Studies on Testosterone Metabolism in

Subjects with Testicular Feminization Syndrome

P. MAuvAIS-JARvIs, J. P. BEmcovici, 0. CREPY, and F. GAUTIR

From the Laboratoire de Chimie Biologique, C.H.U. La Pitie, Paris 13, France

A B S T R A C T The metabolism of radioactive testoster-one simultaneously administered intravenously andeither orally or percutaneously has been studied inseven patients with the syndrome of testicular feminiza-tion and compared with that of normal males and females.This investigation was carried out in order to determinethe relative contribution to urinary 17-oxo and 17p-hydroxy androstane steroids of labeled testosterone, ac-cording to its mode of administration. In normal malesthe yields of urinary 5a-androstane-3a,17,-diol (andros-tanediol) originating from either an intravenous or apercutaneous dose of testosterone were respectively 3and 6 times higher than those arising from an oral dosewhich perfuses the liver directly. These data indicatethat in normal males, testosterone might be 5a-hydroge-nated outside the liver. By contrast in patient withfeminizing testes, because the contribution to androstane-diol of radioactive testosterone is identical whatever itsmode of administration, the extrahepatic 5a-reduction ofthis substrate seems very unlikely.

The metabolic abnormalities observed in patients withtesticular feminization syndrome may be reproduced innormal males by estrogen treatment. Nevertheless, thesensitivity of the patients to estrogen seems to be 10times greater than that of normal males. This sensi-tivity was appreciated from the reduction of radioactivetestosterone intravenously injected to urinary 17P-hy-droxy-5a-androstan-3-one and androstanediol and alsofrom the level of plasma binding for testosterone. Thislevel was significantly higher (P < 0.05) in patientswith feminizing testes than in normal males. The levelincreased dramatically after administration of a lowdose of estrogen whereas this effect was not observedin normal males under the same experimental conditions.

In light of these results the defect of extrahepatic 5a-reduction of testosterone observed in patients with

Part of this work has been published as a preliminarycommunication (14).

Received for publication 9 May 1969 and in revised form22 August 1969.

feminizing testes does not necessarily reflect an enzy-matic impairment but might be related to an abnormalsynthesis of plasma binding protein(s) under the effectof circulating estrogens so that an abnormally smallamount of unbound testosterone may be available intarget cells for 5a-reduction.

INTRODUCTIONIt is well known that the syndrome of testicular femini-zation develops in the presence of blood testosterone1levels sufficiently to cause virilization in normal indi-viduals (1-3). This observation suggests that the clinicalmanifestations of the syndrome are related to a defectin androgen action. Recently, we have reported thatmale sex differentiation is partly associated with astimulation of enzymes allowing the ring A reduction oftestosterone to 17P-hydroxyandrostane steroids (Fig. 1)(4, 5). When normal males and hirsute females are in-travenously injected with radioactive testosterone, thecontribution of this precursor to urinary 5a- and 5P-an-drostane-3a, 17f-diol is higher than in normal femalesand hypogonadal males with gonadotrophin deficiency.The treatment of the latter patients by chorionic gonado-trophin restores a normal male metabolic pattern. Onthe contrary, the administration of synthetic estrogensto normal males decreases the excretion of urinarymetabolites originating from the ring A reduction oftestosterone (6). In all these situations the yield of uri-nary 17P-hydroxyandrostane steroids arising from testos-terone is correlated with the concentration of this an-

'The following trivial names have been used: T = testos-terone; androstanolone = 17#-hydroxy-Sa-androstan-3-one;androstenedione = androsta4-ene-3,17-dione; androstanedione= 5a-androstane-3,17-dione; androsterone (A) = 3a-hy-droxy-5a-androstan-17-one; isoandrosterone = 3pi-hydroxy-5a-androstan-17-one; 5f8-androsterone (5,8-A) = 3a-hydroxy-5p-androstan-17-one; androstanediol (Adiol) = 5a-andro-stane-3a,17fl-diol; Sp-androstanediol (5p-Adiol) = 5p-an-drostane-3a,17,B diol; androstanediols = diols = androstane-diol + 5,B-androstanediol.

