Metabolism and choleretic activity of homochenodeoxycholic acid in the hamster

The hepatic metabolism and the choleretic effect of homochenodeoxycholic acid, the C25 homologue of chenodeoxycholic acid, were investigated in the hamster. After intravenous administration of 3H-labeled homochenodeoxycholic acid into biliary fistula hamsters, more than 80% of the radioactivity was recovered in bile in 4 h. A relatively small proportion of homochenodeoxycholic acid was present in bile as the taurine (22%) or glycine (4%) conjugate. However, more than 70% of the administered compound was biotransformed into C23 bile acids. The major C23 metabolites in bile were norchenodeoxycholic acid (17%), tauronorchenodeoxycholic acid (33%), and a trihydroxy norbile acid (identified as 3 alpha, 5 beta, 7 alpha-trihydroxy-24-nor-5 beta-cholan-23-oic acid, 19%). Small amounts (< 5%) of sulfate(s) and glucuronide(s) were also detected. Homochenodeoxycholic acid, when infused intravenously into the hamster, produced a striking choleresis. The increase in bile flow after infusion of this compound was 6- to 7-times that induced by chenodeoxycholic acid. The apparent choleretic activity of homochenodeoxycholic acid, 181 microliters/mumol, was much greater than that of chenodeoxycholic acid, 11 microliters/mumol. In conclusion, homochenodeoxycholic acid induced a hypercholeresis of the same order of magnitude as norchenodeoxycholic acid, presumably because considerable proportions of this compound were degraded to the hypercholeretic norchenodeoxycholic acid via beta-oxidation in the liver.     

Intestinal absorption of peptides by coupling to bile acids

Poor intestinal absorption of peptides greatly limits their use as drugs for the treatment of chronic diseases. Since bile acids are efficiently absorbed by an active, Na(+)-dependent transport system in the ileum of mammals, model peptides of different chain length were attached to the 3-position of modified 3 beta-(omega-amino-alkoxy)-7 alpha, 12 alpha-dihydroxy-5 beta-cholan-24-oic acid. These peptide-bile acid conjugates inhibited Na(+)-dependent [3H]taurocholate uptake into brush-border membrane vesicles isolated from rabbit ileum in a concentration-dependent manner. Furthermore, photoaffinity labeling of the bile acid-binding proteins of M(r) 93,000 and 14,000, identified as the protein components of the ileal Na(+)-dependent bile acid transport system in rabbit ileum (Kramer, W., Girbig, F., Gutjahr, U., Kowalewski, S., Jouvenal, K., Müller, G., Tripier, D., and Wess, G. (1993) J. Biol. Chem. 268, 18035-18046) by the photoreactive taurocholate analogue, (3,3-azo-7 alpha, 12 alpha-dihydroxy-5 beta [7 beta, -12 beta-3H]cholan-24-oyl)-2-aminoethanesulfonic acid, was inhibited by the peptide-bile acid conjugates. In contrast, the parent peptides and amino acids neither had a significant effect on [3H]taurocholate uptake by ileal brush-border membrane vesicles nor on photoaffinity labeling of the ileal bile acid-binding membrane proteins. The inhibitory effect of peptide-bile acid conjugates on [3H]taurocholate transport and photoaffinity labeling of the bile acid-binding proteins in rabbit ileal vesicles decreased with increasing chain length of the attached peptide radical. By in vivo ileum perfusion in anesthetized rats an intestinal absorption of the bile acid conjugate S3744 of the fluorescent oxaprolylpeptide 4-nitrobenzo-2-oxa-1,3-diazol-beta-Ala-Phe-5-Opr-Gly (S1037) and secretion of the intact compound into bile could be demonstrated, whereas the parent peptide S1037 or its t-butylester S4404 were not absorbed. The intestinal absorption of S3744 showed a similar temperature dependence as [3H]taurocholate absorption and was inhibited by the presence of taurocholate indicating a carrier-mediated uptake of S3744 via the ileal bile acid transporter. In conclusion, these results indicate that oligopeptides can be made enterally absorable by coupling to modified bile acid molecules making use of the specific intestinal absorption pathway for bile acids. This finding may be of great importance for the design and development of orally active peptide drugs.     

