CARDIAC BIOMARKERS:

History  1950’s: Clinical reports that transaminases released from dying myocytes could be detected via laboratory testing, aiding in the diagnosis of myocardial infarction 1  The race to define clinical markers to aid in the diagnosis, prognosis, and risk stratification of patients with potential cardiovascular disease begins 1 Circulation 108:

History  Initial serum markers included AST, LDH, total CK and α -hydroxybutyrate  These enzymes are all released in varying amounts by dying myocytes  Lack of sensitivity and specificity for cardiac muscle necrosis fuels continued research

History: CK and Isoenzymes  CK known to be released during muscle necrosis (including cardiac)  Quantitative assays were cumbersome and difficult to perform  Total CK designed as a fast, reproducible spectrophotometric assay in the late 1960’s

History: CK and Isoenzymes  CK isoenzymes are subsequently described  MM, MB and BB fractions  1970’s: MB fraction noted to be elevated in and highly specific for acute MI 1 1 Clinical Chemistry 50(11):

History: CK and Isoenzymes  CKMB now measured via a highly sensitive monoclonal antibody assay  It was felt for a time that quantitative CKMB determination could be used to enzymatically measure the size of an infarct  This has been complicated by release of additional enzymes during reperfusion

History: CK and Isoenzymes  As CK-MB assays become more sensitive, researchers come to the paradoxical realization that it too is not totally cardiac specific  The MB fraction is determined to be expressed in skeletal muscle, particularly during the process of muscle regeneration  The search for cardiac specificity continues… Clinical Chemistry 50(11):

History  Research turns towards isolation of and development of assays for sarcomeric proteins  Myosin light chains were originally isolated and then subsequently abandoned because of specificity issues

History: Troponin  Troponin I first described as a biomarker specific for AMI in ; Troponin T in  Now the biochemical “gold standard” for the diagnosis of acute myocardial infarction via consensus of ESC/ACC 1 Am Heart J 113: J Mol Cell Cardiol 21:

History  This work encourages development of other clinical assays for diagnosis and prognosis of a wide spectrum of cardiac diseases  Notable examples:  BNP (FDA approved in November 2000 for diagnosis of CHF)  C-reactive protein

MARKERS OF CARDIAC NECROSIS

What is Myocardial Infarction?  Myocardial ischemia results from the reduction of coronary blood flow to an extent that leads to insufficiency of oxygen supply to myocardial tissue  When this ischemia is prolonged & irreversible, myocardial cell death & necrosis occurs ---this is defined as: myocardial infarction

Biochemical Changes in Acute Myocardial Infarction (mechanism of release of myocardial markers) ischemia to myocardial muscles (with low O 2 supply) anaerobic glycolysis increased accumulation of Lactate decrease in pH activate lysosomal enzymes disintegration of myocardial proteins cell death & necrosis release of intracellular contents to blood BIOCHEMICAL MARKERS clinical manifestations (chest pain) ECG changes

Diagnosis of Myocardial Infarction SHOULD depend on THREE items (as recommended by WHO) 1- Clinical Manifestations 2- ECG 3- Biochemical Markers

Markers of Cardiac Necrosis  Cardiac biomarkers an integral part of the most recent joint ACC/ESC consensus statement on the definition of acute or recent MI:

“Perfect” Cardiac Marker  Early appearance  Accurate, specific, precise  Readily available, fast results  Cost-effective

Markers of Cardiac Necrosis Typical rise and gradual fall (troponin) or more rapid rise and fall (CK-MB) of biochemical markers of myocardial necrosis with at least (1) of the following: Ischemic symptoms Development of pathologic Q waves ST segment elevation or depression Coronary artery intervention

Markers of Cardiac Necrosis

Troponins  Troponin T (cTnT) and troponin I (cTnI) control the calcium-mediated interaction of actin and myosin  cTnI completely specific for the heart  cTnT released in small amounts by skeletal muscles, though clinical assays do not detect skeletal TnT

Troponins

 4-6 hours to rise post-infarct, similar to CKMB  6-9 hours to detect pathologic elevations in all patients with infarct  Elevated levels can persist in blood for weeks; the cardiac specificity of troponins thus make them the ideal marker for retrospective diagnosis of infarction

CK-MB  High specificity for cardiac tissue  The preferred marker for cardiac injury for many years  Begins to rise 4-6 hours after infarction but can take up to 12 hours to become elevated in all patients with infarction  Elevations return to baseline within hours, in contrast to troponins  CK-MB is the marker of choice for diagnosis of reinfarction after CABG because of rapid washout

CK and CK-MB

CK-MB: Shortcomings  Concomitant skeletal muscle damage can confuse the issue of diagnosis:  CPR and defibrillation  Cardiac and non-cardiac procedures  Blunt chest trauma  Cocaine abuse

