Ascorbate-mediated electron transfer in protein thiol oxidation in the endoplasmic reticulum.
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Addition of, or gulonolactone oxidase-dependent in situ generation of, ascorbate provoked the oxidation of protein thiols, which was accompanied by ascorbate consumption in liver microsomal vesicles. The maximal rate of protein thiol oxidation was similar upon gulonolactone, ascorbate or dehydroascorbate addition. Cytochrome P450 inhibitors (econazole, proadifen, quercetin) decreased ascorbate consumption and the gulonolactone or ascorbate-stimulated thiol oxidation. The results demonstrate that the ascorbate/dehydroascorbate redox couple plays an important role in electron transfer from protein thiols to oxygen in the hepatic endoplasmic reticulum, even in gulonolactone oxidase deficient species. (+info)
Induction and peroxisomal appearance of gulonolactone oxidase upon clofibrate treatment in mouse liver.
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Various antihyperlipemic peroxisome proliferators are known to be carcinogenic in rodents but not in human, other primates and guinea pig, which species lost their ability to synthesize ascorbate due to mutations in the gulonolactone oxidase gene. Ascorbate synthesis is accompanied by H2O2 production, consequently its induction can be potentially harmful; therefore, the in vivo effect of the peroxisome proliferator clofibrate was investigated on gulonolactone oxidase expression in mouse liver. Liver weights and peroxisomal protein contents were increased upon clofibrate treatment. Elevated plasma ascorbate concentrations were found in clofibrate-treated mice due to the higher microsomal gulonolactone oxidase activities. Remarkable gulonolactone oxidase activity appeared in the peroxisomal fraction upon the treatment. Increased activity of the enzyme was associated with an elevation of its mRNA level. According to the present results the evolutionary loss of gulonolactone oxidase may contribute to the explanation of the missing carcinogenic effect of peroxisome proliferators in humans. (+info)
Random nucleotide substitutions in primate nonfunctional gene for L-gulono-gamma-lactone oxidase, the missing enzyme in L-ascorbic acid biosynthesis.
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Humans and other primates have no functional gene for L-gulono-gamma-lactone oxidase that catalyzes the last step of L-ascorbic acid biosynthesis. The 164-nucleotide sequence of exon X of the gene was compared among human, chimpanzee, orangutan, and macaque, and it was found that nucleotide substitutions had occurred at random throughout the sequence with a single nucleotide deletion, indicating that the primate L-gulono-gamma-lactone oxidase genes are a typical example of pseudogene. (+info)
Different induction of gulonolactone oxidase in aromatic hydrocarbon-responsive or -unresponsive mouse strains.
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The role of aromatic hydrocarbon receptor (AhR)-mediated signal transduction pathways was investigated in the regulation of ascorbate synthesis by using Ah-responsive and Ah-unresponsive mouse strains. In vivo 3-methylcholanthrene treatment increased hepatic and plasma ascorbate concentrations only in the Ah-responsive strain. The mRNA level of gulonolactone oxidase and the microsomal ascorbate production from p-nitrophenyl glucuronide, D-glucuronic acid or gulonolactone in the liver of Ah-responsive and Ah-unresponsive mice were compared. In Ah-responsive mice, these parameters were higher originally, and they further increased upon in vivo addition of 3-methylcholanthrene, while in Ah-unresponsive mice the treatment was not effective. These results suggest that the transcription of gulonolactone oxidase gene is regulated by an Ah receptor-dependent signal transduction pathway. (+info)
Aortic wall damage in mice unable to synthesize ascorbic acid.
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By inactivating the gene for L-gulono-gamma-lactone oxidase, a key enzyme in ascorbic acid synthesis, we have generated mice that, like humans, depend on dietary vitamin C. Regular chow, containing about 110 mg/kg of vitamin C, is unable to support the growth of the mutant mice, which require L-ascorbic acid supplemented in their drinking water (330 mg/liter). Upon withdrawal of supplementation, plasma and tissue ascorbic acid levels decreased to 10-15% of normal within 2 weeks, and after 5 weeks the mutants became anemic, began to lose weight, and die. Plasma total antioxidative capacities were approximately 37% normal in homozygotes after feeding the unsupplemented diet for 3-5 weeks. As plasma ascorbic acid decreased, small, but significant, increases in total cholesterol and decreases in high density lipoprotein cholesterol were observed. The most striking effects of the marginal dietary vitamin C were alterations in the wall of aorta, evidenced by the disruption of elastic laminae, smooth muscle cell proliferation, and focal endothelial desquamation of the luminal surface. Thus, marginal vitamin C deficiency affects the vascular integrity of mice unable to synthesize ascorbic acid, with potentially profound effects on the pathogenesis of vascular diseases. Breeding the vitamin C-dependent mice with mice carrying defined genetic mutations will provide numerous opportunities for systematic studies of the role of antioxidants in health and disease. (+info)
Ascorbic acid synthesis in fetal and neonatal pigs and in pregnant and postpartum sows.
