Metabolism of Chenodeoxycholic Acid in Hamsters TADASHI TATEYAMA and KOUICHI KATAYAMA, Department of Drug Metabolism, Section of Experimental Therapeutics Research,Researchand Development Division, Eisai Co., Ltd., Koishikewa, Bunkyo-ku, Tokyo 112, Japan ABSTRACT
pathogenesis of cholelithiasis (10,11). In this report, the metabolism of CDCA in Golden hamsters was investigated to determine metabolites of CDCA in bile and feces.
The study on the metabolism after oral administration of chenodeoxycholic acid-24-14C was performed by analysis of radioactivity that had appeared in bile and feces of male hamsters. The radioactive bile acids were analyzed by thin layer chromatography and identified by the isotope dilution method. In the bile of the hamsters with bile fistula, radioactivity was originated from unchanged chenodeoxycholic acid for the most part, and 7-ketolithocholic acid, lithocholic acid, and 3-muricholic acid for the remainder. In the feces lithocholic acid, dehydrolithocholic acid, isolithocholic acid, and unchanged form were identified. After the multiple dosing of chenodeoxycholic acid-24-14C for 6 days, 3-muricholic acid was also identified in the feces.
EXPERIMENTAL PROCEDURES
INTRODUCTION
Chenodeoxycholic acid (CDCA), the primary bile acid, has been widely explored as a therapeutic agent for gallstone dissolution because this bile acid decreased the saturation of cholesterol in bile, but cholic acid, the other primary bile acid, did not (1). The metabolism of CDCA has already been studied in the rat (2,3), mouse (4), pig (5), rabbit (6), chicken (7) and man (8,9). However, there has been no report concerning the metabolism of CDCA in the hamster which has been recently used as an experimental animal model for the study of
Chenodeoxycholic acid-24-14 C ( 14 C-CDCA; New England Nuclear Corp., Boston, MA; Specific activity, 54 mCi/mmole) was administered orally (1 mg/kg) to male Golden hamsters (6-9 wk of age) as 0.05% solution of 1% NaHCO3. The bile was collected for 24 hr after the administration of 14C-CDCA in three hamsters with bile fistula. The biliary radioactive bile acids were extracted according to the method of Mahowald et al. (2). The conjugated bile acids were hydrolyzed with 8% NaOH at 80 C for 27 hr in screw-capped tubes. In this method, the conjugated bile acids were completely hydrolyzed and artifacts were not observed. Feces were collected for 7 days after a single administration of 14C-CDCA in three hamsters without bile fistula, and for 12 days in the animals given six equal doses (1 mg/kg) once a day. The feces were dried in vacuo for 24 hr and pulverized. After extracting radioactive bile acids twice by refluxing 0.2 g of the feces in 8% NaOH-ethanol (1:8) for 2 hr, ethanol in the filtrate was evaporated. After the extraction the fecal residue contained radioactivity less than 1% of the total. Before washing out neutral lipids from the fecal extract with petroleum ether, the fecal extract was hydrolyzed at 80 C for 27 hr in screw-capped tubes. The solution was acidified with conc.-HC1 and TABLE II
TABLE I
Nature of Radioactivity Recovered in Bile and Feces after Single Oral Administration of 14C.CDCA
Biliary Excretion of Radioactivity 24 Hours after Single Oral Administration of 14C.CDCA Hamster No.
I
II
III
Recovery in bile (% of dose)
76.5
82.1
75.0
Fraction a
% of total radioactivity
Glyco-CDCA Tauro-CDCA Sum
10.9 78.9 89.9
6.2 81.1 87.3
Compoundsb
6.4 80.0 86.4
aSolvent system: n-butanol/acetic acid/water (10: 1:1).
Dehydrolithocholic acid Isolithocholic acid Lithocholic acid 7-Ketolithocholic acid CDCA Unknown fr.-2 Unknown fr.-I
Bile
%of total Feces
N.D.a N.D. 0.5-0.9 8.0-10.0 75.9-85.2 1.1-5.2 N.D.
