Clinics and Research in Hepatology and Gastroenterology (2011) 35, 281—287

MINI REVIEW

Pathogenesis of cholesterol and pigment gallstones:

An update
Karel Johannes Van Erpecum ∗

Dept of Gastroenterology and Hepatology, University Medical Center Utrecht, HP. F.02.618, PO Box 85500, 3508 GA Utrecht,
The Netherlands

Available online 25 February 2011

Summary Phase separation of cholesterol crystals from supersaturated bile is still consid-
ered the key event in cholesterol gallstone formation. In this review, we will first provide a
basal framework of the interactions between the sterol, bile salts and phospholipids in aqueous
solutions and then summarize new developments. The hepatocytic apical membrane harbours
specific transport proteins for these lipids. Polymorphisms in the gene encoding the cholesterol
transporter ABCG5-G8 have been found to increase overall gallstone risk, whereas functional
mutations in the gene encoding the phospholipid floppase ABCB4 lead to the rare clinical syn-
drome of low phospholipid associated cholelithiasis. Expression of bile salt and phospholipid
transport proteins is regulated bij the bile salt nuclear receptor Farnesoid X receptor (FXR),
while the Liver X Receptor (LXR) ␣ regulates ABCG5-G8. Although data from murine exper-
iments suggest a critical role of FXR in gallstone formation, its role in human lithogenesis
remains controversial. Variants of the gene encoding UGT1A1 (uridine 5 -diphosphate (UDP)-
glucuronosyltransferase 1A1) responsible for bilirubin conjugation were recently associated with
risk of gallstones as well as stone bilirubin content, suggesting common factors in cholesterol
and pigment gallstone pathogenesis.
© 2011 Elsevier Masson SAS. All rights reserved.

Introduction tionally a high prevalence of brown pigment stones residing

in the bile ducts, and causing potentially devastating cholan-
In the Western world, prevalence of cholelithiasis has gitis. The current review will first provide a basal framework
increased considerably in recent years, related to increased of the interactions between the sterol, bile salts and phos-
prevalence of overweight and/or a higher proportion of pholipids in aqueous solutions and then summarize new
elderly subjects in the population. In the Western world, developments since our previous reviews [1,2].
approximately 70% of gallstone carriers exhibit choles-
terol gallbladder stones (cholesterol content > 50%), and 30%
black pigment gallbladder stones. In East Asia, there is tradi- Bile lipids and cholesterol gallstone
pathogenesis
∗ Tel.: +31 88 7557004; fax: +31 88 7555533. Although solubility of cholesterol in aqueous solutions is
E-mail address: k.j.vanerpecum@umcutrecht.nl extremely limited, in gallbladder bile a relatively large

2210-7401/$ – see front matter © 2011 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.clinre.2011.01.009
282 K.J. Van Erpecum

