Difference between cholic acid and chenodeoxycholic acid in dependence upon cholesterol of hepatic and plasmatic sources as the precursor in rats

Some difference in functional pool of cholesterol acting as the precursor of bile acids is pointed out between cholic acid and chenodeoxycholic acid. In order to elucidate this problem further, some experiments were performed with rats equilibrated with [7(n)-3H, 4-(14)C] cholesterol by subcutaneous implantation. The bile duct was cannulated in one series of experiments and ligated in another. After the operation 14C-specific radioactivity of serum cholesterol fell, but reached practically a new equilibrium within three days. 14C-Specific radioactivity of serum cholesterol as well as of biliary bile acids in bile-fistula rats and urinary bile acids in bile duct-ligated rats was determined during a three days-period in the new equilibrated state. The results were as follows: (1) 14C-Specific radioactivity of cholic acid and chenodeoxycholic acid in bile was lower than that of serum cholesterol, and 14C-specific radioactivity of cholic acid was clearly lower than that of chenodeoxycholic acid. (2) 14C-Specific radioactivity of cholic acid and beta-muricholic acid in urine was lower than that of serum cholesterol, and 14C-specific radioactivity of cholic acid was lower than that of beta-muricholic acid. (3) Biliary as well as urinary beta-muricholic acid lost tritium label at 7-position entirely during the course of formation from [7(n)-3H, 4-(14)C]cholesterol.     

Incorporation of 14C-mevalonate into biliary cholesterol and bile acids by perfused rat liver: Effect of plasma and individual lipoproteins

Effect of plasma and individual lipoproteins on the incorporation of ¹⁴C-mevalonate into biliary bile acids and cholesterol by perfused rat liver was investigated. Use of plasma free perfysate (instead of whole blood) gave similar rates of incorporation of ¹⁴ C-mevalonate into biliary bile acids, but showed a decrease in percent distribution in cholic acid and a increase in β-muricholic acid. Addition of VLDL (isolated from Zucker rats) into the plasma free perfusate caused a significant increase in the incorporation of the label into bile acids, but LDL and HDL had no effect. HDL caused an increase in biliary excretion of ¹⁴C-cholesterol.     

Role of endogenous and exogenous cholesterol in liver as the precursor for bile acids in rats

There is considerable evidence suggesting that compartmentalized functional pools of cholesterol in the liver contribute differently to the formation of bile acids as the precursor. The present paper deals with the incorporation of [1-¹⁴C]acetate and of [1,2-³H]cholesterol carried on lipoproteins (LDL and HDL) into biliary bile acids in perfused rat livers and bile-fistula rats. The results showed that endogenous cholesterol synthesized newly from [1-¹⁴C]acetate in the liver was incorporated into both cholic acid and chenodeoxycholic acid in a similar way, while exogenous lipoprotein-[1,2-³H]cholesterol delivered to hepatocytes from hepatic circulation was incorporated into chenodeoxycholic acid at a higher rate.     

Dynamics of the conjugate pattern during the infusion of bile acids into isolated rat liver

The conjugate pattern of biliary [14C]bile acids was investigated in isolated perfused rat livers, which were infused with either [24-14C]cholic acid or [24-14C]chenodeoxycholic acid (40 mumol/h) together with or without taurine or cysteine (80 mumol/h). [14C]Bile acids were chromatographed on a thin-layer plate and the distribution of radioactivity on the plate was measured by radioscanning. The biliary excretion of [14C]bile acids was greater in the infusion with [14C]cholic acid than in the infusion with [14C]chenodeoxycholic acid. Biliary unconjugated [14C]bile acids amounted to about 50% of the total after the infusion with [14C]cholic acid, while only about 10% with [14C]chenodeoxycholic acid. In the initial period of infusion, biliary conjugated [14C]bile acids consisted mostly of the taurine conjugate, which decreased with time and the glycine conjugate increased complementarily. When taurine was simultaneously infused, the decrease in the taurine conjugate was suppressed to some extent. Cysteine infused in place of taurine had a similar influence but was less effective than taurine. The taurine content of liver after the infusion with either of the [14C]bile acids decreased greatly compared with that before the infusion, even when taurine or cysteine was infused simultaneously. The glycine content also decreased after the infusion, but the decrease in glycine was smaller than that in taurine. The results suggest that the conjugate pattern of biliary bile acids in rats depends mainly on the amount of taurine which is supplied to hepatic cells either exogenously from plasma or endogenously within themselves.     

