The Past, Present and Future of Cardiac Biomarkers

By Dr. R Nandakumar, MRCP(UK) FRCP(Ed) FRCP(Lond), Senior Interventional Cardiologist, 34/35, Mt Elizabeth Hospital Novena Hospital Medical Centre & Cardiac Centre International, Gleneagles Hospital

Cardiovascular disease (CVD) is the leading cause of death globally with no geographic, gender or socio-economic boundaries. More people die annually from CVDs than from any other cause. An estimated 17.7 million people died from CVDs in 2015, representing 31 per cent of all global deaths. Of these deaths, an estimated 7.4 million were due to coronary heart disease and 6.7 million were due to stroke.

Coronary heart disease is caused by atherosclerotic plaque build-up in the arteries causing ischemia and heart attacks. Ischemic heart disease accounts for almost half of the increase in the number of cardiovascular deaths. Population aging contributed to an estimated 55 per cent increase in cardiovascular disease deaths globally, and population growth contributed to a 25 per cent increase. Acute coronary syndrome (ACS) refers to a spectrum of clinical presentations ranging from unstable angina to non–ST-segment elevation myocardial infarction (NSTEMI) to ST-segment elevation myocardial infarction (STEMI). It is almost always associated with rupture of an atherosclerotic plaque and partial or complete thrombosis of the infarct-related artery. ACS requires prompt diagnosis and timely initialisation of revascularisation to reduce complications and recurrence. 

While the earliest criteria included ECGs and history, biomarkers were also used quite early on. An ideal biomarker should have sufficient concentration in the myocardium with minimal or no presence in the serum and non-myocardial tissue and be rapidly released into the blood during ischemia. The assay should have analytical precision, with sufficient sensitivity and specificity to distinguish diseased and non-diseased states. In 2001, National Institute of Health (NIH) working group standardised the definition of a biomarker as “a characteristic that is objectively measured and evaluated as an indicator of normal biological processes, pathogenic processes, or pharmacologic responses to a therapeutic intervention.”

History of Biomarkers
The use of biomarkers in the diagnosis of acute myocardial infarction (AMI) began in 1954 when Karmen et al., first reported the elevation of aspartate aminotransferase (AST) in the serum of patients with AMI in 1954. AST (Aspartate aminotransferase) also called as serum glutamic oxalaacetic transaminase (SGOT) increases in blood three to four hours after AMI and reaches maximum value in 15-28 hours and returns to normal around five days. AST however is non-specific as it can be raised in a variety of conditions.

Subsequently, LDH was first published as a marker of MI by Wróblewski and LaDue who observed an increase in LDH activity in serum of patients with AMI, which was subsequently confirmed by Ulmer et al. Since LDH is present in nearly all human tissues, LDH isoenzymes, either as an alpha-hydroxybutyrate dehydrogenase (HBD) or lactate dehydrogenase isoenzyme 1 (LDH-1), were described as possible biomarkers of AMI. A direct enzymatic assay for α-hydroxybutyrate dehydrogenase activity was developed to increase cardiac specificity. LDH and its isoenzyme LDH-1 increases in blood five to 10 hours after AMI, reaches maximum value in blood in 60 –144 hours and returns to normal values in 12 days.

The 1960s marked the beginning of creatine kinase (CK) as a better biomarker as it was demonstrated to be more cardiac-specific and clinically useful due to its kinetics after AMI. Subsequently an effective enzymatic assay for the quantification of creatine kinase (CK) activity was developed by Rosalki and was established as a marker for AMI diagnosis for the next 20 years. While the sensitivity of CK is high (98 per cent within 72 hours) specificity is low. Total CK activity is related to the extent of the myocardial infarction and therefore, prognosis. CK appears in blood three to nine hours after an AMI reaches the maximum value in blood in 10–20 hours and returns to normal in approximately 72 hours.

Myoglobin is a globular, oxygen-carrying heme protein found in skeletal and cardiac with a low molecular weight (17.8 kDa), found in the cytoplasm, which accounts for its early release profile. Myoglobin appears in serum in one to three hours after onset of infarction, peaks at four to seven hours, and returns to normal within 24-36 hours. However, it is cleared rapidly from serum and is not cardio-specific with levels rising in muscular dystrophy, trauma and inflammation or even after strenuous exercise or after intra-muscular injections. The initial RIA assay was followed rapidly by latex-enhanced immunoassays and automated nonisotopic immunoassay.

In the 1970s, radioimmunoassays were developed and revolutionised laboratory medicine including AMI diagnosis. Roe et al in 1972 developed a zone electrophoresis for identification and quantification of the CKMB, an isoenzyme of CK with highest concentration in heart muscle (25-30 per cent vs 1-2 per cent in skeletal muscle). CKMB was shown to help with specific diagnosis in the first few hours after ischemia. 

In 1976, Roberts et al developed a radioimmunoassay for CK isoenzymes and in 1979, the World Health Organization (WHO) recognised the role of CKMB and other enzymes in AMI, and a rise and fall in cardiac enzymes was included in the diagnostic criteria. CK-MB first appears four to six hours after symptom onset, peaks at 24 hours, and returns to normal in 48-72 hours. Its value in the early and late (>72 h) diagnosis of acute MI is limited. 

