Metabolic Profile of Obeticholic Acid and Endogenous Bile Acids in Rats with Decompensated Liver Cirrhosis.

Obeticholic acid (OCA) is a semisynthetic bile acid (BA) analog and potent farnesoid X receptor agonist approved to treat cholestasis. We evaluated the biodistribution and metabolism of OCA administered to carbon tetrachloride-induced cirrhotic rats. This was to ascertain if plasma and hepatic concentrations of OCA are potentially more harmful than those of endogenous BAs. After administration of OCA (30 mg/kg), we used liquid chromatography-mass spectrometry to measure OCA, its metabolites, and BAs at different timepoints in various organs and fluids. Plasma and hepatic concentrations of OCA and BAs were higher in cirrhotic rats than in controls. OCA and endogenous BAs had similar metabolic pathways in cirrhotic rats, although OCA hepatic and intestinal clearance were lower than in controls. BAs' qualitative and quantitative compositions were not modified by a single administration of OCA. In all the matrices studied, OCA concentrations were significantly lower than those of endogenous BAs, potentially much more cytotoxic.

A new gastric-emptying mouse model based on in vivo non-invasive bioluminescence imaging.

BACKGROUND: Different techniques were used to assess gastric emptying (GE) in small animals; most of them require sophisticated equipment, animal sacrifice and are expensive. In the present investigation a simple, non-invasive method based on bioluminescence imaging (BLI) is reported to study GE, using light-emitting Escherichia coli cells as a marker of the gastric content. METHODS: A new thermostable red-emitting luciferase was chosen as reporter gene to transform E. coli cells. Bioluminescent (BL) bacteria were administered to fasting mice, after a solid meal, and in response to different doses of metoclopramide (MET) and hyoscine butylbromide (HY). Bioluminescence imaging allowed to evaluate the real time 2D spatial and temporal distribution of bacteria along the gastrointestinal tract in animals and to calculate GE rate in basal conditions and following pharmacological stimulation. KEY RESULTS: The administered BL bacteria were easily imaged and localized in the stomach and subsequently followed in the duodenum and upper intestine allowing to accurately calculate GE. Gastric emptying after the test meal was significantly slower (T(1/2) 16 ± 3 min) than that obtained in fasting conditions (T(1/2) 2 ± 1 min); administration of HY (1 mg kg(-1) b.w.) significantly (P < 0.05) increased T(1/2) that was delayed up to 25 ± 4 min; MET (1 mg kg(-1) b.w.) significantly (P < 0.05) accelerated T(1/2), that was achieved within 8 ± 2 min. CONCLUSION & INFERENCES: The reported model is simple, inexpensive, reliable, sensitive and accurate; it can detect both acceleration and slowdown of GE. The model is useful in the investigation of new drug-induced alterations of gastric motility allowing to reduce the number of experimental animals.

Generation of a novel antibody probe to the apical sodium-dependent bile acid transporter that inhibits ileal bile acid absorption.

Intestinal bile acid absorption is mediated by a sodium-dependent transporter located in the brush border apical membrane of ileocytes. The transmembrane topology and the role of individual amino acid residues in the bile acid transport process have been investigated by means of various experimental approaches, leading to multiple hypotheses. We raised a monoclonal antibody against a segment of the transporter comprising vicinal cysteine residues, in order to evaluate its functional role. A 14 amino acid peptide, corresponding to amino acids 104-117 of the transporter, was synthesized, and a monoclonal anti-peptide antibody was raised. In vitro uptake-inhibition studies in the presence of the monoclonal anti-peptide antibody were performed using ileal brush border membrane vesicles. Rabbit ileum was perfused in vivo with 5 mM taurocholic acid in the presence of the monoclonal antibody, and bile acid absorption inhibition was evaluated. The anti-peptide monoclonal antibody significantly reduced the in vitro uptake and in vivo absorption of taurocholic acid. The present data demonstrate the functional relevance of the 104-117 peptide segment and report the generation of a novel antibody against the apical sodium-dependent bile acid transporter (ASBT) that may be used as a therapeutic agent in hypercholesterolemia and in cholestatic pruritus.

