Ascorbic Deficiency Research Articles

342 - Ascorbate supplementation increases 5-hydroxymethylcytosine in brain cerebellum DNA of ascorbate knockout mice

5-Hydroxymethylcytosine (5hmC) is a newly characterized epigenetic mark arising from the enzymatic oxidation of 5-methylcytosine (5mC) in DNA by the ten eleven translocation (TET) family enzymes. Whereas 5mC supresses gene expression, the presence of 5hmC correlates with the upregulation of certain genes. Using LC-MS/MS, we compared the global levels of genomic 5mC, 5hmC and 5-formylcytosine (5fC) in the brains of 4-month-old wild-type mice on a standard diet without ascorbate in comparison with Gulo-/- ascorbate knockout mice on a standard diet plus 0, 0.1, and 4.0 g/L ascorbate supplements. In addition, these modifications were measured in 20-month-old wild-type mice. Four regions of the brain were examined, which included cerebellum, cortex, hippocampus and striatum. The total brain weight and particularly the cerebellum weight of Gulo-/- mice were lower than those of wild-type mice. There was also a reduction of both epigenetic marks (5mC and 5hmC) in the cerebellum of Gulo-/- compared to wild-type mice. In contrast, weight, 5mC, 5hmC did not significantly change in other regions of the brain between the age-matched ascorbate deficient Gulo-/- mice. Loss of 5hmC in the cerebellum was increased in going from extreme deficiency (no ascorbate) to ascorbate supplements (4.0 g/L) in Gulo-/- mice. Lastly, similar decreases in weight and the level of 5mC and 5hmC were observed with age when comparing 4-month-old to 20-month-old wild-type mice. These results demonstrate that ascorbate modulates 5hmC in vivo and that the cerebellum is the most sensitive region affected by brain ascorbate deficiency. This work links dietary ascorbate to the epigenetic regulation of pathways that may be involved in a variety of neurological disorders.

In vivo evidence of ascorbate involvement in the generation of epigenetic DNA modifications in leukocytes from patients with colorectal carcinoma, benign adenoma and inflammatory bowel disease

BackgroundA characteristic feature of malignant cells, such as colorectal cancer cells, is a profound decrease in the level of 5-hydroxymethylcytosine, a product of 5-methylcytosine oxidation by TET enzymes. Recent studies showed that ascorbate may upregulate the activity of TET enzymes in cultured cells and enhance formation of their products in genomic DNA.MethodsThe study included four groups of subjects: healthy controls (n = 79), patients with inflammatory bowel disease (IBD, n = 51), adenomatous polyps (n = 67) and colorectal cancer (n = 136). The list of analyzed parameters included (i) leukocyte levels of epigenetic DNA modifications and 8-oxo-7,8-dihydro-2′-deoxyguanosine, a marker of oxidatively modified DNA, determined by means of isotope-dilution automated online two-dimensional ultra-performance liquid chromatography with tandem mass spectrometry, (ii) expression of TET mRNA measured with RT-qPCR, and (iii) chromatographically-determined plasma concentrations of retinol, alpha-tocopherol and ascorbate.ResultsPatients from all groups presented with significantly lower levels of 5-methylcytosine and 5-hydroxymethylcytosine in DNA than the controls. A similar tendency was also observed for 5-hydroxymethyluracil level. Patients with IBD showed the highest levels of 5-formylcytosine and 8-oxo-7,8-dihydro-2′-deoxyguanosine of all study subjects, and individuals with colorectal cancer presented with the lowest concentrations of ascorbate and retinol. A positive correlation was observed between plasma concentration of ascorbate and levels of two epigenetic modifications, 5-hydroxymethylcytosine and 5-hydroxymethyluracil in leukocyte DNA. Moreover, a significant difference was found in the levels of these modifications in patients whose plasma concentrations of ascorbate were below the lower and above the upper quartile for the control group.ConclusionsThese findings suggest that deficiency of ascorbate in the blood may be a marker of its shortage in other tissues, which in turn may correspond to deterioration of DNA methylation-demethylation. These observations may provide a rationale for further research on blood biomarkers of colorectal cancer development.

