To estimate the incidence of variant creatine kinase (CK)isoenzymes, 1,191 unselected patients were screened by means of CK:CK - MB ratio. When CK - MB was higher than 20% of the total CK in the immunoinhibition test, and a myocardial infarction could be excluded, a CK-isoenzyme electrophoresis was performed. We found macro-CK-BB-emia in 13 patients, but only three of them had elevated total CK activity. All patients but two were at the typical age of over 60 years; two males were 2 and 9 years old. The clinical diagnosis in macro-CK-BB-emia was predominantly cardiocerebrovascular insufficiency due to arteriosclerosis. In 16 other patients, the presence of an isoenzyme migrating cathodically to CK-MM was found. In this group, a malignant metastatic tumor was found in eight cases, the rest predominantly suffered from severe liver disease. Presence of both variant CK isoenzymes may lead to diagnostic or therapeutic errors due to the altered CK:CK-MB ratio. According to our studies, a variant CK isoenzyme occurs in 2.6% of hospitalized patients, but this will lead to problems diagnosing acute myocardial infarction only in the group with elevated total CK (0.42% of our patients).

Creatine kinase isoenzyme BB was determined in cerebrospinal fluid (CSF) in 79 preterm neonates using an original enzyme-linked immunosorbent assay. The criterion for inclusion was an Apgar score of 7 or less at 5 min of life. Neurological examination was performed on day 2 and day 5 of life. CSF was obtained on the same days. Lumbar puncture was performed on 41 of these babies on day 2 and in 39 on day 5 of life (one baby underwent lumbar puncture twice). All babies had clinical features of hypoxic-ischemic encephalopathy (HIF) which was classified according to Sarnat and Sarnat. The control group consisted of 90 asphyxiated term babies and 30 adults without CNS pathology. The concentration of CK-BB in cerebrospinal fluid (mean +/- SD) was significantly higher (p < 0.0005) in preterm (168.0 +/- 2) than in term babies (29.0 +/- 3.1) and healthy adults (5.3 +/- 1.2). Our results demonstrate the possibility of using the classification system of Sarnat and Sarnat for assessment of the severity of brain damage not only in term, but also in preterm babies. Neonates with HIE stages II and III showed markedly higher CK-BB values than those with HIE I on day 2 (p < 0.025) and day 5 (p < 0.05) of life. CK-BB values were markedly higher in preterm babies with none of some primitive responses (head turning, Babkin's reflex, palmar grasp). The mean concentration of CK-BB was higher in neonates with retarded psychomotor development compared with those with normal development (p < 0.05) on day 3, and after 6 and 9 months. At 12 months of age no significant difference in median CK-BB concentration was detected between neonates with normal and developmental disturbances.

CONCLUSIONS

The biochemical analyses of the total enzymatic activities of CK. PGAM in innervated cultures with advanced morphological maturation showed a progressive increase up to 60 days after innervation, fetal isozyme patterns of CK-BB and PGAM-BB at early stage of innervation and almost complete transition of CK and PGAM from BB to MM isozymes in more advanced stage of innervation (Figs 1, 2). Time course of G6PD activity in parallel cultures showed a higher activity in early stages of myogenesis(myoblastmyotube: 121.4+/-10 nM/min/mg prot) and in early phase of innervation (30 days after innervation: 109.66+/-26.10 nM/min/mg prot) with a decline of activity in advanced stage of innervation (60 days after innervation: 82.33+/-36.55 nM/min/mg prot) A progressive increase of AM activity in mid (30 days after innervation: 1623.66+/-10.96 pM/min/mg prot) and late stage of innervation (45 days after innervation: 2150.33+/-568.27 pM/min/mg prot) in comparison with aneural (536+/-107.39 pM/min/mg prot) was also found our study suggests these enzymes as biochemical markers of functional innervation of cultured human muscle fibers.

