SARCOIDOSIS, AND CARCINOMA' By FLORENCE B. SEIBERT, MABEL V. SEIBERT, A. JANE ATNO, AND

Transcription

1 VARIATION IN PROTEIN AND POLYSACCHARIDE CONTENT OF SERA IN THE CHRONIC DISEASES, TUBERCULOSIS, SARCOIDOSIS, AND CARCINOMA' By FLORENCE B SEIBERT, MABEL V SEIBERT, A JANE ATNO, AND HAROLD W CAMPBELL (From the Henry Phipps Institute, University of Pennsylvni, Phildelphi) (Received for publiction September 3, 1946) It is well-known tht mrked chnges my occur in the blood proteins in mny diseses, nd these chnges hve usully been expressed s vritions in the lbumin to globulin (A/G) rtio In 1937, Tiselius ( 1 ) showed by mens of the electrophoresis technic tht t lest 3 distinctly different globulins cn be distinguished in serum, nd lter work (2) mde it possible to mesure them quntittively from their electrophoretic digrms Furthermore, it hs been shown tht the reltive mounts of these different protein components vry considerbly in different diseses For exmple, vrious pthologicl ser nd plsms were studied with this technic by Longsworth, Shedlovsky nd McInnes (3); pneumoni ser, by Blix (4); srcoidosis, by Fisher nd Dvis (5), nd lso by Seibert nd Nelson (6); disseminted lupus erythemtosus, by Coburn nd Moore (7); multiple myelom, by Kekwick (8), nd lso Gutmn et l (9); tuberculosis, by Seibert nd Nelson (6); mlri, by Dole nd Kendll (1); rheumtic fever, by Dole, Wtson nd Rothbrd (11), nd lso by Rutstein, Clrke nd Trn (12), nd mny other diseses In certin cses, extr specific serum components (2, 13) with mobilities different from those of the norml globulins pper Such components hve frequently been shown to be ntibodies In most cses, however, increses in the components with norml globulin mobilities occur, indicting n increse in the mount of the norml proteins or the presence of n dditionl substnce such s nother protein, lipoid (14), crbohydrte (15, 14), etc, hving similr mobility s, or ttched to the norml protein Thus, pprently similr chnges, nd therefore similr ptterns, my be seen in different diseses, reflecting similr 'Aided by grnt from the Committee on Medicl Reserch of the Ntionl Tuberculosis Assocition, nd lso by chemicl ssistnce from the Tuberculosis Control Division of the United Sttes Public Helth Service physiologicl disturbnces; but they my lso, on the other hnd, be due to specific cuses However, if sufficient other supporting dt re vilble, the electrophoretic ptterns my become very useful in confirming dignosis, nd certinly re vluble in prognosis For exmple, the present study shows tht there cn, in most cses, be helpful differentition between ser in cses of pulmonry tuberculosis, srcoidosis, nd crcinom A number of studies hve shown tht n bnorml increse in lipoid occurs in the ser in certin diseses (3, 14), nd this increse is seen in the nd f8 globulin components, since the lipoid hs been found usully to ccompny these frctions Less ttention hs been pid to nother importnt colloidl constituent of the serum; nmely, polyscchride While its concentrtion is very much less thn tht of the serum proteins, it is present in s high or higher concentrtion thn glucose, nd my increse s much s 1 per cent or more in some cses Lustig nd Lnger (16) found increses in tuberculosis, crcinom nd pneumoni Nilsson (15) nd Blix, Tiselius nd Svensson (14) lso found n increse in cses of pneumoni Friedmnn nd Sutliff (17) noted the presence of substnce in the serum in certin diseses, probbly polyscchride, from which the pneumococcus is cpble of forming lrge quntities of cid A preliminry report ws published (18) showing tht progressive increse in serum polyscchride prllels dvnce in the tuberculous process It seemed of interest, therefore, to correlte this chnge with the chnges observed by electrophoresis in the different protein components, nd thus to determine Whether the increse in polyscchride could ccount for ny of these chnges In this study, therefore, cses of reltively uncomplicted tuberculosis were crefully selected from the Henry Phipps Institute Clinic by one of 9

