CLIN.CHEM. 32/5, 792-796 (1986) Two MethodsComparedfor MeasuringLD-liTotaJLD Activityin Serum Larry H. Bernstein and Patricia Scinto We presentevidence for the utilityof an improvedassay for the activity of lactate dehydrogenase (EC 1.1.1.27) isoenzymes 1 and 2 in serum, involvinginhibitionof the H-subunit of LD by pyruvate at pH 7.1. Resultscorrelate well with the LD-1/total LD ratio as evaluated by immunologicalassay and, as an indexto infarct, the method is superiorto either the change in CK-MB activityor to the LD-1 activity or to a combinationof these tests, as is the percentage of LD-1 to total LD activity. Moreover, the percentage inhibition ofLD activity by pyruvate may have an advantageover other methods of isoenzyme fractionation because of its smaller populationCV for patients with acute myocardialinfarction than is true of other methods. We also demonstrate how, usinga lineardiscriminantanalysis,we comparedthis method with alternative methods.We determinedthat evaluation of CK-MB isoenzymecontributesno informationin additionto that obtainedfrom the LD-1 isoenzyme. AdditIonal Keyphrases: LD-lltotal LD ratio vs percent inhibition of lactate dehydrogenase by pyruvate - myocardial infarct atlne kinase isoenzyme MB from this laboratory (1-3) have indicated that dehydrogenase (EC 1.1.1.27) isoenzyme-1 (LD-1) activity in serum can be determined by using an assay that exploits the superiority of the pyruvate-to-lactate reaction (4) that is optimized at two concentrations of pyruvate.’ LD-1 activity has Moreover, the importance of determining been reinforced by the observation that it increases during the first 12 h after myocardial infarction and even before creatine kinase (CK; EC 2.7.3.2) isoenzyme MB activity reaches its maximum (5-8), although LD-1 currently is not relied upon as much as CK-MB is for confirming the diagnosis of acute myocardial infarction. That the importance of measuring LD-1 isoenzyme has been underestimated and that its role is not clear (7) is illustrated by studies such as the one supporting measurement of LD-1 activity by the a-hydroxybutyrate dehydrogenase (HBD) method, along with CK-MB, on two consecutive days (9), thus minimizing the importance of the assay or its timing because the combination of tests was considered adequate for clinical use. One may then ask whether the adequacy of CK-MB assay perceived by clinicians accounts for a different attitude toward LD-1 isoenzyme being held by clinicians and by clinical chemists. Jablonsky et al. (7) called attention to a lack of clinical interest in LD-1 isoenzyme because of misperceptions among cardiologists about the methodological and clini#{235}al status of this test, but it is not clear from reviews (10,11) whether the dissatisfaction is related to problems of the logistics of performing the evaluaStudies lactate Department of Pathology, Bridgeport Hospital, Bridgeport, CT 06610. ‘Nonstandard abbreviations: U), lactate dehydrogenase; CK, creatine kinase; AMI, acute myocardial infarction; INH, inhibition; HBD, a-hydroxybutyrate dehydrogenase. Received July 31, 1985; accepted January 792 31, 1986. CLINICALCHEMISTRY,Vol. 32, No. 5, 1986 tion and reporting results; difficulty in interpreting a high cutoff value, resulting in a specificity that is not clinically useful (isoenzyme-1/isoenzyme-2 ratio); or to analytical measurements that are clinically unacceptable or not acceptable as compared with CK-MB. The accuracy of measuring the percentage of LD-1/total LD or the LD-1/LD-2 ratio for the early detection and diagnosis of acute myocardial infarction (AMI) is well documented (6, 7, 12). However, a misperception about its accuracy does not itself appear to be the major reason for disinterest in LD-1. Despite numerous studies defining the efficacy of CK-MB and LD-1 methods, there still is no consensus concerning the advantage of any approach over another, except for the fact that electrophoresis is widely used for CK-MB or LD-1 analyses (13). In addition, when the isoenzyme-1/isoenzyme2 ratio is measured by electrophoresis it is interpreted by use of an inappropriate standard (12). Results by column chromatography (14) and electrophoresis have been comparable, but there have been continuing reports of assays by direct spectrophotometric kinetic assays (15,16). Procedures for separating LD-1 have been based on differences in physical or kinetic properties of the H- and M-type LD, such as heat denaturation, stability of urea (17), inhibition by oxainate or oxalate (18), differences in Michaelis constant (19-22), and differences in the immunological properties of the LD isoenzymes (20, 23), The principal advantage of kinetic methods for determining U) isoenzyme content is their