Bile acid conjugation in the chimpanzee: effective sulfation of lithocholic acid

To characterize the hepatic biotransformation in the chimpanzee of the primary bile acid chenodeoxycholic acid (chenic) and its major bacterial metabolite lithocholic acid (lithocholic) a mixture of tracer amounts of¹⁴C-lithocholic and³H-chenic was injected intravenously into two animals with a bile fistula; the chemical form of radioactivity appearing in bile was inferred using thin layer chromatography. About 80% of chenic, and 70% of lithocholic was recovered in 90 min. Chenic was completely conjugated in bile, appearing predominantly as chenyltaurine (52%) and chenylglycine (37%). An unidentified conjugate (about 11%) was also found. Lithocholic was excreted completely as taurine and glycine conjugates, but the majority (63%) of conjugates was sulfated. Sulfation increased progressively with time, and lithocholylglycine was sulfated more than lithocholyltaurine. We conclude that the chimpanzee is similar to man in that the secondary bile acid lithocholic is efficiently sulfated. The chimpanzee thus differs from the baboon and rhesus monkey which sulfate lithocholic poorly. However, the chimpanzee differs from man and is similar to the baboon and rhesus monkey in showing preferential conjugation of bile acids with taurine. The results imply that hepatotoxicity caused by chenic, which is well documented in the rhesus monkey and baboon and has been related to defective lithocholic sulfation, should not occur in the chimpanzee.The work was supported by GSF, as well as Mayo Foundation, Alexander von Humboldt Foundation and Grant AM 16770     

Effect of ursodeoxycholic acid administration on bile acid composition in hamster bile

The modification in the composition of bile acids in hamster by the administration of high dose of ursodeoxycholic acid (UDCA) was investigated. Male Golden Syrian hamsters were divided into five groups: a control group, two groups that received 0.5 g of UDCA per 100 g of standard diet during 30 and 60 days and another two groups that received 1 g of UDCA per 100 g of standard diet during 30 and 60 days. After ether anaesthesia the gallbladder was removed and bile was immediately aspirated. Bile acids were determined by high performance liquid chromatography (HPLC). Taurolithocholic (TLCA) and glycolithocholic acids (GLCA) increased significantly in all treated groups. The glyco/tauro ratio of 0.69 in controls became more than 1 in treated animals except in the case of lithocholic acid (LCA) conjugates which remained less than 1. UDCA derivatives increased proportionally to the administered dose and the cholic/cheno ratio diminished significantly. A moderate increase of 3- and 7-keto derivatives of chenodeoxycholic acid (CDCA) was observed in all treated groups but the above mentioned increment was especially evident in 3-keto derivatives. A high percentage of UDCA administered in the hamster was likely transformed to CDCA and the glyco conjugates of the bile acids were the predominant species except for the LCA derivatives.     

Further observations on the in vitro metabolism of chenodeoxycholic acid and ursodeoxycholic acid

Human intestinal flora from normal subjects was incubated with chenodeoxycholic acid (CDCA), ursodeoxycholic acid (UDCA) and 7-ketolithocholic acid (7-keto-LCA) for various periods of time and the metabolites thus formed were quantitatively determined by gas chromatography (GC) and identified by gas chromatography-mass spectrometry (GC-MS). Results showed that: a) the 7 alpha-dehydroxylation of CDCA produced more lithocholic acid more rapidly than the 7 beta-dehydroxylation of UDCA; b) the oxidation of CDCA produced more 7-keto-LCA more rapidly than the oxidation of UCDA; and c) the transformation of 7-keto-LCA produced CDCA and UDCA peaks after 8 h of incubation and a lithocholic peak at the end of 24 h of incubation after rising from almost 0% at 4 h. The presence of 3 alpha-hydroxy-delta 6-5 beta-cholenic acid, a hypothetical unsaturated intermediate in the 7-dehydroxylation of CDCA and UDCA, could not be detected by GC-MS.     

