In Vitro Studies of Adrenal Steroid Metabolism in the Skin* Frederick D

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IN VITRO STUDIES OF ADRENAL STEROID METABOLISM IN THE SKIN* FREDERICK D. MALKINSON, M.D., MING W. LEE, B.S. AND I. CUTUKOVIC, CON. EN Sc.

The therapeutic effectiveness of topical hydro-begun within eight to ten hours after the surgical cortisone preparations for certain inflammatory—specimen was obtained. Where cadaver skin was chiefly eczematous—dermatoses has been wellused, incubations were not begun until 10—20 established (1—4). Several studies have alreadyhours after death. Incubations with coriuxu or demonstrated that hydrocortisone penetratesepidermis alone were carried out only with fresh both normal and abnormal skin sites in manskin samples after separation of these layers by (5—7), but to date there is little knowledge ofthe stretch method (11). the subsequent fate of this compound following Four to five grams of skin slices 0.5 to 1.0 mm. its percutaneous absorption (8B). The presentthick were placed in 30 cc. of phosphate-ringer investigation was carried out to determinebuffer solution containing 200 micrograms per cc. whether or not hydrocortisone undergoes activeof hydrocortisone (free alcohol) or cortisone metabolic change in the skin and, if so, in what(free alcohol) 4 The flask was placed under con- part of the skin this change takes place. Instant agitation at 37°C for six hours, following addition, it seemed of interest to conduct similarwhich the incubation was stopped by the addi- studies with cortisone—a relatively ineffectivetion of ethyl acetate. steroid when used topically in dermatologic At the end of the incubation period, extraction therapy (8, 9)—to compare the metabolic altera-of steroids from both the phosphate-ringer buffer tions of this compound and its speed of metabolicsolution and the tissue slices was carried out with change with the findings for hydrocortisone. 100 cc of ethyl acetate for a period of one hour. After repeating this procedure twice again, the METHOD extracts were combined and the ethyl acetate The technic employed consisted of a series ofdistilled off in vacuoat37°C. The residue was in vitro incubations of fresh human skin slicesthen redissolved in a final volume of 2 cc. of with cortisone or hydrocortisonef in phosphate-methanol. ringer buffer solution. These incubations were The apparatus employed for the paper parti- followed by the extraction of steroid substancestion system consisted of a covered glass tank and the separation of these compounds by paperwhich was kept in a closed non-ventilated room chromatography according to the method ofat about 30°C. Because of the volatility of the Bush (10). solvents used, saturation of the atmosphere inside Except for six tissue incubations utilizingthe tank was obtained by covering the tank walls skin samples at post-mortem examination, allwith Whatman ' 3 filter paper which continually incubations were carried out with fresh normalsoaked up both phases of the solyent system. human skin samples removed from surgicalThe chamber was always allowed to saturate specimens. Skin samples were kept in physio-overnight prior to use. logical saline at refrigerator temperatures until The solvent system used was benzene (mobile skin slices were prepared, but all incubations werephase) and equal parts of absolute methanol and *Fromthe Section of Dermatology, Departmentdistilled water (stationary phase). of Medicine, The University of Chicago (Chief of Chromatograms were prepared from 49 cm. Service: Stephen Rothman, M.D.), Chicago, Ill. This study was supported in part by Publicstrips of Whatman 43 filter paper, the flow of Health Service Grant No. E-1444(C2), and in partthe mobile phase for this length of paper requiring by the A. B. Kuppenheimer Endowment Fund. Presented at the Nineteenth Annual Meetingabout four hours. The paper was first allowed to of the Society for Investigative Dermatology,equilibrate for two to three hours before the Inc., San Francisco, California June 22, 1958. f All steroid compounds useI in this study were These concentrations were chosen because kindly supplied by the Upjohn Company, Kala-they are within the limits of water solubility for mazoo, Michigan. these compounds. 