Article pubs.acs.org/crystal
Cholesterol Monohydrate Dissolution in the Presence of Bile Acid Salts Richard S. Abendan‡ and Jennifer A. Swift* Department of Chemistry, Georgetown University, 37th and O Streets NW, Washington, D.C. 20057-1227, United States ABSTRACT: The molecular-level dissolution of cholesterol monohydrate single crystal (001) surfaces was systematically investigated in aqueous solutions of chenodeoxycholate (CDC) and ursodeoxycholate (UDC) using in situ atomic force microscopy (AFM). Dissolution via the layer-bylayer step retreat of monolayers was observed in solutions where the bile salt concentration exceeded the critical micelle concentration (cmc). Studies performed in CDC solution revealed markedly lower cmc values than in UDC. An abrupt transition from sporadic to continuous dissolution between ∼4−7 mM CDC was consistent with a previously proposed 2-step model for micelle formation. In contrast, cholesterol dissolution in UDC was apparent at ∼14 mM with no abrupt change in dissolution rate with increasing UDC concentration. Comparison with previous dissolution studies performed in aqueous ethanol (Cryst. Growth Des. 2005, 5, 2146−2153) reveal key differences in micelle-mediated versus bulk dissolution mechanisms.
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INTRODUCTION Cholesterol serves a number of important biological functions: it is an essential structural component of mammalian cell membranes and a precursor to many hormones, vitamin D, and bile acids. However, elevated levels of cholesterol which exceed the solubilizing capacity of the physiologic medium can lead to deposits which are associated with undesirable medical conditions such as cardiovascular disease and the formation of cholesterol gallstones. A number of intermediates have been identified in the early nucleation and growth stages of cholesterol in saturated model bile solutions;1,2 however, cholesterol monohydrate (ChM) is the sole crystalline form found in human gallstones as confirmed by light microscopy3 and X-ray diffraction.4 Up to 80% by mass of a human gallstone can consist of ChM crystals.3 Cholesterol has low solubility in aqueous solution (3 × 10−8 g/mL).5,6 It is solubilized in human bile solution (which is 97% water7) through the action of mixed micelles and vesicles of dissolved organic compounds such as bile acids and phospholipids.8 Dissolution therapy via oral administration of common bile salts such as chenodeoxycholate (CDC, trade name Chenix) and ursodeoxycholate (UDC, trade name Actigall) is sometimes used to treat gallstones as an alternative to surgery.9−11 Cholesterol solubility in CDC solutions is 1−2 orders of magnitude higher than in UDC solutions,12−15 though these two bile acids differ only in the orientation of the C7 hydroxyl position (Figure 1). Overall, efficacy rates of CDC and UDC treatment are quite different, in part due to undesirable side effects of CDC oral treatment.16 In general, dissolution therapy appears to be a rather slow process requiring several months to years of daily treatment.17−19 Previous bulk dissolution studies of cholesterol in the presence of bile salts and other bile components have relied on traditional methodologies20−22 where the dissolution rates © 2013 American Chemical Society
Figure 1. Molecular structures of cholesterol (top) and bile salts chenodeoxycholate (CDC) and ursodeoxycholate (UDC) (bottom). Note the orientation of the 7-hydroxyl, which is the sole variation between the two bile salts.
of pressed ChM (or anhydrous cholesterol crystal) pellets are determined from monitoring the solution concentration over time. Dissolution rates of gallstones and ChM pellets were found to be similar; however, these bulk methods do not address the molecular-level mechanism(s) of ChM dissolution at the crystal surface. A molecular level understanding of surface dissolution processes within bile salt solutions can provide useful insight into both how pathogenic deposition and/or dissolution of cholesterol might be better controlled. Received: April 16, 2013 Revised: June 12, 2013 Published: June 26, 2013 3596
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The objective of the current study is to examine the molecular-level dissolution of ChM single crystal surfaces in micellar CDC and UDC solutions using in situ atomic force microscopy (AFM) and to compare the observed mechanism with previous dissolution studies23 performed in bulk solvents. Application of in situ AFM techniques to ChM dissolution in the presence of CDC and UDC additionally serves as an alternative method to establish the critical micelle concentration required for cholesterol dissolution.
