Understanding Cardiac Biomarkers

Emerg Med 40(9):28D, 2008

By James M. Gillard, MD, FACEP, FAAEM

Troponins, myoglobin, and creatine kinase can help you make timely decisions about your patient’s true situation. Here’s how to use these enzymes, peptides, and proteins to clarify the clinical picture.

When a patient presents with suspected acute coronary syndrome, the immediate task is to assign him or her to one of four categories: noncardiac diagnosis, chronic stable angina, possible acute coronary syndrome, and definite acute coronary syndrome. To do that, American College of Cardiology (ACC)/American Heart Association (AHA) guidelines recommend immediately taking the patient’s history, performing a physical examination, getting a 12-lead ECG, and doing an initial cardiac marker determination.

Cardiac biomarkers are changing the way we define the criteria for illnesses, as well as how we set the standards for treating them. For example, the World Health Organization’s (WHO’s) nearly 40-year-old criteria for the diagnosis of acute myocardial infarction (AMI) have recently been replaced because they were not sufficiently sensitive or specific. The original criteria required two of the following: typical symptoms of chest pain, ECG evidence evolving into the development of Q waves, and increased plasma enzyme activity. The new definition of AMI by the European Society of Cardiology, WHO, and ACC specifies an elevated cardiac troponin, along with new left bundle branch block, pathologic Q waves on the ECG, ECG changes indicative of ischemia (ST elevation or depression), coronary artery intervention-related marker elevation, or positive imaging for a loss of viable myocardium.

Similarly, Hamm and Brunwald recently revised Brunwald’s criteria for unstable angina. For Brunwald class IIIB, which is angina at rest within the previous 48 hours, they now divide it into troponin-positive and troponin-negative subclasses. A patient in the troponin-positive class IIIB has a 20% risk of dying within 30 days, while one in the troponin-negative subclass has a less than 2% risk. Urgent care physicians now have a marker that can quickly identify one of these high-risk patients, even in the face of a normal ECG.

This article reviews what the current cardiac markers actually are, what they do, and how they are assayed. More important, we’ll discuss what they can tell you and how you can use this information to make important clinical decisions.

MOST COMMON CARDIAC MARKERS

The most commonly assayed cardiac markers—creatine kinase (CK), myoglobin, and troponin—all have unique benefits and drawbacks, but separately or together, they can help you clarify your diagnosis.

Creatine kinase. Creatine kinase is released when any muscle cell undergoes necrosis, which makes it a specific and sensitive marker for any muscle injury. Also known as creatine phosphokinase, CK is an enzyme that catalyzes the muscle reaction between creatine phosphate and adenosine diphosphate (ADP), yielding adenosine triphosphate (ATP) and creatine. Adenosine triphosphate, along with magnesium, provides the energy for muscle contraction. Creatine phosphate, the substrate of CK, is a rather unstable compound that is autohydrolyzed to creatinine. Although creatinine has no real function, its predictable concentration in muscle, as well as its known rate of excretion by the kidney, makes it a valuable marker for renal function measurements. 

The CK enzyme consists of an M (muscle type) subunit and a B (brain type) subunit, which combine for three possible isoforms: CK-MM, CK-BB, and CK-MB. The latter is found in high concentrations within heart muscle but also (in lower concentrations) in smooth and skeletal muscles, which can give a false indication of myocardial injury. In fact, there is now evidence that some “non-Q-wave MIs” (now called “non-ST-elevation MIs”) diagnosed with CK-MB alone never really took place.

Myoglobin. Myoglobin is a heme-containing protein found in all muscle cells—smooth, skeletal, and cardiac. It reversibly binds oxygen passed from hemoglobin in the blood to the muscle cells. It then gives its oxygen up to the cytochrome oxidase of muscle mitochondria. Since myoglobin is a relatively low-molecular-weight protein, it readily diffuses through a damaged muscle’s cell membranes (see box below).

A relatively sensitive marker for myocardial injury, myoglobin is probably the earliest to show up—it is released as early as two hours post-injury and drops to below detectable levels in a little more than 12 hours. Used together with cardiac troponin and CK-MB assays, the myoglobin assay can give valuable clinical information as to the time of injury and the presence of reinjury.

Cardiac troponin. Although CK-MB, myoglobin, and cardiac troponin are all frequently assayed, cardiac troponin, with its better sensitivity and specificity, has become the marker of choice. The troponins are made up of three proteins: troponin C, troponin I, and troponin T. In contrast to enzymes, which may be found free in a cell, troponins are regulatory proteins that are normally tightly bound to the contractile apparatus of muscle proteins, with only a minimal amount in a cytosolic pool. For them to be found in the blood, pathological disruption of the muscle cells must take place first.

