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History |
1 10 Wimpole News, London, United Kingdom.
2 University of Ottawa Heart Institute, Ottawa, Ontario, Canada.
3 Medizinische Universitätsklinik Heidelberg, Abteilung Innere Medizin III, Department of Cardiology, Heidelberg, Germany.
4 Washington University School of Medicine, Division of Laboratory Medicine, Department of Pathology and Immunology, St. Louis, MO.
5 Hennepin County Medical Center and University of Minnesota, Minneapolis, MN.
aAddress correspondence to this author at: Hennepin County Medical Center and University of Minnesota, Minneapolis, MN 55415. E-mail fred.apple{at}co.hennepin.mn.us.
Abstract
Editor’s Note: With great pleasure and anticipation in recognition of Clinical Chemistry’s 50th anniversary, I have been able to arm-twist four talented scientists to document their impressive marks on the science of diagnostics in the field of cardiac biomarkers and detection of myocardial infarction. Their exciting discoveries and applications have dramatically influenced the fields of laboratory medicine and cardiology and have greatly influenced the care and management of thousands of patients suffering from coronary artery disease leading to acute myocardial infarction. As a matter of historical record, I owe a great deal of thanks to each one of the coauthors of this special report because each one has personally influenced my scientific career. I met Dr. Rosalki, during my postdoctoral training, at a national AACC meeting, where he kindly answered my numerous queries regarding creatine kinase enzymology and muscle physiology. Dr. Roberts, while serving as Director of the Coronary Care Unit at Washington University in St. Louis, generously allowed this fledgling fellow into his laboratory and shared many of his clinical and experimental findings with me. Dr. Katus, whom I first met at a scientific meeting sponsored by Boehringer Mannheim in 1986 in Bavaria, where I first became fascinated with cardiac troponin T, has remained a friend and colleague. Lastly, Dr. Ladenson, who as mentor, scientific colleague, and close friend remains ultimately responsible for both my professional growth as a clinical chemist (he was my postdoctoral fellowship advisor) and for stimulating and encouraging my goals and aspirations in the field of cardiac biomarkers. With the descriptions of the ground-breaking science described below, I am extremely excited and optimistic that the future of cardiac biomarkers is secure and open to new discoveries by the Rosalkis, Robertses, Katuses, and Ladensons of the future.
—Fred Apple
Determination of Serum Creatine Kinase Activity
—Sidney B. Rosalki
I had previously described a test for myocardial infarction (
-hydroxybutyrate dehydrogenase) (1). I found that an American company was marketing the procedure with altered substrate concentration so that its diagnostic performance would be impaired, and I wrote complaining to the company president. On a visit to the United Kingdom in November 1964, he invited my wife and me to join him for dinner. During the course of the meal, he suggested that instead of complaining about an existing product, I might propose something novel. At that time, creatine kinase (adenosine triphosphate:creatine phophotransferase; EC 2.7.3.2) methodology was abysmal. The only convenient test (2) frequently gave negative values, and other methods were so prolonged and labor-intensive that few laboratories carried out the determination. On the back of the menu card, which I still possess (Fig. 1
), I sketched out my idea for modification of the Kornberg ATP assay (3) for creatine kinase measurement. This required the addition of creatine phosphate, ADP, and a thiol, and the combination of all reagents in a single lyophilisate that would need only aqueous reconstitution and sample addition. In correspondence, I outlined more fully the required reagent composition, which was prepared and presented in individual gelatin capsules.
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Method optimization and evaluation were carried out under very adverse conditions. At the time, I was a consultant in clinical pathology and responsible for all of the clinical chemistry, hematology, microbiology, and even histopathology for two acute children’s hospitals—all on a part time basis! I had received negligible support from my hospital for my research work. I had no research assistant and no suitable apparatus. I persuaded the American company to donate to me a simple fixed-wavelength (340 nm) single-cuvette spectrophotometer, cost US $250.00. To measure enzyme reaction rates on this instrument, a needle was held at null point by rotating a knob connected to a numbered dial. Each Sunday, my wife would accompany me to the hospital. I would set up the reaction and read out the figures on the dial at 1-min intervals with a stop-watch. My wife would record the readings. Subsequently, I would calculate and plot the change in absorbance per minute. The reaction had a 6-min lag phase, and the linear phase required a further 5-min monitoring period. Each single enzyme determination required 15 min of instrument time. Each reaction mixture contained 10 constituents, each of which had to be individually varied during optimization studies. The labor involved was considerable and can scarcely be imagined in these days of multisample, microprocessor-controlled automated enzyme analyzers.
