Henry Ford Hospital Medical Journal

Volume 30 Number 2 Article 11

6-1982

Isoenzyme Update: Creatine kinase and lactate dehydrogenase

Craig C. Foreback

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Recommended Citation Foreback, Craig C. (1982) "Isoenzyme Update: Creatine kinase and lactate dehydrogenase," Henry Ford Hospital Medical Journal : Vol. 30 : No. 2 , 97-101. Available at: https://scholarlycommons.henryford.com/hfhmedjournal/vol30/iss2/11

This Article is brought to you for free and open access by Henry Ford Health System Scholarly Commons. It has been accepted for inclusion in Henry Ford Hospital Medical Journal by an authorized editor of Henry Ford Health System Scholarly Commons. Henry Ford Hosp Med J Vol 30, No 2,1982

I I "111 illPlI: ""III, .III' i, 111." Ill :oiiii 1/ Isoenzyme Update

Creatine kinase and lactate dehydrogenase

Craig C. Foreback, PhD*

Creatine kinase (CK)and lactate dehydrogenase f LD j are to determine CK-MB activity in serum. New methods to isoenzymes that have been measured in clinical labora­ quantify CK-MB have also been tested, including an tories for over 20 years; their separation has proved automated column technique, the immunoinhibidon/ valuable in the diagnosis of myocardial infarction and immunoprecipitadon technique, and immunoradiome- other cardiac-related diseases. Although, historically, try. In addition, new immunological techniques have electrophoresis was the preferred method to measure recently been developed to analyze LD isoenzymes; the both isoenzymes, there has been controversy over the assay for LD-1 has already replaced electrophoresis. In best method for separating the CK isoenzyme, lon time, specific assays for CK-MB will also be available. exchange chromatography has been used successfully However, new techniques have not eliminated the need to isolate CK isoenzymes, and the procedure was adapted to order CK-MB and LD-1 as a battery and to employ appropriate timing and serial collection of samples.

Elevations of creatine kinase (CK) and lactate dehy­ mobility. The fastest anodally migrating isoenzyme is drogenase (LD) in myocardial infarction are well LD-1 (H4), and the slowest LD-5 (M4). The tissue distribu­ documented, and these enzymes have been measured tion of LD and CK isoenzymes is shown in Tables I and 11, routinely in clinical laboratories for over 20 years. Crea­ respectively. tine kinase is present in striated muscle, brain, smooth muscle, heart muscle, and the prostate. Lactate dehy­ Historically, separation of LD isoenzymes preceded the drogenase is present in the liver, heart, kidney, striated separation of CK isoenzymes as a routine clinical test muscle, red blood cells, and the lung. Because of their (5,6). The classic electrophoretic separation of LD isoen­ widespread tissue distribution, they are also elevated in zymes is effective and relatively accurate. It is still widely other cardiovascular and nonrelated diseases. The obser­ used in most hospital laboratories. Ion exchange chro­ vation that LD and CK existed in isoenzyme forms (1,2) matographic methods have been developed, but these led to speculation that separation and determination of require additional skills and apparatus not always rou­ different isoenzymes in serum might lead to specific tinely available. Therefore, this approach has not achieved markers for various diseases. Indeed, the determination any popularity. After electrophoretic separation, the of CK and LD isoenzymes has proved valuable in diagno­ separation media are "stained" with any substrate spe­ sis and confirmation of acute myocardial infarction (3). cific for LD. The result is five bands of activity corres­ ponding to the five different LD isoenzymes. These Isoenzymes are similar but distinct protein molecules bands are then scanned on a densitometer, and a tracing which catalyze the same chemical reaction. CK is a is produced. Even normal serum contains all five isoen­ dimeric molecule composed of two types of monomers, zymes (Fig. IA). In myocardial infarction, the ratio of the M and B subunits. Varying the combination ofthese LD/LDj becomes > 1, creating the so-called "flipped" subunits results in three isoenzymes, designated the pattern (Fig. IB). The most frequent use of LD isoen- MM (CK-3) skeletal muscle, MB (CK-2) cardiac muscle, and BB (CK-1) brain forms (4). LD exists as a tetramer which is also composed of two types of monomers, the H Submined for publication: May 17, 1982 and M subunits. This results in five different isoenzymes: Accepted for publication: May 24.1982 H4, H3M, H2M2, HM3, and M4. The isoenzymes of LD • Department of Pathology, Division of Cnemistry, Henry Ford Hospital are often referred to in terms of their electrophoretic Address reprint requests to Dr. Foreback, Department of Pathology, Henry Ford Hospital, 2799 W Grand Blvd, Detroit, Ml 48202

