Spectrophotometric assay for serum platelet-activating factor acetylhydrolase activity.
We developed a spectrophotometric assay for serum platelet-activating factor acetylhydrolase (PAF-AH, EC 3.1.1.47.) activity using a platelet-activating factor (PAF) analogue with a 4-nitrophenyl group as substrate. PAF-AH hydrolyzes the sn-2 position of the substrate ¿1-myristoyl-2-(p-nitrophenylsuccinyl)phosphatidylcholine, producing p-nitrophenyl succinate. This liberation was spectrophotometrically monitored and the activity determined from the change in absorption. The assay does not require radioisotopes and is applicable to an automatic analyzer. Utilizing this assay with an automatic analyzer, it is possible to measure the activities of thousands of samples in a few hours with excellent precision (CV 0.5%, n=30) and high correlation (r=0.979, n=100) with the results of a conventional radioisotopic assay. The assay should be particularly useful for clinical diagnostics.
Kosaka
T
T
Diagnostic Research & Development Department, R&D Division, Nesco Company, Azwell Inc., 2-24-3 Sho, Ibaraki, Osaka, Japan. nskrd-06@mbd.sphere.ne.jp
Yamaguchi
M
M
Soda
Y
Y
Kishimoto
T
T
Tago
A
A
Toyosato
M
M
Mizuno
K
K
D043203:1-Alkyl-2-acetylglycerophosphocholine Esterase; D002793:Cholic Acids; D006801:Humans; D006868:Hydrolysis; D009596:Nitrophenols; D010741:Phospholipases A / Q000097:blood*; D010972:Platelet Activating Factor / Q000031:analogs & derivatives; D012680:Sensitivity and Specificity; D013053:Spectrophotometry; D013379:Substrate Specificity
D002793:Cholic Acids; D009596:Nitrophenols; D010972:Platelet Activating Factor; D010741:Phospholipases A; D043203:1-Alkyl-2-acetylglycerophosphocholine Esterase; C028213:3-((3-cholamidopropyl)dimethylammonium)-1-propanesulfonate; C024836:4-nitrophenol