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PubMed1989

Cloning, structure, and expression of the mitochondrial cytochrome P-450 sterol 26-hydroxylase, a bile acid biosynthetic enzyme.

The conversion of cholesterol into bile acids in the liver represents the major catabolic pathway for the removal of cholesterol from the body. In this complex biosynthetic pathway, at least 10 enzymes modify both the ring structure and side chain of cholesterol, resulting in the formation of the primary bile acids, cholic acid, and chenodeoxycholic acid. To gain insight into the details and regulation of this pathway, we have used protein sequencing and molecular cloning techniques to isolate and characterize a cDNA encoding the rabbit mitochondrial sterol 26-hydroxylase. This enzyme catalyzes the first step in the oxidation of the side chain of sterol intermediates in the biosynthesis of bile acids. The structure of the sterol 26-hydroxylase, as deduced by both DNA sequence analysis of the cDNA and protein sequence analysis, reveals it to be a mitochondrial cytochrome P-450. A signal sequence of 36 residues precedes a coding region of 499 amino acids, predicting a molecular weight of 56,657 for the mature protein. The identity of the 26-hydroxylase cDNA was further confirmed by expression in monkey COS cells employing a versatile eukaryotic expression vector. Blotting experiments revealed that the mRNA for this enzyme is expressed in many tissues and that it is encoded by a low copy number gene in the rabbit genome.

Amino Acid Sequence↗

PubMed1989

[An experimental study on the biliary excretion of ceftizoxime in the presence and after relief of biliary obstruction with special reference to the influence of bile acid metabolism on the biliary excretion of the antibiotic].

The biliary excretion of ceftizoxime (CZX) in the presence and after relief of biliary obstruction was experimentally investigated in twelve dogs. In the presence of biliary obstruction, the biliary excretion of CZX was impaired and, immediately after relief of biliary obstruction, the existence of reverse shunt from the vascular system to the biliary tract was suggested. After relief of biliary obstruction, the biliary excretion of CZX and the ratio of [lithocholic acid + deoxycholic acid]/[cholic acid + chenodeoxycholic acid] in the bile were impaired in one group with external drainage as compared to the other group with internal drainage. It was experimentally proved that the biliary excretion of antibiotics seemingly related very closely to the bile acid metabolism and that it was increased by administration of the bile acid after external drainage or by normalization of the enterohepatic circulation of the bile acid after internal drainage.

Animals↗

PubMed1983

Effect of bile acid oxazoline derivatives on microorganisms participating in 7 alpha-hydroxyl epimerization of primary bile acids.

We tested bile acid oxazoline derivatives of chenodeoxycholic (CDC-OX), 7-ketolithocholic (7-KLC-OX), ursodeoxycholic (UDC-OX), and deoxycholic (DC-OX) as inhibitors of the 7-epimerization of the primary bile acids cholic acid (CA) and CDC in cultures of four species of bacteria and the human fecal flora. The organisms tested elaborate a 7 alpha- and/or 7 beta-hydroxysteroid dehydrogenase (HSDH); they were Escherichia coli (7 alpha-HSDH), Bacteroides fragilis (7 alpha-HSDH), Clostridium absonum (7 alpha- and 7 beta-HSDH) and Eubacterium aerofaciens (7 beta-HSDH). None of the oxazolines affected 7 alpha-OH oxidation of CA or CDC by E. coli or the growth of the organism. All the oxazolines (except UDC-OX) inhibited the growth of B. fragilis and its 7 alpha-HSDH. In contrast, only DC-OX blocked 7 alpha-OH epimerization of CA by C. absonum. Surprisingly, the other three oxazolines enhanced 7 alpha-OH epimerization of CA, but not that of CDC, which was inhibited (CDC-OX greater than 7-KLC-OX much greater than UDC-OX). Enzymic data suggest that CDC-OX in the presence of CA can induce a greater level of both 7 alpha- and 7 beta-HSDH than CA or CDC-OX alone, CDC-OX being more toxic in the presence of CDC. Formation of urso-bile acid from 7-keto substrates by E. aerofaciens is totally blocked by the oxazolines (except UDC-OX). Similarly, suppression of urso-bile acid formation from primary bile acids by the human fecal flora was evident with DC-OX greater than 7-KLC-OX greater than CDC-OX much greater than UDC-OX, the last being ineffective. The inhibitory activity of the oxazolines on the 7-dehydroxylation of primary bile acids by human fecal flora followed the same order.

