Kinetics and Mechanism of Gallstone Formation

Here is the web address for a site that explains how gallstones are formed.

http://www.chemeng.drexel.edu/faculty/spw/SPWfacultypage.htm

Kinetics and Mechanism of Gallstone Formation

Gallstones are essentially large crystals of cholesterol found in bile (Gr. chole, bile; stereos, solid), an important biological fluid. Bile is secreted by the liver and contains bile salts, which are needed for digestion of lipids, plus the phospholipid lecithin, a primary constituent of cell membranes. The gallbladder stores bile between meals, and gallstones result when cholesterol precipitates from bile during this storage period.

Cholesterol is insoluble in water, and precipitation from bile at first appears unavoidable. However, bile salts and lecithin are biological surfactants (i.e., they are amphiphilic) that self-assemble in bile and give rise to interesting microstructures that serve as cholesterol transport vehicles. For example, lecithin vesicles enhance the solubility of cholesterol in bile nearly one million fold. Such vesicles are thermodynamically metastable so that cholesterol crystals will ultimately prevail at equilibrium. Attainment of equilibrium is typically slow enough that cholesterol passes through the gallbladder without consequence, but diseased individuals are not so fortunate. Kinetic factors, presumably proteins, influence the rate at which vesicles yield crystals, and the question of who develops gallstones is a matter of chemical kinetics, not thermodynamics. 

Our gallstone research aims at prevention of gallstones in humans by determining the mechanisms by which various proteins influence the rate of gallstone formation.  We recently developed (the first) analytical technique that detects cholesterol nucleation in bile. We are currently using this technique, a fluorescence assay, to study cholesterol nucleation kinetics. Our research deals with thermodynamic and kinetic aspects of the various microstructural transitions in bile and is certainly a molecular level approach. However, we collaborate with clinicians at the University of Washington School of Medicine, and our work has significant practical implications.

NEW! Our work is now featured on-line by several health magazines.

Lecithin-Cholesterol Vesicles

Gallstone Pathway

The pathway to gallstone formation involves five key steps, as described below. Although gallstones affect just 8% of the population, each of the steps is expected to occur in all individuals. This is a consequence of phase behavior studies, which reveal that the bile of both healthy and diseased individuals is supersaturated in cholesterol. It is the rate at which the steps occur that determines who develops gallstones. The kinetics of all steps except cholesterol nucleation (Step 4) have been investigated, but we are currently studying the kinetics of cholesterol nucleation with a recently developed fluorescence assay.

3) Aggregation & Fusion: Lecithin-cholesterol vesicles are thermodynamically metastable and revert to their equilibrium, lamellar sheet form with time. The mechanism by which this occurs involves clustering (or aggregation) of two or more vesicles, followed by fusion (similar to sperm and egg) into larger vesicles.

4) Nucleation: As vesicles decrease in number and increase in size (toward flat sheets), cholesterol clusters called "embryos" may form via lateral diffusion in the bilayer. Small embryos dissolve, but some exceed a critical size and continue to grow. Formation of these larger embryos (called nuclei) is the process of nucleation.

5) Growth: Cholesterol nuclei exit the vesicle and continue to grow into macroscopic crystals (rectangular with a characteristic notch at one corner), which ultimately mature into gallstones.

Phase Behavior in Bile

Lecithin-cholesterol vesicles are thermodynamically metastable and so do not appear on the phase diagram (which gives only equilibrium phases). These vesicles will ultimately attain equilibrium, however, and may land at different points on the phase diagram depending on their composition. The question of who develops gallstones therefore appears to be a simple matter of thermodynamics - the bile of diseased individuals lands in a multi-phase region containing crystals, and the bile of healthy individuals lands in a non-crystalline region. The work of Carey and Small disproves that notion, however, indicating that the composition of bile in (most) all individuals, both healthy and diseased, places it in a multi-phase region containing crystals. Technically speaking, cholesterol supersaturation is a necessary but insufficient condition for gallstone formation, and thermodynamics alone cannot predict who will develop gallstones. The presence/absence of gallstones in individuals depends on the rate at which equilibrium is attained, and it is for this reason that we are studying the kinetics of cholesterol nucleation from vesicles.

1. Carey, M. C. & Small, D. M. J. Clin. Invest. 1978, 61, 998.

Fluorescence Assay to Detect Cholesterol Nucleation

We have recently developed a technique to detect cholesterol nucleation in bile. This technique, which has been featured in several on-line health magazines, employs fluorescence spectroscopy and utilizes fluorescent analogs of the lipids that occur naturally in bile. The idea behind the fluorescence assay is simple - a fluorophore and its quencher are placed into a lecithin cholesterol vesicle, and this leads to a low signal from the fluorophore. Upon nucleation, only the fluorophore (and not the quencher) exits the vesicle, and the fluorescence signal recovers as the separation betweeen the fluorophore and quencher increases. Details of the assay are published in the Journal of Lipid Research.²  For more information on this assay, please contact Steve Wrenn.

2. Wrenn, S.P., Lee, S.P., and Kaler, E.W.  J. Lipid. Res.  1999, 40, 1483.

Applications to Heart Disease

We are currently using the fluorescence assay to characterize several pro- and anti-nucleating agents in bile, including, bile salts, oxysterols, and several proteins.  We are especially interested in the anti-nucleating potency of the lipoproteins Apo A-I and Apo A-II, as these are the proteins most commonly associated with high density lipoproteins (HDLs), the so-called "good cholesterol" in the bloodstream.  The physical chemistry of blood and bile lipids is strikingly similar, and we hypothesize that the protective effects of A-I and A-II in preventing atherosclerosis are related to the protective effects of A-I and A-II in delaying gallstone formation.  By establishing the cholesterol nucleation mechanism from both vesicles and lipoproteins we intend to identify the protective mechanism that is common to both diseases.

http://www.chemeng.drexel.edu/faculty/spw/SPWfacultypage.htm

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