HPLC-fluorimetric assay of phospholipase A2. Application to biological samples with high protein content and various reaction conditions

Phospholipase A2 (PLA2) quantitation in real-time, using (7-nitro-2-1,3-benzoxadiazol-4-yl)amino-derivatives of phosphatidylcholine (NBD-PCs) as substrates, is influenced by high protein content, color or turbidity. The purpose of the study was to overcome such limitations by a complementary reversed-phase HPLC step to separate the substrates from the products of the reaction. Plasma and bronchoalveolar lavage (BAL) fluid were applied as enzymic sources, while standard porcine PLA2 and human plasma PAF-acetylhydrolase (PAF-AH) were employed as positive controls. The method was validated with a radiometric assay and compared with the real-time fluorimetric assay. Regarding PLA2 and PAF-AH determination, the isocratic elution systems CH3OH–H2O (80:20, v/v) and CH3OH–H2O–CH3COOH (60:40:0.2, v/v/v) separated efficiently C12-NBD-FA/C12-NBD-PC and C6-NBD-FA/C6-NBD-PC, with 4.4 and 2.2 resolution, respectively. Analysis time was ∼15 min/injection. The reproducibility, expressed as relative standard deviation, was ≤13% for PLA2 and ≤16% for PAF-AH, respectively. The assay was linear for PLA2 activities extending from 1 pmol up to at least 250 nmol FA/h/mL sample, similar to the radiometric assay. It was appropriate for samples with high protein content, where the real-time fluorimetric assay was insufficient. The HPLC method was also evaluated under elevated temperatures, various pH values and Ca2+ concentrations.