Diseases of the Gallbladder and Bile Ducts - part 9 potx

348 Section 3: Specific conditions
with PBC who are receiving corticosteroids [48]. However,
in a randomized, double-blind, placebo-controlled study in
67 patients with PBC, etidronate did not cause any improve-
ment of bone mass density (BMD). A more recent study, in-
cluding 32 female patients with PBC, showed that both
etidronate and alendronate increase BMD but the positive
effect of alendronate was far superior [49]. So, most likely
alendronate, rather than etidronate, should be recommen-
ded to patients with PBC with osteoporosis, but further stud-
ies are required to establish the role of this agent in the
t re a t men t o f os t e op or os i s i n P BC . I t c a n not b e i g n o r e d t ha t i n
patients with advanced PBC (who are also more likely to have
osteoporosis) and who have esophageal varices, biphospho-
nates may potentially cause esophagitis and increase the risk
of variceal bleeding.
Vitamin K plays a modulatory role on bone metabolism.
Increased BMD and prevention of bone fractures were ob-
served in patients with osteoporosis who were treated with
vitamin K. A randomized study in female patients with PBC
showed a signifi cant increase of BMD in subjects treated with
vitamin K [50]. These results are promising but require con-
firmation in studies including larger cohorts of patients.
After publication of the results of the HERS II trial (heart
and estrogen/progestin replacement study) HRT cannot be
recommended anymore for the treatment of osteoporosis as
it increases the risk of certain malignancies, hip fracture, and
thromboembolism and does not have any signifi cant cardio-
protective effect. Also UDCA and calcitonin seem to be of no
use in the prevention or treatment of osteoporosis in patients
with PBC.

A signifi cant proportion of patients with PBC is deficient of
fat soluble vitamins. This refers mostly to patients with ad-
vanced disease, in particular in those who also have low
serum albumin and cholesterol levels and an elevated biliru-
bin. When the Mayo risk score for PBC is higher or equal to 5,
patients may be found to be vitamin A deficient. It has been
recommended that these patients should be screened for fat-
soluble vitamin deficiencies and adequately supplemented if
necessary.
As with all patients who are found to have large eso-
phageal varices, prophylactic nonselective beta blockade is
recommended.
Specific therapies for PBC
There is no medical therapy that cures PBC. Ursodeoxycholic
acid (UDCA) remains the only US Food and Drug Adminis-
tration approved medication for PBC. Although the mecha-
nisms involved in its hepatoprotective properties have been
extensively studied over last 20 years, the mechanisms of its
action are not yet fully explained. UDCA and its conjugates
stimulate bile salt excretion and protect against bile-salt-
induced mitochondrial damage, oxidative stress, and apop-
tosis and also may have a membrane-stabilizing effect,
protecting against bile-salt-induced solubilization. There is
no doubt that UDCA plays the role of a signaling molecule with
precise regulatory properties in specific signaling pathways
[51]. UDCA causes signifi cant improvement in biochemical
parameters of cholestasis, including bilirubin, alkaline phos-
phatase, and GGT. It also decreases serum cholesterol levels.
UDCA slows down the histological progression of the disease
[52,53]. It may also decrease pruritus in a proportion of pa-

tients (but rarely it may increase pruritus) but has no signifi -
cant effect on fatigue. A beneficial effect on survival was
observed when patients were treated for up to 4 years with
the trial dosage of UDCA [54], although clearly some patients
respond better than others. It has been shown that those pa-
tients with noncirrhotic PBC treated with UDCA appear to
have a 10-year surv ival that is no different from an age- and
gender-matched population [55]. Overall the greatest effect
on short-term survival was seen in those patients with more
severe disease, for whom UDCA treatment may delay the
need for transplantation. Treatment before transplant does
not have a detrimental effect on the patients’ post-transplant
outcome, despite the patients being older when they eventu-
ally come to need a transplant. At the recommended dose of
13 to 15 mg/kg per day UDCA is extremely well tolerated with
only a minority of patients complaining of diarrhea – but
lower doses, that is 10 mg/kg, appear to be less effective.
The beneficial effect of UDCA has been questioned [56]
and no signifi cant impact of this drug on survival or time to
liver transplantation was found, although a signifi cant re-
duction in jaundice and ascites was noted. This report has
been criticized as early studies, where patients were treated
for short periods of time and with subtherapeutic doses of
UDCA, were included in the meta-analysis.
Although the trigger for the development of PBC remains
to be elucidated, autoimmune phenomena may play a role
in the hepatic damage. Thus different immunosuppressive
drugs have been investigated in PBC. In a study published by
Christensen et al, azathioprine was found to prolong survival
by approximately 20 months [57]. A positive effect on sur-

vival was also observed by another European group, in a pla-
cebo controlled trial with cyclosporine A [58]. Unfortunately,
this was associated with signifi cant side-effects of this calci-
neurin inhibitor, including renal impairment and hyperten-
sion. Anecdotal reports suggest that combined therapy of
UDCA and mycophenolate mofetil (MMF) may be of partic-
ular use in patients with a signifi cant infl ammatory compo-
nent on their biopsies. The potential application of MMF in
PBC is now the subject of an ongoing randomized study. With
regard to other treatments, no convincing effect was observed
with colchicine and methotrexate. Budesonide was found to
increase the risk of portal vein thrombosis in cirrhotic pa-
tients with PBC [59] and therefore its use in this subgroup of
PBC patients should be discouraged. Drugs more recently in-
vestigated in PBC include bezafibrate, pranlukast and sulin-
dac [60]. Their role in the management of PBC remains to be
established.
Chapter 21: Primary biliary cirrhosis 349
As PBC progresses very slowly, it is extremely difficult to
prove effi cacy of any therapeutic agent. It has been suggested
that in order to demonstrate a clear effect of any medication
on death rate in patients without liver transplantation, over
200 patients have to be treated for a period of 5 years [58]. For
those who reach end-stage liver disease, liver transplanta-
tion is the only option but, certainly, liver transplantation
can not be reliably used as a primary end-point.
The proportion of patients with PBC who require liver
transplantation, either for end-stage liver failure or for poor
quality of life, is diminishing [61]. PBC used to be the most
common indication for liver transplantation in patients with

end-stage liver disease, comprising up to 55% of all trans-
planted, cirrhotic patients in some centers [45]. This propor-
tion has now decreased to 10%; this is in part due to both
increasing number of patients being transplanted for viral-
and alcohol-related cirrhosis and possibly a protective effect
of UDCA [45]. Indications for liver transplantation in PBC
are summarized in Table 21.4. The Mayo Clinic model and
the more recently introduced MELD score are useful in pre-
dicting survival in patients with end-stage liver disease. It is
recommended that in patients with PBC, once their bilirubin
level has reached 100 µmol/L (5.9 mg/100 mL), they should
be referred for liver transplant assessment [45]. Current
5-year survival after liver transplantation for PBC varies
between 83 and 86%, making this disease an excellent indi-
cation for grafting. Early mortality after surgery is caused
mostly by multiorgan failure and sepsis [61]. Chronic rejec-
tion, which is the most common indication for regrafting
within 1 year of surgery, occurs signifi cantly less commonly
in patients with PBC than in patients transplanted for auto-
immune hepatitis and more commonly than in those trans-
planted for alcohol-related liver disease [62]. PBC does recur
after transplantation and the diagnosis of the recurrence can
only be reliably established byhistological examination [45].
This phenomenon is rarely of clinical signifi cance and
may potentially be associated with tacrolimus rather than
cyclosporine-based immunosupression [63].
A vital issue, which has to be addressed in the near future,
is the identifi cation of patients who are more likely to prog-
ress into end-stage liver disease. Clinical practice clearly
shows that some subjects develop cirrhosis despite being

treated with UDCA whereas others show no clinical or histo-
logical progression over many years, sometimes even with no
treatment. The role of newly identified serum markers of dis-
ease progression has to be validated. Certainly, genetics may
play a crucial role in the natural history of this disease, affect-
ing the rate of progression and response to treatment. This di-
lemma has prompted several leading groups from all over the
world to establish a collaborative project which will allow
complex genetic analyses in a large, shared pool of samples.
Undoubtedly, this effort will enable an unprecedented accel-
eration in our knowledge on the genetic background of PBC
and may have a signifi cant impact on combating this chronic
and potentially lethal disease.
Questions
1. Which sentence is true about the epidemiology of PBC?
a. recent studies have shown that the prevalence of PBC is
decreasing
b. smoking does not increase the risk for developing PBC
c. the rate of AMA seropositivity in the general population is
significantly higher than the prevalence of PBC arising from
epidemiological studies
d. all these statements are false
e. statements (b) and (c) are true
2. In the pathogenesis of PBC
a. there is a clear relationship to herpes zoster infection
b. apoptosis may play a role
c. N. aromaticivorum may play a role in triggering the disease
d. there is a very weak association with possible genetic factors
e. statements (b) and (c) are true
3. With regard to the natural history of PBC which of the following

statements is true?
a. the majority of patients diagnosed today are jaundiced
b. pruritus never precedes the onset of jaundice
c. the asymptomatic phase usually lasts a couple of weeks
d. AMA-positive patients who have normal biochemistry may
have features of PBC on their biopsies
e. the onset of the disease is usually acute
Table 21.4 Indications for transplantation in patients with primary
biliary cirrhosis.
Symptoms
Intractable and intolerable pruritus
Overwhelming lethargy
End-stage disease
Clinical signs
Increasing muscle wasting
Increasing symptomatic osteopenia
Encephalopathy
Intractable ascites
Recurrent, intractable variceal hemorrhage
Spontaneous bacterial peritonitis
Moderate hepatopulmonary syndrome
Early hepatocellular carcinoma
Biochemical
Serum bilirubin > 170 µmol/L > 6 months
Serum albumin < 25 g/L
Patients should be referred to a liver transplant center when their
bilirubin reaches 100 µmol/L. (Reproduced from MacQuillan GC and
Neuberger J. Clin Liver Dis 2003;7:941–56, with permission from
Elsevier.)
350 Section 3: Specific conditions

