Creatine kinase isoenzymes

Creatine kinase (CK), a widely distributed enzyme in the body, has its highest activities in skeletal muscle and myocardium; when serum CK activities are abnormally increased, injury to these organs must be part of the differential diagnosis. The isoenzyme CK-MB is the most important biochemical test in the diagnosis of acute myocardial infarction.     

Creatine kinase in cerebrospinal fluid in newborn infants

L-Alanine was measured by an enzymic micromethod in serum after treatment with perchloric acid, as well as after ultrafiltration through collodium membrane filters. Serum L-alanine was also determined by an automated column Chromatographic, technique. Using the enzymic method, spuriously increased L-alanine levels could be obtained due to the absorbent reaction between hydrazine and NAD-containing reagents. It could be shown that this absorbent reaction depends on the pH value of the test solution. In contrast to supernatant diluted by perchloric acid, ultrafiltrates gave L-alanine data equivalent to those by column chromatography, since conditions of reaction time and pH value could be kept identical for blank, standard and ultrafiltrate solutions.     

Creatine kinase isoenzymes of mitochondrial origin in human serum.

We measured creatine kinase (EC 2.7.3.2) activity in 1009 serum samples from 538 patients in the intensive-care units of the University of Texas Medical Branch hospitals. Creatine kinase isoenzymes migrating cathodal to skeletal muscle creatine kinase (CK-MM) on cellulose acetate electrophoresis were found in sera from 14 of the 538 patients. Creatine kinase, lactate dehydrogenase (EC 1.1.1.27), aspartate aminotransferase (EC 2.6.1.1), and alanine aminotransferase (EC 2.6.1.2) activities were abnormally increased in these 14 patients. Liver lactate dehydrogenase isoenzyme (LDH5) and cardiac creatine kinase isoenzyme (CK-MB) were abnormally increased in 12 and eight of these patients, respectively. Ten of the 14 patients died during their hospital admission. We believe the creatine kinase isoenzymes that migrated cathodal to skeletal muscle creatine kinase (CK-MM) were of mitochondrial origin.     

Creatine kinase isoenzymes in extracts of various human skeletal muscles

We have attempted to "map" creatine kinase (CK; EC 2.7.3.2) anatomically by assaying biopsy specimens of skeletal muscles taken from different locations from 109 surgical patients. Substantial activities of CK-MM were present in every specimen; CK-MB was observed in about 44% of these, at approximately 1 to 6% of the total CK activity.     

Creatine kinase and its isoenzymes in neoplastic disease

The CK-BB isoenzyme is ubiquitous in neoplastic tissue, but with low activity. Accordingly, it might be a nonspecific and insensitive tumor marker. Evaluation of BB isoenzyme in serum might indicate the extent of diseases or the response to therapy. The presence of CK-MB in patients with cancers may cause confusion with AMI. Serial determinations of both CK and lactate dehydrogenase isoenzymes are of great help in differential diagnosis. The presence of mit-CK is a poor prognostic sign in patients with malignancy. The greatest clinical significance of CK-BB and macro-CK isoenzyme lies in their effect on various assays for CK-MB. Macro-CK types 1 and 2 are much more heat stable than are CK-MB and CK-BB, and so by heating samples for 20 min at 45 degrees C the presence of thermostable macro types can be demonstrated. Macro-CK type 2 has a much higher activation energy than macro-CK type 1. If macro-CK is present, determination of the activation energy easily differentiates between types 1 and 2. CK-Bi seems to be glycosylated protein, and it is thought that glycosylation may be a general way of enzyme inactivation. If inactivation inside the cell is postulated, it has to be shown that enzymes indeed pass into the cell compartments where glycosylating enzymes are located. Another possible mechanism is within the circulation. Whether malignant cells themselves produce Ck-Bi or if inactivation occurs in the blood is still unknown. In this connection, one finding is that in plasma of cancer patients, CK-Bi can be reactivated to CK-BB by mercaptoethanol to 95%, whereas in plasma of normal persons there is no reactivation of the much lower CK-Bi concentrations.     

Stabilization of creatine kinase isoenzymes in the cerebrospinal fluid in cranio-cerebral trauma

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.     

Bioluminescence assay of creatine kinase and its isoenzymes in serum and cerebrospinal fluid.

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.     

Creatine kinase isoenzymes in serum from children--especially focusing on CK-BB

In 161 children with ages ranging from 7 months to 18 years the concentration of CK-BB in serum was measurable in 58%. The activity in serum of CK-BB (cCK-BB(S] in children varies with age and is not significantly influenced by seizure, epilepsy or body temperature. The values are most elevated during the first year of life and show a rapid fall, never reaching adult levels. Comparing mean cCK-BB(S) values in children to growth charts the resemblance is striking. CK-BB is the only CK-isoenzyme that changes with age. We postulate that cCK-BB(S) is related to growth as a physiological phenomenon. CK-MB was found in serum in 7% of the children and could not be of cardiac origin. Therefore CK-MB cannot be regarded as cardiac specific in children.     

