79
Biochimica et Biophysica Acta, 399 ( 1 9 7 5 ) 7 9 - - 8 4 © Elsevier Scientific P u b l i s h i n g C o m p a n y , A m s t e r d a m - - P r i n t e d in T h e N e t h e r l a n d s
BBA 27677
E F F E C T OF 1 7 a - E T H I N Y L E S T R A D I O L ON B I L I A R Y E X C R E T I O N OF BILE ACIDS
HISAKO WATANABE
Drug Research Laboratories, Health Protection Branch, Department of National Health and Welfare, Ottawa, Ontario, K I A OL2 (Canada) (Received January 29th, 1975)
Summary The effect of 17a-ethinylestradiol on biliary bile acids has been investigated. The ratio of cholate to c h e n o d e o x y c h o l a t e was diminished by the estrogen in cholestyramine-treated rats. With low doses, this effect was due to increased excretion of chenodeoxycholate. With the highest dose, the decreased ratio was due to a reduction in the levels of cholic acid. In the intermediate dosage range, b o t h factors contributed to the decreased ratio. Prolonged treatment with 500 ;~g daily of 17a-ethinylestradiol produced a reduction in the excretory rate of both bile acids in animals treated or n o t treated with cholestyramine.
Introduction
The precise nature of the estrogen effect on bile acid metabolism and excretion has n o t been clearly established. Total bile acid excretion has been reported to be inhibited by estriol in m o n k e y s [1] and minimally affected by 17a-ethinylestradiol in the rat [ 2 ] . The maximal excretory capacity was markedly reduced by 17a-ethinylestradiol. On the other hand, an elevation in biliary bile acids from radiolabelled cholesterol was observed with estrone in a liver perfusion system [3]. The ratio of bile acids originating from cholic to those from chenodeoxycholic acid was decreased in rats treated with 17~-estradiol [4] whereas the ratio of trihydroxylated to dihydroxylated bile acids was reported to remain unaltered b y estrone with the isolated liver perfusion preparation [3]. *Nomenclature:
choHc
acid,
3a,7~,12a-trihydroxy-5~-cholanoic
acid; c h e n o d e o x y c h o l i c
acid,
3a,7~-dihydroxy-5~-cholanoic acid; 17~-ethinylestradiol, 1,3,5(lO)-estratrien-17a-ethinyl-3,17~diol; estrone, 3-hydroxy-1,3,5(lO)-estratrien-17-one; 17~-estradio1, 103,5(lO)-estxatriene-3,17~-diol; estriol, 1,3,5(1O)-estratxiene-3,16a,17fl-triol; mestranol, 1,3,5(lO)-estratrien-17~-ethinyl-3,17~-diol 3-methyl ether; norethindrone, 17~-ethinyl-17~-hydroxyestra-4-en-3-one; megestrol acetate, 17ahydroxy-6-methyl-4,6-pregnadiene-3,20-dione acetate.
80 Since 17a-ethinylestradiol has been shown to produce cholestasis [ 5--7], which may be due, at least in part, to inhibition of bile salt-dependent flow [1], it was of interest to investigate some of the conditions which may influence the effect of this c o m p o u n d on bile acid excretion and metabolism. Materials and Methods
Materials. Steroids and bile acids were obtained from Steraloids Inc., Pawling, N.Y.; and cholestyramine from Merck, Sharpe and Dohme, West Point, Pa. Treatment o f animals. Female Wistar rats, 165--185 g, maintained on fox cube diets, were variously treated as indicated prior to preparation for bile collections. Under urethane anesthesia, the c o m m o n bile duct was cannulated with polyethylene tubing (PE 10, Clay Adams, Parsipany, N . J . ) a n d samples collected over a period of 3 h. Extraction o f bile acids. Bile acids were extracted and hydrolyzed according to the m e t h o d as described b y Lin et al. [8]. Bile samples, deproteinized with ethanol were extracted with light petroleum before and after acidification with 3 M HC1 to remove neutral lipids and fatty acids. The acidic alcohol phase was dried and dissolved in 5% NaOH. Hydrolysis was carried o u t in sealed tubes at 120°C for 3 h. The hydrolysate was acidified with 3 M HC1, extracted with ethyl ether and