Transformation of cholanic acid derivatives into pharmacologically active esters of phenolic acids by heterogeneous Wittig reaction

Steroid esters of cynnamic acid derivatives have been synthesized by a heterogeneous Wittig reaction under sonochemical conditions from the corresponding triphenylphosphonium bromides and unprotected phenolic aldehyds using K2CO3 as a base. 5 beta-Cholan-3 alpha, 7 alpha, 12 alpha, 24-E-ferulate (11') exhibited a marked inhibitory effect on influenza virus A. The synthetic 3 alpha, 24-E-diferulates of 5 beta-cholan-3 alpha, 24- diol, 5 beta-cholan-3 alpha, 12 alpha, 24-triol and 5 beta-cholan-3 alpha, 7 alpha, 12 alpha, 24-tetrol (8, 9 and 12) showed antitumor activity on leukemia P-388 in mice.     

Formation of varanic acid, 3 alpha, 7 alpha, 12 alpha, 24-tetrahydroxy-5 beta-cholestanoic acid from 3 alpha, 7 alpha, 12 alpha-trihydroxy-5 beta-cholestanoic acid in Bombina orientalis

Varanic acid (3 alpha, 7 alpha, 12 alpha, 24-tetrahydroxy-5 beta-cholestanoic acid; 24-OH-THCA) is almost the sole component of bile acids in the bile of Bombina orientalis. To examine in the mechanism of the formation of 24-OH-THCA, radiolabeled (25R)- and (25S)-3 alpha, 7 alpha, 12 alpha-trihdroxy-5 beta-cholestanoic acids [(25R)- and (25S)-THCA] and (24E)-3 alpha, 7 alpha, 12 alpha-trihdroxy-5 beta-cholest-24-enoic acid (delta 24-THCA) were administered intraperitoneally to B. orientalis, gallbladder bile was collected after 24 h, and bile acids were subsequently extracted. Then the bile acids were analyzed by means of radio thin-layer chromatography and radio high-performance liquid chromatography after conversion to p-bromophenacyl ester derivatives. Although delta 24-THCA was not converted to 24-OH-THCA, (25R)-THCA and (25S)-THCA were transformed to (24R,25R)-24-OH-THCA and (24R,25S)-24-OH-THCA, respectively. These results strongly suggest that 24-OH-THCA was transformed via direct hydroxylation of the saturated side chain of THCA, not via hydration to an alpha, beta-unsaturated acid, delta 24-THCA, in B. orientalis.     

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.     

Non-stereoselective formation of 3 alpha,7 alpha,12 alpha,24-tetrahydroxy-5 beta-cholestan-26-oic acid during cholic acid biosynthesis

Incubation of (25RS)-, (25R)- and (25S)-3 alpha,7 alpha,12 alpha-trihydroxy-5 beta-cholestan-26-oic acid (THCA, 6, 6a, 6b) and (24E)-3 alpha,7 alpha,12 alpha-trihydroxy-5 beta-cholest-24-en-26-oic acid (7) with rat liver mitochondria gave all four stereoisomers (9a,9b,9c,9d) of 3 alpha,7 alpha,12 alpha,24-Tetrahydroxy-5 beta-cholestan-26-oic acid (TeHCA). The corresponding 27-nor analogs (10,11) were also converted non-stereoselectively to a 1:1 mixture of the epimeric 24-hydroxy compounds (12).     

Uptake of 3 alpha, 7 alpha, 12 alpha-trihydroxy-24-nor-5 beta-cholan-23-sulfonate into isolated rat hepatocytes by three transport systems

