Effects of sphincterectomy on bile acid enterohepatic circulation in dogs

Relative rates of bile enterohepatic circulation (EHC) and bile acid pool distribution were compared in intact and sphincterectomized dogs with portacaval shunt. There was no significant difference in the rates of EHC or in the bile acid pool distribution in the groups of animals. Feeding and cholecystokinin administration caused similar increases in bile acid EHC rates in sphincterectomized and intact animals. It was concluded that the sphincter of Oddi has little or no effect on these aspects of bile acid metabolism in dogs.     

Canine bile acid enterohepatic circulation

The enterohepatic circulation (EHC) of bile acids has been studied in fasting dogs with portacaval shunt maintained in the steady state. In such animals the rate of EHC is proportional to systemic blood bile acid concentration. Bile acid EHC was irregular (20 to 100% variation) when measured at 15 minute or hourly intervals. Studies showed that the variations persisted in cholecystectomized and sphincterectomized animals. The irregularities were enhanced by bethanechol chloride which increases intestinal peristalsis and suppressed by diphenoxylate HCl which slows peristalsis. The variations appear to arise from irregular patterns of intestinal peristalsis. This phenomenon may explain some variations in blood bile acid concentration observed in patients with liver disease.     

Short- and long-term effects of biliary drainage on hepatic cholesterol metabolism in the rat.

The present study concerns short- and long-term effects of interruption of the enterohepatic circulation (EHC) on hepatic cholesterol metabolism and biliary secretion in rats. For this purpose, we employed a technique that allows reversible interruption of the EHC, during normal feeding conditions, and excludes effects of anaesthesia and surgical trauma. [3H]Cholesteryl oleate-labelled human low-density lipoprotein (LDL) was injected intravenously in rats with (1) chronically (8 days) interrupted EHC, (2) interrupted EHC at the time of LDL injection and (3) intact EHC. During the first 3 h after interruption of the EHC, bile flow decreased to 50% and biliary bile acid, phospholipid and cholesterol secretion to 5%, 11% and 19% of their initial values respectively. After 8 days of bile diversion, biliary cholesterol output and bile flow were at that same level, but bile acid output was increased 2-3-fold and phospholipid output was about 2 times lower. The total amount of cholesterol in the liver decreased after interruption of the EHC, which was mainly due to a decrease in the amount of cholesteryl ester. Plasma disappearance of LDL was not affected by interruption of the EHC. Biliary secretion of LDL-derived radioactivity occurred 2-4 times faster in chronically interrupted rats as compared with the excretion immediately after interruption of the EHC. Radioactivity was mainly in the form of bile acids under both conditions. This study demonstrates the very rapid changes that occur in cholesterol metabolism and biliary lipid composition after interruption of the EHC. These changes must be taken into account in studies concerning hepatic metabolism of lipoprotein cholesterol and subsequent secretion into bile.     

Cholecystectomy prevents expansion of the bile acid pool and inhibition of cholesterol 7alpha-hydroxylase in rabbits fed cholesterol

To study the effect of cholecystectomy on the regulation of classic and alternative bile acid syntheses, gallbladder-intact (n = 20) and cholecystectomized (n = 20) New Zealand White rabbits were fed either chow or chow with 2% cholesterol (3 g/day). After 10 days, bile fistulas were constructed in half of each rabbit group to recover and measure the bile acid pool and biliary bile acid flux. After cholesterol feeding, the bile acid pool size increased from 268 +/- 55 to 444 +/- 77 mg (P < 0.01) with a 2-fold rise in the biliary bile acid flux in intact rabbits but did not expand the bile acid pool (270 +/- 77 vs. 276 +/- 62 mg), nor did the biliary bile acid flux increase in cholecystectomized rabbits. Ileal apical sodium-dependent bile acid transporter protein increased 46% from 93 +/- 6 to 136 +/- 23 units/mg (P < 0.01) in the intact rabbits but did not change in cholecystectomized rabbits (104 +/- 14 vs. 99 +/- 19 units/mg) after cholesterol feeding. Cholesterol 7alpha-hydroxylase activity was inhibited 59% (P < 0.001) while cholesterol 27-hydroxylase activity rose 83% (P < 0.05) after cholesterol feeding in the intact rabbits but neither enzyme activity changed significantly in cholesterol-fed cholecystectomized rabbits. Fecal bile acid outputs reflecting bile acid synthesis increased significantly in the intact but not in the cholecystectomized rabbits fed cholesterol.Removal of the gallbladder prevented expansion of the bile acid pool after cholesterol feeding as seen in intact rabbits because ileal bile acid transport did not increase. As a result, cholesterol 7alpha-hydroxylase was not inhibited.     

