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PubMed1979

High performance liquid-chromatographic analysis of individual bile acids: free, glycine- and taurine-conjugated bile acids.

High performance liquid-chromatographic analyses of individual bile acids (cholic acid, chenodeoxycholic acid, deoxycholic acid, and lithocholic acid), free and conjugated with glycine and taurine, are described. The analyses of free and glycine-conjugated bile acids are based on esterification of carboxyl group of bile acids with O-(p-nitrobenzyl)-N, N'-diisopropylisourea (PNBDI). Moreover, ursodeoxycholic acid, hyocholic acid, hyodeoxycholic acid and 3beta-hydroxy-5-cholenoic acid also are able to analyse by this method. These bile acids in biological sample were extracted by an Amberlite XAD-2 column, and separated by DEAE-Sepharose CL-6B into free, glycine- and taurine-conjugated bile acids. After the separation, free and glycine-conjugated bile acids were esterified with PNBDI directly. Because taurine-conjugated bile acids are unable to be esterified with PNBDI, these bile acids were hydrolyzed by NaOH in order to make free bile acids, and then they were esterified. Because the p-nitrobenzyl ester of bile acids has characteristic ultraviolet absorption, these compounds were separated to individual bile acids by high performance liquid-chromatography, and detected by an UV-detector. An analysis of individual bile acids in human bile was demonstrated.

Bile Acids and Salts↗
PubMed2006

One-step analysis of major bile components in human bile using 1H NMR spectroscopy.

Human gallbladder bile dissolved in dimethylsulfoxide provides sharp and resolved signals for major bile components in 1H NMR spectra. Characteristic well-resolved marker signals that invariably appear in 1H NMR spectra of bile were identified for cholesterol (H18 methyl signal at 0.643 ppm), lipids (glycerol CH signal at 5.064 ppm), total bile acids (H18 signals in the range 0.520-0.626 ppm), total glycine conjugated bile acids (NH signal at 6.958 ppm), total taurine conjugated bile acids (NH signal at 7.646 ppm), and urea (NH2 signal near 5.48 ppm), which enabled their rapid and accurate analysis. Excellent linearity and precision of quantitative analysis was observed for all the identified bile components (R2 > 0.99 for all). The method was demonstrated on gallbladder bile from 19 patients with gallbladder diseases. Urea in bile was identified by NMR for the first time and its quantitative analysis, along with several other bile components, is presented. The majority of the bile components could be analyzed in a single step. Accurate and rapid quantification of several bile components noninvasively by using the method presented herein may have far-reaching implications in the study of bile acid metabolism and pathophysiology of various hepatobiliary and gastrointestinal diseases.

Bile↗
PubMed1995

Bile acid/phosphatidylcholine interactions in mixed monomolecular layers: differences in condensation effects but not interfacial orientation between hydrophobic and hydrophilic bile acid species.

Monomolecular layers of undissociated bile acids and membrane lipids at the air/water interface serve as useful models for the interactions between fully ionized bile salts and physiological membranes. Employing an automated Langmuir-Pockels surface balance, surface pressures and dipole moments were measured as functions of molecular area for six dihydroxy bile acids: ursodeoxycholic acid (UDCA), deoxycholic acid (DCA), chenodeoxycholic acid (CDCA), hyodeoxycholic acid (HDCA), allodeoxycholic acid (alloDCA), and 7 alpha,12 alpha-dihydroxycholanoic acid (7,12-OH-DCA), individually and in mixed mono-molecular layers with 1-palmitoyl-2-oleoyl-sn-3-glycerophosphatidylcholine (POPC) with and without cholesterol on a 5 M NaCl subphase at pH 2. In general, monolayers of hydrophilic bile acids (UDCA, HDCA) had lower collapse pressures and surface dipole moments than hydrophobic bile acids (CDCA approximately DCA approximately alloDCA < 7,12-OH-DCA). In binary mixtures with POPC, all bile acids including the synthetic 7,12-OH-DCA and the uncommon alloDCA condensed mixed monomolecular layers, with the degree of condensation correlating positively with bile acid hydrophobicity. In contrast, none of the bile acids caused further condensation of condensed POPC/cholesterol monomolecular layers. Surface dipole moments of binary bile acid/POPC +/- cholesterol mixtures demonstrated strict additivity, implying that no change in molecular orientation of the interface occurred over film compositions that varied from 0 to 100% bile acid. We conclude that the long axes of the steroid nuclei of dihydroxy bile acids remain parallel to the interface in mixed bile acid/POPC +/- cholesterol monomolecular layers under all conditions. We infer that fully dissociated hydrophobic and, to a lesser extent, hydrophilic bile salts condense phospholipid monomolecular layers and membranes in a similar fashion.

