Preparation of dehydroepiandrosterone, testosterone and progesterone antigens through 7-carboxymethyl derivatives: Characteristics of the antisera to testosterone and progesterone

235

2459

PREPARATION OF DEHYDROEPIANDROSTERONE,

TESTOSTERONE

AND PROGESTERONE ANTIGENS THROUGH 7-CARBOXYMETRYL DERIVATIVES

: CHARACTERISTICS OF THE ANTISERA

TO

TESTOSTERONE AND PROGESTERONE

Danible Duvall , Bernard Desfosses

and Romeo Emiliozzi

3

1. Laboratoire de Chimie Physique Organique U.E.R. Domaine mediterraneen, Part Valrose 06034 Nice Cedex 2. U.E.R. Bion&dicale des Saints-Peres, 75006 Paris 3. U.E.R. Mddecine et Centre A. Lacassagne 06 Nice

Received :

3-16-v ABSTRACT

Dehydroepiandrosterone, testosterone and progesterone 7-car oxymethyl derivatives were prepared : 7a and 78 epimers were separated and coupled to bovine serumalbumin. Preliminary studies of the antis, ra induced by these antigens showed that they have high affinity and gj od specificity. IMTORDUCTION Since the successful production of anti-steroid antisera (I), more

pecific antibody preparations have been sought. Midgley and Nis-

wende, (2) and then Lindner et al. (3) pointed out that the steroid must be linked to the

immunogen carrier at a site remote from the functional

groups of the steroid. The application of this principle yielded several specific antisera (4,5,18) ; since then numerous reports have supported its validity. We (6-16) and others (17-39) have varied the site of conjugation of the steroid by using positions C-l, C-6, C-7 and C-15. This produced still more specific antibodies , probably because the functional groups which,remain free or the steroid in its entirety act as antigenic determinants. With regard to the 4-en-3-oxo series, we have described a general method (14-16) of putting a carboxymethyl chain on C-7 : bromination by N-bromosuccinimide

(NBS) of the 3-ethyleneketal-5-ene steroid

followed by malonic condensation, saponification and derarboxylation led to 7-carboxymethyl derivatives in both orientations. Epimers (7ctand 7&)

volume 35, Number 3

S

TE&OIDII

March, 1980

236

0

HO 62

III

R=H;

R=Et

IVa;

Ivb

OH

Vlla;

OH

vllb

Villa

; Vlllb

CH3 L,=o

Xla

; Xlb

X.lla;

a

H

CH2C02H

b

CH,CeH

H

Xllb

S

TDROSDrn

237

were separated by chromatographicpartition. After de-blocking, these haptens were coupled to bovine serum albumin (BSA) in order to obtain antigens. This communication describes the preparation of novel haptens derived from dehydroepiandrosterone(DHA), testosterone and progesterone, the preparation of their BSA-conjugates, the generation of the antisera and their application in radio-immunologicassays. EXPERIMENTAL

All melting points were determined on a Leitz microscope with heating stage and have not been corrected. Infrared spectra were obtained on a Perkin Elmer 254 (KBr pellets). Nuclear magnetic resonance spectra were obtained from a Varian A 60A with tetramethylsilaneas the internal standard. Microanalyses were done by Service de Microanalyse du C.N.R.S. 94-Thiais, Prance, Preparation of haptens.

The reaction of ethyleneglycolwith 4-en-3-oxo steroids gave the corresponding cyclic ketal-5-ene. Other functional groups were also blocked and the successive stages were carried out on : - the 3S-acetoxy-5-androsten-17-one-17-ethyleneketal I : m.p. 142"-143°C (44) for the synthesis of DHA derivatives - the 17S-acetoxy-5-androsten-3-one-3-ethyleneketal V : m.p. 203'C (45) for the synthesis of testosterone derivatives - the 5-pregnene-3,20-dione-3,20_bisethyleneketal IX: m.p. 179"-181°C (46) for the synthesis of progesterone derivatives. Bromine was introduced onto position 7, using N-bromosuccinimide (NBS), and was allowed to react immediately. Both epimers were obtained in about the same proportions. They were distinguished and identified by NMR spectra. In fact, thecoupling constant of protons 6 and 7 permitted the assignation of configuration according to Karplus (40). In the 7ctbromo derivative the dihedral angle between H-C6-C7 and C6-C7-H was about 35" and a doublet (.I=5Hz) was observed for the ethylenic H . In the 78 bromo derivative the dihedral angle was about 85" and a larie singlet was observed. In contrast to our results, other authors (41) purified the bromo derivative by crystallizationand they described only the Q epimer. Condensation with the diethyl sodiomalonatewas carried out in anhydrous tetrahydrofuran ; the mechanism proceeded according to SN2. A pure isolated o form of the bromo DHA derivative gave exclusively the corresponding S substituted compound. The mixture of a and $ bromo derivatives in equal proportions gave equal proportions of 01and (3epimers of the reaction products.

