Mass concentration and activity concentration of creatine kinase isoenzyme MB compared in serum after acute myocardial infarction

We compared three current methods (immunoinhibition, "Isomune-CK" immunoprecipitation, and the Tandem-E CKMB II immunoenzymometric assay) for determination of creatine kinase (CK; EC 2.7.3.2) isoenzyme MB in serum. Although results inter-correlated well, the immunoinhibition assay gave higher activity values. Atypical CK forms did not interfere with the immunoprecipitation and immunoenzymometric methods. In acute myocardial infarction the catalytic properties of CK decreased with the enzyme's age, as reflected by a steady increase in activation energy of the catalyzed reaction. In septicemia patients with very low CK and CK-MB catalytic activity, mean CK-MB mass concentration exceeded the upper reference limit, suggesting an increased rate of loss of activity concentration in these patients' sera. Because of the assay's lesser susceptibility to conformational changes at the active site of the enzyme, we suggest that measurement of CK-MB mass concentration is better suited for infarct sizing than measurement of catalytic activity.     

Immunoenzymometric assay of creatine kinase with monoclonal antibodies to the MB isoenzyme compared with electrophoresis

We compare a "second-generation" immunoenzymometric assay (Tandem-E CKMB II) for creatine kinase (EC 2.7.3.2) MB with its electrophoretic (Beckman Paragon system) determination. In the former, two monoclonal antibodies are directed against the B and M subunits. We evaluated 502 samples from 253 patients. Precision, linearity, and analytical recovery for both assays were excellent. The two methods correlated well (r = 0.936). The reference interval for individuals with no suspected cardiac disorder was 0-6.0 micrograms/L; that for non-infarct patients was 0-18.0 micrograms/L. Peak CK-MB values determined by the two assays agreed for 95% of the patients, in terms of exceeding the normal reference interval or not. Diagnostic efficiencies were 86% (Tandem) and 88% (electrophoresis). The immunoenzymometric assay showed no cross reaction with other CK isoenzymes. Both assay methods performed well in detecting CK-MB, although there were some false positives by both methods, as judged from electrocardiographic results. When total CK for the Tandem assay exceeds 2000 U/L, we recommend calculation of a ratio (CK-MB, micrograms/L:total CK, U/L).     

Use of creatine kinase MB isoenzyme for diagnosing myocardial infarction when total creatine kinase activity is high

The usefulness of measuring creatine kinase MB isoenzyme for diagnosing myocardial infarction when activities of total creatine kinase are very high is unclear. We conducted a retrospective study in an urban hospital that serves a largely indigent population. We concentrated on 146 patients whose creatine kinase activity was greater than 1000 U/L (upper limit of normal: 165 U/L for women and 225 U/L for men), with MB isoenzyme greater than 10 U/L and less than 5% of total creatine kinase. The positive predictive value of MB isoenzyme (isoimmune method) values greater than 10 U/L was between 11.6% and 56.8% when the value for total creatine kinase exceeded 1000 U/L. Using different values (MB greater than 4% of total creatine kinase) as positive for myocardial infarction would have resulted in far fewer false-positives, but 10 cases of myocardial infarction would have been missed. The most appropriate cutoff value for MB isoenzyme in this population (total creatine kinase greater than 1000 U/L) was found to be greater than 2% of total creatine kinase.     

Detection of cardiac-specific creatine kinase isoenzyme in sera with normal or slightly increased total creatine kinase activity.

We describe a spectrophotometric kinetic assay for detecting creatine kinase MB isoenzyme activity in the 1 to 10 U/liter range. The MB isoenzyme was isolated [Clin. Chem. 20, 36 (1974)] and assayed (Rosalki method) with an Abbott ABA-100. Good reproducibility was demonstrated for MB isoenzyme activities near 1 U/liter (CV = 2.6%). Sera with normal or slightly increased total creatine kinase activity were evaluated. Sera of 14 patients with acute myocardial infarction contained, per liter, 84 to 236 U of total creatine kinase activity and 4.6 to 28.0 U of isoenzyme MB activity; corresponding ranges for sera from healthy lab technicians and patients with noncardiac disease were 36 to 277 and 0 to 2.6 U. MB isoenzyme activity for infarction patients rose and fell sharply within three days after the infarction. Atypical time-course patterns, MB isoenzyme activity remaining abnormally great for five days, were observed in serum from patients with prolonged atrial fibrillation and congestive heart failure or cardiomyopathy; the BB isoenzyme (1 to 5 U/liter) was also detected in sera of such patients but was absent in sera from infarcation patients. Quantification of column-isolated MB by the assay described is rapid, easy, specific, and extremely sensitive for measuring MB in the 1 to 10 U/liter range.     