Uptake of norcholansulfonate (3 alpha, 7 alpha, 12 alpha-trihydroxy-24-nor-5 beta-cholan-23-sulfonate), an isogeometric analogue of cholate into isolated rat liver hepatocytes occurs only by saturable transport. In order to identify the transport systems involved, uptake of norcholansulfonate was studied using 7 beta-NBD-NCT ({N-[7-(4-nitrobenzo-2-oxa-1,3-diazol)]-7 beta-amino-3 alpha,12 alpha-dihydroxy-5 beta-cholan-24-oyl})-2'-aminoethanesulfonate) as a competing substrate. For transport of both bile salt derivatives, which mutually inhibit their mediated transport competitively, the existence of at least three transport systems must be assumed. Uptake studies using the cloned hepatic Na+/cholyltaurine cotransporting polypeptide stably expressed in CHO cells (Chinese hamster ovary cells) showed that both bile salt derivatives were transported and furnished the definite KT values of this single transport system and the ratio of the maximal uptake velocities. On the basis of these data, uptake of both bile salt derivatives into rat hepatocytes and their mutual competitive inhibition could be analyzed for three transport systems. The maximal flux rates J2 and the half-saturation constants KT2 in the presence of Na+ (143 mM) are for norcholansulfonate: J1(Na+ 143) = 1.0 +/- 0.2 nmol/(min . mg protein), KT1(Na+ 143) = 15 +/- 4 microM, J2(Na+ 143) = 0.5 +/- 0.2 nmol/(min.mg protein), KT2(Na+ 143) = 15 +/- 2 microM, J3(Na+ 143) = 0.5 +/- 0.2 nmol/(min.mg protein), KT3(Na+ 143) = 60 +/- 15 microM, and for 7 beta-NBD-NCT J1(Na+ 143) = 0.14 +/- 0.04 nmol/(min.mg protein), KT1(Na+ 143) = 3.1 +/- 0.5 microM, J2(Na+ 143) = 0.014 +/- 0.005 nmol/(min.mg protein), KT2(Na+ 143) = 21 +/- 2 microM, J3(Na+ 143) = 1.0 +/- 0.1 nmol/(min.mg protein), KT3(Na+ 143) = 190 +/- 25 microM. The kinetic parameters are in accordance with the assumptions that the cloned Na+/cholyltaurine cotransporting polypeptide represents transport system 2 and that the kinetically identified additional transport system 1 is either strictly or partially Na(+)-dependent.     

Comparison of side chain oxidation of potential C27-bile acid intermediates between mitochondria and peroxisomes of the rat liver: presence of beta-oxidation activity for bile acid biosynthesis in mitochondria

The oxidation of the side chains of two potential bile acid intermediates, 3 alpha,7 alpha,12 alpha-trihydroxy-5 beta-cholestanoic acid (THCA) and 3 alpha,7 alpha-dihydroxy-5 beta-cholestanoic acid (DHCA), were investigated in rat liver mitochondria and peroxisomes. Both THCA and DHCA were efficiently oxidized to yield cholic acid and chenodeoxycholic acid, along with 3 alpha,7 alpha,12 alpha-trihydroxy-5 beta-cholest-24-enoic acid and 3 alpha,7 alpha-dihydroxy-5 beta-cholest-24-enoic acid, respectively, in both the mitochondria and peroxisomes. However, the spectrum of the metabolites in the mitochondria differed greatly from those in the peroxisomes. The major products from THCA and DHCA in the mitochondria were 3 alpha,7 alpha,12 alpha-trihydroxy-5 beta-chol-22-enoic acid and 3 alpha,7 alpha-trihydroxy-5 beta-chol-22-enoic acid, respectively, which were tentatively identified from the mass spectral data. However, the formation of these C24-unsaturated bile acids was not observed in the peroxisomes. These results strongly suggest that the cleavage of the side chain of the C27-intermediates for bile acid biosynthesis also occurs independently in the mitochondria, not due to the contamination of peroxisomes.     

Extrahepatic deposition and cytotoxicity of lithocholic acid: studies in two hamster models of hepatic failure and in cultured human fibroblasts

