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 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.
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 MARKER
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
CLINICAL CHARACTERISTICS
fk
Ability to influence therapy
Ability to improve patient outcome

ANALYTICAL
CHARACTERISTICS
Measurable by cost-effective
method
Simple to perform
Rapid turnaround time
Sufficient precision & accuracy
The ideal cardiac
marker does
NOT yet exist!
Scand J Clin Lab Inves 1999;59 (Suppl 230):113-123
CARDIAC MUSCLE CELL
Size and subcellular distribution of myocardial proteins determines time
course of biomarker appearance in the general circulation
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
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
BIOCHEMICAL MARKERS IN
MYOCARDIAL ISCHAEMIA / NECROSIS
RECENT
CK-MB (mass)
c.Troponins (I or T)
Myoglobin
Traditional
AST activity
LDH activity
LDH isoenzymes
CK-Total
CK-MB activity
CK-Isoenzymes

FUTURE:
Ischaemia Modified Albumin
Glycogen Phosphorylase BB
Fatty Acid binding Protein
Highly 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
infarction
It is elevated in other conditions as:
Liver disease: hepatitis, liver cirrhosis, neoplasia
Muscle 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)
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.

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.

CREATINE KINASE
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: 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
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 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
CARDIAC TROPONINS
It consists of 3 subunits troponin C, I, and T.
The complex regulates the contraction of
striated muscle.
1. TnC binds to calcium ions.
2. TnI binds to actin and inhibits actin-myosin
interaction.
3. 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 <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.


cTnI cTnT

cTns
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
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)

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.
BIOCHEMICAL MARKERS IN ACS:
RELEASE, PEAK AND DURATION OF ELEVATION
Marker
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 48 72 h
CK-MB
isoforms
2 3 h 18 h < 24 h
cTnI 6 h 24 h 7 10 days
cTnT 6 h 12 48 h 7 10 days
Myoglobin 2 h 6 7 h 24 h
IMA Few minutes 2 4 h 6 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 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
CARDIAC TROPONINS IN
UNSTABLE ANGINA PECTORIS (UA)
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!
QUESTION:
ACS REDEFINED
If Troponins are not available, best alternative is CK-
MB
mass
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
ACS REDEFINED
Revised Criteria: Acute/Evolving/ Recent MI
Typical myocardial necrosis-associated rise & fall of
Troponin or CK-MB
mass
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



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 (t

= 10 min) Myoglobin is considered the
best re-perfusion marker

Time
M
a
r
k
e
r

L
e
v
e
l

Successful
reperfusion
Unsuccessful
reperfusion
BIOCHEMICAL MARKERS IN ACS
CURRENT RECOMMENDATIONS
AMI Routine diagnosis Troponins (CK-MB
mass
)
Retrospective diagnosis Troponins
Skeletal muscle pathology Troponins
Reinfarction Mb, CK-MB
mass

Reperfusion Mb, Tn, CK-Mb
mass


Infarct size Troponins
Risk stratification in UA Troponins
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
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 ?
Boersma et al, Lancet, 2003:361,p849
++ 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
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