Urinary metabolomics in Fxr-null mice reveals activated adaptive metabolic pathways upon bile acid challenge

Article abstract of DOI:10.1194/jlr.M002923

Farnesoid X receptor (FXR) is a nuclear receptor that regulates genes involved in synthesis, metabolism, and transport of bile acids and thus plays a major role in maintaining bile acid homeostasis. In this study, metabolomic responses were investigated in urine of wild-type and Fxr-null mice fed cholic acid, an FXR ligand, using ultra-performance liquid chromatography (UPLC) coupled with electrospray time-of-flight mass spectrometry (TOFMS). Multivariate data analysis between wild-type and Fxr-null mice on a cholic acid diet revealed that the most increased ions were metabolites of p-cresol (4-methylphenol), corticosterone, and cholic acid in Fxr-null mice. The structural identities of the above metabolites were confirmed by chemical synthesis and by comparing retention time (RT) and/or tandem mass fragmentation patterns of the urinary metabolites with the authentic standards. Tauro-3α,6,7α,12α-tetrol (3α,6,7α,12α- tetrahydroxy-5β-cholestan-26-oyltaurine), one of the most increased metabolites in Fxr-null mice on a CA diet, is a marker for efficient hydroxylation of toxic bile acids possibly through induction of Cyp3a11. A cholestatic model induced by lithocholic acid revealed that enhanced expression of Cyp3a11 is the major defense mechanism to detoxify cholestatic bile acids in Fxr-null mice. These results will be useful for identification of biomarkers for cholestasis and for determination of adaptive molecular mechanisms in cholestasis.

Full text of DOI:10.1194/jlr.M002923

Urinary metabolomics in Fxr-null mice reveals activated
adaptive metabolic pathways upon bile acid challenge
Joo-Youn Cho,* Tsutomu Matsubara,* Dong Wook Kang,† Sung-Hoon Ahn,*
Kristopher W. Krausz,* Jeffrey R. Idle,§ Hans Luecke,† and Frank J. Gonzalez1,*
Laboratory of Metabolism,* Center for Cancer Research, National Cancer Institute, and Laboratory of
Bioorganic Chemistry,† National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes
of Health, Bethesda, MD; and Institute of Pharmacology,§ Charles University, First Faculty of Medicine,
Prague, Czech Republic
Abstract Farnesoid X receptor (FXR) is a nuclear receptor
that regulates genes involved in synthesis, metabolism, and
transport of bile acids and thus plays a major role in main-
taining bile acid homeostasis. In this study, metabolomic
responses were investigated in urine of wild-type and Fxr-
null mice fed cholic acid, an FXR ligand, using ultra-
performance liquid chromatography (UPLC) coupled with
electrospray time-of-flight mass spectrometry (TOFMS).
Multivariate data analysis between wild-type and Fxr-null
mice on a cholic acid diet revealed that the most increased
ions were metabolites of p-cresol (4-methylphenol), cortico-
sterone, and cholic acid in Fxr-null mice. The structural
identities of the above metabolites were confirmed by chem-
ical synthesis and by comparing retention time (RT) and/or
tandem mass fragmentation patterns of the urinary metabo-
lites with the authentic standards. Tauro-3␣,6,7␣,12␣-tetrol
(3␣,6,7␣,12␣-tetrahydroxy-5␤-cholestan-26-oyltaurine), one
of the most increased metabolites in Fxr-null mice on a CA
diet, is a marker for efficient hydroxylation of toxic bile ac-
ids possibly through induction of Cyp3a11. A cholestatic
model induced by lithocholic acid revealed that enhanced
expression of Cyp3a11 is the major defense mechanism
to detoxify cholestatic bile acids in Fxr-null mice. These
results will be useful for identification of biomarkers for
cholestasis and for determination of adaptive molecular
mechanisms in cholestasis.—Cho, J. Y., T. Matsubara, D. W.
Kang, S. H. Ahn, K. W. Krausz, J. R. Idle, H. Luecke, and
F. J. Gonzalez. Urinary metabolomics in Fxr-null mice re-
veals activated adaptive metabolic pathways upon bile acid
challenge. J. Lipid Res. 2010. 51: 1063–1074.
The farnesoid X receptor (FXR; NR1H4) is a nuclear
receptor that is activated by a number of bile acids includ-
ing cholic acid, chenodeoxycholic acid, and lithocholic
acid. Activated FXR forms a heterodimer with retinoid X
receptors (RXR; NR1B) and binds its response elements
usually located upstream of FXR target genes (1, 2). FXR
regulates genes involved in synthesis, metabolism, and
transport of bile acids in liver and gut, thereby maintain-
ing bile acid homeostasis. Bile acids are produced from
cholesterol in liver by bile acid synthetic enzymes, such as
CYP7A1 and CYP8B1 (3), and are excreted into bile
through canalicular bile acid transporters, such as bile salt
export pump (BSEP) and MRP2 (4). However, under the
condition of excessive bile acid loading such as in experi-
mental cholestasis in mice, FXR is strongly activated by
bile acids, downregulates the expression of Cyp7a1 and
Cyp8b1, upregulates bile acid transporters, following which
serum and liver bile acid levels are decreased (5).
The critical role of FXR in bile acid homeostasis was es-
tablished using mice with targeted disruption of Fxr (Fxr-
null mice) (6). Fxr-null mice, under conditions of bile acid
loading such as cholic acid feeding and common bile duct
ligation, had increased serum and liver bile acid levels and
decreased fecal bile acid excretion because of reduced ex-
pression of the bile acid export pump BSEP (6). In addi-
tion, excretion of urinary bile acids was increased in
Fxr-null mice fed diets containing 1% cholic acid, suggest-
Supplementary key words adaptive response • cholic acid • cortico-
sterone • Cyp3a11 • farnesoid X receptor • lithocholic acid • metabolo-
mics • p-cresol
Abbreviations: ALP, alkaline phosphatase; ALT, alanine amino-
transferase; CA, cholic acid; CAR, constitutive androstane receptor;
CYP, cytochrome P450; DHOPA, 11␤,20-dihydroxy-3-oxopregn-4-en-
21-oic acid; FXR, farnesoid X receptor; HDOPA, 11␤-hydroxy-3,20-
dioxopregn-4-en-21-oic acid; LCA, lithocholic acid; MS/MS, tandem
mass spectrometry; OPLS, orthogonal partial least squares; PCA, prin-
cipal components analysis; PLS-DA, partial least-squares discriminant
analysis; PPAR␣, peroxisome proliferator-activated receptor ␣; PXR,
pregnane X receptor; qPCR, quantitative real-time polymerase chain
reaction; RT, retention time; TOFMS, time-of-flight mass spectrometry;
UPLC, ultra-performance liquid chromatography.
This work was supported by the National Cancer Institute Intramural Research
Program. Its contents are solely the responsibility of the authors and do not nec-
essarily represent the official views of the National Institutes of Health. This
work was also supported by Grant A03001 (J.Y.C.) from the Korea Health 21
R&D Project, Ministry of Health and Welfare, Republic of Korea and by a grant
for collaborative research (J.R.I.) from the United States Smokeless Tobacco
Company.
