Androgens in Skin Surface Lipids* Mary Ellen Karunakaran, Ph.D
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THE JOURNAL OF iNVESTIGATIVE DERMATOLOGY Vol. 60 , No.3 Copyrig h t © 1973 by The Williams & Wilkins Co. Printed in U.S.A. ANDROGENS IN SKIN SURFACE LIPIDS* MARY ELLEN KARUNAKARAN, PH.D. , PETER E. POCHI, M.D. , JOHN S. STRAUSS, M.D. , EUGE lA A. VALERIO, M.A., HERBERT H. WOTIZ, PH.D. , AND STANLEY J . CLARK, PH.D ABSTRACT Sebum collected from t high, scalp, back, a nd face skin of adult males was examined for t he presence, in either free or conjugated state, of seven androgens. Estimates of t he quantities present were made by gas chromatographic a nalysis ailer separation of free from conjugated steroids, solvolysis of t he conjugates, preliminary purification by thin-layer c hromatography, and formation of t he t rimethylsil yl derivative of t he hydroxysteroids. Losses during t hese procedures were estimated by measuring t he recovery of 3H -labeled steroids added to t he sebum before treatment. Suggestive evidence was found for t he presence of a ndrostanedione, androstenedione, dihydrotestosterone, androsterone, and dehydroepiandrosterone. The a mounts obtained were much lower t han values that have been reported previously a nd were insufficient for full characterization . Testosterone and etiocholanolone were not detected. A role for t he sebaceous g lands in t he excretion or C 19-steroids in sebum by factors of 3 to 100 of androgenic steroids has been suggested by depending o n t he particular compound. Dubovyi [1] a nd by Oertel and Treiber (2] who analyzed sebum coll ected from t he scalp and MATERIALS AND METHODS thigh , respectively, and reported fi nding micro Solvents. Analytical-grade olvents were distill ed in gram amounts of steroids. S in ce t hese aut hors an all -glass system through a 60-cm Vigreux colu mn . used spectrophotometric methods which m ay Chloroform was distilled once. Cyclohexane, acetone, ov erestimate quantities, we considered i t worth methanol, ethanol, and benzene were distilled twice. while to attempt to confirm t hese resul ts using gas n-B utanol was distilled once over KOH, then redi stilled wi thout KOH. Petroleu m chromatography (GC) and to examine a lso t he ether was washed with con ce ntrated H,SO, unt il the acid no longer turned yell ow, face and back, a reas of m ajor sebaceous gland then was washed successive ly with water, 8% NaHCO,, activity. Sebum from adult m ales was coll ected and water until neutral. It was dried over Na2SO, and from t h ese several sites, and radioactive steroids then disti ll ed tw ice. Diethyl ether was washed with 50 were a dded to m onitor recovery. After preliminary ml/l of a 13% AgN O, solu tion, then with 100 ml/1 of N purification and derivative formation t he q u ant i NaOH. After washing to neutrality with water, the ether ties of severa l androgenic o r C ,9-steroid s were was distil led. Ethyl acetate was stored over K,CO, estimated b y gas chrom atography. Identification ove rnight t hen distilled once over fres h K,CO, and once was based on t he peak augmentation when small without any K,CO, . Radioactive ·steroids. Androstanedione-1 , 2- 'H was amounts of known compounds were chrom ato synthes ized fro m androsterone-!, 2-'H by chromic ox i gr aphed s imultaneously with t he sample. S ugges dation according to Neher, Desaulles, Visher, Wieland, tive evidence was fo und fo r the presence of a and Wettstein [3). Ten 11 Ci of the parent compound we re num ber of a ndrogenic steroids including dissolved in 0.5 ml of pyridine, and 2.0 ml of 1% CrO, in dihydrotestosteronet, a compound which has not pyrid in e were added. The reaction mixture was all owed been recovered previously from sebum. Our quan to stand at room temperature for 16 hr. The pyridine titative results suggest, however, t hat previous solu t ion was diluted with water and extracted with reports overestimate the quan t ities of androgenic chloroform: ether (1: 3) three times, then the organ ic phase was washed wi th 6 N HCl, 0.1 N NaOH, and water un til neutral. The derived product was separated from Manuscript received October 6, 1972; accepted fori the parent by thin -layer chromatography in a system of publication October 27, 1972. benzene: ethyl acetate (7: 3). Other ' H-labeled steroids This wo rk was supported in part by USPHS research were purchased from New England Nuclear Corp. T heir grants AM07084 and CA03135. *From t he Departments of Dermatology and Bio specific activity was approximately 40 Ci/mmole. chemistry, Boston Uni ve rsity School of Medicine, Bos Thin-layer plates. Plates (20 x 5 em) were coated