Accumulation of Vitamin C (Ascorbate) and Its Oxidized Metabolite Dehydroascorbic Acid Occurs by Separate Mechanisms

Welch, Richard W.; Wang, Yaohui; Crossman, Arthur; Park, Jae B.; Kirk, Kenneth L.; Levine, Mark

doi:10.1074/jbc.270.21.12584

Cited by 173 publications

(171 citation statements)

References 35 publications

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“…We first examined whether HepG2 cells accumulated and reduced DHA and whether part of the resulting AH was subsequently released into the extracellular space. As shown previously for other cell types (see, for example, [13,[22][23][24]), incubation of adherent HepG2 cells in itro with 80 µM DHA at 37 mC resulted in detectable intracellular AH. The concentration of AH increased time-dependently to 5 nmol\10' cells over 1 h, concurrently with a decrease in the concentration of DHA in the extracellular medium ( Figure 1A).…”

Section: Resultssupporting

confidence: 76%

“…Previous studies on the uptake of extracellular DHA and its subsequent intracellular conversion to AH have commonly used erythrocytes [13,[22][23][24]. It has been suggested that erythrocytes release AH [22] ; however, the detection of AH efflux from erythrocytes resuspended at 37 mC in HBSS was hampered by haemoglobin leakage (results not shown), as this gives rise to inadvertent oxidation of AH during sample preparation [25].…”

Section: Resultsmentioning

confidence: 99%

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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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Section: Resultssupporting

confidence: 76%

Section: Resultsmentioning

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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“…At concentrations higher than ≈400 μM, relative uptake decreased. These results are consistent with the known active accumulation of ascorbate by cells to concentrations many times more than in the extracellular space [18,65,66]. The ascorbate levels in human neutrophils and monocytes in vivo [16] are similar to the levels of ascorbate taken up by HL-60 and U937 cells, using our approach.…”

Section: Cellular Uptake Of Asch − Is Higher In U937 Cells Compared Tsupporting

confidence: 89%

“…It's concentration in the blood of normal individuals varies from 5 to 90 μM [17]. However, it is actively accumulated in human tissues to a concentration as much as 50-fold greater than plasma [18]. In vitro cancer cells also accumulate ascorbate [19,20].…”

Section: Introductionmentioning

confidence: 99%

Ascorbate enhances the toxicity of the photodynamic action of Verteporfin in HL-60 cells

Kramarenko

Wilke

Dayal

et al. 2006

Free Radical Biology and Medicine

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As a reducing agent, ascorbate serves as an antioxidant. However, its reducing function can in some settings initiate an oxidation cascade, i.e. appear to be a "pro-oxidant". This dichotomy also appears to hold when ascorbate is present during photosensitization. Ascorbate can react with singlet oxygen producing hydrogen peroxide. Thus, if ascorbate is present during photosensitization the formation of highly diffusible hydrogen peroxide could enhance the toxicity of the photodynamic action. On the other side, ascorbate could decrease toxicity by converting highly reactive singlet oxygen to less reactive hydrogen peroxide, which can be removed via peroxide-removing systems such as glutathione and catalase. To test the influence of ascorbate on photodynamic treatment we incubated leukemia cells (HL-60 and U937) with ascorbate and a photosensitizer (verteporfin, VP) and examined: AscH − uptake; levels of glutathione; changes in membrane permeability; cell growth; and toxicity. Accumulation of VP was similar in each cell line. Under our experimental conditions, HL-60 cells were found to accumulate less ascorbate and have lower levels of intracellular GSH compared to U937 cells. Without added ascorbate, HL-60 cells were more sensitive to VP and light treatment than U937 cells. When cells were exposed to VP and light ascorbate acted as an antioxidant in U937 cells, while it was a "pro-oxidant" for HL-60 cells. One possible mechanism to explain these observations is that HL-60 cells express myeloperoxidase activity, while in U937 cells it is below detection limit. Inhibition of myeloperoxidase activity with 4-aminobenzoic acid hydrazide (4-ABAH) had minimal influence on the phototoxicity of VP in HL-60 in the absence of ascorbate. However, 4-ABAH decreased the toxicity of ascorbate on HL-60 cells during VP-photosensitization, but had no affect on ascorbate toxicity in U937 cells. These data demonstrate that ascorbate increases hydrogen peroxide production by VP and light. This hydrogen peroxide activates MPO, producing toxic oxidants. These observations suggest that in some settings, ascorbate may enhance the toxicity of photodynamic action.

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“…Vitamin E was determined at 290 nm. The lymphocyte and neutrophil number obtained from a known blood volume, together with previous data on neutrophil (30.10 À8 ml/neutrophil; Welch et al, 1995) and lymphocyte volume (21.10 À6 ml/lymphocyte; Segel et al, 1981) were used to calculate vitamin E concentration (mM) in lymphocytes and neutrophils. Vitamin E/cholesterol (mmol/mmol) ratio was also calculated.…”

Section: Vitamin E Determinationmentioning

confidence: 99%

Differential response of plasma and immune cell's vitamin E levels to physical activity and antioxidant vitamin supplementation

et al. 2005

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Objective: To assess the differential response of plasma, lymphocyte and neutrophil vitamin E levels to high-intensity physical activity and to vitamin C and E supplementation. Subjects: In all, 14 male trained amateur runners (32-36 y old) were randomly divided in two groups (supplemented and placebo), and participated in a half marathon race. The subjects did not take any other supplements than the ones provided for this study. Intervention: Vitamin C (152 mg/day) and E (50 mg/day) supplementation was administrated to athletes for a month, using a new almond-based isotonic and energetic beverage (supplemented group). The usual dietary habits of participants were assessed using a self-reported 7-day 24-h recall before the day of the study. To avoid the beverage influence, nonenriched vitamin C and E almond-based isotonic and energetic beverage was given to the placebo group. After 1 month, subjects participated in a half marathon race (21 km run). Vitamin E concentration was determined in plasma, neutrophils and lymphocytes before and immediately after the race, and 3 h after finishing the race. Results: Daily energy intake and caloric profile of supplemented and placebo group were not different except for vitamin C and E supplementation. Vitamin supplementation and exercise had no effect on vitamins E levels in plasma. The exercise significantly (Po0.05) increased the lymphocyte vitamin E concentration both in the placebo ( þ 119%) and supplemented groups ( þ 128%), and neutrophil vitamin E content in the supplemented group ( þ 88%). These levels remained significantly (Po0.05) high after the short recovery. After exercise, vitamin E levels in lymphocytes and neutrophils of supplemented subjects were practically twice the levels before exercise, whereas neutrophil vitamin E content of the placebo group was close to those in plasma. Conclusion: After endurance exercise, lymphocytes increased their vitamin E content in the supplemented and placebo subjects whereas this trend in neutrophils was just observed in the supplemented group. The determination of vitamin E content in lymphocytes and neutrophils after exercise is a useful tool to assess the functional status of vitamin E.

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Accumulation of Vitamin C (Ascorbate) and Its Oxidized Metabolite Dehydroascorbic Acid Occurs by Separate Mechanisms

Cited by 173 publications

References 35 publications

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

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

Ascorbate enhances the toxicity of the photodynamic action of Verteporfin in HL-60 cells

Differential response of plasma and immune cell's vitamin E levels to physical activity and antioxidant vitamin supplementation

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