Protective effects of vitamins C and E on the number of micronuclei in lymphocytes in smokers and their role in ascorbate free radical formation in plasma

Cigarette smoke is widely believed to increase free radical concentrations causing subsequent oxidative processes that lead to DNA damage and hence, to several diseases including lung cancer and atherosclerosis. Vitamin C is a reducing agent that can terminate free-radical-driven oxidation by being converted to a resonance-stabilized free radical. To investigate whether short-term supplementation with the antioxidants vitamin C and E decreases free-radical-driven oxidation and thus decreases DNA damage in smokers, we determined the frequency of micronuclei in lymphocytes in 24 subjects and monitored the electron paramagnetic resonance signal of ascorbate free radical formation in plasma. Further parameters comprised sister-chromatid exchanges and thiobarbituric acid-reactive substances. Twelve smokers and twelve non-smokers took 1000 mg ascorbic acid daily for 7 days and then 1000 mg ascorbic acid and 335.5 mg RRR-α-tocopherol daily for the next 7 days. Baseline concentrations of both vitamins C and E were lower and baseline numbers of micronuclei were higher (p < 0.0001) in smokers than in non-smokers. After 7 days of vitamins C and E, DNA damage as monitored by the number of micronulei was decreased in both, smokers and non-smokers, but it was more decreased in smokers as indicated by fewer micronuclei in peripheral lymphocytes (p < 0.05). Concomitantly, the plasma concentrations of vitamin C (p < 0.001) as well as the ascorbate free radical (p < 0.05) were increased. The corresponding values in non-smokers, however, did not change. Our findings show that increased ascorbate free radical formation in plasma after short-term supplementation with vitamins C and E can decrease the number of micronuclei in blood lymphocytes and thus DNA damage in smokers.     

Antioxidant supplementation reduces inter-individual variation in markers of oxidative damage

The aim of this study was to examine the effect of antioxidant supplementation on oxidative damage and chromosome stability in middle-aged men, smokers and non-smokers. A total of 124 men aged 48+/-6 years from Bratislava and from the rural population near Bratislava were investigated; 64 men (22 smokers and 42 non-smokers) were supplemented for 12 weeks with antioxidants, while 60 (25 smokers and 35 non-smokers) were given placebo. The daily antioxidant supplementation consisted of vitamin C (100 mg), vitamin E (100 mg), ss-carotene (6 mg), and selenium (50 microg). Samples of blood were taken on two occasions: At the beginning and at the end of the supplementation trial. Concentrations of dietary antioxidants, ferric reducing ability, malondialdehyde as an indicator of lipid peroxidation in plasma, micronuclei and chromosome aberrations in lymphocytes were measured. Antioxidant supplementation significantly increased the levels of vitamin C, ss-carotene, a-tocopherol and selenium in plasma. The overall antioxidant status of plasma measured as ferric reducing ability of plasma (FRAP) increased significantly (p<0.001) after antioxidant supplementation as well. The increase in antioxidant parameters after supplementation were consistently more pronounced in non-smokers than in smokers. There was a significant decrease of malondialdehyde concentration in the non-smokers, while in smokers the decrease of malondialdehyde concentration was not significant. Antioxidant supplementation did not affect the proportion of lymphocytes with micronuclei or the total number of micronuclei; however, there was a significant positive correlation (p<0.001) between the malondialdehyde concentration at the beginning of the supplementation trial and the difference in number of cells with micronuclei before and after the supplementation. The percent of cells with chromosome aberrations decreased significantly after antioxidant supplementation in smokers. These results indicate that a combined antioxidant supplementation (a) is effective even at very moderate doses; (b) significantly diminishes oxidative damage to lipids when it is high initially; and (c) is effective in decreasing chromosomal instability in lymphocytes of middle-aged men.     

Background level of 8-oxo-2'-deoxyguanosine in lymphocyte DNA does not correlate with the concentration of antioxidant vitamins in blood plasma.

