Metabolic Profiling of Mouse Fecal Samples

Fecal samples were homogenized in 1:12 w/v of saline phosphate buffer (1.9 mM Na₂HPO₄, 8.1 mM NaH₂PO₄, 150 mM NaCl) with 1 mM sodium 3-trimethysilyl-propionate-d4 (TSP-d4) mixed 1:1 v/v with deuterated water (Merck) using ceramic bead beating (Lysing Matrix E, MP Biomedicals), followed by centrifugation at 15,000g, 5 min, and filtration of supernatants at 0.2 μm. ¹H NMR spectra were recorded using a 600-MHz Bruker Avance spectrometer fitted with a 5-mm TCI proton-optimized triple resonance NMR inverse cryoprobe and autosampler (Bruker). Sample temperature was controlled at 300 K. Spectra were transformed with a 0.3-Hz line broadening and zero filling, manually phased, baseline corrected, and referenced by setting the TSP–d4 signal to 0 ppm. Metabolites were identified by comparison with existing literature, an in-house database of mouse fecal metabolite spectra, the Human Metabolome Database (http://www.hmdb.ca/), and by use of 2-dimensional NMR ¹H–¹H correlation spectroscopy, ¹H–¹³C heteronuclear single quantum correlation, and ¹H–¹³C heteronuclear multiple bond correlation spectroscopy [67 (link), 68 (link), 121 (link)]. Concentrations were calculated using Chenomx NMR Suite 7.0, centered (CLR), and log-transformed.