The Journal of Clinical Investigation Volume 49 1970 31

drogen in biological fluids. This is not the case in pa-tients with testicular feminization syndrome in whom,despite normal male testosterone secretion, the con-version of testosterone to androstanediols occurs at avery low rate (5, 7).

These clinical data are ascertained by the fact thatlabeled testosterone is not only taken up by accessorysex tissues but is also quickly metabolized in these tis-sues, particularly to 17P-hydroxy-5a-androstane metabo-lites (8, 9) which are known to be very potent andro-gens (10, 11). More recently, it has been reported that17,8-hydroxy-5a-androstan-3-one (androstanolone) is theonly metabolite present in the prostatic nuclei after in-travenous administration of radiocative testosterone torats or after incubation of rat prostate with the sameprecursor (12, 13). The presence of a highly tissue-specific receptor for androstanolone in the prostatic nu-clear chromatin indicates that this hydrogenated metabo-lite of testosterone might be an active form of androgenin the prostatic nuclei.

Therefore, it was very important to determine whetherthe small conversion of circulating testosterone to uri-nary androstanediols observed in patients with testicu-lar feminization syndrome reflects an enzymatic im-pairment at the level of extrahepatic tissues. In thisinvestigation data are presented which suggest that 5a-hydrogenation of testosterone occurs in the target tis-

o OH

0 0androstenedione, testosterone

o 0H H

5. androstanedione 51 androstanolone

o OH

OH. QH,H H

5 androsterone 52 ancrostanediol13 isoandrosterone 13

FIGURE 1 Different pathways of testosterone metabolism.

sues of normal males and not in those of subjects withfeminizing testes.

METHODSEight patients with testicular feminization syndrome wereinvestigated. There were seven postpubertal untreated caseswith the complete form of the syndrome. The symptoms areno axillary or pubic hair, no clitoris enlargement, and an XYkaryotype. In addition a prepuberal 8 yr old subject (caseM. B.) was studied.

In six cases (five postpubertal cases and the prepuberalcase), 1.0 /Ac of testosterone-4-"C (The Radiochemical Cen-tre, Amersham, specific activity (SA) 29.2 mc/mmole) wasintravenously injected, and simultaneously, 10-12 Ac of tes-tosterone-1,2-3H (Amersham, SA 1000 mc/mmole) wereorally administered. In the prepuberal case a combiped oraland intravenous dose of testosterone was also given aftertreatment with 1 mg of diethylstilbestrol daily for 10 days.The urine was collected for 3 days after the administrationof labeled steroids. In three patients, 1.0 Asc of testosterone-`C was injected, and simultaneously, 23-30 /Ac of testos-

terone-3H dissolved in 100% ethanol were rubbed into theskin as previously reported (14). The urine was collectedover 3 days after the administration of tracers. All theseexperiments were done under the same experimental condi-tions in normal males and females. In a postpubertal case(J. Q.) an intravenous dose of testosterone-'4C was injectedbefore and after castration. The first month was withouthormonal treatment. After that, there was a daily injectionof 25 mg of testosterone propionate for 30 days and then anoral administration of 5 mg of diethylstilbestrol for 20 days.

In addition, 0.5 gc of testosterone-j'C and 3.04.6 uc oftestosterone-17a-8H, synthesized in the laboratory as previ-ously described (15), were injected into one patient withtesticular feminization syndrome and into one normal volun-teer male before and after treatment with 50 mg of diethyl-stilbestrol daily for 20 days. Another patient with testicularfeminization and the same normal male were injected, underthe same experimental conditions, with 1.0 /Ac of testoster-one-"C and 10-11 ,uc of 17,8-hydroxy-5a-androstan-3-one-'H(androstanolone) made in the laboratory according to thepreviously described procedure (16).

