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Metabolism of steroid estrogens in the hen. I. Conversion in vivo of estradiol-17β-4-14C-17α-3H to 16-epiestriol-4-14C-17α-3H
475
METABOLISM
OF
I. C O N V E R S I O N
IN
TO
STEROID VIVO
OF
ESTROGENS
IN
THE
HEN.
ESTRADIOL-17~-4-14C-17~-3H
16-EPIESTRIOL-4-14C-
17o~-3H '~
R . S . M a t h u r and R. H. Common M a c d o n a l d College of McGill University Province of Quebec, Canada. Received August I0, 1968 ABSTRACT A mixture of estradiol-1713-4-14C and estradiol-1713-17oe-3H was injected into a laying hen. The ratios of d. p.m. 3H:d.p.m. 14C of the urinary estradiol-17~, 16-epiestriol and 16-ketoestradiol-17~ w e r e identical. This s h o w e d that 16-epiestriol as well as 16-ketoestradiol-17~ w e r e derived from estradiol-17~ and that estrone was not an intermediate in this conversion. N o tritium activity was observed in estrone or estradiol-17~ fractions and this suggested that estradiol-17~ is derived from estrone.
Supported by the National Institute of Arthritic and Metabolic Disease, U S P H , Grant A M - 0 6 1 3 0 - E N D and by National R e s e a r c h Council of Canada Grant A-56. M a c d o n a l d College Journal series No. 582. The systematic and (in brackets) trivial n a m e s of the steroids mentioned in this paper are:3-hydroxy-estra-l, 3, 5(10)-trien-17-one (estrone): 3, 16~-dihydroxy-estra- i, 3, 5( 10)-trien-17-one (16~-h~droxyestrone): 3, 16~-dihydroxy-estra-l, 3, 5(10)-trien-17-one (16~-hydroxyestrone): 3-hydroxy-estra-l, 3, 5(10)-triene-16, 17-dione (16-ketoestrone): 3, 17~-dihydroxy-estr a- 1 3, 5(10) -trien- 16 -one ( 16 -ketoestr adiol- 17~): estra- I, 3, 5(10)-triene-3 17~-diol (estr adiol- 17~): estra-I, 3, 5( 10)-triene-3 17~-diol (estradiol- 17~): estra- I, 3, 5(10)-triene-3 16~, 17~-triol (estriol): estra-l, 3, 5(10)-triene-3 16~, 17~-triol (16, 17-epiestriol): estra-i, 3, 5(10)-triene-3 16~, 17~-triol (16 epiestriol): estra-l, 3, 5(10)-triene-3 16~, 17~-triol (17-epiestriol).
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ST ER O I D S
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INTRODUCTION Estrone (I), 16-epiestriol (2) and estradiol-17~ (3) have been isolated in crystalline form from laying hens' urine, which contains enough of these steroids to permit of their chemical estimation by classical methods (4, 5, 6). In addition, the presence of estriol and of 16, 17-epiestriol in laying hens' urine has been established chromatographically (7). Information about in vivo conversions of steroid estrogens has been obtained by injecting various 14C-labelled steroid estrogens and then examining the urine for radioactive conversion products.
The steroids injected have included estradiol-17~-16-14C
(8), estradiol-17~-4-14C (9), estrone-16-14C (i0), estriol-16-14C(ll), 16-ketoestradiol-17~-16-14C
(iZ), 16-epiestriol-16-14C (13) and
estradiol-17~-6, 7-3H (14). The radioactive conversion products detected in one or other of these experiments have included estrone, estradiol-17~, estradiol-17oG estriol, 16-epiestriol,
16-ketoestradiol-17~,
16-ketoestrone,
16, 17-epiestriol and 17-epiestriol.
As regards in vitro studies, Mitchell and Hobkirk (15) obtained radioactive estriol upon incubation of liver slices from a laying hen with estradiol-17~-16-14C.
In an extensive study, Ozon and Breuer
(16) incubated estrone, estradiol-17~, estradiol-17~,
16-ketoestradiol-
17~, 16-epiestriol, and 16-hydroxyestrone with chicken liver slices. They demonstrated the presence of the following enzymes in the liver slices:-
16~-, 16~-, 17~- and 17~-hydroxysteroid oxidoreductases,
together with 16~- and 16~-hydroxylases.
They concluded that
Oct. 1968
ST ER O IDS
16-epiestriol was quantitatively a m o r e
477
important metabolite than
estriol. Despite the availability of the foregoing information
on in vivo
and in vitro conversions, there is relatively little information about steroid estrogen metabolic pathways in the hen.
