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Incorporation of stable isotope‐labeled arachidonic acid into cellular phospholipid molecular species and analysis by fast atom bombardment tandem mass spectrometry
Author(s) -
KayganichHarrison Kathleen A.,
Murphy Robert C.
Publication year - 1994
Publication title -
biological mass spectrometry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.475
H-Index - 121
eISSN - 1096-9888
pISSN - 1052-9306
DOI - 10.1002/bms.1200230906
Subject(s) - arachidonic acid , linoleic acid , phospholipid , biochemistry , chemistry , fast atom bombardment , fatty acid , leukotriene , biology , chromatography , membrane , mass spectrometry , enzyme , asthma , immunology
The source of arachidonic acid metabolized to eicosanoids by 5‐lipoxygenase was studied in a cultured neoplastic mast cell using a stable isotope tracer and tandem mass spectrometry strategy. Selected reaction monitoring and fast atom bombardment were used to analyze eight major arachidonate molecular species of glycerophosphocholine, nine major molecular species of glycerophosphoethanolamine, three major species of glycerophosphoinositol, and three major glycerophosphoserine molecular species. Incubation of the mast cells with ( 2 H 8 )arachidonic acid led to a time‐dependent isotopic incorporation in each of these molecular species. Following stimulation with calcium ionophore A23187, the isotope incorporation of leukotriene B 4 (LTB 4 ) was found to be higher than that of the major arachidonate‐containing glycerophospholipid molecular species. The isotope incorporation of LTB 4 was similar to that found for free arachidonic acid present in the unstimulated cell. In order to prevent direct labeling of the intracellular, free arachidonic acid pool, ( 2 H 4 )linoleic acid was added to the culture medium as a biochemical precursor of labeled arachidonic acid. There was a time‐dependent increase of the specific incorporation of labeled arachidonic acid into each of the phospholipid molecular species of each lipid class after incubation with ( 2 H 4 )linoleic acid. Importantly, ( 2 H 4 )linoleic acid incubation also resulted in deuterium‐labeled arachidonic acid in the free arachidonic acid, intracellular pool. The arachidonic acid isotopic incorporation in this pool very closely correlated with the isotopic incorporation of LTB 4 (correlation coefficient 0.97) synthesized after A23187 stimulation, while the isotopic incorporation of the extracellularly released, not esterified arachidonic acid, after stimulation, did not. These studies show that fast atom bombardment and tandem mass spectrometry can be used to follow incorporation of stable isotope‐labeled arachidonic acid into complex mixtures of glycerophospholipids. This approach is relatively sensitive in being able to assess incorporation of 10% or less of the minor lipid pool.