The bile acid composition of gastric contents from neonates with high intestinal obstruction.

The study was designed to identify 'atypical' bile acids in gastric contents from three neonates with high intestinal obstruction on the basis that this was likely to represent a rich source of primary bile acids. Cholic acid was the major component, and related 'atypical' bile acids included its C-3 and C-7 oxidation products, its 3 beta-epimer and 2 beta- and 6 alpha-hydroxylation products. Allocholic acid was the only 5 alpha-cholanic acid derivative identified. 7 alpha, 12 alpha-Dihydroxy-3-oxochol-4-en-24-oic acid was found in all three specimens and might be an intermediate in a biosynthetic pathway from cholesterol to cholic acid in which side-chain oxidation precedes at least some of the nuclear changes. Side-chain-hydroxylated derivatives of trihydroxycoprostanic acid were also detected and these may represent intermediates in biosynthetic pathways from cholesterol to cholic acid via 5 beta-cholestan-3 alpha, 7 alpha, 12 alpha-triol. The most abundant bile acid of this type was (25 epsilon)-3 alpha, 7 alpha, 12 alpha, 25-tetrahydroxy-5 beta-cholestan-26-oic acid, which suggested that C-25 hydroxylation may be an important step in the shortening of the C8 side chain of the cholestane triol to the C5 side chain of cholic acid in the neonatal period. Bile acids lacking a substituent at C-12 included chenodeoxycholic acid, its C-3 and C-7 oxidation products, its 3 beta-epimer and its 6 alpha-hydroxylation product (hyocholic acid).     

Metabolism of bile alcohols in the perfused rabbit liver.

The mechanism and sequence of side chain hydroxylation of cholesterol in bile acid synthesis was studied in the isolated perfused rabbit liver. A comparison was made between the importance of 26- and 25-hydroxylation in cholic acid biosynthesis in the rabbit. The formation of [G-3H]cholic acid was observed when the liver was perfused with 5beta-[G-3H]cholestane-3alpha, 7alpha-diol, 5beta-[G-3H]cholestane-3alpha, 7alpha-12alpha-triol, and 5beta-[G-3H]cholestane-3alpha, 7alpha, 26-triol. No [G-3H]chenodeoxycholic acid was detected in the bile. These findings indicate that potential precursors of chenodeoxycholic acid were hydroxylated at position 12alpha either subsequent to or before hydroxylation of the cholesterol side chain. In addition, no other intermediates (tetrahydroxy or pentahydroxy bile alcohols) were found in the bile when these compounds were perfused in the liver. Bile acid precursors were detected in bile when the rabbit liver was perfused with 5beta-[24-14C]cholestane-3alpha, 7alpha, 25-triol. The 5beta-[24-14C]cholestane-3alpha, 7alpha, 25-triol was hydroxylated in the liver at the 12alpha position to yield the corresponding 5beta-cholestane-3alpha, 7alpha, 12alpha, 25-tetrol. The tetrol was further metabolized to a series of pentols (5beta-cholestane-3alpha, 7alpha, 12alpha, 22, 25-pentol; 5beta-cholestane-3alpha, 7alpha, 12alpha, 23, 25-pentol; 5beta-cholestane-3alpha, 7alpha, 12alpha, 24, 25-pentol; and 5beta-cholestane-3alpha, 7alpha, 12alpha, 25, 26-pentol). The major bile acid obtained from the perfusion of the 5beta-cholestane-3alpha, 7alpha, 25-triol was cholic acid. The experiments indicated that in the rabbit liver 12alpha-hydroxylation can occur after hydroxylation of the cholesterol side chain at either C-25 (5 beta-cholestane-3alpha, 7alpha, 25-triol) or C-26 (5beta-cholestane-3alpha, 7alpha-26-triol). Apparently, the rabbit can form cholic acid via the classical 26-hydroxylation pathway as well as via 25-hydroxylated intermediates.     