2.1-3.7 5.2-8.9 53.1-68.2 Trace 3.3-7.1 2.3-3.8 N.D.
aN.D. = Not detected bSolvent system: benzene/dioxane/acetic acid (75: 20:2.0)
845
846
TADASHI TATEYAMA AND KOUICHI KATAYAMA TABLE llI
TABLE V
Composition Percentage of Radioactivity Recovered in Feces from Day 6 to Day 7 after the Oral Administration of 6 Doses of Equal Dose (1 mg/kg) of 14C-CDCA Once a Day
Identification of/3-Muricholic Acid in Bilea
Compounds a
A
Dehydrolithocholic acid Isolithocholic acid Lithocholic acid 7-Ketolithocholic acid CDCA Unknown fr.-2 Unknown fr.-1
Hamsters
3.0 9.7 29.2 2.2 7.3 3.4 21. S
Recryst..No. B 4.1 6.1 35.2 2.3 12.8 6.7 13.7
aSolvent system: benzene/dioxane/acetic acid (75: 20:2.0) TABLE IV Identification of/3-Murieholic Acid in Fecesa Recryst. No.
Specific activity (x 102 dpm/mg)
1 2 3 4 5
7.04 6.04 4.47 4.50 4.54
a~-Muricholic acid (45.1 mg) was added to 6.08 x 104 dpm (13.5 x 102 dpm/mg) and crystallized from aqueous methanol. the radioactive fecal bile acids were extracted with ether. The biliary and fecal bile acids were analyzed by partition column chromatography (1) or thin layer chromatography (TLC) using Silica Gel 60 plates (E. Merck Co., Darmstadt, Germany). The identification of radioactive bile a c i d s was performed by isotope dilution method. C D C A (Weddel Pharmaceuticals, London, England), lithocholic acid (Tokyo Kasei, Tokyo, Japan), isolithocholic acid (13), dehydrolithocholid (3-keto-5~cholanoic) acid (13), 7-ketolithocholic acid (14), and ~ m u richolic acid (14) were used as standard samples for TLC and isotope dilution methods. The radioactivity was determined by a liquid scintillation counter (Aloka, Model LSC-652, Nihon Musen, Tokyo, Japan). RESULTS A N D DISCUSSION
Table I shows the radioactivity recovered in the bile for 24 hr after a single oral administration of 14C-CDCA in the hamsters with bile fistula, and the percentages of the fractions corresponding to taurochenodeoxycholate and glycochenodeoxycholate. As shown in Table I, LIPIDS, VOL. 11, NO. 12
1 2 3 4 5 6
Specific activity (x 10 2 dpm/mg) 3.53 2.56 2.09 1.77 1.57 1.83
a/~-Muricholic acid (28.6 mg) was added to 3.26 x 104 dpm (11.4 x 102 dpm/mg) and crystallized from aqueous methanol. most of the biliary radioactivity was detected in the fractions of taurine- and glycine- conjugate of CDCA. The hydrolyzates of biliary and fecal bile acids were analyzed by TLC. The percentage composition of CDCA and its metabolites are shown in Table II. The biliary radioactivity was composed mainly of the unchanged 14C-CDCA. This result with hamsters showed a tendency similar to that with rats (2,3), pigs (5), chickens (7) and man (8). The main metabolite of CDCA in the hamster bile was 7-ketolithocholic acid, which has been identified as a metabolite of CDCA in the rat bile (3), the urine of surgically jaundiced mouse (4), and the duodenal fluid of rabbit (6). Lithocholic acid and unknown fr.-2 were observed as minor metabolites in the bile. The unknown fr.-2 corresponded to dihydroxy-bile acid or hyodeoxycholic acid, but not to ursodeoxycholic acid. Fr.-2 was also confirmed to be different from hyodeoxycholic acid by the isotope dilution method. In the feces obtained from the hamsters for a week after the administration of 14C-CDCA, 85-90% of the radioactivity was recovered. Lithocholic acid, isolithocholic acid, and dehydrolithocholic acid were identified as fecal metabolites of CDCA (Table II). 7-Ketolithocholic acid, CDCA, and unknown ft.