Cholesterol Solubility In Bile Research Articles

Effects of Biliary Phospholipids on Cholesterol Crystallization and Growth in Gallstone Formation.

The prevalence of cholesterol gallstone disease is increasing, primarily due to the global epidemic of obesity associated with insulin resistance, and this trend leads to a considerable healthcare, financial, and social burden worldwide. Although phospholipids play an essential role in maintaining cholesterol solubility in bile through both mixed micelles and vesicles, little attention has been paid to the impact of biliary phospholipids on the pathogenesis of cholesterol gallstone formation. A reduction or deficiency of biliary phospholipids results in a distinctly abnormal metastable physical-chemical state of bile predisposing to supersaturation with cholesterol. Changes in biliary phospholipid concentrations influence cholesterol crystallization by yielding both liquid crystalline and "anhydrous" crystalline metastable intermediates, evolving into classical parallelogram-shaped cholesterol monohydrate crystals in supersaturated bile. As a result, five distinct crystallization pathways, A-E, have been defined, mainly based on the prime habits of liquid and solid crystals in the physiological or pathophysiological cholesterol saturation of gallbladder and hepatic bile. This review concisely summarizes the chemical structures and physical-chemical properties of biliary phospholipids and their physiological functions in bile formation and cholesterol solubility in bile, as well as comprehensively discusses the latest advances in the role of biliary phospholipids in cholesterol crystallization and growth in gallstone formation, largely based on the findings from clinical and animal studies and in vitro experiments. The insights gleaned from uncovering the cholelithogenic mechanisms are expected to form a fundamental framework for investigating the hitherto elusive events in the earliest stage of cholesterol nucleation and crystallization. This may help to identify better measures for early diagnosis and prevention in susceptible subjects and effective treatment of patients with gallstones.

Atorvastatin Induces FXR and CYP7A1 Activation as a Result of the Sequential Action of PPARγ/PGC-1α/HNF-4α in Hep3B Cells.

PPARγ, farnesoid X receptor (FXR) and CYP7A1 are associated with solubility of bile. This study was performed to understand a mechanism and interactions of statin-induced PPARγ, PGC-1α and HNF-4α related to the statin-induced activation of FXR and CYP7A1, and verify whether the mevalonate pathway is involved in the mechanism. MTT assays were performed using cultured human Hep3B cells to determine the effect of atorvastatin on the cell proliferation. Expression levels of indicated proteins were measured using Western blotting assays by inhibiting the protein expression or not. Atorvastatin increased expression of PPARγ, PGC-1α, HNF-4α, FXR, and CYP7A1 in Hep3B cells. PPARγ ligand of troglitazone upregulated the expression of PGC-1α, HNF-4α, FXR, and CYP7A1 in Hep3B cells. Silencing of PPARγ, PGC1α, and HNF4α using respective siRNA demonstrated that atorvastatin-induced FXR and CYP7A1 activation required sequential action of PPARγ /PGC-1α/HNF-4α. The silencing of PPARγ completely inhibited atorvastatin-induced PGC-1α expression, and the PGC1α silencing partially inhibited atorvastatin-induced PPARγ expression. The inhibition of HNF4α did not affect atorvastatin-induced PPARγ expression, but partially inhibited atorvastatin-induced PGC-1α expression. Besides, mevalonate completely reversed the effect of atorvastatin on PPARγ, PGC-1α, HNF-4α, FXR, and CYP7A1. Atorvastatin induces FXR and CYP7A1 activation as a result of sequential action of PPARγ/PGC-1α/HNF-4α in human hepatocytes. We propose that atorvastatin enhances solubility of cholesterol in bile by simultaneously activating of FXR and CYP7A1.

