Cls8169

Plasma metabolites extraction was performed based on the methodology previously described (Method 1^{89 (link)}). Briefly, 10 µL of plasma were added to 30 µL of cold methanol containing 1 μg/mL of Phe-¹³C as internal standard and 1 μM BHT as antioxidant. Then, samples were incubated at room temperature for 15 min and centrifuged at 12,000 × g for 3 min. Finally, the supernatant was filtrated through a 0.22-μm organic diameter filter (CLS8169, Sigma, Madrid, Spain) and transferred to Agilent (Barcelona, Spain) vials with glass inserts for further analysis.
Sulphur-containing metabolites were extracted on the bases of the methodology previously described (Method 2^{90 (link)}). Briefly, 2 µL of 5% DTT diluted in methanol (m/v) were added to 10 µL of plasma. The resulting solution was vortexed for 1 min and allowed to stand at room temperature for 10 min. For protein precipitation, 40 µL of acetonitrile containing 0.1% formic acid (v/v), 0.05% trifluoroacetic acid (v/v) and 1 µg/mL of Phe-¹³C as internal standard was added to the sample, and the solution was vortexed for 2 min. Then, samples were incubated at room temperature for 15 min and centrifuged at 12,000 × g for 3 min. Finally, the supernatant was filtrated through a 0.22-μm organic diameter filter (CLS8169, Sigma, Madrid, Spain) and transferred to Agilent (Barcelona, Spain) vials with glass inserts for further analysis.

Plasma metabolites’ extraction was made on the basis of a previously described methodology [23 (link)]. In brief, 10 µL of plasma was mixed with 30 µL of cold methanol containing 1 μg/mL of Phe-13C as the internal standard and 1 μM BHT as the antioxidant. After incubation at 4 °C for 60 min, the samples were centrifuged at 12,000× g for 3 min. Then, the supernatant was filtrated (CLS8169, Sigma, Madrid, Spain) and transferred to vials with glass inserts (Agilent, Barcelona, Spain) for chemical analysis. Samples were decoded and randomized before the LC-MS analysis. To serve as quality control for metabolite extraction, a pool of plasma samples with internal Phe-13C was used and injected every 5 samples.