Ascorbic acid and dehydroascorbic acid measurements in human plasma and serum

Dhariwal, Kuldeep R.; Hartzell, William O.; Levine, Mark

doi:10.1093/ajcn/54.4.712

Cited by 242 publications

(139 citation statements)

References 23 publications

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“…The present study demonstrates that L-ascorbic acid in physiologically relevant concentrations (28,29) potentiates agonistinduced endothelial NO synthesis in a dose-and time-dependent fashion. This was shown by concomitant changes of both the formation of the NO co-product citrulline and the accumulation of the NO effector molecule cGMP, although the latter might additionally indicate an increase in biological activity of NO.…”

Section: Discussionsupporting

confidence: 55%

“…The latter followed a kinetics similar to the ascorbate uptake being about half-maximal after a 6-h incubation of cells with 100 M ascorbic acid and maximal between 18 and 24 h. Moreover, a saturation of the ascorbate uptake into endothelial cells which might occur between 100 and 200 M (27) could explain the lack of further NO synthesis potentiation with ascorbic acid concentrations above 100 M. Interestingly, 100 M is in the range of plasma levels of healthy individuals (28), and similar concentrations have been found in a recent pharmacokinetic study that described the ascorbic acid concentration as a function of dose in healthy volunteers (29).…”

Section: Discussionmentioning

confidence: 91%

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l-Ascorbic Acid Potentiates Nitric Oxide Synthesis in Endothelial Cells

Heller

Münscher-Paulig

Gräbner

et al. 1999

Journal of Biological Chemistry

210

106

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Ascorbic acid has been shown to enhance impaired endothelium-dependent vasodilation in patients with atherosclerosis by a mechanism that is thought to involve protection of nitric oxide (NO) from inactivation by free oxygen radicals. The present study in human endothelial cells from umbilical veins and coronary arteries investigates whether L-ascorbic acid additionally affects cellular NO synthesis. Endothelial cells were incubated for 24 h with 0.1-100 M ascorbic acid and were subsequently stimulated for 15 min with ionomycin (2 M) or thrombin (1 unit/ml) in the absence of extracellular ascorbate. Ascorbate pretreatment led to a 3-fold increase of the cellular production of NO measured as the formation of its co-product citrulline and as the accumulation of its effector molecule cGMP. The effect was saturated at 100 M and followed a similar kinetics as seen for the uptake of ascorbate into the cells. The investigation of the precursor molecule L-gulonolactone and of different ascorbic acid derivatives suggests that the enediol structure of ascorbate is essential for its effect on NO synthesis. Ascorbic acid did not induce the expression of the NO synthase (NOS) protein nor enhance the uptake of the NOS substrate L-arginine into endothelial cells. The ascorbic acid effect was minimal when the citrulline formation was measured in cell lysates from ascorbate-pretreated cells in the presence of known cofactors for NOS activity. However, when the cofactor tetrahydrobiopterin was omitted from the assay, a similar potentiating effect of ascorbate pretreatment as seen in intact cells was demonstrated, suggesting that ascorbic acid may either enhance the availability of tetrahydrobiopterin or increase its affinity for the endothelial NOS. Our data suggest that intracellular ascorbic acid enhances NO synthesis in endothelial cells and that this may explain, in part, the beneficial vascular effects of ascorbic acid. Endothelium-derived nitric oxide (NO)1 exerts several vasoprotective activities including smooth muscle relaxation, inhibition of platelet activation, and regulation of endothelial cell permeability and adhesivity (1-4). NO is generated from the conversion of L-arginine to L-citrulline by the enzymatic action of an NADPH-dependent NO synthase (NOS) that requires tetrahydrobiopterin, FAD, and FMN as cofactors (5). The endothelial NOS isoform (ecNOS) is constitutively expressed and is activated upon an increase of intracellular calcium following cell stimulation with receptor-dependent stimuli such as thrombin and bradykinin or with receptor-independent stimuli like calcium ionophore (6).Since NO interferes with key processes in atherogenesis (7), a lack of NO might promote the development of atherosclerosis. Indeed, clinical studies have confirmed an impairment of vascular NO synthesis in patients with atherosclerosis or with increased atherogenic risk factors (8). The dysfunction of the endothelial NO pathway may involve impaired signal transduction mechanisms, decreased ecNOS activity, reduced intracellular a...