Uptake and killing of Leptospira interrogans and Borrelia burgdorferi, spirochetes pathogenic to humans, by reticuloendothelial cells in perfused rat liver.

In situ-perfused rat livers were infused with a single dose of 1.5 x 10(7) radiolabeled cells of Leptospira interrogans serovar icterohaemorrhagiae, the agent of leptospirosis, or with Borrelia burgdorferi IRS, the agent of Lyme disease. Significant (P<0.0001) differences in the liver uptake of L. interrogans and of B. burgdorferi were observed, the uptakes being 37.4%+/-2.3% for L. interrogans and 60.5%+/-3.1% for B. burgdorferi. Leptospires, in contrast to borreliae, were recovered from the livers when liver samples were cultured in growth medium. Leptospires but not borreliae were recovered in bile within 30 min of infusion. The association of leptospires and borreliae with reticuloendothelial cells of the liver was demonstrated by immunohistochemistry. Leptospires and borreliae were found to be associated with vimentin-positive cells and not with desmin-positive cells. Few leptospires but no borreliae were also seen associated with vimentin- and desmin-negative cells, suggesting the presence of leptospires outside the sinusoidal spaces, in the liver parenchyma.

Competition in liver transport between chenodeoxycholic acid and ursodeoxycholic acid as a mechanism for ursodeoxycholic acid and its amidates' protection of liver damage induced by chenodeoxycholic acid.

BACKGROUND: Ursodeoxycholic acid has been widely used as a therapeutic agent in cholesterol gallstones and liver disease patients, but its mechanism of action is still under investigation. AIMS: The protective effect of ursodeoxycholic acid, both free, taurine and glycine conjugated, against hepatotoxic bile acids such as chenodeoxycholic acid and its taurine amidate was studied in bile fistula rats and compared with the cholic and taurocholic acid effect. METHODS: Tauroursodeoxycholic acid, glycine ursodeoxycholic acid, ursodeoxycholic acid, taurocholic acid and cholic acid were infused iv over 1 hour (8 micromol/min/kg) together with an equimolar dose of either taurochenodeoxycholic acid or chenodeoxycholc acid. Bile flow, total and individual bile acid and biliary lactate dehydrogenase and alkaline phosphatase enzymes were measured. RESULTS: Taurochenodeoxycholic acid and chenodeoxycholc acid caused cholestasis and liver damage associated with a decreased bile flow, total and individual bile acids secretion accompanied by a biliary leakage of lactate dehydrogenase and alkaline phosphatase enzymes. Tauroursodeoxycholic acid, glycine ursodeoxycholic acid, ursodeoxycholic acid and taurocholic acid, on the contrary, were choleretic, inducing an opposite effect on biliary parameters. Simultaneous infusion of taurochenodeoxycholic acid and the protective bile acid resulted in a functional and morphological improvement of the above parameters in the following order: glycine ursodeoxycholic acid > tauroursodeoxycholic acid > ursodeoxycholic acid followed by taurocholic acid; cholic acid was ineffective. CONCLUSIONS: The results show the protective effect of glycine ursodeoxycholic acid, ursodeoxycholic acid and tauroursodeoxycholic acid. This may be due to a facilitated transport of the toxic bile acid into bile; conjugation with taurine is less effective than glycine. Finally, the better protective effect of ursodeoxycholic acid and its amidates with respect to cholic acid and its taurine conjugated form seems to be related to their different lipophilicity and micellar forming capacity.

Systemic antibiotic therapy on multiple intracerebral abscesses of unknown origin.

A 68-year old patient presented with increasing right-sided weakness, dysarthria, pyrexia and a deteriorating general condition. CT and MRI showed about 20 round hyperdense lesions with peripheral enhancement of contrast material up to 1.5 cm in diameter. Antibiotic triple-therapy using ceftriaxone, gentamycin and metronidazole for 39 days and followed by antibiotic double-therapy using ceftriaxone and metronidazole for a further 22 days resulted in a radiologically proven reduction of the abscesses to very small remnants which disappeared completely during the follow-up period of two years. Simultaneously the general condition of the patient improved significantly. Repeated attempts at isolation of bacteria, fungi, protozoa and parasites from a subdural empyema failed. The histological examination of an abscess, which was entirely removed on occipital craniotomy, showed a structure resembling actinomycosis.

