Clinical Cardiology Frontiers

Transcription

1 750 Clinical Cardiology Frontiers Biochemical Markers of Myocardial Injury Is MB Creatine Kinase the Choice for the 1990s? Jesse E. Adams III, MD; Dana R. Abendschein, PhD; Allan S. Jaffe, MD M odern medical practice requires rapid decisions. Patients must be triaged faster, treated more expeditiously, and discharged earlier. Diagnostic tests used to evaluate patients with possible myocardial ischemia/infarction must be simple to perform and must rapidly and accurately accomplish several goals: (1) Differentiate patients with and without acute myocardial infarction (AMI). Only a small percentage (10% to 20%) of patients with chest discomfort or other symptoms compatible with ischemia have infarction.1 There is enthusiasm for the concept that molecular markers might be able to define this group rapidly enough to aid in deciding which patients should receive treatment with thrombolytic agents and which patients are at sufficiently low risk that they could be sent home or cared for in a less intensive and thus less costly facility than an intensive care unit. (2) Distinguish patients who have coronary recanalization after treatment with thrombolytic agents from those who do not so that those in need of additional treatment can be identified. (3) Permit estimation of the extent of myocardial damage, the most reliable predictor of survival after acute myocardial infarction.2 Although tests for the MB isoenzyme of creatine kinase (CK) in plasma have served all of these purposes for nearly three decades, increases in MBCK are not as specific for myocardial injury as first believed and occur too late for optimal early diagnosis of infarction or prompt detection of coronary recanalization. In addition, the complex kinetics of release and clearance after reperfusion confound analyses of infarct size. Because of these limitations, alternative markers have been investigated. Are they better, or will these markers have similar or even more significant limitations than MBCK? Historical Perspective The report of Karmen et al in that serum glutamate oxaloacetate transaminase (SGOT, now aspartate transaminase or AST) activity was increased in the serum of patients with AMI stimulated an explosion in diagnostic testing. Because of greater specificity for heart, measurement of lactate dehydrogenase (now designated LD) and then LD isoenzymes replaced AST for the confirmation of myocardial injury.4 However, the Received December 18, 1992; revision accepted April 2, From the Cardiovascular Division, Washington University School of Medicine, St Louis, Mo. Correspondence to Dr Jaffe, Washington University School of Medicine, Cardiovascular Division, Box 8086, 660 S Euclid, St Louis, MO rapid appearance of CK in serum after AMI5 and the improved specificity for myocardial injury provided by measurement of the MB isoenzyme6 quickly established MBCK as the marker of choice. Nevertheless, it became clear that the ECG could be normal despite biochemical evidence of myocardial damage7 and that marked ECG changes could occur without increased concentrations of cardiac proteins in the blood.8 Thus, the current World Health Organization criteria for the diagnosis of AMI includes the presence of two of the following criteria: clinical symptoms compatible with acute ischemia, ECG abnormalities, and a pattern of enzyme release typical of myocardial injury. It has been suggested that a typical rising and falling pattern of MBCK alone in the proper clinical setting should suffice for confirmation of AMI.9 Characteristics of Markers of Myocardial Injury An ideal marker of myocardial injury would (1) be found in high concentration in myocardium, (2) not be found in other tissues, even in trace amounts or under pathological conditions, (3) be released rapidly and completely after myocardial injury, (4) be released in direct proportion to the extent of myocardial injury, and (5) persist in plasma for several hours to provide a convenient diagnostic time window but not so long that recurrent injury would not be identified. Determining Factors The factors that determine these characteristics and the sensitivity and specificity of each marker are size, cellular localization, solubility, release ratio, clearance, specificity for myocardium, specificity for irreversible injury, and detectability. Size. In general, the smaller the molecular marker, the more rapidly it is released into the circulation.10 Thus, levels of low molecular weight markers such as myoglobin are likely to be elevated earlier than higher molecular weight markers such as LD4 and CK."1 Cellular localization. Cytosolic proteins are released more rapidly than are structural proteins after damage to the sarcolemma, resulting in earlier plasma elevations.12 Solubility. Macromolecules of low solubility, including some cardiac contractile proteins, move slowly out of the myocardium. Release ratio. Some macromolecules, eg, CK, may undergo local degradation after release.13,4 Thus, the amount of marker depleted from the heart may be substantially greater than the amount that can be measured in plasma. The release ratio is rarely 1; some protein usually remains within the central region of

2 Adams et al Biochemical Markers of Myocardial Injury 751 infarction where blood flow is the lowest.'5 Changes in blood flow may alter the release ratio; eg, reperfusion markedly increases the amount of CK depleted from heart that appears in plasma.16 Clearance. Smaller markers generally are cleared more rapidly than those of greater size.10'11 Thus, markers with shorter plasma half-lives are better for characterizing the timing of events such as reperfusion or reinfarction if frequent blood samples can be obtained.17 Markers with longer half-lives are better for integrating events over time with infrequent samples but often will be insensitive to new release, eg, from recurrent myocardial injury. In addition, a marker's clearance is a critical determinant of detectability (see below). Specificity for myocardium. Most macromolecules in the heart also are abundant in skeletal muscle, especially under pathological conditions. After injury, cells alter the molecular species they produce, recapitulating production of proteins produced in utero or early in life (a "return to ontogeny"') Specificity for irreversible injury. It is controversial whether the release of cytosolic proteins indicates cell death. Although the sarcolemma must be damaged for release to occur, it is not clear whether the damage must be so extensive that the cell is irreversibly injured Specificity for irreversible injury has been difficult to define because of limitations in the detectability of marker proteins in plasma, which is related to the plasma levels normally present and the sensitivity of the assay, and because of limitations in the morphological detection of myocardial necrosis. Release of small amounts of proteins could be undetectable after reversible ischemia if baseline levels are too variable, if assays are not sufficiently sensitive, or if the increment in the plasma level does not exceed the upper bound of the physiological range of the marker, leading to the erroneous conclusion that only irreversible injury causes enzyme release. Conversely, if the release ratio is high or levels of the marker in plasma are low and thus detectability high, protein levels in plasma rather than morphological assessment may be more sensitive for the detection of myocardial necrosis, especially if areas of cell death are not contiguous. The majority of the evidence supports the latter hypothesis, in our view. Prolonged release of structural proteins should be highly specific for irreversible injury. Detectability. In addition to the issues noted above, detectability requires accurate and easy-to-use assays with high sensitivity and low variability. In general, markers that under normal circumstances are present in very low levels in plasma provide the best diagnostic accuracy. The assumption that detection of markers by different assays is equally precise or that the results of activity and mass assays are equivalent is not necessarily valid.26 The above considerations must be understood in their clinical context. The accuracy of diagnosis and especially the specificity of any test are dependent in large part on the patient population to which they are applied. Thus, increases in the levels of molecular markers of cardiac injury have better specificity for the diagnosis of AMI when symptoms characteristic of ischemia are present. Conversely, improved analytic sensitivity may The following review summarizes what is known about several conventional and some new molecular markers of myocardial injury to provide a structure within which to consider the biological and clinical importance of markers available in the 1990s. It also summarizes the practical applications of molecular markers for the diagnosis of AMI, for early detection of infarction, for detection of coronary artery recanalization after treatment with thrombolytic agents, and for estimation of infarct size. Finally, it provides a discussion of possible changes in the use of these markers during the 1990s. The markers to be considered are CK isoenzymes, CK isoforms, myoglobin, myosin fragments, LD, troponins, heart fatty acid binding protein, and enolase. Their characteristics are detailed in Table 1. Creatine Kinase The molecular marker of choice for the evaluation of patients with suspected AMI has been the MB isoenzyme of CK. CK isoenzymes are dimers composed of to D subunits synthesized in the cytosol of myocytes. CK catalyzes the transfer of a high-energy phosphate from ATP to creatine, producing creatine phosphate.27 Isoenzymes are composed of two M subunits (MMCK), two B subunits (BBCK), or one M and one B subunit (MBCK). Single subunits are enzymatically inactive. A unique dimeric mitochondrial form not composed of either M or B subunits also exists.28 BBCK is most abundant in brain, MMCK in striated muscle, and MBCK in the heart.19'29 Controversy exists as to whether substantial amounts of MBCK are intrinsic to normal myocardium or whether only trace amounts occur physiologically and that increases are produced in response to recurrent ischemic insults.30 Autopsy data, data from other primates, and the documented ability to diagnose myocardial injury in patients whose myocardium was known to be normal before injury favor the former hypothesis.31 After myocardial injury, the concentration of MBCK in myocardium increases; elevated levels have been observed in humans with hypertension, left ventricular hypertrophy, and coronary artery disease.30 In experimental animals, coronary occlusion appears to induce synthesis of MBCK.32 This may improve the sensitivity for detection of AMI in these groups but does not appear to distort estimates of infarct size, judging from the excellent correlations reported between MBCK estimates and morphology.33'34 CK is inactivated by proteolysis in lymph.14 In experimental animals, CK clearance is not affected by changes in heart rate, blood pressure, or cardiac output. CK is not excreted in urine, and its levels are not influenced by changes in renal or hepatic blood flow in experimental animals.35 Hypothyroidism retards and administration of exogenous thyroid hormone increases clearance of CK.36 Nonspecific reticuloendothelial blockade (with zymozan) also retards clearance.35 Tissue Distribution of CK and MBCK CK is abundant in most tissues. Activities as high as IU/g and as low as 225 IU/g have been reported in striated muscle.19'29 There are approximately 1600 IU of CK activity per gram in myocardium, 15% to 30% of which is MBCK.35 Results with sensitive mass assays reduce specificity of a given assay for AMI. have shown that most skeletal muscles contain small

