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.

Apolipoprotein A-V is present in bile and its secretion increases with lipid absorption in Sprague-Dawley rats.

Apolipoprotein (apo) A-V is a protein synthesized only in the liver that dramatically modulates plasma triglyceride levels. Recent studies suggest a novel role for hepatic apoA-V in regulating the absorption of dietary triglycerides, but its mode of action on the gut remains unknown. The aim of this study was to test for apoA-V in bile and to determine whether its secretion is regulated by dietary lipids. After an overnight recovery, adult male Sprague-Dawley bile fistula rats indeed secreted apoA-V into bile at a constant rate under fasting conditions. An intraduodenal bolus of intralipid (n = 12) increased the biliary secretion of apoA-V but not of other apolipoproteins, such as A-I, A-IV, B, and E. The lipid-induced increase of biliary apoA-V was abolished under conditions of poor lymphatic lipid transport, suggesting that the stimulation is regulated by the magnitude of lipids associated with chylomicrons transported into lymph. We also studied the secretion of apoA-V into bile immediately following bile duct cannulation. Biliary apoA-V increased over time (∼6-fold increase at hour 16, n = 8) but the secretions of other apolipoproteins remained constant. Replenishing luminal phosphatidylcholine and taurocholate (n = 9) only enhanced apoA-V secretion in bile, suggesting that the increase was not due to depletion of phospholipids or bile salts. This is the first study to demonstrate that apoA-V is secreted into bile, introducing a potential route of delivery of hepatic apoA-V to the gut lumen. Our study also reveals the uniqueness of apoA-V secretion into bile that is regulated by mechanisms different from other apolipoproteins.

Conjugated bile acids activate the sphingosine-1-phosphate receptor 2 in primary rodent hepatocytes.

Bile acids have been shown to be important regulatory molecules for cells in the liver and gastrointestinal tract. They can activate various cell signaling pathways including extracellular regulated kinase (ERK)1/2 and protein kinase B (AKT) as well as the G-protein-coupled receptor (GPCR) membrane-type bile acid receptor (TGR5/M-BAR). Activation of the ERK1/2 and AKT signaling pathways by conjugated bile acids has been reported to be sensitive to pertussis toxin (PTX) and dominant-negative Gα(i) in primary rodent hepatocytes. However, the GPCRs responsible for activation of these pathways have not been identified. Screening GPCRs in the lipid-activated phylogenetic family (expressed in HEK293 cells) identified sphingosine-1-phosphate receptor 2 (S1P(2) ) as being activated by taurocholate (TCA). TCA, taurodeoxycholic acid (TDCA), tauroursodeoxycholic acid (TUDCA), glycocholic acid (GCA), glycodeoxycholic acid (GDCA), and S1P-induced activation of ERK1/2 and AKT were significantly inhibited by JTE-013, a S1P(2) antagonist, in primary rat hepatocytes. JTE-013 significantly inhibited hepatic ERK1/2 and AKT activation as well as short heterodimeric partner (SHP) mRNA induction by TCA in the chronic bile fistula rat. Knockdown of the expression of S1P(2) by a recombinant lentivirus encoding S1P(2) shRNA markedly inhibited the activation of ERK1/2 and AKT by TCA and S1P in rat primary hepatocytes. Primary hepatocytes prepared from S1P(2) knock out (S1P(2) (-/-) ) mice were significantly blunted in the activation of the ERK1/2 and AKT pathways by TCA. Structural modeling of the S1P receptors indicated that only S1P(2) can accommodate TCA binding. In summary, all these data support the hypothesis that conjugated bile acids activate the ERK1/2 and AKT signaling pathways primarily through S1P(2) in primary rodent hepatocytes.

The novel formulation design of self-emulsifying drug delivery systems (SEDDS) type O/W microemulsion II: stable gastrointestinal absorption of a poorly water soluble new compound, ER-1258 in bile-fistula rats.

