Engineering Approaches to Cholesterol-Linked Diseases
Steven P. Wrenn
Chemical Engineering
Summer, 2001
I. Introduction
Cholesterol is a Jekyll-and-Hyde molecule. On one hand, cholesterol is essential
to mammalian life. The cells of our body require cholesterol to function properly; in fact,
they cannot survive without cholesterol. This explains why the cholesterol loading of
certain cell membranes exceeds 50 mole%. On the other hand, cholesterol is lethal.
Deposits of cholesterol in atherosclerotic plaques lead to heart attacks, the number one
killer in the previous century and likely to be the number one killer in this century.
This paradoxical nature of cholesterol has fascinated scientists for more than two
centuries, yet we are still far from a complete understanding of why cholesterol is
essential for life and how cholesterol contributes to disease. This article will (with the
exception of a brief summary) leave to the cell biologists the question of why cholesterol
is vital and will focus instead on the issue of how cholesterol influences disease. Two
cholesterol-linked diseases, namely atherosclerosis and gallstone disease, will be
described from an engineering perspective. The cholesterol-link in these diseases refers
to the fact that both diseases involve formation of cholesterol crystals, which develop in
bodily fluids because cholesterol is insoluble in water. Essentially, the question of who
develops disease (i.e., who will suffer a heart attack and who will develop gallstones)
depends on how quickly cholesterol crystallizes in the body. Aside from this common
cholesterol-link, other, striking similarities will arise between the two diseases, which are
rooted in physical chemistry, thermodynamics, and chemical kinetics. Expertise in these
areas is not, however, essential to understanding the concepts to be presented herein.
II. Historical Perspectives and Background
De la Salle (1770) and de Fourcroy (1789) were the first to describe cholesterol,
after isolating a white, easily crystallizable compound from alcohol and ether extracts of
human gallstones. Chevreul later identified the compound as the major component of
gallstones and in 1816 named the substance cholesterine. Cholesterine was renamed
“cholesterol” soon after Berthelot deomonstrated (in 1859) that cholesterine was in fact
an alcohol. More than a century later, cholesterol remains one of the most widely
researched compounds.
Figure 1 shows the structure of cholesterol. An important feature of the molecule
is the aromatic ring network (i.e., the steroid rings), which is planar and relatively
conformationally inflexible. The steroid ring plus the hydrocarbon chain constitute a
region that is highly non-polar. This hydrophobic moiety dominates the hydrophilic
functionality of the single hydroxyl group of cholesterol and accounts for the extremely
low solubility of cholesterol in water (~10-8M). This aqueous insolubility provides the
driving force for cholesterol crystal formation in bodily fluids and is the root cause of
gallstone formation and is a contributing factor in atherosclerosis.
1
HO
Figure 1 – Cholesterol: The chemical structure of cholesterol is given, along with a common cartoon
representation. Cholesterol consists of essentially three parts: the first is a network of steroid rings,
denoted by the gray oval, the second is a short hydrocarbon chain, denoted as the wavy black line, and the
third is a hydroxyl group, denoted by the dark gray circle. The steroid rings, which are relatively planar
and rigid, and the hydrocarbon chain are non-polar. Together, they comprise the hydrophobic portion of
the molecule and account for the very small (~10-8M) aqueous solubility of cholesterol. Owing to the
presence of the small hydroxyl group, which is polar and therefore hydrophilic, cholesterol is, strictly
speaking, amphiphilic. This dual philicity causes cholesterol to orient itself essentially parallel to
phospholipids within the cell membrane.
A. Cholesterol Is Essential for Life
Sterols are found in most plants and animal cells, and the type of sterols used by
plants and animals are different. The most common sterols in the plant kingdom are
sitosterol and stigmasterol, whereas the most common sterol in the animal kingdom,
which includes humans, is cholesterol. That cholesterol is essential to life is clear, for
mammalian cells will not grow in the absence of cholesterol. Moreover, there is
specificity for certain sterols within each species; not any sterol will do. For example,
humans are able to readily absorb cholesterol from the diet yet are effectively unable to
absorb plant sterols (e.g., the absorption efficiency of stigmasterol is just 10% that of
cholesterol). This sterol specificity is illustrated further by the fact that swapping plant
sterols for cholesterol leads to cell death. Such observations suggest that cells require a
particular sterol for proper cellular function.
The importance of cholesterol to cellular function also becomes apparent when
one considers the biochemical, or energetic, cost of producing cholesterol. Cholesterol is
synthesized in the liver via a very long and energetically costly pathway. Nominally 30
reactions, each catalyzed by various enzymes, and 19 sterol intermediates are involved in
the conversion of acetyl-CoA to cholesterol. Such a complicated and energetically
unfavorable process, the purpose of which is to generate a single, specific sterol, is strong
evidence that cholesterol serves a vital function within cells. That cholesterol serves a
vital function is not in dispute. What remains an open question, and one which we will
leave to the cell biologists, is the nature of that function and whether it is rooted in
physical or chemical effects.
2
Before leaving that question, it is worthwhile to summarize what is known about
the physical and chemical effects of cholesterol. First, cholesterol is known to influence
the physical properties of membranes. Here again, cholesterol exhibits a schizophrenic
person(or molecule)ality. Depending on temperature, cholesterol can either make a
membrane more or less fluid. The change in fluidity arises because of the rigidity of the
steroid rings. Recall that cholesterol is conformationally inflexible. As a result, when
cholesterol is placed into a membrane, it restricts the motion of hydrocarbon chains (in
the vicinity of the steroid rings) on adjacent phospholipid molecules. By themselves,
phospholipid molecules exist in either a gel or liquid state (similar to solid or liquid states
with which you are already familiar), which is determined by a gelation temperature
(similar to a melting temperature). If the prevailing temperature is greater than the
gelation temperature, then the phospholipids exist in the liquid state. In this scenario, the
addition of cholesterol decreases the fluidity of the membrane by restricting the liquid
motion of the phospholipid hydrocarbon chains. Conversely, if the temperature is below
the gelation temperature, then the phospholipids exist in the gel state. The gel forms
because of the close packing between hydrocarbon chains. Thus, in this scenario, the
addition of cholesterol increases the fluidity of the membrane by interfering with the
packing (into a gel) of the hydrocarbon chains.
Second, cholesterol is known to exhibit specific and direct interactions with
membrane proteins. Thus, in addition to its modulation of membrane physical properties,
it is speculated that the essentialness of cholesterol stems from chemical effects. This is
demonstrated by the fact that cholesterol affects the activities of enzymes that act
adjacent to, but not within, membranes. This ability of cholesterol to stimulate or inhibit
enzyme activity cannot be due to its alteration of membrane fluidity and is attributed to a
direct (i.e., chemical recognition) interaction between cholesterol and the enzyme. The
bottom line is that cholesterol is essential for life. There is evidence to suggest that the
essential role of cholesterol is physical (i.e., alteration of membrane properties) or
chemical (i.e., recognition of and alteration of activity of enzymes). Certainly, both are
possible, but we now leave those details to our friends in cellular biology.
