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Annals of Clinical & Laboratory Science 32:44-49 (2002)
© 2002 Association of Clinical Scientists

Differences of Creatine Kinase MB and Cardiac Troponin I Concentrations in Normal and Diseased Human Myocardium

Terry M. Welsh1,2,*, Gary D. Kukes1,2 and Lynn M. Sandweiss3
1 Diagnostic & Molecular Medicine Health Care Group, VA Healthcare System, Long Beach, California
2 Department of Pathology, University of California, Irvine, California
3 School of Public Health, University of California, Los Angeles, California

Address correspondence to Gary Kukes, M.D, Ph.D., Pathology and Laboratory Medicine Service (113), Veterans Affairs Healthcare System, 5901 East 7th Street, Long Beach, CA 90822, USA; tel 562 494 2611, ext 4101; fax 562 494 5623; e-mail gary.kukes{at}med.va.gov.


   Abstract
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 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The diagnosis of myocardial infarction (MI) is established in patients with chest pain and equivocal electrocardiogram changes by demonstrating a rise in blood levels of creatine kinase MB (CK-MB) and/or an increase in cardiac troponin I (cTnI) or cardiac troponin T (cTnT). Previous studies have shown that levels of CK-MB are increased in the left ventricle of individuals with heart disease; however, it has not been established whether there are differences in the ventricular myocardium concentrations of cTnI in diseased compared to healthy hearts. Using a simple extraction technique, concentrations of CK-MB and cTnI were measured in the left ventricle (LV) of six hearts obtained at autopsy from individuals ranging in age from 25 to 79 yr, with and without evidence of cardiac disease. The results show an 86-fold higher concentration of CK-MB and 7.7-fold lower concentration of cTnI in left ventricular myocardium of older men with and without cardiac disease, compared to that of younger men (< age 35 yr) without heart disease. These data suggest that age may need to be considered when setting cutoff limits for these markers for the diagnosis of myocardial infarction.

(received 9 August 2001; accepted 8 September 2001)

Keywords: myocardial infarction, cardiac tissue analysis, creatine kinase-MB, cardiac troponin I


   Introduction
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 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
According to the World Health Organization, the diagnosis of acute myocardial infarction requires the presence of at least two of the following criteria: (a) history of characteristic chest pain, (b) evolutionary changes of the electrocardiogram, and (c) elevation of cardiac enzymes in serial serum samples [1]. For a number of years, serial elevation of CK-MB has been the "gold standard" for the laboratory diagnosis of myocardial infarction. As a cardiac marker, serial increase of this enzyme is virtually diagnostic of myocardial infarction in patients with chest pain whose electrocardiogram is equivocal [2]. Because their cardiospecificity is greater than CK-MB, troponin I (cTnI) and T (cTnT) are increasingly used as diagnostic markers for myocardial injury and infarction [3,4]. Since the troponins remain elevated in serum for many days following MI, serum troponin levels are replacing lactate dehydrogenase (LDH) isoenzymes as markers for the diagnosis of late presentation myocardial infarction [5].

While CK-MB as a cardiac marker has depended on its relatively high concentration in heart muscle (>20%) compared to typical skeletal muscle (1–2%) [6], there is evidence that higher concentrations of CK-MB in heart may result from ischemic stress. For example, concentrations of CK-MB have been found to be significantly higher in heart muscle of experimental animals and human myocardium with coronary artery disease, aortic stenosis, or heart failure, compared to normals [7–10]. There are, however, little or no data that compare the concentrations of troponin I in diseased and healthy human ventricular myocardium.

This study measured the concentrations of CK-MB and cTnI in left ventricular myocardium of hearts obtained at autopsy from a group of 6 individuals, with and without heart disease, ranging in age from 25– 79 yr.


   Materials and Methods
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 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Small portions (1–2 g) were taken at autopsy (5–48 hr after death) from a comparable area of the left ventricle of 6 individuals. Two individuals (age <35 yr) were victims of gunshots and had no history of heart disease (specimens from the Los Angeles County Coroner’s Office). Two of 4 older individuals (age >35 yr) had a history of myocardial infarction (specimens taken away from the areas of infarct). The third of the older individuals died of sepsis with significant myocardial hypoperfusion; the fourth had no history of heart disease and died of metastatic squamous cell carcinoma.

Tissue was frozen at -70°C and then ground in a liquid nitrogen-cooled mortar and pestle. The powdered tissue (100–300 mg) was resuspended in 10 ml of buffer (10 mM K2HPO4, 150 mM NaCl, 1 mM EDTA and 1 mM ß-mercaptoethanol, pH 7.2±0.2). The suspension was centifuged at 2000 g for 5 min and an aliquot was recentrifuged at 5000 g for 5 min. An aliquot of the final supernatant was assayed for soluble protein concentration using a modified Lowry protein assay with bovine serum albumin as a standard (BioRad, Hercules, CA). (A small amount of insouluble material remained in the pellet.)

