The relationship between serum creatine kinase brain-specific isoenzyme (CK-BB) activity immediately after birth and neurodevelopmental outcome at two and four years corrected age was studied prospectively in 45 preterm infants (less than 34 weeks gestation). Nine infants died during the neonatal period and one was lost to follow-up. Of the 35 children available for follow-up, seven had motor disabilities: four severe diplegia, two mild to moderate diplegia and one hemiplegia. No relationship existed between these motor disabilities and serum CK-BB activity after birth. There seemed to be a relationship between increased serum CK-BB after birth and low scores on the Bayley Scales of Mental Development, but this did not reach statistical significance. At the age of four years, four of the five survivors with high serum CK-BB activity after birth (greater than 25U/L) needed special schooling because of mental retardation. Increased serum CK-BB activity after birth may be associated with delayed mental development, but further study is needed, especially of asphyxiated infants.

By using highly sensitive enzyme immunoassay method recently developed, concentrations of the three forms of cytoplasmic creatine kinases (CK-BB, CK-MB and CK-MM) were determined in blood plasma samples serially taken from 18 patients, who received mitral valve replacement. Plasma CK-BB levels, that were 0.64 +/- 0.32 ng/ml at the beginning of anesthesia, increased sharply after reperfusion reaching peak levels (23.3 +/- 7.56 ng/ml) at 2 hours after reperfusion, and then decreased rapidly. The response of CK-BB in plasma was more rapid and sensitive than that of CK-BB or CK-MM. The CK-BB concentrations were significantly higher in coronary sinus samples than in arterial samples. These results suggest that the major portion of elevated plasma CK-BB levels at early phase after reperfusion were considered to be derived from the heart muscle.

The localization of creatine phosphokinase-BB (CPK-BB) in the neocortex of persons without mental pathology, who died of cardiovascular diseases (n-15) was investigated immunocytochemically. It has been found that there are several differences in the visualization of CPK-BB in neurons and astrocytes under various conditions of autopsy material treatment. The data obtained may attest to the existence of the immunochemically different forms of CPK-BB in the human brain. The appropriate conditions were defined for satisfactory visualization of CPK-BB on material of pathomorphologic collections.

Serum creatine kinase BB (CK-BB) on the 1st day of life was measured by radioimmunoassay in 37 very low birth weight (VLBW) infants, 14 severely asphyxiated infants and 24 controls. The 31 survivors from the two high-risk groups were followed up for 12 months or more. VLBW non-survivors (n = 14) had significantly higher mean CK-BB levels than survivors (n = 23), (P less than 0.05). However, if only survivors were considered, CK-BB was a poor discriminator of outcome in either study group. First day serum CK-BB is not a useful predictor of neurodevelopmental outcome in surviving high-risk infants.

This paper describes the purification of human creatine kinase BB with high specific activity (1,122 U/mg). The procedure used resulted in a protein yield of 5.4 mg (21% recovery) from 150 g of brain tissue. Two-dimensional electrophoresis and PAGE studies indicated that purified CK-BB might exist as native isoenzyme along with structural aggregates since the multi-banded appearance was reduced to a single band with sodium dodecyl sulfate treatment but not with 2-mercaptoethanol. Investigators are cautioned not to store brain tissue for prolonged periods of time before isolation of the isoenzyme as this may lead to protein redistribution with additional bands becoming evident on PAGE.

The serum concentration of creatine kinase (CK)-BB during and after coronary bypass surgery was measured by a sensitive and highly specific radioimmunoassay technique. All patients showed a distinct and temporary increase in CK-BB concentration shortly after the start of operation. Our results indicate that this increase has no connection with damage of the heart or the brain. Most probably the BB isoenzyme is liberated mainly from the saphenous vein as a consequence of the surgical manipulations during the heart operation.

