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The in vitro metabolism of 3α,17β-androstanediol by liver and kidney
The in Vitro Metabolism of 3cu,l7@Androstanediol by Liver and Kidney1v2 Charles D. Kochakian3 and H. V. Aposhian” From the Department qf Ph:ysiology and Vital Economics, School of Medicine Dentistry, University of Roch.ester, Rochester, New York Received
December
and
10, 1951
INTRODUCTION
Testosterone is metabolized by rabbit liver slices (3) and liver and kidney homogenates (4) to A4-androstene-3,17-dione, epitestosterone, and other unidentified compounds. A4-Androstene-3,17-dione in turn is converted by rabbit liver slices to testosterone, epitestosterone, and a number of other compounds (5). Similar studies now have been made with 3a,l7&androstanediol. EXPERIMEN.TAL
Procedure The procedure for incubation and isolation of the steroids6 was as previously described (3,5) except for a few minor modifications. The liver slices were prepared with the Stadie-Riggs microtome (6) and the homogenates6 in the Waring blendor. The crude extract was freed of cholesterol and lipides by extraction with petroleum ether. The androgens which remained as crystalline material were separated and purified by chromatography. All of the animals were non-fasted adult males. The liver and kidneys were incubated simultaneously. 1 This investigation was supported by a grant-in-aid from the American Cancer Society on recommendation of the Committee on Growth of the National Research Council. * Preliminary reports on parts of these data have been made (1,2). 3 Present address: Oklahoma Medical Research Institute and Hospital, 825 N. E. 13th Street, Oklahoma City 4, Oklahoma. 4 Part of these data was taken from the thesis submitted to the Graduate School of the University of Rochester in partial fulfillment of the MS. degree. 6 The steroids were generously provided by Ciba Pharmaceutical Products, Inc. 6 It is recognized that tissues prepared in the Waring blendor are not the same as those prepared by the Potter-Elvehjem homogenizer (7,8). 442
METABOLISM
OF
ANDROSTANEDIOL
443
All melting points are uncorrected and were obtained on the Fisher-Johns apparatus adapted for use under a microscope. Acid-washed alumina (Harshaw) was used for chromatography. Carbon tetrachloride containing progressively increasing amounts of absolute ethyl alcohol was the eluting agent (9) for the ketonic fractions in which all of the metabolites were found. The non-ketonic fraction was submitted to detailed chromatography, and 3cu,l7fi-androstanediol was the only androgen found.
Pswijkation
of Sa, 17p-Androstanediol
Treatment of the steroid with Girard’s T reagent yielded a trace of oily material in the ketonic fraction which was negative in all color tests. The non-ketonic fraction was adsorbed on a column of alumina and eluted with benzene containing increasing amounts of absolute ethyl alcohol. The recovered material was recrystallized from hot benzene containing 29/, absolute ethyl alcohol, m.p. 233-234”.
The Formation of Androsterone from SCYJ7/3-Androstanediol by Guinea Pig Liver and Kidney Homogenates Liver. The fraction eluted by carbon tetrachloride containing 0.1% ethyl alcohol gave typical color reactions of a l7-ketosteroid (lO,ll), m.p. 175-179”. On admixture with a sample of authentic androsterone (18+187”), the m.p. u-as 183-187”. The infrared spectrum was identical with that of androsterone. Kidney. The fraction eluted with 0.170 absolute ethyl alcohol in carbon tetrachloride gave the typical 17-ketosteroid color reaction (10,ll) but the crystals were not pure and melted at 137-138” with previous softening (androsterone 184-187”). The infrared analysis, however, indicated androsterone with a trace of impurity. Further purification was not attempted because of the small amount of material.
