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Background & Aims: There is no histologic classification system to determine prognoses of patients with alcoholic hepatitis (AH). We identified histologic features associated with disease severity and created a histologic scoring system to predict short-term (90-day) mortality., Methods: We analyzed data from 121 patients admitted to the Liver Unit (Hospital Clinic, Barcelona, Spain) from January 2000 to January 2008 with features of AH and developed a histologic scoring system to determine the risk of death using logistic regression. The system was tested and updated in a test set of 96 patients from 5 academic centers in the United States and Europe, and a semiquantitative scoring system called the Alcoholic Hepatitis Histologic Score (AHHS) was developed. The system was validated in an independent set of 109 patients. Interobserver agreement was evaluated by weighted κ statistical analysis., Results: The degree of fibrosis, degree of neutrophil infiltration, type of bilirubinostasis, and presence of megamitochondria were independently associated with 90-day mortality. We used these 4 parameters to develop the AHHS to identify patients with a low (0-3 points), moderate (4-5 points), or high (6-9 points) risk of death within 90 days (3%, 19%, and 51%, respectively; P < .0001). The AHHS estimated 90-day mortality in the training and test sets with an area under the receiver operating characteristic value of 0.77 (95% confidence interval, 0.71-0.83). Interrater agreement values were 0.65 for fibrosis, 0.86 for bilirubinostasis, 0.60 for neutrophil infiltration, and 0.46 for megamitochondria. Interestingly, the type of bilirubinostasis predicted the development of bacterial infections., Conclusions: We identified histologic features associated with the severity of AH and developed a patient classification system that might be used in clinical decision making., (Copyright © 2014 AGA Institute. Published by Elsevier Inc. All rights reserved.)

The role of acid and duodenogastric reflux (DGR) in the development of esophageal mucosal injury has been extensively investigated using both animal and human models. In this report, clinical and experimental data are reviewed. The mechanisms by which gastric and duodenal contents produce esophageal mucosal injury are also discussed. Acid and pepsin are unquestionably important in causing mucosal damage at low pH values in both animal and human models. Animal models suggest synergistic damaging potential for conjugated bile acids and HCI as well as that of unconjugated bile acids and trypsin in more neutral pH values. Human evidence for the involvement of bile and its constituents has been controversial; however, the advent of better technology to detect DGR is beginning to clarify the role of these constituents. The contribution of each methodology in clarifying the extent of involvement of DGR in esophageal mucosal injury is reviewed. Despite some conflicting results, preliminary human studies support the results from the animal data suggesting synergistic damaging effects for both bile and acid in esophageal mucosal injury. The implication of these studies in treating gastroesophageal reflux disease are discussed.

Phenobarbital response, bile pigment composition, and the fractional biliary excretion ratio of bilirubin were studied in nine children with Crigler-Najjar disease. In five children, serum bilirubin levels decreased during phenobarbital treatment by 26% or more and the pigment composition in bile changed with a decrease in the proportion of unconjugated bilirubin from 33% +/- 12% to 13% +/- 1% and an increase in monoconjugates and diconjugates from 57% +/- 14% and 10% +/- 2%, respectively, to 72% +/- 4% and 16% +/- 3%. In four children, serum bilirubin levels did not change significantly during phenobarbital treatment. In these patients, bile pigments comprised 91% +/- 10% unconjugated bilirubin, 9% +/- 11% monoconjugates, and 1% +/- 1% diconjugates. On the basis of these differences, the former group can be classified as having type 2 Crigler-Najjar disease and the latter, type 1. Bile pigment analysis in parents of patients with Crigler-Najjar disease showed an increased proportion of monoconjugates in at least one of the partners in three of four couples tested, despite normal serum bilirubin levels. Serum bilirubin levels were about the same in type 1 and 2 patients and amounted to 236 +/- 62 mumol/L and 214 +/- 82 mumol/L, respectively. In addition the fractional bilirubin excretion ratio, calculated as the ratio ([bilirubin in bile]/[bilirubin in serum])/([bile acid in bile]/[bile acid in serum]) could not differentiate between these two groups. However, there was a 10-fold and 100-fold difference of this ratio between patients with Crigler-Najjar disease and those with Gilbert's syndrome and between patients with Crigler-Najjar disease and controls. The fractional bilirubin excretion ratio proved an excellent tool to differentiate between Gilbert's syndrome and Crigler-Najjar disease, whereas Crigler-Najjar disease types 1 and 2 could be differentiated on the basis of bile pigment analysis.

