Soluble Epoxide Hydrolase Inhibitor Attenuates …Soluble Epoxide Hydrolase Inhibitor Attenuates...
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Soluble Epoxide Hydrolase Inhibitor Attenuates Inflammation and Airway
Hyperresponsiveness in Mice
Jun Yang1, Jennifer Bratt
2, Lisa Franzi
2, Junyan Liu
1, Guodong Zhang
1, Amir A. Zeki
2,
Christoph F. A. Vogel3,4
, Keisha Williams2, Hua Dong
1, Yanping Lin
1, Sung Hee
Hwang1, Nicholas J. Kenyon
2, Bruce D. Hammock
1 *
1 Department of Entomology and Comprehensive Cancer Center,
2 Department of Internal
Medicine, Division of Pulmonary, Critical Care, and Sleep Medicine, 3 Department of
Environmental Toxicology, 4
Center for Health and the Environment. University of
California, Davis, CA 95616.
Address correspondence to: Bruce D. Hammock
Department of Entomology
University of California at Davis
One Shields Avenue, Davis, CA 95616.
Tel: (530) 752-7519
Fax: (530) 751-1537
E-mail: [email protected]
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Abstract
Rationale: Control of airway inflammation is critical in asthma treatment. The soluble
epoxide hydrolase (sEH) has recently been demonstrated as a novel therapeutic target for
treating inflammation, including lung inflammation. Here, we hypothesize that
pharmacological inhibition of sEH can modulate the inflammatory response in a murine
ovalbumin (OVA) model of asthma.
Methods: BALB/c mice were sensitized and exposed to OVA over 6 weeks. The sEH
inhibitor (sEHI) was administrated for two weeks. Respiratory system compliance,
resistance and forced exhale nitric oxide were measured. Lung lavage cell counts were
performed and a selected panel of cytokines and chemokines in the lung lavage fluid
were measured by a Multiplex immunoassay kit. A liquid chromatography tandem mass
spectrometry method was used to measure 87 regulatory lipids mediators in plasma, lung
tissue homogenates, and lung lavage fluid.
Results: The pharmacological inhibition of sEH increased the concentrations of the anti-
inflammatory epoxy eicosatrienoic acids (EETs) and simultaneously decreased the
concentrations of the pro-inflammatory dihydroxyeicosatrienoic acids (DHETs) and
dihydroxyoctadecenoic acids (DHOMEs). All monitored inflammatory markers, which
including FeNO levels, and total cell and eosinophil numbers in the lung lavage of OVA-
exposed mice were reduced by sEHI. The Th2 cytokines (IL-4, IL-5) and chemokines
(Eotaxin and RANTES) were dramatically reduced after administration of sEHI.
Moreover, the resistance and dynamic lung compliance were also improved by sEHI.
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Conclusions: We demonstrated that the administration of an sEHI attenuates allergic
airway inflammation and airway responsiveness in a murine model. sEHI may have
potential as a novel therapeutic strategy for allergic asthma.
Keywords
Soluble epoxide hydrolase; asthma; inflammation; lipid mediators; Th2 cytokines
Running Title
sEHI attenuates lung inflammation in asthma
Listing of contributions of authors:
Conception and design: BDH, JY, NJK;
Analysis and Interpretation: JY, JB, LF, JYL, GDZ, AAZ, CFAV, KW, HD,
YPL;
Drafting the manuscript: JY, BDH, NJK
Word Count:
Abstract: 250 words
Methods: 459 words
Total: 2959 words
Figures: 6
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INTRODUCTION
Three hundred million people worldwide suffer from asthma either episodically or
persistently (1). The cornerstones of treatment for persistent asthma are inhaled
corticosteroids (ICS) and beta-agonist bronchodilators; however, a significant minority of
asthma patients does not respond well to these therapies (2). Thus, there is ongoing effort
to develop novel treatment strategies (3) such as specific antagonists of Th2 cytokines
and mediators.
Epoxyeicosatrienoic acids (EETs, or EpETrEs according to LIPIDMAPS nomenclature)
are a class of important lipid mediators with critical physiological functions that include
vasodilation, anti-inflammation, anti-hypertension, organ protection, and analgesic
effects (4). Specifically in lung health and disease, EETs are reported to affect lung
epithelial ion transport (5-7), relax the pre-contracted bronchi (8), reduce inflammation
(9, 10), regulate endothelial permeability in the lung (11), and regulate pulmonary
vascular pressures (12, 13). Thus, modulation of endogenous EETs is an attractive
approach to potentially control the symptoms of asthma, which include chronic airway
inflammation and airway hyperresponsiveness (AHR).
The soluble epoxide hydrolase (sEH) hydrolyzes these bioactive EETs to corresponding
diols, which are less beneficial, if not toxic. Using potent inhibitors of sEH to stabilize
endogenous EETs (14-17), sEH has recently been demonstrated in animal models as a
novel therapeutic target (4) for treating cardiovascular diseases (18-20), inflammation (21,
22), pain (23-25), and pulmonary diseases such as pulmonary hypertension (26, 27) and
tobacco smoke-induced chronic obstructive pulmonary disease (COPD) (9, 10).
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In the present study, we hypothesized that pharmacological inhibition of sEH can
modulate the inflammatory response in a well-established murine ovalbumin (OVA)-
exposure asthma model. We found that administration of a sEH inhibitor (t-TUCB)
reduced the total inflammatory cell infiltration into the airway and lung, and inhibited
OVA-induced influx of eosinophil. The profiling of regulatory lipid mediators shows that
administration of t-TUCB not only increased the anti-inflammatory lipid mediators-EETs,
but also increased other anti-inflammatory mediators such as 17-hydroxy
docosahexaenoic acid (17-HDoHE) and decreased the pro-inflammatory lipid mediators
including DHOMEs (DiHOMEs) and LTB4 in the plasma, lung tissue, and lavage.
Stabilization of the anti-inflammatory epoxide lipid mediators through pharmacological
inhibition of metabolism by the enzyme-sEH decreased production of Th2 cytokines at
both protein and mRNA levels after OVA induction. Furthermore, compliance and
resistance of the respiratory system were also improved after the administration of sEHI.
These findings support the hypothesis that sEH is a potential target to treat asthma.
METHODS
Animals
Pathogen free male BALB/c mice, aged 8-10 weeks, were purchased from Charles River
Laboratory (Wilmington, MA). All mice were maintained in a HEPA-filtered laminar
flow cage rack with a 12-hour light/dark cycle and allowed free access to food and water.
Figure 1A shows the animal protocol. All procedures with mice were performed in
accordance with an IACUC approved protocol.
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Drug Solutions and Exposure of Mice to Ovalbumin Aerosol
The sEHI, trans-4-{4-[3-(4-trifluoromethoxyphenyl)-ureido]-cyclohexyloxy}-benzoic
acid (t-TUCB), was synthesized as previously described (17). The sEHI was dissolved in
0.05% Tween-80 water solution. The sEHI solution (1 or 3 mg/Kg) was administered
subcutaneously to the mice every day for 14 days. On the last day, the drugs were
administrated half an hour before the exposure of ovalbumin aerosol. The exposure
procedures of ovalbumin used have been described in detail previously (28).
Lung Compliance and Resistance Measurements
The dynamic lung compliance (Cdyn) and resistance of the respiratory system (Rrs) were
simultaneously measured with a whole body plethysmograph for restrained animals
(Buxco Inc., Troy, NY) 1–3 hours after termination of the final OVA exposure. The
whole procedure for this is described in detail in the supplementary Information (SI).
Measurement of Exhaled NO
A 5-minute sample of exhaled gases was collected from the cannulated mice through the
ventilator exhalation port immediately after insertion of the mouse into a plethysmograph
as previously described (29).
Cytokine and Chemokine Assays
The concentrations of selected cytokines and chemokines (Eotaxin, IFN-γ, IL-1β,IL2,
IL4, IL5, IL6, IL10, IL13, RANTES, TNF-α) from BALF supernatant were measured
with commercially available multiplex immunoassays according to the manufacturer’s
instructions (Millipore, St. Charles, MO).
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Measurement of t-TUCB Concentration
Blood (10 µL) was diluted by with 0.1% aqueous EDTA (50 µL) and mixed vigorously.
Samples were then extracted using 200 µL of ethyl acetate twice and dried by Speedvac.
In the end, the residue was reconstituted to 50 µL of internal standard solution and
measured by LC/MS/MS.
Regulatory Lipid Mediator Profiling
Profiles of regulatory mediators were measured using the LC/MS/MS method as
published previously (30). Aliquots of plasma (250 µL), BAL supernatant (2mL), or lung
tissue (~100 mg) were used for the measurements, respectively. Extraction protocols are
fully described in the SI.
Quantitative Real-time Reverse Transcription–PCR (RT-PCR)
Total RNA was isolated from lung tissue using Trizol and a Quick-RNA Mini prep
isolation kit (Zymo Research, Irvine, CA), cDNA synthesis and the RT-PCR processes
are fully described in the SI.
Statistical Analysis
Data are presented as mean values ± SEM. Data were analyzed using unpaired values
compared by two tailed Student's t-test or one-way or two-way ANOVA with Tukey's
post test where appropriate using the Prism 5.0 software package (GraphPad, Inc., San
Diego, CA), with statistical significance defined as a p value of 0.05 or less.
RESULTS
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sEHI was Successfully Delivered and Well Engaged.
After 14 days of subcutaneous injection of sEHI (t-TUCB), concentrations of t-TUCB in
blood (Figure 2A) reached 55.6 ± 13.2 nM (1mg/Kg) and 213 ± 38 nM (3 mg/Kg), which
are 42.8 times and 164 times higher, respectively, than the IC50 of t-TUCB in mouse on
the murine recombinant sEH (1.3 nM) using trans-diphenylpropene oxide (t-DPPO) as
substrate (17). As a result of enzyme inhibition, the concentrations of 14,15-EpETrE, one
endogenous substrate of sEH, increased by 3-3.6 fold from 0.78 nM to 2.56 or 2.86 nM
with administration of 1 or 3 mg/Kg t-TUCB compared to the vehicle control (Figure
2B). Increased concentrations of 14,15-EpETrE after exposure to t-TUCB confirmed the
efficacy of t-TUCB as an sEHI in this study.
Administration of sEHI Increased Anti-inflammatory Mediator Concentrations and
Decreased Pro-inflammatory Mediator Concentrations.
We analyzed the regulatory lipid mediators from BALF, plasma, and lung tissue
homogenate using LC/MS/MS. Figure 3 shows the results presented as heat maps. Figure
3A shows a simplified arachidonic acid cascade listing the major lipid mediators. Figures
3B-D show the significantly changed regulatory lipid mediators after administration of t-
TUCB. In general, for the P450 and sEH pathways, administration of sEHI increased
epoxides in plasma and BALF, and decreased diols in BALF and lung homogenates. In
plasma, administration of sEHI increased some COX and LOX metabolites (11-HETE, 9-
HETE, 5-HETE, 5-HEPE). The low dose of sEHI significantly reduced the pro-
inflammatory mediators: 6-keto-PGF1α (the metabolite and surrogate of prostacyclin-
PGI2) and LTB4. In BALF, administration of sEHI increased LOX metabolites including
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11-HETE, 9-HETE, 5-HETE, 15-HEPE, 17HDoHE, 8-HETE. In lung homogenates,
administration of sEHI increased the COX metabolites 6-keto-PGF1α, TXB2, PGF2α,
PGE3 and PGJ2, 11-HETE; increased the LOX metabolites including 15-HETE, 15(s)
HETrE, 15-HEPE, 17-HDoHE, and 8-HETE.
