(ACTH) was first demonstrated by Ingle in the

Transcription

1 PLASMA ADRENOCORTICOTROPHIC HORMONE IN ADDISON'S DISEASE AND ITS MODIFICATION BY THE ADMINIS- TRATION OF ADRENAL STEROIDS By JOHN E. BETHUNE,' DON H. NELSON,2 AND GEORGE W. THORN (From the Deprtment of Medicine, Hrvrd Medicl School nd the Peter Bent Brighm Hospitl, Boston, Mss.) (Submitted for publiction July 18, 1957; ccepted August 23, 1957) The depressnt effect of drenl steroids on the relese of pituitry drenocorticotrophic hormone (ACTH) ws first demonstrted by Ingle in the experimentl niml in 1937 (1). This observtion hs been confirmed by mny investigtors (2-7), but not by ll (8). In mn the clinicl, morphologicl nd biochemicl evidence for steroidl inhibition of pituitry secretion is impressive but there re few ctul mesurements of blood ACTH reported. There is substntil evidence for n incresed level of circulting ACTH in Addison's disese (9, 1) nd postopertive bilterlly drenlectomized ptients (11), but n dequte quntittive evlution of this ctivity hs not been vilble. Evidence for n incresed secretion of ACTH in the dreno-genitl syndrome (12) is lso convincing, but similr increses reported in rheumtic fever (13), Cushing's disese (14), pregnncy (14), the erly post-prtum period (15), nd other conditions (16) hve not been confirmed. The present study ws undertken to determine the level of ACTH in peripherl blood in Addison's disese nd to observe the effects of drenl steroids on the ltertion of this ctivity. METHODS The ssy procedure followed in these studies hs been previously described by Nelson nd Hume (17) nd utilizes the production of corticosteroids by the drenl cortex of the hypophysectomized dog s mesure of ACTH ctivity in dministered humn plsm. It ws performed s follows: 4 to 6 milliliters of blood were withdrwn from the ptient into heprinized syringe. The plsm ws immeditely seprted by centrifugtion nd frozen. Within one to six dys the plsm ws injected into the ssy niml. The ssy ws performed 1 Supported by n Americn College of Physicins' Fellowship, , nd the Commonwelth Fund, Investigtor, Howrd Hughes Medicl Institute. in hypophysectomized dog in which ctheter hd been plced in the right drenl vein in such wy s to mke intermittent, complete collections of the drenl venous effluent possible. Physiologicl sline solution contining.1 N hydrochloric cid nd 1 mu of U.S.P. Stndrd ACTH per ml. ws used s reference ginst which the plsm ctivity ws mesured. One, 2, 5 nd 8 ml. of this stndrd, nd 2 to 3 ml. of plsm were injected into the drenl by the femorl vein during two minute period. (The difference in volumes injected did not pper to ffect the result significntly. Although use of whole plsm llows possibly toxic substnces to be injected with the ACTH, cid-cetone, nd oxycellulose purified extrcts of duplicte smples of plsm lwys showed less ACTH ctivity thn ws present in the whole plsm.) One minute lter the drenl venous collection ws begun nd continued for exctly 1 minutes. A 5 ml. liquot ws removed nd the remining blood ws returned to the venous circultion of the dog. The intervl between collections ws 3 minutes. In the performnce of typicl ssy, two control smples of.1 N HC1 in sline nd five unknown plsm smples were lternted with nine ACTH stndrd