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EXPERIMENTAL SECTION
Materials. Cholesterol (Aldrich, 99+%), chenodeoxycholic acid (CDC, Fluka, 98%), and ursodeoxycholic acid (UDC, Sigma, 99%) were used as received. ChM Sample Preparation. Platelike single crystals of ChM were grown by slow evaporation of saturated 95% ethanol (5% water) solutions by volume over a period of 7−10 days. Crystals typically exhibited large well-developed (001) faces and dimensions ranging from a few hundred micrometers to 4 mm wide. In situ Atomic Force Microscopy. All images were obtained in situ in tapping mode with a Nanoscope IIIa Atomic Force Microscope (Digital Instruments, Santa Barbara, CA) equipped with a 30 μL glass fluid cell and temperature stage. Single crystals of ChM were mounted in the fluid cell, as described previously.23 Pure aqueous and bile salt solutions of varying concentrations were held in a custom-built fluid reservoir maintained at 37 °C and introduced into the AFM fluid cell at flow rates of 0−1.0 mL/h using a syringe pump (Kd Scientific, Holliston, MA). The temperature within the fluid cell (34.7 °C) was measured by a directly inserted thermocouple. In each experiment, tapping mode imaging was first performed in pure aqueous solutions, resulting in no discernible changes in the surface over at least one hour. Bile acid solution was subsequently introduced into the fluid cell (∼10 mL/h) where it rapidly exchanged with the aqueous solution. All topographical images presented and discussed herein were collected at least 5 min after the solution exchange. The kinetic rates reported and mechanistic behavior observed under a given set of solution conditions were reproducible over many experiments (all were done in at least triplicate). All images were processed and analyzed with the standard AFM operating software (Nanoscope 4.43r8 and 5.12r3). Step heights were determined from bearing and section analyses. Scion Image (Scion Corporation, Frederick, MD) was used to trace the perimeter of individual topographical features in order to calculate dissolution rates. Area values were measured in (pixels)2, and then converted to nm2 based on the known image size, and finally to the number of cholesterol molecules based on the unit cell volume. Bile Salt Solutions. CDC and UDC solutions ranging from 0.75 to 60 mM were prepared in a sodium carbonate/sodium bicarbonate buffer solution (pH∼10, [Na+] = 0.075M) at room temperature. Higher UDC solution concentrations (40−60 mM) required an additional 2−3 drops of 0.1 N NaOH and >2 h equilibration before the acid was fully dissolved. Solutions were maintained in sealed volumetric flasks and used within 7 days. All solutions were optically clear and free of any precipitates.
Figure 2. Packing diagram of ChM showing the bilayer structure viewed down the a axis. Image was prepared using fractional coordinates reported in ref 24.
that all terraces on the surface must be chemically homogeneous. Chemical force microscopy (CFM) experiments performed with variously functionalized tips revealed that the surface is predominantly terminated with the hydroxyl end of cholesterol when immersed in an aqueous environment, while the alkyl tail groups terminate the plate surface in ethylene glycol solutions. Though crystallographically identical, we use (001) and (002) in order to unambiguously refer to surfaces terminated by alkyl or hydroxyl groups, respectively. ChM has very limited solubility in pure aqueous solution, but in bile salt solutions, dissolution can occur once the critical micelle concentration (cmc) of a given surfactant is reached. The cmc for bile salts has previously been reported using a variety of methods (e.g., surface tension, solution turbidity, spectrophotometric signals from dye probes, potentiometric titrations).13−15,26−28 Micelle formation affects solution properties in different ways, and there is typically some small variation in the reported cmc values based on the technique employed. Because cholesterol dissolution requires the presence of solubilizing micelles, in situ AFM experiments provide a direct means to evaluate cmc. The sodium ion concentration (0.075 M), pH, and temperature used in this study are comparable to the parameters employed in previous cmc studies. Dissolution in CDC Solutions. Previous AFM studies showed that ChM (001) surfaces exhibit no appreciable dissolution in pure aqueous solution,23 with only rare and occasional slow dissolution in isolated areas. In the current study, the onset of clear changes in the ChM surface topography in AFM images at a given bile acid concentration is interpreted as an indication of micelle formation since these solubilizing units are needed for cholesterol dissolution. Series of AFM images were collected on >30 different ChM (001) single crystal surfaces in aqueous CDC solutions with
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RESULTS AND DISCUSSION ChM Structure. ChM single crystals adopt a lamellar structure (P1; a = 12.39 Å, b = 12.41 Å, c = 34.36 Å, α = 91.9°, β = 98.1°, γ = 100.8°)24 in which the hydroxyl groups of the cholesterol and water molecules form a 2D H-bonded network in the ab plane (Figure 2). The crystals grow in a platelike morphology in which the largest macroscopic face is (001). Previous in situ atomic force microscopy (AFM) studies have sought to characterize the molecular level topography and chemical functionality of the (001) surface in different solution environments.25 These experiments revealed that the smallest steps measured 33−34 Å, which is consistent with the calculated bilayer stacking height of 33.9 Å. It also indicated 3597
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Figure 3. Consistent bilayer dissolution observed on a ChM (001) surface in 7 mM CDC solution. Note that practically all bilayer structures exhibit some form of dissolution activity, with layer-by-layer dissolution evident in structures A−D. (Flow rate = 1 mL/h, vertical color scale = 60 nm).
