Development of a radioimmunoassay for 15-hete and its application to 15-hete production by reticulocytes☆

Mono-hydroxy-eicosatetraenoic acids (HETE's) are frequently the principal lipoxygenase-derived products in a number of cell types. This paper describes the development of a selective and sensitive radioimmunoassay procedure for 15-HETE, a metabolite which has previously been shown to be both an activator and inhibitor of leukotriene formation in various cells. Initially, rabbits were immunized with 15-HETE conjugated to bovine serum albumin. After seven months, the anti-plasma showed significant binding of tritiated 15-HETE (40– 45% binding with a 1:600 dilution of the anti-plasma) which was displaceable by cold 15-HETE. The sensitivity of the assay was approximately 20 pg. of 15-HETE. The anti-plasma exhibited very little (<1%) cross-reactivity with arachidonic acid, 5−, 8−, 9−, 11− and 12−HETE's, HHT, TXB2, PGE2 and 6-Keto-PGF1α. Significant cross-reactivity was observed with 5,15-diHETE (53%), 8, 15-diHETE (6.6%), and several other 15-hydroxy-eicosanoids. Rabbit reticulocytes have a very active 15S-lipoxygenase and converted arachidonic acid (final concentration 7 μM) principally to 15-HETE. Unstimulated reticulocytes were found to release negligible amounts of 15-HETE as determined by radio-immunoassay Treatment of these cells with the calcium ionophore A23187 (0.16 to 4.0 μg/ml) elicited a level of 15-HETE release (8 − 14 ng/ml) that was twenty to forty times less than obtained with exogenous arachidonic acid (2.5 μg/ml). The radioimmunoassay reported here may be useful for identifying factors which stimulate cellular release of 15-HETE and other 15-hydroxy-eicosanoids from endogenous arachidonic acid.