Ascorbate Deficiency among Childrens of African Descent with Protein Energy Malnutrition in Sokoto, North Western Nigeria

Protein energy malnutrition is the most widespread nutritional deficiency disorder of mankind and continues to be a major public health burden particularly in developing countries. The aim of this case-control study was to determine the changes in the ascorbic acid levels among children with PEM in Sokoto, North Western Nigeria. The study included a total of 90 children (47 subjects with Protein Energy Malnutrition and 43 apparently healthy controls) aged 6 months-5 years, admitted to the Paediatric units of Usmanu Danfodiyo University Teaching Hospital and Specialist Hospital, Sokoto. Ascorbic acid levels were assayed by a standard chemical method. Nutritional status was determined using the Welcome Trust Classification. Data were analyzed using SPSS 22.0 statistical package. A p-value ≤ 0.05 was considered significant in all statistical comparisons. The mean value of ascorbic acid was significantly lower among subjects (0.82±0.04mg/dl) compared to controls (1.06±0.02 mg/dl) (p=0.0001). Underweight subjects had lower ascorbic acid levels when compared with other types of protein energy malnutrition (p=0.0001). Protein energy malnutrition was more prevalent among children from low socioeconomic class whose mothers have no formal education. Marasmus was the most common type of protein energy malnutrition. Finding from this study seems a justification to monitor the ascorbic acid levels in children with PEM and to possibly offer ascorbic acid supplementation for those that are deficient. There is need for infant feeding practice to be strengthened by promoting exclusive breast feeding. There is also the need for increased enrollment of women in schools, enlightenment on nutritional education and empowerment of women to improve their socioeconomic status

Vitamin C Sensitizes Melanoma to BET Inhibitors.

Bromodomain and extraterminal inhibitors (BETi) are promising cancer therapies, yet prominent side effects of BETi at effective doses have been reported in phase I clinical trials. Here, we screened a panel of small molecules targeting epigenetic modulators against human metastatic melanoma cells. Cells were pretreated with or without ascorbate (vitamin C), which promotes DNA demethylation and subsequently changes the sensitivity to drugs. Top hits were structurally unrelated BETi, including JQ1, I-BET151, CPI-203, and BI-2536. Ascorbate enhanced the efficacy of BETi by decreasing acetylation of histone H4, but not H3, while exerting no effect on the expression of BRD proteins. Histone acetyltransferase 1 (HAT1), which catalyzes H4K5ac and H4K12ac, was downregulated by ascorbate mainly via the TET-mediated DNA hydroxymethylation pathway. Loss of H4ac, especially H4K5ac and H4K12ac, disrupted the interaction between BRD4 and H4 by which ascorbate and BETi blocked the binding of BRD4 to acetylated histones. Cotreatment with ascorbate and JQ1 induced apoptosis and inhibited proliferation of cultured melanoma cells. Ascorbate deficiency as modeled in Gulo-/- mice diminished the treatment outcome of JQ1 for melanoma tumorgraft. In contrast, ascorbate supplementation lowered the effective dose of JQ1 needed to successfully inhibit melanoma tumors in mice. On the basis of our findings, future clinical trials with BETi should consider ascorbate levels in patients. Furthermore, ascorbate supplementation might help reduce the severe side effects that arise from BETi therapy by reducing the dosage necessary for treatment.Significance: This study shows that ascorbate can enhance the efficacy of BET inhibitors, providing a possible clinical solution to challenges arising in phase I trials from the dose-dependent side effects of this class of epigenetic therapy. Cancer Res; 78(2); 572-83. ©2017 AACR.

Mitochondrial dysfunction in the APP/PSEN1 mouse model of Alzheimer’s disease and a novel protective role for ascorbate

Mitochondrial dysfunction is elevated in very early stages of Alzheimer’s disease and exacerbates oxidative stress, which contributes to disease pathology. Mitochondria were isolated from 4-month-old wild-type mice, transgenic mice carrying the APPSWE and PSEN1dE9 mutations, mice with decreased brain and mitochondrial ascorbate (vitamin C) via heterozygous knockout of the sodium dependent vitamin C transporter (SVCT2+/-) and transgenic APP/PSEN1 mice with heterozygous SVCT2 expression. Mitochondrial isolates from SVCT2+/- mice were observed to consume less oxygen using high-resolution respirometry, and also exhibited decreased mitochondrial membrane potential compared to wild type isolates. Conversely, isolates from young (4 months) APP/PSEN1 mice consumed more oxygen, and exhibited an increase in mitochondrial membrane potential, but had a significantly lower ATP/ADP ratio compared to wild type isolates. Greater levels of reactive oxygen species were also produced in mitochondria isolated from both APP/PSEN1 and SVCT2+/- mice compared to wild type isolates. Acute administration of ascorbate to mitochondria isolated from wild-type mice increased oxygen consumption compared with untreated mitochondria suggesting ascorbate may support energy production. This study suggests that both presence of amyloid and ascorbate deficiency can contribute to mitochondrial dysfunction, even at an early, prodromal stage of Alzheimer’s disease, although occurring via different pathways. Ascorbate may, therefore, provide a useful preventative strategy against neurodegenerative disease, particularly in populations most at risk for Alzheimer’s disease in which stores are often depleted through mitochondrial dysfunction and elevated oxidative stress.