Antioxidant vitamins, being effective free radical scavengers, can protect cellular DNA from oxidative damage. Therefore, in the present study we report on the relationship between basal level of 8-oxo-2'-deoxyguanosine in human lymphocyte DNA and the concentration of antioxidant vitamins (A, C and E). The average level of 8-oxo-2'-deoxyguanosine in lymphocytes of the studied group (15 males and 20 females) was 9.57 per 10(6) dG molecules. The endogenous level of ascorbic acid (vitamin C) in the plasma was, on average, 56.78 microM, while the mean concentrations of retinol (vitamin A) and alpha-tocopherol (vitamin E) were 1.24 uM and 25.74,uM, respectively. No correlations were found between individual 8-oxo-2 micro-deoxyguanosine levels in lymphocyte DNA and endogenous concentration of the vitamins.     

Short-term and long-term vitamin C supplementation in humans dose-dependently increases the resistance of plasma to ex vivo lipid peroxidation

To assess the effects of short-term and long-term vitamin C supplementation in humans on plasma antioxidant status and resistance to oxidative stress, plasma was obtained from 20 individuals before and 2h after oral administration of 2g of vitamin C, or from eight subjects enrolled in a vitamin C depletion-repletion study using increasing daily doses of vitamin C from 30 to 2500 mg. Plasma concentrations of ascorbate, but not other physiological antioxidants, increased significantly after short-term supplementation, and increased progressively in the long-term study with increasing vitamin C doses of up to 1000 mg/day. Upon incubation of plasma with a free radical initiator, ascorbate concentrations were positively correlated with the lag phase preceding detectable lipid peroxidation. We conclude that vitamin C supplementation in humans dose-dependently increases plasma ascorbate concentrations and, thus, the resistance of plasma to lipid peroxidation ex vivo. Plasma and body saturation with vitamin C in humans appears desirable to maximize antioxidant protection and lower risk of oxidative damage.     

Antioxidant vitamins C, E and beta-carotene reduce DNA damage before as well as after gamma-ray irradiation of human lymphocytes in vitro

The protective effect of Vitamins C, E and beta-carotene against gamma-ray-induced DNA damage in human lymphocytes in vitro was investigated. Cultured lymphocytes were exposed to increasing concentration of these vitamins either before or after irradiation with 2Gy of gamma-rays and DNA damage was estimated using micronucleus assay. A radioprotective effect was observed when antioxidant vitamins were added to cultured cells before as well after irradiation; the strongest effect was observed when they were added no later than 1h after irradiation. The radioprotective effect of vitamins also depended on their concentration; Vitamins C added at low concentration (1 microg/ml) before exposure of the cells to radiation prevented induction of micronuclei. Vitamin E at the concentration above 2 microg/ml decreased the level of radiation-induced micronuclei when compared to the cells irradiated without vitamin treatment. beta-Carotene was effective at all tested concentrations from 1 to 5 microg/ml and reduced the number of micronuclei in irradiated cells. The vitamins had no effect on radiation-induced cytotoxicity as measured by nuclear division index. The radioprotective action of antioxidant Vitamins C, E and beta-carotene was dependent upon their concentration as well as time and sequence of application.     

Dietary vitamin C down-regulates inflammatory gene expression in apoE4 smokers

The deleterious impact of cigarette smoking on cardiovascular health may be in part attributable to a free radical mediated proinflammatory response in circulating monocytes. In the current investigation, the impact of vitamin C supplementation on monocyte gene expression was determined in apoE4 smokers versus non-smokers. A total of 10 smokers and 11 non-smokers consumed 60mg/day of vitamin C for four weeks and a fasting blood sample was taken at baseline and post-intervention for the determination of plasma vitamin C and monocyte gene expression profiles using cDNA array and real time PCR. In apoE4 smokers, supplementation resulted in a 43% increase in plasma vitamin C concentrations. Furthermore, a number of genes were differentially expressed more than 2-fold in response to treatment, including a downregulation of the proinflammatory mediators tumor necrosis factor (TNF) beta, TNF receptor, neurotrophin-3 growth factor receptor, and monocyte chemoattractant protein 1 receptor. The study has identified a number of molecular mechanisms underlying the benefit of vitamin C supplementation in smokers.     

