Cholylglycine Hydrolase Research Articles

Metabolism of 5-fluorouracil to an N-cholyl-2-fluoro-beta-alanine conjugate: previously unrecognized role for bile acids in drug conjugation.

Recently we demonstrated clinically significant levels of a previously unrecognized metabolite of the anticancer drug 5-fluorouracil (FUra) in bile of cancer patients. In the present study, reanalysis of bile from these patients demonstrated the presence of not one but two previously unrecognized metabolites. The major unrecognized metabolite was purified by reversed-phase HPLC, after which its molecular weight was determined by fast-atom-bombardment mass spectrometry to be 497. The similarity in HPLC retention times and molecular weights of this FUra derivative and the bile acids N-cholylglycine (Mr 465) and N-cholyltaurine (Mr 515), along with the structural similarity of the FUra catabolite 2-fluoro-beta-alanine and the amino acids glycine and taurine, led to the hypothesis that this metabolite could be a conjugate of 2-fluoro-beta-alanine and cholic acid. This hypothesis was tested and confirmed by hydrolyzing the purified metabolite by cholylglycine hydrolase after which: 2-fluoro-beta-alanine was demonstrated by using a sensitive HPLC technique capable of resolving all of the known putative FUra metabolites, and unconjugated cholic acid was identified by both GC and GC-MS. Additionally, chemically synthesized N-cholyl-2-fluoro-beta-alanine was shown to cochromatograph on HPLC and TLC with the purified biliary metabolite. In summary, this study demonstrates a unique, so far as we know, pathway of drug metabolism in man in which an amino acid drug metabolite is conjugated with cholic acid and eliminated into the bile. Furthermore, the finding that 2-fluoro-beta-alanine is conjugated to bile acids may provide some insight into the mechanism of cholestasis that is frequently observed after administration of fluoropyrimidine by hepatic arterial infusion.

Improved enzymatic hydrolysis of conjugated bile acids.

The hydrolysis of conjugated bile acids by cholylglycine hydrolase (EC 3.5.1.24) using the standard procedure for a commercial enzyme preparation was found to be incomplete, as judged by the use of 24-14C-taurocholic acid as a tracer. A method is proposed that incorporates the nonionic detergent Triton X-100 into the reaction mixture to achieve almost complete hydrolysis. It is proposed that the observed enhancement of enzyme activity is due to the formation of micelles by the detergent.

Rapid diagnosis of Zellweger syndrome and infantile Refsum's disease by fast atom bombardment—mass spectrometry of urine bile salts

A method is described for the rapid determination of urinary bile salt profiles by fast atom bombardment-mass spectrometry (FAB-MS). Urine was passed through a reverse-phase octadecylsilane bonded silica cartridge and the bile salts eluted with methanol. Negative ion FAB spectra could be obtained from the equivalent of 10 μl of urine loaded onto the target probe with glycerol as matrix. In samples from normal infants and children bile salt peaks were rarely detectable above the background whereas peaks produced by steroid sulphates and glucuronides and bile alcohol glucuronides could usually be identified. In samples from infants and children with cholestasis the major peaks were produced by the taurine and glycine conjugates of di-, tri- and tetrahydroxycholanoic acids (and their monosulphates). In samples from patients with Zellweger syndrome and infantile Refsum's disease, a unique ion at m/ z 572 indicated the presence of taurine-conjugated tetrahydroxycholestanoic acid(s). The amide linkage to taurine was cleaved by alkaline hydrolysis but not by cholylglycine hydrolase. Capillary gas chromatography-mass spectrometry (GC-MS) of the bile acids liberated by alkaline hydrolysis indicated the presence of at least two nuclear-tetrahydroxylated cholestanoic acids, probably the 6α- and 1β-hydroxylated derivatives of 3α, 7α, 12α-trihydroxy-5β-cholestan-26-oic acid.

