Bile acid structure and bile formation in the guinea pig

The effects of intravenous infusions (1-4 mumol/min/kg) of 14 bile acids, cholic, deoxycholic, ursodeoxycholic, chenodeoxycholic, dehydrocholic, and their glycine and taurine conjugates, on bile flow and composition and on the biliary permeation of inert carbohydrates have been studied in the guinea pig bile fistula. Hydroxy bile acids were eliminated in bile without major transformation, except for conjugation (over 90%) when unconjugated bile acids were infused. During infusion of dehydrocholate and taurodehydrocholate, 77-100% of the administered dose was recovered in bile as 3-hydroxy bile acids, thus indicating that reduction of the keto group in position 3 was virtually complete. All bile acids produced choleresis at the doses employed: the strongest choleretic was deoxycholate (81.78 microliters/mumol), the weakest was taurodehydrocholate (10.2 microliters/mumol). Choleretic activity was directly and linearly related to bile acid hydrophobicity, as inferred by HPLC, both for similarly conjugated bile acids, and for bile acids having the same number, position, or configuration of the hydroxyl groups. In all instances, the rank ordering was: deoxycholate greater than chenodeoxycholate greater than cholate greater than ursodeoxycholate. During choleresis produced by any of the bile acids tested, bicarbonate concentration in bile slightly declined, but the calculated concentration in bile-acid-stimulated bile (45-57 mmol/l) was always higher than that measured in plasma (23-26 mmol/l). Biliary concentrations of cholesterol (20-68 mumol/l) and phospholipid (14-63 mumol/l) were very low during spontaneous secretion, and declined even further following bile acid choleresis. None of the infused bile acids consistently modified biliary excretion of cholesterol and phospholipid. Consistent with a previous observation from this laboratory, all hydroxy bile acids reversibly diminished [14C]erythritol and [14C]mannitol biliary entry during choleresis, while they increased or failed to modify that of [3H]sucrose and [3H]inulin. The rank ordering for the inhibitory effect on [14C]erythritol and [14C]mannitol permeation was: 3 alpha,7 alpha,12 alpha-trihydroxy greater than 3 alpha,7 alpha-dihydroxy greater than 3 alpha,7 beta-dihydroxy greater than 3 alpha,12 alpha-dihydroxy bile acids.(ABSTRACT TRUNCATED AT 400 WORDS)     

Choleretic effects of differently structured bile acids in the guinea pig

The effects of 10 differently structured bile acids on bile flow and composition were studied in anesthetized, bile duct-cannulated guinea pigs. At the infusion rates of 2 and 4 mumole/min/kg, all bile acids produced choleresis. The most potent was chenodeoxycholate, which increased bile flow by an average of 31.25 microliters/mumole of bile acids excreted in bile. The weakest choleretic was tauroursodeoxycholate (11.02 mu/mumole). When the choleretic activity was plotted against bile acid hydrophobicity (high-performance liquid chromatography retention factor, obtained from the literature), linearity was observed with similarly conjugated bile acids. The order of potency was deoxycholate greater than chenodeoxycholate greater than cholate greater than ursodeoxycholate, both for the glycine and taurine conjugates, and for the unconjugated bile acids as well. Conjugation was also important, and the rank ordering for the choleretic activity (unconjugated bile acids greater than glycine-conjugates greater than taurine-conjugates) was the same as that for the hydrophobicity. When the choleretic activity was plotted against bile acid micellar aggregation number (in 0.15 M NaCl at 36 degrees C, obtained from the literature), a linear, direct relationship was observed. All bile acids produced similar effects on bile electrolyte concentrations: both bicarbonate and chloride slightly declined during choleresis, whereas bile acid concentrations increased. These studies suggest that, in the guinea pig the differing choleretic activities of differently structured bile acids are not due to their forming micelles in bile of different sizes; either the more hydrophobic bile acids form vesicles, whereas the more hydrophilic form micelles; or bile acids produce choleresis, in part or exclusively, by stimulating an additional secretory mechanism, possibly an inorganic ion pump; or both.     

