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

Abstract

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. 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). 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. LC/MS/MS can be used to detect the incorporation of plasma palmitate into muscle ceramides in humans, in vivo.