All stools passed on the first 3 days after extracorporeal shock-wave lithotripsy of gallbladder stones in 21 patients were collected and examined for the presence of stone fragments. A total of 555 fragments varying in number per patient (4-69) and in size (maximum diameters from 0.5-8.0 mm) were recovered by sieving aqueous suspensions of the feces. All 482 fragments less than or equal to 3.0 mm left the biliary tract without any clinical symptoms, as did the three largest fragments with maximum diameters of 7.0-8.0 mm and almost all of the 70 fragments measuring 3.5-5.0 mm. During the observation period, four episodes of biliary complaints were recorded in three patients in whom fragments with maximum diameters of 3.5-5.0 mm were found. The only chemical abnormality was a temporary elevation of lipase activity to twice the normal range in 1 case. All fragments were identified as gallbladder stones by infrared spectroscopy on the basis of their (varyingly high) cholesterol content. By macroscopic criteria, most of the fragments were from mixed stones; therefore, provided there is a functioning gallbladder and sufficiently fine fragmentation, successful extracorporeal shock-wave lithotripsy does not seem to be limited to pure cholesterol stones.

Methyl tert-butyl ether rapidly dissolves cholesterol gallstones, although insoluble debris may remain. Total gallstone dissolution could be achieved if safe solvents for these noncholesterol components can be developed. We evaluated the in vitro ability of ethylenediaminetetraacetic acid, citrate, dimethyl sulfoxide, and ionic or nonionic detergents to dissolve the predominantly calcium bilirubinate and calcium carbonate debris remaining after methyl tert-butyl ether gallstone dissolution. Ethylenediaminetetraacetic acid 1% or 2% at pH 9.5 was the most effective of the solvents studied for dissolving calcium and bile pigment. The addition of cholate (25-200 mM) or polysorbate (1%-10%) to ethylenediaminetetraacetic acid 1% at pH 9.5 enhanced pigment dissolution compared to ethylenediaminetetraacetic acid alone. Dissolution of pellets prepared from human gallstones and composed predominantly of either calcium bilirubinate or calcium carbonate was 80% and 85% at 4 h using ethylenediaminetetraacetic acid 1% plus polysorbate-20 1% at pH 9.5. We conclude that ethylenediaminetetraacetic acid, either alone or with a detergent, is an effective solvent for methyl tert-butyl ether-insoluble gallstone debris and deserves assessment in vivo.

During studies on the effect of bile salt-pool depletion in the bile-fistula rat (adult male Sprague-Dawley), the spontaneous formation of an orange-brown precipitate was noted. The nature of this phenomenon and its relationship to BS and calcium concentration was investigated in depth. Bile from 18 animals was collected in the dark into transparent tubes containing sodium azide, ascorbic acid, and glucaro-1,4-lactone. The tubes were flushed with nitrogen, sealed, and incubated at 37 degrees C. The pigmentary precipitate formed in all the bile salt-depleted (less than 3-5 mM) bile samples (i.e., those collected after 5-7 h of external biliary drainage), but not in bile salt-rich biles. It appeared within 30-240 min after collection, both in bile samples collected at room temperature and at 37 degrees C, initially as a pale flocculation and then slowly sedimenting to form, after centrifugation, a solid, dark-orange pellet. There were no pH changes during incubation, and bile cultures were negative. Under polarizing microscopy, the precipitate appeared amorphous, and there was no evidence of birefringence. High-performance liquid chromatography showed that unconjugated bilirubin was the prevalent pigmentary component, but significant amounts of monoconjugated bilirubin also coprecipitated. Lipid chemistry showed the presence of lecithin (80.1% of total lipids), which was rich in palmitoyl and linoleoyl fatty acids, and of fatty acids (predominantly palmitic and oleic). Infrared spectroscopy and x-ray diffraction showed the presence of calcium bilirubinate and palmitate. In-vivo replenishment of the bile salt pool by intravenous infusion of either taurocholate or taurochenodeoxycholate (1 mumol/min) completely prevented the pigmentary precipitation. In vitro experiments showed inhibition of the precipitate formation by the addition of individual bile salt in concentrations approximating their critical micellar concentration. Precipitate formation was hastened by the addition of calcium chloride (4-12 mM), but only in bile salt-depleted biles. As the composition of the precipitate closely resembles that of human brown-pigment stones and sludge, these findings may provide new insights into an understanding of the pathogenesis of pigment gallstone disease.