Effects of acute administration of taurocholic and taurochenodeoxycholic acid on biliary lipid excretion in the rat.

Comparison of the effects of biliary lipid excretion produced by infusion of taurochenodeoxycholate and taurocholate showed no significant difference when the bile acids were infused for a relatively short period of time. Cholesterol excretion rates measured during depletion of the bile acid pool were significantly higher than cholesterol excretion rates measured during infusion of bile acids at various rates. These data indicate that there is some mechanism in addition to bile acid excretion that is responsible for biliary excretion of cholesterol when the enterohepatic circulation is intact.     

Neurotensin elevates hepatic bile acid secretion in chickens by a mechanism requiring an intact enterohepatic circulation

Neurotensin (NT), given intravenously at 10-50 pmol/kg per min to anesthetized female chickens equipped with a bile duct fistula, dose-dependently elevated hepatic bile flow and bile acid output but only when the enterohepatic circulation was maintained by returning the bile to the intestinal lumen. Infusion of NT at 10 and 50 pmol/kg per min increased the average hepatic bile acid output over a 30-min period to 138 +/- 11 and 188 +/- 13% of control, respectively. During infusion of NT, plasma levels of immunoreactive NT (iNT) increased in time from the basal level (14 +/- 1.3 pM) to reach steady state at 30 min. There was a near linear relationship between the dose of NT infused and the increment in plasma iNT. In addition, infusion of NT at 40 pmol/kg min gave a plasma level of iNT (approximately/= 88 pM) which was within the range of those observed during duodenal perfusion with lipid (54-300 pM) and near to that measured in hepatic portal blood from fed animals (52 +/- 5 pM). Perfusion of duodenum with lipid released endogenous NT and increased the rate of hepatic bile flow. When NT antagonist SR48692 was given, bile flow rate decreased to the basal level. These results suggest that intestinal NT, released by lipid, may participate in the regulation of hepatic bile acid output by a mechanism requiring an intact enterohepatic circulation.     

Ethanol has an acute effect on bile acid biosynthesis in man

A single dose of ethanol, 0.4 g/kg body weight, was found to give a 5-15 fold increase of the plasma concentrations of 7 alpha-hydroxy-cholesterol and 7 alpha-hydroxy-4-cholesten-3-one in humans. The rise was maximal 4 h after ethanol ingestion, was dose-dependent and was not seen in a cholecystectomized subject. The effect was selective for these and some other 7 alpha-hydroxylated C27-intermediates in bile acid biosynthesis. The changes are compatible with an acute stimulation of cholesterol 7 alpha-hydroxylase possibly due to an ethanol-induced inhibition of gallbladder contraction resulting in an interruption of the enterohepatic circulation of bile acids. The effect is of interest in relation to the influence of ethanol consumption on cardiovascular and gallstone diseases.     

Metabolic fate of ethynodiol diacetate in the baboon.

The enterohepatic circulation and metabolism of ethynodiol diacetate (3beta,17beta-diacetoxy-17alpha-ethynyl-estr-4-ene) in baboons were studied following the intravenous injection of this contraceptive steroid labeled with 14C (4-position) and with 3H (in either the 3- or 17-acetoxy moieties). Bile and urine from four baboons with biliary fistulas and urine from four intact baboons were collected for 7 hours. On the average, 40% and 44% of the injected dose were excreted in the bile and urine, respectively. Only 48% was recovered in the urine of intact baboons. Analysis of these excretion rates indicates an insignificant enterohepatic circulation of this compound. The steroid was excreted mostly (over 80%) as a glucosiduronate in urine and bile. Very little excretion of the 3-acetoxy compound was detected in the urine or bile at any time interval. 17-Monoacetoxy compounds, however, were detected both in urine and bile, suggesting a difference in the rate of in vivo hydrolysis of the 17beta- vs. the 3beta-acetate.     

