2013 serum bile

The Author 2013. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For permissions, please email: [email protected].

Evaluation of Serum Bile Acid Profiles as Biomarkers of Liver Injury in Rodents

LinaLuo,* ShelliSchomaker,* ChristopherHoule, JiriAubrecht,* and Jennifer L.Colangelo*,1

*Biomarkers of Drug Safety Research and Development and Toxicologic Pathology of Drug Safety Research and Development, Pfizer Inc., Groton, Connecticut 06340

1To whom correspondence should be addressed at Biomarkers of Drug Safety Research and Development, Pfizer Inc., 8274-1429 Eastern Point Road, Groton, CT 06340. Fax: (860) 715-8045. E-mail: [email protected].

Received June 19, 2013; accepted September 9, 2013

Bile acids (BAs) have been studied as potential biomarkers of drug-induced liver injury. However, the relationship between lev-els of individual BAs and specific forms of liver injury remains to be fully understood. Thus, we set out to evaluate cholic acid (CA), glycocholic acid (GCA), and taurocholic acid (TCA) as potential biomarkers of liver injury in rodent toxicity studies. We have developed a sensitive liquid chromatography-tandem mass spec-trometry (LC/MS/MS) assay applicable to rat and mouse serum and evaluated levels of the individual BAs in comparison with the classical biomarkers of hepatotoxicity (alanine aminotransferase [ALT], aspartate aminotransferase [AST], glutamate dehydro-genase (GLDH), alkaline phosphatase, total bilirubin, gamma-glutamyl transferase, and total BAs) and histopathology findings in animals treated with model toxicants. The pattern of changes in the individual BAs varied with different forms of liver injury. Animals with histopathologic signs of hepatocellular necrosis showed increases in all 3 BAs tested, as well as increases in ALT, AST, GLDH, and total BAs. Animals with histopathologic signs of bile duct hyperplasia (BDH) displayed increases in only conju-gated BAs (GCA and TCA), a pattern not observed with the other toxicants. Because BDH is detectable only via histopathology, our results indicate the potential diagnostic value of examining indi-vidual BAs levels in serum as biomarkers capable of differentiat-ing specific forms of liver injury in rodent toxicity studies.

Key Words: bile acids; drug-induced liver necrosis; bile duct hyperplasia; LC/MS/MS; biomarkers.

Adverse drug reactions, especially drug-induced liver injury (DILI), represent a major challenge for drug develop-ment. Hepatotoxicity has been considered the most frequent cause of safety-related drug withdrawals for the past 50years (FDA, 2009; Kola and Landis, 2004; Lazarou et al., 1998; Pirmohamed etal., 2004). Serum enzymatic activity of alanine aminotransferase (ALT) is considered the gold standard clini-cal chemistry biomarker of liver injury in preclinical species and humans (Amacher, 2002; Amacher etal., 1998; Ozer etal.,

2010). However, ALT assessments in preclinical studies may present a challenge, especially when increases in ALT activity do not correlate with histopathology findings (Ennulat etal., 2010). In many cases, these increases can be attributed to induc-tion or extrahepatic injury, such as muscle damage or metabolic state, and might be addressed by additional biochemical param-eters. On the other hand, histopathologic analysis may be the only indicator for certain types of liver injury, such as bile duct hyperplasia (BDH). BDH can occur secondary to other abnor-malities, such as portal inflammation, cholestasis, and biliary and/or hepatocellular injury, and can also occur as a primary lesion. Thus, the development of additional biomarkers capable of facilitating the interpretation of serum ALT increases and differentiating between various histopathologic findings in pre-clinical toxicity studies is important.

Efforts to identify and develop additional biomarkers for DILI and/or to enhance the current biomarker panels have been initiated (Amacher et al., 2005; Ozer et al., 2008). Standard clinical chemistry panels currently include monitoring some combination of ALT, aspartate aminotransferase (AST), alka-line phosphatase (ALP), and gamma-glutamyl transferase (GGT) serum activity, as well as serum concentration of total bilirubin (TBIL). However, these often are of limited use for detecting BDH. Several research groups have employed genomics, proteomics, and metabonomics platforms in hopes of identifying potential biomarkers in this area. Recent atten-tion has been given to bile acids (BAs), which have been identi-fied in several metabonomic studies. These studies indicated that an altered BA profile was a key characteristic of the toxic response in the liver (Beckwith-Hall et al., 1998; Davis and Thompson, 1993; Lin etal., 2009; Yamazaki etal., 2013).

