Cardiac Bio Markers

– “A biomarker is a substance used as an indicator of a biologic state”.

–Accurate repeated measurements at reasonable cost–Must provide additional information– Should aid treatment

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HISTORY OF CARDIAC BIOMARKERS

• 1954 - SGOT (AST)• 1955 - LDH • 1960 - CPK • 1972 - CPK isoforms by Electrophoresis • 1975 - CK - MB by immunoinhibition • 1975 - Myoglobin • 1985 - CK - MB Mass immunoassay • 1989 - Troponin T • 1992 - Troponin I

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CLASSIFICATION OF CARDIAC BIOMARKERS

• Biomarkers of myocardial injury– markers of myocardial necrosis– markers of myocardial ischemia

• Biomarkers of haemodynamic stress

• Inflammatory and prognostic Biomarkers

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Marker for inflammation hsCRP sCD40L Homocysteine

Marker for plaque Destabilization PAPP-A LP-PLA2

Marker for hemodynamic

stress

Natriuretic peptides• ANP• BNP• Pro-BNP• CNP

LDH

• Pyruvate lactate • 100-200 U/L• Concentration in RBC is high than plasma

• 5 iso enzmes • Flipped pattern• Increases by 24-48 hours • Peak in 3-6 days• Normalises in 8-14 days

Aspartate aminotransferase (AST,SGOT)

• 8-20 u/l• Raise by 6–8 h • peak by 18–24 h, • Normalises by 4 to 5 d

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CREATINE KINASE

Function: it catalyses the conversion of creatine to phosphocreatine degrading ATP to ADP

• The CK enzyme consists of two subunits, B (brain type) or M (muscle type), Making three different isoenzymes: CK-MM, CK-BB and CK-MB

• CK-BB occurs mainly in tissues, rarely of any significance in the bloodstream

• Skeletal muscle expresses CK-MM (98%) and low levels of CK-MB (1%)

• The myocardium has CK-MM at 70% and CK-MB at ~30%

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CK-MB• High specificity for cardiac tissue

• Begins to rise 4-6 hours after onset of infarction

• Peaks at about 12 hours

• Returns to baseline at 24-36 hours

• Can be used to indicate early re-infarction if level normalizes and then increases again

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DETECTION

• The CK-MB isoforms may also be analyzed using high-voltage electrophoresis

• The ratio of MB2/MB1 is calculated

• MB2 released from heart muscle and converted to MB1

• A level of MB2 > or = 1 and a ratio of MB2/MB1 > 1.5 indicates myocardial injury

• A result is positive if MB2 is elevated and the ratio is more than 1.5

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• False positive (for MI) CK-MB elevation can be seen in:– Significant skeletal muscle injury– The MB fraction is determined to be expressed

during the process of muscle regeneration– Cardiac injury for reason other than MI• Defibrillation • Blunt chest trauma• Cocaine abuse

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MYOGLOBIN• Small-size heme protein found in all tissues mainly assists in oxygen transport

• It is released from all damaged tissues

• Increases often occur more rapidly than TI and CK

• Released from damaged tissue within 1 hour

• Normal value: 17.4-105.7 ng/ml

• Timing:– Earliest Rise: 1-3 hrs– Peak 6-9 hrs– Return to normal: 12 hrs

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CONDITIONS FOR MYOGLOBIN INCREASE

• Acute myocardial infarction

• Skeletal muscle damage, muscular dystrophy, inflammatory myopathies

• Renal failure, severe uremia

• Shock and trauma

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• Rapid monitor of success of thrombolytic therapy

• Negative predictor of MI

• Due to poor specificity, myoglobin levels do not always predict myocardial injury

• Not utilized often for AMI/cardiac damage

assessment because of its very rapid metabolism

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CARDIAC TROPONINS

• Troponin is a complex of three regulatory proteins that is integral to non-smooth muscle contraction in skeletal as well as cardiac muscle

