r 26 000 and M_r 24 500 membrane polypeptides, respectively. The M_r 26 000 polypeptide was identified as the major subunits of the light-harvesting chlorophyll a/b-protein (LHCP) complex and the M_r 24 500 component seems to be functionally connected with this complex. From the results it is concluded that the chloroplast ATP synthetase consists of the subunit of the α, β, γ, δ, ? and III (proteolipid only and that proteolytically altered LHCP polypeptides bind artifically to the protein complex during isolation.     

Effects of ascorbic acid and ascorbic acid 2-phosphate, a long-acting vitamin C derivative, on the proliferation and differentiation of human osteoblast-like cells

In order to investigate the effect of ascorbic acid (AsA) and ascorbic acid 2-phosphate (Asc 2-P), a long-acting vitamin C derivative, on the growth and differentiation of human osteoblast-like cells, we supplemented the culture medium of MG-63 cells with various concentrations (0.25 to 1 mM) of these factors. Asc 2-P significantly stimulated nascent cell growth at all concentrations in the presence of fetal bovine serum (FBS). On the other hand, AsA showed a growth repressive effect depending on its concentration, and that of FBS. Asc 2-P also increased expression of osteoblast differentiation markers, such as collagen synthesis and alkaline phosphatase (ALP) activity. These stimulative activities of Asc 2-P were attenuated by inhibitors of collagen synthesis, indicating that these effects were dependent on collagen synthesis. Electron micrographs of the cells showed the formation of a three-dimensional tissue-like structure endowed with a mature extracellular matrix in the presence of Asc 2-P.     

Reaction of thymidine and ascorbic acid induced by UV in the presence of salicylic acid

When a neutral solution of thymidine and ascorbic acid was irradiated with UV light of wavelength longer than 300 nm in the presence of salicylic acid as a photosensitizer, six product peaks appeared in an HPLC chromatogram in addition to small amounts of thymidine dimers. The six products were identified as three pairs of diastereomers of 5-(2-deoxy-2-l-ascorbyl)-5,6-dihydrothymidine, 5-(2-l-ascorbyl)-5,6-dihydrothymidine, and 5,6-dihydrothymidine. These results suggest that novel DNA damage may be generated by ascorbic acid with salicylic acid induced by sunlight.     

Uric acid uptake in erythrocytes of beagle and dalmatian dogs

Uric acid uptake by erythrocytes of Beagle and Dalmatian dogs has been measured, using (2-14C) uric acid. Uptake was characterized by a fast and a slow component. Urate uptake was inhibited by certain purine and pyrimidine derivatives and by anion transport inhibitors. It was dependent on intraerythrocyte glycolysis. Temperature only influenced uptake by the slow component (Q10 = 2.6). Urate uptake by the slow component is apparently due to the transport into the erythrocytes by facilitated diffusion (Km = 6.6 mmol/l, Vmax = 390 mumol/l/min), whereas the fast component exhibits an adsorption of urate on erythrocyte surface. No difference of urate uptake by erythrocytes of Beagle and Dalmatian dogs has been observed.     

Acetylsalicylic acid (aspirin) and salicylic acid interaction with the human erythrocyte membrane bilayer induce in vitro changes in the morphology of erythrocytes

Despite the well-documented information, there are insufficient reports concerning the effects of salicylate compounds on the structure and functions of cell membranes, particularly those of human erythrocytes. With the aim to better understand the molecular mechanisms of the interaction of acetylsalicylic acid (ASA) and salicylic acid (SA) with cell membranes, human erythrocyte membranes and molecular models were utilized. These consisted of bilayers of dimyristoylphosphatidylcholine (DMPC) and dimyristoylphosphatidylethanolamine (DMPE), representative of phospholipid classes located in the outer and inner monolayers of the human erythrocyte membrane, respectively. The capacity of ASA and SA to perturb the multibilayer structures of DMPC and DMPE was evaluated by X-ray diffraction while DMPC unilamellar vesicles (LUV) were studied by fluorescence spectroscopy. Moreover, we took advantage of the capability of differential scanning calorimetry (DSC) to detect the changes in the thermotropic phase behavior of lipid bilayers resulting from ASA and SA interaction with PC and PE molecules. In an attempt to further elucidate their effects on cell membranes, the present work also examined their influence on the morphology of intact human erythrocytes by means of defocusing and scanning electron microscopy, while isolated unsealed human erythrocyte membranes (IUM) were studied by fluorescence spectroscopy. Results indicated that both salicylates interact with human erythrocytes and their molecular models in a concentration-dependent manner perturbing their bilayer structures.     