with ton University Medical Center, Boston, Massachusetts a 0.25-mm layer of Silica Gel G (Brinkmann Instru 02 118. ments). T he plates were was hed for 48 hr with methanol, t The followi ng trivial names were used: androstane then for 48 hr with ben ze ne, using descending elution. diane, 5a-androstane-3, 17-dione; androstenedione, The plates we re activated for 1 hr at 100° C, then stored androst-4-en-3, 17-d •one; androsterone, 3a-hydroxy-5a in a desiccator over CaCl,. androstan-17-one; dehydroepiandrosterone, 3{3- hyd roxy androst-5-en-1 7-one; dihydrotestosterone, 17 /3- hydroxy- Collection of sebum. Sebum was co ll ected from the 5a-androstan-3-one; etiocholanolone, 3{3-hydroxy-5{3-a n thi gh and back by swabbing the s kin with cotton or drostan-17 -one; testosterone, 17 /3- hydroxy-a ndrost-4-en- rayon ba lls soaked in solvent. Before use, the ball s were 3-one. extracted wi th several changes of ethanol- acetone ( 1: 1), 121 122 THE JOURNAL OF INVESTIGATIVE DERMATOLOGY and then allowed to air-dry. Each area of the thigh or solved in 100 ml of water. After 50 gm of (NH,) ,SO, were black skin was swabbed twice, first with ether- ethanol dissolved in the aqueo us solution, the conjugates were water (1: 4: 1), and then with ethyl acetate. extracted with two 25-ml portions of ethyl acetate. The Collections from the thighs were carried out in 12 ethyl acetate was removed by evaporation and the normal male subjects. Surface lipid was extracted from residue was redissolved in 50 ml of ethyl acetate contain both anterior thigh surfaces (approximately a 100-crn' ing 0.01 M HClO,. The solvolysis was allowed to proceed area for each} in all subjects and the co llections were at room temperature for 3 _hr; the solution was then pooled. washed with 5 ml of 10% aqueous KOH followed by 1 ml In 11 men, washings were obtained from the entire of water. The ethyl acetate was removed by evaporation, upper back area (approximately a 500-cm' area for each} and the residue was dissolved in 35 ml of 1% ethanolic and again· the collections were pooled for analysis. KOH. The reaction mixture was held at room tempera The solvent-soaked balls were transferred to a large ture for 1 hr in order to hydrolyse any acetates formed Soxhlet extractor and extracted with ethanol- acetone during solvolysis. The solution was then diluted wi th (1: 1). The solvent was removed from the extract by water and the freed steroids were extracted with two evaporation under reduced pressure. 20-ml portions of petroleum ether- benzene (1: 1) . Thin Facial sebum was collected from 10 normal men using layer chromatography revealed that the added 'H-dehy. the same solvents as for the thigh and back. However the droepiandrosterone sulfate was virtually completely hy solvents were pipetted directly onto the skin surface in drolysed by this procedure. No destruction of the dehy glass cups with an internal area of 10 ern'. Both sides of droepiandrosterone was observed when the solvolysis the forehead were sampled and all collections from all time was limited to 3 hr. subjects were combined. Thin-layer chromatography. Free steroids were sepa Scalp sebum was collected by washing the scalp rated from conjugated steroids in a solv ent system of directly with ether. The solvent was removed by evapo t-butanol-ethyl acetate-5 N NH,OH (41: 50: 20) [5]. The ration and the sebum was weighed. free steroids were then separated into four fractions in Addition of radioactive steroids. Radioactive steroids chloroform-ether (95: 5). The fractions and their re were added to the sebum in order to monitor the losses spective Rf values were: I, testosterone and etiocholano incurred during the purification procedures and also to lone, Rf ~ 0. 28; II, androsterone, dehydroepiandroster make it possible to locate zones on thin-layer chromato one, and dihydrotestosterone, Rf = 0.44; III, andros grams by scanning the plates for radioactivity. As each tenedione, Rf ~ 0.62; and IV, androstanedione, Rf = of the steroids was added to the sebum, an equal amount 0.80. For further purification, fractions I and II were was measured into a sc intillation vial to serve as a each rechromatographed in cyclohexane- ethyl acetate standard for the radioactivity added. Approximately 1 '40: 60). Fractions III and IV were each rechromato ~tCi of each of the following steroids was added to the graphed in benzene- ethyl acetate (80 : 20). samples of thigh, back, and face sebum: androstane Derivative formation. In order to increase the sensitiv diane, androstenedione, dihydrotestosterone, and testos ity of the gas chromatography, the hydroxysteroids were terone (all 1, 2- 3H).