Cardiac Markers (Recap and Update)

CARDIAC BIOMARKERS
Recap & Update
Dr. Bikash Chaudhury
HOD-Biochemistry
Vijaya Diagnostic Center

Biomarker
• “A biomarker is a substance used as an
indicator of a biologic state”.
Morrow and de lomos three criteria for biomarkers
• Accurate repeated measurements at reasonable cost
• Must provide additional information
• Should aid treatment

Biomarker
An indicator used for objective
measurement and evaluation of
Response to
therapeutic
intervention
Pathogenic process
Normal biological
process
Dr. Bikash Kr. Chaudhury

Characteristics of an ideal biomarker
• Standardized
• High Sensitivity and Specificity
• Accurate
• Reproducible
• Easy to interpret
• Acceptable to patient
• Consistent and Cost effective
• Has an impact on clinical/risk management

BIOCHEMICAL
CARDIAC MARKERS
WHAT ARE CARDIAC MARKERS?
• 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

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
Risk stratification in apparently healthy persons is not done
with cardiac markers, but by measurement and assessment
of cardiac risk factors
R. Hinzmann, 2002

History of Cardiac
Biomarkers
 1954 - SGOT (AST)
 1955 - LDH
 1960 - CPK
 1972 - CPK isoforms by Electrophoresis
 1975 - CK - MB by immunoinhibition
 1975 - Myoglobin
 1985 - CK - MB Mass immunoassay
 1989 - Troponin T
 1992 - Troponin I

TIME LINE OF MARKERS OF
MYOCARDIAC DAMAGE & FUNCTION
1950 1960 1970 1980 1990 2000 2005
AST
in
AMI
CK in
AMI
Electrophoresis
for CK and LD
CK – MB
Myoglobin
assay
RIA for
ANP
CK-MB
mass
assay
cTnT
assay
RIA for BNP
and
proANP
cTnl
assay
RIA for
proBNP
POCT for myoglobin CK-
MB, cTnI
Immuno assay
for proBNP
IMA
Genetic
Markers
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
Time [years]

Cardiac biomarkers
• Cardiac biomarkers were first developed for assisting the cardiac
events, especially acute myocardial infarction.
• Better understanding of cardiac disease process and advancement
in detection technology has pushed the application of cardiac
biomarkers beyond the diagnosis boundaries.
• Cardiac biomarkers are now used for staging of cardiac disease,
timing of cardiac events and prognostification.

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

Classification
of
Cardiac Biomarkers
according to
variousstages
during cardiac
diseaseprocess

hsCRP
sCD40L
homocysteine
Marker of Inflammation

High-Sensitivity
C-Reactive Protein

• CRP is Pentameric structure consisting of five identical
subunits of 23-kDa.
• Its plasma levels can increase rapidly to 10,000x levels.
• It is the most extensively studied marker of inflammation.
Despite some controversy regarding its clinical use, it appears
to be the most promising to date.
• Although considered to be a general nonspecific marker of
inflammation, elevated baseline levels of hsCRP are correlated
with higher risk of future CV morbidity and mortality among
those with or without clinical evidence of CVD.
High-Sensitivity
C-Reactive Protein

Once ligand-bound, CRP can:
– Activate the classical compliment pathway
– Stimulate phagocytosis
– Bind to immunoglobulin receptors
– Endothelial dysfunction via ↑ NO synthesis
– ↑LDL deposition in plaque by CRP-stimulated
macrophages
More recent data implicate
CRP as an actual mediator of atherogenesis

High-Sensitivity
C-Reactive Protein

Clinical Uses
– Screening for cardiovascular risk in otherwise “healthy”
individuals
– Predictive value of CRP levels for disease severity in pre-
existing Coronary artery disease
Elevated levels are predictive of
• Long-term risk of first MI
• Ischemic stroke
High-Sensitivity
C-Reactive Protein

• Low specificity
• No evidence that lowering CRP levels decreases CV risk
Industry and FDA staff guidelines 2005 had given clinical cut off value as
less than 1 mg/l as safe levels with hs-CRP tests
CRP Risk for CVD
Less than 1.0 mg/L Low
1.0-2.9 mg/L Intermediate
Greater than 3.0 mg/L High
Limitations of CRP

• Soluble fragment CD 40 ligand.
• It is a signalling protein that reflects both inflammatory and platelet interaction.
• ↑ levels of sCD40L is associated with ↑ risk of cardiac events
• However rise is also associated with many other inflammatory conditions like RA,
SCD, SLE etc.
• Furthermore, pre-analytical procedure such as anticoagulant quantity, temperature,
time and centrifugation speed significantly affect the final result, which proves to be
potential barrier to practical application of test.
sCD40L

