Effects of ascorbic acid on high-GSH and normal-GSH dog erythrocytes (II)

Mechanisms of ascorbic acid uptake and oxidative effects were studied in dog erythrocytes with high GSH and normal GSH concentrations. It was noted that ascorbic acid enters dog erythrocytes very slowly in the reduced form compared with the oxidised form (DHA) and by a glucose-independent mechanism. In the absence of glucose, accumulation of ascorbic acid, GSH depletion and increased methaemoglobin formation occurred successively. Compared with erythrocytes with normal GSH levels, those with high GSH levels showed a greater accumulation and prolonged presence of ascorbic acid, considerable delay in GSH depletion and less methaemoglobin formation. This is due to there being a larger reserve of GSH in high-GSH cells, which is exhausted more slowly. GSH-depleted erythrocytes were no longer able to accumulate ascorbic acid. Hydrogen peroxide (H₂O₂) was found to be the sole reactive oxygen species responsible for oxidative effects of ascorbic acid, while superoxide (O ₂ ^– ), hydroxyl radical (OH) and metal ions had relatively minor roles. Overall, these results clearly imply that GSH is essential in accumulating ascorbic acid and resisting oxidative effects in dog erythrocytes and that cells with high GSH levels containing larger reserve of GSH have an advantage with regard to the accumulation of ascorbic acid and its function.     

Effects of ascorbic acid on high GSH and normal dog erythrocytes (I)

Dog erythrocytes with high GSH and normal GSH concentrations were compared under effects of ascorbic acid. In the presence of glucose, these two types of erythrocytes showed a similar increase in intracellular ascorbic acid without change in GSH levels. Addition of iron (Fe³⁺) to the incubation medium enhanced ascorbic acid accumulation. In the absence of glucose, GSH fell markedly in both types of erythrocyte and less ascorbic acid accumulated in normal GSH cells than high GSH cells. With iron, normal cells showed GSH depletion and marked methaemoglobin formation. Lipid peroxidation with ascorbic acid and iron increased at a similar rate in both types of erythrocyte and was inhibited by catalase. Ferricyanide reduction in both erythrocytes loaded with ascorbic acid were similar and increased with glucose or catalase. There was a high correlation between intracellular ascorbic acid content and ferricyanide reduction. These results suggest that high GSH and normal GSH dog erythrocytes have a similar capacity for accumulating ascorbic acid and for reducing ferricyanide. However, normal GSH erythrocytes are more susceptible to the oxidant effect of ascorbic acid than high GSH cells; this is probably due to a smaller GSH reserve, which is exhausted more rapidly under oxidative stress.     

Kinetic properties of dehydroascorbic acid transport in erythrocytes of young and adult beagle dogs

Kinetic properties of dehydroascorbic acid (DHA) transport were studied in erythrocytes of beagle dogs at 1 and 12 months. DHA uptake was much faster in erythrocytes of dogs at 1 than 12 months. Young dogs showed much greater affinity for DHA than adult dogs and the rate of DHA uptake was not affected by glucose. It was due to the presence of fetal erythrocytes with high activity of Glut-1 glucose transporter in young dogs. In adult dogs, DHA uptake was slow and competitively inhibited by physiological concentrations of glucose. Glut-4 was identified as the carrier protein responsible for DHA uptake in erythrocytes of adult dogs. Glut-4 was hardly detectable in neonatal dogs, but increased to adult levels by 3 months as fetal erythrocytes were eliminated and switched to adult cells.     

Distinct mechanisms of transport of ascorbic acid and dehydroascorbic acid in intestinal epithelial cells (IEC-6)

