Metabolomics (2014) 10:1169–1175
DOI 10.1007/s11306-014-0653-y
SHORT COMMUNICATION
De novo synthesize of bile acids in pulmonary arterial
hypertension lung
Yidan D. Zhao • Hana Z. H. Yun • Jenny Peng •
Li Yin • Lei Chu • Licun Wu • Ryan Michalek •
Mingyao Liu • Shaf Keshavjee • Thomas Waddell
John Granton • Marc de Perrot
•
Received: 18 October 2013 / Accepted: 26 March 2014 / Published online: 11 April 2014
Ó The Author(s) 2014. This article is published with open access at Springerlink.com
Abstract Although multiple, complex molecular studies
have been done for understanding the development and
progression of pulmonary hypertension (PAH), little is
known about the metabolic heterogeneity of PAH. Using a
combination of high-throughput liquid-and-gas-chromatography-based mass spectrometry, we found bile acid
metabolites, which are normally product derivatives of the
liver and gallbladder, were highly increased in the PAH
lung. Microarray showed that the gene encoding cytochrome P450 7B1 (CYP7B1), an isozyme for bile acid
synthesis, was highly expressed in the PAH lung compared
with the control. CYP7B1 protein was found to be primarily localized on pulmonary vascular endothelial cells
suggesting de novo bile acid synthesis may be involved in
Electronic supplementary material The online version of this
article (doi:10.1007/s11306-014-0653-y) contains supplementary
material, which is available to authorized users.
Y. D. Zhao H. Z. H. Yun J. Peng L. Yin L. Chu L. Wu M. Liu S. Keshavjee T. Waddell M. de Perrot
Latner Thoracic Surgery Research Laboratories, Division of
Thoracic Surgery, University of Toronto, Toronto, ON, Canada
Y. D. Zhao (&) M. de Perrot (&)
MaRS Centre, Toronto Medical Discovery Tower, 2nd Floor Rm
2-817, 101 College Street, Toronto, ON M5G 1L7, Canada
e-mail: [email protected]
M. de Perrot
e-mail: [email protected]
R. Michalek
Metabolon, Incorporated, 617 Davis Drive, Durham, NC 27713,
USA
J. Granton
Clinical Studies Resource Centre, Toronto General Hospital,
University Health Network, University of Toronto, Toronto, ON,
Canada
the development of PAH. Here, by profiling the metabolomic heterogeneity of the PAH lung, we reveal a newly
discovered pathogenesis mechanism of PAH.
Keywords Bile acid pathway Pulmonary arterial
hypertension Lung
1 Introduction
Pulmonary arterial hypertension (PAH) is a severe vascular
disease characterized by persistent precapillary pulmonary
hypertension (PH) (Stacher et al. 2012; International PPHC
et al. 2000; Zhao et al. 2002; Fujiwara et al. 2008; Nasim
et al. 2011; Olschewski 2010; Bogaard et al. 2012; MMea
and 2013), which can be either be idiopathic (sporadic90 %, familial-10 %). PAH can also be a complication
associated with other conditions such as connective tissue
disease, congenital heart disease, anorexigen use (dexfenfluramine), portal hypertension, and human immunodeficiency virus (Stacher et al. 2012; International PPHC, Lane
KB, Machado RD, Pauciulo MW, Thomson JR, et al. 2000;
MMea et al. 2013). Evidence in the literature suggests that
metabolic pathway abnormalities characterize and may
play a significant role in the development and progression
of PAH (Fessel et al. 2012). For example, pulmonary
arterial endothelial cells (PAECs) in PAH share similar
hyperproliferative characteristics as malignant tumor
transformation that is accompanied by significant metabolic shifts to support anabolic growth and energy metabolism (Xu et al. 2005; Chen et al. 2007). Moreover, it has
been shown that mitochondrial oxidative phosphorylation
with glucose uptake and utilization occurs in PAEC
development. Significant elevation of hemoglobin has been
found in the PAH sample group without a history of
123
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Y. D. Zhao et al.
