Troponins: The New Cardiac Markers of Choice

In the hierarchy of indicators for myocardial infarction, troponins have leapfrogged creatine kinase-MB and myoglobin, says the author. He explains why, breaking down the chemistry that ties troponins to myocardial necrosis and highlighting what physicians should know about troponin assays and their clinical application.

By James M. Gillard, MD, FACEP, FAAEM

Current guidelines for the evaluation of patients with suspected acute coronary syndromes recommend obtaining markers of myocardial necrosis as part of the initial work-up. Troponins, creatine kinase-MB (CK-MB), and myoglobin are the markers most frequently measured. Because of their higher sensitivity and specificity, troponins are now preferred as the markers of choice over CK-MB and myoglobin. This article will review what every clinician should know about the troponins—what they are, what they do in the heart, and how they are assayed.
 

PROBLEMS WITH CK-MB AS A MARKER

Creatine kinase is an enzyme that catalyzes the reaction of creatine phosphate and adenosine diphosphate in muscle, yielding adenosine triphosphate (ATP) and creatine. Along with magnesium, ATP provides the energy for muscle contractions. Creatine kinase-MB, an isoform of CK, is found in high concentrations in heart muscle and is released when myocardial cells undergo necrosis. Until recently, CK-MB was considered the best marker to diagnose myocardial infarction (MI). Unfortunately, CK-MB is also found in smooth muscle, bone, and the brain. Abnormalities or injury to these other organ systems may give a false indication of myocardial injury.

Another problem with the CK-MB marker is that it is present in the blood of healthy individuals in a wide range of normal levels. It takes substantial myocardial damage to raise circulating CK-MB to pathologic levels. To be certain that myocardial necrosis has in fact occurred, one needs an absolutely high CK-MB level or a certain ratio of the CK-MB to the total CK in a blood sample.

Myoglobin, a heme-containing protein found in muscle cells, is not only one of the earliest markers for myocardial injury to appear but a relatively sensitive one as well. It is released as early as two hours post-injury and does not drop to undetectable levels for about 12 hours. Unfortunately, many conditions can provide a positive myoglobin test. Since myoglobin is found in all types of muscle, its absence is an important indicator for ruling out, rather than ruling in, myocardial injury.
 

TROPONINS: SUPERIOR SENSITIVITY AND SPECIFICITY

Because of their superior sensitivity and specificity compared to CK-MB, cardiac troponins are now considered the best marker for myocardial cell destruction. The mere presence of troponins in the blood indicates some type of myocardial injury. The injury could be a frank MI or ongoing microinfarction from platelet emboli being released from an ulcer in a coronary artery. Patients with microinfarction are at high risk for a complete coronary occlusion; the condition is associated with high mortality and fatal outcomes usually occur within a matter of months. In the past, it could not be detected by CK-MB assays, simply because microscopic damage does not raise CK-MB to levels outside of its normal range. More extensive myocardial necrosis is required to raise CK-MB to detectable levels.

Cardiac troponins are detectable in the blood at around the same six-hour post-injury time as CK-MB. In contrast to CK-MB, however, troponins remain detectable for up to 14 days, compared to four or five days with CK-MB. Troponins have also been reported to be elevated in myocarditis, myocardial contusion, myocardial toxicity from chemotherapy, and cardiac transplant rejection.

Troponins have by no means replaced myoglobin and CK-MB assays. The results from all three markers can provide valuable information. Understanding the timing of the rise and fall of these markers makes it possible to differentiate acute injury, subacute injury, and reinjury. The use of lactate dehydrogenase and aspartate aminotransferase, which were once assayed to help diagnose and time myocardial injury, is no longer recommended by current guidelines.
 

STRUCTURE OF CARDIAC MUSCLE

To understand the role troponins play in cardiac activity, it is necessary to review the structure of cardiac muscle. A single muscle fiber is really a large cell formed through the fusion of several smaller cells. These fibers contain several nuclei. Each fiber has its cytoplasm packed full of myofibrils, which contain the contractile elements of the muscle. Cardiac muscle differs from skeletal muscle in that its cells are smaller and its sarcoplasmic reticulum less well developed. The contractile elements within the myofibrils, however, are similar in both cardiac and skeletal muscle. Under the microscope, an isolated cardiac or skeletal muscle fiber has a characteristic banding appearance, which is why they are both termed striated muscle.

Myofibrils are composed of repeating assemblies of thick and thin filaments. Both of these filament types are organized into a highly structured configuration. The smallest single contractile unit of overlapping strands of thin and thick filament is called the sarcomere. In areas of overlapping thick and thin filaments within a sarcomere, dark bands are visible.

At the molecular level, the thick filaments are made up of myosin, a relatively large protein composed of six polypeptide chains with ATP activity. Its ability to split the high-energy phosphate bonds from ATP is what supplies the energy needed to drive muscle contraction.

