期刊
CHEMISTRY AND PHYSICS OF LIPIDS
卷 165, 期 5, 页码 571-576出版社
ELSEVIER IRELAND LTD
DOI: 10.1016/j.chemphyslip.2012.06.003
关键词
Enzymatic measurement; Sphingomyelin; Sphingomyelinase; Cell density
资金
- Japanese Ministry of Education, Culture, Sports, Science, and Technology [22790053]
- Takeda Science Foundation
- Grants-in-Aid for Scientific Research [23659077, 22390029, 22790053] Funding Source: KAKEN
Sphingomyelin (SM) is the most abundant sphingolipid in mammalian cell membranes and plays multiple physiological roles. In this study, we improved the sensitivity of the enzymatic measurement of SM and validated its specificity and accuracy. The enzymatic reaction sequence of the method involves the hydrolysis of SM by sphingomyelinase, dephosphorylation of phosphorylcholine, oxidation of choline, and reaction of hydrogen peroxide with Amplex Red. The calibration curve was shown to be quadratic and linear at low (0-10 mu M) and high (10-100 mu M) concentrations, respectively, and the detection limit was 0.5 mu M (5 pmol in the reaction mixture), which was more sensitive than all other SM assays reported previously. This SM measurement using Triton X-100 detected only SM, but not other choline-containing phospholipids, sphingosylphosphocholine, phosphatidylcholine, and lysophosphatidylcholine, and quantified SM regardless of the length and double bonds of the acyl chain. By using this method, we demonstrated that an increase in the density of HEK293 cells was accompanied by an elevation in the cellular content of SM, and that the treatment of HEK293 cells with tumor necrosis factor a significantly decreased the SM content. This specific and sensitive met hod for measuring SM will be helpful in studying various cellular processes. (C) 2012 Elsevier Ireland Ltd. All rights reserved.
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