Metabolic Perturbations of Postnatal Growth Restriction and Hyperoxia-Induced Pulmonary Hypertension in a Bronchopulmonary Dysplasia Model

Michael R. La Frano*1,2,3, Johannes F. Fahrmann*1, Dmitry Grapov4,Oliver Fiehn1,5, Theresa L. Pedersen6, John W. Newman1,2,6, Mark A. Underwood7, Robin H. Steinhorn8, Stephen Wedgwood7#

1NIH West Coast Metabolomics Center, Davis, CA

2Department of Nutrition, University of California Davis, Davis, CA

3Department of Food Science and Nutrition, California Polytechnic State University, San Luis Obispo, CA

4CDS Creative Data Solutions, Ballwin, MO

5Department of Biochemistry, Faculty of Sciences, King Abdulaziz University, Jeddah 21589, Saudi-Arabia

6USDA-ARS Western Human Nutrition Research Center, Davis, CA

7Department of Pediatrics, University of California Davis Medical Center, Sacramento, CA

8Department of Pediatrics, Childrens National Medical Center, George Washington University, Washington DC

*authors contributed equally

# Corresponding Author

Stephen Wedgwood, PhD.

Department of Pediatrics

UC Davis Medical Center

Research II Building

4625 2nd Avenue

Sacramento, CA 95817

Tel:(916) 734-1518

swedgwood@ucdavis.edu

Supplementary Material

Sample and data processing description

For analysis of primary metabolites, 30 l plasma aliquots or 5g of lung tissue homogenate, which were extracted with 1 ml of degassed acetonitrile:isopropanol:water (3:3:2) at 20C, centrifuged, the supernatant removed and solvents evaporated to dryness under reduced pressure. To remove membrane lipids and triglycerides, dried samples were reconstituted with acetonitrile/water (1:1), decanted and taken to dryness under reduced pressure. Internal standards, C8C30 fatty acid methyl esters (FAMEs), were added to samples and derivatized with methoxyamine hydrochloride in pyridine and subsequently by MSTFA (Sigma-Aldrich) for trimethylsilylation of acidic protons and analyzed by GC-TOF mass spectrometry. An Agilent 7890A gas chromatograph (Santa Clara, CA) was used with a 30 m long, 0.25 mm i.d. Rtx5Sil-MS column with 0.25 m 5% diphenyl film; an additional 10 m integrated guard column was used (Restek, Bellefonte PA)(Weckwerth et al. 2004; Fiehn 2008; Kind et al. 2007). A Gerstel MPS2 automatic liner exchange system (ALEX) was used to eliminate sample cross-contamination during the GC-TOF analysis. 0.5L microliter of sample was injected at 50C (ramped to 250C) in splitless mode with a 25 sec splitless time. The chromatographic gradient consisted of a constant flow of 1 ml/min, ramping the oven temperature from 50C for to 330C over 22 min. Mass spectrometry was done using a Leco Pegasus IV time of flight mass (TOF) spectrometer, 280C transfer line temperature, electron ionization at 70 V and an ion source temperature of 250C. Mass spectra were acquired at 1525 V detector voltage at m/z 85500 with 17 spectra/sec.

All samples were analyzed in one batch, throughout which data quality and instrument performance were monitored using quality control and reference plasma samples (National Institute of Standards and Technology; NIST). Quality controls (n=4), comprised of a mixture of standards and analyzed every 10 samples, were monitored for changes in the ratio of analyte peak heights, and used to ensure equivalent instrumental conditions (p>0.05, t-Test comparing observed to expected ratios of analyte response factors) over the duration of the sample acquisition(Fiehn et al. 2008). Acquired spectra were further processed using the BinBase database(O. Fiehn et al. 2005; Scholz and Fiehn 2007). Briefly, output results(Kind et al. 2007) were filtered based on multiple parameters to exclude noisy or inconsistent peaks. Detailed criteria for peak reporting including: mass spectral matching, spectral purity, signal-to-noise and retention time are discussed in detail elsewhere(Oliver Fiehn et al. 2005). Known artifact peaks such as polysiloxanes or phthalates were excluded from data export in BinBase. Missing values were replaced by investigating the extracted ion traces of the raw data, subtracted by the local background noise. All 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 NIST11 commercial library. Metabolites were reported if present in at least 50% of the PH or control samples. Data reported as quantitative ion peak heights were normalized by the sum intensity of all annotated metabolites and used for further statistical analysis.

