Measurement Of Isoenzymes Research Articles

Significance of Adenosine Deaminase Activity and Its Isoenzymes in Tuberculous Effusions

Adenosine deaminase (ADA) activity is increased in effusions caused by certain clinical conditions including tuberculosis and bacterial infections. In this study, the ADA isoenzyme patterns in tuberculous and parainfective effusions were investigated to determine the isoenzyme responsible for this increase in activity. Fifty-one tuberculous effusions and six parainfective effusions were investigated. All effusions had increased ADA activity (median values of 126 and 127 units/L, respectively). In the tuberculous effusions, ADA2 isoenzyme was found to be primarily responsible for total activity, with a median contribution of 88 percent. The ADA1 (both ADA1m and ADA1c isoenzymes) was the major isoenzyme in the parainfective effusions with a median contribution of 70 percent. The ADA2 isoenzyme activity most likely reflects monocyte-macrophage turnover or activity, while ADA1 probably originates from lymphocytes or neutrophils. It is therefore essential to determine the isoenzyme profile when interpreting ADA activity levels in effusions. The measurement of the individual isoenzymes will enhance the diagnostic utility of ADA activity determinations in pleural effusions.

Monitoring skeletal cancer metastases with the bone isoenzyme of tissue unspecific alkaline phosphatase

The efficacy of bone alkaline phosphatase (ALP) isoenzyme measurement using lectin precipitation in confirming metastatic bone lesions was compared with total ALP and osteocalcin assay in serum. Sixty-five patients with cancer and metastases to bone ( n = 44), liver ( n = 15) or lymph nodes ( n = 6) as well as 33 healthy adults were studied. Assay of bone ALP is as sensitive but more specific than assay of total ALP in the identification of bone metastases. On the other hand, bone ALP did not correlate with osteocalcin, as is the case in other bone diseases. In the serial monitoring of nine patients with skeletal metastases, bone ALP correlated well with the presence of pain and the progression or regression of metastatic spread.

Is determination of creatine kinase-2 after electrophoretic separation accurate?

Since 1991, the Ontario Laboratory Proficiency Testing Program has assessed the analytical performance of creatine kinase (CK; EC 2.7.3.2) isoenzyme-2, using fresh human serum supplemented with purified human CK isoenzymes. In Ontario, the 142 laboratories licensed to analyze CK-2 use a variety of methods: electrophoresis-based, immunoinhibition, and mass assays. During a 1992 survey, duplicate CK-2 samples with different total CK activities showed poorer precision when analyzed after electrophoretic separation than by any other method. A 1993 survey designed to validate these observations conclusively showed that electrophoresis-based assays are subject to a bias proportional to the total CK activity. These survey results were confirmed by studies with selected patients' specimens. We therefore conclude that electrophoresis-based assays may not warrant their reputation as the gold standard for CK isoenzyme measurement.

Analytical and Clinical Evaluation of Creatine Kinase MB Mass Assay by IMx: Comparison with MB Isoenzyme Activity and Serum Myoglobin for Early Diagnosis of Myocardial Infarction

We analytically and clinically evaluated Abbott's IMx assay for creatine kinase (CK) isoenzyme MB (CK-MB) in serum. Over a 1-year period, the method was more specific but less precise than catalytic isoenzyme measurements by electrophoresis or immunoinhibition. Sera from different individuals without electrophoretic evidence of CK-MB but containing macro CK type 1 (n = 20), mitochondrial CK (n = 5), or CK-BB (n = 5) were scored as CK-MB negative by the IMx. Likewise, CK-MB-negative by the sera remained so after addition of purified human CK-MM (< or = 7600 U/L) or CK-BB (< or = 8100 U/L). For 39 patients admitted for suspicion of uncomplicated acute myocardial infarction (precordial pain for < or = 4 h), the diagnostic performance of the IMx CK-MB assay on admission and 4 h later was superior to that of total CK activity and compared well with that of CK-MB activity measured by electrophoresis or immunoinhibition. An admission, myoglobin showed a higher diagnostic sensitivity, specificity, and predictive value than did CK-MB and was the most informative test. Diagnostic performance on admission and 4 h later was further improved by considering positivity for myoglobin and for CK-MB by IMx and for the change in each over the first 4 h of hospitalization as criteria. Twelve hours after admission, diagnostic performance was further improved for all CK and CK-MB methods but began to decline for myoglobin.