RT: 5.04
AA: 100367
BP: 514.4
RT: 4.41 - 5.74 SM: 5G
NL: 6.57E4
m/z= 513.8-514.8 F: ITMS
- c ESI Full ms
[80.00-1000.00] MS ICIS
LTQ2NEG20120924_UNT
100
PPH sample
Relative Abundance
80
Peak for Taurocholate
O0412_LUNG2_17
60
40
20
RT: 4.62
AA: 6010
BP: 514.6
RT: 5.11
AA: 1168
RT: 5.27
BP: 514.7 RT: 5.21 AA: 21807
AA: 6265 BP: 514.5
BP: 514.8
RT: 4.89
AA: 3718
BP: 514.4
RT: 5.48
AA: 18581
BP: 514.5
0
RT: 5.61
AA: 9792
BP: 514.1
RT: 5.49
AA: 23892
BP: 514.5
100
NL: 9.00E3
Relative Abundance
NL sample
80
RT: 5.60
AA: 10278
BP: 514.1
RT: 4.61
AA: 6823
BP: 514.7
60
No peak
40
O0412_LUNG1_29
RT: 5.24
AA: 9787
BP: 514.7
RT: 5.35
AA: 1702
BP: 513.9
RT: 5.09
AA: 2357
BP: 514.6
20
m/z= 513.8-514.8 F: ITMS
- c ESI Full ms
[80.00-1000.00] MS ICIS
LTQ2NEG20120924_UNT
RT: 5.71
AA: 3026
BP: 514.3
0
4.5
4.6
4.7
4.8
4.9
5.0
5.1
5.2
5.3
5.4
5.5
5.6
5.7
Time (min)
496.3
100
353.3
Relative Abundance
PAH sample
60
40
351.3 354.4
497.4
369.3
412.2
453.3
20
495.7
432.4
167.1
0
100
Relative Abundance
NL: 7.92E2
LTQ2NEG20120924_UNTO04
12_LUNG2_17#1262 RT:
5.04 AV: 1 F: ITMS - c ESI d
Full ms2 [email protected]
[130.00-1040.00]
80
187.1 208.1 216.2
243.2
259.3 275.2
302.4
323.2
333.4
357.7
372.5
410.5
415.5
451.5 469.3
476.6
500.2
496.3
353.2
NL: 1.43E4
LTQ220071031N_PLEX106_
HIGH_41#1342 RT: 5.10 AV:
1 F: ITMS - c ESI d Full ms2
[email protected]
[130.00-1040.00]
80
Authentic standard
60
40
371.3
329.2
20
315.3
314.2
165.1 178.2
0
160
180
206.1 215.3
241.3
259.2
200
240
260
220
280
300
320
340
m/z
123
351.2
335.2
342.7
281.2 300.5
412.3
497.3
369.3
495.3
430.4
432.4
368.3
360
372.3 394.3
382.4
380
400
414.4
428.3
420
469.2
450.4
440
460
493.3
482.3
480
498.3
500
De novo synthesize of bile acids
b Fig. 1 MS/MS fragmentation spectrum of taurocholate in control and
PAH lung. Top panel shows a representative negative ion, selected
ion chromatogram (SIC) for taurocholate (m/z 514.3) in normal (NL)
and pulmonary hypertension (PAH) lung tissue. Taurocholate compound identification relied on confirmed experimental MS/MS
fragmentation spectrum matched to the authenticated taurocholate
standard, run separately (bottom panel). Limited peak detection was
observed in NL samples
diabetes or any other obvious metabolic diseases, indicating the impairment of whole-body glucose homeostasis in
PAH (Pugh et al. 2011; Hansmann et al. 2007; Archer et al.
2010). Additionally, vascular changes under chronic hypoxic condition has been directly linked to an imbalance
between glycolysis, glucose oxidation, and fatty acid oxidation (Sutendra et al. 2010), while in vitro pulmonary
arterial endothelial cell culture with disruption of the Bone
Morphogenetic Protein Receptor II (BMPRII) gene showed
significant metabolomic changes (Fessel et al. 2012). Our
recent work showed that disrupted glycolysis, increased
TCA cycle, and fatty acid metabolites with altered oxidation pathways exited in the human PAH lung, indicating
that PAH has specific metabolic pathways contributing to
abnormal ATP synthesis for the vascular remodeling process in pulmonary hypertension (Zhao et al. 2014). Collectively, in vitro, human and animal models suggest that
multiple metabolic pathways are reprogrammed during
PAH vascular remolding and that metabolic heterogeneity
may play an important role in both ATP energy supply and
the molecular pathogenesis of pulmonary hypertension.