Ascorbic acid (AsA) is an essential nutrient for humans as they lack its biosynthesizing key enzyme. Its absorption mechanism in small intestinal epithelial cells still remains to be resolved. In this study, the transport mechanisms functioning on the uptake of AsA and its oxidized form, dehydroascorbic acid (DHA), were investigated using rat small intestinal epithelial cell line IEC-6. Both AsA and DHA were accumulated in the cells in time- and concentration-dependent manners, but their absorption kinetics were apparently different. The saturability of AsA uptake was shown at a considerably lower concentration in IEC-6 cells as well as other mammalian cells, indicating that this absorption was mediated by a specific transporting carrier. The absorption efficiency of AsA was about 1/5-1/10 that of DHA at the same concentration range and, moreover, the uptake of DHA was almost comparable to that of 2-deoxy-D-glucose, an alternative of glucose. The uptake of AsA was diminished by the removal of sodium ion, but not by the addition of glucose, whereas that of DHA was sodium ion-independent and effectively inhibited by glucose. In addition, phlorizin and cytochalasin B, which are blockers of glucose transporters, interfered the uptake of DHA more efficiently than that of AsA. These results indicate that there are at least two distinct transport systems of vitamin C in rat small intestinal epithelial cells; AsA is transported by a specific transporter and DHA is mainly transported by glucose transporter(s).     

The contribution of ascorbic acid and dehydroascorbic acid to the protective role of pleura during inflammatory reactions

It is well-known that parapneumonic effusions lead to the formation of inflammatory exudates which contain an increasing amount of inflammatory cells, especially polymorphonuclear. At these pathological conditions characterized by oxidative stress, ascorbic acid (AA) plays an important role in quenching free radicals, so that it could protect neutrophils and mesothelial cells from oxidative damage. Besides that ascorbic acid and its metabolite dehydroascorbic acid (DHA) alters the sheep visceral and parietal pleura permeability. More specific ascorbic acid as well as dehydroascorbic acid decreases the permeability of pleura after addition on apical and basolateral side in both visceral and parietal pleurae. It seems that, AA and DHA have an opposite action upon pleura from that of the inflammatory mediators, like VEGF, which increases the permeability of pleura and causes mesothelial barrier dysfunction. The decrease of pleura permeability induced by AA and DHA suggest the hypothesis that AA and/or its metabolite DHA during inflammatory reactions not only protects mesothelial cells from oxidative damage, but also contributes to maintaining the mesothelial barrier function. Consequently, the inflammatory pleural fluid may be trapped in pleural space and the inflammation may be restricted, and have extension avoided.     

GSH is required to recycle ascorbic acid in cultured liver cell lines

Liver is the site of ascorbic acid synthesis in most mammals. As human liver cannot synthesize ascorbate de novo, it may differ from liver of other species in the capacity or mechanism for ascorbate recycling from its oxidized forms. Therefore, we compared the ability of cultured liver-derived cells from humans (HepG2 cells) and rats (H4IIE cells) to take up and reduce dehydroascorbic acid (DHA) to ascorbate. Neither cell type contained appreciable amounts of ascorbate in culture, but both rapidly took up and reduced DHA to ascorbate. Intracellular ascorbate accumulated to concentrations of 10-20 mM following loading with DHA. The capacity of HepG2 cells to take up and reduce DHA to ascorbate was more than twice that of H4IIE cells. In both cell types, DHA reduction lowered glutathione (GSH) concentrations and was inhibited by prior depletion of GSH with diethyl maleate, buthionine sulfoximine, and phenylarsine oxide. NADPH-dependent DHA reduction due to thioredoxin reductase occurred in overnight-dialyzed extracts of both cell types. These results show that cells derived from rat liver synthesize little ascorbate in culture, that cultured human-derived liver cells have a greater capacity for DHA reduction than do rat-derived liver cells, but that both cell types rely largely on GSH- or NADPH-dependent mechanisms for ascorbate recycling from DHA.     

Effects of Red Wine Tannat on Oxidative Stress Induced by Glucose and Fructose in Erythrocytes in Vitro

The literature indicates that red wine presents in its composition several substances that are beneficial to health. This study has investigated the antioxidant effects of Tannat red wine on oxidative stress induced by glucose and fructose in erythrocytes in vitro, with the purpose to determine some of its majoritarian phenolic compounds and its antioxidant capacity. Erythrocytes were incubated using different concentrations of glucose and fructose in the presence or absence of wine. From these erythrocytes were determined the production of thiobarbituric acid reactive species (TBARS), glucose consumption, and osmotic fragility. Moreover, quantification of total phenolic, gallic acid, caffeic acid, epicatechin, resveratrol, and DPPH scavenging activity in wine were also assessed. Red wine showed high levels of polyphenols analyzed, as well as high antioxidant potential. Erythrocytes incubated with glucose and fructose had an increase in lipid peroxidation and this was prevented by the addition of wine. The wine increased glucose uptake into erythrocytes and was able to decrease the osmotic fragility of erythrocytes incubated with fructose. Altogether, these results suggest that wine leads to a reduction of the oxidative stress induced by high concentrations of glucose and fructose.     