2 PROTEINS AND POLYSACCHARIDE IN PATHOLOGICAL SERA us on the bsis of the clinicl findings, nd clssified s to the stge of tuberculosis Cses of Boeck's srcoid which were strongly suspected on the bsis of the clinicl exmintions, or proved by biopsy, were studied Ser from ptients with crcinom were obtined, nd lso 6 cses ech of dibetes nd of dibetes complicted with tuberculosis Norml dult nd lso fetl ser were studied All these ser were nlyzed for totl protein, lbumin,,, 2, f8 nd y globulins, glucose, polyscchride, nd lso sedimenttion rte (except the dibetics nd crcinoms, which were not secured in our clinic) METHODS Clinicl pprisl The terms miniml, modertely dvnced, nd fr dvnced pulmonry tuberculosis re used t the Henry Phipps Institute in ccordnce with the definitions of the Ntionl Tuberculosis Assocition (19) In ddition ech of these clsses is subdivided into 3 groups: (1) ctive lesions for which tretment is indicted, (2) lesions of questionble clinicl significnce, nd (3) lesions which re pprently heled nd presumbly without present clinicl significnce The term "ctive lesions" is used in this study to indicte lesions which in x-ry ppernce re soft nd flocculent, or hve shown recent fluctution, nd disese from which tubercle bcilli hve been recently demonstrted in the sputum "Lesions of questionble clinicl significnce" include infiltrtions tht hve n x-ry ppernce suggesting chronicity or stbility, s indicted by fibrosis nd scrring in prt or prts of the lesion, with softer or less well defined shdows elsewhere suggesting tht the disese is in prt unheled "Lesions which re pprently heled nd presumbly without clinicl significnce" include disese tht ppers to be wholly fibrotic or fibroclcific, nd disese tht shows no evidence of fluctution on seril x-ry exmintions For use in the punch crd system, these lesions re given the following code numbers (which re used in the following tbles) 81 ctive miniml pulmonry tuberculosis 82 ctive modertely dvnced pulmonry tuberculosis 83 ctive fr dvnced pulmonry tuberculosis 84 miniml pulmonry tuberculosis of questionble clinicl significnce 85 modertely dvnced pulmonry tuberculosis of questionble clinicl significnce In the srcoidosis series, 4 of the cses hve been verified by study of biopsy mteril The dignoses of the others were bsed upon the presence of extensive pulmonry fibrosis, usully with enlrgement of the lymph nodes t the hilum or medistinum, nd upon the bsence of significnt symptoms or deteriortion of helth, prtic- 91 ulrly in reltion to the extent nd density of the pulmonry shdows roentgenologiclly In these cses, repeted exmintions of the sputum were negtive for tubercle bcilli, nd the rections to stndrd doses of Purified Protein Derivtive were generlly slight or bsent In 6 cses there were skin lesions typicl of srcoid lesions Three of these were confirmed by consulttion elsewhere X-ry exmintions of the bones of the hnds nd feet were not routinely mde The dignoses on the cses of crcinom nd dibetes were confirmed in ech cse in the respective institutions from which they were received The crcinom cses included lesions of the lungs, colon, rectum, stomch, pncres, liver, brin, temple, tongue, brest, uterus nd cervix Dibetic cses were studied in order to determine whether chnge in the polyscchride content of serum my be ssocited with the disturbed crbohydrte metbolism of this disese No ttempt ws mde to obtin fsting bloods, since it hd previously (2) been shown tht the polyscchride content of the serum is lmost independent of the stte of metbolism The bloods were tken, llowed to clot, nd within severl hours the ser were removed nd plced in freezing box which kept them just t the point of freezing until the nlyses could be mde It ws shown (2) tht under these conditions no chnge occurs in the polyscchride or glucose content of serum over long periods of time Sedimenttion A smll smple of the blood, immeditely fter being drwn, ws mixed with oxlte nd used for the sedimenttion test, ccording to the method of Cutler (21), in the routine clinicl lbortory of our institute Polyscchride ws determined by the crbzole rection ccording to the procedure outlined recently (2), using the glucose-glctose-mnnose stndrd In this study, the totl polyscchride of serum ws determined, nd from it the glucose concentrtion, determnined by the Somogyi (22) method, ws subtrcted, giving the true polyscchride concentrtion Totl protein ws determined by the Kingsley (23) biuret method, using the Klett colorimeter The clibrtion curve ws mde ginst serum protein determined by mens of the micro Kjeldhl method Protein ptterns were mde by mens of the Tiselius electrophoresis technic (1) Erlier studies (24, 6) using phosphte buffer, ph 77 u = 1, showed tht progressive rise in 'v globulin prlleled the development of tuberculosis In ddition to this chnge, bnormlities were noted in the form of the lbumin curve, of such nture tht often there seemed to be present component with slightly greter mobility thn the lbumin This bnormlity in the lbumin component does not occur if the electrophoresis is mde in brbiturte buffer, but insted n extr globulin ppers, s first shown by Longsworth (25) It is possible, therefore, tht the extr globulin resolved in this ltter buffer my represent the sme component included in the lbumin frction in the phosphte buffer,

