The effects of bile salt hydrophobicity on model bile vesicle morphology (original) (raw)
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Bile salt-induced cholesterol crystal formation from model bile vesicles: A time course study
The Journal of Lipid Research
Precipitation of cholesterol crystals from vesicles is an important step in the pathogenesis of cholesterol gallstones. Little is known, however, about the kinetics and the mechanisms involved in cholesterol crystallization. Therefore, the time course of cholesterol crystal precipitation and lipid exchange between vesicles and micelles were monitored in a model bile system. Vesicles obtained from supersaturated model bile (cholesterol saturation index (CSI) 1.4; 10 g/dl) by KBr density gradient ultracentrifugation, were incubated with various bile salts: deoxycholate (DC), chenodeoxycholate (CDC), cholate (C), ursodeoxycholate (UDC), and their respective taurine and glycine conjugates. Vesicle integrity was assessed in a leakageassay of carboxyfluorescein-loaded vesicles (0-15 min) and by the change in optical absorbance at 340 nm of a vesicle solution (0-50 min). Fluorescence increased within 1 min after addition of bile salt, and was stable within 5-10 min. After addition of bile salt, absorbance fell immediately and stabilized within 30 min. Fluorescence and absorbance were dependent on bile salt hydrophobicity and concentration. At several timc points after addition of bile salt to vesicles (from 1 to 72 h), the extent of cholesterol nucleation was determined semiquantitatively and incubation mixtures were again subjected to ultracentrifugation to assess the lipid distribution among residual vesicles, de novo formed mixed micelles, and cholesterol crystals. Nucleation occurred within 0.5 h after exposure of vesicles to the hydrophobic bile salts D C or CDC, and the cholesterol/phospholipid (c/p) ratio of the vesicles showed a transient rise from 1.45 to 3-4 (at t = 0.5 h) that coincided with the appearance of mixed micelles. Then the vesicular c/p ratio decreased to 0.6-0.8 (at t = 24 h) concomitantly with increasing precipitation of cholesterol crystals. In the case of UDC, the most hydrophilic bile salt used, < 5% micellization, no nucleation, and a constant vesicular c/p ra-
European Journal of Clinical Investigation, 1993
Cholesterol in bile is solubilized in mixed micelles and cholesterol-phospholipid vesicles. Biliary cholesterol supersaturation and increased concentration of bile in the gallbladder promotes nucleation of cholesterol monohydrate crystals and gallstone formation, possibly by creating unstable vesicles with a high cholesterol/phospholipid ratio. In the present study super-saturated and unsaturated biles (cholesterol saturation index (CSI) 1.4 and 0.8 respectively) were prepared with concentrations typical of gallbladder and more dilute hepatic bile (total lipid concentration (TLCo) 10 and 2.5 g dl-' respectively). The distribution of cholesterol between vesicles and micelles, and vesicular cholesterol/phospholipid ratio were studied using ultracentrifugation and gel-permeation chromatography. The nucleation time of cholesterol crystals was determined in whole model bile, and in the vesicular and micellar peak fractions. Increased CSI and bile dilution led to an increased proportion of cholesterol solubilized in vesicles. The concentration of bile did not influence vesicular cholesterol/phospholipid ratio. The vesicular cholesterol/ phospholipid ratio found in gel-permeation chromatography experiments was similar at high and low CSI, whereas the ratio was significantly higher in supersaturated than in unsaturated biles in ultracentrifugation studies. Nucleation of cholesterol crystals from whole model bile was more rapid at the higher bile concentration and higher cholesterol saturation. Nucleation time in whole model bile correlated significantly with nucleation time in the corresponding vesicular peak fraction obtained by gel-permeation chromatography (r=0,58: P < 0.01) and with the cholesterol concentration in this vesicular peak (r = -0.77; P < 0.002) but not with vesicular peak cholesterol/phospholipid ratio. Highest vesicular peak cholesterol concentrations and shortest nucleation times were found for concentrated supersaturated biles, whereas vesicular cholesterol/phospholipid ratio was not different from Correspondence: K . J. van Erpecum MD, Department of Gastroenterology, University Hospital Utrecht, Postbox 85500, 3508 . GA Utrecht. The Netherlands.
Cholesterol-phospholipid vesicles in human bile: an ultrastructural study
Biochimica et biophysica acta, 1986
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 precipi...
Journal of lipid research, 2001
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 i...