Despite all of the difficulties, method and clinical studies were completed in 1965. Details were submitted and immediately accepted for publication in 1966, and appeared in 1967 (4). With this procedure, the hitherto complex creatine kinase determination was now so simple, requiring only sample addition to a single preprepared substrate mixture, and so sensitive that it was adopted worldwide for creatine kinase determination in clinical biochemistry laboratories. This facilitated wider recognition of the outstanding value of creatine kinase determination in the investigation of heart and muscle disease (5) and, in turn, prompted increased use of the method.
Compared with other enzyme tests for myocardial infarction diagnosis, creatine kinase has the advantage of earlier increases, higher sensitivity, and higher specificity. It invariably shows a rapid increase in serum in the early hours after admission with chest pain from infarction (6). In addition, monitoring of increases in serum creatine kinase remains the most sensitive diagnostic enzyme procedure for the detection of myopathy, for the preclinical detection of muscular dystrophy and for detection of dystrophy carriers, and for the assessment of response of polymyositis to therapy. An important additional use is the identification of muscle damage as a consequence of drug therapy (7).
One Molecule, MBCK, Changed the Diagnostic World
—Robert Roberts
historical perspective
The only form of energy available to all life on this planet is high-energy phosphate, usually in the form of ATP. The enzyme responsible for catalyzing the transfer of high-energy phosphate from creatine phosphate to ATP is creatine kinase (CK).1
It is perhaps somewhat surprising that this molecule would take center stage in the diagnosis of myocardial infarction. However, the heart, a muscle that continually contracts without a break, has a high energy requirement. Thus, when the heart muscle dies during myocardial infarction, it releases many molecules into the bloodstream, one of the more abundant being CK. There is rapid release because of the favorable gradient between the high myocardial content of CK and the low content of the blood (ratio >1000 to 1). CK is a dimeric molecule composed of two subunits (M or B) with a molecular mass of 42 000 Da. In 1959, Ehashi et al. (8) demonstrated that increased plasma CK activity is an extremely sensitive index of skeletal muscle disease. One year later, Dreyfus et al. (9) demonstrated markedly increased plasma CK activity in patients with myocardial infarction. CK was shown to exist in three molecular forms MM, MB, and BB. In 1996, van der Ween and Willebrands (10) showed that MBCK is a highly specific marker of myocardial infarction. The CK content of myocardial tissue was shown to be 
15% MB and the remainder MMCK (11). MBCK was in the right place at the right time. A series of elegant animal studies (12)(13) showed that myocardial damage in association with myocardial infarction occurred over several hours and that the extent of damage is the determinant of mortality and morbidity. These studies provided the impetus to quantify the extent of myocardial infarction in humans by determining the amount of CK released into the blood and relating it back to histologic estimates of infarction. It was recognized that each unit (e.g., gram) of myocardium releases a specific amount of CK into the blood and, thus, that by determining the total amount of CK released in the blood, it was possible to determine the extent of myocardial damage in grams of infarct. A new term was about to enter the world’s languages, namely, enzymatic estimates of infarct size.
Total CK was recognized as nonspecific and, thus, unlikely to provide the desirable accuracy. Development of the first quantitative assay for MBCK was my first research project in Dr. Sobel’s laboratory, which we published in 1974 (14), and for me; it was the beginning of an exciting career. The detection and quantification of myocardial infarction by MBCK would inseminate and stimulate cardiology literature for several decades. It became evident that MBCK would be the most specific, accurate, and cost-effective means of detecting myocardial infarction. The diagnosis of myocardial infarction by use of MBCK required only 12–24 h, and the traditional 3 days in the cardiac critical care unit for those without infarction was reduced to 1 day. CK was to play a major role in an age that would subsequently be known as the golden era for cardiology. A major breakthrough came with the observation that early intervention could prevent or limit the amount of myocardial damage and, as such, markedly reduce the mortality and morbidity associated with myocardial infarction. Today, it is well recognized that reperfusion initiated within the first hour of myocardial infarction is associated with a mortality of 1% as opposed to 10% if initiated at or after 6 h. This, however, is markedly less than the 30–35% hospital mortality of the 1960s. It is somewhat ironic, however, that enzymatic estimates of infarct size played a major role in determining the effect of reperfusion therapy whereas the therapy used routinely today, i.e., thrombolysis or angioplasty, proved to be the death of enzymatic estimates of infarct size. The random onset of reperfusion and induced washout of CK from the myocardium would render estimates of infarct size based on release of MBCK highly variable. The decades of quantifying infarct size with MBCK played into attempts to estimate infarct size from myocardial imaging, but to date, these have not become routine.
