Influence of fever on biliary elements of guinea pigs

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Impaired biliary cholesterol secretion and decreased gallstone formation in apolipoprotein E-deficient mice fed a high-cholesterol diet

BACKGROUND & AIMS: Because apolipoprotein E (apoE) is a key cholesterol transport molecule involved in the hepatic uptake of chylomicron cholesterol, it may play a critical role in controlling bile cholesterol elimination and cholesterol gallstone formation induced by dietary cholesterol. To test this hypothesis, we studied biliary lipid secretion and gallstone formation in apoE-deficient mice fed cholesterol-rich diets. METHODS: Bile lipid outputs and gallstone sequence events were analyzed in apoE-deficient mice fed a high-cholesterol diet or a lithogenic diet compared with control animals. RESULTS: A high-cholesterol diet increased biliary cholesterol secretion and gallbladder bile cholesterol concentration in wild-type mice; the increase in bile cholesterol secretion was significantly attenuated in apoE-deficient mice. ApoE knockout mice fed a high-cholesterol lithogenic diet had a markedly lower frequency of gallbladder bile cholesterol crystal and gallstone formation than wild-type mice, which was most likely a result of the decreased cholesterol saturation index found in gallbladder bile of apoE-deficient mice. CONCLUSIONS: These results show that apoE expression is an important factor for regulating both biliary secretion of diet-derived cholesterol as well as diet-induced cholesterol gallstone formation in mice.     

Biliary lipid secretion,bile acid metabolism,and gallstone formation are not impaired in hepatic lipase-deficient mice

Whereas hepatic lipase (HL) has been implicated in lipoprotein metabolism and atherosclerosis, its role in controlling biliary lipid physiology has not been reported. This work characterizes plasma lipoprotein cholesterol, hepatic cholesterol content, bile acid metabolism, biliary cholesterol secretion, and gallstone formation in HL-deficient mice and C57BL/6 controls fed standard chow, a cholesterol-supplemented diet, or a lithogenic diet. Compared with C57BL/6 controls, HL knockout mice exhibited increased basal plasma high-density lipoprotein (HDL) cholesterol as well as reduced cholesterol levels transported in large lipoproteins in response to cholesterol-enriched diets. Hepatic cholesterol content and biliary cholesterol secretion of chow-fed HL knockout and wild-type mice were not different and increased similarly in both strains after feeding dietary cholesterol or a lithogenic diet. There were no differences in biliary bile acid secretion, bile acid pool size and composition, or fecal bile acid excretion between HL-deficient and control mice. HL knockout mice had a similar prevalence of gallstone formation as compared with control mice when both strains were fed with a lithogenic diet. In conclusion, the deficiency of HL has no major impact on the availability of lipoprotein-derived hepatic cholesterol for biliary secretion; HL expression is not essential for diet-induced gallstone formation in mice.     

Comparison of effects of cholecystokinin and erythromycin on bile chemistry and gallstone formation in aged guinea pigs.

BACKGROUND: There has been considerable interest in gall bladder motility in recent years. We compared the effects of cholecystokinin (CCK) and erythromycin on bile chemistry and gallstone formation in aged guinea pigs. METHODS: Two groups of guinea pigs (1-mo and 3-y old; n=40 each) were studied. Each group was divided into four subgroups of 10 animals each; one subgroup received lithogenic diet, one each received CCK or erythromycin daily in addition to lithogenic diet for 4 weeks, and one received normal diet. After 4 weeks, the presence of gallstones or sludge was recorded and bile composition including concentrations of bile acid, cholesterol, lecithin and protein concentrations was studied. RESULTS: No gallstones were observed in the 1-mo-old animals. In the 3-year-old animals, 9 of 10 guinea pigs on lithogenic diet and 4 of 10 in each treatment subgroup and the normal diet subgroup developed gallstones. CCK and erythromycin had similar effects on bile chemistry and stone formation. CONCLUSIONS: Aging increases the formation of gallstones in guinea pigs. Erythromycin is as effective as CCK in reducing gallstone formation by improving gall bladder motility.     

