 Chairman

Dr. K.V.Venkteswaran, Professor, Dept. of

Veterinary Pharmacology and Toxicology, MVC,
Chennai

 Members
Dr. S.Selvasubramanian, Professor, Dept. of
Veterinary Pharmacology and Toxicology, MVC,
Chennai
Dr.P.S.L.Sesh, Assistant Professor, Dept. o
VeterinaryBiochemistry, MVC, Chennai
 “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
HEART FAILURE
Heart failure a major and growing health problem
appears to result not only from cardiac overload or
injury but also from complex interplay among
genetic, inflammatory and biological changes acting
on cardiac myocytes, the cardiac interstitium or
both.

ACUTE MYOCARDIAL INFARCTION

A sudden occlusion of a coronary artey by
thrombus or by embolisation causes an acute
myocardial infarction.
 Cardiac biomarkers are protein molecules released
into the blood stream from damaged heart muscle

SinceECG…… inconclusive ….biomarkers !!!!!?????

myocardial injury

 These biomarkers have a characteristic rise and fall

pattern

Dr. Jaikanth
 High cardiac specificity
 Pharmacokinetics of cardiac biomarker
 Easy diagnosis
 Marker should play a designed role in the treatment
and management of clinical subject
 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
 Biomarkers of myocardial injury
› markers of myocardial necrosis
› markers of myocardial ischemia

 Biomarkers of haemodynamic stress

 Inflammatory and prognostic Biomarkers

 Markers of myocardial necrosis
› Creatine kinase – MB
› Myoglobin
› Cardiac troponins
• Markers of myocardial ischemia
Ischemia Modified Albumin (IMA)
Heart-type fatty acid binding protein (H-FABP)

Dr. Jaikanth
Zones of Ischemia Injury and Infarction with
Transmural and Subendocardial Infarction
 Creatine kinase (CK/CPK) is an enzyme expressed in a
number of tissues.

Function: it catalyses the conversion of creatine to

phosphocreatine degrading ATP to ADP

The CK enzyme consists of two subunits, B (brain type) or

M (muscle type), Making three different isoenzymes: CK-
MM, CK-BB and CK-MB

 CK-BB occurs mainly in tissues, rarely of any significance in the

bloodstream
 Skeletal muscle expresses CK-MM (98%) and low levels of CK-
MB (1%)
 The myocardium has CK-MM at 70% and CK-MB at ~30%
 High specificity for cardiac tissue

 Begins to rise 4-6 hours after onset of

infarction

 Peaks at about 12 hours

 Returns to baseline at 24-36 hours

 Can be used to indicate early re-infarction if level

normalizes and then increases again
 CK-MB now measured via a highly sensitive monoclonal antibody
assay

 Immunological Sandwich technique using two Abs for different

epitopes of CK –MB molecule

 The first Ab is rendered immobile on a matrix (e.g. CrO2

particles)

 The second Ab conjugate to an enzyme (β- galacto-

sidase)

 Separated bound sandwiches are reacted with their substrate

(e.g. Chlorophenol β- Red Galactopyranoside)

 Liberated end product chlorophenol is measured

spectrophotometrically and is proportionate to CK-MB amount
(not the activity)
 The CK-MB isoforms may also be analyzed using high-
voltage electrophoresis

 The ratio of MB2/MB1 is calculated

 MB2 released from heart muscle and converted to MB1

 A level of MB2 > or = 1 and a ratio of MB2/MB1 > 1.5

indicates myocardial injury

 A result is positive if MB2 is elevated and the ratio is

more than 1.5
 False positive (for MI) CK-MB elevation can be seen in:
› Significant skeletal muscle injury
› The MB fraction is determined to be expressed during the
process of muscle regeneration
› Cardiac injury for reason other than MI
 Defibrillation
 Blunt chest trauma
 Cocaine abuse

