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Plasma vitamin C concentrations fluctuate in response to recent dietary intake; therefore levels are typically determined in the fasting state. Erythrocyte ascorbate concentrations have been shown to be similar to plasma levels, but little is known about the kinetics of ascorbate accumulation in these cells. In this study, we investigated ascorbate uptake into erythrocytes after dietary supplementation with vitamin C and compared it to changes in plasma ascorbate concentrations. Seven individuals with baseline fasting plasma vitamin C concentrations ≥ 50 µmol/L were depleted of vitamin C-containing foods and drinks for one week, and then supplemented with 250 mg vitamin C/day in addition to resuming their normal diet. Fasting or steady-state plasma ascorbate concentrations declined to almost half of their baseline concentration over the week of vitamin C depletion, and then returned to saturation within two days of beginning supplementation. Erythrocyte ascorbate concentrations exhibited a very similar profile to plasma levels, with values ~76% of plasma, and a strong linear correlation (r = 0.89, p < 0.0001). Using a pharmacokinetic study design in six individuals with baseline fasting plasma vitamin C concentrations ≥50 µmol/L, we also showed that, unlike plasma, which peaked between 2 and 4 h following ingestion of 200 mg of vitamin C, erythrocyte ascorbate concentrations did not change in the six hours after supplementation. The data from these two intervention studies indicate that erythrocyte ascorbate concentration provides a stable measure of steady-state plasma ascorbate status and could be used to monitor ascorbate status in healthy non-fasting individuals.

Chronic inflammatory diseases are the major causes of mortality in humans and recent research has improved our understanding of the major impact of life-style factors upon inflammatory diseases and conditions. One of the most influential of these is nutrition, which may drive both pro-inflammatory as well as anti-inflammatory cascades at molecular and cellular levels. There are a variety of model systems that may be employed to investigate the impact of micronutrients and macronutrients upon inflammatory pathways, many of which operate through oxidative stress, either at the level of controlling the redox state of the cell and downstream redox-regulated gene transcription factors, and other acting as free radical generating or scavenging agents. This chapter focuses upon biological sample preparation prior to assay and details methods for analyzing certain antioxidant micronutrients and biomarkers of oxidative stress.

The objective of this study was to investigate the effects of the hot summer season on plasma glucose and oxidative stress markers. For two 14-day experimental periods, namely periods 1 (July-August) and 2 (October-November), 12 and 14 lactating dairy cows, respectively, that were milked using an automatic milking system, were fed diets containing similar ingredients, and their milk production, plasma metabolites and oxidative status markers were investigated. Dry matter intake and milk yield were not affected by the experimental period. Rectal temperature at 18.00 hours and milk protein concentration in period 1 were higher and lower, respectively, than in period 2 (P < 0.05), suggesting that the hot summer season had an effect on the experimental dairy cows. Plasma glucose and the ascorbic acid + dehydroascorbic acid (AA) concentrations in period 1 were lower than in period 2 (P < 0.01). The plasma malondialdehyde (MDA) concentration did not differ between the experimental periods. The increase in the cellular AA uptake in peripheral tissues in period 1 might be a possible compensatory mechanism to balance the occurrence of reactive oxygen species and the antioxidant capacity in the cells, resulting in the absence of an effect of the hot summer season on plasma MDA concentration. © 2016 Japanese Society of Animal Science., (© 2016 Japanese Society of Animal Science.)

Objective: The aim is to evaluate intrapartum fetal oxidative stress in real-time by umbilical cord blood dimethyl sulfate (DMSO)-induced ascorbyl-free radical (AFR) measured by an electron spin resonance (ESR) method., Methods: Seventy-five mothers delivering at gestational age after 37 weeks were recruited. They were divided into three groups: spontaneous vaginal birth (n = 27), elective cesarean section (CS) (n = 34), and emergency CS due to non-reassuring fetal status (n = 14). Umbilical artery (UA) and venous (UV) cord blood gas analysis was performed. Serum levels of DMSO-induced AFR (AFR/DMSO) that reflect vitamin C concentrations were measured by ESR spectroscopy., Results: Blood gas analysis showed no significant differences among the groups. UA-AFR/DMSO level of elective CS group was significantly lower compared with spontaneous delivery group (0.32 ± 0.12 versus 0.46 ± 0.14, p < 0.005). Emergency CS group showed significantly lower levels of UA-AFR/DMSO compared with elective CS group (0.25 ± 0.11 versus 0.32 ± 0.12, p < 0.005). UV-AFR/DMSO levels had no significant difference among the groups., Conclusions: It is suggested that fetal cord blood AFR/DMSO is a sensitive marker to assess fetal oxidative stress during delivery.

Objective: The use of cardiopulmonary bypass (CPB) is suggested to induce oxidative stress, reflected by an imbalance between prooxidant and antioxidant substances. The majority of studies published have either focused on only one aspect (prooxidant or antioxidant side) or covered only a short observation period. Therefore, the aim of this study was to investigate the long-term effects of CPB on the balance of prooxidative markers and antioxidant substances in one single group of patients, being able to estimate the degree of oxidative stress., Methods: Blood samples were taken from 29 patients undergoing cardiovascular surgery beginning the day before surgery through postoperative day 6 (discharge). Plasma concentrations of vitamins C (total ascorbic acid) and E and malondialdehyde were measured by high-performance liquid chromatography. Plasma levels of ascorbyl free radical were determined using electron paramagnetic resonance spectroscopy., Results: The study showed a significant decrease in vitamin C plasma levels during CPB without any recovery of vitamin C up to the time of discharge. Furthermore, CPB induced a significant increase in malondialdehyde plasma concentrations immediately after unclamping, accompanied by a significant increase in the ascorbyl free radical to total ascorbic acid ratio. The latter stayed elevated until the end of observation., Conclusions: Our findings indicate that the oxidative stress event after CPB can be divided into two phases: Immediately after reperfusion, a massive oxidative stress occurs, reflected by the increase in malondialdehyde. During convalescence, there must be an ongoing situation of oxidative stress, especially in the water-soluble compartment, leading to the consumption of vitamin C. Because the main antioxidant substance, vitamin C, did not increase again over the entire observation period, supplementation should be given consideration., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Recently, many clinical reports have suggested that the ascorbyl free radical (Asc(∙)) can be treated as a noninvasive, reliable, real-time marker of oxidative stress, but its generation mechanisms in human blood have rarely been discussed. In this study, we used upstream substances, enzyme inhibitors, and free radical scavengers to delineate the mechanisms of Asc(∙) formation in human platelet-rich plasma (PRP). Our results show that the doublet signal was detected in PRP samples by using electron spin resonance, and the hyperfine splitting of the doublet signal was a(H) = 1.88 gauss and g-factor = 2.00627, which was determined to be the Asc(∙). We observed that the inhibitors of NADPH oxidase (NOX), cyclooxygenase (COX), lipoxygenase (LOX), cytochrome P450 (CYP450), mitochondria complex III, and nitric oxide synthase (NOS), but not xanthine oxidase, diminished the intensity of the Asc(∙) signal dose dependently. All enzyme inhibitors showed no obvious antioxidant activity during a Fenton reaction assay. In summary, the obtained data suggest that Asc(∙) formation is associated with NOX, COX, LOX, CYP450, eNOS, and mitochondria in human PRP.

Intravenous laser blood irradiation (ILBI) is widely applied in the treatment of different pathologies including diabetes mellitus. The aim of this study is to evaluate the effects of ILBI on the metabolites of blood in diabetic type 2 patients using metabolomics. We compared blood samples of nine diabetic type 2 patients, using metabolomics, before and after ILBI with blue light laser. The results showed significant decrease in glucose, glucose 6 phosphate, dehydroascorbic acid, R-3-hydroxybutyric acid, L-histidine, and L-alanine and significant increase in L-arginine level in blood and blood sugar in the patients have reduced significantly (p < 0.05). This study clearly demonstrated a significant positive effect of ILBI on metabolites of blood in diabetic type 2 patients. These findings support the therapeutic potential of ILBI in diabetic patients.