The stabilization effect of the novel self-emulsifying drug delivery systems (SEDDS) type O/W microemulsion on the gastrointestinal absorption of a poorly water soluble new compound, ER-1258 was examined by bile-fistula model rats. In the components of this formulation, medium chain fatty acid triglyceride (MCT), diglyceryl monooleate (DGMO-C), polyoxyethylene hydrogenated castor oil 40 (HCO-40) and ethanol were used as an oil, a lipophilic surfactant, a hydrophilic surfactant and a solubilizer at the mixture ratio of 25/5/45/25 w/w%, respectively. The ratios of AUC in the non-treated rats to that in the bile-fistula rats were 5.1, 12.1 and 3.0 for the suspension, the oily solution and the SEDDS type O/W microemulsion, respectively. The risk from which the difference between individuals of the compound absorption amounts resulting from the flow of the bile secretion serves as the maximum was high in order of oily solution>suspension>SEDDS type O/W microemulsion. Therefore, it was verified that the SEDDS type O/W microemulsion was able to reduce this risk, compared with the other formulations. When short chain fatty acid triglyceride (Triacetin) was used as an oil, the similar effect was demonstrated in the formulation composed of sorbitan sesquioleate (SO-15) as a lipophilic surfactant and polyoxyethylene hydrogenated castor oil 60 (HCO-60) or polyoxyethylene 20 sorbitan monooleate (TO-10M) as a hydrophilic surfactant.

Conjugation with taurine prevents side-chain desaturation of ursodeoxycholic and beta-muricholic acids in bile fistula rats.

The metabolism of intravenously infused bile salts, tauroursodeoxycholate, tauro-beta-muricholate and their corresponding unconjugated forms in the liver was investigated in bile salt-depleted bile fistula rats. The biliary bile salt composition was determined by gas chromatography-mass spectrometry using chemical positive ionization and electron-impact methods. For an infusion rate of 2 micromol/min/kg, all bile salts were efficiently secreted in bile, inducing similar choleresis. Only tauroconjugated bile salts were recovered; no glucuronide or glyco derivatives were detected. The infusion of free ursodeoxycholate led to the appearance of a metabolite identified as a Delta22 derivative (12%). A similar biotransformation rate (11%) was observed following free beta-muricholate infusion. In contrast, no metabolite was observed after infusion of the tauroconjugated form of ursodeoxycholate and beta-muricholate. The unsaturation process probably depends on the availability of the carboxyl group for the starting step of the beta-oxidation mechanism. In conclusion, the current in vivo study demonstrates a hepatic origin for Delta22 bile salts. It also shows that free bile salts were sensitive to Delta22 formation while conjugation with taurine totally prevented the side-chain oxidation of the two 7beta-hydroxylated bile salts.

Is fenestration a safe treatment for adult polycystic liver disease?: A report of refractory complications.

We report a 67-year-old man with highly symptomatic polycystic liver disease. Fenestration was selected to treat symptoms because the cysts were scattered diffusely and the normal liver volume was limited. Although this patient was relieved from symptoms of liver cysts consequently, several severe postoperative complications including disseminated intravascular coagulation, respiratory failure, liver failure, and biliary leakage occurred resulting in a 6-month postoperative hospital stay. Although various treatments for symptomatic adult polycystic liver disease have been advocated, a definitive treatment remains controversial, especially in diffuse adult polycystic liver disease. Fenestration is one of the alternative treatments for the patients whose cysts are difficult to resect. However high morbidity rate should be carefully assessed, if extensive fenestration is needed to treat diffuse adult polycystic liver disease. Further consideration of appropriate treatments for diffuse adult polycystic liver disease is needed.

Sirolimus/cyclosporine/tacrolimus interactions on bile flow and biliary excretion of immunosuppressants in a subchronic bile fistula rat model.

The new immunosuppressive agent sirolimus generally is combined in transplant patients with cyclosporine and tacrolimus which both exhibit cholestatic effects. Nothing is known about possible cholestatic effects of these combinations which might be important for biliary excretion of endogenous compounds as well as of immunosuppressants. Rats were daily treated with sirolimus (1 mg kg(-1) p.o.), cyclosporine (10 mg kg(-1) i.p.), tacrolimus (1 mg kg(-1) i.p.), or a combination of sirolimus with cyclosporine or tacrolimus. After 14 days a bile fistula was installed to investigate the effects of the immunosuppressants and their combinations on bile flow and on biliary excretion of bile salts, cholesterol, and immunosuppressants. Cyclosporine as well as tacrolimus reduced bile flow (-22%; -18%), biliary excretion of bile salts (-15%;-36%) and cholesterol (-15%; -47%). Sirolimus decreased bile flow by 10%, but had no effect on cholesterol or bile salt excretion. Combination of sirolimus/cyclosporine decreased bile flow and biliary bile salt excretion to the same extent as cyclosporine alone, but led to a 2 fold increase of biliary cholesterol excretion. Combination of sirolimus/tacrolimus reduced bile flow only by 7.5% and did not change biliary bile salt and cholesterol excretion. Sirolimus enhanced blood concentrations of cyclosporine (+40%) and tacrolimus (+57%). Sirolimus blood concentration was increased by cyclosporine (+400%), but was not affected by tacrolimus. We conclude that a combination of sirolimus/tacrolimus could be the better alternative to the cotreatment of sirolimus/cyclosporine in cholestatic patients and in those facing difficulties in reaching therapeutic ranges of sirolimus blood concentration.