Deficiency of Niemann‐Pick C1 protein protects against diet‐induced gallstone formation in mice

Background/aims: Receptor‐mediated endocytosis is a critical cellular mechanism for the uptake of lipoprotein cholesterol in the liver. Because Niemann‐Pick C1 (NPC1) protein is a key component for the intracellular distribution of cholesterol originating from lipoprotein endocytosis, it may play an important role in controlling biliary cholesterol secretion and gallstone formation induced by a lithogenic diet. Methods: We studied biliary cholesterol secretion, gallbladder lipid composition and gallstone formation in NPC1‐deficient mice fed a low‐fat lithogenic diet (1.5% cholesterol and 0.5% cholic acid) compared with control animals under the same diet. Results: The lipid secretion response to the lithogenic diet was impaired in NPC1 (?/?) mice, leading to a decreased cholesterol output and an increased hepatic cholesterol concentration compared with the lithogenic diet‐fed wild‐type mice. A decreased cholesterol saturation index was found in the gallbladder bile of NPC1 (+/?) and (?/?) mice after lithogenic diet feeding. Consequently, mice with a partial or a total deficiency of NPC1 had a drastically lower frequency of gallbladder cholesterol crystals and a reduced prevalence of gallstones. Conclusion: Hepatic NPC1 expression is an important factor for regulating the biliary secretion of diet‐derived cholesterol as well as for diet‐induced cholesterol gallstone formation in mice.     

Endogenous Hypercholecystokininemia,But Not Aspirin,Reduces the Gallstone Incidence in the Hamster Model

Borch K, Chu M, Kullman E, Carlsson B, Rehfeld JF. Endogenous hypercholecystokininemia, but not aspirin, reduces the gallstone incidence in the hamster model. Scand J Gastroenterol 1994;29:740-743.

Background: Studies in humans and rodents indicate that gallstone development may be prevented by inhibiting gallbladder mucus hypersecretion with non-steroidal anti-inflammatory drugs or by preventing stasis of gallbladder bile with administration of cholecystokinin. Methods: The effect of oral aspirin and pancreaticobiliary diversion with endogenous hypercholecystokininemia on crystal and gallstone formation was studied in Syrian golden hamsters fed a lithogenic diet for 8 weeks. Results: None of the control animals fed a normal diet developed gallstones or crystals in gallbladder bile. Gallstones developed in 67% of the animals fed a lithogenic diet only. The gallstone prevalence did not differ significantly in animals on a lithogenic diet and a daily aspirin dose of 6 mg/kg (gallstone prevalence, 60%) or 100 mg/kg (gallstone prevalence, 70%), whereas it was significantly lower in animals with endogenous hypercholecystokininemia on a lithogenic diet (gallstone prevalence, 29%). The prevalence of crystals in gallbladder bile did not differ significantly between any of the experimental groups. Conclusions: It is concluded that in hamsters on a lithogenic diet, aspirin does not prevent gallstone formation, whereas endogenous hypercholecystokininemia reduces the prevalence of stones without affecting the occurrence of crystals in gallbladder bile.     

Secretion of biliary calcium is increased in dogs with pigment gallstones

We have previously demonstrated that gallbladder bile is supersaturated with calcium bilirubinate in a canine dietary model of pigment gallstones. Supersaturation resulted from combined increases in the concentrations of both biliary calcium and unconjugated bilirubin. The elevations in biliary calcium and unconjugated bilirubin concentrations remain unexplained but could possibly be due to increases in hepatic or ductular secretion, alterations in bile composition with respect to calcium- or bilirubin-binding affinity, decreases in absorption from the gallbladder lumen, or, in the case of unconjugated bilirubin, production within the lumen by hydrolysis of conjugated bilirubin. Here, we study a single possible cause for the observed increase in biliary calcium concentration during pigment gallstone formation in dogs. Secretion of calcium into bile in dogs with pigment gallstones before and after infusion of the bile salt, taurocholate, was compared to normal dogs. A significant increase in bile acid-independent bile flow and calcium output (CaO) was observed at any given bile acid output. Thus, plots of bile flow and CaO versus bile acid output yielded two separate functions in normal dogs and dogs with pigment gallstones. The slopes of these functions were similar, but intercepts extrapolated to zero bile acid output were markedly different, indicating that bile acid-independent, but not bile acid-dependent, bile flow and CaO was increased. The increase in CaO was not due to secretion of bile with increased concentrations of calcium but rather to the increases in the rate of bile flow. These findings might, in part, explain elevated calcium concentrations since increased amounts of calcium would be presented to the gallbladder in these animals during gallstone formation.This work was supported by a Veteran's Administration Merit Review Award and by NIH Grant 32130, National Institute of Diabetes, Digestive, and Kidney Diseases.     

