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.

Demonstration of 8 S-cytoplasmic oestrogen receptor in rat mullerian duct.

1. High affinity macromolecular binding of the non-steroidal synthetic oestrogen [3H]diethylstilboestrol and of [3H] oestradiol-17beta in cytosol of Müllerian duct and uterus, and in blood plasma of perinatal rats, was investigated by sucrose density gradient sedimentation. 2. While [3H] oestradiol was bound to both the characteristic 8 S uterine cytoplasmic receptor and a 4 S component of uterine cytosol and plasma of 11-day-old rats, [3H] diethylstilboestrol was bound almost exclusively by the 8 S cytoplasmic receptor. 3. The greatly reduced binding of [3H] diethylstilboestrol to the 4 S plasma plasmic receptor in the Müllerian duct (precursor of the uterus) of 20-day-old foetuses.

Increased saturation of the fatty acids in the sn-2 position of phospholipids reduces cholesterol crystallization in model biles.

Changes in the molecular structure of biliary phospholipids were shown to have major effects on cholesterol solubility, carriers and crystallization in human and model biles. This study investigated systematically the effects of varying saturation of the phosphatidylcholine (PC) sn-2 fatty acid on the cholesterol crystallization process in 3 different model biles. Twenty % of the egg PC (EPC) in these biles were replaced by synthetic PC's with 16:0-18:0, 16:0-18:1, or 16:0-18:2 fatty acyl chains. With 18:0 in the sn-2 position, the crystal observation time (COT) was prolonged from 2 days in the control EPC solution to 14 days (p<0.05). The crystal growth rate (CGR) was reduced from 0.1 OD/day to unmeasurable levels, and the total crystal mass on day 14 decreased by 86%. The introduction of one (18:1), and two (18:2) double bonds in the sn-2 fatty acid rapidly reversed these effects. Ultracentrifugal analysis showed precipitable cholesterol as monohydrate crystals. In the 16:0-18:0 test solution, most of the precipitable cholesterol remained in the supersaturated multilamellar vesicles. Saturation of the biliary PC sn-2 fatty acyl chain prolongs the COT, slows the CGR, reduces the crystal mass, and extends cholesterol solubility in multilamellar vesicles. Desaturation of the sn-2 fatty acid reverses these effects.

Phospholipid lamellae are cholesterol carriers in human bile.

Cholesterol solubility and precipitation in bile are major factors in the pathogenesis of cholesterol gallstones. At present, mixed micelles and phospholipid vesicles are considered to be the only cholesterol carriers in bile. In this study we present evidence showing that phospholipid lamellae are major cholesterol carriers in human bile. Lamellae are a known aggregational form in pure phospholipid model systems. In the present study, lamellae were demonstrated by electron microscopy after negative staining and by small-angle X-ray diffraction in all human gallbladder bile samples examined. During diffraction experiments, cholesterol was found to crystallize from these lamellae. Cholesterol carriers in bile were separated by high-resolution chromatography and by prolonged ultracentrifugation. Lamellae were shown to solubilize most of the biliary cholesterol; vesicles solubilized a lesser amount; while micelles solubilized only a minor portion. Our data suggest that phospholipid aggregates are the main cholesterol carriers in bile. Bile salts may control the equilibrium between the various aggregational forms of cholesterol-carrying phospholipids.

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.

A non-micellar mode of cholesterol transport in human bile.

Quasi-elastic light scattering (QELS) was used to measure particle size in fresh human hepatic bile of 14 subjects. Particles with an approximate diameter of 700 A were found in all biles. The particles were almost unchanged after the bile salt concentration was reduced to 0.06 mM by dilution or dialysis against 150 mM NaCl. During dialysis bile salts were removed, while cholesterol and phospholipids remained in solution apparently in the large particles-vesicles. These experiments suggest the presence of a novel, bile salt-independent, mode of cholesterol transport in saturated human bile.

The induction of lamellar stacking by cholesterol in lecithin-bile salt model systems and human bile studied by synchrotron X-radiation.