Effects of bile acids on tissues outside of the enterohepatic circulation may be of major pathophysiological significance under conditions of elevated serum bile acid concentrations, such as in hepatobiliary disease. Two hamster models of hepatic failure, namely functional hepatectomy (HepX), and 2-day bile duct ligation (BDL), as well as cultured human fibroblasts, were used to study the comparative tissue uptake, distribution, and cytotoxicity of lithocholic acid (LCA) in relation to various experimental conditions, such as binding of LCA to low-density lipoprotein (LDL) or albumin as protein carriers. Fifteen minutes after i.v. infusion of [24-(14)C]LCA, the majority of LCA in sham-operated control animals was recovered in liver, bile, and small intestine. After hepatectomy, a significant increase in LCA was found in blood, muscle, heart, brain, adrenals, and thymus. In bile duct-ligated animals, significantly more LCA was associated with blood and skin, and a greater than twofold increase in LCA was observed in the colon. In the hepatectomized model, the administration of LCA bound to LDL resulted in a significantly higher uptake in the kidneys and skin. The comparative time- and concentration-dependent uptake of [14C]LCA, [14C]chenodeoxycholic acid (CDCA), and [14C]cholic acid (CA) in cultured human fibroblasts was nonsaturable and remained a function of concentration. Initial rates of uptake were significantly increased by approximately tenfold, with decreasing hydroxylation of the respective bile acid. After 1 hour of exposure of fibroblasts to LCA, there was a significant, dose-dependent decrease in mitochondrial dehydrogenase activity from 18% to 34% of the control, at LCA concentrations ranging from 1 to 20 micromol/L. At a respective concentration of 100 and 700 micromol/L, CDCA caused a 35% and 99% inhibition of mitochondrial dehydrogenase activity. None of the bile acids tested, with the exception of 700 micromol/L CDCA, caused a significant release of cytosolic lactate dehydrogenase into the medium. In conclusion, we show that bile acids selectively accumulate in nonhepatic tissues under two conditions of impaired liver function. Furthermore, the extrahepatic tissue distribution of bile acids during cholestasis may be affected by serum lipoprotein composition. At a respective concentration of 1 and 100 micromol/L, LCA and CDCA induced mitochondrial damage in human fibroblasts, after just 1 hour of exposure. Therefore, enhanced extrahepatic uptake of hydrophobic bile acids during liver dysfunction, or disorders of lipoprotein metabolism, may have important implications for bile-acid induced cytotoxic effects in tissues of the systemic circulation.     

A new approach to the synthesis of 3 beta, 23-diacetoxy-24-nor-5-cholene from 3 beta, 21-diacetoxy-5-pregnen-20-one

Synthesis of 3 beta, 23-diacetoxy-24-nor-5-cholene in six steps from 3 beta, 21-diacetoxy-5-pregnen-20-one has been achieved by a new approach. The method would allow to label the side chain of a bile acid at carbon-atom 21.     

Composition of biliary lipids and kinetics of bile acids after cholecystectomy in man

Postcholecystectomy biliary lipid composition and bile acid kinetics were studied in 24 women and 4 men. Hepatic bile was collected periodically for as long as 4 months without interrupting the enterohepatic circulation and without infecting the biliary system. In 23 patients with cholesterol gallstones, fasting biliary cholesterol made up 10.2% of total lipids in the steady state; in 5 patients with bilirubinate stones, saturation of fasting hepatic bile with cholesterol was lower (8.7% of total lipids). The percentage of deoxycholic acid after cholecystectomy was not higher than that of seven healthy, noncholecystectomized controls. Postcholecystectomy studies of diurnal variation of biliary lipids (7 patients) showed that postprandial hepatic bile had a significantly lower cholesterol saturation than fasting bile. Pool sizes of cholic and chenodeoxycholic acids were low (average 0.4 g/70 kg, each); total synthesis for both bile acids was normal (average 460 mg/day/70 kg), but fractional turnover rates of the two primary bile acids increased after cholecystectomy, probably due to more frequent recycling of the small bile acid pool.     

A compartmental model for hepatic transport of taurocholic acid in isolated perfused rat liver

In order to characterize the transport of bile acids through the liver and to study the influence of drugs on these processes, a kinetic model for hepatobiliary transport of taurocholic acid (TC) using the isolated perfused liver was developed. After the system was brought to a steady state by infusing TC at a constant rate, a tracer dose of 14C-TC was injected into the medium. The medium disappearance of 14C-TC followed a first-order kinetic with a single rate constant. The plot of the biliary secretion rate of radioactivity versus time revealed a curve composed of at least three exponential components. From the described results and the present knowledge of hepatobiliary transport of bile acids we proposed a three compartment model, composed of a perfusion medium compartment and two liver compartments. Parameters calculated from the model constants agreed well with model-independent estimations. The influence of bromosulfophthalein (BSP) on the kinetic parameters was studied to compare the result with the known effect of BSP on hepatic transport of taurocholic acid. BSP decreased the constant describing the fractional transfer of taurocholic acid from medium into the liver, which is in agreement with the inhibition of hepatic uptake of taurocholic acid by BSP. Thus a three compartment model may adequately define the hepatobiliary transport of taurocholic acid in the isolated perfused rat liver.     

