IgA-CK-BB complex with CK-MB electrophoretic mobility can lead to erroneous diagnosis of acute myocardial infarction

This patient, on admission, presented with a tentative diagnosis of myocardial infarction: the electrocardiogram showed a nonspecific ST-segment and T-wave abnormalities, and total creatine kinase (CK; EC 2.7.3.2) activity was slightly increased (238 U/L). However, a high electrophoretic value for CK-MB (50% of total CK activity) and the electrophoretic pattern of lactate dehydrogenase (EC 1.1.1.27) isoenzymes ruled out myocardial infarction. The isoenzyme migrating as CK-MB was found later to contain no immunologically normal CK-M subunits, and it was bound to IgA. A mixture of the patient's serum and a human serum control containing all CK isoenzymes showed altered electrophoretic mobility only for CK-BB, indicating that the patient's serum contained antibodies to the B unit of CK. Elution from a Sephadex G-200 column showed that the peak at which most of the anodic CK was eluted corresponded to a molecular mass of approximately 200 kDa. Evidently this atypical isoenzyme was an IgA-CK-BB complex. Because this macro CK type 1 can mimic CK-MB, it may therefore be a source of confusion.     

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.     

Erum-induced changes in electrophoretic mobility of creatine phosphokinase isoenzymes?

BB isoenzyme of creatine phosphokinase (CK-BB) obtained from human brain-extract changes its electrophoretic mobility after incubation in human serum at 37° C. No change of electrophoretic mobility of CK-BB is observed after incubation in isotonic saline. We have shown by means of immunoprecipitation with specific antibodies that the structure of CK-BB is not changed. These findings are supported by other authors and make the diagnostic value of electrophoretic separation of CK isoenzymes doubtful as after a 3-h incubation CK-BB migrates similarly to CK-MB and consequently may be misinterpreted.     

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.     

Macro creatine kinase BB in serum, and some data on its prevalence.

We report the case of a patient with persistently above-normal activity of creatine kinase (CK) in serum, a major fraction of which on electrophoresis moved as a band between the MM and MB isoenzymes and on anion-exchange column chromatography eluted in the MB fraction. Measurements in the presence of specific M or B subunit-inhibitory antibodies indicated that 93% of the activity consisted of B-isomers. From these experiments we conclude that the abnormal CK is of BB nature. Gel filtration and immunoglobulin precipitation showed that the CK-BB was complexed with IgG. Normal CK-BB, when mixed with the patient's serum, was converted to macro CK-BB. In vitro stability of 37 degrees C of the abnormal enzyme was much greater than that of normal BB and MM isoenzymes. Following this finding, we then assessed 310 sera, received for enzyme assay by the clinical laboratory, for electrophoretically abnormally migrating CK isoenzymes. Of these, five (1.6%) contained such enzymes, all being of BB nature. They were of increased molecular mass, and at least three of them were complexed with IgG.     

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.     

Creatine kinase and lactate dehydrogenase isoenzymes in serum and tissues of patients with stomach adenocarcinoma

Total activities of creatine kinase (EC 2.7.3.2; CK) and lactate dehydrogenase (EC 1.1.1.27; LD) and their isoenzymes were estimated in serum and tissue samples from patients with stomach adenocarcinomas who were to undergo gastric resection. Total CK activity (U/g protein) appeared to be markedly decreased in neoplastic stomach tissue. CK-BB was the predominant isoenzyme in both neoplastic and normal stomach tissues; however, the CK-BB/total CK ratio was increased in adenocarcinoma tissue. Macro CK type 1 was found in two neoplastic tissues and macro CK type 2 in 11. LD4 and LD5 isoenzymes were predominant in gastric tissues, but LD5 and the LD5/LD1 ratio were higher in adenocarcinoma tissue. At 24 h before surgery, CK-BB was demonstrated in sera of all patients and CK-MB in 69%. The CK-BB probably originated from neoplastic stomach tissue, which contains high CK activity, with BB isoenzyme predominating. After gastrectomy, CK and LD isoenzymes in sera were markedly increased by 24 h postsurgery. These alterations were attributed to release from damaged tissue during gastric resection.     

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.     

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.     

