Aminopropyl solid phase extraction and two-dimensional thin layer chromatography of neutral glycosphingolipids and neutral lyso-glycosphingolipids

Jacques Bodennec, Dori Pelled, and Anthony H. Futerman

Biol. Chem., Weizmann Institute, Rehovot 76-100

Corresponding Author: tony.futerman{at}weizmann.ac.il

Methods for isolation of neutral lyso-glycosphingolipids (n-lyso-GSLs) such as glucosylsphingosine and psychosine (galactosylsphingosine) normally involve mild alkaline or acid hydrolysis to degrade glycerolipids followed by multiple chromatography steps, yielding relatively low recoveries of the n-lyso-GSLs and the parent neutral glycosphingolipids (n-GSLs). We now describe a new technique for isolating and characterizing these compounds using one chromatography step, resulting in quantitative recovery of n-GSLs and n-lyso-GSLs in the same fraction with high yields and without significant contamination. Lipids are extracted using a modified Folch procedure in which recovery is optimized by re-extracting the Folch upper phase with water-saturated butanol. This lipid extract is then applied, without any further chemical steps, to an aminopropyl solid phase column, from which both n-GSLs and n-lyso-GSLs are eluted in the same fraction over a wide range of concentrations and with high yield. Separation of n-GSLs and n-lyso-GSLs is achieved using a new 2D-TLC 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 hippocampal 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. Thus, this simple and rapid technique can be used for the simultaneous analysis of lyso-GSLs and GSLs in the same biological tissue, which may permit determination of their metabolic pathways in normal and in pathological tissues, such as those taken from Gaucher and Krabbe disease patients.

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