Metabolism of the Bile Acid Analogues 7,β-Methyl-Cholic Acid and 7α-MethylUrsocholic Acid

GASTROENTEROLOGY 1987;92:876-84

Metabolism of the Bile Acid Analogues 7,B-Methyl-Cholic Acid and 7 a-MethylUrsocholic Acid SYOJI KUROKI, ERWIN H. MOSBACH, BERTRAM I. COHEN, and CHARLES K. McSHERRY

Departments of Surgery, Beth Israel Medical Center and the Mount Sinai School of Medicine of The City University of New York, New York, New York

The metabolism of two new bile acid analogues, 7f3-methyl-cholate and 7a-methyl-ursocholate, was compared with that of cholate in the hamster. After intraduodenal administration of 14C-labeled compounds into bile fistula hamsters, radioactivity was exclusively recovered in bile; the more hydrophobic bile acid was absorbed more rapidly. Hepatic extraction of intravenously infused compounds was efficient and administered analogues became major biliary bile acids. Amidation of cholate was essentially complete, whereas 39% of 7f3-methyl-cholate and 65% of 7a-methyl-ursocholate were secreted in un conjugated form. After intragastric administration of the compounds, radioactivity was quantitatively recovered in feces. Cho1ate was 7-dehydroxylated to deoxycholate, whereas 31% of 7f3-methyl-cholate and 78% of 7a-methy1-ursocholate were recovered unchanged. Fifty percent of 7f3-methyl-cho1ate and 15% of 7a-methyl-ursocho1ate were transformed into ketonic derivatives, without loss of the 7hydroxyl group. It is concluded that the introduction of the 7·methyl group did not interfere with intestinal absorption, hepatic extraction, and biliary secretion but did affect enzymatic amidation and bacterial 7-dehydroxylation of the analogues. The oral administration of chenodeoxycholic acid (CDA) or ursodeoxycholic acid (UDA) is known to dissolve cholesterol gallstones in a certain percentage of selected patients (1,2). Many studies, both in experimental animals and in humans, have conReceived April 14, 1986. Accepted September 3, 1986. Address requests for repririts to: Erwin H. Mosbach, Ph.D., Department of Surgery, Beth Israel Medical Center, First Avenue at 16th Street, New York, New York 10003. This work was supported iri part by U.S. Public Health Service grant HL 24b61 from the National Heart, Lung, and Blood Institute. © 1987 by the American Gastroenterological Association 0016-5085/87/$3.50

firmed the cholelitholytic properties of these dihydroxy bile acids (1-3). Although the toxicity of CDA and UDA in humans seems to be low, moderate to severe hepatotoxicity, especially with CDA, has been observed in some laboratory animals, such as the rabbit and the rhesus monkey (4,5). This problem has been ascribed to the fact that lithocholic acid (LA). a major metabolite of CDA and UDA, is known to be a hepatotoxin (4,5); LA is also a possible cocarcinogen for colon cancer (6). Lithocholic acid arises from the bacterial 7-dehydroxylation of CDA and UDA in the large intestine; it is relatively insoluble and largely disposed of via fecal excretion, but some intestinal reabsorption takes place so that appreciable proportions of total biliary bile acids may be in the form of this monohydroxy bile acid (7). The dehydroxylation and subsequent fecal excretion of the dihydroxy bile acids in the form of LA implies the loss of the active drug during intestinal passage, as the human liver has little capacity to 7-hydroxylate LA to either CDA or UDA (8). We therefore postulated that inhibition of the bacterial 7-dehydroxylation by introduction of a methyl group in the 7-position of CDA or UDA might prevent the formation of LA and preserve the 7-methyl bile acid for prolonged enterohepatic cycling (9). The availability of 7-methyl-substituted cholanoic acids [3a,7a-dihydroxy-7f3-methyl-5f3-cholanoic acid (7Me-CDA); 3a,7 ,B-dihydroxy-7 a-methyl-5f3-cholanoic acid (7-Me-UDA); and 3a-hydroxy-n-methyl-5f3Abbreviations used in this paper: CA, cholic acid; CDA, chenodeoxycholic acid; DCA, deoxycholic acid; LA, lithocholic acid; 7-Me-CA, 3a,7a,12a-trihydroxy-7f3-methyl-5,B-cholanoic acid; 7Me-CDA, 3a,7 a,-dihydroxy-7 f3-methyl-5f3-choHmoic acid; 7-MeDCA, 3a,12a-dihydroxy-7f.methyl-5f3-cholanoit acid; 7-Me-UCA; 3a,7 f3, 12a-trihydtoxy-7 a-methyl-5,B-cholanoic acid; 7-Me-UDA, 3a,7 f3-dihydroxy-7 a-methyl-5,B-cholanoic acid; R f , retardation factor; TLC, thin-layer chromatography; UCA, 3a,7 f3, 12atrihydroxy-5f3-cholanoic acid; UDA, ursodeoxycholic acid.

April 1987

METABOLISM OF 7-METHYL TRIHYDROXY BILE ACIDS

cholanoic acid] (9) made it possible to test this hypothesis. Studies of the metabolism and pharmacologic effects of these compounds revealed that they are (a) readily absorbed from the intestine, (b) extracted by the liver and secreted in the bile fully conjugated with glycine or taurine, and (c) more resistant to bacterial dehydroxylation than the naturally occurring dihydroxy bile acid (10). In the prairie dog model of cholesterol cholelithiasis, 7-MeCDA was found to be as effective in preventing gallstone formation as the parent compound CDA (unpublished observation). On the other hand, it was recently shown that in humans, cholic acid (CA) can lower the saturation index of bile, provided the formation of deoxycholic acid (DCA) is inhibited by the simultaneous administration of the antibiotic ampicillin (11). 3a,7f3,12aTrihydroxy-5f3-cholanic acid (UCA) is also of interest, because this highly hydrophilic compound could conceivably dissolve cholesterol gallstones via liquid crystal formation, as has been observed with UDA. However, because both CA and UCA are easily dehydroxylated by intestinal bacteria to form DCA (11), it is difficult to evaluate the effect of these trihydroxy bile acids directly. Therefore, to preserve the 7-hydroxyl group of CA and UCA, we recently synthesized the 7-methyl-trihydroxy bile acids, 3a, 7a, 12a-trihydroxy-7 f3-methyl-5f3-cholanoic acid (7~Me-CA) and 3a,7f3,12a-trihydroxy-7a-methyl-5f3cholanoic acid (7-Me-UCA) (12). These compounds are relatively hydrophilic, like UDA, and may have cholelitholytic properties. It was the aim of the present investigation to study the metabolism of 7-Me-CA and 7-Me-UCA in the hamster with respect to intestinal absorption, hepatic transformation, conjugation with taurine and glycine, and resistance to bacterial degradation (7-dehydroxylation).

