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A radioimmunoassay for serum conjugated cholic acid is described using antiserum obtained five weeks after immunization of rabbits with cholic acid bovine serum albumin conjugate. Prior to the radioimmunoassay, extraction of bile acids was performed with Amberlite XAD-2. The displacement curve of glyco[3H]cholic acid was linear on a logit-log plot from 5 to 80 pmol of unlabelled glycocholic acid. Values for 8 normal fasting subjects ranged from 0.18 to 1.25 mumol/1. In 31 fasting subjects with or without liver disease serum values of conjugated cholic acid are related to serum bilirubin. Endoscopic retrograde cholangiopancreatography may influence serum levels of conjugated cholic acid.
The mechanism of the uptake of cholic acids and the interaction of various bile acids on the cholic acid uptake were investigated using isolated rat hepatocytes. The uptake consisted of unsaturable and saturable processes at 0 degrees C and 37 degrees C, respectively. The activation energy found for the saturable process was 26.1 Kcal/mol. In the saturable process the rate of cholic acid uptake followed Michaelis-Menten kinetics with Km: 67 microM and Vmax: 1.43 nmoles/ml protein/min. The uptake was significantly inhibited by 2,4-dinitrophenol, and replacement of extracellular Na+ by choline did not decrease the uptake. The uptake of cholic acid was competitively inhibited by deoxycholic acid, taurocholic acid, glycocholic acid, chenodeoxycholic acid, taurochenodeoxycholic acid and glycochenodeoxycholic acid. It is concluded from the above results that the cholic acid uptake in isolated hepatocytes is mainly mediated by an energy-dependent and sodium-independent carrier-mediated transport process.
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OBJECTIVE: In humans, two primary bile acids are synthesized: cholic acid (CA) and chenodeoxycholic acid (CDCA), the first and rate-limiting enzyme being cholesterol 7alpha-hydroxylase (CYP7A1). CA has one more hydroxyl group at position 12alpha. This hydroxylation is carried out by the sterol 12alpha-hydroxylase (CYP8B1). Earlier, we and others have noticed a marked variation in the ratio between CA and CDCA in human bile. The aim of this study was to investigate whether this marked difference could be due to a genetic polymorphism in the gene of the CYP8B1. MATERIAL AND METHODS: Screening for genetic polymorphisms was carried out in a 2.4-kb-long area including the exon and part of the promoter region in subjects who had undergone cholecystectomy earlier, and where bile acid analysis had been performed. Among these subjects those with very high or low CA/CDCA ratios (ranging from 0.9 to 6.8) were investigated. The subjects were all female, normolipidaemic, having normal weight and a normal thyroid function. RESULTS: No polymorphisms were found in the investigated sequence. However, a statistically significant correlation was found between the activity of the CYP7A1 and the ratio between CA and CDCA. The difference in ratio could, at least in part, be explained by the difference in rate of bile acid synthesis. CONCLUSION: The difference in ratio between CA and CDCA cannot be explained by a polymorphism in the coding area of the CYP8B1.
A method has been developed for simultaneous determination of pool sizes and fractional turnover rates (FTR) of chenodeoxycholic acid (CDCA) and cholic acid (CA) in man by 13C/12C isotope ratio measurements of bile acids in serum after oral administration of 20-50 mg of [24-13C]-labeled bile acids. 13C/12C isotope ratio measurements were performed by capillary gas-liquid chromatography/electron impact mass spectrometry. CA and CDCA kinetics in serum measured by this method exhibited first order kinetics and permitted calculation of pool size and FTR of CA and CDCA. The validity of the measurements in serum was tested by simultaneous measurements in bile in three healthy volunteers and in five patients with various hepatobiliary disorders (three patients with cirrhosis, one with cholecystectomy and sphincterotomy, and one with sphincterotomy only). No consistent differences were found between the pool sizes and FTR's obtained from serum and bile. In a total of five healthy volunteers bile acid kinetics were measured in serum. The values found for the pool sizes and FTR's of CA and CDCA in these subjects were in excellent agreement with data reported in the literature based on 14C or 3H measurements in bile. The pool sizes (mean +/- SD) of CDCA and CA were 32.6 +/- 9.9 and 31.8 +/- 16.0 mumol X kg-1, respectively. The corresponding values for the FTR's were 0.24 +/- 0.13 and 0.48 +/- 0.22 d-1. These data demonstrate that pool sizes and fractional turnover rates of cholic and chenodeoxycholic acid can be measured simultaneously by blood sampling after oral administration of the respective 13C-labeled bile acids.
