Separation of Tissue and Serum Creatine Kinase Isoenzymes by

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1 CLIN. CHEM. 20/1, 6-40 (1974) eprtion of Tissue nd erum Cretine Kinse Isoenzymes by Ion-Exchnge ColumnChromtogrphy Donld W. Mercer I describe simple, rpid nion-exchnge column chromtogrphic technique for seprting the cretine kinse (CK) isoenzymes in humn serum nd tissue. Extrcts of CK-rich tissues (skeletl muscle, crdic muscle, nd brin) were used to determine optimum conditions for seprting CK isoenzymes MM,, nd BB. mples, lyered on mini-columns (0.5 X 6.0 cm) of DEAE-ephdex A-50, were eluted stepwise with Tris-buffered sodium chloride (100, 200, nd 00 mmol/iiter). Column effluents were ssyed by the Roslki CK method. Distribution of totl ctivity mong the eluted frctions ws tissue-specific nd reproducible. Evlution of ser from 71 ptients with myocrdil infrction nd other diseses ssocited with elevted CK ctivity reveled isoenzyme ptterns tht resembled those of either crdic muscle or skeletl muscle. Crdic pttern (presence of isoenzyme) nd clinicl documenttion of myocrdil infrction were 100% correlted in the 5 ptients so studied. Additionl Keyphrses: dignostic ids #{149}isoenzyme seprtion #{149} kit methods The effective use of CK1 s sensitive indictor of cute myocrdil infrction hs recently been dim inished becuse of numerous reports of CK elevtions owing to noncrdic conditions, such s chronic lcoholism (1), crdioversion (2), cerebrovsculr disese (), hypothyroidism (4), intrmusculr injections (5) nd surgicl trum (6). Attempts to enhnce the dignostic specificity of CK ssys hve been stimulted by these reports. The chief im of pst work hs been to seprte serum CK isoenzymes MM,, nd BB by conven- Biochemistry ection, Dept. of Pthology, Monteflore Hospitl, Pittsburgh, P., 1521; nd Dept. of Pthology, chool of Medicine, niversity of Pittsburgh, P Presented t the 25th Ntionl Meeting of the AACC, New York, N.Y., July 16-20, Nonstndrd bbrevitions used: CK, cretine kinse (EC ); Tris, tris(hydroxymethyl)minomethne; DEAE, diethylminoethyl; MM, skeletl-muscle isoenzyme of CK;, crdic-muscle isoenzyme of CK; BB, brin isoenzyme of CK. Received ept. 20, 197; ccepted Oct. 24, 197. tionl electrophoretic techniques. Cliniclly significnt seprtions of MM from hve been reported (7-10). However, routine use of the electrophoretic technique is limited since lborious gel preprtions nd poor stining techniques remin n integrl prt of the electrophoretic procedure. I describe here simple, rpid, nd quntittive nion-exchnge chromtogrphic technique for CK isoenzymes tht elimintes the difficulties ssocited with existing techniques. Mterils nd Methods Tissue nd erum Preprtion Humn tissue used in this study ws tken from utopsy mteril in which no gross ntomicl chnges were evident. Homogentes (1 g diluted to 10 ml) were prepred with Tris-hydrochloride buffer (50 mmol/liter, ph 8.0) contining sodium chloride (100 mmol/liter) nd dithiothreitol (0.1 mmol/liter). After the homogente ws centrifuged ( x g, for 10 mm), the insoluble pellet ws discrded nd the supernte used in subsequent chromtogrphic experiments. er with CK ctivity greter thn six times norml were obtined from generl hospitl popultion, nd kept refrigerted until isoenzyme nlysis, which usully ws done within two dys. Enzyme Activity Anlysis CK ws ssyed with n ultrviolet kinetic test kit (mith Kline Instruments, Plo Alto, Clif. 9404) bsed on the method of Roslski (11). All ssys were conducted either with the DA 564-B (Beckmn Instruments, Fullerton, Clif. 9264) or with the semi-utomted Esklb spectrophotometer (mith Kline). Electrophoresis Isoenzyme electrophoresis ws performed on polycrylmide (7 g/dl), with the use of n nlyticl verticl-gel pprtus (Cnlco, Rockviile, Md ). mples were diluted 1:1 with sucrose (40 6 CLINICAL CHEMITRY, Vol. 20, No. 1, 1974