New insights in the physiology and molecular basis of the intestinal bile acid absorption.

Intestinal bile acid absorption is a fundamental step in the enterohepatic circulation and metabolism of these endogenous compounds. The physiology of the active, sodium coupled transport system for bile acids in the terminal ileum has been extensively studied and characterized. Structure-activity studies have elucidated the requirements for the ileal transport system, and studies with photolabile bile acid derivatives identified the putative ileal bile acid transport proteins. Characterization of the functional sites of the transport system elucidated some of the possible mechanisms which allow the interaction of bile acids and sodium ions with the ileal transporter. Considerable progress has been made during recent years, after the ileal apical and cytosolic bile acid transport proteins have been cloned and characterized. The role of point mutations in bile acid malabsorption has been studied, and the knowledge of the amino acid sequence of the transport proteins will be of help in the investigation of the transport mechanisms.

Relationship between structure and intestinal absorption of bile acids with a steroid or side-chain modification.

UNLABELLED: A structure-activity relationship for bile acid (BA) intestinal absorption is known to exist. To better understand the BA structural requirements for optimal BA intestinal absorption, rabbit ileal perfusion studies were performed. Unconjugated BA: Ursodeoxycholic (UDCA) and ursocholic acid (UCA) with methyl (6MUDCA and 6MUCA) or fluoro group (6FUDCA and 6FUCA) in the 6 position and UCA with a methyl group in 23 position (23MUCA) were compared with unconjugated UDCA, UCA, deoxycholic (DCA), chenodeoxycholic (CDCA), hyocholic (HCA), and hyodeoxycholic (HDCA) acid. BA lipophilicity was evaluated by their octanol-water partition coefficient. Conjugated BA: Taurine-conjugated UDCA and UCA with a methyl group in the 23 position (T23MUDCA and T23MUCA) were compared with the corresponding taurine-conjugated natural analogs. An analog of glycine-conjugated UDCA with the C24 amide bond replaced by a -CO-CH2- in the 24 position (24PUDCA) was studied and results were compared with the natural form (GUDCA). Unconjugated BA absorption was dose dependent (i.e., passive) and followed their lipophilicity: DCA > 6MUDCA > CDCA > HDCA > UDCA > HCA > 6FUDCA > 6MUCA > 6FUCA > UCA. Conjugated BA absorption was active, and Vmax was in the following order: TCA > TUDCA > TUCA > T23MUCA > T23MUDCA > 24PUDCA > GUDCA. 24PUDCA transport was also active and higher than GUDCA. CONCLUSION: Passive transport is dependent on BA lipophilicity. Conjugated BAs are actively transported, and the presence of a 23-C methyl group does not improve transport when compared with the natural analogs. The substitution of the C24 amide bond with a -CO-CH2-still affords interaction of the BA with the intestinal transport carrier.

Uptake and killing of Lyme disease and relapsing fever borreliae in the perfused rat liver and by isolated Kupffer cells.

In situ-perfused rat livers were infused with a single dose of 1.5 x 10(7) radiolabeled borreliae. Significant (P < 0.00005) differences in the liver uptake of the agents of Lyme borreliosis, Borrelia burgdorferi IRS, Borrelia afzelii VS461, and Borrelia garinii PBi, and that of the agents of relapsing fever, Borrelia hermsii, Borrelia parkeri, and Borrelia turicatae, were observed. The liver uptakes ranged between 65.9% for B. burgdorferi IRS and 40.5% for B. turicatae. Neither relapsing fever nor Lyme disease borreliae were recovered from infected livers when the livers were cultured in Barbour-Stoenner-Kelly II medium. The in vitro uptake of B. burgdorferi IRS by isolated rat Kupffer cells was rapid, and within 30 min of the infection, large intracellular aggregates of amorphous material were detectable by immunofluorescence with specific anti-B. burgdorferi antibody. The reculturing of B. burgdorferi IRS from Kupffer cells incubated for 24 h in RPMI medium before inoculation with bacteria was negative. The results obtained in this study indicated that borreliae are efficiently taken up and killed by rat hepatic macrophages in the absence of serum factors.