Bile Acids and Salts↗
PubMed2001

The orphan nuclear receptor, shp, mediates bile acid-induced inhibition of the rat bile acid transporter, ntcp.

BACKGROUND AND AIMS: Hepatic bile acid homeostasis is regulated by negative feedback inhibition of genes involved in the uptake and synthesis of bile acids. Bile acids down-regulate the rate-limiting gene for bile acid synthesis, cholesterol 7alpha-hydroxylase (cyp7a), via bile acid receptor (fxr) activation of an inhibitory nuclear receptor, shp. We hypothesized that shp would also mediate negative feedback regulation of ntcp, the principal hepatic bile acid transporter. METHODS: Primary rat hepatocytes or transfected HepG2 and Cos cells were treated with retinoids with or without bile acids, and effects on bile acid transport and ntcp and shp gene expression and promoter activity were determined. Gel shift assays were performed using synthetic fxr, rxr, and rar proteins. RESULTS: Bile acid treatment of primary rat hepatocytes prevented retinoid activation of ntcp gene expression and function; this corresponded temporally with shp gene activation. Bile acid-mediated down-regulation occurred via fxr-dependent suppression of the ntcp RXR:RAR response element. Moreover, cotransfected shp directly inhibited retinoid activation of the ntcp promoter. CONCLUSIONS: These studies show negative feedback regulation of ntcp by bile acid-activated fxr via induction of shp. This novel regulatory pathway provides a means for coordinated down-regulation of bile acid import and synthesis, thereby protecting the hepatocyte from bile acid-mediated damage in cholestatic conditions.

Animals↗
PubMed2004

Are bile acids involved in the renal dysfunction of obstructive jaundice? An experimental study in bile duct ligated rats.

BACKGROUND: Surgery on patients with obstructive jaundice is associated with a significant risk of postoperative renal failure. Bile acids are implicated as nephrotoxins because they accumulate in the plasma and the kidney becomes their only excretory route in cholestasis. The experimental evidence favoring this proposal is inadequate and unconvincing. Therefore, we designed an animal experiment involving bile duct ligated (BDL) rats in which we could correlate variations in serum and urine bile acids with indices of nephrotoxicity and renal function. HYPOTHESIS: Bile acids are putative nephrotoxins. MATERIALS AND METHODS: Total serum and urine bile acid concentrations and profiles were determined using liquid chromatography/gas chromatography/mass spectrometry selected ion monitoring. Nephrotoxicity was assessed by renal histopathology and by determination of the urinary activities of the following enzymes: muramidase, glutamate dehydrogenase, alkaline phosphatase, N-acetyl-beta-D-glucosaminidase, and lactate dehydrogenase. Renal function was assessed by measuring urine osmolality, daily osmolar excretion, sodium excretion (U(Na)V), potassium excretion (U(K)V), and total protein and albumin excretion. RESULTS: Maximum plasma concentrations and renal clearance of bile acids occurred between the third or fourth postoperative day following BDL. This peak coincided with maximal disruption of proximal convoluted tubule architecture and postoperative changes in renal function-increased urine flow rate and decreases in urine osmolality and sodium excretion. Thereafter, 1) plasma levels of bile acids returned toward normal levels, 2) urinary bile acid clearance declined, 3) normal renal histology was restored, and 4) normal renal function was reestablished. Throughout this period, fluctuations in enzymuria were evident. However, these shifts did not coincide with plasma and urine bile acid concentrations and histological and functional changes. DISCUSSION AND CONCLUSIONS: Transient functional impairment of renal cation and water transport and nonspecific morphological changes in the proximal convoluted tubule occur 3 to 4 days following bile duct ligation in rats. These functional and morphological changes occurred when plasma total and urinary bile acids were at their peaks. Although it is tempting to equate association with causality, we cannot implicate bile acids as being responsible for the aberrations in renal function and structure following BDL. Accordingly, we have concluded that elevated plasma concentrations of bile acids are renal exacerbates acting in concert with other factors, be they prerenal or renal in origin to precipitate a cascade of events leading to postoperative renal failure in cholestasis.

