Trolox-Equivalent Antioxidant Capacity Assay Versus Oxygen Radical Absorbance Capacity Assay in Plasma

Abstract

Because of difficulty in measuring each antioxidant component separately and interactions among antioxidants, methods have been developed to assess the total antioxidant capacity of serum or plasma. The 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox)-equivalent antioxidant capacity (TEAC) assay (1), the oxygen radical absorbance capacity (ORAC) assay (2), and the ferric reducing ability of plasma (FRAP) assay (3) are commonly used and have been extensively evaluated. Although comparable mean results have been obtained with the TEAC and ORAC assays (4)(5), no correlation has been found between ORAC and TEAC values or between FRAP and TEAC values (6). This lack of correlation between TEAC and other assays is likely attributable to underestimation of overall antioxidant capacity. Underestimation may be related to the effects of dilution (7) and to premature measurement of inhibition percentage at a fixed time of 3 min (6). In fact, both the ORAC and TEAC assays are inhibition methods: a sample is added to a free- radical-generating system, and the inhibition of the free radical action is measured. This inhibition is related to the antioxidant capacity of the sample. In addition, both assay methods measure antioxidants in serum or plasma proteins, including albumin (6). In this study we investigated the performance of the TEAC assay, modified the procedure, and then reevaluated the TEAC for comparison with the ORAC assay. The TEAC assay, commercialized by Randox Laboratories Ltd., is based on the suppression of the absorbance of radical cations of 2,2′-azinobis(3-ethylbenzothiazoline 6-sulfonate) (ABTS) by antioxidants in the test sample when ABTS incubates with a peroxidase (metmyoglobin) and H2O2 (1). If the inhibition time is fixed at 3 min, as stated in the manufacturer’s instructions, the added antioxidants quench ABTS radicals in a nonlinear dose–response fashion (6). To optimize the incubation period for the complete inhibition of ABTS radical …