FR\l=E'\D\l=E'\RIQUEKUTTENN, IR\l=E'\NEMOWSZOWICZ,

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1 ANDROGEN PRODUCTION AND SKIN METABOLISM IN HIRSUTISM FR\l=E'\D\l=E'\RIQUEKUTTENN, IR\l=E'\NEMOWSZOWICZ, GILBERT SCHAISON AND PIERRE MAUVAIS-JARVIS Department ofmedical Biochemistry, Faculty ofmedicine, Pitié-Salpêtrière, Paris Cedex 13, France (Received 17 January 1977) SUMMARY The concentrations of testosterone, androstenedione and dihydrotestosterone (DHT) in the plasma and 5\g=a\-androstane-3\g=a\,17\g=b\-diol(androstanediol) in the urine were measured in 40 women with hirsutism of ovarian, adrenal and idiopathic origin. Conversion of [3H]testosterone to DHT, 3\g=a\-and 3\g=b\-androstanediols was also studied in homogenates of pubic skin obtained from 15 of the patients. Results were compared with values obtained from normal men and women. Values for the levels of testosterone, DHT and androstenedione in the plasma and androstanediol in the urine of hirsute women were all above control levels, especially for plasma androstenedione and urinary androstanediol (P < 0\m=.\001).This finding was particularly marked in patients with hirsutism of ovarian origin. Conversion of [3H]testosterone to 5\g=a\-reducedmetabolites by homogenates of skin obtained from hirsute women was significantly greater than by homogenates of skin from normal women (P < 0\m=.\001) but was the same as the value for normal men. The highest values for conversion were obtained from the patients with idiopathic hirsutism. These results indicate that androstenedione is the principal androgen secreted in hirsutism. In sexual skin this steroid may be converted to DHT and 3\g=a\-,and 3\g=b\-androstanediols and the increased activity of testosterone 5\g=a\-reductase may result in an exaggerated 'utilization' of androstenedione in this tissue. The high rate of excretion of androstanediol in the urine of patients with idiopathic hirsutism may be explained by the fact that this steroid is an end-product of testosterone metabolism. INTRODUCTION Hirsutism in women may be defined as excessive hair growth in anatomical sites where such growth is considered to be a secondary male characteristic. This abnormality is related to increased activity of two androgen-dependent structures of human skin: the sebaceous gland and the hair follicle. In these structures the mechanism of the action of testosterone has been studied with particular emphasis on the 5a-reduction of testosterone to dihydrotestosterone (DHT) (Mauvais-Jarvis, Bercovici & Gauthier, 1969; Wilson & Walker, 1969; Voigt, Fernandez & Hsia, 1970) and the binding of DHT to the specific receptor proteins that play a crucial role in mediating the effects of androgens (Adachi & Kano, 1972; Eppenberger & Hsia, 1972; Keenan, Meyer, Hadjian, Jones & Migeon, 1974; Keenan, Meyer, Hadjian & Migeon, 1975). * Present address and address for reprint requests: Hôpital Necker, 149 rue de Sèvres, Paris Cedex 15, France.

