Purification and characterization of an ATPase from human liver which catalyzes ATP hydrolysis in the presence of the conjugates of bilirubin bile acids and glutathione

An ATPase has been purified from the membrane fraction of human liver which catalyzes ATP in the presence of bilirubin ditaurate, lithocholic acid 3-O-sulfate and lithocholic acid 3-O-glucuronide as well as dinitrophenylglutathione and other glutathione conjugates. Its subunit Mr value (38,000) and immunological properties are similar to dinitrophenylglutathione ATPase of human erythrocytes. Kinetic constants of the enzyme for the conjugates of glutathione, bile acids and bilirubin are comparable indicating that this ATPase may mediate active transport of all these anionic conjugates in liver.     

Defective biliary secretion of bile acid 3-O-glucuronides in rats with hereditary conjugated hyperbilirubinemia

Biliary secretion of bile acid glucuronides was studied in control rats and in rats with a congenital defect in hepatobiliary transport of organic anions (GY rats). In control animals, hepatobiliary transport of [3H]lithocholic acid 3-O-glucuronide and [3H]cholic acid 3-O-glucuronide was efficient (greater than 95% in 1 h) and comparable to that of [14C]taurocholic acid. Secretion of both glucuronides was impaired in GY rats (24% and 71% at 1 h), whereas that of taurocholate was similar to control values. However, recovery of the glucuronides in bile was nearly complete within 24 h; virtually no radioactivity was found in urine. In control rats, biliary secretion of lithocholic acid 3-O-glucuronide, but not that of cholic acid 3-O-glucuronide or taurocholate, could be delayed by simultaneous infusion of dibromosulphthalein. In mutant rats, dibromosulphthalein infusion was also able to inhibit secretion of cholic acid 3-O-glucuronide. [3H]Hydroxyetianic acid, a C20 short-chain bile acid, was secreted by control rats as a mixture of 20% carboxyl-linked and 80% hydroxyl-linked (3-O-)glucuronide; secretion was very efficient (99% in 1 h). In GY rats, secretion was drastically impaired (16% at 1 h and 74% over a 24-h period). Initially, the mutant secreted more carboxyl- than hydroxyl-linked glucuronide, but the ratio reached that of control animals after 24 h. The rates of formation of both types of hydroxyetianic acid glucuronide by hepatic microsomes from mutant rats were similar or even slightly higher than those of control microsomes. These findings indicate that bile acid 3-O-glucuronides, but probably not carboxyl-linked glucuronides, are secreted into bile by a transport system shared with organic anions such as conjugated bilirubin and dibromosulphthalein, but different from that for amino acid-conjugated bile acids.     

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.     

Binding of bile acids by 100 000g supernatants from rat liver.

1. The binding of glycocholic acid, chenodeoxycholic acid and lithocholic acid to rat liver 1000 000g supernatants was studied by equilibrium dialysis. 2. The binding characteristics of the bile acids suggest that the binding components are involved in bile acid transport. 3. When mixtures of [14C]lithocholic acid and liver supernatants were eluted from columns of Sephadex G-75, a prominent peak of [14C]lithocholic acid appeared with proteins of mol.wt. approx. 40000. A second, smaller, peak of [14C]lithocholic acid was eluted with proteins of mol.wt. approx. 100000. 4. The inclusion of cholic acid, glycocholic acid or chenodeoxycholic acid in the eluting buffer decreased the amount of [14C]lithocholic acid that was eluted with the higher-molecular-weight component.     

