Quantification of LDL cholesteryl ester contribution to biliary steroids in the rat

The proportion of LDL cholesteryl ester converted to biliary steroids was quantified in the rat. The pre-existing pool of bile was allowed to drain for 10-12 h through a bile duct cannula. A single intravenous pulse injection of LDL labelled with [3H]cholesterol linoleyl ester was made, followed by a constant infusion of the same material in order to maintain constant specific radioactivity in plasma. A new steady state was achieved within 6 h and bile samples were then collected hourly until 12 h. Although substantial amounts (53-61 micrograms/h) of cholesteryl ester were released into the liver during LDL catabolism, only a very small fraction (0.8-1.90 micrograms/h) was found in biliary steroids. The proportion of LDL cholesteryl esters contributing to biliary steroids was only 1-2%. These results perhaps explain why perturbations to accelerate bile acid excretion have no effect on plasma LDL cholesterol concentration in the rat.     

Biliary, fecal and urinary excretion of [3H]-prostaglandins in the rat

The profiles of biliary, fecal and urinary excretion of tritium labeled prostaglandins (PG's) of differing biological activity were investigated in the rat. The PG's (10 micrograms/kg: 2 to 50 microCi/rat, in 1 ml polyethylene glycol-400) were administered intragastrically. Excretion data were expressed as a percentage of the total administered radioactivity. For the orally administered PG's 11R-methyl-16R-fluoro-15R-hydroxy-9-oxoprosta-ci s-5-trans-13-dienoic acid and its methyl ester, excretion was equally divided between urine and feces. The fecal and urinary profile of excretion of 3H after prostacyclin (PGI2) was similar to that following administration of 11R, 16, 16-trimethyl-15R-hydroxy-9-oxoprosta-cis-5-trans-13-dienoic acid (trimoprostil), a PG with antisecretory-antiulcer potential. However, PGI2 was very poorly absorbed from the intestine, while the absorption of trimoprostil was very efficient. Biliary excretion, with little entero-porto-hepatic biliary circulation, was the main route of elimination of trimoprostil, thereby resulting in rapid elimination of drug-related products and diminishing the potential for systemic liability in the rat.     

Bile protein secretion in the rat stimulated by taurocholate: effect of chloroquine

The biliary protein excretion during sodium taurocholate induced choleresis was studied in normal rats and in rats treated with the lysosomotropic agent, chloroquine. The analysis of the protein component in bile was made on SDS-polyacrilamide gel, and the individual polypeptides were quantitated by densitometry. The excretion of bile polypeptides was compared with that of lysosomal acid phosphatase. The biliary excretion of polypeptides of molecular mass lower than and equal to 54 kDa was markedly stimulated by taurocholate-induced choleresis. Chloroquine treatment of rats diminished the biliary excretion of such polypeptides and also inhibited their excretion induced by taurocholate. The behaviour of these polypeptides was well correlated to that of the lysosomal marker. The biliary excretion of polypeptide bands of a higher molecular mass (up to 140 kDa) did not show major changes during taurocholate-induced choleresis in any of the groups. The results indicate that biliary excretion of proteins in the rat may be either stimulated by taurocholate or may be independent of the bile salt. The former requires the functional integrity of chloroquine-sensitive hepatocyte compartments, which may involve the lysosomes.     

Biliary, fecal and uninary excretion of [H]-prostaglandins in the rat

The profiles of biliary, fecal and urinary excretion of tritium labeled prostaglandins (PG's) of differing biological activity were investigated in the rat. The PG's (10 μg/kg: 2 to 50 μCi/rat, in 1 ml polyethylene glycol-400) were administered intragastrically. Excretion data were expressed as a percentage of the total administered radioactivity. for the orally administered PG's 11R-methyl-16R-fluoro-15R-hydroxy-9-oxoprosta- -5- -13-dienoic acid and its methyl ester, excretion was equally divided between urine and feces. The fecal and urinary profile of excretion of ³H after prostacyclin (PGl₂) was similar to that following administration of 11R, 16, 16-trimethyl-15R-hydroxy-9-oxoprosta- -5- -13-dienoic acid (trimoprostil), a PG with antisecretory-antiulcer potential. However, PGl₂ was very poorly absorbed from the intestine, while the absorption of trimoprostil was very efficient. Biliary excretion, with little entero-porto-hepatic biliary circulation, was the main route of elimination of trimoprostil, thereby resulting in rapid elimination of drug-related products and diminishing the potential for systemic liability in the rat.     