An atypical creatine kinase (CK) isoenzyme migrating cathodically to CK-MM in the electrophoresis was observed as response to the embolization of the right hepatic artery in two patients with liver metastasis. The time course of the atypical CK release was compared to that of tumor secretion products, i.e., insulin and 5-hydroxy-in-dol-acetic acid. The CK isoenzyme probably reflects a mitochondrial breakdown of the metastasis in question. The influence of the treatment on liver function was characterized by a marginal augmentation of ASAT and ALAT and by a small but significant leakage of mitochondrial glutamate dehydrogenase. In addition to the atypical CK, CK-BB, and CK-MB isoenzymes were found in the serum of the patient with primary carcinoid carcinoma.

In the present series of experiments, we studied the effects of developmental neural control on morphological differentiation and on the activity of muscle-specific (creatine kinase, CK; phosphoglycerate mutase, PGAM; phosphorylase, PPL; and phosphofructokinase, PFK) and non-specific (acid maltase, AM; glucose-6-phosphate dehydrogenase, G6PD), human muscle enzymes in de novo innervated muscle cultures. Following innervation of muscle cultures, we noted an increase in the activity of CK, PPL, PFK and AM along with a reduction in G6PD activity. There was also a change of the CK isoenzymes present in the myotubes, i.e. BB and MB are the major isoenzymes in non innervated cultures, but MM becomes predominant following innervation. In the case of PGAM, the only isoenzyme present in the non innervated cultures was BB while the MM isoform appeared only after a prolonged innervation period in most cases--with the exception of AM--these changes in enzyme activity and in the type of isoenzymes present, demonstrate that innervated cultures are more similar to mature muscle. This maturation of enzymatic activity correlates well with the morphological maturation of the myotubes observed following innervation. Such innervated cultures therefore represent a better model with which to study the morphological and biochemical abnormalities associated with neuromuscular diseases.

We studied electrophysiological, oxygen metabolic, and histological variables in dogs to establish the reliability and safety of partial brachiocephalic perfusion (PBP) under hypothermic cardiopulmonary bypass (CPB) at 23 degrees-25 degrees C. Sixteen mongrel dogs were divided into two groups. Six (control group) underwent typical hypothermic CPB for 90 min, and 10 (PBP group) underwent PBP under hypothermic CPB for 90 min. During core cooling on the CPB, a progressive reduction in voltage and slowing of frequency of the electroencephalogram (EEG) was observed. At around 23 degrees C nasopharyngeal temperature the tracing became almost flat and remained so throughout the hypothermic CPB or the PBP under hypothermic CPB. Consistent recovery of the EEG was, however, observed during the period of rewarming on the CPB, and the voltage and frequency of the EEG recovered to control levels on weaning off CPB at 36 degrees C in both groups. In the PBP group, the cerebral arteriovenous oxygen (AVO2) difference was 12.4 +/- 4.0 vol% before beginning the CPB, and it was 5.6 +/- 2.7, 5.7 +/- 3.1, 5.4 +/- 3.3, and 4.9 +/- 2.9 vol% at 10, 30, 60, and 90 min respectively after commencement of the PBP under hypothermic CPB. The cerebral AVO2 difference measured 10 min after commencement of the PBP was significantly less than that in the control group (P less than 0.05), but otherwise there were no significant differences between cerebral AVO2 differences in the two groups. Concentration of serum creatine kinase-BB (CK-BB) gradually increased in proportion to the duration of CPB in both groups.(ABSTRACT TRUNCATED AT 250 WORDS)