• Troponin is attached to the tropomyosin sitting in the groove between actin filaments in muscle tissue

• Troponin has three subunits, TnC, TnT, and TnI– Troponin-C has calcium binding ability and has no

diagnostic value– Troponin-T binds the troponin tropomyosin complex, – Troponin-I is an inhibitory protein

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Cardiac Troponin Release after MI

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TROPONIN I

1. Cardiac Troponin I (cTnl) is a cardiac muscle protein with a molecular weight of 24 kilo-Daltons.

2. The cTnl has a additional amino acid residues on its N-terminal that are not exist on the skeletal form.

3. The half life = 2~4 hours.

4. Serum increase = 2-8 hours

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TROPONIN T 1. Cardiac Troponin T (cTnT) is present in fetal

skeletal muscle.

2. In healthy adult skeletal muscle cTnT is absent.

3. The gene of cTnT may be re-expressed in skeletal muscle disease.

4. Biological half life and early serum increases of cTnT are similar to that of cTnI.

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TROPONIN LEVELS

• Less than 5% in cytosol

• Troponin levels begin to rise 2-3 hours after onset of myocardial injury

• Elevations in Troponin-I and Troponin-T can persist for up to 10 days after MI

• CK-MB returns to baseline by 48 hours

• Thus far, studies have failed to find a source of Troponin-I outside the heart, but have found some Troponin-T in skeletal muscle

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Conditions commonly associated with cardiac troponin elevations• Arrhythmias• Congestive heart failure• Coronary artery disease• Coronary vasospasm• Critically ill patient• Hypertension• Myocarditis• Pericarditis• Pulmonary embolism• Pulmonary hypertension, severe• Renal failure• Sepsis/septic shock• Sepsis-related myocardial dysfunction• Systemic inflammatory diseases• Trauma

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Increased Troponins

• Troponin T and I are not detected in healthy individuals

• Significant increase in Troponins reflects myocardial necrosis

• ACC/ESC has defined increase in Troponins as a measurement above 99th percentile value of reference group

• To reduce false-positive outcomes, CV of 10% at decision limit is recommended

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TROPONIN ASSAYS

• TropT (Roche Diagnostics, Germany) • Trop I (Siemens Healthcare Diagnostics)

• Troponin T – 99th percentile limits - 0.01 ng/mL – assay ranges - 0.01-25 ng/mL

• (Troponin I) – 99th percentile limits -0.04 ng/mL – assay range -0.04-40 ng/mL

• Reference limits based on the 99th percentile for a healthy population are 0.01 ng/mL (Troponin T) and 0.04 ng/mL (Troponin I)

Timing Summary

TEST ONSET PEAK DURATION

CK/CK-MB 4-8 hours 18-24 hours 36-48 hours

Troponins 3-12 hours 18-24 hours Up to 10 days

Myoglobin 1-4 hours 6-7 hours 24 hours

LDH 6-12 hours 24-48 hours 6-8 days

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Ischemia Modified Albumin (IMA)

• A novel marker of ischemia, is produced when circulating serum albumin contacts ischemic heart tissues

• IMA can be measured by the albumin cobalt binding (ACB) assay that is based on IMA's inability to bind to cobalt

• Mechanism- due to structural change in the amino terminal end of albumin

• IMA levels rise within 6 hours

• Remains elevated for 12 hours

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• IMA levels raised in non- cardiac ischemia• Modification to n- terminal end may also

be induced by extracellular hypoxia, acidosis etc,

• FDA in 2010 has approved a multimarker approach for using the combination of ECG, the cTnI, and the IMA levels achieving a sensitivity of 95% for ACS

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Heart-type fatty acid binding protein (H-FABP)

• H-FABP is a very stable abundant [138] low molecularweight protein (14–15 kDa) in the cytoplasm of myocardial cells

• Appearing as early as 90 min after symptom onset and peaking within 6 h

• Parameters of kinetic release make it an ideal candidate both for early assessment or exclusion of AMI and for the measurement of a recurrent infarction

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• A study by Puls et al– the negative predictive value (NPV) of H-FABP was an

impressive 100%– its Positive predictive value was 41% which was greater

than that of both cTnT (29%) and NT-proBNP (19%).

