We conducted this study to establish initial base line data for the metabolism of 3H-steroid hormones (testosterone, dehydroepiandrosterone, progesterone, and estradiol) in skin and its appendages, the isolated sebaceous glands. Additionally, we wished to know whether or not changes occurring in aging skin and a relative lack of endogenous hormones alter the skin's capacity to metabolize steroid hormones in vitro. Eighty-seven biopsy samples from the preauricular area of the cheeks w e re

more »

... ed from 13 young women (ages 18 to 30) and 3 postmenopausal women (ages 60 to 68). Values were obtained for the rate of metabolism of tritiated testosterone,. estradiol, progesterone, and dehydroepiandrosterone in the isolated sebaceous ghinds and dermis of the women. Wide individual variation was observed in sebaceous gland metabolism in both young and old women. The patterns of 3 H-steroid metabolites contained in the dermis were similar in the younger and older women. However, the ratios of precursors to metabolites in the sebaceous glands of the older women differed from the ratios of the younger ones. The pattern of in vitro lipid synthesis from I'le-acetate in sebaceous glands of the 2 groups was not significantly different. The most interesting relationships derived from this s tudy were the relative molar uptakes of 3H_ steroids. Tritiate d progesterone was present in much greater quantities than either :IH-androgens or :IH-estrogens, in both dermis and sebaceous glands. On a weight basis the sebaceous glands were much more active than the dermis in the metabolism of :lH-steroid hormones. Initial data of this type may be use d as a conceptual framework within which c3anges associated with various pathologic and physiological states such as aging, acne, hirsutism, and alopecia can be examined. Androgen m etabolis m in whole human s kin and pluc ked hair follicles has been studied by m a ny investigator s [1-5]. Little, however, is known about the m etabolis m of other steroid hormones in the individual compone nts of huma n s kin. This work provides initia l base line data on steroid hormone m etabolism in the isolated human sebaceous gla nds and d ermis. The changes commonly d escribed in aging s kin include an irregularity in the size, shape, and pattern of the epidermal cells; a diminution of the interfibrill ary ground s ubstance of the upper dermis; a nd a d epletion in the numbers of fibroblasts, his tiocytes, perivascular adventitia l cells, and m ast cells [6]. Most in ves tigators have d escribe d cell d e pletion as a characteristic feature of aging s kin . C h a nges in numbe rs and secretory functions of fibroblasts of aged s kin h ave been re pOIted , as we ll Rep rin t req uests to: Gail Sanso ne-Bazzano, Ph .D., Department 01" Medicine, Division of Dermatology, Un iversity of California at Los Ange les School of Med icin e, Los Ange les, CA 90024 . Abbreviations: GLC: gas-li qu id chromatogra phy TLC: thi n-layer chromatog rap hy as a d ecreased cell turnover in sebaceous glands d espite seba· ceous g land hyperplasia [7]. Therefore, we obtaine d data to d etermine whether in pos~ m e nopausal women the d ermis, sebaceo us glands, or both wo ul maintain the capacity to m etabolize steroid hormones at levels som ewhat s imilar to those found in yo ung wom e n . W e also wis h ed to find out whether, as a result of d ecr eased levels of circulating steroid h ormones, the patte rn of in vitro lipid s~n thesis in the sebaceous glands of o lder women would be sign Iiicantly a lter e d . MATERIALS AND METHODS The 87 skin biopsy samples used in this study were obtained from !} youn g women 18 to 30 yr of age and 3 postmenopausal women 60, 60, and 68 yr of age. All were in good health and were no t taking al1~ medica tion. The postmenopausal women had visibly aged skIn a 1l 1 w.ere undergo ing corr~ctive fa cia l ~perations . Samples ~2-mm pun c o ! bIOpSIes) were taken from skll1 eXCIsed from the preau n cular area the cheek, placed in chilled medium (Gibco #243), and transported l~ the labo rato ry in an ice bu cket. The subcutaneo us fat was removed 8 11 : approximately 20 to 30 mg of tissue were routine ly used in ea c I incubation tube. C· The incubation mixt ure consisted of 2 !LCi of I"C-acetate and 10 11 ~ of 1 of the tritiated steroids, Gibco 243 med ium, and penicillin .a l1 strepto mycin (2,500 U) in a total vo lu me of 1.5 ml. The specific activltte~ 01" the tritiated steroids (obtained from, New England Nuclear) wer~:/ fo llows: "H -testosterone-IO CI/ mM ; 3H-estrad lOl-46.6 CI/ mM , prog~stero ne,-10 .Ci/mM.; a nd "H -de hydroe piandrosterone ('H-DHA~ 20 CI/ mM . 1 he tncubatlon tubes were gassed With 95% 0 " and 5%. C 30 I capped, and incubated at 37°C in a Dubnoff metabolic shaker {or min, 1 hI', and 3 hr. The reaction was te rminated by quick-freezing .. The samples were placed in a so lu~i o n of cold 1 M CaC~" for 30 n::~ to 1 hr for easIer nllCrOdlssectlO n. 1 he epIdermiS, haIr follicl es, a g sebaceous glands were stripped away from the dermis under a disse cttJ1 , microsco pe. Figure 1 is a photograph of se baceo us gla nds isolated b.\ this proced ure. . I The possibili ty that diff~sion ca u s~d by soaking in CaCb solu ttf,~ mIgh t a lte r the a moun ts of sterOIds found co mpartmenta lized In t . various skin components was examined in se veral preliminary exper; • ments. S kin sampl es were a llowed to stand in 1 M CaCb solu tIon I D e var yin g periods of time. A ha lf hour to 1 hI' was fin a lly selected as t Ig soaking time necessary to fac ili tate microdissection without ca uSills significant diffusion «4%). Th is in te rval, however, posed pro blen \ with attempts at qu antitative recove ry since it ":as impos~ibl e to di5~:o a ll se baceous glands from the samples. Soakll1g overl1lght proV I Is better yields of se baceous gland s, but it a lso caused unaccepta ble le Ve ll8 of diffusion. )"c Ca re was ta ken to avo id cross-co ntamination . The tissues we i se parated, weighed, and homogenized in Potter glass tissue homOg c1 \ zers; the lipids were extracted by the method of Bl igh and Dyer [~h The chloroform extract W<lS dried und er N". Zero time co ntrols Wi e tissue a nd co ntrols co nta ining the incubation mixture minus tissue w cr run for 3 hr. .•. We used thin-layer chromatography (TLC) and gas-l iq uid chroJ1l< tograph y (GLC) to id e ntif~y and quantify the steroid metabolites. )"1 Aliquots of the lipid fraction co nta ining radioactive steroids we '!' separated chromatographically on TLC a nd GLC along with a mixl.lll co ntaining 20 p.g each of authentic calTi er steroids. Glass plates were coated with sili ca ge l G (Brinkman Instrulnen(~ Inc. , Westbury, N.Y.) a nd activated at 110°C for 1 hr. The plates ' ' 'C \ developed by asce nding chromatog ra phy in 96% benzene:4 metha. llo ; The neutral lipid fract ions were se pa ra ted chromatographically til