3 92 F B SEIBERT, M V SEIBERT, A J ATNO, AND H W CAMPBELL All the experiments in the present study were, therefore, crried out in brbiturte buffer (veronl) ph 85 to 86, =1, fter the serum ws dilyzed for severl dys ginst this buffer t 1 C nd then filtered nd diluted 1: 2 with the sme buffer In ll determintions the sme buffer concentrtion nd ph nd lso the sme protein concentrtion were used, since vrition in these conditions leds to vrition in the percentge of the different components (26, 27) In these experiments the crossed-slit rrngement of Svensson (28) ws used All electrophoreses were continued for exctly 2 hours t bout 81 to 88 volts per cm, during which time the components spred throughout most of the long type cell Erlier reports fvored using only the descending pttern for mesurements, since fewer boundry errors occurred on this side, s evidenced by the smller e pek The e pek represents grdient due to buffer electrolyte lone, wheres the boundry on the scending side is due to concentrtion grdients of both buffer slts nd proteins These boundry nomlies, the 8 nd e effects, cn be gretly reduced by reducing protein concentrtion nd lso by incresing the ionic strength of the buffer (29, 26, 27) In fct, they cn be so much reduced, when totl ionic strength of 2 to 25 is used, tht Svensson now recommends using the ptterns on both sides, nd tking n verge of the results for the corresponding components on the 2 sides However, in the present study, since n ionic strength of 1 ws used, clcultions were mde only from the descending ptterns Since reltive rther thn bsolute vlues re desired, ny error due to this method of clcultion would hve no significnt bering on the conclusions drwn No difficulties were encountered with P nomlies so frequently reported by mny workers Digrms were enlrged 5 times, nd the res due to the different components were lwys resolved by the sme individul into curves, s described by Pedersen (3), nd mesured with plnimeter Since the components re not lwys definitely resolved into isolted peks, certin mount of personl error enters into these resolutions, nd Tble I will show the degree of error involved under the conditions described In some cses, the sme serum ws exmined on 2 successive dys, nd in others on widely different dys, even s much s 3 months prt Different exposures during the sme experiment were lso compred, using different ngles on the digonl slit, in order to obtin ptterns of different sizes No systemtic error seemed to be cused by such vrition, but nevertheless the sme ngle, 3, ws used in ll experiments in the comprtive series reported in this pper In generl, s much s 2 per cent error my be expected in the globulin components, nd 3 per cent in the lbumin, under these conditions Results re reported s percentge of the totl grdient re (exclusive of the e component), s the different components, lbumin, l,, P, nd globulins This method of clcultion involves the lest error, since no committl is mde s to the composition of these com- TABLE I Elkctrophoretic mesurements of sme serum t different times, nd with different ngks t ste time in veronl buffer ph 85 to 87, p = 1 Percentge of totl protein s Nme Dte bled Dte run Angle Alb i 2 SF MG SF FJ MC CR AF JB SS JG SC ponents It is known tht considerble percentge of some of these components is not protein For exmple, it ws emphsized erlier tht considerble lipoid nd crbohydrte ccompny the x nd P components However, even these results do not give n entirely true picture for comprison, since the totl protein nd colloidl concentrtion of different ser vry Therefore, in spite of the obvious errors involved, the method of clculting the ctul grms per cent of ech component from the totl protein concentrtion, s used extensively by other workers (31, 32), is lso used here, nd the results re recorded s the second item under ech component While the ctul content in grms of ech component could lso be clculted directly from the electrophoretic digrms, no more ccurte results would be obtined, since the refrctive index increments of the vrious colloidl constituents necessry in the clcultions re lso not known In spite of the inherent errors indicted, the differences recorded for the different stges of disese nd different diseses re so gret tht the conclusions drwn re, without doubt, vlid

