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-葡萄糖醛酸的结合在胆汁酸的消除中起重要作用。因此，我们已经进行了表征的葡萄糖醛酸苷形成的胆汁酸与雄性Wistar大鼠肝微粒体部分孵育，采用LC/APCI-MS;在这里的胆汁酸的酯型酰基葡萄糖醛酸苷共轭通过24-羧基已被确定为他们的乙酸甲酯衍生物。此外，我们合成了胆酸的酰基葡萄糖醛酸苷作为真实的样品。此外，一个高度敏感的（10 fmol）和可靠的方法分离和测定的酰基葡萄糖醛酸苷使用LC/ESI-MS监测与强烈的去质子化离子[M-H]已开发。应用新开发的方法定量人尿中胆汁酸24-葡萄糖醛酸苷，发现存在鹅脱氧胆酸和脱氧胆酸24-葡萄糖醛酸苷。 ...更多信息 不与蛋白质反应形成共价胆汁酸-蛋白质加合物。因此，通过基质辅助激光解吸电离（MALDI）/飞行时间质谱法（TOFMS）测定溶菌酶与石胆酸24-葡萄糖醛酸苷（LCA-24 G）体外孵育形成加合物和蛋白质上的优选结合位点。将蛋白加合物还原，羧甲基化，并用内切蛋白酶Lys-C消化。通过MALDI-TOFMS在源后衰减模式下对修饰肽进行结构表征。所鉴定的肽含有通过葡萄糖醛酸共价连接至蛋白质赖氨酸基团（赖氨酸1和97）的石胆酸或直接连接至赖氨酸（赖氨酸1和97）的LCA。进行了进一步的研究，以证明胆酸在肝脏中转化为胆酰腺苷酸，优先于胆酰CoA硫酯的形成和胆汁酸-蛋白质共价加合物的体外形成。这些观察结果在胆汁酸-蛋白质加合物诱导肝胆疾病和致癌（如结肠癌）的某些毒性作用方面具有重要意义。少

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 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 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 lkegawa: "Separation and characterization of carboxyl-linked glucuronides of bile acids in incubation mixture of rat liver microsomes"Steroids. 63. 186-192 (1998)

Shigeo Lkekawa：“大鼠肝微粒体孵育混合物中胆汁酸羧基连接的葡萄糖苷酸的分离和表征”类固醇。