Administration of sEHI Reduced Th2 Cytokines and Chemokines
Several inflammatory cytokines were induced after OVA exposure (Figure 4 and
supplementary Figure E1). After administration of sEHI, IL-4 and IL-5 in lung lavage
fluid decreased to almost the base line of the control animals (Figures 4A-B). By contrast,
there were no clear trends for those Th1 and innate immune cytokines assayed
(Supplementary Figure E1). Because of the methodological issues involved in IL-13
detection, we could not make firm conclusions regarding involvement of IL-13 in this
model system. The chemokine eotaxin was induced after OVA exposure, and its levels
were blunted by inhibition of sEH (Figure 4C). These data suggest that inhibition of sEH
could reduce the Th2-specific cytokines and chemokines, which are important in
eosinophil trafficking, recruitment, and maturation in airways. Lung expression of IL-4
and IL-5 also showed that RNA levels of these Th2 cytokines were down-regulated by
the administration of sEHI (Figure 4D-E).
Administration of sEHI Reduced Inflammatory Cell Infiltration in Lung Tissues
and Lavage Fluid.
As Figure 5A shows, sensitization and exposure of mice to OVA induced a significant
inflammatory cell infiltration into the airway. The total cell count in BAL reached
approximately 2.6 × 106 cells / mL. Administration of the inhibitor of sEH, t-TUCB at 1
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mg/Kg dose, decreased the number of inflammatory cells infiltrated into the lavage fluid.
Furthermore, 3 mg/Kg t-TUCB significantly reduced the total cell number in BAL to
approximately 46.2%. Analysis of these results by ANOVA showed that there was
significant reduction of total live cell number after administration of sEHI (p=0.04).
Figure 5B shows the differential cell counts determined by the Hema-3 stain set. After
OVA exposure, eosinophil is the dominant inflammatory cell type in BAL comprising up
to 75% of the total inflammatory cell infiltrate. After administration of sEHI, the
percentage of eosinophil was reduced to 65%. t-TUCB at a dose of 3 mg/Kg significantly
reduced the eosinophil infiltration into lung lavage from 1.63× 106 to 7.05 × 10
5. The
ANOVA analysis shows that there is significant reduction of total live cell number after
administration of sEHI (p=0.049). It indicates that sEHI not only reduced the total
inflammatory cell infiltration into the airway but also altered the ratio of inflammatory
cells present in the BAL (eosinophil/macrophage ratio from 4.39 to 2.30). This result also
corresponds to the reduction of Th2 cytokines in the lavage and lung tissue. Exhaled
nitric oxide (NO) is a biomarker of airway eosinophil inflammation, and was increased
from 5.43 ppb to 15.1 ppb after ovalbumin exposure (Figure 5C). Treatment with
1mg/Kg and 3 mg/Kg of t-TUCB decreased the induction of this inflammatory biomarker
to 3.68 ppb and 6.57 ppb respectively. The ANOVA analysis with Bonferroni post test
shows that there is significant reduction of FeNO after administration of sEHI
(p=0.0006). In lung tissues, there was marked inflammatory cell influx in the peri-
bronchiolar space post-OVA exposure as shown in the H&E stain result (Figures 5, D-E)
consistent with the lung lavage data (Figure 5A-B). After inhibition of sEH,
inflammatory cells in the lung tissue were reduced in a dose dependent manner (Figures
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5, F-G). These figures show that administration of sEHI reduced the lung inflammatory
cell infiltration, which is consistent with the inflammatory cell results in the BAL cell
counts.
Taken together, there is severe eosinophil dominant inflammation in both airway and
alveolar of the mice after exposure of ovalbumin. Administration of sEHI reduced this
inflammation as shown in total live cell number, inflammatory cell differentiation, H&E
stained lung tissue, and FeNO.
sEHI Reduced Methacholine-induced Changes in Resistance and Dynamic
Compliance of the Respiratory System.
Exposure to OVA reduced the baseline compliance from 0.036 ml/cm H2O (FA) to 0.023
mL/cm H2O (OVA + vehicle). Administration of 3 mg/Kg sEHI rescued the baseline
compliance to 0.03 ml/cm H2O (Figure 6A). Although 1 mg/Kg sEHI did not rescue the
baseline compliance, both sEHI treatment groups did help to prevent the change in
compliance with methacholine challenge (p<0.0001 for overall effect by treatment group,
p=0.0005 for the doses, assessed by two-way ANOVA).
OVA exposure increased both baseline resistance and the slope of resistance change in
response to the methacholine challenge, as presented in Figure 6B. Administration of
sEHI rescued the baseline resistance in a dose dependent manner (p=0.0035 for overall
effect by treatment group, p=0.0018 for the doses of methacholine, assessed by two-way
ANOVA). At 1 mg/Kg, sEHI reduced the slope of resistance change along with
methacholine, whereas sEHI at a dose of 3 mg/Kg shows a more complex pattern: a
shallow initial slope with low methacholine dose, then rapid increase in slope with the
higher dose of methacholine.
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DISCUSSION
To summarize briefly, we demonstrated that administration of a soluble epoxide
hydrolase inhibitor markedly attenuates the allergic airway inflammation and AHR
caused by exposing mice to ovalbumin. Specifically, the sEHI reduced the total
inflammatory cell number by 50%. Compared with two clinically available compounds,
montelukast and dexamethasone tested in the similar animal model, sEHI had a greater
effect at reducing the inflammatory cells infiltration more than montelukast and
dexamethasone did (40% and 28%) (31). In addition, sEHI reduced IL-5 levels to 12.5%
of those mice treated with ovalbumin and vehicle. Taken together, sEHI is a promising
potential candidate drug to treat allergic asthma.
We and others have previously shown the anti-inflammatory effects of sEHIs on different
disease models (9, 21, 22, 32). The mechanism is believed to be through stabilizing of the
anti-inflammatory EET, which regulates NFκ-B translocation (33) or reducing production
of pro-inflammatory diols including DHOMEs (34) and dihydroxy eicosatrienoic acid
(DHET) (35). Here, our data show that the concentration of sEHI in the plasma
significantly altered the circulating EETs and DHETs levels present in plasma, BALF
and lung homogenates (Figure 3C, 3D). Node (33) reported that EETs (EpETrEs) can
reduce endothelial cell VCAM-1 expression in response to TNF-α, IL-1α, and LPS. At
the same time, administration of sEHI significantly reduced pro-inflammatory DHETs in
BALF and lung homogenate (Figure 3C, 3D). The DHETs were reported as essential for
monocyte chemotaxis to MCP-1 (35). In particular, sEHI reduced in the lung
homogenates the DHOMEs, metabolites of leukotoxin and iso-leukotoxin. It was found
that these DHOMEs are more toxic than EpOMEs and are associated with multiple organ
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failure and adult respiratory distress syndrome (34). Taken together, the effect of the
sEHI, t-TUCB, on the concentrations of both EETs and DHETs may contribute to the
anti-inflammatory effects of sEHI in this murine asthmatic model.
In this study, we also observed that additional lipid mediators were affected upon
administration of t-TUCB. The pro-inflammatory lipid mediator-LTB4 was decreased in
the plasma after the low dose of administration of sEHI. It was reported that LTB4
participates in the allergic sensitization process in animal models (36). Therefore, the
effect of inhibition of sEH might also benefit from reduction of pro-inflammatory LTB4.
Another lipid mediator, 17HDoHE, which is a precursor to resolvins and possesses
biological activity that inhibits TNFα-induced IL-1β expression (37), was increased in
both lavage and lung homogenates after administration of sEHI. Resolvins have been
reported to promote the resolution of allergic airways response (38).
Our findings add to those reported elsewhere by describing the effects of sEH inhibition
on allergic airway inflammation. Several studies (10, 21) have demonstrated that sEHI
can reduce the inflammatory cytokines IL-1β, IL-6, INF-γ. In the present study, we found
that sEHI reduced Th2 cytokines and chemokines, which are known to play major roles
in the asthmatic immune response (39). Specifically, the pronounced effect of sEHI on
IL-5 and eotaxin-1, a key cytokine and chemokine responsible for the release of
eosinophil from the bone marrow and homing of eosinophil to the lung, is rather
intriguing. Indeed mepolizumab, a monoclonal antibody against IL-5, used for the
treatment of severe asthma, is garnering significant attention in the clinical realm (40).
The inhibition of sEH may be an alternative strategy for decreasing IL-5 levels in concert
with other key mediators of lung inflammation.
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There are reports indicating that some of these regulatory lipid mediators, such as EETs
have direct function on the bronchi (6, 41). We observed that administration of sEHI
increased EETs in the BALF as shown in Figure 5C, which may directly rescue the
airway hyperreactivity. However, inflammation may also play a role in regulating lung
compliance and resistance. The anti-inflammatory effects of sEHI might have contributed
to the improvement of lung function after sEHI administration in this acute model of
asthma. In addition, it is worth keeping in mind that lipid mediators such as HETEs are
reported (42) to have effects on the airways. Alterations in the levels of various lipid
mediators may explain why treatment with 3 mg/kg of sEHI did not show improved
rescue (in comparison to treatment with 1 mg/kg) of the resistance induced by 2.0 mg/mL
of methacholine (Figure 6B). Direct pulmonary administration of an sEH inhibitor would
provide additional evidence on how sEHI regulates lung compliance and resistance. To
date we have not developed an effective system for administering sEHI directly to the
lung.
In our current study, neither COX2 nor 5-LOX in lung homogenates were significantly
suppressed by sEHI administration in OVA-exposed mice (supplementary Figure E2).
These findings suggest, at least in lung homogenates, that the major effects of sEHI are
unlikely due to the NFκ-B pathway.
Taken together, the administration of sEHI increased the anti-inflammatory mediators
both systemically and in the airways, as indicated by the lipid mediator levels from the
lavage, while simultaneously decreased pro-inflammatory mediators in the lung tissues
and airways. These lipid mediators changes influenced the reduction and down-regulation
of Th2 cytokines and chemokines expression in the airways. The reduction of these Th2
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cytokines and chemokines further decreased the recruitment of inflammatory cells
infiltration into the lungs and airways. The reduction in overall lung inflammation and the
increase of EETs in airway contributes to the alleviation of AHR.
The data are consistent with the sEH (EPHX2) being primarily responsible for the
conversion of fatty acid epoxides to the corresponding diols in the lung and with t-TUCB
inhibiting this catalytic activity. The activity of mEH (EPHX1) on this substrate is low
and pulmonary levels of the mEH are low. Also we have found no evidence for catalytic
activity of EH3 (EPHX3) or 4 in the lung. Although there is no evidence of inhibition of
EPHX1, 3, 4 or an unknown enzyme by t-TUCB, we cannot exclude the possibility of off
target effects with absolute certainty.
Among the limitations of this study was that we used only prophylactic and not
therapeutic treatment. Long term pulmonary inflammation including asthma leads to
chronic changes in the lung. However, our short-term model did not assess the effects of
sEHI on fibrotic biomarkers since this would require a longer term exposure to
inflammation, particularly that is associated with chronic diseases involving the use of
multiple drugs with different mechanisms of action. Future studies need to address
possible beneficial and detrimental effects of long term sEHI use.
Acknowledgements
This work was supported by American Asthma Association AAF 09-0269,
NIEHS grant R01 ES002710 and NIEHS Superfund Research Program grant P42
ES004699. Analytical work was partially supported by the NIH and NIDDK grant U24
DK097154. NIH HL105573 provided the NO work equipment. JY was supported by a
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Fellowship from Cystic Fibrosis Research, Inc. BDH is a George and Judy Marcus senior
fellow of the American Asthma Foundation. AAZ was funded by #UL1TR000002 and
CTSC NIH KL2 (K12) Award TR000134. JB was supported by T32HL007013.
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18. Xu D, Li N, He Y, Timofeyev V, Lu L, Tsai HJ, Kim IH, Tuteja D, Mateo RK,
Singapuri A, Davis BB, Low R, Hammock BD, Chiamvimonvat N. Prevention and
reversal of cardiac hypertrophy by soluble epoxide hydrolase inhibitors. Proc Natl Acad
Sci U S A 2006;103:18733-18738.
19. Ai D, Fu Y, Guo D, Tanaka H, Wang N, Tang C, Hammock BD, Shyy JY, Zhu Y.
Angiotensin II up-regulates soluble epoxide hydrolase in vascular endothelium in vitro
and in vivo. Proc Natl Acad Sci U S A 2007;104:9018-9023.
