Male pattern baldness and the metabolism of androgens by human scalp skin

Transcription

1 J. Soc. Cosmet. Chem (1973) 1973 Society of Cosmetic Chemists of Great Britain Male pattern baldness and the metabolism of androgens by human scalp skin K. D. BINGHAM and D. A. SHAW* Presented at the 7th Congress of the International Federation of Societies of Cosmetic Chemists, in Hamburg, Germany, 13th September 1972 Synopsis---Human male scalp skin was incubated with [4-x4C]TESTOSTERONE and the metabolites identified were 5a-androstane-3a, 1713-diol, androsterone, epiandrosterone, dihydrotestosterone, androstenedione, and androstanedione. A non-polar metabolite thought to be a steroid ester was also found. One bald and one hairy SCALP biopsy specimen from each of five subjects were incubated with [4-x4C]testosterone for 1 h and the amounts of each metabolite were measured after separation by thin-layer chromatography. Both the uptake and METABOLISM of testosterone were greater in bald skin than in hairy skin. The percentage of each metabolite in the total metabolite pool was found to be fairly constanthus demonstrating that male pattern BALDNESS was not accompanied by any gross change in the metabolism of testosterone. The main finding was that bald skin contained increased activity of 5a-reductase(s). Four of the metabolites of testosterone, namely dihydrotestosterone, androstenedione, androstanedione, and epiandrosterone were incubated with bald and hairy scalp skin and the metabolites identified. The results from these further studies showed that there was no gross change in the metabolism of these STEROIDS in male pattern baldness and also that the activity of 5ct-reductase(s) was greater in bald skin than in hairy skin from the same subject. INTRODUCTION Since the time of the ancient Egyptians, about years ago (1), the scalp hair has been the subject of considerable attention by both cosmeticians and the medical profession. There are several different types of *Unilever' Research, Isleworth Laboratory, Middlesex, England. 523

2 524 JOURNAL OF THE SOCIETY OF COSMETI CHEMISTS baldness or alopecia but the one which is the subject of these studies is of the male pattern type. This type has been estimated to comprise more than 90 o of all cases of baldness. The term male pattern baldness has been given to this condition because of the characteristic way in which the hair is lost, usually starting with a recession in the fronto-temporal region. Countless numbers of alleged cures for baldness have been formulated ranging from the very old--such as a mixture of crocodile, lion, hippopotamus, and serpent fat--to the very new--such as ultrasonics. However, none of these 'cures' has been successful and at the present time the only effective treatment is the auto-transplantation of small pieces of scalp skin from hairy to bald regions of the head. When the wound has healed the hair on the transplanted skin retains the characteristics of the donor site, i.e. it will continue to grow (2). However, the growth of hair can never spread beyond the boundary of the transplant. The reports that baldness can be arrested or new growth obtained with the male sex hormone testosterone (3), oestrogens (4), or anti-androgens (5) are difficult to substantiate. The use of such substances is often accompanied by undesirable side-effects because of their varied and potent biological activities. On the human scalp, as on the rest of the human body, the formation of hair by individual follicles is intermittent. Thus the growth of hair proceeds in cycles each consisting of an active growth period (anagen, 3-6 years on the scalp) and a resting period (telogen, 2-6 months on the scalp) when the hair is retained as a dead (club) hair in the inactive follicle. Anagen and telogen are separated by a transition phase called catagen which lasts for about 2 weeks. There is a good deal of evidence to show that the hair growth cycle in laboratory animals involves an inherent rhythm which can be slowly modified by systemic factors such as steroid hormones (6) and there seems to be no reason why this explanation should not also apply to human hair. During the balding process the period of anagen becomes progressively shorter while the periods of catagen and telogen do not change to any degree (7). This gradual reduction in the period of the growing phase causes a corresponding progressive reduction in the length of hair grown (and also thickness), an increase in the rate of hair fall, and a decrease in the density of hair on the head (7). Finally the pigmented terminal hairs are replaced by the very short unpigmented vellus hairs which cover the scalp of a bald man. Three factors control the onset and progress of male pattern baldness. These are age, heredity, and androgenic status; normally all of these factors must operate to ensure that baldness will occur. Males under the age of puberty do not go bald, but after puberty the chances of doing so become

