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Journal of Lipid Research, Vol. 44, 1413-1419, July 2003
Copyright © 2003 by American Society for Biochemistry and Molecular Biology

Methods

Simultaneous quantification of lyso-neutral glycosphingolipids and neutral glycosphingolipids by N-acetylation with [³H]acetic anhydride

Jacques Bodennec, Selena Trajkovic-Bodennec and Anthony H. Futerman¹

Department of Biological Chemistry, Weizmann Institute of Science, 76100, Rehovot, Israel

¹ To whom correspondence should be addressed. e-mail: tony.futerman{at}weizmann.ac.il

We describe a new method that permits quantification in the pmol to nmol range of three lyso-neutral glycosphingolipids (lyso-n-GSLs), glucosylsphingosine (GlcSph), galactosylsphingosine (GalSph), and lactosylsphingosine, in the same sample as neutral glycosphingolipids (n-GSLs). Lyso-n-GSLs and n-GSLs are initially obtained from a crude lipid extract using Sephadex G25 chromatography, followed by their isolation in one fraction, which is devoid of other contaminating lipids, by aminopropyl solid-phase chromatography. Lyso-n-GSLs and n-GSLs are subsequently separated from one another by weak cation exchange chromatography. N-GSLs are then deacylated by strong alkaline hydrolysis, and the N-deacylated-GSLs and lyso-n-GSLs are subsequently N-acetylated using [³H]acetic anhydride. An optimal concentration of 5 mM acetic anhydride was established, which gave >95% N-acetylation. We demonstrate the usefulness of this technique by showing an 40-fold increase of both GlcSph and glucosylceramide in brain tissue from a glucocerebrosidase-deficient mouse, as well as significant lactosylceramide accumulation.

The application and optimization of this technique for lyso-n-GSLs and lyso-GSLs will permit their quantification in small amounts of biological tissues, particularly in the GSL storage diseases, such as Gaucher and Krabbe's disease, in which GlcSph and GalSph, respectively, accumulate.

Abbreviations: C2-N-GSL, N-acetyl-GSL; GalSph, galactosylsphingosine; GlcSph, glucosylsphingosine; GSL, glycosphingolipid; LacCer, lactosylceramide; LacSph, lactosylsphingosine; lyso-n-GSL, lyso-neutral glycosphingolipid; n-GSL, neutral glycosphingolipid

Supplementary key words glucosylceramide • galactosylceramide • lactosylceramide • Gaucher disease • Krabbe's disease

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