The interaction of 5,5-dimethyl-1-pyrroline- N-oxide with human myeloperoxidase and its potential impact on spin trapping of neutrophil-derived free radicals

Abstract

Activation of human neutrophils leads to secretion of myeloperoxidase (MPO) with resulting generation of several oxidant species including OCl −. Spin trapping techniques employing 5,5-dimethyl-1-pyrroline- N-oxide (DMPO) are being applied increasingly to the investigation of free radical production by in vitro and in vivo experimental systems which contain neutrophils. Because such knowledge is critical to the interpretation of these data, we examined the impact of MPO and MPO-derived oxidants on DMPO spin adduct formation and stability. Addition of increasing concentrations of OCl − to DMPO yielded a number of EPR-detectable products including DMPO-OH. However, the concentration of OCl − required was in excess of that expected under physiologic conditions. Addition of purified human MPO and H 2O 2 to DMPO yielded EPR spectra consisting of small DMPO-OH peaks. The addition of MPO and H 2O 2 to preformed DMPO-OH and DMPO-CH 3 resulted in rapid destruction of these spin adducts. Thus MPO H 2 O 2 appeared to both generate and destroy DMPO spin adducts. Neutrophils stimulated with phorbol myristate acetate or opsonized zymosan generated large DMPO-OOH and DMPO-OH peaks as well as small DMPO-CH 3 peaks. Addition of the MPO inhibitor azide to the reaction mixture had no effecting on resulting DMPO-OH or DMPO-CH 3 peak amplitudes but increased that of DMPO-OOH. These data suggest that MPO-derived oxidants likely have little impact on the nature of EPR spectra resulting from DMPO spin trapping of free radical species following neutrophil stimulation. Because MPO oxidants did appear to react with DMPO the ability of DMPO to protect a biologic target from in vitro MPO injury was examined. DMPO (>10 m m) significantly decreased MPO/ H 2O 2/Cl −-mediated erythrocyte hemolysis as assessed by 51Cr release. The experimental and/or pharmacologic implications of this observation require further study.