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Brief Report Differential oxidations of estradiol-17 by the chimpanzee in vivo.

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American Journal of Primatoloxy 5971-275 (1983)
BRIEF REPORT
Differential Oxidations of Estradiol-176 by the Chimpanzee
In Vivo
P.I. MUSEY, D.C. COLLINS, K.G. GOULD, AND J.R.K. PREEDY
Departmen&of Medicine and Yerkes Primate Center, Emory University School of MedicincNA
Medical Center. Atlanta
The metabolism of estradiol-170 is primarily a n oxidative process at either
carbon-2 or carbon-16 in the human. The objective of this study was to
determine the relative importance of these two oxygenation pathways in
the chimpanzee. The rate of oxidation of estradiol-17fl at each position was
determined by measuring the release of tritium into body water from carbon-2 or carbon-16. [2-3HJ-Estradiol-17flor [ 16-3H]-estradiol-17fiwas injected
intravenously into three adult male chimpanzees, and blood samples were
obtained at several time intervals between 1 and 48 hr. The blood was
lyophilized, and the release of tritium from the specifically labeled estrogens
into the body fluid pool was determined. The release of tritium from the
16a-position was very low and did not exceed 3% in any animal. The release
of tritium from the carbon-2 was much faster, amounting to 2996, 34%,and
35%, respectively, by 24 hr. The ratio of tritium released from carbon2:carbon-16 was 5.0, 13.2 and 16.9, respectively, at 24 hr after injection of
the specifically labeled estradiol-170. These results demonstrate clearly that
the major pathway for oxidative metabolism of estradiol-170 in the chimpanzee is via oxygenation a t carbon-2, with the formation of catechol estrogens,
as in the human
Key words: chimpanzee, estradiol-Ii'P, metabolism, catechol estrogens, estriols, nonhuman primates, Pan troglodytes
INTRODUCTION
Close similarities in the metabolism of estrogens have previously been reported
between human and nonhuman primates, especially the chimpanzee with respect to
steroid conjugation and excretion [Musey et al, 1973,1976,19821. I n the human, the
metabolism of estradiol-170 (biologically the most active estrogen) is characterized
by a rapid initial oxidation to estrone. Thereafter, estrone, and to a lesser extent
estradiol-170, may be oxidized at either carbon-2 or carbon-16 to form catechol
estrogens or estriols, respectively [Fishman et al, 19601. The physiological consequence of oxygenation a t carbon-2 is a reduction in the estrogenicity of the molecule
Received April 22, 1983; revision accepted June 30, 1983
Address reprint requests to Dr. P.I. Musey, Medical Research Service, VA Medical Center, 1670 Clairmont
Road, Decatur, GA 30033.
0 1983 Alan R. I,iss, Inc.
272
Musey et a1
[Martucci & Fishman, 19771, whereas estrogens oxygenated a t carbon-16 retain
considerable estrogenic activity [Clark et al, 1977; Fishman & Martucci, 19801.
Alterations in the ratio of hydroxylation a t carbon-2 to hydroxylation at carbon-16
have been reported in a number of pathological conditions, such as thyroid dysfunction [Fishman et al, 19621, liver disease [Zumoff et al, 19681, and anorexia nervosa
[Fishman et al, 19751.
The rate of oxidation a t each position can be determined radiometrically by
measuring the release of tritium from estradiol-17/3, specifically labeled at either
carbon-2 or carbon-16, into body water [Fishman et al, 1970, 19801. This approach
has been used to determine the site of oxidation in a number of physiological and
pathophysiological conditions in the human [Fishman et al, 1962, 1975; Zumoff et
al, 1968; Lahita et al, 19811.
The present study was undertaken to determine the relative importance of the
hydroxylation of estrogens a t carbon-2 and a t carbon-16 in the chimpanzee, using
the radiometric approach described above. The results of this study suggest that
hydroxylation a t carbon-2 is the major route for metabolism of estrogens in the
chimpanzee, as well as in the human.
MATERIALS AND METHODS
Subjects
Three healthy, adult male chimpanzees (Pan troglodytes) housed at Yerkes
Regional Primate Research Center were fasted overnight prior to the injection of
the radioactive steroid and collection of blood samples. The injection and blood
collection procedures were conducted under ketamine hydrochloride anesthesia (610 mgkg).
Radioactive Steroids.
