Changes in the ratio of lactate dehydrogenase isoenzymes 1 and 2 during the first day after acute myocardial infarction

It is known that the ratio of isoenzyme 1 to total lactate dehydrogenase (LD, EC 1.1.1.27) in serum is increased in all patients with acute myocardial infarction within 24 h of the infarct. We now show that the LD-1/LD-2 ratio for serum more promptly indicates acute myocardial infarction, being for most patients equivalent to measurement of creatine kinase (EC 2.7.3.2) isoenzyme 2 (CK-2, CK-MB) in serum. Of 128 patients with a confirmed diagnosis of myocardial infarction, 66 had normal values for all "cardiac" enzymes at the time of admission, but greater than 75% of them showed a parallel increase in values for CK-2 and the LD-1/LD-2 ratio. Of the 26 patients who had one or more abnormal values for cardiac enzymes on admission, 95% showed a parallel increase in CK-2 and the LD-1/LD-2 ratio, the median time for the beginning of these changes being 9 h from the onset of chest pain. The remaining 36 patients were excluded from the study because CK-2 decreased after admission or because the time of onset of chest pain was uncertain.     

Two methods compared for measuring LD-1/total LD activity in serum

We present evidence for the utility of an improved assay for the activity of lactate dehydrogenase (EC 1.1.1.27) isoenzymes 1 and 2 in serum, involving inhibition of the H-subunit of LD by pyruvate at pH 7.1. Results correlate well with the LD-1/total LD ratio as evaluated by immunological assay and, as an index to infarct, the method is superior to either the change in CK-MB activity or to the LD-1 activity or to a combination of these tests, as is the percentage of LD-1 to total LD activity. Moreover, the percentage inhibition of LD activity by pyruvate may have an advantage over other methods of isoenzyme fractionation because of its smaller population CV for patients with acute myocardial infarction than is true of other methods. We also demonstrate how, using a linear discriminant analysis, we compared this method with alternative methods. We determined that evaluation of CK-MB isoenzyme contributes no information in addition to that obtained from the LD-1 isoenzyme.     

Increased activities of creatine kinase and lactate dehydrogenase isoenzymes in a patient with metastatic ovarian tumor

A 50-year-old woman with metastatic rhabdomyosarcoma of the ovary had increased activities of creatine kinase (CK; EC 2.7.3.2), CK-MB isoenzyme, lactate dehydrogenase (LD; EC 1.1.1.27), and LD-2 isoenzyme in her serum. The isoenzyme activities did not show a pattern of increasing, then decreasing. Clinical findings, including electrocardiograms, did not support the diagnosis of myocardial infarction. We suggest that high activities of CK-MB and LD-2 in serum may serve as a marker of rhabdomyosarcoma.     

Determination, by radioimmunoassay, of the mass of lactate dehydrogenase isoenzyme 1 in human serum and of its rate of removal from serum after a myocardial infarction

In this radioimmunoassay of lactate dehydrogenase-1 (LD-1; EC 1.1.1.27) in human serum we use a commercial LD-1-selective assay system and a goat antiserum. We have determined the fractional rate of disappearance from serum and the half-life of LD-1, in terms of both enzyme activity and enzyme mass, in 21 myocardial infarction patients. Our evidence suggests that this isoenzyme is inactivated in serum. Furthermore, our data suggest that the conventionally accepted half-life of about 110 h for serum LD-1 activity may grossly overestimate the actual LD-1 half-life in many post-myocardial infarction patients.     

An evaluation of the immunochemical LD-1 method in routine clinical practice

We report our extended clinical experience with the use of an immunochemical method for LD-1 assay in 260 unselected, consecutive patients admitted with the clinical suspicion of recent myocardial infarction (M.I.). We determined on every patient total creatine kinase (CK) and total lactate dehydrogenase (LD) enzyme activity, and performed electrophoresis for LD isoenzymes as well as the heart-specific band of creatine kinase (CK-MB). An immunochemical assay for the heart-specific isoenzyme of LD (LD-1) was also performed. The timing of the samples was determined by the clinicians according to routine clinical protocols in the coronary care units. The diagnosis was based on the usual combination of clinical, electrocardiographic (EKG) and laboratory findings, and was arrived at independently by the clinician. In this extended series, the overall efficiency of the immunochemical LD-1 assay for the proper classification of the patients according with the discharge diagnosis was 92%. For CK-MB it was 90%, for EKG 77% and for LD electrophoresis 76%. The immunochemical LD-1 assay required no special instruments or highly skilled technicians and is probably the method of choice for the stat evaluation of recent M.I.     

