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Androgen metabolism in sheep
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ANDROGEN METABOLISM IN SHEEP Yukio Yamamoto, Ludvik Peric-Golia*, Yoshio Osawa**, Rashad Y. Kirdani and Avery A. Sandberg Roswell Park Memorial Institute, Buffalo, N.Y., 14263, Veterans Hospital, Salt Lake City, Utah* and The Medical Foundation of Buffalo** Buffalo, N.Y. Received 7-l-78 ABSTRACT 3H-Testosterone (%-T) plus "C-androst-4-ene-3,17-dione (A-dione) and 3H-epi-testosterone (17a-hydroxy-4-androsten-3-one)(epiT) plus 14C-T were injected intravenously into two male sheep with bile fistulae, respectively. Urine and bile samples were collected at intervals for 48 hours and analyzed by the use of DEAE-Sephadex A-25 and Lipidex 5000 columns, TLC, and paper chromatography; the aglycones were identified by co-crystallization with authentic standards. Five fractions were obtained from urine and bile: unconjugated, glucosiduronates, sulfates, sulfo-glucosiduronates and disulfates. In urine, the major conjugates were glucosiduronates, while sulfates predominated in bile. About W-90% of recovered radioactivity was found to be either glucosiduronates or sulfates. Among the metabolites identified, epi-T was the principal one, accounting for 10-15X of the administered doses. Conversion to 17ol-hydroxysteroids thus appears to be a major route of metabolism of the androgens administered in sheep. Other metabolites in the glucosiduronate and sulfate fractions were androsterone, etiocholanolone (3a-hydroxy-5$-androstan-17-one), 5@androstane3a,17B-diol, two unknown diols and polar metabolites. The results indicated that androgen metabolism is somewhat unusual in sheep, as compared with other animals and the human.
INTRODUCTION Recently, we have reported on the urinary and/or biliary metabolism of intravenously administered labeled T and A-dione in dogs (l,Z), baboons (3), humans (3) and rhesus monkeys (4).
The results indicated
that androgen metabolism is different in each species studied.
It is
known (5) that in sheep some aspects of estrogen metabolism are somewhat unique when compared to other species, i.e., this animal has a peculiar urinary and biliary excretion pattern and one of the major metabolites was 17a-estradiol.
There is a paucity of information concerning andro-
i/i?lume32, Number 3
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gen metabolism in sheep.
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Osborne -et al. (6) showed in the wether that
the radioactivity of intramuscularly injected 3H-T was recovered preferentially in the urine (41% in 24 hours and 59.6% in 37 days); 55.6% of the radioactivity recovered in the urine was in conjugated form.
Fe-
cal excretion constituted 15% of the injected label in 24 hours and 42% in 37 days.
Androsterone but not etiocholanolone was identified.
The present report deals with results obtained after injecting two male sheep i.v., one with
?l-T and 14C-A-dione (Sheep I) and another with
'H-epiT and 14C-T (Sheep II).
Bile and urine were collected at intervals
for 4 and 8 hours to study the excretion, conjugation and metabolic conversion of the administered steroids.
MATERIALS AND METHODS The experimental procedures, chemical analyses and radioactive measurements were carried out according to methods previously published (l4). All radioactive steroids were obtained from New England Nuclear Co., Inc. and their purities were checked on paper chromatography before use. The two male sheep used were surgically prepared (5). Sheep I (wt. 81 Kg) was injected with a mixture of 38nCi &1,2-T (specific activity 40 Ci/mmole) and 9nCi 14C-4-A-dione (specific activity 57mCi/mM); Sheep II (wt. 116 Kg) was injected trith 88.6uCi 14C-4-epiT (specific activity 51.5Ci/mmole) and 33nCi lr, C-4-T (specific activity 57.7mCi/mmole). Following the intravenous administration of the steroids, dissolved in an alcoholic solution diluted with saline, bile and urine were collected at intervals for 8 hours (Sheep I) and 4 hours (Sheep II). The collected bile and urine samples were stored in the cold room until use. Bile and urine samples were first chromatographed through DEAESephadex A25 columns [three column system: 0.9x60cm., 0.9x30cm. and 0.9x15cm. in series] using 0.6M NaCl linear gradient elution. This chromatography separated the conjugates into groups, which were then hydrolyzed individually either by enzyme (B-glucuronidase), solvolysis procedures according to Burstein and Lieberman (7), or by a sequential combination of both (enzyme hydrolysis followed by solvolysis). The aglycones were chromatographed and separated through Lipidex 5000 (a two column system), and were further isolated and purified using thin layer or paper chromatographies (l-4). The final identification of aglycone metabolites was carried out by co-crystallization methods.
Radioactivity was measured in lOm1 of Aqueous Counting Scintillant (Amersham/Searle Co.) by Packard Tri-Carb Spectromers 2450 or 3375 and corrected to dpm.
RESULTS 1.
Excretion:
Urinary and biliary excretion of radioactivity is
given in Table 1 as percent of the injected amount.
Recovery following
the i.v. injection of 3H-T and r4C-A-dione into Sheep I (Exp. 1) was much higher in urine (3H 58.2%, '*C 46.8%) than bile (3H 5.8%, '*C 9.2%).
There was a high rate of excretion in the initial four hours.
When Sheep II (Exp. II) was injected with 3H-epi-T and 14C-T, more radioactivity was excreted in bile (3W 72.9%, 14C 66.5%) than urine (3H 34.0% and 14C 34.5%).
Even though the quantitative excretion rates in bile
and urine differed in the two animals, the
qualitative aspects of such
excretion were very similar and, hence, they will be described in a combined fashion for both animals. 2.
Conjugation:
Different conjugate groups were obtained upon
DEAF-Sephadex chromatography of urine and bile.
The quantitation of
these conjugates is summarized in Table II. a)
Sheep Urine The elution pattern of the samples is shown in Figs. 1 and
2; a number of fractions was obtained (F-I to F-V and F-VIZ, F-VIII). Peaks corresponding to F-VII and F-VIII of Sheep I urine were not present in that of Sheep II.
F-I (tubes 2-4) was eluted in the distilled water
wash and contained uncharged compounds.