3 752 Circulation Vol 88, No 2 August 1993 TABLE 1. Molecular Markers Used or Proposed for Use in the Diagnosis of Acute Myocardial Infarction Range of times to Mean time to peak initial elevation elevations Time to return to Most common sampling Marker MW (D) (h) (nonthrombolysis) normal range schedule hfabp h 24 h On presentation, then 4 h later Myoglobin h 24 h Frequent; 1-2 h after CP MLC d 6-12 d Once at least 12 h after CP ctni h 5-10 d Once at least 12 h after CP ctnt h-2 d 5-14 d Once at least 12 h after CP MBCK h h Every 12 hx3* MMCK tissue isoform h 38 h min after CP MBCK tissue isoform h Unknown min after CP Enolase h 48 h Every 12 hx3 LD h d Once at least 24 h after CP MHC d 14 d Once at least >2 d after CP hfabp indicates heart fatty acid binding proteins; MLC, myosin light chain; ctni, cardiac troponin I; ctnt, cardiac troponin T; MBCK, MB isoenzyme of creatine kinase (CK); MMCK, MM isoenzyme of CK; LD, lactate dehydrogenase; MHC, myosin heavy chain; CP, chest pain. *Increased sensitivity can be achieved with sampling every 6 or 8 h. amounts (1% to 3%) of MBCK.37 Thus, skeletal muscle damage can result in detectable amounts of MBCK in plasma. This was unappreciated initially because dilution of samples to measure elevated total CK activity reduced MBCK activity below the limit of detectability of electrophoretic assays available at the time.38 In general, small amounts of damage to healthy skeletal muscle does not release enough MBCK into plasma to cause diagnostic confusion.38 For example, intramuscular injections and instrumentation during cardiac catheterization and uncomplicated coronary angioplasty do not result in increases of MBCK despite marked increases in total CK.39,40 When skeletal muscle is injured, it produces increased amounts of B subunits, just as it does during fetal development.18'19 Transient increases in the percentage of MBCK in skeletal muscle and elevations in circulating levels occur after acute muscle injury (including extremely rigorous exercise). For example, increases of the skeletal muscle concentration of MBCK in marathon runners are maximal 24 hours after a marathon.41 Increases of MBCK in plasma also have been documented in professional football players after skeletal muscle injury.42 Persistent increases in MBCK resulting from chronic myopathies also occur and are probably the cause at least in part of the increases in patients with chronic renal failure and hypothyroidism '44 Persistent increases of MBCK in plasma in patients with chronic myopathic insults usually allow differentiation of acute from chronic injury. Whether an occult skeletal myopathy can occasionally lead to transient increases in MBCK is controversial. Increases in MBCK in plasma usually are attributable to release from myocardium in the absence of conditions known to increase MBCK in muscle or abnormalities of clearance. Patients with a rising and falling pattern of MBCK and a peak value that exceeds the upper limit of the reference range should be considered to have had an AMI until shown otherwise. Specificity of CK for Irreversible Injury In most studies, release of MBCK into plasma correlates with other evidence of myocardial injury. In addition, increases in MBCK do not occur after ischemia evoked by stress testing8 or in most patients with severe unstable angina.45'46 However, in some experimental studies, small amounts of MBCK have been detected in lymph, coronary sinus effluent, and plasma in the absence of characteristic histological evidence of myocardial damage. However, it is not possible to exclude the possibility that minute amounts of nonconfluent myocardial damage (that were not detectable by random sampling and light microscopy) could have led to the release of small amounts of MBCK. This argument cannot be adjudicated definitively at present. However, CK release after rigorous exercise is associated with skeletal muscle necrosis. Similar physiology may exist in myocardium as well. This is consistent with data demonstrating that patients with chest pain and minor increases in MBCK indicative of AMI but without marked elevations in total CK are at increased risk for subsequent cardiac events.47 Techniques of Measurement Assays of CK activity and/or mass can be done on either plasma or serum. Samples should be maximally reduced with f8-mercaptoethanol to protect against oxidation of sulfhydryl groups in the active center, which leads to loss of activity.48 Commercial kits contain reagents to inhibit adenylate kinase, which can be released by red blood cells, resulting in artifactual elevations of CK activity.49 Assays for total and MBCK activity and mass have been reviewed extensively elsewhere.50'51 Artifacts can

4 Adams et al Biochemical Markers of Myocardial Injury 753 occur with any assay but are particularly common with electrophoresis. Naturally fluorescing compounds such as bilirubin, benzodiazipines, antidepressants, pyridoxime, and aspirin in high doses can produce artifactual bands by electrophoresis. Patients with renal failure can also manifest artifactual bands that comigrate with MBCK and BBCK.44 Carryover of MM into the MB fraction is a common problem with "column" methods,52 and assays that detect B subunits53 can measure elevated levels that are the results of brain injury; pregnancy; carcinoma of the prostate, lung, or gastrointestinal tract; tuberculosis; or uterine abnormalities. Up to 3.2% of hospitalized patients have detectable circulating levels of BBCK.54 Because BB is cleared rapidly, it usually is only detectable in patients with disease processes that result in continuing release. Assays based on monoclonal antibodies that measure MBCK mass are rapid, highly sensitive and specific, and are now the analytic method of choice.55 However, monoclonal antibodies directed against different epitopes of the same marker may be affected differently by pathological and/or artifactual processes.26 Recently, it was found that elevations in MBCK mass were produced by cross-reactivity of atypical forms of alkaline phosphatase in plasma with the alkaline phosphatase used to stabilize the reagents for the assay.56 Similar artifactual results can occur with all assays using monoclonal antibodies. In general, mass and activity assays yield comparable results, with the occasional exception of samples with very high total and MBCK values.57 Macrokinases, including immunoglobulin G bound to BBCK (macro-ck1) or aggregates of mitochondrial CK (macro-ck2), are detected with many assays.58,59 In a prospective study of 3000 hospitalized patients,58 macrokinases were detected in 4.2% (1.0% had type 1 and 3.4% had type 2), but for patients with cardiovascular disease, this may be a substantial overestimation.26 Macro-CK1 migrates close to and is often indistinguishable from MBCK on electrophoretic separations, thus markedly increasing the percentage of MBCK with respect to total CK. Macro-CK1 is detected most often in chronically ill older women. Macro-CK2 migrates near MMCK, resulting in elevations in total CK activity.59 The presence of macro-ck2 is associated with chronic disease (especially liver cirrhosis and malignancy) and severe acute illness. In contrast to free CK enzyme, macrokinases are heat stabile. The persistence of enzyme activity after incubation for 20 minutes at 45 C has been used to verify the presence of macrokinase activity.26 Although it is hoped that the newer assays based on mass will be less affected by macrokinases, cross-reactivity may still occur. Diagnosis ofami Measurement of MBCK is currently the test of choice to confirm the diagnosis of an AMI."1 (See Table 1.) Increases in plasma levels usually occur between 6 to 10 hours after the onset of infarction (in the absence of thrombolysis), peak at 24 hours, and return to normal by 36 to 72 hours.60 Given these kinetics, measurement of MBCK every 12 hours is an adequate and cost-effective method for diagnosis of AMI. Obtaining values more frequently will increase diagnostic sensitivity.61 Levels disappears slightly more rapidly than total CK. The time to peak enzyme concentration is earlier for small infarctions,62 leading some to hypothesize that non-q wave infarctions, which also tend to be smaller, result from occlusion and reperfusion.63 This is an interesting but unproven speculation. Recanalization increases the rapidity with which MBCK appears in plasma, permitting earlier diagnosis of AMI. Enzyme levels in plasma are abnormal within 2 hours after coronary recanalization in experimental animals and when recanalization is confirmed in patients by cardiac catheterization.64,65 In the absence of reocclusion, there usually is a return to baseline values by 24 hours, representing a shorter period of enzyme release rather than a change in the rate of clearance.66 Processes Associated With Elevations of MBCK in the Absence ofami Myocardial cell death of any cause will result in a rising and falling pattern of MBCK. This has been observed in patients with cardiac contusion after chest wall trauma, electrical injury, and pericarditis with concomitant myocardial involvement As discussed above, sufficient MBCK can be released from damaged skeletal muscle to elevate circulating levels. This can cause diagnostic confusion in patients after surgery or trauma, requiring noninvasive tests (such as echocardiography) to confirm the diagnosis.67'70 Some have proposed that an MBCK/total CK percentage of 3% to 5% or an MBCK mass/ck activity ratio of 80 ng/u or 2.5% differentiates a myocardial from a skeletal muscle source.71,72 However, results of a large study of trauma patients did not support this approach.67 When high levels of total CK are present because of skeletal muscle injury, a large amount of MBCK has to be released from myocardium to meet percentage criteria, decreasing sensitivity, whereas chronically injured muscle may release large amounts of MBCK, decreasing specificity. The failure of percentage criteria to differentiate skeletal from myocardial release of MBCK has been confirmed recently in a large study of patients with acute and chronic skeletal muscle abnormalities.73 In general, when MBCK is released because of skeletal muscle injury, it is released for a longer period of time. Thus, plasma levels decline more slowly than after an AMI. The use of more specific markers (such as cardiac troponin I) in these patients should allow for a more facile determination of whether concomitant cardiac injury is present (see below). Patients with hypothyroidism have increased levels of MBCK in plasma because of skeletal myopathy and a decreased rate of clearance.13 Administration of exogenous thyroid hormone increases the disappearance rate of CK and usually rapidly normalizes circulating levels of CK and MBCK.13 Detection of Reperfusion Early detection of failure of recanalization could allow for mechanical intervention to reperfuse ischemic myocardium. After reperfusion, the release ratio of CK appears to double, and the rate of egress into the circulation increases,16 which aids detection of reperfusion. (See Table 2.) Very early detection is not possible with total CK, but of MBCK peak slightly earlier, and the MB fraction it does have moderate sensitivity for the detection of