-2 were also detected in the feces. Lithocholic acid has been reported as the main fecal metabolite of CDCA in rabbits (6) and man (9), as in hamsters. It is, therefore, suggested that most of CDCA administered is transformed to metabolites less polar than CDCA and excreted in feces. Table III shows the percentage composition of radioactivity recovered in feces from day 6 to day 7 after the termination of multiple dosing of 14C_CDCA" The percentage composition of lithocholic acid in the feces of multiple dosing (Table III) was lower than that in a single dosing (Table II). On the other hand, the percentages of unknown fr.-1 corresponding to
METABOLISM OF CHENODEOXYCHOLIC ACID t r i h y d r o x y - b i l e acids o n t h e T L C a n d u n k n o w n fr.-2 were larger in c o m p a r i s o n w i t h t h o s e in a single dosing. T h e u n k n o w n fr.-1 was f u r t h e r a n a l y z e d b y T L C w i t h t h e solvent s y s t e m of chloroform/methanol/acetic acid ( 8 0 : 1 2 : 3 ) (15). A s m a l l p o r t i o n o f t h e r a d i o a c t i v i t y was d e t e c t e d in t h e f r a c t i o n of /~-muricholic acid a n d i d e n t i f i e d as /~-muricholic acid b y t h e isot o p e d i l u t i o n m e t h o d ( T a b l e IV). A similar analysis was p e r f o r m e d o n t h e bile of h a m s t e r s w i t h bile fistula ( T a b l e V). These results i n d i c a t e t h a t t h e small p o r t i o n o f C D C A a d m i n i s t e r e d is t r a n s f o r m e d t o /~-muricholic acid in h a m s t e r s , w h i c h has b e e n k n o w n as a m e t a b o l i t e o f C D C A in t h e m o u s e ( 4 ) a n d t h e rat (2,3). However, ~ - m u r i c h o l i c acid, a m e t a b olite o f C D C A in t h e m o u s e (4) a n d t h e rat (2,3), was n o t d e t e c t e d i n t h e p r e s e n t experiment.
ACKNOWLEDGMENTS
REFERENCES A.F., and G. Paumgartner, Eds., "Chenodeoxycholic Acid Therapy of Gallstones,"
I. Hofmann,
2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14.
We thank Dr. S. Ohtake, Director, Section of Experimental Therapeutics Research, and Dr. J. Tsutsumi, Chief Researcher in our Department, for their helpful advice, and also Mr. A. Yamagishi, Director, Research and Development Division, for his support of our research.
847
15.
F.IC Schattauer Verlag, Stuttgart-New York, 1975. Mahowald, T.A., J.T. Matschiner, S.L. Hsia, R. Richter, E.A. Doisy, Jr., W.H. Elliott, and E.A. Doisy, J. Biol. Chem. 225:781 (1957). Samuelsson, B., Acta Chem. Seand. 13:976 (1959). Ziboh, V.A., J.T. Matschiner, E.A. Doisy, Jr., S.L. Hisia, W., H. Elliott, A. Thayer, and E.A. Doisy, J. Biol. Chem. 236:387 (1961). BergstriSm, S., Acta Chem. Scand. 13:776 (1959). Hellstr6m, K., and J. Sjbvall, Ibid. 14:1763 (1960). Ahlberg, J.W., V.A. Ziboh, R.C. Sonders, and S.L Hsia, Fed. Proc. 20:283.(1961). HellstriSm, IC, and J. Sjbvall, Acta Physiol. Scand. 51:218 (1961). Danielsson, H., P. Eneroth, K. Hellstrbm, S. Lindstedt, and J. Sjbvall, J. Biol. Chem. 238:2299 (1963). Wheeler, H.O., Gastroenterol. 65:92 (1973). Robins, S.J., and J. Fasulo, Ibid. 65:104 (1973). Eneroth, P., J. Lipid Res. 4:11 (1963). Reindel. F., and K. Niederlander, Ber. Deutsch. Chem. 68:1243 (1935). Hsia, S.L., in "The Bile Acids, Chemistry, Physiology, and Metabolism," Vol. 1, Edited by P.P. Nair and D. Kritchevsky, Plenum Press, New York, NY0 1971, p. 95. Siegfield, C.M., and W.H. Elliott, J. Lipid Res. 9:394 (1968). [ R e c e i v e d J u n e 8, 1 9 7 6 ]
LIPIDS, VOL. 11, NO. 12
pathogenesis of cholelithiasis (10,11). In this report, the metabolism of CDCA in Golden hamsters was investigated to determine metabolites of CDCA in bile and feces.