4 PROTEINS AND POLYSACCHARIDE IN PATHOLOGICAL SERA RESULTS Since it is impossible to include the ctul nlyses of ll cses, becuse of the lrge number, n ttempt hs been mde to give summrizing tbles which will contin dt to show for ech type of cse the men, the stndrd devition, the probble error, the rnge, nd lso the significnce of the devitions from the norml mens The method of clculting this significnce is s follows The stndrd devition (r) = /, where d = the difference from the men, nd N = the number of cses studied Probble error (E) = 6745, - 2 = the difference between the men (,) of the norml group nd tht of the group under study (,) TABLE II Mens nd their significnce Probble error of ( -) = 4E1 + E2- The significnce of the differences from the norml men is shown by the rtio, - 2 1E1' + E22 For exmple, if this rtio is 5, then, ccording to Gvett (33), the odds ginst getting men of similr group which is outside of the probble error re bout 1,3 to 1, nd the difference is therefore highly significnt If the rtio is 3 the odds re only 22 to 1; if the rtio is 4 the odds re 142 to 1; if it is 6 the odds re 2, to 1; if it is 7 the odds re 445, to 1; etc Tble II shows, in the top hlf, the number of cses studied nd their mens for the different nlyses The lower hlf gives the rtios, which show the significnce of the devitions of these mens from the norml The electrophoretic dt Num- Poly- Totl Globulins Digosis ber scch- pro- Albumin - cses ride tein i 2 Albumin y s globulin rtio (A/G) mgm grms grms grms grms grms grms per per per cen per per cen per per cen per per cent per per cen per cent cent cet cent ceni cent cent Mens ~~~~~~~~~~to Norml dult Fetl Tuberculosis Srcoidosis 11 12& Crcinom Dibetes Dibetes tuberculosis - s/!4e, + E,2 (Significnce of vritions from the mens) Norml dult 43 Fetl Tuberculosis Srcoidosis Crcinom Dibetes Dibetes tuberculosis 93

5 94 F B SEIBERT, M V SEIBERT A J ATNO, AND H W CAMPBELL were clculted both on the bsis of percentge of the totl, nd lso s the ctul grms per cent, ssuming tht ll of the components re protein, or tht the error due to the presence of non-protein substnces is negligible Tble III contins the stndrd devitions nd probble errors corresponding to the results in Tble II Tble IV contins the dt,, - 2 4E12 + E2, used for clculting the significnce rtios, nd lso the rnges of the different nlyses, for use in deciding wht to expect from nlyses on individul ser From these results it is redily seen tht there is significnt increse in the men totl protein concentrtion in srcoidosis, nd significnt decrese in crcinom, while in tuberculosis, regrdless of the stge of the disese, neither n increse nor decrese is the rule TABLE III Stndrd devitions nd probble errors Figure 1 shows typicl electrophoretic digrms of ech type nd stge of disese studied; the individul ptterns were chosen becuse their nlyses fitted most closely to the men vlues for ech prticulr type of cse Figures 2, 3 nd 4 show the individul polyscchride concentrtions plotted respectively ginst the 2 globulin, y globulin, nd lbumin, since it ws in these components tht there proved to be significnt vritions In ll cses the dots represent norml ser, the tringles miniml, the circles modertely dvnced, the blck squres fr dvnced tuberculosis, nd the crosses crcinom In miniml ctive tuberculosis (tuberculosis 81) decrese in the men A/G rtio is significnt, nd this is obviously due to the increse in the y globulin, with corresponding decrese in lbumin Therefore, the most significnt chnge found in this erly stge of the disese is the rise in the y globulin As the disese progresses to the mod- Poly- Totl Globulins Dignosis rc- pro- Albumin ride ten AIG rtio mgm grms grms grms grms grms grms per per per cent per per cent per per cent per per cent per per cent per cent cent cent cent cent cent cent Stndrd devitions Norml dult Fetl Tuberculosis Srcoidosis Crcinom Dibetes Dibetes + tuberculosis Probble errors of the mens Norml dult Fetl Tuberculosis Srcoidosis Crcinom Dibetes Dibetes + tuberculosis