About 60% of this fraction was
ether extractable, and because of low levels of radioactivity, no further identification was attempted. F-II (tubes 35-45) was obtained as the first peak after the start
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TABLE I PERCENT RECOVERY OF RADIOACTIVITY IN URINE AND BILE FOLLOWING THE INTRAVENOUS INJECTION OF 3H-T AND "C-A-DIONE (SHEEP I) AND 3~-~P~-~ AND 14c-~ (SHEEP II) TIME URINE BILE lit ratio (hours) 3H 1‘c ratio 3H C Exp. I o-2 23.7 18.6 5.4 0 0.1 2-4 20.4 15.5 5.5 2.1 2.9 3.1 4-6 8.9 7.8 4.8 2.0 3.3 2.6 6-8 5.2 4.9 4.5 1.7 2.9 2.7 TOTAL 58.2 46.8 5.2 5.8 9.2 2.7 Exp. II o-1 39.9 35.2 3.1 l-2 25.5 24.2 2.8 2-4 7.5 7.1 2.9 TOTAL (0 - 4) 34.0 34.5 2.6 72.9 66.5 2.9 Injected ratio = 4.2 (Sheep I), 2.7 (Sheep II)
A
TABLE II HYDROLYSIS OF CONJUGATES IN SHEEP URINE AND BILE F-I F-II F-III F-IV F-V F-VI 3H 86-93** * (-1 (-1 (-) (-) 14c 85-93** 3H
B
89.9 *
83.5 88.0
*
*
*
*
S
S
93.3 75.4
91.3
80.0 S-G
93.8 S-G
*
*
14C G
(-1
89.0
3H C
*
73.3 (-1
88.1
F-VIII
(-1
88.0
*
14C
F-VII
S
DS
Numbers express percent of hydrolysis A = 8-glucuronidase hydrolysis B = Solvolysis C = 8-glucuronidase hydrolysis followed by solvolysis (-) no significant hydrolysis obtained * not carried out ** The hydrolysis of control and enzyme inhibition studies were 3-6% and 9-12% respectively G = glucosiduronate S = sulfate S-G=sulfoglucuronide DS= double sulfate
6T
377
?FBEOXD-
3H “C (x103DPMI 120 30
3H me- ‘%
1
2.m
r-----_----_
Wash
I 60
NaCl
15
0
20
40
80
60
00
I20
140
I60
TUBE NUMBER Fig. 1. DEAE-Sephadex A-25 column chromatographic elution pattern of androgen metabolites in the Sheep I urine, collected following i.v. injection of ?-I-Tand r4C-A-dione. The seven fractions obtained were: F-I uncharged, F-II glucosiduronates, F-III, F-IV and F-V sulfates, F-VII sulfoglucosiduronates, and F-VIII disulfates. of the linear gradient elution.
This fraction was satisfactorily hydro-
lyzed with B-glucuronidase (85%-93%), as is shown in Table II.
This
result together with those obtained upon extraction of the control and enzyme-inhibited incubation, indicate F-II to be composed of glucosiduronates.
The next three peaks, F-III (tubes 46-54), F-IV (tubes 55-
56) and F-V (tubes 67-80), were eluted as three successive peaks in the middle of the gradient.
These peaks could not be hydrolyzed with B-glu-
curonidase, but were successfully solvolyzed (83-90% of the label became extractable, Table II).
Therefore, these three doubly labeled fractions
were tentatively considered to be sulfates. labeled, containing only 3H. linear gradient.
F-VII and F-VIII were singly
F-VII was eluted as the last peak of the
This fraction was not hydrolyzed either by B-glucuroni-
dase or by solvolysis alone, but was satisfactorily hydrolyzed by sequential treatment by the two hydrolytic procedures (75-80% of the label
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became extractable), and was therefore considered to be composed of sulfo-glucosiduronates.
F-VIII was eluted in the 2.OM NaCl wash, and 74 to
93% of the radioactivity in this peak became extractable with organic solvent upon solvolysis; because of the position of the peak in the elution pattern, peak F-VIII was thought to be comprised of disulfates. Thus, five conjugate groups were Sheep:
uncharged (F-I), glucosiduronates
and V), sulfoglucosiduronates b)
obtained in the urine from the (F-II), sulfates (F-III, IV
(F-VII) and disulfates (F-VIII).
Sheep Bile The elution pattern of the bile from Sheep I was almost identical
to that obtained upon fractionation of bile of Sheep II, even though the separation of peaks in the former was not as definite because of the low amounts of radioactivity.
Hence, the description and the results of the
fractionation are based primarily on those of bile of Sheep II. The collected bile samples were separated through DEAE-Sephadex A25 columns, yielding seven doubly labeled fractions, with almost an identical elution pattern being observed upon chromatography of the biles from both sheep.
Fig. 3 depicts this pattern and shows that there are
slight differences when compared to those obtained from urine samples. F-I was eluted in distilled water and comprised uncharged compounds. further analysis of this peak was undertaken.
No
F-II was the first peak
eluted with the linear NaCl gradient and was satisfactorily hydrolyzed with glucuronidase
(85-87%), thus comprising mainly glucosiduronates.
F-III was composed of sulfate conjugates.
No peak corresponding to that
of urinary F-IV was eluted upon chromatography of the bile.
F-V was a
small peak eluted in the same gradient concentration as that of the urine
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3H ___ l4c
F-II
Linear Gradient
20
40
60
80 100 TUBE NUMBER
120
140
I60
Fig. 2. DEAE-Sephadex A-25 column chromatographic elution pattern of androgen metabolites in the Sheep II urine, collected following the i.v. injection of 3H-epi-T and 14C-T. Three conjugate fractions were separated: F-I uncharged, F-II glucosiduronates, F-III, F-IV and F-V sulfates. No diconjugates were eluted.
3-l ‘4c
F-Ill
(xlO%PMI 42 l6-
,__‘____________ 12.OM Naa Wash -0.6M NaCl L.G.S.
21 8-
F-VIII 0
20
40
-
0
80 lO0 60 TUBE NUMBER
Fig. 3. DEAE-Sephadex A-25 column chromatographic elution pattern of androgen metabolites in the Sheep II bile, collected following the i.v. injection of %epi-T and r4C-T. Seven fractions were obtained: F-I uncharged, F-II glucosiduronates, F-III and F-V sulfates, F-VI and F-VII sulfoglucosiduronates, and F-VIII disulfates.
samples, and was determined to be a sulfate.
Peak F-VI, not found in
urine samples, appeared in tubes 78-87 and was a double conjugate hydrolyzed by sequential hydrolysis.
Since fraction F-VII also was hydrolyzed
in an identical manner, both F-VI and F-VII were considered to be sulfoF-VIII, in the 2.OM NaCl wash, was found to be com-
glucosiduronates.
posed of disulfates. Thus, biliary metabolites were separated into five groups: charged (F-I), glucosiduronates foglucosiduronates
c)
un-
(F-II), sulfates (F-III and F-V), sul-
(F-VI and F-VII) and disulfates (F-VIII).
Effect of time on and total excretion of conjugates: After all bile and urine samples had been analyzed, the per-
cent distribution of radioactivity in each conjugate fraction was compared.
The results show that there were no outstanding time dependent
variations.
The distribution of glucosiduronates decreased slightly in
later hour samples and that of diconjugates increased.
The excretion of
sulfates did not vary with time, either in urine or bile. The excretion of radioactivity, as percentage of the administered dose and that of the recovered labels, is given in Tables III and IV for Sheep I and II.
In Sheep I glucosiduronates were the major conjugates
[54% of the total recovery (urine plus bile) of both 3H and 14C] and the radioactivity associated with this conjugate fraction was found to be almost twice as much as that associated with the sulfates of urine and bile (26% of the recovered 3H and 14C),
In Sheep II, the total excretion of
sulfates (3H 49%, 14C 43% of the total recovery) was slightly higher than that of glucosiduronates
(41% of both 3H and l&C).
