How healthy is your heart?

If the answer is “not very” then myeloperoxidase can be used as a useful detector of cardiac disease

MYELOPEROXIDASE (MPO) is an inflammatory biomarker that may show elevated levels in the plasma of patients suffering heart failure and cardiac dysfunction. With a rise in MPO appearing earlier than with other traditional cardiac biomarkers (such as troponin), MPO offers new possibilities for early identification of cardiac events and, potentially, treatment before irreversible cell damage (necrosis) has occurred. Release of MPO can even precede myocardial injury and so identify at-risk patients. Here we examine its clinical use as a predictor of cardiovascular events.

In a typical case of acute coronary syndrome (ACS), a patient is likely to present to the hospital accident and emergency department with chest pains. One possible cause of this pain is a blockage in the coronary artery following the rupture of an atherosclerotic plaque. Plaques typically are build-ups of cholesterol and fatty acids in the lining of an artery, a condition commonly known as atherosclerosis.

Prolonged restriction of blood to the heart resulting from a blocked artery causes heart tissue damage, which can lead to death of heart muscle cells. In medical terms: prolonged ischemia causes infarction, which can lead to myocardial necrosis. As tissue dies the heart’s ventricles can become overloaded, leading to heart failure.

Following myocardial infarction (MI), an electrocardiogram (ECG) can identify damaged heart muscle but only in particular regions. Evaluating an ST segment of an ECG trace allows a distinction to be made between ST elevation myocardial infarction (STEMI) or non-ST elevation myocardial infarction (NSTEMI). The majority of patients with ACS, however, present without ST segment elevation and are the most challenging to diagnose. In these cases ECG results are insufficient and clinicians rely on cardiac biomarkers.

The aim of diagnostic and prognostic biomarkers is to: identify patients with ACS even when there is no evidence of tissue death and predict the risks of further complications to patients following an ACS event.

The ideal biomarker should be released from heart tissue only, have a relatively high concentration in heart tissue, be detectable soon after the onset of a cardiac event, have high clinical sensitivity and specificity, and be tested on an analytical system capable of quick turnaround time. Unfortunately, no single marker fits all these criteria, so the practice of assessing multiple markers has been adopted.

The usual panel of biomarkers used to diagnose acute chest pain includes troponin, myoglobin and creatine kinase-MB (CK-MB). These markers show progression of an event from plaque instability through to necrosis1. To follow up on patients with confirmed ACS, the marker B-type natriuretic peptide (BNP) can be added. Such cardiac biomarker panels provide much more detailed stratification between patients with different conditions for both diagnosis and future risk assessment2. This allows cardiologists to understand individual differences and select the correct drug for each patient.

Now, the addition of the novel cardiac biomarker myeloperoxidase (MPO) to the panel offers new possibilities. Because levels of MPO become elevated even earlier than traditional markers it can be used as an early indicator of plaque destabilisation. It is also provides critical risk stratification information.

MPO is a haemoprotein enzyme stored in the neutrophil granuolcyte of the white blood cell population. Its tetrameric structure is shown in Figure 1. The iron within the haem group of this protein is capable of undergoing oxidation and reduction and is shifted to a higher wavelength (red-shifted) compared to other haem molecules. This causes MPO to appear green in secretions rich in neutrophils, such as pus and some forms of mucus.

When ingesting undesirable microorganisms, neutrophil granulocytes undergo a process called degranulation. This process releases both toxic, oxygen-derived products, such as hydrogen peroxide (H2O2), and MPO enzymes. The MPO behaves as a catalyst to promote peroxidation of chloride anions (Cl-) into hypochlorous acid (HOCl).

"For patients presenting with chest pain, MPO analysis together with troponin enables early cardiac disease detection even before irreversible damage occurs"

MPO also oxidises tyrosine to tyrosyl radical3. While both HOCl and tyrosyl radical are cytotoxins, designed to kill invading bacteria, viruses and fungi, they also promote oxidative damage at sites of inflammation. HOCl, for example, is a weak acid that reacts with a wide variety of biomolecules including DNA, RNA, fatty acid groups, cholesterol and proteins.

