Phenylalanine d8

T-REx-293 cells were seeded to 6 cm plates at 2×10⁶ and allowed to attach and grow for 1 d. They were then washed with warm PBS and treated with serine-free DMEM supplemented with 3 mM D-formate (Sigma) and 10% dialyzed FBS. After 12 hr treatment, cells were aspirated, washed with ice-cold PBS, and metabolites extracted using 1 ml 80% methanol: 20% water. Deuterated serine and methionine were quantitated as described previously (Mascanfroni et al., 2015 (link)). Briefly, cell extracts (10 μL) were diluted using 90 μL of 74.9:24.9:0.2 vol/vol/vol acetonitrile/methanol/formic acid containing stable isotope-labeled internal standards (valine-d8, Isotec; and phenylalanine-d8, Cambridge Isotope Laboratories; Andover, MA). The samples were centrifuged (10 min, 9000 g, 4°C) and the supernatants were injected directly onto a 150 × 2 mm 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 1 min followed by a linear gradient to 40% mobile phase B (acetonitrile with 0.1% formic acid) over 10 min. The electrospray ionization voltage was 3.5 kV and data were acquired using full scan analysis over m/z 70–800 at 70,000 resolution. LC-MS data were processed and visually inspected using TraceFinder 3.1 software (Thermo Fisher Scientific; Waltham, MA).

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).