Species differences in hepatic bile acid uptake: comparative evaluation of taurocholate and tauroursodeoxycholate extraction in rat and rabbit.

Dose-response curves for taurocholate and tauroursodeoxycholate were performed in rat and rabbit livers to get more insight into species differences in the hepatic bile acid uptake. The bile acids showed saturation kinetics in both animals, the Vmax in rat being higher than in rabbit and the Km being lower in the rat than in the rabbit for both the bile acids, with no significant difference in the hepatic cells morphometric parameters. Therefore, it is possible that differences in the kinetic parameters are related to the number and, to a lesser extent, to the affinity of the transporters on the sinusoidal plasma membranes.

Intestinal absorption of bile acids in the rabbit: different transport rates in jejunum and ileum.

BACKGROUND & AIMS: A direct comparison of jejunal and ileal absorption rates of bile acids has not been reported. The aim of this study was to compare the relative transport rates of different bile acids in the jejunum and ileum. METHODS: Jejunal and ileal rabbit intestinal segments were separately perfused with bile acid solutions, and dose-response curves were obtained for taurocholate, ursodeoxycholate, chenodeoxycholate, deoxycholate, and their glycoconjugates. Membrane fluidity and bile acid transport were assessed in brush border membrane vesicles. RESULTS: Taurocholate showed active transport in the ileum and no transport in the jejunum. Unconjugated bile acids showed passive diffusion in the two tracts, whereas glycoconjugated bile acids showed both components of transport in the ileum and passive diffusion in the jejunum (lower in the latter). A higher membrane fluidity and lower cholesterol-to-phospholipid ratio were found in the jejunum. Ursodeoxycholate reduced bile acid uptake into membrane vesicles from both ileum and jejunum. CONCLUSIONS: Active transport is limited to the ileum. Passive diffusion is higher through a less fluid membrane with a higher cholesterol-to-phospholipid ratio in the ileum than in the jejunum. Ursodeoxycholate inhibition may be at the level of a facilitated, sodium-independent diffusion in the jejunum.

Bile acid structure and intestinal absorption in the animal model.

A close structure-activity relationship exists between the transport of bile acids (BA) in the liver and intestine; hepatic and intestinal BA transport can be evaluated and compared by using perfused liver and perfused intestine in the rabbit. The passive intestinal absorption is limited to the unconjugated BA, which occurs throughout the small bowel and colon, and is conditioned by the apical membrane lipid composition. A higher diffusion component is found in the terminal ileum compared to the jejunum, and seems to be related to the higher cholesterol-to-phospholipid ratio of the ileal brush border membranes. The active transport system is well characterized and the brush border membrane receptor, cytosolic BA binding proteins and basolateral anion exchange protein have been identified. Recently, the ileal BA transporter has been cloned from the hamster and human ileum and the main cytosolic BA binding protein was cloned from the rat ileum. In the liver, the active transport predominates on the passive diffusion both for conjugated and unconjugated BA. The maximal transport capacity in the liver is tenfold higher than in the intestine, while the Km values are of the same order of magnitude, i.e. in the millimolar range. Neither system operates at its maximum transport rate with prevalent concentrations of BA in portal blood or luminal content.

Hepatic uptake and intestinal absorption of bile acids in the rabbit.

