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P-217 Epimerization of isoursodeoxycholic acid to ursodeoxycholic acid in the rat liver
Posters/International.Hepatology Communications 3 Suppl. (1995) $37-S169
$91
P-217 EPIMERIZATION OF ISOURSODEOXYCHOLIC ACID TO URSODEOXYCHOLIC ACID IN THE RAT LIVER. K. Nakamura, S. Yokohama, K. Tamori, Y. Sato, M. Fujita, A. Kimura, K. Baba, T. Hasegawa, H. Saito, M.Yoneda and 1. Makino. 2nd Dep. of Medicine, Asahikawa Med. College, Asahikawa, Japan
P-218 EFFECTS OF URSODEOXYCHOLATE (UDC)AND ITS CONJUGATES ON BILIARY GLUTATHIONE EXCRETION K. Kitaura, H. Takikawa, N. Sano, R. Yamazaki, S. Fukumura, Y. Wako, M. Fujiyoshi, Y. Kuyama, K. Miyake, M. Yamanaka !st Dept. of Medicine, Teikyo Univ. Sch. of Medicine, Tokyo
Isoursodeoxycholic acid (isoUDCA), the 3/3-hydroxyepimer of ursodeoxycholic acid (UDCA) was isolated from the serum and the urine of patients treated with UDCA. Since isoUDCA could not be detected in the bile, it was suggested that isoUDCA was metabolized in the liver. The present study was undertaken to investigate the hepatic metabolism of isoUDCA in the rat. Methods: Male Wistar rats (n=3) with the bile fistula were intravenously administered with isoUDCA (50mg/kg, BW) dissolved in 1 ml of carbonate buffer (pH 8.0). Bile was collected evely 10 min and bile acids were determined by GLC. In the second study, the rat livers (n=5) were homogenated with 0.1M PBS (pH 7.4) and the protein concentration of the homogenate was adjusted to 30 mg/ml. IsoUDCA was icubated with 1 ml of the liver homogenate and the effects of incubation time, substrate dose, coenzyme (NADPH or NADH) and HgCI2 were studied. Bile acids in the liver homogenate were eluted and purified by Bond elut and determined by GLC. Results: 67.7 -+ 2,6% of administered isoUDCA (Mean _+ SD) was recovered as UDCA in the bile during 60 rain and ~-2% was recovered during first l0 rain. However, only 0.6 + 0.2% was recovered as isoUDCA. In vitro study, isoUDCA was epimerized to UDCA dose- and timedependently, and this epimefization was promoted by the addition of coenzyme, although NADPH was more effective than NADH. Forethermore, in the addition of HgC12, the epimerization of isoUDCA were decreased by 14%. Conclusion: These results suggest the epimerization of isoUDCA to UDCA in the rat liver.
Biliary excretion of glutathione is considered to be mediated by an ATP-in dependent electrogenie transport system. We previously reported that some organic anions and bile acid conjugates such as UDC-3-O-gluouronide (3-glcA-UDC) inhibited biUaryglutathione excretion. Herein, we studied the effects of UDC, its tauroconjugate (UDC4au) and sulfates (3-suI-UDC and 3,7-suI-UDC) on biliary glutathione excretion in male SD rats. Pdorto the bile duct cannulation, acivicin was retrograde injected. Bile acids were injected as a bolus viathe femoral vein (2 or 10 ~.mol/100g) and bile samples were collected every 15 min. Biliary glutathione levels were determined bythe recycling assay. Although bilellow(l~l/min/100g) was increased by UDC (10) from 3.8 to 7.5 and remained increased, biliary gtutathione excretion was only trensitionally increased to t 24%. Bile flow was increased by UDC4au (10) to 5.7 and biliary glutathione excretion was increased to 154% during the lirst 15 min in parallel to rapid UDC4au excretion. Biliaryexcretien of 3-suI-UDC (2) was peaked at the first 15 min and bile llow increased to 4.1, but biliary glutathione excretion did not change (103%). Biliary excretion of 3,7-suI-UDC was peaked between 15 and 30 rain and bile flow was increased to 5.2. Biliary glutathione excretion was transitionally decreased to 63%. In conclusion, the increase in biliary glutathione excretion was not parallel to UDC excretion, possibly due to the production of 3-glcA-UDC which inhibited glutathione excretion. Although 3-glcA-UDC (2) completely inhibited biliary excretion, UDC sulfates did not or only slightly inhibited glutathione excretion, suggesting th at th e excretory pathways for the g lucuronide and the sulfate are at least partially different.