immunologic detection of myocardial infarction
In the 1970s, it became evident that MBCK was to be the standard for the diagnostic and quantitative assessment of myocardial infarction. Several laboratories pursued a variety of assays to improve the sensitivity and rapidity of assaying for MBCK. Most techniques took advantage of the differential charge between MBCK and that of MM or BBCK (15). In 1976, we developed a RIA (16) for MBCK based on an antibody to the B-subunit. This paved the way for the subsequent development of an antibody specific for MBCK (17). It was the beginning of a movement that would use antibodies to detect biomarkers in the plasma associated with a variety of diseases, including the present assays for detection of troponin I and troponin T. Immunologic detection of biomarkers today is routine and offers the advantage of protein concentration over enzymatic activity.
rapid diagnosis of infarction by mbck subforms
Wevers et al. (18) had shown that, on release into the blood, CK undergoes modification that gives rise to different forms. There was considerable controversy in the literature as to whether these forms really existed. In 1984, we isolated and purified the various subforms and elucidated that the mechanism is cleavage by carboxypeptidase N of the C-terminal lysine from the M subunit (19). Removal of the lysine provided a net increase in negative charge and a means for differential detection, but it also produced a molecule more vulnerable for uptake and thus with a shorter half life in the circulation. It was now possible to detect in a precise and sensitive manner release of new MBCK from the heart into the blood by showing an increase in the uncleaved MBCK and thus provide an earlier diagnosis. The total MBCK would initially remain the same, with the uncleaved MBCK increased. Again the subforms had selected an opportune time to appear on the diagnostic stage. MBCK had by now replaced all markers as the most sensitive and specific diagnostic index of myocardial infarction. The WHO criteria (1957) had all but been eclipsed, and confirmation of infarction was essentially based on MBCK. In addition, it was evident that less than one half of all heart attacks exhibited ST-segment elevation, the major WHO criterion and sine qua non of detecting infarction on the electrocardiogram. This further provided the impetus to rely on MBCK as the marker for infarction. Furthermore, with reperfusion as routine therapy for infarction, an earlier diagnosis was essential. The non-Q-wave infarction is difficult to separate from ischemia without the use of a biomarker. The subforms offered the potential to screen patients with acute coronary syndrome and to separate the group with infarction from those with ischemia.
Use of the MBCK subforms was shown to diagnose myocardial infarction within 6 hours of onset of symptoms, and >60% of infarctions were detectable within the first hour of arriving in the emergency room (20). A prospective multicenter double-blind study, referred to as Diagnostic Marker Cooperative Study for the Diagnosis of Myocardial Infarction (21), was performed to compare myoglobin with the subforms for early diagnosis, and total MBCK, troponin I, and troponin T as markers for late diagnosis of infarction. The study confirmed that MBCK subforms provide a reliable and specific diagnosis with 97% accuracy in the first 6 h of onset of symptoms. The diagnosis of infarction within 24 h was shown to be equally accurate for troponin I, troponin T, and MBCK, each averaging peak detection 16–18 h from onset. The MBCK subforms remain the only demonstrated, reliable, and accurate test for diagnosing patients with myocardial infarction within 6 h of onset of symptoms. It was shown to have a negative predictive value of 99% in excluding individuals with chest pain without infarction in the emergency room. Despite this advantage, it remains much underused.
