Measurement of Enzymatic Activity and by Radioimmunoassay

Transcription

1 CLIN. CHEM. 23/1, (1977) Comprison of Humn Prosttic Acid Phosphtse by Mesurement of Enzymtic Activity nd by Rdioimmunossy Andrs G. Foti,1 Hrvey Herschmn,2 nd J. Fenimore Cooper3 We compred results of mesurement of prosttic cid phosphtse ctivity in serum nd vrious tissues by enzymtic ssy nd rdioimmunossy. By enzymtic ssy, ctivity in serum is lost rpidly, even t room temperture. In contrst, there ws no chnge in ntigenic ctivity during 48 h by rdioimmunossy. The rdioimmunossy ws more specific in 12 tissues nd in serum thn were severl enzymtic ssys tht mke use of inhibitors of the enzyme. The enzymtic ssy resulted in 26.6% (24/9) flse positives from non-prosttic cncer ptients. In contrst, with rdioimmunossy there were only 5.5% (5/9) flse positives. We conclude tht immunologicl detection of prosttic cid phosphtse is the more relible technique. AddItionl Keyphrses:cncer. norml vlues #{149} ssy specificity #{149} dignostic ids Prosttic cid phosphtse (EC ) is n isoenzyme of orthophosphoric-monoester phosphohydrolse (cid optimum). Mesurement of its ctivity in the serum becme cliniclly importnt fter it ws discovered tht prosttic cid phosphtse ctivity usully increses s consequence of prosttic crcinom (1-5) nd tht the ctivity in the serum generlly increses s the disese progresses (6). Mny substrtes hve been used in mesuring this ctivity in ptients serum (7-13). Becuse mny of these substrtes re not specific for the cid phosphtse originting from the prostte, inhibitors hve been introduced to decrese the effect of cid phosphtses from other tissues (14, 15). Deprtment of Reserch, Southern Cliforni Permnente Medicl Group, Los Angeles, Clif Deprtment of Biologicl Chemistry nd Lbortory of Nucler Medicine, UCLA School of Medicine, Los Angeles, Clif Deprtment of Urology, Southern Cliforni Permnente Medicl Group nd Kiser Foundtion Hospitl, Los Angeles, Clif Received Aug. 1, 1976; ccepted Nov. 1, In ddition to the specificity of the substrtes, the stbility of the enzymtic ctivity of the protein is very importnt for relible dignostic test. If enzyme ctivity is lbile under conditions normlly used for serum collection or storge, berrnt results will be obtined (4, 16). To overcome both of these insufficiencies of the enzymtic determintion of prosttic cid phosphtse, we hve introduced rdioimmunossy to quntitte this enzyme in ser from ptients (17, 18). Here, we present comprtive dt concerning the specificity of the rdioimmunossy nd of four enzymtic determintions, s well s dt demonstrting the incresed stbility of prosttic cid phosphtse in the rdioimmunossy reltive to the enzymtic ctivity. Mterils nd Methods Disodium p-nitrophenyl phosphte, thymolphthlein monophosphte (mgnesium slt), phenolphthlein monophosphte (sodium slt), -nphthyl phosphte (potssium slt), tetrzotized o-dinisidine, nd Brij 35 ( surfctnt, polyoxyethylene ether of luryl lcohol; concentrtion, 3 g/liter) were obtined from Sigm Chemicl Co., St. Louis, Mo All other chemicls were nlyticl grde, from J. T. Bker Chemicl Co., Phillipsburg, N. J The purifiction of humn prosttic cid phosphtse nd its iodintion nd the production of rbbit ntiserum to cid phosphtse hve been described (18). Extrction of tissues: The tissue specimens were from utopsy. They were extrcted twice in homogenizer t 4 #{176}C for 1 mm, t top speed, in five volumes of sline (9 g of NCL per liter). Pltelets nd erythrocytes were lysed by thrice-repeted freezing nd thwing. The homogentes were centrifuged for 2 mm t 3 X g. Pltelets nd erythrocytes were obtined from our blood bnk. CLINICAL CHEMISTRY, Vol. 23, No. 1,

