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

作者：Jacques Bodennec, Dori Pelled, Anthony H. Futerman · 发表于：Journal of Lipid Research · 年份：2003 · DOI：10.1194/jlr.d200026-jlr200 · 被引用次数：22 · 研究领域：Sphingolipid Metabolism and Signaling、Lysosomal Storage Disorders Research、Lipid Membrane Structure and Behavior

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-(3)H]ceramide and Glc[4,5-(3)H]sphingosine accumulation in metabolically-labeled neurons treated with an inhibitor of lysosomal glucocerebrosidase. Accurate quantification of both lipids was obtained with Glc[4,5-(3)H]ceramide and Glc[4,5-(3)H]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 an...