Creatine kinase (CK; EC 2.7.3.2) catalytic activity in serum is widely measured in clinical chemistry practice and provides information for diagnosis and follow-up in many pathological conditions affecting heart, muscle, and brain. Depending on the organ involved, the predominant CK isoenzyme in serum varies. However, routine methods measure total CK catalytic activity, and standardized methods for doing so have been recommended by the International Federation of Clinical Chemistry and by several national scientific societies. Many commercial kits for those methods are now available. With use of a reference material for CK, commercial reagents can be compared with standardized methods, improving confidence in the results. Here we present a reference preparation of CK consisting of the BB isoenzyme purified from human placentae. We describe the procedure of purification and the properties of the lyophilized preparation of CK-BB, which has been certified by the Community Bureau of Reference of the Commission of the European Communities under the designation CRM 299. The preparation can be used to calibrate assays of the catalytic activity of CK-MM and CK-MB, as well as CK-BB.

High-resolution real time ultrasound scan is used for early diagnosis of PIVH in neonates. Furthermore, in term infants an increase of serum Creatine-Kinase BB (CK-BB) values has been found to be clinically useful for diagnosis and prognosis of several perinatal cerebrovascular injuries. Eighty-eight preterm infants with different grades of PIVH (I-IV) were studied by serial measurements of CK-BB levels in serum. No statistical correlation was found between enzymatic levels, severity of PIVH and perinatal risk factors (p greater than 0.5).

To increase the sensitivity and specificity and decrease the cost on determining the isoenzymes of creatine kinase (CK), including CK-MM, CK-MB, and CK-BB, we used the electrophoresis bath with different buffers to isolate the isoenzymes and used the fluorescent scanner to determine the content of each isoenzyme. We found that the concordance appeared in sensitivity and specificity in comparison with the MOPS electrophoretic system. There was a positive correlation between our method and MOPS, and the cost was much less than that of the MOPS.

The activity and content of the brain isoenzyme of creatine phosphokinase-BB (CPK-BB) were investigated in the peripheral tissues containing this isoenzyme (in the small intestine and heart) in health and schizophrenia. The decrease of CPK-BB activity and content in schizophrenic brain was shown before. The present work demonstrates the reduction of CPK-BB activity and concentration not only in the brain of mental patients but also in the intestine and heart tissues. A high correlation was discovered between the CPK-BB level in the brain and in the intestine of schizophrenic patients (r = 0.85).

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.

Purified MM and BB isoenzymes of human creatine kinase (CK) were labeled with Bolton-Hunter reagent. Labeling process did not affect their enzymic activity, although the labeled enzymes lost enzymic activity in storage. The labeled BB isoenzyme progressively changed its electrophoretic mobility, while labeled MM isoenzyme did not. Both labeled isoenzymes, however, maintained their immunoreactivity with their respective antisera. These results suggest that the enzymic and the immunoreactive sites of each CK isoenzyme are different and that BB isoenzyme, not MM isoenzyme, is electrophoretically unstable.

The creatine kinase (CK) activity in human skeletal and cardiac muscle submitted to QAE-Sephadex chromatography was found to distribute into three peaks (m and h I, II, III fractions). Characterization according to electrophoretic behaviour, approximate molecular weight, thermal stability and immunological properties unambiguously demonstrated the coincidence of fractions I and III with reference MM-CK and MB-CK isoenzymes, while both cardiac and muscular forms II proved to escape any assignment within the dimeric (M, B) model. A higher molecular weight than for reference CK and the absence of interaction with BB-antiserum resulted for both forms II, which on the contrary were found to diverge as for reactivity to MM-antiserum and stability characteristics. Quantitation of the CK forms, attempted on a limited number of samples, confirmed the expected relative levels of MM-CK and BB-CK and gave evidence for the presence of hII- or mII-CK amounting up to 30% of the total activity in cardiac and skeletal muscle, respectively. The results of the study both confirm a complexity greater than supposed for the CK system and point to the inadequacy of several of the commonly used analytical approaches to derive correct information.

