Cardiac troponin T in the diagnosis of myocardial injury.

Critical Reviews in Clinical Laboratory Sciences, 29( 1):3 1-57 (1992)

Cardiac Troponin T in the Diagnosis of Myocardial Injury Critical Reviews in Clinical Laboratory Sciences Downloaded from informahealthcare.com by CDL-UC Davis on 11/08/14 For personal use only.

Johannes Mair, M.D., Franz Dienstl, M.D., and Bernd Puschendotf, M.D. The Department of Medical Chemistry and Biochemistry and the Department of Internal Medicine', University lnnsbruck School of Medicine, A-6020 Innsbruck, Austria Referee: Dr. Alexander D. Romaschln, Department of Clinical Biochemistry, Toronto General Hospital, 200 Elizabeth Street, Toronto, Ontario, M5G 2C4.

*Correspondence and reprint requests to: Prof. Dr. B. Puschendorf, lnstitut fur Medizinische Chemie und Biochemie, Universitat Innsbruck, Fritz-PreglstraOe 3, A-6020 Innsbruck, Austria.

ABSTRACT: In the last several decades serum levels of cardiac enzymes and isoenzymes have become the final arbiters by which myocardial damage is diagnosed or excluded. Because conventionally used enzymes are neither perfectly sensitive nor specific, there is need for a new sensitive and cardiospecific marker of myocardial damage. Cardiac troponin T (TnT) is a contractile protein unique to cardiac muscle and can be differentiated by immunologic methods from its skeletalmuscle isoform. An enzyme immunoassay specific for cardiac TnT is now available in a commercial kit for routine use. The biggest advantage of this assay is its cardiospecificity. TnT measurements, however, are also highly sensitive in diagnosis of myocardial injury and accurately discern even small amounts of myocardial necrosis. TnT measurements are, therefore, particularly useful in patients with borderline CIS-MB and in clinical settings in which traditional enzymes fail to diagnose myocardial damage efficiently because of lack of specificity - for example, perioperative myocardial infarction or blunt heart trauma. TnT release kinetics reveal characteristics of both soluble, cytoplasmic, and structurally bound molecules. It starts to increase a few hours after the onset of myocardial damage and remains increased for several days. TnT allows late diagnosis of myocardial infarction. The diagnostic efficiency remains at 98% until 6 d after the onset of infarct-related symptoms. TnT is also useful in monitoring the effectiveness of thrombolytic therapy in myocardial infarction patients. The ratio of peak TnT concentration on day 1 to TnT concentration at day 4 discriminates between patients with successful (>1) and failed ( 5 1 ) reperfusion. TnT measurements are very sensitive and specific for the early and late diagnosis of myocardial damage and could, therefore, provide a new criterion in laboratory diagnosis of the occurrence of myocardial damage.

KEY WORDS: myocardial injury, myocardial infarction, diagnosis, troponin T, contractile protein, myocardial reperfusion, creatine kinase, lactate dehydrogenase, enzyme immunoassay , cardiospecific enzymes, contractile proteins, coronary artery bypass grafting

1040-8363/92/$.50 0 1992 by CRC Press, Inc.

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1. THE DIAGNOSIS OF MYOCARDIAL INJURY: CURRENT STANDARDS OF DIAGNOSIS A. The Diagnosis of an Acute Myocardial Infarction (AMI) The diagnosis of an AM1 is generally based upon World Health Organization (WHO) criteria,'-3 which is the result of at least two of three classic findings:

Critical Reviews in Clinical Laboratory Sciences Downloaded from informahealthcare.com by CDL-UC Davis on 11/08/14 For personal use only.

1.

2. 3.

Clinical history of ischemic chest discomfort of more than 30 min duration. Evolution of typical, unequivocal electrocardiographic changes. Rise and fall of serum enzymes indicating myocardial muscle fiber injury (see Table 1).

Myocardial infarctions are subdivided into two groups (Q wave and non-Q wave AMI), based on whether or not new Q waves develop in the patient's electrocardiogram (ECG). The diagnosis of non-Q wave AM1 may be suspected from ECG findings, but must be confirmed by enzyme determinations. Unfortunately, unequivocal clinical symptoms and ECG findings are not present or easily discerned in every AM1 patient. For example, myocardial ischemia, even when prolonged, is not always accompanied by chest pain. The Framingham study4 and the Multiple Risk Factor Intervention Trial (MRFIT)5 have indicated that as many as one in three AMIs are not clinically recognized by either patient or physician because the chest pain is atypical or absent. The incidence of silent myocardial infarction is higher in diabetic patients6 and appears to be more common in women than men.4 Similarly, ECG findings in patients with presumed AM1 are often unresolved. This is true in particular for non-Q wave AMI, where the ECG abnormalities are nonspecific and have to be taken into account with the clinical setting because infarction cannot be established with certainty in the absence of alterations of the QRS complex. When only the

TABLE 1 Commonly Used Criteria for Diagnosing Acute Myocardial Infarction Clinical history Anginal pain, manifestations of excessive autonomic nervous system activity, positive risk factors, signs and symptoms of congestive heart failure ECG criteria (at least two subsequent ECG recordings required) Q-wave AM/: development of new, persistent Q or QS waves (30.04 s duration), R wave reduction (225%), and ST segment changes and/or T wave peaking suggestive of transmural ischemia in at least two leads of the 12-lead standard ECG non-Q wave AM/ ST segment or T wave changes, or both, in at least two leads of the standard 12-lead ECG, persisting for at least 24 h Time-dependent changes of CK and CK-MB Initial rise with subsequent fall

