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Male conventional rats have been treated for five days with benzylpenicillin, neomycin, kanamycin, erythromycin, bacitracintneomycin, succihylsulfathiazole or metronidazole. Total fecal bile acids were analyzed in samples collected during periods of three days during the pretreatment period and during the eight weeks following drug treatment. Metronidazole or succinylsulfathiazole had no or minor effects on the conventional bile acid pattern and the "bile acid index" (ratio beta-muricholic acid/deoxycholic acid) remained low. Benzylpenicillin, neomycin or kanamycin induced a germfree bile acid pattern, i.e. increased the relative amounts of alpha-and beta-muricholic acid in feces and eliminated deoxycholic acid and hyodeoxycholic acid from feces. The high bile acid index was normalized within three weeks after termination of drug treatment but the excretion of alpha- and beta-muricholic acid was not normalized until a normal flora had been established by giving an enema with intestinal contents from intact, oncentional rats. Treatment with eythromycin or bacitracintineomycin also produced a germfree bile acid pattern. In these cases, the bile acid index was not back to normal until after five to eight weeks and the excretion of the muricholic acids was not normalized until an enema with intestinal bacteria had been given. It is suggested that these long-lasting effects of antibiotics on the metabolism of bile acids in the intestinal tract should be considered after short-term antibiotic therapy in humans.

The present study was directed toward providing additional information in man on the nature of a potential alternative pathway to cholic acid not involving an initial 7 alpha-hydroxylation of cholesterol. Two bile fistula patients and one normal subject each received 25-hydroxy[G-3H]cholesterol; [14C]cholic and [14C]chenodeoxycholic acids were also simultaneously administered to one bile fistula patient and normal subject. The labeled 25-hydroxycholesterol was found to be poorly converted to primary bile acids by all three patients; the range of conversion was 9.7 to 18.9%. Cholic acid was favored over chenodeoxycholic acid by a margin of about 1.4/1. It is concluded that a pathway to primary bile acid via the 25-hydroxylation of cholesterol is of minor importance under conditions of normal or accelerated synthesis in man.

Several hydroxylase activities in bile acid biosynthesis were assayed in subcellular fractions of human fetal liver. The livers were obtained at legal abortions performed between gestational weeks 14 and 24. Microsomal 12 alpha-hydroxylase and mitochondrial 12 alpha- and 26-hydroxylase activities were detected from week 14. The microsomal fraction also had capacity for 25-hydroxylation, whereas 7 alpha- and 26-hydroxylase activities were hardly detectable. The variation of the hydroxylase activities between different experiments can be explained by inactivation during the abortion or workup procedure. The results are discussed with respect to earlier studies of bile acid biosynthesis during development and adult life.

The present investigation has aimed at defining the factor responsible for the differences in microsomal metabolism of steroids between germ-free and conventional rats. Cholesterol, cholic acid, taurocholic acid, and chenodeoxycholic acid were fed to conventional and germ-free male rats and the effects on liver microsomal metabolism of 4-[4-14C]androstene-3,17-dione, 5alpha-[4-14C]androstane-3alpha,17beta-diol, [4-14C]-cholesterol, 7alpha-hydroxy-4-[6beta-3H]cholesten-3-one, and [24-14C]lithocholic acid were studied. The most consistent effects were found with dietary cholesterol that stimulated the activities of several of the hydroxylases active on 4-androstene-3,17-dione and 5alpha-androstane-3alpha,17beta-diol and that decreased the 5alpha reduction of 4-androstene-3,17-dione, increased the 7alpha hydroxylation of cholesterol, decreased the 12alpha hydroxylation of 7alpha-hydroxy-4-cholesten-3-one, and increased by 6beta hydroxylation of lithocholic acid. These effects of cholesterol feeding on the microsomal metabolism of steroids in conventional rats made the pattern of microsomal enzyme activities resemble that characteristic of germ-free rats. Cholesterol feeding led to a pronounced increase in the intestinal concentration of beta-muricholic acid in conventional rats. Furthermore, cholesterol feeding to conventional animals led to an intestinal ratio of chenodeoxycholic acid (including its metabolites alpha- and beta-muricholic acid and hyodeoxycholic acid) to cholic acid (including deoxycholic acid) that was almost identical to that in germ-free rats. Conventionalization of germ-free rats for a period of up to 56 days led only to a partial normalization of the liver microsomal metabolism of 5alpha-[4-14C]androstane-3alpha, 17beta-diol and 7alpha-hydroxy-4-[6beta-3H]cholesten-3-one and of the liver microsomal concentration of cytochrome P-450. The concentration of cholesterol was higher in both total liver homogenate and liver microsomal fraction of germ-free rats than in corresponding preparations from conventional rats. In conclusion, it is suggested that cholesterol is one of the factors responsible for the different microsomal metabolism of steroids in germ-free and conventional rats. It is also suggested that cholesterol may play a role as regulator of microsomal enzyme activities.

