ADULTS AND RHEUMATOID ARTHRITIS PATIENTS TREATED WITH ACTH 1, 2

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1 AMNO ACD STUDES AND CLNCAL FNDNGS N NORMAL ADULTS AND RHEUMATOD ARTHRTS PATENTS TREATED WTH ACTH 1, 2 By A. L. BORDEN, E. C. BRODE, E. B. WALLRAFF, W. P. HOLBROOK, D. F. HLL, C. A. L. STEPHENS, JR., R. B. JOHNSON, AND A. R. KEMMERER (From the Southwestern Clinic nd Reserch nstitute, nc., nd The Deprtment of Nutrition, University of Arizon, Tucson, Arizon) (Submitted for publiction August 30, 1951; ccepted Februry 5, 1952) Previous publictions.from this lbortory ( 1, 2) hve reported tht rheumtoid rthritis ptients who were treted with ACTH (drenocorticotrophic hormone) nd cortisone (17-hydroxy-11- dehydrocorticosterone) exhibited chnges in the urinry excretion of certin mino cids. Similr results were found to occur during remission induced by pregnncy (3, 4), nd one rheumtoid rthritis ptient in remission cused by jundice showed chnges even more striking (5). Significnt differences hve been reported to exist in plsm concentrtions of certin "free" mino cids in untreted rheumtoid rthritis ptients nd in norml individuls (6). t ws considered importnt, therefore, to determine whether in this disese chnges occur in plsm concentrtions concomitntly with remission induced by ACTH therpy nd lso to estblish the effect of ACTH on plsm concentrtions nd urinry excretion of mino cids in norml dults. Results for six pprent "free" mino cids in the urinry excretion of norml dults nd for seven "free" mino cids in plsm of norml individuls treted with ACTH re compred with those from rheumtoid rthritis ptients similrly treted. METHODS The norml control group consisted of four mle nd four femle university students. mmeditely before the experiment ech student ws subjected to complete physicl exmintion nd bsence of ny disese or obvious metbolic disorder ws estblished. The students ' This work ws supported in prt by grnts from the United Sttes Public Helth Service nd the Fir Foundtion. ACTH ws provided by the Armour Lbortories. 2A preliminry report of this mteril ws presented before the Federtion of Americn Societies for Experimentl Biology, Clevelnd, Ohio, April were not hospitlized but were kept on closely supervised diet of dequte cloric nd vitmin C intke nd one which supplied one grm of protein per kilogrm of body weight. The source of protein ws the sme for ll subjects nd to tht extent the mino cid intke ws controlled. Following three-dy control period on the diet, 40 mg. of ACTH were dministered intrmusculrly in three divided doses ech dy for seven dys. Thirtyfive ptients were chosen on the bsis of n unquestionble dignosis of rheumtoid rthritis nd hd been under tretment by the investigtors for periods rnging from six months to 15 yers. They were hospitlized for observtion nd study throughout the experiment nd were mintined on the sme controlled diet s tht used for the norml dult group. ACTH ws dministered intrmusculrly in vrible dosge of from 40 to 60 mg. dily except for one ptient who received much lrger dose. t ws determined conclusively tht the size of the dose hd no mesurble effect on the plsm levels of the mino cids ssyed. n n effort to ccelerte or prolong the fvorble effects of ACTH therpy some ptients included in this study received supplementry mediction. During the control period nd/or through the tretment period vrious gents such s ATP (denosine triphosphte) scorbic cid, thyroid, testosterone, spirin, nd mino cid feedings were used. None of these supplements ltered the significnt mino cid increses induced by ACTH. Twenty-four hour urine specimens were collected on two dys prior to the dministrtion of ACTH. During tretment, specimens were collected on lternte dys. Aliquots of the smples were stored t C. By using the method of Hier nd Bergeim (7), tungstic cid filtrtes were prepred from