Mano N, Oda Y, Yamada K, Asakawa N, Katayama K
Section of Analytical Chemistry, Tsukuba Research Laboratories, Eisar Company Ltd., Ibaraki, Japan.
Anal Biochem. 1997 Jan 15;244(2):291-300. doi: 10.1006/abio.1996.9891.
Sphingolipid metabolites ceramide, sphingomyelin, sphingosine, psycosine, sphingosylphosphorylcholine, and dimethylsphingosine were separated and simulataneously quantitated by liquid chromatography/ionspray ionization tandem mass spectrometry (LC/MS/ MS). The use of glassware throughout minimized losses due to adsorption and the pretreatment of this method consisted of simple liquid-liquid extraction procedure with a mixture of chloroform and methanol. After separation on a short C18 silica column eluted in a gradient mode, the metabolites were detected by MS/ MS. This assay allows simultaneously quantification of these metabolites over a range of at least 0.1 to 100 ng/ 10(6) cells. The LC/MS/MS analyses took 10 to 15 min per sample and we could examine up to 50 samples per day. We also detected endogenous sphingosine 1-phosphate in HL-60 cells. The utility of the method was demonstrated by examining changes in metabolites levels in HL-60 cells after treatment with sphingomyelinase. It was found that sphingomyelinase from Bacillus cereus may have selectivity for acyl chain length.
鞘脂代谢产物神经酰胺、鞘磷脂、鞘氨醇、Psychosine、鞘氨醇磷酸胆碱和二甲基鞘氨醇通过液相色谱/离子喷雾电离串联质谱法（LC/MS/MS）进行分离和同时定量。全程使用玻璃器皿可最大程度减少因吸附造成的损失，该方法的预处理包括用氯仿和甲醇的混合物进行简单的液液萃取程序。在以梯度模式洗脱的短C18硅胶柱上分离后，通过MS/MS检测代谢产物。该测定法可同时在至少0.1至100 ng/10⁶细胞的范围内对这些代谢产物进行定量。每个样品的LC/MS/MS分析耗时10至15分钟，我们每天最多可检测50个样品。我们还在HL-60细胞中检测到内源性鞘氨醇-1-磷酸。通过检测用鞘磷脂酶处理后HL-60细胞中代谢产物水平的变化，证明了该方法的实用性。发现蜡样芽孢杆菌的鞘磷脂酶可能对酰基链长度具有选择性。
