N butyric

The faecal short chain fatty acids (SCFA) acetate, propionate and butyrate, major anti-inflammatory and immunomodulatory bacterial metabolites [12 (link)], previously implicated in the aetiology of CD [13 (link)] were measured in diethyl ether extracts by gas chromatography using a TRACE™ 2000 gas chromatograph (ThermoQuest Ltd, Manchester, UK) equipped with a flame ionisation detector (250°C) and Zebron ZB-Wax capillary column (15 m x 0.53 mm x 1 μm), made of polyethylene glycol (Phenomenex, Cheshire, UK) [11 (link)]. The carrier gas was Nitrogen (30 ml/min). Internal standard (86.1 mmol/l, 3-methyl-n-valeric acid, Sigma-Aldrich, UK) and concentrated orthophosphoric acid were added to 50 mg of freeze-dried faecal material stored in 1M NaOH. The mixture was extracted three times with 3 ml diethyl ether, centrifuged and the ether layers pooled. One microlitre of ether extract was automatically injected (230°C, splitless) into the column. The column temperature was held at 80°C for 1 min, increased at 15°C/min until 210°C and held for 1 min. The chromatograms were analysed using Chrom-Card 32 version 1.07β5 (ThermoQuest, Milan, Italy). Authentic external standards were used as calibrators (166.5 mmol/l acetic, 135.0 mmol/l propionic, 113.5 mmol/l n-butyric, Sigma-Aldrich, UK). Results were presented per mass of faecal material (μmol/g) and as proportional ratio (%) to total SCFA.

Short-chain fatty acids were analyzed by dry matter content as described previously by Panasevich et al. (35 (link)), courtesy of the Metabolomics Center at the University of Illinois at Urbana-Champaign. To calculate dry matter percentage, a portion (~0.08–0.15 g) of wet cecal contents were weighed and then heated in oven for 24 h to dry samples. Resulting dry matter percentage was calculated as [weight of dry sample/weight (g) of wet sample (g) × 100]. The rest of the cecal contents (~0.05–0.15 g) were acidified immediately after collection in 6.25% meta-phosphoric acid solution and stored at −20°C until analysis. SCFA concentrations (wet) were determined by gas chromatography by using a gas chromatograph (Hewlett-Packard 5890A Series II) and a glass column (180 cm × 4 mm i.d.), packed with 10% SP-1200/1% H3PO4 on 80/100 + mesh Chromosorb WAW (Supelco, Inc.). Nitrogen was the carrier gas with a flow rate of 75 mL/min. Oven, detector, and injector temperatures were 125°, 175°, and 180°C, respectively. Acetic, n-butyric, and propionic acid solutions (Sigma-Aldrich) were used as standards.