NEN Products. Radiochemical purity of each labeled compound was better than 98% as determined by radio-thinlayer chromatography (TLC). Each labeled compound was diluted with the unlabeled substance (described below) to a specific activity of 5.0 x 10 5 cpm/mg and dissolved in 1% NaHCa 3 solution (4 mg/ml or 10 JLmollml). Unlabeled 7-methyl bile acid analogues (7-Me-CA, 7Me-UCA, 7-Me-DCA, 7-Me-CDA, 7-Me-UDA) were synthesized as described previously (9,12). Glycine or taurine conjugates of cholic, deoxycholic, chenodeoxycholic, and lithocholic acids, were prepared by published procedures (13). Nordeoxycholic acid (Steraloids, Inc., Wilton, N.H.) was purified (99%) by recrystallization from ethyl acetate. Experimental animals. Male golden Syrian hamsters (Harland Sprague-Dawley, Indianapolis, Ind.). weighing 109 ± 10 g, were acclimated for at least 2 wk on a commercial pelleted hamster diet. The animals were given food and water ad libitum and maintained under controlled illumination; the light period was from 6 AM to 6

877

PM.

Surgical procedures. The animals were anesthetized by intramuscular injection with 15 mg of sodium pentobarbital and maintained under anesthesia with diethyl ether. All animals were operated upon between 8 and 9 AM. For intravenous infusion studies, a polyethylene catheter (PE-l0, 0.28 mm ID and 0.61 mm aD; ClayAdams, Parsippany, N.J.) was inserted into the femoral vein and 0.9% NaCI solution was infused at a rate of 1.1 mllh. The abdomen was opened through an upper transverse incision. The cystic duct was occluded with a plastic clip (Absolok MCA; Ethicon Inc., Somerville, N.J.) and a PE-lO catheter was inserted into the common bile duct. For intraduodenal administration studies, a PE-20 catheter (0.38 mm ID and 1.09 mm aD) with a flanged tip (formed by heating the end of the tubing with flame) was introduced into the distal portion of the duodenum via a small duodenotomy; the common bile duct was cannulated as described above. In both series of experiments, the abdominal incision was closed and urine was allowed to collect in the bladder by ligating the urethra. Body temperature was maintained with a heat lamp.

Experimental Design

Materials and Methods Labeled and reference compounds. [7,B-Methyl14C]3a,7 a-dihydroxy-7 ,B-methyl-5,B-cholanoic acid (sp act 1.1 x 10 6 cpm/mg) and [7a-methyl-14C]7-Me-UDA (sp act 1.1 x 10 6 cpm/mg) were prepared as described previously (9). [7 ,B-Methyl-14C]3a,7 a,12a-trihydroxy-7 ,B-methyl-5 ,13- cholanoic acid (sp act 7.1 x 10 5 cpm/mg). [7a-methyl14C]7-Me-UCA (sp act 7.0 x 10 5 cpm/mg) and [7g-methyl14C]3a, 12a-dihydroxy-7 g-methyl-5,B-cholanoic acid (7Me-DCA) (sp act 5.2 x 10 5 cpm/mg) were synthesized by a Grignard reaction between 2-(3a,12a-dihydroxy-7-oxo-24nor-5,B-cholanyl)-4,4-dimethyl-2-oxazoline and [14C]CH3 MgI (prepared from [14 Clmethyl iodide, 1.0 mCi, sp act 13.2 JLCilJLmol, NEN Products, Boston, Mass.) followed by acid hydrolysis, purification by silica gel chromatography, and hydrogenation, as previously reported (12). [2414C]Cholic acid (sp act 96 JLCi/mg) was purchased from

Intravenous infusion. Before infusion of the bile acid or analogue, saline was infused at a rate of 1.1 mllh for 60 min. Each labeled compound was then administered at different concentrations. In one series ("high concentration") the infusion rate was 50 JLg/min for 20 min and in a second series ("low concentration") the rate was 10 JLg/min for 120 min. In the high concentration studies, bile samples were collected every 20 min for a total period of 4.5 h and, in the low concentration studies, bile was collected every 30 min for 5 h. At the end of the infusion, blood and urine samples were obtained by cardiac puncture and by aspiration of the urinary bladder, and the liver was removed. All the specimens were stored at -20°C until analyzed. Intraduodenal administration. After a control period with intraduodenal administration of saline (1.1 mllh) for 60 min, a bolus of the labeled compound (1.0 mg, 5.0 x 10 5 cpm) was administered through the duodenal catheter.

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Intraduodenal saline was then continued at the same rate. Bile samples were collected every 30 min for 4 h. Blood, urine, and liver were examined for radioactivity. Intragastric administration. The labeled compound (1.0 mg, 5.0 x 105 cpm) was introduced directly into the stomach by stomach tube, and feces and urine were collected for 10 days.

