Comparison of biliary excretion and metabolism of lithocholic acid and its sulfate and glucuronide conjugates in rats

Biliary excretion and biotransformation of tracer doses of [14C]lithocholic acid and its sulfate and glucuronide intravenously injected into bile-drainaged rats were compared. Biliary excretion efficiency was in the order of unconjugate sulfate glucuronide and all conjugates were completely excreted into bile within 60 min after injection. Only tracer doses of radioactivity were found in the liver and urine. About 90% of radiolabeled bile acids in bile were conjugated with taurine immediately after injection of lithocholic acid, whereas lithocholic acid-glucuronide was only partly conjugated with taurine all the time (less than 6%) and excreted into bile mainly as native compound. In the first 10 min, 66% of lithocholic acid-sulfate was conjugated with taurine and it gradually proceeded up to 87%. Hydroxylation at C-6 and C-7 positions of lithocholic acid proceeded time-dependently up to 45%. No hydroxylation was observed with lithocholic acid-sulfate or glucuronide. Differences of biliary excretion rate of these conjugates may be one of the reasons for the delayed decrease of sulfated and glucuronidated bile acids in serum after bile drainage to patients with obstructive jaundice of during the recovery of acute hepatitis than non-esterified bile acids.     

High biliary and systemic excretion of a glucuronide during its hepatic synthesis

Using isolated perfused rat liver and ¹⁴C-p-nitrophenol as a model substrate for glucuronidation, evidence was obtained that during synthesis of p-nitrophenyl-glucuronide biliary excretion of the metabolite is much higher than would be expected from concentrations of the metabolite in the circulation. Isotopic dilution experiments indicate that newly synthesized glucuronide is in a pool which is readily available for biliary excretion but is not easily miscible, or diluted, by the glucuronide in the circulation.     

Structure-activity relationship of the cholestatic activity of dihydrotestosterone glucuronide, allo bile acids and lithocholate

Dihydrotestosterone glucuronide (DHTG), a series of 5 alpha-bile acids, or allo-bile acids (3 alpha-hydroxy-5 alpha-cholanic acid, 3-keto-5 alpha-cholanic acid and 3 beta-hydroxy-5 alpha-cholanic acid) and their normal bile acid analogues (3 alpha-hydroxy-5 beta-cholanic acid or lithocholate, 3-keto-5 beta-cholanic acid and 3 beta-hydroxy-5 beta-cholanic acid) were administered intravenously to female rats in order to determine their effects on bile flow. All agents caused a rapid and profound inhibition of bile flow which was dose-dependent. The logarithm of the dose vs the cholestatic response curve for DHTG, the allo-bile acids and lithocholate were all parallel. DHTG was the most potent congener and was two times more potent than 3-keto-5 alpha-cholanic acid and 5 times more potent than lithocholate. These data indicate that the glucuronic acid moiety and the trans configuration of the A and B rings of the steroid nucleus confer the greatest cholestatic potency.     

Alterations induced by fascioliasis and cirrhosis on the biliary excretion of cefmetazole in wistar rats

1. Alterations induced by fascioliasis and cirrhosis on the biliary excretion of cefmetazole have been studied in Wistar rats.2. Both infestation with Fasciola hepatica and experimental cirrhosis originated a significant decrease in the biliary excretion and in bile flow increase induced by the drug.3. Administration of the β-lactam antibiotic induced a lower degree of uncoupling of biliary lipid secretion in the cirrhotic and fasciolotic animals, but the effect was evident in all experimental groups.     

Biliary excretion of warfarin metabolites and their metabolism by rat gut flora.

The bile was determined to be the major excretory route for ¹⁴C-warfarin in the rat with approximately 10% of the administered dose excreted within 5 hours after injection. Relatively little radioactivity appeared in the faces, indicating that considerable enterohepatic recycling was taking place. Less than 4% of the radioactivity in the bile could be extracted with organic solvents. Following incubation of the bile with β-glucuronidase or rat gut flora, however, the bulk of the radioactivity was extractable. The extent of gut flora-mediated hydrolysis of these polar biliary warfarin conjugates was approximately the same as obtained with β-glucuronidase. Approximately $\text{1}\text{3}$ of the gut flora-hydrolyzable conjugates were labile and subject to nonenzymatic hydrolysis at 37 C for 24 h. Chromatographic examination of the extracts from β-glucuronidase-treated bile revealed the presence of warfarin and 4′-, 6-, 7-, and 8-hydroxywarfarin. Warfarin and 7-hydroxywarfarin were the most abundant metabolites in the extracts.     

Cholestasis, metabolism and biliary lipid secretion during perfusion of rat liver with different bile salts.