Tetrahydrobiopterin (BH₄) is an essential co-factor of nitric oxide synthases and is easily oxidized to dihydrobiopterin (BH₂) which promotes endothelial nitric oxide synthase uncoupling and deleterious superoxide production. Vitamin C has been shown to improve endothelial function by different mechanisms, some involving BH₄. The hypothesis of the present study was that vitamin C status, in particular low levels, influences biopterin redox status in vivo. Like humans, the guinea pig lacks the ability to synthesize vitamin C and was therefore used as model. Seven day old animals (n = 10/group) were given a diet containing 100, 250, 500, 750, 1000, or 1500 ppm vitamin C until euthanasia at age 60-64 days. Blood samples were drawn from the heart and analyzed for ascorbate, dehydroascorbic acid (DHA), BH₄ and BH₂ by high-performance liquid chromatography. Plasma BH₄ levels were found to be significantly lower in animals fed 100 ppm vitamin C compared to all other groups (P < .05 or less). BH₂ levels were not significantly different between groups but the BH₂-to-BH₄ ratio was higher in the group fed 100 ppm vitamin C (P < .001 all cases). Significant positive correlations between BH4 and ascorbate and between BH₂-to-BH₄ ratio and DHA were observed (P < .0001 both cases). Likewise, BH₂-to-BH₄ ratio was negatively correlated with ascorbate (P < .0001) as was BH₄ and DHA (P < .005). In conclusion, the redox status of plasma biopterins, essentially involved in vasodilation, depends on the vitamin C status in vivo. Thus, ingestion of insufficient quantities of vitamin C not only leads to vitamin C deficiency but also to increased BH₄ oxidation which may promote endothelial dysfunction., (© 2013 Elsevier Inc. All rights reserved.)

Manufacturers have developed prototype cigarettes yielding reduced levels of some tobacco smoke toxicants, when tested using laboratory machine smoking under standardised conditions. For the scientific assessment of modified risk tobacco products, tests that offer objective, reproducible data, which can be obtained in a much shorter time than the requirements of conventional epidemiology are needed. In this review, we consider whether biomarkers of biological effect related to oxidative stress can be used in this role. Based on published data, urinary 8-oxo-7,8-dihydro-2-deoxyguanosine, thymidine glycol, F2-isoprostanes, serum dehydroascorbic acid to ascorbic acid ratio and carotenoid concentrations show promise, while 4-hydroxynonenal requires further qualification.

Objective: To study the effects and immunoregulation mechanism of the traditional Mongolian medicine Wuweifengshi capsule on adjuvant arthritis (AA)., Method: Wister rats were divided into several groups: normal group, AA model group, Wuweifengshi capsule groups (with low, moderate, high dose of 0.2, 0.4, 0.8 g x kg(-1) x d(-1) respectively), and Zhonglun-5 group (original dose of 1.68 g x kg(-1) x d(-1)). The edema degree, the level of IL-1beta, TNF-alpha, PGE2, NO and MDA and the activity of SOD in serum were detected. Through cell culture, the effects of the medicine on AA rat's splenic cell's multiplication capacity were studied. The influence of celiac macrophage cell culture fluid of AA rats' on C57BL/6J mice thymic cell multiplication capacity under the medicine was evaluated., Result: Wuweifengshi capsule showed an inhibiting function on the level of IL-1beta, TNF-alpha, PGE2, NO and increased the activity of SOD in serum, but showed no significant influence on MDA. It also inhibited the AA rat's splenic cell's multiplication capacity and the influence of celiac macrophage cell culture fluid of AA rat's on C57BL/6J mice thymic cell multiplication capacity., Conclusion: The anti-AA effect of Wuweifengshi capsule is possibly due to its inhibition of relevant cytokines and its adjustment of corresponding enzyme's activity and immunization organ's cell multiplication capacity.

Skeletal muscles are the largest organs in the human body, and several therapeutic trials have been conducted that included stem cell transplantation to regenerate damaged or wasted muscles. It is well known that it is essential to make a favorable microenvironment (stem cell niche) to induce the proper differentiation of the transplanted stem cells. Some drugs, such as losartan (angiotensin II type I blocker), enhance the therapeutic effects of transplanted stem cells by inhibiting fibrosis. In this study, we hypothesized that another substance, vitamin C (ascorbic acid), might improve the niche for stem cell transplantation based on its potent antioxidant effects. In both gross and microscopic observations, vitamin C-depleted mice exhibited more incomplete regeneration of damaged muscles than those treated with vitamin C. Carbonylated protein groups, which are the end products of oxidative stress, were detected in all experimental groups; however, the vitamin C-depleted groups exhibited a more potent positive reaction than that of the vitamin C-supplied groups. The difference is clearer in the presence of transplanted stem cells. Moreover, the serum total vitamin C level and the ascorbic acid (AA) to dehydroascorbic acid (DHA) ratio also were decreased in the presence of transplanted adipose-derived stem cells (ASCs). Taken together, these data can be considered as proof of vitamin C utilization by cells in vivo. The vitamin C-supplied groups displayed more severe fibrosis than that of the vitamin C-depleted groups. Since vitamin C is a major cofactor for the collagen synthesis, its deficiency resulted in reduced fibrosis. In conclusion, we demonstrated that vitamin C not only has a positive effect on adjusting the stem cell niche to boost muscle regeneration but also has an adverse aspect due to its profibrotic effect.

Kinin-vasoactive peptides activate two G-protein-coupled receptors (R), B(1)R (inducible) and B(2)R (constitutive). Their complex role in cardiovascular diseases could be related to differential actions on oxidative stress. This study investigated impacts of B(1)R or B(2)R gene deletion in mice on the cardiac function and plasma antioxidant and oxidant status. Echocardiography-Doppler was performed in B(1)R (B(1)R(-/-)) and B(2)R (B(2)R(-/-)) deficient and wild type (WT) adult male mice. No functional alteration was observed in B(2)R(-/-) hearts. B(1)R(-/-) mice had significantly lowered fractional shortening and increased isovolumetric contraction time. The diastolic E and A waves velocity ratio was similar in all mice groups. Thus B(1)R(-/-) mice provide a model of moderate systolic dysfunction, whereas B(2)R(-/-) mice displayed a normal cardiac phenotype. Plasma antioxidant capacity (ORAC) was significantly decreased in both B(1)R(-/-) and B(2)R(-/-) mice whereas the vitamin C levels were decreased in B(2)R(-/-) mice only. Plasma ascorbyl free radical was significantly higher in B(1)R(-/-) compared to WT and B(2)R(-/-) mice. Therefore, the oxidative stress index, ascorbyl free radical to vitamin C ratio, was increased in both B(1)R(-/-) and B(2)R(-/-) mice. Hence, B(1)R and B(2)R deficiency are associated with increased oxidative stress, but there is a differential imbalance between free radical production and antioxidant defense. The interrelationship between the differential B(1)R and B(2)R roles in oxidative stress and cardiovascular diseases remain to be investigated.

HIV disease results in decreased IL-7 receptor expression and IL-7 responsiveness in T cells. To explore mechanisms of these deficiencies, we compared CD127 expression and IL-7 induction of P-STAT5 in T cells from HIV-infected persons with serum concentrations of cytokines (IL-7, IL-6 and IL-15), markers of microbial translocation (sCD14 and LPS), and with an indicator of oxidative stress (malondialdehyde (MDA) adducts). CD127 expression was directly related to IL-7 responsiveness in most CD8+ T cell subsets but not in CD4+ T cells from HIV-infected persons. MDA adducts were increased in serum of HIV-infected patients and were inversely related to IL-7 responsiveness in CD8+ T cells and in central memory CD4+ T cells. Incubation of T cells from healthy controls with hydrogen peroxide resulted in impairments in IL-7 induction of P-STAT5. These findings suggest that oxidative stress that is characteristic of HIV disease could contribute to impairments in IL-7 responsiveness and disrupt T cell homeostasis.

