Impaired human gallbladder lipid absorption in cholesterol gallstone disease and its effect on cholesterol solubility in bile.

BACKGROUND & AIMS: The role of the gallbladder in gallstone pathogenesis is still unclear. We examined the effects of gallbladder mucosal lipid absorption on lipid composition and cholesterol crystallization in bile. METHODS: The in vitro-isolated, intra-arterially perfused gallbladder model was used (1) to compare the absorption rates of lipids from standard bile by gallbladders obtained from 7 patients with cholesterol gallstones and 6 controls; and (2) to measure the microscopic cholesterol crystal detection time in cholesterol-enriched pig bile before and after lipid absorption by the pig gallbladder. RESULTS: Control gallbladders, but not cholesterol gallstone gallbladders, significantly reduced cholesterol (P < 0.02) and phospholipid (P < 0.01) and increased bile salt (P < 0.01) molar percentages in bile over a 5-hour period by efficient and selective cholesterol and phospholipid absorption. A histomorphometric study of the epithelial cells showed significantly higher values for nuclear density (P < 0.01) and nuclear (P < 0.05) and cytoplasmic (P < 0.05) areas in the cholesterol gallstone than the control group. Sequential microscopy of cholesterol-enriched pig bile showed significantly shorter cholesterol filament (P < 0.01) and typical cholesterol plate (P < 0. 02) detection times before than after exposure of bile to the gallbladder lipid absorption. CONCLUSIONS: In cholesterol gallstone disease, the human gallbladder epithelium loses its capacity to selectively and efficiently absorb cholesterol and phospholipids from bile, even if it is hyperplastic and hypertrophic. This epithelial dysfunction eliminates the positive effect that the normal gallbladder exerts on cholesterol solubility in bile and might be a pathogenetic cofactor for cholesterol gallstone formation.

Glucose intolerance and insulin resistance in cirrhosis are normalized after liver transplantation.

Cirrhosis is often associated with insulin resistance and glucose intolerance. We evaluated if these alterations are restored by liver transplantation (LT). Glucose tolerance (oral glucose tolerance test [OGTT]), peripheral insulin sensitivity (euglycemic insulin clamp technique), glucose oxidation (indirect calorimetry), nonoxidative glucose disposal, and insulin secretion (hyperglycemic clamp technique) were measured in 6 patients (Group 1) before and 6 months after LT, in 12 patients (Group 2) who underwent LT 6 to 30 months previously, and in 6 healthy individuals (controls). In Group 1, glucose tolerance and insulin sensitivity (3.24 +/- 0.37 mg/kg/min) were normalized after LT (8.6 +/- 0.77 mg/kg/min; P <.0001; P = not significant vs. controls). The improved insulin-mediated glucose uptake was the result of a normalization of nonoxidative glucose disposal. Fasting insulin and C-peptide decreased from 24.6 +/- 3.3 microU/mL and 4.37 +/- 0.46 ng/dL, respectively, to 12.7 +/- 1.9 microU/mL and 2.46 +/- 0.5 ng/dL (controls: 10.0 +/- 3 microU/mL and 1.45 +/- 0.34 ng/dL). The glucose-induced increase of insulin concentration, which was higher before LT, showed a significant reduction, although the first phase of beta-cell secretion remained significantly higher compared with that of controls. All these findings were also confirmed in Group 2. The present data indicate that LT normalizes glucose tolerance and insulin sensitivity in cirrhotic patients through an improvement of both hepatic glucose clearance and the peripheral glucose disposal. The latter effect may be the result of the correction of chronic hyperinsulinemia. An increased first-phase beta-cell insulin secretion in response to high glucose levels persists, suggesting that a memory of previous insulin resistance is maintained.

Clotting activation after transjugular intrahepatic portosystemic stent shunt.

BACKGROUND AND AIM: Aim of the study was to investigate the behaviour of clotting system in peripheral circulation of cirrhotic patients undergoing transjugular intrahepatic portosystemic stent shunt (TIPS). METHODS: Clotting variables and endotoxemia were measured 48 h and 30 days after TIPS in patients randomised to receive heparin or not. RESULTS: Forty-eight hours after TIPS, a significant increase of prothrombin fragment F1+2 was observed; such increase was less evident in patients given heparin. Similar findings were observed for endotoxemia, which, however, was not affected by heparin treatment. Thirty days after TIPS procedure prothrombin fragment F1+2 and endotoxemia returned to baseline values independently of the treatment given. CONCLUSION: This study shows that TIPS is associated with an increase of clotting activation which might contribute to acute thrombosis observed after this procedure.