2 g/dl) nd pplied to the top of the polymerized gel (0.5 X.5 cm). Electrophoresis ws performed t 2 ma per gel with the Tris-glycine buffer system s originlly described by Dvis (12). CK isoenzymes were detected on sliced 2.5-mm segments of the polycrylmide gel. Gel segments were plced in seprte test tubes contining 0.2 ml of the Tris-hydrochloride-sodium chloride-dithiothreitol buffer, nd liquots were removed nd ssyed for CK ctivity fter they hd soked overnight t room temperture. Column Chromtogrphy The mini-column consisted of 12.5-cm Psteur pipette (Chse Instruments, Lindenhurst, N.Y ) filled with bout 60 mg of the nion-exchnger DEAE-ephdex A-50 (Phrmci, Pisctwy, N.J ). Column dimensions were 0.5 X 6.0 cm. The ion-exchnger ws prepred for column pcking by slowly mixing 5 g of dry DEAE-ephdex with one liter of Tris-hydrochloride strting buffer (50 mmol/liter, ph 8.0) contining sodium chloride (100 mmol/liter). After sedimenttion nd decnttion the ion-exchnger ws resuspended in nother liter of strting buffer. After repeting the sedimenttion nd decnttion step, slurry of bout one prt ionexchnger to four prts strting buffer ws trnsferred to vcuum flsk, where trpped ir bubbles were removed under decresed pressure. The de-erted slurry ws poured into the column until the finl height of the settled suspension ws 6 cm. Before the smple ws pplied, 2 ml of strting buffer ws pssed through the column. (It ws convenient to prepre lrge numbers of columns t the sme time nd to store them until needed; columns stored t room temperture for two weeks hve performed stisfctorily). Figure 1 is digrmmtic representtion of the chromtogrphic process. A smple volume of 1 ml, contining CK ctivity in the rnge of 600 to 2000 m, ws pplied to the top of the column nd smple effluent ws collected in the first vil. After the mini-column hd drined, the second collection vil ws plced under it. ubsequent elution ws stepwise with eluents of Tris-buffered sodium chloride: AMPLE NCI ph ph7.0 LLHL FRACTION NO. Fig. 1. Digrmmtic representtion of the ion-exchnge chromtogrphic procedure for seprtion of CK isoenzymes FRACTION NO. Fig. 2. Distribution of tissue CK isoenzymes MM,, nd BB on DEAE-ephdex columns Three 1-ml frctions of Tris-hydrochioride strting buffer (50 mmol/liter, ph 8.0) contining sodium chloride (100 mmol/liter) were collected in vils 2,, nd 4. Then three 1-ml frctions of Tris-hydrochloride (50 mmol/liter, ph 8.0) contining sodium chloride (200 mmol/liter, ph 8.0) were collected in vils 5, 6, nd 7. Finlly, three 1-ml frctions of Tris-hydrochloride (50 mmol/liter, ph 7.0) contining sodium chloride (00 mmol/liter) were collected in vils 8, 9, nd 10. A flow rte of 0.5 ml/min ws mintined. Totl elution time ws bout 15 mm. Aliquots (50 to 200 il) of column effluents were ssyed for CK ctivity. Results The ion-exchnge chromtogrphic system ws chrcterized by running extrcts of CK-rich tissues through the column. Figure 2 depicts the behvior of the MM isoenzyme from skeletl muscle, the isoenzyme from crdic muscle, nd the BB isoenzyme from brin. keletl muscle extrct exhibited CK ctivity ssumed to be the MM isoenzyme in frctions 1 nd 2. No ctivity ws found in the other frctions. Crdic extrct exhibited bout 90% of its CK ctivity in the MM isoenzyme region nd bout CLINICAL CHEMITRY. Vol.20, No. 1,