Small angle X-ray scattering (SAXS) with synchroton radiation was used to investigate interactions among lipid particles in lecithin-bile salt model systems and in native gallbladder biles. In model systems in the absence of cholesterol, isotropic, continuous spectra were found, indicating the absence of periodic structures. In the presence of excess cholesterol, interaction in the form of lamellar stacking was detected by the appearance of discrete diffraction peaks. In the supersaturated cholesterol region of the commonly accepted phase diagram [1], where cholesterol crystals were expected, we found lamellar stacking. The high proportion of cholesterol to bile salts seems to be the common denominator of these models. The lamellar stacking was also found in native unprocessed bile. This effect of cholesterol on lipid structure has not been previously described. Lamellar stacking may contribute to cholesterol solubilization. Its influence on the kinetics of cholesterol crystallization is presently unknown.

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.

Gel filtration and quasielastic light scattering studies of human bile.

Analyses of cholesterol carriers in the extremely variable biliary samples were performed by chromatography under standard conditions. Sephacryl columns were eluted with buffer containing 10 mmol/L sodium cholate, 150 mmol/L sodium chloride, 50 mmol/L Tris(hydroxymethyl)aminomethane-hydrochloride, pH 8.0, and 1.5 mmol/L ethylenediaminetetraacetate. Moderate changes in temperature, flow rate or sample size produced only minor differences in the results. Increments in bile salt concentration in the elution buffer caused progressive disappearance of vesicles and a rise in their cholesterol/phospholipid ratio. Using the standard chromatographic technique, analyses of the same bile gave reproducible results, and comparisons among various biles were possible. Quantitative light scattering measurements of bile showed that the amount of vesicular cholesterol in whole unprocessed bile was similar to that measured by Sephacryl chromatography. Analyses of human gallbladder biles using a high-resolution Sephacryl column showed four cholesterol-containing peaks: vesicles, lamellae, mixed micelles and an undefined carrier. This long chromatographic column separated the lamellar from the micellar peaks (which had merged on the short column). Phospholipid lamellae solubilized most of the biliary cholesterol. These findings were confirmed by ultracentrifugation of bile.

Evidence for 24,25-dihydroxycholecalciferol receptors in long bones of newborn rats.

Several reports have appeared that suggest that 24,25-dihydroxycholecalciferol has a possible biological role in bone formation. We have utilized competition studies, saturation analysis, sucrose-density-gradient sedimentation and DEAE-cellulose chromatography to demonstrate that long bones of vitamin D-depleted newborn rats contain cytoplasmic and possibly nuclear receptors that bind 24,25-dihydroxycholecalciferol with specificity and high affinity (Kd = 1.79 nM). Sucrose-density-gradient analysis of the cytoplasmic 24,25-dihydroxycholecalciferol-binding component showed a single binding macromolecule for 24,25-dihydroxycholecalciferol with a sedimentation coefficient of 3.1 S. DEAE-cellulose chromatography showed a [3H]24,25, dihydroxycholecalciferol-macromolecular complex that binds to DEAE-cellulose and elutes between 0.15 and 0.21 M-KCl. The finding of 24,25-dihydroxycholecalciferol receptors in long bones of newborn rats suggests a possible involvement of 24,25-dihydroxycholecalciferol in the metabolism of developing skeletal tissues.

Determination of free polyamines in human bile by high-performance liquid chromatography.

1. Polyamines are widely distributed in the body and may have cholesterol-nucleating activity in model bile and human bile. There are only partial and scant data available on the type of polyamines in human bile. 2. In this study methods for extraction of free polyamines, benzoylation and an h.p.l.c.-based method for the quantitative determination of putrescine, cadaverine, spermidine and spermine in bile are described. The h.p.l.c. methodology was validated and compared with separation on t.l.c. after dansylation. 3. The polyamine content of 11 gallbladder bile samples and 11 T-tube bile samples was analysed, all from patients with gallstones. Polyamines were found in three out of 11 gallbladder bile samples and eight out of 11 T-tube bile samples. Putrescine levels were 0.5-287 mumol/l and cadaverine levels were 2.4-645.4 mumol/l; these were considerably higher than spermine and spermidine levels. 4. As many of these gallstones bile samples were devoid of polyamines, it is questionable whether polyamines play an important role in cholesterol nucleation in human bile.

