Radioimmunoassay of creatine kinase isoenzymes in human serum: isoenzyme MM

Measurement of the mass concentration of serum enzymes by radioimmunoassay provides direct quantitation of specific isoenzymes and may be less subject to some of the limitations of traditional assay procedures for enzymes. We describe the development of a sensitive and specific radioimmunoassay for the muscle isoenzyme of creatine kinase, CM-MM, in human serum. CK-MM, purified from human skeletal muscle, was used to raise high-titer antisera and for iodination by the Chloramine T method. The radioimmunoassay required 50 microliter of sample, utilized a double-antibody separation method, and was completed in 24 h. Cross reactivity with CK-BB was virtually zero, 3--17% with CK-MB. The mass concentration of CK-MM in the serum of healthy subjects ranged from 36 to 1668 microgram/liter and correlated closely with total CK enzymatic activity. Serum concentrations of CK-MM from casually selected patients correlated less well with total CK enzymatic activity, suggesting the existence of other CK isoenzymes or the presence of inactive forms.     

heterogeneity of creatine kinase isoenzyme MM in serum in myocardial infarction: interconversion of the "normal" and "abnormal" sub-bands by glutathione

We used isoelectric focusing (IEF) in polyacrylamide gels to investigate the effects of glutathione on the sub-bands of serum creatine kinase (CK; EC 2.7.3.2) isoenzyme MM in acute myocardial infarction. The intensity of the "abnormal" sub-bands c (pI 7.25), e (pI 6.85), and g (pI 6.50) increased, and that of the "normal" sub-bands 1 (pI 6.91), 2 (pI 6.65), and 3 (pI 6.35) decreased, following serum incubation with reduced glutathione (GSH, final concentration 1.25 mmol/L). Further incubation with oxidized glutathione (GSSG, final concentration 5 mmol/L) reversed this change and restored the original pattern, whereas GSSG at 7.5 mmol/L caused sub-bands c, e, and g to disappear and sub-bands 1, 2, and 3 to be enhanced. Sequential incubation of serum with 2.5 mmol of GSSG and 7.5 mmol of GSH per liter produced the opposite sequence of events; i.e., the "abnormal" sub-bands disappeared then reappeared (and GSH at 10 mmol/L enhanced their reappearance). At higher concentrations, glutathione (GSH or GSSG) impaired the detection of the CK-MM sub-bands after IEF, an effect that was "quenched" by heat-inactivated serum of low CK activity. Likewise, the intensity of tissue CK-MM (corresponding to myocardium extracted into 100 mmol/L Tris HCl buffer, pH 7.4) was greatly enhanced by adding heat-inactivated serum to the tissue extract before IEF. We discuss the significance of these findings for the diagnosis of myocardial infarction.     

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.     

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.     

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.     

Isoforms of creatine kinase MM and MB in acute myocardial infarction: a clinical evaluation

Serum creatine kinase (CK, EC 2.7.3.2) isoenzymes MM and MB were resolved, respectively, into three (MM1, MM2, MM3) and two (MB1, MB2) isoforms (subforms derived from the same isoenzyme) by electrophoresis and the isoform patterns were determined in multiple sequential serum samples, timed from the onset of chest pain, from 58 patients with acute myocardial infarction (AMI). During the first 3 h after the onset of chest pain, the serum isoform activity resembled the pattern seen in normal volunteers. Specimens obtained 6 h after AMI showed predominantly MM3 and MB2 (45% and 11% of the total CK activity, respectively). Between 10 and 72 h, there was a gradual shift in which MM3, MM2 and MB2 decreased, while MM1 and MB1 increased. MB2 and MB1 disappeared from the pattern for samples collected after 24-48 h, while MM1 was always the most prominent band at the end of the observation period (66%, range 41-77%, at 48 h). These data suggest that a single determination of CK isoform pattern, drawn between 6 and 48 h after AMI, may provide an effective means of predicting the time of onset of necrosis. There were no significant differences in the CK isoform patterns according to infarct location and functional status of patients.     