rapidity, their inexpensiveness, and their adaptability to automated instruments. Here we report on further studies in which we measured the H-subunit content of LD activity in serum (2), comparing this method with the immunological assay for LD-1 (23). The method involves two LD assays under different conditions, and does not require separation of the LD-1 fraction before assay. We also construct functions, using the results of CK-MB and LD-1 concentrations measured in serum, to patients with and determine their efficiency in classifying without infarcts, and we rank these tests according to the contribution of each as single or joint predictors in classifying patients. Materials and Methods Enzyme Assays Isoenzymes were quantified with kits for immunological inhibition of CK-MB (SmithKline Beckman Corp., Brea, CA) and for immunological assay of LD-1 (Isomune; Roche Diagnostics, Nutley, NJ). These were compared with the assay of LD-1 activity by the method of optimized pyruvate inhibition (2) with kits manufactured for Bridgeport Hospital by Diagnostic Chemicals, Ltd, Monroe, CT. Human H4, H3M, and M4 isoenzymes of U) were purchased from Sigma Chemical Co., St. Louis, MO. We carried out serial CK-MB analyses at 6-h intervals during the first 24 h after admission of the patient and LD-1 analyses at the peak of CK-MB activity or 6 h thereafter as previously described (8). For immunochemical LD-1 assay, performed at 30#{176}C, we used a Cobas-Bio centrifugal analyz- er (Roche Instruments, Nutley, NJ). LD-1 inhibition was at pH 7.1 and 30 or 37 #{176}C by the method of Bernstein and Everse (2), under the following conditions. Total U) activity is measured in a 0.34 mmol/L solution of the oxidized substrate. This is followed by a second assay in a reagent containing, per liter, 02 mmol of NADH and 5 mmol (30 #{176}C) or 5.5 mmol (37 #{176}C) of pyruvate, respectively. The NADH concentration was increased (from 0.14 mmol/L) for the clinical studies to eliminate substrate depletion at high LD activities, and the standard curve for the reaction is linear to >500 U/L. The modification of pyruvate concentration at 37#{176}C is an empirically determined adjustment for the increased activity at the higher 4 I- a temperature. Subjects The 85 patients in this study were admitted to Bridgeport Hospital for diagnosis or exclusion of AMI. The diagnoses were established by clinical presentation, electrocardiography, serial enzyme measurements, and additional catheterization and angiographic studies as required. After grouping the patients into two categories on the basis of a detailed study of the medical records, we carried out data analyses, using the “Statistical Package for the Social Sciences.” The mean age of the study population was well over 60 years, but congestive heart failure was not a common complication, although it is the most significant factor determining which patients will require an extended stay in the hospital. In the absence of arrhythinias these patients’ cases are considered uncomplicated unless pulmonary infiltrates appear in their chest roentgenograms, appropriate clinical findings are present, and (or) they have decreased ejection fractions, any of which would lead to their condition being classified as in Killip Class LU or IV. LD-5 is massively increased in patients who develop cardiogenic shock as a complication of AM!, but there were no patients in Killip Class IV in this study, which would have been complicated by any effect of severe congestive heart failure on the LD-1 isoenzyme in their serum. We carried out basic statistics to measure central tendency, variation, and skewness, and plotted histograms by group for each variable. This was followed by one-way analyses of variance and analyses, to determine the best model for discriminant predicting membership in either the AMI or non-AM! groups. pH I.. I- a a p. a a a S. PYIUVATI (SS,IIL) 0.876) by the equation: %INH = 25.0 + 0.61378 (% LD-1) Results Figure la, a plot Of Km for pyruvate vs pH for both the H4 and M4 isoenzyme of U), shows the decreasing affinity of U) for pyruvate with increasing pH with an optimum pH at 7. Figure lb is a plot of the percentage of maximum activities of H4 and M4 in human LD as a function of pyruvate concentration at pH 7.0. On the basis of these data, we chose a pair of pyruvate concentrations for assay for U) isoenzyme composition, utilizing the pH-dependent inhibition of U) activity by pyruvate to distinguish the activities of the isoenzymes at defined pH and temperature. Measuring U) activity after the inhibition of U) activity of human 1-14, H3M, and M4 in different combined ratios gives a parabolic relationship between the percent H-subunit activity and