• 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
Homocysteine

• Homocysteine is implicated directly in vascular injury including:
– Intimal thickening
– Disruption of elastic lamina
– Smooth muscle hypertrophy
– Platelet aggregation
• Proposed mechanisms by which it induces vascular injury are leukocyte
recruitment, foam cell formation, and inhibition of NO synthesis.
• Normal levels : 3.7 – 13.9 µmol/L
Homocysteine

• Elevated levels of homocysteine appear to be an
independent risk factor, though less important than
the classic CV risk factors.
• Treatment includes supplementation with folate, B6
and B12.
Homocysteine

Markers of Plaque Destabilization
PAPP-A
LP-PLA₂

• Its high relative stability in plasma, have led to its potential use in the
clinical setting.
• Elevated level of PAPP-A are found in patients presenting with unstable
plaques, aggravated unstable angina and acute MI.
• It is also a reliable predictor of mortality in patients with chronic stable
CAD.
• FREE PAPP-A >1.74 mIU/L is considered abnormal
• Currently there is no standardised assay in widespread clinical use.Dr. Bikash Kr. Chaudhury
Pregnancy associated plasma
protein-A (PAPP-A)

• Lp-PLA₂ is also known as platelate activating factor
acetyl hydrolase.
• This phospholipase enzyme is encoded by PLA2G7
gene.
• It is a 45 kDa protein of 441 amino acids.
Lp-PLA₂
PAF LYSO-PAF + acetate
Lipoprotein-associated
phospholipase A₂

• In the blood it mainly travels with LDL. Less than 20% is associated with HDL.
• It is produced by inflammatory cells and hydrolyzes oxidised phospholipids in LDL
• Two main sources of Lp-PLA₂ are :
1. which is brought from circulation into the intima bound to LDL
2. which is synthesised de novo by plaque inflammatory cells.
• Lp-PLA₂ is involved in the development of atherosclerosis and It is positively correlated
with increased risk of developing coronary artery disease.
• Its level in blood is measured by PLAC test, an assay which uses sandwich ELISA.
• Average value for females is 174 ng/mL
for males is 251 ng/ml

Marker of Mocardial Ischemia
IMA
H-FABP

• Ischemia modified albumin is a marker formed after damage in the N
terminal region of the albumin in ischemic conditions.
• This structural change leads to loss of its ability to bind with transitional
metals (cu/co).
• Endothelial or extracellular hypoxia, acidosis and free oxygen radicals
causes increase in IMA.
• IMA rises within minutes from onset of ischemia and remains elevated for
several hours after cessation of ischemia.
Ischemia
Modified Albumin

it is used as diagnostic criteria for myocardial necrosis
that develops after CABG operation.
It is a non specific marker, since it is also reported to be
elevated in pulmonary infarction, critical limb ischemia
and cerebrovascular disorders.
Basically, it is used to rule out ischemia rather than
diagnosing the occurrence of ischemia. Which is helpful
in differentiating pain of Angina from Myocardial
ischemia. Dr. Bikash Kr. Chaudhury
Clinical uses of IMA :

• Heart type fatty acid binding protein is a very stable low molecular
weight (14-15kDa) in the cytoplasm of myocardial cells.
• FABPs are involved in active fatty acid metabolism where it transports
fatty acid from cell membrane to mitochondria for oxidation.
• Small size of H-FABP facilitates rapid diffusion through interstitial space,
appearing as early as 1-3 hrs after onset and peaking within 6hrs. It
return to normal levels with in 12-24hrs.
• Normal levels : 1.6 – 19 ng/ml
H-FABP

• H-FABP is 20 times more specific to cardiac muscle than myoglobin
• H-FABP is recommended to be measured with troponin to identify MI and
ACS in patient presenting with chest pain.
• In addition to its diagnostic potential H-FABP also has prognostic value.
The risk associated with ↑ H-FABP is dependent upon its concentration.
Patients who were cTnI- but H-FABP+ have more risk of morbidity and
mortality after 1 year follow up than those with cTnI+HFABP-.
H-FABP

Marker for cardiac necrosis
cTn
CK-MB
Myoglobins

• Troponin is a complex of three regulatory
proteins (Troponin C, Troponin I and Troponin T)
that is associated with muscle contraction in
skeletal and cardiac muscle.
• Cardiac troponin is slightly different from
skeletal troponin structurally hence serve as a
potent and specific marker for cardiac disease.
Cardiac Troponins