the extract dried over anhydrous Na: SO4. Chromatographic separations. The bile acids were methylated with freshly prepared diazomethane prior to thin-layer and gas-liquid chromatography. Thin-layer chromatography was performed on 0.4-mm silica gel-coated plates (Kieselgel N, Macherey, Nagel and Co., Germany) using the solvent system, chloroform/acetic acid/methanol (90 : 3 : 5, by vol.). Spots were located by spraying with 5% p h o s p h o m o l y b d i c acid in methanol. Gas-liquid chromatography was carried o u t on a model 810 F and M chromatograph on a 6 ft X 4 mm column of 4% SE-30 ultraphase on Chromosorb G. The oven temperature was 275°C. Quantitation of the peaks was achieved, in most cases, by the m e t h o d of triangulation. In certain instances, peak areas were measured by weighing the c u t o u t peaks. Peak areas were converted to mass by comparison with gas-liquid chromatographic response of reference compounds. Results
In order to facilitate quantitation of effects on the primary bile acids, the enterohepatic circulation was interrupted by treating the animals with cholestyramine daily for 3 days prior to bile collection. The ratio of the t w o major primary acids, cholate and chenodeoxycholate, was determined by comparisons with reference standards of peak areas obtained on gas-liquid chromatography. The effect of 17a-ethinylestradiol dosage on the ratio of the bile acids, excreted during 3 h following treatment is shown in Table I. Up to a dosage level of 25 pg, there was a progressive decline in the ratio. The decline was due to an increase in the amounts of chenodeoxycholate, the cholate levels remaining constant in this dosage range. Between 50 and 250 pg, there was no further increase in the c h e n o d e o x y c h o l a t e levels nor a further decline in the ratio.
81 TABLE I E F F E C T O F 1 7 ~ - E T H I N Y L E S T R A D I O L D O S A G E ON C H O L A T E / C H E N O D E O X Y C H O L A T E
RATIO
D u r i n g 3 d a y s p r i o r to bile c o l l e c t i o n , all a n i m a l s w e r e fed c h o l e s t y r a m i n e a d d e d at 3% level to a g r o u n d f o x c u b e d i e t . 1 7 a - E t h i n y l e s t r a d i o l in 10% e t h a n o l i c saline w a s a d m i n i s t e r e d b y oral i n t u b a t i o n d a i l y f o r 4 d a y s at t h e d o s a g e levels i n d i c a t e d . C o n t r o l a n i m a l s r e c e i v e d e t h a n o l i c saline. 18 h a f t e r t h e last dose o f steroid, bile s a m p l e s w e r e c o l l e c t e d o v e r a p e r i o d o f 3 h a n d bile salts e x t r a c t e d f r o m h a l f t h e v o l u m e o f e a c h s a m p l e as d e s c r i b e d in Materials a n d M e t h o d s . F o l l o w i n g h y d r o l y s i s a n d m e t h y l a t i o n , t h e s a m p l e s w e r e each m a d e up to 3 m l a n d 4-pl a l i q u o t s a n a l y z e d b y gas-liquid c h r o m a t o g r a p h y . R e s u l t s are r e p o r t e d as p m o l e x c r e t e d p e r 3 h. Dose (pg)
0
Bile volume (ml)
Cholate (pmol)
Chenodeoxycholate (~mol)
Cholate + chenodeoxycholate
Cholate/ chenodeoxycholate
1.10" 1.60 1.32
6.33 12.41 ~ 15.02 J
0.92 1.98 ~ 2.55 J
14.39 17.57
6.88 6.27 5.89
13.72
2.27
15.98
6.35
13.37 11.54 14.91
2.70 2.67 3.80
16.07 14.21 18.71
4.95 4.32 3.92
13.27
3.06
16.33
4.40
12.03 16.53 11.04
4.35 4.38 3.00
16.38 20.91 14.04
2.77 3.77 3.68
13.20
3.91
17.11
3.41
8.93 14.18 12.14
3.03 7.20 4.43
11.96 21.38 16.57
2.95 1.97 2.74
11.75
4.89
16.64
2.55
7.32 8 . 4 8 -~ 9.36 ~
3.39 2.93~ 2.34 ~
11.41 11.70
2.16 2.89 4.00
8.92
2.64
11.56
3.02
6.26 11.18
2.70 5.45
16.63
2.32 2.05
1.75 1.40 1.80
10
25
50
1.38 1.32 1.03
1.04 1.50 0.53
0.95** 1.50 1.50
100
0.87** 1.35
2.19 250
1.0
1.48 0.97
6.57 10.73 7.88
2.63 3.69 3.47
9.20 14.42 11.35
2.50 2.91 2.27
8.39
3.26
11.66
2.56
* 3--6-h sample. ** S o m e lost.