20. Ai D, Pang W, Li N, Xu M, Jones PD, Yang J, Zhang Y, Chiamvimonvat N, Shyy
JY, Hammock BD, Zhu Y. Soluble epoxide hydrolase plays an essential role in
angiotensin II-induced cardiac hypertrophy. Proc Natl Acad Sci U S A 2009;106:564-569.
21. Liu JY, Yang J, Inceoglu B, Qiu H, Ulu A, Hwang SH, Chiamvimonvat N,
Hammock BD. Inhibition of soluble epoxide hydrolase enhances the anti-inflammatory
effects of aspirin and 5-lipoxygenase activation protein inhibitor in a murine model. .
Biochem Pharmacol 2010;79:880-887.
22. Schmelzer KR, Kubala L, Newman JW, Kim IH, Eiserich JP, Hammock BD.
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23. Inceoglu B, Jinks SL, Ulu A, Hegedus CM, Georgi K, Schmelzer KR, Wagner K,
Jones PD, Morisseau C, Hammock BD. Soluble epoxide hydrolase and
epoxyeicosatrienoic acids modulate two distinct analgesic pathways. Proc Natl Acad Sci
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20
24. Inceoglu B, Wagner K, Schebb NH, Morisseau C, Jinks SL, Ulu A, Hegedus C,
Rose T, Brosnan R, Hammock BD. Analgesia mediated by soluble epoxide hydrolase
inhibitors is dependent on camp. Proc Natl Acad Sci U S A 2011;108:5093-5097.
25. Inceoglu B, Wagner KM, Yang J, Bettaieb A, Schebb NH, Hwang SH, Morisseau
C, Haj FG, Hammock BD. Acute augmentation of epoxygenated fatty acid levels rapidly
reduces pain-related behavior in a rat model of type I diabetes. Proc Natl Acad Sci U S A
2012;109:11390-11395.
26. Keseru B, Barbosa-Sicard E, Schermuly RT, Tanaka H, Hammock BD,
Weissmann N, Fisslthaler B, Fleming I. Hypoxia-induced pulmonary hypertension:
Comparison of soluble epoxide hydrolase deletion vs. inhibition. Cardiovasc Res
2010;85:232-240.
27. Revermann M, Barbosa-Sicard E, Dony E, Schermuly RT, Morisseau C,
Geisslinger G, Fleming I, Hammock BD, Brandes RP. Inhibition of the soluble epoxide
hydrolase attenuates monocrotaline-induced pulmonary hypertension in rats. J Hypertens
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21
30. Yang J, Schmelzer K, Georgi K, Hammock BD. Quantitative profiling method for
oxylipin metabolome by liquid chromatography electrospray ionization tandem mass
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38. Rogerio AP, Haworth O, Croze R, Oh SF, Uddin M, Carlo T, Pfeffer MA, Priluck
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Figure Legends
Figure 1. The protocol for exposure and treatments. The BALB/c mice were sensitized by i.p.
(intraperitoneal) injection of ovalbumin (OVA) and alum adjuvant solution for 4 weeks and then
were exposed to OVA aerosol six times. Depending on the treatment groups, mice were injected
subcutaneous (s.c.) with vehicle solution, 1 mg/Kg t-TUCB or 3mg/Kg t-TUCB by s.c. every
other day from day 26 to day 38. Previous pharmacokinetic studies with t-TUCB in mice
indicated that these doses will give good exposure.
Figure 2. The concentration of t-TUCB in blood (A) and 14,15-EpETrE (EET) concentration in
plasma (B) suggest that the inhibitor was delivered successfully in vivo and well engaged. Blood
was drawn 2-6 hours after administration of the last dose. * means significant difference from
vehicle group. # means significantly difference between 1 mg/Kg and 3 mg/Kg t-TUCB groups.
All of three groups were exposed to OVA. (n=5 in all groups except n=4 in Air+Vehicle group)
Figure 3. Heatmaps generated from regulatory lipid mediators show that administration of t-
TUCB increased anti-inflammatory lipid mediators and decreased pro-inflammatory lipid
mediators in vivo. (A) A simplified depiction of the arachidonic acid cascade. (B-D) Heat maps
based on regulatory lipid mediators result of plasma (B), Bronchoalveolar lavage fluid (BALF)
(C), and lung homogenates (D). The color is corresponding to the fold change as shown in the
legend. Bright red means increase more than 2 times significantly (p<0.05); dark red means
increase less than 2 times significantly; black means no significant change; dark green means
decrease less than 2 times significantly; bright green means decrease by more than 2 times
significantly. (For plasma and BALF, n=5 in all groups except n=4 in Air+Vehicle group; for
lung homogenates result, n=4 in all groups except n=3 in Air+Vehicle group)
Figure 4. The administration of t-TUCB dramatically decreased the Th2 cytokines (IL-4 and IL-5)
(A and B) and chemokine (Eotaxin) (C) production in BALF and down-regulated the gene
expression of these Th2 cytokines (D and E) in lung homogenates. * means significant difference
from OVA+vehicle group. (n=5 in all groups except n=4 in non-immunized+Vehicle group).
Figure 5. (A) Administration of t-TUCB reduced the infiltrated inflammatory cells (#cell/mL) in
BAL. (B) The result of differentiation cells by Hema-3 stain shows that sEHI reduced the
number of eosinophil (#cell/mL) in BAL. (C) Administration of t-TUCB reduced the
inflammatory marker-FeNO. (D-G). The H&E stain results of lung tissues shows that sEHI
reduces the infiltration of inflammatory cells into lung tissues. (D) is from the air control group;
(E) is from the vehicle control after OVA exposure; (F) is from 1 mg/kg t-TUCB treated group;
(G) is from 3 mg/kg t-TUCB treated group. * means significant difference from OVA+vehicle
group.
Figure 6. Dynamic lung compliance (A) and airway hyperreactivity (B) results show that
administration of t-TUCB rescued OVA-induced asthmatic AHR. (n=4 for OVA+ Vehicle group
Page 23 of 76 AJRCMB Articles in Press. Published on 12-June-2014 as 10.1165/rcmb.2013-0440OC
Copyright © 2014 by the American Thoracic Society
and OVA+ 3 mg/Kg t-TUCB group; n=3 for non-ummunized+Vehicle group and OVA+ 1
mg/Kg t-TUCB group.)
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Figure 1
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Figure 2
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Figure 3
(A)
(B) Plasma
(C) BALF
(D) Lung Homogenate
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Figure 4.
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Figure 5
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(D) (E)
(F) (G)
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Figure 6.
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1
Supplementary Files
Soluble Epoxide Hydrolase Inhibitor Attenuates Inflammation and Airway
Hyperresponsiveness in Mice
Jun Yang1, Jennifer Bratt
2, Lisa Franzi
2, Junyan Liu
1, Guodong Zhang
1, Amir A. Zeki
2,
Christoph F. A. Vogel3,4
, Keisha Williams2, Hua Dong
1, Yanping Lin
1, Sung Hee
Hwang1, Nicholas J. Kenyon
2, Bruce D. Hammock
1 *
1 Department of Entomology and Comprehensive Cancer Center,
2 Department of Internal
Medicine, Division of Pulmonary, Critical Care, and Sleep Medicine, 3 Department of
Environmental Toxicology, 4
Center for Health and the Environment. University of
California, Davis, CA 95616.
Address correspondence to: Bruce D. Hammock
Department of Entomology
University of California at Davis
One Shields Avenue, Davis, CA 95616.
Tel: (530) 752-7519
Fax: (530) 751-1537
E-mail: [email protected]
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2
SUPPLEMENTARY MATERIALS AND METHODS
Lung compliance and resistance measurements
The dynamic lung compliance (Cdyn) and resistance of the respiratory system (Rst) was
measured with a plethysmograph for restrained animals (Buxco Inc., Troy, NY) 1–3
hours after termination of the final (6th) OVA exposure. Mice were deeply anesthetized
and sedated with medetomidine, 0.5 mg/kg (Domitor, Orion Pharma, Finland), and
tiletamine/zolpidem, 50 mg/kg (Telazol, Fort Dodge Laboratories, Fort Dodge, IA) and
surgically cannulated and ventilated at 7–8 cc/kg using a mouse ventilator (MiniVent,
Harvard Apparatus, Cambridge, MA) for the duration of the procedure. Cdyn (mL/cm of
H2O) and Rst (cm of H2O*sec/mL) measurements were made at baseline and
immediately following serial 3 minute nebulizations of saline and methacholine (0.5, 1.0
and 2.0 mg/ml), with 2-minute recovery periods allowed after each exposure.
Lung Tissue Processing
Immediately after lung physiology measurements, mice were killed with an overdose of
Beuthanasia-D. After blood collection, lungs were lavaged twice with 1 ml of PBS (pH
7.4), and then centrifuged at 2,500 rpm on a tabletop centrifuge for 10 minutes. The total
lavage live cell number and differential cell counts were determined.
Mice had their lungs fixed or immediately frozen and stored at -80 °C. Lungs were fixed
for histological evaluation with 1% paraformaldehyde. After fixation and paraffin
embedding, the lungs were stained with hematoxylin and eosin to qualitatively assess
peribronchiolar inflammation. Half of right lungs were immersed in RNA Later solution
(Life Technology, Carlsbad, CA) for the followed RT-PCR measurements.
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3
Airway Inflammatory Cell Populations
Total cell counts in the lung lavage fluid were determined using a hemocytometer and
trypan blue exclusion; 100 µL of the remaining cell suspension was processed onto slides
using a cytocentrifuge at 1650 rpm for 7 minutes for determination of the cell differential
counts. Slides were air-dried, stained with a Hema3 stain set as described in the
manufacturer's instructions (Fisher Scientific, Kalamazoo, MI).
LC/MS/MS analysis for t-TUCB
The liquid chromatography system used for analysis was an Agilent 1200 SL liquid
chromatography series (Agilent Corporation, Palo Alto, CA). The autosampler was kept
at 4 °C. Liquid chromatography was performed on an ACQUITY UPLC BEH C18, 1.7
µm, 2.1× 50 mm column (Waters, Milford, MA). Mobile phase A was water with 0.1%
glacial acetic acid. Mobile phase B consisted of acetonitrile with 0.1% glacial acetic acid.
Gradient elution was performed at a flow rate of 400 µL/min. The gradient began with
50% B and reached 95% B at 3 min. After holding at 95% for 0.5 min, the composition
of mobile phase went back to 50% at 3.5 min for another 0.5 min. The column was
connected to a 4000 QTrap tandem mass spectrometer (Applied Biosystems Instrument
Corporation, Foster City, CA) equipped with an electrospray source (Turbo V). The
instrument was operated in negative MRM mode. Individual analyte standards were
infused into the mass spectrometer and MRM transitions and source parameters
optimized for t-TUCB. The MRM transition for t-TUCB is 437.2/137.1; the transition for
CUDA is 339.2/214.3.
Extraction Protocol for Plasma Oxylipin Profile
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4
The Oasis cartridges (Waters,) were washed with ethyl acetate and methanol, and
equilibrated to the initial condition with the solution containing H2O and methanol.
Diluted supernatant was loaded on the cartridge and washed with SPE solution (H2O with
5% methanol with 0.1% acetic acid) twice. The cartridges were dried by vacuum and
analytes were eluted with 0.5 mL of methanol and 1.5 mL of ethyl acetate to tubes with 6
µL of trapping solution (30% glycerol in methanol). The elutes were dried by vacuum
(SpeedVac) and reconstitute with internal solution (200 nM CUDA methanol solution).
Extraction Protocol for BALF Oxylipin Profile
Ten microLiters of antioxidant solution (0.2 mg/mL of BHT and EDTA) were added to 2
mL BAL fluid supernatant followed by the addition of 10 uL of surrogate solutions, in
which 9 deuterated internal standards were included. BAL fluids were then loaded on the
Oasis cartridges as mentioned above. The followed SPE protocol is same with the plasma
extraction protocol described above.
