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Protective role of biliary cholesterol and phospholipid lamellae against bile acid-induced cell damage

BACKGROUND/AIMS Bile salts (BS) are cytotoxic agents, but cell damage is not observed in the hepatobiliary system. We hypothesized that biliary lipid vesicles (unilamellae and multilamellae) could have a protective role against BS-induced cytotoxicity. METHODS Biliary lipid lamellar secretion was induced by feeding rats with 0.5% diosgenin. Cytoprotection was assessed in bile duct-obstructed rats and by incubating human erythrocytes with sodium taurocholate. RESULTS Biliary cholesterol concentration increased > 300% in diosgenin-fed rats; electron microscopic examination showed a great abundance of lipid lamellar vesicles in bile and within the canaliculi. After bile duct obstruction, serum hepatic enzyme activities were significantly lower in diosgenin-fed rats. Histologically severe and confluent hepatocellular necrosis was only observed in control rats. Biliary lamellar lipid material significantly reduced the BS-induced hemolytic effect in vitro in a concentration-dependent manner. This protective effect correlated to a progressive decrease in the intermicellar BS concentration. Phosphatidylcholine or cholesterol, alone or as lamellar structures, also showed cytoprotective effect in vitro but always less than native biliary lamellae. CONCLUSIONS These results support the concept that native biliary cholesterol phospholipid lamellae represent an important cytoprotective factor for hepatocytes and biliary epithelial cells against BS-induced damage.

Cholesterol saturation, not proteins or cholecystitis, is critical for crystal formation in human gallbladder bile

BACKGROUND & AIMS Biliary proteins are promoters of cholesterol crystallization in artificial model bile. However, their pathogenic importance for cholesterol precipitation in native gallbladder bile (GB) is uncertain. The aim of this study was to evaluate the significance of biliary lipids and proteins on cholesterol crystal detection time (ChCDT) of GB in patients with gallstones. METHODS ChCDT and concentrations of lipids, albumin, mucins, aminopeptidase N, alpha1-acid glycoprotein, haptoglobin, and immunoglobulins (Igs) were measured in GB of 92 patients, 52 of whom had cholesterol gallstones. RESULTS ChCDT was markedly reduced in gallstone patients. Compared with patients without gallstones, they had a significant increase in cholesterol saturation and total protein, albumin, mucin, and IgG biliary concentrations. In univariate analysis, ChCDT of GB was significantly correlated with cholesterol saturation and total lipid, protein, Ig, aminopeptidase N, and alpha1-acid glycoprotein concentrations. However, stepwise logistic regression analysis showed that only cholesterol saturation independently correlated to ChCDT. Gallbladder inflammation correlated with the concentration of Igs, but subtraction of IgG from GB did not modify the ChCDT. CONCLUSIONS Biliary cholesterol transport and saturation, but not proteins, appear critical for the cholesterol crystallization abnormality observed in native bile from patients with gallstones.

Modulation of intrahepatic cholesterol trafficking: evidence by in vivo antisense treatment for the involvement of sterol carrier protein-2 in newly synthesized cholesterol transport into rat bile

Biliary cholesterol represents one of the two major excretory pathways for sterol elimination from the body and plays a central role in cholesterol gallstone formation. Biliary cholesterol originates from a precursor pool of preformed and newly synthesized free cholesterol. Although it has been suggested that newly synthesized and preformed biliary cholesterol are secreted by independent pathways, the specific cellular and molecular mechanisms are unknown. We used male Wistar rats to study the time-course of the appearance of newly synthesized cholesterol, phosphatidylcholine and protein into bile. The specific role of sterol carrier protein-2 (SCP-2) in the transport of newly synthesized biliary cholesterol was evaluated by an in vivo antisense oligonucleotide approach. In contrast to [14C]phosphatidylcholine and [35S]proteins, the time-course of [14C]cholesterol appearance into bile was rapid, and microtubule- and Golgi-independent. In vivo SCP-2 antisense treatment reduced and delayed the appearance of biliary [14C]cholesterol. Furthermore, hepatic SCP-2 expression increased more than 3-fold over control values in rats that had been treated with diosgenin to increase biliary secretion of newly synthesized cholesterol. These results suggest that SCP-2 is necessary for the rapid transport of newly synthesized cholesterol into bile and that hepatocytes can induce SCP-2 expression according to the rate of biliary secretion of newly synthesized cholesterol.

Cholesterol crystallization-promoting activity of aminopeptidase-N isolated from the vesicular carrier of biliary lipids.

Different hydrophobic glycoproteins are associated to native biliary vesicles, which are the major carrier of biliary cholesterol. Some of these proteins promote cholesterol crystallization, a key step in cholesterol gallstone formation. This study was specifically conducted to identify the 130 kDa biliary vesicle‐associated glycoprotein and to determine its in vitro effect on the cholesterol crystal formation time. The 130 kDa vesicular glycoprotein was identified as aminopeptidase‐N by amino acid sequencing and specific enzymatic assay. Polyclonal antibodies raised against aminopeptidase‐N allowed us to determine its concentration in human hepatic bile, which varied from 17.3 to 57.6 μg/ml. Aminopeptidase‐N showed a concentration‐dependent cholesterol crystallization activity when it was added to supersaturated model bile at a concentration range usually found in native bile. Because of this promoting effect on in vitro cholesterol crystal formation, we suggest that biliary aminopeptidase‐N may play a critical role in the pathogenesis of cholesterol gallstone disease.

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.

Biliary aminopeptidase-N and the cholesterol crystallisation defect in cholelithiasis.

Several biliary proteins have cholesterol crystallisation promoting activity. One of these glycoproteins is aminopeptidase-N, a canalicular ectoenzyme. This study attempted to localise aminopeptidase-N along the biliary tree, to assess its concentration in a series of 98 patients subjected to abdominal surgery, 40 of them without gall stones, and to correlate its concentration with cholesterol crystal formation time of gall bladder bile. Aminopeptidase-N was isolated from purified native biliary vesicles. A specific polyclonal rabbit anti-aminopeptidase-N antibody was prepared for quantitative immunoblotting and for immunolocalisation. Tissue was obtained from liver biopsy specimens and from gall bladders removed at surgery because of gall stone disease. Aminopeptidase-N was immunolocalised to the apical membranes of hepatocytes and to the apical pole of ductular and gall bladder mucosal cells. The nucleation time of gall bladder bile was mean (SD) 4 (3) days in the gall stone group, compared with 21 (18) days in the control group (p < 0.001). Total absolute biliary protein and aminopeptidase-N concentrations were similar in both the control and gall stone patients. There was a reciprocal significant correlation, however, between the nucleation time and the relative aminopeptidase-N concentration (r = -0.35, p < 0.01) only in the gall stone group of patients. This study shows that this apical transmembrane ectoenzyme with cholesterol crystallisation promoting activity is present along the biliary tree and the hepatocyte. These findings support the concept that high concentrations or qualitative changes of biliary aminopeptidase-N contribute to cholesterol gall stone formation.