32


admission ECG is considered in patients with prolonged chest pain and no prior infarction, the overall efficiency is 75%.7Retrospective assessment of serial ECGs - by which time it may be too late for therapeutic interventions - increases the efficiency to 94%.* Nonetheless, the ECG is misleading in at least 8% of all AMIs and is indeterminate in an additional 12% of patients, primarily because of the presence of left bundle-branch block or nonspecific ST-T wave abnormalities. Nearly 50% of patients with AM1 will initially have nondiagnostic ECGs on presentation to the emergency de~artment.~ Thus, while clinical history and ECG changes can accurately diagnose AM1 in the majority of patients, there is an important minority in whom this is not true. The third tool available in the diagnosis of AM1 is measurement of serum levels of intramyocardial proteins. This is the most accurate means of diagnosing AMI. When myocytes are damaged, membrane leakage leads to loss of intracellular macromolecules that subsequently appear in the general circulation. Despite the presence of many potential markers, only the enzymes creatine kinase (CK) and lactate dehydrogenase (LDH) and their isoenzymes enjoy widespread clinical use. Over the past 25 years, serial measurements of CK isoenzyme MB in blood have become a cornerstone of diagnosing or excluding AMI. When myocardial injury occurs, blood levels of CK and CK-MB begin to rise within 4 to 8 h. Peak levels occur within 24 h of the onset of AM1 and return to baseline within 2 to 3 d. If the patient’s hospitalization is inordinately delayed, CK-MB may already have returned to normal. Therefore, measurements of LDH and its isoenzymes LDH-1 and LDH-2 are used widely for the late diagnosis of myocardial infarction. The rise in serum total of LDH and LDH-1 begins 8 to 12 h after myocardial cell damage; LDH peaks at 3 to 6 d, and it returns to baseline by 8 to 14 d after the onset of symptoms. B. The Diagnosis of Perioperative Myocardial Infarction 1. The Diagnosis of Perioperative Myocardial infarction in Coronary Artery Bypass Surgery Perioperative myocardial infarction remains a major and frequent complication following coronary artery bypass grafting (CABG) and adversely affects prognosis. l o Reports of its incidence vary from 8 to 35%. However, the diagnosis of perioperative myocardial infarction is difficult because there is no classic presentation. Many potential diagnostic criteria are related not only to myocardial infarction but also to the surgical procedure, cardioplegia, hypothermia, hemolysis, and other factors. Consequently, the reliability of every criterion used for the diagnosis of perioperative myocardial infarction - ECG, myocardial scintigraphy, and enzyme changes - has been criticized. Several recommend the confirmation of at least two positive criteria (ECG, myocardial scintigraphy, or enzyme results) as evidence of perioperative myocardial infarction. After CABG, the usual reference limits of enzyme concentration in serum

33

are invalid as a consequence of inevitable cardiac (e.g., cardioplegia) and extracardiac tissue damage occurring during the surgical procedure. The interpretation of CK-MB elevation is considerably more complex. Only increases in CK-MB of more than 12 to 18 h in duration correlate well with other evidence of myocardial infarction.I


2. The Diagnosis of Perioperative Myocardial Infarction in Patients Having Noncardiac Surgery Diagnostic strategies should be similar to those used with patients not having surgery, with special emphasis on serial sampling to distinguish noncardiac from cardiac sources of increased CK-MB to exclude false positive results." Assays for LDH isoenzymes are less useful because hemolysis associated with surgical trauma can cause an isoenzyme profile similar to that of myocardial infarction. C. Nonischemic Myocardial Damage 1. Myocarditis Myocarditis can be caused by a number of infectious and noninfectious agents. In North America and Europe viruses are the most common agents producing myocarditis. l 2 Since the time course of virus-neutralizing antibodies measured in serum can only support the diagnosis retrospectively, the diagnosis of myocarditis is based mainly on clinical signs and symptoms. A high index of suspicion is often necessary. The clinical presentation shows wide variations, ranging from a total absence of clinical manifestations to severe heart failure or sudden, unexpected death. l 3 The presence of myocarditis is often inferred from electrocardiographic abnormalities, particularly arrhythmias, disturbances of the conducting system, ST segment elevation, and flattening, or inversion of T waves. Rarely, Q waves may be seen.12 Due to lack of sensitivity and specificity (as discussed later), measurements of CK-MB and LDH are often inconclusive as well. Therefore, endomyocardiaI biopsy is a very important diagnostic tool and is often used to confirm a suspected myocarditis. l4 Recognizing its limited therapeutic relevance, overuse of endomyocardial biopsy in patients with suspected myocarditis has been criticized. Thus, despite various efforts to develop diagnostic criteria and techniques, the diagnosis of acute myocarditis remains a dilemma in many

2. Heart Contusion Nonpenetrating injuries of the heart result from the effects of external physical forces and are frequently overlooked. The consequences of nonpenetrating injuries

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to the myocardium vary in intensity from mild contusion to cardiac rupture. Thus, clinical manifestations also vary proportionately, and a high index of suspicion is often necessary for their recognition in all but the most obvious cases.I8 The clinical diagnosis of a heart contusion is generally based on a CK-MB / total CK activity index of more than 5% together with ECG alterations (arrhythmias, conduction disturbances, ST-T segment alterations) or a positive result in twodimensional echocardiography.l9 A number of diagnostic techniques have additionally been tested in the diagnosis of heart contusion. The reliability of all criteria used (e.g ., ECG, technetium-labeled pyrophosphate scanning, thallium SPECT scintigraphy , radionuclide angiography, echocardiography) is discussed controversially in the literature.20*2' In particular, the value of CK-MB measurements as a screening test for heart contusion in trauma victims has been questioned by a study published recently.22

II. LABORATORY DIAGNOSIS OF MYOCARDIAL DAMAGE: NEED FOR A SENSITIVE AND CARDIAC-SPECIFIC MARKER In the last quarter century, serum enzyme and isoenzyme levels - as outlined previously - have become the final arbiters by which myocardial damage is diagnosed or excluded. Serum enzymes, however, are neither perfectly sensitive nor specific. CK (molecular weight 86,000) exists as a dimer of two subunits, B and M. Three isoenzymes of CK exist (CK-MM, CK-BB, and CK-MB). CK-MB is predominately located in the heart muscle (15 to 20% CK-MB with the remainder being CK-MM). CK-MB measurements are an excellent tool for diagnosis of myocardial damage in the majority of patients. While methodological interferences rarely lead to false positive results using immunometric CK-MB mass assays (CK-MM, CK-BB, Macro CK Type I and 11, and adenylate kinase do not interfere with these assays), CK-MB not released from the myocardium can be detected in the serum of certain subgroups of patients (see Table 2), which may be misleading.23-25The CK-MB/total CK index improves the specificity of CK-MB in patients with concomitant skeletal muscle damage but leads to an unacceptable loss of sensitivity. In cases of simultaneous heart and skeletal muscle injury, CK-MB (absolute value or CK-MB index) may be inconclusive over all. The greater the extent of muscle injury, the easier it is to miss changes in CK-MB due to cardiac injury because the cardiac CK-MB is diluted by the large quantities of CK-MM, thereby decreasing the percentage of CK-MB. LDH exists as a tetramer (molecular weight 135,000). There are two subunits, M (muscle) and H (heart). LDH may be fractionated into five isoenzymes. Most tissues contain all five isoenzymes, but LDH-1 (comprised of four H subunits) and, to a lesser extent, LDH-2 predominate in the heart. Skeletal muscle and the liver have mostly LDH-5. LDH-1 is abundant in the myocardium and appears in blood when myocardial cell damage occurs. If LDH-1 exceeds LDH-2, myocardial damage is likely. Erythrocytes, kidneys, the brain, and the pancreas are other 35


