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.     

Accurate separation of vesicles, micelles and cholesterol crystals in supersaturated model biles by ultracentrifugation, ultrafiltration and dialysis.

Gel filtration with bile salts at intermixed micellar/vesicular concentrations (IMC) in the eluant has been proposed to isolate vesicles and micelles from supersaturated model biles, but the presence of vesicular aggregates makes this method unreliable. We have now validated a new method for isolation of various phases. First, aggregated vesicles and - if present - cholesterol crystals are pelleted by short ultracentrifugation. Cholesterol contained in crystals and vesicular aggregates can be quantitated from the difference of cholesterol contents in the pellets before and after bile salt-induced solubilization of the vesicular aggregates. Micelles are then isolated by ultrafiltration of the supernatant through a highly selective 300 kDa filter and unilamellar vesicles by dialysis against buffer containing bile salts at IMC values. Lipids contained in unilamellar vesicles are also estimated by subtraction of lipid contents in filtered micelles from lipid contents in (unilamellar vesicle+micelle containing) supernatant ('subtraction method'). 'Ultrafiltration-dialysis' and 'subtraction' methods yielded identical lipid solubilization in unilamellar vesicles and identical vesicular cholesterol/phospholipid ratios. In contrast, gel filtration yielded much more lipids in micelles and less in unilamellar vesicles, with much higher vesicular cholesterol/phospholipid ratios. When vesicles obtained by dialysis were analyzed by gel filtration, vesicular cholesterol/phospholipid ratios increased strongly, despite correct IMC values for bile salts in the eluant. Subsequent extraction of column material showed significant amounts of lipids. In conclusion, gel filtration may underestimate vesicular lipids and overestimate vesicular cholesterol/phospholipid ratios, supposedly because of lipids remaining attached to the column. Combined ultracentrifugation-ultrafiltration-dialysis should be considered state-of-the-art methodology for quantification of cholesterol carriers in model biles.     

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.     

Structural alterations in lecithin-cholesterol vesicles following interactions with monomeric and micellar bile salts: physical-chemical basis for subselection of biliary lecithin species and aggregative states of biliary lipids during bile formation

Using complementary physical-chemical methods including turbidimetry, quasielastic light scattering, gel filtration, and phase analysis, we examined the interactions between dilute concentrations of the common bile salt, taurochenodeoxycholate (TCDC), and uni- and multilamellar vesicles (MLVs) composed of defined molecular species of lecithin (L) and varying contents of cholesterol (Ch). Dissolution rates of MLVs with micellar TCDC, as assessed by turbidimetry, were more rapid with vesicles composed of sn-1 palmitoyl species, typical of biliary L, compared with those composed of the more hydrophobic sn-1 stearoyl species. Incorporation of Ch retarded MLV dissolution rates in proportion to the Ch content, and only at high Ch contents were dissolution rates appreciably influenced by the sn-2 fatty acid composition of L. When MLVs contained Ch in amounts characteristic of intracellular membranes (Ch/L approximately 0.1), the dissolution rates of the individual L species by TCDC accurately predicted the steady state L composition of human bile. TCDC interacted with small unilamellar L/Ch vesicles (SUVs) at concentrations well below, as well as appreciably above, its critical micellar concentration. In accordance with the TCDC-egg yolk L-H2O phase diagram, perimicellar concentrations of TCDC interacted with SUVs to form aggregates that were approximately twice the size of the SUVs. These were consistent with the formation of a dispersed hexagonal (rod-like) phase, which co-existed with aqueous bile salt (BS) monomers and either micellar or unilamellar SUV phases. Micellar TCDC completely solubilized SUVs as mixed micelles, putatively via this transient hexagonal phase. With modest Ch-supersaturation, dissolution was followed by the reemergence of a new vesicle population that coexisted metastably with mixed micelles. With high Ch supersaturation, TCDC extracted L and Ch molecules from SUVs in different proportions to form Ch-supersaturated mixed micelles and Ch-enriched SUVs, in accordance with the metastable phase diagram. These experiments are consistent with the hypothesis that sn-1 palmitoyl L species are subselected for bile, in part, by physical-chemical interactions of intracellular BS concentrations with Ch-poor membranes and that the subsequent evolution of Ch-rich vesicles and Ch-saturated mixed micelles occurs via a transitional hexagonal (rod) phase. These liquid-crystalline states are likely to be transient in Ch-unsaturated biles, but may persist in Ch-supersaturated human biles because of their high Ch contents which retard or inhibit these phase transitions.     

