بسم الله الرحمن الرحيم

1. بسم الله الرحمن الرحيم

2. Biochemical Markers in Cardiac Disease Dr/Ehsan Mohamed Rizk

3. ACUTE CORONARY SYNDROME (ACS) • Ischemic heart diseases (acute coronary syndrome) includes: 1-Angina 2-Unstable angina 3-Myocardial infarction: most serious form of ischemia that leads to injury or even death of myocardium. • The most common cause of myocardial ischemia is atherosclerosis. • Risk factors for Coronary Artery Disease: 1-Age 2-Gender 3-Family history 4-Hyperlipidemia 5-Smoking 6-Hypertension 7-Diabetes 8-Obesity 9-High plasma homocysteine levels

4. CRITERIA FOR DIAGNOSIS OF ACS • Triad of criteria: • Clinical picture Severe & prolonged chest pain Atypical pain (epigastric) Silent ischemia. • ECG changes consistent with acute MI • Elevated serum cardiac MARKERS • Diagnosis requires at least two of them.

6. CARDIAC MARKERS MUST BE: • Located in the myocardium. • Released in cardiac injury. – Myocardial infarction – Non-Q-wave infarction – Unstable angina pectoris – Other conditions affecting cardiac muscle (trauma, cardiac surgery, myocarditis etc.) • Can be measured in blood samples.

7. THE IDEAL CARDIAC MARKER The ideal cardiac marker does NOT yet exist! HIGH SENSITIVITY High concentration in myocardium Released after myocardial injury: Rapid release for early diagnosis Long half-life in blood for late diagnosis HIGH SPECIFICITY Absent in non-myocardial tissue Not detectable in blood of non-diseased subjects ANALYTICAL CHARACTERISTICS Measurable by cost-effective method Simple to perform Rapid turnaround time Sufficient precision & accuracy CLINICAL CHARACTERISTICSfk Ability to influence therapy Ability to improve patient outcome Scand J Clin Lab Inves 1999;59 (Suppl 230):113-123

8. CARDIAC MUSCLE CELL Size and subcellular distribution of myocardial proteins determines time course of biomarker appearance in the general circulation

9. CLASSIFICATION OF LABORATORY TESTS IN CARDIAC DISEASE • Markers of cardiac tissue damage • Markers of myocardial function • Cardiovascular risk factor markers • Genetic analysis for candidate genes or risk factors

10. PATHOPHYSIOLOGY OF ACS • Proinflammatory Cytokines • IL-6 • Plaque Destabilization • MPO • Plaque Rupture • sCD40L • Acute Phase Reactants • hs-CRP • Ischemia • IMA • Necrosis • cTnT • cTnI • Myocardial Dysfunction • BNP • NT-proBNP

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

12. ASPARATATE AMINOTRANFERASE (AST) • An enzyme that catalysis the transfer of amino group from amino acid to keto acid which is important for providing α keto acid for tricarboxylic acid cycle (energy production) and providing amino acid for urea cycle. • It is widely distributed in hear, liver, skeletal muscle, kidney and RBCs. • AST activity is increased after myocardial infarction • It is elevated in other conditions as: • Liver disease: hepatitis, liver cirrhosis, neoplasia • Muscle diseases: muscular dystrophy and dermatomyositis

13. LACTATE DEHYDROGENASE (LDH) • LDH is a hydrogen transfer enzyme that catalysis the oxidation of L-Lactate to Pyruvate. • It is composed of 4 subunits of 2 types M type encoded by a gene on ch 11 H type encoded by a gene on ch 12. • There are 5 isoenzymes: LD-1 (4 H subunits) LD-2 (3 H and 1 M sumunits) LD-3 (2 H and 2 M sumunits) LD-4 (1 H and 3 M sumunits) LD-5 (4 M subunits)

14. LDH • Both total and LDH isoenzymes are elevated in myocardial injury. • Level of LD-1 are elevated 10 – 12 after acute myocardial infarction, peak in 2 days and return to normal in 7 -10 days • Usually the amount of LD-2 in the blood is higher than amount of LD-1. Patient with AMI have more LD-1 than LD-2 (ratio > 1) this is called "Flipped Ratio". • An elevated level of LD=1 with flipped ratio has a sensitivity and specificity of approximately 75% - 90% for detection of AMI.