Manuscript received 5 October 2009 and in revised form 9 November 2009.
Published, JLR Papers in Press, November 9, 2009
DOI 10.1194/jlr.M002923
1 To whom correspondence should be addressed.
e-mail: fjgonz@helix.nih.gov
This article is available online at http://www.jlr.org
Journal of Lipid Research Volume 51, 2010
1063

ting (San Meteo, CA). HPLC-grade solvents (acetonitrile, metha-
nol, and water) were purchased from Fisher Scientific (Hampton,
NH).
ing that the metabolic pathways of controlling bile acid
levels might be altered in Fxr-null mice (6). Recently, an
adaptive response to bile acids in Fxr-null mice was re-
ported. Loss of FXR leads to increased susceptibility to bile
acid-induced liver injury and results in adaptive changes of
genes involved in regulation of basolateral bile acid up-
take, bile acid metabolism/detoxification, and alternative
basolateral bile acid secretion in an FXR-independent
manner (7, 8).
Animals, diets, and sample collection
Fxr-null mice and the background matched wild-type mice
generated as reported previously (6) were maintained under a
standard 12-h light/12-h dark cycle with water and a normal diet
(NIH-31) provided ad libitum. Groups of 8- to 12-week-old male
mice were put on a synthetic purified diet (AIN-93G, Bio-Serv) at
day 5 before treatment and maintained with water and the same
diet ad libitum. The CA diet and LCA diet consisted of the con-
trol diet (AIN-93G) supplemented with 1% (w/w) CA and 0.6%
(w/w) lithocholic acid, respectively. Fxr-null and wild-type mice
were fed the CA diet or control diet for 7 days and the LCA diet
or control diet for 4 days. Urine samples were collected from
mice placed individually in metabolic cages for 24 h, before treat-
ment and on day 6 of CA treatment or day 3 of LCA treatment.
All urine samples were stored at –80°C until analyzed. At the end
of the study, animals were killed, and serum and liver tissue were
collected and frozen at –80°C for further analysis. Protocols for
all animal studies were approved by the National Cancer Institute
Animal Care and Use Committee and were carried out in accor-
dance with the guidelines of Institute of Laboratory Animal
Resources.
Metabolomics, the global approach for identification
and quantification of metabolites in a living system or bio-
logical sample has been mostly performed using nuclear
magnetic resonance (NMR) spectroscopy or mass spec-
trometry (MS) combined with various multivariate data
analyses (9). Recently, robust metabolomic methods using
the high resolution capability of ultra-performance liquid
chromatography (UPLC) coupled with the accurate mass
determination of time-of-flight (TOF) MS were developed
for detection and characterization of small organic mole-
cules in complex biological matrices (10). A recent me-
tabolomics study, combined with a genetically modified
knockout mouse, has revealed that activation of peroxi-
some proliferator-activated receptor ␣ (PPAR␣) affects
the metabolism of tryptophan, corticosterone, and fatty
acids, and that novel steroid metabolites, 11␤-hydroxy-
3,20-dioxopregn-4-en-21-oic acid (HDOPA) and 11␤,20-
dihydroxy-3-oxopregn-4-en-21-oic acid (DHOPA), in urine
can be biomarkers for activation of PPAR␣ (11). More re-
cently, a metabolomics study on the activation of pregnane
X receptor (PXR) was performed using urine from Pxr-
null mice and their congenic wild-type counterparts
treated with the PXR ligand, pregnenolone 16␣-
carbonitrile (12). A novel vitamin E metabolite, ␥-CEHC
glucoside, and ␣-CEHC glucuronide were strongly de-
creased upon activation of PXR and might serve as bio-
markers for PXR activation. Thus, metabolomics revealed
PXR-activated metabolic pathways associated with vitamin
E metabolism (12). These studies highlight that metabolo-
mics can be useful to identify small molecule signatures
for gene deficiency and activation of nuclear receptors
and/or their pathophysiological stimuli.
Serum chemistry
Serum was prepared by centrifugation at 8,000 rpm for 10 min
and the catalytic activities of alanine aminotransferase (ALT)
and alkaline phosphatase (ALP) were measured in serum. Briefly,
2 ␮l of serum was mixed with 200 ␮l of ALT or ALP assay buffer
(Catachem, Bridgeport, CT) in a 96-well microplate and moni-
tored at 340 nm or 405 nm for 10 min at 37°C. Serum corticoste-
rone levels were determined using a Corticosterone EIA Kit
(Cayman Chemical Co., Ann Arbor, MI) according to the manu-
facturer’s instruction.
UPLC-TOFMS analyses
Urine aliquots were diluted with 4 vols of 50% acetonitrile and
centrifuged at 18,000 g for 20 min at 4°C to remove particles and
proteins. The aliquots (5 ␮l) were injected into a reverse-phase
50 × 2.1 mm ACQUITY® 1.7-␮m BEHC18 column (Waters Corp.,
Milford, MA) using an ACQUITY® UPLC system (Waters) with a
gradient mobile phase comprising 0.1% formic acid (A) and ac-
etonitrile containing 0.1% formic acid (B). Each sample was re-
solved for 10 min at a flow rate of 0.5 ml/min with the gradient
consisted of 100% A for 0.5 min, 20% B for 3.5 min, 95% B for 4
min, 100% B for 1 min, and 100% A for 1 min. The eluent was
introduced by electrospray ionization into the mass spectrome-
ter, Q-TOF Premier® (Waters), operating in either negative ion
(ESIϪ) or positive ion (ESI+) electrospray ionization modes. The
capillary and sampling cone voltages were set to 3000 and 30 V,
respectively. The desolvation gas flow was set to 650 L/h and the
temperature was set to 350°C. The cone gas flow was 50 L/h, and
the source temperature was 120°C. To maintain mass accuracy,
sulfadimethoxine ([M-H]- 309.0658) at a concentration of 250
pg/␮l in 50% acetonitrile was used as a lock mass and injected at
a rate of 30 ␮l/min. Data were acquired in centroid mode from
50 to 800 m/z in MS scanning. Tandem MS collision energy was
scanned from 5 to 35 V.
In the present study, altered metabolomic responses
were investigated in urine of Fxr-null mice administered
cholic acid compared with wild-type mice. UPLC-TOFMS
coupled with multivariate data analyses were used to dis-
tinguish between the urinary metabolites in Fxr-null and
wild-type mice on the CA diet. The structure of novel me-
tabolites was identified and those metabolites were quanti-
fied using synthetic standards. This study further revealed
adaptive molecular mechanisms for activation of alternate
metabolic pathways in the cholestatic Fxr-null mouse.
EXPERIMENTAL PROCEDURES
Reagents
Data processing and multivariate data analysis
Standards for taurocholate, tauro-␤-muricholate and reagents
for synthesis were purchased from Sigma (St. Louis, MO).