5 754 Circulation Vol 88, No 2 August 1993 TABLE 2. Molecular Markers and Criteria Proposed for Noninvasive Detection of Coronary Recanalization Marker Proposed criteria Sensitivity Specificity Myoglobin Minimal rate of rise >2.6 ng ml-'. min-1 at 1-2 h74 92% 60% 4.6-Fold increase over 2 h74 85% 100% Rate/initial value > % 80% CK Time to peak <4 h64 10% 82% Time to peak >16 h64 39% 81% MMCK isoforms Minimal rate of decline of MM3% >3.1%/h66 87% 74-90% MM3%/MM1% >0.35 at 60 min173 60% 100% Rate of rise of MM3% >0.18%/min74 92% 100% MBCK Minimal rate >0.04 IU at 1 h74 L`. min' 92% 20% 2.2-Fold increase over 90 min (inferior MI)65 77% 100% 2.5-Fold increase over 90 min (anterior MI)65 92% 100% Appearance rate constant (k1) > % 100% MBCK isoforms Peak MB2/MB1 ratio >3.8 at 2 hb03 82% 78% Cardiac troponin T Peak concentration/concentration at 32 h >1.1'74 93% 100% Concentration at 14 h/concentration at 32 h > % 100% CK indicates creatine kinase; MMCK, MM isoenzyme of CK; MBCK, MB isoenzyme of CK; MI, myocardial infarction. reperfusion late after treatment. Although a time to peak CK of less than 4 hours is highly predictive of recanalization, most patients have peak CK levels at 4 to 16 hours, when the predictive power is between 40% and 60%.64,74 The usefulness of the measurement can be improved by definition of peak levels and determination of the time required to reach a percentage of it.75 Early detection of reperfusion is possible with measurement of MBCK. An early increase in MBCK levels and a shorter time to peak MBCK have been found to be indicators of reperfusion.65,75 A 2.2-fold increase in MBCK in patients with inferior myocardial infarction and a 2.5-fold increase in those with anterior myocardial infarction during the initial 2 hours of treatment are adequately sensitive (85%) and 100% specific for the detection of recanalization.65,74 However, measurement of MBCK 1 hour after thrombolysis does not differentiate between those in whom recanalization is and those in whom it is not successful.74 Estimation of the Size ofami Infarct size can be estimated after AMI if the amount of enzyme lost from the myocardium, its volume of distribution, and its release ratio are known. Extensive modeling of the volume of distribution and disappearance rate have facilitated calculations.'5 Infarct size as assessed by MBCK closely correlates with the volume of the infarction, the ejection fraction, the incidence of ventricular arrhythmias, and prognosis.2,76 Initially, it was believed that 30% of the MBCK depleted from heart appears in the plasma compartment,16.77 but it has been postulated recently that the release ratio was miscalculated as a result of the use of an incorrect disappearance rate and that 70% of the MBCK depleted from heart appeared in plasma.78 These data at present stand alone against previous results and the known proteolysis of MBCK in lymph.'4 Approximately 15% of CK activity remains in heart even after infarction while some of the remaining 85% is deactivated in lymph.'3 Calculations are further complicated by release of MMCK from skeletal muscle in patients with large infarctions, making estimates based on total CK larger than those based on MBCK.79 Thus, in plasma, the percentage of MBCK with respect to total CK fluctuates, depending on the presence of concomitant skeletal muscle disease, the amount of MBCK in muscle, the size of the infarction, and regional blood flow. After recanalization, the amount of enzyme released into plasma appears to double, limiting the use of calculations of infarct size as defined in its absence.16,77,80 It is unclear whether a continuum exists in the amount of enzyme that reaches the circulation as a function of the total ischemic time and the amount of reflow or whether in some way this response is a "step function." It is likely that the release of smaller cumulative amounts of MBCK in patients undergoing early thrombolysis compared with those not treated with thrombolytic agents is indicative of salvage of myocardium. However, comparisons of enzyme levels between untreated patients and those with late recanalization are less useful. CK Isoforms Isoenzyme subforms with similar enzymatic activity but different isoelectric points (thus isoforms) have been identified for both MMCK and MBCK.81 The tissue distribution and specificity of CK isoforms for irreversible injury are the same as those for CK. Three

6 Adams et al Biochemical Markers of Myocardial Injury 755 isoforms of MMCK are known to exist,81'82 and at least three are known for MBCK83 Isoforms represent posttranslational modifications of the carboxyterminus of the M and B monomers. They are numbered by their relative electrophoretic migration to the anode with those migrating furthest toward the anode having the lowest number.81 In tissue, greater than 95% of MMCK is comprised by the tissue isoform designated MM3,82 ie, it remains near the origin on electrophoresis. Structurally, it has a lysine residue on the carboxyterminus of each subunit.84 Once released into plasma, carboxypeptidase-n sequentially removes the carboxyterminal lysines from MMCK by hydrolytic cleavage, generating an intermediate designated MM2 and an ultimate conversion product designated MM1.85 Each lysine cleaved removes one positive charge from the molecule, resulting in an anodal electrophoretic shift.8' The conversion85 and rapid clearance86 of MM3 result in its swift disappearance from plasma. MBCK isoforms should be more specific for cardiac injury than MMCK isoforms. The MB tissue isoform (designated MB2) has lysines on the carboxyterminals of both M and B monomers. Although four isoforms could exist theoretically, electrophoretic separations have delineated only two forms, and it has been presumed that the second form (designated MB1) represents cleavage of the carboxyterminal lysine from the M monomer by carboxypeptidase-n.87'88 With column chromatography and monoclonal antibodies to the M carboxyterminal lysine, it is now clear that at least three isoforms circulate in vivo; the tissue isoform, a form with a lysine on the M subunit but not on the B subunit, and a form with lysines absent from both carboxyterminals Thus, cleavage of the B monomer lysine is favored. Comigration on electrophoresis of the tissue isoform and the isoform with a lysine removed from the B monomer appears to account for the separation of only two species.83 Techniques of Measurement Samples should be collected in ethylene diamine tetraacetic acid (final concentration 7 to 10 mmol/l) to inhibit in vitro conversion.90 MMCK isoforms vary with respect to charge, allowing separation by prolonged electrophoresis, high-voltage rapid electrophoresis, nonequilibrium ph gradient electrophoresis, isoelectric focusing, or chromatofocusing.51'66,9' The high-voltage electrophoretic methods are the most rapid and least difficult. However, these methods tend to underestimate MM3 and overestimate MM1 compared with chromatofocusing (A.S. Jaffe and D.A. Abendschein, unpublished observations). Electrophoresis also has been used to separate isoforms of MBCK81,87,88 However, as described above, this method only delineates two of the three forms known to exist in plasma in vivo.83 Diagnosis ofami Measurement of MMCK isoforms is highly sensitive for the early detection of AMI The sensitivity of MMCK isoforms is due to the fact that the tissue isoform (MM3) is abundant in myocardium, but because of conversion of MM3 to other forms and its rapid clearance from the circulation, MM3 comprises only 15% of MMCK activity in plasma under physiological conditions.92 The ratio of MM3 to MM1 averages Thus, the release of only small amounts of MM3 will dramatically increase its percentage in plasma and the MM3 to MM1 ratio that has been used for diagnosis. When a ratio of MM3 to MM1 >0.5 is used as the upper limit of the reference range and patients who present late are diagnosed by increases in MBCK, 94% of patients who fulfill subsequent criteria for AMI can be identified by analysis of MMCK isoforms and MBCK in the first sample of blood drawn on presentation.92 By 12 to 16 hours, the conversion and clearance of MM3 return the ratio to normal or near-normal values,92,94'95 but by this time MBCK levels usually are elevated so that a diagnosis can be made early with analysis of MMCK isoforms and later with MBCK.93 Since MMCK isoforms are not specific for myocardium, their utilization for the diagnosis of AMI requires that acute skeletal muscle damage be excluded. Even then, the specificity of MMCK isoforms for the early diagnosis of AMI varies between 77% and 94%, depending on the specific criteria used.92'93 Measurement of MBCK isoforms could be more specific than MMCK isoforms for the diagnosis of AMI. Because the absolute levels of MBCK in plasma are low (much lower than MMCK), the release of small amounts of the tissue isoform (MB2) will result in a marked change in the ratio of MB2/MB1 hours before elevations of circulating MBCK are manifest. However, it may be that the small amounts of MB2 contained in skeletal muscle could deleteriously affect cardiac specificity by the same mechanism. This issue is yet to be resolved. Application of MB isoforms in patients with a high pretest probability of AMI, as with all such tests, improves the cardiac specificity. The presence of elevated levels of MBCK isoforms, defined as a plasma MB2 activity > 1.0 U/L and a MB2/MB1 ratio > 1.5, has a 59% sensitivity for the diagnosis of AMI at 2 to 4 hours and a 92% sensitivity at 4 to 6 hours after the onset of symptoms.96 The early accuracy of these criteria may improve if separation of all the isoforms known to circulate in plasma can be accomplished. Myocardial injury that is not due to AMI may also be detectable by analysis of CK isoforms. The proportion of MB2 to total MBCK in plasma has been found to be elevated in patients with cardiac transplant rejection and cardiomyopathy.97'98 Whether this is due to skeletal muscle or cardiac release is unclear at present. Detection of Reperfusion The presence of the combination of relief of chest pain, normalization of ST-segment changes, and reperfusion arrhythmias has been found to be indicative of coronary recanalization, but these criteria are not sensitive because all three occur in only 15% of patients.99 Results with CK isoforms have been far more promising (Table 2).100,101 An accelerated rate of increase of MM3% in plasma reliably distinguishes animals with and without coronary recanalization by 30 minutes after reflow is established'00 and identifies patients with recanalization within 1 hour of the administration of thrombolytic agents.74"101'102 The rate of rise in MM3% not only permits a very early determination in experimental animals but is less affected by residual high-grade acute or chronic coronary stenoses.100 Currently, the best criteria