Subsequently immunoassays were substantially improved by using monoclonal antibodies, and rapid immunoassays for measuring the so-called CKMB “mass” replaced CKMB activity measurements as the criterion standard for AMI diagnosis.

Present Scenario
Towards the end of the 1980s, cardiac troponins emerged with more sensitivity and specificity and AMI diagnosis moved on from a clinical-electrocardiographic-biochemical centred approach to a biomarker-centred definition of AMI where cardiac troponins played a central role. Troponins are cardiac proteins important for actin and myosin interaction, modulating sarcomeric contractile function in response to cytosolic calcium and protein phosphorylation. The troponin complex is composed of three regulatory proteins: cTnC, cTnI, and cTnT though only cardiac tropnions T and I have unique isoforms and are currently the gold standard for detection of myocardial injury and also play a key role in risk stratification and management strategy.

Lately, high sensitive troponins have emerged, which permits detection of very low levels of cTnT. Using the hs-cTn assay improves the overall diagnostic accuracy in patients with suspected AMI, while a negative result also has a high negative predictive value. These assays are extremely sensitive, allowing for earlier and faster recognition of MI patients, quicker diagnosis and ability to treat patients appropriately. The new assays are precise, having small CV levels even at the 99 per cent in reference populations CRP – C-reactive protein is an acute-phase inflammatory reactant marker that has been extensively studied as indicative of a higher cardiovascular risk among patients with established atherosclerosis. CRP is commonly elevated in ACS. (hsCRP) detects lower levels of CRP and can stratify patients into low, intermediate and high-risk category of future cardiovascular risk. Statins have been shown to be capable of reducing CRP.

Emerging Biomarkers
Myeloperoxidase (MPO) is a member of the heme peroxidase family- a leukocyte enzyme that generates reactant oxidant species, which are released in inflammatory conditions. Myocardial injury involves leukocyte accumulation and activation in the atherosclerotic plaque. These leukocytes undergo degranulation in the coronary circulation in patients with ACS, which leads to the release of MPO and thus accounts for the elevated levels of MPO seen in ACS. MPO is capable of inducing low density lipid (LDL) and ApoA-I oxidation, is involved in plaque formation, plaque instability (mismatch between pro and anti-oxidants) and vasoconstriction from nitrous oxide depletion. Elevated MPO was first linked with coronary artery disease in 2001 and is also an independent predictor of major adverse cardiac events (MACE). Esporcatte et al., who reported a diagnostic sensitivity of 92 per cent and specificity of 40 per cent for identifying AMI patients with acute chest pain.
Ischemia modified albumin (IMA) is a novel marker of ischemia that is produced when circulating serum albumin contacts ischemic heart tissues. IMA can be measured by the albumin cobalt binding assay that is based on IMA’s inability to bind to cobalt. IMA levels rise within minutes of transient ischemia, peak within six hours, and can remain elevated for as long as 12 hours. While sensitivity varies from 71-98 per cent and specificity between 45-65 per cent use of multimarker panels can improve sensitivity to 97 per cent for myocardial ischemia.

Copeptin a glycosylated 39-amino-acid peptide, is a C-terminal part of the precursor pre-provasopressin (pre-proAVP) and is released in the same amount as AVP. Copeptin is stable and has a half-life of days in plasma, as compared to five to 20 min for AVP. Keller et al showed that addition of copeptin to troponin(cTnT) improved sensitivity and negative predictive value to 92.4 per cent whereas the CHOPIN trial improved the negative predictive value to 99 per cent.

Matrixmetalloproteinases (MMPs) are a family of endopeptidases expressed by macrophages, vascular smooth muscle cells, and endothelial cells in response to inflammatory stimuli and oxidative stress and are extracellular matrix degrading enzymes. MMPs are consistently implicated in atherogenesis and plaque destabilisation, responsible for thinning and rupturing of atherosclerotic fibrous cap. MMP-2, MMP-8, and MMP-9 have been recognised as proteases that contribute to atherosclerotic plaque rupture and clinical events by degenerating structural components of the plaque matrix. MMP-2 is elevated post-MI, elevated MMP-2 activity in plaques is associated with higher rate of ischemic cerebrovascular events and is an independent predictor of all-cause mortality in post-ACS. MMP-9 progressively increases with severity of clinical presentation and there is strong association between serum levels of MMP-9 and subsequent cardiovascular complications. This maybe related to increased activity of circulating monocytes related to more extensive myocardial necrosis. Elevated MMP-8 levels in the carotid plaque have been associated with unstable plaque phenotype. 

GDF 15 or Growth differentiation Factor is a member of the transforming growth factor -B cytokine superfamily expressed by activated macrophages associated with cellular oxidative stress and is inducible in the myocardium after ischemia. GDF 15 has been used to identify those pts with ACS who would benefit from an invasive strategy.