Urinary N-acetyl-β-D-glucosaminidase isoenzyme measurement in lead-exposed workers

Urinary N-acetyl-β-D-glucosaminidase isoenzyme measurement in lead-exposed workers

Serum creatine kinase isoenzyme measurements in master male and female marathon runners

This study investigated exercise‐induced muscle enzyme release in master runners by assessing the activity of total serum creatine kinase (total CK) and the concentration of the isoenzyme creatine kinase MB (CK‐MB). Subjects were 12 male (54 ± 9 years) and seven female (50 ± 6 years) master marathon runners. Premarathon (24 hours before) and postmarathon serum samples were collected (24, 48, 72, and 96 hours after the race). Total CK was measured enzymatically and CK‐MB concentration was assayed utilizing a double antibody procedure. The men's and women's 24 hour (1012 U/L, 782 U/L) and 48 hour (581 U/L, 441 U/L) postrace mean total CK activities were significantly (p≤0.05) elevated above prerace values (66 U/L, 119 U/L). CK‐MB concentration 24 hours postrace was also significantly (≤0.05) elevated from prerace values (0.27 ng/ml, 0.0 ng/ml) in both males (34.4 ng/ml) and females (16.7 ng/ml). The results of this study indicate that the pattern of exercise‐induced muscle enzyme release from the stress of ...

Urinary N-acetyl-.BETA.-D-glucosaminidase isoenzyme measurement by the isoelectrofocusing-MCP-NAG method.

Urinary N-acetyl-.BETA.-D-glucosaminidase isoenzyme measurement by the isoelectrofocusing-MCP-NAG method.

Evaluation of two new methods for routine measurement of alkaline phosphatase isoenzymes.

To evaluate the performance of two new methods for the analysis of alkaline phosphatase (ALP) isoenzymes designed for use in the routine chemical pathology laboratory: pre-incubation with neuraminidase before agarose electrophoresis; and selective precipitation of the bone isoenzyme with wheat germ agglutinin (WGA). Serum samples from 39 patients were analysed. Seventeen were from patients with liver disease, eight from patients with bone disease, and 14 from patients with normal ALP activity and no evidence of liver or bone disease. The two new methods were compared with the established method, wheat germ agglutinin affinity electrophoresis. There was good correlation between the neuraminidase and WGA electrophoretic methods. The WGA precipitation method showed negative interference in the measurement of bone isoenzyme activity in samples containing biliary alkaline phosphatase. Both the new methods had advantages of speed and simplicity over the existing method, but cost per test was considerably higher. The neuraminidase electrophoretic method is a satisfactory alternative to the WGA affinity electrophoretic method, although it is more expensive. The WGA precipitation method cannot be recommended for use with serum samples from patients with suspected liver disease.

Creatine kinase isoenzymes in the diagnosis of intestinal infarction

Total creatine kinase and its isoenzymes CK-MB and CK-BB were measured in the serum of patients admitted with acute abdominal pain or signs suggestive of an intraabdominal catastrophe. Total creatine kinase was measured by automated spectrophotometry, CK-MB by chemiluminescent assay, and CK-BB by radioimmunoassay. Patients were grouped according to their final diagnosis: intestinal infarction (N = 8); all other diagnoses (N = 22); controls (N = 20). CK-BB in the infarction group (22.3 +/- 5.3 ng/ml, mean +/- SE) was significantly greater (P less than 0.01) than in the noninfarction or the control groups (11.0 +/- 0.8 ng/ml and 5.8 +/- 0.7 ng/ml, respectively). There were no differences in total creatine kinase and CK-MB in the three groups. Stepwise deletion multiple regression analysis of 26 independent regressors showed that among a cluster of six significant variables (R2 = 0.92, P less than 0.005), CK-BB greater than 20 ng/ml was the best predictor of intestinal infarction. Results of this study indicate that CK-BB isoenzyme measurement may be useful in the diagnosis of intestinal infarction in man.

