Measuring creatine kinase MB isoenzyme in a maintenance hemodialysis population: chemiluminometric immunoassay and electrophoresis compared

In an effort to clarify the issue of potentially false increases in creatine kinase (EC 2.7.3.2) MB isoenzyme (CK-MB) in uremia, we evaluated the CK profile of 84 persons undergoing chronic maintenance hemodialysis. We compared the performance of a new commercial two-site chemiluminometric immunoassay of CK-MB (Magic Lite; Ciba Corning Diagnostics) with that of electrophoresis on agarose gel (Cardio Trak-CK; Corning Medical). Results of the new chemiluminometric immunoassay for samples from hemodialysis patients correlated well with those of the electrophoretic method (r = 0.86, P less than 0.001), showing that neither substances in the serum of uremic patients nor CK-MM isoenzyme give false-positive increases in CK-MB isoenzyme. Our evidence suggests that the chemiluminometric method may be more specific than is electrophoresis in establishing absolute CK-MB values in the diagnosis of suspected myocardial injury in this population.     

Creatine kinase isoenzymes in human cerebrospinal fluid and brain

Extracts of normal brains obtained at autopsy and cerebrospinal fluid (CSF) from patients with global brain ischemia were analyzed for creatine kinase (CK; EC 2.7.3.2) isoenzymes. We used both qualitative and quantitative assays (electrophoresis and immunoinhibition). Brain extracts contained CK-BB isoenzyme and mitochondrial CK. In 54 CSF samples free of blood contamination and with total activities ranging from 7 to 2010 U/L (mean 202 U/L), virtually all of the CK activity was due to CK-BB, and none to CK-MM or CK-MB. We conclude that brain contains CK-BB and mitochondrial CK, but lacks CK-MM and CK-MB. After cardiac arrest, CK-BB is released into the CSF. Any CK-MM in the CSF is probably from blood contamination, in which case immunoinhibition with anti-CK-M antibodies accurately quantifies CK-BB.     

Quantitation of creatine kinase MB using an agarose electrophoresis method; importance of enzyme reagent concentration

Quantification of CK-MB activity in serum using agarose electrophoresis requires that CK enzyme reagent be layered over the gel in order to develop enzyme activity. We have shown that when commercial enzyme reagents were used, and reconstituted in a normal fashion, that the CK-MM isoenzyme activity is underestimated which results in an overestimation of CK-MB activity. Underestimation of CK-MM occurs because the enzyme reagent are diluted approximately five-fold by the agarose, which results in the non-linear development of enzyme activity under sub-optimal conditions. Reconstitution of these commercial enzyme reagents in a concentrated manner (5 times more concentrated) compensates for the dilution of the reagent by the agarose and allows for the linear development with time of CK-MM and CK-MB enzyme activity. However, use of five-fold concentrated enzyme reagent does not totally prevent underestimation of CK-MM enzyme activity, and care must be taken in limiting the amount of enzyme activity placed on the agarose gel.     

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.     

Direct measurement of creatine kinase-MB activity in serum after extraction with a monoclonal antibody specific to the MB isoenzyme

Fusion of splenocytes from A/J mice immunized by creatine kinase (EC 2.7.3.2)-MB isoenzyme (CK-MB) with SP2/0-Ag14 myeloma cell line generated hybridomas producing monoclonal antibodies specific to CK-MB. One of these monoclonal antibodies ("Conan-MB") was used to develop a direct assay for CK-MB activity. In the assay, serum is incubated for 30 min at room temperature with "Conan-MB" immobilized on latex beads. The beads are then washed, and CK-MB activity bound to the antibody is measured after incubation with CK enzyme reagent for 30 min at 37 degrees C. Results with the assay correlated well (r = 0.997) with those for CK-MB concentration as measured by a two-site immunoassay. Neither CK-MM, CK-BB, mitochondrial CK, nor a hemolysate of erythrocytes interfered. Use of this unique monoclonal antibody allows rapid, precise, and direct determination of CK-MB activity.     

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.     