There was a tendency towards a decrease in cholate levels in this dosage range. This reduction in cholate excretion was also apparent in animals treated with 500 pg of 17a-ethinylestradiol (Table II). The effect of duration of treatment with 17a-ethinyl-estradiol on bile acid composition is shown in Table II. A progressive decline in the ratio was noted
82 T A B L E II E F F E C T O F D U R A T I O N O F T R E A T M E N T W I T H 1 7 ~ - E T H I N Y L E S T R A D I O L ON B I L E A C I D COMPOSITION T r e a t m e n t o f a n i m a l s and analysis o f bile s a m p l e s w e r e i d e n t i c a l to t h a t d e s c r i b e d in Table I e x c e p t that 5 0 0 ~g o f 17ot-ethinylestradiol w a s a d m i n i s t e r e d daffy for the t i m e p e r i o d s i n d i c a t e d . R e s u l t s are the averages o f three a n i m a l s per group. Treatment (days)
Cholate (pmol)
Chenodeoxycholate (pmol)
Cholate + chenodeoxycholate
Cholate/ chenodeoxycholate
0 1 2 3 5 7 10
10.55 7.48 9.75 3.80 6.28 5.63 2.46
4.53 3.56 5.31 2.80 4.87 4.70 1.39
15.08 11.04 15.06 6.60 11.15 10.33 3.85
2.33 2.10 1.84 1.36 1.29 1.20 1.77
with increasing periods of treatment. This effect was apparent following just a single dose of the estrogen. Lower cholate levels contributed mainly to the decreased ratio although at 10 days the excretion of both bile acids was dramatically reduced. Cholate and chenodeoxycholate together in the latter 3-h samples was 26% that of control. A visual inspection of the chromatogram in Fig. 1 indicates that excretion of the two primary acids was also reduced in animals, in which the eneterohepatic circulation had not been interrupted with cholestyramine. Quantitation of the sum of the peak areas by weight indicated that the total bile acid excreted in 3 h was 83% of control in the 17~-ethinylestradiol-treated group. A number of other steroids were tested, the results of which are shown in Table III. The progestins were without effect at the dosage tested. When the steroids were administered subcutaneously, no significant differences were observed in the response as compared to oral administration.
8
2
J
,
i
I
.
.
I
Fig. 1. Gas-Hquid c h r o m a t o g r a p h y o f bile acids in a n i m a l s t r e a t e d w i t h 1 7 ~ - e t h i n y l - e s t r a d i o l . ( A ) c o n t r o l . (B) 5 0 0 ,ug e t h m y l e s t r a d i o l a d m i n ~ t e r e d b y oral i n t u b a t i o n d m l y for 1 0 days. 1. c h e n o d e o x y c h o l a t e ; 2, cholate.