3 10% in frctions 6 nd 7. Activity in 6 nd 7 ws ssumed to be the isoenzyme. isoenzyme ws not found in the other tissues. Brin extrct exhibited predominnt pek of CK ctivity in frctions 8, 9, nd 10. Activity in these frctions ws ssumed to be the BB isoenzyme. All three of these tissue isoenzyme ptterns hve been reproduced mny times with number of different tissue preprtions. Evidence tht these ptterns re indeed rel nd not merely rtifcts of the chromtogrphic system is shown in Figure. In this experiment, column frctions 1 nd 2 from crdic muscle (MM isoenzyme), column frctions 6 nd 7 from crdic muscle ( isoenzyme), nd column frctions 8, 9, nd 10 from brin (BB isoenzyme) were rechromtogrphed on DEAE-ephdex. As shown in Figure, the ionexchnge iso#{22}nzymes ppered s single peks with unchnged elution chrcteristics. Results of polycrylmide-gel electrophoresis of the ion-exchnge isoenzymes re shown digrmmticlly in Figure 4. All three ion-exchnge isoenzymes showed single bnds of ctivity with chrcteristic migrtion rtes similr to those reported for MM,, nd BB by other investigtors (7-10, 1). The uniqueness of the ion-exchnge isoenzymes ws lso demonstrted by the results of thermostbility ex- I.- C) 4 F-) (+) BB ORIGIN- MM - () HEART HEART BRAIN FRACTION FRACTION FRACTION ,9,810 DEAE EPHAOEXEFFLENT Fig. 4. Polycrylmide gel electrophoresis of DEAE-ephdex effluents - MINTE Fig. 5. Thermostbility (56 #{176}C) of DEAE-ephdex effluents -j FRACTION Fig.. Rechromtogrphy of DEAE-ephdex effluents NO. periment t 56#{176}C (Figure 5). Figure 6 shows typicl ion-exchnge ptterns of ser obtined from ptients with myocrdil infrction nd from ptients with other diseses tht lso exhibit elevted CK. These ptterns were obtined by pplying 1 ml of serum, contining bout 1000 m of CK, to the column. A close reltionship between serum isoenzyme ptterns nd those of crdic nd skeletl muscle (Figure 2) ws observed. Although MM isoenzyme found in frctions 1, 2, nd ws the predominnt pek of ctivity in both ptterns, only in ser of ptients with myocrdil infrction ws the isoenzyme detected. Tble 1 reltes dignosis (of 71 ptients in whose ser CK ctivity ws more thn six times norml) nd serum isoenzyme distribution. Note tht of the 5 ptients hving known myocrdil infrction, MM nd isoenzymes were detected in ech cse. However, in the remining ptients who hd different dignoses, only the MM isoenzyme ws detected, but not the isoenzyme. BB isoenzyme ws not detected in this group of ptients. Results of quntittive nlysis for MM nd in 20 ptients with myocrdil infrction re shown in Tble 2. Isoenzyme nlysis ws performed on specimens collected during pek ctivity of totl CK without regrd to specified post-infrct time periods. An verge yield of 6% for isoenzyme ws consistent with ctivities (pproximtely 10%) of 8 CLINICAL CHEMITRY, Vol. 20, No. 1, 1974