Bacteria↗

PubMed1995

[Bile acids in liver diseases--current indications].

Ursodeoxycholic acid (UDCA) has beneficial effects in cholestatic diseases such as primary biliary cirrhosis, primary sclerosing cholangitis and cholestasis of pregnancy. In chronic hepatitis and alcoholic hepatitis, an effect of UDCA is uncertain. After organ transplantation (liver, bone, heart), favorable effects of UDCA which await confirmation were observed. UDCA very likely has a beneficial effect in children with Byler's and Alagille's syndrome, extrahepatic biliary atresia after the Kasai procedure and in cholestasis of cystic fibrosis. Unclear is the effect of UDCA in benign intermittent cholestasis (Summerskill-Tygstrup syndrome). Children with cholestasis and inborn errors of bile acid synthesis need additional administration of a primary bile acid (cholic acid, chenodeoxycholic acid). UDCA treatment in general does not lead to definitive cure of the disease but to improvement of laboratory parameters and possibly of symptoms and liver histology, and liver transplantation may be postponed to a later time point.

Bile Acids and Salts↗

PubMed1998

Sensitive analysis of serum 3alpha, 7alpha, 12alpha,24-tetrahydroxy- 5beta-cholestan-26-oic acid diastereomers using gas chromatography-mass spectrometry and its application in peroxisomal D-bifunctional protein deficiency.

The final steps in bile acid biosynthesis take place in peroxisomes and involve oxidative cleavage of the side chain of C27-5beta-cholestanoic acids leading to the formation of the primary bile acids cholic acid and chenodeoxycholic acid. The enoyl-CoA hydratase and beta-hydroxy acyl-CoA dehydrogenase reactions involved in the chain shortening of C27-5beta-cholestanoic acids are catalyzed by the recently identified peroxisomal d-bifunctional protein. Deficiencies of d-bifunctional protein lead, among others, to an accumulation of 3alpha,7alpha,12alpha, 24-tetrahydroxy-5beta-cholest-26-oic acid (varanic acid). The ability to resolve the four C24, C25 diastereomers of varanic acid has, so far, only been carried out on biliary bile acids using p -bromophenacyl derivatives. Here, we describe a sensitive gas chromatography-mass spectrometry (GC/MS) method that enables good separation of the four varanic acid diastereomers by use of 2R-butylester-trimethylsilylether derivatives. This method showed the specific accumulation of (24R,25R)-varanic acid in the serum of a patient with isolated deficiency of the d-3-hydroxy acyl-CoA dehydrogenase part of peroxisomal d-bifunctional protein, whereas this diastereomer was absent in a serum sample from a patient suffering from complete d-bifunctional protein deficiency. In samples from both patients an accumulation of (24S,25S)-varanic acid was observed, most likely due to the action of l-bifunctional protein on Delta24E-THCA-CoA. This GC/MS method is applicable to serum samples, obviating the use of bile fluid, and is a helpful tool in the subclassification of patients with peroxisomal d-bifunctional protein deficiency.

17-Hydroxysteroid Dehydrogenases↗

PubMed1998

Effect of selected substances on heat-induced aggregation of albumin, IgG and lysozyme.

