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 activity and isoenzyme pattern in various normal tissues and neoplasms

I measured total creatine kinase (CK; EC 2.7.3.2) activity and isoenzyme pattern in normal and neoplastic tissues. CK activity was detected in all of them examined. In various tumors it was greater than, less than, or the same as that in normal tissue, no clear correlation being seen between total activity and growth rate or degree of differentiation. In several cases, there was a greater proportion of the CK-MM isoenzyme, and 15 of 53 cases showed an atypical CK-MM band. The atypical CK-MM band, also reported by others, might be an insensitive and nonspecific tumor marker. The CK-BB isoenzyme, ubiquitous in neoplastic tissues, might accordingly be a nonspecific marker. Total CK activity was very low in most tumor tissues. Presumably a bulky tumor or an advanced stage of malignancy is a requisite to release of routinely detectable CK-BB into the circulation.     

Reactivation of serum creatine kinase isoenzyme BB in patients with malignancies

Reactivation of serum creatine kinase isoenzyme BB (CK-BB) with 2-mercaptoethanol and EDTA increased the electrophoretic detection rate of CK-BB from 34% to 78% in 58 hospitalized patients with various malignancies. Patients with solid tumors showed the largest and patients with hematologic malignancies the smallest percentage increase in CK-BB after reactivation. For serum from 50 hospitalized patients without cancer, reactivation resulted in detectable CK-BB in two patients; the CK-BB band was never seen in 15 healthy adults. For reasons unknown, five of eight patients with senile dementia of the Alzheimer's type showed CK-BB in serum after reactivation, as did two of five patients suspected of having this disorder. Serum CK-BB may be a useful tumor marker if reactivation with a thiol and EDTA is used immediately after collection.     

The presence of creatine kinase bb isoenzyme in patients with prostatic cancer

Creatine kinase BB isoenzyme (CK-BB) was detected in abnormal amounts in serum samples from 11 of 46 patients with Stage D carcinoma of the prostate by electrophoresis. Thirteen of 46 Stage D patients had elevated acid phosphatase values and 10 of these 13 had elevated CK-BB. CK-BB elevations were less frequent in earlier stages of prostatic cancer; Stage C: 0 of 35, Stage B: 1 of 26, Stage A: 0 of 3 and none in a group of 35 with BPH, prostatitis and bladder cancer. Results of CK-BB by a specific radioimmunoassay correlated well with those obtained by electrophoresis in most cases. Several patients were followed over time and data on CK-BB is presented for this interval. The origin of the CK-BB is still unclear. The BB isoenzyme predominates in prostatic tissue and CK-BB is the fetal form of the enzyme in human muscle and myocardium. The increase in serum CK-BB may be related to increased release of the isoenzyme, either from the prostate itself or from a metastatic lesion, or may represent a release of the fetal form of the enzyme from dedifferentiated tumor tissue.     

Creatine kinase BB: a new tumor-associated marker.

Creatine kinase isoenzyme BB (CK-BB) is found in the serum of patients with various types of cancer. Using both radioimmunoassay and agarose electrophoresis, we found abnormal amounts of this isoenzyme in the serum of 15 of 17 patients with untreated prostatic carcinoma. Among patients with other types of adenocarcinomas and metastatic disease, serum CK-BB was increased in most of those who were unresponsive to therapy. In benign or malignant prostate tissue and in malignant pleural effusions, cytoplasmic CK-BB as determined by immunoperoxidase staining was found in epithelial cells. Prostatic fluid had high concentrations of CK-BB, as did malignant, but not benign, pleural fluid supernates. We conclude that glandular epithelial cells contain CK-BB, which is released in benign and malignant states and may appear in higher concentrations in the circulation in malignant states. These conclusions are consistent with predictions we have made from a model experimental system concerning characteristics of possible tumor markers. The observations indicate a role for CK-BB as a tumor marker, particularly for adenocarcinoma of the prostate.     