Vitamin C supplementation lowers urinary levels of 4-hydroperoxy-2-nonenal metabolites in humans

The lack of suitable biomarkers of oxidative stress is a common problem for antioxidant intervention studies in humans. We evaluated the efficacy of vitamin C supplementation in decreasing biomarkers of lipid peroxidation in nonsmokers and in cigarette smokers, a commonly studied, free-living human model of chronic oxidative stress. Participants received ascorbic acid (500mg twice per day) or placebo for 17 days in a double-blind, placebo-controlled, randomized crossover design study. The urinary biomarkers assessed and reported herein are derived from 4-hydroperoxy-2-nonenal (HPNE) and include the mercapturic acid (MA) conjugates of 4-hydroxy-2(E)-nonenal (HNE), 1,4-dihydroxy-2(E)-nonene (DHN), and 4-oxo-2(E)-nonenol(ONO). Vitamin C supplementation decreased the urinary concentrations of both ONO-MA (p=0.0013) and HNE-MA (p=0.0213) by ~30%; however, neither cigarette smoking nor sex affected these biomarkers. In contrast, vitamin C supplementation decreased urinary concentrations of DHN-MA (three-way interaction p=0.0304) in nonsmoking men compared with nonsmoking women (p<0.05), as well as in nonsmoking men compared with smoking men (p<0.05). Vitamin C supplementation also decreased (p=0.0092) urinary total of metabolites by ~20%. Thus, HPNE metabolites can be reduced favorably in response to improved plasma ascorbic acid concentrations, an effect due to ascorbic acid antioxidant function.     

Faster plasma vitamin E disappearance in smokers is normalized by vitamin C supplementation

Vitamin E disappearance is accelerated in cigarette smokers due to their increased oxidative stress and is inversely correlated with plasma vitamin C concentrations. Therefore, we hypothesized that ascorbic acid supplementation (500 mg, twice daily; 2 weeks) would normalize smokers' plasma alpha- and gamma-tocopherol disappearance rates and conducted a double-blind, placebo-controlled, randomized crossover investigation in smokers (n=11) and nonsmokers (n=13) given a single dose of deuterium-labeled alpha- and gamma-tocopherols (50 mg each d6-RRR-alpha and d2-RRR-gamma-tocopheryl acetate). During the placebo trial, smokers, compared with nonsmokers, had significantly (P<0.05) greater alpha- and gamma-tocopherol fractional disappearance rates and shorter half-lives. Ascorbic acid supplementation doubled (P<0.0001) plasma ascorbic acid concentrations in both groups and attenuated smokers', but not nonsmokers', plasma alpha- and gamma-tocopherol (P<0.05) fractional disappearance rates by 25% and 45%, respectively. Likewise, smokers' plasma deuterium-labeled alpha- and gamma-tocopherol concentrations were significantly higher (P<0.05) at 72 h during ascorbic acid supplementation compared with placebo. Ascorbic acid supplementation did not significantly change (P>0.05) time of maximal or maximal-labeled alpha- and gamma-tocopherol concentrations. Smokers' plasma F2alpha-isoprostanes were approximately 26% higher than nonsmokers (P>0.05) and were not affected by ascorbic acid supplementation in either group (P>0.05). In summary, cigarette smoking increased plasma alpha- and gamma-tocopherol fractional disappearance rates, suggesting that the oxidative stress from smoking oxidizes tocopherols and that plasma ascorbic acid reduces alpha- and gamma-tocopheroxyl radicals to nonoxidized forms, thereby decreasing vitamin E disappearance in humans.     