P-219
URSODEOXYCHOLIC ACID (UDCA) INCREASES BILIARY SECRETION OF GLUTATHIONE (GSH) H.Yoshida, K . O h m u r a , K.Onuki, K.Okano Div. o f G a s t r o e n t e r o l o g y , I n s t i t u t e for A d u l t Diseases, A s a h i Life F o u n d a t i o n , S h i n j u k u , J a p a n UDCA is n o w w i d e l y u s e d i n t h e t r e a t m e n t of p r i m a r y b i l i a r y c i r r h o s i s (PBC) b u t its m e c h a n i s m o f a c t i o n is p o o r l y u n d e r s t o o d . W e i n v e s t i g a t e d t h e effects of UDCA o n b i l i a r y s e c r e t i o n of e n d o g e n o u s GSH. [METHODS] UDCA w a s a d m i n i s t e r e d i.v. to m a l e S p r a g u e - D a w l e y r a t s t r e a t e d w i t h a c i v i c i n e i t h e r as b o l u s ( 2 0 0 u m o l / k g , G r o u p I ) o r as c o n t i n u o u s i n f u s i o n (5 u m o l / k g / m i n , G r o u p II). In G r o u p III, r a t s w e r e t r e a t e d w i t h c o l c h i c i n e a n d c h o l a t e p r i o r to UDCA ( 2 0 0 u m o l / k g ) . {RESULTS] (I)Biliary GSH s e c r e t i o n r a t e s w e r e 1 8 4 % a n d 152% of its i n i t i a l l e v e l 10 a n d 2 0 r a i n a f t e r UDCA i n j e c t i o n , r e s p e c t i v e l y . (II)GSH s e c r e t i o n r a t e w a s i n c r e a s e d d u r i n g UDCA i n f u s i o n , b e i n g 1 9 9 % o f its i n i t i a l l e v e l b y 7 0 m i n of i n f u s i o n . (III)UDCA a d m i n i s t r a t i o n i n t o c o l c h i c i n e c h o l a t e t r e a t e d r a t s r e s u l t e d i n a r a p i d r e c o v e r y o f GSH s e c r e t i o n . GSH s e c r e t i o n r a t e a t 2 0 r a i n a f t e r UDCA a d m i n i s t r a t i o n w a s 2 1 0 % of t h a t i n c o n t r o l s . [CONCLUSION] UDCA i n c r e a s e s b i l i a r y s e c r e t i o n of GSH f r o m h e p a t o c y t e s , e v e n i n t h e c h o l e s t a t i c r a t s t r e a t e d w i t h c o l c h i c i n e a n d c h o l a t e . UDCA m a y i n c r e a s e b i l i a r y GSH s e c r e t i o n i n p a t i e n t s w i t h c h o l e s t a t i c d i s e a s e s s u c h as PBC a n d t h e bile d u c t cells m a y b e s u p p l i e d w i t h m o r e GSH.
P'~20 ENHANCING EFFECTS OF URSODEOXYCHOLIC ACID (UDCA) ON GLUTATRIONE S-TRANSFERASE (GST) ENZYME ACTIVITIES AND I S O Z Y ~ CONCENTR~IO~S IN MOUSE LIVER CYTOSOL. S. Kanai , K. Kitanl , Dept. ~lin. Physiol., Tokyo Metropolitan Inst. Gerontol. Radioisotope Res. Inst., Faculty of Med., Univ. Tokyo, Tokyo, Japan The authors have previously shown that oral administration of UDCA for 3 wks slgnifleantiy increases GST activities in mouse liver cytosol fractions (Life Sci. 54, 983, 1994). The present study aimed to clarify the possible difference between male and female mice for thls effect and, further, to clarify changes in three GST isozymes (GST-I, II, III). A 3 wk feeding of a UDCA containing diet (0.3 %) significantly increased GST actlvaties towards l-chloro-2,4-dinltrobenzene (CDNB) and 1,2-dlchloro-4-nitrobenzene (DCNB) compared with respective values in control animals, however, the increase was generally greater (4 times) in male than in female (2 times) mice. Immunoblot analysis using three suhunits (m-1,2,3) for three GST isozymes (GST-I, II, III, respectively) further revealed that isozyme III was selectively induced by UDCA treatment. The results suggest that the GST induction by UDCA is unique among inducers known for GST, since it is generally known that GST induction is greater in female mice than in males. Furthermore, this induction was not related to any precancerous lesion which is known to be caused by an induction of GST II.