The present report,
therefore, deals with a study of the pathway leading f r o m estradiol17~ to 16-epiestriol in the hen. EXPERIM
EN TAL
Materials and m e t h o d s The experimental birds w e r e m a t u r e R h o d e Island R e d hens modified surgically (17) to exteriorize the ureteral openings and to permit quantitative collection of urine as described elsewhere (ii). Urine was r e m o v e d f r o m the collection tube several times daily and placed immediately in cold storage at -15°C. The sub-samples w e r e bulked to give Z4-hr samples immediately before analysis. Estradiol- 17~-4- 14C (Radiochemical Centre, A m e r s h a m , England) and estradiol-17~-17~-3H (presented by Dr. D.S. Layne) w e r e purified by thin-layer c h r o m a t o g r a p h y before use. (Reference estradiol-17~, estrone, estradiol-17~, 16-epiestriol and 16-ketoestradiol17~ w e r e obtained f r o m Steraloids Inc., Pawling, N.Y., U.S.A.). Solvents w e r e of analytical grade. Thin-layer chromatography was p e r f o r m e d on silica gel G (Merck) with the use of the solvent systems listed in Table I. F r e e phenolic steroids w e r e visualized on the plates by the reagent of Folin and Ciocalteu or by spraying with 2 % H z S O 4 in ethanol (v/v) and then heating the plates at ii0 ° for about 5 minutes. Methyl ethers and acetates of the phenolic steroids w e r e visualized by m e a n s of the latter reagent. Radioactivities w e r e assayed by m e a n s of a P a c k a r d Tri'Carb liquid scintillation spectrometer, M o d e l 3003. The procedure was that described by Flood et ai.(18). Settings at the counter w e r e adjusted in order completely to eliminate tritium f r o m counts in the 14C channel. A small r e s i d u u m of 14C counts was still counted in the tritium channel, however, so corrections for 'overflow' of 14C w e r e m a d e by use of calibration curves prepared with reference 14C- and 3H-toluene. Methylations w e r e done by Brown's m e t h o d (19) with strict adherence to that author's directions. Acetylations w e r e done by dissol-
478
ST ER O ID S
12:4
ring the steroid in I. 0 m l pyridine, adding i. 0 m l acetic anhydride and leaving the m i x t u r e overnight at ambient temperature. Thereafter the solvents w e r e evaporated under Nz and the residual acetylated product w a s dissolved in methanol. TABLE
I
Thin-layer C h r o m a t o g r a p h i c S y s t e m No.
B C F O X Experimental
Systems
Composition
Reference
Ethyl acetate - n-hexane ethanol (v/v 80:15:5) C y c l o h e x a n e - ethyl acetate
(20)
(I:i)
(z0)
B e n z e n e - m e t h a n o l (9:1) n - h e x a n e - ethyl acetate (75:Z5) CHC13 : diethyl ether (6:4)
(Zl) (20) (Z2)
Procedure
A m i x t u r e of 2. 1 x 106 d p m e s t r a d i o l - 1 7 ~ - 4 - 1 4 C a n d 2. 1 x 107 dprn estradiol-17~-17a-~H was injected intramuscularly into a laying Rhode Island Red hen. Three successive Z4-hr urines were collected thereafter. Each 24-hr sample was assayed for radioactivity. The urine for day 1 was divided into two equal parts and each part was processed separately as follows:Free steroids were extracted with CHC13 (3 x 1 v o l ) . T h e CHC13 w a s e v a p o r a t e d , the residue was taken u p in 10 m l m e t h a n o l a n d 0. 10 m l p o r t i o n s of t h i s s o l u t i o n w e r e a s s a y e d for radioactivity. T h e aqueous phase f r o m the foregoing extraction w a s solvolysed for 48-hr by the m e t h o d of Burstein and L i e b e r m a n (23). Previous experience (24) had s h o w n that 24-hr w a s insufficient to hydrolyse completely the conjugates (mainly sulphates) in the urine. The solvolysed fraction w a s w a s h e d with i. 0 M N a H C O 3 (Z x 0. 1 vol) and then with water (2 x 0. 1 vol). The solvent w a s r e m o v e d f r o m the solvolysed fraction and the residue w a s dissolved in m e t h a n o l and a s s a y e d for radioactivity. T h e C H C I s extract and the solvolysed fraction w e r e purified separately as follows:- T h e material w a s c h r o m a t o g r a p h e d in s y s t e m X with a m i x t u r e of reference estrone, estradiol-17~, estradiol-17~, 16-ketoestradiol-17~ and 16-epiestriol on lateral columns. Zones corresponding in mobilities to the reference steroids w e r e r e m o v e d f r o m the plate, eluted and the eluates r e c h r o m a t o g r a p h e d separately in s y s t e m C. T h e 16-epiestriol fraction w a s further c h r o m a t o g r a p h e d in s y s t e m B to purify it f r o m any residual 17-epiestriol. The 16-
Oct. 1968
S T ER O ID S
479
ketoestradiol-17~, estradiol-17~ and estradiol-17~ subfractions w e r e r e c h r o m a t o g r a p h e d i n system X. E a c h fraction was assayed for radioactivity after each purification step and ratios d p m 3H : dprn 14C w e r e calculated. Portions of the radioactive steroids thus purified w e r e m i x e d with the appr opriate non-r adioactive refer ence steroid and chr omatographed in systems X and C (for estradiols and 16-ketoestradiol-17~) and systems X and B (for 16-epiestriol). After development the estrogens wer e visualized with the HzSO4-ethanol reagent and then the gel was scraped f r o m the plate and assayed. In every case the stain corresponded in position with the radioactivity. Portions of the purified phenolic fraction f r o m the solvolysis wet e m ethylated and the m ethyl ether s wet e m i x e d with appropriate refer ence methyl ether s and c h r o m a t o g r a p h e d in system C bidimensionally and in system F dimensionally. In each case the stained spot corresponded in position with the radioactivity. Further portions of each purified phenolic steroid w e r e acetylated and c h r o m a t o g r a p h e d twice in the s a m e direction in system O and then once at right angles to this in the s a m e system. This procedure afforded a superior separation of the acetates of the two estradiol epirners if either w e r e still contaminated by the other. Attempts to study the estriol and 16, 17-epiestriol of the extracts by similar m e a n s failed because of the small amounts of radioactivity in these fr actions. RESULTS T h e total 14C and 3H radioactivities recovered in the urine w e r e 31. 1% and 13. 18%, respectively, of the amounts injected (Table II). The crude ratio d p m 3H : d p m
14C in the urine for day 1 was 3.45:1.
