Crm46975

A volume of 25 mL of digesta from the colon was used for pH analysis using a digital pH meter (Dostmann electronic, GmbH, Wertheim, Germany) following the manufacturer’s protocol [82 ]. The ammonia concentration was measured using an ammonia selective-ion electrode (Orion 95–12, Orion Research Inc., Franklin, MA, USA) attached to a selective ion meter (710A model, Orion Research Inc., Franklin, MA, USA). The calibration was performed using dilutions at 10, 100, and 1000 mg/L of a 0.1 M standard ammonium chloride solution. In addition, an ISA solution (Ionic Strength Adjuster, Orion Research Inc., Franklin, MA, USA) of 0.5 mL was mixed into every 25 mL sample to ensure a uniform background ionic strength [83 (link)] The final results were reported as µmol/g.
Short-chain fatty acids from the piglet colonic digesta samples were analysed in aqueous extracts by gas chromatography as described by Marin et al. [80 (link)]. Briefly, the aqueous extracts were injected into a gas chromatograph (Varian, 430-GC, Varian Inc., Palo Alto, CA, USA) after centrifugation. The GC instrument is fitted with an Elite-FFAP capillary column (inside diameter 320 mm) (Perkin Elmer, Waltham, MA, USA). Volatile fatty acids obtained from CRM46975, Supelco, Bellefonte, PA, USA, were used as standards. The final results were reported as µmol/g.

Stool pellets from WT and Ern2^–/– mice were collected directly into sterile preweighed tubes, immediately frozen, and stored at –80°C. Pellets were homogenized in HPLC grade water by vortexing. The pH of the cleared homogenate was adjusted to 2-3, 2-methyl pentanoic acid was added as an internal standard (0.1%), and short chain fatty acids (SCFAs) were extracted by adding 1 volume of ethyl ether anhydrous. Samples were vortexed for 2 minutes and centrifuged at 5000g for 2 minutes. The upper ether layer was collected, and SCFA content was analyzed on an Agilent 7890B gas chromatography system with flame ionization detector using a high-resolution capillary column for detection of volatile acids (DB-FFAP, 30 m × 0.25 mm with 0.25 μm film thickness; Agilent Technologies). A standard solution containing 10 mM of acetic, propionic, isobutyric, butyric, isovaleric, valeric, isocaproic, caproic, and heptanoic acids (Supelco CRM46975) was processed and analyzed in the same manner as the stool samples. The retention times and peak heights of the acids in the standard mix were used as references for the unknown samples. Each acid was identified by its specific retention time, and the concentration was determined and expressed as mM per gram of fecal material. Chromatograms and data integration were carried out using OpenLab ChemStation Software (Agilent Technologies).

SCFAs (acetic, propionic, butyric and valeric acids) were quantified in water extracts of pig's colon content sample by gas chromatography. Briefly, colon samples were mixed with distilled water in a proportion of 1:2 (w:v), centrifuged at 12,000 g for 25 min and diluted 1:2 with distilled water. A sample volume of 1 μL from the centrifuged extract was injected under split mode into a gas chromatograph (Varian, 430-GC) equipped with a capillary column Elite-FFAP with a length of 30 m, an inner diameter of 320 μm, and a film thickness of 0.25 μm (Perkin Elmer, USA). The carrier gas was hydrogen; flow, 1.5 mL/per min. The injector was set at 250°C, and the split rate was 1:40. The flame ionization detector (FID) was set to 200°C, and the column oven was set to 110°C. The oven temperature was increased to 170°C at a rate of 12°C/min, where it was held for 9.5 min. The analysis time was 10 min. The sample concentration was calculated referring to a standard commercial mixture of volatile fatty acids (CRM46975, Supelco, USA). Results were expressed as μmol/g for total SCFAs and as a percentage for individual SCFA.