Improving the in vivo predictability of an on-line HPLC stable free radical decoloration assay for antioxidant activity in methanol-buffer medium

The pivotal role of ROS (reactive oxygen species) in various (patho)physiological processes has stimulated research on the potential of intervening in these processes with antioxidants (AO). In vitro model systems to investigate AO activity against the various ROS are a valuable tool in classifying antioxidants. To improve the in vivo predictability of the results obtained, we have modified and characterized the widely used DPPH (2,2'-diphenyl-1-picrylhydrazyl) on-line decoloration assay. Previous investigations using the DPPH reaction in a pure methanolic medium exhibit slow kinetics and a reaction going to completion. In this study, a medium which includes an aqueous buffer at physiological pH has been applied, resulting in the rapid establishment of equilibrium. The results obtained in an aqueous medium at physiological pH are expected to be more relevant for extrapolation to in vivo circumstances than previously published findings. The antioxidants investigated are classified according to the results obtained and the relevance of their behavior to in vivo situations is discussed. Special emphasis is put on the significance of the results for prediction of redox-cycling characteristics and structure-activity relationships.