Valine d8

20–30 mls of venous blood was collected at the time of CTCA. Patients were advised to fast for 2 h prior to the procedure. The blood was processed, aliquoted, and stored at −80 °C. For metabolomic assessment, thawed plasma samples were prepared and analysed with slight modifications as previously described [26 (link),27 (link)]. In brief, plasma samples were deproteinized using acetonitrile/methanol/formic acid (75:25:02; v/v/v) containing deuterated internal standards 10 mM valine-d₈ (98%; Sigma-Aldrich, St Louis, MS, USA) and 25 mM phenylalanine-d₈ (98%; Cambridge Isotope Laboratories, Inc., Tewksbury, MA, USA) for Hydrophilic Interaction Chromatography (HILIC) on an Atlantis^® HILIC column, and acetonitrile/methanol (75:25; v/v) containing 10 mM thymine-d₄, 10 mM L-phenylalanine-d₈ (98%; Cambridge Isotope Laboratories, Inc. Tewksbury, MA, USA). After vortexing, the samples were centrifuged at 20,000× g at 4 °C for 15 min, and the supernatant was transferred to HPLC-grade glass vials with inserts (Waters).

Metabolites were extracted using acetonitrile/methanol (75:25; v/v) containing deuterated internal standards (25 μM thymine-d₄ [Sigma-Aldrich], 10 μM inosine-¹⁵N₄ [Cambridge Isotope Laboratories], 10 μM citrulline-d₇ [Sigma-Aldrich], 25 μM phenylalanine-d₈ [Cambridge Isotope Laboratories], and 10 μM valine-d₈ [Sigma-Aldrich]). The samples were separated using a 2.1 × 100 mm 3.5-μm Xbridge amide column (Waters). Mobile phase A was 95:5 (v/v) water/acetonitrile, with 20 mM ammonium acetate and 20 mM ammonium hydroxide (pH 9.5). Mobile phase B was acetonitrile. For amide-negative mode, the chromatography system consisted of a 1260 Infinity autosampler (Agilent) connected to a 1290 Infinity HLPC binary pump system (Agilent). The eluents were detected in negative mode on a coupled 6490 QQQ mass spectrometry equipped with an electrospray ionization source. The settings were as follows: sheath gas temperature, 400 °C; sheath gas flow, 12 L/min; drying gas temperature, 290 °C; drying gas flow, 15 L/min; capillary, 4000 V; nozzle pressure, 30 psi; nozzle voltage, 500 V; and delta EMV, 200 V. Detailed methods have been described previously⁶² .

LC-MS analysis used a 4000 QTRAP triple quadrupole mass spectrometer (Applied Biosystems/Sciex), coupled to an Acquity UPLC (Waters) as described by Roberts et al. (2012) (link), except that an Aquity UPLC (Waters) was used and multiple reaction monitoring parameters were added as outlined in Table S5. Formic acid, ammonium acetate, LC-MS-grade solvents and valine-d8 were from Sigma-Aldrich. Dried aqueous fractions from HeLa and 2FTGH cells were prepared for LC-MS analyses by the addition of 100 µl of 74.9 : 24.9 : 0.2 (by vol.) acetonitrile/methanol/Formic acid containing the stable isotope-labelled internal standard valine-d8. Samples were vortexed, sonicated and the supernatants were injected directly.

Plasma metabolites were measured using targeted a liquid chromatography tandem mass spectrometry (LC-MS) configured on a 1290 Infinity II U-HPLC coupled to an Agilent 6495 Triple Quadrupole mass spectrometer (Agilent Tech. Santa Clara, CA). Metabolites were extracted from plasma (10 µL) using 90 µL of acetonitrile/methanol/formic acid (74.9:24.9:0.2 v/v/v) containing stable isotope-labeled internal standards (valine-d8, Sigma-Aldrich; St. Louis, MO; and phenylalanine-d8, Cambridge Isotope Laboratories, Andover, MA). The samples were centrifuged (10 min, 9,000 x g, 4°C), and the supernatants were injected directly onto a 150 x 2 mm, 3 µm Atlantis HILIC column (Waters; Milford, MA). The column was eluted isocratically at a flow rate of 250 µL/min with 5% mobile phase A (10 mM ammonium formate and 0.1% formic acid in water) for 0.5 minute followed by a linear gradient to 40% mobile phase B (acetonitrile with 0.1% formic acid) over 10 minutes. Multiple reaction monitoring MS parameters were determined using authentic reference standards for every metabolite. Peak abundances were manually integrated using the MassHunter software provided by the LC-MS manufacturer and visually reviewed to ensure quality.

Methanol (high-performance liquid chromatography grade) was purchased from Merck (Darmstadt, Germany). Ultrapure water was filtered through a Milli-Q water system (EMD Millipore Corporation, Billerica, MA, USA). Derivatization reagents including methoxyamine hydrochloride, pyridine and N-methyl-N-(trimethylsilyl)-trifluoroacetamide (MSTFA) were supplied by Sigma-Aldrich (St. Louis, MO, USA). Valine-d8, succinic acid-d4, phenylalanine-d5, tridecanoic acid, citric acid-d4 and myristic acid-d27 were used as internal standards and obtained from Sigma-Aldrich. Metabolite standards for structure identification were acquired from Sigma-Aldrich, Alfa Aesar (Ward Hill, MA, USA), Fluka (Seelze, Niedersachsen, Germany) and J&K Scientific (Beijing, China).