OPTIMIZATION OF AN ASSAY FOR STUDYING THE EFFECTS OF AGENTS ON CYCLOOXYGENASE AND LIPOXYGENASE METABOLISM OF ARACHIDONIC-ACID IN WASHED HUMAN PLATELETS

Before one can examine the effects of substances on the metabolism of arachidonic acid (AA) by the cyclooxygenase and lipoxygenase pathways, an assay system which allows one to detect increases or decreases in both pathways in needed. In order to develop such a system, we have examined nonaggregating washed human platelets (108 platelets/0.5 ml) incubated for various times with 2 μCi 3H-AA and increasing concentrations of AA. T/B2, HHT, 12-HETE, and AA were extracted and separated using reverse phase-HPLC. We first calculated the mass of AA products formed with 10-7 to 10-4 M AA and found that the cyclooxygenase was saturated with 10-5 M AA whereas the lipoxygenase was not saturated with 10-4 M AA. Cyclooxygenase products were more prevalent than 12-HETE below 10-5 M AA, while lipoxygenase products predominated at 3 × 10-5-10-4 M AA. Using 3 μM AA, which does not saturate the cyclooxygenase, we examined the effect of 0.25-10 minute incubation durations on the distribution of AA metabolites and found AA product formation to increase throughout this period without completely depleting the substrate. Since substrate depletion does not occur and further metabolism could be detected for both pathways with a 5 minute incubation with 3 μM AA, these incubation parameters were chosen in order to further test the assay system. Using these parameters, we found that 10-4 M 5-hydroxytryptamine enhanced platelet 12-HETE formation and decreased T/B2 and HHT formation, thus demonstrating the capacity of this system to simultaneously detect changes in cyclooxygenase and lipoxygenase enzyme metabolism. This defined system should prove useful in order to promote a more standardized approach to the study of arachidonic acid metabolism in platelets. © 1990.