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.     

Contribution of vesicular and micellar carriers to cholesterol transport in human bile

A nonmicellar, bile salt-independent mode of cholesterol transport in human bile involving phospholipid vesicles was recently reported by our group. In the present study, we have investigated the relative contribution of the phospholipid vesicles and mixed bile salt-phospholipid micelles to cholesterol transport in human hepatic and gallbladder biles. The vesicles (ca 800 A diameter) were demonstrated by quasi-elastic light scattering (QELS) in fresh bile and after chromatography. Gel filtration under conditions that preserved micellar integrity demonstrated that biliary cholesterol was associated with both vesicles and micelles. At low bile salt concentration, the vesicular phase was predominant and most of the cholesterol was transported by it. With increasing bile salt concentrations, a progressive solubilization of the vesicles occurred with a concomitant increase in the amount of cholesterol transported by micelles. The vesicular carrier may be of particular biological significance for cholesterol solubilization in supersaturated biles.     

Biliary micellar cholesterol nucleates via the vesicular pathway

Biliary cholesterol nucleates primarily from phospholipid vesicles. In this study, we investigated the mode of nucleation of micellar cholesterol. Ten biles (four human and six model) were examined. The vesicular and micellar fractions of each bile were separated by gel chromatography. The whole biles and their isolated carriers were incubated at 37 degrees C until nucleation time. In whole human biles, the proportion of total cholesterol in vesicles rose throughout the incubation (from zero time to nucleation time) from 15.5 +/- 8.6% to 28.0 +/- 12.5%, and in model biles from 46.8 +/- 22.4% to 75.5 +/- 8.2%. The vesicular isolated fraction remained unchanged throughout incubation. In isolated micelles devoid of vesicles at zero time, new vesicles formed during incubation, carrying increasing proportions of cholesterol. At nucleation time, these vesicles contained 11.0% of originally micellar cholesterol in human biles, and 41.2% in model biles. The new vesicles formed in whole bile and in the micellar fraction were chromatographically and chemically similar to the vesicles originally present in bile. These data suggest that micellar cholesterol nucleates via the neoformation of phospholipid vesicles, which seem to be the final common pathway for cholesterol nucleation in 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.     

Quantitation of phospholipid vesicles and their cholesterol content in human bile by quasi-elastic light scattering

The proportion of biliary cholesterol carried by phospholipid vesicles may be an important determinant of the lithogenicity of bile. The distribution of biliary cholesterol between vesicles and other aggregational forms is often determined by gel filtration under standard conditions. The aim of this study was to measure the proportion of biliary cholesterol in vesicles in native unprocessed bile and to compare it with values obtained by chromatography. A modified quasi-elastic light-scattering method was used to measure vesicular cholesterol in whole bile. It was suitable only for lightly pigmented biles with a relatively monodisperse population of vesicles. In ten human biles examined, the proportion of cholesterol in vesicles by gel filtration was 40 +/- 8.1% (mean +/- S.D.) by chemical measurement, and 38 +/- 7.2% by [3H]cholesterol estimation. Quasi-elastic light-scattering measurements of these biles produced vesicular cholesterol values of 36 +/- 9.4%. Chromatography may affect lipid particles in bile. Nevertheless, it provides a relatively accurate measurement of biliary cholesterol in vesicles.     

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.     

Effect of cholesterol nucleation-promoting activity on cholesterol solubilization in model bile

Human bile contains a factor with cholesterol nucleation-promoting activity that binds to concanavalin A-Sepharose. In this study we have investigated the effect of this activity on the dynamics of lipid solubilization in supersaturated model bile. A concanavalin A binding protein fraction of human bile was mixed with model bile and the effect on the distribution of cholesterol and phospholipid between mixed micelles and phospholipid/cholesterol vesicles was studied by means of density gradient ultracentrifugation. The nucleation-promoting activity containing fraction induced a transfer of cholesterol and phospholipid from the micellar to the vesicular phase. This led to a decrease in the density of the vesicular fraction. We have also studied the effect of promoting activity on the nucleation time of an isolated vesicle fraction. A decrease of the nucleation time of 10.7 +/- 1.3 to 2.3 +/- 0.3 days was observed. In conclusion, a concanavalin A binding protein fraction from human bile stimulated cholesterol nucleation via a double effect; it increased the amount of vesicular cholesterol and phospholipid, and it also directly induced nucleation of cholesterol from the vesicles.     

