Molecular & Cellular Toxicology

The Korean Society of Toxicogenomics and Toxicoproteomics (대한독성유전 단백체학회)
권호 표지

N-oleoyl-D-erythro-sphingosine-based Analysis of Ceramide by High Performance Liquid Chromatography and Its Application to Determination in Diverse Biological Samples

  • Lee, Youn-Sun (College of Pharmacy and CBITRC, Chungbuk National University) ;
  • Choi, Heon-Kyo (College of Pharmacy and CBITRC, Chungbuk National University) ;
  • Yoo, Jae-Myung (College of Pharmacy and CBITRC, Chungbuk National University) ;
  • Choi, Kyong-Mi (College of Pharmacy and CBITRC, Chungbuk National University) ;
  • Lee, Yong-Moon (College of Pharmacy and CBITRC, Chungbuk National University) ;
  • Oh, Sei-Kwan (Department of Neuroscience, College of Medicine, Ewha Womans University) ;
  • Kim, Tack-Joong (College of Pharmacy and CBITRC, Chungbuk National University) ;
  • Yun, Yeo-Pyo (College of Pharmacy and CBITRC, Chungbuk National University) ;
  • Hong, Jin-Tae (College of Pharmacy and CBITRC, Chungbuk National University) ;
  • Okino, Nozomu (Department of Bioscience and Biotechnology, Graduate School of Bioresource and Bioenvironmental Sciences, Kyushu University) ;
  • Ito, Makoto (Department of Bioscience and Biotechnology, Graduate School of Bioresource and Bioenvironmental Sciences, Kyushu University) ;
  • Yoo, Hwan-Soo (College of Pharmacy and CBITRC, Chungbuk National University)
  • Published : 2007.12.31
Download PDF
⟨ Previous Next ⟩

Abstract

Ceramide is involved in cell death as a lipid mediator of stress responses. In this study, we developed an improved method of ceramide quantification based on added synthetic ceramide and thin layer chromatography (TLC) separation, and applied to biological samples. Lipids were extracted from samples spiked with N-oleoyl-D-erythro-sphingosine ($C_{17}$ ceramide) as an internal standard. Ceramide was resolved by TLC, complexed with fatty-acidfree bovine serum albumin (BSA), and deacylated by ceramidase (CDase). The released sphingosine was derivatized with o-phthalaldehyde (OPA) and measured by high performance liquid chromatography (HPLC). The limit of detection for ceramide was about 1-2 pmol and the lower limit of quantification was 5 pmol. Ceramide recovery was approximately 86-93%. Ceramide concentrations were determined in biological samples including cultured cells, mouse tissues, and mouse and human plasma. TLC separation of ceramide provides HPLC chromatogram with a clean background without any interfering peaks and the enhanced solubility of ceramide by BSAceramide complex leads to the increased deacylation of ceramide. The use of an internal standard for the determination of ceramide concentration in these samples provides an accurate and reproducible analytical method, and this method can be applicable to diverse biological samples.