TABLE 2 Causes of CK-MB Increases Not Associated with Myocardial Damage Release of nonmyocardial CK-MB Trauma to muscle Crush injury Burns Electrical injuries Noncardiac surgery Extreme exercise (e.g., marathon running, long-distance skiing) Grand ma1 seizures Various inflammatory and noninflammatory myopathies Chronic renal failure Hypothyroidism Chronic alcoholism Myositis Rhabdomyolysis Hyperthermia and hypothermia Ectopic CK-MB production in tumor patients Decreased clearance of serum CK-MB Hypo- and hyperthyroidism

important sources of LDH-1,26 so that an abnormal serum concentration of LDH-1 may result from irreversible damage to any of these tissues as well. LDH and its isoenzymes are less accurate than CK and CK-MB determinations and add little to the enzymatic diagnosis of myocardial damage when CK and CK-MB rise and fall appropriately.27 Finally, conventionally used enzymes fail to diagnose myocardial damage efficiently in patients with myocardial injury and skeletal muscle damage -e.g., after cardiac surgery, polytrauma, or multiorgan damage. Therefore, clinicians would benefit from the introduction of a new sensitive and cardiac-specific marker of damage to myocardial muscle cells. The contractile and regulatory proteins of the myocardium could provide such a useful diagnostic tool.

111. BIOCHEMISTRY OF TROPONIN T AND TROPONIN COMPLEX Troponin T (TnT) is a structural protein of striated muscle fibers. It is part of the contractile apparatus, which consists of two different filaments, the thick and thin filaments. The thick filaments are surrounded by a hexagonal array of thin filaments. The thick filament consists of myosin molecules. Myosin molecules in their monomeric form consist of,two heavy chains (MHC) and four light chains (MLC), two MLC-1 and two MLC-2. The thin filament comprises actin, tropomyosin, and troponin complex (see Figure 1).

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troponin complex

FIGURE 1. Schematic of a part of the thin filament showing actin helix, tropomyosin coil, and troponin complex.

Troponin complex comprises three protein ~ u b u n i t s : ~ * * ~ ~ 1.

2.

3.

Troponin C (TnC) - TnC binds calcium and is responsible for regulating the process of thin filament activation during skeletal and heart muscle c o n t r a ~ t i o nIts . ~~molecular weight is 18,000 Da. Troponin I ( T i ) - TnI is the 21,000 Da inhibitory subunit that prevents contraction in the absence of calcium and TnC.3’ Troponin T (TnT)-TnT (37,000 Da) is responsible for binding the troponin complex to tropomyo~in.~~ In the human heart muscle, approximately 6% (about 0.025 mg/g wet weight) of the total myocardial TnT is found as a soluble, cytoplasmic pool, which probably serves as a precursor pool for the synthesis of troponin

A. Model of Contractility The sliding filament model of muscle contraction postulates that the force of contraction is generated by cyclic interactions of myosin heads with the actin subunits of the thin filament (cross-bridge formation). Energy for this process is derived from the hydrolysis of ATP by the actomyosin ATPase. In resting muscle, actin-myosin interactions are prevented by the troponidtropomyosin complex. Electrical depolarization of a striated muscle cell leads to an increase in intracellular calcium concentration. Calcium binds to TnC, causing a conformational change of troponin-tropomyosin complex, which leads to derepression of actinmyosin interactions and results in muscle cell contraction. Differences in the activity patterns of slow and fast skeletal muscle, smooth and heart muscle are reflected in the characteristics of their myofibrillar proteins. Myosin, tropomyosin, TnI, TnC, and TnT all exist in polymorphic forms that are characteristic of the muscle type from which the proteins are derived. The

37

polymorphic forms of each protein are different gene products. Myofibrillar proteins of striated muscle are expressed as tissue-specific isoforms. Cardiac TnI and TnT are molecules unique to cardiac muscle and may be differentiated from their skeletal muscle isoforms by immunologic methods.


IV. CARDIAC TROPONIN T ASSAY An enzyme-linked immunosorbent assay (ELISA) specific for cardiac TnT has been developed by Katus et al.34An improved version of this assay is now available in a commercial kit.” This automated ELISA of cardiac TnT is based on a single-step sandwich principle, with streptavidin-coated tubes as the solid phase and two monoclonal anti-human cardiac TnT antibodies. Test results can be obtained 90 min after starting the assay. The assay is immunologically specific for cardiac TnT. However, a cross-reactivity of 1 to 2% with purified skeletal muscle TnT at high concentrations was found due to nonspecific absorption of skeletal muscle TnT to the assay tube^.^^.^^ The clinical experience with the TnT assay,3G38by contrast, suggests that this theoretical cross-reactivity with purified skeletal muscle TnT is not a relevant problem in clinical practice. V. TROPONIN T EVALUATED IN DIAGNOSING MYOCARDIAL INJURY: PRESENT CLINICAL RESULTS AND FUTURE PERSPECTIVES A. Acute Myocardlal Infarction 1. Troponin T Time Course in Acute Myocardial infarction When myocytes are damaged, loss of membrane integrity occurs and intracellular macromolecules diffuse first into the interstitium and subsequently into the intravascular space and lymphatics. The pattern of appearance in blood depends on: The intracellular location and whether molecules are bound or free. Molecular weight (because heavier molecules diffuse at a slower rate). Local blood and lymphatic flow. 4. The rate of elimination from blood. 1.