Extraction Protocol for Lung Homogenates Oxylipin Profile
Ten microLiters of anti-oxidant solution were added to 100mg of lung tissue followed by
the addition of 10 µL of surrogate solution. After addition of 400 µL of ice-cold
methanol with 0.1 % of acetic acid and 0.1% of BHT, the lung tissues were stored at -
80°C freezer for 30 min. Then, the lung tissue samples were homogenized using Mixer
Mill MM301 (Retsch, Haan, Germany) at 30 Hz for 10 min. The homogenates were
stored at -80°C freezer overnight. After centrifuged at 10,000 rpm for 10 min, the
supernatant were collected and the remaining pellets were washed with 100 µL of ice-
cold methanol with 0.1 % of acetic acid and 0.1% of BHT and centrifuged again. The
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5
supernatants of each sample were combined and diluted with 2 mL of H2O and load onto
SPE cartridges. Then the SPE protocol is the same as the one for plasma extraction
protocol.
Quantitative Real-time Reverse Transcription–PCR (RT-PCR)
Total RNA was isolated from tissue using Trizol and a Quick-RNA Mini prep isolation
kit (Zymo Research, Irvine, CA), and cDNA synthesis and RT-PCR process are fully
described in the supplementary files. was done as previously described (1). Quantitative
detection of β-actin and differentially expressed genes was performed with LightCycler
LC480 (Roche, Indianapolis, IN) using the Fast SYBR Green Master Mix (Life
Technologies, Grand Island, NY) according to the manufacturer’s instructions. DNA-free
total RNA (1.0 µg) was reverse-transcribed using 4 U Omniscript reverse transcriptase
(RT; Qiagen) and 1 µg oligo(dT)15 in a final volume of 40 µl. The primers for each gene
were designed on the basis of the respective cDNA or mRNA sequences using OLIGO
primer analysis software provided by Steve Rozen and the Whitehead Institute/MIT
Center for Genome Research (2). So, that the targets were 100–200 bp in length. The
following primer sequences were used: mouse β-actin (forward primer, 5′-
AGCCATGTACGTAGCCATCC-3′; reverse primer, 5′-
CTCTCAGCTGTGGTGGTGAA-3′), mouse IL-4 (forward primer, 5’-
TCAACCCCCAGCTAGTTGTC-3’; reverse primer, 5’-
TGTTCTTCGTTGCTGTGAGG-5’), mouse IL-5 (forward primer, 5’-
GAAGTGTGGCGAGGAGAGAC-3’; reverse primer, 5’-
GCACAGTTTTGTGGGGTTTT-3’) and mouse IL-13 (forward primer:5’-
CAGCTCCCTGGTTCTCTCAC-3’); reverse primer: 5’-
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6
CCACACTCCATACCATGCTG-3’). PCR amplification was carried out in a total
volume of 20 µl containing 2 µl cDNA, 10 µl 2 × Fast SYBR Green Master Mix, and 0.2
µM of each primer. The PCR cycling conditions were 95°C for 30 sec followed by 40
cycles of 94°C for 3 sec, and 60°C for 30 sec. Detection of the fluorescent product was
performed at the end of the 72°C extension period. Negative controls were concomitantly
run to confirm that the samples were not cross-contaminated. A sample with DNase- and
RNase-free water instead of RNA was concomitantly examined for each of the reaction
units described above. To confirm the amplification specificity, the PCR products were
subjected to melting curve analysis.
Western blot method
The lung tissue were solicited in a homogenization buffer containing 0.1% SDS, protease
inhibitor cocktail (Sigma-Aldrich, St. Louis, MO) and 574 µM phenylmethanesulfonyl
fluoride (PMSF) in PBS. Homogenates were centrifuged at 10,000 rpm for 20 minutes
and the resulting supernatant was stored at -80 °C until further use.
Antibodies were purchased from Santa Cruz Biotechnology, Inc (Santa Cruz, CA) unless
otherwise stated. Total protein concentration of homogenate samples was determined
using the Micro BCA Protein Assay Kit (Pierce Biotechnology, Rockford, IL). Samples
containing 20 µg of protein were incubated at 65 °C for 15 minutes, electroporated under
reducing conditions, and transferred to a polyvinylidene difluoride (PVDF) membrane.
Membranes were blocked in 5% dry milk in PBS for 1 hour at 25 °C, then incubated in
0.4 µg/ml of goat, anti-mouse Arg1, 0.4 µg/ml of goat, anti-mouse Arg2, or 0.4 µg/ml of
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7
rabbit anti-human α-actinin overnight at 4 °C, then incubated for 1 hour at 25 °C in 40
ng/ml of horseradish peroxidase (HRP)-conjugated donkey anti-goat IgG (Pierce
Biotechnology, Rockford, IL) or 40 ng/ml of horseradish peroxidase (HRP) conjugated
goat anti-rabbit IgG (Pierce Biotechnology, Rockford, IL). Bands were visualized using
the Immobilon western chemiluminescent HRP substrate kit (Millipore, Billerica, MA)
and images captured using Image Reader LAS-3000 version 2.1 (FUJIFILM, Cypress,
CA).
References
1. Vogel, C. F., Sciullo, E., Park, S., Liedtke, C., Trautwein, C., Matsumura, F.,
2004. Dioxin increases C/EBPbeta transcription by activating cAMP/protein
kinase A. J Biol Chem 279, 8886-94.
2. Rozen, S., Skaletsky, H., 2000. Primer3 on the WWW for general users and for
biologist programmers. Methods Mol Biol 132, 365-86.
SUPPLEMENTARY FIGURE LEGENDS
Figure E1. The inflammatory cytokine panel as well as the IL-4, IL-5, eotaxin shown in
Figure 4 responded differently to the OVA exposure and administration of sEHI.
Figure E2. COX2 (A) and 5-LOX (B) induced by OVA challenge but were not reduced
by administration of sEHI.
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8
Supplementary Table E1. The concentrations of lipid mediators in Plasma, BALF and
lung homogenates. The units in plasma and BALF are in nmol/L. The unit in lung
homogenates is in pmol/g tissue.
Supplementary Table E2. The epoxides to diols ratios supported the engagement of sEHI
in plasma, BALF and lung homegenates.
This table is in Excel format. It can be accessed from this issue’s table of contents online
at www.atsjournal.org.
Figure E1.
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9
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Figure E2.
(A)
(B)
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Copyright © 2014 by the American Thoracic Society
Origin # Group 6-keto-PGF1aPGE2 PGD2 PGF2a PGD1 15-deoxy-PGJ211-HETE
115 Vehicle 0.54 0.14 0.41 0.15 0.09 0.72 0.18
116 Vehicle 0.68 0.11 0.43 0.26 0.07 0.72 0.63
117 Vehicle 0.69 0.10 0.46 0.00 0.05 0.67 0.31
118 Vehicle 0.73 0.08 0.51 0.00 0.03 0.42 0.73
119 Vehicle 0.67 0.08 0.43 0.00 0.10 1.02 0.24
120 Vehicle 0.85 0.09 0.44 0.15 0.42 0.99 0.22
133 Low 1728 0.58 0.15 0.51 0.00 0.08 1.14 0.81
134 Low 1728 0.36 0.44 0.45 0.81 0.06 1.83 0.70
135 Low 1728 0.51 0.16 0.55 0.00 0.08 0.53 1.01
136 Low 1728 0.77 0.11 0.55 0.00 0.09 0.69 0.92
137 Low 1728 0.70 0.09 0.41 0.00 0.05 1.11 0.35
138 Low 1728 0.77 0.06 0.43 0.63 0.34 1.61 0.18
139 Hi 1728 0.73 0.13 0.64 2.12 0.21 0.90 0.75
140 Hi 1728 0.74 0.36 0.62 0.15 0.17 0.79 0.80
141 Hi 1728 0.82 0.07 0.44 0.00 0.10 0.82 0.87
142 Hi 1728 0.56 0.12 0.54 2.91 0.73 0.73 0.71
143 Hi 1728 1.20 0.33 0.60 1.11 0.15 1.37 1.32
144 Hi 1728 0.76 0.12 0.46 0.91 0.08 1.02 0.53
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9-HETE EKODE LTB4 20-COOH-LTB420-OH-LTB46-trans-LTB4LTB3 5-HETE 5-HEPE
0.40 8.12 0.32 0.71 0.12 0.20 0.23 1.25 0.36
0.60 8.15 0.32 0.49 0.13 0.29 0.12 1.65 1.48
0.38 5.00 0.33 0.44 0.14 0.38 0.14 0.98 1.22
0.61 9.50 0.35 0.45 0.07 0.25 0.15 1.65 1.51
0.32 8.33 0.38 0.78 0.10 0.23 0.25 1.54 0.87
0.38 4.53 0.34 1.04 0.15 0.73 0.23 1.25 0.66
0.50 15.42 0.36 0.55 0.05 0.44 0.18 1.77 0.69
0.54 21.37 0.38 0.87 0.07 0.85 0.19 1.59 0.85
0.55 12.71 0.38 0.43 0.08 0.00 0.17 3.27 0.78
0.94 11.53 0.42 0.48 0.10 0.29 0.15 2.94 1.13
0.37 4.23 0.35 0.63 0.08 0.03 0.14 1.40 0.34
0.66 5.60 0.37 0.62 0.23 0.31 0.18 1.97 0.85
0.42 9.02 0.43 0.87 0.10 0.24 0.23 2.68 0.64
0.46 8.29 0.41 1.02 0.12 0.00 0.13 2.62 0.71
0.37 12.45 0.38 0.66 0.05 0.00 0.14 2.82 0.89