B. Cholesterol Contributes to Important and Widespread Diseases
Coronary artery disease is the most important cardiovascular scourge that
mankind has faced in the twentieth century. It will continue to be the leading cause of
morbidity and death in the next century, both in men and women, and in developing and
developed nations alike. Fourteen million people in the United States have coronary
artery disease, and of these one million develop an acute coronary event, and 400,000 die,
each year. Although less morbid, gallstone disease afflicts 12% of the adult US
population, and annual medical expenses relating to gallstones exceed $2 billion.
Although several non-surgical treatments remove stones temporarily (e.g., lithotripsy,
bile salt therapy, and solvent instillation), the only permanent cure for gallstone disease is
surgical removal of the gallbladder. The number of laparoscopic cholecystectomies (i.e.,
the operation to remove the gallbladder) performed each year exceeds 500,000.
The common link between these two widespread diseases is precipitation of
cholesterol crystals from bodily fluids, owing to the extremely low solubility (i.e., 10-8
3
M) of cholesterol in water. Considering atherosclerosis, cholesterol crystals are
recognized as a hallmark of advanced atherosclerotic plaques, and numerous studies
confirm the existence of cholesterol monohydrate crystals within the lipid core of
plaques. Similarly, cholesterol monohydrate crystals are the primary component in
cholesterol gallstones, which account for more than 75% of all gallstones. Given the low
solubility of cholesterol in water and the presence of crystals in disease, an interesting
question arises; namely, why do just certain individuals develop gallstones and why do
only some people suffer heart attacks?
At first glance, the answer might appear simple; people with gallstones and
people who get heart attacks must have abnormally high levels of cholesterol. While the
level of cholesterol does play a role, it does not explain why not everybody develops
cholesterol-related diseases, since the cholesterol level is well above the saturation limit
in (nearly) everyone. To answer the question one must recognize that humans are
inherently non-equilibrium beings. So, the fact that cholesterol crystals constitute an
equilibrium phase in water does not guarantee that cholesterol will precipitate in the body
(a happy fact, which explains why most people do NOT suffer heart attacks at a young
age and why most people do NOT develop gallstones). Precipitation of cholesterol
crystals, and hence disease, occurs only if the rate of cholesterol crystallization (or more
correctly, cholesterol nucleation) is sufficiently rapid. In healthy individuals, the rate of
cholesterol precipitation is so slow that cholesterol is cleared from problematic areas
before crystals have a chance to grow and accumulate. However, the rate of cholesterol
crystallization is fast enough in diseased individuals that crystals appear, accumulate, and
contribute to disease.1
The dependence of disease on the cholesterol crystallization (nucleation) rate is
encouraging, for it suggests the possibility that the diseases can be prevented by
controlling the cholesterol nucleation rate. Turning this possibility into a reality first
requires a detailed understanding of the cholesterol nucleation mechanisms within the
contexts of gallstone formation and heart disease. Unfortunately, very little is known
about the molecular details of cholesterol nucleation from membranes. What is known is
that gallstone cholesterol nucleates from thermodynamically metastable lecithincholesterol vesicles, atherosclerotic plaque cholesterol nucleates from low density
lipoproteins, and there are striking similarities in the physical chemistry associated with
the two systems. Perhaps most striking is the observation that the appearance of crystals
in bile and in plasma is nearly always preceded by aggregation of the vesicles and
lipoproteins, respectively. Moreover, the rate of nucleation from vesicles and
lipoproteins is insufficiently rapid to yield crystals, regardless of the level of cholesterol
supersaturation (provided the value is in the physiologically meaningful range), unless an
aggregation-inducing factor is present. We now take a closer look at each of the two
cholesterol-linked diseases and will examine the striking similarities that emerge.
1
This is definitely the case in gallstone formation, since cholesterol crystals are the precursors to
stones. The situation is less clear in the case of atherosclerosis. Crystals are definitely a part of the plaque
that forms, and are believed to influence the likelihood that the plaque will trigger a heart attack, but this
has not yet been proven.
4
III. Gallstones
A. Bile – The “Water” from which Gallstone Cholesterol Precipitates
Gallstones come in two varieties, cholesterol stones and pigment stones, but
greater than 75% of all stones are cholesterol stones. Cholesterol stones are aggregates of
cholesterol crystals that form in an aqueous fluid called bile, and arise because of the
insolubility of cholesterol in water. The naming of cholesterol, a molecule that was first
isolated from gallstones, reflects this fact (Gr.chole, bile; stereos, solid). Cholesterol
crystal formation is expected when bile becomes supersaturated with cholesterol, a
condition that exists in nearly all individuals. However, just eight percent of the
population develops stones. This seeming paradox raises two important questions:
1) How does any individual with a supersaturated cholesterol level avoid stone
formation? and 2) What factors determine whether an individual develops stones? The
answers to these questions involve the presence of other species in bile that aid in the
solubilization of cholesterol.
Bile is an aqueous fluid, secreted by the liver and stored in the gallbladder, the
purpose of which is to aid in the digestion of fats. Bile contains the following five primary
solutes: sterols, phospholipids, bile salts (of which there are several species), proteins, and
pigments. Table 1.1 gives the relative content of these solutes in the bile of an average
human. Lecithin, or phosphatidylcholine, accounts for more than 95% of the phospholipid
species in bile, and cholesterol accounts for 90% - 95 % of all sterols. The total solute
concentration of fresh bile, which is secreted by the liver, is approximately 3 g/dL. Upon
secretion by the liver, bile travels to the gallbladder for storage between meals, and water
uptake by the gallbladder concentrates the bile solids to approximately 10 g/dL.
Table 1.1
Composition of Average Human Gallbladder Bile
Composition
Water
Cholesterol
Lecithins
Bile Salts
Proteins, Pigments
Weight %
88
1
2
8
1
Bile is an important biological fluid that serves two main purposes. One is the
elimination of excess cholesterol, since hepatic secretion of cholesterol into bile, either
directly or indirectly after conversion to bile salts, provides the only excretion pathway for
cholesterol from the body. The other purpose of bile is to digest fat. After a meal, the
presence of food in the intestines triggers a hormonal response that results in contraction of
the gallbladder and expulsion of bile into the intestines.
The ability of bile to digest fats in the intestine stems from the action of biological
surfactants, molecules with both hydrophobic and hydrophilic moieties. Surfactants act
at the interface between polar and non-polar environments (i.e., they are surface active
5
agents) and provide a means of solubilizing non-polar substances in water. Bile salts and
lecithins are biological surfactants in bile that solubilize cholesterol, effectively
increasing the cholesterol solubility nearly a million-fold over its inherent aqueous
saturation limit. The dissolution of cholesterol in bile occurs inside microscopic
aggregates (to be considered shortly) of the lecithins and bile salts. The type of
aggregate, and the cholesterol solubilizing capacity of the aggregate, differs for lecithin
and for various bile salt species, and examination of the individual molecules in bile
accounts for the differences. The structure of cholesterol was considered in Figure 1 and
is repeated in Figure 2 (a) for comparison with these other biliary molecules.
Figure 2 - Molecular Structures and Cartoon Representations of the Lipid Species in Bile: (a)
Cholesterol, (b) Sodium Cholate, a common bile salt, and (c) Lecithin.
