Saturated Bile Research Articles

The gallbladder in diabetes

The gallbladder in diabetes

Gallbladder bile composition in patients with Crohn’s disease

To further elucidate the pathogenesis and mechanisms of the high risk of gallstone formation in Crohn's disease. Gallbladder bile was obtained from patients with Crohn's disease who were admitted for elective surgery (17 with ileal/ileocolonic disease and 7 with Crohn's colitis). Fourteen gallstone patients served as controls. Duodenal bile was obtained from ten healthy subjects before and after the treatment with ursodeoxycholic acid. Bile was analyzed for biliary lipids, bile acids, bilirubin, crystals, and crystal detection time (CDT). Cholesterol saturation index was calculated. The biliary concentration of bilirubin was about 50% higher in patients with Crohn's disease than in patients with cholesterol gallstones. Ten of the patients with Crohn's disease involving ileum and three of those with Crohn's colitis had cholesterol saturated bile. Four patients with ileal disease and one of those with colonic disease displayed cholesterol crystals in their bile. About 1/3 of the patients with Crohn's disease had a short CDT. Treatment of healthy subjects with ursodeoxycholic acid did not increase the concentration of bilirubin in duodenal bile. Several patients with Crohn's disease, with or without ileal resection/disease had gallbladder bile supersaturated with cholesterol and short CDT and contained cholesterol crystals. The biliary concentration of bilirubin was also increased in patients with Crohn's colitis probably not due to bile acid malabsorption. Several factors may be of importance for the high risk of developing gallstones of both cholesterol and pigment types in patients with Crohn's disease.

Leptin-resistant obese mice do not form biliary crystals on a high cholesterol diet 1,2

Human obesity is associated with leptin resistance and cholesterol gallstone formation. Previously, we demonstrated that leptin-resistant (Lep(db)) obese mice fed a low cholesterol diet have enlarged gallbladders, but a decreased cholesterol saturation index, despite elevated serum cholesterol. Obese humans, however, consume a high cholesterol diet. Therefore, we hypothesized that on a high cholesterol diet, leptin-resistant mice would have cholesterol saturated bile and would form biliary crystals. Eight-week old female lean control (n = 70) and leptin-resistant (n = 72) mice were fed a 1% cholesterol diet for 4 weeks. All animals then had cholecystectomies. Bile was collected, grouped into pools to determine cholesterol saturation index (CSI), and examined for cholesterol crystals. Serum cholesterol and leptin were also measured. Gallbladder volumes for Lep(db) mice were enlarged compared with the lean mice (35.8 microl versus 19.1 microl, P < 0.001), but the CSI for the Lep(db) mice was lower than for the lean animals (0.91 versus 1.15, P < 0.03). The obese animals did not form cholesterol crystals, whereas the lean animals averaged 2.2 crystals per high-powered field (hpf) (P < 0.001). Serum cholesterol and leptin were also elevated (P < 0.001) in the obese animals. These data suggest that Lep(db) obese mice fed a high cholesterol diet have increased gallbladder volume and decreased biliary cholesterol saturation and crystal formation despite elevated serum cholesterol compared with lean control mice. We conclude that the link among obesity, diet, and gallstone formation may not require hypersecretion of biliary cholesterol and may be related to the effects of diabetes, hyperlipidemia, or both on gallbladder motility.

Leptin-resistant obese mice do not form biliary crystals on a high cholesterol diet