4. In the natural history of PBC
a. factors predisposing to progression from asymptomatic to
symptomatic disease include bilirubin and alkaline
phosphatase
b. as many as 90% of initially asymptomatic patients may develop
symptoms of liver disease within 4 years of diagnosis
c. PBC in children usually has a very aggressive course
d. the diagnosis of PBC is most commonly established by the age
of 20
e. all statements are false
5. Fatigue in PBC – which statement is true?
a. the origin of fatigue in PBC is most likely central
b. fatigue is a rare symptom in PBC
c. there is a strong correlation between age and degree of
fatigue
d. there is a strong correlation between fatigue and hepatic
histology
e. fatigue in PBC is usually relieved by rest
6. Pruritus in PBC – which statement is true?
a. pruritus occurs in about 30% of patients
b. pruritus is usually worst in the morning
c. the palms or soles are never affected
d. the classical “butterfly” area delineates the area of most intense
scratching
e. scratching usually does not relieve this symptom
7. Diseases associated with PBC – which statement is true?
a. sicca syndrome occurs in approximately 10% of patients
b. a history of hypothyroidism is present in 90% of patients
c. psoriasis is the commonest associated disease
d. majority of patients are found to have antiendomysial

antibodies
e. all statements are false
8. Diagnosis of PBC – which statement is true?
a. diagnosis is established on the presence of positive
ANA
b. AMA may b e p o s it i v e in abo ut 40 to 5 0 % o f p at i e n t s
c. liver biopsy is essential for establishing the diagnosis
d. immunochemistry for AMA may be falsely positive in patients
with type 2 autoimmune hepatitis
e. the presence of AMA confirms only advanced PBC
9. Treatment of PBC – which statement is true?
a. the curative rate of ursodeoxycholic acid is approximately
60%
b. questran should be avoided in the treatment of pruritus
c. rifampicin is a first-line treatment of pruritus
d. HRT is highly recommended to prevent osteoporosis only in
postmenopausal women with PBC
e. budesonide must not be used in cirrhotic patients with
PBC
10. Liver transplantation in PBC – which statement is true?
a. amongst patients with cirrhosis, PBC is currently the most
common indication for liver transplantation
b. the 5-year survival after liver transplantation for PBC is
approximately 85%
c. treatment with ursodeoxycholic acid before transplant
significantly reduces survival following liver transplantation
d. calcineurin inhibitors (tacrolimus and cyclosporin A) have to be
avoided after liver transplantation as they increase the risk of
acute rejection
e. recurrence of the disease is the most challenging issue after liver

transplantation
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SECTION 3.3
Intrahepatic cholestasis
Diseases of the Gallbladder and Bile Ducts: Diagnosis and Treatment, Second Edition
Edited By Pierre-Alain Clavien, John Baillie
Copyright © 2006 by Blackwell Publishing Ltd
CHAPTER 22
Intrahepatic cholestasis
Andrew Stolz and Neil Kaplowitz
22
OBJECTIVES
• Recognize the major hepatic transporters responsible for the sinusoidal uptake of bile salts or organic anions or the efflux

of bile salts, organic anions, or lipids into bile
• Understand how transcription factors co-ordinate the response of hepatocyte transporters and detoxification enzymes to
cholestatic liver injury
• Learn about the major genetic causes of cholestatic diseases in children and adults
• Identify the clinical features and molecular mechanisms for drug-induced cholestatic liver injury
Multiple etiologies are responsible for cholestatic liver injury,
which require vastly different diagnostic and therapeutic ap-
proaches. In this chapter, we will review a diverse group of
nonsurgical diseases that can mimic the presentation of other
causes of cholestatic liver disease discussed elsewhere in this
book. In order to understand how these diverse disorders can
cause cholestatic liver injury, we will concisely review the
current understanding of the key proteins that constitute the
normal hepatic uptake and biliary excretory pathways. In
human disease and animal models, the molecular regulation
of these key transport proteins in response to cholestatic liver
injury has provided detailed insight into the pathophysiolog-
ical mechanisms in different cholestatic conditions. Clinical
presentation, evaluation, treatment, and medical conditions
associated with cholestasis will follow a review of the normal
biliary physiology. A review of drug-induced cholestasis will
also be provided, as inability to recognize this important eti-
ology can lead to persistent exposure to the offending agent
and unnecessary diagnostic or therapeutic interventions. We
anticipate that enhanced understanding of molecular mech-
anisms of cholestatic liver disease should lead to new thera-
peutic strategies for these widely different diseases in the
future.
Mechanisms for hepatic bile formation
The cellular organization of the liver combined with the

unique endothelial structure of the sinusoids are ideally suit-
ed for the efficient sinusoidal uptake and effl ux of molecules
by the hepatocytes as well as their biliary excretion into the
canaliculus, the most proximal portion of the biliary system
355
[1–4]. The hepatocytes, like other epithelial cells such as the
enterocyte, are polarized cells with distinct membrane do-
mains exposed to either the sinusoidal surface, referred to as
the basal or sinusoidal domain, or to the canaliculus, referred
to as the apical or canalicular domain. The membrane be-
tween these regions forms the lateral domain. Cords of hepa-
tocytes two cell thick are attached at their sinusoidal and
apical domain by gap junctions, generating a physical barrier
between the vascular sinusoidal space and the biliary excre-
tory pathway. Ions and water can travel between these gaps
and disruption of these anchors between hepatocytes can
lead to regurgitation of biliary components into the vascular
space during cholestatic conditions. Bile formation requires
both the maintenance of these cell-to-cell contacts as well as
vectorial uptake at the sinusoidal membrane, followed by se-
cretion of biliary components at the canalicular membrane.
Based on animal studies, the origins of bile flow are divid-
ed into bile acid dependent and independent bile flow. Bile
salts are the predominate solute in bile which, along with
sodium ion, are responsible for water movement into the
canalicular space, predominately by paracellular pathways
between adjacent hepatocytes. Bile salts are also essential for
biliary excretion of phospholipids, which are extracted from
the outer leaflet of the canalicular membrane forming mixed
micelles in the presence of bile salts. An absence of bile salts

leads to signifi cant reduction in bile formation and phospho-
lipid excretion. On the other hand, bile salt excretion into the
canalicular space in the absence of phospholipid excretion
leads to extensive damage to cholangiocytes, due to potent,
detergent and cytotoxic effects of free bile salts. A bile-salt-
independent bile flow also exists in which effl ux of organic
Diseases of the Gallbladder and Bile Ducts: Diagnosis and Treatment, Second Edition
Edited By Pierre-Alain Clavien, John Baillie
Copyright © 2006 by Blackwell Publishing Ltd
356 Section 3: Specific conditions
anions and the important peptide antioxidant, glutathione
(GSH), coupled with its cleavage by γ glutamyl transpepti-
dase and dipeptidases into its component amino acids, gener-
ate an osmotic gradient promoting water movement into the
canalicular space [5]. Bicarbonate secretion is another im-
portant component whose contribution is highly species de-
pendent [6].
The bile canalicular space created by adjacent hepatocytes
eventually empty at the periphery of the lobule into ductules
lined by cholangiocytes which further modify ion and bicar-
bonate content in bile, which is regulated by secretin. A cho-
lehepatic shunt between the hepatocytes and cholangiocytes
has been proposed to explain the bicarbonate-rich biliary ex-
cretion caused by infusion of unconjugated bile acids such as
ursodeoxycholate in animals. The role this plays in normal
bile flow is controversial. It is postulated that certain uncon-
jugated bile acids secreted by hepatocytes are reabsorbed by
cholangiocytes in protonated form (leaving bicarbonate be-
hind) and then returned to the hepatocytes via the ductular
capillaries, emptying into the sinusoids in a repetitive recy-

cling, leading to enhanced bicarbonate secretion associated
with a hypercholeresis [7]. In addition to fl uid and electro-
lyte movement, mechanical contraction of canalicular
membrane by a microfilament network located at the apical
domain of hepatocytes also promote bile flow by a “squeezing
action” on the canalicular space.
Within the last decade, identifi cation of cellular transport
proteins mediating sinusoidal uptake and biliary secretion of
the key solutes has greatly expanded our understanding of
the cellular mechanism for bile formation and its dysregula-
tion in cholestatic conditions. We will review the key trans-
port proteins listed in Table 22.1 and illustrated in Fig. 22.1 at
each domain and the nuclear receptors that regulate their ex-
pression. The altered regulation of these transporters in re-
sponse to cholestatic stimuli are now known to play a key role
in the development and maintenance of cholestasis [3,4].
Table 22.1 Molecular and functional characteristics of human hepatocyte membrane transporters.
Transporter Protein Gene Domain Transport Model substrates
features
Na
+
-taurcholate cotransporting NTCP SLC10A1 Sinusoidal Uni Bile acids
polypeptide
Microsomal epoxide hydrolyase mEH EPHX1 Sinusoidal/ Uni Cholate, taurocholate
intracellular
Organic anion transport peptide OATP1B1 SLCO1B1 Sinusoidal Bi Bile salts, HMG-Co reductase inhibitors,
(OATP-C, LST-1, OATP2) organic anion
Organic anion transport peptide OATP1B3 SLCO1B3 Basolateral Bi Bile salts, digoxin
(OATP-8)
Organic anion transport peptide OATP2B1 SLCO2B1 Basolateral Bi Estrone-3-sulfate, dehydroepiandrosterone-

(OATP-B) sulfate
Bile salt export pump BSEP ABCB11 Canaliculus Uni Taurocholate, bile acids
ABCG5/ABCG8 ABCG5/ABC G8 ABCG5/ Canaliculus Uni Cholesterol
ABCG8
Multidrug resistance 3 MDR3 ABCB4 Canaliculus Uni Phosphatidylcholine
Familial intrahepatic FIC1 ATP8B1 Canaliculus Uni Unknown
cholestasis 1
Multiresistance protein 2 (cMOAT) MRP2 ABCC2 Canaliculus Uni Leukotriene C4, GSH conjugates, conjugated
bilirubin
Multiresistance protein 3 MRP3 ABCC3 Basolateral Uni Bile acids, conjugated sex steroids
Multiresistance protein 4 MRP4 ABCC4 Basolateral Uni Nucleotide analogs, organic anions, bile
salts (with GSH)
Multiresistance protein 5 MRP5 ABCC5 Basolateral Uni Nucleotide analogs, organic anions
Multiresistance protein 6 MRP6 ABCC6 Basolateral Uni Organic anions
Location (domain) of human hepatic transporters and some of their model substrates [3,21,28]. Transport features refers to the ability of the
transporter to function as a unidirectional (Uni) or bidirectional (Bi) transporter.
Chapter 22: Intrahepatic cholestasis 357
Sinusoidal membrane
Uptake of bile acids by the sinusoidal membrane is a key step
in the enterohepatic circulation of bile salts in which approx-
imately 95% of the bile salt pool is efficiently recovered by the
intestine and resecreted by the liver [8]. Initial characteriza-
tion of bile acid and bile salt uptake in isolated hepatocytes
and enriched sinusoidal plasma membrane vesicles identi-
fied both sodium dependent and independent transport sys-
tems for bile salts. In addition, hydrophobic secondary bile
acids or unconjugated bile acids may also enter into the hepa-
tocytes by passive diffusion. Using an expression cloning
strategy, a specific sodium-dependent bile salt transporter
has been identifi ed as well as sodium-independent organic

