Properties of creatine kinase-BB from canine and human brain tissues

Creatine kinase (EC 2.7.3.2) BB isoenzyme (CK-BB) was purified to homogeneity from canine and human brain tissues. The purified protein from both sources exhibits Mr of 84,700 daltons. The canine isoenzyme exhibits several properties similar to human isoenzyme with respect to reactive and total thiol groups, UV spectra, isoelectric points and reaction kinetics. While both canine and human CK-BB isoenzymes are unstable compared to other CK isoenzymes, canine CK-BB is even less stable than the human enzyme, losing most of its activity within 20 h at 4 degrees C at pH 5.0. Addition of 2-mercaptoethanol does not prevent rapid loss of the enzyme activity. Increasing the pH to 9.0, however, increases the stability of both CK-BB isoenzymes. Agarose electrophoresis demonstrated the presence of MM as well as BB isoenzyme in various parts of brain tissues. BB was present at an activity of 90.8-93.3 U/mg and MM at 6.7-9.2 U/mg.     

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.     

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.     

Preparation and stability of a liquid creatine kinase isoenzyme control from rabbit serum.

The MB isoenzyme of creatine kinase (CK) may be prepared in vitro from rabbit serum containing only the MM and BB isoenzymes, by means of a hybridization technique. The MM and BB dimers dissociate in 4 mol/L urea, which allows random recombination of M and B monomers. A liquid CK-isoenzyme control can be made from mixtures of rabbit sera obtained after hybridization and stabilized with glycerol and 25 mmol of 2-mercaptoethanol per liter. A liquid control stored at 4 degrees C showed good stability over a three-month period, declining to a mean residual activity of CK of approximately 90% after three weeks and a mean residual activity of MM, MB, and BB of 80--85% after six weeks. At 25 degrees C, CK activity of the liquid control declined to 75--80% after the fourth week. CK-BB at 25 degrees C was the least stable isoenzyme, declining to 75% after the third week and reaching 60% of activity after 12 weeks. CK-MB and CK-MM showed approximately 10--15% less stability at 25 degrees C than at 4 degrees C.     

Macro creatine kinase BB isoenzyme in serum: most likely an antigen-autoantibody complex

Nine sera containing an abnormal creatine kinase BB isoenzyme, "macro CK-BB", were examined. Immunoglobulin precipitation after addition of radiolabelled CK-BB suggested that in eight of the sera the enzyme was linked to an immunoglobulin G. Results obtained with papain-digested and with pepsin-digested IgG suggested that the binding of CK-BB occurred in the antigen-binding region (the "Fab-region") of IgG. Each of the two antigen-binding fragments of IgG, obtained by papain-digestion, were CK-BB specific, since they complexed this isoenzyme equally well when excess CK-MM and CK-MB was added. From Scatchard plots the affinity constant for binding of CK-BB to IgG and the BB-binding capacity of four of the sera was calculated. The affinity constant was high and differed little between the sera (range 0.7 x 10(11)-1.6 x 10(11)1/mol). The BB-binding capacity differed widely (range 21-900 microgram of CK-BB per litre of serum), but in each serum it roughly paralleled the activity of the macro CK-BB complex. The results suggest that in eight of the nine sera examined the BB-binding IgG is an antibody with activity directed towards CK-BB.     

Macro creatine kinase BB isoenzyme in serum: most likely an antigen-autoantibody complex

Nine sera containing an abnormal creatine kinase BB isoenzyme, “macro CK-BB”, were examined. Immunoglobulin precipitation after addition of radio-labelled CK-BB suggested that in eight of the sera the enzyme was linked to an immunoglobulin G. Results obtained with papain-digested and with pepsin-digested IgG suggested that the binding of CK-BB occurred in the antigen-binding region (the “Fab-region”) of JgG. Each of the two antigen-binding fragments of IgG, obtained by papain-digestion, were CK-BB specific, since they complexed this isoenzyme equally well when excess CK-MM and CK-MB was added. From Scatchard plots the affinity constant for binding of CK-BB to IgG and the BB-binding capacity of four of the sera was calculated. The affinity constant was high and differed little between the sera (range 0.7 ± 10¹¹-^?1.6 ± 10¹¹ l/mol). The BB-binding capacity differed widely (range 21—900 μg of CK-BB per litre of serum), but in each serum it roughly paralleled the activity of the macro CK-BB complex. The results suggest that in eight of the nine sera examined the BB-binding IgG is an antibody with activity directed towards CK-BB.     

A "column-batch" method for separating MB and LD1 in a single fraction

In this "column-batch" method for separating the MB and BB isoenzymes of creatine kinase and the LD1 isoenzyme of lactate dehydrogenase, one can, alternatively, separate MB from BB or obtain a combined fraction containing MB, BB, and LD1. The principal advantage is that the resulting fractions are twofold as concentrated as was the applied sample. Thus, activity can be measured by conventional automated methods, with no need for the modifications to compensate for diluted fractions that are required by other ion-exchange methods. Another advantage is the total absence of interference by the MM isoenzyme. A strong anion exchanger (AG-MP1, Bio-Rad) is used in the acetate form at pH 6.3. There is no retention of MM; retained MB, BB, and LD1 are eluted with a solution of magnesium acetate. Results are compared with those obtained for subunit B and LD1 by immunoinhibition. Results with patients are considered consistent with myocardial infarction if MB exceeds 20 U/L and 3% of the total CK and LD1 exceeds 130 U/L or 28% of the total LD activity.     