Analytical Techniques Determination of radioactivity. Bile from bile fistula animals was collected in a preweighed tube and its volume was calculated using the specific gravity of the bile (1.032 ± 0.007). A 10-MI bile sample was then removed with a Micropet disposable A-pipette (Clay-Adams) for radioactivity determination. Bile acids in the fecal samples were extracted as described previously (14) and an aliquot of the ethanolic extract was analyzed for radioactivity. All of the samples were analyzed for radioactivity using Aquasol-2 (NEN Products) in a Beckman LS 8000 liquid scintillation system (Beckman Instruments, Fullerton, Calif.). Corrections were made for background and quenching. Thin-layer chromatography. Silica gel 60 F 254 precoated TLC aluminum sheets (0.2-mm thickness; Merck, Darmstadt, F.R.G.) were used for thin-layer chromatography (TLC) analysis. The solvent systems were as follows: system 1 (for conjugated bile acids), n-butanolacetic acid-water 85:10:5; system 2 (for free bile acids), benzene-isopropanol-acetic acid 30: 10: 1. Bile samples (5 MI) were streaked on the plates and reference bile salts were applied as spots at each side. After development (15 cm), bile salts were visualized by spraying the plates with 10% phosphomolybdic acid in ethanol followed by heating at 120°C for 1 min. To localize radioactivity in chromatographed samples, each TLC plates was cut into 5-mm segments from origin to solvent front and each segment was put into a scintillation vial. The radioactive compounds were extracted with methanol and counted as described above. Preliminary purification of urine. After taking an aliquot for determining the total radioactivity excreted in urine, samples containing appreciaele radioactivity (>1 % of the given dose) were analyzed by TLC. Bile salts in urine were extracted and partially purified with a reversedphase cartridge (Sep-Pak C18 ; Waters Associates, Milford, Mass.) (15). The methanol eluate was dried under a stream of nitrogen and redissolved in methanol for direct TLC or in buffer solution for enzymatic hydrolysis, as described below. Enzymatic hydrolysis. Bile (10 Ml) or urine samples were diluted with 2.0 ml of sodium acetate buffer (0.025 M, pH 5.6), 0.2 ml of 0.05 M ethylenediamine tetraacetic acid dis odium salt solution, 0.2 ml of 0.1 M 2-mercaptoethanol solution, and 10 U of cholylglycine hydrolase from Clostridium perfringens (Sigma Chemical, St. Louis, Mo.). The mixture was incubated at 37°C for 18 h. Bile salts were extracted with Sep-Pak C18 cartridges. Preliminary purification of fecal samples. The procedure was fundamentally based on the method of Grundy et al. (14) with modifications. The ethanolic ex-

tract of feces was concentrated by evaporation in vacuo, transferred into a test tube, and diluted with an equal volume of 1 N NaHC0 3 solution. Neutral sterols were extracted three times with hexane (5 ml each time). The aqueous layer was diuted with water to decrease the ethanol concentration below 20%, and the bile salts were extracted with a Sep-Pak C18 cartridge. NaBH4 treatment of fecal bile acids. Aliquots of fecal extracts partially purified as described above were dissolved in 200 MI of ethanol. To the ice-cooled solution, 20 mg of NaBH4 (Sigma Chemical) in 1 ml of ethanol was added and the mixture was kept overnight at room temperature. Saturated NH4Cl solution (5 ml) was added and bile acids were extracted with a Sep-Pak C18 cartridge and analyzed by TLC. Analysis of biliary lipids. Bile samples (10 Ml) were hydrolyzed enzymatically as described above. An ethanolic solution containing 10 Mg of nordeoxycholic acid and 5 Mg of 5a-cholestane (internal standards) was added. Cholesterol and hydrolyzed bile acids were analyzed by gas-liquid chromatography using a 6-ft glass column packed with 3% QF-l on 100/120 Gas Chrom Q as their methyl ester-dimethylethylsilyl ether derivatives as described previously (16,17). Relative retention times of methyl ester-dimethylethylsilyl ether derivatives of bile acids were as follows (CA relative retention times, 1.00; absolute retention time, 19,4 min): 5a-cholestane, 0.16; cholesterol, 0.35; nordeoxycholic acid, 0.55; LA, 0.57; DCA, 0.74; CDA, 0.80; CA, 1.00; 3a-hydroxy-7-oxo-5{3cholanoic acid, 1.70; 3a,12a-dihydroxy-7-oxo-5f3-cholanoic acid, 2.72; 7-Me-DCA, 0.82; 7-Me-CDA, 0.92; 7-MeUDA, 1.05; 7-Me-CA, 1.22; 7-Me-UCA, 1.19. Calculations. The results are expressed as mean ± SD. The significance of differences among the group means was calculated using one-way analysis of variance followed by Student's t-test (18). Biologic half-life of the labeled bile acid was determined as described by Lindstedt and Norman (19).

Results Recovery of Labeled Bile Acid Analogues The experimental design and recovery of the labeled compounds administered by three different routes are summarized in Table 1. In most experiments, the recovery of radioactivity in bile, urine, liver, and plasma was nearly quantitative.

Intravenous Infusion of Labeled 7-Methyl Bile Acids Biliary excretion of the radioactivity after the intravenous infusion of CA, 7-Me-CA, and 7-MeUCA is shown in Figure 1. Cholic acid and 7-Me-CA were rapidly excreted into bile and >90% of the radioactivity was recovered within 20 min after the infusion. 3 a, 7 {3, 12a- Trihydroxy-7 a-methyl-5{3cholanoic acid was secreted into the bile somewhat more slowly, and 8% of the infused 7-Me-UCA was

METABOLISM OF 7-METHYL TRIHYDROXY BILE ACIDS

April 1987

879

Table 1. Experimental Design U and Isotope Recoveries Bile acid administered CA 7-Me-CA 7-Me-UCA 7-Me-UCA 7-Me-CDA 7-Me-UDA 7-Me-DCA CA 7-Me-CA 7-Me-UCA CA 7-Me-CA 7-Me-UCA

Route of administration Intravenous Intravenous Intravenous Intravenous Intravenous Intravenous Intravenous Intraduodenal Intraduodenal Intraduodenal Intragastric Intragastric Intragastric

Percent of recovery

Number of animals b

5 6b 4c 2d 2c 2C 2c 4 3 4 4 4 4

Bile 98.3 ± 2.8 99.4 ± 2.8 90.8 ± 2.9 76.6 ± 8.7 96,3 ± 2.6 96.3 ± 4.3 95.9 ± 4.4 92.9 ± 1.3 97.8 ± 2.7 74.2 ± 6.5 ND ND ND