HDOPA and DHOPA were purchased from Anbilaunch consul-
Centroided and integrated chromatographic mass data from
50 to 800 m/z were processed by MarkerLynx® (Waters) to gener-
ate a multivariate data matrix. Pareto-scaled MarkerLynx matri-
1064
Journal of Lipid Research Volume 51, 2010

ces including information on sample identity were analyzed by
principal components analysis (PCA) and partial least-squares
discriminant analysis (PLS-DA) using SIMCA-P+ 12 (Umetrics,
Kinnelon, NJ). To determine which ions contribute to the differ-
ence between wild-type (y = 0) and Fxr-null (y = 1) mice of the
CA-fed group, orthogonal partial least squares (OPLS) was used.
The loadings scatter S-plots and the contribution lists were used
to describe the candidate markers that were significantly differ-
ent between wild-type and Fxr-null mice of CA-fed group.
analyte, and the resultant concentrations were expressed as
␮mol/mmol creatinine (normalized).
Gene expression analysis
Quantitative real-time PCR (qPCR) was performed using
cDNA generated from 1 ␮g total mRNA SuperScript II Reverse
Transcriptase kit (Invitrogen, Carlsbad, CA). Primers were de-
signed for qPCR using the Primer Express software (Applied Bio-
systems, Foster City, CA) based on GenBank sequence data:
Cyp3a11: forward 5′-ttctgtcttcacaaaccggc-3′; reverse 5′-gggggacag-
caaagctctat-3′, Cyp2b10: forward 5′-tcttgcgcctgctgcag-3′; reverse
5′-tggctggagaatgagcttatgag-3′ and Actb: forward 5′-ttctttgcagctcct-
tcgtt-3′, reverse 5′-atggaggggaatacagccc-3′. qPCR reactions were
performed with the cDNA, the primer sets and SYBR Green PCR
Master Mix (Applied Biosystems, Foster City, CA) and demon-
strated by an Applied Biosystems Prism 7900HT Sequence Detec-
tion System. Relative mRNA levels were calculated by the
comparative threshold cycle method using ␤-actin (Actb) as the
internal control.
Identification of metabolites
To identify the structure of high-contribution score metabo-
lites, elemental compositions were generated with MassLynx®
(Waters) based on the exact masses of the top twelve ions. To
confirm the identities of markers, authentic standards at 5–20
␮M in 50% acetonitrile were compared with the urine sample on
the condition of MS/MS fragmentation with collision energy
ramping from 15 to 35 V. Therefore, the MS/MS fragmentation
spectrum of putative urine metabolites was shown to be identical
to that of the authentic standards.
Statistical analysis
Synthesis of p-cresol sulfate and glucuronide
Each group consisted of 6–12 animals for this study. All values
are expressed as the means SD. Statistical analysis was per-
formed by two-way ANOVA combined with Bonferroni posttests
using Prism 5 (GraphPad Software Inc., San Diego, CA). Correla-
tions between relative Cyp3a11 levels and relative abundance of
taurotetrol or ALT activity were calculated using Pearson correla-
tion tests (Prism 5). A P value of less than 0.05 was considered
statistically significant.
p-Cresol sulfate was prepared by treating p-cresol with chloro-
sulfonic acid in methylene chloride. To synthesize p-cresol
glucuronide, p-cresol was reacted with tetraacetylglucuronyl
trichloroimidate in the presence of boron trifluoride diethyl
etherate in methylene chloride to afford p-cresol tetraacetyl glu-
curonide (13). The product was hydrolyzed with 2M potassium
hydroxide in methanol to obtain p-cresol glucuronide.
Synthesis of bile acid metabolites
RESULTS
Synthesis of (3␣,6␣/␤,7␣/␤,12␣)-tetrahydroxy-5␤-cholanic ac-
ids (4 epimers) have been reported (14). Synthesis of 3␣,7␤,12␣-
trihydroxy-5␤-cholanic acid also followed this method. All
stereoisomeric bile acids were synthesized from cholic acid. Tau-
rine conjugates of 5␤-cholanic acids were synthesized by two-step
reactions (15, 16). The first step was to prepare pentafluorophenyl
esters where 5␤-cholanic acids reacted with pentafluorophenol
in the presence of 1-(3-dimethylaminopropyl)-3-ethylcarbodiim-
ide hydrochloride and a catalytic amount of N,N-dimethylforma-
mide in methylene chloride. The second step was the reaction of
5␤-cholanic acid pentafluorophenyl esters and taurine in 1,8-
diazabicyclo[5,4,0]undec-7-ene as a base in methylene chloride
to synthesize taurine conjugates of bile acids. Although most of
5␤-cholanic acid taurine conjugates are very polar compounds,
they can be purified by a general silica gel column chromatogra-
phy method eluting with gradient of 5-25% methanol in methyl-
ene chloride. To isolate taurine conjugates by this general
column chromatography method, 1% acetic acid was added to
the eluting solvent. All 5␤-cholanic acids and their taurine conju-
gates have good solubility in water and methanol.
Phenotypes of Fxr-null mice
Male wild-type and Fxr-null mice fed a control diet
showed no significant differences in body weight, liver-to-
body weight ratio, and serum ALP activity (Table 1). How-
ever, after feeding 1% CA for 7 days, Fxr-null mice exhibited
more severe body weight loss, higher liver-to-body weight
ratios, and higher serum ALP activity than wild-type mice
(Table 1). These phenotypes are similar with those previ-
ously reported for wild-type and Fxr-null mice after feeding
CA (17). In contrast, the typical diagnostic hepatotoxicity
marker, serum ALT activity, was two-fold higher in Fxr-null
mice than in wild-type mice fed control diet but was not
significantly changed between Fxr-null and wild-type mice
TABLE 1. Changes in body weight, liver- to body weight ratio,
and liver toxicity
Control
CA-Fed
Diagnostic
Marker
Quantification of urinary metabolites
Wild-Type Mice Fxr-Null mice Wild-Type Mice Fxr-Null Mice
QuanLynx® (Waters) was used to quantify urinary creatinine,
HDOPA, DHOPA, taurocholate, tauro-7-epicholate, and tauro-
3,6,7,12-tetrol from their peak areas. One ␮M of dehydrocholate
([M-H]- 401.2330) was included as an internal standard. Calibra-
tion curves were constructed from 50 to 1000 ␮M of creatinine
([M+H]+ 114.0670) and from 1 to 100 ␮M of HDOPA ([M-H]-
359.1843), DHOPA ([M-H]- 361.2008), taurocholate ([M-H]-
514.2831 and RT 5.5 min), tauro-7-epicholate ([M-H]- 514.2822
and RT 5.0 min) and tauro-3,6,7,12-tetrol ([M-H]- 530.2779).
The concentration of each analyte in mouse urine was deter-
mined from the calibration curves using linear regression analy-
sis. All determined correlation coefficients were >0.95 for each
BW (%)
LW/BW
ratio
ALT (IU/l)
ALP (IU/l)
101.5 2.7
3.92 0.21
102.7 1.6
3.79 0.12
90.7 8.9
73.3 4.16b
5.08 0.46b
3.61 0.45
71.7 20.1 141.4 62.8a 1240 1110 1810 625
163 18.2 221 102 165 58.0
456 91.0b
Comparison of diagnostic markers between wild-type and Fxr-null
mice in either control or cholic acid-treated groups (n = 4–12). Values
are means
SD. ALP, alkaline phosphatase; ALT, alanine
aminotransferase; BW, body weight; FXR, farnesoid X receptor; LW,
liver weight.
aP < 0.01 compared with wild-type mice.
bP < 0.001 compared with wild-type mice.