6 Cv O- PROTEINS AND POLYSACCHARIDE IN PATHOLOGICAL SERA f e-cf CZ - IC -In - U)'Oo' o ' ooooooooo _ oouc4 rtu )m1 O - - o ooooooooo 11i ~ ' -4OUO- ooxouo66 s U) - " ' )' o ooooo-oxo: - U)-_~ U)U) -e- '-U MC,4 U) - Ier U ) -U ) - - l- e'ue - )-O-U ' o ooo -I oooqoo ~C'~ -- U :) U) U) U) CO- r-:)--o--b C4 wo 4oll (1 -' - C Oe- -C- - - N - C --U )- CO o - '4- ---)U) U U)-,C 1t el 14x m " w z t o o o- UU)4 coooooooo OOX-r_u sco --m ) 'Uf C4 NOt oocooo666 r C C4 - - o ooooooooo G os ocoooxooooo I ooouz - '' 1 '- U C CO oo -e' oooo66 14) o ooooooooo -H bo CS t-> COC C- -4UU)--o-t- CO' U) '-' CS!) -44- U U) r, o oooooo ooo --- n r- I"! titit )~ ~U c U) o U) oooo>eootooo -'t ) --U)t--e ooooooooo (1 O cli nx --- CO uo -R Ct Iut- 2E U)>U)-U)' tq O U) U>>vUU)U)U) : ts : ; et r- 'R Il4- U) -- ' Ot-U)Uuu)U CO oeo C 4)-_ U) H+ H-H+-H oo toca 'u)' 'u) ncct ' '-Of) Oc~ ' Ut1 L U') U)" --4- CO o o o o IoCS _ N U) U), 1-d -t- (u zo-u Cd _- - = O A, u o-oo ooos ID - -'' I'd -4-- ''(7 ' - ts+++ E U)U) MXc>> c-oi ' J X- OC_OE -d -''U Ul) ) Zod-o soco 95 _ C = - (s midv) D 4CO 4U)_ U)4 E:1, U co to E ; n = Zodoo 4

7 96 F B SEIBERT, Al V SEIBERT A J ATNO, AND H W CAMPBELL "', 'k FIG 1 ELECTROPIIORETIC DIAGRA-MS OF SERA EXA-MINED IN VERONAL BUFFER PH 86, A = 1, AND AT A 3 ANGLE Only descending ptterns re shown In ll cses the lbumin, l,, 3 nd y globulin peks nd the e boundry re evident ertely dvnced stge (tuberculosis 82), there ppered lso rise in the, globulin nd polyscchride content With fr dvnced disese (tuberculosis 83) ll globulins, nd especilly 2, s well s polyscchride content, incresed with corresponding decrese in lbumin, even though the totl protein chnged only slightly In those cses of miniml tuberculosis clssified s of questionble clinicl significnce (tuberculosis 84), no devition from the norml ws found In cses of modertely dvnced tuberculosis of questioinble clinicl significnce (tuberculosis 85), the only significnt chnge is decrese in the lbumin nd the corresponding lowered A/G rtio In srcoidosis, lrge increse occurred in the men y globulin, nd lso in the,s globulin,

8 PROTEINS ANID simultneous decrese in lbumin nd consequently in the A/G rtio There ws lso some increse in polyscchride but by no mens s much s found in cses of fr dvnced tuberculosis, which showed similr increses in the 'y globulin The totl protein lso ws lwys incresed In only 4 of the 11 cses reported in this study ws the dignosis proved by biopsy The others were strongly suspected to be srcoidosis from the clinicl dt While the number of cses is comprtively few, it is believed the conclusions re vlid, since previous group of srcoid cses (6) yielded comprble results, nd lso Fisher nd Dvis (5) obtined similr results on group of 12 cses of srcoid, ll of which were proved by biopsy Crcinom ser showed, on the other hnd, no increse in the men y globulin In fct, in the mjority of cses, very much flttened y pek (see Figure 1) occurred, which in further studies POLYSACCHARIDE IN PATHOLOGICAL SERA my be possibly resolved into very smll true y globulin, nd nother smll component with mobility between the y nd f8 globulins Severl ser, especilly from those cses with metstses to the liver nd resulting jundice, showed n bnormlly high y globulin, nd these brought the verge y up to ner the norml men The, globulin, nd lso the polyscchride content, in prcticlly ll cses were conspicuously incresed nd, of course, the lbumin nd A/G rtio were decresed The totl protein in most cses ws reduced The series of dibetic cses is dmittedly too smll to permit relible conclusions, but it is obvious tht no gret chnge occurred, except possibly in the f8 globulin Since 5 out of the 6 cses showed mrked increse in this component, nd since ft metbolism is known to be disturbed in dibetes, further study of this disese my yield helpful results The dibetics with dvnced tuberculosis showed 97 TUKRGULOSIS CARCINOMA *o lb U * so K 'C k )C ' K x x x Ik K 12C x WOC) Sc r A5 A ~ 49' O,e&6S& * : A * Oe 1ṭ * x GCRAMSS GLOBULIN GRAMS OZ GLOULIN 2 2* I IA IE J,5,,7 FIG 2 GRAMS PER CENT GLOBULIN ARE PLOTTED AGAINST MGM PER CENT POLYSACCHARIDE FOR NORMAL ADULT (e), FETAL (*), MINIMAL (A), MODERATELY ADVANCED () AND FAR AD- VANCED TUBERCULOSIS (i*), AND CARCINOMA (X)

