Choline d9

Deuterated isotope tracking was performed by addition of 2 mM choline-d₉ (Sigma-Aldrich) in 50 ml of THB; 3.7 mM GPC-d₉ (Toronto Research Chemicals) was added to 15 ml of THB to yield a final concentration of 130 μM. Cultures were grown in 5% CO₂ at 37°C for 18 h before 10 ml of choline-d₉ culture or 5 ml of GPC-d₉ culture was removed for metabolite extraction and the remaining culture was pelleted for lipid extraction. LysoPC(20:0) was obtained from Avanti Polar Lipids. Cultures were supplemented with 2 mg of lysoPC per 15 ml of THB, unless otherwise stated, and incubated overnight at 37°C in 5% CO₂. Lipid extractions were performed as described above.

L-asparagine (≥98%), choline chloride (≥99%), creatine (≥99.5%), creatinine (≥98%), L-leucine (≥98%), L-methionine (≥98%), L-proline (≥99.5%), taurine (≥99%), L-valine (≥98%), 2-hydroxybutanoic acid, L-lactic acid (≥98%), malic acid (≥99%), and 2-oxo-glutaric acid (≥99%) were purchased from Sigma Aldrich (St. Louis, MO, United States). L-lactic acid-d₃ and choline-d₉ were obtained from Toronto Research Chemicals (Toronto, ON, Canada). L-asparagine ¹³C₄ (≥99%) and L-leucine-d₃ (≥99%) were obtained from Euriso-top (Saint-Aubin, France). Acetonitrile, formic acid, trifluoroacetic acid, isopropanol, methanol, and water (MS-grade) were bought from Biosolve (Valkenswaard, The Netherlands). hemaPEN^® devices were obtained from Trajan Scientific and Medical (VIC, Australia) and Ostro^® 96-well sample preparation plates from Waters Corporation (Dublin, Ireland). MiniCollect^® safety lancets were purchased from Greiner Bio-One (Vilvoorde, Belgium).