Crystalline Cholesterol Monohydrate Research Articles

Regulation of the Gating of BKCa Channel by Lipid Bilayer Thickness

Transmembrane segments of ion channels tend to match the hydrophobic thickness of lipid bilayers to minimize mismatch energy and to maintain their proper organization and function. To probe how ion channels respond to mismatch with lipid bilayers of different thicknesses, we examined the single channel activities of BK(Ca) (hSlo alpha-subunit) channels in planar bilayers of binary mixtures of DOPE (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine) with phosphatidylcholines (PCs) of varying chain lengths, including PC 14:1, PC 18:1, PC 22:1, PC 24:1, and with porcine brain sphingomyelin. Bilayer thickness and structure was measured with small angle x-ray diffraction and atomic force microscopy. The open probability (P(o)) of the BK(Ca) channel was finely tuned by bilayer thickness, first decreasing with increases in bilayer thickness from PC 14:1 to PC 22:1 and then increasing from PC 22:1 to PC 24:1 and to porcine brain sphingomyelin. Single channel kinetic analyses revealed that the mean open time of the channel increased monotonically with bilayer thickness and, therefore, could not account for the biphasic changes in P(o). The mean closed time increased with bilayer thickness from PC 14:1 up to PC 22:1 and then decreased with further increases in bilayer thickness to PC 24:1 and sphingomyelin, correlating with changes in P(o). This is consistent with the proposition that bilayer thickness affects channel activity mainly through altering the stability of the closed state. We suggest a simple mechanical model that combines forces of lateral stress within the lipid bilayer with local hydrophobic mismatch between lipids and the protein to account for the biphasic modulation of BK(Ca) gating.

The epitaxial growth of cholesterol crystals from bile solutions on calcite substrates.

Epitaxial relationships between the surfaces of inorganic and bioorganic crystals can be an important factor in crystal nucleation and growth processes in a variety of biological environments. Crystalline cholesterol monohydrate (ChM), a constituent of both gallstone and atherosclerotic plaques, is often found in association with assorted mineral phases. Using in situ atomic force microscopy (AFM) and well-characterized model bile solutions, the nucleation and epitaxial growth of ChM on calcite (104) surfaces in real-time is demonstrated. The growth rates of individual cholesterol islands formed on calcite substrates were determined at physiological temperatures. Evidence of Ostwald's ripening was also observed under these experimental conditions. The energetics of various (104) calcite/(001) ChM interfaces were calculated to determine the most stable interfacial structure. These simulations suggest that the interface is fully hydrated and that cholesterol hydroxyl groups are preferentially positioned above carbonate ions in the calcite surface. This combination of experimental and theoretical work provides a clearer picture of how preexisting mineral seeds might provide a viable growth template that can reduce the energetic barrier to cholesterol nucleation under some physiological conditions.

Quantification of Cholesteryl Esters in Human and Rabbit Atherosclerotic Plaques by Magic-Angle Spinning 13 C-NMR

Accumulation of cholesteryl esters (CEs) is a key event in the formation of atherosclerotic plaques. More recent work suggests a role for CEs in plaque rupture leading to thrombosis, which can result in an acute event such as myocardial infarction or stroke. In this study, we present nuclear magnetic resonance (NMR) protocols for quantification of CEs in plaques in situ. Total CEs quantified by (13)C magic-angle spinning (MAS) NMR in excised plaques from human carotid arteries and rabbit aortic arteries were in good agreement with the amounts determined by subsequent standard chemical assays. The latter analysis is disadvantageous because it requires that plaque lipids be extracted from the tissue, resulting in the loss of all phase information of CEs as well as other major plaque components. With our MAS-NMR protocol, the plaque components are preserved in their native phases. Combining MAS and off-MAS NMR, we were able to quantitatively distinguish isotropic (liquid) CEs from anisotropic (liquid-crystalline) CEs in plaque tissues. In a recent study, we applied a different (13)C MAS-NMR protocol to quantify crystalline cholesterol monohydrate in plaques. Together, these 2 studies describe a new, noninvasive MAS-NMR strategy for the identification and quantification of the major lipid components in plaques in situ. This approach will be useful for investigation of the relationship between plaque rupture and specific lipids in their biologically relevant phases.

Quantification in situ of crystalline cholesterol and calcium phosphate hydroxyapatite in human atherosclerotic plaques by solid-state magic angle spinning NMR.

