Uptake of dehydroascorbic acid in high-GSH and normal-GSH dog erythrocytes

Uptake of dehydroascorbic acid (DHA) was studied in two types of dog erythrocytes with high GSH and normal GSH levels. Compared with ascorbic acid uptake, DHA produced a much greater ascorbic acid accumulation in dog erythrocytes. Both dog erythrocytes showed a concentration dependence of DHA uptake, and cellular ascorbic acid concentrations were significantly higher in high-GSH cells than in normal-GSH cells. Glucose and cytochalasin B inhibited DHA uptake. This suggests that DHA enters dog erythrocytes predominantly by the facilitated glucose transporter, particularly by the Glut 1 glucose transporter. The rate of glucose uptake was quite similar in the two types of cells. Compared with normal-GSH cells, high-GSH cells were more resistant to oxidative stress induced by high concentration of DHA. As a rapid entry of DHA inflicts on cells a heavy demand for GSH for its reduction to ascorbic acid, high-GSH cells containing a larger reserve of GSH have an advantage over normal-GSH cells in both ascorbic acid accumulation and resisting oxidative stress produced by DHA.     

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.     

维生素C,皮质醇对正常人外周血单核细胞抗原提呈功能的影响

取健康男性献血员的肝素抗凝新鲜静脉血,密度梯度离心获单个核细胞(MNC),借粘附法和尼龙毛柱分离法分别获高纯度的单核细胞(Mon)和T淋巴细胞(T)。将Mon预先与纯化蛋白衍生物(PPD)和不同浓度的维生素C(VC)、皮质醇(HC)、VC HC共育24小时后加入T,借~3H-TdR掺入的T细胞增殖反应观察Mon向T提呈PPD的能力。结果表明,VC(3.75mM)对Mon的抗原提呈功能(APF)有显著的促进作用(P<0.01);而HC(5μg/ml)则有显著的抑制作用(P<0.01);二者并用时(浓度同上),VC能完全对消HC对Mon APF的抑制作用(P>0.05)。结果提示,VC可增强机体的特异性免疫功能,且能对消HC抑制免疫的副作用。     

The effect of ascorbic acid on cutaneous and nasal response to histamine and allergen

The effect of ascorbic acid (AA) on the skin wheal and flare response to histamine and allergen and on the nasal response to allergen was evaluated in eight adults with seasonal allergic rhinitis. The above parameters were measured after 3 days of AA administration (2 gm/day) and again after 3 days of a similar-appearing lactose placebo. An additional study was conducted in which six subjects took 0 (placebo), 1, 2, and 4 gm/day of AA to determine the dose-response effect of AA on histamine skin tests. Overall there was no difference in skin or nasal reactivity between AA and placebo regimens. The findings in this study suggest that AA in relatively high doses would have no beneficial effects on symptoms resulting from allergen exposure and that AA in doses of up to 4 gm/day will not suppress the histamine skin response.     

Sodium-dependent ascorbic acid transporter family SLC23

l-Ascorbic acid (vitamin C) is an effective antioxidant and an essential cofactor in numerous enzymatic reactions. Two Na(+)-dependent vitamin C transporters (SVCT1 and SVCT2) are members of the SLC23 human gene family, which also contains two orphan members. SVCT1 and SVCT2 display similar properties, including high affinity for l-ascorbic acid, but are discretely distributed. SVCT1 is confined to epithelial systems including intestine, kidney, and liver, whereas SVCT2 serves a host of metabolically active and specialized cells and tissues including neurons, the eye, lung, and placenta, and a range of neuroendocrine, exocrine, and endothelial tissues. An SVCT2-knockout mouse reveals an obligatory requirement for SVCT2, but many of the specific roles of this transporter remain unclear.     

Metabolism in the erythrocytes of dogs with low and high glutathione concentrations

Metabolism of the erythrocytes of high potassium and high glutathione (HK-high GSH), high potassium and low GSH (HK-low GSH) and normal low potassium and low GSH (LK-low GSH) dogs were studied with respect to the levels of enzymes associated with GSH synthesis and metabolism, and the capacity of the cells to regenerate GSH. HK-high GSH dogs were found to have significantly higher levels of hexokinase (Hx) and -glutamylcyclotransferase (GCT) than low GSH groups. The rate of GSH regeneration was much greater in high GSH dogs than in those of low GSH groups, and there is a positive correlation between GSH level and its, regeneration rate. Glutathione-S-transfer-ase (GST) activity was high in both HK-high GSH and HK-low GSH cells. There were no significant differences in the activities of -glutamyl cysteine synthetase (GCS), glutathione synthetase (GSHS) and glutathione reductase (GR) among these three groups. Our results indicate that GSH levels and Hx activity influence GSH regeneration rate, and the GSH levels also affect GST activity.     

