TY - JOUR
T1 - 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
AU - Britigan, Bradley E.
AU - Hamill, Danielle R.
N1 - Funding Information:
This work was supported by National Institute of Health Grant RR05372a nd the Veterans Administration Research Service and was performedw hile Bradley E. Britigan was a Pfizer Scholar and a Veterans Administration Research Associate. We thank Drs. Robert Clark and William Nauseef for reagents provided and helpful suggestions;D rs. Garry Buettner, Myron Cohen, Sovitj Pou, and Gerald Rosen for their commentsa nd criticisms; Dr. Harold Goff and his as-sociatesf or making available the EPR spectrometer; and Naomi Erickson for preparation of the manuscript.
PY - 1989/11/15
Y1 - 1989/11/15
N2 - 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 H2O2 to DMPO yielded EPR spectra consisting of small DMPO-OH peaks. The addition of MPO and H2O2 to preformed DMPO-OH and DMPO-CH3 resulted in rapid destruction of these spin adducts. Thus MPO H2O2 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-CH3 peaks. Addition of the MPO inhibitor azide to the reaction mixture had no effecting on resulting DMPO-OH or DMPO-CH3 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 mm) significantly decreased MPO/ H2O2/Cl--mediated erythrocyte hemolysis as assessed by 51Cr release. The experimental and/or pharmacologic implications of this observation require further study.
AB - 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 H2O2 to DMPO yielded EPR spectra consisting of small DMPO-OH peaks. The addition of MPO and H2O2 to preformed DMPO-OH and DMPO-CH3 resulted in rapid destruction of these spin adducts. Thus MPO H2O2 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-CH3 peaks. Addition of the MPO inhibitor azide to the reaction mixture had no effecting on resulting DMPO-OH or DMPO-CH3 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 mm) significantly decreased MPO/ H2O2/Cl--mediated erythrocyte hemolysis as assessed by 51Cr release. The experimental and/or pharmacologic implications of this observation require further study.
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U2 - 10.1016/0003-9861(89)90351-2
DO - 10.1016/0003-9861(89)90351-2
M3 - Article
C2 - 2554813
AN - SCOPUS:0024420946
SN - 0003-9861
VL - 275
SP - 72
EP - 81
JO - Archives of Biochemistry and Biophysics
JF - Archives of Biochemistry and Biophysics
IS - 1
ER -