He Xingxuan, Dagan Arie, Gatt Shimon, Schuchman Edward H
Department of Human Genetics, Mount Sinai School of Medicine, New York, NY 10029, USA.
Anal Biochem. 2005 May 1;340(1):113-22. doi: 10.1016/j.ab.2005.01.058.
Ceramide and sphingosine are sphingolipids with important functional and structural roles in cells. In this paper we report a new enzyme-based method to simultaneously quantify the levels of ceramide and sphingosine in biological samples. This method utilizes purified human recombinant acid ceramidase to completely hydrolyze ceramide to sphingosine, followed by derivatization of the latter with naphthalene-2,3-dialdehyde (NDA) and quantification by reverse-phase high-performance liquid chromatography. The limits of detection for sphingosine-NDA and ceramidase-derived sphingosine-NDA were 9.6 and 12.3 fmol, respectively, and the limits of quantification were 34.2 and 45.7 fmol, respectively. The recovery of sphingosine and ceramide standards quantified by this assay were between 95.6 and 104.6%. The relative standard deviations for the intra- and interday sphingosine assay were 2.1 and 4.5%, respectively, and those for the ceramide assay were 3.3 and 4.1%, respectively. To validate this procedure, we quantified ceramide and sphingosine in mouse plasma, white blood cells, and hemoglobin, the first reported time that the amounts of these lipids have been documented in individual blood components. We also used this technique to evaluate the ability of a novel ceramide analog, AD2646, to inhibit the hydrolytic activity of acid ceramidase. The results demonstrate that this new procedure can provide sensitive, reproducible, and simultaneous ceramide and sphingosine quantification. The technique also may be used for determining the activity and inhibition of ceramidases and may be adapted for quantifying sphingomyelin and sphingosine-1-phosphate levels. In the future it could be an important tool for investigators studying the role of ceramide/sphingosine metabolism in signal transduction, cell growth and differentiation, and cancer pathogenesis and treatment.
神经酰胺和鞘氨醇是鞘脂类物质，在细胞中具有重要的功能和结构作用。在本文中，我们报告了一种基于酶的新方法，用于同时定量生物样品中神经酰胺和鞘氨醇的水平。该方法利用纯化的人重组酸性神经酰胺酶将神经酰胺完全水解为鞘氨醇，然后用萘 - 2,3 - 二醛（NDA）对后者进行衍生化，并通过反相高效液相色谱进行定量。鞘氨醇 - NDA和神经酰胺酶衍生的鞘氨醇 - NDA的检测限分别为9.6和12.3飞摩尔，定量限分别为34.2和45.7飞摩尔。通过该测定法定量的鞘氨醇和神经酰胺标准品的回收率在95.6%至104.6%之间。鞘氨醇测定的日内和日间相对标准偏差分别为2.1%和4.5%，神经酰胺测定的相对标准偏差分别为3.3%和4.1%。为了验证该方法，我们定量了小鼠血浆、白细胞和血红蛋白中的神经酰胺和鞘氨醇，这是首次报道在个体血液成分中记录这些脂质的含量。我们还使用该技术评估了一种新型神经酰胺类似物AD2646抑制酸性神经酰胺酶水解活性的能力。结果表明，这种新方法可以提供灵敏、可重复且同时定量神经酰胺和鞘氨醇的能力。该技术还可用于测定神经酰胺酶的活性和抑制作用，并可适用于定量鞘磷脂和鞘氨醇 - 1 - 磷酸的水平。未来，它可能成为研究人员研究神经酰胺/鞘氨醇代谢在信号转导、细胞生长和分化以及癌症发病机制和治疗中的作用的重要工具。
