- Email: [email protected]
Testosterone metabolites in dog bile
233
2228
TESTOSTERONE METABOLITES IN DOG BILE Y. Yamamoto, Y. Osawa,* R. Y. Kirdani and A. A. Sandberg Roswell Park Memorial Institute and The Medical Foundation of Buffalo* Buffalo, New York 14263 Rec'd. 10-14-77. ABSTRACT Testosterone-1,2-3H was injected intravenously into a male dog with a bile fistula and bile and urine collected. The radioactivity was excreted preponderantly in bile (52% of the injected dose) in 6 hours: only 12% appeared in the urine. Methods to study the biliary metabolites of testosterone in this and other animals were developed. Satisfactory conjugate patterns were obtained by fractionation on DEAE-Sephadex A-25 columns using two different elution systems. In addition to an unchanged fraction, six different monoglucuronide fractions were separated. No other conjugates were isolated. Lipidex 5000 column chromatography, TLC and paper chromatography were used for the isolation and purification of aglycone metabolites, which were further identified by co-crystallization methods. The biliary metabolites of testosterone were epiandrosterone (3B-hydroxy-5ct-androstan-17-one), etiocholanolone (3a-hydroxy-5!3-androstan-17-one), 5o.-androstan-3H,17H-diol, 5@androstan3a,17&diol and 5H-androstan-3H,17@diol. A special feature of testosterone metabolism in dog was the preponderant excretion into bile of almost exclusively monoglucosiduronates. In the case of androstanediols, the glucuronic acid moiety was located at different hydroxyl groups.
INTRODUCTION Even though the dog has been used as a model for human prostatic disease, particularly benign hypertrophy, there is a paucity of reports in the literature concerning the metabolic fate of androgens in this animal.
Work in our laboratory has shown [l] that dogs metabolize
estrogens differently from the other animals, and, hence, by extension we undertook to study the excretory patterns of androgens in this animal.
Evans and Pierrepoint
[2,3] have indicated that the dog differs
from many mammals in that the most active androgens in its prostate have the 17ct configuration.
Vo Fume 32, Nwnber 2
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TDEOXDI
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1978
S
234
TBX&OID~
In the present study we report on methods used to examine biliary patterns of excretion and metabolites in dog following the administration of labeled testosterone (T).
The methods devised in this
work have been used to investigate androgen metabolism in other animals (sheep, baboon, Rhesus monkey) and in the human [4]. Various analytical methods have been devised and used to senarate steroid metabolites.
DEAE-Sephadex, diethylaminoethyl derivative of
Sephadex G-25 or C-50 containing anion exchanger, was first introduced and developed as a column chromatographic method to separate con.jugates by Hahnel [5,6].
More recently, Hobkirk et al. [7,8] and Collins _et al.
[9] satisfactorily fractionated estrogen metabolites as their conjugates utilizing such chromatographic approaches. We have applied this ion-exchange column chromatography to the analysis of androgen metabolites in dog bile.
For further separation
of aglycones, Lipidex 5000 column chromatography was used.
Since the
methods used by us though lengthy are effective in separating conjugates and aglycones, we wish to report methodological details as applied to analysis of dog bile after administration of 3H-T.
In
following reports [4,10] the results of analysis of dog urine and bile and urine of the other animals will be reported. MATERIALS AND METHODS STEROIDS Testosterone-1,2-3H (3H-T) (specific activity 4OCi/mmole, 138mCi/mg) was purchased from the New England Nuclear Co., Boston, Mass. and diluted with carrier for the purposes of the study. The purity of this labeled steroid was checked and found to be more than 95% by paper chromatography in the system, hexane:methanol:water (100:90:10). Non-radioactive steroids were obtained from Steraloids, Inc., Wilton, N.H. and Research Plus Steroid Laboratories, Inc., Denville, New Jersey. These unlabeled compounds were used as standards for TLC, paper chromatography and cocrystallization.
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235
EXPERIMENTAL PROCEDURE: Injection and Collection of Bile: Under intravenous anesthesia with diazepam (Valium, Roche) and phencyclidine hydrochloride (Sernylan, Parke-Davis), an adult male mongrel dog (24kg body weight) was laparotomized and a polyethylene tube inserted into the common bile duct to serve for collection of bile. 3H-T (20.3pCi/ 5ug), dissolved in 0.5 ml of pure ethanol, was diluted to 30 ml with normal saline and injected into the saphenous vein over a period of a few minutes. Bile was collected at time intervals following the sample administration, adjusted to pH9 and stored at 4°C until analyzed. The bile collected between 30 and 120 minutes was used for this study. Lactate-Ringer fluid was infused intravenously during the bile collection. COLUMN CHROMATOGRAPHY: DEAE-Sephadex (DEAE-Sephadex A-25 from Pharmacia Fine Chemicals, Piscataway, New Jersey) was previously swollen and equilibrated in distilled water at least overnight before use. Columns were first filled with distilled water and then packed with DEAE-Sephadex by gravity to the top. Two different multiple column systems were used: System A, Kg/60 (Pharmacia Fine Chemicals, 0.9 cm diameter x 60 cm length) plus K9/30, and System B, K15/90 plus K15/90 plus K15/30. The columns were connected by silicone tubes (0.2-0.3 cm diameter). System A was used for the preliminary separation of conjugates and the columns were eluted with a linear gradient system obtained by mixing 500 ml of distilled water with 500 ml of 0.6M NaCl. A more elaborate System B was also used in which a linear gradient consisted of 1000 ml of O.lM NaCl in the mixing bottle and 1000 ml of 0.4M NaCl in the reservoir bottle. The columns were first washed with 100 to 200 ml of distilled water after sample application in order to eliminate uncharged compounds; following the linear gradient elution the column was washed again with 200 to 400 ml of 2.OM NaCl to elute highly charged conjugates. Ten ml were collected in one tube and 0.3 to 0.5 ml aliquots were used for the determination of radioactivity. Lipidex 5000 (Packard Instrument Co., Inc., Downers Grove, Ill.) was prepared by mixing the suspension with hexane in an ultrasonic bath or by bubbling with nitrogen gas to obtain a slurry. Two connected columns (Pyrex 2145 and 1282) were filled with hexane and packed by gravity to 17 cm and 10 cm heights, respectively. The sample dissolved in 0.5 ml chloroform or benzene, was applied to the 17 cm column. The columns were eluted with 100 to 200 ml hexane, followed by a linear gradient system of 500 ml of hexane and 500 ml of 20% benzene in hexane. The columns were washed out with 150 ml of a polar solvent mixture (hexane:chloroform:methanol = 70:20:10). Aliquots were taken from the 10 ml fraction for the determination of radioactivity. The organic solvents were redistilled before use. Lipidex was used repeatedly after washing with chloroform and methanol (1:l). TLC AND PAPER CHROMATOGRAPHY: For the isolation and purification of aglycones, TLC and paper chromatography were carried out. Silica gel GF plates (250 microns thick, Analtec Inc., Newark, Del.) were developed with a solvent system of chloroform:acetone (37:3). Unlabeled steroids were detected by spraying with 2% sulfuric acid in methanol