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The ontogeny of ascorbic acid synthesis and its concentration in fetal pigs from mid- to late gestation, and the effect of birth order and premature or normal delivery ages were evaluated. In Experiment 1, fetal pigs were collected from three sows at 60, 80, 100, 107 and 111 d of development. Liver L-gulono-gamma-lactone oxidase (GLO) activity and ascorbic acid concentration were measured. High liver GLO activity in fetal liver occurred at 60 d but declined as pregnancy advanced (P < 0.01), whereas ascorbic acid concentration increased (P < 0.01). Experiment 2 evaluated ascorbic acid synthesis and concentration in neonates born early (1st and 2nd) or late (7th and 8th) in the birthing sequence, or when born 2 d prematurely vs. the normal delivery age. Pigs born early in the birthing sequence (P < 0.01) and those born at the natural delivery age (P < 0.05) had higher liver ascorbic acid concentrations, but liver GLO activity did not differ among groups. Sows were killed at each period; liver GLO activity was constant during gestation but increased postpartum (P < 0.01). Liver ascorbic acid concentration was constant during gestation, except for a decline during late gestation, and increased postpartum (P < 0.05). These results suggest that more ascorbic acid was transferred from the dam to the fetuses as pregnancy advanced, possibly suppressing fetal GLO activity. Thus, fetal liver GLO activity was the primary source of ascorbic acid during early fetal development, but more fetal ascorbic acid was transferred from the dam during later pregnancy. (+info)
Liver L-gulonolactone oxidase activity and tissue ascorbic acid concentrations in nursing pigs and the effect of various weaning ages.
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In Experiment 1, we evaluated liver L-gulono-gamma-lactone oxidase (GLO) activity and tissue concentration of ascorbic acid in young pigs from birth to weaning (14 d) and through a 28-d postweaning period; in Experiment 2, we evaluated the effect of three weaning ages on these measurements. Sow colostrum and milk collected in both experiments demonstrated a linear decline (P < 0.01) in ascorbic acid concentration as lactation progressed. In Experiment 1, three pigs were killed at 0, 3, 7, 14, 21, 28, 35 and 42 d of age for determining liver GLO activity and serum and tissue ascorbic acid. Liver GLO activity decreased by 80% from 0 to 3 d of age and remained low until d 14 (weaning). After weaning, liver GLO activity increased linearly (P < 0.01). Tissue ascorbic acid concentrations decreased during the nursing period and again after weaning, but then increased to 42 d of age (P < 0.01). In Experiment 2, pigs were weaned at 10, 17 or 24 d of age. Three pigs from each group were killed at weaning and at each week postweaning until 38 d of age. Liver GLO activity was low during the nursing period but increased linearly (P < 0.01) for each group during the subsequent postweaning period. Pig serum and tissue ascorbic acid concentrations increased postweaning in each group. These results suggest that a factor in sow's milk, possibly ascorbic acid, suppressed liver GLO activity of nursing pigs but upon weaning, liver GLO activity of pigs increased in a linear manner (P < 0.01). (+info)
Vulnerable atherosclerotic plaque morphology in apolipoprotein E-deficient mice unable to make ascorbic Acid.
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BACKGROUND: Oxidative stress is thought to play an important role in atherogenesis, suggesting that antioxidants could prevent coronary artery disease. However, the efficacy of vitamin C in reducing atherosclerosis is debatable in humans and has not been tested rigorously in animals. METHODS AND RESULTS: Gulo(-/-)Apoe(-/-) mice were used to test a hypothesis that chronic vitamin C deficiency enhances the initiation and development of atherosclerosis. These mice are dependent on dietary vitamin C because of the lack of L-gulonolactone-gamma-oxidase and are prone to develop atherosclerosis because of lacking apolipoprotein E. Beginning at 6 weeks of age, the Gulo(-/-)Apoe(-/-) mice were fed regular chow or Western-type diets containing high fat and supplemented with either 0.033 g or 3.3 g/L of vitamin C in their drinking water. This regimen produced mice with chronically low vitamin C (average 1.5 microg/mL in plasma) or high vitamin C (average 10 to 30 microg/mL in plasma). Morphometric analysis showed that within each sex, age, and diet group, the sizes of the atherosclerotic plaques were not different between low vitamin C mice and high vitamin C mice. However, advanced plaques in the low vitamin C mice had significantly reduced amounts of Sirius red-staining collagen (36.4+/-2.2% versus 54.8+/-2.3%, P<0.0001), larger necrotic cores within the plaques, and reduced fibroproliferation and neovascularization in the aortic adventitia. CONCLUSIONS: Chronic vitamin C deficiency does not influence the initiation or progression of atherosclerotic plaques but severely compromises collagen deposition and induces a type of plaque morphology that is potentially vulnerable to rupture. (+info)