The troponin T and troponin I of cardiac muscle differ structurally, and therefore antigenically, from their skeletal muscle counterparts. This is important in that skeletal muscle troponin won’t be detected by cardiac troponin assays. Troponin C is antigenically the same in both types of muscle, making it unsuitable for a cardiac biomarker.

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

THE PEPTIDES

In addition to CK-MB, myoglobin, and troponin, the natriuretic peptides can help reveal what’s going on in the heart. More than 40 years ago, researchers using electron microscopy noticed secretory granules in heart muscle, suggesting a possible endocrine function of the heart. In 1981, Adolfo de Bold and colleagues injected extracts of atrial tissue into rats, resulting in a rapid renal excretion of sodium and water. The 28-amino-acid peptide causing this phenomenon, atrial natriuretic peptide (ANP), was later isolated and purified. Another natriuretic peptide with similar properties was later found in porcine brain; this was named brain natriuretic peptide (BNP). Since it is now known that BNP is mainly produced in the heart, the term B-natriuretic peptide, rather than brain natriuretic peptide, is most often used. C-natriuretic peptide has also been isolated, but BNP is the one used now as a biomarker for congestive heart failure.

Both BNP and proBNP (the precursor of BNP) are found in high concentrations in the blood of patients with CHF. However, since other conditions can raise serum levels of these markers (not to mention rare errors with any mass assay), a clinical evaluation is very important when using them to evaluate a patient.

B-natriuretic peptide and proBNP have found their place with the other biomarkers to determine risk factors. A recent study demonstrated that elevated proBNP in patients with stable coronary artery disease and preserved ventricular function is linked to an increase in the incidence of cardiovascular mortality, heart failure, and stroke. No increase in the incidence of MI was found, however. Both BNP and proBNP demonstrated an increased risk of heart failure.

B-natriuretic peptide determinations are also regarded as a good tool for evaluating possible heart failure in a dyspneic patient with chronic obstructive pulmonary disease, because BNP is released in response to the stretching of cardiac atrial muscle, usually due to ventricular dysfunction or volume overload. The ability of the ventricles to develop this function seems to appear in a failing heart. 

A BNP of less than 100 ng/L on immunoassay makes heart failure unlikely. In patients with coronary artery disease, obstructive sleep apnea, stroke, or diabetes, very high levels of BNP may identify those with a higher risk of death. It is difficult to establish a normal range, though, for these natriuretic peptides. For example, women, older adults, and patients with renal insufficiency seem to have higher normal values. Obese patients seem to have a lower normal range.

It is interesting to note an apparent endocrine paradox in patients with heart failure. Although many have elevated levels of measurable BNP, they still demonstrate sodium retention and edema. To make this more confusing, many of these patients will respond to the therapeutic infusion of synthetic BNP. This can be explained by the fact that immunoassays can detect the biologically inactive precursors. There also appears to be a required maturation of BNP before it becomes biologically active.

LIMITATIONS OF TROPONIN ASSAYS

Since the presence of troponin I or troponin T can identify high-risk patients who could probably benefit from timely percutaneous coronary intervention and glycoprotein (GP) IIb/IIIa inhibitor therapy in an emergency department, urgent care physicians should be familiar with the different cardiac marker assays available.

Several quantitative troponin assays are used in hospital central laboratories. These are mass immunoassays and their results are usually reported in microgram-per-liter or nanogram-per-milliliter concentrations. This gives the impression that they are accurately measuring the weight of something per known volume. This is not really the case.

In myocardial necrosis, troponins are initially released as a large ternary troponin I-troponin T-troponin C complex, along with various already degraded forms of these proteins. What you find in the serum of a patient undergoing myocardial injury is the progressively degrading large complex, along with a smaller binary complex of troponin C and troponin I, free troponin T, or small peptide fragments of any of the above. Since there are proven, multiple troponin molecular changes taking place within the blood of a post-myocardial injury patient, one cannot really put an accurate mass measurement on any of this. These observations are still only part of the complexity of the situation.

When you test the same reference sample using several of the different quantitative troponin assays commercially available, you get up to a 100-fold difference in reported concentrations per milliliter. There also exist a number of positive reports on one quantitative assay with a negative report on another, even when using the same sample.

Some of these specific lab assays can give false positives, usually because the sample is contaminated by heterophile antigens (patient antibodies against the animal antibodies in the assay), fibrin strands, rheumatoid factor, or other substances (such as heparin). This problem is compounded by the fact that each different 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 ongoing chemical processes within the sample, or is cleaved from the molecule by enzymatic degradation, it could be missed altogether.

