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
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 CHARACTERISTICS fk
Ability to influence therapy
Ability to improve patient outcome
Scand J Clin Lab Inves 1999;59 (Suppl 230):

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. BIOCHEMICAL MARKERS IN MYOCARDIAL ISCHAEMIA / NECROSIS
Traditional
AST activity
LDH activity
LDH isoenzymes
CK-Total
CK-MB activity
CK-Isoenzymes
RECENT
CK-MB (mass)
c.Troponins (I or T)
Myoglobin
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 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. 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

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
start
Peak
Duration of elevation
LD-1
24 – 47 h
48 – 72 h
7 – 10 days
Total CK
3 – 8 h
12 – 30 h
3 – 4 days
CK-MB
4 – 6 h
24 h
CK-MB isoforms
2 – 3 h
18 h
< 24 h
cTnI
6 h
cTnT
12 – 48 h
Myoglobin
2 h
6 – 7 h
IMA
Few minutes
2 – 4 h

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. 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

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. 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 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, 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
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