Sulfates were the preponderant conjugates in bile of both animals; in Sheep II, sulfates accounted for 47.2% of ?I and 35-X of 14C of the
TABLE III DISTRIBUTION OF -RADIOACTIVITY IN EACH CONJUGATE FRACTION ( SHEEP I ) BILE* URINE TOTAL (URINE~~ILE)** '4c 3H 3H 3H 14c C % OF ADMINISTERED DOSE 4.7 3.4 0.2 3.2 0.2 4.5 uncharged 34.7 30.2 2.8 27.4 1.8 32.9 glucosiduronates 17.8 13.7 3.3 10.4 2.1 15.7 sulfates 5.0 3.9 1.6 2.3 1.2 3.8 diconjugates % OF RECOVERY 7.3 6.8 2.1 6.8 4.3 7.7 uncharged 54.2 53.9 30.2 58.5 30.6 56.5 glucosiduronates 27.8 24.5 35.6 22.2 36.7 27.0 sulfates 7.8 7.0 16.9 4.9 21.1 6.5 diconjugates * The results are based on the fractionation of only one bile sample ( 6 - 8 hour collection ). **The total recovery of radioactivity (urine + bile) in 8 hours of collection were 3H, 64.0% and 14C, 56.0%.
TABLE IV DISTRIBUTION OF RADIOACTIVITY IN EACH CONJUGATE FRACTION ( SHEEP II ) BILE URINE TOTAL(URINEf~ILE)* 14C 3H C 3H 14C 3H % OF ADMINISTERED DOSE 3.1 1.9 0.2 1.7 0.6 2.5 uncharged 43.6 41.2 17.3 23.9 17.6 26.0 glucosiduronates 51.8 43.6 35.7 7.9 47.2 4.6 sulfates 6.8 12.5 12.5 --6.8 --diconjugates % OF RECOVERY 2.9 1.9 0.3 4.9 0.8 7.3 uncharged 40.9 40.8 26.0 69.3 24.3 76.5 glucosiduronates 48.6 43.2 53.7 22.9 65.1 13.5 sulfates 6.4 12.4 18.8 ---9.4 ---diconjugates * The total recovery of radioactivit y4dut-;;; iybile) in 4 hours of collection were ?I, 106.5% and , . D.
administered dose (Table IV).
Diconjugates were found to be minor com-
ponents, constituting 7% to 12% of the total recovery. 3.
Metabolites:
The aglycones obtained from enzyme hydrolysis of
the glucosiduronate fractions were separated and/or isolated by chroma-
tography and identified by co=crystallization with standards.
Eight
peaks were separated by Lipidex 5000 chromatography, a typical pattern being shown in Fig. 4.
Hexane eluted three monohydroxy metabolite peaks
w4c ( x103DPM) 560
140~
P3
Hex.:20KBenz.
TUBE NUMBER
Fig. 4. Lipidex 5000 column chromatographic separation of androgen metabolites in the glucosiduronate fraction of Sheep I urine following i.v. injection of 3H-T and 14C-A-dione. Metabolites identified in the peaks were: Pl androsterone, P2 etiocholanolone, P3 epi-T, P4 5B-A-36, 17@diol, P5 not identified (Diol Z), P6 5B-A-3a,17B-diol, P7 not identified (Diol 4), P8 Polar Metabolites. (Pl,PZ,P3) which were identified by co-crystallization to be androsterone, etiocholanolone and epi-T, respectively.
Four dihydroxy metabolite
peaks (P4,P5,P6,P7) appeared upon elution with the linear gradient, but their separation was not complete. layer chromatography.
These four peaks were purified by thin
From the Rf values on TLC and from results of co-
crystallization, P4 and P6 (Fig. 4) proved to be 58-androstane-3B,17Bdiol and 5B-androstane-3a,17&-diol,
respectively.
P5 could not be iden-
tified, even though the Rf on thin layer was very similar to that of standard So-diols (3a,17@ and 38,178).
P7, the last peak eluted with
the linear gradient from the Lipidex 5000 column, was more polar than
56-sndrostane-3o,17B_diol
and migrated on thin layer with an Rf similar
to that of standard 6B-hydroxy-T in the system chloroform:ethanol The conclusive identification of this peak was not possible.
(9:l).
pa was
washed out with polar solvent (hexsne:chloroform:methanol,7:2:1)
and
separated upon TLC into two peaks in the system chloroform:ethanol
(9:l);
they were more polar than standard 5~-androstane-3~,16~,17~-triol. The aglycones obtained upon solvolysis of the sulfate fractions (F-III,F-IV,F-V) were separated into aglycone groups either by chromatography on Lipidex 5000 or by thin layer chromatography.
F-III was mostly
comprised of a few polar metabolites, which could not be identified. F-IV was comprised of monohydroxy metabolite peaks which were separated on thin layer; epi-T was the major metabolite identified upon further purification by paper chromatography.
F-V was separated into two dihy-
droxy metabolite peaks on thin layer, which had Rf values similar to those of diols (diol-2 and dial-4); the latter were not identified in the products obtained upon hydrolysis of the glucosiduronates. The aglycones obtained upon sequential hydrolysis of the sulfoglucosiduronate fractions were separated on TLC and a few peaks resulted which coincided in Rf with unidentified dihydroxy metabolites diol-2 and diol-4, and with more polar metabolites.
The disulfate fraction
(F-VIII} also contained unidentified dihydroxy (dial-2) and polar metabolites. The quantitation of aglycone metabolites in Sheep I and II urines and Sheep II bile, expressed as a percent of the administered dose, is given in Tables V,VI and VII, respectively.
Epi-T was the principal
monohydroxy metabolite identified and accounted for 13.6% of 3H and 14.7% of r4C in Sheep I urine (sum of aglycones from glucosiduronates
and sulfates) and 14.2% of k and 10.1% of 14C in Sheep II bile and urine.
In urine, epi-T was excreted both as a glucosiduronate and sul-
fate, but in bile it was present only as a glucosiduronate.
The excre-
tion of monohydroxy metabolites was not observed in the sulfate fraction of bile and this fact explains why fraction P-IV from conjugate separation on DEAE-Sephadex is absent in bile. At least four different dihydroxy metabolites were separated.
Even
though each metabolite individually accounted for a small percentage, collectively conversion to dihydroxy compounds was considerable, i.e., ?I 14.9%, r4C 9.6% in Sheep I urine (Table V) and 3H 5.6% and 14C 5.2% in Sheep 11 bile (Table VII).
Polar unidentified metabolites consti-
tuted a high percentage, particularly in Sheep II bile f3H 45.1% and 14c 34.0%).
DISCUSSION Since the data obtained from Sheep I urine and bile upon injection of 3H-T and 14C-A indicated that epi-T is the major androgen metabolite in sheep, Sheep II was injected with 3H-epi-T and 14C-T.
gven though
the quantitative distribution of radioactivity excreted in bile and urine differed in the two animals, conjugate formation and metabolic conversion were quite similar. Sheep I excreted radioactivity preponderantly in urine, but in Sheep II the excretion in bile was twice as much as that in urine.
In
a published report it was shown that following the injection of labeled T into a wether (6), 60% appeared in urine and 41% in feces.