Clinically, the presence of either troponin I or troponin T at detectable levels means the same thing. A possible exception is in cases of renal dysfunction. Troponin T has been shown to be abnormally elevated in renal failure, in the supposed absence of myocardial necrosis. Since patients with renal failure are at high risk for cardiovascular mortality, anyway, some experts recommend evaluating an elevated troponin T or troponin I as if the renal failure weren’t there. This can present a problem, however, since renal function is a major factor when using angiographic contrast dyes to study these patients in the cath lab. 

Quantitative assays are far from the last word. There have been reports in the literature that give a cutoff for differentiating between unstable angina and non-ST-elevation MI, based on blood concentrations of troponin. This assumption was just a speculative one created by some authors that 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 non-ST-elevation MI category. 

Efforts are under way to standardize all the different troponin tests. This standardization won’t 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 a recombinant DNA-synthesized human troponin I that can be used to standardize the many assays available. As expected, each assay gives a different value. Since this isn’t the way the molecule is really found in pathologic serum, not being stabilized by complexation, its half-life is very short. At present only one assay is on the market for troponin T. As a result, there are no other assays with which to compare its accuracy.

However, since troponins should not be present in the blood of healthy individuals, there is a bottom line. Any reliable qualitative detection of troponin I or troponin T is of extreme clinical significance, especially when the quantitative laboratory assays don’t correlate well with one another.

TURNAROUND TIME MATTERS

Once a sample is drawn from a patient, structural changes in the troponins can take place in serum, mainly through proteolytic cleavage of the peptide bonds. A sample sitting for an extended time at room temperature in the laboratory, waiting to be analyzed, is subject to a loss of detectable troponin. These changes can take place in addition to those occurring in ischemic myocardium, through the phosphorylation of amino acids and the oxidation of sulfhydryl groups. Moreover, it’s important to bear in mind that all currently available troponin assays use an antibody directed towards a specific, three-dimensional part of a targeted molecule. Any change in this target can affect the assay’s accuracy.

A rapid laboratory turnaround time for cardiac markers is essential, especially when the current ACC/AHA guidelines (with participation by the American College of Emergency Physicians and other national and international organizations) specify that results should be available in less than 60 minutes. Their guidelines also state: “Point-of-care systems, if implemented at the bedside, have the advantage of reducing delays due to transportation and processing in a central laboratory and can eliminate delays due to the lack of availability of central laboratory assays at all hours.” Point-of-care devices take their samples directly from the patient, with essentially no time for a sample to degrade in a test tube. A further advantage is that if the test is completed at the bedside, there is no risk of a sample mix-up in a busy lab. 

The central laboratory assays usually have some price advantage over bedside determinations, even though the bedside assays are not really very expensive. However, when viewed in the total context of the cost of care, the benefits of the early institution of myocardial salvage and the avoidance of unnecessary admissions, the bedside devices could create significant savings. Nonetheless, it is yet to be determined whether these new tests can reduce the 4% to 7% missed MIs being sent home from our nation’s emergency departments.

The qualitative measurements for urgent care patients should not be confused with the serial marker determinations used on hospital inpatients. For inpatients, serial quantitative determinations—using the same assay—remain essential. A single determination only shows injury more than several hours old. Serial determinations are needed to rule out an injury occurring now.

Moreover, be careful not to compare a value from a quantitative bedside or point-of-care device with a central laboratory value for a later sample draw. For serial mass assay measurements, the same assay must be used, because of the wide variation possible when the same sample is tested using the many different commercial assays available.

HIGH TROPONIN, HIGH RISK

Just the presence of cardiac troponins identifies someone who has had some type of myocardial injury. This could be a frank MI or ongoing microinfarction from platelet emboli being showered from an ulcer in a coronary artery. These latter patients are at high risk for a complete coronary occlusion and death within months. In the past, these patients went undetected by CK-MB assays, because microscopic damage doesn’t raise the CK-MB to levels outside of the normal range. A larger volume of myocardial necrosis is required to raise CK-MB to abnormal levels when compared with troponin. Cardiac troponin T and troponin I have also been shown to be elevated in myocarditis, myocardial contusion, myocardial toxicity from chemotherapy, microvascular ischemia from cocaine and other drugs, and cardiac transplant rejection.