M etabolites Formed from Scr,l7@-Androstwnediol in a Rabbit Liver Slice System &IT-Androstanedione. The first fraction eluted with carbon tetrachloride plus 0.2y0 absolute ethyl alcohol gave in the Holtorfi-Koch (10) test a deep violet color after 60 min. at 20”, indicating the presence of a 17-ketosteroid group. A deep-violet color after 10 min. at 20” and also at 0” indicated the presence of a a-ketone group on the saturated A-ring (12). The intensity of the color was similar to that of known androstanedione. The infrared spectrum also suggested the presence of a 3,17-diketone. This fraction was rechromatographed on alumina with carbon tetrachloride: benzene: ether:methyl alcohol. The benzene eluate gave impure crystals m.p. 105-125”, but the Holtorff-Koch modification of the Zimmermann color test and the infrared spectrum were identical with those of 3, IT-androstanedione. Androsterone and Epiandrosterone. The remaining fractions eluted with carbon tetrachloride containing 0.270 absolute ethyl alcohol melted at 160-166”. Treatment with digitonin yielded two fractions both of which gave the characteristic l’i-ketosteroid color test. They were both recrystallized from aqueous acetone. The a-fraction melted at 178-182”; on admixture with a sample of authentic androsterone (m.p. 184-187’), the m.p. was 18@181”. The infrared spectrum conformed with that of androsterone.
444
CHARLES
D.
KOCHAKIAN
AND
H.
V.
APOSHIAN
The p-fraction melted at lTl”-174”; on admixture with a sample of authentic epiandrosterone (m.p. 175-177”), the m.p. was 173-17’7”. The infrared spectrum was identical with that of an authentic specimen of epiandrosterone. In addition to the above, an impure sample of androsterone (i.9 mg.) m.p. 118-132” and one of epiandrosterone (1.3 mg.) m.p. 157-167” were obtained. The infrared spectra of these two compounds were identical with that of their respective authentic compounds. Metaboliles Formed from &,I 7&Androsianediol in a Rabbit Liver Homogenate Preparation. Chromatography on alumina did not result in the separation of a single pure substance. Therefore, all the fractions which gave a positive Holtorff-Koch color test were pooled and rechromatographed. All of the 17-ketosteroids were eluted with carbon tetrachloride containing 0.2yo absolute alcohol. The first portion also gave color tests indicative of a 3-ketone group in a saturated A-ring (12). The infrared analysis suggested a 3,17diketone. On rechromatography on alumina with carbon tetrachloride: benzene : ether: methyl alcohol, the benzene eluate gave impure crystals m.p. 95-125” (3,17-androstanedione m.p. 131-132”), but the m-dinitrobenzene color tests and the infrared spectrum were identical with those of 3,17-androstanedione.
RESULTS Comparison of Rat, Guinea Pig and Rabbit Kidney and Liver Homogenates The incubation of 3ql7/3-androstanediol for 3 hr. with homogenates of liver and kidney of rat, guinea pig, and rabbit resulted in the recovery of 82-97% of the added steroid and the formation of less than 1% of ketonic materials (Table I). The positive 17-ketosteroid color test (10) indicated the oxidation of the 17-hydroxyl group to a ketonic group. Another fraction gave a color test similar to that of androstane3,17-dione. The paucity of metabolites precluded isolation and characterization. In the next study (Table II) the incubation was extended to 6 hr. and an atmosphere of oxygen was used. In an attempt to increase the solubility of the 3cql7@androstanediol, it was homogenized in a PotterElvehjem apparatus with approximately 1 g. of tissue and 5 ml. of buffer prior to addition to the Waring blendor. Guinea pig kidney and liver were used. The extracts of the several experiments were pooled according to tissue. The 3cr,l7/3-androstanediol again proved resistant to oxidation by both the kidney and liver homogenates; 84 and 85% of the original material was recovered and a crude ketonic fraction of only 0.9 and 1.3yo was obtained. Androsterone, 0.3 and 0.5yo, however, was isolated by chromatography and identified. Small amounts of other metabolites were indicated but could not‘be isolated for charac-
METABOLISM
OF
445
ANDROSTANEDIOL
terization. Infrared’ analysis, however, suggested the presence of epiandrosterone in a few fractions of the chromatographed ketonic materials. Comparison of Rabbit Liver Slices with Homogenates Since homogenization is known to decrease the respiration rate of tissues, especially if prepared in the Waring blendor [cf. Refs. (7,8)], TABLE The in Vitro
Metabolism
I
of &,17p-Androstanediol
by the Tissues of Different
Liver”
Guinea
Rat
my.
7%’
Kidney
pig
mg.