Compounds capable of inhibiting protein aggregation may find pharmacological applications in the treatment of a number of diseases called protein condensation diseases [Benedek (1997)], which include cataract, biliary and urinary lithiasis and certain rheumatic diseases. We examined the effect of selected compounds on heat-induced aggregation human serum albumin (HSA), IgG and lysozyme. HSA (0.2% w/v in 0.066 M sodium phosphate pH 5.3 at 22 degrees C), IgG (0.5% w/v in 0.066 M Tris pH 8.0 at 22 degrees C), and L (0.2 % w/v in 0.066 M CAPS pH 11.0 at 22 degrees C) were heated for 30 min at 70 degrees C in the presence or absence of different concentrations of the substance under examination and heat-induced aggregation of 100 microl aliquots was evaluated by measuring the absorbance at 595 nm using an automatic microplate reader. In these conditions, inhibition of aggregation could be due to an anti-denaturant effect or to interferences with the aggregation of denatured molecules, as previously described [Saso, Casini et al. (1998)]. However, this distinction may not be pharmacologically relevant when the target of the therapy is the prevention of abnormal phenomena of protein aggregation. Inorganic salts like NaCl and CaCl2 were active on the three proteins (IgG > HSA > L) but many ligands of HSA such as tryptophan, N-acetyl-tryptophan, caprylic acid, capric acid, cholic acid, deoxycholic acid, chenodeoxycholic acid, lithocholic acid and bendazac were active on their carrier but not on IgG and L, indicating that the latter proteins are more difficult to protect and that specific anti-denaturant and/or anti-aggregant compounds should be developed.

Albumins↗

PubMed1979

Altered bile acid metabolism in alloxan diabetic rats.

Changes of cholesterol, phospholipid, triglyceride or bile acid levels in serum liver, bile and feces after the treatment with alloxan were examined in Wistar strain male rats. Serum cholesterol, phospholipid and triglyceride levels and liver cholesterol level markedly increased but liver phospholipid and triglyceride levels remained unchanged. The lipid levels in serum very low density and low density lipoproteins were elevated but those in high density lipoprotein were not. Bile flow was not changed but biliary secretion of cholesterol, phospholipid and bile acids markedly increased. Among the biliary bile acid components, cholic acid markedly increased but the amount of chenodeoxycholic acid was similar to that of normal rats. Fecal excretion of deoxycholic acid increased but that of lithocholic and hyodeoxycholic acids decreased, and alpha, beta- and omega-muricholic acids did not change, thus, the total amount of fecal bile acids remained unchanged. Hepatic cholesterol synthesis was markedly depressed, while cholesterol 7 alpha-hydroxylase activity did not change and cytochrome P-450 content was elevated by about 40%. From such evidence, it was apparent that synthesis of cholic acid increased while that of chenodeoxycholic acid decreased and the total amount of bile acids synthesized did not change in the diabetic rats. Furthermore, marked increase of the pool size of cholic acid and hepatic secretion of cholic acid stimulated the absorption of lipids and produced a hyperlipidemia in the diabetic rats.

Alloxan↗

PubMed1976

Effects of fasting on bile acid metabolism and biliary lipid composition in man.

The effects of a four to six day fast on gallbladder bile lipid composition, bile acid pool size, bile acid composition, and cholic acid metabolism have been determined in normal human subjects. Total bile acid pool size and cholic acid pool size were measured before and after fasting by a one-sample technique previously validated in our laboratory. The rate of synthesis of cholic acid and its fractional turnover rate before fasting were measured using standard techniques. Estimates of fasting cholic acid synthesis rate and fractional turnover rate were calculated as daily averages from the change in cholic acid pool size, in combination with the change in cholic acid specific activity, during the fasting period. Since these estimates are approximate, a maximum value for cholic acid synthesis rate during fasting was also determined by assuming that the entire change in cholic acid specific activity during the fasting period occurred instantaneously. The molar percent of cholesterol in gallbladder bile was reduced in eight of nine subjects after a four to six day fast (p less than .01; mean reduction 30.5%). The molar percents of bile acid and phospholipid were not significantly altered by fasting. The cholesterol saturation index, calculated on the basis of these data, was reduced by an average of 31.0% after a four to six day fast (p less than .02). The average daily cholic acid synthesis rate and the fractional turnover rate were reduced in all six subjects on whom isotope kinetic studies were carried out. The mean decrease in synthesis rate was 68.5% (p less than .05; range 55.2-79.8%) while the mean decrease in fractional turnover rate was 64.4% (p less than .05; range 30.2-100%). Reduction in synthesis rate was confirmed by the determination of maximum fasting synthesis of cholic acid, which averaged 61.1% lower than synthesis in the fed period. Fasting had no consistent effect on total bile acid pool size, cholic acid pool size, or bile acid species composition.