0.46 7.38 0.35 0.53 0.20 0.20 0.21 3.08 1.02
0.57 13.49 0.38 1.41 0.09 1.16 0.19 3.42 1.56
0.49 15.95 0.36 0.80 0.13 0.69 0.14 1.86 0.72
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15-HETE 15(S)-HETrE13-HOTrE 13-HODE 15-HEPE 17-HDoHE 12-HETE 8-HETE 9-HOTrE
1.37 0.30 2.09 24.25 0.40 2.29 19.01 0.55 0.44
2.14 0.62 4.64 74.02 1.25 4.07 33.38 0.88 2.03
1.42 0.40 3.24 46.20 1.23 4.10 8.75 0.51 1.06
1.83 0.63 5.66 77.59 1.57 3.55 33.20 0.81 2.79
1.77 0.54 2.87 34.65 0.88 9.26 32.21 0.64 1.10
1.66 0.33 3.33 27.37 0.48 12.73 13.21 0.47 0.72
1.69 0.72 3.88 51.62 0.70 2.30 20.20 0.83 1.84
2.23 0.81 5.28 70.17 0.96 6.73 27.05 1.33 1.98
5.73 1.08 1.73 51.22 0.44 4.81 111.81 1.45 1.90
4.98 1.02 2.35 71.85 0.50 11.46 64.69 1.25 3.24
1.82 0.42 1.95 19.32 0.31 5.76 26.10 0.54 0.66
2.71 0.38 2.82 33.38 0.77 12.24 14.99 0.75 0.92
3.32 0.65 1.38 25.87 0.29 0.85 8.35 0.77 0.65
3.45 0.71 2.02 41.86 0.48 6.03 28.98 0.94 1.60
3.79 0.82 3.21 54.63 0.49 4.20 32.07 1.05 2.89
3.68 0.88 1.84 37.30 0.50 7.19 39.07 1.01 1.21
4.33 0.95 3.55 59.06 1.57 5.51 77.03 1.14 1.84
1.68 0.71 3.62 42.57 0.58 1.57 44.48 0.90 1.50
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9-HODE 12-HEPE 8-HEPE 12-oxo-ETE15-oxo-ETE13-oxo-ODE9-oxo-ODE 20-HETE 14(15)-EpETrE
10.54 5.32 0.22 13.13 0.12 0.30 3.67 10.29 0.87
24.18 15.60 1.61 113.91 0.38 0.41 3.55 6.88 0.77
16.74 4.25 1.52 12.00 0.10 0.29 2.44 6.22 0.50
27.66 15.16 1.44 21.96 0.24 0.59 3.37 7.32 0.79
15.29 10.08 0.24 25.33 0.28 0.30 5.21 6.23 0.75
13.79 3.22 0.25 10.92 0.25 0.30 4.21 5.04 0.99
18.05 5.98 0.41 40.10 0.26 1.61 5.35 14.08 3.25
25.88 7.69 0.46 35.65 0.49 3.21 10.04 20.17 2.85
24.28 28.44 0.25 28.59 0.61 0.54 7.91 11.77 3.28
32.66 20.45 0.49 12.14 0.35 0.91 7.20 16.78 2.13
7.76 5.85 0.14 32.94 0.40 0.30 2.48 4.08 1.45
14.09 4.10 0.41 32.48 0.39 0.44 3.77 5.85 2.41
11.85 2.34 0.27 3.84 0.49 0.41 4.52 7.20 3.14
19.61 7.08 0.41 10.46 0.59 0.33 5.44 10.56 2.66
25.14 8.42 0.36 11.75 0.31 1.22 5.93 13.40 2.76
16.78 9.58 0.64 10.86 1.13 0.39 6.57 4.58 3.05
28.30 26.25 0.80 19.22 0.85 0.45 8.77 12.21 2.96
17.67 13.44 0.48 91.81 0.45 1.62 6.03 8.87 2.59
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11(12)-EpETrE8(9)-EpETrE5(6)-EpETrE12(13)-EpOME9(10)-EpOME15(16)-EpODE12(13)-EpODE9(10)-EpODE19(20)-EpDPE
0.66 0.42 1.40 14.62 10.65 5.27 0.03 0.36 8.03
1.60 0.37 2.29 33.16 11.98 30.28 0.42 1.08 9.43
0.64 0.47 1.18 27.66 8.11 20.58 0.45 0.58 5.07
0.87 0.63 1.94 34.93 13.14 36.81 0.58 1.16 9.33
0.54 0.75 1.64 14.52 9.34 7.56 0.04 0.53 9.17
1.22 0.28 1.22 13.68 9.19 6.35 0.04 0.61 10.99
1.62 1.19 3.54 153.17 24.10 56.85 2.23 2.85 26.19
2.06 0.88 2.82 168.34 30.37 68.61 2.81 2.77 32.51
2.58 1.07 3.51 76.91 20.27 32.16 1.02 1.72 24.74
1.79 1.40 2.09 104.76 22.74 53.47 1.55 2.28 20.97
1.03 1.00 0.95 19.35 5.99 7.09 0.22 0.41 6.68
1.68 0.97 1.92 65.29 12.91 23.48 0.61 1.40 13.81
1.90 1.42 2.57 58.40 12.55 17.42 0.51 0.43 17.25
2.22 1.82 1.94 84.67 15.21 24.44 1.07 1.35 16.38
1.87 1.20 1.85 97.61 15.09 28.26 1.47 1.43 16.74
2.15 0.84 2.64 80.17 14.43 26.53 0.98 1.66 18.65
2.34 1.05 2.63 98.51 23.70 46.33 0.42 1.53 25.90
1.78 1.11 2.56 97.27 18.79 44.62 1.27 1.52 22.90
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16(17)-EpDPE13(14)-EpDPE10(11)-EpDPE17(18)-EpETE14,15-DiHETrE11,12-DiHETrE8,9-DiHETrE5,6-DiHETrE12,13-DiHOME
0.61 0.42 0.96 1.22 1.49 0.71 0.48 0.23 30.27
0.80 0.61 0.89 3.84 3.78 1.86 0.76 0.26 82.73
0.47 0.22 0.65 3.25 1.45 0.63 0.37 0.15 61.96
0.82 0.46 0.96 3.57 2.60 1.15 0.57 0.18 92.64
0.72 0.53 1.00 1.78 1.30 0.63 0.56 0.33 26.57
0.89 0.50 0.77 1.11 1.25 0.61 0.66 0.19 50.07
3.03 0.95 1.68 7.37 2.44 1.73 0.79 0.14 29.41
2.95 0.86 2.31 8.42 5.70 3.67 1.47 0.22 80.93
3.27 1.50 2.48 6.65 2.17 1.46 0.59 0.12 23.45
1.93 1.16 1.93 7.38 2.43 1.62 0.58 0.12 42.72
0.69 0.28 0.69 2.11 0.85 0.46 0.28 0.13 12.18
1.36 0.94 1.25 4.86 0.79 0.54 0.49 0.22 23.14
2.04 0.80 1.49 5.46 1.23 0.99 0.54 0.17 11.95
2.45 0.91 1.76 4.41 2.05 1.75 0.64 0.25 15.20
2.16 0.89 1.40 5.10 2.23 1.63 0.52 0.12 20.53
2.18 1.09 2.01 5.97 1.79 1.37 0.64 0.20 15.90
2.26 1.21 2.09 8.29 1.20 0.80 0.68 0.24 44.00
1.87 0.57 1.00 5.63 1.19 0.70 0.47 0.15 51.71
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9,10-DiHOME15,16-DiHODE12,13-DiHODE9,10-DiHODE19,20-DiHDPE16,17-DiHDPE13,14-DiHDPE10,11-DiHDPE7,8-DiHDPE
9.14 6.72 0.58 0.30 29.00 2.58 0.70 0.65 1.45
24.03 21.92 3.87 3.10 42.56 13.40 2.37 1.30 1.66
18.38 15.81 3.13 2.39 20.13 5.74 0.85 0.67 1.24
26.39 22.12 4.15 3.16 27.58 8.33 1.53 0.92 1.51
10.40 8.69 0.58 0.62 27.74 2.76 0.77 0.54 1.66
18.24 10.50 1.11 0.83 18.92 2.03 0.62 0.62 1.89
11.69 3.85 1.16 1.93 33.79 7.63 2.12 0.87 1.20
26.27 8.44 2.72 3.57 65.05 16.53 4.57 2.25 2.09
9.79 2.77 1.06 1.17 33.63 4.89 1.22 0.72 0.74
15.09 4.58 1.61 1.90 32.40 5.68 1.32 0.88 0.71
4.32 3.19 0.81 0.52 12.68 1.77 0.47 0.44 0.50
9.11 4.60 0.91 1.22 12.30 1.80 0.57 0.56 0.46
6.79 0.71 0.46 0.57 18.59 2.88 0.96 0.56 0.49
7.04 2.19 0.71 0.94 22.29 5.90 1.74 0.73 0.39
9.46 2.55 0.71 1.33 24.14 6.21 1.77 0.71 0.56
7.81 2.67 0.84 1.09 23.47 4.54 1.22 0.60 0.87
19.85 5.20 1.86 2.11 18.01 2.60 0.91 0.66 1.05
16.77 5.68 1.62 2.16 17.19 2.82 0.81 0.53 0.72
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4,5-DiHDPE17,18-DiHETE14,15-DiHETE11,12-DiHETE11,12-,15-TriHETrE9,12,13-TriHOME9,10,13-TriHOME5,15-DiHETEResolvin E1
2.38 7.49 0.94 0.25 0.33 13.25 7.34 0.10 23.47
1.26 13.74 3.95 0.74 0.35 16.60 7.80 0.10 18.65
0.80 9.77 2.60 0.47 0.53 15.18 5.70 0.10 19.46
1.51 10.69 3.78 0.70 0.16 13.55 4.87 0.10 15.55
1.66 8.81 1.16 0.25 0.63 43.42 23.38 0.11 19.51
1.11 5.10 0.76 0.25 0.97 36.83 18.61 0.14 27.33
3.22 7.36 2.99 0.72 0.15 2.09 0.00 0.11 10.84
5.78 14.87 5.17 1.20 0.77 16.25 6.05 0.10 7.64
3.84 6.70 1.80 0.53 0.65 16.74 5.90 0.11 12.25
4.56 6.97 1.89 0.60 0.45 5.44 0.47 0.12 14.25
0.83 2.98 0.74 0.19 0.27 3.44 0.00 0.09 10.18
2.21 3.44 0.97 0.26 0.59 17.31 8.33 0.12 16.61
3.13 4.08 1.02 0.35 0.54 14.51 9.87 0.13 18.25
3.67 5.86 1.71 0.53 0.54 14.01 4.90 0.10 16.52
2.50 5.76 2.06 0.55 0.14 13.32 4.81 0.10 9.24
7.42 5.80 1.56 0.37 1.03 14.14 15.92 0.16 17.85
3.46 5.79 1.54 0.41 0.85 6.16 3.89 0.20 19.18
1.54 5.03 1.25 0.31 0.27 6.19 0.08 0.11 9.17
Page 51 of 76 AJRCMB Articles in Press. Published on 12-June-2014 as 10.1165/rcmb.2013-0440OC
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LXA4
0.09
0.26
0.11
0.08
0.09
0.08
0.16
0.44
0.09
0.10
0.09
0.25
0.10
0.24
0.05
0.21
0.48
0.31
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Origin # Group 6-keto-PGF1aPGE2 PGD2 PGF2a PGD1 15-deoxy-PGJ211-HETE
115 Vehicle 0.612786 0.218459 0.121927 0.208148 0.013287 0.035531 0.053011
116 Vehicle 0.956194 0.386346 0.158625 0.320304 0.023161 0.039398 0.06692
117 Vehicle 1.376529 0.85117 0.325032 0.396892 0.028867 0.035373 0.099083
118 Vehicle 1.365408 0.562445 0.256395 0.284891 0.029533 0.029159 0.08331
119 Vehicle 0.939118 0.878885 0.273148 0.604412 0.021071 0.032862 0.089597
120 Vehicle 2.660328 1.045844 0.289158 0.883455 0.039807 0.036129 0.160688
133 Low 1728 2.86382 1.51714 0.768938 0.505077 0.725672 0.255651 0.51054
134 Low 1728 0.402519 0.366602 0.163822 0.139071 0.021203 0.044107 0.072778
135 Low 1728 1.134424 1.38821 0.869759 1.243642 0.058021 0.131235 0.343799
136 Low 1728 0.929851 0.933749 0.372035 0.910885 0.027194 0.024349 0.184279
137 Low 1728 0.223406 0.704127 0.177762 0.282541 0.038602 0.032414 0.1122
138 Low 1728 0.344297 1.251326 0.295564 1.743336 0.023832 0.029072 0.255544
139 Hi 1728 0.64107 0.880575 0.357194 0.793268 0.023041 0.028822 0.126731
140 Hi 1728 1.302977 1.243945 0.411332 1.124204 0.031967 0.027863 0.26551
141 Hi 1728 1.259654 0.8524 0.422008 0.280382 0.036814 0.033938 0.217729
142 Hi 1728 0.998102 0.444168 0.171561 0.182186 0.013455 0.023324 0.134411