anion transporters, some of which can also mediate sodium-
independent bile acid uptake.
Sodium-dependent bile acid transporter
The sodium-dependent taurocholate carrier protein, Ntcp, is
a 55-kD glycoprotein composed of 362 amino acids in the rat
which strictly mediates sodium-dependent bile salt transport
favoring taurine-conjugated, trihydroxy-bile acids [9]. This
protein is exclusively expressed in the hepatocyte at the sinu-
soidal domain throughout the hepatic acinus. Like other so-
dium cotransporters, the concentrative uptake of bile salts is
thermodynamically favored by coupling to a signifi cant out-
side-to-inside sodium gradient maintained by the activity of
Na
+
, K
+
ATPase, assuring efficient uptake of bile salts at the
sinusoidal surface. Two sodium ions are transported for each
molecule of bile salt. Activity of the ntcp transporter is regu-
lated by both its levels of expression at the sinusoidal surface
and by gene transcription, which is an active area of investi-
gation [3,4,10]. Increased cAMP rapidly increases the con-
tent of ntcp at the sinusoidal membrane by mobilizing an
intracellular pool of transporters, the translocation being
dependent on microtubule and microfilament activity [11].
The ntcp expression is rapidly lost in primary cultured hepa-
tocytes and all tumor-derived liver cell lines, suggesting tight
regulation of this transporter, but it has been identified in
human hepatocellular carcinoma samples. Elevated serum
levels of bile salts are associated with decreased expression

whereas increased expression of the transporter gene has
been noted postpartum due to prolactin [12,13]. Increased
Figure 22.1 Transport proteins of the basolateral (sinusoidal) and
canalicular surface of human hepatocytes. Proteins involved in the
hepatocellular uptake of organic anions include: Na
+
-taurocholate
cotransporting polypeptide (NTCP) and microsomal epoxide hydrolase
(mEH) (secondary active unidirectional transporters) and organic
anion transporting polypeptides OATP1B1, OATP1B3, and OATP2B1
functioning as bidirectional antiporters. Multidrug resistance proteins
(MRPs), bile salt export pump (BSEP), and multidrug resistant gene
product (MDR) are unidirectional primary active transporters involved
in the efflux of specific substrates listed with their respective
transporters. The substrate for FIC 1 is unknown. Na
+
,K
+
ATPase is
responsible for maintaining the low sodium, high potassium within the
cell. BS
−
= bile s a l t s, OA
−
= organic anions, PC = phosphatidylcholine,
BDG = bilirubin diglucuronide, CHOL = cholesterol.
358 Section 3: Specific conditions
bile salt fl ux does not regulate its expression in the rat [14].
Teleologically, decreased expression of ntcp protects the liver
in cholestasis in the face of elevated serum bile salts by reduc-

ing hepatic accumulation and potential toxicity of increased
concentration of intrahepatic bile salts. Human NTCP is a 349
amino acid protein, which shares 77% sequence identity
with the rat [15]. Of note, NTCP shares 50% sequence identi-
ty with the sodium-dependent ileal bile acid transporter, re-
ferred to as apical sodium dependent bile acid transporter
(ASBT) which is expressed at the lumenal (apical) domain of
the enterocyte [16,17]. ASBT has also been identified in the
apical domain of large cholangiocytes, where it participates
in cholehepatic shunting, and in renal tubules cells [18,19].
Microsomal epoxide hydrolase (mEH), a key enzyme in de-
toxifi cation of reactive epoxides expressed at both the plasma
membrane and endoplasmic reticulum in the rat, has also
been implicated as a sodium dependent bile acid transporter
as its expression in a cell line can confer sodium-dependent
bile salt uptake [20]. mEH expression seems to favor glycine-
conjugated bile salts.
Organic anion and sodium-independent bile salt
transport
In addition to sodium-dependent bile salt transport, dihy-
droxy-bile salts and unconjugated bile acids can enter hepa-
tocytes via a sodium-independent transport mechanism
mediated by members of the organic anion transporting pep-
tides (OATP) superfamily. Members of this large transporter
family are characterized by 12 membrane-spanning do-
mains containing a distinctive, superfamily peptide signa-
ture [21]. These transporters may share common substrates
or be highly selective and are located in diverse tissues, in-
cluding the blood–brain barrier, liver, lung, intestine, and
kidneys. Members of subfamilies are now defined by their se-

quence homology with proteins in humans, designated as
OATPs with the corresponding SLCO gene symbol. As a class,
OATPs function as organic anion exchangers promoting up-
take of organic anions by exchanging them with effl ux of
other anions such as bicarbonate and glutathione [22,23]. In
human liver, three OATP family members are expressed on
the sinusoidal domain [3,21]. OATP1B1 is only expressed in
hepatocytes and facilitates the transport of organic anions
bound to albumin, including bile salts and their conjugates
and bilirubin, into the liver. The restricted site of expression
of OATP1B1 in hepatocytes, coupled with its known poly-
morphisms, are likely to have a major impact on the metabo-
lism of pharmaceutical agents. OATP1B3 is also expressed
in hepatocytes and shares 80% sequence identity with
OATP1B1, but is expressed in multiple tissues as well as in
cancer cell lines. In addition to organic anions, it can
also transport small peptides such as cholecystokinin 8,
which differentiates it from OATP1B1. OATP2B1 is the other
family member expressed in the liver, as well as in spleen,
placenta, and lungs. It has a narrower range of substrates as
compared to OATP1B family members and favors transport
of sulfobromophthalein (SBP) and estrone sulfate. Molecu-
lar cloning of other oatp/OATP family members reveals a
complex pattern of substrate specificity and distinct organ
distribution in different species, suggesting that these trans-
porters are involved with organ-specific uptake of specific
organic anions [3,21].
Transcellular movement
Little is known about the transcellular movement of the key
constituents of bile [24,25]. Intracellular binding proteins

with high affinity for bile acids, organic anions, fatty acids,
and phosphatidylcholine have been identified, but their
physiological function is still speculative [2,26]. These pro-
teins are assumed to target their hydrophobic ligands to dif-
ferent intracellular components of the cell, including their
respective canalicular transporters. Vesicular-mediated
transport of bile acids has also been implicated, based on in-
creased transcellular movement of bile acids in response to
cAMP and enhanced phospholipid excretion in response to
infused bile acids. These effects may also be due to increased
insertion of canalicular transporters into the membrane.
Detailed studies with fluorescent bile acids have failed to
demonstrate evidence for vesicular transport in isolated
hepatocytes although these modified bile acids may not
accurately reflect processing of bile salts [27].
Canalicular excretion
Canalicular membrane transport is the rate-limiting step in
the vectorial movement of biliary constituents from the sinu-
soidal space into bile. Excretion of biliary components occurs
across a relative concentration gradient of 100- to 1000-fold
excess, requiring an active transport process. Prior biochem-
ical characterization of transport activity in canalicular-
enriched plasma membrane vesicles has recently been
advanced by the molecular identifi cation of specific canalic-
ular transporters. Detailed analysis of these individual trans-
porters, coupled with loss of activities in rare human
cholestatic syndromes and genetically engineered mice, has
revealed their role in normal biliary physiology. To date, all
the canalicular transporters involved with biliary excretion
are members of the diverse ATP binding cassette (ABC) class

of membrane transporters, in which transport of substrate is
dependent on ATP hydrolysis [28,29]. These proteins share
either a single or dual magnesium-dependent ATPase region
contained within a six-membrane-spanning domain with
substrate specificity dictated by other regions of the trans-
porters. Major subclasses within this super gene family
whose members play essential roles in biliary excretion in-
clude the multi drug resistant (MDR) proteins, also known as
P-glycoproteins, and the multidrug resistant proteins (MRP).
Besides these ABC transporters with two ATP binding and
catalysis domains, transporters with a single nucleotide
binding domain also exist and function as key effl ux pumps.
Chapter 22: Intrahepatic cholestasis 359
Another key gene involved with bile formation is a recently
identified P type ATPase, referred to as FIC 1 (ATP8B1), which
is responsible for Byler’s disease and benign recurrent intra-
hepatic cholestasis (BRIC) [30,31]. We will review these key
ATP-dependent transporters and their role in the excretion
of biliary components.
Canalicular bile salt transporter
As bile salt transport is increased by ATP hydrolysis, ABC
transporters of unknown function were screened as poten-
tial bile salt transporters [32,33]. Using this strategy, a gene
similar to P-glycoprotein, which is exclusively expressed in
the hepatocyte at the canalicular domain, was considered
and shown to mediate ATP-dependent taurocholate trans-
port in recombinant expression studies [34]. This trans-
porter, now referred to as bile salt excretory peptide (BSEP)
(ABCB11) is composed of 1321 amino acids and shares 70%
identity with the P-glycoprotein [35]. The gene is located on

chromosome 2 q24 in humans, which is identical to a shared
chromosomal region identified in children with a rare pro-
gressive familial intrahepatic cholestasis (PFIC 2) further
confirming its essential role in biliary excretion of bile salts
(see discussion of familial disorders below) [36,37]. Kinetic
features coupled with unique expression at the canalicular
domain of the hepatocytes and its absence in PFIC 2 indicates
that this is the major, if not sole, bile acid transporter in the
canaliculus.
Regulation of BSEP transport activity occurs at both
the transcriptional and more importantly at the post-
translational level, in which its redistribution from the sub-
canalicular endosomal pool to the canalicular domain can be
rapidly enhanced during high bile salt loads. T raffi cking be-
tween these pools is regulated by second messengers includ-
ing cAMP and PI3 kinase products, which enhance insertion
into the membrane [38,39]. This rapid regulation of expres-
sion at the canalicular membrane permits dynamic modula-
tion of transport activity coupled to fl ux of bile salts by
altering the relative density of transporter.
Canalicular phospholipid flippase
In addition to bile salts, phospholipids, such as phosphatidyl-
choline, and cholesterol are signifi cant components of bile.
Elimination of murine Mdr2, which corresponds to MDR3
(ABCB4) in humans, by gene knock-out technology lead to
almost complete elimination of phosphatidylcholine in the
bile with a progressive cholangitis in these animals [26,40].
This ABC transporter is postulated to function as a “flippase”
transferring the energetically unfavorable movement of
phosphatidylcholine from the inner leaflet to the outer leaf-