Separation of CK isoenzymes on DEAE-sephadex A-50 at neutral pH

The MM, MB and BB isoenzymes of human creatine kinase (CK) were separated by elution from micro-columns of DEAE-Sephadex A-50 with Tris buffer containing increasing concentrations of NaCl at pH 7.0, instead of pH 8.0 as has commonly been used. Since pH 7.0 is close to the pH optimum of CK, this allowed the use of four times larger aliquots of the eluates for the estimation of CK activity and, consequently, a 4-fold increase in sensitivity. Using serum specimens from patients with acute myocardial infarction, there was a good correlation of the CK-MM (r = 0.99) and CK-MB (r = 0.93) activities obtained with the two buffer systems. Similarly, normal sera had CK-MB and CK-BB activities of less than 2 U/l with both buffer systems. Comparison of the composition of serum proteins in the eluates by conventional electrophoresis revealed that although the distribution of CK isoenzymes separated by the two buffer systems was similar, the distribution of proteins at pH 7.0 showed an appreciable shift of protein from the MB to the MM eluates.     

Stability and electrophoretic characteristics of creatine kinase BB extracted from human brain and intestine

Creatine kinase (CK; EC 2.7.3.2) isoenzyme BB extracted from brains of rats reportedly undergoes modification at 37 degrees C, leaving an electrophoretic variant that accounts for most of the residual CK activity. This variant, called CK-BB', migrates on electrophoresis similarly to creatine kinase isoenzyme MB. Using electrophoresis and immunoinhibition with antiserum to creatine kinase isoenzyme MM, we found CK-BB to be the only identifiable cytoplasmic isoenzyme in surgical samples from human brain and intestine. In contrast, we found that some samples of brain obtained at autopsy contain CK-BB'. We also found that CK-BB extracted from human brain was converted to CK-BB' upon incubation in serum or plasma at 37 degrees C. We found a similar development of CK-BB' in incubation mixtures of serum or plasma containing CK-BB obtained from surgical samples of human intestine. The development of CK-BB' during infarction of the gastrointestinal system may thus be a source of false-positive CK-MB in the laboratory verification of myocardial infarction when electrophoresis is used as the only method to identify CK isoenzymes.     

A sensitive bioluminescent immunoinhibition test for CK-B subunit activity and a CK-MB specific ELISA compared: correlation with agarose electrophoresis and influence of CK-isoenzyme profile on results

Searching for alternatives to the imprecise spectrophotometric tests for low-concentration creatine kinase (EC 2.7.3.2) isoenzyme MB (CK-MB), we investigated the analytical performance of two potentially superior approaches--a bioluminescent immunoinhibition assay (I, LKB-Wallac) and an ELISA (enzyme-labeled immunosorbent assay) technique (II, Hybritech)--in comparison with an electrophoretic method (III, Beckman). Only I showed good between-day precision (CV 8.3%) at the upper reference limit, allowing reproducible assay of CK-B subunit activity down to at least 3 U/L. In conditions where CK isoenzyme assays remained unaffected by CK-MM concentrations, test results were proportional to the amount of CK-MB in the sample up to at least 50 U/L for I, 120 micrograms/L for II, and 100 U/L for III (r greater than 0.998 by linear regression analysis). For CK-MB-positive samples, the data by I correlated more closely with values by III (n = 24; r = 0.994) than did results by II (n = 15; r = 0.909), but both methods were equally effective in discriminating between samples with or without electrophoretically supranormal CK-MB activity (93% sensitivity). II was entirely CK-MB specific, whereas CK-B activity by I was consistently (18/18) increased in CK-MB-negative samples containing CK-BB (n = 6; r = 0.996) or macro CK, types 1 or 2 (n = 12; r = 0.930). I is highly sensitive for screening for increased non-MM CK activity, the nature of which should be subsequently clarified by electrophoresis.     

Adenylate kinase mimics creatine kinase-MM isoenzyme in a CK isoenzyme electrophoresis assay

Adenylate kinase activity (AK) originating from erythrocytes, present in hemolyzed serum behaves like creatine kinase MM isoenzyme (CK-MM) in some CK electrophoresis assays that employ, in their visualization reagent kits, adenosine monophosphate (AMP) as the sole inhibitor of AK, rather than a combination of AMP and a more potent inhibitor of erythrocyte AK, diadenosine pentaphosphate (Ap5A), to inhibit all contaminating-AK activities in serum and quantify only the CK isoenzyme activities in serum following electrophoretic fractionation on agarose gel. This can spuriously overestimate the CK-MM fraction and thereby result in underestimation of CK-MM or CK-BB isoenzymes if present. A hemolyzed serum sample obtained from an elderly patient was erroneously reported as containing low CK-MB due to such overestimation of CK-MM fraction in the sample. Supplementing the AMP already present in the visualization reagent formulation, used to estimate CK isoenzyme concentration in serum, with Ap5A can eliminate or effectively minimize AK interference, especially that caused by hemolysis, and thereby prevent reporting false-negative CK-MB result obtained with CK isoenzyme electrophoresis assays. © 1994 Wiley-Liss, Inc.     