Formation of mevalonic acid, sterols and bile acids from [1-14C]acetyl-CoA and [2-14C]malonyl-CoA in the liver of rabbits with experimental hypercholesterolemia

The effect of cholesterol diet on the rate of mevalonic acid biosynthesis from 1-14C acetyl-CoA, 2-14C malonyl-CoA and the incorporation of these substrates into sterols and bile acids in rabbit liver were studied. Simultaneously, the activities of 3-hydroxy-3-methylglutaryl-CoA reductase (HMG-CoA reductase) and acetyl-CoA carboxylase and the biosynthesis of fatty acids from acetyl-CoA and malonyl-CoA were measured. Hypercholesterolemia was found to be concomitant with the inhibition of acetyl-CoA carboxylase activity only in cell-free (700 g) and mitochondrial fractions and slightly decreased the incorporation of acetyl-CoA and malonyl-CoA into fatty acids in the postmitochondrial fraction. The HMG-CoA reductase activity in all subcellular fractions except for the postmicrosomal one was inhibited under these conditions. A significant decrease of acetyl-CoA incorporation and an increase in malonyl-CoA incorporation into mevalonic acid in all liver fractions except for microsomal one were observed in rabbits with hypercholesterolemia. These data provide evidence for the existence of two pathways of mevalonic acid synthesis from the above-said substrates that are differently sensitive to cholesterol. Cholesterol feeding resulted in a decreased synthesis of the total unsaponified fraction including cholesterol from acetyl-CoA, malonyl-CoA and mevalonic acid. The rate of incorporation of these substrates into lanosterol was unchanged. All the indicated substrates (acetyl-CoA, malonyl-CoA, mevalonic acid) are precursors of bile acid synthesis in rabbit liver. Cholesterol feeding and the subsequent development of hypercholesterolemia resulted in bile acid synthesis stimulation, preferentially in the formation of the cholic + deoxycholic acids from these precursors.     

Measurement of bile acid synthesis by 14CO2: the metabolism of propionyl CoA.

The relationship between 14CO2 evolution from the catabolism of [26 or 2714C] cholesterol to bile acids was studied in rats with biliary fistulae. When equal quantities of [26 or 2714C] cholesterol and [414C] cholesterol were administered, there was a significant linear relationship between 14CO2 expiration in the breath and [414C] bile acid excreted in the bile. Bile acid synthesis calculated as the ratio of 14CO2: molar specific activity of biliary cholesterol correlated highly with biliary bile acid excretion in the bile acid depleted rat. Phenobarbital, a known inducer of gamma-amino levulenic acid formation from succinyl CoA did not alter the relationship between the 14CO2 estimation of bile acid synthesis and biliary bile acid excretion, indicating that the relationship between [26 or 2714C] cholesterol side chain cleavage and 14CO2 formation was not altered. Phenobarbital, however, did cause a reduction in bile acid synthesis measured by 14CO2 evolution and by biliary bile acid excretion. The 14CO2 method underestimated bile acid excretion. 8.7% in untreated and phenobarbital treated rats respectively. Since 11% of the radioactivity which was expired as 14CO2 was isolated as bile acids, radioactivity cleaved as [1 or 314C] propionyl CoA may enter cholesterol-bile acid biosynthesis resulting in the underestimation of bile acid synthesis. To test whether radioactivity from propionyl CoA enters steroid biosynthesis [114C] propionate and [214C] propionate were given to untreated biliary fistula rats and the biliary lipids excreted in 60 hours were analyzed. Incorporation of radioactivity into cholesterol and bile acids was greater after the administration of [214C] propionate than after [114C] propionate than after [114C] propionate, suggesting that radioactivity from propionyl CoA may enter steroid biosynthesis by metabolic events in which the methylene and carboxyl carbon atoms are differentiated. Although the use of 14CO2 expiration from [26 or 2714C] cholesterol catabolism underestimates the rate of bile acid synthesis, it should have many applications because of the constant relationship between 14CO2 formation and cholesterol side chain cleavage.     