ApoE promotes hepatic selective uptake but not RCT due to increased ABCA1-mediated cholesterol efflux to plasma

ApoE plays an important role in lipoprotein metabolism. This study investigated the effects of adenovirus-mediated human apoE overexpression (AdhApoE3) on sterol metabolism and in vivo reverse cholesterol transport (RCT). In wild-type mice, AdhApoE3 resulted in decreased HDL cholesterol levels and a shift toward larger HDL in plasma, whereas hepatic cholesterol content increased (P < 0.05). These effects were dependent on scavenger receptor class B type I (SR-BI) as confirmed using SR-BI-deficient mice. Kinetic studies demonstrated increased plasma HDL cholesteryl ester catabolic rates (P < 0.05) and higher hepatic selective uptake of HDL cholesteryl esters in AdhApoE3-injected wild-type mice (P < 0.01). However, biliary and fecal sterol output as well as in vivo macrophage-to-feces RCT studied with (3)H-cholesterol-loaded mouse macrophage foam cells remained unchanged upon human apoE overexpression. Similar results were obtained using hApoE3 overexpression in human CETP transgenic mice. However, blocking ABCA1-mediated cholesterol efflux from hepatocytes in AdhApoE3-injected mice using probucol increased biliary cholesterol secretion (P < 0.05), fecal neutral sterol excretion (P < 0.05), and in vivo RCT (P < 0.01), specifically within neutral sterols. These combined data demonstrate that systemic apoE overexpression increases i) SR-BI-mediated selective uptake into the liver and ii) ABCA1-mediated efflux of RCT-relevant cholesterol from hepatocytes back to the plasma compartment, thereby resulting in unchanged fecal mass sterol excretion and overall in vivo RCT.     

Multiple hepatic excretory mechanisms for organic anions. A study with succinylsulfathiazole and taurocholate in the rat.

A study was done to investigate interactions in the biliary excretion of [14-C]succinylsulfathiazole and [3-H]taurocholate after intravenous administration of the two compounds to anesthetized rats. Either compound administered alone increased bile flow and was excreted in the bile. The simultaneous infusion of both significantly increased bile flow above the values seen when either was given alone. However, the biliary-excretion rates of both compounds and their concentrations in bile were reduced when they were administered concomitantly. The simultaneous injection of radioactive taurocholate and succinylsulfathiazole did not alter significantly the plasma concentrations of either compound or their binding to plasma proteins from the values obtained when each was given alone. These results are consistent with a concept of competition between these compounds for the same liver-to-bile transport system. They contrast with previous observations that indicated that the concomitant administration of taurocholate increased the biliary excretion of acidic compounds. In the light of this work, it might be suggested that there are more than one transport system for the biliary excretion of organic anions.     

Effect of 3-methylcholanthrene on the biliary excretion of bromsulphthalein and eosine in newborn rats.

The biliary excretion rates of bromsulphthalein (BSP), bromsulphthalein-glutathione conjugate (BSP-GSH) and eosine have been studied in 3-methylcholanthrene (3-MC)-pretreated (100 mg/kg i.p.) and control rats aged 10 days. Liver weight was invariably increased after 3-MC treatment, associated with enhanced biliary excretion of total BSP. The increase in the biliary excretion of total BSP was due solely to the increased excretion of BSP-GSH. Following 3-MC pretreatment, BSP-GSH and eosine appeared in the bile in the same amount as in the control rats after i.v. administration of BSP-GSH and eosine. Pretreatment with 3-MC increased the ratio of BSP-GSH to BSP in the liver and bile. Our results suggest that the increased biliary excretion of total BSP following 3-MC treatment was due to an enhanced conjugation of BSP with GSH.     

Lack of biliary lipid excretion in the little skate, Raja erinacea, indicates the absence of functional Mdr2, Abcg5, and Abcg8 transporters

The ABC transporters bile salt export pump (BSEP; encoded by the ABCB11 gene), MDR3 P-glycoprotein (ABCB4), and sterolin 1 and 2 (ABCG5 and ABCG8) are crucial for the excretion of bile salt, phospholipid, and cholesterol, respectively, into the bile of mammals. The current paradigm is that phospholipid excretion mainly serves to protect membranes of the biliary tree against bile salt micelles. Bile salt composition and cytotoxicity, however, differ greatly between species. We investigated whether biliary phospholipid and cholesterol excretion occurs in a primitive species, the little skate, which almost exclusively excretes the sulphated bile alcohol scymnolsulphate. We observed no phospholipid and very little cholesterol excretion into bile of these animals. Conversely, when scymnolsulphate was added to the perfusate of isolated mouse liver perfusions, it was very well capable of driving biliary phospholipid and cholesterol excretion. Furthermore, in an erythrocyte cytolysis assay, scymnolsulphate was found to be at least as cytotoxic as taurocholate. These results demonstrate that the little skate does not have a system for the excretion of phospholipid and cholesterol and that both the MDR3 and the two half-transporter genes, ABCG5 and ABCG8, have evolved relatively late in evolution to mediate biliary lipid excretion. Little skate plasma membranes may be protected against bile salt micelles mainly by their high sphingomyelin content.     

