Purification and characterization of bile acid-CoA:amino acid N-acyltransferase from rat liver

An in vitro study of bile acid-CoA:amino acid N-acyltransferase activity of rat liver was undertaken in order to determine whether separate amino acid-specific enzymes catalyzed the formation of glycine and taurine conjugates of bile acids as postulated by others. Polyacrylamide gel electrophoresis of 200-fold purified enzyme localized the glycine- and taurine-dependent activities to a single band. Both activities were optimal at pH 7.8 and showed similar loss of activity at pH 6.0, pH 9.0, in the presence of 5,5'-dithiobis(2-nitrobenzoic acid), and at temperatures exceeding 50 degrees. With the purified fraction, Km for glycine was 31 mM and Km for taurine was 0.8 mM. Km for several bile acid-CoA substrates was approximately 20 micron and independent of the amino acid acceptor. Only amino acids with terminal alpha- or beta-amino groups were active as acyl acceptors. Acyl donors were limited to bile acid-CoA derivatives. The data support the conclusion that the rat has a single bile acid-CoA:amino acid N-acyltransferase. The substrate kinetics are consistent with previous observations that taurine conjugates predominate in rat bile at normal hepatocellular concentrations of glycine and taurine.     

Relative acidity scale of glycine- and taurine-conjugated bile acids through ESI-MS measurements

The most important bile acids, in the form of glycine and taurine conjugates, have been ordered in terms of relative acidity scale. The measurements have been carried out using mass spectrometric techniques. The group of taurine conjugates confirm the superior acidity over the glycine derivatives. Rationale of the differences found in gas-phase and comparison with the data reported in solution-phase are discussed with the support of theoretical calculations. The study has been completed with the acidity sequence of mixed oxo-hydroxy bile acids.     

Biological properties of the 2-fluoro-beta-alanine conjugates of cholic acid and chenodeoxycholic acid in the isolated perfused rat liver

The isolated perfused rat liver was used to examine the hepatic extraction, biliary secretion and effect on bile flow of the 2-fluoro-beta-alanine conjugates of cholic acid and chenodeoxycholic acid. The naturally occurring taurine and glycine conjugates of these bile acids were used for comparisons. The 2-fluoro-beta-alanine conjugates were extracted by the liver to a similar extent as the taurine and glycine conjugates. The biliary secretion rate and increase in bile flow were similar for all the cholic acid conjugates. On the other hand, the maximal biliary secretion rate of the 2-fluoro-beta-alanine conjugate of chenodeoxycholate was similar to that of the glycochenodeoxycholate, but 47% lower than that of taurochenodeoxycholate. In addition, the 2-fluoro-beta-alanine conjugate of chenodeoxycholate produced a decrease in bile flow that was comparable to that observed with the glycochenodeoxycholate (54% vs. 74%), but which was greater than that produced by the taurochenodeoxycholate (12%). In summary, these data demonstrate that the biological properties of the 2-fluoro-beta-alanine conjugates of cholic acid and chenodeoxycholic acid are not markedly different from those of the naturally occurring taurine and glycine conjugates. These data also suggest that the amino acid moiety can influence the biliary secretion and cholestatic properties of chenodeoxycholic acid conjugates.     

Development and validation of a method for measuring the glycine and taurine conjugates of bile acids in bile by high-performance liquid chromatography

We developed and validated a simple method for measuring the individual glycine and taurine conjugates of bile acids in bile by high-performance liquid chromatography with a C₁₈ reversed-phase column using an isocratic solvent system of acidified methanol—potassium phosphate. Without preliminary derivatization or purification, complete separation of the ten major conjugated bile acids present in bile could be achieved in 65 min. Total bile acid concentrations were identical when measured enzymatically and by summing the individual bile acids determined by high-performance liquid chromatography. Bile acid composition determined by gas—liquid chromatography correlated with results by high-performance liquid chromatography. Finally, measurements of individual glycine and taurine conjugates in human bile and in mixtures of bile acid standards by high-performance liquid chromatography and thin-layer chromatography gave similar results. This high-performance liquid chromatographic system permits simultaneous quantification of total and individual bile acids and their glycine and taurine conjugates in bile.     

