Comparison of the binding sites of GSH S-transferases of the Ya- and Yb-subunit classes: effect of glutathione on the binding of bile acids

We have previously observed that the Ya subunit-containing glutathione (GSH) S-transferases from rat liver exhibit a common high affinity binding site for lithocholic acid, bilirubin, and sulfobromophthalein (BSP) (1984. J. Lipid Res. 25: 1177-1183). Subsequently we found that cholic acid and its amidates bound to a site on the Ya subunit separate for the lithocholic acid/bilirubin site (1986. J. Lipid Res. 27: 955-966). We now have extended this work by showing that amidates of lithocholic acid as well as chenodeoxycholic acid and its amidates competitively displace [14C]lithocholic acid from the Ya subunit. GSH did not inhibit binding of any of the ligands to the high affinity Ya site, but did inhibit binding to the cholic acid site on the Ya subunit. We have also defined the binding sites and effects of GSH on the Yb class of subunits. Lithocholic, chenodeoxycholic, and cholic acids (and amidates) shared a common site on the Yb or Y'b subunit, whereas BSP and bilirubin were bound at a different site. Both the bile acid and organic anion sites on the Yb subunit were inhibited by GSH. The inhibition by GSH in all cases (Ya cholic acid site or Yb bile acid or bilirubin sites) was saturable, of the competitive type, and incomplete at maximal GSH concentrations, suggesting that when GSH binds to its distinct substrate site, it induces a conformational change in the proteins affecting the other binding sites.     

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.     

Ursodeoxycholic acid, chenodeoxycholic acid, and 7-ketolithocholic acid are primary bile acids of the guinea pig

Guinea pig gallbladder bile contains chenodeoxycholic acid (62 +/- 5%), ursodeoxycholic acid (8 +/- 5%), and 7-ketolithocholic acid (30 +/- 5%). All three bile acids became labeled to the same specific activity within 30 min after [3H]cholesterol was injected into bile fistula guinea pigs. When a mixture of [3H]ursodeoxycholic acid and [14C]chenodeoxycholic acid was infused into another bile fistula guinea pig, little 3H could be detected in either chenodeoxycholic acid or 7-ketolithocholic acid. But, 14C was efficiently incorporated into ursodeoxycholic and 7-ketolithocholic acids. Monohydroxylated bile acids make up 51% and ursodeoxycholic acid 38% of fecal bile acids. After 3 weeks of antibiotic therapy, lithocholic acid was reduced to 6% of the total, but ursodeoxycholic acid (5-11%) and 7-ketolithocholic (15-21%) acid persisted in bile. Lathosterol constituted 19% of skin sterols and was detected in the feces of an antibiotic-fed animal. After one bile fistula guinea pig suffered a partial biliary obstruction, ursodeoxycholic and 7-ketolithocholic acids increased to 46% and 22% of total bile acids, respectively. These results demonstrate that chenodeoxycholic acid, ursodeoxycholic acid, and 7-ketolithocholic acid can all be made in the liver of the guinea pig.     

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.     

Reduction of 3 alpha-hydroxy-5 beta-chol-6-en-24-oic acid to lithocholic acid in rats

After [24-14C]delta 6-lithocholic acid was injected into the cecum of rats, [14C]lithocholic acid was identified as a metabolite in feces. When the labeled delta 6-bile acid was injected intraperitoneally into bile-fistula rats, radioactivity excreted in bile was contained most abundantly in the taurine-conjugated fraction of bile acids. In the fraction, taurine conjugate of [14C]delta 6-lithocholic acid but of neither [14C]lithocholic acid nor other bile acids was found. The results showed that [24-14C]delta 6-lithocholic acid was reduced to [14C]lithocholic acid by the intestinal flora but not by the liver, which, however, was capable of conjugating delta 6-lithocholic acid with taurine.     

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.     

