Comparative placental steroid synthesis. II. C19 steroid metabolism by guinea-pig placentas and fetal adrenals in vitro

813

COMPARATIVE PLACENTAL STEROID SYNTHESIS. XI. CXp STEROID METABOLISM BY GUINEA-PTG PLAGENTAS AND FETAL ADRENALS -IN VITRO

Stanley B. Grossman and Eric Bloch Departments of Gynecology-Obstetricsand of Biochemistry, and of The Rose F. Kennedy Center for Research in Mental Retardation and Human Development Albert Einstein College of Medicine Bronx, New York 10461 Received: 9129172 ABSTRACT

Placental homogenates from guinea-pigs at L6, 20, 35 and 55 days gestation were incubated with 7~-~-dehydroep~androsterone and 4-"C-androstenedfone and analyzed for conversion products by reverse isotope dilution methods. 14C-3a-Hydroxy-5a-androstan-L7-one, '*C-androstane-3a,l7B-dial and 3Handrost-5-ene-3S,l7S-diolwere isolated from homogenates incubated with substrates for 2 hours. 'H,'*C-Testosteronewas isolated from preparations incubated for 15 minutes or with high substrate:tissueratios. Androst-4ene-3,17-dione, 5a-androstane-3,17-dione,SS-androstanedione derivative and c 1B steroid formation could not be demonstrated. These results demonstrate the capacity of guinea-pig placentas to convert dehydroepiandrosteroneand androstenedione to testosterone and to derivatives reduced in ring A (5a) and at carbon 17. The acttvvityof the As-3$-hydroxysteroiddehydrogenase enzyme system appears to have been rate limiting. Homogenates of adrenals from 44-55 day old fetuses converted 4-14C-pregnenol to androst-4-ene-3,17-dione,and 6S- and ll$-hydroxyandrostenedione. A guin pig fetal-placentalunit is postulated, with steroid metabolic characteristic different from the human unit. Both permit reduction of fetal adrenal corti: production and placental.removal of C,, steroids.

21:6

STEROIDS

814

Previous work from this laboratory established or confirmed the presence of 3B-hydroxysteroid dehydrogenase and 4-5 isomerase activities in placentas of several mammalian species (1).

The demonstration of activity rested on the conversion of

dehydroepiandrosterone vitro.

to androstenedione by placental slices -in

Using this experimental system, androstenedione

(or other

A*-3-keto-C 19 steroid) formation could not be shown in late gestation placentas of guinea-pigs, armadillos and rabbits.

Evidence for

androstenediol synthesis by guinea-pig placentas was obtained; this reduction product of dehydroepiandrosterone metabolism was isolated from incubations with armadillo placentas.

These data suggested that

the capacity for oxidative metabolism of Cl9 steroids by guinea-pig placentas near term is restricted in favor of reductive pathways. In agreement with this suggestion is the absence of aromatizing activity in this tissue as reported by Ainsworth and Ryan (2). detailed examination of C

19

A more

steroid metabolism by guinea-pig placentas,

as extended to periods of early and mid-gestation, appeared warranted and is presented in this paper. These experiments were combined with additional work on the capacity of adrenal homogenates of fetal guinea-pigs to synthesize Cl9 steroids, particularly androstenedione.

In an earlier study, conversion

of pregnenolone to llg-hydroxyandrostenedione dione had been shown (3).

and, possibly, androstene-

Within the context of the concept of the

fetal-placental unit (41, adrenal synthesis of an estrogen precursor in a species lacking placental aromatizing activity is of interest.

