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

✍ Scribed by C. Balestrieri; G. Camussi; A. Giovane; E.L. Iorio; L. Quagliuolo; L. Servillo

• Publisher: Elsevier Science
• Year: 1996
• Tongue: English
• Weight: 83 KB
• Volume: 233
• Category: Article
• ISSN: 0003-2697
• DOI: 10.1006/abio.1996.0021

✦ Synopsis

wide range of biological activities which are relevant A sensitive method for determining platelet-activatin the development of inflammation and endotoxic ing factor acetylhydrolase (PAF-AH) activity in human shock (4, 12). PAF acts via a specific receptor that is serum, using high-performance liquid chromatogracoupled with a G-protein which activates a phosphatiphy (HPLC) with a fluorimetric detection, is described. dylinositol-specific phospholipase C (8). Some of its The method is based on the derivatization with 7-dibiological activities occur at concentrations as low as ethylaminocoumarin-3-carbonylazide of the 2-lyso-10 012 M. It is conceivable that the synthesis and catabo-PAF, by-product of PAF-AH activity, extracted from lism of this potent autacoid is deeply regulated. The the reaction mixture by phase partition into organic concentration of PAF in cells and in extracellular fluids solvents. After 3 h of derivatization, the fluorescent represents the net difference between synthesis and derivatives were analyzed by HPLC on a reverseddegradation. Therefore, it may be important for the phase column. The mobile phase was made up with a understanding of the pathophysiological role of PAF to gradient between head solvent, composed of methastudy changes in the levels and activation states of nol:water (80:20, v/v) containing 0.25 g/liter choline enzymes involved either in the biosynthesis or catabochloride, and chloroform. Fluorescence detection was lism of this mediator. Once synthesized, PAF is readily at excitation wavelength of 400 nm and at emission

wavelength of 480 nm. The described chromatographic catabolyzed by a PAF-specific acetylhydrolase (PAFprocedure is able to resolve and simultaneously quan-AH) (EC 3.1.1.47) (5, 16). This enzyme cleaves the acetitate the fluorescent derivatives of the C:18 and C:16 tyl moiety of PAF to produce acetate and 2-lysoPAF 2-lysoPAF. The comparison with the classical radiowhich is biologically inactive. Changes in the activity metric determination of PAF-AH activity demonof this enzyme in plasma have been related to several strates that the herein described procedure is suitable diseases or physiological states. In particular, it was for study of enzyme kinetics and changes occurring found that a significant reduction in PAF-AH activity in physiological conditions such as pregnancy. ᭧ 1996 precedes delivery in pregnant woman (9) and also oc-Academic Press, Inc. curs in the active phases of systemic lupus erythematosus (18), while an increased activity correlates with essential hypertension (13). Moreover, it was found a correlation between deficiency of serum PAF-AH and Platelet-activating factor (PAF), 1 1-O-alkyl-2-sn-acerespiratory symptoms in asthmatic children (10). Retyl-3-glycerylphosphorylcholine, is a phospholipid mecently it was shown that a substance from cigarette diator that belongs to the family of biologically active smoke inhibits the activity of the PAF-AH suggesting acetylated phosphoglycerides (6,15). PAF possesses a that the observed increase of PAF in plasma of smokers may depend on a reduced catabolism of this mediator (11).