236
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WSlEOXD11
followed by heating (ZOO'C). Whatman No. 1 filter paper, (Fisher Scientific Co., Rochester, N.Y.) was developed in the system hexane: methanol:water (100:90:10). Unlabeled 17-keto-steroids were detected by Zi~erman color reaction and A" -3-ketosteroids by UV fluorescence. ENZYME HYDROLYSIS: The fractions or peaks obtained upon DEAF-Sephadex column chromatography were evaporated to dryness -in vacua and dissolved in 15 ml of O.lM acetate buffer, pH 5. H-glucuronidase from bovine liver (Sigma Chemical Co., St. Louis, MO.), 500-1000 units per ml, was added and the solution incubated at 37°C for 72 hours. It was then extracted with 3 times the volume of water-saturated ether. Inhibition of hydrolysis with saccharolactone (1O-3 to 2.5 x lo-'EI)was used as an index for the specificity of the @-glucuronidase hydrolysis. IDENTIFICATION OF METABOLITES: Following the isolation and purification of metabolites by chromatographic methods, aglycones were identified by co-recrystallation with unlabeled steroid carriers (lo-25 mg, depending on availability) from methanol-water. When a constant specific activity was obtained, the crystals were acetylated overnight at room temperature with acetic anhydride and pyridine. The acetylated aglycones were recrystallized and the specific activity determined. Aliquots of column chromatography samples RADIOACTIVITY MEASUREMENT: and other pipetted samples were dissolved in 10 ml of Aqueous Counting Scintillant (Amersbam/Searle Co.), diluted with toluene (1 part toluene in 2 parts Scintillant). The radioactivity was measured by Packard TriCarb spectrometers models 3375 or 2450 and corrected to dpm by an automatic standardization method. The efficiency of single label counting for 3H was between 30% and 46%. Thin layer plates and paper strips were scanned by a Packard Radiochromatogram Scanner model 7201-C. The radioactivity in samples that produced severe quenching, such as bile, was determined by firs& combusting the sample in a Packard Sample Oxidizer model 306, and then counting in a Packard Tri-Carb spectrometer.
RESULTS The excretion pattern of injected 3H-T is shown in Fig. 1.
Biliary
excretion (52%) exceeded urinary excretion (12%) during the six hours of collection; a high excretion rate was observed in the first two hours. Analysis of the collected bile was performed by DEAE-Sephadex A-25 column chromatography, enzyme hydrolysis, Lipidex 5000 column chromatography, TLC, paper chromatography and co-crystallization specific activity methods.
to constant
Flow charts of the methods used are shown
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237
40 30 20 IO
I
4
2 Time
6
( hours)
Fig. 1. The cumulative excretion pattern into bile (solid line) and urine (dotted line) following the intravenous injection of 3H-testosterone @Cl !_KZjinto a male dog. in Figs. 2 and 3 for the A and B systems (see Methods), respectively. Fractions obtained using the A method are labeled "A", those using the B method as I'D". 1. Fractionation of Conjugates: Separation by System A (Fig. 2): The bile was directly applied to a DEAE-Sephadex column and eluted as described in Methods. in Fig. 4.
The elution pattern of radioactivity is shown
Al (tubes 4-6) was eluted with distilled water and 38% of
this fraction was ether extractable,
A2
(tubes 30-45) and A3 (tubes
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238
IZ?BEOXDI FIGURE
2
BILE DEAE-SEPHADEX COLUMN CHROMATOGRAPHY SYSTEM A A-i
A-2
(Uncha;gedFr.) Ether Eftraction Extrl,crble
A-3
(8-ylucuronidaseHydrolysis) G~~c_o:
Fr.
i
Gl+onide
Fr.
Llpidex Column Chromatography
Unex;Wl;Ic;e
Steroids
5 Aglycone Peaks
Aglycone Peaks
I
I
Etioch'lanolone
Epiand'osterone
SU-Androstane-38,17B-dial 5a-Androstane-3B,175-dial SB-Androstane-3n,l7f+diol Polar Metabolites
DEAE-Sephadex
column
c’hromaro~raphy,
System
B
I I
1 GLUCURONIDEFT.
I UNCHARGEDFr.
Peak 81
I
Peak
82
I Peak B3
Peak
I 8-glucuronidase
84
I Peak B5
I Pesk B6
1 Peak
B7
hy
Lip polar
I dihydroxy
i monohydrory etioc b olan“lone
t
X3-A-Za,l73-dial %-A-f3,118-dial
55-70) were then eluted using a linear NaCl gradient.
erD”e
h-A-8 ,17d-dial ‘B-A-3a,lXT-dial s-A-X3,179-dial
A2 was composed
of two peaks (Fig. 4), the separation of which was not complete, and A3 was composed of three peaks.
No peaks appeared in the 2.OM NaCl wash.
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TBEOIDl
239
x 104dpm 8
r___ _-___-
----*QMN&,
1.
-r e
*M-C cc
4
I
0.6M linear
**--
No’3 gradiwd
Distillad
1
0
20
II-I,
40
I:
60 TUBE
1
80
100
I
120
Wolrr
1
140
NUMBERS
Fig. 4. DEAE-Sephadex A-25 column chromatographic pattern of dog bile by column system 1 (K 9/60 + K 9/60). The column was first washed with 100 ml distilled water after loading the sample, then it was developed with 0 - 0.6M NaCl linear gradient system (1000 ml), and at the end washed out with 400 ml 2.OM NaCl. Ten ml was collected in each tube. Three fractions, A-I, A-II and A-III were obtained; A-I is uncharged, A-II and A-III are both glucuronide conjugates. Diconjugates were not present and would have appeared in tube numbers above 115. Separation by System B (Fig. 3): The bile was fractionated through large columns (see Methods), the elution pattern of which is shown in Fig. 5; seven fractions were separated.
The first fraction, Bl, was eluted in tubes 20-30 (Fig. 3, 5;
compare with fraction Al of system A, Figs, 2, 4).
Fractions B2, B3
and B4 were eluted as a separate group of peaks in tubes 75-115 and represented a more highly resolved fraction, AZ, since no fraction comparable to B3 was found using the A system.