Biliary cholesterol hypersecretion in gallstone-susceptible mice is associated with hepatic up-regulation of the high-density lipoprotein receptor SRBI

Enhanced hepatocellular trafficking of cholesterol to the bile canaliculus and cholesterol hypersecretion appears critical for gallstone formation. Therefore, we studied in more detail the hepatic cholesterol transport pathways in a mouse model of cholesterol gallstone disease. Biliary lipid secretion rates, plasma lipoprotein levels, hepatic expression of lipoprotein receptors, lipid regulatory enzymes, and putative cholesterol transporting proteins were analyzed in gallstone-susceptible C57L/J and gallstone-resistant AKR/J mice, which were fed a lithogenic diet. Biliary cholesterol hypersecretion in C57L mice was associated with decreased plasma high-density lipoprotein (HDL) cholesterol levels and significant hepatic induction of the HDL receptor (SRBI) and cholesteryl ester hydrolase. In response to the lithogenic diet, fatty-acid binding protein of liver (FABPL) was markedly induced in both mouse strains. Caveolin 1 was elevated only in plasma membranes of gallstone-susceptible C57L mice, which also failed to down-regulate cholesterol synthesis. These data suggest a role of the reverse cholesterol transport pathway for genetically determined gallstone susceptibility in the mouse.     

Biliary lipid outputs in young women with cholesterol gallstones

Hepatic secretions of biliary lipids were determined in eight young women with cholesterol gallstones and 14 white women without gallstones. All of the gallstone patients were non-Indian; seven were white and one was black. Hourly outputs of biliary cholesterol were significantly greater in gallstone patients than in white controls. This increased cholesterol output was a major factor in the production of lithogenic bile. The greater cholesterol output in gallstone patients was apparently related to obesity. Despite an increased hepatic secretion of cholesterol, secretion rates of bile acids were relatively low in gallstone patients. However, there was considerable overlap between secretion rates of bile acids in subjects with and without stones, and it was not demonstrated that an absolute deficiency of bile acids existed in gallstone patients. Nevertheless, the contribution of an increased output of biliary cholesterol to the formation of lithogenic bile was clearly evident in our patients.     

Biliary lipids and cholesterol crystal formation in leptin-deficient obese mice

Background. Obesity is often associated with increased biliary cholesterol secretion resulting in cholesterol gallstone formation. We have previously demonstrated that leptin-deficient C57Bl/6J Lep ob obese mice have abnormal biliary motility and are prone to cholesterol crystal formation. In addition, others have demonstrated that leptin-deficient mice when fed a lithogenic diet for eight weeks are not prone to gallstone formation. However, the biliary lipid and in vivo cholesterol crystal response of homozygous and heterozygous leptin-deficient mice to four weeks on a lithogenic diet has not been studied. Therefore, we tested the hypothesis that lithogenic diets influence gallbladder bile composition, serum lipids and cholesterol crystal formation in homozygous and heterozygous leptin-deficient mice compared to normal lean controls. Methods. 319 female lean control mice, 280 heterozygous lep ob obese mice and 117 homozygous lep ob obese mice were studied. Mice were fed either a lithogenic or control non-lithogenic chow diet for four weeks. Gallbladder volumes were measured, and bile was pooled to calculate cholesterol saturation indices. Serum cholesterol, glucose, and leptin levels were determined. Hepatic fat vacuoles were counted, and bile was observed microscopically for cholesterol crystal formation. Results. The lithogenic diet and mouse strain influenced body and liver weights, gallbladder volume, cholesterol crystal formation, serum cholesterol, glucose and leptin levels and hepatic fat vacuole numbers. However, only diet, not strain, altered biliary cholesterol saturation. Conclusion. The association among obesity, leptin, and gallstone formation may be primarily related to altered gallbladder motility and cholesterol crystal formation and only secondarily to biliary cholesterol saturation.     

Modeling plasma lipoprotein-bile lipid relationships: Differential impact of psyllium and cholestyramine in hamsters fed a lithogenic diet

