This report describes the procedures for isolation of creatine kinase BB isoenzyme (CK-BB) from human placenta on preparative polyacrylamide gel electrophoresis. 2.5 mg of CK-BB was purified from a 100-g portion of the human placenta, which had a mean specific activity of 957 kU/g and a mean yield of 16%. The placenta CK-BB exhibited single protein bands on several electrophoretic techniques. In addition, both of the placenta and brain CK-BB preparations were individually iodinated and the identical immunological properties of both the CK-BB preparations were confirmed in radioimmunoassay.

We hypothesized that maternal serum levels of the isoenzyme creatine kinase (CK)-BB, which is highly expressed in the placenta, may be elevated during the early second trimester in gestations destined to deliver prematurely or of a small-for-gestational-age infant (birthweight below 10th percentile). To test this hypothesis, we compared maternal serum CK-BB levels and percentage of CK-BB over total CK, in 69 normal pregnancies (delivering at term of appropriate-for-gestational-age infants) with those of 25 cases complicated by preterm delivery at < or = 34 weeks (n = 14), of a small-for-gestational-age infant (n = 8), or both (n = 3). No differences were present in maternal serum CK BB levels between normal and complicated pregnancies. Moreover, no correlation was found between gestational age at delivery and CK BB levels (r = 0.03; p = 0.7).

We examined the sensitivity of bioluminescence for the determination of very low concentrations of creatine kinase brain-type subunit (CK-BB) in serum and in cerebrospinal fluid. To optimize the sensitivity of CK-isoenzyme assays and eliminate possible sources of error, we separated the isoenzyme fractions by using inhibiting anti-MM and precipitating anti-MM and anti-BB antibodies. The results with the bioluminescence assay correlated with spectrophotometric values such that r = 0.97 for the total CK activity and r = 0.98 for the CK-B activity. The reproducibility of the present method was comparable with the spectrophotometric method and was even better at low enzyme activities. The within-series precision for assay of total CK activity at 2 U/L corresponded to a CV of 9%; at 13 U/L the CV was 5.8%. All the assays were carried out at 25 degrees C. Even at this low temperature, CK activities as low as 0.2 U/L could be determined. In eight patients without any evidence of cerebral cell damage, total CK activity in cerebrospinal fluid was x = 1.05 +/- 0.6 U/L, and CK-BB activity was x = 0.7 +/- 0.4 U/L. In sera of these patients CK-BB activity was x = 0.6 +/- 0.5 U/L. Differences in CK and CK-BB activities in four patients with transient or progressive brain-cell damage are discussed.

Caffeine and retinoic acid were examined for effects upon limb morphogenesis and upon creatine kinase (CK) as a measure of limb myogenesis. Caffeine at 200 mg/kg, i.p., on E11 produced a low level of forelimb (1.2%) and hindlimb (2.0%) defects. Retinoic acid, at 50 mg/kg given orally as an oily suspension, induced a high level of reduction deformities. Hindlimbs (100%) were affected more than forelimbs (88%). Limbs (E16) were examined for CK isoenzymes using DEAE-Sephacel column chromatography. Untreated limbs had 88.04% skeletal muscle (MM), 6.98% hybrid (MB) and 5.08% brain (BB) CK isoenzyme. Caffeine had no effect. However, retinoic acid increased MM-CK to 92.67%, and decreased BB-CK to 2.24%. This is the first evidence that suggests that retinoic acid may modify the phenotypic expression of developing muscle.

We used a new radioimmunological (RIA) kit for the assay of B subunit of creatine kinase enzyme (CK). This RIA system uses a specific antisera against the B subunit as ligant, human CK-BB labelled with 125I as tracer, and purified human CK-BB isoenzyme as standard. The mean (+/- SD) sensitivity obtained was 0.25 +/- 0.16 ng/tube and the between assay variability was about 9-10%. Serum levels of 113 normal subjects was not normally distributed. The 95% of values was found below 5 ng/ml. This new RIA is usefull in clinical practice when serum levels of CK-BB isoenzyme must be determined. This method is quickly and it is characterized by a good degree of precision, but the CK-MB isoenzyme cross-reacts for about 40% in this RIA system. Therefore, for the clinical diagnosis by means of this RIA it is necessary to rule out the concomitant elevations of serum CK-MB values.

