组织神经节苷脂的简易快速薄层分离纯化法

组织神经节苷脂(Gls)传统的分析方法,是用薄层层析,在分析之前要先进行操作步骤繁杂的分离纯化。Harth等于1978年报告用组织粗提液直接点样于层析薄板,再以三种不同的溶剂系统展开的方法,得清晰的薄板层析图谱。它们的展开剂为:1.氯仿;2.氯仿-甲醇-水(70:30:4,V/V/V);3.氯仿-甲醇-0.25%KCl(60:35:8,V/V/V)。此法简便,但未能推广采用。根据我们的经验它未能推广的原因是粗提液颜色较深,对唾液酸测定干扰甚大,薄板上的点样量较难掌握。我们用硅胶G层析薄板(5×20厘米),将组织的氯仿-甲醇粗提液直接点样于其上,以Harth的前两种展开剂展开后,用问苯二酚显色,参考定位,刮下含Gls部分硅胶,经洗脱、离心、氮气吹干后,溶于C:M(1:1),以唾液酸含量为参数,点样于高效层析(HPTLC)薄板上,在氯仿-甲醇-0.15%CaCl_2(60:40:9,V/V/V)展开,可得良好的图谱。这一改良可去掉呈色物质,大大减少HPTLC板的用量,降低了成本,同时本法未经Folch分配,有可能获得比传统方法更为真实的结果。

RAPID AND SIMPLE THIN-LAYER CHROMATOGRAPHIC SEPARATION AND PURIFICATION OF GANGLIOSIDES IN TISSUES

The classic method for determining gangliosides (Gls) species in tissues is thin-layer chromatogrphy, in which complex Preliminary separation and purification of Gls. mixtures are necessary. In 1978, Harth et al published a direct thin-layer chromatography system for separating an aliquot of the total lipid extract. The three successive solvent systems used are: A, chloroform; B, chloroform-methanol-water (C:M:W, 70:30:4 V/V/V);C, Chloroform-methanol-0.25% KC1 (60:35:8, V/V/V). Satisfactory chromatogram has been obtained by this rapid and simple procedure, but it has not been popularized. Why is it not popularized? We think it is because the deep color of the crude extract seriously disturbs the determination of sialic acid (SA). In addition, it is difficult to control the volume of the sample applied onto the thin Plate. We adopted a silica gel coated Plate with crude tissue lipid extract in chloroform-methanol directly applied to it. The plate is developed with Harth's solvent A and B, and located with resorcinol-HCl reagent. The scraped silica gel containing Gls was eluted with methanol and chloroform-methanol mixtures respectively. The eluents are centrifugalized and dried with flow of nitrogen. The residue thus obtained is redissolved in C:M(1:1)and applied on the high-performance thin-layer chromatography (HPTLC) plate with SA concentration as parameter. The plate was developed with the solvent C:M:0.15% CaCl_2 (60:40:9, V/V/V),thus Gls were well separated from each other as well as from the other lipids. The improved method has the advantage of removing the interference caused by colored materials and sparing the HPTLC Plates used in comparison with the Harth's method. The procedure has omitted Folch's partition, and thus results nearer to the natural state could be obtained more readily than the classic method.