percent inhibition (r = 0.8957). We carried out further studies of the 32 AMI and 53 nonAM! patients to compare the percentage of LD-lltotal U) activity and percent inhibition of U) activity (percent INH). The percent INN and percent LD-l are correlated (r = cQCINTATION Fig. 1a Plot of ! in millimolesof pyruvate per litervs pHforH4(dosed squares) and M4 (dosed dries) W isoenzymesof man; b. Plot of percentma,dmumactivityof H4 (‘en squares) and M4(en dries) vs pyruvateconcentration in millimoles per liter LDH4and M4 isoenzymes were providedby J.Everse afteraffinitych,omatographyand crystallinepuflficallon of AM! and non-AM! patients by assay at The comparison 30#{176}C for inhibition of U) activity in the presence of 5.0 mniol of pyruvate per liter showed the population coefficient of variation for percent inhibition to be considerably lower than for percent U)-l isoenzyme, as follows: for AM!, % LD1 = 2626%, %INH = 6.93;for non-AM!, % LD-1 = 33.85%, %INH 16.37%. Similarly, when the assay was modified for assay at 37 #{176}C (55 mmol of pyruvate per liter) we obtained an overall population CV of 50.22% for percent LD1 as compared with 25.98% for percent INN. In Table 1 we compare the patients’ results obtained by inhibiting U) activity at 37#{176}C by use of 5.5 mmol/L pyruvate with the measurements of CK-MB activity and percentage of LD-1/total U) activity in the AM! and the non-AM! groups. The standard deviation for the populations was higher for CK-MB and U)-1 activity than for either percent LD-l or for percent INN. This proved to be important in our other studies. = CLINICAL CHEMISTRY, Vol. 32, No.5, 1986 793 Table 1. PercentLD-1as Measuredby Two Methods Diagnosis CK-MB, U/L Age, yr Trauma %LD.1 12 40 CHFN. FIBC 62 3 22 Cirrhosis, 46 43 36 50 8 24 18 48 22 39 65 2 56 30 335 79 11 22k’ 56 73 78 21 47 21 50 38 39 65 170 we ob- Additionally, the change in CK-MB was found to contribute no additional predictive value in distinguishing the AM! and non-AM! groups, mainly because of shared variance with the other predictors. The results clearly show the discriminating power of 15 30 18 71 However, 47 13 14 thrombocytopenia Angina Angina AM! by pyruvate. tamed the best separations in any combination that included U).1/total LD activity, even using inhibition of subunit content of U) activity for proportion of LD-l activity. 24 Angina and its inhibition hl Ion 20 52 36 Gunshot Trauma activity 47 52 the percent U)-! and reinforce the principle of measuring total U) activity whenever isoenzyme-l U) activity is obtained. Although the results show that the discriminating power of percent INH with U) activity measured with pyruvate as substrate is not as great as that of percent LD-1, the separation is more than adequate. 48 Discussion CHFC 56 59 43 179 45 75 73 75 54 37 57 38 56 57 60 56 60 #{149}ij:) increase to 33% representsearly evoMng AMI. bAnterolateral AMI with absent CK-MB and LD.1 changes. Total LD activity (0.34 mmot/L:P-”L) increased. cngestlve heart failure. In addition to basic statistics, we carried out one-way analyses of variance and established that the differences between the two groups, AM! and non-AM!, were statistically significant (p <0.01) for all of the dependent variables: U)-l, percent U)-l, and percent NH. The F-value for percent U)-1 was the highest at 160.6. The correlation matrix for these predictors showed the highest correlation and U) activity (pyruvate); LD-l between: U)-l activity and percent U)-1; and percent U)-l and percent !NH. We also conducted discriminant analyses, with results significant at the 0.01 level. In Table 2 we compare results of discriminant analyses that best classify patients, using the candidate predictors. We entered the tests one at a time to determine combinations of the fewest predictors that would result in low misclassification rates and to determine the contribution of each predictor to the classification functions. The very high eigenvalue, high canonical correlation, high chi square, and low Wilk’s lambda for the percent LD-l shows good separation with this single predictor variable, somewhat better than the results obtained with a combination of total U) Table 2. Discrlmlnant Analyses of AMI and Non-AMI Elgsn- CanonIcal value corr. Wilk’s lambda CliP %INH 1.99 0.82 0.33 78.9 %LD-1 1.90 0.81 0.34 77.2 1.25 0.74 0.44 58.6 0.93 0.69 0.52 474 Varisbl.s No. 1 %LD-1 No.2 No.3 LD- pyruvate %INH No.4 %INH LD-1 794 CLINICALCHEMISTRY, Vol. 32, No. 5, 1986 We conclude that measuring LD-l either as percentage of isoenzyme-l/total U) (6) or as percent inhibition of Hsubunit activity is superior to measuring the activity of U)1 or CK-MB, either individually or in combination, an important conclusion in view of the current