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Cholic acid (CA), chenodeoxycholic acid (CDCA) and deoxycholic acid (DCA) which are extracted with CH3OH from Bezoar can be separated on HPTLC silica gel plate (made in China) with isooctane-n-butyl acetate-acetic acid (4:2:1), and the three bile acids were determined by TLC densitometry.
Bile acids in serum, urine and dialysate of 8 patients with renal failure in chronic hemodialysis were analyzed by gas chromatography and gas chromatography-mass spectrometry. The following results were obtained: 1. Lithocholic acid, 3 beta-hydroxy-5-cholen-24-oic acid, deoxycholic acid, chenodeoxycholic acid, ursodeoxycholic acid, and cholic acid were identified in hemodialysate as well as in serum and urine. 2. The serum bile acid concentration of the patients was 2.78 +/- 0.57 micrograms/mL before hemodialysis and 1.34 +/- 0.48 micrograms/mL after a 5-h period hemodialysis with cuprophane membrane. The proportions of secondary bile acids in predialysis and postdialysis serum of patients were significantly higher than those of healthy subjects. 3. Two out of 8 patients excreted urine. But the amounts of bile acids in urine of the patients were very small compared to those of healthy subjects. 4. The amount of bile acids removed from blood by hemodialysis was 0.70 +/- 0.25 mg. In dialysate, cholic acid constituted a larger proportion of the total bile acids, and lithocholic acid a smaller proportion, when compared to those in urine of patients and healthy subjects.
Biliary lipid composition was determined in fasting gallbladder bile of 12 young and 12 elderly asymptomatic Chilean women with normal weights and recent, normal cholecystograms. The proportion of biliary cholesterol and the lithogenic index were significantly higher in elderly females. Moreover, supersaturated bile was present in 8.3% of the young and in 41.7% of the older women studied. Obesity enhances the saturation of bile in older females: lithogenic bile was found in 100% of 4 elderly, obese women with radiologically normal gallbladders. The bile acid pool and cholic acid metabolism were studied in 5 young and 4 older normal females. Both groups showed similar values of bile acid pool, cholic acid synthesis, and turnover. These results indicate that aging per se modifies the proportions of biliary lipids in Chilean women, and provide a partial explanation for the frequency of gallstones observed among them. The increment in the lithogenic index of gallbladder bile with age takes place without significant changes in bile acid metabolism, suggesting that the canalicular secretion of cholesterol increases with aging.
To explain the hypercholeretic effect of ursodeoxycholic acid, a cholehepatic shunt circulation has been postulated. This pathway includes secretion by the hepatocyte into bile and absorption by the biliary epithelial cells. To test this possibility, we have attempted to localize ursodeoxycholic acid in hepatocytes and portal bile duct cells by an indirect immunoperoxidase technique using specific polyclonal antibodies against ursodeoxycholic acid and cholic acid conjugates. Rat livers were fixed with paraformaldehyde, incubated with antibodies directed against bile acids and examined by electron microscopy. After infusion of ursodeoxycholic acid and incubation with antibodies against this bile acid, an electron-dense staining was observed on the apical (luminal) membrane of the portal bile ductal cells. In contrast, no staining was observed when no ursodeoxycholic acid was infused, or after infusion with taurocholate and incubation with antibodies against cholic acid conjugates. These observations are consistent with absorption of ursodeoxycholic acid by the portal bile ducts and a cholehepatic circulation of this bile acid.
The purpose of this study was to clarify whether physiological concentrations of bile acids could affect endothelial nitric oxide production. We investigated the relationships between clinical concentrations of individual bile acids observed in patients with hepatobiliary diseases and endothelial nitric oxide production induced by each bile acid. Fifteen serum bile acids were measured using high-performance liquid chromatography combined with enzymatic fluorometry in 8 patients with liver cirrhosis, obstructive jaundice, and 8 healthy subjects. The effects of individual bile acids on nitric oxide production were examined in human umbilical endothelial cells by measuring the concentration of NO2- in the cultured medium. NO release in the blood was also determined by measuring the NO2-/NO3- concentration in these patients. In patients with hepatobiliary diseases, the plasma concentrations of chenodeoxycholic acid, ursodeoxycholic acid and cholic acid (free acid, taurine and glycine conjugates) were markedly elevated. Incubation of cells with chenodeoxycholic acid and deoxycholic acid (free acid, taurine and glycine conjugates) enhanced NO2- production in a concentration-dependent manner, while cholic acid (free and its conjugates) did not. The effects of individual bile acids on nitric oxide production were additive. Patients with liver cirrhosis and obstructive jaundice had higher plasma levels of NO2-/NO3- levels than the control subjects. These results suggest that increased plasma concentrations of chenodeoxycholic acid (free, taurine and glycine conjugates) in patients with hepatobiliary diseases may induce endothelial nitric oxide production. Thus, nitric oxide production induced by bile acids may be involved in the pathogenesis of circulatory abnormalities in patients with liver diseases.