9 98 F B SEIBERT, M V SEIBERT, A J ATNO, AND H W CAMPBELL Jq ft I" * s * i- O 3, A J 3 O O * s A A * ":9 *,Ab A A A&A - A * :* * 4 &* A A A A * A ca S 6 tams -GAMMA GLOBULIN Z22 2A FIG 3 GRAMS PER CENT GLOBULIN ARE PLOTTED AGAINST MGM PER CENT POLYSACCHARIE FOR NORMAL ADULT (e), MINIMAL (A), MODERATELY ADVANCED () AND FAR ADVANCED TUBERcULosIs (U) chnges similr to those found in the tuberculous ptients, except possibly still higher globulin, thus dding significnce to the increse in this component noted in the non-tuberculous dibetics Polyscchride content of fetl serum Ser tken from the cord blood of 6 ptients t prturition showed very much lowered content of polyscchride Even if the lowered totl protein content should be tken s n index of dilution fctor, still the corrected polyscchride content would correspond only to the lowest figures obtined in few dults in the norml group There is n indiction, therefore, tht this constituent tends to increse with ge Only 2 electrophorectic nlyses were mde of fetl ser, since the results obtined corresponded well with those lredy reported (32) The bsolute nd reltive concentrtions of y globulin re higher thn the dult vlues, nd the other globulin concentrtions re considerbly lower, including especilly the 2 globulin Sedimenttion test In generl, with fr dvnced tuberculosis, the sedimenttion rte ws bnorml nd the serum constituents were lso found to be bnorml; but with less dvnced disese (tuberculosis 82), 6 cses out of 2 were found to hve norml sedimenttion rte, while t the sme time their ser showed considerble increse over the men of y globulin nd in some cses of polyscchride Among the group of 43 norml dults in whom no obvious clinicl bnortnlity could be detected, 9 hd sedimenttion rtes of 2 or more, nd in ll of these one of the globulins or polyscchride, or both, ws mong the high rnge of normls There were lso 3 who hd completely norml sedimenttion rtes, but hd the highest vlues for polyscchride (131, 124, 126 mgm per cent) which were included mong our norml group In view of the mny fctors which cn influence the sedimenttion rte, no definite correltions cn be drwn on the bsis of the present work

10 PROTEINS AND POLYSACCHARIDE IN PATHOLOGICAL SERA They do not, however, conflict with the conclusion drwn by Shedlovsky (37) tht there is correltion between the increse in globulin nd increse in sedimenttion rte, except in our group of srcoids where 6 cses showed n incresed sedimenttion rte, but no increse in globulin CONCLUSIONS AND DISCUSSION The dt show slight but significnt rise in the men y globulin component in erly tuberculosis Since ntibodies for most diseses hve been found in the y globulin frction (34), nd, furthermore, since ntibodies in tuberculosis hve lso been found in this frction (35, 36), it is resonble to suspect tht the rise in this component in the erly stge of the disese process my represent n ntibody rection to the orgnism It will be of interest to determine whether the rise in this component hs ny prognostic vlue in differentiting potentilly dngerous cses from the self-heling type Such study is in progress As the disese progresses, the next chnge to pper is n increse in the 2 globulin nd polyscchride content of ser, nd finlly with fr dvnced disese, ll of the globulins, nd especilly the, globulin, increse, with blncing decrese in the lbumin, since the totl protein content does not notbly chnge The polyscchride lso conspicuously increses Therefore, with dvnce in the disese process, it ppers tht the ccompnying tissue destruction my be reflected by this increse in the, globulin nd polyscchride It hs been stted by Longsworth, Shedlovsky nd McInnes (3) tht rise in globulin is found in febrile diseses Shedlovsky nd Scudder (37) concluded tht rise in globulin indicted tissue destruction, nd Perlmnn, Glenn, nd Kufmn (38) found n increse in the globulin component of serum nd of lymph in bums The rgument tht n increse in polyscchride, s well s in ;2 globulin, indictes the presence of tissue destruction is supported by the 99 eoo U toov 16C 14C) 12( lo( p * n El U N Pe 3 3~ * dp 9~~~~~* A A o, A& A *I* A A O A v 4, A, e 8c 61 GRAMS ALBUMINt I _ FIG 4 GRAMS PER CENT ALRUMIN ARE PLOTTED AGAINST Mt M PER CENT POLYSACCHARIDE FOR NoRMAL ADuLT (), MINIMAL (A), MODERATELY ADVANCED (O; AND FAR ADVANCED TUBERCULOSIS (M)