fsting blood smples obtined before nd on lternte dys of tretment. Filtrtes were stored in the frozen stte until time of ssy. A modifiction of the microbiologicl technic of Henderson nd Snell () ws used for the method of ssy. Lctobcillus mesenteroides nd Streptococcus feclis R were the test orgnisms employed. All specimens from one individul were nlyzed in one series to eliminte errors due to vritions in orgnism response. The smples were mesured in duplicte nd t three levels of dilution. n ll other detils the procedures s previously described (7, ) were followed. n n erlier publiction (1) we estblished tht ACTH hs no inhibi- 375

2 376 BORDEN, BRODE, WALLRAFF, HOLBROOK, HLL, STEPHENS, JOHNSON, KEMMERER tory or stimultory effect on the bove mentioned micro- Chemicl orgnisms. n the sme publiction it ws proven tht supplementry mediction does not lter the pttern of uri- Urie: n Tble the comprison is mde benry excretion of mino cids induced by tretment with tween the urinry excretion of six pprent "free" ACTH. Therefore, ptients receiving ACTH with or mino cids in rheumtoid rthritis ptients nd without supplementry mediction were compred to nor- norml dults treted with ACTH. The response ml dults receiving only ACTH. The effect of supple- to the mediction gives "curve" type rection in mentry mediction on mino cid vlues in the plsm which excretion vlues to "pek" or mxiis discussed lter. The results shown in this pper were sttisticlly nlyzed by the Multiple Vrince Method mumilevel gndwle medon ill beinged (9) nd the Student's t Test (10). ministered, grdully level off. All tretment vlues, however, re well bove control levels es- RESULTS tblished before institution of therpy. Previous Clinicl work (1, 2) reported tht in rheumtoid rthritis ptients treted with ACTH, the verge nd mxi- The clinicl findings in the group of rheumtoid mum 24-hour excretion of histidine, lysine, threorthritis ptients were the sme s those reported nine, nd tyrosine showed 1 % significnt inin one of our erlier publictions (1). crese. n the norml dults the urinry excretion nterestingly enough the group of norml dults of none of the bove, mentioned mino cids ws did not experience the findings mnifested by the significntly incresed with the exception of threoptients with the disese. There ws no increse in nine for which there ws significnt 5% inppetite, no indiction of insomni, no increse in crese t the mximum level only. Phenyllnine energy, nor ny mesurble chnge in mentl lert- nd tyrosine were incresed t the 1 %o significnt ness. Weight ws mintined t reltively con- level but only t the time of mximum excretion. stnt level throughout the experiment. There The degree of response to ACTH ws significntly were no indictions of toxicity. n short, upon less in the urinry excretion of norml dults thn creful exmintion it ws impossible to detect ny in ptients with rheumtoid. rthritis. The single difference in the wy the norml dults rected exception ws phenyllnine where the reverse before beginning the mediction nd during its ws true t the mximum level. n neither group dministrtion. ws the excretion of rgirline ltered. Tyrosine TABLE Averge nd mximum 24-hour urinry excretion of six pprent "free" mino cids in rheumtoid rthritis ptients nd in norml dults treted with A CTH Amino cid Type subject No. Before tretment During tretment During tretment (men) (verge men) (mximum) mg./24 hr. mg./24 hr. mg./24 hr. Arginine Arthritis ± 0.94 Histidine Arthritis ± ±12.42*t 169.0±16.5*t 137.0± i i 16.65$ Lysine Arthritis ±: 5.7*t 67.2±.4*t 23.4± ± ±:.52 Phenyllnine Arthritis ± ± 3.07? 9.± t 13.3± Threonine Arthritis ±1: ±i *t 2.52$ 65.2 :±: 12.3*t t Tyrosine Arthritis ± ± ± 14.7± 3.17*t ± 4.06*t 17.4±4 1.14t * Significnt t the 1% level (Anlysis of Vrince).. Significntly different from norml t 1% level (t Test). Significnt t the 5% level (Anlysis of Vrince).