Previously
published results by us of estriol metabolism in sheep (5) showed that biliary and urinary excretion varied from 15% to 57% and 8% to 85%, re-
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TABLEV SHEEP I URINE METABOLIC CONVERSIONS (PERCENT OF ADMINISTERED DOSE) AFTER INTRAVENOUS INJECTION OF S-T AND 14C-A-DIONE GLUCOSIDU~~NATES SULFATT$ METABOLITES 3H Ratio C Ratio 3H C Androsterone 1.1 1.0 4.6 Etiocholanolone 1.7 0.5 14.3 Epi-testosterone 7.7 10.1 3.2 5.9 4.1 6.0 5&androstane2.2 1.1 8.4 36,17@-diol Diol-2 2.8 2.3 5.1 3.7 1.3 12.0 5S-androstane1.9 0.2 39.9 1.0 1.7 2.5 3a,17S-diol Diol-4 2.8 2.9 4.5 Polar 11.2 8.2 5.7 4.3 2.8 6.5 Injected 3H/"C Ratio = 4.2
TABLE VI SHEEP II URINE METABOLIC CONVERSIONS (PERCENT OF ADMINISTERED DOSE) AFTER INTRAVENOUS INJECTION OF %EPI-T AND 14c-~ GLUCOSIDIJRONATES SULFATES METABOLITES 3H ")C Ratio % 14C Ratio Androsterone Etiocholanolone Epi-androsterone (3S-OH-5a-androstan-17-one)0.1 1.1 0.3 Epi-testosterone 9.9 7.6 3.5 0.6 0.6 2.7 5$-androstane3@,178-diol Diol-2 1.8 2.0 1.3 3.5 1.3 1.0 5f3-androstane1.5 1.6 2.5 0.6 0.4 4.1 3a,17@diol Diol-4 4.0 3.6 3.0 0.9 0.5 4.9 Polar 8.7 2.7 1.1 1.4 2.0 Injected %l/"C Ratio ="2.!7 TABLE VII SHEEP II BILE METABOLIC CONVERSIONS (PERCENT OF ADMINISTERED DOSE) AFTER INTRAVENOUS INJECTION OF 3H-EPI-T AND 14C-T GLUCOSIDU~~NATES SULFATY; METABOLITES 3H C Ratio 3H C Ratio Androsterone 0.6 0.4 4.1 Etiocholanolone 0.5 0.9 2.3 Epi-testosterone 3.7 1.9 5.3 Diol-2 1.7 3.9 1.2 [email protected] 2.4 1.8 3a,17@diol Diol-4 2.9 1.9 4.1 1.2 0.9 3.6 Polar 6.0 5.2 3.1 45.1 34.0 3.6 Injected 3H/14C Ratio = 2.7
385
spectively, among three bile-fistula and three intact sheep.
Thus,
there is a suggestion that the mode of excretion may differ considerably among individual sheep, a situation which may be unique to sheep when compared to other animals.
Preponderant urinary excretion of radioacti-
vity after labeled androgen administration has been observed in humans (3), baboons
(3) and rabbits (8), whereas preponderant biliary excretion
has been observed in rat (9), dog (2) and rhesus monkey (4).
In addi-
tion, there was no individual variation observed in the species mentioned (as contrasted to sheep) as far as major route of excretion of radioactivity. The quantitative excretion of various conjugates by sheep also presents an interesting aspect of the study.
While glucosiduronates were
the principal conjugates in urine, sulfates predominated in bile.
Fur-
thermore, the total excretion of conjugates in the sulfate fraction was greater in sheep than in any of the other species studied.
The metabo-
lites contained in these fractions also present a contrasting picture; while urinary glucosiduronates consisted of a variety of metabolites (monohydroxy-, dihydroxy- and polar metabolites), bile sulfates contained only polar metabolites.
This may explain the unique route of excretion
of conjugates in sheep.
Baboons (3), rhesus monkeys (4), dogs (2), rab-
bits (8), rats (9) and humans (3) excrete androgen metabolites preponderantly as glucosiduronates in urine and bile.
In sheep, Miyazaki -et
-al. (6) reported that the 3-sulfate constituted the preponderant conjugate of estriol in urine and the 3-glucosiduronate in bile.
While fur-
ther studies are necessary to ascertain the mechanism of conjugation in sheep, it is likely that these animals have more complicated conjugate systems when compared with other animals.
In common with other animals,
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387
however, diconjugates were quantitatively less frequent than either glucosiduronates or sulfates. The metabolic conversion of either T or A-dione to epi-T is a major route in sheep, the latter being the principal metabolite identified in two experiments.
While injected labeled epi-T was directly conjugated
with glucuronic or sulfuric acid and excreted in urine, labeled T did not appear in bile or urine as such; it underwent further metabolic conversion.
Epi-T has been identified in humans (ll-13), baboons (3) and rhe-
sus monkeys (4), with excretion and metabolic conversion from T and Adione being comparatively very minor.
Interestingly, estradiol-178 is
the principal estrogen excreted by the sheep ovary, whereas the major estrogen excreted in urine and feces of these animals is estradiol-17a, derived from the former via estrone.
Even though the 17a epimer of es-
tradiol-176 is considered to be inactive physiologically, this conversion seems to be important in sheep for the excretion of estrogens.
Wilson
et al. (10) injected labeled epi-T into man and concluded that it is -poorly metabolized to 17-keto or dihydroxy compounds and is excreted largely as unchanged epi-T-glucosiduronate.
In our previous studies in
dog (2), epi-T was metabolized to l'l-keto androgens (15X), 5o-androstane-3i3,17a-diol (17%), lice-androstane-3@,17B-diol(5%), unidentified dihydroxy and to polar compounds (54%). only 2.5%.
Unmetabolized epi-T constituted
Thus, epi-T metabolism seems to be considerably different
among animal species. Androsterone and etiocholanolone were also obtained as metabolic products from T, A-dione and epi-T, but these 17-keto conversions were quantitatively minor in sheep compared with other animals and the human (2,3,4,9).
Unidentified dihydroxy and polar metabolites constituted a
388
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major part of the excretion; their significance is not understood. The overall results indicate that sheep have unique aspects of excretion, conjugation and metabolic conversion of androgens, when compared with those of other animal species, including the human.
ACKNOWLEDGMENTS This study has been supported in part by grant AM-01240 from the National Institute of Health. This paper was presented in part at the 59th Annual Meeting of the Endocrine Society.
REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13.
Yamamoto, Y., Osawa, Y., Kirdani, R.Y. and Sandberg, A.A. STEROIDS 31, 233 (1978). Yamamoto, Y., Osawa, Y., Kirdani, R.Y. and Sandberg, A.A. J. STEROID BIOCHEMISTRY. In press (1978). Yamamoto, Y., Manyon, A.T., Osawa, Y., Kirdani, R.Y. and Sandberg, A.A. J. STEROID BIOCHEMISTRY. In press (1978). Yamamoto, Y., Manyon, A.T., Kirdani, R.Y. and Sandberg, A.A. STEROIDS 2, 711 (1978). Miyazaki, T., Peric-Golia, L., Slaunwhite, Jr., W.R. and Sandberg, A.A. ENDOCRINOLOGY 90, 516 (1972). Osborne, W.B., Wong, P.K. and Garnett, J.L. AUST. J. BIOL. SCI. 19, 1101 (1966). Burstein, S. and Lieberman, S. J. BIOL. CHEM. 233, 331 (1958). Yamamoto, Y. and Okada, H. (unpublished data). Matsui, M., Kinuyama, Y. and Hakozaki, M. STEROIDS 24, 557 (1974). Wilson, H. and Lipsett, M. J. CLIN. ENDOCRINOL. 6, 902 (1966). Korenman, S.G., Wilson, H. and Lipsett, M.B. J. CLIN. INVEST. 42, 1753 (1963). Korenman, S.G., Wilson, H. and Lipsett, M.B. J. BIOL. CHEM. 239, 1004 (1964). Brooks, R.V. and Giualini, G. STEROIDS ft_,101 (1964).