Unexplained increase in serum creatine kinase isoenzyme MB activity in a lung cancer patient

In this case of mixed small cell--large cell cancer of the lung in an elderly woman, creatine kinase (EC 2.7.3.2) isoenzymes were assayed serially because of chest pain. The proportions of serum CK-BB and CK-MB isoenzyme activities were persistently above normal (CK-MB 10-18%, normal less than 5%). Electrocardiograms revealed no signs of ischemia or infarction. At autopsy no gross or microscopic infarction or inflammation of the heart was seen. There was also no infarction of smooth or skeletal muscle. The tumor was the probable source of most of the circulating CK-MB isoenzyme. Future cases may pose a similar diagnostic dilemma: differentiating creatine kinase that is present as a result of myocardial infarction from tumor-related CK-MB. Whether or not CK-MB assay could be useful in detecting tumors remains to be investigated.     

Transient increase in serum creatine kinase isoenzyme BB activity after prostatectomy in a patient with massive benign prostatic hyperplasia

The activity of creatine kinase isoenzyme BB (CK-BB) in serum is rarely abnormally high (i.e., detectable). An increase in immunoreactive CK-BB or CK-BB activity in patients with prostatic disease has been proposed as an indication of prostatic adenocarcinoma. Here we report the case of an elderly man with massive benign prostatic hyperplasia but no clinical or pathological evidence of prostatic adenocarcinoma, whose serum CK-BB activity was found by agarose gel electrophoresis to be 1 U/L (normal: 0%), 10% of his total CK activity. Serum CK-BB activity was further increased to 16 U/L (20% of total CK activity) 1 h after prostatectomy, but became undetectable by the second day after the operation. The findings suggest that: (a) the source of the serum CK-BB activity was the enlarged prostate gland; (b) abnormally high CK-BB activity in serum of men with prostatic disease does not necessarily indicate the presence of prostatic adenocarcinoma; and (c) myocardial injury could be erroneously diagnosed postoperatively in prostatectomy patients if CK isoenzyme methods are used that do not consistently separate "heart-specific" CK-MB from CK-BB.     

Radioimmunoassay of creatine kinase isoenzymes in human serum: isoenzyme BB

We describe a sensitive, specific radioimmunoassay for the BB isoenzyme of creatine kinase (CK-BB) in serum. A sequential saturation assay was used to achieve sufficient sensitivity to detect the isoenzyme in 100-microliter serum samples of all healthy persons and patients tested. Bound and free antigen were separated by a second antibody system. Large excesses of purified isoenzyme MM did not react in the assay. Cross reactivity of two preparations of CK-MB was only 1 to 7+. The 95th percentile of serum CK-BB in 208 healthy adults was 6.2 microgram/liter. Within-assay and between-assay precision ranged from 5.5 to 11.9% and 9.7 to 13.6%, respectively.     

Reassessment of creatine kinase BB as a marker for cancer of the prostate, breast, and lung

We used an enzyme-linked immunoabsorbent assay to measure creatine kinase (CK; EC 2.7.3.2) BB in the sera of 58 cancer patients. A pre-incubation step with an anti-CK-M antibody-coated bead removed M chain components, and CK-BB was quantified with use of an anti-CK-B antibody-coated tube. No cross reactivity was observed with mitochondrial CK or CK-MM; CK-MB cross reacted slightly (1.6%). Macro CK type 1 was measured as CK-BB. Average analytical recovery of purified CK-BB added to serum was 97.7%. Although the enzyme activity of CK-BB is labile, our studies show that this protein is antigenically stable for 12 months when stored frozen. The upper limit of normal for CK-BB concentration was 0.3 micrograms/L (95th percentile, n = 25). Of the 20 cases of breast cancer of various stages, none showed any increases of serum CK-BB. Only two of 18 patients with prostatic carcinoma (stage D), and two of 10 patients with oat-cell carcinoma of the lung had increased concentrations of CK-BB in the serum. Ten patients with squamous cell cancer of the lung had normal concentration of the enzyme. Thus the CK-BB isoenzyme is not frequently increased in cancers of the prostate, lung, and breast, and it has little apparent value as a tumor marker for these diseases.     