In a report based on a large national sampling of Medicare admissions for AMI, the Health Care Financing Administration found a consistent increase in in-hospital mortality based on the presence of troponin alone, irrespective of the presence of CK-MB. 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 this elevation could have been from a noncardiac source, these patients weren’t really at high risk after all.

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 ongoing myocardial necrosis. These patients didn’t demonstrate enough CK-MB release to raise this marker above the wide range of normal. This finding underscores the importance of determining whether a new patient with chest pain is troponin positive or negative.

RISK STRATIFICATION

Troponin I and troponin T are powerful tools for risk stratification. Cardiac troponins have greater specificity and sensitivity than CK-MB, but they require the same four to six hours after myocardial injury to be reliably detected in the blood. The troponins can be detected for up to two weeks post-MI. Using the known timing of the rise and fall of these markers, it’s possible to differentiate between new injury, old injury, and reinjury.

When you’re using troponin to evaluate a patient, the mere presence of troponin at detectable levels is significant. If measured with a qualitative bedside device, relatively free from false positives, this can be as good as many of the central lab assays. Just because the lab will give you a number doesn’t guarantee it will be accurate. On the other hand, the reported lab value doesn’t really have to be accurate to be clinically meaningful if its value is above a known limit for myocardial necrosis.

Myoglobin, as noted, is the earliest and most sensitive marker for myocardial injury. A negative test is useful four to eight hours after the onset of symptoms to rule out myocardial necrosis. But because the same myoglobin is present in all types of muscle, many conditions can produce a positive myoglobin test. The absence of myoglobin is more important as a “rule out,” rather than its presence being a “rule in” for myocardial damage because of its poor specificity. There have been reports of a cardiac-specific myoglobin test, which combines a myoglobin assay with the detection of carbonic anhydrase III, an enzyme found in skeletal muscle but not in cardiac muscle. The presence of myoglobin and the absence of carbonic anhydrase III has been reported as a reliable early indicator of myocardial injury. Unfortunately, a commercial assay using this technology has yet to be developed.

In the past, CK-MB was a reliable marker for MI, but it requires a significant elevation to make a diagnosis. It’s released more slowly than myoglobin and requires about four to six hours after myocardial injury to reach significant levels. It usually drops below significant levels in two to five days.

A problem with CK-MB is that it is normally present in the blood of healthy individuals, at a wide range of normal levels. For example, athletes and others with more muscle mass may have higher levels. It takes a considerable volume of myocardial damage to raise circulating CK-MB to pathological levels. If the total CK is elevated, the CK-MB could rise above the pathological cutoff of many assays. This is why we need to look at the relative index, or the percentage of CK-MB to the total CK level in a blood sample, to determine with higher probability that myocardial necrosis has actually taken place. On the other hand, a small rise in CK-MB from a true myocardial event could go unnoticed, within the broad range of normal values.

The literature reports a more cardiac-specific isoform of CK-MB. This actually is determined by comparing the intact whole CK-MB molecule to a ratio of its early cardiac metabolized form, which lacks its terminal amino acid, leucine. This cardiac-specific isoform of CK-MB is both a specific and a sensitive marker for myocardial necrosis. The problem with using this marker clinically is that it differs by only a single amino acid from the isoform normally assayed for. This makes separation by electrophoresis necessary before assay. Because of technical difficulties, CK-MB subform assays are not readily available and take a significant amount of time to run. This is unfortunate, especially when ACC/AHA guidelines for cardiac markers recommend that when a central laboratory is used, the results should be available within 60 minutes (preferably within 30 minutes).

Some clinicians have felt that a negative troponin with a positive CK-MB rules out a cardiac origin for the CK-MB. But remember that even the best assays have occasional false positives and negatives. A qualitative CK-MB above the cutoff threshold might mean the total CK is elevated from muscle damage. This could suggest a myositis in a patient taking a statin.

Using the three markers together and understanding their properties can improve the sensitivity and specificity for determining myocardial necrosis. There are now abbreviated serial determinations that look at the rate of change in the level of CK-MB or myoglobin over a two-hour period in suspected coronary syndromes. These delta values have shown promise in ruling in myocardial injury before the troponin has a chance to be positive.

PUTTING THE MARKERS TO GOOD USE

Any test must be used with good judgment, experience, and current standards of care in order to make a diagnosis and a sound treatment plan. A negative test is not necessarily a green light to send a suspected high-risk patient home. Remember that all of the markers require a significant time after injury to become positive. In the face of chest pain with the obvious ST elevations of an AMI, it is unwise to wait for biomarkers, which probably haven’t had the time to become positive. These patients need immediate percutaneous coronary intervention, preferably within 90 minutes.

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