Impaired glutathione metabolism in hemolytic anemia

During the delivery of oxygen by erythrocytes, highly reactive oxygen species such as superoxide anion arise. The presence of reactive species damages the cell constituents. Glutathione (GSH) functions to repair cells when they are attacked by oxidative stress. GSH is synthesized in erythrocytes and glutathione disulfide (GSSG) is transported outside the cells to maintain a high GSH/GSSG ratio. The redox cycle of GSH by glutathione reductase and glutathione peroxidase is closely related to G6PD. Hereditary enzyme deficiency related to GSH metabolism, with hemolytic anemia has been reported. G6PD deficiency causes hemolytic anemia due to insufficiency of the redox cycle of GSH. Deficiency of GSH synthesizing enzymes or glutathione reductase also causes hemolysis. Pyrimidine 5'-nucleotidase deficiency causes hemolytic anemia even when there is a high concentration of GSH. Accumulation of nucleotides in red cells causes inhibition of G6PD activity.     

Bovine retinal pericytes are resistant to glucose-induced oxidative stress in vitro

Diabetic retinopathy is a sight-threatening complication of diabetes, and loss of pericytes represents early signs of its development. We tested the hypothesis that high glucose levels may induce signs of oxidative stress in cultured bovine retinal pericytes. Pericytes were exposed to either normal (5.5 mM) or high (22 mM) glucose levels for 1, 3, and 5 days. Signs of oxidative stress were measured by expression of copper/zinc superoxide dismutase, manganese superoxide dismutase, catalase, and glutathione peroxidase using real-time RTPCR. To elucidate the role of oxidative stress, we also measured glutathione (GSH) concentration in the cells and investigated the impact of thiol-reactive metal ions and hydrogen peroxide (H(2)O(2)) on intracellular GSH. Despite the stimulation with high glucose, thiol-reactive metal ions, or H(2)O(2), there was no clear increased expression of antioxidant enzymes or influence of GSH levels. Lipid peroxidation (malondialdehyde level) was increased in bovine aortic smooth muscle cells, but not in bovine retinal pericytes. The data indicate that pericytes do not develop oxidative stress in response to hyperglycemia. However, it is not definitively excluded that oxidative stress may occur after longer time periods of glucose stimulation.     

α-硫辛酸、银杏叶提取物对波动高糖所致人视网膜色素上皮细胞损伤的作用

目的:研究α-硫辛酸(alpha lipoic acid,ALA)、银杏叶提取物(Gingko biloba,EGb761)对波动性高糖所致人视网膜色素上皮细胞(human retinal pigment epithelial,HRPE)氧化应激和炎性损伤的保护作用.方法:培养HRPE细胞株,取生长良好的对数期细胞用于实验,换用不同条件培养液,分10组进行试验.1)5.5 mmol/L葡萄糖对照组(N组);2)33 mmol/L持续高糖组(H组);3)5.5~33 mmol/L波动葡萄糖组(F组);4)33 mmol/L渗透压对照组(P组);5)ALA50干预组(ALA50组):5.5~33 mmol/L波动葡萄糖组+50μmol/L ALA;6)ALA100干预组(ALA100组):5.5~33 mmol/L波动葡萄糖组+100μmol/L ALA;7)ALA200干预组(ALA200组):5.5~33 mmol/L波动葡萄糖组+200μmol/L ALA;8)EGb7610.1干预组(EGb7610.1组):5.5~33 mmol/L波动葡萄糖组+0.1 g/L EGb761;9)EGb7610.5干预组(EGb7610.5组):5.5~33 mmol/L波动葡萄糖组+0.5 g/L EGb761;10)EGb7611.0干预组(EGb7611.0组):5.5~33 mmol/L波动葡萄糖组+1.0 g/L EGb761.各组细胞培养72 h,72 h时检测各组HRPE细胞上清液中SOD,GSH,MDA,TNF-α与ICAM-1的含量.采用SPSS18.0软件进行统计学分析.结果:与正常对照组相比,持续高糖组、波动性高糖组、ALA干预组(ALA50,ALA100,ALA200)、EGb761干预组(EGb7610.1,EGb7610.5,EGb7611.0)的SOD,GSH,MDA,TNF-α与ICAM-1含量差异有统计学意义(分别为FSOD=67.936,FGSH=71.363,FMDA=81.123,FTNF-α=70.684,FICAM-1=80.193;均P<0.05);渗透压对照组与正常对照组相比各指标差异均无统计学意义(P>0.05);波动性高糖组与持续性高糖组相比,SOD活性、GSH含量明显下降,MDA、ICAM-1与TNF-α的表达量明显升高(均P<0.05).ALA干预组(ALA50,ALA100,ALA200)、EGb761干预组(EGb7610.1,EGb7610.5,EGb7611.0)与波动性高塘组相比,各组有统计学差异,且随药物剂量增加,SOD活性、GSH含量增加,MDA,ICAM-1与TNF-α的表达量下降,存在剂量相关性(均P<0.05).结论:ALA,EGb761可能通过降低波动性高血糖对人HRPE所致氧化应激水平,减轻炎性反应,对波动性高血糖所导致的病理损伤有一定保护作用.     

