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Journal of Lipid Research, Vol. 44, 218-226, January 2003
Copyright © 2003 by Lipid Research, Inc.

Methods

Aminopropyl solid phase extraction and 2 D TLC of neutral glycosphingolipids and neutral lysoglycosphingolipids

Jacques Bodennec, Dori Pelled 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

Methods for isolation of neutral lysoglycosphingolipids (n-lyso-GSLs) such as glucosylsphingosine and galactosylsphingosine normally involve mild alkaline or acid hydrolysis followed by multiple chromatography steps, yielding relatively low recoveries of n-lyso-GSLs and neutral glycosphingolipids (n-GSLs). We now describe a new technique for isolating these compounds using one chromatography step, resulting in quantitative recovery of n-GSLs and n-lyso-GSLs. Lipids are extracted using a modified Folch procedure in which recovery is optimized by reextracting the Folch upper phase with water-saturated butanol. The extract is applied to an aminopropyl solid phase column from which both n-GSLs and n-lyso-GSLs elute in the same fraction. Separation is achieved using a new two-dimensional thin-layer chromatography procedure. The usefulness of this technique for biological samples was tested by examining Glc[4,5-³H]ceramide and Glc[4,5-³H]sphingosine accumulation in metabolically-labeled neurons treated with an inhibitor of lysosomal glucocerebrosidase. Accurate quantification of both lipids was obtained with Glc[4,5-³H]ceramide and Glc[4,5-³H]sphingosine accumulating at levels of 20 nmol/mg DNA and 40 pmol/mg DNA, respectively.

This simple and rapid technique can therefore be used for the analysis of lyso-GSLs and GSLs in the same tissue, which may permit the determination of their metabolic pathways in normal and in pathological tissues, such as those taken from Gaucher and Krabbe's disease patients.

Abbreviations: CBE, conduritol-B-epoxide; CDH, ceramide dihexoside; CTH, ceramide trihexoside; GalCer, galactosylceramide; GlcCer, glucosylceramide; GlcSph, glucosylsphingosine; GalSph, galactosylsphingosine; GSL, glycosphingolipid; HPLC, high performance liquid chromatography; LacSph, lactosylsphingosine; n-GSL, neutral glycosphingolipid; n-lyso-GSL, neutral lysoglycosphingolipid; SM, sphingomyelin; SPC, sphingosylphosphorylcholine; 2D-TLC, two-dimensional thin-layer chromatography

Supplementary key words glucosylsphingosine • psychosine • Gaucher disease • Krabbe's disease • Niemann-Pick disease • two-dimensional thin-layer chromatography

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