Bile acid sulfates. II. Formation, metabolism, and excretion of lithocholic acid sulfates in the rat

Sulfate esterification has been shown previously to be a prominent feature of lithocholate metabolism in man. These studies were undertaken to ascertain whether this metabolic pathway is also present in rats, and to investigate the physiological significance of bile acid sulfate formation. Lithocholic acid-24-(14)C was administered to bile fistula rats, and sulfated metabolites were identified in bile by chromatographic and appropriate degradative procedures. They constituted only a small fraction (2-9%) of the total metabolites but a more significant fraction (about 20%) of the secreted monohydroxy bile acids, most of the lithocholate having been hydroxylated by the rat liver. When sulfated glycolithocholate was administered orally, it was absorbed from the intestine without loss of the sulfate, presumably by active transport, and secreted intact into the bile. In comparison with non-sulfated lithocholate, an unusually large fraction (24%) of the sulfated bile acid was excreted in the urine, and fecal excretion took place more rapidly. Both the amino acid and sulfate moieties were extensively removed prior to excretion in the feces. Hydroxylation of bile acid sulfates or sulfation of polyhydroxylated bile acids did not occur to any great extent, if at all.     

Sensitive detection of specific repair endonucleases: radial diffusion assay utilizing differential alkaline denaturation of supercoiled and nicked PM2 DNA in agarose gels

An improved method for separation and quantitation of sulfated neutral and acidic steroids in human feces was developed. The procedure consists of separation of sulfated steroids on Sephadex LH-20 and hydrolysis by cholylglycine hydrolase followed by quantitation and identification of the trimethylsilylether derivatives by gas-liquid chromatography and gas-liquid chromatography-mass spectroscopy. Using this procedure, we detected no sulfated bile acids in human feces. However, sulfated cholesterol was detected in the sulfated bile acid fraction obtained from human fecal extracts. Analysis showed that cholesterol sulfate comprised 12.3, 11.2, and 31.0% of the total neutral sterol fraction in the three fecal samples. Using our procedures, cholesterol sulfate and bile acid sulfates in a biological mixture can be quantitated and identified when they are present.     

Specificity of bile salt sulfatase activity from Clostridium sp. strains S1.

Clostridium sp. strain S1, an unnamed bile acid-desulfating strain from rat intestinal microflora (S.M. Huijghebaert, J. A. Mertens, and H. J. Eyssen, Appl. Environ. Microbiol. 43:185-192, 1982), was examined for its ability to desulfate different bile acid sulfates and steroid sulfates in growing cultures. Clostridium sp. strain S1 desulfated the 3 alpha-monosulfates of chenodeoxycholic, deoxycholic, and cholic acid, but not their 7 alpha- or 12 alpha-monosulfates. Among the 3-sulfates of the 5 alpha- and 5 beta-bile acids, only bile acid-3-sulfates with an equatorial sulfate group were desulfated. Hence, Clostridium sp. strain S1 desulfated the 3-sulfates of bile acids with a 3 alpha, 5 beta-, a 3 beta, 5 alpha- or a 3 beta, delta 5-structure. In contrast, the bile acid-3-sulfates with a 3 beta, 5 beta- or a 3 alpha, 5 alpha-structure were not desulfated. In addition, Clostridium sp. strain S1 did not hydrolyze the equatorial 3-sulfate esters of C19 and C21 steroids and cholesterol or the phenolic 3-sulfate esters of estrone and estradiol. 23-Nordeoxycholic acid with a C-23 carboxyl group was also not desulfated, in contrast to the 5 beta-bile acid 3 alpha-sulfates with a C-24 or C-26 carboxyl group. Therefore, the specificity of the sulfatase of Clostridium sp. strain S1 is related to the location of the sulfate group on the bile acid molecule, the equatorial orientation of the sulfate group, and the structure of the C-17 side chain, its carboxyl group, and chain length.     