Micro-RNAs or MiRNAs are endogenous, small, noncoding RNA molecules, which regulate the expression of approximately 30 per cent of genes in the human genome and are highly stable in the circulation. Several MiRNA’s are found in blood early after MI and are correlated with risk of cardiovascular death. MiRNA-145 is expressed in smooth muscle cells of the blood vessel and is implicated in atherosclerosis along with miR-126 and miR 155. Wang et al., have reported that elevated levels of cardiac specific miR-208a in plasma may represent a new biomarker for early detection of myocardial damage.

PAPP-A or Pregnancy-associated plasma protein A (PAPP-A), a zinc-binding matrix metalloproteinase, was originally identified in pregnant women, produced in the placenta, and has been found to be a useful biomarker for predicting the rupture of unstable atherosclerotic plaques and risk of future cardiac events in patients with CAD. Wu et al., also showed that higher PAPP-A levels are associated with higher 3 vessel thin cap-fibroatheroma burden and instability.

H-FABP or heart-type Fatty acid binding protein is a low molecular weight protein with 132 aminoacids, involved in myocardial fatty acid metabolism. It is released into the cytosol in early AMI and serves as a highly specific marker for ACS and can detect myocardial ischemia and minor myocardial injury even in the absence of myocardial necrosis.

MR-PAMP or mid regional proadrenomedullin is a peptide that is used to indirectly quantify ADM. ADM or Adrenomedullin a potent vasodilator synthesised in the adrenal medulla, endothelial cells and the heart. It is more stable than ADM. It is elevated in patients with early atherosclerotic plaque and subclinical CAD and also has prognostic value as predictor of cardiovascular mortality.

ST2 is a member of the interleukin family and is involved in activation of T helper type2 cells and its cytokines. ST2 helps to predict mortality in low risk patients and can also be used for risk stratification in STEMI patients and in heart failure (ENTIRE-TIMI 23, CLARITY). A highly sensitive ELISA for sST2 has been developed (Presage ST2), which has low imprecision (coefficient of variation <5 per cent) even at very low analyte concentrations.
Neuregulin-1β (NRG-1β) is a member of the neuregulin family of signalling proteins, a myocardial stress activated growth and survival factor released from endocardial and endothelial cells. It acts through ErbB receptors on NRG-responsive cells. NRG-1β exerts paracrine effects on local myocytes and blood vessels. Geisberg et al, found that that NRG-1β levels inversely correlates with CAD severity in stable patients and plasma NRG-1β levels are statistically higher in patients with stress-induced ischemia.

Galectin-3 (Gal-3) belongs to a family of soluble β-galactoside binding lectins and plays an important role in atherogenesis through some of its actions such as monocytes chemoattraction, enhancement of phagocytosis, and induction of vascular smooth muscle cells proliferation. Gal3 has been shown to independently predict CAD occurrence and also cardiovascular mortality in pts at high cardiac risk.

Endothelin -1(ET-1) is a 21 aminopeptide secreted by the vascular endothelial cells with potent vasoconstrictor effects and is considered a surrogate marker of atherosclerosis. Elevated plasma levels of endothelin have been associated with coronary artery disease, essential hypertension and heart failure. Both in stable CAD and AMI patients, CT-proET-1 has been shown to be associated with cardiovascular death and HF independent of clinical variables.

Lipoprotein – associated phospholipase A1 (Lp-PLA2) is a member of the phospholipase A2 superfamily mainly produced by monocytes and macrophages. Lp-PLA2 is an enzyme that hydrolyzes oxidized phospholipids on oxidized LDL particles within the arterial. The localisation of Lp-PLA2 in atherosclerotic lesions and its association with plaque instability support a potential causal role for Lp-PLA2 in cardiovascular disease. Several studies have shown Lp-LPA2 activity to be independently predictive of cardiovascular events.

Future Prospects
Fibrinogen is a 340-kDa acute phase glycoprotein synthesised in the liver and is involved in platelet aggregation, endothelial injury, plasma viscosity, and mediates thrombus formation. Elevated fibrinogen levels are associated with an increased risk of CAD, stroke and mortality.

Uric acid is the end product of purine metabolism in humans. Elevated serum UA has been hypothesised to contribute to CVD development since the 1950s. Uric acid has been found to impair nitric oxide synthesis, resulting in vascular endothelial dysfunction lower flow-mediated vasodilatation, increasing oxidative stress and enhancing inflammation. Here is an independent positive association between UA and cardiovascular mortality. The National Health and Nutrition Examination Survey I Follow-Up Study, in which 5,926 subjects with 16.4 years of follow up were enrolled, demonstrated that increased serum uric acid levels were associated significantly with a higher risk of cardiovascular mortality. Other studies have showed similar results.

In summary, since those early days of non-specific and poorly sensitive biomarkers, we have come a long way with current biomarkers having the ability to diagnose cardiac injury earlier and at very low levels. Further, many of these current markers have the ability to risk stratify and help to apply the appropriate strategy as well for better outcomes. Newer biomarkers continue to emerge opening up new vistas from predicting vulnerable plaques to vascular endothelial activity and will continue to play a central role in cardiac therapy in times to come.