2 Mesurement of cid phosphtse ctivity: The method from Sigm Bulletin 14 (19) ws dpted when p-nitrophenyl phosphte ws used s substrte. The Colemn procedure (2) ws modified when we used phenolphthlein monophosphte. One milliliter of citrte buffer (.1 mol/liter, ph 6.) contining 5 mmol of phenolphthlein monophosphte per liter ws preincubted t 37 #{176}C, nd.2 ml of smple or enzyme solution ws dded. After further 3-mm incubtion, 5 ml of NOH (5 mmol/liter) ws dded. The bsorbnce ws then mesured t 546 nm. When thymolphthlein monophosphte ws used s substrte, the ssy procedure ws tht of Roy et l. (13). The method of Amdor et l. (21) ws used, except tht tetrzotized o-dinisidine ws used (11) insted of dizotized 5-nitro-o -nisidine, when -nphthyl phosphte ws the substrte. Rdioimmunossy procedure for humn pros ttic cid phosphtse: Polypropylene tubes were coted with.5 ml of rbbit ntiserum, diluted 3-fold, for 1 h t 4#{176}C. After the ntiserum ws spirted, the wlls of the tubes were wshed twice with 1 ml of PBS, nd they were llowed to stnd for 45 mm t room temperture with PBS contining 1 g of BSA per liter.4 The contents of the tubes were spirted nd.4 ml of sline,.1 ml of smple, nd 1 il of 1251-lbeled ntigen (2 X 1 cpm) were dded. The tubes were left t 4 #{176}C for 36 h nd, fter the removl of the free ntigen, the wlls of the tubes were wshed twice with PBS nd counted in gmm counter. This ssy hs been described in detil previously (15). Stbility of serum prosttic cid phosphtse: Blood ws obtined from prosttic cncer ptients by venipuncture. The serum (ph 8.4) ws seprted by centrifugtion, divided into three portions, nd incubted t either 23, 36, or 46 #{176}C. Smples for the ssy were tken t the indicted times nd quickly frozen until they were mesured by enzyme ssy (p-nitrophenylphosphte substrte) nd by rdioimmunossy. To test the enzyme s stbility to freezing nd thwing, serum ws frozen (solid C2/ethnol) nd thwed 14 times nd the ctivity then determined by enzymtic ssy nd rdioimmunossy. Results Stbility: The ctivity of serum prosttic cid phosphtse decreses rpidly t room temperture, owing to the incresed ph of the serum (7). However, repeted freezing nd thwing does not decrese either the enzyme ctivity or the immunologicl ctivity of prosttic cid phosphtse in the serum of prosttic cncer ptients. The serum retined ll its originl prosttic cid phosphtse ctivity fter it hd been frozen nd thwed 14 times. Consequently, it is vlid to store series of smples frozen nd lter mesure their ctivity t one time. 4Nonstndrd bbrevitions used: BSA, bovine serum lbumin; PBS, phosphte-buffered sline (15 mmol/liter NCI nd.1 mol/ liter sodium phosphte buffer, ph 7.2). > /4 /2 TIME (Ii 37 C Fig. 1. Het stbility of prosttic cid phosphtse in serum Serum from prosttic cncer ptients ws incubted t , nd 46 #{176}C. Smples tkent the indicted time were subjectedto enzymessy (closed symbols) nd rdioimmunossy (open symbols) Het stbility mesurements were done t ph 8.4, becuse hemolysis does not chnge the ph of the serum substntilly. We sw mrked difference in the stbility of prosttic cid phosphtse when serum