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Metabolism of 4-14C-estradiol-17β by the newborn puppy
723
METABOLISM OF 4-14C-ESTRADIOL-17~ BY THE NEWBORN PUPPY A. Ainsworth Hagen and Paul F. Consroe Department of Pharmacology and Department of Medicine, Section of Endocrinology University of Tennessee, Memphis, Tennessee 38103 Received:
June i0, 1970 ABSTRACT
The metabolites of 4-14C-estradio1-17B, administered intravenously, were i d e n t i f i e d and quantitated in samples of urine, b i l e , and l i v e r from two male and two female newborn dogs. Conjugation of the metabol i t e s was extensive as less than lO percent of the r a d i o a c t i v i t y recovered from b i l e and urine was in an ether soluble form. Following enzyme hydrolysis the majority of the remaining r a d i o a c t i v i t y was rendered ether soluble. The major metaboIite found in urine and b i l e was estrone. Estradio1-17~was the second most important metabolite q u a n t i t a t i v e l y . In the l i v e r estradiol-17~ was q u a n t i t a t i v e l y most important. Two additional metabolites were isolated which have been t e n t a t i v e l y i d e n t i f i e d as 15cz-hydroxyestrone and 15cz-hydroxyestradiol17B. No evidence for the presence of 2-methoxy estrogens or any 16oxygenated estrogens was found. INTRODUCTION The metabolism of estrogens is a complex process and generally the b i o l o g i c a l l y active estrogens are rendered less active by a number of enzymes. These various enzyme systems contribute to the metabolic s i m i l a r i t i e s and differences in estrogen metabolism noted within the animal kingdom.
In addition to a species v a r i a t i o n the age of the
animal is also of importance. Metabolismof estrogens in the adult maybe quite d i f f e r e n t from that seen in the fetus or newborn. Since estrogens are produced during pregnancy, i t
is not unexpected that the
animal fetus can a c t i v e l y p a r t i c i p a t e in estrogen metabolism as does the human fetus (1,2).
In this regard, f e t a l organs and excreta from
newborn animals are known to contain estrogen metabolites (3-6). While estrogen metabolism in the adult dog has been investigated by several groups of investigators (7-13), information on estrogen
724
S T ER O I D S
metabolism
in the newborn dog is essentially nonexistant.
presents data concerning
16:6
This report
the metabolism of 4-14C-estradiol-17~
in the
newborn dog. MATERIALS AND METHODS Purification of Tracer. Prior to use the 4-14C-estradiol-17~, purchased from New England Nuclear Corporation (SA 58.8 mc/mM), was first chromatographed to the front on paper partition chromatography in chloroform/formamide. The area behaving in a manner similar to authentic estradiol-17~ was eluted and rechromatographed on thin layer chromatography in the system ethyl acetate:cyclohexane (1:1)o Formation of Derivatives. To aid in the identification of the metabolites several derivatives were made. The method of Brown (14) was used for methylating the phenolic groups. Acetylation was performed using acetic anhydride-pyridine (1:2) for 15 hours. Reduction of ketone groups was done using potassium borohydride. Acetonide formation was carried out according to the method of Marrian and Bauld (15) with a slight modification (16). Sample Preparation. Two male and two female mongrel puppies from the same litter and between I and 5 days of age were anesthesized with ether. Approximately I pc of 4-14C-estradiol-17~ was dissolved in 0.25 ml each of 0.85% saline and absolute ethanol and injected via the femoral vein. Three hours later the animals were sacrificed. Urine and bile were aspirated from the urinary and gall bladded respectively. In addition, the liver was excised from each puppy and mascerated using a pestle and mortar to which fine sand had been added. Using distilled water to wash each mortar, the tissues were drained into separate 250 ml centrifuge tubes and centrifuged° This procedure was repeated until no additional radioactivity could be extracted from the tissue. Extraction and Separation of the Metabolites. All samples were initially extracted with ether 5 times equal volume. After-removing traces of ether from the aqueous phase, two volumes of acetate buffer pH 4.3 were added. The samples were incubated at 38 C for 48 hours and during this time, eight hundred units per ml of Glusulase (Endo Laboratories, Inc.) were added in four equal parts of 200 units per ml every 12 hours. Following hydrolysis, the samples were reextracted with ether, 5 times equal volume. The greatest percentage of recovered radioactivity was in this fraction. The samples were first chromatographed to the front in hexane, benzene (1:1)/formamide. Two major peaks of radioactivity were present, a polar area (A) near the origin and an area designated B with an Rf corresponding to estrone. Some radioactivity was present in the nonpolar area of chromatogram (C). The polar area (A) was rechromatographed in chloroform/formamide to the front. Three distinct peaks resulted which were designated AI, A2, and A 3. Peak A 3 appeared to be at least two compounds and constituted the majority of-radioactivity in the polar area. Rechromatography of Peak A 3 in hexane, benzene (1:1)/
Dec. 1970
ST ER O I D S
725
formamide for 12 hours separated this area into two distinct peaks. The more polar peak (A~a) behaved similar to authentic estradiol-17~ while the nonpolar pea~ (A3b) had a mobility similar to authentic estradio|-17~. Peak A 2 appeared to be a single compound with a mobility similar to 17-epiestriol. When peak A I was rechromatographed in chloroform/formamide for 48 hours two peaks were resolved. One of these remained at the origin while the second peak had a polarity similar to estriol. At all stages of sample preparation aliquots were taken for liquid scintillation counting to correct for losses during the procedure. RESULTS Primarily five metabolites were present
in all of the specimens.