Pancreatic carboxypeptidase hydrolysis of bile acid-amino acid conjugates: Selective resistance of glycine and taurine amidates

To find a possible explanation for the selective hepatic conjugation of bile acids with glycine or taurine, the N-acyl amidates of cholic acid and a number of amino acids and amino acid analogues were synthesized, and their susceptibility to hydrolysis by pancreatic juice, gastric juice, serum, or small intestinal mucosal enzymes was measured. Deconjugation by pure carboxypeptidase A and B was also examined, and hydrolysis by these tissue fluids and enzymes was compared with that mediated by a bacterial cholylglycine hydrolase. Human pancreatic juice efficiently hydrolyzed cholyl conjugates of all neutral-l-amino acids (cholyl-l-alanine, cholyl-l-valine, cholyl-l-leucine, and cholyl-l-tyrosine), except cholylglycine. The net hourly rate of hydrolysis (in micromales per milligram protein per hour) increased when the terminal residue was aromatic or branched aliphatic, and appeared to be specific for l-α-amino acids as cholyl-β-alanine and cholyl-d-valine were not cleaved. From cholyl glycylglycine, only the terminal glycine was efficiently removed. Cholyltaurine and cholyl conjugates with the methyl and propyl analogues of taurine were resistant to hydrolysis. Two basic amino acid conjugates (cholyl-l-lysine and cholyl-l-arginine) were cleaved, whereas conjugates of acidic amino acids (cholyl-aspartate and cholyl-cysteate) were not cleaved. Studies using pure enzymes showed that bovine carboxypeptidase A hydrolyzed the cholyl conjugates of the neutral l-α-amino acids with similar specificity as observed for the human pancreatic juice, whereas bovine carboxypeptidase B cleaved the basic amino acid conjugates. Cholyl-l-lysine and cholyl-l-arginine were also cleaved by serum and plasma, which are known to possess carboxypeptidase activity. Cholyl conjugates were not cleaved by gastric juice, by trypsin, or by homogenates of rat small intestinal mucosa. In contrast, all cholyl conjugates were cleaved by a bacterial cholylglycine hydrolase. These experiments indicate that glycine and taurine amidates of cholic acid differ from a number of other conjugates with neutral and basic amino acid in being resistant to hydrolysis by pancreatic and plasma carboxypeptidases. These data, together with other data indicating that bile acid conjugation greatly decreases passive intestinal absorption, indicate that a physiologic function of bile acid conjugation with glycine or taurine is to form surfactants that remain indigestible and rather nonabsorbable during digestion in the proximal small intestine.

Fasting and postprandial serum bile acid concentration with special reference to variations in the conjugate profile.

Serum bile acids were group-separated by ion exchange chromatography on diethylaminohydroxypropyl Sephadex LH-20 into unconjugated bile acids and bile acids conjugated with either glycine, taurine, glucuronic acid or sulphuric acid. The conjugate moiety was hydrolysed by treatment with a combination of Helix pomatia and cholylglycine hydrolase and the released bile acids analysed by gas liquid chromatography/mass spectrometry. Analysis of fasting and postprandial serum from six healthy subjects showed that, in addition to the primary bile acids, cholic (C) and chenodeoxycholic acid (CDC), secondary bile acids were present to varying extents. Unconjugated serum bile acids were found in four of the six subjects. Glycine and taurine conjugates of C and CDC and their glucuronides and sulphates were found in all subjects. The postprandial increase of serum bile acids was mainly due to increase of the glycine conjugates of C and CDC. After the meal, the ratio C:CDC in glycine and taurine conjugates shifted to lower values.

Substrate specificity of cholylglycine hydrolase for the hydrolysis of bile acid conjugates.

The substrate specificity of cholylglycine hydrolase has been investigated using bile acid conjugates with modifications in the steroid ring system, the side chain, or the amino acid moiety. Epimerization at C-3 and C-7 did not affect the activity of the enzyme while oxidation of the three nuclear hydroxyl groups reduced the affinity of the enzyme toward the substrate. Elongation of the side chain by one or three carbons inhibited enzyme activity. Conjugates prepared from C24 bile acids and analogs of taurine and glycine with one or two methylene groups were effectively hydrolyzed, whereas conjugates with a tertiary amide group completely resisted hydrolysis. Increasing the length of the bile acid side chain or using a bile acid conjugate with a tertiary amide group may produce compounds that will resist intestinal bacterial destruction.