97 Foreback

TABLE I

Tissue Localization of LD Isoenzymes

Tissue Principal Isoenzymes

Heart Kidney LD-1, LD-2 Brain Red Blood Cells

Thyroid Adrenal Gland LD-3 Lymph Nodes Pancreas Thymus Spleen Leukocytes ^ H A A Liver Skeletal Muscle LD-4, LD-5 Fig. 2

Electrophoresis of LD isoenzymes showing treatment of serum wilh anii M, anii-anti M precipitation agenl. LD is al lop of picture. N= TABLE II normal palieni; A= abnormal palieni (myocardial infarction).

Tissue Localization of CK Isoenzymes TABLE III Tissue Isoenzyme LD Isoenzyme Pallerns in Various Diseases

Brain Disease Paltern Smooth Muscle Thyroid BB Lung Myocardial Infarct* LD-1, LD-2 elevated Prostate Pernicious Anemia Acute Renal Damage Cardiac Muscle Hemolysis Tongue MB Diaphragm Liver Damage LD-5 elevated Skeletal Muscle (Trace Amounts) Myocardial Infarction with LD(1>2) ± LD-5 4^ Skeletal Muscle MM Liver Congestion Cardiac Muscle Lymphoproliferative Disorders LD-2, LD-3 elevated Pulmonary Infarct

Heart Failure All isoenzymes elevated Crush Syndrome LDH ISOENZYMES MYOCARDIAL-INFARCT Neoplastic Disease NORMAL LDH ISOENZYMES

•Generally LD-1/LD-2 greater than 1.0

zymes is in the diagnosis or confirmation of myocardial infarction. The presence of the "flipped" pattern in clin­ ically diagnosed myocardial infarction is highly specific. Although elevations of the other LD isoenzymes (LD-3- LD-5) have been reported in various diseases (Table 111), their value in diagnosing or confirming these diseases is doubtful. There has been considerably more controversy over the preferred method of separating CK isoenzymes. Like LD isoenzymes, CK isoenzymes were first separated by elec­ Fig.1 trophoresis. Of particular interest was the CK-2 or MB

98 Isoenzyme Update

Comparison of LD-1 by Roche Isomune Cy) procedure was adapted to accurately determine CK-MB to LD-1 by Electrophoresis (x) activity in serum. There was intense interest in this procedure because it could provide an accurate, quan­ titative assessment of CK-MB in serum.

200- Originally CK-MB was thought to be specific for myocardial infarction based upon the generally accepted tissue dis­ tribution of the CK isoenzymes. However, elevated CK-

150H MB has been reported in other cardiac-related diseases, such as severe angina, congestive heart failure, and car­ y(IU/L) diomyopathy. CK-MB has also been reported in Reye's

tOO-t syndrome, carbon monoxide poisoning, and various skeletal muscular myopathies. This in part accounts for the demand by clinicians for a quantitative CK-MB / r = 0 994l %^ y- 1 008x- 1 34 method. Varat and Mercer have suggested that the rela­ 50- / • tive percent of CK-MB compared to the total CK activity is more specific for myocardial infarction (9); over 5-8 units of MB activity greater than 2% ofthe total is indica­