The biological effects of bile acids depend largely upon their molecular structure. When bile acid uptake exceeds the maximal biliary secretory rate (SRm) cholestasis occurs. In order to characterize the influence of bile acid structure on its cholestatic potency we systematically studied SRm, maximal bile flow, maximal and cumulative phospholipid and cholesterol secretion with different taurine-conjugated tri-, di- and keto bile acids (Table I) in the isolated perfused rat liver. Bile acids with a high critical micellar concentration (CMC) promoted the greatest bile flow; a positive non-linear correlation between CMC and maximal bile flow was found. 3 alpha-Hydroxylated bile acids with a hydroxyl group in 6 alpha and/or 7 beta position and lacking a 12 alpha hydroxy group had a high SRm. SRm was not related to CMC or maximal bile flow, respectively. Phospholipids and cholesterol were secreted in a nearly fixed ratio of 12:1; a strong linear relationship could be observed. Cumulative phospholipid secretion over 48 min was significantly lower for non and poor micelle forming bile acids (TDHC and TUC) than for those with comparatively low CMC values (TUDC, TC, THC, THDC, TCDC) (70-140 vs. 210-450 nmol/g liver). At SRm all bile acids with good micelle forming properties showed a similar cumulative biliary lipid output. However, when biliary lipid output was related to 1 mumol bile acid secreted bile acids with a low SRm induced the highest lipid secretion (TCDC, TC). These data (1) demonstrate that a 6 alpha and/or a 7 beta hydroxy group on the steroid nucleus reduce cholestatic potency if the 12 alpha hydroxy group is absent, (2) suggest that in the case of micelle forming bile acids the total amount of phospholipids secreted in bile (depletion of cellular phospholipids) is associated with the occurrence of cholestasis whereby bile acids with a low SRm deplete the cellular phospholipid content at much lower bile acid concentrations than those with a higher SRm and (3) imply that bile acids with non and poor micelle forming properties (TDHC, TUC) presumably do not cause cholestasis (solely) by depletion of cellular phospholipids.     

Sodium arsenite induced alterations in bilirubin excretion and heme metabolism

The acute administration of sodium arsenite (AsIII) to rats resulted in a biphasic alteration of the hepatic cytosolic "free" heme pool. The first stage was an increase in the cytosolic "free" heme without significant effects on the content of cytochrome P-450 or on bilirubin excretion. The second stage consisted of a continuous fall of the cytosolic "free" heme and of the content of cytochrome P-450. These changes were concurrent with an eight-fold increase in heme oxygenase activity and associated with marked elevations in the biliary excretion of bilirubin. The bile was collected from chronically cannulated rats to avoid artifacts related to anesthesia or post anesthetic effects. The rapid increase in biliary excretion of labeled heme degradation products indicated an increased breakdown of newly synthesized heme. Immunoelectrophoresis of bile proteins showed an altered pattern of bile protein excretion. The increased biliary haptoglobin suggested some hemolysis, while the reduction in the free immunoglobulin A (IgA) secretory component showed an AsIII-related decreased protein transport across hepatocytes to bile. Further research is required to assess the direct role of an increased heme degradation in the genesis of the hepatotoxic effects of AsIII.     

Inhibition of biliary taurocholate excretion during menadione metabolism in perfused rat liver

In perfused rat liver menadione elicits substantial oxidation in both the NADPH and GSH redox systems. Biliary excretion of GSSG is increased several-fold. Menadione derivatives appear in the bile predominantly as the menadione-S-glutathione conjugate, thiodione (60%), or as conjugates derived therefrom (17%). About 10% appear as menadione glucuronides. The excretion of taurocholate into bile is strongly inhibited upon menadione infusion. The inhibition of taurocholate excretion is small in livers with a low content of Se-GSH-peroxidase and in glutathione-depleted livers. In these livers intracellular GSSG and biliary GSSG release remain at low values, although menadione still imposes oxidative stress as indicated by an oxidation of intracellular NADPH. Under anoxic conditions menadione has little influence on both the NADPH and GSH redox systems and also on biliary taurocholate excretion. The amount of thiodione released into bile is similar to that found under normoxia, whereas the amount of glucuronidated products almost doubled. We conclude (a) that intracellular formation of GSSG by menadione occurs via the generation of hydrogen peroxide; (b) that the inhibition of biliary taurocholate excretion by menadione is related to the increased formation of glutathione disulfide; and (c) that menadione derivatives show little, if any, contribution to the inhibition of taurocholate excretion.     

Human intestinal sulphation of lithocholate: A new site for bile acid metabolism

The liver and intestinal lumen are both important sites in the entero-hepatic circulation of bile salts. Lithocholate, a secondary bile acid and a potent hepatotoxin is probably detoxified in man predominantly by hepatic sulphation. Other sites may also be important. Because steroid sulphating enzymes exist throughout the gastrointestinal tract we have examined the invitro lithocholate sulphation capacity of healthy and diseased ileal and colonic mucosal samples using radio-isotope tracer techniques. Lithocholate sulphation was demonstrated in healthy and diseased ileal mucosa but not in the colonic mucosal samples studied. This new site for lithocholate metabolism acts as a further protective mechanism to prevent toxic unsulphated lithocholate reaching the liver. These findings suggest that the intestinal mucosa may have an important role in the metabolism of other bile acids.     