Cholesterol gallstones in alloxan-diabetic mice

Normal and alloxan-diabetic male mice (Crj-ICR) were fed a diet containing 0.5% cholesterol for 5 and 10 weeks, and gallbladder bile was analyzed for cholesterol, phospholipids and bile acids, feces for sterols and bile acids, and plasma and liver for cholesterol, phospholipids, and triglycerides. Normal mice developed no gallstones but the diabetic mice developed cholesterol gallstones with an incidence of 70% by 5 weeks and 80% by 10 weeks after feeding of the cholesterol diet. Diabetic mice fed the ordinary diet also developed stones (23%) by 10 weeks. In the diabetic mice, the gallbladder was enlarged about threefold, and biliary lipid concentration, diet intake, and fecal excretion of sterols and bile acids increased but body weight decreased. Cholic acid and beta-muricholic acid comprised over 40% each of the total biliary bile acids in normal mice, but cholic acid increased to about 80% and beta-muricholic acid decreased to a few percent in the diabetic mice. Fecal excretion of bile acids increased after cholesterol feeding in both normal and diabetic mice, but the increased bile acid in the normal animals was beta-muricholic acid and that in the diabetic mice was deoxycholic acid. The mice that developed gallstones showed a marked increase in biliary cholesterol value and decreases in gallbladder bile and bile acid concentration, but no difference in biliary and fecal bile acid composition, bile acid synthesis, fecal sterols, or plasma and liver lipid levels. Cholesterol absorption was increased in the diabetic mice when examined by plasma 14C/3H ratio and fecal 14C-labeled sterol excretion after a single oral administration of [14C]cholesterol and a simultaneous intravenous injection of [3H]cholesterol. These data led to the conclusion that cholesterol gallstones developed in alloxan-diabetic mice fed excess cholesterol, due to the hyperphagia and the enhancement of cholesterol absorption caused by increases in the synthesis and secretion of cholic acid.     

The role of conjugation reactions in enhancing biliary secretion of bile acids.

Shortening the five-carbon carboxylic acid side chain of cholic acid by one methylene group gave rise to a bile acid (norcholate) that was not a substrate for the bile acid-conjugating enzymes. The metabolism and biliary secretion of norcholate in intact liver was examined in the isolated perfused rat liver system. When rat livers were perfused with 14-20 microM solutions of norcholate for 10 min, norcholate was found in the unconjugated form in liver, venous effluent and bile. Neither tauronorcholate nor glyconorcholate was detectable by high-pressure liquid chromatography or fast-atom-bombardment mass spectrometry. The kinetics of hepatic uptake and biliary secretion of norcholate was compared with that for cholate, taurocholate and chemically synthesized tauronorcholate. The latter three bile acids were completely cleared from the perfusate and efficiently secreted into the bile. However, norcholate was incompletely extracted from the perfusate, and this was shown to be at least partially due to its relatively lower rate of hepatic uptake. Furthermore, the rate of norcholate secretion into bile was greatly reduced relative to the secretion of cholate or chemically synthesized tauronorcholate, even though the concentration of norcholate in the liver was comparatively high. These data demonstrate that the conjugation of bile acids greatly facilitates their secretion into bile.     