Here, we provide direct evidence of a novel increase in bile
acid metabolites in PAH lung tissue associated with the
elevated expression of bile acid synthesis related transcripts, indicating de novo synthesis of bile acids may
characterize and contribute to the pathogenesis of PAH.
2 Materials and methods
Global biochemical profiles were determined in human
lung tissue and compared across 8 normal (47 ± 15 years
of age, 4 females) and 8 pulmonary arterial hypertension
patients (40 ± 12 years of age, 5 females). Eligibility criteria included end stage PAH patients who went through
lung transplantation. Lung samples were obtained from the
recipient lung at the time of lung transplantation. Control
lung samples were obtained from normal tissue of cancer
patients undergoing surgery (lobectomy). Biospecimens
and associated clinical data related to the study were collected with written consent from the University Health
Network and approved by the Internal Review Board.
Unbiased metabolomic profiling using liquid/gas chromatography coupled to mass spectrometry (LC/GC–MS) was
performed as described (Reitman et al. 2011; Evans et al.
1171
2009). The detail procedure of metabolic analysis has been
documented in the Supplement data.
2.1 Transcriptomic analysis
mRNA samples from the normal (n = 8) and native PAH
lungs (n = 8) were isolated as described (Zhao et al. 2014).
Bile acid related profiles were compared between a control
group and samples with idiopathic pulmonary arterial
hypertension. Briefly, the total RNA analysis in lung tissues was performed using Trizol extraction according to
the manufacturer’s instructions. Biotinylated cRNA was
prepared according to the standard Affymetrix protocol
(Expression Analysis). Following fragmentation, cRNA
were hybridized on GeneChip Genome Array. GeneChips
were scanned using the HuGene-1_0-st-v1 GeneArray
Scanner G2500A. The data were analyzed with Partek
Genomics Suite 6.6 using the Affymetrix default analysis
settings and global scaling as the normalization method.
The value definition was set up using Partek Genomics
Suite 6.6. Significantly changed genes were determined by
t test with a false discovery rate of two fold. The data base
has been submitted to NCBI/GEO and has been approved
and assigned a GEO accession number GSE53408.
2.2 Immunoblotting
Protein concentrations were determined using the BCA
protein assay (Pierce, IL, USA). Equal amounts of the protein lysates were separated by SDS-PAGE and transferred
onto nitrocellulose membranes. The membranes were incubated for overnight at 4 °C with the following antibodies
from AbcamR: anti-CYP7B1(1:1,000). After wash with
TBS-Tween, the blots were incubated for 60 min at room
temperature with horseradish peroxidase-conjugated antibodies, respectively: anti-rabbit antibody (1:15,000; SigmaAldrich, St. Louis, MO). Signals from immunoreactive
bands were visualized by fluorography using an ECL reagent
(Pierce). The intensity of individual bands in immunoblots
were quantified using the NIH Image program.
2.3 Immunohistochemistry
The sections of both PAH and normal lung tissue were
fixed for 4 h at room temperature with PBS made of 4 %
formaldehyde, permeabilized for 30 min in Triton X-100
(0.5 % in PBS), and incubated with 5 % nonfat skim milk
in PBS for 90 min. Sections were incubated for 180 min at
room temperature with antibodies for anti-CYP7B1
(1:1,000). The sections were then incubated with biotinylated secondary antibody and visualized with DAB.
Stained cells and sections were visualized with the Zeiss
LSM 510 confocal microscope.
123
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Y. D. Zhao et al.