143 Hi 1728 0.249087 1.387744 0.326129 1.500634 0.027874 0.016343 0.280939
144 Hi 1728 0.29534 1.327986 0.293624 1.360559 0.029014 0.033823 0.311188
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9-HETE EKODE LTB4 20-COOH-LTB420-OH-LTB46-trans-LTB4LTB3 5-HETE 5-HEPE
0.067853 0.380647 0.064116 0.049453 0.017601 0.20438 0.01587 0.085581 0.014185
0.048073 0.312643 0.0592 0.052563 0.034477 0.023874 0.022486 0.091649 0.022387
0.065941 0.595679 0.071417 0.050735 0.01997 0.177846 0.017253 0.153374 0.020635
0.064296 0.336425 0.072547 0.059757 0.020525 0.062872 0.021029 0.142306 0.016216
0.059516 0.457214 0.058296 0.057807 0.023078 0.04886 0.023905 0.119605 0.029003
0.078542 1.181789 0.059505 0.06126 0.011836 0.045036 0.019524 0.166964 0.034562
0.203744 1.353911 0.207195 1.349274 0.265559 0.355086 0.13916 0.392215 0.073583
0.04139 0.397768 0.047511 0.087986 0.013445 0.029179 0.016266 0.082697 0.016078
0.421167 2.933581 0.231385 0.272204 0.132343 0.380552 0.155808 0.735233 0.138383
0.121184 0.659799 0.07201 0.059682 0.01517 0.178055 0.016679 0.255135 0.040245
0.095262 3.555115 0.037803 0.075867 0.00669 0.071538 0.013188 0.204857 0.054505
0.106914 0.36582 0.051144 0.058514 0.017505 0.099096 0.025318 0.18142 0.032932
0.090926 0.820914 0.068098 0.075709 0.024407 0.205261 0.017799 0.221306 0.024457
0.127235 0.787516 0.07306 0.076836 0.019652 0.09641 0.023756 0.306961 0.037674
0.104315 0.513432 0.049146 0.058433 0.015089 0.091768 0.017592 0.324443 0.036582
0.056135 0.836944 0.051618 0.055703 0.029163 0.154589 0.024834 0.160157 0.04197
0.11675 1.234484 0.0555 0.059743 0.023945 0.219686 0.022178 0.140093 0.061704
0.0978 0.748396 0.064443 0.070246 0.019641 0.120162 0.028746 0.096758 0.029898
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15-HETE 15(S)-HETrE13-HOTrE 13-HODE 15-HEPE 17-HDoHE 12-HETE 8-HETE 9-HOTrE
0.376804 0.064036 0.125627 1.804169 0.12202 1.428552 1.809009 0.070388 0.018619
0.609455 0.13631 0.119471 1.592272 0.17491 1.462823 3.354617 0.089598 0.016675
0.956516 0.216492 0.133204 1.917558 0.291631 2.65702 4.304629 0.125338 0.019957
0.811332 0.205847 0.15818 1.815429 0.253952 2.422955 4.652585 0.149875 0.019636
0.674998 0.113426 0.125057 1.515146 0.237936 1.65332 3.877494 0.138802 0.017756
0.74353 0.111381 0.150987 2.292522 0.398245 2.751634 3.106782 0.090213 0.026262
3.017167 0.769511 0.473171 6.271757 0.963781 8.277269 21.01629 0.651107 0.069111
0.658962 0.1678 0.140871 1.332826 0.193647 1.94745 2.969319 0.086764 0.018962
1.907105 0.384686 0.490881 7.835754 0.664926 5.837052 21.66928 0.570486 0.076797
1.486609 0.33769 0.146631 2.254071 0.514386 4.373995 7.696472 0.238249 0.019057
0.979411 0.087056 0.098176 1.699852 0.367844 4.473531 3.136645 0.116592 0.022583
0.81092 0.09114 0.12553 2.105628 0.568129 6.759525 6.298741 0.187822 0.022248
1.241961 0.215197 0.11222 1.976468 0.307661 3.777612 7.141128 0.226007 0.025342
1.880459 0.27821 0.112721 2.344623 0.537548 6.130197 14.40448 0.305041 0.021092
1.097876 0.255628 0.091389 1.523802 0.252627 2.309894 5.857338 0.24912 0.018512
0.762676 0.119812 0.101421 1.356356 0.28393 1.850825 3.494148 0.125863 0.019253
1.03486 0.137089 0.14389 2.590617 0.527202 4.893099 4.53335 0.165671 0.021714
0.921204 0.123242 0.140178 1.54157 0.526415 3.808423 3.672426 0.134066 0.017526
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9-HODE 12-HEPE 8-HEPE 12-oxo-ETE15-oxo-ETE13-oxo-ODE9-oxo-ODE 20-HETE 14(15)-EpETrE
0.506759 0.177585 0.016738 5.053253 0.089183 0.031301 0.421337 0.064741 0.036176
0.44733 0.254517 0.034789 6.801508 0.102704 0.026052 0.421684 0.057025 0.018426
0.593705 0.263127 0.027603 16.73455 0.276489 0.024667 0.360299 0.061534 0.035012
0.583685 0.17519 0.022024 6.924372 0.136575 0.035898 0.461238 0.085732 0.018827
0.529352 0.247036 0.018614 13.40917 0.276041 0.04308 0.450581 0.091285 0.062836
0.735448 0.418384 0.014631 19.50895 0.395822 0.025603 0.495794 0.11297 0.047425
1.808979 1.171672 0.058912 49.55249 0.557718 0.132862 1.49623 0.6558 0.166199
0.39058 0.190149 0.009892 6.219621 0.104136 0.028382 0.308093 0.240448 0.034332
2.517467 3.522689 0.162334 133.8143 0.736505 0.204568 2.648898 0.322609 0.184179
0.732051 0.707024 0.029148 19.73892 0.430841 0.030066 0.470443 0.178505 0.072144
0.66243 0.421464 0.033924 45.48099 1.630235 0.027737 0.997295 0.084338 0.128147
0.686124 0.923791 0.042923 10.71194 0.282822 0.044479 0.773994 0.297386 0.103081
0.629772 0.325799 0.022644 17.49105 0.302893 0.030147 0.501278 0.096702 0.069343
0.676412 1.430798 0.033453 14.53737 0.329418 0.032562 0.494861 0.082505 0.096057
0.649809 0.432682 0.025293 21.69519 0.242256 0.027228 0.380428 0.06276 0.083443
0.394605 0.391906 0.048427 18.31002 0.373319 0.037715 0.550057 0.150164 0.06517
0.696256 0.493772 0.026045 43.04296 0.726815 0.053316 0.979083 0.26616 0.169921
0.443011 0.325604 0.026796 23.85318 0.415747 0.039573 0.624891 0.289281 0.118303
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11(12)-EpETrE8(9)-EpETrE5(6)-EpETrE12(13)-EpOME9(10)-EpOME15(16)-EpODE12(13)-EpODE9(10)-EpODE19(20)-EpDPE
0.041856 0.064592 0.062325 0.132566 0.100761 0.055698 0.00729 0.007179 0.399098
0.061845 0.073668 0.043012 0.313277 0.11285 0.262864 0.006066 #VALUE! 0.260731
0.079855 0.118755 0.087836 0.320366 0.136424 0.249152 0.01395 0.010821 0.341708
0.043195 0.084106 0.072949 0.418468 0.15323 0.386644 0.011131 0.011869 0.347628
0.123656 0.132043 0.107122 0.198011 0.153576 0.084174 0.002277 0.006641 0.494202
0.117164 0.19915 0.198466 0.203671 0.213165 0.110033 0.004829 0.004585 0.479328
0.162218 0.171603 0.111555 2.942377 0.679201 1.64346 0.227788 0.241215 2.131312
0.050612 0.045873 0.033091 0.904818 0.153856 0.680872 0.051 0.008856 0.555556
0.485403 0.528362 0.537523 2.370386 0.958909 1.01712 0.093826 0.086045 3.377893
0.119985 0.261291 0.216571 0.844317 0.257884 0.546855 0.028812 0.023514 0.676558
0.209549 0.068085 0.249691 0.406008 0.279245 0.14856 0.007068 0.010466 0.710918
0.171177 0.052935 0.190185 0.575995 0.286494 0.143367 0.019866 0.00976 0.58486
0.123663 0.027133 0.157767 0.323585 0.182284 0.098966 0.008801 0.014879 0.286667
0.163308 0.054232 0.233707 0.589147 0.227661 0.211434 0.01647 0.017377 0.35312
0.131229 0.057022 0.155431 0.801629 0.184821 0.371828 0.033894 0.01227 0.414469
0.063811 0.188263 0.168489 0.383995 0.161252 0.151211 0.012013 0.013562 0.369882
0.226185 0.042592 0.285887 0.900976 0.266535 0.418422 0.024677 0.027461 1.146981
0.214473 0.071321 0.156759 0.789114 0.262072 0.419188 0.02711 0.033371 0.950195
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16(17)-EpDPE13(14)-EpDPE10(11)-EpDPE17(18)-EpETE14,15-DiHETrE11,12-DiHETrE8,9-DiHETrE5,6-DiHETrE12,13-DiHOME
0.021211 0.024815 0.036158 0.02427 0.037437 0.027521 0.025654 0.007101 0.190061
0.025916 0.034267 0.040998 0.067664 0.052968 0.034246 0.026904 0.009141 0.662454
0.028658 0.020596 0.027127 0.049325 0.041985 0.026447 0.010531 0.011225 0.447108
0.014538 0.016119 0.030824 0.05143 0.038847 0.027481 0.011603 0.008714 0.895221
0.056538 0.035783 0.069629 0.056381 0.035232 0.024825 0.008464 0.006544 0.126563
0.051297 0.046281 0.107162 0.096688 0.038858 0.028712 0.014163 0.006217 0.282352
0.167116 0.027507 0.165292 0.32892 0.106941 0.174439 0.021345 0.064413 0.923862
0.025296 0.021918 0.041806 0.078094 0.068911 0.04555 0.015444 0.007517 0.453376
0.222644 0.060153 0.26173 0.53203 0.18215 0.154913 0.155967 0.069662 1.260703
0.037592 0.046271 0.086302 0.088507 0.038033 0.032184 0.019611 0.01161 0.071094
0.065578 0.088451 0.125355 0.140146 0.029423 0.015595 0.008783 0.011316 0.041966
0.045941 0.102557 0.141823 0.237828 0.01996 0.018603 0.017214 0.010908 0.07874
0.033654 0.014264 0.094435 0.14038 0.026709 0.023486 0.016296 0.013539 #VALUE!
0.060661 0.06216 0.073265 0.112609 0.032704 0.026799 0.023727 0.005962 #VALUE!