MPO has been shown to accumulate in atherosclerotic plaques. The oxidative products generated by MPO have been attributed to promoting destabilisation of the plaque and its subsequent rupture in atherosclerosis and coronary artery disease4, 5.

While the use of multiple markers for diagnosis is still in its infancy, recent studies have added to the body of evidence supporting the use of MPO as an early marker of cardiac disease. Evidence for its use to provide risk stratification is robust and its potential for risk assessment in healthy individuals has been demonstrated.

Chest pain

Table 1: MPO analysis together with troponin enables cardiac disease detection

For patients presenting with chest pain, MPO analysis together with troponin enables early cardiac disease detection even

Possible diagnosis for a patient presenting with chest pain	Expected results of troponin-l and MPO tests
Non-cardiac (eg pulmonary origin)	Tn -ve MPO* -ve
Cardiac, non-ischemic	Tn -ve, MPO +ve
Stable/unstable angina pectoris	Tn -ve or +ve, MPO +ve
Myocardial infarction	MPO and/or Tn +ve

before irreversible damage occurs (Table 1).

Whereas troponin takes 3-6 hours to rise to measurable circulating levels after myocardial injury, MPO levels have been shown to be significantly elevated (even within two hours of the onset of symptoms) in patients who were initially negative
for troponin6.

MPO serum levels powerfully predict an increased risk for subsequent cardiovascular events and

*MPO may be positive in patients with non-cariac inflammation

extend the prognostic information gained from traditional biochemical markers7. MPO assessment may also be useful in triage in the emergency department.

Heart failure
In a study of patients with chronic heart failure8, higher plasma levels of MPO were associated with an increased likelihood of more advanced heart failure. Moreover, elevated plasma MPO levels within a heart failure subject seem to be predictive of increased adverse clinical outcomes. This is of great prognostic value to the clinician.

Risk assessment
Elevated MPO levels can also be used to predict future risk of coronary artery disease (CAD) in apparently healthy individuals. In a case-control study9, MPO was measured in baseline samples of 1,138 apparently healthy men and women who developed CAD during an 8-year follow-up. Control subjects (n=2,237) remained free of CAD. The MPO levels were significantly higher in case subjects than control subjects and correlated with C-reactive protein and white blood cell count. This study suggests that inflammatory activation precedes the onset of overt CAD by many years.

One technique to determine the presence of MPO in human plasma is an immunoassay that exploits the particular chemiluminescence of the enzyme. The newly launched ARCHITECT MPO assay (Abbott Diagnostics, UK), for example, uses this technique within a two-step immunoassay using chemiluminescent magnetic immunoassay (CMIA) technology with flexible assay protocols, referred to as CHEMIFLEX. The assay has STAT protocol capability. Figure 2 shows the basic steps of the straightforward assaying procedure, designed to facilitate easy implementation into routine clinical test practices10, 11.

In the first step, sample and anti-MPO coated paramagnetic microparticles are

Figure 2: ARCHITECT MPO assay design format

combined. MPO present in the sample binds to the anti-MPO coated microparticles. After incubation and wash, anti-MPO acridinium labelled conjugate is added in the second step. Following another incubation and wash, pre-trigger and trigger solutions are added to the reaction mixture. The resulting chemiluminescent reaction is measured as relative light units (RLUs).

A direct relationship exists between the amount of MPO in the sample and the RLUs detected by the ARCHITECT immunoassay analyser optics. The concentration of MPO is read relative to a standard curve established with calibrators of known MPO concentration from 0 to 10,000pmol/L.

This type of MPO assay technique has demonstrated excellent lot-to-lot consistency, acceptable analytical performance and correlation with the PrognostiX CardioMPO ELISA assay. The precision ranges and detection limits are good and the assay exhibits excellent dilution linearity and spike recovery.

Recent studies have added to the body of evidence supporting the use of MPO as an early marker of cardiac disease. Used together with traditional biomarkers, MPO offers a valuable new tool for the clinician for assessing patients with chest pain or at risk of a cardiac event.