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.     

Transient increase in serum creatine kinase isoenzyme BB activity after prostatectomy in a patient with massive benign prostatic hyperplasia

The activity of creatine kinase isoenzyme BB (CK-BB) in serum is rarely abnormally high (i.e., detectable). An increase in immunoreactive CK-BB or CK-BB activity in patients with prostatic disease has been proposed as an indication of prostatic adenocarcinoma. Here we report the case of an elderly man with massive benign prostatic hyperplasia but no clinical or pathological evidence of prostatic adenocarcinoma, whose serum CK-BB activity was found by agarose gel electrophoresis to be 1 U/L (normal: 0%), 10% of his total CK activity. Serum CK-BB activity was further increased to 16 U/L (20% of total CK activity) 1 h after prostatectomy, but became undetectable by the second day after the operation. The findings suggest that: (a) the source of the serum CK-BB activity was the enlarged prostate gland; (b) abnormally high CK-BB activity in serum of men with prostatic disease does not necessarily indicate the presence of prostatic adenocarcinoma; and (c) myocardial injury could be erroneously diagnosed postoperatively in prostatectomy patients if CK isoenzyme methods are used that do not consistently separate "heart-specific" CK-MB from CK-BB.     

影响肌酸激酶同工酶活性检测结果的因素探讨

目的探讨应用酶免疫抑制法检测血清肌酸激酶（CK）同工酶——CK—MB酶活力单位时,引起临床假阳性的因素,以科学合理地解释每一检测结果及解决方法。方法采用回顾性研究,分析125例非心肌梗死患者,应用酶免疫抑制法检测血清标本中CK、CK—MB酶活力单位,分析可能引起检测值CK—MB酶活力单位假性升高的影响因素。结果86例成人非心肌梗死患者中,总CK活力相对较低,癌症患者、脑梗死患者、变态反应疾病患者CK—MB假阳性比例较大,其中CK—MB占CK总活力大于5％有59例,其中53例在6％～21％,6例大于38％;38例新生儿中CK活性范围为145～1974U／L,其中1例新生儿呼吸窘迫综合征患儿CKMB占CK总活力达90％;另1例溶血标本也引起假阳性。结论应用酶免疫抑制法检测非心肌梗死患者CK—MB酶活力单位假性升高的影响因素,可能是血清中存在巨CK、CK—BB、AK等,影响试剂单克隆抗体与肌酸激酶中的M亚基结合,存在未被抑制的CK—M、M亚基同时与B亚基参加酶促反应,而且因计算时结果乘以2,更加扩大误差,是该方法学缺点造成的错误结果,不能作为诊断依据。     

Macro creatine kinase type 2: A marker of myocardial damage in infants?

The case histories of two children (aged two months) affected by myocarditis showing an atypical band of serum creatine kinase (EC 2.7.3.2; CK) in the CK isoenzyme electrophoretic pattern are reported. The electrophoretic mobility on cellulose acetate of the atypical iso-CK band and its greater relative molecular mass, the lack of binding with immunoglobulins and the result of CK-BB determination by RIA, allowed us to identify the band with an oligomeric form of the mitochondrial isoenzyme. One child died 2 days after admission, while in the other it was possible to demonstrate reduction and disappearance of the atypical band in concomitance with a marked clinical improvement. Our findings suggest that the oligomeric form of mitochondrial-CK is released in conditions of serious heart muscle damage, and that it may be an indicator of myocardial cellular necrosis in pediatric patients.     

Evaluation of hypoxic brain injury with spinal fluid enzymes, lactate, and pyruvate.