Bile Salts: Bile salts (salts of bile acids) share the ring network structure of cholesterol, but
the hydrocarbon chain of bile salts is shorter than that of cholesterol and terminates in a
carboxylic acid group (Figure 2b). Conjugation of the bile acid with one of two amino
6
acids, either taurine (NH2CH2CH2SO3H) or glycine (NH2CH2COOH), typically occurs
prior to secretion into bile. This prevents detrimental protonation at physiological
conditions (pH < 7.4) that would make the bile salts only sparingly soluble in the biliary
pathway.
Bile salts vary in the number, position, and orientation of hydroxyl groups. In
most cases the hydroxyl groups reside at carbon positions 3, 7, or 12 (see Figure 2b).
However, the orientation of a hydroxyl group at a given carbon position can be either below
() or above () the plane of the bile salt molecule. Moreover, the orientation can be either
equatorial (in the plane) or axial (out of the plane) with respect to the ring on which the
carbon resides. An  hydroxyl group at carbon number 3 is equatorial, whereas an 
hydroxyl group at either carbon number 7 or carbon number 12 is axial. Conversely, 
hydroxyl groups are axial at carbon number 3 and equatorial at carbons 7 and 12.
The two primary bile salts in humans, chenodeoxycholate and cholate, are
dihydroxyl and trihydroxyl bile salts, respectively. Both contain the 3 hydroxyl group of
cholesterol plus a hydroxyl group at position 7 on the steroid backbone. The third
hydroxyl group on cholate resides at position 12. The bile salts lithocholate and
deoxycholate form via 7 dehydroxylation of chenodeoxycholate and cholate, respectively,
and constitute the “secondary” bile salts. The dehydroxylation occurs inside the intestine
and is the result of bacterial action.
In the case of chenodeoxycholate, 7 dehydrogenation is also possible and
produces the intermediate compound 7-oxo-lithocholate. Bacterial reduction of this oxointermediate reproduces chenodeoxycholate, the 7 parent compound. However, a second
possibility is the formation of a 7 epimer called ursodeoxycholate. The  hydroxyl group
provides ursodeoxycholate with a decreased hydrophobicity relative to the other bile salts,
resulting in a greater capacity to dissolve cholesterol. Ursodeoxycholate is therefore the
bile salt of choice in bile salt therapy for the dissolution of gallstones.
With the exception of ursodeoxycholate, the multiple hydroxyl groups of most
bile salts adopt orientations that are either all  or all . Along with the carboxylic acid
group, the hydroxyl groups constitute the so-called hydrophilic “face” of the bile salt
molecule. Since the steroid ring system is hydrophobic, bile salts are amphiphilic and
therefore surface active. The hydrophilicity and surface activity of bile salts, and hence the
ability to dissolve cholesterol, varies with the number, position, and orientation of hydroxyl
groups.
Lecithins: Bile contains a variety of phospholipids, including phosphatidylcholines
(lecithins), phosphatidylethanolamines (cephalins), and sphingomyelins. Lecithins (Figure
2c) are the predominant phospholipid species, i.e., glycerolipids in which the substituents
on the acyl carbons are aliphatic hydrocarbon chains and the substituent on the C(3)
hydroxyl group of glycerol is phosphocholine. Typically, a saturated palmitoyl chain
resides at position sn-1 on the glycerol backbone, while an unsaturated oleoyl or
arachidonyl chain occupies the position at sn-2. The chain length and degree of
unsaturation have a significant impact on the physical properties of the lecithins; most
notable is the decrease in chain melting temperature with increased unsaturation.
The dual chains are hydrophobic and constitute the “tail” portion of the
molecule. On the other hand, phosphocholine is hydrophilic and constitutes the
“headgroup” of the lecithin molecule. The hydrophilicity stems from the fact that
7
phosphocholine, while electrically neutral, is zwitterionic. Lecithins are therefore
amphiphilic and, like bile salts, serve as surfactants in bile. A key difference between
lecithin and bile salt surface activity, however, is the type of aggregate each forms. The
lecithin aggregate, called a “vesicle”, is thermodynamically unstable but relatively longlived under normal conditions. Temporary partitioning of cholesterol into the lecithin
aggregate explains the existence of supersaturated bile in healthy individuals.
Pigments and Proteins
Although they account for just a small portion of the total biliary solids content,
pigments and proteins are suspected to play a major role in gallstone pathogenesis. For
example, although gallstones are comprised primarily of cholesterol, the core of most
cholesterol stones is calcium bilirubinate, the calcium salt of the pigment bilirubin.
Bilirubin, along with biliverdin, accounts for the characteristic greenish-yellow color of
bile.
Moreover, a variety of biliary proteins act to either accelerate or inhibit
cholesterol crystal formation. Those proteins that accelerate crystal formation, the “pronucleators”, include mucin and other glycoproteins, concanavalin A-binding proteins,
immunoglobulins, aminopeptidase N, fibronectin, and phospholipase C. Proteins that
inhibit cholesterol crystal formation, the “anti-nucleators”, are apoproteins A-I and A-II.
Albumin, the most prevalent protein in bile, is neither a promoter nor an inhibitor of
cholesterol crystal formation in native or model bile.
It is hypothesized that the relative balance of pro- and/or anti-nucleating
proteins in bile ultimately determines who develops gallstones, but the mechanisms by
which these proteins act are largely unknown. The proteins likely impact the stability and
cholesterol-solubilizing capacity of the lecithin and/or bile salt aggregates present in bile.
B. Surfactant Aggregates in Bile – The Temporary “Carriers” of Cholesterol
(a)
(b)
Figure 3 -Bile Salt Microstructures: (a) Primary Micelle - A grouping of up to ten bile salt monomers in
which the steroid rings are oriented radially inward. The hydrophilic hydroxyl (filled circles) and carboxylic
acid groups (open circles) face outward and are in contact with bulk water. (b) Secondary Micelle - Primary
micelles aggregate to form a larger micelle microstructure.
8
Bile Salt Micelles: Surfactants self-assemble in water to maximize the extent of
hydrogen bonding between water molecules. Self-assembly, which is driven by the
hydrophobic effect, minimizes contact between polar and non-polar regions and gives
rise to interesting surfactant aggregates, or microstructures. For example, bile salts form
globular micelles (Figure 3a) at concentrations slightly exceeding a monomeric solubility
limit termed the critical micellar concentration (CMC). Bile salt CMC values typically
fall in the range 1.6 mM to 15 mM and increase with the number of hydroxyl groups.
Globular bile salt micelles comprise up to ten molecules, each oriented with the
hydrophilic moiety facing outward to shield the aromatic region from water. These
primary micelles aggregate further with increasing concentration into so-called secondary
micelles, which are elongated clusters of the globular micelles (Figure 3b).
Lecithin Vesicles: Whereas bile salt is soluble in water to a CMC, lecithin is insoluble in
water and swells to form alternating stacks of lecithin sheets (called lamellae) and water.
Each lecithin sheet is two molecules thick (a bilayer), in which acyl chains stack end-toend and phosphocholine headgroups point outward in contact with water (Figure 4a).
The sheets ‘wrap up’ spontaneously into spherical particles called liposomes.
