Spectrophotometric monitoring of lipoxygenase and cyclooxygenase pathway activity using ionophore stimulated whole blood

We have employed clotting human blood stimulated with ionophore to develop a system for measuring cyclooxygenase, 5-lipoxygenase, and 12-lipoxygenase pathway products released into the serum fraction. In a single chromatographic run, 5-HETE, 12-HETE, 12-OH-heptadecatrienoic acid (HHT), LTB₄, 20-OH-LTB₄, and 20-COOH-LTB₄ are quantitated by UV monitoring after separation by HPLC. The kinetics of product formation/release of all fatty acid products into the serum show an apparent lag of approximately 2 min, after which time the amounts of HHT, 5-HETE, and LTB₄, respectively, plateau at 10 min while 12-HETE increases over a 60 min period. The system is responsive to standard cyclooxygenase and lipoxygenase inhibitors, and is of value of evaluating prospective blockers of AA metabolism in a whole blood setting.