Comparison of large unilamellar vesicles prepared by a petroleum ether vaporization method with multilamellar vesicles: ESR, diffusion and entrapment analyses.

Large unilamellar vesicles, prepared by a petroleum ether vaporization method, were compared to multilamellar vesicles with respect to a number of physical and functional properties. Rotational correlation time approximations, derived from ESR spectra of both hydrophilic (3-doxyl cholestane) and hydrophobic (3-doxyl androstanol) steroid spin probes, indicated similar molecular packing of lipids in bilayers of multilamellar and large unilamellar liposomes. Light scattering measurements demonstrated a reduction in apparent absorbance of large unilamellar vesicles, suggesting loss of multilamellar structure which was confirmed by electron microscopy. Furthermore, large unilamellar vesicles exhibited enhanced passive diffusion rates of small solutes, releasing a greater percentage of their contents within 90 min than multilamellar vesicles, and reflecting the less restricted diffusion of a unilamellar system. The volume trapping capacity of large unilamellar vesicles far exceeded that of multilamellar liposomes, except in the presence of a trapped protein, soy bean trypsin inhibitor, which reduced the volume of the aqueous compartments of large unilamellar vesicles. Finally, measurement of vesicle diameters from electron micrographs of large unilamellar vesicles showed a vesicle size distribution predominantly in the range of 0.1--0.4 micron with a mean diameter of 0.21 micron.     

Interaction of short-chain lecithin with long-chain phospholipids: characterization of vesicles that form spontaneously

Stable unilamellar vesicles formed spontaneously upon mixing aqueous suspensions of long-chain phospholipid (synthetic, saturated, and naturally occurring phosphatidylcholine, phosphatidylethanolamine, and sphingomyelin) with small amounts of short-chain lecithin (fatty acid chain lengths of 6-8 carbons) have been characterized by using NMR spectroscopy, negative staining electron microscopy, differential scanning calorimetry, and Fourier transform infrared (FTIR) spectroscopy. This method of vesicle preparation can produce bilayer vesicles spanning the size range 100 to greater than 1000 A. The combination of short-chain lecithin and long-chain lecithin in its gel state at room temperature produces relatively small unilamellar vesicles, while using long-chain lecithin in its liquid-crystalline state produces large unilamellar vesicles. The length of the short-chain lecithin does not affect the size distribution of the vesicles as much as the ratio of short-chain to long-chain components. In general, additional short-chain decreases the average vesicle size. Incorporation of cholesterol can affect vesicle size, with the solubility limit of cholesterol in short-chain lecithin micelles governing any size change. If the amount of cholesterol is below the solubility limit of micellar short-chain lecithin, then the addition of cholesterol to the vesicle bilayer has no effect on the vesicle size; if more cholesterol is added, particle growth is observed. Vesicles formed with a saturated long-chain lecithin and short-chain species exhibit similar phase transition behavior and enthalpy values to small unilamellar vesicles of the pure long-chain lecithin prepared by sonication. As the size of the short-chain/long-chain vesicles decreases, the phase transition temperature decreases to temperatures observed for sonicated unilamellar vesicles. FTIR spectroscopy confirms that the incorporation of the short-chain lipid in the vesicle bilayer does not drastically alter the gauche bond conformation of the long-chain lipids (i.e., their transness in the gel state and the presence of multiple gauche bonds in the liquid-crystalline state).     