let of the canalicular membrane. Once at the outer leaflet,
phosphatidylcholine is extracted from the membrane by the
high concentration of bile salts present in the canalicular
space. Inability to translocate the phospholipid to the outer
leaflet in the Mdr2 knock-out mice leads to bile salts freely in-
teracting with the luminal membrane of ductules, causing a
detergent-induced cholangiopathy [41]. In these Mdr2-
deficient mice, progressive injury due to lack of buffering of
bile salts leads to a progressive cholangiopathy and eventual
hepatocellular tumor formation. The human counterpart
(PFIC 3) is discussed below.
Cholesterol transport
Characterization of the molecular basis for sitosterolemia, an
autosomal recessive disorder characterized by the accumula-
tion of both plant-derived (primarily sitosterol) and animal-
derived (cholesterol) sterols in plasma and tissues, lead to the
identifi cation of two ABC transporters responsible for cho-
lesterol effl ux [42]. These ABC transporters are members of
the ABCG subfamily, which are so-called “half-transport-
ers” composed of a single, six-membrane-spanning region
with one ATPase domain. ABCG5 (ABCG5) and ABCG8
(ABCG8) heterodimerize to form a functional pump for ex-
cretion of cholesterol and plant sterols. They are coexpressed
in the canalicular domain and their overexpression in mice is
associated with increasing canalicular excretion of choles-
terol. Like MDR3, these transporters are presumed to pro-
mote movement of cholesterol from the inner to the outer
leaflet of the canalicular domain, thereby functioning as a
flippase.
Canalicular organic anion transport

A multispecific organic anion transporter whose substrates
include conjugated bilirubin, originally referred to as cMOAT
and now as MRP2, was initially characterized in enriched
canalicular membrane preparations whose activity was en-
hanced by ATP hydrolysis [43]. In humans, Dubin–Johnson
syndrome is associated with persistent conjugated hyperbili-
rubinemia and retention of hepatic pigment without other
manifestations of cholestatic liver injury, suggesting specific
loss of MRP2 (ABCC2). These patients have a classical SBP
retention pattern in which a delayed regurgitation of SBP-
conjugates in the serum occurs after initial clearance, indi-
cating a deficiency in biliary effl ux. Rodent models of this
disease also exist, allowing for detailed analysis of bile com-
position [44]. This 1545 amino acid protein is expressed pre-
dominantly in the apical domain of hepatocytes as well as
proximal tubules of the kidney and duodenum and is one of
eight MRP family members cloned to date [28]. A peculiar
feature of the animal models (TR
−
and EHBR rats) is the lack
of biliary GSH, indicating that mrp2 is essential for hepatic
GSH excretion, a major contributor of bile-salt-independent
bile flow [23].
Other members of the MRP family are also implicated in
biliary excretion of organic anions [28,29]. MRP1 (ABCC1) is
minimally expressed in the liver but may be upregulated
during cholestasis at the basolateral domain. mrp3, which is
located at the basolateral surface of hepatocytes, is upregu-
lated in animals lacking mrp2 suggesting an alternative
360 Section 3: Specific conditions

pathway for elimination of organic anions across the basolat-
eral surface [46]. MRP3 (ABCC3) is upregulated in patients
with primary biliary cirrhosis (PBC) as well, suggesting a
compensatory pathway in response to chronic cholestasis
[6]. MRP3 is able to transport bile acids and their sulfated
and glucuronidated conjugates, which provides another
elimination route for retained bile salts in cholestatic disor-
ders. Also, MRP3 is expressed on the basolateral aspect of
human cholangiocytes where it might participate in chole-
hepatic shunting and provide another means for the elimina-
tion of bile salts in cholestatic conditions. In addition to the
liver, MRP3 is extensively expressed in the small intestine
and kidney as well as the adrenal cortex. MRP4 (ABCC4) and
MRP5 (ABCC5) have low levels of expression in the liver with
MRP4 being predominately expressed in the kidney and
prostate, and MRP5 expressed ubiquitously with greatest
levels in the brain and skeletal muscle. Both these transport-
ers lack the amino terminal membrane-spanning domain
and thus are smaller then other MRP family members. They
can both transport cAMP and cGMP nucleotides, which dif-
ferentiates them from other family members, and MRP4
preferentially transports conjugated steroids and estrogen
17β-glucuronide. In the presence of ATP and GSH, monoan-
ionic bile salts are substrates for MRP4-mediated transport
across membranes, providing another route for effl ux of bile
salts out of hepatocytes during cholestasis. MRP 6 is well ex-
pressed in the liver and has been localized to the basolateral
membrane and therefore may mediate the effl ux of organic
anions from the liver into the sinusoidal space during normal
and cholestatic conditions. The substrates of MRP 6 have not

been characterized. Surprisingly, mutations in MRP6
(ABCC6) are associated with pseudoxanthoma elasticum, a
disorder of connective tissues [28]. Differential regulation of
these family members during cholestasis, with reduction of
apical MRP2 expression and induction of other MRPs loca-
ted at the basolateral membrane, provide alternative routes
for the effl ux of biliary-excreted compounds from hepato-
cytes into blood, thereby protecting hepatocytes from toxic
accumulation and allowing these substances to be excreted
by the kidney.
FIC-1 P type aminophospholipid transporter
Rare disorders of progressive familial intrahepatic cholesta-
sis (PFIC-1) provide a unique opportunity to identify genes
associated with bile formation by searching for shared chro-
mosomal regions within selected affected populations. Link-
age analysis of Byler’s disease in an Amish population and
benign recurrent intrahepaticcholestasis (BRIC-1) in a small
Dutch fi shing village revealed that these disorders share
the same chromosomal region at 18q21–22 [36]. Subse-
quent identifi cation of the gene for FIC 1, now referred to
as ATP8B1, at this locus and discovery of mutations within
it in both clinical syndromes reveal that both diseases are
due to varying degrees of loss of activity of the FIC 1 protein
[30]. This protein shares amino acid sequence identity with
an aminophospholipid P-type ATPase, which mediates
transfer of aminophospholipids from the outer to the inner
leaflet of membranes, similar to the “flippase” activity of
MDR3. The protein is expressed in the small intestine and
pancreas with lower expression in the liver, including
cholangiocytes and the canalicular domain of the hepato-

cyte. The mechanism by which the defect in this gene leads
to impairment of bile formation is unknown. Recently, it
has been shown that absence of FIC1 in ileal enterocytes
leads to increased expression of ASBT and, consequently, in-
creased bile acid absorption, which may overload the liver’s
ability to maintain bile formation due to other abnormalities
induced by this mutation. Indeed, partial biliary diversion
can ameliorate the clinical symptoms in some of these
patients.
Regulation of bile acid synthesis,
transport, and metabolism by nuclear
transcription factors
Like other steroid hormones, nuclear receptors play a key role
in regulating the de novo synthesis of primary bile acids from
cholesterol, expression of bile salts, and organic anion trans-
porters in both hepatocytes and enterocytes, and in the re-
sponse of the liver to cholestatic injury. Beside their essential
roles in intestinal fat absorption and bile formation, synthe-
sis of the primary bile acids has a major impact on global lipid
metabolism and fatty acid synthesis in the liver. We will pro-
vide an overview of the current status of those transcription
factors that regulate these diverse processes, which are
largely based on studies performed in knock-out mice. These
receptors are listed in Table 22.2 and depicted in Fig. 22.2. All
these nuclear receptors share typical, modular protein struc-
tures, which are listed from their amino (N) to carboxyl (C)
terminal ends: A/B – contains the N terminal ligand inde-
pendent transcription activator domain (AF1), C – the DNA
binding domain, D – a shared domain and hinge region, and
E – the ligand-binding domain with the ligand dependent

AF2 activation domain (F) located at the C terminal end of
the protein. The AF regions harbor binding sites for coactiva-
tors or corepressors, which modify gene expression by inter-
acting with the transcriptional machinery. Differences in the
amino acid sequence of the binding domain define the unique
substrate specificity for these transcription factors. These
transcriptions factors most often heterodimerize with mem-
bers of the RXR nuclear receptor family (NRB1, 2, 3), whose
ligands include derivatives of retinoic acid [42,47]. The abili-
ty of these factors to function as master co-ordinators for both
metabolism and transport of bile salts and organic anions
makes them ideal targets for the development of selec-
tive agonists guided by the structural features of their
Chapter 22: Intrahepatic cholestasis 361
Table 22.2 Primary and secondary nuclear receptors regulating bile acid synthesis and response to cholestatic injury.
Nuclear receptor Protein Gene Binding Ligand Effect
partner
Farsenoid X receptor FXR NR1H4 RXR Chenodeoxycholate > Induction of ABCB11, ABCC4, OATP1B3
chenodeoxycholate,
cholate > lithocholate
Liver receptor homolog-1 LRH-1 NR5A2 none None Induction of CYP7A1, CYP8B1, ABCC3
Small heterodimer partner SHP NROB2 LRH-1 None Blocks LRH-1 dependent gene expression
Pregnane X receptor PXR NR1I2 RXR Rifampin, natural as Repression of CYP7A1, CYP8B1, induction of
well as synthetic CYP3A11, SULT1A2, ABCC2, SLCO1B1
steroids, xenobiotics
Constitutive androstane CAR NR1I3 RXR Required for Induction of ABCC3, ABCC2, CYP2B, SULT2A1
receptor translocation into
nucleus – phenobarbital,
TCPOBOP
TCPOBOP, 1, 4, bis [2-(3,5 dichloropyridyloxy)] benzene. (Summarized from [3,42,47,50–52,95,96].)

