Global biochemical profiles were determined by using the serum (12 mice/group) from each mouse. Metabolomic profiling analysis was performed by Metabolon (Durham, NC, USA) using ultra-high-performance liquid chromatography–tandem mass spectroscopy (UPLC-MS/MS) platforms, as previously described (15 (link)). In brief, sample (100 µl serum) preparation was conducted by using a proprietary series of organic and aqueous extractions to remove the protein fraction while allowing maximum recovery of small molecules. The resultant extract was divided into 4 fractions: one for analysis by UPLC-MS/MS (positive mode), one for UPLC-MS/MS (negative mode), one for GC-MS, and one for backup. The sample extract was dried and then reconstituted in acidic or basic liquid chromatography–compatible solvents, each of which contained ≥8 injection standards at fixed concentrations to ensure injection and chromatographic consistency. The data were extracted, with peaks and compounds identified by using the local area network backbone and database server running Oracle 10.2.0.1 Enterprise Edition (Oracle, Redwood City, CA, USA).
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