Interactions of combined bile acids on hepatocyte viability: cytoprotection or synergism

Cholestasis results from hepatocyte dysfunction due to the accumulation of bile acids in the cell, many of which are known to be cytotoxic. Recent evidence implicates competitive antagonism of key cytotoxic responses as the mechanism by which certain therapeutic bile acids might afford cytoprotection against cholestasis. In this work, we compare the relative cytotoxicity of bile acids in terms of dose- and time-dependence. To better elucidate the controversy related to the therapeutic use of ursodeoxycholate (UDCA) in cholestatic patients, we also evaluated the effects of bile acid combinations. Viability of Wistar rat hepatocytes in primary culture was measured by LDH leakage after 12 and 24 h exposure of cells to the various bile acids. All unconjugated bile acids caused a dose-dependent decrease in cell viability. The tauro- and glyco-conjugates of chenodeoxycholate (CDCA) and UDCA were all less toxic than the corresponding unconjugated form. Although relatively non-toxic, UDCA caused synergistic cell killing by lithocholate (LCA), CDCA, glyco-CDCA (GCDC) and tauro-CDCA (TCDC). Glycoursodeoxycholate decreased the toxicity of GCDC, but potentiated the toxicity of unconjugated CDCA and LCA. The tauro-conjugate of UDCA had no significant effect. These data suggest that at cholestatic concentrations, bile acid-induced cell death correlates with the degree of lipophilicity of individual bile acids. However, these results indicate that the reported improvement of biochemical parameters in cholestatic patients treated with UDCA is not due to a direct effect of UDCA on hepatocyte viability. Therefore, any therapeutic effect of UDCA must be secondary to some other process, such as altered membrane transport or nonparenchymal cell function.     

Kinetic analysis of bile acid sulfation by stably expressed human sulfotransferase 2A1 (SULT2A1)

1. Human sulfotransferase 2A1 (SULT2A1) is a member of the hydroxysteroid sulfotransferase (SULT2) family that mediates sulfo-conjugation of a variety of endogenous molecules including dehydroepiandrosterone (DHEA) and bile acids. In this study, we have constructed a stable cell line expressing SULT2A1 by transfection into HEK293 cells. The expression system was used to characterize and compare the sulfation kinetics of DHEA and 15 human bile acids by SULT2A1.

2. Formation of DHEA sulfate demonstrated Michaelis–Menten kinetics with apparent K_m and V_max values of 3.8?μM and 130.8 pmol min^?1 mg^?1 protein, respectively. Sulfation kinetics of bile acids also demonstrated Michaelis–Menten kinetics with a marked variation in apparent K_m and V_max values between individual bile acids.

3. Sulfation affinity was inversely proportional to the number of hydroxyl groups of bile acids. The monohydroxy- and most toxic bile acid (lithocholic acid) had the highest affinity, whereas the trihydroxy- and least toxic bile acid (cholic acid) had the lowest affinity to sulfation by SULT2A1. Intrinsic clearance (CL_int) of ursodeoxycholic acid (UDCA) was approximately 1.5- and 9.0-fold higher than that of deoxycholic acid (DCA) and chenodeoxycholic acid (CDCA), respectively, despite the fact that all three are dihydroxy bile acids.

     

Effects of fatty acid esters of cheno- and ursodeoxycholic acids on gallstone formation in hamsters

Fatty acid esters (at the 7 position) of chenodeoxycholic (CDCA) and ursodeoxycholic (UDCA) acids have been tested for their effects on formation and dissolution of gallstones in hamsters. The free bile acids were fed at a level of 0.2% of the diet and esters were fed at equimolar levels. The earlier finding that CDCA does not affect gallstone formation in hamsters fed the Dam and Christensen diet were confirmed. The acetic, butyric and lauric acid esters of CDCA had a very slight inhibitory effect on lithogenesis but CDCA 7 oleate and linoleate completely inhibited gallstone formation. UDCA and its 7 oleate inhibited both formation and progression of gallstones. The observed effects are probably a function of the form of the bile acid and not of the esterifying acid. The observation that ethyl oleate has a slight litholytic effect suggests that the acid moiety of the ester may exert a slight influence.     

Biotransformation of diclofenac sodium (Voltaren) in animals and in man. II. Quantitative determination of the unchanged drug and principal phenolic metabolites, in urine and bile.