Regulation of hepatic connexins in cholestasis: possible involvement of Kupffer cells and inflammatory mediators.

Hepatocyte gap junction proteins, connexins (Cxs) 26 and 32, are downregulated during obstructive cholestasis (OC) and lipopolysaccharide hepatocellular cholestasis (LPS-HC). We investigated rat hepatic Cxs during ethynylestradiol hepatocellular cholestasis (EE-HC) and choledochocaval fistula (CCF) and compared them with OC and LPS-HC. Levels (immunoblotting) and cellular distribution (immunofluorescence) of Cx26, -32, and -43, as well as macrophage infiltration, were studied in livers of rats under each condition. Cx26 and -32 were reduced in LPS-HC, OC, and CCF. However, in EE-HC, Cx26 did not change and Cx32 was increased. Prominent inflammation occurred in LPS-HC, OC, and CCF, which was associated with increased levels of Cx43 in LPS-HC and OC but not CCF. No inflammation nor changes in Cx43 levels occurred during EE-HC. In cultured hepatocytes, dye coupling was reduced by tumor necrosis factor-alpha and interleukins-1beta and -6, whereas reduction induced by LPS required coculture with Kupffer cells. Thus hepatocyte gap junctions are downregulated in forms of cholestasis associated with inflammation, and reduced intercellular communication might be induced in part by proinflammatory mediators.

Zinc salts precipitate unconjugated bilirubin in vitro and inhibit enterohepatic cycling of bilirubin in hamsters.

BACKGROUND: We have evidence for enterohepatic cycling of bilirubin experimentally and in vivo in humans. This study was designed to investigate whether Zn salts might inhibit such cycling of bilirubin. MATERIALS AND METHODS: Micellar bile salt solutions with unconjugated bilirubin were prepared, appropriate concentrations of Zn salts were added, and unconjugated bilirubin precipitation was measured. Hamsters and Wistar rats were fed a chow diet or a chow diet enriched with 1% ZnSO4, and bilirubin secretion rates were monitored. RESULTS: Unconjugated bilirubin was precipitated maximally (90%) after a 10-min incubation with 5 mM Zn salts in the pH range of 6.8-9.0. In control hamsters, total bilirubin secretion rates into bile were 36.0 +/- 2.8 nmol h(-1) 100g(-1) body weight, whereas they were 25.0 +/- 3.3 nmol h-1 100(-1) g in the ZnSO4 group (P < 0.05). CONCLUSIONS: Zn salts that flocculate at physiological pH adsorb unconjugated bilirubin almost completely from unsaturated micellar BS solutions. In addition, Zn salts administered orally suppress biliary bilirubin secretion rates in hamsters. These findings suggest that the administration of Zn salts may inhibit the enterohepatic cycling of unconjugated bilirubin in humans who are predisposed to pigment gallstone formation due to diet, disease or drugs.

Sulindac is excreted into bile by a canalicular bile salt pump and undergoes a cholehepatic circulation in rats.

BACKGROUND & AIMS: Dihydroxy bile acids induce a bicarbonate-rich hypercholeresis when secreted into canalicular bile in unconjugated form; the mechanism is cholehepatic shunting. The aim of this study was to identify a xenobiotic that induces hypercholeresis by a similar mechanism. METHODS: Five organic acids (sulindac, ibuprofen, ketoprofen, diclofenac, and norfloxacin) were infused into rats with biliary fistulas. Biliary recovery, bile flow, and biliary bicarbonate were analyzed. Sulindac transport was further characterized using Tr(-) rats (deficient in mrp2, a canalicular transporter for organic anions), the isolated perfused rat liver, and hepatocyte membrane fractions. RESULTS: In biliary fistula rats, sulindac was recovered in bile in unconjugated form and induced hypercholeresis of canalicular origin. Other compounds underwent glucuronidation and were not hypercholeretic. In the isolated liver, sulindac had delayed biliary recovery and induced prolonged choleresis, consistent with a cholehepatic circulation. Sulindac was secreted normally in Tr(-) rats, indicating that its canalicular transport did not require mrp2. In the perfused liver, sulindac inhibited cholyltaurine uptake, and when coinfused with cholyltaurine, induced acute cholestasis. With both basolateral and canalicular membrane fractions, sulindac inhibited cholyltaurine transport competitively. CONCLUSIONS: Sulindac is secreted into bile in unconjugated form by a canalicular bile acid transporter and is absorbed by cholangiocytes, inducing hypercholeresis. At high flux rates, sulindac competitively inhibits canalicular bile salt transport; such inhibition may contribute to the propensity of sulindac to induce cholestasis in patients.