THE TROPONIN
REGULATORY COMPLEX

Individual subunits serve different functions:
• Troponin C binds to calcium ions to produce a
conformational change in TnI
• Troponin T binds to tropomyosin, interlocking them to
form a troponin-tropomyosin complex
• Troponin I binds to actin in thin myofilaments to hold
the troponin-tropomyosin complex in place
• Usually, Troponin is not detectable in healthy
individual.
Cardiac Troponins

• It is extremely useful in patients who do not seek attention in the 2
to 3 days window when CK-MB is elevated.
Rise : with in few hours after onset of chest pain
Peak : 2 days
returns normal : 7-10 days
• cTnT may show a biphasic release in some patients with a first peak
occurring during first 24 hr of onset of symptom and second peak
on appx. 4th day after admission.
• TnT has cardiac as well as skeletal muscle source.
cTnT :

• It is cardiac specific because it has additional amino acid residue
on its N-terminal that are non existent in skeletal muscle.
Rise : b/w 4-6 hr after onset of pain
Peaks : 12-18 hrs
Returns normal : 6 days
• Its measurement is advantageous over CK-MB as it is not found in
detectable amount in serum of patients with multiple injuries,
renal disease and in those with acute and chronic skeletal muscle
disorders.
cTnI :

• Arrhythmias
• Congestive heart failure
• Coronary artery disease
• Coronary vasospasm
• Critically ill patient
• Hypertension
• Myocarditis
• Pericarditis
• Pulmonary embolism
• Pulmonary hypertension, severe
• Renal failure
• Sepsis/septic shock
• Sepsis-related myocardial
dysfunction
• Systemic inflammatory diseases
• Trauma
Conditionsassociated
withtroponinelevation

• Creatine kinase (CK) is a cytosolic enzyme involved with the
transfer of energy in muscle metabolism. It catalyses the
conversion of creatine to phospho-creatine degrading ATP to ADP.
• CK is a dimer composed of two subunits B (brain type) and M
(muscle type), resulting in three isoenzyme:
CK-BB (CK1) : is of brain origin, found in blood only when BBB is
damaged.
CK-MB (CK2) : it is relatively specific for myocardial origin
CK-MM (CK3) : it is found primarily in skeletal muscle
CREATINE KINASE: CK-MB

CREATINE KINASE: CK-MB
• CK-MB is the most cardiac-specific CK isoenzyme
• Proportion of CK-MB varies in skeletal & cardiac muscle
• In normal population CK-MB < 6% Tot CK
• Sensitive marker with rapid rise & fall
• More specific than Tot CK but has limitations
• “Gold standard” biochemical marker for past few decades
• “There is no place for measurement of CK-MB by
electrophoretic or immunoinhibition methods in the 21st
century laboratory” Jacobs, Lab Test Handbook 5th Ed 2001,157
Only CK-MBmass should be measured

It is a valuable tool for the diagnosis of MI because of its
relative high specificity for myocardial damage.
Rise : 4-6 hrs after onset of symptoms
Peak : 12 hrs
Return to normal : 24-36 hrs
Can be used to indicate early re-infarction if level normalizes
and then increases again.
CK-MB :

CK-MBmass
RELATIVE INDEX (%RI)
% RI = (CK-MBmass / Tot CK activity) x 100
• Increased RI suggests myocardial origin
• RI > 3 – 6 % with Tot CK activity elevated suggests myocardial
necrosis

• Small-size heme protein found in all tissues mainly assists in oxygen transport
• It is released from all damaged tissues
• Its level rises more rapidly than cTn and CK-MB.
• Released from damaged tissue within 1 hour
• Normal value: 17.4-105.7 ng/ml
• Timing:
– Earliest Rise: 1-4 hrs
– Peak 6-9 hrs
– Return to normal: 12 hrs
Myoglobin

• Acute myocardial infarction
• Skeletal muscle damage, muscular dystrophy,
inflammatory myopathies
• Renal failure, severe uremia
• Shock and trauma
CONDITIONS FOR
MYOGLOBIN INCREASE :

*if myoglobin concentration remains within the reference range 8 hours after
the onset of chest pain, AMI can be ruled out essentially.
*because of its rapid clearance by the kidney, a persistently normal Mb
concentration will rule out reinfarction in patient with recurrent chest pain
after AMI
*Rapid monitor of success of thrombolytic therapy
DRAWBACKS
• Due to poor specificity, myoglobin levels do not always predict myocardial
injury
Clinical usefulness of
myoglobin :