TABLE
II
Total Urinary Recoveries of Administered 14C and 3 H 14 C Day
dpm
3H % of dose
dpm
% of dose
1
5.97 x i05
28.21
2 . 0 6 x I06
9.69
2
6. 12 x 104
2.89
4.80 x 105
2.25
3
negligible
-
2.63 x 105
1.24
31. I0
2.80 x 106
13. 18
Sum
i+2+3
6.58 x 105
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ST ER O I D S
12:4
The C H C I 3 extract of the day-/ urine yielded 9.75% of its 14C radioactivity, the corresponding figure for
3
H being 6.2%.
The subsequent 48-hour solvolysis liberated 41.76% of the radioactivity and Z0.73% of the
3
14
C
H radioactivity of the day-i urine.
Examination of the radioactivities of the phenolic steroids of the urinary C H C I 3 extract prior to solvolysis ('free' estrogens), as separated by thin-layer chromatography, estradiol-17~ did not contain 3H.
revealed that the estrone and
The ratios d p m 3H : d p m
the other phenolic steroids are s u m m a r i z e d TABLE
14G for
in Table III.
III
Ratios d p m 3H : d p m 14C for Phenolic Steroids of C H C I 3 Extract of day-I Urine before Solvolysis. C H C I 3 Extract accounted for 9.75% of 14C in day-I Urine. Steroid
d p m 3H : d p m
E str one Estr adiol- 17~ Estr adiol- 17~ 16 -Ketoestr adiol- 1713 16-Epie str iol *
3
14C
~ 4. 03 4.05 4.40
H radioactivity not detectable. The similar examination of the extract of the day-1 urine after
solvolysis yielded the results s u m m a r i z e d
in Table IV.
The 14C radioactivities of the phenolic steroids from the solvolysed urine provided an estimate of the relative proportions of the various radioactive phenols as set out in Table V.
Oct. 1968
ST ER O I D S
TABLE
481
IV
Ratios d p m 3H : d p m 14C for Phenolic Steroids of C H C I 3 Extract of day-i Urine after Solvolysis. Extract after Solvolysis accounted for 4 1 . 7 6 % of 14~ and 2 0 . 7 3 % of 3H in day-i Urine. dpm
Steroid
Phenol
Estrone Estr adiol - 17~ Estradiol-17~
16-Ketoestradiol-17~ 16-Epiestriol *
3
H
: dpm
14
C
Average for phenol and 2 derivatives
Methyl ether
Acetate
* * 4.03
* * 4.05
* * 3.97
4.01
4.00 4.04
4. 30 4. 13
4. i0 3.73
4. 13 3.97
3 H radioactivity too low to be m e a s u r a b l e with any precision.
TABLE
V
Relative Proportions of Radioactive Phenolic Steroids from Extract Solvolysed Day-i Urine. Chromatography on Silica gel G in System
Steroid
of X.
Percentage
Estr one Estr adiol- 17~ Estr adiol- 17~ 16 -Ketoestr adiol- 179 16-Epiestriol A r e a s between spots Total
37. 1 20.9 24.8 0.5 4.9 ii. 8 i00. 00
DISCUSSION Fishrnan et al. (25) have shown that estradiol-17~ is reversibly metabolised to estrone in the human, lying m u c h
in favour of estrone.
with the equilibrium in vivo
They found also that estriol and 16-
~82
STEROIDS
12:4
epiestriol w e r e derived f r o m estrone and not directly f r o m estradiol17~.
R a u d (26) has found that h e n liver h o m o g e n a t e converts estrone
preferentially to estradiol- 17~. Studies in this laboratory (6) have furnished s o m e
evidence that there is m o r e
estradiol-17~ than es£rone
in the urine of non-laying hens w h e r e a s there is roughly as m u c h estradiol-17~ as estrone in the urine of the laying hen. In the course of their incubation experiments with chicken liver slices, O z o n and B r e u e r (16) did not find 16~- or 16~-hydroxy estrone a m o n g the in vitro conversion products of estradiol-17~ or estradiol-17~.
At the s a m e time they did find 16-epiestriol to be one
of the m a j o r in vitro metabolites of estradiol-17~. 16-Epiestriol is f o r m e d f r o m estrone in the h u m a n 16~-hydroxyestrone.
subject via
T h e results of the experiments n o w reported
s h o w that !6-epiestriol w a s f o r m e d f r o m estradiol-17~ in the hen. If any appreciable fraction of the 16-epiestriol had been f o r m e d f r o m the estradiol-17~ via estrone, then the ratio d p m
3H : dpm
14C for the
16-epiestriol w o u l d have b e e n lower than the ratio for the urinary estradiol-17~.
In the upshot, the two ratios w e r e practically identi-
cal, w h i c h suggests strongly that estrone does not, in the hen, lie on the metabolic p a t h w a y f r o m estradiol-17~ to 16-epiestriol. O z o n and B r e u e r (16)incubated 16~-hydroxyestrone with chicken liver slices and identified estriol and 17-epiestriol as the conversion products.
