Effect of natural and structurally modified bile acids on cholesterol metabolizing enzymes in rat liver microsomes

The effect of chenodeoxycholic (CDCA), ursodeoxycholic (UDCA), tauroursodeoxycholic (TUDCA), cholic (CA), ursocholic (UCA) acids, analogues of CDCA and UDCA with a cyclopropyl ring at C22, C23 (cypro-CDCA and cypro-UDCA) and 23-methylursodeoxycholic acid (MUDCA) on cholesterol 7 alpha-hydroxylase was studied in rat liver microsomes. Cypro-analogues consisted of a mixture of four diasteroisomers, while MUDCA was the racemic mixture of two enantiomers. Each steroid was added to liver microsomes at concentrations ranging from 10 to 200 microM. With the exception of UCA and CA, all the bile acids inhibited cholesterol 7 alpha-hydroxylase activity. The inhibition shown by cypro-CDCA and cypro-UDCA was stronger than that observed with the corresponding natural compounds. 22S,23S cypro-UDCA exhibited an inhibitory effect which was more pronounced than that of the diasteroisomer mixture. The isomer 22R,23S was less effective and decreased cholesterol 7 alpha-hydroxylase activity in a manner comparable to that of UDCA. The effect of CDCA, UDCA and the cyclopropyl analogues was also tested with respect to HMG-CoA reductase and acylCoA cholesterol acyltransferase (ACAT) activities. ACAT was stimulated by the isomer 22S,23S cypro-UDCA but not affected by the other bile acids. No effect was observed as regards HMG-CoA reductase.     

Physicochemical and biological properties of natural and synthetic C-22 and C-23 hydroxylated bile acids

In order to define the effect of a side chain hydroxy group on bile acid (BA) physicochemical and biological properties, 23-hydroxylated bile acids were synthesized following a new efficient route involving the alpha-oxygenation of silylalkenes. 22-Hydroxylated bile acids were also studied. The synthesized bile acids included R and S epimers of 3 alpha,7 alpha,23-trihydroxy-5 beta-cholan-24-oic acid (23R epimer: phocaecholic acid), 3 alpha,12 alpha,23-trihydroxy-5 beta-cholan-24-oic (23R epimer: bitocholic acid), and 3 alpha,7 beta,23-trihydroxy-5 beta-cholan-24-oic acid. A 3 alpha,7 alpha,22-trihydroxy-5 beta-cholan-24-oic acid (haemulcholic acid) was also studied. The presence of a hydroxy group on the side chain slightly modified the physicochemical behavior in aqueous solution with respect to common BA: the critical micellar concentration (CMC) and the hydrophilicity were similar to naturally occurring trihydroxy BA such as cholic acid. The pKa value was lowered by 1.5 units with respect to common BA, being 3.8 for all the C-23 hydroxy BA. C-22 had a higher pKa (4.2) as a result of the increased distance of the hydroxy group from the carboxy group. When the C-23 hydroxylated BA were intravenously administered to bile fistula rats, they were efficiently recovered in bile (more than 80% unmodified) while the corresponding analogs, lacking the 23- hydroxy group, were almost completely glycine- or taurine-conjugated. On the other hand, the C-22 hydroxylated BA were extensively conjugated with taurine and less than 40% of the administered dose was secreted without being conjugated. In the presence of intestinal bacteria, they were mostly metabolized to the corresponding 7-dehydroxylated compound similar to common BA with the exception of bitocholic acid which was relatively stable. The presence of a hydroxy group at the C-23 position increased the acidity of the BA and this accounted for poor absorption within the biliary tree and efficient biliary secretion without the need for conjugation. 3 alpha,7 beta-23 R/S trihydroxy-5 beta-cholan-24-oic acids could improve the efficiency of ursodeoxycholic acid (UDCA) for gallstone dissolution or cholestatic syndrome therapy, as it is relatively hydrophilic and efficiently secreted into bile without altering the glycine and taurine hepatic pool.     

The effect of cholesterol feeding on gallbladder bile acids of the rabbit. Evidence that lithocholic acid is a primary bile acid in the rabbit.

