Thymine d4

LC­MS samples were prepared from stool homogenates (30 μL) via protein precipitation with the addition of four volumes of 80% methanol containing inosine­15N4, thymine­d4 and glycocholate­d4 internal standards (Cambridge Isotope Laboratories; Andover, MA). The samples were centrifuged (10 min, 9,000 x g, 4°C) and the supernatants were injected directly onto a 150 × 2.0 mm Luna NH2 column (Phenomenex; Torrance, CA). The column was eluted at a flow rate of 400 μL/min with initial conditions of 10% mobile phase A (20 mM ammonium acetate and 20 mM ammonium hydroxide in water) and 90% mobile phase B (10 mM ammonium hydroxide in 75:25 v/v acetonitrile/methanol) followed by a 10 min linear gradient to 100% mobile phase A. MS analyses were carried out using electrospray ionization in the negative ion mode using full scan analysis over m/z 60­750 at 70,000 resolution and 3 Hz data acquisition rate. Additional MS settings were: ion spray voltage, ­3.0 kV; capillary temperature, 350°C; probe heater temperature, 325 °C; sheath gas, 55; auxiliary gas, 10; and S­lens RF level 40.

Reduced and oxidized glutathione were profiled in negative ionization mode by liquid chromatography tandem mass spectrometry (LC-MS) methods as described previously^{38 (link)}. Data were acquired using an ACQUITY UPLC (Waters Corp, Milford MA) coupled to a 5500 QTRAP triple quadrupole mass spectrometer (AB SCIEX, Framingham MA). Tissue homogenates (30 μL) were extracted using 120 μL of 80% methanol containing 0.05 ng/μL inosine-¹⁵N₄, 0.05 ng/μL thymine-d₄, and 0.1 ng/μL glycocholate-d₄ as internal standards (Cambridge Isotope Laboratories, Inc., Tewksbury MA). The samples were centrifuged (10 min, 9,000 × g, 4ºC) and the supernatants (10 μL) were injected directly onto a 150 × 2.0 mm Luna NH2 column (Phenomenex, Torrance CA). The column was eluted at a flow rate of 400 μL/min with initial conditions of 10% mobile phase A (20 mM ammonium acetate and 20 mM ammonium hydroxide (Sigma-Aldrich) in water (VWR)) and 90% mobile phase B (10 mM ammonium hydroxide in 75:25 v/v acetonitrile/methanol (VWR)) followed by a 10 min linear gradient to 100% mobile phase A. The ion spray voltage was −4.5 kV and the source temperature was 500°C. Raw data were processed using MultiQuant 2.1 software (AB SCIEX, Framingham MA) for automated peak integration. LC-MS data were processed and visually inspected using TraceFinder 3.1 software (Thermo Fisher Scientific; Waltham, MA).

Sample preparation for metabolite profding was performed as previously described (Xiao et al., 2020 (link)). Briefly, tissues were homogenized in 80% methanol containing inosine-¹⁵N₄, thymine-d4 and glycocholate-d4 internal standards (Cambridge Isotope Laboratories; Andover, MA) at a 4:1 volume to wet weight ratio and centrifuged.

This method was used for stable isotopic labeling analysis (SITA) of [¹³C₆]-glucose incorporation into GSH. Human donor islets were label led for 24 h in RPMI media containing 5.8 mM [¹³C₆]-glucose at a density of 100 islets/ml media. Islet were extracted by sonication in 80% methanol containing internal standards; inosine-¹⁵N₄, thymine-d₄ and glycocholate-d₄ (Cambridge Isotope Laboratories). Extracts were centrifuged at 16,000 RPM 4°C for 20 min, supernatants were collected and loaded onto a Luna-HILIC column through an UltiMate-3000 TPLRS LC, with a mobile phase A (20 mM ammonium acetate and 20 mM ammonium hydroxide dissolved in 5:95 v/v acetonitrile/water) and 90% mobile phase B (10 mM ammonium hydroxide in 75:25 v/v acetonitrile/methanol). Metabolites were separated with a 10 min linear gradient up to 99% mobile phase A. Mass analysis was carried out using a Q-Exactive HF-X mass spectrometer (Thermo Fisher Scientific). Negative modes were used with full scan analysis over m/z 70-750 m/z at 60,000 resolution, 1e6 AGC, and 100 ms maximum ion accumulation time. Additional settings were: ion spray voltage of 3.8 kV; capillary temperature of 350°C; probe heater temperature of 320°C; sheath gas of 50; auxiliary gas of 15; and S-lens RF at level 40.