Ascorbate and dehydroascorbic acid are frequently used as biomarkers of oxidative stress, but their lack of stability ex vivo and rapid postsampling interconversion continue to result in erroneous reference values. One problem is the large variety of vacutainer devices used for blood sampling purposes and the basic question of plasma vs serum as matrix. This study acquired blood samples by using 9 different and commonly used vacutainer systems followed by acidic stabilization and analysis by a well-validated method with the purpose of identifying acceptable means of collecting samples for proper ascorbate/dehydroascorbic acid analysis. In comparison, K(3)-EDTA vacutainers were superior in maintaining low ex vivo oxidation of vitamin C., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Background: A possible mechanism underlying cardiovascular morbidity after major vascular surgery may be the perioperative ischaemia-reperfusion with excessive oxygen-derived free-radical production and increased levels of circulating inflammatory mediators. We examined the effect of melatonin infusion during surgery and oral melatonin treatment for 3 days after surgery on biochemical markers of oxidative and inflammatory stress., Methods: Patients received an intra-operative intravenous infusion of 50 mg melatonin or placebo. In addition, all patients received 10 mg melatonin or placebo orally the first 3 nights after surgery. Blood samples for analysis of malondialdehyde (MDA), ascorbic acid (AA), dehydroascorbic acid (DHA) and C-reactive protein (CRP) were collected preoperatively, and at 5 min, 6 h and 24 h after clamp removal (recirculation of the first leg)., Results: Twenty-six patients received melatonin and 24 patients received placebo. No significant differences were observed in any of the oxidative and inflammatory stress parameters. There were significantly more side effects in the melatonin group than in the placebo group., Conclusions: Melatonin treatment in the perioperative period did not reduce the oxidative and inflammatory parameters measured in this study., (Copyright © 2010 European Society for Vascular Surgery. Published by Elsevier Ltd. All rights reserved.)

Background: Sivelestat, a neutrophil elastase inhibitor, has been used to treat acute lung injury (ALI) with varying levels of clinical success. Variable baseline levels of oxidative stress in patients with ALI have been proposed as one explanation for inconsistent results., Methods: Using a bedside electron spin resonance spectrometer, we evaluated electron spin resonance signal intensities of serum ascorbyl free radicals supplemented with dimethyl sulfoxide (AFR/DMSO) in patients with ALI., Results: We found a positive correlation between AFR/DMSO and ascorbate levels, suggesting that serum AFR/DMSO measurements may serve as a surrogate for real-time assessments of oxidative stress. Levels of AFR/DMSO in patients with ALI were significantly lower than those found in healthy controls. Stratified analyses revealed that baseline AFR/DMSO levels were significantly lower in patients with ALI who failed to respond to sivelestat compared with those who did respond., Conclusions: Our results suggest that the clinical efficacy of sivelestat is dependent on baseline oxidative stress levels.

Ascorbic acid (AA) has a strong anti-oxidant function evident as its ability to scavenge superoxide radicals in vitro. Moreover, AA is an essential ingredient for post-translational proline hydroxylation of collagen molecules. Dehydroascorbic acid (DHA), the oxidized form of AA, is generated from these reactions. In this study, we describe an improved method for assessing DHA in biological samples. The use of 35 mM tris(2-carboxyethyl)phosphine hydrochloride (TCEP) as a reductant completely reduced DHA to AA after 2 h on ice in a 5% solution of metaphosphoric acid containing 1 mM ethylenediaminetetraacetic acid (EDTA) at pH 1.5. This method enabled us to measure the DHA content in multiple tissues and plasma of 6-weeks-old mice. The percentages of DHA per total AA differed markedly among these tissues, i.e., from 0.8 to 19.5%. The lung, heart, spleen and plasma had the highest levels at more than 10% of DHA per total AA content, whereas the cerebrum, cerebellum, liver, kidney and small intestine had less than 5% of DHA per total AA content. This difference in DHA content may indicate an important disparity of oxidative stress levels among physiologic sites. Therefore, this improved method provides a useful standard for all DHA determinations.

The potential of free radical formation in serum of beta-thalassemia/Hb E patients receiving a single oral dose of 25 mg/kg body weight of deferiprone, a bidentate orally active iron chelator, was evaluated using EPR/spin trapping technique. In the presence of ascorbic acid and tert-butylhydroperoxide, EPR signals of ascorbyl radical (aH=0.18 mT) and DMPO-carbon centred adduct (aH=2.37 mT, aN=1.65 mT) were detected. Shortly after deferiprone administration, EPR signal intensities decreased concomitant with an increase in serum levels of deferiprone. Unfortunately, enhanced EPR signal intensities were observed at 300 min after dosing in patients with serum molar ratio of deferiprone to iron less than 3, suggesting the formation of incomplete iron-deferiprone complexes and consequently free radical formation. To avoid adverse effects of deferiprone, a dosage regimen should be designed according to iron status of the patients and aimed at maintaining an adequate ratio of serum chelator-to-iron concentration.

A robust and rapid high-pressure liquid chromatography-electrochemical detection (HPLC-ECD) method was developed and validated for the accurate determination of ascorbic acid (AA) and uric acid (UA), in human plasma. Dehydroascorbic acid (DHAA) was indirectly measured by subtracting native ascorbic acid from total ascorbic acid concentrations; the latter was obtained after chemical reduction. A stable electrochemical active internal standard (homogentisic acid) was added for the accurate quantification of the analytes. The analyses were performed on a reverse-phase column with traditional HPLC and ultra-HPLC (UHPLC). The UHPLC method showed increased sensitivity with detection limit of 0.05ng for both AA and UA, 2 times lower compared to conventional HPLC. UHPLC also reduced run times fourfold with less waste generation. Both assays showed good accuracy and precision, the intra- and inter-day CVs of AA and UA analysis are less than 7%.

We performed oral loading of AsA or DAsA (1 mmol) in subjects who had consumed a diet low in vitamin C (C) (C< or =5 mg/d) for 3 d before loading, and measured urinary and blood vitamin C. Since the crossover method was used, the same experiment was repeated after an interval of about 1 mo in each subject. The results of the experiment including a total of 17 subjects for 2005 and 2006, were as follows. (1) There were marked individual differences in urinary C excretion. (2) The C level in 24-h urine after C loading did not differ between the two orally administered C forms (AsA and DAsA). (3) C excretion between 0 and 3 h after C loading was significantly higher (p<0.05) for the DAsA group, while those between 3 and 6, 6 and 9, 9 and 12, and 12 and 24 h after C loading were significantly higher (p<0.05 or p<0.01) for the AsA group. (4) The blood C concentration and the increase in C 1 h after C loading were significantly higher (p<0.05 and p<0.01, respectively) in the DAsA than in the AsA group. (5) Evaluation of the association between C metabolism and the single nucleotide polymorphisms of glutathione S-transferase P (GSTP) 1-1 showed a lower urinary C excretion and a significantly lower C level in 24-h urine (p<0.05) after AsA loading, and a significantly lower urinary C excretion between 0 and 3 h after DAsA loading (p<0.05) for the GA heterozygotes than for the AA homozygotes. Considering the activity of C as DAsA in humans, based on urinary and blood C levels after a single loading of C, the utilization of DAsA is equivalent to that of AsA, although the metabolic turnover time is different. The involvement of polymorphisms in the xenobiotic metabolizing enzyme, GSTP1-1, in C metabolism, particularly urinary C excretion, was also clarified. This demonstrates the necessity of considering gene polymorphisms in determining individual C requirements. An abstract of this paper was reported by the Vitamin C Research Committee (Ochanomizu University) in 2007.