[2-3H]-Estradiol-17/3and [16a-3H]-estradiol-17Pwere provided by Dr. H.L. Bradlow, Rockefeller University, NY. Details of the synthesis, purification, and stability
of these steroids have previously been described [Fishman et al, 1970, 19801. Briefly,
[2-3H]-estradiol-17/3was prepared by catalytic 3H reduction of 2-iodoestradiol in
methylene chloride in the presence of 5% palladiudcharcoal catalyst followed by
NaBH4 reduction. [16a-3H]-Estradiol-17/3was similarly prepared by catalytic trituration of estrone enol diacetate followed by methanoliKHC03 refluxing to generate
[ 16a,17a3H]-estradiol-17@.
The latter product was then subjected to chromic acid
oxidation to yield [16a3H]-estrone,which was then reduced to [ 16a3H]-estradiol-17P
in methanol containing NaBH4. Tritiated water was purchased from New England
Nuclear Corp., Boston, MA.
Radioisotope Administration
An initial control blood sample was collected from the chimpanzees. Then a
known amount of [16a-3H]-17/3(5-7 pCi) in 0.1 ml propylene glycol diluted with 5 ml
10%ethanolic saline was injected into the femoral vein. Blood samples (5 ml) were
taken in heparinized tubes a t 0.5, 1,2, 3,4,24, and 48 hr and stored until analyzed.
After four days, the above protocol was repeated except that [2-3H]-estradiol-17fiwas
injected.
The plasma or whole blood samples were lyophilized to collect body water.
Duplicate aliquots (1ml) of each lyophilate were analyzed for tritium.
Total Body Water Volume
The total body water volumes of the chimpanzees were determined by the body
dilultion of ingested tritiated water, Seven days after the last steroid injection, 0.1
Estrogen Metabolism in the Chimpanzee
273
pCi of tritiated water was added to 200 cc of fruit juice and given to each animal by
mouth. Blood samples were collected 2 and 3 hr after ingestion. The specific activity
( d p d m l ) of the blood lyophilate was used to calculate the volume of distribution by
a simple dilution ratio.
All radioactive samples were counted in a Packard 3255 Liquid Scintillation
Spectrometer with automatic external standardization for dpm calculations [Collins
& Preedy, 19751. Each ml of water was mixed with 5 ml of water-based commercial
scintillation fluid (Hydrocount-Mallinckrodt Chemical Co., St. Louis, MO), and
counted for a t least 90 min or until 10,000 counts were accumulated.
RESULTS
The amount of tritium released into the body water ( d p d m l blood) following
intravenous administration of [2-3H]-estradiol-17~
or [16a-3H]-estradiol-17@
is shown
in Table I. The release of the tritium label from the 16a-position was very low
throughout the study period. The maximum count was achieved only a t 48 hr in one
animal. Release of the tritium label from the 16a-position a t carbon-16 did not
exceed 3% of dose in any animal. These results suggest that the oxidation of
estrogens a t the 1601position is very slow in the adult male chimpanzee.
Tritium released from the carbon-2 position of estradiol-170 reflects 2-hydroxylation and catechol estrogen formation. The release of tritium from this position was
much faster than from the 16a-position (Table I). The release was somewhat faster
in one animal (GR) than in the others. However, comparable results were attained
in all animals by 24 hr following estrogen administration when 29%, 35%, and 34%,
respectively, of the [2-3H]-estradiol-17/3dose had been released (Table 11). The ratio
of tritium released from carbon-2:carbon-16varied between 2 and 32, with the higher
ratios being achieved between 4 and 24 hr after injection.
The rate of tritium release from the administered estrogens is affected by the
rate of water turnover. Since the chimpanzees were sedated without any intravenous
fluid infusion and did not void any urine a t least within the initial 4 hr, the results
in Table I1 only approximate the real-time course of carbon-2 and carbon-16 oxidations for the first 4 hr. The release of tritium from [2-3H]-estradiol-170was relatively
rapid during the initial 4 hr after injection, with 17% and 32% of the dose being
metabolized a t 1 and 4 hr, respectively, in one subject (GR). Comparable values for
the other subjects were 12% and 29%, respectively.
T.4HLE I. The Concentration of Free Tritium Present in the Blood (dpm/ml hlood) at
Different Times After Injection of [:'H]-Estradiol-l7PSpecifically Labeled at Carbon-:! or
Carbon-1601
Subiect
GR
wo
CH
'H (dpmiml) after Injection of:
-
[2-'H]
"H Dose
8.42
~.
x 10-'((dpm)
Time (hr)
1
2
3
4
24
48
73
110
132
142
157
128
[ 16a-"HI
9.81
5
6
6
5
7
15
l2-"H1
7.08
36
47
57
84
100
89
[ 16a-"HI
10.15
5
8
10
12
11
12
.~
[2-:'HI
[16a-"HI
12.88
12.91
~~~
59
103
120
131
196
181
27
33
33
37
41
31
274
Musey et al
TABLE 11. The Percent of Tritium Dose Keleased Into the Blood as "HzO at
Different Times After Injection of ~'H]-EstradioI-l7PSpecifically Labeled at Carbon-:!
and Carbon-ltb
Time (hr)
Subject GR
% 12-"H]
%' [16u-"H]
Ratio 21161~
Subject CH
r/r 12-:'H]
94 116a-'H]
Ratio 2116cu
Subject WO
% 12-"H]
5% [ 1601-3H]
Ratio 2116a
1
2~.