Metabolism of lithocholic acid in the rat: formation of lithocholic acid 3-O-glucuronide in vivo

Milligram amounts of [3 beta-3H]lithocholic (3 alpha-hydroxy-5 beta-cholanoic) acid were administered by intravenous infusion to rats prepared with a biliary fistula. Analysis of sequential bile samples by thin-layer chromatography (TLC) demonstrated that lithocholic acid glucuronide was present in bile throughout the course of the experiments and that its secretion rate paralleled that of total isotope secretion. Initial confirmation of the identity of this metabolite was obtained by the recovery of labeled lithocholic acid after beta-glucuronidase hydrolysis of bile samples. For detailed analysis of biliary metabolites of [3H]lithocholic acid, pooled bile samples from infused rats were subjected to reversed-phase chromatography and four major labeled peaks were isolated. After complete deconjugation, the two major compounds in the combined first two peaks were identified as murideoxycholic (3 alpha, 6 beta-dihydroxy-5 beta-cholanoic) and beta-muricholic (3 alpha, 6 beta, 7 beta-trihydroxy-5 beta-cholanoic) acids and the third peak was identified as taurolithocholic acid. The major component of the fourth peak, after isolation, derivatization (to the methyl ester acetate), and purification by high pressure liquid chromatography (HPLC), was positively identified by proton nuclear magnetic resonance as lithocholic acid 3 alpha-O-(beta-D-glucuronide). These studies have shown, for the first time, that lithocholic acid glucuronide is a product of in vivo hepatic metabolism of lithocholic acid in the rat.     

6alpha-hydroxylation of taurochenodeoxycholic acid and lithocholic acid by CYP3A4 in human liver microsomes

The aim of the present study was to identify the enzymes in human liver catalyzing hydroxylations of bile acids. Fourteen recombinant expressed cytochrome P450 (CYP) enzymes, human liver microsomes from different donors, and selective cytochrome P450 inhibitors were used to study the hydroxylation of taurochenodeoxycholic acid and lithocholic acid. Recombinant expressed CYP3A4 was the only enzyme that was active towards these bile acids and the enzyme catalyzed an efficient 6alpha-hydroxylation of both taurochenodeoxycholic acid and lithocholic acid. The Vmax for 6alpha-hydroxylation of taurochenodeoxycholic acid by CYP3A4 was 18.2 nmol/nmol P450/min and the apparent Km was 90 microM. Cytochrome b5 was required for maximal activity. Human liver microsomes from 10 different donors, in which different P450 marker activities had been determined, were separately incubated with taurochenodeoxycholic acid and lithocholic acid. A strong correlation was found between 6alpha-hydroxylation of taurochenodeoxycholic acid, CYP3A levels (r2=0.97) and testosterone 6beta-hydroxylation (r2=0.9). There was also a strong correlation between 6alpha-hydroxylation of lithocholic acid, CYP3A levels and testosterone 6beta-hydroxylation (r2=0.7). Troleandomycin, a selective inhibitor of CYP3A enzymes, inhibited 6alpha-hydroxylation of taurochenodeoxycholic acid almost completely at a 10 microM concentration. Other inhibitors, such as alpha-naphthoflavone, sulfaphenazole and tranylcypromine had very little or no effect on the activity. The apparent Km for 6alpha-hydroxylation of taurochenodeoxycholic by human liver microsomes was high (716 microM). This might give an explanation for the limited formation of 6alpha-hydroxylated bile acids in healthy humans. From the present results, it can be concluded that CYP3A4 is active in the 6alpha-hydroxylation of both taurochenodeoxycholic acid and lithocholic acid in human liver.     

A comparative study of the sulfation of bile acids and a bile alcohol by the Zebra danio (Danio rerio) and human cytosolic sulfotransferases (SULTs)

The current study was designed to examine the sulfation of bile acids and bile alcohols by the Zebra danio (Danio rerio) SULTs in comparison with human SULTs. A systematic analysis using the fifteen Zebra danio SULTs revealed that SULT3 ST2 and SULT3 ST3 were the major bile acid/alcohol-sulfating SULTs. Among the eleven human SULTs, only SULT2A1 was found to be capable of sulfating bile acids and bile alcohols. To further investigate the sulfation of bile acids and bile alcohols by the two Zebra danio SULT3 STs and the human SULT2A1, pH-dependence and kinetics of the sulfation of bile acids/alcohols were analyzed. pH-dependence experiments showed that the mechanisms underlying substrate recognition for the sulfation of lithocholic acid (a bile acid) and 5α-petromyzonol (a bile alcohol) differed between the human SULT2A1 and the Zebra danio SULT3 ST2 and ST3. Kinetic analysis indicated that both the two Zebra danio SULT3 STs preferred petromyzonol as substrate compared to bile acids. In contrast, the human SULT2A1 was more catalytically efficient toward lithocholic acid than petromyzonol. Collectively, the results imply that the Zebra danio and human SULTs have evolved to serve for the sulfation of, respectively, bile alcohols and bile acids, matching the cholanoid profile in these two vertebrate species.     

