Cholesterol Nucleation Research Articles

Degree of fatty acyl chain unsaturation in biliary lecithin dictates cholesterol nucleation and crystal growth

To clarify factors involved in the formation of cholesterol gallstones, we studied the relationship between the degree of fatty acyl chain unsaturation of biliary lecithin and bile metastability. We used supersaturated model bile solutions (molar taurocholate /lecithin/cholesterol ratio (73:19.5:7.5), total lipid concentration 9 g/dl) that contained equimolar egg yolk or soybean lecithins or a sn-1 palmitoyl, sn-2 linoleoyl phosphatidylcholine. Gel permeation Chromatographie studies showed that the vesicular cholesterol distribution and dimension were inversely related to the degree of unsaturation of the lecithin species, estimated by reverse phase, high-performance liquid chromatography. Differential interference contrast microscopy and assay of cholesterol crystal growth showed that a higher degree of fatty acyl chain unsaturation of the lecithin species was associated with a faster nucleation time and rate of crystal growth. Our results suggest that vesicular lecithins containing more unsaturated fatty acyl chains bind less tightly to cholesterol than lecithins containing predominantly saturated fatty acids, and that the biliary lecithin species dictates, in part, the nucleation and growth of cholesterol crystals in bile.

Calcium affinity for biliary lipid aggregates in model biles: Complementary importance of bile salts and lecithin

Background/Aims : Despite putative roles of calcium in biliary physiology and gallstone formation, quantitative aspects of calcium binding to bile salt (BS) monomers, simple micelles, mixed micelles, and vesicles, which constitute the lipid aggregates in bile, remain unexplored. Methods : Calcium activity was measured using the calcium electrode in pathophysiologically relevant model biles composed of either individual BS species or a physiological mixture of glycine and taurine conjugates, as functions of lecithin and cholesterol contents and total lipid concentration. Results : Calcium binding increased with increasing BS concentrations and lecithin contents and varied with species (dihydroxy > trihydroxy BS) and with conjugation (unconjugated > glycine conjugates > taurine conjugates). Although lecithin/cholesterol vesicles did not bind detectable calcium, when taurocholate was incorporated into membrane bilayers, calcium binding was substantially greater than with equimolar BS alone. Added cholesterol did not alter calcium binding, despite cholesterol saturation of biliary lipid aggregates and induction of liquid crystalline and solid crystalline-phase transitions. Conclusions : In model biles, most calcium is bound to mixed micelles, with minor contributions by BS monomers, simple micelles, and vesicles. It is proposed that BS-induced binding of calcium to vesicles and mixed micelles may be important in nucleation of cholesterol and bilirubinates from native bile.

Fibronectin in human gallbladder bile: cholesterol pronucleating and/or mucin "link" protein?

Some biliary proteins (pronucleators) seem to be essential factors for cholesterol crystal formation and crystal growth in bile. A recent study suggests that fibronectin is such a pronucleator in bile. Fibronectin also seems to closely interact with intestinal mucin. Since biliary mucin plays an important role in gallstone formation, such an interaction in bile may be of relevance in cholesterol gallstone formation. To more clearly elucidate the role of fibronectin in cholesterol gallstone disease, we measured the concentration of fibronectin in native bile of cholesterol gallstone patients and checked its influence on the cholesterol nucleation time of model bile. We further looked for a molecular interaction between biliary fibronectin and gallbladder mucin. We found that fibronectin is present in gallbladder bile of gallstone patients in low concentrations (2.6 +/- 1.2 micrograms/ml). Bile fibronectin did not interact with gallbladder mucin. Moreover, in a wide range of concentrations fibronectin had no influence on the nucleation time of model bile. We conclude that fibronectin does not seem to play a major role in cholesterol gallstone disease.

Bile salt-induced cholesterol crystal formation from model bile vesicles: a time course study.

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 leakage-assay 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 time 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 DC 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 ratio were observed. We conclude that under the conditions used in the present model study, the kinetics of cholesterol crystallization are governed by the hydrophobicity of the added bile salts and their capacity to form mixed micelles. The results emphasize the pivotal role of time, and the dynamic aspects of the processes involved in cholesterol crystal formation.

