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Journal of Lipid Research, Vol. 40, 2346-2356, December 1999
Copyright © 1999 by Lipid Research, Inc.

Methods

Differential determination of phospholipase A₂ and PAF-acetylhydrolase in biological fluids using fluorescent substrates

Correspondence to: Marilena E. Lekka

The purpose of the present study was the development and evaluation of a fluorimetric method for the screening and differential determination of phospholipase A₂ and PAF-acetylhydrolase in bronchoalveolar lavage (BAL) fluid and serum. Phospholipase A₂ was determined using C₁₂-NBD-PC in the presence of Ca²⁺, from the slope of the fluorescence enhancement due to the formation of C₁₂-NBD-fatty acid. PAF-acetylhydrolase was determined using C₆-NBD-PC, from the slope of the curve due to C₆-NBD-fatty acid formation in the absence of Ca²⁺. The results were confirmed after TLC analysis. The method's selectivity was evaluated by comparing to radiometric measurements. Light scattering did not interfere and inner filter effects was not observed under our experimental conditions. The effects of pH, temperature, and Ca²⁺ were investigated. Protein caused an increase in the background fluorescence 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 ml of reaction mixture at the normal pH 7.4. The fluorescence of the NBD-fatty acids remained stable for increasing concentrations of BAL fluid and serum and for BSA up to 100 µg/ml of reaction mixture. Porcine pancreatic PLase A₂ showed preference for C₁₂-NBD-PC in the presence of Ca²⁺, while without Ca²⁺, serum PAF-AcH hydrolyzed only C₆-NBD-PC.

The method is highly sensitivite, accurate, and reproducible and can be applied for the differential determination of phospholipase A₂ and PAF-acetylhydrolase activities in BAL fluid and serum.—Kitsiouli, E. I., G. Nakos, and M. E. Lekka. Differential determination of phospholipase A₂ and PAF-acetylhydrolase in biological fluids using fluorescent substrates. J. Lipid Res. 1999. 40: 2346;–2356.

Supplementary key words: phospholipase A₂, PAF-acetylhydrolase, bronchoalveolar lavage, NBD-lipids, fluorescence, phosphatidylcholines

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