Urine 0.1 0.2 8.2 20.3 0.2 0.6 0.2 d.l 0.1 0.3 0.5 2.3 2.9

± 0.0 ± ± ± ± ± ± ± ± ± ± ± ±

0.1 3.3 4,5 0.1 0.2 0.1 0.1 0.0 0.2 0.2 1.2 1.2

Total

Liver

Plasma

Feces

0 0 0.4 ± 0.4 0.2 ± 0.0 0 0 0 ND ND ND ND ND NO

0 0 0 0 0 0 0 ND ND ND ND ND ND

ND ND ND ND ND ND ND ND ND ND 84.6 ± 3.6 90.6 ± 3.4 89.4 ± 3.8

98.4 99.6 99.4 97.1 96.5 96.9 96.1 93.0 97.9 74.5 85.1 92.9 92.3

± ± ± ± ± ± ± ± ± ± ± ± ±

2.8 2.8 0.6 4.2 2.5 4.5 4.2 1.3 2.7 6.5 3.6 2.5 3.2

CA, cholic acid; CDA, chenodeoxycholic acid; DCA, deoxycholic acid; ND, not determined; UCA, :la,7 {3, 12a-trihydroxy-5{3-cholanoic acid; UDA, ursodeoxycholic acid; The 14C-Ia:beled bile acids listed in the table were administered intravenously at rates of 50 or 10 p.g/min, intraduodenally (l-mg bolus) or intragastrically (l-n1g bolus). Bile fistula animals were used for the intravenous .and inttadubdenal experiments, intact hamsters for intragastric studies. The data are expressed a~ the mean ± SD. b Includes data at two different infusion rates (50 and 10 p.g/min), as the results were identical. C Data for high infusion rate (50 p.g/minJ. d Data for low infusion rate (10 p.g/min). Q

excreted in urine (Figure 1 and Table 1). Wheh 7-Me-UCA was infused at a lower concentration (10 p,g/min) for a longer period (120 min), 20% of the radioactivity was recovered in urine (Table 1). The urinary radioactivity was present mainly as unconjugated 7-Me-UCA (96.3% ± 3.7%). For comparison, 7-methyl dihydroxycholaIioic acids (7-Me-CDA, 7Me-UDA, and 7-Me-DCA) were also infused at the rate of 50 p,g/min. These compounds were secreted

into the bile as rapidly as the naturally occurring CA and with practically no urinary excretion (Table 1). The conjugation profiles of 7-Me-CA and 7~Me­ UCA in bile usihg the high concentration inftision rate are shown in Figure 2, Considerable amounts of 7-Me-CA (39%) and 7-Me-UGA (65%) weresecreted into bile without prior conjugation with glycine or taurine, whereas CA was almost completely conjugated (Table 2). A decrease of the infusion rate from Solven! Fronl

-

7-Me-CA

7-Me'UCA e9 _8 e7"

>\"

e6

... 50

o CA • 7-Me-CA • 7-Me-UCA

L

3

2

o

e5 e4

\==

e3 e2 e' Origin

o

2

3

RADIOACTIVITY (cpm x 10-')

1

0L---6~0~~12~0--~18~0~~2~4~0~~ TIME(mlh)

Figure 1. Biliary excretion of radioactivity in bile fistula hamsters after intravenous infusion of 14C-Iabeled CA, 7Me-CA, and 7-Me-UCA. After a 60-min control period, the bile acids were infused at a rate of 50 p.g/min for 20 min. The vertical bars indicate one standard deviation from the mean. Radioactivity not recovered in bile after the administration of 7-Me-UCA was found in the urine.

Figure 2. Thin-layer chromatography analysis of radioactivity in bile samples from bile fistula hamsters infused with labeled 7-Me-CA (left) and 7-Me-UCA (right). Bile samples were analyzed on precoated silica gel TLC plates developed in solvent system 1 (n-butanbl-acetic acidwater, 85: 10: 5). Reference compounds (1-9) were as foliows: 1-3, sodium saits of taurine-conjugated 7-MeCA (1), 7-Me-UCA (2), and 7-Me-DCA (3); 4-6, sodium salts of glycine-conjugated 7-Me-CA (4), 7-Me-UCA (5), and 7-MecDCA (6); 7-9, sodium salts of free (unconjugated) 7-Me-CA (7), 7-Me-UCA (8), and 7-Me-DCA (9). Bile samples of animals infused with CA (not shawn) contained 65% taurocholate, 31 % glycocholate, and 4% unconjugated CA.

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Table 2. Conjugation Profiles of Intravenously Infused Bile Acids in Bile Fistula Hamsters U Conjugation profile (%) Infused compound

Number of animals

CA 7-Me-CA 7-Me-UCA 7-Me-CDA 7-Me-UDA 7-Me-DCA

3 4 4 3 2 2

Unconjugated 4.4 39.1 64.9 0.5 3.3 1.2

± ± ± ± ± +

1.5 4.5 b 6.7 b

O.Ob 1.3 0.6

Glycine conjugated

Taurine conjugated

30.8 16.8 12.3 28.9 42.6 40.0

64.8 44.1 22.8 70.6 54.1 58.8

± ± ± ± ± ±

4.6 2.5 b 3.1 b

5.4 4.4 4.7

± ± ± ± ± ±

3.7 3.9 b

4.0b 5.3 3.9 5.3

Glycine/taurine ratio 0.48 0.38 0.54 0.41 0.79 0.68

± ± ± ± ± ±

0.10 0.07 0.09 0.10 0.15 0.15

CA, cholic acid; CDA, chenodeoxycholic acid; DCA, deoxycholic acid; UCA, 3a,7,B,12a-trihydroxY-5,B-cholanoic acid; UDA, urs~deoxy­ cholic acid. a 14C-Labeled bile acids were administered at a rate of 50 ~g/min. Hepatic bile samples were analyzed by thIll-layer chromatography with the solvent system n-butanol-acetic acid-water, 85:10: 5, and by liquid scintillation counting. All data are expressed as the mean ± SD. b Significantly different from the CA conjugation profile (p < 0.05).