2. 3.

Figure 2 shows TnT changes compared with myoglobin, LDH, total CIS-, and CK-MB activity time courses in a representative AM1 patient. Most striking is the much higher relative increase in TnT compared with other laboratory parameters. Concentrations of TnT in serum start increasing within a few hours after the onset of symptoms (median: 4, range: 1 to 10 h36). A plateau exists from the second to the fifth day. The sensitivity of TnT for detecting myocardial

38

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100

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CKMB activity LDH

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FIGURE 2. Time-dependent patterns of cardiac troponin T,myoglobin, total creatine kinase, creatine kinase-MBactivity, and lactate dehydrogenasein a 45-year-old man with anterior 0wave infarction receiving intravenousthrombolytic therapy. Relative increaseswere calculated by dividing each value by the respective upper limit of the reference interval of each parameter. Note: logarithmic scale of y-axis.

infarction is 100% from 10 h to at least 5 d after the onset of chest pain. TnT concentrations identify all Q and non-Q wave myocardial infarction during this time i n t e r ~ a l .TnT ~ ~ .release ~ ~ in non-Q wave AM1 is similar to that occurring in Q wave AMI. Thus, TnT also provides reliable diagnosis of non-Q wave AMI. In many AM1 patients, increased TnT concentrations beyond the seventh day allow the diagnosis based on this biochemical marker even during the second week after admission. Concentrations are increased for up to 3 weeks in some patients with late and/or high peak values. Consequently, TnT determinations are particularily useful for diagnosing late myocardial infarction in AM1 patients who do not seek medical attention within the 2- to 3-d window during which CK and CK-MB are elevated.

a. Early Myocardial infarct Detection TnT is an early marker of myocardial injury as well. Figure 3 shows a comparison of diagnostic sensitivities of myoglobin, CK, CK-MB activity, and cardiac TnT during the early hours after the onset of AMI. TnT is significantly more sensitive than CK-MB activity (differences between TnT and CK or myoglobin are not significant). 50% of all AM1 patients investigated had increased TnT concentrations at 3 h3’ and 4 h36(see Figure 3) after the onset of chest pain,

39

Occurrence of first increased values after AM1

8

0

0

+ j % c-1 9 0

8


8

8

myoglobin

0

CK

CKMBactivity

troponinT

FIGURE 3. A comparison of sensitivities of marker proteins in the early phase of acute myocardial infarction. The occurrences of first increased values (given in hours after the onset of chest pain) of myoglobin, total creatine kinase (CK), CK-MB activity, and cardiac troponin T were compared in 25 patients with acute myocardial infarction (AMI). All patients were admitted within 4 h of chest pain to the coronary care unit (median: 2.13 h) and all received intravenous thrombolytic therapy. The differences between TnT and CK-MB activity, myoglobin and CK, myoglobin and CK-MB activity are statistically significant (no significant difference between TnT and myoglobin or CK). Blood samples were drawn every hour during the first 10 h after admission to the coronary care unit.

respectively. TnT is 100% sensitive from 10 h after the onset of infarct-related symptoms onward until the fifth to sixth day after AMI.36.37Receiver-operatingcharacteristic (ROC) curves of TnT and CK-MB activity to discriminate between AM1 and no AM1 on admission to the emergency department in patients with chest pain but without trauma are compared in Figure 4. These ROC curves do not differ markedly. Thus, in the early diagnosis of AMI, the efficiency of both markers is about the same in this patient population. The clinical implication of this finding is of limited value until more rapid determination of TnT (stat measurement) is available (current assay time is 90 min35).However, TnT concentrations measured after a 12-h observation period in patients without recurrent chest pain could provide a simple, sensitive, and specific diagnostic aid to exclude AM1 and identify candidates for the early “step down’’ transfer from the coronary care unit,30resulting in more efficient and economic use of the critical care facility. One of the most striking features of TnT following AM1 is its high relative increase compared with TnT concentrations measured in healthy controls. A comparable increase in AM1 patients has been described for only CK-MB mass concentrations.40*41 Cardiac TnT shows an average 30- to 40-fold increase above

40

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0,l 0,2 0,3 0,4 0,s 0,6 0,7 0.8 0,9 1.0

specificity FIGURE 4. Receiver-operating-characteristic curves (ROC) for the discrimination between AM1 and no AM1 in emergency room chest pain patients without trauma. ROC curves are based on TnT and CK-MB activity measurements in blood samples that were drawn immediately after admission to the emergency department. The equation sensitivity = specificity is represented as a straight diagonal line and indicates a worthless test. The larger the deviation from this line (and the larger the area under the ROC curve), the better the discriminative power of the test under scrutiny.

the upper limit of the reference interval, significantly greater than the relative increases of total CK, CK-MB activity, myoglobin, and LDH in the same patients (see Figure 2, note logarithmic scale of Y-axis), which partly explains the high diagnostic sensitivity of TnT. In patients with small AMI, the time course of TnT contrasts more strongly with the normal range than all other markers except for CK-MB mass c o n ~ e n t r a t i o n . ~Thus, ~ * ~ 'TnT measurements are particularly useful in the 9% of patients with borderline CK-MB activity in whom the diagnosis of AM1 cannot be made or excluded with certainty.42The overall efficiency of TnT in diagnosing AM1 is marginally greater than that of CK-MB (98 vs. 97%).37 However, in contrast to the transient usefulness of CK-MB measurements, the efficiency of TnT remains 98% until 6 d after admission. 2. Effects of Infarct-Related Artery Reperfusion on the Release Kinetics of Troponin T in Acute Myocardial Infarction Patients Reperfusion obviously influences the release of TnT in AM1 patient^.^^^^^ In 41

patients who undergo thrombolytic treatment, changes of TnT concentration on the first day are very variable (see Figure 5). The appearance of TnT in serum on day 1 after the onset of AM1 depends strongly on reperfusion and on the duration of ischemia until reperfusion occurs.33 By contrast, the kinetics of TnT release later than the first day after AM1 are unaffected by reperfusion.


a. Early Reperfusion In patients in whom early perfusion was successful, TnT reaches a peak within 24 h after admission. A distinct peak in TnT concentrations with a sub-

A 50

40

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hours after the onset of chest pain

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FIGURE 5. Cardiac troponin T (A) and total CK (B) release profile in three representative patients with Q wave myocardial infarction. E,R: patient receiving thrombolytic therapy leading to early reperfusion (note: distinct maximum 12 h after the onset of AMI), LR: patient with thrombolytic therapy without early reperfusion (late reperfusion), PO: patient with permanent occlusion (no thrombolytic therapy).