Identification and characterization of an ascorbic acid transporter in human granulosa-lutein cells.

Ascorbic acid serves a vital role as a pre-eminent antioxidant. In animals, it has been shown to be concentrated in granulosa and theca cells of the follicle, in luteal cells of the corpus luteum, and in the peripheral cytoplasm of the oocyte. We have previously identified hormonally-regulated ascorbic acid transporters in rat granulosa and luteal cells, and herein present preliminary evidence for the presence of a transporter for ascorbic acid in human granulosa-lutein cells. Granulosa-lutein cells were obtained from the follicular fluid of patients undergoing in-vitro fertilization. Following an overnight incubation, the cells were incubated with [14C]-ascorbic acid (0.15 microCi; 150 microM) and ascorbic acid uptake was determined. The uptake of ascorbic acid was saturable with a Michaeli's constant (Km) and maximum velocity (Vmax) of 21 microM and 3 pmol/10(6) cells/min respectively. Ouabain, low Na+ medium, and dinitrophenol significantly inhibited ascorbic acid uptake (P<0.05). Neither the presence of insulin, human chorionic gonadotrophin (HCG), insulin-like growth factor (IGF)-I, nor IGF-II affected the uptake of ascorbic acid in a statistically significant fashion. Following saturation of cellular uptake, the ascorbic acid level was estimated to be 1.04 pmoles/10(6) cells or approximately 1 mM, a high concentration similar to that seen in rat luteal cells. Active ascorbic acid transport in human granulosa-lutein cells appears to occur via a Na+ - and energy-dependent transporter, with high levels of ascorbic acid being accumulated in these cells.     

DHA通过抑制PI3K通路和GLUT2表达而诱导NSCLC细胞毒性

目的:研究二氢青蒿素(dihydroartemisinin,DHA)诱导NSCLC细胞毒性的分子机制。方法:将不同浓度的DHA作用NSCLC细胞系A549和NCI-H1650细胞不同时间,运用MTT法、克隆形成实验、Annexin V染色和流式细胞术测定DHA对细胞活力、克隆形成能力和细胞凋亡的影响;同时测定DHA对细胞中葡萄糖水平、ATP和乳酸含量的影响;Western blot检测PI3K通路活性和GLUT2表达变化;通过细胞转染实现葡萄糖转运体2(GLUT2)和Rheb在A549和NCI-H1650细胞中高表达,测定和分析DHA作用下细胞活力、细胞凋亡、葡萄糖水平、ATP含量和PI3K通路活性的变化;分析葡萄糖缺乏对DHA诱导NSCLC细胞毒性的影响。结果:与对照组比较,DHA显著抑制A549和NCI-H1650细胞活力和克隆形成能力以及诱导细胞凋亡,同时降低ATP和乳酸含量以及抑制细胞对葡萄糖的摄取,具有时间和剂量依赖效应。Western blot结果显示,DHA能抑制PI3K通路活性和GLUT2的表达。上调GLUT2的表达和激活PI3K通路能减弱DHA对NSCLC细胞的毒性作用;葡萄糖缺乏能增强DHA对NSCLC细胞的毒性;相反,高浓度葡萄糖则抑制DHA对NSCLC细胞的毒性。结论:DHA能抑制NSCLC细胞活力和克隆形成,诱导细胞凋亡,该作用是通过降低PI3K活性和GLUT2的表达进而抑制细胞糖酵解代谢实现的。     