Occurrence of sulfated 5alpha-cholanoates in rat bile

Bile acids in bile from male and female rats with cannulated bile ducts have been analyzed by repetitive scanning gas-liquid chromatography-mass spectrometry after initial fractionation of conjugate classes on diethylaminohydroxypropyl Sephadex LH-20. Sex differences were observed in the amounts and types of bile acids in the sulfate fraction. The proportion of total bile acids excreted as sulfates was higher in female (0.9-1.3%) than in male (0.1-0.2%) rats. Most of the sulfated bile acids had a 5alpha configuration, allochenodeoxycholic acid being the major compound in bile from female rats. This bile acid was also present in the nonsulfate fraction but could not be found in bile from male rats. The results indicate that gas-liquid chromatography-mass spectrometry has to be used to provide sufficient specificity in the bile acid analyses. Thus, compounds from the sulfate fraction having the retention times of cholic and chenodeoxycholic acid derivatives were found to be due to derivatives of the 3beta,5alpha-isomers of these bile acids.     

Determination of sulfated and nonsulfated bile acids in serum by mass fragmentography

A Sep-Pak C18 cartridge was used for purification of bile acids from serum. Three kinds of deuterium labeled internal standards were required for accurate measurement of individual sulfated and nonsulfated bile acids. These internal standards were added to the serum before its application to the cartridge. Separation of sulfated and nonsulfated bile acids was performed on piperidinohydroxypropyl Sephadex LH-20 column chromatography. The nonsulfate fraction was submitted to alkaline hydrolysis, and the sulfate fraction to solvolysis followed by alkaline hydrolysis. Each fraction was converted to the hexafluoroisopropyl-trifluoroacetyl derivatives and quantitated by mass fragmentography. The recovery of each bile acid sulfate was quite satisfactory. In fasting healthy subjects the mean of total nonsulfated bile acids in serum was 1.324 micrograms/ml, and that of total sulfated bile acids was 0.450 micrograms/ml. Sulfated lithocholic acid comprised a large part of sulfated bile acids in healthy subjects.     

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.     

Bile acid sulfates. III. Synthesis of 7- and 12-monosulfates of bile acids and their conjugates using a sulfur trioxide-triethylamine complex.

The 7- and 12-monosulfates of chenodeoxycholic acid, deoxycholic acid, and cholic acid were prepared by sulfation of the protected bile acids with sulfur trioxide-triethylamine in pyridine overnight and were isolated by precipitation as the p-toluidinium salt after removing the protecting group(s). The taurine conjugates were obtained by conjugating the bile acid sulfates with taurine in hot dimethylformamide (DMF) in the presence of N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ). A new procedure of preparing glycine conjugated bile acid sulfates by direct conjugation of the bile acid sulfate triethylammonium salt with ethyl glycinate in boiling chloroform in the presence of EEDQ is also described. The advantage of these procedures over other procedures are their simplicity and their higher yields (tyically above 90%) The thin layer chromatographic mobilities of these sulfates are presented. The influence of side chain and hydroxyl group configurations on the properties of bile acid sulfates is briefly discussed.     

The synthesis of diazo, halo, and sulfoxy bile acid derivatives: potential affinity labels.

Bile acid derivatives, with and without C-3 sulfate groups, and having either the diazo- or halomethylketone moieties, have been synthesized in good yield and purity. The synthetic sequence, COOH leads to COC1 leads to COCHN2 leads to COCH2X, was used with deoxycholic and cholic acids, which requires carefully controlled quench, work-up, and purification procedures, especially for the 3-sulfate esters (made from deoxycholic acid derivatives only). The pure title compounds are anticipated to be useful chemical probes (affinity labels), especially the completely water soluble sulfates, toward our studies of ileal active transport of bile salts. A new use for Sephadex LH-20 as a sulfate ester protecting group is reported. Also developed were the use of acetamide hydrochloride complex as a mild hydrochlorination reagent and a neutral desalting method for sulfate esters of deoxycholic acid derivatives.     

