CARDIAC MARKERS

PRESENTED: BY RUBAINA ALI

PRESENTED TO: Dr. BLESSY K GEORGE, DEPARTMENT OF
PHARMACY PRACTICE
 CARDIAC BIOMARKERS
• Cardiac biomarkers are substance that are releases into the blood when the heart is
damaged or stressed.
• Cardiac biomarkers are used for diagnosis, treatment monitoring, staging of
cardiac disease, timing of cardiac events and prognostification.
• Measurement of these are used to help diagnose acute coronary syndrome (ACS),
ischemia (condition associated with insufficient blood flow to the heart.
• Test for biomarkers can also be used to help determine a persons risk of having
these conditions or to help monitor and manage someone with suspected ACS and
cardiac ischemia and in patient presenting with chest pain
 HISTORY OF CARDIAC MARKERS
• 1954 - SGOT (AST)
1955 - LDH
1960 - CPK
1972 - CPK isoforms by Electrophoresis
1975 - CK - MB by immuno inhibition
1975 - Myoglobin
1985 - CK - MB Mass immunoassay
1989 - Troponin T
1992 - Troponin I
 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
 • INFLAMMTORY:
• ENDOTHELIAL IL- 6,1B,10,12,18
TNF- alpha, sCD40L, INFy, CRP
LDL- C, oxidized LDL-C, RPS homocysteine
PLAQUE
FORMATION
• PLAQUE DESTABILISATION
Cytokine, adiponectin, LP PLA2, PAPP-A

Classification of Cardiac
Biomarkers according to MYOCARDIAL ISCHEMIA
various stages during cardiac IMA, H-FABP, BNP/ NT pro-BNP, HSP
disease process

