A radiochemical assay for detection of leukotriene B4 production from isolated cells

A radiochemical procedure for quantitating the effect of inhibitors of leukotriene B4 (LTB4) biosynthesis is described. Rat peritoneal cells were labeled with 3H-arachidonic acid and stimulated with the calcium ionophore A23187. 3H-LTB4 was isolated by processing on C18-Sep Pak cartridges followed by reverse-phase high pressure liquid chromatography (HPLC). The identity of this product, as well as other arachidonic acid metabolites, was verified by using silicic acid column chromatography followed by straight-phase HPLC and thin layer chromatography. Using this assay, LTB4 release by ionophore A23187 has been shown to be both time- and concentration-dependent. Indomethacin enhanced, while NDGA and ETYA inhibited, the A23187-induced production of LTB4. This procedure is both simple and direct and is capable of assessing the ability of novel compounds to alter LTB4 production.