3 MALE PATTERN BALDNESS 525 greater with increasing age (8) and by the age of 50, 60 per cent of all Caucasian males have at least some baldness of the temples and vertex. Examination of the genetic aspects of male pattern baldness has shown that the condition is inherited as an incomplete dominant trait (9). The studies of Hamilton (10, 11) have shown that male pattern baldness does not develop in subjects who have been castrated before puberty or in those who fail to reach sexual maturity due to gonadal insufficiency. Those subjects who were castrated when already partially bald showed no expansion of the bald areas even 18 years after castration. Perhaps the most convincing demonstration of the involvement of testosterone in baldness was not only that the administration of testosterone propionate to eunuchs caused loss of their scalp hair, provided that there was a suitable genetic background, but that the progress of the hair loss was halted on cessation of the androgenic treatment (10). There is no evidence for a relationship between the levels of plasma or urinary testosterone or related steroids and the degree of baldness. RATIONALE FOR THE STUDY OF ANDROGEN METABOLISM The action of systemic testosterone can be considerably altered at its site of action by local metabolism and relatively small chemical changes in the molecule can produce large changes in biological activity. Thus protein synthesis in the prostate gland, which is an androgen target tissue, is stimulated by dihydrotestosterone, one of the metabolites of testosterone (12). Skin from the chin, forehead, and back of acne sufferers has been demonstrated to convert testosterone to dihydrotestosterone faster than skin from a similar group of subjects without acne (13). We have examined the metabolism of testosterone and related androgenic steroids in human scalp skin in order to determine whether there are differences in this metabolism between bald and hairy areas of the scalp. Model systems Initially the use of animals was considered but the only candidates who suffer the equivalent of human male pattern baldness are certain primates, principally the stump-tailed macaque (14) and to a lesser extent the Kenyan wattled starling (Creatophora carunculata). Hamilton (15) has shown that male wattled starlings lose their scalp feathers in the mating season in response to increased secretion of endogenous testosterone and regrow

4 526 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS these feathers at the end of the mating season when levels of circulating testosterone fall. Female starlings remain fully leathered throughout the year. We have established a colony of Kenyan wattled starlings at Isleworth and have examined the metabolism of [4-4C]testosterone in vitro in male starling scalp skin. Unfortunately the pattern of metabolism obtained was unlike that obtained with human scalp skin under identical conditions. METABOLISM OF TESTOSTERONE BY HUMAN SCALP SKIN The incubation experiments with testosterone consisted of two main studies. The first was to identify the products of metabolism in scalp skin and the second was to determine any differences in this metabolism between bald and hairy skin. The trivial names for testosterone and most of its metabolites are used throughout this paper. The corresponding systematic names are: testosterone--17 [3-hydroxyandrost-4-en-3-one androsterone--3a-hydroxy-sa-androstan-17-one epiandrosterone--3 [3-hydroxy-5 a-androstan- 17-one dihydrotestosterone--17 [3-hydroxy-5 a-androstan-3-one androstenedione--androst-4-ene-3,17-dione andr ostanedione--5 a-androstane- 3,17-dione Identification The first experiment was performed with skin taken (under general anaesthesia) from the semi-bald scalp of a healthy male subject by means of a 3.5-mm diameter biopsy punch. The thickness of these specimens was 3-4 mm in order to include the hair bulbs. The tissue was stored at 0 ø for 4-5 h until its incubation with [4-4C]testosterone for 5 h at 37øC with shaking under air in tissue culture medium 199. The relevant control incubations were also performed. An aliquot of the acetone extract of skin was chromatographed on a thin-layer plate with standards and autoradiographed. Further aliquots were then separated into their individual radioactive components by preparative layer chromatography. Identification of these components was accomplished by thin-layer chromatography, gasliquid chromatography, and crystallization to constant specific activity after dilution with inactive material. This procedure demonstrated the presence of unchanged testosterone, 5a-androstane-3a, 17 [3-diol, androsterone, epiandrosterone, dihydrotestosterone, androstenedione, androstanedione, and