83 T A B L E III E F F E C T O F V A R I O U S S T E R O I D S ON C H O L A T E / C H E N O D E O X Y C H O L A T E
RATIO
T r e a t m e n t o f a n i m a l s a n d analysis o f bile s a m p l e s w e r e i d e n t i c a l to t h a t d e s c r i b e d in T a b l e I e x c e p t t h a t the d o s a g e o f e s t r o g e n s w a s a d j u s t e d t o a m o l a r e q u i v a l e n c e o f 10/~g o f 17¢~°ethinylestradiol daily and, for t h e p r o g e s t i n s , o f 1 m g o f n o r e t h i n d r o n e d a i l y . A n i m a l s w e r e t r e a t e d w i t h t h e s t e r o i d s f o r 4 d a y s . Results are t h e a v e r a g e s o f t h r e e a n i m a l s p e r g r o u p . Steroid treatment
Cholate/chenodeoxycholate
None 17fl-Estradiol 17c~-Ethinylestradiol Mestranol Norethindrone Megestrol acetate N o r e t h i n d r o n e + 17c~-ethinylestradiol
2.93 2.49 2.12 1.73 2.53 2.32 2.12
Discussion 17~-Ethinylestradiol consistently reduced the ratio of cholate to chenodeoxycholate in cholestyramine-treated animals. Up to a 25 pg dose, the reduced ratio was due mainly to a progressive increase in the rate of excretion of chenodeoxycholate. Between 50 and 250 pg, although still elevated, there was no further increase. It should be noted that, with cholestyramine treatment, bile acid synthesis may already be occurring at near maximal rates. Thus, it is possible that, without the added stimulation by cholestyramine, the capacity for a further progressive increase in chenodeoxycholate would have been greater at the higher dosages. In animals treated with daily doses of 17~-ethinylestradiol of 50 pg or more, a reduced rate of excretion of cholate contributed to the decrease in the ratio. With the 500 pg dose, it was the major factor in the altered proportions of the t w o primary bile acids excreted. These results are in agreement with those of Uchida et al. [4] who reported a reduction in the ratio of bile acids originating from cholic acid to those from chenodeoxycholie acid with 17~-estradiol treatment. Although Saini and Patrick [3] found no difference in the ratio of trihydroxylated to dihydroxylated bile acids with estrone, their m e t h o d would n o t have separated the bile acids according to their origins from the t w o main primary acids. Greim et al. [9] have recently reported a stimulation in the formation of metabolites of c h e n o d e o x y c h o l a t e in bile duct-ligated rats. They suggested that this effect served a protective function by preventing large accumulation of the strongly detergent chenodeoxycholic acid. Saini and Patrick [3] observed significantly increased formation of radiolabelled bile acids from [4 -14 C] cholesterol when estrone was added to a liver perfusion system. In the present study, the rate of excretion of chenodeoxycholate was increased by 17~-ethinylestradiol up to a dose of 25/~g contributing to a slight increase in the excretory rate of the t w o primary acids. It is possible that the estrogen effect m a y have been masked to some extent by a near maximal rate of bile acid synthesis with cholestyramine treatment. With a prolonged daily dose of 500 pg of 17~-ethinylestradiol, there was a
84 substantial reduction in the rate of bile acid excretion in cholestyraminetreated animals. At 10 days, the amount excreted was 23% of control values. In animals not treated with cholestyramine, the corresponding figure was 83% of control. The latter result is closer to that obtained by Gumucio and Valdivieso [2] in rats similarly treated. Total bile acid excretion was only minimally reduced in their study. Since cholestyramine, which was administered for 3 days prior to bile collection, would be expected to stimulate bile acid synthesis, the difference between animals treated and not treated with this compound may be due to the additive effect of a lack of, or lesser cholestyramine-induced stimulation of bile acid synthesis. In summary, it appears that 17~-ethinylestradiol in low dose stimulates chenodeoxycholate excretion rates, whereas in high dose, it reduces cholate excretion. The possibility that the latter effect is related to the reported lowering of the maximal secretory capacity by 17~-ethinylestradiol [2,10] cannot be excluded. Acknowledgement The excellent technical assistance of Mr J. A. Menzies is gratefully acknowledged. References 1 2 3 4 5 6 7 8 9 10
Lynn, J., Williams, L., O'Brien, J., Wittenberg, J. and Egdhal, R.H., (1973) Ann. Sur~ 178, 514 Gumucio, J.J. and Valdivieso, V.D. (1971) Gastroenterology 61, 339 Saini, V.C. and Patrick, S.J. (1970) Biochim. Biophys. Acta 202, 556 Uchida, K., Nomura, Y., Kadowaki, M., Miyata, K. and Miyake, T. (1970) Endocrinol. Jap. 17, 107 Kreek, M.J., Peterson, R.E., Sleisenger, M.H. and Jeffries, G.H. (1969) Proc. Soc. Exp. Biol. Med. 131, 646 Heikel, T.A.J. and Lathe, G.H. (1970) Br. J. PharmacoL 38, 593 Watanabe, H. (1971) Biochim. Biophys. Acta 2 3 1 , 3 9 9 Lin, T.H., Rubinstein, R. and Holmes, W.L. (1963) J. Lipid Res. 4, 63 Greim, H., Trulzsch, D., Roboz, J., Dressler, K., Czygan, P., Hutterer, F., Schaffner, F. and Popper, H. (1972) Gastroenterology 63, 837 Javitt, N.B. and Harkavy, M. (1969) Gastroenterology 56, 400
Biochimica et Biophysica Acta, 399 ( 1 9 7 5 ) 7 9 - - 8 4 © Elsevier Scientific P u b l i s h i n g C o m p a n y , A m s t e r d a m - - P r i n t e d in T h e N e t h e r l a n d s
BBA 27677
E F F E C T OF 1 7 a - E T H I N Y L E S T R A D I O L ON B I L I A R Y E X C R E T I O N OF BILE ACIDS
HISAKO WATANABE
Drug Research Laboratories, Health Protection Branch, Department of National Health and Welfare, Ottawa, Ontario, K I A OL2 (Canada) (Received January 29th, 1975)
Summary The effect of 17a-ethinylestradiol on biliary bile acids has been investigated. The ratio of cholate to c h e n o d e o x y c h o l a t e was diminished by the estrogen in cholestyramine-treated rats. With low doses, this effect was due to increased excretion of chenodeoxycholate. With the highest dose, the decreased ratio was due to a reduction in the levels of cholic acid. In the intermediate dosage range, b o t h factors contributed to the decreased ratio. Prolonged treatment with 500 ;~g daily of 17a-ethinylestradiol produced a reduction in the excretory rate of both bile acids in animals treated or n o t treated with cholestyramine.
