Abstract
A simple gas chromatographic method for the assay of phospholipase A 2 (PLA 2) has been described in which arachidonic acid released from endogenous phospholipid pools is measured following its extraction and derivatization to pentafluorobenzyl esters. Using this assay, PLA 2 activities in control and calcium ionophore-stimulated human neutrophils, as well as in control, thrombin, and calcium ionophore stimulated human platelets, have been measured. These values are compared with those obtained by monitoring the release of radioactivity from [ 3H]- or [ 14C]arachidonic acid prelabeled cells. While the radiometric assay measures only the release of exogenously incorporated radioactive arachidonic acid, the gas chromatographic assay measures arachidonic acid released from all the endogenous pools. Thus, the apparent increase in PLA 2 activity in stimulated cells measured by the gas chromatographic assay is four- to fivefold higher than that by the radiometric assay. Inclusion of fatty acid free bovine serum albumin in the reaction buffer significantly increases the amount of arachidonic acid that is measured by gas chromatography. The gas chromatographic method has also been successfully utilized for measuring PLA 2 activity in cell-free preparations derived from physically disrupted human neutrophils.
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