101 102 THE JOURNAL OF INVESTIGATIVE DERMATOLOGY mobile phase was added. All papers were runextracts from all of these incuhations yielded two with both the incubate extracts and standardspots, one corresponding in RF value to the solutions of hydrocortisone (free alcohol) andreference standard for hydrocortisone and the cortisone (free alcohol) in methanol. Afterother—representing unchanged substrate—cor- running and drying, the sheets were exposed toresponding to the reference standard for corti- an ultraviolet light source emitting nearly all ofsone. RF values for the spots corresponding to its energy in the region of 2540A (cold quartzhydrocortisone deviated from the RF value of mercury vapor lamp with Corning 9-54 filter).the standard hydrocortisone solution by less than Steroids were detected by the appearance of0.05. dark spots on the exposed paper. The sensitivity Two 6-hour incubations with 125—150 of the method permitted certain identificationmilligrams of epidermis alone added to the of steroids in amounts as low as 6 microgramscortisone-phosphate-ringer buffer solution yielded per spot. RF values were 0.24 for hydrocortisoneno detectable steroid spots on chromatography and 0.40 for cortisone. except for the substrate cortisone. Two incubations of whole skin slices with RESULTS cortisone-phosphate-ringer buffer solution were Four 6-hour ineubations of fresh whole skinrun for three hours instead of six hours. On paper or corium slices from three skin samples werechromatoraphy steroid extracts from these carried out in phosphate-ringer buffer solutionincubations yielded two spots, one corresponding containing hydrocortisone (free alcohol) 200in RF value to the reference standard for hydro- micrograms per cc. On paper chromatography,cortisone and the other to the reference standard steroid extracts from all of these incubationsfor the substrate cortisone. yielded two spots, one corresponding in RF Three 6-hour incubations in cortisone-phos- value to the reference standard for cortisone andphate-ringer buffer solution were carried out with the other—representing unchanged substrate—whole skin slices obtained from three cadaver corresponding to the reference standard forskin specimens. Following extraction of steroids, hydrocortisone. RF values for the spots cor-no other spots other than those corresponding to responding to cortisone deviated from the RFcortisone were detectable on paper chromatog- value obtained from the standard cortisoneraphy. solution by less than 0.05. Three incubations of fresh whole skin slices Two incubations of whole skin slices withfrom three skin specimens were carried out in hydrocortisone-phosphate-ringer buffer solutionphosphate-ringer buffer solution containing pred- were run for three hours instead of six hours.nisone 200 micrograms per cc. Three similar Paper chromatographs of the steroid extractsincubations were carried out in phosphate-ringer showed no detectable spots except for the sub-buffer solution containing prenisolone 200 strate hydrocortisone. micrograms per cc. These incubations were Two 6-hour ineubations with 125—150 milli-stopped after 5, 6, and 9 hours. Following grams of epidermis alone added to the hydro-extraction of steroids, chromatography yielded cortisone-phosphate-ringer bufer solution yieldedno spots other than those corresponding to ref er- no detectable steroid spots on chromatographyence standards for the substrates. except for the substrate hydrocortisone. From two papers run with steroid extracts of Three 6-hour incubations in hydrocortisone-fresh skin incubations, the spots corresponding to phosphate-ringer buffer solution were carried outthe metabolic transformation products of hydro- with whole skin slices obtained from three cadavercortisone and cortisone were eluted with meth- skin specimens. Following steroid extraction, noanol. The eluates were then rerun on paper to- spots other than those corresponding to hydro-gether with standard solutions of hydrocortisone cortisone were detectable on chromatography. and cortisone according to the method