Increased lactate dehydrogenase isoenzyme 1 in serum and tumor tissue of a patient with small-cell carcinoma

Histological examination of supraclavicular lymph node tissue obtained at biopsy from a 63-year-old man disclosed metastatic small-cell carcinoma. On admission and for four days subsequently, total lactate dehydrogenase (LD; EC 1.1.1.27) activity in serum was 6.5 times normal; studies of LD isoenzyme showed persistently increased LD-1, with LD-1 greater than LD-2. Isoenzyme electrophoresis of tissue homogenates prepared from the patient's tumor also showed the LD-1 greater than LD-2 pattern. Isoenzyme studies for supraclavicular lymph node tissue from five control subjects showed contrasting isoenzyme patterns as compared with the patients in whom LD-2, LD-3, and LD-4 predominated. Because these abnormalities were persistent, they differ from the temporal sequence for LD usually seen in myocardial infarction. This emphasizes the importance of repetitive sampling for clinical interpretation of data on this enzyme.     

"Flipped" patterns of lactate dehydrogenase isoenzymes in serum of elite college basketball players

We kinetically measured total lactate dehydrogenase (LD, EC 1.1.1.27), total creatine kinase (CK, EC 2.7.3.2), and aspartate aminotransferase (AST, EC 2.6.1.1.) in 16 elite college basketball players, before the competition season and not in close temporal relation to near-maximal exercise, and in 17 healthy non-athlete controls. LD isoenzymes were determined by both electrophoretic and immunoprecipitation methods. CK-MB isoenzyme was measured electrophoretically. We found significantly higher mean LD-1 values and LD-1/LD-2 ratios in the players than the controls: 31.6 (SD 3.7)% vs 25.8 (SD 3.2)% (P less than 0.005) and 1.1 (SD 0.13) vs 0.87 (SD 0.16) (P less than 0.001), respectively. A "flipped" LD pattern (LD-1 greater than LD-2) was found in half the players and in six of the eight black athletes, but in only two of the control group and in none of the black controls. Mean CK activity in serum exceeded normal values in the serum of the athletes and was higher in comparison with the control group [274 (SD 156) vs 103 (SD 82) U/L]. Mean CK was significantly higher in the eight athletes with the flipped LD pattern than in those with LD-1 less than LD-2 [322 (SD 163) vs 180 (SD 98) U/L; P = 0.05], and also in comparison with CK in the two controls with flipped LD pattern. We saw no significant difference in mean CK between the nine players with normal immunochemical LD-1/LD ratios and the seven players with above-normal ratios. CK-MB was not detected in either athletes or controls. None of the players had any clinical or electrocardiographic evidence for myocardial ischemia or infarction. Evidently the flipped LD pattern usually found in patients with acute myocardial infarction and reported in some athletes after extreme exercise such as ultra-marathon running may also be found in athletes who are in their "basal fitness shape" but who are not involved in competitive physical activity.     

Lactate dehydrogenase isoenzyme 1: Its usefulness as a screening test in diagnosis of myocardial infarction

Immunological assay of LD-1 activity provides a quantitative measurement of the type of lactate dehydrogenase (LD, EC 1.1.1.27) activity characteristic of myocardial origin. Using this test, a laboratory diagnosis of myocardial infarction can be either ruled out or confirmed in approximately 75% of patients in whom this diagnosis is suspected, without electrophoretic separation of creatine kinase (CK, EC 2.7.3.2.) and LD isoenzymes. Normal total CK and LD activities cannot be used to rule out myocardial infarction since CK-MB and LD-1 may have increased although total activities remain within their reference ranges. LD-1 activity increases as quickly as CK-MB following the onset of pain in the majority of patients but it remains elevated longer giving a greater period of time during which the diagnosis of myocardial infarction can be confirmed.     