2 From these studies it can be argued that hirsutism may arise from two different causes : an increased production of active androgens by the adrenal glands or ovaries, and/or an increased utilization of androgens in the blood by the target cells of the skin. The first possibility has already been extensively reviewed by several authors (Bardin & Lipsett, 1967; Kirschner & Jacobs, 1971). The raised metabolic clearance rate of testosterone reported by Bardin & Lipsett (1967) in hirsutism supports the second possibility. It results mainly from an increased metabolism of testosterone by extrahepatic tissues (Kirschner & Bardin, 1972) as shown by the higher rate of conversion of testosterone to DHT and 5a-androstane-3a,-17/?- diol (androstanediol) in the blood of hirsute women than in that of normal women (Mahoudeau, Bardin & Lipsett, 1971). This increased metabolic clearance of testosterone is facilitated by the low concentration of testosterone binding globulin in the plasma (Dray, Sebaoun, Delzant, Ledru & Mowszowicz, 1968; Southren, Gordon, Tochimoto, Olivo, Sherman & Pinzón, 1969; Vermeulen, Verdonck, Van der Straeten & Orie, 1969), and a concentration of unbound testosterone in the plasma which is higher than normal (Rosenfield, 1972; Clark, Marcellus, de Lory & Bird, 1975). The aim of this study was to examine the roles of overproduction of active androgens and excessive transformation of testosterone to 5a-reduced metabolites in the skin in the production of female hirsutism. The levels of testosterone and androstenedione in the plasma (which mainly reflect excessive production of androgens by the adrenal glands or ovaries) and the levels of DHT in the plasma and androstanediol in the urine (which in part originate from the metabolism of testosterone and androstenedione within the androgen target cells) were investigated in 40 women with hirsutism of different origins. The results are compared with conversion values obtained from the incubation of pubic skin samples from 15 of these patients with [3H]testosterone. MATERIALS AND METHODS Forty hirsute women aged between 14 and 40 years were classified into the following three groups according to their clinical features : ovarian hirsutism (OH), adrenal hirsutism (AH) and idiopathic hirsutism (IH). Table 1 gives full details. Forty normal volunteers ( men and women) aged between 18 and 40 years acted as controls. Blood samples were drawn between and h on day 4 of the luteal phase in control women and patients with ovulatory cycles, and collected on heparin. Plasma was stored at C until the steroids were assayed. Androstanediol was measured in urine collected over a 24 h period. One determination was made for each subject. Ovarian hirsutism (polycystic ovaries at coelioscopy) Table 1. Clinicalfeatures ofhirsute patients No. of patients 14 Mean age and range (years) 24-8 (18-38) Degree of hirsutism* Slight 3 Adrenal hirsutism Marked 6 Severe 5 Acne, seborrhoea, alopecia 9 (congenital adrenal hyperplasia, 3 ; (18-40) Cushing's syndrome, 3) Idiopathic hirsutism (no Menstrual abnormalities 12 symptoms of ovarian (14-39) or adrenal dysfunction) * Hirsutism was evaluated as slight, marked or severe. Slight: face only; marked: thighs, legs, breast, abdomen (but not face), or face, breast, arms, abdomen; severe: generalized.

3 80 The concentrations of steroids in the plasma were determined by radioimmunoassay techniques. Testosterone and DHT were assayed using [3H]testosterone (New England Nuclear Corporation, sp. act. 85 Ci/mmol) as tracer and a commercially available antitestosterone antiserum (A/79550 Institut Pasteur, Paris); this antiserum was specific for testosterone except for a 65 % cross-reaction with DHT, which allowed the use of the same antiserum for both assays. After the addition of appropriate tracers ([3H]testosterone and [3H]DHT: 1500 d.p.m. each) to monitor recovery, plasma samples (2 ml) were extracted with 10 ml cyclohexane : ethyl acetate (1:1, v/v); the aqueous phase was frozen rapidly at C and the supernatant fluid poured into a conical siliconed tube, and taken to dryness. The dry sample was dissolved in 2 ml heptane : benzene (98:2, v/v) and applied to the top of a small Celite column (0-5 g Celite:0-25 ml formamide) prepared according to Abraham, Tulchinsky & Korenman (1970). The column was eluted with three successive phases: 4 ml heptane : benzene (98:2, v/v), 5 ml heptane : benzene (which elutes DHT), and 2-5 ml benzene alone (which elutes testosterone). The assay itself