Biochemical Markers in Cardiac Disease

Dr/Ehsan Mohamed Rizk

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

CRITERIA FOR DIAGNOSIS OF ACSTriad of criteria: Clinical picture Severe & prolonged chest pain Atypical pain (epigastric) Silent ischemia.ECG changes consistent with acute MIElevated serum cardiac MARKERSDiagnosis requires at least two of them.

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.

THE IDEAL CARDIAC MARKERHIGH SENSITIVITYHigh concentration in myocardium Released after myocardial injury:Rapid release for early diagnosisLong half-life in blood for late diagnosisHIGH SPECIFICITYAbsent in non-myocardial tissueNot detectable in blood of non-diseased subjectsCLINICAL CHARACTERISTICS fkAbility to influence therapyAbility to improve patient outcomeANALYTICAL CHARACTERISTICSMeasurable by cost-effective method Simple to perform Rapid turnaround time Sufficient precision & accuracyThe ideal cardiacmarker does NOT yet exist!Scand J Clin Lab Inves 1999;59 (Suppl 230):113-123

CARDIAC MUSCLE CELLSize and subcellular distribution of myocardial proteins determines time course of biomarker appearance in the general circulation

CLASSIFICATION OF LABORATORY TESTS IN CARDIAC DISEASEMarkers of cardiac tissue damage

Markers of myocardial function

Cardiovascular risk factor markers

Genetic analysis for candidate genes or risk factors

PATHOPHYSIOLOGY OF ACSProinflammatory CytokinesIL-6Plaque DestabilizationMPOPlaque RupturesCD40LAcute Phase Reactants hs-CRP IschemiaIMANecrosis cTnTcTnIMyocardial DysfunctionBNPNT-proBNP

BIOCHEMICAL MARKERS IN MYOCARDIAL ISCHAEMIA / NECROSISRECENTCK-MB (mass)c.Troponins (I or T)MyoglobinTraditionalAST activityLDH activityLDH isoenzymesCK-TotalCK-MB activityCK-Isoenzymes

FUTURE:Ischaemia Modified AlbuminGlycogen Phosphorylase BBFatty Acid binding ProteinHighly sensitive CRP.

ASPARATATE AMINOTRANFERASE (AST)

An enzyme that catalysis the transfer of amino group from amino acid to keto acid which is important for providing 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 infarctionIt is elevated in other conditions as:Liver disease: hepatitis, liver cirrhosis, neoplasiaMuscle diseases: muscular dystrophy and dermatomyositis

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)

LDHBoth 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 daysUsually 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.

CREATINE KINASECK 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 tissueCK3 (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.

CREATINE KINASENORMAL VALUES:

Vary according to agesexracephysical conditionmuscle mass

PATHOLOGICAL INCREASES:

Myocardial infarction or injurySkeletal muscle injury or diseaseHypothyroidismIM injectionsGeneralised convulsionsCerebral injuryMalignant hyperpyrexiaProlonged hypothermia

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 -HypothyroidismMB Index = (CKMB /total CK) x 100Combined use with MB Index helps to rule-out patients with skeletal muscle injury

CK-MB RELATIVE INDEX AND CK-MB mass:

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

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).

MYOGLOBIN (Mb)Low MW proteinSkeletal & cardiac muscle Mb identicalSerum levels increase within 2h of muscle damagePeak at 6 9hNormal by 24 36hExcellent NEGATIVE predictor of myocardial injury2 samples 2 4 hours apart with no rise in levels virtually excludes AMIRapid, quantitative serum immunoassays

CARDIAC TROPONINSIt 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.