373
ANDROGEN METABOLISM IN SHEEP Yukio Yamamoto, Ludvik Peric-Golia*, Yoshio Osawa**, Rashad Y. Kirdani and Avery A. Sandberg Roswell Park Memorial Institute, Buffalo, N.Y., 14263, Veterans Hospital, Salt Lake City, Utah* and The Medical Foundation of Buffalo** Buffalo, N.Y. Received 7-l-78 ABSTRACT 3H-Testosterone (%-T) plus "C-androst-4-ene-3,17-dione (A-dione) and 3H-epi-testosterone (17a-hydroxy-4-androsten-3-one)(epiT) plus 14C-T were injected intravenously into two male sheep with bile fistulae, respectively. Urine and bile samples were collected at intervals for 48 hours and analyzed by the use of DEAE-Sephadex A-25 and Lipidex 5000 columns, TLC, and paper chromatography; the aglycones were identified by co-crystallization with authentic standards. Five fractions were obtained from urine and bile: unconjugated, glucosiduronates, sulfates, sulfo-glucosiduronates and disulfates. In urine, the major conjugates were glucosiduronates, while sulfates predominated in bile. About W-90% of recovered radioactivity was found to be either glucosiduronates or sulfates. Among the metabolites identified, epi-T was the principal one, accounting for 10-15X of the administered doses. Conversion to 17ol-hydroxysteroids thus appears to be a major route of metabolism of the androgens administered in sheep. Other metabolites in the glucosiduronate and sulfate fractions were androsterone, etiocholanolone (3a-hydroxy-5$-androstan-17-one), 5@androstane3a,17B-diol, two unknown diols and polar metabolites. The results indicated that androgen metabolism is somewhat unusual in sheep, as compared with other animals and the human.
INTRODUCTION Recently, we have reported on the urinary and/or biliary metabolism of intravenously administered labeled T and A-dione in dogs (l,Z), baboons (3), humans (3) and rhesus monkeys (4).
The results indicated
that androgen metabolism is different in each species studied.
It is
known (5) that in sheep some aspects of estrogen metabolism are somewhat unique when compared to other species, i.e., this animal has a peculiar urinary and biliary excretion pattern and one of the major metabolites was 17a-estradiol.
There is a paucity of information concerning andro-
i/i?lume32, Number 3
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gen metabolism in sheep.
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Osborne -et al. (6) showed in the wether that
the radioactivity of intramuscularly injected 3H-T was recovered preferentially in the urine (41% in 24 hours and 59.6% in 37 days); 55.6% of the radioactivity recovered in the urine was in conjugated form.
Fe-
cal excretion constituted 15% of the injected label in 24 hours and 42% in 37 days.
Androsterone but not etiocholanolone was identified.
The present report deals with results obtained after injecting two male sheep i.v., one with
?l-T and 14C-A-dione (Sheep I) and another with
'H-epiT and 14C-T (Sheep II).
Bile and urine were collected at intervals
for 4 and 8 hours to study the excretion, conjugation and metabolic conversion of the administered steroids.
MATERIALS AND METHODS The experimental procedures, chemical analyses and radioactive measurements were carried out according to methods previously published (l4). All radioactive steroids were obtained from New England Nuclear Co., Inc. and their purities were checked on paper chromatography before use. The two male sheep used were surgically prepared (5). Sheep I (wt. 81 Kg) was injected with a mixture of 38nCi &1,2-T (specific activity 40 Ci/mmole) and 9nCi 14C-4-A-dione (specific activity 57mCi/mM); Sheep II (wt. 116 Kg) was injected trith 88.6uCi 14C-4-epiT (specific activity 51.5Ci/mmole) and 33nCi lr, C-4-T (specific activity 57.7mCi/mmole). Following the intravenous administration of the steroids, dissolved in an alcoholic solution diluted with saline, bile and urine were collected at intervals for 8 hours (Sheep I) and 4 hours (Sheep II). The collected bile and urine samples were stored in the cold room until use. Bile and urine samples were first chromatographed through DEAESephadex A25 columns [three column system: 0.9x60cm., 0.9x30cm. and 0.9x15cm. in series] using 0.6M NaCl linear gradient elution. This chromatography separated the conjugates into groups, which were then hydrolyzed individually either by enzyme (B-glucuronidase), solvolysis procedures according to Burstein and Lieberman (7), or by a sequential combination of both (enzyme hydrolysis followed by solvolysis). The aglycones were chromatographed and separated through Lipidex 5000 (a two column system), and were further isolated and purified using thin layer or paper chromatographies (l-4). The final identification of aglycone metabolites was carried out by co-crystallization methods.
Radioactivity was measured in lOm1 of Aqueous Counting Scintillant (Amersham/Searle Co.) by Packard Tri-Carb Spectromers 2450 or 3375 and corrected to dpm.
RESULTS 1.
Excretion:
Urinary and biliary excretion of radioactivity is
given in Table 1 as percent of the injected amount.
Recovery following
the i.v. injection of 3H-T and r4C-A-dione into Sheep I (Exp. 1) was much higher in urine (3H 58.2%, '*C 46.8%) than bile (3H 5.8%, '*C 9.2%).
There was a high rate of excretion in the initial four hours.
When Sheep II (Exp. II) was injected with 3H-epi-T and 14C-T, more radioactivity was excreted in bile (3W 72.9%, 14C 66.5%) than urine (3H 34.0% and 14C 34.5%).
Even though the quantitative excretion rates in bile
and urine differed in the two animals, the
qualitative aspects of such
excretion were very similar and, hence, they will be described in a combined fashion for both animals. 2.
Conjugation:
Different conjugate groups were obtained upon
DEAF-Sephadex chromatography of urine and bile.
The quantitation of
these conjugates is summarized in Table II. a)
Sheep Urine The elution pattern of the samples is shown in Figs. 1 and
2; a number of fractions was obtained (F-I to F-V and F-VIZ, F-VIII). Peaks corresponding to F-VII and F-VIII of Sheep I urine were not present in that of Sheep II.
F-I (tubes 2-4) was eluted in the distilled water
wash and contained uncharged compounds.