described Five 6-hour ineubations of fresh whole skinabove. The RF value of the transformation or corium slices from four skin samples wereproduct of hydrocortisone, which previously carried out in phosphate-ringer buffer solutionhad been identical to that of cortisone, again containing cortisone (free alcohol) 200 micro-corresponded to that of the standard cortisone grams per cc. On paper chromatography steroidsolution. Similarly, the R value of the trans- IN VITRO STUDIES OF ADRENAL STEROID METABOLISM 103 formation product of cortisone, which previouslyspecific hormone with its possible chemical had been identical to that of hydrocortisone,transformations has always been of interest. The again corresponded to that of the standard hy-anti-inflammatory effectsof cortisone and drocortisone solution. In addition, samples ofhydrocortisone are not markedly different when these same eluates were then run in a differentthese compounds are administered systemically. solvent system (mobile phase: light benzene andYet, despite apparently comparable absorption petroleum; stationary phase: methanol andof these hormones through the skin (5, 17) water) (10). The steroid transformation producttopical hydrocortisone is impressively anti- previously recovered following tissue incubationinflammatory in the treatment of certain eczem- with hydrocortisone again corresponded in Ratous dermatoses (1—4) while locally applied value to the control solution of cortisone. Thecortisone is relatively ineffective (8, 9). It has steroid transformation product recovered frombeen postulated that the difference in topical tissue incubations with cortisone alsocor-therapeutic efficacy of these compounds might responded in RF value to the control solution ofbe related to differences either in the pathways of hydrocortisone. metabolism or in the speed of metabolic inctiva- Several control incubations were run at 370Ction of these steroids in the skin. These alterna- with 30 cc. of phosphate-ringer buffer solutiontive explanations, however, have been further alone and with the same solution containingbased on the premise that hydrocortisone and four to five grams of whole skin without thecortisone actually undergo metabolic alteration addition of hydrocortisone or cortisone. Thesein the skin before passage into the systemic circu- incubations were "stopped" at the end of sixlation. The present study was undertaken to hours by addition of ethyl acetate, followingexamine both of these problems more closely. which they were "extracted" with ethyl acetate That adrenal steroid metabolism may actually as previously described. No spots were detectabletake place in the skin has been shown by thee on paper chromatography following these controlin vitro studies described. Since metabolic change procedures. Similar control "incubations" carriedwas demonstrable only with tissue slices of whole out with phosphate-ringer buffer solution con-skin or corium it can be concluded that alteration taining either hydrocortisone or cortisone, 200of hydrocortisone or cortisone occurs mainly in micrograms per cc., failed to yield any spot onthe connective tissue. Nonetheless, while incu- paper chromatography other than that corre-bation studies with epidermis were negative, sponding to the steroid orginally added. epidermal specimens with tissue weights com- Several papers obtained from extracts of freshparable to those of the corium must be evaluated skin incubations with either hydrocortisone orbefore concluding that metabolic alteration of cortisone were sprayed with a solution of NaOHthese compounds occurs in the dermis alone. in methanol. This technic, recommended by Bush Some inferences can now be drawn concerning (10) for increasing the sensitivity of the methodthe metabolic pathways of hydrocortisone and used for the detection of aB-unsaturated keto-cortisone in human skin. The six hour in vitro steroids, failed to show additional steroid spotsincubation studies indicate that hydrocortisone on any of the papers examined. (free alcohol) is partly converted into a compound possessing the paper chromatographic rnscussloN characteristics of cortisone. Similarly, under