Simultaneous determination of hydroxyeicosanoid (HETE) binding to cells and its cellular metabolism

In order to study the mechanism(s) through which certain biologically active lipids, such as the hydroxyeicosanoids (HETEs), exert their effects, it is necessary to distinguish between binding of these lipids to cells and their cellular metabolism. A novel and simple method is described for the simultaneous determination of [3H]15-hydroxyeicosanoid (15-HETE) binding to cells and cellular [3H]15-HETE metabolism. The method involves initial separation of radiolabeled cells by filtration, filter extraction of cellular lipids by methanol, and thin-layer chromatography (or high performance liquid chromatography) determination of both nonesterified 15-HETE bound to cells and 15-HETE incorporation into cellular phospholipids. The method was applied to both PT-18 mast/basophil cells and rat basophilic leukemia (RBL-1) cells and should be applicable to other cells as well as other metabolizable hydroxy fatty acids or lipids.