Development of assay method of novel metabolites of endogenous prostaglandins and its application

内源性前列腺素新代谢物检测方法的建立及其应用

• 批准号: 62870011
• 负责人: KATORI Makoto
• 金额: $ 7.74万
• 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: 1987
• 资助国家: 日本
• 起止时间: 1987 至 1989
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-62870011/
• 关键词: Thromboxane A_2; 11-dehydro-thromboxane B_2; Immunoaffinity-columu; 11ーDHーTXB_2; 抗体固定化カラム; 尿; 血漿; 動物種差; オ-プン体; 11ーデヒドローTXB_2; 抗体固相化カラム; 2,3ージノールーTXB_2

TXB2 had been used as an index of TXA2 generation in the body, but cannot be used anymore, because of its artifactual generation during blood collection. Instead, 11- dehydro(DH)-TXB2 was chosen and its enzyme immunoassay method was developed by a research member. The subsequent usage of the commercially available RIA kits prompted us to proceed analyses of the determination method of 11-DH-TXB2 and we realized that the extraction and purification was an unavoidable step before assay. Then, the simpler extraction and purification procedure by an immunoaffinity column was explored icolaboration with Sankyo group of this research group. The monoclonal antibody against 11- DH-TXB2, produced by Dr. Yamamoto's group, Tokushima University, was used for this immunoaffinity column and the standard extraction method was established by a workshop of this research group. Each member of this research group measured the 11-DH-TXB2 content in the distributed known samples and the column was applied … More to measure ll-DH- TXB2 levels in pathological models and in clinical patients. It was confirmed that this immunoaffinity columns was useful for assay of 11-DH-TXB2 not only in urine, but also in plasma. There are, however, many difficulties for taking 11-DH-TXB2 as an index of TXA2; the large variety in animal species differences, showing that dogs scarcely produce ll-DH- TXB2 from TXB2. In contrast, 11-DH-TXB2 is produced largely in human from TXB2 and its half life is approximately 60 min, indicating that this metabolite can be taken as an index of TXA2 generation in clinical field. Moreover, we found many difficulties in this assay, such as use of the open type of 11-DH-TXB2, method to exclude substances, disturbed in RIA procedures, reliability of isotope labeled standards, available commercially (large variation of the contentents in individual lots) and others. It is clear that three years studies of this research group established the assay method of 11-DH-TXB2 including extraction and purification using this immunoaffinity column and disclosed many difficulties during the extraction. Less

TXB2曾被用作体内TXA2生成的指标，但由于其在采血过程中的人为生成而不能再使用。相反，选择了11-脱氢(DH)-TXB2，其酶联免疫分析方法是由一名研究成员开发的。随后市售RIA试剂盒的使用促使我们对11-DH-TXB2的测定方法进行了分析，我们意识到提取和纯化是测定前不可避免的步骤。随后，与本课题组三共课题组合作，探索了更简单的免疫亲和柱提取纯化工艺。该免疫亲和柱使用德岛大学山本博士课题组生产的针对11-DH-TXB2的单克隆抗体，标准提取方法是由该课题组的研讨会制定的。该研究小组的每位成员测量了分发的已知样品中的 11-DH-TXB2 含量，并应用该色谱柱测量病理模型和临床患者中的 ll-DH-TXB2 水平。经证实，该免疫亲和柱不仅可用于检测尿液中的 11-DH-TXB2，还可用于检测血浆中的 11-DH-TXB2。然而，以11-DH-TXB2作为TXA2的指标存在很多困难；动物物种差异很大，表明狗几乎不能从 TXB2 产生 ll-DH-TXB2。相比之下，11-DH-TXB2在人体中主要由TXB2产生，其半衰期约为60分钟，表明该代谢物可以作为临床领域TXA2生成的指标。此外，我们在该测定中发现了许多困难，例如使用开放型11-DH-TXB2、排除物质的方法、RIA程序中的干扰、同位素标记标准品的可靠性、商业上可用的（各个批次中的含量变化很大）等。显然，该课题组经过三年的研究，建立了使用该免疫亲和柱提取和纯化11-DH-TXB2的测定方法，并揭示了提取过程中的许多困难。较少的

项目成果

K.Takeuchi,K.Abe,M.Sato,M.Yasujima,T.Hagino,S.Fang and K.Yoshinaga: "Effect of in vitro aging on prostaglandin synthesis in cultured rat vascular smooth muscle cell." Vasodepressor Hormones. 49-54 (1987)

K.Takeuchi、K.Abe、M.Sato、M.Yasujima、T.Hagino、S.Fang 和 K.Yoshinaga：“体外衰老对培养的大鼠血管平滑肌细胞前列腺素合成的影响”。

K.Ymamaki and S.Oh-ishi: "Fluorometric high performance liquid chromatographic analysis of prostaglandin(PG)metabolites:Application to the catabolism of PGE_2 and PGF_2 α in rat and rabbit kidney homogenates." J.Phrmacobio-Dynamics. 12. 74-79 (1988)

K.Ymamaki 和 S.Oh-ishi：“前列腺素 (PG) 代谢物的荧光高效液相色谱分析：在大鼠和兔肾匀浆中 PGE_2 和 PGF_2 α 分解代谢中的应用”，J.Phrmacobio-Dynamics。 -79 (1988)

澤田正文、無量林啓子、新各哲也、宮本茂敏、桶川忠夫、川崎晃義: "Radioimmunoassay of 11-dehydro-TXB_2" 1988年台北PGカンファレンスで発表(投稿準備中),Abstract book. P.12.

Masafumi Sawada、Keiko Muryobayashi、Tetsuya Arata、Shigetoshi Miyamoto、Tadao Okekawa、Akiyoshi Kawasaki：“11-deHydro-TXB_2 的放射免疫分析”发表于 1988 年台北 PG 会议（准备提交），摘要书第 12 页。

大内和雄、平沢典保、武田裕子、浅野薫、渡辺雅子、鶴藤丞: "Mechanism of antiallergic action of β-agonists in allergic inflammation of air pouch type inrats." Int.Arch.Allergy,Apple.Immunol.82. 26-32 (1987)

Kazuo Ouchi、Noriyasu Hirasawa、Yuko Takeda、Kaoru Asano、Masako Watanabe、Jo Tsuruto：“β-激动剂在大鼠气囊型过敏性炎症中的抗过敏作用机制。” Int.Arch.Allergy，Apple.Immunol。 -32 (1987)

大内和雄、渡辺雅子、平沢典保、鶴藤丞、尾関恒雄、藤木博太: "Inhibition by gossypol of tumor promoter-induced arachidonic acid metabolism in rat peritoneal macrophages." Biochim.Biophys.Acta. 971. 85-91 (1988)

Kazuo Ouchi、Masako Watanabe、Noriyasu Hirasawa、Tsuru Tsuruto、Tsuneo Ozeki、Hirota Fujiki：“棉酚对大鼠腹膜巨噬细胞中肿瘤促进剂诱导的花生四烯酸代谢的抑制”Biochim.Biophys.Acta。