In addition to multi-lamellar liposomes, it is possible to obtain uni-lamellar
liposomes called vesicles. Figure 4b illustrates the structure of a lecithin vesicle, which is a
spherical, single-bilayer, closed shell that encapsulates an aqueous interior. Such
unilamellar vesicles typically range from 20 to 150 nm in diameter, although it is also
possible to make much larger, so-called giant vesicles. Although they form spontaneously
in vivo, vesicles rarely form in vitro without the input of significant mechanical energy.
Typical means of vesicle preparation include sonication, high pressure filtration, detergent
dialysis and reverse phase evaporation. The stability of vesicles formed by any of these
methods is limited because multi-lamellar liposomes or flat, lamellar sheets are the
equilibrium form of aggregation. Thus, vesicles are thermodynamically metastable and
revert to multi-lamellar liposomes with time. Geometric arguments explain the nonequilibrium nature of lecithin vesicles. Simply put, lecithin molecules are cylindrical and
prefer to form flat bilayer sheets or large, multi-lamellar liposomes with little curvature.
The metastability of small, unilamellar vesicles therefore results from excessive curvature
of the lecithin bilayer.
In molecular terms, there exists an optimal area per headgroup, ao, for any
given surfactant, the value of which is set by two opposing forces. An attractive force,
driven by the hydrophobic effect, favors self-assembly and competes with a steric or
electrostatic repulsive force between headgroups. The hydrophobic effect minimizes free
energy by eliminating contact between non-polar and polar regions, giving rise to the
formation of a new phase (i.e., an infinite aggregate) in aqueous mixtures. However,
aggregates of finite size form in mixtures of water and surfactant, and a dimensionless
group termed the packing parameter, P, determines the size and shape of the resulting
aggregates.
The packing parameter is the ratio of surfactant tail volume, vT, to the product
of extended chain length, lc, and optimal headgroup area: P = vT/(lcao). Lecithin is a
double tailed surfactant with a neutral headgroup, and the optimal headgroup area is
sufficiently small that the packing parameter is approximately one. The shape of a lecithin
9
Figure 4 - Lecithin Microstructures: (a) Stacked lamellae - Bilayers of lecithin are stacked vertically upon
one another and separated by a thin film of water. Lecithin molecules are oriented such that their hydrophilic
head groups protect the acyl chains from the aqueous region. (b) Unilamellar vesicle - A metastable
microstructure consisting of a spherical lecithin bilayer that encloses an aqueous core.
molecule is therefore cylindrical so that the equilibrium state of lecithin aggregation is a flat
bilayer. Formation of closed spherical vesicles requires the surfactant shape to approximate
a truncated cone, which is the case for packing parameter values in the range 1/2 < P < 1.
The discovery of equilibrium vesicles that form spontaneously from mixtures of cationic
and anionic surfactants supports this claim.
At physiological water:lecithin molar ratios, the lecithin lamellae adopt a
spherical configuration to minimize contact of hydrocarbon chains and water (i.e., to
eliminate edge effects). The resulting aggregates, called multi-lamellar liposomes, form by
simply hydrating lecithin in excess water. Bangham first demonstrated liposome formation
10
in 1965, and utilization of liposomes as models for cell membranes is now a common
practice.
Bile Salt-Lecithin Mixed Micelles: It is also possible to form aggregates comprised of both
bile salt and lecithin. Small angle neutron scattering (SANS) studies revealed that the
microstructure of such “mixed micelles” containing bile salt and lecithin is rod-like. Figure
5a shows the rod-like mixed micelle architecture, in which lecithin molecules are aligned
radially with acyl chains protruding into the interior of the rod and phosphocholine
headgroups coating the exterior. Bile salt molecules lie on the surface of the rod with the
steroid rings sunken into the oily interior, and hydroxyl groups and carboxylic acid groups
face outward in contact with water.
What is remarkable about bile salt-lecithin mixed micelles is that they are
equilibrium structures. They form spontaneously and do not aggregate; they are stable over
long periods of time. Moreover, due to the non-polar environment they provide, bile saltlecithin mixed micelles accommodate nearly 10 mole% cholesterol on a lipid basis. The
otherwise insoluble cholesterol partitions into the oily interior of the rod, thereby avoiding
contact with water. The resulting bile salt-lecithin-cholesterol micelle is thus a “carrier” of
cholesterol in bile.
The Cholesterol Carriers
For decades it was believed that the bile salt-lecithin-cholesterol mixed micelle
was the only cholesterol carrier in bile. This belief led to a simple hypothesis concerning
gallstone formation that is based solely on thermodynamics, namely that healthy
individuals possess enough bile salt to fully dissolve cholesterol in the form of micelles,
whereas in diseased individuals cholesterol cannot be fully dissolved and results in stones.
The latter scenario might be attributed to excessive cholesterol levels, deficient bile salt
levels, or both. However, the observation that the bile of many healthy individuals contains
cholesterol in amounts greater than can be carried by micelles alone contradicted this
hypothesis.
The discovery of lecithin-cholesterol vesicles as a second carrier of cholesterol
in bile explained the existence of non-micellar cholesterol in healthy individuals and shed
new light on the mechanism for gallstone formation. Such lecithin-cholesterol vesicles
accommodate a cholesterol:lecithin molar ratio as high as 2:1 and increase the solubility of
cholesterol in bile by nearly a million fold. A lecithin-cholesterol vesicle is therefore much
more efficient at dissolving cholesterol than the bile salt-lecithin mixed micelle and is the
predominant cholesterol carrier in bile. Figure 5b shows the lecithin-cholesterol vesicle, in
which cholesterol resides parallel to lecithin inside the vesicle bilayer with the hydroxyl
group protruding into the lecithin headgroup region.
Like the parent lecithin vesicle, lecithin-cholesterol vesicles are
thermodynamically metastable and revert to a lamellar phase with time. A key distinction
between lecithin vesicles and lecithin-cholesterol vesicles, however, is the fate of
cholesterol at equilibrium. The maximal cholesterol:lecithin molar ratio in the lamellar
phase of lecithin-cholesterol mixtures is unity, and any excess vesicular cholesterol
nucleates into a cholesterol crystalline phase at equilibrium. The cholesterol crystals are
the precursors to gallstones.
11
(a)
125 A
24 A
(b)
Water
Core
~50 nm
Figure 5 - Cholesterol Carriers in Bile: (a) The rod-like microstructure of the bile salt-lecithin mixed
micelle and (b) the unilamellar lecithin-cholesterol vesicle.
12
C. Phase Behavior Versus Kinetics
Although the kinetics of cholesterol nucleation, rather than the mere fact that
cholesterol crystals represent an equilibrium phase, determine whether an individual
develops gallstones, it is worthwhile to examine the equilibrium phase behavior of human
bile. This is because the kinetics of nucleation depend, at least in part, on the
thermodynamics of the process. Figure 6 shows what appears to be the ternary phase
diagram for a lecithin-cholesterol-bile salt system. However, the real system of interest
includes water, and Figure 6 is in fact a horizontal slice, at ~80% water, through the phase
tetrahedron (see inset). This solute concentration approximates that of physiological bile,
and Figure 6 is therefore a phase map that shows the “pseudo-phase behavior” in the fourcomponent system water- lecithin-cholesterol-bile salt. Traditional phase rules do not
apply in such a phase map. For example, the crystals in the large three-phase region of the
diagram are cholesterol monohydrate crystals rather than the pure cholesterol crystals that
would be expected if Figure 6 was a phase diagram.