Creatine kinase (CK) activity and isozyme patterns were assessed in newborn and adult rat anterior tibial muscle in response to denervation. Total CK activity was low in the control neonatal muscle, gradually increasing to the adult level within 1 month. Denervation prevented this normal increase, and, therefore, CK activity was reduced to 25% of control at 2 months. In the denervated adult muscle, total CK activity decreased to 50% of control within 3 weeks and remained at that level. Denervation of neonatal muscle resulted in a greater conservation of MB isozyme compared with controls. The alteration in BB isozyme expression was even more dramatic with a 33-fold difference expressed at 2 months in terms of percent total CK in denervated vs. control muscle. In denervated adult muscle, MB and BB isozyme activities increased gradually, attaining levels 3-fold and 13-fold, respectively, above control muscle at the end of the experimental period.

This work describes the action of the lysosomal enzymes arylsulfatase A (EC 3.1.6.1) and sialidase (EC 3.2.1.18) on human creatine kinase (CK, EC 2.7.3.2) isoenzyme BB. The isoenzyme, which gives a positive reaction with the periodic acid-Schiff reagent, contains 12 molecules of sulfate and two molecules of sialic acid per molecule. On treatment with arylsulfatase, CK-BB lost enzyme activity but retained immunoreactivity, its isoelectric point was altered, and it was partly bound to a "Glyco-gel" affinity column. On treatment with sialidase, the isoenzyme lost activity, its immunoreactivity was decreased by 70%, and the inactivated CK-BB would not bind to either "Glyco-gel" or concanavalin A. We propose that the sulfate groups are involved in maintaining the integrity of the active site of the enzyme but are not involved in antigenic recognition sites on the molecule. Sialic acid plays an important role in both the structural pattern of the antigenic determinant and the active site of CK-BB.

Creatine kinase (CK) is a marker of muscle damage and pathology present as multiple tissue-specific circulating isoforms. CK is often measured using enzyme activity assays that are unable to distinguish these isoforms. We have developed an immunoassay specific for the MM isoform of CK, found predominantly in skeletal muscle, which uses very small volumes of plasma (1-2 microL). A sandwich enzyme-linked immunosorbent assay (ELISA) for CK-MM was developed using isoform-specific antibodies. Cross-reactivity with CK-BB and MB isoforms was also assessed. The ELISA was validated using plasma samples from a group of athletes, and the measured CK-MM concentrations were correlated with CK enzyme activity assays measured by a contractor using the same samples. The CK-MM ELISA has a limit of detection of 0.02 ng/mL, an IC(50) of 2.3 ng/mL, and 5.8% cross-reactivity with CK-MB. CK-MM concentrations measured using this assay correlate well (p<0.0001, Spearman r=0.89) with enzyme activity assays. The CK-MM-specific ELISA can be used to help assess skeletal muscle damage independent of enzyme activity or interference from other CK isoforms, leading to more precise studies of muscle biology.

OBJECTIVE

We hypothesized that the anaerobic ATP synthesis mediated by the creatine kinase/phosphocreatine (CK/PCr) system is sexually dimorphic during maturation and aging of the rat heart.

BACKGROUND

Gender-related morphological and functional differences in cardiovascular aging seem to explain the greater longevity of mammalian females, including women.

METHODS

By means of heart CK specific activity and cytosolic CK isoenzyme analyses we studied 46 male and female Wistar rats of similar weight divided in groups of 200, 250, and 300 g of body weight.

RESULTS

No sex differences were observed in heart weight and post 27,000 x g heart protein content at any studied weight. Heart/body weight ratios did not show any significant gender difference along the study. Differences of heart CK specific activity were found only at 257 +/- 6 g of rat body weight due to a decrease of the male enzyme activity. The female heart showed a larger variety of cytosolic CK isoenzymes at any studied weight. Heavily catalytically stained BB-CK type cytosolic isoenzymes were consistently found in the heart of rats of either sex at the studied weights, contrarily to the accepted view of CK tissue specificity.

