Quantifying Short-Chain Fatty Acids in Biological Samples

Colonic luminal content samples were weighed into 1.5 ml tubes, crushed and homogenized in 100 µl of distilled water. Subsequently, 40 mg of sodium chloride, 20 mg of citric acid, 40 µl of 1 M hydrochloric acid, and 200 µl of butanol were added. The tubes were vortexed for 2 min and centrifuged at 18,000×g for 15 min. The supernatant was transferred to microtubes, and 1 µl was injected into the gas chromatograph. For serum measurements, 20 mg of sodium chloride, 10 mg of citric acid, 20 µl of 1 M hydrochloric acid, and 100 µl of butanol were added to 100 µl of serum samples. Tubes were vortexed and centrifuged as previously described and 1 µl was injected into the gas chromatograph. To quantify SCFAs, a calibration curve for the concentration range of 0.015–1 mg/ml was constructed. SCFAs measurements were performed following a recently published protocol^{41 (link)}: chromatographic analyses were performed using an Agilent 6850 system with ExChrom software, equipped with a 7683B automatic liquid sampler, a flame ionization detector (FID) (Agilent Technologies, USA), and a fused-silica capillary RTX-WAX (Restec Corporation, U.S.) with dimensions of 60 m × 0.25 mm internal diameter (i.d.) coated with a 0.15-µm thick layer of polyethylene glycol. The initial oven temperature was 100 °C (hold 2 min), which was increased to 200 °C at a rate of 15 °C/min (hold 5 min). The FID temperature was maintained at 260 °C, and the flow rates of H₂, air, and the make-up gas N₂ were 35, 350, and 25 ml/min, respectively. Sample volumes of 1 µl were injected at 260 °C using a split ratio of approximately 25:1. Nitrogen was used as the carrier gas at 25 ml/min. The runtime for each analysis was 12.95 min.