Quantification of Serum Creatine Phosphokinase

lsoenzyme Activity

ROBERT ROBERTS, MD PHILIP D. HENRY, MD SAKE A. G. J. W~EVEEN, MD BURTDN E. SOBEL, MD, FACC

La Jo& California St. Louis, Missouri

Three serum creatine phosphokinase (CPK) isoenrymes (MM, MB and BB) have been recognized. My~ardium is richly endowed with MB CPK. Accordingly, Increased serum MB CPK activity reflects myocar- dial injury. Although recent advances have facilitated detection of ac- tivity of CPK isoenzymes, available techniques have unavoidable quan- titative limitations. Accordingly, we used a new procedure to quantify CPK Noenzymes in serum samples wlh normal or increased total CPK activity. Samples were dlluted or concentrated so that total CPK activi- ty was 0.100 to 1.200 IlJ/ml and then subjected to electrophoresis on cellulose acetate. Regions of the strips encompassing each isoenzyme were cut out, immersed in 0.5 to 3 ml of assay medium in which the re- duced form of nkotinamide adenine dkuckotide phosphate (NADPH) was generated as a fu~t~n of tsoenzyme activity. NADPH was de- tected by serial determinations of fluorescence in the medium. 7he assay was linear wtth respect to time and isoentyme activity (0.010 to 1.2 W/ml, no. = 100) and recovery averaged 83 f 1.8 percent (stan- dard deviation). Reproductbillty was within 2 percent. Accuracy of the method was validated with selected mixtures of BB and MM koen- zymes. Serum MB CPK activity averaged onty 0.002 f 0.001 W/ml in normal persons (no. = 271, compared to 0.084 f 0.043 W/ml in 30 patients with myocardtal infarction. Serum MB CPK activity was not in- creased in 23 patients after intramuscular injectlons or abdomkal sur- gery. The procedure described permfts quantitative determination of activtty of individual CPK isoenxymes in serum samples with or wtthout increased total CPK activity and thus Is of potential value in the quanti- tative assessment of myocardial infarction by serum enzyme analysis.

From the Department of Medicine, University of California, San Diego, La Jolla, Calif. and the Department of Medicine, Washington University. St. Louis, MO. This research was supported in part by U. S. Public Health Service Myocardiil Infarction Research Unit Contract PH 43- §8~~~1332, and National Heart and Lung Insti- tute U. S. Public Health Service Research and Career Development Award (l-KCHL-50, 179- 01) (Dr. Sobel), and Ontario Training Grant and Ontario Heart Foundation (Dr. Roberts). Manu- script accepted September 9, 1973.

Address for reprints: Burton E. Sobei, MD, Cardiovascular Diilsion, Wa~i~on Un~ers~, 880 South Euclid Ave., St. Louis, MO. 63110.

Serum creatine phosphokinase (CPK) de~rminations are becoming increasingly important in the diagnosis1 and quantificatjon2-~ of acute myocardial infarction. Three serum CPK isoenzymes, each a dimer (MM, MB, and BB), have been recognized. Extracts of human myocardium contain both MB and MM in contrast to extracts of skeletal muscle, which contain primarily MM. Thus, ischemic myo- cardial injury is reflected by increased serum MB activity.5-7 Recent improvements in procedures for assay of CPK isoenzyme activity in- volve separation of isoenzymes by electrophoresis and scanning of the electrophoretic medium to detect fluorescence of the reduced form of nicotinamide adenine dinucleotide phosphate (NADPH).5*8 However, quantitative results obtained with all methods dependent upon scan- ning tend to be inaccurate for several reasonsealO: (1) Isoenzymes may be distributed asymmetrically in the electrophoretic supporting me- dium so that selection of the position used for scanning may influence results. (2) Activity of individual isoenzymes may be underestimated because of limited diffusion of substrate into the supporting medium or loss of NADPH or dye from the medium. (3) Apparent activity of specific isoenzymes may deviate from linearity with respect to time or

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QUANTlFlCATlON OF SERUM CPK ISOENZYMES-ROBERTS ET AL.

actual activity because of disparate activities of one or more isoenzymes in a mixture. Accordingly, we de- veloped and evaluated a procedure for quantitative analysis of CPK isoenzymes designed to eliminate these difficulties and used it to measure activity of CPK isoenzymes in serum from hospitalized subjects with and without acute myocardial infarction.