June 1973

STEROIDS

815

MATERIALS AND METHODS

Steroids (10.4 mCi/pM), 4-14C7-SH-Dehydroepiandrosterone androstenedione (58 pCi/pM), (both from New England Nuclear Corp.,Boston), and 4-l*C-pregnenolone (55.7 pCi/pM) (Amersham-Searle, Chicago), were found to be radiochemically homogeneous as determined by crystallization of aliquots with crystalline, carrier steroid (5). Nonradioactive, crystalline steroids, obtained commercially, migrated as a single zone upon thin-layer chromatography. In addition, the purity of most crystalline steroids was further verified by melting point determinations. Radioactivity Determinations Specific activities and SH/14C ratios were determined by removing duplicate aliquots for counting and for weight determinations (5,6). Radioactivity was determined in a Nuclear-Chicago Mark I liquid scintillation spectrometer, model 6860, adjusted to simultaneously count both 3H and 14C with respective efficiencies of about 36% and 65%. Radioactivity in aqueous fractions was measured by the use of Bray's solution (7) with counting efficiencies of 31% and 54% for 3H and 14C, respectively. Sufficient counts were accumulated to assure a counting precision of + 3%. Chromatography Thin-layer, and paper chromatography were performed by conventional techniques. Silica-gel coated plates were used in all thin-layer chromatoplates. Chromatographic zones containing 14C were located either by autoradiography (8) or by scanning paper chromatograms with a Nuclear-Chicago Actigraph III Scanning System. Quantitation Steroids possessing an a,$-unsaturated carbonyl system in ring A were scanned in a Zeiss spectrophotometer over the range of 225-260 nm, and their concentrations determined by absorbancy at 240 The concentrations of both saturated and As-3B-hydroxysteroids zre estimated by reacting with concentrated sulphuric acid (9,10), and measuring the resulting chromogen at its absorption maximum (between 280 and 370 nm). Tissue Preparation and Incubation Conditions Guinea-pigs were sacrificed, and the desired tissues were immediately placed into ice-cold 0.9% saline solution. Tissues from

21:6

STEROIDS

816

several animals at the same gestational age were combined, gestational age being determined by fetal length, weight and gross development (11,lZ). Placentas, adrenals or endometria were prepared as homogenates in 0.25M sucrose-0.08M nicotinamide0.05M phosphate buffer, according to the procedure of Ryan (13). Two ml aliquots of tissue homogenates were placed into 10 ml Erlenmeyer flasks, followed by the addition of 10 umoles NADP, 5 pmoles NAD, 0.1 mmole glucose-6-phosphate and 4 units of glucose6-phosphate dehydrogenase (Type XV, Sigma Chem. Corp., St. Louis). Tritium or l&C-labeled steroid, suspended in 0.2 ml propylene glycol, was added to the preparation. The final tissue concentrations are given in Table I. Incubation was carried out in a 95% oxygen: 5% carbon dioxide atmosphere. EXPERIMENTAL AND RESULTS A.

Placental C

Steroid Metabolism

-19

In two separate experiments, placental homogenates (endometrial-placental homogenate, flask 1) were incubated with 7-3H-dehydroepiandrosterone plus 4-"C-androstenedione hours (Table I). (sample 4-15).

for two

In flask 4, a 15 minute aliquot was removed The incubated preparations were extracted three

times with purified methylene dichloride.

The total recoveries

of radioactivity added as substrate, with respect to both 3H and '*C, were 42 to 56% in flasks 3 and 4, and 71 to 87% in flasks 1,2, and 5.

The reasons for this variation are unknown.

More

than 85% of the recovered radioactivity was extracted into the organic phase.

The radioactivity remaining in the aqueous phase

plus tissue residues was not further investigated. Phenolic steroids The extracts were partitioned between 20 ml toluene and four times 5 ml N NaOH.

The residues of the NaOH fractions, contain-

ing 0.4-2.0% of the radioactivity in the extracts, upon thin-layer

June 1973

STEROIDS

817

chromatography in the solvent system chloroform:ethanol (19:1), yielded eluates corresponding to estrone and to estradiol-l?g in chromatographicmobflity devoid of radioactivity. Neutral steroids The residues of the toluene fractions were developed on thin-layer chromatoplates in the solvent system benzene:chloroform: methanol (3:7:1). Radioautography of the chromatoplates showed major areas of carbon-24 activity. These and selected other areas were eluted. The sum of the radioactlvities in the eluates from each flask represented 74 to 97% of the applied radioactivity. The residue of each eluate was submitted to chromatography on paper in the solvent system hexanef96% methanol. The data of the two ~hromatographic resolutions suggested the presence of labeled androsterone, epiandrosterone, dehydroepiandrosterone,androstanediol and S-androstenediolin the neutral fractions derived from flasks 2 to 5.