247 1.4 0.9
83 “0:;
252 1.7 1.5
Rabbit
Guinea
Rat
pig
Rabbit
%’
Steroid Crystalline steroidsd Ketonic (as 17ketosteroid) Holtorff-Koch (IO) Pinem (11)
Animals”
recovered 84
254
0.6 0.5
85
1.4 1.4
0.5 0.4
145 0.5 0.3
97
192
0.3 0.2
96
0.5 0.2
0.3 0.1
174
87
0.7 0.4
0.4 0.2
0 Adult non-fasted male animals used. Steroids, tissues and 50 ml. of Ringerphosphate buffer mixed in Waring blendor for 2 min. Mixture transferred quantitatively by washing with 50 ml. of buffer to Fernbach flash. Incubation at 37” for 3 hr.; air was the gaseous phase. All equipment and buffer cold and sterile. b Two separate experiments for each animal. The incubation mixtures were pooled prior to extraction. c Per cent of added 3a,l7,%androstanediol. d This fraction was separated from the total extract by dissolving the lipides and cholesterol in petroleum ether.
liver slices were compared (Table III) with homogenates prepared simultaneously from the same animals. The liver slices were only slightly more effective than the homogenate. The degree of oxidation of the 3a,l78-androstanediol, however, 7 The infrared analyses were kindly Institute for Cancer Research.
performed
by Dr. K. Dobriner,
Sloan-Kettering
446
CHARLES
was so small in The metabolites color tests and but only those satisfactory for
D.
KOCHAKIAN
AND
H.
V.
dPOSHIAN
both instances that the difference was not significant. isolated in pure form or indicated as being present by infrared analysis were identical in both experiments, in the slice experiments were obtainable in a state full characterization.
Disuccinate The Incubation of Sql7/3-Androstanediol with Rabbit Liver Homogenate Since 3a,l7&androstanediol was quite insoluble in tissue fluids (7) the disuccinate was prepared and dissolved in the calculated amount of 0.1% sodium hydroxide solution prior to addition to the homogenate. The ester, however, was not hydrolyzed during the incubation period; 92yo of the added 3a,l7&androstanediol disuccinate was recovered in pure form. An impure ketonic fraction of only 5 mg. was obtained which gave color reactions suggestive of the presence of a 3-keto group, but no material could be isolated for identification. TBBLE The in Vitro
II
Metabolism of Sru,l’?‘pAndrostanediol of Guinea Pig Tissue
by Homogenates
Liverb
Kidney
ml.
sz
mg.
%
Incubation Tissue 301,17/.%Androstanediol
4 x 4,800 4 x 150
8 x 15,000 8 x 200 Steroids recovered
Crystalline steroid& 301,17j+Androstanediol Ketonic As 17-ketosteroid [Holtorff-Koch (lo)] Androsterone
1,391 1,348
14.1 5.8
87 84
0.9 0.4
539 509
7.6 3.0
90 85
1.3 0.5
n Procedure as in Table I except that approximately 1 g. of tissue, steroid, and 5 ml. buffer homogenized in Potter-Elvehjem apparatus prior to addition to Waring blendor in an attempt to increase solubility of steroid. *The incubation mixtures of eight separate experiments were pooled prior to extraction. Each experiment contained approximately 15 g. of liver, 200 mg. of steroid, and 100 ml. buffer. c The incubation mixtures of four separate experiments were pooled prior to extraction. Each experiment contained the two kidneys (4.2-5.2 g.), 150 mg. of steroid, and 100 ml. of buffer. d Petroleum ether-insoluble fraction of total fats.
METABOLISM
OF
TABLE In Vitro
Metabolism
447
ANDROSTANEDIOL
III
of Sor,lT’fi-Androstanediol
by Rabbit LiveF
(Slices vs. homogenates) Slices” mu.
4,o
Homogenate nw.
