Analysis of fecal Short Chain Fatty Acid (SCFA) concentrations

Fecal SCFA concentrations were determined using gas chromatography (3800 Varian GC, Agilent Technologies, Santa Clara, CA). For each sample, 2.5 mL of distilled water was added to one gram of thawed feces in a conical tube and vortexed. Samples were centrifuged at 4°C for 10 min at 2,000 × g for supernatant removal. One mL of supernatant was then transferred into a 1.5 mL centrifuge tube and mixed with 0.2 mL of metaphosphoric acid for deproteinizing and 0.1 mL of isocaproic acid as an internal standard (48.3 mM; Sigma-Aldrich, Saint Louis, MO). A standard curve was generated using five concentrations of acetate, butyrate, propionate, valerate, isovalerate, and isobutyrate (Sigma-Aldrich, Saint Louis, MO). The tubes were then centrifuged at 12,000 × g at 4°C for 25 min. Aliquots of the supernatant (1 mL) from standard and fecal samples were transferred to 1.5 mL GC vials and duplicate injections of 100 uL were used by the GC for analysis. A flame ionization detector was used with an oven temperature of 60–200°C. The Nukol capillary column (15 m x 0.25 mm x 0.25 μm; Sigma-Aldrich‎, Bellefonte, PA) was operated with highly purified He, as the carrier gas, at 1 mL/min. Concentrations of acetate, butyrate, propionate, valerate, isovalerate, and isobutyrate were calculated using the ratio of the peak area of each compound to the internal standard and standard curve regression. Molar proportions of SCFA (%) were calculated as the individual SCFA / total SCFA concentration × 100.