Testosterone was

indicated 2n the fractions from flasks 1, 2 and 4-15. The compounds in each of these eluates were further purified by reverse isotope dilution or chromatographicprocedures. The rigorous identificationof each steroid by purification to radiochemical homogeneity was sought in samples derived from two incubated preparations (Tables 1,24). Whenever crystallizationwas employed, 10 - 40 mg crystalline carrier steroid were added followed by a minimum of three recrystallizations carried out from drfferent non-aqueous solvents. Metabolites Identified Testosterone from flask I was present in the paper chromatographic eluate with a 3H/'4C ratio of 0.39. Forty mg. of

21%

STEROIDS

818

crystalline testosterone were added to the eluate, and the mixture recrystallized to radiochemical homogeneity as the free, acetylated and regenerated free compound (Table 2). The testosterone samples from flasks 2 and 4-15 were acetylated and regenerated as free testosterone prior to carrier addition, crystallization and oxidation (Table 3).

The pre-crystal-

lization steps were based on the purification of testosterone isolated from flask T_, in which radiochemical homogeneity with respect to 'H was achieved after the hydrolysis of testosterone-acetate 2).

(Table

The two placental testosterone samples were purified to radio-

chemical homogeneity with respect to both 3H and 14C (Table 3).

The

rather wide range of analytical values obtained with testosterone from flask 4-15 is explained by the low 3H content (and, therefore, low 'H/14C ratios) in this sample. 14

C-Androsterone from flasks 3 and 4 were brought to experimentally constant specific activities during 7 to 8 crystallizations as free, acetylated and regenerated free androsterone (Table 4).

In contrast, the presence of 3H-androsterone could not

be established after repeated crystallization.

The ratios of dpm

'H mother 1iquor:crystalline fractions during the repeated recrystallizations in each of three analytic steps slightly but consistently exceeded unity (1.06-1.21). "C-Androstanediol

and 3H-5-androstenediol from flasks 3 and

4 were recovered as radiochemically purified steroids (Table 4).

When

an aliquot of 14C-androstanediol was reacted with hydroxysteroid dehydrogenase (from Ps. testosteroni, Worthington Biochemical Co.,

June 1973

STEROIDS

819

Freehold, N.J.), (17,18), 71 and 74% (flask 3 and 4, respectively), of the reacted radioactivity was recovered as androstanedione-X4C* The presence of 'H-androstanedioland of 14C-5-androstenediolcould not be established after repeated crystallization. Other Metabolites The specific activities and SHf'*C ratios of epiandrosterone from flasks 3 and 4 during three recrystallizatfonsand oxidation to androstanedioneprovided presumptive evidence for the presence of this steroid. Insufficient carrier addition and radioactivity precluded conclusive identification. The chromatographiccharacteristicsof labeled steroids separated from the neutral fraction of flask 5 suggested a pattern of metabolites similar to that isolated from flasks 3 and 4.

'H-Dehydroepiandrosteronewas recovered in chromato-

graphic eluates from all placental preparations. The synthesis of ICYandrostanedioneor the presence of residual f4C-androstenedionecould not be demonstrated. As control experiments, boiled placental tissue was incubated in duplicate with 7-SH-dehydroepiandrosterone.Of the recovered "H-radioactivity ~80,85%), 93 and 95% were reisolated as 3H-dehydroepiandrosterone. Epfandrosteronewas the only steroid tested which retained radioactivity after crystallization,representing 0.9% or less of the starting material. In control incubations with 4-14Candrostenedione,reisolated 4-"C-androstenedione represented more than 98% of the recovered L4C-radioactivity(80,87X). B* Adrenal 2x9 Steroid Synthesis An adrenal homogenate (80 mg;30 q/ml) from fetal guinea-pigs

STEROIDS

820

21:6

at 44-55 days gestation was incubated with 2.2 x 10' dpm (5.7 ug) 4-l&C-pregnenolone.

The incubation was for three hours, with

removal of a single aliquot at 30 minutes (designated preparation F-30). Upon extraction, 64% of the radioactivity added as substrate was recovered as free, methylene dichloride soluble steroids.

A

further 22% was extractable into ethyl acetate following hydrolysis with 3,000 units S-glucuronidase (Sigma Chem. Corp., St. Louis) per ml aqueous residue.

