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Department of Internal Medicine, Division of Gastroenterology and Hepatology, University of California Davis, Davis, CA
2To whom correspondence should be addressed. E-mail: clbowlus{at}ucdavis.edu.
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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KEY WORDS: • oxidative stress • ascorbic acid • iron • antioxidant status
Iron is an essential nutrient for a number of cellular activities including oxygen transport, electron transfer, and gene regulation. However, excess cellular iron can be toxic by production of reactive oxygen species such as O2- and HO· that damage proteins, lipids, and DNA (1–3). The liver, which is the primary site for iron storage, is at particular risk for iron-induced oxidative stress and tissue damage.
Ascorbic acid is a potent water-soluble antioxidant that scavenges reactive oxygen species and reactive nitrogen species (4). In these processes, ascorbic acid is reduced to dehydroascorbic acid through an ascorbyl radical intermediary. Ascorbic acid deficiency is characterized by increased oxidative stress and tissue injury (5–7). However, in vitro ascorbic acid can also maintain iron and other transition metals in a reduced state, leading to the production of hydroxyl radicals and lipid alkoxyl radicals (8,9). In addition, ascorbic acid enhances the bioavailability of nonheme iron, potentially leading to greater body iron stores. Whether ascorbic acid supplementation has a prooxidant effect in vivo continues to be debated (10,11).
We hypothesize that in a sufficient state, whether ascorbic acid has a net prooxidant or antioxidant effect depends on the concentration of ascorbic acid as well as the iron concentration and redox state of the cell. In the present study, we investigated this hypothesis by studying the effects of iron and ascorbic acid on hepatic oxidative stress in mice using a 2 x 2 factorial design varying dietary iron with or without ascorbic acid supplementation. Because mice synthesize endogenous ascorbic acid, we assumed that mice were sufficient in ascorbic acid.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Animals, feeding protocol and sample collection. C3H/HeJ mice were originally obtained from the Jackson Laboratory, but later generations were bred in-house. All of the experiments were carried out with weanling C3H/He mice (n = 31). From birth to 3 wk, mice were housed in plastic cages with a 12-h light:dark cycle and free access to a standard laboratory diet (Lab Diet 5K52, Nutrition International) and water. Mice were randomly assigned in a 2 x 2 study design to receive one of four diets supplemented with iron to 100 mg/kg diet or 300 mg/kg diet and with ascorbic acid to 15 g/kg diet or without ascorbic acid (Table 1). In separate experiments, we observed that consumption of these diets did not differ significantly and that the calculated mean intake of ascorbic acid in the supplemented diets was 54 mg/d (data not shown). Food and demineralized water were consumed ad libitum for 3 wk, after which the mice were killed; livers were excised, rinsed with ice-cold PBS, blotted, and stored at -80°C. All experiments were performed in compliance with federal veterinary guidelines, and were approved by the Animal Use and Care Administrative Advisory Committee at the University of California Davis. Each experimental group consisted of eight mice (4 males and 4 females) except group 2 (4 males and 3 females).
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Biochemical assays. Assays for MDA, GSH, GST, GPx, SOD, and CAT were performed according to the manufacturer’s protocol (Calbiochem). Briefly, MDA was assayed by its reaction with 10.3 mmol/L N-methyl-2-phenylindole in acetonitrile and measuring absorbance at 586 nm (12). GSH was estimated on the basis of the transformation of thioesters with GSH into a chromophoric thione measured at 400 nm. The activity of GST was assayed by the conversion of 1-chloro-2,4-dinitrobenzene by GSH to a dinitrophenyl thioether and measuring absorbance at 340 nm over time. GPx activity expressed as the oxidation of NADPH by oxidized glutathione, measured by the decrease in absorbance at 340 nm over time. One unit is equivalent to the enzyme activity consuming 1 µmol of NADPH/min. SOD activity was measured by the autooxidation of 5,6,6a,11b-tetrahydro-3,9,10-trihydroxybenzo(c)fluorene in the presence and absence of tissue homogenates. The ratios of the rates of change in absorbance at 525 nm were calculated from the linear ranges of the curves. CAT activity was measured on the basis of the rate of dismutation of hydrogen peroxide (H2O2) to water and molecular oxygen. Homogenates were incubated at 37°C in 10 mmol/L H2O2 for 1 min and the reaction stopped by the addition of sodium azide. The remaining H2O2 was determined by the conversion of 4-aminophenazone and 3,5-dichloro-2-hydroxybenzenesulfonic acid to quinoneimine dye in the presence of horseradish peroxidase and measuring absorbance at 520 nm. Concentrations and enzyme activities were corrected for dilution and normalized to total protein concentration of the homogenates, which was determined using the BCA protein assay reagent kit (Pierce).
Tissue iron analysis. Liver was digested in 16 mol/L HNO3 for 7 d and analyzed for iron by atomic absorption spectrophotometry (Thermo Elemental 4000) (13).
Statistical analysis. Treatment effects were analyzed by two-way ANOVA using GraphPad Prism version 4.0 (GraphPad Software). When the P-value obtained from ANOVA was significant, Tukey’s test was applied to test for differences among groups. ANOVA was performed grouping animals by sex and diet (2 x 4). Sex did not have an effect on any endpoint. Differences were considered significant if P < 0.05. Values in the text are means ± SD.