The existence of transporters for bile acids (BA) in liver and intestine has been well documented, but information is still needed as to their respective transport capacity. In the present investigation, we compared the hepatic and intestinal transport rates for BA, using perfused livers and intestines. The livers and intestines were separately perfused and dose-response curves (0.25-10 mM) for tauroursodeoxycholate, taurocholate and taurodeoxycholate were obtained. The intestinal and mesenteric concentration and bile acid pattern were also evaluated in six non-fasting rabbits. Taurocholic, tauroursodeoxycholic and taurodeoxycholic acid ileal absorption showed saturation kinetics in the intestine as in the liver; the maximal uptake velocity for each bile acid in the liver was tenfold higher than the respective maximal transport velocity in the intestine; the Km values obtained in the liver were of the same order of magnitude, i.e. in the millimolar range. Taurocholic, tauroursodeoxycholic and taurodeoxycholic acid transport differences in the liver paralleled those in the intestine. Although the intestine was not homogeneously filled, the bile acid concentration in the ileal content fell into the range of the Km for the three studied bile acids, while the portal blood total bile acid concentration was inferior to the observed Kms of liver uptake. Therefore, both the hepatic and intestinal systems do not operate at their maximal transport rates at the prevailing concentrations in portal blood and luminal content, and the hepatic transport occurs at its highest efficiency (below the Km values) in physiological conditions.

Ursodeoxycholic acid administration on bile acid metabolism in patients with early stages of primary biliary cirrhosis.

Ursodeoxycholic acid has been proposed for the treatment of primary biliary cirrhosis. The aim of this study was to evaluate the effect of ursodeoxycholic acid administration on bile acid metabolism in patients with early-stage primary biliary cirrhosis. Biliary bile acid composition, primary bile acid pool sizes, synthesis, and fractional turnover rate were measured before and after four weeks of ursodeoxycholic acid administration (600 mg/day) in nine patients with biopsy-proven primary biliary cirrhosis (stages I-III). Molar percentages of chenodeoxycholic, cholic, and deoxycholic acids in bile were significantly decreased by ursodeoxycholic acid administration, while its biliary concentration increased to 34.2% at the end of the same four-week period. The cholic and chenodeoxycholic acid pools decreased, although not significantly, while the deoxycholic acid pool was reduced by 60% (from 0.7 +/- 0.12 to 0.29 +/- 0.07 mmol, P < 0.002). Primary bile acid synthesis was slightly increased, and fractional turnover rate was significantly increased. The conversion rate of cholic to deoxycholic acid was measured and found to be significantly increased (P < 0.05) after ursodeoxycholic acid administration; however, serum levels of both free and conjugated deoxycholic acid were significantly decreased (from 23.2 +/- 9.7 to 3.8 +/- 1.9 mumol/liter, P < 0.001). We conclude that in patients with primary biliary cirrhosis, ursodeoxycholic acid administration replaces endogenous bile acids in the enterohepatic circulation by increasing bile acid fractional turnover rate without significant increments of their hepatic synthesis.

Treatment of hepatic encephalopathy with non-absorbable antibiotics.

Hepatic encephalopathy represents a well known neuropsychiatric syndrome in patients with either acute or chronic impaired liver function and is characterized by disturbance of consciousness, personality and intellectual capacity, altered neuromuscular activity and electroencephalographic abnormalities. The pathogenesis of the syndrome is still unknown, although important roles are ascribed to circulating gut-derived toxins of nitrogenous origin and to changing in central neurotransmission. Therefore, treatment is aimed to reduce the production and absorption of gut-derived toxins and to modify central neurotransmission balance. Among the different therapeutic approaches proposed for the management of hepatic encephalopathy, antimicrobial agents, alone or in combination with non-absorbable disaccharides, represent an important step, being able to reduce the production and absorption of ammonia, a compound of key importance in the pathogenesis of hepatic encephalopathy.

Bile acid active and passive ileal transport in the rabbit: effect of luminal stirring.