HPLC法同时测定对乙酰氨基酚、咖啡因、盐酸麻黄碱和维生素C的含量

目的：建立同一色谱条件下对乙酰氨基酚、咖啡因、盐酸麻黄碱和维生素C含量的HPLC测定法。方法：采用高效液相色谱法,以Capcell-pak C18为色谱柱,流动相为乙晴/庚烷磺酸钠溶液缓冲液（30/70）,检测波长为210nm,流速1.0ml.min-1,采用外标法计算含量。结果：对乙酰氨基酚、咖啡因、盐酸麻黄碱和维生素C的分离度好,乙酰氨基酚在15mg.100ml-1～120mg.100ml-1范围内线性关系良好（r=0.9999）;咖啡因在6.25mg.100ml-1～50mg.100ml-1范围内线性关系良好（r=0.9994）;盐酸麻黄碱在6.25mg.100ml-1～50mg.100ml-1范围内线性关系良好（r=0.9998）;维生素C在40mg.100ml-1～320mg.100ml-1范围内线性关系良好（r=0.9994）。实验的精确度和稳定性良好,样品回收率高。结论：本方法操作简单,结果准确,可以有效地控制该类制剂的质量。     

Blood cholesterol profile in guineapigs: Effect of ascorbic acid ingestion on hypercholesterolemia induced by manganese deficiency or overdosage of an anti-thyroid agent in diet

Effects of ascorbic acid ingestion (0.25 g/day) on serum cholesterol and total lipid levels were compared in three hyperlipidemic guineapig models. The first model received a diet rich in atherogenic and thrombogenic agents (e.g. cholesterol, butter-fat, cholic acid, vitamin D₂ etc.). The second (hypothyroid) and third (deficient manganese) models were created by feeding excess propyl-thiouracil (a potent goitrogen) and low-level manganese without added dietary cholesterol. The extreme rapidity with which guineapigs of Wistar strain develop hypercholesterolemia and early atherosclerotic lesions makes them an attractive animal model for studying the early development of atherosclerosis in man.

Decreased serum and tissue cholesterol levels despite more dietary cholesterol and butter-fat indicate that ascorbic acid is a good hypolipidemic/hypocholesterolemic agent. As the lipid-lowering property of ascorbic acid is obliterated due to hypothyroidism, it is most probable that vitamin C may act through the thyroid gland. Interaction with thyroid hormones seems not unlikely for ascorbic acid under physiological conditions. Since ascorbic acid has been suggested to provide protection against atherosclerosis induced by atherogenic/thrombogenic agents and proved incoherent in the face of thyroid dysfunction and manganese deprivation, the precise relation of these effects to lipid metabolism warrants investigation in some other fields.     

ATP levels and glutathione regeneration in canine erythrocytes

The effect of ATP levels on GSH regeneration was examined in canine erythrocytes. The main findings were: (1) The GSH regeneration was dependent on glucose and ATP; (2) cytochalasin B and polymyxin B, both glucose transport inhibitors, reduced ATP synthesis and GSH regeneration; (3) inosine, a substrate of the salvage pathway, was not effective for ATP synthesis and GSH regeneration. These results indicate that glucose transport and its metabolism play an important role in oxidant defence systems in general and GSH regeneration in particular in canine erythrocytes.     

The protective role of ascorbic acid on hippocampal CA1 pyramidal neurons in a rat model of maternal lead exposure

Oxidative stress is a major pathogenic mechanism of lead neurotoxicity. The antioxidant ascorbic acid protects hippocampal pyramidal neurons against cell death during congenital lead exposure; however, critical functions like synaptic transmission, integration, and plasticity depend on preservation of dendritic and somal morphology. This study was designed to examine if ascorbic acid also protects neuronal morphology during developmental lead exposure. Timed pregnant rats were divided into four treatment groups: (1) control, (2) 100 mg/kg ascorbic acid once a day via gavage, (3) 0.05% lead acetate in drinking water, and (4) 0.05% lead + 100 mg/kg oral ascorbic acid. Brains of eight male pups (P25) per treatment group were processed for Golgi staining. Changes in hippocampal CA1 pyramidal neurons’ somal size were estimated by cross-sectional area and changes in dendritic arborization by Sholl’s analysis. One-way ANOVA was used to compare results among treatment groups. Lead-exposed pups exhibited a significant decrease in somal size compared to controls (P < 0.01) that was reversed by cotreatment with ascorbic acid. Sholl’s analysis revealed a significant increase in apical dendritic branch points near cell body (P < 0.05) and a decreased total dendritic length in both apical and basal dendritic trees of CA1 neurons (P < 0.05). Ascorbic acid significantly but only partially reversed the somal and dendritic damage caused by developmental lead exposure. Oxidative stress thus contributes to lead neurotoxicity but other pathogenic mechanisms are also involved.     