Regulation of Bile Salt Pool Size in Man

When the enterohepatic circulation is intact the size of the bile salt pool is largely determined by the frequency of its enterohepatic circulation. This hypothesis, derived from studies of cholate kinetics in coeliac, cholecystectomy, and gall stone patients, represents the simplest interpretation of the data. A corollary is that daily cholate secretion is likely to be normal in these conditions and that therefore the propensity of bile to form cholesterol gall stones is not likely to be directly related to bile salt pool size.     

Influence of time, type of diet and ursodeoxycholic acid on biliary secretion in rats with intestinal resection

The effects of time and the type of dietary fat on biliary physiology in rats with 50% resection of the distal small intestine was investigated. The effects of ursodeoxycholic acid as an exogenous source in bile acid added to the diet were also studied. The fat composition of all diets was the same in quantitative terms (4%), and differed only in the type of lipid supplied: olive oil (diet A) or 1/3 medium chain triglycerides, 1/3 sunflower seed oil and 1/3 olive oil (diet B). Three months after intestinal resection, neither group of resected rats showed significant changes in bile flow or bile acid output in comparison to controls, whereas the slope of the regression line was notably increased, indicating that enterohepatic circulation failed to adapt to the different dietary fats during recovery from intestinal resection. The addition of ursodeoxycholic acid to diet B significantly raised bile acid flow and output in comparison to controls, as a result of the recovery of enterohepatic circulation. This was reflected in the lower slope of the regression line, thus suggesting that de novo synthesis in resected animals was less intensely stimulated.     

Bile-salt-dependent and independent choleresis induced by bucolome in the rat.

Choleresis induced by bucolome (BC) (1-cyclohexyl-5-n-butyl-2,4,6-trioxoperhydropyrimidine) was studied in male Wistar rats. [14C]Erythritol and mannitol clearance studies indicated this choleresis to be of canalicular origin. In 1-h continuous bile collection studies, immediately after the interruption of enterohepatic circulation (acute interruption), both bile flow and bile salt excretion rates were significantly increased in rats administered BC. However, the bile salt excretion rate fell rather rapidly in BC-administered rats, while the bile flow rate was fairly constant during this 1-h period. Thus, unlike the situation in control rats, bile flow rate was not significantly correlated with the bile salt excretion rate in BC-administered rats. In rats that had an external bile fistula open for 16-20 h (chronic interruption of enterohepatic circulation) the bile flow rate was also significantly increased by BC administration, while the bile salt excretion rate was not changed after BC administration. It is suggested that BC induced bile-salt-independent choleresis in both experimental rat groups (acute and chronic interruption of enterohepatic circulation). In addition, BC appeared to increase the bile-salt-dependent fraction of bile in rats with acute interruption of enterohepatic circulation, possibly by mobilizing the bile salt pooled in the intestinal content and (or) intestinal wall.     

Administration of phosphatidylcholine-cholesterol liposomes partially reconstitutes fat absorption in chronically bile-diverted rats

BACKGROUND AND AIMS: Intestinal bile deficiency in cholestatic patients leads to fat malabsorption. We addressed the potency of model bile, bile salts and phosphatidylcholine (PC)-cholesterol (CH) liposomes to reconstitute fat absorption in permanently bile-diverted (BD) rats. METHODS: The plasma appearance of 13C-labeled palmitic acid (13C-16:0) and linoleic acid (13C-18:2) was determined after their enteral administration to BD or to control rats with an intact enterohepatic circulation (EHC) (13C-16:0 and 13C-18:2 dissolved in 25% olive oil-75% medium chain triacylglycerol oil mixture). BD rats were intraduodenally infused with buffer, model bile [consisting of 60 mM taurocholate (TC), 8 mM PC and 1 mM CH], buffer with TC, buffer with PC and CH liposomes, or buffer with lyso-PC and CH. RESULTS: Plasma concentrations of 13C-16:0 and 13C-18:2 were consistently three- to eightfold higher in control rats than those in buffer-infused BD rats (P < 0.01). ID administration of either model bile or TC to BD rats restored plasma appearance of 13C-fatty acids at least to concentrations observed in control rats. Administration of PC + CH liposomes to BD rats partially reconstituted the plasma appearance of 13C-16:0, but did not affect that of 13C-18:2. Compared with control rats, the area under the curve (AUC) of plasma 13C-16:0 concentrations was 13.0 +/- 6.9% in buffer-infused rats and 40.9 +/- 3.1% in liposome-infused rats (P < 0.005). CONCLUSIONS: Enteral administration of PC + CH liposomes to BD rats partially corrects the absorption of palmitic acid. Present data suggest that administration of PC + CH liposomes could enhance fat absorption in clinical conditions of cholestasis in which bile salt supplemention is contraindicated.     

