Sphingomyelin synthase activity measurement by the fluorescent product in cell culture medium or animal plasma

doi:10.5246/jcps.2014.04.036

Journal of Chinese Pharmaceutical Sciences ›› 2014, Vol. 23 ›› Issue (4): 256-261.DOI: 10.5246/jcps.2014.04.036

• Original articles • Previous Articles Next Articles

Sphingomyelin synthase activity measurement by the fluorescent product in cell culture medium or animal plasma

Tingbo Ding^*, Jibin Dong, Bin Lou, Xian-Cheng Jiang

1. School of Pharmacy, Fudan University, Shanghai 201203, China
2. Department of Cell Biology, SUNY Downstate Medical Center, Brooklyn, New York 11203, USA

Received:2013-12-13 Revised:2014-01-26 Online:2014-04-28 Published:2014-02-14
Contact: *Corresponding author. Tel.: 86-21-51980031; E-mail: tbding@shmu.edu.cn
About author:*Corresponding author. Tel.: 86-21-51980031; E-mail: tbding@shmu.edu.cn
Supported by:
Shanghai Natural Science Fund (Grant No. 09ZR140430), and partially supported by grants National Institute of Health (Grant No. HL69817), VA Merit 000900-01.

Abstract

Abstract:

Sphingolipids, a new class of lipid mediators, are involved in a variety of important physiological and pathological processes. Sphingomyelin synthase (SMS) is an enzyme to convert the ceramide (Cer.) and phosphatidylcholine into sphingomyelin (SM) and diacylglycerol, which plays a key role in sphingolipid biosynthesis. Two SMS isoforms, SMS1 and SMS2, have been identified with different subcellular localizations and expression level in tissues. Previous studies have shown that SMS may serve as a potential therapeutic target for the treatment of various diseases, such as cardiovascular and metabolic diseases. Thus, there is an urgent need for a rapid and sensitive method for SMS activity analysis. In our study, we developed a novel method for SMS activity by monitoring the appearance of the product, NBD-SM, in the tissue culture medium or bloodand applied this method in cells and mice. In Huh7 cells, the interassay coefficient of variation of the SMS activity assay was (3.60±0.07)%. In wild type (WT) mice, we observed accumulation of NBD-SM in blood in a time dependent fashion. In SMS2 KO mice, NBD-SM in plasma collected at 5- (0%, P<0.01), 30- (16%, P<0.01), and 60 min (21%, P<0.01) after injection of fluorescence liposome solution was significantly decreased compared with WT mice. However, in SMS1 KO mice, NBD-SM in plasma collected 5- and 30 min is similar to that in WT mice. Our results suggest that this method could be used for SMS activity measurement in vitro and in vivo.

Key words: Sphingomyelin synthase, SMS activity assay, SMS inhibitor screening, Sphingomyelin, Ceramide

CLC Number:

R963

Supporting:

Tingbo Ding, Jibin Dong, Bin Lou, Xian-Cheng Jiang. Sphingomyelin synthase activity measurement by the fluorescent product in cell culture medium or animal plasma[J]. Journal of Chinese Pharmaceutical Sciences, 2014, 23(4): 256-261.

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Sphingomyelin synthase activity measurement by the fluorescent product in cell culture medium or animal plasma

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