Development of Peroxyoxalate Chemiluminescence Assay Method for Platelet Activationg Factor

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[1993.4-1994.3] A preliminary study on a flow-injection analysis (FIA) has been performed for the determination of platelet activating factor (PAF) and choline-containing phospholipids. 1) Phospholipase D and sholine oxidase were immobilized to alkylamine glass beads by a glutaraldehyde method, packed into a small stainless steel column to prepare column reactor. 2) FIA conditions are as follows : carrier solution was 10 mM imidazole buffer (pH 8) containing 10 mM Co (NO_3)_2 and 0.1% Triton-X ; chemiluminescence reagent was a mixture of 0.2 mM TCPO and 0.25mM TMP in acetonitrile ; flow rate was 1.0ml/min. 3) Calibration curves were linear up to 200 pmol with the detection limits of lower pmol levels.[1994.4-1995.3] HPLC separation of PAF and phospholipids were examined. 1) The eluent corresponding to each choline-containing phospholipid was evaporated and dissolved in 0.1% Triton-X,and an aliquot of the resultant was applied to the FIA system. Calibration curves were linear up to 180 pmol on column with the detection limits of lower pmol levels. 2) Total choline concentrations in normal human sera were 102-168 mg/dL,which were well corresponded to those obtained by the commercially available kits. 4) The ratio of phosphatidylcholine, sphingomyeline, and lyso-phosphatidylcholine in normal serum was 80 : 15 : 5.As the proposed method is convenient and sensitive, it should be useful for the determination of PAF and related compounds in biomedical and clinical studies.