Introduction: Human obesity is associated with leptin resistance, elevated serum lipids, hepatic steatosis, and cholesterol gallstone formation. We have previously demonstrated that leptin-resistant (Lepdb) obese mice fed a low cholesterol diet have enlarged gallbladders, but a decreased cholesterol saturation index, despite marked hepatic steatosis and elevated serum cholesterol. However, obese humans typically consume a high cholesterol diet and are prone to form gallstones. Therefore, we tested the hypothesis that on a high cholesterol diet, leptin-resistant obese mice would have large gallbladders, cholesterol saturated bile, and would readily form biliary crystals. Methods: Eight week old female lean control (C57BL/6J, n = 70) and leptin-resistant (Lepdb, n = 72) mice were fed a 1% cholesterol diet for four weeks. At 12 weeks, all animals were fasted overnight and underwent cholecystectomies. Gallbladder volume (μl) was measured and grouped into 7 lean control and 12 Lepdb pools for determination of cholesterol saturation index (CSI). Bile was examined for cholesterol crystals under polarized light microscopy (#/hpf). Serum cholesterol (mg/dl) and leptin levels (ng/ml) also were measured. Results were analyzed using the Student’s t-test. TABLE—ABSTRACT P57GB volCSICrystalsSerum XOLLeptinLean19.1 ± 0.91.15 ± .102.2 ± 0.5131 ± 76 ± 0Lepdb35.8 ± 2.2∗0.91 ± .10†0.0 ± 0.0∗278 ± 10∗229 ± 8∗∗P < 0.001.†P < 0.03 vs C57b lean controls.Conclusions: These data suggest that leptin-resistant obese mice fed a high cholesterol diet have 1) increased gallbladder volume, 2) decreased biliary cholesterol saturation and crystal formation despite 3) elevated serum cholesterol compared to lean control mice. Therefore, we conclude that the link among obesity, diet, and gallstone formation does not require hypersecretion of biliary cholesterol and may be related to gallbladder stasis.

Bile composition in inflammatory bowel disease: ileal disease and colectomy, but not colitis, induce lithogenic bile.

Inflammatory bowel disease is a risk factor for gall-bladder stones, but there is controversy about the composition of these stones and whether such patients develop lithogenic bile. In 54 gallstone-free inflammatory bowel disease patients and 13 non-inflammatory bowel disease patients with cholesterol-rich gallstones, we measured the biliary cholesterol saturation indices, nucleation times and bilirubin concentrations, and determined the bile acid composition and molecular species of phosphatidylcholine, in gall-bladder bile. Patients with Crohn's colitis or ulcerative colitis had less saturated bile (mean cholesterol saturation index, 0.9) and longer nucleation times (median, 21 days) than those with ileal Crohn's disease (1.5; 14 days) or those who had undergone colectomy (1.6; 5 days). In patients with ileal Crohn's disease, the mean biliary bilirubin concentration was two- to three-fold higher than that in the other groups, and was associated with a decrease in the percentage of biliary deoxycholate and an increase in the percentage of ursodeoxycholate, compared with disease controls, but phosphatidylcholine species were similar. Patients with small bowel Crohn's disease, or who have undergone colonic resection, have supersaturated bile and an increased risk of cholesterol gallstone formation. In patients with ileal disease, the presence of high biliary bilirubin concentrations and low percentage of deoxycholic acid may also favour the formation of mixed, pigment-rich, gallstones.

Nucleation of biliary cholesterol, arachidonate, prostaglandin E2, and glycoproteins in postmenopausal women

BACKGROUND & AIMS: Among dieting obese patients, cholesterol gallstone formation is preceded by increases in levels of biliary cholesterol saturation, arachidonate, prostaglandin E2, total glycoproteins, and rapid nucleation of cholesterol. The aim of this study was to determine if similar increases occur among postmenopausal women with cholesterol crystals in their bile. METHODS: In 101 postmenopausal women without gallstones, gallbladder bile was sampled via nasoduodenal tube and analyzed. RESULTS: Nineteen of the women had saturated bile and crystals. Levels of cholesterol saturation, arachidonate, prostaglandin E2, and total glycoprotein were highest among women with cholesterol- saturated bile and cholesterol crystals and lowest among women with unsaturated bile. Levels were intermediate among women with saturated bile but no crystals. CONCLUSIONS: Among postmenopausal women, increases in levels of biliary cholesterol saturation, arachidonate, prostaglandin E2, and total glycoproteins may be important pathophysiologically in the rapid nucleation of cholesterol crystals. (Gastroenterology 1997 Apr;112(4):1271-6)

