title: Analysis of fluorescent ceramide and sphingomyelin analogs: a novel approach for in vivo monitoring of sphingomyelin synthase activity.
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.
authors.affiliation: School of Pharmacy, Fudan University, No. 826, Zhangheng Road, Shanghai, 201203, People's Republic of China.
authors.forename: Taomin
authors.initials: T
authors.lastname: Huang
authors.forename: Xiaoxia
authors.initials: X
authors.lastname: Li
authors.forename: Shuang
authors.initials: S
authors.lastname: Hu
authors.forename: Bo
authors.initials: B
authors.lastname: Zhao
authors.forename: Ping
authors.initials: P
authors.lastname: Chen
authors.forename: Xiao
authors.initials: X
authors.lastname: Liu
authors.forename: Deyong
authors.initials: D
authors.lastname: Ye
authors.forename: Nengneng
authors.initials: N
authors.lastname: Cheng
mesh_terms: D009327:4-Chloro-7-nitrobenzofurazan / Q000031:analogs & derivatives* / Q000097:blood; D000097:Acetonitriles / Q000097:blood; D000818:Animals; D001774:Blood Chemical Analysis / Q000379:methods; D001952:Bridged-Ring Compounds / Q000494:pharmacology; D002518:Ceramides / Q000097:blood*; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D003199:Computer Systems; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005456:Fluorescent Dyes / Q000032:analysis; D008297:Male; D009636:Norbornanes; D010069:Oxadiazoles / Q000097:blood*; D051381:Rats; D017207:Rats, Sprague-Dawley; D013050:Spectrometry, Fluorescence; D013109:Sphingomyelins / Q000097:blood*; D013859:Thiocarbamates; D013871:Thiones / Q000494:pharmacology; D017855:Transferases (Other Substituted Phosphate Groups) / Q000037:antagonists & inhibitors / Q000097:blood*
chemical_list: D000097:Acetonitriles; D001952:Bridged-Ring Compounds; D002518:Ceramides; D004791:Enzyme Inhibitors; D005456:Fluorescent Dyes; C104907:N-6-(7-nitro-2,1,3-benzoxadiazol-4-yl)aminohexanoysphingomyelin; D009636:Norbornanes; D010069:Oxadiazoles; D013109:Sphingomyelins; D013859:Thiocarbamates; D013871:Thiones; C046498:tricyclodecane-9-yl-xanthogenate; C037974:N-(7-(4-nitrobenzo-2-oxa-1,3-diazole))-6-aminocaproyl sphingosine; D017855:Transferases (Other Substituted Phosphate Groups); C070186:phosphatidylcholine-ceramide phosphocholine transferase; D009327:4-Chloro-7-nitrobenzofurazan
references.citation: J Clin Invest. 1996 Sep 15;98(6):1455-64
references.pmid: 8823312
references.citation: Arterioscler Thromb Vasc Biol. 2009 Jun;29(6):850-6
references.pmid: 19286635
references.citation: Trends Cardiovasc Med. 2002 Jan;12(1):37-42
references.pmid: 11796243
references.citation: J Agric Food Chem. 2009 Oct 28;57(20):9469-74
references.pmid: 20175585
references.citation: J Org Chem. 2013 Apr 5;78(7):3367-73
references.pmid: 23442124
references.citation: Biochim Biophys Acta. 2008 Oct;1781(10):610-7
references.pmid: 18694848
references.citation: Arterioscler Thromb Vasc Biol. 2008 Aug;28(8):1519-26
references.pmid: 18566297
references.citation: Med Hypotheses. 2007;69(4):908-12
references.pmid: 17433563
references.citation: Drugs Exp Clin Res. 1996;22(6):287-94
references.pmid: 9034754
references.citation: Cell. 1992 Nov 27;71(5):765-76
references.pmid: 1330325
references.citation: J Pharmacol Exp Ther. 1998 May;285(2):800-4
references.pmid: 9580629
references.citation: Mol Neurobiol. 2012 Jun;45(3):455-64
references.pmid: 22415444
references.citation: J Biol Chem. 2007 Jun 15;282(24):17537-47
references.pmid: 17449912
references.citation: J Biol Chem. 1998 Jun 5;273(23):14550-9
references.pmid: 9603970
references.citation: PLoS One. 2011;6(8):e23852
references.pmid: 21909365
references.citation: Circ Res. 2009 Jul 31;105(3):295-303
references.pmid: 19590047
references.citation: J Lipid Res. 2011 Mar;52(3):471-9
references.pmid: 21217100
references.citation: Lipids. 2008 Sep;43(9):867-75
references.pmid: 18629562
references.citation: Bioorg Med Chem Lett. 2006 Sep 15;16(18):4780-3
references.pmid: 16872828
references.citation: Chem Phys Lipids. 2012 Oct;165(7):760-8
references.pmid: 23063490
references.citation: Biochim Biophys Acta. 2007 Sep;1771(9):1186-94
references.pmid: 17616479
references.citation: J Biol Chem. 1997 Dec 19;272(51):32108-14
references.pmid: 9405408
references.citation: Eur J Med Chem. 2014 Feb 12;73:1-7
references.pmid: 24374347
references.citation: Arterioscler Thromb Vasc Biol. 2012 Jul;32(7):1577-84
references.pmid: 22580896
references.citation: Arch Pharm Res. 2011 Feb;34(2):229-36
references.pmid: 21380806
references.citation: J Lipid Res. 2008 Feb;49(2):376-85
references.pmid: 17982138
references.citation: Circulation. 2004 Nov 30;110(22):3465-71
references.pmid: 15545514
references.citation: J Lipid Res. 2006 Jun;47(6):1307-14
references.pmid: 16508036
references.citation: Neurochem Res. 2012 Apr;37(4):671-9
references.pmid: 22101393
references.citation: Arterioscler Thromb Vasc Biol. 2000 Dec;20(12):2607-13
references.pmid: 11116060
references.citation: Arterioscler Thromb Vasc Biol. 2010 Mar;30(3):628-33
references.pmid: 20032291
references.citation: Arterioscler Thromb Vasc Biol. 1996 Jan;16(1):4-11
references.pmid: 8548424
references.citation: Arterioscler Thromb Vasc Biol. 2000 Dec;20(12):2614-8
references.pmid: 11116061