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Analysis of C-3 epimerization mechanism in vitamin D_3 to develop a new osteoporosis drug

Research Project

Project/Area Number 14572030
Research Category

Grant-in-Aid for Scientific Research (C)

Allocation TypeSingle-year Grants
Section一般
Research Field Physical pharmacy
Research InstitutionKanazawa University

Principal Investigator

HIGASHI Tatsuya  Kanazawa University, Graduate School of Natural Science and Technology, Associate Professor, 自然科学研究科, 助教授 (90272963)

Project Period (FY) 2002 – 2004
Project Status Completed (Fiscal Year 2004)
Budget Amount *help
¥3,400,000 (Direct Cost: ¥3,400,000)
Fiscal Year 2004: ¥1,000,000 (Direct Cost: ¥1,000,000)
Fiscal Year 2003: ¥1,000,000 (Direct Cost: ¥1,000,000)
Fiscal Year 2002: ¥1,400,000 (Direct Cost: ¥1,400,000)
Keywordsvitamin D_3 / osteoporosis / C-3 epimerization / LC / MS / derivatization / hydroxysteroid dehydrogenase / in vitro assay for bone metabolism / ultramicro analysis / 24,25-ジヒドロキシビタミンD_3 / キンギョウロコ / 骨芽細胞 / 破骨細胞 / 電子捕獲APCI-MS / ヒドロキシステロイド脱水素酵素 / 中間体
Research Abstract

First, highly sensitive LC/MS methods for analysis of vitamin D_3 drug and metabolites were developed. Cookson-type reagents, which quantitatively react with vitamin D_3 compounds to form the Diets-Alder adducts, were employed to enhance their detection responses in APCI-MS. Pharmacokinetic study of 1α-hydroxyvitamin D_3 in humans was carried out by the LC/MS method combined with derivatization with a proton-affinitive Cookson-type reagent, DMEQTAD. Using this method, urinary vitamin D_3 metabolites in human under physiological conditions were also detected and characterized. Next, an electron-affinitive Cookson-type reagent, NPTAD, was applied to the assay of 25-hydroxyvitamin D_3 in human plasma using LC/electron capture APCI-MS. This method was superior to the commercially available RIA in sensitivity and sample throughput. Furthermore, a new derivatization reagent having the boronic acid as the reacting group was developed for the assay of 24,25-dihydroxyvitamin D_3 [24,25(OH)_2D_3].
Second, studies on the C-3 epimerization in 24,25(OH)_2D_3 were performed. In consequence, we found the 3-epimer[3-epi-24,25(OH)_2D_3] is formed from 24,25(OH)_2D_3 by the catalysis of 3α-and β-hydroxysteroid dehydrogenase (HSD) and the HSD-catalyzed epimerization is reversible. In addition, 3-oxo-form was also isolated from the reaction mixture, which strongly suggested that the formation of the 3-epimer by HSD involves a dehydrogenation and reduction processes.
In third study, osteoclastic and osteoblastic activities of some vitamin D_3 compounds were examined using a culture system of the goldfish scale. It was observed that 3-epi-24,25(OH)_2D_3 activated osteoblastic cell at 10^<-8>M, which was different from the effects of 24,25(OH)_2D_3 and active vitamin D_3, 1,25-dihydroxyvitamin D_3, in bone metabolism.

Report

(4 results)
  • 2004 Annual Research Report   Final Research Report Summary
  • 2003 Annual Research Report
  • 2002 Annual Research Report
  • Research Products

    (23 results)

All 2004 2003 2002 Other

All Journal Article (16 results) Publications (7 results)

  • [Journal Article] 電子捕獲大気圧化学イオン化を利用する生理活性ステロイドの高速液体クロマトグラフィー/質量分析2004

    • Author(s)
      東 達也
    • Journal Title

      分析化学 53(7)

      Pages: 645-655

    • NAID

      110002905410

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2004 Annual Research Report 2004 Final Research Report Summary
  • [Journal Article] Analysis of C-3 epimerization in (24R)-24,25-dihydroxyvitamin D_3 catalyzed by hydroxysteroid dehydrogenase2004

    • Author(s)
      Tatsuya Higashi
    • Journal Title

      Journal of Pharmaceutical and Biomedical Analysis 36(3)

      Pages: 429-436

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2004 Annual Research Report 2004 Final Research Report Summary
  • [Journal Article] High-performance liquid chromatography/electron capture atmospheric pressure chemical ionization-mass spectrometric determination of biologically active steroids2004

    • Author(s)
      Tatsuya Higashi
    • Journal Title

      Bunseki Kagaku 53-7

      Pages: 645-655

    • NAID

      110002905410

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2004 Final Research Report Summary
  • [Journal Article] Analysis of C-3 epimerization in (24R)-24,25-dihydroxyvitamin D_3 catalyzed by hydroxysteroid dehydrogenase2004

    • Author(s)
      Tatsuya Higashi
    • Journal Title

      Journal of Pharmaceutical and Biomedical Analysis 36-3

      Pages: 429-436

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2004 Final Research Report Summary
  • [Journal Article] Development of analyses of biological steroids using chromatography -special reference to vitamin D compounds and neurosteroids-2003

    • Author(s)
      Kazutake Shimada
    • Journal Title

      Chromatography 24(1)

      Pages: 1-6

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2004 Final Research Report Summary
  • [Journal Article] Application of 4-(4-nitrophenyl)-1,2,4-triazoline-3,5-dione to analysis of 25-hydroxyvitamin D_3 in human plasma by liquid chromatography/electron capture atmospheric pressure chemical ionization-mass spectrometry2003

    • Author(s)
      Tatsuya Higashi
    • Journal Title

      Analytical Sciences 19(6)