Fractions B5, B6 and B7
were also eluted as a separate zone in tubes 145-205 and were comparable to A3.
S
240
T=EOIDI
82 x IO4 dpm
IO
B5
_-0.4M N&l Linaor Grodhnl System
5 B-I ___----__-II
60
0. IM No’3
90
120
150
180
210
240
TUBE NUMBERS
Fig. 5. DEAE-Sephadex A-25 chromatographic pattern of dog bile by column system 2 (K 15/90 + K 15/90 + K 15/90). The column was eluted with distilled water after loading, then linear gradient O.lM - 0.4M NaCl (2000 ml) was applied. F-I is uncharged fraction. Glucuronide fractions F-II and F-III were separated respectively into three peaks (Bl to B7); B2 and B5 - polar, B3 and B6 - monohydroxy, B4 and B7 dihydroxy. 2.
Enzymatic Hydrolysis of Conjugate Fractions:
Follow ng DEAE-Sephadex column chromatography, all conjugate fractions were hydrolyzed with 8-glucuronidase. with the A fractions hydrolysis
(8%and
are given in Table I.
The results obtained
The high percentage of
95%) and the efficient enzyme inhibition by sac-
charolactone indicate that A2 and A3 are glucuronide conjugates.
The
individual fractions obtained from System B elution were also hydrolyzed individually and these data a .so indicate the conjugates to be glucuronides.
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WBEOXDI
241
TABLE I The Hydrolysis of Fractions A2 and A3 by $-glucuronidase Fraction F-II F-III
Enzyme Hydrolysis
Control
82.3% 95.0%
Enzyme Inhibition 7.0% 7.0%
7.4% 2.7%
enzyme hydrolysis = substrate + acetate buffer f 8-glucuronidase control = substrate + acetate buffer enzyme inhibition = substrate + acetate buffer + 8-glucuronidase + saccharolactone (enzyme inhibitor), 72 hours incubation at 3J'C (buffer pH 5.0).
3.
Separation and Identification of Agylcones:
I.
From System A.
The aglycone mixture obtained upon hydrolysis
with 8-glucuronidase of fractions A2 and A3, by chromatography on Lipidex-5000. each resolved into 5 fractions.
was
initially separated
The hydrolysates from A2 and A3
Since it was found, using standards,
that Lipidex chromatography did not, at times, resolve compounds closely related in polarity, monohydroxy androgens obtained by hydrolysis of biliary conjugates were further purified and tentatively identified by chromatography on paper (see Methods).
In the system used, etiocho-
lanolone and epiandrosterone had Rf values of 0.31 and 0.29, respectively.
For comparison, typical Rf values of standard monohydroxy
compounds in the same system are as follows: hydroxy-4-androsten-3-one),
T, epitestosterone
dihydrotestosterone
(17~
(lJ@hydroxy-5a-andro-
Stan-3-one) and androsterone 0.12, 0.17, 0.31 and 0.41, respectively. The dihydroxyandrogens
isolated from bile were further separated and
tentatively identified by chromatography on silica gel (see Methods). In the system used, 5a-androstan-3B,lJ&diol, diol, 58-androstan-38,178-diol respectively.
58-androstan-3a,lJR-
had Rf values of 0.19, 0.11 and 0.35,
By comparison, Rf values of some standards in the same
system are as follows:
5a-androstane-3a,l7B_diol
3c1,17a-diol 0.09, 5o-androstane-3B,17a-diol
0.16, 5a-androstan-
0.17.
Admixture of the above tentatively identified compounds with appropriate carriers and crystallization to constant specific activity resulted in identification of the following compounds in the two peaks, A2 and A3: -AZ:
Monohydroxy compound:
5&androstan-3@,17&diol, 3a,17&diol.
etiocholanolone.
Dihydroxy compounds:
Su-androstane-3$,17&diol
and SB-androstan-
Also present was a polar metabolite which was not
further
identified. A3: -
Monohydroxy compound:
5&-androstane-3@,17@-diol, 3a,17&diol.
Epiandrosterone.
Dihydroxy compounds:
5o-androstane-3B,17a_diol
and 5R-androstan-
Also present was a polar metabolite which was not identi-
fied further. Fig. 6 is an example of an elution pattern following Lipidex chromatography and depicts separation of aglycones obtained after O-glucuronidase hydrolysis of fraction A2. II.
From System
B.
The aglycone mixture obtained upon hydrolysis
of fractions B2 through B7 were identified using the above procedure as: B2: -
Unidentified polar compound.
-B3:
Monohydroxy compound:
-B4:
Dihydroxy compounds: 3a,17&diol
etiocholanolone. So-androstane-3!3,17B_diol, 5&androstan-
and 5&androstan-3@,17&diol.
B5* _.
Unidentified polar metabolite.
-B6:
Monohydroxy compound:
-B7:
Dihydroxy compounds: 3n,17&diol
epiandrosterone. 5a-androstan-3@,17B-diol,
and 5&androstan-3B,17&diol.
SB-androstane-
S
%?1SEOfDI
c
E
x lO*dpm
I2
243
1
3
----Porn so~nl
Waam:Chlorolorm: Ythami 7aP lo)
2
20% Gmnr*m Hmon*. Litmar Grodiont !Syahm ___.,.e--
I
0
2040
6080
IO0
120
140
TUBE NUMBERS
Lipidex 5000 column chromatography of A2 aglycones obFig. 6. tained after &-glucuronidase hydrolysis. The column was eluted with 200 ml hexane following sample loading, then hexane - 20% benzene hexane linear gradient system (1000 ml) was used. The column was washed out finally with a polar solvent mixture (hexane:chloroform: methanol = (70:20:10). Five peaks were eluted: Peak A consists of monohydroxy, Peaks B, C, D of dihydroxy and Peak E of polar metabolites. Table II presents details of co-crystallization with carriers of the compounds described above obtained from the B system analysis. From the above results, it appears that after administration of 3H-T , only monoglucuronides are found in dog bile.
These conjugates
were separable into two major groups by fractionation in systems A or B.
In each of these two groups, monohydroxy compounds were found.
How-
ever, in each group the three dihydroxy androgens were also present.
DISCUSSION It appears that the major excretory route for T metabolites in the dog is the bile.