Characterization of phospholipid transfer between mixed phospholipid-bile salt micelles

Concentration-dependent self-quenching of the fluorescent phospholipid N-(7-nitro-2,1,3-benzoxadiazol-4-yl)phosphatidylethanolamine (N-NBD-PE) was used to measure the rate of N-NBD-PE transfer between phosphatidylcholine-bile salt mixed micelles. In a previous study using the same technique, the rate of N-NBD-PE transfer between phosphatidylcholine-taurocholate mixed micelles was found to be several orders of magnitude faster than its transfer between phosphatidylcholine vesicles as a result of an increased rate of transfer through the water at low micelle concentrations and an increased rate of transfer during transient micelle collisions at higher micelle concentrations [Nichols, J. W. (1988) Biochemistry 27, 3925-3931]. In this study we have determined the influence of bile salt structure, incorporation of cholesterol, and temperature on the rate and mechanism of phospholipid transfer between mixed micelles. We found that both transfer pathways were a common property of mixed micelles prepared from a series of different bile salts and that the rates of transfer by both pathways increased as a function of the degree of bile salt hydrophobicity. Cholesterol incorporation into phosphatidylcholine-taurocholate mixed micelles displaced taurocholate from the micelles and resulted in an increased rate of transfer through the water and a decreased rate of transfer during micelle collisions. The temperature dependence of the transfer rates was used to calculate the activation free energy, enthalpy, and entropy for both mechanisms. The activation enthalpy was the major barrier to transfer by both mechanisms.(ABSTRACT TRUNCATED AT 250 WORDS)     

Molecular dynamics simulations of mixed micelles modeling human bile

Extensive molecular dynamics (MD) simulations of binary systems of phospholipids and bile salts, a model for human bile, have been performed. Recent progress in hardware and software development allows simulation of the spontaneous aggregation of the constituents into small mixed micelles, in agreement with experimental observations. The MD simulations reveal the structure of these micelles at atomic detail. The phospholipids are packed radially with their headgroups at the surface and the hydrophobic tails pointing toward the micellar center. The bile salts act as wedges between the phospholipid headgroups, with their hydrophilic sides exposed to the aqueous environment. The structure of the micelles strongly resembles the previously proposed radial shell model. Simulations including small fractions of cholesterol reveal how cholesterol is solubilized inside these mixed micelles without changing their overall structure.     

Solubilization of lipids from hamster bile-canalicular and contiguous membranes and from human erythrocyte membranes by conjugated bile salts.

We have demonstrated in vitro the efficacy of the taurine-conjugated dihydroxy bile salts deoxycholate and chenodeoxycholate in solubilizing both cholesterol and phospholipid from hamster liver bile-canalicular and contiguous membranes and from human erythrocyte membrane. On the other hand, the dihydroxy bile salt ursodeoxycholate and the trihydroxy bile salt cholate solubilize much less lipid. The lipid solubilization by the four bile salts correlated well with their hydrophobicity: glycochenodeoxycolate, which is more hydrophobic than the tauro derivative, also solubilized more lipid. All the dihydroxy bile salts have a threshold concentration above which lipid solubilization increases rapidly; this correlates approximately with the critical micellar concentration. The non-micelle-forming bile salt dehydrocholate solubilized no lipid at all up to 32 mM. All the dihydroxy bile acids are much more efficient at solubilizing phospholipid than cholesterol. Cholate does not show such a pronounced discrimination. Lipid solubilization by chenodeoxycholate was essentially complete within 1 min, whereas that by cholate was linear up to 5 min. Maximal lipid solubilization with chenodeoxycholate occurred at 8-12 mM; solubilization by cholate was linear up to 32 mM. Ursodeoxycholate was the only dihydroxy bile salt which was able to solubilize phospholipid (although not cholesterol) below the critical micellar concentration. This similarity between cholate and ursodeoxycholate may reflect their ability to form a more extensive liquid-crystal system. Membrane specificity was demonstrated only inasmuch as the lower the cholesterol/phospholipid ratio in the membrane, the greater the fractional solubilization of cholesterol by bile salts, i.e. the total amount of cholesterol solubilized depended only on the bile-salt concentration. On the other hand, the total amount of phospholipid solubilized decreased with increasing cholesterol/phospholipid ratio in the membrane.     

Gender Differences in Cholesterol Nucleation in Native Bile: Estrogen Is a Potential Contributory Factor

The incidence of gallstone disease is two to three times higher in women than in men, and female sex hormones, particularly estrogens, have been implicated as contributory factors. Cholesterol nucleation is the initial step in gallstone pathogenesis and proceeds from cholesterol-rich phospholipid vesicles. The aim of this study was to investigate if there is a difference in cholesterol nucleation rates in male and female bile and whether estrogen influences nucleation rates by interacting with cholesterol-rich regions known as “lipid rafts” that exist within the cholesterol-phospholipid vesicles of the bile. Cholesterol nucleation from native prairie dog bile and the interaction of estrogens with lipid rafts in model bile solutions were investigated using Förster resonance energy transfer (FRET). Female native bile samples showed a greater reduction in energy transfer than did male native bile, indicating that cholesterol nucleation occurred more readily in female bile than in male bile. Model bile experiments demonstrated that the addition of estrogen has a significant effect, either cholesterol nucleation or raft disruption, but only in samples containing cholesterol-rich rafts. These results suggest that estrogen interacts with cholesterol-rich rafts in vesicles within bile to promote cholesterol nucleation and predispose females to gallstone formation.     

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.     

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.     

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.     

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.     