Comparison of large unilamellar vesicles prepared by a petroleum ether vaporization method with multilamellar vesicles: ESR,diffusion and entrapment analyses

Large unilamellar vesicles, prepared by a petroleum ether vaporization method, were compared to multilamellar vesicles with respect to a number of physical and functional properties. Rotational correlation time approximations, derived from ESR spectra of both hydrophilic (3-doxyl cholestane) and hydrophobic (3-doxyl androstanol) steroid spin probes, indicated similar molecular packing of lipids in bilayers of multilamellar and large unilamellar liposomes. Light scattering measurements demonstrated a reduction in apparent absorbance of large unilamellar vesicles, suggesting loss of multilamellar structure which was confirmed by electron microscopy. Furthermore, large unilamellar vesicles exhibited enhanced passive diffusion rates of small solutes, releasing a greater percentage of their contents within 90 min than multilamellar vesicles, and reflecting the less restricted diffusion of a unilamellar system. The volume trapping capacity of large unilamellar vesicles far exceeded that of multilamellar liposomes, except in the presence of a trapped protein, soy bean trypsin inhibitor, which reduced the volume of the aqueous compartments of large unilamellar vesicles. Finally, measurement of vesicle diameters from electron micrographs of large unilamellar vesicles showed a vesicle size distribution predominantly in the range of 0.1–0.4 μm with a mean diameter of 0.21 μm.     

Lipid-DNA complex formation: reorganization and rupture of lipid vesicles in the presence of DNA as observed by cryoelectron microscopy.

Cryoelectron microscopy has been used to study the reorganization of unilamellar cationic lipid vesicles upon the addition of DNA. Unilamellar DNA-coated vesicles, as well as multilamellar DNA lipid complexes, could be observed. Also, DNA induced fusion of unilamellar vesicles was found. DNA appears to adsorb to the oppositely charged lipid bilayer in a monolayer of parallel helices and can act as a molecular "glue" enforcing close apposition of neighboring vesicle membranes. In samples with relatively high DNA content, there is evidence for DNA-induced aggregation and flattening of unilamellar vesicles. In these samples, multilamellar complexes are rare and contain only a small number of lamellae. At lower DNA contents, large multilamellar CL-DNA complexes, often with >10 bilayers, are formed. The multilamellar complexes in both types of sample frequently exhibit partially open bilayer segments on their outside surfaces. DNA seems to accumulate or coil near the edges of such unusually terminated membranes. Multilamellar lipid-DNA complexes appear to form by a mechanism that involves the rupture of an approaching vesicle and subsequent adsorption of its membrane to a "template" vesicle or a lipid-DNA complex.     

Morphology and phase behavior of two types of unilamellar vesicles prepared from synthetic phosphatidylcholines studied by freeze-fracture electron microscopy and calorimetry

Differential scanning calorimetry and freeze-fracture electron microscopy have been used to characterize the phase behavior and morphology of two types of unilamellar vesicles composed of synthetic phosphatidylcholines. The first type displayed an average diameter of roughly 100 nm and was formed by slow dilution and dialysis of octylglucoside-solubilized lipid. These large, unilamellar vesicles were termed dialyzed, octylglucoside vesicles and could be obtained as a fairly well defined and uniform population of vesicles. The second vesicle type was prepared by a unique procedure involving dialysis of deoxycholate-solubilized lipid at its pre-transition temperature. This procedure produced a much more heterogeneous distribution of vesicle sizes (500 to 4000 nm in diameter) and left some dilamellar and oligolamellar species which could not be conveniently separated from the giant, unilamellar vesicles constituting the major portion of the sample. Both populations of vesicles displayed phase behavior similar, but not identical to that of large, multilamellar vesicles (LMV). Fracture-face morphology of the gel phase was also observed to differ between the two unilamellar and the multilamellar species. LMV have previously been shown to have clear undulated or banded fracture-faces in the P beta phase, while octylglucoside vesicles are shown here to have facetted fracture-faces. Giant, unilamellar vesicles displayed a faint banded morphology similar to but less distinct than that of the LMV P beta phase. These results have demonstrated that bilayer apposition is not required to support the banded fracture-face morphology characteristic of the P beta phase but that a limiting curvature is necessary.     