A novel CK isoenzyme migrating between CK-MB and CK-BB

A CK isoenzyme migrating between CK-MB and CK-BB was detected in the serum of three patients with metastatic prostatic carcinoma. CK-BB was detected in the serum of all three patients and mitochondrial CK in two of the patients. Total CK activity was either normal or elevated, and the atypical CK isoenzyme, CK-BB and the mitochondrial CK isoenzyme were present in serum for up to 1.5 months. This atypical CK isoenzyme was not CK-MB, an albumin-ligand complex, or adenylate kinase, and was not bound to an immunoglobulin. This atypical CK isoenzyme did not contain immunologically normal CK-M subunits but had some CK-B subunits and could be a variant CK-BB or CK-MB isoenzyme. Its appearance in serum could be indicative of a serious illness.     

Quantifying the MB isoenzyme of creatine kinase with the Abbott "IMx" immunoassay analyzer

In this two-step automated assay of the MB isoenzyme of creatine kinase (CK-MB), developed for the Abbott "IMx" immunoassay analyzer, monoclonal anti-CK-MB antibody immobilized onto latex microparticles and polyclonal anti-CK-MM antibody coupled to alkaline phosphatase are used. Within-run CVs ranged from 3.9% to 9.0%, between-run CVs from 0.0% to 5.6%, and the sensitivity was 0.2 microgram/L. Twenty-four results can be obtained in about 37 min. Analytical recovery of CK-MB added to human serum or plasma ranged from 89% to 109%. Icteric, lipemic, or hemolyzed samples did not interfere with CK-MB recovery. Cross-reactivity with CK-MM and CK-BB was 0.012% and 0.001%, respectively. The normal reference interval was 0-5 micrograms/L. IMx CK-MB results correlated well with CK-MB enzyme activity as determined by electrophoresis (n = 203; r = 0.97; slope = 0.90; y-intercept = -4.29) and with commercial immunoassays. We think that this assay will be useful for confirmation of acute myocardial infarction, both in critical-care units and in the clinical laboratory.     

Analytical and clinical evaluation of creatine kinase MB mass assay by IMx: comparison with MB isoenzyme activity and serum myoglobin for early diagnosis of myocardial infarction.

We analytically and clinically evaluated Abbott's IMx assay for creatine kinase (CK) isoenzyme MB (CK-MB) in serum. Over a 1-year period, the method was more specific but less precise than catalytic isoenzyme measurements by electrophoresis or immunoinhibition. Sera from different individuals without electrophoretic evidence of CK-MB but containing macro CK type 1 (n = 20), mitochondrial CK (n = 5), or CK-BB (n = 5) were scored as CK-MB negative by the IMx. Likewise, CK-MB-negative by the sera remained so after addition of purified human CK-MM (< or = 7600 U/L) or CK-BB (< or = 8100 U/L). For 39 patients admitted for suspicion of uncomplicated acute myocardial infarction (precordial pain for < or = 4 h), the diagnostic performance of the IMx CK-MB assay on admission and 4 h later was superior to that of total CK activity and compared well with that of CK-MB activity measured by electrophoresis or immunoinhibition. An admission, myoglobin showed a higher diagnostic sensitivity, specificity, and predictive value than did CK-MB and was the most informative test. Diagnostic performance on admission and 4 h later was further improved by considering positivity for myoglobin and for CK-MB by IMx and for the change in each over the first 4 h of hospitalization as criteria. Twelve hours after admission, diagnostic performance was further improved for all CK and CK-MB methods but began to decline for myoglobin.     

Monoclonal antibody inhibiting creatine kinase MM3 but not isoform MM1

Monoclonal antibody CKM-G01 inhibited greater than 99% of the activity of porcine and human creatine kinase(CK)-MM isoenzyme purified from muscle. However, it inhibited only 54% of CK-MM in human serum. Chromatofocusing of serum CK-MM showed that CKM-G01 inhibited 100% of MM3 but not isoform MM1. CKM-G01 inhibited CK-MM2 by 57%. CKM-G01 specifically inhibited only the original CK-M subunit and not the subunit modified by removal of C-terminal lysine by carboxypeptidase N. CKM-G01 can be used for assay of CK isoforms. We devised a new diagnostic reagent involving it, which requires no analytical separation of isoforms, based on the immunoinhibition method, and applied it to early diagnosis of acute myocardial infarction. The "inhibition index," (inhibited CK activity/total CK activity) x 100, increased more rapidly than did total CK and CK-MB. Evidently this diagnostic reagent can be used for easy, early diagnosis of acute myocardial infarction.     