The work presented shows that DEAE-Sephadex column
chromatography can be used to separate androgen conjugates, similar to
AcCR
RA ; XL ; ML ; CR ; AV ; AcCAL
2700 1100
3000
300
2800
RA/mg
3800 2300 900
2600 5400
250 460
2600 3700
CR L
2787 2272 875
2342 2777
250 250
2300 2300
CR 2
2600 2300 900
2200 2250
250 250
2400 2450
CR 3
2550 24.50 800
2300 2200
2350 2200
CR 4
2331 858
2250
250
2400
AV
ISO0 750
1750
200
AcCAL
2000 1800 700
1800 1700
ZOO 200
AcCR
ML 1400 950 850 900 700 radioactivity crystal mother liquor cocrystallization from methanol water, expressed as specific activity (dpmfmg) average specific activity of two or three constant numbers ; calculated specific activity of the diacetates obtained from the last crystallizations of the diols, i.e., C3 or C4 ; cocrystallization of acetylated compounds
64300 29700
29300
5ct-Androstane- XL 3@,17B-dial ML
3a,17$-diol ML 5&-Androstane- XL Epiandrosterone XL
6600
XL ML
52800
Etiocholanolone XL ML
5&Androstane38,17&diol
TOTAL RA
IDENTIFICATIONOF EfETAEiOLITES BY COCRYSTALLIZATLON (EXPRESSED IN DPM)
METABOLITES
TABLE II.
; a
0
:
a
the separation of estrogen conjugates as first reported by Hahnel and co-workers [4,5f, developed further by Hobkirk -et al. [6,7] and used extensively in this laboratory [9-X]. In the present study is described methodology for investigation of androgen conjugates and metabolites in dog bile after administrationof 3H-T. This methodology has now been used routinely by us in investigation of androgen metabolism in dog, other animals and man [4]. System A (see Methods above) is initially applied to the bile or urine for preliminary separation and identificationof the types of conjugates present (e.g., monoglucuronides,monosulfates or diconjugates). In the present case, in addition to a very small (ether extractable radioactivity = 1% of the excreted dose) uncharged fraction Al of system A (Figs. 2,4), which appeared early with water elution, six different monoglucuronideswere found. System B was subsequently used to achieve a more elaborate fractionation,but in the present case gave about the same results as those obtained with system A. Identificationof the glucuronides in the present work was achieved by noting the elution sequence of the peaks as compared to those of standards, almost quantitative enzyme hydrolysis and inhibition. After hydrolysis, Lipidex 5000 column chro~tography gave initial separation of the aglycones. Final separation and tentative identification of the androgens was achieved by paper chromatography or TLC. Final identificationwas accomplished by co-crystallizationwith the appropriate carrier to constant specific activity. In either system A or B, two separate groups of monoglucuronide peaks were discernible, their separation being related to differences in the configuration of rings A and B.
In each group a glucuronide of
a monohydroxy-androgen was present.
In the first group, this androgen
was etiocholanolone and in the second epiandrosterone. 17-ketoandrostanol,
In addition to
there were three -dihydroxyandrogens, which were the
same in both groups.
Since each of these later androgens was conjugated
to a single glucuronic acid moiety, it followed that the conjugating group was attached to a different hydroxyl in the steroid molecule and, therefore, the monoglucuronide was eluted with a different volume from the DEAE-Sephadex column. Only a few studies have appeared on androgen metabolism in dog. Harri -et al. [17] reported on -in vitro and -in vivo studies of androgen metabolism in canine intestine and found 4-androstendione and at least eight different metabolites including 5a-androstan-3,17-dione, 3B-hydroxy-So-androstan-17-ones androstanediols
0 19
It
(the latter predominant), and two
(5~~,-3B,17B and 5&3~~,17B).
analyzed endogenous C
3o- and
Recently, Martin -et al. [lg]
metabolites in bile and feces of the beagle. 2
was demonstrated that the biliary-fecal axis is a major excretory
route for androgen metabolites in this animal; two striking features in the pattern of metabolites was the predominance of glucosiduronate conjugates and of 58, 3o and 5a,3B-isomers within the glucosiduronate fraction.
The four major metabolites identified were the same ones
as obtained in the present study.
They also isolated a small quantity
of monosulfates, which we did not find.
We wish to thank Mrs. Diane Smith, Mrs. Susan Ash and Mrs. Cathy Russin for their excellent technical and clerical assistance. This study has been supported in part by grant (AM-012401 from the National Institutes of Health. This paper was presented in part at the 59th Annual Meeting of the Endocrine Meeting, Chicago, Ill., June, 1977.
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REFERENCES 1.
2. 3. 4. 5. 6. 7. 8. 9. 10. 11.
12. 13. 14. 15. 16. 17.
18.
Kirdani, R.Y. and Sandberg, A.A. STEROIDS 23, 667 (1974). Evans, C.R. and Pierrepoint, C.G. J. ENDOCR. 3, 539 (1975). Evans, C.R. and Pierrepoint, C.G. J. ENDOCR. 70, 31 (1976). Yamamoto, Y. Osawa, Y., Kirdani, R.Y. and Sandberg, A.A. in preparation, 1977. Hahnel, R. ANAL. BIOCHEM. lo, 184 (1965). Hahnel, R. and Abdul Rahman, M.G.B. BIOCHEM. J. 105, 1047 (1967). Hobkirk, R., Musey, P. and Nilsen, M. STEROIDS l4_, 191 (1969). Hobkirk, R. and Nilsen, M. STEROIDS 14, 533 (1969). Collins, D.C., Musey, P.I. and Preedy, J.R.K. STEROIDS 8, 67 (1976). Yamamoto, Y., Osawa, Y., Kirdani, R.Y. and Sandberg, A.A. in preparation (1977). Ishihara, M., Osawa, Y., Kirdani, R.Y. and Sandberg, A.A. J. STEROID BIOCHEM. 5, 1213 (1975). Ishihara, M., Osawa, Y., Kirdani, R.Y. and Sandberg, A.A. STEROIDS 2, 829 (1975). Ishihara, M., Kirdani, R.Y., Osawa, Y. and Sandberg, A.A. J. STEROID BIOCHEM. 1, 65 (1976). Honjo, H., Ishihara, M., Osawa, Y., Kirdani, R.Y. and Sandberg, A.A. STEROIDS 1, 79 (1976). Honjo, H., Ishihara, M., Osawa, Y., Kirdani, R.Y. and Sandberg, A.A. ENDOCRINOLOGY 99, 1054 (1976). Honjo, H., Barua, N.R., Osawa, Y., Kirdani, R.Y. and Sandberg, A.A. J. Cl. ENDOCR. METAB. 2, 1294 (1976). Harri, M.P., Nienstedt, W. and Hartiala, K. ACTA CHEM. FENN. B 2, 385 (1970). Martin, F., Bhargava, A.S. and Adlercreutz, H. J STEROID BIOCHEM. 8, 753 (1977).
