Detection and identifi cation of oxidants formed during (NO)-N-center dot/O-2(center dot-) reaction: A multi- well plate CW-EPR spectroscopy combined with HPLC analyses

摘要

New techniques and probes are routinely emerging for detecting short-lived free radicals such as superoxide radical anion (O-2(center dot-)), nitric oxide ((NO)-N-center dot), and transient oxidants derived from peroxynitrite (ONOO-/ONOOH). Recently, we reported the profi les of oxidation products (2-hydroxyethidium, ethidium, and various dimeric products) of the fl uorogenic probe hydroethidine (HE) in the (NO)-N-center dot/O-2(center dot-) system (Zielonka et al. 2012). In this study, we used HPLC analyses of HE oxidation products in combination with continuous wave electron paramagnetic resonance (CW-EPR) spin trapping with 5-tert -butoxycarbonyl-5-methyl-1-pyrroline N-oxide (BMPO) to defi ne the identity of the oxidizing species formed in the (NO)-N-center dot/O-2(center dot-) system. EPR spin-trapping technique is still considered as the gold standard for characterization of free radicals and their intermediates. We monitored formation of BMPO-superoxide (BMPO-(OOH)-O-center dot) and BMPO-hydroxyl (BMPO-(OH)-O-center dot) radical adducts. Simultaneous analyses of results from EPR spin-trapping and HPLC measurements are helpful in the interpretation of the mechanism of formation of products of HE oxidation.