CARDIAC NECROSIS
cTn, CK-MB, H-FABP, myoglobin

MYOCARDIAL HEART FAILURE

STRETCH/ STRESS CADIAC DAMAGE
CAM1, BNP/ NT
ADM, ANP, BNP/ pro-BNP
NT pro-BNP
 MARKER OF INFLAMMATION
• hsCRP
sCD40L
homocysteine
• CRP: CRP plays a pivotal role in many aspects of atherogenesis including,
activation of complement pathway, lipids uptake by macrophage, release of
proinflammatory cytokines, induces the expression of tissue
factor in monocytes, promotes the endothelial dysfunction and inhibits nitric
oxide production.
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
High-Sensitivity C-Reactive Protein
PROINFLAMMATORY
CYTOKINES
 HS CRP
• 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.
Clinical Uses
– Screening for cardiovascular risk in otherwise “healthy”
individuals
– Predictive value of CRP levels for disease severity in preexisting Coronary artery
disease
Elevated levels are predictive of
• Long-term risk of first MI
• Ischemic stroke
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
 sCD40L
• Soluble fragment CD 40 ligand.
• It is a signalling protein that reflects both inflammatory and platelet interaction.
• It is present in the platelet granules and thus its presence in blood is marker of platelet
activation. By interacting with CD40 found on endothelial and smooth muscle cells,
sCD4OL may trigger the release of inflammatory mediators, lead to increased activity of
matrix metalloproteinases and activate the coagulation cascade.
• ↑ levels of sCD40L is associated with ↑ risk of cardiac events
• However rise is also associated with many other inflammatory conditions like RA,
SCD(SPONDYLOCOSTAL DYSOSTOSIS), 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.
 Schematic overview of the regulation of platelet CD40L and the role of sCD40L in signaling
after binding to platelet CD40 and αIIbβ3 inducing an auto-amplification loop. Synaptosomal-
associated protein 23 (SNAP23), mitogen-activated protein kinase (MAPK), nuclear factor
kappa B (NF-κB), protein kinase B (AKT) matrix metalloproteinase-2 (MMP-2), TNF receptor
associated factor 2 (TRAF2).
CD40L and its receptors: the binding of
CD40L to CD40, αIIbβ3, α5β1, or Mac-1
(αMβ2) induces different inflammatory
pathologies. Systemic lupus erythematosus
(SLE), transfusion-related acute lung injury
(TRALI), reactive oxygen and nitrogen
species (RONS), Myeloperoxidase (MPO).
 HOMOCYSTEIN
• 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 (hardening of artery) and venous thrombosis (clot formation)
• Can result from genetic defects, drugs, vitamin deficiencies (associated with low
levels of vitamin B6, vitamin B12, folate), as well as renal disease. And get mostly
from eating red meat.
• 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.
 HOMOCYSTEIN
• • Normal levels : 3.7 – 13.9 µmol/L
• Treatment includes supplementation with folate, B6
and B12.
HOMOCYSTE
IN
METABOLIS
M
Blood reference ranges for homocysteine:
Therapeuti
Sex Age Lower limit Upper limit Unit Elevated
c target
3.3 7.2 μmol/L
12–19 years > 10.4
45 100 μg/dL μmol/L
Female
4.9 11.6 μmol/L or
>60 years > 140 μg/dl < 6.3
66 160 μg/dL μmol/L
4.3 9.9 μmol/L > 11.4 or
12–19 years μmol/L < 85 μg/dL
60 130 μg/dL
Male or
5.9 15.3 μmol/L > 150
>60 years
80 210 μg/dL μg/dL
• A high level of homocysteine in the blood (hyperhomocysteinemia) makes a
person more prone to endothelial cell injury, which leads to inflammation in
the blood vessels, which in turn may lead to atherogenesis, which can result
in ischemic injury. Hyperhomocysteinemia is therefore a possible risk factor
for coronary artery disease. Coronary artery disease occurs when an
atherosclerotic plaque blocks blood flow to the coronary arteries, which
supply the heart with oxygenated blood.
• Hyperhomocysteinemia has been correlated with the occurrence of blood
clots, heart attacks and strokes, though it is unclear whether
hyperhomocysteinemia is an independent risk factor for these conditions.
• Hyperhomocysteinemia has also been associated with early pregnancy
loss[4] and with neural tube defects.
• Abnormally high levels of homocysteine in the serum, above 15 µmol/L,
are a medical condition called hyperhomocysteinemia.This has been claimed
to be a significant risk factor for the development of a wide range of
diseases, including thrombosis, neuropsychiatric illness, and fractures. It
is also found to be associated with microalbuminuria which is a strong
indicator of the risk of future cardiovascular disease and renal
dysfunction.
 Markers of Plaque Destabilization
• PAPP-A
LP-PLA₂
 Pregnancy associated plasma
protein-A (PAPP-A)

• • 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.
• Pappalysin-1, also known as pregnancy-associated plasma protein A, is
a protein encoded by the PAPPA gene in humans. PAPPA is a secreted protease
whose main substrate is insulin-like growth factor binding proteins. Pappalysin-1 is
also used in screening tests for Down syndrome.[5][6]
• FUNCTION:
This gene encodes a secreted metalloproteinase which cleaves insulin-like growth
factor binding proteins (IGFBPs). PAPPA's proteolytic function is activated upon
collagen binding. It is thought to be involved in local proliferative processes such
as wound healing and bone remodeling. Low plasma level of this protein has been
suggested as a biochemical marker for pregnancies with aneuploid fetuses (fetuses
with an abnormal number of chromosomes).[6] For example, low PAPPA may be
commonly seen in prenatal screening for Down syndrome.[5] Low levels may
alternatively predict issues with the placenta, resulting in adverse complications such
as intrauterine growth restriction, preeclampsia, placental abruption, premature
birth, or fetal death.
 Lipoprotein-associated
phospholipase A₂

• Lp-PLA₂ is also known as platelet 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
• REDUCED RISK < 225 nmol/min/mL
• INCREASED RISK >255 nmol/min/mL
• 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 Myocardial Ischemia
• IMA
H-FABP
IMA(Ischemia Modified Albumin )
• 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.
 Clinical uses of IMA :