Cost analysis for the prevention of variceal rebleeding: a comparison between transjugular intrahepatic portosystemic shunt and endoscopic sclerotherapy in a selected group of Italian cirrhotic patients.

The aim of the present study was to compare the cumulative cost of the first 18-month period in a selected group of Italian cirrhotic patients treated with transjugular intrahepatic portosystemic shunt (TIPS) versus endoscopic sclerotherapy (ES) to prevent variceal rebleeding. Thirty-eight patients enrolled in a controlled trial were considered (18 TIPS and 20 sclerotherapy). The number of days spent in the hospital for the initial treatment and during the follow-up period were defined as the costs of hospitalization. ES sessions, TIPS procedures, angioplasty or addition of a second stent to maintain the shunt patency, were defined as the costs of therapeutic procedures. The two groups were comparable for age, sex, and Child-Pugh score. During the observation period 4 patients died in the TIPS group, and 2 died and 1 was transplanted in the sclerotherapy group. The rebleeding rate was significantly higher in the sclerotherapy group. Despite this, the number of days spent in the hospital was similar in the two groups. This was because of a higher number of hospital admissions for the treatment of hepatic encephalopathy and shunt insufficiency in the TIPS group. The therapeutic procedures were more expensive for TIPS. Consequently, the cumulative cost was higher for patients treated with TIPS than for those treated with sclerotherapy. The extra cost was because of the initial higher cost of the procedure and the difference was still maintained at the end of the 18-month follow-up. When the cumulative costs were expressed per month free of rebleeding, the disadvantage of TIPS disappeared. In conclusion, a program of prevention of variceal rebleeding with TIPS, despite the longer interval free of rebleeding, is not a cost-saving strategy in comparison with sclerotherapy.

The human gallbladder increases cholesterol solubility in bile by differential lipid absorption: a study using a new in vitro model of isolated intra-arterially perfused gallbladder.

In this study, we first developed and validated a new in vitro isolated, intra-arterially perfused, gallbladder model and then applied the method to investigate the absorption of biliary lipids by the gallbladder wall and the effect of this process on the composition of human bile. Oxygenated and glucose-added buffer was perfused through the cystic artery to maintain organ viability. A standard pooled natural bile, radiolabeled with H3-cholesterol and C14-palmitoyl-linoleoyl-phosphatidylcholine, was instilled in the lumen via a cystic duct catheter. Changes in bile volume and lipid concentrations were monitored at time intervals to evaluate the disappearance of lipids from bile caused by gallbladder absorptive function. Organ viability was demonstrated by stable lactate dehydrogenase (LDH) organ release and oxygen consumption throughout the experiments. In the pig, disappearance rates of lipids from bile were similar in vitro and in vivo, demonstrating the validity of the isolated in vitro model for functional studies. By applying our in vitro isolated preparation to the human gallbladder, we found that 23% of cholesterol and 32% of phosphatidylcholine, but only 9% of bile salts, disappeared from bile in 5 hours. As a consequence, at the end of the experiments, cholesterol (P < .05) and phospholipid (P < .05) molar percentages were significantly reduced, while the bile salt (P < .05) molar percentage was significantly increased with respect to values at the beginning of the studies. Our findings are of pathophysiological relevance and support the concept that the human gallbladder modifies the relative composition of biliary lipids in such a way as to increase cholesterol solubility in bile.

Human gallbladder mucosal function: effects on intraluminal fluid and lipid composition in health and disease.