In control experiments with two flasks of boiled

maternal adrenal tissue, 86 and 90% of the 14C-activities were extracted into methylene dichloride with less than 1% remaining in the aqueous phase. The residue of the methylene dichloride fraction was developed on thin-layer chromatoplates in the solvent system benzene:chloroform: methanol (3:7:1).

Metabolites of "C-pregnenolone

were located by

radioautography and eluted from the chromatoplate. Two zones (zones 1 and 3), derived from the extracts of the 30 minute (F-30) and 3 hour preparations and absorbing ultra-violet light, migrated with mobilities corresponding to androstenedione and IlS-hydroxyandrostenedione,

respectively.

Rechromatography of the

eluate of zone 1 on paper in the system hexane/96% methanol yielded one zone moving as standard androstenedione.

The identity of zone 1

as androstenedione was confirmed by crystalline carrier addition and crystallization to constant specific activity (Table 5).

Zone 3, upon

rechromatography on paper in the same solvent system, separated into four zones, two migrating as the reference compounds llB- and 6!3-hydroxy-

June1973

STEROIDS

821

androstenedione,respectively. The appropriate crystalline carrier was added to each of the two presumptive hydroxyandrostenediones, and the identification confirmed by crystallization and chromic acid oxidation to the corresponding androstenetrione (Table 5). Dehydroepiandrosteroneor androstenediol formation could not be demonstrated. Several more polar, ultraviolet light absorbing zones were noted on the chromatoplate. Rechromatography on thin-layer chromatoplates or on paper, both as free and acetylated derivatives, and comparison of mobilities with that of authentic compounds on the same plate suggested the presence of cortisol, ll-deoxycortisoland 2cthydroxycortisone. The distribution of radioactivity was androstenedione = 1.06 x 10' dpm, llS-hydroxyandrostenedione= 5.6 x 10' dpm, 613hyc$oxyandrostenedione= 1.18 x 10' dpm, "cortisol" zone = 8.4 x lo4 dpm, "11-deoxycortisol"zone = 2.0 x 10' dpm, "68-hydroxycortisol" zone = 3.3 x lo5 dpm and "2o-hydroxycortisone"zone = 2.4 x 10' dpm. The ethyl acetate soluble extracts obtained after Fglucuronidase hydrolysis, contained chiefly material of "polarity" greater than that of cortisol. A adrenal homogenate (200 mg; 76 mg/ml) from the mothers of these fetuses, incubated with 4-"C-pregnenolone and analyzed for conversio! products exactly as the fetal adrenal preparation, failed to yield C 19

steroids. The chromatographicpattern of presumed C

steroids was 21

similar to that found with the fetal preparation. DISCUSSION 14C-Androsterone, "C-androstanediol and 3H-5-androstenediol were isolated from placental homogenates of 20 (flask 3) and 35 day

STEROIDS

822

21:6

(flask 4) pregnant guinea-pigs incubated with 7-'H-dehydroepiandrosterone and 4- "C-androstenedione.

These metabolites were isolated as radio-

chemically homogenous compounds by reverse isotope dilution procedures. 3H-Androsterone and 3H, x&C-epiandrosterone were probably formed although unequivocal radiochemical purity was not established.

The

distribution of radioactivity upon chromatography and some identification steps indicated the metabolism of 7-'H-dehydroepiandrosterone

and 4-“'C-

androstenedione by 16 and 55 day old placental tissue to be the same as that of 20 and 35 day old tissue. 3H, "C-Testosterone

was isolated from the incubation media

of preparations with mixed endometrial placental tissue (flask l), diminished tissue:substrate ratio (flask 2), or reduced period of incubation (preparation 4-15).

Testosterone synthesis could not be

demonstrated in placental preparations incubated for 2 hours, except for flask 2.