The bile acid in gallbladder bile of rabbits fed a normal diet or one containing 2% (w/w) cholesterol have been determined by gas chromatography-mass spectrometry. The predominant bile acids in normally fed rabbits were 3 alpha, 7 alpha, 12 alpha-trihydroxy-5 beta-cholan-24-oic acid (cholic acid), 3 alpha, 12 alpha-dihydroxy-5 alpha-cholan-24-oic acid (allodeoxycholic acid) and 3 alpha, 12 alpha-dihydroxy-5 beta-cholan-24-oic acid (deoxycholic acid) with very much smaller amounts of 3 alpha-hydroxy-5 beta-cholan-24-oic acid (lithocholic acid) and 3 alpha, 12 beta-dihydroxy-5 beta-cholan-24-oic acid. In the cholesterol-fed animals the lithocholate became a predominant bile acid. Sulphated bile acids accounted for less than 1% of the total bile acids. It is proposed that lithocholic acid may be a primary bile acid in the cholesterol-fed rabbit, formed by an alternative pathway of biosynthesis involving hepatic mitochondria.     

Synthesis of 3 alpha,6 beta,7 alpha,12 beta- and 3 alpha,6 beta,7 beta,12 beta-tetrahydroxy-5 beta-cholanoic acids.

Chemical synthesis of 3 alpha,6 beta,7 alpha,12 beta- and 3 alpha,6 beta,7 beta,12 beta-tetrahydroxy-5 beta-cholan-24-oic acids is described. 3 alpha,12 beta-Dihydroxy-5 beta-chol-6-en-24-oic acid used as the starting material in the synthesis was prepared via oxidation of 3 alpha,12 alpha-dihydroxy-5 beta-chol-6-en-24-oic acid 3-hemisuccinate at C-12 followed by reduction with potassium/tertiary amyl alcohol. alpha-Epoxidation of the ester diacetate of 3 alpha,12 beta-dihydroxy-5 beta-chol-6-en-24-oic acid with m-chloroperbenzoic acid followed by cleavage of the epoxide with acetic acid and alkaline hydrolysis yielded 3 alpha,6 beta,7 alpha,12 beta-tetrahydroxy-5 beta-cholan-24-oic acid (overall yield 25%). N-Methylmorpholine-N-oxide-catalyzed osmium tetroxide oxidation of the ester diacetate of 3 alpha,12 beta-dihydroxy-5 beta-chol-6-en-24-oic acid followed by alkaline hydrolysis yielded 3 alpha,6 beta,7 beta,12 beta-tetrahydroxy-5 beta-cholan-24-oic acid (overall yield 33%). The structures of the synthesized bile acids were confirmed from their proto nuclear magnetic resonance and mass spectral fragmentation patterns.     

Regulation of bile acid synthesis. IV. Interrelationship between cholesterol and bile acid biosynthesis pathways

Under most experimental conditions, the activities of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA reductase) and cholesterol 7 alpha-hydroxylase, change together in parallel directions. It has been suggested that newly synthesized cholesterol may be the preferred substrate for cholesterol 7 alpha-hydroxylase, which may account for the observed synchronous behavior of the two enzymes. To test this hypothesis, mevinolinic acid, a potent competitive inhibitor of HMG-CoA reductase, was administered as a single intravenous bolus (10 mg/kg) to rats with a chronic bile fistula. Bile acid synthesis was determined following inhibition of HMG-CoA reductase by mevinolinic acid over a 27-h time course and specific activities of HMG-CoA reductase and cholesterol 7 alpha-hydroxylase were determined in liver microsomes. At 3, 6, and 27 h after a bolus dose of mevinolinic acid, bile acid synthesis was reduced by 54 +/- 5%, 42 +/- 8%, and 23 +/- 13%, respectively, from preinfusion baseline. Within 30 min after administration of mevinolinic acid, HMG-CoA reductase activity was inhibited by at least 87%. At 0.5, 1.5, 3, 6, and 27 h after mevinolinic acid injection, cholesterol 7 alpha-hydroxylase activity was decreased by 6%, 25%, 54%, 41%, and 17%, respectively. By 27 h, the activities of both enzymes had returned to baseline levels. The reduction of bile acid synthesis correlated closely with the observed changes in the activities of cholesterol 7 alpha-hydroxylase. In vitro addition of mevinolinic acid (up to 20 microM) to rat liver microsomes failed to inhibit cholesterol 7 alpha-hydroxylase activity, suggesting no direct effect of mevinolinic acid on enzyme activity. When a bolus dose of mevinolinic acid was coupled with a continuous infusion of mevalonate, the product of the reaction catalyzed by HMG-CoA reductase, the mevinolinic acid-induced decrease in cholesterol 7 alpha-hydroxylase activity and bile acid synthesis was prevented. The results of this study provide evidence that, under the experimental conditions described, there is a linkage between the rates of cholesterol synthesis and the activities of cholesterol 7 alpha-hydroxylase. The data also emphasize the importance of the newly synthesized cholesterol in the regulation of cholesterol 7 alpha-hydroxylase activity.     