Influence of dilution on the physical state of model bile systems: NMR and quasi-elastic light-scattering investigations

Multinuclear (1H and 31P) nuclear magnetic resonance (NMR) spectroscopy and quasi-elastic light scattering have been used to characterize molecular aggregates formed in dilute sodium taurocholate--egg lecithin solutions. When mixed micelles (1.25 g/dL) are diluted with 150 mM aqueous sodium chloride, light-scattering measurements suggest a transformation from mixed micelles to unilamellar vesicle species. Decreased 1H NMR line widths for bile salt resonances are consistent with predominance of a monomer form. The concurrent appearance of a second phospholipid choline methyl resonance indicates two types of phospholipid environment in slow chemical exchange: this behavior is consistent with small unilamellar vesicles. The appearance of bilayer vesicles in dilute model bile solutions is confirmed by addition of a lanthanide shift reagent (Pr3+), which splits the 1H or 31P head-group peak into two components with distinct chemical shift sensitivities. These mixed micelle and vesicle aggregates are also distinguished by their susceptibility to the lipolytic enzyme phospholipase A2 from cobra venom.     

Comparison of the enthalpy state of vesicles of different size by their interaction with alpha-lactalbumin

The characteristics of small unilamellar, large unilamellar and large multilamellar vesicles of dimyristoylphosphatidylcholine and their interaction with alpha-lactalbumin are compared at pH 4. (1) By differential scanning calorimetry and from steady-state fluorescence anisotropy data of the lipophilic probe 1,6-diphenyl-1,3,5-hexatriene it is shown that the transition characteristics of the phospholipids in the large unilamellar vesicles resemble more those of the multilamellar vesicles than of the small unilamellar vesicles. (2) The size and composition of the lipid-protein complex formed with alpha-lactalbumin around the transition temperature of the lipid are independent of the vesicle type used. Fluorescence anisotropy data indicate that in this complex the motions of the lipid molecules are strongly restricted in the presence of alpha-lactalbumin. (3) The previous data and a comparison of the enthalpy changes, delta H, of the interaction of the three vesicle types with alpha-lactalbumin allow us to derive that the enthalpy state of the small unilamellar vesicles just below 24 degrees C is about 24 kJ/mol lipid higher than the enthalpy state of both large vesicle types at the same temperature. The abrupt transition from endothermic to exothermic delta H values around 24 degrees C for large vesicles approximates the transition enthalpy of the pure phospholipid.     

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.     

Fusogenic activity of delta-haemolysin from Staphylococcus aureus in phospholipid vesicles in the liquid-crystalline phase

A study has been conducted of the interaction of the lytic toxin delta-haemolysin with vesicles of phospholipid, using electron microscopy, fluorescence depolarisation and excimer fluorescence. The peptide is shown to be a fusogen towards phosphatidylcholine vesicles in fluid phases. In the presence of gel phase lipid, fusion between fluid and gel phases is not seen. Fluid phase lipid vesicles are fused together to form large multilamellar structures, and initial vesicle size does not appear to be important since small unilamellar vesicles and large unilamellar vesicles are similarly affected. Fusogenic activity of delta-haemolysin is compared to that of melittin. The former is a progressive fusogen for fluid phase lipid, while the latter causes vesicle fusion in a manner related to occurrence of a lipid phase transition.     

Structural characterization of the micelle-vesicle transition in lecithin-bile salt solutions.

Small-angle neutron scattering (SANS) and dynamic light scattering (QLS) are used to characterize the aggregates found upon dilution of mixed lecithin-bile salt micelles. Molar ratios of lecithin (L) to taurocholate (TC) studied varied from 0.1 to 1, and one series contained cholesterol (Ch). Mixed aggregates of L and taurodeoxycholate (TDC) at ratios of 0.4 and 1 were also examined. In all cases the micelles are cylindrical or globular and elongate upon dilution. The radius of the mixed micelles varies only slightly with the overall composition of lecithin and bile salt which indicates that the composition of the cylindrical micelle body is nearly constant. The transition from micelles to vesicles is a smooth transformation involving a region where micelles and vesicles coexist. SANS measurements are more sensitive to the presence of two aggregate populations than QLS. Beyond the coexistence region the vesicle size and degree of polydispersity decrease with dilution. Incorporation of a small amount of cholesterol in the lipid mixture does not affect the sequence of observed aggregate structures.     