(In a preparation of placental homogenate from 20 day

pregnant guinea-pigs incubated with 4-'*C-androstenedione,

the 14C-

testosterone fraction comprised 36% of the extracted radioactivity after 15 minutes of incubation, but less than 3% after 2 hours). The results demonstrate the capacity of placental tissue of guinea-pigs to convert dehydroepiandrosterone

and androstenedione to

testosterone and to derivatives reduced in ring A or at carbon-17. The synthesis of 3a-hydroxy-5a-androstane

compounds, the absence of

identifiable amounts of 5a-androstanedione and of residual androstendione, and the isolation of testosterone only under conditions of relatively increased substrate concentrations suggest marked 3a- and 17&hydroxysteroid dehydrogenase and Sa-steroid reductase activities in these

June

STEROIDS

1973

823

preparations. In contrast, the activity of the As-3$-hydroxysteroid dehydrogenase enzyme system appears to have been limited:
21:6

STEROIDS

824

metabolic pattern seen in flask 1 as probably primarily that of endometrial tissue.

The guinea-pig uterus placental unit has the

potential capacity to convert C 19 steroidal androgens to reduced and probably conjugated metabolites. "C-Androstenedione,

and 6$- and ll$-hydroxyandrostenedione

(3) were isolated as radiochemically pure conversion products of 4l'+C-pregnenolone incubated with fetal adrenal homogenates.

These

findings confirm the synthesis of llg-hydroxyandrostenedione and establish that of androstenedione, suggested in earlier work (3). The presence of 6g-hydroxyandrostenedione had not been sought in the earlier incubation study (3).

Androstenedione constituted 32% of

the activity in the extract from preparation F-30. The adrenal cortex of the fetal guinea-pig has the capacity for significant C

steroid production from pregnenolone.

This capacity

19

appears to be characterized by the formation of A&-3-keto-C19 steroids. Adrenal cortical preparations from adult (19-22) and fetal (3) guineapigs suggest formation of these C 1g steroids either from 17o-hydroxyprogesterone or the corresponding 17a-hydroxycorticoids.

Our data does

not distinguish between the two possibilities. The results of this study with guinea-pig placentas and fetal adrenals invite comparison with data obtained from other species. extensive and stereo-specific

The

(5a) reduction of androstenedione found

here has been observed in a variety of mammalian placentas, with either testosterone-androstenedione

or progesterone as substrates (23-28).

The

human placenta may be an exception in that reductive metabolism occurs

June 1773

STEROIDS

825

only to a very limited extent (29,30). In common with other shortgestation mammals, and as previously demonstrated by Ainsworth and Ryan (2), aromatization could not be demonstrated in the guinea-pig placentas. The capacity for C1, steroid synthesis found in fetal guinea-pig adrenals has not been shown in such other short-gestation mammals as have been examined (3). It is speculated that a fetal-placentalunit is operative in the pregnant guinea-pig. In relation to C,, steroid metabolism, the unit is characterizedby (a) the production by the fetal adrenal cortex of androstenedione,and hydroxylated androstenedionederivatives, derived either from 17a-hydroxyprogesterone,or 17a-hydroxycorticoids, and (b) the reductive metabolism of these Ao-3-keto-CIg steroids to largely 5a-androstane derivatives by the placenta. If this speculative suggestion is correct, then the characteristicsof this fetal-placental unit differ from those associated with long-gestation (31), including human (4) models. In the latter, CL8 steroids of fetal adrenal origin are aromatized to Cle steroids by the placenta. Both types of units have the capacity to regulate the production of adrenal cortisol, either by diminished synthesis or increased catabolism. This regulatory capacity may play a role in determining the length of gestation (32).

STEROIDS

826

21:(,

ACKNOWLEDGEMENTS

This National Institutes thesis submitted to partial fulfillment Science.

study was supported by Research Grant HD-00420, of Health, U.S.P.H.S. This work is part of a Fairleigh Dickinson University by S.B.G. in of the requirements for the degree of Master of

Trivial names used, in addition to those listed by the 1UPAC:pregnenolone (38-hydroxy-S-pregnen-20-one), androstenedione (4-androstene-3,17-dione), dehydroepiandrosterone (38-hydroxy-5androsten-17-one), 5-androstenediol (5-androstene-38,17B-dial), androstanediol (5a-androstane-3a,l78-dial), androsterone (3c+hydroxy5a-androstan-17-one), epiandrosterone (38-hydroxy-5a-androstan-17-one), androstanedione (5a-androstane-3,17-dione). Other trivial names are substituted derivatives of steroids listed above or by IUPAC (e.g., 6S-hydroxycortisol).

References

1)

Bloch, E. and Newman, E., Endocrinol., 2,

524 (1966).

