Monitoring eicosanoid biosynthesis via lipoxygenase and cyclooxygenase pathways in human whole blood by single HPLC run

Eicosanoids play an important role as lipid mediators for physiological and pathological processes. Inhibitors of their biosynthesis have been developed as drugs for various diseases with major health political relevance. The search for more efficient inhibitors of eicosanoid formation requires simultaneous monitoring of various metabolic pathways. We developed an HPLC-based assay system, which quantifies lipoxygenase metabolites leukotriene B4 (LTB4), 5-hydroxyeicosatetraenoic acid (5-HETE), 12-hydroxyeicosatetraenoic acid (12-HETE), 15-hydroxyeicosatetraenoic acid (15-HETE) and cyclooxygenase metabolite 12-hydroxy-5,8,10-heptadecatrienoic acid (12-HHT) in whole human blood. Eicosanoid formation in blood is initiated with calcium ionophore A23187, arachidonic acid and calcium and magnesium ions. After solid phase extraction the different eicosanoids were separated by isocratic RP-HPLC using prostaglandin B1 as authentic standard. To verify the assay we determined the IC50 of known inhibitors of eicosanoid biosynthesis (zileuton, indomethacin, nordihydroguaiaretic acid). The test system is simple. It does not require extensive methodological experience and can be carried out in any biochemical laboratory. The analytical procedure can be robotized and thus, the assay appears suitable for medium-throughput testing of drugs.