It s e e m s probable, therefore, that estrone is not
converted to its 16-hydroxy derivatives in the hen as it is in h u m a n s ,
Oct. 1968
ST ER O ID S
483
but that estrone m e r e l y remains in meta]~olic equilibrium with the two estr adiol
epimer
The
s.
results
for ratios
suggest that, in the hen, estradiol-17~.
dpm
3H : d p m
14G (Tables III and IV)
16-ketoestradiol-17~ is also derived f r o m
It is already k n o w n that the hen can convert estriol
into 16-ketoestradiol-17~ in vivo (11) and that 16-ketoestradiol-17~ gives rise to 16-epiestriol (12), hence the m a i n in vivo interconversions of these three steroids m a y Estriol--~
be s u m m a r i z e d
16-ketoestradiol-17~ ~
as 16-epiestriol.
If estriol w e r e not derived from estradiol-17~ but w e r e f o r m e d via estrone, then the ratio d p m 3H : d p m
14C would have been zero
for the urinary 16-epiestriol in the experiments n o w reported.
In
point of fact, the ratios for the estradiol-17~ and 16-epiestriol w e r e virtually identical, and estriol itself m u s t arise from estradiol-17~. If this be granted, then it b e c o m e s possible to visualize the in vivo metabolism
of estradiol-17~ in the hen as follows:Estradiol-17~"
,L
) Estrone I
, Estradiol-17~
I
s
16-Epiestrioi ¢
) 16-Ketoestradiol-1713 {
Estriol
ACKNOWLEDGMENTS We e x p r e s s o u r t h a n k s to t h e N a t i o n a l I n s t i t u t e of A r t h r i t i c a n d M e t a b o l i c D i s e a s e , U . S . D e p a r t m e n t of N a t i o n a l H e a l t h a n d W e l f a r e , a n d t h e N a t i o n a l R e s e a r c h C o u n c i l of C a n a d a f o r t h e f i n a n c i a l s u p p o r t that made this work possible. We a l s o t h a n k D r . D . S . L a y n e , D e p a r t m e n t of Biochemistry, University of Ottawa, for providing estradiol17~-17~-3H and Dr. N. Nikolaiczuk, M a c d o n a l d College, for cooperation in supplying suitable experimental hens.
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ST ER O I D S
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REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. I0. Ii. 12. 13. 14. 15. 16. 17. 18.
19. 20. 21. 22. 23. 24.
Ainsworth, L., and C o m m o n , R.H. N A T U R E , 195, 77 (1962). Hertelendy, F., and C o m m o n , R.H. C A N . J. BIOCHF-aXd., 42, 1177 (1964). Hertelendy, F., Taylor, T.G., Mathur, R.S. and C o m m o n , R.H. C A N . J. BIOCHEIVI., 43, 1379 (1965). C o m m o n , R.H., Ainsworth, L., Hertelendy, F., and Mathur, R.S. C A N . J. BIOCHF-~., 43, 539 (1965). Mahhur, R.S., Anastassiadis, P.A., and C o m m o n , R.H. POULTRY SCI., 45, 946 (1966). Mathur, R.S., and C o m m o n , R.H. P O U L T R Y SCI. in press. Mathur, R.S., and C o m m o n , R.H. CAN. J. BIOCH~Vl., 45, 531 (1967). Hertelendy, F., and C o m m o n , R.H. P O U L T R Y SCI., 44, 1379 (1965). Mulay, S., and C o m m o n , R.H. CAN. J. B I O C H ~ I . , 46, (in press) (1968). Ainsworth, L., Carter, A.L., and C o m m o n , R.H. C A N . J. BIOCHI~I. and PHYSIOL., 40, 123 (1962). MacRae, H.F., Dale, D.G., and C o m m o n , R.H. CAN. J. B I O C H E M . and PHYSIOL., 38, 523 (1960). Ainsworth, L., and C o m m o n , R.H. C A N . J. B I O C H ~ M . and P H Y S I O L . , 4!, 2045 (1963). Ainsworth, L., Carter, A.L., and C o m m o n , R.H. C A N . J. B I O C H E M . , 42, 521 (1964). Mulay, S., Henneberry, G.O. and C o m m o n , R.H. S T E R O I D S , (in press) (1968). Mitchell, J.E., and Hobkirk, R. B I O C H E M . B I O P H Y S . RES. C O M M . , i, 72 (1959). Ozon, R., and Breuer, H. ZEIT. P H Y S I O L . C H E M . , 341, 239 (1965). Ainsworth, L. P O U L T R Y SCI., 44, 1561 (1965). Flood, C., Layne, D.S., Ramcharan, S., Rossipal, E., Tait, J.F., and Tait, S.A.S. A C T A E N D O C R I N O L . , 36, 237 (1961). Brown, J.B. BIOCH~VI. J., 60, 185 (1955). Lisboa, B.P., and Diczfalusy, E. A C T A E N D O C R I N O L . , 40, 60 (1962). Ladany, S., andFinkelstein, M. STEROIDS, 2, 297(1963). Sobrevilla, L., Hagermann, D., and Villee, C. B I O C H I M . B I O P H Y S . A C T A , 93, 665 (1964). Burstein, S., and Lieberman, S. J. BIOL. C H E M . , 233, 331 (1958). Mathur, R.S., C o m m o n , R.H., Collins, D. C~ and Layne, D.S. unpublished observations.