Quantitative estimations of the contribution of different bile acid pathways to total bile acid synthesis in the rat.

BACKGROUND & AIMS: Cholesterol degradation to bile acids occurs via "classic" or "alternative" bile acid biosynthetic pathways. The aim of this study was to assess the contributions of these two pathways to total bile acid synthesis in vivo. METHODS: Rats with biliary fistulas were infused with squalestatin for 24 and 48 hours; specific activities of cholesterol 7 alpha-hydroxylase (C7 alpha H) and sterol 27-hydroxylase (S27H) and rates of bile acid synthesis were determined. RESULTS: Continuous squalestatin infusion (15 micrograms/h) decreased C7 alpha H specific activities to 4% and 12% of paired biliary fistula controls at 24 and 48 hours, respectively (P < 0.05) without any changes in S27H specific activities (82% and 95% of controls). At 24 hours, bile acid synthesis decreased to 43% (P < 0.05) but returned to 87% at 48 hours (P = NS). Cholic acid synthesis decreased at 24 hours but returned to control levels at 48 hours. Similar changes in C7 alpha H, S27H, and bile acid synthesis were observed in primary rat hepatocytes after addition of squalestatin (1.0 mumol/L). CONCLUSIONS: In the face of persistent suppression of C7 alpha H and the classic pathway, an alternative pathway becomes a main pathway of bile acid synthesis capable of generating cholic and chenodeoxycholic acids. The observed induction of bile acid synthesis via an alternative pathway or pathways represents an important mechanism for maintenance of cholesterol homeostasis in the rat.

Effect of side chain length on biotransformation, hepatic transport, and choleretic properties of chenodeoxycholyl homologues in the rodent: studies with dinorchenodeoxycholic acid, norchenodeoxycholic acid, and chenodeoxycholic acid.

To assess the effect of side chain length on the metabolism and physiological effects of homologues of chenodeoxycholic acid (CDCA), dinorCDCA, the C22 homologue, was synthesized and its hepatic biotransformation, transport kinetics, and choleretic properties were defined in rat and hamster biliary fistula and in isolated perfused rat liver. Results were compared with those of norCDCA, the C23 homologue, and of CDCA, the natural C24 homologue. In the rat, dinorCDCA was secreted mostly in unconjugated form (the majority as dinor-alpha-muricholic acid); the remainder was glucuronidated. In the hamster, glucuronidation was greater, and the unconjugated fraction contained equal parts of dinorCDCA and 5beta-hydroxy-dinorCDCA. NorCDCA was glucuronidated extensively (70%, rat; 40%, hamster). CDCA, in contrast, was efficiently amidated with taurine or glycine. In the perfused liver, the initial uptake rate of all three homologues was identical; later, regurgitation and/or cholehepatic shunting of dinorCDCA and norCDCA, but not of CDCA, occurred. In rats and hamsters with biliary fistulas, dinorCDCA and norCDCA, but not CDCA, induced a bicarbonate-rich hypercholeresis of canalicular origin. Hypercholeresis was not induced by the taurine conjugate of dinorCDCA. Hepatobiliary retention of both dinorCDCA and norCDCA occurred, consistent with efficient ductular absorption (calculated to be 94%) and cholehepatic cycling of the unmetabolized bile acids. It is concluded that dinorCDCA, as norCDCA, is inefficiently amidated, is metabolized as a xenobiotic, and induces hypercholeresis. DinorCDCA is the first dihydroxy bile acid to be identified that is secreted largely in unconjugated form in bile.

Expression of the rat liver Na+/taurocholate cotransporter is regulated in vivo by retention of biliary constituents but not their depletion.