smples (ph 8.4) of prosttic cncer ptients were first incubted t 23, 36, or 46 #{176}C nd the ctivity of the enzyme ws then mesured by enzyme ssy (p-nitrophenyl phosphte s substrte) nd by rdioimmunossy. There ws no mesurble loss of ctivity of the prosttic cid phosphtse by rdioimmunossy when serum ws incubted t 23 #{176}C for s long s 72 h. In contrst, by enzymtic ssy of the sme smples there ws 3% decrese in the ctivity in 3 h, nd by 72 h ll ctivity of the enzyme ws gone. At higher tempertures (36 or 46 #{176}C) the enzymtic nd immunologicl ctivities deteriorted much fster but the difference between the two rtes ws still substntil (Figure 1). Specificity of enzymtic ctivity ssy nd rdioimmunossy: We ssyed extrcts from pltelets, erythrocytes, nine tissues, nd serum fom norml mles for prosttic cid phosphtse ctivity, with p -nitrophenyl phosphte, thymolphthlemn monophosphte, phenolphthlein monophosphte, or -nphthyl phosphte s substrtes. The ctivity of the enzyme ws determined in the bsence (totl cid phosphtse) nd in the presence of L-trtrte inhibitor. (When substrtes re not specific for prosttic cid phosphtse, L-trtrte is incorported to determine the contribution of other phosphtses.) Thymolphthlein monophosphte is the most nerly specific substrte for prosttic cid phosphtse of those studied; with L-trtrte present, the results were generlly little chnged (Tble 1). Simultneously, the smples were mesured by rdioimmunossy for prosttic cid phosphtse. Tble 1 shows the results of the nonspecific hydrolysis of the substrtes by vrious tissues. For ech enzyme ssy, the third column shows the reltive specific ctivities 96 CLINICAL CHEMISTRY, Vol. 23, No. 1, 1977

3 C. In o - I- >.e ill O#{149}E C >3 C, U, u cc. -. cc.c C,).. o e 4- >,._.c.. >. N C E I->.. 4- ) - Oi-. L..2 2 C ) L <qo,q.-o.- o -. C..J 8 - do2. o<nc )-O) tc )O -. C ) L C i L C l -) C-. COCO ; c-. c.- -: CiC < -.. J C ) NCOL..- o o.l)-c)c.iococoo, I-.- c 1NOLCOC.O - - CO ( 4 CO L LU ( 4 L - ( 4 N O. -Orcc ).- -CD t(qc )C ) <Q...Q..-. o N (N CO - L C ) ( ( 4. < C ) CO ( 4 ( N I- N C ) L ( LU ( ( #{149}oooooc( )o. I O-OQOOOoo.- <..-.cio.o. - (.N C )L.-LC -,- LU. e<nl.-olulu(oconcn COO I--)COO).-N.-( NLC) OQC )C )C ),-Q ) -..)NLC ))) 2 <q(n-c )C )cnoo C ) - t C ) COO) C ) L- LU.- <-LCOLU( I- -.- b ) C ; C ; of prosttic nd totl cid phosphtse. Totl cid phosphtse ctivity in the different extrcts is in ech cse quite low, but the rtio of prosttic to totl cid phosphtse in serum smples vries from 1 to 25%. Thus, it is essentil to be sure one is mesuring only prosttic cid phosphtse when ssying for this ctivity in serum. The use of L-trtrte to inhibit the prosttic frction of cid phosphtse is essentil when p-nitrophenyl phosphte or phenolphthlein monophosphte is used s substrte. The phosphtse ctivity of nonprosttic phosphtses in tissue extrcts is high when p-nitrophenyl phosphte or phenolphthlein monophosphte re used, in comprison with thymolphthlein monophosphte. The rnge of inhibition by L-trtrte is 3 to 9% for phenolphthlein monophosphte nd p-nitrophenyl phosphte. - Nphthylphosphte is more specific, with less inhibition by L-trtrte. We found thymolphthlein monophosphte to be the most nerly specific substrte for prosttic cid phosphtse. The bldder nd the pncres contin the gretest mount of cross-recting protein nd totl cid phosphtse. The rdioimmunossy gve the lest mount of cross