Cardiac biomarker past, today and future

CARDIAC BIOMARKER

Dr Anurag YadavPostGraduate

BIOMARKER

“A biomarker is a substance used as an indicator of a biologic state”.

Accurate repeated measurements at reasonable cost

Must provide additional information

Should aid treatment

CARDIAC BIOMARKERS

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

CHARACTERISTICS

High cardiac specificity

Pharmacokinetics of cardiac biomarker

Easy diagnosis

Marker should play a designed role in the treatment and management of clinical subject

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

CLASSIFICATION OF CARDIAC BIOMARKERS

Biomarkers of myocardial injury• markers of myocardial necrosis• markers of myocardial ischemia

Biomarkers of haemodynamic stress

Inflammatory and prognostic Biomarkers

CLASSIFICATION OF

CARDIAC BIOMARKERS

ACCORDING TO VARIOUS STAGES DURING CARDIAC DISEASE PROCESS

BIOMARKERS OF MYOCARDIAL INJURY

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

FABP

Marker for inflammatio

nhsCRP sCD40L Homocystei

n

Marker for plaque

DestabilizationPAPP-A LP-PLA2

Marker for hemodynamic

stress

Natriuretic peptides• ANP• BNP• Pro-BNP• CNP• DNP

Time of increase

Peak Return to normal

CK-MB 4-8 h 12-24h 72-96h (3-4D)

LDH 2-5 D 10 D

Myoglobin 2-4 h 8-10h 24 h

cTnI 4-6 h 12 h 3-10 D

cTnT 4-6 h 12-48 h 7-10 D

0 4 8 12 16 20 24 28 32 36 40 44 480

1

2

3

4

5

6

7

myoglobinCK-MBcTnTcTnI

COMPARISON OF CTN, CK-MB , MB

Time after onset of AMI (hours)

Χ up

per l

imit

of re

fere

nce

inte

rval

OTHER CARDIAC MARKERS

miRNA

IMA

MPO

MMP-9

FFAU

sCD40L

PIGF

PAPP-A

Choline

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

TROPONIN ASSAYS

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)

Creatine kinase (CK) is a cytosolic enzyme

CK-MB

CK-MB :

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 hrsReturn to normal : 24-36 hrs

Can be used to indicate early re-infarction if level normalizes and

then increases again.

CK-MBRelative Index = χ

100 Total CK

*The relative index allows the distinction between increased total CK due to myocardial damage and

that due to skeletal or neural damage.

*A relative index exceeding 3 is indicative of AMI

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

• Earliest Rise: 1-4 hrs• Peak 6-9 hrs• Return to normal: 12 hrs

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:

Myoglobin

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

0 4 8 12 16 20 24 28 32 36 40 44 480

1

2

3

4

5

6

7

myoglobinCK-MBcTnTcTnI

COMPARISON OF CTN, CK-MB , MB

Time after onset of AMI (hours)

Χ up

per l

imit

of re

fere

nce

inte

rval

Ischemia Modified Albumin (IMA)

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 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

NATRIURETIC PEPTIDES

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

OTHER CARDIAC MARKERS

miRNA

IMA

MPO

MMP-9

FFAU

sCD40L

PIGF

PAPP-A

Choline

hsCRP

Homocysteine

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, or smoking

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

Homocysteine

Elevated levels appear to be an independent risk factor,

Screening recommended in patients with premature CV disease (or unexplained DVT) and absence of other risk factors

Treatment includes supplementation with folate, B6 and B12

Homocysteine

C-REACTIVE PROTEIN

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 specificityNo 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 Low1.0-2.9 mg/L IntermediateGreater than 3.0 mg/L High

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 ehibits 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.

CYTOKINES

Variety of interleukins (TNF, IL-1, IL-6, IL-8, IL-12, IL-18) are responsible for the atherosclerosis events.Regulated through the T cell-mediated and monocytes.

At present there are no standardized assay, reference interval studies.

MYELOPEROXIDASE (MPO)

Released from aggregated neutrophils.

Increased in CAD, ACS.

Is one of the best prognostic marker

At present no standardized assay method, nor consistency validation of the test.

Further, Type of specimen collect have shown variations.

Normal level: 640 pmol/L

Previously known as PAF ( platelet activating factor)

Synthesised from monocytes and lymphocytes.

Known to form lipid fragments which are most atherogenic which increase endothelial adhesion.

FDA approved for the assay.

Threshold for high risk: >200μg/L

It is an metaloprotimase.

Produced from the plaque which are prone to rupture.

Marker for the adverse CV events.

At present no standardized assay, consistency validation is present.

By two approach, median value for free PAPP-A is 0.18 mIU/L.

CHOLINE

Is released after stimulation by phospholipase D.

Touted as a test of prognosis in pt with chest discomfort.

Not available

UNBOUND FREE FATTY ACID (FFA-U)

Marker for ischemia.

Ischemia increase the small unbound fraction.

Studies revealed mixed result.

Not available

NOURIN

Small protein released by stressed myocytes.

Induce changes of inflammatory cytokines and attract neutrophils.

Preliminary studies have made attempt to validate its use.

30 a.a glycoprotein.

Prognostic marker in hemorrhagic and sepsis.

Recently, early biomarker for rule out the AMI with normal cTn.

The test method has to undergo many trials and head-to-head comparison with cTn.

COPEPTIN