Head of Cardiology Department , 6th October University Head of Egypt Hearts Society

Pathophysiology of ACS and biochemichal markers release

Biochemical profile in ACS patients: vascular inflammation to plaque rupture to ischemia to cell death to myocardial dysfunction Apple, F. S. et al. Clin Chem 2005;51:810-824 Copyright ©2005 American Association for Clinical Chemistry

Interdependence of Cardiac Biomarkers Pathophysiology Biochemical Markers Coronary artery disease Risk factors (eg, cholesterol) Coronary inflammation CRP, Lp-PLA2*, homocysteine, MPO Plaque instability/disruption MPO, Lp(a), Lp-PLA2 Myocardial ischemia/necrosis Cardiac troponins, CK-MB, myoglobin Ventricular overload BNP, Nt-proBNP Adapted from Panteghini. Eur Heart J. 2004;25:1187-1196. * Lipoprotein associated phospholipase A 2

TIME LINE OF MARKERS OF MYOCARDIAC DAMAGE & FUNCTION Myoglobin assay RIA for BNP and proANP RIA for proBNP CK – MB CK-MB mass assay cTnl assay Electrophoresis for CK and LD POCT for myoglobin CK-MB, cTnI AST in AMI CK in AMI RIA for ANP cTnT assay Immuno assay for proBNP IMA Genetic Markers 1950 1960 1970 1980 1990 2000 2005 Time [years] Timeline history of assay methods for markers of cardiac tissue damage and myocardial function. AST: aspartate aminotransferase ANP: atrial natriuretic peptide CK: creatine kinase BNP: brain natriuretic peptide LD: lactate dehyydrogenase POCT: point-of-care testing cTn: cardiac-specific troponin IMA: ischaemia-modified albumin

CLINICAL CHARACTERISTICS AND UTILIZATION OF BIOCHEMICAL MARKERS IN ACS USE OF BIOCHEMICAL MARKERS IN THE INITIAL EVALUATION OF ACS MANAGEMENT OF NSTEACS MANAGEMENT OF STEMI

USE OF BIOCHEMICAL MARKERS IN THE INITIAL EVALUATION OF ACS A. Diagnosis of myocardial infarction 1. Biochemical markers of myocardial necrosis 2. Optimal timing of sample acquisition 3. Criteria for diagnosis of MI 4. Additional considerations in the use of bio-markers for diagnosis of MI B. Early Risk Stratification 1. Biochemical markers of cardiac injury 2. Natriuretic peptides 3. Biochemical markers of inflammation 4. Biochemical markers of ischemia 5. Multimarker approach 6. Other novel markers

QUESTIONS ANSWERED BY CARDIAC MARKERS Rule in/out an acute MI Confirm an old MI (several days) Monitor the success of thrombolytic therapy Risk stratification of patients with unstable angina pectoris N.B. Risk stratification in apparently healthy persons is not done with cardiac markers, but by measurement and assessment of cardiac risk factors R. Hinzmann, 2002

BIOCHEMICAL MARKERS IN MYOCARDIAL ISCHAEMIA / NECROSIS CK-MB (mass) c.Troponins (I or T) Myoglobin OUT: AST activity LDH activity LDH isoenzymes CK-MB activity CK-Isoenzymes ?CK-Total FUTURE: Ischaemia Modified Albumin Glycogen Phosphorylase BB Fatty Acid binding Protein

“CARDIAC ENZYMES” are Obsolete!

KINETICS OF CARDIAC MARKERS AFTER AMI MARKER DETECTION PEAK DISAPPEARANCE Myoglobin 1 – 4 h 6 – 7 h 24 h CK-MB mass 3 – 12 h 12 – 18 h 2 – 3 days Total CK 4 – 8 h 12 – 30 h 3 – 4 days cTnT 4 – 12 h 12 – 48 h 5 – 15 days cTnI 4 – 12 h 12 – 24 h 5 – 7 days These values represent averages. IMA (ischaemia) few minutes 2 – 4 h 6 hours

Marker of Ischemia

ISCHAEMIA-MODIFIED ALBUMIN (IMA) Serum albumin is altered by free radicals released from ischaemic tissue Angioplasty studies show that albumin is modified within minutes of the onset of ischaemia. IMA levels rise rapidly, remain elevated for 2-4 h + return to baseline within 6h Clinically may detect reversible myocardial ischaemic damage Not specific (elevated in stroke, some neoplasms, hepatic cirrhosis, end-stage renal disease) Thus potential value is as a negative predictor FDA approved as a rule-out marker in low risk ACS patients (2003).