Chemical synthesis of 3β-sulfooxy-7β-hydroxy-24-nor-5-cholenoic acid: An internal standard for mass spectrometric analysis of the abnormal Δ-bile acids occurring in Niemann-Pick disease

In Niemann-Pick disease, type C1, increased amounts of 3β,7β-dihydroxy-5-cholenoic acid are reported to be present in urinary bile acids. The compound occurs as a tri-conjugate, sulfated at C-3, N-acetylglucosamidated at C-7, and N-acylamidated with taurine or glycine at C-24. For sensitive LC-MS/MS analysis of this bile acid, a suitable internal standard is needed. We report here the synthesis of a satisfactory internal standard, 3β-sulfooxy-7β-hydroxy-24-nor-5-cholenoic acid (as the disodium salt). The key reactions involved were (1) the so-called “second order” Beckmann rearrangement (one-carbon degradation at C-24) of hyodeoxycholic acid (HDCA) 3,6-diformate with sodium nitrite in a mixture of trifluoroacetic anhydride and trifluoroacetic acid, (2) simultaneous inversion at C-3 and elimination at C-6 of the ditosylate derivatives of the resulting 3α,6α-dihydroxy-24-nor-5β-cholanoic acid with potassium acetate in aqueous N,N-dimethylformamide, and (3) regioselective sulfation at C-3 of an intermediary 3β,7β-dihydroxy-24-nor-Δ⁵ derivative using sulfur trioxide-trimethylamine complex. Overall yield of the desired compound was 1.8% in 12 steps from HDCA.     

Comparison of biliary excretion and metabolism of lithocholic acid and its sulfate and glucuronide conjugates in rats

Biliary excretion and biotransformation of tracer doses of [14C]lithocholic acid and its sulfate and glucuronide intravenously injected into bile-drainaged rats were compared. Biliary excretion efficiency was in the order of unconjugate sulfate glucuronide and all conjugates were completely excreted into bile within 60 min after injection. Only tracer doses of radioactivity were found in the liver and urine. About 90% of radiolabeled bile acids in bile were conjugated with taurine immediately after injection of lithocholic acid, whereas lithocholic acid-glucuronide was only partly conjugated with taurine all the time (less than 6%) and excreted into bile mainly as native compound. In the first 10 min, 66% of lithocholic acid-sulfate was conjugated with taurine and it gradually proceeded up to 87%. Hydroxylation at C-6 and C-7 positions of lithocholic acid proceeded time-dependently up to 45%. No hydroxylation was observed with lithocholic acid-sulfate or glucuronide. Differences of biliary excretion rate of these conjugates may be one of the reasons for the delayed decrease of sulfated and glucuronidated bile acids in serum after bile drainage to patients with obstructive jaundice of during the recovery of acute hepatitis than non-esterified bile acids.     

Preferential utilization of newly synthesized cholesterol as substrate for bile acid biosynthesis. An in vivo study using 18O2-inhalation technique.

Incorporation of 18O in cholic anc chenodeoxycholic acid was determined after inhalation of 18O2 by rats with biliary fistula. After a 30-min inhalation, the maximal incorporation of 18O in the three hydroxyl groups of cholic acid was about 1.8 atoms, and in the two hydroxyl groups of chenodeoxycholic acid about 1.1 atoms. About 0.4 atom of 18O in the cholic and chenodeoxycholic acid isolated was present at C-3. It was calculated that at least 50% of the biosynthesized bile acids were derived from newly synthesized cholesterol. The time course for the incorporation of 18O at C-3 of chenodeoxycholic acid was slightly different from that of cholic acid, indicating that a small part of chenodeoxycholic acid might have been synthesized from a pool of cholesterol different from that utilized in the biosynthesis of cholic acid. Incorporation of 18O in biliary cholesterol was less than 0.05 atom, indicating that the major part of this cholesterol is derived from a pool different from that utilized in bile acid biosynthesis.     

Specificity of bile salt sulfatase activity from Clostridium sp. strains S1.

Clostridium sp. strain S1, an unnamed bile acid-desulfating strain from rat intestinal microflora (S.M. Huijghebaert, J. A. Mertens, and H. J. Eyssen, Appl. Environ. Microbiol. 43:185-192, 1982), was examined for its ability to desulfate different bile acid sulfates and steroid sulfates in growing cultures. Clostridium sp. strain S1 desulfated the 3 alpha-monosulfates of chenodeoxycholic, deoxycholic, and cholic acid, but not their 7 alpha- or 12 alpha-monosulfates. Among the 3-sulfates of the 5 alpha- and 5 beta-bile acids, only bile acid-3-sulfates with an equatorial sulfate group were desulfated. Hence, Clostridium sp. strain S1 desulfated the 3-sulfates of bile acids with a 3 alpha, 5 beta-, a 3 beta, 5 alpha- or a 3 beta, delta 5-structure. In contrast, the bile acid-3-sulfates with a 3 beta, 5 beta- or a 3 alpha, 5 alpha-structure were not desulfated. In addition, Clostridium sp. strain S1 did not hydrolyze the equatorial 3-sulfate esters of C19 and C21 steroids and cholesterol or the phenolic 3-sulfate esters of estrone and estradiol. 23-Nordeoxycholic acid with a C-23 carboxyl group was also not desulfated, in contrast to the 5 beta-bile acid 3 alpha-sulfates with a C-24 or C-26 carboxyl group. Therefore, the specificity of the sulfatase of Clostridium sp. strain S1 is related to the location of the sulfate group on the bile acid molecule, the equatorial orientation of the sulfate group, and the structure of the C-17 side chain, its carboxyl group, and chain length.     

