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 CO 3 •− 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 formation by autoxidation of luminol is 13%, not negligible, but that of ONOOH homolysis is negligible. The •NO2 is predominantly formed from the decomposition of the ONOO−–carbon dioxide adduct and only 0.5% directly from •NO oxidized by molecular oxygen. But the contribution of the latter pathway depends strongly on the •NO and superoxide formation rate ratio, at a ratio of 2:1, it would increase to 14%. The measured time interval of the initial increase of LCL intensity complies with the time needed luminol aorta outside and inside concentrations in the sample to be equalized by diffusion, the 7 × 10−3 s−1 rate constant obtained by modeling enabled to estimate 5 × 10−7 cm2 s−1 as the diffusion coefficient of luminol in the diabetic rat aorta.