50 to 10 JLg/min did not significantly influence the conjugation pattern of the 7-methyl-trihydroxy bile acids (4% of CA, 31% of 7-Me-CA, and 62% of 7-Me-UCA were found unconjugated). Cholylglycine hydrolase treatment of the bile samples followed by radio TLC revealed a single peak of radioactivity at the retardation factor (Hf ) value of unconjugated 7-Me-CA or 7-Me-UCA. In contrast, 7-methyldihydroxy bile acids (7-Me-CDA, 7-Me-UDA, and 7-Me-DCA) were almost completely conjugated even at the high infusion rate (Table 2). Enzymatic hydrolysis of these bile samples also released the unchanged dihydroxY-7-methyl-cholanoic acids. In the hamsters used in the present studies, the ratio of glycine- to taurine-conjugated bile acid analogues (G/T ratio) lies between 0.4 and 0.8 (high rates, 50 JLg/min) (Table 2). The biliary bile acid composition of bile fistula hamsters infused with labeled CA, 7-Me-CA, and 7-Me-UCA at the high infusion rate (50 JLg/min) is shown in Table 3. Hepatic bile during the control period (0-60 min) was composed of CA (65%), CDA (19%), DCA (10%), and 3a,12a-dihydroxy-7-oxo-513cholanoic acid (5%). Lithocholic acid (0.5%) and 3a-hydroxy-7-oxo-513-cholanoic acid (0.5%) were also detected as minor constituents. During the bile acid infusion period (time 60-80 min) and the following 20-min period (time 80-100 min) the proportions of the infused compound in bile increased to about 30% to 40% of the total bile acids (Table 3). The infused bile acids disappeared rapidly from the bile during the next 40 min (time 100-140 min), so that the proportions of the naturally occurring bile acids returned to the control levels during the last infusion period. The proportion of secondary bile acids (DCA and 3a,12a-dihydroxy-7-oxo-513-cholanoic acid) did not change significantly (Table 3) during 4 h of biliary drainage.

Intraduodenal Administration The biliary secretion rate of labeled compounds after intraduodenal administration is shown in Figure 3. More than 90% of administered CA was secreted in the bile within 3 h. 3a,7a,12a-Trihydroxy-713-methyl-513-cholanoic acid was secreted much faster than CA, and nearly 90% of 7-Me-CA was recovered within 1 h. In contrast, 7-Me-UCA appeared in bile much more slowly than CA; recovery was only 74% within 3 h (also see Table 1). Little 7-Me-UCA was excreted in urine after duodenal administration (Table 1). Amidation patterns of these compounds were similar to those found in the intravenous infusion experiments (Table 2). Unconjugated starting material was detected in the bile after the administration of all three bile acids: 3.2% ± 1.5% of CA, 31.8% ± 9.8% of 7-Me-CA, and 66.9% ± 10.9% of 7-Me-UCA were present as the free cholanoic acids. Intragastric Administration Cumulative fecal excretion of radioactivity after the intragastric administration of labeled bile acids is depicted in Figure 4. After CA administration, the radioactivity was excreted with a half-life of 2.1 ± 0.3 day. Cumulative fecal excretion of labeled CA within 10 days was 85% (Table 1). Labeled 7-Me-CA and 7-Me-UCA were excreted more rapidly than CA. Urinary excretion of these compounds was almost identical, amounting to <3% of the administered dose (Table 1). Radio-thin-Iayer chromatography analysis of the fecal metabolites of the labeled compounds administered via stomach tube are shown in Figures 5-7. The major metabolites of CA had Hf values consistent with those of DCA (65% ± 4%) and monohydroxy monoketo-cholanoic acids (3-hydroxy-12-oxo-513cholanoic acid and/or 12a-hydroxy-3-oxo-5{3-

METABOLISM OF 7-METHYL TRIHYDROXY BILE ACIDS

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Table 3. Biliary Bile Acid Composition of Bile Fistula Hamsters Infused Intravenously With Labeled Cholic Acid, 3a,7a,12a-Trihydroxy-7f3-Methyl-5f3-Cholanoic Acid, and 3a,7f3,12a-Trihydroxy-7a-Methyl-5f3-Cholanoic Acid Q Bile acid infused

Infusion time (min)

Bile acid composition(%) DCA

CA

0-20 20-40 40-60 60-80 80-100 100-140 140-200 200-260

9 10 13 6 5 11 13 11

7-Me-CA

0-20 20-40 40-60 60-80 80-100 100-140 140-200 200-260

10 ± 7 13 ± 4 13 ± 7 8±4 8 ± 4 12 ± 6 13 ± 6 12 ± 7

7-Me-UCA

0-20 20-40 40-60 60-80 80-100 100-140 140-200 200-260

6 6 6 5 4 8 8 8

± ± ± ± ± ± ± ±

± ± ± ± ± ± ± ±

2 6 5 2b 2b 1 6 4

8 5 5 5 4 7 8 8

CDA

7-Me-CA

CA

17 17 19 10 8 16 16 16

± ± ± ± ± ± ± ±

3 2 5 3h 3b 8 1 2

65 64 58 80 83 66 63 64

± ± ± ± ± ± ± ±

3 6 4 3h 3b 8 7 5

15 17 19 13 12 16 14 12

± ± ± ± ± ± ± ±

2 2 2 2b 3b

± ± ± ± ± ± ± ±

8 9 9 3b 8h

4 5 5b

70 63 62 40 37 60 65 69

23 21 22 15 12 19 17 17

± ± ± ± ± ± ± ±

12 11 8 3 4h 5 2 2

68 69 68 53 44 60 67 70

± ± ± ± ± ± ± ±

12 13 8 10 6b 5 8 7

5 7 7

7-Me-UCA

7KDCA 9 9 10 4 4 7 8 9

35 40 6 1 1

± ± ± ± ±

6 13 3 1 0

24 37 8 2 1

± ± ± ± ±

13 11 3 1 1

± ± ± ± ± ± ± ±

1 0 1 lb lb 1 1 1

5 ± 7 ± 6 ± 4 ± 3 ± 6 ± 7 ± 6 ±

1 2 2 2 2 4 4 5

3 4 4 3 3 5 6 4

2 3 3 1 1 3 3 3

± ± ± ± ± ± ± ±

DCA, deoxycholic acid; CDA, chenodeoxycholic acid; CA, cholic acid; UCA, 3a,7/3,12a-trihydroxy-5/3-cholanoic acid; 7KDCA, 3a,12a-dihydroxy-7-oxo-5/3-cholanoic acid a After a 60-min control period (time 0-60 min) the 14C-labeled bile acids were infused at a rate of 50 J.Lg/min for 20 min. The bile acids were extracted from the bile and were analyzed by GLC as the methyl ester-dimethylethylsilyl ether. Small proportions «1 % of total bile acids) of lithocholic acid and 3a-hydroxy-7-oxo-5/3-cholanoic acid were detected but are not shown in the table. All data are expressed as the mean ± SD. h Significantly different from the 40-60-min control period by Student's t-test (p < 0.05).