Critical evaluation of the existence of so-called tissue-bound lithocholate in human liver tissue by selected ion monitoring.

Monohydroxy bile acids in liver tissue may be of importance because of their hepatotoxicity and strong cholestatic effects. Recently, the existence of lithocholate in liver tissue in two forms was suggested by Nair et al. (Lipids. 1977. 12: 922-929) i.e., either in free form or as so-called tissue-bound lithocholate released exclusively by cholylglycine hydrolase treatment. The presence of the latter aroused much interest in relation to its hepatotoxicity and possible role in tumor induction. In the present investigation lithocholyl-epsilon-L-lysine, proposed as the predominant tissue-bound bile acid, was synthesized and its metabolic behavior was tested. Lithocholyl-epsilon-lysine was not deconjugated by cholylglycine hydrolase treatment but only by alkaline hydrolysis. Bile acids in seven cirrhotic and three noncirrhotic liver samples were extracted with 95% ethanol-0.1% ammonium hydroxide. The bile acids in the extract and residue were quantified by glass capillary gas-liquid chromatography using selected ion monitoring. The presence of so-called tissue-bound lithocholate could not be substantiated in either cirrhotic or noncirrhotic liver tissues. Nearly complete extraction of lithocholate was achieved by the use of organic solvent alone. Therefore, tissue-bound lithocholate, if it exists at all, may be attached to tissue by a physical linkage which can be disrupted by the use of conventional organic solvent.

Inability of cholylglycine hydrolase to cleave the amide bond of tauronorcholic acid

High pressure liquid chromatography, glass capillary gas chromatography and liquid secondary ion mass spectrometry have been used to demonstrate that the amide bond of the taurine conjugate of norcholic acid, a homolog of cholic acid which contains one less methylene group in the side chain, cannot be hydrolyzed enzymatically by cholylglycine hydrolase. Conventional alkaline hydrolysis cleaved the amide bond of both taurocholate and tauronorcholate, producing the free acids. Treatment with cholylglycine hydrolase yielded the free acid for taurocholate but failed to affect tauronorcholate, which was recovered quantitatively.

The nature of urinary bile acid conjugates in patients with extrahepatic cholestasis.

Urinary bile acids from patients with extrahepatic cholestasis were extracted with Sep-pak C18 cartridges and group separated on diethylaminohydroxypropyl Sephadex LH-20. The nature of the different conjugates of cholic and chenodeoxycholic acid in the fractions was studied after further separation by preparative thin-layer chromatography. The free and glycine-conjugated bile acids were quantified by capillary gas chromatography and identified by gas chromatography-mass spectrometry (GC/MS). Taurine conjugates were split with cholylglycine hydrolase and the liberated free bile acids analysed by GC/MS. Sulphate esters were hydrolysed with Helix pomatia and the resulting bile acid derivatives were analysed as above. After hydrolysis with cholylglycine hydrolase, the glucuronides of the unconjugated bile acids were separated and identified by GC/MS. Amino acid analysis of the different fractions revealed that glycine and taurine were the only amino acids present in connection with cholic and chenodeoxycholic acid. Large amounts of monosulphated bile acid conjugates were present but no disulphates. Only 3-sulphates were found. Both sulphates and glucuronides were found exclusively as glycine or taurine conjugates and no such derivatives of unconjugated bile acids were isolated. The isolated conjugates were split either by a combination of acid solvolysis and alkaline hydrolysis or by Helix pomatia and cholylglycine hydrolase.

Energy transport in dc and ac vortex stabilized arcs: L E Gettel and F L Curzon, J Phys D: Appl Phys, 15 (5), 1982, 845–865