Activity concentration and mass concentration (monoclonal antibody immunoenzymometric method) compared for creatine kinase MB isoenzyme in serum

Results of the "Tandem-E CKMB" immunoenzymometric procedure (y) for creatine kinase (CK; EC 2.9.3.2) were compared with electrophoresis (x) for 160 serum samples from patients suspected of having sustained myocardial infarctions. The results correlated well: y, microgram/L (Tandem assay) = 1.3x-6.3 U/L(electrophoresis) (r = 0.95). CK-MB mass measurement was more stable than enzyme activity after storage and appeared to be more sensitive. Sera from 86 other people, which had no detectable CK-MB upon electrophoresis, gave a mean CK-MB value of 1.1 microgram/L (SD 1.3, range 0-8) with the Tandem assay. To determine whether these low values represented actual isoenzyme, we tested for possible interference by heterophile antibodies in the patients' sera by preincubating the samples with mouse serum before the Tandem assay. The mouse serum did not interfere with the assay of sera that had substantial quantities of CK-MB by electrophoresis. However, in five of six samples that were negative by electrophoresis, the CK-MB values were substantially smaller, indicating that the values measured were false-positives caused by the presence of heterophile antibodies directed against mouse proteins, an interference that could be eliminated by pretreatment with mouse serum.     

Creatine kinase and lactate dehydrogenase isoenzymes in serum of patients suffering burns, blunt trauma, or myocardial infarction

Medical records of 53 burn and trauma patients were reviewed to assess the possibility of myocardial damage. Except for electrophoretically detectable creatine kinase MB isoenzyme, none showed evidence of myocardial injury. Lactate dehydrogenase isoenzyme tests, electrocardiograms, myocardial pyrophosphate scans, clinical course, and results of (two) autopsies were all negative for myocardial necrosis or ischemia. Types of patient, number, mean peak value (U/L) for serum creatine kinase, and ranges of percentage MB isoenzyme were as follows. Burns from direct electrical contact: 28, 16 600, 0-29; electrical flash or other thermal burns: 10, 4340, 0-22; blunt trauma (mostly from automobile accidents): 15, 3430, 0-18; myocardial infarction: 57, 1520, 4-46. Evidently creatine kinase MB isoenzyme is nonspecific in burn and trauma patients and should not be the only test result used to assess myocardial involvement.     

Study of a myoglobin test in patients hospitalized for suspected myocardial infarction

An immunoagglutination latex test was studied in comparison with a plasma myoglobin radioimmunoassay in 103 subjects with suspected myocardial infarction. The test provided an early and reliable indication of raised plasma myoglobin (greater than 85 micrograms/l), a biochemical marker for the early phase (12 h) of myocardial infarction. The diagnostic values (sensitivity and specificity) studied over a 36 h period were the same as those for the plasma myoglobin assay. The sensitivity was similar to that of creatine kinase activity and better than that of the creatine kinase MB/creatine kinase ratio; the lower specificity was due to false-positive results in some subjects with angina. The myoglobin test, which provides rapid results, may be substituted in early diagnosis of myocardial infarction for the plasma myoglobin assay which is unsuitable for emergency analysis.     

Determination of creatine kinase isoenzyme MB activity in serum using immunological inhibition of creatine kinase M subunit activity. Activity kinetics and diagnostic significance in myocardial infarction.

This is a new method for the determination of creatine kinase isoenzyme MB activity in serum. The method uses direct activity measurement of creatine kinase B subunit activity after blocking of CK-M subunit activity by inhibiting antibodies. The test takes no longer than 15 min. The method yields an intra-serial C.V. of 2.0-12.9%, and a C.V. from day to day of 5.5%. The detection limit is 3.4 U/l creatine kinase MB. In the 95 cases with proven myocardial infarction several types of creatine kinase MB activity kinetics could be determined. The percentage of creatine kinase MB of peak CK-total is 6-25%, with a mean of 11.1%. The amount of creatine kinase MB with respect to total CK activity after reinfarction is higher than the amount after initial infarction.     