Bile acids and bile alcohols in a child with hepatic 3 beta-hydroxy-delta 5-C27-steroid dehydrogenase deficiency: effects of chenodeoxycholic acid treatment

Duodenal bile, urine, plasma, and feces from a child with hepatic 3 beta-hydroxy-delta 5-C27-steroid dehydrogenase deficiency were analyzed by fast atom bombardment mass spectrometry and gas chromatography-mass spectrometry to investigate the formation and excretion of abnormal bile acids and bile alcohols. The biliary bile salts consisted of glycocholic acid (25%) and of sulfated and glycine conjugated di- and trihydroxycholenoic acids (55%), two C27 bile acids, and eleven sulfated bile alcohols (mainly tetrols, 20%), all having 3 beta,7 alpha-dihydroxy-delta 5 or 3 beta,7 alpha,12 alpha-trihydroxy-delta 5 ring structures. In plasma, sulfated cholenoic acids constituted 65% and unconjugated 3 beta,7 alpha-dihydroxy-5-cholestenoic acid 25% of the total level, 71 micrograms/ml. The urinary excretion of the former was 30.4 mg/day and that of unsaturated bile alcohol sulfates, mainly pentols, 7 mg/day. The predominant bile acid in feces was an unconjugated epimer of 3 beta,7 alpha,12 alpha-trihydroxy-5-cholenoic acid, and small amounts of cholic acid were present. The minimum total excretion was 11.3 mg/day. Treatment with chenodeoxycholic acid resulted in marked clinical improvement and normalized liver function tests. Further studies are needed to define the mechanism of action. Plasma bile acids decreased to 1.6 micrograms/ml and urinary excretion to 3.4 mg/day. Chenodeoxycholic and ursodeoxycholic acids became predominant in all samples. The fecal excretion of unsaturated cholenoic acid sulfates increased to 40 mg/day compared to 89 mg/day of saturated bile acids. The results provide further support for a defective hepatic 3 beta-hydroxy-delta 5-C27-steroid dehydrogenase deficiency, and indicate that the 3 beta-hydroxy-delta 5 bile acids are formed via 7 alpha-hydroxycholesterol. The formation of glycocholic acid may be due to an incomplete enzyme defect or to transformation of the 3 beta-hydroxy-delta 5 structure by bacterial and hepatic enzymes during an enterohepatic circulation.

Intrahepatic cholesterol stones: A rationale for dissolution therapy

A case of primary cholesterol hepatolithiasis is reported. Stone composition was documented by infrared spectroscopy, and the presence of cholesterol saturated bile was demonstrated using standard biochemical techniques. The patient was treated with operative stone extraction, choledochoscopy, biliary enteric anastomosis, and oral dissolution therapy. The administration of oral dissolution agents has altered the composition of the patient's bile and may prevent further stone formation. We advocate the use of both stone and biliary biochemical analysis for patients with primary hepatolithiasis to facilitate optimal therapy.

Hepatobiliary effects of obesity and weight-reducing surgery.

Obesity clearly increases the risk of gallstone formation. The role of nucleating factors may be most decisive in transforming merely saturated bile into a gallstone-forming solution. Weight loss in the short run may promote stone formation but in the long run may be beneficial if it is maintained. UDCA appears to be a promising prophylactic agent against gallstone formation during weight loss regardless of whether it is achieved by diet alone, by jejunoileal bypass, or by gastric restriction surgery.