Animals↗
PubMed2003

Effects of microflora status, dietary bile salts and guar gum on lipid digestibility, intestinal bile salts, and histomorphology in broiler chickens.

The aim of the present study was to reexamine the effects of intestinal viscosity, microflora, and bile salts, and their interactions in order to clarify the mechanisms that explain the effect of intestinal viscosity on lipid digestibility, especially those that could involve microflora. Effects of intestinal viscosity, microflora status, and dietary bile salts on lipid digestibility, intestinal bile salts, and intestinal histomorphology were tested in a 2 x 2 x 2 factorial experiment. The effect of microflora status was examined by comparing conventional chickens to chickens with limited microflora obtained by rearing birds in sterilized conditions. Viscosity and dietary bile salts were tested with guar gum (0 or 0.5% in diets) and sodium taurocholate (0 or 0.3% in diets), respectively. Guar gum was autoclaved and added to the gamma-irradiated diets by mixing inside the sterile isolators. The intestinal concentration of lactic acid and cecal concentration of short-chain fatty acids were both very low in birds with limited microflora compared to conventional birds (P = 0.0001). Chickens with limited microflora had higher gain:feed ratios (P = 0.002), higher fecal lipid digestibility (P = 0.0001), more intestinal conjugated bile salts (P = 0.0001), less intestinal unconjugated bile salts (P = 0.0001), and their gastrointestinal compartments were smaller (P < 0.05) than those of conventional chickens. Addition of bile salts in diets increased the lipid digestibility (P = 0.0001) with a more pronounced effect in conventional birds (P = 0.0001) and in birds fed on guar gum diets (P = 0.002). Feeding the guar gum diets increased the intestinal supernatant viscosity (P = 0.0001) for both microflora status. Guar gum addition increased (P < 0.05) the lactic acid concentration in the small intestine of conventional chickens. Guar gum decreased the fecal lipid digestibility (P = 0.0001) and the intestinal conjugated bile salts (P = 0.0001) for both microflora status. However, the magnitude of lipid digestibility decrease due to guar gum was lower (P = 0.05) in birds with limited microflora than in conventional birds, and the negative effect of guar gum on intestinal conjugated bile salts was more pronounced (P = 0.02) in birds with limited microflora. Bile salt addition reduced the negative effect of guar gum on lipid digestibility (P = 0.02) for both microflora status. The mean lipid digestibilities were negatively correlated (P = 0.0001) with the ratio [Ln(viscosity)/total bile salt] measured in intestinal digesta. Except for gizzard, size of digestive compartments and villus heights increased (P < 0.05) with feed efficiency and digestibility decreased. In conclusion, the results provided evidences that the negative effect of guar gum on lipid digestibility was mainly mediated by its direct effect on intestinal bile salt concentration and efficiency. The small interaction between guar gum and microflora status effects on lipid digestibility had to be accounted for by the low basal level of intestinal bile salts in conventional chickens.

Animal Feed↗
PubMed2005

A PrfA-regulated bile exclusion system (BilE) is a novel virulence factor in Listeria monocytogenes.

The ability to colonize the gall bladder has recently been shown to be an important feature of virulent Listeria monocytogenes (J. Hardy, K. P. Francis, M. DeBoer, P. Chu, K. Gibbs, C. H. Contag. Science 303: 851-853, 2004). We suggest that the cytotoxic effects of bile may be increased upon release from the gall bladder into the upper small intestine, and report the identification of a novel bile exclusion system which plays an essential role in intestinal colonization and virulence of L. monocytogenes. In silico analysis of the L. monocytogenes EGDe genome revealed a two-gene operon (formerly opuB) exhibiting significant sequence similarity to members of the betaine carnitine choline transporter (BCCT) family. The operon, herein designated bilE (bile Exclusion) is preceded by consensus sigmaA- and sigmaB-dependent promoter-binding sites and is transcriptionally upregulated at elevated osmolarities and reduced temperatures (stresses known to induce sigB). Furthermore, a significant reduction in the level of bilE transcription was observed in the absence of sigmaB. In addition, we demonstrate an important role for PrfA, the master regulator of virulence potential in L. monocytogenes, in coordinating bilE expression. Computational structural analysis suggests that, rather than functioning as a compatible solute uptake system as was previously believed, BilE is more likely to be an exclusion system, a conclusion substantiated by radiolabelled bile accumulation studies. In addition, functionally inactivating BilE resulted in a five-log reduction in the ability of the bacterium to tolerate lethal concentrations of bovine bile (oxgall) and also significantly increased sensitivity to physiological concentrations of human bile, a phenotype which translates to a significant reduction in virulence potential when administered to a murine model by the oral route. Thus, this novel bile exclusion locus bilE, coordinately regulated by sigmaB and PrfA, represents a new and important virulence factor in L. monocytogenes.

