Coupled assay of sphingomyelin and ceramide molecular species by gas liquid chromatography.
Coupled assay of sphingomyelin and ceramide molecular species by gas liquid chromatography.
复制标题
通过气相色谱法耦合测定鞘磷脂和神经酰胺分子种类。
作者：
C. Vieu;F. Tercé;F. Chevy;C. Rolland;R. Barbaras;H. Chap;C. Wolf;B. Perret;X. Collet
本研究报告了一个单步分析的内源性神经酰胺的分子种类和神经酰胺部分的鞘磷脂在生物样品中，使用气液色谱法（GLC）。甲硅烷基化鞘磷脂定量转化为单甲硅烷基化神经酰胺注射到GLC后，而游离神经酰胺二甲硅烷基化的伯和仲醇功能，如质谱法所证实。单甲硅烷基化衍生物和二甲硅烷基化衍生物之间保留时间的可重现偏移使得能够同时定量多种鞘磷脂和神经酰胺分子种类。首先通过对脂质提取物进行温和的碱性处理来去除重叠的二酰基甘油。最低检测限（5 pmol）不允许鉴定人血浆中的游离神经酰胺，但对源自鞘磷脂的17种神经酰胺分子种类进行了定量，从NC 16：0到NC 24：1。相比之下，在HEPG 2和中国仓鼠卵巢（CHO）细胞中定量了三种主要的游离神经酰胺（NC 16：0、NC 24：0和NC 24：1）。通过C6-神经酰胺诱导CHO细胞凋亡后，我们可以观察到C6-神经酰胺的消失，其部分转化为C6-鞘磷脂，以及NC 16：0神经酰胺的显著增加。因此，我们的方法代表了一个独特的程序，同时分析鞘磷脂和神经酰胺分子种类，能够监测生物样品中不同池的变化。
This study reports a single-step analysis of the molecular species of endogenous ceramides and of the ceramide moiety of sphingomyelins in biological samples, using gas liquid chromatography (GLC). Silylated sphingomyelins were quantitatively converted to monosilylated ceramide upon injection into GLC, whereas the free ceramides were di-silylated on the primary and secondary alcohol function, as confirmed by mass spectrometry. The reproducible shift of the retention times between the mono- and di-silylated derivatives enables simultaneous quantification of the variety of sphingomyelin and ceramide molecular species. Overlapping diacylglycerols were first removed by a mild alkaline treatment of the lipid extract. The lowest detection limit (5 pmol) did not allow for identification of free ceramides in human plasma, but 17 molecular species of ceramides derived from sphingomyelins were quantified, from NC16:0 up to NC24:1. By contrast, three major free ceramides (NC16:0, NC24:0, and NC24:1) were quantified in HEPG2 and Chinese hamster ovary (CHO) cells. Upon induction of apoptosis in CHO cells by C6-ceramide, we could follow the disappearance of the C6-ceramide, its partial conversion to C6-sphingomyelin, and the prominent increase of NC16:0 ceramide. Thus, our method represents a unique procedure of simultaneous analysis of sphingomyelin and ceramide molecular species able to monitor the variation of the different pools in biological samples.
DOI：
10.1007/s13361-017-1829-2
发表时间：
2018-01-01
影响因子：
3.2
作者：
Gallego, Sandra F.;Hojlund, Kurt;Ejsing, Christer S.
通讯作者：
Ejsing, Christer S.