87

CONVERSION OF ESTRADIOL-17a TO ESTRONE

BY THE HEN IN VIVO

S . M u l a y , G . O . H e n n e b e r r y and R . H. C o m m o n

M a c d o n a l d C o l l e g e of M c G i l l U n i v e r s i t y P r o v i n c e of Q u e b e c , C a n a d a

Received March 6, 1968.

ABSTRACT

E s t r a d i o l - 1 7 a - 6 , 7-3H was i n j e c t e d in to a l a y i n g h e n . T h e u r i n e s u b s e q u e n t l y e x c r e t e d c o n t a i n e d e s t r o n e - ~ r I , w h i c h was i d e n t i f i e d b y i t s c h r o m a t o g r a p h i c b e h a v i o u r and by c r y s t a l l i z a t i o n w i th r e f e r e n c e c a r r i e r e s t r o n e to c o n s t , s p e c . a c t . The e s t r o n e r a d i o a c t i v i t y r e - c o v e r e d f r o m the u r i n e r e p r e s e n t e d 1 .94% of t he r a d i o a c t i v i t y i n j e c t e d and Z4 .0% of the r a d i o a c t i v i t y e x t r a c t e d by CHCI~ f r o m u r i n e tha t h a d b e e n i n c u b a t e d w i t h a ~ - g l u c u _ r o n i d a s e p r e p a r a t i o n . T h e p o s s i b i l i t y of f o r 1 ~ a t i o n of t he r a d i o a c t i v e e s t r o n e by m i c r o b i o l o g i c a l a c t i o n a f t e r e x c r e t i o n of the u r i n e was e x c l u d e d b y ad hoc e x p e r i m e n t a t i o n .

It was c o n c l u d e d t ha t the hen can c o n v e r t e s t r a d i o l - 1 7 a to e s t r o n e in v i vo . S o m e i m p l i c a t i o n s of th is f i nd ing in r e l a t i o n to u r i n a r y s t e r o i d e s t r o g e n e x c r e t i o n by the fowl a r e d i s c u s s e d b r i e f l y .

IN TRODUC TION

O z o n and B r e u e r (1) h a v e shown t ha t c h i c k e n l i v e r s l i c e s c a n i n t e r -

c o n v e r t (a) e s t r o n e and e s t r a d i o l - 1 7 a and (b) e s t r o n e and e s t r a d i o l - 1 7 ~ .

Others have shown that the hen can effect the interconversion of

e s t r o n e and e s t r a d i o l - 1 7 ~ in v i vo (2, 3) and the c o n v e r s i o n of e s t r o n e

a n d e s t r a d i o l - 1 7 ~ to e s t r a d i o l 17a in v ivo (4) . T h e s e o b s e r v a t i o n s

prompted an investigation of the in vivo urinary conversion products

of estradiol-17~ in the hen and this investigation is described below.

88 S T E R O I D S 12:1

EXPERIMENTAL

M a t e r i a l s and m e t h o d s

The experimental birds were mature Rhode Island hens. These hens were in lay when operated surgically to exteriorize the ureteral openings by Ainsworth's technique (5), but were not in lay at the times when they were used for experiment. Urine was collected as described elsewhere (6) except that (a) the urine was removed from the collection tube twice each day and (b) penicillin G (IZ5, 000 units) was dusted into the collection tube before each collection. Immediately after collection of urine, it was placed in cold storage at

-15°C pending analysis. Solvents, purification of solvents and thin-layer chromatog-

raphy (TLC) were as described elsewhere (7). The solvent systems used are listed in Table I.

TABLE I

Composition of solvent systems

System Reference Cornpositibn in parts by vol.

A (8) C (8) X (9) Y (I0) F 1 (i0) o (11)

CHCI 3

Cyclohexane: ethyl acetate: ethanol (45:45: I0) Cyclohexane: ethyl acetate (50: 50) Chloroform: diethyl ether (60:40) Benzene: methanol (95:5) Benzene: methanol (99: i) n-Hexane: ethyl acetate (75:Z5) Chloroform

When necessary, estrogens were visualized on chromato- plates either by spraying with the reagent of Folin and Ciocalteu or with HzSO4-ethanol (2:98 v/v) and then heating the plate in an oven at I05°C.