For analysis of complex lipids, plasma aliquots (20L) or lung tissue homogenate (5g), stored at 80C, were thawed on ice and extracted using a modified liquid-liquid phase extraction approach purposed by Matyash et al (Matyash et al. 2008). Briefly, 225l of chilled methanol containing an internal standard mixture (PE(17:0/17:0); PG(17:0/17:0); PC(17:0/0:0); C17 Spingosine; C17 Ceramide; SM (d18:0/17:0); Palmitic Acid-d3; PC (12:0/13:0); Cholesterol-d7; TG (17:0/17:1/17:0)-d5; DG (12:0/12:0/0:0); DG (18:1/2:0/0:0); MG (17:0/0:0/0:0); PE (17:1/0:0); LPC (17:0); LPE (17:1)) and 750L of chilled MTBE (Methyl Tertiary Butyl Ether, Sigma Alrich) containing the internal standard 22:1 cholesteryl ester was added to 20L aliquots of sample. Samples were shaken for 6 minute at 4C using an Orbital Mixing Chilling/Heating Plate (Torrey Pines Scientific Instruments) where after 188L of ultrapure water was added. Samples were vortexed, centrifuged and the upper layer was transferred to a new 1.5mL eppendorf tube. The upper layer was dried under reduced pressure, resuspended in methanol:toluene (90:10) containing 50ng/mL CUDA ((12- [[(cyclohexylamino)carbonyl]amino]- dodecanoic acid, Cayman Chemical), sonicated, centrifuged and subsequently transferred an amber glass vial (National Scientific-C4000-2W) with a micro-insert (Supelco 27400-U).

Resuspended samples were analyzed on an Agilent 1290A Infinity Ultra High Performance Liquid Chromatography system with an Agilent Accurate Mass-6530-QTOF in both positive and negative mode. The column (65C) was a Waters Acquity UPLC CSH C18 (100mm length x 2.1mm internal diameter; 1.7M particles) coupled with a Waters Acquity VanGuard CSH C18 1.7M Pre-column. For positive mode acquisition, the solvent system included A) 60:40 v/v acetonitrile:water (LCMS grade) containing 10mM ammonium formate and 0.1% formic acid and B) 90:10 v/v isopropanol:acetonitrile containing 10 mM ammonium formate and 0.1% formic acid. For negative mode acquisition, the solvent system consisted of A) 60:40 v/v acetonitrile:water (LCMS grade) containing 10mM ammonium acetate and B) 90:10 v/v isopropanol:acetonitrile containing 10 mM ammonium acetate. The gradient started from 0 min 15% (B), 0-2 min 30% (B), 2-2.5 min 48% (B), 2.5-11 min 82% (B), 11-11.5 min 99% (B), 11.5-12 min 99% (B), 12-12.1 min 15% (B), and 12.1-15 min 15% (B). The flow rate was 0.6 mL/min and with an injection volume of 5L for ESI (+/-) mode acquisitions. ESI capillary voltage was +3.5 kV and -3.5 kV with collision energies of 25eV and 40eV for MSMS collection in positive and negative acquisition modes, respectively. Data was collected at a mass range of m/z 60-1700 Da with a spectral acquisition speed of 2 spectra per second. Data quality and instrument performance was monitored throughout the data acquisition using quality control (internal STDS), method blanks and reference pooled plasma samples.

Data was processed using MZmine 2.10. All peak intensities are representative of peak heights. Annotations were completed by matching experimental accurate mass MS/MS spectra to MS/MS libraries, including Metlin-MSMS, NIST12, and LipidBlast(Kind, T, 2013). Spectral matching was automated using the MSPepSearch tool, and manually curated using The NIST Mass Spectral Search Program Version 2.0g. Metabolite libraries were created, in positive and negative ionization modes, containing all confirmed identified compounds. MZmines Custom Database Search tool was used to assign annotations based on accurate mass and retention time matching. Data, reported as peak heights for the quantification ion (m/z) at the specific retention time for each annotated and unknown metabolite, was normalized to the class-specific internal standard (annotated) or to the internal standard which had the closest retention time (unknowns). Pooled Bioreclamation plasma (BioreclamationIVT) and method blanks were used to assess data quality.

For analysis of biogenic amines, including targeted analysis of arginine, citrulline and ornithine, half of the polar (bottom) layer from the lipid extract were dried under reduced pressure and resuspended in 60L of 80:20 ACN/H2O containing the internal standards CUDA, 2g/mL L-arginine-15N2 (Cambridge Isotope Laboratory, Inc), and Val-Tyr-Val (Sigma Aldrich). Resuspended samples were analyzed on an Agilent 1290A Infinity Ultra High Performance Liquid Chromatography system with an Agilent Accurate Mass-6550-QTOF in both positive. The column (45C) was a Waters Acquity UPLC BEH (150mm length x 2.1mm internal diameter; 1.7M particles) coupled with a Waters Acquity VanGuard BEH C18 (50mm length x 2.1 mm internal diameter; 1.7M particles) Pre-column. The solvent system included A) 100% water (LCMS grade) containing 10mM ammonium formate and 0.125% formic acid and B) 95:5 v/v acetonitrile:water containing 10 mM ammonium formate and 0.125% formic acid. The gradient started from 0 min 100% (B), 0-2 min 100% (B), 2-7.7 min 70% (B), 7.7-9.5 min 40% (B), 9.5-10.25 min 30% (B), 10.25-12.75 min 100% (B), and 12.75-16.75 min 100% (B). The flow rate was 0.4 mL/min and with an injection volume of 5L. ESI capillary voltage was +3.5 kV with collision energies of 20eV MSMS collection in positive acquisition mode. Data was collected at a mass range of m/z 60-1700 Da with a spectral acquisition speed of 4 spectra per second.

