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.
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.
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 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 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 present. We now take a closer look at each of the two cholesterol-linked diseases and will examine the striking similarities that emerge.
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 1.1Composition of Average Human Gallbladder Bile
Composition / Weight %Water / 88
Cholesterol / 1
Lecithins / 2
Bile Salts / 8
Proteins, Pigments / 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 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 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 oxo-intermediate 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.