FIC1, the protein affected in two forms of hereditary cholestasis, is localized in the cholangiocyte and the canalicular membrane of the hepatocyte.

BACKGROUND/AIMS: FIC1 (familial intrahepatic cholestasis 1) is affected in two clinically distinct forms of hereditary cholestasis, namely progressive familial intrahepatic cholestasis type 1 (PFIC1) and benign recurrent intrahepatic cholestasis. Here we examined the subcellular localization of this protein within the liver. METHODS: Antibodies raised against different epitopes of human FIC1 were used for immunoblot analysis and immunohistochemical detection of FICI. RESULTS: Immunoblot analysis of intestine and liver tissue extracts from human, rat and mouse origin indicated that the antibodies raised against FIC1 specifically detected FIC1 as a 140-kDa protein. In the liver homogenate of a PFIC1 patient, FIC1 could not be detected. Analysis of isolated rat liver membrane vesicles indicated that this protein is predominantly present in the canalicular membrane fraction. Immunohistochemical detection of the protein in liver sections confirmed that FIC1 was present in the canalicular membrane, whereas no staining was observed in the PFIC1 patients liver. Double label immunofluorescence of murine liver revealed that FIC1 colocalized with cytokeratin 7 in cholangiocytes. CONCLUSIONS: The localization of FIC1 in the canalicular membrane and cholangiocytes suggests that it may directly or indirectly play a role in bile formation since mutations in FICI are associated with severe symptoms of cholestasis.

Increased activity in the biliary Con A-binding fraction accounts for the difference in crystallization behavior in bile from Chilean gallstone patients compared with Dutch gallstone patients.

Chile has one of the highest prevalences of cholesterol gallstone disease in the world. Recent data indicate that this is partly caused by genetic (Indian) factors. However, the causal factors inducing increased gallstone formation have not been elucidated. The aim of this study was to compare biliary composition and cholesterol crystallization in bile from patients of high and moderate risk areas (Chile and The Netherlands) for gallstone disease. Bile was sampled at cholecystectomy from 30 Chilean and 26 Dutch gallstone patients. The Con A-binding fraction (CABF) was extracted from fresh native bile samples by incubation with Con A-sepharose. Reconstitution of the CABF to the Con A-extracted native bile induced almost full recovery of crystallization confirming the validity of this technique. There was no difference between the two groups regarding sex and age. Chilean bile nucleated significantly faster (3.5 +/- 0.6 vs. 7.9 +/- 1.5 days) despite the fact that Dutch bile had a significantly higher cholesterol saturation index (CSI) (1.6 vs. 1.2, P = .01). The total lipid content was not different. Chilean bile contained more total protein (5 vs. 2.9 mg/mL, P = .008). IgG, IgM, Haptoglobin and alpha-1-acid glycoprotein were not different between the two groups. IgA, though, was significantly higher in the Chilean samples (0.44 vs. 0.19 mg/mL, P < .001). Extraction of CABF increased crystal observation time (COT) and decreased crystal growth in both groups. However, the effects were much more pronounced in the Chilean samples. Compared with Dutch bile, Chilean bile crystallizes much faster despite a lower CSI. Chilean bile contains an increased content of Con A-binding nucleation promoting activity.

Concanavalin A-binding cholesterol crystallization inhibiting and promoting activity in bile from patients with Crohn's disease compared to patients with ulcerative colitis.

BACKGROUND/AIMS: Crohn's disease is a risk factor for gallstone formation. In contrast, patients with ulcerative colitis have an incidence of gallstone formation comparable to the general population. The reason for this difference is not known. The aim of this study was to elucidate the factors controlling cholesterol crystallization in gallbladder bile of Crohn's disease and ulcerative colitis patients. METHODS: Gallbladder bile was obtained by aspiration during bowel resections (26 Crohn's disease patients, 20 ulcerative colitis patients). Biliary lipid composition, crystal detection time and the effect of extraction of the concanavalin A-binding fraction on crystal formation were determined. RESULTS: Cholesterol crystals were present in seven of the 26 bile samples of Crohn's disease-patients and one of the 20 ulcerative colitis patients. Four of the bile samples of Crohn's disease patients were fast nucleating. None of the 20 ulcerative colitis patients had fast nucleating bile. Lipid composition, total lipid concentration and CSI were not significantly different between the two groups. In Crohn's disease patients extraction of concanavalin A-binding fraction decreased crystallization in 10 bile samples but accelerated crystallization in one bile sample. In eight bile samples from ulcerative colitis patients crystallization increased after concanavalin A-binding fraction extraction. CONCLUSIONS: Compared to ulcerative colitis patients, gallbladder bile of Crohn's disease patients showed increased cholesterol crystallization despite comparable lipid composition and cholesterol saturation index. This difference is caused by increased cholesterol crystallization-promoting activity. Bile from ulcerative colitis patients contains a Con A-binding factor which inhibits cholesterol crystallization.

