Metabolism of obeticholic acid in brown bullhead (Ameiurus nebulosus).

Analysis of brown bullhead (Ameiurus nebulosus) bile by ultra performance liquid chromatography high-resolution mass spectrometry (UPLC/HRMS) revealed a series of bile acids similar to those found in humans. Accordingly, we chose this fish as a model organism to examine the metabolism of obeticholic acid, a bile acid used to treat a number of human liver diseases and the one that has the potential to occur as an environmental contaminant. The taurine and glycine conjugates of obeticholic acid and keto-obeticholic acid were identified, as well as the D-cysteinolic acid conjugate of obeticholic acid, likely a metabolite specific to fish. In addition, metabolites of obeticholic acid (sulphate and glucuronide) and several hydroxy-obeticholic acid derivatives were found, representing typical pathways of primary and secondary steroid metabolism. Brown bullhead exposed to obeticholic acid at a dose of 100 mg/kg gave no overt signs of distress or toxicity.

Gas chromatography-mass spectrometry-based simultaneous quantitative analytical method for urinary oxysterols and bile acids in rats.

A simultaneous quantitative assay method for urinary oxysterols and bile acids using GC-MS was developed to investigate the mechanism of liver toxicity induced by drugs or chemicals. Sample preparations were optimized by exploring various extraction solvents, derivatization reagents, and hydrolysis methods to achieve reliable and maximum sensitivity for these two different compound classes. As a result, satisfactory accuracy, precision, and sensitivity were obtained in the validation. The method was then applied to quantify urinary oxysterols and bile acids produced from liver toxicity induced by atorvastatin (250 mg/kg/day). From the results, increases in bile acid levels and decreases in the concentration ratio between cholic acid and chenodeoxycholic acid, which are the distinguishing phenomena observed in serum or bile for liver toxicity, were also observed in urine. Additionally, the mechanism of liver toxicity was investigated with the urinary concentration ratio of product to precursor in the metabolic pathway from cholesterol to bile acids. The results indicated that enzyme activities related to the production and degradation of bile acids, not oxysterols, were significantly changed from liver toxicity. Thus, it was concluded that urinary levels of oxysterols and bile acids could be useful tools for checking liver toxicity and investigating its mechanism.

Bile acid levels are increased in the liver of patients with steatohepatitis.

BACKGROUND/AIMS: The pathogenesis of steatohepatitis remains largely unknown; however, bile acids may play a role as potential mediators of liver damage. The aim of this study was to characterize bile acid profiles in liver tissue of patients with steatohepatitis. METHODS: Bile acid composition was determined by gas-liquid chromatography in liver tissue from patients with nonalcoholic steatohepatitis (NASH; n=15), patients with alcoholic steatohepatitis (ASH; n=14), and controls (n=8). Liver biopsies were graded for steatosis, inflammation, and fibrosis. RESULTS: Bile acids were moderately increased in liver tissue of steatohepatitis patients compared with controls (P<0.05). Deoxycholic, chenodeoxycholic, and cholic acids were elevated by 92, 64, and 43%, respectively, in patients with steatohepatitis (P<0.05). Cholic acid was the prevailing bile acid in NASH patients and in controls. More hydrophobic bile acid species were elevated in ASH patients compared with controls (P<0.05). Significant correlations were found in NASH patients between hepatic chenodeoxycholic acid and fibrosis, and between cholic acid and trihydroxy/dihydroxy bile acids and inflammation (P<0.05). In patients with ASH, cholic acid and trihydroxy/dihydroxy bile acids were correlated with steatosis (P<0.01). CONCLUSION: This study shows a distinct pattern of bile acids in the liver of patients with steatohepatitis. Further, the association between bile acids and histological liver injury suggests an association of specific bile acids and disease progression, possibly through bile acid-induced liver injury.

Apoptotic cell death does not parallel other indicators of liver damage in chronic hepatitis C patients.