Development and clinical evaluation of a microcentrifugal analyzer method for determining creatine kinase MB isoenzyme

We have adapted to a microcentrifugal analyzer an immunoinhibition assay for measuring the activity of creatine kinase MB by using an inhibitory antibody for the M monomer. The method actually measures half the MB activity, but results are not multiplied by two because atypical isoenzymes of creatine kinase, including BB, IgG-BB, and the isoenzyme derived from mitochondria, are also detected, if they are present. Results correlated well with an electrophoresis method for 36 serum samples. Myocardial infarction was assessed in 175 patients admitted to our coronary-care unit, with respect to sensitivity (100%) and specificity (98%) when a decision point of 100 U/L (30 degrees C) was chosen for total creatine kinase activity (dithiothreitol-activated) and 6 U/L (30 degrees C) for the isoenzyme (by immunoinhibition). Atypical isoenzymes are easily recognized and confirmed by electrophoresis when the MB activity (by immunoinhibition) exceeds 6 U/L and 20% of the total creatine kinase activity.     

Chromatographic and electrophoretic separation of creatine kinase isoenzymes compared.

We compared two techniques for separating and evaluating serum creatine kinase isoenzymes--fluorometric agarose electrophoresis and Sephadex chromatography--in 50 patients, 25 of whom had confirmed acute myocardial infarction. In every case isoenzyme MB (heart isoenzyme) was detected with equal sensitivity by either procedure. Evidently, only the presence or absence of MB is clinically significant; none of the 25 patients without infarction had detectable MB activity in their serum. Columns connected to a continuous-flow sample line for analyses of the eluting stream without further modification produced satisfactory results.     

New manual and automated method for determining activity of creatine kinase isoenzyme MB, by use of dithiothreitol: clinical applications.

The method is based on the selective activating capacity of dithiothreitol on creatine kinase isoenzyme MB, after isoenzyme MM is activated by glutathione. Isolated isoenzymes MM and MB of human and canine origin were assayed individually and in mixtures of known activities. When glutathione was present in the assay medium the activity of each isoenzyme could be measured individually, but glutathione did not activate isoenzyme MB if it was present in a mixture with MM. Dithiothreitol, added to the serum before assay, activated the isoenzyme MB in the mixture. Values for MB activities obtained for isolated isoenzyme MB and for the isoenzyme mixture after dithiothreitol was added averaged 110 and 111 U/liter, respectively (r = 0.998; y = 1.007 x + 0.298; n = 10). In the serum of 40 patients with documented acute transmural myocardial infarction, the mean proportion of isoenzyme MB activity measured in this way was 5.5% (coefficient of variation, 7.7%). Isoenzyme MB activities measured by use of dithiothreitol compared well with those obtained by conventional electrophoresis/spectrophotometry (r = 0.998; y = 1.09x -0.65) and spectrofluorometry (r = 0.996; y = 1.10 x + 0.80). The assay of MB activity by the dithiothreitol method was automated, by use of an Abbott Bichromatic Analyser and a Calbiochem Super-Stat Pack Kit. In 60 isoenzyme MB determinations the manual and automated method correlated well (r = 0.990; y = 1.0x -1.36). The simplicity of isoenzyme MB determination by use of dithiothreitol and its ease of automation allow routine monitoring of the isoenzyme activity in patients with ischemic heart disease.     

Creatine kinase isoenzyme MB assay by electrophoresis

A method for determination of the creatine kinase isoenzyme MB (CK-MB) is reported: separation of the isoenzymes was done by electrophoresis and the activity of the isoenzyme bands quantitated by scanning fluorometry. Total CK activity was used for calculation of CK-MB level. The precision of the method was satisfactory: coefficient of variation 5-10%. Its accuracy good: CK-MB was consistently found in high concentrations in tissue extracts of myocardium, but was virtually absent in skeletal muscle and could not be demonstrated in serum from patients with skeletal muscle damage. The sensitivity of the method fitted its clinical use: CK-MB was undetectable (less than 5 U/l) in normal sera, below 30 U/l in seventy-six out of seventy-seven patients in whom the diagnosis of acute myocardial infarction (AMI) was disproved, and above 30 U/l in all seventy-two patients with AMI according to WHO criteria. The CK-MB concentration in serum rises to a maximum about 20 h after onset of clinical symptoms of AMI and reaches baseline levels 20-30 h later. The electrophoretic CK-MB method is easy, fast and reliable and is considered as an important diagnostic test for AMI.     