concentrations ranging from 0.75−25 mM. As in the pure aqueous solution, AFM images collected over 1−2 h periods in 0.75−3.5 mM CDC showed no consistent signs of observable dissolution. We interpret this as an indication that solubilizing bile salt micelles have not yet formed. These concentrations are in the lower range of the previously reported cmc values (1.5− 3.2 mM)13 for CDC. In 4 to 5 mM CDC solutions, modest yet slow dissolution activity becomes apparent on a majority of crystal surfaces. Typically dissolution occurs at the outer edges of some bilayers, while others remain resistant to dissolution. A few image series collected at this concentration range showed no significant dissolution activity at all. This suggests at least some solubilizing micelles are present; however, their capacity to solubilize cholesterol appears limited. Further increasing the CDC concentration to 7 mM resulted in consistent and robust dissolution on the ChM surface in all cases. Mechanistically, dissolution was dominated by the retreat of bilayer steps. Figure 3 illustrates the typical behavior observed under these conditions in which edges of bilayers labeled A−D retreat fairly consistently over time, with bilayer A fully disappearing after ∼60 min. Increasing the CDC concentration to 7−13 mM resulted in faster dissolution. In about half of all experiments performed in this concentration range, well-defined pits 14−18 Å deep and ranging from 20 nm
to a few hundred nanometers in diameter also appear on the flat terraces (Figure 4). These features, which are apparent typically ∼15−60 min after the introduction of solution, correspond to monolayers (the theoretical monolayer height is 17 Å).24 In general, the monolayers are stable enough to be imaged over long time periods, though monolayer regions often combine with other expanding monolayer pits nearby to become bilayers or multilayers or are eliminated when other multilayer steps recede over the area. Additional increases in the surfactant concentration resulted in faster dissolution rates but no discernible change in mechanism. In 25 mM CDC, dissolution becomes so fast that it is difficult to track the rapidly changing positions of individual step edges in sequential images. That the minimum step height corresponds to monolayers is significant. Given the bilayer crystal structure of ChM, adjacent (001) and (002) monolayer steps must be terminated with different chemical functionalities. In all previous studies done in pure aqueous, aqueous ethanol, and ethylene glycol solutions, the minimum step height always corresponded to bilayers; monolayers were never observed.23,25 The coexistence of (001) and (002) surfaces in the >7 mM CDC solution indicates that the difference in surface energy of hydroxy- and alkylterminated surfaces must be smaller than in bulk solvents. 3598
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Figure 4. Image of monolayer pits formed on the ChM (001) surface during dissolution in 7 mM CDC solution (top). Section analysis of AFM image showing the depths of both the bilayer and monolayer features (bottom).
This must be a direct consequence of interactions with the amphiphilic surfactants. It is also significant that in CDC dissolution studies, the main mechanism for dissolution is via a layer-by-layer step retreat. Previous work revealed that during ChM (001) dissolution in ethanol,23 the expansion of deep multilayer pits and voids occurred much faster than layer by layer dissolution from smoother terraces. Multilayer pits and voids originate from long-range defects created during growth. (ChM crystals used in both the previous and current studies were prepared under identical conditions and should therefore have similar defect densities.) Fast dissolution in aqueous ethanol can occur from these higher-energy defect sites which penetrate deep into the crystal because cholesterol molecules released from the surface can readily diffuse into the bulk solvent. In CDC solution, cholesterol molecules must presumably be incorporated into the micelle before being transported away from the ChM surface. The larger aggregate size and/or slower diffusion of these solubilizing micelles likely results in a reduction in their ability to access narrow deep void spaces. It therefore becomes energetically more feasible in micellar solutions for dissolution to occur from step sites on the topmost surface layers. Figure 5 is representative of the topological changes that occur on a ChM single crystal surface when the CDC concentration is increased in real time. Quantitative dissolution rates were determined from changes in the areas of three distinct bilayers (labeled A, B, and C) over time. Initially, the ChM surface was exposed to a flowing 3.5 mM CDC solution, and quantitative tracking of the perimeter of the bilayers shows only a very slight decrease in the area over the first 170 min. When the solution was changed to 7 mM CDC (red vertical line), an immediate increase in the dissolution rate ensued. Features A, B, and C were completely dissolved less than 80
Figure 5. Select topographical images of a ChM (001) surface initially exposed to flowing 3.5 mM CDC solution (t = 0−170 min) and 7.0 mM CDC solution (t > 170 min) (top) . The area of three bilayers (A, B, C) monitored over time (bottom).
