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: re-examination of optimum reaction conditions.

I have re-examined optimum reaction conditions for measurement of creatine kinase (EC 2.7.3.2). The optimum pH is 6.45, and 2,2-bis(hydroxymethyl)-2,2',2'-nitrotriethanol acetate, 200 mmol/liter, is the buffer of choice. Thioglycerol, 20 mmol/liter, is superior for both in-assay reactivation and for storage stability of sera. Fluoride, 25 mmol/liter, a broad inactivator of adenylate kinase (EC 2.7.4.3), has little effect on creatine kinase and is superior to AMP for adenylate kinase inhibition in the assay of creatine kinase. Magnesium ion, ADP, and buffer concentrations are interdependent and their optima must be determined together. The hexokinase/glucose-6-phosphate dehydrogenase activity ratio should not exceed 1.6. The range of linearity is limited by the glucose-6-phosphate dehydrogenase and NAD+ concentrations. Glucose-6-phosphate dehydrogenase, ADP, and NAD+ are the constituents most likely to result in unacceptable blanks. Creatine kinase is inhibited noncompetitively by anions: acetate and fluoride inhibit slightly, but sulfates, nitrates, and excessive chlorides should be avoided.     

Creatine kinase in serum: 1. Determination of optimum reaction conditions.

To establish optimum conditions for creatine kinase (EC 2.7.3.2) activity measurement with the creatine phosphate in equilibrium creatine reaction, we re-examined all kinetics factors relevant to an optimal and standardized enzyme assay at 30 and 25 degrees C. We determined the pH optimum in vaious buffers, considering the effect of the type and concentration of the buffer, as well as the influence of various buffer anions on the activity. The relation between activity and substrate concentration was shown and the apparent Michaelis constants of creatine kinase for creatine phosphate and ADP were evaluated. We tested the effect on creatine kinase measurement of the concentration of substrates (glucose and NADP+) in the auxillary and indicator reactions, especially the influence of the added auxiliary (hexokinase) and indicator (glucose-6-phosphate dehydrogenase) enzymes on the lag phase, at different temperatures. The NADP+ concentration proved to be the factor limiting the duration of constant reaction rate. We studied the inhibition of creatine kinase and adenylate kinase by AMP and established a convenient AMP concentration. For reactivation of creatine kinase, N-acetyl cysteine as sulfhydryl compound was introduced. Finally, we examined the relationship between activity and temperature.     

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 isoforms in ischemic heart disease

The MM and MB isoenzymes of creatine kinase exist in serum as a collection of at least three major MM and two major MB isoforms. Each of these are derived from single tissue MM and MB isoforms, which are converted to these other forms by carboxypeptidase N after their release from necrotic skeletal and myocardial tissue. Measurement of the MM isoforms in ischemic heart disease is useful for early diagnosis of acute myocardial infarction and for the noninvasive determination of coronary artery reperfusion for infarction patients receiving thrombolytic therapy. Because MM is also released in acute skeletal-muscle disease, MB isoform measurements may have the highest clinical sensitivity. These determinations are important for providing objective information to cardiologists who need to make critical decisions concerning the management of these patients. I review the procedures for treating patients with myocardial infarction, the potential role of CK isoforms, and the methods currently available for isoform analysis, including high-resolution electrophoresis, isoelectric and chromatofocusing, and liquid chromatography. Rapid and highly sensitive methods are needed for implementation of CK-MM and MB isoforms for prospective emergency determinations for patients with acute myocardial infarction.     

Creatine kinase MB isoforms for early diagnosis and monitoring of acute myocardial infarction.

