Preparation of [3H]-12-L-hydroxyeicosatetraenoic acid and its use in radioimmunoassay.

An efficient method for the enzymic preparation of high specific radioactivity [3H]-12-L-hydroxyeicosatetraenoic acid (12-L-HETE) is described. This compound was used as a radiolabeled ligand in the radioimmunoassay (RIA) of 12-L-HETE. The accuracy of the RIA was checked by incubating [14C]arachidonic acid with platelet lipoxygenase, and measuring enzyme activity in the presence of the inhibitor, 1-phenyl-3-pyrazolidinone (phenidone). The amount of 12-L-HETE synthesized, determined by RIA, was found to be in agreement with that analyzed by radiochemical assay after thin layer plate chromatography.