Urinary bile acids in population screening for inapparent liver disease

BACKGROUND/AIMS: We performed this study in order to evaluate the diagnostic potential of bile acids in random samples of urine for detection of latent liver disease and to compare a radioimmunoassay for urine bile acids with an enzymatic method that detects bile acid sulfates. This was a prospective cohort study carried out at the VA Medical Center involving 151 adults who attended a Community Health Fair at the hospital and wanted to know if they had liver disease. METHODOLOGY: Urinary bile acids in random specimens of 5-10 ml urine were measured. Radioimmunoassay for primary bile acids and an enzymatic assay with or without sulfated bile acids, all corrected with creatinine for urine flow were performed. Serum primary bile acids were determined by radioimmunoassay. In addition, routine liver profile and clinical examination were carried out. RESULTS: In 78 of 151 subjects there was at least one recent liver profile to match with the urine bile acids. Of these 78, 52 subjects with normal urine bile acids had a normal liver profile. In 11 subjects abnormal urine bile acids were associated with an abnormal liver profile. Nine of these 11 subjects were anti HCV positive, one was HIV positive. Urine bile acids correctly predicted the outcome of routine liver tests in 89% of 78 subjects. In nine cases there was a discordance between urinary bile acids and the liver profile. Failure to correctly predict the liver profile using urine, was reduced from nine subjects to three when urine bile acids were obtained twice at separate intervals. Urine bile acids predicted the outcome of anti HCV testing in 37 subjects with similar accuracy as serum ALT or AST. Urine bile acids correlated with serum bile acids at r=0.96, 0.88 and 0.76 for the radioimmunoassay, enzymatic assay that included sulfated bile acids and enzymatic assay without the sulfates, respectively. CONCLUSION: Bile acids in a random sample of urine are useful for population screening for latent liver disease. Prediction of sub-clinical hepatitis C is comparable to that of serum ALT or AST. Inclusion of bile acid sulfates mildly increases the predictive value of urine. Urine bile acids highly correlate with serum bile acids, indicating their surrogate diagnostic value.     

Oxidation of corticosteroids to steroidal carboxylic acids by an enzyme preparation from hamster liver

Enzyme activity which catalyzes the oxidation of 11-deoxycorticosterone to 21-oic acids accompanies the "detritiating" enzyme (isomerase) of hamster liver recently isolated by Martin, K. O., et al. ((1977) Biochemistry 16 (preceding paper in this issue)). The metabolites isolated were 20alpha- and 20beta-hydroxy-3-oxo-pregn-4-en-21-oic acid and 3,20-dioxo-pregn-4-en-21-oic acid. When 21-hydroxy[4-14C, 21-3H]pregn-4-en-3,20-dione was the substrate, about half of the tritium was retained in position 20 of the hydroxy acids. The system which catalyzes the conversion of the ketol side chain of corticosteroids to acid metabolites appears to be a cluster of closely related enzymes. As a result of these studies, we believe that the hamster liver enzyme preparation provides a useful model system for studies on the biosynthesis of acid metabolites of the corticosteroids in man.     

Comparative studies of metabolism of simultaneously administered chenodeoxycholic acid and ursodeoxycholic acid in hamsters

We present the comparative studies of metabolism of chenodeoxycholic acid and ursodeoxycholic acid and their taurine conjugates in the liver and fecal culture from hamsters. When [24-14C]chenodeoxycholic acid and [11,12-3H]ursodeoxycholic acid were simultaneously instilled into the jujunal loop of bile fistula hamsters, both bile acids administered were recovered mainly as their conjugates with taurine and glycine in the fistula bile. The recovery of chenodeoxycholic acid was slightly but significantly higher than that of ursodeoxycholic acid. Chenodeoxycholic acid was more efficiently conjugated with glycine than ursodeoxycholic acid. The glycine/taurine ratio in the biliary chenodeoxycholic acid was 1.9, and that in ursodeoxycholic acid was 1.6. In addition, as much as 6.2% of ursodeoxycholic acid was excreted as the unconjugated form; on the other hand only 2.4% of unconjugated chenodeoxycholic acid was excreted. When [24-14C]chenodeoxycholyltaurine and [11,12-3H]ursodeoxycholyltaurine were simultaneously administered into the ileum loop of bile fistula hamsters, both bile salts were absorbed and secreted efficiently into the bile at the same rate. These results indicate that slightly lower recovery of ursodeoxycholic acid in the bile could be due to the less effective conjugation of ursodeoxycholic acid than chenodeoxycholic acid in the liver. Deconjugation by fecal culture from a hamster proceeded more rapidly in chenodeoxycholyltaurine than ursodeoxycholyltaurine. 7-Dehyroxylation to form lithocholic acid by fecal culture was also faster in chenodeoxycholic acid than ursodeoxycholic acid. The formation of 7-oxolithocholic acid from ursodeoxycholic acid was lesser than from chenodeoxycholic acid. In summary, bacterial deconjugation followed by 7-dehydroxylation to form lithocholic acid seems to be achieved more efficiently with chenodeoxycholic acid than ursodeoxycholic acid.     