Obeticholic acid may increase the risk of gallstone formation in susceptible patients

The nuclear farnesoid X receptor (FXR) agonist obeticholic acid (OCA) has been developed for the treatment of liver diseases. We aimed to determine whether OCA treatment increases the risk of gallstone formation. Twenty patients awaiting laparoscopic cholecystectomy were randomized to treatment with OCA (25 mg/day) or placebo for 3 weeks until the day before surgery. Serum bile acids (BAs), the BA synthesis marker C4 (7α-hydroxy-4-cholesten-3-one), and fibroblast growth factor 19 (FGF19) were measured before and after treatment. During surgery, biopsies from the liver and the whole bile-filled gallbladder were collected for analyses of gene expression, biliary lipids and FGF19. In serum, OCA increased FGF19 (from 95.0 ± 8.5 to 234.4 ± 35.6 ng/L) and decreased C4 (from 31.4 ± 22.8 to 2.8 ± 4.0 nmol/L) and endogenous BAs (from 1,312.2 ± 236.2 to 517.7 ± 178.9 nmol/L; all p <0.05). At surgery, BAs in gallbladder bile were lower in patients that received OCA than in controls (OCA, 77.9 ± 53.6 mmol/L; placebo, 196.4 ± 99.3 mmol/L; p <0.01), resulting in a higher cholesterol saturation index (OCA, 2.8 ± 1.1; placebo, 1.8 ± 0.8; p <0.05). In addition, hydrophobic OCA conjugates accounted for 13.6 ± 5.0% of gallbladder BAs after OCA treatment, resulting in a higher hydrophobicity index (OCA, 0.43 ± 0.09; placebo, 0.34 ± 0.07, p <0.05). Gallbladder FGF19 levels were 3-fold higher in OCA patients than in controls (OCA, 40.3 ± 16.5 ng/L; placebo, 13.5 ± 13.1 ng/ml; p <0.005). Gene expression analysis indicated that FGF19 mainly originated from the gallbladder epithelium. Our results show for the first time an enrichment of FGF19 in human bile after OCA treatment. In accordance with its murine homolog FGF15, FGF19 might trigger relaxation and filling of the gallbladder which, in combination with increased cholesterol saturation and BA hydrophobicity, would enhance the risk of gallstone development. Obeticholic acid increased human gallbladder cholesterol saturation and bile acid hydrophobicity, both decreasing cholesterol solubility in bile. Together with increased hepatobiliary levels of fibroblast growth factor 19, our findings suggest that pharmacological activation of the farnesoid X receptor increases the risk of gallstone formation. Clinical trial number: NCT01625026.

Lack of a liver‐specific apolipoprotein (apo)A‐V in bile promotes cholesterol gallstone formation by disrupting biliary cholesterol homeostasis in mice