THE TROPONIN REGULATORY COMPLEX

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

TROPONIN SUMMARYRegulatory complex of striated muscle contractionEarly release ex cytosolic poolProlonged release due degradation of myofilamentsDistinct skeletal & myocardial muscle formsHigh specificity for myocardial injurySensitive to minor myocardial damage

ISCHAEMIA-MODIFIED ALBUMIN (IMA)Serum albumin is altered by free radicals released from ischaemic tissueAngioplasty 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 6hClinically may detect reversible myocardial ischaemic damageNot specific (elevated in stroke, some neoplasms, hepatic cirrhosis, end-stage renal disease) Thus potential value is as a negative predictorSpectrophotometric assay for IMA adapted for automated clinical chemistry analysers FDA approved as a rule-out marker in low risk ACS patients (2003)

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 contractionThree GP isoenzymes are found in human tissues: oGP-LL in liver oGP-MM in muscle oGP-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.

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

Marker start PeakDuration of elevation LD-1 24 47 h 48 72 h7 10 daysTotal CK 3 8 h12 30 h3 4 daysCK-MB 4 6 h24 h48 72 hCK-MB isoforms 2 3 h18 h< 24 hcTnI 6 h 24 h7 10 dayscTnT6 h12 48 h7 10 daysMyoglobin2 h6 7 h24 hIMAFew minutes2 4 h6 h

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

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 formationVariable degree of ischaemia resulting in reversible or irreversible injuryNon-occlusive plaques may produce sufficient ischaemia for release of low molecular weight markerscTnI & 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

CARDIAC TROPONINS IN UNSTABLE ANGINA PECTORIS (UA)Does an elevated Troponin level in the absence of other signs reflect irreversible myocardial damage?

Epidemiological studiesAnimal experimentsClinical trialsSensitive imaging techniquesSay YES!MI must be REDEFINED!QUESTION:

ACS REDEFINEDIf Troponins are not available, best alternative is CK-MBmassDegree of elevation of the marker is related to clinical riskCK(total), AST & LDH (Cardiac Enzymes) should NOT be used!Combine early (myoglobin) & late (Troponins) markersSerial testing: admission, 6 9 h, 12 24 hAn elevated Troponin level in the absence of clinical evidence of ischaemia should prompt searching for other causes of cardiac damage

ACS REDEFINEDRevised Criteria: Acute/Evolving/ Recent MI

Typical myocardial necrosis-associated rise & fall of Troponin or CK-MBmass

PLUSOne of:Cardiac Ischaemia symptomsQ waves on ECGST segment changes indicative of ischaemiaCoronary artery imaging (stenosis/obstruction)

OR Pathologic findings of an acute MI

BIOCHEMICAL MARKERS IN AMI ASSESSMENT OF REPERFUSIONWashout phenomenon enzymes & proteins have direct vascular access when occluded coronary circulation becomes patentPeak concentrations earlier & at higher levels if reperfusion successfulDue to short plasma half life (t = 10 min) Myoglobin is considered the best re-perfusion marker

BIOCHEMICAL MARKERS IN ACS CURRENT RECOMMENDATIONSAMI Routine diagnosisTroponins (CK-MBmass)Retrospective diagnosisTroponinsSkeletal muscle pathologyTroponinsReinfarctionMb, CK-MBmassReperfusionMb, Tn, CK-Mbmass Infarct sizeTroponinsRisk stratification in UATroponins

BIOCHEMICAL MARKERS OF MYOCARDIAL FUNCTIONCARDIAC NATRIURETIC PEPTIDES:(ANP, BNP & pro-peptide forms)

Family of peptides secreted by cardiac atria (+ ventricles) with potent diuretic, natriuretic & vascular smooth muscle relaxing activityLevels of these neuro-hormonal factors can be measured in bloodClinical usefulness (especially BNP/N-terminal pro-BNP)Detection of LV dysfunctionScreening for heart diseaseDifferential diagnosis of dyspnea

CARDIOVASCULAR RISK FACTORS

ESTABLISHED RISK FACTORSEVIDENCE 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 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?Boersma et al, Lancet, 2003:361,p849

GENETIC ANALYSIS OF CANDIDATE GENES OR RISK FACTORS FOR CARDIOVASCULAR DISEASERecent 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