Taurine increases bile acid pool size and reduces bile saturation index in the hamster

There is evidence that increased availability of taurine enhances the proportion of taurine-conjugated bile acids in bile. To explore the possibility that taurine treatment could also influence hepatic cholesterol and bile acid metabolism, we fed female hamsters for 1 week and measured both the biliary lipid content and the microsomal level of the rate-limiting enzymes of cholesterol and bile acid synthesis. In these animals the cholesterol 7 alpha-hydroxylase activity was significantly greater in respect to controls (P less than 0.05). The total HMG-CoA reductase activity, as well as that of the active form, was similarly increased. The stimulation of 7 alpha-hydroxycholesterol synthesis was associated with an expansion of the bile acid pool size in taurine-fed animals. Taurine feeding was observed to induce an increase in bile flow as well as in the rate of excretion of bile acids, whereas the secretion rate of cholesterol in bile was decreased. As a consequence, the saturation index was significantly lower in taurine-fed animals (P less than 0.05). The possible mechanisms through which taurine exhibits the modification of the enzyme activities and of the biliary lipid composition are discussed.     

Regulatory effects of sterols and bile acids on hepatic 3-hydroxy-3-methylglutaryl CoA reductase and cholesterol 7alpha-hydroxylase in the rat

Specific activities of the hepatic microsomal enzymes 3-hydroxy-3-methylglutaryl CoA (HMG CoA) reductase and cholesterol 7alpha-hydroxylase were studied in rats fed sterols and bile acids. The administration of bile acids (taurocholate, taurodeoxycholate, taurochenodeoxycholate) at a level of 1% of the diet for 1 wk reduced the activity of HMG CoA reductase. Taurocholate and taurodeoxycholate, but not taurochenodeoxycholate, inhibited cholesterol 7alpha-hydroxylase. Dietary sitosterol produced increases in the specific activity of HMG CoA reductase (3.6-fold) and cholesterol 7alpha-hydroxylase (1.4-fold), and biliary cholesterol concentrations in this group more than doubled. Compared with controls fed the stock diet, the simultaneous administration of sitosterol and taurochenodeoxycholate resulted in a 60% decrease of HMG CoA reductase activity and no change in cholesterol 7alpha-hydroxylase activity or biliary cholesterol concentration. Rats fed sitosterol plus taurocholate had nearly normal HMG CoA reductase activity, but cholesterol 7alpha-hydroxylase was inhibited and biliary cholesterol remained high. Bile acid secretion rates and biliary bile acid composition were similar in controls and sterol-fed animals. In all groups receiving bile acids, biliary secretion of bile acids was nearly doubled and bile acid composition was shifted in the direction of the administered bile acid. It is concluded that the composition of the bile acid pool influences the hepatic concentrations of the rate-controlling enzymes of bile acid synthesis.     

Bile formation and hepatic plasma membrane composition in guinea-pigs and rats

We compared bile formation, and biliary and liver plasma membrane composition in guinea-pigs and rats in an attempt to explain the observation that the bile flow rate and the bile acid independent fraction of bile flow (BAIF) in guinea-pigs is about five to seven times higher than in rats. Analysis of electrolytes in bile showed that bicarbonate was significantly [acid] higher in guinea-pigs while Cl⁻, phosphate and Ca²⁺ were markedly lower than in rats. High bile independent secretion in guinea-pigs was associated with a significantly lower concentration of total bile acid, phospholipid and cholesterol than in rats. Bile acid distribution studies showed that glycine conjugated chenodeoxycholate and ketolithocholate were the main bile acids in guinea-pigs, while taurine conjugated cholate and muricholate were the predominant bile acids in rats. Total fatty acid analysis of bile indicated that in rats the major fatty acids were palmitic acid (C16:0) and linoleic acid (C18:2, n-6). In guinea-pigs, the contribution of these fatty acids was lower than in rats and compensated with a significantly higher percentage of oleic acid (C18:1, n-9). Concentrations of anionic polypeptide fraction (APF), an acidic calcium binding apoprotein closely associated with biliary phospholipid and cholesterol secretion was also significantly lower in guinea-pigs. Canalicular plasma membrane analysis showed that as compared with rats, specific activities of Na⁺,K⁺ ATPase, and cholesterol and phospholipid content were markedly lower in guinea-pigs. Total fatty acid analysis of the membrane revealed that palmitic acid (C16:0), stearic acid (C18:0) and linoleic acid (C18:2, n-6) were the predominant fatty acids in guinea-pigs, while palmitic acid (C16:0), stearic acid (C18:0) and arachidonic acid (C20:4, n-6) were the most important in rats. Thus, high bile flow rate and BAIF in the guinea-pig may be attributed to the low bile acid concentration (below the critical micellar concentration), secretion of hypercholeretic bile acids (e.g. ketolithocholate) and high bicarbonate output.     