Established biomakrkers: Creatinin Kinase-MB (mass) Myoglobin. Troponin.

CREATINE KINASE NORMAL VALUES: PATHOLOGICAL INCREASES: Vary according to – age sex race physical condition muscle mass PATHOLOGICAL INCREASES: Myocardial infarction or injury Skeletal muscle injury or disease Hypothyroidism IM injections Generalised convulsions Cerebral injury Malignant hyperpyrexia Prolonged hypothermia

CREATINE KINASE: CK-MB CK-MB is the most cardiac-specific CK isoenzyme Sensitive marker with rapid rise & fall “Gold standard” biochemical marker for ~ 2 decades Only CK-MBmass should be measured

Sensitivity Specificity Myoglobin Troponins Currently earliest marker Like total CK it is by no means cardio-specific Troponins Kinetics comparable with total CK and CK-MB Cardio-specific Sensitivity Specificity R. Hinzmann, 2002

MYOGLOBIN (Mb) Peak at 6 – 9h Normal by 24 – 36h Excellent NEGATIVE predictor of myocardial injury 2 samples 2 – 4 hours apart with no rise in levels virtually excludes AMI

CARDIAC TROPONINS Striated and cardiac muscle filaments consist of: Actin Myosin Troponin regulatory complex Troponin consists of 3 sub-units TnC, TnT & TnI

TROPONIN SUMMARY High specificity for myocardial injury Sensitive to minor myocardial damage

THE DUAL APPROACH LEAVES AN OPEN QUESTION Troponin concentration ? normal acute MI 97.5 th percentile Acute MI cut-off value

Troponin IN UA Several studies have investigated the role of TnT/I in risk stratification of unstable angina (UA) Of importance is that UA patients with elevated Tn showed same incidence of cardiac death or AMI at 6 months as did patients with pre-existing AMI (15%) Risk of AMI in UA patients with normal Tn was 4 %. Irreversible minor myocardial injury detected by TnT/I may stratify UA patients as high risk for progression to AMI

INCIDENCE OF DEATH OR MI IN ACS PATIENTS Baseline levels of troponin have been shown to predict the risk of adverse cardiac events in patients with non-ST elevation ACS From: NEJM 1997;337:1648 (Study 1);JACC 1998;32:8 (Study 2); Circulation 1997;95:2053 (Study 3); Am J Cardiol 2002;89:1035 (Study 4).

CLINICAL OUTCOME AT DIFFERENT FOLLOW-UP PERIODS The prognostic information of an elevated cTnI upon presentation is maintained over time. From: JACC 2000;36:1812 and Am J Cardiol 2002;89:1035

CARDIAC TROPONINS IN UNSTABLE ANGINA PECTORIS (UA) QUESTION: Does an elevated Troponin level in the absence of other signs reflect irreversible myocardial damage? Epidemiological studies Animal experiments Clinical trials Sensitive imaging techniques Say YES! MI must be REDEFINED!

New criteria for acute, evolving or recent MI Either one of the following criteria satisfies the diagnosis for an acute, evolving or recent MI : 1. Typical rise and gradual fall (troponin) or more rapid rise and fall (CK-MB) of biochemical markers of myocardial necrosis with at least one of the following: a) ischemic symptoms; b) development of pathologic Q waves on the ECG; c) ECG changes indicative of ischemia (ST segment elevation or depression); or d) coronary artery intervention (e.g. coronary angioplasty). 2. Pathologic findings of an acute MI

Etiologies for Cardiac Troponin Increases AMI NSTEMI False +ve (eg heterophilic antibodies) Pericarditis Pulmonary Embolism Sepsis Shock Acute LVF Trauma Hypertension/Hypotension Drug Toxicity Iatrogenic Cardiac Surgery PCI Cardioversion Cardiotoxin Drugs EP Ablation

TROPONIN AND MI DIAGNOSIS "It is estimated that about 30% of patients who present with chest pain without ST-segment elevation and would otherwise be diagnosed as having unstable angina because of a lack of CK-MB elevation actually have NSTEMI when assessed with cardiac-specific troponin assays" From:JACC and Circulation 2002

PREDICTION OF RISK/PROGNOSIS Troponin can be used to efficiently categorise patients into high and low risk groups for appropriate management pathways. Adapted from: ACC/AHA Guideline Update for the Management of patients with UA and NSTEMI. 2002