CONCLUSIONS

In this work, significant gender differences were mainly found in the patterns and number of catalytical cytosolic CK cardiac isoforms. Regarding the alternate anaerobic mechanism of ATP production, these differences may explain in part the sex differential susceptibility to hemodynamic compromise in response to cardiovascular stress, in favor of females.

We have developed a method for identifying IgG-complexed creatine kinase (CK; EC 2.7.3.2) (IgG-CK) and IgA-complexed CK (IgA-CK) in serum. We used immobilized Protein G to bind IgG-CK and immobilized jacalin to bind IgA-CK, leaving noncomplexed CK in solution. The noncomplexed CK and total CK were measured kinetically. The results are reported as CK bound to immobilized Protein G and CK bound to immobilized jacalin. We validated the method by using sera determined immunochemically to contain IgG-CK, IgA-CK, mitochondrial CK (CKmt), and free CK-BB. We demonstrated concomitant binding of CK and approximately 99% of IgG, and of CK and approximately 87% of IgA. For CK bound to immobilized Protein G and to immobilized jacalin, intra- and interassay precisions ranged from 2.5% to 9.6%, and detection limits were less than 9 318 U/L in 40 sera containing IgG-CK, and CK bound to immobilized jacalin ranged from 10 to 59 U/L in eight sera containing IgA-CK. These ranges represent the activities of immunoglobulin-bound CK in the sera. In 13 sera containing CKmt and in eight sera containing free CK-BB, the binding of CK was less than 9 U/L. Evidently, this method is useful for identifying IgG-CK and IgA-CK in serum.

OBJECTIVE

The aim of this study was to test the utility of serum creatine kinase (CK) isoenzyme determinations as a marker of tissue injury in preterm newborns with respiratory distress syndrome (RDS).

METHODS

Two groups of neonates were studied, 26 suffering from RDS who required mechanical ventilation and 20 healthy newborns with gestational ages, hours of life and birth weights similar to the first group. The activity of CK and its isoenzymes was determined in the bronchial aspirate and serum samples that were obtained before and 24 hours after exogenous surfactant therapy. The isoenzymes were separated by electrophoresis on agarose gel and their activity expressed as a percentage of the total CK. Total proteins were quantified in the bronchial aspirate and CK enzymatic activity expressed in U/mg of protein x 10-3.

RESULTS

The CK-BB isoenzyme was significantly increased (p < 0.001) in the serum of infants with RDS compared with the control group. In the bronchial aspirate, the isoenzymatic study showed that the CK-BB isoenzyme represented 98-100% of the total enzymatic CK activity.

CONCLUSIONS

The study shows significant differences in the CK isoenzyme patterns of neonates with RDS compared to controls. An increase in serum levels of the CK-BB isoenzyme could be an effective marker of tissue injury in lung disease in the newborn.

The purpose of this study is to investigate the relationship of creatine kinase (CK) and its isoenzyme levels in the newborn to the mode of delivery, time interval from birth (divided in four 6-hour time periods), parity and sex of the neonates. During the 1st postpartum day, serum levels of CK and its isoenzymes (CK-MM, CK-MB, CK-BB) were determined from 115 healthy full-term neonates born consecutively either by spontaneous vaginal delivery (VD, n = 85) or by elective cesarean section (CS, n = 30). The multiple regression analysis was applied. Total CK levels were positively correlated with VD (p < 0.0003). This was mainly attributed to a rise in the CK-MM activity which presented a similar pattern to CK. CK-MB activity was also positively correlated with VD. In contrast, CK-BB was negatively correlated to the postpartum time period. Neonatal sex and parity did not influence CK and its isoenzyme levels significantly. In conclusion, VD contributes significantly to an increase in CK levels during the 1st day of extrauterine life.