A rhesus monkey model for continuous infusion of drugs into cerebrospinal fluid

A new rhesus monkey model with two intraventricular catheter systems was developed to examine the pharmacokinetics and neurotoxicity of chemotherapeutic agents administered by continuous intraventricular infusion. A lateral ventricular catheter system implanted in the lateral ventricle and attached to a subcutaneous access port on the animal's back is used for infusion of drugs into the ventricle. A Pudenz catheter implanted in the fourth ventricle and connected to a subcutaneous Ommaya reservoir permits repetitive CSF sampling in unanesthetized animals. The model was evaluated in five animals for over 12 months for catheter patency, surgical complications, and utility in studying the pharmacokinetics of continuous intraventricular infusion of methotrexate. There were no perioperative complications. Three of the five monkeys maintained both systems successfully. The other two animals developed staphylococcal ventriculitis, one at 7 days as a result of manipulation of the incision by the animal leading to cellulitis around the catheter site and subsequent ventriculitis, the other at 5 months. Both animals were treated successfully with antibiotics and catheter removal. An infusion of 0.05 mg of methotrexate over 24 hours maintained ventricular drug concentrations of 1 mol/L without evidence of neurotoxicity. This new model has applications both for the development of continuous intraventricular infusion as a therapeutic approach for the treatment of meningeal cancers in humans and as a research tool to study the distribution and elimination of drugs from the CSF.     

Effects of bile salts on bile formation in rabbits

The effects of different species of bile salts: deoxycholate, taurochenodeoxycholate, ursodeoxycholate, glycodeoxycholate, tauroursodeoxycholate, chenodeoxycholate and cholate (DCA, TCDC, UDCA, GDCA, TUDC, CDCA, CA) on bile secretion were examined in anesthetized rabbits using two different infusion routes. When bile salts were infused intravenously, all bile salts (except for TCDC) significantly increased the volume of bile and bile salt excretion, but their respective efficiency for bile formation was different. The concentration of bicarbonate ion in the bile significantly increased during the choleretic periods induced by DCA, UDCA, GDCA and CDCA but remained unchanged with the other bile salts (CA, TCDC, TUDC). In rabbits, where a bile salt solution was infused in the duodenum and then drained from the intestine through an incision in the distal part of duodenum, none of these bile salts affected bile secretion. The effects of intravenously administered bile salts on rabbit bile secretion are different in terms of their choleretic potency and bicarbonate excretion depending on the species of bile salts used. Furthermore, it was concluded that the intraduodenal infusion of UDCA, which was found to stimulate the pancreatic exocrine function, did not affect bile secretion.     