The search for cardiac specificity continues…

 Small-size heme protein found in all tissues mainly assists in
oxygen transport

 It is released from all damaged tissues

 Increases often occur more rapidly than TI and CK

 Released from damaged tissue within 1 hour

 Normal value: 17.4-105.7 ng/ml

 Timing:
› Earliest 1-3 hrs
Peak
›Rise: 6-9 hrs
› Return to normal: 12 hrs
 Acute myocardial infarction

 Skeletal muscle damage, muscular

dystrophy, inflammatory myopathies

 Renal failure, severe uremia

 Shock and trauma

 Rapid monitor of success of thrombolytic
therapy

 Negative predictor of MI

DRAWBACKS
 Due to poor specificity, myoglobin levels do
not always predict myocardial injury
 Not utilized often for AMI/cardiac damage
assessment because of its very rapid metabolism
 Troponin is a complex of three regulatory proteins that is
integral to non-smooth muscle contraction in skeletal as well as
cardiac muscle

 Troponin is attached to the tropomyosin sitting in the groove

between actin filaments in muscle tissue

 Troponin has three subunits, TnC, TnT, and TnI

› Troponin-C has calcium binding ability and has no diagnostic
value
› Troponin-T binds the troponin tropomyosin complex,
› Troponin-I is an inhibitory protein
 Less than 5% in cytosol

 Troponin levels begin to rise 2-3 hours after onset of

myocardial injury

 Elevations in Troponin-I and Troponin-T can persist for up

to 10 days after MI

 Remember, CK-MB returns to baseline by 48 hours

 Thus far, studies have failed to find a source of

Troponin-I outside the heart, but have found some
Troponin-T in skeletal muscle
NEJM 2002;Vol.346,No.26:2079-82
Conditions commonly associated with cardiac troponin
elevations
 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
 Troponin T and I are not detected in healthy
individuals
 Significant increase in Troponins reflects
myocardial necrosis
 ACC/ESC has defined increase in Troponins
as a measurement above 99th percentile value of
reference group
 To reduce false-positive outcomes, CV of
10% at
decision limit is recommended
• TropT (Roche Diagnostics, Germany)
• Trop I (Siemens Healthcare Diagnostics)

• Troponin T
› 99th percentile limits - 0.01 ng/mL
› assay ranges - 0.01-25 ng/mL
 (Troponin I)
› 99th percentile limits -0.04 ng/mL
› assay range -0.04-40 ng/mL

• Reference limits based on the 99th percentile for a

healthy population are 0.01 ng/mL (Troponin T) and
0.04 ng/mL (Troponin I)
 They used the concentration of 0.04 ng per mL
as the upper reference limit and established the
diagnosis of myocardial infarction if one value of more
than 0.04
ng per mL was documented, combined with a rise or
fall
in the value of 30% or more within 6 hours after
admission.

Patients with troponin rises benefit more from

GlycoproteinIIb IIIa inhibitors such as,
 Abciximab
 Eptifibatin
 Clopidogrel
Most commonly used in dogs and cats
Clinical conditions
 Congestive heart failure
 Percardial disease
 Doxorubicin toxicity
 Gastric dilatation and volvulus etc,.
120 animals were examined in emergency
with cardiac troponin assays,

 First group= ctni less than .15ng/ml

 Second group= ctni .15-1 ng/ml
 Third group= ctni more than 1 ng/ml

Prognosis grave in second and third

group
 A novel marker of ischemia, is produced when circulating
serum albumin contacts ischemic heart tissues

IMA can be measured by the albumin cobalt binding (ACB)

assay that is based on IMA's inability to bind to cobalt

 Mechanism- due to structural change in the amino terminal

end of albumin

 IMA levels rise within 6 hours

 remain elevated for 12 hours

Drawbacks
IMA levels raised in non- cardiac ischemia

Modification to n- terminal end may also be

induced by extracellular hypoxia, acidosis etc,

Conclusion
FDA in 2010 has approved a multimarker approach
for using the combination of ECG, the cTnI, and the
IMA levels achieving a sensitivity of 95% for ACS
Heart-type fatty acid binding protein (H-FABP)

 H-FABP is a very stable abundant [138] low molecularweight

protein (14–15 kDa) in the cytoplasm of myocardial cells

 Appearing as early as 90 min after symptom onset and peaking

within 6 h

 Parameters of kinetic release make it an ideal candidate both for

early assessment or exclusion of AMI and for the measurement
of a recurrent infarction
 A study by Puls et al
› the negative predictive value (NPV) of H-FABP was an
impressive
100%
› its Positive predictive value was 41% which was greater than
that of both cTnT (29%) and NT-proBNP (19%).