Effect of bile acid analogs on 7 alpha-dehydroxylase activity in Eubacterium sp. V.P.I. 12708

7 beta-Methyl-chenodeoxycholic acid (7-MeCDC, 3 alpha, 7 alpha-dihydroxy-7 beta-methyl-5 beta-cholan-24-oic acid), 7 alpha-methyl-ursodeoxycholic acid (7-MeUDC, 3 alpha, 7 beta-dihydroxy-7 alpha-methyl-5 beta-cholan-24-oic acid), 7 xi-methyl-lithocholic acid (7-MeLC, 3 alpha-hydroxy-7 xi-methyl-5 beta-cholan-24-oic acid) and ursodeoxycholylsarcosine (UDCS) were tested as inhibitors of bacterial bile acid 7 alpha-dehydroxylase activity. At a concentration of 50 microM, 7-MeCDC and 7-MeUDC inhibited enzyme activity by 66% and 12%, respectively. 7 alpha-Dehydroxylase activity was not inhibited in the presence of 7-MeLC and UDCS. None of the four bile acid analogs tested inhibited the growth of Eubacterium sp. V.P.I. 12708 at concentrations up to 100 microM.     

Synthesis of four diastereoisomers at carbons 24 and 25 of 3 alpha,7 alpha,12 alpha,24-tetrahydroxy-5 beta-cholestan-26-oic acid, intermediates of bile acid biosynthesis

The synthesis of four stereoisomers at C-24 and C-25 of 3 alpha,7 alpha,12 alpha,24-tetrahydroxy-5 beta-cholestan-26-oic acid is described. Pyridium chlorochromate oxidation of 3 alpha,7 alpha,12 alpha-triacetoxy-5 beta-cholan-24-ol (II) prepared from cholic acid (I) afforded 3 alpha,7 alpha,12 alpha-triacetoxy-5 beta-cholan-24-al (III) which was converted to a mixture of the four stereoisomers (IV-VII) by a Reformatsky reaction with ethyl DL-alpha-bromopropionate followed by alkaline hydrolysis. Separation of these isomers (IV-VII) was achieved by silica gel column chromatography, and subsequent reversed-phase partition column chromatography. The configurations at C-24 were elucidated by conversion of each isomer into (24R)- or (24S)-5 beta-cholestane-3 alpha,7 alpha,12 alpha,24-tetrol (XII or XI) by Kolbe electric coupling, the C-24 configurations of which were determined by modified Horeau's method and 13C-nuclear magnetic resonance spectroscopy. The stereochemistries at C-25 were deduced by comparison of IV-VII with the products of the hydroboration followed by oxidation with alkaline hydrogen peroxide of (24E)-3 alpha,7 alpha,12 alpha-trihydroxy-5 beta-cholest-24-en-26-oic acid (XIII).     

Synthesis of 24-nor-5 beta-cholan-23-oic acid derivatives: a convenient and efficient one-carbon degradation of the side chain of natural bile acids

An efficient procedure for obtaining nor-bile acids from natural (C24) bile acids is described. Treatment of formylated bile acids with sodium nitrite in a mixture of trifluoroacetic anhydride with trifluoroacetic acid gives, through a "second order" Beckmann rearrangement, 24-nor-23-nitriles. These compounds, on alkaline hydrolysis, afford the corresponding nor-bile acids in high yields. The sequence was successfully applied to the synthesis of 3 alpha-hydroxy-24-nor-5 beta-cholan-23-oic (norlithocholic) acid, 3 alpha,6 alpha- (norhyodeoxycholic), 3 alpha,7 alpha- (norchenodeoxycholic), 3 alpha,7 beta- (norursodeoxycholic), and 3 alpha,12 alpha-dihydroxy-24-nor-5 beta-cholan-23-oic (nordeoxycholic) acids, as well as 3 alpha,7 alpha,12 alpha-trihydroxy-24-nor-5 beta-cholan-23-oic (norcholic) acid. 13C-NMR spectra of their methyl esters are reported. The procedure provides a more rapid alternative to the Barbier-Wieland degradation for shortening by one methylene group the side chain of natural (C24) bile acids.     