Methods Blood samples were collected in 0.010 molar neutralized

EGTA, and serum was separated by centrifugation. Super- natant fractions of 16,000 g were prepared from human pa- pillary muscles obtained freshly during the course of mitral valve replacement, and from brain obtained at autopsy.” Total CPK activity was measured spectrophotometrically after samples were diluted with 0.01 molar Tris, pH 7.4 containing 0.2 percent bovine serum albumin (Tris-BSA), so that CPK activity was approximately 0.1 IU/ml and thus within the limits of linearity of the assay.” CPK isoen- zymes were separated by cellulose acetate electrophore- sis. 7~12 Samples were diluted with Tris-BSA or concentrat- ed with Aquaside II (Calbiochem) to produce total CPK ac- tivity values of 0.100 to 1.200 IU/ml. Control experiments were performed by omitting creatine phosphate as sub- strate.

In preliminary experiments (see Results) we found that detection of electrophoretically separated isoenzymes by fluorescence scanning5*s was inadequate for quantification. Accordingly, the following approach was employed:

1. CPK isoenzymes were separated by cellulose acetate electrophoresis.

2. After electrophoresis, sections of the cellulose acetate strips containing each isoenzyme were cut out, immersed in 0.5 to 3 ml of the CPK assay medium in a disposable cu- vette, and incubated at 25’ C with constant shaking. Incu- bation time and volume were chosen to provide linear reac- tion rates and sufficient fluorescence from NADPH gener- ated- by isoenzymes on each selected portion of the strip. Analysis of 100 samples each of sections containing the three isoenzymes indicated that the enzymes were con- tained within the following regions: MM, 4 mm cathodal to 12 mm anodal; MB, 12 mm anodal to 37 mm anodal; BB, 37 mm anodal to 47 mm anodal.

3. NADPH generated was detected by serial measure- ment of fluorescence in the aqueous medium in which the selected portions of the cellulose acetate strips were being incubated.

To determine whether NADPH was eluted quantita- tively under these conditions we applied 10 ~1 aliquots of samples containing known quantities of NADPH to moist cellulose acetate membranes, allowed the samples to dry for 15 minutes, eluted the NADPH from the strips under the same conditions as those used in the isoenzyme assay procedure and determined fluorescence in the aqueous me- dium with use of spectrophotometry.

To determine whether the isoenzyme assay was linear with respect to time and enzyme activity, aliquots of rab- bit skeletal muscle CPK were applied to cellulose acetate strips but not separated by electrophoresis. Selected re- gions of the strip were cut out and incubated in CPK reac- tion medium. Fluorescence in the aqueous medium was de- termined serially with an Aminco-Bowman fluorometer while the cellulose acetate strip was removed transiently from the solution.

To assess recovery of isoenzyme activity from cellulose acetate strips, fluorescence detected with aliquots added to the medium itself was compared with fluorescence detected

INFARCTION NO INFARCTION FIGURE 1: Total serum CPK activity and MB CPK activity in patients with myocardial infarction and in hospitalized patients without infarc- tion. CPK activity and MB CPK activity were assayed in samples ob- tained from patients with myocardial infarction and selected on the basis of peak CPK activity for each patient. Results expressed rep- resent means plus 1 standard deviation indicated by the vertical bars. Serum MB CPK activity in patients with infarction averaged 0.064 IU/ml, compared with 0.002 IU/ml in samples from hospital- ized patients without infarction.

when corresponding aliquots were assayed in cellulose ace- tate strips immersed in incubation medium.

To determine whether electrophoresis altered recovery of CPK actiuity, aliquots of rabbit skeletal muscle CPK were subjected to electrophoresis before fluorometric assay of CPK activity in appropriate regions of the cellulose ace- tate membrane. Percent recovery was calculated by com- parison with activity observed when corresponding aliquots were assayed in solution. Recovery of MM and MB isoen- zymes in serum samples was determined in the original sample and in serial dilutions.

Reliability of the assay was examined with human myo- cardial supernatant fractions containing MM and MB CPK. Activities of each were determined 10 times from ali- quots of the initial sample and 10 times each from aliquots of the sample diluted to contain 0.2 and 0.1 IU/ml.

