Measurement of Vitamin C in Blood Components by High‐Performance Liquid Chromatography

Omaye, Stanley T.; Schaus, Ellen E.; Kutnink, Mark A.; Hawkes, Wayne Chris

doi:10.1111/j.1749-6632.1987.tb23776.x

Cited by 53 publications

(44 citation statements)

References 29 publications

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“…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. Although the molar concentration of ascorbate is higher in the cells compared to the media, at 1 × 10 6 cell mL −1 there will be ≈50 -200 times more moles of ascorbate in the media than in the cells.…”

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

confidence: 89%

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

confidence: 89%

“…As a vitamin, it must be taken up from the diet; it is found in almost all tissues [16]. It's concentration in the blood of normal individuals varies from 5 to 90 μM [17].…”

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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“…It is also a co-factor in the biosynthesis and post-translational modification of collagen due to interaction with proline hydroxylase [12,13], and has been demonstrated to affect immune function [14], platelet activation [15], prostaglandin synthesis [16] and the polyol pathway [3]. These observations suggest apossible close interrelationship between AA metabolism and pathways known to be influenced by the diabetic process.Although leucocytes act as a storage reservoir for ascorbic acid and thus are considered by some workers to reflect overall vitamin C status, leucocyte concentrations do not correlate well with intake [17], are technically more difficult to measure, and are subject to a variety of influences such as stress, surgery, tobacco smoking, and exercise [18]. In comparison, plasma and serum ascorbate levels have been shown to reflect metabolic turnover of the vitamin [19], respond more rapidly to supplementation [20], and are relatively simple and convenient to measure.…”

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confidence: 99%

“…Although leucocytes act as a storage reservoir for ascorbic acid and thus are considered by some workers to reflect overall vitamin C status, leucocyte concentrations do not correlate well with intake [17], are technically more difficult to measure, and are subject to a variety of influences such as stress, surgery, tobacco smoking, and exercise [18]. In comparison, plasma and serum ascorbate levels have been shown to reflect metabolic turnover of the vitamin [19], respond more rapidly to supplementation [20], and are relatively simple and convenient to measure.…”

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confidence: 99%

Disturbed handling of ascorbic acid in diabetic patients with and without microangiopathy during high dose ascorbate supplementation

et al. 1991

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Summary. Abnormalities of ascorbic acid metabolism have been reported in experimentally-induced diabetes and in diabetic patients. Ascorbate is a powerful antioxidant, a cofactor in collagen biosynthesis, and affects platelet activation, prostaglandin synthesis and the polyol pathway. This suggests a possible close interrelationship between ascorbic acid metabolism and pathways known to be influenced by diabetes. We determined serum ascorbic acid and its metabolite, dehydroascorbic acid, as indices of antioxidant status, and the ratio, dehydroascorbate/ascorbate, as an index of oxidative stress, in 20 matched diabetic patients with and 20 without microangiopathy and in 22 age-matched control subjects. Each study subject then took ascorbic acid, 1 g daily orally, for six weeks with repeat measurements taken at three and six weeks. At baseline, patients with microangiopathy had lower ascorbic acid concentrations than those without microangiopathy and control subjects (42.1+19.3 vs 55.6 _+ 20.0, p < 0.01, vs 82.9 _+ 30.9 gmol/1, p < 0.001) and elevated dehydroascorbate/ascorbate ratios (0.87+0.46 vs 0.61 + 0.26, p < 0.01, vs 0.38 + 0.14, p < 0.001). At three weeks, ascorbate concentrations rose in all groups (p < 0.0001) and was maintained in control subjects (151.5 + 56.3 ~tmol/1), but fell in both diabetic groups by six weeks (p<0.01). Dehydroascorbate/ascorbate ratios fell in all groups at three weeks (p < 0.0001) but rose again in the diabetic groups by six weeks (p < 0.001) and was unchanged in the control subjects. Dehydroascorbate concentrations rose significantly from baseline in all groups by six weeks of ascorbic acid supplementation (p < 0.05). No significant changes were observed in fructosamine concentrations in any group during the study. Diabetes mellitus is associated with a major disturbance of ascorbic acid metabolism which is only partially corrected by ascorbate supplementation.Key words: Ascorbic acid, dehydroascorbic acid, diabetes mellitus, free radical activity, oxidative stress.Reduced levels and altered metabolic turnover of ascorbic acid (AA, vitamin C) have been reportedin severaltissues in experimen tally induced diabetes [1-3] and in diabeticpatients [4]. There are reports that high dose vitamin C regimens are associated with reversal of early signs of retinopathy [5] and normalisation of capillary strength in diabetes mellitus [6]. In the elderly non-diabetic population AA is often deficient and may be correctable by supplementation [7] -this problem might be of particular significance in elderly diabetic patients who are reported to show more rapid progress of some diabetic complications [8].Ascorbic acid functions as an important component of cellular defence against oxygen toxicity and lipid peroxidation caused by free radical mechanisms [9,10]. During scavenging of free radicals it is converted to dehydroascorbic acid, DHA, in serum and in the mitochondrial fractions of various tissues [11]. It is also a co-factor in the biosynthesis and post-translational modification of collagen...