Metabolism of deoxycholic acid in bile fistula patients

Although it has been assumed that the secondary bile acid deoxycholic acid is not rehydroxylated by the human liver, little direct evidence is available to support this assumption. To investigate the metabolism of deoxycholic acid in man, deoxycholic acid-(14)C was given intravenously to two patients with complete external bile fistulas. After hydrolysis of the bile salts and chromatographic separation of bile acids, more than 94% of the radioactivity was found in deoxycholic acid and the remainder was scattered in several small unidentified peaks, none of which was cholic acid. Approximately 85% of deoxycholate was excreted as glycine conjugates and 13% as taurine conjugates in this experiment. No detectable sulfate esters were found. These results indicate that the metabolism of deoxycholic acid in man involves only the reconjugation with glycine and taurine without rehydroxylation to cholic acid or sulfation.     

Radioassay of bile acid coenzyme A:glycine/taurine: N-acyltransferase using an n-butanol solvent extraction procedure

A rapid, specific, and sensitive radioassay for measuring bile acid CoA:glycine/taurine: N-acyltransferase (EC 2.3.1) has been developed. In this assay, 3H-labeled amino acids (glycine or taurine) are conjugated with unlabeled bile acid CoA derivatives to form 3H-labeled bile acid amidates. Following incubation, the 3H-labeled bile acid amidate is separated from the unreacted amino acid by an n-butanol extraction method. The extraction procedure was developed by evaluating the effects of buffer concentration and pH on the recovery of radiolabeled bile acid amidate standards in the presence of human hepatic cytosol. Highest recovery (greater than 90%) of bile acid amidate standards occurred under acidic conditions (pH 2) in the presence of 1% (w/v) SDS. When the radioassay and accompanying n-butanol extraction procedure were utilized to study the amidation of glycine or taurine with cholic acid in human hepatic cytosol, a single peak of radioactivity corresponding with either authentic glycocholate or taurocholate was detected in the n-butanol phase by high-performance liquid chromatography. This assay for bile acid CoA:glycine/taurine: N-acyltransferase activity was linear with incubation time and protein concentration. This assay should be useful in the biochemical studies of this enzyme, as well as in the examination of bile acid amidation in clinical liver specimens.     

Quantitative determination of bile acids in bile with reversed-phase high-performance liquid chromatography

Separation and quantitation of glycine and taurine conjugates of commonly occurring bile acids in bile, i.e. lithocholic, deoxycholic, chenodeoxycholic, ursodeoxycholic and cholic acids in their naturally occurring states have been successfully accomplished using high-performance liquid chromatography. No preliminary purification of bile acids is required except ethanol extraction of bile. A μ Bondapak C₁₈ column and acetonitrile—methanol—phosphate buffer and ultraviolet detector at 200 nm were used. Detection limit was 0.05 μg and linearity was observed in the range up to 16 μg. Bile acid composition of ten randomly chosen normal human gallbladder bile samples is given. A large difference in bile acid composition between glycine and taurine conjugates was found to be present.     

Rat liver bile acid CoA:amino acid N-acyltransferase: expression,characterization, and peroxisomal localization

Bile acid CoA:amino acid N-acyltransferase (BAT) is responsible for the amidation of bile acids with the amino acids taurine and glycine. Rat liver BAT (rBAT) cDNA was isolated from a rat liver lambdaZAP cDNA library and expressed in Sf9 insect cells using a baculoviral vector. rBAT displayed 65% amino acid sequence homology with human BAT (hBAT) and 85% homology with mouse BAT (mBAT). Similar to hBAT, expressed rBAT was capable of forming both taurine and glycine conjugates with cholyl-CoA. mBAT, which is highly homologous to rBAT, forms only taurine conjugated bile acids (Falany, C. N., H. Fortinberry, E. H. Leiter, and S. Barnes. 1997. Cloning and expression of mouse liver bile acid CoA: Amino acid N-acyltransferase. J. Lipid Res. 38: 86-95). Immunoblot analysis of rat tissues detected rBAT only in rat liver cytosol following homogenization and ultracentrifugation. Subcellular localization of rBAT detected activity and immunoreactive protein in both cytosol and isolated peroxisomes. Rat bile acid CoA ligase (rBAL), the enzyme responsible for the formation of bile acid CoA esters, was detected only in rat liver microsomes. Treatment of rats with clofibrate, a known peroxisomal proliferator, significantly induced rBAT activity, message, and immunoreactive protein in rat liver. Peroxisomal membrane protein-70, a marker for peroxisomes, was also induced by clofibrate, whereas rBAL activity and protein amount were not affected. In summary, rBAT is capable of forming both taurine and glycine bile acid conjugates and the enzyme is localized primarily in peroxisomes in rat liver.     

