ESR detection of intraphagosomal superoxide in polymorphonuclear leukocytes using 5-(diethoxyphosphoryl)-5-methyl-1-pyrroline-N-oxide

We applied a spin trap, 5-(diethoxyphosphoryl)-5-methyl-1-pyrroline-N-oxide (DEPMPO), to detect O-2(.-) generation during phagocytosis in human polymo rphonuclear leukocytes (PMNs). PMNs were activated with serum-opsonized zym osan (sOZ) in the presence of DEPMPO. The ESR spectra mainly consisted of C u,Zn-SOD-sensitive DEPMPO-OOH spin adducts. To clarify where these spin-add ucts were present, cells after stimulation were separated from extracellula r fluid by brief centrifugation and resuspended in Hanks' balanced salt sol ution. ESR examination showed that DEPMPO-OOH adducts were present in both fractions. When cells were stimulated by phorbol myristate acetate (PMA), t he DEPMPO-OOH was detected in extracellular fluid but not in the cell fract ion. Furthermore, DEPMPO-OOH adducts were quickly converted into ESR-silent compounds by addition of cell lysate of PMNs. These results indicate that DEPMPO is useful to detect O-2(.-) of extracellular space including the int raphagosome but not that of intracellular space in sOZ-stimulated phagocyte s.