The epigenetic role of vitamin C in health and disease.

Recent advances have uncovered a previously unknown function of vitamin C in epigenetic regulation. Vitamin C exists predominantly as an ascorbate anion under physiological pH conditions. Ascorbate was discovered as a cofactor for methylcytosine dioxygenases that are responsible for DNA demethylation, and also as a likely cofactor for some JmjC domain-containing histone demethylases that catalyze histone demethylation. Variation in ascorbate bioavailability thus can influence the demethylation of both DNA and histone, further leading to different phenotypic presentations. Ascorbate deficiency can be presented systematically, spatially and temporally in different tissues at the different stages of development and aging. Here, we review how ascorbate deficiency could potentially be involved in embryonic and postnatal development, and plays a role in various diseases such as neurodegeneration and cancer through epigenetic dysregulation.

Famine From Feast: Low Red Cell Vitamin C Levels in Diabetes

Famine From Feast: Low Red Cell Vitamin C Levels in Diabetes

Effects produced by different types of laser in cornea of Guinea pigs: Identification of a laser capable of producing superficial lesions without leaving scars

PurposeClimatic droplets keratopathy (CDK) is closely associated with superficial corneal erosions and lack of protective mechanisms against the harmful effects of ultraviolet radiation (UVR) during a prolonged period of time. One of the difficulties in studying the pathogenic mechanisms involved in this human disease is the lack of an experimental animal model. In this paper, a study is conducted on the effects of 4 types of lasers at various powers and time conditions on the normal guinea pig corneas in order to select only one laser condition that reversibly injures the epithelium and superficial stroma, without leaving scarring. MethodsDamage was induced in the cornea of guinea pigs using different powers and exposure times of 4 types of laser: argon, CO2, diode and Nd–Yag, and any injuries were evaluated by biomicroscopy (BM) and optical microscopy. Corneas from other normal animals were exposed to argon laser (350mW, 0.3s, 50μm of diameter), and the induced alterations were studied at different times using BM, optical coherence tomography (OCT) and transmission electron microscopy (TEM). ResultsOnly argon laser at 350mW, 0.3s and 50μm of diameter produced epithelium and superficial stroma lesions. Some leukomas were observed by BM, and they disappeared by day 15. Corneal thickness measured by OCT decreased in the eyes treated with argon laser during the first week. Using TEM, different ultrastructural alterations in corneal epithelium and stroma were observed during the early days, which disappeared by day 15. ConclusionsIt was possible to develop reproducible corneal epithelium and anterior stroma injuries using argon laser at 350mW, 0.3s and 50μm of diameter. In vivo and in vitro studies showed that injured corneas with these laser conditions did not leave irreversible microscopic or ultrastructural alterations. This protocol of corneal erosion combined with exposure to UVR and partial deficiency of ascorbate in the diets of the animals for an extended period of time has been used to try to develop an experimental model of CDK.

Efectos producidos por diferentes tipos de láser en córnea de cobayos: identificación de un láser capaz de provocar lesiones superficiales sin dejar cicatrices