METABOLISM
OF
I. C O N V E R S I O N
IN
TO
STEROID VIVO
OF
ESTROGENS
IN
THE
HEN.
ESTRADIOL-17~-4-14C-17~-3H
16-EPIESTRIOL-4-14C-
17o~-3H '~
R . S . M a t h u r and R. H. Common M a c d o n a l d College of McGill University Province of Quebec, Canada. Received August I0, 1968 ABSTRACT A mixture of estradiol-1713-4-14C and estradiol-1713-17oe-3H was injected into a laying hen. The ratios of d. p.m. 3H:d.p.m. 14C of the urinary estradiol-17~, 16-epiestriol and 16-ketoestradiol-17~ w e r e identical. This s h o w e d that 16-epiestriol as well as 16-ketoestradiol-17~ w e r e derived from estradiol-17~ and that estrone was not an intermediate in this conversion. N o tritium activity was observed in estrone or estradiol-17~ fractions and this suggested that estradiol-17~ is derived from estrone.
Supported by the National Institute of Arthritic and Metabolic Disease, U S P H , Grant A M - 0 6 1 3 0 - E N D and by National R e s e a r c h Council of Canada Grant A-56. M a c d o n a l d College Journal series No. 582. The systematic and (in brackets) trivial n a m e s of the steroids mentioned in this paper are:3-hydroxy-estra-l, 3, 5(10)-trien-17-one (estrone): 3, 16~-dihydroxy-estra- i, 3, 5( 10)-trien-17-one (16~-h~droxyestrone): 3, 16~-dihydroxy-estra-l, 3, 5(10)-trien-17-one (16~-hydroxyestrone): 3-hydroxy-estra-l, 3, 5(10)-triene-16, 17-dione (16-ketoestrone): 3, 17~-dihydroxy-estr a- 1 3, 5(10) -trien- 16 -one ( 16 -ketoestr adiol- 17~): estra- I, 3, 5(10)-triene-3 17~-diol (estr adiol- 17~): estra-I, 3, 5( 10)-triene-3 17~-diol (estradiol- 17~): estra- I, 3, 5(10)-triene-3 16~, 17~-triol (estriol): estra-l, 3, 5(10)-triene-3 16~, 17~-triol (16, 17-epiestriol): estra-i, 3, 5(10)-triene-3 16~, 17~-triol (16 epiestriol): estra-l, 3, 5(10)-triene-3 16~, 17~-triol (17-epiestriol).
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ST ER O I D S
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INTRODUCTION Estrone (I), 16-epiestriol (2) and estradiol-17~ (3) have been isolated in crystalline form from laying hens' urine, which contains enough of these steroids to permit of their chemical estimation by classical methods (4, 5, 6). In addition, the presence of estriol and of 16, 17-epiestriol in laying hens' urine has been established chromatographically (7). Information about in vivo conversions of steroid estrogens has been obtained by injecting various 14C-labelled steroid estrogens and then examining the urine for radioactive conversion products.
The steroids injected have included estradiol-17~-16-14C
(8), estradiol-17~-4-14C (9), estrone-16-14C (i0), estriol-16-14C(ll), 16-ketoestradiol-17~-16-14C
(iZ), 16-epiestriol-16-14C (13) and
estradiol-17~-6, 7-3H (14). The radioactive conversion products detected in one or other of these experiments have included estrone, estradiol-17~, estradiol-17oG estriol, 16-epiestriol,
16-ketoestradiol-17~,
16-ketoestrone,
16, 17-epiestriol and 17-epiestriol.
As regards in vitro studies, Mitchell and Hobkirk (15) obtained radioactive estriol upon incubation of liver slices from a laying hen with estradiol-17~-16-14C.
In an extensive study, Ozon and Breuer
(16) incubated estrone, estradiol-17~, estradiol-17~,
16-ketoestradiol-
17~, 16-epiestriol, and 16-hydroxyestrone with chicken liver slices. They demonstrated the presence of the following enzymes in the liver slices:-
16~-, 16~-, 17~- and 17~-hydroxysteroid oxidoreductases,
together with 16~- and 16~-hydroxylases.
They concluded that
Oct. 1968
ST ER O IDS
16-epiestriol was quantitatively a m o r e
477
important metabolite than
estriol. Despite the availability of the foregoing information
on in vivo
and in vitro conversions, there is relatively little information about steroid estrogen metabolic pathways in the hen.