Adolescent↗

PubMed1989

Serum lipoprotein and apoprotein concentrations in 4-(4-chlorophenyl)-2-hydroxytetronic acid and clofibrate-treated cholesterol and cholic acid-fed rats.

Influence of clofibrate and an aci-reductone, 4-(4-chlorophenyl)-2-hydroxytetronic acid (CHTA) on lipoproteins and apoproteins was studied in cholesterol- plus cholic acid-fed rats. CHTA (0.4 mmol/kg body wt, twice daily) significantly lowered serum total cholesterol and triglyceride concentrations at both 10 and 16 days, whereas clofibrate at the same dose did not alter serum cholesterol levels, but elevated serum triglyceride concentrations at 16 days. The abnormal cholesterol-rich very low density lipoproteins (VLDL), intermediate density lipoproteins (IDL) and low density lipoproteins (LDL) produced by cholesterol plus cholic acid were significantly reduced in their cholesterol content by treatment with CHTA, a compound having an oxidation reduction potential. Conversely, clofibrate administration increased VLDL-cholesterol with concomitant decreases in IDL- and LDL-cholesterol concentrations. Administration of CHTA to cholesterol- plus cholic acid-fed rats significantly increased concentrations of VLDL and IDL, but had no effect on HDL protein. Both CHTA and clofibrate administration to cholesterol- plus cholic acid-fed rats significantly lowered IDL protein concentrations. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) studies of apoproteins revealed that clofibrate treatment significantly reduced apoC-III and C-II in VLDL, C-II in IDL, and apoA-IV and A-I in HDL. Rats treated with CHTA significantly raised apoC-II and C-III in HDL.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1990

Differential induction of peroxisomal oxidation of palmitic acid and 3 alpha,7 alpha,12 alpha-trihydroxy-5 beta-cholestanoic acid in rat liver.

The effect of feeding rats 20% partially hydrogenated marine oil (PHMO), 20% soybean oil, or clofibrate on the conversion of 3 alpha,7 alpha,12 alpha-trihydroxy-5 beta-cholestanoic acid to cholic acid was studied in light mitochondrial (L) fractions prepared from liver. 20% PHMO gave a doubling both of the specific and of the total activity of the cholic acid formation compared to those found in the L-fraction from animals given standard pellets. 20% soybean oil induced the specific and the total activity to a lesser extent, 1.4- and 1.2-fold, respectively. The specific and total activity of the peroxisomal beta-oxidation of palmitic acid were induced 2.4- and 2.7-fold, respectively, by PHMO feeding. Soybean oil gave a smaller increase, 2-fold, in both specific and total activity. Clofibrate, a known peroxisomal proliferator, induced the specific and total activity of the peroxisomal fatty acid beta-oxidation 5.2- and 5.7-fold, respectively, whereas the specific activity of the cholic acid formation remained unchanged compared to standard pellet feeding. The same pattern was found in the postnuclear supernatants (E-fractions), excluding the possibility that different treatments caused different distributions of organelles between the fractions. This differential induction of two similar peroxisomal reaction sequences suggests that at least two mechanisms for peroxisomal induction exist.

Acyl-CoA Oxidase↗

PubMed1985

Defective peroxisomal cleavage of the C27-steroid side chain in the cerebro-hepato-renal syndrome of Zellweger.

