Liver Ascorbate Research Articles

Vitamin C modulates the levels of several proteins of the mitochondrial complex III and its activity in the mouse liver

Ascorbate is a crucial antioxidant and essential cofactor of biosynthetic and regulatory enzymes. Unlike humans, mice can synthesize ascorbate thanks to the key enzyme gulonolactone oxidase (Gulo). In the present study, we used the Gulo−/− mouse model, which cannot synthesize their own ascorbate to determine the impact of this vitamin on the liver proteome of specific subcellular organelles. We performed label-free Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS) global quantitative proteomic profiling to identify and quantify proteins in microsomal enriched liver extracts (MEE) from Gulo−/− mice treated with 0–0.4% (w/v) ascorbate in drinking water until the age of four months. Using a principal component analysis on normalized and imputed data of the label-free protein quantifications, a sex-based difference in MEE proteome profiles was observed for all the different ascorbate treated mice. Suboptimal hepatic ascorbate concentrations affected the levels of more proteins and hence biochemical processes in females than in males. Nevertheless, Pearson correlation analyses revealed that the MS intensities of various proteins involved in complement activation inversely correlated with liver ascorbate concentrations in both Gulo−/− males and females. Moreover, the correlation analyses also indicated that several proteins in the mitochondrial complex III of the electron transport chain positively correlated with liver ascorbate concentrations in both Gulo−/− females and males. Consequently, the mitochondrial complex III activity in Gulo−/− female and male mice treated with suboptimal hepatic concentrations of ascorbate was significantly lower than Gulo−/− mice treated with optimal ascorbate concentration. Finally, the whole liver of ascorbate-deficient Gulo−/− mice exhibited lower ATP levels and increased reactive oxygen species. These findings provide new information on how ascorbate deficiency potentially induces mitochondrial dysfunction in the liver of mice.

305 - High-Dose Ascorbate Administration Increases Tumor Ascorbate Levels and Decreases Hypoxia-Inducible Factor-1 Activity and Tumor Growth in Mice

The activation of the transcription factor hypoxia-inducible factor (HIF)-1 is associated with an aggressive tumor phenotype. HIF-1 is regulated by hydroxylase enzymes that require vitamin C (ascorbate) as cofactor and analyses of clinical tumor samples and dietary supplementation studies in mice have demonstrated an inverse correlation between tumor ascorbate levels and HIF-1 activity. In this study we investigated whether pharmacological doses of ascorbate can affect tumor ascorbate levels and exert an anti-tumor effect in tumor-bearing Gulo-/- mice, a model of human ascorbate dependency. Ascorbate concentrations peaked within an hour after a single intra-peritoneal injection (1g/kg), with millimolar levels measureable in plasma, and increased levels in liver and tumor tissues. Plasma and liver ascorbate returned to baseline within 16h, but tumor levels remained elevated for 48h. Modelling ascorbate uptake and stability in tumors suggests that increased stability under hypoxia may account for its prolonged presence within the tumor tissue. The expression of HIF-1 and its target proteins was down-regulated during the first 4h post-injection, and increased again after 16–48h. Increased tumor ascorbate levels could be maintained with repeated daily or alternate day administration. HIF-1 and vascular endothelial growth factor proteins were inversely correlated with tumor ascorbate and were significantly down-regulated in tumors when ascorbate was administered daily. Increased tumor ascorbate was associated with reduced microvessel density, tumor hypoxia and tumor growth. These results support the suppression of the hypoxic response as being a possible anti-tumor activity of ascorbate that may be useful in a clinical setting.

