The Impact of Vitamin C on Different System Models of Werner Syndrome.

Significance: Werner syndrome (WS) is a rare autosomal recessive malady typified by a pro-oxidant/proinflammatory status, genetic instability, and by the early onset of numerous age-associated illnesses. The protein malfunctioning in WS individuals (WRN) is a helicase/exonuclease implicated in transcription, DNA replication/repair, and telomere maintenance. Recent Advances: In the last two decades, a series of important biological systems were created to comprehend at the molecular level the effect of a defective WRN protein. Such biological tools include mouse and worm (Caenorhabditis elegans) with a mutation in the Wrn helicase ortholog as well as human WS-induced pluripotent stem cells that can ultimately be differentiated into most cell lineages. Such WS models have identified anomalies related to the hallmarks of aging. Most importantly, vitamin C counteracts these age-related cellular phenotypes in these systems. Critical Issues: Vitamin C is the only antioxidant agent capable of reversing the cellular aging-related phenotypes in those biological systems. Since vitamin C is a cofactor for many hydroxylases and mono- or dioxygenase, it adds another level of complexity in deciphering the exact molecular pathways affected by this vitamin. Moreover, it is still unclear whether a short- or long-term vitamin C supplementation in human WS patients who already display aging-related phenotypes will have a beneficial impact. Future Directions: The discovery of new molecular markers specific to the modified biological pathways in WS that can be used for novel imaging techniques or as blood markers will be necessary to assess the favorable effect of vitamin C supplementation in WS. Antioxid. Redox Signal. 34, 856-874.

The effect of low ascorbic acid content on tomato fruit ripening.

MAIN CONCLUSION: The oxidant/antioxidant balance affects the ripening time of tomato fruit. Ripening of tomato fruit is associated with several modifications such as loss of cell wall firmness and transformation of chloroplasts to chromoplasts. Besides a peak in H2O2, reactive oxygen species (ROS) are observed at the transition stage. However, the role of different components of oxidative stress metabolism in fruit ripening has been scarcely addressed. Two GDP-L-galactose phosphorylase (GGP) Solanum lycopersicum L. cv Micro-Tom mutants which have fruit with low ascorbic acid content (30% of wild type) were used in this work to unravel the participation of ascorbic acid and H2O2 in fruit maturation. Both GGP mutants show delayed fruit maturation with no peak of H2O2; treatment with ascorbic acid increases its own concentration and accelerates ripening only in mutants to become like wild type plants. Unexpectedly, the treatment with ascorbic acid increases H2O2 synthesis in both mutants resembling what is observed in wild type fruit. Exogenous supplementation with H2O2 decreases its own synthesis delaying fruit maturation in plants with low ascorbic acid content. The site of ROS production is localized in the chloroplasts of fruit of all genotypes as determined by confocal microscopy analysis. The results presented here demonstrate that both ascorbic acid and H2O2 actively participate in tomato fruit ripening.

Mapping of quantitative trait loci for antioxidant molecules in tomato fruit: Carotenoids, vitamins C and E, glutathione and phenolic acids.

The nutritional value of a crop lies not only in its protein, lipid, and sugar content but also involves compounds such as the antioxidants lycopene, ß-carotene and vitamin C. In the present study, wild tomato Solanum pimpinellifolium LA 1589 was assessed for its potential to improve antioxidant content. This wild species was found to be a good source of alleles for increasing ß-carotene, lycopene, vitamin C and vitamin E contents in cultivated tomato. Characterization of an LA 1589 interspecific inbred backcross line (IBL) mapping population revealed many individuals with transgressive segregation for the antioxidants confirming the usefulness of this wild species for breeding of these traits. Molecular markers were used to identify QTLs for the metabolites in the IBL population. In total, 64 QTLs were identified for the antioxidants and their locations were compared to the map positions of previously identified QTLs for confirmation. Four (57 %) of the carotenoid QTLs, four (36 %) of the vitamin QTLs, and 11 (25 %) of the phenolic acid QTLs were supported by previous studies. Furthermore, several potential candidate genes were identified for vitamins C and E and phenolic acids loci. These candidate genes might be used as markers in breeding programs to increase tomato's antioxidant content.

Transcriptome mining and in silico structural and functional analysis of ascorbic acid and tartaric acid biosynthesis pathway enzymes in rose-scanted geranium.