Newly admitted psychotic patients often have elevations of serum creatine kinase (CK) enzymatic activity. Previous studies indicate that this increase consists of the muscle (MM) isozyme, and increases in the brain (BB) isozyme have not been observed. Using sensitive and specific radioimmunoassays that detect both active and inactive enzyme, we measured CK-MM and CK-BB in the serum and CSF of 100 patients with schizophrenia who were not newly admitted but whose conditions varied from acute to chronic to determine whether CK-MM or CK-BB appears in the CSF and whether CK-BB can be found in the serum of these patients. We found no unusual concentrations of either isozyme in CSF. We did observe a few elevations in serum CK-BB levels, but this test does not appear to be of diagnostic value for schizophrenic patients who are not newly admitted.

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.

The cerebrospinal fluid (CSF) creatine kinase (CK) activity and isozymic spectrum (EC 2.7.3.2) have been examined in patients with craniocerebral injuries of varying severity. The CK activity has been elevated in all the patients. Three isoforms have been detected: CK-BB, CK-MB, and CK-MM. CK-BB has been detected in all the patients in the presence of the total CK activity; this is explained by the isozyme release from the brain tissue during the injury and as a result of functional and structural impairment of the cellular membranes in intensification of lipid peroxidation. The CK-MM activity is due to blood admixture in the CSF and to impaired hematoencephalic barrier during the injury. The presence of CK-MB in the CSF of patients without cardiac symptoms probably results from a recombination of CK-BB and CK-MM isoforms and is of no diagnostic significance. Measurements of the total and isozymic CK activity in the CSF of patients with craniocerebral injuries may become a test for the laboratory diagnosis of the trauma severity and course.

Although euryhaline teleosts can adapt to environmental fluctuation of salinity, their energy source for responding to changes in salinity and osmolarity remains unclear. This study examines the cellular localization of creatine kinase (CK) expression in branchia of tilapia (Oreochromis mossambicus). Western blot analysis of muscle-type CK (MM form) revealed a high association with salinity changes, but BB and MB forms of CK in the gills of fish adapted to seawater did not change. With the use of immunocytochemistry, three CK isoforms (MM, MB, and BB) were localized in mitochondria-rich (MR) cells and other epithelial cells of tilapia gills. In addition, staining intensity of MM-form CK in MR cells increased after seawater transfer, whereas BB and MB forms did not significantly change. To our knowledge, this work presents the first evidence of CK expression in MR cells of tilapia gills, highlighting the potential role of CK in providing energy for ion transport.

Creatine kinase isoform CK-MB has been widely applied as a biomarker of myocardial injury. While a variety of methods have been used to measure CK-MB activity or mass in clinical laboratories, a CK-MB standard is needed to eliminate between-method bias. Because the in vitro expression of human creatine kinase generates three isoenzymes, CK-MM, CK-MB, and CK-BB, it is important to establish an effective method to purify the isoform CK-MB from the mixture. In this study, we aimed at using tandem affinity purification (TAP) to purify recombinant CK-MB protein and evaluate its value in clinical laboratories. After the optimized sequence coding CK-M and CK-B were synthesized, they were combined with TAP tags (6His and SBP) and inserted into a pRSFDuet vector; then, the constructed 6His-CK-M-SBP-CK-B-pRSF plasmid was transformed into Escherichia coli BL21 (DE3) for expression. After TAP, we obtained purified CK-MB protein. We also did recovery testing using the engineered CK-MB and standard CK-MB (Randox) at different concentrations, and the results suggested that the engineered CK-MB could be used as the reference material. Moreover, the stability study of recombinant CK-MB showed high stability during long-term storage at -80 °C. In conclusion, the TAP-purified recombinant CK-MB protein may be a much better and cheaper standard or reference material for clinical laboratories.