Appearance of atypical 3 alpha,6 beta,7 beta,12 alpha-tetrahydroxy-5 beta-cholan-24-oic acid in spgp knockout mice

Bile formation and its canalicular secretion are essential functions of the mammalian liver. The sister-of-p-glycoprotein (spgp) gene was shown to encode the canalicular bile salt export protein, and mutations in spgp gene were identified as the cause of progressive familial intrahepatic cholestasis type 2. However, target inactivation of spgp gene in mice results in nonprogressive but persistent cholestasis and causes the secretion of unexpectedly large amounts of unknown tetrahydroxylated bile acid in the bile. The present study confirms the identity of this tetrahydroxylated bile acid as 3 alpha,6 beta,7 beta,12 alpha-tetrahydroxy-5 beta-cholan-24-oic acid. The data further show that in serum, liver, and urine of the spgp knockout mice, there is a significant increase in the concentration of total bile salts containing a large amount of tetrahydroxy-5 beta-cholan-24-oic acid. The increase in total bile acids was associated with up-regulation of the mRNA of cholesterol 7 alpha-hydroxylase in male mice only. It is suggested that the lower severity of the cholestasis in the spgp knockout mice may be due to the synthesis of 3 alpha,6 beta,7 beta,12 alpha-tetrahydroxy-5 beta-cholan-24-oic acid, which neutralizes in part the toxic effect of bile acids accumulated in the liver.     

Studies on the link between HMG-CoA reductase and cholesterol 7 alpha-hydroxylase in rat liver

Under most experimental conditions, there is a covariation between the rate-limiting enzyme in cholesterol biosynthesis, HMG-CoA reductase, and the rate-limiting enzyme in bile acid biosynthesis, cholesterol 7 alpha-hydroxylase. The most simple explanation for the coupling between the two enzymes is that newly synthesized cholesterol is a substrate for an unsaturated cholesterol 7 alpha-hydroxylase and that substrate availability is of major regulatory importance for this enzyme. The following results seem, however, to rule out that such a simple regulatory mechanism is of major importance and that HMG-CoA reductase activity per se is of importance in the regulation of cholesterol 7 alpha-hydroxylase. 1) The apparent degree of saturation of cholesterol 7 alpha-hydroxylase, as measured in vitro in rat liver microsomes, was found to be relatively high (70-90%) under most experimental conditions, including starvation, cholestyramine treatment, and cholesterol treatment. A significant decrease in the degree of saturation was obtained first after a drastic reduction of total concentration of cholesterol in the microsomes by treatment with high doses of triparanol, an inhibitor of cholesterol biosynthesis. 2) The stimulatory effect of cholesterol feeding on cholesterol 7 alpha-hydroxylase activity in rats seems to be an effect on the enzyme activity (enzyme induction?) rather than an effect on substrate availability. Thus, the stimulatory effect of cholesterol feeding was retained also after almost complete removal of the endogenous cholesterol by extraction with acetone.(ABSTRACT TRUNCATED AT 250 WORDS)     

Identification of 3 beta, 7 beta-dihydroxy-5 beta-cholan-24-oic acid in serum from patients treated with ursodeoxycholic acid

An unknown bile acid was found by gas-liquid chromatography in the serum of patients who were administered ursodeoxycholic acid for the treatment of cholesterol gallstones. Identification of the chemical structure of the unknown bile acid was performed by the use of gas-liquid chromatography-mass spectrometry. Mass spectrum analysis of the methyl ester trimethylsilyl ether of the bile acid showed explicitly that this is dihydroxy-5 beta-cholanoic acid, since peaks at m/e 460 and 370 characteristic of methyl ester trimethylsilyl ether of dihydroxy bile acid were clearly exhibited. Sites of the two hydroxyl groups on the steroid nucleus were determined to be at the 3- and 7-positions by conversion of the bile acid to the corresponding dioxo-cholanoic acid and by comparison of the gas-liquid chromatographic behavior with those of authentic dioxo bile acids. Four authentic 3,7-dihydroxy-5 beta-cholan-24-oic acids were chemically synthesized and retention times and mass spectra of their methyl ester trimethylsilyl ether derivatives compared precisely with that of the unknown bile acid. The results indicate that the unknown bile acid is 3 beta, 7 beta-dihydroxy-5 beta-cholan-24-oic acid. Preliminary experiments suggest that 3 beta, 7 beta-dihydroxy-5 beta-cholan-24-oic acid is absent as amino acid-conjugated forms in serum. It is also suggested that the bile acid is excreted into urine but not into bile.     

