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Glycobiology Advance Access originally published online on May 1, 2009
Glycobiology 2009 19(7):767-775; 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 Zama2,6, Yasuhiro Hayashi2,6, Shinya Ito3, Yoshio Hirabayashi4,6, Takehiko Inoue5, Kousaku Ohno5, Nozomu Okino2,6 and Makoto Ito1,2,6,7

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

1 To whom correspondence should be addressed: Tel: +81-92-642-2898; Fax: +81-92-642-2898; e-mail: makotoi{at}agr.kyushu-u.ac.jp

Received on December 24, 2008; revised on March 21, 2009; 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 the 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 2-fold when an acid glucocerebrosidase inhibitor CBE was injected into zebrafish embryos. Interestingly, the 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: high-performance liquid chromatography / galactosylceramide / glucosylceramide / glycosphingolipid / sphingolipid ceramide N-deacylase


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