Studies of the stereochemistry of the intermediates, 3 alpha,7 alpha,12 alpha-trihydroxy-5 beta-cholest-24-en-26-oic acid and 3 alpha,7 alpha,12 alpha,24-tetrahydroxy-5 beta-cholestan-26-oic acid, in the biosynthetic sequence between 3 alpha,7 alpha,12 alpha-trihydroxy-5 beta-cholestan-26-oic acid and cholic acid have been undertaken. (25R)- or (25S)-3 alpha,7 alpha, 12 alpha-Trihydroxy-5 beta-cholestan-26-oic acid was incubated with rat liver homogenates. The reaction products were converted to p-bromophenacyl ester derivatives and the esters were analyzed by high-performance liquid chromatography. By comparison with authentic samples of two (24E)- and (24Z)-isomers of the alpha, beta-unsaturated acid and of four isomers at C-24 and C-25 of the beta-hydroxy acid, (24E)-3 alpha,7 alpha, 12 alpha-trihydroxy-5 beta-cholestan-26-oic acid and (24R,25S)-3 alpha,7 alpha,12 alpha,24-tetrahydroxy-5 beta-cholestan-26-oic acid were found to be formed from either (25R)- or (25S)-3 alpha,7 alpha, 12 alpha-trihydroxy-5 beta-cholestan-26-oic acid. No formation of the (24Z)-isomer of the trihydroxycholestenoic acid or the other three isomers of the tetrahydroxycholestanoic acid was detected. The findings are discussed in relation to the assumed pathway for side chain cleavage in cholic acid biosynthesis.
Cholic and deoxycholic acids can be converted into 3-keto derivatives in 75-80% yield, by a four-step synthesis consisting of formylation, selective deformylation of the 3-formoxyl group, oxidation, then deformylation of the remaining formoxyl groups. The intermediate 3-keto formoxyl acids in this sequence were shown to be suitable starting compounds for the synthesis of 4-ene-3-keto acids, in 55-60% yield, via bromination, dehydrobromination, and deformylation. By extending the dehydrobromination reaction, the 7 alpha-formoxyl group of the intermediate 4-ene-3-keto-7 alpha,12 alpha-diformoxyl acid is also lost, hence providing a useful synthetic route to 4,6-diene-3-keto bile acids.
The ionization behavior of carboxylic acids including aliphatic chain fatty acids and bile acids in solutions, micelles, membranes and proteins is of considerable biological interest. The 13C nuclear magnetic resonance chemical shift of carboxyl carbon of a variety of acids has been shown to be a linear function of the state of ionization of the carboxyl group. Thus, by measuring the chemical shift as a function of the amount of acid or base added to a solution, the state of ionization and the apparent pKa of the carboxyl group may be determined. The method is illustrated in this paper using butyric acid, and results (given as apparent pKa values) are tabulated for a variety of fatty acids and cholic acid in different environments such as monomeric solutions, micelles, membranes and bound to albumin. The apparent pKa varies greatly depending upon the environment. For instance, the apparent pKa of oleic acid is about 4.2 when bound to albumin whereas it is about 7.5 when incorporated in the bilayer of a phospholipid vesicle. Cholic acid shows similar changes in the apparent pKa, being approximately 5.2 in sodium taurocholate micelles and at least 6.8 in phospholipid vesicles. The use of 13C nuclear magnetic resonance (NMR) and, in particular, 13C NMR of 13C-enriched carboxyl groups provides a method for direct observation of the state of ionization of the carboxyl group in biologically interesting acids. Such techniques may be adapted for use in biological systems, such as cells or tissues.