Serum bile acids and ursodeoxycholic acid treatment in cystic fibrosis-related liver disease

Increased circulating levels of hepatotoxic bile acids may contribute to the cholestasis characteristic of cystic fibrosis-related liver disease. The aims of this study were to compare serum bile acid profiles in patients with cystic fibrosis with and without liver disease, and to evaluate the effect of treatment with ursodeoxycholic acid, a non-hepatotoxic bile acid, on liver biochemistry and serum bile acids in patients with cystic fibrosis-related liver disease. Fasting and postprandial serum bile acid levels were analysed in 15 patients (nine males; median age 18 years) with cystic fibrosis-related liver disease and compared with serum bile acid levels in 18 cystic fibrosis patients (12 males; median age 22 years) without liver disease and 10 control subjects. Fasting and postprandial serum levels of primary and secondary serum bile acids were analysed using high-performance liquid chromatography. Liver biochemistry and serum bile acids were measured in six cystic fibrosis patients with liver disease before and 6 months after treatment with ursodeoxycholic acid 20 mg/kg/day and compared with six control patients with cystic fibrosis-related liver disease. Total fasting and postprandial serum bile acid levels were significantly (P < 0.01) elevated in patients with liver disease compared to those without liver disease and controls. The fasting glycine conjugates of cholic acid, chenodeoxycholic acid and deoxycholic acid, and the fasting and postprandial taurine conjugates of cholic acid and chenodeoxycholic acid were significantly (P < 0.05) elevated in liver disease patients compared to patients without liver disease and controls. After 6 months' treatment with ursodeoxycholic acid, although the serum was significantly saturated with ursodeoxycholic acid and significant improvements in liver biochemistry were observed in the treatment group, there was no significant reduction in the levels of individual serum bile acids. Although circulating levels of potentially hepatotoxic serum bile acids are elevated in patients with cystic fibrosis-related liver disease, improvements in liver biochemistry associated with ursodeoxycholic acid treatment cannot be attributed solely to alterations in levels of endogenous bile acids.     

Effect of bile duct ligation on bile acid metabolism in rats

The effect of bile duct ligation on the quantitative and qualitative changes of bile acids in serum, liver, urine, and feces, and the concentration of cholesterol and phospholipids in serum and liver were examined in male rats. The concentration of bile acids in serum increased over 100-fold on day 5 but was lower than the 5-day level on days 10 and 15. The concentration in the liver also increased about 10-fold. beta-Muricholic acid predominantly increased but the secondary bile acids, deoxycholic acid and hyodeoxycholic acid, decreased. The urinary excretion of bile acids increased to about 40 mg/day per rat on the first day of bile duct ligation but this increase was reduced on day 2 to about half and remained at that level until day 24. These values exceeded that of fecal bile acids, 12 mg/day per rat, before bile duct ligation. The amount of bile acid sulfates in the urine was as low as 1% of the total. The urinary non-sulfated bile acids consisted mainly of beta-muricholic acid (60%) and cholic acid (20%), while the sulfates contained a considerable amount of unidentified acidic substances (40%) in addition to cholic acid and beta-muricholic acid. The concentration of cholesterol and phospholipids in serum markedly increased on day 5 but declined gradually thereafter. The liver cholesterol concentration did not change but the phospholipid concentration decreased. Fecal sterols did not change in both the total amount and composition. These data indicated that daily synthesis of bile acids, especially beta-muricholic acid, was accelerated in bile duct-ligated rats.     