Based on in vitro work with rat liver, we recently suggested that the peroxisomal fraction is most important for the oxidation of 3 alpha, 7 alpha, 12 alpha-trihydroxy-5 beta-cholestanoic acid (THCA) into cholic acid. The cerebro-hepato-renal syndrome of Zellweger is a fatal recessive autosomal disorder, the most characteristic histological feature of which is a virtual absence of peroxisomes in liver and kidneys. This disease offers a unique opportunity to evaluate the relative importance of peroxisomes in bile acid biosynthesis. A child with Zellweger syndrome was studied in the present work. In accordance with previous work, there was a considerable accumulation of THCA, 3 alpha, 7 alpha, 12 alpha, 24-tetrahydroxy-5 beta-cholestanoic acid (24-OH-THCA), 3 alpha, 7 alpha, 12 alpha-trihydroxy-27-carboxymethyl-5 beta-cholestan-26-oic acid (C29-dicarboxylic acid), and 3 alpha, 7 alpha-dihydroxy-5 beta-cholestanoic acid in serum. In addition, a tetrahydroxylated 5 beta-cholestanoic acid with all the hydroxyl groups in the steroid nucleus was found. 3H-Labeled 5 beta-cholestane-3 alpha, 7 alpha, 12 alpha-triol was administered intravenously together with 14C-labeled cholic acid. There was a rapid incorporation of 3H in THCA and a slow incorporation into cholic acid. The specific radioactivity of 3H in THCA was about one magnitude higher than that in cholic acid. The conversion was evaluated by following the increasing ratio between 3H and 14C in biliary cholic acid. The rate of incorporation of 3H in cholic acid was considerably less than previously reported in experiments with healthy subjects, and the maximal conversion of the triol into cholic acid was only 15-20%. About the same rate of conversion was found after oral administration of 3H-THCA. Both in the experiment with 3H-5 beta-cholestane-3 alpha, 7 alpha, 12 alpha-triol and with 3H-THCA, there was an efficient incorporation of 3H in the above unidentified tetrahydroxylated 5 beta-cholestanoic acid. There was only slow incorporation of radioactivity into 24-OH-THCA and into the C29-dicarboxylic acid. From the specific activity decay curve of 14C in cholic acid obtained after intravenous injection of 14C-cholic acid, the pool size of cholic acid was calculated to be 24 mg/m2 and the daily production rate to 9 mg/m2 per d. This corresponds to a reduction of approximately 85 and 90%, respectively, when compared with normal infants. It is concluded that liver peroxisomes are essential in the normal conversion of THCA to cholic acid. In the Zellweger syndrome this conversion is defective and as a consequence the accumulated THCA is either excreted as such or transformed into other metabolites by hydroxylation or side chain elongation. The accumulation of THCA, as well as the similar rate of conversion of 5 beta-cholestane-3 alpha,7 alpha.12 alpha-triol and THCA into cholic acid, support the contention that the 26-hydroxylase pathway with intermediate formation of THCA is the most important pathway for formation of cholic acid in man.

Bile↗

PubMed1999

Bile acid patterns in meconium are influenced by cholestasis of pregnancy and not altered by ursodeoxycholic acid treatment.

BACKGROUND: Data on meconium bile acid composition in newborn babies of patients with intrahepatic cholestasis of pregnancy (ICP) are relatively scant, and changes that occur on ursodeoxycholic acid (UDCA) administration have not been evaluated. AIMS: To investigate bile acid profiles in meconium of neonates from untreated and UDCA treated patients with ICP. Maternal serum bile acid composition was also analysed both at diagnosis and delivery to determine whether this influences the concentration and proportion of bile acids in the meconium. PATIENTS/METHODS: The population included eight healthy pregnant women and 16 patients with ICP, nine of which received UDCA (12.5-15.0 mg/kg body weight/day) for 15+/-4 days until parturition. Bile acids were assessed in the meconium by gas chromatography-mass spectrometry and in maternal serum by high performance liquid chromatography. RESULTS: Total bile acid and cholic acid concentrations in the meconium were increased (p<0.01) in newborns from patients with ICP (13.5 (5.1) and 8.4 (4.1) micromol/g respectively; mean (SEM)) as compared with controls (2.0 (0.5) and 0.8 (0.3) micromol/g respectively), reflecting the total bile acid and cholic acid levels in the maternal serum (r = 0.85 and r = 0.84, p<0.01). After UDCA administration, total bile acid concentrations decreased in the mother ( approximately 3-fold, p<0. 05) but not in the meconium. UDCA concentration in the meconium showed only a 2-fold increase after treatment, despite the much greater increase in the maternal serum (p<0.01). Lithocholic acid concentration in the meconium was not increased by UDCA treatment. CONCLUSIONS: UDCA administration does not influence the concentration and proportion of bile acids in the meconium, which in turn are altered by ICP. Moreover, this beneficial treatment for the mother does not increase meconium levels of potentially toxic metabolites of UDCA such as lithocholic acid.