The recovery of radioactivity from aliquots of urine col-lections as androsterone (sulfate + glucuronide), 5,8-andro-sterone (sulfate + glucuronide), androstanediol, and 5p8-an-drostanediol (glucuronides) was measured. Details of theanalysis procedure are as previously described (5, 15). The'H:4C ratio of each urinary metabolite was constant

throughout gradient elution, paper and thin-layer chroma-tography, and crystallization. After injection of testosterone-4C and androstanolone-3H, 8H:14C androstanolone was re-

covered in the urine as a ketonic glucuroconjugated steroid.As previously published (16), this metabolite was isolated inthe radioactive isoandrosterone fraction after gradient-elu-tion chromatography. It was further purified by paper chro-matography (ligroin-propanediol system) and thin-layerchromatography (ethyl acetate: benzene, 1: 1) which sepa-rated completely isoandrosterone, Sa- and 5,6-androstanolone.The final product was crystallized in two different systemsof solvents. Its radiochemical homogeneity was confirmedby a constant 8H :4C ratio and specific activity in crystalsand mothers' liquors.

The relative binding affinity of serum protein for testos-terone was determined by the semimicro method of Pearlman

land Crepy (17). This technique is based on the principle ofequilibrium dialysis with use of Sephadex G-25 (Pharmacia)

32 P. Mauvais-Jarvis, J. P. Bercovici, 0. Crepy, and F. Gauthier

in a batchwise fashion. With such a technique, it is possibleunder precise experimental conditions to determine theamount of serum proteins necessary to obtain 50% of bind-ing with a given and very low concentration of radioactivesteroid (1 X 10' mole). The relative binding activity maybe expressed as Sb/(Su X P), Sb and Su representing re-spectively the bound and unbound steroid concentration andP, the amount of protein.

When Sb/Su = 1.0, the binding affinity is equal to 1/P.Expressed in liters per gram, this value is a good index ofthe relative binding affinity of testosterone for various sera.

This determination was performed in six cases of testicu-lar feminization, five normal males, and eight females. Inaddition, this was repeated in four patients with feminizingtestes and two volunteer normal males after administrationof 5 mg of diethylstilbestrol daily for 20 days.

RESULTSSimultaneous administration of testosterone-H orally

and testosterone-14C intravenously (Table I, Fig. 2). In

normal males, females, or patients with feminizing testes,the recoveries of urinary 5fP-androsterone originatingfrom radioactive testosterone either orally or intra-venously administered were not significantly different(P < 0.50 for the three groups of- subjects). Similarlyin males, the per cent conversions of the two doses oftestosterone to 5f-androstanediol did not differ signifi-cantly (P < 0.10). However in females, there was less5fi-androstanediol recovered in urine from the intra-venously injected testosterone (1.55 +0.15% SE) thanfrom the orally administered compound (1.88 ±0.22%SE). These values are significantly different (P < 0.05).In the case of patients with feminizing testes, the dif-ference was more significant (P

ences may also be expressed by calculating the propor-tion of the intravenous compared with the oral dose ofradioactive testosterone excreted as 5#-androstanediol,i.e., the R value of 5P-androstanediol. In the present ex-periments, this R value was, calculated by dividing the8H: 1'C ratio of the injected steroids by the 'H: 14C ratioin a purified sample of a metabolite. The mean R valuesfor 5P-androstanediol were 1.15 in males, 0.80 in females,and 0.60 in patients with feminizing testes. The threegroups differ from one another (P < 0.01).

5R-A A 5R-Adio Adiol3.0 r.

2X10

1.0

4,I

(O

a

Q

Al

I4'

4)

INI

0

2.0

o 1.0

' o

,44

4)

:t2.0

1.0

0

MALES

(4-)

La

FEMALES

_ (3)

TESTICULAR

(5)

T --T- L

As regards androstanediol, the per cent conversionof the intravenously injected dose of testosterone was1.60 ±0.20% SE in males. This value was significantlyhigher (P < 0.05) than the per cent conversion of theoral dose of testosterone to the same metabolite (0.68±0.17% SE). In females the difference was less impor-tant but still significant (P < 0.05). In patients withfeminizing testes the yields of urinary androstanedioloriginating from either the intravenously or the orallyadministered testosterone were respectively 0.50 ±0.15%

A 5A-AdiaI Adiol

(3)