11 1 F B SEIBERT, M V SEIBERT, A J ATNO, AND H W CAMPBELL following fcts Isolted 2 globulin contins higher percentge of polyscchride thn do the other norml serum proteins (these results will be reported in lter communiction), nd these 2 constituents of serum lwys simultneously increse They re both low in fetl ser nd, furthermore, increse in tuberculosis s well s in crcinom, diseses where there is known to be tissue destruction, wheres in srcoidosis, without tissue destruction, no comprble rise in 2 globulin occurs, nd, lso, the rise in polyscchride is much less In this ltter cse the increse tht is found in the polyscchride is probbly due to the rise in 'y globulin which lso does contin some polyscchride, but possibly of different nture As cses of tuberculosis tend to hel nd re then viewed s of questionble clinicl significnce, there is return to the norml serum picture If the disese hs been dvnced, then decrese in the lbumin still persists In other words, the lbumin is the lst component to return to norml, nd this is especilly significnt in view of the fct tht Zeldis nd Alling (39) found the restortion of lbumin to be slower thn tht of the other serum proteins following cute depletion by plsmpheresis It is of considerble importnce tht 3 diseses, which frequently re difficult to differentite cliniclly or from the x-ry picture, nmely, pulmonry tuberculosis, srcoidosis, nd crcinom, present such different nlyticl pictures s here described Such nlyses could, therefore, be of considerble help in dignosis SUMMARY A sttisticlly significnt rise in the y globulin occurs in the serum in miniml ctive tuberculosis, with corresponding decrese in lbumin In modertely dvnced disese, the 2 globulin nd polyscchride content lso increse These chnges re still further emphsized in fr dvnced tuberculosis, nd ll the globulins re lso incresed The men totl protein, however, does not differ from the norml No devition from the norml occurs in miniml tuberculosis of questionble clinicl significnce, except possibly in slightly lowered A/G rtio Modertely dvnced tuberculosis of questionble clinicl significnce shows only decrese in lbumin, indicting tht it is the lst component to return to norml Ser from cses of srcoidosis show n increse in totl protein, proportiontely lrge increse in y globulin, nd only moderte increse in polyscchride content In crcinom there is decrese in totl protein, lrge increse in 2 globulin nd polyscchride content Fetl ser show low totl protein nd polyscchride content nd low globulins, except y globulin, which is high It is suggested tht the increse in y globulin in miniml tuberculosis my indicte ntibody formtion, wheres the rise in 2 globulin nd simultneous rise in polyscchride in dvnced tuberculosis nd crcinom my represent tissue destruction Fetl ser s well s ser in srcoidosis re chrcterized by high y globulin Of protein frctions isolted from norml plsm, the 2 globulin hs the highest polyscchride content The increses in this component in the diseses studied pprently ccount for most of the increse in the polyscchride of the ser Some increse my be due lso to the increse in y globulin, s in srcoidosis We express our pprecition to Dr Ktherine R Boucot, of the Phildelphi Tuberculosis nd Helth Assocition, for referring number of the tuberculous ptients to us For some of the cses of suspected srcoid s well s the cses of dibetes complicted with tuberculosis we re indebted to Dr S C Stein of the Henry Phipps Institute We express our sincere thnks to Dr T Grier Miller, Dr Ruth Breitwiesser, Dr Jennette McConnell, nd Dr Cecili Riegel of the University of Pennsylvni Hospitl, nd to Dr Ctherine Mcfrlne of Womn's Medicl College, for securing the cses nd dignoses of crcinom To Dr Fried Bumnn of Womn's Medicl College we express thnks for the cses of dibetes uncomplicted with tuberculosis Dr Henry Lee of Children's nd Lnkenu Hospitls kindly supplied us with the fetl ser We express our thnks to Misses Mry Mullhy nd Emm DuFour for tking the blood smples nd mking the sedimenttion determintions BIBLIOGRAPHY 1 Tiselius, Arne, Electrophoresis of serum globulin II Electrophoretic nlysis of norml nd immune ser Biochem J, 1937, 31, 1464

12 PROTEINS AND POLYSACCHARIDE IN PATHOLOGICAL SERA 11 2 Tiselius, Arne, nd Kbt, E A, An electrophoretic study of immune ser nd purified ntibody preprtions J Exper Med, 1939, 69, Longsworth, Lewis G, Shedlovsky, T, nd Mc- Innes, D A, Electrophoretic ptterns of norml nd pthologicl humn blood serum nd plsm J Exper Med, 1939, 7, Blix, Gunnr, Quntittive bestimmung von electrophoretisch getrennten serum globulin Zeits fur ges Exper Med, 1939, 15, Fisher, A M, nd Dvis, B D, The serum proteins in srcoid: electrophoretic studies Bull Johns Hopkins Hosp, 1942, 71, Seibert, F B, nd Nelson, J W, Electrophoresis of serum Serum proteins in tuberculosis nd other chronic diseses Am Rev Tuberc, 1943, 47, 66 7 Coburn, A