A liquid chromatography/tandem mass spectrometry method for measuring the in vivo incorporation of plasma free fatty acids into intramyocellular ceramides in humans

Agnieszka U. Blachnio-Zabielska, Xuan Mai T. Persson, Christina Koutsari, Piotr Zabielski, Michael D. Jensen

Research output: Contribution to journalArticlepeer-review

61 Scopus citations

Abstract

RATIONALE 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- 13C]palmitate into intramyocellular ceramides. METHODS We used liquid chromatography/tandem mass spectrometry (LC/MS/MS) to measure the concentrations of different sphingolipid species and 13C-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 [ 13C 16]16:0-ceramide was monitored as [M+16+H] +. By monitoring two different transitions of the [ 13C 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- 13C]palmitate infusion. CONCLUSIONS LC/MS/MS can be used to detect the incorporation of plasma palmitate into muscle ceramides in humans, in vivo.

Original languageEnglish (US)
Pages (from-to)1134-1140
Number of pages7
JournalRapid Communications in Mass Spectrometry
Volume26
Issue number9
DOIs
StatePublished - May 15 2012

ASJC Scopus subject areas

  • Analytical Chemistry
  • Spectroscopy
  • Organic Chemistry

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