A normally functioning vascular endothelium is required for vascular tone modulation and blood fluidity. Systemic and local circulatory and coagulation alterations, even to the point of renal failure, may be observed in obstructive jaundice; bile salts are included among the potential pathogenetic factors. To assess the effects of taurocholic acid, glycocholic acid, and cholic acid on the integrity and properties of the endothelium, cultured human endothelial cells (HUVEC) were studied. Taurocholic and glycocholic acids (up to 2000 mumoles/L) did not exhibit any significant effect on 51Cr release from HUVEC after 6 h incubation. Following HUVEC exposure to 2000 mumoles/L of the unconjugated compound, cholic acid, a significant discharge of the radiolabel and LDH leakage in the supernatant were observed, to some extent prevented by the presence of human plasma or albumin (physiologic carrier). Prostacyclin spontaneous release from HUVEC was significantly depressed by both taurocholic and glycocholic acid; the action was related to bile salt concentration (200-1000 mumoles/L) and to the time of exposure (1 to 24 h); the reduced production of PGI2 was demonstrated to be reversible. Conversely, spontaneous TxA2 generation and fVIII release were not affected by the presence of bile salts in culture medium. Previous investigations showed that experimental obstructive jaundice could impair prostacyclin release from rat aortic tissue. The same effect was also demonstrated after in vitro exposure of the vessel wall to jaundiced serum and bile salts alone; furthermore, bile salts exert toxic effects on the integrity of several cells and impair the prostaglandin system of different tissues.(ABSTRACT TRUNCATED AT 250 WORDS)
BACKGROUND: Inborn errors of bile acid synthesis are newly recognized disorders that may cause the phenotypic appearance of neonatal hepatitis or neonatal cholestasis. METHODS: This is a clinicopathologic study of two sets of siblings with cholestatic neonatal liver failure. RESULTS: In 3 of the infants, diagnostic evaluation, including analysis of urinary bile salts, revealed a predominance of 7 alpha-hydroxy-3-oxo-4-cholenoic and 7 alpha, 12 alpha-dihydroxy-3-oxo-4-cholenoic acids, a pattern consistent with delta 4-3-oxosteroid 5 beta-reductase deficiency, which could be primary or secondary. The fourth infant died before such testing could be carried out. In addition, all 4 infants had histologically disseminated hemochromatosis and met diagnostic criteria for neonatal hemochromatosis. In the 3 infants studied, histologic examination of the liver disclosed giant cell hepatitis with extensive loss of hepatic parenchyma and rapid progression to cirrhosis. Early treatment with ursodeoxycholic acid and cholic acid, previously reported as effective therapy, was given to 2 siblings; it failed to reverse or halt the liver damage, and both infants died. One infant, with the original diagnosis of neonatal hemochromatosis, was treated with a variety of antioxidants and chelation therapy, as recently reported. No improvement was demonstrated, and he went on to liver transplantation. CONCLUSIONS: The presentation of delta 4-3-oxosteroid 5 beta-reductase deficiency as neonatal hemochromatosis may represent a distinct subset of this disorder with an accelerated course, no response to therapy and poor prognosis.
Bile acid-induced inhibition of DNA synthesis by the regenerating rat liver in the absence of other manifestation of impairment in liver cell viability has been reported. Because in experiments carried out on in vivo models bile acids are rapidly taken up and secreted into bile, it is difficult to establish steady concentrations to which the hepatocytes are exposed. Thus, in this work, a dose-response study was carried out to investigate the in vitro cytotoxic effect of major unconjugated and tauro- (T) or glyco- (G) conjugated bile acids and to compare this as regards their ability to inhibit DNA synthesis. Viability of hepatocytes in primary culture was measured by Neutral red uptake and formazan formation after 6 h exposure of cells to bile acids. The rate of DNA synthesis was determined by radiolabeled thymidine incorporation into DNA. Incubation of hepatocytes with different bile acid species - cholic acid (CA), deoxycholic acid (DCA), chenodeoxycholic acid (CDCA) and ursodeoxycholic acid (UDCA), in the range of 10-1000 microM - revealed that toxicity was stronger for the unconjugated forms of CDCA and DCA than for CA and UDCA. Conjugation markedly reduced the effects of bile acids on cell viability. By contrast, the ability to inhibit radiolabeled thymidine incorporation into DNA was only slightly lower for taurodeoxycholic acid (TDCA) and glycodeoxycholic acid (GDCA) than for DCA. When the effect of these bile acids on DNA synthesis and cell viability was compared, a clear dissociation was observed. Radiolabeled thymidine incorporation into DNA was significantly decreased (-50%) at TDCA concentrations at which cell viability was not affected. Lack of a cause-effect relationship between both processes was further supported by the fact that well-known hepatoprotective compounds, such as tauroursodeoxycholic acid (TUDCA) and S-adenosylmethionine (SAMe) failed to prevent the effect of bile acids on DNA synthesis. In summary, our results indicate that bile acid-induced reduction of DNA synthesis does not require previous decreases in hepatocyte viability. This suggests the existence of a high sensitivity to bile acids of cellular mechanisms that may affect the rate of DNA repair and/or proliferation, which is of particular interest regarding the role of bile acids in the etiology of certain types of cancer.