Subcellular characterization of glycoproteins in the principal cells of human gallbladder. A lectin cytochemical study.

Gallbladder mucus is mainly composed of glycoproteins, which seem to play a critical role in cholesterol nucleation during gallstone formation. The biosynthetic pathway and sequential processing as well as the characterization of the oligosaccharide side-chains of human gallbladder secretory glycoproteins have not been completely defined. The aim of the present study is the subcellular characterization of the glycoproteins in the principal cells of human gallbladder. Principal cells of normal human gallbladder were studied by means of a variety of cytochemical techniques, including lectin histochemistry, enzyme and chemical treatments, immunocytochemistry and lectin-gold technology. Fucose, galactose, N-acetylglucosamine, N-acetylgalactosamine and N-acetylneuraminic acid residues were detected in mucous granules, Golgi apparatus and apical membrane of principal cells. Mannose residues were only observed in dense bodies. Oligosaccharide side-chains of the glycoproteins contained in the biliary mucus are synthesized in the Golgi apparatus of the principal cells of the gallbladder epithelium and are also contained in the mucous granules of these cells. Terminal N-acetylneuraminic acid(alpha 2-3)galactose(beta 1-3)N-acetylgalactosamine, N-acetylneuraminic acid(alpha 2-3)galactose(beta 1-4)N-acetylglucosamine and galactose(beta 1-4)N-acetylglucosamine sequences are contained in the oligosaccharide chains of gallbladder mucus glycoproteins. The dense bodies detected in the cytoplasm of the principal cells contained N-linked glycoproteins. Mucin-type O-linked glycoproteins were the main components of the mucous granules although some N-linked chains were also detected.

Cholesterol nucleates rapidly from mixed micelles in the prairie dog

Current theory suggests that the nucleation of cholesterol in human bile requires the aggregation and fusion of cholesterol-enriched phospholipid vesicles. This theory is based on observations which do not exclude the precipitation of cholesterol from mixed micelles. The present study examines the role of mixed micelles and vesicles in the formation of cholesterol monohydrate crystals in the prairie dog. The intermicellar bile salt concentration of prairie dog gallbladder bile was determined using equilibrium dialysis. Model bile equivalent to gallbladder bile from cholesterol-fed prairie dogs was used as dialysant yielding the intermicellar (dialysate) concentration of 9 mM. Cholesterol carriers in gallbladder bile from 11 cholesterol-fed animals were then separated by Sephacryl S200 gel filtration chromatography using eluant buffer containing the intermicellar bile salt concentration. Gel filtration chromatography of fresh bile demonstrated that 100% of cholesterol was carried in the mixed micellar fraction with no vesicles observed in any of the 11 animals. The gallbladder bile nucleation time was 2.0 ± 0.3 days for the cholesterol-fed animals. Gel filtration chromatography immediately after nucleation again revealed a single mixed micellar peak. These data indicate that cholesterol is carried exclusively in and nucleates rapidly from mixed micelles in the cholesterol-fed prairie dog and that cholesterol-phospholipid vesicles are not required in this process.

Heterogeneity of Human Biliary Mucin: Functional Implications

1. Human gallbladder mucin has been implicated as playing a role in the pathogenesis of gallstones. In previous studies no differences have been found in the content or composition of mucins derived from control bile or cholesterol gallstone bile. Until now, no differences were also found between these two groups of mucins with regard to their ability to cause cholesterol nucleation. In the accompanying paper we have reported that there is a strong heterogeneity of gallbladder mucins derived from individual patients (M. J. A. van Wijland, J. H. Klinkspoor, L. Th. de Wit, R. P. J. Oude Elferink, G. N. J. Tytgat and A. K. Groen, Clin Sci 1994; 86: 67-74). In the present study we further investigated a possible patient to patient heterogeneity of mucin by means of immunological and functional characterization of mucins isolated from hepatic bile of six different patients with gallstones. 2. Considerable heterogeneity was found. Two of the mucins barely reacted with a polyclonal anti-mucin antibody, whereas the other four mucins reacted very strongly. Lectin-binding studies indicated that the glycans of these two mucins expressed less D-N-acetylgalactosamine residues than the other four mucins. This was confirmed by analyses of the glycan compositions. These studies furthermore indicated that the glycans were of the O-linked type, contained alpha-D-N-acetylglucosamine and were fucosylated, sialylated and sulphated to different extents. Except for a strong heterogeneity in the sugar composition of the mucins, heterogeneity was also found in the biological activity of the mucins.(ABSTRACT TRUNCATED AT 250 WORDS)

The relation between biliary lipids, nucleation time, and number of gallbladder stones after percutaneous gallbladder puncture.