BackgroundApoA‐V is synthesized exclusively by the liver and secreted into the circulation, involving in plasma lipoprotein and lipid metabolism. Although its concentration in human plasma is extremely low (114–258 ng/mL), 1,000 to 10,000‐fold lower than that of apoB‐100 and apoA‐I, respectively, human and mice lacking functional apoA‐V suffer from hypertriglyceridemia. Because our new results found that apoA‐V is also secreted into bile, we hypothesize that it plays an important role in regulating biliary cholesterol metabolism, and its deficiency reduces cholesterol solubility in bile, promoting cholesterol crystallization and gallstone formation.MethodsTo explore the lithogenic mechanisms of biliary apoA‐V deficiency, male apoA‐V knockout (KO) and wild‐type (WT) mice (n=10 per group) were fed a lithogenic diet for 4 wk. Gallstones and cholesterol crystallization in bile were examined by physical‐chemical methods. Hepatic lipid output was determined by biliary secretion studies. Gene expression and protein content were analyzed by qRT‐PCR, Western blots, and ELISA.ResultsCompared to the chow diet, feeding the lithogenic diet significantly (P&lt;0.01) reduced hepatic apoA‐V mRNA and protein levels by 55% and its bile concentrations from 45.7±12.4 ng/mL to 16.7±6.0 ng/mL in WT mice. ApoA‐V was associated only with vesicles, but not micelles, in bile of WT mice. Biliary cholesterol, but not phospholipid or bile salt, output was higher (P&lt;0.05) in KO mice (29.0±8.5 μmol/h/kg) than in WT mice (20.4±5.5 μmol/h/kg), leading to cholesterol‐supersaturated bile. The deficiency of apoA‐V in bile reduced biliary cholesterol solubility and promoted cholesterol crystallization through the liquid crystalline pathway by enhancing biliary unilamellar vesicle aggregation and fusion to form unstable multilamellar vesicles in KO mice. After 50 ng/mL of apoA‐V protein isolated from WT mice was added to supersaturated model bile, cholesterol crystallization was significantly delayed than that added the protein at 0 ng/mL. This implies that apoA‐V plays a key role in regulating cholesterol solubility in bile by modulating the physical state of cholesterol carriers. In addition, the lack of apoA‐V impaired gallbladder emptying and refilling function, thus promoting the accumulation of excess mucin gel and the formation of biliary sludge, a precursor of gallstones. These abnormalities enhanced the growth and agglomeration of solid cholesterol crystal into microlithiasis. After 4 wk of feeding, gallstone prevalence was higher (P&lt;0.05) in KO mice (60%) than in WT mice (25%).ConclusionsThese findings clearly demonstrate that the liver‐specific protein apoA‐V plays a critical role in the regulation of biliary cholesterol metabolism and transport, and its lack in bile dramatically enhances cholelithogenesis by disrupting biliary cholesterol homeostasis. Increasing biliary apoA‐V may be a novel and efficient approach for the prevention of cholesterol gallstones.Support or Funding InformationThis work was supported in part by research grants DK101793, DK106249 and AA03557, all from the National Institutes of Health (US Public Health Service).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Combination of curcumin and piperine prevents formation of gallstones in C57BL6 mice fed on lithogenic diet: whether NPC1L1/SREBP2 participates in this process?

BackgroundA disruption of cholesterol homeostasis characterized by the physical-chemical imbalance of cholesterol solubility in bile often results in formation of cholesterol gallstones. Our earlier studies revealed that curcumin (1000 mg/kg) could prevent formation of gallstones. It has been proved that curcumin is poorly absorbed while piperine is a bioavailability-enhancer. Nevertheless, whether curcumin combined with piperine could enhance the effect of curcumin in preventing gallstones is still awaited.MethodC57BL6 mice were fed on a lithogenic diet concomitant with curcumin at 500 or 1000 mg/kg and/or piperine at 20 mg/kg for 4 weeks. The ratio of gallbladder stone formation was recorded and samples of blood, bile, gallbladder, liver and small intestine were also collected. The volume of gallbladder and weight of liver were calculated, and blood and bile samples were analyzed through biochemical methods. Intestinal NPC1L1 and SREBP2 mRNA and protein expression were detected by real-time PCR and Western blot.ResultCombining with piperine can significantly enhance the effect of curcumin, thus preventing the development of gallbladder stones, lowering the saturation of blood lipids and cholesterol in bile, as well as decreasing the expression of NPC1L1 and SREBP2 in both mRNA and protein levels.ConclusionCurcumin can prevent the formation of cholesterol gallstones induced by high fat diet in mice and SREBP2 and NPC1L1 may participate in this process. Piperine can increase curcumin’s bioavailability, thereby enhancing the effect of curcumin.

Prevention of cholesterol gallstones by inhibiting hepatic biosynthesis and intestinal absorption of cholesterol

