Metabolic profiling and quantification of ceramide by liquid chromatography mass spectrometry

Abstract

ObjectiveCeramides are putative mediators of insulin resistance and lipotoxicity; an up‐regulation of ceramide biosynthesis has been well described in rodent models of metabolic disease. A precise, robust and specific LC/MS method was developed to quantify ceramide profiles and support determinations of ceramide flux. The LC/MS method was used to determine the effects of diet‐induced obesity (DIO) in C57B1/6J mice ±treatment with myriocin (1mg/kg, over 7 days), an inhibitor of ceramide synthesis.MethodPlasma and tissue samples were collected from mice (lean, high‐fat fed ± myriocin) at various times following administration of stable isotope tracers. The ceramides were extracted with organic solvents and analyzed using electrospray ionization tandem mass spectrometry; concentrations were determined by normalization to an internal standard (d31‐c16:0 ceramide).ResultsIn contrast to lean mice, DIO‐mice demonstrated an increased concentration and synthesis of circulating ceramides. Myriocin treatment inhibited ceramide synthesis and led to a normalization of circulating levels.ConclusionLC/MS analyses can enable characterization of circulating concentrations of ceramide subspecies and enable stable isotope determinations of in vivo kinetics.Support or Funding InformationAll authors are employed by Merck.