RT: 5.06
AA: 100063
BP: 464.4
RT: 4.48 - 5.67 SM: 5G
RT: 5.42
AA: 230796
BP: 464.3
100
NL: 7.06E4
m/z= 463.9-464.9 F: ITMS
- c ESI Full ms
[80.00-1000.00] MS ICIS
LTQ2NEG20120924_UNT
O0412_LUNG1_09
Relative Abundance
PAH sample
80
60
Peak for Glycocholate
RT: 5.56
AA: 62313
BP: 464.4
40
20
RT: 4.63
AA: 10135
RT: 4.58 BP: 464.4
AA: 1223
BP: 464.2
0
RT: 5.20
AA: 17465
BP: 464.2
RT: 4.90
AA: 3952
BP: 464.4
Relative Abundance
100
RT: 5.06
AA: 5267
BP: 464.7
80
RT: 5.24
AA: 22719
BP: 464.5
NL sample
RT: 5.54
AA: 6129 RT: 5.58
BP: 464.3 AA: 2707
BP: 464.3
RT: 5.33
AA: 2890
BP: 464.1
60
NL: 3.05E3
m/z= 463.9-464.9 F: ITMS
- c ESI Full ms
[80.00-1000.00] MS ICIS
LTQ2NEG20120924_UNT
O0412_LUNG1_29
RT: 5.66
AA: 1694
BP: 464.4
RT: 5.44
AA: 2449
BP: 464.6
40
20
0
4.5
4.6
4.7
4.8
4.9
5.0
5.1
5.2
5.3
5.4
5.5
Time (min)
NL: 4.67E3
LTQ2NEG20120924_UNTO0
412_LUNG1_09#1268 RT:
5.06 AV: 1 F: ITMS - c ESI d
Full ms2 [email protected]
[115.00-940.00]
402.3
100
Relative Abundance
5.6
PAH sample
80
60
40
420.4
446.3
403.5
20
400.3
147.9
0
100
166.1
190.2 198.2 210.1 221.0 237.2
281.0 294.8 310.4 321.6
257.2
341.3 353.3
371.4
431.0
447.3
419.4
418.3
384.4
389.3
461.4
402.4
80
NL: 6.59E3
LTQ2NEG20080627_PLEX1
09_1_37#1550 RT: 5.45 AV:
1 F: ITMS - c ESI d Full ms2
[email protected]
[115.00-940.00]
Authentic standard
60
40
446.4
403.4
420.4
20
447.4
400.5
177.2
0
140
160
180
193.1 212.2 221.0
200
220
244.2
240
274.4 286.3 295.3
260
280
314.9 321.5
300
m/z
123
320
353.4 366.2
340
360
431.2
384.4
380
419.5
400
420
440
448.6
453.9
465.4
460
De novo synthesize of bile acids
1173
b Fig. 2 MS/MS fragmentation spectrum of glycocholate in control
a
glycocholate
taurocholate
8
a.
9.0
*
8.5
8.0
10
4
NL
5
0
PAH
b
0
mannitol
glycolithocholate sulfate
1400
3
2
700
1
0
0
glycochenodeoxycholate
taurochenodeoxycholate
c
10
20
CYP7B1
5
10
GAPDH
0
0
NL
PAH
NL
NL
PAH
Fig. 3 PAH lung has a unique bile acids metabolic pathway.
Intermediates in the bile acids pathway revealed significantly elevated
levels of multiple glycine and taurine conjugated bile acids in the
PAH lung. Data for normal lung (NL, n = 8) are represented in green
boxes, while data for pulmonary hypertension lung (n = 8) are shown
in pink boxes. Quantities are in relative arbitrary units specific to the
internal standards for each quantified metabolite and normalized to
protein concentration (PAH with red frame indicates *p \ 0.05
compared to NL
3 Results and discussion
We explored and characterized the metabolomic signature
of pulmonary hypertension (PAH) to enhance our understanding of disease progression. Using untargeted metabolic profiling, we found that PAH lung (n = 8) possessed
significantly higher levels of multiple bile acid metabolites,
including the primary bile acids taurocholate (Fig. 1),
glycocholate (Fig. 2), taurochenodeoxycholate, and glycochenodeoxycholate (Fig. 3). Bile acids are normally
synthesized in the liver and gallbladder from cholesterol by
7-alpha-hydroxylase, also called cytochrome P450
(CYP7A1), as a rate-limiting enzyme in the synthesis of
bile acid via the classic pathway (Nishimoto et al. 1993;
Cohen et al. 1992; Crestani et al. 1993; Wang and Chiang
1994). Although the presence of bile acids in lung tissue
may partially reflect reflux in these patient (D’Ovidio et al.