3.0

2.0,

1.0 Smf

ao (3)

1.0

FEMINIZATION(3)

T-14-C intravenouslyT-3H orally

T-3H percutaneouslyT-14G intravenously

FIGURE 2 Proportion of an intravenous compared with an oral dose oftes-tosterone and proportion of a percutaneous compared with an intravenous doseof testosterone excreted as urinary 17-ketosteroids and androstanediols (Rvalue, mean and range). In the case of experiments using the simultaneous ad-ministration of testosterone-3H orally and testosterone-14C intravenously, the

3H :14C ratio of the radioactive precursorsR value =.. ...As regards the3H :14C ratio in a purified sample of a metabolitesimultaneous administration of testosterone-3H percutaneously and testos-terone-14C intravenously, the R value =

14C :3H ratio of the radioactive precursors14C:3H ratio in a purified sample of a metabolite

(see also Results). T, testosterone; 5j3-A, 5,6-androsterone; A, androsterone;5,6-Adiol, 5jB-androstanediol; Adiol, androstanediol.

34 P. Mauvais-Jarvis, J. P. Bercovici, 0. Crepy, and F. Gauthier

TestosteroneAdministered -

TABLE IIRecovery of Radioactivity in Urine after Simultaneous Administration of Testosterone-3H by

Percutaneous Route and Testosterone-14C Intravenously

Ratio 50-Androsterone Androsterone 50-Androstanediol AndrostanediolH :14C

of cpm-'H cpm 14C cpm-JH cpm-14C cpm-'H cpm-"C cpm-3H Cm-14CSubjects precursors X 108 X 108 5H:14C* X 10' X 10' $H:14C* X 10' X 103 3H:14C* X108 X 108 3H:14C*

Testicular feminizationN. G. 6.8 105 114 0.92 154 160 0.96 10.7 12.5 0.86 5.6 4.9 1.15J. Q. 7.3 184 107 1.71 190 118 1.61 15.8 10.1 1.56 6.9 4.2 1.64G. P. 7.2 190 132 1.44 273 175 1.56 16.4 11,7 1.40 9.0 4.9 1.82

Normal malesA. H. 8.1 140 143 0.98 114 135 0.85 25.4 21.7 1.17 26.6 10.0 2.66P. M. 8.8 121 120 1.01 102 106 0.96 27.4 25.9 1.05 36.4 14.2 2.56F. D. 8.6 85 83 1.03 188 186 1.01 21.7 20.2 1.07 34.7 11.7 2.96

Normal femalesN. J. 7.7 117 113 1.04 152 141 1.08 14.6 14.5 1.01 12.1 6.3 1.92M. C. 6.6 142 148 0.96 92 88 1.05 11.1 12.2 0.91 12.6 6.9 1.84A. M. 7.2 136 126 1.08 129 116 1.12 12.1 12.7 0.96 9.6 5.1 1.89

* This ratio was obtained from a purified sample of the urinary metabolite.

SE and 0.35 +0.06% SE. These values are not significantlydifferent (P < 0.0). If one considers the proportion ofintravenous compared to the oral dose of radioactivetestosterone excreted as androstanediol, the R valuesobtained were respectively 2.34 in males, 1.85 in females,and 1.45 in patients with feminizing testes. These meanR values are significantly different (P < 0.01) if onegroup of subjects is compared with another.

In a prepuberal patient (case M.B., Table I, Fig. 4),there was a greater proportion of androstanediol arisingfrom the intravenously injected testosterone (2.41 %)than from the orally administered precursor (0.77%).The R value of androstanediol was therefore elevated(3.10). This pattern is quite similar to that observedin normal males and very different from that of patientsstudied after puberty. In addition when the prepuberalpatient was treated with 1 mg of diethylstilbestrol foronly 10 days, there was a dramatic decrease in the con-tribution to androstanediol of testosterone injected in theperipheral circulation and not of testosterone orally ad-ministered. The relative contribution to androstanediolof the two doses of testosterone (R = 1.50) was thencomparable to that of adult patients.