2)

Ainsworth, L. and Ryan, K.J., Endocrinol., 79, 875 (1966).

3)

Bloch, E., Steroids, 13, 589 (1969).

4)

Diczfalusy, E., Fed. Proc., 23, 791 (1964).

5)

Bloch, E., Steroids, 2, 415 (1967).

6)

Bloch, E., Endocrinol., 74,

7)

Bray, G.A., Anal. Biochem., 1, 279 (1960).

8)

Richardson, G.S., Weliky, I., Batchelder, W., Griffith, M. and Engel, L.L., J. Chromatogr., 12, 115 (1963).

9)

Zaffaroni, A., J. Amer. Chem. Sot., 72, 3828 (1950).

833 (1964).

10)

Smith, L.L. and Bernstein, S. in "Physical Properties of the Steroid Hormones", L.L. Engel, ed., Macmillan Co., N.Y.; 1963, p. 321.

11)

Scott, J.P., Amer. J. Anat., 60, 397 (1936).

12)

Draper, R.L., Anat. Rec., 18, 369 (1920).

13)

Ryan, K.J., J. Biol. Chem., 234,

268 (1959).

June 1773

STEROIDS

827

14)

Bush, I.E., "The Chromatography of Steroids", Pergamon Press, London; 1961, p. 358.

15)

Bush, I.E. and Willoughby, M.L.N., Biochem. J., 67, 689 (1957).

16)

Pincus, G., J. Clin. Endocrinol., 3_, 195 (1943).

17)

Marcus, P.I. and Talalay, P., J. Biol. Chem., 2,

18)

Worthington Enzyme Manual, Worthington Biochem. Corp., Freehold, N.J., 1972, p. 15.

19)

Lipsett, M.B., and Hoekfelt, B., Experientia, 2,

20)

Deshpande, N., Carson, P., and Harley, S., J. Endocrinol., 50, 467, (1971).

21)

Billiar, R.B. and Eik-Nes, K.B., Biochim. Biophys. Acta, l&, 503 (1965).

22)

Spats, and Hoffmann, F., Endocrinol., 2,

23)

Townsend, L. and Ryan, K.J., Endocrinol., 87, 151 (1970).

24)

Rembiesa, R., Marchut, M. and Warchol, A., Experientia, 27, 1081 (1971).

25)

Ainsworth, L. and Ryan, K.J., Endocrinol., 8l, 1349 (1967).

26)

Ainsworth, L. and Ryan, K.J., Endocrinol., 84, 91 (1969).

27)

Ainsworth, L. and Ryan, K.J., Steroids, 14, 301 (1969).

28)

Ainsworth, L., Daenen, M. and Ryan, K.J., Endocrinol., 84, 1421 (1969).

29)

Solomon, S., J. Clin. Endocrinol., Metabolism, 3,

30)

Smith, S.W. and Axelrod, L.R., Steroids, 12, 67S (1968).

31)

Ryan, K.J. in "The Foeto-Placental Unit", A. Pecile and C. Finzi, eds., Excerpta Medica Found., Amsterdam; 1969, p. 120.

661 (1965).

449 (1961).

971 (1966).

762 (1966).

46.9 26.6

l4C

n.d. n.d. n.d. 0.23 2.66

15.7 5.3

-3H

Testosterone

14 16 20 35 55

Gestational age (days)

sH

1.92 1.51 2.34 2.34 1.92

14

C

a4

C

A-diol

-3H

5-A-diol

1.51 1.07 0.98 1.32 1.50 0.155

-3H

14

C

18.6 15.8

+

-3H

DHA

0.86 0.54 0.65 0.86 0.081

0.90

Extract (dpm x 10e6) l4C =H

Epiandrosterone

1.19 1.06 1.19 1.19 1.07

Substrate (dpm x lo-") 111 3H C

+ ( not detected ) (not detected) (chromatographic pattern as in flasks 3,4) 16.2 5.2 (1.24) 8.9 (8.9) (3.2) (14.2) 18.6 4.9 4.9 (1.28) (4.7) (chromatographic pattern as in flasks 3,4) (analyzed for testosterone only )

-

0.55 0.12 0.55 0.58 0.58

Tissue cont.(C) (g/ml)