The mechanisms of hepatocyte damage and the events that lead to high rates of chronic liver disease in hepatitis C virus (HCV) infection remain unclear. Recent in vitro studies have suggested that the HCV core protein may disrupt specific signalling pathways of apoptosis. This prompted us to study patients with chronic HCV infection to: determine the extent of apoptosis in the liver; evaluate whether clinical and biochemical data are correlated with histological findings; and to investigate if apoptosis is related to the histological activity of the disease. Twelve patients with chronic hepatitis C were included in the study. Liver histology was scored by using the histological activity index (HAI) of Knodell et al. DNA fragmentation was assessed in liver tissue by the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end-labelling (TUNEL) assay. Routine methods were used to determine serum markers of liver disease. Bile acids were measured in serum and liver by gas chromatography. Patients were placed, according to their HAI score, into group A (3.8 +/- 0.3) or group B (7.8 +/- 0.8) (P < 0.01). Liver enzymes tended to be higher in group B patients than in patients of group A. Levels of toxic bile acids in serum were greater in patients than in controls (P < 0.01). Chenodeoxycholic acid values were slightly higher in serum and liver of patients in group A. Liver biopsies with low HAI scores showed an increased rate of apoptosis (18.0 +/- 4.0 apoptotic cells per field) compared to those with higher HAI scores (6.6 +/- 2.1, P < 0.05) or to controls (3.5 +/- 0.4, P < 0.01). Hence, less severe liver disease, associated with lower histological grades and biochemistries, as well as increased levels of chenodeoxycholic acid, induces an expanded apoptotic response. The lower apoptotic rate in advanced liver disease may be associated with the high incidence of hepatocellular dysplasia/neoplasia.

Biliary bile acid composition in gastric cancer.

Bile reflux into the stomach has been considered carcinogenic. Secondary bile acids, and in particular deoxycholic acid, have been shown to act experimentally as co-carcinogens in the colon and are increased in patients with colorectal adenocarcinoma. No information is available with respect to biliary bile acid composition in patients with gastric cancer. We studied biliary bile acid composition in 11 patients with gastric cancer and 23 healthy controls. Bile acids were measured using high-performance liquid chromatography. The site of gastric cancer was the antrum in 6 patients and body in 5. There were 6 intestinal-type and 5 diffuse adenocarcinomas. Only 2 patients had Helicobacter pylori infection. Deoxycholic acid constituted 24% +/- 2% of biliary bile acid in gastric cancer patients versus 22% +/- 2% in healthy controls (NS). Similarly, no differences were found between the two groups for all other bile acids. Deoxycholic acid constituted 23% +/- 3% of biliary bile acid (NS vs. controls) in patients with antral adenocarcinoma and 25% +/- 2% (NS vs. controls) in patients with intestinal-type gastric adenocarcinoma. Gastric adenocarcinoma is not associated with an increase in the more-toxic secondary bile acids, and deoxycholic acid in particular. This reduces the importance of bile acid composition as a promotor in gastric carcinogenesis.

Determination of fetal bile acids in biological fluids from neonates by gas chromatography-negative ion chemical ionization mass spectrometry.

A method has been developed for microanalysis of fetal bile acids in biological fluids from neonates by capillary gas chromatography-mass spectrometry using negative-ion chemical ionization of pentafluorobenzyl ester-dimethylethylsilyl ether derivatives of bile acids. Calibration curves for the bile acid derivatives are useful over the range 0.1-100 pg and the detection limit for bile acids was 1 fg (S/N = 5) using isobutane as a reagent gas. Recoveries of the bile acids and their glycine and taurine conjugates from bile acid-free serum and dried blood discs ranged from 92 to 101% and from 93 to 108%, respectively, of the added amounts of their standard samples. The analysis of bile acids on a dried blood disc, meconium and urine from infants, exhibited significant hydroxylation at the 1 beta-, 2 beta-, 4 beta- and 6 alpha-positions of the usual bile acids, cholic and chenodeoxycholic acids, for the urinary or fecal excretion of bile acids in the fetal and neonatal periods. The present method was applied clinically to analyze bile acids on a dried blood disc from neonatal patients with congenital biliary atresia and hyper-bile-acidemia.

[Analysis of duodenal bile acids in patients with colorectal tumors and control subjects].

Bile acids are believed to play a role in the etiology of colorectal cancer. To investigate a possible relationship between bile acids and colorectal tumors, duodenal bile acids were analyzed in 18 patients with colorectal adenomas, 18 patients with colorectal carcinoma and 18 control subjects. Using high performance liquid chromatography and immobilized 3 alpha-hydroxysteroid dehydrogenase in column form, significant increases in the proportion of chenodeoxycholic acid and significant decreases in the proportion of deoxycholic acid and lithocholic acid were found in the bile of patients with colorectal adenoma or carcinoma compared with the control subjects. The data support the concept that bile acids have a role to play in the development of large bowel tumors.