The reference steroids used were as follows:- Estrone (Steroid Laboratories, Montreal) was recrystalli~ed to chromato- graphic homogeneity (three systems) and hadm.m.p. Z60°C. Estradiol-17~ (Signaa Chemical Co., St. Louis, Mo.) was recrystal- lized and the purified material was homogeneous as judged by TLC (three systems). Estradiol-17~ (Sigma Chemical Co., St. Louis, Mo.) w_as purified by TLC in system C. 16-Epiestriol (donated by Dr. D.S. Layne, Department of Biochemistry, University of Ottawa, Ottawa, Canada) was chromatographically homogeneous (three systems) and hadm.m.p. 274°C. 17-Epiestriol was donatedby Dr. Jack Fishman,

89 July 1968 S T E R O I D S

Montefiore Hospital, New York, N.Y. It was used as received. Estriol (Steraloids, Pawling, N.Y.) was purified by fractional micro-

sublimation to chromatographic homogeneity and hadm. m.p. 280°C.

Estradiol-17~-6, 7-23-I was donated by Dr. O.S. Layne, Department of Biochemistry, University of Ottawa, Ottawa, Canada. It was

purified immediately before use by TLC in system X (double develop-

ment), The purified material was pure as tested by TLC and approp- riate examinations of the distribution of radioactivity on chromato-

plates (See Fig. i). Radioactivity was measured with a Packard Scintillation Spectrometer Model 3003. Measurements of separation

effected by TLC were performed by scraping serial strips (either 3 mm or 5 mm wide) from the plates and suspending the gel samples

thus obtained in scintillation fluid to which had been added 4 g Cab-O-

Sil (Cabot Corporation, Boston, Mass.) per i00 ml. Measurements on crude urine and aqueous extracts were made by the procedure of

Okita et al. (12).

b

E d. d

v

>

o

n,-

2 4

1 8

12

6

0

s.f.

$

e • I I

D i s t a n c e (cm~

FIG. l.Thin-layer chromatogram (system X) of estradiol-17~-6,

7-3H used for injection, s.f. = solvent front, e = position

of reference estradiol-17~.

The bulked urine for each 24 hr collection period was sub- jected to enzymic hydrolysis with ~-glucuronidase (Ketodase, Warner-

Chilcott, Toronto, Ont. ) and subsequent extraction as follows: The

urine was diluted and filtered. The solid urates were ground up with

water several times and the aqueous extracts were added to the main

90 S T E R O I D S 12:1

f i l t r a t e . T h e v o l u m e w a s m a d e u p to 500 m l a n d to t h i s s o l u t i o n w e r e a d d e d 2 0 . 5 g s o d i u m a c e t a t e ( a n h y d r o u s ) a n d 0 . 6 0 5 g K z H P O 4 . T h e s o l u t i o n w a s a d j u s t e d to p H 4 . 5 w i t h g l a c i a l a c e t i c a c i d , a n d ~ - g l u c u r o n i d a s e w a s a d d e d a t t h e r a t e of 500 F . U . p e r m l s o l u t i o n a n d t h e s o l u t i o n w a s t h e n i n c u b a t e d f o r 24 h r . a t 3 7 ° C . a n d s u b s e q u e n t l y e x t r a c t e d w i t h CHC13 (2 x 1 v o l . ) T h e a q u e o u s p h a s e w a s r e - a d j u s t e d to p H 4. 5, a f u r t h e r a d d i t i o n of ~ - g l u c u r o n i d a s e w a s m a d e , a n d i n c u b a t i o n a n d e x t r a c t i o n w e r e d o n e a s b e f o r e . T h i s c y c l e of i n - c u b a t i o n a n d e x t r a c t i o n w a s r e p e a t e d f o r a t h i r d t i m e . T h e b u l k e d CHC13 e x t r a c t w a s f r e e d f r o m CHC13 u n d e r r e d u c e d p r e s s u r e a n d t h e r e s i d u e w a s d r i e d in a v a c u u m d e s i c c a t o r o v e r f r e s h CaC12 ( a n h y d r . ) T h e r e s i d u e w a s t a k e n u p in m e t h a n o l a n d a l i q u o t s w e r e t a k e n f o r T L C , t h e a p p l i c a t i o n s to t h e c h r o m a t o p l a t e b e i n g m a d e b y a s t r e a k i n g t e c h - n i q u e .