Kidneys from both normal and vitamin D-deficient rats were found to show changes in responsiveness to vitamin D metabolites during postnatal development, correlated with the concentrations of the specific receptor for 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] or the specific binding protein for 24R,25-dihydroxyvitamin D3 [24,25(OH)2D3]. Cytosol preparations from kidneys of vitamin D-deficient rats, in the second week of life, contained specific binding proteins for 24,25-(OH)2D3. From the fourth week of life, specific receptors for 1,25(OH)2D3 were predominant. In the third week after birth, both the receptor for 1,25(OH)2D3 and the 24,25(OH)2D3 binding protein were present. We have used a sensitive parameter for vitamin D action, the stimulation of creatine kinase BB (CKBB) activity, to measure the response of kidneys from vitamin D-deficient or normal rats. In the first days of life of vitamin D-deficient rats, the kidneys did not respond to either vitamin D metabolite; in the second week of life, there was stimulation of renal CKBB only by 24R,25(OH)2D3; beginning in the fourth week of life, only 1,25(OH)2D3 stimulated renal CKBB. However, during the third week of life, CKBB activity was increased by both metabolites. In normal animals, which showed a lower CK activity at all ages, the response was similar to that in vitamin D-deficient animals but the peak was achieved a few days later. The stimulation of CKBB by vitamin D metabolites occurred in all the zones of the kidneys. An increase in renal CKBB by 1,25(OH)2D3 was also detected immunohistochemically. The increase of CKBB activity caused by the two vitamin D metabolites at different stages of development, closely correlated with changes in the presence of the 1,25(OH)2D3 receptor or the 24,25(OH)2D3 binding protein, suggests a specific role for each metabolite during renal development.

Creatine kinase is a key enzyme for energy metabolism of contraction and relaxation in skeletal muscle. This enzyme also correlated to mitochondrial oxidative phosphorylation. Distribution of this enzyme is quite wide, including skeletal muscle, myocard, central nervous system, and smooth muscle. Creatine kinase has three main isoenzymes; CK-MM, CK-MB, and CK-BB. Mitochondrial isoenzyme (CKm) is the fourth isoenzyme migrating electrophoretically toward the cathode. Moreover serum creatine kinase isoenzyme (mainly CK-MB and CK-BB) and lesions in the myocard, skeletal muscle, central nervous system, gastrointestinal system, renal and urogenital systems, or acute psychosis, intoxication, pregnancy, labor, and cord blood are also explained. A compendium on CK-BB is also done.

Creatine kinase activity and its isoenzymatic profile in rat intestinal mucose during normal development have been studied. Creatine kinase enzymatic activity increased stepwise during fetal development and the first week of life. An isoenzymatic pattern of exclusively CK-BB types occurred in all segments of the digestive tract during the early fetal stage. The isoenzyme profile of creatine kinase in the esophagic tissue with advancing maturation of the fetus shifted in the same way as in adults, with preferential concentration of CK-MM. However, CK-BB continued to be the main isoenzyme in the rest of the digestive tract. Our results show that rats are particularly suitable for experimental studies of intestinal creatine kinase isoenzymes.

Creatine kinase (CK; E.C. 2.7.3.2) is an important enzyme that catalyzes the reversible transfer of a phosphoryl group from ATP to creatine in energy homeostasis. The brain-type cytosolic isoform of creatine kinase (BB-CK), which is found mainly in the brain and retina, is a key enzyme in brain energy metabolism, because high-energy phosphates are transferred through the creatine kinase/phosphocreatine shuttle system. The recombinant human BB-CK protein was overexpressed as a soluble form in Escherichia coli and crystallized at 22 degrees C using PEG 4000 as a precipitant. Native X-ray diffraction data were collected to 2.2 A resolution using synchrotron radiation. The crystals belonged to the tetragonal space group P43212, with cell parameters of a=b=97.963, c= 164.312 A, and alpha=beta=gamma=90 degrees. The asymmetric unit contained two molecules of CK, giving a crystal volume per protein mass (Vm) of 1.80 A3 Da-1 and a solvent content of 31.6%.

The mitochondrial isoenzyme of creatine kinase (MiMi-CK) was separated by affinity chromatography on Cibachrome-Blue-Sepharose (Sepharose-Blue, Pharmacia). While the soluble CK isoforms (BB-CK and MM-CK) were specifically eluted by raising the pH of the column buffer from pH 6.0 to pH 8.0, MiMi-CK remained bound under these conditions but was specifically eluted by subsequent addition of ADP to the pH 8.0 buffer. This one-step method allows a fast and efficient separation of MiMi-CK from MM-and BB-CK isoenzymes and at the same time an enrichment of MiMi-CK by about 50-fold. Since MiMi-CK can be assayed separately after isolation by affinity chromatography on Sepharose-Blue, this method may be of clinical importance.

