Increased biliary cholesterol secretion in alloxan diabetic mice

Plasma and liver cholesterol levels and biliary cholesterol, phospholipid and bile acid concentrations were examined in normal and alloxan diabetic mice fed ordinary and 0.5 per cent cholesterol diets. The plasma and liver cholesterol levels markedly increased in the diabetic mice, and the cholesterol diet further increased the liver cholesterol level but not that in the plasma. The gallbladder bile weight increased in the diabetic mice, but not after the cholesterol diet. The biliary lipid concentrations markedly increased in the diabetic mice, and the increases of the cholesterol and phospholipids exceeded that of the bile acids, resulting in increases of the cholesterol molar concentration ratio (mole percent) and the lithogenic index. The cholesterol diet increased the biliary cholesterol concentration and slightly the phospholipid, but not the bile acids. Therefore, the cholesterol mole percent and the lithogenic index increased. Among the biliary bile acid composition, cholic and deoxycholic acids increased and β-muricholic acid decreased in the diabetic mice, whereas the cholesterol diet feeding decreased cholic acid and increased chenodeoxycholic and α-muricholic acids. These data suggest that the mechanism of the increase of biliary cholesterol secretion in diabetic mice is different from that after cholesterol diet.     

The prevention of experimental cholesterol gallstones by ileectomy in mice

After a lithogenic diet containing 0.5 per cent cholesterol and 0.25 per cent sodium cholate was fed to a group of normal Crj-ICR male mice for 10 days, cholesterol gallstones developed. No formation of gallstones occurred, however, in a group of mice from which 20 cms of terminal ileum had been removed prior to the feeding of the lithogenic diet. The biliary concentrations of cholesterol, phospholipids and bile acids were markedly lower in the ileectomized mice, with the decrease in cholesterol concentration being most significant. On the other hand, fecal excretion of sterols and bile acids increased in the ileectomized mice. The pool size of bile acids increased after the feeding of the lithogenic diet, but ileectomy decreased the pool size in mice fed the ordinary or lithogenic diets. The biliary concentration of cholic acid increased after the feeding of the lithogenic diet, but decreased with ileectomy. The biliary concentration and fecal excretion of deoxycholic acid markedly increased, while those of beta-muricholic acid and its secondary bile acids, omega-muricholic acid and hyodeoxycholic acid, decreased. The increase in plasma and liver cholesterol levels after the feeding of the lithogenic diet was prevented by ileectomy. These data suggest that ileectomy prevents the formation of cholesterol gallstones after the feeding of a lithogenic diet due to a decrease in cholic acid absorption.     

The prevention of experimental cholesterol gallstones by ileectomy in mice

After a lithogenic diet containing 0.5 per cent cholesterol and 0.25 per cent sodium cholate was fed to a group of normal Crj-ICR male mice for 10 days, cholesterol gallstones developed. No formation of gallstones occurred, however, in a group of mice from which 20 cms of terminal ileum had been removed prior to the feeding of the lithogenic diet. The biliary concentrations of cholesterol, phospholipids and bile acids were markedly lower in the ileectomized mice, with the decrease in cholesterol concentration being most significant. On the other hand, fecal excretion of sterols and bile acids increased in the ileectomized mice. The pool size of bile acids increased after the feeding of the lithogenic diet, but ileectomy decreased the pool size in mice fed the ordinary or lithogenic diets. The biliary concentration of cholic acid increased after the feeding of the lithogenic diet, but decreased with ileectomy. The biliary concentration and fecal excretion of deoxycholic acid markedly increased, while those of β-muricholic acid and its secondary bile acids, ω-muricholic acid and hyodeoxycholic acid, decreased. The increase in plasma and liver cholesterol levels after the feeding of the lithogenic diet was prevented by ileectomy. These data suggest that ileectomy prevents the formation of cholesterol gallstones after the feeding of a lithogenic diet due to a decrease in cholic acid absorption.     