In vivo, continuous and automatic monitoring of extracellular ascorbic acid by microdialysis and on-line liquid chromatography

A system for in vivo, automatic, continuous monitoring of organ extracellular ascorbic acid in anesthetized rat is described. This system involves microdialysis perfusion and a LC system equipped with an electrochemical detector. Microdialysate, eluted from a microdialysis probe implanted in the brain cortex or in the left ventricular myocardium of anesthetized rats was collected in the sample loop of an on-line injector for direct injection onto the LC system. This automated method provides a shortened sample processing time. This system was utilized to investigate the effect of cerebral ischemia on cortex extracellular ascorbic acid and the effect of myocardial ischemia on left ventricular myocardium extracellular ascorbic acid in anesthetized rats. Basal ascorbic acid concentrations in the cortex and left ventricular myocardium ranged from 9.7 to 15.4 μM (mean±S.D. 12.7±2.5 μM from the results of eight rats) and from 9.3 to 36.0 μM (mean±S.D., 24.3±8.9 μM from the results of twelve rats), respectively. Cerebral ischemia significantly elevated ascorbic acid levels in the cortex extracellular space, while myocardial ischemia did not significantly alter ascorbic acid levels in the left ventricular myocardium extracellular space.     

Uric acid determination in the presence of ascorbic acid using self-assembled submonolayer of dimercaptothiadiazole-modified gold electrodes

This article reports the determination of uric acid (UA) in the presence of ascorbic acid (AA) using a self-assembled submonolayer of heteroaromatic dithiol, 2,5-dimercapto-1,3,4-thiadiazole (DMcT), on gold (Au) electrode. Submonolayer to multilayers of DMcT can be prepared on Au electrode by varying the soaking time of Au electrode in 1mM aqueous solution of DMcT. The formation of submonolayer, monolayer, and multilayers of DMcT on Au electrode was confirmed from its reductive desorption measurements and electrochemical blocking behavior toward ferricyanide. Interestingly, submonolayer of DMcT separates the voltammetric signal of UA from AA by 210 mV, whereas monolayer and multilayers of DMcT fail to separate them. The voltammetric signals of AA and UA are highly stable and reproducible at submonolayer of DMcT. Fast electron transfer, weak hydrogen bonding interactions with AA and UA, and prevention of fouling effect caused by oxidized product of AA can be achieved at submonolayer of DMcT, and thus it successfully separates the voltammetric signals of AA and UA. The practical application of the current system is demonstrated by measuring the concentration of UA in human urine samples without any treatment.     

Chemometric-assisted kinetic-spectrophotometric method for simultaneous determination of ascorbic acid, uric acid, and dopamine

A chemometric-assisted kinetic spectrophotometric method has been developed for simultaneous determination of ascorbic acid (AA), uric acid (UA), and dopamine (DA). This method relies on the difference in the kinetic rates of the reactions of analytes with a common oxidizing agent, tris(1,10-phenanthroline) and iron(III) complex (ferritin, [Fe(phen)₃]³⁺) at pH 4.4. The changes in absorbance were monitored spectrophotometrically. The data obtained from the experiments were processed by chemometric methods of artificial neural network (ANN) and partial least squares (PLS). Acceptable techniques of prediction set, randomization t test, cross-validation, and Y randomization were applied for the selection of the best chemometric method. The results showed that feedforward artificial neural network (FFANN) is more efficient than the other chemometric methods. The parameters affecting the experimental conditions were optimized, and it was found that under optimal conditions Beer’s law is followed in the concentration ranges of 4.3–74.1, 4.3–78.3, and 2.0–33.0 μM for AA, UA, and DA, respectively. The proposed method was successfully applied to the determination of analytes in serum and urine samples.     