Effect of colectomy with ileo-anal anastomosis on the biliary lipids.

Total colectomy with ileo-anal anastomosis is an effective treatment for ulcerative colitis and familial adenomatous polyposis. The absence of the colon and the coexistence of bile acid malabsorption may increase bile lithogenicity, but data on biliary lipid composition in patients with this operation is lacking. Our aim was to assess bile lithogenicity, bile composition and mass of biliary lipids within the gallbladder. We studied 11 patients with total colectomy and ileo-anal anastomosis and 16 healthy controls. We measured the percentage composition of conjugated bile acids and the masses within the gallbladder of the three main biliary lipids. This method, in contrast with measurement of cholesterol saturation index, can determine the cause of bile lithogenicity in terms of absolute modifications of the biliary lipids. There was no difference in the cholesterol saturation index between patients and controls. Colectomy patients had reduced masses of all three biliary lipids (medians and ranges, mmol): cholesterol 0.11 (0.03-0.24) vs. 0.36 (0.02-0.96), P < 0.02; bile acid 1.62 (0.75-5.21) vs. 3.95 (1.27-8.70), P < 0.01; phospholipids 0.35 (0.07-0.69) vs. 1.14 (0.14-3.00), P < 0.002. They also had reduced per cent deoxycholic acid: 3.8 (0.0-27.6) vs. 17.4 (6.4-44.7), P < 0.005, and increased percent cholic acid: 44.9 (23.3-71.4) vs. 34.3 (19.2-57.9), P < 0.05. We conclude that, despite having bile acid malabsorption, patients with colectomy and ileo-anal anastomosis have a normal cholesterol saturation index, caused by a concomitant reduction in the masses of all three biliary lipids. The reduced per cent biliary deoxycholic acid may help explain the reduced cholesterol and phospholipid masses in these patients. Total colectomy with ileo-anal anastomosis does not seem to predispose to the formation of cholesterol gallstones.

Breast-gut connection: origin of chenodeoxycholic acid in breast cyst fluid.

The notion that a breast-gut connection might modulate the microenvironment of breast tissue was supported by the finding that breast cyst fluid contains bile acids that are characteristically found in the intestines. To establish that the gut, rather than circulating steroid precursors, is the source of bile acids in breast cyst fluid, we gave two patients deuterium-labelled chenodeoxycholic acid (three 200 mg doses by mouth), starting 9 days before aspiration of breast cysts. The chenodeoxycholic acid concentration of seven samples of aspirated cyst fluid ranged from 42 to 94 mumol/L. The corresponding serum concentrations of chenodeoxycholic acid on the same day were 0.8 and 2.9 mumol/L, of which the labelled compound comprised 13.0% (0.38 mumol/L) and 28.2% (0.23 mumol/L). The deuterated chenodeoxycholic acid concentrations in cyst fluid were 0.79 and 1.26 mumol/L in two samples from patient 1 and 3.22 mumol/L in patient 2; these values are equivalent to 11-17% of the serum concentrations [corrected]. This study shows that intestinal bile acids rapidly gain access to cyst fluid. Further studies should investigate the mechanisms that govern the exchange processes and the maintenance of the high cyst fluid to plasma concentration gradients, and the biological half-lives of individual constituents.

Biliary bile acids in cholelithiasis and carcinoma of the gall bladder.

The role of biliary deoxycholate as an endogenous carcinogen and the possible association between cholelithiasis and the subsequent development of carcinoma of the gall bladder is unclear. This paper describes biliary bile acid analysis performed on three groups of patients, 10 with cholelithiasis, 10 with carcinoma of the gall bladder and 10 control patients. This is the first report of bile acid changes in carcinoma of the gall bladder. In these 30 patients the total bile acids concentration was highly variable (11.44-53.68 mg/ml). The mean ratio of primary to secondary bile acids was 3.5:1. This ratio was, however, significantly higher in cholelithiasis than in the control group (5.34:1; P < 0.001); patients with carcinoma of the gall bladder had significantly higher secondary bile acids (1:1; P < 0.001). This is due to a marked increase in the secondary bile acids and indicates that raised biliary deoxycholate concentrations are present in patients with carcinoma of the gall bladder and therefore may well be a factor in carcinogenesis.

