595 BILIARY J.

METABOLITES

OF

ESTRIOL

IN T H E

RAT

A l l a n M e n z i e s and H i s a k o W a t a n a b e * Drug Research Laboratories Health Protection Branch O t t a w a , O n t . , K I A OL2 Canada

Received: 12/22/75 ABSTRACT _ F o l l o w i n g the s u b c u t a n e o u s administration of e s t r i o l 6 , 7 - 3 H to r a t s , b i l i a r y m e t a b o l i t e s w e r e i d e n t i f i e d and quantitated. Approximately 70% of the m e t a b o l i t e s were e x c r e t e d in the f o r m of " g l u c o s i d u r o n a t e " conjugates. 3,17B-Dihydroxy-2-methoxy-l,3,5(lO)-estratrien-16-one was the m a j o r m e t a b o l i t e in t h i s c o n j u g a t e f r a c t i o n . Signific a n t a m o u n t s of 3 , 1 7 ~ - d i h y d r o x y - l , 3 , 5 ( 1 0 ) - e s t r a t r i e n - 1 6 - o n e and 2 , 3 , 1 7 B - t r i h y d r o x y - l , 3 , 5 ( 1 0 ) - e s t r a t r i e n - 1 6 - o n e , as w e l l as s m a l l e r q u a n t i t i e s of 1 , 3 , 5 ( 1 0 ) - e s t r a t r i e n e - 2 , 3 , 1 6 a , 1 7 B tetrol and 2-methoxy-l,3,5(lO)-estratriene-3,16a,17B-triol, w e r e a l s o f o u n d . In 1 7 a - e t h i n y l e s t r a d i o l - treated animals, the r a t e of e x c r e t i o n of r a d i o a c t i v i t y and the p r o p o r t i o n of 16-oxo-17~-oi metabolites f o u n d in the " g l u c o s i d u r o n a t e " fraction were reduced. INTRODUCTION Although detected

in

16~-hydroxylated

large

hydroxylation

can

demonstrated estrone (3)

(4).

It

further

by

occur

the

in

rat

in v i v o

isolation

in

of

have

excreta this

not

been

(1,2),

species

that

has

l~-

been

16~-hydroxy-2-methoxy-

(3,16a-dihydroxy-2-methoxy-l,3,5(lO)-estratrien-17-

one)

tively

quantities

estrogens

from is

bile

following

possible

more

that

significant

metabolism the

vestigated.

Since

estrone

(2),

metabolism

increase the

failure

metabolism

i~

of

by

to

in

effect

the of

compound

To

estriol

of

estrone

quantitais

due

to

examine was

been

in-

shown

metabolites on

estriol

a

this

(l~-ethinyl-l,3,5-

previously

unidentified this

route

products.

exogenous

has

detect

this

-ethinylestradiol

(10)-estratriene-3,17~-diol) an

the

administration

to u n i d e n t i f i e d

possibility,

produce

the

to

of meta-

B

596

bolism

was

report

on

appeared

also the

-l" "~ It,. 0

studied.