6L

238

'SFTD&OXD#B

Saponificationby KOH gave a mixture of the corresponding substituted malonic diacids (when the B epimer was predominant) and malonic acid monoethyl ester (when the a epimer was predominant). Decarboxylation of this mixture gave an acid fraction in which the quantities of 7a and 78 epimers were in a ratio of If9 to 218, and an ethyl ester fraction in which their ratio was approximately 7/3 ; this fraction was saponified. The carboxymethyl derivatives were separated by chromatography,and each of them was identified by NMR ; as seen above, the coupling constant of protons 6 and 7 determined the configuration. A doublet (J=5 Hz) was observed for the 7a-carboxymethyl derivative and a large singlet for the 7@- carboxymethyl derivative. Treatment by dilute acids permitted the return to the functional groups initially blocked. Preparation Of antigens. Each hapten was condensed with BSA according to the method of Vaughan (42). The number of steroids incorporated per mole of BSA was measured by ultraviolet absorption at 248 nm and for DHA by utilising nitrotropone according to Tamaoki et al. (43). Between 20 and 25 moles'of steroid for each mole of BSA were generally incorporated.

7a-and 7pCARBOXYMETHYL

DEHYDROEPIANDROSTERONE

1

3@-Acetoxy-7a-and7~-bromo-5-androsten-17-one-17-ethyleneketal (II) A solution of I (10.7 mmoles) in Ccl4 (IOOml) was refluxed. NBS (13 mmoles) was rapidly added, and the mixture was heated and irradiated with a photoflood lamp of 500W for 10 minutes. After cooling, SUCcinimide was filtered out and CC1 evaporated under vacuum. 4 N.M.R. (CC14) ppm : 7o epimer

:

0.83 (s, 3H, 18-CH3), 1.05 (s, 3H, 19-CH3), 1.97 (s, 3H, acetoxy CH3),3.80 (s, 4H, ketal-CH2-CH2-), 4.4 (m, IH C-3H), 5.70 (d, J=5 Hz, IH, C-6 H)

78 epimer

:

0.85 (s, 3H, 1%CH3), 1.10 (s, 3H, 19-CH3), 1.97 (s, 3H, acetoxy CH3), 3.80 (s, 4H, ketal-CH2-CH2-), 4.4 (m, IH, C-3H), 5.83 (s, lH, c-6 H).

and 3B-Hydroxy-7-carboxymethyl-5-androsten-l'I-one-J7-ethyleneketal (III R=Et, R=H). In an apparatus with reflux condenser and protected frommoisture, NaH (0.025 mole) (50/60 weight % in oil) and anhydrous tetrahydrofuran (THF) (40ml) were introduced, Freshly distilled ethylmalonate (0.03 mole) was slowly added, and the mixture was agitated at room temperature for l/2 an hour. Then the crude bromo compound, dissolved in anhydrous

