Abstract
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 and Krabbe's disease patients.
Highlights
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)
Been difficult to distinguish their functions in vivo from those of their parent neutral glycosphingolipids (n-GSLs) [i.e., galactosylceramide (GalCer) and glucosylceramide (GlcCer)]
When applied to aminopropyl solid phase extraction cartridges, n-lyso-GSLs were recovered in one fraction, namely fraction 3 (Fig. 3A), the same fraction as that in which the parent n-GSLs are eluted [46], with only a small amount of GalSph and LacSph lost to fraction 4
Summary
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). 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. The most common method of isolating n-lyso-GSLs is by strong cation exchange chromatography, followed by desalting on a C18 reverse phase column [25], a method recently adapted to commercially available solid phase extraction cartridges [22].
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