Human liver steroid sulphotransferase sulphates bile acids.

The sulphation of bile acids is an important pathway for the detoxification and elimination of bile acids during cholestatic liver disease. A dehydroepiandrosterone (DHEA) sulphotransferase has been purified from male and female human liver cytosol using DEAE-Sepharose CL-6B and adenosine 3',5'-diphosphate-agarose affinity chromatography [Falany, Vazquez & Kalb (1989) Biochem. J. 260, 641-646]. Results in the present paper show that the DHEA sulphotransferase, purified to homogeneity, is also reactive towards bile acids, including lithocholic acid and 6-hydroxylated bile acids, as well as 3-hydroxylated short-chain bile acids. The highest activity towards bile acids was observed with lithocholic acid (54.3 +/- 3.6 nmol/min per mg of protein); of the substrates tested, the lowest activity was detected with hyodeoxycholic acid (4.2 +/- 0.01 nmol/min per mg of protein). The apparent Km values for the enzyme are 1.5 +/- 0.31 microM for lithocholic acid and 4.2 +/- 0.73 microM for taurolithocholic acid. Lithocholic acid also competitively inhibits DHEA sulphation by the purified sulphotransferase (Ki 1.4 microM). No evidence was found for the formation of bile acid sulphates by sulphotransferases different from the DHEA sulphotransferase during purification work. The above results suggest that a single steroid sulphotransferase with broad specificity encompassing neutral steroids and bile acids exists in human liver.     

The effect of cholesterol feeding on gallbladder bile acids of the rabbit. Evidence that lithocholic acid is a primary bile acid in the rabbit.

The bile acid in gallbladder bile of rabbits fed a normal diet or one containing 2% (w/w) cholesterol have been determined by gas chromatography-mass spectrometry. The predominant bile acids in normally fed rabbits were 3 alpha, 7 alpha, 12 alpha-trihydroxy-5 beta-cholan-24-oic acid (cholic acid), 3 alpha, 12 alpha-dihydroxy-5 alpha-cholan-24-oic acid (allodeoxycholic acid) and 3 alpha, 12 alpha-dihydroxy-5 beta-cholan-24-oic acid (deoxycholic acid) with very much smaller amounts of 3 alpha-hydroxy-5 beta-cholan-24-oic acid (lithocholic acid) and 3 alpha, 12 beta-dihydroxy-5 beta-cholan-24-oic acid. In the cholesterol-fed animals the lithocholate became a predominant bile acid. Sulphated bile acids accounted for less than 1% of the total bile acids. It is proposed that lithocholic acid may be a primary bile acid in the cholesterol-fed rabbit, formed by an alternative pathway of biosynthesis involving hepatic mitochondria.     

Regulation and ligand-binding specificities of two sex-specific bile acid-binding proteins of rat liver cytosol