Figure 22.2 Primary and secondary nuclear receptor for bile acids in
hepatocytes. Representative genes regulated by the primary bile acid
receptor, FXR, and the secondary receptors, PXR and FXR, are illustrated.
When bound to bile acids, FXR directly induces expression of the gene
involved in canalicular bile salt transport, ABCB11, which encodes BSEP,
and canalicular organic anion transporter, ABCC2, which encodes MRP2.
By induction of SHP, FXR can down regulate the first steps of primary
bile acid synthesis in both the neutral pathway, catalyzed by the gene
product of CYP7A1, and the acidic pathway, catalyzed by the gene
product of CYP8B1. SHP acts as a dominant negative receptor blocking
the transcriptional activity of LRH-1, a potent inducer of these two
genes. The secondary bile acid receptors, PXR and CAR, have a lower
binding affinity for bile acids as compared to FXR. In addition,
cholestatic bile acids, such as lithocholate and deoxycholate, as well as
xenobiotics, are potent ligands for these transcription factors. PXR and
CAR induce expression of genes that encode efflux pumps, such as
ABCC2 (MRP2) and ABCC3 (MDR3) as w e ll as t h e gene tha t encodes a
bile acid sulfotransferase, SULT2A1. Each nuclear receptor can also
induce a subset of specific genes. When bound to its ligand, PXR induces
CYP3A family members, which can hydroxylate cholestatic bile acids.
CAR ligands can activate expression of CYP2Bfamily members while
inducing expression of SULT2A1 and ABCC3.
362 Section 3: Specific conditions
ligand-binding domains. In the near future, it is anticipated
that highly selective ligands for these factors will be used to
enhance cellular defense against cholestatic injury and ame-
liorate clinical symptoms. The major nuclear receptors are
divided into the primary bile acid receptor, the farsenoid X
receptor (FXR), and secondary bile salts receptors, pregnane
X receptor (PXR) and the constitutive androstane receptor

(CAR), which are induced by xenobiotics as well as choles-
tatic bile acids.
Farsenoid X receptor (FXR) (NR1H4)
In 1999, the primary bile acids, chenodeoxycholate and cho-
late, were found to be the endogenous ligands for the previ-
ously identified FXR receptor [42,47]. FXR is responsible for
mediating bile-acid-induced down regulation of its own
synthesis, by indirectly reducing the expression of CYP 7A1,
the rate-limiting enzyme for the synthesis of primary
bile acids in the neutral pathway [42,47]. FXR induces the
expression of SHP (NR0B2), a transcription factor that lacks a
DNA-binding domain, and binds with LRH-1 (NR5A2), a
potent promoter activator of the CYP 7A1 gene. In the
absence of bile acids, LRH-1 potentiates the effects of LXRα
(NR1H3) thereby up regulating expression of CYP7A1. In
humans, chenodeoxycholate is the most potent natural
ligand while in mice, cholate is, and this may account for the
species-specific effects of FXR. The reader is referred to Ory
and Edwards et al. [42,47] for an in-depth review of how FXR
and these other nuclear receptors regulate global lipid
metabolism.
In addition to down regulating CYP7A1, FXR bound with
bile acids increases expression of BSEP, which encodes the
major canalicular transporter of bile salts as well as enzymes
responsible for conjugating primary bile acids with taurine
or glycine. Induction of these enzymes promotes formation
of bile salts from primary bile acids and ensures the availabil-
ity of canalicular transporters required for their enterohe-
patic circulation. MDR3 is also up regulated, permitting the
co-ordinated excretion of bile salts and phospholipids into

bile. FXR can also induce expression of MRP2, which trans-
ports bilirubin diglucuronides and other organic anions.
FXR also regulates expression of the sodium-dependent bile
salt transporter on the ileal apical domain, ASBT, and the in-
tracellular bile acid binder, the intestinal fatty acid binding
protein (I-FABP). As FXR regulates expression of these bile
salts transporters, increased FXR signaling in the hepatocyte
is predicted to be protective against cholestatic injury by pro-
moting bile salt excretion. The bile acid, ursodeoxycholate, is
routinely used for treatment of primary biliary cirrhosis, but
it is a poor FXR activator. However, GW4064, a potent FXR
agonist, has been shown to decrease injury in a bile duct li-
gated model of cholestasis and to reduce the incidence of gall-
stone formation in mice [48,49]. FXR agonists therefore
constitute a promising new class of agents for the medical
treatment of chronic, cholestatic conditions.
Pregnane X receptor (PXR, NR1I2) and constitutive
androgen receptor (CAR, NR1I3)
Beside FXR, studies in knock-out mice have demonstrated
the importance of two well-recognized nuclear receptors,
PXR and CAR, that enhance the metabolism of xenobiotics
by induction of cytochrome P450 (CYP) family members
[3,50,51]. PXR responds to a wide number of lipophilic com-
pounds, including rifampin, due to its large ligand-binding
domain, and is responsible for induction of the CYP3A gene
family. This family of enzymes catalyzes a monoxygenase ac-
tivity for diverse substrates, which is often the first step in the
conversion of lipophilic compounds to more polar derivates.
In humans, the CYP3A gene family is responsible for metabo-
lizing approximately half of the prescribed drugs as well as

endogenous and exogenous compounds. Thus, PXR is an
important factor in hepatic drug metabolism. Recently, it
has been shown that secondary, and especially cholestatic,
bile salts, such as lithocholate, are potent PXR agonists, link-
ing bile acid metabolism to their previous, well-established
roles in xenobiotic metabolism. CY P3A4 is able to hydroxyl-
ate the cholestatic bile acid, lithocholate, at the 6 position,
generating a more hydrophilic and less cholestatic bile acid.
Ursodeoxycholate is another PXR ligand. In addition to the
CYP3Afamily, PXR inhibits CYP7A1expression by a non-SHP
dependent pathway, demonstrating multiple sites for regula-
tion of the CYP7A1 gene. Besides CYP3A members, PXR also
induces expression of a sulfotransferase (SULT2A1), which
catalyzes sulfation of cholestatic bile salts and thus facilitates
their renal excretion. Members of the ABC transporter
family, including MRP2 and MRP3, are also induced,
allowing for the effl ux of conjugated bile acids out of hepato-
cytes and into the vascular compartment for their eventual
excretion in urine. The effectiveness of rifampin to treat
pruritus is likely due to induction of these PXR-dependent
pathways.
Besides PXR, CAR also participates in the metabolism and
transport of cholestatic bile salts. CAR’s mechanism of action
differs from PXR and FXR, which are typically located in the
nucleus, and activated by binding to their respective ligands.
In contrast, CAR is activated in the absence of ligand and nor-
mally resides in the cytosol. Some ligands directly bind to
CAR in the cytosol whereas others, such as phenobarbital,
lead to exposure of its nuclear localization domain causing it
to migrate into the nucleus where it binds at xenobiotic re-

sponsive elements. Although some of its ligands overlap with
those that bind to PXR and both receptors can induce similar
genes, only CAR is able to selectively induce CYP2B family
members. Studies in knock-out mice have demonstrated dif-
ferences in response to lithocholate with CAR inducing
Cyp3a11 and AbcC3 whereas PXR induces Slco1B3, the sinusoi-
dal transporter, which is thought to enhance uptake of cho-
lestatic bile acids from the circulation [52]. Sulfotransferases
are also induced by CAR and catalyze production of sulfated
bile salts, which can be excreted in urine. These receptors
Chapter 22: Intrahepatic cholestasis 363
represent additional targets for future therapeutic interven-
tions to treat cholestatic disorders.
Regulation of key hepatic transporters in
experimental cholestatic conditions
Animal models have been developed to mimic different types
of clinical cholestatic syndromes which can occur with
either sepsis, bile duct obstruction, or high estrogen levels as
found in pregnancy [6], although species-specific responses
have been noted.
Effect of estrogens
High doses of circulating estrogens have been implicated
in both intrahepatic cholestasis of pregnancy and oral
contraceptive-associated cholestasis [53]. As the concentra-
tion of the synthetic estrogen, ethinyl estradiol, has been re-
duced in current oral contraceptive pills, this is a now a rare
cause of drug-induced cholestatic liver injury. However, in-
t r a he p a t ic c ho l e s t a s i s o f p r e g n a nc y i s l i k el y t o b e d ue , i n pa r t ,
to high circulating estrogens or their metabolites, such as the
estradiol 17β glucuron ide, wh ich is known to be cholestatic.

Treatment with the synthetic estrogen, ethinyl estradiol, in
rats leads to decreased bile flow and reduced excretion of or-
ganic anions, including bile salts and bilirubin, in the absence
of any morphologic changes, referred to as bland cholestasis.
Estrogen-induced cholestasis is complicated by multiple
effects on membrane fl uidity, expression and location of
key transporters, as well as decreased enzymatic activity of
the Na
+
,K
+
ATPase, a key regulator of sodium-dependent
cotransport processes. Increased permeability across tight
junctions has also been implicated in cholestasis [53].
Estrogens affect transport activity at both the sinusoidal
and canalicular domain. Decreased ATP-dependent trans-
port of organic anions and bile acids occurs in canalicular
membranes from ethinyl estradiol-treated animals. De-
creased mrp2 and bsep protein expression in rats without
altering mRNA levels were found after ethinyl estradiol
treatment, indicating a post-translational dysregulation
[53,54]. At the sinusoidal domain, decreased Na
+
,K
+
ATPase
activity was noted without a corresponding decrease in
mRNA or protein levels, indicating a functional change in
enzymatic activity which may be due to a secondary increase
in membrane fl uidity. Decreased sodium-dependent tauro-

cholate uptake activity was associated with a progressive loss
of both ntcp protein and gene expression as well as Slco3a1
protein and gene expression [6,55]. These effects observed in
rats may mirror similar mechanisms that contribute to
cholestasis of pregnancy in humans.
Effect of lipopolysaccaride / endotoxin / cytokines
Cholestasis associated with sepsis is a well-described clinical
entity, which can be reproduced in animal models by single-
dose administration of endotoxin, the lipopolysaccaride
(LPS) component of the outer membrane of Gram-negative
bacteria. In rats treated with LPS, bile-salt-independent bile
flow decreases with reduced expression of mrp2 in the cana-
licular membrane which slowly recovers after 3 to 4 days.
Uptake of organic anions from the basolateral and canalicu-
lar membrane fractions isolated from LPS-treated animals
are reduced without altered transport kinetics, indicating a
decrease in the total number of transporters [10,56]. No
changes in P-glycoprotein were found, indicating a selective
dow n r eg u lat i on of m r p2 c a na l ic u la r t r a ns p or te r. D ec r ea se d
expression of ntcp protein and mRNA were also found, which
was mediated by a proximal regulatory element in the NTCP
gene.
Regulation of different transporters in response to LPS in-
jection is mediated by cytokine response. Pretreatment of
animals with corticosteroids prior to LPS prevents the reduc-
tion in mrp2 expression by decreasing activation of NF-kB
pathway, a key regulator for the induction of cytokines.
Treatment with anti-TNF antibody also abrogates the choles-
tatic response. Treatment with IL-1 or TNF-α can also mimic
effects of LPS on Ntcp gene expression whereas IL-6 treat-

ment leads to reduced taurocholate uptake by reduction of
Na
+
,K
+
ATPase activity without effecting Ntcp expression
[56]. Thus, specific cytokines can contribute to cholestasis by
modulating different components of the normal excretory
pathway and provide targets for future treatments for choles-
tatic disorders.
Effect of bile duct obstruction
Animal models of acute bile duct obstruction mimic biliary
tract obstruction due to stone, tumors, or stricture. In rats,
acute obstruction of the common bile duct leads to retention
of biliary content within the hepatocytes, further contribut-
ing to cholestatic syndrome [14]. Accumulation of second-
ary, hydrophobic bile acid species can also reduce bile
formation, increasing the cholestatic insult. Absence of bile
salts in the intestinal lumen can also promote translocation
of bacterial LPS. NTCPgene expression is inversely correlated
with serum bilirubin levels [12], suggesting increased biliary
content in the serum and liver can down regulate gene ex-
pression. Down regulation of sinusoidal transporters in this
setting would protect the hepatocyte from further accumu-
lation of toxic bile acid. In primary biliary cirrhosis, OAT-
P1B1 was decreased whereas BSEP and MRP2 was preserved
and MDR3 and MRP2 were increased [57]. There was a posi-
tive correlation between the fractional percentage of ursode-
oxycholate in the total hepatic bile salt pool and expression of
OATP1B1 and MRP2.

