Adaptation of the phosphotungstate method to determine reduced and oxidized vitamin C in blood plasma.
The phosphotungstate reagent (PTR) was used for quantitative spectrophotometric determination of physiological forms of vitamin C in blood plasma. An immediate action of PTR on the first half of the tested samples allowed to determine reduced vitamin C concentrations (I) at 700 nm. 10 mM dithiothreitol added to the second half of the samples reduced oxidized vitamin C in it--hence the total amount of this vitamin was reduced with a concentration (II) determined as above (remains of dithiothreitol were removed with N-ethylmaleimide). The difference of results (II) and (I) gave the concentration of oxidized vitamin C. The method is characterised by fault-less analytical parameters: correlation coefficients of analytical curves > 0.99, recovery factor 100.5%, variation coefficients intra- and inter-serial < 3% and < 5%, respectively, detection limit 0.05 microM. The simplicity of the method enables an easy control of the ratio of oxidized and reduced vitamin C concentrations in blood plasma--the biomarker of the level of oxidative damage to cells.
Rutkowski
Maciej
M
Department of Clinical Biochemistry and Chemistry, Institute of Physiology and Biochemistry, Medical University, Pl. J. Hallera 1, 90-647 łódź, Poland. bio-chemialodz@wp.pl
Grzegorczyk
Krzysztof
K
Greger
Janusz
J
D000818:Animals; D001205:Ascorbic Acid / Q000097:blood*; D002417:Cattle; D003545:Cysteine / Q000097:blood; D004229:Dithiothreitol / Q000097:blood; D005978:Glutathione / Q000097:blood; D006801:Humans; D007202:Indicators and Reagents; D008623:Mercaptoethanol / Q000097:blood; D010084:Oxidation-Reduction; D010772:Phosphotungstic Acid; D013053:Spectrophotometry / Q000379:methods
D007202:Indicators and Reagents; D010772:Phosphotungstic Acid; D008623:Mercaptoethanol; D005978:Glutathione; D003545:Cysteine; D001205:Ascorbic Acid; D004229:Dithiothreitol