Fecal metabolites were extracted from fecal pellets (50 mg) using 500 μl of 4°C 50% methanol followed by homogenization with ceramic beads using a Bead Ruptor 4 (Omni). Insoluble debris was separated by centrifugation at 10,000g × 15 min at 4°C, and 300 μl was collected. The remaining material was reextracted in an additional 300 μl of 4°C 50% methanol following the same procedure, and supernatants from the two-stage extraction were combined. Metabolites present in whole blood were extracted by diluting samples 1:20 (v/v) in 4°C 50% methanol. Insoluble material was removed by centrifugation at 14,600g × 5 min at 4°C, and the supernatant was harvested. All metabolite extracts were stored at −80°C until quantitative analyses could be completed. All MS analyses were conducted at the Calgary Metabolomics Research Facility (CMRF). Metabolites in the extracts were quantified using UHPLC MS. Fecal metabolites were separated by hydrophilic interaction liquid chromatography (Syncronis HILIC column, Thermo Fisher Scientific), and serum metabolites were separated using reversed-phase chromatography (Accucore Vanquish C18+, Thermo Fisher Scientific). MS analyses were conducted using a Q-Exactive HF Mass Spectrometer (Thermo Fisher Scientific) in negative ion full-scan mode. Metabolites were assigned by high-resolution MS and coelution of the metabolite with a commercial metabolite standard (MPA and GA from Thermo Fisher Scientific, and MPAG and AcMPAG from Toronto Research Chemicals). Metabolite concentrations were measured by fitting peak intensities of extracted ion chromatograms to calibration reference curves prepared using standards. Data were analyzed using the MAVEN software package (38).

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