Determination of sphingosine kinase activity in biological samples by liquid chromatography–tandem mass spectrometry |
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Authors: | Tian Lan Huichang Bi Suowen Xu Kang Le Zhiyong Xie Yiwei Liu Heqing Huang |
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Affiliation: | 1. Laboratory of Pharmacology and Toxicology, School of Pharmaceutical Sciences, Sun Yat‐sen University, Guangzhou 510006, China;2. Institute of Clinical Pharmacology, School of Pharmaceutical Sciences, Sun Yat‐sen University, Guangzhou 510006, China;3. Laboratory of Pharmaceutical Analysis and Quality Assessment, School of Pharmaceutical Sciences, Sun Yat‐sen University, Guangzhou 510006, China |
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Abstract: | Sphingosine kinase (SphK) is a key enzyme in modulating the levels of sphingosine 1‐phosphate (S1P) as well as an important enzyme in numerous biological responses. Using C17‐sphingosine as a substrate, we established a rapid, sensitive and highly efficient method for determination of SphK activity by analyzing the product C17‐sphingosine 1‐phosphate (C17‐S1P) using liquid chromatography–tandem mass spectrometry. The standard curve for C17‐S1P was linear over a wide range (10–1000 ng/mL) with correlation coefficient (r2) greater than 0.999. The lower limit of quantification for C17‐S1P was 10 ng/mL. The Km values for C17‐sphingosine and ATP were determined to be 28.17 and 188.5 mM, respectively. More importantly, the SphK activity dramatically increased in cultured HEK 293 cells expressing wild‐type SphK1 as well as cells treated with tumor necrosis factor‐a, a sphingosine kinase activator. In contrast, the SphK activity decreased in cultured HEK 293 cells treated with dimethylsphngosine, a sphingosine kinase inhibitor. In conclusion, this method was sensitive and rapid in the determination of SphK acitivity, providing striking utilities in exploring the sphingosine kinase signaling pathway and screening active compounds targeting SphK activity. Copyright © 2010 John Wiley & Sons, Ltd. |
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Keywords: | sphingosine kinase C17‐sphingosine C17‐sphingosine 1‐phosphate sphingosine 1‐phosphate liquid chromatography– tandem mass spectrometry (LC‐MS/MS) |
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