PurposeClimatic droplets keratopathy (CDK) is closely associated with superficial corneal erosions and lack of protective mechanisms against the harmful effects of ultraviolet radiation (UVR) during a prolonged period of time. One of the difficulties in studying the pathogenic mechanisms involved in this human disease is the lack of an experimental animal model. In this paper, a study is conducted on the effects of 4 types of lasers at various powers and time conditions on the normal guinea pig corneas in order to select only one laser condition that reversibly injures the epithelium and superficial stroma, without leaving scarring. MethodsDamage was induced in the cornea of Guinea pigs using different powers and exposure times of 4 types of laser: argon, CO2, diode and Nd-Yag, and any injuries were evaluated by biomicroscopy (BM) and optical microscopy. Corneas from other normal animals were exposed to argon laser (350 mW, 0.3s, 50μm of diameter), and the induced alterations were studied at different times using BM, optical coherence tomography (OCT) and transmission electron microscopy (TEM). ResultsOnly argon laser at 350 mW, 0.3s, 50μm of diameter produced epithelium and superficial stroma lesions. Some leukomas were observed by BM, and they disappeared by day 15. Corneal thickness measured by OCT decreased in the eyes treated with argon laser during the first week. Using TEM, different ultra structural alterations in corneal epithelium and stroma were observed during the early days, which disappeared by day 15. ConclusionsIt was possible to develop reproducible corneal epithelium and anterior stroma injuries using Argon laser at 350 mW, 0.3s, 50μm of diameter. In vivo and in vitro studies showed that injured corneas with these laser conditions did not leave irreversible microscopic or ultra structural alterations. This protocol of corneal erosion combined with exposure to UVR and partial deficiency of ascorbate in the diets of the animals for an extended period of time has been used in order to try to develop an experimental model of CDK.

Both the concentration and redox state of glutathione and ascorbate influence the sensitivity of arabidopsis to cadmium.

Background and Aims Cadmium (Cd) is a non-essential trace element that elicits oxidative stress. Plants respond to Cd toxicity via increasing their Cd-chelating and antioxidative capacities. They predominantly chelate Cd via glutathione (GSH) and phytochelatins (PCs), while antioxidative defence is mainly based on the use and recycling of both GSH and ascorbate (AsA), complemented by superoxide dismutase (SOD) and catalase (CAT). In addition, both metabolites act as a substrate for the regeneration of other essential antioxidants, which neutralize and regulate reactive oxygen species (ROS). Together, these functions influence the concentration and cellular redox state of GSH and AsA. In this study, these two parameters were examined in plants of Arabidopsis thaliana exposed to sub-lethal Cd concentrations.Methods Wild-type plants and mutant arabidopsis plants containing 30–45 % of wild-type levels of GSH (cad2-1) or 40–50 % of AsA (vtc1-1), together with the double-mutant (cad2-1 vtc1-1) were cultivated in a hydroponic system and exposed to sub-lethal Cd concentrations. Cadmium detoxification was investigated at different levels including gene expression and metabolite concentrations.Key Results In comparison with wild-type plants, elevated basal thiol levels and enhanced PC synthesis upon exposure to Cd efficiently compensated AsA deficiency in vtc1-1 plants and contributed to decreased sensitivity towards Cd. Glutathione-deficient (cad2-1 and cad2-1 vtc1-1) mutants, however, showed a more oxidized GSH redox state, resulting in initial oxidative stress and a higher sensitivity to Cd. In order to cope with the Cd stress to which they were exposed, GSH-deficient mutants activated multiple alternative pathways.Conclusions Our observations indicate that GSH and AsA deficiency differentially alter plant GSH homeostasis, resulting in opposite Cd sensitivities relative to wild-type plants. Upon Cd exposure, GSH-deficient mutants were hampered in chelation. They experienced phenotypic disturbances and even more oxidative stress, and therefore activated multiple alternative pathways such as SOD, CAT and ascorbate peroxidase, indicating a higher Cd sensitivity. Ascorbate deficiency, however, was associated with enhanced PC synthesis in comparison with wild-type plants after Cd exposure, which contributed to decreased sensitivity towards Cd.

Vitamin C In Neuropsychiatry

Abstract Vitamins are necessary factors in human development and normal brain function. Vitamin C is a hydrosoluble compound that humans cannot produce; therefore, we are completely dependent on food intake for vitamin C. Ascorbic acid is an important antioxidative agent and is present in high concentrations in neurons and is also crucial for collagen synthesis throughout the body. Ascorbic acid has a role in modulating many essential neurotransmitters, enables neurogenesis in adult brain and protects cells against infection. While SVCT1 enables the absorption of vitamin C in the intestine, SVCT2 is primarily located in the brain. Ascorbate deficiency is classically expressed as scurvy, which is lethal if not treated. However, subclinical deficiencies are probably much more frequent. Potential fields of vitamin C therapy are in neurodegenerative, cerebrovascular and affective diseases, cancer, brain trauma and others. For example, there is some data on its positive effects in Alzheimer’s disease. Various dosing regimes are used, but ascorbate is safe, even in high doses for protracted periods. Better designed studies are needed to elucidate all of the potential therapeutic roles of vitamin C.