Polyamines--potential nucleating factors in bile.

Lithogenicity of human bile is dependent not only on cholesterol saturation, but also on the presence of nucleating and antinucleating factors. Most of the research in this field is directed toward biliary proteins, particularly glycoproteins. In the present study we have shown that spermine, spermidine, cadaverine and putrescine have a nucleating effect in model bile as well as in native human bile. These findings are based on 183 mixing experiments using biles from 10 patients and model biles. The effect seems to be dose dependent at concentrations up to 10 mmol/l. It is not accompanied by a shift in cholesterol distribution between its vesicular and micellar carriers. It is at present uncertain whether these effects are pharmacologic or physiologic. These findings emphasize, however, the potential importance of non-protein compounds in the cholesterol nucleation process in bile.

A new method for the rapid measurement of cholesterol crystallization in model biles using a spectrophotometric microplate reader.

Measurements of the cholesterol crystal observation time, and particularly the crystal growth rate in model biles, are important in biliary pathophysiology. The aim of this study was to develop a semi-automated method permitting multiple, simultaneous, and precise measurements of the crystal growth rate in model biles. Incubated model biles were mixed with a high concentration of NaTDC to solubilize non-crystalline turbidity and spectrophotometric measurements were performed. In parallel, samples were observed by light microscopy. The absorbance correlated linearly with the crystal mass and permitted quantitation of the crystal growth rate. Polarized light microscopy was more sensitive than spectrophotometry for determining the initial crystal observation time, while spectrophotometry was more precise and quantitative for measuring the crystal growth rate.

Endoscope-induced colitis: description, probable cause by glutaraldehyde, and prevention.

Six cases of acute, self-limited colitis that occurred after screening flexible sigmoidoscopy were deduced to be iatrogenic, probably caused by glutaraldehyde residues in the endoscopes after disinfection in an automatic disinfecting machine. The concentration of these residues was indirectly estimated by conductivity measurements of electrolyte concentration in the rinse-water tank of the disinfecting machine during and after five cycles and also in fluid remaining on the surface of the endoscopes and in the endoscope channels. After five cycles, residues in both the rinse water and fluid in the endoscope channels contained the equivalent of 0.2% glutaraldehyde. This was the result of a combination of technical and human errors. It can be prevented by changing the rinse water after each cycle, using forced air to dry the instruments, and washing the endoscopes before use. Users of the automatic disinfecting machine should be aware of the potential accumulation of toxic levels of residues that remain in endoscopes after disinfection.

The effects of phospholipid molecular species on cholesterol crystallization in model biles: the influence of phospholipid head groups.

BACKGROUND/AIMS: Variations in the molecular species of biliary phospholipids have been shown to exert major effects on cholesterol solubility and carriers in model and human biles. The aim of this study was to explore systematically the effects of various phospholipid head groups on the cholesterol crystallization process in model biles. METHODS: Three different control model biles were prepared using varying proportions of egg lecithin, cholesterol and Na taurocholate. In the test biles, 20% of the egg lecithin was replaced with synthetic phosphatidylserine, phosphatidylethanolamine, phosphatidylglycerol or phosphatidylcholine, keeping the phospholipid acyl chains and other biliary lipids constant in each experiment. RESULTS: Phosphatidylserine and phosphatidylglycerol significantly prolonged the crystal observation time, from 2 days to 10 and 6 days, respectively (p<0.02), while phosphatidylethanolamine had little and phosphatidylcholine no effect. The crystal growth rate was significantly slowed down with 20% phospholipid replacement in the following order: phosphatidylglycerol >phosphatidylserine >phosphatidylethanolamine. The total crystal mass after 14 days, as measured by chemical analysis, was reduced by 59% with phosphatidylserine (p<0.05), and by 73% with phosphatidylglycerol (p<0.05); while phosphatidylethanolamine had little effect. The precipitable cholesterol crystal fractions after 14 days were significantly reduced with phosphatidylserine (54%) and phosphatidylglycerol (37%), but not with phosphatidylethanolamine or phosphatidylcholine. CONCLUSIONS: Variations in the head groups of biliary phospholipids may markedly slow down the cholesterol crystallization process in model biles.