Plasma non-esterified oxylipins were isolated using a Waters Ostro Sample Preparation Plate (Milford, MA). Aliquots of 50L plasma were extracted and added to the plate wells and spiked with a 5 L anti-oxidant solution (0.2 mg/ml solution BHT/EDTA in 1:1 MeOH:water) and 5 L 1000nM analytical deuterated surrogates. Acetonitrile (150 L) with 1% formic acid was forcefully added to the sample and eluted into glass inserts containing 10 L 20% glycerol with vacuum and dried under reduced pressure. Samples were re-constituted with the internal standards 1-cyclohexyl ureido, 3-dodecanoic acid (CUDA) and 1-phenyl 3-hexadecanoic acid urea (PHAU) at 100 nM (50:50 MeOH:ACN), and filtered at 0.1 m before analysis.

For the targeted analysis of total alkaline stable oxylipins (i.e. esterified and non-esterified species) lung tissue was processed as previously reported (Gladine et al. 2014). Briefly, rat lung samples (~25 mg) were extracted with 10:8:11 cylcohexane: 2- propanol:1 M ammonium acetate, incubated with 100 L 0.5M sodium methoxide for 1hr at 60 C to trans esterify esterified oxylipins, and diluted with 100 L H2O and incubated 30 min at 60C to yield oxylipin free acids which were isolated using 10mg Oasis HLB solid phase extraction column (Waters Corp, Milford Mass) prior to analysis.

Analytes in 50L extract aliquots from plasma and lung extractions were separated utilizing a Waters Acquity UPLC 1.7m, 3.0 X 150mm (Waters, Milford, MA) with a solvent gradient using modifications of a previously published protocols for oxylipins (Strassburg et al. 2012; D. Grapov et al. 2012). Separated residues were detected by negative mode electrospray ionization using multiple reaction monitoring on an API 4000 QTrap (AB Sciex, Framingham, MA, USA). Analytes were quantified using internal standard methods and 5 to 9point calibration curves (r2 0.997). Calibrants and isotopically labeled surrogates were either synthesized or purchased from Cayman Chemical (Ann Arbor, MI), or Larodan Fine Chemicals (Malmo, Sweden). Data was processed with AB Sciex MultiQuant version 3.0.

Data Analysis

Univariate statistical analyses were performed using one-way analysis of variance (ANOVA) on log10 transformed values. The significance levels (i.e. p-values) were adjusted for multiple hypothesis testing according to Benjamini and Hochberg(Benjamini and Hochberg 1995) at a false discovery rate (FDR) of 0.05. Tukey HSD posthoc test was used to determine pairwise group differences. All univariate analysis was performed using DeviumWeb (DeviumWeb 2014).

Multivariate modeling was conducted using principal component analysis (PCA) and orthogonal signal correction partial least squares discriminant analysis (O-PLS-DA)(O. Svensson 2002). Only metabolites with known annotations were included in the multivariate modeling. For both PCA and O-PLS-DA, values were mean centered and scaled to unit-variance. The first two principal components were used for PCA analysis. Colored ellipses (based on the Hotellings T2 95% confidence interval) were used to display experimental group scores. Model latent variable number and orthogonal number was selected using leave-one-out-validation (LOOV). For O-PLS-DA, two latent variables (LV) and one orthogonal LV was selected to discriminate between lung tissue metabolites for the 4 experimental groups; whereas two LVs were selected to differentiate between circulating metabolites for the 4 experimental groups. The probability of achieving the models Q2 (cross-validated fit to the training data) and root mean squared error of prediction (RMSEP, errors of predicting class labels of the test data) was determined based on 100 Monte Carlo cross-validations. For each run the full data set was split into 1/3 test and 2/3 training sets. The training set was used to fit the model (calculate Q2) and to predict the class labels for the tests set (calculate RMSEP). This procedure was repeated for 100 randomly permuted class label models (permutation testing). Model significance (permutation test p-value) was determined based on the comparison of the models performance statistics to that of the permuted models. Multivariate modeling was carried out in DeviumWeb(DeviumWeb 2014).

A Venn diagram was used to illustrate the number of unique and similar metabolic differences (Tukey HSD p-value