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Amino Acids

, Volume 6, Issue 1, pp 57–63 | Cite as

Rapid determination of desmosine and isodesmosine in tissue hydrolysates by isocratic high performance liquid chromatography and precolumn derivatization

  • P. Charpiot
  • R. Calaf
  • C. Chareyre
  • P. H. Rolland
  • D. Garçon
Article

Summary

A rapid and sensitive isocratic high performance liquid chromatographic method has been developed for the single and specific determination of low concentrations of desmosine (Des) and isodesmosine (Ide), the major specific crosslink aminoacids in elastin.

Samples of isolated elastin or whole tissue were hydrolysed in 6N HCl, and the hydrolysates were prefractionated on cellulose CF1. Des, Ide,γ-glutamyl-glutamic acid as internal standard were dansylated and derivatives were extracted from reaction mixture by ethylacetate. Their separation on a Lichrosphere 100-NH2 column, using methanol-water as mobile phase containing acetic acid and 0.25 M sodium acetate, final pH 6.5, was followed by fluorescence detection (340–510 nm). The overall reproducibility was 5.9% for Des and 5.0% for Ide. The limits of detection were 2.2 pmol and 2.5 pmol, respectively. The method was successfully applied for the determination of Des and Ide in normal pig aortas.

Keywords

Amino acids Elastin crosslinks Desmosine High-performance liquid chromatography Isocratic 

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References

  1. Bayer E, Grom E, Kaltenegger B, Uhmann R (1976) Separation of amino acids by high performance liquid chromatography. Anal Chem 48: 1106–1109Google Scholar
  2. Faris B, Ferrera R, Glembourtt P, Mogayzel PJ, Crombie G, Franzblau C (1981) Rapid quantitation of desmosine content in tissue hydrolysates by high-performance liquid chromatography. Anal Biochem 114: 71–74Google Scholar
  3. Gray WR (1972) End group analysis using Dansyl chloride. Methods Enzymol. 25: 121–138Google Scholar
  4. Gunja-Smith Z (1985) An enzyme-linked immunosorbent assay to quantitate the elastin crosslink desmosine in tissue and urine samples. Anal Biochem 147: 258–264Google Scholar
  5. King GS, Mohan VS, Starcher BC (1980) Radioimmunoassay for desmosine. Conn Tissue Res 7: 263–267Google Scholar
  6. Lansing AI, Rosenthal TB, Alex M, Dempsey EW (1952) The structure and chemical characterization of elastic fibers as revealed by elastase and electron microscopy. Anat Rec 144: 555Google Scholar
  7. Lunte SM, Mohabbat T, Wong OS, Kuwana T (1989) Determination of desmosine, isodesmosine, and other amino acids by liquid chromatography with electrochemical detection following precolumn derivatization with naphtalenedialdehyde/cyanide. Anal Biochem 178: 202–207Google Scholar
  8. Negro A, Garbisa S, Gotte L, Spina M (1987) The use of reverse-phase high performance liquid chromatography and precolumn derivatization with Dansyl chlorid for quantitation of specific aminoacids in collagen and elastin. Anal Biochem 160: 39–46Google Scholar
  9. Sandberg LW, Soskel NT, Leslie JG (1981) Elastin structure, biosynthesis, and relation to disease states. N Engl J Med 304: 566–579Google Scholar
  10. Schwartz E, Cruickshank FA, Lebwohl M (1990) Determination of desmosine in elastinrelated skin disorders by isocratic high-performance liquid chromatography. Exp Mol Pathol 52: 63–68Google Scholar
  11. Seiler N (1970) Use of the dansyl reaction in biochemical analysis. Methods Biochem Anal 18: 259–337Google Scholar
  12. Skinner SJM (1982) Rapid method for the purification of the elastin crosslinks, desmosine and isodesmosine. J Chromatogr 229: 200–204Google Scholar
  13. Soskel NT (1986) High performance liquid chromatographic quantitation of desmosine plus isodesmosine in elastin and whole tissue hydrolysates. Anal Biochem 160: 98–104Google Scholar
  14. Tapuhi Y, Schmidt DE, Lindner W, Karger BL (1981) Dansylation of amino acids for high-performance liquid chromatography analysis. Anal Biochem 115: 123–129Google Scholar
  15. Tapuhi Y, Miller N, Karger BL (1980) Practical considerations in the chiral separation of Dns-amino acids by reversed-phase liquid chromatography using metal chelate additives. J Chromatogr 205: 325–337Google Scholar
  16. Yamaguchi Y, Haginata J, Kunitomo M, Yasuda H, Bando Y (1987) High-performance liquid chromatographic determination of desmosine and isodesmosine in tissues and its application to studies of alteration of elastin induced by atherosclerosis. J Chromatogr 422: 53–59Google Scholar
  17. Zarkadas CG, Zarkadas GC, Karatzas CN, Khalili AD, Nguyen Q (1986) Rapid method for determinating desmosine, isodesmosine, 5-hydroxylysine, tryptophan, lysinoalanine and the amino sugars in proteins and tissues. J Chromatogr 378: 67–76Google Scholar

Copyright information

© Springer-Verlag 1994

Authors and Affiliations

  • P. Charpiot
    • 1
  • R. Calaf
    • 1
  • C. Chareyre
    • 1
  • P. H. Rolland
    • 1
  • D. Garçon
    • 1
  1. 1.School of PharmacyLaboratory of Biological Chemistry (PC, RC, CC, DG) and INSERM (PHR)Marseille Cedex 5France

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