Simultaneous administration of testosterone-'H by per-cutaneous route and of testosterone-'4C by intravenousinjection (Table II). Only 4-6% of radioactive testos-terone percutaneously administered was recovered in theurine as 17-ketosteroids and androstanediols. However,as long as the radioactive doses of testosterone wereadministered with an elevated 'H: 'C ratio (6.6-8.8), itwas possible to determine with good precision the 'H: SCratio in the recovered urinary metabolites. In normalmales, females, and patients with feminizing testes, the'H: "C ratios of androsterone, 5j3-androsterone, and 5f-

androstanediol were very similar for all subjects. How-ever in males, the 'H: "C ratios of androstanediol were3 times the 'H: 'C ratios of other metabolites. This dif-ference was less important in females whereas in patientswith feminizing testes the 'H: "C ratios of androstane-diol did not differ from those of other metabolites.These data are emphasized by comparing for each groupof subjects the proportion of percutaneous compared tointravenous dose of radioactive testosterone excreted asthe same metabolite, i.e., R value. This R value was cal-culated for each metabolite by dividing the "C: 'H ratioof the injected compounds by the "C: 'H ratio in a puri-fied sample of the metabolite (Fig. 2). The values ob-tained have been arbitrarily multiplied by 10 becausethe radioactivity recovered in urinary metabolites origi-nating from percutaneous testosterone-'H was only the8th to the 10th part of radioactivity recovered in themetabolites arising from testosterone-J'C intravenouslyinjected. Then, it was possible to compare the mean Rvalues of the same metabolites arising from a combineddose of testosterone administered either percutaneouslyand intravenously or intravenously and orally.

Moreover, the contribution to androstanediol of eachadministered dose of testosterone was also appreciatedby comparing the different 5a: 5fi ratios calculated fromthe radioactivity recovered in the urine as androstane-diols (Fig. 3). In males the mean 5a: 5f ratio of an-drostanediols arising from the percutaneous dose oftestosterone is 3 and 6 times the corresponding ratio ofdiols originating respectively from the intravenous andthe oral dose. In patients with feminizing testes the5a: 5p ratios of diols are very similar when testosteroneis either intravenously or percutaneously administered.

Testosterone Metabolism in Testicular Feminization 35

XDMALES~FEMALES

1.2

TESTICULAR FEMINIZATION~

1.0

0.8

K0.6.

(4-) (3) (5) (4.) (3) (5) (3) (3) (3)Testosterone. Orally Intravenously PercutaneouslyAdministered-

FIGURE 3 Mean and range of 5a: 5,3 ratio calculated from the radioactivity recoveredas urinary androstanediols (androstanediol/5,8-androstanediol) after administration ofradioactive testosterone by various routes (oral, intravenous, and percutaneous).

Longitudinal studies in a case of testicular feminim-tion after castration (Fig. 4). After castration of thepatient J.Q., there was a significant increase in theconversion rate to androstanediols (especially to the5a-compound) of radioactive testosterone intravenouslyinjected. The yields of urinary diols were then similarto those of normal males. In addition, the treatment ofthis castrated patient with testosterone propionate didnot modify the conversion rate of testosterone to diols,whereas his treatment with 5 mg of diethylstilbestrolfor 20 days was followed by a fall in urinary metabolitesoriginating from the ring A reduction of testosterone.

Metabolism of 17a-'H4-"4C-testosterone (Table III).In the normal male studied, the 'H: 14C ratios of an-drostanediols and of testosterone glucuronide were veryclose and similar to the 'H: 14C ratio of the injectedtestosterone. This was not the case in a patient with

Case M.B. 8yr

Before Pubertyr--&--i nrc

feminizing testes where the 'H: 14C ratio of androstane-diol differed significantly from the 'H: 14C ratios of5,3-androstanediol and testosterone glucuronide. In thecase of a male treated with 50 mg of diethylstilbestrolfor 20 days, a decrease in the 'H: 14C ratio of androstane-diol was only observed. These results mean that in thesetwo subjects and contrarily to what is observed in theuntreated normal male, more than 50% of androstane-diol-14C recovered in urine after injection of 17a-'H-14C-testosterone are formed via a "17-ketonic pathway,"such as, testosterone -> androstenedione -* androstane-dione -* androsterone -> androstanediol, and not fromthe 5a-reduction of testosterone (see Fig. 1).