G i r a r d s e p a r a t i o n s w e r e d o n e b y t h e p r o c e d u r e of G i v n e r e t a l . (13).

RESULTS AND DISCUSSION

Estradiol-17a-6, 7-3H (5.492 x 108 d.p.m. ) in solution in

p r o p y l e n e g l y c o l w a s i n j e c t e d i n t r a m u s c u l a r l y i n t o t he h e n . T h e 2 4 -

h o u r u r i n e s a m p l e s w e r e c o l l e c t e d o v e r s i x o f t h e s u c c e e d i n g s e v e n

d a y s . T h e r e c o v e r i e s o f r a d i o a c t i v i t y in t h e s e s a m p l e s a r e s u m n ~ a r -

i z e d in T a b l e I I .

R e c o v e r i e s

T A B L E I I

of i n j e c t e d r a d i o a c t i v i t y in u r i n e

Day Total urinary, radioactivity

as d.p.m. As % of radioactivity injected

l 1.65 x l08 29.9 Z 6.45 x 10. b 1.2 3 2.37 x l0 b 0.4

5 0.78 x l06 0.14 6 0.21 x 106 0.04 7 0.09 x I0 b 0.02

Sum 1.74x 108 31.7

Urine collection omitted on day 4.

July 1968 s T E R O I D S 91

The urinary recovery of radioactivity (31.7%) was of the

same order as those reported for injections of 16-ketoestradiol-17~

(14), 16-epiestriol (15), estradiol-17~ (16) and estrone (16) under

similar conditions. Gomparison of the percentage partitions of

urinary radioactivity for the first three Z4-hr. urine samples did not

reveal any differences as between these three samples. Accordingly,

the data for the first three days were pooled and are summarized in

Table III. The loss of ZZ.9~, of the radioactivity in the course of the

partitions was high but not exceptionally so for experiments of this

kind.

TABLE III

Partition of urinary radioactivity after enzymatic hydrolysis Fir st three Z4-hr collections

Fraction d.p.m. percent total

urinary radioactivity

Total radioactivity in urine 173.3 x 106 I00.0

CHCI 3 extr. after ist incubation Z6. 1 x 106 " Znd " 3.6 x 10,6 " 3rd " 3.2 x 100

6 CHCI 3 extr., total 32.9 x 106 Et. Acet. extr. after CHCI 3 extr. 20.6 x 106 Aqueous residue 80.0 x 10

19.0

11.9 46. Z

Total radioactivity recovered in partition fractions

6 133.5 x i0 77.1

The pooled CHCI 3 extract for the first three Z4-hr samples

(19.0 percent of total urinary radioactivity) was freed from solvents,

dried over CaClz, taken up in methanol and aliquots subjected to TLC

9 2 S T E R O I D S 12:1

in system X. The distribution of radioactivity on a typical chromato-

plate is shown in Fig. 2.

'0

E 0

>

0

"0

O/

6

4

0

s. f .

$ a bc d e

i l I I I I I I I l l I I I l I f l I J l I l J

0 5 10 15 D i s t a n c e (cm)

FIG. 2. Distribution of radioactivity on thin-layer chromatograrn

(system X) of CHCI 3 extract of enzyrne-hydrolyzed urine.

s.f. = solvent front.

indicated as follows:

a = estriol

c = 17-epiestriol

e = estradiol-17~

Positions of reference estrogens

b = 16-epiestriol

d = estradiol-17~

f = estrone

Note: 16-ketoestrone is slightly less polar than estradiol-

17o/ in sysLem X.

