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

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-C-13] palmitate into intramyocellular ceramides. METHODS: We used liquid chromatography/ tandem mass spectrometry ( LC/ MS/ MS) to measure the concentrations of different sphingolipid species and C-13-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)C16] 16:0-ceramide was monitored as [M+16+H](+). By monitoring two different transitions of the [(13)C16]16:0-ceramide (554/536 and 554/264) we could indirectlymeasure 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-C-13] palmitate infusion. CONCLUSIONS: LC/ MS/ MS can be used to detect the incorporation of plasma palmitate into muscle ceramides in humans, in vivo. Copyright (C) 2012 John Wiley & Sons, Ltd.