F, nd Moore, D H, The plsm proteins in disseminted lupus erythemtosus Bull Johns Hopkins Hosp, 1943, 73, Kekwick, R A, The serum proteins in multiple myelomtosis Biochem J, 194, 34, Gutmn, A B, Moore, D H, Gutmn, E B, Mc- Clelln, V, nd Kbt, E A, Frctiontion of serum proteins in hyperproteinemi with specil reference to multiple myelom J Clin Invest, 1941, 2, Dole, Vincent P, nd Emerson, K, Jr, Electrophoretic chnges in the plsm protein ptterns of ptients with relpsing mlri J Clin Invest, 1945, 24, Dole, Vincent P, Wtson, R F, nd Rothbrd, S, Electrophoretic chnges in the serum protein ptterns of ptients with scrlet fever nd rheumtic fever J Gin Invest, 1945, 24, Rutstein, D D, Clrke, F H, nd Trn, L M, Electrophoretic studies in rheumtic fever Science, 1945, 11, vn der Scheer, J, Lgsdin, J B, nd Wyckoff, R W G, Electrophoretic nd ultrcentrifugl nlyses of ntipneumococcl horse ser J Immunol, 1941, 41, Blix, G, Tiselius, A, nd Svensson, H, Lipids nd polyscchrides in electrophoreticlly seprted blood serum proteins J Biol Chem, 1941, 137, Nilsson, Ivr, tqber den glucosmingehlt des blutserums bei gesunden und pneumoniekrnken Biochem Z, 1937, 291, Lustig, B, nd Lnger, A, tuber die Bestimmung und den Gehlt n freiem, Eiweiss- und gebundenem Nichteiweisszucker in normlen und pthologischen Serum, Liquor und Hrn Biochem Z, 1931, 242, 32 Lustig, B, nd Nssu, E, The crbohydrte content of the proteins in blood serum, cnthridin blisters nd pleurl effusions Am Rev Tuberc, 1941, 43, Friedmnn, T E, nd Sutliff, W D, Appernce of fermentble polyscchride in the blood nd simple method of its detection Science, 1939, 9, Seibert, F B, Nelson, J W, nd Seibert, M V, Correltion of extent of tuberculosis with mount of polyscchride in the serum Proc Soc Exper Biol nd Med, 1943, '52, Ntionl Tuberculosis-Assocition, Dignostic Stndrds nd Clssifiction of Tuberculosis, 194 Edition, New York City 2 Seibert, F B, nd Atno, J, Determintion of polyscchride in serum J Biol Chem, 1946, 163, Cutler, J W, Stndrdiztion of sedimenttion rte Am Rev Tuberc, 1943, 48, Somogyi, M, A regent for the copper-iodometric determintion of very smll mounts of sugt J Biol Chem, 1937, 117, Kingsley, G R, The direct biuret method for the determintion of serum proteins s pplied to photoelectric nd visul colorimetry J Lb nd Clin Med, 1942, 27, Seibert, F B, nd Nelson, J W, Electrophoretic study of the blood protein response in tuberculosis J Biol Chem, 1942, 143, Longsworth, L G, Recent dvnces in the study of proteins by electrophoresis Chem Rev, 1942, 3, Perlmnn, G E, nd Kufmn, D, The effect of ionic strength nd protein concentrtion in the electrophoretic nlysis of humn plsm J Am Chem Soc, 1945, 67, Svensson, Hrry, Electrophoresis by the Moving Boundry Method A theoreticl nd experimentl study Arkiv for Kemi, Minerlogi och Geologi, 1946, 22A, No 1 (Thesis) 28 Svensson, H, Direkte photogrphische Aufnhme von Elektrophorese-Digrmmen Kolloid-Z, 1939, 87, 181 Theorie der Beobchtungsmethode der gekreuzten Splte Kolloid-Z, 194, 9, Longsworth, L G, nd McInnes, D A, Electrophoresis of proteins by the Tiselius method Chem Rev, 1939, 24, Svedberg, T, nd Pedersen, K O, The Ultrcentrifuge, p 296 Oxford University Press, London, Dole, Vincent P, The electrophoretic ptterns of norml plsm J Clin Invest, 1945, 23, Longsworth, L G, Curtis, R M, nd Pembroke, R H, Jr, The electrophoretic nlysis of mteril nd fetl plsms nd ser J Clin Invest, 1945, 24, Gvett, G I, A First Course in Sttisticl Method, p 188 McGrw-Hill Book Co, Inc, New York, Enders, J F, The concentrtion of certin ntibodies in globulin frctions derived from humn blood plsm J Clin Invest, 1944, 23, Seibert, F B, nd Nelson, J W, Proteins of tuberculin J Am Chem Soc, 1943, 65, Emmrt, E W, nd Seibert, F B, The effect of tuberculous nd sensitized ser nd serum frctions on the development of tubercles in the chorio-

13 12 F B SEIBERT, M V SEIBERT, A J ATNO, AND H W CAMPBELL llntoic membrne of the chick J Immunol, 1945, 5, Shedlovsky, T, nd Scudder, J, A comprison of erythrocyte sedimenttion rtes nd electrophoretic ptterns of norml nd pthologicl humn blood J Exper Med, 1942, 75, 119 3& Perlmnn, G E, Glenn, W W L, nd Kufmn, D, Chnges in the electrophoretic pttern in lymph nd serum in experimentl burns J Clin Invest, 1943, 22, Zeldis, L J, nd Ailing, E L, Plsm protein metbolism-electrophoretic studies Restortion of circulting proteins following cute depletion by plsmpheresis J Exper Med, 1945, 81, 515