A continuous fluorescence assay for the determination of calcium-dependent secretory phospholipase A₂ activity in serum

Background: Calcium-dependent secretory phospholipase A₂-IIA (sPLA₂-IIA) in the circulation is a marker of inflammation, associated with acute and chronic disease processes. We describe a quick, sensitive and reliable microplate continuous fluorescence assay for determining sPLA₂ activity in serum. Methods: Liposomes composed of a fluorescent probe and varying amounts of l-α-phosphatidylglycerol (PG) and 1,2-dioleoyl-l-α-phosphatidylcholine (DOPC) were used as substrates to determine the optimal protocol for sPLA₂ activity determination without interference from serum albumin and lipoproteins. Results: Hydrolysis of the labeled substrate by sPLA₂-IIA, characterized by increase in fluorescence intensity (FI) and confirmed by end-product analysis, occurred in a time-, calcium-, and protein-dependent manner. Liposomes containing 100% PG were most suitable for measurement of sPLA₂ activity without interference from serum components; LDL produced a Ca²⁺-independent increase in FI when liposomes containing DOPC were used. The assay determined that sPLA₂ activity in serum spiked with sPLA₂-IIA and illustrated that endogenous sPLA₂ activity was markedly higher in sera from patients with sepsis than in healthy subjects. Intra-assay and inter-assay CVs were in the ranges of 1.6-8.8% and 3.0-11.5%, respectively. Conclusions: The described method has potential for rapid and sensitive screening of sPLA₂ activity in both clinical and research settings.