Influence of tauroursodeoxycholic and taurodeoxycholic acids on hepatic metabolism and biliary secretion of phosphatidylcholine in the isolated rat liver

Studies were carried out using an isolated rat liver system to define: the contribution of exogenous phosphatidylcholine (PC) to biliary phospholipid secretion; and its hepatic metabolism during perfusion of the livers with conjugated bile salts with different hydrophilic/hydrophobic properties. A tracer dose of sn-1-palmitoyl-sn-2-[14C]linoleoylPC was injected as a bolus into the recirculating liver perfusate, under constant infusion of 0.75 mumol/min of tauroursodeoxycholate or taurodeoxycholate. The effects on bile flow, biliary lipid secretion, 14C disappearance from the perfusate and its appearance in bile, as well as hepatic and biliary biotransformation were determined. With both the bile salts, about 40% of the [14C]PC was taken up by the liver from the perfusate over 100 min. During the same period less than 2% of the given radioactivity was secreted into bile. More than 95% of the 14C recovered in bile was located within the identical injected PC molecular species. The biliary secretion of labeled as well as unlabeled PC, however, was significantly higher in livers perfused with taurodeoxycholate than tauroursodeoxycholate, while the reverse was observed with respect to bile flow and total bile salt secretion. The exogenous PC underwent extensive hepatic metabolization which appeared to be influenced by the type of bile salt perfusing the liver. After 2 h perfusion, the liver radioactivity was found, in decreasing order, in PC, triacylglycerol, phosphatidylethanolamine and diacylglycerol. In addition, the specific activity of triacylglycerol was significantly higher in tauroursodeoxycholate than in taurodeoxycholate-perfused livers (P less than 0.025), while the reverse was true for the specific activity of hepatic PC (P less than 0.01). Because taurodeoxycholate and tauroursodeoxycholate showed opposite effects on both biliary lipid secretion and hepatic PC biotransformations, we conclude that the hepatic metabolism of glycerolipids is influenced by the physiochemical properties of bile salts.     

Biosynthetic studies on aromatic carotenoids. Biosynthesis of chlorobactene

1. The incorporation of [2-¹⁴C]mevalonic acid by Chloropseudomonas ethylica strain 2K into chlorobactene was studied. 2. Oxidative degradation of chlorobactene of constant specific radioactivity produced labelled benzenecarboxylic acids and indicated that the benzene ring originates from mevalonic acid. 3. Decarboxylation studies demonstrated a stereospecific methyl migration in the formation of the 1,2,5-trimethylphenyl group of chlorobactene. The migrating methyl group was derived from the C-3′ position of mevalonic acid.     

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.     

7-Methyl bile acids: 7 beta-methyl-cholic acid inhibits bacterial 7-dehydroxylation of cholic acid and chenodeoxycholic acid in the hamster

The effect of dietary 7 beta-methyl-cholic acid [0.075% in rodent chow (6.4 mg/animal per day)] on cholesterol and bile acid metabolism was studied and compared with that of cholic acid in the hamster. Following oral administration of 7 beta-methyl-cholic acid for 3 weeks, the glycine-conjugated bile acid analog became a major constituent of gallbladder bile. Biliary cholic acid concentration decreased significantly, while that of chenodeoxycholic acid remained unchanged. Serum and liver cholesterol levels were increased by dietary 7 beta-methyl-cholic acid and by cholic acid. Hepatic microsomal HMG-CoA reductase activity was inhibited (30% of the control value) by both bile acids; cholesterol 7 alpha-hydroxylase activity was not affected. In chow controls and cholic acid-fed animals, bacterial 7-dehydroxylation of [14C]chenodeoxycholic acid and [14C]cholic acid was nearly complete. In contrast, dietary 7 beta-methyl-cholic acid effectively prevented the 7-dehydroxylation of the two primary bile acids. These results show that dietary 7 beta-methyl-cholic acid is preserved in the enterohepatic circulation and has an effect on serum and liver cholesterol concentrations similar to those produced by the naturally occurring cholic acid. 7 beta-Methyl-cholic acid is an efficient inhibitor of the bacterial 7-dehydroxylation of the primary bile acids in the hamster.     