Gallbladder mucosal absorption of fluid during fasting is a well-known process. Indirect in vivo and recent in vitro evidence for physiologically relevant gallbladder absorption of cholesterol and phospholipids from bile has been observed in humans. The present study explored and compared by indirect means the relative efficiences of human gallbladder mucosal absorption of fluid and lipids in health and disease. Biliary lipids and pigment content were measured in fasting gallbladder bile samples obtained from gallstone-free controls and from four study groups: multiple and solitary cholesterol gallstone patients, and morbidly obese subjects with and without gallstones. Bile salts and pigment content were significantly greater in gallstone-free controls than in all other disease study groups. This was interpreted as evidence of more effective gallbladder mucosal fluid absorption in nonobese gallstone-free controls compared to that in all other groups. Correlation plot analyses of biliary lipids showed lower concentrations of phospholipids than expected from the index bile salt concentrations. The same was found for cholesterol concentrations but only in supersaturated samples. These findings were much more pronounced in gallstone free-controls and were accordingly interpreted as evidence of more efficient gallbladder absorption of both phospholipids and cholesterol in controls compared with that found in each of the disease study groups. Moreover, impaired gallbladder mucosal function, while invariably associated with cholesterol gallstone disease, was not found to be a necessary consequence of the physical presence of stones. It is concluded that efficient gallbladder mucosal absorption of both fluid and apolar lipids from bile is a normal physiological process that is often seriously impaired in the presence of either cholesterol gallstone disease or at least one of its precursor forms.

Transjugular intrahepatic portosystemic shunt versus endoscopic sclerotherapy for the prevention of variceal bleeding in cirrhosis: a randomized multicenter trial. Gruppo Italiano Studio TIPS (G.I.S.T.).

Transjugular intrahepatic portosystemic shunt (TIPS), a new technique for the treatment of portal hypertension, has been successful in preliminary studies to treat acute variceal hemorrhage and to prevent variceal rebleeding. The purpose of this multicenter, randomized controlled trial is to compare the efficacy of TIPS with that of endoscopic sclerotherapy in the prevention of variceal rebleeding in cirrhosis. Eighty-one cirrhotic patients, with endoscopically proven variceal bleeding, were randomized to either TIPS (38 patients) or endoscopic sclerotherapy (43 patients). Randomization was stratified according to the following: if bleeding occurred < 1 week (stratum I); if bleeding occurred 1 to 6 weeks (stratum II); and if bleeding occurred 6 weeks to 6 months (stratum III) before enrollment. Follow-up included clinical, biochemical, Doppler Ultrasound, and endoscopic examinations every 6 months. During a mean follow-up of 17.7 months, 51% of the patients treated with sclerotherapy and 24% of those treated with TIPS rebled (P = .011). Mortality was 19% in sclerotherapy patients and 24% in TIPS patients (P = .50). Hepatic encephalopathy (HE) developed in 26% and 55%, respectively (P = .006). A separate analysis of the three strata showed that TIPS was significantly more effective than sclerotherapy (P = .026) in preventing rebleeding only in stratum I patients. TIPS is significantly better than sclerotherapy in preventing rebleeding only when it is performed shortly after a variceal bleed; however, TIPS does not improve survival and is associated with a significantly higher incidence of HE. The overall performance of TIPS does not seem to justify the adoption of this technique as a first-choice treatment to prevent rebleeding from esophageal varices in cirrhotic patients.

Effects of isosorbide-5-mononitrate compared with propranolol on first bleeding and long-term survival in cirrhosis.

BACKGROUND & AIMS: Isosorbide-5-mononitrate (Is-5-Mn) exerts beneficial hemodynamic effects in portal hypertension, yet the long-term clinical value is uncertain. The aim of this study was to determine the long-term effects of Is-5-Mn vs. propranolol (Pro) on first bleeding, complications, and death in cirrhosis. METHODS: One hundred eighteen patients included in a previously published randomized trial comparing Is-5-Mn (20 mg three times daily) with Pro were followed up for up to 7 years (range, 2-91 months). Fifty-seven patients received Is-5-Mn and 61 received Pro. RESULTS: Thirty episodes of first upper bleeding occurred; 16 were in the Is-5-Mn group. Actuarial probability of bleeding did not differ between the two groups. Endoscopic variceal red signs were the only independent predictors of early bleeding. Of the 52 patients who died, 28 were in the Is-5-Mn group. The likelihood of death was greater among patients assigned to Is-5-Mn than to Pro, but only in patients older than 50 years (72% vs. 48% at 6 years; P = 0.006). Child-Pugh score, bleeding, age, and assignment to Is-5-Mn were independent predictors of death. The likelihood of death without bleeding was also higher (P = 0.05) in the Is-5-Mn group. CONCLUSIONS: Is-5-Mn is as effective as Pro in preventing early bleeding but is associated with higher long-term mortality.