Biliary lipids and nucleation time are increasingly of importance in the understanding of the cholesterol nucleation process in gallstone patients. Biliary lipids, total lipid concentration (TLC), cholesterol saturation index (CSI) and nucleation time (NT) were studied in 221 bile samples from patients with solitary (n = 120) and multiple (n = 101) gallbladder stones. Biliary cholesterol concentration and CSI did not differ between patients with solitary or multiple stones; however, it was positively correlated with the CSI (r = 0.93; p < 0.01). We found a negative correlation between CSI and TLC (r = -0.77 for solitary stones and r = -0.79 for multiple stones; p < 0.01). Furthermore, levels of total bile acids and phospholipids were similar in cases with solitary and multiple gallbladder stones. TLC did not correlate with single or multiple stones, whereas NT was determined to be negatively correlated with the number of gallstones (r = -0.39; p < 0.01). Patients with solitary stones had a significantly (p < 0.01) longer NT than those with multiple gallbladder stones (7.5 +/- 4.2 days versus 2.3 +/- 1.5 days). Our findings suggest that there exists a nucleation-promoting activity, which seems to be more pronounced in patients with multiple gallbladder stones than in those with solitary stones, indicating a major risk factor for the higher recurrence rate seen in these patients.

Effect of Nicotine on Gallbladder Bile

Several studies have shown that symptomatic gallstones are largely a disease of nonsmokers, which raises the possibility that nicotine may protect against the formation of gallstones. To examine the effect of nicotine on the gallbladder, 32 rabbits were allocated to four groups: controls and three treatment groups in which nicotine tartarate at low, medium and high doses was administered subcutaneously via an osmotic minipump. After 14 days’ treatment the gallbladder was removed and measurements made of gallbladder mucin synthesis, bile mucin concentration, bile acid concentration and cholesterol saturation. Serum nicotine concentrations (ng/mL) were (± SE) 0.4±0.1, 3.5±0.4, 8.8±0.8 and 16.2±1.8 in the controls and three treatment groups, respectively. Total bile acid concentration increased significantly in all three treated groups with the greatest increase in the group given low dose nicotine (P&lt;0.001). Cholesterol saturation did not differ significantly in any group but soluble mucin concentration in gallbladder bile was significantly reduced (P=0.013, 95% CI: 16 to 111) with high dose nicotine. Gallbladder mucin synthesis, measured by3H-glucosamine incorporation, did not change significantly with nicotine treatment. Subcutaneous nicotine 2.0 mg/kg/day for 14 days significantly reduced the concentration of biliary mucin, which could potentially reduce cholesterol nucleation and subsequent gallstone formation. This may be one of the mechanisms responsible for the relative reduction in gallstone disease among smokers.

The distribution of the biliary-anionic polypeptide fraction between cholesterol carriers in bile and its effect on nucleation

The small (7 kD) biliary phospholipid and calcium binding polypeptide (anionic polypeptide fraction/calcium binding protein) has been found in higher concentrations in the bile of patients with pigment stones than in controls. In different model systems it was variously found to promote or retard cholesteral crystalization. In the present study we investigated its distribution between cholesterol carriers in bile and its effect on cholesterol crystalization in native and model biles. On gel chromatography anionic polypeptide fraction/calcium binding protein was found predominantly in three areas: in the vesicular fraction, in the non-vesicular lipid fraction and in another fraction unassociated with biliary lipids. It was much more concentrated in the vesicular than in the non-vesicular fraction, the mean anionic polypeptide fraction/phospholipid molar ratio being 219 +/- 181 vs. 30.4 +/- 16, respectively. Anionic polypeptide fraction/calcium binding protein was added at three dose levels, 0.14, 0.28, 0.42 mg/ml (representing approximately 18%-55% of the physiologic biliary concentration), to 19 human and five model biles. This did not produce any significant changes in the nucleation time. The addition of anionic polypeptide fraction/calcium binding protein at a dose level of 0.42 mg/ml to 13 different human biles did not induce changes in the distribution of cholesterol among its carriers. The present experiments do not support a role for anionic polypeptide fraction/calcium binding protein in the process of cholesterol nucleation in bile. Qualitative changes in the protein molecule, as demonstrated in other human secretions, cannot be excluded.