 The myoglobin/heart FABP ratio has been used to differentiate

between heart muscle and skeletal muscle injury

 Cardiodetect

Dr. Jaikanth
 The natriuretic peptides (NP) are a group of
structurally similar but genetically distinct
peptides.

 NPs are identified as regulatory diuretic-natriuretic

substances responsible for salt and water
homeostasis

 Lowers blood pressure.

 The NP family 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.)
Fig. Schematic representation of the ANP and BNP precursors with
sequence numbering defining low-molecular-mass forms, N-terminal forms
and high- molecular-mass precursors
 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
 originally isolated from porcine brain

 Subsequently also isolated from human heart

 Circulating levels of BNP are raised in patients

with cardiovascular or renal disease

 More important than ANP in heart failure

 Greatest proportion of circulating BNP is

thought to come from the ventricles (left)

Synthesis
Three receptors for natriuretic peptides :
Natriuretic peptide receptor -A
Natriuretic peptide receptor -B
Natriuretic peptide receptor -C

NPR-A and NPR-B

 NPR-A and NPR-B are particulate guanylyl cyclases that
catalyses the conversion of GTP to c-GMP

 NPR-C
They lack the guanyl cyclase domain and may influence
the target cell function through inhibitory guanine
nucleotide (Gi) protein, and they likely also act as clearance
receptors for circulating peptides.
NPR-A and NPR-B
 NPR- A is the most abundant type in
large blood vessels
 NPR-B predominate in the brain
 Both receptors are present in the
adrenal glands and the kidney
 Affinity
for NPR-A : ANP > BNP > CNP
for NPR-B : CNP > BNP
> ANP
Action of Atrial Natriuretic Peptide at Target Cells
 ANP and BNP concentrations increase in
response to volume expansion and pressure
overload of the heart

 ANP and BNP have been shown to be

physiological antagonists of the effects of
(1) angiotensin II on vascular tone
(2) aldosterone secretion
(3) renal-tubule sodium reabsorption
(4) vascular-cell growth
Figure . Physiology of the natriuretic-peptide family
 Clearance factors for natriuretic peptides
NPR-C and neutral endopeptidase

Endopeptidase

 Neutral endopeptidase inactivates all

three natriuretic peptides

 Present within renal tubular cells and

vascular cells
Conditions or factors commonly associated with B-type
natriuretic peptide or N-terminal-pro-B-type natriuretic
peptide
elevations
 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
 Prospective study of 1586 patients presenting to the emergency
department with acute dyspnea

Outcomes of the study

 The predictive value of BNP much superior to previous standards including

radiographic, clinical exam, or Framingham Criteria

 Bnp cut point was fixed at 100pg/ml and it showed 90% sensitivity and 80%
specificity for diagnosing heart failure

Veterinary field
 Chronic left ventricular systolic and diastolic dysfunction

 Cardiac vs non- cardiac dyspnoea

 Recent development of ELISA kits for canine and feline NTproBNPby

Guildhay Ltd (www. guildhay. Co.uk)
ANP and BNP infusion
 Decrease renin and
aldosterone concentration
 Increase urinary sodium and water
excretion
Neutralendopeptidase inhibitor
 Nesiritide is a new drug that is a synthetic
BNP that vasodilates vessels and serves
as a potent diuretic agent
 BNP for Rx of decompensated heart
failure Nesiritide (h-BNP)

R I S
D SS
M S
K G
L
R H
GF
S CG R
C S K V L R
G
S P K M V Q G S
32 amino acid sequence
Recombinant technology using E-c
ays
C-reactive protein