Clinical approaches to intrahepatic
cholestasis and specific conditions
Cholestatic disease clinically means hepatobiliary disease
predominantly caused by, and manifested by, impaired bile
364 Section 3: Specific conditions
secretion, usually without major liver destruction. Thus,
acute and chronic viral hepatitis and alcoholic hepatitis and
cirrhosis commonly cause jaundice which undoubtedly rep-
resents failure of bile secretion but this occurs in the setting
of marked parenchymal cell death (elevated ALT) and abnor-
mal synthetic function (coagulopathy and low serum albu-
min). Cholestatic conditions typically are associated with
increased serum bile acids, markedly abnormal serum alka-
line phosphatase, and variable conjugated hyperbilirubine-
mia. When bilirubin is normal to about 3 mg/dL, we refer to
this as anicteric cholestasis which is typical of PBC, primary
sclerosing cholangitis (PSC), chronic pancreatitis-associated
common bile duct stricture, and infiltrative diseases of the
liver. Otherw ise, when jaund ice is present, it is referred to as
icteric cholestasis. Since bile salts are the major solute that
determines osmotic bile secretion, retention and elevation of
bile salts in serum is a hallmark of cholestasis. Selective de-
fects in bilirubin metabolism and transport, such as MRP2-
deficiency of Dubin–Johnson syndrome, cause nonchole-
static jaundice, which is not accompanied by increased serum
bile acids.
Conceptually, cholestasis can occur because of mechanical
obstruction due to diseases of bile ducts: macroscopic
extrahepatic ducts (stones, stricture, intrinsic, or extrinsic
neoplasms) or destruction of microscopic intrahepatic ducts

(primary biliary cirrhosis, autoimmune cholangiopathy,
sarcoidosis, drug-induced vanishing duct syndrome). Alter-
natively, cholestasis can occur at the level of the hepatocana-
liculus as a result of a selective impairment in bile secretion,
which is the focus of the remainder of this chapter (see Table
22.3).
The work-up of cholestasis is covered in Chapter 4. Suffice
it to say that once extrahepatic duct obstruction has been ex-
cluded, one is working in the realm of intrahepatic cholesta-
sis. In adults, a limited number of associated or causative
conditions are pertinent, most of which will be obvious on
clinical grounds: pregnancy, BRIC, sepsis, alcohol, postviral
hepatitis, drugs, total parenteral nutrition (TPN), paraneo-
plastic syndrome, and amyloidosis, along with the vanishing
duct diseases (e.g. PBC, sarcoid). From a diagnostic point of
view, patients presenting with intrahepatic cholestasis
should have blood cultures, viral hepatitis serology panel,
serum mitochondrial antibody, and, if there is suspicion of
infiltrative processes, imaging of the liver and abdomen. It is
important to remember that neoplasms that infiltrate the
liver produce anicteric cholestasis but rarely cause jaundice
on the basis of infiltration.
Genetic disorders presenting with
cholestatic syndrome
Rare genetic disorders of progressive familial intrahepatic
cholestasis clearly demonstrate the key function of these
transporters and their clinical presentation due to loss of
activity. Subtle changes in functional activity of these key
transporters, which leads to reduced but not absent activity,
or loss of a normal copy of the gene (heterozygous conditions)

are being increasingly recognized as important risk factors
for the development of cholestatic syndrome in response to
clinical conditions such as sepsis, intrahepatic cholestatsis of
pregnancy, or possibly drug-induced cholestatic liver injury.
Accumulation of genotyping information in these various
conditions may ultimately identify genetic polymorphisms
that could identify those patients at increased risk for devel-
oping these cholestatic disorders.
Progressive familial intrahepatic cholestatic (PFIC) syn-
dromes commonly present in early childhood with distinc-
tive pathological and laboratory features and a normal biliary
anatomy [2,58]. Biliary atresia is the major cause of cholesta-
sis in this age group whereas PFIC syndromes represent a dis-
tinct minority. If untreated, these patients may develop
progressive liver injury requiring transplantation. It is im-
portant to identify genetic disorders in the bile acid synthetic
pathway, in which toxic accumulation of normal precursors
occur and are poorly transported by the canalicular trans-
port system. Molecular identifi cation of accumulated bile
acid precursors by mass spectroscopy of the urine is highly
effective for identifying these rare patients. The PFICs
have recently been classified according to the identity of the
specific genes mutated in these disorders, as listed in Table
22.4 along with other disorders of the excretory pathway. It
is now increasingly recognized that adults presenting with
benign, recurrent intrahepatic cholestasis (BRIC) carry mu-
tations in the same transporters with less severe functional
consequences.
PFIC-1: Byler’s syndrome / disease
Byler’s disease, initially observed in descendants of the large

Amish family of Jacob Byler, is characterized by recurrent
Table 22.3 Intrahepatic cholestasis, exclusive of intrahepatic duct
disease: PBC, sarcoid, Caroli’s disease, PSC, autoimmune
cholangiopathy.
Congenital/inherited: PFIC 1, 2, 3
BRIC-1, BRIC -2
Pregnancy
Sepsis
Viral hepatitis
Alcoholic fatty liver
Total parenteral nutrition
Lymphoma and paraneoplastic syndrome
Amyloidosis
Drugs
Chapter 22: Intrahepatic cholestasis 365
episodes of jaundice which become persistent, with severe
pruritus along with elevated serum and reduced biliary lev-
els of bile salts [58,59]. Patients with this disease who are not
direct descendants of Byler are referred to as having Byler’s
syndrome. Patients have intermittent diarrhea and failure to
develop due to fat malabsorption. A unique feature of these
patients is the normal level of serum γ glutamyl transpepti-
dase, which differentiates this from other progressive choles-
tatic syndromes in children [58]. In general, patients are
compound heterozygotes with missense, chromosomal dele-
tions, insertions, or splice site mutations. Patients with the
same mutations may have different presentations or can be
asymptomatic, indicating that other genes or environmental
factors may modify clinical presentation [60]. These patients
may develop progressive liver disease within the first decade

of life and require liver transplantation. In some patients,
partial external biliary diversion results in a substantial im-
provement in symptoms and regression of hepatic injury,
suggesting that cholestatic liver injury is a consequence of
cholestatic factors produced in the intestine. Loss of FXR
activity in the intestine of PFIC11 patients has been observed
as well, which may contribute to cholestatic liver injury. It
is speculated that loss of FIC 1 alters signaling leading to
decreased nuclear FXR. As a consequence of decreased SHP
expression, ileal ASBT expression increases, leading to in-
creased bile acid absorption. Furthermore, decreased FIC 1 in
the liver might decrease FXR, leading to decreased BSEP and
increased NTCP, but this remains to be shown.
PFIC-2
PFIC 2 was originally identified in rare familial pediatric
cholestatic disorders which presented with similar serum
chemistry as Byler’s disease but affected individuals lacked
the commonly shared PFIC 1 chromosomal region [61].
These patients presented with a more aggressive cholestatic
syndrome soon after birth with rapidly developing liver
failure requiring transplantation and were unresponsive to
ursodeoxycholate therapy [62]. Linkage analysis revealed
shared chromosomal region in afflicted patients at position
2q24, the same location as the BSEP gene [36,37]. Subse-
quent studies in these patients confirmed mutations in
ABCB11 leading to absence of protein expression [63]. The
absence of the characteristic elevation in the serum γ gluta-
myl transpeptidase in severe cholestasis and minimal ductal
injury observed in liver biopsies indicates that bile salts with-
in the biliary tree are essential for duct injury associated with

increased serum γ glutamyl transpeptidase.
PFIC-3
Patients who present with a similar pattern of cholestatic
liver disease as PFIC 1 and 2 but with elevated serum γ gluta-
myl transpeptidase were shown to have mutations in both al-
leles of the human ABCB4 gene which encodes MDR3, a
transporter with phospholipid flippase activity [58,64].
These patients, as in the corresponding Mdr2knock-out mice,
have severe duc t a l injur y due to lac k of bu f feri ng of bi le sa lts
by phospholipids [41]. Histologically, these patients have
portal fibrosis with ductular proliferation and infl ammatory
infiltrate despite patency of the bile ducts. Clinically, these
patients present at later stages than PFIC 1 and 2 with more
symptoms related to cirrhosis, such as portal hypertension.
Interestingly, some cases of cholestasis of pregnancy have
been observed in heterozygote carriers of these same MDR3
mutations (see below).
Benign recurrent intrahepatic cholestasis (BRIC)
The diagnosis of BRIC should always be considered in pa-
tients with recurrent episodes of cholestasis in the absence of
bile duct obstruction, or infiltrative or chronic liver disease.
Genetic analysis of the transporters responsible for the PFIC
syndromes have now revealed the molecular mechanism for
some of these disorders. Initially, mutations in the FIC1 gene,
now referred to as ATP8B1, were found in both patients with
BRIC and PFIC-1. The severity of cholestatic liver caused by
mutations in ATP8B1 disease refl ected the relative activity of
the FIC 1 transporter [30,60]. Both PFIC-1 and this form of
BRIC, now referred to as BRIC-1, are inherited as autosomal
recessive diseases. BRIC-1 initially presents in childhood or