reliance on serial CK and CK-MB isoenzyme measurements in many institutions. Indeed, the findings make it extremely difficult to explain or justify the declining use of U) isoenzyme-1 evaluation for the diagnosis of AM! (7). Perhaps there are factors related to untimely reporting rather than accuracy of laboratory results that have provoked this disinterest in LD1. Moreover, there is a need for early recognition of AM! in to limit the progression of order to initiate treatments infarct and avoid late complications. This has created greater interest in excluding non-AM! patients who have equivocal CK-MB changes. There is a lack of agreement about the best method for measuring LD-l, but the studies of Leung and Henderson (12) provide some explanation for the popularity of electrophoresis over all other methods. The value of electrophoretically separating CK-MB and LD-l may be compared with that of measuring CK-MB immunochemically combined with HBD on the first and second day (9), but the better efficiency of such an approach has not adequately been validated with respect to errors of sensitivity estimations (25). Nevertheless, the study (9) supports measuring the activity of LD-l after 12 hand kinetically, mainly because it is used with CK-MB isoenzyme in addition to electrocardiographic and clinical criteria. Moreover, the study (9) places greater importance in the value of LD-1 used for a late marker for AM! than in the method of the U)-l assay. We present evidence for the adequacy of an U)-l assay that is nearly as accurate as the immunological LD-1 assay but is as inexpensive as the HBD assay. The advantages of this approach have not been previously identified. Other assays have recently been reported for measuring the activities of U)-! and LD-2 isoenzyme by automated kinetic procedures (15, 16). While we do not report as high a correlation with inhibition by pyruvate and U)-1/total U) determined by immunological assay as that reported for alkaline inactivation, the manipulation required in the latter method is as great as that required for immunological assay. We here substantiate by its clinical performance the use of substrate inhibition for measuring LD-1 activity. This kinetic method for determining the isoenzyme contents of activity is based on the fact that H-type U) is significantly inhibited by high concentrations of pyruvate (1) associated with the formation at pH 7 of an abortive complex between enzyme, NAD, and pyruvate. This assay takes advantage of the known superiority of the pyruvateto-lactate reaction (3), and it is simpler than those recently reported (15, 16). The comparison of the pyruvate-inhibition assay with the iinniunological assay for U)-1 fraction shows a slight superiority of the latter despite the lower CVs of the inhibition method within the study populations. The differences between these methods are largely found in examining the means and variances between the AM! and non-AM! populations. These differences may not be substantiated in studies with a larger population. The measurement of LD-1 by inhibition of U) subunit activity is dependent on a set of variables that includes the differences in Km for pyruvate of the H and M subunits. The means of LD isoenzyme inhibition in non-AM! and patients with small AM! tend to cluster in a range of between 45 and 48% inhibition. Comparable results in the U)-1 immunological assay would be consistently less than 29% of the total U) activity. This would be similar to the population with an isoenzyme-1/isoenzyme-2 ratio in a range from 0.69-0.85 by electrophoresis, currently under investigation by Henderson and coworkers (7, 12). For this group it becomes very difficult to assess the accuracy of these methods because of the limited size of the subpopulation under investigation. However, reduction of the population variances becomes a desirable feature in determining their sensitivities and for comparing the performance of these methods in the clinical subpopulation of borderline AM! patients. The method of inhibition of subunit content, though it is biased in estimating the H-type U) activity because it is affected by the presence of U)-2 isoenzyme, does appear to accurately measure differential increases in LD-1 isoenzyme activity in serum. What significance is there to the distinct and subtle differences between the immunological and the enzymatic assay for LD-1 isoenzyme composition? The results suggest that the significance is more apparent than real, as judged from the extremely good efficiency obtained with either method. The apparent problem of concern with either method has been the issue of dealing with U)-! activities in the presence of congestive heart failure. This only