“In vitro” improvement of defective monocyte chemotaxis in intravenous drug abusers after incubation with ascorbic acid

Summary Intravenous drug abusers have a deficit of monocyte chemotaxis. In vitro ascorbic acid restores the impaired chemotaxis.Abbreviations CI Chemotactic index - EAS Endotoxin-activating serum - IDA Intravenous drug abusers - PBM Peripheral blood monocytes     

The role of ascorbic acid on titanium dioxide-induced genetic damage assessed by the comet assay and cytogenetic tests

Titanium dioxide (TiO₂) is used in several commercial products such as cosmetics, sunscreen, toothpaste and pharmaceuticals. However, some recent investigations have revealed that titanium particles generate potential harmful effects on the environment and humans. Because of its strong antioxidant activity, ascorbic acid (AA) is admitted to act as an anti-mutagenic agent. The present study was undertaken to investigate the protective effect of AA against TiO₂-induced genotoxicity. Sister chromatid exchange (SCE), micronucleus (MN) and the comet assays were used to assess TiO₂-induced genotoxicity and to establish the protective effects of AA. There were significant increases (P<0.05) in both SCE and MN frequencies of cultures treated with TiO₂ as compared to controls. However, co-application of AA (4.87 and 9.73 μM) and TiO₂ resulted in decreases of SCE and MN rates as compared to the group treated with titanium alone. Besides, significant reductions of primary DNA damage (comet assay) were determined when the AA was added to the cell culture medium simultaneously with TiO₂. In conclusion, the preventive role of AA in alleviating TiO₂-induced DNA damage was indicated for the first time in the present study.     

Transient erythrocytosis (Primary polycythaemia) in a dog

The clinical findings in a dog with transient erythrocytosis (primary polycythaemia) are described. The dog was initially presented for neurological signs induced by exercise. The diagnosis was based on PCV (74%), clinically normal hydration, normal arterial blood gas determination, and exclusion of reported causes of secondary polycythaemia. Serum erythropoietin concentration was normal. Phlebotomy decreased the PCV to 45%. During the next 18 months, the PCV remained in the normal range without further treatment, and there was no recurrence of clinical signs.     

维生素C对大鼠肋骨生长板软骨细胞增殖和胶原合成的影响

探讨不同浓度的维生素C(AscorbicAcid ,Asc)对培养大鼠肋生长板软骨细胞 (ratcostochondralgrowthplatechondrocyte,RGC)增殖和胶原合成的影响。分离、培养RGC ,以组织化学和免疫组织化学的方法鉴定第 2代RGC的表型 ;分别用3 H TdR和3 H Proline掺入法检测 2 5、5 0和 10 0mg/LAsc对第 2代RGC增殖和胶原合成的影响。3种浓度的Asc均能促进RGC胶原合成 (P <0 0 5 ) ,2 5mg/L和 5 0mg/LAsc的促胶原合成作用明显强于 10 0mg/L(P <0 0 5 ) ;2 5mg/L和 5 0mg/L的Asc促进RGC增殖 (P <0 0 5 ) ,10 0mg/L的Asc抑制RGC的增殖 (P <0 0 5 )。因此一定浓度的Asc具有促进RGC增殖和胶原合成的作用 ,2 5mg/L～ 5 0mg/L是较为合适的添加浓度。     

Ascorbic acid alleviates pancreatic damage induced by dibutyltin dichloride (DBTC) in rats

