Biochemical Markers in

Cardiovascular Disease

Maimun Z Arthamin
Tinny R Indra
Dept of Clinical Pathology
Heart physiology
Classification of laboratory tests in
cardiac disease

Cardiovascular risk factor markers

(Cholesterol, Trygliceride, LDL, HDL,
Obesity, DM, Hypertension, smoking,
Lp(a), Homocystein, CRP….)
Markers of cardiac tissue damage
(SGOT, LDH, Myoglobin, CK,
CKMB,Troponin…..)
Markers of myocardial function
(Congestive Heart Failure)
(BNP)
Cardiovascular Disease: (5% from
mortality)
1. Myocarditis
2. Hypertension
3. Congenital heart disease
4. Rheumatoid heart disease
5. Coronary heart disease
(Coronary arterial disease)
1. Stable Angina pectoris
2. Unstable Angina pectoris
3. Myocardial infarction
6. Congestive Heart failure
 Historical criteria for diagnosis of
MI (WHO, 1980)

• Triad of criteria (WHO)

• Diagnosis requires two of:

– Severe & prolonged chest pain

– Unequivocal ECG changes
consistent with acute MI
– Elevated serum cardiac markers
Coronary Heart disease/Coronary arterial
Disease
Heart disease caused by arterial
coronary obstruction so that result
dysfunction cardiac organ
Cause: coronary atherosclerosis
Atherosclerosis = the inner layers of
artery walls are made thick and
irregular by deposits of a fatty
substance (plaque); the internal
channels of arteries become narrowed
and blood supply is reduced
 Atherosclerosis
– Atherosclerosis stages:
1. Fatty streak
2. Fibrous plaque
3. Plaque Rupture

Plaque rupture influenced by:

1. Plaque morphology and
component
2. Hemodynamic
3. Inflammation
 Atherosclerosis: Stages of Plaque Development

Plaque buildup In response to the Chemicals release The narrowed

begin when damage, platelet by cells in and artery is
endothelial cells and other types of around the plaque vulnerable to
lining the arteries cells collect at the cause further blockage by clots.
are damaged by site; a fibrous cap inflammation and The risk of
smoking, high forms, isolating buildup; an blockage & heart
blood pressure, the plaque within advanced plaque attack rises if the
and oxidizied LDL the artery wall. An contains LDL, fibrous cap cracks
and other causes; early-stage plaque white blood cells, (probably due to
destructive
excess cholesterol is called a fatty connective tissue
enzymes released
particles beneath streak smooth muscle
by WBC within
these cells. cells, platelets, and the plaque)
other compound
 Pathogenesis

– Stage 1: Asymptomatic, non

obstructive atherosclerotic plaque
development & the progression
depends on the risk factors.
– Stage 2: Rapid thrombogenesis due to
plaque rupture  stimulate platelet
aggregation, fibrin development 
thrombus occlusion.
• Stable Angina pectoris
• Unstable Angina pectoris
• Myocardial Infarction
 Pathophysiology of myocardial infarction
The pathophysiology of acute coronary syndromes and biomarkers
released into blood
Continuum of AMI risk

Plaque rupture –

Unstable angina
Asymptomatic

Myocardial Infarction
Intracoronary thrombus –

Reduced blood flow –

Myocardial ischemia – (Reversible damage)

Myocardial necrosis – Troponin, myoglobin, CK-MB (Irreversible

Damage)

Acute coronary syndromes are due to an acute or sub acute primary

reduction of myocardial oxygen supply provoked by disruption of an
atherosclerotic plaque associated with inflammation, thrombosis,
vasoconstriction and microembolization. Finite process. (>4-6 h for necrosis
to develop).
Typical Progression of Coronary Atherosclerosis
Chronic Stable Angina
 Myocardial infarction

Left coronary artery

Anterior left ventricle

Right coronary artery
 ECG changes in myocardial
infarction
R
Normal
T
U
P
Q
S

Myocardial infarction R

S-T elevation
T

Q
S
 cutoff for cutoff for
specific nonspecific
cardiac marker cardiac marker

Normal Stable Unstable Myocardial

individuals angina angina infarction

no injury no injury little to significant

moderate injury
injury

increasing
Concentration of cardiac marker
 Patients with AMI can be categorized into 4
group based on time of presentation (Apple, 2010)
1. Present early to the ED within 1 – 4 hours after the onset
of chest pain, diagnostic ECG evidence of AMI (-).
Biomarker of MI must be released rapidly from the
heart into the circ
2. 4 – 48 h after the onset : evidence of AMI on ECG (-),
requires serial monitoring of both cardiac biomarkers &
ECG changes
3. > 48 h after the onset & non specific ECG change: ideal
biomarker of myocardial injury would persist in the circ
for several days to provide a late diagnostic time
window
4. Present at anytime after the onset & clear ECG evidence
of AMI: biomarker of MI is theorytical not necessary
Markers for cardiac
tissue damage
 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
 The ideal cardiac marker
High sensitivity
High concentration in High specificity
myocardium released after Absent in non-myocardial
myocardial injury: tissue
Rapid release for early Not detectable in blood of
diagnosis non-diseased subjects
The
Long half-life in blood for ideal cardiac
late diagnosis marker does
NOT yet exist!
Analytical characteristics Clinical characteristics
Measurable by cost-effective Ability to influence therapy
method Ability to improve patient
Simple to perform outcome
Rapid turnaround time
Sufficient precision &
accuracy

