Different interactions of egg-yolk phosphatidylcholine and sphingomyelin with detergent bile salts

To examine physical-chemical aspects of bile salt-phospholipid interactions that could contribute to preferential phosphatidylcholine (PC) secretion into bile, we have compared transitions between vesicles and micelles in model systems containing taurocholate (TC) and either egg-yolk PC (EYPC), egg-yolk sphingomyelin (EYSM), buttermilk SM (BMSM) or dipalmitoyl PC (DPPC). Phase transitions from micelles to vesicles were observed at 4-fold dilution of serially diluted EYPC/TC systems, but not earlier than at 16-fold dilution of SM/TC or DPPC/TC systems, indicating lower concentrations of the detergent required for micellization in the case of SM or DPPC. Cryo-transmission electron microscopy of phase transitions initiated by addition of TC to phospholipid vesicles revealed extremely long SM-containing intermediate structures, but shorter EYPC-containing intermediate structures. Again, larger amounts of bile salt were required to induce phase transitions in the case of EYPC compared to SM. Sizes of TC-phospholipid micelles increased progressively upon increasing phospholipid contents in the rank order: DPPC-TC相似文献   

Biliary cholesterol crystallization characterized by single-crystal cryogenic electron diffraction

Cholesterol crystals are the building blocks of cholesterol gallstones. The exact structure of early-forming crystals is still controversial. We combined cryogenic-temperature transmission electron microscopy with cryogenic-temperature electron diffraction to sequentially study crystal development and structure in nucleating model and native gallbladder biles. The growth and long-term stability of classic cholesterol monohydrate (ChM) crystals in native and model biles was determined. In solutions of model bile with low phospholipid-to-cholesterol ratio, electron diffraction provided direct proof of a novel transient polymorph that had an elongated habit and unit cell parameters differing from those of classic triclinic ChM. This crystal is exactly the monoclinic ChM phase described by Solomonov and coworkers (Biophysical J., In press) in cholesterol monolayers compressed on the air-water interface. We observed no evidence of anhydrous cholesterol crystallization in any of the biles studied. In conclusion, classic ChM is the predominant and stable form in native and model biles. However, under certain (low phospholipid) conditions, transient intermediate polymorphs may form. These findings, documenting single-crystal analysis in bulk solution, provide an experimental approach to investigating factors influencing biliary cholesterol crystal nucleation and growth as well as other processes of nucleation and crystallization in liquid systems.     

Physical-chemical behavior of dietary and biliary lipids during intestinal digestion and absorption. 1. Phase behavior and aggregation states of model lipid systems patterned after aqueous duodenal contents of healthy adult human beings