Cholesterol cholelithiasis is a multifactorial disease influenced by a complex interaction of genetic and environmental factors and represents a failure of biliary cholesterol homoeostasis in which the physical-chemical balance of cholesterol solubility in bile is disturbed. The primary pathophysiologic event is persistent hepatic hypersecretion of biliary cholesterol, which has both hepatic and small intestinal components. The majority of the environmental factors are probably related to Western-type dietary habits, including excess cholesterol consumption. Laparoscopic cholecystectomy, one of the most commonly performed surgical procedures in the United States, is nowadays a major treatment for gallstones. However, it is invasive and can cause surgical complications, and not all patients with symptomatic gallstones are candidates for surgery. The hydrophilic bile acid, ursodeoxycholic acid (UDCA), has been employed as first-line pharmacological therapy in a subgroup of symptomatic patients with small, radiolucent cholesterol gallstones. Long-term administration of UDCA can promote the dissolution of cholesterol gallstones. However, the optimal use of UDCA is not always achieved in clinical practice because of failure to titrate the dose adequately. Therefore, the development of novel, effective and noninvasive therapies is crucial for reducing the costs of health care associated with gallstones. In this review, we summarize recent progress in investigating the inhibitory effects of ezetimibe and statins on intestinal absorption and hepatic biosynthesis of cholesterol, respectively, for the treatment of gallstones, as well as in elucidating their molecular mechanisms by which combination therapy could prevent this very common liver disease worldwide.

Concentration-dependent displacement of cholesterol in micelles by hydrophobic rice bran protein hydrolysates

Rice bran, containing about 100-150 g kg(-1) protein, is a by-product of rice milling that has only become an available ingredient in recent years owing to the centralisation of rice milling. Rice bran, but not its protein fraction or hydrolysates, has been shown to have a hypocholesterolaemic effect. Peptides from soy, milk and other foods have been proposed to have hypocholesterolaemic effects based on their ability to lower cholesterol solubility in bile acid/phosphotidyl choline micelles. Rice bran protein hydrolysates (RBPHs) were prepared and investigated for their potential to lower cholesterol concentration in micelles. The RBPHs were produced by digestion using four different peptidases, alcalase 2.4L(®), neutrase 0.8L(®), papaya latex papain and porcine pancreas trypsin, and then fractionated by hydrophobicity using styrene/divinylbeneze resins. Alcalase 2.4L(®) produced the highest degree of hydrolysis, and the resulting hydrolysates had the highest micellar cholesterol inhibition ability in an in vitro hypocholesterolaemic test. The adsorption dynamics of four different macroporous resins, DA201-C, Sepabeads SP207 and SP825 and Diaion HP20, were determined using the Langmuir isotherm model. DA201-C had the highest adsorption capacity with an equilibrium concentration of 220 mg g(-1). The hydrolysates eluted with 25, 50, 75 and 95% (v/v) ethanol lowered the micellar cholesterol concentration by 11.88, 14.76, 19.37 and 7.56% respectively. A hydrophobic fraction of RBPH had the highest inhibitory activity on micellar cholesterol, which suggests that it may have hypocholesterolaemic properties.

ABCB4 sequence variations in young adults with cholesterol gallstone disease

Mutations in the gene encoding the ABCB4 [adenosine triphosphate (ATP)-binding cassette, sub-family B (MDR/TAP), member 4] transporter lower phosphatidylcholine output into bile and contribute to cholesterol gallstone formation by decreasing the solubility of cholesterol in bile. Mutations in ABCB4 have been identified in patients with low phospholipid-associated cholelithiasis. The aim of the present study was to determine the types and frequencies of ABCB4 mutations in cholecystectomized patients aged <40 years. Hundred and four patients (mean age 30.6 years, range 12-39) were included in the study and the ABCB4 gene was sequenced. The frequency of missense mutations found in the patient material was measured in 95 healthy controls. The potential functional implications of the ABCB4 missense variations were assessed by computerized analysis (BLOSUM62 and Grantham substitution matrices, polymorphism phenotyping and sorting intolerant from tolerant). One patient was heterozygous for a frameshift mutation (c.1399_1400ins10/p.Y467F fsX25). Another patient was heterozygous for a nonsense mutation (c.3136C>T/p.R1046X). These two mutations are considered detrimental to ABCB4 protein function. In addition, six missense mutations were found in the ABCB4 gene, and three of these were only present in patients. In our study, <2% of young gallstone patients were found to be heterozygous for detrimental ABCB4 mutations. The functional implication of several missense mutations remains to be clarified. Thus, mutations in the ABCB4 gene are a rare cause of gallstone disease.

Role of ABC transporters in secretion of cholesterol from liver into bile.

