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

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.