The second highest peak on Fig. Z corresponded chromato-

graphically to estrone and accounted for 24.0% of the radioactivity

on the chromaLoplate. This area was eluted and a portion of the

eluate was subjected to crystallization with reference carrier estrone

with the results set out in Table IV.

July 1968 s T E R O I D S 93

TABLE IV

Crystallization of radioactive urinary

estrone with reference estrone

Purification step -3

d.p.m, x i0 rag. Specific activity d.p.m, x 10-3/rag.

Ist crystallization 197.6 17.5 1 I. Z9 Mother pool 96. Z 6. Z 15.5Z Znd crystallization 154.9 15.3 i0. 13

Mother pool ZI. 0 Z. 0 10.49 3rd crystallization 10Z. 7 I0. Z i0.07

Mother pool 37.5 3.7 i0. 14

O t h e r s a m p l e s of the e l u a t e w e r e s u b j e c t e d to T L G in a d m i x t u r e

with suitable quantities of non-radioactive reference material. The

positions of the radioactive materials were ascertained by counting

serial strips as described above. In each case the mobility of the

radioactive (test) sample corresponded with that of its reference mat-

erial. The observed RF values are summarized in Table V.

TABLE V

RF values of estrone-3H obtained from urine and of estrone-3H-acetate obtained from the estrone-~-I

E s t r o n e - 3 H

E s t r o n e - 3 H - a c e t a t e

Solvent system

A C Y F i O CHGI 3

0.7Z 0.50 0.43 - -

- - - 0.42 0. Z9 0.50

The foregoing results demonstrated the identity of the urinary

radioactive estrone and hence established the presence of radioactive

estrone in the urine.

9 4 S T E R O I D S 12:1

The highest peak (Fig. Z) corresponded to the positions of

e s t r a d i o l - 1 7 ~ , e s t r a d i o l - 1 7 a a n d 1 6 - k e t o e s t r o n e w i t h t h e h i g h e s t p o i n t

c o r r e s p o n d i n g to e s t r a d i o l - 1 7 a . T h e e n t i r e p e a k (5. 5 c m to 7 . 5 can

on t h e p l a t e a n d c o n t a i n i n g 5 4 . 8 % of t he r a d i o a c t i v i t y on t h e p l a t e )

was eluted and the eluate was subjected to Girard separation. The

K e t o n i c f r a c t i o n a c c o u n t e d f o r 1 9 . 9 % of t h e r a d i o a c t i v i t y , h e n c e a n y

16-ketoestrone present could not have accounted for more than 19.9%

of t h i s f r a c t i o n o r 10. 1% of t h e t o t a l r a d i o a c t i v i t y on t h e c h r o m a t o -

plate. The non-ketonic fraction was subjected to TLC in system X

with results shown in Fig. 3.

FIG. 3.

~ 8

Jo

0

>

U

~ 2 o

"(3

n- O

j] ( 28.8

&£

a bc de f 1 I I I I I I I

I I I I I I I l I I l I I I I I I I

0 5 10 15 D i s t ance ( cm)

Distribution of radioactivity on thin-layer chromatograrn

(system X), double development of the non-ketonic fraction

from the major peak obtained by TLC of urinary CHCI 3 extr- act (see Fig. Z). s.f. = solvent front.

Positions of reference estrogens indicated as follows:-

a = estriol b = 16-epiestriol c = 17-epiestriol d = estradiol-17~

e = estradiol-17~ f = estrone

July 1968 s T E R O I D S 95

There was a very high peak of radioactivity precisely in the

position of estradiol-17~ with a shoulder in roughly the position of

estradiol-17~, but resolution of the diols was not attempted.