PURPOSE: Because previous studies have reported depleted antioxidant capacity in patients with chronic pancreatitis (CP), prevention of free radical production has gained importance in antifibrotic treatment strategies for CP. The aim of this study was to investigate the effects of ascorbic acid on oxidative capacity and pancreatic damage in experimental CP. MATERIALS AND METHODS: CP was induced in male Sprague-Dawley rats by infusion of dibutyltin dichloride (DBTC) into the tail vein. Ascorbic acid was given intraperitoneally at a daily dose of 10 mg/kg body weight. The treatment groups were as follows: group 1, DBTC plus intraperitoneal physiologic saline; group 2, DBTC plus intraperitoneal ascorbic acid; group 3, solvent plus intraperitoneal physiologic saline; group 4, no operation plus intraperitoneal physiologic saline. Each group contained 15 animals. Treatment was started after CP was established. After 4 weeks of treatment, serum hyaluronic acid and laminin levels were determined by radioimmunoassay, pancreatic tissue oxidative stress was analyzed, and the degree of pancreatic damage was determined. RESULTS: Ascorbic acid treatment markedly increased superoxide dismutase (SOD) activity and decreased malondialdehyde (MDA) concentrations in pancreatic tissue (p < 0.01 for both). Significant serum hyaluronic acid and laminin reductions were observed in group 2 as compared with group 1 (p < 0.05). However, the serum hyaluronic acid and laminin levels remained elevated when compared with those of groups 3 and 4 (p < 0.05). Histopathologic scores were also lower in animals with CP that underwent ascorbic acid-treatment (p < 0.05). CONCLUSION: Ascorbic acid treatment alleviated the degree of oxidative stress and pancreatic damage in rat CP. Antioxidant treatment might be considered a potential option to improve the pathologic process in CP.     

Effects of fasting on haematology and clinical chemistry values in the rat and dog

Many regulatory toxicity guidelines and the recommendation of AACC-DACC/ASVCP joint task force of the USA on clinical pathology testing require overnight fasting for rats and non-rodents before blood sampling. However, the reason why animals must be fasted before blood sampling is unclear in toxicology studies.Fasting, one of many preanalytical conditions, can lead to false low or high values, which in turn may lead to misinterpretation of test compound effects in toxicological studies. This paper reviews the literature with respect to fasting, and reports on our own studies, in the hope of increasing the awareness among investigators of these problems.Haematocrit values and plasma chemistry values in blood obtained from rats and dogs following fasting were compared with unfasted animals. In male F344 rats, after 16 h fasting, body weight decreased. Increases in aspartate aminotransferase (AST)/glutamic oxaloacetic transaminase (GOT) and decreases of plasma alkaline phosphatase (ALP), total cholesterol (CHO), triglycerides (TG), phospholipids (PL), urea nitrogen (UN) and calcium were observed. Haematocrit, plasma alanine aminotransferase (ALT)/glutamic pyruvic transaminase (GPT), total proteins (TP), glucose, and inorganic phosphorus (IP) were unchanged. In male beagle dogs after 16 h fasting, TG, PL, UN, calcium and IP were decreased. Haematocrit, ALP, TP, albumin, glucose, CHO, creatinine, AST/GOT, ALT/GPT, LDH and CPK were not changed.Our own studies show that in order to avoid excessive stress to test animals, the fasting period should be decided case by case, and not made uniform in toxicology studies. It would be useful if regulatory guidelines made some mention of both the effect of feeding, and of stress caused by fasting.     

Comparison of glutamate metabolism in low K (LK), high K and high glutathione (HK/HG) and high K and low glutathione (HK/LG) dog red blood cells

The reasons for the high accumulation of glutamate (Glu), aspartate (Asp) and glutamine (Gin) in high K and high glutathione (HK/HG) dog red blood cells (DRBCs) have been explained as due to enhanced Glu/Asp influxes. However, in our study, Glu/Asp influxes in high K and low glutathione (HK/LG) DRBCs were low, whereas their cellular Asp and Gin contents were high. In low K (LK) DRBCs, there were also other variant cells with high Asp accumulation, but extremely low Glu/Asp influxes. So, the high amino acid accumulation in DRBCs of these new variants might not be due to Glu/Asp influxes. To examine the high accumulation of these amino acids in these variant DRBCs, first, LK and HK/LG DRBCs were classified into two subgroups with their Na-dependent Glu/Asp influxes; one had clear Na-dependent Glu/Asp transport (GAT⁺), and the other failed to have any transport (GAT⁻). The influxes of both Glu and Asp in HK/HG DRBCs were the highest, and the order was HK/HG>LK/GAT⁺>HK/LG/GAT⁺>>LK/GAT⁻=HK/LG/GAT⁻. LK/GAT⁺ cells represented normal DRBCs. Glu/Asp influxes were only trace in both LK/GAT⁻ and HK/LG/GAT⁻ cells, but Glu and Asp concentration was high in HK/LG/GAT⁻ cells whereas Asp concentration was high in LK/GAT⁻ cells. In HK/HG cells, the conversion of Glu into Gin in whole cells was several fold higher than in the other cell groups due to the differing amount of the substrate of glutamine synthetase, Glu, but glutamine synthetase activity itself was not different among these cell groups. Furthermore, glutamine synthetase and glutaminase activities were not different among the cell groups. Therefore, these enzymes were not involved in the high amino acid accumulation.     