was similar to that described by Barberia & Thorneycroft (1974) except that incubation was carried out at room temperature for 30 min and at 0 C for a further 15 min. Dextran-coated charcoal was used to separate bound from free steroids. Radioactivity was counted in 5 ml Jensen scintillation fluid (Pearlman, Crepy & Murphy, 1967) with 5 ml aqueous phase. Androstenedione was assayed in a similar way using [3H]androstenedione (Amersham, sp. act. 83 Ci/mmol) as tracer and an anti-androstenedione antiserum (A , Institut Pasteur, Paris), which cross-reacted slightly with dehydroepiandrosterone (8-5 %) and testosterone (14 %); androstenedione was purified from these two steroids on a Celite column (1 g Celite: 0-5 ml propanediol). The extract was dissolved in 1 ml iso-octane and applied to the top of the column, which was then eluted once with 4 ml iso-octane, and twice with 4 ml iso-octane : benzene (92:8, v/v). The second fraction of this mixture eluted andro stenedione. The assay technique was otherwise similar to that used for testosterone and DHT; extraction was carried out with 0-4 ml plasma. Mean recovery was 83-5 ±9-5 (s.d.) % for testosterone (69 determinations), % for DHT (77 determinations) and 66-3 ± 9-5 % for androstenedione (56 determinations). Intra-assay and interassay coefficients of variation were between 6 and 8 % for all steroids. Blank values were constantly 0. The sensitivity of the assay allowed the detection of 0-1 ng testosterone or DHT/ml plasma and 0-05 ng androstenedione/ml plasma. Androstanediol in the urine was assayed by gas chromatography as already described (Charransol, Bobas- Masson, Guillemant & Mauvais-Jarvis, 1972). Measurement of testosterone 5cc-reductase activity ofskin Testosterone 5a-reductase activity was measured in a 0 mg sample of pubic skin as described previously by Mauvais-Jarvis, Kuttenn & Baudot (1974). In some instances, however, the two separations by paper chromatography were replaced by a single step involving thin-layer chromatography on silica gel in chloroform : methanol (97-5:2-5, v/v). This system allowed adequate separation of androstenedione, testosterone, DHT and the 3a- and 3/?-androstanediols in one step. When duplicate samples were studied by both methods, similar results were obtained. Thereafter thin-layer chromatography was used routinely. The results are expressed as percentages of 5a-reduced metabolites relative to the total amount of radioactivity eluted. RESULTS Plasma steroids In 40 hirsute women, the mean concentration of testosterone in the plasma was 72-9 ± 9-0 (s.e.m.) ng/100 ml, which was significantly higher than the value for normal women (33-2 +

4 FRÉDÉRIQUE KUTTENN AND OTHERS o o ß íoo e % 50 ' fiòl h Testosterone XXX o < -1 Androstenedione ill 14 6 O < " DHT Fig. 1. Mean concentrations (± s.e.m.) of testosterone, androstenedione and dihydrotestosterone (DHT) in the plasma of (a) 40 hirsute women (solid bars), normal women (double hatched bars) and normal men (single hatched bars) and (b) women in the three subgroups of hirsutism: ovarian (OH), adrenal (AH) and idiopathic (IH). The number of subjects studied is given at the base of each bar. 2-7 ng/100 ml, Fig. 1). In the group of OH women, the mean level of testosterone in the plasma was 92-4 ± 15-2 ng/100 ml, which was significantly higher than the value for AH (58-0 ± 10-5 ng/100 ml;p< 0-02) and IH (51-0 ±50 ng/100 ml;p < 0-001) women. There was no significant difference between the levels of testosterone in the plasma ofah and IH women. In hirsute women, the mean concentration of androstenedione in the plasma was ± 18-6 ng/100 ml; this value was significantly higher (P < 0-001) than that found in normal women (133-3 ±6-8 ng/100 ml) and normal men ( ng/100 ml, Fig. 1). In OH women, the mean level of androstenedione in the plasma ( ng/100 ml) significantly higher was than the level in AH ( ng/100 ml; < 0-05) or IH (3-7± 22-4 ng/100 ml; < 0001) women. However, there was no significant difference between the levels of androstenedione in the plasma of the AH and IH women. The mean concentration of DHT in the plasma of hirsute women was 34-9 ± 2-4 ng/100 ml, which was significantly higher (P < 0-001) than the value for normal women (17-9 ± 2-9 ng/ 100 ml) and lower (P < 0-01) than the value for normal men (58-6 ± 3-9 ng/100 ml, Fig. 1). There was no significant difference between the three groups of hirsute women ; the con centrations of DHT were 30-8 ± 2-6, and 34-2 ± 3-6 ng/100 ml in OH, AH and IH women respectively.