Three of these have been identified as estrone, estradiol-17~, estradiol-17~.
and
The two additional metabolites have characteristics
similar to 15~-hydroxyestrone and 15~- hydroxyestradiol-17~. Identification of Estrone, Estradiol-17~ and Estradiol-17~: During the initial chromatographic metabolites,
designated,
separation three radioactive
B, A3a and A3b, were isolated.
The mobilities
of these estrogens were similar to authentic estrone, estradiol-17~, and estradiol-17~,
respectively.
Additional
identification was accom-
plished by derivative formation and rechromatography systems.
in different
In all cases the Rf values agreed with the authentic standard.
In addition, an aliquot of each was recrystallized to constant specific activity, Table I.
These results confirm that metabolite B is estrone,
metabolite A3a is estradiol-17~, Identification of Metabolite A 2.
and metabolite A~b is estradiol-17~. The chromatographic mobility of this
metabolite was similar to 17-epiestriol.
However,
it soon became
apparent that this metabolite was not 17-epiestriol°
Derivative form-
ation indicated that reduction could occur suggesting the presence of a ketone°
It could be methylated,
acetylated.
Thus,
and following methylation could be
it appears that this metabolite has two hydroxyl
groups and one ketone.
The possibility that this may be one of the
726
ST ER O I D S
16:6
TABLE I RECRYSTALLIZATION OF A MIXTURE OF METABOLITE B WITH ESTRONE METABOLITE A3a WITH ESTRADIOL-17~, AND METABOLITE A3b WITH ESTRADIOL-17~
CRYSTALS (DPM/MG) STEROID
1 METHANOL/WATER
2 ETHANOL/WATER
3 BENZENE/ACETONE
Estrone
121
139
127
Estradiol-17~
157
161
158
Estradiol-17a
139
158
140
1 6 ~ - h y d r o x y e s t r o n e or 16-ketoestradiol-17~
ketols, 16¢~-hydroxyestrone, was ruled out.
Comparing the mobility of this metabolite and several
derivatives with 6-ketoestradiol-17~ estrogen. that
The polarity of this metabolite suggested the possibility
this
may be a 15~-hydroxy e s t r o g e n ,
metabolite
and i t s
derivatives
cated a c l o s e s i m i l a r i t y Table
II.
in a l l
These data s t r o n g l y
be 1 5 ~ - h y d r o x y e s t r o n e . recrystallization Identification ly
indicated that it was not this
in a l l
with authentic
suggest t h a t t h i s
i t may be e s t r i o l
Initially,
as a u t h e n t i c constant
is summarized
metabolite
the p o l a r i t y
but chromatography
of t h i s
for
is d e s i r a b l e .
of t h i s
metabolite
in c h l o r o f o r m : e t h y l The p o s s i b i l i t y
m e t a b o l i t e were i d e n t i c a l
15~-hydroxyestradiol-17~,
specific
activity
Table I I I ,
suggested
acetate that
(5:1)/
this The
t o reduced A 2 as w e l l Recrystallization
was not done because of a l i m i t e d
of 1 5 ~ - h y d r o x y e s t r a d i o l - 1 7 ~ .
in
may indeed
is t h e reduced form of m e t a b o l i t e A 2 was c o n s i d e r e d ,
characteristics
indi-
This m e t a b o l i t e was found c o n s i s t e n t -
formamide s e p a r a t e d t h e s e two e s t r o g e n s . metabolite
This
standard was not a v a i l a b l e
confirmation
o f M e t a b o l i t e A1b.
samples,
15~-hydroxyestrone
systems s t u d i e d .