Pharmacokinetic and anti-cancer properties of high dose ascorbate in solid tumours of ascorbate-dependent mice

Despite recent evidence for an anti-tumour role for high-dose ascorbate, potential mechanisms of action are still unclear. At mM concentrations that are achieved with high-dose intravenous administration, autoxidation of ascorbate can generate cytotoxic levels of H2O2. Ascorbate is also a required co-factor for the hydroxylases that suppress the transcription factor hypoxia-inducible factor (HIF-1). HIF-1 supports an aggressive tumour phenotype and is associated with poor prognosis, and previous studies have shown that optimizing intracellular ascorbate levels down-regulates HIF-1 activation. In this study we have simultaneously measured ascorbate concentrations and the HIF-1 pathway activity in tumour tissue following high dose ascorbate administration, and have studied tumour growth and physiology. Gulo-/- mice, a model of the human ascorbate dependency condition, were implanted with syngeneic Lewis lung tumours, 1g/kg ascorbate was administered into the peritoneum, and ascorbate concentrations were monitored in plasma, liver and tumours. Ascorbate levels peaked within 30min, and although plasma and liver ascorbate returned to baseline within 16h, tumour levels remained elevated for 48h, possibly reflecting increased stability in the hypoxic tumour environment. The expression of HIF-1 and its target proteins was down-regulated with tumour ascorbate uptake. Elevated tumour ascorbate levels could be maintained with daily administration, and HIF-1 and vascular endothelial growth factor protein levels were reduced in these conditions. Increased tumour ascorbate was associated with slowed tumour growth, reduced tumour microvessel density and decreased hypoxia. Alternate day administration of ascorbate resulted in lower tumour levels and did not consistently decrease HIF-1 pathway activity. Levels of sodium-dependent vitamin C transporters 1 and 2 were not clearly associated with ascorbate accumulation by murine tumour cells in vitro or in vivo. Our results support the suppression of the hypoxic response by ascorbate as a plausible mechanism of action of its anti-tumour activity, and this may be useful in a clinical setting.

Ameliorative Effect of Vitamin C on the Altered Tissue Ascorbate Levels in Wistar Rats Exposed to Chlorpyrifos and Lead Acetate for 98 Days

The present study was designed to evaluate the effects of chlorpyrifos, lead acetate, vitamin C alone and in combination on tissue ascorbate levels in Wistar rats. Rats of 150–200 g body weight were divided into eight groups of six animals each and were subjected to various daily oral treatment regimens for 98 days. Group C served as control receiving only corn oil, group CP received chlorpyrifos at 5.5 mg/kg in corn oil, group L received lead acetate at 100 ppm in water, whereas animals in group CP + L received a combination of chlorpyrifos at 5.5 mg/kg in corn oil and lead acetate at 100 ppm in water. Group VC received vitamin C at 100 mg/kg in water, group CP + VC received a combination of chlorpyrifos at 5.5 mg/kg and vitamin C at 100 mg/kg, group L + VC received lead acetate at 100 ppm in water and vitamin C at 100 mg/kg and group CP + L + VC received chlorpyrifos at 5.5 mg/kg, lead acetate at 100 ppm in water and vitamin C at 100 mg/kg. After 98-day exposure of chlorpyrifos and lead acetate, the concentration of ascorbate in liver, kidney and lung homogenates was decreased significantly as compared to the control group, besides a significant decrease was also observed in the homogenates of brain and heart tissues of chlorpyrifos and lead treatment groups, respectively. In groups receiving both chlorpyrifos and lead acetate, the concentration of ascorbate was significantly decreased in kidney, lung and liver homogenates. The study demonstrated that treatment of chlorpyriphos and lead treated rats with vitamin C significantly altered some of the important haematological parameters revealing the protective effect of this vitamin against haematological alterations induced by chlorpyriphos and lead.

Evaluation of Beta-Carotene, Iron and other Micronutrient Status of Rats Fed Sun or Shade-Dried Fluted Pumpkin (Telfariaoccidentalis) Leaves and its Product, the Leaf Curd