Creatine kinase isoenzymes in the diagnosis of acute cranio-cerebral trauma

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.     

Creatine kinase and lactate dehydrogenase isoenzymes in stage D prostatic carcinoma

Serum creatine kinase (CK) and lactate dehydrogenase (LD) isoenzymes were determined electrophoretically, along with various other biochemical markers of malignancy, in 19 patients with metastatic carcinoma of the prostate. Mitochondrial CK appeared in 15 patients, the CK-BB isoenzyme in 6. As a result, CK activity not inhibited by anti-M-subunit antibodies, CK non-M, was above the reference value in altogether 17 patients. There was a cathodic shift among the LD isoenzymes, significantly more prominent with increasing total LD, and a positive correlation between elevations of CK non-M and LD-5, suggesting a relation to tumour burden for both. An LD 'flip' (LD-1 greater than LD-2) was present in 10/15 patients. The frequency of CK non-M elevations was similar to--but not quantitatively correlated with--elevations of prostatic acid phosphatase and alkaline phosphatase. Thus, changes in CK and LD patterns are frequent in patients with prostatic cancer and must be taken into consideration when acute cardiac symptoms are evaluated in such patients.     

Diagnostic value of lactate dehydrogenase isoenzymes in cerebrospinal fluid

To evaluate the diagnostic value of lactate dehydrogenase (LD) isoenzymes in cerebrospinal fluid (CSF), 93 consecutive CSF specimens were analyzed. These specimens were from patients of four categories: tumors, infections, hemorrhages, and others. It was found that the isoenzyme patterns overlapped among different categories, but they differed within each category and were thus helpful in differential diagnosis. For instance, metastatic tumors showed prominent LD-5, whereas a primary brain tumor demonstrated an increase in all fractions. Viral encephalitis revealed an increase in the first three isoenzymes and bacterial meningitis, the last two. In acquired immune deficiency syndrome (AIDS) cases, however, LD isoenzyme changes were demonstrated in CSF when only cryptococcal meningitis and not when encephalitis was present. Both subdural and subarachnoid hemorrhages showed elevation of all fractions in our study. Elevation of the first three fractions was usually due to brain tissue damage or hemorrhage, as proven by our isoenzyme study of hemolysate mixed with CSF. The prominence of the last two fractions was related to anaerobic metabolism in the central nervous system or to granulocytic infiltration. In conclusion, LD isoenzyme analysis in CSF is helpful in differential diagnosis of various CNS disorders, although its sensitivity awaits further improvement.     

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.     

Creatine kinase and lactate dehydrogenase isoenzymes in serum and tissues of patients with stomach adenocarcinoma

Total activities of creatine kinase (EC 2.7.3.2; CK) and lactate dehydrogenase (EC 1.1.1.27; LD) and their isoenzymes were estimated in serum and tissue samples from patients with stomach adenocarcinomas who were to undergo gastric resection. Total CK activity (U/g protein) appeared to be markedly decreased in neoplastic stomach tissue. CK-BB was the predominant isoenzyme in both neoplastic and normal stomach tissues; however, the CK-BB/total CK ratio was increased in adenocarcinoma tissue. Macro CK type 1 was found in two neoplastic tissues and macro CK type 2 in 11. LD4 and LD5 isoenzymes were predominant in gastric tissues, but LD5 and the LD5/LD1 ratio were higher in adenocarcinoma tissue. At 24 h before surgery, CK-BB was demonstrated in sera of all patients and CK-MB in 69%. The CK-BB probably originated from neoplastic stomach tissue, which contains high CK activity, with BB isoenzyme predominating. After gastrectomy, CK and LD isoenzymes in sera were markedly increased by 24 h postsurgery. These alterations were attributed to release from damaged tissue during gastric resection.     

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.     

Creatine kinase (EC-No.2.7.3.2) and creatine kinase isoenzymes during pregnancy and labor and in the cord blood

Total CK and CK isoenzyme activity in serum was investigated during pregnancy, labor and after delivery as well as in cord blood. Total CK was decreased in the second trimester of pregnancy but increased in late pregnancy. Low CK-MB activity in serum was found in patients with early labor pains. CK-BB activity could never be detected during pregnancy. Total CK and isoenzyme activity increased after delivery. The rise of total CK and CK-MB in maternal serum is directly correlated with the following: type of delivery, duration of labor, parity of the mother, and birth weight. From this it can be deduced that postpartum CK levels depend on skeletal muscle activity as well as on the activity of uterine muscle. Prematures and infants "small for date" have significantly lower total CK and slightly more elevated CK-BB activity in cord blood than children of normal maturity. CK-BB activity is much more pronounced in high risk patients with low Apgar score.