1. Quantitative determinations of unchanged diclofenac and two of its major phenolic metabolites were made by reverse isotope dilution analysis on urine of rat, dog, rhesus monkey, baboon and man and on bile of rat, dog and man. Isotope dilution analysis was performed before and after various methods of enzymic and chemical hydrolysis. 2. The same samples were also analysed by two-dimensional t.l.c. and subsequent autoradiography, to estimate the remaining phenolic metabolites. 3. In contrast to rat, rhesus monkey, baboon and man, which excrete mainly hydroxylated metabolites, the dog does not oxidize diclofenac. Dog urine contained a relatively stable taurine conjugate of diclofenac, and in the bile an ester glucuronide was excreted, which decomposed even in weakly alkaline soln. 4. The unstable ester glucuronide found in dog bile was also demonstrable in rat bile. It presumably hydrolyses in the duodenum, releasing diclofenac which undergoes enterohepatic circulation.     

Differing effect of chenodeoxycholic acid and ursodeoxycholic acid on bile acids in rat colonic wall and contents

Bile acids may promote experimental colonic cancer. Many studies correlate fecal bile acids and colorectal carcinomas. Little is known on bile acids in the colonic mucosa and their relation to luminal bile acids. We, therefore, studied bile acids in colonic wall and contents of normal female Wistar rats and after 14 days' administration of chenodeoxycholic acid or ursodeoxycholic acid (90 mg/kg daily), two bile acids used in medicamentous cholelitholysis. Both regimens increase total bile acids in colonic contents, ursodeoxycholic acid produces a higher rise in toxic lithocholic acid. In the colonic wall, only ursodeoxycholic acid causes an increase of most nonsulfated bile acids including lithocholic acid. Bile acid patterns do not correlate in colonic wall and contents. We conclude that increased colonic wall bile acids after ursodeoxycholic acid administration warrant control in man. In future colorectal carcinoma studies, not only fecal, but also mucosal bile acid concentrations should be correlated to carcinogenesis.     

鹅去氧胆酸对胆汁淤积孕鼠血生化指标、肝脏病理及胎鼠预后的影响

目的以熊去氧胆酸(UDCA)为对比,研究法尼醇X受体(FXR)激动剂——鹅去氧胆酸(CDCA)对妊娠期肝内胆汁淤积(ICP)孕鼠血生化指标、肝脏病理及胎鼠预后的影响。方法建立ICP孕鼠模型,将孕17d的SD大鼠40只随机分成对照组、ICP组、UDCA组和CDCA组。比色法检测用药前后各组丙氨酸转氨酶(ALT),天冬氨酸转氨酶(AST),碱性磷酸酶(ALP)和总胆酸(TBA)水平,光镜下观察孕鼠肝脏病理学变化,记录胎鼠身长、尾长、体重及死胎数。结果ICP组ALT、AST、ALP、TBA明显高于对照组(P<0.008 3);与ICP组比较,UDCA组ALT、AST、ALP明显降低(P<0.008 3),但TBA无明显改善(P>0.008 3);CDCA组ALT、AST、ALP无明显改善(P>0.008 3),但TBA显著降低(P<0.008 3);2治疗组胎鼠的身长、尾长、体重及死亡率与ICP组比较均无统计学差异(P>0.008 3)。光镜下见ICP组肝细胞肿胀变性,肝血窦变窄,胆管扩张;UDCA组肝细胞脂肪变性明显,小叶结构正常;CDCA组小部分肝细胞脂肪变性,小叶结构正常。结论UDCA能改善ICP孕鼠肝功能指标,但不能有效降低血胆汁酸水平及胎鼠死亡率;CDCA能明显降低胆汁酸,促进胆汁酸代谢,但其安全性还有待确定。     

A novel constitutive androstane receptor-mediated and CYP3A-independent pathway of bile acid detoxification

Cytosolic sulfotransferase (SULT)-mediated sulfation plays an essential role in the detoxification of bile acids and is necessary to avoid pathological conditions, such as cholestasis, liver damage, and colon cancer. In this study, using transgenic mice bearing conditional expression of the activated constitutive androstane receptor (CAR), we demonstrate that activation of CAR is both necessary and sufficient to confer resistance to the hepatotoxicity of lithocholic acid (LCA). Surprisingly, the CAR-mediated protection is not attributable to the expected and previously characterized CYP3A pathway; rather, it is associated with a robust induction of SULT gene expression and increased LCA sulfation. We have also provided direct evidence that CAR regulates SULT expression by binding to the CAR response elements found within the SULT gene promoters. Interestingly, activation of CAR was also associated with an increased expression of the 3'-phosphoadenosine 5'-phosphosulfate synthetase 2 (PAPSS2), an enzyme responsible for generating the sulfate donor 3'-phosphoadenosine-5'-phosphosulfate. Analysis of gene knockout mice revealed that CAR is also indispensable for ligand-dependent activation of SULT and PAPSS2 in vivo. Therefore, we establish an essential and unique role of CAR in controlling the mammalian sulfation system and its implication in the detoxification of bile acids.     