By 1968, the basic principles underlying cholesterol solubility in bile had been established (1, 2). Free cholesterol (C) had been found to be almost entirely insoluble in water and very poorly soluble in bile salt solutions. Cholesterol was however very soluble in phosphotidylcholine (PC) lamellar liquid crystals (bilayers), and bile salts could solubilize the PC/C liquid crystal bilayers into mixed micelles, thus carrying the C into solution. In 1970, I speculated (3) that the molecular mechanism for biliary lipid secretion was “that bile salts penetrate the cannilicular membrane from the interior of the liver cell and dissect out specifically lecithin and cholesterol leaving the membrane protein and other structural lipids intact.” This hypothesis, based on the physical chemistry of the lipid systems, has now been proven to be almost entirely incorrect. It is now clear that all biliary lipids are secreted in a controlled way by ABC transporters, and that the physical chemistry of the lipid interactions probably occurs within the lumen of the canniliculus and biliary ducts.

Impaired human gallbladder lipid absorption in cholesterol gallstone disease and its effect on cholesterol solubility in bile

Background & Aims: The role of the gallbladder in gallstone pathogenesis is still unclear. We examined the effects of gallbladder mucosal lipid absorption on lipid composition and cholesterol crystallization in bile. Methods: The in vitro–isolated, intra-arterially perfused gallbladder model was used (1) to compare the absorption rates of lipids from standard bile by gallbladders obtained from 7 patients with cholesterol gallstones and 6 controls; and (2) to measure the microscopic cholesterol crystal detection time in cholesterol-enriched pig bile before and after lipid absorption by the pig gallbladder. Results: Control gallbladders, but not cholesterol gallstone gallbladders, significantly reduced cholesterol ( P < 0.02) and phospholipid ( P < 0.01) and increased bile salt ( P < 0.01) molar percentages in bile over a 5-hour period by efficient and selective cholesterol and phospholipid absorption. A histomorphometric study of the epithelial cells showed significantly higher values for nuclear density ( P < 0.01) and nuclear ( P < 0.05) and cytoplasmic ( P < 0.05) areas in the cholesterol gallstone than the control group. Sequential microscopy of cholesterol-enriched pig bile showed significantly shorter cholesterol filament ( P < 0.01) and typical cholesterol plate ( P < 0.02) detection times before than after exposure of bile to the gallbladder lipid absorption. Conclusions: In cholesterol gallstone disease, the human gallbladder epithelium loses its capacity to selectively and efficiently absorb cholesterol and phospholipids from bile, even if it is hyperplastic and hypertrophic. This epithelial dysfunction eliminates the positive effect that the normal gallbladder exerts on cholesterol solubility in bile and might be a pathogenetic cofactor for cholesterol gallstone formation. GASTROENTEROLOGY 2000;118:912-920

The human gallbladder increases cholesterol solubility in bile by differential lipid absorption: a study using a new in vitro model of isolated intra-arterially perfused gallbladder.

In this study, we first developed and validated a new in vitro isolated, intra-arterially perfused, gallbladder model and then applied the method to investigate the absorption of biliary lipids by the gallbladder wall and the effect of this process on the composition of human bile. Oxygenated and glucose-added buffer was perfused through the cystic artery to maintain organ viability. A standard pooled natural bile, radiolabeled with H3-cholesterol and C14-palmitoyl-linoleoyl-phosphatidylcholine, was instilled in the lumen via a cystic duct catheter. Changes in bile volume and lipid concentrations were monitored at time intervals to evaluate the disappearance of lipids from bile caused by gallbladder absorptive function. Organ viability was demonstrated by stable lactate dehydrogenase (LDH) organ release and oxygen consumption throughout the experiments. In the pig, disappearance rates of lipids from bile were similar in vitro and in vivo, demonstrating the validity of the isolated in vitro model for functional studies. By applying our in vitro isolated preparation to the human gallbladder, we found that 23% of cholesterol and 32% of phosphatidylcholine, but only 9% of bile salts, disappeared from bile in 5 hours. As a consequence, at the end of the experiments, cholesterol (P < .05) and phospholipid (P < .05) molar percentages were significantly reduced, while the bile salt (P < .05) molar percentage was significantly increased with respect to values at the beginning of the studies. Our findings are of pathophysiological relevance and support the concept that the human gallbladder modifies the relative composition of biliary lipids in such a way as to increase cholesterol solubility in bile.

