2002 Fiscal Year Annual Research Report

脳内胆汁酸の生理的意義解明に関する研究

Research Project

Project/Area Number	14370726
Research Institution	Tohoku University
Principal Investigator	後藤順一東北大学, 医学部附属病院, 教授 (80006337)
Co-Investigator(Kenkyū-buntansha)	飯田隆日本大学, 文理学部, 教授 (60060125) 眞野成康東北大学, 大学院・薬学研究科, 講師 (50323035) 小林典裕神戸薬科大学, 教授 (90205477)
Keywords	脳内胆汁酸 / 脳内胆汁酸の生合成 / ケノデオキシコール酸 / LC / ESI-MS / トレーサー法 / MALDI-TOFMS / アフィニティイー抽出 / ディファレンシャルディスプレー
Research Abstract	胆汁酸は、肝においてコレステロールより生合成される炭素数24個のカルボン酸であり、腸肝循環しているため、通常血中や末梢組織中にはごく僅かにしか存在しない。最近当研究グループにおいて、LC/ESI-MSを用いてラット脳組織中の胆汁酸の検索を行ったところ、3種の遊離型胆汁酸が存在し、しかもそれらは、ある種の蛋白質と強くバンドしていることを世界に先駆けて証明した。そこで今回、脳内胆汁酸の由来を解明するとともに、プロテオミクス手法を用いてその結合蛋白質の解析を試みることとした。まず、側鎖カルボキシル基に重酸素を標識した3β-ヒドロキシ-5-コレン酸を調製し、ラット脳ミクロソーム画分とインキュベートした結果、7α位に水酸化を受けることが判明した。次いで、生成した3β,7α-ジヒドロキシ-5-コレン酸の重酸素標識体を調製し、先と同様にラット脳ミクロソーム画分とインキュベートすることにより、7α-ヒドロキシ-3-オキソ-4-コレン酸が生成することが判明した。さらにその重酸素標識体を調製してラット脳細胞質画分とインキュベートしたところ、ケノデオキシコール酸(CDCA)が経時的に生成することが明らかとなった。以上の結果から、脳内に3β-ヒドロキシ-5-コレン酸が存在すると、それが酵素的にCDCAに変換される可能性が強く示唆された。一方、脳内においてCDCAに結合する蛋白質を特定するため、CDCAを固定化したアガロースゲルを用い、ラット脳細胞質画分中の結合蛋白質の抽出を試みた。得られた蛋白質混合物を二次元ゲル電気泳動法を用いて分離したところ、数種の蛋白質が濃縮されることが判明した。ゲル内消化後、MALDI-TOFMSを用いてそれらの構造を解析したところ、ビタミンD依存性Ca結合蛋白質の他、チューブリンや14-3-3蛋白質などが同定された。

Research Products

(15 results)

All Other

All Publications (15 results)

[Publications] Nariyasu Mano: "Characterization of rat liver bile acid acyl glucuronosyltransferase"Steroids. 67. 257-262 (2002)
[Publications] Takashi Iida: "Potential bile acid metabolites. 24. An efficient synthesis of carboxyl-linked glucosides"Lipids. 37. 101-110 (2002)
[Publications] Norihiro Kobayashi: "A monoclonal antibody-based enzyme-linked immunosorbent assay of glycolithocholic acid sulfate in human urine for liver function test"Steroids. 67. 827-833 (2002)
[Publications] Norihiro Kobayashi: "Single-chain Fv fragments derived from an anti-11-deoxycortisol antibody : Affinity, specificity, and idiotype analysis"Steroids. 67. 733-742 (2002)
[Publications] Nariyasu Mano: "Rapid and simple quantitative assay method for diastereomeric flurbiprofen glucuronides in the incubation mixture"Journal of Chromatography B. 776. 125-131 (2002)
[Publications] Toshiaki Kume: "Isolation and a novel diterpenoid substance with potent neuroprotective activity from fetal calf serum"Proceedings of National Academy of Sciences in USA. 99. 3288-3293 (2002)
[Publications] Nariyasu Mano: "Separation and determination of diastereomeric flurbiprofen acyl glucuronides in human urine by LC/ESI-MS with a simple column-switching technique"Drug Metabolism and Pharmacokinetics. 17. 142-149 (2002)
[Publications] Sookie La: "Enantioseparation of chiral aromatic acids by capillary electrophoresis in neutral and charged cyclodextrin selector modes"Electrophoresis. 23. 4123-4131 (2002)
[Publications] Takashi Iida: "Potential bile acid metabolites. 25. Synthesis and chemical property of stereoisomeric 3α, 7α, 16-and 3α, 7α, 15-trihydroxy-5β-cholan-24-oic acids"Chemical and Pharmaceutical Bulletin. 50. 1335-1340 (2002)
[Publications] Norihiro Kobayashi: "Generation of a novel monoclonal antibody against cortisol-[C-4]-bovine serum albumin conjugate : Application to enzyme-linked immunosorbent assay for urinary and serum cortisol"Analytical Sciences. 18. 1309-1314 (2002)
[Publications] Takashi Iida: "Direct capillary gas chromatographic analysis and thermal stability of carboxy-linked glucosides of bile acids"Chromatographia. 56. 489-494 (2002)
[Publications] Norihiro Kobayashi: "Idiotype-anti-idiotype-based noncompetitive enzyme-linked immunosorbent assay of ursodeoxycholic acid 7-N-acetylglucosaminides in human urine with subfemtomole range sensitivity"Journal of Immunological Methods. 272. 1-10 (2003)
[Publications] Norihiro Kobayashi: "Monoclonal anti-idiotype antibodies recognizing the variable of region a high-affinity antibody against 11-deoxycortisol. Production, characterization and application to a sensitive noncompetitive immunoassay"Journal of Immunological Methods. 274. 63-75 (2003)
[Publications] Takashi Iida: "^1H and ^<13>C signal assignments of carboxyl-linked glucosides of bile acids"Magnetic Resonance in Chemistry. (in press).
[Publications] Takashi Iida: "Functionalization of unactivated carbons in 3α, 6-and 3α, 24-dihydroxy-5β-cholan derivatives by dimethyldioxirane"Lipids. (in press).