Fecal bile acid excretion and composition in response to changes in dietary wheat bran, fat and calcium in the rat.

The effect and possible interactive influence of different dietary amounts of wheat bran, fat and calcium on the fecal excretion, concentration and composition of bile acids was studied in Fischer-344 rats. The fecal bile acids were analyzed using gas-liquid chromatography. Dietary wheat bran increased both total bile acid excretion and fecal weight without changes in fecal bile acid concentration. The proportion of fecal hyodeoxycholic acid decreased with increasing dietary fiber, whereas that of lithocholic and deoxycholic acids increased significantly with fiber intake. The percent content of fecal chenodeoxycholic acid did not change. Increasing dietary fat led to an increase in bile acid excretion without changes in either fecal weight or bile acid concentration. In contrast, the level of dietary calcium did not affect the total excretion of bile acids. However, since calcium increased the fecal weight, it consequently diluted bile acids and decreased their fecal concentration. Dietary fat and calcium had no influence on fecal bile acid composition. There were no interactive effects of wheat bran, fat and calcium on fecal bile acids. The finding in this study that dietary fiber, fat and calcium induce significant changes in fecal bile acids may be of relevance to the potential of bile acids to promote carcinogenesis.

Faecal unconjugated bile acids in patients with colorectal cancer or polyps.

The unconjugated faecal bile acid profiles of 14 patients with colorectal cancer, nine patients with polyps and 10 controls were compared using gas liquid chromatography, controlling for such confounding variables as cholecystectomy, gall stones and hepatic function. Patients with adenomatous polyps had a higher concentration of faecal bile acids (5.23 mumol/g, 2.16-13.67 (median, range) v 1.96, 0.91-6.97; p = 0.016) lithocholic acid (2.41, 0.88-3.22 v 1.07, 0.38-2.03; p = 0.013) and total secondary bile acids (5.23, 2.16-13.4 v 1.96, 0.73-6.63; p = 0.02) compared with control subjects. Patients with colorectal cancer had an increased (p = 0.029) proportion of secondary faecal bile acids (mol%) compared with controls (100, 96.5-100 v 95.19, 81.73-100) and the ratios of the primary bile acids, cholic and chenodeoxycholic acid, to their respective derivatives (secondary bile acids) were significantly lower in cancer patients compared with control and patients with polyps (p = 0.034 to 0.004). This study lends further support to the theory that bile acids may play a role in the development of polyps and colorectal cancer.

Cholesterol metabolism in human gallbladder mucosa: relationship to cholesterol gallstone disease and effects of chenodeoxycholic acid and ursodeoxycholic acid treatment.

The objective of this study was to investigate cholesterol metabolism in human gallbladder mucosa, especially in relation to hepatic cholesterol metabolism, gallstone disease and treatment with bile acids. Gallbladder mucosa and liver tissue samples were collected in 44 patients undergoing cholecystectomy; 30 had cholesterol gallstones and the rest were stone free. Ten of the gallstone patients were treated with chenodeoxycholic acid and eight received ursodeoxycholic acid, with a daily dose of 15 mg/kg body wt, for 3 wk before surgery. The 3-hydroxy-3-methylglutaryl coenzyme A reductase activity, governing cholesterol synthesis, was considerably lower in the gallbladder mucosa than in liver tissue (28 +/- 6 and 120 +/- 40 pmol/min/mg protein). The acyl coenzyme A:acyltransferase activity in the gallbladder mucosa catalyzing the esterification of cholesterol was, on the other hand, several times higher than corresponding activity in the liver (92 +/- 23 and 11 +/- 2 pmol/min/mg protein). In the presence of exogenous cholesterol, the acyl coenzyme A:acyltransferase activity increased about twofold in the gallbladder mucosa. The acyl coenzyme A:acyltransferase activity of the gallbladder mucosa from untreated gallstone patients was not stimulated further by the addition of exogenous cholesterol. Otherwise, there were no significant differences in acyl coenzyme A:acyltransferase and 3-hydroxy-3-methylglutaryl coenzyme A reductase activities in the gallbladder mucosa of gallstone patients compared with gallstone-free controls. Treatment with chenodeoxycholic and ursodeoxycholic acids did not affect the 3-hydroxy-3-methylglutaryl coenzyme A reductase activity of the gallbladder mucosa but reduced the acyl coenzyme A:acyltransferase activity by 60% to 65%.(ABSTRACT TRUNCATED AT 250 WORDS)

Cholesterol and bile acid synthesis: utilization of D2O for metabolic studies.