The foregoing results established the presence in the urine of

radioactive conversion products of the injected estradiol-17~-3H, a

major product being radioactive estrone. Despite the circumstances

that antibiotic was added to the collection tubes and every care was

excercised to reduce the risk of microbial action after excretion of the

urine, there remained a possibility that the conversion might be due

to such microbial action. Accordingly, estradiol-17~-3H was added

to the collection tube of a comparable hen that had not received any

injection of radioactive material and the 24-hour urine was collected,

extracted with chloroform and the extract examined as had been the

extract of the experimental urine. TLC in system X yielded no evid-

ence that any of the estradiol-17~-31-1 had been converted to estrone

(see Fig. 4) or to any other product. It was concluded that the urinary

radioactive estrone in the urine of the bird that had received an in-

jection of estradiol-17~-3H (see Fig. 2) was not formed by microbiolog-

ical conversion after excretion. This did not exclude the possibility

of conversion by the action of intestinal microorganisms on estradiol-

17~-3H excreted into the intestine, with subsequent absorption from

the intestine and re-excretion via the urine, but this possibility is in-

herent in in vivo experiments unless germ-free animals are used.

96 S T E R O I D S 12:1

2 0

'O1 5 ~j

rS d

> , 1 0

U

m 5 o

tll

0

< 30.2

s.f $

a bc d e f I I II I I I I

I I i i I i / I i I I I I I I I l ]

0 5 10 15 D i s t a n c e ( cm)

FIG. 4. Distribution of radioactivity on thin-layer chromatogram

(system X) of CHCI 3 extract of urine control from uninjected

bird (see text), s.f. = solvent front.

Positions of reference estrogens denoted as in Fig. 2.

Since estradiol-17~ gives rise to estrone in vivo in the hen,

and since estrone is known to give rise to all four estriol epirners in

vivo in the hen (17), it might be expected that estradiol-17~ would

give rise to the four estriols. Inspection of Fig. Z shows that there

was in fact a minor peak of radioactivity in a position corresponding to

those of 16-epiestriol and 17-epiestriol. This peak was eluted and the

eluate was rechromatographed along with reference 16-epiestriol and

17-epiestriol in two systems. The mobility of the radioactivity was

consistent with the presence of radioactive 16-epiestriol on the plate.

Although this evidence for the presence of the cis-estriols is admit-

July 1968 S T E R O I D S 97

redly meagre, it does provide incidental support for the thesis that

estradiol-17~ is converted in vivo to other steroid estrogens in

addition to estrone.

DISCUSSION

The results demonstrated that radioactive estrone appears

in the urine after the injection of radioactive estradiol-17~, and that

this conversion did not take place in the urine subsequent to its ex-

cretion. The conversion of estradiol-17~ to estrone had taken

place in vivo, therefore, either in the tissues or in consequence of

conversion in the intestine with subsequent reabsorption and re-

excretion in the urine. The facts (a) that chicken tissue slices can

convert estradiol-17~ to estrone (i) and (b) that chicken liver has

recently been shown (18) to contain the necessary enzymes are con-

sistent with the view that the radioactive estrone found in the urine

in the present experiments was mainly, if not entirely, the product

of metabolic conversion in the tissues of the hen; and if estradiol-

17~ thus gives rise to estrone in the hen, the estrone thus formed

will give rise to the other known in vivo conversion products (5) of

estrone.

ACKNOWLEDGEM EN TS

We thank Dr. D.S. Layne and Dr. Jack Fishman for gifts of

steroid estrogens. We also thank Dr. R.S.Mathur, Macdonald

College, Que., for surgical operation of the hens used in the study;

and the National Institute of Arthritis and Metabolic Diseases, U.S.

9 8 S T E R O I D S 12:1

Department of National Health and Wel£are, for Grant AM-06130-END and the National Research Council of Canada (Grant A-56), which grants supported the work. We are indebted to the National Research Council of Canada for a Graduate Scholarship awarded to one of us

(S.M.)

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3.

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