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.     

AMI患者早期CK同工酶亚型的高压电泳图谱分析

目的探讨CK同工酶亚型在AMI胸痛发作后24h内的变化规律,为AMI患者的早期诊断提供依据。方法采用REP全自动高压电泳仪检测AMI胸痛发作后不同时间以及对照组的CK-MM1、CK-MM2、CK-MM3、CK-MB1、CK-MB2等指标并进行荧光扫描,同时在Olympus2700全自动生化分析仪上测定CK、CK-MB的总活性,对所得数据进行恰当的统计分析。结果在AMI患者胸痛发作24h内,CK同工酶亚型有一特殊的变化规律:4~6h内,CK-MB2、CK-MM3开始升高,8~12h达高峰,92%的病人CK-MB2/CK-MB1>1.5,同时91%的病人CK-MM3/CK-MM1>0.5。结论CK同工酶亚型CK-MM3/CK-MM1、CK-MB2/CK-MB1的检测可作为AMI早期诊断的指标。     

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.     

Increased activity of creatine kinase isoenzyme MB in a theophylline-intoxicated patient

A 78-year-old woman had increased activities of creatine kinase (CK; EC 2.7.3.2) and CK-MB isoenzyme in her serum, associated with severe theophylline intoxication. The time course for CK-MB activity was similar to that from an acute myocardial infarction. Clinical findings, however, including electrocardiograms, did not support the diagnosis of myocardial infarction. We suggest caution in interpreting CK-MB results in severe theophylline intoxication.     

Clinical role of recurrently elevated macro creatine kinase type 1

The typical creatine kinase (CK) isoenzymes include CK-BB, CK-MB, and CK-MM. Macro CK type 1, one of the atypical CK enzymes, has been identified in human serum, but the clinical significance still remains uncertain. In our laboratory, 105 patients who expressed serum macro CK isoenzyme type 1 were identified from March 2004 to March 2007. We found that macro CK type 1 recurred after at least one month in 16 patients. Clinical diagnoses were myopathy in 14 patients, sepsis in one, and acute coronary syndrome in one. The averages of serum total CK and macro CK type 1 were 9,132 and 1,925 (U/L), respectively. The linear regression analysis between recurrent macro CK type 1 and total CK revealed a good correlation (y=3.5054x+2381.3, R(2)=0.7822, P<0.001). Three patients had critical illness, including one respiratory failure and two mortalities. Good linear correlation is documented between total CK and recurrent macro CK type 1. In conclusion, the macro CK type I isoenzyme recurred primarily in patients with myopathy.     

Appearance of a cathodic band in the electrophoretogram of blood creatine kinase isoenzyme-MM fraction during hypoxia in rats

In electrophoretograms of creatine kinase (CK; EC 2.7.3.2) in patients' blood, a band, presumably of mitochondrial origin, is occasionally observed on the cathodic side of the CK-MM fraction. We studied the implications of this phenomenon in rats exposed to hypoxic conditions. In the hypoxic cardiac muscle, the proportions of CK-MB and CK-MM were not significantly different from controls, but that of the mitochondrial CK was lower. In the corresponding blood, the cathodic mitochondrial CK band appeared, but disappeared as the animals recovered from hypoxia. The CK-MB isoenzyme was increased in the blood of the control rats, as obtained by heart puncture, but no mitochondrial fraction was detected. We believe that changes in myocardial mitochondria during hypoxia are related to the appearance of the cathodic band. Cytoplasmic CK-MB, unlike mitochondrial CK, markedly increased in the rats' blood during the recovery stage rather than during the hypoxia.     