min later. The area decrease over time for each bilayer differs slightly, but in every case the reduction in area follows a smooth and consistent process. In aqueous ethanol, the fastest dissolution occurred at deep voids; however, it was also possible to track the dissolution of bilayers and multilayers. Under aqueous ethanol conditions, similar plots tracking the area of dissolving bilayers showed significantly more erratic and highly variable rates over time.23 From the slope of plots made from bilayer area tracking in Figure 5, it is possible to obtain absolute dissolution rates. However, such values reflect local rates and are not an accurate measure of the absolute dissolution rate over the entire surface. Absolute values obtained from tracking individual bilayers from multiple crystals undergoing dissolution in 7 mM and 13 mM CDC showed higher overall dissolution rates obtained at the higher concentration; however, they were accompanied by a broad range of absolute rates. Eight different experiments performed in 7 mM CDC yielded specific dissolution rates ranging from 45−484 molecules/min (average = 191 ± 148), where six experiments performed in 13 mM CDC had rates of 105−2246 molecules/min (average = 1058 ± 923). Critical Micelle Concentration. CDC micelle formation has been proposed to occur in two steps.29−31 Hydrophobic interactions first drive a back-to-back binding of the steroid backbone (in which the hydrophilic groups project out toward the aqueous phase); aggregation of these globular primary 3599
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Figure 6. Continuous dissolution of bilayer structures observed in 60 mM UDC solution. Note the appearance of several small monolayer structures (the largest one is labeled X) which in this sequence appear after ∼30 min.
higher concentration of UDC was always required. This is consistent with bulk solubility studies which show lower cholesterol solubility in UDC solutions. Unlike aliphatic surfactants which typically have a charged headgroup and long hydrophobic tail, bile acids are rigid sterols and the α (bottom) and β (top) faces differ in their hydrophilicity. It has been hypothesized that the presence of the α-OH at C7 in CDC enables it to form large secondary micelles more easily than UDC (with a β-OH at C7) and that this imparts it with a greater capacity for solubilizing cholesterol.13 UDC solutions also exhibit minimal increases in aggregate size at higher concentrations. Four independent experiments performed in 7 mM UDC showed no significant dissolution activity, and only one series showed a small amount of bilayer dissolution. The lack of significant dissolution activity is noteworthy given that some of the reported values for the cmc of UDC are in the range of 1−7 mM.13,34 Others have reported much higher cmc values ranging from 10−17 mM UDC.26,27,33 Notably, whereas fluorescence methods identified a two-stage model for micelle formation in CDC,14 analogous cmc studies showed only one inflection point at ∼12 mM.27 Fifteen additional experiments performed under higher UDC concentrations showed that the apparent onset of dissolution activity occurs over a much broader concentration range than in
micelles then form larger, rodlike secondary micelles at higher concentrations. However, except for one study using pyrene probes,14 only one cmc value is typically reported for CDC. Inflection points at this single value are usually interpreted as the formation of primary micelles. In these earlier studies, there were no reported solution property changes arising from the secondary aggregation of micelles. Carey et al.13 reported the mean hydrodynamic radii of CDC bile salt micelles to be 13.0 Å. With increasing CDC concentrations above the cmc, an increase in the mean hydrodynamic radii (R̅ h) was reported, though this was not directly attributed to secondary aggregation. In situ AFM observations of limited dissolution at ∼4 mM compare favorably to the lower range of reported cmc values for CDC, which were obtained through fluorescent probes (5.0 mM)28 and potentiometric titration (4.6 mM)26 studies. Microcalorimetric titration32 and fast chromatography33 studies reported cmc values closer to ∼7 mM. Pyrene fluorescence studies reported a second cmc value of 6.5 mM.14 In our AFM studies, the observed onset of continuous dissolution at ∼7 mM is consistent with these higher cmc values. Dissolution in UDC Solutions. In situ dissolution experiments were similarly performed in UDC solutions of varying concentration. In order to attain a dissolution activity comparable to that observed in CDC solutions, a significantly 3600
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CDC. Under in situ AFM conditions, dissolution activity in 13 mM UDC resulted in inconsistent behavior, with no apparent dissolution in some image series and a combination of spurious dissolution and growth over small (∼100 nm) length scales in others. The inconsistent minor topography changes suggest that if micelles are present in 13 mM UDC solutions, they are not of sufficient size or stability to solubilize cholesterol in an appreciable way. To achieve consistent dissolution on ChM (001), UDC concentrations of 40−60 mM were required. Dissolution rates in 40 mM and 60 mM UDC were not appreciably different. Figure 6 is representative of the type of continuous bilayer dissolution observed in 60 mM UDC solution. Monolayer steps were observed in some image sequences obtained in this concentration range (one monolayer is labeled X in Figure 6), and such features typically become apparent ∼20 min or more after the surfactant solution was first introduced. Monolayer features tend to expand rather slowly, and their tracking is often obscured by faster dissolving bilayer steps. In contrast, monolayer features were never observed in the less concentrated 7−13 mM UDC solutions. Mechanistically, the consistent dissolution behavior observed in 40−60 UDC was qualitatively similar to that observed in 7 mM CDC. These observations and others at lower concentrations reinforce previous studies which indicated that CDC is a more powerful solubilizing agent for cholesterol and that a two-stage cmc model for this surfactant may be appropriate. There was no distinctive transition from limited to consistent dissolution in UDC solutions with increasing concentration, which appears consistent with the hypothesis that secondary micelles are not formed in UDC.