Numerous investigations have demonstratedcomparable experimental conditions, cortisone the capacity of various tisues to effect the chemi-(free alcohol) is partly converted into a com- cal transformation of steroid hormones added topound possessing the paper chromatographic in vitro systems. Much of this work has beencharacteristics of hydrocortisone. Although no summarized in two comprehensive reviews (12,attempts were made in either case to determine 13). In addition Wilson et al have published aquantitatively the precise extent of this con- series of reports on the in vivo metabolism ofversion, it could be estimated from the known hydrocortisone and cortisone in the humanlimits of sensitivity of the method used that at synovial cavity (14—16). least 10% of the steroid substrate had undergone As regards steroid metabolism in the skin, themetabolic alteration. relationship of the physiological activity of the While further studies are necessary to establish 104 THEJOURNAL OF INVESTIGATIVE DERMATOLOGY

absorption curve (29) from cortisone. Subsequent investigations have confirmed the fact that cortisone is not found among the synovial tissue metabolites of hydrocortisone (16). Our own studies with human skin have shown that in tissue incubations with hydrocortisone the compound formed possessing the chromatographic mobility of cortisone also yields a sulfuric acid chromogen absorption curve which differs from that of both cortisone and hydrocortisone (Fig. 1). In fact, the curve for this compound strorigly resembles the absorption curve for the compound isolated by McEwen et al (14) following injection of hydrocortisone into the synovial cavity. Additional studies will be required to identify this substance further. The striking differences between the anti-inflammatory effects of hydrocortisone and cortisone applied locally to the skin remain unex- plained. As Wilson and her collaborators have emphasized for the therapeutic disparity in synovial tissue (16), the simplest explanation FIG. 1. Sulfuric acid chromogen absorptionwould be that hydrocortisone itself is the effective spectra of hydrocortisone (a), cortisone (b), andanti-inflammatory agent and that inadequate hydrocortisone metabolite (c). quantities of cortisone are transformed into hydrocortisone in situ. As these same authors the identity of the cortisone conversion product,emphasize, this explanation is unsatisfactory, it would not be surprising to find that hydro-however, since the systemic administration of cortisone occurs as a metabolic product of corti-cortisone is followed by only a relatively small sone in the skin. Metabolic conversion of cortisoneyield of hydrocortisone while numerous additional in part to hydrocortisone has already beenmetabolites of cortisone can also be recovered described following systemic administration offrom the urine (16). The comparable anti- cortisone to humans (18—21). Similar findingsinflammatory effects of hydrocortisone and have also been recorded following in vitroincuba-cortisone following systemic administration tions of certain animal tissues or organs withrather suggest that in regard to local application, cortisone (22, 23). It is of interest, too, thatthere are perhaps certain tissues capable of trans- hydrocortisone is found among the products offorming hydrocortisone into a low yield of one cortisone metabolism both in the rabbit eye (24)or more potent anti-inflammatory agents which and in the human synovial cavity (14—16),these same tissues cannot produce—or produce although in the eye, cortisone is an effectivein inadequate amounts—from the substrate anti-inflammatory agent (25) while in the jointcortisone (16). The well known anti-inflammatory cavity its anti-inflammatory properties are unim-effects of cortisone injected intradermally (8, 8A) pressive in contrast to the action of hydrocorti-could also be explained by such a theory on the sone (26, 27). basis that the skin is presented with such a The occurrence of cortisone among the urinarylarge amount of substrate that the ordinarily metabolic products of hydrocortisone in man haspoor yield of anti-inflammatory compounds is been described by Burstein