injections. Studies in this lbortory (18) nd by Grnirer (15) hve shown tht ACTH will retin its potency in frozen plsm for 171 t lest three or four weeks. The content of 17-hydroxycorticosteroids in the liquot of whole blood removed from the drenl vein of the dog ws determined by slight modifiction of the method of Nelson nd Smuels (19). Ech determintion required the withdrwl of t lest 4 ml. of blood from the ptient nd it ws therefore usully impossible to perform the determintions for ACTH in duplicte. Vlues of ACTH in this pper re expressed in mu per 1 ml. of plsm. The ACTH concentrtion of the plsm smple nd its stndrd error were estimted in logrithmic terms by the use of the usul formule from comprison of the effect of n injected plsm smple on the relese of 17-hydroxycorticosteroids with the response given by grded doses of U.S.P. Stndrd ACTH (2). By conversion to ntilogrithms, the desired vlues re obtined in mu. Since the ddition of logrithms implies multipliction of ntilogrithms, the plus-minus symbols (±) usully used for stndrd errors re chnged to the multipliction-division symbols (X) for the mu (ntilogrithms), s shown in

2 172 JOHN E. BETHUNE, DON H. NELSON, AND GEORGE W. THORN the following exmple: In logs In ntilogs Estimte of potency.93 8 mu Stndrd error X1.3 In this exmple, the stndrd error my lso be cited s 3 per cent of the potency estimte: (1.3-1.) 1 equls 3 per cent. Squring the ntilogrithm of the stndrd error prior to multipliction nd division will give the pproximte 95 per cent confidence limits of ech vlue. Differences between groups of vlues were evluted by Student's "t" test; the two til test being pplied to the differences between groups of vlues in different ptients nd the one til test for differences in the sme ptient (21). The lower limit of sensitivity of the ssy. vries somewht depending on the individul responsiveness of the drenl glnd of the ssy niml. In generl, significnt increse in 17-hydroxycorticosteroid content of the drenl venous blood ws seen with.75 to 1.S mu of ACTH. Therefore, depending on the mount of plsm dministered, the miniml detectble level expressed per 1 ml. of plsm is bout 2 to 5 mu per 1 ml. nd vlues shown in the tbles s "zero" could thus be somewht elevted over norml if they should fll below this lower limit of sensitivity. Such vlues re more correctly expressed s "nondetectble" thn s zero or negtive vlues, but for convenience the zero symbol is retined in the tbles. It ws impossible to clculte stisfctorily n exct upper limit of these zero vlues. TABLE I Plsm A CTH in norml subjects, hospitlized ptients with norml drenl function, nd ptients with Addison's disese X nd + by X nd + by ACTH ntilog. ACTH ntilog. Ptient mu/1 ml. S.E.M. Ptient mu/1 ml. S.E.M. Norml subjects J.B. * D.M. J. F. N. McW. D.N. E.R. D.S. R.F. Hospitlized ptients R.A. H. B. M.S. A.P. M.U. J.G. E.U. R. I. Addisonin ptients N. P J. G E. V A. R G. R. * D. McG D. R. D. McG D. R D. McG W. L A. K.t W. L A. K W. L A. K M. K W. D. M. K W. D M. K K. H. J. H.t L. W J. H. L. W J. H. L. W J. H. C. M J.C. C. G J.C. W. G L. H W. G L.H. W. G E. C P. T M. C J. S H.W. G.S H. McC E. M H. McC J. O'S. H. McC M. L R. S S8 L. L S. B L. L * Limits cnnot be clculted for these vlues. t Adrenl insufficiency due to bilterl drenlectomy.