Risk of gallstone disease is associated with serum level of alpha-1-acid glycoprotein.

We explored the association between serum level of alpha-1-acid glycoprotein (AGP, an acute phase protein) and the risk of gallstones. AGP was determined in stored serum samples from a case-control study (cases: 113 patients who underwent cholecystectomy for gallstone disease; controls: 184 surgery patients screened by ultrasound, showing no gallstones). Serum AGP correlated negatively with HDL-cholesterol and positively with triglycerides. Presence of gallstones was positively associated with serum AGP, more strongly so for cholesterol gallstones than for pigment gallstones, but not differently between solitary and multiple cholesterol stones. Asymptomatic gallstones (13 surgery patients with gallstones found by screening) were also associated with serum AGP levels. A causal role of elevation of serum AGP (or underlying immune activation) in gallstone formation is discussed.

Lack of enteral nutrition during critical illness is associated with profound decrements in biliary lipid concentrations.

BACKGROUND: Food in the intestine drives the enterohepatic circulation of bile components. OBJECTIVE: We investigated whether parenteral or enteral delivery of nutrients alters serum and biliary lipids in critically ill patients. DESIGN: Eight intensive care unit (ICU) patients who had received >/= 5 d of total parenteral nutrition (TPN) were compared with 8 ICU patients who had fasted for >/=5 d. Both groups were studied before and after 5 d of enteral nutrition (EN). Each patient served as his or her own control. Duodenal bile was analyzed for biliary lipid content and serum lipids were determined simultaneously. Duodenal bile samples from 18 healthy persons served as controls. RESULTS: Bile salt concentrations in all ICU patients were 17% of control values before EN (P < 0.005) and 34% of control values after 5 d of EN (P < 0.005). Phospholipid concentrations were 12% of control before EN (P < 0. 0005) but increased almost 4-fold after EN (P < 0.0005). Biliary cholesterol concentrations were 20% of control values before EN (P < 0.001) and did not improve afterward. No difference in bile composition was observed between fasted ICU patients and those who received TPN. The inverse correlation between the severity of illness and biliary lipid concentrations observed before EN disappeared with enteric stimulation. The low serum concentrations of HDL cholesterol and apolipoprotein A-I increased significantly with EN in all ICU patients. CONCLUSION: Lack of EN during critical illness was associated with profound decrements in biliary lipid concentrations that normalized partially after 5 d of EN. We hypothesize that loss of enteric stimulation in ICU patients impairs hepatic lipid metabolism.

Enterohepatic cycling of bilirubin: a putative mechanism for pigment gallstone formation in ileal Crohn's disease.

BACKGROUND & AIMS: Patients with ileal disease, bypass, or resection are at increased risk for developing gallstones. In ileectomized rats, bilirubin secretion rates into bile are elevated, most likely caused by increased colonic bile salt levels, which solubilize unconjugated bilirubin, prevent calcium complexing, and promote its absorption and enterohepatic cycling. The hypothesis that ileal disease or resection engenders the same pathophysiology in humans was tested. METHODS: Sterile gallbladder bile samples were obtained intraoperatively from 29 patients with Crohn's disease and 19 patients with ulcerative colitis. Bilirubin, total calcium, biliary lipids, beta-glucuronidase activities, and cholesterol saturation indices in bile were measured, and markers of hemolysis and ineffective erythropoiesis in blood were assessed. RESULTS: Bilirubin conjugates, unconjugated bilirubin, and total calcium levels were increased 3-10-fold in bile of patients with ileal disease and/or resection compared with patients with Crohn's colitis or ulcerative colitis. Biliary bilirubin concentrations correlated positively with the anatomic length and duration of ileal disease. Endogenous biliary beta-glucuronidase activities were comparable in all groups, and both the hemogram and serum vitamin B12 levels were normal. CONCLUSIONS: This study establishes that increased bilirubin levels in bile of patients with Crohn's disease are caused by lack of functional ileum, supporting the hypothesis that enterohepatic cycling of bilirubin occurs.

Mucin secretion by the human colon cell line LS174T is regulated by bile salts.