Metabolomics in Fxr-null mice
1065

fed CA, which suggested that Fxr-null mice under bile acid-
loading conditions exhibited FXR-dependent cholestasis,
but showed similar sensitivity to bile acid-induced hepato-
toxicity compared with CA fed wild-type mice.
plot providing a clear separation between wild-type (+/+
)
and Fxr-null (Ϫ/Ϫ) mice of CA-fed groups in component 1
(Y-axis) but not in the control diet group. This result re-
vealed that more specific metabolic phenotypes were
changed in Fxr-null mice than in wild-type mice by CA
loading. After OPLS analysis of wild-type and Fxr-null mice
in the CA-fed groups, a loadings S-plot showed ions with
the highest confidence and greatest contribution to sepa-
ration between wild-type and Fxr-null mice in the CA-fed
groups (Fig. 1B). The significant ions increased in Fxr-null
mice after CA loading were in the upper-right quadrant
and those decreased were in the lower-left quadrant. The
increased negative ions were labeled with a number from
1 to 12 according to the highest confidence and greatest
contribution to separation between wild-type and Fxr-null
mice in CA-fed groups in Fig. 1B. Three types of metabo-
lites, specifically p-cresol, corticosterone, and cholic acid
metabolites, were confirmed from the data (Table 2). To
identify the structure of the above ions, accurate mass val-
ues of each ion was used for chemical formula calculations
using elemental composition and mass-based searches in
variousdatabase/literaturewereperformed.Both187.0060
(1; [M-H]Ϫ) and 397.0009 (9; [2M-2H+Na]Ϫ) ions corre-
spond to p-cresol sulfate, and the 283.0812 (3; [M-H]Ϫ)
Metabolomic analysis of mouse urine
Urine samples for 0–24 h were collected before and af-
ter six days of CA feeding and analyzed by UPLC-TOFMS
operating in both positive and negative ionization modes.
Bile acids showed several fragment ions derived from only
one bile acid in positive ion mode and thus multivariate
data analysis was performed on the data matrix from the
negative ionization mode. A large data matrix containing
approximately 6000 ions was produced by MarkerLynx
and subjected to both PCA and PLS-DA multivariate data
analyses. Unsupervised PCA yielded a good separation of
the data sets from the control and CA-fed groups in both
wild-type and Fxr-null mice, with fitness (R2 value) of 0.52
and prediction power (Q2 value) of 0.28 (data not shown).
A supervised PLS-DA model with six components success-
fully discriminated the differences between all four groups
of mice, having cumulative fitness (R2X and R2Y values) of
0.65 and 0.96, respectively, and cumulative prediction
power (Q2 value) of 0.80. Fig. 1A shows a PLS-DA scores
Fig. 1. Metabolomic analysis of control diet- and
CA diet-fed mouse urine. Wild-type and Fxr-null mice
(n = 6–12) were fed with control diet or 1% CA con-
taining diet for 7 days. Urinary metabolites were ana-
lyzed by UPLC-TOFMS and data processing and
multivariate data analysis conducted using Marker-
Lynx and SIMCA-P+ software, respectively. A: Scores
scatter plot of PLS-DA model of urine from the con-
trol and CA-treated groups in both wild-type (+/+
)
and Fxr-null (Ϫ/Ϫ) mice. A six-component PLS-DA
model was constructed to characterize the relation-
ship among four mouse groups, including wild-type
(control, open circle; CA-fed, closed circle) and Fxr-
null (control, open triangle; CA, closed triangle).
The t(1) and t(2) values represent the scores of each
sample in principal components 1 and 2, respectively.
The cumulative fitness (R2X and R2Y values) and pre-
diction power (Q2 value) of this six-component
model are 0.65, 0.96, and 0.80, respectively. B: OPLS
loadings S-plot comparing Fxr-null mice versus wild-
type mice in CA-fed groups. The x axis is a measure of
the relative abundance of the ions, and the y axis is a
measure of the correlation of each ion to the model.
Negative ions located in the upper right quadrant are
increased when comparing Fxr-null mice with wild-
type mice in CA-fed groups. Labeling of significant
ions increased (closed square) is the same as in Table
1. CA, cholic acid; FXR, farnesoid X receptor;
TOFMS, time-of-flight mass spectrometry; UPLC,
ultra-performance liquid chromatography.
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Journal of Lipid Research Volume 51, 2010

TABLE 2. Summary of negative metabolite ions showing significant increase in urine concentration
of CA-fed Fxr-null mice
Mass Error
(ppm)
Fold Change
(Fxr-null/WT)
Ion Rank
RT (min)
Mass (m/z)
Identity
Empirical Formula
p-Cresol metabolites
1
3
9
3.41
187.006
p-Cresol sulfate
C7H8O4S
C13H16O7
2.5
2.0
4.6
5
8
9
3.53
3.40
283.0812 p-Cresol glucuronide
397.0009 p-Cresol sulfate_dimer+Na C14H16O8S2Na
Corticosterone metabolites
4
5.29
5.39
4.66
4.54
361.2008 DHOPA
C21H30O5
C21H28O5
C21H30O6
C21H28O6
1.9
4.4
5.5
2.3
18
5
6
359.1843 HDOPA
50<
50<
50<
377.1943 Hydroxylated DHOPAa
375.1799 Hydroxylated HDOPAa
10
Cholic acid metabolites
2
4.79
5.50
5.37
5.00
5.15
530.2779
C26H45NO8S
C26H45NO7S
C30H50O10
C26H45NO7S
C32H55NO12S
1.6
1.5
3.5
3.2
1.9
50<
5
12
50<
50<
Tauro-3␣,6,7␣,12␣-tetrol
7
8
514.2831 Taurocholate
569.3306 Cholate glucosidea
514.2822 Tauro-7-epicholate
676.3354 Taurocholate glucosidea
11
12
CA-fed Fxr-null mice compared with CA-fed wild-type mice (n = 12). Conditions for UPLC-TOFMS analysis are
described in “Experimental Procedures.” Ion rank from a loading S-plot of OPLS analysis showed the rank of ions
with the highest confidence and greatest contribution to separation between wild-type and Fxr-null mice. Ion rank
is the same as labeling of ions in Fig. 1B. Fold change equals the fold increase in the level observed in Fxr-null mice
group compared with wild-type mice group. CA, cholic acid; FXR, farnesoid X receptor; RT, retention time; TOFMS,
time-of-flight mass spectrometry; UPLC, ultra-performance liquid chromatography; WT, wild type.
a Structural identities of these metabolites were not elucidated.