Selective determination of lactate dehydrogenase isoenzyme 1 in serum after inhibition by 1,6-hexanediol

A rapid selective method for measuring the activity of lactate dehydrogenase isoenzyme LD-1 in serum by using 1,6-hexanediol as an inhibitor of the M-subunit was developed. Hexanediol was added to serum at a final concentration of 0.7 mol/l. After incubation at 30 degrees C for 15 min, the activity was measured with an automatic analyser. The inter-assay coefficient of variation was 6.9% for the lactate dehydrogenase isoenzyme LD-1 measurement. The results obtained from the sera of 100 patients analysed by the proposed selective method and by the conventional electrophoretic method, respectively, showed an excellent correlation. This selective method was used to determine the lactate dehydrogenase isoenzyme LD-1 activity of sera from patients with acute myocardial infarction, and the results were correlated well with those obtained by the immunological, Roch Isomune method. Addition of 1,6-hexanediol did not affect the measurement of activities of other enzymes such as alkaline phosphatase, gamma-glutamyltransferase, aspartate aminotransferase and alanine aminotransferase.     

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.     

Cardiac enzymes. How to use serial determinations to confirm acute myocardial infarction.

Although acute myocardial infarction can be diagnosed on the basis of clinical history, electrocardiographic (ECG) findings, and abnormalities of creatine kinase (CK) and lactate dehydrogenase (LDH) enzyme levels, measurement of cardiac enzyme levels is the most reliable way to confirm or exclude the diagnosis. If the MB isoenzyme of creatine kinase (CK-MB) remains normal during the 48 hours after the suspected clinical event, acute myocardial infarction can be reliably ruled out; if CK-MB values become elevated and the LDH isoenzyme pattern (LDH2:LDH1 ratio) becomes "flipped," the diagnosis can reliably be made. However, if CK-MB values become elevated but the LDH isoenzyme pattern remains normal, the diagnosis is less firm and ECG and myocardial imaging techniques may be needed to confirm or exclude myocardial infarction.     

Improved column method for separating lactate dehydrogenase isoenzymes 1 and 2

Lactate dehydrogenase (LD) isoenzymes 1 and 2 in human serum were separated on a column of diethylaminoethyl-Sephadex. Samples layered on mini-columns were eluted with buffered sodium chloride (100, 150, and 200 mmol/liter). Lactate dehydrogenase activity in column effluents was measured by the Wacker method, and their isoenzyme content was evaluated by electrophoresis on polyacrylamide gel. Results for column-fractionated LD-1 and LD-2 were expressed in two ways: LD-1/LD-2 ratios and total LD-1 + LD-2 activities. The former is a more specific indicator of myocardial infarction than the latter. Sera from 10 patients with acute myocardial infarction (increased creatine kinease isoenzyme MB activity) exhibited ratios in the range of 0.92 to 1.56, ratios for 10 patients without heart disease (normal creatine kinase MB) ranged from 0.33 to 0.69.     

Atypical patterns of lactate dehydrogenase isoenzymes in acute myocardial infarction

Total lactate dehydrogenase (LD; EC 1.1.1.27) activity in serum and LD isoenzymes were quantified in 190 patients with acute myocardial infarction (AMI) 24, 48, and 72 h after admission. In 90% of the 570 blood specimens an LD isoenzyme pattern typical of AMI (LD-1/LD-2 greater than 0.76) was found. The other 56 blood specimens showed an LD isoenzyme pattern atypical of AMI (LD-1/LD-2 less than 0.76). They were divided into three groups: 28 specimens with isomorphic pattern (relative increase in all five LD isoenzymes); 18 with relatively increased LD-3 proportion (greater than 35%); and 10 specimens with increased LD-5 proportion (greater than 10%). No difference was found in mean total LD activity in serum between the typical isoenzyme group and the three atypical groups. The LD isomorphic pattern was found in 60% of AMI patients complicated by cardiogenic shock. Fifty percent of AMI patients admitted with pulmonary edema showed increased LD-3 proportion and half of the patients with AMI and congestive heart failure, predominant right, demonstrated increased LD-5 proportion. We conclude that although most patients with AMI present at diagnosis with a typical LD isoenzyme pattern, it is important to recognize that some may present with atypical LD isoenzyme patterns, which may be associated with specific AMI complications.     

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.     

Laboratory diagnosis of patients with acute chest pain.