Myeloperoxidase

Homocysteine
 CRP is an acute-phase protein produced by
the liver

 Pentameric structure consisting of five 23-

kDa identical subunits

 Plasma levels can increase rapidly to 10000x

levels
 High-sensitivity CRP (hs-CRP) assays
 CRP previously known to be a marker of high
risk in cardiovascular disease

 More recent data may implicate CRP as an

actual mediator of atherogenesis

Mechanism of CRP-mediated
atherogenesis:
 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
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 predictive of

• Long-term risk of first MI
• Ischemic stroke
 Limitations of CRP
 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 High
mg/L
 MPO is an enzyme that aids white blood cells
in destroying bacteria and viral particles

 MPO catalyzes the conversion of hydrogen

peroxide and chloride ions (Cl-) into
hypochlorous acid

 MPO is released in response to infection and

inflammation

 EPIC Norfolk Study

Sugiyama Am J Pathology 2001

 MPO leads to oxidized LDL cholesterol
› Oxidized LDL is phagocytosed by
macrophages producing foam cells
 MPO leads to the consumption of nitric oxide
› Vasoconstriction and endothelial
dysfunction
 MPO can cause endothelial denuding and
superficial platelet aggregation
 MPO indicates activated immune cells
› Activated immune cells and inflammation lead
to unstable plaque
 Inflammatory plaque is inherently less stable
› Thin fibrous cap/fissured/denuded
 In august 2005, FDA approved the first
MPO assay, Cardio MPO tm developed by
Prognostix,inc.

 The test is a sandwich enzyme immuno

assay

 Normal plasma MPO levels are 51-

633pmol/ml
 Progression of Biomarkers in ACS

STABLE CAD PLAQUE RUPTURE UA/NSTEMI STEMI

MPO MPO MPO TnI

ICAM D-dimer TnT
CRP sCD40L IMA Myoglobin
IL-6 PAPP- FABP CKMB
A

Inflammation has been linked to the development of

vulnerable plaque and to plaque rupture
ACS, acute coronary syndrome; UA, unstable angina; NSTEMI, non–ST-segment elevation myocardial infarction; STEMI, ST-segment
elevation myocardial infarction
Adapted from: Apple Clinical Chemistry March 2005
 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 implicated directly in
vascular injury including:
› Intimal thickening
› Disruption of elastic lamina
› Smooth muscle hypertrophy
› Platelet aggregation

 Vascular injury induced by leukocyte

recruitment, foam cell formation, and
inhibition of NO synthesis
 Normal levels less than 6micro mol/l
 Elevated levels appear to be an
independent risk factor, though less
important than the classic CV risk factors

 Treatment includes supplementation with

folate, B6 and B12
Stefan Blankenberg, MD; Renate Schnabel, MD; Edith Lubos, MD, et al., Myeloperoxidase Early Indicator of Acute Coronary Syndrome and
Predictor of Future Cardiovascular Events 2005
 Send the comments of this ppt to
jaipersie@gmail.com for further
enhancement of my presentations

Dr. Jaikanth
 Moraes DL, Colucci WS, Givertz MM. Secondary pulmonary hypertension in chronic heart failure: the role of the endothelium in
pathophysiology and management. Circulation 2000;102:1718-23.
 Hulsmann M, Stanek B, Frey B, et al. Value of cardiopulmonary exercise testing and big endothelin plasma levels to predict short-term
prognosis of patients with chronic heart failure. J Am Coll Cardiol 1998;32:1695-700.
 Latini R, Masson S, Anand I, et al. Thecomparative prognostic value of plasma neurohormones at baseline in patients with heart failure
enrolled
 in Val-HeFT. Eur Heart J 2004;25:292-9.
Zannad F, Alla F, Dousset B, Perez A, Pitt B. Limitation of excessive extracellular matrix turnover may contribute to survival benefit of
 spironolactone therapy in patients with congestive heart failure insights from the Randomized Aldactone
Morrow DA, Cannon CP, Jesse RL, Newby LK, Ravkilde J, Storrow AB, Wu AH, Christenson RH. National Academy of Clinical Biochemistry
Laboratory Medicine Practice Guidelines: clinical characteristics and utilization of biochemical markers in acute coronary syndromes.
 Circulation. In press.
Morrow DA, Braunwald E. Future of biomarkers in acute coronary syndromes: moving toward a multimarker strategy. Circulation. 2003;
 108:250