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.

Monitoring cholesterol nucleation and crystallization from physiologically relevant model bile by time-lapse density gradient ultracentrifugation (UC)

Monitoring cholesterol nucleation and crystallization from physiologically relevant model bile by time-lapse density gradient ultracentrifugation (UC)

Effect of phospholipids and bile acids on cholesterol nucleation time and vesicular/micellar cholesterol in gallbladder bile of patients with cholesterol stones

Supersaturation and rapid nucleation of cholesterol in bile are of key importance in the pathogenesis of cholesterol gallstones. While the effects of bile acids and phospholipids on cholesterol saturation of bile have been extensively studied, their influence on the cholesterol nucleation time has not been compared. We, therefore, investigated whether increases of bile acid or phospholipid concentrations in bile by in vitro supplementation affect the cholesterol nucleation time. Bile samples were obtained at surgery from patients with cholesterol gallstones. Prior to the nucleation assay the bile samples were divided into 0.5-ml aliquots and supplemented with 1.25, 2.5, 5.0, and 10.0 mumol/ml of different phosphatidylcholines (PC-dimyristoyl, PC-dipalmitoyl, PC-distearoyl, and extracted biliary PCs) or with 5.0, 10.0, and 20.0 mumol/ml of bile acids (glycine or taurine conjugates of cholic acid, deoxycholic acid, or chenodeoxycholic acid). The increase of phosphatidylcholine or bile acid concentration decreased the mean cholesterol saturation index to a similar extent (PC: 0.1-0.3; BA: 0.1-0.2). Supplementations of bile with increasing amounts of synthetic or biliary PCs caused a marked prolongation of the nucleation time in bile from 1.5 +/- 0.2 up to > or = 21 days or 2.5 +/- 0.7 up to > or = 21 days. Concurrently, biliary cholesterol was shifted from vesicles to mixed micelles and the cholesterol/phospholipid ratio of the remaining vesicles was progressively lowered. In contrast, the addition of bile acids to gallbladder bile did not affect the cholesterol nucleation time (2.2 +/- 0.3 days), the percentage of vesicular cholesterol, or the cholesterol/phospholipid ratio of vesicles and micelles.(ABSTRACT TRUNCATED AT 250 WORDS)

Reduced cholesterol metastability of hepatic bile and its further decline in gall bladder bile in patients with cholesterol gall stones.

The reduced metastability of biliary cholesterol in the gall bladder bile of patients with cholesterol gall stones has been well shown. The purpose of this study was to examine the hypothesis that such a difference in metastability already exists in hepatic bile. Paired hepatic and gall bladder bile samples were collected from 10 patients with cholesterol gall stones and six patients without gall stones. Cholesterol nucleation time, biliary lipid concentration, vesicular cholesterol distribution, and biliary protein concentration were measured and compared. The nucleation time in the hepatic bile of patients with cholesterol gall stones was significantly shorter than the gall stone free patients (8.2 (7.2) v 15.7 (5.8) days, p < 0.05), and was associated with a greater concentration of biliary lipid despite the lack of a difference in the cholesterol saturation index (CSI) and total protein concentration. During the storage of bile in the gall bladder, the nucleation time became quicker in the patients with cholesterol gall stone (2.9 (1.7) days) while it was similar in the gall stone free patients (17.3 (5.7) days) compared with that of the corresponding hepatic bile. These differences were associated with a higher CSI (1.44 (0.33) v 1.13 (0.14), p < 0.05) and a greater vesicular cholesterol distribution (19.7 (11.9) v 4.4 (1.4)%, p < 0.01) in the patients with cholesterol gall stones than the gall stone free patients. The concentrations of total lipid and protein in gall bladder bile were not significantly different between the two groups. In conclusion, patients with cholesterol gall stones produce less metastable hepatic bile by the evidence of shorter nucleation time. During the storage of the bile in the gall bladder, the metastability is reduced further only in the cholesterol gall stone patients but not in the gall stone free patients.