BAs are a class of structurally similar compounds that play essential roles in cholesterol homeostasis, lipid absorption, and intestinal signaling (Xiang etal., 2010). Synthesized in the liver from cholesterol, BAs are excreted into the small intestine via the bile duct mainly as glycine or taurine conjugates and then

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Evaluation of SErum BilE acid ProfilES

undergo enterohepatic circulation with further metabolism by bacterial and hepatic enzymes (Martin etal., 2007). Liver and gastrointestinal diseases often disturb the hepatic synthesis and clearance of individual BAs giving rise to quantitative changes in the pattern of serum BAs (Burkard etal., 2005; Meng etal., 1997; Thompson et al., 1987). In contrast, the measurement of total BAs may provide a general overall assessment of liver function, particularly in advanced stages of liver disease, but provide little insight into specific liver pathology especially early on in the disease process (Berg etal., 1986; Reyes and Sjvall, 2000). Because the total BA test measures the sum of all serum BAs, which is over 20 BAs in most species, and can be influenced by the presence of other endogenous molecules, the analysis of individual BAs has been proposed to provide valuable information regarding the pathogenesis of toxic liver injury and disease (Alnouti etal., 2008; Bentayeb etal., 2008; Ducroq etal., 2010; Turley and Dietschy, 1978).

Numerous analytical methods have emerged to determine BA concentrations in plasma and serum (Gatti etal., 1997; Lee etal., 1997; Street etal., 1985; Thompson etal., 1987). The complexity of metabolism, the typically low concentration of BAs in biological fluids, and the existence of multiple isobaric structural isomers make BA separation and quantitation chal-lenging (Janzen et al., 2010; Ostrow, 1993). In recent years, liquid chromatography coupled with mass spectrometry (LC/MS) has become an ideal option for the analysis of individ-ual BAs due to the high sensitivity and selectivity of the plat-form (Alnouti etal., 2008; Ando etal., 2006; Bentayeb etal., 2008; Bobeldijk etal., 2008; Griffiths and Sjvall, 2010; Hagio etal., 2009; Scherer etal., 2009). Other benefits of utilizing the LC/MS platform for these analyses include simple sample preparation, low sample volume requirements, and relatively low cost for reagents. Of the 2 primary BAs, cholic acid (CA) and chenodeoxycholic acid (CDCA), CA is more abundant in rats. Because multiplexing individual BAs into a single LC/MS assay often impairs accuracy and precision (Suzuki etal., 2013), we selected the most relevant primary BA to rat, CA, and its direct conjugates, glycocholic acid (GCA) and tauro-cholic acid (TCA), for our studies.

Goals of this study were to develop a sensitive LC/MS method for detection of CA, GCA, and TCA in rodent serum and to evaluate the potential of BA serum profiles as a bio-marker of DILI with the capability of differentiating biliary and hepatocellular damage in rodents.

MATeriALS AnD MeTHoDS

Chemicals and reagents. CA, GCA, and TCA were purchased from Sigma-Aldrich (St Louis, Missouri). d

4-CA and d

4-GCA were purchased

from C/D/N ISOTOPES Inc. (Quebec, Canada). d4-TCA was purchased from

Toronto Research Chemicals Inc. (Ontario, Canada). HPLC grade metha-nol, acetonitrile, water, and formic acid were purchased from Honeywell Burdick & Jackson (Muskegon, Michigan). Charcoal-stripped serum was from Bioreclamation (Westbury, NewYork).

Galactosamine (GalN), microcystin-LR (MC), -naphthylisothiocyanate (ANIT), acetaminophen (APAP), and isoproterenol (ISO) were purchased from

Sigma. Drug candidates A, B, C, and D were obtained from Pfizer (Groton, Connecticut).

Animals. All studies were approved and conducted under the oversight of the Institutional Animal Care and Use Committee. Studies were conducted on male Sprague Dawley rats (approximately 69 weeks old) or male CD-1 mice (approximately 68 weeks old), with the exception of the ISO study that used Hanover Wistar rats (approximately 69 weeks old). Hanover Wistar was selected for the ISO study because we had conducted full characterization of the cardiac toxicity in this strain, and no differences in serum concentrations of individual BAs have been observed between the 2 strains of rats (data not pub-lished). Drinking water and a standard commercial laboratory certified rodent diet were provided ad libitum throughout the studies. All rats were in the fasted state prior to necropsy.

