Aminopropyl solid phase extraction and 2 D TLC of neutral glycosphingolipids and neutral lysoglycosphingolipids
Aminopropyl solid phase extraction and 2 D TLC of neutral glycosphingolipids and neutral lysoglycosphingolipids
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DOI：
10.1194/jlr.d200026-jlr200
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发表时间：
2003-01-01
影响因子：
6.5
通讯作者：
Futerman, AH
中科院分区：
文献类型：
--
作者：
Bodennec, J;Pelled, D;Futerman, AH
中性溶酶体糖鞘脂（n - lyso - GSLs）如葡糖基鞘氨醇和半乳糖基鞘氨醇的分离方法通常包括温和的碱水解或酸水解，随后进行多个色谱步骤，n - lyso - GSLs和中性糖鞘脂（n - GSLs）的回收率相对较低。我们现在描述一种使用一个色谱步骤分离这些化合物的新技术，可实现n - GSLs和n - lyso - GSLs的定量回收。使用改良的Folch法提取脂质，通过用水饱和的丁醇对Folch上层相进行再提取来优化回收率。提取物上样到氨丙基固相柱，n - GSLs和n - lyso - GSLs在同一组分中洗脱。使用一种新的二维薄层色谱法进行分离。通过检测用溶酶体葡糖脑苷脂酶抑制剂处理的代谢标记神经元中[4,5 - H - 3]葡糖基神经酰胺和[4,5 - H - 3]葡糖基鞘氨醇的积累，测试了该技术对生物样品的有效性。对这两种脂质进行了准确的定量，[4,5 - H - 3]葡糖基神经酰胺和[4,5 - H - 3]葡糖基鞘氨醇分别在20 nmol/mg DNA和40 pmol/mg DNA的水平上积累。因此，这种简单快速的技术可用于分析同一组织中的溶酶体糖鞘脂和糖鞘脂，这可能有助于确定它们在正常组织和病理组织（如从戈谢病和克拉伯病患者获取的组织）中的代谢途径。
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-H-3]ceramide and Glc[4,5-H-3]sphingosine accumulation in metabolically-labeled neurons treated with an inhibitor of lysosomal glucocerebrosidase. Accurate quantification of both lipids was obtained with Glc[4,5-H-3]ceramide and Glc[4,5-H-3]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.