Endotoxin pp 203-213 | Cite as

Development of a New Quantitative Method for Detection of Endotoxin by Fluorescence Labeling of 3-Hydroxy Fatty Acid

  • K. Tanamoto
Part of the Advances in Experimental Medicine and Biology book series (AEMB, volume 256)


New quantitative method for the detection of minute amounts of endotoxin has been developed using 3-hydroxytetradecanoic acid as a chemical marker. After converting 3-hydroxyteatradecanoic acid to methyl ester, it was coupled with a fluorescent probe, anthracene-9-carboxyl chloride, obtained by chlorization of 9-anthroic acid with oxalyl chloride. The resulting ester was isolated by HPLC on silica column. The purified product, methyl-3-0-(9-carboxy-anthracenyl) tetradecanoate (M/Z 462), was highly responsive to a fluorescence spectrophotometer, showing maximum emission with excitation wavelength at 257 nm and emission wavelength at 458 nm in dichloromethane, the limit of detection being as little as 10 f mol. Using this method it is currently possible to detect Salmonella abortus equi endotoxin in aqueous solution at a level of 100 pg.


High Performance Liquid Chromatography Oxalyl Chloride Anhydrous Dichloromethane Synthetic Lipid Limulus Amoebocyte Lysate Assay 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. 1.
    Blair, E., Wise, A., and Mackay, A.G., 1969. Gram-negative bacteremic shock. J. Am. Med. Assoc. 207: 333.Google Scholar
  2. 2.
    Cheim, S., Chen, C., Dellenback, J., Usami, S. and Gregerson, M. I., 1966, Hemodynamic changes in endotoxin shock. Am. J. Physiol. 210: 1401.Google Scholar
  3. 3.
    Freudenberg, M. and Galanos, C., 1985, Alteration in rats in vivo of the chemical structure of lipopolysaccharide from Salmonella abortus equi. Eur. J. Biochem. 152: 353.Google Scholar
  4. 4.
    Galanos, C., Lehmann, V., Lüderitz, O., Rietschel, E. T., Westphal, 0., Brade, H., Brade, L., Freudenberg, M. A., Hansen-Hagge, T., Lüderitz, T., McKenzie, G., Shade, U., Strittmatter, W., Tanamoto, K., Zähringer, U., Imoto, M., Yoshimura, H., Yamamoto, M., Shimamoto, T., Kusumoto, S., and Shiba, T., 1984, Endotoxic properties of chemically synthesized lipid A part structures. Eur. J. Biochem. 140: 221.Google Scholar
  5. 5.
    Goto, J., Goto, N., Shamsa, F., Saito, M., Komatsu, S., Suzaki, K., and Nambara, T., 1983, New sensitive derivatization of hydrosteroids for high-performance liquid chromatography with fluorescence detection. Analitica Chimica Acta 147: 397.CrossRefGoogle Scholar
  6. 6.
    Hall, C. L. and Munford, R. S., 1983, Enzymatic deacylation of the lipid A moiety of Salmonella typhimurium lipopolysaccharides by human neutrophils. Proc. Natl. Acad. Sci. 80: 6671.Google Scholar
  7. 7.
    Homma, J. Y., Matsuura, M., Kanegasaki, S., Kawakubo, Y., Shibukawa, N., Kumazawa, Y., Yamamoto, A., Tanamoto, K., Yasuda, T., Imoto, M., Yoshimura, H., Kusumoto, S., and Shiba, T., 1985, Structural requirement of lipid A responsible for the function: A study with chemically synthesized lipid A and its analogues. J. Biochem. 98: 395.Google Scholar
  8. 8.
    Imoto, M., Yoshimura, H., Yamamoto, M., Shimamoto, T., Kusumoto, S., and Shiba, T., 1984, Chemical synthesis of phosphorylated tetraacyl disaccharide corresponding to a biosynthetic precursor of lipid A. Tetrahedron Lett. 25: 2667.CrossRefGoogle Scholar
  9. 9.
    Imoto, M., Yoshimura, H., Sakaguchi, N., Kusumoto, S., and Shiba, T., 1985, Total synthesis of Escherichia coli lipid A. Tetrahedron Lett. 26: 1545.CrossRefGoogle Scholar
  10. 10.