General study design. Test articles (N=9) were selected to induce liver injury or injury to other organs. These compounds included classic hepato-toxicants, a cardiac toxicant, a testicular toxicant, a compound known to elicit BDH, and a nontoxic comparator compound. Within each study, a vehicle con-trol group was utilized with the same number of animals as treatment groups. Treatments of test article or vehicle were administrated to rats or mice for a period of time by oral (PO) gavage or subcutaneous (SC) or intraperitoneal (IP) injection (Table1). Blood samples were collected prior to necropsy at the end of treatment.

Sample collection. Blood samples were collected via the vena cava at necropsy, unless otherwise noted. Serum was collected in tubes containing no anticoagulant and was obtained from all rats dosed with vehicle or compound. Terminal serum samples were collected at the time of necropsy. All serum sam-ples were stored at 80C until analysis.

Clinical chemistry and histopathology. ALT, AST, ALP, GGT, GLDH, TBIL, and total bile acids (TBAs) were analyzed by standard clinical chemis-try techniques on the Siemens Advia 2400 platform. Cerner HNA Millennium Laboratory Information System was used to acquiredata.

Histopathologic examination of the liver was performed in all cases with additional evaluation of other major organs when appropriate. Organ samples were fixed in 10% buffered formalin, embedded in paraffin, sectioned, and stained with hematoxylin and eosin. Liver pathology was generally graded using a 4-point scheme as follows: (1) minimal, (2) mild, (3) moderate, and (4) marked.

Sample preparation for LC/MS/MS analysis. Fifty microliters of each serum sample was placed into an individual well of a Sirocco protein precipita-tion plate on the top of a 96-well collection plate. Three hundred microliters of methanol was added to each well for protein precipitation. Deuterated stand-ards of CA, GCA, and TCA were used as internal standards (ISs). Ten microlit-ers of the 1.0g/ml working solution of IS mixture was added, followed by vortex mixing. After 10 min of centrifugation, the Sirocco plate was removed, the supernatant was evaporated to dryness under a steady stream of N

2 at 37C.

The samples were reconstituted in 50l methanol/water (1/1, vol/vol) and then 3l was injected onto the LC/MS system.

Calibration and quality control standard preparation. One milligram per milliliter of BA stock solutions or IS stock solutions were prepared by dissolv-ing each BA reference in methanol. The individual BA stock solutions were combined and diluted to achieve the concentration of 20g/ml for a working standard solution or 1g/ml for a working IS solution. Nine calibration stand-ard solutions ranging from 5 to 5000 ng/ml were prepared by serially diluting the working standard solution into charcoal-stripped serum. Quality control (QC) standards were prepared in the same manner at 20, 500, and 4000 ng/ml in stripped serum. The calibration standards and QC standards then went through the sample preparation process described above.

Individual bile acid analysis. Individual bile acid (IBA) analysis was performed by LC/MS/MS. The mass spectrometer was an ABSciex 5500 QTrap equipped with Turbo Spray ion source, operating in negative mode. An Acquity UPLC system was interfaced to the front end of the MS system. All chromatographic separations were performed by gradient elution with an

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Acquity Shield RP18 column, 50 2.1 mm, 1.7m, maintained at 55C at a flow rate of 400l/min. The gradient program started at 65% mobile phase A(0.05% formic acid in water) and 35% mobile phase B (5% acetonitrile in methanol), increased to 90% of B in 7 min, decreased to 35% B in 0.5 min, and then held at 35% B for 2.5 min. The mass spectrometer was operated with the source and desolvation temperatures set at 120C and 350C, respectively. The curtain gas was 40 psi; the ion spray voltage was 4500 V; probe temperature was 600C; and ion source gas 1 and ion source gas 2 were 40 and 30 psi, respectively. Deuterated standards d

4-TCA, d

4-GCA, and d

4-CA were used as

ISs for TCA, GCA, and CA, respectively. Analysis in the mass spectrometer was performed in multiple reaction monitoring (MRM) mode. The MRM tran-sitions (m/z) were 407.3>407.3 for CA, 464.3>74.3 for GCA, 514.3>80.3 for TCA, 411.3>411.3 for d

4-CA, 468.2>73.8 for d

4-GCA, and 518.1>79.7 for d

4-

TCA. Peak integration and quantification were performed using Analyst 1.5.1 software. Individual standard curves for each BA were constructed by plotting the ratio of the BA peak area to its deuterated standard peak area versus con-centration. Slope and y-intercept were calculated using a linear curve fit with 1/x2 weighting. The concentrations of BAs in study samples were calculated relative to the regressionline.