recurrent and future use of mbck
Plasma MBCK remains the standard against which biomarkers will continue to be compared for their accuracy in detection and quantification of myocardial infarction. In addition, it remains the only biomarker whose release has been shown to depend on irreversible myocardial injury (22). It is highly likely that troponin I and T reflect necrosis; but because they are much smaller molecules of 23 000 or 39 000 kDa, proof is still required to show that necrosis is necessary and not just ischemia. Despite the competition from troponin I and T, MBCK remains a diagnostic marker worldwide. Measurement of MBCK subforms remain the most specific and reliable test for detection of infarction within the first 4–6 h and could conceivably be used in the future as a means of detecting non-Q-wave myocardial infarction before reperfusion therapy. Total CK also remains a marker for detection of myocardial infarction throughout the world because, in many countries, MBCK assays are not routinely used. Detection of reinfarction within 3–10 days requires MBCK subforms because total MBCK is increased for 3–4 days and the troponins for 10–14 days. The ratio of cleaved to uncleaved MBCK subforms returns to normal within 18–30 h.
Cardiac Troponin T: The Past, the Present, and the Future
—Hugo A. Katus and Evangelos Giannitsis
the past
In 1978, we started our work on diagnostic assays for myofibrillar proteins. This work was driven by five important pieces of evidence. First, in clinical cardiology, it became apparent that unstable coronary artery plaque and thrombosis generate a wide spectrum of disease manifestations, today classified as acute coronary syndromes (23). Furthermore, in chest pain patients in whom acute myocardial infarction (AMI) was ruled out by electrocardiography and cardiac enzymes, 1-year outcomes were similar to those of patients with definite AMI (24)(25), indicating that the available diagnostic methods were not useful for risk prediction and guidance of treatment. Second, the isoenzymes of CK and lactate dehydrogenase had been introduced into clinical practice (26)(27) and had been shown to have enhanced specificity and sensitivity compared with conventional assays. The lack of cardiospecificity of these tests, however, still did not allow the definite distinction of skeletal and cardiac muscle injuries. Third, immunochemical methods (RIA and enzyme-linked immunoassays) were successfully used to detect in blood minute amounts of molecules independently of their enzymatic or biological activity (15). The hybridoma technology for generation of monoclonal antibodies and in vitro selection of antibody molecules according to their predefined specificity promised great potential for development of well-standardized assays (28). Fourth, more refined analyses of contraction characteristics revealed differences in sarcomeric proteins of cardiac and skeletal muscle. It was reported that different isoforms of sarcomeric proteins may explain these findings (29)(30). Finally, first reports, particularly from Dr. Habers’s group at the Massachusetts General Hospital in Boston had indicated the usefulness of isoforms of sarcomeric proteins as targets for diagnosis of myocardial injury either by anti-myosin antibody imaging or by RIA for myosin light chains in experimental AMI (31)(32).
On the basis of this knowledge, we decided to take advantage of the large sarcomeric protein pool to develop highly specific and sensitive assays useful for routine clinical testing. This required multiple time-consuming and technically demanding steps such as (a) the development of purification protocols for the subunits of the human sarcomere, (b) the generation of polyclonal antisera and monoclonal antibodies, (c) the development of RIAs and enzyme-linked immunoassays, (d) preclinical testing, and finally (e) evaluation in large multicenter trials.
The initial work on myosin light chains was abandoned in 1982 when it became apparent that the cardiac isoform of myosin light chain was also produced in slow skeletal muscle, explaining the observed cross-reactivity in clinical samples (30)(33)(34). Because Western blot analyses revealed that antibodies against the cardiac isoform of troponin T do enable a definite distinction of cardiac and skeletal muscle, further work was focused on cardiac troponin T (cTnT) (35)(36)(37)(38)(39). To translate the work to a clinically useful product, cooperation was initiated with Boehringer Mannheim (now Roche Diagnostics) for the development of a cTnT assay. Interestingly, the original work (40)(41)(42) was not accepted for publication by Circulation or by Clinical Chemistry because the isoenzymes of CKMB were deemed to be the perfect marker and to remain the standard for AMI diagnosis.