S

TIIROSDb

THF (lOOm1) was added. After one day of magnetic agitation at ZOO, THF was evaporated under vacuum. The residue was dissolved in ethanol (170 ml), KOH (16g) and water (2Oml) were added and the mixture was shaken at 45' for 48 hours. After evaporation of ethanol, water was added (500 ml), and extracted with ether (3 x lOOm1). The aqueous phase was acidified with hydrochloric acid, then extracted with ethyl acetate (4 x 15Oml). The extracts were washed with a saturated aqueous solution of sodium chloride until the wash-water was neutral, then dried over sodium sulfate, and evaporated under vacuum. The residue (a mixture of diacid and monoacid ethyl ester) was dissolved in pyridine (lOOm1). After 48 hours refluxing, the pyridine was evaporated ; a 5% aqueous solution of potassium hydroxide (3OOml) was added, and extracted with ether (3 x 100ml). The fractions of the ethereal phase, washed with a dilute solutionof hydrochloricacid,then withater,dried, and evaporated under vacuusn,gave 2.3g (yield 52% from I) of a mixture of 7ol-and7S-ethoxycarbonylmethyl steroids (III R=Et) (7OWaand 30% @)_ The aqueous phase was acidified with hydrochloric acid, then extracted with ethyl acetate (4 x 150x11).The fractions of the organic phase were washed with a saturated aqueous solution of sodium chloride until the wash-water was neutral, then dried, and evaporated under vacuum ; this led to the isolation of 1.2g (yield 28% from I) of a mixture of 7a-and 7@-carboxymethylsteroids (III R=H) (lS%cl and 85% g).

3~-H~droxy-7~-carbox~ethyl-S-androsten-l7~ne (LVa). Saponificationof asters (III R=Et) by alcoholic potassium hydroxide for 12 hours at 45" gave a mixture of the corresponding acids. These products were dissolved in acetone (18Oml) and a 4% aqueous solution of sulfuric acid (3Oml) and refluxed for 20 minutes . After evaporation of acetone the residue was taken up with ethyl acetate (2OOml), washed with water, dried and evaporated to dryness. Several crystallizations from methanol gave IVa m.p. 280-281'C. Anal. Calcd. for

I.R. 7 max.cm-1

C21H3004

C 72.79

found

72.3

H

8.74

0

18.47

8.5

0

18.9

: 3550-2500 (OH and carboxyl OH), 1740 (C=O) 1690 (carboxyl C=O)

N.M.R. (DMSO d6) 6 ppm : 0.80 (s, 31-1, 18-CH3), 0.98 (s, 3R, 19-CH3), 3.25 (m, lH, 3~R&5.42

(d,.I=5Hz,lH,C-6H).

3f3-Hydroxy-7g-carboxymethyl-5-androsten-l7-one (IVb). The acidic fraction (III R-H) was deketalised as described above (reflux 20 minutes). Several crystallizationsfrom a mixture of methylene chloride-chloroformgave IVb m.p. 247-255'C.

Anal. Calcd. for

1.R.T max.cm"1 N&R.

'2YH3004

C 72.79

found

72.5

B

8.74 8.5

0

18.47 18.6

: 3600-2400 (OH and carboxyl OH), 1745 (C=O), 1680 (carboxyl C=O)

(DMSO d6) 6 ppm : 0.80 (s, SI& 18-(X3), 0.95 (s, 3R, 19'cR3), 3.25 (m, lH, 3a-R), 5.15 (s, TEE,C-6 H)

__ 7wand 7B-~ARBOX~ETHYL TESTOSTERONE

17~-Acetoxy-7-bxomo-S-androsten-3-one-3-ethyleneketal (VI). To a solution of V (10.7 nraofes)in CC14 (lOOml), anhydrous potassium acetate (8g)waa added, then the mixture was refluxed, NBS (13 moles) was rapidly added, and the mixture was heated and irradiated with a photoflood lamp of SOOW for 10 minutes. After cooling, succinimide and potassium acetate were filtered out. The CC1 solution was washed with NaHC03 and dried over R2C03. The solution w,4sevaporated to dryness under vacuum,

lf&-Hydroxy-7ol-carbox~etkyl-5-andros~en-3-one-3-~thyleneketal (VIIa) and 17B-nydroxr7t3-carboxymethyl-5-androsten-3-one-3-ethyleneketal (VIIb) The condensation of the bromoderivative,withthe diethyl sodiomalonate, the saponificationand decarboxylationwwe carried out according to the method described for the preparation of the DRA derivatives. The following products were isolated : l.lg (yield 24% from V) of the mixture of i&and 7@-ethoxycarbonylmethy1 steroids (7OXa and 30% 6). Saponificationof these esters by alcoholic potassium hydroxide for 12 hours at 45' gave a mixture of the corresponding acids, and 0.67g (yield 16% from V) of the mixture of &-and -I$-carboxymethyl steroids (VIIa and VIIb) (ZO%ol and 80% 8). The epimers were separated by chromatography (of each of the two acid fractions) over a silica gel column (activity II), Benzene-ether 8/2 elution was used. (VIIa)

