Pack fa column

The concentration of lactic acid and butyric acid in the serum was measured by HPLC. Briefly, standard lactic acid and butyric acid and the serum samples, including 4-methylvaleric acid as an internal standard, were labeled with an SCFA labeling kit (YMC Co., Ltd., Kyoto, Japan), separated on a YMC-Pack FA column (250 × 6.0 mm ID; YMC Co. Ltd.,) and detected by UV absorbance at 400 nm.

One hundred mg of caecum contents from each were homogenized in MQ water (100 mg/mL) by vortex for 30 s, and then centrifuged at 8000 rpm for 10 min at room temperature to get supernatant. A mixture of 100 μL of the caecum supernatant and 200 μL of 0.25 mM 2-ethylbutyric acid as internal standard were labelled with 2-nitrophenyl hydrazide (2-NPH) using a short- and long-chain fatty acid analysis kit (YMC Co., Ltd., Kyoto, Japan) according to the manufacturer’s manual. 2-NPH labelled caecum supernatant was filtered through a 0.45 μm syringe filter (Millex Syringe Driven Filter Unit, MERCK, Darmstadt, Germany), and then injected into a high-performance liquid chromatography (HPLC) system (JASCO Corporation, Tokyo, Japan) with a YMC-Pack FA column (250 × 4.5 mm; YMC Co., Ltd. Tokyo, Japan). Mobile phase was acetonitrile-methanol-water (30:16:54, v/v/v), which adjusted to pH 4.5 by 0.01 M of hydrochloric acid. The injection volume was 20 μL and flow rate was kept at 1 mL/min and column oven temperature was set as 50 °C.