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

Kota Zama, Yasuhiro Hayashi, Shinya Ito, Yoshio Hirabayashi, Takehiko Inoue, Kousaku Ohno, Nozomu Okino, Makoto Ito

Research output: Contribution to journalArticle

19 Citations (Scopus)

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 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.

Original languageEnglish
Pages (from-to)767-775
Number of pages9
JournalGlycobiology
Volume19
Issue number7
DOIs
Publication statusPublished - Jun 8 2009

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Galactosylceramides
Glucosylceramides
High Pressure Liquid Chromatography
Derivatives
ceramide glucosyltransferase
Cerebrosides
Zebrafish
Embryonic Structures
Chemical beam epitaxy
Glucosylceramidase
sphingolipid ceramide N-deacylase
Gaucher Disease
Fibroblasts
Labeling
Cyclosporine
Proteins
Therapeutics

All Science Journal Classification (ASJC) codes

  • Biochemistry

Cite this

Simultaneous quantification of glucosylceramide and galactosylceramide by normal-phase HPLC using O-phtalaldehyde derivatives prepared with sphingolipid ceramide N-deacylase. / Zama, Kota; Hayashi, Yasuhiro; Ito, Shinya; Hirabayashi, Yoshio; Inoue, Takehiko; Ohno, Kousaku; Okino, Nozomu; Ito, Makoto.

In: Glycobiology, Vol. 19, No. 7, 08.06.2009, p. 767-775.

Research output: Contribution to journalArticle

Zama, Kota ; Hayashi, Yasuhiro ; Ito, Shinya ; Hirabayashi, Yoshio ; Inoue, Takehiko ; Ohno, Kousaku ; Okino, Nozomu ; Ito, Makoto. / Simultaneous quantification of glucosylceramide and galactosylceramide by normal-phase HPLC using O-phtalaldehyde derivatives prepared with sphingolipid ceramide N-deacylase. In: Glycobiology. 2009 ; Vol. 19, No. 7. pp. 767-775.
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AU - Ito, Shinya

AU - Hirabayashi, Yoshio

AU - Inoue, Takehiko

AU - Ohno, Kousaku

AU - Okino, Nozomu

AU - Ito, Makoto

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AB - 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.

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