MB isoforms of creatine kinase (ATP:creatine N-phosphotransferase, EC 2.7.3.2, CK) in 848 sera obtained from 80 patients with acute myocardial infarction (AMI) were studied by agarose gel isoelectric focusing. In 173 sera (20%) from 25 patients (31%), a new isoform designated as MB3 (pI 5.4) was detected at the cathodal side of MB2 (pI 5.2) in addition to the previously known MB2 and MB1 (pI 5.1). The new isoform MB3 was found in the extract of the cardiac muscle. MB3 was dominant in the sera at an earlier stage and a shorter period of time after AMI: 1-14 h (range), 1-29.5 h and 4-154 h for MB3, MB2 and MB1 dominant, respectively. MB3 was therefore found to be an earlier and a shorter phase indicator for AMI than MB2 or MB1. However, MB2 greater than 1 was the most prevalent pattern at the time of admission to the hospital. In AMI, specificity was 96.2%, 92.4% and 90.6%, and sensitivity was 20.4%, 88.9% and 97.6%, for MB3, MB2 and MB1 isoforms, respectively. CK-MB isoform patterns were biased to MB1 dominant in the deceased group, and to MB2 dominant in the surviving group. Therefore determination of CK-MB isoforms is also useful in the course of observation of AMI. The fourth isoform, MB0 (pI 5.0), was detected at the anodal side of MB1. MB0 was a minor band of the CK-MB isoform which appeared when serum CK-MB activity increased.     

Creatine kinase and its CK-MB isoenzyme: the conventional marker for the diagnosis of acute myocardial infarction

Biochemical markers are commonly used for detection, diagnosis, and management of various diseases. Creatine kinase (CK) is an enzyme found in most tissues, and is the best known marker for the identification of acute myocardial infarction (AMI). We review the most common techniques used to quantify creatine kinase and its cardiac specific fraction, CK-MB. Electrophoresis, immunoinhibition, monoclonal immunoassay, and CK-MB isoform assays are considered for efficacy, sensitivity and specificity, and timeliness in the setting of acute non-traumatic chest pain. Limitations of the various techniques for identifying AMI are identified. Current recommendations for CK-MB panels, including combinations with other cardiac markers, are included.     

Bioptic microbiology in the differential diagnosis of enterocolitis

Parameters in the differential diagnosis of enterocolitis have been poorly evident for many years. Development and profitable employment of endoscopic instruments were the first step towards advancing the diagnostic facilities in inflammatory bowel disease. The microbiologic examination of mucosal biopsies creates a new diagnostic dimension, and it distinctly seems to increase the diagnostic sensitivity for pathogens. Within fifteen months 152 patients admitted to the gastroenterologic unit with acute, or symptoms of exacerbated, bowel disease were examined for the aetiologic agents. Compared with former reports, idiopathic inflammatory bowel disease (IIBD) such as Crohn's disease (32.2%) and ulcerative colitis (18.4%) were decreased. Infectious colitis (22.3%), mostly Campylobacter or Yersinia infections, was, sometimes exclusively, diagnosed by bioptic microbiology, non-classifiable forms of colitis (21.7%), and rare forms (5.4%) were diagnosed more often. It proved to be important that IIBD was frequently superinfected by Campylobacter, Yersinia and Chlamydia, and the differential diagnosis was complicated, since these microorganisms can mimic IIBD. The results suggest that coloileoscopy combined with bioptic microbiologic investigation additional to faecal samples should include a search for Campylobacter and Yersinia. It appears indispensable that the final diagnosis "Crohn's disease" or "ulcerative colitis" should be confirmed by sequential coloileoscopy and microbiologic examination.     

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 isoenzyme--antibody reactions in immuno-inhibition and immunonephelometry

I have raised, in rabbits, antibodies against MM and BB isoenzymes of creatine kinase. The antibodies produced were homogeneous by Ouchterlony double immunodiffusion and did not cross react with their opposite antigens. Both antisera, however, appeared to be mixtures of antibodies, displaying different equivalences for activation, inactivation, and, possibly, precipitation. Inactivation studies indicated the presence of antibodies effective against dimer only and antibodies effective against monomer or dimer. Both antisera cross reacted with MB and displayed antibodies that appeared to block only half of the activity as well as antibodies that blocked all of the activity. The antisera produced were useful for measuring MB by both immuno-inhibition and immunonephelometry, but neither appears to be advantageous over current electrophoresis or ion-exchange methods. A comparison of decay of MB in patients between activity and mass measurements indicated that activity decay is about 12-fold faster than mass decay.