Unlabelled: Circulation on blood extracorporeally through plastic tubing activates several pathways including systemic inflammation and oxidative stress. These phenomena are suspected to participate to neurological and cardiovascular side effects observed in the patients under cardiopulmonary bypass (CPB). A direct relationship, in diabetic patients, between hyperglycemia and morbidity and mortality has been established. However, it is still unclear whether perioperative hyperglycemia has a direct effect on adverse events in cardiac surgery. The purpose of this study was to determine the influence of hyperglycemia on inflammation and oxidative stress in patients under CPB during cardiac surgery., Material and Methods: Control patients (n=17) and diabetic (type 2) patients (n=13) were included in this study. Blood samples were drawn before, during and after the CPB. Oxidative stress was evaluated in the plasma by direct and indirect approaches. Direct detection of ascorbyl radicals was assessed by electron spin resonance spectroscopy. An index: ascorbyl radical/vitamin C ratio is an indicator of the degree of oxidative stress taking place in the plasma. Oxygen radical absorbing capacity (ORAC) values were used as measurement of antioxidant capacity of the plasma. To determine inflammation profile of patients, we measure the evolution of plasma concentration of interleukin 8 (IL-8)., Results: During cross clamping and post-CPB, the index ascorbyl radical/vitamin C is increased; the value of the index is more significant in diabetic patients. Concomitantly, ORAC values decreased in all the patients during cross clamping (p<0.05). Results concerning inflammatory index showed that IL-8 levels increased during the CPB., Conclusion: In conclusion, the current study indicates that a systemic oxidative stress occurs during CPB and post-CPB periods and that in patients with type 2 diabetes mellitus, the systemic oxidative stress was increased.

Lack of post-sampling stability of ascorbate and dehydroascorbic acid and failure to block their in vivo equilibrium have lowered their value as biomarkers of oxidative stress and limited the ability to further investigate their possible role in disease prevention. In the present article, analytic reproducibility was tested by repeated analysis of plasma aliquots from one individual over 4 years. The plasma was subjected to acidic deproteinization with an equal volume of 10% meta-phosphoric acid containing 2 mmol/L of EDTA and analyzed for ascorbate and dehydroascorbic acid by high-performance liquid chromatography with coulometric detection. In a parallel experiment, the stability of human plasma samples treated as above and stored at -80 degrees C for 5 years was tested in a cohort of 131 individuals. No degradation or shift in the equilibrium between ascorbate and dehydroascorbic acid was observed in either of the experiments. In conclusion, ascorbate and dehydroascorbic acid could be adequately preserved in plasma stored at -80 degrees C following acidic deproteinization with meta-phosphoric acid containing 2 mmol/L of EDTA.

Background and Purpose: Cigarette smoking has been associated with the development of cardiovascular disease and cancer. Even though the molecular mechanism(s) are not clear, the pathology has been related to oxygen free radicals present in cigarette smoke. Thus, the main objective of this study was to establish the changes in the oxidation/antioxidation balance induced by cigarette smoking., Methods: Thirty healthy subjects (15 smokers and 15 nonsmokers) of both sexes were studied. The smokers group had smoked a mean of 14 cigarettes per day for an average of 4.5 years. Fasting serum levels of malondialdehyde (MDA), a marker of oxidative stress, nitric oxide (NO), reduced glutathione (GSH), and vitamin C (ascorbic and dehydroascorbic acids) were measured., Results: Fasting NO concentration was significantly higher in smokers (51.3 +/- 5.3 microM) than in nonsmokers (35.2 +/- 4.8 microM, P < 0.05). The smokers had significantly higher serum dehydroascorbic acid levels (2.4 +/- 0.5 mg/dL, P < 0.03) than the nonsmokers (1.08 +/- 0.08 mg/dL). No significant differences were observed in the levels of ascorbic acid, MDA, and GSH between the smokers and nonsmokers., Conclusions: Our results suggest that exposure to cigarette smoke increases NO synthesis, such that NO may act in a compensatory way as an inhibitor of lipid peroxidation. Smoking also activates other antioxidative mechanisms such as involving vitamin C. These protective mechanisms appear to be enough in preventing accumulation of oxidative products such as MDA and avoiding oxidative damage.

Objectives: The purpose of this study is to assess the effect of beta1-selective blocker on coronary vasospasm., Materials and Methods: Balloon epicardial coronary artery endothelial denudation was performed at the left anterior descending coronary artery every 2 weeks for a total of 4 times in pigs. Changes in denuded site diameter and left anterior descending coronary artery blood flow caused by acetylcholine or serotonin were assessed before each endothelial denudation and at week 8 in untreated pigs (ED group) and in those treated with metoprolol (Meto group) or propranolol (Pro group)., Results: In the ED group, decreased blood flow response to acetylcholine enhanced from -20+/-10% before the first ED to -100% (i.e. zero flow) at week 8 without denuded site narrowing, suggesting microvascular spasm, and serotonin-induced left anterior descending coronary artery diameter reduction at week 8 was -92+/-15%. In the Pro group, blood flow reduction by acetylcholine and left anterior descending coronary artery diameter reduction by serotonin did not change compared with those of the ED group. In the Meto group however, blood flow reduction by acetylcholine (week 8, -70+/-16%) and left anterior descending coronary artery diameter reduction by serotonin (week 8, -64+/-15%) were blunted (P<0.01) compared with those of ED and Pro groups., Conclusion: The beta1-selective blocker metoprolol was effective to prevent coronary vasospasm.

Background: Human erythrocytes can take up dehydroascorbate on the glucose transporter 1 (GLUT 1) and reduce it to ascorbate. Intraerythrocyte ascorbate was proved to be directly responsible for decreased oxidation of extraerythrocytic ascorbate. In addition to spontaneous and irreversible loss of ascorbate in plasma, the hemodialysis (HD) process itself consumes plasma ascorbate. However, intraerythrocyte ascorbate status in uremic patients during HD has yet to be reported., Methods: Plasma and intraerythrocyte ascorbate, dehydroascorbate, GLUT 1 expression on erythrocyte membranes, and in vitro studies of "erythrocyte ascorbate recycling" were investigated in age- and sex-matched healthy subjects (control group) and HD patients (HD group)., Results: Intraerythrocyte ascorbate concentrations decreased after 1 HD session compared with pre-HD and recovered to pre-HD values 2 days later, whereas plasma ascorbate concentrations did not recover. In vitro studies suggested that erythrocytes of HD patients have a stronger ability to maintain intracellular ascorbate concentrations compared with healthy subjects. This ability could be inhibited by cytochalasin B (GLUT 1 inhibitor). We also found increased GLUT 1 expression (P = 0.002) on erythrocyte membranes in the HD group compared with the control group., Conclusion: Erythrocytes of uremic patients lost large amounts of ascorbate during HD, but regained it to the pre-HD level 2 days later. Enhanced GLUT 1 expression on erythrocyte membranes for HD patients may contribute to better preservation of intracellular ascorbate compared with healthy subjects.

Background: Estrogens, apart from their classic role as steroid hormones, also possess significant antioxidant properties. The present study was undertaken to assess the antioxidant potential of the female during the various menstrual phases and to investigate the correlation between ascorbic acid, dehydroascorbic acid plasma levels, total antioxidant plasma status, and estradiol levels., Design and Methods: Thirteen eumenorrhoic women were studied. Ascorbic acid and dehydroascorbic acid plasma levels, total antioxidant plasma status, estradiol, progesterone, luteinizing hormone, and follicle-stimulating hormone during time of menstruation, midfollicular phase, time of ovulation, and midluteal phase were determined. Ascorbic-dehydroascorbic acid ratio was also calculated., Results: A progressive significant rise in ascorbic acid plasma levels (p < 0.01), ascorbic-dehydroascorbic acid ratio (p < 0.001), and total antioxidant plasma status (p < 0.05) from menstruation to ovulation was observed. Moreover, a significant decrease in dehydroascorbic acid was found at the same phases (p < 0.05). Changes of estradiol levels during the menstrual cycle correlated positively with the changes of ascorbic acid levels and total antioxidant plasma status (p < 0.05). Furthermore, estradiol levels correlated positively with ascorbic acid levels (p < 0.05, r < 0.5), ascorbic-dehydroascorbic acid ratio (p < 0.05, r < 0.5), and total antioxidant plasma status (p < 0.05, r < 0.8) in all menstrual phases., Conclusions: An elevated antioxidant protection during ovulation and the midluteal phase appears to be present in eumenorrhoic women. Moreover we observed a cyclic variation in the antioxidant parameters we assayed in the females in the present study, which could be due to cyclic changes in estradiol levels.