Altered bile acid metabolism in nonobese, spontaneously diabetic (NOD) mice

Cholesterol and bile acid metabolism was examined in nonobese, spontaneously diabetic (NOD) female mice before and after the development of diabetes. After the development of glucosuria, the plasma and liver cholesterol levels, gallbladder bile weight after 5-h fasting, biliary cholesterol, phospholipid and bile acid concentrations, the lithogenic index, the pool size of bile acids, and fecal sterol excretion markedly increased, but fecal bile acid excretion and fractional turnover rates for the cholic acid and chenodeoxycholic acid groups decreased. The distribution percentage of bile acids in the small intestine did not change, but it increased in the gallbladder and decreased in the large intestine. One striking finding was a change in the bile acid composition: increases were recorded in cholic and deoxycholic acids while decreases occurred in bile acids derived from chenodeoxycholic acid, such as beta-muricholic and ursodeoxycholic acids in the bile and alpha-muricholic, beta-muricholic, omega-muricholic, and hyodeoxycholic acids in the feces. Therefore, the cholic acid group/chenodeoxycholic acid group (CA/CDCA) ratio increased in the bile, feces, and small and large intestines after the development of diabetes. These changes were very similar to those observed in alloxan-treated mice, suggesting that the changes found in NOD mice are caused by insulin deficiency.     

Biliary cholesterol and lithogeneity of bile in patients after ileal resection

For determination of the factors that regulate biliary cholesterol secretion and the lithogenity of bile in ileal dysfunction, plasma and biliary lipids and fecal excretion of bile acids were studied in 29 patients who had undergone ileal resection. Seven patients with ileal resection had normal bile acid excretion (less than 10 mg/kg/day), and 22 had various degrees of bile acid malabsorption. None of the patients had gallstones when examined with abdominal sonography. LDL cholesterol levels were decreased in bile acid malabsorption and demonstrated a positive correlation with the molar percentage of biliary cholesterol. Biliary cholesterol (mol percent) was inversely correlated with fecal bile acid excretion. This finding suggests that biliary cholesterol secretion decreases with increasing loss of bile acids to feces in ileal dysfunction, leading to an actual decrease in the lithogenic index and to hyposaturation of cholesterol in bile. The reduction in biliary cholesterol, regarded as protecting the gallbladder mucosa against the detergent properties of bile acids, may play an important role in the pathogenesis of increased gallstone formation in ileal dysfunction.     

Biliary lipid output in the early stage of acute liver failure induced by 90% hepatectomy in the rat

BACKGROUND: Differences in biliary lipid output were compared in rats after 70% or 90% hepatectomy (Hx) to evaluate a possible index of the early stage of acute liver failure. METHODS: Male Sprague-Dawley (SD) rats weighing 300 to 350 g were randomly divided into two groups for 70% Hx or 90% Hx, and animals were sacrificed at 0, 6, 24, and 48 h after Hx. Before sacrifice, a polyethylene tube was cannulated into the bile duct and bile was collected for 1 h. Outputs of total bile acids, phospholipid, and total cholesterol in serum and bile were determined. Biliary total cholesterol, bile acid concentrations, and bile acid component levels were determined using gas liquid chromatography. Hepatic microsomal cholesterol 7alpha-hydroxylase and sterol 12alpha-hydroxylase activities were also determined using high performance liquid chromatography. RESULTS: The 3-day survival rate after 90% Hx was 50%. In the 90% Hx group, the serum total bile acid concentration at each point was significantly higher than it was in the 70% Hx group. The bile flow rate and biliary outputs of cholesterol, phospholipid, and bile acids were significantly lower at 6 h after 90% Hx than after 70% Hx. Among bile acid species, cholic and chenodeoxycholic acid outputs into bile were significantly less at 6 h after 90% Hx. The activities of cholesterol 7alpha-hydroxylase and sterol 12alpha-hydroxylase were decreased after 90% Hx. CONCLUSIONS: Our results suggest that determinations of the bile flow rate and biliary lipid outputs are supposed to be useful for early detection of hepatic failure after extensive hepatectomy.     