Since 2-fluoro-β-alanine (FBAL) conjugates of bile acids (BA), the primary biliary metabolites of fluoropyrimidine (FP) drugs, have been suggested to be related to the hepatotoxicity which develops in patients receiving FP chemotherapy by intrahepatic arterial infusion (Proc. Natl. Acad. Sci. USA 84, 5439–5443, 1987), it was important to determine whether they undergo enterohepatic circulation and hence accumulate in the liver and biliary system. In initial studies, sensitivity of FBAL-BA conjugates to hydrolysis by pancreatic enzymes was examined. In subsequent in vivo studies, a model FBAL-BA conjugate, FBAL-chenodeoxycholate (FBAL-CDC), was introduced into the lumen of the small intestine of anesthetized rats with biliary fistulas to quantitate the intestinal absorption, metabolism and tissue distribution of the conjugate. The results indicated that: (1) FBAL-BA conjugates were resistant to hydrolysis by pancreatic enzymes (carboxypeptidase A, carboxypeptidase B and trypsin) and by human pancreatic juice, but were completely hydrolyzed by cholylglycine hydrolase. (2) At least one-half of the administered FBAL-CDC was deconjugeted during the process of intestinal absorption, as shown by HPLC analysis of the radioactivity in portal venous blood. (3) Deconjugated FBAL or CDC was reconjugated in liver with other bile acids or amino acids (glycine and taurine), respectively, as shown by radiochromatography of bile. (4) FBAL, formed as a result of hydrolysis of FBAL-CDC, had a wide tissue distribution. In conclusion, FBAL-CDC has a rapid turnover during its enterohepatic circulation due to deconjugation in the intestine and reconjugation in the liver.

Fluorescence high-performance liquid chromatographic determination of free and conjugated bile acids in serum and bile using 1-bromoacetylpyrene as a pre-labeling reagent

A fluorescence high-performance liquid chromatographic method is described for the determination of free and conjugated bile acids in serum and bile. Free and conjugated bile acids are extracted from serum or bile using a Sep-Pak C 18 cartridge and then fractionated on a piperidinohydroxypropyl Sephadex LH-20 column. Free and glycine-conjugated bile acids are labeled with 1-bromoacetylpyrene in acetonitrile using dicyclohexyl-18-crown-6-ether as catalyst. Taurine-conjugated bile acids are hydrolyzed by cholylglycine hydrolase and then derivatized by the same reagent. Derivatized bile acids are separated stepwise on a reversed-phase column (Radial Pak A) using acetonitrile—methanol—water (A) (100 : 50 : 40) and (B) (100 : 50 : 20) as mobile phase. The eluate is monitored by a fluorophotometer at 370 nm (excitation) and 440 nm (emission). Linearities of fluorescence intensities (peak heights) with the amounts of free and conjugated bile acids were obtained between 50 pmol and 200 pmol for free bile acids and between 25 pmol and 100 pmol for glycine-conjugated bile acids, respectively. Recoveries from serum and bile samples are not less than 90%. This method is sensitive, reliable and useful for the simultaneous determination of free and conjugated bile acids in serum and bile.

Isolation and quantitation of sulfated and unsulfated steroids in human feces

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.

An improved gas-chromatographic method for the determination of sulfated and unsulfated bile acids in serum

This study has been aimed at improving some steps in the gas-liquid Chromatographic determination of sulfated bile acids. The best conditions for the enzymatic hydrolysis with cholylglycine hydrolase of sulfolithocholylglycine and sulfolithocholyltaurine are described. Recoveries of more than 85% were obtained after prolonging the incubation to 12 h. A single-step procedure for solvolysis and methylation of bile acids was achieved by using, as a water scavenger, 2,2-dimethoxypropane added directly to the hydrolysis mixture. This procedure avoided the difficulty of the extraction of sulfated bile acids from aqueous solutions.

Determination of bile acids in serum by capillary gas-liquid chromatography

A glass capillary column and an appropriate relatively simple procedure for sample preparation have been developed for determination of serum bile acids. Sample preparation involved extraction with Amberlite XAD-2, solvolysis of sulfates, enzymatic hydrolysis with cholylglycine hydrolase, methylation and silylation. Because of complete Chromatographie separation of bile acid trimethylsilylether derivatives from cholesterol on the capillary column, an additional step for elimination of cholesterol could be omitted. Trimethylsilylether derivatives were separated on a 20 meter × 0.3 mm i.d. glass capillary column covered with a crystal layer of barium carbonate and coated with polyethyleneglycol 20 000 as liquid phase according to Grob, K. and Grob, G. (1976) J. Chromatogr. 125, 471–485, and Grob, K., Grob, G. and Grob, Jr., K., (1977) Chromatographia 10, 181–187. Overall recovery of the major human conjugated bile acids ranged from 86 to 89%. Reproducibility of bile acid determination was satisfactory in both normal and pathological serum with elevated bile acid concentrations (coefficient of variation 7.6 to 10.0%). The mean concentrations of cholic, deoxycholic, chenodeoxycholic and lithocholic acid in the serum of healthy subjects were 0.9, 1.0, 1.7 and 0.2 μmol/l in males, and 1.0, 0.8, 1.4 and 0.2 Jμmol/l in females.

