creatine kinase isoenzymes in the assessment of heart disease

Fundamentals of clinical cardiology

Creatine kinase isoenzymes in the assessment

of heart disease

Robert Roberts, M.D. Burton E. Sobel, M.D. St. Louis, MO.

Because chest pain and transitory electrocardiographic changes do not differentiate patients with coronary insufficiency from those with myocardial infarction, objective confirmation of myocardial necrosis by analysis of plasma enzymes has’ assumed increasing importance. Enzymes such as serum glutamic oxalacetic transaminase @GOT), lactate dehydrogenase (LDH), and creatine kinase (CK)* may be released into blood from organs besides the heart. However, delineation of isoenzyme profiles improves diagnostic specificity substantially. Since elevated plasma activity of one isoenzyme of CK, MB CK, appears to be the most sensitive and specific enzymatic index of acute myocardial infarction,‘, 2* 3 MB CK will be the subject of this selective review.

Ch8r8CteliStiCS of creatine kinase and creatine

kinase isoenzymes

Creatine kinase is a dimeric molecule with molecular weight of approximately 86,006 dal- tons, consisting of two subunits of either the B or M type.4 The M subunit is predominant in skeletal muscle; the B subunit in brain. CK partici- pates in a reversible reaction transferring high energy phosphate from ATP to creatine phosphate. Arginine in each monomer binds the

From the Cardiovascular Division, Washington University School of Medicine, St. Louis, MO.

Supported in part by National Institutes of Health Grant HL 17646, SCOR in Ischemic Heart Disease.

Received for publication Feb. 18, 1977.

Reprint requests: Burton E. Sobel, M.D., Director, Cardiovascular Division, Washington University School of Medicine, 660 South Euclid Ave., St. Louis, MO. 63110.

l Creating kinase is the preferred term, rather than creatine phosphokinase, since by definition kineses mediate the transfer of high energy phosphate from substrate to product.

magnesium ATP or ADP complex necessary for the reaction. Each monomer contains one sulfhy- dry1 group necessary for enzymatic activity. Thus, a thiol activator is needed to elicit maximal activity in uit~o.~-’ CK dimers are subject to dissociation into subunits, particularly when exposed to freezing and thawing or to high concentrations of urea.4

Three CK isoenzymes have been recognized in plasma: BB, MM, and MB. Because of the nega- tive charge of the B subunit at pH 8.0, MM is neutral, MB intermediate, and BB most nega- tively charged and hence most mobile in an electrophoretic field.”

Measured CK activity is similar in corre- sponding serum and plasma samples and not affected by therapeutic concentrations of heparin or coumadin compounds. However, plasma samples should be collected in EGTA rather than EDTA since magnesium required for enzymatic activity may be sequestered by EDTA. Stability of CK during storage varies with the isoenzyme profile in the sample.9 MM CK collected in EGTA and mercaptoethanol is stable at room temperature for 48 hours, but MB and BB are stable for only 2 hours under these conditions.‘” However, BB and to some extent MB are unstable at room temperature in the absence of a reducing agent, which must be added promptly, since loss of activity due to dissociation into subunits cannot be restored completely. With refrigeration, MM is stable for at least 6 days and BB and MB for 24 hours.‘O With fast freezing and storage at -20° or -70” C. in the presence of mercaptoethanol and EGTA, MM and MB are stable for years and BB is stable for at least six months.‘O

The half-lives of circulating CK isoenzymes in

0002-8703/78/0495-0521$00.80/O 0 1978 The C. V. Moeby CO. American Heart Journal 521

Roberts and Sobel

uiuo are: 10 to 12 hours (MM) and 6 to 8 hours (MB). BB appears only rarely in plasma in part because of an apparently short half-life.”