2228
TESTOSTERONE METABOLITES IN DOG BILE Y. Yamamoto, Y. Osawa,* R. Y. Kirdani and A. A. Sandberg Roswell Park Memorial Institute and The Medical Foundation of Buffalo* Buffalo, New York 14263 Rec'd. 10-14-77. ABSTRACT Testosterone-1,2-3H was injected intravenously into a male dog with a bile fistula and bile and urine collected. The radioactivity was excreted preponderantly in bile (52% of the injected dose) in 6 hours: only 12% appeared in the urine. Methods to study the biliary metabolites of testosterone in this and other animals were developed. Satisfactory conjugate patterns were obtained by fractionation on DEAE-Sephadex A-25 columns using two different elution systems. In addition to an unchanged fraction, six different monoglucuronide fractions were separated. No other conjugates were isolated. Lipidex 5000 column chromatography, TLC and paper chromatography were used for the isolation and purification of aglycone metabolites, which were further identified by co-crystallization methods. The biliary metabolites of testosterone were epiandrosterone (3B-hydroxy-5ct-androstan-17-one), etiocholanolone (3a-hydroxy-5!3-androstan-17-one), 5o.-androstan-3H,17H-diol, 5@androstan3a,17&diol and 5H-androstan-3H,17@diol. A special feature of testosterone metabolism in dog was the preponderant excretion into bile of almost exclusively monoglucosiduronates. In the case of androstanediols, the glucuronic acid moiety was located at different hydroxyl groups.
INTRODUCTION Even though the dog has been used as a model for human prostatic disease, particularly benign hypertrophy, there is a paucity of reports in the literature concerning the metabolic fate of androgens in this animal.
Work in our laboratory has shown [l] that dogs metabolize
estrogens differently from the other animals, and, hence, by extension we undertook to study the excretory patterns of androgens in this animal.
Evans and Pierrepoint
[2,3] have indicated that the dog differs
from many mammals in that the most active androgens in its prostate have the 17ct configuration.
Vo Fume 32, Nwnber 2
S
TDEOXDI
February,
1978
S
234
TBX&OID~
In the present study we report on methods used to examine biliary patterns of excretion and metabolites in dog following the administration of labeled testosterone (T).
The methods devised in this
work have been used to investigate androgen metabolism in other animals (sheep, baboon, Rhesus monkey) and in the human [4]. Various analytical methods have been devised and used to senarate steroid metabolites.
DEAE-Sephadex, diethylaminoethyl derivative of
Sephadex G-25 or C-50 containing anion exchanger, was first introduced and developed as a column chromatographic method to separate con.jugates by Hahnel [5,6].
More recently, Hobkirk et al. [7,8] and Collins _et al.
[9] satisfactorily fractionated estrogen metabolites as their conjugates utilizing such chromatographic approaches. We have applied this ion-exchange column chromatography to the analysis of androgen metabolites in dog bile.
For further separation
of aglycones, Lipidex 5000 column chromatography was used.
Since the
methods used by us though lengthy are effective in separating conjugates and aglycones, we wish to report methodological details as applied to analysis of dog bile after administration of 3H-T.
In
following reports [4,10] the results of analysis of dog urine and bile and urine of the other animals will be reported. MATERIALS AND METHODS STEROIDS Testosterone-1,2-3H (3H-T) (specific activity 4OCi/mmole, 138mCi/mg) was purchased from the New England Nuclear Co., Boston, Mass. and diluted with carrier for the purposes of the study. The purity of this labeled steroid was checked and found to be more than 95% by paper chromatography in the system, hexane:methanol:water (100:90:10). Non-radioactive steroids were obtained from Steraloids, Inc., Wilton, N.H. and Research Plus Steroid Laboratories, Inc., Denville, New Jersey. These unlabeled compounds were used as standards for TLC, paper chromatography and cocrystallization.
S
TBEOXDI
235
EXPERIMENTAL PROCEDURE: Injection and Collection of Bile: Under intravenous anesthesia with diazepam (Valium, Roche) and phencyclidine hydrochloride (Sernylan, Parke-Davis), an adult male mongrel dog (24kg body weight) was laparotomized and a polyethylene tube inserted into the common bile duct to serve for collection of bile. 3H-T (20.3pCi/ 5ug), dissolved in 0.5 ml of pure ethanol, was diluted to 30 ml with normal saline and injected into the saphenous vein over a period of a few minutes. Bile was collected at time intervals following the sample administration, adjusted to pH9 and stored at 4°C until analyzed. The bile collected between 30 and 120 minutes was used for this study. Lactate-Ringer fluid was infused intravenously during the bile collection. COLUMN CHROMATOGRAPHY: DEAE-Sephadex (DEAE-Sephadex A-25 from Pharmacia Fine Chemicals, Piscataway, New Jersey) was previously swollen and equilibrated in distilled water at least overnight before use. Columns were first filled with distilled water and then packed with DEAE-Sephadex by gravity to the top. Two different multiple column systems were used: System A, Kg/60 (Pharmacia Fine Chemicals, 0.9 cm diameter x 60 cm length) plus K9/30, and System B, K15/90 plus K15/90 plus K15/30. The columns were connected by silicone tubes (0.2-0.3 cm diameter). System A was used for the preliminary separation of conjugates and the columns were eluted with a linear gradient system obtained by mixing 500 ml of distilled water with 500 ml of 0.6M NaCl. A more elaborate System B was also used in which a linear gradient consisted of 1000 ml of O.lM NaCl in the mixing bottle and 1000 ml of 0.4M NaCl in the reservoir bottle. The columns were first washed with 100 to 200 ml of distilled water after sample application in order to eliminate uncharged compounds; following the linear gradient elution the column was washed again with 200 to 400 ml of 2.OM NaCl to elute highly charged conjugates. Ten ml were collected in one tube and 0.3 to 0.5 ml aliquots were used for the determination of radioactivity. Lipidex 5000 (Packard Instrument Co., Inc., Downers Grove, Ill.) was prepared by mixing the suspension with hexane in an ultrasonic bath or by bubbling with nitrogen gas to obtain a slurry. Two connected columns (Pyrex 2145 and 1282) were filled with hexane and packed by gravity to 17 cm and 10 cm heights, respectively. The sample dissolved in 0.5 ml chloroform or benzene, was applied to the 17 cm column. The columns were eluted with 100 to 200 ml hexane, followed by a linear gradient system of 500 ml of hexane and 500 ml of 20% benzene in hexane. The columns were washed out with 150 ml of a polar solvent mixture (hexane:chloroform:methanol = 70:20:10). Aliquots were taken from the 10 ml fraction for the determination of radioactivity. The organic solvents were redistilled before use. Lipidex was used repeatedly after washing with chloroform and methanol (1:l). TLC AND PAPER CHROMATOGRAPHY: For the isolation and purification of aglycones, TLC and paper chromatography were carried out. Silica gel GF plates (250 microns thick, Analtec Inc., Newark, Del.) were developed with a solvent system of chloroform:acetone (37:3). Unlabeled steroids were detected by spraying with 2% sulfuric acid in methanol