The enzyme activities of creatine kinase (CK), its isoenzyme MB (CK-MB) and of lactate dehydrogenase isoenzyme 1 (LD-1) have been used for years in diagnosing patients with chest pain in order to differentiate patients with acute myocardial infarction (AMI) from non-AMI patients. These methods are easy to perform as automated analyses, but they are not specific for cardiac muscle damage. During the early 90's the situation changed. First creatine kinase MB mass (CK-MB mass) replaced the measurement of CK-MB activity. Subsequently cardiac-specific proteins troponin T (cTnT) and troponin I (cTnI) appeared on the scene, displacing LD-1 analysis. However, troponin concentrations in blood increase only from four to six hours after onset of chest pain. Therefore a rapid marker such as myoglobin, fatty acid binding protein or glycogen phosphorylase BB could be used in early diagnosis of AMI. On the other hand, CK-MB isoforms alone may also be useful in rapid diagnosis of cardiac muscle damage. Myoglobin, CK-MB mass, cTnT and cTnI are nowadays widely used in diagnosing patients with acute chest pain. Myoglobin is not cardiac-specific and therefore requires supplementation with some other analyses such as troponins to support the myoglobin value. Troponins are very highly cardiac-specific. Only the sera of some patients with severe renal failure, which requires hemodialysis, have elevated cTnT and/or cTnI without there being any evidence of cardiac damage. On the other hand, the latest studies have shown that elevated troponin levels in sera of hemodialysis patients point to an increased risk of future cardiac events in a similar manner to the elevated troponin values in sera of patients with unstable angina pectoris. In addition, the bedside tests for cTnT and cTnI alone or together with myoglobin and CK-MB mass can be used instead of quantitative analyses in the diagnosis of patients with chest pain. These rapid tests are easy to perform and they do not require expensive instrumentation. For routine clinical laboratory practice we suggest that in diagnosis of patients with chest pain, myoglobin and CK-MB mass measurements should be performed whenever they are requested (24 h/day) and cTnT or cTnI on admission to the hospital and then 4-6 and 12 hours later.     

Differentiating muscle damage from myocardial injury by means of the serum creatine kinase (CK) isoenzyme MB mass measurement/total CK activity ratio

We immunoenzymometrically measured creatine kinase (CK) isoenzyme MB in extracts of myocardium and in homogenates of five different skeletal muscles. CK-MB concentrations in the former averaged 80.9 micrograms/g wet tissue; in the skeletal muscles it varied widely, being (e.g.) 25-fold greater in diaphragm than in psoas. CK-MB in skeletal muscles ranged from 0.9 to 44 ng/U of total CK; the mean for myocardium was 202 ng/U. In sera from 10 trauma and 36 burn patients without myocardial involvement, maximum ratios for CK-MB mass/total CK activity averaged 7 (SEM 1) ng/U and 18 (SEM 6) ng/U, respectively. Except for an infant (220 ng/U), the highest ratio we found for serum after muscular damage was 38 ng/U. In contrast, the mean maximum ratio determined in 23 cases of acute myocardial infarction exceeded 200 ng/U. Among seven determinations performed 8 to 32 h after onset of symptoms, each infarct patient demonstrated at least one ratio greater than or equal to 110 ng/U. Ratios observed after infarct were unrelated to treatment received during the acute phase. We propose a CK-MB/total CK ratio of 80 ng/U as the cutoff value for differentiating myocardial necrosis from muscular injury.     

Production of recombinant human creatine kinase (r-hCK) isozymes by tandem repeat expression of M and B genes and characterization of r-hCK-MB

BACKGROUND: Serum creatine kinase-MB isoenzyme (CK-MB) is widely used as a marker of myocardial injury. We prepared recombinant human CK (r-hCK) MB isoenzyme and examined its potential for use as a control material for assay of CK-MB in serum. METHODS: cDNAs encoding CK-M and CK-B subunits were inserted into the same plasmid vector, followed by transformation of Escherichia coli. The resulting three types of CK isoenzymes were purified by conventional chromatography. RESULTS: The ratio of MB to MM to BB was 50:40:10 on the basis of CK activity. Highly purified CK-MB with a specific activity of 533 U/mg was produced in a yield of 5.7 mg/g of packed cells. Purified r-hCK-MB had the isoelectric point (pI 5.3) and molecular size (46 kDa for the subunit) of native CK-MB. Its immunoreactivity in an ELISA using antibody against native heart enzyme was similar to that of cardiac CK-MB. The r-hCK-MB retained >90% activity for at least 4 months at 11 degrees C in a delipidated serum matrix in a liquid form at a concentration of 118 U/L. CONCLUSIONS: r-hCK-MB shows key properties of the native cardiac isoenzyme and may be useful as a control and calibrator for serum assays of CK-MB.     