70
Incubation Tissue 3a, 17&4ndrostanediol
8 x 12,000 8 X 200 Steroids
8 X 12,000 8 X 200 recovered
2,049 Crystalline steroidsc 1,890 $3.9 1,342 3q 17&4ndrostanediol 1,047 87.8 5.6 0.4 Androsterone (3.7)d 0.2 0.2 Epiandrosterone (1.3)d 0.1 (i::)d 0.1 3,174ndrostanedione (l.O)d 0.1 154’ Cholesterol 116 (1Procedure as in Table I except that oxygen was the gaseous phase during the incubation. The slices and homogenates were prepared and run simultaneously. There were eight separate experiments for each. b The steroid was homogenized (3) in 5 ml. of the rabbits’ own serum, the rest (11-21 ml.) of the serum was added, and the final fluid volume made to 100 ml. with the Ringer’s phosphate buffer. c Petroleum ether-insoluble fraction of total lipides. d Estimated by the Holtorff-Koch (10) color test. c The greater amount of cholesterol obtained from the experiment with slices was due to the addition of serum to the incubation medium (cf. footnote b).
Control Experiments Rabbit liver (425 g.) and kidney (158 g.) were homogenized and fractionated as in the incubation experiments. The only identified steroid was cholesterol; liver, 751 mg. ; and kidney, 302 mg. DISCUSSION
These experiments indicate that 3a,l7&androstanediol can be oxidized in essentially the same manner by both the liver and kidney of the rat, guinea pig, and rabbit. In every instance, however, the degree of metabolism is small compared with the effect of these tissues on testosterone and A4-androstene-3,17-dione. The integrity of the cell, furthermore, does not make a great difference in the ability of 3ru,17/3androstanediol to resist the in vitro action of at least the liver. A comparison of the rates of metabolism of various androgens in the rabbit liver slice system indicates a rough parallelism with the solubilities of these steroids in tissue fluids (13).
448
CHARLES
D. KOCHAKIAN
AND
H. V. APOSHIAN
The small yields of ketonic metabolites might be due to the fact that t,he chemical equilibria among these compounds favor the formation of the completely reduced steroid, 3a,l7&androstanediol. Androsterone is 3cr,17/GAndrostanediol
ti Androsterone s 3,17-Androstanedione
$ Epiandrosterone
converted by rabbit liver slices to only small amounts of 3,17-androstanedione, and epiandrosterone (14). 3q(,17@-Androstanediol is the main metabolite. Thus, the predominant enzymatic action of the tissues seems to be reduction of the 17-ketone group. This same phenomenon is seen in the relationship of 3,17-androstenedione with testosterone (3,5) and dehydroisoandrosterone with A5-androstene-3P,17/3-diol (15). SUMMARY
3a,178-Androstanediol was metabolized in vitro by homogenates of rat, guinea pig, and rabbit liver and kidneys. The metabolites isolated or indicated as present were androsterone, epiandrosterone, and 3,17androstanedione. The total amount of conversion to these substances was less than 5y0;, and more than 90% of the original material ‘was accounted for. Rabbit liver slices were no more effective than the homogenates. REFERENCES 1. KOCHAKIAN, University 2. KOCHAKIAN,
C. D., in GORDON, E. S., Symposium on Streoid Hormones. p. 93. Wisconsin Press, Madison, 1950. C. D., PARENTE, N., AND APOSEIIAN, H. V., Federation Proc. 8, 214
(1949). 3. CLARK, L. C., JR., AND KOCHAKIAN, C. D., J. Biol. Chem. 170, 23 (1947). 4. GONGORA, J., AND KOCHAKIAN, C. D., Federation Proc. 7, 42 (1948). 5. CLARK, L. C., JR., KOCHAKIAN, C. D., AND LOBOTSKY, J., J. Biol. Chem. 171,493 (1947). 6. STADIE, W. C., AND RIGGS, B. C., J. Biol. Chem. 164, 687 (1944). 7. POTTER, V. R., RECKNAGEL, R. O., AND HURLBERT, R. B., Federation Proc. [3], 10, 646 (1951). 8. SCHNEIDER, W. C., AND HOGEBOOM, G. H., Cancer Research [l], 11, 1 (1951). 9. CALLOW, N. H., Biochem. J. 33, 559 (1939). 10. HOLTORFF, A. F., AND KOCH, F. C., 1. Biol. Chem. 136, 377 (1940). 11. PINCUS, G., Endocrinology 32, 176 (1943). 12. KOCHAKIAN, C. D., AND STEDDIAN, L. A., Am. J. Physiol. 126, 556 (1939). 13. KOCHAKIAN, C. D., Am. J. Physiol. 142, 315 (1944). 14. SCHNEIDER, J. J., AND MASON, H. L., J. Biol. Chem. 175, 231 (1948). 15. SCHNEIDER, J. J., AND MASON, H. L., J. Biol. Chem. 172, 771 (1948).