236
S
WSlEOXD11
followed by heating (ZOO'C). Whatman No. 1 filter paper, (Fisher Scientific Co., Rochester, N.Y.) was developed in the system hexane: methanol:water (100:90:10). Unlabeled 17-keto-steroids were detected by Zi~erman color reaction and A" -3-ketosteroids by UV fluorescence. ENZYME HYDROLYSIS: The fractions or peaks obtained upon DEAF-Sephadex column chromatography were evaporated to dryness -in vacua and dissolved in 15 ml of O.lM acetate buffer, pH 5. H-glucuronidase from bovine liver (Sigma Chemical Co., St. Louis, MO.), 500-1000 units per ml, was added and the solution incubated at 37°C for 72 hours. It was then extracted with 3 times the volume of water-saturated ether. Inhibition of hydrolysis with saccharolactone (1O-3 to 2.5 x lo-'EI)was used as an index for the specificity of the @-glucuronidase hydrolysis. IDENTIFICATION OF METABOLITES: Following the isolation and purification of metabolites by chromatographic methods, aglycones were identified by co-recrystallation with unlabeled steroid carriers (lo-25 mg, depending on availability) from methanol-water. When a constant specific activity was obtained, the crystals were acetylated overnight at room temperature with acetic anhydride and pyridine. The acetylated aglycones were recrystallized and the specific activity determined. Aliquots of column chromatography samples RADIOACTIVITY MEASUREMENT: and other pipetted samples were dissolved in 10 ml of Aqueous Counting Scintillant (Amersbam/Searle Co.), diluted with toluene (1 part toluene in 2 parts Scintillant). The radioactivity was measured by Packard TriCarb spectrometers models 3375 or 2450 and corrected to dpm by an automatic standardization method. The efficiency of single label counting for 3H was between 30% and 46%. Thin layer plates and paper strips were scanned by a Packard Radiochromatogram Scanner model 7201-C. The radioactivity in samples that produced severe quenching, such as bile, was determined by firs& combusting the sample in a Packard Sample Oxidizer model 306, and then counting in a Packard Tri-Carb spectrometer.
RESULTS The excretion pattern of injected 3H-T is shown in Fig. 1.
Biliary
excretion (52%) exceeded urinary excretion (12%) during the six hours of collection; a high excretion rate was observed in the first two hours. Analysis of the collected bile was performed by DEAE-Sephadex A-25 column chromatography, enzyme hydrolysis, Lipidex 5000 column chromatography, TLC, paper chromatography and co-crystallization specific activity methods.
to constant
Flow charts of the methods used are shown
S
T1BBtOXDI
237
40 30 20 IO
I
4
2 Time
6
( hours)
Fig. 1. The cumulative excretion pattern into bile (solid line) and urine (dotted line) following the intravenous injection of 3H-testosterone @Cl !_KZjinto a male dog. in Figs. 2 and 3 for the A and B systems (see Methods), respectively. Fractions obtained using the A method are labeled "A", those using the B method as I'D". 1. Fractionation of Conjugates: Separation by System A (Fig. 2): The bile was directly applied to a DEAE-Sephadex column and eluted as described in Methods. in Fig. 4.
The elution pattern of radioactivity is shown
Al (tubes 4-6) was eluted with distilled water and 38% of
this fraction was ether extractable,
A2
(tubes 30-45) and A3 (tubes
S
238
IZ?BEOXDI FIGURE
2
BILE DEAE-SEPHADEX COLUMN CHROMATOGRAPHY SYSTEM A A-i
A-2
(Uncha;gedFr.) Ether Eftraction Extrl,crble
A-3
(8-ylucuronidaseHydrolysis) G~~c_o:
Fr.
i
Gl+onide
Fr.
Llpidex Column Chromatography
Unex;Wl;Ic;e
Steroids
5 Aglycone Peaks
Aglycone Peaks
I
I
Etioch'lanolone
Epiand'osterone
SU-Androstane-38,17B-dial 5a-Androstane-3B,175-dial SB-Androstane-3n,l7f+diol Polar Metabolites
DEAE-Sephadex
column
c’hromaro~raphy,
System
B
I I
1 GLUCURONIDEFT.
I UNCHARGEDFr.
Peak 81
I
Peak
82
I Peak B3
Peak
I 8-glucuronidase
84
I Peak B5
I Pesk B6
1 Peak
B7
hy
Lip polar
I dihydroxy
i monohydrory etioc b olan“lone
t
X3-A-Za,l73-dial %-A-f3,118-dial
55-70) were then eluted using a linear NaCl gradient.
erD”e
h-A-8 ,17d-dial ‘B-A-3a,lXT-dial s-A-X3,179-dial
A2 was composed
of two peaks (Fig. 4), the separation of which was not complete, and A3 was composed of three peaks.
No peaks appeared in the 2.OM NaCl wash.
S
TBEOIDl
239
x 104dpm 8
r___ _-___-
----*QMN&,
1.
-r e
*M-C cc
4
I
0.6M linear
**--
No’3 gradiwd
Distillad
1
0
20
II-I,
40
I:
60 TUBE
1
80
100
I
120
Wolrr
1
140
NUMBERS
Fig. 4. DEAE-Sephadex A-25 column chromatographic pattern of dog bile by column system 1 (K 9/60 + K 9/60). The column was first washed with 100 ml distilled water after loading the sample, then it was developed with 0 - 0.6M NaCl linear gradient system (1000 ml), and at the end washed out with 400 ml 2.OM NaCl. Ten ml was collected in each tube. Three fractions, A-I, A-II and A-III were obtained; A-I is uncharged, A-II and A-III are both glucuronide conjugates. Diconjugates were not present and would have appeared in tube numbers above 115. Separation by System B (Fig. 3): The bile was fractionated through large columns (see Methods), the elution pattern of which is shown in Fig. 5; seven fractions were separated.
The first fraction, Bl, was eluted in tubes 20-30 (Fig. 3, 5;
compare with fraction Al of system A, Figs, 2, 4).
Fractions B2, B3
and B4 were eluted as a separate group of peaks in tubes 75-115 and represented a more highly resolved fraction, AZ, since no fraction comparable to B3 was found using the A system.