the present
The cTnT assay has undergone several technical improvements and is now available as a chemiluminescence assay for the ELECSYS analyzer (Roche Diagnostics) and for a point-of-care test device (40)(43)(44)(45)(46). These tests have undergone thorough clinical investigations, not commonly performed in evaluation of novel diagnostic methods, and were also evaluated in large multicenter trials. These well-controlled clinical investigations confirmed the hypotheses derived from biology of cTnT. Thus, cTnT allows, in contrast to CKMB, the differentiation of skeletal and cardiac muscle injury. Most importantly, in chest pain patients, cTnT is increased in 30% of all patients despite normal CKMB (35). This minor increase in cTnT has been heavily disputed as "troponin diseases" or "troponin bumps". However, consistently, all multi- and single-center trials indicate the prognostic impact of theses minor increases in cTnT (47). Furthermore, it was observed in large clinical trials that these patients derive significant benefit from potent platelet inhibition and immediate percutaneous coronary interventions (47). This convincing evidence of the significance of microinfarction has led to the redefinition of AMI by committees of the European Society of Cardiology (ESC) and American College of Cardiology (ACC) (48). Today, cardiac troponin measurements serve as the "standard" for diagnosis of AMI, representing a paradigm shift in the way an AMI is detected.
Clinical investigations also revealed that increases in cTnT may result not only from ischemic but also from any myocardial cell injury. The diagnostic and prognostic power of cTnT is well described in patients with pulmonary embolism, myocarditis, toxic cardiac damage, and end-stage renal disease (49)(50)(51). Thus, it is the clinical circumstance that determines the specific diagnosis, whereas cTnT indicates severe and, most probably, irreversible myocardial cell injury.
the future
The detection limits of all troponin assays do not yet allow the demonstration of normal cardiac troponin in healthy controls. Thus, the analytical characteristics of the cardiac troponin assay determine its decision limits and, consequently, the diagnostic classification of an individual patient. For comparability and standardization, the joint committee of the ESC and ACC defined myocardial infarction as any cardiac troponin concentration exceeding the 99th percentile of a normal reference group that has <10% imprecision in the assay (48). Clinical trials and registries suggest that a lower decision limit would improve risk assessment and the number of patients classified as suffering from AMI (52)(53)(54). Because the current precision requirements at the cutoff are not met by the majority of commercial troponin assay, it will be a major effort to improve assay sensitivity and precision even further to finally detect normal concentrations of cTnT. When this goal is achieved, substantial work must be done to investigate the clinical implications and diagnostic power of such very minor increases.
Remembering CK-MB and Troponin I
—Jack H. Ladenson
I have been asked to comment on the cardiac biomarkers CK-MB mass and cardiac troponin I (cTnI) on the occasion of the 50th anniversary of Clinical Chemistry and the role my laboratory had in their evolution. I would first like to apologize to any individuals whose contributions I do not note, as this field is vast. A Medline search from 1966 to the present showed 23 661 different articles whose keywords included creatine kinase or troponin.
The major use of cardiac biomarkers until very recently has been the detection of myocardial infarction (MI). The rationale of using the measurement of a protein in blood for this purpose is straightforward. The myocyte is the major cell in the heart, and the heart’s purpose is to pump blood. Because myocytes essentially cannot be regenerated, if heart cells die, then cardiac function has a high probability of being impaired. When the cell dies, the proteins inside the cell will be released, with proteins in the cytoplasm leaving the cell more rapidly than ones in membranes or fixed cell elements. The most sensitive markers should be those in highest abundance in the cell, and because the major function of the heart is contraction, the proteins involved in contraction and producing the energy to support it should be good candidates for biomarkers in blood. If such proteins have cardiac-specific forms, then specificity might be achievable as well as sensitivity.
Although not necessarily for the above reasons, this is how the tests used for biochemical detection of heart damage have evolved. The first practical test was the measurement of transaminases described in 1954 (55). CK later supplanted aspartate aminotransferase, and for many years CK and lactate dehydrogenase and their isoenzymes were the mainstay of biomarkers for MI. My involvement in this field began in the era of measurement of CK and lactate dehydrogenase isoenzymes.