The present report,
therefore, deals with a study of the pathway leading f r o m estradiol17~ to 16-epiestriol in the hen. EXPERIM
EN TAL
Materials and m e t h o d s The experimental birds w e r e m a t u r e R h o d e Island R e d hens modified surgically (17) to exteriorize the ureteral openings and to permit quantitative collection of urine as described elsewhere (ii). Urine was r e m o v e d f r o m the collection tube several times daily and placed immediately in cold storage at -15°C. The sub-samples w e r e bulked to give Z4-hr samples immediately before analysis. Estradiol- 17~-4- 14C (Radiochemical Centre, A m e r s h a m , England) and estradiol-17~-17~-3H (presented by Dr. D.S. Layne) w e r e purified by thin-layer c h r o m a t o g r a p h y before use. (Reference estradiol-17~, estrone, estradiol-17~, 16-epiestriol and 16-ketoestradiol17~ w e r e obtained f r o m Steraloids Inc., Pawling, N.Y., U.S.A.). Solvents w e r e of analytical grade. Thin-layer chromatography was p e r f o r m e d on silica gel G (Merck) with the use of the solvent systems listed in Table I. F r e e phenolic steroids w e r e visualized on the plates by the reagent of Folin and Ciocalteu or by spraying with 2 % H z S O 4 in ethanol (v/v) and then heating the plates at ii0 ° for about 5 minutes. Methyl ethers and acetates of the phenolic steroids w e r e visualized by m e a n s of the latter reagent. Radioactivities w e r e assayed by m e a n s of a P a c k a r d Tri'Carb liquid scintillation spectrometer, M o d e l 3003. The procedure was that described by Flood et ai.(18). Settings at the counter w e r e adjusted in order completely to eliminate tritium f r o m counts in the 14C channel. A small r e s i d u u m of 14C counts was still counted in the tritium channel, however, so corrections for 'overflow' of 14C w e r e m a d e by use of calibration curves prepared with reference 14C- and 3H-toluene. Methylations w e r e done by Brown's m e t h o d (19) with strict adherence to that author's directions. Acetylations w e r e done by dissol-
478
ST ER O ID S
12:4
ring the steroid in I. 0 m l pyridine, adding i. 0 m l acetic anhydride and leaving the m i x t u r e overnight at ambient temperature. Thereafter the solvents w e r e evaporated under Nz and the residual acetylated product w a s dissolved in methanol. TABLE
I
Thin-layer C h r o m a t o g r a p h i c S y s t e m No.
B C F O X Experimental
Systems
Composition
Reference
Ethyl acetate - n-hexane ethanol (v/v 80:15:5) C y c l o h e x a n e - ethyl acetate
(20)
(I:i)
(z0)
B e n z e n e - m e t h a n o l (9:1) n - h e x a n e - ethyl acetate (75:Z5) CHC13 : diethyl ether (6:4)
(Zl) (20) (Z2)
Procedure
A m i x t u r e of 2. 1 x 106 d p m e s t r a d i o l - 1 7 ~ - 4 - 1 4 C a n d 2. 1 x 107 dprn estradiol-17~-17a-~H was injected intramuscularly into a laying Rhode Island Red hen. Three successive Z4-hr urines were collected thereafter. Each 24-hr sample was assayed for radioactivity. The urine for day 1 was divided into two equal parts and each part was processed separately as follows:Free steroids were extracted with CHC13 (3 x 1 v o l ) . T h e CHC13 w a s e v a p o r a t e d , the residue was taken u p in 10 m l m e t h a n o l a n d 0. 10 m l p o r t i o n s of t h i s s o l u t i o n w e r e a s s a y e d for radioactivity. T h e aqueous phase f r o m the foregoing extraction w a s solvolysed for 48-hr by the m e t h o d of Burstein and L i e b e r m a n (23). Previous experience (24) had s h o w n that 24-hr w a s insufficient to hydrolyse completely the conjugates (mainly sulphates) in the urine. The solvolysed fraction w a s w a s h e d with i. 0 M N a H C O 3 (Z x 0. 1 vol) and then with water (2 x 0. 1 vol). The solvent w a s r e m o v e d f r o m the solvolysed fraction and the residue w a s dissolved in m e t h a n o l and a s s a y e d for radioactivity. T h e C H C I s extract and the solvolysed fraction w e r e purified separately as follows:- T h e material w a s c h r o m a t o g r a p h e d in s y s t e m X with a m i x t u r e of reference estrone, estradiol-17~, estradiol-17~, 16-ketoestradiol-17~ and 16-epiestriol on lateral columns. Zones corresponding in mobilities to the reference steroids w e r e r e m o v e d f r o m the plate, eluted and the eluates r e c h r o m a t o g r a p h e d separately in s y s t e m C. T h e 16-epiestriol fraction w a s further c h r o m a t o g r a p h e d in s y s t e m B to purify it f r o m any residual 17-epiestriol. The 16-
Oct. 1968
S T ER O ID S
479
ketoestradiol-17~, estradiol-17~ and estradiol-17~ subfractions w e r e r e c h r o m a t o g r a p h e d i n system X. E a c h fraction was assayed for radioactivity after each purification step and ratios d p m 3H : dprn 14C w e r e calculated. Portions of the radioactive steroids thus purified w e r e m i x e d with the appr opriate non-r adioactive refer ence steroid and chr omatographed in systems X and C (for estradiols and 16-ketoestradiol-17~) and systems X and B (for 16-epiestriol). After development the estrogens wer e visualized with the HzSO4-ethanol reagent and then the gel was scraped f r o m the plate and assayed. In every case the stain corresponded in position with the radioactivity. Portions of the purified phenolic fraction f r o m the solvolysis wet e m ethylated and the m ethyl ether s wet e m i x e d with appropriate refer ence methyl ether s and c h r o m a t o g r a p h e d in system C bidimensionally and in system F dimensionally. In each case the stained spot corresponded in position with the radioactivity. Further portions of each purified phenolic steroid w e r e acetylated and c h r o m a t o g r a p h e d twice in the s a m e direction in system O and then once at right angles to this in the s a m e system. This procedure afforded a superior separation of the acetates of the two estradiol epirners if either w e r e still contaminated by the other. Attempts to study the estriol and 16, 17-epiestriol of the extracts by similar m e a n s failed because of the small amounts of radioactivity in these fr actions. RESULTS T h e total 14C and 3H radioactivities recovered in the urine w e r e 31. 1% and 13. 18%, respectively, of the amounts injected (Table II). The crude ratio d p m 3H : d p m
14C in the urine for day 1 was 3.45:1.