OBJECTIVE

The glycogen phosphorylase isoenzyme BB (GPBB), as an ischemic marker, has not yet been investigated after elective percutaneous coronary intervention (PCI). ose aim of the study was to monitor GPBB, creatine kinase myocardial isoform (CK-MB) mass) and troponin I (TnI) value after PCI in correlation with ischemic incidents.

METHODS

Forty-two consecutive patients undergoing elective PCI were included in the study. Baseline blood samples and two more after the PCI (3 and 24 hours) were taken. The significance of cardiac markers in twenty-ththe stable patients with baseline values of CK-MB mass and TnI below the upper reference limit (URL) was evaluated based on ischemic incidents after PCI.

RESULTS

TnI value was the only biomarker that was statistically significant at 3 and 24 hours after PCI in group of 23 stable patients. An overall comparisonthe biomarkers of 18 patients without and five patients with ischemic incidents displayed significant differences only for the baseline GPBB (p=0.019) and CK-MB mass 24 hours after PCI (p=0.034). Ischemic incidents were independently predictable only based on overall CK-MB mass measurements (OR=1.680, p=0.041) and particularly GPBB at baseline (OR=1.899, p=0.008) and CK-MB mass 24 hours after PCI (OR=2.111, p=0.022).

CONCLUSIONS

Only significant increases in TnI were observed after elective PCI with ischemic incidents predicted using GPBB and CK-MB mass measurements.

The present study aimed to clarify the effects of Agaricus brasiliensis KA21 (i.e., Agaricus blazei) mushroom on circulatory function. Spontaneously hypertensive rats (SHRs) were fed 10% A. blazei-containing pellets (agaricus group) or normal pellets (control group) for 5 weeks from 6 to 11 weeks of age. For Experiment 1, tail blood pressure and heart rate were measured in the conscious SHRs. For Experiment 2, echocardiographic and blood biochemical measurements were performed in the anesthetized SHRs. In Experiment 1, blood pressure and heart rate were significantly lower in the agaricus group compared with the control group throughout the observation period. In Experiment 2, the agaricus group also showed a significant decrease in cardiac output accompanied by a decrease in heart rate and an increase in early and late ventricular filling velocity (E/A ratio). Moreover, levels of escape enzymes such as creatine kinase (CK), CK-BB, CK-MB, asparate aminotransferase, lactate dehydrogenase, and aldolase were significantly lower than in the control group. We concluded that the ingestion of feed containing A. brasiliensis KA21 can improve hypertensive cardiovascular hemodynamics by decreasing the working load of the heart, presumably by lowering the sympathetic nervous tone in SHRs.

CK-BB activity increases after hypoxic conditions associated with central nervous system disease. Fifty-four high-risk newborn babies were studied with serial measurements of CK-BB activities during the first 3 postnatal days. These values were correlated to their early neurologic outcome at 5 months of age. Data were carried out using a new and very sensitive method for estimating CK-BB activity and showed a good correlation between enzymatic values and the presence of persistent neurologic abnormalities (P less than .05). We conclude that early CK-BB determinations can be used as indicator of neonatal brain damage.

Chemical parameters comprising urea and creatinine nitrogen, cations (Na+, K+, and Ca2+), chloride, phosphorus, protein, cholesterol and enzymes, aminotransferases, alkaline and prostatic acid phosphatases, gamma-glutamyltransferase, creatine kinase, and lactate dehydrogenase were ascertained for semen from groups A (vasectomized), B (oligospermic), and C (normospermic) men, 19 to 55 years of age. Of the parameters, the vasectomized group underwent definite depressions in potassium ion, alkaline phosphatase, aspartate aminotransferase, gamma-glutamyltransferase, and lactate dehydrogenase as compared with the normospermic group; the last three enzymes and, possibly, the urea-creatinine ratio were decreased for the oligospermic group vs. the normospermic men. In the comparison of groups A and B, only the decrements in alanine aminotransferase and lactate dehydrogenase were statistically significant. In corroboration of past reports, CK-BB comprised the main isoenzyme of semen creatine kinase.