Comparison of glutamate metabolism in low K (LK), high K and high glutathione (HK/HG) and high K and low glutathione (HK/LG) dog red blood cells

The reasons for the high accumulation of glutamate (Glu), aspartate (Asp) and glutamine (Gin) in high K and high glutathione (HK/HG) dog red blood cells (DRBCs) have been explained as due to enhanced Glu/Asp influxes. However, in our study, Glu/Asp influxes in high K and low glutathione (HK/LG) DRBCs were low, whereas their cellular Asp and Gin contents were high. In low K (LK) DRBCs, there were also other variant cells with high Asp accumulation, but extremely low Glu/Asp influxes. So, the high amino acid accumulation in DRBCs of these new variants might not be due to Glu/Asp influxes. To examine the high accumulation of these amino acids in these variant DRBCs, first, LK and HK/LG DRBCs were classified into two subgroups with their Na-dependent Glu/Asp influxes; one had clear Na-dependent Glu/Asp transport (GAT⁺), and the other failed to have any transport (GAT⁻). The influxes of both Glu and Asp in HK/HG DRBCs were the highest, and the order was HK/HG>LK/GAT⁺>HK/LG/GAT⁺>>LK/GAT⁻=HK/LG/GAT⁻. LK/GAT⁺ cells represented normal DRBCs. Glu/Asp influxes were only trace in both LK/GAT⁻ and HK/LG/GAT⁻ cells, but Glu and Asp concentration was high in HK/LG/GAT⁻ cells whereas Asp concentration was high in LK/GAT⁻ cells. In HK/HG cells, the conversion of Glu into Gin in whole cells was several fold higher than in the other cell groups due to the differing amount of the substrate of glutamine synthetase, Glu, but glutamine synthetase activity itself was not different among these cell groups. Furthermore, glutamine synthetase and glutaminase activities were not different among the cell groups. Therefore, these enzymes were not involved in the high amino acid accumulation.     

Sensory defects caused by lesions of the first (SI) and second (SII) somatosensory areas of the dog

Summary Dogs were trained to respond to light tactile stimulation of one of two places on either hindlimb and to flashes of light. One stage or sequential, uni- and/or bilateral lesions were then made in SI and/or SII. The lesions encompassed the hindlimb projection areas, and were placed on the basis of electrophysiological mapping made during surgery. Defects attributable to specific somatosensory deficits were observed after lesions involving SII. No such defects were observed after lesions of SI alone. The defects caused by SII lesions were enhanced by previous or simultaneous lesions of SI. The results suggest that SII rather than SI is of primary importance for the recognition of tactile stimuli, but that SI also may be involved in the tactile conditioned reflex, perhaps as a focus for sensory-motor feedback loops.     

Effects of topical allicin on second-intention wound healing in dogs (histological aspects)

Alllicin is one of the pharmacologically active garlic sulfur compounds that have antimicrobial (antibacterial, antiviral, antifungal and antiparasitic) and vasodilating effects. Five normal, male, mixed-breed dogs were selected to investigate the effects of allicin (5 mg/ml in methyl cellulose gel) as a topical treatment for full-thickness, excisional wounds. The dogs were approximately 3 years old. The histological aspects of second-intention wound healing were studied. Eight full-thickness skin wounds (20×20 mm) were created on the back of each dog. On days 0, 7, 14 and 21, each dog received two wounds, symmetrically, and were assigned to one of two groups: control (methyl cellulose gel) or test (allicin 5 mg/ml methyl cellulose gel). Wounds were treated once daily for a week. Left-side wounds were treated with allicfin (test group) and right-side wounds were treated with methylcellulose gel (control group). At day 28 (4 weeks) after initial wounding, biopsies were taken from wounds for histological examination. The density of inflammatory cells in the center of the day 7 wounds was significantly lower in test group (P=0.041), but the density of fibrocytes and fibroblasts in the center of day 7 wounds was significantly higher in the test group (P=0.042). No significant differences were observed in the amount of collagen and fibrin between the test and control wounds (P>0.05).