Current concepts of hepatic uptake, intracellular transport and biliary secretion of bile acids: physiological basis and pathophysiological changes in cholestatic liver dysfunction

Hepatic sinusoidal uptake of bile acids is mediated by defined carrier proteins against unfavourable concentration and electrical gradients. Putative carrier proteins have been identified using bile acid photoaffinity labels and more recently using immunological probes, such as monoclonal antibodies. At the sinusoidal domain, proteins with molecular weights of 49 and 54 kDa have been shown to be carriers for bile acid transport. The 49 kDa protein has been associated with the Na(+)-dependent uptake of conjugated bile acids, while the 54 kDa carrier has been involved in the Na(+)-independent bile acid uptake process. Within the hepatocyte, cytosolic proteins, such as the glutathione S-transferase (also designated the Y protein), the Y binders and the fatty acid binding proteins, are able to bind bile acids and possibly facilitate their movement to the canalicular domain. At the canalicular domain a 100 kDa carrier protein has been isolated and it has been shown by several laboratories that this particular protein is concerned with canalicular bile acid transport. The system is ATP-dependent and follows Michaelis-Menten kinetics. Interference with bile acid transport has been demonstrated by several chemicals. The mechanisms by which these chemicals inhibit bile acid transport may explain the apparent cholestatic properties observed in patients and experimental animals treated with these agents. Several studies have shown that Na+/K(+)-ATPase activity is markedly decreased in cholestasis induced by ethinyloestradiol, taurolithocholate and chlorpromazine. However, other types of interference have been described and the cholestatic effects may be the result of several mechanisms. Cholestasis is associated with several adaptive changes that may be responsible for the accumulation of bile acids and other cholephilic compounds in the blood of these patients. It may be speculated that the nature of these changes is to protect liver parenchymal cells from an accumulation of bile acids to toxic levels. However, more detailed quantitative experiments are necessary to answer questions with regard to the significance of these changes and the effect of various hepatobiliary disorders in modifying these mechanisms. It is expected that the mechanisms by which bile acid transport is regulated and efforts to understand the molecular basis for these processes will be among the areas of future research.     

Chemical constituents of Rosa laevigata Michx

Six compounds were isolated from Rosa laevigata. Five of them were obtained from the ethanolic extract and identified as 2 alpha, 3 beta, 19 alpha, 23-tetrahydroxyurs-12-en-28-oic acid, 2 alpha, 3 alpha, 19 alpha, 23-tetrahydroxyurs-12-en-28-oic acid, euscaphic acid, beta-sitosterol and daucosterol. The other one was obtained from the acetate of emulsive layer of the petroleum ether and elucidared as 2 alpha, 3 beta-dihydrolup-28-methyl ester diacetate.     

Effect of protein content on low temperature inactivation of the extracellular proteinase from Pseudomonas fluorescens 22F

The formation of cholic acid and chenodeoxycholic acid through cleavage of the side chains of CoA esters of 3alpha,7alpha,12alpha-trihydroxy-5beta-choles tan-26-oic acid and 3alpha,7alpha-dihydroxy-5beta-cholestan-26-oic acid is believed to occur in peroxisomes. Recently, we found a new peroxisomal enzyme, D-3-hydroxyacyl-CoA dehydratase/D-3-hydroxyacyl-CoA dehydrogenase bifunctional protein, and suggested that this bifunctional protein is responsible for the conversion of 3alpha,7alpha,12alpha-trihydroxy-5beta-cholest-2 4-en-26-oyl-CoA and 3alpha,7alpha-dihydroxy-5beta-cholest-24-en-26-oyl-CoA to their 24-oxo-forms. In the present study, the products of this bifunctional protein reaction were analyzed by gas chromatography-mass spectrometry, and the formation of 24-oxo-27-nor-cholestanes was confirmed. Previously, we found a new thiolase in Caenorhabditis elegans, P-44, and suggested that P-44 and sterol carrier protein x, a peroxisomal protein, constitute a second group of 3-oxoacyl-CoA thiolases. The production of cholic acid and chenodeoxycholic acid from the precursors on incubation with the bifunctional protein and sterol carrier protein x or P-44 was confirmed by gas chromatography.