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)会议化验工作坊的发言者组成，根据工作坊的结论，重点研究了以下事项。净化方法的建立：以自身方法(RIA、EIA、GC-MS有无净化)测定已知含量(1 Ng)的人血浆中6-酮-lt；PGF_(1a)&Gt；为盲样，比较净化方法。这些数值的变化很小，但提出了以下批评：(1)大多数成员用于分离PGs的Sep-Pak C18，有机溶剂材料释放的PGs，干扰了RIA和EIA。(2)Sep-Pak C18应与高效液相色谱法或薄层层析法联用，以消除杂质。(3)用于测定回收率的放射性标记化合物的纯度以及标准的校准曲线之间的差异也引起了数值的变化。11-脱氢-lt；TXB_2&gt酶免疫测定法的研究：建立了人为产生的血栓素B_2代谢物--dH-lt；TXB_2&gt；的酶免疫测定法。用本试剂盒测定结果表明，采血过程中未见血药浓度升高，血药浓度持续时间长于血栓素B_2血药浓度。高血压病患者尿dH-&lt；TXB_2&gt；与尿-lt；TXB_2&gt；呈正相关，而TXA合成酶抑制剂对大鼠尿dH-&lt；TXB_2&gt；值影响不大。白三烯(LT)测定方法：在使用商业RIA试剂盒之前必须进行清理，样品在pH 3.5下酸化，然后用Sep-Pak C18分离，使我们能够轻松地测定LT；LTB_4和GT；以及免疫活性LT。值得注意的是，12-HETE在高效液相色谱上与乙醇中的不纯物质共同迁移，这表明当检测到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。