Determination of gangliosides as 2,4-dinitrophenylhydrazides by high-performance liquid chromatography.
Determination of gangliosides as 2,4-dinitrophenylhydrazides by high-performance liquid chromatography.
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高效液相色谱法测定 2,4-二硝基苯酰肼形式的神经节苷脂。
DOI：
10.1042/bj2350755
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发表时间：
1986
期刊：
影响因子：
--
通讯作者：
Lee,YC
中科院分区：
文献类型：
--
作者：
Miyazaki,K;Okamura,N;Kishimoto,Y;Lee,YC
建立了一种特异、灵敏、简便的测定组织中神经节苷脂的方法。去除组织中的水溶性化合物后，提取组织中的总脂，然后在0℃的二甲基甲酰胺中用2，4-二硝基苯肼和二环己基碳二亚胺处理形成神经节苷酰肼。用硅酸柱层析去除多余试剂后，将神经节苷脂2，4-二硝基苯肼洗脱出来，用高效毛细管电泳法进行分析。使用硅胶正相柱，用等度氯仿/甲醇/水/醋酸体系洗脱。CaCl2的加入促进了含N-乙酰神经氨酸的GM3神经节苷脂与含N-乙醇基神经氨酸的GM3神经节苷脂的分离。在342 nm处测定了2，4-二硝基苯肼的吸收峰。神经节苷脂GM3、GM2、GM1、GD1a、GD1b、GT1b和LM1在0.02-1.6nmol范围内呈线性关系。GM4、GD3、GT1a和GQ1b神经节苷脂也有明显的峰，但没有考察线性范围。将该方法应用于大鼠脑和肝细胞总脂的分析。
A specific, sensitive and easily performed method for the determination of gangliosides in tissue was developed. After removal of water-soluble compounds, total lipids were extracted from tissue and then treated with 2,4-dinitrophenylhydrazine hydrochloride and dicyclohexylcarbodi-imide in dimethylformamide at 0 degrees C to form ganglioside hydrazides. After removal of excess reagents by column chromatography on silicic acid, the ganglioside 2,4-dinitrophenylhydrazides were eluted from the column and analysed by h.p.l.c. with the use of a silica-gel normal-phase column eluted with an isocratic chloroform/methanol/water/acetic acid system. The addition of CaCl2 improved the separation of GM3 ganglioside containing N-acetylneuraminic acid from that containing N-glycollylneuraminic acid. 2,4-Dinitrophenylhydrazide peaks were measured by the absorbance at 342 nm. Quantification of GM3, GM2, GM1, GD1a, GD1b, GT1b and LM1 gangliosides was linear in a range 0.02-1.6 nmol. GM4, GD3, GT1a and GQ1b gangliosides also yielded distinct peaks, although the range of linearity was not examined. This method was applied to the analysis of the total lipids of rat brain and hepatocytes.