42

sequent rapid decrease is found at a median time of 14 h after the onset of chest pain in all patients with AM1 reperfused within 3.5 h after the onset of pain.33 A second, much smaller peak is observed at about the fourth day after admission to the hospital. TnT appears and disappears in serum significantly earlier in patients with early reperfused AMI.


b. Permanent Occlusion (No Thromboiytic Treatment) In patients without thrombolytic therapy and no spontaneous early reperfusion, there is no distinct TnT peak on day 1. In these patients, maximal TnT concentrations occur several days after admission on or about the fourth day after the onset of symptoms. c. Late Reperfusion TnT time courses of patients receiving thrombolytic therapy not leading to early reperfusion resemble those of patients without thrombolytic treatment. The early peak is absent in patients with AM1 reperfusion occumng >5.5 h after the onset of pain. The effects of reperfusion on the kinetics of TnT release in AM1 can be used to assess noninvasively the effectiveness of thrombolytic therapy. All patients with AM1 reperfusion C 5 . 5 h after the onset of pain had ratios of peak TnT concentration at day 1 to TnT concentration at day 4 > 1.O due to a marked TnT “washout phenomenon”, whereas in patients with nonreperfused AM1 this ratio is ~ 1 . 0 . ~ ~ The serum concentration changes of TnT after AM1 are most probably explained by the intracellular compartmentation of this contractile protein (see section on biochemistry). The TnT time course in AM1 reveals characteristics of free cytosolic molecules on day 1 after the onset of symptoms, and those of structurally bound constituents thereafter. AM1 reperfusion obviously leads to “washout” of the free cytosolic TnT pool, resulting in a rapid increase in serum TnT. By contrast, kinetics of TnT released after day 1 are not affected by reperfusion. Because the biological half-life of TnT in the general circulation is 120 min3’, the later peak around day 4 most probably reflects ongoing damage to the contractile apparatus. Thus, the biphasic release profile of TnT is probably the result of a rapid loss of the cytoplasmic pool superimposed on the prolonged myofibrillar degradation that results in a long plateau effect several days after the onset of pain.

3. Noninvasive Estimation of hfarct Size The reperfusion-dependent release hampers attempts to calculate the size of

43


the infarct by use of cytosolic TnT, by contrast, reveals the characteristics of both a cytoplasmic and a structurally bound protein. The early rise in TnT may be due to leakage from the cytoplasm of ischemic but partly viable myocytes, whereas the later peak, usually around day 4, reflects irreversible damage to the contractile apparatus. Thus, TnT measurements may be more accurate in quantifying infarct size. The rapid decrease in TnT concentrations and the significantly decreased concentrations from the second day onward after successful reperfusion may reflect a reduced size of myocardial infarction. Late TnT peaks may therefore depend on the size of AMI, an assumption that is currently being tested in experimental and clinical studies. It remains to be shown whether cumulative TnT release provides a more useful tool in noninvasive estimation of infarct size than commonly used cumulative CK-MI3 or LDH-1 release.

B. Diagnosis of Perioperative Myocardial Infarction 1. Diagnosis of Perioperative Myocardial Infarction in Noncardiac Surgery The biggest advantage of TnT is its cardiospecificity. Thus, measurements are especially helpful in the assessment of patients with myocardial ischemia and skeletal muscle injury, for example, after surgery. The ratio of CK-MB/total CK offers better specificity than measurements of CK-MB alone, but with an unacceptable loss of sensitivity. Therefore, TnT should replace CK-MB measurements in the laboratory diagnosis of perioperative myocardial infarction, because TnT is the most useful diagnostic test to assess the presence or absence of cardiac injury in patients with skeletal muscle damage.37*38 2. Diagnosis of Perioperative AM1 in CABG Surgery

In cardiac surgery patients, the ECG and CK activity are often unhelpful.44 As for other cardiac enzymes and proteins after CABG, the significance of the usual reference limits of TnT are invalid as a consequence of inevitable cardiac tissue damage (e.g., cardioplegia) occurring during the surgical p r o ~ e d u r e . ~ ~ , ~ Figure 6 shows the TnT time course after CABG in a representative patient with perioperative AM1 and in another representative uncomplicated patient. In perioperative AMI, TnT concentrations start increasing during reperfusion of the cardioplegic heart, peak on or about the fourth postoperative day, and remain increased until at least the seventh day after CABG surgery. In patients without perioperative complications, TnT concentrations do not exceed 2.5 pg/l .45 TnT in uncomplicated patients also significantly increases after reperfusion over concentrations before bypass, which probably reflects myocardial cell damage from ischemia during ~ardioplegia.~~ In these patients, TnT concentrations (peak Values, duration of increase) are associated with the duration of cardiac arrest and

44

A

3

i 1

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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19

SURGERY

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-

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0

SURGERY

FIGURE 6.