Transfer of heavy metals from sediment to fish,and their biliary excretion

Uptake and biliary excretion of metals were studied in rainbow trout, Oncorhynchus mykiss, exposed through spiked sediment to a mixture of seven heavy metals. Metal concentrations and toxicity of bile and blood plasma were used as indicators of exposure. Among the seven metals (Cd, Cr, Cu, Hg, Ni, Pb, and Zn) only three (Cu, Hg, and Pb) were concentrated in the bile (bile-plasma ratio >1). Bile-plasma ratios in the rainbow trout were similar to those found in rats for Cu and Hg. Daphnia magna bioassays were used to determine toxicity of bile and blood plasma in the same trout. Toxicity of bile and blood plasma increased after treatment with acid. An analysis of variance (ANOVA) showed that toxicity of bile and blood plasma to D. magna in metal-exposed trout was significantly correlated with (1) bile and blood plasma test concentration, (2) acid treatment of bile and blood plasma (hydrolysis of metal-plasma and metal-bile complexes) and (3) sediment concentration of metals during exposure of trout. In order to significantly detect the magnitude of the exposure to a xenobiotic the biomarker must respond in a dose- or time-dependent manner. Therefore, the potential use of bile toxicity as a biomarker of heavy metal exposure in fish is probably limited by the low bioconcentration of many of these toxicants in bile.     

Enantioselective inhibition of the binding of rac-profens to human serum albumin induced by lithocholate

The reversible binding of lithocholate to human serum albumin determines a decrease of the binding of rac-ketoprofen. The process was followed by displacement chromatography using increasing concentrations of the competitor, i.e., lithocholate, in the mobile phase. The inhibition of rac-ketoprofen binding resulting was enantioselective and greater displacement was observed for the (S) enantiomer. The displacement process resulting was competitive in nature, the two enantiomers of ketoprofen binding to the same binding site as the modifier. The investigation was extended to other nonsteroidal antiinflammatory drugs. The enantioselective binding inhibition was larger in the case of rac-naproxen and rac-suprofen with respect to the phenomenon observed in the case of rac-ketoprofen. The difference in circular dichroism spectroscopy was also used to characterize the binding of lithocholate to human serum albumin. This bile acid was proven to bind to site II on human serum albumin. The results, as obtained by displacement chromatography and difference circular dichroism spectroscopy, strongly support the hypothesized role of bile acids in inducing the enantioselective inhibition of ketoprofen binding to human serum albumin in patients suffering from liver diseases.     

Direct quantitative estimation of several iodothyronines in rat bile by radioimmunoassay and basal data on their biliary excretion

A method was developed for the hydrolysis of conjugated iodothyronines in bile with the aid of β-glucuronidase / arylsulfatase and for subsequent direct estimation of total and free iodothyronines with the aid of specific radioimmunoassay. The amount of conjugated fraction could then be calculated from the difference. Thus, basal biliary excretion of several iodothyronines was measured in 31 normal, fed rats in which the bile duct was drained with polyethylene tubing under pentobarbiturate anesthesia and the bile was collected for 2 h. The free fraction of thyroxine, 3,5,3′-triiodothyronine and 3,3′-diiodothyronine was approx. 30% of total content, while that of 3,3′,5′-triiodothyronine and 3,5-diiodothyronine was approx. 20% and that of 3′,5′-diiodothyronine was less than 10%. This suggests some considerable diffrences in the conjugation of individual iodothyronines was about 3–8 times higher than in plasma. This shows close interrelations between the iodothyronine deiodinating pathway in liver cells in vivo and the spectrum of iodothyronine in bile. The average ratio of T₃/rT₃ as found in bile was about 4.     

Cholestasis induced nephrotoxicity: The role of endogenous opioids

AimsElevated levels of endogenous opioids play a pivotal role in several deleterious consequences of cholestasis. Renal dysfunction occurs in cholestasis but its exact mechanism is still unknown. In this study, we investigated the role of endogenous opioids in cholestasis induced nephrotoxicity.Main methodsThirty-five rats were divided into five groups. In groups 1 and 2 BDL rats received either daily subcutaneous 20 mg/kg of naltrexone or its vehicle, for 7 days after BDL. In groups 3 and 4, BDL or Sham rats received no injections. In group 5, normal rats received subcutaneous injections of 20 mg/kg/day of naltrexone for 7 days. At the 7th day, 24 h urine was collected to measure urinary N-acetyl-β-D-glucosaminidase (NAG) as an early marker of renal tubular injury. Kidney samples were then collected for light and electron microscopic studies.Key findingsBDL significantly increased NAG activity compared to sham groups. Naltrexone significantly reversed NAG activity to normal levels in BDL animals. Naltrexone treatment in BDL animals also significantly reversed ALT and AST to their normal levels. In light and electron microscopic studies, there were significant structural alterations in BDL samples, which were mostly prevented in naltrexone treated BDL animals.SignificanceSignificant changes in urinary NAG activity and renal morphology of cholestatic rats were reversed by naltrexone treatment. These results suggest a possible role for endogenous opioids in inducing cholestatic nephrotoxicity.