The effects of dietary phospholipids enriched with phosphatidylethanolamine on bile and red cell membrane lipids in humans.

The role of phospholipids in biliary cholesterol solubilization and crystallization has only recently begun to be appreciated. Phospholipid vesicles are believed to be the metastable carrier from which cholesterol nucleates. Cholesterol crystallization is influenced by the phospholipid species in bile. Feeding rats and hamsters with diets enriched in phospholipids or their precursors, especially ethanolamine, resulted in reduced cholesterol saturation of bile. Although whole phospholipids are normal dietary constituents, the effects and safety of phospholipid components have not been tested in humans. In the present study, we have evaluated the effects of a dietary phospholipid mixture, enriched with phosphatidylethanolamine, on human bile and red blood cell membrane lipid composition. Five ambulatory volunteers having a chronic indwelling T-tube, with an intact enterohepatic circulation, were investigated. Thirty-six grams of phospholipids (54% phosphatidylethanolamine, 54% linoleyl acyl chains) were added to their daily diet for fourteen days. Biliary nucleation time, cholesterol carriers, as well as plasma, red blood cell membrane, and bile lipid compositions, were monitored. Following phospholipid supplementation, the proportion of linoleyl chains (18:2) in biliary phospholipids increased significantly from 31.1 +/- 1.2 to 37.7 +/- 5.3%, while that of oleyl chains (18:1) decreased from 11.4 +/- 1.6 to 9.6 +/- 1.1%. These changes were accompanied by an increase of linoleate and its metabolite, arachidonate, in red cell membranes. Phospholipid feeding did not cause any side effects, and no significant changes in biliary nucleation time, cholesterol, phospholipid, or bile salt concentrations, or in the distribution of cholesterol within micelles or vesicles. We conclude that phospholipid feeding is safe, and can be effective as a vehicle for lecithin fatty acyl chain modulation of bile and lipid membranes. These findings may provide a basis for a controlled modulation of biliary phospholipids to increase cholesterol solubility in bile.

Bile acids in health and disease.

Over the last quarter of a century Danish research on bile acids has comprised studies of their physical and chemical properties, their physiology, pathophysiology, metabolism, and kinetics, and their clinical applicability. In the beginning of the period a major contribution was made to the understanding of the factors involved in the solubility of cholesterol in bile. The growing international understanding of the potential importance of the bile acids in health and disease gave raise to a substantial Danish contribution in the 1970s and 1980s in parallel with international achievements. Emphasis was on the possible clinical implications of bile acids. Studies on physiology and pathophysiology were in focus. Patients who have had an intestinal bypass operation for obesity served as a model for obtaining new knowledge on various aspects of the properties of the bile acids. Also the analytical methods were improved. Important physiological research on the mechanisms of hepatic bile flow was conducted. An intestinal perfusion model served as a tool providing information on absorption kinetics and on transmucosal water and electrolyte movements. The gallstone disease, liver diseases, inflammatory bowel disease, fat malabsorption, and other intestinal disorders were studied. The 'idiopathic ileopathy' as a cause for bile acid malabsorption causing diarrhoea was established as a new disorder. Thus, in the time period concerned, substantial Danish contributions emerged on major and minor topics of the bile acid field.