Sufficient
so a d d i t i o n a l
Indeed, chromatography of t h i s
quantity
to
Dec. 1970
ST ER O I D S
727
TABLE II CHROMATOGRAPHIC DATA OF CHEMICAL DERIVATLVES OF COMPOUND A 2 AND AUTHENTIC 15C:Z-HYDROXYESTRONE
STEROID
DERIVATIVE
Compound A 2 15~-Hydroxyestrone
Rf
SYSTEM PAPER PARTITION
0.20 0.20
Chloroform/Formamide
Hexane:Benzene/Formamide (1:1)
Compound A 2 15~-Hydroxyestrone
Methylation
0.49 0.49
Compound A 2 15~-Hydroxyestrone
Methylatjon & Acetylation
0.52 Hexane/Formamide N.Do*
Compound A 2 15~-Hydroxyestrone
Methylation, Acetylation & Reduction
0.15 O.13
Hexane/Formamide
Compound A 2 15~-Hydroxyestrone
Methylation, Acetylation, Reduction & Acetylation
0.82 0.82
Hexane/Formamide
THIN LAYER
Compound A2 15~-Hydroxyestrone Compound A 2 15~-Hydroxyestrone
Reduction
0.40 0.40
Ethyl acetate:Hexane Ethanol (80:15:5)
0.20 0.20
Ethyl acetate:Hexane Ethanol (80:15:5)
*N.D. - not detected Some of the radioactivity in the unidentified fraction was nonpolar.
Attempts to demonstrate the presence of 2-metho×yestrone, 2-
metho×yestradiol-17~ or 2-hydro×yestrone, however, were unsuccessful. Quantitation of the Metabolites: Estrone was the major metabolite of 4-14C-estradio1-17~ in urine. Some 31-49 percent of the radioactivity recovered in urine was identified as this estrogen, Table IV. second in importance.
Quantitatively, estradiol-17~ was
Both of the metabolites tentatively identified
as 15~-hydro×yestrogens were present in all samples with 15~-hydro×yestrone accounting for as much as 25 percent in one animal.
728
16:6
S T ER O I D S TABLE III CHROMATOGRAPHIC DATA OF CHEMICAL DERIVATIVES OF COMPOUND A1b AND AUTHENTIC 15a-HYDROXYESTRADIOL-17~ STEROIDS
DERIVATIVES
Rf
Compound A1b 15~-Hydroxyestradiol-17~
SYSTEM
0.04 0.04
Chloroform/Formamide
Compound A1b 15a-Hydroxyestradiol-17~
Methylation
0.39 0.39
Benzene/Formamide
Compound A1b 15~-Hydroxyestradiol-17~
Methylation & Acetylation
0.79 0.79
Hexane/Formamide
Compound A1b 15~-Hydroxyestradiol-17~ Compound A 2 15a-Hydroxyestrone
Reduction
0.77* 0.77* 0.77* 0.77*
Chloroform:Ethyl acetate (5:1)/ Formamide
*R s Standard = Estriol
TABLE IV PERCENT DISTRIBUTION OF RADIOACTIVITY IN THE URINE OF NEWBORN DOGS
FRACTION Ether Soluble Ether Soluble AH* Ether Insoluble AH*
MALES DOG 3 DOG 4
FEMALES DOG I DOG 2 7 88 5
2 94 4
Estradiol-17~ Estradio]-17~ Estrone 15CZ-Hydroxyestrone 15CZ-Hydroxyestradiol-17B Unidentified
3 21 49 8 2 5
11 23 31 25 2 2
*AH - A f t e r h y d r o l y s i s ~-::The m e t a b o l i t e s are those
from the ether
I
2
• 94 5
97 I
7 24 47 12 3
13 28 34 16 4
I
2
METABOLITES-:~t-
soluble
AH f r a c t i o n .
Dec. 1970
ST ER O I D S
729
A sizable fraction of the injected radioactivity was recovered bile.
in
Analysis of the distribution of metabolites demonstrated a
similar pattern to that seen in the urine.
Although some quantitative
differences were noted, these were small. In contrast to urine and bile, however, estradiol-17~ was the major metabolite
in the liver.
appreciable quantities.
Both 15~-hydroxyestrogens were present
in
In fact, the quantity of these metabolites
in
liver was greater than was estrone. DISCUSSION Estrone and estradiol-17~ were the major metabolites of estradiol17~ in the newborn dog. adult dog (10,11,13).
This pattern
is similar to that seen in the
No evidence was seen for the presence of 16-
oxygenated metabolites.
This was somewhat surprising as several
metabolites have been suggested to be present dogs, these being primarily estriol epiestriol present
(5,6).
(5,7)
in the urine of adult
17-epiestriol
(7) and 16-
Two additional metabolites of estradiol-17~ were
in the newborn dog though and these have been tentatively
identified as 15~-hydroxyestrone and 15~-hydroxyestradiol-17B. suggests a different pattern of estrogen metabolism in comparison with that reported that the difference grams
such
in the adult.
This
in the newborn dog
It is not felt, however,
is as marked as it may appear,
fln most chromato-
it is not possible to separate 15~-hydroxyestrone from 17-
epiestriol
and in some cases 16-epiestriol.
Similarly,
difficult to separate 15~-hydroxyestradiol-17B
from estriol.
it is very possible that these metabolites are present have not been recognized.
it is quite
The failure of Siegal et al.