Metabolic pathways involved in the conversion of cholesterol to cholic and chenodeoxycholic acids have been investigated in bile fistula patients treated with a number of labeled potential bile acid intermediates. The findings of the present report indicate that the human liver cell has the capacity to synthesize both primary bile acids via multiple routes from cholesterol and 7 alpha-hydroxycholesterol. Evidence has been obtained for the existence of a major pathway to chenodeoxycholic acid via the 26-hydroxylation of 7 alpha-hydroxycholest-4-en-3-one. Cholic acid is synthesized preferentially via pathways from 5 beta-cholestane 3 alpha, 7 alpha-diol and a pathway from cholesterol not involving an initial 7 alpha-hydroxylation.

Hydroxylation of 5 beta-[7 beta-3H]cholestane-3 alpha, 7 alpha-diol was studied in mitochondrial preparations from human fetal livers. The livers were obtained at legal abortions between weeks 14 and 24. In addition to hydroxylation in the 26-position, 5 beta-cholestane-3 alpha, 7 alpha-diol was hydroxylated in the 12 alpha-position. In one experiment, mitochondrial protein was solubilized and partially purified. Material with such chromatographic properties as those of cytochrome P450 showed 12 alpha-hydroxylase activity when combined with adrenodoxin and adrenodoxin reductase from bovine adrenal mitochondria. Because adrenodoxin and adrenodoxin reductase are components specific for mitochondrial hydroxylase systems, the results exclude microsomal contamination as the origin of this 12 alpha-hydroxylase activity. Further, there was no hydroxylase activity when NADPH-cytochrome P450 reductase from rat liver microsomes was added instead of adrenodoxin and adrenodoxin reductase. The microsomal fraction of fetal liver was also shown to possess 12 alpha-hydroxylase activity. Microsomal and mitochondrial hydroxylase activities per milligram of protein towards 5 beta-cholestane-3 alpha, 7 alpha-diol were of the same order of magnitude. The occurrence of an efficient sterol nucleus hydroxylase activity in liver mitochondria appears to be unique for fetal liver.

The present report has been directed toward providing additional information on the major defects in the bile acid pathways present in patients with cirrhosis and its relevance to the problem of how bile acid synthesis is regulated in man. An unusual patient with severe liver disease and a completely broken enterohepatic circuit was studied. The synthesis of cholic and chenodeoxycholic acids was examined over a 5-d period. The secretion rats and the incorporation of [3H]7 alpha-hydroxycholesterol and [3H]26-hydroxycholesterol into both primary bile acids in the cirrhotic bile fistula patient was cross compared to earlier data obtained on patients with and without liver disease and an intact enterohepatic circuit and patients with no liver disease and a bile fistula. The daily synthesis rate of cholic acid increased 7-fold and chenodeoxycholic acid 2-fold in the cirrhotic bile fistula patient. The incorporation of [3H]7 alpha- hydroxycholesterol into bile acids in the cirrhotic bile fistula patient was efficient (75%) and equal to bile fistula patients with no cirrhosis (76%); chenodeoxycholic acid synthesis was favored over cholic acid particularly in the cirrhotic patient. [3H]26-hydroxycholesterol was poorly incorporated in patients with no cirrhosis (25%) and the cirrhotic patient (20%); chenodeoxycholic acid was favored by a wide margin. It is concluded from this and previous reports that the profound reduction in bile acid synthesis present in patients with cirrhosis is not caused singly by a failure in the metabolic pathways from 7 alpha-hydroxycholesterol to cholic and chenodeoxycholic acid (i.e., 12 alpha-hydroxylation step), but rather due to a defect in the feedback control regulating bile acid synthesis.