Determination of fetal bile acids in biological fluids from neonates by gas chromatography-negative ion chemical ionization mass spectrometry

A method has been developed for microanalysis of fetal bile acids in biological fluids from neonates by capillary gas chromatography-mass spectrometry using negative-ion chemical ionization of pentafluorobenzyl ester-dimethylethylsilyl ether derivatives of bile acids. Calibration curves for the bile acid derivatives are useful over the range 0.1–100 pg and the detection limit for bile acids was 1 fg (S/N=5) using isobutane as a reagent gas. Recoveries of the bile acids and their glycine and taurine conjugates from bile acid-free serum and dried blood discs ranged from 92 to 101% and from 93 to 108%, respectively, of the added amounts of their standard samples. The analysis of bile acids on a dried blood disc, meconium and urine from infants, exhibited significant hydroxylation at the 1β-, 2β-, 4β and 6α-positions of the usual bile acids, cholic and chenodeoxycholic acids, for the urinary or fecal excretion of bile acids in the fetal and neonatal periods. The present method was applied clinically to analyze bile acids on a dried blood disc from neonatal patients with congenital biliary atresia and hyper-bile-acidemia.     

Modulation of gamma-glutamyl transpeptidase activity by bile acids

The free bile acids (cholate, chenodeoxycholate, and deoxycholate) stimulate the hydrolysis and transpeptidation reactions catalyzed by gamma-glutamyl transpeptidase, while their glycine and taurine conjugates inhibit both reactions. Kinetic studies using D-gamma-glutamyl-p-nitroanilide as gamma-glutamyl donor indicate that the free bile acids decrease the Km for hydrolysis and increase the Vmax; transpeptidation is similarly activated. The conjugated bile acids increase the Km and Vmax of hydrolysis and decrease both of these for transpeptidation. This mixed type of modulation has also been shown to occur with hippurate and maleate (Thompson, G.A., and Meister, A. (1980) J. Biol. Chem. 255, 2109-2113). Glycine conjugates are substantially stronger inhibitors than the taurine conjugates. The results with free cholate indicate the presence of an activator binding domain on the enzyme with minimal overlap on the substrate binding sites. In contrast, the conjugated bile acids, like maleate and hippurate, may overlap on the substrate binding sites. The results suggest a potential feedback role for bile ductule gamma-glutamyl transpeptidase, in which free bile acids activate the enzyme to catabolize biliary glutathione and thus increase the pool of amino acid precursors required for conjugation (glycine directly and taurine through cysteine oxidation). Conjugated bile acids would have the reverse effect by inhibiting ductule gamma-glutamyl transpeptidase.     

The hydrolysis of bile acid conjugates

Studies were made of a) the relationship of bile acid structure and analytical recoveries (measured by 3-hydroxysteroid oxidoreductase) following vigorous alkaline hydrolysis of bile acid conjugates and b) the relationship of structure and hydrolysis time of taurine- and glycine bile acid conjugates in a reaction catalyzed by glycocholic acid hydrolase. Alkaline hydrolysis resulted in good recoveries of hydroxy and 7 and 12- oxo-bile acids but poor recoveries of 3-oxo-bile acids. Borohydride reduction of the 3-oxo-acids prevented these losses. Complete enzymatic hydrolysis of glycine conjugated bile acids was about five times more rapid than that of taurine conjugates. Hydrolysis of conjugates containing oxo groups was slow. Borohydride reduction of oxoacids corrected this and did not inhibit enzymatic hydrolysis. It was concluded that both vigorous alkaline and enzymatic hydrolysis are satisfactory in bile acid assays if borohydride reduction is instituted before the hydrolytic step. However, due to the presence of possible enzyme inhibitors and solubility difficulties, strong alkaline hydrolysis is preferable to enzymatic hydrolysis in fecal bile acid determinations at this time.     