My first experimental experience with these enzymes came via a biochemical teaching laboratory for medical students and involved David Bruns, the current editor of Clinical Chemistry, then a resident at Barnes Hospital. We had set up an example of enzyme reactions using the assay of CK in patient serum. One of the students looking at the chart recorder (remember those) said, "This thing isn’t working", or something to that effect. It turned out that the sample came from a patient with metastatic cancer and had a highly prolonged lag phase in the enzymatic reaction for detecting CK (56). Around the same time, I had also developed a friendship with Gabor Szasz from Giessen, Germany, who had done beautiful analytical work in optimizing the measurement of CK activity (described in a series of six papers published in this journal between 1976 and 1979). Because we had published our experience with the lag phase in the CK reaction noted above and some follow-up articles, Gabor always thought I must have his passion for the assay. In addition, in the mid- to late 1970s, one of the best enzymologists I have ever known, Dave Dietzler, joined our division, and it was hard to avoid his love of enzymology.
In the 1980s, Washington University became involved in an academic-industrial research arrangement to use the then-new technique of making monoclonal antibodies (57) for use in diagnostics and therapy of human disease. The original agreement was with both the Monsanto Company (now part of Pfizer) and Mallinckrodt (now part of Tyco Healthcare). To avoid conflicts among the industrial partners, there was a clear delineation of project areas, such as lipoproteins, enzymes, and coagulation, with proportional funding of core facilities such as a monoclonal antibody fusion center and an immunoassay center. The agreements were modified after a few years, with Mallinckrodt ending their involvement and Monsanto (Pfizer) broadening theirs into efforts more targeted at novel drug discovery. This arrangement between Monsanto (Pfizer) and Washington University continues today.
The work that led to the development of a monoclonal antibody reactive only with the CK-MB isoenzyme (Conan MB) and quantitative tests for CK-MB (17) began as one of the Monsanto projects. The work evolved rapidly after Hemant Vaidya and Sharon Porter joined our laboratory as a postdoctoral fellow and research technologist, respectively. The patent describing the use of Conan was granted in the US on March 27, 1990 (US patent number 4,912,033).
The name of the antibody came about as follows. We had several proteins and antibodies we were working with in the mid-1980s. A system of numbering was in use, which allowed tracing a variety of technical variables, such as the antigen preparation, fusion number, ELISA plate, and well number. When reviewing data concerning the various antibodies at laboratory meetings, it became difficult to follow which was which. Vonnie Landt (nee, Maynard), my research associate, was asked to give the antibodies names, to make it easier to follow the various experiments. Various names, such as Jack, Hem, Yvonne, and Fred, were used. The antibody that was identified to have the unique reactivity for CK-MB was then named Conan. Vonnie was known to be a fan of the Conan movies made by Arnold Schwarzenegger (current Governor of California), and it is "presumed" that this was the source of the name.
Based in part on the royalties earned by a nonexclusive license of the Conan MB antibody, two endowed chairs in Laboratory Medicine have been established at Washington University: the Oree M. Carroll and Lillian B. Ladenson Chair of Clinical Chemistry, created in 1993, which I still occupy (Oree M. Carroll was my father-in-law and Lillian B. Ladenson was my mother), and the Conan Professor of Laboratory Medicine created in 2002. This chair is currently vacant but was occupied by Sam Santoro, now Chair of Pathology at Vanderbilt School of Medicine (Fig. 2
).
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Conan MB, a conformationally sensitive antibody, was first used as a capture antibody for CK-MB, followed by CK activity measurement (17). Later it was paired with an antibody to the B subunit of CK-MB (named Mr. Bill; remember the television show "Saturday Night Live") to make a convenient, two-site mass immunoassay. The assay was first made commercially available by Dade International in 1988, and shortly thereafter by several other manufacturers. Its arrival was timely because the demand for CK-MB analysis was climbing rapidly, probably as a result of the advent of new therapeutic interventions for MI, such as streptokinase (Food and Drug Administration-approved in 1982) and tissue plasminogen activator (Food and Drug Administration-approved in 1987). We were doing multiple runs of CK-MB activity by electrophoresis, followed by overlay with substrate, and the laboratory people and the cardiologists were getting frustrated with each other. A side bar of the use of a facile and sensitive CK-MB mass assay was increased realization that CK-MB was not totally heart-specific. When performing electrophoresis, we had to dilute samples to an activity of 300 U/L to do the electrophoresis and did not detect CK-MB very often from non-MI patients. With direct analysis of CK-MB irrespective of the total activity, it was quickly realized that CK-MB can come from skeletal muscle, and we then realized that this occurred in a variety of situations as a result of skeletal muscle regeneration. The CK-B gene is an embryonic gene, and its production is increased in regenerating skeletal muscle. Recognition of the occasional lack of specificity of CK-MB for MI accelerated the desire for a more specific test than CK-MB.