Finally, 1:10–1:100 dilutions of the supernatant were assayed for CK-MB and cTnI with the Status II immunoassay analyzer. In this system, quantitation of CK-MB mass is based on a two-site sandwich fluorimetric immunoassay in which enzyme-labeled anti-CK-BB antibody binds the B antigenic site of CK-MB, which is immobilized by anti CK-MB capture antibody. Troponin I is measured by a two-site immunoassay that uses a monoclonal capture antibody specific for the cardiac isotype of troponin I and an anti-troponin I monoclonal detection antibody conjugated to alkaline phosphatase.

CK-MB and cTnI mass concentrations were measured as µg/ml and expressed after conversion as µg/mg of total soluble protein.


   Results
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 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
The mean concentration of CK-MB in post-mortem left ventricular myocardium of younger indivduals, (<age 35 yr ) was 0.06 µg/mg total soluble protein (n=2) compared to the mean of 4.71 µg/mg total soluble protein (n=4) in older individuals (> age 35 yr). This represents an 86-fold greater concentration in the myocardium of older individuals.

The mean concentration of cTnI in µg/mg total soluble protein in left ventricular myocardium from younger individuals (n=2) was 3.65 compared to 0.46 (n=4) in the older population. The older population had a 7.7-fold lower concentration of troponin I in left ventricular myocardium, compared to younger individuals without a history of heart disease (Table 1Go). Due to small sample sizes, non-parametric statistics were used for data analysis. Using Spearman’s rank correlation test [11,12], the correlation between both age groups is high for CK-MB (Spearman’s rho =0.83) and cTnI (Spearman’s rho = -0.83). Both are significant at p =0.04.


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  		Table 1. Total soluble protein (µg/mg) of CK-MB and troponin I in postmortem left ventricular myocardium in young (<35 yr) and older (>35 yr) patients.
 

   Discussion
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A number of studies have shown that the concentration of CK-MB is higher in ventricular myocardial tissue in animal models of hypertrophy or ischemia and in humans with a variety of cardiac conditions, compared to controls or young individuals without cardiac disease [7–10]. In human myocardial biopsy material, concentrations of CK-MB have been reported to be 100-fold greater in hearts from patients with aortic stenosis, coronary artery disease, and coronary artery disease with left ventricular hypertrophy compared to patients without such findings [9]. Autopsy material has shown about 30% of the concentration of CK-MB in normal hearts, compared to those from patients who died of myocardial infarction [13]. Enzymatic activities of CK-MB from accident victims’ hearts were reported to be only 14–16% of those from donor hearts on life support systems and from failing hearts [10]. Such isoform swithching to higher levels of CK-MB induced by hypoxic stress is probably adaptive, since CK-MB has a higher affinity for its substrate than CK-MM [14].

Studies reporting the concentration of CK-MB specifically in human left ventricular myocardium from individuals with no history of cardiac disease range from 0.014 µg/mg to 0.77 µg/mg myocardial protein [9,13]. Our finding of an average of 0.06 µg/mg protein from autopsy tissue of two young gunshot victims represents an intermediate value. Our findings of 86-fold higher concentration of CK-MB in 4 patients, 3 of whom had known cardiac disease including 1 with sepsis, prolonged hypotension, and cardiac ischemia (patient #6), compared to young healthy controls, are in agreement with the significantly higher amounts of myocardial CK-MB in patients with the common pathophysiologic state of myocardial hypoxia [9,13].

Cardiac troponin I (cTnI), a 24 kD polypeptide that inhibits actomyosin ATPase activity, is found in the thin filament of human cardiac muscle and exists in a small (2–8%) cytosolic fraction and a large (92–98%), more insoluble, myofibrillar-bound fraction [15–17]. Reported concentrations of troponin I in human ventricular myocardium include values of 5.98 µg/mg protein, or 0.39 µg/ mg wet weight, from left ventricular tissue at autopsy [17] and 4.0 µg/mg tissue (±0.9 SD) from human heart at autopsy or from explanted heart at transplantation [16]. A troponin I level of 4.35 µg/mg tissue (±1.36 SD) in human heart has also been reported [18]. In the latter two studies, the anatomic site of the tissue sample was not stated. Recently, Swaanenberg et al [19] reported values of troponin I of 1.54 and 64.1 µg/mg protein in human left ventricle taken at autopsy and measured by two different assays (Access, Beckman Instruments; AxSYM, Abbott Diagnostics).