Bile acids of snakes of the subfamily Viperinae and the biosynthesis of C-23-hydroxylated bile acids in liver homogenate fractions from the adder, Vipera berus (Linn.).

1. Analysis of bile salts of four snakes of the subfamily Viperinae showed that their bile acids consisted mainly of C-23-hydroxylated bile acids. 2. Incubations of 14C-labelled sodium cholate (3 alpha, 7 alpha, 12 alpha-trihydroxy-5 beta-cholan-24-oate) and deoxycholate (3 alpha, 12 alpha-dihydroxy-5 beta-cholan-24-oate) with whole and fractionated adder liver homogenates were carried out in the presence of molecular oxygen and NADPH or an NADPH-generating system. The formation of C-23-hydroxylated bile acids, namely bitocholic acid (3 alpha, 12 alpha, 23xi-trihydroxy-5 beta-cholan-24-oic acid) and 3 alpha, 7 alpha, 12 alpha, 23 xi-tetrahydroxy-cholanic acid (3 alpha, 7 alpha, 12 alpha, 23 xi-tetrahydroxy-5 beta-cholan-24-oic acid), was observed mainly in the microsomal fraction and partly in the mitochondrial fraction. 3. Biosynthetic pathways of C-23-hydroxylated bile acids are discussed.     

Effect of long-term administration of estrogens on the subcellular distribution of cholesterol and the activity of rate-limiting enzymes of cholesterol biosynthesis and degradation in pigeon liver

To determine the mechanism of the hypocholesteremic effect of estrogens noted in pigeon, studies were done on the subcellular distribution of cholesterol and the activity of beta-hydroxy-beta-methylglutaryl CoA reductase (HMG-CoA reductase) and and cholesterol 7alpha-hydroxylase in pigeon liver after long-term (6 months) estrogen administration. Estrogens significantly reduced free cholesterol concentration in microsomes and mitochondrial fraction. The concentration of cholesteryl ester was reduced in the supernatant fraction. The activity of HMG-CoA reductase was significantly reduced in the estrogen-treated birds, while cholesterol 7alpha-hydroxylase activity showed no changes. Thus, the hypocholesteremic effect of estrogen noted in pigeons could be mainly due to the inhibition of cholesterol biosynthesis in the liver.     

Effects of ursodeoxycholic acid, analogues of ursodeoxycholic acid and combination of bile acids on bile acid synthesis in cultured rat hepatocytes

The effect of individual 7 beta-hydroxy bile acids (ursodeoxycholic and ursocholic acid), bile acid analogues of ursodeoxycholic acid, combination of bile acids (taurochenodeoxycholate and taurocholate), and mixtures of bile acids, phospholipids and cholesterol in proportions found in rat bile, on bile acids synthesis was studied in cultured rat hepatocytes. Individual steroids tested included ursodeoxycholate (UDCA), ursocholate (UCA), glycoursodeoxycholate (GUDCA) and tauroursodeoxycholate (TUDCA). Analogues of UDCA (7-methylursodeoxycholate, sarcosylursodeoxycholate and ursooxazoline) and allochenodeoxycholate, a representative of 5 alpha-cholanoic bile acid were also tested in order to determine the specificity of the bile acid biofeedback. Each individual steroid was added to the culture media at concentrations ranging from 10 to 200 microM. Mixtures of taurochenodeoxycholate (TDCA) and taurocholate in concentrations ranging from 150 to 600 microM alone and in combination with phosphatidylcholine (10-125 microM) and cholesterol (3-13 microM) were also tested for their effects on bile acid synthesis. Rates of bile acid synthesis were determined as the conversion of added lipoprotein [4-14C]cholesterol or [2-14C]mevalonate into 14C-labeled bile acids and by GLC quantitation of bile acids secreted into the culture media. Individual bile acids, bile acid analogues, combination of bile acids and mixture of bile acids with phosphatidylcholine and cholesterol failed to inhibit bile acid synthesis in cultured hepatocytes. The addition of UDCA or UCA to the culture medium resulted in a marked increase in the intracellular level of both bile acids, and in the case of UDCA there was a 4-fold increase in beta-muricholate. These results demonstrate effective uptake and metabolism of these bile acids by the rat hepatocytes. UDCA, UCA, TUDCA and GUDCA also failed to inhibit cholesterol-7 alpha-hydroxylase activity in microsomes prepared from cholestyramine-fed rats. The current data confirm and extend our previous observations that, under conditions employed, neither single bile acid nor a mixture of bile acids with or without phosphatidylcholine and cholesterol inhibits bile acid synthesis in primary rat hepatocyte cultures. We postulate that mechanisms other than a direct effect of bile acids on cholesterol-7 alpha-hydroxylase might play a role in the regulation of bile acid synthesis.     