Vitamin C plays a central role in the body. One of its important functions is its role as an antioxidant, and accurate measurements are important for interpretations of this role. However, its reactive nature and instability complicates the assessment, especially in biological samples. A high-throughput chromatographic method using monolithic column and UV-detection was developed for the assessment of plasma ascorbic acid and total ascorbic acid. The method showed excellent analytical sensitivity, specificity, precision, recovery and linearity during the validation study. The method was used for the assessment of ascorbic acid and total ascorbic acid during several clinical studies.

Background & Aims: Oxygenated water with an oxygen concentration of 30-120 mg/l water is believed to improve the immune status, without any toxicological effects. The purpose of this clinical study was to assess the effects of long-term drinking of oxygenated water on the immune status., Methods: In this prospective, double-blinded, randomised study 24 volunteers of either sex (age 18-63 years) drank daily 3 times 500 ml either oxygenated (Verum-group: n=12) or normal mineral water (Placebo-group: n=12) for 28 days. On day 1 and day 28 standard laboratory tests, IgG, IgA and IgM, lymphocyte subpopulations and functional analysis of T-cells by flow cytometry, were done. Furthermore, the oxygen radicals were determined by the detection of the ascorbyl radicals., Results: Drinking of normal or oxygenated water had no effect on whole blood count or the liver enzymes. Interestingly the volunteers in the Verum-group showed a significant increase in ascorbyl radicals after drinking oxygenated water for 14 and 21 days. CD4+ and CD4+CD45RA+ lymphocytes as well as lymphocyte activation marker (CD69) and soluble IL-2 receptor increased in both groups, in contrast T-helper2 cells and IgG decreased during the study. The only differences between the two groups were a significant decrease of NK-cells form 13.42%+ or -5.04 to 10.83%+ or -4.82 (P<.002) and an increase of the Th1/Th2-ratio from 2.77%+ or -1.07 to 6.68%+ or -5.33 (P<.03) in the Verum-group., Conclusion: Long-term consumption of oxygenated water has no apparent harmful effect on the liver, blood and the immune system. Moreover it leads to a transient moderate increase of oxygen radicals in the blood. An interesting observation is the increase of the Th1/Th2-ratio in the Verum group, whereas in both groups T-cell activation after mitogen stimulation, the soluble IL-2 receptor, the CD4+ and the naive CD4+CD45RA+ cells increased.

Objective: To examine the oxidation or reduction products in patients with rheumatism arthritis (RA), and investigate the relationship between oxidation or reduction products and occurrence and development of RA., Methods: The serum levels of total ascorbic acid (TAA), dehydroascorbic acid (DHAA)/TAA, vitamin E, advanced oxidation protein products (AOPP) and malondialdehyde (MDA) were detected by high-performance liquid chromatography with electrochemical detection in 83 RA patients and 30 healthy adults. Correlation analysis of AOPP, MDA and hs-CRP was performed., Results: Compared with normal control group, significantly higher serum MDA, DHAA/TAA, and AOPP levels were detected in RA patients (P<0.05), but vitamin E showed no significant difference (P<0.05). Linear regression analysis showed that MDA (P<0.01) was positively but AOPP (P>0.05) negatively correlated to hs-CRP., Conclusions: Oxidation or reduction products in serum of RA patients increases significantly, which may be an important mechanism for the occurrence and development of RA. Serum AOPP and MDA levels can reflect the oxidation status in RA patients.

Objective: The objective of these studies was to determine the role of ascorbate deficiency in HIV infection in the defective detoxification of sulfamethoxazole-nitroso, the metabolite thought to mediate sulfonamide hypersensitivity reactions., Methods: Fifty-one HIV-infected patients and 26 healthy volunteers were evaluated. Vitamin supplementation histories were obtained, and blood samples were collected for determination of plasma ascorbate, dehydroascorbate, and cysteine concentrations, erythrocyte glutathione concentrations, and plasma reduction of sulfamethoxazole-nitroso in vitro., Results: Plasma ascorbate concentrations were significantly lower in HIV-positive patients not taking vitamin supplements (29.5 +/- 22.3 microM) than in healthy subjects (54.8 +/- 22.3 microM; P = 0.0005) and patients taking 500-1000 mg of ascorbate daily (82.5 +/- 26.3 microM; P < 0.0001). Plasma ascorbate deficiency was strongly correlated with impaired reduction of sulfamethoxazole-nitroso to its hydroxylamine (r = 0.60, P < 0.0001), and during in vitro reduction, the loss of plasma ascorbate was strongly associated with the amount of nitroso reduced (r = 0.70, P < 0.0001). Ascorbate added ex vivo normalized this reduction pathway. Erythrocyte glutathione concentrations were significantly lower in HIV-positive patients (0.98+/-0.32 mM) than in healthy subjects (1.45+/-0.49 mM; P = 0.001), but this finding was unrelated to ascorbate supplementation. There was trend toward lower plasma cysteine concentrations in patients (8.4+/-3.9 microM) than in controls (10.3+/-4.3 microM), but this trend was similarly unrelated to ascorbate supplementation. Dehydroascorbate concentrations were not significantly higher in HIV-positive patients (7.4+/-10.5%) than in healthy controls (4.0+/-6.2%), even in the subset of patients taking ascorbate (8.4+/-9.4%)., Conclusions: Ascorbate deficiency is common in HIV-positive patients and is associated with impaired detoxification of sulfamethoxazole-nitroso, the suspected proximate toxin in sulfonamide hypersensitivity. Patients taking daily ascorbate supplements (500-1000 mg) achieved high plasma ascorbate concentrations and did not show this detoxification defect. Ascorbate deficiency (or supplementation) was not associated with changes in glutathione or cysteine concentrations. These data suggest that ascorbate deficiency, independent of thiol status, may be an important determinant of impaired drug detoxification in HIV infection.

Under the cell-free condition, copper is known to oxidize ascorbic acid (the active form of vitamin C) and the event leads to the loss of vitamin C. However, the biological consequence of this interaction was never examined in the presence of cells. We demonstrated in intestinal epithelial cells that dehydroascorbic acid (the oxidized form of ascorbic acid), when generated from ascorbic acid in the presence of copper, can be efficiently transported into the cells and reduced back to ascorbic acid. We also observed in other types of cells the transport and intracellular reduction of dehydroascorbic acid in the presence of copper. In the presence of iron, a metal that also oxidizes ascorbic acid, we observed similar oxidation-related accumulation in intestinal cells. Other metals that do not interact with ascorbic acid had little effect on vitamin C transport. A nonmetal pro-oxidant, hydrogen peroxide, is known to oxidize ascorbic acid and we observed that the oxidation is also accompanied by an increased intracellular accumulation of vitamin C. The efficient coupling between dehydroascorbic acid transport and intracellular reduction could help to preserve the important nutrient when facing oxidative metals in the intestine.

Our aim was to assess the plasma free 8-epi-prostaglandin F(2alpha) (8-isoprostane) and ascorbyl radical as risk indicators for oxidative damage in extremely low birth weight infants (ELBWIs) and the effect of N-acetylcysteine (NAC) on these markers. Plasma samples were collected on days 3 and 7 of life from infants who were enrolled in a randomized, controlled trial in which i.v. NAC or placebo was administered to ELBWIs during the first week of life, with the aim of preventing bronchopulmonary dysplasia (BPD). Plasma 8-isoprostane was analyzed in 83 infants using an enzyme immunoassay kit. Ascorbyl radical concentration was measured in 61 infants with electron spin resonance spectroscopy. The 8-isoprostane concentrations were similar in the NAC and placebo groups. In infants who later developed BPD or died (n = 29), the median (range) 8-isoprostane concentration was significantly higher (p = 0.001) on day 3 and day 7 [50.0 pg/mL (19-360) and 57.0 pg/mL (14-460), respectively] than in survivors without BPD [n = 54; 34.5 pg/mL (5-240) and 39.5 pg/mL (7-400), respectively]. The 8-isoprostane levels increased significantly more (p < 0.05) in infants who later developed periventricular leukomalacia. NAC treatment or the later development of BPD was not related to the ascorbyl radical levels. The ascorbyl radical level decreased significantly in all groups from day 3 to day 7, but the difference between the groups was not significant. The mean (SD) ascorbyl radical level on day 3 was significantly higher (p < 0.01) in infants who later developed periventricular leukomalacia [287 (124) versus 194 (90)]. These data suggest that plasma 8-isoprostane could serve as a marker in assessing the risk for BPD development in ELBWIs.