2005; Blondeau et al. 2009), microarray analysis
CYP7B1 (%)
relative metabolite abundance
Relitive CYP7B1
expression
and PAH lung. Representative negative ion is selected ion chromatogram (SIC) for glycocholate (m/z 464.4) in normal (NL) and
pulmonary hypertension (PAH) lung tissue (top panel). Glycocholate
compound identification relied on confirmed experimental MS/MS
fragmentation spectrum matched to the authenticated glycocholate
standard, run separately (bottom panel)
150
PAH
*
100
50
0
NL
PAH
Fig. 4 a Microarray data showed that the gene encoding cytochrome
P450, family 7, subfamily B, polypeptide 1 (Oxysterol 7a-hydroxylase) was significantly highly expressed in PAH lung.
(p = 0.000187299). b Western blot analysis of CYP7B1 expression
in normal and PAH lungs. Lung lysate was loaded and immunoblotted
with antibody against CYP7B1 and GAPDH (loading control).
Consistent with a significant increase of CYP7B1 gene expression
in PAH, the enzyme protein for CYP7B1 (37KD) was significantly
increased in PAH lungs compared with NL lungs. Densitometric
analysis of CYP7B1 was normalized to the intensity of the respective
GAPDH band. Data are expressed as mean ± SD (n = 4). *p \ 0.05
versus NL. c CYP7B1 positive immunostaining in newly formed
small blood vessels (arrows) in the plexiform lesions of occluded
pulmonary small vessel in PAH lung. Representative micrographs of
immunostaining of PAH lung sections are shown with anti–CYP7B1
in the pulmonary vascular endothelial cells. (ratio 1:200)
surprisingly revealed that the gene encoding cytochrome
P450 B1 (CYP7B1), but not CYP7A1, had a significantly
higher expression in PAH lung (Fig. 4a). This finding was
also confirmed by Real time RTPCR. Further molecular
123
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Y. D. Zhao et al.
cholesterol
CYP7A1
liver
CYP7B1
lung
7-a-hydroxycholesterol-4-en-3-one
propionyl CoA
cholate
chenodeoxycholate
glycine
taurine
taurine
glycine
conjugates conjugates conjugates conjugates
(Liver)
(lung)
biliary and
urinary
excretion
+ SO4
bile acid sulfation
Fig. 5 Major intermediates in the classical bile acids pathway
through Cholesterol 7 alpha-hydroxylase, also known or cytochrome
P450 7A1 (CYP7A1), are shown in blue. Our finding suggests that
PAH lung has a specific bile acids pathway though CYP7B1, as
shown in red
analysis using Western blot showed that the expression of
CYP7B1enzyme was higher in PAH lung (Fig. 4b). These
results suggest that increased bile acid metabolites may not
solely be due to reflux from the esophagus (D’Ovidio et al.
2005; Blondeau et al. 2009) but come from the lung itself.
Thus, PAH lung tissue may have the capacity for de novo
synthesis of bile acids. Notably, increased bile acids
metabolites could potentially serve as biomarkers for disease progression. By applying immunohistochemistry,
CYP7B1positive immunostaining was found in pulmonary
vascular endothelial cells, specifically in newly formed
vascular endothelial cells in plexiform lesions of occluded
pulmonary arteries (Fig. 4c), suggesting that CYP7B1may
also be involved in the vasculogenesis during the vascular
remodeling process of PAH (Fig. 5). This hypothesis needs
to be further tested by additional functional analyses. In
summary, we have shown direct evidence that a de novo
synthesis of bile acid may be involved in pathogenesis of
PAH, suggesting that bile acids in lavage fluid may serve as
ideal biomarkers for the diagnosis and prognosis of PAH.
Open Access This article is distributed under the terms of the
Creative Commons Attribution License which permits any use, distribution, and reproduction in any medium, provided the original
author(s) and the source are credited.
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