Cholic acid uptake was studied in isolated rat hepatocytes using a centrifugal filtration technique to allow rapid sampling. Hepatocytes were found to adsorb as well as to transport cholic acid. The adsorption was characterized by a capacity of 24 nmol X mg cell protein-1 and an association constant of 0.59 X 103 M-1. Cholic acid uptake was linear with respect to concentration at or below 10 degree C, suggesting a unsaturable uptake process which was considered to represent simple diffusion and is quantitated by a diffusion coefficient of 1.76 pmol cholic acid X min-1 X mg protein-1 X muM-1. Above 10 degrees C the uptake curve was biphasic. After subtracting the unsaturable component from uptake rates at higher temperatures, a curve showing saturable kinetics resulted. The apparent Km and V values at 37 degrees C were calculated to be 31muM and 0.8 nmol X min-1 X mg protein-1 respectively. This saturable uptake process was temperature-dependent with an activation energy of 13 kcal X mol-1 (5.44 X 104 J X mol-1) and was inhibited by oligomycin and KCN. Countertransport was demonstrated with cholic, taurocholic and chenodeoxycholic acids. The results suggest that cholic acid is transported by an energy-dependent carrier-mediated process in addition to simple diffusion by hepatocytes, and that the postulated carrier has affinity for other bile acids.
Cholic acid biosynthesis is defective in individuals with cerebrotendinous xanthomatosis (CTX) and is associated with the excretion of 5beta-cholestane-3alpha,7alpha, 12alpha,25-tetrol, an intermediate in the 25-hydroxylation pathway of cholic acid in CTX. To define the enzymatic defect in CTX, two suspected precursors of cholic acid, namely 5beta-[7beta-(3)H]cholestane-3alpha,7alpha, 12alpha-triol and 5beta-[24-(14)C]cholestane-3alpha,7alpha, 12alpha,24S,25-pentol were examined by both in vivo and in vitro experiments. A third precursor, 5beta-[7beta-(3)H]-cholestane-3alpha,7alpha, 12alpha,25-tetrol, was compared with them in vitro. In the in vivo experiments, each one of the labeled precursors was administered intravenously to two CTX and two control subjects. In the controls, 5beta-[7beta-(3)H]cholestane-3alpha,7alpha, 12alpha-triol as well as 5beta-[24-(14)C]-cholestane-3alpha,7alpha, 12alpha,24S,25-pentol were rapidly converted to labeled cholic acid. Maximum specific activity values were reached within 1 d after pulse labeling, followed by exponential decay of the cholic acid specific activity curves. In contrast, these two precursors differed widely when administered to two CTX patients. While 5beta-[24-(14)C]cholestane-3alpha,7alpha, 12alpha,24S,25-pentol was rapidly converted to [24-(14)C]cholic acid and yielded identical decay curves with those obtained in the control subjects, maximum specific activity values in [7beta-(3)H]cholic acid were much lower and peaked only on the second day after the injection of 5beta-[7beta-(3)H]cholestane-3alpha,7alpha, 12alpha-triol. Furthermore, an appreciable amount of (3)H label was present in the 5beta-cholestane-3alpha,7alpha, 12alpha,25-tetrol isolated from the bile of the subjects with CTX. In the in vitro experiments, three enzymes on the 25-hydroxylation pathway of cholic acid were examined in both control and CTX subjects. The rate of the 25-hydroxylation of 5beta-cholestane-3alpha,7alpha, 12alpha-triol in CTX patients was comparable to that in the controls. Similarly, the transformation of 5beta-cholestane-3alpha,7alpha, 12alpha,24S,25-pentol to cholic acid, catalyzed by soluble enzymes, proceeded at approximately equal rates in CTX and in control individuals. On the other hand, the rate of 5beta-cholestane-3alpha,7alpha, 12alpha,24S,25-pentol formation was about four times greater in the control subjects than in the CTX patients.The results of the in vivo as well as the in vitro experiments suggest that the site of the enzymatic defect in CTX is at the 24S-hydroxylation of 5beta-cholestane-3alpha,7alpha, 12alpha,25-tetrol. The relative deficiency of this hydroxylase in CTX patients, accompanied by the accumulation of its substrate in bile and feces, probably accounts for the subnormal production of bile acids in CTX patients.
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.