Role of hydrophilic bile acids and of sterols on cholelithiasis in the hamster

The effect of various dietary additions such as cholesterol, beta-sitosterol, bile acids, and bile acid analogs on gallstone formation was studied in the hamster. Gallstones were formed in 50% of the animals fed a high glucose, fat-free diet. Administration of 0.2% cholesterol or 1% beta-sitosterol had no effect on the incidence of gallstones. Ursodeoxycholic acid (0.5%) and its analog ursodeoxy-oxazoline [2-(3 alpha, 7 beta-dihydroxy-24-nor-5 beta-cholanyl)-4,4-dimethyl-2- oxazoline] were ineffective in preventing gallstones. Hyodeoxycholic acid and hyodeoxy-oxazoline [2-(3 alpha,6 alpha-dihydroxy-24-nor-5 beta-cholanyl)-4,4-dimethyl-2- oxazoline] at the same dosage effectively prevented gallstones, while the trihydroxy bile acid, hyocholic acid, was not effective. Of all the dietary regimens tested, only hyodeoxycholic acid significantly lowered serum cholesterol. The lithogenic diet produced a five-fold increase in hepatic HMG-CoA reductase activity; this activity was not affected by dietary cholesterol or beta-sitosterol. Hyodeoxycholic acid and hyocholic acid feeding increased the reductase activity by an additional 50% while the other bile acids had no effect. beta-Sitosterol doubled the cholesterol 7 alpha-hydroxylase activity whereas hyodeoxy-oxazoline lowered it. Hyodeoxycholic acid-fed animals had significantly lower cholesterol absorption than the animals on the lithogenic diet alone. Biliary cholesterol content increased dramatically in the animals fed the lithogenic diet and was increased still further by ursodeoxycholic acid, hyodeoxycholic acid, and hyodeoxy-oxazoline. These data show that hyodeoxycholic acid and hyodeoxy-oxazoline do not prevent gallstones by inhibiting hepatic cholesterol synthesis or biliary cholesterol secretion.     

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.     

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.     

Incorporation of the 1-pro-R and the 1-pro-S hydrogen atoms of ethanol into steroids and phosphatidylcholines in vivo.

The transfer of deuterium from chiral 1-monodeuteroethanols to various metabolites formed in the liver was studied in order to investigate the coupling of metabolic reductions to the alcohol dehydrogenase and the aldehyde dehydrogenase reactions. The ethanols were administered to female bile fistula rats for 10 h. The hydrogen at C-2 in the glycerol moiety of newly formed phosphatidylcholine molecules in bile, liver and plasma was derived to 22-25% from the 1-pro-R position and to 5-6% from the 1-pro-S position in the ethanol. sn-Glycerol 3-phosphate isolated from liver had a lower deuterium content at C-2. The ratio between the contributions from the two positions in ethanol to C-2 of free sn-glycerol 3-phosphate was the same as in the phosphatidylcholines. This indicates that the higher degree of labelling of this position in phosphatidylcholines is not due to a specific coupling between alcohol dehydrogenase and the formation of a phosphatidylcholine precursor. Cholesterol and chenodeoxycholic acid in bile became increasingly labelled, and the ratio between the incorporations from the 1-pro-S and the 1-pro-R positions of ethanol was about 0.37 in cholesterol and 0.46 in chenodeoxycholic acid. Thus, these NADPH-dependent reactions utilized hydrogen from the 1-pro-S position to a larger extent than NADH-dependent reactions.     

Thyroid hormone differentially augments biliary sterol secretion in the rat. II. The chronic bile fistula model.