Enhanced Gallbladder Absorption During Gallstone Formation: The Roles of Cholesterol Saturated Bile and Gallbladder Stasis

Cholesterol saturated bile and gallbladder stasis are important factors in the pathogenesis of cholesterol gallstones. The degree to which either or both of these factors affect gallbladder transport of fluid remains obscure. The authors tested the hypothesis that both cholesterol saturated bile and gallbladder stasis, and not stasis alone, promotes gallbladder fluid absorption. Prairie dogs were maintained for 2 weeks on either a control chow, total parenteral nutrition (TPN), or a 1.2% cholesterol enriched chow. The bile acid pool was labeled with 14C-cholic acid and indexes for cholesterol saturation (CSI) and gallbladder stasis (Rsa) were determined. Fluid transport was indirectly measured by calculating the ratio of gallbladder to hepatic bile concentrations of individual and total biliary lipids. Despite evidence of stasis in prairie dogs maintained on TPN, bile was unsaturated, and gallbladder absorption was not appreciably changed. In contrast, cholesterol-fed animals had cholesterol supersaturated bile, gallbladder stasis, and altered gallbladder absorption, as manifested by a significant change in the ratio of gallbladder to hepatic bile concentrations of individual and total biliary lipids. These data suggest that both cholesterol saturated bile and gallbladder stasis, and not stasis alone, are essential in promoting the enhanced gallbladder absorption, which has previously been observed during early cholesterol gallstone formation.

Biliary lipid composition in patients with nonoperated Crohn's disease.

Lipid composition of fasting duodenal bile was studied in 56 patients with nonoperated Crohn's disease, 21 normals matched for age and sex, 13 patients with cholesterol cholelithiasis, and 9 patients with ileal resections. Crohn's patients had a significantly higher mean saturation index, calculated according to Thomas (0.84 +/- 0.51) when compared to normal (0.63 +/- 0.25). Patients with ileocolonic Crohn's disease and patients with severe bile acid malabsorption, particularly, showed an increased incidence of cholesterol saturated bile. Saturation in patients with nonoperated Crohn's disease was not increased to the levels found in patients with ileal resection or cholesterol gallstones. Bile acid composition of gallbladder bile was characterized by a significant decrease of the deoxycholate fractions in patients with Crohn's ileocolitis and colitis as well as in ileal resected patients. These qualitative changes of bile acid composition may influence cholesterol solubility. It is concluded that patients with nonoperated Crohn's disease may have an increased risk of developing cholesterol gallstones.

Biliary Lipid Output and Bile Acid Kinetics in Cholesterol Gallstone Disease

Abstract Sweden has one of the highest incidences of gallstone disease in the Western world. It is therefore important to characterize the mechanisms responsible for the formation of cholesterol gallstones in this population. In the present study, we have determined the kinetics of the two primary bile acids, cholic acid and chenodeoxycholic acid, and the hepatic secretion rates of the biliary lipids in 21 normolipidemic, nonobese gallstone patients (13 with functioning and 8 with nonfunctioning gallbladder) and in 23 healthy controls. The cholesterol saturation of fasting gallbladder bile averaged 110% in the gallstone patients with functioning gallbladder and 82% in the controls. The pool sizes of cholic acid and chenodeoxycholic acid were reduced by about 40% in the two groups of gallstone patients, whereas the rates of synthesis were close to normal. The fractional catabolic rate of both bile acids was increased in both groups of gallstone patients. The gallstone patients with functioning gallbladder had an increased (about 50%) cholesterol secretion but normal bile acid and phospholipid secretion rates. In the gallstone patients with nonfunctioning gallbladder the secretion rates of biliary lipids were not significantly different from those of the controls. The ratio between cholesterol and bile acids was about 50% higher in the gallstone patients with functioning gallbladder than in the controls or in those with nonfunctioning gallbladder. The results indicate that the hepatic secretion of cholesterol is an important determinant for the development of saturated gallbladder bile in Swedish gallstone patients.