Accuracy of the method was assessed with solutions for- mulated with selected quantities of BB and MM isoen- zymes.

Case material: Isoenzymes were assayed quantitatively in serum samples containing MM and MB isoenzymes and increased total CPK activity’ from 30 patients with acute myocardial infarction, 27 patients without infarction and normal total CPK activity, 23 patients with elevated total CPK activity after intramuscular injections (16 patients) and abdominal surgery (7 patients). Isoenzymes were as- sayed also in 16 skeletal muscle extracts obtained at opera- tion or at autopsy.

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QUANTlFlCATlON OF SERUM CPK IBGENZYMEB -ROBERTS El AL.

TABLE I

Total CPK and CPK lsoenryme Activity in Serum Samples and Skeletal Muscle Extracts (means h 1 standard deviation)

Category and no. Total CPK MM CPK MB CPK

A. Serum Samples (lU/ml)*

Hospitalized control subjects (27)

Patients with acute myocardial

infarction (30) Patients receiving intramuscular

injections (deltoid) Diazepam (5) Morphine sulfate (7) Ampicillin (4)

Patients undergoing abdominal

surgery (7)

0.036 f. 0.013 0.033 It 0.012 0.002 f 0.001 0.860 f 0.120 0.797 f 0.112 0.063 f 0.043

0.454 f 0.051 0.451 Z!Z 0.052 0.003 * 0.001 0.347 i 0.089 0.345 Z+Z 0.089 0.003 ZjZ 0.002 0.595 i 0.121 0.593 XIZ 0.121 0.002 f. 0.001 1.024 f 0.232 1.022 & 0.232 0.003 * 0.002

B. Skeletal Muscle Extracts (IU/g)t

Obtained at autopsy (10) 2.630 i 0.260 2.630 f 0.260 0 Obtained at operation (6) 1.430 & 0.080 1.430 l 0.000 0

* Serum samples analyzed were those exhibiting peak total CPK in patients who had serial samples obtained at 3 hour intervals. t Extracts of skeletal muscle were prepared as 1:25 homogenates in 0.25 M sucrose, 1 mM EGTA and 10 mM potassium phosphate

buffer, pH 7.4. Total CPK and CPK isoenzymes were assayed in 16,000 g supernatant fractions. Tissue samples were obtained from the rectus abdominis muscle in patients undergoing abdominal surgery and from the deltoid, pectoralis major and psoas muscles in

patients who had died 4 to 6 hours before the study.

In studies designed to determine the effects of storage media on preservation of isoenzyme activity, papillary muscle extracts were diluted with human serum, divided into aliquots and protected with 0.005 mercaptoethanol, neutralized 0.010 molar sodium ethyleneglycol tetraacetic acid (EGTA) or 2.5 molar sucrose or a combination of the three. Samples were frozen on dry ice and ethanol, stored at -20” C and assayed serially after fast thawing.

Results MB and MM activity in serum samples: MM

and MB activities in serum samples with peak total CPK activity from 30 patients with acute myocardial infarction and in serum samples from 27 patients without increased total serum CPK are shown in Fig- ure 1. Both total CPK and MB CPK activity were in- creased in samples from patients with acute myocar- dial infarction. Furthermore, the average absolute ac- tivity of MB CPK was more than 20 times greater in samples from patients with infarction. Increased MB CPK activity after infarction has been identified in previous studies. 5+%syg However, the sensitivity of the present method permits detection of some MB CPK in serum from patients without acute myocardial in- farction who have normal total serum CPK activity.

Total serum CPK activity may increase after intra- muscular injections or surgery, presumably because of release of CPK from skeletal muscle, and frequent- ly the diagnosis of acute myocardial infarction must be considered in patients in this setting. Accordingly, we assayed CPK isoenzymes in samples with in- creased total CPK activity from hospitalized patients without myocardial infarction who had received in- tramuscular injections of diazepam, morphine sulfate

or ampicillin. Total CPK, MB CPK and MM CPK activity in these samples are tabulated in Table I and compared with results in samples from patients who had recently had abdominal surgery or myocardial infarction. MB CPK activity was not appreciably in- creased in samples from patients after intramuscular injections or surgery even when total serum CPK values were as high as those in patients with acute myocardial infarction.

