PUBLICATION
Simultaneous quantification of glucosylceramide and galactosylceramide by normal phase HPLC using O-phtalaldehyde derivatives prepared with sphingolipid ceramide N-deacylase
- Authors
- Zama, K., Hayashi, Y., Ito, S., Hirabayashi, Y., Inoue, T., Ohno, K., Okino, N., and Ito, M.
- ID
- ZDB-PUB-090505-21
- Date
- 2009
- Source
- Glycobiology 19(7): 767-775 (Journal)
- Registered Authors
- Keywords
- glycosphingolipid, glucosylceramide, galactosylceramide, sphingolipid ceramide N-deacylase, high-performance liquid chromatography, zebrafish
- MeSH Terms
-
- Amidohydrolases/metabolism*
- Animals
- Cell Line
- Chromatography, High Pressure Liquid
- Cricetinae
- Cricetulus
- Fibroblasts/chemistry
- Galactosylceramides/analysis*
- Galactosylceramides/chemistry
- Glucosylceramides/analysis*
- Glucosylceramides/chemistry
- Humans
- Nanotechnology
- Time Factors
- Zebrafish/embryology
- o-Phthalaldehyde/analogs & derivatives
- o-Phthalaldehyde/chemistry*
- PubMed
- 19411660 Full text @ Glycobiology
Citation
Zama, K., Hayashi, Y., Ito, S., Hirabayashi, Y., Inoue, T., Ohno, K., Okino, N., and Ito, M. (2009) Simultaneous quantification of glucosylceramide and galactosylceramide by normal phase HPLC using O-phtalaldehyde derivatives prepared with sphingolipid ceramide N-deacylase. Glycobiology. 19(7):767-775.
Abstract
We report here a method of simultaneously quantifying glucosylceramide (GlcCer) and galactosylceramide (GalCer) by normal-phase HPLC using o-phtalaldehyde derivatives. Treatment with sphingolipid ceramide N-deacylase which converts the cerebrosides in the sample to their lyso-forms was followed by the quantitative labeling of free NH(2) groups of the lyso-cerebrosides with o-phtalaldehyde. Using this method, 14.1 pmol of GlcCer and 10.4 pmol of GalCer, and 108.1 pmol of GlcCer and 191.1 pmol of GalCer were detected in zebrafish embryos and RPMI 1864 cells, respectively, while 22.2 pmol of GlcCer but no GalCer was detected in CHOP cells using cell lysate containing 100 mug of protein. Linearity for the determination of each cerebroside was observed from 50 to 400 mug of protein under the conditions used, which corresponds to approximately 10(3) to 10(5) RPMI cells and 5 to 80 zebrafish embryos. The present method clearly revealed that treatment of RPMI cells with a GlcCer synthase inhibitor P4 resulted in a marked decrease in GlcCer but not GalCer, concomitantly with a significant decrease in the GlcCer synthase activity. On the other hand, GlcCer but not GalCer increased two fold when an acid glucocerebrosidase inhibitor CBE was injected into zebrafish embryos. Interestingly, treatment of CHOP cells with ciclosporin A increased GlcCer possibly due to the inhibition of LacCer synthase. A significant increase in levels of GlcCer in fibroblasts from patients with Gaucher disease was clearly shown by the method. The proposed method is useful for the determination of GlcCer and GalCer levels in various biological samples.
Genes / Markers
Expression
Phenotype
Mutations / Transgenics
Human Disease / Model
Sequence Targeting Reagents
Fish
Orthology
Engineered Foreign Genes
Mapping