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| ASCORBIC ACID FUNCTION AND METABOLISMSummaryPrincipal Investigator: James M May Affiliation: Vanderbilt University Country: USA Abstract: Ascorbic acid, or vitamin C, is the most important water-soluble antioxidant in the human circulation. Although humans cannot synthesize ascorbate de novo, it can be efficiently recycled within cells from its oxidized product, dehydroascorbate. Our work has suggested that such ascorbate recycling is particularly important in the human erythrocyte, where such recycling can contribute both to circulating levels of the vitamin, and to protecting the erythrocyte membrane against lipid peroxidative damage. The goals of this proposal are to determine the mechanisms by which erythrocyte ascorbate recycling contributes to the defense against lipid peroxidation in blood, and to establish the extent to which this protection occurs. To achieve these goals, we will utilize techniques of fluorescence spectroscopy, EPR spectroscopy, molecular cloning, and novel biochemical assays. Whether and how ascorbate functions to protect cell membranes and lipoproteins from oxidation is relevant to mechanisms of inflammation, ageing, and atherosclerosis, especially given the fact that dietary supplementation can more than double circulating and tissue levels of the vitamin. In the first specific aim, we will address the question of how reducing equivalents from intracellular ascorbate are transferred across or into the cell membrane. In erythrocytes such transfer appears to be mediated via a transmembrane ascorbate-dependent oxidoreductase enzyme. We will first complete our studies of the kinetic mechanism of this enzyme activity, purify it from human erythrocytes, and clone its cDNA sequence from a bone marrow-derived cDNA library. We will also evaluate the role of ubiquinol-10 (coenzyme Q10) as a cofactor necessary for electron transfer by the enzyme as well as whether the enzyme is functions similarly in a cultured monocyte-derived cell. In the second specific aim-, we will determine whether and to what extent membrane-bound lipid hydroperoxides, alpha-tocopherol and ubiquinone-10 are natural acceptors of electrons derived from ascorbate within erythrocytes. In the third specific aim we will determine whether extracellular ascorbate or its lipophilic derivatives can prevent oxidant-mediated damage in erythrocyte membranes, either directly or through recycling of alpha tocopherol. We will also test the hypothesis that ascorbate-dependent recycling of alpha tocopherol in erythrocytes can replenish depleted stores of alpha tocopherol in human LDL. Funding Period: 1995-09-30 - 2000-08-31 more information: NIH RePORT Top Publications
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Transport and intracellular accumulation of vitamin C in endothelial cells: relevance to collagen synthesisJames M May
Department of Medicine, Vanderbilt University School of Medicine, Nashville, TN 37232 6303, USA
Arch Biochem Biophys 434:178-86. 2005..Since such concentrations can only be generated by the ascorbate transporter, these results show the necessity of transport for this crucial function of the vitamin in endothelium...
Transfer of ascorbic acid across the vascular endothelium: mechanism and self-regulationJames M May
Department of Medicine, Vanderbilt Univ School of Medicine, Nashville, TN 37232 0475, USA
Am J Physiol Cell Physiol 297:C169-78. 2009..Since the latter effect occurred over the physiological range of ascorbate plasma concentrations, it could reflect a role for the vitamin in control of endothelial barrier function in vivo...- Cobalt-induced oxidant stress in cultured endothelial cells: prevention by ascorbate in relation to HIF-1alphaHuan Qiao
Department of Medicine, Vanderbilt University School of Medicine, Nashville, TN 37232 0475, USA
Biofactors 35:306-13. 2009.... 