The effects of differently processed leaves and the curd of fluted pumpkin (Telfariaoccidentalis)on beta carotene, iron, ascorbate, zinc, copper and calcium status of rats were evaluated. Fluted pumpkin leaf was divided into four (4) portions. One was shade-dried, another was sun-dried and the other was used to produce leaf curd. The last portion was not processed and served as the control. All the processed samples were milled to fine flour andeach of the pumpkin leaf flour was incorporated into rat chow for a 28-day study period. Twenty (20) male weanling rats were divided into four (4) groups of five (5) rats each. They were housed in individual metabolism cages and fed diets and water ad libitum. Blood samples were drawn before and after the experiment by ocular puncture and were used for biochemical analysis. Blood plasma was used to determine haemoglobin while serum was used to determine pro- vitamin A, ascorbate, ferritin, iron, copper, zinc and calcium. The liver was individually removed and analysed for liver ferritin, ascorbate and other micronutrients. The results showed that the rats fed rat chow supplemented with dried leaf curd had higher serum beta carotene, ascorbate, and ferritin than those of the other groups. The rats fed rat chow supplemented with shade dried-fluted pumpkin leaf had higher (p<0.05) haemoglobin level, liver beta-carotene and ascorbate than the other groups. The result showed that processing fluted pumpkin leaf into leaf curd improved serum beta-carotene, serum and liver ascorbate, serum ferritin and liver iron of rats more than other processing methods.

Different effects of ascorbate deprivation and classical vascular nitrate tolerance on aldehyde dehydrogenase‐catalysed bioactivation of nitroglycerin

Vascular tolerance to nitroglycerin (GTN) may be caused by impaired GTN bioactivation due to inactivation of mitochondrial aldehyde dehydrogenase (ALDH2). As relaxation to GTN is reduced but still sensitive to ALDH2 inhibitors in ascorbate deficiency, we compared the contribution of ALDH2 inactivation to GTN hyposensitivity in ascorbate deficiency and classical in vivo nitrate tolerance. Guinea pigs were fed standard or ascorbate-free diet for 2 weeks. Reversibility was tested by feeding ascorbate-deficient animals standard diet for 1 week. Nitrate tolerance was induced by subcutaneous injection of 50 mg x kg(-1) GTN 4 times daily for 3 days. Ascorbate levels were determined in plasma, blood vessels, heart and liver. GTN-induced relaxation was measured as isometric tension of aortic rings; vascular GTN biotransformation was assayed as formation of 1,2- and 1,3-glyceryl dinitrate (GDN). Two weeks of ascorbate deprivation had no effect on relaxation to nitric oxide but reduced the potency of GTN approximately 10-fold in a fully reversible manner. GTN-induced relaxation was similarly reduced in nitrate tolerance but not further attenuated by ALDH inhibitors. Nitrate tolerance reduced ascorbate plasma levels without affecting ascorbate in blood vessels, liver and heart. GTN denitration was significantly diminished in nitrate-tolerant and ascorbate-deficient rings. However, while the approximately 10-fold preferential 1,2-GDN formation, indicative for active ALDH2, had been retained in ascorbate deficiency, selectivity was largely lost in nitrate tolerance. These results indicate that nitrate tolerance is associated with ALDH2 inactivation, whereas ascorbate deficiency possibly results in down-regulation of ALDH2 expression.

Protective Effects of Coenzyme Q10 on Decreased Oxidative Stress Resistance Induced by Simvastatin

The effects of simvastatin, an inhibitor of 3-hydroxy-3-methylglutaryl CoA reductase (HMG-CoA reductase), on oxidative stress resistance and the protective effects of coenzyme Q (CoQ) were investigated. When simvastatin was administered orally to mice, the levels of oxidized and reduced CoQ9 and CoQ10 in serum, liver, and heart, decreased significantly when compared to those of control. The levels of thiobarbituric acid reactive substances induced by Fe2+-ascorbate in liver and heart mitochondria also increased significantly with simvastatin. Furthermore, cultured cardiac myocytes treated with simvastatin exhibited less resistance to oxidative stress, decreased time to the cessation of spontaneous beating in response to H2O2 addition, and decreased responsiveness to electrical field stimulation. These results suggested that oral administration of simvastatin suppresses the biosynthesis of CoQ, which shares the same biosynthesis pathway as cholesterol up to farnesyl pyrophosphate, thus compromising the physiological function of reduced CoQ, which possesses antioxidant activity. However, these undesirable effects induced by simvastatin were alleviated by coadministering CoQ10 with simvastatin to mice. Simvastatin also reduced the activity of NADPH-CoQ reductase, a biological enzyme that converts oxidized CoQ to the corresponding reduced CoQ, while CoQ10 administration improved it. These findings may also support the efficacy of coadministering CoQ10 with statins.