Muscle enzymes and isoenzymes in Emery-Dreifuss muscular dystrophy

Emery-Dreifuss muscular dystrophy (EDMD) is a rare X-linked muscular dystrophy. Creatine kinase (CK) activity usually is increased in serum of affected males, but results for aldolase and lactate dehydrogenase (LD) in serum have been inconsistent, as have those for CK in carrier females. There have been few studies of CK-MB or LD isoenzyme-1 (LD-1) in EDMD. We measured CK, CK-MB, LD, LD-1, and aldolase activity in sera of 84 members of two large families with EDMD. DNA analysis had been carried out on all subjects. Although CK, LD, and aldolase activities were significantly increased in affected males, CK activity was the most consistently increased and was the least subject to artifactual increases. Mean CK-MB in serum was mildly increased, but LD-1 was within the normal reference interval, suggesting that CK-MB is increased in skeletal muscle in EDMD, as has been found in other forms of dystrophy. CK decreased with age in affected males. We saw no significant increases of muscle enzymes or isoenzymes in 33 EDMD carriers studied, of whom 19 were obligate carriers and 14 had been identified by DNA analysis.     

Clinical effectiveness of the Du Pont aca measurement of creatine kinase MB in serum from patients in a coronary-care unit

We evaluated the clinical effectiveness of measuring creatine kinase (CK; EC 2.7.3.2) isoenzyme MB and lactate dehydrogenase (LD; EC 1.1.1.27) isoenzymes in diagnosis of acute myocardial infarction. We used an agarose electrophoresis method to measure CK and LD isoenzymes and the Du Pont aca column method to measure CK-MB. Serial blood specimens were drawn from 100 patients consecutively admitted to our Coronary Care Unit. Because of the low diagnostic specificity for CK-MB measurements by both agarose electrophoresis and the discrete-analysis method, as compared with reported values, we re-evaluated our isoenzyme data by using Receiver Operating Characteristic curves. Such analysis of the data established optimal decision levels of greater than or equal to 25 U/L and greater than or equal to 18 U/L plus greater than or equal to 6% of total CK for serum CK-MB measured by the agarose electrophoresis and the aca methods, respectively, and an optimal decision level of greater than or equal to 0.92 for the ratio of LD 1/2 measured after agarose electrophoresis. At these decision levels we obtained a sensitivity of 100%, 100%, and 95% and a specificity of 94%, 92%, and 90% for CK-MB (agarose electrophoresis), CK-MB (aca), and the LD 1/2 ratio, respectively.     

IgA-CK-BB complex with CK-MB electrophoretic mobility can lead to erroneous diagnosis of acute myocardial infarction

This patient, on admission, presented with a tentative diagnosis of myocardial infarction: the electrocardiogram showed a nonspecific ST-segment and T-wave abnormalities, and total creatine kinase (CK; EC 2.7.3.2) activity was slightly increased (238 U/L). However, a high electrophoretic value for CK-MB (50% of total CK activity) and the electrophoretic pattern of lactate dehydrogenase (EC 1.1.1.27) isoenzymes ruled out myocardial infarction. The isoenzyme migrating as CK-MB was found later to contain no immunologically normal CK-M subunits, and it was bound to IgA. A mixture of the patient's serum and a human serum control containing all CK isoenzymes showed altered electrophoretic mobility only for CK-BB, indicating that the patient's serum contained antibodies to the B unit of CK. Elution from a Sephadex G-200 column showed that the peak at which most of the anodic CK was eluted corresponded to a molecular mass of approximately 200 kDa. Evidently this atypical isoenzyme was an IgA-CK-BB complex. Because this macro CK type 1 can mimic CK-MB, it may therefore be a source of confusion.