Standarization of assay methods of endogenous prostaglandins and leukotrienes

内源性前列腺素和白三烯测定方法的标准化

• 批准号: 59870087
• 负责人: KATORI Makoto
• 金额: $ 5.25万
• 依托单位: Kitasato University School of Medicine
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Developmental Scientific Research
• 财政年份: 1984
• 资助国家: 日本
• 起止时间: 1984 至 1986
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-59870087/
• 关键词: Assay methods for prostaglandins; Elimination of disturbing substances for assay; Assay for 11-dehydro-Thromboxane <B_2>; leukotriene; 12-HETE; 6-keto-PG【F_(1α)】; 11-dehydro-thromboxane【B_2】; leukotriene【B_4】; leukotriene【C_4】; leukotriene【D_4】 /

This project team, composed of speakers of assay workshop in Kyoto Prostaglandin (PG) Conference, 1984, has focused the research on the following items, according to the conclusion of the workshop.1. Establishment of clean-up method: 6-Keto- <PGF_(la)> in a test sample of human plasma containing a known amount (1 ng) was measured as a blind sample by own method of the team members (RIA, EIA, GC-MS with or without cleaning-up) to compare cleaning method. Variation of the values was rather small, but the following criticisms were made: (1) Sep-Pak C18, which was used by most members for separation of PGs, releases by organic solvent materials, which disturb RIA and EIA. (2) Thus, Sep-Pak C18 should be used in combination with HPLC or TLC for elimination of impure substances. (3) Purity of radio-labelled compounds for testing recovery as well as a difference between calibration curves of the standard also caused variation of the values.2. Development of enzyme-immunoassay of 11-dehydro- <TXB_2> (DH- <TXB_2> ): Enzyme-immuno-assay of DH- <TXB_2> , a metabolite of <TXB_2> , has been developed, as <TXB_2> is generated artificially. Determination with the present kit revealed that an increase in DH- <TXB_2> values during blood collection was not observed and duration of blood concentration of DH- <TXB_2> was longer than that of <TXB_2> . DH- <TXB_2> value in urine from patients of essential hypertension was well correlated to that of <TXB_2> , but TXA synthetase inhibitor attenuated DH- <TXB_2> value in rat urine only slightly.3. Leukotriene (LT) assay method: Cleaning-up must be made before using commercial RIA kits; acidification of samples at pH 3.5, followed by separation of LTs by Sep-Pak C18, enabled us to determine <LTB_4> and immunoreactive LTs without difficulty. It should be noticeable that 12-HETE on HPLC co-migrates with that of impure materials from ethanol, indicating that caution should be taken when 12-HETE is detected.

该项目组由1984年京都前列腺素（PG）会议测定研讨会的发言者组成，根据研讨会的结论，重点研究了以下项目： 1．净化方法的建立：通过团队成员自己的方法（RIA、EIA、 GC-MS（有或没有净化）来比较清洁方法。数值变化相当小，但提出了以下批评：（1）大多数成员用于分离PG的Sep-Pak C18，有机溶剂材料释放，干扰RIA和EIA。 (2) 因此，Sep-Pak C18 应与 HPLC 或 TLC 结合使用，以消除不纯物质。 (3)回收率测试中放射性标记化合物的纯度以及标准品校准曲线的差异也会引起数值的变化。2． 11-脱氢-<TXB_2>（DH-<TXB_2>）酶联免疫分析的开发：<TXB_2>的代谢物DH-<TXB_2>的酶联免疫分析已经开发出来，因为<TXB_2>的生成人为地。用本试剂盒测定表明，在采血过程中没有观察到DH-<TXB_2>值的增加，并且DH-<TXB_2>的血药浓度持续时间比<TXB_2>的血药浓度持续时间长。原发性高血压患者尿液中DH-<TXB_2>值与<TXB_2>值有良好的相关性，但TXA合成酶抑制剂仅轻微减弱大鼠尿液中DH-<TXB_2>值。3．白三烯（LT）测定方法：使用市售RIA试剂盒前必须进行清洗；将样品在 pH 3.5 下酸化，然后通过 Sep-Pak C18 分离 LT，使我们能够毫无困难地确定 <LTB_4> 和免疫反应性 LT。值得注意的是，HPLC 上的 12-HETE 与乙醇中的不纯材料共迁移，表明检测到 12-HETE 时应谨慎。

项目成果

Isono: Biochem.Biophys.Res.Comm.130. (1985)

矶野：Biochem.Biophys.Res.Comm.130。

M. Katori; six authors Eds. O. Hayaishi; S. Yamamoto: Raven Press. Seven articles in "Assay--Utility and Difficulty" in Advances in Prostagalndin, Thromboxane,and Leukotirene Research, 21-102 (1985)

M.香取；

佐藤牧人: 臨床検査 MooK. 24. 97-114 (1986)

佐藤真人：临床测试 MooK。24. 97-114 (1986)

M.Sato: Hypertension. 8. 762-771 (1986)

佐藤先生：高血压。

M.Sato: Agents and Actions. (1987)

佐藤先生：代理人和行动。