Effect of indomethacin on the uptake, metabolism and excretion of 3-oxocholic acid: studies in isolated hepatocytes and perfused rat liver

3 alpha-Hydroxysteroid dehydrogenase catalyzes the reduction of 3-oxo-bile acids and binds 3 alpha-hydroxy bile acids. Indomethacin is a competitive inhibitor of the enzyme. In incubations of isolated rat hepatocytes, indomethacin delayed the intracellular reduction and the initial uptake of 3-oxocholic acid. Following a tracer dose of 3-oxocholic acid in perfused rat liver, rapid biliary excretion was observed mainly as taurocholic acid. Only 1.1% of the dose was recovered in the caval outflow and nearly all appeared in the first 5 min collection. When the tracer dose was given after initiating a constant infusion of indomethacin (50 microM), a dramatic decrease in biliary excretion was observed, still mainly as taurocholic acid, and 14% of the dose was recovered in the caval effluent: 10% in the first 5 min collection, mainly as 3-oxocholic acid, followed by a steady, slow release of mainly taurocholic acid. The increased intrahepatic retention of bile acids and slow release into perfusate and bile in response to indomethacin are consistent with displacement of bile acids from cytosolic protein.     

Effect of organic anions and bile acid conjugates on biliary excretion of taurine-conjugated bile acid sulfates in the rat

Biliary organic anion excretion is mediated by an ATP-dependent primary active transporter, canalicular multispecific organic anion transporter/multidrug resistance protein 2. On the other hand, a multiplicity of canalicular organic anion transporter/multidrug resistance protein 2 has been suggested. Therefore, to examine the effect of hydrophobicity on the substrate specificity of canalicular multispecific organic anion transporter/multidrug resistance protein 2, we examined the effect of organic anions and bile acid conjugates on biliary excretion of three taurine-conjugated bile acid sulfates with different hydrophobicity, taurolithocholate-3-sulfate, taurochenodeoxycholate3-sulfate, and taurocholate-3-sulfate in rats. Biliary excretions of these bile acid conjugates were delayed in Eisai hyperbilirubinemic rats. Biliary excretion of these bile acid conjugates was inhibited by sulfobromophthalein, whereas biliary excretion and taurocholate-3-sulfate was not inhibited by phenolphthalein glucuronide. Taurolithocholate-3-sulfate and ursodeoxycholate-3-glucuronide decreased biliary excretion of taurochenodeoxycholate-3-sulfate and taurocholate-3-sulfate, but ursodeoxycholate-3,7-disulfate did not affect biliary excretion of taurochenodeoxycholate-3-sulfate and taurocholate-3-sulfate. These findings indicate that very hydrophilic organic anions are not good substrates of canalicular multispecific organic anion transporter/multidrug resistance protein 2.     

A Delayed Nonlinear PBPK Model for Genistein Dosimetry in Rats

Genistein is an endocrine-active compound (EAC) found in soy products. It has been linked to beneficial effects such as mammary tumor growth suppression and adverse endocrine-related effects such as reduced birth weight in rats and humans. In its conjugated form, genistein is excreted in the bile, which is a significant factor in its pharmacokinetics. Experimental data suggest that genistein induces a concentration-dependent suppression of biliary excretion. In this article, we describe a physiologically based pharmacokinetic (PBPK) model that focuses on biliary excretion with the goal of accurately simulating the observed suppression. The mathematical model is a system of nonlinear differential equations with state-dependent delay to describe biliary excretion. The model was analyzed to examine local existence and uniqueness of a solution to the equations. Furthermore, unknown parameters were estimated, and the mathematical model was compared against published experimental data. This research was supported by the American Chemistry Council (formerly the Chemical Manufacturers Association, CMA Agreement Reference Number 9121).     

Copper transport and metabolism are normal in aceruloplasminemic mice.