5 Urinary excretion ofandrostanediol The mean rate of excretion of androstanediol in the urine of 34 hirsute women was (s.e.m.) /tg/24 h (Fig. 2). This value was significantly higher (P < 0-001) than that found in normal women (16-5 ± 2-2 /tg/24 h), but there was no overlap with the range found in normal men (1330 ± 7-5 /tg/24 h, Fig. 2). The highest rate of excretion ofandrostanediol was observed in the OH patients ( /tg/24 h). The values for IH and AH women were 58-0±8-3 and 30-0 ± 4-9 /tg/24 h respectively. These values are significantly different (P < 0-05) from each other , («) 150 (*) X! 3 ö loo H e W F* //.Va 0J o-j OH AH è- Fig. 2. Mean rate of excretion of androstanediol (± s.e.m.) in the urine of (a) 34 hirsute women (solid bars), eight normal women (double hatched bars) and 11 normal men (single hatched bars) and (b) women in the three subgroups of hirsutism : ovarian (OH), adrenal (AH) and idiopathic (IH). The number of subjects studied is given at the base of each bar. Testosterone 5oc-reductase activity in skin The activity of testosterone 5a-reductase in skin samples was investigated in 15 hirsute patients. In these women, the conversion of radioactive testosterone to DHT + 3a- and 3/?-androstanedioIs was 14-8 ± 2-4 (s.e.m.) % which was similar to the mean value for normal men (14-9 ±1-0%) and strikingly higher than that for normal women (3-6 ±0-5%; < 0-001; see Fig. 3). The highest conversion of testosterone to DHT + 3a- and 3/?-androstanediols was observed in IH patients (18-0 ±3-0 %), but this was not significantly greater than the value for OH patients (14-4 ±3-2%). In contrast, in patients with adrenal dys function, the recovery of DHT + 3a- and 3/?-androstanediols from testosterone in skin was only slightly raised (5-3 ± 0-7 %).

6 (ß) , o 15 - k > 15 II Fig. 3. Percentage (mean ± s.e.m.) of testosterone converted to 5a-reduced metabolites (dihydro testosterone + 3a- and 3/?-androstanediols) in homogenates of pubic skin from (a) 15 hirsute women (solid bars), eight normal women (double hatched bars) and 11 normal men (single hatched bars) and (b) women in the three subgroups of hirsutism : ovarian (OH), adrenal (AH) and idiopathic (IH). The number of subjects studied is given at the base of each bar. DISCUSSION In normal women about 70 % of the testosterone in the plasma is derived from plasma prehormones, essentially dehydroepiandrosterone and androstenedione. Androstenedione is by far the most important prehormone and serves as precursor for at least 50 % of the testosterone found in the blood (Horton & Tait, 1966; Bardin & Lipsett, 1967), whereas dehydroepiandrosterone accounts for only 15% (Horton & Tait, 1967). The remaining 30 % of the testosterone in the plasma is apparently secreted by the adrenal glands and ovaries (Kirschner & Jacobs, 1971 ; Kirschner & Bardin, 1972) and thus the concurrent determination of the concentrations of androstenedione and testosterone in the plasma gives useful information about the production of active androgens in hirsute patients. Androstanediol and DHT are two potent androgens that do not seem to be secreted directly by the gonads or adrenal glands. In fact, DHT in the plasma is produced mainly by hepatic and extrahepatic 5a-reduction of testosterone and androstenedione (Ito & Horton, 1971 ; Mahoudeau et al. 1971), and the same assumption has been made regarding andro stanediol in the urine (Mauvais-Jarvis, Charransol & Bobas-Masson, 1973). However, the various studies on the sources of plasma DHT and plasma and urinary androstanediol cannot distinguish between the synthesis of these androgens by liver or by target tissues.

