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
[email protected]
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