NEW GENERATION
CARDIAC MARKERS
• Myoglobin
– 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

0
1
2
3
4
5
6
7
0 4 8 12 16 20 24 28 32 36 40 44 48
myoglobin
CK-MB
cTnT
cTnI
Comparison of
cTn, CK-MB , Mb
Time after onset of AMI (hours)
Χupperlimitofreferenceinterval

Marker for haemodynamic stress
natriuretic peptides

• The natriuretic peptides (NP) are a group of structurally similar but genetically distinct peptide hormone.
It includes :
ANP : -atrial natriuretic peptide (28 a.a.)
N-terminal proANP (98 a.a.)
BNP : brain natriuretic peptide (32 a.a.)
N-terminal proBNP (76 a.a.)
CNP : C-type natriuretic peptide (22 and 53 a.a.)
DNP : D- type natriuretic peptide
• The NPs play important role in regulation of salt and water balance (CV homeostasis)

• ANP is released primarily in response to atrial wall stretching and
intravascular volume expansion.
• BNP is mainly secreted by the ventricles
• CNP is found predominantly in the brain and also synthesized by vascular
endothelial cells

• Circulating levels of BNP are raised in patients with
cardiovascular or renal disease
• BNP is More important than ANP in heart failure
• Greatest proportion of circulating BNP is thought to
come from the ventricles (left)
BRAIN NATRIURETIC
PEPTIDE (BNP)

Synthesis in myocyte

• BNP and the terminal fragment of its prohormone (NT-proBNP) are
released on ventricular stretch or stress to the myocyte in the
absence of the necrosis.
• Therefore, BNP is increased in diseases characterised by an
expanded fluid volume (e.g. CHF, renal failure,hepatic cirrhosis etc.)
• BNP has circulating T₁/₂ of 20 minutes, so it is indicative of snapshot
of myocardial function, while NT-proBNP has T₁/₂ of 90 minutes
giving a longer view of myocyte .
BRAIN NATRIURETIC
PEPTIDE (BNP)

• Age
• Arrhythmias
• Cardiomyopathy: hypertrophic,
ischemic, or dilated
• Congestive heart failure
• Coronary artery disease
• Gender
• Hypertension
• Left ventricular diastolic
dysfunction
• Pulmonary embolism
• Renal failure
• Right heart failure
• Right ventricular overloading:
fluid, or pressure overloading
• Sepsis or septic shock
• Sepsis-related myocardial
dysfunction
Conditions or factors commonly associated
with B-type natriuretic peptide or N-terminal-
pro-B-type natriuretic peptide elevations

Future Cardiac Biomarker
miRNA

• miRNAs are appx. 20-25 nucleotide long non coding RNAs, that
negatively regulate or inhibit gene expression by binding to sites
in the untranslated regions of targeted messenger RNAs.
miRNA

• miRNA are found to be involved in almost every biological
process, from cellular differentiation and proliferation to
cell death and apoptosis
• Many different types of miRNA can be detected in
circulating blood and these miRNA are present in
remarkably stable form that even withstand repetitive
freezing/thawing cycle and are protected against Rnases.
• Thousands of miRNAs have been described in human to
date which exhibits tissue specific pattern of expression.
miRNA

• miRNAs that regulates cardiovascular system can be divided into 4 groups :
1. miRNA regulating endothelium function and angiogenesis : miR126, miR17-92
cluster, miR130a, miR221, miR21
2. cardiac myocyte specific mRNA : miR208a
3. cardiac myocyte and skeletal muscle miRNA : miR1, miR133a, miR499
4. smooth muscle miRNAs :miR143, miR145
miRNAs hold promise as very specific and accurate marker of cardiac
dysfunction.
miRNA

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
Theideal cardiac
markerdoes
NOTyetexist!

“CARDIAC ENZYMES”
are
Obsolete!

BIOCHEMICAL MARKERS IN MYOCARDIAL ISCHAEMIA
/ NECROSIS
IN:
• 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

SUMMARY
• “Cardiac Enzymes” are obsolete
• Medical & laboratory progress has required a redefinition of Cardiac
Events
• 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
• Need for rapid TAT & reliable cardiac markers
• Additional roles for cardiac markers in:
– Reperfusion monitoring
– Infarct size/prognosis
– Intra/post-operative MI (non-cardiac/cardiac surgery)
• Evolving laboratory role in the evaluation of cardiac disease particularly in
the areas of cardiac dysfunction & general biochemical or genetic risk
factors

Cardiac Markers (Recap and Update)

Cardiac Markers (Recap and Update)

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