PDZK1敲除对小鼠肝脏胆固醇代谢和胆囊结石形成影响的实验研究

目的探讨敲除PDZK1(Postsynaptic density-95,disks-large,ZO-1-domain K1,PDZK1)基因对小鼠肝脏胆固醇代谢调节和胆囊结石形成的影响。方法雄性成年PDZK1基因敲除小鼠(PDZK1 knockout,KO组)和野生型小鼠(wild type,WT组),每组各10只,以成石饲料分别喂养4周,观察胆囊成石情况,并收集肝脏和胆囊组织。采用蛋白印迹法测定肝脏PDZK1和清道夫受体B族1型(scavenger receptor B type 1,SRB1)表达。采用胆总管插管收集肝胆汁,测定胆汁分泌率和胆汁胆固醇含量。采用试剂盒酶法测定胆囊胆汁成分并计算胆汁胆固醇饱和指数(cholesterol saturation index,CSI)。以实时定量PCR检测肝脏脂质代谢相关基因的mRNA表达。结果成石饲料喂养4周后,WT组小鼠全部成石(10/10),KO组小鼠则为40%(4/10)成石。两组小鼠肝胆汁分泌率差异无统计学意义,但KO组小鼠肝胆汁胆固醇含量显著降低(P0.05),胆汁酸含量增加(P0.05),且CSI降低(P0.05)。KO组小鼠肝脏SRB1蛋白表达降低(P0.05),甾醇氧-酰基转移酶基因1/2mRNA表达降低(P0.05),而肝型脂肪酸结合蛋白1和胆汁酸转运相关蛋白(ATP结合盒b11)表达则显著增加(P0.05)。结论 PDZK1影响SRB1在小鼠肝脏中表达,降低对高密度脂蛋白胆固醇摄取,减少胆汁胆固醇分泌,继而降低胆囊结石形成。     

Cholelithiasis in mice: effects of different chemicals upon formation and prevention of gallstones

Prevention of gallstones induced in mice by 1% cholesterol and 0.5% cholic acid (lithogenic diet) for 8 weeks was obtained by simultaneously feeding sulfaguanidine (1.5%) and dodecyl sodium sulfate (0.5–1.0%) along with the lithogenic diet. Citrus pectin (3%), egg lecithin (1%), n-octyl alcohol (1%), neomycin sulfate (0.2–0.5%) and l-ascorbic acid (5%) added to the lithogenic diet did not prevent gallstone formation. The condition of the liver, fatty or normal, in the experiment could not be correlated with the stone formation. Lower serum and liver cholesterol levels and an elevation of lecithin concentrations in serum was noticed in mice fed the sulfaguanidine and dodecyl sodium sulfate diet.     

Altered bile composition during cholesterol gallstone formation: cause or effect?

Gallbladder stasis, increased gallbladder absorption, and elevated biliary levels of calcium, hydrogen ion, and bilirubin have been implicated as factors potentially critical to cholesterol crystal precipitation. Previous studies, however, have analyzed bile only when crystals or gallstones have already formed. Therefore, we tested the hypothesis that changes in bile composition are a late effect, occurring only after crystal formation. Adult male prairie dogs were fed a standard nonlithogenic control diet (n = 7) or a lithogenic 1.2% cholesterol diet for 5, 9, or 14 days to cause cholesterol saturation (n = 7), cholesterol monohydrate crystals (n = 7), or gallstones (n = 7). Gallbladder bile was examined microscopically for crystals, and analyzed for ionized calcium, bilirubin, pH, total protein, and biliary lipids. The ratio of gallbladder to hepatic bile radiolabeled cholic acid specific activity (Rsa) was calculated as an index of gallbladder stasis. Cholesterol saturation index was calculated. The results demonstrate that increased gallbladder bile cholesterol saturation and total protein concentration precede cholesterol monohydrate crystal precipitation. However, changes in gallbladder ionized calcium, unconjugated bilirubin, pH, stasis, and absorption were noted only after crystals and gallstones had already formed. These data indicate that alterations in gallbladder bile calcium, bilirubin, pH, stasis, and absorption are not early changes, but occur simultaneously with or after crystal formation. Increased biliary protein, however, which was elevated prior to nucleation, may be an important mediator of cholesterol precipitation in cholesterol-saturated bile.     

