Spectrophotometric determination of dehydroascorbic acid in biological samples.
We describe a method for accurately and precisely measuring dehydroascorbic acid in perchloric acid extracts prepared from human plasma, lymphocytes, and mammalian cells. Samples were assayed by spectrophotometrically monitoring the kinetics of the concentration-dependent absorbance changes of dehydroascorbic acid with phosphate-methanol-containing buffers. The lowest detectable dehydroascorbate concentration using this assay is estimated to be below 0.1 mumol/liter. Total analysis time is less than 10 min and allows the simultaneous measurement of numerous samples. The calibration curve is linear (r > 0.995) over the range 0-200 mumol/liter. The dehydroascorbic acid concentrations measured in supplemented samples agree with known concentrations. Interference of ascorbic acid and 2,3-diketogulonic acid with this assay was excluded. The correlation with a highly specific chromatographic procedure gave comparable results over the range of physiologically relevant concentrations. The procedure avoids the most commonly applied method of measuring the native ascorbic acid, then reducing the dehydroascorbic acid, and finally measuring the total ascorbic acid and determining dehydroascorbic acid by the difference. Stabilization of ascorbic acid during assay was achieved by addition of desferrioxamine.
Moeslinger
T
T
Institute for Medical Physiology, Vienna, Austria.
Brunner
M
M
Spieckermann
P G
PG
D016475:3T3 Cells; D000818:Animals; D001205:Ascorbic Acid; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D003676:Deferoxamine; D003683:Dehydroascorbic Acid / Q000032:analysis* / Q000097:blood; D004492:Edetic Acid; D005347:Fibroblasts / Q000737:chemistry; D006801:Humans; D007202:Indicators and Reagents; D008214:Lymphocytes / Q000737:chemistry*; D008322:Mammals; D051379:Mice; D012016:Reference Values; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013053:Spectrophotometry / Q000379:methods
D007202:Indicators and Reagents; D004492:Edetic Acid; D003676:Deferoxamine; D001205:Ascorbic Acid; D003683:Dehydroascorbic Acid