During

identification

I

the

D

~

progress

of m e t a b o l i t e s

of of

this

work,

estriol

a

has

(5). EXPERIMENTAL

Materials Estriol-6,7-3H (sp.a. 1 5 C i / m m o l e ) , obtained from New E n g l a n d N u c l e a r C o r p . , B o s t o n , M a s s . , w a s p u r i f i e d by t h i n layer chromatography in the s y s t e m c h l o r o f o r m - e t h a n o l ; 9:1 p r i o r to use. 1 7 ~ - E t h i n y l e s t r a d i o l and 1 6 - o x o e s t r a d i o l (3,17B-dihydroxy-l,3,5(lO)-estratrien-16-one) were purchased from Steraloids Inc., P a w l i n g , N.Y. 2-Hydroxyestriol (l,3,5(10)-estratriene-2,3,16~,17B-tetrol) and 2 - m e t h o x y e s t riol (2-methoxy-l,3,5(lO)-estratriene-3,16~,17B-triol) were synthesized according to a p r e v i o u s l y p u b l i s h e d m e t h o d (4). 2-Hydroxy-16-oxoestradiol (2,3,17B-trihydroxy-l,3,5(lO)estratrien-16-one) and 2 - m e t h o x y - 1 6 - o x o e s t r a d i o l (3,17Bdihydroxy-2-methoxy-l,3,5(lO)-estratrien-16-one) were o b t a i n e d by t r e a t m e n t of 2 , 1 6 ~ - d i h y d r o x y e s t r o n e (2,3,16~trihydroxy-l,3,5(10)-estratrien-17-one) and its 2 ~ m e t h y l e t h e r (4) w i t h a l k a l i u n d e r n i t r o g e n . Fragmentations characteristic of e s t r o g e n s (6) w e r e s e e n in the m a s s s p e c tra and r e a r r a n g e m e n t to the 1 6 - o x o - 1 7 B - o l structure was i n d i c a t e d by a r e v e r s a l of the r e l a t i v e p r o m i n e n c e of the f r a g m e n t ion M - 7 2 o v e r M - 7 3 f o l l o w i n g a l k a l i t r e a t m e n t . Methods F o l l o w i n g the s u b c u t a n e o u s administration of 1 ~Ci of estriol-6,7-3H (sp. a. 1 5 C i / m m o l e or 2 . 8 8 m C i / m m o l e ) to m a t u r e f e m a l e W i s t a r r a t s , b i l e s a m p l e s w e r e c o l l e c t e d as previously described (2). Three animals were treated orally with 17~-ethinylestradiol at a d o s a g e of 0.5 mg (in 10% e t h a n o l i c s a l i n e ) d a i l y for n i n e d a y s p r i o r to b i l e c o l l e c tion. C o n t r o l a n i m a l s r e c e i v e d an e q u a l v o l u m e of e t h a n o l i c saline. The b i l e s a m p l e s w e r e m a d e 80% w i t h r e s p e c t to e t h a n o l and the p r e c i p i t a t e s washed with ethanol. The p o o l e d s u p e r n a t a n t s w e r e d r i e d , m a d e up in w a t e r and e x t r a c t e d w i t h e t h y l a c e t a t e to o b t a i n the f r e e s t e r o i d f r a c t i o n . The r a d i o a c t i v e m a t e r i a l c o l l e c t e d in the a q u e o u s p h a s e w a s i n c u b a t e d w i t h 500 u n i t s of K e ~ o d a s e per ml ( W a r n e r - C h i l c o t t L a b s . , N e w Y o r k , N . Y . ) in 0.i M s o d i u m a c e t a t e b u f f e r (pH 5.0) at 37 ° for 24 h. F o l l o w i n g e x t r a c t i o n of the i n c u b a t e w i t h e t h y l a c e t a t e , the a q u e o u s p h a s e w a s f u r t h e r i n c u b a t e d , with phenolsulfatase ( M y l a s e P, M a n n R e s e a r c h L a b s . , N e w Y o r k , N . Y . ) for 24 h in 0.i M s o d i u m a c e t a t e b u f f e r (pH 6.0) at 37 ° . For the q u a n t i t a t i v e experiments, aglycone carriers

597

in q u a n t i t i e s of i 0 0 ~ g e a c h w e r e a d d e d to e a c h of the incubation mixtures to minimize decomposition of the l i b e r a t e d steroids. The a q u e o u s residue, obtained after ethyl acetate extraction, w a s t h e n t r e a t e d w i t h IN H C I at i i 0 ° in a s e a l e d t u b e for 2 hr. The r a d i o a c t i v e material released by t h i s reaction was extracted with ethyl acetate. The e t h y l a c e t a t e extracts were dried over sodium s u l f a t e a n d the m e t a b o l i t e s were identified by c o m p a r i s o n s of t h e i r T L C and p a p e r c h r o m a t o g r a p h i c Rf v a l u e s , and GLC retention t i m e s and m a s s s p e c t r a of t r i m e t h y l s i l y l ether derivatives w i t h t h o s e of s t a n d a r d s . Thin-layer chromatography was performed on 0.4 m m s i l i c a g e l p l a t e s (Kieselgel N, M a c h e r e y , N a g e l and Co., G e r m a n y ) in the s y s t e m c h l o r o form - cyclohexane - acetic acid; 3:1:1. Paper chromatography was performed in a c h l o r o f o r m - formamide system. Gas-liquid chromatography w a s c a r r i e d out w i t h a m o d e l 810 F and M chromatograph equipped with a stream-splitter on a c o l u m n of 5% OV - 210 w i t h a c o l u m n t e m p e r a t u r e of 210 ° . The t r i m e t h y l s i l y l ether derivatives of all of the m e t a bolites were well separated by g a s - l i q u i d chromatography. Estriol and 2-hydroxy-16-oxoestradiol were not separated by the t h i n - l a y e r and paper chromatographic systems used. For purposes of q u a n t i t a t i o n , GLC s e p a r a t i o n of t h e s e two m e t a bolites were achieved following elution from thin-layer plates and trimethylsilyl ether derivatization of p o o l e d s a m p l e s f r o m the c o n t r o l a n d t r e a t e d g r o u p s . All quantit a t i o n s of r a d i o a c t i v i t y w e r e m a d e by l i q u i d s c i n t i l l a t i o n spectrometry (Packard Liquid Scintillation Spectrometer, M o d e l 3375, P a c k a r d Instrument Co., D o w n e r s G r o v e , Ill.) using Bray's reagent (5). The counting effeciency was approximately 30%. RESULTS In