About 60% of this fraction was
ether extractable, and because of low levels of radioactivity, no further identification was attempted. F-II (tubes 35-45) was obtained as the first peak after the start
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376
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TABLE I PERCENT RECOVERY OF RADIOACTIVITY IN URINE AND BILE FOLLOWING THE INTRAVENOUS INJECTION OF 3H-T AND "C-A-DIONE (SHEEP I) AND 3~-~P~-~ AND 14c-~ (SHEEP II) TIME URINE BILE lit ratio (hours) 3H 1‘c ratio 3H C Exp. I o-2 23.7 18.6 5.4 0 0.1 2-4 20.4 15.5 5.5 2.1 2.9 3.1 4-6 8.9 7.8 4.8 2.0 3.3 2.6 6-8 5.2 4.9 4.5 1.7 2.9 2.7 TOTAL 58.2 46.8 5.2 5.8 9.2 2.7 Exp. II o-1 39.9 35.2 3.1 l-2 25.5 24.2 2.8 2-4 7.5 7.1 2.9 TOTAL (0 - 4) 34.0 34.5 2.6 72.9 66.5 2.9 Injected ratio = 4.2 (Sheep I), 2.7 (Sheep II)
A
TABLE II HYDROLYSIS OF CONJUGATES IN SHEEP URINE AND BILE F-I F-II F-III F-IV F-V F-VI 3H 86-93** * (-1 (-1 (-) (-) 14c 85-93** 3H
B
89.9 *
83.5 88.0
*
*
*
*
S
S
93.3 75.4
91.3
80.0 S-G
93.8 S-G
*
*
14C G
(-1
89.0
3H C
*
73.3 (-1
88.1
F-VIII
(-1
88.0
*
14C
F-VII
S
DS
Numbers express percent of hydrolysis A = 8-glucuronidase hydrolysis B = Solvolysis C = 8-glucuronidase hydrolysis followed by solvolysis (-) no significant hydrolysis obtained * not carried out ** The hydrolysis of control and enzyme inhibition studies were 3-6% and 9-12% respectively G = glucosiduronate S = sulfate S-G=sulfoglucuronide DS= double sulfate
6T
377
?FBEOXD-
3H “C (x103DPMI 120 30
3H me- ‘%
1
2.m
r-----_----_
Wash
I 60
NaCl
15
0
20
40
80
60
00
I20
140
I60
TUBE NUMBER Fig. 1. DEAE-Sephadex A-25 column chromatographic elution pattern of androgen metabolites in the Sheep I urine, collected following i.v. injection of ?-I-Tand r4C-A-dione. The seven fractions obtained were: F-I uncharged, F-II glucosiduronates, F-III, F-IV and F-V sulfates, F-VII sulfoglucosiduronates, and F-VIII disulfates. of the linear gradient elution.
This fraction was satisfactorily hydro-
lyzed with B-glucuronidase (85%-93%), as is shown in Table II.
This
result together with those obtained upon extraction of the control and enzyme-inhibited incubation, indicate F-II to be composed of glucosiduronates.
The next three peaks, F-III (tubes 46-54), F-IV (tubes 55-
56) and F-V (tubes 67-80), were eluted as three successive peaks in the middle of the gradient.
These peaks could not be hydrolyzed with B-glu-
curonidase, but were successfully solvolyzed (83-90% of the label became extractable, Table II).
Therefore, these three doubly labeled fractions
were tentatively considered to be sulfates. labeled, containing only 3H. linear gradient.
F-VII and F-VIII were singly
F-VII was eluted as the last peak of the
This fraction was not hydrolyzed either by B-glucuroni-
dase or by solvolysis alone, but was satisfactorily hydrolyzed by sequential treatment by the two hydrolytic procedures (75-80% of the label
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378
IcZlB;OXDI
became extractable), and was therefore considered to be composed of sulfo-glucosiduronates.
F-VIII was eluted in the 2.OM NaCl wash, and 74 to
93% of the radioactivity in this peak became extractable with organic solvent upon solvolysis; because of the position of the peak in the elution pattern, peak F-VIII was thought to be comprised of disulfates. Thus, five conjugate groups were Sheep:
uncharged (F-I), glucosiduronates
and V), sulfoglucosiduronates b)
obtained in the urine from the (F-II), sulfates (F-III, IV
(F-VII) and disulfates (F-VIII).
Sheep Bile The elution pattern of the bile from Sheep I was almost identical
to that obtained upon fractionation of bile of Sheep II, even though the separation of peaks in the former was not as definite because of the low amounts of radioactivity.
Hence, the description and the results of the
fractionation are based primarily on those of bile of Sheep II. The collected bile samples were separated through DEAE-Sephadex A25 columns, yielding seven doubly labeled fractions, with almost an identical elution pattern being observed upon chromatography of the biles from both sheep.
Fig. 3 depicts this pattern and shows that there are
slight differences when compared to those obtained from urine samples. F-I was eluted in distilled water and comprised uncharged compounds. further analysis of this peak was undertaken.
No
F-II was the first peak
eluted with the linear NaCl gradient and was satisfactorily hydrolyzed with glucuronidase
(85-87%), thus comprising mainly glucosiduronates.
F-III was composed of sulfate conjugates.
No peak corresponding to that
of urinary F-IV was eluted upon chromatography of the bile.
F-V was a
small peak eluted in the same gradient concentration as that of the urine
S
379
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3H ___ l4c
F-II
Linear Gradient
20
40
60
80 100 TUBE NUMBER
120
140
I60
Fig. 2. DEAE-Sephadex A-25 column chromatographic elution pattern of androgen metabolites in the Sheep II urine, collected following the i.v. injection of 3H-epi-T and 14C-T. Three conjugate fractions were separated: F-I uncharged, F-II glucosiduronates, F-III, F-IV and F-V sulfates. No diconjugates were eluted.
3-l ‘4c
F-Ill
(xlO%PMI 42 l6-
,__‘____________ 12.OM Naa Wash -0.6M NaCl L.G.S.
21 8-
F-VIII 0
20
40
-
0
80 lO0 60 TUBE NUMBER
Fig. 3. DEAE-Sephadex A-25 column chromatographic elution pattern of androgen metabolites in the Sheep II bile, collected following the i.v. injection of %epi-T and r4C-T. Seven fractions were obtained: F-I uncharged, F-II glucosiduronates, F-III and F-V sulfates, F-VI and F-VII sulfoglucosiduronates, and F-VIII disulfates.
samples, and was determined to be a sulfate.
Peak F-VI, not found in
urine samples, appeared in tubes 78-87 and was a double conjugate hydrolyzed by sequential hydrolysis.
Since fraction F-VII also was hydrolyzed
in an identical manner, both F-VI and F-VII were considered to be sulfoF-VIII, in the 2.OM NaCl wash, was found to be com-
glucosiduronates.
posed of disulfates. Thus, biliary metabolites were separated into five groups: charged (F-I), glucosiduronates foglucosiduronates
c)
un-
(F-II), sulfates (F-III and F-V), sul-
(F-VI and F-VII) and disulfates (F-VIII).
Effect of time on and total excretion of conjugates: After all bile and urine samples had been analyzed, the per-
cent distribution of radioactivity in each conjugate fraction was compared.
The results show that there were no outstanding time dependent
variations.
The distribution of glucosiduronates decreased slightly in
later hour samples and that of diconjugates increased.