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Section: Discussionsupporting

confidence: 55%

Section: Discussionmentioning

confidence: 91%

l-Ascorbic Acid Potentiates Nitric Oxide Synthesis in Endothelial Cells

Heller

Münscher-Paulig

Gräbner

et al. 1999

Journal of Biological Chemistry

210

106

View full text Add to dashboard Cite

show abstract

“…Increased plasma levels of DHA, the two-electron oxidation product of AH, have been reported in diseases such as diabetes [7] and might be formed under acute inflammatory conditions in which phagocytes become activated and release oxidants [8]. However, in normal subjects, plasma vitamin C is primarily found in its fully reduced, antioxidant form (AH) [9]. Thus it seems that, under normal physiological conditions, effective mechanism(s) exist that eliminate DHA.…”

Section: Introductionmentioning

confidence: 99%

Efflux of hepatic ascorbate: a potential contributor to the maintenance of plasma vitamin C

Upston

Karjalainen

Bygrave

et al. 1999

Biochemical Journal

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Ascorbate (AH, the reduced form of vitamin C) is an important radical scavenger and antioxidant in human plasma; the resulting ascorbyl radical can disproportionate to AH and dehydroascorbic acid (DHA). Here we address potential maintenance mechanism(s) for extracellular AH by examining the ability of cells to convert extracellularly presented DHA to AH. DHA was rapidly transported into human liver (HepG2), endothelial and whole blood cells invitro by plasma membrane glucose transporters and reduced intracellularly. Liver cells displayed the highest capacity to release the intracellularly accumulated AH. The proteins responsible for DHA uptake and AH release could be distinguished by inhibitor studies. Thus, unlike DHA uptake, AH efflux was largely insensitive to cytochalasin B and thiol-reactive agents but was inhibited by phloretin, 4,4′-di-isothiocyanostilbene-2,2′-disulphonate and isoascorbate. Efflux of AH from cells was temperature-sensitive and saturable with a low affinity (millimolar, intracellular) for AH. In addition to isolated liver cells, perfusion of intact rat and guinea-pig liver with DHA resulted in AH in the circulating perfusate. Our results show that hepatocytes take up and reduce DHA and subsequently release part of the AH formed, probably via a membrane transporter. By converting extracellular DHA to extracellular AH, the liver might contribute to the maintenance of plasma AH, a process that could be important under conditions of oxidative stress.

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“…Thus, DHA must compete with Dglucose for uptake on the GLUT-type transporters. Since plasma D-glucose concentrations are 5 mM and those of DHA are less than 2 μM (Dhariwal et al, 1991), DHA uptake will not be favored in cells in the vascular bed. Further, human erythrocytes, which contain abundant GLUT1 (Allard & Lienhard, 1985), but no SVCT2 (May et al, 2007), have intracellular ascorbate concentrations very close to those in the blood from which they were prepared (Evans et al, 1982;Mendiratta et al, 1998).…”

Section: Cellular Uptake Of Ascorbatementioning

confidence: 99%

Inflammation in the vascular bed: Importance of vitamin C

Aguirre

May

2008

Pharmacology & Therapeutics

110

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Despite decreases in atherosclerotic coronary vascular disease over the last several decades, atherosclerosis remains a major cause of mortality in developed nations. One possible contributor to this residual risk is oxidant stress, which is generated by the inflammatory response of atherosclerosis. Although there is a wealth of in vitro, cellular, and animal data supporting a protective role for antioxidant vitamins and nutrients in the atherosclerotic process, the best clinical trials have been negative. This may be due to the fact that antioxidant therapies are applied "too little and too late." This review considers the role of vitamin C, or ascorbic acid in preventing the earliest inflammatory changes in atherosclerosis. It focuses on the three major vascular cell types involved in atherosclerosis: endothelial cells, vascular smooth muscle cells, and macrophages. Ascorbate chemistry, recycling, and function are described for these cell types, with emphasis on whether and how the vitamin might affect the inflammatory process. For endothelial cells, ascorbate helps to prevent endothelial dysfunction, stimulates type IV collagen synthesis, and enhances cell proliferation. For vascular smooth muscle cells, ascorbate inhibits dedifferentiation, recruitment, and proliferation in areas of vascular damage. For macrophages, ascorbate decreases oxidant stress related to their activation, decreases uptake and degradation of oxidized LDL in some studies, and enhances several aspects of their function. 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.

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Ascorbic acid and dehydroascorbic acid measurements in human plasma and serum

Cited by 242 publications

References 23 publications

l-Ascorbic Acid Potentiates Nitric Oxide Synthesis in Endothelial Cells

l-Ascorbic Acid Potentiates Nitric Oxide Synthesis in Endothelial Cells

Efflux of hepatic ascorbate: a potential contributor to the maintenance of plasma vitamin C

Inflammation in the vascular bed: Importance of vitamin C

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