2.5. Metabolomics

Analysis of short-chain fatty acids (SCFAs) in fecal samples was performed based on a method developed by Borchers and coworkers [29]. This method was previously modified for semitargeted analysis by liquid chromatography–mass spectrometry (LC-MS) and described in detail [30]. Metabolite extraction of fecal samples was performed with 50:50 acetonitrile:water at a ratio of 10 µL extraction solvent:1 mg fecal sample. Samples were manually homogenized, mixed at 2000 revolutions per minute (RPM) for 20 min at 4 °C, and centrifuged for 15 min at 18,000× g and 4 °C. Metabolite extracts (25 µL) were derivatized with 3-nitrophenylhydrazine (3-NPH). Extracts were mixed with 10 µL of 200 mM 3-NPH·HCl in 50:50 acetonitrile:water (ACN:H2O) solution and 10 µL of 120 mM N-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide·HCl (EDC) in 50:44:6 ACN:H2O:pyridine solution. Derivatization was performed at 40 °C with 800 RPM mixing for 30 min. Derivatization was quenched by addition of 960 µL of 50:50 ACN:H2O at 4 °C.

Relative quantitation of SCFAs was performed by LC-MS on an Agilent 1290 Infinity II LC coupled to a 6560 Q-TOF operated in negative ionization mode as previously described in detail [30]. Briefly, chromatography was performed with a Waters Acquity UPLC BEH C18 column (1.7 µm, 2.1 mm × 100 mm). Mobile phases A and B were composed of 0.01% formic acid in H20 and 0.01% formic acid in ACN, respectively.

Feature extraction, retention time alignment, and relative quantitation of SCFAs was performed using MS-DIAL version 4.60 [31]. Identities of SCFAs were verified by m/z and retention time matching using pure 3-NPH-derivatized SCFA standards. SCFA abundances were normalized in MS-DIAL to the total ion chromatogram (TIC) in order to account for any differences in metabolite content of fecal samples from different groups. Normalized abundances are reported in Table S1. Donor values from 3 replicates were averaged.