5 MALE PATTERN BALDNESS 527 an unidentified non-polar metabolite X. The percentage of each of these components in the skin extract is shown in Table L With the exception of the non-polar metabolite X, these steroids have all been found previously in incubations of testosterone with human skin from abdominal and sexual sites (16-18). There was no sign of a metabolite possessing the 5 [3-configuration in this or any other of the incubations described here. Table The metabolism of [4-x4C]testosterone by human male scalp skin. 101aCi of [4-x4C]testosterone (specific activity 58.2 mci/mmol) was incubated with mg of semibald male scalp skin for 5 h at 37øC with shaking under air in 5 ml tissue culture medium 199. I Metabolite Percentage Polar material (origin) 0.5 5a-Androstane-3a,1713-diol + other diol(s) 2 Unchanged testosterone 46 Androsterone 4 Epiandrosterone 2 Dihydrotestosterone 23 Androstenedione 8 An d ro stanedione 13 Unidentified non-polar metabolite X 1 Ancillary experiments In all experiments, hairy skin samples were taken from the occipital region of the scalp which in the majority of balding men remains hairy throughout life. In some preliminary work one hairy skin sample and one sample from a bald region of the scalp were cut parallel to the skin surface at a depth of about 2 mm so that the upper parts contained all the epidermis and substantially all the sebaceous glands and dermis while the lower parts contained mainly adipose tissue and the hair bulbs. Each piece of skin was incubated separately with testosterone under the conditions described above. Thin-layer chromatography of the skin extracts showed that both the upper and lower portions of the hairy biopsy specimen metabolized testosterone to give the metabolites found previously, both halves giving similar proportions of the metabolites. Some differences were observed in the percentages of the various metabolites in the upper and lower portions of the bald biopsy specimens but the experiment served to demonstrate that the metabolism of testosterone is not confined to sebaceous glands or any

6 528 JOURNAL OF THE SOCIETY OF COSMETI CHEMISTS one skin stratum. That steroid-metabolizing enzymes are not confined to the sebaceous glands has also been shown by Wilson and Walker (17) using skin from the leg and the sole of a foot. Whilst skin from only one subject was used by us, it is worth noting that the unidentified metabolite X comprised a greater proportion of the metabolites in the extracts of the lower portions of the skin than in the extracts of the upper portions. Also another unidentified metabolite Y, of polarity intermediate between that of androstenedione and androstanedione was found in the extracts of the lower, but not the upper, portions of each biopsy specimen. A brief investigation of the metabolism of testosterone as a function of time showed that there was considerable metabolism after 1 h and this period was therefore used for the quantitative studies. Quantification of metabolism The second main experiment was carried out using bald and hairy skin samples taken from five balding males under local anaesthesia. The skin specimens were between 3 and 5 mm in diameter and approximately 4 mm deep. These samples were incubated with [4-4C]testosterone for 1 h at 37øC under air within 30 min of being removed from the scalp. The bald biopsy specimen from subject PFW (see Table I1) was taken from an area which had been rubbed daily with an aqueous-alcoholic solution of testosterone (1 o) for 61 days immediately prior to the removal of the skin sample. This Table II Specific uptakes of [4-x4C]testosterone by bald and hairy human male scalp skin in vitro. Incubation of [4-x4C] testosterone with bald and hairy human scalp skin for 1 h at 37øC under air. Donor Uptake of [4-11C] testosterone in Ratio of pmol/mg extracted skin uptakes Bald Hairy (bald/hairy) PFW 318' PA JZ AC CB *This biopsy specimen was taken from the site to which the non-radioactive testosterone had been applied.

7 MALE PATTERN BALDNESS 529 was to determine whether high concentrations of non-radioactive testosterone could alter the metabolism of radioactive testosterone by skin. Each piece of skin was extracted with acetone as before. The uptake oftestosterone by skin per extracted weight of skin is shown in Table II. For donors PA, JZ, AC, and CB, the uptake of testosterone was greater for bald skin than for hairy skin. This situation is reversed for donor PFW and is undoubtedly due to the presence of the unlabelled testosterone that this subject had been applying to his scalp at the site o ' the biopsy. It has been reported that bald scalps contain larger sebaceous glands than hairy scalps (19) and since sebaceous glands are believed to be target organs for androgens (20) this may in part account for the greater uptake of testo~ sterone by bald skin. Other reasons for this difference in uptake might be alteration in the water and/or lipid content of the skin which would affect our data since we have used the weight of acetone extracted skin in the calculation of uptake. The acetone extracts of skin were chromatographed on thin-layer plates with standard steroids and autoradiographed. The standards were then visualized with iodine vapour and the silica gel of both the standards and the areas macenat was eluted and counted. Table III shows the percentage of testosterone, Table III. Metabolism of [4-uC]testosterone by bald and hairy human male scalp skin in vitro. Incubation of [4-xaC]testosterone with bald and hairy human scalp skin for 1 h at 37øC under air. Donor Type of Uptake of Percentage of Amount of Ratio of site testosterone testosterone testosterone amounts (pmol/mg metabolized metabolized (bald/hairy) extracted skin) (pmol/mg extracted skin) PFW Bald* Hairy PA Bald Hairy JZ Bald Hairy AC Bald Hairy CB Bald Hairy! *See footnote to Table II.

