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<Sample processing and extraction>&l … <Sample processing and extraction><br />
Each sample was extracted at a concentration of 5 mg flesh weight (FW) of tissues per ml extraction medium (methanol / chloroform/water [3:1:1 v/v/v]) containing 10 stable isotope reference compounds:<br />
• [2H4]-succinic acid<br />
• [13C5,15N]-glutamic acid<br />
• [2H7]-cholesterol<br />
• [13C3]-myristic acid<br />
• [13C5]-proline<br />
• [13C12]-sucrose<br />
• [13C4]-hexadecanoic acid<br />
• [2H4]-1,4-butanediamine<br />
• [2H6]-2-hydoxybenzoic acid<br />
• [13C6]-glucose<br />
using a Retsch mixer mill MM310 at a frequency of 30 Hz for 3 min at 4°C. Each isotope compound was adjusted to a final concentration of 15 ng µl-1 for each 1-µl injection. After 5-min centrifugation at 15,100 × g, a 100 µl aliquot of the supernatant was drawn and transferred into a glass insert vial. The extracts were evaporated to dryness in an SPD2010 SpeedVac® concentrator from ThermoSavant (Thermo Electron Corporation, Waltham, MA, USA). For methoximation, 30 µl of methoxyamine hydrochloride (20 mg/ml in pyridine) was added to the sample. After 24 h of derivatization at room temperature, the sample was trimethylsilylated for 1 h using 30 µl of MSTFA with 1% TMCS at 37°C with shaking. For methoximation, 30 µl of methoxyamine hydrochloride (20 mg ml-1 in pyridine) were added to the sample. After 24 h of derivatization at room temperature, the sample was trimethylsilylated for 1 h using 30 µl of MSTFA at 37°C with shaking. After silylation 30 µl of n-heptane were added. All derivatization steps were performed in the vacuum glove box VSC-100 (Sanplatec, Japan) filled with 99.9995% (G3 grade) of dry nitrogen.<br /><br />
<GC-TOF/MS conditions><br />
Using the splitless mode by an CTC CombiPAL auto-sampler (CTC analytics, Zwin-gen, Switzerland), 1 µl of each sample was injected into an Agilent 6890N gas chromatograph (Agilent Technologies, Wilmingston, USA) featuring a 30 m x 0.25 mm inner diameter fused-silica capillary column with a chemically bound 0.25-µl film Rtx-5 Sil MS stationary phase (RESTEK, Bellefonte, USA) for metabolome analysis. Helium was delivered as the carrier gas at a constant flow rate of 1 ml min-1. The temperature program for metabolome analysis started with a 2-min isothermal step at 80°C followed by temperature ramping of 30°C to a final temperature of 320°C, which was maintained for 3.5 min. The transfer line and the ion source temperatures were 250 and 200°C, respectively. Ions were generated with a 70-eV electron beam at an ionization current of 2.0 mA. Data acquisition was on a Pegasus IV TOF MS instrument (LECO, St. Joseph, MI, USA) at an acquisition rate of 30 spectra s-1 in the mass range of a mass-to-charge ratio of m/z = 60-800. The solvent delay was 237 sec.
Alkane standard mixtures (C8-C20 and C21-C40) were purchased from Sigma-Aldrich (Tokyo, Japan) and used for calculating the retention index (RI). The normalized response for calculating the signal intensity of each metabolite from the mass-detector response was obtained with each selected ion current unique in each metabolite MS spectrum to normalize the peak response. For quality control, we injected methylstearate in every 6 samples. Quality control (QC) samples were prepared by mixing 100 μl of extracts of each wild-type sample. We run six QC samples per batch (30 samples in total). C samples per batch (30 samples in total).
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