86

14

0 ; TOTAl

85 07 07

15 I3 13 FIGURE 2. Activity of total serum CPK, MM CPK and MB CPK in ali- quots from serial dilutions of the same initiil serum sample. The pro- portion of MM and MB isoenzymes remained constant in aliquots from serial dilutions of the initial sample with total CPK activity levels ranging from 1.120 to 0.120 Wml. Results are averages of dupli- cate determinations with each dilution.

652 May 1974 The American Journal d CARDIOLOGY Volume 33

Both total CPK and activity of each isoenzyme (BB, MM, and MB) remained constant for at least 2 weeks when samples were stored with EGTA, mer- captoethanol or sucrose as described under Methods.

Evaluation of the quantitative method: Recov- ery of NADPH from cellulose acetate membranes ranged from 97.5 to 99.5 percent (no. = 10). Fluores- cence increased linearly with time for at least 5 min- utes in each case when CPK in rabbit skeletal muscle samples (0.010 to 1.2 III/ml) was assayed after elec- trophoresis (no. = 100).

Average recovery was 97 f 2.5 percent (standard deviation) (no. = 100) when rabbit skeletal muscle samples were applied to cellulose acetate but not sep- arated by electrophoresis. After electrophoresis re- covery was virtually constant, averaging 83 f 1.8 per- cent (no. = 100). Determinations of MM activity after electrophoresis varied by 4~3 percent (no. = I00 samples with activity of 0.010 to I.2 IU/ml). Results with serial dilutions of serum samples containing more than one isoenzyme (Fig. 2) indicated consis- tent proportions of MB and MM CPK in aliquots with total CPK activity values ranging from 1.120 to 0.120 IIJlml.

When each of three papillary muscle extracts (MB CPK activity = 0.018,0.037 and 0.256 III/ml) was as- sayed 10 times, apparent activity of each isoenzyme varied by only 4~1.5 percent (range). When selected mixtures of BB from human brain and MM CPK from rabbit skeletal muscle were assayed, the ob- served and expected values for each isoenzyme agreed within ~t3 percent (three mixtures with re- spective total CPK activity levels of 0.2, 0.5 and 1.0 IU/ml).

Difficulties encountered with conventional scanning procedures: Although fluorescence scan- ning is a sensitive technique,s*13 in our hands it had serious quantitative limitations. For example, when the fluorometer was used to scan five different posi- tions on the same strip, the apparent MB CPK isoen- zyme activity ranged from 12 to 38 mIU/ml. When al- iquots of the same sample were applied to 10 differ- ent strips and each strip was subsequently scanned, the apparent activity of MB CPK in the sample ranged from 11 to 39 mIU/ml. When selected mixtures of BB and MM CPK were applied to eight different strips and fluorometric scanning was per- formed, the apparent ratio of BB to MM activity dif- fered from that expected by 20 to 80 percent (no. = 8) despite vigorous efforts to apply the samples uni- formly. In preliminary experiments (data not shown) in which electrophoresis was performed on agarose5t8 rather than on cellulose acetate, we encountered sim- ilar difficulties. Thus, despite the sensitivity exhib- ited by fluorescence scanning, we found quantitative

limitations that were minimized or avoided by the procedure used in this study.

Discussion

Increased total serum CPK activity is a hallmark of acute myocardial infarction. However, total CPK activity increases after intramuscular injections, trauma and in association with many disorders. Re- lease of CPK from organs other than the heart could influence estimation of infarct size based on serum CPK analysis. Thus, quantification of serum CPK isoenzymes is of particular importance in delineating the source of CPK in serum.

