MEASUREMENT OF PLATELET-ACTIVATING-FACTOR ACETYLHYDROLASE ACTIVITY BYQUANTITATIVE HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHY DETERMINATION OF COUMARIN-DERIVATIZED 1-O-ALKYL-2-SN-LYSOGLYCERYL-3-PHOSPHORYLCHOLINE

A sensitive method for determining platelet-activating factor acetylhy drolase (PAF-AH) activity in human serum, using high-performance liqui d chromatography (HPLC) with a fluorimetric detection, is described. T he method is based on the derivatization with 7-diethylaminocoumarin-3 -carbonylazide of the 2-lysoPAF, by-product of PAF-AH activity, extrac ted from the reaction mixture by phase partition into organic solvents . After 3 h of derivatization, the fluorescent derivatives were analyz ed by HPLC on a reversed-phase column. The mobile phase was made up wi th a gradient between head solvent, composed of methanol:water (80:20, v/v) containing 0.25 g/liter choline chloride, and chloroform. Fluore scence detection was at excitation wavelength of 400 nm and at emissio n wavelength of 480 nm. The described chromatographic procedure is abl e to resolve and simultaneously quantitate the fluorescent derivatives of the C:18 and C:16 2-lysoPAF. The comparison with the classical rad iometric determination of PAF-AH activity demonstrates that the herein described procedure is suitable for study of enzyme kinetics and chan ges occurring in physiological conditions such as pregnancy. (C) 1996 Academic Press, Inc.