Analysis of Fluorescent Ceramide and Sphingomyelin Analogs: A Novel Approach for in Vivo Monitoring of Sphingomyelin Synthase Activity

作者：Huang Taomin; Li Xiaoxia; Hu Shuang; Zhao Bo; Chen Ping; Liu Xiao; Ye Deyong; Cheng Nengneng^*

来源：Lipids, 2014, 49(10): 1071-1079.

DOI：10.1007/s11745-014-3940-5

• Sphingomyelin
• Sphingomyelin synthase activity
• Ceramide
• High-performance liquid chromatography-fluorescence detection
• Inhibitor screening
• D2
• D609

A novel sensitive high-performance liquid chromatography-fluorescence detection (HPLC-FLD) method was developed for real-time monitoring of relative sphingomyelin synthase (SMS) activity based on the measurement of a fluorescent ceramide (Cer) analog and its metabolite, a fluorescent sphingomyelin (CerPCho) analog, in plasma. Analyses were conducted using HPLC-FLD following a protein precipitation procedure. The chromatographic separations were carried out on an Agilent C18 RP column (150 x 4.6 mm, 5 mu m) based on a methanol-0.1 % trifluoroacetic acid aqueous solution (88:12, by vol) elution at a flow-rate of 1 mL/min. The limit of quantification in plasma was 0.05 mu M for both the fluorescent Cer analog and its metabolite. Significant differences in the fluorescent Cer analog and its metabolite concentration ratio at 5 min were found between vehicle control group and three D2 (a novel SMS inhibitor) dose groups (P < 0.05). Dose-dependent effects (D2 doses: 0, 2.5, 5, 10 mg/kg) were observed. Our method could be used to detect relative SMS activity in biochemical assays and to screen potential SMS inhibitors in vivo. D2 was found to be a potent SMS inhibitor in vivo, and may have a potential antiatherosclerotic effect, which is under further study. D609 was also selected as another model SMS inhibitor to validate our newly developed method.

• 出版日期2014-10
• 单位复旦大学