7 Therefore, the evaluation of the capacity of sexual skin to metabolize testosterone into DHT and androstanediol in vitro seems the most reliable method for determining the actual rate of ' utilization ' of androgens in the blood by target cells. The 5a-reductase enzyme of human skin uses unbound testosterone as substrate, which may originate either from testosterone in the blood or from the transformation of prehormones, notably andro stenedione, within the target cells (Ito & Horton, 1971). In this study, the production of androgens seemed to be increased in all hirsute women, since the levels of testosterone and androstenedione were abnormally high in all the patients studied. As the major active androgen in the bloodstream is testosterone, it is interesting to note that the concentration of testosterone was especially high in the plasma of patients with either ovarian or adrenal dysfunction, whereas it was close to the normal range in patients with idiopathic hirsutism. In fact, the only androgen with a constantly and signifi cantly increased concentration in all cases of hirsutism was androstenedione. This is con sistent with data reported elsewhere (Bardin & Lipsett, 1967; André & James, 1974). With regard to the parameters of peripheral androgen metabolism, it appears clear that in hirsute patients the rate of excretion of androstanediol in the urine is a better discriminant of hirsutism than the level of DHT in the plasma. This observation is probably best explained as follows. Since most of the DHT synthesized in tissues is reduced in situ to 3a- and 3ßandrostanediols, only a small fraction of DHT enters the blood. By contrast, almost all of the 3a- and 3^-androstanediols formed in tissues are excreted into the urine. This con clusion is substantiated by the fact that the estimated rate of production of 3a- and 3ßandrostanediols in the blood (Bird, Choong, Knight & Clark, 1974; Kinouchi & Horton, 1974) is similar to the daily excretion rate. These observations support the assumption that 3a- and 3/?-androstanediols are end products of androgen metabolism. In so far as androstenedione appears to be the major source of DHT in women (Ito & Horton, 1971 ; Mahoudeau et al. 1971), the raised concentration of DHT in the plasma of hirsute patients could be due to the peripheral conversion of this prehormone. The raised excretion rate of androstanediol observed in hirsute patients reflects an increase in both the amount of androstenedione produced and its peripheral conversion. Indeed, the rate of production of androstenedione, which is around 3-0 mg/24 h in normal women, may rise to 8-0 mg/24 h in hirsute women (Bardin & Lipsett, 1967). The contribution of andro stenedione to the androstanediol found in the urine was calculated after i.v. injection of radioactive androstenedione (Baulieu & Mauvais-Jarvis, 1964) and ranges from 0-5 % in normal women to 1 0% in hirsute patients (P. Mauvais-Jarvis, unpublished results). The variations in these two parameters may account for the striking difference in androstanediol and the excretion observed between normal and hirsute women (from 12 to 100/tg/24 h) wide range of variation within the group of hirsute women in the present study. The same calculation of the contribution of testosterone in the blood to the level of androstanediol in the urine of hirsute patients leads to the conclusion that at most, /tg of the urinary andro stanediol excreted/24 h could arise from testosterone. The rate of excretion of androstanediol in the urine therefore gives a good estimate of the peripheral metabolism of androstenedione. This is of particular importance since, contrary to what was observed for testosterone, an increase in androstenedione metabolism by extrahepatic tissues is not reflected by an increase in the metabolic clearance rate for androstenedione (MCRA). There is, in fact, no difference between the MCRA of normal and hirsute women (Bardin & Lipsett, 1967; Kirschner, Zucker & Jespersen, 1976). The differences observed between normal and hirsute women in the contribution made by testosterone and androstenedione to the level of androstanediol in the urine in vivo are directly related to the differences observed in the potential capacity of sexual skin to trans form testosterone into DHT and 3a-, and 3/?-androstanediols in vitro (Kuttenn & Mauvais-

