title: A liquid chromatography/tandem mass spectrometry method for measuring the in vivo incorporation of plasma free fatty acids into intramyocellular ceramides in humans.
abstract: BACKGROUND
  Sphingolipids are important components of cell membranes that serve as cell signaling molecules; ceramide plays a central role in sphingolipid metabolism. De novo ceramide biosynthesis depends on fatty acid availability, but whether muscle uses circulating free fatty acids or pre-existing intracellular stores is unknown. Our goal was to develop a method to detect the incorporation of intravenously infused [U-(13)C]palmitate into intramyocellular ceramides.
  
  
  METHODS
  We used liquid chromatography/tandem mass spectrometry (LC/MS/MS) to measure the concentrations of different sphingolipid species and (13)C-isotopic enrichment of 16:0-ceramide. Chromatographic separation was performed using ultra-performance liquid chromatography. The analysis was performed on a triple quadrupole mass spectrometer using a positive ion electrospray ionization source with selected reaction monitoring (SRM).
  
  
  RESULTS
  The sphingolipids ions, except enriched ceramide, were monitored as [M+2+H](+). The [(13)C(16)]16:0-ceramide was monitored as [M+16+H](+). By monitoring two different transitions of the [(13)C(16)]16:0-ceramide (554/536 and 554/264) we could indirectly measure enrichment of the palmitate that is not a part of the sphingoid base. Concentration and enrichment could be measured using 20 mg of muscle obtained from volunteers receiving a low dose [U-(13)C]palmitate infusion.
  
  
  CONCLUSIONS
  LC/MS/MS can be used to detect the incorporation of plasma palmitate into muscle ceramides in humans, in vivo.
authors.affiliation: Endocrine Research Unit, Mayo Clinic, Rochester, MN 55905, USA.
authors.forename: Agnieszka U
authors.initials: AU
authors.lastname: Blachnio-Zabielska
authors.forename: Xuan-Mai T
authors.initials: XM
authors.lastname: Persson
authors.forename: Christina
authors.initials: C
authors.lastname: Koutsari
authors.forename: Piotr
authors.initials: P
authors.lastname: Zabielski
authors.forename: Michael D
authors.initials: MD
authors.lastname: Jensen
mesh_terms: D002247:Carbon Isotopes; D002518:Ceramides / Q000032:analysis / Q000737:chemistry / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D006801:Humans; D016014:Linear Models; D018482:Muscle, Skeletal / Q000737:chemistry / Q000378:metabolism*; D010168:Palmitates / Q000008:administration & dosage / Q000097:blood / Q000737:chemistry / Q000378:metabolism*; D053719:Tandem Mass Spectrometry / Q000379:methods*
chemical_list: D002247:Carbon Isotopes; D002518:Ceramides; D010168:Palmitates