The status of ascorbic acid (AA) in dialysis patients is the subject of debate. Some reports have found AA to be deficient in dialysis patients, while others have found that AA is not deficient. In an attempt to confirm AA serum concentrations in dialysis patients, we analyzed the concentrations of AA as well as its metabolites using the specific determination of AA with chemical derivatization and the HPLC method. We studied 131 patients under maintenance hemodialysis therapy (HD), 23 patients with chronic renal failure (CRF) and 48 healthy controls (C). Serum concentrations of AA and the AA metabolites dehydroascorbic acid (DHA) and 2, 3-diketogulonate (DKG) were measured by HPLC. Nine HD patients were taking AA supplements. Seventy-six (62.3%) of the 122 HD patients not taking AA supplements exhibited deficient levels of AA (< 20 microM), while 13 (56.5%) of the 23 CRF patients and 9 (18.8%) of the 48 C showed deficient levels of AA. Analysis of AA metabolites in the normal-range AA (20-80 microM) group revealed that the DHA/AA ratio in HD patients was significantly higher than in C (3.3 +/- 2.6% and 1.2 +/- 2.2%, respectively). The DKG/AA ratio in HD patients was higher than in CRF patients (3.6 +/- 5.2% vs. 0.9 +/- 1.9%), whereas DKG was not detected in C. When compared to serum levels before the start of dialysis, serum AA, DHA and DKG concentrations at the end of the dialysis session decreased by an average of 74.2, 84.0 and 78.8% respectively. In HD patients, serum levels of thiobarbituric reactive substances (TBARS) were significantly lower in the higher AA (> 80 microM) group than in the deficient and normal-range AA groups. In 12 AA-deficient patients, after 1 month of taking AA supplements (200 mg/day), serum AA levels rose to 79.9 microM, while serum TBARS level declined when compared with levels before supplementation. In conclusion, the frequency of AA deficiency in dialysis patients is extremely high. AA deficiency in HD patients may result in high TBARS levels, which reflect increased oxidative stress. Adequate AA supplementation should therefore be considered in such patients.

We present a fast to perform spectrophotometric method for the quantification of ascorbic acid and its oxidized form dehydroascorbic acid in biological samples. The assay detects a chromophore formed during the reaction of dehydroascorbic acid with methanol in phosphate/citrate buffer. This reaction can also be employed for the determination of ascorbate (vitamin C) in the presence of ascorbate oxidase. The major advantage of the developed protocol for the determination of both forms of vitamin C is a simple spectrophotometrical single end point determination. It is demonstrated that the methanol method is an improvement compared with a commercially available test kit for the determination of vitamin C. Using the methanol method, a dose-dependent increase in intracellular ascorbic acid was determined upon incubation of L-929 cells and RAW 264.7 macrophages with increasing concentrations of extracellular ascorbate. In blood serum, vitamin C was determined at concentrations between 46 and 97 microM. Supplementation with different amounts of ascorbate showed satisfying recovery. In L-929 cells, even unphysiologically high amounts of reactive nitrogen species were unable to completely oxidize intracellular vitamin C.

Ascorbic acid plays a major role in pulmonary antioxidant defense. Sufficient amounts of ascorbic acid are necessary to maintain normal metabolic processes in the lung. We measured the levels of ascorbic, dehydroascorbic and diketogulonic acids in blood serum of patients with pulmonary tuberculosis (PTB) and pneumonia. The serum levels of ascorbic acid were decreased in PTB and pneumonia, and those of dehydroascorbic acid were decreased in PTB, but not in pneumonia. The serum diketogulonic acid levels were not significantly changed in either PTB or pneumonia. The ratio of ascorbic to dehydroascorbic acid levels in serum were increased in PTB, but in pneumonia we observed a significant decrease in this index. The ratio of dehydroascorbic to diketogulonic acid in PTB was decreased, but in pneumonia this index did not significantly differ from the control value. Thus, in PTB the rate of ascorbic acid oxidation is decreased and the rate of dehydroascorbic acid oxidation is increased. By contrast, in pneumonia the rate of ascorbic acid oxidation is increased, but the rate of dehydroascorbic acid oxidation did not differ from control values.

Plasma and milk concentrations of ascorbic acid and dehydro-L-ascorbic acid (DHAA) were measured before and after 21 Holstein cows (approximately 26 DIM) were given an intramammary infusion of Escherichia coli. Blood, milk from the unchallenged quarters, and milk from the challenged gland were sampled immediately before challenge (d 0) and 24 h and 7 d postchallenge. Plasma vitamin C (ascorbic acid + DHAA) concentrations decreased 39%, and concentrations of vitamin C and ascorbic acid in milk from the challenged quarter decreased 52 and 62%, respectively, in samples taken 24 h postchallenge. No change was observed in vitamin C concentrations in milk from unchallenged quarters. The concentration of DHAA in milk from challenged quarters increased 67% 24 h postchallenge. The duration of clinical mastitis, peak body temperature, number of colony-forming units of E. coli isolated from the infected gland, and loss in milk yield were associated with a change in concentration of vitamin C in milk from the challenged quarter. Increased severity of clinical signs was associated with large decreases in concentration of vitamin C in milk from the challenged quarter. Similar, but statistically weaker, relationships were observed for changes in plasma vitamin C concentrations.

Background: Extensive exercise may promote the formation of reactive oxygen species and subsequently contribute to tissue damage. A compound which can protect cells and tissues is vitamin E. The vitamin E radical, formed during the radical scavenging process, can be reduced by vitamin C resulting in a higher level of the vitamin C radical (semidehydroascorbate free radical). An increase of the vitamin C radical, however, is assumed to exert prooxidative effects., Aim of the Study: To elucidate whether supplementation of vitamin E and exhaustive exercise lead to an increase of the vitamin C radical in human plasma., Methods: A placebo controlled, cross over study with 13 male volunteers was carried out. After an 8 day supplementation period with 500 I.U. D-alpha-tocopherol, the subjects performed two exhaustive treadmill runs. Blood samples were collected at rest, 0, 0.25, 1, 3, 24 and 48 h after exercise. Serum was separated and concentrations of D-alpha-tocopherol and ascorbic acid were determined by HPLC. Vitamin C radical levels in plasma were assessed by electron paramagnetic resonance (EPR)., Results: Vitamin E and C both showed a tendency to decrease between 3 h and 24 h after exercise. Vitamin C radical level remained stable during the whole period. Neither vitamin E supplementation nor exercise had any influence on the plasma concentration of the vitamin C radical., Conclusions: Vitamin E supplementation under conditions of mild oxidative stress does not result in an increased vitamin C radical concentration.

