Journal of Hepatoiogy 1995; 22: 239-246
5
Printed in Denmark All rights reserved
Munksgaard Copenhagen
Review
Biliary cholesterol transport and the nucleation defect in cholesterol
gallstone formation
Daniel I? O’Leary
Department of Surgery, Southmead Hospital, Bristol, UK
C
HOLESTEROL is solubilized in bile by bile salts and
phospholipids (principally lecithin). Early
physicochemical studies described the cholesterol con-
tent of human bile as a percentage saturation or chol-
esterol saturation index (CSI) relative to the solubility
limits at equilibrium in model micellar systems of simi-
lar lipid composition (14). Gallbladder bile from chol-
esterol gallstone patients was invariably supersaturated
with cholesterol. However, normal hepatic and
gallbladder biles which did not contain crystals, were
also often supersaturated according to the limits of mi-
cellar solubility (2,5,6). This evidence indicated that
cholesterol supersaturation is not a sufficient cause for
cholesterol gallstones, nor does it explain their pre-
dominant occurrence in the gallbladder, rather than
throughout the biliary tree. In 1979, Holan et al. de-
scribed the “nucleation time” assay, which measured
the time taken to form new solid cholesterol monohy-
drate crystals in human bile, as judged by light micro-
scopy (7). Gallbladder bile from cholesterol gallstone
patients nucleated significantly faster than equally su-
persaturated controls. This crucial observation, con-
firmed by several groups (8-l l), prompted an intense
research effort into the “nucleation defect” which pro-
motes cholesterol nucleation in the gallbladder bile of
cholesterol gallstone patients. This article presents cur-
rent concepts of cholesterol transport and nucleation
in bile, with particular regard to the role of nucleating
agents which promote this vital step in cholesterol gall-
stone formation.
Cholesterol Transport in Bile
Mixed micelles are globular particles (40-80 A”) com-
posed of cholesterol, bile salts and phospholipids.
Correspondence: Daniel I? O’Leary, Department of
Surgery, Southmead Hospital, Bristol BSlO 5NB, UK
Copyright 0 Journal of Hepadogy 199.
Journalof Hepatology
ISSNO168-8278
They are important cholesterol carriers in human bile
and are responsible for cholesterol solubilization in
model biles that have been allowed time to equilibrate
(1,2). Cholesterol-phospholipid vesicles are an ad-
ditional cholesterol carrier in fresh human bile (12-
16). Vesicles are unilamellar (single-layered), spherical
particles (500-1000 A”) composed of cholesterol and
phospholipid, with little or no bile salt (12). Chol-
esterol enters bile from hepatocytes by cosecretion
with phospholipid in the form of vesicles (17-19).
Formation of mixed micelles requires bile salts, which
are secreted separately Vesicles have greater chol-
esterol carrying capacity per mole of phospholipid
than mixed micelles. Whereas the cholesterol : phos-
pholipid molar ratio in mixed micelles does not ex-
ceed 1 : 2, it may be more than 2 : 1 in vesicles
(14,16). The amount of cholesterol that can be sol-
ubilized in bile will vary, therefore, according to the
proportions of vesicles and micelles present. The cru-
cial point is that these proportions vary Low bile salt
concentration, low total lipid concentration, a raised
cholesterol saturation index and a low bile salt : leci-
thin ratio all promote cholesterol transport in vesicles
rather than micelles in model biles (20,21). Fasting
hepatic bile is dilute and has low bile salt and total
lipid concentrations (22). Cholesterol is carried most-
ly in vesicles (13,15). The gallbladder concentrates
bile, raising its total lipid and bile salt concentrations.
This leads to leaching of cholesterol and phospho-
lipid from vesicles to form mixed micelles (micell-
ation), probably via a hexagonal rod-like intermediate
(13-16, 23). Proportionately more phospholipid than
cholesterol is removed. If the cholesterol load is high
a state of “micellar insufficiency” arises and remain-
ing vesicles become heavily supersaturated with chol-
esterol and unstable (2426).
Recently, “phospholipid lamellae” have been de-
scribed in the form of stacked discs and multiple paral-
239
D. O’Leary
lel curved sheets (27,28), and have been proposed as
a third important cholesterol carrier in bile. However,
reviewing the methodology used, Cohen et al. conclude
that they may be an artifactual representation of mixed
micelles and multilamellar vesicles (vi& infra) caused
by negative stain electron microscopy (29).