during early adolescence and reoccurs throughout adult-
hood. During cholestatic episodes, patients present with a
1- to 2-week prodrome of pruritus and anorexia before onset
of jaundice, which can last from 1 to 3 months and spontane-
ously resolves. Viral illness, pregnancy, or winter have been
temporally associated with the onset of cholestatic episodes
but often no precipitating event can be identified. Diets rich
in fatty acids and oral contraceptives have also been im-
plicated as precipitating factors. In between episodes, pa-
tients have a contracted bile acid pool, which is enriched with
conjugates of secondary bile acids[65]. Recently, 20 individ-
uals with BRIC symptoms but no identified mutations in
ATP8B1 were evaluated for mutation in the ABCB11 gene,
which encodes for BSEP [66]. Homozygous or compound
Table 22.4 Hereditary disorders of liver transporters causing jaundice.
Syndrome Protein Gene Chromosome
PFIC 1 (Byler’s disease/ FIC1 ATP8B1 18q21–22
syndrome)
PFIC 2 BSEP ABCB11 2q24
PFIC 3 MDR3 ABCB4 7q21
BRIC-1 FIC 1 ATP8B1 18q21–22
BRIC-2 BSEP ABCB11 7q21
Dubin–Johnson MRP2 ABCC2 10q23 –24
syndrome
366 Section 3: Specific conditions
heterozygote mutations were detected in 10 of these 20 pa-
tients and in one patient, only a single mutation was found.
These patients, now referred to as BRIC-2, differed from those
with mutations in ATP8B1 (BRIC-1) by their absence of wa-
tery diarrhea, pancreatitis, or hearing loss. Some patients

developed permanent cholestasis with age and liver biopsies
typically demonstrated histologic features of cholestasis
without fibrosis. During cholestatic episodes, serum bile salts
were elevated. In two women, cholestasis developed during
pregnancy. Treatment with rifampin, cholestryamine, or ur-
sodeoxycholate may hasten resolution of cholestatic episode
but not prevent their occurrences [62]. Like PFIC-1 and PFIC-
2 patients, these BRIC-1 and BRIC-2 patients have elevation
of serum alkaline phosphatase levels with normal serum γ
glutamyl transpeptidase, which differentiates BRIC from
other cholestatic syndromes in adults. In the future, genetic
testing may be used to identify the etiology and confirm the
diagnosis of BRIC.
Total parenteral nutrition associated
cholestatic syndromes
Long-term total parenteral nutrition (TPN), especially in ne-
onates, is associated with a nonobstructive cholestatic syn-
drome and can lead to liver failure. Correlation between
incidence of cholestasis and TPN in adults is complicated by
underlying conditions and the different indications for TPN
support [67,68]. In adults, cholestasis is the predominant he-
patic abnormality, which occurs after 3 weeks or longer of
therapy and presents with increased alkaline phosphatase,
occasionally associated with serum bilirubin elevation. Liver
biopsy typically reveals canalicular bile plugs in periportal or
perivenous zones in combination with mild portal triaditis.
The cholestasis is attributed to multiple etiological factors ei-
ther directly related to the TPN solution or the underlying
clinical conditions. Increased uptake of amino acids by the
liver in combination with decreased excretion of normal bili-

ary constituents and steatosis, secondary to the high carbo-
hydrate concentration of the TPN solution, are risk factors
considered to be responsible for cholestasis. Reduced mdr2
expression preceding liver injury was noted in mice treated
with TPN, suggesting loss of normal canalicular function as a
cause for this cholestatic condition [69]. Lack of oral intake
with loss of the normal enterohepatic circulation of bile salts
is associated with a 10-fold increase in TPN-associated cho-
lestasis. In these patients, greater production of cholestatic
secondary bile acids is another factor. Patients with ileal dys-
function, such as with Crohn’s disease, are also at greater
risk, further indicating that the lack of a normal enterohepat-
ic circulation is an important factor. Intercurrent sepsis is
another risk factor and by itself may cause cholestasis as
previously described. Cessation of TPN leads to resolution of
the cholestasis in most cases. Alternatively, reduction in car-
bohydrate content of TPN, treatment with oral antibiotics,
and small enteral feeding to promote the enterohepatic cir-
culation can be tried while closely monitoring cholestatic
markers. In anecdotal case reports, UDCA treatment im-
proved TPN-associated cholestasis. It is important to recog-
nize that these patients are also at increased risk for formation
of biliary sludge and gallstone formation leading to cholecys-
titis and/or cholangitis which can present with right upper
quadrant pain and fever associated with increased alkaline
phosphatase and jaundice. Prompt recognition and imaging
of the liver and biliary system is required to make the diagno-
sis and initiate treatment.
Cholestasis of pregnancy
Intrahepatic cholestasis of pregnancy (ICP) is a rare disease

of unknown cause which may be recurrent in 40 to 60% of
cases [70]. Patients typically present in the second half of
their pregnancy, initially with pruritus occasionally accom-
panied by jaundice which rapidly resolves in the postpartum
period. Increased serum bile acids, which are predominately
conjugated cholic acid, bilirubin, and moderately increased
alkaline phosphatase and γ glutamyl transpeptidase are
found. No long-term sequelae have been noted for the mother
but the fetus is at increased risk of premature delivery, fetal
distress, and perinatal mortality. In a study from Sweden,
fetal complications were only identifi ed in those mothers
whose serum bile salts were greater then 40 µmol/L [71],
leading the authors to recommend that serum bile salts
should be routinely monitored in patients with ICP. Treat-
ment with UDCA in clinical studies was associated with im-
proved fetal outcome [72]. These patients are also at risk for
oral contraceptive-induced jaundice. Etiology for the disease
is unknown but increased incidence of ICP in multiparous
births which have higher estrogen levels suggest a possible
defect in estrogen metabolism, leading to an increased pro-
duction of the cholestatic estradiol 17β-glucuronide, as a po-
tential mechanism for this disorder. Widely different rates of
ICP are noted in different countries, with Chile and Sweden
having the highest rates, suggesting genetic factors are
clearly important. Recently, the incidence of ICP has de-
creased in both these countries. In anecdotal cases, muta-
tions in ABCC3 have been found in patients with ICP. I n one
patient presenting with recurrent cholestatic episodes, ICP
and eventually PBC, a novel mutation in ABCC3 encoding for
MDR3 was found [73]. Linkage analysis in a nonrelated

group of Finnish women with ICP revealed an association
between ICP and a single nucleotide polymorphism in
ABCB11, which encodes for BSEP [74]. However, another
study from Finland found no common regions in either the
ABCB11, ATP8B1, or ABCC3 genes in these women, suggesting
a genetically diverse group of disorders.
Viral hepatitis and cholestastic liver disease
Jaundice with acute or chronic viral hepatitis is caused by
loss of parenchymal function due to hepatocellular necrosis
and/or fibrosis. Rarely, viral hepatitis may present with cho-
Chapter 22: Intrahepatic cholestasis 367
lestasis as the predominant clinical feature and is most fre-
quently found in adults with hepatitis A viral (HAV) infection
[75]. Unlike children, in whom HAV is often a subclinical
and anicteric illness, adults can present with severe choles-
tatic syndrome mimicking chronic bile duct obstruction. Pa-
tients complain of pruritus, fever, diarrhea, and weight loss
and symptoms may last 1 to 4 months. A relapsing course can
occur in which an initial apparent improvement, lasting 3 to
5 weeks with normal serum chemistries, is followed by re-
currence of cholestatic serum markers and symptoms for an
additional 4 to 6 weeks. Liver biopsy reveals intraductal
cholestasis and portal tract infl ammation associated with
paucity of bile ducts.
In anecdotal case reports of HAV, treatment with oral pred-
nisone decreased jaundice and improved cholestatic symp-
toms. This treatment is not recommended for other viral
h e p at i t id e s . H AV i n a d u l t s s ho u ld a lw ay s b e c o n s i de r e d i n t h e
differential diagnosis of intrahepatic cholestasis. Rarely,
other causes of acute and chronic viral hepatitis can present

with predominant cholestatic features. Case reports of cho-
lestasis in chronic hepatitis C virus infection, acute cytomeg-
alovirus in immunocompetent individuals, and Epstein–Barr
virus infection have also been reported [76,77].
Cholestatic syndrome with ethanol
Occasionally, patients with alcoholic liver disease may pres-
ent with a cholestatic syndrome in the absence of cirrhosis or
alcoholic hepatitis. Initial reports associated alcoholic intra-
hepatic cholestasis with marked steatosis [78,79]. Patients
present with malaise, anorexia, and hepatomegaly with a
cholestatic pattern of serum liver tests. Compared to patients
presenting with alcoholic hepatitis, these patients tended to
have poorer nutritional status, greater alcohol consumption,
and a worse prognosis. Treatment consists of nutritional sup-
plementation and supportive care after imaging of the liver to
eliminate biliary obstruction or infiltrative disease. Chief
histologic features of alcoholic cholestatic liver are macrove-
sicular fat with some microvescicular fat in centrolobular he-
patocytes, portal tracts infiltrated with infl ammatory cells
and proliferating bile ducts. Fibrosis extending from the por-
tal tract into the liver acinus is routinely found without Mal-
lory bodies and minimal focal necrosis. In one large Veteran’s
Administration cooperative study, a retrospective review of
patients with cholestatic features on liver biopsy was as-
sociated with increased serum alkaline phosphatase and
signifi cantly decreased survival compared to those without
cholestatic features [80].
Some patients with a cholestatic presentation have pre-
dominantly microvesicular steatosis (alcoholic foamy fatty
disease). They may have jaundice and variable, sometimes

markedly elevated, serum alkaline phosphatase and aspar-
tate aminotansferase (AST) (up to 1000). Liver biopsies re-
veal cholestasis, predominant centrilobular microvesicular
fat, and little infl ammation or necrosis. This picture has been
associated with deletions of mitochondrial DNA. Although
occasionally fatal, most patients improve rapidly after alco-
hol is withdrawn.
Sepsis-associated cholestatic syndromes
Cholestatic liver disease accompanying systemic sepsis is a
well-recognized clinical syndrome and is hypothesized to be
due to increased cytokine production. The development of
jaundice with sepsis is a poor prognostic indicator due to the
gravity of the underlying infection. Cholestatic liver disease
can be caused by Gram-positive and Gram-negative bacterial
and fungal infections. Sepsis is the predominant clinical fea-
ture in these patients, who do not complain of right upper
quadrant pain or pruritus. Serum alkaline phosphatase is
routinely elevated (but only mildly) with conjugated hyper-
bilirubinemia occurring to a variable degree. Occasionally,
10-fold or greater increased serum alkaline phosphatase in
the absence of jaundice has been reported [81]. In one study,
cholestasis and increasing hyperbilirubinemia were associ-
ated with multisystem failure and increased mortality [82].
No specific therapy for the cholestasis is necessary in these
patients, after excluding the liver or biliary system as the site
of infection, and cholestasis resolves with treatment of the
underlying infection. Jaundice without increased alkaline
phosphatase may be a heralding sign for impending sepsis
[83]. Liver biopsy in severe sepsis-associated cholestasis re-
veals inspissated bile with dilated and proliferating portal