Contribution of insulin to the ascorbate recycling system in the chicken liver

ABSTRACTThe effect of insulin on the ascorbate recycling system in the chicken liver was examined. First, insulin was injected subcutaneously into the chicken, and liver glutathione‐dependent dehydroascorbate reductase (GSH‐DHAR) activity was determined. Insulin increased liver GSH‐DHAR activity, but did not affect plasma and liver ascorbate concentration. Dehydroascorbate increased plasma and liver ascorbate levels, and liver GSH‐DHAR activity. However, distinct changes in plasma insulin level were not observed by dehydroascorbate injection. In addition, reduction of external dehydroascorbate in cultured chicken hepatocytes could not be observed in an insulin‐deprived culture, although the cells reduced external dehydroascorbate in a serum‐free culture with insulin. We concluded that insulin affects the ascorbate recycling system as an essential factor in the chicken liver.

Protective role of dehydroascorbate in rat liver ischemia-reperfusion injury

Oxidative stress plays an important role in liver ischemia/reperfusion (I/R) injury. Thus, enhancing the liver antioxidant capacity could be a promising therapeutic strategy. Ascorbate (AA) is considered the perfect antioxidant, but its therapeutic efficacy is greatly limited by its slow achievement of high intracellular levels. This might be circumvented by administering dehydroascorbate (DHA), which presents a several-fold greater uptake than AA, and undergoes rapid intracellular reduction to AA. Thus, our aim was to assess the protective role of DHA in liver I/R injury. Wistar rats (200-300 g bw) were pretreated iv with different doses of AA or DHA 20 min before liver ischemia, followed by 6 h reperfusion. Liver damage was assessed by biochemical and morphological indices. DHA pretreatment induced a rapid increase in liver ascorbate levels, significantly higher than findings for AA, without any significant reduction in glutathione levels. Liver damage during I/R in controls showed significant increases in serum transaminases and hepatic thiobarbituric acid reactive substances with alterations of liver morphology. DHA administration induced a clear, significant protection against I/R injury, whereas liver damage was only moderately prevented by AA. DHA might represent a simple, effective therapeutic option to prevent liver damage associated with ischemia/reperfusion.

Requirements of vitamin C (L-ascorbyl-2-monophosphate-Mg and L-ascorbyl-2-monophosphate-Na) and its effects on immune responses of grouper, Epinephelus malabaricus

An 8-week feeding trial was conducted to evaluate two vitamin C derivatives, L-ascorbyl-2-monophosphate-Mg (C2MP-Mg) and L-ascorbyl-2-monophosphate-Na (C2MP-Na), to satisfy the vitamin C requirement and to test their effects on the immune responses of juvenile grouper, Epinephelus malabaricus. C2MP-Mg and C2MP-Na were each supplemented at 20, 50, 80, 150, 250, and 400 mg kg−1 diet in the basal diet providing of 7, 18, 31, 51, 93, 145 mg ascorbic acid (AA) equivalent of C2MP-Mg kg−1 diet and 4, 10, 17, 31, 47, 77 mg ascorbic acid (AA) equivalent of C2MP-Na kg−1 diet, respectively. Basal diet without AA supplementation was included as control. Each diet was fed to triplicate groups of grouper (mean initial weight 3.20 ± 0.05 g). Fish fed diets supplemented with either C2MP-Mg or C2MP-Na had significantly (P < 0.05) greater weight gain (WG), feed efficiency and survival than those fed the unsupplemented control diet. Liver ascorbate concentrations in fish generally increased as dietary C2MP-Mg or C2MP-Na supplementation level increased. Haemolytic complement activity was higher in fish fed diets supplemented with 92 mg AA equivalent of C2MP-Mg kg−1 or 10–17 mg AA equivalent of C2MP-Na kg−1 than fish fed the unsupplemented control diet. Lysozyme activity was higher in fish fed ≥51 mg AA equivalent of C2MP-Mg kg−1 or ≥47 mg AA equivalent of C2MP-Na kg−1 than fish fed the unsupplemented control diet. Analysis by broken-line regression of WG indicated that the adequate dietary vitamin C concentration from each vitamin C derivative in growing grouper is 17.9 mg AA equivalent of 2MP-Mg kg−1 and 8.3 mg AA equivalent of C2MP-Na kg−1, and it also indicated that C2MP-Mg is about 46% as effective as C2MP-Na in meeting the vitamin C requirement of grouper.