ATP-Binding Cassette Transporters↗
PubMed2005

Contribution of three bile-associated loci, bsh, pva, and btlB, to gastrointestinal persistence and bile tolerance of Listeria monocytogenes.

Listeria monocytogenes must resist the deleterious actions of bile in order to infect and subsequently colonize the human gastrointestinal tract. The molecular mechanisms used by the bacterium to resist bile and the influence of bile on pathogenesis are as yet largely unexplored. This study describes the analysis of three genes--bsh, pva, and btlB--previously annotated as bile-associated loci in the sequenced L. monocytogenes EGDe genome (lmo2067, lmo0446, and lmo0754, respectively). Analysis of deletion mutants revealed a role for all three genes in resisting the acute toxicity of bile and bile salts, particularly glycoconjugated bile salts at low pH. Mutants were unaffected in the other stress responses examined (acid, salt, and detergents). Bile hydrolysis assays demonstrate that L. monocytogenes possesses only one bile salt hydrolase gene, namely, bsh. Transcriptional analyses and activity assays revealed that, although it is regulated by both PrfA and sigma(B), the latter appears to play the greater role in modulating bsh expression. In addition to being incapable of bile hydrolysis, a sigB mutant was shown to be exquisitely sensitive to bile salts. Furthermore, increased expression of sigB was detected under anaerobic conditions and during murine infection. A gene previously annotated as a possible penicillin V amidase (pva) or bile salt hydrolase was shown to be required for resistance to penicillin V but not penicillin G but did not demonstrate a role in bile hydrolysis. Finally, animal (murine) studies revealed an important role for both bsh and btlB in the intestinal persistence of L. monocytogenes.

Animals↗
PubMed2001

Fatty acid bile acid conjugates (FABACs)--new molecules for the prevention of cholesterol crystallisation in bile.

BACKGROUND: Cholesterol gall stones are a frequent disease for which at present surgery is the usual therapy. Despite the importance of bile acids it has become evident that phospholipids are the main cholesterol solubilisers in bile. Even phospholipid components, such as fatty acids, have anticrystallising activity. AIM: To synthesise fatty acid bile acid conjugates (FABACs) and study their effects on cholesterol crystallisation in bile in vitro and in vivo. METHODS: FABACs were prepared by conjugation of cholic acid at position 3 with saturated fatty acids of variable chain length using an amide bond. Cholesterol crystallisation and its kinetics (crystal observation time, crystal mass) were studied in model bile, pooled enriched human bile, and fresh human bile using FABACs with saturated fatty acids of varying chain length (C-6 to C-22). Absorption of FABACs into blood and bile was tested in hamsters. Prevention of biliary cholesterol crystallisation in vivo was tested in hamsters and inbred mice. RESULTS: FABACs strongly inhibited cholesterol crystallisation in model as well as native bile. The FABACs with longer acyl chains (C-16 to C-22) were more effective. At a concentration of 5 mM, FABACs almost completely inhibited cholesterol crystallisation in fresh human bile for 21 days. FABACs were absorbed and found in both portal and heart blood of hamsters. Levels in bile were 2-3 times higher than in blood, indicating active secretion. Appreciable levels were found in the systemic circulation 24-48 hours after a single administration. Ingested FABACs completely prevented the formation of cholesterol crystals in the gall bladders of hamsters and mice fed a lithogenic diet. CONCLUSIONS: FABACs are potent inhibitors of cholesterol crystallisation in bile. They are absorbed and secreted into bile and prevent the earliest step of cholesterol gall stone formation in animals. These compounds may be of potential use in cholesterol gall stone disease in humans.