Metabolomic mapping is an emerging discipline geared at providing information on a large number of metabolites as a complement to genomics and proteomics. Here we have probed ascorbic acid homeostasis and degradation in diabetes using 6-deoxy-6-fluoro ascorbic acid (F-ASA) and 750 MHz (19)F-nuclear magnetic resonance (NMR) spectroscopy with proton decoupling In vitro, Cu(2+)-mediated degradation of F-ASA revealed the formation of 4 major stable degradation products at 24 hours. However, when normal or diabetics rats were injected with F-ASA intraperitoneally (IP) for 4 days, up to 20 fluorine-labeled compounds were observed in the urine. Their composition resembled, in part, metal catalyzed degradation of F-ASA and was not explained by spontaneous degradation in the urine. Diabetes led to a dramatic increase in urinary F-ASA loss and a relative decrease in most other urinary F-compounds. Diabetes tilted F-ASA homeostasis toward oxidation in liver (P <.01), kidney (P <.01), spleen (P <.01), and plasma (P <.01), but tended to decrease oxidation in brain, adrenal glands, and heart. Surprisingly, however, besides the major oxidation product fluoro-dehydroascorbic acid (F-DHA), no F-ASA advanced catabolites were detected in tissues at 5 micromol/L sensitivity. These findings not only confirm the key role of the kidney in diabetes-mediated loss of ascorbic acid, but demonstrate that only selected tissues are prone to increased oxidation in diabetes. While the structure of most degradation products needs to be established, the method illustrates the power of high resolution (19)F-NMR spectroscopy for the mapping of complex metabolomic pathways in disease states.

The objective of this work was to evaluate the use of vitamin C as a biomarker in the inflammatory phase of the rat adjuvant arthritis and to correlate it with other parameters used for disease evaluation. Paw swelling was used for physical evaluation and the levels of ascorbate and dehydroascorbate in the serum of male rats, before and after adjuvant arthritis induction, were quantified by a high-performance liquid chromatography method (HPLC). The optimised HPLC assay enabled the quantification of both forms of the vitamin in rat sera, with the same extraction method and using different detectors, instead of obtaining dehydroascorbate by subtraction of the total ascorbate measurement. This method was used to follow the severity of adjuvant arthritis and the results were correlated with other already established disease activity parameters. A decrease of ascorbic acid and dehydroascorbic acid was observed with the increase of right paw circumference during the course of adjuvant arthritis. The disease associated changes in the serum concentrations of ascorbic acid, from biosynthesis and from recycling, can be evaluated by the direct quantification of dehydroascorbic acid. This provides some evidence for the potential of the quantification of these biomarkers to study the disease activity, and as a tool for the establishment of therapeutic protocols, to evaluate the anti-inflammatory effect of new drugs or formulations.

An imbalance between oxidative damage and antioxidative protection in association with the pathophysiology of atherosclerosis has been suggested. The aim of our study was to investigate the relationship between plasma lipids, the antioxidant system and oxidative damage in Thai patients with stable coronary artery disease (CAD). Sixty-one patients (40 males, 21 females), who were angiographically defined as having CAD and were clinically stable, participated in this study. Thirty-two healthy subjects (20 males, 12 females) served as normal controls. The investigation included the measurements of plasma lipid profiles and plasma total antioxidative status (TAS) such as plasma vitamin E erythrocyte glutathione (GSH) and glutathione peroxidase (GPx), as well as malondialdehyde (MDA) and total plasma total protein thiols (P-SH). In patients with CAD, erythrocyte GSH and GPx were significantly lower than those found in controls. However plasma TAS and vitamin E were not significantly different between groups. Patients with CAD also had higher MDA and lower P-SH levels than the controls, which represents the oxidative damage products of lipid and proteins. Multiple regression analysis revealed negative correlations between GSH and cholesterol, GSH and low density lipoprotein (LDL), vitamin E and MDA, as well as P-SH and MDA. This study demonstrated the status of oxidative stress in patients with stable CAD. Since oxidative stress is the imbalance between the total oxidants and antioxidants in the body, any single oxidant/antioxidant parameter may not reflect the overall oxidative stress system. Thus, in patients with CAD, diets with various types of antioxidants may be more beneficial in increasing antioxidant activity than any particular antioxidant supplementation.

Background: The amount of dehydroascorbic acid contained within total ascorbic acid (oxidized as well as non-oxidized forms) in plasma, hereafter referred to as the dehydroascorbic acid fraction, may be a measure of oxidative stress during haemodialysis. In the present study, we determined this fraction in chronic haemodialysis patients., Methods: Using high performance liquid chromatography, dehydroascorbic acid and total ascorbic acid levels were measured in 80 maintenance haemodialysis patients for a period of > 2 years as well as in 49 controls, to examine a possible association of these compounds with clinical parameters and/or drugs taken by the patients., Results: Dialysis patients who had an increased plasma urate level (P < 0.05) and had been taking allopurinol (P < 0.05) or NSAID (non-steroid anti-inflammatory drugs) (P < 0.01), and dialysis patients who were younger (< or = 55 years), as compared with older dialysis patients (P < 0.01), were found to have a lower dehydroascorbic acid fraction by multivariate analysis. Mean plasma dehydroascorbic acid levels and dehydroascorbic acid fractions were significantly lower in the younger haemodialysis patients (4.8 +/- 0.7 micromol/l and 28.4 +/- 3.9%) than in healthy younger controls (13.3 +/- 1.1 micromol/l and 41.1 +/- 1.8%) (P < 0.0001 and P < 0.01, respectively). Moreover, a correlation was found between plasma dehydroascorbic acid fraction and plasma lipid peroxide (r = 0.66, P < 0.01) in patients who had not been taking allopurinol and/or NSAID., Conclusion: We found that dehydroascorbic acid fraction was related to patients' age, plasma urate level and to taking allopurinol or NSAID. Dehydroascorbic acid fraction may be another indirect index of oxidative stress.

Reactive oxygen species have been implicated in the pathogenesis of acute pancreatitis. Few studies have focused on the loss of endogenous antioxidants and molecular oxidative damage. Two acute pancreatitis models in rats; taurocholate (3% intraductal infusion) and cerulein (10 microg/kg/h), were used to study markers of oxidative stress: Glutathione, ascorbic acid, and their oxidized forms (glutathione disulfide and dehydroascorbic acid), malondialdehyde, and 4-hydroxynoneal in plasma and pancreas, as well as 7-hydro-8-oxo-2'-deoxyguanosine in pancreas. In both models, pancreatic glutathione depleted by 36-46% and pancreatic ascorbic acid depleted by 36-40% (p <.05). In the taurocholate model, plasma glutathione was depleted by 34% (p <.05), but there were no significant changes in plasma ascorbic acid or in plasma and pancreas dehydroascorbic acid, malondialdehyde, and 4-hydroxynoneal, and no significant changes in the pancreas glutathione disulfide/glutathione ratio. While pancreas glutathione disulfide/glutathione ratio increased in the cerulein model, there were no significant changes in plasma glutathione, plasma, or pancreas ascorbic acid, dehydroascorbic acid, 4-hydroxynoneal, and malondialdehyde, or in pancreas 7-hydro-8-oxo-2'-deoxyguanosine. Reactive oxygen species have a minor role in the intermediate stages of pancreatitis models.

Ascorbyl radical (ASR) in human cerebrospinal fluid (CSF) of various patients using electron paramagnetic resonance (EPR) at ambient temperature was investigated. Also, effect of chemotherapy on ASR as well as ascorbate (ASA) in CSF and serum of acute lymphoblastic leukemia (ALL) was studied. EPR spectra of various CSF samples showed a characteristic doublet, which was attributed to ASR. ASR in CSF and serum was directly measured without any chemical modification. ASA and ASR concentration in CSF were approximately two times higher than those in serum. ASA and ASR concentrations in CSF and serum were statistically analyzed. The analyses showed that ASR and ASA in CSF and serum had good correlation for patients undergoing chemotherapy but not for patients after the therapy. The correlation for ASR and ASA suggests that ascorbate may play an important role during chemotherapy. In addition, dynamic aspects of ASA and ASR in CSF and serum are discussed.

Ascorbate is readily oxidized in aqueous solution by ascorbate oxidase. Ascorbate radicals are formed, which disproportionate to ascorbate and dehydroascorbic acid. Addition of erythrocytes with increasing intracellular ascorbate concentrations decreased the oxidation of ascorbate in a concentration-dependent manner. Concurrently, it was found, utilizing electron spin resonance spectroscopy, that extracellular ascorbate radical levels were decreased. Control experiments showed that these results could not be explained by leakage of ascorbate from the cells, inactivation of ascorbate oxidase, or oxygen depletion. Thus, this means that intracellular ascorbate is directly responsible for the decreased oxidation of extracellular ascorbate. Exposure of ascorbate-loaded erythrocytes to higher levels of extracellular ascorbate radicals resulted in the detection of intracellular ascorbate radicals. Moreover, efflux of dehydroascorbic acid was observed under these conditions. These data confirm the view that intracellular ascorbate donates electrons to extracellular ascorbate free radical via a plasma membrane redox system. Such a redox system enables the cells to effectively counteract oxidative processes and thereby prevent depletion of extracellular ascorbate.