BIOCHEMICAL MARKERS IN ACS CLINICAL DECISION POINTS Unstable Angina AMI Infarct size Prognosis Thrombolysis and Reperfusion Peri-operative infarcts Coronary surgery complications Transplant rejection

BIOCHEMICAL MARKERS IN AMI ASSESSMENT OF REPERFUSION Time Marker Level Successful reperfusion Unsuccessful reperfusion “Washout” phenomenon – enzymes & proteins have direct vascular access when occluded coronary circulation becomes patent Peak concentrations earlier & at higher levels if reperfusion successful Due to short plasma half life (t½ = 10 min) Myoglobin is considered the best re-perfusion marker

BIOCHEMICAL MARKERS IN ACS CURRENT RECOMMENDATIONS AMI – Routine diagnosis Troponins (CK-MBmass) Retrospective diagnosis Troponins Skeletal muscle pathology Troponins Reinfarction Mb, CK-MBmass Reperfusion Mb, Tn, CK-Mbmass Infarct size Troponins Risk stratification in UA Troponins

Problems with the current biochemical markers

The perfect marker Marker for myocardial necrosis, and also for cardiac ischemia Linear relationship between blood levels and extent of myocardial injury (and prognosis) 100% sensitive 100% specific Immediate increase (+ constant blood level for hours to days) Test kits : reliable, rapid, universally available and inexpensive

What about troponin T and I ? Very high sensitivity for myocardial necrosis Related to prognosis Not 100% specific for atherosclerotic coronary artery disease myocarditis, cardiomyopathy, myocardial contusion, ... renal failure, auto-immune diseases, ...) Up to 6 hours before raised blood levels no early MI diagnosis possible Raised blood levels for many days troublesome diagnosis of re-infarction BUT

Role for myoglobin ? Initial elevation : 1 to 4h after onset better early marker than troponins BUT : early myoglobin is less sensitive and less specific (due to skeletal muscle trauma) than late troponin decisions mainly based on clinical skills, ECG and late troponin (except rarely for reperfusion therapy) Duration of elevation : 24 – 48h useful for re-infarction diagnosis

Role for CK-MB ? Initial elevation comparable with troponins Less sensitive than troponins High specificity (comparable with troponins) Rapid rise and fall (instead of gradual fall for troponins) allowing more accurate estimation of MI extent

OTHER MARKERS CURRENTLY UNDER INVESTIGATION Free fatty acids Fibrin peptide A Fatty acid binding protein Glycogen phosphorylase BB

Markers of myocardial function

BIOCHEMICAL MARKERS OF MYOCARDIAL FUNCTION CARDIAC NATRIURETIC PEPTIDES: (ANP, BNP & pro-peptide forms) Family of peptides secreted by cardiac atria (+ ventricles) with potent diuretic, natriuretic & vascular smooth muscle relaxing activity Levels of these neuro-hormonal factors can be measured in blood Clinical usefulness (especially BNP/N-terminal pro-BNP) Detection of LV dysfunction Screening for heart disease Differential diagnosis of dyspnea Stratification of CCF patients New generation markers currently under development

SOME COMMON DISEASES IN WHICH PLASMA CARDIAC NATRIURETIC PEPTIDES HAVE BEEN FOUND TO BE ALTERED, COMPARED TO HEALTHY SUBJECTS DISEASES ANP/BNP LEVELS Cardiac diseases Heart failure AMI (first 2 – 3 days) Essential hypertension with CMP Pulmonary diseases Acute dyspnea Obstructive pulmonary disease Endocrine & metabolic diseases Hyperthyroidism Hypothyroidism Cushing’s syndrome Primary aldosteronism Addison’s disease Diabetes mellitus Liver cirrhosis with ascites Renal failure (acute or chronic) Greatly increased Greatly increased Increased Increased Increased Increased Decreased Increased Increased Normal or increased Normal or increased Increased Greatly increased AMI = acute myocardial infarction; CMP = cardiomyopathy with left ventricular hypertrophy Clarico; Clin Chem Lab Med, 2003; 41 (17) p876