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.     

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.     

Creatine kinase in serum: 4. Differences in substrate affinity among the isoenzymes

The goal of this work was to find out whether it is possible to measure all three creatine kinase isoenzymes under the same reaction conditions in spite of their different kinetic properties. We found the tightest substrate binding for purified human BB, followed by the MB And MM isoenzyme preparations for both creatine phosphate and ADP. An increase in substrate concentration usually resulted in an inhibition. Nevertheless, it was possible with a method optimized for the MM isoenzyme also to measure the BB and MB isoenzymes at a rate of inhibition of only 6 and 3%, respectively. Marked differences in the apparent Km values between purified and native MM isoenzyme in human serum may indicate that the enzyme declined in substrate affinity during the isolation procedure. The use of enzyme preparations for standardization purposes, therefore, is only suitable if their kinetic properties are close to those of the enzyme in serum. Difficulties in the calculation of the apparent Km values are discussed and the graphical procedures of Lineweaver and Burk and of Eisenthal and Cornish-Bowden compared.     

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.     

The origin of the elevated activities of creatine kinase and other enzymes in the sera of patients with myxoedema.

An attempt has been made to establish the origin of the elevated serum creatine kinase which occurs in most patients with myxoedema. Parallel determinations of a number of other serum enzymes were made but the incidence of elevated values was appreciably less than in the case of creatine kinase. Rather surprisingly, the serum amylase activity was found to be increased in more than 50% of the patients studied. Creatine kinase isoenzymes were separated by starch-gel electrophoresis of the sera of 26 patients with myxoedema. In 25 the MM isoenzyme only could be identified while the remaining serum also contained a trace of the MB fraction. Similar isoenzyme studies were made with the sera of normal and thyroidectomized rats, all of which are shown to contain all three isoenzymes. (MM, MB and BB) irrespective of thyroid functional status. No consistent difference was apparent between the patterns exhibited by the thyroidectomized and control groups, and it was concluded that thyroidectomized rats cannot be regarded as a suitable experimental model for the study of this aspect of human hypothyroidism. It is suggested that enzyme release in myxoedema is a non-specific effect, possibly die to diminution in the ATP content of tissues generally. The greater incidence of creatine kinase elevation is probably due to the relatively high concentrations of this enzyme in skeletal muscle, the mass of which is much greater than that of any other tissue.     

Creatine kinase isoenzyme variants in human serum

In serum from about 800 patients, total creatine kinase and its subunit B activities were determined by the recommended Scandinavian creatine kinase method in the absence and presence of a creatine kinase M subunit inhibitory antibody. Eight patients had supranormal subunit B activities, but normal or near-normal values for total creatine kinase activity. Electrophoresis of sera from these eight patients showed, in addition to the normally migrating isoenzyme MM, one or two abnormally migrating creatine kinase isoenzyme bands, located between normally migrating isoenzymes MM and MB. Experimental data suggest that these abnormal bands may be isoenzyme BB with changed electrophoretic mobility. The eight patients had no particular disorder in common.     

Reassessment of creatine kinase BB as a marker for cancer of the prostate, breast, and lung

We used an enzyme-linked immunoabsorbent assay to measure creatine kinase (CK; EC 2.7.3.2) BB in the sera of 58 cancer patients. A pre-incubation step with an anti-CK-M antibody-coated bead removed M chain components, and CK-BB was quantified with use of an anti-CK-B antibody-coated tube. No cross reactivity was observed with mitochondrial CK or CK-MM; CK-MB cross reacted slightly (1.6%). Macro CK type 1 was measured as CK-BB. Average analytical recovery of purified CK-BB added to serum was 97.7%. Although the enzyme activity of CK-BB is labile, our studies show that this protein is antigenically stable for 12 months when stored frozen. The upper limit of normal for CK-BB concentration was 0.3 micrograms/L (95th percentile, n = 25). Of the 20 cases of breast cancer of various stages, none showed any increases of serum CK-BB. Only two of 18 patients with prostatic carcinoma (stage D), and two of 10 patients with oat-cell carcinoma of the lung had increased concentrations of CK-BB in the serum. Ten patients with squamous cell cancer of the lung had normal concentration of the enzyme. Thus the CK-BB isoenzyme is not frequently increased in cancers of the prostate, lung, and breast, and it has little apparent value as a tumor marker for these diseases.     

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.     

Appearance of creatine kinase BB isoenzyme in the serum of a patient suffering from infarction of the colon.

A case is described of multiple pathologies which was associated with very high levels of total serum creatine kinase activity. Electrophoretic analysis showed the circulating enzyme to be made up of all three isoenzyme fractions; MM, MB and BB. Acute necrosis of a portion of large intestine seems the most likely explanation for the transient appearance of the BB fraction. The implications of these findings with regard to creatine kinase isoenzyme analysis techniques are discussed.     

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.     

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.     

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.     

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.