0.036672 0.006496 0.063949 0.090012 0.053879 0.055658 0.034971 0.059845 0.039946
0.064775 0.046141 0.0629 0.101919 0.031915 0.03108 0.011912 0.012658 0.047634
0.079699 0.098018 0.111727 0.180696 0.032565 0.028888 0.01283 0.01963 0.218392
0.120387 0.053201 0.084314 0.207787 0.031846 0.023024 0.006369 0.012683 0.311589
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9,10-DiHOME15,16-DiHODE12,13-DiHODE9,10-DiHODE19,20-DiHDPE16,17-DiHDPE13,14-DiHDPE10,11-DiHDPE7,8-DiHDPE
0.176349 0.091326 0.043101 0.011931 0.640611 0.059075 0.008788 0.012117 0.011751
0.334853 0.218458 0.107922 0.062382 0.704312 0.130096 0.02624 0.021612 0.013643
0.202828 0.210427 0.056162 0.043432 0.528366 0.107808 0.022895 0.01545 0.013647
0.42587 0.24123 0.079569 0.06593 0.574224 0.085517 0.023219 0.007078 0.011893
0.164588 0.112834 0.049701 0.018985 0.590848 0.07207 0.014267 0.013744 0.009219
0.134358 0.099411 0.039348 0.018408 0.518187 0.073935 0.025467 0.026033 0.030779
0.64366 0.043979 0.140122 0.059886 1.478486 0.200424 0.034745 0.03104 0.07511
0.352286 0.064319 0.040246 0.055391 0.66695 0.140037 0.04639 0.02007 0.029883
1.947561 0.102318 0.204985 0.222567 2.96003 0.3506 0.081946 0.069232 0.147295
0.200016 0.039401 0.034811 0.052269 0.620168 0.06184 0.020449 0.018268 0.016787
0.064107 0.060872 0.04193 0.022371 0.360402 0.052802 0.013219 0.027591 0.022149
0.211005 0.059107 0.051712 0.028239 0.365433 0.041813 0.007245 0.009919 0.024697
0.163622 0.015277 0.038709 0.016563 0.337395 0.036007 0.011664 0.017103 0.016164
0.124041 0.030307 0.058596 0.021846 0.397673 0.074608 0.020674 0.014191 0.027706
0.201951 0.025296 0.044684 0.033654 0.515453 0.095253 0.042232 0.026571 0.016943
0.21013 0.032831 0.066216 0.024123 0.530208 0.053335 0.017223 0.01128 0.015336
0.301452 0.058656 0.033106 0.03509 0.533379 0.051818 0.024029 0.02635 0.02388
0.223922 0.040527 0.039201 0.036278 0.442819 0.049657 0.013149 0.019381 0.018399
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4,5-DiHDPE17,18-DiHETE14,15-DiHETE11,12-DiHETE11,12-,15-TriHETrE9,12,13-TriHOME9,10,13-TriHOME5,15-DiHETEResolvin E1
0.159971 0.139166 0.009551 0.002954 0.070323 0.233561 0.451246 0.027029 2.400569
0.152152 0.19187 0.045334 0.015585 0.213228 0.535334 #VALUE! 0.021164 2.872838
0.12093 0.193139 0.026531 0.014882 0.256382 1.382336 0.261222 0.020862 1.455278
0.100924 0.179596 0.029363 0.013078 0.324716 1.29722 0.900576 0.023463 2.518766
0.179379 0.16433 0.015365 0.00766 0.163037 0.713734 0.099865 0.019555 2.370417
0.129011 0.120548 0.011539 0.009465 0.525087 1.665844 0.403568 0.013161 1.142784
0.549976 0.291188 0.054472 0.035656 0.781394 4.671805 11.61565 0.076897 74.26371
0.273097 0.176065 0.034852 0.012649 0.075892 0.16127 #VALUE! 0.011872 1.557754
1.658988 0.528423 0.069361 0.057379 1.092276 2.026949 1.914944 0.101326 20.73341
0.223264 0.112439 0.021794 0.010719 0.712472 1.727044 0.679307 0.026155 1.440972
0.189016 0.12334 0.02062 0.00823 0.455702 1.898317 0.905803 0.014498 1.832499
0.311859 0.098184 0.013421 0.004135 0.266897 1.1724 1.447146 0.022295 3.263252
0.199075 0.088202 0.011337 0.008337 0.584571 0.559811 0.789125 0.026089 2.690301
0.22517 0.0916 0.018109 0.008977 0.597867 0.785996 0.409221 0.031179 2.507613
0.318555 0.106999 0.022126 0.010737 0.684737 0.703331 0.515799 0.022352 1.556333
0.197042 0.121676 0.012225 0.004944 0.104209 1.558571 1.568063 0.017877 2.864938
0.376026 0.120857 0.011832 0.009783 0.194804 1.150577 1.342292 0.017404 2.98628
0.243658 0.134026 0.016757 0.008029 0.261296 1.415231 0.907535 0.021038 3.458414
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LXA4
0.011358
0.024215
0.034912
0.01612
0.029532
0.032401
0.091628
0.007697
0.069406
0.029676
0.147651
0.034244
0.028164
0.021972
0.05251
0.03644
0.045767
0.038891
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Original # Group 6-keto-PGF1aTXB2 PGE2 PGD2 PGF2a PGE1 PGD1
115 OVA+Vehicle 3663.073 376.0273 7863.451 564.8455 845.29 196.12 1308.33
116 OVA+Vehicle 3039.464 236.4339 4659.682 549.9922 525.07 103.83 673.14
117 OVA+Vehicle 2657.946 468.2563 7669.167 449.8852 681.60 170.06 1201.32
118 OVA+Vehicle 3671.499 461.7956 9177.225 979.0713 1033.54 228.45 1264.31
133 Low 1728 5897.612 705.8463 9783.181 741.215 1342.91 229.92 1536.85
134 Low 1728 4331.293 647.3931 8215.391 498.1015 1277.92 176.35 932.15
135 Low 1728 4812.217 403.6614 8130.968 613.7957 921.30 181.70 1086.05
136 Low 1728 4159.56 671.5802 9708.752 721.8982 1075.63 231.89 1382.13
139 Hi 1728 3963.685 347.6451 6075.597 469.4399 612.20 140.79 893.62
140 Hi 1728 3486.334 349.9203 5752.099 483.2927 478.98 138.54 997.28
141 Hi 1728 5365.061 613.5994 10500.13 745.0418 1042.81 200.49 1386.26
142 Hi 1728 5962.055 880.4157 12574.05 1147.543 1879.40 272.15 1639.63
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PGE3 PGD3 PGJ2 PGB2 11-HETE 9-HETE EKODE LTB4 LTB5
238.84 11.56 14.86 9.09 1020.90 32.89 44.86 6.83 2.10
221.21 14.46 9.36 3.92 744.34 0.00 35.74 2.05 2.09
221.46 7.48 13.74 8.33 1320.04 163.85 59.27 3.05 5.28
299.87 13.26 16.02 8.99 1737.99 98.42 50.00 7.89 2.19
340.67 15.00 15.10 9.30 2849.31 200.06 44.92 9.33 3.91
496.77 18.09 18.73 11.76 2046.41 113.14 74.53 3.31 2.28
630.48 24.01 19.16 8.64 2100.74 153.53 70.45 2.46 2.60
387.82 0.56 15.96 11.63 2809.20 206.80 50.51 4.47 1.87
297.66 12.28 12.60 7.82 2537.36 118.87 56.81 1.12 2.32
254.22 8.63 12.13 8.27 2085.85 118.16 36.17 2.98 1.61
515.02 12.80 22.19 11.76 2852.78 137.53 87.42 9.61 4.16
615.77 10.21 33.60 18.33 5282.73 212.02 79.81 21.25 8.71
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6-trans-LTB45-HETE 5-HEPE 15-HETE 15(S)-HETrE13-HOTrE 13-HODE 15-HEPE 17-HDoHE
33.37 84.79 6.44 2100.64 390.01 68.65 3559.39 260.84 2026.29
9.85 61.65 9.11 1852.28 276.96 114.86 2543.12 439.64 1207.59
73.96 50.83 4.23 2511.49 556.59 157.61 4711.99 338.41 2408.57
25.77 127.48 10.93 2422.06 324.09 44.37 2008.38 302.39 1261.70
52.07 81.47 5.36 5908.71 1090.68 197.31 6232.62 1145.34 5064.08
21.33 68.96 6.55 3772.53 544.94 143.75 3632.39 1099.99 3157.10
28.19 43.79 6.05 6269.63 812.54 213.13 4879.48 3559.33 5546.76
27.07 64.19 6.46 5727.57 700.34 111.18 3349.83 1144.56 5768.76
24.36 87.98 7.81 7285.98 915.43 98.05 3818.37 2029.40 7234.27
26.91 54.05 4.15 4992.70 753.41 106.70 3929.21 1099.88 5404.31
71.48 140.87 7.37 5737.73 797.16 121.24 3166.56 1611.62 6336.93
175.25 217.87 13.74 10869.59 1334.92 133.02 4014.07 2528.11 8828.08
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12-HETE 8-HETE 9-HOTrE 9-HODE 12-HEPE 8-HEPE 5-oxo-ETE 12-oxo-ETE15-oxo-ETE
15818.56 342.67 4.46 2432.42 1186.43 7.58 16.23 3574.90 66.60
12258.27 210.13 3.33 1428.60 2075.46 13.90 30.03 2042.81 27.22
46555.84 535.76 5.23 2373.57 4553.51 7.88 69.90 13195.46 80.33
22712.36 331.88 3.31 1958.54 2149.10 7.37 48.45 5548.21 73.22
56969.27 734.02 5.89 3315.98 4229.02 6.78 38.83 9755.20 124.48
33380.23 353.87 4.32 2500.21 4017.44 6.31 20.76 6685.23 65.77
35555.42 463.36 4.81 2594.42 5591.80 17.05 14.90 5763.28 78.97
40773.99 487.25 3.21 2275.65 3463.70 8.71 37.38 6533.13 123.55
32405.77 708.58 3.02 1785.55 1994.75 14.00 12.51 5864.63 119.00
26797.13 514.63 2.91 2194.78 1662.00 6.01 15.45 6901.99 109.25
38831.14 476.13 2.85 1866.91 2837.79 10.71 40.53 10270.68 130.94
76010.82 1203.41 3.77 1889.34 5844.13 13.98 59.60 13420.11 203.62
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13-oxo-ODE9-oxo-ODE 20-HETE 14(15)-EpETrE11(12)-EpETrE8(9)-EpETrE5(6)-EpETrE12(13)-EpOME9(10)-EpOME
7.18 72.66 25.31 11.09 20.97 21.05 29.87 25.55 20.83
3.38 20.13 10.42 9.46 15.86 9.31 18.80 17.25 12.70
14.20 46.19 36.59 12.67 21.37 51.72 28.52 21.35 21.77
5.96 37.40 21.05 17.41 33.07 24.69 54.78 20.76 22.32
19.02 43.67 0.00 10.60 17.73 57.99 29.08 31.18 18.73
9.00 21.26 7.46 12.15 23.37 28.19 35.46 27.69 18.30
6.78 29.62 6.87 11.65 18.90 24.67 27.61 20.30 16.77
16.53 40.73 17.94 10.67 22.17 38.09 18.52 26.52 15.66
10.04 59.92 12.51 15.00 20.18 23.07 18.85 34.12 23.76
6.48 51.94 3.65 9.64 20.65 16.80 15.76 27.59 17.57
11.41 54.79 1.39 16.78 35.21 20.59 51.43 29.91 19.93
13.74 42.99 10.13 10.44 35.73 118.63 41.17 22.28 17.17
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15(16)-EpODE9(10)-EpODE19(20)-EpDPE16(17)-EpDPE13(14)-EpDPE10(11)-EpDPE7(8)-EpDPE 17(18)-EpETE14,15-DiHETrE
3.52 0.00 6.00 3.53 3.02 3.35 30.35 3.12 1.46
12.37 0.00 8.91 0.53 1.20 0.71 22.63 2.79 0.90
10.13 0.00 10.01 3.62 2.46 3.08 106.05 3.36 1.00
10.17 0.00 7.72 4.24 2.54 7.18 76.94 2.96 1.54
15.20 0.00 3.31 1.37 1.49 3.47 68.79 3.64 0.80
17.17 0.08 7.19 1.49 2.12 5.15 48.92 2.77 1.17
10.43 0.25 6.20 2.42 1.83 3.23 30.53 4.01 0.88
11.50 0.54 11.82 3.17 3.07 4.00 73.68 5.15 0.77
11.16 0.41 6.99 3.01 1.31 2.83 36.05 6.16 0.59
10.43 0.39 5.83 1.91 1.74 3.14 58.57 2.98 0.51
12.33 0.00 7.01 2.34 3.60 5.92 107.66 3.26 0.93
6.93 0.00 6.75 6.13 5.34 2.59 40.54 3.44 1.21
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11,12-DiHETrE12,13-DiHOME9,10-DiHOME15,16-DiHODE12,13-DiHODE9,10-DiHODE19,20-DiHDPE16,17-DiHDPE13,14-DiHDPE
1.55 16.49 15.84 3.62 1.62 0.46 30.04 64.27 0.48
0.61 12.86 5.95 5.03 1.85 0.12 15.54 44.17 0.55
1.47 12.77 7.60 4.50 1.35 0.51 15.46 82.14 0.71
0.89 19.31 9.81 4.52 1.72 0.82 23.92 27.73 0.51
0.74 7.08 7.84 2.46 1.31 0.21 26.34 109.25 1.06
0.77 8.83 6.65 1.80 0.86 0.24 25.48 44.41 1.04
0.36 4.87 5.06 2.68 0.94 0.00 27.95 86.79 0.93
1.03 8.53 6.36 2.78 1.22 0.47 31.15 61.07 0.58
0.64 7.08 4.22 1.38 1.22 0.00 22.09 64.75 0.54
0.42 4.55 3.47 1.51 0.82 0.00 24.14 65.88 0.49
0.67 5.64 5.40 2.29 1.26 0.37 29.05 72.01 1.02
1.00 5.45 5.43 2.02 1.52 0.00 41.96 103.28 1.00
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10,11-DiHDPE7,8-DiHDPE4,5-DiHDPE17,18-DiHETE14,15-DiHETE5,6-DiHETE 11,12-,15-TriHETrE9,12,13-TriHOME9,10,13-TriHOME
1.52 0.89 3.41 4.40 2.40 0.08 50.11 862.46 330.16
1.36 0.01 1.81 4.30 0.64 0.37 62.46 130.37 45.78
1.51 0.40 2.00 1.40 9.21 0.44 63.74 308.25 98.25
1.24 0.56 2.10 3.03 1.77 1.62 70.93 262.63 101.96
1.68 0.53 4.06 2.52 12.67 0.43 91.26 294.11 101.75
1.47 0.05 2.76 3.22 2.54 0.00 102.27 156.62 57.11
1.65 0.50 2.11 1.91 3.73 0.21 118.65 215.20 53.65
1.39 0.65 3.09 2.55 5.62 0.29 100.05 193.58 60.45
1.74 0.57 2.68 1.89 2.59 0.20 70.48 169.84 54.46
1.14 0.32 2.45 2.66 4.72 0.11 60.44 189.06 61.97
1.34 0.28 1.88 3.67 5.20 1.01 115.05 214.98 77.68
2.00 0.81 2.90 3.80 11.13 2.80 339.45 402.64 132.09
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8,15-DiHETE5,15-DiHETELXA4
106.65 17.77 7.44
35.76 8.33 5.91
104.08 19.46 5.09
49.54 9.74 4.76
94.28 43.15 4.06
32.00 11.08 4.16
55.03 16.69 5.35
33.88 16.15 3.76
57.23 18.34 3.56
70.32 24.25 2.61
87.18 32.85 6.44
175.70 59.92 11.99
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Table E2. The epoxides to diols ratios supported the engagement of sEHI in plasma, BALF and lung homegenates.