Since the development of Rosalki’s modifica- tion of the original Oliver assay for total CK activity,5 uniform and reproducible results have been obtained with the back reaction in which creatine phosphate is converted to ATP. Rea- gents required are supplied from several manufac- turers in kit form and provide reliable results as long as reasonable precautions are taken for quality control. Activity is expressed in international units/liter with 1 IU defined as the activity required to convert 1 pmole of substrate to 1 wale of product under reaction conditions at 30” C. With assays performed at 30“ (the temperature recommended by the International Union of Boochemists), the upper limit of normal for total CK is 65 IU/L. and 50 IU/L. for male and female subjects.

Although some CK activity is present in most human tissues obtained surgically (as opposed to necropsy specimens), appreciable quantities are found in only four.3. I2 Skeletal muscle is the most richly endowed source with 3200 IU/Gm. Human myocardium contains 1600 IU/Gm., brain 200 IU/Gm., and gastrointestinal tract approximately 150 IU/Gm. Other tissues such as lung (13 IU/ Gm., spleen and liver (< 1 IU/Gm.), and kidney (13 IU/Gm.) contain almost negligible amounts. No CK is detectable in human erythrocytes.

Plasma CK as an index of myocardial infarction

Elevated CK activity as a criterion of myocardial infarction was first described by Dreyfus and co-workers in 196013 and found soon after to be a sensitive index of acute myocardial injury with positive results in 95 to 100 per cent of patients.14. I5 Results of studies with large numbers of patients with comparison of several enzymatic indices indicated that CK was the most sensitive.‘4. I5

Total plasma CK activity generally increases 4 to 8 hours after the onset of chest pain, peaks within 12 to 24 hours, and returns to within the normal range within 72 to 96 hours.3 However, despite its sensitivity, elevation of total plasma CK activity lacks specificity for the diagnosis of acute myocardial infarction with a false positive incidence of approximately 15 per cent.‘l This is not surprising since total plasma CK activity increases in association with many noncardiac

disorders including: muscular dystrophy, inflammatory disease of muscle, trauma or intramuscular injections (particularly of morphine, pheno- thiazines, and barbiturates even without overt signs of injury), cerebral disease, alcohol intoxication, diabetes mellitus with and without ketosis, convulsions, and psychosis-often due to CK release from skeletal muscle. In addition, increases in plasma CK occur with shock, myxe- dema, pulmonary embolism, pneumonia, radio- therapy, chronic lung disease, surgery, and exer- c~e.‘6-32

High concentrations of barbiturates, Valium, morphine, and anesthetics decrease the rate of disapparence of CK from the circulation in experimental animals and may therefore be associated with elevated plasma CK even under conditions in which release is not augmented.33 Increases have been seen as well after oral administration of aminocaproic acid, clofibrate, carbenoxolone, imipramine, and glutethimide.3”-3K

Several cardiac conditions besides myocardial infarction may give rise to elevated total plasma CK activity. Although plasma CK does not generally increase in patients with mild congestive heart failure, it may in patients with pulmonary edema and severe hepatic congestion.3Y Pericardi- tis, myocarditis, electrical cardioversion, and cardiac catheterization may lead to increased CK dependent upon CK release from skeletal muscle or other organs besides the heart.“9e’2

Plasma CK isoenzymes and myocardial infarction

Beginning in 1966, analysis of plasma CK isoenzyme profiles was utilized to provide more specific diagnostic, information regarding myocardial infarction.8 However, technical limitations precluded general use of this approach until much later despite progress by Sherwin and associates,43 Trainer and colleagues,q4 and others.‘. *. 45 Quanti- tative analysis of CK isoenzyme profiles in human tissues, necessary to establish both diagnostic specificity and sensitivity of elevated plasma MB CK, was hampered by lack of quantitative techniques46 for assay of plasma CK isoenzymes until recently when a kinetic, fluorometric technique was developed.” Although laborious, this method served as a useful standard for development of more convenient approaches and facilitated delineation of tissue isoenzyme profiles.

