Metabolomics dataset of PPAR-pan treated rat liver

This article contains mass spectrometry (MS) data investigating small molecule changes as an effect of a triple peroxisome proliferator-activated receptor (PPAR-pan) agonist GW625019 in the liver as described in the manuscript (Ament et al., 2016) [1]. Samples were measured using gas chromatography-mass spectrometry (GC–MS) for total fatty acid content, and liquid chromatography-mass spectrometry (LC–MS) to measure intact lipids, carnitines and selected aqueous metabolites and eicosanoids. Data files comprise of Excel (Microsoft, WA, USA) spreadsheets of identified metabolites and their area ratio values for total fatty acids, carnitines, aqueous metabolites, and eicosanoids where the intensity of the analytes were normalised to the intensity of the internal standard. In the case of open profiling intact lipid data, the Excel file contains area ratio values of retention time and mass to charge ratio pairs; again, the area ratio values were calculated by normalising to the intensity of the internal standard. It should be noted that several metabolic changes are potentially indirect (secondary, tertiary and ensuing changes).


a b s t r a c t
This article contains mass spectrometry (MS) data investigating small molecule changes as an effect of a triple peroxisome proliferatoractivated receptor (PPAR-pan) agonist GW625019 in the liver as described in the manuscript (Ament et al., 2016) [1]. Samples were measured using gas chromatography-mass spectrometry (GC-MS) for total fatty acid content, and liquid chromatography-mass spectrometry (LC-MS) to measure intact lipids, carnitines and selected aqueous metabolites and eicosanoids. Data files comprise of Excel (Microsoft, WA, USA) spreadsheets of identified metabolites and their area ratio values for total fatty acids, carnitines, aqueous metabolites, and eicosanoids where the intensity of the analytes were normalised to the intensity of the internal standard. In the case of open profiling intact lipid data, the Excel file contains area ratio values of retention time and mass to charge ratio pairs; again, the area ratio values were calculated by normalising to the intensity of the internal standard. It should be noted that several metabolic changes are potentially indirect (secondary, tertiary and ensuing changes

Value of the data
These data provide a broad survey of rat liver metabolite changes due to PPAR-pan agonist treatment.
The different datasets can be used to explore challenges of data merging and integration across analysis platforms.
The study has both a dose response and drug recovery aspect allowing others to model these types of data.

Data
This article contains mass spectrometry data of small molecules, including open profiling assays for total fatty acids (GC-MS) and intact lipids (LC-MS) as well as targeted LC-MS assays for the detection of a range of carnitines, aqueous metabolites and eicosanoids [1]. The carnitine, aqueous and eicosanoid datasets are available in raw data form through MetaboLights (MTBLS278, MTBLS303).

Study design
All animal studies were ethically reviewed and carried out in accordance with Animals (Scientific Procedures) Act 1986 and the GSK Policy on the Care, Welfare and Treatment of Animals. GW625019, a PPAR-pan activator was administered to male Sprague-Dawley rats (Crl:CD (SD) strain), 12 animals per group, by daily oral gavage at 30,100, 300, and 1000 mg/kg/day for 13 weeks. A separate satellite group of animals (6 per group) were kept for a 4 week treatment free period in the control, intermediate 2 (300 mg/kg/day) and high (1000 mg/kg/day) dose groups ( Table 1).
All samples were analysed for total fatty acid, intact lipids, carnitine and aqueous metabolite content. Eicosanoids were measured from a subset of the samples including the control, intermediate 2 (300 mg/kg/day) and high (1000 mg/kg/day) dose groups.

Sampling
Tissue samples were collected following an overdose of anaesthetic (halothane Ph. Eur. Vapour). Samples of the liver were immediately removed, weighed, and sections snap-frozen in liquid nitrogen. Samples were maintained at À80°C until further analysis.

Extraction of total fatty acids, intact lipids, carnitines and aqueous metabolites
Methanol: chloroform solution (2:1, 600 mL) was added to approximately 50 mg of frozen tissue and homogenised with a tissue lyser. Chloroform and water (200 mL each) was added, samples were sonicated for 15 min and centrifuged (13,500 rpm, 20 min). The resulting aqueous and organic layers were separated and the extraction procedure was repeated. Samples were dried under nitrogen and processed for mass spectrometry.

GC-MS analysis of fatty acid methyl esters (FAMEs)
Organic fractions were reconstituted in 1 mL of methanol:chloroform 2:1 and a fifth of each sample (200 mL) was dried under nitrogen. Chloroform:methanol (1:1, 100 μl), boron trifluoride in methanol (10%, 125 μl) and 150 mL D-25-tridecanoic acid (200 mM in chloroform) were added to the dried extracts. Samples were vortex mixed and heated to 80°C for 90 min. After cooling, 300 mL water and 600 mL hexane were added, samples were vortex mixed, the lower aqueous layer was discarded and the remaining organic layer dried under nitrogen. The samples were reconstituted in 150 μl hexane and transferred to autosampler vials prior to analysis using a Trace GC Ultra coupled to a DSQ II single-quadrupole mass spectrometer (Thermo Scientific, Hemel Hempstead, Hertfordshire). Samples were injected onto a Zebron™ ZB-WAX column (100% polyethylene glycol 30 m Â 0.25 mm ID, 0.25 mm film thickness). The injector temperature was 230°C and the flow rate of helium was 1.2 mL/ min. The column was held at 60°C for 2 min, after which the temperature was increased to 150°C at a rate of 15°C/min, and finally increased to 240°C at a rate of 2.5°C/min. The transfer line temperature was maintained at 240°C, while the ion source was at 250°C, operating at 70 eV for electron ionisation (EI). The detector was initiated after 240 s, and full scan spectra were collected over a range of 50-650 m/z [2,3]. GC-MS chromatograms were processed using Xcalibur™ (version 2.0; Thermo Electron, Waltham, Massachusetts) (Supplementary Data 1).