TABLE
II
Total Urinary Recoveries of Administered 14C and 3 H 14 C Day
dpm
3H % of dose
dpm
% of dose
1
5.97 x i05
28.21
2 . 0 6 x I06
9.69
2
6. 12 x 104
2.89
4.80 x 105
2.25
3
negligible
-
2.63 x 105
1.24
31. I0
2.80 x 106
13. 18
Sum
i+2+3
6.58 x 105
~8o
ST ER O I D S
12:4
The C H C I 3 extract of the day-/ urine yielded 9.75% of its 14C radioactivity, the corresponding figure for
3
H being 6.2%.
The subsequent 48-hour solvolysis liberated 41.76% of the radioactivity and Z0.73% of the
3
14
C
H radioactivity of the day-i urine.
Examination of the radioactivities of the phenolic steroids of the urinary C H C I 3 extract prior to solvolysis ('free' estrogens), as separated by thin-layer chromatography, estradiol-17~ did not contain 3H.
revealed that the estrone and
The ratios d p m 3H : d p m
the other phenolic steroids are s u m m a r i z e d TABLE
14G for
in Table III.
III
Ratios d p m 3H : d p m 14C for Phenolic Steroids of C H C I 3 Extract of day-I Urine before Solvolysis. C H C I 3 Extract accounted for 9.75% of 14C in day-I Urine. Steroid
d p m 3H : d p m
E str one Estr adiol- 17~ Estr adiol- 17~ 16 -Ketoestr adiol- 1713 16-Epie str iol *
3
14C
~ 4. 03 4.05 4.40
H radioactivity not detectable. The similar examination of the extract of the day-1 urine after
solvolysis yielded the results s u m m a r i z e d
in Table IV.
The 14C radioactivities of the phenolic steroids from the solvolysed urine provided an estimate of the relative proportions of the various radioactive phenols as set out in Table V.
Oct. 1968
ST ER O I D S
TABLE
481
IV
Ratios d p m 3H : d p m 14C for Phenolic Steroids of C H C I 3 Extract of day-i Urine after Solvolysis. Extract after Solvolysis accounted for 4 1 . 7 6 % of 14~ and 2 0 . 7 3 % of 3H in day-i Urine. dpm
Steroid
Phenol
Estrone Estr adiol - 17~ Estradiol-17~
16-Ketoestradiol-17~ 16-Epiestriol *
3
H
: dpm
14
C
Average for phenol and 2 derivatives
Methyl ether
Acetate
* * 4.03
* * 4.05
* * 3.97
4.01
4.00 4.04
4. 30 4. 13
4. i0 3.73
4. 13 3.97
3 H radioactivity too low to be m e a s u r a b l e with any precision.
TABLE
V
Relative Proportions of Radioactive Phenolic Steroids from Extract Solvolysed Day-i Urine. Chromatography on Silica gel G in System
Steroid
of X.
Percentage
Estr one Estr adiol- 17~ Estr adiol- 17~ 16 -Ketoestr adiol- 179 16-Epiestriol A r e a s between spots Total
37. 1 20.9 24.8 0.5 4.9 ii. 8 i00. 00
DISCUSSION Fishrnan et al. (25) have shown that estradiol-17~ is reversibly metabolised to estrone in the human, lying m u c h
in favour of estrone.
with the equilibrium in vivo
They found also that estriol and 16-
~82
STEROIDS
12:4
epiestriol w e r e derived f r o m estrone and not directly f r o m estradiol17~.
R a u d (26) has found that h e n liver h o m o g e n a t e converts estrone
preferentially to estradiol- 17~. Studies in this laboratory (6) have furnished s o m e
evidence that there is m o r e
estradiol-17~ than es£rone
in the urine of non-laying hens w h e r e a s there is roughly as m u c h estradiol-17~ as estrone in the urine of the laying hen. In the course of their incubation experiments with chicken liver slices, O z o n and B r e u e r (16) did not find 16~- or 16~-hydroxy estrone a m o n g the in vitro conversion products of estradiol-17~ or estradiol-17~.
At the s a m e time they did find 16-epiestriol to be one
of the m a j o r in vitro metabolites of estradiol-17~. 16-Epiestriol is f o r m e d f r o m estrone in the h u m a n 16~-hydroxyestrone.
subject via
T h e results of the experiments n o w reported
s h o w that !6-epiestriol w a s f o r m e d f r o m estradiol-17~ in the hen. If any appreciable fraction of the 16-epiestriol had been f o r m e d f r o m the estradiol-17~ via estrone, then the ratio d p m
3H : dpm
14C for the
16-epiestriol w o u l d have b e e n lower than the ratio for the urinary estradiol-17~.
In the upshot, the two ratios w e r e practically identi-
cal, w h i c h suggests strongly that estrone does not, in the hen, lie on the metabolic p a t h w a y f r o m estradiol-17~ to 16-epiestriol. O z o n and B r e u e r (16)incubated 16~-hydroxyestrone with chicken liver slices and identified estriol and 17-epiestriol as the conversion products.