cholanoic acid, 24% ± 5%). Treatment with NaBH4 reduced the ketonic cholanoic acids to DCA (Figure 5). Thin-layer chromatography analysis of fecal extracts of animals fed 7-Me-CA. showed at least six radioactive peaks (Figure 6). Unmetabo.lized 7-MeCA accounted for 31% ± 2% of total radioactivity. Peaks having polarities between 7-Me-CA and 7-MeDCA (32% ± 1% and 7% ± 2%) and a peak having a polarity less than that of 7-Me-DCA (12% ± 3%) gave the original 7-methyl trihydroxy bile acid (Figure 6) after NaBH4 reduction. Two peaks, more polar than 7-Me-CA, were also present and were neither reduced by Na:BH4 treatment nor altered by enzymatic hydrolysis with cholylglycine hydrolase. Because of the small quantities of these metabolites in the biologic samples, we were unable to establish the structure of these compounds. As shown in Figure 7, 7-Me-UCA was metabolized to a lesser extent than 7-Me-CA; 79% ± 9% was recovered unchanged in the feces. The major metabolite (probably a 7methyl-dihydroxy monoketo cholanoic acid, 15% ±

6%) had an Rfvalue between those of 7-Me-UCA and 7-Me-DCA and was reduced by NaBH4 to 7-Me-UCA (Figure 7).

Discussion This study has shown some interesting similarities and differences between the naturally occurring CA and its 7-methyl analogues in the hamster. The intestinal absorption of unconjugated bile acids is thought to take place via nonionic passive diffusion (20), and relatively hydrophobic cholanoic acids are more readily absorbed than the hydrophilic compounds (21). After intra duodenal administration, 7-Me-CA was absorbed more rapidly and 7-MeUCA was absorbed more slowly than CA (Figure 3) so that the rates of absorption of these compounds appeared to decrease with increasing hydrophilicity (12). When conjugated with glycine or taurine, these analogues should be absorbed by the active transport mechanism of the terminal ileum (22).

882

GASTROENTEROLOGY Vol. 92, No.4

KUROKI ET AL.

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Figure 3. Biliary excretion of radioactivity in bile fistula hamsters after intraduodenal administration of labeled bile acids. A bolus (1 mg) of labeled CA, 7-Me-CA, or 7-Me-UCA was administered into the duodenum of bile fistula hamsters, and bile samples were analyzed for radioactivity for 3 h. Urinary excretion of the radioactivity after intraduodenal administration of each compound was minirhai so that the biliary excretion of radioactivity reflects the rate of intestinal absorption of the labeled compounds.

Hepatic extraction of bile acids is very efficient and >90% of the naturally occurring bile acids, both in conjugated or unconjugated form, are extracted by the hepatocytes during a single passage (23). The present study with various 7-methyl bile acids has shown that the methyl group at C-7 will not interfere with the hepatic extraction of these compounds (Figure 1 and Table 1). It is thought that the extraction mechanism of bile acids by hepatocytes involves special carrier proteins and the active transport system of hepatocytes (23,24), Therefore, it might be concluded that these uptake mechanisms have broad substrate specificity and function efficiently even when there is im additional methyl group at the 7-position. The length of the side chain may have a greater effect upon conjugation with glycine or taurine than the structure of the nucleus (25). When a 7c methyl substituent was introduced into CDA and UDA, ami dation was practically complete (10). Consequently, it was expected that introduction of a 7methyl substituent in CA and UCA would riot affect amidation of the carboxyl side chain. However, 7-Me-CA and 7-Me-UCA were not conjugated completely (Figure 2 and Table 2). It was considered that this incomplete conjugation was due to the presence of both the 7-methyl group and the 12a-hydroxyl group. Infusion of 7-Me-DCA, however, at the same concentration revealed that a compound that has both a 7-methyl and a 12a-hydroxyl group but no

Figure 4. Fecal excretion of radioactivity after intragastric administration of labeled bile acids in hamsters. Each bile acid (1 rhg) was administered via stomach tube, and feces were collected for 10 days. Total urinary excretion of each coinpound was <3%.

7-hydroxyl group can be almost completely conjugated (Table 2). A decrease of the infusion rate of 7-Me-CA and 7~Me-UCA to one-fifth (10 /Lg/min) and changing the route of admiilistration (intraduodenal) still resulted in incomplete amidation of these bile acids (Table 2). It is hypothesized that in the C24 trihydroxy bile acids the presence of two substituents in the 7-position plus the 12a-hydroxyl group inhibited enzymatic conjugation. The ratio of glycine- to taurine-conjugated bile acids was higher in 7-Me-UCA treated animals than those receiving CA and 7-Me-CA (Table 2). It is known that the relative rates of formation of glycine and taurine conjugates vary with changes in bile acid structure (25). Glycine might be a preferred substrate of cholanoyl coenzyme A: glycine/taurine-N-acyl-

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Figure 5. Radio-thin-Iayer chromatography analysis of fecal metabolites of [24- '4 C]cholic acid after intragastric adrhinistratioh (1 mg). The fecal bile acids were chromatographed before NaBH4 reduction (left) and after NaBH4 reduction (right). Solvent system 2: benzeneisopropanol-acetic acid, 30: 10: 1. The labeled metabolite having the Rf value of 12a-hydroxy-3-oxo-5f3cholanoic acid (3K) was reduced to DCA by NaBH4.