Therefore,
in the adult but (11) to be able
to recrystallize estriol may have been due to the fact that it was a metabolite similar to 15~-hydroxyestradiol-17~
rather than estriol.
73o
ST ER O I D S
16:6
Preliminary evidence in our laboratory does, in fact, show the presence of these same two metabolites
in urine and bile of adult animals follow-
ing the administration of estradiol-17B. If additional proof of structure substantiates the present identification of these two metabolites as 15~-hydroxyestrone and 15~-hydroxyestradiol-17~, this would demonstrate the in vivo formation of 155hydroxyestrogens
in a species other than the human (17, 18)°
formation of such metabolites has already been demonstrated
The in vitro by
incubation of ox adrenal tissue (19) or by perfusion of bovine adrenal glands (20).
Thus, it is not unreasonable to expect that other species
may also have the capacity to 15~-hydroxylate estrogens.
What role such
metabolites may play, if any, remains to be elucidated.
ACKNOWLEDGEMENTS The excellent technical assistance of Mrs. Renee Ellis is gratefully acknowledged. The authors would also like to express their gratitute to Dr. Samuel Solomon for a generous supply of authentic 155hydroxyestrone and 15~-hydroxyestradiol-17~. This work was supported by grant AM-11201 from the United States Public Health Service.
REFERENCES I. Diczfalusy, E., Cassmer, 0., Alonso, C., and DeMiquel, M., RECENT PROGR. HORMONE RES. 17: 147, (1961). 2. Diczfalusy, E., In: Proceedings, International Symposium, The Foeto-Placental Unit, Milan 1968, p. 65. Editors: A. Pecile and C. Finzi. ICS 183, Excerpta Medica, Amsterdam. 3. Velle, W., ACTA CHEM. SCAND. I__~I:1793, (1957). 4. Velle, W., and Pigon, H., ACTA ENDOCRINOLOGICA Suppl
(196o).
5. Velle, W., ACTA ENDOCRINOLOGICA 29: 381,
(1958).
6. Velle, W., GEN. COMP. ENDOCRINOLOGY~: 621, (1963).
~51: 1117,
Dec. 1970
ST ER O I D S
731
7. Cantarow, A., Rakoff, A. E., Paschkis, K. E., Hansen, L. P., and Walkling, A. A., END0CRINOLOGY31: 515, (1942). 8. Pearlman, W. H., Rakoff, A. E., Paschkis, K. E., Cantarow, A., and Walkling, A. A., J. BIOL. CHEM. 173: 175, (1948). 9. Kristofferson,
J., and Velle, W., NATURE
(London)
]85: 253, (1960).
10. Siegel, E. T., and Derfman, R. I., STEROIDS ~: 409,
(1963).
11. Siegel, E. T., Dorfman, R. I., Brodey, R. S., and Friedman, PROC. SOC. BIOL. MED. 111: 533, (1962).
12. Metzler, F., J r . , E]eftheriou, BIOL. MED. 121: 374, (1966).
M. H. F.,
B. E., and Fox, M., PROC. SOC. EXP.
13. Balikian, H., Southerland, J., Howard, C. M., and Preedy, J. R. K., ENDOCRINOLOGY 82: 500, (1968). 14. Brown, J. B., BIOCHEM. J. 6__00: 185, (1955). 15. Marrian,
G. F., and Bauld, W. S., BIOCHEM.
16. Hagen, A. A., Barr, M., and Diczfalusy, 49: 207, (1965). 17. Schwers, J., Eriksson, G., Wiqvist, BIOPHYS. ACTA 100: 313, (1965).
J. 5_,9: 136,
(1955).
E., ACTA ENDOCRINOLOGICA
N., and Diczfalusy,
E., BIOCHIM.
18. Zucconi, G., Lisboa, B. P., Simonitsch, E., Roth, L., Hagen, A. A., and Diczfalusy, E., ACTA ENDOCRINOLOGICA 56: 413, (1967).
19. Knuppen, R., and Breuer, H., HOPPE-SEYLER'S Z. PHYSIOL. CHEM. 337: 159, (1964). 20. Levy, H., Hood, B., Cha, C. H., and Carlo, J. J., STEROIDS ~: 677, (1965).