Bile Acids and Salts↗

PubMed1987

Biliary lipid secretion in the rat during infusion of increasing doses of unconjugated bile acids.

The aim of the present study was to examine the secretion of biliary components in rats during infusion of increasing doses of either deoxycholic acid, chenodeoxycholic acid or cholic acid and to test the hypothesis that biliary phospholipids may regulate the hepatic bile acid secretory capacity. Analysis of bile samples, collected every 10 min throughout the infusion period showed that there was an elevation of bile acid, phospholipid, cholesterol and alkaline-phosphodiesterase secretion, with all the bile acids, peaking and then gradually declining. Their secretory rates maximum differed and were inversely related to their detergent strength. However, the secretory rates maximum and total output of phospholipids and cholesterol were similar for all bile acids infused. The per cent contribution of phosphatidylcholine to total bile acid-dependent phospholipid secretion was reduced from 84% (in the pre-infusion period) to 59, 46 and 13% at the end of the cholic acid, chenodeoxycholic acid and deoxycholic acid infusions, respectively. This decrease in the per cent contribution of phosphatidylcholine was associated with an increase in the contribution of both sphingomyelin and phosphatidylethanolamine. The biliary phospholipid fatty acid pattern corroborated these changes in the phospholipid classes. Since sphingomyelin and phosphatidylethanolamine are major phospholipids in bile canalicular and other hepatocellular membranes, the marked increase in their secretion in bile during the infusion of high doses of bile acids may indicate solubilization of membrane phospholipids, resulting in membrane structural changes responsible for the reduced excretory function of the liver.

Animals↗

PubMedDate not supplied

Age-related changes of bile acid metabolism in rats.

Cholesterol and bile acid leves were examined in young (8 weeks), middle-aged (12 months) and old (24 months) germ-free male rats, and young (8 weeks) and middle-aged (12 months) conventional male rats. The plasma cholesterol levels were higher in the aged rats, being more marked in the conventional rats. The liver cholesterol levels also increased with age and the increases were almost identical for both groups. No age-related changes were found in the biliary bile acid secretion, the pool size and distribution of bile acids in the bile, small intestine and large intestine, nor in the turnover frequency of bile acids, but the pool size in the young and middle-aged germ-free rats was much larger than that in the conventional rats. The turnover frequency was less in the germ-free rats. The bile acid synthesis presumed from the fecal bile acid excretion decreased in the aged germ-free rats but not in the conventional rats. A most remarkable age-related change was found in the bile acid composition; cholic acid increased and beta-muricholic acid derived from chenodeoxycholic acid in the rat decreased by aging, resulting in an increase of the CA/CDCA ratio (bile acids belonging to the cholic acid group/bile acids to the chenodeoxycholic acid group) in the bile, feces and pool. These results suggest that cholic acid synthesis increases while chenodeoxycholic acid synthesis is impaired by aging in rats.

Journal Article↗

PubMed1981

Quantitative determination of individual non-sulfated bile acids and sulfated lithocholic acid in serum by mass fragmentography.

Individual non-sulfated bile acids and sulfated lithocholic acid in serum were determined by mass fragmentography. A hexafluoroisopropyl ester-trifluoroacetyl derivative of bile acid was prepared by the method of Imai et al. (J. Chromatogr. 120, 181, 1976). Deuterium labeled deoxycholic acid was used as an internal standard monitoring at m/z 623. Lithocholic acid, deoxycholic acid, chenodeoxycholic acid, ursodeoxycholic acid, and cholic acid were determined by monitoring the intensities of m/z 622, m/z 620, m/z 620, m/z 620, ad m/z 618, respectively. A serum sample of 200 microliters including 500 ng of internal standard was hydrolyzed with strong alkali, then acidified to pH 1 with 2N HCl under cooling on ice, and extracted with diethyl ether immediately. Ether extracts were derivatized without further purification. Besides this assay of non-sulfated bile acids, total serum lithocholic acid including the sulfated form was determined as follows: extraction was performed after mixing the acidified (pH 1 with 2 N HCl) hydrolysate with ether and incubation at 40 degrees C for 2 h. Bile acid peaks in the mass fragmentogram were not affected by other materials in these serum extracts. The average values of individual non-sulfated bile acids in sera from healthy fasting subjects (n=15) were as follows: lithocholic acid, 0.049 micrograms/ml; deoxycholic acid, 0.462 micrograms/ml; chenodeoxycholic acid, 0.671 micrograms/ml; ursodeoxycholic acid, 0.070 icrograms/ml; and cholic acid, 0.217 micrograms/ml. Total lithocholic acid (non-sulfated and sulfated) in sera was 0.166 microgram/ml.