Determination of dehydroascorbic acid in biological samples most commonly involves indirect measurement. The concentration is calculated by subtraction of the measured ascorbic acid concentration from that of total ascorbic acid analyzed after reduction of the dehydroascorbic acid present; a methodology also referred to as subtraction methods. Consequently, successful determination of dehydroascorbic acid is dependent on proper sample handling, quantitative reduction of the compound, and accurate quantification of both ascorbic acid and total ascorbic acid. In this paper, the recently introduced reductant tris[2-carboxyethyl]phosphine (TCEP) is evaluated as a reliable alternative to the commonly used reducing agent dithiothreitol (DTT). The results show that TCEP offers a more efficient reduction of dehydroascorbic acid at low pH compared to that of DTT. Moreover, while DTT maintains a reducing sample environment for less than 24 h, TCEP show complete protection from oxidation of ascorbic acid for at least 96 h following sample preparation. Removal of TCEP prior to analysis is unnecessary. A revised HPLC-EC method incorporating TCEP as reductant as well as the coanalysis of isoascorbic acid and uric acid is presented. The within- and between-day coefficients of variation for the complete assay are less than 1.5 and 3.5% for all analytes. As a whole, the method presented here is simpler and more reliable than existing methods., (Copyright 2000 Academic Press.)

13C-NMR spectroscopy was used to record time courses of the metabolism of [1-(13)C]-L-ascorbic acid (AA) and [2-(13)C]-L-ascorbic acid and their dehydro-counterparts (DHAA) by human erythrocytes. Under a range of experimental conditions, but most notably in the absence of glucose in the incubation medium, no (13)C-NMR signal for lactate emerged during any of the 5 h time courses. The NMR resonances that did emerge over time were assigned to diketogulonic (DKG) acid and CO(2). Only very minor resonances from degradation products of DKG appeared from samples that contained physiologically high concentrations of DHAA. These results are in contrast with those in a recent report that lactate is derived from AA in human erythrocytes. However, an explanation for this possible artifact is given.

We investigated the possibility that human erythrocytes can reduce extracellular ascorbate free radical (AFR). When the AFR was generated from ascorbate by ascorbate oxidase, intact cells slowed the loss of extracellular ascorbate, an effect that could not be explained by changes in enzyme activity or by release of ascorbate from the cells. If cells preserve extracellular ascorbate by regenerating it from the AFR, then they should decrease the steady-state concentration of the AFR. This was confirmed directly by electron paramagnetic resonance spectroscopy, in which the steady-state extracellular AFR signal varied inversely with the cell concentration and was a saturable function of the absolute AFR concentration. Treatment of cells N-ethylmaleimide (2 mM) impaired their ability both to preserve extracellular ascorbate, and to decrease the extracellular AFR concentration. These results suggest that erythrocytes spare extracellular ascorbate by enhancing recycling of the AFR, which could help to maintain extracellular concentrations of the vitamin., (Copyright 2000 Academic Press.)

Background and Purpose: Total body irradiation (TBI) is a routine preconditioning procedure for the treatment of leukemia and aplastic anemia, prior to bone marrow transplantation (BMT). Ionizing radiation generates reactive oxygen derived species (ROS) that can be removed by antioxidants. Our purpose is to determine the antioxidant status of patients undergoing TBI by evaluating the oxidant stress and their antioxidant capacity., Material and Methods: We evaluated by cyclic voltammetry (CV) the total antioxidant capacity (TAC) in plasma of 14 patients undergoing TBI prior to BMT. The levels of the antioxidants, ascorbic acid (AA) and uric acid (UA) were determined by HPLC-ECD. The oxidant stress level was calculated by the ratio [dehydro ascorbic acid]/total ascorbic acid]., Results: TAC was reduced by 36% (p < 0.02) but after 4 months recovered to a level 22% higher than before the treatment (p < 0.05). Both, AA and UA, decreased following irradiation by 84% (p < 0.02) and 24% (p < 0.05) respectively, but returned to a level of 21% and 320% after 4 months compared to baseline values. The changes in [UA] were affected by Allopurinol (xanthine oxidase inhibitor), given as a routine pretransplant therapy until day -1. The [dehydroascorcbic acid]/[total ascorbic acid] (%) was 45% (range of normal controls = 13.2 +/- 1.5%) and increased by 69% following TBI. In order to obtain a decrease in the TAC of plasma in vitro, comparable to that in vivo, a 1000 fold higher dose of irradiation was required., Conclusions: TBI caused a pronounced decrease in antioxidant capacity and an excessive increase in oxidant stress. We assume that TBI alters antioxidant homeostasis greatly enhancing the stress damage. CV measurements may lead to a better understanding of the balance between oxidant stress and antioxidant utilization, and to a reconsideration of the routine use of Allopurinol as pretreatment for TBI, and antioxidant support before and/or after TBI.

Background: Vitamin C is currently considered a potent water-soluble antioxidant and it appeared reasonable to study the metabolic changes of vitamin C in uraemia and during haemodialysis., Methods: We measured the levels of ascorbic, dehydroascorbic and diketogulonic acids in sera of uraemic patients before and during haemodialysis, using the 2,4-dinitrophenylhydrazine method., Results: The results indicate that the levels of ascorbic and dehydroascorbic acids in uraemic sera are low in comparison with controls, but the levels of diketogulonic acid are higher than in healthy persons. The comparison of ratios between levels of these substances in uraemic sera and in normal sera indicate that the oxidation of ascorbic acid to dehydroascorbic in uraemia proceeds much slower than in controls. We propose that uraemic patients are characterized by pronounced deficiency of dehydroascorbic acid. A marked decrease of ascorbic and diketogulonic acid plasma levels, and a pronounced increase of dehydroascorbic acid levels during haemodialysis was observed. Ratios between plasma levels of ascorbic and dehydroascorbic acids following haemodialysis are the same as in healthy persons., Conclusion: Uraemic patients are characterized by marked deficiency of dehydroascorbic acid and this deficiency can be partially corrected by haemodialysis.

Impaired glucose transport across brain tissue barriers causes infantile seizures, developmental delay and acquired microcephaly. Since the first report in 1991 (De Vivo et al, NEJM, 1991) 17 patients have been identified with the glucose transporter protein syndrome (GTPS). The diagnostic feature of the syndrome is an unexplained hypoglycorrhachia in the clinical setting of an infantile epileptic encephalopathy. We review our clinical experience by highlighting one illustrative case: a 6-year old girl who presented at age 2 months with infantile seizures and hypoglycorrhachia. The CSF/blood glucose ratio was 0.33. DNA sequencing identified a missense mutation in exon 7 (C1108T). Erythrocyte GLUT1 immunoreactivity was normal. The time course of 3-O-methyl-glucose (3OMG) uptake by erythrocytes of the patient was 46% that of mother and father. The apparent Km was similar in all cases (2-4 mmol/L), but the apparent Vmax in the patient was only 28% that of the parents (500 versus 1,766 fmol/s/10(6)RBC; p < 0.004). In addition, a 3-month trial of oral thioctic acid also benefited the patient and increased the Vmax to 935 fmol/s/10(6) RBC (p < 3 x 10(-7)). Uptake of dehydroascorbic acid by erythrocytes of the patient was impaired to the same degree as that of 3OMG (Vmax was 38% of that of the mother's), which supports previous observations of GLUT1 being multifunctional. These studies confirm the molecular basis of the GTPS and the multifunctional role of GLUT1. The need for more effective treatment is compelling.