Scand J Clin Lab Inves 1999;59 (Suppl 230):113-123

 Ideal cardiac markers should have:
• Highly sensitivity & specificity to the heart tissue:
provide early detection of small myocardial injury
• Be detectable relatively early after the onset of chest
pain
• Serve as a risk stratification tool in ACS patient
• The ability to be measured at low concentration of the
marker
• Extended diagnostic window: remain elevated in the
blood for several days to provide a late diagnostic
• Detect reocclusion & recurrent infarction
• Determined the timing of infarction & infarct size
• Asses the success of reperfusion after thrombolytic
therapy
 Myocardial proteins

Myoglobin

Actin, CK, AST

Myosin
Troponin

LDH
• Myocardium contains cardiac muscle
cells. Actions as cardiac pump:
contraction and relaxation.
• Cardiac muscle cells consist of protein:
Specific contractile: actin & myosin
Regulators: troponin &
tropomyosin
Energy: myoglobin
Enzymes: SGOT, CK, CK-MB, LDH
 Enzyme markers

Aspartate transaminase (AST=SGOT)

Lactate dehydrogenase
Five isoenzymes, composed of
combinations of H (heart) and M
(muscle) subunits
Creatine kinase
Three isoenzymes, composed of
combinations of M (muscle) and B
(brain) subunits
 Time line of markers of myocardiac
damage & function

Myoglobin assay RIA for BNP RIA for

and proANP proBNP
CK – MB CK-MB cTnl assay
Electrophoresis POCT for myoglobin CK-
mass assay
for CK and LD MB, cTnI
AST in
AMI CK in Immuno assay for
RIA for cTnT assay
AMI proBNP
ANP
IMA
Genetic
Markers

1950 1960 1970 1980 1990 2000 2005

Time [years]

Notes:
AST: aspartate aminotransferase ANP: atrial natriuretic peptide
CK: creatine kinase BNP: brain natriuretic peptide
LD: lactate dehydrogenase POCT: point-of-care testing
cTn: cardiac-specific troponin IMA: ischaemia-modified albumin
 AST (Aspartic aminotransferase)

Enzyme released by myocard infarction

Increase after 2-4 hours infarction, maximum
at 24 hours and decline to normal levels at
about 48 hours post infarction
Not specific for cardiac disease in that it may
reflect disease of the lung, liver, and skeletal
muscle.
Not reliable marker and now rarely used in
diagnosis
Normal value: 9-25 units/L
 Lactate Dehidrogenase (LDH) 1956

Levels do not begin to rise until 12 -24 h, do not

peak until 48 -72 hours post infarction and are
therefore of little help in early diagnosis
Primarily used if myocard infarction occurred more
than 24 h prior to admission, when the CK levels are
declining. Remain elevated up to 10 days post
infarction.
Normal: 110-210 units/L
There are 5 isoenzymes (LDH1-LDH5) –
electrophoresis. MW: 134.000 kDa
Elevated in hemolysis, megaloblastic anemia, liver,
renal, and skeletal muscle disease.
 Tissue specificity of LD isoenzymes

LD isoenzyme Tissues
LD-1 Heart (60%), RBC, Kidney
LD-2 Heart (30%), RBC, Kidney
LD-3 Brain, Kidney
LD-4 Liver, Skeletal muscle, Brain, Kidney
LD-5 Liver, Skeletal muscle, Kidney
 LD isoenzyme electrophoresis (normal)

LD-2 > LD-1 > LD-3 > LD-4 > LD-5

LD-2

LD-1
LD-3
LD-4
LD-5

Cathode (-) Anode (+)

 LD isoenzyme electrophoresis (abnormal)

LD-1 > LD-2

LD-1

LD-2

LD-3
LD-4
LD-5
Cathode (-) Anode (+)
Creatine phosphokinase (CPK/CK)
(1960)

Normal value: 40-150 units/L

There are 3 subunits : CKMM, CKBB, CKMB
(1970)
CKMB mass as Gold Standard for Dx Myocardial
Infarction since 1980 (WHO).
Normal value : 0-5 ng/ml
CK or CKMB levels increase 4-6 h post attack,
maximum level after 24 h and decline to normal
level after 48-72 h.
CPK/CK...