Expression and function of the hepatic Na+/taurocholate cotransporter (ntcp) are down-regulated in several models of experimental cholestasis. To test whether retention and/or depletion of biliary constituents are involved in ntcp regulation, ntcp expression was quantified in several animal models with altered levels of these constituents. In choledochocaval fistula rats (CCF) (retention model), ntcp mRNA expression specifically declined after 1 and 3 days by 76 +/- 4% (P < .005) and 31 +/- 9% (P < .05), respectively, returning to control levels by 7 days. However, protein expression as assessed by Western blotting remained unchanged for up to 7 days of CCF. In rats with bile fistulas (depletion model) for 0.5, 1, 2, 4, and 7 days, both ntcp protein and mRNA expression remained unaltered. Infusion of either taurocholate or taurochenodeoxycholate for 12 hours also did not effect ntcp mRNA expression in intact animals, probably because of its inability to increase serum and intrahepatic bile acid levels. In rats with selective bile duct ligation (SBDL), ntcp mRNA levels were down-regulated by 40 +/- 10% (P < .05) only after 12 and 24 hours in ligated lobes, and mRNA levels returned to control values in these lobes after 2 and 4 days. ntcp mRNA expression remained unchanged in the nonobstructed lobes at any time. When data from CCF and SBDL rats were combined, serum bile acids correlated linearly with ntcp mRNA (r = .62, P < .0005) over a 0 to 110-micromol/L range. Our results indicate that ntcp is constitutively expressed and remains uneffected by either depletion or increased flux of biliary constituents. However, retention of biliary constituents results in rapid down-regulation of ntcp mRNA, consistent with the concept that hepatocytes may be protected from bile acid toxicity during cholestasis by this mechanism.

Disposition of [3H]fluvastatin following single oral doses in beagle dogs and rhesus monkeys with bile fistulae.

The disposition of [3H]fluvastatin was examined following single oral doses in dogs (12.4 mg kg-1) and monkeys (0.48 and 45.5 mg kg-1) with bile fistulae. Serial plasma and complete urine, feces, and bile were collected at designated intervals for 3 or 4 d, and were analyzed for total radioactivity and unchanged fluvastatin. In the dog, peak radioactivity concentrations (Cmax) averaged 7260 ng equiv. mL-1 and the mean time to peak (tmax) was approximately 9 h. In the monkey, the mean radioactivity tmax values were approximately 5 and 13 h following the low and high doses, the respective Cmax values being 116 and 10,400 ng equiv. mL-1. The mean AUC of total radioactivity was proportional to the dose while that of fluvastatin was overproportional to dose, suggesting dose independent absorption but saturable first-pass effect. The AUC ratio of unchanged fluvastatin versus total radioactivity was approximately 63% in the dog, and 9% and 13% for the low and high doses, respectively in the monkey. The bile was the major excretory route of radioactivity (dog, 56%; low-dose monkey, 73%; high-dose monkey, 69%) whereas the renal pathway accounted for < 5% of the dose in both species. Approximately 12% of the biliary radioactivity in the dog was due to intact fluvastatin, compared with 0% and 7.5% after the low and high doses in the monkey. These results showed a smaller extent of fluvastatin metabolism in the dog than in the monkey, and suggested that metabolism in the monkey was saturable in the dose range studied.

Failure of intravenous infusion of taurocholate to down-regulate cholesterol 7 alpha-hydroxylase in rats with biliary fistulas.

BACKGROUND/AIMS: The decrease in cholesterol 7 alpha-hydroxylase induced by intraduodenal infusion of taurocholate in bile fistula rats may be indirect, i.e., mediated through release or absorption of an intestinal factor in response to the presence of bile salts in the intestine. The aim of this study was to determine if negative feedback regulation of cholesterol 7 alpha-hydroxylase can be shown when equimolar concentrations of taurocholate are administered intravenously, thus bypassing the intestine. METHODS: After 96 hours of biliary diversion, taurocholate (36 mumol.h-1.100 g, rat-1) was infused into the rats either intravenously or intraduodenally for the final 24 hours. Livers were then harvested for analysis of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase specific activity, cholesterol 7 alpha-hydroxylase specific activity, messenger RNA levels, and transcriptional activity. RESULTS: Intraduodenally administered taurocholate significantly decreased HMG-CoA reductase and cholesterol 7 alpha-hydroxylase specific activity by more than 50% and cholesterol 7 alpha-hydroxylase steady-state messenger RNA levels and transcriptional activity by 50%-75%. In contrast, intravenous administration of taurocholate failed to down-regulate either cholesterol 7 alpha-hydroxylase or HMG-CoA reductase. CONCLUSIONS: Passage of taurocholate through the intestine strongly potentiates negative feedback regulation of cholesterol 7 alpha-hydroxylase. A putative intestinal factor, released or absorbed in the presence of bile acids in the intestinal lumen, may play a role in the regulation of bile acid synthesis.