CARDIOVASCULAR RISK FACTORS EMERGING RISK FACTORS Inflammatory Markers Sensitive C-reactive protein + Interleukins + Serum amyloid A + Pregnancy-associated plasma protein A ? Chronic infection (Chlamydia pneumoniae, ? Helicobacter pylori, etc) Procoagulant Markers Plasma Homocysteine + Tissue plasminogen activator + Plasminogen activator inhibitor + Lipoprotein A + Process Markers Fibrinogen + D-dimer ? Coronary artery calcification ? + Clear evidence, but less clear whether modification of the risk factor decreases the risk of cardiovascular disease ? Risk factor under scrutiny Boersma et al, Lancet, 2003:361,p849

GUIDELINES: USE OF CARDIAC MARKERS IN PATIENTS WITH CHEST PAIN Serial sampling is critical for accurate diagnosis Do NOT discharge patients on the basis of negative results on a single (admission) specimen If onset of chest pain >9-12 h before admission only Troponin is necessary CK-MBmass is most useful in assessing a recent vs an older MI or to confirm reinfarction (occurs in 17% of AMI’s). Repeat CK-MBmass if chest pain recurs in AMI patients

GUIDELINES: USE OF CARDIAC MARKERS IN PATIENTS WITH CHEST PAIN Mb, CK-MBmass, Troponin POSITIVE AMI Mb ONLY POSITIVE Possible early infarction or skeletal muscle injury Repeat markers (NB importance of Mb is as a Negative Predictor) Mb + CK-MB POSITIVE Probable early infarction A rising CK-MB.

GUIDELINES: USE OF CARDIAC MARKERS IN PATIENTS WITH CHEST PAIN TnI < 0.06 ng/mL OR TnT < 0.03 ng/mL on two specimens > 6 hours apart Unstable Angina Troponin I > 0.06 OR TnT > 0.1 ng/mL (TnT levels > 0.03 and < 0.1 ng/mL are equivocal and should be repeated) ? High risk ACS(AMI) or non-ischaemic myocardial damage depending on clinical cardiac ischaemia These patients require follow-up!! Troponin I > 0.4 ng/mL “traditional” AMI

GUIDELINES: USE OF CARDIAC MARKERS IN PATIENTS WITH CHEST PAIN FOR ASSESSMENT OF: Reperfusion Mb, CK-MBmass Intra- or post-operative AMI Troponin MI after percutaneous Troponin ( in 30 - 40 % patients) coronary artery intervention CK-MB ( in 5 - 30 % patients) (compare with baseline or use 5-15 fold higher cut-off level) Reinfarction serial CK-MBmass determinations

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

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 1st two hours) Also identifies patients at increased risk of CV event in the 6 months following a negative troponin NEJM 349: 1595-1604

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

So much information! What does it all mean?!?

The Future of Cardiac Biomarkers Many experts are advocating the move towards a multimarker strategy for the purposes of diagnosis, prognosis, and treatment design As the pathophysiology of ACS is heterogeneous, so must be the diagnostic strategies

Multiple Markers Are Needed for Diagnosis and Prognosis of ACS No single ideal marker exists for ACS Complicated diseases are not likely to be associated with single markers Multiple markers define disease categories Multi-marker panels can aid in differential diagnosis 57

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markers of inflammation Leukocyte chemoattractants (MCP-1, IL-8, PDGF, MC-SF) Procoagulant activity (tissue factor) Cytokines (TNFa, FAS,CD40L) NO ET-1 Adhesion molecules E-selectin, ICAM-1, VCAM-1 Permeability Apoptosis

Markers of protection?

T T markers of inflammation IL-10 Leukocyte chemoattractants (MCP-1, IL-8, PDGF, MC-SF) Procoagulant activity (tissue factor) Cytokines (TNFa, FAS,CD40L etc.) NO ET-1 Adhesion molecules E-selectin, ICAM-1, VCAM-1 Permeability Apoptosis T hepatocyte growth factor

SUMMARY “Cardiac Enzymes” are obsolete Medical & laboratory progress has required a redefinition of Myocardial Infarction Cardiac Troponins & Myoglobin now play a pivotal role in the diagnosis of AMI Cardiac Troponins play an important role in the risk stratification of ACS patients Elevated Troponin levels in patients without ECG changes & with normal CK-MB levels may identify patients at increased risk of cardiac events

SUMMARY Elevated Troponins in the absence of clinical signs of ischaemic heart disease require consideration of other causes of cardiac injury Additional roles for cardiac markers in: Reperfusion monitoring Infarct size/prognosis Intra/post-operative MI (non-cardiac/cardiac surgery).

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