et at (28). Althoughnow adequate. Altogether, this attractive hypoth- McEwen and his collaborators (14) isolated fromesis certainly bears further investigation as it synovial fluid a compound with the paperrelates to steroid metabolism in the skin. chromatographic characteristics of cortisone The present studies indicate that a second following the intraarticular injection of hydro-explanation may also be advanced for the con- cortisone, further studies showed this substancetrast in local effects of hydrocortisone and corti- to have a different sulfuric acid chromogensone in the skin. The three hour incubation IN VITRO STUDIES OF ADRENAL STEROID METABOLISM 105 studies have shown that there is some metabolicExtraction and chromatographic separation of alteration of cortisone during this time intervalsteroids from tissue incubates have shown but that no transformation of hydrocortisone isthat: demonstrable. Similar in vivo differences in the 1. Hydrocortisone (free alcohol) and cortisone rate of metabolism of hydrocortisone and corti-(free alcohol) are actively metabolized by the sone have been described previously. Malkinsonskin, and that this metabolic transformation et at (17), working with human volunteers,occurs mainly in the corium. found that after topical application of cortisone- 2. The single metabolic product of hydro- 4-C'4 acetate to normal human skin sites, peakcortisone detectable by the technics employed urinary excretion of radioactive metabolites waspossess the paper chromatographic characteris- demonstrable within the first 24-hour period.tics of cortisone. Sulfuric acid chromogen absorp- In comparable studies with hydrocortisone-4-tion curves indicate, however, that this substance C'4 (free alcohol) the peak urinary excretion ofis not cortisone, and further studies are required radioactive material occurred in the second ratherto establish the identity of this compound. than the first 24-hour period. Similarly Sandberg 3. The single metabolic product of cortisone et at (30) found that after oral administration ofdetectable by the technic employed possesses cortisone or hydrocortisone, the steroid degrada-the paper chromatographic characteristics of tion products of cortisone appeared in the urinehydrocortisone. Additional studieswill be at a more rapid rate than the degradation prod-necessary to establish firmly the identity of this ucts of hydrocortisone. The present in vitrocompound. findingsindicate, then, that the skin metabolizes 4. Under the experimental conditions described, cortisone more readily and perhaps more rapidlythe quantitative yield of the metabolic conver- than it does hydrocortisone, suggesting thatsion products of hydrocortisone or cortisone is speedier inactivation of cortisone or cortisoneat least 10%. metabolites may taken place. Obviously if this 5. The metabolic alteration of cortisone occurs rate of metabolic inactivation in the skin weremore rapidly than the metabolic alteration of sufficiently rapid, the anti-inflammatory effecthydrocortisone in the skin. of this steroid could be drastically reduced. The significance of these findings is discussed, In regard to the speed of adrenal steroidparticularly in relation to the striking differences metabolism in the skin, it also seems worthwhilein the anti-inflammatory potency of hydrocorti- to comment briefly on the fact that three skinsone and cortisone following local application incubations carried out with prednisone andto the skin. prenisolone showed no metabolic changes in these REFERENCES 6, compounds during incubation periods of 51,, 1. SULZBERGER, M. B. AND WrrrBN, V. H.: The and9 hours. These preliminary findings for both effect of topically applied compound F in compounds are of interest in the light of recent selected dermatoses. J. Invest. Dermat., 19: investigations (31) which have shown that the 101,1952. 