Bile acid sulfates. I. Synthesis of lithocholic acid sulfates and their identification in human bile

Sulfate esters of lithocholic, glycolithocholic, and taurolithocholic acids were synthesized using sulfur trioxide in pyridine; they were purified by crystallization from methanol or ethanol as the diammonium salts, and their chemical compositions, infared spectra, and chromatographic behavior were determined. Strong alkaline hydrolysis of these sulfates, as commonly performed during quantitative and qualitative analyses of conjugated bile salts, was found to result in a number of degradation products, presumably through disruption of the C-O bond of the hydroxyl group and conversion of the original steroid to isolithocholate and other (possibly olefinic) compounds. After oral administration of lithocholate-(14)C to three patients with cholelithiasis, radioactive metabolites having the chromatographic properties of sulfated lithocholates were isolated from bile and, confirming a preliminary report (1), were identified as sulfated glycolithocholate and taurolithocholate by their characteristic chromatographic mobilities during a series of specific hydrolytic procedures and by crystallizing them to constant specific activities with the synthetic sulfates. The fraction of endogenous lithocholate present in bile as the sulfate was calculated for two patients by isotope dilution and was shown to be 41% and 75% of the total. Sulfation can be expected to affect the physiological and pharmacological properties of lithocholates and may, therefore, influence the toxic properties of these compounds.     

Bile acids and bile alcohols in a child with hepatic 3 beta-hydroxy-delta 5-C27-steroid dehydrogenase deficiency: effects of chenodeoxycholic acid treatment

Duodenal bile, urine, plasma, and feces from a child with hepatic 3 beta-hydroxy-delta 5-C27-steroid dehydrogenase deficiency were analyzed by fast atom bombardment mass spectrometry and gas chromatography-mass spectrometry to investigate the formation and excretion of abnormal bile acids and bile alcohols. The biliary bile salts consisted of glycocholic acid (25%) and of sulfated and glycine conjugated di- and trihydroxycholenoic acids (55%), two C27 bile acids, and eleven sulfated bile alcohols (mainly tetrols, 20%), all having 3 beta,7 alpha-dihydroxy-delta 5 or 3 beta,7 alpha,12 alpha-trihydroxy-delta 5 ring structures. In plasma, sulfated cholenoic acids constituted 65% and unconjugated 3 beta,7 alpha-dihydroxy-5-cholestenoic acid 25% of the total level, 71 micrograms/ml. The urinary excretion of the former was 30.4 mg/day and that of unsaturated bile alcohol sulfates, mainly pentols, 7 mg/day. The predominant bile acid in feces was an unconjugated epimer of 3 beta,7 alpha,12 alpha-trihydroxy-5-cholenoic acid, and small amounts of cholic acid were present. The minimum total excretion was 11.3 mg/day. Treatment with chenodeoxycholic acid resulted in marked clinical improvement and normalized liver function tests. Further studies are needed to define the mechanism of action. Plasma bile acids decreased to 1.6 micrograms/ml and urinary excretion to 3.4 mg/day. Chenodeoxycholic and ursodeoxycholic acids became predominant in all samples. The fecal excretion of unsaturated cholenoic acid sulfates increased to 40 mg/day compared to 89 mg/day of saturated bile acids. The results provide further support for a defective hepatic 3 beta-hydroxy-delta 5-C27-steroid dehydrogenase deficiency, and indicate that the 3 beta-hydroxy-delta 5 bile acids are formed via 7 alpha-hydroxycholesterol. The formation of glycocholic acid may be due to an incomplete enzyme defect or to transformation of the 3 beta-hydroxy-delta 5 structure by bacterial and hepatic enzymes during an enterohepatic circulation.     

Electrophysiological evidence for detection and discrimination of pheromonal bile acids by the olfactory epithelium of female sea lampreys (<Emphasis Type="Italic">Petromyzon marinus</Emphasis>)