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Table I. Creatine kinase isoenzyme distribution in human skeletal muscle

Van der Veen and f +++ Wilbbrands, 1966*

Dawson and Fine, 1967’ + +++ Sherwin et al., 1967’” rt +++ Trainer and Gruening, 1966” - +++ Magalhaea, 1970”” + +++ Konttinen and Somer, 1972’ +++ Roe et al., 1972* +++ Smith, 1972’* -+ +++ Klein et al., 1973’5 +++

Legend: + present inconsistently; - absent consistently; + present consistently.

Results of qualitative CK isoenzyme assays

Plasma CK isoenzymes were first separated on ’ the basis of electric charge.’ With electrophoresis of samples at alkaline pH, MM remains at I the origin, BB exhibits the greatest electrophoretic mobility, and the mobility of MB is intermediate. Supporting media for electrophoresis include agar,8 agarose,* cellulose acetate,45 and polyacrylamide gel.‘” After separation of the isoenzymes by electrophoresis, the supporting medium is incu- bated with reagents necetiry to generate NADPH (detectable by fluorescence or dye reduction) in regions where CK’ isoenzyme activity is present. When CK isoenzyme profiles were delineated in human tissues with these qualitative techniques, brain was found to contain BB and heart MM and MB. However, observations with extracts from skeletal muscle were conflicting (Table I). Although in most studies only MM was detected in skeletal muscle, MB was reported in some as well. In view of limitations of assays based on electrophoretic techniques, stability of CK isoenzymes, and recent information obtained with quantitative CK isoenzyme assays, these results must be inter- preted with caution. In all but one44 of these studies, postmortem material was used. Since MB is much less stable than MM, it is possible that MB present initially in the tissue could have been overlooked because of tissue autolysis under these circumstances. Since tissue samples were often analyzed after repetitive freezing and thawing, now known to induce conformational changes in the molecule and alteration of electrophoretic mobility, results may have been distorted.48

Table II. Creatine kinase isoenzyme distribution in human tissue*

Tissue CK (IU/Gm.) MM MB BB

Muscle 3,200 k 200 3,200 + 200 0 0 Heart 1,600 k 160 1,370 + 120 230 + 30 0 Brain 200 + 30 0 0 200 k 30 GI tract 140 +- 20 0 4.2 e’l 136 -c 20 Adrenal 50+- 6 0 0 50+ 6 Lung 13+- 2 1 + 0.2 0 12 k 0.5 Kidney 9+- 2 1 f 0.1 0 8 f 0.5

*Data obtained from references No. 3, 12, 47, and unpublished observations.

Nonspecific fluorescence from moieties other than CK was often not excluded by performing assays both with and without creatine phosphate, the-substrate specific for CK. Thus, apparent MB in the skeletal muscle extract may have been an artifact unrelated to any CK isoenzyme. Since electrophoretic scanning techniques are not quantitative, they may have grossly overesti- mated the amount of MB CK activity present, as was the case in early reports of MB CK content in canine myocardium representing as much as 40 per cent of total CK activity5” and -therefore twentyfold more than the 2. per cent actually present and measurable with quantitative techniques.” However, even a small proportion of MB in skeletal muscle could be associated with release of a significant amount of MB into the circulation after intramuscular injectians, muscle trauma, surgery, or shock potentially clouding the diagnosis of myocardial infarction. Thus, resolution of the question of how much MB, if any, is present in skeletal muscle was needed.