8 530 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS taken up by the skin, that was metabolized and also the amount of testosterone metabolized per weight of extracted skin. Comparison of the amounts of testosterone metabolized shows that for one subject (PA) these amounts were virtually equal while for the other three subjects the amount metabolized by the bald skin was greater than that metabolized by the hairy skin. As expected the amount of radioactive testosterone metabolized by the bald skin from PFW was reduced due to the presence of excess unlabelled testosterot e within the tissue. Table IV Distribution of radioactivity among the metabolites of [4-x C]testosterone by bald and hairy human male scalp skin invitro. Radioactivity of the major metabolites separated by thin-layer chromatography as percentages of the total activity on the plate less that due to testosterone. Donor Metabolite PFW PA JZ AC CB Bald* Hairy Bald Hairy Bald Hairy Bald Hairy Bald Hairy Androsterone epiandrosterone Dihydrotestosterone Androstenedione Metabolite Y Androstanedione Metabolite X *See footnote to Table II. The radioactivity of each metabolite expressed as a percentage of the sum of the radioactivities of the metabolites is shown in Table IV. The very low activities associated with the origin, the solvent front, and products more polar than testosterone which comprise less than 1 o of the total activity are not shown. The results show that the percentages of androsterone + epiandrosterone, dihydrotestosterone, and androstanedione remain fairly constant. The percentage of androstenedione however shows more variation, but it can be seen that low formation of this compound is usually coupled with the appearance of the metabolite Y. No one metabolite appeared as the major product in all skin extracts in contrasto skin from other body sites in which dihydrotestosterone appears as the major metabolite. Overall we can say that the patterns of metabolism of testosterone are similar in bald and hairy sites from the same scalp and also in scalp skin from different subjects. Thus we did not obtain evidence for any gross abnormality of testosterone metabolism in male pattern baldness.

9 MALE PATTERN BALDNESS 531 A+E DHT A -AD 5(x-AD Amount of metabolite formed in pmol/ rng extracted tissue Subject JZ Key: Subject AC- Bald Subject CB Figure I. Formation of metabolites from testosterone. A-t-E, Androsterone-lepiandrosterone; DHT, dihydrotestosterone; A 4-AD, androstenedione; 5 -AD, androstanedione. Fig. 1 shows the conversion of radioactive testosterone to the various metabolites expressed in terms of pmol/mg extracted weight of skin (PFW omitted). The conversions of testosterone into androsterone + epiandrosterone, androstenedione, and androstanedione were greater in bald skin than in hairy skin for all subjects. The conversion of testosterone to dihydrotestosterone was greater in bald skin than in hairy for three out of the four subjects whilst no conclusions can be drawn for the conversions to the two unidentified metabolites. In general, the rate of formation in vitro of dihydrotestosterone from testosterone is greater in skin from sexual sites (382 pmol/100 mg tissue/h for prepuce and 534 pmol/100 mg tissue/h for scrotum) (17) than for non-sexual sites (1-46 pmol/100 mg tissue/h) (17, 21). In the present work we have found that scalp skin converts testosterone to dihydrotestosterone at a rate of pmol/100 mg extracted tissue/h. These figures must be reduced by a factor of 3-4 in order to allow for the water and lipid removed from the tissue by the acetone extraction but they are still high enough to indicate that scalp skin is a target tissue for testosterobe. The relative activities of 5e-reductase(s) in the scalp specimens can be estimated by summing the amounts of 5e-reduced products (androsterone,

10 532 JOURNAL OF THE SOCIETY OF COSMETI CHEMISTS epiandrosterone, dihydrostestosterone, and androstanedione) formed per hour per milligramme of extracted tissue. Since the structure of metabolite X has not been determined this sum has been calculated both including and excluding this compound. These data (Table V) show that for one subject (PA) there was about the same amount of 5a-reduced metabolites in the bald and hairy skin specimens but for the other three subjects more 5areduction had occurred in bald skin than in hairy skin. With the reservation that we have examined only a small number of subjects we suggest that these increased levels of 5a-reductase are an expression of the genetic factor in male pattern baldness. Table V Ratio of formation (pmol/h) of 5a-reduced metabolites* of [4- C]testosterone per mg extracted tissue in bald scalp skin to that in hairy scalp skin Donor Ratio (bald/hairy) Ratio (bald/hairy) assuming that assuming that metabolite X has a metabolite X does not 5a-reduced structure have a 5a-reduced structure PA JZ AC CB *Dihydrotestosterone, androsterone, epiandrosterone and androstanedione. The data that we have here concern the rate of formation of the metabo- lites and not the pool size for any one metabolite in the skin. However, the apparently increased activity of 5a-reductase in bald skin could lead to an increased pool size either for dihydrotestosterone, which Adachi (22) has implicated in the balding process, or indeed any other product of 5a- reduction. Unidentified metabolites 3[ and Y Metabolite X was not acetylated by acetic anhydride-pyridine at room temperature overnight but when it was treated with 2,4-dinitrophenylhydrazine it formed a derivative whose polarity indicated that X possessed one carbonyl group. These results taken together with the chromatographic