3 PLASMA ACTH IN ADDISON'S DISEASE.Effet Of Hydro;!rtkmmue fin Ac~sn D e ses...i On PIEE ACTI rn Adis L,..s.D.i... D 173 Is I.- I 2 * 21..-'~ tomea. -W 4_.. i]. i _ - I 3 I.. go-i. ".3, < *HYDROCORTISONE L.V. - * FIG. 1. INFUSION OF HYDRcORTItSONE FOR EIGHT HOURS ON EIGHT OCCA- SIONS To FOUR ADDISONIAN PATIENTS The 5 mg. infusion ws for four hours in fifth ptient. A distinctive line nd symbol is shown for ech ptient, the symbol indicting the plsm level of ACTH expressed in mu per 1 ml. of plsm. Vlues t the zero line indicte ctivity ws not detectble. Comprison of initil nd finl vlues by one til "t" test gives p of <.25 for 2 mg. dose nd <.2 for 1. mg. dose. RESULTS Tble 1 shows the results of ACTH determintions performed on plsm smples from 29 ptients with Addison's disese, three bilterlly drenlectomized ptients, eight hospitlized ptients without drenl dysfunction, nd eight norml subjects. All smples were withdrwn in the fsting stte between 7:3 nd 9: A.M. The ptients with drenl insufficiency received no cortisone for t lest 24 hours prior to the venous collection nd most were on monthly injections of 25 to 5 mg. of desoxycorticosterone trimethylcette. A few ptients were given dded slt during the period of study. In greement with most in igtors, ithoug in disgreement with some, no mesurble level of ACTH ws found in the norml subjects s well s in the hospitlized ptients with no drenl disese (12, 22, 23). In contrst to this group, only 2 of 28 ptients with drenl insufficiency filed to hve ACTH ctivity if more thn one plsm smple were ssyed. The ACTH vlue for the 32 ptients with drenl insufficiency ws found to be 8.4 ± 7.4 (S.D.) mu per 1 ml. This vlue ws significntly different from the group of control subjects (p <.1). There were nine ptients with Addison's disese in whom ACTH ws not initilly detectble. In three of these, repet vlues showed detectble levels of ctivity, in four no repet determintions could be obtined, nd in two repetition gve negtive result on two nd four occsions, respectivey. One ptient who hd prtil drenl insufficiency hd ACTH ctivity present on the three occsions on which it ws mesured. Figures 1, 2, nd 3 illustrte the ACTH vlues obtined in 1 ptients with Addison's disese given 16 infusions of 2.5 to 2 mg. of free hydrocortisone in norml sline for periods vrying from two minutes to eight hours. Smples of plsm for ACTH ssy were obtined t intervls prior to, during, nd up to 12 hours following the

4 174 JOHN E. BETHUNE, DON H. NELSON, AND GEORGE W. THORN Effect Of Hydrocortisone On Plosmo ACTH In Addison's Disese Hydrocort isoe Smg. I.w. 2 hours o IE 1 - l. -o -U - IE *1 mg. dose Hours FIG. 2. INFUSION OF S MG. OF HYDROCORTISONE OVER A Two HoUR PEOD To THREE PATIENTS AND 1 MG. TO A FOURTH PATIENT Plsm ws ssyed up to eight hours following the end of the infusion. P for significnce of difference between initil nd lowest vlues is <.1. 3mgo. RESPONSE OF PLASM CTH AND CORTICOSTEROIDS TO \ HYDROCORTISONE INJECTION 17-HydroxWorticolds - per loom l.plsm.i Pȯ _IW 2 MI Milkilts of ACTH \7 per 11. of Piesmo + x, -'7- Q1 4 5S6 91O 12 Hours Following Injection FIG. 3. TWENTY MG. OF HYDROCORTISONE GNvEN INTRAVENOUSLY To TErn ADDISONIANS IN A Two MINUTE PERIOD Blood corticosteroids nd ACTH ctivity were mesured concurrently up to 12 hours following this infusion. P for significnce of difference between initil nd finl vlues t one hour is <.1.