$91
P-217 EPIMERIZATION OF ISOURSODEOXYCHOLIC ACID TO URSODEOXYCHOLIC ACID IN THE RAT LIVER. K. Nakamura, S. Yokohama, K. Tamori, Y. Sato, M. Fujita, A. Kimura, K. Baba, T. Hasegawa, H. Saito, M.Yoneda and 1. Makino. 2nd Dep. of Medicine, Asahikawa Med. College, Asahikawa, Japan
P-218 EFFECTS OF URSODEOXYCHOLATE (UDC)AND ITS CONJUGATES ON BILIARY GLUTATHIONE EXCRETION K. Kitaura, H. Takikawa, N. Sano, R. Yamazaki, S. Fukumura, Y. Wako, M. Fujiyoshi, Y. Kuyama, K. Miyake, M. Yamanaka !st Dept. of Medicine, Teikyo Univ. Sch. of Medicine, Tokyo
Isoursodeoxycholic acid (isoUDCA), the 3/3-hydroxyepimer of ursodeoxycholic acid (UDCA) was isolated from the serum and the urine of patients treated with UDCA. Since isoUDCA could not be detected in the bile, it was suggested that isoUDCA was metabolized in the liver. The present study was undertaken to investigate the hepatic metabolism of isoUDCA in the rat. Methods: Male Wistar rats (n=3) with the bile fistula were intravenously administered with isoUDCA (50mg/kg, BW) dissolved in 1 ml of carbonate buffer (pH 8.0). Bile was collected evely 10 min and bile acids were determined by GLC. In the second study, the rat livers (n=5) were homogenated with 0.1M PBS (pH 7.4) and the protein concentration of the homogenate was adjusted to 30 mg/ml. IsoUDCA was icubated with 1 ml of the liver homogenate and the effects of incubation time, substrate dose, coenzyme (NADPH or NADH) and HgCI2 were studied. Bile acids in the liver homogenate were eluted and purified by Bond elut and determined by GLC. Results: 67.7 -+ 2,6% of administered isoUDCA (Mean _+ SD) was recovered as UDCA in the bile during 60 rain and ~-2% was recovered during first l0 rain. However, only 0.6 + 0.2% was recovered as isoUDCA. In vitro study, isoUDCA was epimerized to UDCA dose- and timedependently, and this epimefization was promoted by the addition of coenzyme, although NADPH was more effective than NADH. Forethermore, in the addition of HgC12, the epimerization of isoUDCA were decreased by 14%. Conclusion: These results suggest the epimerization of isoUDCA to UDCA in the rat liver.
Biliary excretion of glutathione is considered to be mediated by an ATP-in dependent electrogenie transport system. We previously reported that some organic anions and bile acid conjugates such as UDC-3-O-gluouronide (3-glcA-UDC) inhibited biUaryglutathione excretion. Herein, we studied the effects of UDC, its tauroconjugate (UDC4au) and sulfates (3-suI-UDC and 3,7-suI-UDC) on biliary glutathione excretion in male SD rats. Pdorto the bile duct cannulation, acivicin was retrograde injected. Bile acids were injected as a bolus viathe femoral vein (2 or 10 ~.mol/100g) and bile samples were collected every 15 min. Biliary glutathione levels were determined bythe recycling assay. Although bilellow(l~l/min/100g) was increased by UDC (10) from 3.8 to 7.5 and remained increased, biliary gtutathione excretion was only trensitionally increased to t 24%. Bile flow was increased by UDC4au (10) to 5.7 and biliary glutathione excretion was increased to 154% during the lirst 15 min in parallel to rapid UDC4au excretion. Biliaryexcretien of 3-suI-UDC (2) was peaked at the first 15 min and bile llow increased to 4.1, but biliary glutathione excretion did not change (103%). Biliary excretion of 3,7-suI-UDC was peaked between 15 and 30 rain and bile flow was increased to 5.2. Biliary glutathione excretion was transitionally decreased to 63%. In conclusion, the increase in biliary glutathione excretion was not parallel to UDC excretion, possibly due to the production of 3-glcA-UDC which inhibited glutathione excretion. Although 3-glcA-UDC (2) completely inhibited biliary excretion, UDC sulfates did not or only slightly inhibited glutathione excretion, suggesting th at th e excretory pathways for the g lucuronide and the sulfate are at least partially different.