Impaired Gallbladder Emptying Before Gallstone Formation in the Prairie Dog

Several human and experimental observations suggest that gallbladder stasis is an important link between the hepatic secretion of cholesterol saturated bile and the formation of cholesterol gallstones. In the cholesterol-fed prairie dog model, gallbladder stasis occurs before gallstone formation. In this study we sought to determine the specific defects in extrahepatic biliary physiology responsible for gallbladder stasis in this model. Adult male prairie dogs were fed either a trace cholesterol or a 0.4% cholesterol-enriched diet. In acute terminal experiments, gallbladder contents were examined for cholesterol crystals and gallstones, and gallbladder function was determined at rest and in response to intravenous cholecystokinin-octapeptide. The following alterations in gallbladder function developed concurrently with biliary cholesterol crystallization, but before gallstone formation: (a) decreased gallbladder emptying, (b) increased intragallbladder pressure in response to cholecystokinin-octapeptide, (c) increased cystic duct closing pressure, and (d) increased resistance to outflow through the cystic duct.

3-hydroxy-3-methylglutaric acid (HMGA) reduces dietary cholesterol induction of saturated bile in hamster

3-Hydroxy-3-methylglutaric acid (HMGA) is a competitive inhibitor of 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCoAR) and strongly reduces cholesterol biosynthesis both in vitro and in vivo . Since the effects of this compound on biliary lipid composition are hitherto unknown, we have investigated whether it prevents dietary cholesterol induction of saturated bile in the hamster. Female Golden Syrian hamsters have been divided into four groups and treated for 10 weeks as follows: I) Standard diet, containing 0.8 mg cholesterol/g food; II) Standard diet plus HMGA (100 mg/kg b.w./day per os); III) Lithogenic diet containing 2.4 mg cholesterol/g food; IV) Lithogenic diet plus HMGA as above. The results indicate that HMGA is effective in reducing both bile cholesterol supersaturation and hypercholesterolemia. Inhibition of hepatic cholesterogenesis at the level of mevalonate synthesis by HMGCoAR and reduction of intestinal cholesterol absorption may be responsible for these effects.

Analysis and structure determination of unsaturated 5 beta - cholanoic acids.

Unsaturated cholanoic acids are known to arise as artifacts of chemical transformation processes and during storage and high-temperature gas liquid chromatography (GLC) of various derivatives of saturated bile acids. Nevertheless, there is evidence for their natural occurrence and isolation under conditions where artifactual formation of unsaturated bile acids would be unlikely. Since structural identification of such compounds is often complicated by a lack of knowledge of their analytical properties, a representative series of monounsaturated cholanoic acids with double bonds in rings A, B and C were prepared by POCl3 and ZnCL2 dehydration of saturated bile acids with selectively blocked hydroxyl functions. The cholenoic acids were indistinguishable from their saturated analogs by thin layer chromatography (TLC) on plain silica gel, but those compounds with sterically exposed double bonds were resolved by AgNO3-TLC, using chloroform/methanol solvent systems. The synthetic 5 beta-cholenoic acids obeyed the general rules of GLC mobility based on the overall shape of the molecule and the number and configuration of the functional groups. Constant retention factors attributable to the double bond were observed for all of the double bond types on several GLC phases, and theoretical retention times could be calculated for combinations of double bonds and functional groups not specifically represented among the synthetic standards. With gas chromatography-mass spectrometry (GC-MS), the unsaturated bile acids gave several characteristic fragments, which, in conjunction with the chromatographic properties of the parent compounds, permitted an unambiguous distinction among different unsaturated acids, and between unsaturated and saturated bile acids of the same number and configuration of functional groups. For complete structural identification of saturated and unsaturated bile acids, capillary GC-MS represents the ideal state of the art, but the less expensive combination of AgNO3-TLC and GLC also can yield much useful information concerning the structure of natural and synthetic 5 beta-cholenoic acids. This study emphasizes the need for special precautions in the isolation and derivatization of bile acids intended for studies of unsaturated components.

Circular dichroism studies of keto-bile acids

Circular dichroism curves for various saturated bile acid derivatives with carbonyl groups at carbon atoms 3,7 and 12 are presented and it is demonstrated that this method offers a convenient solution for the location of mono and diketo carbonyl groups in the bile acid molecule. From the shapes (n → II ∗ ∗ Dayal, B., Toome, V., Tint, G.S., Shefer, S. and Salen, G., Presented in part at the ACS/CSJ Chemical Congress meeting in Honolulu, Hawaii, April, 1979. )II transition) of these CD curves the precise localization of the carbonyl groups and the stereochemistry of the A/B ring juncture can be determined.