llll 1 1 1 p 1 20 40 60 80 100 120 140 160 ISO tive of myocardial necrosis. Other workers have quanti­ X (lU/L) tated CK-MB and attempted to relate it to histological infarct size. Progress in this area has recently been Fig. 3 reviewed (10,11). Quantitation of CK-MB may also be useful in evaluating the effectiveness of streptokinase in myocardial infarction (LeeTG, personal communication). Comparison of LD-1 by Roche Isomune to LD-1/LD-2 by Electrophoresis Other quantitative methods are immunoinhibition (12) and radioimmune assay (RIA) for the CK-B subunit (13). Key: t. MB band (+) In terms of efficiency of diagnosing or confirming • Specimen later tlipped • Specimen * Flipped on earlier specimen myocardial infarction, none ofthe methods are superior to electrophoresis, which directly detects the presence of the CK-MB isoenzyme visually. The immunoinhibition techniques are insensitive, while the CK-B subunit RIA techniques require long incubation times and can be

tolal 2673 run economically only in batches. Both suffer from BB I LD3, LD4, LD5 dilution (5:1) interference. Although oncethought to be uncommon, CK-BB is found in about 1% of all CK isoenzymes requested at Henry Ford Hospital. 1?0 160 200 240 lsomune-LD-1 In our laboratory, we have been evaluating several new Value lU/L CK-MB proced ures. The first is an automated column technique which can easily be performed 24 hoursa day, Fig.4 seven days a week (14). The method works quite well to analyze serum, providing at least three samples are col­ lected serially. Specimens should be collected at 8, 16, band, because its presence in human serum was reported 24, and 36 hours following suspected infarction. In many to have a high degree of specificity in the diagnosis of cases, the method is not sensitive enough to be diagnos­ myocardial infarction (7). tically accurate on a single specimen. The procedure may not be useful on patients who have recently under­ The development of ion exchange chromatography to gone cardiac surgery such as coronary artery bypass. successfully isolate and purify tissue isoenzymes of CK led to clinical applications in the diagnosis of myocardial Another approach currently being tested is the im- infarction, as reported to Mercer (8). The advantage of munoinhibition/immunoprecipitation technique (15). thistechnique was that column effluents could be quan­ This method is more specific for CK-MB because a two- titated by the conventional assay for total CK. Thus, the tube procedure is employed. One tube provides immu-

99 Foreback

noinhibition of CK M-subunit and results in a CK-MB -F becomes elevated before the flipped pattern becomes CK-BB assay. In the second tube, an immunoprecipita- apparent on electrophoretic separation (Fig. 5). tion of CK-MM and MB is carried out, and the remaining In summary, electrophoretic separation of CK and LD enzyme activity is due to CK-BB and other endogenous isoenzymes is no longer the state ofthe art in laboratory kinases which may be present. The subtraction of the testing. The assay for LD-1 has already replaced electro­ second tube from the first results in a very specific assay phoretic separations of LD-1 isoenzymes, and it is only a for CK-MB. Again, the problem is sensitivity due to the matter of time until specific assays for CK-MB will be low amounts of CK-MB present. However, serial assays available. It must be emphasized, however, that new with this procedure can give reasonable information techniques have not eliminated the need to order CK- about the rise and fall of CK-MB in serum and confirma­ MB and LD-1 together as a battery and to employ tion of myocardial infarction. appropriate timing and serial collection of samples. The third method being investigated is immunoradi- ometry (16). In this procedure serum is reacted with anti B bound to solid phase. MB and BB bind to the solid support, and CK-MM can be decanted. Then, radiola­ Rise and Fall of Serum Enzyme Activity beled anti CK-M is added to the tube which will attach Following Myocardial Infarction only to the bound CK-MB but not to CK-BB. Excess CK tracer is removed, and the resulting activity is a measure CK MB of the amount of CK-MB originally present. Modifica­ '~ Total LD LD 1 tions are being made to increase the analytical sensitivity of the method with the hope that diagnostic sensitivity o will be correspondingly increased. It should also be < m noted that techniques which employ radio-tracers mea­ E sure enzyme mass and do not rely on its catalytic activity. >. N C Recently, immunological techniques have been employed m to analyze LD isoenzymes (17). As with CK isoenzymes, antibodies developed against LD isoenzymes are sub- unit specific. Thus, anti M antibody would react with all 24 32 40 48 54 LD forms except LD-1. This is the approach currently Time in Hours used to determine LD-1 at Henry Ford Hospital.

