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Glycobiology Advance Access published online on May 1, 2009
Glycobiology, doi:10.1093/glycob/cwp047
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© The Author 2009. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oxfordjournals.org

Simultaneous quantification of glucosylceramide and galactosylceramide by normal phase HPLC using O-phtalaldehyde derivatives prepared with sphingolipid ceramide N-deacylase

Kota zama1,5, Yasuhiro Hayashi1,5, Shinya Ito2, Yoshio Hirabayashi3,5, Takehiko Inoue4, Kousaku Ohno4, Nozomu Okino1,5 and Makoto Ito1,5,6

1 Department of Bioscience and Biotechnology, Graduate School of Bioresource and Bioenvironmental Sciences, Kyushu University, Fukuoka 812-8581, Japan
2 Hitachi High-Technologies Corporation, Minato-ku, Tokyo 105-8717, Japan
3 Brain Science Institute, The Institute of Physical and Chemical Research, Wako 351-0918, Japan
4 Department of Child Neurology, Tottori University Faculty of Medicine, Yonago 683-8503, Japan
5 CREST-JST, Chiyoda-ku, Tokyo 102-0075, Japan
6 Bio-Architecture Center, Kyushu University, Fukuoka 812-8581, Japan

Corresponding author: Tel/Fax: +81-92-642-2898, E-mail: makotoi{at}agr.kyushu-u.ac.jp

Received on December 24, 2008; accepted on March 23, 2009

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 NH2 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 µg of protein. Linearity for the determination of each cerebroside was observed from 50 to 400 µg of protein under the conditions used, which corresponds to approximately 103 to 105 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.

Key words: glycosphingolipid / glucosylceramide / galactosylceramide / sphingolipid ceramide N-deacylase / high-performance liquid chromatography / zebrafish


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