Introduction
The precise nature of the estrogen effect on bile acid metabolism and excretion has n o t been clearly established. Total bile acid excretion has been reported to be inhibited by estriol in m o n k e y s [1] and minimally affected by 17a-ethinylestradiol in the rat [ 2 ] . The maximal excretory capacity was markedly reduced by 17a-ethinylestradiol. On the other hand, an elevation in biliary bile acids from radiolabelled cholesterol was observed with estrone in a liver perfusion system [3]. The ratio of bile acids originating from cholic to those from chenodeoxycholic acid was decreased in rats treated with 17~-estradiol [4] whereas the ratio of trihydroxylated to dihydroxylated bile acids was reported to remain unaltered b y estrone with the isolated liver perfusion preparation [3]. *Nomenclature:
choHc
acid,
3a,7~,12a-trihydroxy-5~-cholanoic
acid; c h e n o d e o x y c h o l i c
acid,
3a,7~-dihydroxy-5~-cholanoic acid; 17~-ethinylestradiol, 1,3,5(lO)-estratrien-17a-ethinyl-3,17~diol; estrone, 3-hydroxy-1,3,5(lO)-estratrien-17-one; 17~-estradio1, 103,5(lO)-estxatriene-3,17~-diol; estriol, 1,3,5(1O)-estratxiene-3,16a,17fl-triol; mestranol, 1,3,5(lO)-estratrien-17~-ethinyl-3,17~-diol 3-methyl ether; norethindrone, 17~-ethinyl-17~-hydroxyestra-4-en-3-one; megestrol acetate, 17ahydroxy-6-methyl-4,6-pregnadiene-3,20-dione acetate.
80 Since 17a-ethinylestradiol has been shown to produce cholestasis [ 5--7], which may be due, at least in part, to inhibition of bile salt-dependent flow [1], it was of interest to investigate some of the conditions which may influence the effect of this c o m p o u n d on bile acid excretion and metabolism. Materials and Methods
Materials. Steroids and bile acids were obtained from Steraloids Inc., Pawling, N.Y.; and cholestyramine from Merck, Sharpe and Dohme, West Point, Pa. Treatment o f animals. Female Wistar rats, 165--185 g, maintained on fox cube diets, were variously treated as indicated prior to preparation for bile collections. Under urethane anesthesia, the c o m m o n bile duct was cannulated with polyethylene tubing (PE 10, Clay Adams, Parsipany, N . J . ) a n d samples collected over a period of 3 h. Extraction o f bile acids. Bile acids were extracted and hydrolyzed according to the m e t h o d as described b y Lin et al. [8]. Bile samples, deproteinized with ethanol were extracted with light petroleum before and after acidification with 3 M HC1 to remove neutral lipids and fatty acids. The acidic alcohol phase was dried and dissolved in 5% NaOH. Hydrolysis was carried o u t in sealed tubes at 120°C for 3 h. The hydrolysate was acidified with 3 M HC1, extracted with ethyl ether and the extract dried over anhydrous Na: SO4. Chromatographic separations. The bile acids were methylated with freshly prepared diazomethane prior to thin-layer and gas-liquid chromatography. Thin-layer chromatography was performed on 0.4-mm silica gel-coated plates (Kieselgel N, Macherey, Nagel and Co., Germany) using the solvent system, chloroform/acetic acid/methanol (90 : 3 : 5, by vol.). Spots were located by spraying with 5% p h o s p h o m o l y b d i c acid in methanol. Gas-liquid chromatography was carried o u t on a model 810 F and M chromatograph on a 6 ft X 4 mm column of 4% SE-30 ultraphase on Chromosorb G. The oven temperature was 275°C. Quantitation of the peaks was achieved, in most cases, by the m e t h o d of triangulation. In certain instances, peak areas were measured by weighing the c u t o u t peaks. Peak areas were converted to mass by comparison with gas-liquid chromatographic response of reference compounds. Results