Exercise-induced endotoxemia: the effect of ascorbic acid supplementation

Strenuous, long-duration aerobic exercise results in endotoxemia due to increased plasma levels of lipopolysaccharide (LPS) leading to cytokine release, oxidative stress, and altered gastrointestinal function. However, the effect of short-term strenuous aerobic exercise either with or without antioxidant supplementation on exercise-induced endotoxemia is unknown. A significant increase in the concentration of bacterial LPS (endotoxin) was noted in the venous circulation of healthy volunteers following maximal acute aerobic exercise (0.14(-1) pre-exercise vs. 0.24(-1) postexercise, p <0.01). Plasma nitrite concentration also increased with exercise (0.09 +/- 0.05 nM x ml(-1) vs. 0.14 +/- 0.01 nM x ml(-1), p <0.05) as did ascorbate free radical levels (0.02 +/- 0.001 vs. 0.03 +/- 0.002 arbitrary units, p <0.05). Oral ascorbic acid supplementation (1000 mg) significantly increased plasma ascorbic acid concentration (29.45 mM x l(-1) to 121.22 mM x l(-1), p <0.05), and was associated with a decrease in plasma LPS and nitrite concentration before and after exercise (LPS: 0.01(-1); nitrite: 0.02 +/- 0.02 nM x ml(-1) vs. 0.02 +/- 0.03 nM x ml(-1)). Ascorbic acid supplementation led to a significant increase in ascorbate free radical levels both before (0.04 +/- 0.01 arbitrary units) and after exercise (0.06 +/- 0.02 arbitrary units, p <0.05). In conclusion, strenuous short-term aerobic exercise results in significant increases in plasma LPS levels (endotoxemia) together with increases in markers of oxidative stress. Supplementation with ascorbic acid, however, abolished the increase in LPS and nitrite but led to a significant increase in the ascorbate radical in plasma. The amelioration of exercise-induced endotoxemia by antioxidant pretreatment implies that it is a free radical-mediated process while the use of the ascorbate radical as a marker of oxidative stress in supplemented systems is limited.     

Vitamin C for DNA damage prevention

The ability of vitamin C to affect genetic damage was reviewed in human studies that used molecular epidemiology methods, including analysis of DNA adducts, DNA strand breakage (using the Comet assay), oxidative damage measured as levels of 8-oxo-7,8-dihydroxy-2'-deoxyguanosine (8-oxodG), cytogenetic analysis of chromosomal aberrations and micronuclei, and the induction of DNA repair proteins. The protective effect of vitamin C was observed at plasma levels>50μmol/l. Vitamin C supplementation decreased the frequency of chromosomal aberrations in groups with insufficient dietary intake who were occupationally exposed to mutagens, and also decreased the sensitivity to mutagens as assessed using the bleomycin assay. High vitamin C levels in plasma decreased the frequency of genomic translocations in groups exposed to ionizing radiation or c-PAHs in polluted air. The frequency of micronuclei was decreased by vitamin C supplementation in smokers challenged with γ-irradiation, and higher vitamin C levels in plasma counteracted the damage induced by air pollution. The prevalence of DNA adducts inversely correlated with vitamin C levels in groups environmentally exposed to high concentrations of c-PAHs. Increased vitamin C levels decreased DNA strand breakage induced by air pollution. Oxidative damage (8-oxodG levels) was decreased by vitamin C supplementation in groups with plasma levels>50μmol/l exposed to PM2.5 and c-PAHs. Modulation of DNA repair by vitamin C supplementation was observed both in poorly nourished subjects and in groups with vitamin C plasma levels>50μmol/l exposed to higher concentrations of c-PAHs. It is possible that the impact of vitamin C on DNA damage depends both on background values of vitamin C in the individual as well as on the level of exposure to xenobiotics or oxidative stress.     

Docosahexaenoic acid supplementation-increased oxidative damage in bone marrow DNA in aged rats and its relation to antioxidant vitamins

We compared the influence of docosahexaenoic acid (DHA) supplementation on oxidative DNA damage in bone marrow between young and aged rats. As a marker of oxidative DNA damage, 8-hydroxydeoxy-guanosine (8-OHdG) in DNA was analyzed. Young (5-week-old) and aged (100-week-old) female Wistar rats were given DHA (300mg/kg body weight/day) or vehicle (control) orally for 12 weeks. The 8-OHdG in the bone marrow in the aged DHA group was significantly higher than that in the other groups. Vitamin E concentrations, however, did not differ among the groups regardless of the DHA supplementation. Vitamin C (ascorbic acid) concentrations in the aged control group were approximately 1/2 those in the young control group. The concentrations of vitamin C tended to be higher in the young DHA group and lower in the aged DHA group when compared to their respective control groups. Changes in the concentrations of vitamin C and vitamin E in plasma were similar to those in the bone marrow. The activity of hepatic l-gulono- γ -lactone oxidase, an enzyme responsible for vitamin C synthesis, corresponded well to the concentrations of vitamin C in the bone marrow and the plasma. These results suggest that in aged rats, but not young rats, excess supplementation of DHA induces oxidative DNA damage in bone marrow and that the decrease in vitamin C synthesis in aged rats is involved in the mechanisms of DNA damage.     

