| Project/Area Number |
10672017
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Physical pharmacy
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| Research Institution | TOHOKU UNIVERSITY |
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Principal Investigator |
IKEGAWA Shigeo GRADUATE SCHOOL OF PHARMERCEUTICAL SCIENCES, TOHOKU UNIVERSITY ASSOCIATE PROFESSOR, 大学院・薬学研究科, 助教授 (90111301)
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| Project Period (FY) |
1998 – 1999
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| Project Status |
Completed (Fiscal Year 1999)
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| Budget Amount *help |
¥3,200,000 (Direct Cost: ¥3,200,000)
Fiscal Year 1999: ¥1,100,000 (Direct Cost: ¥1,100,000)
Fiscal Year 1998: ¥2,100,000 (Direct Cost: ¥2,100,000)
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| Keywords | 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 |
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| Research Abstract |
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
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