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

• Published in: PloS one Vol. 12; no. 5; p. e0177953
• Main Authors: Patel, Daxesh P., Krausz, Kristopher W., Xie, Cen, Beyoğlu, Diren, Gonzalez, Frank J., Idle, Jeffrey R.
• Format: Journal Article
• Language: English
• Published: United States Public Library of Science 16.05.2017; Public Library of Science (PLoS)
• Subjects: a-Ketoglutaric acid; Amino acids; Animals; Assaying; Biology and Life Sciences; Blood Chemical Analysis - methods; Cancer; Chromatography; Chronic diseases; Citric acid; Citric Acid - blood; Coenzyme A; Diet; Diet, High-Fat - adverse effects; Energy Metabolism; Engineering and Technology; Fat metabolism; Fatty acids; Fatty Acids - metabolism; Gas chromatography; Gas Chromatography-Mass Spectrometry - methods; Gene expression; Glucose transporter; Glutamic Acid - blood; Glycolysis; Health aspects; Hepatology; High fat diet; Insulin resistance; Intermediates; Ketoglutaric acid; Laboratories; Lactic acid; Lactic Acid - blood; Liver; Malate; Male; Mammals; Mass spectrometry; Mass spectroscopy; Medical research; Medicine and Health Sciences; Metabolism; Metabolites; Metabolome; Mice; Mice, Inbred C57BL; Obesity; Obesity - blood; Obesity - etiology; Oxidation; Pantothenic acid; Pantothenic Acid - blood; Physical Sciences; Proteins; Pyruvic acid; Pyruvic Acid - blood; Reagents; Risk factors; Scientific imaging; Studies; Symporters - genetics; Symporters - metabolism; Tricarboxylic acid cycle; Urine; Massachusetts
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0177953

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 β-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 β-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.