A fluorescence-based method for measuring total plasma antioxidant capability

Abstract

The Total Radical-Trapping Antioxidant Parameter (TRAP) of 10 freshly prepared human plasmas was measured by a new fluorometric assay. In this method, the rate of peroxidation induced by 2,2'-diazobis (2-amidinopropane) dihydrochloride (ABAP) was monitored through the loss of fluorescence of the protein R-Phycoerythrin (R-PE). The lag-phase induced by plasma was compared to that induced by 6-hydroxy-2,5,7,8-tetramethylchroman-2- carboxylic acid (Trolox, a water-soluble analogue of vitamin E). Proteins (but not their sulphydryl groups) interfere with the analysis, partially protecting R-PE when all plasma antioxidants are exhausted. A Trolox-induced lag-phase must therefore be measured on each plasma sample. We found that ascorbate (2.5-5.3%), alpha-tocopherol (2.9-8.5%), urate (19.6-61.0), and thiol groups (17.3-42.3%) jointly explain up to 70% of TRAP. Thus, either other compounds present in plasma are likely to exert antioxidant action, or a marked synergistic action between antioxidants should be postulated to exist. This latter hypothesis is supported by the finding that the simultaneous inactivation of ascorbate and thiol groups produces a loss in antioxidant capacity of plasma greater (26%) than the sum of the decreases produced by the separate inactivation of each of the two compounds. The proposed method appears simple, reliable, and allows the rapid handling of a reasonable number of freshly prepared plasma samples. Given the rapid loss of TRAP upon storage, the latter characteristic is crucial in studies on humans, involving a large number of subjects.