METABOLISM OF 7-METHYL TRIHYDROXY BILE ACIDS

April 1987

After NaBH4

1.5 RADIOACTIVITY (cpm x 10-')

Figure 6. Radio-thin-layer chromatography analysis of fecal metabolites of hamsters given labeled 7-Me-CA (1 mg) by gastric intubation (solvent system 2). The metabolites consisted of unchanged 7-Me-CA (about 30%) and a considerable proportion of compounds less polar than 7-Me-CA. Most of the less polar material was reduced by NaBH. to a compound having the mobility of 7-MeCA, suggesting that the fecal metabolites of 7-Me-CA were probably 3- or 12-ketones, or both. 3a,12adihydroxy-7 (;-methyl-5f3-cholanoic acid was not formed in appreciable amounts.

transferase of the hamster liver for both 7-Me-UCA and 7-Me-UDA (10). It has been reported that in the hamster certain bile acid analogues (nor-bile acids) are conjugated with glucuronate or sulfate, or both (26). However, the properties of the radioactive metabolites of the 7methyl bile acids were consistent with those expected of unconjugated or amidated bile acids, both in bile (Figure 2) and in urine. Cholylglycine hydrolase treatment of the bile and the urine resulted in the quantitative recovery of unconjugated 7-methyl bile acids. These findings indicate that sulfation or glucuronidation of the analogues may not be a major metabolic pathway in the present acute experiments. After infusion of 7-Me-CA and 7-Me-UCA, the proportion of the administerea bile acid analogue amounted to 30%-40% of total biliary bile acids (Table 3). Because of the presence of the endogenous bile acid pool and endogenous bile acid synthesis, the proportion of the infused exogenous bile acids did not exceed 50% in the present acute experiments. Long-term feeding studies are needed to find out whether the proportions of 7-Me-CA and 7-MeUCA in bile can be maintained at levels of 50% or higher. Generally, the main route for bile acid excretion from the body is via the intestinal tract unless there are some mechanical or pathological obstructions such as bile duct ligation (27), obstructive jaundice (28), or cirrhosis (28-30). In the present study, 7-MeUCA was excreted into the urine after intravenous infusion (8%-20%, Table 1); almost all of the radioactivity in urine was in the form of free (unconju-

883

gated) 7-Me-UCA. In contrast, urinary excretion of 7-Me-UCA was quite low when it was administered into the duodenum or the stomach (Table 1). In the intravenous infusion experiment, 7-Me-UCA administered via the femoral vein goes directly to the heart and enters the systemic circulation before passing through the liver. However, after intraduodenal or intragastric administration, absorbed 7-Me-UCA is transferred through the portal vein to the liver and is extracted efficiently by the liver. Thus, although unchanged 7-Me-UCA can be excreted in the urine due to its hydrophilicity, this is not an important pathway in the hamster when the analogue is administered orally. After intragastric administration of labeled bile acids, the radioactivity was quantitatively recovered in the feces, as the excretion rate of 7-Me-CA and 7-Me-UCA was faster than that of CA (Figure 4). Because of endogenous CA synthesis, the turnover of 7-methyl bile acids cannot be directly compared with that of CA. Chronic feeding of 7-methyl bile acids will be necessary to determine their enterohepatic circulation. To test the hypothesis that 7-methyl bile acids are resistant to bacterial 7-dehydroxylation, the fecal metabolites of CA, 7-Me-CA, and 7-Me-UCA were analyzed after intragastric administration of labeled bile acids. As depicted in Figure 5, CA was 7dehydroxylated to DCA and part of the latter was further oxidized to 12a-hydroxy-3-oxo-513-cholanoic acid or 3a-hydroxy-12-oxo-513-cholanoic acid, or both. Chemical reduction of the ketonic derivatives with NaBH4 yielded DCA. In contrast, 7-Me-CA was resistant to bacterial 7-dehydroxylation. After chemical reduction of the fecal extract, 7-Me-DCA, a potential bacterial metabolite formed by 7-dehydroxylation of 7-Me-CA was not detectable (Figure 6). The major fecal metabolites of 7-Me-CA were thought to be ketonic derivatives as NaBH4 reduction of these Solvent Front Before NaBH4

After

NaBH4

LCA

• •

7'Me-DCA

•

7-Me-CA

Origin

3

2

0

0

2

3

RADIOACTIVITY (cpm x 10-'1

Figure 7. Radio-thin-layer chromatography analysis of fecal metabolites of animals after intragastric administration of 7-Me-UCA (solvent system 2). Most of the administered 7-Me-UCA was recovered unchanged and ketonic metabolite(s) less polar than 7-Me-UCA were reduced to 7-Me-UCA by NaBH. treatment.

884

KUROKI ET AL.

compounds gave 7-Me-CA. 3a,7f3,12a-trihydroxy7a-methyl-5f3-cholanoic acid was more resistant to bacterial transformation; ketonic compounds were produced to a much smaller extent than in the case of 7-Me-CA (Figure 7). The polar metabolites found in the feces of the animals treated with 7-Me-CA were inert to NaBH4 reduction and cholylglycine hydrolase. On the basis of TLC mobilities, these compounds were thought to be neither sulfated nor glucuronidated (31,32). They are probably isomers of trihydroxy-7-methyl bile acids and perhaps tetrahydroxy-7-methyl bile acids (10,33), but further studies are needed to verify this point. In conclusion, trihydroxycholanoic acids having 7-methyl groups were absorbed efficiently by the intestine, rapidly extracted by the liver, and excreted into the bile partly conjugated with taurine or glycine and partly in the free form. The 7-methyl group rendered the cholanoic acids resistant to bacterial 7-dehydroxylation.