Influence of autoantibodies to creatine kinase-BB on assays for MB isoenzyme

We describe the influence of autoantibodies that bind creatine kinase BB (CK-BB) on the methods for MB isoenzyme. If these autoantibodies are present in patients' sera, they cause the formation of macro CK type 1 (immunoglobulin-linked CK-BB). In some of these cases they can bind not only endogenous CK-BB but also CK-MB without significantly affecting enzyme activity. Although these antibodies show distinctly less affinity for CK-MB than for CK-BB, they nevertheless bind CK-MB in these particular sera, because their concentration exceeds that of CK-BB isoenzyme. If a person with such autoantibodies has an acute myocardial infarction, the immunoinhibition method for CK-MB, which does not discriminate between CK-MB and CK-BB, will recognize the increase and peak of CK-MB with time, although persistent macro CK activity will be superimposed on the typical isoenzyme pattern. However, isoenzyme electrophoresis and recently introduced immunoenzymometric assays for CK-MB in these cases may be less sensitive for detecting myocardial infarctions, because the typical increase in CK-MB activity may be identified later in the progression of symptoms, or even be missed.     

Comparison of catalytic activity and mass concentration of serum creatine kinase MB isoenzyme in the detection of coronary reperfusion in acute myocardial infarction after therapeutic thrombolysis

Serum creatinine kinase MB isoenzyme time-activity curves are useful for the assessment of coronary reperfusion after acute myocardial infarction. The purpose of this study was to compare serum creatine kinase MB catalytic activity with mass concentration for the determination of coronary reflow after therapeutic thrombolysis. Creatine kinase MB mass was determined immunoenzymometrically. Creatinine kinase MB catalytic activity concentration was determined by electrophoresis. Serum was collected every 4 hours for 96 hours in two groups of myocardial infarction patients: A (n = 10), urokinase induced reperfusion; B (n = 10), conventional therapy without urokinase. Peaks of mass and activity occurred at similar times in groups A and B. Both were significantly earlier in the urokinase treated patients. The maximal rate of increase of creatine kinase MB (based on either mass or catalytic activity) was threefold greater in the urokinase group. There are no important differences between the behaviour of creatine kinase measured as catalytic concentration or as mass concentration. Mass concentration is therefore equally useful as an indicator of coronary reperfusion.     

At what level of serum total creatine kinase activity can measurement of serum creatine kinase MB isoenzyme activity be omitted in suspected myocardial infarction?

The purpose of this study was to establish a discriminatory limit for serum total creatine kinase activity (CK activity) below which CK isoenzyme fractionation is unnecessary. We looked at 2610 serum samples from 1077 consecutive patients with suspected acute myocardial infraction (AMI). The CK activity was determined according to the Scandinavian recommended method. Isoenzymes of CK were separated by agarose gel electrophoresis, followed by fluorometric scanning. When the threshold for CK activity was 150 U/l, none of the samples had a creatine kinase MB isoenzyme activity (CK-MB activity) equal to or higher than 30 U/l (the diagnostic level), which has been found to differentiate between patients with AMI and those without AMI. Only 14 patients (1.3% of all patients investigated) had CK-MB activity peaks between 10 U/l (detection limit) and 30 U/l. Of these, AMI was only diagnosed in one. We recommend that CK-MB activity should be measured only when CK activity is higher than 150 U/l. This would make about 50% of all CK-MB measurements unnecessary.     

Etude de la myoglobinemie et de son evolution au cours de l'infarctus du myocardeStudy on myoglobinemia and its development during myocardial infarction

Thirty six patients suffering from myocardial infarction were investigated by assay of their serum myoglobin, total creatine kinase and creatine kinase isoenzyme MB activities. Determination of serum myoglobin presents, with regard to creatine kinase MB, two major advantages: a very early increase after the onset of the pain (about three hours later) and a very quick clearance, allowing the diagnosis of a second episode of necrosis after about one day.     

