High-performance liquid chromatographic assay with ultraviolet spectrometric detection for the evaluation of inhibitors of secretory phospholipase A2

A non-radioactive spectrometric assay for the evaluation of inhibitors of pancreatic group IB and non-pancreatic group IIA secretory phospholipase A2 (sPLA2) is described. Mixed-micelles consisting of 1 mM of a 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol and 6 mM of sodium deoxycholate were used as substrate. The enzyme activity was determined directly without any sample clean-up by measuring the sPLA2-mediated oleic acid release with reversed-phase HPLC and UV-detection at 200 nm. The known sPLA2 inhibitors MJ33 and AR-C 67047MI were analyzed in this assay for their inhibitory potency. While MJ33 revealed only a very weak inhibition of group IB and IIA sPLA2 at the highest test concentration (33 μM), AR-C 67047MI proved to be a potent inhibitor of both enzymes with IC50-values of 0.36 and 0.14 μM, respectively.