Plamsa
Origin # Group
14(15)EpETrE/1
4,15DiHETrE
11,12
EpETrE/11,12
DiHETrE
8,9EpETrE/8,9
DiHETrE
5,6 EpETrE/5,6
DiHETrE
115 Vehicle 0.58 0.93 0.88 6.23
116 Vehicle 0.20 0.86 0.48 8.69
117 Vehicle 0.35 1.02 1.30 8.09
118 Vehicle 0.30 0.76 1.10 10.69
119 Vehicle 0.58 0.86 1.33 4.90
120 Vehicle 0.79 1.98 0.43 6.35
133 Low 1728 1.33 0.94 1.50 25.34
134 Low 1728 0.50 0.56 0.60 13.05
135 Low 1728 1.51 1.77 1.82 29.82
136 Low 1728 0.88 1.11 2.41 17.02
137 Low 1728 1.71 2.23 3.55 7.10
138 Low 1728 3.05 3.12 1.97 8.57
139 Hi 1728 2.56 1.92 2.61 15.30
140 Hi 1728 1.30 1.27 2.85 7.66
141 Hi 1728 1.24 1.14 2.33 15.66
142 Hi 1728 1.71 1.56 1.32 12.92
143 Hi 1728 2.46 2.92 1.53 10.91
144 Hi 1728 2.19 2.54 2.35 16.73
BALF
Origin # Group
14(15)EpETrE/1
4,15DiHETrE
11,12
EpETrE/11,12
DiHETrE
8,9EpETrE/8,9
DiHETrE
5,6 EpETrE/5,6
DiHETrE
115 Vehicle 0.97 1.52 2.52 8.78
116 Vehicle 0.35 1.81 2.74 4.71
117 Vehicle 0.83 3.02 11.28 7.83
118 Vehicle 0.48 1.57 7.25 8.37
119 Vehicle 1.78 4.98 15.60 16.37
120 Vehicle 1.22 4.08 14.06 31.92
133 Low 1728 1.55 0.93 8.04 1.73
134 Low 1728 0.50 1.11 2.97 4.40
135 Low 1728 1.01 3.13 3.39 7.72
136 Low 1728 1.90 3.73 13.32 18.65
137 Low 1728 4.36 13.44 7.75 22.06
138 Low 1728 5.16 9.20 3.08 17.44
139 Hi 1728 2.60 5.27 1.67 11.65
140 Hi 1728 2.94 6.09 2.29 39.20
141 Hi 1728 1.55 2.36 1.63 2.60
142 Hi 1728 2.04 2.05 15.81 13.31
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143 Hi 1728 5.22 7.83 3.32 14.56
144 Hi 1728 3.71 9.32 11.20 12.36
Lung Homegenates
Original # Group
14(15)EpETrE/1
4,15DiHETrE
11,12
EpETrE/11,12
DiHETrE
8,9EpETrE/8,9
DiHETrE
5,6 EpETrE/5,6
DiHETrE
115 OVA+Vehicle 7.62 13.57
116 OVA+Vehicle 10.48 26.07
117 OVA+Vehicle 12.63 14.51
118 OVA+Vehicle 11.31 37.02
133 Low 1728 13.18 23.82
134 Low 1728 10.40 30.23
135 Low 1728 13.16 52.03
136 Low 1728 13.87 21.46
139 Hi 1728 25.39 31.74
140 Hi 1728 19.05 49.28
141 Hi 1728 18.06 52.38
142 Hi 1728 8.64 35.79
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Table E2. The epoxides to diols ratios supported the engagement of sEHI in plasma, BALF and lung homegenates.
12,13
EpOME/12,13
DiHOME
9,10EpOME/9,1
0 DiHOME
15,16
EpODE/15,16Di
HODE
12,13
EpODE/12,13
DiHODE
9,10
EpODE/9,10
DiHODE
19,20
EpDPE/19,20
DiHDPE
0.48 1.16 0.78 0.05 1.20 0.28
0.40 0.50 1.38 0.11 0.35 0.22
0.45 0.44 1.30 0.14 0.24 0.25
0.38 0.50 1.66 0.14 0.37 0.34
0.55 0.90 0.87 0.07 0.85 0.33
0.27 0.50 0.60 0.03 0.73 0.58
5.21 2.06 14.76 1.92 1.48 0.77
2.08 1.16 8.13 1.03 0.78 0.50
3.28 2.07 11.60 0.96 1.47 0.74
2.45 1.51 11.69 0.96 1.20 0.65
1.59 1.38 2.23 0.28 0.78 0.53
2.82 1.42 5.10 0.67 1.15 1.12
4.89 1.85 24.58 1.12 0.75 0.93
5.57 2.16 11.17 1.50 1.43 0.74
4.75 1.59 11.08 2.06 1.07 0.69
5.04 1.85 9.96 1.17 1.53 0.79
2.24 1.19 8.91 0.22 0.73 1.44
1.88 1.12 7.86 0.78 0.70 1.33
12,13
EpOME/12,13
DiHOME
9,10EpOME/9,1
0 DiHOME
15,16
EpODE/15,16Di
HODE
12,13
EpODE/12,13
DiHODE
9,10
EpODE/9,10
DiHODE
19,20
EpDPE/19,20
DiHDPE
0.70 0.57 0.61 0.17 0.60 0.62
0.47 0.34 1.20 0.06 #VALUE! 0.37
0.72 0.67 1.18 0.25 0.25 0.65
0.47 0.36 1.60 0.14 0.18 0.61
1.56 0.93 0.75 0.05 0.35 0.84
0.72 1.59 1.11 0.12 0.25 0.93
3.18 1.06 37.37 1.63 4.03 1.44
2.00 0.44 10.59 1.27 0.16 0.83
1.88 0.49 9.94 0.46 0.39 1.14
11.88 1.29 13.88 0.83 0.45 1.09
9.67 4.36 2.44 0.17 0.47 1.97
7.32 1.36 2.43 0.38 0.35 1.60
#VALUE! 1.11 6.48 0.23 0.90 0.85
#VALUE! 1.84 6.98 0.28 0.80 0.89
20.07 0.92 14.70 0.76 0.36 0.80
8.06 0.77 4.61 0.18 0.56 0.70
Page 73 of 76 AJRCMB Articles in Press. Published on 12-June-2014 as 10.1165/rcmb.2013-0440OC
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4.13 0.88 7.13 0.75 0.78 2.15
2.53 1.17 10.34 0.69 0.92 2.15
12,13
EpOME/12,13
DiHOME
9,10EpOME/9,1
0 DiHOME
15,16
EpODE/15,16Di
HODE
12,13
EpODE/12,13
DiHODE
9,10
EpODE/9,10
DiHODE
19,20
EpDPE/19,20
DiHDPE
1.55 1.32 0.97 0.20
1.34 2.13 2.46 0.57
1.67 2.86 2.25 0.65
1.08 2.28 2.25 0.32
4.40 2.39 6.17 0.13
3.13 2.75 9.55 0.28
4.17 3.31 3.88 0.22
3.11 2.46 4.14 0.38
4.82 5.63 8.06 0.32
6.06 5.06 6.93 0.24
5.30 3.69 5.39 0.24
4.09 3.16 3.42 0.16
Page 74 of 76 AJRCMB Articles in Press. Published on 12-June-2014 as 10.1165/rcmb.2013-0440OC
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16,17
EpDPE/16,17
DiHDPE
13,14
EpDPE/13,14
DiHDPE
10,11
EpDPE/10,11
DiHDPE
7,8 EpDPE/7,8
DiHDPE
17,18
EpETE/17,18Di
HETE
0.24 0.60 1.47 0.16
0.06 0.26 0.69 0.28
0.08 0.25 0.96 0.33
0.10 0.30 1.04 0.33
0.26 0.70 1.86 0.20
0.44 0.80 1.24 0.22
0.40 0.45 1.94 1.00
0.18 0.19 1.03 0.57
0.67 1.22 3.43 0.99
0.34 0.88 2.19 1.06
0.39 0.60 1.54 0.71
0.76 1.66 2.24 1.41
0.71 0.83 2.64 1.34
0.42 0.52 2.43 0.75
0.35 0.50 1.98 0.89
0.48 0.90 3.36 1.03
0.87 1.32 3.16 1.43
0.66 0.70 1.87 1.12
16,17
EpDPE/16,17
DiHDPE
13,14
EpDPE/13,14
DiHDPE
10,11
EpDPE/10,11
DiHDPE
7,8 EpDPE/7,8
DiHDPE
17,18
EpETE/17,18Di
HETE
0.36 2.82 2.98 0.17
0.20 1.31 1.90 0.35
0.27 0.90 1.76 0.26
0.17 0.69 4.35 0.29
0.78 2.51 5.07 0.34
0.69 1.82 4.12 0.80
0.83 0.79 5.33 1.13
0.18 0.47 2.08 0.44
0.64 0.73 3.78 1.01
0.61 2.26 4.72 0.79
1.24 6.69 4.54 1.14
1.10 14.16 14.30 2.42
0.93 1.22 5.52 1.59
0.81 3.01 5.16 1.23
0.38 0.15 2.41 0.84
1.21 2.68 5.58 0.84
Page 75 of 76 AJRCMB Articles in Press. Published on 12-June-2014 as 10.1165/rcmb.2013-0440OC
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1.54 4.08 4.24 1.50
2.42 4.05 4.35 1.55
16,17
EpDPE/16,17
DiHDPE
13,14
EpDPE/13,14
DiHDPE
10,11
EpDPE/10,11
DiHDPE
7,8 EpDPE/7,8
DiHDPE
17,18
EpETE/17,18Di
HETE
0.05 6.24 2.21 34.01 0.71
0.01 2.18 0.52 1901.52 0.65
0.04 3.45 2.04 268.38 2.41
0.15 5.01 5.81 137.43 0.98
0.01 1.41 2.06 130.34 1.45
0.03 2.03 3.51 951.10 0.86
0.03 1.97 1.95 60.67 2.09
0.05 5.33 2.86 112.99 2.02
0.05 2.41 1.62 63.75 3.25
0.03 3.53 2.75 184.29 1.12
0.03 3.53 4.41 387.72 0.89
0.06 5.34 1.30 50.14 0.91
Page 76 of 76 AJRCMB Articles in Press. Published on 12-June-2014 as 10.1165/rcmb.2013-0440OC
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