The utility of the bile phase map is to evoke the kinetic basis of gallstone
formation. The apexes in Figure 6 denote pure components, and any point within the
triangle represents the composition (in mole%) of the three solutes. Four zones emerge
from the diagram, three of which contain bile salt-lecithin-cholesterol rod-like micelles and
lecithin-cholesterol lamellae and/or cholesterol monohydrate crystals. In addition to these
multi-phase regions, a single-phase region that consists of rod-like micelles exists along the
majority of the bile salt-lecithin edge of the diagram. The micelles in this region are the
classical carriers of cholesterol in bile, and the region itself is of historical importance
because it defines the classical limit of cholesterol solubility.
Carey and Small determined experimentally the micellar phase boundary as a
function of total solute concentration, solute composition, temperature, bile salt type, and
NaCl concentration. These researchers fit the micellar solubility data with fifth order
polynomials and tabulated the maximal mole percentage of cholesterol that can be
dissolved solely by the micellar phase. They defined a Cholesterol Saturation Index (CSI)
as the actual cholesterol mole percentage in a given bile sample divided by the maximal
mole percentage of cholesterol that can be dissolved in micelles. Thus was born the
thermodynamic hypothesis for gallstone formation, namely that any bile with a CSI greater
than unity will, owing to cholesterol supersaturation, generate cholesterol crystals at
equilibrium and hence yield stones. In the context of the bile phase map (Figure 6) this
argument states that the compositions of healthy biles lie within the one-phase micellar
zone, whereas diseased biles fall in a multi-phase zone that contains cholesterol crystals. In
short, no crystals means no stones.
The CSI was a poor predictor of gallstone formation, for it was soon realized
that the majority of healthy biles possess a CSI greater than unity. Thus, cholesterol
supersaturation is a necessary but insufficient condition for gallstone formation, and
thermodynamics alone cannot predict the occurrence of gallstones. The recognition that
bile is not at equilibrium within the body explains this failure of thermodynamics. Bile is a
metastable liquid, and kinetic factors must play a role.
13
Figure 6 - Kinetic Versus Thermodynamic Basis For Gallstone Formation: A phase map of bile is
shown for a solute concentration of ~20 g/dL. Cholesterol-enriched vesicles are metastable and do not appear
within the equilibrium phase triangle. However, when vesicles mix with bile salts in the gallbladder, the
resulting bile composition falls somewhere within the triangular composition space. Gallstones require
cholesterol crystals, and the question of who develops stones was originally believed to be a simple matter of
thermodynamics: The bile composition of healthy individuals was assumed to fall within the one-phase
micellar region (A), whereas the bile composition of diseased individuals was assumed to fall in a multi-phase
region that includes cholesterol crystals (B). It is now known that the bile composition of nearly all
individuals is such that cholesterol crystals are expected at equilibrium. The question of who develops stones
is therefore a matter of kinetics: Stones occur in individuals for whom the rate of cholesterol nucleation from
lecithin-cholesterol vesicles is sufficiently rapid to generate crystals during the short residence time of bile
within the biliary tract.
Kinetic Basis of Gallstone Disease: The liver secretes cholesterol in the form of lecithincholesterol vesicles and secretes bile salts by an independent pathway. Since they are nonequilibrium structures, vesicles do not appear on the bile phase map of Figure 6. After
secretion by the liver, lecithin-cholesterol vesicles encounter bile salts within the
gallbladder, and the interaction with bile salt induces mixed micelle formation. If sufficient
bile salt is present to fully micellize the vesicles, then attainment of equilibrium places the
bile within the single-phase region of the phase map (Path A). This is seldom the case,
however, and micellization of vesicles is typically incomplete. Moreover, the micellization
process favors transport of lecithin over cholesterol (by a factor of four) so that the
remaining vesicles are enriched in cholesterol relative to the hepatic vesicles. Subsequent
14
equilibration of the cholesterol-enriched vesicles then yields both a lecithin-cholesterol
lamellar phase and cholesterol monohydrate crystals, the precursors to stones (Path B).
Supersaturated lecithin-cholesterol vesicles are thus the source of gallstone
cholesterol, and the presence of such vesicles in the bile of nearly all individuals indicates
that gallstone formation is a matter of kinetics. Specifically, gallstone formation depends
on the rate at which metastable lecithin-cholesterol vesicles attain equilibrium and expel
excess cholesterol. Studies involving video-enhanced microscopy indicate that the
transition from vesicles to crystals proceeds by a multi-step mechanism consisting of
vesicle aggregation and fusion, cholesterol nucleation, and crystal growth, and the rates of
the individual steps depend on a variety of physiological factors.
D. Overall Gallstone Pathway
Figure 7 summarizes the above discussion of gallstones in terms of a five-step
mechanism for gallstone formation. First, the liver secretes cholesterol in the form of
metastable lecithin-cholesterol vesicles and secretes bile salt micelles by an independent
pathway. The vesicles and micelles migrate to the gallbladder, where mixing and water
absorption induce a vesicle-to-micelle transition. The transition to micelles is incomplete
and leaves vesicles that are enriched in cholesterol. The cholesterol-enriched vesicles then
aggregate and fuse to form larger, multi-lamellar liposomes. Through an unknown
mechanism, perhaps including lateral phase separation of cholesterol within a single bilayer
and/or alignment of cholesterol domains among multiple bilayers, excess cholesterol
nucleates from the aggregated vesicles. Finally, growth of the nascent micro-crystalline
phase into macroscopic crystals leads to gallstones.
The above pathway holds for all individuals, but the speed with which it occurs
varies. The primary reason for the variation in speed relates to differences in the types and
concentrations of certain factors in bile that alter the kinetics of nucleation. Some factors,
termed “pro-nucleators” increase the rate of nucleation, while others, termed “antinulceators,” slow the nucleation rate. The pro-nucleators typically act by increasing the
rate of vesicle aggregation. Noteworthy among the list of pro- and anti-nucleating factors
in bile are the apoproteins (e.g., A-I) and oxysterols (cholestan-3,5,6-triol). Apo A-I,
which is present in bile as an intact peptide, is the predominant protein associated with
HDL in the bloodstream. The ability of this human plasma lipoprotein to inhibit gallstone
formation might be related to its ability to solubilize and transport serum lipids. Thus, there
is a potential link between the protective effect of A-I in the bloodstream (i.e., inhibiting
atherosclerosis) and the anti-nucleating effect of A-I in bile (i.e., inhibiting gallstone
formation). Similarly, it is now well established that oxidation of cholesterol is an
important factor in the pathogenesis of atherosclerosis, and many studies confirm the
presence of oxysterols in the bloodstream and atherosclerotic plaques. Moreover, foam cell
formation from the macrophage is considered a hallmark of the atherosclerotic lesion, yet
macrophages metabolize LDL at a rate that is insufficient to produce foam cells. Generation
of foam cells requires oxidized LDL, the uptake of which proceeds via the scavenger
receptor. It is possible, if not likely, that the role of oxysterols in bile might also be related
to the function of these compounds in the bloodstream. We now turn our attention to
consider these details of atherosclerosis more closely.