We developed equilibrium phase diagrams corresponding to aqueous lipid compositions of upper small intestinal contents during lipid digestion and absorption in adult human beings. Ternary lipid systems were composed of a physiological mixture of bile salts (BS), mixed intestinal lipids (MIL), principally partially ionized fatty (oleic) acid (FA) plus racemic monooleylglycerol (MG), and cholesterol (Ch), all at fixed aqueous-electrolyte concentrations, pH, temperature, and pressure. The condensed phase diagram for typical physiological conditions (1 g/dL total lipids, FA:MG molar ratio of 5:1, pH 6.5, 0.15 M Na+ at 37 degrees C) was similar to that of a dilute model bile [BS/lecithin (PL)/Ch] system [Carey, M. C., & Small, D. M. (1978) J. Clin. Invest. 61, 998-1026]. We identified two one-phase zones composed of mixed micelles and lamellar liquid crystals, respectively, and two two-phase zones, one composed of Ch monohydrate crystals and Ch-saturated micelles and the other of physiologic relevance composed of Ch- and MIL-saturated mixed micelles and unilamellar vesicles. A single large three-phase zone in the system was composed of Ch-saturated micelles, Ch monohydrate crystals, and liquid crystals. Micellar phase boundaries for otherwise typical physiological conditions were expanded by increases in total lipid concentration (0.25-5 g/dL), pH (5.5-7.5), and FA:MG molar ratio (5-20:1), resulting in a reduction of the size of the physiological two-phase zone. Mean particle hydrodynamic radii (Rh), measured by quasielastic light scattering (QLS), demonstrated an abrupt increase from micellar (less than 40 A) to micelle plus vesicle sizes (400-700 A) as this two-phase zone was entered. With relative lipid compositions within this zone, unilamellar vesicles formed spontaneously following coprecipitation, and their sizes changed markedly as functions of time, reaching equilibrium values only after 4 days. Further, vesicle Rh values were influenced appreciably by MIL:mixed bile salt (MBS) ratio, pH, total lipid concentration, and FA:MG ratio, but not by Ch content. In comparison, micellar systems equilibrated rapidly, and their Rh values only slightly influenced by physical-chemical variables of physiological importance. In contrast to the BS-PL-Ch system [Mazer, N. A., & Carey, M. C. (1983) Biochemistry 22, 426-442], no divergence in micellar sizes occurred as the micellar phase boundary was approached. The ionization state of FA at simulated "intestinal" pH values (5.5-7.5) in the micellar and physiologic two-phase zones was principally that of 1:1 sodium hydrogen dioleate, an insoluble swelling "acid soap" compound. By phase separation and analysis, tie-lines for the constituent phase in the two-phase zone demonstrated that the mixed micelles were saturated with MIL and Ch and the coexisting vesicles were saturated with MBS, but not with Ch.(ABSTRACT TRUNCATED AT 400 WORDS)     

Incorporation of cholesterol in sphingomyelin- phosphatidylcholine vesicles has profound effects on detergent-induced phase transitions

Vesicle <--> micelle transitions are important phenomena during bile formation and intestinal lipid processing. The hepatocyte canalicular membrane outer leaflet contains appreciable amounts of phosphatidylcholine (PC) and sphingomyelin (SM), and both phospholipids are found in the human diet. Dietary SM enrichment inhibits intestinal cholesterol absorption. We therefore studied detergent-induced vesicle --> micelle transitions in SM-PC vesicles. Phase transitions were evaluated by spectrophotometry and cryotransmission electron microscopy (cryo-TEM) after addition of taurocholate (3-7 mM) to SM-PC vesicles (4 mM phospholipid, SM/PC 40%/60%, without or with 1.6 mM cholesterol). After addition of excess (5-7 mM) taurocholate, SM-PC vesicles were more sensitive to micellization than PC vesicles. As shown by sequential cryo-TEM, addition of equimolar (4 mM) taurocholate to SM-PC vesicles induced formation of open vesicles, then (at the absorbance peak) fusion of bilayer fragments into large open structures (around 200 nm diameter) coexisting with some multilamellar or fused vesicles and thread-like micelles and, finally, transformation into an uniform picture with long thread-like micelles. Incorporation of cholesterol in the SM/PC bilayer changed initial vesicular shape from spherical into ellipsoid and profoundly increased detergent resistance. Disk-like micelles and multilamellar vesicles, and then extremely large vesicular structures, were observed by sequential cryo-TEM under these circumstances, with persistently increased absorbance values by spectrophotometry. These findings may be relevant for bile formation and intestinal lipid processing. Inhibition of intestinal cholesterol absorption by dietary SM enrichment may relate to high resistance against bile salt-induced micellization of intestinal lipids in presence of the sphingolipid.     

Cholesterol crystallization in model biles: effects of bile salt and phospholipid species composition