Because of renewed interest in the progression, stabilization, and regression of atherosclerotic plaques, it has become important to develop methods for characterizing structural features of plaques in situ and noninvasively. We present a nondestructive method for ex vivo quantification of 2 solid-phase components of plaques: crystalline cholesterol and calcium phosphate salts. Magic angle spinning (MAS) nuclear magnetic resonance (NMR) spectra of human carotid endarterectomy plaques revealed (13)C resonances of crystalline cholesterol monohydrate and a (31)P resonance of calcium phosphate hydroxyapatite (CPH). The spectra were obtained under conditions in which there was little or no interference from other chemical components and were suitable for quantification in situ of the crystalline cholesterol and CPH. Carotid atherosclerotic plaques showed a wide variation in their crystalline cholesterol content. The calculated molar ratio of liquid-crystalline cholesterol to phospholipid ranged from 1.1 to 1.7, demonstrating different capabilities of the phospholipids to reduce crystallization of cholesterol. The spectral properties of the phosphate groups in CPH in carotid plaques were identical to those of CPH in bone. (31)P MAS NMR is a simple, rapid method for quantification of calcium phosphate salts in tissue without extraction and time-consuming chemical analysis. Crystalline phases in intact atherosclerotic plaques (ex vivo) can be quantified accurately by solid-state (13)C and (31)P MAS NMR spectroscopy.

The inflammatory effects of crystalline cholesterol monohydrate in the guinea pig gallbladder in vivo

Background: The etiologic role of crystalline material in inflammatory arthritis is well established. The role of crystals in cholecystitis is unclear. We hypothesized that crystalline cholesterol monohydrate stimulates guinea pig gallbladder inflammation in vivo. Methods: Crystalline cholesterol monohydrate, lipopolysaccharide (LPS), lysolecithin, polystyrene latex spheres (noninflammatory particles), and saline were instilled into guinea pig gallbladders for 24 to 72 hours after cystic duct ligation. Water transport across gallbladder mucosa was measured. Gallbladder tissue was analyzed for mucus layer thickness, myeloperoxidase, prostaglandin E2 (PGE2), prostaglandin F-1α (PGF-1α), and interleukin-1. Luminal fluid was also examined for PGE2 and PGF-1α. Values for each test were compared with saline controls by using Student's t test (p < 0.05). Results: Crystalline cholesterol, LPS, and lysolecithin caused significant reduction in mucus layer thickness, reversed water absorption to secretion across the gallbladder mucosa, caused significant increases in myeloperoxidase and interleukin-1 in gallbladder tissue, and caused significant increases in PGE2 and PGF-1α in luminal fluid. These effects were generally dose- but not time-dependent. Polystyrene latex particles caused no difference in outcomes compared with saline controls. Conclusions: Crystalline cholesterol monohydrate has dose-dependent inflammatory effects in the guinea pig gallbladder in vivo that are not simply due to mechanical irritation of the gallbladder wall by crystalline particles. Crystals in the gallbladder may have an etiologic role in cholecystitis. (Surgery 1998;123:258-63.)

The inflammatory effects of crystalline cholesterol monohydrate in the guinea pig gallbladder in vivo

Background: The etiologic role of crystalline material in inflammatory arthritis is well established. The role of crystals in cholecystitis is unclear. We hypothesized that crystalline cholesterol monohydrate stimulates guinea pig gallbladder inflammation in vivo. Methods: Crystalline cholesterol monohydrate, lipopolysaccharide (LPS), lysolecithin, polystyrene latex spheres (noninflammatory particles), and saline were instilled into guinea pig gallbladders for 24 to 72 hours after cystic duct ligation. Water transport across gallbladder mucosa was measured. Gallbladder tissue was analyzed for mucus layer thickness, myeloperoxidase, prostaglandin E 2 (PGE 2) , prostaglandin F-1α (PGF-1α), and interleukin-1. Luminal fluid was also examined for PGE 2 and PGF-1α. Values for each test were compared with saline controls by using Student's t test ( p < 0.05). Results: Crystalline cholesterol, LPS, and lysolecithin caused significant reduction in mucus layer thickness, reversed water absorption to secretion across the gallbladder mucosa, caused significant increases in myeloperoxidase and interleukin-1 in gallbladder tissue, and caused significant increases in PGE 2 and PGF-1α in luminal fluid. These effects were generally dose- but not time-dependent. Polystyrene latex particles caused no difference in outcomes compared with saline controls. Conclusions: Crystalline cholesterol monohydrate has dose-dependent inflammatory effects in the guinea pig gallbladder in vivo that are not simply due to mechanical irritation of the gallbladder wall by crystalline particles. Crystals in the gallbladder may have an etiologic role in cholecystitis. (Surgery 1998;123:258-63.)