Lithocholic acid in human liver: identification of epsilon-lithocholyl lysine in tissue protein.

AbstractHuman liver had been shown to contain two forms of lithocholic acid. One form is extractable by 95% ethanol‐ammonia, 1000∶1, v/v (soluble lithocholate, SL) and the other form is firmly bound to the tissue residue. The latter, tissue‐bound lithocholic acid (TBL), can be enzymatically released by means of the specific clostridial peptide bond hydrolase, cholylglycine hydrolase (cholanoyl amino acid hydrolase, EC no. 3.5). Solvolytic procedures for the analysis of hepatic lithocholic acid sulfate revealed that almost all of the TBL was non sulfated, while in the SL fraction there was an apparent preponderance of the sulfated form. Cholylglycine hydrolase liberates free labeled lithocholic acid from synthetic [24‐14C] lithocholyl‐bovine serum albumin and from [24‐14C]lithocholyl polylysine. By analogy, the enzyme releases lithocholic acid from tissue protein in which the bile acid is conjugated through amino groups of basic side chains. Hydrolysis of [24‐14C] lithocholyl polylysine with 6n HC1 yielded ∈‐[24‐14C] lithocholyl lysine, which was chromatographically similar to the product obtained by acid hydrolysis of TBL. Chromatographic, infrared, and mass spectroscopic studies with synthetic N‐α‐lithocholyl lysine, N‐∈‐lithocholyl lysine, and N‐α‐∈‐Bis lithocholyl lysine showed the hydrolytic product from TBL to be N‐∈‐lithocholyl lysine. Since monohydroxy bile acids, such as lithocholic acid, are known to show unusual cytotoxic properties, the identification of TBL in human liver poses important questions regarding the role of lithocholate in liver injury.

Purification and characterization of bile salt hydrolase from Bacteroides fragilis subsp. fragilis

A high-molecular-weight (250 000) bile salt hydrolase (cholylglycine hydrolase, EC 3.5.-.-) was isolated and purified 128-fold from the “spheroplast lysate” fraction prepared from Bacteroides fragilis subsp. fragilis ATCC 25285. The intact enzyme had a molecular weight of approx. 250 000 as determined by gel filtration chromatography. One major protein band, corresponding to a molecular weight of 32 500, was observed on 7% sodium dodecyl sulfate polyacrylamide gel electrophoresis of pooled fractions from DEAE-cellulose column chromatography (128-fold purified). The pH optimum for the 64-fold purified enzyme isolated from Bio-Gel A 1.5 M chromatography was 4.2 and bile salt hydrolase activity measured in intact cell suspensions had a pH optimum of 4.5. Substrate specificity studies indicated that taurine and glycine conjugates of cholic acid, chenodeoxycholic acid and deoxycholic acid were readily hydrolyzed; however, lithocholic acid conjugates were not hydrolyzed. Substrate saturation kinetics were biphasic with an intermediate plateau (0.2–0.3 mM) and a complete loss of enzymatic activity was observed at high concentration for certain substrates. The presence or absence of 7-α-hydroxysteroid dehydrogenase was absolutely correlated with that of bile salt hydrolase activity in six to ten strains and subspecies of B. fragilis.

