Low nanogram range quantitation of diglycerides and ceramide by high-performance liquid chromatography.
Low nanogram range quantitation of diglycerides and ceramide by high-performance liquid chromatography.
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通过高效液相色谱法对甘油二酯和神经酰胺进行低纳克范围定量。
DOI：
10.1006/abio.1996.0014
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发表时间：
1996
影响因子：
2.9
通讯作者：
S. Capitani
中科院分区：
文献类型：
--
作者：
M. Previati;L. Bertolaso;M. Tramarin;V. Bertagnolo;S. Capitani
建立了神经酰胺（CER）和甘油二酯（DG）的正相高效液相色谱分离测定方法。使用4-二甲基氨基吡啶和N，N '-二环己基碳二亚胺作为催化剂，将神经酰胺和二氢甘油的游离oxydril基团偶联到荧光标记（+）-6-甲氧基-α-甲基-2-萘乙酸（NAP）的羧基上。使用不含NAP的酸代替卤化物活化的形式确保了试剂的更高稳定性、更低的反应温度以及改进的产率和再现性。在-20 ℃下反应3小时后，反应产率大于90%。在HPLC分离结束时回收了超过85%的起始材料。CER的检测下限低于5 ng，DG的检测下限低于150 ng。在试验所用条件下，甘油三酯、鞘脂或磷脂未发生显著水解，酯化反应不受粗脂质提取物组分的影响。由于不需要分离和/或纯化步骤，因此可以容易且快速地测量CER和DG的细胞水平。
A method for ceramide (CER) and diradylglycerol (DG) determination after normal-phase HPLC separation was developed. The free oxydril group of ceramide and diradylglycerol is coupled to the carboxylic group of the fluorescent label (+)-6-methoxy-alpha-methyl-2 naphthaleneacetic acid (NAP), using as catalytic agents 4-dimethylaminopyridine and N,N'-dicyclohexylcarbodiimide. The use of NAP-free acid instead of the halide-activated form ensures higher stability of the reagent, lower reaction temperatures, and improved yield and reproducibility. The yield of the reaction is greater than 90% after a period of 3 h at the temperature of -20 degrees C. Over 85% of the starting material is recovered at the end of HPLC separation. The lower detection limit is below 5 ng for CER and 150 ng for DG. Under the conditions employed in the assay, no significant hydrolysis of triglycerides, sphingolipids, or phospholipids occurs and the esterification reaction is not affected by components of crude lipid extracts. Since separation and/or purification steps are not required, cellular levels of CER and DG can be easily and rapidly measured.