title: Attenuation of Slc27a5 gene expression followed by LC-MS measurement of bile acid reconjugation using metabolomics and a stable isotope tracer strategy.
abstract: The purpose of this study was to evaluate the use of high resolution LC-MS together with metabolomics and D(4)-cholic acid (D(4)-CA) as a metabolic tracer to measure the metabolism and reconjugation of bile acids (BAs) in vitro and in vivo. Metabolic tracers are very important because they allow for the direct detection (substrate-to-product) of small and significant biological perturbations that may not be apparent when monitoring "static" endogenous levels of particular metabolites. Slc27a5, also known as fatty acid transport protein 5 (FATP5), is the hepatic BA-CoA ligase involved in reconjugating BAs during enterohepatic BA recycling. Using Slc27a5-cKD mice, silencing of ∼90% gene expression was achieved followed by reduction in the reconjugation of D(4)-CA to D(4)-taurocholic acid (D(4)-TCA), as well as other conjugated BA metabolites in plasma (p = 0.0031). The method described allowed a rapid measure of many D(4) and endogenous BA. Analysis of bile resulted in the detection of 39 BA metabolites from a 13 min analytical run. Finally, the utilization of a novel high resolution mass spectrometry method in combination with metabolomics and a stable isotope metabolic tracer allowed for the detection of targeted and untargeted BAs following silencing of the Slc27a5 gene in primary hepatocytes and in mice.
authors.affiliation: Merck Research Laboratories, Rahway, New Jersey, United States. jose_castro-perez@merck.com
authors.forename: Jose M
authors.initials: JM
authors.lastname: Castro-Perez
authors.forename: Thomas P
authors.initials: TP
authors.lastname: Roddy
authors.forename: Vinit
authors.initials: V
authors.lastname: Shah
authors.forename: Sheng-Ping
authors.initials: SP
authors.lastname: Wang
authors.forename: Xuesong
authors.initials: X
authors.lastname: Ouyang
authors.forename: Anthony
authors.initials: A
authors.lastname: Ogawa
authors.forename: David G
authors.initials: DG
authors.lastname: McLaren
authors.forename: Marija
authors.initials: M
authors.lastname: Tadin-Strapps
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Robinson
authors.forename: Steven R
authors.initials: SR
authors.lastname: Bartz
authors.forename: Brandon
authors.initials: B
authors.lastname: Ason
authors.forename: Ying
authors.initials: Y
authors.lastname: Chen
authors.forename: Stephen F
authors.initials: SF
authors.lastname: Previs
authors.forename: Kenny K
authors.initials: KK
authors.lastname: Wong
authors.forename: Rob J
authors.initials: RJ
authors.lastname: Vreeken
authors.forename: Douglas G
authors.initials: DG
authors.lastname: Johns
authors.forename: Brian K
authors.initials: BK
authors.lastname: Hubbard
authors.forename: Thomas
authors.initials: T
authors.lastname: Hankemeier
authors.forename: Lyndon
authors.initials: L
authors.lastname: Mitnaul
mesh_terms: D000818:Animals; D001647:Bile Acids and Salts / Q000378:metabolism*; D002853:Chromatography, Liquid / Q000379:methods*; D050612:Fatty Acid Transport Proteins / Q000378:metabolism*; D020868:Gene Silencing; D006801:Humans; D008099:Liver / Q000473:pathology*; D013058:Mass Spectrometry / Q000379:methods*; D055432:Metabolomics / Q000379:methods*; D051379:Mice; D008810:Mice, Inbred C57BL; D008822:Mice, Transgenic; D021241:Spectrometry, Mass, Electrospray Ionization
chemical_list: D001647:Bile Acids and Salts; D050612:Fatty Acid Transport Proteins; C113098:Slc27a5 protein, mouse