Electro-olfactograms were used to determine sensitivity and specificity of olfactory organs of female sea lampreys (Petromyzon marinus) to four bile acids: 3-keto petromyzonol sulfate and 3-keto allocholic acid from spermiating males and petromyzonol sulfate and allocholic acid from larvae. Spermiating male bile acids are thought to function as a mating pheromone and larval bile acids as a migratory pheromone. The response threshold was 10^–12 mol l^–1 for 3-keto petromyzonol sulfate and 10^–10 mol l^–1 for the other bile acids. At concentrations above 10^–9 mol l^–1, the sulfated bile acids showed almost identical potency, as did the non-sulfated bile acids. The two sulfated bile acids were more potent than the two non-sulfated ones. In addition, 3-keto petromyzonol sulfate and water conditioned with spermiating males induced similar concentration-response curves and response thresholds. Cross-adaptation experiments demonstrated that the sulfated and non-sulfated bile acids represent different odors to the olfactory epithelium of females. Further exploration revealed that 3-keto petromyzonol sulfate represents a different odor than petromyzonol sulfate, while 3-keto allocholic acid and allocholic acid represent the same odor. Results indicate that male-specific bile acids are potent and specific stimulants to the female olfactory organ, supporting the previous hypothesis that these bile acids function as a pheromone.Abbreviations 3kACA 3-keto allocholic acid - 3kPZS 3-keto petromyzonol sulfate - ACA allocholic acid - ANOVA analysis of variance - ELISA enzyme-linked immunosorbent assay - EOG electro-olfactogram - PIR percent initial response - PZS petromyzonol sulfate - SMW spermiating male washings     

Hepatic bile acid metabolism during early development revealed from the analysis of human fetal gallbladder bile

A detailed study of the qualitative and quantitative composition of bile acids in human fetal gallbladder bile is described. Bile was collected during early gestation (weeks 16-19) and analyzed by gas chromatography and mass spectrometry, fast atom bombardment ionization mass spectrometry, and high performance liquid chromatography. Bile acids were separated into different conjugate groups by chromatography on the lipophilic anion exchange gel, diethylaminohydroxypropyl Sephadex LH-20. Quantitatively more than 80% of the bile acids were secreted into bile conjugated to taurine. Unconjugated bile acids and glycine conjugates accounted for 5-10% of the total biliary bile acids. Bile acid sulfates were present only in trace amounts indicating that quantitatively sulfation is not an important pathway in bile acid metabolism during development. Total biliary bile acid concentrations were low (0.1-0.4 mM) when compared to reported values for adult bile (greater than 10 mM). Chenodeoxycholic acid was the major biliary bile acid and exceeded cholic acid concentrations by 1.43-fold indicating either a relative immaturity in 12 alpha-hydroxylase activity during early life or a dominance of alternative pathways for chenodeoxycholic acid synthesis. A relatively large proportion of the biliary bile acids comprised metabolites not found in adult bile. The presence of relatively high proportions of hyocholic acid (often greater than cholic acid) and several 1 beta-hydroxycholanoic acid isomers indicates that C-1 and C-6 hydroxylation are important pathways in bile acid synthesis during development. We describe, for the first time, evidence for the existence of a C-4 hydroxylation pathway in the metabolism of bile acids, which may be unique to early human development. Mass spectrometry was used to confirm the identification of 3 alpha,4 beta,7 alpha-trihydroxy-5 beta-cholanoic and 3 alpha,4 beta-dihydroxy-5 beta-cholanoic acids. Quantitatively, these C-4 hydroxylated bile acids accounted for 5-15% of the total biliary bile acids of the fetus, suggesting that C-4 hydroxylation is quantitatively an important pathway in the bile acid metabolism during early life.     

Formation of delta 2- and delta 3-cholenoic acids from bile acid 3-sulfates by a human intestinal Fusobacterium strain

We isolated two strains of an unnamed Fusobacterium species from human intestinal microflora, which stereospecifically transformed bile acid 3-sulfates into C-3-unsubstituted, ring A-unsaturated bile acids. Both 3 alpha- and 3 beta-sulfates of 5 beta-bile acids were metabolized to delta 3-5 beta-cholenoic acids; 3 beta-sulfates of 5 alpha-bile acids were converted into a mixture of delta 2-5 alpha-bile acids and 3 alpha-hydroxy-5 alpha-bile acids, whereas 3 alpha-sulfates of 5 alpha-bile acids were left intact. Unsulfated bile acids were not transformed into unsaturated derivatives. These strains differ from previously isolated intestinal bacteria, which desulfated bile acid sulfates without further transformation.     

Formation of delta 2- and delta 3-cholenoic acids from bile acid 3-sulfates by a human intestinal Fusobacterium strain.