AZT induces oxidative damage to cardiac mitochondria: protective effect of vitamins C and E

AZT (zidovudine) is a potent inhibitor of HIV replication and a major antiretroviral drug used for AIDS treatment. A major limitation in the use of AZT is the occurrence of severe side effects. The aim of this work was to test whether AZT causes oxidative damage to heart mitochondria and whether this can be prevented by supranutritional doses of antioxidant vitamins. An experimental animal model was used in which mice were treated with AZT for 35 days (10 mg/kg/day) in drinking water. Animals treated with antioxidant vitamins were fed the same diet as controls but supplemented with vitamins C (ascorbic acid, 10 g/ kg diet) and E (alpha-dl-tocopherol, 0.6 g/kg diet) for 65 days before sacrifice. This resulted in a daily intake of 1250 mg/kg/day (vitamin C) and 75 mg/kg/day (vitamin E). Cardiac mitochondrial DNA (mtDNA) of mice treated with AZT had over 120% more oxo-dG (8-oxo-7,8-dihydro-2'-deoxyguanosine, which is a biomarker of oxidative damage to DNA) in their mitochondrial DNA than untreated controls. AZT treatment also caused an increase in mitochondrial lipid peroxidation and an oxidation of mitochondrial glutathione. Dietary supplementation with supranutritional doses of the antioxidant vitamins C and E protected against these signs of mitochondrial oxidative stress. The oxidative effects of AZT are probably due to an increase in production of reactive oxygen species by mitochondria of AZT-treated animals, raising the possibility that oxidative stress may play an important role in the cardiotoxicity of AZT.     

Interaction of vitamin C and vitamin E during free radical stress in plasma: An ESR study

To study the interaction of the antioxidant vitamins C and E in a biological system, we used electron spin resonance (ESR) spectroscopy to make serial measurement of ascorbate tocopheroxyl free radicals in plasma subjected to continuous free radical-mediated oxidative stress. Upon initiation of a continuous oxidative stress, we observed an immediate increase in the concentration of ascorbate radical, which reached a peak, and then steadily declined. Only after the virtual disappearance of the ascorbate radical did we observe the appearance of the tocopheroxyl radical. These data are consistent with the hypothesis that ascorbate is the terminal small-molecule antioxidant in biological systems. This is the first experimental demonstration that the predicted thermodynamic hierarchy of ascorbate, -tocopherol, and their free radicals holds in a biological system containing endogenous levels of these antioxidant vitamins.     

Phospholipid Hydroperoxides and Lipid Peroxidation in Liver and Plasma of ODS Rats Supplemented with α-Tocopherol and Ascorbic Acid

Forty-five mutant male ODs rats, unable to synthesize ascorbic acid, were fed nine diets containing 5, 50 or 250 mg of vitamin E/kg diet and 150,300 or 900 mg of vitamin C/kg diet for 21 days. The concentrations of vitamins C and E increased in liver and plasma in relation to the level of these vitamins in the diet. Vitamin C dietary supplementation increased the plasma vitamin E content at low levels of vitamin E intake, supporting the concept of an in vivo synergism between both antioxidant vitamins. Vitamin C, at the dietary levels studied, did not affect the lipid peroxidation. Vitamin E decreased liver and plasma endogenous levels of thiobarbituric acid-reactive substances and liver sensitivity to non-enzymatic lipid peroxidation. This was confirmed by a highly specific assay of lipid hydroperoxides using high performance liquid chromatography with chemiluminescence detection. The hepatic concentration of both phosphatidylcholine and phosphatidylethanolamine hydroperoxides decreased as the vitamin E content of the diet increased. The results show for the first time the capacity of vitamin E to protect against peroxidation of major phospho-lipids in vivo under basal unstressed conditions.     