15
Figure 7 - Overall Gallstone Pathway: Gallstone formation proceeds by a five-step sequence. 1) The
liver secretes lecithin-cholesterol vesicles and bile salt micelles via independent pathways. 2) Vesicles and
micelles travel to the gallbladder, where mixing and water uptake occur. 3) Bile salts partially dissolve
vesicles, giving rise to rod- mixed micelles. The preferential transport of lecithin into the micelles leaves
cholesterol-enriched vesicles. 4) Metastable, cholesterol-enriched vesicles revert to an equilibrium lamellar
phase and nucleate excess cholesterol in the form of microscopic, plate-like crystals. 5) Cholesterol
crystals grow and ultimately yield gallstones.
16
IV. Atherosclerosis
A. The Nucleation is from Blood, not Bile, but is Similar to Gallstones Nonetheless
Coronary artery disease is the most important cardiovascular scourge that
mankind has faced in the twentieth century. It will continue to be the leading cause of
morbidity and death in the next century, both in men and women, and in developing and
developed nations alike. The management of coronary artery disease has almost always
been based on demonstration of the severity of luminal stenosis (i.e., progressive
narrowing of arteries) using such techniques as angioscopy, intravascular
ultrasonography, and optical coherence tomography. This approach does not consider
plaque morphology, which is the major determinant of clinical outcome. An alternative,
and more desirable, approach is to identify those atherosclerotic lesions (i.e., unstable
plaques) with the morphological characteristics associated with an increased risk of
clinical events.
Fourteen million people in the United States have coronary artery disease.
Whereas identification of each one of them might seem ideal, it is most desirable to
identify that subset of patients at greatest risk of developing an acute coronary event.
This is because coronary atherosclerosis without thrombosis is generally benign;
coronary lesions occur in most individuals after just two decades of life, and mature
plaques merely narrow the arteries and limit arterial blood flow. It is now well
recognized that progressive luminal stenosis of the coronary artery is often not associated
with an acute event, and atherosclerotic plaques become life-threatening only if they
rupture, leading to arterial occlusion and heart attack. Thus, of the fourteen million
people with coronary artery disease, just one million develop an acute coronary event
(although 400,000 die) each year.
An important question then arises, namely why do certain atherosclerotic plaques,
which are otherwise stable for many years, suddenly rupture and lead to loss of life?
Recent observations indicate that plaque composition is a key factor. As the name
suggests, “atherosclerotic” plaques consist of two primary moieties: a soft, lipid-rich
“atheromatous” core and a hard, collagen-rich “sclerotic” cap. However, the relative
amounts of these two moieties, along with the composition within each moiety, vary from
plaque to plaque. Plaques with large lipid cores and thin fibrous caps are the most
susceptible to rupture and are deemed unstable. Thus, identification of those patients at
risk of an impending heart attack merely requires detection of the unstable plaques.
Now what is very interesting is that recent evidence indicates that plaque stability
correlates with the amount of cholesterol crystals in the plaque. Hence, the question of
who will suffer a heart attack also relates to the kinetics of cholesterol nucleation!
Moreover, the nucleation of cholesterol in atherosclerosis also involves “carriers” of
cholesterol and the same chemical species as in bile (i.e., phospholipids, apoproteins, and
oxysterols). These are just two of the striking similarities between these two cholesterollinked diseases.
17
B. The Atherosclerosis “Pathway”
Atherosclerosis results from a complex interplay between components of
the blood vessel wall and components within the blood. The process begins with the
development of atherosclerotic lesions, an immunoinflammatory response of the intima
(i.e., the inner lining of the blood vessel wall) to injury. The injury is initiated by
oxidized low density lipoproteins (LDLs, i.e., “bad” cholesterol) that permeate through
the endothelium (i.e., the outer lining of the blood vessel wall) and lead to recruitment of
monocyte-derived macrophages. The interaction of endothelial cells, macrophages, and
oxidized lipids leads to proliferation of smooth muscle cells, which, along with the
macrophages, migrate through the endothelial layer. Once in the intima, macrophages
and smooth muscle cells ingest LDLs and evolve into so-called foam cells. Collections
of the lipid-filled foam cells appear macroscopically as yellowish “fatty streaks,” which
are the precursors to plaques.
Maturation of the early lesions into atherosclerotic plaques involves three
processes: 1) continued recruitment, migration, and proliferation of macrophages and
smooth muscle cells within the intima, 2) formation of a connective tissue matrix by the
smooth muscle cells that comprises fiber proteins, collagen, and proteoglycans, and 3)
accumulation of lipids within the macrophages and smooth muscle cells and also in the
surrounding extracellular matrix. The latter processes determine the vulnerability of
plaques to rupture, since the extent of connective tissue affects the integrity of the fibrous
cap and the amount and type of accumulated lipids set the size of the lipid core.
The properties of unstable plaques that make them susceptible to rupture include a
large lipid core and a thin fibrous cap. Of the constituents in the plaque (which include
proteins in the cap region and macrophages or lipids in the core region), the main
component is the large lipid pool in the core. This is because LDLs transport cholesterol
primarily in the form of cholesteryl esters, which is contained within the LDL lipid core.
There is mounting evidence that cholesterol monohydrate crystals constitute a significant
fraction of the plaque core, although the origin of the crystalline cholesterol (i.e., free
cholesterol or deesterified cholesteryl esters) is unknown.
C. The Carriers of Cholesterol in Blood
Cholesterol is commonly mentioned in the media, and the layman might be
familiar with the terms “good” and “bad” cholesterol. Actually, cholesterol is a
molecule; there are no two kinds of cholesterol, let alone good or bad. The terms “good
cholesterol” and “bad cholesterol” refer to the type of cholesterol carrier in the
bloodstream. Just as vesicles and micelles transport cholesterol through bile, there are
similar particles that transport cholesterol in blood. Unlike the carriers in bile, in which
vesicles and micelles are fundamentally different microstructures, the carriers in blood
are all structurally similar. These carriers, termed lipoproteins, are essentially spherical
in shape. Each consists of a lipid (rather than aqueous in the case of a vesicle) core of
cholesteryl esters. Since the core is lipid, the lipoprotein contains just a single
(mono)layer (rather than a bilayer as in the case of a vesicle) of phospholipid and
18
cholesterol. Embedded in the monolayer is a protein (hence the name lipoprotein),
which, like the surfactants you’ve already learned about, is amphiphilic. The
hydrophobic portion of the protein rests in the lipid monolayer where it is shielded from
water, and the hydrophilic portion faces outward in contact with bulk water (or in this
case blood plasma).
The lipoproteins in blood are distinguished based on two properties; one is the
relative amounts of lipid core, monolayer, and protein and another is the type of protein
contained in the monolayer. The first of these properties affects the density of the carrier
particle and gives rise to the name of the particle. Hence the carriers of cholesterol in
blood, given in order of increasing density, are the Very Low Density Lipoprotein
(VLDL), Intermediate Density Lipoprotein (IDL), Low Density Lipoprotein (LDL), and
High Density Lipoprotein (HDL). The primary proteins associated with each particle are
C-I & C-II, C-III & E, B-100, and A-I & A-II, respectively.