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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50%. Similar degrees of inhibition of hepatic GPx activity were reported previously in iron overload induced by intraperitoneal iron injection (15). GPx functions to reduce lipid hydroperoxides into lipid alcohols. Thus, iron-induced lipid peroxidation may be exacerbated by the reduction in GPx activity. Four GPx genes have been identified but only Gpx1 is expressed in the mouse liver. Selenium and iron deficiencies are known to downregulate Gpx1 mRNA in rodent liver, but the effect of iron loading on Gpx1 mRNA is unknown (16–18). Our study also demonstrated that ascorbic acid can have prooxidant effects under certain conditions. Surprisingly, however, the prooxidant effect occurred only in mice fed the low-iron diet. In mice fed the high-iron diet, there was a small antioxidant effect with an increase in GPx activity. These findings do not support the recommendation of several investigators to avoid ascorbic acid supplementation in individuals at risk of iron overload (19–22). These recommendations are based on two theoretical concerns. First, test meal studies show that ascorbic acid promotes iron absorption (23). However, few direct data exist on the long-term effects of ascorbic acid on dietary iron absorption. In fact, the data available do not indicate a significant increase in body iron stores with long-term ascorbic acid supplementation, suggesting the existence of a tightly regulated mechanism restricting iron absorption (24,25). Second, ascorbic acid may have prooxidant properties in the presence of iron. Several mechanisms of ascorbic acid–induced oxidant stress were suggested. As an electron donor, ascorbic acid can maintain iron in a reduced state that may then react with H2O2 to form OH· (26). Alternatively, ascorbic acid may be oxidized to an ascorbyl radical. However, whether cellular iron is available in a reactive form to interact with ascorbic acid is not known.
Podmore and colleagues (10) first suggested that ascorbic acid supplementation could have prooxidant effects. In their study in humans, 500 mg/d of ascorbic acid decreased levels of 8-oxo-guanine but increased levels of 8-oxo-adenine from baseline. However, studies of co-supplementation of iron and ascorbic acid did not show any interaction or prooxidant effects of ascorbic acid on DNA base damage. Rehman et al. (27) compared co-supplementation of iron with either 60 or 260 mg/d of ascorbic acid on DNA base damage in humans. They found that at 6 wk, both combinations had increased total base damage, but this effect was lost at 12 wk. In a placebo-controlled, crossover study comparing placebo, 260 mg/d ascorbic acid, and 260 mg/d ascorbic acid plus 14 mg/d iron, the same authors found no effect of any treatment on DNA base damage after 6 wk.
Limitations in human studies to the use of peripheral blood have prompted others as well as us to perform studies in animals. Collis et al. (28) found that in guinea pigs, orally administered iron resulted in greater MDA during autooxidation of liver microsomes and that ascorbic acid co-supplementation was protective against this effect. Chen et al. (29) also compared lipid peroxidation in the liver of guinea pigs fed high or low doses of ascorbic acid with or without iron loading by i.p. injection of iron dextran. The guinea pigs administered the high ascorbic acid dose had reduced liver F2-isoprostanes, regardless of iron treatment. It should be noted, however, that in that study, the lower ascorbic acid dose was designed to produce ascorbic acid deficiency. Therefore, the effects of ascorbic acid deficiency may have confounded the lipid peroxidation induced by iron loading and any interaction between iron and ascorbic acid.
Our study was aimed at determining whether ascorbic acid in the setting of ascorbic acid sufficiency has prooxidant properties in the liver. One important difference between the guinea pig studies and our study is the ability of mice to synthesize endogenous ascorbic acid. Thus our study is not confounded by the prooxidant state of ascorbic acid deficiency. Furthermore, although we did not measure tissue ascorbic acid levels, a dose of ascorbic acid similar to that used in this study was shown to increase liver and plasma ascorbic acid concentrations in mice (30,31).
Our results are consistent with the findings of Podmore and colleagues (10) and support the hypothesis that excessive dietary ascorbic acid can promote oxidative stress. The mechanism of this increase in oxidative stress is not clear but does not appear to depend on liver iron concentration. Although there was a trend toward a greater liver iron concentration in mice supplemented with ascorbic acid (P = 0.07), this does not explain the prooxidant effects of ascorbic acid. Thus, the prooxidant effects of ascorbic acid cannot be attributed to an enhancement of dietary iron absorption.
A higher intracellular ascorbic acid concentration could lead to a greater reducing capacity. This in turn could lead to the mobilization of iron from ferritin. This redistribution of iron to a more reactive form could potentially increase oxidative stress without changing iron concentrations. Alternatively, ascorbic acid may lead directly to the decomposition of lipid hydroperoxides independently of iron. Lee and colleagues (32) showed that ascorbic acid can decompose the (n-6) lipid, hydroperoxide 13-hydroperoxy-9,11-octadecadienoic acid, in a dose-dependent manner. The lack of a prooxidant effect of ascorbic acid supplementation on lipid peroxidation in mice fed the high-iron diet illustrates the complexity of the interaction between iron and ascorbic acid. It also suggests that ascorbic acid does not augment iron-induced oxidative stress.
In conclusion, our results suggest that ascorbic acid can have prooxidant properties, reflected particularly by increased lipid peroxidation and decreased GPx activity, but ascorbic acid does not increase the oxidative stress induced by iron. Further studies are required to determine the mechanisms by which ascorbic acid can act as a prooxidant.
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FOOTNOTES |
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3 Abbreviations used: CAT, catalase; GPx, glutathione peroxidase; GSH, glutathione; GST, glutathione S-transferase; MDA, malondialdehyde; SOD, superoxide dismutase.
Manuscript received 25 July 2003. Initial review completed 6 September 2003. Revision accepted 4 November 2003.
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LITERATURE CITED |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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