Metabolites (dpm x lo-') (d) Androsterone

1.15 0.15 1.15 1.50 1.50

Tissue weight per flask (g)

= Homogenate of endometrium containing placental tissue, with placenta less than 5% of entire mass. ;;; = An aliquot from flask 4 removed at 15 minutes of incubation; analyzed only for testosterone. (c) = Final tissue concentration in complete incubation medium. (d) = Minimum values based on final specific activities and not corrected for losses prior to carrier steroid addition and recrystallization procedure. Value for tentatively identified metabolites shown in parenthesis. A-diol=Sa-androstane-3u,178-diol, 5-A-diol=5-androstene-3B,17B-diol,DHA = dehydroepiandrosterone.

2 3 4 5 4-15

1

l(a) 2 3 4 5 4-15(b)

Flask

METABOLISM OF 7-'H-DEHYDROEPIANDROSTERONE AND 4-14C-ANDROSTENEDIONE BY GUINEA-PIG PLACENTAL HOMOGENATES

TABLE I

; v,

0

cn -I M W

1.77

1.70

1.76

1.68

1.74

1.75

1.66

1.74

1.80

1.73

1.74

1.75

1.86

1.60

0.33

0.34

0.39

0.39

0.39

0.38

0.42

0.37

0.44

0.32

0.36

0.39

0.38

0.42

0.58

0.47

CrO oxidation (6,14) to 4-androstene-3,17-dione carried out on separate aliquot of testosterone fraction obtiined from paper chromatogram; paper chromatographic purification in hexane/96% methanol.

0.56

0.64

0.68

0.67

0.73

1.09

0.75

1.92

M.L.

(b)

0.85

Cryst.

=H/'"C

Crystallization of testosterone, of testosterone-acetate (6,14) and of testosterone regeneraged after alkaline hydrolysis (14,15).

PC

0.58

0.68

4th

2nd

0.66

3rd

0.58

0.64

2nd

1st

0.66

0.69

1st

3rd

talc.

M.L.

Cryst.

Cryst.

M.L.

dpm I4C/vmole x lo-'

dpm 3H/umole x 10-9

(a)

Androstenedione(b)

Testosterone

Testosterone acetate

Testosterone

Crystallization

PURIFICATION OF 3H-, l"C-TESTOSTERONE ISOLATED FROM FLASK I (ENDOMETRIAL-PLACENTAL PREPARATION)

TABLE 2

W

,"

CA

er

-

0

:

Z

670

580

600

Second

Third

n.&(c)

550

560 0.19

0.18

0.24

0*20

0.18

0.19

18

19 19 25

23 25

0.084

0.046

0.070 0.083

0.054

0.055

E; m

(a) Placental preparations incubated with 7-Ha-dehydroepiandrosterone and 4-l*C-androstenedionein 3H/"4C ratios of 2.0 (flask 2) and 1.4 (flask 4). (I-J)Aliquot remaved from flask 4 after 15 minutes of incubation. (c) Paper chromatography(PC> of testosterone in hexanef96% methanol; thin-layer chromatography ITLC) of testosteroneacetate /6,14) in chloroform:ethylacetate (4:l); crystallizationsof testosterone regenerated after alkaline hydrolysis (14,151, and TLC of 4-androstene-3,17-dione,obtained after oxidation with chromic s acid (6,141. 3\ (Cl) Crystalline (Cxyst.)and mother-liquor (M.L.) fractions (e) not determined (n,d,)

CrO oxidation; T&Z

590

First

0

:

0,074

0.073

Cryst. M.L.

Crystallization: Calculated 25

M,L.

3H/1"C ratio

:

0.17

Ctyst*

dpmaH/pmole

Flask 4-15 @)

CXI bJ 0

Testosterone

650

0.20

M.L.

Testosterone acetate;TLC

Cry&.