Bile acid synthesis. Metabolism of 3 beta-hydroxy-5-cholenoic acid to chenodeoxycholic acid

Metabolism of 3 beta-hydroxy-5-cholenoic acid to chenodeoxycholic acid has been found to occur in rabbits and humans, species that cannot 7 alpha-hydroxylate lithocholic acid. This novel pathway for chenodeoxycholic acid synthesis from 3 beta-hydroxy-5-cholenoic acid led to a reinvestigation of the pathway for chenodeoxycholic acid from 3 beta-hydroxy-5-cholenoic acid in the hamster. Simultaneous infusion of equimolar [1,2-3H]lithocholic acid and 3 beta-hydroxy-5-[14C]cholenoic acid indicated that the 14C enrichment of chenodeoxycholic acid was much greater than that of lithocholic acid. Thus, in all these species, a novel 7 alpha-hydroxylation pathway exists that prevents the deleterious biologic effects of 3 beta-hydroxy-5-cholenoic acid.     

Hydroxylation, conjugation and sulfation of bile acids in primary monolayer cultures of rat hepatocytes

Hydroxylation of lithocholic, chenodeoxycholic, deoxycholic and cholic acids was studied in monolayers of rat hepatocytes cultured for 76 h. The majority of added lithocholic and chenodeoxycholic acids was metabolized to beta-muricholic acid (56-76%). A small part of these bile acids (9%), however, and a considerable amount of deoxycholic and cholic acids (21%) were converted into metabolites more polar than cholic acid in the first culture period. Formation of these compounds decreased during the last day of culture. Bile acids synthesized after addition of [4-14C]-cholesterol were almost entirely (97%) sulfated and/or conjugated, predominantly with taurine (54-66%), during culture. Sulfated bile acids were mainly composed of free bile acids. The ability of hepatocytes to sulfurylate bile acids declined with culture age. Thus, rat hepatocytes in primary monolayer culture are capable to sulfurylate bile acids and to hydroxylate trihydroxylated bile acids, suggesting formation of polyhydroxylated metabolites.     

Evidence for a lack of regulatory importance of the 12 alpha-hydroxylase in formation of bile acids in man: an in vivo study

The possibility that the 12 alpha-hydroxylase involved in formation of bile acids is of regulatory importance for the ratio between cholic acid and chenodeoxycholic acid in bile was studied with an in vivo technique. [4-14C]7 alpha-Hydroxy-4-cholesten-3-one and [6 beta-3H]7 alpha, 12 alpha-dihydroxy-4-cholesten-3-one were synthesized, and a mixture of these two bile acid intermediates was administered intravenously in five healthy subjects and in one patient with severe liver cirrhosis. The patient with liver cirrhosis was included in the study because of a considerable reduction in biosynthesis of cholic acid. Since the [4-14C]-labeled steroid is an intermediate just proximal to and since the [6 beta-3H]-labeled steroid is an intermediate just distal to the 12 alpha-hydroxylase step, the 3H/14C ratio in the cholic acid formed should reflect the relative 12 alpha-hydroxylase activity. The 3H/14C ratio varied between 1.8 and 3.9 in the cholic acid isolated from the healthy subjects and was 3.6 in the cholic acid isolated from the patient with liver cirrhosis. The ratio between cholic acid and chenodeoxycholic acid varied between 0.6 and 3.9 in the bile from the control subjects and was only 0.4 in the bile from patients with liver cirrhosis. There was no correlation between the 3H/14C ratios and the ratios between cholic acid and chenodeoxycholic acid in bile.(ABSTRACT TRUNCATED AT 250 WORDS)     

Dynamics of the conjugate pattern during the infusion of bile acids into isolated rat liver

The conjugate pattern of biliary [14C]bile acids was investigated in isolated perfused rat livers, which were infused with either [24-14C]cholic acid or [24-14C]chenodeoxycholic acid (40 mumol/h) together with or without taurine or cysteine (80 mumol/h). [14C]Bile acids were chromatographed on a thin-layer plate and the distribution of radioactivity on the plate was measured by radioscanning. The biliary excretion of [14C]bile acids was greater in the infusion with [14C]cholic acid than in the infusion with [14C]chenodeoxycholic acid. Biliary unconjugated [14C]bile acids amounted to about 50% of the total after the infusion with [14C]cholic acid, while only about 10% with [14C]chenodeoxycholic acid. In the initial period of infusion, biliary conjugated [14C]bile acids consisted mostly of the taurine conjugate, which decreased with time and the glycine conjugate increased complementarily. When taurine was simultaneously infused, the decrease in the taurine conjugate was suppressed to some extent. Cysteine infused in place of taurine had a similar influence but was less effective than taurine. The taurine content of liver after the infusion with either of the [14C]bile acids decreased greatly compared with that before the infusion, even when taurine or cysteine was infused simultaneously. The glycine content also decreased after the infusion, but the decrease in glycine was smaller than that in taurine. The results suggest that the conjugate pattern of biliary bile acids in rats depends mainly on the amount of taurine which is supplied to hepatic cells either exogenously from plasma or endogenously within themselves.     