Human cecal bile acids: concentration and spectrum

To obtain information on the concentration and spectrum of bile acids in human cecal content, samples were obtained from 19 persons who had died an unnatural death from causes such as trauma, homicide, suicide, or drug overdose. Bile acid concentration was measured via an enzymatic assay for 3alpha-hydroxy bile acids; bile acid classes were determined by electrospray ionization mass spectrometry and individual bile acids by gas chromatography mass spectrometry and liquid chromatography mass spectrometry. The 3alpha-hydroxy bile acid concentration (mumol bile acid/ml cecal content) was 0.4 +/- 0.2 mM (mean +/- SD); the total 3-hydroxy bile acid concentration was 0.6 +/- 0.3 mM. The aqueous concentration of bile acids (supernatant after centrifugation) was identical, indicating that most bile acids were in solution. By liquid chromatography mass spectrometry, bile acids were mostly in unconjugated form (90 +/- 9%, mean +/- SD); sulfated, nonamidated bile acids were 7 +/- 5%, and nonsulfated amidated bile acids (glycine or taurine conjugates) were 3 +/- 7%. By gas chromatography mass spectrometry, 10 bile acids were identified: deoxycholic (34 +/- 16%), lithocholic (26 +/- 10%), and ursodeoxycholic (6 +/- 9), as well as their primary bile acid precursors cholic (6 +/- 9%) and chenodeoxycholic acid (7 +/- 8%). In addition, 3beta-hydroxy derivatives of some or all of these bile acids were present and averaged 27 +/- 18% of total bile acids, indicating that 3beta-hydroxy bile acids are normal constituents of cecal content. In the human cecum, deconjugation and dehydroxylation of bile acids are nearly complete, resulting in most bile acids being in unconjugated form at submicellar and subsecretory concentrations.     

Precipitation and 13C-NMR relaxation enhancement measurements of the interactions of bile acids with synthetic cationic bile acid derivatives, and with spin labelled fatty acids.

In an investigation of novel potential bile acid sequestrants, the affinities of the sodium salts of the glycine and taurine conjugates of naturally occurring bile acids (cholate, deoxycholate, chenodeoxycholate and lithocholate) for several cationic ammonium bile acid derivatives have been investigated by measurements of the extent to which the derivatives are able to precipitate the bile acids. This is roughly proportional to the lipophilicity of the interacting species. Thus, amino and ammonium derivatives of cholic acid do not precipitate taurocholate or glycocholate to any great extent, whereas ammonium derivatives of deoxycholate and lithocholate are much more effective. To complement the precipitation measurements, high resolution 13C-NMR has been applied to investigate the weaker interactions between the ammonium cholate derivative and glycocholate, glycodeoxycholate and glycochenodeoxycholate. Addition of either of the latter two bile acids to the cationic ammonium compound results in considerable broadening of the 13C resonances of both species, indicating the formation of relatively rigid structures. In addition, we have used T2 relaxation enhancement induced by spin-labelled fatty acids to examine the mechanism of interaction with bile acids of amphiphilic anions, which might compete with bile acids for sites on bile acid sequestrants. Low concentrations of 16-DOXY L-Stearate dramatically broaden the 13C-NMR resonances of deoxycholate carbons 19, 18 and 7 in particular, while 5-DOXY L-Stearate exerts much less specific effects. These results have been incorporated into a snapshot model of bile acid-fatty acid interactions.     