2002 Fiscal Year Annual Research Report

脳内胆汁酸の生理的意義解明に関する研究

Principal Investigator

後藤 順一 東北大学, 医学部附属病院, 教授 (80006337)

Research Products

[Publications] Nariyasu Mano: "Characterization of rat liver bile acid acyl glucuronosyltransferase"Steroids. 67. 257-262 (2002)

[Publications] Takashi Iida: "Potential bile acid metabolites. 24. An efficient synthesis of carboxyl-linked glucosides"Lipids. 37. 101-110 (2002)

[Publications] Norihiro Kobayashi: "A monoclonal antibody-based enzyme-linked immunosorbent assay of glycolithocholic acid sulfate in human urine for liver function test"Steroids. 67. 827-833 (2002)

[Publications] Norihiro Kobayashi: "Single-chain Fv fragments derived from an anti-11-deoxycortisol antibody : Affinity, specificity, and idiotype analysis"Steroids. 67. 733-742 (2002)

[Publications] Nariyasu Mano: "Rapid and simple quantitative assay method for diastereomeric flurbiprofen glucuronides in the incubation mixture"Journal of Chromatography B. 776. 125-131 (2002)

[Publications] Toshiaki Kume: "Isolation and a novel diterpenoid substance with potent neuroprotective activity from fetal calf serum"Proceedings of National Academy of Sciences in USA. 99. 3288-3293 (2002)

[Publications] Nariyasu Mano: "Separation and determination of diastereomeric flurbiprofen acyl glucuronides in human urine by LC/ESI-MS with a simple column-switching technique"Drug Metabolism and Pharmacokinetics. 17. 142-149 (2002)

[Publications] Sookie La: "Enantioseparation of chiral aromatic acids by capillary electrophoresis in neutral and charged cyclodextrin selector modes"Electrophoresis. 23. 4123-4131 (2002)

[Publications] Takashi Iida: "Potential bile acid metabolites. 25. Synthesis and chemical property of stereoisomeric 3α, 7α, 16-and 3α, 7α, 15-trihydroxy-5β-cholan-24-oic acids"Chemical and Pharmaceutical Bulletin. 50. 1335-1340 (2002)

[Publications] Norihiro Kobayashi: "Generation of a novel monoclonal antibody against cortisol-[C-4]-bovine serum albumin conjugate : Application to enzyme-linked immunosorbent assay for urinary and serum cortisol"Analytical Sciences. 18. 1309-1314 (2002)

[Publications] Takashi Iida: "Direct capillary gas chromatographic analysis and thermal stability of carboxy-linked glucosides of bile acids"Chromatographia. 56. 489-494 (2002)

[Publications] Norihiro Kobayashi: "Idiotype-anti-idiotype-based noncompetitive enzyme-linked immunosorbent assay of ursodeoxycholic acid 7-N-acetylglucosaminides in human urine with subfemtomole range sensitivity"Journal of Immunological Methods. 272. 1-10 (2003)

[Publications] Norihiro Kobayashi: "Monoclonal anti-idiotype antibodies recognizing the variable of region a high-affinity antibody against 11-deoxycortisol. Production, characterization and application to a sensitive noncompetitive immunoassay"Journal of Immunological Methods. 274. 63-75 (2003)

[Publications] Takashi Iida: "^1H and ^<13>C signal assignments of carboxyl-linked glucosides of bile acids"Magnetic Resonance in Chemistry. (in press).

[Publications] Takashi Iida: "Functionalization of unactivated carbons in 3α, 6-and 3α, 24-dihydroxy-5β-cholan derivatives by dimethyldioxirane"Lipids. (in press).

後藤順一東北大学, 医学部附属病院, 教授 (80006337)