Enterohepatic circulation in man. A simple method for the determination of duodenal bile acids

A method has been developed for easy sampling of duodenal bile acids. For this purpose Entero-Test was used, an encapsulated nylon thread originally used to estimate enteral parasites. This capsule is swallowed by a fasting subject and one end of the thread is taped at a corner of the month. Four hours after swallowing the thread, it is withdrawn and bile acids are eluted with buffer. The solution is applied to a Sep-Pak C18 cartridge to extract bile acids, which are subsequently analyzed by capillary gas-liquid chromatography and liquid chromatography. In vitro analyses showed that there was no preferential binding to the thread of any bile acid and that binding was pH-independent. A high correlation (r = 0.98) was found between direct analyses of bile and analyses by Entero-Test after in vitro incubation. The values obtained by the Entero-Test were similar to those of duodenal bile simultaneously collected with the normal intubation technique (r = 0.99). Duodenal bile acid composition showed a daily variation. In 11 healthy volunteers the following bile acid composition of unstimulated duodenal juice was found (mean +/- SD; %): choleate 44 +/- 12 (glycine/taurine ratio 1.8), chenodeoxycholate: 29 +/- 6 (G/T ratio 2.3); deoxycholate: 25 +/- 11 (G/T ratio 5.7), lithocholate: 1, ursodeoxycholate: less than 1. The described technique turned out to be an easily applicable method for determination of duodenal bile acids in man. This enables longitudinal studies concerning the factors that determine the bile acid pool composition and its relevance to various diseases.     

Another method to prevent venous thrombosis in microsurgery: an in situ venous catheter

Free-flap failure is in the order of 4 to 10 percent. Heparin is more effective at preventing venous thrombosis than arterial thrombosis. This study was undertaken to investigate the efficacy of delivering heparin at a high dose locally but low dose systemically (heparin infusion via a catheter placed proximal to the venous anastomosis) to prevent venous thrombosis in microsurgery. A model of venous thrombosis was first established by a venous inversion graft in the rat femoral vein (this was performed in seven animals and resulted in 100 percent thrombosis). Saline and heparin were delivered proximal to the inverted vein graft to assess the effect of each in preventing venous thrombosis. Flow/patency distal to the inverted vein graft was assessed by observation under the microscope, the milk test, and rate of flow (flowmeter). Saline infused via a catheter proximal to the venous inversion graft resulted in 100 percent thrombosis in 10 animals. Heparin (100 U/ml at 2 to 3 ml/hour) infused through a catheter for 2 hours proximal to the anastomosis resulted in flow in all 10 animals during the infusion. Blood was also taken before beginning the procedure (control) and after the heparin infusion distal to the anastomosis (local partial thromboplastin time) as well as in the contralateral femoral vein (systemic). The control for all animals that received heparin was <3 minutes. The systemic partial thromboplastin time after heparin infusion was <3 minutes in seven animals, 3.3 minutes in two animals, and >7 minutes in one animal. The local partial thromboplastin time distal to the inverted vein graft was >10 minutes in nine animals and 3.7 minutes in one animal. The study also had a clinical component, in which a catheter was placed in a vein of the free flap, and heparin was infused over 5 days. This technique has been used in 83 consecutive free flaps. In three recent free flaps performed on the limbs, the local partial thromboplastin time (close to the anastomosis) was raised but the systemic time was normal. This technique offers a method in preventing venous thrombosis in microsurgery. It is simple to implement and is not associated with the systemic complications of heparin.     

Nitrogen sparing effect of a branched-chain amino acid infusion during an acute fast in guinea pigs

Thirty male guinea pigs (350–600 g) were fasted for 48–72 hours while receiving lactated Ringers solution through a catheter in the internal jugular vein which had been implanted just before the start of the experiment under halothane anesthesia. Ten of the animals also received leucine, isoleucine, and valine in their infusions at a level approximating their usual daily requirement for these amino acids. Eight of the animals received glucose in their infusion at a level which was isocaloric to the branched-chain amino acid infusion. There was a 37% improvement (p < .01) in nitrogen balance in the animals supplemented with the branched-chain amino acids compared to the completely fasted animals. Nitrogen balance was increased by 27% (p < .05) in the amino acid treated animals relative to the glucose treated group. These results may relate to the specific regulatory role of leucine, isoleucine, and valine on muscle protein turnover. In addition, the preferential oxidation of these amino acids in muscle may be a limiting factor in the overall reutilization of essential amino acids during early fasting.     

Metabolism of intravenously administered 7 alpha-hydroxycholesterol-3 beta-stearate in the hamster.