Radioimmunoassay of creatine kinase isoenzymes in human serum: isoenzyme BB

We describe a sensitive, specific radioimmunoassay for the BB isoenzyme of creatine kinase (CK-BB) in serum. A sequential saturation assay was used to achieve sufficient sensitivity to detect the isoenzyme in 100-microliter serum samples of all healthy persons and patients tested. Bound and free antigen were separated by a second antibody system. Large excesses of purified isoenzyme MM did not react in the assay. Cross reactivity of two preparations of CK-MB was only 1 to 7+. The 95th percentile of serum CK-BB in 208 healthy adults was 6.2 microgram/liter. Within-assay and between-assay precision ranged from 5.5 to 11.9% and 9.7 to 13.6%, respectively.     

Diagnosis of acute myocardial infarction from two measurements of creatine kinase isoenzyme MB with use of nonparametric probability estimation

By using bivariate probability estimation for the diagnosis of acute myocardial infarction (AMI) we show how to overcome the difficulties encountered for patients whose clinical presentation is atypical and those encountered when multiple isoenzyme determinations are treated by univariate methods. We use the values for creatine kinase isoenzyme MB measured at the time of admission and 12 h later to estimate the Bayes factors in favor of AMI. The Bayes factors are compiled into a table that the clinician can use to estimate the posterior probability that a patient has AMI. The table of Bayes factors is based on data for a sample of 802 non-AMI patients and 180 AMI patients. Further to validate the method, we randomly chose 200 of the non-AMI and 50 of the AMI patients as an evaluation sample, then used the remaining 602 non-AMI and 130 AMI patients to recompute the Bayes factors. These Bayes factors were used to find the probability of AMI for each of the 250 patients in the evaluation sample. The method resulted in only one false positive and no false negatives. For the misclassified patient the measurements at admission and 12 h later were 1 and 11 U/L; the posterior odds were 15 to 1 in favor of AMI, but in fact the patient was non-AMI.     

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.     

Serum creatine kinase MM sub-band determination by isoelectric focusing: a potential method for the monitoring of myocardial infarction

Fresh myocardium homogenates analyzed by thin-layer isoelectric focusing revealed the presence of two prominent creatine kinase (CK; EC 2.7.3.2) sub-bands, MMO (pI 7.10) and MM1 (pI 6.88), in approximately equal proportion. While these forms represented together as much as 85% of the cellular MM fraction, they accounted only for viz. 2.2 and 27.7% of the total serum MM activity when measured 8 h before the CK peak in patients with myocardial infarction. Incubation of the isolated MMO and MM1 with normal human serum demonstrated that the former turned to MM1 within 5 h at 37°C; further changes affecting MM1 gave rise to other sub-bands, MM2 (pI 6.70), MM3 (pI 6.45), and MM4 (pI 6.25). In our patient population, these three forms represented more than 75% of the serum CK-MM activity at the CK peak; hence, soon after the enzyme release, the serum MM isoenzyme mainly consists of degradation products arising from the labile MMO and MMl. Among the two cellular forms, MMO was the best related to the total enzyme activities and the most efficient for differentiating the patients with left ventricular failure from the others during the entire survey period (F = 3.8, p < 0.05). Because its presence in the blood provides evidence for a very recent CK release from the tissues, serum CK-MMO determinations might be proposed for following the extension of the lesion after a myocardial infarct.     

Comparison of serum creatine kinase and creatine kinase MB activities post marathon race versus post myocardial infarction

Serial total creatine kinase (CK) and CK MB activities were determined in the serum of seven runners following a marathon race and compared to enzyme activities in the sera from five patients following acute myocardial infarction (AMI). In the runner's sera, total CK and CK MB activities were significantly elevated at 1, 24, 48 and 72 hours post marathon race when compared to the 1 hour pre-marathon samples (p < 0.01). Serum CK MB activities peaked at 24 hours in both groups of subjects. The MB activities 24 hours following the marathon were substantially higher (91 ± 30 U/l; mean ± SD) than the MB activities 24 hours following AMI (46 ± 38 U/l). However, the percentages of CK MB 24 hours following the marathon and AMI were almost identical (7.0 ± 2.4% and 7.2 ± 2.3%, respectively). Furthermore, CK and CK MB clearances were significantly prolonged (p < 0.02 and p < 0.001, respectively) following the marathon race (T $\text{1}\text{2}$ CK, 49 hours; T $\text{1}\text{2}$ CK MB, 29 hours) as compared to following AMI (T $\text{1}\text{2}$ CK, 27 hours; T $\text{1}\text{2}$ CK MB, 12 hours). These results suggest release of CK MB from the skeletal muscle of marathon runners. Therefore, we recommend that elevation of CK MB in the range indicative of myocardial damage be interpreted with caution in long-distance runners.     