In order to facilitate quantitation of effects on the primary bile acids, the enterohepatic circulation was interrupted by treating the animals with cholestyramine daily for 3 days prior to bile collection. The ratio of the t w o major primary acids, cholate and chenodeoxycholate, was determined by comparisons with reference standards of peak areas obtained on gas-liquid chromatography. The effect of 17a-ethinylestradiol dosage on the ratio of the bile acids, excreted during 3 h following treatment is shown in Table I. Up to a dosage level of 25 pg, there was a progressive decline in the ratio. The decline was due to an increase in the amounts of chenodeoxycholate, the cholate levels remaining constant in this dosage range. Between 50 and 250 pg, there was no further increase in the c h e n o d e o x y c h o l a t e levels nor a further decline in the ratio.
81 TABLE I E F F E C T O F 1 7 ~ - E T H I N Y L E S T R A D I O L D O S A G E ON C H O L A T E / C H E N O D E O X Y C H O L A T E
RATIO
D u r i n g 3 d a y s p r i o r to bile c o l l e c t i o n , all a n i m a l s w e r e fed c h o l e s t y r a m i n e a d d e d at 3% level to a g r o u n d f o x c u b e d i e t . 1 7 a - E t h i n y l e s t r a d i o l in 10% e t h a n o l i c saline w a s a d m i n i s t e r e d b y oral i n t u b a t i o n d a i l y f o r 4 d a y s at t h e d o s a g e levels i n d i c a t e d . C o n t r o l a n i m a l s r e c e i v e d e t h a n o l i c saline. 18 h a f t e r t h e last dose o f steroid, bile s a m p l e s w e r e c o l l e c t e d o v e r a p e r i o d o f 3 h a n d bile salts e x t r a c t e d f r o m h a l f t h e v o l u m e o f e a c h s a m p l e as d e s c r i b e d in Materials a n d M e t h o d s . F o l l o w i n g h y d r o l y s i s a n d m e t h y l a t i o n , t h e s a m p l e s w e r e each m a d e up to 3 m l a n d 4-pl a l i q u o t s a n a l y z e d b y gas-liquid c h r o m a t o g r a p h y . R e s u l t s are r e p o r t e d as p m o l e x c r e t e d p e r 3 h. Dose (pg)
0
Bile volume (ml)
Cholate (pmol)
Chenodeoxycholate (~mol)
Cholate + chenodeoxycholate
Cholate/ chenodeoxycholate
1.10" 1.60 1.32
6.33 12.41 ~ 15.02 J
0.92 1.98 ~ 2.55 J
14.39 17.57
6.88 6.27 5.89
13.72
2.27
15.98
6.35
13.37 11.54 14.91
2.70 2.67 3.80
16.07 14.21 18.71
4.95 4.32 3.92
13.27
3.06
16.33
4.40
12.03 16.53 11.04
4.35 4.38 3.00
16.38 20.91 14.04
2.77 3.77 3.68
13.20
3.91
17.11
3.41
8.93 14.18 12.14
3.03 7.20 4.43
11.96 21.38 16.57
2.95 1.97 2.74
11.75
4.89
16.64
2.55
7.32 8 . 4 8 -~ 9.36 ~
3.39 2.93~ 2.34 ~
11.41 11.70
2.16 2.89 4.00
8.92
2.64
11.56
3.02
6.26 11.18
2.70 5.45
16.63
2.32 2.05
1.75 1.40 1.80
10
25
50
1.38 1.32 1.03
1.04 1.50 0.53
0.95** 1.50 1.50
100
0.87** 1.35
2.19 250
1.0
1.48 0.97
6.57 10.73 7.88
2.63 3.69 3.47
9.20 14.42 11.35
2.50 2.91 2.27
8.39
3.26
11.66
2.56
* 3--6-h sample. ** S o m e lost.