Rat liver cytosolic proteins were photoaffinity labeled with the synthetic steroid [3H]methyltrienolone in order to identify and characterize hepatic proteins that may participate in the intracellular binding and transport of steroid hormones and other sterols. A male-specific and a female-specific sterol-binding protein (SBP) that migrated to the 4 S region of a sucrose gradient and had similar molecular weights (male-specific 34-kDa protein (SBP34), female-specific 31-kDa protein (SBP31] were thus identified. Experiments were undertaken to determine the biochemical basis for the sex-specific expression of these two proteins. In vivo hormonal manipulations established that the female-specific expression of SBP31 could, in part, be accounted for by the suppressive effects of androgen on SBP31 levels in male rats. In contrast, androgen stimulated expression of the male-specific SBP34, while estrogen and the estrogen-regulated continuous plasma growth hormone profile that is characteristic of adult female rats were suppressive toward this protein. Unlike several other androgen-dependent hepatic proteins, however, SBP34 did not require an intact pituitary for androgen-stimulated expression, nor was its expression stimulated by the intermittent pulses of plasma growth hormone that are characteristic of adult male rats. SBP34 and SBP31 were not induced but were suppressed to various extents by dexamethasone, phenobarbital, and clofibrate, drugs that are known to induce other hepatic proteins involved in steroid binding and metabolism. Competition experiments revealed that SBP31 has a relatively broad ligand specificity, with significant competition for [3H]methyltrienolone binding exhibited by bile acids (chenodeoxycholic acid and lithocholic acid) and a range of steroid hormones (progesterone, estradiol, testosterone, and 5 alpha-dihydrotestosterone) when present in the low micromolar range. No binding was detected with this protein toward cholesterol, triamcinolone acetonide, 5 alpha-androstan-3 alpha,17 beta-diol, cholic acid, and deoxycholic acid. In contrast, SBP34 exhibited greater binding specificity, with competition for [3H]methyltrienolone binding observed only with primary bile acids (cholic acid and chenodeoxycholic acid) and their metabolites (deoxycholic acid and lithocholic acid). On the basis of these binding specificities and the relatively high concentration of bile acids found in the liver, it is proposed that SBP31 and SBP34 function in the intracellular binding and/or transport of bile acids.     

Characterization of rat liver bile acid acyl glucuronosyltransferase

Recent studies have suggested that bile acid acyl glucuronides form covalently bound protein adducts which may cause hypersensitivity reactions and increased morbidity in patients. Although the preferential biosynthesis of the acyl glucuronides has been known, the characterization of hepatic bile acid acyl glucuronosyltransferase has not yet been clearly elucidated. We have investigated the substrate specificity of the hepatic bile acid acyl glucuronosyltransferase using five common bile acids as substrates. The glucuronidation rate was dependent on the number of the hydroxy group on the steroid nucleus and mono-hydroxylated lithocholic acid, the more lipophilic common bile acid, was most effectively metabolized into its acyl glucuronide. The tri-hydroxylated cholic acid, the more water-soluble common bile acid, barely transformed into its glucuronide. Results showed decreasing of the initial velocity of the acyl glucuronidation with increasing of the concentration of substrate, lithocholic acid, owing to the substrate inhibition of the hepatic bile acid acyl glucuronosyltransferase. The substrate analogues, glycine and taurine conjugated bile acids, which exist in the body fluids in high concentrations, also inhibited the enzyme's activity. In addition, enzymatic reaction products, bile acid acyl glucuronides, also inhibited the activity. These inhibitory mechanisms may be responsible for the low concentration of bile acid acyl glucuronides in urine and may be an important detoxification system in the body.     

Newly identified bile acid binders in rat liver cytosol. Purification and comparison with glutathione S-transferases

Gel filtration of male rat liver cytosol preincubated with radiolabeled lithocholic, chenodeoxycholic, and glycochenodeoxycholic acids, and taurocholic acid revealed two major peaks of radioactivity, one co-eluting with the glutathione S-transferases and the other with a separate fraction, respectively. Chromatofocusing of the pooled fractions containing the new bile acid binding activity resulted in a separation of bile acid binding from the previously described organic anion binding activity in this fraction. Two binding peaks for lithocholic acid (pI 5.6, Binder I, and pI 5.5, Binder II) were identified on chromatofocusing and were further purified to apparent homogeneity by hydroxyapatite chromatography. The two Binders were monomers having identical molecular weight (33,000) and similar amino acid compositions. Bile acid binding to purified Binders I and II and glutathione S-transferases A, B, and C was studied by inhibition of the fluorescence of bound 1-anilino-8-naphthalenesulfonate (ANS). Confirmatory experiments using equilibrium dialysis produced comparable results. Glutathione S-transferase B had greater affinity for bile acids than transferases A or C. Binder II, which had greater affinity than Binder I for most bile acids, had greater affinity for chenodeoxycholic acid than transferase B but comparable or lower affinities for the other bile acids. All bile acids studied diminished ANS fluorescence with Binder II. Taurocholic and cholic acids increased ANS fluorescence with Binder I without affecting KANS, whereas lithocholic and chenodeoxycholic acids diminished ANS fluorescence with Binder I. In summary, we have identified and isolated two proteins (Binders I and II) which, along with glutathione S-transferase B, are the major hepatic cytosol bile acid binding proteins; these proteins have overlapping but distinct specificities for various bile acids.     