Although we are not interested in the details here, the different carriers play
different roles in cholesterol transport. Essentially, LDL delivers cholesterol to the cells,
and HDL is involved in the so-called reverse transport of cholesterol from the cells.
Because crystalline cholesterol, which contributes to atherosclerosis, orginates from LDL
this particle (not the cholesterol molecule itself) is termed “bad cholesterol.” Conversely,
HDL plays a protective role by scavenging free cholesterol for return the liver.
Accordingly, HDL is termed “good cholesterol.”
Aside from structural and chemical similarities, LDL is similar to hepatic vesicles
in that LDL aggregation is necessary to induce cholesterol nucleation. Oxidatively
modified LDL is suspected to play an important role in the development of
atherosclerosis, and LDL oxidation products, which have been shown to induce
aggregation of LDL particles, are prevalent in human lesions. Moreover, sphingomyelin
hydrolysis, caused by the enzyme sphingomyelinase, yields ceramide and choline
phosphate. Generation of the former induces LDL aggregation and elevates the molar
ratio of cholesterol to phospholipid in the LDL monolayer.
D. The Source of Nucleated Cholesterol: Directly from LDL or Macrophage or Both?
Cholesterol crystals are recognized as a hallmark of advanced atherosclerotic
plaques. Numerous studies confirm the existence of cholesterol monohydrate crystals,
which are presumed to play an important role in cell disruption and necrosis, within the
lipid core of plaques. Moreover, recent studies point to the importance of crystalline
cholesterol as a determinant of plaque morphology, which has been demonstrated to be
directly related to the incidence of plaque rupture and thrombosis. The formation of
cholesterol crystals in atherosclerosis is therefore an important issue in vascular biology
that desperately needs to be addressed.
19
Figure 8 – Cholesterol Nucleation from LDL, the Carrier of Cholesterol in Blood: A low density
lipoprotein (LDL) serves as the carrier of cholesterol from the liver to cells. The LDL particle is quite
similar to the lecithin-cholesterol vesicle, in that it is spherical and consists of an outer lecithin-cholesterol
monolayer. The primary difference between LDL and vesicles is that LDL contains a lipid (cholesteryl
ester) core and is draped by a protein (Apo B). It is known that LDL is the ultimate source of cholesterol
crystals within atherosclerotic plaques. However, it is uncertain whether the crystals are comprised
primarily of free cholesterol in the outer monolayer or contain deesterified cholesteryl esters from the lipid
core. A related issue is the question of whether LDL particles nucleate cholesterol crystals directly or if
uptake of the LDL by macrophages (and/or smooth muscle cells) and subsequent foam cell formation is a
prerequisite.
Cholesterol crystals nucleate in the plaques because the concentration of free
cholesterol greatly exceeds its solubility limit. The crystals, which are inert, form
sequential layers in which newly deposited crystals enter from the luminal side of the
lesion. However, the origin of the crystals within the plaque, and the mechanism by
which they nucleate, are largely unknown. Several studies indicate that the crystals
originate from intracellular lipid accumulated by foam cells, while others suggest that the
crystals originate from extracellular lipid that is trapped within the lesion. While useful,
these studies utilize crystal detection methods that are at best semi-quantitative (i.e.,
microscopy) and fail to provide any molecular information concerning the cholesterol
nucleation event.
V. NUCLEATION
Cholesterol supersaturation is a necessary, but insufficient, condition for
cholesterol crystal formation in the body. The kinetics of cholesterol nucleation, rather
than the thermodynamics of the process, determine whether an individual develops crystals.
In particular, crystal formation depends on the rates at which cholesterol nucleates from
either lecithin-cholesterol vesicles (gallstones) or low density lipoproteins (atherosclerosis).
It is suspected that the rate of cholesterol nucleation varies with physiological factors such
as the type and amount of bile salts, the relative balance of pro- and anti-nucleating
proteins, the existence of oxidized sterols, and more recently, the presence of certain
bacteria. However, the impact of such factors on nucleation kinetics has not yet been
characterized. Here we consider the classical nucleation theory as a starting point for
nucleation of cholesterol in the presence of more complex systems such as the ones
described above.
20
CLASSICAL NUCLEATION THEORY:
Nucleation is the term given to the formation of a new, stable phase in a
metastable system. Unlike spinodal decomposition, which refers to the spontaneous
formation of a new phase from an unstable state, nucleation is an activated process. Small
clusters, or embryos, of the new phase form within the bulk metastable phase, and a free
energy barrier must be overcome to form clusters, called nuclei, of a critical size,
subsequent to which growth of the new phase proceeds spontaneously. This is the process
of homogeneous nucleation.
Gibbs was the first to treat the thermodynamics of cluster formation (in 1876)
when he described the energy change (G) associated with the formation of a small globule
of phase B within a bulk phase A:
G  (GB  GA )  VB    AB
(1)
where GA and GB are the bulk (Gibbs) free energies of phases A and B, per unit volume,
respectively, VB is the volume of the globule, AB is the interfacial area between the globule
and the bulk phase, and  is the interfacial tension. The derivation of eqn 1, which is
known as the capillarity approximation, assumes that the radius of curvature of the interface
is much larger than molecular dimensions.
If the cluster is a sphere of radius rB, eqn 1 becomes
G  (G B  G A )(4 / 3)rB 3  4rB 2  
(2)
The difference in free energies, (GB - GA), is merely the difference in the chemical
potential, , per molecule between the stable and metastable states:
(G B  G A )  ( B   A ) / 
(3)
where  is the molecular volume and
μ φ  μ φ o  k B T ln
aφ
(4)
where the subscript  denotes the phase, A or B, the superscript o denotes the standard
state, kB is the Boltzmann constant, T is the absolute temperature, and a is the activity.
Defining the supersaturation, S, as the ratio of activities, aA/aB, eqn 2 becomes
G  (4 / 3)k B T ln( S)  rB 3  4 rB 2
(5)
The supersaturation is always greater than unity because phase B has the lower free energy,
and the interfacial tension is always positive. Thus in eqn 5 the volume term is negative,
21
the surface term is positive, and a plot of G versus rB exhibits a maximum (Figure 9). It
will be convenient to recast eqn 5 in terms of the number of molecules in a cluster, n, where
VB = n, to give
G  k BT ln( S)  n  (36)1 / 3   2 / 3    n 2 / 3
(6)
Clusters that yield the maximal change in free energy are deemed critical clusters, or nuclei,
and setting the derivative of G with respect to n equal to zero gives the number of
molecules in the critical cluster, n*, as
n* 
50
32   2   3
(7)
3  (k B T ) 3  ln 3 (S)
Surface
Term
G
*
Energy
G
30
10
0
-10 0
-30
10
20
30
40
50
60
70
Volume
Term
-50
n*

n
Cluster radius, rB
Figure 9 - Graphical Representation of the Gibbs Capillarity Approximation: The overall change in free
energy (G) associated with the formation of a small cluster of a stable phase within a metastable phase, as
given originally by Gibbs (eqn 1), is plotted as a function of the cluster radius. The overall energy is
comprised of a negative volume term and a positive surface term, and the plot of G exhibits a maximum
(G*) at a cluster radius corresponding to n* molecules. The free energy does not become negative until the
cluster contains n molecules.