Systematic I. 2. 3. 4. 5. 6. 7.
names of steroids used in this study are:
estradiol-17~ = 1,3,5(10)-estratriene-3,17B_dlol estrone = 3-hydroxy-l,3,5(10)-estratriene-17-one estradiol-17~ = 1,3,5(lO)-estratriene-3,17~-diol estrio] = 1,3,5(lO)-estratriene-3,16~,]7B-trio I 15~-hydroxyestrone = 3,15£~-dihydroxy-1,3,5(10)estratriene-17-one 15~-bydroxyestradio]-17~ = 1,3,5(10)-estratriene3,15~,17~-trioI 17-eplestriol = 1,3,5(10)-estratriene-3,16c~,17~-triol
METABOLISM OF 4-14C-ESTRADIOL-17~ BY THE NEWBORN PUPPY A. Ainsworth Hagen and Paul F. Consroe Department of Pharmacology and Department of Medicine, Section of Endocrinology University of Tennessee, Memphis, Tennessee 38103 Received:
June i0, 1970 ABSTRACT
The metabolites of 4-14C-estradio1-17B, administered intravenously, were i d e n t i f i e d and quantitated in samples of urine, b i l e , and l i v e r from two male and two female newborn dogs. Conjugation of the metabol i t e s was extensive as less than lO percent of the r a d i o a c t i v i t y recovered from b i l e and urine was in an ether soluble form. Following enzyme hydrolysis the majority of the remaining r a d i o a c t i v i t y was rendered ether soluble. The major metaboIite found in urine and b i l e was estrone. Estradio1-17~was the second most important metabolite q u a n t i t a t i v e l y . In the l i v e r estradiol-17~ was q u a n t i t a t i v e l y most important. Two additional metabolites were isolated which have been t e n t a t i v e l y i d e n t i f i e d as 15cz-hydroxyestrone and 15cz-hydroxyestradiol17B. No evidence for the presence of 2-methoxy estrogens or any 16oxygenated estrogens was found. INTRODUCTION The metabolism of estrogens is a complex process and generally the b i o l o g i c a l l y active estrogens are rendered less active by a number of enzymes. These various enzyme systems contribute to the metabolic s i m i l a r i t i e s and differences in estrogen metabolism noted within the animal kingdom.
In addition to a species v a r i a t i o n the age of the
animal is also of importance. Metabolismof estrogens in the adult maybe quite d i f f e r e n t from that seen in the fetus or newborn. Since estrogens are produced during pregnancy, i t
is not unexpected that the
animal fetus can a c t i v e l y p a r t i c i p a t e in estrogen metabolism as does the human fetus (1,2).
In this regard, f e t a l organs and excreta from
newborn animals are known to contain estrogen metabolites (3-6). While estrogen metabolism in the adult dog has been investigated by several groups of investigators (7-13), information on estrogen
724
S T ER O I D S
metabolism
in the newborn dog is essentially nonexistant.
presents data concerning
16:6
This report
the metabolism of 4-14C-estradiol-17~
in the
newborn dog. MATERIALS AND METHODS Purification of Tracer. Prior to use the 4-14C-estradiol-17~, purchased from New England Nuclear Corporation (SA 58.8 mc/mM), was first chromatographed to the front on paper partition chromatography in chloroform/formamide. The area behaving in a manner similar to authentic estradiol-17~ was eluted and rechromatographed on thin layer chromatography in the system ethyl acetate:cyclohexane (1:1)o Formation of Derivatives. To aid in the identification of the metabolites several derivatives were made. The method of Brown (14) was used for methylating the phenolic groups. Acetylation was performed using acetic anhydride-pyridine (1:2) for 15 hours. Reduction of ketone groups was done using potassium borohydride. Acetonide formation was carried out according to the method of Marrian and Bauld (15) with a slight modification (16). Sample Preparation. Two male and two female mongrel puppies from the same litter and between I and 5 days of age were anesthesized with ether. Approximately I pc of 4-14C-estradiol-17~ was dissolved in 0.25 ml each of 0.85% saline and absolute ethanol and injected via the femoral vein. Three hours later the animals were sacrificed. Urine and bile were aspirated from the urinary and gall bladded respectively. In addition, the liver was excised from each puppy and mascerated using a pestle and mortar to which fine sand had been added. Using distilled water to wash each mortar, the tissues were drained into separate 250 ml centrifuge tubes and centrifuged° This procedure was repeated until no additional radioactivity could be extracted from the tissue. Extraction and Separation of the Metabolites. All samples were initially extracted with ether 5 times equal volume. After-removing traces of ether from the aqueous phase, two volumes of acetate buffer pH 4.3 were added. The samples were incubated at 38 C for 48 hours and during this time, eight hundred units per ml of Glusulase (Endo Laboratories, Inc.) were added in four equal parts of 200 units per ml every 12 hours. Following hydrolysis, the samples were reextracted with ether, 5 times equal volume. The greatest percentage of recovered radioactivity was in this fraction. The samples were first chromatographed to the front in hexane, benzene (1:1)/formamide. Two major peaks of radioactivity were present, a polar area (A) near the origin and an area designated B with an Rf corresponding to estrone. Some radioactivity was present in the nonpolar area of chromatogram (C). The polar area (A) was rechromatographed in chloroform/formamide to the front. Three distinct peaks resulted which were designated AI, A2, and A 3. Peak A 3 appeared to be at least two compounds and constituted the majority of-radioactivity in the polar area. Rechromatography of Peak A 3 in hexane, benzene (1:1)/
Dec. 1970
ST ER O I D S
725
formamide for 12 hours separated this area into two distinct peaks. The more polar peak (A~a) behaved similar to authentic estradiol-17~ while the nonpolar pea~ (A3b) had a mobility similar to authentic estradio|-17~. Peak A 2 appeared to be a single compound with a mobility similar to 17-epiestriol. When peak A I was rechromatographed in chloroform/formamide for 48 hours two peaks were resolved. One of these remained at the origin while the second peak had a polarity similar to estriol. At all stages of sample preparation aliquots were taken for liquid scintillation counting to correct for losses during the procedure. RESULTS Primarily five metabolites were present
in all of the specimens.