Freshly prepared standard curve and QC samples were included in each analysis run. Arun was deemed acceptable if the QC samples were 15% of the nominal concentrations and the coefficient of variance (CV) did not exceed 10%.

Method validation. The method was validated using QC samples at 3 concentration levels (20, 200, and 2000 ng/ml) from the calibration curve. Four replicates of each QC sample were analyzed in a single run to determine the intraassay accuracy and precision. This process was repeated 4 times over 4days in order to determine the interassay accuracy and precision. Accuracy and precision were calculated from the % relative error (RE) [%(meas-ured theoretical)/theoretical concentration] and relative standard deviation [%RSD=% standard deviation/mean], respectively. The assay recovery was evaluated by comparing the mean detector response of extracted QC samples at low, medium, and high concentrations (20, 200, and 1000 ng/ml) in 4 rep-licates to those of postextracted serum blanks spiked at equal concentrations. The matrix effect was estimated by comparing the extracted serum residue and the neat solution.

Statistical analysis. Data are presented as individual animals or group mean SD. Statistical analyses were conducted by 2-tailed Students t test to compare drug treatment groups with vehicle control groups. Values signifi-cantly different from control are indicated as **p < .01 and *p < .05.

reSuLTS

Development of an LC/MS Assay for Detection of CA, GCA, and TCA

We have developed a sensitive method for the quantifica-tion of BAs using LC/MS. Because BAs are endogenous molecules and already present in rodent serum, we used char-coal-stripped rat serum for the standard curve and QC sample preparations. To increase the method accuracy and precision, deuterium-labeled ISs for each corresponding BA were used for the quantification. The accuracy and precision of TCA have been reported to be acceptable only when d

4-TCA was used

as the IS (Xiang et al., 2010). All BAs gave excellent linear response over a 103 dynamic range of 55000 ng/ml, with coef-ficients of determination (R2) above 0.999. The lower detection limit for these BAs was 0.5 ng/ml. Mean intraassay accuracy was 95%109% for the 3 IBAs, with a mean CV of 3%8%; mean interassay accuracy was 97%106%, with a mean CV

of 4%10% (Table2). The mean recovery rates of the extrac-tion procedures were between 103% and 105%. No significant matrix effect was observed, and the signal difference was less than 5%. These values were within the acceptable range, and the method was judged to be suitably accurate and precise for the analytes.

Currently, no published data can be found on the reference ranges of individual serum BA levels. The 3 IBA serum con-centrations from various vehicle-treated rodents in toxicology studies were generated here to provide baseline endogenous lev-els. Endogenous levels of TCA, GCA, and CA in various vehi-cle-treated rats (n=46) measured by this LC/MS assay were 0.184 0.153g/ml, 0.559 0.333g/ml, and 5.455 2.196g/ml, respectively; the serum levels of TCA, GCA, and CA in various vehicle-treated mice (n = 18) were 0.470 0.464g/ml, 0.004 0.002g/ml, and 0.091 0.11g/ml, respectively.

Serum BA Profiles After Treatment With Model Liver Toxicants

As expected, the treatment of rats with model liver toxicants GalN, MC, ANIT, and APAP caused a wide degree of hepato-cellular/hepatobiliary effects detected via histopathology and serum biochemical analyses (Table3). As expected, no histo-pathologic or serum biomarker changes were observed in vehi-cle-treated animals.