The time course and dose-response to proteolysis of three dimeric isozymes of creatine kinase, CK-MM (muscle), CK-BB (brain), and CK-MB (heart) and the homologous monomer, arginine kinase were compared. Chymotrypsin and trypsin cause a rapid and significant loss of intact CK-BB, but limited hydrolysis of CK-MM. After 1h of hydrolysis by chymotrypsin, 80% of CK-MM is intact as judged by quantification of monomers after electrophoresis in sodium dodecyl sulfate. While 50% of the intact monomers of CK-MB remain under these conditions, no CK-BB monomers are detected. These results indicate that treatment with chymotrypsin leads to a CK-MB devoid of the B-subunit. When treated with trypsin for 1h, CK-MM is totally resistant to hydrolysis and all CK-BB is highly degraded. However, CK-MB exhibits approximately 90% intact monomers, indicating survival of intact B-subunit in CK-MB. This suggests that heterodimerization of a B-subunit with an M-subunit may have a protective effect against hydrolysis by trypsin. In view of the considerably larger number of potentially tryptic sensitive sites on the muscle isozyme, the resistance of CK-MM and susceptibility of CK-BB dimers to trypsin implies that differences in subunit tertiary structure are a factor in proteolysis of the homodimeric isozymes. Arginine kinase is rapidly degraded by trypsin, but is minimally affected by chymotrypsin. The finding that both a monomeric (arginine kinase) and dimeric (CK-BB) phosphagen kinase are highly susceptible to proteolysis by trypsin indicates that quaternary structure is not, in and of itself, an advantage in resistance to proteolysis. Since both arginine kinase and muscle creatine kinase are resistant to chymotryptic hydrolysis, it seems unlikely that in general, the increased packing density, which may result from dimerization can account for the stability of CK-MM towards trypsin.

We tested the hypothesis that the renin-angiotensin system (RAS) protects the contractile function of the myocardium against the damaging effect of hypoxia-reoxygenation. For this purpose, the contractility of isolated papillary muscles from wild-type (WT) rats and from rats expressing human renin and angiotensinogen as transgenes (TGR) was compared. After 15 min of hypoxia, peak force (PF) was decreased to 24 +/- 5% of the normoxic values in TGR (n = 10) and to 18 +/- 1% in WT rats (n = 12). PF and relaxation rates recovered completely in TGR but not in WT rats during 45 min of reoxygenation. Improved contractility of the papillary muscles from TGR during hypoxia-reoxygenation correlated with increased glutathione peroxidase activities and creatine kinase (CK)-MB and CK-BB isoenzyme levels. On the other hand, inhibition of the RAS with ramipril (1 mg/kg body wt for 3 wk) in WT animals resulted in deterioration of the contractile function of the papillary muscles during reoxygenation compared with untreated rats. These findings suggest that activation of the RAS protects contractile function of the cardiac muscle against hypoxia-reoxygenation, possibly through changes in CK isoenzymes and enhanced antioxidant capacity.

We present two patients who were hospitalized due to thrombo-embolic disease. Both patients had an increase in total creatine kinase activity with the creatine kinase MB fraction value exceeding the total creatine kinase activity. We determined that the high values for creatine kinase MB fraction in the immunoinhibition assay were due to the existence of macro creatine kinase type I in one patient and a highly elevated creatinine kinase BB fraction in the other patient. The patient with macro CK type I had ulcerative colitis and the other patient with elevated CK BB fraction was diagnosed with prostatic carcinoma.

OBJECTIVE

To investigate the serum creatine kinase isoenzyme pattern, specific biochemical markers of bone metabolism, and cytokines in a Chinese family with osteopetrosis, and correlate abnormalities with the pathophysiology of this condition.

METHODS

A Chinese female baby was diagnosed with malignant infantile osteopetrosis at the age of 3 weeks by clinical history and biochemical investigations. We studied the laboratory and radiological manifestations of this index case and her family members.

