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Development and Application of High-performance Liquid Chromatography/Chemiluminescence Determination of Biological Thiols

Research Project

Project/Area Number 63571023
Research Category

Grant-in-Aid for General Scientific Research (C)

Allocation TypeSingle-year Grants
Research Field Physical pharmacy
Research InstitutionNagasaki University

Principal Investigator

NAKASHIMA Kenichiro  Nagasaki University, School of Pharmaceutical Sciences, Associate Professor, 薬学部, 助教授 (30039656)

Co-Investigator(Kenkyū-buntansha) AKIYAMA Shuzo  Nagasaki University, School of Pharmaceutical Sciences, Professor, 薬学部, 教授 (50028148)
Project Period (FY) 1988 – 1989
Project Status Completed (Fiscal Year 1989)
Budget Amount *help
¥2,000,000 (Direct Cost: ¥2,000,000)
Fiscal Year 1989: ¥1,000,000 (Direct Cost: ¥1,000,000)
Fiscal Year 1988: ¥1,000,000 (Direct Cost: ¥1,000,000)
KeywordsBiological thiols / High-performance liquid chromatography / Chemiluminescence detection / Aryl oxalate / Lipoperoxide / Malondialdehyde / Peroxyoxalate chemiluminescence / 生体チオ-ル / 生体チオール / 化学発光 / 高速液体クロマトグラフィー / アリールオキザレート / ピリミドピリミジン誘導体
Research Abstract

The results obtained in this study are follows:
1. A high-performance liquid chromatography (HPLC)-chemiluminescence (CL) determination method of biological thiols using N-[4-(6-dimethylamino-2-benzofuranyl)phenyl]maleimide (DBPM) as a fluorogenic label has been developed and applied to the assay of reduced glutathione and cysteine in rat liver. The method was very sensitive and could detect fmol levels of thiols. Next, another HPLC-CL method using 4-fluoro-7-nitrobenzo-2,1,3-oxadiazole (NBD-F) as a label was developed. However, the sensitivity of the method was lower and pmol level. In the course of this study, it was found that CL detection was not always sensitive than fluorescence detection for some thiols. It might be caused by the suitabilities of the chemiluminogenic reagents used.
2. To evaluate oxalates and fluorescers as peroxyoxalate CL reagents, a simple and rapid method by flow system has been developed. By the method, newly synthesized aryl oxalates and fluorescent compounds were evaluated and found some useful compounds as CL reagents. The method should be suitable for the selection of HPLC-peroxyoxalate CL reagents.
3. By the method of 2, CL efficiencies of condensates of malondialdehyde (MDA) with thiobarbituric acids were determined. As the result, MDA-1,3-diethyl-2-thiobarbituric acid (DETBA) condensate showed the largest CL efficiency and could be detect as low as 10 fmol. This suggests that DETBA would be an excellent derivatization reagent for MDA. By using DETBA, a high sensitive assay method for lipoperoxide (MDA) should be developed.

Report

(3 results)
  • 1989 Annual Research Report   Final Research Report Summary
  • 1988 Annual Research Report
  • Research Products

    (25 results)

All Other

All Publications (25 results)

  • [Publications] Kenichiro Nakashima: "Determination of D-Penicillamine in Serum by High-performance Liquid Chromatography Using N-(4-(6- Dimethylamino-2-benzofuranyl)phenyl)maleimide" Jap.J.Clin.Chem.17. 51-54 (1988)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1989 Final Research Report Summary
  • [Publications] Kenichiro Nakashima: "High-performance liquid Chromatography/Chemilumiluminescence Determination of Biological Thiols with N-(4-(6-Dimethyl-amino-2-benzofuranyl)phenyl)maleimide" Biomed.Chromatogr.3. 39-42 (1989)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1989 Final Research Report Summary
  • [Publications] W.R.G.Baeyens: "Development of Chemiluminescence in Biomedical Analysis" J.Pharm.Biomed.Anal.7. 407-412 (1989)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1989 Final Research Report Summary
  • [Publications] Kenichiro Nakashima: "Synthesis and Evaluation of Oxalates As Peroxyoxalate Chemiluminescence Reagents" Analyst. 114. 1413-1416 (1989)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1989 Final Research Report Summary
  • [Publications] Kenichiro Nakashima: "Synthesis of Pyrimido(5、4-d)pyrimidine Derivatives and Their Ultraviolet Absorption and Fluorescence Spectral Properties" Dyes and Pigments. 12. 21-26 (1990)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1989 Final Research Report Summary
  • [Publications] Kenichiro Nakashima: "Evaluation of Pyrimido(5、4-d)pyrimidine Derivatives As Peroxyoxalate Chemiluminescence Reagents Using Flow In jection System" Biomed.Chromatogr.(1990)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1989 Final Research Report Summary
  • [Publications] 中島憲一郎: "生物発光と化学発光、基礎と実験(今井一洋編集)" 廣川書店(東京), 32 (1989)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1989 Final Research Report Summary
  • [Publications] R.S.Givens: "ACS Symposium Series 383(Chapter 8);Peroxyoxalate Chemiluminescence Reaction; A Versatile Method for Activation of Fluorophore Luminescence" American Chemical Society, 28 (1989)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1989 Final Research Report Summary
  • [Publications] K. Nakashima: "Determination of D-Penicillamine in Serum by High-performance Liquid Chromatography Using N-[4-(6-Dimethylamino-2-benzofuranyl)phenyl]maleimide" Jap. J. Clin. Chem.17(2). 51-54 (1988)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1989 Final Research Report Summary
  • [Publications] K. Nakashima: "High-performance Liquid Chromatography/Chemiluminescence Determination of Biological Thiols with N-[4-(6-Dimethylamino-2-benzofuranyl)phenyl]maleimide" Biomed. Chromatogr., 3(1), 39-42, 1989.