Carcass selenium loss as an indicator of stress in barge transported chinook salmon (Oncorhynchus tshawytscha Walbaum) smolts

Two salmon smolt barge transport experiments were conducted to measure tissue trace element selenium (Se) loss during the 30 h transport 500 km downriver past seven hydroelectric dams on the Columbia River. Carcass Se was measured before and after the barge trip. Liver glutathione peroxidase (GTPX) activity and total ascorbate concentrations were assayed to correlate Se loss with GTPX levels. Hatchery-reared smolt chinook salmon, Oncorhynchus tshawytscha (Walbaum), were tested before and after barging. Liver ascorbate concentrations, measured by high-performance liquid chromatography techniques, showed levels adequate to support GTPX activity. Salmon smolts lost up to 20% of carcass tissue Se during the 30 h barge confinement. Selenium was analysed by analytical polarography. Samples collected after two barging episodes showed Se loss and concomitant elevated liver GTPX activity after the transport. Tissue Se loss may be useful to measure stress in fish, and increasing tissue Se in hatchery salmon smolts prior to release and transport may be warranted.

Oxidative DNA damage in vitamin C-supplemented guinea pigs after intratracheal instillation of diesel exhaust particles

The health effects of diesel exhaust particles (DEP) are thought to involve oxidative damage. We have investigated the effect of intratracheal DEP instillation to guinea pigs in three groups of 12 animals each given 0, 0.7, or 2.1 mg. Five days later guinea pigs exposed to DEP had increased levels of oxidized amino acids (γ-glutamyl semialdehyde), DNA strand breaks, and 7-hydro-8-oxo-2′-deoxyguanosine (8-oxodG) in the lung. Bulky DNA ad- ducts were not significantly elevated in the lung. The antioxidant enzyme activity of glutathione reductase was increased in the lung of DEP-exposed guinea pigs, whereas glutathione peroxidase and superoxide dismutase enzyme activities were unaltered. There was no difference in DNA strand breaks in lymphocytes or urinary excretion of 8-oxodG at the two doses tested. Protein oxidations in plasma and in erythrocytes were not altered by DEP exposure. The concentrations of ascorbate in liver, lung, and plasma were unaltered by the DEP exposure. The results indicate that in guinea pigs DEP causes oxidative DNA damage rather than bulky DNA adducts in the lung. Guinea pigs, which are similar to humans with respect to vitamin C metabolism, may serve as a new model for the study of oxidative damage induced by particulate matter.

Increased Oxidative Damage in Vitamin C Deficiency is Accompanied by Induction of Ascorbic Acid Recycling Capacity in Young But Not Mature Guinea Pigs