December
and
10, 1951
INTRODUCTION
Testosterone is metabolized by rabbit liver slices (3) and liver and kidney homogenates (4) to A4-androstene-3,17-dione, epitestosterone, and other unidentified compounds. A4-Androstene-3,17-dione in turn is converted by rabbit liver slices to testosterone, epitestosterone, and a number of other compounds (5). Similar studies now have been made with 3a,l7&androstanediol. EXPERIMEN.TAL
Procedure The procedure for incubation and isolation of the steroids6 was as previously described (3,5) except for a few minor modifications. The liver slices were prepared with the Stadie-Riggs microtome (6) and the homogenates6 in the Waring blendor. The crude extract was freed of cholesterol and lipides by extraction with petroleum ether. The androgens which remained as crystalline material were separated and purified by chromatography. All of the animals were non-fasted adult males. The liver and kidneys were incubated simultaneously. 1 This investigation was supported by a grant-in-aid from the American Cancer Society on recommendation of the Committee on Growth of the National Research Council. * Preliminary reports on parts of these data have been made (1,2). 3 Present address: Oklahoma Medical Research Institute and Hospital, 825 N. E. 13th Street, Oklahoma City 4, Oklahoma. 4 Part of these data was taken from the thesis submitted to the Graduate School of the University of Rochester in partial fulfillment of the MS. degree. 6 The steroids were generously provided by Ciba Pharmaceutical Products, Inc. 6 It is recognized that tissues prepared in the Waring blendor are not the same as those prepared by the Potter-Elvehjem homogenizer (7,8). 442
METABOLISM
OF
ANDROSTANEDIOL
443
All melting points are uncorrected and were obtained on the Fisher-Johns apparatus adapted for use under a microscope. Acid-washed alumina (Harshaw) was used for chromatography. Carbon tetrachloride containing progressively increasing amounts of absolute ethyl alcohol was the eluting agent (9) for the ketonic fractions in which all of the metabolites were found. The non-ketonic fraction was submitted to detailed chromatography, and 3cu,l7fi-androstanediol was the only androgen found.
Pswijkation
of Sa, 17p-Androstanediol
Treatment of the steroid with Girard’s T reagent yielded a trace of oily material in the ketonic fraction which was negative in all color tests. The non-ketonic fraction was adsorbed on a column of alumina and eluted with benzene containing increasing amounts of absolute ethyl alcohol. The recovered material was recrystallized from hot benzene containing 29/, absolute ethyl alcohol, m.p. 233-234”.
The Formation of Androsterone from SCYJ7/3-Androstanediol by Guinea Pig Liver and Kidney Homogenates Liver. The fraction eluted by carbon tetrachloride containing 0.1% ethyl alcohol gave typical color reactions of a l7-ketosteroid (lO,ll), m.p. 175-179”. On admixture with a sample of authentic androsterone (18+187”), the m.p. u-as 183-187”. The infrared spectrum was identical with that of androsterone. Kidney. The fraction eluted with 0.170 absolute ethyl alcohol in carbon tetrachloride gave the typical 17-ketosteroid color reaction (10,ll) but the crystals were not pure and melted at 137-138” with previous softening (androsterone 184-187”). The infrared analysis, however, indicated androsterone with a trace of impurity. Further purification was not attempted because of the small amount of material.
M etabolites Formed from Scr,l7@-Androstwnediol in a Rabbit Liver Slice System &IT-Androstanedione. The first fraction eluted with carbon tetrachloride plus 0.2y0 absolute ethyl alcohol gave in the Holtorfi-Koch (10) test a deep violet color after 60 min. at 20”, indicating the presence of a 17-ketosteroid group. A deep-violet color after 10 min. at 20” and also at 0” indicated the presence of a a-ketone group on the saturated A-ring (12). The intensity of the color was similar to that of known androstanedione. The infrared spectrum also suggested the presence of a 3,17-diketone. This fraction was rechromatographed on alumina with carbon tetrachloride: benzene: ether:methyl alcohol. The benzene eluate gave impure crystals m.p. 105-125”, but the Holtorff-Koch modification of the Zimmermann color test and the infrared spectrum were identical with those of 3, IT-androstanedione. Androsterone and Epiandrosterone. The remaining fractions eluted with carbon tetrachloride containing 0.270 absolute ethyl alcohol melted at 160-166”. Treatment with digitonin yielded two fractions both of which gave the characteristic l’i-ketosteroid color test. They were both recrystallized from aqueous acetone. The a-fraction melted at 178-182”; on admixture with a sample of authentic androsterone (m.p. 184-187’), the m.p. was 18@181”. The infrared spectrum conformed with that of androsterone.
