Method of colored model radicals for assessment of oxidative equilibrium in biologic samples

Abstract

The most specific method of the recording of the rate offree radical reactions is the method of electron paramagnetic resonance (EPR) spectroscopy, but it is rarely used in applied biology due to expensive equipment and complexity of the execution of measurements. However chemists have found a number of colored organic radicals which lose the coloring under transition into diamagnetic form. In the given paper there are presented results of our studies on the development of methods for the assessment of oxidant equilibrium in biological media with a use of stable radical 2,2-diphenyl-1-picrylhydrazyl (DPPH) and cation-radicals of N,N-diethyl-p-phenylenediamine (DEPPD). We have developed the new modification of DPPH test, replacing methanol-based incubation medium by non-ionic detergent solution, compatible with native blood serum. Modified DPPH test conserved typical biphasic kinetics of the origin variant, had the similar sensitivity to model antioxidants (IC values 49, 38 and 13 mkMfor ascorbate, a-tocopherol and quercetine, correspondingly) and was applied in experiments on laboratory animals treated with nano- and ionic silver, carbon nanotubes, microfine coal and electrolytic dust. We have tried also the assay of serum lipid hydroperoxides based on Fe-initiated DEPPD oxidation (Alberti et al., 2000). The comparison of kinetics of DEPPD oxidation in model (HO/Fe) and biologic (rat serum/Fe) systems, before and after Fe addition, seems to be an evidence that ceruloplasmin (CP) was involved in the resulting process, but failed to determine its polynomial kinetics, at least for the rat serum and DEPPD excess. The use of CP monoclonal antibodies seems to be the best way for the clarification of the mechanism of this reaction.