rectivity with other proteins in the extrcts of vrious orgns. The best overll result ws obtined by rdioimmunossy. The norml vlues for serum prosttic cid phosphtse re 12 ± 4 Sigm units per liter nd 48 ± 8 csg/liter by enzyme ssy nd rdioimmunossy, respectively. For this study we used the men vlue plus two stndrd devitions to define bove-norml vlues; i.e., enzyme vlues of >2 Sigm units/liter or rdioimmunossy vlues >64 gig/liter (22). When cncer ptients with other thn prosttic cncer (37 men, 53 women) were evluted for serum prosttic cid phosphtse, 26.6% (24/9) hd bovenorml prosttic cid phosphtse vlues by enzymtic ssy nd 5.5% (5/9) hd bove-norml vlues by rdioimmunossy. Tble 2 shows the distributions of tumors with bove-norml serum prosttic cid phosphtse by rdioimmunossy nd by enzyme ssy. None of the women hd bove-norml prosttic cid phosphtse ctivity by rdioimmunossy. Three men hd bove-norml vlues by both methods: one with phryngel crcinom hd n extremely high vlue; one with epidermoid crcinom of the lung, which metstsized to the prietl re of the brin, hd modertely incresed vlues; one with denocrcinom of the lung hd smller increse. For the rdioimmunossy, 13.5% (5/37) of the mle nonprosttic cncer ptients hd flsely positive ssys for prosttic cid phosphtse by rdioimmunossy, number tht concurs with preliminry study tht showed 8 to 11% flse-positive tests for norml mles (22). #{149} 3 )....! 2 Discussion The level of serum prosttic cid phosphtse is n importnt clinicl dignostic test for cncer of the prostte. Until recently, enzymtic ssys hve been CLINICALCHEMISTRY,Vol. 23, No. 1,

4 Tble 2. Prosttic Acid PhosphtseActivities in Ser of Nonprosttic Cncer Ptients Lung Site of mlignncies Cervix Brest Endometrium Lrynx Bldder Hodgkins disese Lymphom Nsl Brin Phrynx Leukemi Pituitry Others: (renl; uterus; ovry; etc.) Totl Above-norml by Above-norml by rdioimmunossy enzyme y Mle Femle Mle Femle 3/11 /8 5/11 3/8 - /9-1/9 - /9-1/9 - /7-4/7 /6 /1 2/6 1/1 /3 /2 /3 1/2, /1 /4 1/1 /4 used exclusively for this dignostic test. However, incresed temperture inctivtes the enzyme t the ph of norml serum; therefore, results obtined by these methods might not reflect the true ctivity of this enzyme in serum if scrupulous precutions hve not been followed in prepring the serum. Such considertions, s well s questions of substrte specificity, hve led to n immunologicl quntittion of prosttic cid phosphtse. The immunologicl rectivity of prosttic cid phosphtse is pprently more consistent thn enzymtic ctivity.5 Therefore, serum smples do not require specil tretment (e.g., ph djusting) nd hndling. Norml serum contins severl cid phosphtses of different origin. Consequently, specific ssy tht detects enzymes of prosttic origin is required. The cid phosphtse derived from the prostte-s opposed to extrprosttic sources-tht is present in the serum evidently comprises only bout 1 to 25% of the totl prosttic cid phosphtse. Extrcts from pltelets, erythrocytes, nd vrious tissues were evluted for the totl nd prosttic frctions of cid phosphtse s defined by enzyme inhibitor studies nd for prosttic cid phosphtse s ssyed by rdioinununossy. The highest specificity for the enzyme ws demonstrted by rdioimmunossy. Among the enzyme substrtes, thymolphthlein monophosphte hs the highest specificity for prosttic cid phosphtse-no inhibitor hs to be used with this substrte. However, there