5 PLASMA ACTH IN ADDISON'S DISEASE Effect Of Vrious Steroids On Plosm ACTH In Addison's Diseose 175. IL qo. 4IL 9 l ft Progesterone: 5mg. (Sor. i.v.) DOC : 5mog. (hr. i.v.) DOCA: 25m. DOC : 5mg. Hours * PIoSMo soemple token 7hours fter orol odministrotion of 9t4Fluorohydrocortisone FIG. 4. INFUSION OF PROGESTERONE, FREE DESOXYCORTICOSTERONE (DOC), AND DESOXYCORTICOSTEONE ACETATE (DOCA) IN FOUR ADDISONIANS Prednisone (A 1 E) ws given intrvenously in two hours nd 9- fluorohydrocortisone (9 FF) ws given by mouth. P for significnce of difference between initil nd finl vlues for DOC nd DOCA is <.5. beginning of the infusion. Plsm 17-hydroxycorticosteroids were determined t the sme time s ACTH in three of these studies s shown in Figure 3. The infusions were mintined t constnt rte nd, therefore, smples tken t intermedite points represent the effect of the frction of the totl steroid dose dministered t tht prticulr time. For exmple, one-fourth of the 2 mg. dose shown in Figure 1 hd suppressed ACTH ctivity nd represents the effect of 5 mg. given for two hours t the sme infusion rte. Anlysis of the ACTH vlues obtined prior to nd t the lowest point during or following the infusion of hydrocortisone in the studies t 5, 1, nd 2 mg. dose levels revels tht significnt fll in ACTH concentrtion hd occurred (p <.5 >.25). Further nlysis of the smller groups seprted by dosge nd durtion of infusion is given in the legend of ech tble. The groups in these smller dose rnges were too smll to lend significnce to observed chnges; however, it would pper tht the smller doses were less effective in suppressing ACTH thn ws the 2 mg. dose. The effect of intrvenous desoxycorticosterone, prednisone, nd orl 9- fluorohydrocortisone on the suppression of plsm ACTH re shown in Figure 4. DISCUSSION Although ttempts hve been mde to mesure the levels of ACTH found in the plsm of ptients with Addison's disese, the mjority of reports del only with the qulittive presence or bsence of ctivity. Sydnor, Syers, Brown, nd Tyler hve reported vlues of 2 to 4 mu ACTH per 1 ml. of whole blood in two Addisonin ptients (24). Prrott hs reported similr ptient with level of 14 mu ACTH per 1 ml. of plsm while on tretment with 25 mg. of cortison (1). Vlues for circulting ACTH reported previously by Prrott for norml subjects, nd prticulrly those found in ptients with vrious diseses, hve been criticized becuse they seemed unresonbly high (11). The methods used by these uthors nd others require the injection of t lest 2 ml. of humn plsm or its equivlent into the rt for relible estimtion of ACTH potency in the dministered plsm or plsm extrct. The rt, the ssy niml used in the scorbic cid

6 176 JOHN E. BETHUNE, DON H. NELSON, AND GEORGE W. THORN depletion method, my be dversely ffected if it receives more thn 2 to 4 ml. of humn plsm intrvenously. Therefore, vrious procedures for the extrction or concentrtion of ACTH hve been pplied to the plsm prior to the ctul ssy by tht method. Vritions in recovery by the methods employed to extrct ACTH before dministrtion to the test niml my explin discrepncies in results found by different groups of workers (25, 11 ). The method used in this study llows the direct injection of 2 to 4 ml. of plsm into the ssy niml nd should obvite the difficulties in the extrction inherent in other procedures. Twenty-six of 28 ptients with Addison's disese exmined by this procedure hd mesurble level of ACTH when dequte numbers of plsm smples were ssyed. The men vlue found for this group of 28 ws mu per 1 ml. (this excludes those four ptients on whom repet determintions were not done nd which if included give vlue of 8.4 ± 7.4). This vlue is significntly different (p <.1) from the results found in the control group of 16 persons. A similr rnge of ctivity hs been found in two previously reported ptients (24) nd in the drenlectomized