M“ I.R.

at m/e 3max.cm -1 :

390 3600-240~ (OH and carboxyl OH), 1700 (carboxyl C-O)

N.M.R. (CDC13) 6 ppm : 0.77 (s, 3R, 18+X3), 1.05 (s, 3R, 19-CS3), 3.67 &I, ID.,17a-R), 3.97 (s, 4H, ketal-CH2-CH2-), 5.52 (d, .I=5Hz, lH, C-6 R).

S (VIIb)

YW?D=OIDI

241

390

M+' at m/e -1 I.R. 7 max.cm :

3550-2400 (OH and carboxyl OH), 1705 (carboxyl C=O>

N.M.R. (CDC13) 6 ppm :

0.77 (s, 3H, 18-CH3), 1.00 (s, 3H, 19-CH3), 3.67 (m, lH, 17~~H), 3.97 (s, 4H, ketal-CH2-CH2-), 5.25 (s, lH, c-6 H).

178-Hydroxy-7a-carboxymethyl-4-androsten-3-one (VIIIa). The deketalisation of VIIa, carried out as described above (reflux 80 minutes), gave VIIIa, after several recrystallizationsin methanol, m.p. 226-229°C. Anal. Calcd. for -1 1.R.T max.cm

C21H3004 found

c 72.79

H

72.6

8.74

0

18.47 18.7

8.7

: 3600-2400 (OH and carboxyl OH), 1720 (carboxyl C=C), 1680 (C=O), 1620 (C=C>

N.M.R. (DMSO d6) S ppm : 0.70 (s, 3H, 18-CH3), 1.17 (s, 3H, 19-CH3), 3.42 (m, lH, 17~H), 5.55 (s, lH, C-4 H).

178-Hydroxy-78-carboxymethyl-4-androsten-3-one (VIIIb). The same treatment was applied to VIIb, and after several recrystallizationsin benzene gave VIIIb, m.p. 200-205'C. Anal. Calcd. for -1 I.R. T max.cm

'21H3004 found

c 72.79 72.5

H

8.74 8.7

0

18.47 18.6

: 3600-2500 (OH and carboxyl OH), 1725 (carboxyl C=O), 1600 (C=O), 1620 (C=C)

N.M.R. (DMSO d6) S ppm : 0.67 (s, 3H, 18-CH3), 1.15 (s, 3H, 19-CH3), 3.38 (m, IH, 17o-H), 5.59 (s, lH, C-4 H) 7a-and 7pCARBOXYMETHYL

PROGESTERONE

The same procedure was used as for the preparation of testosterone derivatives. Yields were of the same ordre, 7a-and 78-Bromo-5-pregnene-3,20-dione3,20-bis-ethyleneketal(X). N.M.R. (CC14) 6 ppm : 7a epimer

: 0.77 (s, 3H, 18-CH3), 1.02 (s, 3H, 19-CH3) 1.21 (s, 3H, 27-CH3), 3.83 (s, 8H, ketal 2-CH2-CH2-), 5.6 (d,J=5Hz,lH, C-6 H).

S

242

78 epimer

T=EOSDCI

: 0.77 (s, 3H, 18-CH3), 1.08 (s, 3H, J9-CH3), 1.21 (e, 3H, 21-CH3), 3.83 (s, 8H, ketal 2-CH2-CH2-), 5.42 (S, IH, C-6 H).

7~-Carboxymethyl-5--pregnene-3,20-dione-3-ethyleneketal 416

M+* at m/e I.R. vmax.cm

(XIa).

-1

: 3600-2550 (OH and carboxyl OH),1725 (carboxyl C=O>, 1700 (C=O)

N.M.R. (CDC13) S ppm : 0.65 (s, 3H, 18-CH3), 1.04 (s, 3H, 19-CH3), 2.13 (s, 3H, 21-CH3), 3.95 (s, 4H, ketal-CH2CH2), 5.53 (d, J=5 Hz, IH, C-6 H). 78-Carboxymethyl-5-pregnene-3,20-dione-3-ethyleneketal 416

M'. at m/e I.R. 7 max.cm

(XIb).