Briefly, the procedure employs a first antibody to react Fig. 5 with all M subunits of LD. Then a second antibody bound to a solid phase is added to precipitate all isoen­ zymes that contain the M subunit (Fig. 3). A commercial References kit for this test is available from Roche Diagnostic under the trade name of Isomune LD. In our laboratory evalua­ 1. Deal DH, von Breeman JFL, Electrophoresis of CPK from various tions, LD-1 determined by the Isomune LD assay cor­ organs. Clin Chem Acta 1964; 10:276.

relates well with LD-1 determined by electrophoretic 2. CohnRD,KaplanNO,Levin L.Zwilling F. Nature and development separation (Fig.4). As can be seen in Fig. 5, an elevation of of lactic dehydrogenases. Science 1962; 136:962. of LD (reference range upper limit 80 U/L) corresponds 3. Galen RS, Relffce JA, Gambino SR. Diagnosis of acute myocardial to the presence or absence of the flipped pattern on the infarction, relative efficiency of serum enzyme and isoenzyme electrophoretic tracings. measurements. JAMA 1975;232:145.

LD isoenzymes are primarily ordered to confirm or rule 4. Dawson DM, Eppenbergh HM, Kaplan NO. Creatine kinase: Evi­ out myocardial infarction. Since the LD-1 assay has been dence for a dimeric structure. Biochem Biophys Res Comm 1965:21:346. shown to correlate so well with electrophoresis in con­ firming cardiac disease, it has replaced complete isoen­ 5. Van der Veen KF, Wulebiauds AF. Isoenzymes of creatine phos­ zyme analysis. This assay is easier to perform in the phokinase in tissue extracts and in normal pathological serum. Clin laboratory, and it is easier to interpret results since LD-1 Chem Acta 1966;13:312. values are compared to a reference range. An added 6. Van der Helm HJ. Simple method of demonstrative lactic acid advantage is the fact that in myocardial infarction LD-1 dehydrogenase isoenzymes. Lancet 1961;2:108-9.

100 Isoenzyme Update

7. Roberts R, Henry RD, Witteveen SAGJ, Sabel BF, Quantitation of 13. Williamson JT, Stone MJ, Ting R, Mukherjee A, Gomez-Sauchez serum creatine kinase isoenzyme activity. Am J Cardiol 1974;33:650. CE, Lewis P, Hersh LB. Radioimmunoassay of creatine kinase: B 8. Mercer DW. Separation of tissue and serum creatine kinase isoen­ isoenzyme in human sera: Results in patients with acute myocardial zyme by ion exchange chromatography. Clin Chem 1974;20:36. infarction. Proc Natl Acad Sci USA 1977;74:1711.

9. Varat MA, Mercer DW, Cardiac specific creatine phosphokinase 14. El DuPont de Nemours & Co. Unpublished report, Wilmington, DE isoenzyme in the diagnosis of acute myocardial infarction. Circula­ 19898. tion 1975:51:855. 15. Wicks RW, Ustequi-Gomez M, Miller M, Warshaw M. Immuno­ chemical determination of CK-MB isoenzyme in human serum. II. 10. Sobel BE, Morham J, Roberts R. Factors influencing enzymatic An enzymatic approach. Clin Chem 1982:28:54. estimates of infarct size. Am J Cardiol 1977;39:130. 16. Embria CK-MB package insert. International Immunoassay Labs, 11. Rae CR. Validity of estimating myocardial infarct size from serial Santa Clara, CA 95050. measurements of enzyme activity in the serum. Clin Chem 1977;23:1807. 17. Usatequi-Gomez M, Wicks RW, Warshaw M. Immunochemical determination of the heart isoenzyme of lactate dehydrogenase 12. Gerhardt W, Waldenstrom J. Creatine kinase B-subunit activity in (LDH) in human serum. Clin Chem 1979;25:729. serum after immunoinhibition of M-subunit activity. Clin Chem 1979:25:1274.

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