Although fluorescence scanning has been useful for detecting CPK isoenzymes,8 several factors may limit its value as a quantitative tool including (1) asym- metric distribution of isoenzymes along the axis of scanning,gJ0113-15 (2) 1 oss of NADPH from the elec- trophoreti~ medium, l6 (3) limi~tion of penetration of substrate into the supporting medium,sJs and (4) deviation of the reaction from linearity with respect to one or more isoenzymes in a mixture because of disparate activities of each.gJ6

Advantages of method: The method described was designed to avoid these difficulties and to facili- tate accurate quantitative determinations of the ac- tivity of each individual isoenzyme in samples con- taining a mixture. Since isoenzymes are assayed sep- arately, reaction conditions can be adjusted to achieve linearity with respect to time and activity of each isoenzyme. All NADPH generated contributes to fluorescence since it is eluted into the aqueous reaction medium. Availability of substrate to each isoenzyme in the supporting medium is faciliated by constant shaking and immersion of the supporting medium into the reaction solution. The problem of nonuniform distribution of the sample is eliminated since the entire region of the supporting medium en- compassing each isoenzyme is utilized. Reactions are performed kinetically. The method detects individual isoenzymes with an activity level of less than 0.002 IUlml, and hence is capable of qu~tifying isoen- zymes even when total serum CPK activity is not in- creased. Results appear to be reproducible.

Clinical application: The precision, accuracy and sensitivity of this method make it a potentially useful adjunct in delineating the source of CPK contribut- ing to total serum CPK activity in patients and in quantifying myocardial injury by analysis of serial changes in activity of serum enzymes.4 The technique should prove particularly useful in detecting and quantifying myoeardial injury in patients with con- comitant injury to skeletal muscle, such as those undergoing coronary bypass grafting and other surgi- cal procedures.

References

1. Sobel BE, She6 WE: Serum enzyme ~t~rn~tions in the dig- s~ni~nce of regional ~s~n~~n ping acute ~~r~~l in- nosis and assessment of myocardiai infarction. Circulation 45: far&ion. Circulation 45441-456, 1972 47%462,1972 3. Shell WE, Kjekshus JK, Sobel BE: Quantitative assessment of

2. Zeret BL, Pitt 8, Ross RS: Determination of the site, extent, and the extent of myocardial infarction in the conscious dog by

May 1974 7he American Journal of CARDtOLOGY Vokmo 33 333

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means of analysis of serial changes in serum creatine phos- phokinase (CPK) activity. J Clin invest 50:2614-2625, 1971

4. Sobel BE, Breenahan OF, Shell WE, et al: Estimation of infarct size in man and its relation to prognosis. Circulation 46:640- 646, 1972

5. Wagner GS, Roe CR, Llmblrd LE, et al: The importance of iden- tification of the myocardhl-specific isoenzyme of creatine phos- phokinase (MB form) of acute myocardial infarction. Circulation 47:263-269. 1973

6. Konttlnen A, Somer H: Determination of serum creatine kinase isoenzymes in myocardial infarction. Am J Cardiol 29:817-820. 1972

7. Klein MS, Shell WE, Sobel BE: Serum creatine phosphokinase (CPK) isoenzymes following intramuscular injections, surgery, and myocardiil infarction: experimental and clinical studies. Cardiovasc Res 7:412-418, 1973

8. Roe CR, Limbird LE, Wagner GS, et al: Combined isoenzyme analysis in the diagnosis of myocardiil injury: application of etectrophoretic methods for the detection and quantification of

the creatine phosphokinase MB isoenzyme. J Lab Clin Med 80: 577-590, 1972

9. Latner AL, Sklllen AW: lsoenzymes in Biology and Medicine. New York, Academic Press, 1968, p 187

10. Whipple HE: Gel electrophoresis. Ann NY Acad Sci 121:305- 650, 1964

11, Rosalkl SB: An improved procedure for serum creatine phos- phokinase determination. J Lab Clin Med 69:696-705, 1967

12. Sobel BE, Shell WE, Klein MS: An isoenzyme of creatine phos- phokinase in mitochondria from rabbi myocardium. J Molec Cell Cardiol4:367-380, 1972

13. Pert JH, Plnterlc L: Quantification of starch gel electrophoresis. Ann NY Acad Sci 121:310-320. 1964

14. Ornsteln L: Disc electrophoresis. Part 1: Background and theo- ry. Ann NY Acad Sci 121:321-349. 1964

15 WhHe JW, Kushnlr I: Enzyme resolution in starch gel electropho- resis. Anal Biochem 16:302-313, 1966

16. Brewer GJ: An Introduction to lsozyme Techniques. New York, Academic Press, 1970, p 161

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