Formation and secretion of glycolithocholate-3-sulfate in primary hepatocyte cultures

Bile acid sulfation was studied in primary hepatocyte cultures. The primary hepatocyte cultures formed glycolithocholate-3-sulfate (GLC-S) when glycolithocholate (GLC) was added to the medium. The relative percentage of GLC-S formation increased when the GLC concentration was increased from 10 microM to 100 microM. GLC-S formation was linear to 60 min. GLC-S secretion into the medium was detectable at 75 min and linear to 8 hr. In contrast to the effect of GLC concentration, there was no difference in GLC-S formation or secretion when inorganic sulfate in the medium was increased 16-fold (100 microM-1600 microM). We conclude that the rate of bile acid sulfate formation in cultured primary hepatocytes is primarily controlled by bile acid, but not inorganic sulfate, concentration.     

Quantitative Determination of 14C-Ethylene Produced by Plant Tissues

Pseudomonas testosteroni ATCC 11996 was found to produce a novel bile acid sulfate sulfatase that hydrolyzes the sulfate ester bond in lithocholic acid sulfate (LCA-S). The enzyme synthesis was induced by several kinds of bile acids including LCA-S. Mn²⁺ functioned as an essential component for the enzyme synthesis and suppressed it. This sulfatase hydrolyzes LCA-S to isolithocholic acid and sulfuric acid with inversion of α- to β-configuration of the hydroxyl group at the third position of lithocholic acid.     

A bile alcohol sulfate as a major component in the bile of the small skate (Raja erinacea)

The nature of bile alcohols and bile acids in gall-bladder and hepatic bile from perfused livers of the small skate (Raja erinacea) has been investigated. The main bile alcohol sulfate was isolated by thin-layer chromatography and analyzed by fast atom bombardment mass spectrometry and 13C NMR. Following solvolysis and purification on Lipidex-DEAP, the bile alcohol profile was measured by capillary gas-liquid chromatography-electron impact mass spectrometry. Based on these studies and on comparison with authentic scymmnol sulfate and scymnol, the main bile alcohol was identified as 5 beta-cholestane-3 alpha,7 alpha,12 alpha,24 xi,26,27-hexol sulfate. The mean +/- SD concentration in gallbladder bile from five different skates was 24.6 +/- 8.7 mmol/l. Only 0.1 mmol/l of cholic acid and its conjugates was found in a pool of skate bile. The main bile alcohol sulfate in the bile of the small skate seems to be a metabolic end product, present in a concentration comparable to that of bile salts in mammals.     

An active type IV secretion system encoded by the F plasmid sensitizes Escherichia coli to bile salts

F(+) strains of Escherichia coli infected with donor-specific bacteriophage such as M13 are sensitive to bile salts. We show here that this sensitivity has two components. The first derives from secretion of bacteriophage particles through the cell envelope, but the second can be attributed to expression of the F genes required for the formation of conjugative (F) pili. The latter component was manifested as reduced or no growth of an F(+) strain in liquid medium containing bile salts at concentrations that had little or no effect on the isogenic F(-) strain or as a reduced plating efficiency of the F(+) strain on solid media; at 2% bile salts, plating efficiency was reduced 10(4)-fold. Strains with F or F-like R factors were consistently more sensitive to bile salts than isogenic, plasmid-free strains, but the quantitative effect of bile salts depended on both the plasmid and the strain. Sensitivity also depended on the bile salt, with conjugated bile salts (glycocholate and taurocholate) being less active than unconjugated bile salts (deoxycholate and cholate). F(+) cells were also more sensitive to sodium dodecyl sulfate than otherwise isogenic F(-) cells, suggesting a selectivity for amphipathic anions. A mutation in any but one F tra gene required for the assembly of F pili, including the traA gene encoding F pilin, substantially restored bile salt resistance, suggesting that bile salt sensitivity requires an active system for F pilin secretion. The exception was traW. A traW mutant was 100-fold more sensitive to cholate than the tra(+) strain but only marginally more sensitive to taurocholate or glycocholate. Bile salt sensitivity could not be attributed to a generalized change in the surface permeability of F(+) cells, as judged by the effects of hydrophilic and hydrophobic antibiotics and by leakage of periplasmic beta-lactamase into the medium.     