2. SULZBRRGEIi, M. B., WIrrEN, V. H. AND increased anti-inflammatory potency of predni- SMrru, C. C.: Hydrocortisone (Compound sone and prednisolone in humans may be related F) acetate ointment in dermatological therapy. J. A. M. A., 151: 468, 1953. to the much slower metabolism of these com- 3. Rornrsor, H. M., JR. AND ROBINSON, R. C. V.: pounds in body tissues as contrasted with corti- Treatment of dermatoses with local applica- sone and hydrocortisone. Further support for this tion of hydrocortisone acetate. J. A. M. A., 155: 1213, 1954. hypothesis as it concerns the treatment of skin 4. Muz1iNsoN, F. D. AND WELLs, G. C.: Clinical diseases may be obtained from the observation experience with hydrocortisone ointment. Brit. J. Dermat., 66: 300, 1954. of Frolow, Witten, and Sulzberger (32) topical 5. MALKINSON, F. D. AND FERGUSON, E. H.: prednisone—the analogue of cortisone—is equally Preliminary and short report: Percutaneous as effective as topical prednisolone—the ana- absorption of hydrocortisone-4-C'4 in two logue of hydrocortisone—in its anti-inflammatory human subjects. J. Invest. Dermat., 25: 281, 1955. effects. 6. SCOTT, A. AND KALZ, F.: The penetration and distribution of C'4-hydrocortisone in human SUMMARY skin after its topical application. J. Invest. Dermat., 26: 149, 1956. Studies have been conducted on the in vitro 7. LIDDLE, GRANT, W.: ,'9a-fluorohydrocorti- metabolism of adrenal steroids in the skin. sone: A new investigative tool in adrenal 106 THE JOURNAL OF INVESTIGATIVE DERMATOLOGY

physiology. Letters to the Editor, J. Clin. ACTH in man. Federation Proc., 10: 216, Endocrinol., 16: 557, 1956. 1951. 8. GOLDMAN, L., Taorn'soN, R. G. AND TRIeR, 20. BURSTEIN, S., SAVARD, K. AND DORFMAN, E. R.: Cortisone acetate in skin disease; R. I.: The in vivo metabolism of cortisone. local effect in skin from topical application Endocrinology, 52: 448, 1953. and local injection. Arch. Dermat. & Syph.,21. BURTON, R. B., KEUTMAN, E. H. AND WATER- 65: 177, 1952. ROUSE, C.: The conversion of cortisone 8A. GOLDMAN, L. Discussion of Malkinson, et acetate to other alpha-ketolic steroids. J. al. Ref. 17. Olin. Endocrinol. and Metab., 13: 48, 1953. 8B. GOLDMAN, L., FLATT, R., MIHINA, J. S.,22. EISENSTEIN, A.: Steroid compounds resulting Mnm, J. AND DASRALAKIS, E. G.: Studies in, from incubation of cortisone with surviving local action of corticosteroids at cellular liver slices. Proc. Soc. Exper. Biol. & Med., level in the skin of man. VIII. Partition 83: 27, 1953. paper chromatography of skin extracts. J.23. AMELUNG, D., HtTBENER, H. J., ROKA, L. Invest. Dermat., 29: 1, 1957. AND MEYERIIEIM, G.: Conversion of corti- 9. STJLZBERGER, M. B. AND BARR, R. L.: Present sone to compound F (Letter to the Editor). status of ACTH, cortisone, and compound J. Olin. Endocrinol. and Metab., 13: 1125, F in dermatologic management: A guide for 1953. the general practitioner. In: Year Book of24. HAMASHIGE, S. AND POTTS, A. M.: The pene- Dermatology and Syphilology, 1952, pp. tration of cortisone and hydrocortisone into 7—21. Chicago, The Year Book Publishers, the ocular structures. Am. J. Ophth., 40 1953. (part 2): 211, 1955. 10. BusH, I. E.: Methods of paper chromatog-25. STEFFENSEN, E. H., OLSON, J. A., MARGULIS, raphy of steroids applicable to the study of R. R., SMITH, R. W. AND WHITNEY, E. L.: steroids in mammalian blood and tissues. The experimental use of cortisone in inflam- Biochem. J., 50: 370, 1952. matory eye disease. Am. J. Ophth., 33: 1033, 11. VAN SCOTT, E. J.: Mechanical separation of 1950. the epidermis from the corium. J. Invest.26. ZIFF, M., SCULL E., F0RP, D., MCEWEN, C. Dermat., 18:377, 1952. AND BUNIM, i.J.:Effects in rheumatoid 12. LIEBERMAN, S. AND TRIeR, S.: Recent trends arthritis of hydrocortisone and cortisone in the biochemistry of the steroid hormones. injected intra-articularly. Arch. mt. Med., Pharmacol. Revs., 5:285, 1953. 90: 774, 1952. 13. ROBERTS, S. AND SZEGO, C. M.: Biochemistry 27. HOLLANDER, J. L., BROWN, E. M., Ja., JESSAR, of the steroid hormones. Ann. Rev. Bio- R. A. AND BROUN, C. Y.: Hydrocortisone chem., 24: 543, 1955. and cortisone injected into arthritic joints: 14. MCEwEN, C., WILsoN, H. AND ZIFF, M.: Comparative effects of and use of hydro- Studies on the metabolism of adrenal corti- cortisone as a local antiarthritic agent. cal steroids in the synovial cavity in rheum- J. A. M. A., 147: 1629, 1951. atoid arthritis. Tr. A. Am. Physicians, 67:28. BURSTEIN, S., SAVARD, K. AND DORFMAN, R. 97, 1954. I.: The in vivo metabolism of hydrocorti- 15. WILSON, H., GLYN, J., SCULL, E., MCEWEN, sone. Endocrinology, 53: 88, 1953. C. AND ZIFF, M.: Metabolites of hydrocorti-29. ZAFFARONI, A.: Absorption spectra of sulfuric sone and cortisone in the synovial cavity acid chromogens obtained from adrenal in rheumatoid arthritis. Proc. Soc. Exper. steroids and related compounds. J. Am. Biol. & Med., 83: 648, 1953. Chem. Soc., 72: 3828, 1950. 