Cecal Short-Chain Fatty Acid Quantification

Contents of cecum were extracted as described previously and the concentration of acetate and butyrate in each extract was measured via high-performance liquid chromatography. Briefly, distilled water was added to 20-50 mg of cecal content to a final weight of 200 mg. Following incubation at 80°C for 15 minutes, each sample was centrifuged at 13 000 rpm for 10 minutes, after which the supernatant was filtered through a membrane filter (pore size 0.45 μm). Acetate and butyrate in each sample was then separated and measured using an Agilent 1100 series instrument (Agilent Technologies, Inc, Santa Clara, CA, USA) equipped with a C18 column (ZORBAX Eclipse XDB-C18, analytical 4.6 × 150 mm, 5-Micron; Agilent) and a UV detector (210 nm). The mobile phase consisted of 90% 10 mM KH²PO^{4 (link)} and 10% acetonitrile.