Metabolism of androstanolone and testosterone (Ta-ble IV). With respect to the normal male, androstano-lone-'H and testosterone-"C contribute to urinary an-drostanediol and androstanolone glucuronides in the

Case J.Q. 30yrAL

BeforeCastrationI

After Castration. . I _---

13 Loontrol utb Control HCCALontrol T

A)

~~~~~~~~~~~~2.52.6

r, ON.2.0.)20iat 2,

DES

I IE H1.2 1.212

IK110.7~~~~~~~~~~~~~~~~~~~~~~~.

&l ILLIL0IJfFIGURE 4 A longitudinal study on testosterone metabolism in two cases of testicularfeminization syndrome. M. B., prepuberal case and J. Q., postpubertal case. Theresults are expressed as per cent conversion of radioactive testosterone intravenouslyinjected to urinary androstanediol and 5,8-androstanediol. HCG=values after treat-ment with 1500 UI HCGfor 10 days. T = values after injection of testosterone pro-pionate (25 mg daily X 30 days). DES= values after treatment with diethylstilbestrol(1 mg daily X 10 days in the case M. B. and 5 mg daily X 20 days in the case J. Q.).

36 P. Mauvais-Jarvis, J. P. Bercovici, 0. Crepy, and F. Gauthier

TABLE IIIRecovery of Radioactivity in Urine after Intravenous Injection of 17a-3H-4-14C-testosterone

(T-17a-3H and T-14C)

5#-Androstanediol Androstanediol Testosterone*

Ratio Per cent con- Per cent con- Per cent con-H :14C version version version

of pre-Subjects cursors T-17a-sH T-14C aH:14CT T-17a-'H T-4C 'H:14CT T-17a-'H T-"4C 'H:4C4

Normal male J. H.)Control 1.79 3.50 3.51 1.76 1.62 1.71 1.70 0.91 0.91 1.80DES§ 1.68 3.24 3.21 1.64 0.14 0.40 0.59 0.50 0.5 1 1.65

Testicular femi-nization (G. P.) 2.82 0.86 1.32 1.84 0.16 0.31 1.40 0.50 0.52 2.79

* Testosterone isolated in the glucuronide fraction of the urine.This ratio was obtained from a purified sample of the urinary metabolite.

§ After treatment with 50 mgof diethylstilbestrol daily for 20 days.

same proportions since the 'H: 14C ratios of these metab-olites are very close. In the patient with feminizingtestes as in the male treated with diethylstilbestrol, prac-tically no androstanolone glucuronide was recovered inthe urine from the injected radioactive testosterone, andthe 'H: 14C ratios of urinary androstanediol and andros-tanolone glucuronides were very different. These resultsconfirm that in the latter subjects androstanolone is notan important metabolic intermediate between testosteroneand androstanediol.

Testosterone binding levels in serum (Fig. 5). Therelative binding affinity of serum protein for testosteronewas significantly higher in patients with feminizingtestes than in normal males (P < 0.05) and very simi-lar to that of normal females. The mean values (inliters/gram of serum protein) were, respectively, 1.56+0.59 SE for testicular feminization syndrome cases(n = 6 determinations), 0.90 0.21 SE for males (n = 5

TABRecovery of Radioactivity in Urine after .

and Testo

determinations), and 1.43 +0.30 SE for females (n = 8determinations) .

After administration of diethylstilbestrol (5 mg dailyfor 20 days), there was a very striking increase in tes-tosterone binding levels in the case of patients withfeminizing testes but not in the case of normal males.The values obtained in patients with feminizing testeswere then similar to those observed in females during thefirst trimester of pregnancy.

DISCUSSIONAs far as experiments using the intravenous injectionof radioactive testosterone are concerned, it seems likelythat in the adult patients with untreated feminizingtestes circulating testosterone is largely oxidized to an-drostenedione and then reduced to androsterone, iso-androsterone, and 59-androsterone (4-7). Unlike normalmales, these patients show ring A reduction of testos-

LE IVIntravenous Injection of Androstanolone-5Htosterone-14C

Ratio'H: 14C Androstanediol Androstanolone*of pre-

Subjects cursors cpm-8H cpm-'4C 'H:14C cpm-$H cpm-14C 'H:14C

Normal male (P. M.)Control 3.1 229,032 20,211 11.3 9380 700 13.4DESt 3.4 101,109 3,348 30.2 2250 30 75

Testicular femi-nization (F. L.) 3.4 92,400 7,000 13.2 1050 15 70