8 Jarvis, 1975). Since androstenedione may be converted into testosterone by human skin (Gomez & Hsia, 1968; Flamigny, Collins, Koullapis, Craft, Dewhurst & Sommerville, 1971), the level of 5a-reductase activity present in this tissue is the major factor controlling the amount of androstenedione that is effectively metabolized into DHT and 3a- and 3ßandrostanediols. In IH patients the increase in the concentration of androstenedione in the plasma reflects an excessive secretion of androstenedione by the ovaries as reported by Kirschner & Jacobs (1971) and Kirschner et al. (1976), but it is reasonable to suggest that the abnormally high conversion of androstenedione into testosterone and DHT by sexual skin plays a major role in the production of hirsutism. In other words, the observation in IH patients of a very high 5a-reductase activity, contrasting with only a moderate increase in the levels of test osterone and androstenedione in the plasma, supports the thesis that IH is caused by an increase in the sensitivity of the target organs to androgens (Bardin & Lipsett, 1967). How ever, in ovarian and adrenal hirsutism, the increased production of androgens is the most important factor and in some cases can be responsible itself for the increase in the amount of androstanediol excreted. In conclusion, the determination of both the level of androgens in the plasma and the capacity of skin to transform testosterone into 5a-reduced metabolites is necessary to elucidate the respective roles of abnormal androgen secretion and/or skin 'utilization' in the production of hirsutism. The constant he'p of Drs Pierre Desgrez and Jean-Claude Legrand is gratefully acknow ledged. The technical assistance of Nicole Baudot, Jean-Marc Chauffournier, Jeanine Bullard, Abiba Doukani and Geneviève Maistre is greatly appreciated. This work was supported in part by a grant of the Fondation pour la Recherche Médicale Française and B.I.R.E.C. REFERENCES Abraham, G. E., Tulchinsky, D. & Korenman, S. G. (1970). Chromatographie purification of estradiol-17/? for use in radioligand assay. Biochemical Medicine 3, Adachi, K. & Kano, M. (1972). The role of receptor proteins in controlling androgen action in the sebaceous gland of hamsters. Steroids 19, André, C. M. & James, V. H. T. (1974). Plasma androgens in idiopathic hirsutism. Steroids 24, Barberia, J. M. & Thorneycroft, I. H. (1974). Simultaneous radioimmunoassay of testosterone and dihydro testosterone. Steroids 23, Bardin, C. W. & Lipsett, M. B. (1967). Testosterone and androstenedione blood production rates in normal women and women with idiopathic hirsutism or polycystic ovaries. Journal of Clinical Investigation 46, Baulieu, E. E. & Mauvais-Jarvis, P. (1964). Studies on testosterone metabolism. II. Metabolism of testosterone-4-14c and androst-4-ene-3,17-dione-l,2-3h. Journal of Biological Chemistry 239, Bird, C. E., Choong,., Knight, L. & Clark, A. F. (1974). Kinetics of 5ac-androstan-3a,17/?-diol metabolism in normal men and women. Journal of Clinical Endocrinology and Metabolism 38, Charransol, G., Bobas-Masson, F., Guillemant, S. & Mauvais-Jarvis, P. (1972). Determination of urinary androstanediol and testosterone in normal men by gas liquid chromatography. Journal of Chromatography 66, Clark, A. F., Marcellus, S., de Lory, B. & Bird, C. E. (1975). Plasma testosterone free index : a better indicator of plasma androgen activity? Fertility and Sterility 26, Dray, F., Sebaoun, J., Delzant, G., Ledru, M. J. & Mowszowicz, I. (1968). Activité de liaison de la testo sterone dans le sérum des femmes présentant un virilisme pilaire idiopathique. Revue Française d'etudes Cliniques et Biologiques 13, Eppenberger, U. & Hsia, S. L. (1972). Binding of steroid hormones by the 105,000 x g supernatant fraction from homogenates of rat skin and variations during the hair cycle. Journal of Biological Chemistry TAT, Flamigny, C, Collins, W. P., Koullapis, E. N., Craft, I., Dewhurst, C. J. & Sommerville, I. F. (1971). Androgen metabolism in human skin. Journal of Clinical Endocrinology and Metabolism 32, Gomez, E. C. & Hsia, S. L. (1968). In vitro metabolism of testosterone-4-"c and A4-androstene-3,17- dione-4-14c in human skin. Biochemistry 7,