11 MALE PATTERN BALDNESS 533 mobility of X suggesthat this metabolite is an ester of asteroid and one or more fatty acids. Further work on the identification of this compound is continuing. The metabolite Y was not found in all extracts and its identification not pursued. was FURTHER TRANSFORMATIONS OF TESTOSTERONE METABOLITES Having incubated bald and hairy scalp skin with [4-x4C]testosterone, we decided to examine the further transformations of its metabolites in scalp tissue. Since, as has been stated above, dihydrotestosterone is believed to be the active form of testosterone at least in the prostate (12, 23) this metabolite was examined first. [4-x4C]Dihydrotestosterone was prepared by reduction of [4-x C] testosterone with lithium in liquid ammonia and then subsequent purification by thin-layer chromatography. One bald and one hairy scalp biopsy specimen from each of three subjects were incubated with [4-x C]dihydro - testosterone under the same conditions used for [4- C]testosterone. The work-up procedure was also identical to that used for the testosterone study. The uptake of dihydrotestosterone by skin was of the same order as that of testosterone but the amount of dihydrotestosterone metabolized, whilst being very similar in all the skin specimenstudied, was less than that of testosterone. The products of metabolism (identified by the techniques described previously), were androstanedione, a mixture of the two isomeric diols 5a-androstane-3,17 -diol and 5a-androstane-3a, 17 -diol in the ratio of about 3: 2, and an unidentified non-polar product. Neither androsterone nor epiandrosterone was found in any of the incubations. There were no consistent differences between bald and hairy sites in the uptake of dihydrotestosterone or in their qualitative and quantitative patterns of metabolism. Thin-layer chromatography in several systemshowed that the unidentified metabolite was a single compound which had the same chromatographic mobilityas compound X from the testosterone incubations and like X it did not form an acetate under standard conditions. The next radioactive steroid substrate studied was [4-x C]androstene - dione. Again one bald and one hairy biopsy from each of three subjects were incubated under the conditions used previously. The uptake of [4-4C]androstenedione by skin was similar to that of testosterone. The main product of metabolism was androstanedione and its formation was 1.06,

12 534 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS 2.8 and 3.2 times greater in bald skin than in hairy skin for the three subjects studied. Since the enzyme which converts androstenedione to androstanedione is a 5a-reductase, these results demonstrate that two out of three subjects have a higher 5a-reductase activity in bald skin than in hairy skin. This is in agreement with the results obtained from the incubations of testosterone. Small amounts of androsterone and epiandrosterone were identified but we were unable to detect the presence of testosterone which would be formed by reduction of the 17-oxo-group of androstenedione. The third metabolite to be studied, [4- x4c]androstanedione, was prepared by reduction of [4-x C]androstenedione with lithium in liquid ammonia. One bald and one hairy biopsy specimen from each of three subjects were incubated with [4- C]androstanedione under the conditions used for the other experiments. The uptake of this steroid by skin was similar to those of the other androgens. The amount of metabolism was small, androsterone and epiandrosterone being the only products. There was no evidence that reduction of the 17-oxo-group had occurred to give either dihydrotestosterone or a 3,17-diol. O, Ar rostendione! I.(;}H 1 0 Androstanedione i0 I Testosterone ' And sterone 1. H. > B Epiandroste Figure 2. OH HO o< Androstane H Metabolism of testosterone in human male scalp skin.