15. CREATINE KINASE • CK is a dimeric enzyme that regulates high energy phosphate production and utilization in contractile tissues. • It is composed of two subunits: M subunit encoded by a gene on chromosome 14. B subunit encoded by a gene on chromosome 19. • There are different isoenzymes: • CK1 (CK-BB): the predominant isoenzyme found in brain. • CK2 (CK-MB): represent 20 – 30 % of total CK in diseased cardiac tissue • CK3 (CK-MM): 98% in skeletal muscles and 1% in cardiac muscles. • CK-mitochondrial (CK-Mt): located in mitochondria and encoded by a different gene on chromosome 15. • Macro-CK: CK complexed with Igs.

16. NORMAL VALUES: 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

17. CREATINE KINASE: CK-MB • In normal population CK-MB < 6% Total CK • Sensitive marker with rapid rise & fall: • Serum CK-MB levels rise within 2~8 hours after AMI. • CK-MB values return to normal 2~3 days after the event. • More specific than total CK but has limitations: • False elevations in: • -perioperative patients without cardiac injury • -Skeletal muscle injury • -Marathon runners • -Chronic renal failure • -Hypothyroidism • MB Index = (CKMB /total CK) x 100 • Combined use with MB Index helps to rule-out patients with skeletal muscle injury

18. CK-MB RELATIVE INDEX AND CK-MB mass: • MB Index = (CKMB /total CK) x 10 Combined use with MB Index helps to rule-out patients with skeletal muscle injury • CK-MB MASS: • Measure the concentration of CK-MB protein is now available using sandwich technique with a detection limit < 1µg/dl. • More sensitive than measurement of activity.

19. CK-isoforms: • Both M and B subunits have N-terminal lysine residues but only M subunit is hydrolyzed by carboxypeptidase-N enzyme found in blood. • CK-MM is present in three isoforms: CK-MM3: tissue form. CK-MM2: (one lysine residue is removed). CK-MM1: (both lysine residue are removed) • CK-MB has two isoforms: CK-MB2: tissue form. CK-MB1: circulating form. • The ratio of tissue isoforms and plasma modified isoforms are used as markers of recent myocardial damage (elevated CK-MM3/CK-MM2 and CK-MB2/CK-MB1/CK-MB1 indicates a rise in tissue isoforms caused by recent release).

20. MYOGLOBIN (Mb) • Low MW protein • Skeletal & cardiac muscle Mb identical • Serum levels increase within 2h of muscle damage • 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 • Rapid, quantitative serum immunoassays

21. CARDIAC TROPONINS • It consists of 3 subunits troponin C, I, and T. • The complex regulates the contraction of striated muscle. • TnC binds to calcium ions. • TnI binds to actin and inhibits actin-myosin interaction. • TnT binds to tropomyosin, attaching to thin filament.

22. THE TROPONIN REGULATORY COMPLEX

23. cTns cTnI cTnT 1. Cardiac Troponin I (cTnl) is a cardiac muscle protein with a molecular weight of 24 kilo-Daltons. 2. The human cTnl has a additional amino acid residues on its N-terminal that are not exist on the skeletal form. 3. The half life of cTnI is estimated to be 2~4 hours. 4. Serum increase is found between 2-8 hours and returns to normal 7~10 days after AMI. 5. Cardiac TnI levels provide useful prognostic information. 6. Reference range: cTnI <2 ng/ml 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. 5. Peak between 12~96 hours and return to normal 14 days after AMI.

