Vnmrs spectrometer

The reagents and indicator molecules were purchased from Sigma-Aldrich (St. Louis, MO, USA) and Alfa Aesar (Haverhill, MA, USA) and used without further purification. Solvents were freshly distilled prior to use and were dried over anhydrous Na₂SO₄. ¹H and ¹³C-NMR spectra were recorded on a 600-MHz Varian VNMRS spectrometer (Varian, Inc., NMR Systems, Palo Alto, CA, USA, Varian is now part of Agilent Technologies) in DMSO-d₆ or D₂O solutions; δ is given in ppm relative to tetramethylsilane (TMS) as internal standard. ¹H and ¹³C-NMR signals were assigned on the basis of one- and two-dimensional homo- and heteronuclear experiments (COSY, TOCSY, HMBC and HSQC). Melting points were taken on a Stuart SMP-3 apparatus (Global Science NZ Ltd., Auckland, New Zealand). The high-resolution accurate masses were determined with a Dionex Ultimate 3000 UHPLC system hyphenated with an Orbitrap Q Exactive Focus Mass Spectrometer equipped with electrospray ionization (ESI) (Thermo Fischer Scientific, Waltham, MA, USA). Reaction progress was observed by thin-layer chromatography on commercial silica gel plates (Merck silica gel F254 on aluminum sheets, Darmstadt, Germany) using different mobile phases. For column chromatography, Kieselgel 60 (particle size 0.040–0.063 mm, ordered from VWR Chemicals, Radnor, PA, USA) was employed.

All one-dimensional (1D) ¹H-NMR spectra were collected at 25 °C on a 600 MHz Varian VNMRS spectrometer equipped with a cryogenic probe according to our previously published method [38 (link)]. Each 1D spectrum was accumulated for 1028 scans, with an acquisition time of ~2.5 s (24,576 complex points) and a 3 s repetition delay for a total collection time of ~2 h [38 (link)]. 1D ¹H-NMR spectra were referenced to 0.5 mM 4,4-dimethyl-4-silapentane-1-sulfonic acid (DSS). NMR signals arising from small water-soluble metabolites (<1000 Da) were identified and quantified relative to formate (1 mM) as the internal reference by Chenomx software version 6 (http://www.chenomx.com). All metabolite concentrations are reported as values relative to formate.

The rat was placed inside the magnet holder in supine position with a custom-built quadrature ¹H/single loop ¹³C surface coil placed on top of its shaved liver region. The exact position of the coil relative to the liver was adjusted and verified with ¹H images acquired using a gradient echo sequence (8 slices, slice thickness = 2 mm, TR = 50 ms, TE = 3 ms, field of view = 30 × 30 mm, Resolution= 128 × 128, flip angle = 60°). The static magnetic field was homogenized for a selected voxel to reduce the proton linewidth using the FASTESTMAP protocol. A 1 ml bolus of HP [1-¹³C]pyruvate solution at various concentrations was injected through the femoral vein over ~9 s. In vivo ¹³C MR spectra were recorded with a repetition time of ~3 s using a VNMRS spectrometer (Varian, Palo Alto, CA coupled to the 9.4 T horizontal-bore magnet. 30° BIR4 adiabatic RF excitation pulses were applied, and spectra were acquired with proton decoupling. All imaging, shimming and ¹³C MRS acquisitions were respiration gated and cardiac triggered. For the PEPCK inhibition study with 3-MPA administration, fed and fasted rats were injected with 0.066 ± 0.007 mmol/kg of HP [1-¹³C]pyruvate and compared to untreated rats receiving a comparable dose.