Human fibroblasts and hepatoma (Hep G2) cells were grown in media containing 25% D2O. Cholesterol extracted from the cells and bile acids obtained from the media were analyzed by gas chromatography/mass spectrometry (GC/MS). Fibroblasts that were transferred serially in media containing D2O continued to grow and to synthesize cholesterol enriched in deuterium. The observed distribution of deuterium-enriched species of cholesterol corresponded to a distribution that was calculated based on C = 27, 13C = 1.107%, D2O/H2O = 0.25, hydrogen derived from water = 20, and is in agreement with the concept that deuterium incorporation occurs randomly and represents mostly the NADPD/NADPH ratio in the medium. The deuterium enrichment of cholesterol from hepatoma cells indicated a shift of the most abundant species from m/z 373 to m/z 375, which corresponds more closely to the derivation of 25 hydrogens from water and implies the formation of deuterated acetate in the medium. Analysis of chenodeoxycholic acid, the predominant bile acid synthesized by Hep G2 cells in vitro, indicates its derivation from both pre-formed and newly synthesized cholesterol and that A ring transformation from cholesterol utilizes deuterium derived from water. Analysis of the bile acids derived from hamster bile following the administration of D2O confirms that similar events occur in vivo.

Distribution of tissue bile acids in the human alimentary tract and colon polyps.

To determine the characteristic distribution of tissue-bound bile acids in the human alimentary tract and colon polyps, we measured the concentration of bile acids in the mucosal tissues of the alimentary tract obtained at autopsy and polyps obtained by endoscopic polypectomy, using enzymatic fluorimetry and gas-liquid chromatography. The concentration of tissue-bound bile acid, especially chenodeoxycholic acid, was significantly higher in the ileum or ascending colon than in the other portions of the alimentary tract. The bile acid level of polyps was also higher in the ascending colon than in the other portions of the colon. These results suggest that the high concentration of tissue-bound bile acids is obtained at the site of absorption of bile acids in the alimentary tract.

Fecal and biliary bile acid composition after partial ileal bypass operation.

Biliary and fecal bile acid composition was studied in 13 patients 3-12 years after a partial ileal bypass operation and in 10 unoperated controls, all with heterozygous familial hypercholesterolemia. Three operated patients were taking cholestyramine. The relative amount of cholic acid in the bile was decreased at the expense of chenodeoxycholic acid in the operated subjects. Chenodeoxycholic acid content of the bile correlated negatively with the fractional cholesterol absorption, suggesting that in compromised absorption chenodeoxycholic acid is absorbed more efficiently than cholic acid. Despite a ninefold increase in total bile acid synthesis the cholic/chenodeoxycholic acid synthesis ratio was not significantly different in the operated and control subjects. However, the lower the chenodeoxycholic acid synthesis the higher was the proportion of deoxycholic acid in the bile and feces, suggesting regulation of chenodeoxycholic acid synthesis by deoxycholic acid. Ileal exclusion had increased the proportion of primary bile acids in the feces from below 10 to over 50%. Despite increased fecal water excretion the concentration of fecal total and dihydroxy bile acids was higher in the operated than in control subjects. However, the fecal concentration of the potentially cancer-promoting bile acids, deoxycholic acid and lithocholic acid, was not increased in the operated subjects. In the operated subjects, fecal water output was positively correlated with total bile acid and chenodeoxycholic acid synthesis. It is concluded that the severe bile acid malabsorption caused by ileal exclusion activates the synthesis of both primary bile acids in similar amount. However, after ileal exclusion the relative amount of cholic acid in the bile is decreased, obviously because loss of ileal absorption predominantly affects the absorption of cholic acid.