High-performance liquid chromatographic determination of ursodeoxycholic acid after solid phase extraction of blood serum and detection-oriented derivatization

Ursodeoxycholic acid (3, 7β-dihydroxy-5β-cholanoic acid, UDCA) is a therapeutically applicable bile acid widely used for the dissolution of cholesterol-rich gallstones and in the treatment of chronic liver diseases associated with cholestasis. UDCA is more hydrophilic and less toxic than another therapeutically valuable bile acid, chenodeoxycholic acid (CDCA), the 7-epimer of UDCA. Procedures for sample preparation and HPLC determination of UDCA in blood serum were developed and validated. A higher homologue of UDCA containing an additional methylene group in the side chain was synthetized and used as an internal standard (IS). Serum samples with IS were diluted with a buffer (pH=7). The bile acids and IS were captured using solid phase extraction (C18 cartridges). The carboxylic group of the analytes was derivatized using 2-bromo-2′-acetonaphthone (a detection-oriented derivatization), and reaction mixtures were analyzed (HPLC with UV 245 nm detection; a 125–4 mm column containing Lichrospher 100 C18, 5 μm; mobile phase: acetonitrile–water, 6:4 (v/v)). Following validation, this method was used for pharmacokinetic studies of UDCA in humans.     

Metabolism of arylacetic acids. 2. The fate of [14C]hydratropic acid and its variation with species.

1. (+/-)-[methyl-14C]-Hydratropic acid was administered to man, rhesus monkey, cat, rabbit and fruit bat. 2. All species excreted 60-100% of administered 14C in the urine in 24 h, and unchanged hydratropic acid accounted for 0-17% of the dose. 3. In man, the urinary 14C consisted of a very small quantity (1%) of unchanged hydratropic acid with the remainder as hydratropylglucuronide. 4. Hydratropylglucuronide was the major urinary excretion product in the 4 animal species, while the glycine conjugate was present in the urine of cat and rat. Additionally, cats excreted the taurine conjugate of hydratropic acid. 5. Bile-duct cannulated rats excreted 20-30% of an injected dose of [14C] hydratropic acid in the bile in 3 h mainly as hydratropylglucuronide.     

Efficacy of bile acid therapy for gallstone dissolution: a meta-analysis of randomized trials

To define better the efficacy of bile acid therapy for dissolution of radiolucent gallstones, we performed a meta-analysis of published trials from January 1966 to September 1992. Studies were identified using a MEDLINE computer search followed by an extensive manual search. The inclusion criteria used were: randomized trial, radiolucent gallstones in a visualizing gallbladder on oral cholecystography, and complete stone dissolution confirmed by oral cholecystography or ultrasound. Study results were pooled into 6 groups: placebo: high- and low-dose chenodeoxycholic acid (CDCA) (≥10 mg.kg/day and < 10 mg.kg/day); high- and low-dose ursodeoxycholic acid (UDCA) (< 7 mg. kg/day and < 7 mg. kg/day) and combined CDCA plus UDCA. Homogeneity calculations were performed and the percentage of complete stone dissolution calculated for each group with 95% confidence intervals. Of 66 trials identified, 23 comprising 1949 patients met the inclusion criteria. A total of 1062 patients were treated with CDCA, 819 with UDCA and 78 combination therapy. In studies > 6 months' duration, high-dose UDCA completely dissolved stones in 37.3% of patients (95% C.I. 33–42%), low-dose UDCA in 20.6%) and high-dose CDCA 18.2% (95% C.I. 15–21%). Based on only two studies, combination therapy achieved dissolution in 62.8% (95% C.I. 51–74%) of patients. Stones less than 10 mm dissolved significantly more frequently than stones larger than 10 mm. This analysis shows that UDCA in doses greater than 7 mg. kg/day taken for greater than 6 months will dissolve radiolucent gallstones in 38% of patients. The combination of UDCA and CDCA may be more efficacious but this observation is based upon only 78 patients and requires confirmation in further randomized trials.     