Calcium binding by bile acids: in vitro studies using a calcium ion electrode

In this study, we compared in vitro calcium binding by the taurine and glycine conjugates of the major bile acids in human bile: cholic (CA), chenodeoxycholic (CDCA) and deoxycholic (DCA) acids, together with the cholelitholytic bile acids ursodeoxycholic (UDCA) and ursocholic (UCA) acids. At physiological total calcium (CaTOT) (1-15 mM) and bile acid (BA) (10-50 mM) concentrations, all the bile acids caused concentration-dependent falls in [Ca2+], suggesting calcium binding. Except for glycine-conjugated CDCA, all the other calcium-bile acid complexes were soluble in 150 mM NaCl. The calcium binding affinities followed the pattern: dihydroxy (CDCA, UDCA and DCA) greater than trihydroxy (CA and UCA) bile acids, and glycine conjugates greater than taurine conjugates. The glycine conjugate of UDCA, which increases during UDCA treatment, had the highest calcium binding affinity. Ten-20 mM phospholipid modestly increased calcium binding by CA conjugates, but not by CDCA, UDCA, and DCA conjugates. Phospholipid also prevented the precipitation of glyco-CDCA in the presence of calcium. Bile acid-calcium biding was pH-independent over the range 6.5-8.5. The different calcium binding affinities of the major biliary bile acids may partly explain their varying effects on biliary calcium secretion. The results also suggest that neither precipitation of calcium-bile acid complexes nor impaired calcium binding by bile acids is important in the pathogenesis of human calcium gallstone formation.     

BILIARY BILE ACID COMPOSITION OF THE PHYSETERIDAE (SPERM WHALES)

Abstract: The bile acid composition of bile obtained from the hepatopancreatic ducts of three species of sperm whales (Cetacea: Physeteridae) was investigated. Bile acids were isolated by adsorption chromatography and analyzed by sequential HPLC, SIMS, and GLC-MS. In each species the dominant bile acids were deoxycholic acid (a secondary bile acid formed by bacterial 7α-dehydroxylation of cholic acid), and chenodeoxycholic acid (a primary bile acid) which together composed more than 86% of biliary bile acids in all three species. In Physeter catodon (sperm whale) deoxycholic acid constituted 79%, and in Kogia breviceps (pygmy sperm whale) it was 61% of biliary bile acids. The sperm whale, which differs from other whales in having a remnant of a large intestine, is the second mammal identified to date in which deoxycholic acid is the predominant bile acid. The high proportion of deoxycholic acid indicates that in the Physeteridae, anaerobic fermentation occurs in its cecum, and that bile acids undergo enterohepatic cycling. Also found were minor proportions of cholic acid, as well as bacterial derivatives of chenodeoxycholic acid (ursodeoxycholic acid, lithocholic acid, and the 12β-epimer of allo-deoxycholic acid). Bile acids were conjugated with taurine in all species; however, in the sperm whale ( Physeter ) glycine conjugates were present in trace proportions. The bile acid hydroxylation pattern (12α- but not 6α-hydroxylation), lack of primary 5α- (allo) bile acids, and presence of glycine conjugated bile acids suggests the possibility that sperm whales originated from ancient artiodactyls.     

N-acetylglucosaminides. A new type of bile acid conjugate in man

Bile acids were extracted from human urine and were separated into groups of nonamidated and glycine- and taurine-conjugated compounds. Each group was subfractionated in a reversed-phase high performance liquid chromatography system, and the fractions were analyzed by negative ion fast atom bombardment mass spectrometry and also by gas chromatography-mass spectrometry after enzymatic removal of glycine and taurine moieties. The major glycosides of the non-amidated bile acids were more polar than reference bile acid glucosides and gave quasimolecular ions at m/z 592, 594, and 610 consistent with N-acetylglucosaminides of unsaturated dihydroxy and saturated di- and trihydroxy bile acids. Gas chromatography-mass spectrometry analyses of methyl ester trimethylsilyl ether derivatives showed fragments typical for N-acetylglucosaminides (m/z 173 and 186) in addition to those also given by glucosides (m/z 204 and 217). The N-acetylglucosaminides were inert toward alpha- and beta-glucosidase but were cleaved completely with N-acetylglucosaminidase. The released sugar moiety was identified as N-acetylglucosamine. One of the liberated bile acids was identified as ursodeoxycholic acid. The other acids were not identical to any known primary or secondary bile acid in humans. Fast atom bombardment mass spectrometry analyses of the glycine-and taurine-conjugated bile acid glycosides only showed ions consistent with the presence of glucosides (m/z 626 and 676). These compounds were sensitive only toward beta-glucosidase which liberated a trihydroxy bile acid as the major compound. Based on the recover of 13C- and 14C-labeled chenodeoxycholic acid glucoside added as internal standard, the daily excretion of nonamidated bile acid glycosides was estimated to be about 137 micrograms or 0.29 mumol, N-acetylglucosaminides constituting about 90%. The daily excretion of the glucosides of amidated bile acids was about 150 micrograms or 0.25 mumol, glycine conjugates constituting about 90%.     