Cholesterol in human bile is solubilized in micelles by (relatively hydrophobic) bile salts and phosphatidylcholine (unsaturated acyl chains at sn-2 position). Hydrophilic tauroursodeoxycholate, dipalmitoyl phosphatidylcholine, and sphingomyelin all decrease cholesterol crystal-containing zones in the equilibrium ternary phase diagram (van Erpecum, K. J., and M. C. Carey. 1997. Biochim. Biophys. Acta. 1345: 269-282) and thus could be valuable in gallstone prevention. We have now compared crystallization in cholesterol-supersaturated model systems (3.6 g/dl, 37 degrees C) composed of various bile salts as well as egg yolk phosphatidylcholine (unsaturated acyl chains at sn-2 position), dipalmitoyl phosphatidylcholine, or sphingomyelin throughout the phase diagram. At low phospholipid contents [left two-phase (micelle plus crystal-containing) zone], tauroursodeoxycholate, dipalmitoyl phosphatidylcholine, and sphingomyelin all enhanced crystallization. At pathophysiologically relevant intermediate phospholipid contents [central three-phase (micelle plus vesicle plus crystal-containing) zone], tauroursodeoxycholate inhibited, but dipalmitoyl phosphatidylcholine and sphingomyelin enhanced, crystallization. Also, during 10 days of incubation, there was a strong decrease in vesicular cholesterol contents and vesicular cholesterol-to-phospholipid ratios (approximately 1 on day 10), coinciding with a strong increase in crystal mass. At high phospholipid contents [right two-phase (micelle plus vesicle-containing) zone], vesicles were always unsaturated and crystallization did not occur. Strategies aiming to increase amounts of hydrophilic bile salts may be preferable to increasing saturated phospholipids in bile, because the latter may enhance crystallization.     

Influence of total lipid concentration, bile salt:lecithin ratio, and cholesterol content on inter-mixed micellar/vesicular (non-lecithin-associated) bile salt concentrations in model bile.

We modified classic equilibrium dialysis methodology to correct for dialysant dilution and Donnan effects, and have systematically studied how variations in total lipid concentration, bile salt (taurocholate):lecithin (egg yolk) ratio, and cholesterol content influence inter-mixed micellar/vesicular (non-lecithin-associated) concentrations (IMC) of bile salts (BS) in model bile. To simulate large volumes of dialysant, the total volume (1 ml) of model bile was exchanged nine times during dialysis. When equilibrium was reached, dialysate BS concentrations plateaued, and initial and final BS concentrations in the dialysant were identical. After corrections for Donnan effects, IMC values were appreciably lower than final dialysate BS concentrations. Quasielastic light scattering was used to validate these IMC values by demonstrating that lipid particle sizes and mean scattered light intensities did not vary when model biles were diluted with aqueous BS solutions of the appropriate IMC. Micelles and vesicles were separated from cholesterol-supersaturated model bile, utilizing high performance gel chromatography with an eluant containing the IMC. Upon rechromatography of micelles and vesicles using an identical IMC, there was no net transfer of lipid between micelles and vesicles. To simulate dilution during gel filtration, model biles were diluted with 10 mM Na cholate, the prevailing literature eluant, resulting in net transfer of lipid between micelles and vesicles, the direction of which depended upon total lipid concentration and BS/lecithin ratio. Using the present methodology, we demonstrated that inter-mixed micellar/vesicular concentrations (IMC) values increased strongly (5 to 40 mM) with increases in both bile salt (BS):lecithin ratio and total lipid concentration, whereas variations in cholesterol content had no appreciable effects. For model biles with typical physiological biliary lipid compositions, IMC values exceeded the critical micellar concentration of the pure BS, implying that in cholesterol-supersaturated biles, simple BS micelles coexist with mixed BS/lecithin/cholesterol micelles and cholesterol/lecithin vesicles. We believe that this methodology allows the systematic evaluation of IMC values, with the ultimate aim of accurately separating micellar, vesicular, and potential other cholesterol-carrying particles from native bile.     