The thin filaments are made up of repeating units of the globular protein actin. Wound through the clefts formed in this long strand of actin molecules is the regulatory protein tropomyosin, a rod-shaped molecule that forms an alpha-helical strand through seven units of actin. Tropomyosin prevents actin's interaction with the thick filament's protein, myosin, by simply getting in the way. It is the transient ATP-activated crossbridging between actin and myosin that causes muscle contraction.
 

THE TROPONIN COMPLEX

Tropomyosin's interaction with the contractile proteins actin and myosin is regulated by the troponin complex, which is made up of three distinct subunits: troponin-I, troponin-T, and troponin-C. The troponin-I and troponin-T of cardiac muscle differ structurally and therefore antigenically from their skeletal muscle counterparts. Troponin-C is the same in both types of muscle, making it an unsuitable marker for detecting cardiac muscle injury.

Troponin-C is the calcium ion receptor. Troponin-I is the inhibitory subunit that shuttles between tight binding to calcium-bound troponin-C and tight binding to actin when there is no calcium bound to troponin-C. Troponin-T acts as a kind of cement, binding itself to tropomyosin, troponin-C, and troponin-I.

What actually happens during the cardiac cycle is a very complicated sequence of events involving transient binding of calcium ions to troponin-C, transient binding of phosphate to serine residues in troponin-I, and the actual movement of tropomyosin within the grooves of actin. The final result is that the troponin complex, through transient conformational changes, inhibits or allows the direct interaction of actin and myosin, thus allowing crossbridging between these molecules to take place. In short, the presence of calcium releases troponin-I's inhibition and allows actual muscle contraction.

Recent information shows that crossbridging is not a simple "all or none" phenomenon. In reality, it is a dynamic state of weak and strong interactions. There is also an array of pumps, exchangers, and calcium channels that fine-tune these interactions. It is through knowledge of these complex phenomena that cardiac stunning, reperfusion injury, and heart failure may be better understood.
 

QUANTITATIVE ASSAYS

Today, most cardiac marker determinations employ an immunoassay that uses an antibody directed against a certain area or epitope of the targeted protein's molecule. These are usually monoclonal or polyclonal rabbit, mouse, or goat antibodies. These animal antibodies may be tagged with a dye or another indicator to detect a specific cardiac marker. Even though these assays are very sensitive, they are still subject to errors.

The presence of troponin-I or troponin-T can identify chest pain patients at high risk for mortality who could possibly benefit from timely angiographic evaluation and glycoprotein IIb/IIIa inhibitor therapy. Therefore, the clinician should be aware of the differences between the troponin assays available.

Several quantitative assays are on the market and are used in hospital laboratories. These are mass immunoassays, and their results are usually reported in micrograms per liter or nanograms per milliliter concentrations. This gives the impression of an accurate measurement of the weight of the troponin per known volume. This is not actually the case. Quantitative troponin laboratory results are not really an accurate measurement of troponin concentration. They do, however, indicate the troponin's relative presence in a sample. There is no normal range of troponin levels in blood. It is either present or it is not. The central laboratory's reported range simply gives a threshold for troponin's detection, valid only for its particular assay. This is done to dilute out artifacts and other sources of false positives. Any number above the threshold may be considered a reliable detection of cardiac troponin.

In myocardial necrosis, troponins are initially released as a large ternary troponin-I/troponin-T/troponin-C complex, along with various degraded forms of these proteins. The serum of a patient with myocardial injury reveals progressive degradation of the large complex, with smaller binary complexes of troponin-C and troponin-I, free troponin-T, or small peptide fragments of any of the troponins. Since there are proven, multiple troponin molecular changes taking place within the blood of a post-myocardial injury patient, it is not possible to put an accurate mass measurement on any of this.

When the same reference sample is run on several of the quantitative troponin assays that are commercially available, a big difference may be found in the reported concentrations. Differences of 20- to 100-fold have been reported, even when assays were run on the same sample. There may also be a positive report on one quantitative assay and a negative report on another, even when using the same sample. Some of these lab assays can provide false positive results. Usually this is because of the presence of heterophile antigens (patient antibodies against the animal antibodies in the assay), fibrin strands, rheumatoid factor, or other substances that contaminate the sample. The difficulty is compounded by the fact that each immunoassay uses a different antibody to target a different epitope on the troponin molecule. If the targeted epitope is hidden by complexation, is changed by chemical processes occurring within the sample, or is removed from the molecule by enzymatic degradation, it can be missed altogether.

Quantitative assays should not be considered the last word with troponins. Reports in the literature provide a cutoff point, based on blood concentrations of troponins, for distinguishing between unstable angina and non-ST-elevation myocardial infarction (NSTEMI). This determination was only speculative, created by certain authors who found the cutoff point was valid for them only when using a particular assay. With the newer consensus definitions, any troponin detected now can place previously diagnosed unstable angina patients into the acute NSTEMI category.
 

STANDARDIZING THE TROPONIN-I TESTS

There is an ongoing effort to attempt to standardize all the different troponin-I tests. This standardization will not simply be a recalibration of the assays, since many of the assays use an entirely different antibody system to look at a different part of the targeted antigen. There is no currently accepted standard for all the commercially available troponin-I assays.