We recently reported that bile salts play a role in the regulation of mucin secretion by cultured dog gallbladder epithelial cells. In this study we have examined whether bile salts also influence mucin secretion by the human epithelial colon cell line LS174T. Solutions of bile salts were applied to monolayers of LS174T cells. Mucin secretion was quantified by measuring the secretion of [3H]GlcNAc labeled glycoproteins. Both unconjugated bile salts as well as taurine conjugated bile salts stimulated mucin secretion by the colon cells in a dose-dependent fashion. Hydrophobic bile salts were more potent stimulators than hydrophilic bile salts. Free (unconjugated) bile salts were more stimulatory compared with their taurine conjugated counterparts. Stimulation of mucin secretion by LS174T cells was found to occur at much lower bile salt concentrations than in the experiments with the dog gallbladder epithelial cells. The protein kinase C activators PMA and PDB had no stimulatory effect on mucin secretion. We conclude that mucin secretion by the human colon epithelial cell line LS174T is regulated by bile salts. We suggest that regulation of mucin secretion by bile salts might be a common mechanism, by which different epithelia protect themselves against the detergent action of bile salts, to which they are exposed throughout the gastrointestinal tract.

Hepatic bile versus gallbladder bile: a comparison of protein and lipid concentration and composition in cholesterol gallstone patients.

Many studies have demonstrated that gallbladder bile (but not hepatic bile) of animals or patients with cholesterol gallstones contains higher protein concentrations than does gallbladder bile of control patients without stones or with pigment stones. The underlying defect has not been elucidated. To establish whether there is net production or net absorption/degradation of protein by gallbladder epithelium for different classes of protein, paired samples of hepatic and gallbladder bile were obtained from fourteen patients with cholesterol gallstones during elective cholecystectomy. In these paired samples, lipid and protein composition were determined. To obtain the concentration ratio (CR) of protein and lipid, its concentration in the gallbladder was divided by the concentration determined in the paired hepatic bile sample. The CR of bile salts was used as a parameter for water absorption in the gallbladder. Of the biliary proteins that were determined only mucin, albumin, immunoglobulin (Ig) G, and aminopeptidase N appeared to increase in the gallbladder from another cause than water absorption. A strong correlation was found between mucin, albumin, and IgG. Haptoglobin, alpha1-acid glycoprotein, IgM, and IgA appeared to be absorbed by gallbladder epithelium in the majority of patients. In cholesterol gallstone patients, total protein concentration in gallbladder bile of cholesterol gallstone patients is increased when compared with hepatic bile. The increase in protein concentration cannot be explained for all bile samples solely by water absorption. In this study we show that the defect is largely caused by a selective increase in albumin, mucin, and IgG. All other proteins which were investigated are taken up by the gallbladder.

Analysis of occluded pancreatic stents and juices in patients with chronic pancreatitis.

BACKGROUND: Pancreatic stents may occlude with time, and there is little information available on the nature of the clogging process. METHODS: We analyzed the contents of occluded pancreatic polyethylene stents in nine patients with chronic pancreatitis. In the same patients, the protein patterns in the corresponding pancreatic juices were analyzed. The stents had been in place for a mean of 9 weeks (range 2 to 17). RESULTS: All stents were occluded at both ends, especially around side holes, with thick creamy-white precipitate. The average dry weight of occluding debris was 3 mg per 3.25 cm 10F stent. Total protein content was 50% (SD 16.3) and total calcium 0.8% of dry weight (SD 0.6). Light microscopy showed that proteinaceous material completely filled the stent lumen. Yeasts and plant material were seen in two stents. A variable number of bacteria of mixed species, sometimes in clumps, were patchily scattered in the protein matrix. Cultures of stent contents grew several species of Gram-positive and negative bacteria. Scanning and transmission electron microscopy showed an amorphous protein matrix in all stents, arranged as a network in some areas, but in layers in other areas. Sodium dodecylsulfate polyacrimide gel electrophoresis showed that protein patterns of stent contents were remarkably different from the protein patterns of the juice samples of the same patient. A 66 kD band, identified as albumin, appeared in the protein patterns of stent content, whereas it was lacking in most juice samples. CONCLUSIONS: Adherence of protein, especially albumin, plays an important role in the process of pancreatic stent clogging. Other factors, such as bacteria, refluxed duodenal contents, and calcium seem to be of less importance.

The role of the Concanavalin A-binding fraction in cholesterol crystallization in native human bile.