Bile Acids and Salts↗

PubMed1986

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.

Animals↗

PubMed1986

In vitro formation of bile acids from di- and trihydroxy-5 beta-cholestanoic acid in human liver peroxisomes.

The conversion of 3 alpha,7 alpha,12 alpha-trihydroxy-5 beta-[3H]cholestanoic acid into cholic acid and 3 alpha,7 alpha-dihydroxy-5 beta-[3H]cholestanoic acid into chenodeoxycholic acid has been studied in subcellular fractions of human liver. The products were separated from the substrates by high-pressure liquid chromatography and identified by combined gas chromatography-mass spectrometry. The highest rates of conversion were found in the light mitochondrial fraction. This fraction also contained the highest amount of the marker enzymes for peroxisomes. The maximal rates of cholic acid and chenodeoxycholic acid formation were 1.3 and 1.8 nmol/mg protein per h, respectively. The presence of KCN in the incubation medium stimulated the formation of bile acids. Peroxisomes were prepared from the light mitochondrial fraction by sucrose-gradient centrifugation. By use of different marker enzymes, it was confirmed that the major part of the activity for cholic acid formation in the light mitochondrial fraction was located in the peroxisomes. It is concluded that liver peroxisomes are important for the oxidative cleavage of the C27 steroid side chain in bile acid formation in man.

Acetylglucosaminidase↗

PubMed1993

Human hepatoblastoma cells (HepG2) and rat hepatoma cells are defective in important enzyme activities in the oxidation of the C27 steroid side chain in bile acid formation.

We have examined the ability of HepG2 human hepatoblastoma cells and 7800 C1 Morris rat hepatoma cells to convert 3 alpha, 7 alpha, 12 alpha-trihydroxy-5 beta-cholestanoic acid (THCA) and 3 alpha, 7 alpha-dihydroxy-5 beta-cholestanoic acid (DHCA) to cholic acid and chenodeoxycholic acid, respectively. Cell extracts from both these cell lines could neither form cholic acid from THCA nor from the activated form, THCA-CoA. This suggests that both cell lines are defective in two enzyme activities involved in the pathway, the microsomal THCA-CoA ligase and the peroxisomal THCA-CoA oxidase. Furthermore, we show that the subsequent enzymes are active in the conversion to bile acids, because the product of the THCA-CoA oxidase, 3 alpha, 7 alpha, 12 alpha-trihydroxy-5 beta-cholest-24-enoyl-coenzyme A (delta 24-THCA-CoA) or delta 24-THCA in the presence of THCA-CoA ligase, are converted to cholic acid by both cell lines. HepG2 cells were able to slowly form chenodeoxycholic acid and cholic acid from 5 beta-cholestane-3 alpha, 7 alpha-diol and 5 beta-cholestane-3 alpha, 7 alpha, 12 alpha-triol, respectively, in 24- and 96-h incubations. The rate of cholic acid formation was lower than the rate for chenodeoxycholic acid and there was a clear accumulation of THCA. 7800 C1 Morris cells had no ability to form cholic acid or chenodeoxycholic acid after 96 h incubation. We conclude that these two cell lines have defects in two enzyme activities involved in the peroxisomal oxidation in bile acid formation, the microsomal THCA-CoA ligase and the peroxisomal THCA-CoA oxidase.

Animals↗