Hamsters fed a lithogenic diet become hyperlipemic with elevated very-low-density lipoprotein (VLDL) and high-density lipoprotein 2 (HDL₂) cholesterol pools and develop lithogenic bile in which chenodeoxycholate (cheno) typically predominates. The relationship between these distorted lipoprotein and bile lipid profiles and gallstone induction was investigated in male Syrian hamsters fed for 5 weeks a gallstone-inducing purified diet (5% butter, 0.4% cholesterol) or the same diet supplemented with 5% psyllium or 1% cholestyramine, agents known to alter bile acid metabolism. The gallstone diet essentially doubled plasma cholesterol level, whereas psyllium decreased it to near normal, and cholestyramine to a subnormal level, while correcting the distorted distribution of cholesterol among lipoproteins. Both the gallstone diet and psyllium produced cholesterol-laden livers, in contrast to subnormal values produced by cholestyramine. Fecal bile acid excretion was increased eightfold with cholestyramine and fourfold with psyllium relative to the value produced by the gallstone diet and a literature control value. Supersaturated bile developed with the gallstone diet (lithogenic index [LI], 2.3 ± 0.6), whereas the LI was decreased by psyllium (1.2 ± 0.4) and cholestyramine (0.7 ± 0.3). The gallstone diet decreased the concentration of bile acids in gallbladder bile, but greatly increased the percentage of taurochenodeoxycholic acid, whereas psyllium preferentially decreased all taurine-conjugated bile acid levels and expanded glycocholate output. Cholestyramine greatly decreased the secretion of biliary cholesterol and cheno independent of its conjugation. Accordingly, psyllium increased the glycine to taurine ratio of gallbladder bile fivefold, whereas cholestyramine did not affect this ratio, but increased the cholate to cheno ratio dramatically (25-fold) as compared with a threefold increase with psyllium. This combination of biliary lipid and bile acid alterations induced coordinated responses in the LI and the hydrophobicity index (HI) such that cholesterol gallstones developed in 11 of 12 hamsters fed the gallstone diet, whereas only one of 11 of the psyllium-fed and none of 12 cholestyramine-fed hamsters had cholesterol stones. Thus, psyllium and cholestyramine differentially increased bile acid excretion, which improved the lipoprotein profile and inhibited cholesterol gallstone formation. Both agents operated by different means to decrease biliary cholesterol secretion and the percentage of cheno, which decreased the LI and HI, respectively.     

Lincomycin treatment of guinea pigs causes formation of pigmented phosphate containing gallbladder sludge and stones

We studied the mechanism of gallbladder sludge formation in guinea pigs (n = 30) treated with lincomycin (80 mg/kg/day) for 7 consecutive days. At sacrifice (day 8) gallbladders of treated animals contained turbid bile, sludge and in one animal a single gallstone. The precipitates were amorphous on X-ray diffraction. Infra-red spectroscopy revealed calcium phosphate as the major component. Compared to saline-treated controls (n = 15) concentrations of total protein, total phosphate and total bilirubin in gallbladder bile were significantly increased (P less than 0.05). The increase in total phosphate was due to the inorganic component, since phospholipid phosphorus was unchanged. The relative amounts of unconjugated bilirubin and of bilirubin mono- and diconjugates in gallbladder bile were unaffected by treatment as was beta-glucuronidase activity. However, sludge was enriched in unconjugated bilirubin compared to gallbladder bile. This was most probably caused by alkaline hydrolysis of bilirubin monoconjugates. To some extent, disproportionation of bilirubin monoconjugates in bile or sludge, either in vivo or during sample preparation, might also have led to increased unconjugated pigment.     

Apolipoprotein A‐I deficiency does not affect biliary lipid secretion and gallstone formation in mice

Background/Aims: Apolipoprotein A‐I (apo A‐I) is the main protein component of plasma high‐density lipoproteins (HDL) and a key determinant of HDL cholesterol levels and metabolism. The relevance of HDL in controlling the traffic of cholesterol from plasma into bile has been partially addressed. The aim of this study was to evaluate the role of apo A‐I expression in controlling the secretion of biliary lipids as well as the risk of gallstone disease in vivo. Methods: We evaluated biliary lipid secretion and bile acid homeostasis in mice deficient for apo A‐I compared with wild‐type animals when fed with low‐ or high‐cholesterol diets. In addition, we assessed the importance of murine apoA‐I expression for gallstone formation after feeding a lithogenic diet. Results: Bile acid pool size and faecal excretion were within the normal range in chow‐ and cholesterol‐fed apo A‐I knockout (KO) mice. Basal biliary cholesterol secretion was comparable and increased similarly in both murine strains after cholesterol feeding. Lithogenic diet‐fed apo A‐I KO mice exhibited an impaired hypercholesterolaemic response owing to a lower increase in cholesterol levels transported in large lipoproteins. However, the lack of apo A‐I expression did not affect biliary cholesterol precipitation or gallstone formation in lithogenic diet‐fed mice. Conclusions: These findings indicate that biliary lipid secretion, bile acid metabolism and gallstone formation are independent of apo A‐I expression and plasma HDL cholesterol levels in mice.     

