Lipoxygenase metabolism of endogenous and exogenous arachidonate in leukocytes: GC-MS analyses of incubations in H2(18)O buffers.

Fatty acids are labeled with 18O in the carboxyl group during ester hydrolysis in H2(18)O. We utilized this principle to develop a novel mass spectrometric method for study of the turnover of arachidonic acid in intact cell systems. Analysis of 18O incorporations was by negative ion chemical ionization GC-MS. The use of deuterium-labeled exogenous substrates allowed the metabolic fate of exogenous and intrinsic compounds to be distinguished. Thus, it was shown that the preferred route of 5-lipoxygenase metabolism of exogenous arachidonic acid in ionophore-stimulated human neutrophils is via direct reaction with the enzyme and not via incorporation into cellular lipids. The re-uptake of 5-HETE was also studied. For investigation of intracellular reactions which involve ester hydrolysis, the 18O labeling method is unique in providing direct evidence of the intermediate metabolic pathway of endogenous and exogenous products associated with the arachidonic acid cascade.