Total parenteral nutrition solution increases bile lithogenicity in rat

The effect of parenteral and enteral nutrition on bile composition was studied in rats. Bile flow, bile fractional composition, bile component output, and lithogenic index were determined in five groups of rats: (A) preoperative control, (B) freely feeding rats infused with normal saline, (C) parenterally fed rats with balanced total parenteral nutrition (TPN) formula (4% amino acids, 22.5% dextrose, 1% Intralipid), (D) constantly intragastrically fed rats with the same TPN formula, and (E) rats fed intragastrically with the same TPN formula but only during 10 hr at night. After 12 days of infusion, bile and blood were collected. There were no differences between the two parenterally and enterally continuously fed groups. Both Groups C and D showed decreased bile flow, decreased bile components output, increased molar percentage concentrations of biliary cholesterol (2.2 ± 0.3 and 1.8 ± 0.1 in Groups C and D, respectively, control, 1.4 ± 0.1), and increased lithogenic index. The cyclically enterally fed group had a normal lithogenic index in the postabsorptive period. Plasma cholesterol and triglycerides were higher in the formula fed groups. There were no changes in plasma liver function tests between the groups. We conclude, therefore, that continuous administration of TPN solution induces an increase in lithogenicity of the bile, independent of the route of administration.     

Hyodeoxycholic acid: a new approach to gallstone prevention

Hyodeoxycholic acid and its isomer, 6 beta-hyodeoxycholic acid, when added to a lithogenic diet prevented the formation of cholesterol gallstones and crystals in prairie dogs. This beneficial effect occurred in the presence of bile supersaturated with cholesterol. Hyodeoxycholic acid abolished the feedback inhibition of hepatic hydroxymethylglutaryl coenzyme A reductase activity, the rate-limiting enzyme of cholesterol synthesis, and prevented elevations in serum and liver cholesterol observed in animals fed a 0.4 percent cholesterol diet. The gallbladder bile of the animals fed hyodeoxycholic acid and 6 beta-hyodeoxycholic acid contained abundant liquid crystals. This suggests that these bile acids prevented the transition of cholesterol from its liquid crystalline phase to solid crystals and stones.     

An animal model of pigment cholelithiasis

Pigment stones of high calcium content were induced in male hamsters of the Harlan Sprague-Dawley strain fed a nutritionally adequate semipurified diet for a period of 14 weeks. The diet contained moderate amounts of cholesterol (0.30 percent) and ethinyl estradiol (15 micrograms/day per animal). At sacrifice, the incidence of pigment stones was 50 percent. When stones were present, they were in the form of numerous black amorphous rods about 0.1 to 0.4 mm in length. Infrared analysis of the dried stones indicated the following composition: calcium phosphate 26.7 percent, calcium bilirubinate 12.8 percent, cholesterol 15.1 percent, and protein 45.4 percent. Pigment stones were associated with an elevated biliary total calcium level (probably induced by the dietary cholesterol) and a paradoxic decrease in the biliary total bilirubin level. The lithogenic diet produced marked elevations in liver and plasma cholesterol levels and cholesterol saturation of bile, but no cholesterol crystals or stones were observed. The accumulation of elevated levels of cholesterol in the livers of the experimental animals produced mild to moderate hepatotoxicity. The precise mechanism of the dietary induction of pigment stones in this hamster model remains to be elucidated.     

Bile composition and bile acid pool size. Comparison after truncal, selective, and highly selective vagotomy

We studied biliary lipid composition and bile acid pool size in 29 patients surgically treated for duodenal ulcer. Fourteen were examined both before and after surgery, the rest postsurgically only. They were divided into three groups according to type of vagotomy. With duodenal fluid obtained via nasogastric tube, we determined bile acid pool size, bile concentrations, and lithogenic index. We found no significant differences in bile composition and bile acid pool size among the three types of vagotomy, postsurgically. However, patients studied before surgery, compared with the entire post-vagotomy group, had a significant increase in relative cholesterol content and lithogenic index, most pronounced in the truncal vagotomy group. Bile acid pool size was also increased postsurgically. Vagotomy may predispose to gallstone development by increasing the bile's relative cholesterol concentration and thus the lithogenic index. However, the slightly expanded bile acid pool size may improve cholesterol solubility in certain patients.     