The specific radioactivity of the tissue free amino acid pool as a basis for measuring the rate of protein synthesis in the rat in vivo

1. Rats were infused in vivo with [U-(14)C]glycine for periods of 2-6h, during which time the specific radioactivity of the free glycine in plasma and tissue approached a constant value. 2. Free serine also became labelled. The ratio of specific radioactivity of serine to that of glycine in the protein of liver, kidney, brain, jejunum, heart, diaphragm and gastrocnemius muscle was closer to the ratio in the free amino acid pool of the tissue than that of the plasma. 3. The kinetics of incorporation of [(14)C]glycine and [(14)C]serine into the protein of gastrocnemius muscle further suggested that the plasma free amino acids were not the immediate precursors of protein. 4. Infusion of rats with [U-(14)C]serine resulted in labelling of free glycine. The ratio of specific radioactivity of glycine to serine in the protein of liver, kidney, brain, jejunum and heart again suggested incorporation from a pool similar to the free amino acid pool of the tissue. 5. Rates of tissue protein synthesis calculated from the incorporation into protein of both radioactive glycine and serine, either infused or derived, were very similar when the precursor specific radioactivity was taken to be that in the total free amino acids of the tissue. Except for gastrocnemius muscle and diaphragm during the infusion of radioactive serine, the rates of tissue protein synthesis calculated from the specific radioactivity of the free glycine and serine in plasma differed markedly.     

Role of peroxisomal fatty acyl-CoA beta-oxidation in phospholipid biosynthesis

We have already reported that peroxisomal beta-oxidation has an anabolic function, supplying acetyl-CoA for bile acid biosynthesis [H. Hayashi and A. Miwa, 1989, Arch. Biochem. Biophys. 274, 582-589]. The anabolic significance of peroxisomal beta-oxidation was further investigated in the present study by using clofibrate, a peroxisome proliferator, as an experimental tool. Clofibrate suppressed 3-hydroxymethylglutaryl-CoA reductase activity (the key enzyme of cholesterol synthesis) and enhanced fatty acyl-CoA oxidase activity (the rate-limiting enzyme of beta-oxidation). Rats were fed a chow containing 0.25% clofibrate for 2 weeks, and then a bile duct fistula was implanted. [1-14C]lignoceric acid, which is degraded exclusively by peroxisomal FAOS, was injected into the rats 24 h after the operation. By this time, the secondary bile acids and pooled cholesterol which would normally be secreted into the bile are considered to have been exhausted from the liver. Clofibrate significantly decreased the incorporations of radioactivity into biliary bile acid (40% of the control) and cholesterol (50%), but did not affect biliary lipid contents. [14C]Acetyl-CoA formed by peroxisomal beta-oxidation of [1-14C]lignoceric acid was preferentially utilized for syntheses of long-chain fatty acids and phospholipids rather than synthesis of cholesterol or triglyceride. The radioactivities incorporated into the former two lipids were increased 2-fold over the control by administration of clofibrate, while the incorporation into triglyceride was decreased to approximately half. In particular, the incorporation into phosphatidylethanolamine was increased as much as 3.5-fold over the control. The contents of these lipids in the liver were not affected by clofibrate. The results suggest that peroxisomal beta-oxidation plays an important role in the biosynthesis of functional lipids such as phospholipids (this work), in addition to bile acids and cholesterol (previous report) by supplying acetyl-CoA.     

Studies on the origin of biliary phospholipid. Effect of dehydrocholic acid and cholic acid infusions on hepatic and biliary phospholipids.

The correlation between the secretion of biliary phospholipid (PL) and bile acid suggests a regulatory effect of bile acid on PL secretion. Bile acids may influence PL synthesis and/or the mobilization of a preformed PL pool. The objective of this study was to determine the contribution of these two sources to biliary PL, by using an experimental protocol in which dehydrocholic acid (DHCA) and cholic acid (CA) were infused to manipulate biliary PL secretion. In control rats, there was a steady state in bile flow. PL secretion and the biliary secretion of newly synthesized phosphatidylcholine (PC). The specific radioactivity of PC in bile was significantly higher than in plasma, microsomes and canalicular membranes. DHCA infusion decreased biliary PC secretion rate by 80%, and secretion returned to normal values at the transport maximum of CA. The specific radioactivity of biliary PC was decreased by 30% by DHCA infusion and reached normal values during CA infusion. There were no significant changes in the specific radioactivity of PC in plasma or cellular organelles during infusion of bile acids. These data indicate that: (1) newly synthesized PC contributes a small percentage to biliary PC; thus a preformed pool (microsomal and extrahepatic) is a major source of biliary PL; (2) the contribution of the extrahepatic pool to the biliary PL may be more important than the microsomal pool.     

