Ascorbic acid and dehydroascorbic acid analyses in biological samples

Washko, Philip W.; Welch, Richard W.; Dhariwal, Kuldeep R.; Wang, Yaohui; Levine, Mark

doi:10.1016/0003-2697(92)90131-p

Cited by 258 publications

(190 citation statements)

References 92 publications

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“…Samples stored at À201C should not be used for AA analysis, as substantial degradation of AA are observed after only one day of storage. A chromatographic method should be used, as the enzymatic or spectrophotometric assays have been associated with substantial interferences in several reviews on vitamin C methodology (Pachla et al, 1985;Washko et al, 1992;Levine et al, 1999). It is also preferable that the method provides a high throughput of samples, as the stability of AA is limited in prepared samples (Karlsen et al, 2005).…”

Section: Resultsmentioning

confidence: 99%

“…The ratio between DHAA and AA may serve as an important plasma biomarker of oxidative stress. AA is the most abundant form in plasma and sensitive to oxidation and degradation during blood sampling, handling, storage, and analysis (Washko et al, 1992;Levine et al, 1999). Various factors such as choice of anticoagulant, temperature, light, pH, dissolved oxygen, solvent, ionic strength, trace metals as ferric ions or the presence of oxidizing enzymes have been reported to affect AA stability (Bradley et al, 1973;Lunec and Blake, 1985;Galan et al, 1988;Margolis and Davis, 1988;Comstock et al, 1995;Key et al, 1996;Margolis and Duewer, 1996;Chung et al, 2001;Ching et al, 2002).…”

Section: Introductionmentioning

confidence: 99%

“…It is essential to avoid degradation and oxidation of AA during sampling, sample handling and analysis, as this may skew the ratio between DHAA and AA. As DHAA is unstable at biological conditions, the ratio between DHAA and AA is usually measured as total AA (TAA) with the use of subtraction methods (Washko et al, 1992;Levine et al, 1999;Lykkesfeldt, 2000). The stability of TAA and DHAA are not discussed herein, as it is the preservation of AA in its reduced form that is the essential aspect of accurate DHAA and TAA measurements.…”

Section: Introductionmentioning

confidence: 99%

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Stability of whole blood and plasma ascorbic acid

2007

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The objective of this study was to evaluate the impact of pre-analytical factors on the short and long term stability of ascorbic acid (AA), the main form of vitamin C in whole blood and plasma. The effects of various anticoagulants, acidification, storage temperature and time were tested. A recently developed fast and sensitive HPLC method was used to measure AA levels. AA baseline values observed in heparin plasma were significantly higher than values observed in EDTA, citrate and Stabilyte plasma, as well as in serum. pH and temperature were identified as additional critical pre-analytical factors during the short, medium and long term handling and storage. Thus, assessment of reliable and accurate AA status in biological samples demonstrates to be highly dependent on whether the initial conditions during sample handling are controlled. In conclusion, heparin tubes should be used for blood sample collection. As AA is rapidly degraded, sample collection should be followed by immediate centrifugation and plasma acidification. To avoid further degradation during sample handling, samples should be stored at À701C without delay and analyzed within 80 days.

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Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Stability of whole blood and plasma ascorbic acid

2007

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“…To determine intracellular AH, cells were washed three times in HBSS to remove extracellular AH and DHA. Cells were extracted with ice-cold 90 % (v\v) methanol\1 mM EDTA followed by centrifugation (16 000 g, 2 min), as described [17]. The clear supernatant was removed and stored on solid CO # for less than 8 h before analysis.…”

Section: Cellular Uptake Of Dha and Efflux Of Ahmentioning

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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“…Plasma ascorbate was measured by high-performance liquid chromatography (HPLC) isocratic delivery using a reversephase column Prevail C18 (Alltech, Deerfield, IL, USA) with 2 mM KCl mobile phase and electrochemical detection at 1000 mV (Washko et al, 1992). Plasma a-tocopherol and retinol were determined by HPLC (Shimadzu, Kyoto, Japan) with a reversed-phase C18 column LiChrospher RP C18 (Alltech, Deerfield, IL, USA) with 100% methanol mobile phase and detection at 292 and 325 nm, respectively (CayeVaugien et al, 1990).…”

Section: Laboratory Analysismentioning

confidence: 99%

Does vitamin C supplementation influence the levels of circulating oxidized LDL, sICAM-1, sVCAM-1 and vWF-antigen in healthy male smokers?

et al. 2004

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Objective: To examine the effects of vitamin C supplementation on the concentration of oxidation markers, in particular, circulating oxidized LDL (OxLDL) and on endothelial activation markers. Design: Randomized double-blind, placebo-controlled crossover trial. Setting: Belgian population of the city of Leuven. Subjects: A total of 34 healthy male smokers aged 26-73 y. Intervention: Smokers were randomly assigned to receive either vitamin C (250 mg twice daily) or placebo capsules, each to be taken for 4 weeks. After a 1-week washout period, participants then crossed over to the alternative capsules for further 4 weeks. Mean outcome measures: Markers of oxidation (bilirubin, uric acid, a-tocopherol, retinol, malondialdehyde, circulating Oxidized LDL (OxLDL)) and markers of endothelial activation (sICAM-1, sVCAM-1, vWF-antigen) were analysed. Results: Plasma ascorbate concentrations significantly increased from 46.6717.6 to 70.1721.2 mmol/l after a 4-week treatment with 500 mg vitamin C per day. The other plasma antioxidants concentrations, including bilirubin, uric acid, a-tocopherol and retinol, were similar in both treatment periods. Vitamin C did not change plasma malondialdehyde and circulating OxLDL compared with placebo (vitamin C 0.7370.25 mg/dl OxLDL; placebo 0.7270.21 mg/dl OxLDL). After vitamin C supplementation, neither sICAM-1 and sVCAM-1 levels nor the concentration of vWF-antigen significantly differed from placebo condition. Conclusions: Oral supplementation of vitamin C is not associated with changes in markers of oxidation or endothelial activation in healthy male smokers.

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Ascorbic acid and dehydroascorbic acid analyses in biological samples

Cited by 258 publications

References 92 publications

Stability of whole blood and plasma ascorbic acid

Stability of whole blood and plasma ascorbic acid

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

Does vitamin C supplementation influence the levels of circulating oxidized LDL, sICAM-1, sVCAM-1 and vWF-antigen in healthy male smokers?

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