Varian 600 mhz spectrometer

NMR spectra were recorded on a Varian 400 MHz and/or Varian 600 MHz spectrometer (Varian, Palo Alto, CA, USA ) using CDCl₃ or CDCl₃/methanol-d₄ (4:1) as the solvent, unless otherwise stated. MS analyses were performed on an Agilent Series 1100 SL equipped with an ESI source (Agilent Technologies, Palo Alto, CA, USA). Column chromatography was carried out on silica gel 60 (230–400 mesh) (Sigma-Aldrich, St. Louis, MO, USA) and Sephadex LH-20 (105 × 3 and 69 × 2 cm²) (GE Healthcare Bio-Science, Marlborough, MA, USA). HPLC analysis was carried out on a Hewlett Packard 1100 series instrument with Luna C18 columns (10µ C₁₈ 250 × 4.6 mm², 10 micron; 10μ C₁₈ 250 × 2 mm², 10 micron; Phenomenex (Torrance, CA, USA) as the stationary phase and methanol-water (1:4) as the mobile phase. TLC spots were detected under UV light and by heating after spraying with anisaldehyde reagent.

Plasma and urine samples were sent to the Pusan National University (Busan, Republic of Korea) for nontargeted quantitative metabolome analysis by ¹H-NMR spectroscopy using a Varian 600 MHz spectrometer (Varian, Palo Alto, CA, USA), as detailed by Kim et al. [12 (link)]. The resulting ¹H-NMR dataset contained 31 metabolites in plasma samples and 63 metabolites in urine samples.

The culture medium of each experimental group was collected before and after the treatment (0 and 24 h) for ¹H-NMR analysis. The procedure used to obtain the spectra was similar to the one described by Alves and collaborators [23 (link)]. Briefly, spectra were obtained using samples of 220 µL run in a Varian 600 MHz spectrometer (Varian, Inc., Palo Alto, CA, USA), equipped with a 3 mm indirect detection probe (Varian, Inc., Palo Alto, CA, USA) at 25 °C. As an internal reference, sodium fumarate with a final concentration of 2 mM (singlet, 6.50 ppm) was used to quantify the metabolites in the extracellular medium: H1-α-glucose (duplet, 5.22 ppm), acetate (singlet, 1.9 ppm), alanine (duplet, 1.45 ppm), lactate (duplet, 1.33 ppm), 2-hydroxyisobutyrate (singlet, 1.35 ppm), 3-hydroxyisobutyrate (duplet, 1.08 ppm), and isobutyrate (duplet, 1.05 ppm). Spectra were manually phased, baseline corrected, and quantified by measuring the relative areas of ¹H-NMR resonances using the curve fitting routine in the NUTSpro^TM spectral analysis program (Acorn NMR, Inc., Fremont, CA, USA). Results were normalized in relation to the quantities of the metabolites in the treatment medium before cell incubation and in relation to the number of seeded cells. The resulting values were expressed as metabolite consumption or production (µmol/million of cells).