Pitfalls of peroxynitrite determination by luminescent probe in diabetic rat aorta

Abstract Pitfalls of peroxynitrite (ONOO−) formation in diabetic rat aorta on luminol-induced chemiluminescence (LCL) are investigated based on a detailed reaction mechanism in a case where 1.0 × 10−7 M s−1 superoxide formation rate and nitric oxide (•NO) formation were measured by electron paramagnetic resonance, while ONOO− formation by LCL. Modeling ONOO− formation at equimolar reactant ratio at pH 7.4 and 37 °C predicts 2.0 nM ONOO− and 2.1 × 10−6 M steady-state •NO concentrations, which are both biologically relevant. Comparison of steady-state concentrations to those obtained by modeling the LCL intensity at pH 10 shows that ONOO− concentration increases with 10% while peroxynitrous acid (ONOOH) concentration decreases complying with the pH shift. Evaluation of steady-state reaction rates reveals that the contribution of CO3•− radicals to the formation of luminol radicals is 76%, that of •NO2 is 24%, considerable, but that of •OH radicals negligible. The contribution of additional superoxide formati...