Fractions B5, B6 and B7
were also eluted as a separate zone in tubes 145-205 and were comparable to A3.
S
240
T=EOIDI
82 x IO4 dpm
IO
B5
_-0.4M N&l Linaor Grodhnl System
5 B-I ___----__-II
60
0. IM No’3
90
120
150
180
210
240
TUBE NUMBERS
Fig. 5. DEAE-Sephadex A-25 chromatographic pattern of dog bile by column system 2 (K 15/90 + K 15/90 + K 15/90). The column was eluted with distilled water after loading, then linear gradient O.lM - 0.4M NaCl (2000 ml) was applied. F-I is uncharged fraction. Glucuronide fractions F-II and F-III were separated respectively into three peaks (Bl to B7); B2 and B5 - polar, B3 and B6 - monohydroxy, B4 and B7 dihydroxy. 2.
Enzymatic Hydrolysis of Conjugate Fractions:
Follow ng DEAE-Sephadex column chromatography, all conjugate fractions were hydrolyzed with 8-glucuronidase. with the A fractions hydrolysis
(8%and
are given in Table I.
The results obtained
The high percentage of
95%) and the efficient enzyme inhibition by sac-
charolactone indicate that A2 and A3 are glucuronide conjugates.
The
individual fractions obtained from System B elution were also hydrolyzed individually and these data a .so indicate the conjugates to be glucuronides.
S
WBEOXDI
241
TABLE I The Hydrolysis of Fractions A2 and A3 by $-glucuronidase Fraction F-II F-III
Enzyme Hydrolysis
Control
82.3% 95.0%
Enzyme Inhibition 7.0% 7.0%
7.4% 2.7%
enzyme hydrolysis = substrate + acetate buffer f 8-glucuronidase control = substrate + acetate buffer enzyme inhibition = substrate + acetate buffer + 8-glucuronidase + saccharolactone (enzyme inhibitor), 72 hours incubation at 3J'C (buffer pH 5.0).
3.
Separation and Identification of Agylcones:
I.
From System A.
The aglycone mixture obtained upon hydrolysis
with 8-glucuronidase of fractions A2 and A3, by chromatography on Lipidex-5000. each resolved into 5 fractions.
was
initially separated
The hydrolysates from A2 and A3
Since it was found, using standards,
that Lipidex chromatography did not, at times, resolve compounds closely related in polarity, monohydroxy androgens obtained by hydrolysis of biliary conjugates were further purified and tentatively identified by chromatography on paper (see Methods).
In the system used, etiocho-
lanolone and epiandrosterone had Rf values of 0.31 and 0.29, respectively.
For comparison, typical Rf values of standard monohydroxy
compounds in the same system are as follows: hydroxy-4-androsten-3-one),
T, epitestosterone
dihydrotestosterone
(17~
(lJ@hydroxy-5a-andro-
Stan-3-one) and androsterone 0.12, 0.17, 0.31 and 0.41, respectively. The dihydroxyandrogens
isolated from bile were further separated and
tentatively identified by chromatography on silica gel (see Methods). In the system used, 5a-androstan-3B,lJ&diol, diol, 58-androstan-38,178-diol respectively.
58-androstan-3a,lJR-
had Rf values of 0.19, 0.11 and 0.35,
By comparison, Rf values of some standards in the same
system are as follows:
5a-androstane-3a,l7B_diol
3c1,17a-diol 0.09, 5o-androstane-3B,17a-diol
0.16, 5a-androstan-
0.17.
Admixture of the above tentatively identified compounds with appropriate carriers and crystallization to constant specific activity resulted in identification of the following compounds in the two peaks, A2 and A3: -AZ:
Monohydroxy compound:
5&androstan-3@,17&diol, 3a,17&diol.
etiocholanolone.
Dihydroxy compounds:
Su-androstane-3$,17&diol
and SB-androstan-
Also present was a polar metabolite which was not
further
identified. A3: -
Monohydroxy compound:
5&-androstane-3@,17@-diol, 3a,17&diol.
Epiandrosterone.
Dihydroxy compounds:
5o-androstane-3B,17a_diol
and 5R-androstan-
Also present was a polar metabolite which was not identi-
fied further. Fig. 6 is an example of an elution pattern following Lipidex chromatography and depicts separation of aglycones obtained after O-glucuronidase hydrolysis of fraction A2. II.
From System
B.
The aglycone mixture obtained upon hydrolysis
of fractions B2 through B7 were identified using the above procedure as: B2: -
Unidentified polar compound.
-B3:
Monohydroxy compound:
-B4:
Dihydroxy compounds: 3a,17&diol
etiocholanolone. So-androstane-3!3,17B_diol, 5&androstan-
and 5&androstan-3@,17&diol.
B5* _.
Unidentified polar metabolite.
-B6:
Monohydroxy compound:
-B7:
Dihydroxy compounds: 3n,17&diol
epiandrosterone. 5a-androstan-3@,17B-diol,
and 5&androstan-3B,17&diol.
SB-androstane-
S
%?1SEOfDI
c
E
x lO*dpm
I2
243
1
3
----Porn so~nl
Waam:Chlorolorm: Ythami 7aP lo)
2
20% Gmnr*m Hmon*. Litmar Grodiont !Syahm ___.,.e--
I
0
2040
6080
IO0
120
140
TUBE NUMBERS
Lipidex 5000 column chromatography of A2 aglycones obFig. 6. tained after &-glucuronidase hydrolysis. The column was eluted with 200 ml hexane following sample loading, then hexane - 20% benzene hexane linear gradient system (1000 ml) was used. The column was washed out finally with a polar solvent mixture (hexane:chloroform: methanol = (70:20:10). Five peaks were eluted: Peak A consists of monohydroxy, Peaks B, C, D of dihydroxy and Peak E of polar metabolites. Table II presents details of co-crystallization with carriers of the compounds described above obtained from the B system analysis. From the above results, it appears that after administration of 3H-T , only monoglucuronides are found in dog bile.
These conjugates
were separable into two major groups by fractionation in systems A or B.
In each of these two groups, monohydroxy compounds were found.
How-
ever, in each group the three dihydroxy androgens were also present.
DISCUSSION It appears that the major excretory route for T metabolites in the dog is the bile.