Mechanism of enhanced bioavailability and diuretic effect of azosemide by ascorbic acid in rats

To increase the extent of comparative oral bioavailability (F) value and the diuretic and natriuretic effects of orally administered azosemide, ascorbic acid was coadministered to rats. The rationales for this study are that ascorbic acid might inhibit intestinal first-pass effect of azosemide and might increase the unionized fraction of azosemide at the receptor sites. After oral administration of azosemide (20 mg/kg) with 100 mg of ascorbic acid, the F value (138% vs. 100%), 8-h urinary excretion of azosemide (5.18% vs. 1.32% of oral dose), 8-h urine output (41.3 vs. 23.0 ml), and 8-h urinary excretion of sodium (24.6 vs. 15.3 mmol/kg) were greater than controls (without ascorbic acid). The amount of spiked azosemide remaining after 30 min incubation of 50 mug of azosemide with the 9000 g supernatant fraction of rat small intestine was significantly greater by 100 microg of ascorbic acid (45.3 vs. 40.9 microg) than controls (without ascorbic acid). After oral administration of azosemide with NH4Cl, the urine pH decreased by 0.5 U, and 8-h urine output (25.8 vs. 11.0 ml) and 8-h urinary excretion of sodium (13.3 vs. 6.89 mmol/kg) were significantly greater than controls (without NH4Cl). The increase in F value and diuretic and natriuretic effects of azosemide with coadministration of ascorbic acid seemed to be due to reduced intestinal first-pass metabolism of azosemide, increased urinary excretion of azosemide, and increased unionized fraction of azosemide at the renal tubular receptor sites.     

Effects of ascorbic acid supplementation on male reproductive system during exposure to hypoxia

Two groups of male rats were exposed to simulated altitudes of 6060 m and 7576 m for 6 h/day for 7 days (intermittent exposure). In two additional groups of animals exposed to the same altitude, 100 mg of ascorbic acid (AA) was fed daily for 5 days prior to the exposure period and also during the exposure period. Rats that did not receive AA showed loss of body weight and weight of reproductive organs after exposure. Sex organs showed atrophy on histological examination and there was a deterioration in spermatozoal quality. There was an increase in alkaline and acid phosphatase, and decrease in protein, sialic acid and glyceryl phosphorylcholine content in various reproductive tissues after exposure. All the above changes in histology and biochemical composition could be partially prevented by AA supplementation. AA supplementation can therefore protect the male reproductive system from deleterious effects of hypoxia. The probable mechanism of action of AA is discussed.     

In vitro effect of ascorbic acid on neutrophil–endothelial cell interaction

The effect of different concentrations (0.06, 0.6 and 6.0 mmol/L) of ascorbic acid on neutrophil–endothelial interaction was studied using an in vitro model of human umbilical cord vein endothelial cells and human neutrophils. The aim of the study was to determine changes in chemiluminescence response of neutrophils during adherence to endothelial cells. Because adherence of neutrophils to endothelial cells is an essential component in inflammatory processes leading to endothelial cell injury, the influence of ascorbic acid on adherence and endothelial cell injury have been investigated. Production of oxygen-derived metabolites, measured by chemiluminescence response of neutrophils, decreased significantly in the presence of 6 mmol/L ascorbic acid during coincubation of neutrophils and endothelial cells (p < 0.025). The adherence of neutrophils to endothelial cells was significantly decreased at a concentration of 6 mmol/L (p < 0.0005). The inhibition of neutrophil adherence to endothelial cells was correlated with a diminished neutrophil-mediated endothelial cell injury during incubation with 6 mmol/L ascorbic acid (p < 0.0005). The present results indicate that ascorbic acid might exert a protective effect on neutrophil-mediated endothelial cell injury by decreasing adherence of neutrophils to endothelial cells and by scavenging reactive oxygen metabolites. Moreover, the current investigation points to probable protective effect of ascorbic acid on oxidant-mediated cell damage in diseases (e.g., Adult Respiratory Distress Syndrome).     