Ceruloplasmin is an abundant serum glycoprotein containing greater than 95% of the copper found in the plasma of vertebrate species. Although this protein is known to function as an essential ferroxidase, the role of ceruloplasmin in copper transport and metabolism remains unclear. To elucidate the role of ceruloplasmin in copper metabolism, the kinetics of copper absorption, transport, distribution, and excretion were examined utilizing (64)Cu in wild-type and aceruloplasminemic mice. No differences in gastrointestinal absorption, hepatic uptake, or biliary excretion were observed in these animals. Furthermore, steady state measurements of tissue copper content utilizing (64)Cu and atomic absorption spectroscopy revealed no differences in the copper content of the brain, heart, spleen, and kidney. Consistent with these findings, the activity of copper-zinc superoxide dismutase in these tissues was equivalent in wild-type and ceruloplasmin-deficient mice. Hepatic iron was elevated 3.5-fold in aceruloplasminemic mice because of the loss of ferroxidase function. Hepatic copper content was markedly increased in aceruloplasminemic mice. As no differences were observed in copper absorption or biliary copper excretion, these data suggest that in these animals, hepatocyte copper intended for ceruloplasmin incorporation is trafficked into a compartment that is less available for biliary copper excretion. Taken together, these data reveal no essential role for ceruloplasmin in copper metabolism and suggest a previously unappreciated complexity to the subcellular distribution of this metal within the hepatocyte secretory pathway.     

Studies in the rat on the hepatic subcellular distribution and billary excretion of lithocholic acid.

1. A compartmental model has been used to derive the in vivo subcellular distribution of lithocholic acid in rat liver. The model is based on the values of the partition coefficients for the distribution of lithocholic acid between subcellular fractions and buffer. It also permits calculation of the amount of lithocholic acid which is in free solution in cytosol. 2. The hypothesis that the rate of biliary excretion of a bile acid depends on the proportion in free solution was investigated by comparing the rates of biliary excretion of lithocholic acid and glycocholic acid. The rate for lithocholic acid was substantially less than for glycocholic acid while the percentages of each bile acid in free solution were 0.8% and 10%, respectively. 3. The validity of the model was supported by the observation that the amounts of lithocholic acid predicted to be present in the nuclear and cytosolic fractions were similar to the amounts found after intravenous injection of the bile acid.     

Biliary excretion of foreign compounds. Biphenyl, stilboestrol and phenolphthalein in the rat: molecular weight, polarity and metabolism as factors in biliary excretion

1. The extent of biliary excretion of biphenyl, tetralin, stilboestrol and phenolphthalein was studied in the rat. 2. Biphenyl and its 4-hydroxy and 4,4′-dihydroxy derivatives are extensively excreted in the bile as glucuronides in amounts increasing in order of molecular weight. 3. Stilboestrol and its glucuronide are excreted almost quantitatively in the bile mainly as the monoglucuronide, as are also phenolphthalein and its glucuronide. 4. Tetralin is excreted to the extent of about 13% of the dose, mainly as ac-tetralyl glucuronides. 5. The results and those of Abou-El-Makarem, Millburn, Smith & Williams (1967) are discussed and it is concluded that the extent of biliary excretion of foreign compounds in rats depends on their molecular weight and their possessing a strongly polar anionic group. There appears to be a minimum value of this molecular weight below which little biliary excretion (i.e. not more than 5–10% of the dose) occurs. There is some latitude in the choice of this molecular weight, which is about 325±50. The necessary molecular weight and polar group can be acquired by metabolism. Above this minimum value biliary excretion increases with molecular weight. It is suggested that the mechanism of the biliary excretion of foreign compounds may be similar to that of conjugated bile acids, which are highly polar and whose molecular weights exceed 400.     

Studies on the metabolism of testosterone trans-4-n-butylcyclohexanoic acid in the cynomolgus monkey, Macaca fascicularis

Metabolism of intravenously administered testosterone trans-4-n-butylcyclohexanoate (T bucyclate), a potent, long-acting androgen, was studied in cynomolgus monkeys (Macaca fascicularis). About 5% of the radioactivity of a dose of doubly labeled ester (¹⁴C, ³H) was excreted via the gastrointestinal tract. Most of the administered radioactivity was excreted in the urine within 120 h. No intact T bucyclate was recovered from either compartment. Tritium attributed to bucyclic acid and its metabolites was excreted rapidly (peak excretion was at 6 h after injection), while ¹⁴C excretion, attributed to testosterone and its metabolites, extended over 4 days. Testosterone metabolites were excreted predominantly as sulfate esters. Analysis of urinary products derived from the bucyclic acid moiety of T bucyclate showed no products susceptible to glucuronidase treatment, and showed a mixture of unidentified solvolyzable and unconjugated products. No unmetabolized trans-4-n-butylcyclohexanoic acid was detected in urine or feces. It is concluded that metabolism of testosterone bucyclate is initiated in vivo in cynomolgus monkeys by hydrolysis of ester to testosterone and bucyclic acid. The bucyclate side chain is rapidly cleared, and the testosterone is retained in the circulation.     