I

I

I 2 3 4 5 6 7 1 8 9 10 11 12 13 14115 I6 17 I8 I

DAYl

DAYS24

Cardiac troponin T (A) and CK-MB (B) in

two representative patients after coronary artery bypass grafting surgery. Abbreviations: CPBP = cardiopulmonary bypass, AM1 = acute myocardial infarction. Time: before induction of anesthesia (l), prior to surgery (2), before (3) and hourly after (4 to 8) CPBP, subsequently every 4 h (8 to 14), and daily thereafter (15to 19).

aortic cross-clamping.46After CABG surgery there is a third group of patients with minor perioperative myocardial damage.4s.46TnT in these patients is moderately increased and exceeds 2.5 pg/l. However, these patients do not meet the standard enzymatic and ECG criteria of penoperative AMI. Nonspecific ECG changes are frequent in those patients. Perioperative myocardial cell damage seems to be much more common than has been previously recognized by changes of ECG and serum enzymes. The clinical relevance of minor penoperative myocardial damage after CABG is the topic of a current clinical study. In summary, TnT reliably identifies penoperative infarction during CABG and is superior to CK-MB activity, especially in diagnosing minor perioperative myocardial damage. TnT may be very useful in assessing the effectiveness of various cardioprotective measures.46

45

C. Coronary Artery Disease 7. Diagnosis of Minor Myocardial Damage (Microinfarction) in Patients with Unstable Angina Pectoris Unstable angina pectoris comprises patients with: 1.


2. 3.

Chest pain at rest or provoked by minimal exertion. A new pattern of increasing chest pain in a previously chronic angina. Both, with or without ST segment alterations or T wave inversion in the ECG .

These patients do not meet the standard enzymatic and ECG criteria for myocardial infarction. Stable and unstable angina and myocardial infarction represent a continuum, from stable lumen-restricting coronary artery plaques, to plaque fissuring with overlying but nonocclusive thrombus, to more substantive plaque disruption with adjacent occlusive thrombus composed of varying amounts of erythrocytes, fibrin, and platelets.47The distinction between small non-Q wave infarction and unstable angina pectoris is only a shade of gray. For appropriate therapy and preventive measures, the challenge is not always simply to rule in or rule out myocardial infarction, but rather to distinguish patients with acutely unstable coronary lesions from those with either stable coronary disease or none. The lack of sensitivity of CK and CK-MB activity assays makes it very difficult to determine whether or not small amounts of myocardial necrosis are present in patients with chest pain. TnT concentrations, by contrast, might provide a very sensitive marker of necrosis of small amounts of cardiac muscle. In fact, increased TnT concentrations have been described in a subgroup of unstable angina pectoris patients (minor myocardial damage or microinfarction) who did ~ . ~concentrations ' in patients with unstanot meet the standard AM1 c ~ i t e r i a . ~TnT ble angina correlated with ECG signs of myocardial ischemia and with the inhospital development of AM1 and death.37Katus et a1.,37however, emphasize that the TnT test cannot be used to screen (negative predictive value: 0.26) for the presence of severe coronary artery disease (>75% obstruction of a major coronary artery). In patients with unstable angina normal TnT test results in serially drawn blood samples rule out AMI, but do not preclude the presence of severe coronary artery disease and a poor prognosis for the patient. A protein marker can only indicate acute myocardial ischemia and thus only indirectly indicates the state of a coronary vessel. On the other hand, increased TnT concentrations in patients with unstable angina indicate recently developed severe While it might be temptcoronary artery narrowing with high specificity (0.85).37 ing to conclude that TnT determinations are of limited usefulness in the identification of angina pectoris patients at risk or with a poor prognosis, a final judgment will require further prospective evaluation.

46


2. TnT Concentrations after Percutaneous Transluminal Coronary Angioplasty (PTCA) PTCA is a method used frequently to dilate stenotic coronary arteries. After PTCA, TnT is a sensitive marker for determining whether myocardial injury is present or not.48 TnT time courses of three representative patients after PTCA are shown in Figure 7. In uncomplicated PTCA patients, TnT does not increase in serum. Angiographically visible occlusion of smaller side-branches, however, although not accompanied by ST segment changes or chest pain, leads to an increase in TnT above the normal range. Reocclusion of a successfully dilated stenosis causes a marked rise in TnT (see Figure 7). The extent of myocardial damage after PTCA can be reliably estimated by cardiac TnT measurements, which could be valuable diagnostic aids in deciding when to discharge patients from the hospital.

D. Myocarditis The high sensitivity and cardiospecificity of the TnT assay suggest that TnT measurements might discern myocardial muscle damage in myocarditis patients. In an experimental model of myocarditis in mice, we found a close correlation between TnT serum concentrations and the histopathological signs of degeneration of myocytes (preliminary unpublished observations). In addition, we found in-

6 reocclusion 5

9 b

3

4

3

i 2

side-branch occlusion

1

0 0

48

24

72

96

hours after PTCA

FIGURE 7. Cardiac troponin T in three representative patients after percutaneous transluminalcoronary angioplasty (PTCA). Time: 0 = before PTCA. Side-branch occlusion in this patient was not accompanied by ST segment changes or chest pain.

47


creased TnT concentrations in patients with confirmed myocarditis, and elevated TnT has also been reported in patients with perimyocarditis by Katus et TnT concentrations and CK and CK-MB activities in a patient with viral perimyocarditis are shown in Figure 8. A systematic evaluation of TnT measurements in diagnosing myocarditis has not yet been published. Strategies based on TnT measurements, however, will require widespread prospective evaluation before they are adopted as part of routine care. Another subtle aspect of myocardial damage is rejection of the transplanted heart. At present, various groups are evaluating cardiac TnT measurements in diagnosing rejection in heart-transplanted patients. Although the results of these studies have not yet been published, TnT measurements might help to reduce the frequency of percutaneous right ventricular endomyocardial biopsies in these patients.

E. Heart Contusion Troponin T measurements are particularly useful in the assessment of cardiac injury in the presence of skeletal muscle da~nage.~’.~* Serial cardiac TnT measurements are a valuable aid in the diagnosis of heart contusion.49Although these results must be confirmed in a larger series, it is reasonable to suggest that TnT should replace CK-MB measurements as a criterion in the laboratory diagnosis

r

CKhlB activity

0

6

12

18 hours after admission

24

30

FIGURE 8. Troponin T, total CK and CK-MB activity in a patient with viral perirnyocarditis. Relative increases were calculated by dividing each value by the respective upper limit of the reference interval for that parameter.

48

of blunt heart trauma. Total CK, CK-MB activity and troponin T concentrations in a polytraumatized patient with heart contusion are shown in Figure 9. VI. CARDIAC TROPONIN T IN DIAGNOSIS OF MYOCARDIAL


INJURY COMPARED WITH OTHER STRUCTURAL PROTEINS OF HEART MUSCLE FIBERS During the last decade, different contractile proteins have been assayed in the sera of patients after AM1 as an alternative to cytoplasmic enzymes. The purpose of this review is to compare the TnT assay with previously described assays for other contractile proteins.