Highly sensitive enzyme immunoassay for human creatine kinase MM and MB isozymes

A sensitive enzyme immunoassay system for measurement of MM and MB forms of human creatine kinase (CK) was developed using purified monospecific antibodies to the M subunit and to the B subunit of CK. The CK-MM assay system consisted of polystyrene balls with immobilized F(ab')2 fragments of anti-M and the same antibody Fab' fragments labeled with beta-D-galactosidase from Escherichia coli. The CK-MB was assayed with the polystyrene balls with either antibody (anti-M or anti-B) F(ab')2 fragments and the other antibody (anti-B and anti-M, respectively) Fab' fragments labeled with galactosidase. The assays were highly sensitive and 3 pg of CK-MM and CK-MB were measurable. The CK-MM assay was specific to the M subunit of creatine kinase, and it cross-reacted about 15% with CK-MB, but not with CK-BB. The CK-MB assay did not cross-react with CK-MM nor CK-BB. Therefore, concentrations of CK-MM could be estimated by subtracting the cross-reacting values of CK-MB. Coefficients of variation in within-run and between-run precision studies for serum CK-MM and CK-MB were less than 9%. Serum levels in healthy adults of various ages (16-59 yr old) were ranged from 35.2-132 ng/ml for CK-MM and from 0.40-1.77 ng/ml for CK-MB. There was apparently no statistical significance among the sex- and age-related differences. Concentrations of CK-MM and CK-MB in various human tissues were also determined. The CK-MM was present abundantly in the heart and the tissues composed of striated muscles (skeletal muscle, diaphragm and proximal esophagus). The CK-MB was distributed not only in the heart but also in the striated muscle tissues at a relatively high level.     

Creatine kinase isoenzymes of mitochondrial origin in human serum.

We measured creatine kinase (EC 2.7.3.2) activity in 1009 serum samples from 538 patients in the intensive-care units of the University of Texas Medical Branch hospitals. Creatine kinase isoenzymes migrating cathodal to skeletal muscle creatine kinase (CK-MM) on cellulose acetate electrophoresis were found in sera from 14 of the 538 patients. Creatine kinase, lactate dehydrogenase (EC 1.1.1.27), aspartate aminotransferase (EC 2.6.1.1), and alanine aminotransferase (EC 2.6.1.2) activities were abnormally increased in these 14 patients. Liver lactate dehydrogenase isoenzyme (LDH5) and cardiac creatine kinase isoenzyme (CK-MB) were abnormally increased in 12 and eight of these patients, respectively. Ten of the 14 patients died during their hospital admission. We believe the creatine kinase isoenzymes that migrated cathodal to skeletal muscle creatine kinase (CK-MM) were of mitochondrial origin.     

Macro creatine kinase type 1 with electrophoretic mobility identical to that of the MB isoenzyme

We describe the case of an elderly woman whose symptoms and electrocardiographic pattern initially suggested acute myocardial infarction. The value for total serum creatine kinase (EC 2.7.3.2; CK) was 737 U/L (reference interval: 22-269 U/L), and electrophoresis for CK isoenzymes demonstrated two bands, the more anodal migrating to the CK-MB region and the second migrating between the CK-MB and CK-MM regions. The above-normal total CK and electrophoretic pattern persisted during her 11-day hospital course. The QuiCK-MB (International Immunoassay Labs.) and Tandem-E CK-MB (Hybritech) immunoassays, however, showed CK-MB mass measurements within the normal range. In further investigation with a mixture of patient's serum and human-serum-based control containing all CK isoenzymes, the electrophoretic mobility of only CK-BB was altered, proving that the patient had antibody to the B unit of CK in her serum. Immunofixation revealed the more anodal band to be a CK-IgA lambda complex, and the more cathodal band, a CK-IgG kappa complex. Mixing the patient's serum with polyclonal antibody specific for CK-B slowed the electrophoretic mobility of only the more anodal band. Polyclonal antibody specific for CK-M had no effect on either band. Evidently, this patient had two different types of macro CK type 1, both containing CK-BB. We conclude that macro CK type 1 can mimic CK-MB and be a source of confusion.     

Cathodic isozyme of serum creatine kinase in a case of stomach cancer complicated by disseminated intravascular coagulation

A rare isozyme of serum creatine kinase (CK) migrating cathodic to CK-MM on electrophoresis was found in a 30-year-old male with stomach cancer complicated by disseminated intravascular coagulation leading to massive upper gastrointestinal bleeding and marked anemia. Serum CK activity rose to a maximum of 374 U/l without detectable CK-MB isozyme. The patient was also characterized by a marked increase in serum lactate dehydrogenase (all isozymes elevated) and by preferential leakage of mitochondrial aspartate aminotransferase and glutamate dehydrogenase, indicating the presence of extensive tissue damage involving mitochondria. Skeletal muscle mitochondria were considered the most likely source of the additional CK isozyme.