At the present time there is only one assay on the market for troponin T. As a result, there are no other assays that can be used to compare its accuracy. Clinically, the presence of either troponin-I or troponin-T at detectable levels means virtually the same thing. A possible exception in the past was that troponin-T had been shown to be abnormally elevated in renal failure, in the absence of myocardial necrosis. This was reported with the earlier assays and probably is not much of a problem now, using the second- and third-generation systems. However, before the unexpected presence of troponin is attributed to a false positive, it is prudent to consider that myocardial necrosis might be taking place, as a complication of another illness. This is especially true when using the new, more reliable assays available today.

Once a blood sample is drawn from a patient who has had an acute MI, there are structural changes in the troponins that can take place in serum as a result of proteolytic cleavage of the peptide bonds. These changes take place in addition to those that have been demonstrated to occur in an ischemic myocardium, through the phosphorylation of amino acids and the oxidation of sulfhydryl groups. A sample sitting at room temperature in the laboratory for an extended period of time is subject to a loss of detectable troponin. All currently available troponin assays use an antibody directed toward a specific, three-dimensional part of a targeted molecule. Any change in this target could affect the assay's accuracy.
 

USE OF POINT-OF-CARE DEVICES

A fast laboratory turnaround time is important for cardiac markers. Current guidelines from the American Heart Association and the American College of Cardiology specify that results should be available within one hour and preferably within 30 minutes. The time it takes to receive central laboratory results in the emergency department has frequently been an issue between emergency physicians and clinical pathologists. This observation explains why the use of point-of-care devices to measure important values has become so popular. Point-of-care devices for cardiac markers take their samples directly from the patient, with essentially no time for a sample to degrade in a tube. Also, the sample does not have to leave the patient's side, reducing the risk of a mix-up in a busy laboratory. The early detection of high-risk patients with bedside marker devices is now well documented in the medical literature.

At least three manufacturers have developed bedside devices that detect CK-MB, myoglobin, and troponins on the same platform. One group of researchers is currently developing a point-of-care, cardiac-specific myoglobin test. With its proven sensitivity and rapid appearance after myocardial injury, cardiac-specific myoglobin might someday replace the troponins as the preferred marker for myocardial necrosis.

In the evaluation of an emergency department patient, the presence of troponins at detectable levels is significant. If this can be confirmed with a qualitative bedside device, relatively free from false positives, that can be as good as, if not better than, many of the central laboratory assays. Qualitative measurements for emergency department patients should not be confused with serial marker determinations used for in-patients. For in-patients, serial quantitative determinations using the same assay remain essential.
 

PROPOSED DIAGNOSTIC GUIDELINES

New guidelines for the diagnosis of acute MI have been proposed by the European Society of Cardiology and the American College of Cardiology. These guidelines state that to confirm the diagnosis there must be a characteristic rise and gradual fall of troponin or CK-MB, along with at least one of the following: ischemic symptoms, pathologic Q waves on ECG, changes on ECG indicative of ischemia (such as ST-elevation or depression), or history of coronary artery intervention. This is replacing the almost four-decades-old World Health Organization's criteria for the diagnosis of acute MI, which specified that two out of the following three criteria needed to be present: classic symptoms of chest pain, pathologic Q waves on ECG, and increased plasma enzyme activity. The revised definition has been adopted because the former criteria were neither sufficiently sensitive nor specific.

A recent report based on a large national sampling of Medicare admissions for acute MI from the Centers for Medicare and Medicaid Services (formerly the Health Care Financing Administration) found a consistent increase in in-hospital mortality associated with the presence of troponin alone, irrespective of CK-MB's presence. This probably could be explained by the fact that many patients in the past were diagnosed as having a non-Q-wave infarction based solely on an elevated CK-MB. However, since elevated CK-MB can result from a noncardiac source, many of these patients were most likely misdiagnosed. On the other hand, high-risk patients in the past were ruled out for MI using CK-MB assays when, in fact, they were experiencing microscopic myocardial necrosis. These patients did not demonstrate enough CK-MB release to raise this marker above the wide ranges of normal. This finding underscores the importance of determining whether or not a new chest pain patient is simply troponin-positive or -negative.
 

REVISED CRITERIA

Hamm and Brunwald recently revised Brunwald's criteria for class IIIB unstable angina, which is angina at rest within the past 48 hours. This is now divided into troponin-positive or troponin-negative subclasses. Troponin-positive class IIIB carries a 20% mortality within 30 days while the troponin-negative subclass carries a risk of less than 2%. Thus, a marker is now available that can quickly identify a high-risk patient, even one who has a normal ECG.

Although cardiac markers are useful tools in the evaluation of patients with suspected acute coronary syndrome, there is still no substitute for clinical judgment. Any test result must be evaluated in the context of the patient's history, physical examination, risk factors, and current standards of care. A negative result is not a green light to send a suspected high-risk patient home. It is important to remember that all cardiac markers require a significant time after injury to become positive and that they only show that some form of myocardial damage has already taken place.

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