Development of Peroxyoxalate Chemiluminescence Assay Method for Platelet Activationg Factor

过氧草酸盐化学发光测定血小板活化因子方法的建立

基本信息

批准号：
05671785
负责人：
NAKASHIMA Kenichiro
金额：
$ 1.34万
依托单位：
Nagasaki University
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (C)
财政年份：
1993
资助国家：
日本
起止时间：
1993 至 1994
项目状态：
已结题

项目摘要

[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.

[1993.4-1994.3]已经对流动注射分析（FIA）进行初步研究，以确定血小板激活因子（PAF）和含胆碱的磷脂。 1）通过戊二醛法将磷脂酶D和肖林氧化酶固定在烷基胺玻璃珠中，并填充到一个小的不锈钢柱中，以制备色谱柱反应器。 2）FIA条件如下：载体溶液为10 mM咪唑缓冲液（pH 8），其中包含10 mm CO（NO_3）_2和0.1％Triton-X；化学发光试剂是乙腈中0.2 mM TCPO和0.25mm TMP的混合物。流速为1.0ml/min。 3）校准曲线的线性最高为200 pmol，其检测限为较低的PMOL水平。[1994.4-1995.3]检查了PAF和磷脂的HPLC分离。 1）将与每个含胆碱磷脂相对应的洗脱液蒸发并溶解在0.1％Triton-X中，并将其等分试样应用于FIA系统。校准曲线在柱上的线性最高为180 pmol，其检测限为较低的PMOL水平。 2）正常人血清中的总胆碱浓度为102-168 mg/dL，这与可获得的试剂盒获得的胆碱浓度非常相对应。 4）正常血清中磷脂酰胆碱，鞘脂线和溶血 - 磷脂酰胆碱的比率为80：15：5。由于所提出的方法很方便且敏感，因此对于测定PAF和相关化合物应该在生物医学和临床研究中有用。