* Androstanolone was recovered as a glucuronide in the ketonic fraction of the urine. The valuesexpressed in the table are those obtained after two crystallizations to constant specific activity.t After treatment with 50 mg diethylstilbestrol daily for 20 days.

Testosterone Metabolism in Testicular Feminization 37

terone to androstanediols only to a small extent. Mostof the androstanediol recovered in the urine of these pa-tients is formed via a "17-ketonic intermediate" suchas androsterone. The results obtained from experimentsusing a combined injection of testosterone-14C and ofeither testosterone-17a-3H or androstanolone-'H empha-sized this possibility. However, these experiments can-not give any information on the respective role of liverand target tissues in the metabolic abnormalities ob-served in patients with feminizing testes. Therefore, itwas interesting to study the fate of radioactive testos-terone according to its mode of administration (oral,intravenous, or percutaneous). In the case of oral ad-ministration, the steroid first enters the liver by way ofthe portal vein. When testosterone is intravenously in-jected into the peripheral circulation, it perfuses thetarget tissues before entering the liver by the hepaticartery. In the case of percutaneous administration, tes-tosterone must pass through the skin and eventuallythrough the muscle before reaching the peripheral cir-culation. With such experimental models, it is possibleto compare the metabolism of testosterone circulatingin the peripheral blood with that of the same precursordirectly brought into the liver or into the target tissues.

In normal males whatever its mode of administration,radioactive testosterone contributes identically to its 5#-hydrogenated metabolites. Such a result is compatiblewith data indicating that in normal males 5p8-reductionof testosterone does not occur to any significant extentin extrahepatic tissues (9, 18, 19).

In contrast the yield of androstanediol originatingfrom testosterone injected in the peripheral circulation

3

(b

,k 2

K1

1I

FIGURE 5 Testosterone binding levels in the serum of nor-mal subj ects and patients with the syndrome of testicularfeminization. Control values and values observed aftertreatment with 5 mg diethylstilbestrol daily for 20 days.

is 2 times higher than that originating from testosteronewhich enters the liver directly by the portal vein,whereas the yield of androstanediol arising from testos-terone percutaneously applied is 2.5 times higher thanthat arising from testosterone injected in the peripheralcirculation. These results are in agreement with in vitrodata suggesting that a characteristic of cutaneous tes-tosterone metabolism is the stereospecific reduction ofthe ring A leading to the formation of 5a-androstanesteroids (18, 19). The actual importance of testosteronemetabolism by an organ as large as the skin is difficultto establish. However, from our in vivo results it maybe postulated that at least 50% of androstanediol re-covered in the urine of males arise from the 5a-reduc-tion of testosterone outside the liver. Contrary to whatis observed in males, the extrahepatic 5a-reduction oftestosterone seems to be negligible in patients with femini-zing testes since identical yields of urinary androstane-diol result whatever the mode of administration of radio-active testosterone. These in vivo data are in agreementwith those obtained by Wilson and Walker (20) and byNorthcutt, Island, and Liddle (21) from in vitro ex-periments. From these reports, there could be a lack oftestosterone 5a-reduction in the skin of patients withtesticular feminization syndrome. Furthermore in thelight of our results, the hepatic 5p8-reduction of testos-terone seems to be hampered if one considers the lowyield of 5,8-androstanediol originating from testosteroneintravenously injected to these patients.

