Pitfalls of peroxynitrite determination by luminescent probe in diabetic rat aorta Andra ´s Ne ´meth • Krisztia ´n Stadler • Judit Jakus • Tama ´s Vido ´czy Received: 15 November 2011 / Accepted: 20 January 2012 / Published online: 9 February 2012 Ó Akade ´miai Kiado ´, Budapest, Hungary 2012 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 9 10 -7 Ms -1 superoxide formation rate and nitric oxide ( • NO) formation were measured by electron paramagnetic reso- nance, while ONOO - formation by LCL. Modeling ONOO - formation at equi- molar reactant ratio at pH 7.4 and 37 °C predicts 2.0 nM ONOO - and 2.1 9 10 -6 M steady-state • NO concentrations, which are both biologically rele- vant. 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 • NO 2 is 24%, consid- erable, 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 • NO 2 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 A. Ne ´meth (&) Á K. Stadler Á J. Jakus Institute of Biomolecular Chemistry, Chemical Research Center, Hungarian Academy of Sciences, Pusztaszeri u ´t 59-67, Budapest 1025, Hungary e-mail: anemeth@chemres.hu Present Address: K. Stadler Oxidative Stress and Disease Laboratory, Pennington Biomedical Research Center, Baton Rouge, LA 70808, USA T. Vido ´czy Institute of Structural Chemistry, Chemical Research Center, Hungarian Academy of Sciences, Pusztaszeri u ´t 59-67, Budapest 1025, Hungary 123 Reac Kinet Mech Cat (2012) 106:1–10 DOI 10.1007/s11144-012-0427-3