Metabolic profiling by gas chromatography-mass spectrometry of energy metabolism in high-fat diet-fed obese mice.

A novel, selective and sensitive single-ion monitoring (SIM) gas chromatography-mass spectrometry (GCMS) method was developed and validated for the determination of energy metabolites related to glycolysis, the tricarboxylic acid (TCA) cycle, glutaminolysis, and fatty acid ß-oxidation. This assay used N-tert-butyldimethylsilyl-N-methyltrifluoroacetamide (MTBSTFA) containing 1% tert-butyldimethylchlorosilane (TBDMCS) as derivatizing reagent and was highly reproducible, sensitive, specific and robust. The assay was used to analyze liver tissue and serum from C57BL/6N obese mice fed a high-fat diet (HFD) and C57BL/6N mice fed normal chow for 8 weeks. HFD-fed mice serum displayed statistically significantly reduced concentrations of pyruvate, citrate, succinate, fumarate, and 2-oxoglutarate, with an elevated concentration of pantothenic acid. In liver tissue, HFD-fed mice exhibited depressed levels of glycolysis end-products pyruvate and lactate, glutamate, and the TCA cycle intermediates citrate, succinate, fumarate, malate, and oxaloacetate. Pantothenate levels were 3-fold elevated accompanied by a modest increased gene expression of Scl5a6 that encodes the pantothenate transporter SLC5A6. Since both glucose and fatty acids inhibit coenzyme A synthesis from pantothenate, it was concluded that these data were consistent with downregulated fatty acid ß-oxidation, glutaminolysis, glycolysis, and TCA cycle activity, due to impaired anaplerosis. The novel SIM GCMS assay provided new insights into metabolic effects of HFD in mice.