Three of these have been identified as estrone, estradiol-17~, estradiol-17~.
and
The two additional metabolites have characteristics
similar to 15~-hydroxyestrone and 15~- hydroxyestradiol-17~. Identification of Estrone, Estradiol-17~ and Estradiol-17~: During the initial chromatographic metabolites,
designated,
separation three radioactive
B, A3a and A3b, were isolated.
The mobilities
of these estrogens were similar to authentic estrone, estradiol-17~, and estradiol-17~,
respectively.
Additional
identification was accom-
plished by derivative formation and rechromatography systems.
in different
In all cases the Rf values agreed with the authentic standard.
In addition, an aliquot of each was recrystallized to constant specific activity, Table I.
These results confirm that metabolite B is estrone,
metabolite A3a is estradiol-17~, Identification of Metabolite A 2.
and metabolite A~b is estradiol-17~. The chromatographic mobility of this
metabolite was similar to 17-epiestriol.
However,
it soon became
apparent that this metabolite was not 17-epiestriol°
Derivative form-
ation indicated that reduction could occur suggesting the presence of a ketone°
It could be methylated,
acetylated.
Thus,
and following methylation could be
it appears that this metabolite has two hydroxyl
groups and one ketone.
The possibility that this may be one of the
726
ST ER O I D S
16:6
TABLE I RECRYSTALLIZATION OF A MIXTURE OF METABOLITE B WITH ESTRONE METABOLITE A3a WITH ESTRADIOL-17~, AND METABOLITE A3b WITH ESTRADIOL-17~
CRYSTALS (DPM/MG) STEROID
1 METHANOL/WATER
2 ETHANOL/WATER
3 BENZENE/ACETONE
Estrone
121
139
127
Estradiol-17~
157
161
158
Estradiol-17a
139
158
140
1 6 ~ - h y d r o x y e s t r o n e or 16-ketoestradiol-17~
ketols, 16¢~-hydroxyestrone, was ruled out.
Comparing the mobility of this metabolite and several
derivatives with 6-ketoestradiol-17~ estrogen. that
The polarity of this metabolite suggested the possibility
this
may be a 15~-hydroxy e s t r o g e n ,
metabolite
and i t s
derivatives
cated a c l o s e s i m i l a r i t y Table
II.
in a l l
These data s t r o n g l y
be 1 5 ~ - h y d r o x y e s t r o n e . recrystallization Identification ly
indicated that it was not this
in a l l
with authentic
suggest t h a t t h i s
i t may be e s t r i o l
Initially,
as a u t h e n t i c constant
is summarized
metabolite
the p o l a r i t y
but chromatography
of t h i s
for
is d e s i r a b l e .
of t h i s
metabolite
in c h l o r o f o r m : e t h y l The p o s s i b i l i t y
m e t a b o l i t e were i d e n t i c a l
15~-hydroxyestradiol-17~,
specific
activity
Table I I I ,
suggested
acetate that
(5:1)/
this The
t o reduced A 2 as w e l l Recrystallization
was not done because of a l i m i t e d
of 1 5 ~ - h y d r o x y e s t r a d i o l - 1 7 ~ .
in
may indeed
is t h e reduced form of m e t a b o l i t e A 2 was c o n s i d e r e d ,
characteristics
indi-
This m e t a b o l i t e was found c o n s i s t e n t -
formamide s e p a r a t e d t h e s e two e s t r o g e n s . metabolite
This
standard was not a v a i l a b l e
confirmation
o f M e t a b o l i t e A1b.
samples,
15~-hydroxyestrone
systems s t u d i e d .