444
CHARLES
D.
KOCHAKIAN
AND
H.
V.
APOSHIAN
The p-fraction melted at lTl”-174”; on admixture with a sample of authentic epiandrosterone (m.p. 175-177”), the m.p. was 173-17’7”. The infrared spectrum was identical with that of an authentic specimen of epiandrosterone. In addition to the above, an impure sample of androsterone (i.9 mg.) m.p. 118-132” and one of epiandrosterone (1.3 mg.) m.p. 157-167” were obtained. The infrared spectra of these two compounds were identical with that of their respective authentic compounds. Metaboliles Formed from &,I 7&Androsianediol in a Rabbit Liver Homogenate Preparation. Chromatography on alumina did not result in the separation of a single pure substance. Therefore, all the fractions which gave a positive Holtorff-Koch color test were pooled and rechromatographed. All of the 17-ketosteroids were eluted with carbon tetrachloride containing 0.2yo absolute alcohol. The first portion also gave color tests indicative of a 3-ketone group in a saturated A-ring (12). The infrared analysis suggested a 3,17diketone. On rechromatography on alumina with carbon tetrachloride: benzene : ether: methyl alcohol, the benzene eluate gave impure crystals m.p. 95-125” (3,17-androstanedione m.p. 131-132”), but the m-dinitrobenzene color tests and the infrared spectrum were identical with those of 3,17-androstanedione.
RESULTS Comparison of Rat, Guinea Pig and Rabbit Kidney and Liver Homogenates The incubation of 3ql7/3-androstanediol for 3 hr. with homogenates of liver and kidney of rat, guinea pig, and rabbit resulted in the recovery of 82-97% of the added steroid and the formation of less than 1% of ketonic materials (Table I). The positive 17-ketosteroid color test (10) indicated the oxidation of the 17-hydroxyl group to a ketonic group. Another fraction gave a color test similar to that of androstane3,17-dione. The paucity of metabolites precluded isolation and characterization. In the next study (Table II) the incubation was extended to 6 hr. and an atmosphere of oxygen was used. In an attempt to increase the solubility of the 3cql7@androstanediol, it was homogenized in a PotterElvehjem apparatus with approximately 1 g. of tissue and 5 ml. of buffer prior to addition to the Waring blendor. Guinea pig kidney and liver were used. The extracts of the several experiments were pooled according to tissue. The 3cr,l7/3-androstanediol again proved resistant to oxidation by both the kidney and liver homogenates; 84 and 85% of the original material was recovered and a crude ketonic fraction of only 0.9 and 1.3yo was obtained. Androsterone, 0.3 and 0.5yo, however, was isolated by chromatography and identified. Small amounts of other metabolites were indicated but could not‘be isolated for charac-
METABOLISM
OF
445
ANDROSTANEDIOL
terization. Infrared’ analysis, however, suggested the presence of epiandrosterone in a few fractions of the chromatographed ketonic materials. Comparison of Rabbit Liver Slices with Homogenates Since homogenization is known to decrease the respiration rate of tissues, especially if prepared in the Waring blendor [cf. Refs. (7,8)], TABLE The in Vitro
Metabolism
I
of &,17p-Androstanediol
by the Tissues of Different
Liver”
Guinea
Rat
my.
7%’
Kidney
pig
mg.