Studies on the fatty acid binding proteins in cytosol of rat liver.

Gel filtration on Sephadex G-75 of crude rat liver supernatant preincubated with [1-14C]oleic acid yields three peaks of radioactivity which are attributed to the presence in these fractions of fatty acid binding proteins. We have confirmed these observations with binding assays by phase partition, polyacrylamide gel electrophoresis, and thin layer electrofocusing. Peak I (mol. wt. 60,000 pI 5.01 was shown to be albumin, which mainly arises from a contamination of the liver preparation by blood. Peak II (mol. wt. 10,000, pI 5.9) is a fatty acid binding protein. Finally peak III (mol. wt. 1500, pI 5.7) is a fatty acid binding component, the chemical nature of which was not elucidated. These fatty acid binding fractions have no effect on the reaction of acyl-CoA synthetase whereas the crude liver supernatant does stimulate the activation of fatty acid as shown earlier. In consequence, the physiological role of these fatty acid binding fractions is not yet elucidated.     

The formation of lithocholic acid, chenodeoxycholic acid and α- and β-muricholic acids from cholesterol incubated with rat-liver mitochondria

1. When rat-liver mitochondria were incubated with [4-(14)C]cholesterol in the presence of a soluble supernatant fraction, various steroids more polar than cholesterol were formed. These included 3beta-hydroxycholest-5-en-26-oic acid, 3beta-hydroxychol-5-enoic acid, lithocholic acid, chenodeoxycholic acid and alpha- and beta-muricholic acids. 2. All the radioactive C(24) bile acids recovered were in conjugated form, probably as taurine conjugates. 3. The formation of 3beta-hydroxychol-5-enoic acid from cholesterol shows that liver mitochondria are capable of carrying out the oxidative removal of the isopropyl unit of the side chain before any modification has occurred in the ring system.     

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.     

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.     

Role of endogenous and exogenous cholesterol in liver as the precursor for bile acids in rats

There is considerable evidence suggesting that compartmentalized functional pools of cholesterol in the liver contribute differently to the formation of bile acids as the precursor. The present paper deals with the incorporation of [1-¹⁴C]acetate and of [1,2-³H]cholesterol carried on lipoproteins (LDL and HDL) into biliary bile acids in perfused rat livers and bile-fistula rats. The results showed that endogenous cholesterol synthesized newly from [1-¹⁴C]acetate in the liver was incorporated into both cholic acid and chenodeoxycholic acid in a similar way, while exogenous lipoprotein-[1,2-³H]cholesterol delivered to hepatocytes from hepatic circulation was incorporated into chenodeoxycholic acid at a higher rate.     

7-Methyl bile acids: 7 beta-methyl-cholic acid inhibits bacterial 7-dehydroxylation of cholic acid and chenodeoxycholic acid in the hamster

The effect of dietary 7 beta-methyl-cholic acid [0.075% in rodent chow (6.4 mg/animal per day)] on cholesterol and bile acid metabolism was studied and compared with that of cholic acid in the hamster. Following oral administration of 7 beta-methyl-cholic acid for 3 weeks, the glycine-conjugated bile acid analog became a major constituent of gallbladder bile. Biliary cholic acid concentration decreased significantly, while that of chenodeoxycholic acid remained unchanged. Serum and liver cholesterol levels were increased by dietary 7 beta-methyl-cholic acid and by cholic acid. Hepatic microsomal HMG-CoA reductase activity was inhibited (30% of the control value) by both bile acids; cholesterol 7 alpha-hydroxylase activity was not affected. In chow controls and cholic acid-fed animals, bacterial 7-dehydroxylation of [14C]chenodeoxycholic acid and [14C]cholic acid was nearly complete. In contrast, dietary 7 beta-methyl-cholic acid effectively prevented the 7-dehydroxylation of the two primary bile acids. These results show that dietary 7 beta-methyl-cholic acid is preserved in the enterohepatic circulation and has an effect on serum and liver cholesterol concentrations similar to those produced by the naturally occurring cholic acid. 7 beta-Methyl-cholic acid is an efficient inhibitor of the bacterial 7-dehydroxylation of the primary bile acids in the hamster.     