CK:CK-MB Ratio  Proposed to improve specificity for use in diagnosis of AMI  Ratios have been proposed  Significantly reduces sensitivity in patients with both skeletal muscle and cardiac injury  Also known to be misleading in the setting of hypothyroidism, renal failure, and chronic skeletal muscle diseases

Myoglobin  Heme protein rapidly released from damaged muscle  Elevations can be seen as early as one hour post- infarct  Much less cardiac specific; meant to be used as a marker protein for early diagnosis in conjunction with troponins

NATRIURETIC PEPTIDES

Natriuretic Peptides  Present in two forms, atrial (ANP) and brain (BNP)  Both ANP and BNP have diuretic, natriuretic and hypotensive effects  Both inhibit the renin-angiotensin system and renal sympathetic activity  BNP is released from the cardiac ventricles in response to volume expansion and wall stress

BNP Assay  Approved by the FDA for diagnosis of cardiac causes of dysnpea  Currently measured via a rapid, bedside immunofluorescence assay taking 10 minutes  Especially useful in ruling out heart failure as a cause of dyspnea given its excellent negative predictive value

BNP  Came to market in 2000 based on data from many studies, primarily the Breathing Not Properly (BNP) study  Prospective study of 1586 patients presenting to the ER with acute dyspnea  The predictive value of BNP much superior to previous standards including radiographic, clinical exam, or Framingham Criteria

BNP  BNP has also shown utility as a prognostic marker in acute coronary syndrome  It is associated with increased risk of death at 10 months as concentration at 40 hours post-infarct increased  Also associated with increased risk for new or recurrent MI

PROGNOSTIC MARKERS AND MARKERS OF RISK STRATIFICATION

Prognostic Markers and Markers of Risk Stratification  C-reactive protein  Myeloperoxidase  Homocysteine  Glomerular filtration rate

C-Reactive Protein  Multiple roles in cardiovascular disease have been examined  Screening for cardiovascular risk in otherwise “healthy” men and women  Predictive value of CRP levels for disease severity in pre-existing CAD  Prognostic value in ACS

C-Reactive Protein  Pentameric structure consisting of five 23-kDa identical subunits  Produced primarily in hepatocytes  Plasma levels can increase rapidly to 1000x baseline levels in response to acute inflammation  “Positive acute phase reactant”

C-Reactive Protein  Binds to multiple ligands, including many found in bacterial cell walls  Once ligand-bound, CRP can:  Activate the classical compliment pathway  Stimulate phagocytosis  Bind to immunoglobulin receptors

C-Reactive Protein: Risk Factor or Risk Marker?  CRP previously known to be a marker of high risk in cardiovascular disease  More recent data may implicate CRP as an actual mediator of atherogenesis  Multiple hypotheses for the mechanism of CRP- mediated atherogenesis:  Endothelial dysfunction via ↑ NO synthesis  ↑ LDL deposition in plaque by CRP-stimulated macrophages

CRP and CV Risk  Elevated levels predictive of:  Long-term risk of first MI  Ischemic stroke  All-cause mortality

Myeloperoxidase  Released by activated leukocytes at elevated levels in vulnerable plaques  Predicts cardiac risk independently of other markers of inflammation  May be useful in triage of ACS (levels elevate in the 1 st two hours)  Also identifies patients at increased risk of CV event in the 6 months following a negative troponin NEJM 349:

Homocysteine  Intermediary amino acid formed by the conversion of methionine to cysteine  Moderate hyperhomocysteinemia occurs in 5-7% of the population  Recognized as an independent risk factor for the development of atherosclerotic vascular disease and venous thrombosis  Can result from genetic defects, drugs, vitamin deficiencies, or smoking

Homocysteine  Homocysteine implicated directly in vascular injury including:  Intimal thickening  Disruption of elastic lamina  Smooth muscle hypertrophy  Platelet aggregation  Vascular injury induced by leukocyte recruitment, foam cell formation, and inhibition of NO synthesis

Homocysteine  Elevated levels appear to be an independent risk factor, though less important than the classic CV risk factors  Screening recommended in patients with premature CV disease (or unexplained DVT) and absence of other risk factors  Treatment includes supplementation with folate, B6 and B12

Glomerular Filtration Rate  The relationship between chronic kidney disease and cardiovascular risk is longstanding  Is this the result of multiple comorbid conditions (such as diabetes and hypertension), or is there an independent relationship?

Glomerular Filtration Rate  Recent studies have sought to identify whether creatinine clearance itself is inversely related to increased cardiovascular risk, independent of comorbid conditions

Glomerular Filtration Rate  Go, et al performed a cohort analysis of 1.12 million adults in California with CKD that were not yet dialysis-dependent  Their hypothesis was that GFR was an independent predictor of cardiovascular morbidity and mortality  They noted a strong independent association between the two NEJM 351:

Glomerular Filtration Rate  Reduced GFR has been associated with:  Increased inflammatory factors  Abnormal lipoprotein levels  Elevated plasma homocysteine  Anemia  Arterial stiffness  Endothelial dysfunction