Animals↗
PubMed2005

Weanling, but not adult, rabbit colon absorbs bile acids: flux is linked to expression of putative bile acid transporters.

Intestinal handling of bile acids is age dependent; adult, but not newborn, ileum absorbs bile acids, and adult, but not weanling or newborn, distal colon secretes Cl(-) in response to bile acids. Bile acid transport involving the apical Na(+)-dependent bile acid transporter (Asbt) and lipid-binding protein (LBP) is well characterized in the ileum, but little is known about colonic bile acid transport. We investigated colonic bile acid transport and the nature of the underlying transporters and receptors. Colon from adult, weanling, and newborn rabbits was screened by semiquantitative RT-PCR for Asbt, its truncated variant t-Asbt, LBP, multidrug resistance-associated protein 3, organic solute transporter-alpha, and farnesoid X receptor. Asbt and LBP showed maximal expression in weanling and significantly less expression in adult and newborn rabbits. The ileum, but not the colon, expressed t-Asbt. Asbt, LBP, and farnesoid X receptor mRNA expression in weanling colon parallel the profile in adult ileum, a tissue designed for high bile acid absorption. To examine their functional role, transepithelial [(3)H]taurocholate transport was measured in weanling and adult colon and ileum. Under short-circuit conditions, weanling colon and ileum and adult ileum showed net bile acid absorption: 1.23 +/- 0.62, 5.53 +/- 1.20, and 11.41 +/- 3.45 nmol x cm(-2) x h(-1), respectively. However, adult colon secreted bile acids (-1.39 +/- 0.47 nmol x cm(-2) x h(-1)). We demonstrate for the first time that weanling, but not adult, distal colon shows net bile acid absorption. Thus increased expression of Asbt and LBP in weanling colon, which is associated with parallel increases in taurocholate absorption, has relevance in enterohepatic conservation of bile acids when ileal bile acid recycling is not fully developed.

Aging↗
PubMed1979

Regulation of hepatic transport of bile salt. Effect of protein synthesis inhibition on excretion of bile salts and their binding to liver surface membrane fractions.

The overall transport of bile salts across the hepatocyte is characterized as a carrier-mediated process whose rate-limiting step is biliary secretion. Specific bile salt binding proteins have been identified in liver surface membrane fractions and were postulated to represent the initial interaction in bile salt translocation across both the sinusoidal and canalicular membranes. To test this hypothesis, cycloheximide was administered to rats to inhibit hepatic protein synthesis. 16 h after cycloheximide administration [14C]leucine incorporation into hepatic protein was inhibited by 93% at 1 h and 47% at 12 h. However, values of liver function tests were not increased, although serum albumin, serum alanine amino-transferase, and alkaline phosphatase were significantly decreased. Light and electron microscopy did not demonstrate necrosis or fat accumulation. The latter demonstrated minimal disorganization of rough endoplasmic reticulum and occasional lamellar whorls. 16 h after cycloheximide administration bile salt independent bile flow, basal bile salt excretion, and basal bile flow were unaltered, but the maximum bile salt transport capacity was reduced to 62% of control and 24 h later to 38%. Decreased bile salt transport was reversible, for it returned to control values after 48 h, when hepatic protein synthesis was also normal. Maximum bromosulfophthalein (BSP) transport, on the other hand, was reduced after 16 h to only 85% of control. Both bile salt and BPS maximum transport capacities decreased with time during inhibition of protein synthesis, apparently following first order kinetics. It was estimated that their half-lives are 20 h for bile salt transport and 55 h for BSP transport. These different turnover rates suggest that cycloheximide does not decrease active transport through generalized hepatic dysfunction or alteration of high energy sources possibly required for transport. The maximum number of [14C]cholic acid binding sites in liver surface membrane fractions was determined by an ultrafiltration assay. They were reduced to 68% of control after 16 h of cycloheximide and to 25% after 24 h. This reduction in the number of binding sites is apparently selective, for the activities of the liver surface membrane enzymes (Na+-K+)ATPase, Mg++-ATPase, and 5'-nucleotidase were not significantly changed. The associated alterations in bile salt transport and the maximum number of binding sites after cycloheximide administration suggests that these receptors may be the bile salt carriers.