ion corresponds to p-cresol glucuronide. Generation of
the isomeric o-cresol and m-cresol sulfates in situ by the
sulfation of o- and m-cresol with fuming sulfuric acid re-
vealed that the three isomeric sulfates could not be re-
solved by UPLC and that they had virtually identical MS/
MS spectra (data not shown). Accordingly, urines were hy-
drolyzed with 5M HCl at 100°C, neutralized, subjected to
solid phase extraction, eluted and then, together with au-
thentic o-, m-, and p-cresol, derivatized with BSTFA to yield
the trimethylsilyl derivatives of the cresol isomers. GCMS
analysis revealed that the cresol present in urine as its sul-
fate and glucuronide was almost exclusively p-cresol (data
not shown). The 361.2008 (4; [M-H]Ϫ) and 359.1843 (5;
[M-H]Ϫ) ions correspond to DHOPA and HDOPA, respec-
tively, according to a previous report (11). The 377.1943
(6; [M-H]Ϫ) and 375.1799 (10; [M-H]Ϫ) ions are novel
ions that are proposed to be hydroxylated DHOPA and
HDOPA, respectively. The 530.2779 (2; [M-H]Ϫ), 514.2831
(7; [M-H]Ϫ), 569.3306 (8; [M-H]Ϫ), 514.2822 (11; [M-H]Ϫ)
and 676.3354 (12; [M-H]Ϫ) ions correspond to tauro-
3␣,6,7␣,12␣-tetrol, taurocholate, cholate glucoside, tauro-
7-epicholate, and taurocholate glucoside, respectively.
Because the basal levels of the metabolite ions, 2, 5, 6, 10,
11, and 12 were very low, the values of fold change in each
metabolite were over 50. However, the chemical structures
still required experimental verification using the authen-
tic compounds.
ter ions 107 and 79.9 in negative ion mode were interpreted
in the inlaid structural diagram. In addition, the MS/MS
structural elucidation of p-cresol glucuronide ([M-H]Ϫ
=
283.0812) in negative ion mode was performed (Fig. 2B).
The major daughter ions 175 and 107 in negative ion mode
were interpreted in the inlaid structural diagram. Because
of the insufficient purity of synthetic standards, proper cali-
bration curves could not be constructed for both p-cresol
sulfate and p-cresol glucuronide. Therefore, relative abun-
dance of p-cresol sulfate and p-cresol glucuronide were
measured in each urine sample and expressed as response/
mmol creatinine, a variable independent of urine volume.
Although the relative abundance of p-cresol sulfate and
p-cresol glucuronide were decreased significantly in both
wild-type and Fxr-null mice after CA feeding (P < 0.001),
the wild-type mice on the CA diet exhibited a significant
and dramatic depletion of urinary p-cresol sulfate (8.3% of
control, P < 0.001, Fig. 2C) and p-cresol glucuronide (4.5%
of control, P < 0.001, Fig. 2D). Therefore, the Fxr-null mice
on the CA diet exhibited significantly greater than wild-
type mice on the CA diet by 4-fold (P < 0.01) or 10-fold
(P < 0.001) in either p-cresol sulfate or p-cresol glucuronide
urine levels, respectively (Fig. 2C, D).
Identification and quantification of glucocorticoid
metabolites
To identify the structure of hydroxylated HDOPA
and DHOPA, authentic compounds of 19-hydroxylated
HDOPA and 19-hydroxylated DHOPA were synthesized
and tandem MS fragmentation was performed. The pat-
terns of MS/MS spectra between the synthetic compounds
and the hydroxylated metabolites in mouse urine were dif-
ferent (data not shown), suggesting that the hydroxylated
HDOPA and DHOPA is not 19-hydroxyl HDOPA and
19-hydroxyl DHOPA. The identities of HDOPA and
DHOPA were established by comparison of MS/MS frag-
Identification and quantification of p-cresol metabolites
Authentic p-cresol sulfate and p-cresol glucuronide were
required to confirm the structural identification of these
metabolites by UPLC-MS/MS and to quantify the levels of
these metabolites. The p-cresol sulfate and p-cresol
glucuronide were synthesized and the MS/MS structural
elucidation of p-cresol sulfate ([M-H]Ϫ = 187.006) in nega-
tive ion mode was carried out (Fig. 2A). The major daugh-
Metabolomics in Fxr-null mice
1067

Fig. 2. Identification and characterization of p-cresol sulfate and p-cresol glucuronide. A: MS/MS spec-
trum of authentic p-cresol sulfate and metabolite 1 from mouse urine in negative ion mode. MS/MS frag-
mentation was conducted with collision energy ramping from 5 to 35 eV. Major daughter ions from MS/MS
fragmentation were interpreted in the inlaid structural diagrams. B: MS/MS spectrum of authentic p-cresol
glucuronide and metabolite 3 from mouse urine in negative ion mode. MS/MS fragmentation was con-
ducted with same condition as p-cresol sulfate. C: Relative abundance of p-cresol sulfate in the 24-h urine of
wild-type and Fxr-null mice treated with 1% CA or control for 7 days. Relative abundance of p-cresol sulfate
(mean SD) was determined by the peak area responses of p-cresol sulfate normalized by the creatinine
concentration in urine. D: Relative abundance of p-cresol glucuronide (mean SD) was determined by the
peak area responses of p-cresol glucuronide normalized by the creatinine concentration in urine. Open and
filled bars represent wild-type and Fxr-null mice groups, respectively. The P values were calculated by ANOVA
with Bonferroni posttests. *P < 0.01 and **P < 0.001 compared with wild-type mice. CA, cholic acid; FXR,
farnesoid X receptor; MS/MS, tandem mass spectrometry.
mentation of authentic standards and the urinary com-
pounds as demonstrated in the previous report (11). In
addition, the concentrations of HDOPA and DHOPA in
urine were determined using calibration curves. Whereas
both wild-type and Fxr-null mice fed control diet had un-
detectable HDOPA and DHOPA in their urine, Fxr-null
mice fed CA diet only had 229 92 ␮mol/mmol creati-
nine in HDOPA and 110 25 ␮mol/mmol creatinine in
DHOPA (Fig. 3A, B). Moreover, the relative abundance of
hydroxylated HDOPA and DHOPA was significantly in-
creased in Fxr-null mice fed CA diet as a similar pattern to
the concentration of HDOPA and DHOPA (Fig. 3C, D).
Since corticosterone metabolites are elevated in urine, se-
rum corticosterone levels would be expected to increase in
the Fxr-null mice fed CA diet, and thus corticosterone lev-
els were determined in serum (Fig. 3E). Indeed, Fxr-null
mice showed a robust increase in corticosterone level after
treatment with the CA diet (changed 24.0 8.3 to 149 76
ng/ml, P < 0.01). On the other hand, wild-type mice did
not exhibit a significant change in corticosterone levels
(changed 13.0 7.0 to 29.0 17.0 ng/ml).
Identification of cholic acid metabolites
Authentic cholic acid metabolites were required for
elucidatingstructure.Tauro-3␣,7␤,12␣-trihydroxycholate
(tauro-7-epicholate) and
4 epimers of tauro-3␣,6(␣/
␤),7(␣/␤),12␣-tetrahydroxycholate were synthesized as
described in “Experimental Procedures.” However, two novel
metabolites, cholate glucoside (C30H50O10, mass error =
3.5 ppm) and taurocholate glucoside (C32H55NO12S, mass
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Journal of Lipid Research Volume 51, 2010

Fig. 3. Quantification of urinary corticosterone metabolites and serum corticosterone in wild-type and Fxr-
null mice treated with 1% CA or control for 7 days. A: Creatinine-normalized concentration of HDOPA.