Substrate-dependent inhibition of human cytochrome P450 3A catalytic activity by specific isomers of vitamin E

OBJECTIVE Lithocholic acid,which is a secondary bile acid,has been reported to be hepatotoxic and carcinogenic.It is metabolized by human cytochrome P450 3A(CYP3A)to form 3-ketocholanoic acid.A previous study suggests that vitamin E isomers(tocotrienols and tocopherols)are metabolized by CYP3 A.Given that substrates of an enzyme may competitively inhibit the enzyme,we determined whether alpha-tocotrienol,gamma-tocotrienol,delta-tocotrienol,tocotrienol-rich mixture(a mixture consisting of 25.7% d-α-tocotrienol,2.6% d-β-tocotrienol,28.6% d-γ-tocotrienol,8.4% d-δ-tocotrienol,25.6% d-α-tocopherol,and 4.3% d-α-tocomonoenol),and alpha-tocopherol inhibit human liver microsomal CYP3Aactivity,as assessed by the enzymatic conversion of lithocholic acid to 3-ketocholanoic acid and of testosterone to6β-hydroxytestosterone.METHODS Enzymatic formation of 3-ketocholanoic acid via lithocholic acid 3-oxidation was determined in pooled human liver microsomes and recombinant CYP3A4 and CYP3A5.Enzyme inhibition assay was conducted in a mixture containing potassium phosphate buffer(pH 7.4),human liver microsomes,NADPH,lithocholic acid,and various concentrations of a test chemical.The amount of 3-ketocholanoic acid formed was quantified by a novel,validated ultra-high performance liquid chromatography-tandem mass spectrometry(UPLC-MS-MS)method.RESULTS Lithocholic acid was metabolized to 3-ketocholanoic acid by human recombinant CYP3A4 and CYP3A5enzymes and human liver microsomes.Alpha-tocotrienol,gamma-tocotrienol,delta-tocotrienol,and tocotriernol-rich mixture,but not alpha-tocopherol,inhibited 3-ketocholanoic acid formation in human liver microsomes.Concentration-response experiments indicated that tocotrienol-rich mixture and delta-tocotrienol inhibited 3-ketocholanoic acid formation with IC50 values of 6.6±2.1μg·mL-1 and 19.0±1.0μmol·L-1,respectively.CONCLUSION Tocotrienols inhibited CYP3A-catalyzed lithocholic acid 3-oxidation but not CYP3A-catalyzed testosterone 6-beta-hydroxylation.This suggests that lithocholic acid and testosterone bind to different CYP3 Abinding sites and that tocotrienols preferentially inhibit the lithocholic acid binding site on CYP3 Aenzymes.     

Chenodeoxycholic acid-mediated activation of the farnesoid X receptor negatively regulates hydroxysteroid sulfotransferase

Hydroxysteroid sulfotransferase catalyzing bile acid sulfation plays an essential role in protection against lithocholic acid (LCA)-induced liver toxicity. Hepatic levels of Sult2a is up to 8-fold higher in farnesoid X receptor-null mice than in the wild-type mice. Thus, the influence of FXR ligand (chenodeoxycholic acid (CDCA) and LCA) feeding on hepatic Sult2a expression was examined in FXR-null and wild-type mice. Hepatic Sult2a protein content was elevated in FXR-null and wild-type mice fed a LCA (1% and 0.5%) diet. Treatment with 0.5% CDCA diet decreased hepatic Sult2a to 20% of the control in wild-type mice, but increased the content in FXR-null mice. Liver Sult2a1 (St2a4) mRNA levels were reduced to 26% in wild-type mice after feeding of a CDCA diet, while no decrease was observed on Sult2a1 mRNA levels in FXR-null mice after CDCA feeding. A significant inverse relationship (r(2)=0.523) was found between hepatic Sult2a protein content and small heterodimer partner (SHP) mRNA level. PCN-mediated increase in Sult2a protein levels were attenuated by CDCA feeding in wild-type mice, but not in FXR-null mice. Human SULT2A1 protein and mRNA levels were decreased in HepG2 cells treated with the FXR agonists, CDCA or GW4064 in dose-dependent manners, although SHP mRNA levels were increased. These results suggest that SULT2A is negatively regulated through CDCA-mediated FXR activation in mice and humans.     