We began looking at troponin I and myosin light chain as possible cardiac biomarkers in the 1980s. Interest in troponin I was prompted by the work of Cummings et al. (58) and myosin light chain by the reports of Katus et al. (41). There was no cardiac specificity with the myosin light chain assay developed by Emad Daoud in my laboratory, and it now appears that the results attributed to myosin light chain by Katus were probably attributable to cTnT, and this was developed into a useful cardiac biomarker (40). The work with cTnI, performed in part by a postdoctoral fellow in the laboratory, Geza Bodor, was successful (59), and these antibodies are now used in the various cTnI assays available through Dade-Behring, Inc. Around the same time, David Silva, a postdoctoral fellow, developed a two-site immunoassay for myoglobin (60), which has also been widely used.
Our clinical studies with cTnI led to two surprises. One was the time course after MI. The initial appearance of cTnI over time after an MI is similar to that of CK-MB, as is the initial decrease after the peak value. CK-MB returned to baseline after a few days (61), but there was some increase of TnI values for 7–10 days, although the half-life of TnI or the entire troponin complex is similar to that of CK-MB. The current thought about this phenomenon is that the initial increase in cTnI is attributable to release of cytosolic TnI and that the extended increase is attributable to TnI release from the myofibrils as the repair process occurs in the heart. This turned out to be an advantage because it allowed troponin testing to replace lactate dehydrogenase isoenzymes for detecting cardiac damage in patients presenting late after their MI. Lactate dehydrogenase isoenzyme testing, in hindsight, was not a very good test in patients without electrocardiographic evidence of MI (61). The second surprise was the prolonged increase in troponin over time in some patients with angina. Initially, some of us thought that this represented delayed presentation of patients with MI, but it is now clear that a subset of patients with a clinical diagnosis of unstable angina have low persistent increases in troponins and that this subset of patients has a prognosis similar to that of patients with non-Q-wave MI.
The troponin work after assay development was actually very challenging because we thought we might have a test that performed better than the gold standard of CK-MB. Fortunately, I had excellent clinical researchers as collaborators. In our Cardiology group, Al Jaffe and Victor Davila-Roman devised a means of detecting MI by serial echocardiograms, which kept us out of the logic loop of simply comparing two blood tests and trying to prove one is better by shouting louder.
So what is next? Now that troponin is established as a highly specific, if not absolute, indicator of heart damage, standardization efforts are ongoing at both the laboratory and clinical level, and often this means something new is due. Regarding damage indicators, I think it unlikely that troponin will be replaced for awhile. A better marker would be one that was equally sensitive and specific but was altered in blood much sooner. Because the major reason for the delay in the increase in blood values is blockage of blood flow, it seems hard to visualize how to readily get around this problem with any blood test. Given the high diagnostic power of more invasive cardiovascular tests such as stress tests and angiography, a simple blood test that indicates problems a year or so before a possible MI would be a very useful screening test. There is no shortage of candidates, somewhat reminiscent of the 1981 survey of 246 suggested coronary risk factors (62). At any rate, time and careful evaluation will sort out what will be useful for medical practice.
The 1980s and 1990s were a lot of fun for those of us involved with cardiac biomarkers, and I am sure that the next 20 years will be for those who follow. One of those who might choose to follow is shown in Fig. 3
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Acknowledgments
J.H.L. and Washington University may receive income on licensing of technology discussed in this report (CK-MB, troponin, myoglobin). In addition, J.H.L. is a consultant to the Dade-Behring Co. and receives research funding from Dade Behring, Inc. The terms of the above arrangements are being managed by Washington University in accordance with its conflict of interest policies.
Footnotes
1 Nonstandard abbreviations: CK, creatine kinase; MBCK and CK-MB, creatine kinase MB isoenzyme; AMI, acute myocardial infraction; cTnT and cTnI, cardiac troponin T and I, respectively; ESC, European Society of Cardiology; and ACC, American College of Cardiology. 
References
-ketobutyrate by human serum. Nature 1960;188:1110-1111.[CrossRef][Medline]
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