16. WILSoN, H., FAIRBANKS, R., SCIALABBA, D., 30. SANDBERG A. A., NELSON, D. H., GLENN, MCEWEN, C. AND ZIFF, M.: Metabolites of M. E. +YLER, F. H. AND SAMUELS, L. T.: hydrocortisone and cortisone in synovial 17-hydroxycorticosteroids and 17-ketoster- fluid in rheumatoid arthritis. J. Olin. Endo- oids in urine of human subjects: Clinical crinol. and Metab., 16:86, 1956. application of a method employing -glu- 17. MALKIN5ON, F. D., FERGUSON, E. H. AND curonidase hydrolysis. J. Olin. Endocrinol. WANG, M. C.: Percutaneous absorption of cortisone4-C'4 through normal human and Metab., 13: 1445, 1953. skin. J. Invest. Dermat., 28:211, 1957. 31. SLAUNWHITE, W. R., JR. AND SANDBERG, A. A.: 18. MASON, H. L.: Isolation of adrenal cortical The metabolism of 1-dehydro-17-hydroxy- hormones from urine: 17-hydroxycorticos- corticosteroids in human subjects. J. Olin. terone and 17-hydroxy-il-dehydrocorticos- Endocrinol., 17:395, 1957. terone. J. Biol. Chem., 182: 131, 1950. 32. FROLOW, G. R., WITTRN, V. H. AND SULZBER- 19. LIEBERMAN, S. HARITON, L. B., SPOKEM, M. GER, M. B.: Topically applied prednisone B., STUDER, P. E. AND DOBRINER, K.: in the treatment of selected dermatoses. Steroid excretion after administration of Arch. Dermat., & Syph., 76: 185, 1957.

DISCUSSION

DR. JOHN H. EPSTEIN (San Francisco, Calif.): inactive steroid products. Perhaps cortisone is I enjoyed Dr. Malkinon's presentation very rapidly metabolized to this substance, accounting much. I would just like to ask whether tetra-for its lack of clinical efficacy. hydrocortisone was considered as one of the DR. CLARENCE S. LIVINGOOD (Detroit, Michi- IN VITJO STUDIES OF ADRENAL STEROID METABOLISM 107 gan): It would be interesting for Dr. Malkinsonis concerned we have done some preliminary to compare these results with studies of the otherstudies which are included in the text for publica- steroids which are effective topically. tion but were omitted from the presentation. In Dn. FREDiRICK D. MALKINSON (in closing): I tissue incubations with prednisone and with would like to thank those who discussed thisprednisolone for periods of 5, 6, and 9 hours paper. Since precise identification of the metabolicwe were unable to demonstrate formation of any conversion products of cortisone and hydrocorti-conversion products. This is of interest in the sone has not been completed I cannot say whetherlight of recent findings by Slaunwhite and Sand- tetrahydrocortisone is formed in the skin. It isberg that the metabolism of prednisone and generally assumed that this inactive metaboliteprednisolone in humans proceeds much more is formed in the liver and it has been shown in theslowly than that of cortisone and hydrocortisone. case of other peripheral tissues—especiallyit is assumed that the slower rate of metabolism synovial tissue—that formation of tetrahydro-may be responsible for the enhanced anti-inflam- cortisone does not take place locally. We do knowmatory effects of these newer compounds. Our from previous studies with C14 labeled hormones preliminary findings of delayed metabolism of that tetrahydrocortisone and tetrahydrohydro-prednisone and prednisolone in the skin are also cortisone can be recovered from the urine follow- of interest in relation to the observations of ing application of cortisone and hydrocortisone respectively to normal human skin. The site ofFrolow, Witten, and Sulzberger that the topical this metabolic change following percutaneousapplication of prednisone—the analogue of corti- absorption has not yet been identified but Isone-is as effective in eczematous dermatoses suspect it is chiefly the liver. as the local application of prednisolone, the As far as in vitro metabolism of other steroidsanalogue of hydrocortisone.