It s e e m s probable, therefore, that estrone is not
converted to its 16-hydroxy derivatives in the hen as it is in h u m a n s ,
Oct. 1968
ST ER O ID S
483
but that estrone m e r e l y remains in meta]~olic equilibrium with the two estr adiol
epimer
The
s.
results
for ratios
suggest that, in the hen, estradiol-17~.
dpm
3H : d p m
14G (Tables III and IV)
16-ketoestradiol-17~ is also derived f r o m
It is already k n o w n that the hen can convert estriol
into 16-ketoestradiol-17~ in vivo (11) and that 16-ketoestradiol-17~ gives rise to 16-epiestriol (12), hence the m a i n in vivo interconversions of these three steroids m a y Estriol--~
be s u m m a r i z e d
16-ketoestradiol-17~ ~
as 16-epiestriol.
If estriol w e r e not derived from estradiol-17~ but w e r e f o r m e d via estrone, then the ratio d p m 3H : d p m
14C would have been zero
for the urinary 16-epiestriol in the experiments n o w reported.
In
point of fact, the ratios for the estradiol-17~ and 16-epiestriol w e r e virtually identical, and estriol itself m u s t arise from estradiol-17~. If this be granted, then it b e c o m e s possible to visualize the in vivo metabolism
of estradiol-17~ in the hen as follows:Estradiol-17~"
,L
) Estrone I
, Estradiol-17~
I
s
16-Epiestrioi ¢
) 16-Ketoestradiol-1713 {
Estriol
ACKNOWLEDGMENTS We e x p r e s s o u r t h a n k s to t h e N a t i o n a l I n s t i t u t e of A r t h r i t i c a n d M e t a b o l i c D i s e a s e , U . S . D e p a r t m e n t of N a t i o n a l H e a l t h a n d W e l f a r e , a n d t h e N a t i o n a l R e s e a r c h C o u n c i l of C a n a d a f o r t h e f i n a n c i a l s u p p o r t that made this work possible. We a l s o t h a n k D r . D . S . L a y n e , D e p a r t m e n t of Biochemistry, University of Ottawa, for providing estradiol17~-17~-3H and Dr. N. Nikolaiczuk, M a c d o n a l d College, for cooperation in supplying suitable experimental hens.
g8q
ST ER O I D S
12:4
REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. I0. Ii. 12. 13. 14. 15. 16. 17. 18.
19. 20. 21. 22. 23. 24.
Ainsworth, L., and C o m m o n , R.H. N A T U R E , 195, 77 (1962). Hertelendy, F., and C o m m o n , R.H. C A N . J. BIOCHF-aXd., 42, 1177 (1964). Hertelendy, F., Taylor, T.G., Mathur, R.S. and C o m m o n , R.H. C A N . J. BIOCHEIVI., 43, 1379 (1965). C o m m o n , R.H., Ainsworth, L., Hertelendy, F., and Mathur, R.S. C A N . J. BIOCHF-~., 43, 539 (1965). Mahhur, R.S., Anastassiadis, P.A., and C o m m o n , R.H. POULTRY SCI., 45, 946 (1966). Mathur, R.S., and C o m m o n , R.H. P O U L T R Y SCI. in press. Mathur, R.S., and C o m m o n , R.H. CAN. J. BIOCH~Vl., 45, 531 (1967). Hertelendy, F., and C o m m o n , R.H. P O U L T R Y SCI., 44, 1379 (1965). Mulay, S., and C o m m o n , R.H. CAN. J. B I O C H ~ I . , 46, (in press) (1968). Ainsworth, L., Carter, A.L., and C o m m o n , R.H. C A N . J. BIOCHI~I. and PHYSIOL., 40, 123 (1962). MacRae, H.F., Dale, D.G., and C o m m o n , R.H. CAN. J. B I O C H E M . and PHYSIOL., 38, 523 (1960). Ainsworth, L., and C o m m o n , R.H. C A N . J. B I O C H ~ M . and P H Y S I O L . , 4!, 2045 (1963). Ainsworth, L., Carter, A.L., and C o m m o n , R.H. C A N . J. B I O C H E M . , 42, 521 (1964). Mulay, S., Henneberry, G.O. and C o m m o n , R.H. S T E R O I D S , (in press) (1968). Mitchell, J.E., and Hobkirk, R. B I O C H E M . B I O P H Y S . RES. C O M M . , i, 72 (1959). Ozon, R., and Breuer, H. ZEIT. P H Y S I O L . C H E M . , 341, 239 (1965). Ainsworth, L. P O U L T R Y SCI., 44, 1561 (1965). Flood, C., Layne, D.S., Ramcharan, S., Rossipal, E., Tait, J.F., and Tait, S.A.S. A C T A E N D O C R I N O L . , 36, 237 (1961). Brown, J.B. BIOCH~VI. J., 60, 185 (1955). Lisboa, B.P., and Diczfalusy, E. A C T A E N D O C R I N O L . , 40, 60 (1962). Ladany, S., andFinkelstein, M. STEROIDS, 2, 297(1963). Sobrevilla, L., Hagermann, D., and Villee, C. B I O C H I M . B I O P H Y S . A C T A , 93, 665 (1964). Burstein, S., and Lieberman, S. J. BIOL. C H E M . , 233, 331 (1958). Mathur, R.S., C o m m o n , R.H., Collins, D. C~ and Layne, D.S. unpublished observations.