Journal
JOURNAL OF BIOCHEMICAL AND BIOPHYSICAL METHODS
Volume 58, Issue 3, Pages 207-218Publisher
ELSEVIER SCIENCE BV
DOI: 10.1016/j.jbbm.2003.10.010
Keywords
ascorbate; dehydroascorbic acid; vitamin C; L-929; RAW 264.7; methanol
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We present a fast to perform spectrophotometric method for the quantification of ascorbic acid and its oxidized form dehydroascorbic acid in biological samples. The assay detects a chromophore formed during the reaction of dehydroascorbic acid with methanol in phosphate/citrate buffer. This reaction can also be employed for the determination of ascorbate (vitamin Q in the presence of ascorbate oxidase. The major advantage of the developed protocol for the determination of both forms of vitamin C is a simple spectrophotometrical single end point determination. It is demonstrated that the methanol method is an improvement compared with a commercially available test kit for the determination of vitamin C. Using the methanol method, a dose-dependent increase in intracellular ascorbic acid was determined upon incubation of L-929 cells and RAW 264.7 macrophages with increasing concentrations of extracellular ascorbate. In blood serum, vitamin C was determined at concentrations between 46 and 97 muM. Supplementation with different amounts of ascorbate showed satisfying recovery. In L-929 cells, even unphysiologically high amounts of reactive nitrogen species were unable to completely oxidize intracellular vitamin C. (C) 2004 Elsevier B.V. All rights reserved.
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