Gas Chromatograph

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.

Hippocampal tissue samples were homogenized in aqueous buffered solution on ice with a Bead Ruptor 12 (Omni International, Kennesaw, GA, USA). Aliquots of the homogenates (20 μL, which corresponded to tissue equivalents of 1 mg) were subjected to lipid extraction with 1.5 mL methanol/chloroform (2:1, v:v). The extraction solvent contained C₁₇-ceramide and C₁₆-d₃₁-sphingomyelin (both Avanti Polar Lipids, Alabaster, AL, USA) as internal standards. Extraction was facilitated by incubation at 48 °C with gentle shaking (120 rpm) overnight. Pellets of purified cell organelles (lysosomes, Golgi bodies, and ER) were loosened by ultrasonication on ice for 15 min, followed by lipid extraction as described for hippocampal tissue. After lipid extraction, samples were saponified with 150 μL 1 M methanolic KOH for 2 h at 37 °C with gentle shaking (120 rpm). Samples were then neutralized with 12 μL glacial acetic acid and centrifuged at 2200 × g for 10 min at 4 °C. Organic supernatants were evaporated to dryness through vacuum centrifugation with a Savant SpeedVac concentrator (Thermo Fisher Scientific, Dreieich, Germany). Dried residues were reconstituted in 200 μL of a 95:5 (v:v) mixture of HPLC eluents B:A (see below), thoroughly vortexed for 10 min at 1500 rpm, centrifuged at 2200 × g for 10 min at 4 °C, and subjected to mass spectrometric sphingolipid quantification. All analyses were conducted with a 1200 series high-performance liquid chromatograph (HPLC) coupled to a quadrupole time-of-flight (QTOF) 6530 mass spectrometer (Agilent Technologies, Waldbronn, Germany) operating in the positive electrospray ionization (ESI+) mode.
Chromatographic separations were achieved on a ZORBAX Eclipse Plus C8 column (2.1 × 150 mm, 3.5 µm; Agilent Technologies) at 30 °C. The injection volume per sample was 10 μL. A mobile phase system consisting of water (eluent A) and acetonitrile/methanol (1:1, v:v; eluent B), both acidified with 0.1% formic acid, was used for gradient elution at an initial composition of 10:90 (A:B, v:v) and a flow rate of 0.7 mL/min. The total run time for one analysis, including re-equilibration of the HPLC system, was 34 min. For mass spectrometric measurements, the following ion source settings were adjusted: sheath gas temperature, 380 °C; sheath gas flow, 12 L/min of nitrogen; nebulizer pressure, 45 psig; drying gas temperature, 360 °C; drying gas flow, 10 L/min of nitrogen; capillary voltage, 4500 V; fragmentor voltage, 155 V; and nozzle voltage, 2000 V. Ceramides and sphingomyelins, both eluting at various retention times depending on their chain length, were analyzed in tandem mass spectrometry (MS/MS) mode using the fragmentation of the precursor ions into the product ion m/z 264.270 (for ceramides) or m/z 184.074 (for sphingomyelins) [59 (link)]. A collision energy of 25 eV was applied for collision-induced dissociation (CID) of all sphingolipid species investigated. Quantification was performed by means of external calibration with the MassHunter software (Agilent Technologies). Calibration curves of reference ceramides and sphingomyelins were performed from 1 to 100 pmol per injection and were constructed by linear fitting using the least-squares linear regression calculation. The resulting slope of the calibration curve was used to calculate the concentration of the respective analyte in the samples. Determined sphingolipid amounts were normalized to the actual protein content of the homogenate or cell organelle pellet used for extraction.

The analysis of the constituents of the pumpkin seed extract was performed using GC-MS. A 0.2 g sample of pumpkin seed extract was mixed with 2 mL of a methylation mixture (MeOH:benzene:2,2-dimethoxy-propane:H₂SO₄ = 39:20:5:2) and 1 mL of heptane in a 4 mL vial fitted with a Teflon cap, extracted at 80 °C for 2 h, and then filtered for experiment use. Analyses were performed using an Agilent 7890A gas chromatograph equipped with a 5977B mass selective detector (GC/MS) (Agilent Technologies, Santa Clara, CA, USA), with chromatographic separation on a DB-23 column (120 mm × 0.25 mm × 0.25 μm; Agilent). The temperature of injector was set at 250 °C, into which a 1 μL volume of sample was injected in the split mode with a ratio of 10:1. The carrier gas was helium, circulated at a constant flow rate of 2 mL/min. The GC oven temperature was initially set to 80 °C for 1.5 min, and thereafter increased to 110 °C for 2 min at 30 °C/min and then to 200 °C for 8 min at 15 °C/min, to 215 °C for 8 min at 1 °C/min, and finally to 250 °C, at which it was held for 3 min [27 (link)]. Scan mode was used in the range of 29–550 m/z for 62 min. Single compounds were identified by comparing mass spectra with NIST17 mass spectral libraries (National Institute of Standards, 2017 version).

0.1 g feces sample was weight and mixed with 1 mL methanol. After vortexing, ultrasonic treatment and centrifugation, the supernatant was transferred into a new tube. The pellet was further extracted with 1 mL methanol. The supernatant from the two extractions were combined and dried. Then 800 µL 0.5 mmol KOH‐methanol was added, shaked to dissolve and incubated in the dark for 1 h. 800 µL n‐hexane was further added and mixed for 1 min, and then incubated to separate layers. The upper layer of n‐hexane was transfer to a new tube, extract for 3 times. The n‐hexane phases were combined, dried and dissolved in n‐hexane, which was further analyzed using the Agilent 6890N gas chromatograph equipped with PEG‐20 M cross‐linked polyethylene glycol column.