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Metabolic profiling and quantification of ceramide by liquid chromatography mass spectrometry
Author(s) -
Chen Ying,
McLaren David,
Liu Jinqi,
Yin Wu,
Mayoral Rafael,
Petrov Aleksandr,
Bojic Lazar,
Berejnaia Olga,
Previs Stephen,
Kelley David
Publication year - 2016
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.30.1_supplement.684.17
Subject(s) - ceramide , sphingolipid , chemistry , chromatography , mass spectrometry , lipidomics , electrospray ionization , biochemistry , apoptosis
Objective Ceramides 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. Method Plasma 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). Results In 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. Conclusion LC/MS analyses can enable characterization of circulating concentrations of ceramide subspecies and enable stable isotope determinations of in vivo kinetics. Support or Funding Information All authors are employed by Merck.