13 MALE PATTERN BALDNESS 535 The last steroid to be incubated was [4-4C]epiandrosterone which was prepared in the radioactive form by the hydrogenation of [4-4C]dehydro - epiandrosterone over Adam's catalyst. One bald and one hairy skin sample from each of two subjects were incubated under the standard conditions used previously. The uptake by skin of this steroid was higher than that for testosterone. The products of metabolism were androstanedione and a nonpolar metabolite. This non-polar metabolite had a similar chromatographic mobility to the mobilities of the non-polar metabolites from the testosterone and dihydrotestosterone incubations. An unidentified non-polar metabolite has therefore been found in the incubation of steroids possessing a hydroxyl group (either in the 3- or 17-position) but not in those where the substrate is a diketone. This fact, coupled with the chemical and chromatographic properties of metabolite X, suggests that all three unidentified metabolites are steroid esters. The identification of these metabolites is continuing. The data from all the incubation studies reported here can best be summarized in the form of a metabolic scheme shown in Fig. 2. Although we have not incubated androsterone with scalp skin it seems likely that its metabolism will be as indicated in the figure. The unidentified metabolites have been omitted for the sake of clarity. (Received: 9 February 1973) REFERENCES (1) Byron, C. P. (translation) The papyrus ebers (1931)(Appleton& Co., New York). (2) Orentreich, N. Hair transplants. Arch. Otolaryngol (1970). (3) Papa, C. M. and Kligman, A.M. Stimulation of hair growth by topical application of androgens. J. Amer. Med. Ass (1965). (4) Shapiro, I. Premature baldness. Preliminary report on treatment with topically applied estrogen lotion. J. Med. Soc. N.J (1953). (5) Sunday Times, London, 31st May, p. 53 (1970). (6) Ebling, F. J. and Hale, P. A. The control of the mammalian moult. Mere. Soc. Endocrinol. 18 (1970). (7) Barman, J. M., Pecoraro, ¾. and Astore, I. Biological basis of the inception and evolution of baldness. J. Gerontol (1969). (8) Hamilton, J. B. The role of testicular secretions as indicated by the effects of castration in man and by studies of the pathological conditions and the short lifespan associated with malehess. Recent Progr. Hotre. Res (1948). (9) Snyder, L. H. and Yingling, H. C. Studies in human inheritance. XII. The application of the gene-frequency method of analysis to sex. Influence factors, with specific reference to baldness. Hum. Biol.! (1935). (10) Hamilton, J. B. Male hormone stimulationis prerequisite and an incitant in common baldness. Amer. J. Anat (1942). (11) Hamilton, J. B. Effect of castration in adolescent and young males upon further changes in the proportions of bare and hairy scalp. J. Clin. Endocrinol. Metab (1960).

14 536 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS (12) (13) (14) (15) (16) (17) (18) (19) (20) (21) (22) (23) Bruchovsky, N. and Wilson, J. D. The conversion of testosterone to 5a-androstan-17 -ol- 3-one by rat prostate in vivo and in vitro. J. Biol. Chem (1968). Sansone, G. and Reisner, R. M. Differential rates of conversion of testosterone to dihydrotestosterone in acne and in normal human skin--a possible pathogenic factor in acne. J. Invest. Dermatol (1971). Montagna, W. and Uno, H. Baldness in non-human primates. J. Soc. Cosmet. Chem (1968). Hamilton, J. B. A male pattern baldness in wattled starlings resembling the condition in man. Ann. N.Y. Acad. $ci (1959). Gomez, E. C. and Hsia, S. L. In vitro metabolism of testosterone-4-x4c and A 4-androstene - 3,17-dione-4-4C in human skin. Biochemistry (1968). Wilson, J. D. and Walker, J. D. The conversion of testosterone to 5a-androstan-1713-ol- 3-one (dihydrotestosterone) by skin slices of man. or. Clin. Invest (1969). Flamigni, C., Collins, W. P., Koullapis, E. N., Craft, I., Dewhurst, C. J. and Sommerville, I. F. Androgen metabolism in human skin. J. Clin. Endocrinol. Metab (1971). Ellis, R. A. Ageing of the human male scalp. The biology of hair growth eds W. Montagna and R. A. Ellis (1958). (Academic Press, New York). Calman, K. C., Muir, A. V., Milne, J. A. and Young, H. Survey of the distribution of steroid dehydrogenases in sebaceous glands of human skin. Brit. J. Dermatol (1970). Jenkins, J. S. and Ash, S. The metabolism of testosterone by skin in normal subjects and in testicular feminization. J. Endocrinol (1971). Adachi, K. and Kano, M. Adenyl cyclase in human hair follicles: its inhibition by dihydrotestosterone. Blochem. Biophys. Res. Commun (1970). Anderson, K. M. and Liao, S. Selective retention of dihydrotestosterone by prostatic nuclei. Nature (London) (1968).