Rats dosed with a single dose of 1000 mg/kg GalN for 24 h revealed moderate to marked panlobular hepatocellular necro-sis that was randomly distributed throughout the liver (Fig.1A). Statistically significant, treatment-related increases in serum ALT, AST, and GLDH activity levels, together with increased concentrations of total BAs, were also noted. The LC/MS anal-ysis of CA, GCA, and TCA levels from the GaIN-treated rats showed statistically significant elevations of serum concentra-tions for all measured BAs. CA, as one of the primary BAs in rats, remained the most abundant BA in GalN-treated ani-mals (Fig.2). Interestingly, the BA proportions changed after treatment with GalN with CA accounting for 51% of the total 3 BAs measured as compared with 93% in the control group. Conversely, TCA accounted for 41% of the total 3 BAs in the profile after treatment compared with 2% in controls.

Rats dosed with a single dose of 1000 mg/kg APAP for 24 h produced characteristic hepatocellular necrosis that was accom-panied by significant increases in ALT, AST, ALP, and GLDH activity and elevation of total BA concentrations (Fig.1B). No other clinical chemistry parameters, such as GGT and TBIL, exhibited significant changes. LC/MS analysis revealed sta-tistically significant changes in serum concentrations of the 3 IBAs when compared with their corresponding control groups (Table3). APAP treatment resulted in an IBA profile similar to GalN treatment with CA remaining the major BA (77% of the total 3 BAs) as shown in Figure2.

Treatment of rats with 30 or 100 mg/kg of ANIT for 24 h caused mild to moderate hepatobiliary portal inflamma-tion and biliary degeneration and necrosis in most animals

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(Fig.1C). These changes were slightly more extensive in rats given 100 mg/kg of ANIT, as compared with rats given 30 mg/kg. Focal or multifocal, minimal, randomly scattered areas of hepatocellular necrosis were observed in some rats given each dose. The biomarker analysis showed statistical increases in levels of all tested biomarkers (ALT, AST, ALP, GLDH, GGT, TBIL, and total BAs) at both dose levels. The LC/MS analy-sis of CA, GCA, and TCA concentrations in ANIT-treated rats revealed statistically significant elevations. Furthermore, the proportions of the 3 IBAs were altered markedly. Conjugated BAs, especially TCA, were substantially elevated in both the 30 and 100 mg/kg groups (682 and 856 fold increases over the control group) and became the predominate BA in the treated groups. The unconjugated BA (CA) exhibited a comparatively small, yet statistically significant increase (Fig. 2). The ratio of the taurine conjugate over the total 3 BAs was higher in the 100 mg/kg group as compared with the 30 mg/kg group (data not shown). Paradoxically, ALT, AST, and GLDH had larger fold changes in the 30 mg/kg dose group (4.1, 5.7, and 64 control, respectively) than in the 100 mg/kg group (1.9, 3.4, and 35 control, respectively). The fold increases of ALP, GGT, and TBIL, which are generally considered indicators of hepatobiliary toxicity, were slightly increased in the 100 mg/kg group as compared with the 30 mg/kg group. This was consist-ent with TCA levels, the predominant BA here, which showed dose-dependent increases from 30 to 100 mg/kg (Table3).

Treatment of rats with a single dose of 0.2 mg/kg MC for 24 h caused moderate to marked centrilobular necrosis with fre-quent involvement of adjacent portal tracts, which include bil-iary tracts. These findings were accompanied by significantly increased activity levels for ALT, AST, GLDH, ALP, TBIL, and total BAs (Fig. 1D). The LC/MS analysis of CA, GCA, and TCA levels from the rats treated with MC showed statistically significant elevations of serum concentrations of all measured BAs. Again, the BA proportions changed dramatically. TCA had the largest fold increases of 903 over the control (Table3) and became the major BA accounting for 58% of the total 3 BAs compared with 1.4% in control (Fig.2).

Serum BA Profiles and BDH

To evaluate the BA profile in response to BDH, we treated rats with a well-characterized drug candidate (compound A) that was discontinued from development due to prevalent BDH detected in rat safety studies. To assess specificity of the bio-marker response, we used a structurally similar discontinued drug candidate of the same class (compound B) that did not cause BDH in rat safety studies. As expected, histopathologic examination of rats dosed with compound Arevealed mild to moderate BDH in the 3 highest dose groups, 300, 500, and 1000 mg/kg (Fig. 1E). In agreement with previous studies, the classical biomarkers of liver injury measured in our stud-ies (ALT, AST, ALP, GLDH, GGT, TBIL, and TBAs) failed to detect BDH. Although total BA levels detected by an enzyme cyclingbased assay was unchanged, the LC/MS analysis of

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90.

91.

41.

01.