Lipid composition of bile in diabetics and obesity-matched controls.

Duodenal bile from 27 diabetes was compared with samples from healthy subjects matched for age, sex, and body mass index. Cholesterol saturation and the molar percentages of bile acids, phospholipids, and cholesterol were not significantly different. Most bile samples were supersaturated in both groups. The maturity onset diabetics who were almost all obese had more saturated bile than the slimmer juvenile onset patients. Body fatness and plasma triglyceride levels were both positively correlated with the cholesterol saturation of bile in the controls but not in the diabetics. Bile was less concentrated in female diabetics than in controls, which is consistent with impaired gallbladder emptying. It is possible that the increased prevalence of gallstones in diabetics is due not so much to diabetes itself as to the frequently associated obesity.

Resolution of saturated and unsaturated 5 beta-cholanoic acids by gas-liquid and thin-layer chromatography.

The thin-layer and gas-liquid chromatographic properties of the methyl ester acetates were determined for a series of 20 monounsaturated 5 beta-cholanoic acids representing the simple chemical and enzymic dehydration products of the common bile acids. The unsaturated acids were generally indistinguishable from their saturated analogues by thin-layer chromatography on plain silica gel, but resolution was achieved on silica gel impregnated with silver nitrate for compounds having sterically exposed double bonds. The gas-liquid chromatographic behaviour of the unsaturated bile acids on the OV-225, SE-30, and Poly-S-179 liquid phases was closely similar to that observed for the saturated bile acids. The 5 beta-cholenoic acids obeyed the general rules of chromatographic mobility based on the overall shape of the molecule and the number and configuration of functional groups, with a constant retention factor attributable to the olefinic bond. The structural information provided by the chromatographic behaviour of the standard unsaturated bile acids allows a distinction to be made among most of the isomeric 5 beta-cholenoates. A complete identification of all isomeric olefins is possible when chromatographic and mass spectrometric data are combined.

Development of Lithogenic Bile during Puberty in Pima Indians

To determine whether highly saturated bile is a congenital or acquired characteristic of Pima Indians and to elucidate the basis of the rapid postpubertal increase in gallstones in Pimas, we studied the bile of 66 Pimas nine to 21 years of age. Highly saturated bile is not prevalent among Pimas under the age of 13, but bile saturation increases significantly (P less than 0.05) in both sexes during pubertal growth and development. Bile saturation was 15 per cent higher in females than males. Bile acid pools increased with age in the young men, but not in women. Bile cholesterol saturation correlated with obesity (r = 0.41; P less than 0.001) and urinary estrogen excretion (r = 0.44; P less than 0.001). Highly saturated bile may be present for several years before the onset of cholesterol cholelithiasis.

Preparation and mass spectral behaviour of some 5β-cholenoic acids

Monounsaturated 5 beta-cholanoic acids with double bonds in rings A, B, and C were prepared by POCl3 and ZnCl2 dehydration from natural bile acids with selectively blocked hydroxyl functions. The yields ranged from 15 to 100%. The products were purified by thin-layer and AgNO3 thin-layer chromatography and the structures were confirmed by nuclear magnetic resonance spectroscopy and mass spectrometry. The methyl ester acetates of the unsaturated 5 beta-cholanoic acids possessed chromatographic properties closely similar to those of the corresponding saturated bile acids. Several characteristic fragments were seen in the mass spectra which, in conjunction with the chromatographic properties, permitted an unambiguous distinction between different monounsaturated acids, and between saturated and unsaturated bile acids of the same number and configuration of functional groups. The 20 5 beta-cholenoic acids examined represent all of the simple chemical and enzymatic dehydration products of natural bile acids and can be completely identified by their combined chromatographic and mass spectral properties.