Isolation of mucin from human hepatic bile and its induced effects on precipitation of cholesterol and calcium carbonatein vitro

Biliary mucin was isolated from human hepatic bile, and its induced effects on the appearance time of cholesterol monohydrate crystals (nucleation time) and on the precipitation of calcium carbonate were studied in vitro to examine the possible significance of mucin for ductular gallstone formation. Mucin was isolated by gel filtration on Sepharose CL-4B and a subsequent CsCl density gradient ultracentrifugation. Mucin thus obtained had a high purity as shown by a high-molecular-weight band on SDS-polyacrylamide gel electrophoresis and by the compatible amino acid composition with mucin purified from the gallbladder. The mucin at as low a concentration as 100 micrograms/ml significantly shortened the cholesterol nucleation time in the supersaturated model bile, mimicking human hepatic bile. On the other hand, the addition of mucin inhibited calcium carbonate precipitation in vitro. Taking account of that both cholesterol and calcium salts are major constituents of ductular gallstones, we conclude that biliary mucin is likely to play an important regulating role in the formation of ductular stones.

Histological evaluation of the intrahepatic biliary tree in intrahepatic cholesterol stones, including immunohistochemical staining against apolipoprotein A-1

Apolipoprotein A-1 is known to be one of inhibiting factors of cholesterol nucleation in bile, and decreased activity of apolipoprotein A-1 is considered to predispose cholesterol-supersaturated bile to formation of cholesterol crystals. To study the pathogenesis of the intrahepatic formation of cholesterol stones, we examined surgically resected liver specimens from six patients with intrahepatic cholesterol stones and compared the characteristic histopathological features with those of intrahepatic calcium bilirubinate stones, using morphological examination and immunohistochemical staining against apolipoprotein A-1. Morphologically, in all six patients with cholesterol stones the severity of chronic proliferative cholangitis with proliferation of the mucus-producing glandular elements in the walls of the large bile duct or periductal tissues was less extensive than that seen with calcium bilirubinate stones, and cholesterol crystals had formed in the septal and interlobular bile ducts. Immunohistochemically, unlike the normal liver and calcium bilirubinate stone-containing lobes, the hepatocytes and the epithelial lining of the bile ducts and peribiliary glands of the cholesterol stone-containing lobes did not react completely (some of the epithelial cells reacted only faintly) with apolipoprotein A-1 antibody. These findings suggest that an abundance of mucous substance and bacterial infection of the biliary tree may not be necessary for the formation of cholesterol stones, compared with findings in cases of calcium bilirubinate stones. We suggest that cholesterol crystals may be produced in the septal and interlobular bile ducts in the microenvironment of cholesterol-supersaturated bile and decreased activity of apolipoprotein A-1.

Cholesterol crystal appearance time of gallbladder bile in middle-aged women.

The aim of our study was to examine the hypothesis that the cholesterol nucleation time of gallbladder bile might be rapid in women in their fourth and fifth decades, which increases their risk of cholesterol gallstones. Fifty-four gallbladder bile samples were collected from gallstone-free patients and patients with cholesterol gallstones in a functioning gallbladder. We examined the relations of nucleation time to age and sex in each group. The nucleation time of gallbladder bile tended to be prolonged with age, but there was no correlation between nucleation time and age in each group. Cholesterol nucleation time was not significantly different between age-matched females and males in each group, nor was it faster in middle-aged females than in older females. Thus, nucleation time was not rapid in middle-aged females even in the gallstone-free group. These results indicate that cholesterol nucleation time is not significantly influenced by either sex or age, and cannot therefore account for the higher frequency of cholesterol gallstone disease in females in the fourth and fifth decades.

Effect of human biliary immunoglobulins on the nucleation of cholesterol.