Measurement of protein synthesis in rat lungs perfused in situ

Compartmentalization of amino acid was investigated to define conditions required for accurate measurements of rates of protein synthesis in rat lungs perfused in situ. Lungs were perfused with Krebs–Henseleit bicarbonate buffer containing 4.5% (w/v) bovine serum albumin, 5.6mm-glucose, normal plasma concentrations of 19 amino acids, and 8.6–690μm-[U-¹⁴C]phenylalanine. The perfusate was equilibrated with the same humidified gas mixture used to ventilate the lungs [O₂/CO₂ (19:1) or O₂/N₂/CO₂ (4:15:1)]. [U-¹⁴C]Phenylalanine was shown to be a suitable precursor for studies of protein synthesis in perfused lungs: it entered the tissue rapidly (t_½, 81s) and was not converted to other compounds. As perfusate phenylalanine was decreased below 5 times the normal plasma concentration, the specific radioactivity of the pool of phenylalanine serving as precursor for protein synthesis, and thus [¹⁴C]phenylalanine incorporation into protein, declined. In contrast, incorporation of [¹⁴C]histidine into lung protein was unaffected. At low perfusate phenylalanine concentrations, rates of protein synthesis that were based on the specific radioactivity of phenylalanyl-tRNA were between rates calculated from the specific radioactivity of phenylalanine in the extracellular or intracellular pools. Rates based on the specific radioactivities of these three pools of phenylalanine were the same when extracellular phenylalanine was increased. These observations suggested that: (1) phenylalanine was compartmentalized in lung tissue; (2) neither the extracellular nor the total intracellular pool of phenylalanine served as the sole source of precursor for protein; (3) at low extracellular phenylalanine concentrations, rates of protein synthesis were in error if calculated from the specific radioactivity of the free amino acid; (4) at high extracellular phenylalanine concentrations, the effects of compartmentalization were negligible and protein synthesis could be calculated accurately from the specific radioactivity of the free or tRNA-bound phenylalanine pool.     

The role of peroxisomal fatty acyl-CoA beta-oxidation in bile acid biosynthesis

The physiological role of the peroxisomal fatty acyl-CoA beta-oxidizing system (FAOS) is not yet established. We speculated that there might be a relationship between peroxisomal degradation of long-chain fatty acids in the liver and the biosynthesis of bile acids. This was investigated using [1-14C]butyric acid and [1-14C]lignoceric acid as substrates of FAOS in mitochondria and peroxisomes, respectively. The incorporation of [14C]lignoceric acid into primary bile acids was approximately four times higher than that of [14C]butyric acid (in terms of C-2 units). The pools of these two fatty acids in the liver were exceedingly small. The incorporations of radioactivity into the primary bile acids were strongly inhibited by administration of aminotriazole, which is a specific inhibitor of peroxisomal FAOS in vivo [F. Hashimoto and H. Hayashi (1987) Biochim. Biophys. Acta 921, 142-150]. Aminotriazole inhibited preferentially the formation of cholate, the major primary bile acid, from both [14C]lignoceric acid and [14C]butyric acid, rather than the formation of chenodeoxycholate. The former inhibition was about 70% and the latter was approximately 40-50%. In view of reports that cholate is biosynthesized from endogenous cholesterol, the above results indicate that peroxisomal FAOS may have an anabolic function, supplying acetyl CoA for bile acid biosynthesis.     

Quantitative aspects of the conversion of 5 beta-cholestane intermediates to bile acids in man.