B: Creatinine-normalized concentration of DHOPA. Concentrations of creatinine, HDOPA, and DHOPA
were determined from calibration curve of each metabolite. All concentrations for quantification were nor-
malized to the creatinine concentration (␮mol/mmol creatinine). C: Relative abundance of hydroxylated
HDOPA was determined by the peak area responses of hydroxylated HDOPA normalized by the creatinine
concentration in urine (response/mmol creatinine). D: Relative abundance of hydroxylated DHOPA was
determined by the peak area responses of hydroxylated DHOPA normalized by the creatinine concentration
in urine (response/mmol creatinine). Data were represented as mean value SD (n = 6–12). E: Serum cor-
ticosterone levels. The corticosterone levels were measured as described in “Experimental Procedures.” Data
were expressed as mean value and SD (n = 4–5). Significant difference was determined by one-way ANOVA
following Bonferroni’s test. *P < 0.01 compared with wild-type mice. Open and filled bars represent wild-type
and Fxr-null mice groups, respectively. CA, cholic acid; DHOPA, 11␤,20-dihydroxy-3-oxopregn-4-en-21-oic
acid; FXR, farnesoid X receptor; HDOPA, 11␤-hydroxy-3,20-dioxopregn-4-en-21-oic acid.
error = 1.9 ppm), could not be synthesized and confirmed
by UPLC-MS/MS.
acid metabolites are too stable in negative ion mode to
generate their tandem mass spectra (Fig. 4). Taurocholate
The identities of tauro-3␣,7␣,12␣-trihydroxycholate
(taurocholate), tauro-7-epicholate, and tauro-3␣,6,7␣,12␣-
tetrahydroxycholate (tauro-3␣,6,7␣,12␣-tetrol) were con-
firmed by comparison of retention time from authentic
compounds and the urinary constituent because cholic
and tauro-7-epicholate have identical m/z ([M-H]Ϫ
=
514.283) but separable retention time, i.e., 5.39 min (Fig.
4A) and 4.97 min (data not shown), respectively. Among
the urinary ions with 514.283 m/z ([M-H]Ϫ), Peak a had a
4.97 min retention time and was revealed to be identical to
Metabolomics in Fxr-null mice
1069

Fig. 4. Identification of tauro-7-epicholate and tauro-3␣,6(␣/␤),7␣,12␣-tetrol. A: Chromatograms of syn-
thetic tauro-7-epicholate (upper panel) and mouse urine (lower panel). Both ion chromatograms were ex-
tracted in the 50 ppm mass range of tauro-7-epicholate (514.2839 m/z) in the negative ion mode. B:
Chromatograms of synthetic tauro-3␣,6(␣/␤),7␣,12␣-tetrol (upper panel) and mouse urine (lower panel).
Both ion chromatograms were extracted in the 50 ppm mass range of tauro-3␣,6(␣/␤),7␣,12␣-tetrol
(530.2788 m/z) in negative ion mode. The values of retention time for several peaks are listed above their
respective peak. In mouse urine, several peaks confirmed with authentic compounds are labeled: tauro-7-
epicholate (a), tauro-␤-muricholate (b), taurocholate (c), and tauro-3␣,6(␣/␤),7␣,12␣-tetrol (d).
tauro-7-epicholate. Peaks b and c in Fig. 4A were con-
firmed with standard compounds of tauro-3␣,6␣,7␤-
trihydroxycholate(tauro-␤-muricholate)andtaurocholate,
respectively. Among the four epimers of tauro-
3␣,6(␣/␤),7(␣/␤),12␣-tetrol synthesized, both tauro-
3␣,6␣,7␣,12␣-tetrol and tauro-3␣,6␤,7␣,12␣-tetrol had
identical retention times (4.69 min) under the chromato-
graphic conditions employed. Peak d, the highest among
concentration of tauro-7-epicholate and tauro-3␣,6,7␣,12␣-
tetrol in wild-type mice on the CA diet, their concentrations
in Fxr-null mice on the CA diet was 82.8 68.3 ␮mol/mmol
creatinine for tauro-7-epicholate and 444
322 ␮mol/
mmol creatinine for tauro-3␣,6,7␣,12␣-tetrol (Fig. 5B, C).
Hepatic gene expression
Metabolomic differences in urine between wild-type and
Fxr-null mice after feeding the CA diet suggested that in-
creased urinary metabolic phenotypes resulted from an
adaptive response of Fxr-null mice to CA-induced toxicity
through alteration of gene expression for metabolic en-
zymes. Specifically, the highly increased cholic acid metab-
olite in Fxr-null fed CA, tauro-3␣,6,7␣,12␣-tetrol, was a
6-hydroxylated form of taurocholate, which suggested that
bile acid-oxidation enzymes such as CYP3A11 and CYP2B10
were induced in Fxr-null mice (18). The hepatic expression
of Cyp3a11, a representative PXR target gene, was elevated
significantly by approximately 6-fold in Fxr-null mice fed
both control and CA diets, compared with wild-type mice
fed a control diet (data not shown). The hepatic expres-
sion of Cyp2b10, a representative constitutive androstane
receptor (CAR) target gene, was elevated by approximately
10-fold and 4-fold in Fxr-null mice fed control and CA di-
ets, respectively, compared with wild-type mice fed a con-
trol diet (data not shown), but these differences were not
statistically significant. These expression levels for Cyp3a11
and Cyp2b10 were similar to a previous report (18).
the ions in the extracted chromatogram ([M-H]Ϫ
=
530.278) of mouse urine sample, had identical retention
time (4.69 min) with the above authentic compounds.
Therefore, the identity of taurotetrol that was greatly in-
creased in Fxr-null mice after CA feeding was tauro-
3␣,6(␣/␤),7␣,12␣-tetrahydroxycholate (Fig. 4B). The other
two epimers, tauro-3␣,6␣,7␤,12␣-tetrahydroxycholate and
tauro-3␣,6␤,7␤,12␣-tetrahydroxycholate did not show the
identical retention time to the other peaks extracted with
530.278 m/z ([M-H]Ϫ).
