Hepatobiliary delivery of polyaminopolycarboxylate chelates: synthesis and characterization of a cholic acid conjugate of EDTA and biodistribution and imaging studies with its indium-111 chelate

A conjugate in which the steroid nucleus of cholic acid was linked to EDTA via an 11-atom spacer was obtained by reacting the succinimidyl ester of cholic acid with the amine formed by reaction of a benzyl isothiocyanate derivative of EDTA with N-(tert-butoxycarbonyl)ethylenediamine and subsequent deprotection. Potentiometric titration studies with model complexes showed that the EDTA moiety retained the ability to form 1:1 chelates of high thermodynamic stability, although formation constants were some 3-4 log K units lower for complexes of the conjugate than for the analogous chelates with underivatized EDTA. A complex formed between the cholic acid-EDTA conjugate and 111InIII was clearly rapidly into the liver when injected iv into mice, with subsequent excretion from the liver into the gastrointestinal tract being complete within 1 h of injection. Radioscintigraphic imaging studies conducted in a rabbit given the 111In-labeled conjugate also showed early liver uptake followed by rapid clearance from the liver into the intestine, with good visualization of the gallbladder in images obtained at 20-25 min postinjection. It is concluded that conjugation to cholic acid provides a useful means for the hepatobiliary delivery of EDTA chelates that otherwise exhibit predominantly extracellular distribution and renal clearance.     

Hepatocyte-hepatoma cell hybrids. Characterization and demonstration of bile acid synthesis

Hybrids were created by fusion of primary rat hepatocytes with well-differentiated Reuber H35 rat hepatoma cells. Seventeen hybrids were screened for bile acid synthesis using [26-14C]cholesterol. As [26-14C]cholesterol was converted to bile acid, 14CO2 was released. Using this assay, four hybrids (8B, 12C, 13C, and 13D) were identified which synthesized bile acid. These four hybrids also incorporated [14C]taurine into bile acid. Bile acids were identified by capillary gas chromatography/mass spectrometry, and their rates of synthesis were quantitated by isotope dilution. Reuber H35 cells synthesized little or no bile acid. However, hybrids 8B, 12C, 13C, and 13D synthesized chenodeoxycholic acid, alpha-muricholic acid, and cholic acid and secreted them into the media. The rates of synthesis of individual bile acids varied among these hybrids. For example, the relative percentage of cholic acid ranged from 11.1% (hybrid 8B) to 50.4% (hybrid 13C). The bile acids synthesized and secreted by the most active hybrid, 12C, were greater than 93% conjugated. In summary, hybrids were created that retain the capacity to synthesize, conjugate, and secrete three major rat bile acid species. Such hybrids are unique model systems that will allow the study of the biochemical and genetic regulation of bile acid synthesis.     

Equilibrium-dialysis studies of the interaction between cholic acid and 100000g-supernatant preparations from the rat liver.

1. The binding of cholic acid to 100000g supernatants from rat livers was investigated by equilibrium dialysis and gel-exculsion chromatography. 2. Supernatants were found to contain at least two classes of binding site for cholic acid. 3. These recptor molecules are probably proteins since incubation with proteolytic enzymes resulted in complete loss of cholic acid binding. 4. Supernatants were added to columns of Sephadex G-75, and two groups of fractions were shown to bind cholic acid. One of these contained low-affinity binding sites and the other contained both low- and high-affinity binding sites. 5. Feeding cholestyramine had no effect on cholic acid binding. 6. Increased cholic acid binding occurred after injection of phenobarbitone. There was an increase in the amount of the low-affinity component but no change in the high-affinity component. 7. The dissociation constants of the binding of cholic acid suggest that the binding proteins may be involved in bile acid transport.     

Synthesis of the specific monosulfates of cholic acid.