Cholesterol-transfer protein located in the intestinal brush-border membrane. Partial purification and characterization

Cholesterol absorption by small intestinal brush border membrane vesicles from taurocholate mixed micelles is a second-order reaction. From a comparison of reaction rates and order before and after proteinase K treatment of brush-border membrane vesicles, it is concluded that cholesterol absorption is protein-mediated. It is shown that the desorption of cholesterol from taurocholate mixed micelles is by a factor of about 10(4) faster than that from egg phosphatidylcholine bilayers. When brush border membrane vesicles are stored at room temperature, intrinsic proteinases are activated and proteins are liberated from the brush border membrane. These proteins collected in the supernatant catalyze cholesterol and phosphatidylcholine exchange between two populations of small unilamellar phospholipid vesicles. One of the active proteins present in the supernatant is purified by a two-step procedure involving gel filtration on Sephadex G-75 SF and affinity chromatography on a Nucleosil-phosphatidylcholine column. The protein thus obtained is pure by polyacrylamide gel electrophoresis in sodium dodecyl sulfate. It has an apparent molecular weight of slightly less than 14,000 as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis and a value of 11,500 determined by gel filtration on Sephadex G-75 SF.     

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.     

Separation of micelles and vesicles within lumenal aspirates from healthy humans: solubilization of cholesterol after a meal

Understanding the physico-chemical relationship of lumenal lipids to one another is critical when elucidating the mechanism of components known to impact cholesterol absorption. Presently, there are no studies that describe the proportion of cholesterol carried as micelles or vesicles within human lumenal contents. Part of the reason for the scarceness of data is because of the lack of appropriate methodology required for reproducible sample collection and analysis. Thus, the object of the present studies was to develop a method to measure the amount of cholesterol carried as micelles or vesicles in human lumenal samples. The method includes the collection of lumenal samples from the ligament of Trietz through a Fredrick Miller tube, separation of the aqueous subphase from the nondigested lipids, separation of micelles and vesicles on Sepharose 4B columns within 48 h of collection using elution buffers consisting of the intermicellar bile acid composition, and finally quantitation of cholesterol eluted off of the columns.The distribution of cholesterol between micelles and vesicles obtained under different concentrations of bile acids and various lipids was comparable to results obtained from phase diagrams using the lumenal molar percentages of lipids obtained from the same samples.     

Use of 1H-NMR to determine the distribution of lecithin between the micellar and vesicular phases in model bile

Biliary cholesterol/phospholipid vesicles play an important role in the pathogenesis of gallstone disease. A prerequisite for the study of the lipid composition and stability of these vesicles is a reliable method to quantify the amount of vesicular lipid. In the present report we show that NMR can be used to determine the distribution of biliary lecithin between the micellar and vesicular phases. The relatively large size of the vesicles leads to such a broadening of the lipid resonances that they are no longer visible in high resolution 1H-NMR spectra. Since micelles are much smaller, lipid present in the micellar phase does give rise to sharp peaks in 1H-NMR spectra. Micellar lecithin can easily be quantified in these spectra. The resonances of cholesterol are masked by the closely related bile acid that is present in a much higher concentration. By determining the difference between chemically and NMR estimated lecithin, the distribution of this phospholipid between the micellar phase and vesicular phase can be assessed. We have compared the results of NMR with gel permeation and density gradient ultracentrifugation. Using standard fractionation conditions, both gel permeation and density gradient ultracentrifugation lead to an underestimation of vesicular lecithin, the difference being minor at relatively high total lipid concentrations (10 g/dl) but large in diluted model bile. We conclude that 1H-NMR can be used to determine the distribution of lecithin in model bile.(ABSTRACT TRUNCATED AT 250 WORDS)     

A phylogenetic survey of biliary lipids in vertebrates

Biliary lipids (bile salts, phospholipids, cholesterol, plant sterols) were determined in 89 vertebrate species (cartilaginous and bony fish, reptiles, birds, and mammals), and individual phospholipid classes were measured in 35 species. All samples contained conjugated bile salts (C(27) bile alcohol sulfates and/or N-acyl amidates of C(27) and/or C(24) bile acids). Phospholipids were generally absent in the bile of cartilaginous fish and reptiles and were present in low amounts relative to bile salts in bony fish and most birds. In mammals, the phospholipid-bile salt ratio varied widely. The bile from species with low biliary phospholipid-bile salt ratios often contained a high proportion of sphingomyelin, confirmed by HPLC-MS. In species with a high phospholipid-bile salt ratio, the predominant biliary phospholipid was phosphatidylcholine (PC). The phospholipid-bile salt ratio correlated weakly with the calculated weighted hydrophobic index value. Cholesterol was present in the bile of virtually all species, with plant sterols uniformly being present in only trace amounts. The cholesterol-bile salt ratio tended to be higher in mammals than in non-mammals, but bile of all species was unsaturated. Thus, most nonmammalian vertebrates have relatively low levels of biliary phospholipid and cholesterol, suggesting that cholesterol is eliminated predominantly as bile salts. Mammals have a higher phospholipid and cholesterol to bile salt ratio, with the dominant phospholipid being PC.