Abstract 229: Restoring physiological levels of ascorbate alleviates HIF-1 activation and reduces tumour growth in Gulo-/- mice

Abstract Background: Hypoxia-inducible factor-1 (HIF-1) upregulates the expression of hundreds of genes involved in adaption of tumours to the hypoxic microenvironment. HIF-1 levels and transcriptional activity are regulated by hydroxylase enzymes, which require ascorbate as a cofactor. Adequate supplementation of cells with ascorbate has been shown to reduce HIF-1 activation in vitro, and we hypothesise that a similar activity could affect tumour growth in vivo. In this study we investigated the effect of dietary ascorbate on tumour growth in Gulo-/- mice, a model of the human ascorbate deficiency condition. Methods: C57/Bl6 Gulo-/- mice were supplemented with 3300 mg/L, 330 mg/L or 33mg/L of ascorbate in their drinking water for one month before subcutaneous, syngeneic tumour implantation with either B16-F10 melanoma, or Lewis lung carcinoma (LL/2), and then for duration of the experiment. Levels of HIF-1α, and its targets, carbonic anhydrase-IX (CA-IX), and glucose transporter-1 (GLUT-1) were analysed by western blotting, and vascular endothelial growth factor (VEGF) levels by ELISA. Ascorbate concentrations of tissue, plasma and tumours were measured using high pressure liquid chromatography with electrochemical detection (HPLC-EC). Results: Across all organs and plasma samples, ascorbate content was in the physiological range (up to levels seen in wild-type mice) and was correlated with dietary ascorbate intake. Tumour ascorbate also reflected ascorbate intake and optimal ascorbate levels (3300 mg/L) significantly increased the time for LL/2 tumours to reach 200 mm3 (lag growth) (13.3±2.6 days), compared to these same tumours in mice supplemented with either 330 mg/L (9.1±2.6 days) or 33 mg/L (9.8±1.39 days) of ascorbate (p<0.001). Similarly, tumours took significantly longer to grow from 200-800 mm3 (log phase) when mice were supplemented with 3300 mg/L compared to lower supplementation (p<0.05). B16-F10 tumours showed no difference in the lag growth period between ascorbate treatments (p=0.49). However, supplementing mice with 3300 mg/mL of ascorbate lead to a significant reduction in the tumour growth rate during log phase (3.9±1.1 days), compared to mice supplemented with either 330 mg/L (2.0±0.90 days) or 33mg/L (2.1±0.71 days) (p<0.001). Levels of HIF-1α protein in tumours decreased as dietary ascorbate supplementation increased for both LL/2 (p<0.001) and B16F10 (p<0.001). Tumour ascorbate was significantly correlated with low levels of CA-IX, GLUT-1 and VEGF levels in both LL/2 and B16 tumours (all p<0.05). Conclusions: The data from the current study support the hypothesis that restoration of optimal intracellular ascorbate levels is associated with reduced HIF-1 levels and slower tumour growth. This data may have implications for the management of cancer, although clinical trials are required to assess whether human tumour levels of ascorbate can similarly be manipulated. Citation Format: Elizabeth J. Campbell, Stephanie M. Bozonet, Bridget A. Robinson, Margreet CM Vissers, Gabi U. Dachs. Restoring physiological levels of ascorbate alleviates HIF-1 activation and reduces tumour growth in Gulo-/- mice. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 229. doi:10.1158/1538-7445.AM2014-229

Ascorbate deficiency influences the leaf cell wall glycoproteome in Arabidopsis thaliana

The cell wall forms the first line of interaction between the plant and the external environment. Based on the observation that ascorbate-deficient vtc mutants of Arabidopsis thaliana have increased cell wall peroxidase activity, the cell wall glycoproteome of vtc2-2 was investigated. Glycoproteins were purified from fully expanded leaves by Concanavalin A affinity chromatography and analysed by liquid chromatography quadrupole time-of-flight mass spectrometry. This procedure identified 63 proteins with predicted glycosylation sites and cell wall localization. Of these, 11 proteins were differentially expressed between vtc2-2 and wild type. In particular, PRX33/34 were identified as contributing to increased peroxidase activity in response to ascorbate deficiency. This is the same peroxidase previously shown to contribute to hydrogen peroxide generation and pathogen resistance. Three fasciclin-like arabinogalactan proteins (FLA1, 2 and 8) had lower abundance in vtc2-2. Inspection of published microarray data shows that these also have lower gene expression in vtc1 and vtc2-1 and are decreased in expression by pathogen challenge and oxidative stresses. Ascorbate deficiency therefore impacts expression of cell wall proteins involved in pathogen responses and these presumably contribute to the increased resistance of vtc mutants to biotrophic pathogens.