Metabolism of sodium 3 alpha,7 alpha-dihydroxy-5 beta-cholane-24-sulfonate in hamsters

Metabolism of sodium 3 alpha,7 alpha-dihydroxy-5 beta-cholane-24-sulfonate, the sulfonate derivative of chenodeoxycholic acid, was studied in hamsters. In bile fistula hamsters, the sulfonate analogue was efficiently absorbed from the ileum and secreted rapidly into the bile without any modification such as conjugation. However, absorption from the jejunum was smaller than that observed for the ileum. After oral administration, the sulfonate analogue of chenodeoxycholic acid was recovered quantitatively in the feces as the unchanged form in contrast to simultaneously administered chenodeoxycholic acid, which was entirely converted to lithocholic acid during its passage through the intestinal tract. These results demonstrate that the sulfonate analogue is absorbed mainly from the ileum by active transport, enters the enterohepatic circulation like the endogenous conjugated bile acids, and completely resists bacterial degradation.     

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.     

Lithocholic acid-cholesterol interactions in rat liver plasma membrane fractions

This study was designed to elucidate the steps involved in the incorporation of lithocholic acid and the increase in cholesterol in liver plasma membranes after lithocholic acid injection. In vitro, cholesterol incorporation or binding to liver plasma membrane fractions enriched in bile canalicular structures occurred only when cholesterol was added simultaneously with lithocholic acid. The addition of cholic acid did not prevent the incorporation or binding of lithocholic acid and of cholesterol. However, when cholic acid was incubated with membranes already containing lithocholic acid and cholesterol, the ratio of cholesterol to lithocholic acid increased from 2 to more than 3 via a reduction of lithocholic acid. The binding of lithocholic acid and cholesterol to membranes rose 5-fold in the presence of cytosolic proteins. By electron microscopy the canalicular membrane structures with a high cholesterol content exhibited few microvilli, and their lumen appeared to have collapsed. These data suggest that simultaneous interaction of lithocholic acid and cholesterol, and not prior incorporation or binding of lithocholic acid to the membrane, may be a prerequisite to cholesterol accumulation in the membrane.     

Bile acid synthesis: down-regulation by monohydroxy bile acids

The regulation of bile acid synthesis was studied in rabbits after interruption of the enterohepatic circulation by choledochoureteral anastomosis. Total daily bile acid output was 772 +/- 130 (SD) mumol/24 h, of which greater than 95% was glycocholic acid. Administration of deoxycholic or cholic acid or their conjugates (300-800 mumol) or gall-bladder bile failed to down-regulate endogenous bile acid synthesis. In contrast, chenodeoxycholic acid administration did down-regulate bile acid synthesis, but this effect was related to the formation and excretion of lithocholic acid. This observation was confirmed by the finding that i.v. infusion of 10-20 mumol of either lithocholic acid or 3 beta-hydroxy-5-cholenoic acid significantly reduced cholic acid synthesis. Thus monohydroxy bile acids, derived from either hepatic or intestinal sources, participate in the down-regulation of bile acid synthesis.     