7 756 Circulation Vol 88, No 2 August 1993 for the detection of reperfusion with MMCK isoforms is a rate of rise of MM3%.0.18%/min.74 Criteria based on both the rate of rise and absolute values of MM3% in plasma may be capable of distinguishing patients with persistent recanalization from those with transient reperfusion followed by reocclusion. MB isoforms also have been used to detect coronary recanalization. A ratio of MB2 to MB1.3.8 has been established as the upper limit of the reference range.103 Preliminary results suggest that substantial overlap exists between levels of this marker in patients with and without recanalization.103 Conjoint use of the rates of rise of MM3% and either the ratio of MB2 to MB1104 or the rate of rise of MB2%105 may produce more accurate results by detecting more patients who recanalize. For isoform assessment of recanalization, samples should be obtained before treatment and at 60 to 90 minutes after treatment. Early sampling and rapid turnaround for the assays are essential if additional interventions are to be implemented based on the results. Myoglobin Myoglobin is a low-molecular-weight heme protein ( D) that is abundant in cardiac and skeletal but not smooth muscle. Thus, it is a sensitive but not specific marker of AMI, requiring the exclusion of skeletal muscle injury.'06 Myoglobin is rapidly released from necrotic myocardium, with subsequent rapid renal clearance.'0 Decreased renal function can result in elevated levels.06 Because of its rapid kinetics, myoglobin can define the timing of myocardial events if frequent samples are obtained.'7" Myoglobin is often detectable 2 hours after coronary occlusion, with peak levels occurring at 3 to 15 hours. Sixty-five percent of patients with AMI have elevated levels of myoglobin in their initial samples.'08"109 A pattern of discontinuous release of myoglobin consisting of multiple peaks, or the "staccato phenomenon," can occur in patients who have suffered AMI.'7"10 This pattern of myoglobin elaboration may reflect cyclical spontaneous coronary reocclusion and reperfusion of the ischemic myocardium, leading to bursts of myoglobin release. Early treatment with either nifedipine (within 6 hours of the onset of symptoms) or thrombolytic therapy has been shown to decrease the number of staccato peaks.""",112 When used for the early detection of coronary recanalization after thrombolysis, the lack of specificity of myoglobin for myocardium is less problematic. In both experimental and clinical studies, a rate of change of myoglobin concentration >2.6 ng * ml- * min-1 over 60 minutes or a 4.6-fold rise over 2 hours allows detection of coronary recanalization with an accuracy comparable to MMCK isoforms.74"1'3 In animals, this determination can be made as early as 30 minutes.74 However, because of the rapid clearance of myoglobin, criteria based on myoglobin concentrations are less precise at 120 minutes. The rapid release of myoglobin also seems to make criteria with it more prone to be met by increases associated with transient rather than sustained restoration of blood flow.74"13 Elevated levels of myoglobin have been reported in patients with unstable angina, perhaps reflecting small areas of otherwise undetected myocardial cell death or the release of myoglobin from skeletal muscle in critically ill patients."14"'15 Myosin Fragments Myosin consists of six proteins: two heavy chains (MW, D each) and two pairs of dissimilar proteins (MW, to D each) designated myosin light chain I and II (MLC-I and MLC-II). Both participate in regulation of the interaction of actin and myosin predominantly by modulating changes in calcium flux. Myosin Light Chains At least two forms of MLCs exist in the ventricles and atria, and at least three forms of MLCs are found in skeletal muscle. These forms have similar structures, and the amino acid sequences of ventricular MLC-II and slow skeletal MLC-II are identical."16"'7 Given the close or identical structures of cardiac and skeletal muscle forms of MLCs, the specificity of MLCs for myocardial injury both in normal and pathological situations is limited.1"7"'8 This problem has been accentuated in some studies by the use of polyclonal antibodies that cross-react with troponin T.1"9 Both types of MLC fragments (MLC-I and MLC-II) dissociate from the contractile apparatus after cell necrosis.'20 Although most of the MLC fragments are noncovalently linked to myosin heavy chains, a smaller pool (perhaps present in the cytosol after synthesis) is available for early release.'2"'4 Thus, MLC fragments generally are detectable in plasma within 6 hours of the onset of infarction.'8 Elevated levels are present for more than 7 days and are thought to represent continuing dissociation and release from the contractile apparatus.'2"'122 MLCs are cleared by the kidneys.123 MLC fragments are sensitive markers of myocardial injury, in part because prolonged elevations permit retrospective detection for up to 2 weeks."1 The contention that measurement of MLC is more sensitive for the detection of AMI than either total CK45 or MBCK14 probably is due to this prolonged diagnostic window rather than a greater amount of protein release per gram of injured tissue. Patients with unstable angina who have elevated levels of MLC, perhaps indicating myocardial injury that occurred before admission, have more severe coronary artery disease and a higher incidence of subsequent AMI and/or death.45"'4 Because of their long half-life, monitoring of MLC levels is not a sensitive way to detect recurrent AMI. Measurement of MLC can be used to estimate infarct size. The release ratio is close to unity if samples are obtained until values return to baseline and it is unaffected by reperfusion.122"124 However, the need to obtain multiple samples over at least a 7-day period lessens the attractiveness of this technique. In addition, crossreactivity between antibodies with both skeletal muscle and cardiac forms of MLC may also impair the accuracy of such estimates. Myosin Heavy Chains Myosin heavy chains (MHCs) are larger (MW, D)"16 fragments that dissociate from other structural proteins before release.'25 Levels of MHC are not present in plasma until 2 days after the event; peak levels occur 5 to 6 days later.126 Elevations persist for up to 10 days, allowing for a prolonged retrospective determination of myocardial necrosis.126 In contrast to

8 Adams et al Biochemical Markers of Myocardial Injury 757 other structural proteins, an early releasable pool of this protein does not appear to exist. Although monoclonal antibodies to MHCs have been developed, the existence of multiple variants in atria, ventricles, and skeletal muscle with similar structures has limited cardiac specificity.116,125,127 All patients have elevated levels of MHCs after cardiovascular surgery, but patients with clinical evidence of perioperative AMI have significantly greater elevations.125 Lactate Dehydrogenase LD is a tetramer composed of "M" (muscle; MW, D) and "H" (heart; MW, D) subunits. The two subunits are encoded by different genes and give rise to five distinct isoenzymes that provide LD with an element of tissue specificity.51 The molecular weight of the LD tetramers is approximately D. LD-1 contains four H subunits; LD-2, three H subunits and one M subunit; LD-3, two H and two M subunits; LD-4, one H subunit and three M subunits; and LD-5, four M subunits.51 Individual chains lack biochemical activity. LD is responsible for the interconversion of pyruvate and lactate as the final step in glycolysis. LD-1 is the predominant form in heart but also occurs in erythrocytes, brain, pancreas, kidney, and stomach. LD-2 is also abundant in heart; LD-3, LD-4, and LD-5 are found in only trace amounts. All LD isoenzymes are abundant in many tissues, whereas LD-5 is the predominant isoenzyme in skeletal muscle. Clearance of LD is via the reticuloendothelial system.128 Techniques of Measurement Differences in amino acid composition of M and H chains result in alterations in charge that can be detected by electrophoresis.129 The isoenzymes are designated according to electrophoretic migration, with the lowest number moving closest to the anode. Total LD usually is measured as enzymatic activity, and levels of its isoenzymes can be quantified after separation by electrophoresis. LD isoenzymes (especially those rich in M subunits) are inactivated when chilled or frozen but are stable at room temperature for several days.130 Care must be taken to avoid hemolysis, which will markedly increase LD activity. The details of the various techniques used for the assay of LD and its isoenzymes have been reviewed recently.51 Diagnosis ofami In human plasma, LD-2 is normally found in greater abundance than LD-1. A normal LD-1/LD-2 ratio is <0.76. When myocardial necrosis occurs, more LD-1 than LD-2 is released, and the percentage of LD-1 relative to LD2 increases. Ratios >0.76 are considered by some to be suggestive of AMI, but others require a value of >1, which appears to improve specificity.1'4'13' Alternatively, an LD-1/LD-4 ratio >3.0 (when LD-1 is <40% of total LD) or >4.0 (when LD-1 is >40% of total LD) has been proposed as a more accurate and earlier criterion for AMI than LD-1/LD Plasma LD is increased above the upper limit of the reference range 8 to 12 hours after the onset of AMI, peaks at 28 to 48 hours, and is normal by day A persistent abnormality in the MA/LD-2 ratio may indicate reinfarction. Processes Associated With Increased LD-J/LD-2 Ratio in the Absence ofami Although an elevated LD-1/LD-2 ratio may indicate AMI, there are other possible causes of an abnormal ratio. Erythrocytes are an abundant source of LD-1 and LD-2, and intravascular or extravascular hemolysis will increase LD-1 and LD-2 levels. In patients with chronic muscle disease or recurrent skeletal muscle injury, LD-1 and LD-2 are reexpressed in skeletal muscle133; elevated LD-1/LD-2 ratios occur in healthy, well-trained athletes.'34 Other causes of abnormal LD-1/LD-2 ratios include germ cell tumors (testicular seminomas and dysgerminomas) and diseases of the pancreas, stomach, and kidney.135 The presence of macroenzymes of LD, which are probably complexes of LD with IgG or IgA, increase total LD.58 Estimation of Size ofami A good correlation has been found between LD (and a-hydroxybutyrate dehydrogenase [HBDH], which is equivalent to LD-1 plus LD-2) and anatomic estimates of infarct size.136 It is possible that the release ratios of LD and HBDH are less affected by reperfusion, and estimates of infarct size by these markers thus are superior to those based on CK and MBCK in patients treated with thrombolytic agents.'37'38 Definitive studies are lacking. Troponins Troponins are a complex of regulatory proteins uniquely present in striated muscle that regulate the calcium-mediated interaction of actin and myosin. Troponin T binds the tri-protein complex to tropomyosin, troponin I inhibits coupling of actin and myosin, and troponin C binds to calcium, inducing a steric shift and reversing the inhibitory activity of troponin I. There are tissue-specific isoforms of troponin I, T, and C. Different isoforms of troponin I and T are expressed in slow-twitch, fast-twitch, and cardiac muscle, whereas troponin C has only a fast-twitch and a slow-twitch/ cardiac isoform. Troponin isoforms are the products of different genes; they have unique structures and different regulatory effects on calcium binding Cardiac troponins are tightly complexed to the contractile apparatus. Circulating levels are normally low, but they rise relatively rapidly after AMI, suggesting the presence of a more accessible pool, perhaps troponin synthesized in the cytosol.143 This hypothesis has not been proven definitively. If a cytosolic pool of troponin does exist, then its usefulness as a marker is subject to questions about whether increases are specific for myocyte death. That continuing release for days caused by dissociation of the contractile apparatus indicates cell death is less controversial. The existence of tissuespecific isoforms with unique structures has allowed the development of tissue-specific assay methods. Cardiac troponin I (ctni) is not expressed in skeletal muscle throughout ontogeny.144 Fetal rat, chick, and human heart contain both slow-twitch and ctni initially. After the ninth postnatal month, only ctni is expressed in myocardium Thus, the presence of ctni in the circulation above the reference limit is highly specific for myocardial injury and allows for MBCK elevations resulting from skeletal muscle damage to be distin-