Bile acids with a cyclopropyl-containing side chain. IV. Physicochemical and biological properties of the four diastereoisomers of 3 alpha,7 beta-dihydroxy-22,23-methylene-5 beta-cholan-24-oic acid

To define the influence of the side chain modification on physicochemical and biological properties of bile acids, 3 alpha,7 beta-dihydroxy-22,23-methylene-5 beta-cholan-24-oic acid, a cyclopropyl analog of ursodeoxycholic acid (UDCA) was synthesized in both unconjugated and taurine-conjugated form. The presence of a cyclopropyl ring at C-22, C-23 position introduces chirality generating four diasteroisomers (A, B, C, and D) which greatly differ for the hydrophilicity and critical micellar concentration: A and B are more hydrophilic (K' = 0.21, 0.80 and CMC = 25,20 mM, respectively) than UDCA (K' = 0.95; CMC = 19 mM) while C and D are more hydrophobic and with lower CMC (K' = 1.30, 2.05; CMC = 14, 10 mM, respectively) than UDCA. Differences in these properties are related to the orientation of the C-25 carboxyl which in isomers A and B is oriented toward the back of the steroid body, reducing the continuity of the hydrophobic area. Using the isolated perfused rat liver we found that the isomers inhibited [3H]UDCA uptake differently. Isomer D (noncompetitive) was the most potent (51%) while isomer A (competitive) was the least potent (15%). When infused intravenously to rats, only D isomer and UDCA were quantitatively recovered in bile. They were secreted predominantly as taurine and glycine conjugates. Isomers A, B, C are not conjugated and only partially recovered in bile as unconjugates (less than 15% of the administered dose). The increase in bile flow per unit increase in bile acid secretion induced by isomers A, B, and C, was much greater than that induced by isomer D which is similar to that of UDCA (0.32 +/- 0.04 and 0.22 +/- 0.01, respectively) while it is reduced during infusion of the other isomers. When infused as taurine conjugates they behaved similarly to tauroursodeoxycholic acid. When incubated in anaerobic conditions with human stools only isomer D is partially 7-dehydroxylated (t/2 = 18 hr) even though slower than UDCA (t/2 = 5 hr). The substrate specificity of the taurine conjugated toward cholyglycine hydrolase is very poor, only isomers C and D are partially deconjugated with a kinetics much slower than that of UDCA (10 hr vs. 0.2 hr). By using molecular models it is possible to explain these differences due to the conformation of the side chain that, in the case of isomer D, is quite similar to UDCA. These data are useful to explain the metabolism of dihydroxy bile acids and to design new analogs with enhanced cholelitholytic activity.     

Identification of bile acids in the serum and urine in cholestasis. Evidence for 6alpha-hydroxylation of bile acids in man.

In this qualitative study of the pattern of bile acid excretion in cholestasis, methods are described for the isolation of bile acids from large volumes of urine and plasma. The bile acids were subjected to a group separation and identified by combined gas chromatography-mass spectrometry. The techniques were developed to allow identification of the minor components of the bile acid mixture. Four bile acids that have not previously been described in human urine and plasma were detected, namely 3beta, 7alpha-dihydroxy-5beta-cholan-24-oic acid, 3alpha, 6alpha-dihydroxy-5beta-cholan-24-oic acid (hyodeoxycholic acid), 3alpha, 6alpha, 7alpha-trihydroxy-5beta-cholan-24-oic acid (hyocholic acid) and 3alpha, 7beta, 12alpha-trihydroxy-5beta-cholan-24-oic acid. In addition three C27 steroids were found; 26-hydroxycholesterol and a trihydroxy cholestane, probably 5 beta-cholestane-3alpha, 7alpha, 26-triol were found in the sulphate fraction of plasma and urine. In the plasma sample, a sulphate conjugate of 24-hydroxycholesterol was found. The presence of these compounds probably reflects the existence of further pathways for bile acid metabolism. It is not yet known whether this is a consequence of the cholestasis or whether they are also present in normal man, at much lower concentrations.     