In order to investigate the metabolic fate of serum esterified 7 alpha-hydroxycholesterol, [4-14C]7 alpha-hydroxycholesterol-3 beta-stearate was synthesized from labeled cholesterol and administered to bile fistula hamsters intravenously. Bile samples were collected at every 20 min for 7 h. Radioactivity was detected in bile 40 min after the beginning of the infusion of the labeled compound and 56.5 +/- 5.7% (48.7-66.0%) of the administered radioactivity was recovered in bile during 7 h. The liver contained appreciable radioactivity (19.5 +/- 7.6% of the administered dose) at the time of sacrifice. Only a trace amount of radioactivity was detected in urine and blood. Cumulative recovery of the radioactivity was 76.3 +/- 8.6% (63.3-90.4%). Major radioactive metabolites in the bile samples were identified to be taurine- and glycine-conjugated cholic acid and chenodeoxycholic acid by radioactive thin-layer chromatographic analysis of the bile samples before and after enzymatic hydrolysis and 3 alpha-hydroxysteroid dehydrogenase treatment. The conversion was nearly complete and we could not detect neutral metabolites, such as the mother compound, free 7 alpha-hydroxycholesterol and bile alcohols, as well as glucuronidated or sulfated bile acids. It is concluded that serum esterified 7 alpha-hydroxycholesterol could be effectively taken up by the liver, hydrolyzed by cholesterol esterase and metabolized via the normal biosynthetic pathway to taurine- or glycine-conjugated primary bile acids to be excreted into bile.     

The bile flow and biliary excretion of ursocholate in the rat

Several studies reported that ursodeoxycholate (but not its conjugates), when administered intravenously, increased the biliary bicarbonate concentration in the rat (1–3). At the same time, a complete dissociation between bile flow and the bile salt excretion rate was produced in the second hr of infusion (2). In order to examine whether this property was due to the 7β-hydroxy group in its molecular structure, the choleretic property of ursocholate (3α, 7β, 12α-trihydroxy-5β-cholanoic acid) was investigated in male Wistar rats. Immediately after the start of iv infusion of ursocholate at a rate of 1.2 μmole/min/100 g b. wt., both the bile flow and bile salt excretion rate began to increase. However, unlike with ursodeoxycholate, the bile salt excretion rate continued to be high in the second and third hr of infusion, while the bile flow rate gradually increased. Furthermore, the bicarbonate concentration in the bile fell slightly 10 min after the start of ursocholate infusion. Although the concentration tended to return to the baseline value before the bile salt infusion in the later period of observation, no significant increase in bicarbonate concentration was observed during the whole observation period. These properties were quite similar to those of cholate rather than those of ursodeoxycholate. However, a cholate infusion at the same rate of 1.2 μmole/min/100 g b.wt. caused a cholestasis as early as 20 to 30 min after the start of an infusion. These results suggest that the previously reported properties of ursodeoxycholate (that it causes a complete dissociation between the bile flow and bile salt excretion rate in the second hr and that it increases the biliary bicarbonate concentration) were not due to the 7β-hydroxy group in its steroidal structure, and that the choleretic property of ursocholate is similar to its 7α-hydroxy epimar, cholate. However, the much lower cytotoxicity of ursocholate compared to cholate appears to be due to the 7β-hydroxy group that ursocholate has.     

Biochemical site of regulation of bile acid biosynthesis in the rat

The production of bile salts by rat liver is regulated by a feedback mechanism, but it is not known which enzyme controls endogenous bile acid synthesis. In order to demonstrate the biochemical site of this control mechanism, bile fistula rats were infused intravenously with (14)C-labeled bile acid precursors, and bile acid biosynthesis was inhibited as required by intraduodenal infusion of sodium taurocholate. The infusion of taurocholate (11-14 mg/100 g of rat per hr) inhibited the incorporation of acetate-1-(14)C, mevalonolactone-2-(14)C, and cholesterol-4-(14)C into bile acids by approximately 90%. In contrast, the incorporation of 7alpha-hydroxycholesterol-4-(14)C into bile acids was reduced by less than 10% during taurocholate infusion. These results indicate that the regulation of bile acid biosynthesis is exerted via cholesterol 7alpha-hydroxylase provided that hepatic cholesterol synthesis is adequate.     

