Measurement of arachidonic acid release from human polymorphonuclear neutrophils and platelets: comparison between gas chromatographic and radiometric assays.
a simple gas chromatographic method for the assay of phospholipase A2 (PLA2) 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, PLA2 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 PLA2 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 PLA2 activity in cell-free preparations derived from physically disrupted human neutrophils.
Ramesha
C S
CS
Department of Inflammation Biology, Syntex Research, Palo Alto, California 94303.
Taylor
L A
LA
D000098:Acetophenones / Q000494:pharmacology; D001095:Arachidonic Acids / Q000097:blood*; D001792:Blood Platelets / Q000187:drug effects / Q000378:metabolism*; D000001:Calcimycin / Q000494:pharmacology; D002849:Chromatography, Gas; D005231:Fatty Acids, Unsaturated / Q000494:pharmacology; D006801:Humans; D007700:Kinetics; D009504:Neutrophils / Q000187:drug effects / Q000378:metabolism*; D010741:Phospholipases A / Q000494:pharmacology*; D054467:Phospholipases A2; D012710:Serum Albumin, Bovine / Q000494:pharmacology; D014316:Tritium
D000098:Acetophenones; D001095:Arachidonic Acids; D005231:Fatty Acids, Unsaturated; D014316:Tritium; D012710:Serum Albumin, Bovine; D000001:Calcimycin; C059778:7,7-dimethyl-5,8-eicosadienoic acid; D010741:Phospholipases A; D054467:Phospholipases A2; C006821:4-bromophenacyl bromide