01.

41.

727

*18

**

1000

mg/

kg

(5d

ays)

No

(2)

1.4

0.8

0.9

1.3

1.0

1.0

1.3

1.5

69**

10**

BD

H n

egat

ive

cont

rol

B15

0 m

g/kg

(5

day

s)N

oN

o1.

01.

20.

90.

81.

01.

00.

831.

10.

60.

6

300

mg/

kg

(5d

ays)

No

No

0.9

0.8

1.0

0.8

1.0

1.0

0.84

0.7

1.6

1.6

17



ownloaded from

Luo etaL.

Neg

ativ

e co

ntro

ls

(oth

er o

rgan

to

xica

nts)

ISO

c10

0g

/kg

(6 h

)N

oN

o1.

21.

00.

82.

31.

01.

01.

21.

21.

30.

7

500g

/kg

(6 h

)N

oN

o1.

21.

9*0.

81.

11.

01.

02.

8*2.

01.

70.

5

4000

g/

kg

(6 h

)N

oN

o1.

23.

2**

0.9

1.2

1.0

1.0

1.0

1.3

0.7

0.1

100g

/kg

(24

h)N

oN

o1.

01.

01.

01.

11.

01.

02.

11.

71.

21.

3

500g

/kg

(24

h)N

oN

o1.

01.

30.

91.

21.

01.

01.

41.

21.

11.

8

4000

g/

kg

(24

h)N

oN

o1.

01.

51.

01.

41.

01.

01.

61.

30.

80.

6

Cd

5 m

g/kg

(7

day

s)N

oN

o1.

3*1.

10.

9N

P1.

01.

2N

P0.

60.

51.

1

50 m

g/kg

(7

day

s)N

oN

o1.

8**

0.9

1.1

NP

1.0

1.2

NP

0.7

0.8

0.4

500

mg/

kg

(7d

ays)

No

No

3.2*

*3.

5**

1.1

NP

1.0

1.1

NP

1.1

0.8

0.6

De

75 m

g/kg

(2

1da

ys)

No

No

1.7*

*1.

11.

3*N

P1.

01.

0N

P1.

21.

21.

2

150

mg/

kg

(21

days

)N

oN

o2.

1**

1.2

1.3*

NP

1.0

1.0

NP

0.8

0.8

0.2*

*

a HC

, hep

atoc

yte;

BD

, bile

duc

t or

hepa

tobi

llary

; the

num

ber

in th

e pa

rent

hese

s st

ands

for

the

seve

rity

of

liver

his

topa

th fi

ndin

gs: (

1) m

inim

al; (

2) m

ild; (

3) m

oder

ate;

(4)

mar

ked.

b Val

ues

repr

esen

t fol

d ch

ange

s co

mpa

red

with

thei

r co

rres

pond

ing

cont

rols

; NP,

not

per

form

ed.

c At

the

6-h

time

poin

t ch

ange

s in

the

hea

rt c

onsi

sted

pre

dom

inan

tly o

f m

inim

al t

o m

ild s

ubep

icar

dial

hem

orrh

age/

edem

a at

all

dose

lev

els;

by

24 h

, thi

s pr

ogre

ssed

to

also

inc

lude

min

imal

to

mild

m

yoca

rdia

l deg

ener

atio

n/ne

cros

is a

nd in

flam

mat

ion

at a

ll do

se le

vels

.d M

inim

al m

esen

teri

c lip

id d

eple

tion

in th

e 50

0 m

g/kg

gro

up; m

oder

ate

to m

arke

d zy

mog

en d

ecre

ase

in p

ancr

eas

and

mes

ente

ric

lipid

dep

letio

n in

the

500

mg/

kg g

roup

.e M

inim

al e

pidy

dim

al s

perm

atic

gra

nulo

mas

in th

e 75

mg/

kg g

roup

with

mild

epi

dydi

mal

inte

rstit

ial i

nflam

mat

ion

and

skel

etal

mus

cle

infla

mm

atio

n in

the

150

mg/

kg g

roup

.St

atis

tical

ly s

igni

fican

t cha

nges

are

indi

cate

d by

*p

< .0

5, *

*p

2013 serum bile

Documents

potential biomarkers

druginduced liver injury

total bas

individual bas

bile acids bas

extrahepatic injury

adverse drug reactions

related drug withdrawals