Filamentous Crystallization of Cholesterol and Its Dependence on Lecithin Species in Bile

Abstract Cholesterol crystallization is a poorly understood obligatory step in cholesterol gallstone formation. We have studied early stages of cholesterol crystallization in a model bile system composed of an aqueous solution of taurocholate/cholesterol/lecithin (97.5/1.7/0.8 moles%). Nucleation of cholesterol crystals was induced by dilution of a micellar solution to achieve supersaturation. Following spontaneous vesicle formation, thin filamentous structures were observed by electron and light microscopy. The filaments were composed of a core of >95% cholesterol and a surface layer of < 5% lecithin, which was preferentially enriched in hydrophobic molecular species. The filaments had a buoyant density of 1.03 g/ml, and revealed an x-ray diffraction pattern compatible with crystalline cholesterol monohydrate with an additional Bragg reflection at 4.9A, suggesting a polymorph of cholesterol monohydrate, or early anhydrous crystallization. The filaments grew and transformed via intermediate spiral, helica...

Atomic structural images of crystalline cholesterol monohydrate in human atheroma

To gain further insight regarding the micromechanisms of progression and regression of human atherosclerotic plaques the present study was designed for characterization of crystal lattices of cholesterol monohydrate, demonstration of cholesterol crystallization processes, and identification of crystal disorders of cholesterol monohydrate by means of high resolution electron microscopy (HREM).Clusters of cholesterol clefts containing crystalline cholesterol monohydrate were obtained from atherosclerotic human aortas under a dissecting microscope at autopsy. Cholesterol clefts were milled to a powder with a porcelain pestle. The powdered samples without staining dispersed in distilled water were spread on the copper-grid and dried at room temperature. The air-dried specimens were directly examined by a Hitachi H-9000 high resolution electron microscope operated at 300 KV. High resolution lattice images were taken under an optimal defocus condition. Selected area images and electron diffraction patterns were simultaneously taken for the analysis of crystalline characteristics.

High-resolution electron microscopic investigation of crystalline cholesterol monohydrate in human atheroma at the atomic level

High-resolution electron microscopic investigation of cholesterol monohydrate crystals obtained from human atheroma was carried out for the purpose of characterization of the crystal lattice, demonstration of crystallization processes and identification of crystal disorders. By high-resolution electron microscopy the crystal structures of perfect cholesterol monohydrate crystals were characterized as regular lattice arrays which consisted of stacks of repetitive rod-shaped substructures ca 1.58 nm long and 0.16 nm wide, with the total thickness of bilayered substructures ca 3.36 nm. These substructures were in an end-to-end arrangement of approximately side-to-side parallel packing, with a centre-to-centre spacing ca 0.32 nm. At the atomic level the lattice arrays were made up of regularly spaced rows of dots ca 0.28 nm × 0.16 nm in size. These dots possessed a six-fold ring-like shape, and were arranged in a hexagonal structure with an additional dot in the centre. High-resolution electron microscopic observations of the partially crystallized particles of cholesterol monohydrate showed various stages of cholesterol crystallization, from very small short-ordered segment of lattice arrays to different sized nano- and microcrystallites in the amorphous matrix of the crystals. Furthermore, crystal growth was also demonstrated from detailed examination of the crystal surfaces, the interfaces between the crystals and the boundary structures between the amorphous and crystalline phases. In addition, high-resolution electron microscopy could clearly identify various kinds of crystal defect in the cholesterol monohydrate crystals, including considerable variations of lattice spacings with focal fragmentation of lattice fringes, derangement of atom-sized dots along the lattice fringes and marked alterations of the morphology of atom-sized dots with the vacancies along the lattice arrays. It is hoped that such information obtained from high-resolution electron microscopic observations of the crystalline cholesterol in human atheroma at the atomic or near-atomic level may be helpful by providing a more complete understanding of the pathogenetic mechanisms responsible for the formation, progression and regression of the acellular lipid-rich cores of advanced atherosclerotic plaques.