Automated measurement of amylase isoenzymes by a double kinetic assay with "blocked" beta-2-chloro-4-nitrophenyl maltopentaoside as substrate and with wheat germ inhibitor

We evaluated the enzymic mechanism by which 3-keto butylidene-beta-2-chloro-4-nitrophenyl maltopentaoside (3KB-G5-CNP) serves as a substrate for serum pancreatic (p-) and salivary (s-) amylases. In aliquots of the reaction mixture, three kinds of beta-2-chloro-4-nitrophenyl oligosaccharides (glucose, maltoside, and maltotrioside) were separated from the substrate by high-performance liquid chromatography. Both isoenzymes behaved nearly identically and produced almost the same products. We automated a double kinetic procedure for determining total (t-) and p-amylase with use of a selective inhibitor from wheat germ in a single channel on the Hitachi 7050 analyzer. Within- and between-run CVs were, respectively, 0.5% and 1.7% for t-amylase (240 U/L), and 0.7% and 2.3% for p-amylase (230 U/L). The test results varied linearly with concentrations up to approximately 2000 U/L for t- and p-amylase activities. p/s ratios varied from 0.2 to 5.0. Results correlated well with those obtained by the monoclonal inhibition method (r = 0.992).

Measurement of carbonic anhydrase isoenzymes in early human placental tissues

S. ALIAKBAR,*t P. R. BROWN,* E. JAUNIAUX,t D. E. BIDWELLS and K. H. NlCOLAIDESt *Department of Biochemistry, King’s College London, Strand, London WC2R 2L.S. tllarris Birthright Research Centre for Fetal Medicine, Department of Obstetrics and Gynaecology, King’s College Hospital, Denmark Hill, London SE5 8RX, and $ Nufield Laboratories of Comparative Medicine, The Zoological Society of London, Regent ’s Park, London NWl4RY, U.K.

Aspartate aminotransferase isoenzymes

Aspartate aminotransferase (AST, EC 2.6.1.1) exists in human tissues as two distinct isoenzymes, one located in the cytoplasm (c-AST), and the other in mitochondria (m-AST). Striated muscle, myocardium, and liver tissues are the main sources of AST. A growing body of information suggests that determination of AST isoenzymes in human serum is useful in evaluating damage to some of these organs. In hepatic disease, the test is used to assess liver necrosis and for determining prognosis. It may also assist in identifying patients with active alcoholic liver disease. In patients with acute myocardial infarction, measurement of AST isoenzymes provides diagnostic information that differs from that obtained by determination of total creatine kinase and lactate dehydrogenase enzymes, and their isoenzymes.

The Impact of Technology on the Management of Pancreatic Pseudocyst

The records of 299 patients with 357 admissions for pancreatic pseudocysts seen between 1960 and 1989 were studied; 233 patients underwent operation. The natural history of pancreatic pseudocysts has been clarified by newer technology, such as ultrasonography, computer tomography, amylase isoenzyme measurements, and endoscopic retrograde cholangiopancreatography. All have influenced diagnosis, nonoperative management, and surgical operation. Differences between pancreatic pseudocysts associated with acute pancreatitis in contrast with chronic pancreatitis, and the complications of obstruction, hemorrhage, rupture, pancreatic ascites, infection, and jaundice can now be more rationally treated. Pancreatic pseudocysts and pancreatic ductal changes are now revealed earlier, especially by endoscopic retrograde cholangiopancreatography. Paradoxically, this information has encouraged nonoperative conservative therapy and also larger operations, eg, resection and adjunctive pancreaticojejunostomy. Partial resection of the pancreas together with the pancreatic pseudocysts was performed in 58 (25%) of the 233 patients. Recent technology permits cautious exploration of selective pancreatic pseudocyst drainage percutaneously or transgastroduodenally avoiding laparotomy.

