OH-radical-type reactive oxygen species derived from superoxide and nitric oxide: A sensitive method for their determination and differentiation

Reactive oxygen species are involved in many diseases where the radical species OH-, peroxynitrite and the non-radical, hypochlorous acid, play an outstanding role. The formation of OH-type oxidants is essentially confined to a few types of reactions. The most prominent ones are the one-electron reduction of hydrogen peroxide by Fe²⁺ or Cu⁺- ions (Fenton-type reactions), reaction of hypochlorite with superoxide and finally formation and decay of peroxynitrite (ONOOH), formed from superoxide and NO. In this communication we wish to report on a simple model system allowing to differentiate between these ROS: ethene formation from ACC is only detectable in the presence of hypochlorite (v. Kruedener et al., 1995) and not detectable with Fenton-type oxidants or SIN-1 (3-morpholinosydnonimine, a peroxynitrite generator by releasing sequentially superoxide and NO) at 10 μM concentrations. On the other hand, ethene formation from KMB is negligible in the presence of hypochlorite but proceeds rapidly with Fenton-type oxidants (4 μM H₂O₂; 4 μM Fe²⁺) as well as with 1 μM SIN-1. Stimulation of Fenton-type oxidants and not of SIN-1 by EDTA and characteristic patterns of inhibition by SOD, catalase, hemoglobin and uric acid allow a differentiation between these two potential precursors of OH-radicals. Synthetic ONOOH shows different reaction kinetics as compared to SIN-1. Inhibition of ONOOH-dependent ethene formation by different compounds occurs more or less 'random' indicating an unspecific influence of proteins and also small molecules. Comparism of the individual inhibition types of several selected compounds allows a differential analysis as to the generation pathway of the final oxidants, OH- radical or peroxynitrite.