rt (26). It hs not been possible to correlte the presence or bsence of ACTH ctivity with the degree of pigmenttion or with the durtion of the disese. However, the fct tht ll but one of the ptients hd been on cortisone tretment prior to the study would perhps minimize differences which would hve been otherwise pprent since it hs been shown tht steroid therpy decreses skin pigmenttion in this disese (27). It ws of interest tht one of the ptients with prtil drenl insufficiency hd level of ACTH comprble to the group s whole. This ptient hd pigmenttion typicl of Addison's disese s did ll the ptients in this group of 32. Vn Buchem nd Arends hve recently reported two ptients with Addison's disese without pigmenttion in whom they were unble to demonstrte the presence of either ACTH or melnophore stimulting hormone ctivity (28). The depressnt effect of drenl steroids on plsm ACTH hs been shown in the rt, the dreno-genitl syndrome, nd in the postopertive drenlectomized ptient. The present studies hve shown tht similr reduction in plsm ACTH occurs in the humn with Addison's disese upon the dministrtion of hydrocortisone. The degree of reduction cnnot be directly relted to the dose of dministered steroid, but it would seem tht 5 mg. of hydrocortisone given intrvenously during two hours is the smllest dose which will usully produce fll in plsm ACTH. Infusion of hydrocortisone t the sme rte (2.5 mg. per hour) over longer periods of time (4 to 8 hours) will effect more predictble results, nd lrger dose (1 to 2 mg.) given over shorter period will lso produce more constnt reduction in ACTH concentrtion. The suppression of ACTH ctivity persisted for 8 to 1 hours following the infusion of steroid in four of six ptients during time when there ws lmost complete disppernce of 17-hydroxycorticosteroids from the plsm. Two of the six hd return of ACTH ctivity t six nd eight hours following the end of the hydrocortisone infusion t time when the steroid levels were eqully low. There ppers to be vribility in the responsiveness of the ACTH regulting mechnism in regrd to both the ese of suppression nd the rpidity of reppernce of plsm ctivity following suppression. Fctors other thn the plsm level of 17-hydroxycorticosteroids probbly re required to ccount for the differences noted in the behvior of plsm ACTH in these ptients. Most steroids which hve hydrocortisone-like ctivity suppress pituitry ACTH to vrying degrees nd in generl this hs been shown to correlte with their crbohydrte regulting ctivity rther thn with their slt retining effects (4). The reltive ACTH suppressing ctivity of progesterone nd desoxycorticosterone found in this study in mn re in pproximte greement with those observed in the rt (4). The reltively potent synthetic steroids 9- fluorohydrocortisone nd prednisolone lso ppered to be inhibitors of ACTH secretion in mn. SUMMARY A biossy utilizing the drenl venous blood content of 17-hydroxycorticosteroids in the hypophysectomized dog s mesure of ACTH ctivity hs been pplied to study of the ACTH content of humn plsm. Fifty-one determintions in 32 ptients with drenl insufficiency reveled men ctivity of mu ACTH per 1 ml. of plsm. This ws significntly ele-

7 vted over tht found for 16 control plsm smples obtined from eight norml subjects nd from eight hospitlized ptients without endocrine disese in whom no demonstrble ctivity ws found. The elevted level of ACTH found in group of 1 of the Addisonin ptients ws significntly reduced by the intrvenous infusion of hydrocortisone. The extent of ACTH suppression ws dependent upon the mount of steroid dministered, lthough individul exceptions were noted. Delt- 1-hydrocortisone nd 9- fluorohydrocortisone lso ppered to be potent inhibitors of humn ACTH secretion, wheres desoxycorticosterone hd little nd progesterone no such effect in one subject. ACKNOWLEDGMENT