Hypoxic risk in twins assessed by serum creatine kinase brain isoenzyme measurement

A marked intrapair discordance in placentas and in many body and organ measurements are risk factors influencing perinatal mortality and morbidity in twins. Asphyxia is the single most important perinatal cause of neurologic morbidity in newborn infants. The higher hypoxic risk for the second twin arises, however, from conclusions based on studies that did not consider the new diagnostic possibility of using blood measurements of the brain-type isoenzyme of creatine kinase (CK-BB) as a marker of perinatal asphyxia. CK-BB levels were measured in cord blood of 60 preterm infants (mean birth weight 1670 ± 390 g, and mean gestational age 33 ± 1.9 weeks) born of twin gestation in the last 3 years. The mean CK-BB values were 48 ± 40 U/1 versus 29 ± 31 U/1 (p > 0.5). Skilful antepartum and perinatal care are the keys for optimal management of both babies, as demonstrated by similar CK-BB values obtained in their cord-blood specimens after birth.

Biochemical timing of peri-intraventricular hemorrhage assessed by perinatal CPK-BB isoenzyme measurements.

Precise diagnosis of peri-intraventricular hemorrhage (PIVH) requires brain real-time ultrasound imaging procedure (US). However, maximal diagnostic efficiency of US lies between day 4 and 14 since fresh blood may initially appear sonolucent. Because of this supposed interval required for clot formation to become visible on US, serum CPK-BB estimations were performed in the first 60 hours of life to determine precise biochemical timing of PIVH. A group of 50 preterm infants less than 1500 g birth weight (1120 +/- 320 g) and 34 weeks gestation (30 +/- 3.7 weeks) was studied. Serial CPK-BB measurements were performed in serum immediately after birth (T0), then serially at time T1 (6-10 h), T2 (20-30 h), T3 (40-60 h). The incidence of PIVH diagnosed on the third day of life was 30%. Total CPK-BB values at T0 in infants who developed PIVH were significantly higher than those of patients without cerebral bleeding (70.8 +/- 30.5 vs 20.9 +/- 10.7 U/l) (p less than 0.05). The same statistically significant results were not observed analysing the CPK-BB values at T1, T2 and T3. These results suggest that most pathological conditions responsible for enzyme release occur in the pre- or perinatal period.

Early diagnosis of myocardial infarction by timed sequential enzyme measurements.

Serum samples from patients admitted to a coronary care unit with a history of acute chest pain suggestive of myocardial infarction in the previous 12 h were obtained on admission and at 6 and 12 h, thereafter. Creatine kinase (CK), CK-MB isoenzyme, CK-MM sub-bands, myoglobin, and lactate dehydrogenase (LD) isoenzymes were examined. Changes were evaluated in relation to the diagnosis obtained from clinical examination, serial electrocardiography and 'routine' cardiac enzymes (CK, aspartate transaminase and alpha-hydroxy butyrate dehydrogenase daily for 3 days following admission). The slope of the logarithms of CK, CK-MB activity and CK-MB concentration in the early post infarct period fully distinguished between infarct and non-infarct patients. Measurement of myoglobin and lactate dehydrogenase isoenzymes was less sensitive. Serial estimation of CK-MM sub-band patterns allowed the time from infarction to be estimated. Serial estimation of CK in the 12 h following admission can be substituted for conventional daily enzyme estimations for the diagnosis of acute myocardial infarction in patients with onset of chest pain within the previous 12 h. This could reduce laboratory and in-patient costs.

Regional creatine kinase, adenylate kinase, and lactate dehydrogenase in normal canine brain.