RESULTS

Serum CK-BB fraction of our index patient was elevated to 18.0% (normal 1.6-7.6%). Her biochemical markers of bone resorption including serum C-terminal telopeptide concentration and urine N-terminal telopeptide to creatinine ratio were decreased to 0.54 microg/L (normal 0.72-1.56 microg/L) and 159 x 10(-6) (normal 372-900 x 10(-6)), respectively. Serum cytokines including soluble receptor activator of nuclear factor kappa-B ligand (sRANKL) concentration was suppressed to 0.11 pmol/L (normal 0.23-0.82 pmol/L) and osteoprotegerin (OPG) concentration was 4.9 pmol/L (normal 2.8-4.9 pmol/L), resulting in an elevated OPG to sRANKL ratio of 44.5 (normal 3.8-19.4) in favour of bone formation.

CONCLUSIONS

If left untreated, this condition is usually fatal within the first year of life. With early diagnosis, management including bone marrow transplantation can be planned ahead and will result in a better survival.

The creatinine kinase (CK)-MB assay can be used for the early diagnosis of acute coronary syndrome. We describe the case of an 82-year-old male with lung adenocarcinoma who presented with chest pain. While laboratory findings showed elevated CK-MB levels, there was no cardiac injury. A chest computed tomography scan revealed pleural carcinomatosis. Later, electrophoretic analysis of CK showed a normal CK-MB range but increased CK-BB levels and the presence of macro CK type 2. We determined that the patient's chest pain originated from the visceral pleural invasion of lung cancer. Because of the methods used to measure the CK-MB isozyme, the CK-MB level appeared elevated.

OBJECTIVE

To observe the effect of aminophylline on physiological and pathological changes in acute exposure to high altitude in rats.

METHODS

A total of 21 male Wistar rats were randomly divided into a plain group (altitude 55 m), a high altitude hypoxia group (altitude 4 300 m), and a high altitude hypoxia plus aminophylline group. After 5 days, blood from orbital venous was collected for analyzing biochemical parameters. Blood from abdominal aorta was collected for analyzing the parameters of blood gas. The tissues of brain, lung, and kidney were dissected for pathological observation.

RESULTS

Compared with the plain group, the parameters of LDH, ALP, Urea and cCl? in the hypoxia group or the aminophylline treatment group were significantly increased (P<0.01), while the parameters of ALB, Cr, SatO₂, Hb, Hct, PaCO₂, PaO₂2, BB and BE were significantly reduced (P<0.01). Compared with the hypoxia group, the parameters of Cr, pH, Hct, cNa⁺, cCl⁻ in the aminophylline treatment group were significantly increased (P<0.01), while AST, ALT, ALB, PaCO₂ and cK⁺ were significantly decreased (P<0.01 or P<0.05). Pathological results showed the brain, lung and liver tissues were obviously damaged in the hypoxia group compared with that in the plain group. These damages were significantly attenuated by aminophylline.

CONCLUSIONS

Aminophylline can improve blood gas and biochemical parameters in acute exposure to high altitude in rats. It can protect rat brain, lung and liver from the damage caused by acute high altitude, which may be related its effects on relaxation of bronchial smooth muscle and inhibition of inflammation.

The diagnosis of a heart infarct in the early postoperative period is often difficult. In the present study, the levels of serum CK, its isoenzymes (MM, MB and BB), aspartate aminotransferase (ASAT) and alanine aminotransferase (ALAT) were followed up after coronary arteriography (12 patients), cardiac surgery (23 patients) and non-cardiac thoracotomy (28 patients). Elevation of MB was not detected after coronary arteriography, except in two patients, who had had even before the examination a mildly positive MB finding and electrocardiographic changes indicative of subendocardial infarction. After cardiac surgery the MB findings were positive in all but two patients, who had undergone aortic valve surgery. However, the average MB level was lower than in the 10 heart infarct patients who served as controls. After non-cardiac thoracotomy, six patients had a positive serum MB, but the value of MB was quite low as compared with values after cardiac surgery and nearly insignificant in terms of heart infarct diagnosis.