To further define thyroid hormone effects on bile acid synthesis and biliary lipid secretion, studies were done in chronic bile fistula rats. Euthyroid and methimazole-hypothyroid rats, with and without triiodothyronine (T3) injection, had total bile diversion for timed bile collections. With interrupted enterohepatic circulation, cholesterol absorption is negligible and bile acid secretion equals bile acid synthesis rate. Hypothyroid rats had diminished levels of bile acid synthesis and biliary secretion of cholesterol and phospholipid. Single dose T3 injection produced a 13-fold increase in bile cholesterol secretion and a 3-fold increase in phospholipid secretion, both initiated 12 h after T3. Bile acid synthesis increased by 50%, but the increase did not begin until 24 h after T3. Neither hypothyroidism nor T3 treatment abolished diurnal rhythms of bile acid synthesis and biliary lipid secretion. Inhibition of cholesterol synthesis with lovastatin resulted in a persistent 33% decrease in bile acid synthesis in euthyroid and hypothyroid rats, while bile cholesterol secretion only transiently decreased. Inhibition of cholesterol synthesis did not alter T3-induced bile cholesterol secretion, with a 10-fold increase seen. However, bile acid synthesis was not stimulated by T3 in the presence of lovastatin. We conclude that facilitated bile acid synthesis and biliary cholesterol secretion are early effects of T3 and may account for the hypocholesterolemia of T3. Cholesterol synthesis does not appear to be required for the T3-induced bile cholesterol secretion.     

Proton magnetic resonance assay of total and taurine-conjugated bile acids in bile.

Biliary bile acids, coexisting with phospholipid and cholesterol, are partly conjugated with taurine. In the present report we show that total and taurine-conjugated bile acids in bile can be simultaneously and quantitatively measured by high-resolution (1)H-nuclear magnetic resonance ((1)H-NMR) spectroscopy. We used a 7.05-Tesla NMR spectrometer to obtain the (1)H-NMR spectra of model and biological biles. Only addition of trimethylsilyl-3-propionic acid sodium salt-D(4) (TSP) to each sample as an internal standard was required in preparation for (1)H-NMR measurement. In (1)H-NMR spectra of rat bile, peaks of C-18 methyl protons of bile acids and of C-25 methylene protons on the taurine moiety of taurine-conjugated bile acids were detected at 0.7 ppm and 3.1 ppm, respectively. Peak areas, of C-18 and C-25 peaks, increased in proportion to the concentrations of bile acids or taurine-conjugated bile acids, even in the presence of phospholipid and cholesterol. The accuracy of NMR measurement of total and taurine-conjugated bile acids was confirmed by comparing the results of NMR with those of enzyme-fluorimetry.The results clearly demonstrate that (1)H-NMR spectroscopy can be applied to the quantitative determination of total and taurine-conjugated bile acids in bile without troublesome preparative steps.     

Formation of novel C21-bile acids from cholesterol in the rat. Structure identification of the major Di- and trihydroxylated species

We recently showed that previously unknown di- and trihydroxylated C21-bile acids are major degradation products of sitosterol and campesterol in bile-fistulated female Wistar rats. Using a mixture of 4-14C- and 22-3H-labeled cholesterol it was shown that such C21-bile acids are formed also from cholesterol in amounts up to about 25% of the total formation of bile acids. The C21-bile acids were formed from labeled cholesterol also in perfused rat liver, demonstrating that the liver is the site of synthesis. The major trihydroxylated C21-bile acids in bile were identified, by means of mass spectrometry, NMR, stereospecific dehydrogenases, and reagents, as 5 beta-pregnan-3 alpha, 11 beta, 15 beta-triol-21-oic acid and 5 beta-pregnan-3 alpha, 11 beta, 15 alpha-triol-21-oic acid. The corresponding 11-oxo-isomers were also present. A minor trihydroxylated C21-bile acid was identified as 5 beta-pregnan-3 alpha, 11 beta, 16-triol-21-oic acid. The major dihydroxylated C21-bile acid was identified by the same means as 5 alpha-pregnan-3 alpha, 12 alpha-diol-21-oic acid. Male rats converted 4-14C-cholesterol into C21-bile acids less efficiently than did female rats. None of the C21-bile acids from male rats contained a 15-hydroxyl group. It is speculated that the novel C21-bile acids are formed both from cholesterol and from plant sterols by an initial hydroxylation at C21 followed by peroxisomal or mitochondrial beta-oxidation. The presence of a hydroxyl group at C15 may facilitate this reaction. The above formation of C21-bile acids shows that mammalian liver is able to degrade the side chain of cholesterol beyond the C24 stage, even in the absence of a blocking group at C24. C21-bile acids, or one of their precursors, are hydroxylated in the liver by a hitherto unknown 11 beta-hydroxylase. The possible physiological importance of the C21-bile acids is discussed.     