The three isomeric cholic acid-monosulfates were synthetized and characterized. Cholic acid-3-sulfate was obtained by reacting cholic acid for 2 min with chlorosulfonic acid in pyridine and chromatography of the resulting bile salt mixture on Sephadex LH-20. The 7- and the 12-monosulfate were prepared by sulfation of the corresponding monohydroxy-diacetates followed by removal of the acetyl groups by alkaline hydrolysis and purification by chromatography on Sephadex LH-20. On TLC in n-butanol-acetic acid-water (10:1:1, v/v) the Rf values were 0.59 for cholic acid-3-sulfate, 0.52 for cholic acid-7-sulfate and 0.48 for cholic acid-12-sulfate. The time required for complete solvolysis at 37 degrees C in acid methanol-acetone (1:9) was 3 h for cholic acid-3-sulfate, 12 h for the 12-monosulfate and 18 h for the 7-monosulfate.     

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.     

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.     

Molecular cloning of a gene encoding a 45,000-dalton polypeptide associated with bile acid 7-dehydroxylation in Eubacterium sp. strain VPI 12708.

Eubacterium sp. strain VPI 12708 is an intestinal anaerobic bacterium which possesses an inducible bile acid 7-dehydroxylation activity. Two cholic acid-induced polypeptides with apparent molecular weights of 27,000 and 45,000, respectively, coeluted with bile acid 7-dehydroxylation activity upon anaerobic high-performance gel filtration chromatography of crude cellular protein extracts. The 45,000-dalton polypeptide was purified to greater than 95% homogeneity by high-performance liquid chromatography gel filtration and high-performance liquid-DEAE chromatography. The first 28 amino acid residues of the N terminus of this polypeptide were determined by gas-phase sequencing, and a corresponding mixed oligonucleotide (20-mer) was synthesized. Southern blot analysis of EcoRI total digests of chromosomal DNA showed a 2.6-kilobase fragment which hybridized to the 32P-labeled 20-mer. This fragment was enriched for by size fractionation of an EcoRI total digest of genomic DNA and ligated into bacteriophage lambda gt11. Recombinant phage containing the putative gene encoding the 45,000-dalton polypeptide were detected with the 32P-labeled 20-mer by plaque hybridization techniques. The insert was 2.6 kilobases in length and may contain the entire coding sequence for the 45,000-dalton polypeptide. The 2.6-kilobase insert was subcloned into pUC8 and transformed into Escherichia coli DH5 alpha. However, the 45,000-dalton polypeptide was not detected in cell extracts of this organism when specific antibody was used. Preliminary nucleic acid sequence data correlated exactly with the amino acid sequence. A cholic acid-induced mRNA species of greater than 6 kilobases in size was identified by Northern (RNA) blot analysis of total RNA, suggesting that the gene coding for this polypeptide is part of a larger operon.     

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.     

Formation of cholic acid from 3 alpha, 7 alpha, 12 alpha-trihydroxy-5 beta-cholestanoic acid in human skin fibroblasts.

Whether 3 alpha, 7 alpha, 12 alpha-trihydroxy-5 beta-cholestanoic acid (THCA) was converted into cholic acid in human skin fibroblasts was examined. THCA was incubated with subcellular fractions of cultured skin fibroblasts in the presence of NAD+, ATP, CoA, and Mg2+. The reaction products were analyzed by thin-layer chromatography and high-performance liquid chromatography after p-bromophenacyl ester derivatization. The highest specific activity was found in the light mitochondrial fraction (2.71 nmol/mg protein/h). The specific activity was about 9-fold higher than that in heavy mitochondrial fraction. The peroxisomal fraction prepared from the light mitochondrial fraction by sucrose gradient centrifugation was also able to catalyze the conversion of THCA into cholic acid. The specific activity in this fraction was a further 2.2-fold higher than that in the light mitochondrial fraction. These results suggest that cultured human skin fibroblasts are able to convert THCA into cholic acid, and that the activity exists in peroxisomes.     

