SPECTROPHOTOMETRIC ASSAY FOR TOTAL PEROXYL RADICAL-TRAPPING ANTIOXIDANT POTENTIAL IN HUMAN SERUM

Antioxidants prevent modification of low density lipoprotein (LDL) by free radicals and possibly also atheroma formation. The capacity of hu man serum to resist attacks by free radicals is measured by the total peroxyl radical-trapping potential (TRAP). Its measurement has thus fa r 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-diace tate tate (DCFH-DA), described before in studies of respiratory burst in inflammatory cells. Its oxidation by radicals from thermal decompos ition of 2,2'-diazobis(2-amidinopropane) dihydrochloride (AAPH) conver ts this compound to highly fluorescent dichlorofluorescein (DCF). The DCF also has absorbance at 504 nm thus enabling the determination of T RAP 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 inter assay coefficients of variation of this assay are 3.4% and 4.6%, respe ctively. The mean value for serum TRAP of healthy subjects is 1155 mu mol/l (n = 38). The TRAP in human serum can be increased by adding var ious antioxidant substances to the assay in vitro or by dietary supple mentation of healthy subjects with vitamin E in vivo (P < 0.025). An i ncrease was also found in serum vitamin E levels (P < 0.0001) and in t he length of the time human LDL is able to resist oxidation (P < 0.05) ). Thus the determination of TIW by this method, which requires only c ommercially available chemicals, can be used for the evaluation of phe nomena associated with lipid accumulation in human artery wall.