applies to the patient with Killip Class ffl-IV with reduced ejection fractions and severe circulatory impairment associated with increased U)-5 activity in cardiogenic shock or with increased U)-2 and-3 isoenzyme in some patients with pulmonary edema. When patients are found to have a total U) activity that is out of proportion to the U)-1 activity by any measurement, they are usually found to have an increased LD-5 activity associated with generalized hypoperfusion. In these patients there is no need for a superior LD-1 assay because of the likelihood that there are electrocardioconsistent with AM! with graphic and clinical findings massive increase in CK-MB isoenzyme. The HBD would also be high because of massive increases in U) activity in the serum, originating not only from the heart but also from the liver. The most important clinical problem for the patient is management of cardiogenic shock. The present studies coniirm the validity and discriininating power of LD-1 subunit content measured as percent inhibition of U) activity by pyruvate. More importantly, they demonstrate how we can evaluate these tests and of each to a vector of observations establish the contribution in forming a joint predictor for classifying patients. We found that, by comparison with LD-1 activity as percent of U) activity or as percent inhibition, CK-MB and U)-1 activities provide no additional discrimination beyond that of percent inhibition because of the large population variance of enzyme activities in the AM! and non-AM! populations. Finally, we think that the more rapid and accurate analyses of LD-1 here demonstrated may contribute to the improved treatment of AM! because of the current emphasis on more rapid diagnosis and treatment to reduce late complications, especially in those patients who have small infarcts with equivocal changes in the concentration of CKMB activity in their serum. total We appreciate the competent statistical assistance provided by Dr. Susan Carroll, of Words and Numbers, Torrington, CT. Human IL, and M4 purified by affinity chromatography was kindly provided for determining assay conditions by Professor Johannes Everse, Department of Biochemistry, Texas Tech University Health Sciences Center, Lubbock, TX. References 1. Bernstein LH, Everse J, Shioura N, Russell PJ. Detection of cardiac damage using a steady state assay for lactate dehydrogenase isoenzymes in serum. J Molec Cell Cardiol 1974;6:297-315. 2. Bernstein LH, Everse J. Determination of the isoenzyme levels of lactate dehydrogenase. In: Wood WA, ed. Carbohydrate metaboham. New York, NY: Academic Press, 1975;47-53. (Colowick SP, Kaplan NO, eds. Methods in Enzymology, Vol XLI, Part B). 3. Bernstein LII. Automated kinetic determination of lactate dehydrogenase isoenzymes in serum. Clin Chem 197723:1928-30. 4. Howell BF, McCune S, Schaffer R. Lactate-to-pyruvate or pyruvate-to-lactate assay for lactate dehydrogenase: A reexamination. Clin Chem 1979;25:269-72. 5. Roe CR. 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The two out of three criteria for the diagnosis of infarction. Is it pass#{233}? Chest 1984;86:511-3. 12. Leung FY, Henderson AR. Thin-layer agaroee electrophoresis of lactate dehydrogenase isoenzymes in serum: a note on the method of reporting and on the lactate dehydrogenase iaoenzyme1/isoenzyme-2 ratio in acute myocardial infarction. Clin Chem 1979;25:209-11. 13. Boone J, Sampson &J, Lewis 5, et al. An interlaboratory study of creatine kinase and creatune kinase isoenzymes. Clin Chem 198026:513-9. 14. Vasudevan G, Mercer DW, Varat MA. Lactate dehydrogenase in the diagnosis of acute myocardial isoenzyme determination infarction. Circulation 1978;57:1055-7. 15. Eckfeldt Jil, Kershaw MJ, Lewis LA. Automated kinetic assay for lactate dehydrogenase isoenzymes by centrifugal analysis after alkaline inactivation. Clin Chem 1984;30:1821-4. 16. Tanishima K, Hayashi T, Matsushima M, Mochikowa Y. 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Lane RS, Dekker EE. 2-Keto-4-hydroxybutyrate. Synthesis chemical properties, and as a substrate for lactate dehydrogenase of rabbit muscle. Biochem 1969;8:2958-66. 23. Usategui-Gomez M, Wicks RW, Warshaw M. Immunochemical determination of the heart isoenzyme of lactate dehydrogenase (LI) 1) in human serum. Cliii Chem 1979;25:729-34. 24. Bernstein LII, Reynoso G. Creatine kinase B-subunit activity in serum in cases of suspected myocardial infarction: a prediction model based on the slope of MB increase and percentage CK-MB activity [Letter]. Clin Chem 1983;29:591-2. 25. Linnet K. Precision of sensitivity estimations in diagnostic test evaluations. Power functions for comparisons of sensitivities of two tests. Cliii Chem 1985;31:574-80.