Hereditary liver disease: gallstones

Gallstones are common in Western countries and due to pain and complications pose a substantial burden on health care systems. In general, cholesterol gallstones are distinguished from bilirubin gallstones. Bilirubin gallstones form if the ion product of unconjugated bilirubin and calcium in gallbladder bile exceeds the solubilisation capacities of mixed micelles and vesicles. Cholesterol gallstones develop if the amount of cholesterol in gallbladder bile exceeds the maximum concentration that is soluble at the given concentration of bile salts and phospholipids. In addition, cholesterol gallstone formation requires hypomotility of the gallbladder and a mucin gel as nucleation matrix for monohydrate crystals. The individual risk of gallstone formation is determined by interactions of lithogenic alleles of gallstone susceptibility genes and multiple environmental factors. For asymptomatic gallstones, expectant management is recommended, whereas an episode of gallstone-associated pain substantially increases the risk of complications such as cholecystitis, cholangitis and pancreatitis and therefore necessitates cholecystectomy.     

Effects of sphingolipid synthesis inhibition on cholesterol gallstone formation in C57BL/6J mice

Background: Sphingolipids play a very important role in cell membrane formation, signal transduction and plasma lipoprotein metabolism. The first rate‐limiting step in the sphingolipid biosynthetic pathway is catalyzed by serine palmitoyltransferase (SPT), and myriocin is a potent and specific inhibitor of SPT. We investigated the impact of SPT inhibition on cholesterol gallstone formation in C57BL/6J mice. Methods: Three groups of eight‐week‐old C57BL/6J mice were utilized. Each group consisted of 20 mice; group A, B, and C were fed normal chow, lithogenic diet with phosphate buffered saline, and lithogenic diet with myriocin (0.3 mg/kg), respectively, for 6 weeks. The ceramide levels in both serum and bile were assessed by high performance liquid chromatography analysis. Protein expression of ERK, JNK and p38 in the extracted gallbladder were determined by Western‐blot analysis. Results: Myriocin treatment caused a significant decrease in the rate of cholesterol gallstone formation. The lithogenic diet mice (group B) showed the highest ceramide activities in both the serum and bile among all the tested groups and there was significant suppression of the ceramide levels in both the serum and bile of the myriocin‐treated mice (group C, p < 0.05). Phosphorylation of p38 in the gallbladder was increased in the lithogenic‐diet mice and the expression of phosphorylated p38 was significantly suppressed in the myriocin treated mice. Conclusions: SPT inhibition by myriocin suppressed gallstone formation and the levels of ceramide in both the serum and bile. p38 in the cellular signaling pathways might be associated with cholesterol gallstone formation.     

High-performance liquid chromatography of bilirubin conjugates in bile: effect of beta-glucuronidase on the bile pigments

A simple, specific, and technically easy high-performance liquid chromatography (HPLC) method for the separation and quantification of unconjugated bilirubin, bilirubin monoglucoside-monoglucuronide, bilirubin diglucuronide, and bilirubin monoglucuronide has been developed. The method was used to determine the bilirubin compounds of bile obtained endoscopically from the common bile duct in 43 patients with gallstone disease and in 6 subjects without gallstones or liver disease. The bile samples were also assessed for the presence of beta-glucuronidase-producing bacteria. The amount of unconjugated bilirubin was significantly higher (p less than 0.01) in bile containing bacteria producing beta-glucuronidase than in bile without such bacterial strains. In six 'normal' bile samples the following quantities of bilirubin conjugates and unconjugated bilirubin were found (median and range): bilirubin monoglucoside-monoglucuronide (mixed conjugate), 61 (27-80) mumol/l; bilirubin diglucuronide, 632 (512-861) mumol/l; bilirubin monoglucuronide, 113 (70-175) mumol/l; and unconjugated bilirubin, 3 (1-7) mumol/l. These results are in good agreement with those obtained with other HPLC methods. The concentrations of unconjugated bilirubin were lower than those found when using conventional diazo methods and thin-layer chromatography. HPLC proved to be a useful tool in gallstone pathogenesis studies. Our results support bacterial glucuronidase as a possible pathogenic factor in pigment gallstone disease.     

Gallbladder mucosal blood flow increases during early cholesterol gallstone formation.