Creatine kinase isoenzymes in human cerebrospinal fluid and brain

Extracts of normal brains obtained at autopsy and cerebrospinal fluid (CSF) from patients with global brain ischemia were analyzed for creatine kinase (CK; EC 2.7.3.2) isoenzymes. We used both qualitative and quantitative assays (electrophoresis and immunoinhibition). Brain extracts contained CK-BB isoenzyme and mitochondrial CK. In 54 CSF samples free of blood contamination and with total activities ranging from 7 to 2010 U/L (mean 202 U/L), virtually all of the CK activity was due to CK-BB, and none to CK-MM or CK-MB. We conclude that brain contains CK-BB and mitochondrial CK, but lacks CK-MM and CK-MB. After cardiac arrest, CK-BB is released into the CSF. Any CK-MM in the CSF is probably from blood contamination, in which case immunoinhibition with anti-CK-M antibodies accurately quantifies CK-BB.     

Stability and electrophoretic characteristics of creatine kinase BB extracted from human brain and intestine

Creatine kinase (CK; EC 2.7.3.2) isoenzyme BB extracted from brains of rats reportedly undergoes modification at 37 degrees C, leaving an electrophoretic variant that accounts for most of the residual CK activity. This variant, called CK-BB', migrates on electrophoresis similarly to creatine kinase isoenzyme MB. Using electrophoresis and immunoinhibition with antiserum to creatine kinase isoenzyme MM, we found CK-BB to be the only identifiable cytoplasmic isoenzyme in surgical samples from human brain and intestine. In contrast, we found that some samples of brain obtained at autopsy contain CK-BB'. We also found that CK-BB extracted from human brain was converted to CK-BB' upon incubation in serum or plasma at 37 degrees C. We found a similar development of CK-BB' in incubation mixtures of serum or plasma containing CK-BB obtained from surgical samples of human intestine. The development of CK-BB' during infarction of the gastrointestinal system may thus be a source of false-positive CK-MB in the laboratory verification of myocardial infarction when electrophoresis is used as the only method to identify CK isoenzymes.     

Influence of autoantibodies to creatine kinase-BB on assays for MB isoenzyme

We describe the influence of autoantibodies that bind creatine kinase BB (CK-BB) on the methods for MB isoenzyme. If these autoantibodies are present in patients' sera, they cause the formation of macro CK type 1 (immunoglobulin-linked CK-BB). In some of these cases they can bind not only endogenous CK-BB but also CK-MB without significantly affecting enzyme activity. Although these antibodies show distinctly less affinity for CK-MB than for CK-BB, they nevertheless bind CK-MB in these particular sera, because their concentration exceeds that of CK-BB isoenzyme. If a person with such autoantibodies has an acute myocardial infarction, the immunoinhibition method for CK-MB, which does not discriminate between CK-MB and CK-BB, will recognize the increase and peak of CK-MB with time, although persistent macro CK activity will be superimposed on the typical isoenzyme pattern. However, isoenzyme electrophoresis and recently introduced immunoenzymometric assays for CK-MB in these cases may be less sensitive for detecting myocardial infarctions, because the typical increase in CK-MB activity may be identified later in the progression of symptoms, or even be missed.     

Properties of creatine kinase-BB from canine and human brain tissues

Creatine kinase (EC 2.7.3.2) BB isoenzyme (CK-BB) was purified to homogeneity from canine and human brain tissues. The purified protein from both sources exhibits Mr of 84,700 daltons. The canine isoenzyme exhibits several properties similar to human isoenzyme with respect to reactive and total thiol groups, UV spectra, isoelectric points and reaction kinetics. While both canine and human CK-BB isoenzymes are unstable compared to other CK isoenzymes, canine CK-BB is even less stable than the human enzyme, losing most of its activity within 20 h at 4 degrees C at pH 5.0. Addition of 2-mercaptoethanol does not prevent rapid loss of the enzyme activity. Increasing the pH to 9.0, however, increases the stability of both CK-BB isoenzymes. Agarose electrophoresis demonstrated the presence of MM as well as BB isoenzyme in various parts of brain tissues. BB was present at an activity of 90.8-93.3 U/mg and MM at 6.7-9.2 U/mg.     