Oxidized lipoprotein induces the macrophage ascorbate transporter (SVCT2): protection by intracellular ascorbate against oxidant stress and apoptosisXiumei Chi
Department of Medicine, Vanderbilt University School of Medicine, 7465 Medical Research Building IV, Nashville, TN 37232 6303, USA
Arch Biochem Biophys 485:174-82. 2009..These results suggest that ascorbate protects macrophages against oxLDL-induced oxidant stress and subsequent apoptotic death without impairing their function...- Macrophage differentiation increases expression of the ascorbate transporter (SVCT2)Huan Qiao
Department of Medicine, Vanderbilt University School of Medicine, Nashville, TN 37232 6303, USA
Free Radic Biol Med 46:1221-32. 2009.... - Vitamin C function in the brain: vital role of the ascorbate transporter SVCT2Fiona E Harrison
Department of Medicine, Vanderbilt University School of Medicine, Nashville, TN 37232 6303, USA
Free Radic Biol Med 46:719-30. 2009.... - Ascorbic acid efflux and re-uptake in endothelial cells: maintenance of intracellular ascorbateJames M May
Department of Medicine, Vanderbilt University School of Medicine, 7465 Medical Research Building IV, Nashville, TN 37232 0475, USA
Mol Cell Biochem 325:79-88. 2009..These results support the hypothesis that ascorbate efflux reflects trans-endothelial cell ascorbate movement out of the blood vessel... - Inflammation in the vascular bed: importance of vitamin CRene Aguirre
Department of Medicine, Vanderbilt University School of Medicine, Nashville, TN 37232 6303, USA
Pharmacol Ther 119:96-103. 2008..Although further studies of ascorbate function in these cell types and in novel animal models are needed, available evidence generally supports a salutary role for this vitamin in ameliorating the earliest stages of atherosclerosis... - Ascorbic acid uptake and regulation of type I collagen synthesis in cultured vascular smooth muscle cellsHuan Qiao
Department of Medicine, Vanderbilt University School of Medicine, Nashville, Tennessee 37232, USA
J Vasc Res 46:15-24. 2009..Since inflammation is associated with oxidant stress, we examined the uptake and cellular effects of the antioxidant vitamin ascorbic acid in cultured A10 vascular smooth muscle cells... - Development of ascorbate transporters in brain cortical capillary endothelial cells in cultureHuan Qiao
Department of Medicine, Vanderbilt University School of Medicine, Nashville, TN 37232 0475, USA
Brain Res 1208:79-86. 2008.... - alpha-Lipoic acid and ascorbate prevent LDL oxidation and oxidant stress in endothelial cellsAnup K Sabharwal
Department of Medicine, Vanderbilt University School of Medicine, 7465 Medical Research Building IV, Nashville, TN 37232 0475, USA
Mol Cell Biochem 309:125-32. 2008..Both LA and ascorbate protect endothelial cells and LDL from either intra- or extracellular oxidant stress, but that LA does not spare ascorbate in oxidatively stressed cells... - Maturational loss of the vitamin C transporter in erythrocytesJames M May
Department of Medicine, Vanderbilt University School of Medicine, 715 Preston Research Building, Nashville, TN 37232 6303, USA
Biochem Biophys Res Commun 360:295-8. 2007..Rates of specific ascorbate transport correlated with the presence of the SVCT2. These results show that mature erythrocytes fail to concentrate ascorbate due to the loss of SVCT2 during maturation in the bone marrow... - Human erythrocyte membranes contain a cytochrome b561 that may be involved in extracellular ascorbate recyclingDan Su
Department of Biochemistry, University of Nebraska, Lincoln, Nebraska 68588, USA
J Biol Chem 281:39852-9. 2006..These results suggest that the DCytb present in human erythrocytes may contribute to their ability to reduce extracellular monodehydroascorbate... - Macrophage uptake and recycling of ascorbic acid: response to activation by lipopolysaccharideJames M May
Department of Medicine, 715 Preston Research Building, Vanderbilt University School of Medicine, Nashville, TN 37232 6303, USA
Free Radic Biol Med 39:1449-59. 2005.... - Ascorbate uptake and antioxidant function in peritoneal macrophagesJames M May
Department of Medicine, Vanderbilt University School of Medicine, Nashville, TN 37232 6303, USA
Arch Biochem Biophys 440:165-72. 2005..These results suggest that activated macrophages can use ascorbate to lessen self-generated oxidant stress and spare alpha-tocopherol, which may protect these long-lived cells from necrosis or apoptosis... - Ascorbic acid decreases oxidant stress in endothelial cells caused by the nitroxide tempolJames M May
Department of Medicine, 754 Preston Research Building, Vanderbilt University School of Medicine, Nashville TN 37232 6303, USA
Free Radic Res 39:195-202. 2005..These results show that whereas Tempol may scavenge other more toxic radicals, care must be taken to ensure that it does not itself induce an oxidant stress, especially with regard to depletion of ascorbic acid... - Assessing the reductive capacity of cells by measuring the recycling of ascorbic and lipoic acidsJames M May
Department of Medicine, Vanderbilt University Medical Center, Nashville, TN, USA
Methods Mol Biol 610:229-43. 2010..Together, these assays provide an estimate of the ability of different cell types to recycle ascorbate and to generate intracellular reducing equivalents required to maintain the redox status of the cells...