Biliary lipids, water and cholesterol gallstones

Cholesterol supersaturation, hydrophobic bile salts, pronucleating proteins and impaired gall-bladder motility may contribute to gallstone pathogenesis. We here show that both gallstone-susceptible C57L and gallstone-resistant AKR male inbred mice exhibit supersaturated gall-bladder biles during early lithogenesis, whereas bile-salt composition becomes hydrophobic only in susceptible C57L mice. In vitro, cholesterol crystallization occurs depending on relative amounts of lipids; excess cholesterol may exceed solubilizing capacity of mixed bile salt-phospholipid micelles, whereas excess bile salts compared with phospholipids leads to deficient cholesterol-storage capacity in vesicles. In vivo, bile lipid contents are mainly determined at the level of the hepatocyte canalicular membrane, where specific transport proteins enable lipid secretion [ABCG5/G8 (ATP-binding cassette transporter G5/G8) for cholesterol, MDR3 (multi-drug resistant 3) for phospholipid, BSEP (bile salt export pump)]. These transport proteins are regulated by farnesoid X and liver X nuclear receptors. After nascent bile formation, modulation of bile water contents in biliary tract and gall-bladder exerts critical effects on cholesterol crystallization. During progressive bile concentration (particularly in the fasting gall-bladder), cholesterol and, preferentially, phospholipid transfer occurs from cholesterol-unsaturated vesicles to emerging mixed micelles. The remaining unstable cholesterol-enriched vesicles may nucleate crystals. Various aquaporins have recently been discovered throughout the biliary tract, with potential relevance for gallstone formation.     

Measurement of cholesterol gallstone growth in vitro

Methods to study growth of gallstones in the laboratory have not been reported. We here present such a method. Human cholesterol gallstones were harvested from patients with multiple nearly identical stones. The gallstones were washed and added to supersaturated model biles and the formation of cholesterol crystals and the increases in mass of human cholesterol gallstones were studied concurrently, over a period of weeks, using nephelometry and a microbalance, respectively. All stones incubated in model biles supersaturated with cholesterol increased in mass. Increases in the degree of supersaturation of cholesterol in the model biles resulted in increased growth of stones. The mass increases, the growth rates, and the spatial orientation of accreted crystalline cholesterol differed among various stone types. The kinetics and structures of stone growth were similar when the stones were incubated in supersaturated, native, human gallbladder biles. The structure of accreted cholesterol was the same as found on the surface of some human gallstones that were harvested during apparent active growth in situ. This simple method allows accurate measurements of stone growth in vitro, in patterns that mimic stone growth in vivo, and is useful for studies on the relationships of gallstone growth and the kinetics of cholesterol crystallization.     

No pathophysiologic relationship of soluble biliary proteins to cholesterol crystallization in human bile

This study explores the pathophysiologic effects of soluble biliary glycoproteins in comparison to mucin gel and cholesterol content on microscopic crystal and liquid crystal detection times as well as crystallization sequences in lithogenic human biles incubated at 37 degrees C. Gallbladder biles from 13 cholesterol gallstone patients were ultracentrifuged and microfiltered (samples I). Total biliary lipids were extracted from portions of samples I, and reconstituted with 0.15 m NaCl (pH 7.0) (samples II). Portions of samples II were supplemented with purified concanavalin A-binding biliary glycoproteins (final concentration = 1 mg/mL) (samples III), or mucin gel (samples IV), respectively, isolated from the same cholesterol gallstone biles. Samples V consisted of extracted biliary lipids from uncentrifuged and unfiltered bile samples reconstituted with 0.15 m NaCl (pH 7.0). Analytic lipid compositions of samples I through IV were identical for individual biles but, as anticipated, samples V displayed significantly higher cholesterol saturation indexes. Detection times of cholesterol crystals and liquid crystals were accelerated in the rank order of samples: IV > V > I = II = III, indicating that total soluble biliary glycoproteins in pathophysiologic concentration had no appreciable effect. Crystallization sequences (D. Q-H. Wang and M. C. Carey. J. Lipid Res. 1996. 37: 606-630; and 2539-2549) were similar among samples I through V. Crystal detection times and numbers of solid cholesterol crystals were accelerated in proportion to added mucin gel and the cholesterol saturation of bile only.For pathophysiologically relevant conditions, our results clarify that mucin gel and cholesterol content, but not soluble biliary glycoproteins, promote cholesterol crystallization in human gallbladder bile.     

