A simple assay for a human serum phospholipase A(2) that is associated with high-density lipoproteins

Phospholipase A(2) (PLA(2)) activity is usually assayed with expensive radi oactive or chromogenic substrates unsuitable for performing large numbers o f assays. We have designed a simple microplate assay for human serum PLA(2) using the chromogenic substrate 4-nitro-3-octanoyloxy-benzoic acid. Using this substrate, serum PLA(2) activity was similar to that measured with the previously characterized chromogenic phospholipid substrate 1,2-bis-heptan oylthio-glycerophosphocholine. However, the assay described here appears to be more sensitive. The mean PLA(2) activity in serum from healthy voluntee rs (n = 30) measured by this assay was 10.4 +/- 1.6 mu mol (.) h(-1) (.) ml (-1). The assay is reproducible and is suitable for the analysis of large n umbers of samples in a clinical setting. We have also demonstrated that 94% of the PLA(2) activity in normal human serum is associated with high-densi ty lipoproteins and that serum PLA(2) activity is positively correlated wit h the lipoprotein parameters total triglyceride (P < 0.0001), total cholest erol (P < 0.0001), and atherogenic index (P = 0.008). The serum PLA(2) acti vity was calcium dependent and was inhibited by the serine protease inhibit or 3,4-dichloroisocoumarin (EC50 = 0.4 mM). jlr The PLA(2) activity charact erized here is unlikely to be due to plasma platelet-activating factor acet ylhydrolase or low molecular weight His-Asp sPLA(2), and may represent a ne w sPLA(2) type.