24-hydroxycholesterol is a substrate for hepatic cholesterol 7alpha-hydroxylase (CYP7A)

(24S)-Hydroxycholesterol is formed from cholesterol in the brain and is important for cholesterol homeostasis in this organ. Elimination of (24S)-hydroxycholesterol has been suggested to occur in the liver but little is known about the metabolism of this oxysterol. In the present investigation, we report formation of 7alpha, 24-dihydroxycholesterol in pig and human liver. 7alpha-hydroxylase activity toward both isomers of 24-hydroxycholesterol [(24S) and (24R)] was found in a partially purified and reconstituted cholesterol 7alpha-hydroxylase (CYP7A) enzyme fraction from pig liver microsomes. In contrast, a purified enzyme fraction of pig liver oxysterol 7alpha-hydroxylase with high activity toward 27-hydroxycholesterol did not show any detectable activity toward 24-hydroxycholesterol. 7alpha-Hydroxylation of 24-hydroxycholesterol was strongly inhibited by 7-oxocholesterol, a known inhibitor of CYP7A. Human CYP7A, recombinantly expressed in Escherichia coli and in simian COS cells, showed 7alpha-hydroxylase activity toward both cholesterol and the two isomers of 24-hydroxycholesterol, with a preference for the (24S)-isomer. Our results show that 24-hydroxycholesterol is metabolized by CYP7A, an enzyme previously considered to be specific for cholesterol and cholestanol and not active toward oxysterols. Because CYP7A is the rate-limiting enzyme in the major pathway of bile acid biosynthesis, the possibility is discussed that at least part of the 24-hydroxycholesterol is converted into 7alpha-hydroxylated bile acids by the enzymes involved in the normal biosynthesis of bile acids.     

Studies on the regulation of cholesterol 7 alpha-hydroxylase and HMG-CoA reductase in rat liver: effects of lymphatic drainage and ligation of the lymph duct

Lymphatic drainage leads to a significant stimulation of both the cholesterol 7 alpha-hydroxylase and HMG-CoA reductase activity in rats (Bj?rkhem et al. 1978. Biochem. Biophys. Res. Commun. 85: (532-540). This finding was confirmed here and it was also shown that ligation of the lymph duct leads to a similar but less pronounced effect. Ligation of the lymph duct or lymph fistulation of bile duct-ligated or cholestyramine-treated rats did not further increase 7 alpha-hydroxylase or the HMG-CoA reductase activity. However, treatment of lymph fistula rats with cholestyramine led to a significant further stimulation of both 7 alpha-hydroxylase and HMG-CoA reductase activity. Intravenous infusion of lymph into bile fistula rats led to a significant inhibition of both cholesterol 7 alpha-hydroxylase activity and HMG-CoA reductase activity. A corresponding infusion of cholesterol-enriched Intralipid led to inhibition of HMG-CoA reductase without effect on cholesterol 7 alpha-hydroxylase activity. The results show that cholesterol 7 alpha-hydroxylase is feedback-regulated by bile acids in a situation where the flux of cholesterol to the liver is interrupted also. The possibility is discussed that there is a factor in the lymph that down-regulates cholesterol 7 alpha-hydroxylase. If such a factor exists, it requires an intact enterohepatic circulation for its effect. The stimulatory effect of cholestyramine on HMG-CoA reductase also in lymph fistula rats shows that the previously demonstrated suppressive effect of bile acids on HMG-CoA reductase is not only due to the effect of bile acids on intestinal absorption of cholesterol.(ABSTRACT TRUNCATED AT 250 WORDS)     

Classical bile acids in animals, beta-phocaecholic acid in ducks

1. Bile samples of different animals were analysed and the percentage content of classical bile acids was determined. 2. Herbivorous birds mostly excreted a large proportion of chenodeoxycholic acid. 3. The anteater (Myrmecophaga tridactyla) excreted deoxycholic acid most probably as a primary bile acid. 4. In the bile of ducks (Anas platyrhynchos) a large amount of (23R)3 alpha, 7 alpha, 23-trihydroxy-5 beta-cholan-24-oic acid (beta-phocaecholic acid) was found.