Cholesterol 7 alpha-hydroxylase activity and bile acid synthesis in hepatocytes of unweaned and weaned pigs in monolayer culture

Activity of cholesterol 7 alpha-hydroxylase (EC 1.14.13.17) in freshly isolated hepatocytes from unweaned piglets (2 to 3 weeks old) was 16-times lower as compared to hepatocytes from weaned piglets (7 to 8 weeks old). The monolayer culture activity of the enzyme remained low in unweaned piglet hepatocytes. In contrast, in cultured hepatocytes from weaned piglets, cholesterol 7 alpha-hydroxylase activity declined during the first day of culture, but was restored during the next 2 culture days, provided that fetal bovine serum (10%) was added to the culture medium. Addition of dexamethasone (50 nM) and insulin (135 nM) to the medium, further enhanced cholesterol 7 alpha-hydroxylase activity to values similar to those in freshly isolated hepatocytes and retarded the decline of enzyme activity after the 3rd culture day. Cultured hepatocytes from weaned and unweaned piglets synthesized similar types of bile acids from [14C]cholesterol, among which hyocholic acid (the most prominent), hyodeoxycholic acid, chenodeoxycholic acid, murocholic acid and lithocholic acid could be identified. 95% of radiolabelled bile acids synthesized was conjugated, mainly with glycine, but also with taurine, sulfate and glucuronic acid. The rate of mass production of bile acids by cultured hepatocytes of weaned piglets (as measured by gas-chromatography) parallelled cholesterol 7 alpha-hydroxylase activity, and was low in the absence of serum, but increased in medium containing fetal bovine serum, dexamethasone and insulin to a rate lying in the range of 75% of the in vivo bile acid production during the 3rd culture day. Bile acid production by unweaned piglet hepatocytes was 3-times lower under these conditions. It is concluded that hepatocytes from young weaned pigs cultured in medium containing 10% fetal bovine serum, offer a suitable in vitro model for the study of bile acid synthesis, in view of the high cholesterol 7 alpha-hydroxylase activities and bile acid production rates.     

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     

Physicochemical and physiological properties of 5alpha-cyprinol sulfate,the toxic bile salt of cyprinid fish

5alpha-Cyprinol sulfate was isolated from bile of the Asiatic carp, Cyprinus carpio. 5alpha-Cyprinol sulfate was surface active and formed micelles; its critical micellization concentration (CMC) in 0.15 M Na+ using the maximum bubble pressure device was 1.5 mM; by dye solubilization, its CMC was approximately 4 mM. At concentrations >1 mM, 5alpha-cyprinol sulfate solubilized monooleylglycerol efficiently (2.1 molecules per mol micellar bile salt). When infused intravenously into the anesthetized rat, 5alpha-cyprinol sulfate was hemolytic, cholestatic, and toxic. In the isolated rat liver, it underwent little biotransformation and was poorly transported (Tmax congruent with 0.5 micromol/min/kg) as compared with taurocholate. 5alpha-Cyprinol, its bile alcohol moiety, was oxidized to its corresponding C27 bile acid and to allocholic acid (the latter was then conjugated with taurine); these metabolites were efficiently transported. 5alpha-Cyprinol sulfate inhibited taurocholate uptake in COS-7 cells transfected with rat asbt, the apical bile salt transporter of the ileal enterocyte. 5alpha-Cyprinol had limited aqueous solubility (0.3 mM) and was poorly absorbed from the perfused rat jejunum or ileum. Sampling of carp intestinal content indicated that 5alpha-cyprinol sulfate was present at micellar concentrations, and that it did not undergo hydrolysis during intestinal transit. These studies indicate that 5alpha-cyprinol sulfate is an excellent digestive detergent and suggest that a micellar phase is present during digestion in cyprinid fish.     

Cholesterol 7α-hydroxylase activity and bile acid synthesis in hepatocytes of unwearied and weaned pigs in monolayer culture