Cholesterol Nucleates from the Vesicular Phase
Cholesterol crystals form in pathological gallbladder
bile following aggregation or fusion of cholesterol-rich
unilamellar vesicles to form multivesicular aggregates
or multilamellar vesicles, respectively (30). The mech-
anism is not yet understood at the molecular level. The
importance of vesicles in nucleation is supported by
the observations that it is the vesicular phase which
loses cholesterol once crystals begin to form (15), that
the nucleation time of whole bile correlates with the
speed of nucleation of vesicles from the same bile (31),
and that if vesicles are removed leaving only micelles,
nucleation occurs through “neoformation” of vesicles
(32). The speed of nucleation is proportional to the
degree of cholesterol saturation of vesicles, as distinct
from the CSI of the whole bile (21,33). Nucleation
from micelles is possible in the laboratory (34), but is
not thought to be important under physiologic con-
ditions. Crystals appear first as thin filamentous struc-
tures which evolve into helical and then tubular forms
before breaking into characteristic flat cholesterol
monohydrate plates (35).
Measurement of Nucleating Activity
“Nucleation” properly refers to formation of the
crystal nucleus - the smallest mass of solid chol-
esterol monohydrate (36). The nucleation time assay
(7), which measures the time to form new cholesterol
monohydrate crystals (after removing existing ones
by ultracentrifugation), is limited by the resolution of
light microscopy (>2000 A’). Its end point inevitably
includes both cholesterol nucleation and the early
stages of crystal growth, and is better described as
the crystal observation time (37,38). Nucleating activ-
ity has also been assessed by determining the greatest
dilution of a test agent which will promote nu-
cleation - the “nucleation promoting activity titre”
(9) or by determining the numbers of crystals at suc-
cessive time points (39). Compared with the nu-
cleation time assay, these techniques may be less de-
pendent upon complete removal of microcrystals at
the start. The Cleveland group has used serial meas-
urements of photometric turbidity to assess the mass
of cholesterol crystals present in model biles after
spontaneous crystal formation, or after seeding with
cholesterol crystals (40). This assay generates “crystal
240
growth curves” which should allow differentiation be-
tween truly pronucleating, versus growth promoting
effects. Assays based on the speed of vesicular fusion
are being evaluated (41).
The Lithogenic Gallbladder is the Source of
Rapid Nucleation
Gallbladder bile from cholesterol gallstone patients nu-
cleates significantly more rapidly than hepatic bile (8).
Addition of gallbladder bile from cholesterol gallstone
patients to their own hepatic bile, or to hepatic or
gallbladder biles of non-stone formers, causes rapid
nucleation of these biles also (42). These reports indi-
cate that the stone-containing gallbladder alters bile in
a way that hastens cholesterol nucleation. Theoretic-
ally, this rapid nucleation could be secondary to the
presence of stones. However, in people who have no
gallstones gallbladder bile nucleates faster if chol-
esterol crystals are present than if they are not (26).
More significantly, Marks et al. have demonstrated re-
cently that rapid nucleation preceded crystal formation
in three patients forming gallstones during rapid
weight loss (11).
Nucleating and Anti-Nucleating Agents
In order to be implicated in cholesterol nucleation dur-
ing gallstone formation, any proposed nucleating or
antinucleating agent must not only possess such activ-
ity but must also be found in the gallbladder, in effec-
tive concentrations, at the time that crystals are form-
ing. Many agents have been identified which fulfil the
first criterion, further work is required to see if they
meet the second.
Anti-Nucleating Agents
Human bile contains agents which prevent cholesterol
crystallization. Supersaturated human biles from
people without gallstones nucleate cholesterol con-
siderably (3-40 times) more slowly than model biles of
similar lipid composition (43). Antinucleating activity
has been detected in a low molecular weight fraction
of biliary proteins (43). Apolipoproteins Al and A2,
present in this fraction (44), prolonged nucleation by
up to 2.7 fold (45). However, levels of these antinucleat-
ing apolipoproteins were no lower in gallbladder biles
from stone formers than controls (4546). This suggests
that these agents do not have a major role in regulating
nucleation. An antinucleating glycoprotein (120 kDa)
has been isolated from the gallbladder bile of normals
and stone-formers using Helix Pomatia lectin (47,48).