is significnt disdvntge to this method (23). The men vlue for norml mles is 26 ± 12 Sigm units per liter (24). Using.2 ml of serum, s described for routine nlysis of prosttic cid ) phosphtse, one would obtin n bsorbnce reding /1 /1 /1 of.6 to.17 t 59 nm. Experimentl error with 1/1 / 1/1 / such rnge of redings is extremely high (24). There- /1 /1 /1 1/ i fore, smll chnges in the serum cid phosphtse (e.g., / /2 / 1/2 t stge I or II of the disese) cnnot be ccurtely /9 /5 /9 /5 mesured becuse of this limited sensitivity. An increse in the prosttic cid phosphtse ctivity from 4 to 5 Sigm units per liter, for exmple, results in 5/37 /53 1/37 14/53 chnge in bsorbnce of only.5. Similrly, in the cse of p-nitrophenyl phosphte, the norml vlue for mles is 13 to 63 Sigm units per liter (19), corresponding to n bsorbnce of.5 to.18 t 41 nm. Becuse prosttic cid phosphtse is inhibited by L-trtrte, the rtio prosttic cid phosphtse/totl cid phosphtse vries from 1/1 to 1/4, with bsorbnce redings in the presence of L-trtrte rnging from 4 to 145 Sigm units/liter. The level of prosttic cid phosphtse is estimted by subtrcting these two vlues, which gives n even smller number; such procedure introduced gret del of error into the ssy. Certin nticogulnts (e.g., oxlte, fluoride) inhibit prosttic cid phosphtse ctivity. Mny such compounds tht inhibit the enzymtic ctivity of the prosttic frction of cid phosphtse, hve no effect on the ntibody rection (Foti et l., in preprtion). Similrly, drugs nd other substnces my ffect the ssy. An extended study is under wy to determine wht types of drugs nd other pthologicl conditions my interfere with the detection of prosttic crcinom by rdioimmunossy. The enzymtic ctivity of prosttic cid phosphtse is completely different from its immunologicl ctivity. One site(s) of the protein is responsible for the enzymtic ctivity nd different site(s) is responsible for the immunologicl ctivity. The quntittion of prosttic cid phosphtse cn be done by enzyme nd by immunologicl determintion. It is shown (Figure 1) tht the immunologicl ctivity of prosttic cid phosphtse is more stble thn enzyme ctivity t ll tempertures. After 48 ht 23#{176}C, prosttic cid phosphtse could not be mesured by enzyme ssy becuse of the dectivtion of the enzyme ctivity. However, the immunologicl ctivity towrds the ntibody did not decrese. The concentrtion of prosttic cid phosphtse ws found to be the sme s before. This is the min dvntge of the rdioimmunossy for the detection of prosttic cid phosphtse. If we use enzyme ssy nd rdioimmunossy to mesure the concentrtion of prosttic cid phosphtse in fresh serum, there is close reltionship between the results of the two mesurements. Serum left t room temperture will show decresed concentrtion of prostticcid phosphtse by enzyme ssy nd the originl concentrtion of prostticid phosphtse by rdioimmunossy. We pln to report these findings in detil lter. This work ws supported in prt by Southern Cliforni Permnente Medicl Group nd by contrct E (4-I) Gen-12 between the U. S. ERDA nd the Regents of the University of Cliforni. References 1. Bonner, C. D., Homburger, F., Smithy, G. B., nd Borges, P. R. F., Prosttic serum cid phosphtse level in cncer of the prostte. J. Am. Med. Assoc. 164, 17 (1957). 2. Gutmn, A. B., nd Gutmn, E. B., An cid phosphtse occurring in the serum of ptients with metstsizing crcinom of the prostte glnd. J. Clin. Invest. 17, 473 (1938). 3. Cook, W. B., Fishmn, W. H., nd Clrke, B. G., Serum cid phosphtse of prosttic origin in the dignosis of prosttic cncer: Clinicl evlution of 248 tests by the Fishmn-Lerner method. J. Urol. 88, 281 (1962). 4. Herbert, F. K., The estimtion of the prosttic phosphtse in serum nd its use in the dignosis of prosttic crcinom. Q. J. Med. 15, 221 (1946). 98 CLINICAL CHEMISTRY. Vol. 23, No. 1, 1977

