Quantification of Indole Metabolites

Serum samples (100 µL) and prechilled methanol (400 µL) were mixed by well vortexed. Cecal contents, liver, and adipose tissues (≈10 mg) were individually grounded with liquid nitrogen and the homogenate was resuspended with prechilled 80% methanol and 0.1% formic acid by well vortexed. The samples were incubated on ice for 5 min and then were centrifuged at 15 000 rpm, 4 °C for 5 min. The supernatants were diluted to final concentration containing 53% methanol by LC‐MS/MS grade water. The samples were subsequently transferred to a fresh Eppendorf tube and then were centrifuged at 15 000 g, 4 °C for 10 min. The supernatant was analyzed for tryptophan, I3AA, and other indole derivatives using 4000 Q TRAP LC‐MS/MS (AB Sciex) analysis according to a previously described method (Table S5, Supporting Information).^[33 (link)
^] Specifically, L‐tryptophan (T0254, Sigma), I3AA (45533, Sigma), indole (442619, Sigma), 3‐indoleacrylic acid (I2273, Sigma), indole‐3‐carboxaldehyde (129445, Sigma), and indole‐3‐lactic acid (I157602, Aladdin) were used as standards in the current study. The absolute concentration of metabolites was calculated according to the standard curves, which were created using six appropriate serial dilutions of the corresponding standards.