Measurement of platelet-activating factor acetylhydrolase activity by quantitative high-performance liquid chromatography determination of coumarin-derivatized 1-O-alkyl-2-sn-lysoglyceryl-3-phosphorylcholine.
A sensitive method for determining platelet-activating factor acetylhydrolase (PAF-AH) activity in human serum, using high-performance liquid chromatography (HPLC) with a fluorimetric detection, is described. The method is based on the derivatization with 7-diethylaminocoumarin-3-carbonylazide of the 2-lyso-PAF, by-product of PAF-AH activity, extracted from the reaction mixture by phase partition into organic solvents. After 3 h of derivatization, the fluorescent derivatives were analyzed by HPLC on a reversed-phase column. The mobile phase was made up with a gradient between head solvent, composed of methanol:water (80:20, v/v) containing 0.25 g/liter choline chloride, and chloroform. Fluorescence detection was at excitation wavelength of 400 nm and at emission wavelength of 480 nm. The described chromatographic procedure is able to resolve and simultaneously quantitate the fluorescent derivatives of the C:18 and C:16 2-lysoPAF. The comparison with the classical radiometric determination of PAF-AH activity demonstrates that the herein described procedure is suitable for study of enzyme kinetics and changes occurring in physiological conditions such as pregnancy.
Balestrieri
C
C
Dipartimento di Biochimica e Biofisica, II Università degli Studi di Napoli, Italy.
Camussi
G
G
Giovane
A
A
Iorio
E L
EL
Quagliuolo
L
L
Servillo
L
L
D043203:1-Alkyl-2-acetylglycerophosphocholine Esterase; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D003374:Coumarins / Q000737:chemistry*; D005260:Female; D006801:Humans; D010741:Phospholipases A / Q000097:blood*; D010767:Phosphorylcholine / Q000031:analogs & derivatives* / Q000737:chemistry; D011247:Pregnancy; D012680:Sensitivity and Specificity; D013050:Spectrometry, Fluorescence
D003374:Coumarins; D010767:Phosphorylcholine; C030123:coumarin; D010741:Phospholipases A; D043203:1-Alkyl-2-acetylglycerophosphocholine Esterase