In vivo studies were carried out on three bile fistula patients to further elucidate the side chain oxidation pathways from C-27 bile acids to cholic acid in man. Two patients each received (25-R)- and (25-S)-3 alpha, 7 alpha,-12 alpha-trihydroxy-5 beta-[7 beta-3H]cholestanoic acid (THCA) on consecutive days and three patients wee administered 3 alpha, 7 alpha, 12 alhpa, 24 xi-tetrahydroxy-5 beta-[7 beta-3H]cholestanoic acid (varanic acid). The varanic acid was biosynthetically prepared with rat liver microsomes and was probably the 24 alpha isomer. The patients efficiently (84 to 97%) converted both (R)- and (S)-THCA to cholic acid. There was no apparent significant difference in the ability of either (R)- or (S)-THCA to form cholic acid. Varanic acid was poorly converted (20 to 27%) to cholic acid in all three patients. From 49 to 75% of the administered 3H activity was recovered in the bile as other labeled products. The bulk (30 to 35%) of this 3H activity was identified by thin layer chromatography as varanic acid. The rate of conversion of (R)-THCA, (S)-THCA, and varanic acid was extremely rapid in all three patients with a t 1/2 of 35 to 74 min. The findings suggest that (a) the stereospecific configuration at C-25 of THCA has no significant effect on the efficiency of side chain oxidation to cholic acid; and (b) side chain cleavage pathways may exist which do not pass through varanic acid, or the oxidation of varanic acid in man is highly stereospecific with respect to the hydroxyl group at C-24. To prove the latter, it will be necessary to compare the metabolism of the 24 alpha and 24 beta isomers of varanic acid.

The stereospecificity of microsomal "26" -hydroxylation in bile acid biosynthesis was studied. Cholesterol was biosynthesized from [2-14C] mevalonate by a rat liver preparation. The cholesterol was converted stepwise into 3alpha, 7alpha, 12alpha-trihydroxy-5beta-cholestan-26-oic acid by microsomal and soluble fractions of rat liver homogenate. The 3alpha, 7alpha, 12alpha-trihydroxy-5beta-cholestan-26-oic acid was decarboxylated chemically and the carbon dioxide was assayed for 14C. The amount of radioactivity in the liberated carbon dioxide was assayed for 14C. The amount of radioactivity in the liberated carbon dioxide was such as to indicate complete stereospecificity of the microsomal "26" -hydroxylase system. The system hydroxylates the methyl group in position C-26 (the 25-pro-R methyl group) and its stereospecificity is opposite that of the mitochondrial "26" -hydroxylase system which hydroxylates the methyl group in position C-27 (the 25-pro-S methyl group).

Two siblings with Zellweger's cerebro-hepato-renal syndrome are reported. The two children both had multiple anomalies associated with Zellweger's syndrome such as characteristic facial appearance, cerebral dysfunction, muscular hypotonia, liver abnormalities, failure to thrive, marasm and early death. One of the children, a girl, lacked several anomalies that were present in her brother. In one of the children bile acid analysis was performed by use of gas chromatography-mass spectrometry. 3 alpha,7 alpha,12 alpha-trihydroxy-5 beta-cholestanoic acid (THCA) and varanic acid, both precursors of cholic acid, were found. The defect bile acid synthesis may be due both to liver mitochondrial abnormalities and to the absence of liver peroxisomes, conditions known to occur in Zellweger's syndrome.

The first step in the degradation of the steroid side chain during biosynthesis of bile acids from cholesterol in man was studied in microsomal and mitochondrial fraction of homogenate of livers from 14 patients. The microsomal fraction was found to catalyze an efficient 25-hydroxylation of 5,8-cholestane-3a,7a,12atriol. A small extent of 23-, 24-, and 26-hydroxylation of the same substrate was observed. 53-Cholestane-3a,7adiol was hydroxylated in the 25-position only to a very small extent. The mitochondrial fraction was found to catalyze 26-hydroxylation of cholesterol, 5-cholestene-3P,7a-diol, 5P-cholestane-3a,7a-diol, 7a-hydroxy-4-cholesten-3-one, and 5,0-cholestane-3a,7a,12a-triol. Addition of Mg++ stimulated the 26-hydroxylation of cholesterol but had no effect or an inhibitory effect on 26-hydroxylation of the other substrates, indicating a heterogeneity of the mitochondrial 26-hydroxylating system. The level of 26-hydroxylase activity towards different substrates varied considerably with different mitochondrial preparations. The roles of the microsomal and mitochondrial 26- hydroxylations as well as the microsomal 25-hydroxylation in biosynthesis of bile acids in man are discussed. The results indicate that microsomal 26-hydroxylation is less important than mitochondrial 26-hydroxylation under normal conditions. The possibility that microsomal 25-hydroxylation is important cannot be ruled out.