It prolongs the onset time of crystal detection by 60%
at concentrations well below those estimated to occur
in bile (48).

Nucleating Agents
UnspecQied biliary proteins
Gallbladder bile from cholesterol gallstone patients
contains more protein than controls (7,26,49), the
highest levels being found in patients with coexisting
crystals and stones (50). The nucleation time of
gallbladder biles from cholesterol gallstone patients
correlates with protein concentration (49). Moreover,
among people without gallstones protein concen-
trations are significantly higher (mean 3.5 vs 2.2 mg/
ml), and nucleation times are significantly shorter (117
vs 4.2 days), in gallbladder biles which contain chol-
esterol crystals than in those which do not (26). Quali-
tative differences may be important since it has been
reported that protein from gallbladder biles of chol-
esterol gallstone patients accelerates cholesterol nu-
cleation in control bile, whereas protein from control
gallbladder bile has little effect (49). Given this evi-
dence, it is perhaps surprising that the rapid nucleation
of gallbladder biles from cholesterol gallstone patients
is apparently not slowed by proteolytic treatment with
pronase (51). However, at least one potent cholesterol
nucleating protein in bile is known to be resistant to
proteolysis using pronase (52).
Gallbladder much
Gallbladder mucus is a viscoelastic gel secreted by the
gallbladder epithelium. Mucin glycoprotein is its most
characteristic feature. Increased mucin concentrations
have been reported in gallbladder bile from cholesterol
gallstone patients compared with controls in most
(46,53,54) but not all studies (55). Gallbladder mucin
promotes cholesterol nucleation in model and human
biles in vitro (39,40,56). In cholesterol supersaturated
model bile, human gallbladder mucin (2-4 mg/ml)
shortened cholesterol nucleation time by one third
(39). More impressively, mucin (2-16 mg/ml) increased
the number of crystals seen on successive days in a con-
centration-dependent manner, viz. 2327 crystals/mm3
at 4 mgml compared with 51/mm3 in controls on day
10 (39). Although lower mucin concentrations (0.2-0.5
mg/ml) have been reported in human gallbladder bile
(57) much higher concentrations, e.g. 20 mg/ml, would
be expected in the mucus gel which coats the gallbladd-
er mucosa (39). More specifically, gallbladder mucin
binds selectively to cholesterol and lecithin in vesicles
rather than mixed micelles, and promotes a 32-fold in-
crease in the number of crystals formed from them at
day 2 (58). Mucin also promoted growth of existing
crystals in model biles (59). The nucleating action of
gallbladder mucin probably depends on hydrophobic
binding sites on the non-glycosylated portion of its
Cholesterol nucleation
peptide core; proteolytic digestion impairs both lipid
binding and nucleating activity (60,61).
Although gallbladder mucin is clearly a potent chol-
esterol nucleating agent in model biles, Gallinger et al.
have provided evidence that it is not responsible for the
rapid nucleation of gallbladder bile from cholesterol
gallstone patients (56). Such bile continued to nucleate
rapidly after removing all mucin by ultracentrifugation
and ultrafiltration (56). Nor was there any correlation
between the mucin concentration in gallbladder bile
and nucleation time (55). Whether gallbladder mucin
is responsible for cholesterol nucleation during chol-
esterol gallstone formation is a separate issue which
can only be addressed by investigating the earliest
stages of the lithogenic process.
Increased gallbladder mucin secretion precedes chol-
esterol crystal formation in several animal models of
cholesterol gallstone formation (62-65). Recently, an
(18-fold) increase in mucin concentrations has also
been reported in the gallbladder bile of humans with
newly formed cholesterol gallstones (66). Despite ex-
tensive research (67-70), the cause of gallbladder mu-
tin hypersecretion during lithogenesis remains un-
known. Cholesterol crystals first appear to aggregate
within a thickened mucus gel on the gallbladder mu-
cosa (62). In humans, gallbladder sludge, a reversible
pre-gallstone phase (71) consists of cholesterol crystals
and bilirubin granules in a mesh of mucus (72). Mucin
is a constituent of the core and non-cholesterol matrix
of cholesterol gallstones (73,74). Given the weight of
circumstantial evidence, it is highly likely that
gallbladder mucin is actively involved at a number of
stages in cholesterol gallstone formation, although this
remains to be proved.