Isoenzymes of creatine kinase (CK-MM and CK-BB) of samples of tissues from human skeletal muscle and human brain tissues were highly purified. Anti-CK-M and anti-CK-B were prepared by immunization of goats. The obtained antibodies were used in a modified immunoprecipitation method for measuring the creatine kinase subunits of CK-M and CK-B in serum.

The creatine kinase isoenzymes CK-MM, CK-MB and CK-BB from Rhesus monkey heart and brain tissue homogenates were separated chromatographically. Thereafter it could be demonstrated that the activity of the simian subunit CK-M is completely inhibited by anti-inhibitory-CK-M serum. Thus control sera from simian tissue are in principle suited for quality control in an immunological determination of creatine kinase-MB. The intra-assay variance and interassay variance were n = 56, -/x = 29.2 U/1, SD = 3.2 U/1, CV = 11.1% and n= 12, -/x = 166.7 U/1, SD = 5.0 U/1, CV = 3.0% respectively. It is desirable to develop control sera with catalytic concentrations of creatine kinase-MB in a lower range.

Sera from eight patients with biopsy-proven rectal carcinoma were assayed for creatine kinase (CK). Two of the patients had enzyme activity: one with the BB and the other with an MB isoenzyme. We think that our results may be of interest because the detection of anomalous isoenzymes of CK in serum ma serve as a diagnostic marker or as an indicator of the effectiveness of treatment.

Creatine kinase BB is the main CK isoenzyme expressed in murine hybridoma cells as assessed by agarose gel electrophoresis whereas it was found neither in splenocytes nor in myeloma cells. The presence of CK BB is a constant finding in all 10 murine hybridomas examined to date irrespective of the specificity of the secreted antibodies.

A sensitive sandwich-type enzyme immunoassay method for measurement of brain-type isozyme of human creatine kinase (CK-BB) was developed using purified antibodies specific to the B subunit. The assay system consisted of polystyrene balls with immobilized antibody F(ab')2 fragments and the same antibody Fab' fragments labelled with beta-D-galactosidase from Escherichia coli. The assay was highly sensitive and 1 pg of CK-BB was measurable. The assay was specific to the B subunit of creatine kinase (CK-B), and it cross-reacted about 25% with CK-MB, the heart-type isozyme. However, the assay showed no cross-reactivity with CK-MM, the muscle type-isozyme or with neuron-specific gamma gamma enolase. Coefficients of variation in within-run and between-run precision studies for serum CK-B were less than 8%. Serum CK-B levels in healthy adults of various ages (16-59 yr old) ranged from 0.25-1.44 ng/ml, whereas the CK-B concentrations in children (less than 10 yr old) were relatively high, ranging from 1.3-7.4 ng/ml. The CK-B levels in the cerebrospinal fluids (CSF) could be determined by the present method, and they ranged from 0.10-0.76 ng/ml in the samples from patients with non-neuronal disorders. Determination of immunoreactive CK-B in the extracts of various human tissues confirmed previous reports that CK-B was distributed at high concentrations in the central nervous tissue, prostate, uterus, bladder, gastrointestinal tract and heart muscle.

A improved method for isolation and purification of CK-BB from human brain is introduced. CK-BB of fraction IV was purified to approximately 306.9 fold as compared with homogenate of human brain. The specific activity was 322.2 IU/mg protein. The recovery of creatine kinase activity was 52.8%. This fraction was analyzed by SDS-PAGE. It showed a single major band with an estimated molecular weight of 43000 when it stained with coomassie blue. Purity of fraction V was much better than fraction IV, but some CK activity was lost.

Serum creatine kinase activity was measured during the first post-natal days in healthy full-term and premature infants. The CK isoenzymes (CK-MM, MB and BB) were separated using ion-exchange column chromatography. Total CK activity is lower for premature infants than for full-term infants at the same time-periods. However the separation of the CK isoenzymes shows that the same normal values for the CK-BB (expressed as U/l) may be used for the two groups of infants.