OBJECTIVE: To investigate the prognostic importance in neurologic recovery of the lumbar cerebrospinal fluid (CSF) variables creatine kinase (CK) and brain-type creatine kinase isoenzyme (CK-BB), lactate dehydrogenase (LDH) and its isoenzymes (LDH 1-5), CSF acid phosphatase, beta-D-N-acetylglucosaminidase activity, and CSF lactate, pyruvate, sodium, potassium, and calcium concentrations in patients who experienced cardiac arrest. DESIGN: Prospective clinical study with blood and CSF samples collected 4, 28, 76, and 172 hrs after resuscitation. SETTING: Medical ICU in a university hospital. PATIENTS: Twenty consecutive victims of out-of-hospital cardiac arrest. Eight patients recovered neurologically and 12 patients remained comatose or neurologically disabled until death. MEASUREMENTS AND MAIN RESULTS: CSF CK, CK-BB, LDH, and LDH isoenzyme 1-3 concentrations in all disabled patients were markedly increased at 76 hrs after the resuscitation. However, these variables were not changed in the recovered subjects. Patients (n = 7) with a mean CSF CK level of 25 +/- 33 (SD) U/L, CK-BB 23 +/- 33 U/L, and CSF lactate 3.8 +/- 0.9 mmol/L at 28 hrs after cardiac arrest remained unconscious and died. In the recovered patients, the mean CSF CK concentration was 2.0 +/- 1.5 U/L (p less than .001) and CSF lactate concentration 2.5 +/- 0.5 mmol/L (p less than .002). The lactate concentration was highest at 4 hrs after resuscitation, declining thereafter. Patients with a mean CSF total LDH level of 609 +/- 515 U/L and acid phosphatase 2.4 +/- 1.2 U/L 76 hrs after resuscitation died without regaining consciousness. In the recovered patients, the mean total CSF LDH activity was 82 +/- 58 U/L (p = .003) and CSF acid phosphatase was 0.8 +/- 0.5 U/L (p = .01) 76 hrs after resuscitation. CONCLUSIONS: CSF CK, CK-BB, and CSF lactate concentrations reflect a patient's outcome most reliably when measured within 28 to 76 hrs of the cardiac arrest. Similarly, CSF LDH, its isoenzymes 1-3, and CSF acid phosphatase concentrations, when measured at 76 hrs, can be used to monitor the patient's outcome after cardiac arrest. When correlated with Glasgow Coma Scale scores, the closest negative correlation was again seen in CSF CK and CK-BB at 28 and 76 hrs, as well as in LDH, LDH1-3, and acid phosphatase values at 76 hrs. The negative correlation between CSF lactate and Glasgow Coma Scale scores was most distinct at 28 hrs.     

Specificity of autoantibodies to lactate dehydrogenase isoenzyme subunits

We studied serum from 12 unrelated patients with variant electrophoretic patterns for lactate dehydrogenase (EC 1.1.1.27; LD) isoenzymes characteristic of LD-immunoglobulin complexes. Immunoglobulin class was identified in four cases. In all 12 cases, mixing the patient's serum with control serum altered the mobility of normal LD isoenzymes to that of the variant. To determine antigenic specificity, we mixed ammonium sulfate-precipitated fractions of patient's samples with purified LD1 (H tetramer) or LD5 (M tetramer) prepared by anion-exchange chromatography. In six cases the fractionated antibody displayed a pure M-subunit specificity. In four cases it acted against both M tetramers and H tetramers, and in two cases the antibody affected the migration only of LD isoenzymes containing both M and H subunits (i.e., hybrid isoenzymes). Depending on the relative excess of antibody or LD5 in such mixtures, various different anomalous electrophoretic patterns were produced. These results indicate that apparent differences between variant LD isoenzyme patterns in whole serum of different individuals can arise from autoantibodies with similar reactivities being present in various ratios of antigen to antibody.     

Production of recombinant human creatine kinase (r-hCK) isozymes by tandem repeat expression of M and B genes and characterization of r-hCK-MB

BACKGROUND: Serum creatine kinase-MB isoenzyme (CK-MB) is widely used as a marker of myocardial injury. We prepared recombinant human CK (r-hCK) MB isoenzyme and examined its potential for use as a control material for assay of CK-MB in serum. METHODS: cDNAs encoding CK-M and CK-B subunits were inserted into the same plasmid vector, followed by transformation of Escherichia coli. The resulting three types of CK isoenzymes were purified by conventional chromatography. RESULTS: The ratio of MB to MM to BB was 50:40:10 on the basis of CK activity. Highly purified CK-MB with a specific activity of 533 U/mg was produced in a yield of 5.7 mg/g of packed cells. Purified r-hCK-MB had the isoelectric point (pI 5.3) and molecular size (46 kDa for the subunit) of native CK-MB. Its immunoreactivity in an ELISA using antibody against native heart enzyme was similar to that of cardiac CK-MB. The r-hCK-MB retained >90% activity for at least 4 months at 11 degrees C in a delipidated serum matrix in a liquid form at a concentration of 118 U/L. CONCLUSIONS: r-hCK-MB shows key properties of the native cardiac isoenzyme and may be useful as a control and calibrator for serum assays of CK-MB.