• The myoglobin/heart FABP ratio has been used to differentiate between heart muscle and skeletal muscle injury

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• The NP family includes

ANP : -atrial natriuretic peptide (28 a.a.) N-terminal proANP (98 a.a.)

BNP : brain natriuretic peptide (32 a.a.) N-terminal proBNP (76 a.a.)

CNP : C-type natriuretic peptide (22 and 53 a.a.)

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NATRIURETIC PEPTIDES

• ANP is released primarily in response to atrial wall stretching and intravascular volume expansion.

• BNP is mainly secreted by the ventricles

• CNP is found predominantly in the brain and also synthesized by vascular endothelial cells

N-terminal proBNP interpretation in the patients with acute dyspnea without severe renal failure

N-terminal proBNP interpretation in the patients with acute dyspnea without severe renal failure

37Stefan Blankenberg, MD; Renate Schnabel, MD; Edith Lubos, MD, et al., Myeloperoxidase Early Indicator of Acute Coronary Syndrome and Predictor of Future Cardiovascular Events 2005

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sCD40 Ligand

• CD40 ligand is a transmembrane protein related to TNF.

• It has multiple prothrombotic and proatherogenic effects.

• What is usually measured is the soluble form of the receptor, sCD40 ligand, which has been shown to be a predictor of events after acute presentation.

• At present, standardized assays, reference interval studies, nor consistent assay validations are not available.

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Serum Amyloid Protein A

• Serum amyloid protein A, an acute-phase protein and an apolipoprotein, has been used with hsCRP in cross-sectional studies.

• It can be synergistic with hsCRP326 but is much less commonly used.

• At present, no standardized assays, reference interval studies, nor consistent assay validations are available.

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Cytokines• A variety of stimulatory and inhibitory interleukins (TNF, IL-1,

IL-6, IL-8, IL-12, IL-18) are thought to help mediate the elaboration of CRP and the development of atherosclerosis and acute events.121 These cytokines may stimulate or inhibit leukocytes, often through T cell–mediated processes and effects on monocytes, which are indigenous to atherogenesis.

• In some studies, IL-6 is more prognostic than hsCRP.• These cytokines often have inhibitors and/or binding proteins

that modulate their effects. At present, standardized assays, reference intervals studies, and consistent assay validations are not available.

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Lipoprotein-Associated Phospholipase A2

• Phospholipase A2 (Lp-PLA2) is a phospholipase associated with LDL that is thought to be an inflammatory marker.

• It was previously known as platelet-activating factor (PAF) acetyl hydrolase.

• It is synthesized by monocytes and lymphocytes and is thought to cleave oxidized lipids to produce lipid fragments that are more atherogenic and that increase endothelial adhesion.

• An FDA-approved assay for this analyte includes obligatory reference intervals.

• It has been shown to be predictive of events in a primary prevention cohort, even when hsCRP is present in the model, suggesting that it measures something different from what is measured by the acute-phase reactants associated with hsCRP.

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Pregnancy-Associated Plasma Protein A

• Pregnancy-associated plasma protein A (PAPP-A) is a metalloproteinase that is thought to be expressed in plaques that may be prone to rupture.

• The literature in this regard is mixed at present concerning its use.

• At present, standardize assays, reference interval studies, and consistent assay validations are not available.

• Recent data suggest that heparin administration in MI patients is associated with increased PAPP-A concentrations; this may limit its prognostic role.

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Oxidized LDL• Oxidized LDL has been attributed a key role in the

development of atherosclerosis.• Several methods have been used to measure it, but they

yield potentially different data.• Some have correlated malondialdehyde LDL with the

development of atherosclerosis and short-term events.• Direct identification with antibodies suggests that

oxidized LDL may be released from vessels and may colocalize with lipoprotein a [Lp(a)] after acute events.