Quantification of cholic acid metabolites
The concentrations of taurocholate, tauro-7-epicholate,
and tauro-3␣,6,7␣,12␣-tetrol as well as creatinine were
measured in each urine sample using dehydroxycholate as
an internal standard and expressed as µmol/mmol creati-
nine. Urinary concentrations of taurocholate, tauro-7-
epicholate,andtauro-3␣,6,7␣,12␣-tetrolwereundetectable
in both wild-type and Fxr-null mice fed control diet
(Fig. 5). After feeding the CA diet, taurocholate concen-
trations were elevated to 46.4 59.5 ␮mol/mmol creati-
nine in wild-type mice and to 139 64.4 ␮mol/mmol
creatinine in Fxr-null mice, which were statistically signifi-
cantly different (P < 0.001). In contrast to undetectable
Cholestatic hepatotoxicity model
Recent reports revealed that Fxr-null mice detoxify ac-
cumulating bile acids in the liver by enhanced hydroxyla-
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Journal of Lipid Research Volume 51, 2010

hydroxy bile acid ([M-H]Ϫ = 530.278), which may be de-
rived from LCA, was measured individually in addition to
analysis of the relative Cyp3a11 level and serum ALT activ-
ity. A clear correlation (Pearson’s correlation coefficient, r =
0.63, P = 0.005) was observed between individual relative
abundance of the taurotetrol and Cyp3a11 expression lev-
els (Fig. 6A). The relative abundance of the taurotetrol
was significantly 4.3-fold higher in Fxr-null than in wild-
type mice (P = 0.009). In addition, relative Cyp3a11 expres-
sion levels in liver exhibited a significant increase by
3.8-fold in Fxr-null mice on the LCA diet compared with
those in wild-type mice on LCA diet (P < 0.0001). More-
over, an inverse correlation (r = Ϫ0.85, P < 0.0001) was
observed between ALT activity and relative CYP3A11 levels
in liver of both wild-type and Fxr-null mice (Fig. 6B). Se-
rum ALT activity was 4860 1680 IU/L in wild-type mice
and 1960 1090 IU/L in Fxr-null mice on LCA diet (40%
lower than wild-type mice, P = 0.0003). However, serum
ALP activity, a typical diagnostic cholestatic marker, was
not significantly different between wild-type and Fxr-null
mice on LCA diet (569 169 and 444 195 IU/L, respec-
tively). Taken together, these data suggested that Fxr-null
mice eliminate the systemic and hepatic bile acid load
more rapidly than wild-type mice by increasing the expres-
sion of CYP3A11, resulting in increased hydroxylation of
bile acids and less hepatotoxicity than wild-type mice un-
der LCA-induced cholestasis.
DISCUSSION
Fig. 5. Quantification of taurocholate, tauro-7-epicholate and
tauro-3␣,6(␣/␤),7␣,12␣-tetrol in urine of wild-type and Fxr-null
mice treated with 1% CA or control for 7 days. Concentrations of
creatinine, taurocholate, and tauro-7-epicholate and tauro-
3␣,6(␣/␤),7␣,12␣-tetrol were determined from calibration curve
of each metabolite and normalized to the creatinine concentration
(␮mol/mmol creatinine). A: Creatinine-normalized concentration
of taurocholate. B: Creatinine-normalized concentration of tauro-
7-epicholate. C: Creatinine-normalized concentration of tauro-
3␣,6(␣/␤),7␣,12␣-tetrol. Open and filled bars represent wild-type
and Fxr-null mice groups, respectively. Data were represented as
mean value SD (n = 6–12). The P values were calculated by
ANOVA with Bonferroni posttests. **P < 0.001 compared with wild-
type mice. CA, cholic acid; FXR, farnesoid X receptor.
Global analyses of urinary metabolomes in Fxr-null mice
compared with their wild-type littermates under condi-
tions of CA loading revealed separation of three groups in
a scores plot: control wild-type and Fxr-null mice; CA-fed
wild-type mice; and CA-fed Fxr-null mice. These results
suggest that CA feeding markedly changes urinary metab-
olites in Fxr-null mice and, to a lesser degree, in wild-type
mice. OPLS analysis between wild-type and Fxr-null mice
on the CA diet further showed that most of the increased
ions were metabolites of p-cresol, corticosterone, and CA.
Urinary p-cresol comes from dietary polyphenols or ty-
rosine residues generated by intestinal microorganisms
including Proteus vulgaris, Clostridium difficile, and unidenti-
fied species of Lactobacillus (20, 21). Urinary p-cresol was
reported to be one of the dysbiosis markers, including
benzoate, hippurate, phenylacetate, and hydroxybenzo-
ate, which are associated with microbial overgrowth (22).
More recently, metabolomics analysis revealed p-cresol sul-
fate as being unique to plasma samples of conventional
mice compared with samples of germ-free mice (23). In
this study, attenuated p-cresol conjugates after CA feeding
suggested that oral administration of bile acids inhibits the
intestinal microbial overgrowth (24). Specifically, CA feed-
ing markedly attenuated urinary p-cresol metabolites in
wild-type mice but to a lesser extent in Fxr-null mice, indi-
cating that CA feeding substantially increases fecal bile
acid excretion through induction of bile acid transporters
in wild-type mice but to a lesser extent in Fxr-null mice, as
tion reactions, some of which are catalyzed by CYP3A11, a
P450 typically involved in drug metabolism (18). To char-
acterize the effect of enhanced hydroxylation of bile acids
on hepatoprotection in Fxr-null mice, LCA-induced chole-
static model was used. Because BSEP, regulated by FXR, is
highly expressed in wild-type mice but not in Fxr-null mice,
the CA-induced cholestasis is found only in Fxr-null mice.
Therefore, administration of LCA, a hydrophobic second-
ary bile acid, causes intrahepatic cholestasis in wild-type
mice as well as in Fxr-null mice (19). Male wild-type and
Fxr-null mice were fed a diet containing 0.6% LCA for 4
days, and their urine was collected for 24 h both prior to
and after 3 days on the diet. Blood and liver samples were
taken for biochemical assay after 4 days on the diet. The
relative abundance of a urinary taurine-conjugated tetra-
Metabolomics in Fxr-null mice
1071

Fig. 6. Correlation plots of hepatic Cyp3a11 expression with formation of taurotetrol or hepatotoxicity in
the cholestatic mouse model induced by LCA. Fxr-null and wild-type mice were fed a 0.6% LCA diet or con-
trol diet for 4 days and urine and serum samples were collected. A: Correlation between relative CYP3A11
levels and relative abundance of taurotetrol. B: Correlation between relative CYP3A11 levels and ALT activity
in serum. Relative CYP3A11 levels were determined by real-time PCR analysis using gene-specific primers
and ␤-actin as the internal control. Relative abundance of taurotetrol was determined by the peak area re-
sponses of taurotetrol normalized by the creatinine concentration in urine (response/mmol creatinine).
ALT activities were determined as described in “Experimental Procedures.” Correlation analysis was con-
structed in wild-type (open circle) and Fxr-null (closed circle) mice fed with LCA. Correlation coefficient, r
values and P values were calculated by Pearson correlation test. ALT, alanine aminotransferase; FXR, farne-
soid X receptor; LCA, lithocholic acid.
revealed in the fecal bile acid excretion rate documented
in an earlier study (6).
activated mice, possibly mediated by induction of hydroxy-
lation by enzymes such as CYP3A11. However, further
investigation should be performed to establish the specific
metabolicpathwaysofcorticosterone,HDOPA,andDHOPA
in cholestatic mice.