Role of vitamin C in the function of the vascular endothelium.

Vitamin C, or ascorbic acid, has long been known to participate in several important functions in the vascular bed in support of endothelial cells. These functions include increasing the synthesis and deposition of type IV collagen in the basement membrane, stimulating endothelial proliferation, inhibiting apoptosis, scavenging radical species, and sparing endothelial cell-derived nitric oxide to help modulate blood flow. Although ascorbate may not be able to reverse inflammatory vascular diseases such as atherosclerosis, it may well play a role in preventing the endothelial dysfunction that is the earliest sign of many such diseases. Beyond simply preventing scurvy, evidence is mounting that ascorbate is required for optimal function of many dioxygenase enzymes in addition to those involved in collagen synthesis. Several of these enzymes regulate the transcription of proteins involved in endothelial function, proliferation, and survival, including hypoxia-inducible factor-1α and histone and DNA demethylases. More recently, ascorbate has been found to acutely tighten the endothelial permeability barrier and, thus, may modulate access of ascorbate and other molecules into tissues and organs. The issue of the optimal cellular content of ascorbate remains unresolved, but it appears that low millimolar ascorbate concentrations are normal in most animal tissues, in human leukocytes, and probably in the endothelium. Although there may be little benefit of increasing near maximal cellular ascorbate concentrations in normal people, many diseases and conditions have either systemic or localized cellular ascorbate deficiency as a cause for endothelial dysfunction, including early atherosclerosis, sepsis, smoking, and diabetes. A key focus for future studies of ascorbate and the vascular endothelium will likely be to determine the mechanisms and clinical relevance of ascorbate effects on endothelial function, permeability, and survival in diseases that cause endothelial dysfunction.

Ascorbate Induces Ten-Eleven Translocation (Tet) Methylcytosine Dioxygenase-mediated Generation of 5-Hydroxymethylcytosine

Tet methylcytosine dioxygenase converts 5-mC to 5-hmC in DNA. Ascorbate significantly and specifically enhances Tet-mediated generation of 5-hmC. Our findings suggest that ascorbate enhances 5-hmC generation, most likely by acting as a co-factor for Tet methylcytosine dioxygenase to generate 5-hmC. The availability of ascorbate could have significant consequences for health and diseases by modulating the epigenetic control of genome activity. Ascorbate (vitamin C) is best known for its role in scurvy, in which the hydroxylation of collagen catalyzed by dioxygenases is incomplete due to ascorbate deficiency. Here, we report a novel function of ascorbate in the hydroxylation of 5-methylcytosine (5-mC) to 5-hydroxymethylcytosine (5-hmC) in DNA catalyzed by Tet (ten-eleven translocation) methylcytosine dioxygenase. The content of 5-hmC is extremely low in mouse embryonic fibroblasts cultured in ascorbate-free medium. Additions of ascorbate dose- and time-dependently enhance the generation of 5-hmC, without any effects on the expression of Tet genes. Treatment with another reducer glutathione (GSH) does not change the level of 5-hmC. Further, blocking ascorbate entry into cells by phloretin and knocking down Tet (Tet1, Tet2, and Tet3) expression by short interference RNAs (siRNA) significantly inhibit the effect of ascorbate on 5-hmC. These results suggest that ascorbate enhances 5-hmC generation, most likely by acting as a co-factor for Tet methylcytosine dioxygenase to hydroxylate 5-mC. Thus, we have uncovered a novel role for ascorbate in modulating the epigenetic control of genome activity.