2000
Cholesterol in human bile is solubilized in micelles by (relatively hydrophobic) bile salts and phosphatidylcholine (unsaturated acyl chains at sn -2 position). Hydrophilic taurour- sodeoxycholate, dipalmitoyl phosphatidylcholine, and sphingo- myelin 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 -2 82) and thus could
Lipid solubilization in human gallbladder versus hepatic biles
Journal of Hepatology, 1999
Background/Aims: Cholesterol crystallizes more rapidly in gallbladder than in hepatic biles, supposedly due to formation of cholesterol-supersaturated vesicles in concentrated gallbladder biles because of preferential micellization of phospholipids compared to cholesterol. We therefore aimed to compare lipid solubilization in hepatic and gallbladder biles. Methods: Mixed micellar and vesicular phases were separated from hepatic and associated gallbladder biles of seven cholesterol gallstone patients by using state-of-the-art gel filtration with bile salts at intermixed micellarlintervesicular compositions and concentrations in the eluant. Results: Vesicles were found in 6 out of 7 hepatic biles, but only in 2 of the corresponding gallbladder biles. Both percentage (7.825.1 vs. 36.3+7.6%; p= 0.01) and amount (0.920.2 vs. 1.720.3 mM,p=0.06) of vesicular cholesterol were lower in gallbladder biles. RYSTALLIZATION C of cholesterol from supersaturated bile is a prerequisite for gallstone formation (1,2). The sterol is poorly soluble in an aqueous environment, and is solubilized in bile in mixed micelles, together with bile salts and phospholipids (mainly phosphatidylcholine). In the case of cholesterol supersaturation, the sterol may also be solubilized in vesicles together with phospholipids (3-S). It is generally assumed that cholesterol crystals originate from cholesterol-enriched vesicles after their aggregation and fusion (9-11). The fact that cholesterol crystallization and gallstone formation generally occur in the gallbladder but not in the bile ducts, points to a crucial
Biochimica et biophysica acta, 1987
Bile was obtained from patients with and without cholesterol gallstones at surgery. Biliary vesicles were separated from micelles by gel filtration. The cholesterol/phospholipid ratio in vesicles was much higher than in micelles. Cholesterol crystals nucleated from vesicular fractions, but nucleation from the micellar fractions was slow or did not occur at all. Cholesterol nucleated from vesicles obtained from bile of control patients as rapidly (2.4 days +/- 0.7) as from patients with stones (2.4 days +/- 0.9) and there was no difference in the vesicular cholesterol/phospholipid ratio. The effect of alteration of the bile salt environment was studied by changing the concentration of sodium cholate in the eluting buffer. At low concentrations (5 mM) only vesicles were eluted from the column. These vesicles had a relatively low cholesterol/phospholipid ratio and cholesterol nucleated slowly from these vesicles. At higher concentrations the proportion of micelles increased. The propor...
Effect of cholesterol nucleation-promoting activity on cholesterol solubilization in model bile
Journal of lipid research, 1989
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 bi...
Pathways of cholesterol crystallization in model bile and native bile
Digestive and Liver Disease, 2003
Hypersecretion of hepatic cholesterol, chronic supersaturation of bile with cholesterol and rapid precipitation of cholesterol crystals in the gallbladder from cholesterol-enriched vesicles represent the primum movens in cholesterol gallstone formation. Physical-chemical factors and pathways leading to cholesterol crystallization can be investigated in artificial model biles and ex vivo in fresh human bile. Depending on modulatory factors (i.e., lipid concentration, bile salt or phospholipid species, humidity, mucins, etc.), cholesterol can precipitate in several forms (i.e., monohydrate, anhydrous) and habits (i.e., plate-like, needle-like, intermediate arcs, filaments, tubules, spirals). Careful analysis of biliary cholesterol crystals includes biochemical analysis of precipitated crystals, polarizing quantitative light microscopy, and turbidimetric methods. In this paper, recent concepts on cholesterol crystallization in artificial model biles as well as in human bile will be reviewed.
Biochimica et Biophysica Acta (BBA) - Lipids and Lipid Metabolism, 1995
Time-sequential enzymatic determination of cholesterol (CH) crystals harvested by ultrafiltration, and concomitant polarizing light microscopy observations corroborated the striking importance of the bile salts (BS) species in determining CH crystals formation rate from supersaturated model biles incubated in vitro. The more hydrophilic tauroursodeoxycholate, taurohyocholate, glycohyocholate, taurohyodeoxycholate, glycohyodeoxycholate and glyco-3a, hydroxy-6 oxo-5/3-cholanate inhibited CH precipitation through the formation of a stabilized liquid-crystalline phase. In contrast, in all hydrophobic systems (taurine (T) and glycine (G) conjugates of cholate (C), deoxycholate (DC) and chenodeoxycholate (CDC)), CH crystals precipitated with time. When crystallized CH concentrations were plotted vs. time, the figures showed a sigmoidal pattern, consistent with the transition from metastable systems to stable equilibrium states. Over the equilibration period, the nucleation kinetics (as inferred from enzymatic measurements) and all crystallization events (as microscopically observed) were both shifted in time, depending on the BS species: they were earliest in CDC systems, then in DC systems, and finally in C systems. In the latter, the delay was clearly due to the formation of a transient labile liquid-crystalline phase. G-conjugation also induced a significant delay in CH precipitation, compared to T-conjugation. At last, maximum crystallized CH concentrations at equilibrium were in the decreasing order: C > CDC > DC and T-conjugates > G-homologues. All data are discussed in connection with BS hydrophobicities, with predictions from the phase equilibria of aqueous biliary lipid systems and with new insights into CH crystal habits.