Effect of glutathione administration on hepatic biliary and plasmatic glutathione levels in the rat.

BACKGROUND: Since the effect of exogenous glutathione (GSH) on overall hepatic GSH homeostasis is not known, the present study investigated the changes in the hepatic, biliary, and plasmatic GSH levels during GSH administration in intact rats. METHODS: An exteriorized biliary-duodenal fistula was established, and GSH (1 mmol/kg over 2 h) or saline was administered intraperitoneally to rats with or without pretreatment with 5 mmol/kg L-serine borate, an inhibitor of gamma-glutamyltransferase (GGT). RESULTS: Three hours after GSH administration, biliary GSH efflux and bile flow rose from 104.7 +/- 5.6 to 290.6 +/- 8.6 micrograms/ml bile and from 20.2 +/- 1.3 to 30.2 +/- 2.1 microliters/min, respectively; GSH-treated rats also showed increased liver (35%) and posthepatic vein plasma (68%) GSH concentrations compared with controls. By contrast, in rats pretreated with the GGT inhibitor GSH administration appeared to be devoid of any effect, except for a modest biliary GSH increase. CONCLUSIONS: This study indicates that significant changes occur in the hepatic GSH homeostasis after intraperitoneal GSH administration. The activity of hepatic GGT, most likely through degradation of circulating GSH, followed by an increase in cysteine availability, seems to account, at least partially, for the reported effects.

Cholesterol kinetics in subjects with bile fistula. Positive relationship between size of the bile acid precursor pool and bile acid synthetic rate.

Our aim was to identify and quantitate cholesterol pools and transport pathways in blood and liver. By studying bile fistula subjects, using several types of isotopic preparations, simultaneous labeling of separate cholesterol pools and sampling all components of blood and bile at frequent intervals, we developed a comprehensive multicompartmental model for cholesterol within the rapidly miscible pool. Data in six components (bile acids, esterified cholesterol in whole plasma, and free cholesterol in blood cells, bile, alpha lipoproteins, and beta lipoproteins) were modeled simultaneously with the SAAM program. The analysis revealed extensive exchange of free cholesterol between HDL and liver, blood cells, and other tissues. There was net free cholesterol transport from HDL to the liver in most subjects. The major organ that removed esterified cholesterol from blood was the liver. A large portion (4,211 mumol) of total hepatic cholesterol comprised a pool that turned over rapidly (t1/2 of 72 min) by exchanging mainly with plasma HDL and was the major source of bile acids and biliary cholesterol. Only 6% of hepatic newly synthesized cholesterol was used directly for bile acid synthesis: the analysis showed that 94% of newly synthesized cholesterol was partitioned into the large hepatic pool (putative plasma membrane free cholesterol) which exchanged rapidly with plasma lipoproteins. Bile acid synthetic rate correlated directly with the size of the large hepatic pool. In conclusion, hepatic and blood cholesterol pools and transports have been quantitated. HDL plays a central role in free cholesterol exchange/transport between all tissues and plasma. In humans, the metabolically active pool comprises a large portion of total hepatic cholesterol that, in part, regulates bile acid synthesis.

Effect of cholylsarcosine on hepatic cholesterol and bile acid synthesis and bile secretion in rats.