P-219
URSODEOXYCHOLIC ACID (UDCA) INCREASES BILIARY SECRETION OF GLUTATHIONE (GSH) H.Yoshida, K . O h m u r a , K.Onuki, K.Okano Div. o f G a s t r o e n t e r o l o g y , I n s t i t u t e for A d u l t Diseases, A s a h i Life F o u n d a t i o n , S h i n j u k u , J a p a n UDCA is n o w w i d e l y u s e d i n t h e t r e a t m e n t of p r i m a r y b i l i a r y c i r r h o s i s (PBC) b u t its m e c h a n i s m o f a c t i o n is p o o r l y u n d e r s t o o d . W e i n v e s t i g a t e d t h e effects of UDCA o n b i l i a r y s e c r e t i o n of e n d o g e n o u s GSH. [METHODS] UDCA w a s a d m i n i s t e r e d i.v. to m a l e S p r a g u e - D a w l e y r a t s t r e a t e d w i t h a c i v i c i n e i t h e r as b o l u s ( 2 0 0 u m o l / k g , G r o u p I ) o r as c o n t i n u o u s i n f u s i o n (5 u m o l / k g / m i n , G r o u p II). In G r o u p III, r a t s w e r e t r e a t e d w i t h c o l c h i c i n e a n d c h o l a t e p r i o r to UDCA ( 2 0 0 u m o l / k g ) . {RESULTS] (I)Biliary GSH s e c r e t i o n r a t e s w e r e 1 8 4 % a n d 152% of its i n i t i a l l e v e l 10 a n d 2 0 r a i n a f t e r UDCA i n j e c t i o n , r e s p e c t i v e l y . (II)GSH s e c r e t i o n r a t e w a s i n c r e a s e d d u r i n g UDCA i n f u s i o n , b e i n g 1 9 9 % o f its i n i t i a l l e v e l b y 7 0 m i n of i n f u s i o n . (III)UDCA a d m i n i s t r a t i o n i n t o c o l c h i c i n e c h o l a t e t r e a t e d r a t s r e s u l t e d i n a r a p i d r e c o v e r y o f GSH s e c r e t i o n . GSH s e c r e t i o n r a t e a t 2 0 r a i n a f t e r UDCA a d m i n i s t r a t i o n w a s 2 1 0 % of t h a t i n c o n t r o l s . [CONCLUSION] UDCA i n c r e a s e s b i l i a r y s e c r e t i o n of GSH f r o m h e p a t o c y t e s , e v e n i n t h e c h o l e s t a t i c r a t s t r e a t e d w i t h c o l c h i c i n e a n d c h o l a t e . UDCA m a y i n c r e a s e b i l i a r y GSH s e c r e t i o n i n p a t i e n t s w i t h c h o l e s t a t i c d i s e a s e s s u c h as PBC a n d t h e bile d u c t cells m a y b e s u p p l i e d w i t h m o r e GSH.
P'~20 ENHANCING EFFECTS OF URSODEOXYCHOLIC ACID (UDCA) ON GLUTATRIONE S-TRANSFERASE (GST) ENZYME ACTIVITIES AND I S O Z Y ~ CONCENTR~IO~S IN MOUSE LIVER CYTOSOL. S. Kanai , K. Kitanl , Dept. ~lin. Physiol., Tokyo Metropolitan Inst. Gerontol. Radioisotope Res. Inst., Faculty of Med., Univ. Tokyo, Tokyo, Japan The authors have previously shown that oral administration of UDCA for 3 wks slgnifleantiy increases GST activities in mouse liver cytosol fractions (Life Sci. 54, 983, 1994). The present study aimed to clarify the possible difference between male and female mice for thls effect and, further, to clarify changes in three GST isozymes (GST-I, II, III). A 3 wk feeding of a UDCA containing diet (0.3 %) significantly increased GST actlvaties towards l-chloro-2,4-dinltrobenzene (CDNB) and 1,2-dlchloro-4-nitrobenzene (DCNB) compared with respective values in control animals, however, the increase was generally greater (4 times) in male than in female (2 times) mice. Immunoblot analysis using three suhunits (m-1,2,3) for three GST isozymes (GST-I, II, III, respectively) further revealed that isozyme III was selectively induced by UDCA treatment. The results suggest that the GST induction by UDCA is unique among inducers known for GST, since it is generally known that GST induction is greater in female mice than in males. Furthermore, this induction was not related to any precancerous lesion which is known to be caused by an induction of GST II.