Regulation of vitamin C transporter in the type 1 diabetic mouse bone and bone marrow

A number of studies have revealed that Type I diabetes (T1D) is associated with bone loss and an increased risk of fractures. T1D induces oxidative stress in various tissues and organs. Vitamin C plays an important role in the attenuation of oxidative stress; however, little is known about the effect of T1D induced oxidative stress on the regulation of vitamin C transporter in bone and bone marrow cells. To investigate this, T1D was induced in mice by multiple low dose injections of streptozotocin. We have demonstrated that endogenous antioxidants, glutathione peroxidase (GPx) and superoxide dismutase (SOD) are down-regulated in the bone and bone marrow of T1D. The vitamin C transporter isoform SVCT2, the only known transporter expressed in bone and bone marrow stromal cells (BMSCs), is negatively regulated in the bone and bone marrow of T1D. The μCT imaging of the bone showed significantly lower bone quality in the 8 week T1D mouse. The in-vitro study in BMSCS showed that the knockdown of SVCT2 transporter decreases ascorbic acid (AA) uptake, and increases oxidative stress. The significant reversing effect of antioxidant vitamin C is only possible in control cells, not in knockdown cells. This study suggested that T1D induces oxidative stress and decreases SVCT2 expression in the bone and bone marrow environment. Furthermore, this study confirms that T1D increases bone resorption, decreases bone formation and changes the microstructure of bones. This study has provided evidence that the regulation of the SVCT2 transporter plays an important role not only in T1D osteoporosis but also in other oxidative stress-related musculoskeletal complications.     

Japanese Shiba dogs possessing erythrocytes with high Glut-1 activity and high ascorbic acid recycling capacity

Glut-1 was discovered in erythrocytes of some Japanese Shiba dogs. They have Glut-1 as well as Glut-4 in erythrocytes, while other Shiba dogs and other breeds have only Glut-4. Dog erythrocytes with Glut-1 showed much higher affinity for entry of glucose and oxidized ascorbic acid and greater capacity for ascorbic acid (AA) recycling than those with only Glut-4. Dog is among numerous AA-synthesizing species that normally do not have Glut-1 in erythrocytes. However, this variant of dog is the first example of an AA-synthesizing animal which maintains Glut-1 in erythrocytes throughout life. Shiba dogs can be a new animal model for further understanding the mechanism of regulation and functions of AA in the organism.     

血氧饱和度降低可促进红细胞氧化损伤

研究氧化应激条件下,血氧饱和度(SO2)对红细胞氧化损伤的影响。体外将红细胞孵育至SO2分别为0.3、0.5、0.7、0.9及0.98等状态,加入终浓度为0.15mmol/L叔丁基过氧化氢(BHP)使其发生氧化应激反应。测量胞内还原性谷胱甘肽(GSH)含量反映细胞抗氧化力,以胞内高铁血红蛋白(MetHb)、膜质过氧化(TBARS)及膜上沉淀的变性珠蛋白链水平体现细胞氧化损伤程度。结果显示,BHP存在的条件下,GSH含量随SO2上升而增加;MetHb、TBARS及膜上珠蛋白链水平则随SO2的上升而下降。上述结果表明,氧化应激条件下,红细胞抗氧化力和氧化损伤与SO2密切相关,SO2的降低可促进红细胞氧化损伤。     

Lutein and docosahexaenoic acid prevent cortex lipid peroxidation in streptozotocin-induced diabetic rat cerebral cortex

The mechanisms underlying diabetic encephalopathy, are largely unknown. Here, we examined whether docosahexaenoic acid (DHA) and lutein could attenuate the oxidative changes of the diabetic cerebral cortex. The levels of malondialdehyde (MDA) were significantly increased and glutathione (GSH) and glutathione peroxidase activity (GPx) were decreased in diabetic rats. The number of 4-hydroxynonenal (4-HNE) positive cells was increased. Treatment with insulin, lutein or DHA and the combination of each antioxidant with insulin, significantly restored all markers concentrations mentioned above, and the increase in 4-HNE inmunofluorescence. We combined 4-HNE immunofluorescence with NeuN (Neuronal Nuclei) staining. The latter demonstrated extensive overlap with the 4-HNE staining in the cortex from diabetic rats. Our findings demonstrate a clear participation of glucose-induced oxidative stress in the diabetic encephalopathy, and that the cells suffering oxidative stress are neurons. Lowering oxidative stress through the administration of different antioxidants may be beneficial for the central nervous tissue in diabetes.     