Enterohepatic circulation in hamsters with an extracorporeal bile duct.

The present study describes a novel technique for investigations of the enterohepatic circulation in the hamster with an extracorporeal bile duct that allows long-term bile collection in the free-moving animal. The animals recovered for 7 days after the operation before the external loop was cut and bile was collected over a period of 78 h. Under these optimal conditions, initial bile flow (651 +/- 89 microliters per 100 g.h-1) and the secretion rates of biliary lipids were several-fold higher than reported in an earlier study using the acute fistula hamster. Biliary cholesterol secretion amounted to 369 +/- 32 nmol per 100 g.h-1, phospholipid secretion was 2.6 +/- 0.3 mumol per 100 g.h-1, and total bile acid secretion was 31.9 +/- 2.2 mumol per 100 g.h-1. A clearcut diurnal rhythm was demonstrated for bile flow and all biliary constituents. After 9 h the depletion of the bile acid pool was complete and cholic acid synthesis derepressed 1.4-fold from a basal rate of 818 nmol per 100 g.h-1, whereas the derepression of chenodeoxycholic acid synthesis was even less pronounced. Biliary cholesterol output increased 2.2-fold, but the phospholipid secretion was constant during the full experiment. It may be concluded that the technique of an extracorporeal bile duct in the free-moving animal allows studies of bile secretion under optimal conditions. Most likely the bile secretion rates given above approach the physiological rates in the hamster.     

Enterohepatic circulation of chloramphenicol and its glucuronide in the rat by microdialysis using a hepato-duodenal shunt

A system consisting of a hepato-duodenal shunt in which the bile of a drug-treated donor rat was diverted to the duodenum of an untreated recipient rat via a bile cannula was used to assess the role of hepatic metabolism and enterohepatic circulation in the pharmacokinetics of chloramphenicol. Blood concentrations of unbound chloramphenicol and its glucuronide were measured by on-line microdialysis coupled to a microbore liquid chromatographic system. Results indicated that chloramphenicol and its glucuronide were detected in the blood of both donor and recipient rats following an intravenous 100 mg/kg dose of chloramphenicol succinate to the donor rat. Our finding suggests that although enterohepatic circulation contributed only to a minor extent (approximately 1.8%) was involved in the disposition of unbound chloramphenicol in the rat on-line microdialysis techniques were applicable for such studies.     

The contribution of newly synthesized cholesterol to bile salt synthesis in rats quantified by mass isotopomer distribution analysis

A new stable isotope procedure has been developed and validated in rats, applying [1-(13)C]acetate infusion to quantify the production of bile salts from de novo synthesized cholesterol making use of the mass isotopomer distribution analysis (MIDA) principle. Ions (m/z) 458-461, 370-373 and 285-288 were monitored by GC/MS (EI-mode) for the methyl trimethylsilylether derivatives of cholate, chenodeoxycholate and beta-muricholate, respectively. Rats with intact exteriorized enterohepatic circulation and rats with chronic bile diversion were infused with [1-(13)C]acetate for up to 14 h. After 10 h of infusion the enterohepatic circulation of the intact group was interrupted to deplete the existing bile salt pool (acute bile diversion). The fractions of biliary cholesterol and individual bile salts derived from newly synthesized cholesterol were determined by MIDA at t=14 h. In rats with acute bile diversion, these fractions were 20, 25, 27 and 23% for biliary cholesterol, cholate, chenodeoxycholate and beta-muricholate, respectively. After bile diversion for 8 days to induce hepatic cholesterol and bile salt synthesis, these fractions increased significantly to 32, 47, 41 and 47%, respectively. Calculated enrichments of the acetyl-CoA precursor pools were similar for all bile salts and biliary cholesterol within the two rat groups. However, chronic enterohepatic interruption decreased the acetyl-CoA pool size almost two-fold. We conclude that MIDA is a validated new stable isotope technique for studying the synthetic pathway from acetyl-CoA to bile salts. This technique provides an important new tool for studying bile salt metabolism in humans using stable isotopes.     