247 1.4 0.9
83 “0:;
252 1.7 1.5
Rabbit
Guinea
Rat
pig
Rabbit
%’
Steroid Crystalline steroidsd Ketonic (as 17ketosteroid) Holtorff-Koch (IO) Pinem (11)
Animals”
recovered 84
254
0.6 0.5
85
1.4 1.4
0.5 0.4
145 0.5 0.3
97
192
0.3 0.2
96
0.5 0.2
0.3 0.1
174
87
0.7 0.4
0.4 0.2
0 Adult non-fasted male animals used. Steroids, tissues and 50 ml. of Ringerphosphate buffer mixed in Waring blendor for 2 min. Mixture transferred quantitatively by washing with 50 ml. of buffer to Fernbach flash. Incubation at 37” for 3 hr.; air was the gaseous phase. All equipment and buffer cold and sterile. b Two separate experiments for each animal. The incubation mixtures were pooled prior to extraction. c Per cent of added 3a,l7,%androstanediol. d This fraction was separated from the total extract by dissolving the lipides and cholesterol in petroleum ether.
liver slices were compared (Table III) with homogenates prepared simultaneously from the same animals. The liver slices were only slightly more effective than the homogenate. The degree of oxidation of the 3a,l78-androstanediol, however, 7 The infrared analyses were kindly Institute for Cancer Research.
performed
by Dr. K. Dobriner,
Sloan-Kettering
446
CHARLES
was so small in The metabolites color tests and but only those satisfactory for
D.
KOCHAKIAN
AND
H.
V.
dPOSHIAN
both instances that the difference was not significant. isolated in pure form or indicated as being present by infrared analysis were identical in both experiments, in the slice experiments were obtainable in a state full characterization.
Disuccinate The Incubation of Sql7/3-Androstanediol with Rabbit Liver Homogenate Since 3a,l7&androstanediol was quite insoluble in tissue fluids (7) the disuccinate was prepared and dissolved in the calculated amount of 0.1% sodium hydroxide solution prior to addition to the homogenate. The ester, however, was not hydrolyzed during the incubation period; 92yo of the added 3a,l7&androstanediol disuccinate was recovered in pure form. An impure ketonic fraction of only 5 mg. was obtained which gave color reactions suggestive of the presence of a 3-keto group, but no material could be isolated for identification. TBBLE The in Vitro
II
Metabolism of Sru,l’?‘pAndrostanediol of Guinea Pig Tissue
by Homogenates
Liverb
Kidney
ml.
sz
mg.
%
Incubation Tissue 301,17/.%Androstanediol
4 x 4,800 4 x 150
8 x 15,000 8 x 200 Steroids recovered
Crystalline steroid& 301,17j+Androstanediol Ketonic As 17-ketosteroid [Holtorff-Koch (lo)] Androsterone
1,391 1,348
14.1 5.8
87 84
0.9 0.4
539 509
7.6 3.0
90 85
1.3 0.5
n Procedure as in Table I except that approximately 1 g. of tissue, steroid, and 5 ml. buffer homogenized in Potter-Elvehjem apparatus prior to addition to Waring blendor in an attempt to increase solubility of steroid. *The incubation mixtures of eight separate experiments were pooled prior to extraction. Each experiment contained approximately 15 g. of liver, 200 mg. of steroid, and 100 ml. buffer. c The incubation mixtures of four separate experiments were pooled prior to extraction. Each experiment contained the two kidneys (4.2-5.2 g.), 150 mg. of steroid, and 100 ml. of buffer. d Petroleum ether-insoluble fraction of total fats.
METABOLISM
OF
TABLE In Vitro
Metabolism
447
ANDROSTANEDIOL
III
of Sor,lT’fi-Androstanediol
by Rabbit LiveF
(Slices vs. homogenates) Slices” mu.
4,o
Homogenate nw.
70
Incubation Tissue 3a, 17&4ndrostanediol
8 x 12,000 8 X 200 Steroids
8 X 12,000 8 X 200 recovered
2,049 Crystalline steroidsc 1,890 $3.9 1,342 3q 17&4ndrostanediol 1,047 87.8 5.6 0.4 Androsterone (3.7)d 0.2 0.2 Epiandrosterone (1.3)d 0.1 (i::)d 0.1 3,174ndrostanedione (l.O)d 0.1 154’ Cholesterol 116 (1Procedure as in Table I except that oxygen was the gaseous phase during the incubation. The slices and homogenates were prepared and run simultaneously. There were eight separate experiments for each. b The steroid was homogenized (3) in 5 ml. of the rabbits’ own serum, the rest (11-21 ml.) of the serum was added, and the final fluid volume made to 100 ml. with the Ringer’s phosphate buffer. c Petroleum ether-insoluble fraction of total lipides. d Estimated by the Holtorff-Koch (10) color test. c The greater amount of cholesterol obtained from the experiment with slices was due to the addition of serum to the incubation medium (cf. footnote b).
