"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 patterns in serum of patients with metastatic liver disease

Total lactate dehydrogenase (LD, EC 1.1.1.27) and LD isoenzymes were determined in serum of 170 patients with metastatic liver disease, 35 of whom had multiple metastatic sites. Overall, values of LD were above normal for 78% of the 170 patients. Half of the patients had an isomorphic pattern of LD isoenzymes (i.e., relative increase in all five isoenzymes); the other half had an increased LD-4,5 pattern, mostly a solitary increase in LD-5 only. Of those patients with normal LD values, 92% had the increased LD-4,5 pattern, whereas 70% of patients with LD values greater than 350 U/L had an isomorphic pattern of LD isoenzymes. All 35 patients with multiple metastatic sites had LD activity greater than 350 U/L; in the majority of them (74%) it was greater than 500 U/L; in 31 (89%), the increase was isomorphic. The diagnostic efficiency of the combined LD greater than 225 U/L (upper limit of normal) and increased LD-4,5 test results was much better than that of LD greater than 225 U/L alone (93% vs 74%). We conclude that serum LD and LD isoenzymes should be determined in every patient with suspected liver metastatic disease. The isomorphic pattern of LD isoenzymes is apparently associated with higher values for total LD and was common among the patients with multiple metastatic sites.     

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.     

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.     

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.     

Mechanism of differential inhibition of lactate dehydrogenase isoenzymes in the BMC LD-1 assay

The mechanism of inhibition of lactate dehydrogenase (LD) isoenzymes by guanidinium thiocyanate (GSCN) used in the LD-1 assay developed by Boehringer Mannheim Corporation (BMC) was investigated. Michaelis-Menten inhibition kinetics for the individual isoenzymes revealed that GSCN competitively inhibited LD-1 in the presence of lactate and NAD+, but is a noncompetitive inhibitor of LD-5. LD-2 and LD-3 exhibited mixed inhibition kinetics. The inhibition constants were two- to threefold smaller for LD-5 than for LD-1. Time-dependent studies also showed that the isoenzymes underwent a different rate of inactivation by GSCN. LD-5, LD-3, and LD-2 were rapidly inactivated within 1 min under the BMC assay conditions, whereas LD-1 lost only about 20% of activity after 10 min. The presence of lactate further protects LD-1, but not other isoenzymes. Under this condition, LD-1 was not inactivated during the initial 6 min of reaction. Separate experiments demonstrated that both guanidinium and thiocyanate ions are responsible for the inactivation process that was found to be irreversible. We speculate that GSCN selectively denatures the M subunit of LD. The H subunit is less susceptible to denaturation and is further stabilized by lactate.     

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.     

Lactate dehydrogenase isoenzymes in serum during recent acute myocardial infarction

Lactate dehydrogenase (LD, EC 1.1.1.27) isoenzymes 1 and 2 and the LD 1:2 ratio were determined in 62 patients with recent myocardial infarction 24, 48, and 72 h after total serum LD activity had returned to normal values. From the results we could define two groups of patients. The first, 40 patients in whom proportions of LD-1 and LD-2 isoenzymes in serum and the LD 1:2 ratio were all within the normal reference interval, all had an uncomplicated course of recovery from myocardial infarction. In the remaining 22 patients, LD-1 still exceeded LD-2 24 to 72 h after total LD activity returned to normal values; i.e., the ratio was similar to that in patients with myocardial infarction. Seven of these 22 patients (32%) had a complicated course, with re-infarction in all seven. Thus, even in the presence of normal total LD activity, a high LD 1:2 ratio may reflect a consistent focal myocardial necrosis in some patients with recent myocardial infarction and may serve as an early marker for further re-infarction.     

Immunochemical properties of immunoglobulin G conjugated with lactate dehydrogenase

Quantitative binding affinities of immunoglobulin G (IgG) for the five isoenzymes of lactate dehydrogenase (LD; EC 1.1.1.27) were determined for IgG isolated from three patients' sera that contained LD-IgG complexes. These three IgGs formed soluble complexes with four (LD 1, 2, 3, 5) of the five LD isoenzymes in two of the patients and in the third they bound with three (LD 2, 3, 4) of the five LD isoenzymes without inhibiting the enzyme activity or precipitating with the enzyme molecules. When rabbit antibody to human IgG was added to these sera, the complexes between the LD isoenzymes and the patients' isolated IgG were completely precipitated. The equilibrium constants for the respective isolated IgG complexes with LD-3 were 0.102, 2.58, and 7.49 X 10(8) L/mol. In these three cases, LD-3 evoked the strongest response from the prepared IgG, demonstrating that the site of antigen recognition of LD-linked IgG was not associated with the structure of individual H and M subunits.     