3
4
24
48
16.7
0.9
18.6
24.8
1.2
20.7
29.8
1.3
22.9
32.0
1.0
32.0
35.4
2.1
16.9
28.9
2.9
9.9
12.3
1.2
10.3
19.5
1.9
8.4
24.5
2.4
8.1
28.7
2.9
9.9
34.2
2.6
13.2
30.4
2.9
10.5
8.6
3.7
2.3
14.9
4.7
3.2
17.5
4.7
3.7
19.0
5.3
3.6
28.5
5.7
5.0
26.4
4.4
6.0
The ratio 2/16m was calculated from the ratio of t he percent of tritium released at each time.
DISCUSSION
The results of this study suggest that the chimpanzee oxidizes estradiol-170
predominantly a t carbon-2, leading to catechol estrogen formation as a major pathway for metabolism of estradiol-17P. The release of tritium from the 16a-position to
form 16a-hydroxyestrone and estriols appears to be a minor pathway in this primate.
These findings are similar to the metabolism of estrogens in the rhesus monkey
[Musey et al, 19791,the pigmy chimpanzee [Musey et al, 19791, as well as the human
[Kraychy & Gallagher, 1957; Fishman et al, 19801 in whom 2-hydroxy and 2methoxy estrogens are the major metabolites of estradiol. However, when compared
to the human, nonhuman primates seem to convert a greater percentage of estrogens
to catechol derivatives [Musey et al, 19821.
The metabolism of estradiol-17p is primarily oxidative in nature, occurring
almost exclusively a t carbon-2, carbon-16, and carbon-17 positions. Oxidation at
carbon-17, resulting in estrone formation, is the most rapid pathway, with a halftime of less than 5 min [Fishman et al, 1970). Subsequent metabolism of estrone
depends upon the competition between carbon-2 and carbon-16 hydroxylases. Oxidations at carbon-2 and carbon-16 are, to a n extent, mutually exclusive, leading to
either carbon-2 oxygenated compounds (2-hydroxy and 2-methoxy estrogens) or to
carbon-16 oxygenated products (16a-hydroxyestrone and estriol).
In the human, oxidation of estradiol-17P at carbon-2 is reported to be complete
within 6 hr, with a half-time of 2 hr, while the 16a-hydroxylation of estradiol-170 is
fairly slow [Fishman et al, 19801. Our results suggest that a similar pattern of rapid
oxidation a t carbon-2 and slow oxidation a t the 16a-position occurs in the male
chimpanzee.
The radiometric approach to estradiol-170 metabolism used in this study provides very useful information on the effects of metabolism on the possible physiologic
consequences of circulating estrogens in a n individual. The 2- and 16-hydroxylated
metabolites of estradiol-l7b have different physiological properties since estriols and
16a-hydroxyestrone are potent uterotrophic estrogens in vivo, while the catechol
estrogens appear to be devoid of estrogenic activity [Martucci & Fishman, 19771.
The catechol estrogens, however, are reported to have effects on the pituitaryhypothalamic axis [Schinfeld et al, 1980; Martucci & Fishman, 19791, and may also
act a s anti-estrogens. Thus, the metabolism of estradiol-17P via these two competitive pathways may affect the physiological response of the animal.
Estrogen Metabolism in the Chimpanzee
275
Our results clearly indicate that the major pathway for the metabolism of
estradiol-17P in the chimpanzee is by oxidation at carbon-2. Thus, the chimpanzee is
similar to the human and the rhesus monkey in the oxidation of estradiol-17P in
that the catechol estrogen pathway also predominates the estriol pathway. It is clear
that studies of estrogen biosynthesis and metabolism must include a measure of
these important catechol estrogen metabolites which have been ignored in this
species.
ACKNOWLEDGMENTS
Supported in part by NIH grants 5-RO1-CA-24616and RR-00165 and VA project
7176-001. Presented in part a t the 64th Annual Meeting of the Endocrine Society in
San Francisco, 1982.
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