Chemical synthesis of the 3-sulfooxy-7-N-acetylglucosaminyl-24-amidated conjugates of 3beta,7beta-dihydroxy-5-cholen-24-oic acid, and related compounds: unusual, major metabolites of bile acid in a patient with Niemann-Pick disease type C1

The chemical synthesis of 3beta,7beta-dihydroxy-5-cholen-24-oic acid, triply conjugated by sulfuric acid at C-3, by N-acetylglucosamine (GlcNAc) at C-7, and by glycine or taurine at C-24, is described. These are unusual, major metabolites of bile acid found to be excreted in the urine of a patient with Niemann-Pick disease type C1. Analogous double-conjugates of 3beta-hydroxy-7-oxo-5-cholen-24-oic acid were also prepared. The principal reactions involved were: (1) beta-d-N-acetylglucosaminidation at C-7 of methyl 3beta-tert-butyldimethylsilyloxy (TBDMSi)-7beta-hydroxy-5-cholen-24-oate with 2-acetamido-1alpha-chloro-1,2-dideoxy-3,4,6-tri-O-acetyl-d-glucopyranose in the presence of CdCO(3) in boiling toluene; (2) sulfation at C-3 of the resulting 3beta-TBDMSi-7beta-GlcNAc with sulfur trioxide-trimethylamine complex in pyridine; and (3) direct amidation at C-24 of the 3beta-sulfooxy-7beta-GlcNAc conjugate with glycine methyl ester hydrochloride (or taurine) using 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride as a coupling agent in DMF. The structures of the multi-conjugated bile acids were characterized by liquid chromatography-mass spectrometry with an electrospray ionization probe under the positive and negative ionization modes.     

Suitability of primary monolayer cultures of adult rat hepatocytes for studies of cholesterol and bile acid metabolism

Monolayer cultures of hepatocytes isolated from cholestyramine-fed rats and incubated in serum-free medium converted exogenous [4-14C]cholesterol into bile acids at a 3-fold greater rate than did cultures of hepatocytes prepared from untreated rats. Cholic acid and beta-muricholic acid identified and quantitated by gas-liquid chromatography and thin-layer chromatography were synthesized by cultured cells for at least 96 h following plating. The calculated synthesis rate of total bile acids by hepatocytes prepared from cholestyramine-fed animals was approximately 0.058 micrograms/mg protein/h. beta-Muricholic acid was synthesized at approximately a 3-fold greater rate than cholic acid in these cultures. Cultured hepatocytes rapidly converted the following intermediates of the bile acid pathway; 7 alpha-hydroxy[7 beta-3H]cholesterol, 7 alpha-hydroxy-4-[6 beta-3H] cholesten-3-one, and 5 beta-[7 beta-3H]cholestane-3 alpha, 7 alpha, 12 alpha-triol into bile acids. [24-14C]Chenodeoxycholic acid and [3H]ursodeoxycholic acid were rapidly biotransformed to beta-muricholic acid. 3-Hydroxy-3-methylglutaryl-coenzyme A reductase activity measured in microsomes of cultured hepatocytes decreased during the initial 48 h following plating, but remained relatively constant for the next 72 h. In contrast, cholesterol 7 alpha-hydroxylase activity appeared to decrease during the first 48 h, followed by an increase over the next 48 h. Despite the apparent changes in enzyme activity in vitro, the rate of bile acid synthesis by whole cells during this time period remained constant. It is concluded that primary monolayer cultures of rat hepatocytes can serve as a useful model for studying the interrelationship between cholesterol and bile acid metabolism.     

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.     

Effect of biliary obstruction on formation and metabolism of bile acids in rat

The effect of biliary obstruction in the rat on several hydroxylations involved in the formation and metabolism of bile acids was studied. The hydroxylations studied were all catalyzed by the microsomal fraction of liver homogenate fortified with NADPH. The rate of 7α-hydroxylation of cholesterol increased two- to threefold between 24 and 48 hours after ligation of the bile duct and remained at this level the next 48 hours. During the first 24 hours of obstruction the rates of 1 2α-hydroxylation of 7α-hydroxy-4-cholesten-3-one and 7α-hydroxylation of taurodeoxycholic acid decreased but returned to control levels between 24 and 48 hours after operation. The rate of 6β-hydroxylation of lithocholic acid and taurochenodeoxycholic acid increased gradually and reached a plateau between 24 and 48 hours at which time the rate was two to three times faster than in the controls. The increase in 6β-hydroxylase activity was reflected in the pattern of the bile acids excreted in urine. After 48 hours of obstruction β-muricholic acid accounted for 50% or more of the bile acids in urine.     