0.20

M*L*

3H/14C ratio

Testosterone;PC

CrystY)

dpm 3H/ltmole

Flask 2

PURXFlCCATIONOF 'H, "'C-TESTOSTERONEISOLATED FROM FLASKS 2 AND 4-15(a)

TABLE 3

1st 2nd 3rd

Acetate

TLC

(700)

(750) (e)

1.01

1.00

1.14 1.03 (228) (222)

Ce)

(420)

1.00 1.03 (400) (390)

(390)

0.82 1.00 0.83

1.02 0.95 1.05 1.00 1.02 0.95

(480)

Flask 3

(197)

Flask 4

0.82 0.93 0.74

0.85 0.95 0.97

(360)

(400)ce) 0.91 1.00 (370) (370)

0.95 0.82 0.98 (440) (450)

digitonide separation")

(500)

Flask 3

(500)

Flask 4

1.00 0.96 0.91 (1000) (1100)

_(d)

(870)

Flask 4

(a) = data presented as ratio of specific activities (S.A.) (dpm radioactivity per umole) in crystalline (Cryst.) to mother liquor (M.L.) fraction. These ratios are to be distinguished from numbers in parenthesis,which are specific activity values. (b) = derivative formation as in Tables 2 and 3. n = 2 for androsterone; n = 3 for diols. 5a-AD = 5aandrostane-3,17-dione. (c) = reacted with 1% alcoholic digitonide solution (16); crystallized androstanediol from o-fraction, and androstenediol from S-fraction after regeneration from digitonide complex with pyridine (16). (d) = not crystallizedprior to digitonide formation. (e) = mother-liquor fractions lost.

So-AD

Alcohol

n-2 n-l n Final (Cryst) S.A. (M.L.)

0.85 0.99 0.96

1st 2nd 3rd

Alcohol

0.92 0.95

(810)

CalculatedS.A.

Flask 3

'H-Androstenediol

PURIFICATION OF "C-ANDROSTERONE, 14C-50-ANDROSTANE-3a,17S-DIOL AND 3H-5-ANDROSTENE-3S,17S-DIOL DURING CRYSTALLIZATION (RATIOS OF SPECIFIC ACTIVITIES)(a) I

TABLE 4

W c1

co

5 'UJ

& !z 3 n

510 510 500

1340 1310 1210

ML (n-l) (dpmlmg)

Cryst (n) (dpm/mg)

ML (n) (dpm!mg)

970(d)

1030

1020

1000

980

1060

6300(e)

6600

6100(')

6900

5.87~10"

WI

u

co ,.I Es

Eluates from sequential chromatography on thin-layer chromatoplates (TLC;benzene:chloroform: methanol (3:7:1)) and on paper (PC-l, PC-2; hexane/96% methanol). Recoveries not corrected for aliquots taken for radioactivity and weight determinations. Used 50% of androstenedione and 6$-hydroxyandrostenedione eluates for crystallization. Carrier steroid addition: 20 mg androstenedione and ll@hydroxyandrostendione, and 3.82 mg GS-hydroxyandrostenedione. Specific activities (dpm/mg) of crystalline (tryst) and mother liquor (ML) fractions during final two (n-l, n) recrystallizations. Crystallized twice from ethyl-acetate; ethanol, once from benzene: ethanol and from methanol_, each. (b) not carried out. Single crystallization N (cl Cd) Oxidized to 4-androstene-3,11,17-trione (6,14) ;recovered from TLC developed in chloroform:ether:ethanol 'g (90:10:2) (e) Oxidized to 4-androstene-3,6,17-trione; recovered as in (d).

(a)

_(b)

470

1300

Cryst (n-l) (dpm/mg)

CrOB oxidation (dpm!mg)

450

1310

2.83~10~

1,.21x10'

1.92x104 -

1.55x105

2.71~10~

68-Hydroxyandrcr stenedione

3 hour incubation

llB-Hydroxyandrostenedione

30 minute Incubation

Carrier (talc. dpm/mg)

_(b)

5.34x104

PC-1 eluate (dpm):

PC-2 eluate (dpm)

7.56~10"

3 hour Incubation

TLC eluate (dpm):

Procedure(a)

Androstenedione

IDENTIFICATION BY CHROMATOGRAPHY AND CRYSTALLIZATION OF ANDROSTENEDIONE, 11BHYDROXYANDROSTENEDIONE AND 68-HYDROXYANDROSTENEDIONE ISOLATED FROM FETAL ADRENAL HOMOGENATES INCUBATED WITH 4-i&C-PREGNENOLONE

TABLE 5