Factors associated with gallstone disease in the MICOL experience. Multicenter Italian Study on Epidemiology of Cholelithiasis.

The epidemiological associations of gallstone disease were evaluated in a general population sample of 29,584 individuals (15,910 men and 13,674 women; age range, 30-39 years) belonging to 14 cohorts examined between December 1984 and April 1987. Subjects were screened for the presence of gallstones by gallbladder ultrasonography, completed a questionnaire, and underwent a physical examination and blood chemistry tests. Participants were considered to have gallstone disease if they had already had cholecystectomy or gallstones. Statistical associations were established by univariate analysis of the age-standardized data and by stepwise multiple logistic regression. Increasing age and body mass index and a maternal family history of gallstone disease were the most consistent associations (both at univariate and multivariate analysis and in both sexes) found in this study. Personal history of dieting was associated with gallstone disease in men, and at univariate analysis, in women. Decreasing serum total cholesterol levels and increasing serum triglycerides were associated with gallstone disease in both sexes in the multivariate analysis. In women, associations were also found with a number of pregnancies and paternal family history of gallstone disease. A slight but negative association with contraceptive pill use was identified only at multivariate analysis. Associations (investigated at univariate analysis) were also found with diabetes, cirrhosis, angina or myocardial infarction, and peptic ulcer. There was no association with smoking habits and use of aspirin or antirheumatic drugs.

Regulation of endocytic-transcytotic pathways and bile secretion by phosphatidylinositol 3-kinase in rats.

BACKGROUND & AIMS: Phosphatidylinositol 3-kinases (PI3-K) are a family of enzymes that play key roles in control of cell growth, membrane recycling, and vesicular endoexocytotic processes. The aim of this study was to investigate the effect of a specific PI3-K inhibitor, wortmannin, on bile secretion, cytoskeleton organization, and endotranscytotic pathways in rats. METHODS: Isolated perfused rat liver (IPRL) and isolated rat hepatocyte couplets (IRHCs) were used. RESULTS: Wortmannin induced a 25% inhibition of basal bile flow in IPRL (P < 0.01). Horseradish peroxidase biliary excretion in the IPRL was markedly decreased by wortmanin. In IRHC incubated with 25 nmol/L wortmannin for 10 minutes at 37 degrees C, morphological studies showed early significant dilatation of bite canalicular lumen (P < 0.001). At short intervals (3 minutes), uptake of the fluid-phase marker, Lucifer yellow, was markedly decreased by exposure to wortmannin (P < 0.001). At longer times (20 minutes), Lucifer yellow was retained in basolateral area of IRHC as compared with control cells, where the marker was rapidly transported to the pericanalicular area. In IRHC, wortmannin induced a marked disorganization of microfilaments. CONCLUSIONS: Wortmannin inhibits basal bile flow, endocytosis, and transcytotic transport of fluid-phase markers in the liver, and causes an early dilatation of the canalicular lumen and disorganization of microfilaments. These findings suggest that PI3-K is involved in the regulation of vesicle trafficking, cytoskeleton organization, and the process of bile formation.

Iron overload in patients with chronic viral hepatitis: how common is it?

OBJECTIVES: To establish the prevalence of increased hepatic iron content in patients with hepatitis C virus-related chronic hepatitis and to assess the accuracy of serum iron and ferritin in detecting tissue iron overload. METHODS: Serum iron, serum ferritin, and hepatic iron content were determined in 81 consecutive patients undergoing liver biopsy for chronic ALT elevation and hepatitis C virus infection. Moreover, in a subgroup of 28 patients, outcome of a 6-month course of interferon (IFN) treatment (6 million U of recombinant IFN, three times weekly) was determined after a mean follow-up of 24 +/- 6 months and the outcome was compared with the pretreatment values of hepatic iron content. RESULTS: Elevated serum iron or ferritin levels were detected in approximately 40% of patients, but elevated hepatic iron content was observed in only eight patients (10%). One of these patients had a hepatic iron index > 1.9, indicating hemochromatosis. Liver iron content and serum iron levels were not correlated. No differences in hepatic iron content were observed among patients with a sustained response to IFN (seven patients), short-term responders (seven patients), or nonresponders (14 patients). CONCLUSIONS: Ten percent of patients with chronic hepatitis C have elevated hepatic iron content. These patients cannot be identified using serum markers of iron status. The relationship between liver iron and response to IFN treatment requires further prospective investigations.