Cryoelectron microscopy of a nucleating model bile in vitreous ice: formation of primordial vesicles

Because gallstones form so frequently in human bile, pathophysiologically relevant supersaturated model biles are commonly employed to study cholesterol crystal formation. We used cryo-transmission electron microscopy, complemented by polarizing light microscopy, to investigate early stages of cholesterol nucleation in model bile. In the system studied, the proposed microscopic sequence involves the evolution of small unilamellar to multilamellar vesicles to lamellar liquid crystals and finally to cholesterol crystals. Small aliquots of a concentrated (total lipid concentration = 29.2 g/dl) model bile containing 8.5% cholesterol, 22.9% egg yolk lecithin, and 68.6% taurocholate (all mole %) were vitrified at 2 min to 20 days after fourfold dilution to induce supersaturation. Mixed micelles together with a category of vesicles denoted primordial, small unilamellar vesicles of two distinct morphologies (sphere/ellipsoid and cylinder/arachoid), large unilamellar vesicles, multilamellar vesicles, and cholesterol monohydrate crystals were imaged. No evidence of aggregation/fusion of small unilamellar vesicles to form multilamellar vesicles was detected. Low numbers of multilamellar vesicles were present, some of which were sufficiently large to be identified as liquid crystals by polarizing light microscopy. Dimensions, surface areas, and volumes of spherical/ellipsoidal and cylindrical/arachoidal vesicles were quantified. Early stages in the separation of vesicles from micelles, referred to as primordial vesicles, were imaged 23-31 min after dilution. Observed structures such as enlarged micelles in primordial vesicle interiors, segments of bilayer, and faceted edges at primordial vesicle peripheries are probably early stages of small unilamellar vesicle assembly. A decrease in the mean surface area of spherical/ellipsoidal vesicles was correlated with the increased production of cholesterol crystals at 10-20 days after supersaturation by dilution, supporting the role of small unilamellar vesicles as key players in cholesterol nucleation and as cholesterol donors to crystals. This is the first visualization of an intermediate structure that has been temporally linked to the development of small unilamellar vesicles in the separation of vesicles from micelles in a model bile and suggests a time-resolved system for further investigation.     

Effect of gallbladder hypomotility on cholesterol crystallization and growth in CCK-deficient mice

We investigated the effect of gallbladder hypomotility on cholesterol crystallization and growth during the early stage of gallstone formation in CCK knockout mice. Contrary to wild-type mice, fasting gallbladder volumes were enlarged and the response of gallbladder emptying to a high-fat meal was impaired in knockout mice on chow or the lithogenic diet. In the lithogenic state, large amounts of mucin gel and liquid crystals as well as arc-like and tubular crystals formed first, followed by rapid formation of classic parallelogram-shaped cholesterol monohydrate crystals in knockout mice. Furthermore, three patterns of crystal growth habits were observed: proportional enlargement, spiral dislocation growth, and twin crystal growth, all enlarging solid cholesterol crystals. At day 15 on the lithogenic diet, 75% of knockout mice formed gallstones. However, wild-type mice formed very little mucin gel, liquid, and solid crystals, and gallstones were not observed. We conclude that lack of CCK induces gallbladder hypomotility that prolongs the residence time of excess cholesterol in the gallbladder, leading to rapid crystallization and precipitation of solid cholesterol crystals. Moreover, during the early stage of gallstone formation, there are two pathways of liquid and polymorph anhydrous crystals evolving to monohydrate crystals and three modes for cholesterol crystal growth.     

Guanidination of notexin alters its membrane-damaging activity in response to sphingomyelin and cholesterol