Deoxycytidine glyoxal: lesion induction and evidence of repair following vitamin C supplementation in vivo

Oxidative DNA damage is postulated to be involved in carcinogenesis, and as a consequence, dietary antioxidants have received much interest. A recent report indicates that vitamin C facilitates the decomposition of hydroperoxides in vitro, generating reactive aldehydes. We present evidence for the in vivo generation of glyoxal, an established product of lipid peroxidation, glucose/ascorbate autoxidation, or free radical attack of deoxyribose, following supplementation of volunteers with 400 mg/d vitamin C. Utilizing a monoclonal antibody to a deoxycytidine-glyoxal adduct (gdC), we measured DNA lesion levels in peripheral blood mononuclear cells. Supplementation resulted in significant (p =.001) increases in gdC levels at weeks 11, 16, and 21, with corresponding increases in plasma malondialdehyde levels and, coupled with previous findings, is strongly suggestive of a pro-oxidative effect. However, continued supplementation revealed a highly significant (p =.0001) reduction in gdC levels. Simultaneous analysis of cyclobutane thymine dimers revealed no increase upon supplementation but, as with gdC, levels decreased. Although no single mechanism is identified, our data demonstrate a pro-oxidant event in the generation of reactive aldehydes following vitamin C supplementation in vivo. These results are also consistent with our hypothesis for a role of vitamin C in an adaptive/repair response and indicate that nucleotide excision repair specifically may be affected.     

Influence of vitamin E and C supplementation on lipoprotein oxidation in patients with Alzheimer's disease.

Because increased oxidation is an important feature of Alzheimer's disease (AD) and low concentrations of antioxidant vitamins C and E have been observed in cerebrospinal fluid (CSF) of AD patients, supplementation with these antioxidants might delay the development of AD. Major targets for oxidation in brain are lipids and lipoproteins. We studied whether supplementation with antioxidative vitamins E and C can increase their concentrations not only in plasma but also in CSF, and as a consequence decrease the susceptibility of lipoproteins to in vitro oxidation. Two groups, each consisting of 10 patients with AD, were for 1 month supplemented daily with either a combination of 400 IU vitamin E and 1000 mg vitamin C, or 400 IU vitamin E alone. We found that supplementation with vitamin E and C significantly increased the concentrations of both vitamins in plasma and CSF. Importantly, the abnormally low concentrations of vitamin C were returned to normal level following treatment. As a consequence, susceptibility of CSF and plasma lipoproteins to in vitro oxidation was significantly decreased. In contrast, the supplementation with vitamin E alone significantly increased its CSF and plasma concentrations, but was unable to decrease the lipoprotein oxidizability. These findings document a superiority of a combined vitamin E + C supplementation over a vitamin E supplementation alone in AD and provide a biochemical basis for its use.     

Increased frequency of micronuclei in B and T8 lymphocytes from smokers

The frequency of micronuclei was measured in peripheral B lymphocytes and some T lymphocyte subpopulations from 5 medium-tar cigarette smokers, and 5 non-smokers with no regular exposure to tobacco smoke. The peripheral lymphocytes were stimulated in vitro with phytohemagglutinin and B lymphocytes and the various T lymphocyte subsets were classified by a recently developed MAC (Morphology, Antibody, Chromosomes) method which allows the immunologic identification of different cell lineages. An increased frequency of micronuclei was observed in B and especially in the suppressor/cytotoxic T8 lymphocytes from smokers, as compared with non-smoker values. In non-smoker cultures, no differences in the frequency of micronuclei were observed among the different T lymphocyte subsets. In these cultures, B cells tended to have a higher frequency of micronuclei than did T cells. The proportions of B cells and the various T subpopulations among mitotic and interphasic lymphocytes from smokers and non-smokers were also determined. The proportions of B cells and T cell subsets among all mitotic lymphocytes were similar in smokers and non-smokers. Contrarily, a significant decrease in the proportion of T8 lymphocytes among all interphasic lymphocytes was observed in cultures derived from smokers.     