Results of quantitative CK isoenzyme sssays

The kinetic, fluorometric quantitative assay for CK isoenzyme developed in 1974*’ had a sensitivity of 2 IU/L. and producibility of -+ 3 per cent. Human tissues removed at the time of surgery and extracted immediately prior to freezing, were assayed with this technique both with and without creatine phosphate to exclude apparent activity from moieties other than CK.” As shown in Table II, myocardium was found to contain predominantly MM with MB representing approximately 15 per cent of total CK activity. Skeletal muscle analyzed included deltoid, pecto- ralis major and minor, gastrocnemius, and rectus abdominous, all of which contained MM CK

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exclusively. Lung, kidney, and spleen contained predominantly BB CK with no MB, and red blood cells, rich in LDH and LDH,, an isoenzyme present in myocardium, was devoid of appreciable CK activity. Although prostate and the mucosa of the small intestine contained traces of MB (< 3 per cent), myocardium was the only normal human tissue containing appreciable quantities of MB CK. Subsequent development of more rapid and convenient quantitative assays for CK isoenzymes confirmed the impression that human myocardium contains between 15 and 20 per cent MB CK and that skeletal muscle contains only MM 51-53

Since analysis of the CK isoenzyme profile of each skeletal muscle group is not practical, possible heterogeneity of skeletal muscle isoenzyme profiles cannot be excluded easily. Another approach has entailed analysis of plasma CK isoenzymes after spontaneous, surgical, or experi- mentally-induced injury to skeletal muscle. Plasma MB CK is not elevated after intramuscular injections despite utilization of a wide variety of sites for injection and despite marked elevations of total plasma CK.‘“, i7 When plasma CK isoenzyme profiles were analyzed at six-hour intervals for 24 hours after surgery”l involving the head and neck, ocular muscles, thorax, abdomen, prostate, urinary bladder, or extremities, marked elevations in total plasma CK and MM CK were observed, but MB CK remained normal. Analysis of CK isoenzymes by electrophoresis on agarose,55 polyacrylaminde gel,56 and cellulose acetateA demonstrated the absence of elevation of MB CK after noncardiac surgery; and serial analyses of plasma MB CK with quantitative techniques based on column and batch adsorption chromatography with Sephadex5’ 52 and glycophase glass beads”’ corroborated the absence of elevated plasma MB CK after surgery.

Among 183 patients undergoing cardiac catheterization,57 total plasma CK was often elevated, but the elevation was due exclusively to MM CK (presumably due to skeletal muscle and soft tissue trauma) in all but two cases. In the two patients with MB CK elevations, transmural myocardial infarction was the cause. In cases of rhabdomyolysis despite total CK elevations of several thousandfold, MB CK remained nor- ma1.2. 4o Elevated total plasma CK after exercise has been attributed to MM CK exclusively. These results of analyses of CK isoenzyme profiles in

human tissues and in plasma after injury to skeletal muscle suggest that elevated plasma MB CK is a virtually specific index of injury to myocardium.5”

Sensitivity of qualitative electrophoretic techniques for detection of MB CK is of the order of 5 to 10 IU/L.56. 57. Since plasma from normal subjects contains only 1 to 2 III/L. of MB CK, modest increases in MB CK are not recognized easily with these techniques. Nevertheless, increased activity has been detected consistently in patients with acute myocardial infarction.‘, ?. 8. ll. 48 In contrast, among patients with angina and only transient, nonspecific electrocardiographic changes, MB CK did not increase. Operative mortality associated with coronary bypass grafting in 47 patients with unstable angina without elevated plasma MB CK was less than four per cent, similar, though slightly greater than mortality associated with this procedure in patients with stable angina but markedly less than mortality (as high as 40 per cent) in patients undergoing surgery during evolving myocardial infarction. Thus, selection of candi- dates for surgery by exclusion of apparent infarction based on a lack of elevated MB CK avoids the excess mortality associated with surgery in patients with evolving infarction and suggests that absence of elevated MB reflects absence of infarction.59 Elevated MB CK has been observed in samples from 16 of 111 patients with unstable angina characterized by recurring, prolonged episodes of chest pain. However, each of these 16 patients exhibited independent evidence of acute myocardial infarction. MB CK reported in numerous studies of patients with angina without infarction as well as its absence after transitory coronary occlusion in experimental animals subjected to &hernia insufficient to produce infarction, supports the view that MB CK is released from myocardium only when necrosis occurs.‘. z 3. 47. J7 In addition, ischemia alone induced by treadmill exercise and documented electrocardiographically in patients with coronary artery diseaseeO does not lead to increased plasma MB CK despite elevated total CK, presumably from noncardiac sources.