5 5. Gutmn, E. B., Sproul, E. E., nd Gutmn, A. B., Significnce of incresed phosphtse ctivityof bone t the siteof osteoplstic metstses secondry to crcinom of the prostte glnd. Am. J. Cncer 28, 485 (1936). 6. Murphy, G. P., Reynoso, G., Kenny, G. M., nd Get, J. F., Comprison of totl nd prosttic frction serum cid phosphtse levelsin ptientswith differentitednd undifferentitedprosttic crcinom. Cncer (Phildelphi) 23, 139 (1969). 7. King, E. J., nd Jegtheesn, K. A.,Method forthe determintion of trtrte-lbile, prottic cid phosphtse in serum. J. Clin. Pt hol. 12,85 (1959). 8. Hudson, P. B., Brendler, H., nd Scott, W. W., A simple method for the determintion of serum cid phosphtse. J. Urol. 58, 89 (1947). 9. Bodnsky, A., II Determintion of serum phosphtse. Fctors influencing the ccurcy of the determintion. J. BioS. Chem. 11,93 (1933). 1. Huggins, C., nd Tlly, P., Sodium phenolphthlein phosphte s substrte for phosphtse tests. J. Biol. Chem. 159, 399 (1945). 11. Bbson, A. L., nd Red, P. A., A new ssy for prosttic cid phosphtse in serum. Am. J. Clin. PthoS. 32,88 (1959). 12. Seligmn, A. M., Chuncey, H. H., Nchls, M. M., et l., The colorimetric determintion of phosphtses in humn serum. J. BioS. Chem. 19,7 (1951). 13. Roy, A. V., Brower, M. E., nd Hyden, J. E., Sodium thymolphthlein monophosphte: A new cid phosphtse substrte with greter specificity for the prosttic enzyme in serum. Clin. Chem. 17, 193 (1971). 14. Abul-FdI, M. A. M., nd King, E. J., Properties of the cid phospht.ses of erythrocytes nd of the humn prostteglnd.biochem. J. 45,51(1949). 15. Jcobsson, K., The determintion of trtrte-inhibited phosphtse in serum. Scnd. J. Clin. Lb. Invest. 12,367 (196). 16. Woodrd, H. Q., A note on the inctivtion by het of cid glycerophosphtse in lkline solution. J. Urol. 65,688 (1951). 17. Cooper, J. F., nd Foti, A., A rdioimniunossy for prosttic cid phosphtse. I. Methodology nd rnge of norml mle serum vlues. Invest. Urol. 12,98 (1974). 18. Foti, A.., Herschmn, H., nd Cooper, J. F., A solid-phse rdioimmunossy for humn proettic cid phosphtse. Cncer Res. 35, 2446 (1975). 19. Sigm Bulletin No. 14 (1961), Sigm Chemicl Co., St. Louis, Mo Colemn, C. M., Chromogenic cid phosphtse substrtes. Clin. Chem. 12, 529 (1966). 21. Amdor, E.,Price, J. W., nd Mrshll, G., Serum cid -nphthyl phosphtse ctivity. Am. J. Clin. Pthol. 51,22 (1969). 22. Cooper, J. F., Foti, A. G., Herschmn, H., et l. A solid-phse rdioimmunossy for prosttic cid phosphtse. Comprison with p.-nitrophenylphosphte enzymtic technique. (submitted for publiction) 23. Ewen, L. M., nd Spitzer, R. W., Improved determintion of prosttic cid phosphtse (sodium thymolphthlein monophosphte substrte). Clin. Chem. 22,627 (1976). 24. Sthl, E. L, nd Simon, G. E., Acid phosphtse-clinicl studies using sodium thymolphthlein s substrte. Proceedings of the Kimbrough Urologicl Seminr, 2th Annul Meeting, Eton Lbortories, Norwich, N. Y , 1972, p 252. CLINICALCHEMISTRY, Vol. 23, No. 1,