Bile-mediated aminoglycoside sensitivity in Lactobacillus species likely results from increased membrane permeability attributable to cholic acid

Few studies have been conducted on antimicrobial resistance in lactobacilli, presumably because of their nonpathogenic nature as anaerobic commensals. We assessed resistance in 43 type strains and isolates representing 14 species by using agar disk diffusion and MIC analysis in MRS medium. Most noteworthy were two general phenotypes displayed by nearly every strain tested: (i) they were more susceptible (up to 256-fold in some cases) to the deconjugated bile acid cholic acid than to the conjugate taurocholic or taurodeoxycholic acid, and (ii) they became susceptible to aminoglycosides when assayed on agar medium containing 0.5% fractionated bovine bile (ox gall). Two-dimensional MIC analyses of one representative strain, Lactobacillus plantarum WCFS1, at increasing concentrations of ox gall (0 to 30.3 mg/ml) displayed corresponding decreases in resistance to all of the aminoglycosides tested and ethidium bromide. This effect was clinically relevant, with the gentamicin MIC decreasing from >1,000 to 4 mug/ml in just 3.8 mg of ox gall per ml. In uptake studies at pH 6.5, [G-3H]gentamicin accumulation increased over control levels when cells of this strain were exposed to bile acids or reserpine but not when they were exposed to carbonyl cyanide m-chlorophenylhydrazone. The effect was dramatic, particularly with cholic acid, increasing up to 18-fold, whereas only modest increases, 3- and 5-fold, could be achieved with taurocholic acid and ox gall, respectively. Since L. plantarum, particularly strain WCFS1, is known to encode bile salt hydrolase (deconjugation) activity, our data indicate that mainly cholic acid, but not taurocholic acid, effectively permeabilizes the membrane to aminoglycosides. However, at pHs approaching neutral conditions in the intestinal lumen, aminoglycoside resistance due to membrane impermeability may be complemented by a potential efflux mechanism.     

Identification of a novel conjugate in human urine: bile acid acyl galactosides

We report a novel conjugate, bile acid acyl galactosides, which exist in the urine of healthy volunteers. To identify the two unknown peaks obtained in urine specimens from healthy subjects, the specimens were subjected to solid phase extraction and then to liquid chromatographic separation. The eluate corresponding to the unknown peaks on the chromatogram was collected. Following alkaline hydrolysis and liquid chromatography (LC)/electrospray ionization (ESI)-mass spectrometric (MS) analysis, cholic acid (CA) and deoxycholic acid (DCA) were identified as liberated bile acids. When a portion of the alkaline hydrolyzate was subjected to a derivatization reaction with 1-phenyl-3-methyl-5-pyrazolone, a derivative of galactose was detected by LC/ESI-MS. Finally, the liquid chromatographic and mass spectrometric properties of these unknown compounds in urine specimens were compared to those of authentic specimens and the structures were confirmed as CA 24-galactoside and DCA 24-galactoside. These results strongly imply that bile acid 24-galactosides, a novel conjugate, were synthesized in the human body.     

Hepatic bile acid metabolism during early development revealed from the analysis of human fetal gallbladder bile

A detailed study of the qualitative and quantitative composition of bile acids in human fetal gallbladder bile is described. Bile was collected during early gestation (weeks 16-19) and analyzed by gas chromatography and mass spectrometry, fast atom bombardment ionization mass spectrometry, and high performance liquid chromatography. Bile acids were separated into different conjugate groups by chromatography on the lipophilic anion exchange gel, diethylaminohydroxypropyl Sephadex LH-20. Quantitatively more than 80% of the bile acids were secreted into bile conjugated to taurine. Unconjugated bile acids and glycine conjugates accounted for 5-10% of the total biliary bile acids. Bile acid sulfates were present only in trace amounts indicating that quantitatively sulfation is not an important pathway in bile acid metabolism during development. Total biliary bile acid concentrations were low (0.1-0.4 mM) when compared to reported values for adult bile (greater than 10 mM). Chenodeoxycholic acid was the major biliary bile acid and exceeded cholic acid concentrations by 1.43-fold indicating either a relative immaturity in 12 alpha-hydroxylase activity during early life or a dominance of alternative pathways for chenodeoxycholic acid synthesis. A relatively large proportion of the biliary bile acids comprised metabolites not found in adult bile. The presence of relatively high proportions of hyocholic acid (often greater than cholic acid) and several 1 beta-hydroxycholanoic acid isomers indicates that C-1 and C-6 hydroxylation are important pathways in bile acid synthesis during development. We describe, for the first time, evidence for the existence of a C-4 hydroxylation pathway in the metabolism of bile acids, which may be unique to early human development. Mass spectrometry was used to confirm the identification of 3 alpha,4 beta,7 alpha-trihydroxy-5 beta-cholanoic and 3 alpha,4 beta-dihydroxy-5 beta-cholanoic acids. Quantitatively, these C-4 hydroxylated bile acids accounted for 5-15% of the total biliary bile acids of the fetus, suggesting that C-4 hydroxylation is quantitatively an important pathway in the bile acid metabolism during early life.     

