Erythrocyte Ascorbate Is a Potential Indicator of Steady-State Plasma Ascorbate Concentrations in Healthy Non-Fasting Individuals.

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.

Antioxidant Micronutrients and Oxidative Stress Biomarkers.

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.

A comparison of plasma glucose and oxidative status in lactating dairy cows in summer and autumn.

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.

Mechanisms of ascorbyl radical formation in human platelet-rich plasma.

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.

Massive and long-lasting decrease in vitamin C plasma levels as a consequence of extracorporeal circulation.

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.

New evaluation of fetal oxidative stress: measurement of the umbilical cord blood dimethyl sulfate-induced ascorbyl-free radical by an electron spin resonance method.

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.

Guinea pig ascorbate status predicts tetrahydrobiopterin plasma concentration and oxidation ratio in vivo.

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.

Antioxidant/oxidant status and cardiac function in bradykinin B(1)- and B(2)-receptor null mice.

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.

[Study on effects and mechanism of traditional Mongolian medicine wuweifengshi capsule on adjuvant arthritis in rats].

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.

Lung cancer biomarkers for the assessment of modified risk tobacco products: an oxidative stress perspective.

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.

Decreased IL-7 responsiveness is related to oxidative stress in HIV disease.

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.

A metabolomic study on the effect of intravascular laser blood irradiation on type 2 diabetic patients.

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.

Effects of vitamin C on cytotherapy-mediated muscle regeneration.

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.

Ascorbate and dehydroascorbic acid as biomarkers of oxidative stress: validity of clinical data depends on vacutainer system used.

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.

No effect of melatonin to modify surgical-stress response after major vascular surgery: a randomised placebo-controlled trial.

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.

Determination of dehydroascorbic acid in mouse tissues and plasma by using tris(2-carboxyethyl)phosphine hydrochloride as reductant in metaphosphoric acid/ethylenediaminetetraacetic acid solution.

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 impact of oxidative stress levels on the clinical effectiveness of sivelestat in treating acute lung injury: an electron spin resonance study.

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.

Pharmaco/ferrokinetic-related pro-oxidant activity of deferiprone in beta-thalassemia.

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.

Fast HPLC-ECD analysis of ascorbic acid, dehydroascorbic acid and uric acid.

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%.

Vitamin C activity of dehydroascorbic acid in humans--association between changes in the blood vitamin C concentration or urinary excretion after oral loading.

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.