Activity of cholesterol 7α-hydroxylase (EC 1.14.13.17) in freshly isolated hepatocytes from unweaned piglets (2 to 3 weeks old) was 16-times lower as compared to hepatocytes from weaned piglets (7 to 8 weeks old). The monolayer culture activity of the enzyme remained low in unweaned piglet hepatocytes. In contrast, in cultured hepatocytes from weaned piglets, cholesterol 7α-hydroxylase activity declined during the first day of culture, but was restored during the next 2 culture days, provided that fetal bovine serum (10%) was added to the culture medium. Addition of dexamethasone (50 nM) and insulin (135 nM) to the medium, further enhanced cholesterol 7α-hydroxyease activity to values similar to those in freshly isolated hepatocytes and retarded the decline of enzyme activity after the 3rd culture day. Cultured hepatocytes from weaned and unweaned piglets synthesized similar types of bile acids from [¹⁴C]cholesterol. among which hyocholic acid (the most prominent), hyodeoxycholic acid, chenodeoxycholic acid, murocholic acid and lithocholic acid could be identified. 95% of radiolabelled bile acids synthesized was conjugated, mainly with glycine, but also with taurine, sulfate and glucuronic acid. The rate of mass production of bile acids by cultured hepatocytes of weaned piglets (as measured by gas-chromatography) parallelled cholesterol 7α-hydroxylase activity, and was low in the absence of serum, but increased in medium containing fetal bovine serum, dexamethasone and insulin to a rate lying in the range of 75% of the in vivo bile acid production during the 3rd culture day. Bile acid production by unweaned piglet hepatocytes was 3-times lower under these conditions. It is concluded that hepatocytes from young weaned pigs cultured in medium containing 10% fetal bovine serum, offer a suitable in vitro model for the study of bile acid synthesis, in view of the high cholesterol 7α-hydroxylase activities and bile acid production rates.     

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.     

A novel sulfatase from Pseudomonas testosteroni hydrolyzing lithocholic acid sulfate.

Pseudomonas testosteroni ATCC 11996 was found to produce a novel bile acid sulfate sulfatase that hydrolyzes the sulfate ester bond in lithocholic acid sulfate (LCA-S). The enzyme synthesis was induced by several kinds of bile acids including LCA-S. Mn2+ functioned as an essential component for the enzyme synthesis and SO4(2-) suppressed it. This sulfatase hydrolyzes LCA-S to isolithocholic acid and sulfuric acid with inversion of alpha- to beta-configuration of the hydroxyl group at the third position of lithocholic acid.     

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.     

Bile acid sulfates in serum bile acids determination.

Some bile acid sulfates were synthesized and characterized. The configuration of sulfate groups at C-3, C-7 and C-12 positions was confirmed by Nuclear Magnetic Resonance analysis. These sulfates were utilized in a study of their chemical behaviour in different analytical procedures currently used for serum bile acids determination. Procedures for bile acids extraction from serum with ethanol or Amberlite XAD-2 result in an important loss of the most polar sulfated bile acids. Complete separation of unsulfated from sulfated bile acids on Sephadex LH-20 is not achieved when deconjugation of the most polar bile acid sulfate is slow but does not produce artifacts. Enzymatic determination of bile acids gives positive response with some bile acid sulfates. The current procedures of serum bile acids determination are discussed in consideration of these results.     

Highly independent olfactory receptor sites for naturally occurring bile acids in the sea lamprey, Petromyzon marinus

(1) Electro-olfactogram recording was used to determine whether the olfactory epithelium of adult sea lamprey is specifically sensitive to bile acids, some of which have been hypothesized to function as pheromones. Ten bile acids were selected from 38 which had already been pre-screened for olfactory activity. These compounds were first tested on their own, then as adapting stimuli, and finally as components of mixtures (2) The lamprey-specific bile acids, petromyzonol sulfate and allocholic acid, were the most potent compounds tested. Five other bile acids were also detectable at picomolar concentrations. Petromyzonol sulfate had a distinctive dose-response curve. (3) Cross-adaptation demonstrated that sensitivity to bile acids is attributable to at least four independent classes of olfactory receptor sites and that both the nature and position of conjugating group(s) are critical to receptor specificity. Notably, petromyzonol sulfate has its own highly specific and independent receptor site. The situation for unconjugated bile acids was more complex and there appeared to be several sub-classes of receptor sites for these compounds. (4) Mixture studies largely confirmed the cross-adaptation results, describing receptor site independence for the same four sets of odorants. Mixture enhancement was also seen when expected and there was no evidence of mixture suppression. (5) Together, these data demonstrate that conspecific bile acids are discriminated by the olfactory epithelium of the sea lamprey, supporting the possibility that these compounds may function as migratory pheromones. Accepted: 23 November 1996     

