Hepatic Metabolite and Fatty Acid Profiling

Hepatic metabolite profiles were analysed using an Agilent 1290 Infinity Liquid Chromatography System (Agilent Technologies) equipped with a 2.1 × 100 mm C18 reverse-phase column with 1.8-μm particle size (Waters Corp., Milford, MA, USA) as described previously40 (link). Mass spectrometry was performed on an Agilent 6530 Accurate-Mass QTOF/MS (Agilent Technologies) equipped with an electrospray ionisation source. Data for each ionisation technique were acquired in positive and negative ion modes. LC data were acquired and processed using Mass Hunter Qualitative Analysis Software (version B.03.01; Agilent Technologies). The MS analysis system was used to identify metabolites corresponding to those in the METLIN database (http://metlin.scripps.edu). SIMCA-P+ 11.0 software (Umetrics AB, Umea, Sweden) and online tool MetaboAnalyst 3.0 (http://www.metaboanalyst.ca/MetaboAnalyst) were used for PCA, partial least squares discriminant analysis (PLS-DA) and OPLS-DA analyses. A t-test was used to identify those candidate metabolites obtained from PLS-DA modelling that were statistically different from those in the control group.
Fatty acids in liver tissue were measured by gas chromatography as previously described41 (link)42 (link). TCA cycle metabolites in liver tissue were assayed with a Shimadu QP-2010 ultra GC/MS43 (link).