Isolation of creatine kinase BB isoenzyme with high specific activity and adequate purity for radioimmunoassay from human placenta on preparative polyacrylamide gel electrophoresis

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.     

Serum creatine kinase isoenzyme BB is a poor index to the size of various brain lesions

We divided patients with brain lesions into three groups: (a) patients with primary or metastatic brain cancer, (b) brain infarctions, and (c) brain contusion(s). We analyzed each patient's sera for creatine kinase isoenzyme BB (CK-BB), using a monoclonal antibody kit (Impres-BB; International Immunoassay Laboratories). Computerized axial tomography (CAT) scans were performed on each patient. The size of the various lesions was measured from the CAT scan and recorded in milliliters. Total CK, CK-BB, and their ratios were compared with the volume of damaged brain tissue. We found no correlation between any of the variables and the various brain lesions. We attribute this lack of correlation to an intact blood-brain barrier, the rapid elimination or inactivation of CK-BB, or some combination of these factors.     

Immunochemiluminometric assay of creatine kinase MB with a monoclonal antibody to the MB isoenzyme

Previous two-site immunometric assays for creatine kinase (CK; EC 2.7.3.2) MB isoenzyme have been based on formation of a "sandwich" complex involving CK-MB and antibodies that recognize the CK-MM and the CK-BB isoenzymes. Single-incubation model assays of CK-MB with these antibodies were susceptible to interferences by CK-MM and CK-BB. We produced two anti-CK-MB monoclonal antibodies and studied their suitability for two-site assays. Both antibodies were compatible with anti-CK-MM and anti-CK-BB, but not with each other. Using anti-CK-MB as the tracer antibody eliminated the interference by both CK-MM and CK-BB. Labeling anti-CK-MB with acridinium ester and immobilizing anti-CK-BB on paramagnetic particles, we developed a rapid and highly sensitive chemiluminescent/magnetic separation CK-MB assay. As little as 1 microgram of CK-MB per liter was detectable after 10- or 30-min incubation at room temperature, and the standard curve was linear up to 400 micrograms/L. Results for serum samples by the new assay correlated well (r = 0.94) with those by Corning electrophoretic and the Hybritech Tandem-E immunoenzymometric CK-MB methods. Sera containing macro CK-1 or high concentrations of CK-MM and CK-BB did not interfere. The combined advantages of a more-specific antibody, paramagnetic solid phase, and chemiluminescent label endow this two-site CK-MB assay with performance characteristics and ease of use superior to those of previous assays.     

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.     

The purification of human creatine kinase BB with high specific activity

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.     

Determination of creatine kinase isozymes in sera and tissues of patients with various lung carcinomas

Three creatine kinase isozymes (CK-BB, CK-MB and CK-MM) were estimated by immunoassay in tumor tissues and in sera of patients with various lung carcinomas. CK-BB was increased in small cell carcinoma, but not in other lung carcinomas. CK-MM and CK-MB were not increased in any types of carcinoma. Serum CK-BB was increased in all types of lung carcinoma examined, while serum CK-MM and CK-MB were within normal limits in all patients. Serum CK-BB of healthy adults was estimated as 0.32 +/- 0.14 (mean +/- SD) ng/ml, ranging from 0.11-0.68 ng/ml. If CK-BB values above 1.0 ng/ml were considered abnormal, elevation occurred in 28/40 (70%) of patients with small cell carcinoma, 25/67 (37%) with adenocarcinoma, 21/51 (41%) with squamous cell carcinoma, 4/11 (36%) with other carcinoma of the lung and 10/42 (24%) with lung tuberculosis. Since serum CK-BB with lung cancer changed in parallel with the clinical course, this isozyme may be a marker for monitoring the clinical course, especially in small cell carcinoma of the lung.     

Macro creatine kinase BB isoenzyme in serum: most likely an antigen-autoantibody complex

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.