Depletion of CK from myocardium in experimental animals correlates with morphological criteria of infarction,61, 62 the magnitude of ST- segment elevationG3 decreased blood flow measured by radioactively labelled microspheres,G1

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and alterations in frequency-dependent attenuation of ultrasound indicative of infarction.64 Esti- mates of infarct size based on plasma CK time-activity curves correlate closely with prognosis,65 the severity of angiographically demon- strable wall motion disorders,@ and morphological estimates of infarct size in patients.67 Congestive heart failure uncomplicated by myocardial necrosis, and tachycardia are not associated with elevated plasma MB CK even when total CK is increased.S6, 68 Thus, elevated plasma MB CK, reflecting release virtually exclusively from the heart in man, appears to differentiate myocardial infarction from coronary insufficien- CY.

MB CK is particularly useful as an index of myocardial infarction occurring in patients after noncardiac surgery.54 Conventionally measured enzymes are elevated,@ and LDH isoenzyme analysis may not be helpful since hemolysis leads to increases in LDH, and LDH, simulating the isoenzyme pattern resulting after myocardial infarction and present in myocardium itself.‘O Mortality” associated with infarction after noncardiac surgery is high (sometimes as high as 40 per cent), possibly reflecting delay in initiating appropriate therapy because of delayed recognition of infarction. Because postoperative infarction is most common in elderly patients and those with cardiac disease, groups in whom definitive electrocardiographic diagnosis is often most die- cult, differentiation between ischemia and infarction within the first few hours after operation is difficult and may be best achieved by analysis of plasma MB CK activity.“’

On the other hand, analysis of plasma MB CK activity after cardiac surgery does not help to establish the presence or absence of intra- or perioperative infarction since MB activity is invariably elevated as a result of even minor surgical trauma to the heart.72 Furthermore, since the proportion of myocardial MB CK appearing in the circulation may be much greater after surgical trauma than after infarction, even quantitative evaluation of MB activity in plasma may not permit differentiation of the two. Appearance of q-waves on the electrocardiogram and localized positive findings on myocardial infarct scintigrams with sg*Tc-pyrophosphate appear to be the most useful generally available diagnostic criteria of infarction in this setting.72

Plasma MB CK appears to remain normal in

patients with pneumonia, chronic lung disease, and pulmonary emboli even when total plasma CK is increased,56 although elevated MB CK would be anticipated if severe right ventricular failure and &hernia led to right ventricular infarction. Pericarditis has not been associated with elevated MB CK,‘” but extensive associated epicarditis would be expected to liberate MB CK into the circulation.

Increased total CK activity is common in patients with hypothyroidism, primarily because of increased MM CK that presumably accumu- lates due to decreased clearance. However, on occasion MB CK may be elevated also. It has recently been shown that hypothermia is associated with elevated plasma MM but not MB CK, presumably because of enzyme release from skeletal muscle.74. 75

Both MM and MB plasma CK are elevated consistently in patients with muscular dystrophy.‘6 This appears to result from failure of the normal differentiation or dedifferentiation of skeletal muscle with increasing fetal maturity and hence failure of the normal progression of‘ isoenzyme profiles within the tissue from BB initially, to MM and MB at or before term, and MM alone by birth or during the neonatal inter- val.” In addition, MB CK in plasma in patients with muscular dystrophy may reflect release from the dystrophic heart. Among patients with poly- myositis, elevated plasma MB CK though recognized, appears to be much less consistent.78

Available MB CK assays

Conventional clinical assays for MB CK generally employ electrophoresis of samples on agarose, cellulose acetate, or polyacrylamide gels-with visualization procedures capable of detecting 5 to 10 IU/L. With these assays, myocardial infarction can be detected with a sensitivity and specificity exceeding 95 per cent. Sampling at intervals of 6 to 12 hours will usually lead to detection of even small subendocardial infarcts. Exclusion of nonspecific fluorescence (and hence false positive results) with control samples run without creatine phosphate as substrate is important particularly since com- monly used drugs such as tetracycline, aspirin, and chlorpromazine can give rise to this phenom- enon (Unpublished results).