-1

: 3500-2500 (OH and carboxyl OH), 1725 (carboxyl GO), 1704 (C=O)

N.M.R. (CDC13) 6 ppm : 0.65 (s, 3H, 18-CH3), 0.98 (s, 3H, 19-CH3), 2.13 (s, 4H, 21-CH3), 3.95 (s, 4H, ketal-CH2CH2), 5.25 (s, lH, C-6 H). 7a-Carboxymethyl-4-pregnene-3,20-dione m.p.

(XIIa).

206-210Q C (benzene)

Anal. Calcd. for

C23H3204 found

I.R. j max.cm

-1

c 74.15

H

74.0

8.67

0

8.7

17.18 17.2

: 3200-2450 (OH and carboxyl OH), 1730 (carboxyl C=O), 1705 (C=O), 1655 (conjugated C=O), 1615 (C=C)

N.M.R. (DMSO d6) 6ppm:

0.61 (s, 3H, 18-CH3), 1.18 (s, 3H, 19-CH3), 2.07 (s, 3H, 21-CH3), 5.57 (s, lH, C-4 H).

7B-Carboxymethyl-4-pregnene-3,20_dione m.p.

(XIIb).

195-198°C (acetone-hexane)

Anal. Calcd. for

C23H3204 found

I.R. 3 max.cm

-1

c 74.15 74.1

H

8.67 8.6

0

17.18 17.3

: 3600-2550 (OH and carboxyl OH), 1730 (carbtixyl C=O>, 1710 (C=O), 1645 (conjugated GO),

N.M.R. (DMSO d6)6ppm:

1620 (C-C)

0.58 (s, 3H, 18-CH3), 1.15 (s, 3H, 19-CH3), 2.07 (s, 3H, 21-CH3), 5.6 (s, IH, C-4 H).

IM!WNOLOGICAL PROCEDURES - Immunization : Antisera against antigens of testosterone and progesterone were obtained from rabbits, utilizing the technique of Vaitukaitis et al. (47). Blood samples were drawn from the marginal ear vein one week after each injection. Studies were performed on serum obtained 24 weeks after the first injection. Undiluted sera were stored at -2O'C. - Characterization of antisera. Radioinmunoassay : Conventional immunoassay procedure was followed : the buffer used in the assays was a O.lM sodium phosphate buffer pH7 containing 0.9% sodium chloride, 0.1% sodium azide and 0.1% gelatin. Into each assay tube lOOn antiserum diluted as required to bind 50% tritiated homologous steroid (19,000 dpm = 45 pg for testosterone and 20,000 dpm = 34 pg for progesterone) was incubated with varying amounts of the unlabeled hormone or heterologous steroid. To this mixture was added 1OOul of labeled homologous steroid (38,000 dpm for testosterone and 40,000 dpm for progesterone) and lOOn phosphate buffer. After mixing, the assay tube was kept at 4°C for 3 hours, 30 minutes. The bound and free steroids were separated by dextran-coated charcoal by the following procedure : Iml of phosphate buffer solution pH7 with 0.25% w/v Norit A activated charcoal and 0.025% w/v dextran was added to each tube. The mixture was stirred for 30 sec. then incubated for 70 minutes at 4'C, centrifuged at 2,500g at 4'C for 10 minutes ; the supernatant was decanted into a counting vial containing lOm1 of Bray's solution (48), and counted for 10 minutes in a liquid scintillation spectrometer (Packard Tri-Carb Model 3375). Determinations were done in duplicate. Standard curves were plotted as percentages of labeled hormone at each steroid dose compared to the labeled hormone bound when no radioinert steroidwas added. Association constants were determined from Scatchard plot (49) or at equilibrium by a non-graphical method (50).