Circulating autoantibodies directed at creatine kinase (CK) BB isozyme are detected in plasma in the form of an immune complex (immunoglobulin CK BB) termed macro-CK type 1. Fourteen patients presented a falsely elevated CK MB isozyme fraction as measured by the immunoinhibition method; they were found to have IgG-CK BB complexes, which was considered to be indirect evidence of circulating anti-CK BB autoantibodies. No evident clinical association between the detection of this autoantibody in complexed form and autoimmune disease could be established, there was no significantly increased incidence of other autoantibodies, and there was no specific alteration in immunoglobulin and complement levels; however, the HLA haplotype A1,B8,DR3, which is known to be associated with autoimmunity, was present in five patients.

Myofibrils of chicken heart cells do not contain the electron-dense material of the so-called m-bridges which transverse the sarcomere in the M-region. It has been shown that the M-isoform of creatine kinase (MM-CK) which is mainly responsible for the m-bridge material is not expressed during differentiation of chicken heart cells. No transition from the embryonic BB-CK to the muscle-specific MM-CK takes place in chicken heart, thus no MM-CK is available for m-bridge formation. Here we report on chicken heart cells microinjected with either MM-CK protein or with poly(A+)RNA enriched for M-CK message. In both cases appearance of MM-CK within the M-band of heart myofibrils could be observed by immunofluorescence, indicating translation of the injected message as well as specific binding of the translation product to the M-band of myofibrils. The M-band protein myomesin which is regularly found in heart myofibrils served as specific marker for assembled myofibrils in double immunofluorescence experiments.

We measured creatine kinase (CK) levels of cord blood and evaluated them in relation to the degree of maturity and distress. The reference value of CK of cord blood in normal neonates was 209 +/- 99 U/l (mean +/- SD) and the percent values of the isoenzymes, CK-MM, CK-MB and CK-BB, were 86.7 +/- 6.2%, 5.1 +/- 2.6%, and 8.1 +/- 5.4%, respectively. There was a positive correlation between the birth weight and CK, CK-MM, CK-MB and CK-BB levels of cord blood. The CK and CK-MM levels of cord blood in the neonates who were prematurely delivered and small-for-date were significantly lower than those in normal controls. The CK and CK-MM levels of cord blood in the neonates with low Apgar scores, 6-4 and 3-0, were 172 +/- 74, 145 +/- 73 and 104 +/- 63, 92 +/- 55 U/l, respectively, which were significantly lower than those in the neonates with the high scores (10-7), 208 +/- 104, 183 +/- 92 U/l. The neonates with distress showed the low CK and CK-MM values, 76 +/- 12 and 61 +/- 8 U/l. The CK and CK-MM levels of cord blood tended to decrease with prolongation of labor, but did not differ from each other among the neonates delivered by different modes. These results suggest that the CK and its isoenzyme levels are good indicators for the degree of maturity of neonates and the severity of neonatal distress.

Stabilization with dithiothreitol, together with optimization of Mg2+ and EDTA concentrations in the reaction mixture and storage of the CSF samples for 24 hrs at +4 degrees C, has been carried out for the first time to improve the sensitivity of the technique for measuring the activities of creatine kinase (CK) and its isozymes in the CSF of 38 patients during the first 24 hrs of craniocerebral injury. Dithiothreitol promoted mostly an increase of the CK-BB isozyme; the content of this isozyme in the cerebral tissue is rather high, and it is considered as the cerebral tissue marker. Generally stabilization augmented the CSF total CK activity by 2.2 times on an average (from 50.6 to 113.2 U/l), and the CK-BB activity by 3.5 times on an average (from 21.6 to 74.8 U/l). The method used in this work will help improve the diagnostic sensitivity of the CSF CK-BB measurements, this being significant for the detection of the cerebral tissue minute injuries after traumas and neurosurgery.

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.

By means of the immunoprecipitation method significantly higher activities of creatine kinase BB isoenzyme were measured in the sera of neonates with CNS symptoms than in the sera of healthy or sick neonates without CNS symptoms. The activity of CK-BB inversely correlated with the one-minute Apgar score. These results suggest a leakage of CK-BB from the damaged CNS tissue into the blood circulation. Determination of CK-BB might be helpful in the assessment of perinatal brain damage.