To elucidate the contribution of phospholipase A₂ (PLA₂) activity of notexin to its ability to perturb membranes, comparative studies on the interaction of notexin and guanidinated notexin (Gu-notexin) with egg yolk phosphatidylcholine (EYPC), EYPC/egg yolk sphingomyelin (EYSM) and EYPC/EYSM/cholesterol vesicles were conducted. EYSM notably reduced the membrane-damaging activity of notexin against EYPC vesicles, but had an insignificant influence on that of Gu-notexin. Unlike the effects noted with notexin, inactivation of PLA₂ activity by EDTA led to a reduction in the ability of Gu-notexin to induce EYPC/EYSM vesicle leakage and to increase Gu-notexin-induced membrane permeability of EYPC/EYSM/cholesterol vesicles. The geometrical arrangement of notexin and Gu-notexin in contact with either EYPC/EYSM vesicles or EYPC/EYSM/cholesterol vesicles differed. Moreover, global conformation of notexin and Gu-notexin differed in either Ca²⁺-bound or metal-free states. These results indicate that notexin and Gu-notexin could induce membrane permeability without the involvement of PLA₂ activity, and suggest that guanidination alters the membrane-bound mode of notexin on damaging phospholipid vesicles containing sphingomyelin and cholesterol.     

Cholesterol-phospholipid vesicles in human bile: an ultrastructural study

Phospholipid vesicles, a newly described (bile salt independent) mode of cholesterol transport in human bile, were previously characterized by quasi-elastic light scattering and gel filtration. In the present study the ultrastructure of these vesicles was investigated by electron microscopy using freeze-fracture and negative-staining techniques. Vesicles of varying size were found in all 14 hepatic and 3 gallbladder biles examined. The diameter of the vesicles ranged from 25 to 75 nm by electron microscopy after freeze fracture and from 54 to 94 nm by quasi-elastic light scattering. They had a spherical shape and appeared to be unilamellar. The appearance of the vesicles in fresh hepatic and gallbladder biles as well as in chromatographic fractions was similar. Vesicles were dissolved by the addition of exogenous bile salts. Cholesterol is transported in human bile by both vesicles and micelles. The role of the vesicles may be particularly important in preventing cholesterol precipitation in dilute and supersaturated biles.     

Rapid (1 hour) high performance gel filtration chromatography resolves coexisting simple micelles, mixed micelles, and vesicles in bile

We describe the use and validation of Superose 6, a high performance gel filtration medium for rapid, high resolution separation and sizing of coexisting simple micelles, mixed micelles, and vesicles in bile. We fractionated model biles (1.7-4.2 g/dl total lipid concentration, 0.15 M NaCl) composed of lecithin (L), cholesterol (Ch), and the common bile salt taurocholate (TC) using Superose 6 gel filtration columns (1.0 cm diameter, 30 cm length, 0.5 ml model bile application, 1.0 ml fractions) pre-equilibrated and eluted with 2.5-10.0 mM TC. Lipid particle sizes were determined by quasielastic light scattering and lipid compositions by conventional analyses. In the absence of L and Ch, pure TC "biles" (32.2 mM), when eluted in the presence of 7.5 mM TC, yielded a single peak of particles (mean hydrodynamic radii, Rh values of 11-15 A), consistent with simple TC micelles. Model biles containing L and TC ([L] = 13.8 mM, [TC] = 32.2 mM) were fractionated with baseline resolution into TC-L mixed micelles, (Rh values of 30-40 A) and simple TC micelles. In agreement with the ternary TC-L-H2O phase diagram (Mazer, N. A., et al. 1980. Biochemistry. 19: 601-615), the proportions of simple and mixed micelles were inversely related to L concentrations ([L] = 0-32.2 mM) and correlated positively with eluant TC concentration. Superose 6 gel fractionation of model biles "super-saturated" with Ch (TC:L:Ch molar ratio 27:63:10, total lipid concentration 3 g/dl) yielded high resolution separation of vesicles (Rh value of 320 A) from mixed micelles of TC-L-Ch (Rh values of 40-50 A) and simple TC micelles (Rh values of 11-15 A). At an eluant TC concentration of 7.5 mM, Ch-rich vesicles (Ch/L molar ratio = 1.6) separated that contained 40% of total Ch, 9% of total L, and no TC, accurately reflecting predictions of the quaternary L-Ch-TC-H2O metastable phase diagram (Mazer, N. A., and M. C. Carey. 1983 Biochemistry. 22: 426-442). This suggested that a 7.5 mM TC concentration approximated the intermicellar concentration under the experimental conditions. We also fractionated an identical model bile using conventional Sephacryl S-300, a medium generally used to study model and native biles. Compared with Superose 6, the Sephacryl S-300 column of equivalent size yielded particle separations with lower resolution and speed (30 h v l h).(ABSTRACT TRUNCATED AT 400 WORDS)     