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Placental Growth Factor• Placental growth factor is an angiogenic factor related

to vascular endothelial growth factor (VEGF), which stimulates smooth muscle cells and macrophages.

• It also increases TNF and MCP-1. A novel assay for this analyte is thought to provide additional prognostic information on patients who present with ACS.

• At present, standardized assays, reference interval studies, and consistent assay validations are not available.

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Matrix Metalloproteinases• Matrix metalloproteinases (MMPs) can degrade the collagen matrix in

coronary artery or myocardium.• They are integral to remodeling of the coronary artery and/or the heart

after acute events.• Elaboration of MMP-9, a gelatinase, is thought to be important in plaque

destabilization; thus some have tried to measure it as a prognostic index.• Other MMPs participate in the elaboration of extracellular matrix in the

heart.• Many MMPs also have inhibitors [tissue inhibitors of metalloproteinase

(TIMPs)] that modulate their effects.• At present, standardized assays, reference intervals studies, and• consistent assay validations are not available.

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Monocyte Chemotactic Protein

• Monocyte chemotactic protein (MCP-1) is a chemokine that is thought to be responsible for the recruitment of monocytes into atherosclerotic plaque.

• It has been reported to be elevated in patients with ACS and to have long-term predictive value.

• However, at present, standardized assays, reference interval studies, and consistent assay validations are not available

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Tissue Plasminogen Activator Antigen (t-PA) andPlasminogen Activator Inhibitor 1 (PAI-1)

• t-PA is the body’s physiologic fibrinolytic activator.• PAI-1, its endogenous inhibitor, binds to t-PA.• Inhibition of fibrinolysis has been suggested to be a reason

for recurrent infarction; the fact that maximal inhibition usually occurs in the early morning hours provides a potential explanation for the circadian variability of AMI.

• It may also be the reason why persons with diabetes have such unstable disease; the growth factor properties of insulin stimulate increases in PAI-1.

• An accurate assessment of this system includes both t-PA and PAI-1, along with some assessment of bound versus free levels.

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Secreted Platelet Granular Substances

• Both platelet factor 4 (PF4) and beta thromboglobulin (BTG) are secreted when platelets aggregate.

• PF4 has a short half-life and is released by heparin.• BTG is not released by heparin and has a longer half-life.• Both markers have been used to assess platelet

aggregation.• BTG is by far the more reliable.• At present, standardized assays, reference interval

studies, and consistent assay validations are not available.

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Unbound Free Fatty Acid

• Unbound free fatty acid (uFFA)39 has also been touted as a marker of ischemia.

• Most fatty acid is bound, and ischemia is thought to increase the small unbound fraction.

• Initial studies have reported mixed results.• At present, standardized assays, reference

interval studies, and consistent assay validations are not available.

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Nourin

• Nourin I is a small protein released rapidly by “stressed myocytes.”

• It induces changes in a variety of inflammatory cytokines and attracts neutrophils.

• Preliminary studies have been done to attempt to validate its use.

• At present, standardized assays, reference interval studies, and consistent assay validations are not available.

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Copeptin• Copeptin, a 30 amino acid glycoprotein constituting the C-

terminal portion of arginine vasopressin, has been shown to be a prognostic biomarker in hemorrhagic and septic sepsis.

• More recently, data have shown that measurement of copeptin serves as a rapid and early rule-out biomarker for AMI at presentation in patients with symptoms suggestive of ACS with a normal cTn value.

• An assay measuring copeptin (CT-proAVP) has been described using the Brahms Kryptor Immunology Analyzer, Diamond Diagnostics Holliston, MA.

• Additional clinical and analytical validation studies will be necessary, especially head-to-head comparisons of copeptin versus the new hs-cTn assays.