Impaired nonoxidative glucose metabolism in patients with liver cirrhosis: effects of two insulin doses.

Glucose intolerance is encountered in the majority of cirrhotic patients. This alteration has been attributed to a defective insulin-mediated glucose uptake in peripheral tissue, where nonoxidative glucose disposal seems to be chiefly impaired. To further investigate insulin action under euglycemic conditions, we studied how physiological (100 microU/mL) and pharmacological (1,000 microU/mL) plasma insulin concentrations affect whole-body insulin-mediated glucose uptake, as well as oxidative and nonoxidative glucose disposal, in cirrhotic patients and controls. To this aim, a sequential two-step insulin euglycemic clamp combined with indirect calorimetry was performed in eight cirrhotic patients and six control subjects. During the first step of the clamp, total glucose uptake was reduced by 40% in cirrhotic patients versus controls (4.42 +/- 1.39 v 7.63 +/- 1.60 mg/kg/min, P = .002). By increasing insulin to pharmacological levels, glucose disposal increased in both groups. However, the maximum rate of glucose metabolism achieved in cirrhotic patients was lower than in controls at all times (10.29 +/- 2.04 v 12.82 +/- 0.51 mg/kg/min, P = .012). Glucose oxidation was lower in cirrhotics in the basal state, but similar in both groups during insulin/glucose infusion. On the other hand, the reduced nonoxidative glucose disposal observed in cirrhotic patients was not normalized even by increasing insulin to pharmacological levels. In conclusion, in liver cirrhosis a reduced insulin sensitivity is associated with a reduced insulin responsiveness that is mainly caused by defective nonoxidative glucose disposal.

Hormonal regulation of bicarbonate secretion in the biliary epithelium.

Bicarbonate excretion in bile is a major function of the biliary epithelium. It is driven by the apically located Cl-/HCO3- exchanger which is functionally coupled with a cAMP-dependent Cl- channel (CFTR). A number of hormones and/or neuropeptides with different mechanisms and at different intracellular levels regulate, in concert, the processes underlying bicarbonate excretion in the biliary epithelium. Secretin induces a bicarbonate rich choleresis by stimulating the activity of the Cl-/HCO3- exchanger by cAMP and protein kinase A mediated phosphorylation of CFTR regulatory domain. Protein phosphatase 1/2A are involved in the run-down of secretory stimulus after secretin removal. Acetylcholine potentiates secretin-choleresis by inducing a Ca(++)-calcineurin mediated "sensitization" of adenyl cyclase to secretin. Bombesin and vasoactive intestinal peptide also enhance the Cl-/HCO3- exchanger activity, but the intracellular signal transduction pathway has not yet been defined. Somatostatin and gastrin inhibit basal and/or secretin-stimulated bicarbonate excretion by down-regulating the secretin receptor and decreasing cAMP intracellular levels induced by secretin.

Gallstones and diabetes: a case-control study in a free-living population sample.

Data on the association between cholelithiasis and diabetes often are controversial and are mostly based on autopsies or on hospital series. Therefore, we designed a case-control study to determine the prevalence of diabetes mellitus in a group of subjects with gallstones or having undergone cholecystectomy (cases) and compared these with a control group of subjects without gallstones, selected during an epidemiological study performed on a free-living population sample. The subjects were matched for sex, age, and body mass index. We enlisted 336 cases and 336 controls, aged 30 to 69 years. All subjects with fasting glycemic levels of < 140 mg/dL and without a documented history of diabetes were submitted to a simplified oral glucose tolerance test (OGTT). All subjects who underwent OGTT were classified according to the National Diabetes Data Group (NDDG) criteria. The prevalence of diabetes in the subjects affected by gallstone disease was significantly higher than that in controls (11.6% vs. 4.8%; odds ratio [OR], 2.55; 95% confidence interval [CI], 1.39-4.67). Diabetes was more frequent in subjects with gallstone disease than in the control group, even according to sex (18.3% vs. 9.9% for men: OR, 2.03; 95% CI, 0.99-4.2; 9.3% vs. 2.6% for women: OR, 3.85; 95% CI, 1.4-10.6). We conclude that an altered glucose metabolism may increase the risk of developing cholelithiasis in certain subjects.