Ascorbic acid (AA) recycling, i.e. the intracellular regeneration of AA from its oxidized forms semidehydroascorbyl radical and dehydroascorbic acid (DHA), presumably has a key function in maintaining redox homeostasis. Like humans, guinea pigs cannot synthesize AA. In the present paper, the effects of severe AA deficiency on the AA recycling capacity in erythrocytes (RBCs) and liver homogenates were studied in young and mature guinea pigs. Twelve animals of each age category were divided into weight-matched groups of six animals and fed either an AA deficient or sufficient diet. After 5 weeks, they were sacrificed and RBC and liver ascorbate recycling was estimated along with glutathione, tocopherols, AA, SOD, and malondialdehyde (MDA). For young animals, AA recycling capacity was significantly increased in RBCs from the deficient group as compared to the controls ( p <0.001). RBC MDA was not increased by incubation with t -butylhydroperoxide (TBH) while the initial MDA level was significantly elevated ( p <0.001). In mature animals, neither RBC recycling nor MDA levels depended on AA status. Liver recycling capacity was not affected by age or diet, while liver MDA was significantly higher in young but not in mature deficient animals compared to respective controls ( p <0.01). In young animals, incubation with TBH resulted in significant MDA formation in the deficient compared to sufficient animals in both liver and RBCs ( p <0.05). RBC glutathione was not significantly changed by age or diet indicating that the observed changes in recycling capacity are enzyme dependent. The results suggest that young guinea pigs may have a more adaptable antioxidant defense system compared to mature animals while also being more susceptible to oxidative stress.

Interaction between dietary levels of iron and vitamin C on growth, hematology, immune response and resistance of channel catfish (Ictalurus punctatus) to Edwardsiella ictaluri challenge

Nine egg-white-based diets supplemented with three levels of vitamin C from l-ascorbyl-2-polyphosphate (0, 50 and 3000 mg/kg) for each of the three levels of iron from iron methionine (0, 30 and 300 mg/kg) were each fed to juvenile channel catfish in triplicate aquaria twice daily to apparent satiation for 14 weeks. Fish fed with iron-deficient diets had decreased weight gain, feed conversion and survival. Supplementation of ascorbic acid to the iron-deficient diets further decreased weight gain and survival. Feed conversion was not significantly affected by dietary level of vitamin C or iron and vitamin C interaction. No gross signs of vitamin C deficiency were observed. Total cell count (TCC), red blood cell count (RBC), hematocrit (HCT) and hemoglobin (Hb) were significantly lower for fish fed the iron deficient diet. TCC and RBC significantly increased when 3000 mg of vitamin C/kg was added. However, in the absence of dietary iron, supplementation of ascorbic acid resulted in significant decrease in HCT and Hb values. Hepatic iron concentration increased with increasing dietary level of iron. Supplementation of high level of ascorbic acid to the diet containing 300 mg/kg of supplemental iron significantly increased the liver iron content. Liver ascorbate increased with increasing dietary level of ascorbic acid. Dietary level of iron and the interaction between iron and vitamin C had no effect on liver content of vitamin C. Mean macrophage migration in the absence or presence of Edwardsiella ictaluri exoantigen was significantly higher for fish fed the iron supplemented diets. The significant effect of vitamin C was obtained only when high level (3000 mg/kg) was used. Neither dietary levels of iron nor vitamin C or their interaction influenced survival of juvenile channel catfish against E.ictaluri 14-day post challenge. However, the onset of mortality was earlier for fish fed the iron-deficient diet.

Dietary vitamin C requirement of red drum Sciaenops ocellatus

A feeding trial was conducted to determine the minimum dietary vitamin C requirement of juvenile red drum and characterize signs of vitamin C deficiency. Semipurified diets containing 400 g crude protein kg-1 from lyophilized red drum muscle and crystalline amino acids were used in the feeding trial. The basal diet without supplemental vitamin C contained approximately equal to 6 mg vitamin C kg-1 and was fed for a 1-week conditioning period. Red drum, initially averaging approximately equal to 3.6 g each, were fed diets supplemented with 0, 10, 20, 30, 45, 60, 75, or 150 mg vitamin C kg-1 as ascorbate polyphosphate for a period of 10 weeks. Fish fed the basal diet began to exhibit overt signs of vitamin C deficiency, including reduced weight gain, lordosis, scoliosis and loss of equilibrium after 8 weeks. Total ascorbate was undetectable in liver of fish fed the basal diet while liver ascorbate levels in fish fed the other diets generally reflected dietary supplementation. Regression analysis of weight gain data using the broken-line model resulted in a minimum vitamin C requirement (+/-SE) of 15 +/- 3 mg kg-1 diet.