A dramatic increase of urinary corticosterone metabo-
lites HDOPA and DHOPA, as well as serum corticoster-
one, was observed only in cholestatic mice (i.e., Fxr-null
mice fed CA for 7 days). In cholestatic animal models and
humans, the hypothalamic-pituitary-adrenal axis was acti-
vated by endotoxin and cytokines, such as tumor necrosis
factor-␣, resulting in an elevation of plasma glucocorticoid
concentration (25). Most of the corticosterone present in
the blood is excreted into urine and feces as more polar
metabolites of corticosterone through an intensive steroid
metabolism in liver and gut (26), although there has been
only limited information about metabolic pathways of glu-
cocorticoids (27). Therefore, highly increased urinary ex-
cretion of HDOPA and DHOPA in acute cholestasis may
result from secretion and extensive metabolism of corti-
costerone. Recently, HDOPA and DHOPA were reported
as highly specific biomarkers for activation of PPAR␣ (11).
However, high levels of HDOPA and DHOPA in CA-fed
Fxr-null mice may not be associated with activation of
PPAR␣ because the expression of PPAR␣-target genes
such as Cpt1a and Cyp4a10 were not induced by CA feed-
ing (data not shown). Furthermore, a previous study
reported that bile acids interfere with transactivation of
PPAR␣ at least in part by impairing the recruitment of
transcriptional coactivators (28). Hydroxylated metabo-
lites of HDOPA and DHOPA were also highly increased in
CA-fed Fxr-null mice to an extent similar to that observed
with HDOPA and DHOPA. However, an increase of these
hydroxylated metabolites were not found in urine from
mice treated with the PPAR␣ agonist Wy-14,643 (11). This
result indicates that CA-fed Fxr-null mice have different
metabolic pathways of corticosterone from PPAR␣-
The metabolomics data revealed various CA metabolites
as potential markers of cholestasis: taurocholate; tauro-7-
epicholate; tauro-3␣,6,7␣,12␣-tetrol; cholate glucoside;
and taurocholate glucoside. However, the structures of
cholate glucoside and taurocholate glucoside could not be
confirmed in this study, thus warranting further investiga-
tion. Tauro-7-epicholate and tauro-3␣,6,7␣,12␣-tetrol were
identified and quantified as a form of free bile acids in
previous reports (18, 29, 30), but their structures in this
study were confirmed by comparison with each synthetic
conjugated compound. Because bile acids are generally
too stable in negative ion mode to generate their tandem
mass spectra, identification of structures was performed by
comparison of retention time. Although this method is
not ideal because of similar structures and similar reten-
tion times with each CA metabolite, three compounds
with 514.283 m/z including taurocholate, tauro-7-epi-
cholate, and tauro-␤-muricholate could be distinguished
using retention time. In the case of four epimers of tauro-
3␣,6(␣/␤),7(␣/␤),12␣-tetrol synthesized in the present
study, different retention times were observed between
tauro-3␣,6␣,7␤,12␣-tetrol and tauro-3␣,6␤,7␤,12␣-tetrol,
whereastauro-3␣,6␣,7␣,12␣-tetrolandtauro-3␣,6␤,7␣,12␣-
tetrol had identical retention times under the chro-
matographic conditions performed here. Nevertheless,
tauro-3␣,6(␣/␤),7␣,12␣-tetrols were observed in urine,
plasma, bile or liver tissue of both Fxr-null mice and Bsep-
null mice in previous studies (18, 29). The other taurotet-
rols, except for tauro-3␣,6,7␣,12␣-tetrol, also existed in a
1072
Journal of Lipid Research Volume 51, 2010

chromatogram extracted for 530.278 m/z, which may be
hydroxylated forms of CA in positions other than the 6
position. Marschall et al. had revealed significantly en-
hanced hydroxylation at the 1␤, 2␤, 4␤, 6␣, 6␤, 22, or 23
positions of CA in Fxr-null mice with biliary obstruction
(18). They also exhibited elevated expression of CYP3A11
in Fxr-null mice as compared with wild-type mice with bil-
iary obstruction. Human CYP3A4, a homolog to mouse
CYP3A11, plays a role of hydroxylation of bile acids at the
6␣ and 6␤ positions (31, 32). Therefore, highly expressed
CYP3A11 in cholestatic condition was involved in en-
hanced urinary excretion of hydroxylated bile acids in-
cluding tauro-3␣,6,7␣,12␣-tetrol.
corticosterone and CA had been highly elevated in Fxr-
null mice by CA loading but not in wild-type mice. Tauro-
3␣,6,7␣,12␣-tetrol, one of the most increased metabolites
in Fxr-null mice on a CA diet, is a marker for efficient hy-
droxylation of toxic bile acids possibly by high induction
of CYP3A11. Furthermore, LCA-induced toxic models
proved that the enhanced expression of Cyp3a11 is the ma-
jor defense mechanism to detoxify cholestatic bile acids in
Fxr-null mice. These results will be useful for identification
of biomarkers for cholestasis and for determination of
adaptive molecular mechanisms in cholestasis.
REFERENCES
Several previous reports have clearly demonstrated that
induction of CYP3A expression enhances the formation of
hydroxylated LCA and protects against severe liver dam-
age induced by LCA (32, 33). In the present study, high
expression of CYP3A11 and enhanced urinary excretion
of tetrols in Fxr-null mice under CA loading suggested that
Fxr-null mice had an adaptive defense mechanism to de-
toxify accumulating bile acids in the liver by enhanced hy-
droxylation probably catalyzed by CYP3A11. However,
because wild-type mice under CA loading had increased
expression of BSEP and enhanced biliary excretion of bile
acids but did not show cholestasis, we could not prove the
adaptive hepatoprotective effects against bile acid-induced
liver damage in Fxr-null mice using the CA loading model.
Because LCA feeding induces segmental bile duct obstruc-
tion and destructive cholangitis in wild-type mice (34), we
observed that serum ALT activity in wild-type mice was sig-
nificantly higher than that in Fxr-null mice using LCA-
induced cholestatic mouse model, suggesting that Fxr-null
mice have a higher capacity to attenuate bile acid-induced
hepatotoxicity than do wild-type mice. In addition, the
correlation plot of relative abundance of taurotetrol or se-
rum ALT activities with CYP3A11 expression reveals that
Fxr-null mice are protected against LCA-induced liver
damage, possibly mediated by induction of Cyp3a11 and
production of hydroxylated bile acids.
In addition to hydroxylation by CYP3A11, several adap-
tive mechanisms were activated in cholestatic liver injury
and resulted from induction of phase II enzymes and
transporters such as SULT2A, OST␣/␤, MRP2, MRP3, and
MRP4, possibly through activation of PXR, CAR, and VDR
(7, 35, 36). Despite various defense responses, the urinary
metabolomic data provide an explanation that the en-
hanced hydroxylation of bile acids by CYP3A11 is one of
the major adaptive pathways to detoxify toxic bile acids in
cholestatic Fxr-null mice. Although the underlying molec-
ular mechanisms of the intrinsic adaptive response to bile
acids in Fxr-null mice is not fully understood, disruption of
FXR, a key regulator for bile acids, increases intrahepatic
bile acid concentrations which activate more extensively
other nuclear receptors, such as PXR, CAR, and VDR, to
enhance the hepatic defense against toxic bile acids (7).
In summary, a metabolomic investigation was conducted
on Fxr-null mice with CA loading compared with their
wild-type counterparts. OPLS analysis of mass spectromet-
ric data matrices revealed that the several metabolites of
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