4 ,. Tble 1. erum CK lsoenzymes in 71 Ptients Exhibiting ixfold Norml (or Greter) erum CK Activity Dignosis Myocrdil infrction Acute renl filure l1ypothyroidism Cerebrovsculr disese Pulmonry disese Chronic lcoholic Musculr injections Postopertive recovery Muscle crmps After crdioversion Accident victims Otherdignoses No. CPK Iso.nzymes ptients MM BB FRACTION Fig. 6. Typicl distribution of serum CK isoenzymes. on DEAE-ephdex columns NO HOR AFTER INFARCT 250 -j ? IŪ too 4 50 Fig. 7. Typicl course of chnge in totl CK nd ctivities fter myocrdil infrction found in crdic tissue. Correltion between yields nd totl CK ws not observed. A typicl time course for post-infrct ctivities of totl CK nd is shown in Figure 7. These ctivities prlleled one nother until 6 h fter infrct, when could no longer be detected. Column reproducibility ws evluted by repeted nlysis of serum pool fortified with isoenzyme Tble 2. Quntittive Anlysis of CK lsoenzymes in 20 Ptients with Dignosis of Myocrdil Infrction MM PtIent /liter % (/MM + ) X 100. Tble. Within-Run Vrition Isoenzyme Distribution of Results for in pecil -Fortified erum Control MM from n extrct of hert tissue. A smple volume of 1 mple no. /liter ml, contining 748 m of CK, ws pplied to ech of 10 ion-exchnge columns. The results (Tble ) proved to be stisfctory, nd excellent recoveries of totl CK ctivity from the columns were lso observed (Tble ). Column cpcity for MM isoenzyme ws exceeded by CK smples with ctivities ner 000 /liter. To : prevent cross contmintion of effluents by ex cess MM isoenzyme, I diluted high-ctivity CK sm ples with Tris-hydrochioride strting buffer to CK Men ctivity ner 1000 /liter before they were pplied to the column Recovery, % CLINICAL CHEMITRY, Vol. 20, No. 1,

5 Discussion erum isoenzyme determintions hve long been considered importnt dignostic tests, especilly when the tissue source of n elevted enzyme is not cliniclly pprent. But becuse complex electrophoretic techniques re used, isoenzyme determintions re considered to be highly specilized tests nd re performed by only few lbortories. As n lterntive to electrophoresis, selective inhibition techniques with ure, het, nd specific chemicl inhibitors hve been ttempted. Results from these studies re useful if only one isoenzyme is involved, but yield equivocl results if more thn one isoenzyme is present, s is frequently the cse for serum. Although ion-exchnge chromtogrphy hs been used successfully to isolte nd purify tissue isoenzymes (14-16), clinicl ppliction of this technique hs been neglected. Recent ttempts hve been mde to use ion-exchnge s method to seprte cytoplsmic nd mitochondril sprtte minotrnsferse (EC ) in ser of ptients with liver disese (17, 18). The results were cliniclly significnt, but routine clinicl use is limited, primrily becuse of the long elution time (2 to 4 h) nd the long dilysis period (4 to 5 h) required before the serum is chromtogrphed. The present ion-exchnge method for seprting CK isoenzymes is convenient for clinicl use. The elution times usully ssocited with column chromtogrphy hve been eliminted by the use of minicolumns nd stepwise elution techniques. Need for pre-tretment of smple (dilysis) hs lso been eliminted by setting the column s exchngeble chloride concentrtion equl to the chloride concentrtion of serum. nder these conditions, MM isoenzyme of skeletl muscle quickly psses through the ion-exchnger while the isoenzyme of crdic muscle nd BB isoenzyme of brin remin ttched until specified increse in chloride concentrtion decreses their ionic interction with the ion-exchnger. Quntittive ssy of column effluents by technique usully used to ssy totl CK completes the CK isoenzyme nlysis. Clinicl results for the present technique confirm previous observtions mde with electrophoretic techniques tht is quite