The excretion of
sulfates did not vary with time, either in urine or bile. The excretion of radioactivity, as percentage of the administered dose and that of the recovered labels, is given in Tables III and IV for Sheep I and II.
In Sheep I glucosiduronates were the major conjugates
[54% of the total recovery (urine plus bile) of both 3H and 14C] and the radioactivity associated with this conjugate fraction was found to be almost twice as much as that associated with the sulfates of urine and bile (26% of the recovered 3H and 14C),
In Sheep II, the total excretion of
sulfates (3H 49%, 14C 43% of the total recovery) was slightly higher than that of glucosiduronates
(41% of both 3H and l&C).
Sulfates were the preponderant conjugates in bile of both animals; in Sheep II, sulfates accounted for 47.2% of ?I and 35-X of 14C of the
TABLE III DISTRIBUTION OF -RADIOACTIVITY IN EACH CONJUGATE FRACTION ( SHEEP I ) BILE* URINE TOTAL (URINE~~ILE)** '4c 3H 3H 3H 14c C % OF ADMINISTERED DOSE 4.7 3.4 0.2 3.2 0.2 4.5 uncharged 34.7 30.2 2.8 27.4 1.8 32.9 glucosiduronates 17.8 13.7 3.3 10.4 2.1 15.7 sulfates 5.0 3.9 1.6 2.3 1.2 3.8 diconjugates % OF RECOVERY 7.3 6.8 2.1 6.8 4.3 7.7 uncharged 54.2 53.9 30.2 58.5 30.6 56.5 glucosiduronates 27.8 24.5 35.6 22.2 36.7 27.0 sulfates 7.8 7.0 16.9 4.9 21.1 6.5 diconjugates * The results are based on the fractionation of only one bile sample ( 6 - 8 hour collection ). **The total recovery of radioactivity (urine + bile) in 8 hours of collection were 3H, 64.0% and 14C, 56.0%.
TABLE IV DISTRIBUTION OF RADIOACTIVITY IN EACH CONJUGATE FRACTION ( SHEEP II ) BILE URINE TOTAL(URINEf~ILE)* 14C 3H C 3H 14C 3H % OF ADMINISTERED DOSE 3.1 1.9 0.2 1.7 0.6 2.5 uncharged 43.6 41.2 17.3 23.9 17.6 26.0 glucosiduronates 51.8 43.6 35.7 7.9 47.2 4.6 sulfates 6.8 12.5 12.5 --6.8 --diconjugates % OF RECOVERY 2.9 1.9 0.3 4.9 0.8 7.3 uncharged 40.9 40.8 26.0 69.3 24.3 76.5 glucosiduronates 48.6 43.2 53.7 22.9 65.1 13.5 sulfates 6.4 12.4 18.8 ---9.4 ---diconjugates * The total recovery of radioactivit y4dut-;;; iybile) in 4 hours of collection were ?I, 106.5% and , . D.
administered dose (Table IV).
Diconjugates were found to be minor com-
ponents, constituting 7% to 12% of the total recovery. 3.
Metabolites:
The aglycones obtained from enzyme hydrolysis of
the glucosiduronate fractions were separated and/or isolated by chroma-
tography and identified by co=crystallization with standards.
Eight
peaks were separated by Lipidex 5000 chromatography, a typical pattern being shown in Fig. 4.
Hexane eluted three monohydroxy metabolite peaks
w4c ( x103DPM) 560
140~
P3
Hex.:20KBenz.
TUBE NUMBER
Fig. 4. Lipidex 5000 column chromatographic separation of androgen metabolites in the glucosiduronate fraction of Sheep I urine following i.v. injection of 3H-T and 14C-A-dione. Metabolites identified in the peaks were: Pl androsterone, P2 etiocholanolone, P3 epi-T, P4 5B-A-36, 17@diol, P5 not identified (Diol Z), P6 5B-A-3a,17B-diol, P7 not identified (Diol 4), P8 Polar Metabolites. (Pl,PZ,P3) which were identified by co-crystallization to be androsterone, etiocholanolone and epi-T, respectively.
Four dihydroxy metabolite
peaks (P4,P5,P6,P7) appeared upon elution with the linear gradient, but their separation was not complete. layer chromatography.
These four peaks were purified by thin
From the Rf values on TLC and from results of co-
crystallization, P4 and P6 (Fig. 4) proved to be 58-androstane-3B,17Bdiol and 5B-androstane-3a,17&-diol,
respectively.
P5 could not be iden-
tified, even though the Rf on thin layer was very similar to that of standard So-diols (3a,17@ and 38,178).
P7, the last peak eluted with
the linear gradient from the Lipidex 5000 column, was more polar than
56-sndrostane-3o,17B_diol
and migrated on thin layer with an Rf similar
to that of standard 6B-hydroxy-T in the system chloroform:ethanol The conclusive identification of this peak was not possible.
(9:l).
pa was
washed out with polar solvent (hexsne:chloroform:methanol,7:2:1)
and
separated upon TLC into two peaks in the system chloroform:ethanol
(9:l);
they were more polar than standard 5~-androstane-3~,16~,17~-triol. The aglycones obtained upon solvolysis of the sulfate fractions (F-III,F-IV,F-V) were separated into aglycone groups either by chromatography on Lipidex 5000 or by thin layer chromatography.
F-III was mostly
comprised of a few polar metabolites, which could not be identified. F-IV was comprised of monohydroxy metabolite peaks which were separated on thin layer; epi-T was the major metabolite identified upon further purification by paper chromatography.
F-V was separated into two dihy-
droxy metabolite peaks on thin layer, which had Rf values similar to those of diols (diol-2 and dial-4); the latter were not identified in the products obtained upon hydrolysis of the glucosiduronates. The aglycones obtained upon sequential hydrolysis of the sulfoglucosiduronate fractions were separated on TLC and a few peaks resulted which coincided in Rf with unidentified dihydroxy metabolites diol-2 and diol-4, and with more polar metabolites.
The disulfate fraction
(F-VIII} also contained unidentified dihydroxy (dial-2) and polar metabolites. The quantitation of aglycone metabolites in Sheep I and II urines and Sheep II bile, expressed as a percent of the administered dose, is given in Tables V,VI and VII, respectively.
Epi-T was the principal
monohydroxy metabolite identified and accounted for 13.6% of 3H and 14.7% of r4C in Sheep I urine (sum of aglycones from glucosiduronates
and sulfates) and 14.2% of k and 10.1% of 14C in Sheep II bile and urine.
In urine, epi-T was excreted both as a glucosiduronate and sul-
fate, but in bile it was present only as a glucosiduronate.
The excre-
tion of monohydroxy metabolites was not observed in the sulfate fraction of bile and this fact explains why fraction P-IV from conjugate separation on DEAE-Sephadex is absent in bile. At least four different dihydroxy metabolites were separated.
Even
though each metabolite individually accounted for a small percentage, collectively conversion to dihydroxy compounds was considerable, i.e., ?I 14.9%, r4C 9.6% in Sheep I urine (Table V) and 3H 5.6% and 14C 5.2% in Sheep 11 bile (Table VII).
Polar unidentified metabolites consti-
tuted a high percentage, particularly in Sheep II bile f3H 45.1% and 14c 34.0%).