Effect of diabetes on the metabolism of chenodeoxycholic acid in isolated perfused rat liver

The formation of alpha-muricholic acid and beta-muricholic acid from chenodeoxycholic acid was comparatively investigated in livers isolated from normal, streptozotocin-diabetic, and insulin-treated diabetic rats. [24-14C]Chenodeoxycholic acid or [24-14C]alpha-muricholic acid was infused into the perfused livers. There was no difference in biliary excretion of 14C among the different groups of rats after the infusion of each 14C-labelled bile acid. Biliary [14C]bile acids were chromatographed on a thin-layer plate and the distribution of radioactivity on the plate was measured by radioscanning. In the diabetic group, the formation ratio of alpha-muricholic acid and beta-muricholic acid from [24-14C]chenodeoxycholic acid and also that of beta-muricholic acid from [24-14C]alpha-muricholic acid were much smaller than in the normal group. Treatment of the diabetic group with insulin cancelled the difference in the infusion of each [24-14C]bile acid. The results indicate that not only 6 beta-hydroxylation of chenodeoxycholic acid to alpha-muricholic acid but also 7-epimerization of the latter acid to beta-muricholic acid is suppressed in an insulin-deficient state in rats.     

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.     

The synthesis of bile acids in perfused rat liver subjected to chronic biliary drainage

1. Isolated rat liver was perfused with heparinized whole blood under physiological pressure resulting in the secretion of bile at about the rate observed in vivo. 2. The preparation remained metabolically active for 4h and was apparently normal in function and microscopic appearance. 3. When the perfusate plasma and liver cholesterol pool was labelled by the introduction of [2-(14)C]mevalonic acid the specific radioactivity of the perfusate cholesterol increased. The biliary acids (cholic acid and chenodeoxycholic acid) were labelled and had the same specific radioactivity. 4. Livers removed from rats immediately after, and 40h after, the start of total biliary drainage, were perfused; increased excretion rates of both cholic acid and chenodeoxycholic acid were found when the liver donors had been subjected to biliary drainage. 5. The incorporation of [2-(14)C]mevalonic acid or rat lipoprotein labelled with [(14)C]cholesterol into bile acids was studied. 6. A dissociation between the mass of bile acid excreted and the rate of incorporation of (14)C was found. This was attributed to the changing specific radioactivity of the cholesterol pool acting as the immediate bile acid precursor.     

Radioactive labelling and characterization of the products of activated mouse lymphocytes.

For chemical characterization of the products of activated lymphocytes a radioactive double-label technique was developed which allows one to distinguish those products synthesized either de novo or in increased amounts by the stimulated culture. Spleen cells from Balb/c mice were cultured in serum-free medium in the presence or absence of concanavalin A and simultaneously labelled with radioactive leucine. Optimal culture conditions were established by determining parameters such as cell density, mitogen concentration, and kinetics of protein synthesis following stimulation. Combined supernatants of stimulated and unstimulated cultures each labelled with either [3H]leucine or [14C]leucine were fractionated on Sephadex G-75. Materials derived from control or stimulated supernatants both yielded a qualitatively similar radiolabelled profile. The isotope ratio of stimulated to nonstimulated culture, however, showed a broad peak at KD 0--.35 (approx. mol. wt 75000-20000) which was further analyzed by isoelectric focusing. Pools of every two fractions were focused in polyacrylamide gels at pH 3.5-10. By determining the isotope ratio, the isoelectric point, and the KD (mol wt), it was possible to distinguish at least 24 molecules which had been produced only, or in greater degree, by the stimulated culture.