3 AMNO ACD STUDES N NORMALS AND RHEUMATOD ARTHRTS 377 ACS GASC N URNR VWMON Or AN0 OTWATMENT ON WH ACTH et MTHMn m NORMAL to o #DOffee Slgfl11 1% LO lW %M 6*~ RAQ196 TYU05 ---TN L ft-f so 20 3 NUG-, i FG. 1 C "'~~. A MNE PHENYLALAM nd histidine vlues before tretment re significntly different for norml individuls nd ptients with the disese. The mximum increses in urinry excretion of mino cids in the two treted groups re shown in Figure 1. Plsm: The sme "curve" type response which ws noted in the urinry excretions occurred lso in the plsm estimtions. As seen in Tble, t the mximum plsm concentrtions observed during tretment, the group of rheumtoid rthritis ptients showed significnt increse for ech of the seven mino cids. The verge vlues during tretment were incresed significntly for rginine, lysine, nd threonine. TABLE Averge nd mximum plsm concentrtions for seven pprent "free" mino cids in rheumtoid rthritis ptients nd norml dults treted with A CTH Amino cid Type subject No. Before tretment During tretment During tretment (men) (verge men) (mximum) gmm/ml. gmm/ml. gmm/ml. Arginine Arthritis ± t1.71f *t 15.0± ± ± 1.04 Histidine Arthritis ± ± ±0.99$ 14.0± 1.02 Lysine Arthritis ± ± *t 24.1± ±2.69*t 26.94±1.0 Phenyllnine Arthritis ± *t 12.1 ±0.4 Proline Arthritis ± ± i ± ±2.39* Threonine Arthritis ± ± *t ±:1.29*t 15.4±0.41 Tyrosine Arthritis ± t 9.6± :±1.37tt 10.4±0.7 5Significnt t the 1% level (Anlysis of Vrince). t Significntly different from norml t 1% level (t Test). t Significnt t the 5% level (Anlysis of Vrince).

4 37 BORDEN, BRODE, WALLRAFF, HOLBROOK, HLL, STEPHENS, JOHNSON, KEMMERER MAXMUM NCREASE N AMNO ACDS N PLASMA ON TREATMENT WTH ACTH. St AR~hTC v. NOMAL l0 * odsfs Sy OWtA 1% Lv 4c 2-1l ' fpwfib 34 e MSTDONE TYROSE THREONus LYoE FG PROLNE ARGNNE PtNYAANN While the plsm vlues for the group of norml individuls showed the sme generl trend s the rheumtoid rthritis ptients, the chnges observed, both t the time of mximum increse nd t the verge level, were not significntly incresed over the control vlues obtined before tretment. The behvior of the treted rheumtoid rthritis group s compred with the group of norml dults ws significntly different in severl instnces. At the mximum plsm response the difference in the wy the groups rected ws highly significnt for rginine, lysine, phenyllnine, threonine, nd tyrosine. The mximum vlue response ws not significntly different for histidine or proline. The verge tretment plsm vlues for the two groups were significntly different t the 1%o level for rginine, lysine, threonine, nd tyrosine nd not significntly ltered for histidine, phenyllnine, or proline. The mximum increses in plsm mino cids for the two groups treted with ACTH re shown in Figure 2. Our own investigtions, s well s the work of others (11), hve estblished tht plsm vlues for the mino cids re reltively constnt in n individul. t seems, therefore, tht these significnt differences due to therpy with ACTH re worthy of considertion. The smll but significnt plsm increses, when considered in terms of totl circulting blood volume, indicte n pprecible chnge. By mens of n nlysis with the Multiple Vrince Test (9), the results from ptients with rheumtoid rthritis treted simultneously with supplementry mediction nd ACTH were compred with those from the group whose tretment ws uncomplicted. t ws conclusively proven tht those plsm mino cid concentrtions which were significntly incresed by ACTH therpy were not significntly ffected by supplementry mediction. DSCUSSON We hve no dequte explntion for the chnges in mino cid concentrtions in plsm levels nd in urinry excretions. A number of possibilities hve been considered to explin the mechnism t work but we hve s yet no ctul proof to substntite them. All indictions re tht we re mesuring chnges in intrinsic protein metbolism. Although the mechnism of mino ciduri is not understood, lowered renl threshold does not pper to be involved. Tolernce tests with histidine nd threonine, now in progress in this lbortory, hve shown tht even when n orl dose of from 2 to 10 grms is employed, only 2%b to 5%o of the mino cid is recovered in the urinry excretion (12). t is interesting to note tht the urinry excretion of rginine is not incresed in either group while the plsm levels for rginine in the rheumtoid rthritis group re significntly incresed.

