Mass spectrometric analysis of arachidonyl-containing phospholipids in human U937 cells

The human histiocytic lymphoma U937 cell line contains a rich source of the 85 kDa cytosolic phospholipase A(2) (cPLA(2)). DMSO-differentiated U937 ce lls were used as a model to investigate the free arachidonic acid release, the arachidonate distribution and the phospholipid source of arachidonate u pon Ca2+ ionophore stimulation. A combination of several chromatographic an d mass spectrometric techniques was employed in this study. The amount of f ree arachidonic acid (AA) released upon stimulation, the arachidonate conte nt in total lipids and in each of the phospholipid classes were determined by gas chromatography/mass spectrometry (GC/MS). Glycerophosphoethanolamine (GPE) was found to be the major pool of arachidonate in differentiated hum an U937 cells (55%) and glycerophosphocholine (GPC) and glycerophosphoinosi tol (GPI) contributed 22 and 8%, respectively. Upon Ca2+ ionophore stimulat ion, GPE class lost the largest amount of arachidonate, followed by GPC cla ss. GPI class, however, gained a substantial amount of arachidonate. Most o f the arachidonate depleted from GPE and GPC was recovered as free AA, some of which was rapidly esterified into GPI species. GC/MS with electron capt ure negative chemical ionization provided excellent sensitivity for the mea surement of arachidonic acid which was derivatized to its pentafluorobenzyl ester. Intact phospholipid molecular species including the arachidonyl-con taining phospholipid species were identified using capillary high-performan ce liquid chromatography/continuous-flow liquid secondary ion mass spectrom etry (CF-LSIMS). No specificity was found for releasing free AA among the a rachidonyl-containing GPE and GPC species upon Ca2+ ionophore stimulation. CF-LSIMS provided a sensitive and effective means of detecting intact phosp holipid species. Copyright (C) 1999 John Wiley & Sons, Ltd.