    Iwanaga, S., Morita, T., Harada, T., Nakamura, S., Niwa, N., Tanaka, K., Kimura, T., and Sakakibara, S., 1978, Chromogenic substrates for horseshoe crab clotting enzyme. Its application for the assay of bacterial endotoxins. Hameostasis 7: 183.Google Scholar
  11. 11.
    Kakinuma, A., Asano, T., Torii, and Sugini, Y., 1981, Gelation of Limulus amoebocyte lysate by an antitumor (1–3)- ß-D-glucan. Biochem. Biophys. Res. Commun. 101: 434.CrossRefGoogle Scholar
  12. 12.
    Kanagasaki, S., Tanamoto, K., Yasuda, T., Homma, J. Y., Matsuura, M. Nakatsuka, M., Kumazawa, Y., Yamamoto, A., Shiba, T., Kusumoto, S., Imoto, M., Yoshimura, H., and Shimamoto, T., 1986, Structure-activity relationship of lipid A: Comparison of biological activities of natural and synthetic lipid A’s with different fatty acid compositions. J. Biochem. 99: 1203.Google Scholar
  13. 13.
    Levin, J., Tomasulo, P. A., and Oser, R. S., 1970, Detecton of endotoxin in human blood and demonstration of an inhibitor. J. Lab. Clin. Med. 75: 903.Google Scholar
  14. 14.
    Luderitz, 0., Freudenberg, M., Galanos, C., Lehmann, V., Rietschel, E. T., and Show,D. W., 1982, Lipopolysaccharides of gram-negative bacteria. in: “Microbial membrane lipids,” Vol. 17, pp 79–151, C. Razin and S. Rottem, eds. Academic Press, London. New York.Google Scholar
  15. 15.
    Morita, T., Tanaka, S., Nakamura, T., and Iwanaga, S., 1981, A new (13)- ß -D-glucan mediates coagulation pathway found in Limulus amoebocytes. FEBS Lett. 129: 318.CrossRefGoogle Scholar
  16. 16.
    Maitra, S. K., Schotz, M. C., Yoshikawa, T. T., and Guze, A. B., 1978, Determination of lipid A and endotoxin in serum by mass spectroscopy. Proc. Natl. Acad. Sci. 8: 3993.Google Scholar
  17. 17.
    Peterson, A. A. and Munford, R. S., 1987, Dephosphorylation of the lipid A moiety of Escherichia coli Lipopolysaccharides by mouse macrophages. Infect. Immun. 55: 974.Google Scholar
  18. 18.
    Rietschel, E. T., Brade, H., Brade, L., Brandeburg, K., Shade, U., Seydel, U., Zähringer, U., Galanos, C., Lüderitz, O., Westphal, O. Labischinski, H., Kusumoto, S., and Shiba, T., 1987, Lipid A, the endotoxic center of bacterial Lipopolysaccharides: Relation of chemical structure to biological activity. in: Progr. Clin. Biol. Res. Vol. 231, pp 25–53. Alan R. Liss, Inc. New Yor.Google Scholar
  19. 19.
    Soneson, A., Larsson, L., Westerdahl, G., and Odham, G., 1987, Determination of endotoxins by gas chromatography: Evaluation of electron-capture and negative-ion chemical-ionization mass spectrometric detection of halogenated derivates of ß - hydroxymyristic acid. J. Chromatography 417: 11.Google Scholar
  20. 20.
    Takagi, K., Moriya, A., Tamura, H., Nakahara, C., Tanaka. S., Fujita, Y., and Kawai, T., 1981, Quantitative measurement of endotoxin in human blood using synthetic chromogenic substrate for horseshoe crab clotting enzyme. A comparison of methods of blood sampling and treatment. Thromb. Res. 23: 51.Google Scholar
  21. 21.
    Tanamoto, K., Zähringer, U., McKenzie, G. R. Galanos, C., Rietschel, E. T., Lúderitz, O., Kusumoto, S. and Shiba, T., 1984, Biological activities of synthetic lipid A analogues: Pyrogenicity, lethal toxicity, anticomplemental activity, and induction of gelation of Liiulus amoebocyte lysate. Infect. Immun. 44: 421.Google Scholar
  22. 22.
    Ulevitch, R. J., 1985, Interaction of bacterial lipopolysaccharide and plasma high density lipoproteins, in: Handbook of Endotoxin. L. J. Berry, ed., Vol. 3, pp. 372–388, Elsevier, Amsterdam.Google Scholar

Copyright information

© Springer Science+Business Media New York 1990

Authors and Affiliations

  • K. Tanamoto
    • 1
  1. 1.National Institute of Hygienic SciencesSetagayaku, Tokyo 158Japan

Personalised recommendations