- Specificity studies. The test investigated was the classical test described by Abraham (51) where the degree of cross-reaction is expressed numerically on the basis of the mass of each steroid required to produce 50% displacement of labeled hormone. Percent cross-reaction equals (x/y) x 100 where : x = mass of hormone required to displace 50% of bound homologous 3H hormone , y = mass of cross reacting steroid required to displace 50% of bound 3H hormone.

S

244

%?DP&OXDI RESULTS

For each antigen the sera of two rabbits were tested as follows : - antiserum dilution at which 50% of labeled hormone is bound - mass of radioinert hormone inhibiting 50% of bound radioactivity - equilibrium association constant KA at 4°C - specificity determined as described above. The upper parameters are mentioned in Table I and II for testosterone antibodies and Table III and IV for progesterone antibodies. DISCUSSION

The antigens derived from haptens on which the carboxymethyl chain at position 7 is orientated in both directions gave rise to antisera with fairly good specificity. The characteristicsof these antisera were, moreover, similar to those of the antisera describedbylindneretal. (22,26) i.e., derived form 7a-carboxymethylthioetherand 7a_carboxyethylthioether steroids-. Where testosterone is concerned, the percentages of the crossreactions are lower in our series for the androstanediols (l-3% as compared with 8-17%), and higher for androstenedione (Y-12% as compared to the percentages of cross-reac1%). With 17B-hydroxy-5a-androstan-3-one, tions are the same, and only when the chain was in position 15 were they greatly diminished (12,36). Where progesterone is concerned, the percentages of the cross-, reactions were lower in our series for 17-hydroxyprogesterone(0.3 and 0.7% as compared to IS%), and for 21-hydruxy-4-pregnene-3,2r)-dione (2% as compared with 7%) and higher for 5a-pregnane-3,20-dione(33-53X as compared with II%), and 5f3-pregnane-3,20-dione (6.7-23% as compared with 2%).

S TABLEI

-

245

TIEIIROXDI)

&HA~~TERXSTICSOF ANTISERA (INDUCEDBY 7a AND 78 CARBOXYMETHYLTESTOST~RONE-BSA)

"antisera 7cl"

"antisera 76" rabbit

Antiserum dilution at which 50% of (3H) tracer is bound

inhibitipg 50% of (3H) tracer binding

constant at 4'C

TABLE II

-

PERCENTAGE CROSS-REACTION OF ANTITESTOSTERONEANTISERA

STEROIDS

Testosterone 17$-Rydroxy-So-androstan-3-one 4-Androstene-3,17-dione 17&=Hydroxy-4-androsten-3-one 5&-Androstane-3& 17g-diol So-Androstane-3o, 17$-diol ~ebyd~oe~iaudros~ero~e Androsterone Progesterone 17-I$ydroxyprogesterone 5E+Pregnane-3os,2Oo,-diol Cortisol Estradiol-17B

"antisera 7a" rabbit 11 14 100 41 9

44 11 < 0.1

1.7 3.2

1.1 1.5 < < < < < < <

0.1 0.1 0.1 0.1 0.1 0.1 0.1

"antisera 78" rabbit 18 17

S

246

TABLE III -

-l?BEOIDS

CHARACTERISTICS OF ANTISERA (INDUCED BY 703 AND 7~ CARBOXYMETHYLPROGESTERONE-BSA)

"antisera 70" rabbit 22 25

"antisera 76" rabbit 26 28

Antiserum dilution at which 50% of (3H> tracer is bound

Mass of unlabeled progesterone inhibiting 50% of (3H> tracer binding

1/11,000

l/5,000 l/9,400

180 pg

94 Pg

113,500

190 Pg

80 pg

Equilibrium association constant at 4°C

TABLE IV

-

2 .7 lOgM-l

2.8 10'6'

PERCENTAGE CROSS-REACTION OF ANTIPROGESTERONE ANTISERA

STEROIDS

"antisera 7cl" rabbit 22 25

Progesterone 5u-Pregnane-3,20-dione 48 5B-Pregnane-3,20-dione 23 3S-Hydroxy-5-pregnene-ZO-one 7 lla-Hydroxy-4-pregnene-3,20-dionc6 17-Hydroxyprogesterone 0.3 21-Hydroxy-4-pregnene-3,20-diont2 Corticosterone 17a,21-Dihydroxy-4-pregnene3,20-dione Cortisone Cortisol 5S-Pregnane-3o,ZOu-diol Testosterone Estradiol-178