Characterization of liver cholic acid coenzyme A ligase activity. Evidence that separate microsomal enzymes are responsible for cholic acid and fatty acid activation.

Investigations on the cholic acid CoA ligase activity of rat liver microsomes were made possible by the development of a rapid, sensitive radiochemical assay based on the conversion of [3H]choloyl-CoA. More than 70% of the rat liver cholic acid CoA ligase activity was associated with the microsomal subcellular fraction. The dependencies of cholic acid CoA ligase activity on pH, ATP, CoA, Triton WR-1339, acetone, ethanol, magnesium, and salts were investigated. The hypothesis that the long chain fatty acid CoA ligase activity and the cholic acid CoA ligase activity are catalyzed by a single microsomal enzyme was investigated. The ATP, CoA, and cholic (palmitic) acid kinetics neither supported nor negated the hypothesis. Cholic acid was not an inhibitor of the fatty acid CoA ligase and palmitic acid was not a competitive inhibitor of the cholic acid CoA ligase. The cholic acid CoA ligase activity utilized dATP as a substrate more effectively than did the fatty acid CoA ligase activity. The cholic acid and fatty acid CoA ligase activities appeared to have different pH dependencies, differed in thermolability at 41 degrees, and were differentially inactivated by phospholipase C. Moreover, fatty acid CoA ligase activity was present in microsomal fractions from all rat organs tested while cholic acid CoA ligase activity was detected only in liver microsomes. The data suggest that separate microsomal enzymes are responsible for the cholic acid and the fatty acid CoA ligase activities in liver.     

Isolation and structural characterization of chicken hypothalamic luteinizing hormone releasing hormone

Studies on partially purified chicken hypothalamic luteinizing hormone releasing hormone (LHRH) utilizing chromatography, radioimmunoassay with region-specific antisera, enzymic inactivation, and chemical modification established that the peptide is structurally different from mammalian hypothalamic LHRH. These studies demonstrated that arginine in position 8 is substituted by a neutral amino acid. On the basis of conformational criteria and evolutionary probability of amino acid interchange for arginine, the most likely substitution was glutamine. We therefore synthesized [Gln8]-LHRH and established that it had identical chromatographic, immunologic, and biological properties to the natural chicken peptide. In concurrent studies, purification of 17 micrograms of an LHRH from 249,000 chicken hypothalami was achieved using acetic acid extraction, immuno-affinity chromatography, and cation exchange and reverse phase high performance liquid chromatography. Amino acid composition and sequence analyses confirmed the structure of this form of chicken LHRH as pGlu-His-Trp-Ser-Tyr-Gly-Leu-Gln-Pro-Gly-NH2.     

Preparation of radioactive iodinated cholylhistamine for use in the radioimmunoassay of cholic acid

A major handicap in the development of simple and accurate radioimmunoassay procedures for bile acids has been the lack of a radioactive standard of high specific activity. To provide such a compound, we first synthesized cholylhistamine using the carbodiimide reaction. The hypothesized structure was confirmed by elemental analysis, thin-layer chromatography, infrared and mass spectral analysis. The cholylhistamine was then iodinated with 125I, using the choloramine-T method. The 125I-cholylhistamine was bound by antisera raised against a cholic acid-bovine serum albumin conjugate. This procedure should prove useful in preparing radioactive conjugates for all of the bile acids.     

