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Cardiac biomarkers
1.
2. 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
3. “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
4. 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.
5. Cardiac biomarkers are protein molecules released
into the blood stream from damaged heart muscle
Since ECG…… inconclusive ….biomarkers !!!!!?????
myocardial injury
These biomarkers have a characteristic rise and fall
pattern
Dr. Jaikanth
6. High cardiac specificity
Pharmacokinetics of cardiac biomarker
Easy diagnosis
Marker should play a designed role in the treatment
and management of clinical subject
7. 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
8. Biomarkers of myocardial injury
› markers of myocardial necrosis
› markers of myocardial ischemia
Biomarkers of haemodynamic stress
Inflammatory and prognostic Biomarkers
9.
10. 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
11.
12.
13. Zones of Ischemia Injury and Infarction with
Transmural and Subendocardial Infarction
14. 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%
15. 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
16. 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)
17.
18. 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
19. 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…
20. 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 Rise: 1-3 hrs
› Peak 6-9 hrs
› Return to normal: 12 hrs
22. 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
23. 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
24.
25.
26.
27.
28. 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
32. 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
33. • 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)
34. 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
Glycoprotein IIb IIIa inhibitors such as,
Abciximab
Eptifibatin
Clopidogrel
35. Most commonly used in dogs and cats
Clinical conditions
Congestive heart failure
Percardial disease
Doxorubicin toxicity
Gastric dilatation and volvulus etc,.
36. 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
37.
38.
39. 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
40. 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
41. 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
42. 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
43.
44.
45. 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.
46. 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.)
47. 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
48. 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
49. 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)
51. 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.
52. 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
54. 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
57. 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
58. 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
59. 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)
60. 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
61. BNP for Rx of decompensated heart
failure Nesiritide (h-BNP)
R I S S
D S
M S
K G
R L
G G H
F R
S C R
C S K V L
G
S P K M V Q G S
32 amino acid sequence
Recombinant technology using E-co
NOTE: hBNP affects assay for BNP, but can still use proBNP or one of the proANP assays
62.
63. C-reactive protein
Myeloperoxidase
Homocysteine
64.
65. 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
66.
67. 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
68.
69.
70. 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
71. 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 mg/L High
72. 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
73. 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
74.
75. 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
76. Progression of Biomarkers in ACS
STABLE CAD PLAQUE RUPTURE UA/NSTEMI STEMI
MPO MPO MPO TnI
CRP ICAM D-dimer TnT
IL-6 sCD40L IMA Myoglobin
PAPP-A FABP CKMB
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
77. 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
78. 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
79. 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
80. 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
81. Send the comments of this ppt to
jaipersie@gmail.com for further
enhancement of my presentations
Dr. Jaikanth
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