A. Myosin Heavy Chains (MHC) The incomplete identity of cardiac and skeletal muscle MHC i s o f ~ r m s ~ ~ ~ ~ ' and the coexpression of cardiac beta-MHC isoforms of myosin in slow skeletal muscle5' make it very difficult (but theoretically feasible) to develop cardiac specific monoclonal anti-MHC ,antibodies. All MHC assays described in the literature so farj2-54 strongly cross-reacted with MHC from skeletal muscle and were, therefore, not superior to CK-MB with respect to specificity. MHC frag-

'

l

o

w

l

CK

upper limit of reference interval

0

24

48

72

96

hours after admission FIGURE 9. Troponin T, CK and CK-MB activity in a polytraumatized patient with heart contusion. Relative increases were calculated by dividing each value by the respectiveupper limit of the referenceintervalforthat parameter. Note: logarithmic scale of y-axis.

49


ments are detectable in serum from 2 to 10 d after the onset of myocardial infarction. This delay in plasma release is longer than for any other cytoplasmic or contractile protein so far assayed, which supports the hypothesis that within muscle fibers all MHC is assembled in the contractile filaments (no cytoplasmic MHC precursor pool). This appearance of MHC in the bloodstream precludes its use in the early phase of AMI. The time course is monophasic and MHC peaks several days after the onset of myocardial infarction. Figure 10 shows a comparison of cardiac beta-type MHC and cardiac TnT release in a 68-year-old female patient with inferior wall myocardial infarction. In an experimental canine myocardial infarction model, the necrosed myocardial mass correlated very closely with the cumulative MHC MHC is also a sensitive indicator of myocardial necrosis after a cardiac operation.56In perioperative AMI, MHC starts to increase on postoperative day 3 and reaches maximal levels on day 7. In contrast to TnT, MHC only allows late diagnosis of myocardial damage.

B. Myosin Light Chains (MLC) It has been reported that - although many structural features are shared cardiac and skeletal-muscle MLC are each characterized by a unique amino acid ~equence.~’ On the other hand, there is evidence that the myosin alkali light chains (MLC-1) of mouse ventricular and slow skeletal muscle are indistinguishable and are encoded by the same gene.58Human MLC isoforms (in particular MLC-2)

days after the onset of infarction

FIGURE 10. Cardiac beta type myosin heavy chain and cardiac troponin T release in a patient with inferior wall myocardial infarction. Abbreviations: myosin heavy chain (MHC), troponin T (TnT); methods: cardiac beta-type MHC (immunoradiometricassay, E.R.I.A. Diagnostics Pasteur, Marnes la Coquette, France), cardiac TnT (enzymeimmunoassay, Boehringer Mannheim, Mannheim, Germany).

50


may provide a unique cardiac specific antigen, and cardiac MLC measurements have the potential for very high specificity for cardiac damage. Although this potential has not been realized in the assays described in the literature so far,59-62 because they showed varying degrees of cross-reactivity with skeletalmuscle MLC, monoclonal antibodies in combination with a sandwich technique may lead to a desirable low cross-reactivity (1-2%).61 At present, such a MLC assay is not commercially available. Only a minor fraction of MLC (approximately 1% of the total MLC content) exists as a soluble cytoplasmic precursor pool for myosin s ~ n t h e s i s .After ~ ~ . ~AMI, MLC begins to rise in serum 6 h after the onset of the attack. Peak values are observed several days (about day 3 to 4) after infarction regardless of r e p erfu~ion.~*~’ Despite a serum half-life of 75 min,66 MLC remains elevated up to 14 d in patients with a large AMI. MLC is useful in quantifying infarct ~ i z e .A~subgroup , ~ ~ of patients with unstable angina pectons showed increased MLC serum concentrations.&2 The cytosolic pool of TnT is approximately 50 times larger compared with MLC-1.33Thus, MLC determinations have some drawbacks compared with TnT measurements: 1.

2.

Pathologically increased concentrations occur earlier in TnT than in MLC and TnT is, therefore, superior to MLC in the early diagnosis of myocardial injury. The appearance of MLC in serum is to a much lesser extent dependent on early infarct reperfusion.

The release of MLC was not significantly changed by recanalization of the infarctrelated artery compared with that in nonreperfused AMI.64Thus, only TnT measurements are useful to assess noninvasively whether reperfusion of the infarctrelated artery has occurred.

C. Troponin I (Tnl) The cardiac troponin inhibitory protein (TnI) is a cardiac specific p r ~ t e i n . ~ ’ Cardiac TnI is the only TnI isotype present in myocardium. Experimental results indicate the presence of a cytoplasmic precursor pool of unassembled TnL6* Previous clinical data on TnI release after myocardial damage suggest that all benefits outlined for the TnT assay are also valid for a cardiac specific TnI asThe release profile of TnI is similar to that of TnT, although there are, so far, no data on the reperfusion-dependent release of TnI.69TnI starts to increase parallel to CK-MB, peaks at 18 h (mean) after the onset of infarction, and has increased concentrations in serum for several days.69TnI usually shows a second smaller peak between 60 to 80 h after infarction. Cardiac specific TnI assays have been developed by Cummins et al.69*72Up to now, however, no such assay is commercially available. Thus, at present, TnT is the only cardiac specific marker available for widespread use. 51

D. Troponin C (TnC)


In the adult, the expression of the cardiac TnC isoform is usually restricted to cardiac and slow skeletal The two TnC isoforms are encoded by distinct single copy mammalian genes.3oConsequently, it is not possible to develop a cardiac TnC assay without cross-reactivity with TnC from skeletal muscle. No data on a cardiac TnC assay or the release of TnC after cardiac damage have been published yet. E. Tropomyosin The alpha skeletal form of tropomyosin (molecular weight 66,000) is identical to the cardiac form in mammals, including man,73which prohibits the development of a cardiac specific tropomyosin assay. The release kinetics of tropomyosin after myocardial infarction are similar ‘to those of TnT or TnI.74

F. Actin Differences between the primary sequences of skeletal and cardiac actin forms have been reported.75However, cardiac hypertrophy is associated with the reactivation of skeletal alpha-actin gene expre~sion,’~ which might limit the utility of cardiac actin measurements to diagnose cardiac damage in these patients. Neither data on a cardiac actin assay nor on the actin release after cardiac damage have been published yet.