The regulatory and secretory properties of cholylsarcosine (C-sar), a synthetic conjugated bile acid analogue that resists deconjugation and dehydroxylation, were compared with those of the natural conjugates of cholic acid. After continuous intraduodenal infusion of cholylsarcosine (C-sar), cholyltaurine (C-tau), or cholylglycine (C-gly) at 36 mumol/100 g.h, the infused bile acid in each case became the predominant biliary bile acid. After 48 hours, infusion of C-sar, C-tau, and C-gly suppressed the activity of cholesterol 7 alpha-hydroxylase (C7 alpha H; rate-limiting for bile acid synthesis) by 65%, 78%, and 92%, respectively, compared with biliary fistula controls. After C-sar infusion, levels of C7 alpha H protein, messenger RNA, and transcriptional activity were depressed to the same extent as specific activity, indicating that C-sar, like C-tau, down-regulates C7 alpha H principally at the level of gene transcription. All three bile acids also suppressed activity of 3-hydroxy-3-methylglutaryl-coenzyme A reductase (rate-limiting for cholesterol synthesis). Both short- and long-term, the three cholyl conjugates caused similar increases in bile flow and in biliary secretion of cholesterol and phospholipid. It is concluded that in the rat, cholyl conjugates per se can suppress cholesterol and bile acid biosynthesis without prior conversion to deoxycholate. The effects of C-sar on hepatic cholesterol and bile acid synthesis as well as on induced bile flow and biliary lipid secretion are essentially identical to those of the naturally occurring cholyl conjugates.

Effect of intraduodenal administration of 23-methyl-UDCA diastereoisomers on bile flow in hamsters.

3 alpha,7 beta-Dihydroxy-23-methyl-5 beta-cholan-24-oic acid (MUDCA) and its two diastereoisomers, alpha- and beta-MUDCA, were infused intraduodenally in biliary fistula hamsters in order to evaluate the effect on bile flow and their hepatic biotransformation processes compared with the natural analog ursodeoxycholic acid (UDCA). In addition, the corresponding glycine conjugates were compared. The bile acids were administered at different doses (0.7-6 mumol/min/kg) over periods of 90 min. The results indicate that the racemic mixture exhibits a potent choleretic effect at both low and high doses, while the two individual diastereoisomers show this effect only at high doses. The presence of a C-23 methyl group in the side chain prevents hepatic amidation and alternative conjugations occur, such as glucuronidation, in order to facilitate their biliary secretion. Biotransformation of the methyl derivatives of UDCA occurred mainly by conversion to more polar glucuronide conjugates. There was little alteration to the molecule and, unlike UDCA, very little amidation occurred. These data indicate that the presence of a C-23 methyl group prevents the usual side-chain amidation common to the most naturally occurring bile acids and that glucuronidation is a requisite for efficient biliary excretion.

Sugar absorption by the biliary ductular epithelium of the rat: evidence for two transport systems.

Sugar absorption by the biliary ductular epithelium under steady-state conditions was examined using isolated perfused rat liver. The test sugar and mannitol (as a putative marker of paracellular entry) were added to the glucose-free recirculating perfusate each at a concentration of 5 mmol/L, and apparent active biliary ductular absorption equated with the change in concentration of the test sugar relative to that of mannitol. A metabolizable hexose (D-glucose), pentose (D-xylose), and three nonmetabolizable hexoses (alpha-methyl-glucoside, 3-o-methyl-glucose, and L-glucose) were used. All five monosaccharides were well absorbed at constant rates for 2 hours with apparent rates of absorption (mumol.kg body weight-1.min-1, mean +/- SE) of D-glucose, 0.24 +/- 0.01; L-glucose, 0.20 +/- 0.02; 3-o-methyl-glucose, 0.19 +/- 0.02; alpha-methyl-glucoside, 0.16 +/- 0.03; and D-xylose, 0.10 +/- 0.04. The addition of phloridzin to the perfusate inhibited D-glucose absorption in part but did not inhibit L-glucose absorption. When perfusate Na+ was replaced by N-methylglucamine, the bile-plasma ratio of mannitol remained unchanged, as did the apparent absorption rate of D-glucose and 3-o-methyl-glucose. In contrast, absorption of L-glucose and alpha-methyl-D-glucoside gradually ceased. The addition of 15 mmol/L glucose to the perfusate caused decreased bile flow and increased taurocholate concentration in bile, suggesting that glucose absorption by the biliary ductules induced water reabsorption. It is concluded that sugars are absorbed by the biliary ductular system by Na(+)-dependent and Na(+)-independent transport systems, the substrate affinities of which differ from those reported for apical membrane hexose transport systems in renal tubular and intestinal epithelia. Ductular absorption of solutes such as glucose that enter bile passively may have biological use, because ductular absorption decreases the concentration of substrates for bacterial growth in gallbladder bile. On the other hand, ductular absorption of solutes induces reabsorption of biliary water, resulting in decreased bile flow; this might contribute to cholestasis during prolonged hyperalimentation with solutions containing glucose.