specific for the detection of myocrdil infrction if specimens re collected nd ssyed between 12 nd 6 h fter the infrct (19). It is difficult to detect in lte post-infrct specimens, either by electrophoresis or ion-exchnge, becuse the ctivity becomes negligible. However, it ppers tht the ion-exchnge technique is idelly suited for detecting trce ctivities of, becuse one cn collect nd concentrte effluents from series of ion-exchnge columns on which the specimen in question hs been run. tudies directed towrd the detection of in lte post-infrct specimens nd in other specimens in which totl CK ctivity is norml or slightly elevted re now in progress. References 1. Nygren, A., erum cretine phosphokinse in chronic lcoholism.act Med. cnd. 182,8(1967). 2. Mndecki, T., Leszek, G., nd Krgul, W., erum enzyme ctivities fter crdioversion. Brit. Hert J. 2, 600 (1970).. Acheson, J., Jmes, D. C., Hutchinson, E. C., nd Westhed, R., erum cretine kinse levels in cerebrl vsculr disese. Lncet i, 106 (1965). 4. Griffiths, P. D., Cretinephosphokinse levels in hypothyroidism. Lncet i, 4(196). 5. Meltzer, H. V., Mrozk,., nd Boyer, M., Effect of intrmusculr injections on serum cretine phosphokinse ctivity. Amer. J. Med. ci. 259,42(1970). 6. Dixon,. H., Fuchs, J. C. A., nd Ebert, P. A., Chnges in serum cretine phosphokinse ctivity following thorcic, crdic nd bdominl opertions. Arch. urg. (Chicgo) 10, 66 (1971). 7. mith, A. R., eprtion of tissue nd serum cretine kinse isoenzymes on polycrylmide gel slbs. Clin. Chim. Act 9, 51 (1972). 8. Konttinen, A., nd Hnnu,., Determintion of serum cretine kinse isoenzymes in myocrdil infrction. Amer. J. Crdiol. 29,817(1972). 9. Elevitch, F. R., Isoenzymes. In Fluorometric Techniques in Clinicl Chemistry. Little, Brown nd Co., Boston, Mss., 197, p Roe, C. R., Limbird, L. E., Wrner, G.., nd Nerenberg,. T., Combined isoenzyme nlysis in the dignosis of myocrdil injury: ppliction of electrophoretic methods for the detection nd quntittion of the cretine phosphokinse iosenzyme. J. Lb. Clin. Med. 80, 577 (1972). 11. Roslki,. B., An improved procedure for serum cretine phosphokinse determintion, J. Lb. Clin. Med. 69,696(1967). 12. Dvis, B. J., Disc electrophoresis. Method nd ppliction to humn serum proteins, Ann. N. Y. Acd. ci. 121,404(1964). 1. Dwson, D. M. nd Fine, I. H., Cretine kinse in humn tissues. Arch. Neurl. (Chicgo) 16, 175 (1967). 14. Wood, T., Adenosine triphosphte-cretine phosphotrnsferse from ox brin: Purifiction nd isoltion. Biochem. J. 87, 45 (196). 15. Wchsmuth, E. D., nd Pleiderer, G., Biochemische ntersuchungen n kristllinen Isozymen der lctt Dehydrogense us menschlichen Orgnen. Biochem. Z. 6, 545 (196). 16. Tkhshi, K., shikubo,., Oimomi, M., nd hinko, T., Cretine phosphokinse isoenzyme of humn hert muscle nd skeletl muscle. Clin. Chim. Act 8, 285 (1972). 17. chmidt, E., chmidt, F. W., nd Mohr, J., An improved simple chromtogrphic method for seprting the isoenzymes of mlic dehydrogense nd glutmic oxlocetic trnsminse. Clin. Chim. Act 15,7 (1967). 18. Ideo, G., Frnchis, R, Bellobuono, A., nd Tornghi, G., Asprtte minotrnsferse isoenzymes in humn serum in vrious liver diseses. Enzymes 12,529(1971). 19. Wgner, G.., Roe, C. R., Limbird, L. E., Rosti, R. A., nd Wllce, A. G., The importnce of identifiction of the myocrdil-specific isoenzyme of cretine phosphokinse ( form) in the dignosis of cute myocrdil infrction. Circultion 47, 26 (197). 40 CLINICAL CHEMITRY, Vol. 20, No.1,1974