Bovine gallbladder mucin promotes cholesterol crystal nucleation from cholesterol-transporting vesicles in supersaturated model bile

This study examined the ability of purified gallbladder mucin to accelerate the nucleation of cholesterol monohydrate crystals from the cholesterol-transporting particles in supersaturated model bile. Mixed lipid micelles and cholesterol-phosphatidylcholine vesicles in supersaturated model bile were separated by Sephadex G-200 column chromatography. Mixed lipid micelles prepared by column chromatography had a low cholesterol-phosphatidylcholine ratio (0.30) and did not spontaneously nucleate cholesterol monohydrate crystals. In contrast, vesicles prepared by column chromatography had a cholesterol-phosphatidylcholine ratio of 1.00 and nucleated cholesterol crystals rapidly (P less than 0.001). Nucleation of cholesterol crystals was significantly accelerated in a concentration- and time-dependent manner by purified bovine gallbladder mucin in cholesterol containing vesicles, but not in mixed lipid micelles (P less than 0.001). A rapid filtration binding assay demonstrated significant binding of cholesterol and phosphatidylcholine in vesicles to gallbladder mucin but only minimal binding of cholesterol and phosphatidylcholine in mixed micelles. These data indicate that gallbladder mucin binds cholesterol and phosphatidylcholine in vesicles and accelerates the nucleation of cholesterol monohydrate crystals from these cholesterol-transporting particles in supersaturated model bile.     

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.     

Factors affecting cholesterol monohydrate crystal nucleation time in model systems of supersaturated bile

We explored the influence of several compositional factors considered capable of influencing the nucleation time of model biles supersaturated in cholesterol. In addition to the classical techniques, e.g., electron microscopy and quasielastic light scattering, employed for size measurement and structural assessment, we employed a novel technique, i.e., video-enhanced microscopy, for particle evaluation in these polydisperse systems which often may simultaneously contain isolated small vesicles, their complex aggregates, and small cholesterol monohydrate crystals. The factors we studied included dilution, degree of cholesterol supersaturation, bile salt/lecithin molar ratio, and Ca2+ concentration. Dilution markedly raised the degree of cholesterol saturation, prolonged nucleation time for cholesterol monohydrate crystals, and favored formation of metastable small unilamellar vesicles. Increasing the degree of cholesterol supersaturation as an independent variable in more concentrated systems both shortened the nucleation time and favored spontaneous formation of a relatively small number of isolated vesicles. A decrease in bile salt/lecithin molar ratio within the physiologically relevant range was accompanied by a prolonged nucleation time and favored spontaneous vesicle formation. Large numbers of small unilamellar vesicles were observed even in concentrated model bile solutions (total lipids: 20 g/dl) when the bile salt/lecithin molar ratio was 1.9 or less. At physiological concentrations, Ca2+ promoted nucleation of cholesterol monohydrate crystals only in vesicle-containing solutions. Taken together, the following conclusions can be drawn. First, spontaneous vesicle formation in dilute systems prolongs solid cholesterol crystal nucleation. It can thus provide a supplementary non-micellar mode of cholesterol transport in micellar systems of supersaturated human bile. Second, dilution, degree of cholesterol supersaturation, and a decrease in bile salt/lecithin ratio prolong cholesterol crystal nucleation time and favor spontaneous vesicle formation. With increasing calcium concentrations, opposite effects are observed. Third, the presence of vesicles may help to account for the frequently observed and otherwise unexplained remarkable degree of metastable supersaturation and prolonged metastability (delayed nucleation time) for cholesterol in human bile.     