Despite the fact that most human subjects synthesize about twice as much cholic acid as chenodeoxycholic acid, available evidence suggests that 7 alpha-hydroxycholesterol, the first intermediate in the major pathway for bile acid synthesis, is converted about equally to these two bile acids. Synthesis through the main alternate pathway can not explain this discrepancy because 27-hydroxycholesterol, the first intermediate in that pathway, is converted preferentially to chenodeoxycholic acid. To examine the validity of these contradictory observations, we administered (24-(14)C)-cholic acid and (24-(14)C)-chenodeoxycholic acid together with (7 beta-(3)H)-7 alpha-hydroxycholesterol on one occasion and (22,23-(3)H)-27-hydroxycholesterol on a separate occasion to eight normal human subjects. Synthesis of the two primary bile acids was determined by means of standard isotope dilution kinetics of the carbon 14-specific activities of biliary bile acids. Conversion of (7 beta-(3)H)-7 alpha-hydroxycholesterol and (22,23-(3)H)-27-hydroxycholesterol to bile acid was calculated from the tritium/carbon 14 ratio in cholic and chenodeoxycholic acid. For synthesis, the mean +/- SEM cholic/chenodeoxycholic ratio was 1.82 +/- 0.26. For apparent conversion of (7 beta-(3)H)-7 alpha-hydroxycholesterol to bile acid, the mean +/- SEM cholic/ chenodeoxycholic ratio was 1.02 +/- 0.09, whereas for (22,23(3)H)-27-hydroxycholesterol, the mean +/- SEM cholic/chenodeoxycholic ratio was 0.38 +/- 0.03. These data imply that, on average, more than 40% of cholic acid in these subjects was synthesized through a pathway that bypassed initial 7 alpha-hydroxylation. However, consideration of all potential candidates for such a pathway raises doubts that any of them contributes substantially to bile acid synthesis.
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The aim of this work was to evaluate and compare the effects of a low calorie diet (1026 kcal), simvastatin and ursodeoxycholic acid administration on biliary lipid secretion and cholic acid kinetics in dieting obese subjects. We studied 6 obese subjects before and after four weeks of a hypocaloric diet alone, after four weeks of diet plus ursodeoxycholic acid (900 mg/day) and after four weeks of diet plus simvastatin (40 mg/day), according to a Latin square design. The cholesterol saturation index was increased after diet alone, significantly reduced with diet plus ursodeoxycholic acid (p < 0.01), and unchanged during simvastatin administration. While the cholesterol output was reduced by all three regimens, diet plus ursodeoxycholic acid caused a significantly greater decrease than diet alone (p < 0.01). Cholic acid synthesis and bile acid secretion were decreased by diet and diet plus simvastatin (p < 0.05), but neither was affected by ursodeoxycholic acid. For cholic acid, all three treatments, but especially diet alone and diet plus simvastatin (p < 0.05), reduced the pool size; all three regimens also increased the turnover rate, but this was significant only for ursodeoxycholic acid (p < 0.01). Our study shows that, in obese patients, a hypocaloric diet reduces cholesterol-holding biliary lipid output and consequently increases the cholesterol saturation index. The addition of simvastatin to a hypocaloric dietary regimen reduces cholesterol secretion, but without variation in bile acid and phospholipid output thus the cholesterol saturation index remains unchanged. When ursodeoxycholic acid is added to the dietary regimen, it reduces cholesterol secretion, while maintaining bile acid output and, thus, lowers the cholesterol saturation index. Unlike simvastatin, ursodeoxycholic acid prevents the drop in cholic acid synthesis induced by a low calorie diet.
The metabolism of cholic acid by Arthrobacter simplex was investigated. This organism effected both ring a cleavage and elimination of the hydroxyl groups at C-7 and C-12 and gave a new metabolite, (4R)-4-[4alpha-(2-carboxyethyl)-3aalpha-hexahydro-7abeta-methyl-5-oxoindan-1beta-yl]valeric acid, which was isolated and identified through its partial synthesis. A degradative pathway of cholic acid into this metabolite is tentatively proposed, and the possibility that the proposed pathway could be extended to the cholic acid degradation by other microorganisms besides A. simplex is discussed. The possibility that the observed reactions in vitro could occur during the metabolism of bile acids in vivo is considered.