amount (∼20 × 10−3 M) can be kept in solution, due to incor-

poration of the sterol in mixed micelles, together with bile
salts and phospholipids (mainly phosphatidylcholine). The
traditional view is that supersaturation and cholesterol crys-
tallization (the earliest step to gallstone formation) occur
when either too much cholesterol or not enough solubilizing
bile salt and phosphatidylcholine molecules are secreted to
allow complete micellar solubilization of cholesterol. Nev-
ertheless, the key role of relative amounts of bile salts
versus phospholipids for the process of cholesterol crystal-
lization should be appreciated: Excessive cholesterol may
be kept in vesicles (i.e. spherical bilayers of cholesterol and
phospholipids) and vesicle formation is promoted by excess
phospholipid over bile salt. Vesicles are particular suitable
to prevent excess cholesterol from crystallization (vesic-
ular supersaturation above cholesterol/phospholipid ratio
of 1, compared to micellar supersaturation above choles-
terol/phospholipid ratio of ∼0.25 depending on conditions).
Nevertheless, in case of vesicular cholesterol/phospholipid
ratios above 1, cholesterol crystal nucleation will eventually
occur. In fact, in human gallstone pathogenesis, cholesterol
Figure 1 Equilibrium bile salt-phospholipid-cholesterol phase
crystals are thought to nucleate exclusively from supersat-
diagram. The components are expressed in mol percent.
urated vesicles rather than from supersaturated micelles.
Depicted are a one-phase (micellar) zone at the bottom, a left
Pivotal information on the process of cholesterol crystal
two-phase zone (containing micelles and crystals), a central
nucleation has been obtained from model bile systems. The
three-phase zone (containing micelles, vesicles and crystals)
equilibrium bile salt-phospholipid-cholesterol ternary phase
and a right two-phase zone (containing micelles and vesicles).
diagram allows to predict behaviour of mixtures of the three
Phospholipid/(bile salt + phospholipid) ratios are given at the
biliary lipids when present in various proportions [3]. As
bottom axis, and going from left to right indicates increased
shown in Fig. 1, the phase diagram contains a bottom one-
relative amounts of phospholipids compared to bile salts, with
phase zone (only micelles), a left two-phase (micelles and
increased vesicular cholesterol solubilization. In the phase dia-
cholesterol crystals-containing) zone, a central three-phase
gram, relative amounts of phospholipids compared to bile salts
(micelles, vesicles and cholesterol crystals-containing) zone
are expressed as phospholipid/(bile salt + phospholipid) ratios
and a right two-phase (micelles and vesicles-containing)
since this allows easy (linear) plotting along the bottom axis of
zone. Going from baseline to top in the phase diagram the
the phase diagram. Any line connecting the bottom axis with
relative percentage cholesterol increases, with progressive
the top of the triangle (100% cholesterol) represents identi-
cholesterol crystallization. Secondly, a shift from left to
cal phospholipid/(bile salt + phospholipid) ratio, with increased
right in the phase diagram increases relative amounts of
relative amounts of cholesterol when moving from bottom
phospholipids compared to bile salts, thus allowing more
to top. For example, in the figure, all biles plotting on the
solubilization of cholesterol in vesicles, lower vesicular
coarse interrupted line exhibit identical phospholipid/(bile
cholesterol/phospholipid ratios and less cholesterol crystal-
salt + phospholipid) ratio of 0.8. In the Figure, fine dotted lines
lization.
indicate zones in case of diluted bile, hydrophilic bile salts or
Three additional factors strongly affect the ternary
saturated phospholipid acyl chains. Continuous lines indicate
equilibrium bile salt-phospholipid-cholesterol ternary phase
zones in case of concentrated bile, hydrophobic bile salts or
diagram: bile concentration; bile salt hydrophobicity and
unsaturated phospholipid acyl chains. Under the latter circum-
phospholipids containing unsaturated acyl chains all pro-
stances, cholesterol crystallization is promoted by an expansion
mote cholesterol crystallization. Corresponding effects on
of the crystal-containing zones to the right. The bile sample
the ternary equilibrium bile salt-phospholipid-cholesterol
initially plotting in the right two-phase zone (indicated by the
ternary phase diagram are in all three cases, an increase
dot) now plots in the central three-phase (cholesterol crystal-
of the bottom one-phase (micellar) zone, expansion of
containing zone), despite identical relative lipid composition.
the cholesterol crystal-containing zones to the right and
decrease of the vesicles-containing zones (Fig. 1 interrupted
vs. continuous lines) [3,4]. Pathofysiologic correlations with (i.e. cholesterol-phospholipid ratio > 1) with cholesterol
human gallstone formation in vivo are as follows: significant crystal nucleation. This sequence of events explains why
net water absorption occurs during bile transfer through gallstones are generally formed in the gallbladder rather
the bile ducts and during prolonged storage in the gall- than in the bile ducts. In gallbladder bile of cholesterol
bladder. Mixed cholesterol-phospholipid-bile salt micelles gallstone patients, increased amounts of the hydrophobic
are increasingly formed, because bile salt concentrations bile salt deoxycholate are associated with fast crystalliza-
now progressively exceed critical micellar concentrations tion [5]. In selected patients with cholesterol gallstones,
required for micelle formation. Since solubilizing capac- treatment with the hydrophilic bile salt ursodeoxycholate
ity of micelles for phospholipids is much higher than for may desaturate bile and dissolve the stones. In in vitro
cholesterol, there is preferential phospholipid transfer, and studies with model biles, phospholipid class and phos-
remaining vesicles may become cholesterol supersaturated pholipid acyl chain composition exert profound effects
Pathogenesis of cholesterol and pigment gallstones: An update 283

