Pitfalls in a method for assessment of total antioxidant capacity

A relatively simple and widely applied method for quantitating the total antioxidant capacity of body fluids and drug solutions based on the absorbance of the ABTS radical cation was evaluated. In this assay, the end-point is an antioxidant-induced decrease in absorbance at a fixed time. This decrease is used as an index of total antioxidant capacity. It is shown that Trolox, potassium cyanide and quercetin all decrease the absorbance of ABTS radical cations at a fixed time, but by different mechanisms. Trolox scavenges the ABTS radical, potassium cyanide inhibits radical formation, while quercetin acts by both mechanisms. Using this method antioxidant capacity may be overestimated, due to both a scavenger effect and an effect on the rate of ABTS oxidation. To distinguish between these effects, a post-addition assay was used in which the sample is added when the formation of radicals is stable. Using post- addition assay conditions enables discrimination between effects on radical scavenging and on the radical formation, two major mechanisms for antioxidant action. In extrapolating the results to an in vivo situation it should be questioned: (i) whether the peroxidase process does indeed mimic the process of radical formation in vivo, and (ii) whether the ABTS radicals do resemble the radical species involved in an in vivo situation. Results obtained in the ABTS radical-based methods should therefore be reviewed critically before the antioxidant capacity can be assessed.