In order to evaluate more definitively the observed aberrations in the synthesis of cholic and chenodeoxycholic acids in patients with advanced cirrhosis, two bile acid biosynthesis pathways were examined by determining the efficiency of conversion of [3H]7 alpha-hydroxycholesterol and [3H] 26-hydroxycholesterol to primary bile acids. Bile acid kinetics were determined by administration of [14C]cholic and [14C]chenodeoxycholic acids. Cholic acid synthesis in cirrhotic patients was markedly depressed (170 vs. 927 mumoles per day)( while chenodeoxycholic acid synthesis was reduced to a much lesser degree (227 vs. 550 mumoles per day). The administration of [3H]7 alpha-hydroxycholesterol allowed for an evaluation of the major pathway of bile acid synthesis via the 7 alpha-hydroxylation of cholesterol. This compound was efficiently incorporated into primary bile acids by the two normal subjects (88 and 100%) and two cirrhotic patients (77 and 91%). However, the recovery of the label in cholic acid was slightly less in cirrhotic patients than in normal subjects. [3H]26-hydroxycholesterol was administered to ascertain the contribution of the 26-hydroxylation pathway to bile acid synthesis. All study subjects showed poor conversion (9 to 22%) of this intermediate into bile acids. The results of this study suggest that a major block in the bile acid synthesis pathway in cirrhosis is at the level of 7 alpha-hydroxylation of cholesterol (impairment of 7 alpha-hydroxylase) and /or in the feedback triggering mechanism regulating bile acid synthesis. The data also suggest that the 26-hydroxylation pathway in normal subjects and patients with cirrhosis is a minor contributor to synthesis of the primary bile acids. Therefore, the relative sparing of chenodeoxycholic acid synthesis observed in cirrhotic patients is not due to preferential synthesis of this bile acid via the 26-hydroxylation pathway.

The effect of biliary obstruction on side chain hydroxylations in the biosynthesis and metabolism of bile acids was studied in the rat. For comparison, several other hydroxylation reactions in bile acid biosynthesis and metabolism were assayed. Biliary obstruction inhibited microsomal 26-hydroxylation of 5beta-cholestane-3alpha,7alpha-diol and microsomal 25- and 26-hydroxylation of 5beta-cholestane-3alpha,7alpha-12alpha-triol. Microsomal 7alpha-hydroxylation of cholesterol and 6beta-hydroxylation of lithocholic acid acid increased significantly, whereas the increase in microsomal 12alpha-hydroxylation of 5beta-cholestane-3alpha,7alpha-diol was less. Mitochondrial 26-hydroxylation of cholesterol, 5-cholestene-3beta,7alpha-diol, and 7alpha-hydroxy-4-cholesten-3-one was stimulated, whereas 26-hydroxylation of 5beta-cholestane-3alpha,7alpha-diol was not affected and that of 5beta-cholestane-3alpha,7alpha,12alpha-triol was markedly inhibited. The results indicate that mitochondrial 26-hydroxylation, particularly of substrates that primarily are precursors of chenodeoxycholic acid, plays a more important role in bile acid biosynthesis under conditions of biliary obstruction than under normal conditions.

The metabolism of [24-14C]lithocholic acid was studied in the microsomal fraction of fetal human liver homogenates. Fetal livers were obtained at legal abortions performed between wk 14 and 24. Product formation was analyzed by thin-layer chromatography, liquid chromatography, and gas chromatography-mass spectrometry. From wk 14 lithocholic acid was hydroxylated in several positions. Hyodeoxycholic acid (3 alpha,6 alpha-dihydroxy-5 beta-cholanoic) and 1 beta,3 alpha-dihydroxy-5 beta-cholanoic acid were identified among the products. The 3 beta-isomer of the former acid, 6 alpha-hydroxy-3-oxo-5 beta-cholanoic acid and a 2-hydroxylithocholic acid were tentatively identified. Two other metabolites carried a hydroxyl group in an unknown position, one of them probably at an angular methyl group. The highest total conversions per mg of microsomal protein were obtained with preparations from 18 wk fetuses. The results are discussed with particular reference to the role of lithocholic acid in fetal hepatotoxicity.