The uthors wish to cknowledge the id of Drs. P. L. Munson nd M. C. Sheps with the sttisticl nlysis of the dt. They re indebted to Misses Norm Mderi nd Anit Eisold nd Mrs. Nncy Meegn for technicl ssistnce. REFERENCES PLASMA ACTH IN ADDISON S DISEASE 1. Ingle, D. J., The time for the occurrence of corticodrenl hypertrophy in rts during continued work. Am. J. Physiol., 1938, 124, Abelson, D., nd Bron, D. N., The effect of cortisone cette on drenl scorbic-cid depletion following stress. Lncet, 1952, 2, Renzi, A. A., Gilmn, M., nd Gunt, R., ACTH-suppressing ction of ldosterone. Proc. Soc. Exper. Biol. & Med., 1954, 87, Syers, G., nd Syers, M. A., Regultion of pituitry drenocorticotrophic ctivity during the response of the rt to cute stress. Endocrinology, 1947, 4, Sydnor, K L., Burks, R., nd Brown, D., Blood ACTH in the stressed drenlectomized rt fter intrvenous injection of hydrocortisone. Endocrinology, 1955, 56, Gry, W. D., nd Munson, P. L., The rpidity of the drenocorticotropic response of the pituitry to the intrvenous dministrtion of histmine. Endocrinology, 1951, 48, Frrell, G. L., nd Lqueur, G., Reduction of pituitry content of ACTH by cortisone. Endocrinology, 1955, 56, Fortier, C., Yrrrzvl, S., nd Selye, H., Limittions of the ACTH regulting effect of corticoids. Am. J. Physiol., 1951, 165, Upson, M., Jr., Studies on drenocorticotrophic hormone in humn serum. Thesis, Grdute School, University of Minnesot, Prrott, D. M. V., The ssy of drenocorticotrophic ctivity in plsm extrcts. J. Endocrinol., 1955, 12, Syers, G., Editoril: Blood ACTH. J. Clin. Endocrinol. & Metb., 1955, 15, Sydnor, K. L., Kelley, V. C., Rile, R. B., Ely, R. S., nd Syers, G., Blood drenocorticotrophin in children with congenitl drenl hyperplsi. Proc. Soc. Exper. Biol. & Med., 1953, 82, Brill, A. B., Ely, R. S., Done, A. K., Ainger, L. E., nd Kelley, V. C., Blood drenocorticotropin (ACTH) in children with rheumtic fever. J. Clin. Endocrinol. & Metb., 1956, 16, Gry, C. H., nd Prrott, D. M. V., Observtions on method of mesuring drenocorticotrophic hormone in plsm. J. Endocrinol., 1953, 9, Grnirer, L. W., ACTH nd corticl steroids in postprtum plsm; preliminry report. New York Stte J. Med., 1951, 51, Pris, J., Upson, M., Jr., Sprgue, R. G., Slss, R. M., nd Albert, A., Corticotropic ctivity of humn blood. J. Clin. Endocrinol. & Metb., 1954, 14, Nelson, D. H., nd Hume, D. M., Corticosteroid secretion in the drenl venous blood of the hypophysectomized dog s n ssy for ACTH. Endocrinology, 1955, 57, Bethune, J. E., nd Nelson, D. H., Unpublished observtions. 19. Nelson, D. H., nd Smuels, L. T., Method for the determintion of 17-hydroxycorticosteroids in blood: 17-hydroxycorticosterone in the peripherl circultion. J. Clin. Endocrinol., 1952, 12, Dixon, W. J., nd Mssey, F. J., Introduction to Sttisticl Anlysis, 2nd ed. New York, McGrw- Hill, Bliss, C. I., The Sttistics of Biossy. New York, Acdemic Press Inc., Fujit, T., Determintion of corticotropin (ACTH) in humn blood nd urine by modified oxycellulose method. J. Clin. Endocrinol., 1957, 17, Tylor, A. B., Albert, A., nd Sprgue, R. G., Adrenotrophic ctivity of humn blood. Endocrinology, 1949, 45, Sydnor, K. L., Syers, G., Brown, H., nd Tyler, F. H., Preliminry studies on blood ACTH in mn. J. Clin. Endocrinol. & Metb., 1953, 13, Sydnor, K. L., nd Syers, G., Blood ACTH in rt nd mn (bstrct). J. Clin. Endocrinol. & Metb., 1952, 12, Sydnor, K. L., nd Syers, G., Blood nd pituitry ACTH in intct nd drenlectomized rts fter stress. Endocrinology, 1954, 55, Johnsson, S., nd H6gberg, B., The occurrence of the melnophore hormone in serum from humn subjects in different pthologicl conditions nd in reltion to corticotrophin. Act endocrinol., 1953, 13, vn Buchem, F. S. P., nd Arends, A., Skin pigmenttion in ptients with hypofunction of the drenl glnds. Act med. Scndinv., 1955, 152, 81.