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Establishment of Direct detection of crosslinks of collagen and elastin in human connective tissues using single-column high performance liquid chromatography.

Research Project

Project/Area Number 12671406
Research Category

Grant-in-Aid for Scientific Research (C)

Allocation TypeSingle-year Grants
Section一般
Research Field Orthopaedic surgery
Research InstitutionHamamatsu University School of Medicine

Principal Investigator

TAKANASHI Masaaki  Hamamatsu University School of Medicine, Lecturer, 医学部・附属病院, 助手 (40211346)

Co-Investigator(Kenkyū-buntansha) NAGANO Akira  Hamamatsu University School of Medicine, Professor, 医学部, 教授 (50272547)
HOSINO Hironobu  Hamamatsu University School of Medicine, Lecturer, 医学部・附属病院, 助手 (70293636)
Project Period (FY) 2000 – 2001
Project Status Completed (Fiscal Year 2001)
Budget Amount *help
¥1,700,000 (Direct Cost: ¥1,700,000)
Fiscal Year 2001: ¥800,000 (Direct Cost: ¥800,000)
Fiscal Year 2000: ¥900,000 (Direct Cost: ¥900,000)
Keywordscrosslinks / collagen / elastin / yellow ligament / high performance liquid chromatography / pyridinoline / desmosin / connective tissues / HPLC / advanced glycation endproducts
Research Abstract

Collagen and elastin are recognized as two major connective tissue proteins of human connective tissues. In both collagen and elastin there are many kinds of intra- or inter-molecular crosslinks. Pyridinoline (Pyr) and deoxypyridinoline (Dpyr) are mature crosslinks which maintain the structure of the collagen fibril. Desmosine (Des) and isodesmosine (Isodes) represent the major crosslinking components of elastin. Pentosidine (Pen), which is a senescent crosslink and one of the advanced glycation endproducts, accumulates with age in tissue proteins including collagen. We developed a direct and one-injection HPLC method to measure Pyr, Dpyr, Des, Isodes and Pen in the hydrolysate of human yellow ligament. This method used one column and two detectors. Recovery rates of Pyr, Dpyr, Pen, Des and Isodes were 86.4-98.3%, 83.6-96.8%, 78.7-95.6%, 83.6-97.9% and 85.6-99.3% respectively. The intraassay coefficients of variation for Pyr, Dpyr, Pen, Des and Isodes were 3.7%, 4.1%, 5.4%, 4.5%, and 4 … More .7% respectively. Linear regression analysis showed the linearity (r=0. 99, p=0. 0001) of calibration line for each Pyr, Dpyr, Pen, Des and Isodes. Using this HPLC method, we further tried to measure semi-quantitatively the content of collagen and elastin in connective tissues. Part of the connective tissues such as cortical bone, cancellous bone, cartilage, vertebral disc, meniscus, ligament, tendon, joint capsule, synovium, nerve, and skin were obtained. After hydrolysis of the samples, we measured crosslinks of collagen and elastin such as pyridinoline, deoxypyridinoline, desmosine, isodesmosine by HPLC at once. For calculating the contents of collagen and elastin, we estimated that 1 mole of collagen contains 0.2 moles of pyridinoline in bone ; 1.5 moles in cartilage, meniscus and disc, 0.4 moles in capsule, tendon, synovium, ligament and nerve, and 1 mole of elastin contains 3 moles of desmosine. In the ligament, capsule, synovium, and nerve, both collagen and elastin crosslinks were detected and thus the contents of collagen and elastin were semiquantified. The elastin crosslinks were not measured in the bone, cartilage, meniscus, disc, or tendon. The collagen crosslinks were not measured in the skin. Capsule had more collagen and less elastin, and the ligament had more elastin and less collagen. In the capsule, an old subject had the greater proportion of collagen to elastin than a young subject. We believe that this semi-quntitative HPLC method is useful for the investigation of the connective tissues which have significant amount of collagen and elastin. Less

Report

(3 results)
  • 2001 Annual Research Report   Final Research Report Summary
  • 2000 Annual Research Report
  • Research Products

    (7 results)

All Other

All Publications (7 results)

  • [Publications] Chen JR, Takahashi M, et al.: "Direct detection of crosslinks of collagen and elastin in the hydrolysates of human yellow ligament using single-column high performance liquid chromatography"Anal Biochem. 278. 99-105 (2000)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2001 Final Research Report Summary
  • [Publications] 高橋正哲, ほか: "各種結合組織のコラーゲンとエラスチン濃度の高速液体クロマトグラフィー半定量法による検討"日本整形外科学会雑誌. 75・8. S1071 (2001)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2001 Final Research Report Summary
  • [Publications] Jin Ran Chen, Masaaki Takahashi, Kazuhiro Kushida, Motohiro Suzuki, Kazutaka Suzuki, Kentaro Horiuchi, and Akira Nagano: "Direct detection of crosslinks of collagen and elastm in the hydrolysates of human yellow ligament using single column high performance liquid chromatography"Anal Biochem. 278. 99-105 (2000)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2001 Final Research Report Summary
  • [Publications] Masaaki Takahashi, et al.: "Semi-quantification of collagen and elastin in the connective tissue by high performance liquid chromatography. (Japanese)"NIPPON SEIKEIGEKAGAKKAI ZASSHI. 75-8. S1071 (2001)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2001 Final Research Report Summary
  • [Publications] Chen JR, Takahashi M, et al.: "Direct detection of crosslinks of collagen and elastin in the hydrolysates of human yellow ligament using single-column high performance liquid chromatography"Anal Biochem. 278. 99-105 (2000)

    • Related Report
      2001 Annual Research Report
  • [Publications] 高橋正哲, ほか: "各種結合組織のコラーゲンとエラスチン濃度の高速液体クロマトグラフィー半定量法による検討"日本整形外科学会雑誌. 75・8. S1071 (2001)

    • Related Report
      2001 Annual Research Report
  • [Publications] Chen JR,Takahashi M, et al.: "Direct detection of crosslinks of collagen and elastin in the hydrolysates of human yellow ligament using single-column high performance liquid chromatography"Analytical Biochemistry. 278. 99-105 (2000)

    • Related Report
      2000 Annual Research Report

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Published: 2000-04-01   Modified: 2016-04-21  

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