Species differences in bile acids I. Plasma and urine bile acid composition

Maintenance of bile acid (BA) homeostasis is essential to achieve their physiologic functions and avoid their toxic effects. The marked differences in BA composition between preclinical safety models and humans may play a major role in the poor prediction of drug‐induced liver injury using preclinical models. We compared the composition of plasma and urinary BAs and their metabolites between humans and several animal species. Total BA pools and their composition varied widely among different species. Highest sulfation of BAs was observed in human and chimpanzee. Glycine amidation was predominant in human, minipig, hamster and rabbit, while taurine amidation was predominant in mice, rat and dogs. BA profiles consisted primarily of tri‐OH BAs in hamster, rat, dog and mice, di‐OH BAs in human, rabbit and minipig, and mono‐OH BA in chimpanzee. BA profiles comprised primarily hydrophilic and less toxic BAs in mice, rat, pig and hamster, while it primarily comprised hydrophobic and more toxic BAs in human, rabbit and chimpanzee. Therefore, the hydrophobicity index was lowest in minipig and mice, while it was highest in rabbit, monkey and human. Glucuronidation and glutathione conjugation were low in all species across all BAs. Total concentration of BAs in urine was up to 10× higher and more hydrophilic than plasma in most species. This was due to the presence of more tri‐OH, amidated, sulfated and primary BAs, in urine compared to plasma. In general, BA profiles of chimpanzee and monkeys were most similar to human, while minipig, rat and mice were most dissimilar to human.     

Role of FXR in regulating bile acid homeostasis and relevance for human diseases

Recent studies reveal that bile acids are signalling molecules that activate several nuclear receptors and regulate many physiological pathways and processes to maintain bile acid and cholesterol homeostasis. Analysis of orphan receptor expression patterns in enterohepatic tissues identified bile acids as ligands for farnesoid X receptor (FXR). The primary bile acid chenodeoxycholic acid (CDCA) was shown to be the most potent FXR ligand in vitro at an EC50 of 10-50 microM. FXR can also be activated by the secondary bile acids lithocholic acid (LCA) and deoxycholic acid (DCA). Upon activation FXR heterodimerises with 9-cis retinoic X receptor (RXR) and regulates a cohort of genes involved in cholesterol catabolism and bile acids biosynthesis. Thus bile acid-activated FXR directly induces expression of Small Heterodimer Partner (SHP), a nuclear receptor that suppresses bile acid biosynthesis down-regulates the Na+ taurocholate cotransport peptide (NTCP), a pump depicted to transport bile acids from the lumen into hepatocyte, and induces expression of bile salt export pump (BSEP), the principal bile acid efflux transporter in the liver. As demonstrated by the Fxr null mice, FXR defends the liver against cholestasis. The 6-ethyl derivative of CDCA (6-ECDCA) is approximately 100 fold more potent than CDCA in activating FXR in vitro. In vivo administration of 6-ECDCA protects against cholestasis induced by estrogen and LCA in rats providing evidence that development of potent FXR agonists might represent a new approach for the treatment of cholestastic disorders.     

Metabolism and excretion of a chromone carboxylic acid (FPL 52757) in various animal species

1. The disposition of the chromone carboxylic acid (FPL 52757) in several species has been investigated. The compound is extensively metabolized by hydroxylation in rat, mouse, ferret, squirrel monkey, cynomolgus monkey, rabbit, hamster, stumped-tailed macaque and baboon (e.g. 50–100° in rat, cynomolgus monkey and squirrel monkey).

2. The plasma clearance of the chromone in rat, rabbit and squirrel monkey was 138, 44 and 59 ml/kg per?h respectively. Plasma clearance by the dog was slower (13 ml/kg per?h) and due mainly to elimination of unchanged compound.

3. As the dog is particularly susceptible to FPL 52757-induced hepatotoxicity the parent compound may be responsible. Species which clear the compound rapidly compared with the dog did not show a hepatotoxic response.

4. Although metabolism occurs in man the clearance is still slow (15 ml/kg per?h) and may be one reason for human susceptibility to a mild hepatotoxic effect with the compound.