Sufficient
so a d d i t i o n a l
Indeed, chromatography of t h i s
quantity
to
Dec. 1970
ST ER O I D S
727
TABLE II CHROMATOGRAPHIC DATA OF CHEMICAL DERIVATLVES OF COMPOUND A 2 AND AUTHENTIC 15C:Z-HYDROXYESTRONE
STEROID
DERIVATIVE
Compound A 2 15~-Hydroxyestrone
Rf
SYSTEM PAPER PARTITION
0.20 0.20
Chloroform/Formamide
Hexane:Benzene/Formamide (1:1)
Compound A 2 15~-Hydroxyestrone
Methylation
0.49 0.49
Compound A 2 15~-Hydroxyestrone
Methylatjon & Acetylation
0.52 Hexane/Formamide N.Do*
Compound A 2 15~-Hydroxyestrone
Methylation, Acetylation & Reduction
0.15 O.13
Hexane/Formamide
Compound A 2 15~-Hydroxyestrone
Methylation, Acetylation, Reduction & Acetylation
0.82 0.82
Hexane/Formamide
THIN LAYER
Compound A2 15~-Hydroxyestrone Compound A 2 15~-Hydroxyestrone
Reduction
0.40 0.40
Ethyl acetate:Hexane Ethanol (80:15:5)
0.20 0.20
Ethyl acetate:Hexane Ethanol (80:15:5)
*N.D. - not detected Some of the radioactivity in the unidentified fraction was nonpolar.
Attempts to demonstrate the presence of 2-metho×yestrone, 2-
metho×yestradiol-17~ or 2-hydro×yestrone, however, were unsuccessful. Quantitation of the Metabolites: Estrone was the major metabolite of 4-14C-estradio1-17~ in urine. Some 31-49 percent of the radioactivity recovered in urine was identified as this estrogen, Table IV. second in importance.
Quantitatively, estradiol-17~ was
Both of the metabolites tentatively identified
as 15~-hydro×yestrogens were present in all samples with 15~-hydro×yestrone accounting for as much as 25 percent in one animal.
728
16:6
S T ER O I D S TABLE III CHROMATOGRAPHIC DATA OF CHEMICAL DERIVATIVES OF COMPOUND A1b AND AUTHENTIC 15a-HYDROXYESTRADIOL-17~ STEROIDS
DERIVATIVES
Rf
Compound A1b 15~-Hydroxyestradiol-17~
SYSTEM
0.04 0.04
Chloroform/Formamide
Compound A1b 15a-Hydroxyestradiol-17~
Methylation
0.39 0.39
Benzene/Formamide
Compound A1b 15~-Hydroxyestradiol-17~
Methylation & Acetylation
0.79 0.79
Hexane/Formamide
Compound A1b 15~-Hydroxyestradiol-17~ Compound A 2 15a-Hydroxyestrone
Reduction
0.77* 0.77* 0.77* 0.77*
Chloroform:Ethyl acetate (5:1)/ Formamide
*R s Standard = Estriol
TABLE IV PERCENT DISTRIBUTION OF RADIOACTIVITY IN THE URINE OF NEWBORN DOGS
FRACTION Ether Soluble Ether Soluble AH* Ether Insoluble AH*
MALES DOG 3 DOG 4
FEMALES DOG I DOG 2 7 88 5
2 94 4
Estradiol-17~ Estradio]-17~ Estrone 15CZ-Hydroxyestrone 15CZ-Hydroxyestradiol-17B Unidentified
3 21 49 8 2 5
11 23 31 25 2 2
*AH - A f t e r h y d r o l y s i s ~-::The m e t a b o l i t e s are those
from the ether
I
2
• 94 5
97 I
7 24 47 12 3
13 28 34 16 4
I
2
METABOLITES-:~t-
soluble
AH f r a c t i o n .
Dec. 1970
ST ER O I D S
729
A sizable fraction of the injected radioactivity was recovered bile.
in
Analysis of the distribution of metabolites demonstrated a
similar pattern to that seen in the urine.
Although some quantitative
differences were noted, these were small. In contrast to urine and bile, however, estradiol-17~ was the major metabolite
in the liver.
appreciable quantities.
Both 15~-hydroxyestrogens were present
in
In fact, the quantity of these metabolites
in
liver was greater than was estrone. DISCUSSION Estrone and estradiol-17~ were the major metabolites of estradiol17~ in the newborn dog. adult dog (10,11,13).
This pattern
is similar to that seen in the
No evidence was seen for the presence of 16-
oxygenated metabolites.
This was somewhat surprising as several
metabolites have been suggested to be present dogs, these being primarily estriol epiestriol present
(5,6).
(5,7)
in the urine of adult
17-epiestriol
(7) and 16-
Two additional metabolites of estradiol-17~ were
in the newborn dog though and these have been tentatively
identified as 15~-hydroxyestrone and 15~-hydroxyestradiol-17B. suggests a different pattern of estrogen metabolism in comparison with that reported that the difference grams
such
in the adult.
This
in the newborn dog
It is not felt, however,
is as marked as it may appear,
fln most chromato-
it is not possible to separate 15~-hydroxyestrone from 17-
epiestriol
and in some cases 16-epiestriol.
Similarly,
difficult to separate 15~-hydroxyestradiol-17B
from estriol.
it is very possible that these metabolites are present have not been recognized.
it is quite
The failure of Siegal et al.