Physical-Chemical Basis of Gallstone Formation

The broad outlines of the physical chemistry of cholesterol solubilization are well understood, but minor components of native biles probably alter the equilibrium solubility of cholesterol in bile and, more importantly, profoundly affect the kinetics of cholesterol crystal formation. The physical chemistry of UCB solubilization is much less clear; two decades after the limits of cholesterol solubilization in bile were reported, we still cannot provide accurate information on the limits of UCB solubility in bile. Although UCB is predominantly solubilized by the same biliary aggregates as cholesterol, the ionic nature of UCB implies that other, quantitatively minor biliary components profoundly alter UCB solubilization. Understanding UCB solubilization in bile and the prevention of black pigment stone formation await further studies. An understanding of the physical chemistry of cholesterol and UCB, both as solubilized in bile and as precipitated in gallstones, is basic to an understanding of the pathogenesis of gallstone disease, as well as current and potential chemical methods of gallstone dissolution and prevention.

The study on factors affecting cholesterol solubility in bile

The study on factors affecting cholesterol solubility in bile

Effect of albumin on the solubility of cholesterol in bile

Despite the fact that a considerable amount of albumin is present in bile, little is known about the effect of albumin on micellar solubility of cholesterol. The effect of albumin on solubility of cholesterol in various micellar bile salt solutions was studied using Millipore filtration after equilibration. In addition, partitioning of cholesterol from micellar solution was studied using a polyethylene disc method. Decrease of the solubility of cholesterol by the presence of albumin was observed only in unconjugated bile salt solution. The lowering effect of albumin on the cholesterol solubility was found to be proportional to the hydrophobicity of bile salt. In contrast, albumin had almost no effect on cholesterol solubility, either in conjugated bile salt solution or in micellar bile salt solution containing phosphatidylcholine. Addition of albumin enhanced the partitioning of cholesterol out of the micelles in sodium chenodeoxycholate solution as a result of decreased micellar solubility and increased the aqueous solubility of cholesterol in the presence of albumin. Therefore, conjugated bile salt and phosphatidylcholine exert a buffering action on the albumin-induced adverse effect on cholesterol solubility, thus stabilising bile against inadvertent precipitation of cholesterol.

Effects of fenofibrate on biliary lipids and bile acid pool size in patients with type IV hyperlipoproteinemia

Lipid-lowering drugs, notably clofibrate, may induce a supersaturation of bile with cholesterol, thus favouring the development of cholelithiasis. In order to see whether or not fenofibrate, a clofibrate analogue, has any influence on biliary cholesterol saturation, we determined the lipid composition of gallbladder bile and the bile acid pool size in 15 patients with type IV hyperlipoproteinemia before and after 6–8 weeks of treatment with a daily dose of 300 mg of the drug. At the end of treatment plasma triglycerides were markedly decreased, whereas no detectable influence on liver cell integrity or bile excretory function could be demonstrated in any patient by comparing the pre- and post-treatment serum levels of liver enzymes, bilirubin and bile acids. The mean bile cholesterol saturation index did not significantly change and cholic acid was the only bile acid to increase significantly. In the 3 patients with an initial saturation index of less than 1, bile became supersaturated with cholesterol. However, in no case were the limits of the metastable phase for cholesterol solubility in bile exceeded. Though only long-term prospective studies may give a definitive answer about the lithogenic potential of fenofibrate, our data on gallbladder bile composition in patients with type IV hyperlipoproteinemia indicate that it is not very likely that fenofibrate administration will increase the risk of gallstone formation in severely hyperlipidemic patients.

Vitamin B 6 deficiency decreases lipoprotein 3H-cholesterol incorporation into biliary sterols in cebus monkeys