The effect of cyclosporine A on bile secretion in dogs

Cyclosporine A is reported to cause cholestasis, but the evidence is confounded by anesthesia and surgery used in acute experiments. To better investigate the effect of cyclosporine on the liver, bile output was directly measured in three cholecystectomized dogs by cannulating the common duct through a chronic duodenal fistula. Control studies were done 1 month after surgery. Cyclosporine in oral doses of 5, 15, and 50 mg.kg-1.d-1 was then given for consecutive 1-week periods. Twice during each study period, bile output was measured for 5 h in fasted, awake animals: 3 h to establish basal conditions, followed by 2 h of taurocholate infusions at 1 and then 2 mumols.kg-1.min-1. Under basal conditions, bile flow rose with each dose of cyclosporine, increasing 63, 127, and 179% above control with cyclosporine 5, 15, and 50 mg.kg-1,d-1, respectively. Bile flow increased similarly during taurocholic acid stimulation. Cyclosporine had no effect on bile salt or bilirubin secretion. In this chronic dog model isolated from other causes of cholestasis, cyclosporine did not induce cholestasis but rather caused a dose-related choleresis without any change in bile salt secretion.     

Biliary response to food in chickens with intact or interrupted enterohepatic circulation

The biliary secretion in response to food has been studied in chicken. In this species, a well defined biliary postprandial response with a clear vesicular component can be estimated. The bile salt independent fraction increases after feeding. Our results show that gallbladder bile has a lower osmotic power. The biliary response to food when the enterohepatic circulation is blocked, diminishes. The relationship between bile salt independent fraction values, whether the vesicular duct is tied or not, remains constant when the enterohepatic circulation is interrupted.     

3-Diazirine-derivatives of bile salts for photoaffinity labeling

New carbene-generating photolabile bile salt derivatives, 3,3-azo-7 alpha,12 alpha-dihydroxy-5 beta [7 beta-3H]cholan-24-oic acid and (3,3-azo-7 alpha,12 alpha-dihydroxy-5 beta [7 beta-3H]cholan-24-oyl)-2- aminoethanesulfonic acid were synthesized with high specific radioactivity. These 3-diazirine-derivatives could be activated to the corresponding carbenes by irradiation with ultraviolet light at 350 nm with a half-life time of 2 min. The 3-diazirine derivatives behaved in enterohepatic circulation like the natural bile salts. The uptake of [3H]taurocholate into isolated hepatocytes was competitively inhibited by (3,3-azo-7 alpha,12 alpha-dihydroxy-5 beta-cholan-24-oyl)-2- aminoethanesulfonic acid indicating that the 3,3-azo-derivative of taurocholate shares the hepatic transport systems for natural bile salts. It was demonstrated that the radioactively labeled 3-diazirine bile salt derivatives are useful probes for photoaffinity labeling of bile salt binding proteins especially in intact cells and tissues.     

Modulation of low density lipoprotein receptor activity by bile acids: differential effects of chenodeoxycholic and ursodeoxycholic acids in the hamster

Hamsters were fed chenodeoxycholic acid (CDC), ursodeoxycholic acid, (UDC), or no bile acid. [14C]Sucrose-labeled hamster low density lipoprotein (LDL) and methylated human LDL were infused intravenously to study LDL receptor-dependent and LDL receptor-independent organ uptake, respectively, of LDL. Biliary CDC increased during both CDC and UDC treatment. The UDC enrichment of bile after UDC feeding was relatively small. Bile acid synthesis was suppressed after both bile acid treatments. Under the condition of an acute bile fistula, the hamster LDL uptake increased in the liver, heart, and adrenals in the CDC-treated animals. During an intact enterohepatic circulation, the hepatic uptake of hamster LDL, which accounted for a major portion of the total uptake, was increased after UDC treatment. The hamster LDL uptake in the colon, which represented only a small fraction of the total uptake, increased after CDC treatment. When hamster LDL was infused at increasing concentrations, its uptake was significantly higher in the UDC-treated than in the control and CDC-treated animals. The methylated human LDL uptake showed no significant changes in the different treatment groups under either experimental condition. The study shows significantly different effects of CDC and UDC on LDL receptor activity. Since these differences are expressed in spite of a similar suppression of bile acid synthesis, UDC may directly influence LDL receptor activity.