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1989 Final Research Report Summary
  • [Publications] W.R.G.Baeyens: "Development of Chemiluminescence in Biomedical Analysis" J. Pharm. Biomed. Anal. 7(4), 407-412, 1989.

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1989 Final Research Report Summary
  • [Publications] K. Nakashima: "Synthesis and Evaluation of Oxalates As Peroxyoxalate Chemiluminescence Reagents" Analyst, 114(11) 1413-1416, 1989.

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1989 Final Research Report Summary
  • [Publications] K. Nakashima: "Synthesis of Pyrimido[5, 4-d]pyrimidine Derivatives and Their Ultraviolet Absorption and Fluorescence Spectral Properties" Dyes and Pigments 12, 21-26, 1990.

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1989 Final Research Report Summary
  • [Publications] K. Nakashima: "Evaluation of Pyrimido[5, 4-d]pyrimidine Derivatives As Peroxyoxalate Chemiluminescence Reagents Using Flow Injection System" Biomed. Chromatogr., 1990.

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1989 Final Research Report Summary
  • [Publications] K. Nakashima: Kagakuhakko (Chapter 2). Seibutuhakko to Kagakuhakko (Ed. by K. Imai, Hirokawa Shoten (Japan), 75-108, 1989,

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1989 Final Research Report Summary
  • [Publications] R. S. Givens: Peroxyoxalate Chemiluminescence Reaction: A Versatile Method for Activation of Fluorophore Luminescence. ACS Symposium Series 383, Chapter 8, 127-154, 1989,

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1989 Final Research Report Summary
  • [Publications] Kenichiro Nakashima: "Synthesis and Evaluation of Aryl Oxalates As Peroxyoxalate Chemiluminescence Reagents" Analyst. 114. 1413-1416 (1989)

    • Related Report
      1989 Annual Research Report
  • [Publications] W.R.G.Baeyens: "Development of Chemiluminescence in Biomedical Analysis" J.Pharm.Biomed.Anal.7. 407-412 (1989)

    • Related Report
      1989 Annual Research Report
  • [Publications] Kenichiro Nakashima: "Synthesis of Pyrimido(5,4-d)pyrimidine Derivatives and Their Ultraviolet Absorption and Fluorescence Spectral Properties" Dyes and Pigments. 12. 21-26 (1990)

    • Related Report
      1989 Annual Research Report
  • [Publications] Kenichiro Nakashima: "Evaluation of Pyrimido(5,4-d)pyrimidine Derivatives As Peroxyoxalate Chemiluminescence Reagents Using Flow Injection System" Biomed.Chromatogr.(1990)

    • Related Report
      1989 Annual Research Report
  • [Publications] Kenichiro Nakashima: Jap.J.Clin.Chem.17. 51-54 (1988)

    • Related Report
      1988 Annual Research Report
  • [Publications] Kenichiro Nakashima: Biomedical Chromatography. (1989)

    • Related Report
      1988 Annual Research Report
  • [Publications] Kenichiro Nakashima: Dyes and Pigments. (1989)

    • Related Report
      1988 Annual Research Report
  • [Publications] 中島憲一郎: "生物発光と化学発光" 廣川書店, 77-108 (1989)

    • Related Report
      1988 Annual Research Report
  • [Publications] Richard S.Givens: "Luminescence Applications in Biological,Chemical,Environmental,and Hydrological Sciences" American Chemical Society, 127-154 (1989)

    • Related Report
      1988 Annual Research Report

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Published: 1988-04-01   Modified: 2016-04-21  

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