Estrogens seem to be importantly involved in themetabolic abnormalities observed in patients with testicu-lar feminization syndrome. In subjects without estrogenproduction, such as prepuberal or castrated patients, theyield of androstanediol arising from testosterone is com-parable to that of normal males. In males it is possibleto inhibit with diethylstilbestrol the reduction of testos-terone to androstanolone and androstanediol (6). Inother words, testosterone is metabolized in the sameway in males treated with diethylstilbestrol as it is inpatients without estrogen deprivation. However, the in-hibitory effect of estrogen upon testosterone 5a-reductionmay be obtained in castrated and prepuberal patientswith a dose which is 10 times smaller than in normalmales. One can therefore wonder if the decrease intestosterone 5a-reduction observed in patients withfeminizing testes reflects an enzymatic impairment oris only the result of the abnormal sensitivity of a specificenzyme to estrogens originating either directly fromtestes or indirectly from the peripheral conversion oftestosterone and androstenedione (22, 23). Estrogenmay act by (a) inhibition of the enzyme allowing intarget cells the hydrogenation of testosterone to an-drostanolone as the nuclear enzyme described by Bru-chovsky and Wilson (12) and Anderson and Liao (13)

38 P. Mauvais-Jarvis, J. P. Bercovici, 0. Crepy, and F. Gauthier

____CONTROLVALUES AFTER ESTROGENSMALES FEMALES TEST. FEMIN. MALES TEST FEMIN.

,8

0

1~~~~~~00

00

* 0

I 0* 0 0

or (b) increase of specific plasma protein binding tes-tosterone (24, 25) so that almost no unbound testos-terone may be available in target cells for 5ca-reduction.This last hypothesis is sustained by the high bindinglevels for testosterone observed in the plasma of pa-tients, and furthermore, seems very consistent regardingthe hepatic 5P-reduction of testosterone which is verylow when the steroid is injected intravenously but isnormal when testosterone is orally administered. In thecase of oral administration, testosterone probably entersthe liver before being bound to a specific protein (26)contrarily to what is observed when the steroid is in-jected in the peripheral circulation.

Although data concerning estrogen production inpatients with feminizing testes do not permit a firmconclusion (1, 27, 28), it seems likely that this produc-tion does not exceed that of normal males. Thus, thereseems to exist in subjects with testicular feminizationsyndrome an abnormal synthesis of plasma-bindingprotein under the effect of circulating estrogens. Thishypothesis is supported by the fact that with the samelow dose of diethylstilbestrol plasma-binding levels fortestosterone were not modified in males but did increasedramatically in patients with feminizing testes. Thatcastrated patients do not respond to testosterone doesnot exclude such an hypothesis. In males as in patientswith feminizing testes, the main part of circulating es-trogens originates from peripheral conversion of tes-tosterone (22, 23). Therefore, treatment with testos-terone of castrated patients may maintain an elevatedbinding level in plasma.

However, it has not actually been proven that the syn-thesis of specific protein(s) binding testosterone inplasma is altered in testicular feminization syndrome.Thus, further investigation must be undertaken beforeit can be said if the metabolic abnormalities observed inpatients with this genetic disease are due to the absenceof specific enzyme(s), or to an elevated binding of -tes-tosterone to proteins which prevents the penetration ofthis androgen in target cells, or to both. Furthermore,it has not definitively been proven that the lack ofmasculinization observed in patients with feminizingtestes depends upon the absence of biotransformation oftestosterone to androstanolone in target tissues.

ACKNOWLEDGMENTSThe technical assistance of Mrs. N. Baudot is gratefullyacknowledged. Weare indebted to Professors Laplane, Bri-caire, and Musset, and Doctors Henrion, Lasfargues, LutonRoy, and Sebaoun for allowing us to study their patients.Dr. J. A. Guichard and Miss D. Ravelet are acknowledgedfor their help in writing this manuscript.

This work was supported in part by a grant of the InstitutNational de la Sante et de la Recherche Medicale (IN-SERM).

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40 P. Mauvais-Jarvis, J. P. Bercovici, 0. Crepy, and F. Gauthier