9 758 Circulation Vol 88, No 2 August 1993 guished from those resulting from cardiac injury.147 Elevations in ctni do not occur in marathon runners or patients with acute or chronic muscle disease or in patients with renal failure unless concomitant acute cardiac damage has occurred The specificity of cardiac troponin T (ctnt) for myocardium has not been fully delineated. Skeletal muscle troponin T (stnt) and ctnt are coexpressed in fetal heart muscle, with the skeletal muscle form being suppressed during ontogeny and reexpressed in stressed human heart.150'151 In contrast to ctni, ctnt is expressed in fetal skeletal muscle and is reexpressed in adult rat skeletal muscle after injury or denervation.152 Since the troponin system is highly conserved across species, ctnt could be reexpressed in injured human skeletal muscle, with a decrease in its specificity for AMI. Most'51'153 but not all investigators154 have found multiple isoforms of ctnt, some of which are expressed in skeletal muscle.151 In two studies in which assays were based on polyclonal antibodies with a 1% to 3% crossreactivity with stnt, 16% of patients with skeletal muscle disease and 15% of patients with chronic muscle disease had elevations of ctnt without any evidence of myocardial injury.155'156 There appears to be less analytic cross-reactivity with the monoclonal antibody used by Katus et al.119 In a study of patients undergoing surgery either for forearm fractures or noncardiac thoracic surgery, no elevations were detected despite obvious acute skeletal muscle damage.157 However, in another study using the same monoclonal antibody-based assay for ctnt, three of 14 patients with normal ECGs and low levels of MBCK relative to total CK had elevated levels of ctnt.119 This could be due to increased sensitivity or decreased specificity, since no other cardiac evaluations were performed. In addition, 27% of patients with polymyositis/dermatomyositis have been reported to have elevations of ctnt in the absence of detectable cardiac injury.158 Although elevations of ctnt have not been found in athletes with elevations in MBCK, important details regarding how well trained the subjects were, the exercise involved, and the methods have not been reported.159 A proper understanding of the cardiac specificity of ctnt will be necessary to interpret recent findings that patients with unstable angina and increased levels of ctnt have a poorer prognosis than similar patients without elevations of ctnt.160 It is likely that the measurement of ctnt permitted detection of patients with AMI before presentation to the hospital (since MBCK was not elevated) and that patients with unstable angina after infarction are at greater risk than those with unstable angina alone. However, it is also possible that ctnt is released from poorly perfused skeletal muscle in some critically ill patients. Detection ofami The sensitivity of measurement of ctni has been reported to be comparable to that of MBCK for the diagnosis of ANMI in canine studies and in patients admitted with chest pain; elevations of ctni above the upper bound of the reference range occur a few hours later than those of MBCK.149,161 Increased levels persist for up to 1 to 2 weeks after AMI, facilitating retrospective identification of AMI.143'156,161 In addition, the short half-life of ctnl in plasma and the small amounts present in plasma in the absence of infarction result in very low circulating levels (undetectable: <1 ng/ml for ctni) in normal volunteers, enhancing sensitivity of elevations for detection of infarction.161 ctnt also is a sensitive marker for the detection of AMI in humans156'157,162; some have claimed it to be more sensitive than MBCK.160 This apparent superiority probably is due to a longer diagnostic window rather than to more release of protein per gram of damaged tissue. The cardiac specificity of ctnt in patients with skeletal muscle disease and therefore the accuracy of measurement of ctnt for the diagnosis of AMI in this population has not been defined (see above). Heart Fatty Acid Binding Proteins Heart fatty acid binding proteins (FABPs) are abundant cytosolic proteins of low molecular weight ( to D) that are thought to be important intracellular fatty acid carrier proteins. Three different FABPs are found in heart, liver, and intestine, although each is also present in other tissues that use fatty acids as metabolic substrates.163 Heart FABP (h-fabp) has a unique structure and is abundant in myocardium, but detectable amounts also are present in skeletal muscle and kidney.163 Serum h-fabp is elevated after myocardial injury in rats, and serum and urine levels are elevated in humans after myocardial infarction.1t4,165 Initial and peak elevations occur earlier than increases in MBCK consistent with the smaller size of FABP. Measurement of h-fabp may have comparable sensitivity to that of myoglobin for the detection of reperfusion after thrombolytic therapy.166 The tissue distribution for h-fabp requires determination, but because of its abundance and potential sensitivity, this marker could have exciting clinical applicability if it is adequately specific. Enolase Enolase is an abundant glycolytic enzyme present in all tissues. It catalyzes the interconversion of 2-phosphoglycerate and phosphoenolpyruvate. The enzyme exists as a dimeric structure composed of three distinct subunits, a, 13, and y, with an approximate molecular weight of D. Each subunit is the product of a different gene.'67 Five isoforms of enolase (aa, ca13, 1,1,, ay, and yy) have been described. ca13-enolase is the predominant form in myocardium, 813-enolase represents the predominant form in skeletal muscle, and the y-subunit isoforms (cry and y-y) are present in neuronal tissue.168 The 1-subunit represents 90% of total enolase in humans.'68 Total and "muscle-type" (cr1 and 131) enolase can be measured with assays based on polyclonal antibodies and are elevated 12 to 48 hours after AMI.169,170 Levels of 13-enolase also are elevated after open heart surgery, which some investigators interpret as being indicative of myocardial injury.17' Enolase undergoes postsynthetic modifications similar to those of MMCK.172 At present, it appears that measurement of 13-enolases will not enable differentiation of skeletal muscle from myocardial damage. An assay specific for cr13- enolase might be more useful. However, even this strategy may not result in a method with adequate specificity if pathological states that affect either the myocardium or skeletal muscle alter the distribution of

10 Adams et al Biochemical Markers of Myocardial Injury 759 isoforms. Since the diagnostic window of enolase is comparable to that of MBCK, enolase probably will not have significant clinical utility. Recommendations for the 1990s Acute Myocardial Infarction Routine diagnosis. The measurement of MBCK remains the test of choice for the confirmation or exclusion of AMI and probably will remain the test of choice throughout early 1990s. Samples obtained every 12 hours provide adequate sensitivity and are cost-effective. Retrospective diagnosis. LD still is useful, but it probably will be replaced by more specific long-lived markers (eg, the troponins) in the near future. Samples for these long-lived markers can be done daily. Diagnosis when skeletal muscle injury is present. More specific assays (eg, cardiac troponin I) will in the future be useful conjointly with MBCK. Because cardiac troponin I persists in plasma for prolonged periods of time, it cannot replace the use of MBCK when timing of AMI is necessary. Very early diagnosis. Several approaches are possible. The conjoint use of MBCK isoforms and myoglobin or MMCK isoforms could be a very sensitive approach and will compete with MBCK. Serial measurements over several hours are likely to be most useful; however, it is unclear how earlier diagnosis based on multiple samples would impact on patient care. The application of serial measurements to the large number of patients in whom one might wish to exclude AMI may not be costeffective and would delay the initiation of early treatment. In addition, decisions regarding who should be admitted and who should be discharged should not be based on the presence or absence of these markers of infarction alone. The most promising role at present for this approach is in distinguishing patients with possible AMI who may need admission to an intensive care unit from those who can be treated in a less intensive facility. Whether use of markers other than MBCK to shorten the time to diagnosis to < 12 hours is clinically necessary and/or cost-effective remains to be shown. Detection of Coronary Recanalization After Thrombolysis Criteria based on isoforms of MM and MBCK and myoglobin appear to hold the most promise. It is possible that conjoint use of two markers, eg, myoglobin or MBCK isoforms combined with MMCK isoforms, will emerge as the best strategy. Estimation of Infarct Size Longer-lived structural protein markers such as the troponins or MLC, whose release ratios are less affected by alterations in blood flow, probably will replace MBCK for this use. However, because of the prolonged release of these markers, blood samples will have to be obtained for an extended period of time for these studies. These techniques require further validation. Acknowledgments This study was supported in part by grant HL-17646, SCOR in Coronary and Vascular Diseases. References 1. Lee TH, Goldman L. Serum enzyme assays in the diagnosis of acute myocardial infarction. Ann Intern Med. 1986;105: Geltman EM, Ehsani AA, Campbell MK, Schechtmann K, Roberts R, Sobel BE. The influence of location and extent of myocardial infarction on long-term ventricular dysrhythmia and mortality. Circulation. 1979;60: Karmen A, Wroblewski F, Ladue JS. Transaminase activity in human blood. J Clin Invest. 1954;34: Vasudevan G, Mercer DW, Varat MA. Lactic dehydrogenase isoenzyme determination in the diagnosis of acute myocardial infarction. Circulation. 1978;57: Hess JW, MacDonald RP. Serum creatine phosphokinase activity. Mich Med. 1963;62: Van der Veen KJ, Willebrands AF. Isoenzymes of creatine phosphokinase in tissue extracts and in normal and pathological sera. Clin Chim Acta. 1966;13: Pozen MW, D'Agostino RB, Selker HP, Sytkowski PA, Hood WB. A predictive instrument to improve coronary care units admission practices in acute ischemic heart disease: a prospective multicenter clinical trial. N EngI J Med. 1984;310: Klein MS, Weiss AN, Roberts R, Coleman RE. Technetium-99 stannous pyrophosphate scintigrams in normal subjects, patients with exercise-induced ischemia and patients with a calcified valve. JAm Coll Cardiol. 1977;39: Roberts R. The two out of three criteria for the diagnosis of infarction: Is it passe? Chest. 1984;86: Klocke FJ, Copley DP, Krawszyk JA, Reichlin M. Rapid renal clearance of immunoreactive canine plasma myoglobin. Circulation. 1982;65: Ellis AK. Serum protein measurements and the diagnosis of acute myocardial infarction. Circulation. 1991;83: Samarel AM, Ferguson AG, Vander Heide RS, Davison R, Ganote CE. Release of unassembled rat cardiac myosin light chain I following the calcium paradox. Circ Res. 1986;58: Sobel BE, Markham RP, Karlsberg RP, Roberts R. The nature of disappearance of creatine kinase from the circulation and its influence on enzymatic estimation of infarct size. Circ Res. 1977; 41: Clark GL, Robison AK, Gnepp DR, Roberts R, Sobel BE. Effects of lymphatic transport of enzyme on plasma creatine kinase time activity curves after myocardial infarction in dogs. Circ Res. 1978; 43: Roberts R, Henry PD, Sobel BE. An improved basis for enzymatic estimation of infarct size. Circulation. 1975;52: Vatner SF, Baig H, Manders WT, Maroko PR. Effects of coronary artery reperfusion on myocardial infarct size calculated from creatine kinase. J Clin Invest. 1978;61: Kagen L, Scheidt S, Butt A. Serum myoglobin in myocardial infarction: the "staccato phenomenon."am JMed. 1977;62: Fontanet HL, Trask RV, Haas RC, Strauss AW, Abendschein DR, Billadello JJ. Regulation of expression of M, B, and mitochondrial creatine kinase mrnas in the left ventricle after pressure overload in rats. Circ Res. 1991;68: Trask RV, Billadello JJ. Tissue-specific distribution and developmental regulation of M and B creatine kinase mrnas. Biochim Biophys Acta. 1990;1049: Keshgegian AA, Feinberg NV. Serum creatine kinase MB isoenzyme in chronic muscle disease. Clin Chem. 1984;30: Jockers-Wretou E, Grabert K, Muller E, Pfleiderer G. Serum creatine kinase isoenzyme pattern in nervous system atrophies and neuromuscular disorders. Clin Chim Acta. 1976;73: Siegel RJ, Said JW, Shell WE. Identification and localization of creatine kinase B and M in normal, ischemic and necrotic myocardium: an immunohistochemical study. JMol Cell Cardiol. 1984; 16: Heyndrickx GR, Amano J, Kenna T, Fallon JT, Patrick TH, Manders WT. Creatine kinase release not associated with myocardial necrosis after short periods of coronary artery occlusion in conscious baboons. JAm Coll Cardiol. 1985;6: Piper HM, Schwartz P. Spahr R, Hutter JF, Spiekermann PG. Early enzyme release from myocardial cell is not due to irreversible cell damage. J Mol Cell Cardiol. 1984;16: Geft IL, Fishbein MC, Ninomiya K, Hashida J, Chaux E, Yano J, Y-Rit J, Genov T, Shell W, Ganz W. Intermittent brief episodes of ischemia have a cumulative effect and may cause myocardial necrosis. Circulation. 1982;66: Kohse KP, Wisser H. Antibodies as a source of analytical errors. J Clin Chem Clin Biochem. 1990;28:

11 760 Circulation Vol 88, No 2 August Bessman 5, Carpenter C. The creatine-creatine phosphate energy shuttle. Annu Rev Biochem. 1985;54: Payne RM, Haas RC, Strauss A. Structural characterization and tissue-specific expression of the mrnas encoding isoenzymes from two rat mitochondrial creatine kinase genes. Biochim Eiophys Acta. 1991;1089: Neumeier D. Tissue specific and subcellular distribution of creatine kinase isoenzymes. In: Lang H, ed. Creatine Kinase Isoenzymes. Berlin/Heidelberg: Springer-Verlag; 1981: Ingwall JS, Kramer MF, Fifer MA, Lorell BH, Shemin R, Grossman W, Allen PD. The creatine kinase system in normal and diseased human myocardium. NEngi JMed. 1985;313: Baraka M, Deveaux N, Frank R, Tonet JL, Fontaine G, Grosgogent Y. Creatine kinase MB isoenzyme activity after endocardial catheter fulgration. Circulation. 1987;76(suppl IV):IV-174. Abstract. 32. Sharkey SW, Murakami MM, Smith SA, Apple FS. Canine myocardial creatine kinase isoenzymes after chronic coronary artery occlusion. Circulation. 1991;84: Hakel DB, Reimer KA, Ideker RE, Mikat EM, Hartwell TD, Parker CB, Braunwald EB, Buja M, Gold H, Jaffe AS, Muller JE, Raabe DS, Rude RE, Sobel BE, Stone PH, Roberts R, MILIS Study Group. Comparison of enzymatic and anatomic estimates of myocardial infarct size in man. Circulation. 1984;70: Shell WE, Kjekshus JK, Sobel BE. Quantitative assessment of the extent of myocardial infarction in the conscious dog by means of analysis of serial changes in serum creatine phosphokinase. J Clin Invest. 1971;50: Roberts R. Enzymatic estimation: creatine kinase. In: Wagner GS, ed. Myocardial Infarction: Measurement and Intervention. Hague: Martinus NiJhoff; 1982: Karlsberg RP, Roberts R. Effect of altered thyroid function on plasma creatine kinase clearance in the dog. Am J Physiol. 1978; 235:E614-E Tsung JS, Tsung SS. Creatine kinase isoenzymes in extracts of various human skeletal muscles. Clin Chem. 1986;32: Roberts R, Gowda KS, Ludbrook PA, Sobel BE. Specificity of elevated serum MB creatine phosphokinase activity in the diagnosis of acute myocardial infarction. Am J Cardiol. 1975;36: Rosenblum AM, Ludbrook PA, Jaffe AS. Significance of elevated MB creatine kinase in patients after cardiac catheterization. Cathet Cardiovasc Diagn. 1984;10: Spadaro JJ, Ludbrook PA, Tiefenbrunn AJ, Jaffe AS. Paucity of subtle myocardial injury after angioplasty delineated with MB CK. Cathet Cardiovasc Diagn. 1986;12: Siegel A, Silverman L, Evans W. Elevated skeletal muscle creatine kinase MB isoenzyme levels in marathon runners. JAMA. 1983;250: Jaffe AS, Garfinkel BT, Ritter CS, Sobel BE. Plasma MB creatine kinase after vigorous exercise in professional atheletes. Am J Cardiol. 1984;53: Schuyler GT, Yarbrough LR. Changes in myosin and creatine kinase mrna levels with cardiac hypertrophy and hypothyroidism. Basic Res Cardiol. 1990;85: Jaffe AS, Ritter C, Meltzer V, Harter H, Roberts R. Unmasking artifactual increases in creatine kinase isoenzymes in patients with renal failure. J Lab Clini Med. 1984;104: Katus H, Diederich KW, Hoberg E, Kubler W. Circulating cardiac myosin light chains in patients with angina at rest: identification of a high risk subset. JAm Coil Cardiol. 1988;11: Klein MS, Ludbrcok PA, Mimbs JW, Gafford F, Gillespie TA, Weldon CS, Sobel BE, Roberts R. Perioperative mortality rate in patients with unstable angina selected by exclusion of myocardial infarction. J Thorac Cardiovasc Surg. 1977;73: Clyne CA, Medeiros U-, Marton KI. The prognostic significance of immunoradiometric CK-MB assay (IRMA) diagnosis of myocardial infarction in patients with low total CK and elevated MB isoenzymes. Am Heart J. 1989;118: Morin LG. Creatine kinase: stability, inactivation, reactivation. Clin Chern. 1977;23: Greenson JK, Farber SJ, Dubin SB. The effect of hemolysis on creatine kinase determination. Arch Pathol Lab Med. 1989;113: Horder M, Elser RC, Gerhardt W, Mathieu M, Sampson EJ. Approved recommendation on IFCC methods for the measurement of catalytic concentration of enzymes: part 7. IFCC method for cratn kias (ATP cre atin N-hopotanfrae EC ). Eur J Clin Chem Clin Biochem_ ;29: Marshall T, Williams J, Williams, KM. Electrophoresis of serum enzymes and proteins following acute myocardial infarction. J Chromatogr. 1991;569: Mercer DW. Separation of tissue and serum creatine kinase isoenzymes by ion-exchange column chromatography. Clin Ctern. 1974;20: Henry PD, Roberts R, Sobel BE. Rapid separation of plasma creatine kinase isoenzymes by batch absorption on glass beads. Clini Ctern. 1975;21: Apple FS, Greenspan NS, Dietzier DN. Elevation of creatine kinase BB CK in hospitalized patients. Ann Clin Lab Sci. 1982; 12: Landt TY, Vaida HC, Porter SE, Whalen K, McClellan A, Amyx C, Parvin CA, Kessler G, Nahm M, Dietzler DW, Ladenson JH. Semiautomated direct colorimetric measurement of creatine kinase isoenzyme-mb activity after extraction from serum by use of a CK-MB specific monoclonal antibody. Clin Chem. 1988;34: Butch AW, Goodnow TT, Brown WS. STRATUS automated creatine kinase-mb assay evaluated: identification and elimination of falsely elevated results associated with a high molecular-mass form of alkaline phosphatase. Clin Ctern. 1989;35: Eisenberg PR, Shaw D, Schaab C, Jaffe AS. Concordance of creatine kinase-mb activity and mass. Clini Chem. 1989;35: Pesce MA. The CK and LD macroenzymes. Lab Management. 1984;22: 'Urdal P, Landaas S. Macro creatine kinase BB in serum and some data on its prevalence. Clin C/tern. 1979;25: Sobel BE, Shell WE. Serum enzyme determinations in the diagnosis and assessment of myocardial infarction. Circulation. 1972; 45: Jaffe AS, Klein AJ, Patel B, Siegel B, Roberts R. Abnormal technetium-99m pyrophosphate images in unstable angina: ischemia versus infarction? Am J Cardiol. 1979;44: Cox DA, Stone PH, Muller JE, Parker C, Hartwell TD, Rutherford JD, Roberts R, Jaffe AS, Hackel DB, Passamani ER, Robertson T, Sobel BE, Willerson JT, Braunwald EB, MILIS Study Group. Prognostic implications of an early peak in plasma MB creatine kinase in patients with acute myocardial infarction. JAm Coil Cardiol. 1987;10: Ong L, Reiser P, Coromilas J, Scherr L, Morrison J. Left ventricular function and rapid release of creatine kinase MB in acute myocardial infarction. N Engl J Med. 1983;309: Gore JM, Roberts R, Ball SP, Montero A, Goldberg RJ, Dalen JE. Peak creatine kinase as a measure of effectiveness of thrombolytic therapy in acute myocardial infarction. Am J Cardiol. 1987;59: Garabedian HD, Gold HK, Yasuda T, Johns JA, Finkelstein DM, Gaivin RI. Detection of coronary artery reperfusion with creatine kinase-mb determination during thrombolytic therapy: correlation with acute angiography. JAm Coil Cardiol. 1988;11: Puleo PR, Perryman MB, Bresser MA, Rokey R, Pratt CM, Roberts R. Creatine kinase isoform analysis in the detection and assessment of thrombolysis in man. Circulation. 1987;75: Potkin RT, Werner J, Trobaugh GB, Chestnut CH III, Carrico Cl, Hallstrom A, Cobb LA. Evaluation of noninvasive tests of cardiac damage in suspected cardiac contusion. Circulation. 1982; 66: Shahangian 5, Ash OW, Wahlstrom NO, Warden GD), Safile JR, Taylor A, Green LS. Creatine kinase and lactate dehydrogenase isoenzymes in serum of patients suffering burns, blunt trauma, or myocardial infarction. Clin Chem. 1984;30: Ehsani A, Ewy GA, Sobel BE. Effects of electrical countershock on serum creatine phosphokinase isoenzyme activity. Am J Cardiol. 1976;37: Lenke LG, Bridwell KH, Jaffe AS. Increases in creatine kinase MB isoenzyme levels following spinal surgery. J Spinal Disorders. In press. 71. El Allaf M, Chapelle J, El Allaf D, Adam A, Faymonville M, Laurent P, Heusghem C. Differentiating muscle damage from myocardial injury by means of the serum creatine kinase (CK) isoenzyme MB mass measure/total CK activity ratio. Clin Chem. 1986;32: Wolfson D, Lindberg E, Su L, Farber 5, Dubin SB. Three rapid immunoassays for the determination of creatine kinase MB: an analticl, cinial nd ineprtve vluiation.n Am Heart ; 122:

12 Adams et al Biochemical Markers of Myocardial Injury Adams JE, Bodor G, Divila-Romfin V, Ladenson J, Jaffe AS. Cardiac troponin I: a marker with high specificity for cardiac injury. Circulation. 1993;88: Abendschein DR, Ellis AK, Eisenberg PR, Klocke FJ, Sobel BE, Jaffe AS. Prompt detection of coronary recanalization by analysis of rates of change of concentrations of macromolecular markers in plasma. Coron Artery Dis. 1991;2: Grande P, Granborg J, Clemmensen P, Sevilla D, Wagner N, Wagner G. Indices of reperfusion in patients with acute myocardial infarction using characteristics of the CK-MB time-activity curve. Am Heart J. 1991;122: Rogers WJ, McDaniel HG, Smith LR, Mantle JA, Russell RO, Rackley CE. Correlation of CPK-MB and angiographic estimates of infarct size in man. Circulation. 1977;56: Roberts R. Enzymatic estimation of infarct size: Thrombolysis induced its demise: Will it now rekindle its renaissance? Circulation. 1990;81: Hermens W, van der Veen FH, Willems GM, Mullers-Boumans ML, Schendel A, Schrijvers VAN, Reneman RS. Complete recovery in plasma of enzymes lost from the heart after permanent coronary artery occlusion in the dog. Circulation. 1990;81: Roberts R, Ambos HD, Sobel BE. Estimation of infarct size with MB rather than total CK Int J Cardiol. 1983;2: Roberts R, Ishikawa Y. Enzymatic estimation of infarct size during reperfusion. Circulation. 1983;68(suppl I):I-83-I Wevers RA, Delsing M, Klein Gebbink JA, Soons JBJ. Postsynthetic changes in creatine kinase isoenzymes. Clin Chim Acta. 1978;86: George S, Ishikawa Y, Perryman MB, Roberts R. Purification and characterization of naturally occurring and in vitro induced forms of MM creatine kinase. J Biol Chem. 1984;259: Prager NP, Suzuki T, Jaffe AS, Sobel BE, Abendschein DR. The nature and time course of generation of isoforms of MB creatine kinase in vivo. JAm Coll Cardiol. 1992;20: Billadello JJ, Roman DG, Grace AM, Sobel BE, Strauss AW. The nature of post-translational formation of MM creatine kinase isoforms. J Biol Chem. 1985;260: Abendschein DR, Serota H, Plummer TH, Amiraian K, Strauss AW, Sobel BE, Jaffe AS. Conversion of MM creatine kinase isoforms in human plasma by carboxypeptidase N. J Lab Clin Med. 1987;110: Abendschein DR, Fontanet HL, Markham J, Sobel BE. Physiologic modeling of MM creatine kinase isoforms. Mathl Comput Modeling. 1988;11: Puleo PR, Guadagno PA, Roberts R, Perryman MB. Sensitive, rapid assay of subforms of creatine kinase MB in plasma. Clin Chem. 1989;35: Christenson RH, Ohman EM, Clemmensen P, Grande P, Toffalettti J, Silverman LM, Vollmer RT, Wagner GS. Characteristics of creatine kinase-mb and MB isoforms in serum after reperfusion in acute myocardial infarction. Clin Chem. 1989;35: Billadello JJ, Fontanet HL, Strauss AW, Abendschein DR. Characterization of MB creatine kinase isoform conversion in vitro and in vivo in dogs. J Clin Invest. 1989;83: Abendschein DR, Fontanet HL, Nohara R. Optimized preservation of isoforms of creatine kinase MM isoforms in plasma samples and their rapid quantification by semi-automated chromatofocusing. Clin Chem. 1991;36: Nohara R, Sobel BE, Jaffe AS, Abendschein DR. Quantitative analysis of isoforms of creatine kinase MM in plasma by chromatofocusing with online monitoring of enzyme activity. Clin Chem. 1988;34: Abendschein D, Seacord LM, Nohara R, Sobel BE, Jaffe AS. Prompt detection of myocardial injury by assay of creatine kinase isoforms in initial plasma samples. Clin Cardiol. 1988;11: Jaffe AS, Serota H, Grace A, Sobel BE. Diagnostic changes in plasma creatine kinase isoforms early after the onset of acute myocardial infarction. Circulation. 1986;74: Panteghini M, Cuccia C, Malchiodi A. Isoforms of cre~atine kinase MM and MB in acute myocardial infarction: a clinical evaluation. Clin Chim Acta. 1986;155: Morelli RL, Carlson CJ, Emilson B, Abendschein D, Rapaport E. Serum creatine kinase MM isoenzyme sub-bands after acute myocardial infarction in man. Circulation. 1983;67: Puleo PR, Guadagno PA, Roberts R, Scheel MV, Marion AJ, Churchill D, Perryman B. Early diagnosis of acute myocardial infarction based on assay for subforms of creatine kinase-mb. Circulation. 1990;82: Caforio ALP, Hossein-Nia M, Keeling PJ, Brown P, Bottazzo GF, Holt DW, McKenna WJ. Creatine kinase isoforms as markers of myocardial damage in dilated cardiomyopathy: their association with organ-specific cardiac autoantibodies. Circulation. 1992; 86(suppl I):I-794. Abstract. 98. Jennison S, Hassein-Nia M, Brown P, Murday A, Holt D, McKenna W. Creatine kinase (CKMB) isoforms as an early marker of myocardial cell injury during acute allograft rejection. Circulation. 1992;86(suppl I):I-841. Abstract. 99. Kircher BJ, Topol EJ, O'Neill WW, Pitt B. Prediction of infarct coronary artery recanalization after intravenous thrombolytic therapy. Am J Cardiol. 1987;59: Nohara R, Myears D, Sobel BE, Abendschein DR. Optimal criteria for rapid detection of myocardial reperfusion by creatine kinase MM isoforms in the presence of residual high grade stenosis. J Am Coll Cardiol. 1989;14: Seacord LM, Abendschein DR, Nohara R, Hartzler G, Sobel BE, Jaffe Al. Detection of reperfusion within one hour after coronary recanalization by analysis of isoforms of the MM creatine kinase isoenzyme in plasma. Fibrinolysis. 1988;2: Schofer J, Ress-Grigolo G, Voigt KD, Mathey DG. Early detection of coronary artery patency after thrombolysis by determination of the MM creatine kinase isoforms in patients with acute myocardial infarction. Am Heart J. 1992;123: Puleo PR, Perryman B. Noninvasive detection of reperfusion in acute myocardial infarction based on plasma activity of creatine kinase MB subforms. JAm Coll Cardiol. 1991;17: Abendschein DR, Puleo PR, Cannon CP, TIMI IV and V Investigators. Noninvasive detection of early coronary artery patency based on plasma MM and MB creatine kinase (CK) isoforms. Circulation. 1992;86(suppl I):I-267. Abstract Jaffe AS, Eisenberg PR, Abendschein DR. New criteria for detection of coronary recanalization with MB CK isoforms. Circulation. 1992;86(suppl I):I-267. Abstract Roberts R. Myoglobinemia as an index to myocardial infarction. Ann Intern Med. 1977;87: Kagen L, Scheidt S, Roberts L, Paul H. Myoglobinemia following acute myocardial infarction. Am J Med. 1975;58: Cairns JA, Missirlis E, Walker WHC. Usefulness of serial determinations of myoglobin and creatine kinase in serum compared for assessment of acute myocardial infarction. Clin Chem. 1983; 29: Isakov A, Shapira I, Burke M, Almog C. Serum myoglobin levels in patients with ischemic myocardial insult. Arch Intern Med. 1988;148: Drexel H, Dworzak E, Kirchmair W, Milz MM, Puschendorf B, Diemstel F. Myoglobinemia in the early phase of acute myocardial infarction. Am Heart J. 1983;105: Gasser RNA, Hauptlorenz S, Dworzak E, Moll W, Puschendorf B, Dienstl F. The typical staccato phenomenon of myoglobin in acute myocardial infarction disappears under thrombolytic treatment. Fibrunolysis. 1987;1: Loogna E, Sylven C, Groth T, Mogensen L. Complexity of enzyme release during acute myocardial infarction in a controlled study with early nifedipine treatment. Eur Heart J. 1985;6: Katus HA, Diederich KW, Scheffold T, Uellner M, Schwartz F, Kubler W. Non-invasive assessment of infarct reperfusion: the predictive power of the time to peak value of myoglobin, CKMB, and CK in serum. Eur Heart J. 1988;9: Hoberg E, Katus HA, Diederich KW, Kubler W. Myoglobin, creatine kinase-b isoenzyme, and myosin light chain release in patients with unstable angina pectoris. EurHeart J. 1987;8: Almog C, Isakov A, Ayalon D, Burke M, Shapira I. Serum myoglobin in detection of myocardial necrosis in patients with "coronary insufficiency." Clin Cardiol. 1987;10: Bouvagnet P, Leger JOC, Pons F, Dechesne C, Leger JJ. Fiber types and myosin types in human atrial and ventricular myocardium. Circ Res. 1984;55: Collins JH, Theibert JL, Libera LD. Amino acid sequence of rabbit ventricular myosin light chain-2: identity with the slow skeletal muscle isoform. Biosci Rep. 1986;6: Katus HA, Yasuda T, Gold H, Lienbach RC, Strauss HW, Waksmonski C, Haber E, Khaw BA. Diagnosis of acute myocardial infarction by detection of circulating cardiac myosin light chains. Am J Cardiol. 1984;54: Katus HA, Looser S, Hallermayer K, Remppis A, Scheffold T, Borgya A, Essig U, Geuss U. Development and in vitro characterization of a new immunoassay of cardiac troponin T. Clin Chem. 1992;38: Weeds AG, Pope B. Chemical studies on light chains from cardiac and skeletal muscle myosin. Nature. 1971;234:85-88.

