Plasmatic antioxidant capacity due to ascorbate using TEMPO scavenging and electron spin resonance

Abstract

Background Ascorbate is the most effective water-soluble antioxidant and its plasma concentration is usually measured by different methods including colorimetric assays, HPLC or capillary electrophoresis. Plasma antioxidant capacity is determined by indexes such as total reactive antioxidant potential, total antioxidant reactivity, oxygen radical absorbance capacity, etc. We developed an alternative method for the evaluation of the plasma antioxidant status due to ascorbate. Methods TEMPO kinetics scavenging analyzed by ESR spectroscopy was performed on plasma samples in different antioxidant situations. Plasma ascorbate concentrations were determined by capillary electrophoresis. Ascorbyl radical levels were measured by ESR. Results Plasma reactivity with TEMPO (PR-T) reflected plasma ascorbate levels. Average PR-T for normal plasmas resulted 85 ± 27 μmol/l ( n = 43). PR-T during ascorbic acid intake (1 g/day) increased to an average value of 130 ± 20 μmol/l ( p < 0.001, n = 20). PR-T correlated with the plasmatic ascorbate levels determined by capillary electrophoresis ( r = 0.92), presenting as an advantage the avoiding of the deproteination step. Plasma ascorbyl radical levels increase from 16 ± 2 to 24 ± 3 nmol/l ( p < 0.005, n = 14) after ascorbate intake. Conclusions PR-T could be considered as a measure of the plasmatic antioxidant capacity due to the plasma ascorbate levels and could be useful to investigate different antioxidant situations.