Bile-Mediated Aminoglycoside Sensitivity in Lactobacillus Species Likely Results from Increased Membrane Permeability Attributable to Cholic Acid

Few studies have been conducted on antimicrobial resistance in lactobacilli, presumably because of their nonpathogenic nature as anaerobic commensals. We assessed resistance in 43 type strains and isolates representing 14 species by using agar disk diffusion and MIC analysis in MRS medium. Most noteworthy were two general phenotypes displayed by nearly every strain tested: (i) they were more susceptible (up to 256-fold in some cases) to the deconjugated bile acid cholic acid than to the conjugate taurocholic or taurodeoxycholic acid, and (ii) they became susceptible to aminoglycosides when assayed on agar medium containing 0.5% fractionated bovine bile (ox gall). Two-dimensional MIC analyses of one representative strain, Lactobacillus plantarum WCFS1, at increasing concentrations of ox gall (0 to 30.3 mg/ml) displayed corresponding decreases in resistance to all of the aminoglycosides tested and ethidium bromide. This effect was clinically relevant, with the gentamicin MIC decreasing from >1,000 to 4 μg/ml in just 3.8 mg of ox gall per ml. In uptake studies at pH 6.5, [G-³H]gentamicin accumulation increased over control levels when cells of this strain were exposed to bile acids or reserpine but not when they were exposed to carbonyl cyanide m-chlorophenylhydrazone. The effect was dramatic, particularly with cholic acid, increasing up to 18-fold, whereas only modest increases, 3- and 5-fold, could be achieved with taurocholic acid and ox gall, respectively. Since L. plantarum, particularly strain WCFS1, is known to encode bile salt hydrolase (deconjugation) activity, our data indicate that mainly cholic acid, but not taurocholic acid, effectively permeabilizes the membrane to aminoglycosides. However, at pHs approaching neutral conditions in the intestinal lumen, aminoglycoside resistance due to membrane impermeability may be complemented by a potential efflux mechanism.     

HepG2. A human hepatoblastoma cell line exhibiting defects in bile acid synthesis and conjugation

We used capillary gas chromatography/mass spectrometry to demonstrate that a cell line derived from a well differentiated human hepatoblastoma, HepG2, synthesized and secreted the following bile acids (ng/10(7) cells/h): chenodeoxycholic acid (131.4), cholic acid (3.3), 3 alpha, 7 alpha-dihydroxy-5 beta-cholestan-26-oic acid (DHCA; 4.5), and 3 alpha, 7 alpha, 12 alpha-trihydroxy-5 beta-cholestan-26-oic acid (THCA; 32.0). Deuterium from [7 beta-2H]7 alpha-hydroxycholesterol, which was added to the media, was incorporated into newly synthesized chenodeoxycholic acid, DHCA, and THCA, but not into cholic acid. Since THCA is a known precursor of cholic acid, these data suggest that HepG2 is specifically deficient in the side chain cleavage that transforms THCA into cholic acid. Greater than 90% of the bile acids synthesized and secreted by HepG2 were unconjugated. Conjugation could not be stimulated by the addition of glycine or taurine to the media. Approximately 30% of newly synthesized DHCA and THCA were sulfated. Chenodeoxycholic acid and cholic acid were not appreciably sulfated. In summary, cultured HepG2 cells synthesize bile acid, but in a pattern distinct from that of adult human liver. This cell line may be a model for studying pathways of human bile acid synthesis, conjugation, and sulfation.     

Towards Squalamine Mimics: Synthesis and Antibacterial Activities of Head‐to‐Tail Dimeric Sterol?Polyamine Conjugates

Four dimeric sterol? polyamine conjugates have been synthesized from the homo‐ and hetero‐connection of monomeric sterol? polyamine analogs in a head‐to‐tail manner. These dimeric conjugates show strong antibacterial activity against a broad spectrum of Gram‐positive bacteria, whereas their corresponding activities against Gram‐negative bacteria are relatively moderate. Though no significant difference was observed in the activities of these conjugates, cholic acid‐containing dimeric conjugates generally exhibit higher activities than the corresponding deoxycholic acid‐derived analogs. This is in contrast to the finding that a monomeric deoxycholic acid‐spermine conjugate was more active than the corresponding cholic acid‐derived analog.     

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 Bacteroids fragilis subsp. fragilis ATCC 25285. The intact enzyme had a molecular weight of approx. 250 000 as determined by gel infiltration 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-alpha-hydroxysteroid dehydrogenase was absolutely correlated with that of bile salt hydrolase activity in six to ten strains and subspecies of B. fragilis.