References 1. Fromm H. Roat JW. Gonzalez V. et al. Comparative efficacy and side effects of ursodeoxycholic and chenodeoxycholic acids in dissolving gallstones. Gastroenterology 1983;85: 1257-64. 2. Tint GS. Salen G. Colalillo A. et al. Ursodeoxycholic acid: a safe and effective agent for dissolving cholesterol gallstones. Ann Intern Med 1982;97:351-6. 3. Cohen BI. Singhal AK. Stenger RJ. et al. Effects of chenodeoxycholic acid and ursodeoxycholic acid on lipid metabolism and gallstone formation in the prairie dog. Hepatology 1984;4:300-7. 4. Miyai K. Javitt NB. Gochman N. Jones HM. Baker D. Hepatotoxicity of bile acids in rabbits. Ursodeoxycholic acid is less toxic than chenodeoxycholic acid. Lab Invest 1982;46:428-37. 5. Sarva RP. Fromm H. Farivar S. et al. Comparison of the effects between ursodeoxycholic and chenodeoxycholic acids on liver function and structure and on bile acid composition in the Rhesus monkey. Gastroenterology 1980;79:629-36. 6. Turjman N. Nair PP. Nature of ti~sue bound lithocholic acid and its implications in the role of bile acids in carcinogenesis. Cancer Res 1981;41:3761-3. 7. Fedorowski T. Salen G. Colallilo A. Tint GS. Mosbach EH. Hall JC. Metabolism of ursodeoxycholic acid in man. Gastroenterology 1977;73:1131-7. 8. Carey JB Jr. Williams G. Metabolism of lithocholic acid in bile fistula patients. J Clin Invest 1963;42:450-5. 9. Une M. Cohen BI. Mosbach EH. New bile acid analogs: 3a.7 a-dihydroxy-7 ,8-methyl-5,8-cholanoic acid. 3a. 7,8dihydroxy-7a-methyl-5,8-cholanoic acid. and 3a-hydroxy-7methyl-5,8-cholanoic acid. J Lipid Res 1984;25:407-10. 10. Une M. Singhal AK. McSherry CK. May-Donath P. Mosbach EH. Metabolism of 3a.7a-dihydroxy-7,8-methyl-5,8-cholanoic acid and 3a.7,8-dihydroxy-7a-methyl-5f3-cholanoic acid in hamsters. Biochim Biophys Acta 1985;833:196-202. 11. Carulli N. Ponz De Leon M. Loria p. et al. Effect of the selective expansion of cholic acid pool on bile lipid composition: possible mechanism of bile acid induced bilary cholesterol desaturation. Gastroenterology 1981;81:539-46.

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12. Kuroki S. Une M. Mosbach EH. Synthesis of potential cholelitholytic agents: 3a, 7 a.12a-trihydroxy-7 f3-methyl-5f3cholanoic acid. 3a.7 f3.12a-trihydroxy-7 a-methyl-5f3-cholanoic acid. and 3a,12a-dihydroxy-7 g-methyl-5f3-cholanoic acid. J Lipid Res 1985;26:1205-11. 13. Tserng K-Y. Hachey DL. Klein PD. An improved procedure for the synthesis of glycine and taurine conjugates of bile acids. J Lipid Res 1977;18:404-7. 14. Grundy SM, Ahrens EH Jr, Miettinen TA. Quantitative isolation and gas-liquid chromatographic analysis of total fecal bile acids. J Lipid Res 1965;6:397-410. 15. Setchell KDR. Worthington J. A rapid method for the quantitative extraction of bile acids and their conjugates from serum using commerCially available reverse-phase octadecylsilane bonded silica cartridges. Clin Chim Acta 1982;125:135-44. 16. Kuroki S. Muramoto S. Kuramoto T, Hoshita T. Sex differences in gallbladder bile acid composition and hepatic steroid 12a-hydroxylase activity in hamsters. J Lipid Res 1983;24:1543-9. 17. Yanagisawa J. Itoh M, Ishibashi M. Miyazaki H, Nakayama F. Microanalysis of bile acid in human liver tissue by selected ion monitoring. Anal Biochem 1980;104:75-86. 18. Snedecor GW. In: Statistical methods. Ames. Iowa: Iowa State University Press. 1948;54-81. 19. Lindstedt S, Norman A. The turnover of bile acids in the rat. Bile acids and steroids 39. Acta Physiol Scand 1956;38:121-8. 20. Dietschy JM. Salomon HS. Siperstein MD. Bile acid metabolism. 1. Studies on the mechanism of intestinal transport. J Clin Invest 1966;45:832-46. 21. Schiff ER. Dietschy JM. Current concepts of bile acid absorption. Am J Clin Nutr 1969;22:273-8. 22. Lack L. Weiner 1M. Intestinal bile salt transport: structureactivity relationships and other properties. Am J Physiol 1966;210:1142-52. 23. Reichen J. Paumgartner G. Uptake of bile acids by perfused rat liver. Am J PhysioI1976;231:734-42. 24. Ohkuma S. Kuriyama K. Uptake of cholic acid by freshly isolated rat hepatocytes: presence of a common carrier for bile acid transports. Steroids 1982;39:7-19. 25. Cruza B. Vessey DA. The effect of bile acid structure on the activity of bile acid-CoA: glycine/taurine-N-acyltransferase. J Bioi Chern 1982;257:8761-5. 26. Yoon YB. Hagey LR. Hofmann AF, Gurantz 0, Michelotti EL. Steinbach JH. Effect of side-chain shortening on the physiologic properties of bile acids: hepatic transport and effect on biliary secretion of 23-nor-ursodeoxycholate in rodents. Gastroenterology 1986;90:837-52. 27. Galeazzi R, Javitt NB. Bile acid excretion: the alternate pathway in the hamster. J Clin Invest 1977;60:693-701. 28. Summerfield JA. Cullen J. Barnes S. Billing BH. Evidence for renal control of urinary excretion of bile acids and bile acid sulfates in the cholestatic syndrome. Clin Sci Mol Med 1977;52:51-65. 29. Stiehl A. Raedsch R, Rudolph G. Gundert-Remy U. Senn M. Biliary and urinary excretion of sulfated, glucuronidated and tetrahydroxylated bile acids in cirrhotic patients. Hepatology 1985;5:492-5. 30. Bremmelgaard A. Sj6vall J. Bile acid profiles in urine of patients with liver diseases. Eur J Clin Invest 1979;9:341-8. 31. Parmentier G. Eyssen H. Thin-layer chromatography of bile acid sulfates. J Chromatogr 1978;152:285-9. 32. Back p. Bowen DV. Chemical synthesis and characterization of glucuronic acid coupled mono-. di- and trihydroxy bile acids. Hoppe-Seyler's Z Physiol Chern 1976;357:219-24. 33. Tateyama T. Katayama K. Metabolism of chenodeoxycholic acid in hamsters. Lipids 1976;11:845-7.