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.     

High dose of ascorbic acid induces cell death in mesothelioma cells

Malignant mesothelioma is an asbestos-related fatal disease with no effective cure. Recently, high dose of ascorbate in cancer treatment has been reexamined. We studied whether high dose of ascorbic acid induced cell death of four human mesothelioma cell lines. High dose of ascorbic acid induced cell death of all mesothelioma cell lines in a dose-dependent manner. We further clarified the cell killing mechanism that ascorbic acid induced reactive oxygen species and impaired mitochondrial membrane potential. In vivo experiment, intravenous administration of ascorbic acid significantly decreased the growth rate of mesothelioma tumor inoculated in mice. These data suggest that ascorbic acid may have benefits for patients with mesothelioma.     

The role of ascorbic acid role in the differentiation of sclerotia in Sclerotinia minor

Sclerotinia minor in culture produces ascorbic acid in levels dependent on oxidative growth conditions and stage of development. During differentiation reduced/oxidized ascorbate ratio decreased by 12 and 6 fold at high and low oxidative stress, respectively. Exogenous ascorbate caused a concentration-dependent decrease of oxidative stress (lipid peroxidation), inhibition of sclerotial differentiation (up to 100%) and delay of differentiatlon (up to 10 days). Ascorbic acid may be produced to help the fungus reduce oxidative stress during growth. The data of this study support our theory proposing that oxidative stress is the inducing factor of sclerotial differentiation in fungi.This revised version was published online in October 2005 with corrections to the Cover Date.     

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.     

抗坏血酸对幽门螺杆菌相关萎缩性胃炎小鼠的干预

目的研究不同浓度抗坏血酸(ascorbic acid)溶液对幽门螺杆菌(Helicobacter pylori,H.pylori)相关萎缩性胃炎小鼠模型的作用。方法将C57BL/6小鼠随机分为5组,每组10只。组1为空白对照组,余4组小鼠用幽门螺杆菌悉尼菌株(Helicobacter pylori-Sydney strain 1,H.pylori-SS1)感染2周,快速尿素酶法和免疫组织化学法检测H.pylori-SS1的定植;组3、组4和组5小鼠分别给予抗坏血酸低倍剂量(0.1 g/L)、中倍剂量(0.5 g/L)和高倍剂量(1.0 g/L)灌胃22周。灌胃结束后,处死所有小鼠取胃组织和血清,H&E染色观察组织学变化,ELISA检测血清胃泌素(gastrin)和白介素8(IL-8)水平。结果与H.pylori组相比,抗坏血酸干预组胃组织萎缩程度减轻、血清胃泌素水平升高、炎症因子水平降低(组2 vs组3:F=21.730,P=0.029;组2 vs组3:F=42.781,P=0.015;组2 vs组3:F=35.720,P=0.021),中倍剂量组较低倍剂量组有更显著的作用(组3 vs组4:F=21.730,P=0.041;组3 vs组4:F=42.781,P=0.034;组3 vs组4:F=35.720,P=0.043),但高倍剂量组与中倍剂量组差异无统计学意义(组4 vs组5:F=21.730,P=0.125;组4 vs组5:F=42.781,P=0.391;组4 vs组5:F=35.720,P=0.450)。结论抗坏血酸干预可减轻小鼠H.pylori相关萎缩性胃炎症状,高剂量抗坏血酸与中低剂量比无显著差异。