Skip to main content
PMC home page
Biochemical Journal logoLink to Biochemical Journal
. 1986 May 1;235(3):755–761. doi: 10.1042/bj2350755

Determination of gangliosides as 2,4-dinitrophenylhydrazides by high-performance liquid chromatography.

K Miyazaki, N Okamura, Y Kishimoto, Y C Lee
PMCID: PMC1146752  PMID: 3753443

Abstract

A specific, sensitive and easily performed method for the determination of gangliosides in tissue was developed. After removal of water-soluble compounds, total lipids were extracted from tissue and then treated with 2,4-dinitrophenylhydrazine hydrochloride and dicyclohexylcarbodi-imide in dimethylformamide at 0 degrees C to form ganglioside hydrazides. After removal of excess reagents by column chromatography on silicic acid, the ganglioside 2,4-dinitrophenylhydrazides were eluted from the column and analysed by h.p.l.c. with the use of a silica-gel normal-phase column eluted with an isocratic chloroform/methanol/water/acetic acid system. The addition of CaCl2 improved the separation of GM3 ganglioside containing N-acetylneuraminic acid from that containing N-glycollylneuraminic acid. 2,4-Dinitrophenylhydrazide peaks were measured by the absorbance at 342 nm. Quantification of GM3, GM2, GM1, GD1a, GD1b, GT1b and LM1 gangliosides was linear in a range 0.02-1.6 nmol. GM4, GD3, GT1a and GQ1b gangliosides also yielded distinct peaks, although the range of linearity was not examined. This method was applied to the analysis of the total lipids of rat brain and hepatocytes.