Tolerance to nitroglycerin through proteasomal down‐regulation of aldehyde dehydrogenase‐2 in a genetic mouse model of ascorbate deficiency

Background and PurposeL-gulonolactone oxidase-deficient (Gulo(-/-)) mice were used to study the effects of ascorbate deficiency on aortic relaxation by nitroglycerin (GTN) with focus on changes in the expression and activity of vascular aldehyde dehydrogenase-2 (ALDH2), which catalyses GTN bioactivation.Experimental ApproachAscorbate deficiency was induced in Gulo(-/-) mice by ascorbate deprivation for 4 weeks. Some of the animals were concomitantly treated with the proteasome inhibitor bortezomib and effects compared with ascorbate-supplemented Gulo(-/-), untreated or nitrate-tolerant wild-type mice. Aortic relaxation of the experimental groups to GTN, ACh and a NO donor was studied. Changes in mRNA and protein expression of vascular ALDH2 were quantified by qPCR and immunoblotting, respectively, and aortic GTN denitration rates determined.Key ResultsLike GTN treatment, ascorbate deprivation induced vascular tolerance to GTN that was associated with markedly decreased rates of GTN denitration. Ascorbate deficiency did not affect ALDH2 mRNA levels, but reduced ALDH2 protein expression and the total amount of ubiquitinated proteins to about 40% of wild-type controls. These effects were largely prevented by ascorbate supplementation or treating Gulo(-/-) mice with the 26S proteasome inhibitor bortezomib.Conclusions and ImplicationsOur data indicate that ascorbate deficiency results in vascular tolerance to GTN via proteasomal degradation of ALDH2. The results support the view that impaired ALDH2-catalysed metabolism of GTN contributes significantly to the development of vascular nitrate tolerance and reveal a hitherto unrecognized protective effect of ascorbate in the vasculature.

Tolerance to nitroglycerin through proteasomal down-regulation of aldehyde dehydrogenase-2 in a genetic mouse model of ascorbate deficiency: Nitrate tolerance in ascorbate deficiency

Tolerance to nitroglycerin through proteasomal down-regulation of aldehyde dehydrogenase-2 in a genetic mouse model of ascorbate deficiency: Nitrate tolerance in ascorbate deficiency

Pyrophosphate levels strongly influence ascorbate and starch content in tomato fruit

Ascorbate (vitamin C) deficiency leads to low immunity, scurvy, and other human diseases and is therefore a global health problem. Given that plants are major ascorbate sources for humans, biofortification of this vitamin in our foodstuffs is of considerable importance. Ascorbate is synthetized by a number of alternative pathways: (i) from the glycolytic intermediates D-glucose-6P (the key intermediates are GDP-D-mannose and L-galactose), (ii) from the breakdown of the cell wall polymer pectin which uses the methyl ester of D-galacturonic acid as precursor, and (iii) from myo-inositol as precursor via myo-inositol oxygenase. We report here the engineering of fruit-specific overexpression of a bacterial pyrophosphatase, which hydrolyzes the inorganic pyrophosphate (PPi) to orthophosphate (Pi). This strategy resulted in increased vitamin C levels up to 2.5-fold in ripe fruit as well as increasing in the major sugars, sucrose, and glucose, yet decreasing the level of starch. When considered together, these finding indicate an intimate linkage between ascorbate and sugar biosynthesis in plants. Moreover, the combined data reveal the importance of PPi metabolism in tomato fruit metabolism and development.

Methemoglobinemia and ascorbate deficiency in hemoglobin E β thalassemia: metabolic and clinical implications

AbstractDuring investigations of the phenotypic diversity of hemoglobin (Hb) E β thalassemia, a patient was encountered with persistently high levels of methemoglobin associated with a left-shift in the oxygen dissociation curve, profound ascorbate deficiency, and clinical features of scurvy; these abnormalities were corrected by treatment with vitamin C. Studies of erythropoietin production before and after treatment suggested that, as in an ascorbate-deficient murine model, the human hypoxia induction factor pathway is not totally dependent on ascorbate levels. A follow-up study of 45 patients with HbE β thalassemia showed that methemoglobin levels were significantly increased and that there was also a significant reduction in plasma ascorbate levels. Haptoglobin levels were significantly reduced, and the high frequency of the 2.2 haptoglobin genotype may place an additional pressure on ascorbate as a free-radical scavenger in this population. There was, in addition, a highly significant correlation between methemoglobin levels, splenectomy, and factors that modify the degree of globin-chain imbalance. Because methemoglobin levels are modified by several mechanisms and may play a role in both adaptation to anemia and vascular damage, there is a strong case for its further study in other forms of thalassemia and sickle-cell anemia, particularly when splenic function is defective.

Tolerance to nitroglycerin through proteasomal degradation of aldehyde dehydrogenase-2 in a genetic mouse model of ascorbate deficiency

Tolerance to nitroglycerin through proteasomal degradation of aldehyde dehydrogenase-2 in a genetic mouse model of ascorbate deficiency