Uptake of ascorbic acid by freshly isolated cells and secretory granules from the intermediate lobe of ox hypophyses

Mechanically isolated cells from the intermediate lobe of ox hypophyses contained 40.6 +/- 3.7 nmol mg-1 protein (mean +/- SE, n = 5) of ascorbic acid. They accumulated radioactivity time dependently, on incubation with L-[14C]ascorbic acid in ionic medium dominated by NaCl. No definite saturation of uptake occurred when mechanically isolated cells were incubated with increasing ascorbic acid concentrations up to 0.6 mM. But if such cells were purified on a Percoll gradient, a clear saturation of uptake could be observed. Acetylsalicylic acid reduced the uptake markedly. When cells loaded with L-[14C]ascorbic acid were homogenized and placed on a Percoll gradient, the radioactivity was recovered in several subcellular fractions. Decrease of the Na+ concentration or presence of ouabain in the medium did not cause noticeable changes in uptake by non-purified cells, whereas uptake by purified cells was clearly sodium-dependent. Phloridzin inhibited uptake. Secretory granules from pars intermedia contained 40.0 +/- 3.8 nmol mg-1 protein of ascorbic acid (mean +/- SE, n = 3) and could accumulate L-[14C]ascorbic acid rapidly in a KCl-dominated medium. The uptake was not saturable with ascorbic acid concentration and was not influenced by the presence of I mM ATP + I mM Mg2+ in the medium. The concentration of copper and iron in isolated cells was comparable to that in isolated neurohypophysial nerve terminals, whereas the concentration of zinc was considerably higher in the pars intermedia cells. The concentration of Cu, Zn, Fe and Co in secretory granules from pars intermedia was higher than in secretory granules from neurohypophyses.     

Glutathione and ascorbic acid enhance recovery of Guinea pig spinal cord white matter following ischemia and acrolein exposure.

OBJECTIVE: We have shown that acrolein, a lipid peroxidation byproduct, can inflict significant damage in isolated spinal cord white matter following oxygen glucose deprivation (OGD). The mechanism of such acrolein-induced damage is unclear. The aim of this study was to examine whether glutathione (GSH) and ascorbic acid, two reactive oxygen species (ROS) scavengers, can alleviate functional and anatomical damage due to acrolein. METHODS: We used an OGD injury model with isolated guinea pig spinal cord white matter. Sucrose gap recording was used to monitor axonal impulse conduction, and a horseradish peroxidase exclusion test was employed to determine membrane integrity. The functional and anatomical parameters were compared in three groups: acrolein, acrolein/GSH and acrolein/ascorbic acid. RESULTS: We found that while GSH resulted in an 87% recovery of compound action potential conductance, ascorbic acid produced a 97% recovery, compared with a 69% recovery in an injured group without treatment. It is noted that GSH, and to a lesser extent ascorbic acid, preferentially enhanced functional recovery in smaller axons. CONCLUSION: Acrolein-induced neuronal damage is likely mediated by ROS. Furthermore, GSH and ascorbic acid are effective in suppressing acrolein and free radical-induced injury in spinal cord white matter.     

Effects of Oxidative Agents on the Erythrocytes from High GSH and Low GSH Sheep

The responses of sheep erythrocytes with high and low reduced glutathione (GSH) content were investigated under extracellular oxidative stress induced by 2,2′-azobis(amidinopropane) dihydrochloride (AAPH) and ferric ion. In terms of GSH depletion, haemolysis, lipid peroxidation and methaemoglobin formation, the extent of oxidant damage to high and low GSH sheep erythrocytes was remarkably similar. These results suggest that red cell GSH content per se is relatively unimportant in susceptibility of sheep erythrocytes to extracellular oxidative insults.