The line asymmetry of electron spin resonance spectra as a tool to determine the cis:trans ratio for spin-trapping adducts of chiral pyrrolines N-oxides:: The mechanism of formation of hydroxyl radical adducts of EMPO, DEPMPO, and DIPPMPO in the ischemic-reperfused rat liver

Nonstereospecific addition of free radicals to chiral nitrones yields cis/trans diastereoisomeric nitroxides often displaying different electron spin resonance (ESR) characteristics. Glutathione peroxidase-glutathione (GPx-GSH) reaction was applied to reduce the superoxide adducts (nitrone/(OOH)-O-center dot) to the corresponding hydroxyl radical (HO center dot) adducts (nitrone/(OH)-O-center dot) of two nitrones increasingly used in biological spin trapping, namely 5-diettioxyphosphoryl-5-methyl-l-pyrroline N-oxide (DEPMPO) and 5-ethoxycarbonyl-5-methyl-l-pyrroline N-oxide, and of 5-diisopropoxyphosphoryl-5-methyl-l-pyrroline N-oxide (DIPPMPO), a sterically hindered DEPMPO analogue. The method offered improved conditions to record highly resolved ESR spectra and by accurate simulation of line asymmetry we obtained clear evidence for the existence of previously unrecognized isomer pairs of cis- and trans-[DEPMPO/center dot OH] and [DIPPMPO/(OH)-O-center dot]. Additional nitrone/(OH)-O-center dot generation methods were used, i.e. photolysis of hydrogen peroxide and the Fenton reaction. We developed a kinetic model involving first- and second-order decay and a secondary conversion of trans to cis isomer to fully account for the strongly configuration-dependent behavior of nitrone/center dot OH. In the reductive system and, to a lower extent, in the Fenton or photolytic systems cis-nitrone/(OH)-O-center dot was the more stable diastereoisomer. In various biologically relevant milieu, we found that the cis: trans-nitrone/(OH)-O-center dot ratio determined right after the spin adduct fort-nation significantly differed upon the GPx-GSH vs (Fenton or photolytic) systems of formation. This new mechanistic ESR index consistently showed for all nitrones that nitrone/(OH)-O-center dot signals detected in the postischemic effluents of ischemic isolated rat livers are the reduction products of primary nitrone/(OOH)-O-center dot. Thus, ESR deconvolution of cis/trans diastereoisomers is of great interest in the study of HO center dot fort-nation in biological systems. (c) 2006 Elsevier Inc. All rights reserved.