DISCUSSION Since the data obtained from Sheep I urine and bile upon injection of 3H-T and 14C-A indicated that epi-T is the major androgen metabolite in sheep, Sheep II was injected with 3H-epi-T and 14C-T.
gven though
the quantitative distribution of radioactivity excreted in bile and urine differed in the two animals, conjugate formation and metabolic conversion were quite similar. Sheep I excreted radioactivity preponderantly in urine, but in Sheep II the excretion in bile was twice as much as that in urine.
In
a published report it was shown that following the injection of labeled T into a wether (6), 60% appeared in urine and 41% in feces.
Previously
published results by us of estriol metabolism in sheep (5) showed that biliary and urinary excretion varied from 15% to 57% and 8% to 85%, re-
s
WEEOXDI
TABLEV SHEEP I URINE METABOLIC CONVERSIONS (PERCENT OF ADMINISTERED DOSE) AFTER INTRAVENOUS INJECTION OF S-T AND 14C-A-DIONE GLUCOSIDU~~NATES SULFATT$ METABOLITES 3H Ratio C Ratio 3H C Androsterone 1.1 1.0 4.6 Etiocholanolone 1.7 0.5 14.3 Epi-testosterone 7.7 10.1 3.2 5.9 4.1 6.0 5&androstane2.2 1.1 8.4 36,17@-diol Diol-2 2.8 2.3 5.1 3.7 1.3 12.0 5S-androstane1.9 0.2 39.9 1.0 1.7 2.5 3a,17S-diol Diol-4 2.8 2.9 4.5 Polar 11.2 8.2 5.7 4.3 2.8 6.5 Injected 3H/"C Ratio = 4.2
TABLE VI SHEEP II URINE METABOLIC CONVERSIONS (PERCENT OF ADMINISTERED DOSE) AFTER INTRAVENOUS INJECTION OF %EPI-T AND 14c-~ GLUCOSIDIJRONATES SULFATES METABOLITES 3H ")C Ratio % 14C Ratio Androsterone Etiocholanolone Epi-androsterone (3S-OH-5a-androstan-17-one)0.1 1.1 0.3 Epi-testosterone 9.9 7.6 3.5 0.6 0.6 2.7 5$-androstane3@,178-diol Diol-2 1.8 2.0 1.3 3.5 1.3 1.0 5f3-androstane1.5 1.6 2.5 0.6 0.4 4.1 3a,17@diol Diol-4 4.0 3.6 3.0 0.9 0.5 4.9 Polar 8.7 2.7 1.1 1.4 2.0 Injected %l/"C Ratio ="2.!7 TABLE VII SHEEP II BILE METABOLIC CONVERSIONS (PERCENT OF ADMINISTERED DOSE) AFTER INTRAVENOUS INJECTION OF 3H-EPI-T AND 14C-T GLUCOSIDU~~NATES SULFATY; METABOLITES 3H C Ratio 3H C Ratio Androsterone 0.6 0.4 4.1 Etiocholanolone 0.5 0.9 2.3 Epi-testosterone 3.7 1.9 5.3 Diol-2 1.7 3.9 1.2 [email protected] 2.4 1.8 3a,17@diol Diol-4 2.9 1.9 4.1 1.2 0.9 3.6 Polar 6.0 5.2 3.1 45.1 34.0 3.6 Injected 3H/14C Ratio = 2.7
385
spectively, among three bile-fistula and three intact sheep.
Thus,
there is a suggestion that the mode of excretion may differ considerably among individual sheep, a situation which may be unique to sheep when compared to other animals.
Preponderant urinary excretion of radioacti-
vity after labeled androgen administration has been observed in humans (3), baboons
(3) and rabbits (8), whereas preponderant biliary excretion
has been observed in rat (9), dog (2) and rhesus monkey (4).
In addi-
tion, there was no individual variation observed in the species mentioned (as contrasted to sheep) as far as major route of excretion of radioactivity. The quantitative excretion of various conjugates by sheep also presents an interesting aspect of the study.
While glucosiduronates were
the principal conjugates in urine, sulfates predominated in bile.
Fur-
thermore, the total excretion of conjugates in the sulfate fraction was greater in sheep than in any of the other species studied.
The metabo-
lites contained in these fractions also present a contrasting picture; while urinary glucosiduronates consisted of a variety of metabolites (monohydroxy-, dihydroxy- and polar metabolites), bile sulfates contained only polar metabolites.
This may explain the unique route of excretion
of conjugates in sheep.
Baboons (3), rhesus monkeys (4), dogs (2), rab-
bits (8), rats (9) and humans (3) excrete androgen metabolites preponderantly as glucosiduronates in urine and bile.
In sheep, Miyazaki -et
-al. (6) reported that the 3-sulfate constituted the preponderant conjugate of estriol in urine and the 3-glucosiduronate in bile.
While fur-
ther studies are necessary to ascertain the mechanism of conjugation in sheep, it is likely that these animals have more complicated conjugate systems when compared with other animals.
In common with other animals,
S
TEEOTDl
387
however, diconjugates were quantitatively less frequent than either glucosiduronates or sulfates. The metabolic conversion of either T or A-dione to epi-T is a major route in sheep, the latter being the principal metabolite identified in two experiments.
While injected labeled epi-T was directly conjugated
with glucuronic or sulfuric acid and excreted in urine, labeled T did not appear in bile or urine as such; it underwent further metabolic conversion.
Epi-T has been identified in humans (ll-13), baboons (3) and rhe-
sus monkeys (4), with excretion and metabolic conversion from T and Adione being comparatively very minor.
Interestingly, estradiol-178 is
the principal estrogen excreted by the sheep ovary, whereas the major estrogen excreted in urine and feces of these animals is estradiol-17a, derived from the former via estrone.
Even though the 17a epimer of es-
tradiol-176 is considered to be inactive physiologically, this conversion seems to be important in sheep for the excretion of estrogens.
Wilson
et al. (10) injected labeled epi-T into man and concluded that it is -poorly metabolized to 17-keto or dihydroxy compounds and is excreted largely as unchanged epi-T-glucosiduronate.
In our previous studies in
dog (2), epi-T was metabolized to l'l-keto androgens (15X), 5o-androstane-3i3,17a-diol (17%), lice-androstane-3@,17B-diol(5%), unidentified dihydroxy and to polar compounds (54%). only 2.5%.
Unmetabolized epi-T constituted
Thus, epi-T metabolism seems to be considerably different
among animal species. Androsterone and etiocholanolone were also obtained as metabolic products from T, A-dione and epi-T, but these 17-keto conversions were quantitatively minor in sheep compared with other animals and the human (2,3,4,9).
Unidentified dihydroxy and polar metabolites constituted a
388
S
?#?BEOXDI
major part of the excretion; their significance is not understood. The overall results indicate that sheep have unique aspects of excretion, conjugation and metabolic conversion of androgens, when compared with those of other animal species, including the human.
ACKNOWLEDGMENTS This study has been supported in part by grant AM-01240 from the National Institute of Health. This paper was presented in part at the 59th Annual Meeting of the Endocrine Society.
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