The study on the metabolism after oral administration of chenodeoxycholic acid-24-14C was performed by analysis of radioactivity that had appeared in bile and feces of male hamsters. The radioactive bile acids were analyzed by thin layer chromatography and identified by the isotope dilution method. In the bile of the hamsters with bile fistula, radioactivity was originated from unchanged chenodeoxycholic acid for the most part, and 7-ketolithocholic acid, lithocholic acid, and 3-muricholic acid for the remainder. In the feces lithocholic acid, dehydrolithocholic acid, isolithocholic acid, and unchanged form were identified. After the multiple dosing of chenodeoxycholic acid-24-14C for 6 days, 3-muricholic acid was also identified in the feces.
EXPERIMENTAL PROCEDURES
INTRODUCTION
Chenodeoxycholic acid (CDCA), the primary bile acid, has been widely explored as a therapeutic agent for gallstone dissolution because this bile acid decreased the saturation of cholesterol in bile, but cholic acid, the other primary bile acid, did not (1). The metabolism of CDCA has already been studied in the rat (2,3), mouse (4), pig (5), rabbit (6), chicken (7) and man (8,9). However, there has been no report concerning the metabolism of CDCA in the hamster which has been recently used as an experimental animal model for the study of
Chenodeoxycholic acid-24-14 C ( 14 C-CDCA; New England Nuclear Corp., Boston, MA; Specific activity, 54 mCi/mmole) was administered orally (1 mg/kg) to male Golden hamsters (6-9 wk of age) as 0.05% solution of 1% NaHCO3. The bile was collected for 24 hr after the administration of 14C-CDCA in three hamsters with bile fistula. The biliary radioactive bile acids were extracted according to the method of Mahowald et al. (2). The conjugated bile acids were hydrolyzed with 8% NaOH at 80 C for 27 hr in screw-capped tubes. In this method, the conjugated bile acids were completely hydrolyzed and artifacts were not observed. Feces were collected for 7 days after a single administration of 14C-CDCA in three hamsters without bile fistula, and for 12 days in the animals given six equal doses (1 mg/kg) once a day. The feces were dried in vacuo for 24 hr and pulverized. After extracting radioactive bile acids twice by refluxing 0.2 g of the feces in 8% NaOH-ethanol (1:8) for 2 hr, ethanol in the filtrate was evaporated. After the extraction the fecal residue contained radioactivity less than 1% of the total. Before washing out neutral lipids from the fecal extract with petroleum ether, the fecal extract was hydrolyzed at 80 C for 27 hr in screw-capped tubes. The solution was acidified with conc.-HC1 and TABLE II
TABLE I
Nature of Radioactivity Recovered in Bile and Feces after Single Oral Administration of 14C.CDCA
Biliary Excretion of Radioactivity 24 Hours after Single Oral Administration of 14C.CDCA Hamster No.
I
II
III
Recovery in bile (% of dose)
76.5
82.1
75.0
Fraction a
% of total radioactivity
Glyco-CDCA Tauro-CDCA Sum
10.9 78.9 89.9
6.2 81.1 87.3
Compoundsb
6.4 80.0 86.4
aSolvent system: n-butanol/acetic acid/water (10: 1:1).
Dehydrolithocholic acid Isolithocholic acid Lithocholic acid 7-Ketolithocholic acid CDCA Unknown fr.-2 Unknown fr.-I
Bile
%of total Feces
N.D.a N.D. 0.5-0.9 8.0-10.0 75.9-85.2 1.1-5.2 N.D.