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.     

Molecular weight as a factor in the excretion of monoquaternary ammonium cations in the bile of the rat, rabbit and guinea pig

1. The excretion in the bile and urine of intraperitoneally injected (14)C-labelled monoquaternary ammonium or pyridinium cations was measured in bile-duct-cannulated rats (ten compounds) and in guinea pigs and rabbits (six compounds). 2. Seven of these, namely N-methylpyridinium, tetraethylammonium, trimethylphenylammonium, diethylmethylphenylammonium, methylphenyldipropylammonium, dibenzyldimethylammonium and tribenzylmethylammonium, were excreted largely unchanged in the bile and urine. 3. 3-Hydroxyphenyltrimethylammonium, 3-bromo-N-methylpyridinium and cetyltrimethylammonium were metabolized to an appreciable extent in the rat. 4. In intact rats intraperitoneally injected trimethylphenylammonium (mol.wt. 136) was excreted mainly in the urine, dibenzyldimethylammonium (mol.wt. 226) was excreted in roughly equal amounts in the urine and faeces, and tribenzylmethylammonium (mol.wt. 302) was excreted mainly in the faeces. The faecal excretion of these compounds corresponded to their biliary excretion in bile-duct-cannulated rats. About 3-4% of tribenzyl[(14)C]methylammonium was eliminated as (14)CO(2). 5. In rats the extent of biliary excretion of four cations with molecular weights in the range 94-164 was less than 10% of the dose, whereas that of five cations with molecular weights 173-302 was greater than 10%. These results and other data from the literature suggested that the molecular weight needed for the biliary excretion of such cations to an extent of 10% or more of the dose was about 200+/-50. Studies with six cations in guinea pigs and rabbits suggest that this value applies also to these species. 6. The results suggest that the threshold molecular weight for the appreciable (>10%) biliary excretion of monoquaternary cations is different from that for anions (Millburn et al., 1967a; Hirom et al., 1972b). With rats, guinea pigs and rabbits, no significant species difference was noted, whereas with anions there is a marked species difference.     

Effect of ML-236B (compactin) on biliary excretion of bile salts and lipids, and on bile flow, in the rat

To assess the importance of de novo cholesterol synthesis for bile salt formation, the effects of ML-236B (an inhibitor of 3-hydroxy-3-methylglutaryl-coenzyme A reductase) on biliary excretion of bile salts and lipids were studied in rats with permanent catheters in bile duct, heart and duodenum. In rats having their bile diverted continuously for 8 days, duodenal administration of ML-236B (50 mg/kg) caused an immediate transient choleresis, which subsided after 2 h. Concomitant with the choleresis concentrations of bile salt, phospholipid and cholesterol fell, but this decrease was maintained for 6 h. Consequently, ML-236B inhibited biliary output salts and lipids from the second till the sixth hour after injection. The kinetics of biliary excretion of intravenously injected [14C]taurocholate were not affected by ML-236B administration. In rats having their biliary catheter connected to the duodenal catheter, or in rats with prolonged bile diversion but treated with mevalonolactone, ML-236B again caused a transient choleresis (having subsided after 2 h), but now did not affect biliary excretion of bile salts and lipids. It is concluded that (1) ML-236B causes a transient bile salt-independent choleresis, (2) ML-236B depresses excretion of bile salts and lipids by blocking mevalonate synthesis and not by blocking the bile salt or lipid transport, (3) biliary excretions of phospholipids and cholesterol partly depend on excretion of bile salt, and (4) in rats with a prolonged total bile diversion newly formed mevalonate is a major substrate for bile salt synthesis.     

Postnatal development of the hepatobiliary transport of phenolsulfonphthalein in rats

1. The postnatal development of the biliary excretion of phenolsulfonphthalein (PSP) was studied in male Wistar rats. 2. Following i.v. injection of PSP at 200 mumol/kg body wt, a maximal biliary excretion of 175 +/- 10 nmol/min/100 g body wt and 32 +/- 5 nmol/min/100 g body wt was reached for unconjugated and conjugated PSP, respectively, in the adult group. 3. The maximal biliary excretion of conjugated PSP was significantly lower in the 20-, 30- and 40-day-old groups as compared to the adults. The excretion of unconjugated dye was also significantly lower in 20- and 30-day-old rats. 4. The postnatal development of PSP excretion was unrelated to changes in the activity of UDP-glucuronosyltransferase. The importance of other factors is also discussed.