Rose-scented geranium (Pelargonium sp.) is widely known as aromatic and medicinal herb, accumulating specialized metabolites of high economic importance, such as essential oils, ascorbic acid, and tartaric acid. Ascorbic acid and tartaric acid are multifunctional metabolites of human value to be used as vital antioxidants and flavor enhancing agents in food products. No information is available related to the structural and functional properties of the enzymes involved in ascorbic acid and tartaric acid biosynthesis in rose-scented geranium. In the present study, transcriptome mining was done to identify full-length genes, followed by their bioinformatic and molecular modeling investigations and understanding of in silico structural and functional properties of these enzymes. Evolutionary conserved domains were identified in the pathway enzymes. In silico physicochemical characterization of the catalytic enzymes revealed isoelectric point (pI), instability index, aliphatic index, and grand average hydropathy (GRAVY) values of the enzymes. Secondary structural prediction revealed abundant proportion of alpha helix and random coil confirmations in the pathway enzymes. Three-dimensional homology models were developed for these enzymes. The predicted structures showed significant structural similarity with their respective templates in root mean square deviation analysis. Ramachandran plot analysis of the modeled enzymes revealed that more than 84% of the amino acid residues were within the favored regions. Further, functionally important residues were identified corresponding to catalytic sites located in the enzymes. To, our best knowledge, this is the first report which provides a foundation on functional annotation and structural determination of ascorbic acid and tartaric acid pathway enzymes in rose-scanted geranium.

Solanum diploconos fruits: profile of bioactive compounds and in vitro antioxidant capacity of different parts of the fruit.

Solanum diploconos is an unexploited Brazilian native fruit that belongs to the same genus of important food crops, such as tomato (Solanum lycorpersicum) and potato (Solanum tuberosum). In this study, we determined, for the first time, the profile of bioactive compounds (phenolic compounds, carotenoids, ascorbic acid and tocopherols) of the freeze-dried pulp and peel of Solanum diploconos fruits, as well as of an extract obtained from the whole fruit. Additionally, the antioxidant potential of the whole fruit extract was evaluated in vitro, against reactive oxygen species (ROS) and reactive nitrogen species (RNS). Eighteen phenolic compounds were identified in the peel and pulp and 6 compounds were found in the whole fruit extract. Coumaric, ferulic and caffeic acid derivatives were revealed to be the major phenolic constituents. All-trans-ß-carotene was the major carotenoid (17-38 µg g(-1), dry basis), but all-trans-lutein and 9-cis-ß-carotene were also identified. The peel and pulp presented <2 µg per mL of tocopherols, and ascorbic acid was not detected. The whole fruit extract exhibited scavenging capacity against all tested ROS and RNS (IC50 = 14-461 µg mL(-1)) with high antioxidant efficiency against HOCl. Thus, Solanum diploconos fruits may be seen as a promising source of bioactive compounds with high antioxidant potential against the most physiologically relevant ROS and RNS.

Vitamin C prevents stress-induced damage on the heart caused by the death of cardiomyocytes, through down-regulation of the excessive production of catecholamine, TNF-α, and ROS production in Gulo(-/-)Vit C-Insufficient mice.

It is thought that vitamin C has protective roles on stress-induced heart damage and the development of cardiovascular diseases, but its precise role and mechanisms are unclear. In the present study, we investigated the specific mechanisms by which vitamin C leads to protecting the heart from stress-induced damage in the Gulo(-/-) mice which cannot synthesize vitamin C like humans. By exposure to stress (1h/day), the heartbeat and cardiac output in vitamin C-insufficient Gulo(-/-) mice were definitely decreased, despite a significant increase of adrenaline (ADR) and noradrenaline (NA) production. A change of cardiac structure caused by the death of cardiomyocytes and an increased expression of matrix metalloprotease (MMP)-2 and -9 were also found. Moreover, lipid peroxidation and the production of tumor necrosis factor-alpha (TNF-α) in the heart were increased. Finally, all vitamin C-insufficient Gulo(-/-) mice were expired within 2 weeks. Interestingly, all of the findings in vitamin C-insufficient Gulo(-/-) mice were completely prevented by the supplementation of a sufficient amount of vitamin C. Taken together, vitamin C insufficiency increases the risk of stress-induced cardiac damage with structural and functional changes arising from the apoptosis of cardiomyocytes.

Behavioral and monoamine changes following severe vitamin C deficiency.