Animals↗
PubMed1982

Regulation of bile salt transport in rat liver. Evidence that increased maximum bile salt secretory capacity is due to increased cholic acid receptors.

Expansion of the bile salt pool size in rats increases maximum excretory capacity for taurocholate. We examined whether increased bile salt transport is due to recruitment of centrolobular transport units or rather to adaptive changes in the hepatocyte. Daily sodium cholate (100 mg/100 g body wt) was administered orally to rats. This treatment was well tolerated for at least 4 d and produced an 8.2-fold expansion of the bile salt pool. This expanded pool consisted predominently (99%) of cholic and deoxycholic acids. Significantly increased bile salt transport was not observed until 16 h after bile acid loading, and maximum elevations of transport capacity to 2.3-fold of control required approximately 2 d. In contrast, maximum sulfobromophthalein excretion rates increased 2.2-fold as early as 4 h and actually fell to 1.5-fold increase at 4 d. We studied the possibility that this adaptive increase in bile salt secretory transport was due to changes in canalicular surface membrane area, lipid composition, or increased number of putative carriers. Canalicular membrane protein recovery and the specific activities of leucine aminopeptidase, Mg(++)-ATPase and 5'-nucleotidase activities were unaltered by bile salt pool expansion. The content of free and esterified cholesterol and total phospholipids was unchanged in liver surface membrane fractions compared with control values. In contrast, sodium cholate administration selectively increased specific [(14)C]cholic acid binding sites twofold in liver surface membrane fractions. Increased numbers of [(14)C]cholic acid receptors (a) was associated with the time-dependent increase in bile salt transport, and (b) was selective for the taurine conjugate of cholate and (c) was reduced by chenodeoxycholate. Changes in bile acid binding sites 16 h following taurocholate and chenodeoxycholate and the lack of change with glycocholate was associated with comparable changes in bile salt transport. In conclusion, selective bile salts increase bile salt transport in the liver through an adaptive increase in the density of putative bile acid carriers in liver surface membrane.

Animals↗
PubMed2001

A non-icteric cholecystectomized patient with recurrent attacks of right epigastric pain and dilated common bile duct--do liver function tests predict bile duct stones?

Cholecystectomized patients with recurrent attacks of right epigastric pain and with dilated common bile duct are a clinical challenge. In a number of these patients dilatation of the common bile duct is explained as a normal postoperative state following cholecystectomy and the recurrent pain attacks are of origin other than bile disorder, but in some cases dilatation of the common bile duct and attacks are caused by bile duct stones. The aim of the present work was to study the value of common plasma liver function tests in predicting bile duct stones in the group of non-icteric cholecystectomized patients with recurrent attacks of right epigastric pain and with dilated common bile duct. The study population comprised 24 consecutive non-icteric cholecystectomized patients admitted for elective endoscopic retrograde cholangiopancreatography because of attacks of right epigastric pain and dilated common bile duct in ultrasonography. All the liver function tests seemed to assist in separating patients with bile duct stones (n=11) from those without (n=13). Alanine aminotransferase levels were significantly higher (p=0.05) in patients with bile duct stones than in those without, but also alkaline phosphatase (p=0.07), gamma-glutamyl transferase (p=0.09) and bilirubin (p=0.09) levels seemed to be higher in patients with bile duct stones than in those without, although the differences in these values did not reach statistical significance. In conclusion, common plasma liver function tests assist in separating patients with bile duct stones from those without in this small but clinically important group of non-icteric cholecystectomized patients with recurrent attacks of right epigastric pain and with dilated common bile duct. However, the actual value of these measurements is limited in clinical decision making since overlapping of values occured.

Adult↗
PubMed1993

Cholesterol nucleation time measurement in nasobiliary or nasoduodenal bile. Comparison with surgical bile.