BACKGROUND/AIMS: Many Concanavalin A-binding glycoproteins have been proposed to influence cholesterol crystallization in human bile. This has been studied mainly by addition of the Concanavalin A-binding fraction to model bile. The physiological relevance of the proteins in native bile is not yet known. The aim of this study was to establish the role of the Concanavalin A-binding fraction in cholesterol crystallization in native human gallbladder bile. METHODS: To determine the effects of the removal of Concanavalin A-binding fraction, fresh human gallbladder bile was incubated with either Concanavalin A-Sepharose or Sepharose alone. Beads were sedimented and crystallization was studied in the supernatant. RESULTS: Extraction of Concanavalin A-binding fraction decreased crystallization in fast-nucleating biles (Crystal Detection Time < or =4 days). Slow-nucleating biles were not affected. The effect could not be related to the content of known pronucleating proteins (IgA, IgM, haptoglobin, aminopeptidase N and alpha1-acid glycoprotein), since the slow-nucleating biles contained similar amounts of these proteins. CONCLUSIONS: Although Concanavalin A-binding fraction always accelerated crystallization when added to model bile, removal of the same fraction from native bile often had no effect. We conclude that slow-nucleating biles in particular contain undetermined factors which regulate the activity of pronucleators.

Characterization of the cholesterol crystallization-promoting low-density particle isolated from human bile.

BACKGROUND & AIMS: Biliary concanavalin A-binding glycoprotein (CABG) contains cholesterol crystallization-promoting activity that is not accounted for by the pronucleators that have been characterized in this fraction. The aim of this study was to isolate and characterize the missing activity. METHODS: Biliary glycoprotein was isolated using concanavalin A-Sepharose. Promoting activity in CABG was purified using density gradient ultracentrifugation. RESULTS: Activity in CABG separated into two fractions at low (1.08) and high (1.29) density, which showed different crystallization kinetics in a model bile. The high-density fraction had a late onset time (49.2 +/- 17.8 hours) but a high crystal growth rate (13.4 +/- 5.2 micrograms. mL-1.h-1). The low-density fraction had a rapid onset time (33.9 +/- 20.9 hours) but a slower growth rate (6.5 +/- 3.8 micrograms.mL-1 .h-1). The high-density fraction was not further characterized in this study. The low-density fraction contained solid particles consisting of lipid and very little protein, and the activity was fully pronase resistant. Delipidation of the low-density fraction removed all activity. CONCLUSIONS: A potent pronase-resistant nucleation-promoting activity was activated from human bile and characterized. The low-density fraction may be responsible for the rapid nucleation in bile from typical patients with fast-nucleating gallstones.

Immunoglobulins and alpha 1-acid glycoprotein do not contribute to the cholesterol crystallization-promoting effect of concanavalin A-binding biliary protein.

Human bile contains cholesterol crystallization-stimulating proteins that can be isolated by concanavalin A-Sepharose chromatography. In the past few years an increasing number of different pronucleating proteins have been identified in the concanavalin A-binding fraction. In this study we attempted to estimate the relative contribution of a number of these proteins to total concanavalin A-binding pronucleating activity. For this purpose, concanavalin A-binding glycoproteins were isolated from gallbladder bile samples from 12 patients with gallstones. The role of IgA, IgG and IgM and alpha 1-acid glycoprotein was investigated by means of immunoextraction. No decrease in crystallization-promoting activity was observed after precipitation of more than 98% of the different immunoglobulins. In addition, removal of more than 95% of alpha 1-acid glycoprotein from different concanavalin A-binding fractions had no significant effect on cholesterol crystallization-promoting activity. The influence of fibronectin was estimated by addition of physiological concentrations to a model bile system. At these concentrations fibronectin did not promote crystallization. From these data we conclude that immunoglobulins, alpha 1-acid glycoprotein and probably also fibronectin do not significantly contribute to total concanavalin A-binding activity.

An evaluation of immune competence in different swine breeds.

The purpose of this investigation was to establish parameters for the evaluation of immune competence in different swine breeds. Cellular and humoral immune reactivity against keyhole limpet haemocyanin (KLH) was analysed using the lymphocyte stimulation test (LST), a skin allergy test and the IgG response. Various characteristics of the KLH-specific immune response were studied in 988 sows of four breeds. KLH-specific immune responses showed considerable variability. The applied statistical model explained 63 to 73 per cent of this variation. Genetic influences, expressed as the heritability estimate, were rather high for the IgG response (0.33), as well as for the skin reaction (0.26) and the LST (0.41-0.45). A positive correlation between the various immune parameters was found. Selective breeding for immune responsiveness seems to be feasible, but selection for cellular as well as humoral immune reactivity also seems to be possible with KLH as the antigen.