direct mechanistic implications for how crystal surfaces can interact with all types of biomolecules. Conventional methods used to determine cmc values rely on measuring some change in the solution properties in response to micelle formation. Some variation in the reported cmc values is expected based on the fact that micelle formation affects solution properties in different ways. In situ AFM provides an alternative analytical approach to establishing accurate cmc values. Because cholesterol has almost negligible solubility in aqueous solution, dissolution can only occur when solubilizing micelles are present (i.e., when CDC and UDC concentrations exceed the cmc). Our data supports the hypothesis that micelle formation in CDC is a two-step process. Evidence for micelle formation is apparent ∼4 mM with an abrupt change to consistent dissolution observed at ∼7 mM. This is consistent with a distinct change in the micelle structures in solution. Evidence for micelle formation in UDC was apparent at higher concentrations of ∼13 mM, with modest and then consistent dissolution achieved with increasing UDC concentration. That there was no obvious concentration where robust dissolution suddenly occurred is consistent with a one-step process in which micelle sizes increase with solution concentration and eventually result in consistent observable dissolution on ChM surfaces. Lastly, although the present study shows that cholesterol is more readily solubilized in CDC than UDC solutions, we note that oral administration of UDC is typically regarded as more effective for gallstone dissolution.16 Not only does UDC treatment have fewer side effects but also factors aside from simple micellar solubilization are likely involved in the enhanced dissolution of gallstones. Some studies suggest that secondary structures such as vesicles35 and liquid crystal spherulites36 formed with UDC in model bile solutions may have a role in increasing the solubility of cholesterol. UDC treatment also results in lower biliary cholesterol secretion and reduced intestinal cholesterol absorption,37 indicating that various biological modes of action can work in concert to create an undersaturated cholesterol environment in the gallbladder.
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CONCLUSION This AFM study of ChM (001) single-crystal surfaces in bile acid solutions serves to illustrate some important molecularlevel aspects of dissolution in micellar solutions. Previous studies on ChM dissolution in aqueous ethanol (Cryst. Growth Des. 5(6), 2146−2153, 2005) revealed that the dominant dissolution mechanism was the rapid expansion of deep pits/ voids (from long-range defects). Dissolution via bilayer-bybilayer step retreat, in general, occurred much more slowly. One expects that cholesterol molecules occupying high-energy defect sites should more readily be removed from the surface than those at other surface sites, via simple diffusion into the bulk solution. In contrast, dissolution in CDC and UDC solutions occurred exclusively via layer-by-layer step retreat, which is a direct consequence of micelle sterics. Though deep narrow void spaces are present on the ChM (001) surface, CDC and UDC micelles are presumably either too large to access them effectively or experience limited transport and/or diffusion in and out. For cholesterol molecules to be solubilized and transported away from the ChM crystal surface requires direct micelle−surface interactions, which restricts dissolution to the most accessible sites. That the smallest step heights observable on ChM (001) surfaces in 7 mM CDC and 40 mM UDC solutions corresponded to monolayers (17 Å) is also significant. All previous studies in bulk solution showed that the largest ChM surface is chemically homogeneous, expressing either (001)alkyl or (002)-hydroxyl terminal groups. The fact that both (001) and (002) surfaces coexist in surfactant solutions has
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AUTHOR INFORMATION
Corresponding Author
*Email:
[email protected]. Present Address ‡
Department of Chemistry, Ateneo de Manila University, Quezon City 1108, Philippines. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS The authors are grateful for the financial support provided by the National Science Foundation (Grant DMR-00393096) and the Camille & Henry Dreyfus Foundation.
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