Neutrophil-mediated phospholipid peroxidation assessed by gas chromatography-mass spectroscopy

Abstract

Disease pathophysiology frequently involves manifestations of the systemic inflammatory response syndrome. Oxyradicals represent key inflammatory mediators, and neutrophils are one important source of oxyradicals. This investigation examined neutrophil-mediated peroxidation of dilinoleoyl phosphatidyl-choline (DLPC) liposomes by monitoring the appearance of monohydroxyl linoleic acid with the use of gas chromatography-mass spectroscopy (GC-MS), compared with traditional assessment of thiobarbituric acid-reactive species (TBARS) and phosphatidylcholine-specific conjugated dienes. DLPC was peroxidized in a system using activated neutrophils in balanced salt solution containing chelated iron. 9-Monohydroxyl linoleic acid and 13-monohydroxyl linoleic acid were readily identified in neutrophil-mediated peroxidized DLPC with the use of GC-MS. Neutrophil NADPH oxidoreductase specific activity correlated highly with total ion current or specific ion monitoring of integrated peak areas for peroxidized linoleic acid but correlated poorly with DLPC-derived TBARS or conjugated dienes. These results ascertain that activated neutrophils mediate phosphatidylcholine lipid peroxidation to specific products, which may be precisely monitored with the use of GC-MS. The extent of this peroxidation is highly correlated with the magnitude of the neutrophil respiratory burst.