Incorporation of bile acid of low concentration into model and biological membranes studied by 2H and 31P NMR

We have analyzed the manner of incorporation of bile acid into lipid bilayers and resultant perturbation of the bilayer structure with lower bile acid/lipid ratios relevant to the physiological conditions (approximately 1 mM) by 2H and 31P NMR methods, as an aid to understanding the possible role as an endogenous tumor promoter in colon cancer besides the primary physiological function of solubilizing lipids. On the basis of the 2H quadrupole splittings of [6,6,7,7,8-2H5]deoxycholate and [11,11,12,12-2H4]chenodeoxycholate in the presence of lamellar multibilayers of egg yolk lecithin, these bile acids were found to be incorporated in such a manner that the B-D rings lie parallel with the normal of the bilayers when the ratio of the bile acid to lipid is low (less than 0.11). When the ratio is increased, these bile acid molecules are not dispersed entirely in the bilayer but aggregate to form micelles with lipids. Further, we studied the resultant perturbation of the multibilayers of egg yolk lecithin analyzed by using the 2H quadrupole splitting of [18,18,18-2H3]stearic acid as a probe and by 31P chemical shift anisotropy. We found that the bilayer structure is retained even at the bile acid-to-lipid ratio of 0.25, although a small amount of an isotropic phase appeared such as small vesicles and micelles. The molecular ordering of fatty acyl chains was rather enhanced by the presence of 1 mM deoxycholate in erythrocyte ghosts as seen from the 2H quadrupole splitting of [16,16,16-2H3]palmitic acid, although deoxycholate caused hemolysis in this condition. The former observation can be explained by the way the lipid-protein interaction is modified by deoxycholate located in the interface between the lipids and proteins.     

Bile acid biosynthesis: the metabolism of 7 alpha-hydroxy-4-cholesten-3-one in the bile fistula rat.

The two primary bile acids, cholic acid (3α,7α,12α-tri-hydroxy-5β-cholan-24-oic acid) and chenodeoxycholic acid (3α,7α-dihydroxy-5β-cholan-24-oic acid), are initially synthesized by way of identical precursors, and the point of divergence of this pathway is thought to occur at the intermediate 7α-hydroxy-4-cholesten-3-one. In order to test this hypothesis, bile fistula rats received simultaneous intra-venous infusions of ³H-7α-hydroxy-4-cholesten-3-one and ¹⁴C-cholesterol (5-cholesten-3β-ol). Assays of equal specific activities of the two bile acids from an infusion of ¹⁴C-cholesterol demonstrated the achievement of a steady state, and assays of equal specific activities from an infusion of ³H-7α-hydroxy-4-cholesten-3-one would-validate the above postulate. However, the infusion of ³H-7α-hydroxy-4-cholesten-3-one resulted in unequal specific activities in the bile acids of the rats investigated, with cholic acid always of a lower value. These results suggest that either 7α-hydroxy-4-cholesten-3-one is not the last common intermediate in the production of cholic acid and chenodeoxycholic acid, or that the infused bile acid intermediate was not metabolized in a fashion similar to that formed in the liver from cholesterol.     

Coordinated induction of bile acid detoxification and alternative elimination in mice: role of FXR-regulated organic solute transporter-alpha/beta in the adaptive response to bile acids