order

conjugates

animals

ministration 73%

animals.

initially

excreted,

untreated

and

to

of

of the

bile

over a

tracer

dose

However,

was in

activity

found

in

various

shown

Table

i.

The

liberated

determine samples

dose

excreted

considerably

by

of

Ketodase,

in

part

the

2 h

3

of

the

H.

in

the

the

hydrolysis

bile

biliary being

two ad-

Seventy

these

proportion the

the

from

2 h following

The

of

of

collected

experiments,

extracts

the

nature

estriol-6,7-

less.

major

the

were

of

subsequent

were

was

DISCUSSION

a period

excreted

in

AND

two

amounts of

radio-

samples

are

radioactivity inhibited

598

~~-ffioxx~m

in the p r e s e n c e

of

sulfates

were

residue,

60% was

of i00

~g of

detected.

Of

liberated

estriol

of r a d i o a c t i v i t y in these

saccharolactone.

per

the r e m a i n i n g by acid

was m a r k e d l y

reduced,

there

amounts

in

hydrolysis. the

the a q u e o u s With

rate

of

doses

of e x c r e t i o n

was

no alteratlol

proportions. I

IN V A R I O U S

The

metabolites

shown

in T a b l e

possessing

the

found

2.

Rat

no.

of estriol. were

also

found.

were

excreted

and

the

2- and

and

2-hydroxyestriol

bile

following

(5).

Although

the no

3-methyl

to be p r e s e n t

lesser

have

ethers

ethers

recently

was

was

made

made

minor

fraction

that m e t a b o l i t e s predominate, biliary

2-

metabolite

2-hydroxy-16-oxoestradiol and

of

its

2-methyl

of

ether

16-Oxo-estradiol

2-hydroxy-16-oxoestradi~

been

isolated

a large

at p r e c i s e

in the p r e s e n t

in r e l a t i v e l y

No a t t e m p t

show

quantities.

administration attempt

~0.4

"glucosiduronate"

of

2

3.8 65.3 2.6

the m a j o r

amounts

3-monomethyl

Rat

2.7 69.2 2.3 25.8

2-Hydroxyestriol

in r a t h e r

i

structure

being

Significant

no.

results

16-oxo-17B-ol

OF B I L E Excreted

in the

The

methoxy-16-oxoestradiol

EXTRACTS

% of R a d i o a c t i v i t y

Unconjugated Ketodase hydrolyzed Phenolsulfastase hydrolyzed Aqueous residue

the

28%

although

RADIOACTIVITY

of

small

rat,

Table

are

Only

study,

dose

from of

rat estriol

quantitation they

appeared

amounts.

to i d e n t i f y

the m e t a b o l i t e s

in the

599

sulfate

fraction

conjugates recently

which

excreted. isolated

formed

a minor

However,

part

Nambara

and

2-hydroxy-16-epiestrioI

hydroxyestriol

2-sulfate

administration

(8).

from

rat

Table PROPORTION

OF

bile

Kawarada

following

2.9 3.8 7.9

32.4

19.9 15.7 44.3

a reduction

= 45.5%,

treated

excreted

with

=

of

± 0.09

17.2 16.4 30.1

± 2.6

30.9%)

in

the

the m e t a b o l i c

proportions

of

the

in

products of

amount

17e-ethinylestradiol in

proportions

± 0.9 + 2.1"

63.7

approximately

observed

these

± 0.64* ± 0.81"*

± standard error.

0.05 0.001

was

a reduction

estriol

8.2 6.5 17.7

± 0.69 ± 0.25

Results are reported as means of 3 animals per group

the

2-

17~ -ethinylestradioltreated

79.9

effect

and

FRACTION

14.6

There

have

IN T H E

Control

* P (diff.) < ** P (diff.)