Concanavalin-A binding non-mucin glycoproteins
Groen et al. isolated a non-mucin glycoprotein (mw
approx. 130 kDa) from gallbladder bile using Con-
canavalin-A (Con-A) affinity chromatography (52,75).
It shortens nucleation time by as much as 70%. Unlike
mucin (61), its activity was resistant to proteolytic di-
gestion with pronase (52). Similar levels of activity were
present in gallbladder biles from controls and patients
with solitary gallstones, but higher levels were observed
in patients with multiple gallstones, who as a group
are more likely to form further stones (52,76). In sub-
sequent research, Offner et al. identified aminopeptid-
ase-N as a (possibly the) 130 kD Con-A binding pro-
tein in gallbladder bile, and found that it accounted
for much of the pronucleating activity of the Con-A
binding fraction (77).
Pronucleating immunoglobulins have also been
identified among the Con-A binding glycoproteins in
241
D. O’Leary
bile (78,79). They promoted nucleation in the order
IgM>IgG>IgA (79). The immunoglobulin-containing
fraction in gallbladder bile from cholesterol gallstone
patients was enriched in IgG and considerably more
potent than that from controls, especially with regard
to the number of crystals formed (78,79). Immuno-
globulins might promote nucleation by binding to
vesicle phospholipids causing vesicles to aggregate, or
by acting as autoantibodies against antinucleating
substances, but these possibilities remain speculative.
It is not clear whether they are actually involved in
cholesterol gallstone formation or whether they re-
flect an inflammatory response to gallstones or
lithogenic bile.
A 42 kDa Con-A binding glycoprotein with pro-
nucleating activity has recently been isolated from the
gallbladder bile of normals and cholesterol gallstone
patients (80). It appears to be a biliary alpha-i-acid
glycoprotein and it promotes cholesterol nucleation at
concentrations found in bile. In addition, Pattinson
described phospholipase C activity among the Con-A
binding proteins in gallbladder bile and suggested
that by degrading biliary phospholipid this enzyme
might impair cholesterol transport and promote nu-
cleation (81). Subsequently, phospholipase C has been
found to promote cholesterol nucleation in model
biles (82,83). This involves growth and fusion of chol-
esterol-phospholipid vesicles, and appears to depend
on enzymatic action (84). It is not clear whether a
pronucleating effect is likely at the concentrations of
phospholipase C found in human bile (82,83).
Abei et al. compared the non-mucin Con-A bind-
ing pronucleating glycoproteins and ranked their po-
tencies in the order IgM>IgA= alpha-, -acid glyco-
protein >IgG (85). Contrasted with other reports
(77,82,83), aminopeptidase-N, and phospholipase C
produced no effect. Removal of alpha-i-acid glyco-
protein reduced the pronucleating effect of the Con-
A binding fraction from human bile by 33%, but re-
moval of the other specified glycoproteins was with-
out effect. Interestingly, much of the pronucleating
activity of this fraction appeared to be due to as yet
unidentified agents.
Calcium
Calcium salts and ionized calcium have each been pro-
posed as cholesterol nucleating agents. Calcium salts,
principally calcium carbonate, are found in the core
and matrix of cholesterol gallstones (86). However,
crystalline calcium carbonate does not hasten chol-
esterol nucleation in model bile (87). In patients with
established cholesterol gallstones ionized calcium levels
in gallbladder bile are not significantly higher than in
242
controls (8,88). However, Shiffman et al. have reported
substantial increases in biliary ionized calcium concen-
trations in association with newly formed cholesterol
gallstones following surgery for morbid obesity (66).
High concentrations of ionized calcium promote chol-
esterol nucleation in dilute model biles (20), but there is
no evidence that physiological concentrations of Caf+
promote cholesterol nucleation in human gallbladder
bile.
