Alterations in phospholipid and fatty acid lipid profiles in primary neocortical cells during oxidant-induced cell injury

Specific phospholipids and fatty acids altered during oxidant-induced neuronal cell injury were determined using electrospray ionization mass spectrometry (ESI-MS) and ion trapping. The oxidants hydrogen peroxide (H2O2, 0–1000 μM) and tert-butylhydroperoxide (TBHP, 0–400 μM) induced time- and concentration-dependent increases in reactive oxygen species in primary cultures of mouse neocortical cells as determined by 2′,7′-dichlorofluorescein diacetate staining and thiobarbituric acid formation. ESI-MS analysis of 26 m/z values, representing 42 different phospholipids, demonstrated that H2O2 and TBHP increased the abundance of phospholipids containing polyunsaturated fatty acids, but had minimal affect on those containing mono- or di-unsaturated fatty acids. These increases correlated to time-dependent increase in 16:1-20:4, 16:0-20:4, 18:1-20:4 and 18:0-20:4 phosphatidylcholine. Oxidant exposure also increased mystric (14:0), palmitic (16:0), and stearic (18:0) acid twofold, oleic acid (18:1) two- to threefold, and arachidonic acid (20:4) fourfold, compared to controls. Increases in arachidonic acid levels occurred prior to increases in the phospholipids, but after increases in ROS, and correlated to increases in oxidized arachidonic acid species, specifically [20:4-OOH]–H2O–, 20:4-OH–, and Tri–OH-20:4-arachidonic acid. Treatment of cells with methyl arachidonyl flourophosphonate an inhibitor of Group IV and VI PLA2, decreased oxidant-induced arachidonic acid release, while bromoenol lactone, an inhibitor of Group VI PLA2, did not. Collectively, these data identify phospholipids and fatty acids altered during oxidant treatment of neurons and suggest differential roles for Group IV and VI PLA2 in oxidant-induced neural cell injury.