The work presented shows that DEAE-Sephadex column
chromatography can be used to separate androgen conjugates, similar to
AcCR
RA ; XL ; ML ; CR ; AV ; AcCAL
2700 1100
3000
300
2800
RA/mg
3800 2300 900
2600 5400
250 460
2600 3700
CR L
2787 2272 875
2342 2777
250 250
2300 2300
CR 2
2600 2300 900
2200 2250
250 250
2400 2450
CR 3
2550 24.50 800
2300 2200
2350 2200
CR 4
2331 858
2250
250
2400
AV
ISO0 750
1750
200
AcCAL
2000 1800 700
1800 1700
ZOO 200
AcCR
ML 1400 950 850 900 700 radioactivity crystal mother liquor cocrystallization from methanol water, expressed as specific activity (dpmfmg) average specific activity of two or three constant numbers ; calculated specific activity of the diacetates obtained from the last crystallizations of the diols, i.e., C3 or C4 ; cocrystallization of acetylated compounds
64300 29700
29300
5ct-Androstane- XL 3@,17B-dial ML
3a,17$-diol ML 5&-Androstane- XL Epiandrosterone XL
6600
XL ML
52800
Etiocholanolone XL ML
5&Androstane38,17&diol
TOTAL RA
IDENTIFICATIONOF EfETAEiOLITES BY COCRYSTALLIZATLON (EXPRESSED IN DPM)
METABOLITES
TABLE II.
; a
0
:
a
the separation of estrogen conjugates as first reported by Hahnel and co-workers [4,5f, developed further by Hobkirk -et al. [6,7] and used extensively in this laboratory [9-X]. In the present study is described methodology for investigation of androgen conjugates and metabolites in dog bile after administrationof 3H-T. This methodology has now been used routinely by us in investigation of androgen metabolism in dog, other animals and man [4]. System A (see Methods above) is initially applied to the bile or urine for preliminary separation and identificationof the types of conjugates present (e.g., monoglucuronides,monosulfates or diconjugates). In the present case, in addition to a very small (ether extractable radioactivity = 1% of the excreted dose) uncharged fraction Al of system A (Figs. 2,4), which appeared early with water elution, six different monoglucuronideswere found. System B was subsequently used to achieve a more elaborate fractionation,but in the present case gave about the same results as those obtained with system A. Identificationof the glucuronides in the present work was achieved by noting the elution sequence of the peaks as compared to those of standards, almost quantitative enzyme hydrolysis and inhibition. After hydrolysis, Lipidex 5000 column chro~tography gave initial separation of the aglycones. Final separation and tentative identification of the androgens was achieved by paper chromatography or TLC. Final identificationwas accomplished by co-crystallizationwith the appropriate carrier to constant specific activity. In either system A or B, two separate groups of monoglucuronide peaks were discernible, their separation being related to differences in the configuration of rings A and B.
In each group a glucuronide of
a monohydroxy-androgen was present.
In the first group, this androgen
was etiocholanolone and in the second epiandrosterone. 17-ketoandrostanol,
In addition to
there were three -dihydroxyandrogens, which were the
same in both groups.
Since each of these later androgens was conjugated
to a single glucuronic acid moiety, it followed that the conjugating group was attached to a different hydroxyl in the steroid molecule and, therefore, the monoglucuronide was eluted with a different volume from the DEAE-Sephadex column. Only a few studies have appeared on androgen metabolism in dog. Harri -et al. [17] reported on -in vitro and -in vivo studies of androgen metabolism in canine intestine and found 4-androstendione and at least eight different metabolites including 5a-androstan-3,17-dione, 3B-hydroxy-So-androstan-17-ones androstanediols
0 19
It
(the latter predominant), and two
(5~~,-3B,17B and 5&3~~,17B).
analyzed endogenous C
3o- and
Recently, Martin -et al. [lg]
metabolites in bile and feces of the beagle. 2
was demonstrated that the biliary-fecal axis is a major excretory
route for androgen metabolites in this animal; two striking features in the pattern of metabolites was the predominance of glucosiduronate conjugates and of 58, 3o and 5a,3B-isomers within the glucosiduronate fraction.
The four major metabolites identified were the same ones
as obtained in the present study.
They also isolated a small quantity
of monosulfates, which we did not find.
We wish to thank Mrs. Diane Smith, Mrs. Susan Ash and Mrs. Cathy Russin for their excellent technical and clerical assistance. This study has been supported in part by grant (AM-012401 from the National Institutes of Health. This paper was presented in part at the 59th Annual Meeting of the Endocrine Meeting, Chicago, Ill., June, 1977.
s
TBEOXD-
247
REFERENCES 1.
2. 3. 4. 5. 6. 7. 8. 9. 10. 11.
12. 13. 14. 15. 16. 17.
18.
Kirdani, R.Y. and Sandberg, A.A. STEROIDS 23, 667 (1974). Evans, C.R. and Pierrepoint, C.G. J. ENDOCR. 3, 539 (1975). Evans, C.R. and Pierrepoint, C.G. J. ENDOCR. 70, 31 (1976). Yamamoto, Y. Osawa, Y., Kirdani, R.Y. and Sandberg, A.A. in preparation, 1977. Hahnel, R. ANAL. BIOCHEM. lo, 184 (1965). Hahnel, R. and Abdul Rahman, M.G.B. BIOCHEM. J. 105, 1047 (1967). Hobkirk, R., Musey, P. and Nilsen, M. STEROIDS l4_, 191 (1969). Hobkirk, R. and Nilsen, M. STEROIDS 14, 533 (1969). Collins, D.C., Musey, P.I. and Preedy, J.R.K. STEROIDS 8, 67 (1976). Yamamoto, Y., Osawa, Y., Kirdani, R.Y. and Sandberg, A.A. in preparation (1977). Ishihara, M., Osawa, Y., Kirdani, R.Y. and Sandberg, A.A. J. STEROID BIOCHEM. 5, 1213 (1975). Ishihara, M., Osawa, Y., Kirdani, R.Y. and Sandberg, A.A. STEROIDS 2, 829 (1975). Ishihara, M., Kirdani, R.Y., Osawa, Y. and Sandberg, A.A. J. STEROID BIOCHEM. 1, 65 (1976). Honjo, H., Ishihara, M., Osawa, Y., Kirdani, R.Y. and Sandberg, A.A. STEROIDS 1, 79 (1976). Honjo, H., Ishihara, M., Osawa, Y., Kirdani, R.Y. and Sandberg, A.A. ENDOCRINOLOGY 99, 1054 (1976). Honjo, H., Barua, N.R., Osawa, Y., Kirdani, R.Y. and Sandberg, A.A. J. Cl. ENDOCR. METAB. 2, 1294 (1976). Harri, M.P., Nienstedt, W. and Hartiala, K. ACTA CHEM. FENN. B 2, 385 (1970). Martin, F., Bhargava, A.S. and Adlercreutz, H. J STEROID BIOCHEM. 8, 753 (1977).