The bile acid receptor farnesoid X receptor (FXR) is a key regulator of hepatic defense mechanisms against bile acids. A comprehensive study addressing the role of FXR in the coordinated regulation of adaptive mechanisms including biosynthesis, metabolism, and alternative export together with their functional significance is lacking. We therefore fed FXR knockout (FXR(-/-)) mice with cholic acid (CA) and ursodeoxycholic acid (UDCA). Bile acid synthesis and hydroxylation were assessed by real-time RT-PCR for cytochrome P-450 (Cyp)7a1, Cyp3a11, and Cyp2b10 and mass spectrometry-gas chromatography for determination of bile acid composition. Expression of the export systems multidrug resistance proteins (Mrp)4-6 in the liver and kidney and the recently identified basoalteral bile acid transporter, organic solute transporter (Ost-alpha/Ost-beta), in the liver, kidney, and intestine was also investigated. CA and UDCA repressed Cyp7a1 in FXR(+/+) mice and to lesser extents in FXR(-/-) mice and induced Cyp3a11 and Cyp2b10 independent of FXR. CA and UDCA were hydroxylated in both genotypes. CA induced Ost-alpha/Ost-beta in the liver, kidney, and ileum in FXR(+/+) but not FXR(-/-) mice, whereas UDCA had only minor effects. Mrp4 induction in the liver and kidney correlated with bile acid levels and was observed in UDCA-fed and CA-fed FXR(-/-) animals but not in CA-fed FXR(+/+) animals. Mrp5/6 remained unaffected by bile acid treatment. In conclusion, we identified Ost-alpha/Ost-beta as a novel FXR target. Absent Ost-alpha/Ost-beta induction in CA-fed FXR(-/-) animals may contribute to increased liver injury in these animals. The induction of bile acid hydroxylation and Mrp4 was independent of FXR but could not counteract liver toxicity sufficiently. Limited effects of UDCA on Ost-alpha/Ost-beta may jeopardize its therapeutic efficacy.     

Effects of monosulfate esters of taurochenodeoxycholate on bile flow and biliary lipids in hamsters

The effect of the 3 alpha- and 7 alpha-monosulfate esters of taurochenodeoxycholate on bile flow and biliary lipids was compared to the effect of unsulfated taurochenodeoxycholate. Test bile salts were infused directly into the portal circulation through a catheter introduced into the splenic pulp. Recovery of unsulfated and sulfated bile salts was complete; no biotransformation of any of the administered compounds was noted. Equivalent choleresis was noted in response to administration of each of the test bile salts. Of particular interest, the biliary cholesterol and phospholipid content was tightly linked to biliary bile salt monosulfates; the slope of the line describing the relationship between bile salts and lipids was similar to that for the unsulfated bile salt. The critical micellar concentration of the 3 alpha- and 7 alpha-monosulfate esters was 19 mM and 18 mM, respectively. Sulfation of taurochenodeoxycholate, therefore, does not impair its bile secretory function. Despite a higher critical micellar concentration, biliary lipid excretion with monosulfate esters is equivalent to that seen with unsulfated bile salt. The role of hydrophobic/hydrophilic balance in the promotion of biliary lipid excretion may need to be redefined.     

Excretion of cholate glucuronide

[3-3H]Cholic acid glucuronide [7 alpha,12 alpha-dihydroxy-3 alpha-O-(beta-D-glucopyranosyluronate)-5 beta- cholan-24-oate] was synthesized and administered to rats prepared with either an external biliary fistula or a ligated bile duct. When bile fistula animals were given either microgram or milligram amounts of the glucuronide, biliary secretion of label was rapid and efficient: greater than 90% of the administered label was secreted within 60 min and total recovery of label in bile was 98.6 +/- 1.2%. Studies in which [14C]taurocholate was included in the dose indicated that this bile acid was secreted into bile significantly more rapidly than was the glucuronide. In animals with ligated bile ducts, urinary excretion was the major route of elimination: after 20 hr, 83.4 +/- 9.3% of the administered dose had been excreted in urine. Urinary excretion of cholate glucuronide was significantly more rapid than that of taurocholate. Gas-liquid chromatographic analysis of the methyl ester acetate derivatives of labeled compounds isolated from bile and urine by chromatography established that the bulk (greater than 70%) of the administered material was secreted in bile or excreted in urine as the intact cholate glucuronide. From these results, we conclude that the glucuronidation of cholic acid produces a derivative which is rapidly and effectively cleared from the circulation and excreted.     