13 762 Circulation Vol 88, No 2 August Katus HA, Diederich KW, Schwartz F, Vellner M, Scheffold T, Kubler W. Influence of reperfusion on serum concentration of cytosolic creatine kinase and structural myosin light chains in acute myocardial infarction. Am J Cardiol. 1987;60: Isobe M, Nagai R, Yamoki K, Nakaoka H, Takaku F, Yazaki Y. Quantification of myocardial infarct size after coronary reperfusion by serum cardiac myosin light chain II in conscious dogs. Circ Res. 1989;65: Zak R, Martin AF, Prior G, Rabinowitz M. Comparison of turnover of several myofibrillar proteins and critical evaluation of double isotope method. J Biol Chem. 1977;252: Yazaki Y, Nagai R, Yamaoki K, Ueda S. Myocardial infarct size from serum cardiac myosin light chain. Adv Myocardiol. 1983;4: Seguin JR, Saussine M, Ferrierre M, Leger JJ, Leger J, Larue C, Grolleau R. Myosin: a highly sensitive indicator of myocardial necrosis after cardiac operations. J Thorac Cardiovasc Surg. 1989; 98: Leger JOC, Larue C, Ming T, Calzolari C, Gautier P, Mouton C, Grolleau R, Louisot P, Puech P, Peperstraete B, Staroukine M, Telerman M, Pau B, Leger JJ. Assay of serum cardiac myosin heavy chain fragments in patients with acute myocardial infarction: determination of infarct size and long-term follow-up. Am Heart J. 1990;120: Billeter R, Heizmann CW, Howald H, Jenny E. Analysis of myosin light and heavy chain types in single human skeletal muscle fibers. Eur J Biochem. 1981;116: Smit MJ, Duursma AM, Bouma JMW, Gruber M. Receptormediated endocytosis of lactate dehydrogenase M by liver macrophages: a mechanism for elimination of enzymes from plasma. J Biol Chem. 1987;262: Wieland T, Pfleiderer G. Chemical differences between multiple forms of lactic acid dehydrogenase. Ann N YAcad Sci U S A. 1961;94: Kreutzer HH, Fennis WHS. Lactic dehydrogenase isoenzymes in blood serum after storage at different temperatures. Clin Chim Acta. 1964;9: Jablonsky G, Leung FY, Henderson AR. Changes in LD1/LD2 ratio during the first day after myocardial infarction. Clin Chem. 1985;31: Loughlin JF, Krijnen PM, Jablonsky G, Leung FY, Henderson AR. Diagnostic efficiency of four lactate dehydrogenase isoenzyme-1 ratios in serum after myocardial infarction. Cln Chem. 1988;34: Emery AEH. Electrophoretic pattern of lactic dehydrogenase in carriers and patients with Duchenne muscular dystrophy. Nature. 1964;201: Rotenburg Z, Selp R, Wolfe LA, Burns DE. Flipped patterns of lactate dehydrogenase isoenzymes in serum of elite college basketball players. Clin Chem. 1988;34: Von Eyben FE, Blaabjerg 0, Petersen PH, Horder M, Nielsen HV, Kruse-Anderson S, Parley E. Serum lactate dehydrogenase isoenzyme 1 as a marker of testicular germ cell tumor. J Urol. 1988;140: Mortelmans L, Vanhaecke J, Lesaffre E, Arnold A, Urbain JL, Hermens W, DeRoo M, DeGeest H, Verstraete M, Van de Werf F. Evaluation of the effect of thrombolytic treatment on infarct size and left ventricular function by enzymatic, scintigraphic, and angiographic methods: the European Cooperative Study Group for Recombinant Tissue Type Plasminogen Activator.Am Heart J. 1990;119: van der Laarse A, Kerkhof PLM, Vermeer G, Serruys PW, Hermens WT, Verheugt FWA, Bar FW. Relation between infarct size and left ventricular performance assessed in patients with first acute myocardial infarction randomized to intracoronary thrombolytic therapy or to conventional treatment. Am J CardioL 1988; 61: Karagounis L, Moreno F, Menlove RL, Ipsen S, Anderson J. Effects of early thrombolytic therapy (anistreplase versus streptokinase) on enzymatic and electrocardiographic infarct size in acute myocardial infarction. Am J Cardiol. 1991;68: MacGeoch C, Barton PJ, Vallins WJ, Bhavsar P, Spurr NK. The human cardiac troponin I locus: assignment to chromosome 19p q13.2. Hum Genet. 1991;88: Wilkinson JM, Grand RJA. Comparison of amino acid sequence of troponin I from different striated muscles. Nature. 1978;271: Wade R, Eddy R, Shows TB, Kedes L. cdna sequence, tissuespecific expression, and chromosomal mapping of the human slow-twitch skeletal muscle isoform of troponin I. Genomics. 1990; 7: Schreier T, Kedes L, Gahlmann R. Cloning, structural analysis, and expression of the human slow twitch skeletal muscle/cardiac troponin C gene. J Biol Chem. 1990;265: Katus HA, Remppis A, Scheffold T, Diederich KW, Kuebler W. Intracellular compartmentation of cardiac troponin T and its release kinetics in patients with reperfused and nonreperfused myocardial infarction. Am J Cardiol. 1991;67: Toyota N, Shimada Y. Differentiation of troponin in cardiac and skeletal muscles in chicken embryos as studied by immunofluorescence microscopy. J Cell Biol. 1981;91: Saggin L, Gorza L, Ausoni S, Schiaffino S. Troponin I switching in the developing heart. J Biol Chem. 1989;264: Bhavsar P, Dhoot GK, Cumming DVE, Butler-Browne GS, Yacoub MH, Barton OJR. Developmental expression of troponin I isoforms in fetal human heart. FEBS Lett. 1991;292: Adams JE, Bodor GS, Divila-Romin VG, Sicard GA, Ladenson JH. Improved detection of perioperative myocardial infarction with cardiac troponin I. JAm Coil Cardiol. 1993;21:88A. Abstract Cummins P, Yourn A, Auckland ML, Michie CA, Stone PCW, Shepstone BJ. Comparison of serum cardiac specific troponin-i with creatine kinase, creatine kinase-mb isoenzyme, tropomyosin, myoglobin and C-reactive protein release in marathon runners: cardiac or skeletal muscle trauma? Eur J Clin Invest. 1987; 17: Cummins B, Auckland M, Cummins P. Cardiac-specific troponin-i radioimmunoassay in the diagnosis of acute myocardial infarction. Am Heart J. 1987;113: Schwartz K, de la Bastie D, Bouveret P, Oliviero P, Alonso S, Buckingham M. a-skeletal muscle actin mrna accumulate in hypertrophied adult rat hearts. Circ Res. 1986;59: Anderson PAW, Malouf NN, Oakeley AE, Pagani ED, Allen PD. Troponin T isoform expression in humans: a comparison among normal and failing adult heart, fetal heart, and adult and fetal skeletal muscle. Circulation. 1991;69: Saggin L, Gorza L, Ausoni S, Schiaffino S. Cardiac troponin T in developing, regenerating, and denervated rat skeletal muscle. Development. 1990;110: Anderson PA, Oakeley AE. Immunological identification of five troponin T isoforms reveals an elaborate maturational troponin T profile in rabbit myocardium. Circ Res. 1989;65: Wilkinson JM, Taylor RD. Expression of troponin T in atria and ventricles of avian and mammalian hearts. J Mol Cell Cardiol. 1986;18: Mair J, Artner-Dworzak E, Lechleitner P, Smidt J, Wagner I, Dienstl F, Puschendorf B. Cardiac troponin T in diagnosis of acute myocardial infarction. Clin Chem. 1991;37: Katus HA, Remppis A, Neumann FJ, Scheffold T, Diederich KW, Vinar G, Noe A, Matern G, Kuebler W. Diagnostic efficiency of troponin T measurements in acute myocardial infarction. Circulation. 1991;83: Katus HA, Schoeppenthau M, Tanzeem A, Bauer HG, Saggau W, Diederich KW, Hagl S, Kuebler W. Noninvasive assessment of perioperative myocardial cell damage by circulating cardiac troponin T. Br Heart J. 1991;65: Kobayashi S, Tanaka M, Tamura N, Hashimoto H, Hirose S. Serum cardiac troponin T in polymyositis/dermatomyositis. Lancet. 1992;340:726. Letter Collinson PO, Stubbs PJ. The prognostic value of serum troponin T in unstable angina. N Engl J Med. 1992;327: Hamm CW, Ravkilde J, Gerhardt W, Jorgensen P, Peheim E, Ljungdahl L, Goldman B, Katus HA. The prognostic value of serum troponin T in unstable angina. NEngl JMed. 1992;327: Bodor GS, Porter S, Landt Y, Ladenson JH. The development of monoclonal antibodies and an assay for cardiac troponin-i with preliminary results in suspected myocardial infarction. Clin Chem. 1992;11: Katus HA, Remppis A, Looser S, Hallermeier K, Sheffold T, Kubler W. Enzyme linked immunoassay of cardiac troponin T for the detection of acute myocardial infarction in patients. JMol Cell Cardiol 1989;21: Crisman TS, Claffey KP, Saouaf R, Hanspal J, Brecher P. Measurement of rat heart fatty acid binding protein by ELISA: tissue distribution, developmental changes and subcellular distribution. J Mol Cell Cardiol. 1987;19: Knowlton AA, Apstein CS, Saouf R, Brecher P. Leakage of heart fatty acid binding protein with ischemia and reperfusion in the rat. J Mol Cell Cardiol. 1989;21:

14 Adams et al Biochemical Markers of Myocardial Injury Tanaka T, Hirota Y, Sohmiya K-I, Nishimura S, Kawamura K. Serum and urinary human heart fatty acid binding protein in acute myocardial infarction. Clin Biochem. 1991;24: Abe S, Okino H, Lee S, Toda H, Miyata M, Nomoto K, Arima S, Yamashita T, Nakoa S, Tanaka H, Tanaka T, Kawamura K Human heart fatty acid binding protein, a sensitive and specific marker of coronary reperfusion. Circulation. 1991;85(suppl II):II Abstract Chen S-H, Giblett ER. Enolase: human tissue distribution and evidence for three different loci.ann Hum Genet. 1976;39: Kato K, Ishiguro Y, Ariyoshi Y. Enolase isozymes as disease markers: distribution of three enolase subunits (a, js, and y) in various human tissues. Dis Markers. 1983;1: Nomura M, Kato K, Nagasaka A, Shiga Y, Miyagi Y, Fukui R, Nakano H, Abo Y, Okajima S, Nakai A, Ohyama T, Hishida H, Watanabe Y, Mizuno Y. Serum P-enolase in acute myocardial infarction. Br Heart J. 1987;58: Herraez-Dominguez M, Goldberg D, Anderson A, Fleming JS, Rider CC, Taylor B. Serum enolase and pyruvate kinase activities in the diagnosis of myocardial infarction. Enzyme. 1976;21: Usui A, Kato K, Abe T, Murase M, Tanaka M, Takeuchi E. P-Enolase in blood plasma during open heart surgery. Cardiovasc Res. 1989;23: van Landeghem AAJ, Soons JBJ, Wever RA, Mul-Steinbusch MWFJ, Antonissen-Zijda T. Purification and determination of the modifying protein responsible for the post-synthetic modification of creatine kinase (EC ) and enolase (EC ). Clin Chim Acta. 1985;153: Laperche T, Steg GP, Bennessiano J, Dehoux M, Juliard J, Himbert D, Gourgon R. Patterns of myoglobin and MM creatine kinase isoforms release early after intravenous thrombolysis or direct percutaneous transluminal coronary angioplasty for acute myocardial infarction and implications for the early diagnosis of reperfusion. Am J Cardiol. 1992;70: Katus HA, Scheffold T, Remppis A, Kerth P, Ruedinger W. Troponin T: a marker of reperfusion of the infarct zone. Circulation. 1992;84(suppl II):II-116. Abstract. KEY WORDS * creatine kinase * myocardial injury