There was a tendency towards a decrease in cholate levels in this dosage range. This reduction in cholate excretion was also apparent in animals treated with 500 pg of 17a-ethinylestradiol (Table II). The effect of duration of treatment with 17a-ethinyl-estradiol on bile acid composition is shown in Table II. A progressive decline in the ratio was noted
82 T A B L E II E F F E C T O F D U R A T I O N O F T R E A T M E N T W I T H 1 7 ~ - E T H I N Y L E S T R A D I O L ON B I L E A C I D COMPOSITION T r e a t m e n t o f a n i m a l s and analysis o f bile s a m p l e s w e r e i d e n t i c a l to t h a t d e s c r i b e d in Table I e x c e p t that 5 0 0 ~g o f 17ot-ethinylestradiol w a s a d m i n i s t e r e d daffy for the t i m e p e r i o d s i n d i c a t e d . R e s u l t s are the averages o f three a n i m a l s per group. Treatment (days)
Cholate (pmol)
Chenodeoxycholate (pmol)
Cholate + chenodeoxycholate
Cholate/ chenodeoxycholate
0 1 2 3 5 7 10
10.55 7.48 9.75 3.80 6.28 5.63 2.46
4.53 3.56 5.31 2.80 4.87 4.70 1.39
15.08 11.04 15.06 6.60 11.15 10.33 3.85
2.33 2.10 1.84 1.36 1.29 1.20 1.77
with increasing periods of treatment. This effect was apparent following just a single dose of the estrogen. Lower cholate levels contributed mainly to the decreased ratio although at 10 days the excretion of both bile acids was dramatically reduced. Cholate and chenodeoxycholate together in the latter 3-h samples was 26% that of control. A visual inspection of the chromatogram in Fig. 1 indicates that excretion of the two primary acids was also reduced in animals, in which the eneterohepatic circulation had not been interrupted with cholestyramine. Quantitation of the sum of the peak areas by weight indicated that the total bile acid excreted in 3 h was 83% of control in the 17~-ethinylestradiol-treated group. A number of other steroids were tested, the results of which are shown in Table III. The progestins were without effect at the dosage tested. When the steroids were administered subcutaneously, no significant differences were observed in the response as compared to oral administration.
8
2
J
,
i
I
.
.
I
Fig. 1. Gas-Hquid c h r o m a t o g r a p h y o f bile acids in a n i m a l s t r e a t e d w i t h 1 7 ~ - e t h i n y l - e s t r a d i o l . ( A ) c o n t r o l . (B) 5 0 0 ,ug e t h m y l e s t r a d i o l a d m i n ~ t e r e d b y oral i n t u b a t i o n d m l y for 1 0 days. 1. c h e n o d e o x y c h o l a t e ; 2, cholate.