      Pages: 941-943

    • NAID

      80015993364

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2004 Final Research Report Summary
  • [Journal Article] Development of analyses of biological steroids using chromatography -special reference to vitamin D compounds and neurosteroids-2003

    • Author(s)
      Kazutake Shimada
    • Journal Title

      Chromatography 24-1

      Pages: 1-6

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2004 Final Research Report Summary
  • [Journal Article] Application of 4-(4-nitrophenyl)-1,2,4-triazoline-3,5-dione to analysis of 25-hydroxyvitamin D_3 in human plasma by liquid chromatography/electron capture atmospheric pressure chemical ionization-mass spectrometry2003

    • Author(s)
      Tatsuya Higashi
    • Journal Title

      Analytical Sciences 19-6

      Pages: 941-943

    • NAID

      80015993364

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2004 Final Research Report Summary
  • [Journal Article] Liquid chromatography-mass spectrometric method combined with derivatization for determination of 1α-hydroxyvitamin D_3 in human plasma2002

    • Author(s)
      Tatsuya Higashi
    • Journal Title

      Journal of Chromatography B 772(2)

      Pages: 229-238

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2004 Final Research Report Summary
  • [Journal Article] Characterization of urinary metabolites of vitamin D_3 in man under physiological conditions using liquid chromatography-tandem mass spectrometry2002

    • Author(s)
      Tatsuya Higashi
    • Journal Title

      Journal of Pharmaceutical and Biomedical. Analysis 29(5)

      Pages: 947-955

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2004 Final Research Report Summary
  • [Journal Article] Cookson型誘導体化試薬を用いるビタミンDの高速液体クロマトグラフィー/質量分析2002

    • Author(s)
      島田和武
    • Journal Title

      分析化学 51(7)

      Pages: 487-493

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2004 Final Research Report Summary
  • [Journal Article] Electron-capturing derivatization of neutral steroids for increasing sensitivity in liquid chromatography/negative atmospheric pressure chemical ionization-mass spectrometry2002

    • Author(s)
      Tatsuya Higashi
    • Journal Title

      Analytical Science 18(12)

      Pages: 1301-1307

    • NAID

      10011309621

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2004 Final Research Report Summary
  • [Journal Article] Liquid chromatography-mass spectrometric method combined with derivatization for determination of 1α-hydroxyvitamin D_3 in human plasma2002

    • Author(s)
      Tatsuya Higashi
    • Journal Title

      Journal of Chromatography B 772-2

      Pages: 229-238

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2004 Final Research Report Summary
  • [Journal Article] Characterization of urinary metabolites of vitamin D_3 in man under physiological conditions using liquid chromatography-tandem mass spectrometry2002

    • Author(s)
      Tatsuya Higashi
    • Journal Title

      Journal of Pharmaceutical and Biomedical Analysis 29-5

      Pages: 947-955

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2004 Final Research Report Summary
  • [Journal Article] High-performance liquid chromatography/mass spectrometry of vitamin D compounds employing derivatization with Cookson-type reagents2002

    • Author(s)
      Kazutake Shimada
    • Journal Title

      Bunseki Kagaku 51-7

      Pages: 487-493

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2004 Final Research Report Summary
  • [Journal Article] Electron-capturing derivatization of neutral steroids for increasing sensitivity in liquid chromatography/negative atmospheric pressure chemical ionization-mass spectrometry2002

    • Author(s)
      Tatsuya Higashi
    • Journal Title

      Analytical Sciences 18-12

      Pages: 1301-1307

    • NAID

      10011309621

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2004 Final Research Report Summary
  • [Publications] Tatsuya Higashi: "Application of 4-(4-nitrophenyl)-1,2,4-triazoline-3,5-dione to analysis of 25-hydroxyvitamin D_3 in human plasma by liquid chromatography/electron capture atmospheric pressure chemical ionization-mass spectrometry"Analytical Sciences. 19(7). 941-943 (2003)

    • Related Report
      2003 Annual Research Report
  • [Publications] Kazutake Shimada: "Development of analyses of biological steroids using chromatography -Special reference to vitamin D compounds and neurosteroids-"Chromatography. 24(1). 1-6 (2003)

    • Related Report
      2003 Annual Research Report
  • [Publications] 東 達也: "LC/MSへの誘導体化の導入と生体内超微量ステロイド分析への応用"金沢大学機器分析センターNEWS. 2. 8-12 (2003)

    • Related Report
      2003 Annual Research Report
  • [Publications] Tatsuya Higashi: "Liquid chromatography-mass spectrometric method combined with derivatization for determination of 1α-hydroxyvitamin D_3 in human plasma"Journal of Chromatography B. 772. 229-238 (2002)

    • Related Report
      2002 Annual Research Report
  • [Publications] Tatsuya Higashi: "Characterization of urinary metabolites of vitamin D_3 in man under physiological conditions using liquid chromatography-tandem mass spectrometry"Journal of Pharmaceutical Biomedical Analysis. 29. 947-955 (2002)

    • Related Report
      2002 Annual Research Report
  • [Publications] 島田和武: "Cookson型誘導体化試薬を用いるビタミンDの高速液体クロマトグラフィー/質量分析"分析化学. 51. 487-493 (2002)

    • Related Report
      2002 Annual Research Report
  • [Publications] Tatsuya Higashi: "Electron-capturing derivatization of neutral steroids for increasing sensitivity in liquid chromatography/negative atmospheric pressure chemical ionization-mass spectrometry"Analytical Sciences. 18. 1301-1307 (2002)

    • Related Report
      2002 Annual Research Report

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Published: 2002-04-01   Modified: 2016-04-21  

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