Kinetic simulations of the effect of antioxidants on the metmyoglobin reactions with hydrogen peroxide and their relevance and application to the Trolox equivalent equivalent antioxidant assay

Using software based on a stochastic approach to the kinetic simulation of complex reaction schemes having a wide dynamic range, existing experimental data on the reactions of ascorbate and Trolox C (a soluble form of vitamin E) on the metmyoglobin/hydrogen peroxide reactions have been used for simulation. To match both kinetic and static data, a scheme of 15 reactions was required of which five were either significant revisions of existing rate constants or the proposal of a new mechanism. In this way, new rate constants for the reactions of (center dot)MbFeIVO and MbFeIVO with both ascorbate and Trolox C have been estimated together with that for MbFeIVO + TrolO(center dot). Simulations of the metmyoglobin/hydrogen peroxide reaction scheme in the presence of both ascorbate and Trolox C in the presence of ABTS (ABTS(+)(center dot)) were also used to establish an overall mechanism for the time-resolved evolution of the ABTS cation radical, which can provide both a static or kinetic endpoint of the assay. Rate constants have thus been estimated for the reactions of the ABTS cation radical with both ascorbate and Trolox C, these fast reactions being a prerequirement for the appearance of distinct lag phases for formation of ABTS(+)(center dot) . In the case of the latter reaction with Trolox C, it is proposed that TrolO(center dot) reacts with the ABTS(+)(center dot) It is also clear from simulations that the reactions of the ABTS cation radical with antioxidants must have rate constants 10(4) M-1 s(-1) or greater to produce well-defined lag phases. Lower values result in either no or indistinct lag phases, as sometimes observed with natural antioxidants. Consequences for factors affecting the sensitivity of the TEAC assay and the determination of inhibition factors are discussed.

Automated summary

A stochastic software approach was used to simulate the complex reaction schemes involving the reactions of ascorbate and Trolox C with metmyoglobin/hydrogen peroxide. New rate constants for key reactions were estimated and an overall mechanism for the evolution of the ABTS cation radical was established. The study highlights the importance of fast reactions with antioxidants in producing distinct lag phases in assays.