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)     

Comparison of the enthalpy state of vesicles of different size by their interaction with α-lactalbumin

The characteristics of small unilamellar, large unilamellar and large multilamellar vesicles of dimyristoylphosphatidylcholine and their interaction with α-lactalbumin are compared at pH 4. (1) By differential scanning calorimetry and from steady-state fluorescence anisotropy data of the lipophilic probe 1,6-diphenyl-1,3,5-hexatriene it is shown that the transition characteristics of the phospholipids in the large unilamellar vesicles resemble more those of the multilamellar vesicles than of the small unilamellar vesicles. (2) The size and composition of the lipid-protein complex formed with α-lactalbumin around the transition temperature of the lipid are independent of the vesicle type used. Fluorescence anisotropy data indicate that in this complex the motions of the lipid molecules are strongly restricted in the presence of α-lactalbumin. (3) The previous data and a comparison of the enthalpy changes, $\text{ΔH}$, of the interaction of the three vesicle types with α-lactalbumin allow us to derive that the enthalpy state of the small unilamellar vesicles just below 24°C is about 24 kJ/mol lipid higher than the enthalpy state of both large vesicle types at the same temperature. The abrupt transition from endothermic to exothermic $\text{ΔH}$ values around 24°C for large vesicles approximates the transition enthalpy of the pure phospholipid     

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.     

Liposomal Hexadecylphosphocholine: Characterization and Effects on Adherent Tumor Cells

Abstract

We describe the preparation of small unilamellar and multilamellar vesicles from hexadecylphosphocholine, cholesterol and 1,2-dipalmitoyl-sn-glycero-phosphoglycerol in the molar ratio 4/5/1. Particle size and chemical stability of two types of liposomes, small unilamellar vesicles and lyophilized, freshly resuspended multilamellar vesicles were proved to be stable for at least 12 months. Compared to hexadecylphosphocholine in free form, liposomal hexadecylphosphocholine showed remarkably reduced hemolysis which did not change during storage. Fluorescence microscopy showed the uptake of propidium iodide containing hexadecylphosphocholine liposomes by KB and MDA-MB 231 tumor cells. Free propidium iodide was not incorporated into these cells. Although cytotoxicity seemed to be reduced in liposomal preparations, hexadecylphosphocholine liposomes still affected cultured tumor cells to a great extent. In relatively low concentrations they induced shape alteration, smoothing of the cell surface and blebbing.     

Formation of multilamellar vesicles by addition of tannic acid to phosphatidylcholine-containing small unilamellar vesicles

Tannic acid induces aggregation and formation of multilamellar vesicles when added to preparations of small unilamellar vesicles, specifically those containing phosphatidylcholine. Aggregation and clustering of vesicles was demonstrated by cryo-electron microscopy of thin films and by freeze-fracture technique. Turbidity measurements revealed an approximately one-to-one molar ratio between tannic acid and phosphatidylcholine necessary for a fast and massive aggregation of the small unilamellar vesicles. When tannic acid-induced aggregates were dehydrated and embedded for conventional thin-section electron microscopy, multilamellar vesicles were retrieved in thin sections. It is concluded from morphological studies, as well as previous tracer studies, that tannic acid, at least to a great extent, prevents the extraction of phosphatidylcholine. Multilamellar vesicles were also observed in tannic acid-treated vesicles prepared from total lipid extracts from either rabbit or rat hearts. Substantially more multilamellar vesicles were retrieved in the rabbit vesicle preparation. This difference can probably be explained by the difference in the proportion of the plasmalogen phosphatidylcholine, and possibly the content of sphingomyelin, in lipid extracts of rabbit and rat hearts. It is concluded that the dual effect (reduced extraction and aggregation) of tannic acid on phosphatidylcholines should be taken into consideration when tannic acid is used in tissue preparation.     

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.