Characterization of model bile using fluorescence energy transfer from dehydroergosterol to dansylated lecithin

Fluorescence energy transfer from dehydroergosterol (DHE) to dansylated lecithin (DL) was used to characterize lecithin-cholesterol vesicles in the presence of the bile salt, sodium taurocholate. At lipid concentrations approximating physiological levels, exposure of fluorescently labeled vesicles to the bile salt led to a dose-dependent increase in the DHE-to-DL fluorescence ratio during the first 24 h after mixing. The initial changes in the fluorescence ratio correlated well with conventional turbidity measurements that quantify partial micellization of vesicles as a function of bile salt loading. In addition, fluorescence energy transfer from DHE to DL revealed cholesterol enrichment of vesicles and re-vesiculation of micelles at bile salt loadings for which vesicles and micelles coexisted. Samples containing the cholesterol-enriched vesicle fraction exhibited further increases in the DHE-to-DL fluorescence ratio during a 4-week observation period but only after a significant lag period of several days. The lag period decreased with cholesterol loading, and the increase in the fluorescence ratio always preceded the appearance of microscopic, birefringent, either needlelike or platelike, cholesterol crystals, in samples that were initially supersaturated with cholesterol. Cholesterol crystals were not observed, and the fluorescence ratio did not increase, for any sample that was undersaturated with cholesterol.Taken together, these results suggest that the latter changes in fluorescence are the result of cholesterol nucleation. Fluorescence energy transfer from DHE to DL is therefore a promising technique for the characterization of model bile and, possibly, provides a direct measurement of cholesterol nucleation.     

Influence of Cholesterol and ��-Sitosterol on the Structure of EYPC Bilayers

The influence of cholesterol and β-sitosterol on egg yolk phosphatidylcholine (EYPC) bilayers is compared. Different interactions of these sterols with EYPC bilayers were observed using X-ray diffraction. Cholesterol was miscible with EYPC in the studied concentration range (0-50 mol%), but crystallization of β-sitosterol in EYPC bilayers was observed at X ≥ 41 mol% as detected by X-ray diffraction. Moreover, the repeat distance (d) of the lamellar phase was similar upon addition of the two sterols up to mole fraction 17%, while for X ≥ 17 mol% it became higher in the presence of β-sitosterol compared to cholesterol. SANS data on suspensions of unilamellar vesicles showed that both cholesterol and β-sitosterol similarly increase the EYPC bilayer thickness. Cholesterol in amounts above 33 mol% decreased the interlamellar water layer thickness, probably due to "stiffening" of the bilayer. This effect was not manifested by β-sitosterol, in particular due to the lower solubility of β-sitosterol in EYPC bilayers. Applying the formalism of partial molecular areas, it is shown that the condensing effect of both sterols on the EYPC area at the lipid-water interface is small, if any. The parameters of ESR spectra of spin labels localized in different regions of the EYPC bilayer did not reveal any differences between the effects of cholesterol and β-sitosterol in the range of full miscibility.     

Cholesterol crystal formation and growth in model bile solutions

Cholesterol monohydrate crystal formation was studied in supersaturated model bile solutions, containing unlabeled cholesterol, sodium cholate and soybean phosphatidylcholine, and tracer amounts of [3H]cholesterol. Solutions were either seeded with cholesterol crystals to initiate growth, or not seeded to allow self-nucleation and subsequent crystal growth to occur. Crystal growth at 37 degrees C was measured by two methods. First, radioactive cholesterol crystals were isolated by filtration, and the mass of cholesterol that had precipitated was calculated. In unseeded solutions, there was a long lag period before crystal growth was detected. This lag time was decreased by increases in the cholesterol concentration, temperature, and lipid concentration. In seeded solutions, crystal growth also was dependent on the cholesterol concentration, temperature, and lipid concentration. The second method used to measure crystal growth involved the Coulter Counter. At 37 degrees C, reproducible results were not obtained using unseeded solutions due to blocking of the counter aperture with large crystals. In seeded solutions, crystal growth could be measured as an increase in total particle volume. However, comparison of growth rate estimates from the Coulter Counter with those obtained radiochemically revealed poor agreement between the two methods. It is probable that the Coulter Counter is inaccurate in measuring the volume of cholesterol monohydrate crystals due to their anisometric shape.