Stability of lactate dehydrogenase at different storage temperatures

The effect of storing human serum, cord blood serum or heparinized plasma at 25 degrees C, 4 degrees C & -20 degrees C on the activity and isoenzyme distribution of lactate dehydrogenase (LD) was studied. Cellulose acetate and agarose electrophoresis, as well as an immunochemical inhibition technique, were used for isoenzyme quantification. In contrast to previous reports, cryo-instability was found only in specimens stored at 4 degrees C. Serum specimens stored at 25 degrees C and -20 degrees C retained 74% and 87% of total activity after 45 days of storage. LD-1 was stable at all three temperatures, with a maximum loss of 10%. LD-2, LD-3, LD-4, & LD-5 were most labile at 4 degrees C. Specimens that are to be analyzed for total LD or LD isoenzymes should be stored frozen or, if necessary, at room temperature, but not in a refrigerator. Thus, separate storage of specimens for cardiac isoenzymes (LD & creatine kinase) is not necessary. This may eliminate a possible source of falsely elevated LD-1/LD-2 ratios, as well as reducing the labor factor and the corresponding cost of cardiac isoenzyme determinations.     

Increased lactate dehydrogenase in serum in measles infection

We determined lactate dehydrogenase (LD; EC 1.1.1.27) in serum from 75 sequential measles patients and 105 control patients. In the measles patients, LD was increased to 1147.9 +/- 43.8 (mean +/- SE) U/L, markedly more than in the control group (517.2 +/- 15.0 U/L, p less than 0.001). Both LD-3 and LD-4 isoenzymes were increased, but the concentration of LD-5 was normal. Patients with high LD values (greater than 1500 U/L) had longer hospital stays than did those with lower values (p less than 0.01). Our results suggest that increased LD in serum is a common finding in measles infection and presumably originates from lymphocytes.     

Changes in serum enzymes, lactate, and haptoglobin following acute physical stress in international-class athletes

Skeletal muscle is rich in creatine kinase (CK), lactate dehydrogenase (LD), and other enzymes. Many reports describe changes in serum CK and LD following exercise. In our study, 11 male international-class medium-distance runners were followed over a 10-month period prior to the 1984 US Olympic Trials. Cardiorespiratory fitness, evaluated through repetitive treadmill testing, was unchanged in our athletes. Total CK increased significantly during the course of training, and the CK-MB activity was higher than that of sedentary individuals; CK-MB never rose to more than 3% of the total CK. Total LD also rose following acute exercise; however, the proportions of the five isoenzymes were unaltered. There was no change in the LD-1/LD-2 ratio from normal. The origin of the increased serum enzymes was believed to be primarily skeletal muscle. A decrease of serum haptoglobin following acute stress was attributed to intravascular hemolysis and binding of hemoglobin. As expected, serum lactate was dramatically increased immediately postexercise.     

Serum lactate dehydrogenase isoenzyme 4/5 ratio discriminates between hepatocarcinoma and secondary liver neoplasia.

Total lactate dehydrogenase (LD; EC 1.1.1.27) and its five isoenzymes were determined in sera from (a) 98 cases of cirrhosis at various stages classified according to Child and Turcotte; (b) 37 cases of hepatocarcinoma (HC) at different stages of the Okuda classification; (c) 17 patients with secondary liver neoplasia (SLN), mainly from an abdominal primary site; and (d) 19 cases of abdominal neoplasia without liver metastasis, in an attempt to contribute to the differential diagnosis between these conditions. LD-4 was enhanced in SLN and LD-5 in HC, thus indicating the LD-4/LD-5 ratio as a potential index with which to differentiate between HC and SLN patients. At a cutoff value of 1.05, 91% of these patients were correctly classified (82% for SLN and 95% for HC). Consequently, this biochemical index appears to be an efficient and rapid indicator to distinguish HC from SLN. On the other hand, the LD isoenzymes are unable to discriminate between HC and cirrhosis or between abdominal neoplasia with and without liver metastases.     

Inhibition of lactate dehydrogenase isoenzymes by sodium perchlorate evaluated

We evaluated a method of measuring lactate dehydrogenase isoenzyme 1 (LD-1) selectively (Clin Chem 1987;33:991-2), in which all other LD isoenzymes were inhibited by adding sodium perchlorate to the reaction medium to a final concentration of 0.825 mol/L. In this study we used the different isoenzymes purified from human autopsy tissue and found that the originally published amount of inhibitor (a) increased the original LD-1 activity and (b) did not eliminate all enzyme activity of LD-2 and LD-3. Interference by LD-2 was further demonstrated. Thus we conclude that this method cannot be used for the selective determination of LD-1 because its results do not accurately reflect the original LD-1 activity.     

