13c11 tryptophan

¹³C₃-lactic acid, ¹³C₂-oxalic acid, ²H₃-sacrosine, ²H₈-valine, ¹³C₃-dihydroxyacetone, ²H₁₀-isoleucine, ¹³C₄-fumaric acid, ¹³C₄-malic acid, ²H₃-aspartic acid, ¹³C₅-glutamic acid, ¹³C₆-4-hydroxybenzoic acid, ²H₃-lauric acid, ¹³C₅-ribose, ¹³C₂-taurine, ²H₄-citric acid, ²H₇-ornithine, ¹³C₆-tyrosine, ¹³C₆-dopa, ²H₆-kynurenine, ²H₈-cystamine, and ¹³C₁₁-tryptophan were purchased from Cambridge Isotope Laboratories, Inc. (MA, USA). ²H₃-2-hydorxybutyric acid and 2-isopropylmalic acid were purchased from CDN isotopes (Quebec, CA) and Sigma Aldrich (Tokyo, Japan), respectively. The compounds were dissolved in methanol, as shown in Supplementary Table 3, and then the obtained solution was used as an extraction solution. A standard alkane series mixture (C7 to C33) and octafluoronaphthalene (OFN) were purchased from Restek Co. (PA, USA) and Shimadzu Co. (Kyoto, Japan), respectively. Methoxyamine hydrochloride and N-methyl-N-trimethylsilyl-trifluoroacetamide (MSTFA), which were used for the derivatization, were obtained from Sigma-Aldrich and GL Science (Tokyo, Japan), respectively.

Extraction buffer was prepared by adding 2:2:1 methanol:acetonitrile:water to internal standards at 1 μg/mL each (D4-Citric Acid, ¹³C5-Glutamine, ¹³C5-Glutamic Acid, ¹³C6-Lysine, ¹³C5-Methionine, ¹³C3-Serine, D4-Succinic Acid, ¹³C11-Tryptophan, and D8-Valine; Cambridge Isotope Laboratories). Plasma volume was included in the calculations for the water portion of the buffer. A total of 720 µL of extraction buffer was then added to 40 μL of each plasma sample. Samples were placed on a rotating platform at –20 °C for 1 h and centrifuged at 4 °C for 10 min at 21,000× g. A total of 300 µL of the metabolite extracts was transferred into fresh microcentrifuge tubes. An equal volume of each extract was pooled to serve as a quality control (QC) sample, which was analyzed at the beginning, end, and after every tenth sample throughout the instrument run. Extraction buffer alone was analyzed as a processing blank sample to account for carryover or background contamination from the sample extraction process. Metabolite extracts, the QC sample, and the processing blank were evaporated to dryness using a speed-vacuum. All samples were reconstituted in 30 μL of acetonitrile/water (1:1, v/v), vortexed for 10 min, and incubated at –20 °C for 18 h. Samples were then centrifuged at 4 °C for 2 min at 21,000× g and the supernatant was transferred into autosampler vials for analysis.

For metabolite extraction, samples were extracted in ice cold 2:2:1 methanol/acetonitrile/water which contained a mixture of 9 internal standards (d4‐citric acid, 13C5‐glutamine, 13C5‐glutamic acid, 13C6‐lysine, 13C5‐methionine, 13C3‐serine, d4‐succinic acid, 13C11‐tryptophan, d8‐valine; Cambridge Isotope Laboratories) at a concentration of 1 μg/ml each.  The ratio of extraction solvent to sample volume was 18:1. Tissue samples were lyophilised overnight prior to extraction. Tissues were homogenised using a ceramic bead mill homogeniser, after the addition of extraction buffer. Samples were then incubated at −20°C for 1 h followed by a 10 min centrifugation at maximum speed. Four hundred microliters of supernatants were transferred to fresh tubes.  Pooled QC samples were prepared by adding an equal volume of each sample to a fresh 1.5 ml microcentrifuge tube. Processing blanks were utilised by adding extraction solvent to microcentrifuge tubes. Samples, pooled QCs, and processing blanks were evaporated using a speed‐vac. The resulting dried extracts were reconstituted in 40 μl of acetonitrile/water (1:1, V/V), vortexed and samples, blanks, and pooled QCs were then analysed using liquid chromatographymass spectrometry (LC–MS).