Metabolomic Analysis of Plasma Samples

Plasma samples for metabolomics assays were thawed on ice, aliquoted, re-frozen on dry ice, and stored at −80°C prior to delivery to the Fiehn lab. Plasma aliquots (15 µL) were extracted and derivatized as reported previously [29] (link) using 1 mL of degassed acetonitrile:isopropanol:water (3∶3∶2; v/v/v) at −20°C, centrifuged and decanted with subsequent evaporation of the solvent to complete dryness. A clean-up step with 500 µL acetonitrile/water (1∶1; v/v) removed membrane lipids and triglycerides and the supernatant was dried down again. A set of 13 C8–C30 fatty acid methyl ester internal standards were added and samples were derivatized by 10 µL methoxyamine hydrochloride in pyridine followed by 90 µl MSTFA (1 mL bottles, Sigma-Aldrich) for trimethylsilylation of acidic protons. A Gerstel MPS2 automatic liner exhange system (Mülheim an der Ruhr, Germany) was used to inject 0.5 µL of sample at 50°C (ramped by to 250°C) in splitless mode with 25 s splitless time. Analytes were separated using an Agilent 6890 gas chromatograph (Santa Clara, CA) equipped with a 30 m long, 0.25 mm i.d. Rtx5Sil-MS column with 0.25 µm 5% diphenyl film and additional 10 m integrated guard column (Restek, Bellefonte PA). Chromatography was performed with constant flow of 1 mL/min while ramping the oven temperature from 50°C for to 330°C with 22 min total run time. Mass spectrometry was done by a Leco Pegasus IV time of flight mass spectrometer (St. Joseph, MI) with 280°C transfer line temperature, electron ionization at −70eV and an ion source temperature of 250°C. Mass spectra were acquired from m/z 85–500 at 17 spectra s⁻¹ and 1850 V detector voltage. Result files were exported to our servers and further processed by our metabolomics BinBase database [32] . All database entries in BinBase were matched against the Fiehn mass spectral library of 1,200 authentic metabolite spectra using retention index and mass spectrum information or the NIST05 commercial library. Identified metabolites were reported if present within at least 50% of the samples per study design group (as defined in the SetupX database) [33] . Peak heights of quantifier ions defined for each metabolite in BinBase were normalized to the sum intensities of all known metabolites and used for statistical investigation. External 5-point calibration curves established with quality control mixtures containing 30 metabolites controlled for instrument sensitivity. Each chromatogram was further controlled with respect to the total number of identified metabolites and total peak intensities to ensure that outliers did not confound the subsequent statistical analysis.