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.     

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.     

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.     

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.     

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.     

Decreased storage stability of creatine kinase in a cardiac reperfusion solution

Creatine kinase (CK; EC 2.7.3.2) has been used as an indicator of myocardial cellular damage. In this study we used a Krebs-Henseleit (KH) solution to reperfuse isolated rat hearts after 24 h of cold preservation and collected the KH reperfusate for assay of CK to assess cellular damage. We wanted to determine the stability of CK in the KH solution at different cold-storage temperatures and albumin concentrations. CK activity (mean +/- SEM) after one week of refrigeration (5 degrees C) was 93% +/- 1% of control values, whereas CK activity in nitrogen-frozen (-200 degrees C) samples was only 1.6% +/- 1% of control values, and that in samples frozen at moderately low temperatures (-10 degrees C) was 63% +/- 1% of control values. To enhance stability, we added albumin at several concentrations (49, 25, 12, and 6 g/L) to reperfusion collections in which CK had been previously determined. Specimens were frozen (-10 degrees C), then re-analyzed for CK weekly for three weeks. CK activity was maintained (100% +/- 5%) only in samples containing 25 g/L or more albumin. These data suggest that refrigeration (5 degrees C) for one week maintains normal CK activity in KH solution; however, if prolonged storage is necessary, a stabilizer such as albumin (greater than or equal to 25 g/L) will maintain analyte stability in frozen storage (-10 degrees C) for at least three weeks.     

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.     

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 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.     

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.     

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.     

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.     

Impairment of energy metabolism in intact residual myocardium of rat hearts with chronic myocardial infarction.

The purpose of this study was to test the hypothesis that energy metabolism is impaired in residual intact myocardium of chronically infarcted rat heart, contributing to contractile dysfunction. Myocardial infarction (MI) was induced in rats by coronary artery ligation. Hearts were isolated 8 wk later and buffer-perfused isovolumically. MI hearts showed reduced left ventricular developed pressure, but oxygen consumption was unchanged. High-energy phosphate contents were measured chemically and by 31P-NMR spectroscopy. In residual intact left ventricular tissue, ATP was unchanged after MI, while creatine phosphate was reduced by 31%. Total creatine kinase (CK) activity was reduced by 17%, the fetal CK isoenzymes BB and MB increased, while the "adult" mitochondrial CK isoenzyme activity decreased by 44%. Total creatine content decreased by 35%. Phosphoryl exchange between ATP and creatine phosphate, measured by 31P-NMR magnetization transfer, fell by 50% in MI hearts. Thus, energy reserve is substantially impaired in residual intact myocardium of chronically infarcted rats. Because phosphoryl exchange was still five times higher than ATP synthesis rates calculated from oxygen consumption, phosphoryl transfer via CK may not limit baseline contractile performance 2 mo after MI. In contrast, when MI hearts were subjected to acute stress (hypoxia), mechanical recovery during reoxygenation was impaired, suggesting that reduced energy reserve contributes to increased susceptibility of MI hearts to acute metabolic stress.     

Quantitation of creatine kinase isoenzymes in human tissues and sera by an immunological method

Antisera against the crystallized creatine kinase isoenzymes from human skeletal muscle (MM) and from human brain (BB) were produced in rabbits. Both the MM and BB isoenzymes were precipitated quantitatively by their homologous antisera. No cross-reaction was observed. The hybrid MB from human heart muscle could not be precipitated completely by either of the two antisera. In artifical mixtures the concentrations of individual creatine kinase isoenzymes were determined from the percentage of non-precipitable activity in the supernatant after reaction with each of the antisera.This immunotitration assay was applied to study the quantitative distribution of creatine kinase isoenzymes in extracts of human tissues. The isoenzyme patterns obtained were compared with those determined by electrophoretic analysis.In sera of patients with myocardial infarction, the immunotitration assay allowed the sensitive and rapid quantitation of creatine kinase isoenzymes, especially of the “infarct-specific” hybrid MB, even in sera with low total activity. This indicates that the method is of diagnostic value.     

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.     

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.     

Transient creatine kinase-BB activity in serum or plasma after cardiac or respiratory arrest

We studied the transient appearance of creatine kinase (EC 2.7.3.2) isoenzyme BB, as measured by electrophoresis, in serum or plasma from 19 patients who had just experienced cardiac or respiratory arrest. Creatine kinase BB activity was greatest 0.5 to 3 h after the arrest, with values (measured at 30 degrees C) ranging from 3 to 27 U/L (mean, 7.8 U/L) in 18 patients who were successfully resuscitated. Elimination time for the isoenzyme ranged from 8 to 48 h (mean, 20 h). Elimination t1/2 varied from 4.6 to 16 h for 13 patients from whom adequate serial blood specimens were obtained. We could find no correlation between peak BB activity and eventual case outcome. We attribute this to the near impossibility of drawing a blood specimen exactly when the isoenzyme activity peaks, the instability of creatine kinase BB activity at 37 degrees C, and the fact that nine of these patients died after second or multiple arrests.     

Both systolic and diastolic dysfunction characterize nonischemic inhibition of myocardial energy metabolism: An experimental strain rate echocardiographic study

BACKGROUND: Ischemia is primarily a metabolic event. However, regional functional changes can be affected by structural alterations. We developed an experimental model of sole myocardial energy metabolism inhibition and characterized the resulting regional dysfunction. METHODS: In 12 pigs, we regionally inhibited creatine kinase (CK) and, consequently, myocyte high-energy phosphate transfer by intracoronary administration of iodoacetamide. Myocardial biopsies for CK activity and structural analyses and strain rate (SR) echocardiography scans were obtained at baseline and 60 minutes after iodoacetamide administration. Plasma levels of the CK isoenzyme MB and troponin I were assessed to determine possible myocardial damage. RESULTS: CK activity in the iodoacetamide-perfused myocardium decreased to 0.5% of the original value and was accompanied by a reduction in peak systolic SR ( P < .0001), end-systolic strain ( P < .0001), and peak SRs of myocardial early and late filling waves ( P < .0001). Microscopy showed contracture without sarcomere disruption. Plasma levels of CK isoenzyme MB and troponin I did not change. CONCLUSIONS: Regional inhibition of myocyte energetics leads to both systolic and diastolic dysfunction by SR echocardiography, but the presence of a residual phosphotransfer protects microstructural integrity.     

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.     

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.