Gallbladder absorption increases during early cholesterol gallstone formation and is influenced by the intraluminal presence of lithogenic bile. The effect of lithogenic bile on gallbladder mucosal blood flow is unknown. The current study tested the hypothesis that the presence of lithogenic gallbladder and hepatic bile enhances gallbladder mucosal blood flow in cholesterol-fed (0.4%) prairie dogs, as determined by hydrogen gas clearance. Gallbladder mucosal blood flow in control animals was 35.57 +/- 3.9 mL.min-1.100 g-1. In contrast, basal gallbladder mucosal blood flow in cholesterol-fed animals was significantly (P less than 0.01) increased to 64.94 +/- 8.7 mL.min-1.100 g-1. In crossover studies, the addition of lithogenic gallbladder bile to control animals (n = 6) resulted in a significant (P less than 0.025) 26% increase in gallbladder mucosal blood flow, whereas the addition of nonlithogenic gallbladder bile into gallbladders of cholesterol-fed prairie dogs resulted in a significant (P less than 0.025) 58% decrease in gallbladder mucosal blood flow. Similarly, hepatic bile crossover studies showed that the addition of lithogenic hepatic bile to control gallbladders significantly increased (P less than 0.025) gallbladder blood flow by 30%, whereas instillation of nonlithogenic hepatic bile in gallbladders of cholesterol-fed animals significantly (P less than 0.025) decreased gallbladder mucosal blood flow by 29%. These results suggest that alterations in gallbladder mucosal blood flow, influenced by the presence and absence of lithogenic bile, may play a role in cholesterol gallstone formation.     

Increased production, hepatic conjugation, and biliary secretion of bilirubin in the rat after chronic ethanol consumption

Disturbances of bilirubin metabolism such as jaundice or pigment gallstone formation, or both, occur in alcoholic cirrhosis of the liver. We have studied the influence of chronic ethanol consumption on bilirubin metabolism as well as on biliary calcium and bile acids in 16 pair-fed male rats. The animals received nutritionally adequate liquid diets containing 36% of total calories either as ethanol or isocaloric carbohydrates for 4 wk. Bile flow was significantly enhanced after chronic ethanol feeding (p less than 0.05 after 90-min bile collection) and was found to be mainly bile acid-independent. The biliary output and concentration of bilirubin monoconjugates, bilirubin diconjugates, and total calcium was significantly increased (p less than 0.01) in alcohol-fed rats compared with controls. This was not the case for unconjugated bilirubin and for the calcium/bile acid ratio. Hepatic bilirubin uridine-5'-diphosphate-glucuronosyltransferase activity (p less than 0.01), serum total bilirubin (p less than 0.01), and serum free hemoglobin (p less than 0.001) were significantly increased after ethanol consumption. These data provide evidence for enhanced bilirubin production, probably due to hemolysis, after alcohol ingestion. The enhanced bile production is associated with an increased hepatic conjugation and subsequent biliary secretion of bilirubin conjugates. In advanced alcoholic liver disease, these compensatory mechanisms may fail and contribute to the development of jaundice.     

Bilirubin conjugate changes in the bile of gallbladders containing gallstones

Gallbladder bile was obtained at laparoscopic cholecystectomy from 31 patients with gallstones, and duodenal aspirates from 18 normal controls. Bile pigments (9 conjugates and unconjugated bilirubin) were analyzed by high-performance liquid chromatography. The average proportional composition of the bile pigments from the patients with gallstones was characteristically different from the controls. Whereas the average values for the principal conjugates in the controls were bilirubin diglucuronide 83.4%, bifirubin monoglucuronide 10.1%, bifirubin monoglucuronide monoglucoside 4.5%, and bilirubin monoglucuronide monoxyloside 1.0%, the corresponding values in the biles from the patients with gallstones were 66.3%, 20.6%, 6.5%, and 2.8%, respectively. Values from the more minor conjugates and unconjugated bilirubin were less than 2% in either data set. In samples obtained in 9 of the gallstone patients early and late in the procedure, no significant change was found. Over the spectrum of findings in the gallstone patients, as the proportion of bilirubin diglucuronide became smaller, that of bilirubin monoglucuronide increased substantially, whereas those of bilirubin monoglucuronide monoglucoside and bilirubin monoglucuronide monoxyloside increased to a small extent. The findings suggest that bilirubin diglucuronide hydrolysis occurs in the gallbladder bile of gallstone patients, with the production of bilirubin monoglucuronide, and that if further hydrolysis of bilirubin monoglucuronide occurs with the formation of unconjugated bilirubin, the latter does not ordinarily increase because it is being absorbed. Stasis with increased gallbladder residence time was likely present in some of the patients. The hydrolytic activity is hypothesized to arise from the gallbladder wall, or the process to be spontaneous, and its effects to be amplified by any increase in gallbladder residence time.