Full text

PDF
755

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Ando S., Chang N. C., Yu R. K. High-performance thin-layer chromatography and densitometric determination of brain ganglioside compositions of several species. Anal Biochem. 1978 Sep;89(2):437–450. doi: 10.1016/0003-2697(78)90373-1. [DOI] [PubMed] [Google Scholar]
  2. Bremer E. G., Gross S. K., McCluer R. H. Quantitative analysis of monosialogangliosides by high-performance liquid chromatography of their perbenzoyl derivatives. J Lipid Res. 1979 Nov;20(8):1028–1035. [PubMed] [Google Scholar]
  3. FOLCH J., LEES M., SLOANE STANLEY G. H. A simple method for the isolation and purification of total lipides from animal tissues. J Biol Chem. 1957 May;226(1):497–509. [PubMed] [Google Scholar]
  4. Gazzotti G., Sonnino S., Ghidoni R., Kirschner G., Tettamanti G. Analytical and preparative high-performance liquid chromatography of gangliosides. J Neurosci Res. 1984;12(2-3):179–192. doi: 10.1002/jnr.490120206. [DOI] [PubMed] [Google Scholar]
  5. Kundu S. K., Scott D. D. Rapid separation of gangliosides by high-performance liquid chromatography. J Chromatogr. 1982 Oct 8;232(1):19–27. doi: 10.1016/s0378-4347(00)86003-x. [DOI] [PubMed] [Google Scholar]
  6. Lee W. M., Westrick M. A., Macher B. A. High-performance liquid chromatography of long-chain neutral glycosphingolipids and gangliosides. Biochim Biophys Acta. 1982 Sep 14;712(3):498–504. doi: 10.1016/0005-2760(82)90277-6. [DOI] [PubMed] [Google Scholar]
  7. Nakabayashi H., Iwamori M., Nagai Y. Analysis and quantitation of gangliosides as p-bromophenacyl derivatives by high-performance liquid chromatography. J Biochem. 1984 Oct;96(4):977–984. [PubMed] [Google Scholar]
  8. SVENNERHOLM L. CHROMATOGRAPHIC SEPARATION OF HUMAN BRAIN GANGLIOSIDES. J Neurochem. 1963 Sep;10:613–623. doi: 10.1111/j.1471-4159.1963.tb08933.x. [DOI] [PubMed] [Google Scholar]
  9. SVENNERHOLM L. Quantitative estimation of sialic acids. II. A colorimetric resorcinol-hydrochloric acid method. Biochim Biophys Acta. 1957 Jun;24(3):604–611. doi: 10.1016/0006-3002(57)90254-8. [DOI] [PubMed] [Google Scholar]
  10. Seglen P. O. Preparation of isolated rat liver cells. Methods Cell Biol. 1976;13:29–83. doi: 10.1016/s0091-679x(08)61797-5. [DOI] [PubMed] [Google Scholar]
  11. Seyfried T. N., Ando S., Yu R. K. Isolation and characterization of human liver hematoside. J Lipid Res. 1978 Jul;19(5):538–543. [PubMed] [Google Scholar]
  12. Shigematsu H., Okamura N., Shimeno H., Kishimoto Y., Kan L., Fenselau C. Purification and characterization of the heat-stable factors essential for the conversion of lignoceric acid to cerebronic acid and glutamic acid: identification of N-acetyl-L-aspartic acid. J Neurochem. 1983 Mar;40(3):814–820. doi: 10.1111/j.1471-4159.1983.tb08052.x. [DOI] [PubMed] [Google Scholar]
  13. Shimomura K., Yahara S., Kishimoto Y., Benjamins J. A. Metabolism of cerebrosides and sulfatides in subcellular fractions of developing rat brain. Biochim Biophys Acta. 1984 Sep 12;795(2):265–270. doi: 10.1016/0005-2760(84)90074-2. [DOI] [PubMed] [Google Scholar]
  14. Sonnino S., Ghidoni R., Marchesini S., Tettamanti G. Cytosolic gangliosides: occurrence in calf brain as ganglioside--protein complexes. J Neurochem. 1979 Jul;33(1):117–121. doi: 10.1111/j.1471-4159.1979.tb11713.x. [DOI] [PubMed] [Google Scholar]
  15. Svennerholm L., Fredman P. A procedure for the quantitative isolation of brain gangliosides. Biochim Biophys Acta. 1980 Jan 18;617(1):97–109. doi: 10.1016/0005-2760(80)90227-1. [DOI] [PubMed] [Google Scholar]
  16. Tjaden U. R., Krol J. H., Van Hoeven R. P., Oomen-Meulemans E. P., Emmelot P. High-pressure liquid chromatography of glycosphingolipids (with special reference to gangliosides). J Chromatogr. 1977 Jun 11;136(2):233–243. doi: 10.1016/s0021-9673(00)86275-5. [DOI] [PubMed] [Google Scholar]
  17. Traylor T. D., Koontz D. A., Hogan E. L. High-performance liquid chromatographic resolution of p-nitrobenzyloxyamine derivatives of brain gangliosides. J Chromatogr. 1983 Jan 14;272(1):9–20. doi: 10.1016/s0378-4347(00)86098-3. [DOI] [PubMed] [Google Scholar]
  18. Ullman M. D., McCluer R. H. Quantitative analysis of brain gangliosides by high performance liquid chromatography of their perbenzoyl derivatives. J Lipid Res. 1985 Apr;26(4):501–506. [PubMed] [Google Scholar]
  19. Yahara S., Singh I., Kishimoto Y. Cerebroside and cerebroside III-sulfate in brain cytosol: evidence for their involvement in myelin assembly. Biochim Biophys Acta. 1980 Aug 11;619(2):177–185. doi: 10.1016/0005-2760(80)90066-1. [DOI] [PubMed] [Google Scholar]
  20. Yu R. K., Ledeen R. W. Gas--liquid chromatographic assay of lipid-bound sialic acids: measurement of gangliosides in brain of several species. J Lipid Res. 1970 Nov;11(6):506–516. [PubMed] [Google Scholar]

Articles from Biochemical Journal are provided here courtesy of The Biochemical Society

RESOURCES