We have previously identified that either biliary immunoglobulin IgA or IgM is a pronucleating protein which can accelerate the precipitation of cholesterol from bile. In this study we purified the biliary immunoglobulins (IgA, IgG, and IgM) to homogeneity by affinity chromatography to investigate the relative cholesterol nucleating potency of each immunoglobulin. Each immunoglobulin was added to slow nucleating heated abnormal biles in a dose-response manner to give a final concentration of protein in the range of 62.5-625 micrograms/ml bile. Cholesterol-nucleating activity was measured by noting the first day of cholesterol crystal formation as well as the number of crystals formed over the observation period. Biliary IgM and IgG appear to be more potent pronucleators than IgA. Isolated serum IgM from patients with Waldenstrom's macroglobulinemia as well as serum IgG from patients with and without cholesterol gallstones were shown to have pronucleating activity and acted in a dose-response manner. Commercial IgG unlike commercial IgM retains nucleating activity. The concentration of biliary immunoglobulins was measured by an enzyme-linked immunoassay (ELISA) in the gallbladder bile of patients with and without cholesterol gallstones. Biliary IgG concentrations in bile were higher in cholesterol gallstones patients than in pigmented gallstone patients and controls. We conclude that immunoglobulins particularly IgG and IgM are important pronucleating proteins and could play a role in the pathogenesis of cholesterol gallstones.

Isolation and partial characterization of cholesterol pronucleating hydrophobic glycoproteins associated to native biliary vesicles

Cholesterol is transported both in unilamellar phosphatidylcholine vesicles and in bile salts-mixed micelles in native bile. The vesicular carrier of biliary lipids apparently has a well defined protein profile with a potent cholesterol crystallization-promoting activity. This study was conducted to identify and further characterize these vesicular proteins and to test the effect of isolated vesicular proteins on the cholesterol crystal formation in supersaturated model bile. The results confirmed that proteins are a constant component of highly purified biliary vesicles both in hepatic and gallbladder bile. Immunoglobulins (IgA, IgG and IgM) and albumin are associated to the purified hepatic biliary vesicles. Furthermore, four different hydrophobic glycoproteins with a molecular mass of 130, 114, 86, and 62–67 kDa were isolated. These glycoproteins showed no reactivity with anti-human whole serum or anti-immunoglobulin antibodies, suggesting that these proteins are biliary-specific. Isolated 130, 114 and 62–67 kDa vesicular glycoproteins significantly decreased the cholesterol nucleation time in artificial model bile. We concluded that some, but not all, vesicular-bound hydrophobic glycoproteins have cholesterol pronucleating activity and they may be involved in the pathogenesis of cholesterol gallstone disease.

Human gallbladder bile becomes lithogenic during short-term intravenous hyperalimentation

Biliary sludge formation is reported to be one of the complications of intravenous hyperalimentation (IVH); however, the change in human biliary lithogenicity during IVH treatment has been little studied. To clarify the pathogenesis of IVH-induced biliary sludge, we determined biliary lipid composition, vesicular cholesterol concentration, and nucleation time using gallbladder bile samples collected from three groups: The IVH group comprised 9 patients who received IVH with fasting for a period of 2–8 days prior to surgery for gastrointestinal diseases. The control group comprised 10 patients operated after overnight fasting for gastro-intestinal diseases. The cholesterol gallstone group comprised 14 patients surgically treated for cholesterol gallstone disease after overnight fasting. The nucleation time in the IVH group was significantly shorter (7.8 ± 5.3 days, mean ± SD) than that in the control group (17.3 ± 5.5 days), while it did not reach the value in the cholesterol gallstone group (3.1 ± 3.3 days). The cholesterol saturation index in the IVH group (1.01 ± 0.27) was higher but not significantly different compared with the control group (0.80 ± 0.21). The concentrations of biliary lipids and individual bile acid were similar in the IVH and control groups. The vesicular cholesterol concentration in the IVH group (3.0 ± 2.1 m M) was significantly higher than that in the control group (0.9 ± 1.0 m M). In conclusion, IVH with fasting causes a rapid cholesterol nucleation time and a higher vesicular cholesterol concentration, thereby inducing an initial stage of gallbladder sludge formation.