Transport of fluorescent chenodeoxycholic acid via the human organic anion transporters OATP1B1 and OATP1B3

This study sought to clarify the contributions of organic anion-transporting polypeptide (OATP) 1B1 and 1B3 to the liver uptake of chenodeoxycholic acid (CDCA). We synthesized a fluorescent version of CDCA, chenodeoxychilyl-(Nepsilon-NBD)-lysine (CDCA-NBD), to characterize transporter-mediated uptake. CDCA-NBD is efficiently transported by OATP1B1 and OATP1B3 with high affinities. The Michaelis-Menten constants for CDCA-NBD uptake by OATP1B1 and OATP1B3 were 1.45 +/- 0.39 microM and 0.54 +/- 0.09 microM, respectively. By confocal laser scanning microscopy, CDCA-NBD, which is taken up by OATP1B1 and OATP1B3, was observed to localize to the cytosol. We also examined the transport of newly synthesized fluorescent bile acids. NBD-labeled bile acids, including cholic acid, deoxycholic acid, lithocholic acid, and ursodeoxycholic acid, were all transported by OATP1B1 and OATP1B3. CDCA-NBD exhibited the highest rate of transport of the five NBD-labeled bile acids examined in OATP1B1- and OATP1B3-expressing cells. Our results suggest that OATP1B1 and OATP1B3 play important roles in CDCA uptake into the liver. Fluorescent bile acids are useful tools to characterize the uptake properties of membrane transporters.     

Lithocholic acid induces two different calcium-dependent inner membrane permeability systems in liver mitochondria

The effect of the most hydrophobic bile acid–lithocholic–as an inducer of two different Ca²⁺-dependent inner membrane permeability systems was studied on isolated rat liver mitochondria. It is shown that the addition of lithocholic acid at a concentration of 20 μM to the Ca²⁺-loaded mitochondria leads to swelling of the organelles, rapid release of Ca²⁺ from the matrix and almost complete collapse of Δψ. Mitochondrial pore blocker cyclosporin A (CsA) eliminates mitochondrial swelling but has no effect on the process of Ca²⁺ release and Δψ collapse. In the absence of Ca²⁺ lithocholic acid causes only a transient decrease of Δψ with subsequent complete recovery. Ruthenium red, inhibitor of mitochondrial Ca²⁺ uniporter, which blocks Ca²⁺ influx into the matrix, prevents mitochondrial swelling induced by lithocholic acid. At the same time, ruthenium red, which is added before lithocholic acid to the Ca²⁺-preloaded mitochondria, does not affect the swelling of the organelles but reduces the CsA-insensitive drop in Δψ. It is concluded that lithocholic acid is able to induce two Ca²⁺-dependent energy dissipation systems in the inner membrane of liver mitochondria: CsA-sensitive mitochondrial pore and CsA-insensitive permeability, which exhibits sensitivity to ruthenium red. It is found that the effect of this bile acid as an inductor of CsA-sensitive mitochondrial pore is not associated with the modulation of Pi effects. It is assumed that CsA-insensitive action of lithocholic acid is associated with the induction of Ca²⁺ efflux from the matrix in exchange for protons. In this case, the energy-dependent Ca²⁺ transport in the opposite direction with the participation of mitochondrial calcium uniporter sensitive to ruthenium red leads to the formation of calcium cycle and thereby to energy dissipation.     

Synthesis of bile acid monosulphates by the isolated perfused rat kidney.

Perfusion of an isolated rat kidney with labelled bile acids, in a protein-free medium, resulted in the urinary excretion of the labelled bile acid, 3% being converted into polar metabolities in 1h. These metabolities were neither glycine nor taurine conjugates, nor bile acid glucuronides, and on solovolysis yielded the free bile acid. On t.l.c. the metabolite of [24-14C]lithocholic acid had the mobility of lithocholate 3-sulphate. The principal metabolite of [24-14C]chenodeoxycholic acid had the mobility of chenodeoxycholate 7-sulphate; trace amounts appeared as chenodeoxycholate 3-sulphate. [35S]sulphate was incorporated in chenodeoxycholic acid by the kidney, resulting in a similar pattern of sulphation. No disulphate salt of chenodeoxycholic acid was detected. These findings lend support to the hypothesis that renal synthesis may account for some of the bile acid sulphates present in urine in the cholestatic syndrome in man.