The peroxisome proliferator-activated receptor-alpha (PPARalpha) is a ligand-activated transcription factor that regulates the expression of a number of genes critical for fatty acid beta-oxidation. Because a number of substrates and intermediates of this metabolic pathway serve as ligand activators of this receptor, homeostatic control of fatty acid metabolism is achieved. Evidence also exists for PPARalpha-dependent regulation of genes encoding critical enzymes of bile acid biosynthesis. To determine whether the primary products of bile acid biosynthesis, cholic acid and chenodeoxycholic acid, were capable of modulating PPARalpha function, a variety of in vivo and in vitro approaches were utilized. Feeding a bile acid-enriched diet significantly reduced the degree of hepatomegaly and induction of target genes encoding enzymes of fatty acid beta-oxidation caused by treatment with the potent PPARalpha ligand Wyeth-14,643. Convergent data from mechanistic studies indicate that bile acids interfere with transactivation by PPARalpha at least in part by impairing the recruitment of transcriptional coactivators. The results of this study provide the first evidence in favor of the existence of compounds, normally found within the body, that are capable of antagonizing the physiological actions of PPARalpha. The impact of PPARalpha antagonism by endogenous bile acids is likely to be limited under normal conditions and to have only minimal effects on bile acid homeostasis. However, during certain pathophysiological states where intracellular bile acid concentrations are elevated, meaningful effects on PPARalpha-dependent target gene regulation are possible.
The effects of the free bile acids (FBAs) cholic acid (CA), deoxycholic acid (DCA), and chenodeoxycholic acid on the bioenergetics and growth of lactobacilli and bifidobacteria were investigated. It was found that these FBAs reduced the internal pH levels of these bacteria with rapid and stepwise kinetics and, at certain concentrations, dissipated DeltapH. The bile acid concentrations that dissipated DeltapH corresponded with the MICs for the selected bacteria. Unlike acetate, propionate, and butyrate, FBAs dissipated the transmembrane electrical potential (DeltaPsi). In Bifidobacterium breve JCM 1192, the synthetic proton conductor pentachlorophenol (PCP) dissipated DeltapH with a slow and continuous kinetics at a much lower concentration than FBAs did, suggesting the difference in mode of action between FBAs and true proton conductors. Membrane damage assessed by the fluorescence method and a viability decrease were also observed upon exposure to CA or DCA at the MIC but not to PCP or a short-chain fatty acid mixture. Loss of potassium ion was observed at CA concentrations more than 2 mM (0.4x MIC), while leakage of other cellular components increased at CA concentrations more than 4 mM (0.8 x MIC). Additionally, in experiments with membrane phospholipid vesicles extracted from Lactobacillus salivarius subsp. salicinius JCM 1044, CA and DCA at the MIC collapsed the DeltapH with concomitant leakage of intravesicular fluorescent pH probe, while they did not show proton conductance at a lower concentration range (e.g., 0.2x MIC). Taking these observations together, we conclude that FBAs at the MIC disturb membrane integrity and that this effect can lead to leakage of proton (membrane DeltapH and DeltaPsi dissipation), potassium ion, and other cellular components and eventually cell death.
The application of a sensitive gas liquid chromatography (GLC) assay has enabled patients in different diagnostic groups to be differentiated on the basis of the ratio of the primary bile acids, cholic: chenodeoxycholic acid. Because this method is unsuitable for a routine clinical laboratory, the primary bile acid ratio (PBR) is determined by radioimmunoassay techniques (RIA), and the results were compared with GLC analysis. RIA provided comparable results in control subjects and patients with large-duct obstruction, but significantly higher PBR values were found in cirrhotic subjects.