We isolated two strains of an unnamed Fusobacterium species from human intestinal microflora, which stereospecifically transformed bile acid 3-sulfates into C-3-unsubstituted, ring A-unsaturated bile acids. Both 3 alpha- and 3 beta-sulfates of 5 beta-bile acids were metabolized to delta 3-5 beta-cholenoic acids; 3 beta-sulfates of 5 alpha-bile acids were converted into a mixture of delta 2-5 alpha-bile acids and 3 alpha-hydroxy-5 alpha-bile acids, whereas 3 alpha-sulfates of 5 alpha-bile acids were left intact. Unsulfated bile acids were not transformed into unsaturated derivatives. These strains differ from previously isolated intestinal bacteria, which desulfated bile acid sulfates without further transformation.     

Partial characterization of the steroidsulfatases in Peptococcus niger H4.

The strictly anaerobic intestinal Peptococcus niger H4 synthesizes three different steroidsulfatase enzymes: a constitutive arylsulfatase and two inducible alkylsteroidsulfatases. The arylsulfatase desulfates estrogen-3-sulfates and phenylsulfates. The two alkylsteroidsulfatases desulfate, respectively, 3 alpha-sulfates and 3 beta-sulfates of delta 5, 5 alpha, and 5 beta androstanes, pregnanes, and bile acids. Cholesterol-3 beta-sulfate was not desulfated by the alkylsteroidsulfatases nor were steroids or bile acids that were sulfated in positions other than the 3 position. The alkylsteroidsulfatases were induced by their substrates; bile acid sulfates, however, were poor inducers of the 3 beta-sulfatase and did not induce the 3 alpha-sulfatase activity. In intact bacterial cells, taurine and sulfite suppressed the induction of the alkylsteroidsulfatases and inhibited the activity of the arylsulfatase and alkylsteroidsulfatases. In cell homogenates, the arylsulfatase and alkylsteroidsulfatases activities were inhibited by sulfite and sulfate but not by taurine. Our results support the hypothesis that the main function of the steroidsulfatases in P. niger H4 is to provide the bacteria with sulfur for dissimilatory purposes.     

Partial characterization of the steroidsulfatases in Peptococcus niger H4

The strictly anaerobic intestinal Peptococcus niger H4 synthesizes three different steroidsulfatase enzymes: a constitutive arylsulfatase and two inducible alkylsteroidsulfatases. The arylsulfatase desulfates estrogen-3-sulfates and phenylsulfates. The two alkylsteroidsulfatases desulfate, respectively, 3 alpha-sulfates and 3 beta-sulfates of delta 5, 5 alpha, and 5 beta androstanes, pregnanes, and bile acids. Cholesterol-3 beta-sulfate was not desulfated by the alkylsteroidsulfatases nor were steroids or bile acids that were sulfated in positions other than the 3 position. The alkylsteroidsulfatases were induced by their substrates; bile acid sulfates, however, were poor inducers of the 3 beta-sulfatase and did not induce the 3 alpha-sulfatase activity. In intact bacterial cells, taurine and sulfite suppressed the induction of the alkylsteroidsulfatases and inhibited the activity of the arylsulfatase and alkylsteroidsulfatases. In cell homogenates, the arylsulfatase and alkylsteroidsulfatases activities were inhibited by sulfite and sulfate but not by taurine. Our results support the hypothesis that the main function of the steroidsulfatases in P. niger H4 is to provide the bacteria with sulfur for dissimilatory purposes.     

Solution properties of sulfated monohydroxy bile salts. Relative insolubility of the disodium salt of glycolithocholate sulfate

Physical-chemical properties of the major sulfated monohydroxy bile salts of man are described. In general, the sulfates are significantly more water-soluble than the non-sulfated species as a result of lower critical micellar temperatures, high aqueous monomeric solubilities and critical micellar concentrations. Nevertheless, at 37 degrees C the disodium salt of glycolithocholate sulfate, the major monohydroxy bile salt of man is not more soluble than its non-sulfated form. Since aqueous solubility correlates inversely with the cholestatic potential of bile salts, our results suggest that this sulfate may be potentially hepatoxic. Micellar solubility of phosphatidylcholine and cholesterol by the majority of non-sulfated and sulfated monohydroxy bile salts is slight. Nonetheless, phosphatidylcholine is very well solubilized by taurolithocholate sulfate but cholesterol solubility is not increased appreciably. Cholesterol saturation in model bile systems of taurochenodeoxycholate and phosphatidylcholine is impaired by the addition of sulfated lithocholate conjugates but with physiological bile salt compositions this reduction is not significant.     