"antisera 78" rabbit 26 28

100

100 43 6.7 3.6 2 Q.2

(1.8

33 17 7 2 0.7 2

53 17 10 2 0.3 1.3

< 0.2

< 0.2

c < < < < <

< < < < < <

0.2 0.2 0.2 0.2 0.2 0.2

0.2 0.2 0.2 0.2 0.2 0.2

S

TmEIBOIDS

247

Surprisingly, there was little difference in the specificity of antisera derived from haptens where carboxymethyl chains were epimeric. In this connection, one must remember the results already published concerning the same carboxymethyl chain at the position 6 in its two epimeric orientations

(28, 34, 35).

ACKNOWLEDGMENTS The authors are grateful to Pr E.E. Baulieu who suggested this work. We thank Pr M. Azzaro, Pr M.F. Jayle (t), Dr M. Rouillard, and the Scientific Council of the Faculty of Medicine of Nice for their material help, and Roussel-Uclaf for generous gifts of steroids. This work is a part of the Ph.D. of one of us (D.D.) and it has been supported by Grants of Institut National de la Sant6 et de la Recherche M6dicale (No 76-1-134-4 and No 58-78-90-006) and of F6ddration Nationale des centres de lutte contre le cancer. REFERENCES 1. 2.

3.

4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17.

Erlanger,B.F., Borek,F., Beiser,S.M., and Lieberman,S., J.BIOL. CHEM. 228, 713 (1957). Midgley,A.R., and Niswender,G.D., STEROID ASSAY BY PROTEIN BINDING (Geneve 2nd Karolinska Symposium on Research Methods in Reproductive Endocrinology, Editor Diczfalluzy) ; ACTA ENDOCRIN. KBH, ]47, 320 (1970). Lindner,H.R., Perel,E., Friedlander,A., RESEARCH ON STEROIDS, Editors M.Finkelstein, C.Conti, A.Klopper and C.Cassano, Pergamon Press, 4, 197 (1970). Dray,F., Terqui,M., Desfosses,B., Chauffournier,J.M., Mowszowicz, I ., Kahn,D., and Jayle,M.F., C.R.ACAD.SC.PARIS, 273, 2380 (1971). Exley,D., Johnson,M.G., and Dean,P.D.G., STEROIDS, 2, 605 (1971). Condom,R., Duval,D., and Emiliozzi,R., C.R.ACAD.SC.PARIS, 276, Serie C, 303 (1973). Condom,R., and Emiliozzi,R., STEROIDS, 23, 483 (1974). Serie C, Linder,M., and Emiliozzi,R., C.R.ACAD.SC.PARIS, 2, 807, (1974). Condom,R., C.R.ACAD.SC.PARIS, 281, Serie C, 139, (1975). Linder,M., Desfosses,B., and Emiliozzi,R., STEROIDS, 2, 161 (1977). Condom,R., and Desfosses,B., J.STEROID BIOCHEM., 8, 1085 (1977). Condom,R., and Desfosses,B., J.STEROID BIOCHEM., 4, 1165 (1977). Condom,R., and Desfosses,B., and Habrioux,G., J.STEROID BIOCHEM., 8, 1263 (1977). Duval,D., Azzaro,M., and Emiliozzi,R., C.R.ACAD.SC.PARIS, 284, Serie C, 77 (1977). Duval,D., C.R.ACAD.SC.PARIS, 284, Serie C, 1033 (1977). C.R.ACAD.SC.PARIS, 285, Duval,D., Condom,R., and EmilEzi,R., Serie C, 281 (1977). De Lauzon,S., Desfosses,B., Rombauts,P., Dray,F., Uhrich,F., and Jayle,M.F., C.R.ACAD.SC.PARIS, 274, Serie D, 3023 (1972).

18.