Severe vitamin C deficiency (ascorbic acid; AA) was induced in gulo-/- mice incapable of synthesizing their own AA. A number of behavioral measures were studied before and during the deprivation period, including a scorbutic period, during which weight loss was observed in the mice. Mice were then resuscitated with AA supplements. During the scorbutic period, gulo-/- mice showed decreased voluntary locomotor activity, diminished physical strength, and increased preference for a highly palatable sucrose reward. These behaviors all returned to control levels following resuscitation. Altered trial times in subordinate mice in the tube test for social dominance in the AA-deprived mice persisted following resuscitation and may signify a depressive-like behavior in these mice. Biochemical analyses were undertaken following a second deprivation period. AA deficiency was accompanied by decreased blood glucose levels, oxidative damage to lipids and proteins in the cortex, and decreases in dopamine and serotonin metabolites in both the cortex and striatum. Given the reasonably high proportions of the population that do not consume sufficient AA in the diet, these data have important implications for physical and psychological function in the general population.

Comparative transcriptomic profiling of two tomato lines with different ascorbate content in the fruit.

In recent years, interest in tomato breeding for enhanced antioxidant content has increased as medical research has pointed to human health benefits from antioxidant dietary intake. Ascorbate is one of the major antioxidants present in tomato, and little is known about mechanisms governing ascorbate pool size in this fruit. In order to provide further insights into genetic mechanisms controlling ascorbate biosynthesis and accumulation in tomato, we investigated the fruit transcriptome profile of the Solanum pennellii introgression line 10-1 that exhibits a lower fruit ascorbate level than its cultivated parental genotype. Our results showed that this reduced ascorbate level is associated with an increased antioxidant demand arising from an accelerated oxidative metabolism mainly involving mitochondria, peroxisomes, and cytoplasm. Candidate genes for controlling ascorbate level in tomato fruit were identified, highlighting the role of glycolysis, glyoxylate metabolism, and purine breakdown in modulating the ascorbate pool size.

Effect of vitamin C deficiency during postnatal development on adult behavior: functional phenotype of Gulo-/- knockout mice.

Organisms using oxygen for aerobic respiration require antioxidants to balance the production of reactive oxygen species during metabolic processes. Various species--including humans and other primates--suffer mutations in the GULO gene encoding L-gulono-γ-lactone oxidase; GULO is the rate-limiting enzyme in the biosynthesis of ascorbate, an important cellular antioxidant. Animals lacking the ability to synthesize vitamin C develop scurvy without dietary supplementation. The Gulo-/- knockout (KO) mouse requires oral supplemental vitamin C; without this supplementation the animal dies with a scorbutic condition within several weeks. Vitamin C is known to be most abundant in the brain, where it is believed to play important roles in neuroprotection, neurotransmission and neuromodulation. We therefore hypothesized that ascorbate deficiency in Gulo-/- KO mice might lead to an abnormal behavioral phenotype. We established the amount of ascorbate in the drinking water (220 ppm) necessary for generating a chronic low-ascorbate status in the brain, yet clinically the mice appeared healthy throughout 100 days postpartum at which time all behavioral-phenotyping tests were completed. Compared with Gulo+/+ wild-type littermates, ascorbate-deficient Gulo-/- mice were found to be less active in moving in their environment; when in water, these mice swam more slowly in some tests, consistent with a mild motor deficit. We found no evidence of cognitive, anxiety or sensorimotor-gating problems. Despite being less active, Gulo-/- mice exhibited exaggerated hyperactivity to the dopaminergic agonist methamphetamine. The subnormal movement, combined with hypersensitivity to a dopamine agonist, point to developmental ascorbate deficiency causing long-term striatal dysfunction.

Ascorbate improves metabolic abnormalities in Wrn mutant mice but not the free radical scavenger catechin.

Werner syndrome (WS) is a premature aging disorder caused by mutations in a RecQ-like DNA helicase. Mice lacking the helicase domain of the WRN homologue exhibit many phenotypic features of WS. Importantly, mutant Wrn(Deltahel/Deltahel) mice show abnormal increases in visceral fat deposition and fasting blood triglyceride levels followed by insulin resistance and high blood glucose levels. These mice also exhibit increased heart and liver tissue reactive oxygen species concomitantly with oxidative DNA damage, indicating a pro-oxidant status. We treated mice with either ascorbate or catechin hydrate for 9 months. Vitamin C supplementation reduced oxidative stress in liver and heart tissues and reversed hypertriglyceridemia, hyperglycemia, and insulin resistance and reduced fat weight in mutant Wrn(Deltahel/Deltahel) mice. Although the free scavenger catechin hydrate also reduced oxidative DNA damage in heart and liver tissues, it did not reverse any of the metabolic phenotype aspects in treated mutant mice. Finally, vitamin C and catechin hydrate did not affect the metabolic status of wild-type mice. These results indicate that vitamin C supplementation could be beneficial for WS patients.

