Hilic column

All subjects fasted for 12 hours and refrained from heavy exercise, smoking, and alcohol consumption for 24 hours before the visit. The metabolic and anthropometric measurements were described earlier [17 (link)]. Participants had a frequently sampled glucose tolerance test for which insulin sensitivity (S_I) and acute insulin response (AIR) were calculated. Four isomers of F2-isoprostanes—iPF2α-III, 2,3-dinor-iPF2α-III, iPF2α-VI, and 8,12-iso-iPF2α-VI—are quantified in morning spot urine samples (stored at −70°C) by liquid chromatography with tandem mass spectrometry detection (LC-MS/MS) as previously described. F2-isoprostane levels are corrected by urinary creatinine to account for differences in urine dilution [3 (link)].
Acylcarnitines are also measured by LC-MS/MS. Deuterium-labeled internal standards were added to 25 microliters of serum and the mixture was solubilized in methanol followed by a crash extraction and then injected onto an Atlantis HILIC Column connected to a Waters Xevo triple quadrupole mass spectrometer (Waters, MA). Acylcarnitines were ionized via positive electrospray and the mass spectrometer was operated in the tandem MS mode. The absolute concentration of each acylcarnitine is determined by comparing the corresponding peak to that of the relevant internal standard.

After treatment, one million cells were collected in 1 mL of -80 °C 80% methanol. Samples were vigorously vortexed and frozen, followed by thaw on ice. The freeze and thaw were repeated three times. Then, the supernatant was collected for HPLC analysis of GABA after centrifugation at 13,000 × g for 15 min.
One million cells were cultured with a DMEM medium (1mM Gln -13C (Sigma)) of 10% dialyzed FBS to trace the flux of metabolites. After 24 h, the cell culture medium and cells were collected separately, and the content of glutamine metabolite GABA in cells or the cell culture medium was detected. The samples were lysed as described above. The supernatant was evaporated, and the resulting metabolites were resuspended for LC-MS analysis (Thermo Fisher Q Exactive). The HILIC column (150 × 2.1 mm, 3 μm particle size; Waters Inc) was eluted 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 80% mobile phase B (acetonitrile with 0.1% formic acid) over 25 min. The raw data were processed using Thermo Xcalibur 3.0 software (Thermo Fisher).

MtdL and AtRGP2-mediated reactions were analyzed by ultrahigh-performance liquid chromatography-electrospray ionization-tandem mass spectrometry (UPLC/ESI-Q-TOF-MS) to confirm the correct molecular weights for each substrate and product species. This process was conducted on a 1290 Infinity II LC coupled with a 6545XT AdvanceBio LC/Q-TOF system equipped with a Waters HILIC column (ACQUITY UPLC BEH Amide 2.1 × 100 mm, 1.7 μm). Each procedure, including gradients and detection for chromatographic separation, was carried out as analytical UPLC as noted above.

The crude sample was redissolved in H₂O and then sonicated at 0 °C for 5 min. The sample was injected into the system for UPLC–HRMS detection (Waters) with a HILIC column (2.1 × 50 mm 1.7 μm). The gradient was: 0–5 min, 100% B; 5–15 min, 100% B–40% B, 15–20 min, 40% B–100% B; 20–35 min 100% B at a flow rate of 0.2 Ml min⁻¹. Solvent A: 10 mM NH₄HCO₃; Solvent B: 10 mM NH₄HCO₃, 90% Acetonitrile (ACN). Samples were analyzed by precursor ion screening in negative mode (monitoring m/z 50).

See Supplementary material for serum pretreatment. For organic acid and free-fatty acid detection with gas chromatography-mass spectrometry (GC-MS), chromatographic conditions were tested using the TRACE1310 gas chromatograph and the TSQ9000Evo mass spectrometer (Thermo Finnigan, Austin, TX, United States). The column used was the capillary TG-WAX (30 m × 0.25 mm, 0.25 μm film thickness). For amino acid detection with ultra-performance liquid chromatography-mass spectrometry (UPLC-TQ-MS), the ACQUITYTM UPLC system (Waters Corporation, Milford, CT, United States) was used with a HILIC column (100 mm × 2.1 mm × 1.7 μm, Waters Corporation, Milford, CT, United States). In the above three tests, the quality control (QC) sample was a mixture of equal volumes of samples to be tested. One QC sample was injected at every tenth sample injection to control for batch effects.