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

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Lipids

Abstract

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 × 4.6 mm, 5 μm) 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 μM 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.

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Abbreviations

AS:

Atherosclerosis

C6-NBD-Cer:

6-((N-(7-nitrobenz-2-oxa-1.3-diazol-4-yl) amino) hexanoyl)-sphingosine

C6-NBD-CerPCho:

N-(6-(7-nitro-2-1, 3-benzoxadiazol-4-yl) amino) hexanoyl)-sphingosine -1-phosphocholine

D2:

(2-(2-(benzyloxy)phenyl)-2-(phenylamino) acetonitrile)

DAG:

Diacylglycerol

HPLC-FLD:

High-performance liquid chromatography-fluorescence detection

PtdCho:

Phosphatidylcholine

CerPCho:

Sphingomyelin

SMS:

Sphingomyelin synthase

D609:

Tricyclodecane-9-yl-xanthogenate

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Acknowledgments

We gratefully acknowledge the financial support from the grant of the Science and Technology Research Key Projects, the Science and Technology Commission of Shanghai Municipality (No.11431920102).

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Authors and Affiliations

  1. School of Pharmacy, Fudan University, No. 826, Zhangheng Road, Shanghai, 201203, People’s Republic of China

    Taomin Huang, Xiaoxia Li, Shuang Hu, Bo Zhao, Ping Chen, Xiao Liu, Deyong Ye & Nengneng Cheng

  2. Department of Pharmacy, Eye and ENT Hospital of Fudan University, No. 83, Fenyang Road, Shanghai, 200031, People’s Republic of China

    Taomin Huang

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  1. Taomin Huang
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Correspondence to Nengneng Cheng.

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Huang, T., Li, X., Hu, S. et al. Analysis of Fluorescent Ceramide and Sphingomyelin Analogs: A Novel Approach for in Vivo Monitoring of Sphingomyelin Synthase Activity. Lipids 49, 1071–1079 (2014). https://doi.org/10.1007/s11745-014-3940-5

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  • DOI: https://doi.org/10.1007/s11745-014-3940-5

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