The pathway via D-galacturonate/L-galactonate is significant for ascorbate biosynthesis in Euglena gracilis: identification and functional characterization of aldonolactonase.

We have previously proposed that Euglena gracilis possesses a pathway for the production of ascorbate (AsA) through d-galacturonate/L-galactonate as representative intermediates ( Shigeoka, S., Nakano, Y., and Kitaoka, S. (1979) J. Nutr. Sci. Vitaminol. 25, 299-307 ). However, genetic evidence proving that the pathway exists has not been obtained yet. We report here the identification of a gene encoding aldonolactonase, which catalyzes a penultimate step of the biosynthesis of AsA in Euglena. By a BLAST search, we identified one candidate for the enzyme having significant sequence identity with rat gluconolactonase, a key enzyme for the production of AsA via d-glucuronate in animals. The purified recombinant aldonolactonase expressed in Escherichia coli catalyzed the reversible reaction of L-galactonate and L-galactono-1,4-lactone with zinc ion as a cofactor. The apparent K(m) values for L-galactonate and L-galactono-1,4-lactone were 1.55 +/- 0.3 and 1.67 +/- 0.39 mm, respectively. The cell growth of Euglena was arrested by silencing the expression of aldonolactonase through RNA interference and then restored to the normal state by supplementation with L-galactono-1,4-lactone. Euglena cells accumulated more AsA on supplementation with d-galacturonate than d-glucuronate. The present results indicate that aldonolactonase is significant for the biosynthesis of AsA in Euglena cells, which predominantly utilize the pathwayviad-galacturonate/L-galactonate. The identification of aldonolactonase provides the first insight into the biosynthesis of AsA via uronic acids as the intermediate in photosynthetic algae including Euglena.

Effect of iron and ascorbate on uroporphyria in ascorbate-requiring mice as a model for porphyria cutanea tarda.

UNLABELLED: Excess hepatic iron is known to enhance both porphyria cutanea tarda (PCT) and experimental uroporphyria. Since previous studies have suggested a role for ascorbate (AA) in suppressing uroporphyria in AA-requiring rats (in the absence of excess iron), the present study investigated whether AA could suppress uroporphyria produced by excess hepatic iron. Hepatic URO accumulation was produced in AA-requiring Gulo(-/-) mice by treatment with 3,3',4,4',5-pentachlorbiphenyl, an inducer of CYP1A2, and 5-aminolevulinic acid. Mice were administered either sufficient AA (1000 ppm) in the drinking water to maintain near normal hepatic AA levels or a lower intake (75 ppm) that resulted in 70 % lower hepatic AA levels. The higher AA intake suppressed hepatic URO accumulation in the absence of administered iron, but not when iron dextran (300-500 mg Fe/kg) was administered. This effect of iron was not due to hepatic AA depletion since hepatic AA content was not decreased. The effect of iron to prevent AA suppression of hepatic URO accumulation was not observed until a high hepatic iron threshold was exceeded. At both low and high AA intakes, hepatic malondialdehyde (MDA), an indicator of oxidative stress, was increased three-fold by high doses of iron dextran. MDA was considerably increased even at low iron dextran doses, but without any increase in URO accumulation. The level of hepatic CYP1A2 was unaffected by either AA intake. CONCLUSION: In this mouse model of PCT, AA suppresses hepatic URO accumulation at low, but not high hepatic iron levels. These results may have implications for the management of PCT.

Extraction of total RNA from leaves of Eucalyptus and other woody and herbaceous plants using sodium isoascorbate.

Rapid extraction of total RNA from Eucalyptus leaves is difficult due to the high content of polyphenolics and polysaccharides. A rapid and simple method was developed by using an extraction buffer containing sodium isoascorbate at a concentration of 500 mM. This method consisted of one or two chloroform extractions, one acid guanidium-phenol-chloroform extraction, and isopropanol precipitation alone. The yields of the RNA fractions were 246-1750 micrograms/g fresh weight when leaves of Eucalyptus, five other woody plants, and four herbaceous plants were used as samples. The contamination of the RNA fractions by proteins and polysaccharides was very limited as judged spectrophotometrically. When the RNA fractions were subjected to agarose gel electrophoresis, intact rRNA bands were detected. The RNA fractions could be used for RT-PCR. These results indicate that our new method achieves a simple and rapid preparation of high-quality RNA from leaves of Eucalyptus and other plant species.

Vulnerable atherosclerotic plaque morphology in apolipoprotein E-deficient mice unable to make ascorbic Acid.

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.