Effect of Dietary Vitamin C on Weight Gain, Tissue Ascorbate Concentration, Stress Response, and Disease Resistance of Channel Catfish Ictalurus punctatus1

AbstractJuvenile channel catfish Ictalurus punctatus (average initial weight, 6.5 g/fish) were fed twice daily to apparent satiation with practical‐type diets containing 0, 50, 150, or 250 mg supplemental vitamin C/kg from L‐ascorbyl‐2‐polyphosphate for 10 wk under laboratory conditions. At the end of the feeding period, one half of the fish were stressed for 2 h by confinement and both stressed and nonstressed fish were exposed to a virulent strain of Edwardsiella ictaluri. Weight gain and feed conversion efficiency were lower for fish fed the basal diet than those fed diets containing supplemental vitamin C. No differences were observed in weight gain and feed conversion among fish fed diets containing supplemental vitamin C. There were no differences in feed consumption and survival (prior to experimental infection) among treatments. No vitamin C deficiency signs except reduced weight gain were observed in fish fed the basal diet. Serum cortisol concentrations were higher in stressed fish than in non‐stressed fish. Dietary vitamin C level had no effect on serum cortisol concentration. As dietary vitamin C increased, ascorbate concentration in serum and liver increased. Confinement stress had no effect on serum and liver ascorbate concentrations. Cumulative mortality of channel catfish 21 d subsequent to experimental infection with E. ictaluri was higher for stressed fish than for nonstressed fish. Regardless of stress or nonstress, overall mortality for fish fed the basal diet was lower than the fish fed diets containing supplemental vitamin C. There were no differences in post‐infection antibody levels among treatments or between stressed and nonstressed fish. Results from this study indicate that channel catfish require no more than 50 mg/kg dietary vitamin C for normal growth, stress response, and disease resistance.

Endotoxin Increases Oxidative Injury to Proteins in Guinea Pig Liver: Protection by Dietary Vitamin C

Current information suggests that oxidative damage plays a key role in septic shock induced by endotoxin. This raises the possibility that dietary antioxidant vitamins could protect against endotoxin damage. In this study, the effects of endotoxin administration on protein and lipid oxidative damage and endogenous antioxidants were studied in the liver of guinea pigs previously supplemented with marginal or optimum levels of dietary vitamin C, vitamin E or both. Vitamins C and E inhibited in vitro lipid peroxidation in endotoxin-treated animals. Endotoxin significantly increased oxidative damage to liver proteins in animals receiving low doses of both vitamins, a result described here for the first time. This increase was totally prevented in guinea pigs supplemented with vitamin C alone or in combination with vitamin E, a treatment which strongly increased liver ascorbate. Vitamin C caused small significant increases in superoxide dismutase and glutathione, increased uric acid, and synergically increased alpha-tocopherol levels in vitamin E-supplemented animals treated with endotoxin. The results show that dietary vitamin C protects against endotoxin-induced oxidative damage to proteins in the guinea pig liver. This seems mainly due to a direct protective effect of the increased hepatic ascorbate levels present in vitamin C-supplemented animals.

Reduction of Dehydroascorbate to Ascorbate by the Selenoenzyme Thioredoxin Reductase

