Spectrophotometric assay for total peroxyl radical-trapping antioxidant potential in human serum

Abstract

Antioxidants prevent modification of low density lipoprotein (LDL) by free radicals and possibly also atheroma formation. The capacity of human serum to resist attacks by free radicals is measured by the total peroxyl radical-trapping potential (TRAP). Its measurement has thus far required equipment not available in many clinical laboratories such as a thermostated oxygen electrode cell or a luminometer. To develop a simpler method we used a free radical probe, dichlorofluorescin-diacetate (DCFH-DA), described before in studies of respiratory burst in inflammatory cells. Its oxidation by radicals from thermal decomposition of 2,2'-diazobis(2-amidinopropane)dihydrochloride (AAPH) converts this compound to highly fluorescent dichlorofluorescein (DCF). The DCF also has absorbance at 504 nm thus enabling the determination of TRAP either fluorometrically or spectrophotometrically. Increasing the concentration of AAPH enables the measurement of DCF formation and its inhibition by serum samples at room temperature. The intra- and interassay coefficients of variation of this assay are 3.4% and 4.6%, respectively. The mean value for serum TRAP of healthy subjects is 1155 mumol/l (n = 38). The TRAP in human serum can be increased by adding various antioxidant substances to the assay in vitro or by dietary supplementation of healthy subjects with vitamin E in vivo (P < 0.025). An increase was also found in serum vitamin E levels (P < 0.0001) and in the length of the time human LDL is able to resist oxidation (P < 0.05). Thus the determination of TRAP by this method, which requires only commercially available chemicals, can be used for the evaluation of phenomena associated with lipid accumulation in human artery wall.