Unconjugated bile acids such as cholic acid cause diarrhoea, mucosal irritation and toxicity. We sought to define the mechanism of cholate permeation across intestinal mucosal cells to understand how cellular exposure and accumulation are deleterious to mucosal function. Human intestinal Caco-2 and T84 cell monolayers were prepared by high-density seeding and cultured for >14 days on permeable culture supports. Cholate transport and cellular accumulation were determined using [3H]cholic acid. Epithelial barrier function was assessed by measuring transepithelial electrical resistance (Rt) and [14C]mannitol fluxes. Exposure of Caco-2 epithelia to serosal cholate caused a dose- and time-dependent disruption of barrier function. Apical exposure was without disruptive effect. Similar responses were observed for T84 epithelia. Cholate was preferentially accumulated across the basolateral surfaces in both Caco-2 and T84 cells, but was subject to active transepithelial secretion in Caco-2 monolayers only. Net secretion was substantially reduced by ATP depletion, showed saturation kinetics, and was subject to competitive inhibition by other bile acids. Cholate secretion was also sensitive to inhibition by the leukotriene antagonist MK-571 but not by digoxin, suggesting that MRP2, not MDR1, was responsible. RT-PCR and Western blotting confirmed MRP2 expression in Caco-2 epithelia but indicated its apparent absence from T84 cells.
The effect of intravenous administration of cholic acid on sphincter of Oddi (SO) and gallbladder motility was studied. Bolus doses of cholic acid, 20 to 60 mg/kg, produced inhibition of SO wave frequency, a fall in gallbladder pressure and enhanced bile flow. However, hydrocortisone, 10 and 20 mg/kg, produced comparable elevation in bile flow with no effect on SO and gallbladder motility. The effect of cholic acid on SO motility was not influenced by prior treatment with atropine. Phentolamine or propranolol administration did not influence SO wave frequency SO wave frequency, but subsequent injection of cholic acid resulted in a decrease in SO wave frequency. Gallbladder pressure was not influenced by atropine, phentolamine, or propranolol, and these agents did not influence the cholic acid-induced fall in gallbladder pressure. These findings suggest that bile acids influence the motility of the biliary tract.
The molecular mechanics of cholic acid micelle formation were simulated using the Sybyl energy minimization program (MAXIMIN2), developed by Tripos Associates, interfaced with micro-Vax. Before energy minimization, the molecular dimensions of the cholic acid dodecamer C24H40O6, in terms of the unit cell axes a, b, and c in the cubic crystal class, had values of 13, 18, and 6.7 A, respectively. After energy minimization, at 9370 kcals/dodecamer, these values had increased to 21.6, 42.8 and 20.9 A. At an energy minimization level of 21,626 kcals/dodecamer, the micelle structure is stabilized by hydrophobic interaction, forming distinct horizontal channels along the b-axis, directing the carboxyl and hydroxyl groups toward the surface. These structural changes remain relatively constant as the process of energy minimization continues, down to the lowest energy level we considered, 9370 kcals/dodecamer. The cholic acid layers are highly dissimilar, forming channels of irregular size and shape in a somewhat helical structure. The carboxyl groups and phenanthrene rings are in a puckered orientation, which permits compact packing of the sandwiched multilayers. From the dimension of the channels, it is apparent that guest molecules, such as phospholipid, cholesterol, or inorganic calcium, can be incorporated into the micelle through more than one channel, forming inclusion complexes, such as gallstones.
We examined the effect of the type of cholic acid conjugation (taurine-conjugated, glycine-conjugated, or unconjugated cholic acid) on cholic acid 7 alpha-dehydroxylation by intestinal flora. Cholic acid 7 alpha-dehydroxylation in fecal cultures, in cultures of a defined limited flora consisting of a mixture of seven bacterial species isolated from the intestinal tract, and in a binary culture of a 7 alpha-dehydroxylating Clostridium species plus a cholic acid-deconjugating Bacteroides species was studied. We found that tauroconjugation of cholic acid significantly (P < 0.05) increased bacterial 7 alpha-dehydroxylation of cholic acid into deoxycholic acid from 34 to 55% in fecal cultures, from 45 to 60% in defined limited fecal cultures, and from 75 to 100% in binary cultures. Equimolar concentrations of free taurine did not stimulate 7 alpha-dehydroxylation in fecal cultures or in the defined limited flora, but free taurine did stimulate 7 alpha-dehydroxylation in the binary culture. In the binary culture of Clostridium species strain 9/1 plus Bacteroides species strain R1, the minimal flora capable of increased 7 alpha-dehydroxylation of taurocholic acid, strain R1 deconjugated taurine and rapidly reduced it to H2S. Bacteroides species strain R1 did not grow unless taurine or another appropriate reducible sulfur source was present. Clostridium species strain 9/1 did not grow or 7 alpha-dehydroxylate unless H2S or another source of reduced sulfur was present. We conclude that the increased 7 alpha-dehydroxylation of tauroconjugated cholic acid depends on the reduction of taurine to H2S, which is a necessary growth factor for the 7 alpha-dehydroxylating bacteria.