Recycling of ascorbate from its oxidized forms is essential to maintain stores of the vitamin in human cells. Whereas reduction of dehydroascorbate to ascorbate is thought to be largely GSH-dependent, we reconsidered the possibility that the selenium-dependent thioredoxin system might contribute to ascorbate regeneration. We found that purified rat liver thioredoxin reductase functions as an NADPH-dependent dehydroascorbate reductase, with an apparent Km of 2. 5 mM for dehydroascorbate, and a kcat of 90 min-1. Addition of 2.8 microM purified rat liver thioredoxin lowered the apparent Km to 0.7 mM, without affecting the turnover (kcat of 71 min-1). Since thioredoxin reductase requires selenium, we tested the physiologic importance of this enzyme for dehydroascorbate reduction in livers from control and selenium-deficient rats. Selenium deficiency lowered liver thioredoxin reductase activity by 88%, glutathione peroxidase activity by 99%, and ascorbate content by 33%, but did not affect GSH content. NADPH-dependent dehydroascorbate reductase activity due to thioredoxin reductase, on the basis of inhibition by aurothioglucose, was decreased 88% in dialyzed liver cytosolic fractions from selenium-deficient rats. GSH-dependent dehydroascorbate reductase activity in liver cytosol was variable, but typically 2-3-fold that of NADPH-dependent activity. These results show that the thioredoxin system can reduce dehydroascorbate, and that this function is required for maintenance of liver ascorbate content.

ESTROGEN ADMINISTRATION DEPLETES TISSUE ASCORBATE IN EXERCISED RATS 543

Estrogen has been implicated in protecting muscle from exercise induced oxidative stress. Ascorbate acts as an antioxidant and has been reported to be lost from tissues from exercised or vitamin E deprived animals. It was hypothesized that estrogen administration prior to exercise could protect tissue ascorbate levels. Female and male rats served as controls or were vitamin E deprived for 8 wk and/or injected daily with 40μg/kg BW estradiol for 2 wk prior to a 60 min treadmill run. Animals were sacrificed immediately post exercise, and muscle, heart and liver samples removed. We previously reported that exercise alone lowered reduced ascorbate (50-75%) (P<0.05) but not dehydro-ascorbate levels in heart while not affecting plantaris or liver ascorbate status. Surprisingly, estradiol administration in combination with exercise resulted in further depletion in plantaris, heart and liver reduced- & dehydro-ascorbate (40-100%)(P<0.05). To determine if estrogen interfered with substrates and cofactors associated with ascorbate synthesis in liver, we determined liver glutathione and glycogen status. Correlations between liver glycogen and ascorbate status were not significant(P>0.05). However, exercise and estrogen administration significantly affected liver glutathione status (P<0.05) and liver glutathione status was negatively related to ascorbate level (P<0.05). Previous studies suggest that glutathione depletion stimulates liver ascorbate synthesis. It was concluded that depletion of tissue ascorbate by estrogen administration and exercise is not associated with liver glycogen level but may by related to changes in liver glutathione.

Effects of Co-Supplementation of Iron with Ascorbic Acid on Antioxidant—Pro-Oxidant Balance in the Guinea Pig

The relationship between intake of iron with ascorbic acid and their uptake into the plasma and liver of guinea pigs was studied. The influence on the antioxidant/pro-oxidant balance of liver microsomes was also determined. Animals were fed a standard pelleted diet low in iron and ascorbic acid for 35 days. The pellet diet was supplemented by oral dosing with a solution containing either maintenance dietary levels of ascorbic acid and iron, or one of three regimens that increased the dosage of these substances ten fold. There were no significant differences in animal growth rate or food intake between these regimens. Liver and plasma total ascorbate levels were significantly increased (p < 0.05) in animals receiving either ascorbic acid alone (liver 126 ± 36 lg/g tissue wet wt. and plasma 51.7 ± 17.0 pM; n = 9) or ascorbic acid and iron (105 ± 18 μg/g and 40.3 ± 15.3.0 μM; n = 8) compared to controls (84 ± 36 μg/g and 15.3 ± 8.5 μM; n = 11). Total iron levels in the liver (76.7 ± 7.3 μg/g; control; n = 6) and plasma (2.4 ± 0.03 mg/l; control) were not significantly raised in animals under these conditions of iron or ascorbate intake. Liver microsomes isolated from animals receiving iron had a greater susceptibility to oxidative stress in terms of malondialdehyde production during auto-oxidation compared to those from control animals under the same conditions. This effect was eliminated on combining ascorbic acid with the iron supplementation, suggesting that oral administration of vitamin C has a protective rather than a pro-oxidant effect under these circumstances.