STUDIES ON STRUDTURE CHARACTERIZATION OF BILE ACIDPROTEIN ADDUCTS

胆汁酸蛋白加合物的结构表征研究

• 批准号: 10672017
• 负责人: IKEGAWA Shigeo
• 金额: $ 2.05万
• 依托单位: TOHOKU UNIVERSITY
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 1998
• 资助国家: 日本
• 起止时间: 1998 至 1999
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-10672017/
• 关键词: Bile acid; Ester glucuronide; MALDI-TOFMS; Protein Adduct; Lysozyme; Cholyl adenylate; Glucuronyltrasferase; アシルアデニレート; MALDI; TOFMS; サブスタンスP; 修飾ペプチド

Conjugation of bile acids with D-glucuronic acid plays an important role in the elimination of bile acids. Therefore, we have been carried out to characterize the glucuronides formed by incubation of bile acids with a male Wistar rat liver microsomal fraction, employing LC/APCI-MS; here the ester-type acyl glucuronides of bile acid conjugated through the 24-carboxy group have been identified as their acetate methyl ester derivatives. In addition, we have synthesized the acyl glucuronides of common bile acids as authentic specimens. Moreover, a highly sensitive (10 fmol) and reliable method for the separation and determination of the acyl glucuronides using LC/ESI-MS monitored with an intense deprotonated ion[M-H] has been developed. Application of the newly developed method to the quantitation of bile acid 24-glucuronides in human urine revealed the presence of chenodeoxycholic and deoxycholic acid 24-glucuronides.The acyl glucuronides are intrinsically reactive metabolites to be capab … More le of reacting with protein to form a covalent bile acid-protein adduct. Accordingly, the formation of adducts by in vitro incubation of lysozyme with lithocholic acid 24-glucuronide (LCA-24G) and the preferred binding sites on the protein were determined by matrix-assisted laser desorption ionization (MALDI)/time-of-flight mass spectrometry (TOFMS). The protein adducts were reduced, carboxymethylated, and digested with endoproteinase Lys-C. The modified peptides were structurally characterized by MALDI-TOFMS in the post-source decay mode. The identified peptides contained lithocholic acid linked covalently via glucuronic acid to a protein lysine groups (lysined 1 and 97) or LCA directly linked to lysines (lysines 1 and 97). Further studies were carried out to demonstrate the hepatic transformation of cholic acid into cholyl-adenylate in preference to the formation of cholyl CoA thioester and in vitro formation of a covalent bile acid-protein adduct. These observations are of great interest in relation of some toxic effects of bile acid-protein adducts to induce hepatobiliary disorders and carcinogenesis such as colon cancer. Less

胆汁酸与d-谷氨酸的结合在消除胆汁酸中起重要作用。因此，我们已经进行了表征，以用LC/APCI-MS孵育胆汁酸与雄性Wistar大鼠肝微粒体馏分形成的谷氨酸烯剂。在这里，通过24-羧基偶联的胆汁酸的酯型酰基谷氨酸酯已被鉴定为其乙酸甲酯衍生物。此外，我们将普通胆汁酸的酰基谷氨酸苷合成为正宗标本。此外，已经开发了使用强烈的去质子化离子[M-H]监测的LC/ESI-MS分离和测定酰基谷氨酸盐的高度敏感（10 FMOL）和可靠的方法。新开发的方法在人类尿液中定量24-氯丁醛酰定量时的应用揭示了Chendoxycholic和脱氧胆酸24-葡萄糖醛酸的存在。酰基葡萄糖醛酸酰葡萄糖醛酸是本质上反应性的代谢物是Capab…与蛋白质反应形成纤维酸性蛋白质的蛋白质的更多作用。根据通过基质辅助激光吸收电离（MALDI）/飞行飞机质谱法（TOFM）（TOFM）（TOFM）确定溶菌酶与岩性酸24-葡萄糖醛酸（LCA-24G）（LCA-24G）（LCA-24G）（LCA-24G）（LCA-24G）（LCA-24G）（LCA-24G）（LCA-24G）的形成。蛋白质加合物降低，羧甲基化并用内蛋白酶Lys-C消化。在后源衰减模式下，修饰的肽在结构上以MALDI-TOFM进行了特征。鉴定出的肽含有通过谷氨酸共价连接到蛋白质赖氨酸基（裂解1和97）或LCA或LCA与赖氨酸（赖氨酸1和97）直接相关的岩性酸。进行了进一步的研究，以证明胆酸向胆碱 - 丙烯酸酯的肝转化，优先于形成胆囊壳菌的形成，并在体外形成共价胆汁酸蛋白质加合物。这些观察结果在对胆汁酸蛋白加合物的某些毒性作用的关系中引起了极大的兴趣，以诱导肝疾病和癌变（例如结肠癌）。较少的

项目成果

Shigeo lkegawa: "Separatory determination of diastereomeric ibuprofen glucuronides in human urine by liquid chromatography/electrospray ionization-mass spectrometry"Biomed. Chromatogr.. 12. 1-5 (1998)

Shigeo Lkekawa：“通过液相色谱/电喷雾电离-质谱法分离测定人尿液中的非对映体布洛芬葡萄糖醛酸苷”Biomed。

Shigeo lkegawa: "Synthesis of bile acid 24-acyl glucuronides"Steroids. 63. 180-185 (1998)

Shigeo lkekawa：“胆汁酸24-酰基葡萄糖醛酸苷的合成”类固醇。

Shigeo Ikegaw: "Separatory determination of diastereomeric ibuprofen glucuronides in human urine by liquid chromatography / electrospray ionization-mass spectrometry" Biomed.Chromatogr.12. 1-5 (1998)

Shigeo Ikegaw：“通过液相色谱/电喷雾电离质谱法分离测定人尿液中的非对映体布洛芬葡萄糖醛酸”Biomed.Chromatogr.12。

Shigeo Ikegawa, Hiroshi Ishikawa, Hiroyuki Oiwa, Masanori Nagata, Junichi Goto, Teruya Kozaki, Masaharu Gotowda and Naoki Asakawa: "Characterization of cholyl-adenylate in rat liver microsome by liquid chromatography/electrospray ionization mass-spectrome

Shigeo Ikekawa、Hiroshi Ishikawa、Hiroyuki Oiwa、Masanori Nagata、Junichi Goto、Teruya Kozaki、Masaharu Gotowda 和 Naoki Asakawa：“通过液相色谱/电喷雾电离质谱表征大鼠肝微粒体中的胆酰腺苷酸

Shigeo Ikegawa, Naoaki Murao, Masanori Nagata, Satomi Ohba and Junichi Goto: "Covalent binding of bile acid acyl glucuronide with protein."Anal. Sci.. 15. 213-215 (1999)

Shigeo Ikekawa、Naoaki Murao、Masanori Nagata、Satomi Ohba 和 Junichi Goto：“胆汁酸酰基葡萄糖醛酸与蛋白质的共价结合。”肛门。