Metabolism of Chenodeoxycholate by Intestinal Mucosa

The metabolism of 14C-labeled bile salts was studied in vitro during their absorption from infused segments of rat intestine. The transported bile salts were recovered in transudates which were collected from the serosal surface of infused segments. In the jejunum, 36% of transported [14C]deoxycholate and 48% of transported [14C]chenodeoxycholate were recovered as metabolites which migrated as a seemingly single polar compound on thin layer chromatography; 52 to 72% of these metabolites could be deconjugated by cholylglycine hydrolase to yield original [14C]deoxycholate or [14C]chenodeoxycholate, respectively. The jejunum metabolized 9 times more [14C]chenodeoxycholate than the ileum (38.1 nmoles per hr per g compared with 4.2 nmoles per hr per g), despite the fact that both segments transferred the same amount of radioactivity. In contrast, [14C]cholate, [14C]glycocholate, and [14C]glycodeoxycholate were transferred intact by the jejunum, ileum, and colon. Human jejunal and rectal mucosa were able to metabolize [14C]chenodeoxycholate to polar metabolites in vitro. Polar metabolites were also found in the portal plasma and jejunal wall 20 min after the feeding of [14C]chenodeoxycholate to bile fistula rats. It is suggested that these results complicate the interpretation of in vitro experiments on absorption of dihydroxy bile salts, and that they indicate that the human small intestine may be able to metabolize chenodeoxycholate.

Influence of Synthetic Conjugates of Cholic Acid on Cholesteremia in Rats

The effects on serum and liver cholesterol levels in rats of two naturally occurring conjugates of cholic acid ( taurocholic and glycocholic acids ) and four syn thetic conjugates (glutamocholic, aspartocholic, cysteocholic and cysteinocholic acids) (0.5% diet), in combination with cholesterol (0.5% of diet) were investigated. Hy drolysis of these conjugates by cholylglycine hydrolase (EC 3.5) was also measured. Cholesterol alone did not cause cholesteremia but when fed with cholic acid or any of its conjugates, except aspartocholate, the animals had significantly higher serumâ€liver cholesterol pools (15 to 70%). The aspartocholic acid-fed group had serum and liver cholesterol levels significantly lower than the cholic acid:cholesterol-fed animals but similar to control animals. When the degree of hydrolysis of each of the conjugates by cholyiglycine hydrolase was measured, all conjugates were hydrolyzed to a similar extent (77 to 87%) except aspartocholic (36%) and cysteinocholic acids (42%). Apparently there is a relationship between the ability of a cholic acid conjugate to pro duce elevated serum and/or liver cholesterol levels in rats and the degree to which it is hydrolyzed by the intestinal microfiora. J. Nutr. 104: 1185â€1188, 1974.

Determination of bile acids in needle biopsies of human liver

A microquantitative procedure is described to determine bile acids in 5–15 mg liver tissue, which can be obtained from the specimen of needle biopsy performed for histological examination. The tissue cylinder is solubilized in KOH and the bile acids are deconjugated by cholylglycine hydrolase, purified by thin-layer chromatography, methylated, and trimethylsililated for gas chromatographic separation and quantitation. Recovery of taurine- and glycine-conjugated bile acid standards is 73–86%; the experimental error of the procedure is less than 15%. The technique permits investigation of hepatic bile acids in liver diseases without requiring surgical exploration.

Determination of individual conjugated bile acids in human bile

A method has been developed and validated for the determination of the six major conjugated bile acids, cholesterol, and total phospholipids in bile of human subjects previously injected with 4-(14)C-cholesterol. The procedure is designed for use with 5-10 ml of duodenal or T-tube bile and eliminates difficulties associated with existing methods for bile acid determination, in particular the requirement for preliminary saponification under pressure or the use of paper chromatography. Saponification under pressure is employed only in steps where partial destruction of the steroid moiety of conjugated bile acids is not a crucial matter. A preliminary Folch extraction and washing step separated free cholesterol and phospholipids (bottom layer) from the six major conjugated bile acids (top layer). The conjugated bile acids were then fractionated cleanly by thin-layer chromatography to give four groups, the (14)C content of each of which was determined. A second aliquot of the top layer was used to determine (after deconjugation) the radioactivity ratio of deoxycholic acid to chenodeoxycholic acid for the two unresolved groups (dihydroxycholanoic acid conjugates with glycine and taurine, respectively). A third aliquot was used for determination of specific activities of the methyl esters of cholic, chenodeoxycholic, and deoxycholic acids derived from the total bile salts. Appropriate calculations yielded the concentration in bile of all six major bile acid conjugates.