A continuous fluorescence assay for the determination of calcium-dependent secretory phospholipase A 2 activity in serum

• Published in: Clinica chimica acta Vol. 379; no. 1; pp. 119 - 126
• Main Authors: Tsao, Francis H.C., Shanmuganayagam, Dhanansayan, Zachman, Derek K., Khosravi, Mehdi, Folts, John D., Meyer, Keith C.
• Format: Journal Article
• Language: English
• Published: Elsevier B.V 2007
• Subjects: BODIPY; Fluorescence; Inflammation; Liposomes; Secretory phospholipase A 2; Serum; FFA; PBS; BODIPY; FI; Fluorescence; TLC; Inflammation; EDTA; sPLA 2; EGTA; BSA; NBD; lyso-PC; Secretory phospholipase A 2; LDL; HSA; PG; Serum; Liposomes; DOPC
• ISSN: 0009-8981; 1873-3492
• DOI: 10.1016/j.cca.2006.12.023

Calcium-dependent secretory phospholipase A 2-IIA (sPLA 2-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 sPLA 2 activity in serum. 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 sPLA 2 activity determination without interference from serum albumin and lipoproteins. Hydrolysis of the labeled substrate by sPLA 2-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 sPLA 2 activity without interference from serum components; LDL produced a Ca 2+-independent increase in FI when liposomes containing DOPC were used. The assay determined that sPLA 2 activity in serum spiked with sPLA 2-IIA and illustrated that endogenous sPLA 2 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. The described method has potential for rapid and sensitive screening of sPLA 2 activity in both clinical and research settings.