Since vitamin B 6 is required for taurine synthesis, this study intended to reduce taurine bile acid conjugation in the cebus monkey, an obligate taurine conjugator, through dietary restriction of vitamin B 6. Subsequently, the effect of vitamin B 6 deficiency on lipoprotein 3H-cholesterol incorporation into biliary sterols was evaluated. Two groups of cebus monkeys were each fed a taurine-free soy-protein semi-purified diet for 9 months with (n=5) or without (n=5) 4mg of vitamin B 6 per kg of diet. Bile acid conjugation, pool size and basal synthesis rate, and cholesterol solubility in bile were determined. After total bile acid pool washout, monkeys were injected with a mixture of 3H-cholesterol LDL and HDL, and the specific activity (SA) of biliary cholesterol and primary bile acids was calculated with the percent radioactivity retained by the liver and spleen. The cebus monkey remained an obligate taurine conjugator regardless of taurine and vitamin B 6 status. Neither bile acid conjugation profile or pool size, cholesterol solubility in bile, nor the percent radioactivity retained in liver and spleen were affected by vitamin B 6 deficiency. However, lower B 6-dependent enzyme activity (γ-cystathionase and cysteinesulfinic acid decarboxylase), elevated cystathione, and reduced taurine levels were demonstrated in the B 6-deficient monkeys. Further, the basal synthesis rate of primary bile acids, and the SA of all biliary sterols were significantly reduced in B 6-deficient monkeys. In addition, vitamin B 6 deficiency tended to elevate the plasma cholesterol concentration, which inversely correlated with the incorporation of lipoprotein 3H-cholesterol into biliary cholesterol.

Bile lipid alterations in taurine-depleted monkeys

Newborn cebus and cynomolgus monkeys, differing in their inherent taurine-glycine conjugation of bile acids, were fed taurine-free soy protein infant formula (Isomil) with or without added taurine (500 mg/kg dry diet). After 5 months, monkeys were anesthetized, their enterohepatic circulation interrupted, and their bile pool drained for 4.5 h. Bile acid conjugation, total bile acid pool size, biliary lipid composition, and theoretical maximal cholesterol solubility in bile were determined. Taurine depletion in cebus monkey, an obligate taurine conjugator (97%), did not reduce bile acid conjugation with taurine, or did it alter bile acid pool size, biliary lipid composition, or theoretical maximal cholesterol solubility in bile. Conversely, taurine depletion in cynomolgus, a species normally conjugating with some glycine (15 to 20%), significantly reduced conjugation of taurine with bile acids from 84 to 64%, essentially doubling that of glycine from 16 to 36%. Furthermore, theoretical maximal cholesterol solubility in cynomolgus bile improved significantly as a result of taurine depletion. This improvement was associated with increased percentage distribution of biliary phospholipid from 17 to 33%, in turn reflecting an increase in the taurochenodeoxycholate to taurocholate ratio from 0.7 to 2.9. Concomitant increase in biliary cholesterol concentration associated with increased glycine conjugation precluded any changes in the percent saturation of bile which remained constant at 130% for both dietary groups of cynomolgus. Taurochenodeoxycholate uniquely conserved taurine in the face of body taurine depletion. Taurine availability thus potentially has a substantial influence on bile acid characteristics and cholesterol solubility in a glycine conjugating primate.

Factors affecting the cholesterol dissolution ability of human bile.

Bile samples were obtained from 13 patients with cholesterol gallstones at laparotomy. Control bile samples were obtained from 15 patients with gastric cancer or gastric ulcer. One mixed stone was cut in half and serial thin sections, 10 micrometers in thickness, were prepared from the cut surface. The solubility of cholesterol in these gallstone sections after immersion in the bile samples was observed microscopically after 1, 3, 5 and 24 hr. Of 15 control bile samples, 9 showed a marked cholesterol dissolution after 1 hr, and 6 showed it after 3 and 5 hr. Three out of 13 bile samples from the patients with cholesterol stones dissolved the gallstone section after 1 hr, and 7 after 3 and 5 hr. The dissolution was not observed in the remaining 3 bile samples. Cholesterol and total bile acid concentrations of the control bile were significantly higher than those of the bile from the cholesterol gallstone patients (p less than 0.05 and p less than 0.02, respectively). Correlative studies of the composition of bile and its ability to dissolve cholesterol revealed that bile samples with a high total bile acid concentration possessed a higher dissolution ability, even when the ratios of total bile acid to cholesterol were almost the same in control and cholesterol gallstone bile. On the basis of these findings, it may be concluded that the solubility of cholesterol in bile cannot simply be explained in terms of relative concentrations of cholesterol, phospholipids and total bile acid, because the absolute concentration of total bile acid also plays a significant role for the dissolution ability.