Evidence for bile acid glucosides as normal constituents in human urine

A glucosyltransferase catalysing formation of bile acid glucosides was recently isolated from human liver microsomes. In order to investigate the potential occurrence of such bile acid derivatives in vivo, a method was devised for their isolation and purification from urine. Conditions were established with the aid of glucosides of radiolabelled, unconjugated glycine and taurine conjugated bile acids prepared enzymatically using human liver microsomes. Analysis by gas chromatography and mass spectrometry of methyl ester trimethylsilyl ether derivatives indicated the excretion of glucosides of nonamidated hyodeoxycholic, chenodeoxycholic, deoxycholic, ursodeoxycholic and cholic acids and of glycine and taurine conjugated chenodeoxycholic and cholic acids. Additional compounds were present giving mass spectral fragmentation patterns typical ofdi- and trihydroxy bile acid glycosides. Semiquantitative estimates indicated a total daily excretion of about 1 μmol.     

Alkaline solvent systems for thin-layer chromatography of bile acids

Thin-layer chromatographic separation of the common bile acids and their taurine and glycine conjugates in chloroform-methanol-ammonia is reported. An alkaline system offers advantages for the separation and nondestructive staining of bile acid conjugates.     

High pressure liquid chromatographic analysis of conjugated bile acids in human bile: simultaneous resolution of sulfated and unsulfated lithocholyl amidates and the common conjugated bile acids

A reversed phase high pressure liquid chromatography (HPLC) system capable of simultaneously separating four lithocholyl species (sulfated and unsulfated forms of lithocholylglycine and lithocholyltaurine) as well as the eight other major conjugated bile acids present in human bile is described. The system uses a C18 octadecylsilane column and isocratic elution with methanol phosphate buffer, pH 5.35. Relative bile acid concentration is determined by absorbance at 200 nm. Retention times relative to chenodeoxycholylglycine are reported for the four lithocholic acid forms, the glycine and taurine amidate of the four major bile acids present in human bile (cholic, chenodeoxycholic, ursodeoxycholic, and deoxycholic), and for their corresponding unconjugated forms. Retention times are also reported for the glycine and taurine amidates as well as the unconjugated form of the C23 norderivatives of these bile acids. Maximal absorbance of bile acid amidates is at 200 nm and is very similar for the (unsulfated) glycine and taurine amidates. Sulfated lithocholyl amidates exhibit molar absorptivities at 200 nm which are 1.4 times greater than that of non-sulfated lithocholyl amidates. Unconjugated bile acid absorbance at 200 nm or 210 nm is 20 to 30 times less than that of corresponding peptide conjugates. The method has been applied to samples of gallbladder bile obtained from 14 healthy subjects to define the pattern of conjugated bile acids present in human bile.     

A comparative study of bile acid CoA:amino acid:N-acyltransferase (BAT) from four mammalian species.

1. Bile acid CoA:amino acid:N-acyltransferase (BAT) was partially purified from dog, human, pig and rat livers. The interspecies variation in substrate specificity and kinetics were determined for glycine and taurine. 2. BAT activity from dog liver formed bile acid conjugates with taurine exclusively, whereas BAT activity from each of the other species formed conjugates with both taurine and glycine. 3. Biliary composition of glycine and taurine bile acid conjugates could partly be accounted for by substrate affinity (Km) and turnover number (Vmax) of BAT activity. 4. A monospecific anti-human BAT polyclonal antibody reacted on Western blot analysis with a 40 kDa band in a 100,000 g supernatant fraction from rat liver. 5. Immunoabsorption chromatography using an anti-human BAT antibody-Sepharose affinity column showed that both the immunoreactive protein band and BAT activity were removed from the 100,000 g supernatant fraction from human and rat livers.