Following acute stroke, creatine kinase and other enzymes are released into the cerebrospinal fluid and blood from injured brain tissue. To determine whether regional differences in brain enzyme activity might exist and therefore affect the amount of enzyme released, we quantified the levels of creatine kinase, adenylate kinase, and lactate dehydrogenase in 12 regions of normal canine brain (n = 4). Adenylate kinase activity varied the least among regions (49 +/- 7 units/g), followed by lactate dehydrogenase activity (122 +/- 28 units/g). The pattern for both adenylate kinase and lactate dehydrogenase was higher activity in predominantly gray matter areas, lower activity in white matter, and intermediate activity in mixed regions. The distribution of creatine kinase brain isoenzyme and mitochondrial creatine kinase in canine brain was less predictable, showing wider variations among regions (isoenzyme, 462 +/- 116 units/g; mitochondrial, 42 +/- 20 units/g). Even cerebral gray matter demonstrated substantial regional variations in creatine kinase brain isoenzyme, ranging from 606 units/g in the parietal cortex to 329 units/g in the temporal cortex. We conclude that the content of creatine kinase brain isoenzyme varies more than twofold among areas of brain. This regional variation may be important in the interpretation of creatine kinase brain isoenzyme measurements in cerebrospinal fluid and serum used to assess neurologic injury following stroke.

Coronary and skeletal muscle enzyme changes during a 14 km run.

Increasing coronary enzyme values have previously been demonstrated after physical exercise. In the present study serum values of aspartate aminotransferase (ASAT), lactate dehydrogenase (LDH), LDH isoenzymes, creatinine kinase (CK), and the myocardial-bound fraction of this enzyme (CK-MB) were measured in a random sample of healthy non-smoking, physically fit men before and immediately after a competitional 14 km run. All enzyme values increased during the run, ASAT by 7%, LDH by 25%, CK by 38%, and CK-MB by 53%. Measurement of LDH isoenzymes showed special increments of the non-cardiac fractions. However, absolute increments of the cardiac fractions of the LDH isoenzymes together with other serum enzyme elevations do not exclude a cardiac source of enzyme release during and following physical exercise.

A New Method for Reporting the Sources of Abnormal Activities of Lactate Dehydrogenase in Serum

We have developed and tested a new method to increase the diagnostic usefulness of measurements of lactate dehydrogenase (LDH; EC 1.1.1.27) isoenzymes. The method estimates the separate contributions from enzymatically distinct organ clusters (e.g., heart/kidney/erythrocyte, liver/muscle, lung) to the total activity of LDH in serum. To test this method, we monitored serum LDH isoenzymes over the entire hospital course of 73 patients admitted to the intensive-care unit with chest pain, myocardial infarction, or serious hemodynamic disturbances. The organ-specific estimates provided useful information beyond measurements of the original isoenzymes. The sensitivity and specificity of this new method in detecting acute myocardial infarction, as well as concomitant disorders involving the liver or lung, are significantly greater than those of other diagnostic indices or pathologists' judgments. Serial plots of the organ-specific estimates may provide additional insight into evolving pathophysiological processes.

Neuron-specific enolase. Assessment by ELISA in patients with small cell carcinoma of the lung.

Measurement of tissue-specific enolase isoenzymes may be of assistance in identifying small cell carcinomas of the lung and in distinguishing them from other pulmonary tumors. Enolase (E.C. 4.2.1.11) is a dimeric enzyme composed of various permutations of three immunologically distinct subunits alpha, beta, and gamma. Five isoenzymes alpha alpha, beta beta, gamma gamma, alpha beta, and alpha gamma have been identified. Immunohistochemical studies using antibodies to the gamma subunit have localized alpha gamma and gamma gamma specifically within neuronal and neuroendocrine tissues. Because of this limited distribution, neuron-specific enolase (NSE) can function as a biochemical marker for neuroendocrine tumors. The authors developed an enzyme-linked immunosorbent assay (ELISA) using the double antibody sandwich method. The sandwich is composed of rabbit antirat enolase that cross-reacts to the human gamma monomer, making the test specific for the gamma gamma isoenzyme. The avidin-biotin-peroxidase complex system is used to provide increased assay sensitivity. Serum samples from patients with histologically diagnosed small cell carcinoma have concentration of NSE 20- to 30-fold greater than that found in normal serum. Studies were conducted on patients with a variety of malignant pulmonary lesions and compared with controls to determine the value of NSE as a tumor marker.