on cholesterol crystallization. Human biliary phospholipid spp.-infected and -uninfected BALB/c and congenic immun-
composition is tighly regulated, and almost exclusively com- odeficient Rag2(−/−) mice confirmed the critical role of
posed of phosphatidylcholine with unsaturated acyl chains, helicobacter in murine lithogenesis and pointed to the
thus contributing to human vulnerability for gallstone for- relevance of T-cell proinflammatory response [25]. As a
mation [6]. Although modification of biliary phospholipids result, hypomotility and increased mucin secretion may
towards a more saturated acyl chain composition would be occur [22,23]. Although various helicobacter species have
in theory attractive, dietary modifications to accomplish this been detected in human gallbladders and bile, and anti-
have not been successful so far. For an extensive review on bodies to helicobacter hepaticus were found at increased
biliary lipids we refer to references [1,2]. frequency in gallstone patients [26], the role of helicobac-
The ternary bile salt-phospholipid-cholesterol phase dia- tor in human gallstone pathogenesis remains to be defined.
gram is based on equilibrium conditions. Nevertheless, time Also the extensive literature available on murine Lith genes
to reach equilibrium is essential for human gallstone for- is now difficult to interpret without knowledge on status
mation, since bile is stored in the gallbladder only for a of Helicobacter infection of the various strains applied.
limited period of time. A shift from left to right in the Although impaired motility is generally secondary to bil-
phase diagram is associated with an increase in time to reach iary cholesterol supersaturation, it may still facilitate the
equilibrium (from a few hours to more than 30 days, depend- process of gallstone formation. Gallbladder motility is often
ing on experimental conditions). Numerous biliary proteins impaired in high-risk situations for gallstone formation such
have previously been suggested to shorten or lengthen as pregnancy, obesity, diabetes mellitus, gastric surgery,
time to equilibrium and cholesterol crystallization in gall- treatment with the somatostatin analogue octreotide, very
bladder bile. Various pronucleating proteins are present in low calorie dieting and total parenteral nutrition.
much higher concentrations in the fast nucleating bile of
patients with multiple cholesterol stones. Immunoglobulins
M and G [7,8], haptoglobin [9], ␣1-acid glycoprotein [10,11], Enterohepatic circulation of bile salts,
aminopeptidase-N [12], ␣1-antichymotrypsin and mucin [13]
intestinal cholesterol absorbtion, lipid
are regarded as pro-nucleating proteins and apolipoprotein
A-I [14] and IgA [15] have been postulated to exert anti- transport proteins and cholesterol gallstone
nucleating activity. Nevertheless, in more recent years, a formation
growing number of publications have marshalled experimen-
tal evidence arguing against a role of most of these biliary In a polygenetic disorder as cholesterol gallstone disease,
proteins in human cholesterol gallstone formation [16,17]. several underlying mechanisms may be involved in patho-
In line with these publications, it was recently reported that genesis and their relevance may differ between different
bile composition and risk of gallstones were not altered in populations. Increased bile salt and cholesterol synthesis
apolipoprotein A1 ‘‘knockout’’ mice [18]. Mucin remains one have been reported in Chilean patients [27]. The defect
of the few candidate proteins with a potential role in human was supposed to be secondary to increased intestinal loss of
gallstone formation. bile salts, and preceded gallstone formation. Interestingly,
decreased expression of ileal bile salt transport proteins api-
cal sodium-dependent bile acid transporter (ASBT), cytosolic
The gallbladder and cholesterol gallstone ileal lipid binding protein (ILBP) and basoloateral organic
pathogenesis solute transporter (OST)␣ and ␤ were recently described
in female non-obese patients, as a possible explanation of
Impaired postprandial gallbladder emptying, often present these findings [28,29]. In line with these findings, a variant
in cholesterol gallstone patients, may prolong residence of the SLC10A2 gene encoding the apical sodium-dependent
of bile in the gallbladder, thus allowing more time for bile acid transporter was recently identified as a risk factor
nucleation of cholesterol crystals from supersaturated bile for gallstone disease in a large group of German gallstone
and their growth/aggregation into macroscopic stones. Sig- patients [30].
nificant absorbtion of cholesterol appears to occur from It has also been reported, that a high cholesterol diet
supersaturated bile in the gallbladder [19]. Excess choles- increases biliary cholesterol secretion and decreases bile
terol is then incorporated within the sarcolemmal plasma salt synthesis and bile salt pool in cholesterol gallstone
membrane of the gallbladder smooth muscle cell, with subjects but not in controls [31]. These findings point
decreased membrane fluidity, impaired contractility and to the importance of intestinal cholesterol absorbtion in
impaired relaxation as a result. Recent in vitro data indicate gallstone pathogenesis. Interestingly, increased expression
that reduced contraction is caused by a lower chole- of the intestinal cholesterol uptake protein NPC1L1 (Nie-
cystokinin binding to CCK-1 receptors due to caveolar mann Pick C1 Like protein 1) and ACAT2 (Acyl Coenzyme
sequestration of the receptors [20,21]. Gallbladder wall A-cholesterol acyl transferase: enzyme involved in choles-
inflammation may also be critical in gallstone formation The terol esterification) were recently reported in jejunum
gallbladder wall is exposed to detergent bile salts, unes- of Chinese gallstone patients [32]. These findings suggest
terified cholesterol and bacteria, which all could induce that increased intestinal uptake and esterification of the
inflammation [22,23]. The murine gallstone model which sterol could be important in gallstone pathogenesis. Also,
was employed by the group of Carey in Boston so extensively inhibiting cholesterol absorbtion with etezimibe prevents
to study gallstone pathogenesis, proved to be dependent on gallstone formation in the mouse model and decreases bil-
gallbladder infection with helicobacter species [24]. Sub- iary cholesterol saturation in gallstone patients with slower
sequent studies utilizing immunocompetent Helicobacter crystallization as a result [33].
284 K.J. Van Erpecum