The usual technique of collecting gallbladder bile at laparotomy is not suitable for sequential studies of cholesterol nucleation time (NT) in patients receiving therapy to prevent or dissolve cholesterol gallstones. Our aim was to study the feasibility of measuring NT in bile obtained by nasobiliary or nasoduodenal intubation. We studied a total of 10 cholesterol gallstone patients; in 8 bile was collected by nasobiliary drainage, in 7 it was collected by nasoduodenal intubation, and in 3 it was collected at laparotomy the next day. Three patients developed abdominal pain and increased serum amylase after endoscopic retrograde cannulation. All three biles obtained at operation nucleated quickly (NT, 1-4 days), whereas duodenal biles were all beyond the expected range (NT, > 21 days). Chymotrypsin activity, as a marker of pancreatic juice contamination, was detected in five of eight nasobiliary biles and in all seven duodenal biles but in none of the surgical biles. Free fatty acids (reflecting lipolysis) were significantly higher in duodenal than in surgical biles, with nasobiliary bile showing intermediate values. Nasobiliary bile showed either a rapid (median NT, 3 days) or a slow (median NT, 22 days) NT, depending on whether chymotrypsin activity was absent or present (p < 0.05). We conclude that duodenal bile is never suitable for NT determination because of contamination by pancreatic enzymes, and that nasobiliary bile, if not contaminated by pancreatic enzymes, may be suitable for NT determination but that its collection via a nasobiliary tube after cholecystokinin injection carries a risk of pancreatitis.

Adolescent↗
PubMed1993

Effect of phospholipids and bile acids on cholesterol nucleation time and vesicular/micellar cholesterol in gallbladder bile of patients with cholesterol stones.

Supersaturation and rapid nucleation of cholesterol in bile are of key importance in the pathogenesis of cholesterol gallstones. While the effects of bile acids and phospholipids on cholesterol saturation of bile have been extensively studied, their influence on the cholesterol nucleation time has not been compared. We, therefore, investigated whether increases of bile acid or phospholipid concentrations in bile by in vitro supplementation affect the cholesterol nucleation time. Bile samples were obtained at surgery from patients with cholesterol gallstones. Prior to the nucleation assay the bile samples were divided into 0.5-ml aliquots and supplemented with 1.25, 2.5, 5.0, and 10.0 mumol/ml of different phosphatidylcholines (PC-dimyristoyl, PC-dipalmitoyl, PC-distearoyl, and extracted biliary PCs) or with 5.0, 10.0, and 20.0 mumol/ml of bile acids (glycine or taurine conjugates of cholic acid, deoxycholic acid, or chenodeoxycholic acid). The increase of phosphatidylcholine or bile acid concentration decreased the mean cholesterol saturation index to a similar extent (PC: 0.1-0.3; BA: 0.1-0.2). Supplementations of bile with increasing amounts of synthetic or biliary PCs caused a marked prolongation of the nucleation time in bile from 1.5 +/- 0.2 up to > or = 21 days or 2.5 +/- 0.7 up to > or = 21 days. Concurrently, biliary cholesterol was shifted from vesicles to mixed micelles and the cholesterol/phospholipid ratio of the remaining vesicles was progressively lowered. In contrast, the addition of bile acids to gallbladder bile did not affect the cholesterol nucleation time (2.2 +/- 0.3 days), the percentage of vesicular cholesterol, or the cholesterol/phospholipid ratio of vesicles and micelles.(ABSTRACT TRUNCATED AT 250 WORDS)

Bile Acids and Salts↗
PubMed1996

Characterization of crystallization pathways during cholesterol precipitation from human gallbladder biles: identical pathways to corresponding model biles with three predominating sequences.

In model biles, five crystallization sequences are present as functions of bile salt/lecithin (egg yolk) ratio and their positions on phase diagrams are influenced by bile salt hydrophobicity, temperature, and total lipid concentration (D. Q-H. Wang and M.C. Carey. J. Lipid Res. 1996.37: 606-630). To determine whether the same pathways occur ex vivo during cholesterol precipitation from human gallbladder biles, we examined 22 cholesterol gallstone (CSI = 1.56 +/- 0.26), 4 pigment gallstone (0.69 +/- 0.06), and 4 control biles (0.85 +/- 0.22) by microscopy and lipid analytic techniques for 30 days. Temperature was varied (4-45 degrees C) to move relative compositions into adjacent pathways or supersaturated zones to test whether the same bile could be forced to crystallize in different sequences. Sequences in native bile were identical to those in model systems composed of mixed bile salts-lecithin-cholesterol mixtures, and three corresponding pathways (B, C, D; op. cit.) were observed at 37 degrees C. With increasing lecithin content, we found i) B: plate-like cholesterol monohydrate crystals appeared before arc-shaped (putatively anhydrous cholesterol) crystals which transformed via helices and tubules into plate-like crystals and no liquid crystals formed; ii) C: lamellar liquid crystals, typified by birefringent multilamellar vesicles, were detected before cholesterol monohydrate crystals, and subsequently arc, helical and tubular crystals appeared; and iii) D: precipitation of lamellar liquid crystals was followed by cholesterol monohydrate crystals and no arc crystals were detected. Added EDTA prevented calcium bilirubinate formation, but crystallization sequences in these biles were identical to those without EDTA. We conclude that i) cholesterol crystallization pathways and sequences in human gallbladder biles are identical to model biles matched for appropriate physical-chemical conditions; ii) three of the five sequences observed in model biles were found in native bile; and iii) calcium bilirubinates neither promote biliary cholesterol crystallization nor influence crystal growth.