Therefore,
in the adult but (11) to be able
to recrystallize estriol may have been due to the fact that it was a metabolite similar to 15~-hydroxyestradiol-17~
rather than estriol.
73o
ST ER O I D S
16:6
Preliminary evidence in our laboratory does, in fact, show the presence of these same two metabolites
in urine and bile of adult animals follow-
ing the administration of estradiol-17B. If additional proof of structure substantiates the present identification of these two metabolites as 15~-hydroxyestrone and 15~-hydroxyestradiol-17~, this would demonstrate the in vivo formation of 155hydroxyestrogens
in a species other than the human (17, 18)°
formation of such metabolites has already been demonstrated
The in vitro by
incubation of ox adrenal tissue (19) or by perfusion of bovine adrenal glands (20).
Thus, it is not unreasonable to expect that other species
may also have the capacity to 15~-hydroxylate estrogens.
What role such
metabolites may play, if any, remains to be elucidated.
ACKNOWLEDGEMENTS The excellent technical assistance of Mrs. Renee Ellis is gratefully acknowledged. The authors would also like to express their gratitute to Dr. Samuel Solomon for a generous supply of authentic 155hydroxyestrone and 15~-hydroxyestradiol-17~. This work was supported by grant AM-11201 from the United States Public Health Service.
REFERENCES I. Diczfalusy, E., Cassmer, 0., Alonso, C., and DeMiquel, M., RECENT PROGR. HORMONE RES. 17: 147, (1961). 2. Diczfalusy, E., In: Proceedings, International Symposium, The Foeto-Placental Unit, Milan 1968, p. 65. Editors: A. Pecile and C. Finzi. ICS 183, Excerpta Medica, Amsterdam. 3. Velle, W., ACTA CHEM. SCAND. I__~I:1793, (1957). 4. Velle, W., and Pigon, H., ACTA ENDOCRINOLOGICA Suppl
(196o).
5. Velle, W., ACTA ENDOCRINOLOGICA 29: 381,
(1958).
6. Velle, W., GEN. COMP. ENDOCRINOLOGY~: 621, (1963).
~51: 1117,
Dec. 1970
ST ER O I D S
731
7. Cantarow, A., Rakoff, A. E., Paschkis, K. E., Hansen, L. P., and Walkling, A. A., END0CRINOLOGY31: 515, (1942). 8. Pearlman, W. H., Rakoff, A. E., Paschkis, K. E., Cantarow, A., and Walkling, A. A., J. BIOL. CHEM. 173: 175, (1948). 9. Kristofferson,
J., and Velle, W., NATURE
(London)
]85: 253, (1960).
10. Siegel, E. T., and Derfman, R. I., STEROIDS ~: 409,
(1963).
11. Siegel, E. T., Dorfman, R. I., Brodey, R. S., and Friedman, PROC. SOC. BIOL. MED. 111: 533, (1962).
12. Metzler, F., J r . , E]eftheriou, BIOL. MED. 121: 374, (1966).
M. H. F.,
B. E., and Fox, M., PROC. SOC. EXP.
13. Balikian, H., Southerland, J., Howard, C. M., and Preedy, J. R. K., ENDOCRINOLOGY 82: 500, (1968). 14. Brown, J. B., BIOCHEM. J. 6__00: 185, (1955). 15. Marrian,
G. F., and Bauld, W. S., BIOCHEM.
16. Hagen, A. A., Barr, M., and Diczfalusy, 49: 207, (1965). 17. Schwers, J., Eriksson, G., Wiqvist, BIOPHYS. ACTA 100: 313, (1965).
J. 5_,9: 136,
(1955).
E., ACTA ENDOCRINOLOGICA
N., and Diczfalusy,
E., BIOCHIM.
18. Zucconi, G., Lisboa, B. P., Simonitsch, E., Roth, L., Hagen, A. A., and Diczfalusy, E., ACTA ENDOCRINOLOGICA 56: 413, (1967).
19. Knuppen, R., and Breuer, H., HOPPE-SEYLER'S Z. PHYSIOL. CHEM. 337: 159, (1964). 20. Levy, H., Hood, B., Cha, C. H., and Carlo, J. J., STEROIDS ~: 677, (1965).
Systematic I. 2. 3. 4. 5. 6. 7.
names of steroids used in this study are:
estradiol-17~ = 1,3,5(10)-estratriene-3,17B_dlol estrone = 3-hydroxy-l,3,5(10)-estratriene-17-one estradiol-17~ = 1,3,5(lO)-estratriene-3,17~-diol estrio] = 1,3,5(lO)-estratriene-3,16~,]7B-trio I 15~-hydroxyestrone = 3,15£~-dihydroxy-1,3,5(10)estratriene-17-one 15~-bydroxyestradio]-17~ = 1,3,5(10)-estratriene3,15~,17~-trioI 17-eplestriol = 1,3,5(10)-estratriene-3,16c~,17~-triol