In vivo and in vitro ultrasonic characteristics of gallstones were compared with stone size and composition with the aim of understanding the basis for these features. In vivo and in vitro ultrasonic features of the same stone were identical. Acoustic shadowing was not due to stone type, radiodensity, or calcium content. However, all stones larger than 4 mm in diameter produced a distinct sonic shadow. Gallbladder sludge produced internal echoes without a sonic shadow. These echoes shifted slowly when the patient was reexamined in a different position. Four of seven cholesterol stones containing more than 88% cholesterol floated and produced a sonic shadow without internal echoes or with an area of internal echoes within the gallbladder at a distance from the posterior wall. This last feature may identify patients that are good candidates for a trial of gallstone dissolution with chenodeoxycholic acid.

During the National Cooperative Gallstone Study, chenodiol (chenodeoxycholate), 750 or 375 mg/day, resulted in complete gallstone dissolution in only 13.5% and 5.2% of patients, respectively. The purpose of this study was to analyze the composition and morphology of gallstones from patients who underwent cholecystectomy during the National Cooperative Gallstone Study to determine if calcium salts on the gallstone surface could have been responsible for failure of dissolution. Total gallstone calcium content was not different between the treated and placebo groups; however, surface calcium levels were different, being greater than 1.0% in 47.6% of stones from chenodiol-treated patients (n = 63) but in only 16.7% of those from placebo-treated patients (n = 18), p less than 0.02. Pigmented outer rims were found in 52.4% of the stones from the chenodiol-treated group compared with only 16.7% of stones from the placebo group, p less than 0.01. The rim calcium content of 36 stones with pigmented outer rims was 3.7% +/- 1.0%, whereas that of 45 stones with nonpigmented outer rims was only 1.0% +/- 0.3%, p less than 0.01. We conclude that the presence of rings of increased concentrations of calcium salts on the gallstone surface may impair dissolution by chenodiol.

We previously reported that nb/nb mice with hereditary hemolytic anemia spontaneously developed calcium bilirubinate pigment gallstones. To assess the extent to which gallstone formation and bile composition is gene dependent, we transferred the hemolytic process by transplanting bone marrow from nb/nb mice into a nonhemolytic, but histocompatible genotype, W/Wv mice. Hematologic parameters of transplanted W/Wv mice were nearly identical to those of nb/nb mice. Like nb/nb mice, the percentage of transplanted mice with gallstones increased with the duration of the hemolysis and occurred twice as often in female mice as in male mice (37% vs. 19%; p less than 0.05). However, the rate of gallstone formation in transplanted mice was one-third less than that in nb/nb mice (3.6% per month vs. 5.5%; p less than 0.05). Analysis of hepatic bile revealed that (a) marrow-transplanted mice had higher concentrations of unconjugated bilirubin due to hemolysis (p less than 0.05) and of total bile acids determined by the W/Wv genotype (p less than 0.001) than their respective nb/nb counterparts and (b) transplanted mice with stones had a significantly lower proportion of cholic acid (p less than 0.005) and higher proportion of keto-bile acids (p less than 0.005) than transplanted mice without stones, suggesting that the cholic acid concentration may retard stone formation. These data indicate that the hemolytic process is the primary determinant of pigment gallstone formation in these mice and is influenced by the following factors: (a) duration of the hemolytic process, (b) gender, and (c) the genotype that regulates the composition of biliary components like bile acids.

To study the microscopic and biochemical composition of biliary sludge, we subjected hepatic and gallbladder bile samples to microscopic examination, and analysis of lipids and mucus glycoprotein. These were compared with samples of hepatic and gallbladder bile obtained from normal persons and patients with gallstones. Biliary sludge was found to be sediment composed of cholesterol monohydrate crystals and bilirubin granules embedded in a matrix of mucus gel. There was a striking increase in the amount of mucus glycoprotein in the gallbladder bile of the sludge group compared with the gallstone and control group. The gallbladder biliary mucus content was also higher in the gallstone group compared with normal controls. These differences in mucus concentrations were not observed in hepatic bile samples from the three groups, suggesting that they had occurred within the gallbladder. In addition, gallbladder histology and epithelial mucin histochemical staining reaction were studied. Mucus hypersecretion and early glandular metaplasia in the epithelium were observed in the gallbladders harboring sludge, whereas those having stones had further changes in the mesenchymal tissues, with thickening and fibrosis. These results indicate that in patients with sludge the gallbladder is abnormal, showing mucus hypersecretion and glandular metaplasia. This leads to an increase in the mucus content of gallbladder bile, which in turn may result in nucleation of cholesterol crystals and may be regarded as the embryonic stage of gallstone disease.