5 AMNO ACD STUDES N NORMALS AND RHEUMATOD ARTHRTS Of further interest is the fct tht the vlue for histidine, the mino cid excreted in such lrge mounts in the urine in remissions induced by pregnncy, jundice, cortisone, or ACTH, is not ltered to lrge degree in the plsm. Becuse the dt here presented show such significnt differences between the rections of the two groups to ACTH therpy, dditionl evidence seems provided of correltion between mino cid metbolism nd the remission fctor in rheumtoid rthritis. SUMMARY n group of norml individuls treted with ACTH, microbiologicl ssys for six "free" mino cids were determined on 24-hour urine smples, nd for seven mino cids on plsm filtrtes. The vlues hve been compred with those found in rheumtoid rthritis ptients similrly treted. Unlike the clinicl findings in ptients with rheumtoid rthritis, the treted norml dults mnifested no improvement in ppetite, incresed energy, mentl lertness, or sense of generl wellbeing. Urinry excretion nd plsm vlues for the rheumtoid rthritis group exhibited pprecible increses during the tretment with ACTH. The most significnt chnges occurred for histidine, lysine, threonine, nd tyrosine in the urine, nd for rginine, lysine, nd threonine in the plsm. The results for the norml dults followed the sme generl trend in response but to strikingly less degree. n the urinry findings only phenyllnine nd tyrosine were significntly incresed during tretment nd then only t the time of mximum excretion. None of the plsm mino cids ssyed ws significntly incresed t ny time during the tretment. A highly significnt difference in mino cid response of the two groups occurred for histidine, lysine, threonine, nd tyrosine in the urine, nd for rginine, lysine, threonine, nd tyrosine in the plsm. t is interesting to note tht the histidine chnges re limited to the urinry excretion findings while the rginine chnges re peculir to the plsm. ACKNOWLEDGMENTS The uthors wish to express their pprecition to Dr. M. G. Vvich for his criticisms nd suggestions nd to Mr. Joel E. Fletcher for his vluble help with the sttisticl nlyses nd chrts. Further pprecition is extended to the group of students for their excellent coopertion in mking this study possible. REFERENCES Stephens, C. A. L., Jr., Wllrff, E. B., Borden, A. L., Brodie, E. C., Holbrook, W. P., Hill, D. F., Kent, L. J., nd Kemmerer, A. R., Apprent free histidine plsm nd urine vlues in rheumtoid rthritis treted with cortisone nd ACTH. Proc. Soc. Exper. Biol. & Med., 1950, 74, Brodie, E. C., Wllrff, E. B., Borden, A. L., Holbrook, W. P., Stephens, C. A. L., Jr., Hill, D. F., Kent, L. J., nd Kemmerer, A. R., Urinry excretion of certin mino cids during ACTH nd cortisone tretment of rheumtoid rthritis. Proc. Soc. Exper. Biol. & Med., 1950, 75, Unpublished dt. 4. Wllrff, E. B., Brodie, E. C., nd Borden, A. L., Urinry excretion of mino cids in pregnncy. J. Clin. nvest., 1950, 29, Borden, A. L., Wllrff, E. B., Brodie, E. C., Holbrook, W. P., Hill, D. F., Stephens, C. A. L., Jr., Johnson, R. B., nd Kemmerer, A. R., Effect of drenocorticotrophic hormone on mino cid content of blood nd urine in norml individuls nd ptients with rheumtoid rthritis. Federtion Proc., 1951, 10, Borden, A. L., Wllrff, E. B., Brodie, E. C., Holbrook, W. P., Hill, D. F., Stephens, C. A. L., Jr., Kent, L. J., nd Kemmerer, A. R., Plsm levels of free mino cids in norml subjects compred with ptients with rheumtoid rthritis. Proc. Soc. Exper. Biol. & Med., 1950, 75, Hier, S. W., nd Bergeim, O., Microbiologicl determintion of free leucine, isoleucine, vline, nd threonine in dog plsm. J. Biol. Chem., 1945, 161, Henderson, L. M., nd Snell, E. E., A uniform medium for determintion of mino cids with vrious microorgnisms. J. Biol. Chem., 194, 172, Snedecor, G. W., Sttisticl Methods Applied to Experiments in Agriculture nd Biology. Collegite Press, nc., Ames, ow, 1946, Ed. 4, Section Snedecor, G. W., bid., Section Kirsner, J. B., Sheffner, A. L., nd Plmer, W. L., Studies on mino cid excretion in mn.. Amino cid levels in plsm nd urine of norml men fed diets of vrying protein content. J. Clin. nvest., 1949, 2, Unpublished dt.