The intestinal absorption of bile acids (BA) with different chemical structure has been evaluated in the rabbit, after intestinal infusion of different concentrations (0.25-30 mM) of BA, by mesenteric blood sampling. Cholic (CA), chenodeoxycholic (CDCA), ursodeoxycholic (UDCA) acid, free and taurine (T-) conjugated, together with glycocholic (GCA) acid and deoxycholic acid (DCA) were studied. The apparent uptake parameters were calculated. All conjugated BA showed active transport (T max, nmol min-1 cm-1 int.), with Tmax values in the following order: TCA > TUDCA > TCDCA; unconjugated BA showed passive uptake, with values in the following order: DCA > CDCA > UDCA > CA. GCA and CA showed both passive uptake and active transport. For all BA studied the % uptake in the ileal segment considered was less than 10%, BA uptake being thus limited by transport and/or diffusion kinetics, rather than by flow velocity. The liquid resistance to BA radial diffusion inside the lumen was evaluated, and the infusate-to-blood uptake parameters corrected for it, in order to get the uptake parameters from the epithelium-to-liquid interface to mesenteric blood: the apparent Km decreased, passive uptake coefficient increased, while Tmax was unchanged. The passive component of the uptake, corrected for the luminal resistance, correlated with the BA hydrophobicity (r = 0.963; P < 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of simvastatin, ursodeoxycholic acid and simvastatin plus ursodeoxycholic acid on biliary lipid secretion and cholic acid kinetics in nonfamilial hypercholesterolemia.

It has been recently shown that the newest hypocholesterolemic agent, simvastatin, lowers the biliary cholesterol saturation index and that its association with ursodeoxycholic acid renders it more effective. To determine the mechanism by which simvastatin decreases the biliary cholesterol saturation index, we evaluated hepatic secretion rates of cholesterol, bile acids and phospholipids, and cholic acid pool size, turnover and synthesis in eight hyperlipidemic patients (five women and three men, age range = 38 to 65 yr). These assessments were conducted before treatment, after 4 wk of simvastatin (40 mg/day), after 4 wk of ursodeoxycholic acid (600 mg/day) and after a further 4 wk of a combination therapy of simvastatin (40 mg/day) plus ursodeoxycholic acid (600 mg/day). The cholesterol saturation index was significantly reduced with simvastatin (from 1.51 +/- 0.10 to 0.94 +/- 0.05, mean +/- S.E.; p less than 0.02), with ursodeoxycholic acid (from 1.51 +/- 0.10 to 0.86 +/- 0.03, mean +/- S.E.; p less than 0.02) and with the combination of simvastatin plus ursodeoxycholic acid (from 1.51 +/- 0.01 to 0.70 +/- 0.05, p less than 0.02). The cholesterol saturation index during combination therapy was significantly lower (p less than 0.02) than that reached during the use of simvastatin and ursodeoxycholic acid. Both simvastatin and ursodeoxycholic acid significantly reduced the hepatic secretion rate of cholesterol (from 130 +/- 14 mumols/hr to 81 +/- 12 mumols/hr, p less than 0.01, and 70 +/- 9 mumols/hr, p less than 0.01) without affecting bile acid and phospholipid outputs.(ABSTRACT TRUNCATED AT 250 WORDS)

Pathophysiology and pharmacotherapy of cholelithiasis.

Several factors are involved in the development of gallstone formation: formation of supersaturated bile; nucleation; formation, retention and adhesion of cholesterol crystals and eventually stone growth. The dynamics of the gallbladder may play a key role in the overall process. The pathophysiologic theory of cholesterol gallstone formation and the knowledge of their physico-chemical properties support the modern concept of gallstone therapy. Chenodeoxycholic and ursodeoxycholic have been widely used as cholesterol gallstone dissolving agents and evaluated in terms of efficacy and safety.

Hepatic uptake and biliary secretion of bile acids in the perfused rat liver.

Hepatic uptake and biliary secretion have been evaluated in the isolated perfused rat liver for cholic, chenodeoxycholic, ursodeoxycholic acid, both free and taurine-conjugated; the physicochemical properties of the bile acids have also been calculated and related to these experimental parameters. Cholic acid disappearance rate from the perfusate was the fastest, followed by that of ursodeoxycholic and chenodeoxycholic; it was also faster for taurine-conjugated bile acids than for their respective unconjugated forms. The recovery in bile was higher for conjugated than for unconjugated bile acids, and among each class, was higher for cholic than for chenodeoxycholic and ursodeoxycholic. The hepatic uptake correlated negatively (r = -0.99) with the bile acid lipophilicity, while the biliary secretion correlated with the solubility of the molecules. These results show the effect of the physicochemical properties of BA on their hepatic handling, at the physiological concentration of BA in the portal blood.