2.1-3.7 5.2-8.9 53.1-68.2 Trace 3.3-7.1 2.3-3.8 N.D.
aN.D. = Not detected bSolvent system: benzene/dioxane/acetic acid (75: 20:2.0)
845
846
TADASHI TATEYAMA AND KOUICHI KATAYAMA TABLE llI
TABLE V
Composition Percentage of Radioactivity Recovered in Feces from Day 6 to Day 7 after the Oral Administration of 6 Doses of Equal Dose (1 mg/kg) of 14C-CDCA Once a Day
Identification of/3-Muricholic Acid in Bilea
Compounds a
A
Dehydrolithocholic acid Isolithocholic acid Lithocholic acid 7-Ketolithocholic acid CDCA Unknown fr.-2 Unknown fr.-1
Hamsters
3.0 9.7 29.2 2.2 7.3 3.4 21. S
Recryst..No. B 4.1 6.1 35.2 2.3 12.8 6.7 13.7
aSolvent system: benzene/dioxane/acetic acid (75: 20:2.0) TABLE IV Identification of/3-Murieholic Acid in Fecesa Recryst. No.
Specific activity (x 102 dpm/mg)
1 2 3 4 5
7.04 6.04 4.47 4.50 4.54
a~-Muricholic acid (45.1 mg) was added to 6.08 x 104 dpm (13.5 x 102 dpm/mg) and crystallized from aqueous methanol. the radioactive fecal bile acids were extracted with ether. The biliary and fecal bile acids were analyzed by partition column chromatography (1) or thin layer chromatography (TLC) using Silica Gel 60 plates (E. Merck Co., Darmstadt, Germany). The identification of radioactive bile a c i d s was performed by isotope dilution method. C D C A (Weddel Pharmaceuticals, London, England), lithocholic acid (Tokyo Kasei, Tokyo, Japan), isolithocholic acid (13), dehydrolithocholid (3-keto-5~cholanoic) acid (13), 7-ketolithocholic acid (14), and ~ m u richolic acid (14) were used as standard samples for TLC and isotope dilution methods. The radioactivity was determined by a liquid scintillation counter (Aloka, Model LSC-652, Nihon Musen, Tokyo, Japan). RESULTS A N D DISCUSSION
Table I shows the radioactivity recovered in the bile for 24 hr after a single oral administration of 14C-CDCA in the hamsters with bile fistula, and the percentages of the fractions corresponding to taurochenodeoxycholate and glycochenodeoxycholate. As shown in Table I, LIPIDS, VOL. 11, NO. 12
1 2 3 4 5 6
Specific activity (x 10 2 dpm/mg) 3.53 2.56 2.09 1.77 1.57 1.83
a/~-Muricholic acid (28.6 mg) was added to 3.26 x 104 dpm (11.4 x 102 dpm/mg) and crystallized from aqueous methanol. most of the biliary radioactivity was detected in the fractions of taurine- and glycine- conjugate of CDCA. The hydrolyzates of biliary and fecal bile acids were analyzed by TLC. The percentage composition of CDCA and its metabolites are shown in Table II. The biliary radioactivity was composed mainly of the unchanged 14C-CDCA. This result with hamsters showed a tendency similar to that with rats (2,3), pigs (5), chickens (7) and man (8). The main metabolite of CDCA in the hamster bile was 7-ketolithocholic acid, which has been identified as a metabolite of CDCA in the rat bile (3), the urine of surgically jaundiced mouse (4), and the duodenal fluid of rabbit (6). Lithocholic acid and unknown fr.-2 were observed as minor metabolites in the bile. The unknown fr.-2 corresponded to dihydroxy-bile acid or hyodeoxycholic acid, but not to ursodeoxycholic acid. Fr.-2 was also confirmed to be different from hyodeoxycholic acid by the isotope dilution method. In the feces obtained from the hamsters for a week after the administration of 14C-CDCA, 85-90% of the radioactivity was recovered. Lithocholic acid, isolithocholic acid, and dehydrolithocholic acid were identified as fecal metabolites of CDCA (Table II). 7-Ketolithocholic acid, CDCA, and unknown ft.