Bile acids in the rat bile were fractionated into unconjugated, glycine- and taurine-conjugated fractions by employing piperidino-hydroxypropyl Sephadex LH-20 ion-exchange chromatography. Subsequently, these fractions were analyzed by gas-liquid chromatography (GLC) and GLC-mass spectrometry using a Silicone AN-600 column. Not only lithocholic acid, deoxycholic acid, chenodeoxycholic acid, hyodeoxycholic acid, ursodeoxycholic acid and cholic acid, but also alpha- and beta-muricholic acids were quantitatively and simultaneously detectable in conjugated and unconjugated fractions, respectively. In the unconjugated and conjugated fractions, varying amounts of the unidentified bile acid were detected upon GLC. The electron impact and ammonia chemical ionization mass spectrometric results and catalytic hydrogenation on the compound indicate that this bile acid seems to be a derivative of beta-muricholic acid having a double bond in the side chain. The present method is suitable to the simultaneous and quantitative determination of unconjugated and glycine- and taurine-conjugated bile acids in the rat bile.
The effects of lithocholic acid (LCA), chenodeoxycholic acid (CDCA), deoxycholic acid (DOCA), cholic acid (CA) and dehydrocholic acid (DHCA) on the biliary excretion of amaranth (AM) in rats and on the respiration of isolated liver mitochondria were investigated. Bile acids diminished the biliary excretion of AM, and in vitro they enhanced the state 4 mitochondrial respiration and inhibited DNP- and ADP-stimulated oxygen consumption. The ID50 values (mumol/kg i.v.) and I50 concentrations (X 10(-5) M) of the bile acids for inhibiting AM excretion and ADP-stimulated mitochondrial respiration, respectively, were as follows: for LCA 16 and 3.3, for CDCA 160 and 24, for DOCA 230 and 31, for CA 680 and 105, and for DHCA 700 and 260. LCA, CDCA and DOCA inhibited the biliary excretion of AM uncompetitively. It is concluded that the toxic effect of LCA, CDCA and DOCA on mitochondria might play a role in their inhibitory action on the biliary transport of AM.
In a series of in vitro experiments, the influence of bile salts on platelet aggregation by ADP or by collagen and on serotonin-14C release by collagen, was studied. Sodium salts of the following bile acids showed a clear inhibitory effect: glycochenodeoxycholic acid, taurochenodeoxycholic acid, glycocholic acid, taurocholic acid and cholic acid. Lowering the pH of the platelet-rich plasma resulted in decrease platelet aggregation by ADP and by collagen. Bile salts further enhanced the inhibitory effect of pH change. In contrast the sodium salt of chenodeoxycholic acid was the sole aggregation inducing bile salt we studied. Relating the above studies to the clinical situation of upper gastroduodenal mucosal haemorrhage, we suggest that biliary reflux as well as the acidic environment may contribute to a poor haemostatic response by impaired platelet aggregation in the upper gastrointestinal tract of otherwise normal human subjects.
A gram-positive, anaerobic, chain-forming, rod-shaped anaerobe (isolate G20-7) was isolated from normal human feces. This organism was identified by cellular morphology as well as fermentative and biochemical data as Eubacterium aerofaciens. When isolate G20-7 was grown in the presence of Bacteroides fragilis or Escherichia coli (or another 7 alpha-hydroxysteroid dehydrogenase producer) and chenodeoxycholic acid, ursodeoxycholic acid produced. Time course curves revealed that 3 alpha-hydroxy-7-keto-5 beta-cholanoic acid produced by B. fragilis or E. coli or introduced into the medium as a pure substance was reduced by G20-7 specifically to ursodeoxycholic acid. The addition of glycine- and taurine-conjugated primary bile acids (chenodeoxycholic and cholic acids) and other bile acids to binary cultures of B. fragilis and G20-7 revealed that (i) both conjugates were hydrolyzed to give free bile acids, (ii) ursocholic acid (3 alpha, 7 beta, 12 alpha-trihydroxy-5 beta-cholanoic acid) was produced when conjugated (or free) cholic acid was the substrate, and (iii) the epimerization reaction was at least partially reversible. Corroborating these observations, an NADP-dependent 7 beta-hydroxysteroid dehydrogenase (reacting specifically with 7 beta-OH-groups) was demonstrated in cell-free preparations of isolate G20-7; production of the enzyme was optimal at between 12 and 18 h of growth. This enzyme, when measured in the oxidative direction, was active with ursodeoxycholic acid, ursocholic acid, and the taurine conjugate of ursodeoxycholic acid (but not with chenodeoxycholic, deoxycholic, or cholic acids) and displayed an optimal pH range of 9.8 to 10.2