Additional Potential Nucleating Agents
There have been isolated reports that a variety of other
agents promote cholesterol nucleation. Fibronectin, a
cell surface binding glycoprotein, hastens nucleation of
model biles (89). However, nucleation times of human
biles showed no correlation with fibronectin levels. Pal-
mitic, oleic and linoleic acids, which may occur in in-
fected bile, reduced the nucleation time of model biles
(90). Unlike human bile, however, nucleation was has-
tened without an increase in vesicular cholesterol satu-
ration. Polyamines in bile promote cholesterol nu-
cleation in model biles, but the concentrations required
are probably unphysiological (91). Analyses of trace
elements in bile have revealed no relationship to nu-
cleation time (92).
Other Factors Influencing Cholesterol
Nucleation in Bile
Concentration
An early increase in the gallbladder’s ability to concen-
trate bile (26,93) may promote cholesterol nucleation
during lithogenesis. Thus, nucleation is faster in chol-
esterol gallstone patients whose gallbladder bile is well
concentrated than in those with more dilute bile (94).
Among gallstone-free people, those whose gallbladder
bile contains cholesterol crystals have more concen-
trated bile and shorter nucleation times than those
without crystals (26). These findings are consistent
with the observation that concentration facilitates for-
mation of cholesterol-enriched vesicles (above).
Motility defect
Impaired gallbladder emptying is observed in a high
proportion of patients with gallstones (95,96). Experi-
ments in the cholesterol-fed prairie dog suggest that
this defect occurs early during lithogenesis inasmuch
as reduced gallbladder contractility (97) and emptying
(98) precede formation of cholesterol crystals. In-
creased periods of stasis may promote cholesterol nu-
cleation by allowing bile to become more concentrated,
and more enriched in nucleating agents produced by
the gallbladder. Stasis should also facilitate crystal
growth and aggregation.

Alterations in molecular species of bile salts and
lecithins
The role of individual molecular species of lecithins in
cholesterol transport and nucleation is unclear. Early
studies suggested that gallbladder bile from cholesterol
gallstone patients contained a decreased proportion of
linoleoyl lecithins and increased proportions of oleoyl
and arachidonyl lecithins compared with controls
(99,100). Later, in vitro work suggested that such alter-
ations influence the distribution of cholesterol between
vesicles and micelles, as well as vesicular saturation and
nucleation time (10 1,102). However, Hay et al. have re-
ported recently that ardchidonyl lecithins are no higher
in the bile of cholesterol gallstone patients than in con-
trols, which suggests that they do not, in fact, contrib-
ute to the rapid nucleation of gallstone-containing
biles (103).
An increased proportion of deoxycholate, a highly
hydrophobic bile salt, is found in the gallbladder bile
of cholesterol gallstone patients (104,105). In model
biles the cholesterol solubilizing capacity of mixed mi-
celles increases with increasing bile salt hydrophobicity
(19). Conversely, nucleation in model systems is sig-
nificantly faster when vesicles are exposed to deoxy-
cholate than to less hydrophobic bile salts (e.g. cheno-
deoxycholate, ursodeoxycholate) (106). Definitive
studies are required to determine whether the increased
proportion of deoxycholate contributes to the rapid
nucleation of lithogenic human gallbladder bile.
Current Challenges in Nucleation Research
Rapid nucleation of cholesterol is crucial for chol-
esterol gallstone formation. Identification of the deter-
minants of rapid nucleation may therefore unlock new
approaches to gallstone prevention. Gallbladder bile
from patients with established cholesterol gallstones
contains several potential nucleating and anti-nucleat-
ing agents, and more are being discovered. The import-
ant nucleating agents are probably proteins, most
likely glycoproteins. Prospective investigations are now
necessary in groups at high risk for gallstones to deter-
mine which pronucleating agents are actually present
in gallbladder bile during cholesterol nucleation.
Existing and evolving techniques should then be used
to define the relative importance of such agents in bile
and to determine whether they primarily affect vesicu-
lar aggregation/fusion (41), cholesterol crystallization
or crystal growth (40). Each of these processes needs
to be investigated further in order to describe accu-
rately the mechanism of action of nucleating and anti-
nucleating agents at a molecular level, Ultimately, it
may be possible to prevent gallstones by stimulating
production of anti-nucleating factors, by inhibiting
Cholesterol nucleation
production of nucleating factors or by interfering with
the nucleation process itself.
Acknowledgements
The author gratefully acknowledges the helpful advice
of Dr. M. Carey, MD, and Dr. W. LaMorte, MD, Bos-
ton, and of Dr. E Murray, MD, Dundee.
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