Variations in pigment and carbohydrate content of gallbladder bile affect accurate quantitation of total protein when using the fluorescamine method.

BACKGROUND: Despite solute dilution and reduced total lipid concentrations, an unexplained increase in protein concentration has been reported to occur in the gallbladder bile of cholesterol gallstone patients. METHODS: Solutes in gallbladder bile from gallstone-free controls and from four study groups were measured using standard methods. Total proteins were measured using amino acid analysis and a conventional fluorescamine method. RESULTS: Bile salts and pigment content were greater in gallstone-free controls than in all other study groups, including morbidly obese gallstone-free subjects. Total biliary protein concentration, as determined by amino acid analysis in the gallstone-free control group was higher than in non-obese gallstone patients with multiple stones and in morbidly obese gallstone-free subjects. Total biliary proteins as measured with fluorescamine, however, did not show intergroup differences. A major problem of the conventional fluorescamine assay is shown to be an artefact arising from the high pigment content of the more concentrated samples. CONCLUSIONS: Very dilute gallbladder bile samples are often found in the presence of gallstone disease. This also occurs in morbidly obese subjects, even in the absence of gallstones. Although the contribution of protein secretion/absorption by the gallbladder can also be relevant, especially in the presence of morbid obesity, the protein concentration in gallbladder bile, when accurately measured, generally parallels the concentrations of non-absorbed biliary solutes, reflecting the efficiency of fluid absorption. Measurement of biliary proteins by the conventional fluorescamine method is unreliable in clinical studies in which intergroup differences in pigment content are commonly present.

Chronic liver disease in the Alexandria governorate, Egypt: contribution of schistosomiasis and hepatitis virus infections.

BACKGROUND/AIMS: In Egypt chronic liver disease is customarily attributed to Schistosoma mansoni infection. Anti-HCV antibodies are highly prevalent among Egyptian blood donors, yet little is known about the risk factors, pathogenicity and virological features of HCV and its association with schistosomiasis. We studied 135 adult patients with chronic liver disease living in the Alexandria governorate, mostly in rural areas of the Nile Delta. METHODS: Evaluation included abdominal ultrasonography; detection of anti-HCV antibodies and markers of HBV and HDV infection; HCV-RNA assay by 5' untranslated region nested polymerase-chain-reaction and HCV genotyping by a line probe assay; serologic (anti-soluble egg antigen, anti-SEA) and parasitological examinations for Schistosoma mansoni infection; and liver biopsy, if not contraindicated. RESULTS: Ninety-one (67%) patients had anti-HCV and 107 (85%) anti-SEA, 32 (30%) of whom excreted schistosomal eggs in stools. In addition, 21 (16%) patients had HBsAg, 86 (64%) anti-HBc and four (3%) anti-delta. Thus, many patients had evidence of multiple infections, double in 66% (anti-HCV and anti-SEA), triple in 33% (anti-HCV HBsAg and anti-SEA). Based on our diagnostic criteria, 25 (19%) patients had schistosomal portal fibrosis (anti-HCV positive in eight), 24 (18%) chronic hepatitis (anti-HCV positive in 19), 76 (56%) cirrhosis (anti-HCV positive in 58) and 10 hepatic tumors (anti-HCV positive in six). At multivariate analysis, the presence of anti-HCV was independently associated with previous parenteral anti-schistosomal therapy, a history of hematemesis and seropositivity for anti-HBc. Fifty (55%) of 91 anti-HCV positive sera had HCV-RNA, in 41 cases classified as genotype 4a. Detection of HCV-RNA was associated with a more severe liver disease and occurred less frequently in patients with a history of schistosomiasis. CONCLUSIONS: HCV infection with genotype 4a is the main cause of severe chronic liver disease in Egypt, where it is highly associated with schistosomiasis.