22
Substitution of eqn 7 into eqn 6 gives the (maximal) energy associated with formation of
the critical cluster, G*, as
G * 
16   2   3
3  (k B T) 2  ln 2 (S)
(8)
The above results can be summarized as follows: When n < n*, the energy
penalty due to creation of surface area outweighs the benefit of a lower bulk phase free
energy and gives rise to a positive value of G. Formation of sub-critical clusters is
therefore energetically unfavorable. Strictly speaking, this is also true for critical clusters
and for any cluster containing fewer than n molecules (Figure 9). Supposing, however, the
existence of a critical cluster (i.e., n = n*), any fluctuation in the size of that cluster leads to
a favorable lowering of the free energy. The removal of a molecule from the critical cluster
leads to further dissolution, but the addition of one molecule leads to sustained growth.
Thus, clusters comprising (n* + 1) molecules will only grow, and G* represents an energy
barrier that must be surmounted to form the new phase.
Kinetic Aspects: Volmer and Weber were the first (in 1926) to recognize nucleation as a
kinetic process. They argued that the rate of nucleation is proportional to the probability of
having a critical cluster, which they assumed is proportional to exp( G * / k BT) . Thus,
the Volmer and Weber expression for nucleation reads
J  A o  exp( G * / k B T)
(9)
where J is the nucleation rate and the pre-exponential factor, Ao is a constant of
proportionality. Although eqn 9 resembles the classical expression used to calculate
nucleation rates, Volmer and Weber did not determine the pre-exponential factor nor did
they provide a kinetic model for nucleation.
What is considered “classical” nucleation theory derives from the 1935 work of
Becker and Döring. The basis for the Becker-Döring kinetic model is the recognition that,
because sub-critical cluster formation is energetically unfavorable, cluster-cluster
interactions are unlikely. Growth and dissolution of clusters therefore proceed via addition
and removal of individual molecules, respectively, and nucleation is achieved via accretion.
The classical nucleation theory yields a non-zero, steady-state accretion rate, which is
identical in form to the Volmer & Weber expression. The only difference is that they
define the pre-exponential term, Ao, explicitly in terms of a rate constant and concentration.
23
VI. Future Directions
The nucleation of cholesterol crystals is now recognized as a critical step in the
pathogenesis of gallstone formation and atherosclerosis. Accordingly, efforts are
underway to elucidate the molecular details of nucleation in the context of both diseases.
The motivation is clear; if the nucleation mechanism can be understood, this will lead to
strategies to slow the nucleation rate so as to prevent the formation of cholesterol
crystals. By preventing the crystals from forming, it should be possible to prevent the
diseases.
Controlling the cholesterol nucleation rate therefore offers the possibility of
preventing atherosclerosis and gallstone disease. Turning this possibility into a reality,
which is a long-term goal, first requires a detailed understanding of the cholesterol
nucleation mechanism. Unfortunately, very little is known about the molecular details of
cholesterol nucleation from membranes. What is known is that gallstone cholesterol
nucleates from metastable lecithin-cholesterol vesicles, atherosclerotic plaque cholesterol
nucleates from low density lipoproteins, and there are striking similarities in the physical
chemistry associated with the two systems. Perhaps most striking is the observation that
the appearance of crystals in bile and in plasma is nearly always preceded by aggregation
of the vesicles and lipoproteins, respectively. Moreover, the rate of nucleation from
vesicles and lipoproteins is insufficiently rapid to yield crystals, regardless of the level of
cholesterol supersaturation (provided the value is in the physiologically meaningful
range), unless an aggregation-inducing factor is added.
Figure 10 – Fluorescence Assay for Measuring Cholesterol Nucleation in Bile: Lecithin-cholesterol
vesicles are labeled with dehydroergosterol (DHE) and dansylated lecithin (DL), and energy transfer from
DHE to DL proceeds readily when both fluorophores are in the vesicle bilayer. DHE co-nucleates with
cholesterol into a crystalline phase, leaving lecithin and DL in the vesicle bilayer. The removal of DHE
from the bilayer increases the separation between DHE and DL, thereby alleviating energy transfer (which
depends heavily on the separation between fluorophores). This is observed as an increase in DHE
fluorescence intensity with a concomitant decrease in DL intensity.
24
Current efforts are therefore addressing those factors that are known or suspected
to enhance vesicle and LDL aggregation. Part of the reason that these factors are
presently so poorly understood relates to the lack of analytical tool that can detect the
earliest molecular events of nucleation. Recently, a fluorescence assay was developed by
researchers at Drexel University to overcome this difficulty (see Figure 10). Essentially,
the assay involves decorating the vesicles (and later LDL) with fluorescent analogs of
cholesterol and lecithin. The interaction between the two fluorophores depends heavily
(i.e., 1/r6) on the distance, r, between them. Thus, as cholesterol nucleates from the
vesicle (or LDL), the value of r increases sharply and is evidenced by a change in the
fluorescence properties (intensity and wavelength). The fluorescence assay is being (and
will continue to be) used to provide an understanding of the mechanisms by which
various kinetic factors in bile influence nucleation kinetics. Efforts are also underway to
extend the assay to examine cholesterol nucleation from LDL in plasma systems.
VII. Suggested Additional Reading
Gallstones:
1. Lee, S.P. and J. Sekijima. In Textbook of Gastroenterology; T. Yamada, Ed., Vol 2.,
1966. J.B. Lippincott Company, Philadelphia, 1996.
2. Wang, D. Q.-H. and M. C. Carey. 1996. Complete mapping of crystallization
pathways during cholesterol precipitation from model bile: influence of physicalchemical variables of pathophysiologic relevance and identification of a stable liquid
crystalline state in cold, dilute and hydrophobic bile salt-containing systems. J. Lipid
Res. 37: 606-630.
3. Carey, M.C. and J.T. Lamont. Cholesterol Gallstone Formation. 1. PhysicalChemistry of Bile and Biliary Lipid Secretion. Prog. Liver Dis. 10, 139-163. 1992.
4. Carey, M.C. Critical tables for calculating the cholesterol saturation of native bile.
Journal of Lipid Research, 19, 945-955 (1978).
Atherosclerosis:
1. Small, D. 1988. Progression and Regression of Atherosclerotic Lesions. Insights
from Lipid Physical Biochemistry. Arteriosclerosis 8: 103-129.
2. Kaplan, M., K.J. Williams, H. Mandel, and M. Aviram. 1998. Role of Macrophage
Glycosaminoglycans in the Cellular Catabolism of Oxidized LDL by Macrophages.
Arterioscler. Thromb. Vasc. Biol. 18: 542-553.
3. Carpenter, K.L.H., S.E. Taylor, C. Vanderveen C, B.K. Williamson, J.A. Ballantine,
and M.J. Mitchinson. 1995. Lipids and oxidized lipids in human atherosclerotic lesions
at different stages of development. Biochimica et Biophysica Acta-Lipids and Lipid
Metabolism 1256 (2): 141-150.
25