胆酸对肝硬化大鼠肝大部分切除术后肝脏功能的影响

目的探讨胆酸对肝硬化大鼠肝切除术后肝脏功能的影响。方法24只肝硬化大鼠肝切除术后随机分为胆酸组和对照组喂养1周,每组12只。对照组给以标准饲料喂养,胆酸组给以含0.2%胆酸的饲料喂养。1周后检测大鼠胆汁分泌速度及胆汁总胆汁酸（TBA）含量,检测肝功能、有丝分裂指数（MI）、增殖细胞核抗原（PCNA）标记指数、细胞核DNA含量。结果胆酸组胆汁分泌速度、胆汁TBA含量、血清ALB含量、MI、PCNA标记指数、细胞核DNA含量均高于对照组（P〈0.01）,血清谷草转氨酶（AST）、谷丙转氨酶（ALT）含量均低于对照组（P〈0.01）。结论胆酸可以增加肝硬化大鼠肝切除术后胆汁分泌速度及胆汁TBA含量,促进大鼠肝功能恢复和肝再生。     

Stasis before gallstone formation: Altered gallbladder compliance or cystic duct resistance?

Gallbladder stasis occurs before gallstone formation and provides the link between the hepatic secretion of supersaturated bile and cholesterol cholelithiasis. We recently observed that cystic duct resistance increases while sphincter of Oddi resistance is unchanged in the presence of lithogenic bile without gallstones. Whether alterations in gallbladder function also lead to gallbladder stasis has been unclear. Therefore, we tested the hypothesis that before gallstone formation, stasis results from increased cystic duct resistance and altered gallbladder compliance. Adult, male prairie dogs were fed either a trace cholesterol (control) or a 0.4 percent cholesterol-enriched diet. Cystic duct resistance increased but gallbladder compliance was unchanged before gallstone formation. A significant correlation (p < 0.001) was found between the lithogenic index and cystic duct resistance in pregallstone animals. We conclude that increased resistance to flow across the cystic duct, and not altered gallbladder compliance, is etiologically related to bile stasis, an important event in gallstone formation.     

小鼠胆囊胆固醇结石模型的建立

目的建立简单、可靠、高效的小鼠胆囊胆固醇结石模型,为研究胆石成因及防治提供重要手段。方法C57BL／6小鼠随机分为对照组和模型组,对照组喂饲基础饲料,模型组喂饲致石饲料（基础饲料加10％猪油、1％胆固醇及0．5％胆酸）。两组小鼠分别于喂养4周和8周后,计算小鼠存活率,同时各取一半数量小鼠在乙醚吸入麻醉下手术取胆囊及血液标本,分别检测成石率、血脂浓度及胆汁胆固醇饱和度。结果对照组小鼠8周存活率100％,模型组小鼠死亡1只,存活率95％。对照组8周成石率为零,模型组4周成石率80％,8周成石率100％。血脂分析表明,与对照组比较,模型组4周和8周总胆固醇及低密度脂蛋白显著升高（P〈0．01）,甘油三酯浓度轻度升高（P〈0．05）,高密度脂蛋白显著降低（P〈0．01）。4周和8周时胆汁胆固醇饱和度测定,对照组分别为0．48±0．29和0．58±0．21,模型组分别为1．36±0．36和1．52±0．37,模型组胆固醇浓度处于过饱和状态。结论本模型方法简单、成石率高、动物死亡率低,可作为研究胆石成因及防治的备选模型。     

Ezetimibe ameliorates cholecystosteatosis

BACKGROUND: Cholecystosteatosis is the accumulation of gallbladder wall fats leading to decreased gallbladder emptying. Ezetimibe inhibits intestinal fat absorption and prevents murine gallstone formation. However, the influence of ezetimibe on gallbladder emptying and cholecystosteatosis has not been studied. Therefore, we tested the hypothesis that ezetimibe would improve gallbladder motility by preventing the buildup of fats in the gallbladder wall. METHODS: Forty lean female mice were fed either a control diet or a lithogenic diet for 6 weeks. Half of the mice on each diet received ezetimibe. At 11 weeks of age, all mice were fasted overnight and underwent gallbladder ultrasonography to determine ejection fraction. One week later, the mice were fasted and underwent cholecystectomy. Bile was examined for cholesterol crystals. The gallbladders were snap-frozen for lipid analysis. RESULTS: The lithogenic diet significantly (P < 0.05) increased serum cholesterol, biliary crystals, gallbladder wall cholesterol and cholesterol/phospholipid ratio, and decreased gallbladder ejection fraction. All of these abnormalities were reversed (P < 0.05) by the addition of ezetimibe to the diet. CONCLUSIONS: These data suggest that ezetimibe lowers serum cholesterol, prevents biliary crystals, and normalizes gallbladder wall fat and function. We conclude that ezetimibe ameliorates cholecystosteatosis and may be an effective agent for gallstone prevention.     