Electron microprobe analysis, x-ray diffraction, and infrared spectroscopy have been used to investigate the nature and arrangement of materials present in gallstones. Calcium phosphate and carbonate deposits present centrally in many stones were mixed with a pigmented phase containing copper and protein. In several stones, the central calcium deposits were surrounded by discrete adjacent layers of calcium bilirubinate and cholesterol. The calcium bilirubinate layer was distinguished from other pigmented layers by its higher protein content and increased iron/copper ratio. This sequence of calcium salt and cholesterol deposition occurred in the outer layers of some stones. The phase relationships shown by the present study are consistent with a biphasic mechanism of stone growth in which calcium salts and lipid were alternately deposited. Calcium salt precipitation has been inferred to initiate the sequence as calcium salts were found at the stone centers. The layer sequence has been discussed in terms of some possible effects of damage to the gallbladder wall caused by the calcium salt deposits.

Pigment gallstones are defined as any dark brown-to-black stone, consisting of calcium salts of bilirubin, phosphate, carbonate and other anions, and can be separated into carbonate- and noncarbonate-containing groups. Pigment stones predominate in the rural Orient, in cirrhosis, and in elderly United States patients undergoing cholecystectomy. Clinical associations include bile duct obstruction, stasis, and possibly hemolysis. Of pigment stones, 50% are radioopaque and account for two-thirds of all opaque stones. The concentrations of bile salts, phospholipids,, cholesterol, and total bilirubin in bile are similar to normal levels, but the concentration of unconjugated bilirubin is increased in the bile of some patients. Increased unconjugated bilirubin in bile may be caused by increased hydrolysis of excreted conjugated bilirubin. Unconjugated bilirubin is solubilized by bile salts, but the interaction is primarily nonmicellar. Ionized calcium and pH are important determinants of solubility. Sulfated glycoproteins, excreted in increased amounts in patients with cholelithiasis, may be the site of pigment stone precipitation because these compounds bind calcium salts tightly. E coli is frequently cultured from pigment stones in Japan but not in the United States; thus, bacterial beta-glucuronidase may be important in stone formation in Japan but probably not in the West. Stasis leads to increased calcium secretion and to increases in the concentration of sparingly soluble compounds that may then precipitate. Incomplete emptying of the gallbladder may result in the same concentration process. Unsaturated fats and chronic vagal stimulation cause pigment stone formation in animals. At present, surgery is the only treatment for pigment lithiasis.

The influence of gastric emptying of nutrients on plasma cholecystokinin and pancreatobiliary functions is poorly understood. We therefore temporally related the emptying of fat, protein, and glucose of a mixed meal to release of the gut hormones cholecystokinin, pancreatic polypeptide, and peptide YY and outputs of trypsin, lipase, bilirubin, and bile salts. Five healthy volunteers with a multilumen duodenal tube ingested a mixed meal with phase-specific markers for the aqueous phase, liquid fat, solid fat, and solid protein phases. Duodenal passage was determined by intraduodenal infusion of a second set of phase-specific nonabsorbable markers. Plasma cholecystokinin levels and pancreatobiliary secretions rose to a maximum at 30-60 min and then gradually declined (p less than 0.01) despite continued entry of protein and fat into the duodenum throughout the whole 4-h experimental period. High levels of both pancreatic polypeptide and peptide YY were observed in the last 2 h of the experiment. Release of factors capable of inhibiting cholecystokinin release and subsequently pancreatobiliary secretion may be responsible for the observed time-course.

The chronic antral ulcer developed spontaneously in (WB X C57BL/6)F1-W/WV mice. Natural history of the ulcer was investigated to make clear the mechanisms involved in the development of ulcer in this mutant mouse. Although no histopathologic abnormalities of the stomach were detected at the fifth day after birth, erosions developed at the tenth day and the severity of the ulcerative lesions advanced with age. Antral pH of the W/WV mice was higher than that of the congenic +/+ mice, and the content of gastrin in the glandular stomach of the former was lower than that of the latter. Therefore, hyperacidity cannot be the cause of the ulcer in the W/WV mice. Bilirubin was demonstrable in the stomach contents of the suckling W/WV mice. After subcutaneous injection of [35S]sulfobromophthalein, the 35S radio-activity in the stomach contents of the 10-day-old W/WV mice was about 40 times as great as that observed in the +/+ littermates. As the peak of the bile reflux occurred shortly before the development of obvious ulcer that penetrated beyond the muscularis mucosae, the bile reflux could be a cause rather than a result of the ulcer.