Differential determination of phospholipase A(2) and PAF-acetylhydrolase in biological fluids using fluorescent substrates

The purpose of the present study was the development and evaluation of a fl uorimetric method for the screening and differential determination of phosp holipase A(2) and PAF-acetylhydrolase in bronchoalveolar lavage (BAL) fluid and serum. Phospholipase A(2) was determined using C-12-NBD-PC in the pres ence of Ca2+, from the slope of the fluorescence enhancement due to the for mation of C-12-NBD-fatty acid. PAF-acetylhydrolase was determined using C-6 -NBD-PC, from the slope of the curve due to C-6-NBD-fatty acid formation in the absence of Ca2+, The results were confirmed after TLC analysis. The me thod's selectivity was evaluated by comparing to radiometric measurements. Light scattering did not interfere and inner filter effects was not observe d under our experimental conditions. The effects of pH, temperature, and Ca 2+ were investigated. Protein caused an increase in the background fluoresc ence of both NBD-PCs. The standard curves of both NBD-fatty acids exhibited the same slope. Linearity extended at least up to 4.5 nmoles per mi of rea ction mixture at the normal pH 7.4, The fluorescence of the NBD-fatty acids remained stable for increasing concentrations of BAT, fluid and serum and for BSA up to 100 mu g/ml of reaction mixture. Porcine pancreatic PLase Ap showed preference for C-12-NBD-PC in the presence of Ca2+, while without Ca 2+, serum PAF-AcH hydrolyzed only C-6-NBD-PC. The method is highly sensitiv ite, accurate, and reproducible and can be applied for the differential det ermination of phospholipase A(2) and PAF-acetylhydrolase activities in BAL fluid and serum.-Kitsiouli, E. I., G. Nakos, and M. E. Lekka. Differential determination of phospholipase A(2) and PAF-acetylhydrolase in biological f luids using fluorescent substrates.