Behavior of cholesterol and spin-labeled cholestane in model bile systems studied by electron spin resonance and synchrotron x-ray.

The behavior of mixed bile salt micelles consisting of sodium taurocholate, egg phosphatidylcholine, and cholesterol has been studied by ESR spin labeling and synchrotron x-ray scattering. Consistent with published phase diagrams, pure and mixed bile salt micelles have a limited capacity to incorporate and, hence, solubilize cholesterol. Excess cholesterol crystallizes out, a process that is readily detected both by ESR spin labeling using 3-doxyl-5 alpha-cholestane as a probe for cholesterol and synchrotron x-ray scattering. Both methods yield entirely consistent results. The crystallization of cholesterol from mixed bile salt micelles is indicated by the appearance of a magnetically dilute powder spectrum that is readily detected by visual inspection of the ESR spectra. Both the absence of Heissenberg spin exchange and the observation of a magnetically dilute powder spectrum provide evidence for the spin label co-crystallizing with cholesterol. In mixed bile salt micelles containing egg phosphatidylcholine, the solubility of cholesterol is increased as detected by both methods. With increasing content of phosphatidylcholine and increasing mole ratio cholesterol/phosphatidylcholine, the anisotropy of motion of the spin probe increases. The spin label 3-doxyl-5 alpha-cholestane is a useful substitute for cholesterol provided that it is used in dilute mixtures with excess cholesterol: the cholesterol/spin label mole ratio in these mixtures should be greater than 100. Despite the structural similarity between the two compounds, there are still significant differences in their physico-chemical properties. These differences come to the fore when cholesterol is totally replaced by the spin-label: 3-doxyl-5a-cholestane is significantly less soluble in bile salt and mixed bile salt micelles than cholesterol and, in contrast with cholesterol, it interacts only very weakly, if at all,with phosphatidylcholine. The potential of the ESR method for detecting cholesterol crystal growth in human bile is discussed.

Fatty acid bile acid conjugates (FABACs)--new molecules for the prevention of cholesterol crystallisation in bile.

BACKGROUND: Cholesterol gall stones are a frequent disease for which at present surgery is the usual therapy. Despite the importance of bile acids it has become evident that phospholipids are the main cholesterol solubilisers in bile. Even phospholipid components, such as fatty acids, have anticrystallising activity. AIM: To synthesise fatty acid bile acid conjugates (FABACs) and study their effects on cholesterol crystallisation in bile in vitro and in vivo. METHODS: FABACs were prepared by conjugation of cholic acid at position 3 with saturated fatty acids of variable chain length using an amide bond. Cholesterol crystallisation and its kinetics (crystal observation time, crystal mass) were studied in model bile, pooled enriched human bile, and fresh human bile using FABACs with saturated fatty acids of varying chain length (C-6 to C-22). Absorption of FABACs into blood and bile was tested in hamsters. Prevention of biliary cholesterol crystallisation in vivo was tested in hamsters and inbred mice. RESULTS: FABACs strongly inhibited cholesterol crystallisation in model as well as native bile. The FABACs with longer acyl chains (C-16 to C-22) were more effective. At a concentration of 5 mM, FABACs almost completely inhibited cholesterol crystallisation in fresh human bile for 21 days. FABACs were absorbed and found in both portal and heart blood of hamsters. Levels in bile were 2-3 times higher than in blood, indicating active secretion. Appreciable levels were found in the systemic circulation 24-48 hours after a single administration. Ingested FABACs completely prevented the formation of cholesterol crystals in the gall bladders of hamsters and mice fed a lithogenic diet. CONCLUSIONS: FABACs are potent inhibitors of cholesterol crystallisation in bile. They are absorbed and secreted into bile and prevent the earliest step of cholesterol gall stone formation in animals. These compounds may be of potential use in cholesterol gall stone disease in humans.