Quantitative assays offer numerous advantages including comparison of activity in all serial


Roberts and Sobel

samples from the same patient in view of their ability to detect some activity in plasma from normal subjects.47. 51. 52 Since development of a kinetic, fluorometric technique in 1974,47 several sensitive and more rapid quantitative procedures have been implemented, Many utilize Sepha- dex51-53 or cellulose” to separate individual CK isoenzymes in a sample by chromatography or batch adsorption. These techniques provide assays more sensitive than those based on electrophoresis and obviate the problem of nonspecific fluorescence. However, incomplete separation and limited sensitivity due to dilution as well as nonspecific binding or denaturation of the enzyme on chromatographic media may pose difficulties.

Recently, quantitative assay of MB CK has been accomplished with a radioimmunoassay specific for the B subunit.79. a0 Lability of enzymatic activity of the antigen during the required radioactive labelling procedure and dissociation of the enzyme into subunits during incubation had -precluded previous radioimmunoassay of isoenzymes of CK or other enzymes.80. 81 The methods developed to overcome these difficulties should be applicable to development of radioim- munoassays for multiple forms of other clinically important isoenzymes as well as for use in an improved quantitative assay for MB CK.

The CK isoenzyme radioimmunoassayuu de- tects MB CK reliably with no cross reactivity with MM despite twenty thousandfold molar excess. It is more sensitive than other available assays and capable of detecting as little as 0.01 lU/L. of MB CK.“’ Since results are not dependent on enzyme activity but on binding of immu- noreactive MB CK protein by the antibody, the assay measures the concentration of enzyme protein. Accordingly, it should be useful in clar- ifying rates of turnover and denaturation of MB CK and factors influencing clearance of enzymes from the circulation after infarction. Since the radioimmunoassay is so sensitive and since it can detect enzymatically inactive MB CK in the circulation, it is not surprising that it permits detection of infarction earlier than other techniques, usually within three hours of the onset of chest pain.“’ Because radioimmunoassay is readily adaptable for automated analysis of large numbers of samples, the MB CK radioimmunoassay should be generally useful for detec-

tion and assessment of severity of myocardial infarction.

Some advantages of MB CK as a marker of infarction

CK is found in the heart in large quantities and confined virtually exclusively to myocardial cells as opposed to fibroblasts and other components. Since the enzyme is not present in erythrocytes or leukocytes, it is not released from inflammatory exudate in the heart associated with infarction. After myocardial infarction, CK increases in blood within 4 to 6 hours and generally peaks within 12 to 20 hours, permitting rapid diagnosis. The time course of elevation of MB CK is similar, but in contrast to total CK, MB CK elevations are virtually specific criteria of myocardial injury. The prompt diagnostic sensitivity and specificity provided by analysis of MB CK activity has important therapeutic as well as economic impli- cations. Since many patients with chest pain are admitted to coronary care units in major medical centers and community hospitals, and subjected to sometimes extensive and expensive evalua- tions, prompt exclusion of infarction is a desirable goal. When serial analyses of MB CK in plasma indicate no ‘elevations of activity for 12 to 24 hours, early transfer of the patient from the intensive care unit can be justified often, permitting more efficient and economical utilization of these specialized facilities and their highly trained contingents of personnel.

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528 April, 1978, Vol. 95, No. 4

creatine kinase isoenzymes in the assessment of heart disease

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