The in vivo conversion of several 5 beta-cholestane intermediates to primary bile acids was investigated in three patients with total biliary diversion. The following compounds were administered intravenously: 5 beta-[G-3H]-cholestane-3 alpha, 7 alpha-diol, 5 beta-[G-3H]cholestane-3 alpha, 7alpha, 26-triol, and 5 beta-[24-14C]cholestane-3 alpha, 7 alpha-25-triol. Bile was then collected quantitatively at frequent intervals for the next 21 to 28 h. The administered 5 beta-[G-3H]cholestane-3alpha, 7alpha, 26-triol was found to be efficiently converted to cholic and chenodeoxycholic acids in two patients; 61 and 75% of the administered label was found in primary bile acids. The proportion of labeled cholic to chenodeoxycholic acid was 1.20 and 1.02 in the bile of these patients, indicating that the C-26 triol was efficiently converted to cholic acid. The ratio of cholic to chenodeoxycholic acid (mass) in the bile of these patients was 1.23 and 2.32. The 5 beta-cholestane-3alpha, 7alpha-diol intermediate was also efficiently converted (71%) to both primary bile acids. The cholic to chenodeoxycholic acid ratios by mass and label were similar (2.97 versus 2.23). By contrast, the 5beta-cholestane-3alpha, 7alpha, 25-triol was poorly converted to bile acids in three patients. Following the administration of this compound almost all of the administered radioactivity found in the bile acid fraction was in cholic acid (5 to 19%) and very little (less than 5%) was found in chenodeoxycholic acid. These findings indicate that ring hydroxylation at position 12 is not materially hindered by the presence of a hydroxyl group on the side chain at C-26 in patients with biliary diversion. The labeled C-26-triol which was efficiently converted to both primary bile acids in a proportion similar to that which was observed for the bile acids synthesized by the liver suggests that this 5beta-cholestane derivative may be a major intermediate in the synthesis of both cholic and chenodeoxycholic acids.     

Bile acids of marsupials. 2. Hepatic formation of vulpecholic acid (1 alpha,3 alpha,7 alpha-trihydroxy-5 beta-cholan-24-oic acid) from chenodeoxycholic acid in a marsupial, Trichosurus vulpecula (Lesson).

Free vulpecholic acid (1 alpha,3 alpha,7 alpha-trihydroxy-5 beta-cholan-24-oic) is the major biliary component of the Australian opossum (Trichosurus vulpecula), accompanied only by a few percent of its taurine conjugate. In order to exclude a microbial involvement in its formation (i.e., secondary origin) four sets of experiments were performed. It was found that a) the level of vulpecholic acid remained unchanged in the bile of opossums fed with neomycin and kanamycin for 7 days prior to bile collection; b) it also remained unchanged after long bile drainage; c) in opossums prepared with biliary cannula, intraportally injected [24-14C]chenodeoxycholic acid was transformed to [24-14C]vulpecholic acid; and d) in a similar experiment, the detectable transformation of [1 alpha,2 alpha-3H2]cholesterol to vulpecholic acid was observed. In experiment c) 28-66% of the administered radioactivity was secreted in 2 h in the form of free biliary vulpecholic and chenodeoxycholic acids. Only a trace amount of the corresponding taurine conjugates (approximately 0.4%) was formed. Moreover, rapidly declining specific radioactivity of the unconjugated chenodeoxycholic acid indicated its probable participation in the native formation of vulpecholic acid.     

Biosynthesis of the farnesyl moiety of heme a from exogenous mevalonic acid by cultured chick liver cells

Chick embryo liver cells, when cultured for 41 h in the presence of [2-14C]mevalonic acid, took up label and incorporated radioactivity into heme a, but not into protoheme. Incubation of cells with delta-[4-14C]aminolevulinic acid (ALA) resulted in uptake of label and incorporation of radioactivity into both protoheme and heme a. These results show that both protoheme and heme a are synthesized during the incubation period, and that mevalonic acid is a specific precursor of the farnesyl moiety of heme a. Incubation of cells with [1,2-14C]acetate plus N-methyl mesoporphyrin IX, an inhibitor of heme synthesis, resulted in negligible incorporation of label into protoheme and heme a, although cellular lipids were highly labeled. This result indicates that the heme purification methods employed were capable of separating hemes from lipids, and that the measured incorporation of label into hemes from [14C]mevalonic acid and [14C]ALA was not due to lipid contamination.