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“…It has been suggested that increased free radicals and decline of antioxidant defense mechanisms induce diabetic micro-and macrovascular complications (1)(2)(3). Vitamin C is one of the major antioxidants and is detected in various blood components (4). However, measurements of vitamin C levels have shown inconsistent results, and the interpretation of vitamin C levels in diabetes as an antioxidant biomarker has not been clarified (5)(6)(7)(8).…”

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Lymphocyte and Plasma Vitamin C Levels in Type 2 Diabetic Patients With and Without Diabetes Complications

Yamada

Waki

et al. 2004

Diabetes Care

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Diabetes has been considered to be associated with oxidative stress. It has been suggested that increased free radicals and decline of antioxidant defense mechanisms induce diabetic micro-and macrovascular complications (1-3). Vitamin C is one of the major antioxidants and is detected in various blood components (4). However, measurements of vitamin C levels have shown inconsistent results, and the interpretation of vitamin C levels in diabetes as an antioxidant biomarker has not been clarified (5-8). In this study, we investigated the lymphocyte and plasma vitamin C levels in type 2 diabetic patients with and without diabetes complications. men and 8 women, 137 Ϯ 45 mg/dl, and 7.2 Ϯ 1.0% for diabetic patients with complications compared with 64 Ϯ 7.5 years, 7 men and 8 women, 137 Ϯ 42 mg/dl, and 6.8 Ϯ 0.8% for diabetic patients without complications, respectively). The duration of illness was longer in the diabetic patients with complications than in diabetic patients without complications (13 Ϯ 9.1 vs. 7.7 Ϯ 5.2 years, respectively, P ϭ 0.051). For the normal control subjects, 50 age-and sexmatched healthy volunteers (63 Ϯ 5.7 years, 31 men and 19 women) were recruited. The participants taking vitamin supplements were excluded from the study. All participants gave informed consent before entering the study. The study was conducted in accordance with the Declaration of Helsinki and was approved by the ethics committee at the hospital. RESEARCH DESIGN AND METHODSBlood samples were obtained by vein puncture in the morning while the patients were in the fasting state. Lymphocytes and plasma were prepared by centrifugation and the Ficoll gradients method, then immediately treated with metaphosphoric acid (final 5% wt/wt) to stabilize vitamin C (9,10). These processes were performed within 2 h under cooled conditions on ice to obtain reliable data. The vitamin C samples were stored at Ϫ80°C until analyzed, and the vitamin C (ascorbic acid, reduced form) levels were measured by high-performance liquid chromatography with the electrochemical detector method (11). All samples were handled and stored similarly in both diabetic patients and control subjects.The lymphocyte and plasma vitamin C levels in type 2 diabetic patients were compared with those of the control subjects. The differences between the vitamin C levels in type 2 diabetic patients with and without diabetes complications were also studied. Statistical analysis was performed with the unpaired Student's t test to compare the data between diabetic patients and control subjects and between type 2 diabetic patients with and without diabetes complications. A P value Ͻ0.05 was considered significant. RESULTS -The lymphocyte vitamin C level in diabetic patients was significantly lower than in control subjects (18 Ϯ 4.5 vs. 28 Ϯ 7.9 nmol/mg protein, P Ͻ 0.0001), whereas the plasma vitamin C level was not different (59 Ϯ 19 vs. 53 Ϯ 18 mol/l, P ϭ 0.17) (Fig. 1A and B). There were no significant linear correlations between the lymphocyte and plasma vitamin C levels...

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Measurement of Vitamin C in Blood Components by High‐Performance Liquid Chromatography

Cited by 53 publications

References 29 publications

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

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

Disturbed handling of ascorbic acid in diabetic patients with and without microangiopathy during high dose ascorbate supplementation

Lymphocyte and Plasma Vitamin C Levels in Type 2 Diabetic Patients With and Without Diabetes Complications

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