Control Experiments Rabbit liver (425 g.) and kidney (158 g.) were homogenized and fractionated as in the incubation experiments. The only identified steroid was cholesterol; liver, 751 mg. ; and kidney, 302 mg. DISCUSSION
These experiments indicate that 3a,l7&androstanediol can be oxidized in essentially the same manner by both the liver and kidney of the rat, guinea pig, and rabbit. In every instance, however, the degree of metabolism is small compared with the effect of these tissues on testosterone and A4-androstene-3,17-dione. The integrity of the cell, furthermore, does not make a great difference in the ability of 3ru,17/3androstanediol to resist the in vitro action of at least the liver. A comparison of the rates of metabolism of various androgens in the rabbit liver slice system indicates a rough parallelism with the solubilities of these steroids in tissue fluids (13).
448
CHARLES
D. KOCHAKIAN
AND
H. V. APOSHIAN
The small yields of ketonic metabolites might be due to the fact that t,he chemical equilibria among these compounds favor the formation of the completely reduced steroid, 3a,l7&androstanediol. Androsterone is 3cr,17/GAndrostanediol
ti Androsterone s 3,17-Androstanedione
$ Epiandrosterone
converted by rabbit liver slices to only small amounts of 3,17-androstanedione, and epiandrosterone (14). 3q(,17@-Androstanediol is the main metabolite. Thus, the predominant enzymatic action of the tissues seems to be reduction of the 17-ketone group. This same phenomenon is seen in the relationship of 3,17-androstenedione with testosterone (3,5) and dehydroisoandrosterone with A5-androstene-3P,17/3-diol (15). SUMMARY
3a,178-Androstanediol was metabolized in vitro by homogenates of rat, guinea pig, and rabbit liver and kidneys. The metabolites isolated or indicated as present were androsterone, epiandrosterone, and 3,17androstanedione. The total amount of conversion to these substances was less than 5y0;, and more than 90% of the original material ‘was accounted for. Rabbit liver slices were no more effective than the homogenates. REFERENCES 1. KOCHAKIAN, University 2. KOCHAKIAN,
C. D., in GORDON, E. S., Symposium on Streoid Hormones. p. 93. Wisconsin Press, Madison, 1950. C. D., PARENTE, N., AND APOSEIIAN, H. V., Federation Proc. 8, 214
(1949). 3. CLARK, L. C., JR., AND KOCHAKIAN, C. D., J. Biol. Chem. 170, 23 (1947). 4. GONGORA, J., AND KOCHAKIAN, C. D., Federation Proc. 7, 42 (1948). 5. CLARK, L. C., JR., KOCHAKIAN, C. D., AND LOBOTSKY, J., J. Biol. Chem. 171,493 (1947). 6. STADIE, W. C., AND RIGGS, B. C., J. Biol. Chem. 164, 687 (1944). 7. POTTER, V. R., RECKNAGEL, R. O., AND HURLBERT, R. B., Federation Proc. [3], 10, 646 (1951). 8. SCHNEIDER, W. C., AND HOGEBOOM, G. H., Cancer Research [l], 11, 1 (1951). 9. CALLOW, N. H., Biochem. J. 33, 559 (1939). 10. HOLTORFF, A. F., AND KOCH, F. C., 1. Biol. Chem. 136, 377 (1940). 11. PINCUS, G., Endocrinology 32, 176 (1943). 12. KOCHAKIAN, C. D., AND STEDDIAN, L. A., Am. J. Physiol. 126, 556 (1939). 13. KOCHAKIAN, C. D., Am. J. Physiol. 142, 315 (1944). 14. SCHNEIDER, J. J., AND MASON, H. L., J. Biol. Chem. 175, 231 (1948). 15. SCHNEIDER, J. J., AND MASON, H. L., J. Biol. Chem. 172, 771 (1948).