9 Horton, R. & Tait, J. F. (1966). Androstenedione production and interconversion rates measured in peri pheral blood and studies on the possible site of its conversion to testosterone. Journal of Clinical Investi gation 45, Horton, R. & Tait, J. F. (1967). In vivo conversion of dehydroisoandrosterone to plasma androstenedione and testosterone in man. Journal of Clinical Endocrinology and Metabolism 27, Ito, T. & Horton, R. (1971). The source of plasma dihydrotestosterone in man. Journal of Clinical Investiga tion 50, Keenan, B. S., Meyer, W. J., Ill, Hadjian, A. J., Jones, H. W. & Migeon, C. J. (1974). Syndrome of androgen insensitivity in man: absence of 5œ-dihydrotestosterone binding protein in skin fibroblasts. Journal of Clinical Endocrinology and Metabolism 38, Keenan, B. S., Meyer, W. J., Ill, Hadjian, A. J. & Migeon, C. J. (1975). Androgen receptor in human skin fibroblasts: characterization of a specific 17/?-hydroxy-5<x-androstan-3-one-protein complex in cell sonicates and nuclei. Steroids 25, Kinouchi, T. & Horton, R. (1974). 3<x-Androstanediols in man. Journal of Clinical Investigation 54, Kirschner,.. & Bardin, C. W. (1972). Androgen production and metabolism in normal and virilized women. Metabolism 21, Kirschner,.. & Jacobs, J.. (1971). Combined ovarian and adrenal vein catheterization to determine the site(s) of androgen overproduction in hirsute women. Journal of Clinical Endocrinology and Metabolism 33, Kirschner,.., Zucker, I. R. & Jespersen, D. (1976). Idiopathic hirsutism. An ovarian abnormality. New England Journal ofmedicine 294, Kuttenn, F. & Mauvais-Jarvis, P. (1975). Testosterone 5a-reduction in the skin of normal subjects and of patients with abnormal sex development. Acta Endocrinologica 79, Mahoudeau, J.., Bardin, C. W. & Lipsett,. B. (1971). The metabolic clearance rate and origin of plasma dihydrotestosterone in man and its conversion to the 5<x-androstanediols. Journal of Clinical Investigation 50, Mauvais-Jarvis, P., Bercovici, J. P. & Gauthier, F. (1969). In vivo studies on testosterone metabolism by skin of normal males and patients with the syndrome of testicular feminization. Journal of Clinical Endo crinology and Metabolism 29, Mauvais-Jarvis, P., Charransol, G. & Bobas-Masson, F. (1973). Simultaneous determination of urinary androstanediol and testosterone as an evaluation of human androgenicity. Journal ofclinical Endocrinology and Metabolism 36, Mauvais-Jarvis, P., Kuttenn, F. & Baudot, N. (1974). Inhibition of testosterone conversion to dihydro testosterone in men treated percutaneously by progesterone. Journal of Clinical Endocrinology and Meta bolism 38, Pearlman, W. H., Crepy, O. & Murphy, M. (1967). Testosterone binding levels in the serum of women during the normal menstrual cycle, pregnancy and the post-partum period. Journal of Clinical Endo crinology and Metabolism 27, Rosenfield, R. L. (1972). Plasma testosterone binding globulin and indexes of the concentration of unbound plasma androgens in normal and hirsute subjects. Journal of Clinical Endocrinology and Metabolism 32, Southren, A. L., Gordon, G. G., Tochimoto, S., Olivo, J., Sherman, D. H. & Pinzón, G. (1969). Testo sterone and androstenedione metabolism in the polycystic ovary syndrome: studies of the percentage binding of testosterone in plasma. Journal of Clinical Endocrinology and Metabolism 29, Vermeulen,., Verdonck, L., Van der Straeten, M. & Orie, N. (1969). Capacity of the testosterone binding globulin in human plasma and influence of specific binding of testosterone on its metabolic clearance rate. Journal of Clinical Endocrinology and Metabolism 29, Voigt, W., Fernandez, E. P. & Hsia, S. L. (1970). Transformation of testosterone into 17/?-hydroxy-5<xandrostan-3-one by microsomal preparations of human skin. Journal of Biological Chemistry 245, Wilson, J. D. & Walker, J. D. (1969). The conversion of testosterone to 5ot-androstan-17/?-ol-3-one (dihydro testosterone) by skin slices of man. Journal of Clinical Investigation 48,