Figure 2 Nascent bile formation at the hepatocytic canalicular membrane, biliary cholesterol solubilization and gallstone forma-
tion. ABCG5-G8 transports cholesterol into bile, and is regulated by nuclear receptor LXR. ABCB11 and ABCB4 transport bile salts
and phosphatidylcholine into bile, and are regulated by nuclear receptor FXR. Bile cholesterol is solubilized in mixed micelles or
kept in cholesterol-phospholipid vesicles. Cholesterol crystallization and gallstones occur in most patients because of excess biliary
cholesterol secretion and subsequent crystallization from supersaturated vesicles (continuous lines). In patients with LPAC (low
phospholipid associated cholelithiasis) there are insufficient amounts of phospholipids in bile due to loss of function mutations in
the ABCB4 gene. In patients with BRIC (benign recurrent intrahepatic cholestasis) type 2, there are insufficient amounts of bile salts
in bile due to loss of function mutations in the ABCB11 gene. In patients with LPAC and BRIC type 2, there is an increased risk of
gallstone formation. It is unknown, whether cholesterol crystallization in LPAC and BRIC type 2 (fine resp. coarse interrupted lines)
occurs from supersaturated vesicles or supersaturated micelles.

The process of nascent bile formation is maintained there is a strongly increased frequency of associated gall-
by an elaborate network of ATP-binding cassette (ABC) stones, supposedly due to insufficient amounts of biliary
transporters in the hepatocyte canalicular membrane which bile salts [39]. The human Multi Drug Resistance 3 (MDR3)
enable biliary secretion of cholesterol, bile salts and phos- P-glycoprotein (current nomenclature ABCB4) functions as
pholipids (Fig. 2). The ABCG5/G8 genes encode protein a ‘‘floppase’’, translocating phosphatidylcholine molecules
half-transporters which heterodimerize to form the func- from the inner to the outer leaflet of the canalicular mem-
tional transporter localized in the canalicular membrane brane thus enabling their secretion into bile (Fig. 2). A subset
of hepatocytes and facilitating cholesterol secretion into of gallstone patients has been identified with intrahepatic
bile [34]. Recently, variants of ABCG5/8 were linked to and bile duct stones at young age (< 40 years) and high risk
gallstone disease; ABCG8 D19H in Caucasians and ABCG5 of recurrent biliary symptoms after cholecystectomy. A sub-
Q604E in Chinese populations [35,36]. Similar findings were group of these patients exhibits a severe phenotype with
recently reported in a Swedish twin study [37]. ABCB5-B8 large uni- or multifocal spindle-shaped dilations of the intra-
may also provide a mechanistic link between gallstones and hepatic bile ducts without any bile duct stenosis, and filled
the metabolic syndrome. Mice solely with hepatic insulin of gallstones [40]. The underlying pathogenetic mechanism
resistance, created by liver-specific disruption of the insulin of this so-called ‘‘low phospholipid associated cholelithia-
receptor (LIRKO mice) were found to be markedly pre- sis’’ is thought to be relative biliary phospholipid deficiency
disposed toward cholesterol gallstone formation due to at due to a missense mutation in the MDR3 gene [41]. Nev-
least two distinct mechanisms. Disinhibition of the forkhead ertheless, recent data from sib pairs with gallstones and
transcription factor FoxO1, increased expression of the bil- control do not support a link between ABCB4 and ABCB11
iary cholesterol transporters Abcg5 and Abcg8, resulting in polymorphisms and gallstone formation in the large major-
an increase in biliary cholesterol secretion. Hepatic insulin ity of patients [42]. Also, there appears to be no increased
resistance also decreased expression of the bile salt syn- frequency of mutations in ABCB4 and ABCB11 genes in paedi-
thetic enzymes, particularly Cyp7b1, and produced partial atric gallstone patients with gallstone disease and a positive
resistance to the farnesoid X receptor (see below), leading family history of gallstones [43].
to a lithogenic bile profile [38].
The bile salt export pump (BSEP: current nomencla- Nuclear receptors and gallstone formation
ture ABCB11) pumps bile salts over the membrane into
bile (Fig. 2). In patients with benign recurrent cholesta- Farnesoid X receptor (FXR: NR1H4) is a member of the
sis type 2 (BRIC2) due to mutations in the ABCB11 gene nuclear receptor superfamily and functions as a bile salt
Pathogenesis of cholesterol and pigment gallstones: An update 285