Relationship between duodenal bile acids and colorectal neoplasia.

To investigate a possible relationship between bile acids and colorectal neoplasia duodenal bile acids were analysed in 50 patients with colorectal adenomas and 14 with carcinoma. Using gas liquid and high performance liquid chromatography a small, but significant increase in the proportion of chenodeoxycholic acid was found in the bile of adenoma patients compared with controls (mean % +/- SD 31.0 +/- 10.8, 26.4 +/- 8.3, p = 0.01). The difference in the proportions of chenodeoxycholic acid correlated with increasing malignant potential of the adenomas as determined by increasing size, histological type, degree of dysplasia and number present. In carcinoma patients an increase in the proportion of chenodeoxycholic acid was also observed compared with controls (mean % +/- SD, 47.2 +/- 9.6, 28.0 +/- 4.5, p less than 0.01). The proportions of other bile acids in those with adenoma or carcinoma were normal.

Effects of partial ileocolectomy and Crohn's disease on biliary lipid secretion.

The effects of interruption of the enterohepatic circulation of bile acids on biliary lipid secretion have only been studied experimentally, and quantitative data in patients are lacking. Therefore, biliary lipid secretion during steady-state meal perfusion of the duodenum was studied in six patients with partial ileocolectomy, five patients with Crohn's disease, and five normal subjects. Bile acid outputs in the resection patients were significantly lower than in normal controls, (6.87 +/- 2.10 mmol/6 hr and 13.5 +/- 2.16, respectively; P less than 0.001) and were also decreased in two of the five Crohn's disease patients. Bile acid outputs in patients with resection progressively decreased in the course of the perfusion study; phospholipid and cholesterol secretion did not decrease to the same extent, and cholesterol saturation gradually increased. Bile of these patients, therefore, was frequently supersaturated due to uncoupling of bile acid secretion and outputs of the other biliary lipids. Bile acid outputs, although decreased, did not reach very low values, which shows that the enterohepatic circulation was not totally interrupted. Chenodeoxycholic acid was the main bile acid component of bile in patients with ileocolonic resection. Deoxycholic acid was absent from bile of four resected patients and two Crohn's patients. Two patients with active Crohn's disease had low bile acid outputs despite only moderate fecal bile acid losses. Therefore, decreased outputs may be caused by decreased bile acid pool not compensated for by increased bile acid synthesis in severely ill patients.

Malabsorption of liposoluble vitamins in a child with bile acid deficiency.

A male born to first cousins presented at 12 months with hypocalcemic convulsions, rickets, epistaxis due to vitamin K deficiency, and extremely low serum levels of beta-carotene and vitamin A. Liver function was altered moderately (glutamic-oxaloacetic transaminase, 55 U/L; glutamic-pyruvic transaminase, 37 U/L; lactate dehydrogenase, 255 U/L; alkaline phosphatase, 437 U/L). To correct the deficiencies, 8,000 IU vitamin D/day, 10,000 IU vitamin A/day, and intramuscular administration of vitamin K1 were required. At 9 years, he presented signs of neuromuscular affection, and the serum vitamin E level (measured for the first time) was extremely low. Classic lipid malabsorption syndromes (abetalipoproteinemia, chronic cholestasis, mucoviscidosis, coeliac disease, Whipple's disease) were excluded by appropriate examinations. Composition of duodenal bile acids was characterized by undetectable levels of cholic acid metabolites, and only chenodeoxycholic acid metabolites were present. Serum total bile acid concentration was normal, with an atypical low cholic acid/chenodeoxycholic acid ratio and abnormal presence of 3 beta-OH-delta 5-cholenic acid and 6-OH-bile acids. Urinary bile acid composition was also characterized by elevated 6-OH-bile acids. Known enzymopathies of the bile acid synthetic pathway were excluded (cerebrotendinous xanthomatosis, cerebro-hepato-renal syndrome of Zellweger, coprostanic acidemia). Bile acid pool sizes were determined by using stable isotopes: cholic acid pool size [2.90 (N, 32 +/- 16) microM/kg] and chenodeoxycholic acid pool size [10.8 (N, 32.6 +/- 9.9) microM/kg] were extremely low; fractional turnover rates of both bile acids were in a normal range.(ABSTRACT TRUNCATED AT 250 WORDS)