Cholesterol gallstone dissolution in bile. Dissolution kinetics of crystalline cholesterol monohydrate by conjugated chenodeoxycholate-lecithin and conjugated ursodeoxycholate-lecithin mixtures: dissimilar phase equilibria and dissolution mechanisms

Using compressed discs and microcrystals of cholesterol monohydrate, we evaluated the mechanisms and kinetics of dissolution in conjugated bile salt-lecithin solutions. In stirred conjugated ursodeoxycholate-lecithin and cheno-deoxycholate-lecithin solutions, dissolution of 10,000-psi discs was micellar and linear with time for 10 hours. The dissolution rate constants (k) decreased in proportion to the lecithin content and dissolution rates and k values were appreciably smaller in conjugated ursodeoxycholate-lecithin solutions. After dissolution for 5 to 10 days the discs incubated with ursodeoxycholate-lecithin systems became progressively transformed into macroscopic liquid crystals. Unstirred dissolution of 3,000-psi discs in "simulated" human bile containing physiological lecithin concentrations gave apparent k values that decreased in the following order: ursodeoxycholate-rich >/= chenodeoxycholate-rich > normal. In most cases the discs incubated with ursodeoxycholate-rich bile became covered with a microscopic liquid-crystalline layer. With 20-25 moles % lecithin, these layers eventually dispersed into the bulk solution as microscopic vesicles. During dissolution of microcrystalline cholesterol in conjugated ursodeoxycholate-lecithin systems, a bulk liquid-crystalline phase formed rapidly (within 12 hours) and the final cholesterol solubilities were greater than those in conjugated chenodeoxycholate-lecithin micellar systems. Prolonged incubation of cholesterol microcrystals with pure lecithin or lecithin plus bile salt liposomes did not reproduce these effects. Condensed ternary phase diagrams of conjugated ursodeoxycholate-lecithin-cholesterol systems established that cholesterol-rich liquid crystals constituted an equilibrium precipitate phase that coexisted with cholesterol monohydrate crystals and saturated micelles under physiological conditions. Similar phase dissolution-relationships were observed at physiological lecithin-bile salt ratios for a number of other hydrophilic bile salts (e.g., conjugated ursocholate, hyocholate, and hyodeoxycholate). In contrast, liquid crystals were not observed in conjugated chenodeoxycholate-lecithin-cholesterol systems except at high (nonphysiological) lecithin contents. Based on these and other results we present a molecular hypothesis for cholesterol monohydrate dissolution by any bile salt-lecithin system and postulate that enrichment of bile with highly hydrophilic bile salts will induce crystalline cholesterol dissolution by a combination of micellar and liquid crystalline mechanisms. Since bile salt polarity can be measured and on this basis the ternary phase diagram deduced, we believe that the molecular mechanisms of cholesterol monohydrate dissolution as well as the in vivo cholelitholytic potential of uncommon bile salts can be predicted.-Salvioli, G., H. Igimi, and M. C. Carey. Cholesterol gallstone dissolution in bile. Dissolution kinetics of crystalline cholesterol monohydrate by conjugated chenodeoxycholate-lecithin and conjugated ursodeoxycholate-lecithin mixtures: dissimilar phase equilibria and dissolution mechanisms.

On the nature of dilute aqueous cholesterol suspensions

Cholesterol is commonly supposed to form a micelle in aqueous solution. We have reassessed this finding using a variety of techniques. Centrifugation sedimented cholesterol at all concentrations above 10(-8) M. Density gradient analysis of more concentrated solutions revealed two bands whose densities corresponded to crystalline anhydrous cholesterol and crystalline cholesterol monohydrate. The monohydrate was characterized by light microscopy and phase transition. This band is also claimed to contain micelles, but no enhancement of perylene or diphenylhexatriene fluorescence could be detected. Some crystals of monohydrate could pass through 0.7-microns filters but not through 1.5-nm filters. Crystals of monohydrate were detected on the latter filters when solutions with cholesterol concentrations as low as 2.6 X 10(-8) M were filtered. Thus, under all conditions where micelles might be expected, we have detected micro-crystals of cholesterol monohydrate and we can find no independent evidence to support the existence of a cholesterol micelle.