83 T A B L E III E F F E C T O F V A R I O U S S T E R O I D S ON C H O L A T E / C H E N O D E O X Y C H O L A T E
RATIO
T r e a t m e n t o f a n i m a l s a n d analysis o f bile s a m p l e s w e r e i d e n t i c a l to t h a t d e s c r i b e d in T a b l e I e x c e p t t h a t the d o s a g e o f e s t r o g e n s w a s a d j u s t e d t o a m o l a r e q u i v a l e n c e o f 10/~g o f 17¢~°ethinylestradiol daily and, for t h e p r o g e s t i n s , o f 1 m g o f n o r e t h i n d r o n e d a i l y . A n i m a l s w e r e t r e a t e d w i t h t h e s t e r o i d s f o r 4 d a y s . Results are t h e a v e r a g e s o f t h r e e a n i m a l s p e r g r o u p . Steroid treatment
Cholate/chenodeoxycholate
None 17fl-Estradiol 17c~-Ethinylestradiol Mestranol Norethindrone Megestrol acetate N o r e t h i n d r o n e + 17c~-ethinylestradiol
2.93 2.49 2.12 1.73 2.53 2.32 2.12
Discussion 17~-Ethinylestradiol consistently reduced the ratio of cholate to chenodeoxycholate in cholestyramine-treated animals. Up to a 25 pg dose, the reduced ratio was due mainly to a progressive increase in the rate of excretion of chenodeoxycholate. Between 50 and 250 pg, although still elevated, there was no further increase. It should be noted that, with cholestyramine treatment, bile acid synthesis may already be occurring at near maximal rates. Thus, it is possible that, without the added stimulation by cholestyramine, the capacity for a further progressive increase in chenodeoxycholate would have been greater at the higher dosages. In animals treated with daily doses of 17~-ethinylestradiol of 50 pg or more, a reduced rate of excretion of cholate contributed to the decrease in the ratio. With the 500 pg dose, it was the major factor in the altered proportions of the t w o primary bile acids excreted. These results are in agreement with those of Uchida et al. [4] who reported a reduction in the ratio of bile acids originating from cholic acid to those from chenodeoxycholie acid with 17~-estradiol treatment. Although Saini and Patrick [3] found no difference in the ratio of trihydroxylated to dihydroxylated bile acids with estrone, their m e t h o d would n o t have separated the bile acids according to their origins from the t w o main primary acids. Greim et al. [9] have recently reported a stimulation in the formation of metabolites of c h e n o d e o x y c h o l a t e in bile duct-ligated rats. They suggested that this effect served a protective function by preventing large accumulation of the strongly detergent chenodeoxycholic acid. Saini and Patrick [3] observed significantly increased formation of radiolabelled bile acids from [4 -14 C] cholesterol when estrone was added to a liver perfusion system. In the present study, the rate of excretion of chenodeoxycholate was increased by 17~-ethinylestradiol up to a dose of 25/~g contributing to a slight increase in the excretory rate of the t w o primary acids. It is possible that the estrogen effect m a y have been masked to some extent by a near maximal rate of bile acid synthesis with cholestyramine treatment. With a prolonged daily dose of 500 pg of 17~-ethinylestradiol, there was a
84 substantial reduction in the rate of bile acid excretion in cholestyraminetreated animals. At 10 days, the amount excreted was 23% of control values. In animals not treated with cholestyramine, the corresponding figure was 83% of control. The latter result is closer to that obtained by Gumucio and Valdivieso [2] in rats similarly treated. Total bile acid excretion was only minimally reduced in their study. Since cholestyramine, which was administered for 3 days prior to bile collection, would be expected to stimulate bile acid synthesis, the difference between animals treated and not treated with this compound may be due to the additive effect of a lack of, or lesser cholestyramine-induced stimulation of bile acid synthesis. In summary, it appears that 17~-ethinylestradiol in low dose stimulates chenodeoxycholate excretion rates, whereas in high dose, it reduces cholate excretion. The possibility that the latter effect is related to the reported lowering of the maximal secretory capacity by 17~-ethinylestradiol [2,10] cannot be excluded. Acknowledgement The excellent technical assistance of Mr J. A. Menzies is gratefully acknowledged. References 1 2 3 4 5 6 7 8 9 10
Lynn, J., Williams, L., O'Brien, J., Wittenberg, J. and Egdhal, R.H., (1973) Ann. Sur~ 178, 514 Gumucio, J.J. and Valdivieso, V.D. (1971) Gastroenterology 61, 339 Saini, V.C. and Patrick, S.J. (1970) Biochim. Biophys. Acta 202, 556 Uchida, K., Nomura, Y., Kadowaki, M., Miyata, K. and Miyake, T. (1970) Endocrinol. Jap. 17, 107 Kreek, M.J., Peterson, R.E., Sleisenger, M.H. and Jeffries, G.H. (1969) Proc. Soc. Exp. Biol. Med. 131, 646 Heikel, T.A.J. and Lathe, G.H. (1970) Br. J. PharmacoL 38, 593 Watanabe, H. (1971) Biochim. Biophys. Acta 2 3 1 , 3 9 9 Lin, T.H., Rubinstein, R. and Holmes, W.L. (1963) J. Lipid Res. 4, 63 Greim, H., Trulzsch, D., Roboz, J., Dressler, K., Czygan, P., Hutterer, F., Schaffner, F. and Popper, H. (1972) Gastroenterology 63, 837 Javitt, N.B. and Harkavy, M. (1969) Gastroenterology 56, 400