Gastrointestinal absorption of estrone sulfate in germfree and conventional rats

Steroids are extensively excreted in the bile of rats. There was no significant difference in biliary excretion of steroid following administration of [3H]-estrone sulfate into the proximal small intestine (PSI) of conventional (CVL; 17.8 +/- 62%; mean +/- SD) or germfree (GF; 28.2 +/- 5.3) rats. A similar finding resulted from administration into the distal small intestine (DSI)-CVL, 22.3 +/- 11.8%; GF, 11.4 +/- 3.7%. However, when the drug was given into the caecum, excretion in the bile of CVL rats after 5 h was 59.1% whereas in GF rats it was only 1.7%. When estrone was injected into the PSI and DSI of CVL and GF rats, absorption (as judged by excretion in bile) was more rapid than that seen with estrone sulfate. Five hours after injection into the PSI, biliary excretion was, in CVL 88.2% and in GF 81.7% and after injection into the DSI excretion was, in CVL 84.7% and in GF 83.6%. Absorption of estrone from the caeca of GF rats was apparently reduced (49.0% and 25.3% excreted in the bile of CVL and GF rats respectively). There was no significant difference in bile flow rate between CVL and GF rats. These results give unequivocal evidence of intact absorption of estrone sulfate from the small intestine of the rat. The rate of absorption is however very much reduced compared to the non-sulphated steroid. Estrone sulfate is not absorbed intact in the caecum but is hydrolysed by the gut microflora prior to absorption.     

Taurine and glycine conjugation and sulfation of lithocholate in primary hepatocyte cultures

Rat primary liver cells were used to study taurine and glycine conjugation and sulfation of lithocholate. After addition of [14C]lithocholate to the tissue culture medium, synthesis and excretion of amidated and/or sulfated products were investigated for up to 24 h. After incubation for 1 h, more than 83% of the labeled bile salt was amidated but not sulfated and between 5 and 11% was sulfated, with more than 80% of the sulfated bile salts being also amidated. After 24 h, the proportion of sulfated lithocholate had increased to about 23% and more than 99% of the lithocholate sulfate was additionally conjugated with glycine or taurine. Both sulfates and non-sulfates were preferably amidated with taurine. We conclude that in primary rat hepatocytes, (1) lithocholate is rapidly and almost completely conjugated with glycine or taurine (amidated), whereas sulfation of lithocholate (and its amidates) proceeds slowly and even after 24 h represents only a small proportion of the total lithocholate metabolites, and (2) sulfated and unsulfated bile salts are both preferably amidated with taurine.     

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.     

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.     

Biliary protein output by isolated perfused rat livers. Effects of bile salts.

The output of proteins into bile was studied by using isolated perfused rat livers. Replacement of rat blood with defined perfusion media deprived the liver of rat serum proteins (albumin, immunoglobulin A) and resulted in a rapid decline in the amounts of these proteins in bile. When bovine serum albumin was incorporated into the perfusion medium it appeared in bile within 20 min and the amount in the bile was determined by the concentration of the protein in the perfusion medium. The use of a defined perfusion medium also deprived the livers of bile salts and the amounts of these, and of plasma-membrane enzymes [5'-nucleotidase (EC 3.1.3.5) and phosphodiesterase I], in bile declined rapidly. Introduction of micelle-forming bile salts (taurocholate or glycodeoxycholate) to the perfusion medium 80 min after liver isolation markedly increased the output of plasma-membrane enzymes but had no effect on the other proteins. The magnitude of this response was dependent on the bile salt used and its concentration in bile; there was little effect on plasma-membrane enzyme output until the critical micellar concentration of the bile salt had been exceeded in the bile. A bile salt analogue, taurodehydrocholate, which does not form micelles, did not produce the enhanced output of plasma-membrane enzymes. This work supports the view that the output of plasma-membrane enzymes in bile is a consequence of bile salt output and also provides evidence for mechanisms by which serum proteins enter the bile.     

Shedding of sulfated lipids into gastric fluid and inhibition of pancreatic DNase I by cholesterol sulfate in concert with bile acids

Cholesterol sulfate (CS) and sulfatides in the epithelium of the digestive tract were found in the 1000xg supernatants of digestive fluid, particularly in gastric juices containing the duodenal contents and bile acids, there being 14-131 microg of CS and 3-54 microg of sulfatides per mg of protein in the fluid, respectively. CS and sulfatides dissolved in detergents including bile acids inactivated pancreatic trypsin to the same level as by DMSO-solubilized sulfated lipids at 37 degrees C. Similarly, pancreatic DNase I was inhibited by CS solubilized with DMSO or bile acids, but not by sulfatides or other membrane lipids at 37 degrees C. Both the sulfate group and the hydrophobic side chain of CS were indispensable structures for the inhibition of DNase I. Also, the optimum molar ratio of bile acids to CS was important for expression of the inhibitory activity of CS toward DNase I, it being 0.18 of the optimum ratio for sodium taurocholate, and the molar ratio of CS to DNase I for complete inhibition was 342:1. Thus, CS was shown to play a role as an epithelial inhibitor of DNase I in concert with bile acids.     

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.