Resonance energy transfer was measured between the active site domains of the brain isozyme of creatine kinase (CK-BB). The reactive thiol near the active sites, one on each subunit of the dimeric protein, was derivatized using 5-[2-[iodoacetyl)amino)ethyl]aminonaphthalene-1-sulfonic acid (AED), 2-[4'-iodoacetamidoanilino]naphthalene-6-sulfonic acid (AANS) and 5-iodoacetamidofluorescein (AF). Suitable donor/acceptor protein conjugated hybrids were prepared by controlled kinetics producing CK-BB-AED/AF and CK-BB-AANS/AF. Transfer efficiencies, measured from the quenching of the donor lifetime and steady-state sensitized acceptor emission, ranged from 0.10 to 0.17. From determination of the donor/acceptor overlap integrals, donor quantum yields and attempts to delimit the orientation factor using steady-state and phase-resolved anisotropy measurements, it was found that a suitable estimate of the range between the active sites was between 45 and 57 A. This range is similar to that reported previously for the muscle isozyme of creatine kinase (Grossman, S.H. (1989) Biochemistry 28, 4894-4902) but is a significantly greater distance than detected for the hybrid, myocardial specific isozyme (Grossman, S.H. (1983) Biochemistry 22, 5369-5375).

32 newborn infant's umbilical cord blood CK-BB activity was tested at birth. 14 of them had a superior to 7 Apgar score at first and fifth minutes, whereas 18 newborn infant Apgar score was inferior to 7. The CK-BB activity was significantly higher in newborn infants having a low Apgar score at birth. A low Apgar score at first and fifth minutes together with a high CK-BB activity must be considered a nervous trouble signal and a warning in order to look after children's psychomotor skills more strictly and individual neurological damage.

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.

1. We compare the Roche ion-exchange column chromatography and the Merck antibody inhibition reaction for CK-MB fractionation in 51 sera. Measurements of total CK and CK-MB activities must be done under the same conditions for each method in order to correlate the results. 2. A decisional value must be used for the interpretation of CK-MB results. We have used 10% for the inhibition assay and 3% for the chromatography procedure. The use of a percentage should be preferred to use of CK-MB activity alone. 3. When the % of CK-MB was established for the 51 patients only 4 results disagreed between the two methods. Three of these could be explained by a lack of sensitivity of the column chromatography procedure. 4. The antibody assay produces reliable results. Since CK-BB and CK-MB are simultaneously measured, the method is therefore prone to interference by CK-BB when present in serum. The assay is greatly affected by the presence of adenylate kinase in serum. It is not necessary to run a serum blank with this procedure when the serum in pre-incubated for 7 minutes with the reagents. 5. The Roche method also produces reliable results but offers less sensitivity when total CK remains in the normal range. The procedure is much less affected by the presence of adenylate kinase.

In the chicken, two creatine kinase-type B (B-CK) isoproteins, Ba- and Bb-CK, both of which are derived from a single copy gene by alternative splicing, dimerize in neural tissues. The two isoproteins contain distinct N-terminal portions, but their functional difference remains unknown. We investigated the binding affinities of Ba- and Bb-CK to heparin, hyaluronan and chondroitin sulfates, and examined the influence of these glycosaminoglycans on enzyme activity. Chicken retinal samples analyzed by Western blotting and amino acid sequence study after two-dimensional gel electrophoresis showed that heparin binds Bb-CK, but not Ba-CK, while hyaluronan and chondroitin sulfates showed no interaction with either isoprotein. Using fusion proteins covering the distinct N-terminal portions, we also showed that heparin did not react with the N-terminus of Ba-CK, but did react with that of Bb-CK. Site-directed mutagenesis of basic amino acids found in the N-terminal portion of Bb-CK identified three basic amino acids critical for this binding. Furthermore, heparin dose-dependently inhibited the enzymatic activities of Ba-CK; Bb-CK activities were less intensely inhibited. Hyaluronan and chondroitin sulfates had no effects on the activities of these enzymes. Thus, the N-terminal portion of B-CK is critical to mediate its affinity to heparin and control enzyme activity, which may be important for regulating energy metabolism in neural tissues such as brain and retina, unique organs abundant in heparan sulfates.