Ten electrophoretic methods compared with a selected method for quantifying lactate dehydrogenase isoenzymes in serum

Using the Selected Method of McKenzie and Henderson (Selected Methods Clin Chem 1983;10:59-67) as a reference method, we compared the performance of 10 commercially available methods for determination of lactate dehydrogenase (LD, EC 1.1.1.27) isoenzymes. Results were expressed as percentage of total LD activity, as determined with two different types of densitometers shown to have an average difference less than 1.4% for each isoenzyme. All methods gave generally comparable results, as judged by Bland-Altman plots and correlation analyses. However, in general, estimates by the commercial methods for LD-1, LD-2, and LD-3 were lower, and for LD-4 and LD-5 were higher than with the Selected Method. The overall CV was less than 20% for all methods and isoenzymes, except for LD-4 and LD-5 by the Beckman Paragon, Helena LD-VIS, Gel LDH, Gel PC, and Iso Dot, Gelman LDH Isozyme, and Sebia Hydragel assays, for which it was greater than 20%. Overall, accuracy was best with the Helena Iso Dot and LD-VIS assays, followed by the Corning LD Flur assay; accuracy was poorest with the Gelman LDH Isozyme, Sebia Hydragel, and Beckman Paragon assays.     

Significance of isolated increases in total lactate dehydrogenase and its isoenzymes in serum of patients with bacterial pneumonia

Total lactate dehydrogenase (LD, EC 1.1.1.27) activity in serum and LD isoenzymes were quantified at the time of diagnosis in 320 patients with bacterial pneumonia. In eighty, LD activity was increased, but this was accompanied by either other pathological results for liver-function tests or associated diseases that could explain it. The remaining 240 patients were divided into four groups, based on their total serum LD values: group A, less than 225 U/L (normal limit); group B, 226-350 U/L; group C, 351-499 U/L; and group D, greater than 500 U/L. Total LD was above normal at diagnosis in 40% of the patients. Recovery time was twice as long in group D as in groups A, B, and C. In five patients from group D, the pneumonia reflected underlying lung cancer. In groups B and C, the LD-3 ratio was increased in comparison with group A; in group D, LD-4 and LD-5 were increased up to twice the normal limit. Evidently nearly half of patients with bacterial pneumonia may show isolated increases in total LD activity (mostly LD-3) in serum. In cases with high activity, prolonged recovery time is expected. Intensive follow-up and extensive investigation are warranted in these patients, because some may have underlying lung cancer.     

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.     

Non-M CK--a practical measure of creatine kinase isoenzymes in cancer patients

The individual creatinine kinase (CK) isoenzymes CK-BB and CK-Macro II have previously been investigated as potential tumour markers. We believe there is a need for a system to measure those CK forms not usually present in serum. We have studied a CK-MB immunoinhibition kit which measures all residual CK activity following inactivation of M-subunit activity. In 162 patients with cancer we found no difference in grading (+ or -) between detailed isoenzyme studies and the simple non-M assay. In 33 samples with elevated non-M CK, detailed analysis showed BB alone (45%), Macro II alone (9%), or both (36%). Raised activities were mainly found in patients with small cell lung cancer (SCLC) (17/40; 43%) and GI Tumours (6/11; 55%). In patients with SCLC, elevated activities were associated with disseminated disease. Preliminary evidence indicates that Non-M CK may also be a simple means of monitoring initial treatment response.     

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.     

Lactate dehydrogenase isoenzymes in serum during unstable angina

Values for total lactate dehydrogenase (LD, EC 1.1.1.27) activity in serum, LD isoenzymes 1 and 2, and the LD 1:2 ratio in 25 patients with unstable angina were compared with the same variables in 25 patients whose angina was stable 24, 48, and 72 h after admission. Mean total LD activity and mean LD-2 activity were found to be within the normal range, both in the unstable angina and stable angina groups of patients. In the unstable angina group the mean LD-1 was significantly higher (p less than 0.01) than in the stable angina group at each time studied. The mean LD 1:2 ratio was also significantly different (p less than 0.001) between the two groups of patients. In the unstable angina group of patients the ratio was increased (0.85, SD 0.09), as in patients with acute myocardial infarction, whereas in the stable angina group of patients the ratio was normal (0.60, SD 0.06). We conclude that a high LD 1:2 ratio, even in the presence of normal total LD activity, may indicate myocardial damage in some patients with unstable angina and could therefore help in the clinical and functional evaluation of patients with unstable angina.