Evaluation of the accuracy of antioxidant competition assays: incorrect assumptions with major impact

The activity of antioxidants is frequently determined in competition assays. In these assays an antioxidant (A) and a detector molecule (D) compete for the reactive species (R). The competitive inhibitory effect of A on the reaction of D with R is a measure of the antioxidant activity of A. In determining the activity of A, it is in general incorrectly assumed that the concentrations of A and D remain equal to the initial concentration. However, the principle of the assay is that some A and D is consumed and consequently the concentrations of A and D will decrease during a competition assay, resulting in a deviation in the observed antioxidant activity. Computer modeling was used to obtain a graphical tool to estimate the extent of the deviation caused by the incorrect assumption that the concentrations of A and D do not decrease. Several competition assays were evaluated using this graphical tool, demonstrating that frequently inaccurate antioxidant activities have been reported. In general, differences between antioxidants are underestimated and the activity of all antioxidants shifts toward the antioxidant activity of D. A strategy is provided to improve the accuracy of a competition assay. To obtain accurate results in a competition assay, the reaction rate constant of the detector molecule with the reactive species should be comparable to that of the antioxidant. In addition, the concentration of the reactive species should be as low as possible.