–252.
Jaffe AS, Babuin L, Apple FS. Biomarkers in acute cardiac disease: the present and the future. J Am Coll Cardiol. 2006;48:1–11.
Wollert KC, Kempf T, Peter T, Olofsson S, James S, Johnston N, Lindahl B, Horn-Wichmann R, Brabant G, Simoons ML, Armstrong PW, Califf
 RM, Drexler H, Wallentin L. Prognostic value of growth-differentiation factor-15 in patients with non–ST-elevation acute coronary syndrome.
 Circulation. 2007;115:962–971.
Ridker PM. Evaluating novel cardiovascular risk factors: can we better predict heart attacks? Ann Intern Med. 1999;130:933–937.
 Halldorsdottir AM, Stoker J, Porche-Sorbet R, Eby CS. Soluble CD40 ligand measurement inaccuracies attributable to specimen type,
processing time, and ELISA method. Clin Chem. 2005;51:1054 –1057.
The Joint European Society of Cardiology/American College of Cardiology Committee for the redefinition of myocardial infarction. Myocardial
 infarction redefined: a consensus document of the Joint European Society of Cardiology/American College of Cardiology Committee for the
Redefinition of Myocardial Infarction. J Am Coll Cardiol. 2000;36: 959–969.
 Danesh J, Wheeler JG, Hirschfield GM, Eda S, Eiriksdottir G, Rumley A, Lowe GD, Pepys MB, Gudnason V. C-reactive protein and other
circulating markers of inflammation in the prediction of coronary heart disease. N Engl J Med. 2004;350:1387–1397.
Wiviott SD, Cannon CP, Morrow DA, Murphy SA, Gibson CM, McCabe CH, Sabatine MS, Rifai N, Giugliano RP, DiBattiste PM, Demopoulos LA,
Antman EM, Braunwald E. Differential expression of cardiac biomarkers by gender in patients with unstable angina/non–ST-elevation
 myocardial infarction: a TACTICS-TIMI 18 (Treat Angina with Aggrastat and Determine Cost of Therapy with an Invasive or Conservative

Strategy-Thrombolysis In Myocardial Infarction 18) substudy. Circulation. 2004;109:580 –586.
Cook NR. Use and misuse of the receiver operating characteristic curve in risk prediction. Circulation. 2007;115:928 –935.
James SK, Armstrong P, Barnathan E, Califf R, Lindahl B, Siegbahn A, Simoons ML, Topol EJ, Venge P, Wallentin L. Troponin and C-reactive
 protein have different relations to subsequent mortality and myocardial infarction after acute coronary syndrome: a GUSTO-IV substudy. J
Am Coll Cardiol. 2003;41:916 –924.
 Ridker PM, Cannon CP, Morrow D, Rifai N, Rose LM, McCabe CH, Pfeffer MA, Braunwald E. C-reactive protein levels and outcomes after statin
therapy. N Engl J Med. 2005;352:20 –28.
Sabatine MS, Morrow DA, de Lemos JA, Gibson CM, Murphy SA, Rifai N, McCabe C, Antman EM, Cannon CP, Braunwald E. Multimarker approach
 to risk stratification in non–ST elevation acute coronary syndromes: simultaneous assessment of troponin I, C-reactive protein, and B-type
natriuretic peptide. Circulation. 2002;105:1760 –1763.
Kempf T, Horn-Wichmann R, Brabant G, Peter T, Allhoff T, Klein G, Drexler H, Johnston N, Wallentin L, Wollert KC. Circulating concentrations