• 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.
 H-FABP
• 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 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-.
 Marker for cardiac necrosis
• cTn
CK-MB
Myoglobins
 TROPONIN (I OR T)

• The troponins are regulatory proteins found in skeletal and cardiac muscle.
• Three subunits have been identified: troponin I (TnI), troponin T(TnT), and
troponin C (TnC).
• The genes that encode for the skeletal and cardiac isoforms of TnC are
identical: this, no structural difference exists between them. However, the
skeletal and cardiac subforms for TnI and TnT are distinct, and
immunoassay have been designed to differentiate between then.
 Functions

• 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.
• According to European society of cardiology (ESC)/ American college of
cardiology foundation (ACCF)/ American heart association (AHA)/ world
health federation (WHF) guidelines, MI refers specifically to myocardial
necrosis due to myocardial ischemia. However, although elevations in the
serum levels of TnI, TnT and CK-MB indicate the presence of injury
associated necrosis of myocardial cells, such elevation do not point to the
underlying mechanism of the necrosis occurs in MI, it can also be a product
of predominantly nonischemic myocardial injury, as occurs in association
with heart failure, arrhythmias, myocarditis, renal failure, pulmonary
embolism, and percutaneous or surgical coronary procedures.
 TROPONIN
• Troponins are family of protein found in skeletal and cardiac muscle fibers
that produce muscular contraction.
• When person has a heart attack, levels of cardiac specific troponins I & T
can become elevated in the blood within 3 or 4 hours and peaks in approx.
24hr after injury and may remain elevated for 10 -14 days(1-2wk).
• Cardiac troponin I is currently considered to be the gold standard biomarker
test for myocardial infarction. Moreover, cTnI measurements by a new
generation of high-sensitivity cTnI assays could be helpful for long-term risk
stratifcation of different patient groups, including patients with heart failure
or stable coronary artery disease
Conditions associated with troponin elevation

• Arrhythmias • Pulmonary embolism

• Congestive heart failure
• Coronary artery disease • Pulmonary hypertension,
• Coronary vasospasm severe
• Critically ill patient • Renal failure
• Hypertension
• Myocarditis • Sepsis/septic shock
• Pericarditis • Sepsis-related myocardial
• Trauma dysfunction
• Systemic inflammatory
diseases
 CREATINE KINASE: CK-MB
• 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
• 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 Only CK-MBmass should be measured
• CLINICAL UTILITY
• Acute myocardial infarction (AMI)
• Unstable angina
• AMI prognosis
• Cardiac muscle injury and cell death
• 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-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
 Myoglobin
• Small-size heme protein found in all tissues mainly assists in oxygen
transport (Oxygen binding protein of cardiac and skeletal muscle)
• 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
 CONDITIONS FOR
MYOGLOBIN INCREASE :
• Acute myocardial infarction
• Skeletal muscle damage, muscular dystrophy,
inflammatory myopathies
• Renal failure, severe uremia
• Shock and trauma
 Clinical usefulness of
myoglobin :
• 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
 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 (released on ventricular
stretch or stress to the myocyte in the
absence of the necrosis.
• CNP is found predominantly in the brain and also synthesized by
vascular endothelial cells
natriuretic peptides
• 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)
Conditions or factors commonly associated with B-type natriuretic peptide or
N-terminalpro-B-type natriuretic peptide elevation
• 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
•Future Cardiac Biomarker
miRNA
 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 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.
• 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.
 FUTURE:
Ischaemia Modified
Albumin
Glycogen
Phosphorylase BB
OUT: Fatty Acid binding
• AST activity Protein
• LDH activity
• LDH isoenzymes
• CK-MB activity
• CK-Isoenzymes
IN:
• ?CK-Total
• CK-MB (mass)
• c.Troponins (I or T)
• Myoglobin
THANK YOU