Incorporation of [2-14C,(5R)-5-3H1]mevalonic acid into cholesterol by a rat liver homogenate and into β-sitosterol and 28-isofucosterol by Larix decidua leaves

1. Incubation of a rat liver homogenate with 3R-[2-(14)C,(5R)-5-(3)H(1)]mevalonic acid gave cholesterol with (3)H/(14)C atomic ratio 6:5. 2. Conversion of the labelled cholesterol into 3beta-acetoxy-6-nitrocholest-5-ene or cholest-4-ene-3,6-dione resulted in the loss of one tritium atom from C-6. 3. These results show that during cholesterol biosynthesis the 6alpha-hydrogen atom of a precursor sterol is eliminated during formation of the C-5-C-6 double bond. 4. Incorporation of 3R-[2-(14)C,(5R)-5-(3)H(1)]mevalonic acid into the sterols of larch (Larix decidua) leaves gave labelled cycloartenol and beta-sitosterol with (3)H/(14)C atomic ratios 6:6 and 6:5 respectively. 5. One tritium atom was lost from C-6 on conversion of the labelled beta-sitosterol into either 3beta-acetoxy-6-nitrostigmast-5-ene or stigmast-4-ene-3,6-dione, demonstrating that formation of the C-5-C-6 double bond of phytosterols also involves the elimination of the 6alpha-hydrogen atom of a precursor sterol. 6. The 3R-[2-(14)C,(5R)-5-(3)H(1)]mevalonic acid was also incorporated by larch (L. decidua) leaves into a sterol that co-chromatographed with 28-isofucosterol. Confirmation that the radioactivity was associated with 28-isofucosterol was obtained by co-crystallization with carrier 28-isofucosterol and ozonolysis of the acetate to give radioactively labelled 24-oxocholesteryl acetate. 7. The significance of these results to phytosterol biosynthesis is discussed.     

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

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

Evidence for a common biliary cholesterol and VLDL cholesterol precursor pool in rat liver.

Hepatic free cholesterol levels are influenced by cholesterol synthesis and ester formation, which, in turn, might regulate cholesterol secretion into bile and plasma. We manipulated the rates of hepatic cholesterol synthesis and esterification and measured biliary and very low density lipoprotein (VLDL) cholesterol secretion, and bile acid synthesis. Mevalonate decreased HMG CoA reductase by 80%, increased acyl coenzyme A: cholesterol acyltransferase (ACAT) by 60% and increased [3H]oleate incorporation into microsomal and VLDL cholesteryl esters by 174% and 122%, respectively. Microsomal and biliary free cholesterol remained constant at the expense of increased microsomal and VLDL cholesteryl ester content. Mevalonate did not change bile acid synthesis. 25-OH cholesterol decreased HMG-CoA reductase by 39%, increased ACAT by 24%, but did not effect 7 alpha-hydroxylase. 25-OH cholesterol increased [3H]oleate in microsomal and VLDL cholesterol esters by 71% and 120%. Biliary cholesterol decreased by 40% and VLDL cholesteryl esters increased by 83%. A small and unsustained decrease in bile acid synthesis (14CO2 release) occurred after 25-OH cholesterol. After orotic acid feeding, HMG-CoA reductase increased 352%, and [3H]oleate in microsomal and VLDL cholesteryl esters decreased by 43% and 89%. Orotic acid decreased all VLDL components including free cholesterol (68%) and cholesteryl esters (55%), and increased biliary cholesterol by 160%. No change in bile acid synthesis occurred. Hepatic cholesterol synthesis and esterification appear to regulate a cholesterol pool available for both biliary and VLDL secretion. Changing cholesterol synthesis and esterification did not alter bile acid synthesis, suggesting that either this common bile/VLDL secretory pool is functionally distinct from the cholesterol pool used for bile salt synthesis, or that free cholesterol availability in this precursor pool is not a major determinant of bile acid synthesis.