Dean,P.D.G., Exley,D., and Johnson,M.W., STEROIDS, 18, 503 & 605 (1971). 19. Riley;W.J., Smith,E.R., Robertson,D.M., and Kellie,A.E., J.STEROID BIOCHEM., 2, (1972). 20. Spieler,J.M.,Webb,R.L., Saldarini,R.J.,and Coppola,J.A., STEROIDS, 2, 751 (1972). 21. Lindner,H.R., Perel,A., Friedlander,A., and Zeitlin,A., STEROIDS, 19, 357 (1972). 22. %nstein,A., Lindner,H.R., Friedlander,A., and Bauminger,S., STEROIDS, 2, 789 (1972). 23. Jeffcoate,S.L., and Searle,J.E., STEROIDS, 19, 181 (1972). 24. Kuss,E., and Goebel,R., STEROIDS, E, 509 (1972). 25 Kutas,M., Chung,A., Bartos,D., and Castro,A., STEROIDS, 20, 697 (1972). 26. Bauminger,S., Lindner,H.R., and Weinstein,A., STEROIDS, 1, 847 (1973). 27. Hillier,S.G., Brownsey,B.G., and Cameron,E.H.D., STEROIDS, 21, 735 (1973). 28. Jones,C.D., and Mason,N.R., STEROIDS, c, 323 (1974). Den Hollander,F.C.,Van Weemen,B.K., and Woods,G.F., STEROIDS, 29. 2, 549 (1974). Bosh, A.M.G., Den Hollander,F.C., and Woods,G.F., STEROIDS, 23, 30. 699 (1974). Bauminger,S., Kohen,F., and Lindner,H.R., J.STEROID BIOCHEM., 5, 31. 739 (1974). Raynaud,J.P., Azadian-Boulanger,G.,and Bucourt,R., J.PHARMACOL. 32. PARIS, 51, 1, 27 (1974). Narashiz Rao,P., Shain,S.A., and Axelfrod,L.R., J.STEROID BIO33. CHEM., 5, 433 (1974). 34.a. Tsuji,A., Smulowitz,M.,Liang,J.S.C., and Fukushima,D.K., STEROIDS, 2, 739 (1974). 34.b. Nishina,T., Tsuji,A., and Fukushima,D.K., STEROIDS, 2, 861 (1974). Jones,C.D., and Mason,N.R., STEROIDS, 25, 23 (1975). 35. Narashima Rao,P., Moore Jr,P.H., STEROIDS, 28, 101 & 110 (1976). 36. Exley,D., and Baker,T.S., J.STEROID BIOCHEM., 1, 109 (1976). 37. Narashima Rao,P., Kahn,A.H., and Moore Jr,P.H., STEROIDS, 2, 171 38. (1977). Baker,T.S., and Exley,D., STEROIDS, 2, 429 (1977). 39. Karplus,M., J.CHFM. PHYS., 2, 11 (1959). J. AMER. CHEM.SOC., 2, 40. 2870 (1963). Djerassi,C., Von Mutzenbecher,G.,Fajkos,J., Williams,D.H., and 41. Budzikiewicz,H.,J. AMER. CHEM.SOC., 87, 817 (1965). f3, 3547 (1951). Vaughan,J.R., J.AMER.C'HEM.SOC., 42. Tamaoki,H., Murase,Y., Minato,S., and Nakanishi,K., J.BIOL.CHEM., 43. 62, 1 (1967). Kelly,R.W., and Sykes,P.J., J.CHEM. SOC. (C), 416 (1968). 44. Antonucci,R., Bernstein,S., Littell,R., Sax,K.J., and Williams, 45. J.H., J. ORG. CHEM., 17, 1341 (1952). Antonucci,A., BernsteG,S., Lenhard,R., Sax,K.J., and Williams, 46. J.H., J. ORG. CHEM., 17, 1369 (1952). Vaitukaitis,J., RabbiG,J.B., Nieschlag,E., and Ross,G.T., J.CLIN. 47. ENDOCR. METAB., 22, 988 (1971).

48. 49, so. 51.

Bray,G,A., ANALYTICAL BIOCHEMISTRY,1, 279 (1960). Scatchard,G.,ANN. N.Y. ACAD. SC,, 5l_, 660 (1949)" Goertz,G., Longchamp,J., Crepy,O., Judas,O., and Jayle,M.F.,BIOCHIM. & BTQP~S. ACTA, 451, 287 (1976). Abraham,G.E., J. CLIN. =OCR., 2, 866 (1969).