The influence of dietary vitamin C and E supplementation on the physiological response of pirarucu, Arapaima gigas, in net culture

This study evaluated the efficacy of dietary vitamin C (ascorbic acid or AA), vitamin E (alpha-tocopherol or alpha-T), and C+E supplementation on the blood parameters of Arapaima gigas grown in net cages for 45 days. Four treatments were tested: control (commercial feed); C800; E500 and C+E (800+500) with supplementation of 800 mg AA kg(-1), 500 mg alpha-T kg(-1) and 800+500 mg AA+alpha-T kg(-1), respectively. Hematocrit (Ht), red blood cells (RBC), and hemoglobin concentration (Hb) (oxidative status indicators), thrombocytes and leukocytes (immunological indicators), plasma protein and glucose were evaluated. Fish fed vitamin C and C+E supplemented diets showed greater weight gain and survival. Dietary vitamin C and C+E diet supplementation resulted in increased Ht, Hb, RBC, MCHC, total leukocytes, total proteins, thrombocytes and eosinophils compared to the control and alpha-T. The alpha-tocopherol-supplemented diet reduced the number of total thrombocytes, lymphocytes and neutrophils and increased glucose and eosinophils relatively to the control. In general, leukocytes and thrombocytes were good indicators of the efficiency of vitamin on the defense mechanism of the A. gigas reared in cages. Results indicate that high alpha-T diet supplementation provides no benefit for the maintenance of the oxidative or the immunological status of A. gigas. However, it was demonstrated that high dietary AA improves A. gigas immunological status. Red blood cell indices and immune system indicators showed no synergistic effect between the vitamins after supplementing the A. gigas diet with alpha-T+AA.     

Vitamin C - vitamin E interaction in juvenile lake sturgeon (Acipenser fulvescens R.), a fish able to synthesize ascorbic acid

The hypothesis that vitamin C interacts with vitamin E in vivo was investigated in juvenile lake sturgeon. Ten-month old lake sturgeon were fed diets supplemented with either 0 or 1250 mg ascorbic acid/kg diet concomitantly with either 0 or 200 mg α tocopherol/kg diet for 7 weeks at 17°C. Dietary vitamin C supplement resulted in significant increases of ascorbate concentrations in the posterior kidney and liver of sturgeon. Dietary vitamin E omission affected liver concentrations of α-tocopherol (10.0 ± 4.5 μg/g) in comparison to sturgeon fed a diet supplemented with vitamin E and vitamin C (99.5 ± 22.9 μg/g). Dietary vitamin C supplement decreased liver α-tocopherol concentration in vitamin E-deprived sturgeon. Also, vitamin E supplement lowered posterior kidney and liver ascorbic acid concentrations in vitamin C-deprived sturgeon. Gulonolactone oxidase and dehydroascorbic acid reductase activities were stimulated in groups fed vitamin C. Thiobarbituric acid-reactive substances concentrations (an indicator of lipid peroxidation) were higher in sturgeon fed either of vitamins as compared to sturgeon deprived of both vitamins. The results suggested that large doses of vitamins C and E may be prooxidant in vivo.     

Phycoerythrin fluorescence-based assay for peroxy radicals: a screen for biologically relevant protective agents

Under the conditions of this assay, antioxidants that react rapidly with peroxy free radicals (e.g., ascorbate, vitamin E analogs, urate), protect phycoerythrin completely from damage by such radicals generated by thermal decomposition of 2,2'-azobis(2-amidinopropane); other compounds provide partial concentration-dependent protection. Change in phycoerythrin fluorescence emission with time provides a measure of the rate of free radical damage. The assay exploits the unusual reactivity of phycoerythrin toward these peroxy radicals. On a molar basis, phycoerythrin reacts with these radicals over 100-fold slower than do ascorbate or vitamin E analogs, but over 60-fold faster than other proteins. Applications of this assay to the estimation of the peroxy radical scavenging capacity of human plasma are described, and to the comparison of the scavenging properties of several proteins and of DNA, of vitamins and their derivatives, of catecholamine neurotransmitters, and of a variety of other low molecular weight biological compounds.