Keywords

References

  1. Obeid, L. M., Linardic, C. M., Karolak, L. A. & Hannun, Y. A. Programmed cell death induced by ceramide. Science 259:1769-1771 (1993) https://doi.org/10.1126/science.8456305
  2. Hannun, Y. A. & Luberto, C. Ceramide in the eukaryotic stress response. Trends Cell Biol 10:73-80 (2000) https://doi.org/10.1016/S0962-8924(99)01694-3
  3. Lu, F. G. & Wong, C. S. Radiation-induced apoptosis of oligodendrocytes and its association with increased ceramide and down-regulated protein kinase B/Akt activity. Int J Radiat Biol 80:39-51 (2004) https://doi.org/10.1080/09553000310001642876
  4. Yu, M. U. et al. Altered de novo sphingolipid biosynthesis is involved in the serum deprivation-induced cell death in LLC-PK1 cells. J Toxicol Environ Health A 67:2085-2094 (2004) https://doi.org/10.1080/15287390490515065
  5. Colombaioni, L. et al. Serum deprivation increases ceramide levels and induces apoptosis in undif-ferentiated HN9.10e cells. Neurochem Int 40:327-336 (2002) https://doi.org/10.1016/S0197-0186(01)00090-0
  6. Kok, J. W. & Sietsma, H. Sphingolipid metabolism enzymes as targets for anticancer therapy. Curr Drug Targets 5:375-382 (2004) https://doi.org/10.2174/1389450043345452
  7. Perry, D. K., Bielawska, A. & Hannun, Y. A. Quantitative determination of ceramide using diglyceride kinase. Methods Enzymol 312:22-31 (2000) https://doi.org/10.1016/S0076-6879(00)12897-6
  8. Liebisch, G. et al. Quantitative measurement of different ceramide species from crude cellular extracts by electrospray ionization tandem mass spectrometry (ESI-MS/MS). J Lipid Res 40:1539-1546 (1999)
  9. Merrill, A. H. Jr. et al. Sphingolipidomics: highthroughput, structure-specific, and quantitative analysis of sphingolipids by liquid chromatography tandem mass spectrometry. Methods 36:207-224 (2005) https://doi.org/10.1016/j.ymeth.2005.01.009
  10. He, X., Dagan, A., Gatt, S. & Schuchman, E. H. Simultaneous quantitative analysis of ceramide and sphingosine in mouse blood by naphthalene-2, 3- dicarboxyaldehyde derivatization after hydrolysis with ceramidase. Anal Biochem 340:113-122 (2005) https://doi.org/10.1016/j.ab.2005.01.058
  11. Satoi, H. et al. Astroglial expression of ceramide in Alzheimer's disease brains: a role during neuronal apoptosis. Neuroscience 130:657-666 (2005) https://doi.org/10.1016/j.neuroscience.2004.08.056
  12. Cutler, R. G. et al. Involvement of oxidative stressinduced abnormalities in ceramide and cholesterol metabolism in brain aging and Alzheimer's disease. Proc Natl Acad Sci USA 101:2070-2075 (2004)
  13. Kolesnick, R. The therapeutic potential of modulating the ceramide/sphingomyelin pathway. J Clin Invest 110:3-8 (2002) https://doi.org/10.1172/JCI0216127
  14. Bielawska, A., Perry, D. K. & Hannun, Y. A. Determination of ceramides and diglycerides by the diglyceride kinase assay. Anal Biochem 298:141-150 (2001) https://doi.org/10.1006/abio.2001.5342
  15. Van Veldhoven, P. P., Bishop, W. R., Yurivich, D. A. & Bell, R. M. Ceramide quantitation: evaluation of a mixed micellar assay using E. coli diacylglycerol kinase. Biochem Mol Biol Int 36:21-30 (1995)
  16. Groener, J. E. et al. HPLC for simultaneous quantification of total ceramide, glucosylceramide, and ceramide trihexoside concentrations in plasma. Clin Chem 53:742-747 (2007) https://doi.org/10.1373/clinchem.2006.079012
  17. Previati, M. et al. Low nanogram range quantitation of diglycerides and ceramide by high-performance liquid chromatography. Anal Biochem 233:108-114 (1996) https://doi.org/10.1006/abio.1996.0014
  18. Okino, N. et al. Molecular cloning, sequencing, and expression of the gene encoding alkaline ceramidase from Pseudomonas aeruginosa. Cloning of a ceramidase homologue from Mycobacterium tuberculosis. J Biol Chem 274:36616-36622 (1999) https://doi.org/10.1074/jbc.274.51.36616
  19. Schiffmann, R. et al. Enzyme replacement therapy in Fabry disease: a randomized controlled trial. Jama 285:2743-2749 (2001) https://doi.org/10.1001/jama.285.21.2743
  20. Rahman, P., Gladman, D. D., Wither, J. & Silver, M. D. Coexistence of Fabry's disease and systemic lupus erythematosus. Clin Exp Rheumatol 16:475-478 (1998)
  21. Ullrich, R. et al. Mucosal HIV infection. Pathobiology 66:145-150 (1998) https://doi.org/10.1159/000028012
  22. Ariga, T., Jarvis, W. D. & Yu, R. K. Role of sphingolipid- mediated cell death in neurodegenerative diseases. J Lipid Res 39:1-16 (1998)