-2 were also detected in the feces. Lithocholic acid has been reported as the main fecal metabolite of CDCA in rabbits (6) and man (9), as in hamsters. It is, therefore, suggested that most of CDCA administered is transformed to metabolites less polar than CDCA and excreted in feces. Table III shows the percentage composition of radioactivity recovered in feces from day 6 to day 7 after the termination of multiple dosing of 14C_CDCA" The percentage composition of lithocholic acid in the feces of multiple dosing (Table III) was lower than that in a single dosing (Table II). On the other hand, the percentages of unknown fr.-1 corresponding to
METABOLISM OF CHENODEOXYCHOLIC ACID t r i h y d r o x y - b i l e acids o n t h e T L C a n d u n k n o w n fr.-2 were larger in c o m p a r i s o n w i t h t h o s e in a single dosing. T h e u n k n o w n fr.-1 was f u r t h e r a n a l y z e d b y T L C w i t h t h e solvent s y s t e m of chloroform/methanol/acetic acid ( 8 0 : 1 2 : 3 ) (15). A s m a l l p o r t i o n o f t h e r a d i o a c t i v i t y was d e t e c t e d in t h e f r a c t i o n of /~-muricholic acid a n d i d e n t i f i e d as /~-muricholic acid b y t h e isot o p e d i l u t i o n m e t h o d ( T a b l e IV). A similar analysis was p e r f o r m e d o n t h e bile of h a m s t e r s w i t h bile fistula ( T a b l e V). These results i n d i c a t e t h a t t h e small p o r t i o n o f C D C A a d m i n i s t e r e d is t r a n s f o r m e d t o /~-muricholic acid in h a m s t e r s , w h i c h has b e e n k n o w n as a m e t a b o l i t e o f C D C A in t h e m o u s e ( 4 ) a n d t h e rat (2,3). However, ~ - m u r i c h o l i c acid, a m e t a b olite o f C D C A in t h e m o u s e (4) a n d t h e rat (2,3), was n o t d e t e c t e d i n t h e p r e s e n t experiment.
ACKNOWLEDGMENTS
REFERENCES A.F., and G. Paumgartner, Eds., "Chenodeoxycholic Acid Therapy of Gallstones,"
I. Hofmann,
2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14.
We thank Dr. S. Ohtake, Director, Section of Experimental Therapeutics Research, and Dr. J. Tsutsumi, Chief Researcher in our Department, for their helpful advice, and also Mr. A. Yamagishi, Director, Research and Development Division, for his support of our research.
847
15.
F.IC Schattauer Verlag, Stuttgart-New York, 1975. Mahowald, T.A., J.T. Matschiner, S.L. Hsia, R. Richter, E.A. Doisy, Jr., W.H. Elliott, and E.A. Doisy, J. Biol. Chem. 225:781 (1957). Samuelsson, B., Acta Chem. Seand. 13:976 (1959). Ziboh, V.A., J.T. Matschiner, E.A. Doisy, Jr., S.L. Hisia, W., H. Elliott, A. Thayer, and E.A. Doisy, J. Biol. Chem. 236:387 (1961). BergstriSm, S., Acta Chem. Scand. 13:776 (1959). Hellstr6m, K., and J. Sjbvall, Ibid. 14:1763 (1960). Ahlberg, J.W., V.A. Ziboh, R.C. Sonders, and S.L Hsia, Fed. Proc. 20:283.(1961). HellstriSm, IC, and J. Sjbvall, Acta Physiol. Scand. 51:218 (1961). Danielsson, H., P. Eneroth, K. Hellstrbm, S. Lindstedt, and J. Sjbvall, J. Biol. Chem. 238:2299 (1963). Wheeler, H.O., Gastroenterol. 65:92 (1973). Robins, S.J., and J. Fasulo, Ibid. 65:104 (1973). Eneroth, P., J. Lipid Res. 4:11 (1963). Reindel. F., and K. Niederlander, Ber. Deutsch. Chem. 68:1243 (1935). Hsia, S.L., in "The Bile Acids, Chemistry, Physiology, and Metabolism," Vol. 1, Edited by P.P. Nair and D. Kritchevsky, Plenum Press, New York, NY0 1971, p. 95. Siegfield, C.M., and W.H. Elliott, J. Lipid Res. 9:394 (1968). [ R e c e i v e d J u n e 8, 1 9 7 6 ]
LIPIDS, VOL. 11, NO. 12