Bile acid metabolism in early life: studies of amniotic fluid

Bile acid metabolism of the human fetus was examined in early gestation (weeks 13-19) and compared with the full-term fetus from the analysis of amniotic fluid collected from healthy pregnant women. Total individual bile acids were determined by gas-liquid chromatography-mass spectrometry after solvolysis and hydrolysis of bile acid conjugates. Additionally, bile acids were separated according to their mode of conjugation by lipophilic anion exchange chromatography. Qualitatively the bile acid profiles of amniotic fluid in early gestation were similar and markedly different from those of full-term fetuses. Chenodeoxycholic acid was the major bile acid identified in early gestation and concentrations exceeded those of cholic acid, but by full term this relationship was reversed. Over 50 bile acids were identified in the amniotic fluids, these included C-1, C-4, and C-6 hydroxylated species and reflected primary hepatic synthesis by the fetus. At full term, 7 alpha,12 alpha-dihydroxy-3-oxo-4-cholenoic acid was one of the major bile acids identified in amniotic fluid. The monohydroxy bile acids lithocholic and 3 beta-hydroxy-5-cholenoic acids were present in significant proportions during early gestation, but by full term these accounted for only a few percent of the total bile acids. Quantitatively the total bile acid concentration of amniotic fluid was less than 4 mumol/l. The majority of bile acids were found to be glyco-, tauro-, and sulfate-conjugates. The more hydrophobic bile acids tended to be preferentially sulfated. These data indicate that significant and major changes in bile acid metabolism take place between early and late gestation in the human fetus.     

Heparan sulfates from Swiss mouse 3T3 and SV3T3 cells: O-sulfate difference

A difference in the extent of sulfation between the heparan sulfate isolated from Swiss 3T3 mouse cells and that from Swiss 3T3 cells transformed by the DNA virus SV40 has been reported previously. This variance is manifested by different chromatographic and electrophoretic properties. Heparan sulfates from the two cell types were treated with nitrous acid under conditions that gave selective deaminative cleavage of glucosaminyl residues with sulfated amino groups in order to define the nature of the difference in sulfation further. The O-sulfate containing fragments from the heparan sulfates were compared by gel filtration and ion-exchange chromatography. The results showed that the 3T3 heparan sulfate contains 8% more O-sulfate than does the SV3T3 heparan sulfate. Analysis of uronic acids revealed that both types of heparan sulfates contain 45% L-iduronic acid and 55% D-glucuronic acid. These and other observations indicate that the primary difference in sulfation between the 3T3 and SV3T3 heparan sulfates lies in the extent of O-sulfation.     

Effect of bile duct ligation on bile acid and cholesterol metabolism in rats

The effects of bile duct ligation on bile acid and cholesterol metabolism were examined in male Wistar strain rats. Quantitative and qualitative changes of bile acids and cholesterol in serum and urine occurred; beta-muricholic acid predominantly increased in serum and urine and the ratio of urinary cholic acid and beta-muricholic acid changed from about 5:3 on day 1 to about 1:8 on day 5 under biliary obstruction. The form of the increased urinary bile acids was mainly taurine-conjugated and partly sulfated. Under conditions of bile duct ligation on day 5, 14C-labeled 3 beta-hydroxy-5-cholenoic, lithocholic, and chenodeoxycholic acids were intragastrically administered to the rats after pretreatment with antibiotics and the metabolites of these three acids were investigated. 3 beta-Hydroxy-5-cholenoic acid was most efficiently converted to beta-muricholic acid. The present study strongly suggested the presence of an alternative metabolic pathway induced by bile duct ligation, which caused the change in composition of urinary bile acids, and especially the marked increase in beta-muricholic acid formation. A possible alternative pathway for bile acid biosynthesis under biliary obstruction in rats is postulated.