The effects of ethinylestradiol, a glucose diet and streptozotocin induced diabetes mellitus on gallstone formation and biliary lipid composition in the hamster

Possible risk factors for gallstone formation were examined and the concentrations of biliary lipids and each bile acid in the hepatic and gallbladder bile of hamsters were quantified. Forty female golden Syrian hamsters were divided into 4 groups according to diet; Group I, given control chow, Group II, given an ethinylestradiol and cholesterol supplemented diet, Group III, given a glucose rich diet without induced diabetes mellitus, and Group IV, given a glucose rich diet with diabetes mellitus induced by streptozotocin injection. The formation of cholesterol crystals but not gallstones was induced in Group II associated with a significantly decreased total bile acid concentration in the gallbladder bile but not in the hepatic bile. The formation of cholesterol gallstones and crystals with significantly higher concentrations of cholesterol and phospholipid was observed in Group III, while neither the formation of gallstones nor lithogenicity was enhanced by diabetes mellitus. However, a quite different lithogenicity was evident between the hepatic and gallbladder bile of the Group IV animals. These results suggest that neither the consumption of oral contraceptives nor diabetes mellitus induces gallstone formation, but that these factors can be responsible for dysfunction of the gallbladder.     

The effects of ethinylestradiol,a glucose diet and streptozotocin induced diabetes mellitus on gallstone formation and biliary lipid composition in the hamster

Possible risk factors for gallstone formation were examined and the concentrations of biliary lipids and each bile acid in the hepatic and gallbladder bile of hamsters were quantified. Forty female golden Syrian hamsters were divided into 4 groups according to diet; Group I, given control chow, Group II, given an ethinylestradiol and cholesterol supplemented diet, Group III, given a glucose rich diet without induced diabetes mellitus, and Group IV, given a glucose rich diet with diabetes mellitus induced by streptozotocin injection. The formation of cholesterol crystals but not gallstones was induced in Group II associated with a significantly decreased total bile acid concentration in the gallbladder bile but not in the hepatic bile. The formation of cholesterol gallstones and crystals with significantly higher concentrations of cholesterol and phospholipid was observed in Group III, while neither the formation of gallstones nor lithogenicity was enhanced by diabetes mellitus. However, a quite different lithogenicity was evident between the hepatic and gallbladder bile of the Group IV animals. These results suggest that neither the consumption of oral contraceptives nor diabetes mellitus induces gallstone formation, but that these factors can be responsible for dysfunction of the gallbladder.     

Biliary secretion of bile acids, lipids, and bilirubin by the transplanted liver. A quantitative study in patients on cyclosporine.

To investigate biliary secretion in liver-transplanted patients on cyclosporine treatment we used duodenal perfusion with a nonabsorbable marker. After an overnight fast, 4 women and 5 men were studied for 6 hr at least 6 weeks after orthotopic liver transplantation. The data were compared with those obtained in 6 healthy controls. All transplanted patients received immunosuppressive therapy (corticosteroids, azathioprine, and cyclosporine). Biliary secretion rates of healthy controls were: bile acids 1.58 +/- 0.67 mmol/hr, phospholipids 0.27 +/- 0.11 mmol/hr, cholesterol 0.11 +/- 0.01 mmol/hr, and bilirubin 18 +/- 0.7 mumol/hr (mean +/- SEM). Liver-transplanted patients excreted 2.60 +/- 0.4 mmol/hr bile acids, 0.56 +/- 0.08 mmol/hr phospholipids, 0.18 +/- 0.04 mmol/hr cholesterol, and 22.0 +/- 4.5 mumol/hr bilirubin. Analysis of individual bile acids revealed that in the bile of liver transplant patients the percentage of cholic acid was elevated, whereas that of deoxycholic acid was reduced as compared with controls. These findings indicate that in the transplanted liver under immunosuppressive therapy with cyclosporine biliary secretion of bile acids, lipids, and bilirubin is not reduced.