Improved separation of creatine kinase cardiac isoenzyme in serum by batch fractionation.

I describe a simple, single-tube batch fractionation procedure for separating MM and MB isoenzymes of creatine kinase on a macroporous strong anion exchanger (AG MP-1, Bio-Rad Laboratories). The isoenzymes can be separated in less than 3 min, with a resulting dilution of the serum with no more than an equal volume of buffer. Without sample concentration or spectrofluorometric measurement, the procedure detects 4 U of MB isoenzyme per liter. Sensitivity is limited by the sensitivity and precision of the method of measurement. The CV for the fractionation can be held to less than 4.0% at 65 U of MB per liter. Current fractionation methods are compared to the proposed procedure. With use of a discrete analyzer (Du Pont aca) the mean MB activity in a population free of heart disease was 3.2 +/- 3.0 U/liter (range, 0 to 8 U/liter). The kinetics and stability of isolated isoenzymes are reported, indicating that advisability of storing or pre-incubating samples with mercaptoethanol.     

Increased concentrations of free fatty acids and glycerol and altered creatine kinase MM isoenzyme patterns in certain diabetic patients.

We report unusually high concentrations of free fatty acids and glycerol in sera of patients with adult-onset diabetes, and the accompanying alterations in creatine kinase isoenzyme MM patterns associated with such patients. The serum samples with increased free fatty acids also showed increased electrophoretic movement of the MM isoenzyme on cellulose acetate membranes. Fatty acid concentrations found in such samples averaged 9.88 +/- 5.65 (SD) meq/L and the average glycerol concentration was 153 +/- 115 (SD) mg/L. The serum glycerol concentrations correlated with those of the free fatty acids (r = 0.886, slope = 0.309, intercept = 4.76).     

Isoelectric focusing of creatine kinase MM isoforms and its application for diagnosis of acute myocardial infarction

CK MM isoforms (MM 3 having the highest isoelectric point, followed by MM 2, MM 1, and MM X) were measured in 35 patients with acute myocardial infarction (AMI) by isoelectric focusing on agarose gel. Blood samples were analysed every 2 h for the first 12 h, then every 4-8 h until 72 h after AMI. In the first sample, obtained 2.1 h after the onset of chest pain, the ratio of the isoforms MM 3:1 was 0.7 (range 0.2-1.8), equivalent to a normal value. Before the total CK exceeded normal, in 86% of the patients the ratio MM 3:1 rose to 2.2 (range 0.3-3.3). The maximal individual ratio MM 3:1 was 4 (range 0.9-12) after 7 h. It fell below 1 again after 27 h. Thus, the ratio MM 3:1 was useful in the early diagnosis of AMI by enzymatic methods and to estimate the time elapsed since the onset of infarction. Twenty patients with an open infarct vessel (angiographic data after thrombolytic therapy) showed similar peak enzyme activities as ten non-reperfused patients. They differed significantly in the time to the peak activity, mostly for CK MM 3 and CK MB (p less than 0.0005). A higher ratio CK MM 3:1 and a shorter time to the maximum CK MM 3 activity in reperfused patients helps to assess the success of thrombolytic therapy.     

Assay of creatine kinase isoenzyme MB in serum with time-resolved immunofluorometry

We describe the first time-resolved immunofluorometric assay for creatine kinase (EC 2.7.3.2) isoenzyme MB (CK-MB) in serum. The assay is based on the formation of the complex: solid-phase anti-CK-MB-CK-MB-biotinylated anti-CK-BB-streptavidin-BCPDA-Eu3+, where anti-CK-MB and anti-CK-BB are monoclonal antibodies against the CK isoenzymes MB and BB, respectively, and BCPDA is the europium chelator 4,7-bis(chlorosulfophenyl)-1,10-phenanthroline-2,9-dicarboxylic acid. The solid-phase complex is fluorescent and is measured on the dry solid-phase (microtiter well) in a specially designed time-resolved fluorometer that uses laser excitation. The assay requires 25 microL of serum and is not affected by the presence of either CK-MM (up to 5000 micrograms/L) or CK-BB (up to 1000 micrograms/L) in the sample. Precision and accuracy indices for the assay were satisfactory.