receptor that regulates transcription of numerous genes excretion of bilirubin may increase ten-fold with increased
involved in maintaining cholesterol and bile salt homeosta- risk of calcium bilirubinate precipitation. This phenomenon
sis. FXR has been shown to regulate hepatic expression of explains the high prevalence of black pigment stones in
ABCB11 and ABCB4, thus affecting amounts of solibilizing chronic hemolytic disorders such as sickle cell anemia,
bile salts and phospholipids in bile (Fig. 2). As expected, FXR hereditary spherocytosis and Gilbert syndrome. Conco-
‘‘knockout’’ mice are highly susceptible to gallstone forma- mitant presence of Gilbert syndrome is associated with
tion on a lithogenic diet, due to low relative amounts of increased gallstone prevalence in sickle cell disease [50].
biliary bile salts and phospholipids. Also, gallstone formation Enterohepatic cycling of bilirubin may contribute to high
can be prevented in ‘‘wild type’’ mice by the synthetic FXR frequency of pigment stones in patients with ileal Crohn dis-
agonist GW4064, because increased amounts of solubilizing ease (especially in case of ileal resection) and cystic fibrosis
bile salts and phospholipids prevent cholesterol supersatura- [51]. The proposed mechanism is that increased amounts
tion [44]. FXR is also expressed in the ileal cell, and regulates of bile salts reach the caecum and solubilize unconjugated
activity of transport proteins involved in bile salt reabsorb- bilirubin thus allowing their reabsorbtion with subsequent
tion into the enterohepatic circulation, which are altered in hyperbilirubinobilia [52—54]. Prevalence of Gilbert syn-
subgroups of gallstone patients (see above) [28,29]. Never- drome is increased in patients with cystic fibrosis and
theless, data on gene polymorphisms for FXR have revealed gallstones, which may contribute to pigment stone forma-
controversial results for different human gallstone popu- tion [55]. Interestingly, a recent report on a large cohort
lations [45]. For an in-dept review on the role of FXR in of (both cholesterol and pigment) gallstone patients found
gallstone and other hepatobiliary and intestinal diseases we that variants of the UTG1A1 gene (encoding for uridine 5 -
refer to [46]. diphosphate (UDP)-glucuronosyltransferase 1A1 responsible
Liver X receptor (LXR) regulates expression of ABCG5/G8 for bilirubin conjugation) and the SLO1B1 gene (encoding for
cholesterol transport protein (Fig. 2). In the murine model, a basolateral hepatocytic membrane transporter for various
activation of LXR increases risk of gallstone formation [47]. exogenous and endogenous compounds including bilirubin
In a small group of Chinese non-obese normolipidemic gall- and its glucuronides) determined not only serum bilirubin
stone patiens, hepatic mRNA expression of ABCG5, ABCG8, levels, but also stone bilirubin content and risk of gallstone
and liver X receptor alpha (LXRalpha) were significantly formation [56]. Of note, the combination of UTG1A1 and
increased and correlated with the molar percentage of ABCG5/8 variants as major genetic risk factors explained
biliary cholesterol and cholesterol saturation index [48]. 11% of gallstone risk in males of this German cohort. Impor-
Further research is needed on the role of nuclear recep- tantly, the increased gallstone risk applied to both pigment
tors in gallstone pathogenesis and the therapeutic potential and cholesterol stones, consistent with the classic patho-
of the potent nuclear receptor agonists currently available. physiological model of bilirubin serving as a nucleation core
for gallstone formation in general. These findings could pro-
Pathogenesis of pigment gallstones vide a potential link between pathogenesis of pigment and
cholesterol gallstones.
Brown pigment stones
Conflict of interest statement
Brown pigment stones are formed in the bile ducts [49].
They are primarily composed of calcium salts of uncon- The author has no relevant financial disclosures.
jugated bilirubin and varying amounts of cholesterol and
protein. Brown pigment stones are associated with chronic
bacterial or parasitic infection of the bile ducts. Bacteria References
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