Immunochemiluminometric assay of creatine kinase MB with a monoclonal antibody to the MB isoenzyme

Previous two-site immunometric assays for creatine kinase (CK; EC 2.7.3.2) MB isoenzyme have been based on formation of a "sandwich" complex involving CK-MB and antibodies that recognize the CK-MM and the CK-BB isoenzymes. Single-incubation model assays of CK-MB with these antibodies were susceptible to interferences by CK-MM and CK-BB. We produced two anti-CK-MB monoclonal antibodies and studied their suitability for two-site assays. Both antibodies were compatible with anti-CK-MM and anti-CK-BB, but not with each other. Using anti-CK-MB as the tracer antibody eliminated the interference by both CK-MM and CK-BB. Labeling anti-CK-MB with acridinium ester and immobilizing anti-CK-BB on paramagnetic particles, we developed a rapid and highly sensitive chemiluminescent/magnetic separation CK-MB assay. As little as 1 microgram of CK-MB per liter was detectable after 10- or 30-min incubation at room temperature, and the standard curve was linear up to 400 micrograms/L. Results for serum samples by the new assay correlated well (r = 0.94) with those by Corning electrophoretic and the Hybritech Tandem-E immunoenzymometric CK-MB methods. Sera containing macro CK-1 or high concentrations of CK-MM and CK-BB did not interfere. The combined advantages of a more-specific antibody, paramagnetic solid phase, and chemiluminescent label endow this two-site CK-MB assay with performance characteristics and ease of use superior to those of previous assays.     

Automated quantification of creatine kinase MB isoenzyme in serum by radial partition immunoassay, with use of the Stratus analyzer

We evaluated the analytical and clinical performances of a new radial partition immunoassay for measuring the mass concentration of creatine kinase (CK)-MB in serum. All pipetting, washes, incubations and data reduction were performed in 8 min by the Stratus (Dade) fluorometric analyzer. Within-assay and between-assay CVs were respectively 5.5% and 8.4% at 21 micrograms/L, and 4.2% and 3.4% at 48 micrograms/L. Assaying serial dilutions of serum samples with high CK-MB concentrations demonstrated excellent linearity. Results of the Stratus technique correlated well (n = 115, r = 0.98) with those of the Tandem-E CKMB II assay. There was no interference from hemolysis, bilirubin, rheumatoid factor, or added CK-MM (up to 3500 U/L); consequently, CK-MB can be determined in undiluted serum, even in the presence of high total CK activity. The mean CK-MB concentration in 105 blood donors was 1.9 (SD 1.3) micrograms/L. For seven myocardial infarction patients who received prompt fibrinolytic therapy, the mean CK-MB concentration was 4.5 (SD 1.8) micrograms/L at admission, and maximum concentrations, 119 (SD 94) micrograms/L, were recorded 16 h later. CK-MB returned to concentrations less than 10 micrograms/L within 72 h.     

Secretion of creatine kinase MB isoenzyme by an immature teratoma with predominant rhabdomyosarcomatous elements

A 37-year-old man with metastatic immature (malignant) teratoma with prominent rhabdomyosarcomatous elements had markedly increased activity of creatine kinase (EC 2.7.3.2) MB in serum. There was no electrocardiographic evidence of infarction or ischemia, and autopsy revealed no myocardial infarction, significant coronary atherosclerosis, myocarditis, or invasion of the heart by tumor. A high proportion of the creatine kinase activity in a homogenate of the tumor was attributable to the MB isoenzyme. Persistent increases of creatine kinase-MB and an unusually high MB isoenzyme activity, out of proportion to total creatine kinase activity, may indicate a nonmyocardial origin of this isoenzyme.     

Improved column method for separating lactate dehydrogenase isoenzymes 1 and 2

Lactate dehydrogenase (LD) isoenzymes 1 and 2 in human serum were separated on a column of diethylaminoethyl-Sephadex. Samples layered on mini-columns were eluted with buffered sodium chloride (100, 150, and 200 mmol/liter). Lactate dehydrogenase activity in column effluents was measured by the Wacker method, and their isoenzyme content was evaluated by electrophoresis on polyacrylamide gel. Results for column-fractionated LD-1 and LD-2 were expressed in two ways: LD-1/LD-2 ratios and total LD-1 + LD-2 activities. The former is a more specific indicator of myocardial infarction than the latter. Sera from 10 patients with acute myocardial infarction (increased creatine kinease isoenzyme MB activity) exhibited ratios in the range of 0.92 to 1.56, ratios for 10 patients without heart disease (normal creatine kinase MB) ranged from 0.33 to 0.69.