Document Detail


Specific and sensitive enzymatic measurement of sphingomyelin in cultured cells.
MedLine Citation:
PMID:  22727956     Owner:  NLM     Status:  Publisher    
Abstract/OtherAbstract:
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 (0-10μM) and high (10-100μM) concentrations, respectively, and the detection limit was 0.5μM (5pmol 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 α significantly decreased the SM content. This specific and sensitive method for measuring SM will be helpful in studying various cellular processes.
Authors:
Shin-Ya Morita; Kuriko Soda; Reiko Teraoka; Shuji Kitagawa; Tomohiro Terada
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Publication Detail:
Type:  JOURNAL ARTICLE     Date:  2012-6-19
Journal Detail:
Title:  Chemistry and physics of lipids     Volume:  -     ISSN:  1873-2941     ISO Abbreviation:  -     Publication Date:  2012 Jun 
Date Detail:
Created Date:  2012-6-25     Completed Date:  -     Revised Date:  -    
Medline Journal Info:
Nlm Unique ID:  0067206     Medline TA:  Chem Phys Lipids     Country:  -    
Other Details:
Languages:  ENG     Pagination:  -     Citation Subset:  -    
Copyright Information:
Copyright © 2012. Published by Elsevier Ireland Ltd.
Affiliation:
Department of Pharmacy, Shiga University of Medical Science Hospital, Otsu City, Shiga 520-2192, Japan.
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