24. TROPONIN SUMMARY • Regulatory complex of striated muscle contraction • Early release ex cytosolic pool • Prolonged release due degradation of myofilaments • Distinct skeletal & myocardial muscle forms • High specificity for myocardial injury • Sensitive to minor myocardial damage

25. 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 • Spectrophotometric assay for IMA adapted for automated clinical chemistry analysers • FDA approved as a rule-out marker in low risk ACS patients (2003)

26. Glycogen phosphorylase BB (GPBB): • Glycogen phosphorylase (GP) is a glycolytic enzyme which plays an essential role in the regulation of carbohydrate metabolism. • It functions to provide energy supply for muscle contraction • Three GP isoenzymes are found in human tissues: o GP-LL in liver o GP-MM in muscle o GP-BB in brain. • GP-BB is the predominant isoenzyme in myocardium. With the onset of tissue hypoxia when glycogen is broke down, GP-BB is converted from structurally bound to cytoplasmic form. • In AMI GP-BB: Increases 1 – 4 after onset of chest pain • Peaks before CK-MB and cTnT • Return to reference interval 1 – 2 days after AMI. • However it is not cardiac specific.

27. BIOCHEMICAL MARKERS IN ACS: RELEASE, PEAK AND DURATION OF ELEVATION

28. BIOCHEMICAL MARKERS IN ACS: RELEASE, PEAK AND DURATION OF ELEVATION

29. BIOCHEMICAL MARKERS IN ACS UNSTABLE ANGINA PECTORIS (UA) • Characterised by chest pain at rest • ? Caused by disruption of liquid-filled atherosclerotic plaque with platelet aggregation & thrombus formation • Variable degree of ischaemia resulting in reversible or irreversible injury • Non-occlusive plaques may produce sufficient ischaemia for release of low molecular weight markers • cTnI & cTnT are often elevated in patients with unstable angina pectoris without additional clinical signs (ECG) or classical laboratory signs of acute MI (elevated CK-MB) • These patients have a very high risk of cardiac events

30. 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!

31. “ACS REDEFINED” • If Troponins are not available, best alternative is CK-MBmass • Degree of elevation of the marker is related to clinical risk • CK(total), AST & LDH (Cardiac Enzymes) should NOT be used! • Combine early (myoglobin) & late (Troponins) markers • Serial testing: admission, 6 – 9 h, 12 – 24 h • An elevated Troponin level in the absence of clinical evidence of ischaemia should prompt searching for other causes of cardiac damage

32. ACS REDEFINED Revised Criteria: Acute/Evolving/ Recent MI • Typical myocardial necrosis-associated rise & fall of Troponin or CK-MBmass PLUS • One of: • Cardiac Ischaemia symptoms • Q waves on ECG • ST segment changes indicative of ischaemia • Coronary artery imaging (stenosis/obstruction) • OR Pathologic findings of an acute MI

33. Successful reperfusion Marker Level Unsuccessful reperfusion Time BIOCHEMICAL MARKERS IN AMI ASSESSMENT OF 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

34. 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

35. 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

36. ++Clear evidence, and modification of the risk factor decreases the risk of cardiovascular disease + Clear evidence, but less clear whether modification of the risk factor decreases the risk of cardiovascular disease ? Risk factor under scrutiny CARDIOVASCULAR RISK FACTORS ESTABLISHED RISK FACTORS EVIDENCE Raised serum low density lipoprotein cholesterol ++ Decreased serum high density lipoprotein cholesterol ++ Smoking ++ High Blood pressure ++ Increased plasma glucose concentrations + Physical inactivity + Obesity + Advanced age + EMERGING RISK FACTORSInflammatory MarkersSensitive C-reactive protein + Interleukins + Serum amyloid A + Pregnancy-associated plasma protein A ? Chronic infection (Chlamydia pneumoniae, ? Helicobacter pylori, etc) Procoagulant MarkersPlasmaHomocysteine + Tissue plasminogen activator + Plasminogen activator inhibitor + Lipoprotein A + Process Markers Fibrinogen + D-dimer ? Coronary artery calcification ? Boersma et al, Lancet, 2003:361,p849

37. GENETIC ANALYSIS OF CANDIDATE GENES OR RISK FACTORS FOR CARDIOVASCULAR DISEASE • Recent explosion of genetic analysis & micro-array technology • Common cardiovascular diseases are polygenic. Multiple susceptibility loci interact with lifestyle & environment • Single gene defects may account for some of the cardiomyopathies, inherited cardiac arrhythmias • Possible genetic cardiovascular risk factors under assessment • Technology is still complex & expensive but is developing very rapidly

38. Thank you