and periportal bile ductules. Cholestasis has also been re-
ported in toxic shock syndrome with increase serum bile
acids, mild transaminases, and hypoalbuminemia.
Paraneoplastic cholestasis
Cytokines play a key role in mediating cholestasis associated
with sepsis. Increased cytokine production associated with
neoplastic diseases is now presumed to be responsible for rare
cholestatic disorders associated with malignancy in which
there is no bile duct obstruction or infiltration of the liver pa-
renchyma. Anecdotal case reports in Hodgkin’s disease and
other lymphomas have noted an increase in alkaline phos-
phatase and bilirubin in the absence of infiltrative disease
[84]. Stauffer’s syndrome, a paraneoplastic syndrome asso-
ciated with renal cell carcinoma, which presents with cho-
lestasis, fever, increased acute phase reactants and anemia, is
associated with increased serum IL-6 levels. Experimental
treatment with anti- IL-6 antiserum in these patients led to a
temporary decrease in alkaline phosphatase, which returned
to their elevated levels after cessation of treatment indicating
that IL-6 was responsible for cholestasis [85]. Patients treated
w i t h I L -2 we r e a l s o fo u n d t o d e ve lo p r e ve r s i bl e c h o l e s t a s i s , a s
evidence by increased serum bile acids, bilirubin, and alka-
line phosphatase which returned to normal after completion
of treatment [86]. Prostate cancer may also present with a
cholestatic pattern of liver involvement without evidence for
biliary obstruction, which resolves with implementation of
368 Section 3: Specific conditions
antiandrogen therapy [87]. It is likely that excess cytokines
will also be responsible for other paraneoplastic cholestatic
syndromes.

Amyloidosis
Although amyloid infiltrates are commonly found in the
liver, patients rarely present with jaundice. Hepatomegaly
associated with amyloid has been attributed to the associated
congestive heart failure and not liver infiltration. Rarely, am-
yloidosis may initially present as intrahepatic cholestasis
[88]. Histologically, these patients present with perisinusoi-
dal deposition of amyloid paralleled by advanced hepatocel-
lular atrophy with varying degrees of bile stasis. Patients with
amyloidosis exhibit a low serum gamma globulin in contrast
to the elevated serum globulin levels found in chronic liver
disease.
Drug-induced cholestasis
A wide variety of drugs can cause cholestatic liver disease.
Aside from the bland cholestasis (no infl ammation) seen
with androgens and estrogens, which probably occurs in in-
dividuals with a genetic predisposition to cholestasis, drug-
induced cholestasis tends to exhibit portal tract infl ammation
and bile duct injury along with variable parenchymal in-
fl ammation and necrosis/ apoptosis. Indeed, many of the
drugs that induce clinical cholestasis with jaundice, pruritus,
and marked increased alkaline phosphatase also are associ-
ated with moderate to marked parenchymal destruction, as
reflected in elevated alanine aminotransferase (ALT). Thus,
the same drug in some individuals will produce predomi-
nantly cholestasis, in some mixed cholestasis/ hepatitis, and
in some predominantly hepatitis [89]. Table 22.5 lists drugs
that either usually produce predominantly cholestasis or
most often produce cholestasis but with variable and some-
times predominant hepatitis. The jaundice in this condition

tends to resolve very slowly after discontinuing the drug,
sometimes lasting for months and even evolving into a van-
ishing duct/ biliary cirrhosis picture (Table 22.6).
A very high proportion of the drugs that produce clinical
c ho l e s t at ic d i se as e s ee m t o do s o o n a n i m m u no a l le rg i c b a s i s ;
this is based on early onset, in the first few weeks of therapy,
and the concomitant appearance of fever, rash, eosinophilia,
and, in some instances, positive rechallenge. In the case of
certain drugs, for example antibiotics such as erythromycins
or Augmentin®, following a course of 1 to 2 weeks, the chole-
static syndrome appears up to 3 to 4 weeks after discontinu-
ing the drug. A recent analysis of HLA polymorphisms in a
large group of patients with drug-induced liver injury identi-
fi ed an association with cholestatic reactions, supporting the
predominantly allergic nature of this adverse phenomenon
with many drugs [90].
Whether or not features of allergy are present, the patho-
physiology is uncertain. However, the target of many of these
immunological reactions appears, on circumstantial evi-
dence, to be the bile ductules, which are often associated with
infl ammation and not infrequently with progressive de-
struction (Fig. 22.3). However, parenchymal infl ammation
and cytokines may contribute by down regulating the vari-
ous hepatic transporters, leading to impaired bile acid and
organic anion secretion. In theory, this would be more likely
to be clinically manifest in patients with a genetic predisposi-
tion, such as heterozygotes for defects in the transporters.
This might account for the fact that many of the drugs
commonly induce mild anicteric liver test abnormalities but
rarely cause overt liver disease. In addition, BSEP-mediated

Table 22.5 Some drugs that can lead to cholestasis. (Reproduced from
Zimmerman [89], with permission from Lippincott Williams and
Wilkins.)
Cholestatic injury Cholestatic or
characteristic hepatocellular injury
Ajmaline Allopurinol
Amoxicillin-clavulanate Antidepressants (Tricyclic, tetracyclic)
Anabolic steroids Captopril
Benoxaprofen Carbamazepine
Benzodiazepines Cimetidine
Butyrophenones Clozapine
Carbimazole Droxicam
Cloxacillin Enalapril
Cyproheptadine Fluconazole
D-Propoxaphene Gold compounds
Danazol Hydralazine
Dicloxacillin Itraconazole
Erythromycins Ketoconazole
Flucloxacillin Naproxen
Griseofulvin Nitrofurantoin
Methimazole Phenylbutazone
Oral contraceptives Phenytoin
Penicillamine Piroxicam
Phenothiazines Ranitidine
Sulfonylureas (most) Sulfonamides
Thiabendazole Sulfamethoxazole-trimethoprim
Thioxanthines Sulindac
Ticlopidine Zidovudine
Troleandomycin
Xenalamine

Chapter 22: Intrahepatic cholestasis 369
Table 22.6 Drugs incriminated in chronic cholestasis. (Reproduced
from Zimmerman [89], with permission from Lippincott Williams and
Wilkins.)
Aceprometazine (with meprobamate) Erythromycins
Ajmaline and related drugs Estradiol
Amineptine Flucoxacillin
Amitriptyline Glycyrrhiza
Amoxycillin-clavulanic acid Haloperidol
Ampicillin Imipramine
Arsenicals, organic Methyltestosterone
Azathioprine Norethandrolone
Barbiturates Phenytoin
Carbamazepine Prochlorperazine
Carbutamide Sulfamethoxazole-
Chlorothiazide Trimethoprim
Chlorpromazine Terbinafine
Cimetidine Tetracycline
Clindamycin Thiabendazole
Cyclohexylpropionate Ticlopidine
Cyproheptadine Tiopronin
Cyamemazine Troleandomycin
Xenalamine
Figure 22.3 Pathogenesis of drug-induced
cholestasis. Drugs can inhibit canalicular
BSEP (e.g. cyclosporin A, rifampicin), be
converted to toxic metabolites which might
elicit an immune response directed at
hepatocytes or bile ducts (via secretion), or
an associated inflammatory response which

might lead to cytokine-mediated down
regulation of hepatocyte export pumps.
bile acid transport is competitively inhibited by cyclosporin
A [91–93], rifamycin SV, rifampicin, glibenclamide, bosen-
tan, troglitazone, erythromycin, and estolate, whereas only
cyclosporin among this group inhibits MRP2 [91]. Except for
mild cholestasis with cyclosporin, it is doubtful that the inhi-
bition of BSEP by the other drugs causes clinical cholestasis.
Sulindac also inhibits canalicular bile acid transport [94].
Interestingly ethinylestradiol-17β-glucuronide is secreted
into bile by MRP2 and then transinhibits BSEP [91]. Another
intriguing possibility,with limited proof, isthat toxic but sta-
ble metabolites of certain drugs, that are secreted into bile,
exert toxic effects on the bile duct system. The best experi-
mental support for this mechanism derives from studies of α-
naphthylisothiocyanate (ANIT). ANIT forms a labile GSH
adduct in hepatocytes. After concentrative pumping into
bile, the adduct dissociates in alkaline bile and ANIT, at high
concentrations, attacks cholangiocytes. Rats with mutation
in mrp2 do not pump the adduct into bile and are protected
from ANIT-induced cholestatic injury.
Questions
1. Which serum test can be used to differentiate the etiology of
progressive familial intrahepatic cholestasis?
a. alkaline phosphatase
b. cholesterol
c. gamma glutamyl transpeptidase
d. indirect bilirubin
e. bile acids
370 Section 3: Specific conditions

2. Which of the following transport mechanisms are not
operational at the sinusoidal membrane of hepatocytes?
a. sodium-dependent cotransport
b. anion exchange antiport
c. passive diffusion
d. vesicular transport
3. In patients with MDR3 mutations, the mechanism for hepatic
injury is due to
a. inability to synthesize primary bile acids
b. lack of active transport of bile acids
c. extraction of lipids from the apical domain of hepatocytes and
cholangiocytes
d. inability to secrete cholesterol into bile
e. increased SHP expression
Suggested readings
Kullak-Ublick GA, Stieger B, Meier PJ. Enterohepatic bile salt trans-
porters in normal physiology and liver disease. Gastroenterology
2004;126:322–42.
Excellent review of the current understanding of bile formation, fea-
tures of hepatic transporters and their regulation in human and animal
models of cholestatic liver disease.
Jansen PL, Sturm E. Genetic cholestasis, causes and consequences
for hepatobiliary transport. Liver Int 2003;23:315–22.
Update and concise review of clinical features and the genetic muta-
tions in liver transporters responsible for progressive familial intrahe-
patic cholestatic syndromes in children.
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