Our average value of 3.65 µg/mg protein is comparable to the value of Mair et al [17] and much lower than the value of Swaanenberg et al [19], when factoring the correlation slope bias between the Access and Stratus methods [20]. Assuming that the left ventricular human myocardium contains 50–100 µg protein/mg wet weight tissue [17,19], values of troponin I as µg/mg protein reported by Adams et al [16] and Larue et al [18] would be several times larger than our values and those of Mair et al [17].

Some variation in troponin I concentrations in these studies arises from assays that employ detection or capture antibodies that recognize different epitopes on the cTnI molecule. Currently there is no standard calibrating material [21–24]; concentrations of cTnI obtained with different commercial analyzers can differ by a factor of >= 20-fold [19,25]. Differences may also result from non-uniform detection of the different chemical forms of troponin I. It has been shown that cTnI formes complexes with both cTnI and troponin C (cTnC) and is released into the blood following myocardial infarction predominantely as a binary complex of cTnI-C, but also as a ternary complex [26–28]. It is also known that cTnI exists in human serum in oxidized and reduced forms and can also be phosphorylated in tissue [26,29]. Furthermore, our method, unlike some studies to which our values are compared, was not designed for complete extraction of troponin I from cardiac muscle.

These considerations aside, we contend that our observation of a 7.7-fold higher concentration of cTnI in the left ventricular myocardium of young versus older individuals is valid because: (a) the method of tissue extraction was the same for all samples, (b) the tissue samples were measured on a calibrated immunoassay analyzer (Stratus II), (c) the samples were measured in buffer that gave linear serial dilution curves when spiked with serum containing troponin I (performed on the Access analyzer, data not shown), (d) our EDTA-containing buffer would have reduced cTnI in the samples to the free subunit, which is one of the isoforms recognized by the Status II antibodies [27], and (e) the extraction and tissue handling gave results that agree with several studies showing lower CK-MB concentration in younger than in older individuals with heart disease. Our findings also suggest that the antigenicities of CK-MB and of cTnI, which are not significantly degraded in serum samples taken 6–40 hr after death [30], show comparable stability in tissue extracts from myocardium taken 5–48 hr postmortem.

It is common to observe increased left ventricular muscle mass due to individual fiber hypertrophy in hearts of older individuals who have had ischemic stress (personal observation). One interpretation of our finding of decreased cTnI levels with age is that non-troponin I soluble protein in hypertrophied fibers may increase differentially in comparison to the contractile proteins, a hypothesis that deserves follow-up.

Our finding of lower cTnI in ischemic cardiac muscle is also supported by other studies. Dogs who had myocardial infarction induced by coronary artery occlusion showed a 42–92% reduction in cTnI concentration 3 wk later in the areas of infarct, compared to non-infarcted areas of the same left ventricle. cTnI from non-infarcted areas in these animals was similar to controls [31]. Pigs with infarcts induced by coronary artery occlusion had a 42% decrease of cTnI concentration in left ventriclar myocardium away from the infarct, compared to non-diseased pigs [32].

In human heart, it has been shown that the cytosolic concentration of troponin T is decreased 55% in the left ventriclar myocardium of diseased hearts (patients with coronary artery disease and fatal myocardial infarction) compared to normals [13]. Since there is a stoiochometric relationship between cTnT and cTnI levels in heart muscle [33], our finding of decreased troponin I in diseased myocardium is consistent with lower concentrations of cTnT in similar patients.

Measurement of cTnI in serum is established as a sensitive, specific diagnostic marker for acute myocardial infarction [34,35], for assessment of subclinical cardiac injury [36], and for risk stratification in patients with unstable angina [37,38].

The finding of lower concentration of cTnI in hearts with greater ischemic insult raises the clinical question of whether age-related cutoff values might increase the sensitivity and specificity of cTnI as a marker of cardiac injury. Can myocardial infarction be excluded in a 35-yr-old cocaine abuser presenting with chest pain, when the plasma cTnI concentration falls just above the cutoff level? Do similar plasma elevations of troponin I reflect the same degree of cardiac injury in a 40-yr-old with respiratory arrest, compared to a 75-yr-old with chronic congestive heart failure? Can the predictive value of risk stratification by cTnI be increased by age stratification? Answers to these questions await validation of our findings in larger studies and also further information about the kinetics and chemistry of release of troponin I from damaged myocardium.


   Footnotes
 
* Current address: Department of Pathology, Cedars-Sinai Medical Center, Los Angeles, California Back


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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