Open profiling LC-MS/MS analysis of intact lipids
A 10 mL aliquot, comprising one hundredth of the organic fraction, was diluted into 90 mL of methanol-chloroform (2:1) containing 20 mM 1,2-diheptadecanoyl-sn-glycero-3-phosphocholine (PC palmitoyl-carnitine, Cambridge Isotope Laboratories, Andover, MA, USA) was added to 40 mL of the organic fraction of the methanol: chloroform extraction and the resulting mixture were dried down under nitrogen and derivatised with 100 mL of 3 M butanolic-HCl (Sigma-Aldrich, Louis, Missouri, USA). Samples were evaporated under nitrogen, re-constituted and sonicated in 4:1 acetonitrile: 0.1% formic acid in water before transferring them to autosampler vials. Samples were analysed using an AB Sciex 5500 QTRAP mass spectrometer (AB Sciex UK Limited, Warrington, Cheshire) coupled to an Acquity UPLC system. Mobile phase A consisted of 0.1% formic acid in water, while mobile phase B was acetonitrile. Two microliters of each sample was injected onto a Synergi Polar RP phenyl ether column (100 mm Â 2.1 mm, 2.5 mm) supplied by Phenomenex (Macclesfield, Cheshire, UK). The analytical gradient started at 30% B, followed by a linear increase to 100% B over 3 min. The gradient was then held at 100% B for 5 min, after which it was returned to the re-equilibration level of 30% B for 2 min. A flow rate of 0.5 mL/min was used throughout [4]. Data were analysed using the Quantitation Wizard within Analyst™ version 1.6 by AB Sciex Ltd. (Warrington, Cheshire, UK) (Supplementary Data 3).

Targeted analysis of aqueous metabolites
The entire aqueous fraction was dissolved in 300 ml of 70:30 acetonitrile: water containing 20 mM universally 13 C-and 15 N-labelled glutamate. Samples were vortex mixed, sonicated, centrifuged, (17,000g, 5 min) pipetted into auto sampler vials and analysed using an AB Sciex 5500 Qtrap mass spectrometer (AB Sciex UK Limited, Warrington, Cheshire) coupled to a SIL20-A LC system (Shimadzu Corp., Kyoto, Japan). Mobile phase A consisted of 100 mM ammonium acetate, mobile phase B was acetonitrile, and the flow rate was 0.3 mL/min. Two microliters of each sample was injected, and analytes separated using a 100 mm ZIC-HILIC column with 2.1 mm ID and 3.5 mm particle size (Sequant, Umeå, Sweden). A linear gradient was used, starting at 20% A for 2 min, followed by an increase to 50% A over 10 min, and finally a 3 min re-equilibration. Metabolites of interest were measured in positive ionisation mode with unscheduled multiple reaction monitoring events (MRMs) ( Table 2)

Extraction and analysis of eicosanoids
Eicosanoids were extracted using solid phase extraction (SPE) Waters Oasis-HLB cartridges (Waters Ltd., Elstree, Hertfordshire, UK) [4]. SPE columns were washed with ethyl acetate (2 mL), methanol (2 Â 2 mL), and 15% methanol with 0.1% acetic acid (2 mL). Approximately 100 mg liver tissue samples were homogenised on a TissueLyser (Qiagen Ltd., Manchester, UK; 10 min at 30 Hz) in 1.5 mL 15% methanol with 0.1% acetic acid. The samples were centrifuged (17,000g, 2 min) and the supernatant loaded onto the SPE columns. Cartridges were washed with 1 mL 15% methanol with 0.1% acetic acid. Analytes of interest were eluted with 0.5 mL of methanol followed by 1 mL of ethyl acetate and immediately dried under nitrogen. Samples were finally reconstituted in 40 mL methanol containing 70 nM PGE2-d4 internal standard and transferred to autosampler vials. Analysis was performed using a 4000 QTRAP mass spectrometer (AB Sciex UK Limited, Warrington, Cheshire) coupled to an Acquity ultra performance liquid chromatography (UPLC) system (Waters Corp., Milford, MA).
The autosampler was maintained at 4°C, LC separation was achieved using a Luna, 3 μm particle size, 150 Â 2 mm column (Phenomenex Macclesfield, Cheshire, UK). The gradient of mobile phase A (0.1% acetic acid in water) and B (0.1% acetic acid in acetonitrile: methanol 80:20) is detailed in Table 3. The flow rate was held at 0.4 mL/min. Metabolites of interest were measured in negative ionisation mode with unscheduled multiple reaction monitoring events (MRMs) (