Metabolism of 3α, 7α-dihydrexy-7β-methyl-5β-cholanoic acid and 3α,7β-dihydroxy-7α-methyi-5β-cholanoic acid in hamsters

The metabolic fate of the bile add analogs, 3α,7α-dihydroxy-7β-methyl-5β-cholanoic acid and 3α,7β-dihydroxy-7α-methyl-5β-cholanoic acid, was investigated and compared with that of chenodeoxycholic acid in hamsters. Both bile acid analogs were absorbed rapidly from the intestine and excreted into bile at similar to that of chenodeoxycholic acid. In the strain of hamster studied, the biliary bile were conjugated with both glycine and taurine. After continuous intravenous infusion, chenodeoxycholic acid the analogs became the major bile acid constituents in bile. After oral administration of a single dose of these compounds, fecal analysis revealed the existence of unchanged material (25–35%) as well as considerable amounts of metabolites (65–75%). The major metabolites excreted into feces were more polar than the starting material and were tentatively identified as trifaydroxy-7-methyl compounds by radioactive thin-layer chromatography. However, monohydroxy compounds were also found in the fecal extracts. These results show that chenodeoxycholic acid and ursodeoxycholic acid with a methyl group at the 7-position are resistant to bacterial 7-dehydroxylation than the normally occurring bile acids and that a certain proportion of these analogs is hydroxylated to give the corespondiag trihydroxy compound(s), In a control experiment, about 5% of administered chenodeoxychoulic acid was metabolized to a trihydroxy feile acid, but most of the compound (95%) was transformed into lithocholic acid.     

Regulation of cholesterol 7 alpha-hydroxylase by hepatic 7 alpha-hydroxylated bile acid flux and newly synthesized cholesterol supply

We measured hepatic cholesterol 7 alpha-hydroxylase activity, mass, and catalytic efficiency (activity/unit mass) in bile fistula rats infused intraduodenally with taurocholate and its 7 beta-hydroxy epimer, tauroursocholate, with or without mevalonolactone to supply newly synthesized cholesterol. Enzyme activity was measured by an isotope incorporation assay and enzyme mass by densitometric scanning of immunoblots using rabbit anti-rat liver cholesterol 7 alpha-hydroxylase antisera. Cholesterol 7 alpha-hydroxylase activity increased 6-fold, enzyme mass 34%, and catalytic efficiency 5-fold after interruption of the enterohepatic circulation for 48 h. When taurocholate was infused to the bile acid-depleted animals at a rate equivalent to the hepatic bile acid flux (27 mumol/100-g rat/h), cholesterol 7 alpha-hydroxylase activity and enzyme mass declined 60 and 61%, respectively. Tauroursocholate did not significantly decrease cholesterol 7 alpha-hydroxylase activity, mass and catalytic efficiency. The administration of mevalonolactone, which is converted to cholesterol, modestly increased cholesterol 7 alpha-hydroxylase activity and enzyme mass in the bile acid-depleted rats. However, when taurocholate was infused together with mevalonolactone, cholesterol 7 alpha-hydroxylase activity and catalytic efficiency were markedly depressed while enzyme mass did not change as compared with bile acid-depleted rats. These results show that (a) hepatic bile acid depletion increases bile acid synthesis mainly by activating cholesterol 7 alpha-hydroxylase with only a small rise in enzyme mass, (b) replacement with taurocholate for 24 h decreases both cholesterol 7 alpha-hydroxylase activity and mass proportionally, (c) when cholesterol is available (mevalonolactone supplementation), the infusion of taurocholate results in the formation of a catalytically less active cholesterol 7 alpha-hydroxylase, and (d) tauroursocholate, the 7 beta-hydroxy epimer of taurocholate, does not inhibit cholesterol 7 alpha-hydroxylase. Thus, bile acid synthesis is modulated by the catalytic efficiency and mass of cholesterol 7 alpha-hydroxylase. The enterohepatic flux of 7 alpha-hydroxylated bile acids and the formation of hepatic cholesterol apparently control cholesterol 7 alpha-hydroxylase by different mechanisms.