Bile Acids and Salts↗
PubMed2006

Bile salt toxicity aggravates cold ischemic injury of bile ducts after liver transplantation in Mdr2+/- mice.

Intrahepatic bile duct strictures are a serious complication after orthotopic liver transplantation (OLT). We examined the role of endogenous bile salt toxicity in the pathogenesis of bile duct injury after OLT. Livers from wild-type mice and mice heterozygous for disruption of the multidrug resistance 2 Mdr2 gene (Mdr2+/-) were transplanted into wild-type recipient mice. Mdr2+/- mice secrete only 50% of the normal amount of phospholipids into their bile, leading to an abnormally high bile salt/phospholipid ratio. In contrast to homozygous Mdr2-/- mice, the Mdr2+/- mice have normal liver histology and function under normal conditions. Two weeks after OLT, bile duct injury and cholestasis were assessed by light and electron microscopy, as well as through molecular and biochemical markers. There were no signs of bile duct injury or intrahepatic cholestasis in liver grafts from wild-type donors. Liver grafts from Mdr2+/- donors, however, had enlarged portal tracts with cellular damage, ductular proliferation, biliostasis, and a dense inflammatory infiltrate after OLT. Parallel to this observation, recipients of Mdr2+/- livers had significantly higher serum transaminases, alkaline phosphatase, total bilirubin, and bile salt levels, as compared with recipients of wild-type livers. In addition, hepatic bile transporter expression was compatible with the biochemical and histological cholestatic profile found in Mdr2+/- grafts after OLT. In conclusion, toxic bile composition, due to a high biliary bile salt/phospholipid ratio, acted synergistically with cold ischemia in the pathogenesis of bile duct injury after transplantation.

ATP Binding Cassette Transporter, Subfamily B↗
PubMed1999

In liver transplantation, T tube bile represents total bile flow: physiological and scintigraphic studies on biliary secretion of organic anions.

The present study was performed to clarify the recovery of hepatocellular uptake and the biliary secretion of bile acids during the first 14 days after orthotopic liver transplantation (OLT) and to determine the fraction of bile flow appearing outside through the T tube and entering the duodenum. Therefore, we determined primary and secondary bile acids in bile samples obtained from the T tube at day 5 after OLT, while the T tube was permanently open, and at days 10 and 14 after OLT, i.e., 4 and 9 days after closure of the T tube, respectively, thus restoring enterohepatic bile acid circulation. In addition, we performed hepatobiliary scintigraphy using technetium 99m-labeled [2,4,6 trimethyl-3-bromo]imino-diacetic acid (technetium 99m-BRIDA) in 12 patients between days 4 and 17 after OLT. Chromatographic analyses of biliary bile acids showed no secondary bile acids during the first 5 days after OLT, as opposed to 10 and 14 days after OLT when enterohepatic circulation was restored. Eleven patients with an uncomplicated postoperative course after OLT showed a significantly reduced hepatic uptake and biliary secretion of 99mTc-BRIDA during the first days after OLT with progressive recovery. One patient with an acute allograft rejection episode showed almost no uptake and only minimal secretion. The bile fraction appearing outside through the inserted T tube represented 94.6% +/- 6.2% of the injected 99mTc-BRIDA. We conclude that OLT results in markedly impaired hepatocellular uptake and biliary secretion of organic anions. Simultaneously, bile acid synthesis is significantly reduced, which, in addition, diminishes bile secretion of the graft. We show that T tube bile is a valid tool for bile physiological studies in patients in whom transplantation was successfully performed.

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