Targeted Metabolomic Analysis of Plasma Samples

Plasma samples were obtained at baseline (n = 325), stored at −80°C and analyzed at 15–18 years post-enrollment depending on the enrollment date. We quantified free plasma levels of downstream metabolites of the KP, tryptophan, kynurenine, 3-hydroxykynurenine, kynurenic acid, anthranilic acid, 3-hydroxyanthranilic acid and quinolinic acid, in addition to indole metabolites indoxyl sulfate and indole-3-acetate (Fig. 1), using a targeted liquid chromatography-mass spectrometry (LC-MS) platform. Tryptophan ¹⁵N₂, kynurenic acid D₅, anthranilic acid ¹³C₆, indoxyl sulfate ¹³C₆, indoxyl acetate D₅ and all authentic standards were purchased from Sigma Aldrich (St. Louis, MO, USA). A total of 50 µL of plasma was extracted with 200 µL of chilled acetonitrile spiked with internal standards, vortexed and centrifuged. The supernatant was transferred to glass vials that were vacufuged. The dried samples were reconstituted in 50 µL of water with 0.1% formic acid. The samples were then analyzed with a 6490 QQQ mass spectrometer with a 1290 Liquid Chromatography machine attached (Agilent, Santa Clara, CA, USA) [14 (link)].