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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]用流动注射分析法（FIA）测定血小板活化因子（PAF）和含胆碱磷脂进行了初步研究。1)采用戊二醛法将磷脂酶D和胆碱氧化酶固定在烷基胺玻璃微珠上，装入小型不锈钢塔中制备塔式反应器。2) FIA条件为：载体溶液为10 mM咪唑缓冲液（pH 8），含10 mM Co (NO_3)_2和0.1% Triton-X；化学发光试剂为0.2 mM TCPO与0.25mM TMP在乙腈中的混合物；流速1.0ml/min。3)校正曲线在200 pmol以内呈线性，较低pmol水平检出限。[1994.4-1995.3]对PAF与磷脂的HPLC分离进行了研究。1)将每种含胆碱磷脂对应的洗脱液蒸发，溶解于0.1% Triton-X中，并将所得的等分液加入FIA体系。在180 pmol的柱上，校正曲线呈线性，低pmol的检出限。2)正常人血清中总胆碱浓度为102 ~ 168 mg/dL，与市售试剂盒的测定结果吻合较好。4)正常血清中磷脂酰胆碱、鞘磷脂酰胆碱、溶磷脂酰胆碱的比值为80:15:5。该方法简便、灵敏，可用于生物医学和临床研究中PAF及相关化合物的测定。