VII. SUMMARY AND CONCLUSION In the last quarter-century, serum levels of cardiac enzymes and isoenzymes have become the final arbiters by which myocardial damage is diagnosed or excluded. Since conventionally used enzymes are neither perfectly sensitive nor specific and in particular fail to diagnose myocardial damage in patients with myocardial injury and skeletal muscle damage (e.g., after surgery, polytrauma, or multi-organ damage), there is need for a new sensitive and cardiac specific marker of myocardial injury. Cardiac TnT is a contractile protein unique to cardiac muscle and it can be differentiated by immunologic methods from its skeletal muscle isoform. An enzyme-linked immunosorbent assay specific for cardiac TnT is now available in a commercial kit for routine use. The biggest advantage of this TnT assay is its cardiac-specificity. In human heart muscle, approximately 6% of the total myocardial TnT is found as a soluble cytoplasmic pool. The serum concentration changes of TnT after myocardial damage are most probably explained by the intracellular compartmentation of this protein. TnT starts to

52


increase within a few hours after the onset of myocardial damage and remains increased for several days, reflecting continuous release of protein from disintegrating myofilaments. TnT release reveals characteristics of both free cytosolic molecules on day 1 and of structurally bound constituents thereafter and, therefore, TnT reflects changes in both free and bound proteins. TnT allows early and late diagnosis of myocardial damage (long diagnostic window). Measurement of TnT concentrations appears to be quite accurate, in part because baseline levels are normally very low and elevations are substantial when myocardial damage occurs. TnT provides a very sensitive index of small amounts of necrosis of cardiac muscle. The following proposed indications for usage of cardiac TnT measurements are derived from current clinical results with the new TnT assay: 1.

2.

3.

4.

5. 6.

Acute myocardial infarction - During the early stages of myocardial infarction, TnT measurements are particularly useful to assess the extent of myocardial damage in patients with concomitant skeletal muscle injury (e.g., after cardiopulmonary resuscitation, direct-current countershock therapy, or in patients presenting with chest pain after heavy physical exercise) and in patients with borderline CK-MB. Late diagnosis of myocardial infarction - TnT measurements are useful in myocardial infarction patients who do not seek medical attention within the 48 to 72 h period during which total CK and CK-MB are elevated. The diagnostic efficiency of TnT remains 98%until 6 d after the onset of infarctrelated symptoms. Monitoring the efectiveness of thrombolytic therapy in AMI patients -The ratio of peak TnT concentration on day 1 to TnT level on day 4 allows discrimination between patients with successful (>1) and failed (51) reperfusion. Exclusion of AM1 in patients with unstable angina pectoris, and exclusion of myocardial injury after PTCA Diagnosis of perioperative Ah4I including CABG surgery Diagnosis of blunt heart trauma

Whether a gradation of unstable angina on the basis of serum TnT concentrations can efficiently identify patients at risk remains to be demonstrated in further prospective clinical evaluation. It also remains to be seen whether measurements of TnT can efficiently reveal some of the more subtle aspects of myocardial damage - for example, in myocarditis in cardiotoxicity or in assessment of infarct size. Finally, TnT measurements are sensitive and cardiac-specific and could, therefore, provide a new criterion in laboratory diagnosis of myocardial injury.77 Traditional methods for assessing myocardial damage, however, are well-established. Thus, despite its scientific merits, much work will have to be done with TnT before it gains widespread acceptance.

53


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57

Diagnosis of myocardial infarction: cardiac troponin I or troponin T?

Cardiac troponin T in the diagnosis of heart contusion.

Cardiac troponin I as compared to troponin T for the detection of myocardial damage in horses.

Troponin T and myocardial damage.

Comparison of plasma microRNA-1 and cardiac troponin T in early diagnosis of patients with acute myocardial infarction.

Undetectable high-sensitivity cardiac troponin T level in the emergency department and risk of myocardial infarction.

High sensitivity cardiac troponin and the under-diagnosis of myocardial infarction in women: prospective cohort study.

Cardiac troponin T in forensic autopsy cases.

Impact of Aging on High-sensitivity Cardiac Troponin T in Patients Suspected of Acute Myocardial Infarction.

Cardiac troponin testing and myocardial infarction.

Diagnosis of acute myocardial infarction in patients with renal insufficiency using high-sensitivity troponin T.

Diagnostic significance of high sensitivity troponin in diagnosis of blunt cardiac injury.

Non-invasive assessment of perioperative myocardial cell damage by circulating cardiac troponin T.

Diagnostic Role of High-Sensitivity Cardiac Troponin T in Acute Myocardial Infarction and Cardiac Noncoronary Artery Disease.

Cardiac troponin T elevation associated with transient global amnesia: another differential diagnosis of 'troponosis'.

Cardiac troponin assays: a review of quantitative point-of-care devices and their efficacy in the diagnosis of myocardial infarction.

Origin of Cardiac Troponin T Elevations in Chronic Kidney Disease.

Diagnostics on acute myocardial infarction: Cardiac troponin biomarkers.

High-sensitivity cardiac troponin t concentrations below the limit of detection to exclude acute myocardial infarction: a prospective evaluation.

Hospital Admission and Myocardial Injury Prevalence after the Clinical Introduction of a High-Sensitivity Cardiac Troponin I Assay.

Kinetics of Highly Sensitive Troponin T after Cardiac Surgery.

Use of copeptin and high-sensitive cardiac troponin T for diagnosis and prognosis in patients with diabetes mellitus and suspected acute myocardial infarction.

Troponin I and T in relation to cardiac injury detected with electrocardiography in a population-based cohort - The Maastricht Study.

Intracellular compartmentation of cardiac troponin T and its release kinetics in patients with reperfused and nonreperfused myocardial infarction.