CKMB levels increase in skeletal muscle

disease and post exercise
There are 2 CKMB isoenzymes (1997): MB1
and MB2
– more sensitive and specific than CKMB.
– Increase 1-4 h post attack.
 CK-MBmass Relative index (%RI)

% RI = (CK-MBmass / Tot CK activity) x 100

Increased RI suggests myocardial origin

Not absolute – lack of CK-MBmass assay
standardization and tissue variability
RI > 4% with tot CK activity elevated
(preferably > 2x UR limit) suggests
myocardial necrosis
 Myoglobin (Mb)

Low MW protein
Skeletal & cardiac muscle myoglobin identical
Serum levels increase within 2 h of muscle
damage
Peak at 6 – 9 h
Normal by 24 – 36 h
Excellent negative predictor of myocardial injury
2 samples 2 – 4 hours apart with no rise in
levels virtually excludes AMI
Rapid, quantitative serum immunoassays
Myoglobin...

Myoglobin is sensitive and early cardiac

marker
Myoglobin as oxygen binding protein in
cardiac muscle and skeletal muscle
Small MW = 17,8 kDa so loss easily in blood
circulation when cardiac necrosis
Detect in blood after 1-4 h post attack,
maximal in 4-12h and normal return after
24h
Myoglobin...

Normal: < 50 ng/ml

Myoglobin: high sensitivity cardiac marker,
if not increase during the first 3 - 6 h 
not cardiac infarction.
Also increase in shock, renal, skeletal
muscle disease and post exercise
 Troponin

Tropomyosin Actin TnT (42 Kd)

TnC TnI (23 Kd)

Myosin

Thick Filament
 Cardiac troponins

Normal: < 0.1 - 0.5 ng/ml

Striated and cardiac muscle filaments consist of:
– Actin
– Myosin
– Troponin regulatory complex
Troponin consists of 3 sub-units TnC, TnT & TnI
– TnT MW = 37 000
– TnI MW = 24 000
A fraction of total troponin is found free
dissolved in the cytosol
Tissue specificity of Troponin subunits

Troponin C is the same in all muscle tissue

Troponins I and T have cardiac-specific forms,
cTnI and cTnT
Circulating concentrations of cTnI and cTnT are
very low
cTnI and cTnT remain elevated for several days
Hence, troponins would seem to have the
specificity of CK-MB (or better), and the long-
term sensitivity of LD-1
Troponin subunits...

Sensitive to minor cardiac damage

cTn values are particularly useful at ruling
out MI when the value is negative at 8
hours after the onset of chest pain
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 level may identify patients at
increased risk of cardiac events
 Biochemical markers in AMI: release,
peak and duration of elevation
100 – cTnT

Myoglobin and MB isoforms

Multiples of the Upper Reference Limit

50 –

cTnI

15 –
MLC

10 –
CK-MB
LD1
5 –

Reference interval
     
 

0 1 2 3 4 5 6 7 10
Days After Onset AMI
 Kinetics of cardiac markers after AMI

Marker Detection Peak Disappearance

Myoglobin 1–4h 6–7h 24 h

CK-MB mass 3 – 12 h 12 – 18 h 2 – 3 days
Total CK
cTnT 4–8h 12 – 30 h 3 – 4 days
cTnI 4 – 12 h 12 – 48 h 5 – 15 days
IMA 4 – 12 h 12 – 24 h 5 – 7 days
(ischemia) few minutes 2–4h 6 hours

These values represent averages.

 National Academy of Clinical Biochemistry (NACB)
Recommendations for Cardiac Markers in CAD
(1999)
• Rule in/out of AMI cannot be made on the
basis of data from a single blood sample
Serial determinations recommended
• Use of two markers:
– Early marker (rising 2-4hr after pain
Myoglobin
onset)

– Definitive marker (rising 4-6 h after pain

onset) Cardiac
• High sensitivity and specificity troponins
• Remains abnormal several days
 Plaque disruption

Thrombus formation with or without embolization

Acute cardiac ischaemia

No ST Elevation ST Elevation

Markers of myocardial Elevated markers of Elevated markers of

necrosis not elevated myocardial necrosis myocardial necrosis

Unstable Angina MI (NSTEMI) MI (STEMI)

Pectoris

No Q-wave Q-wave

Acute Coronary Syndrome (ACS)

Spectrum of ACS in patients with acute cardiac chest pain
 Cardiac Marker in Chest Pain
Myoglobin-CK-CKMB

0-4 h >4 h

Myoglobin-CK-CKMB CK-CKMB

Negative or positive

4 hours

CK-CKMB

CK-MB Index >4 CK-MB Index <4

MI Positive MI Negative
 Troponin
0-4 h >4 h

Troponin Troponin

Negative or positive

4 hours

Troponin

> 2.5 ng/ml < 0.5 ng/ml

0.6 – 2.5 ng /ml
MI Positive MI Negative
Suspect MI
 “New” generation cardiac markers

• Myoglobin
– Currently earliest marker
– Like total CK it is by Sensitivity
no means cardio-specific

• Troponins
– Kinetics comparable with Specificity
total CK and CK-MB
– Cardio-specific
 Major Forms of Cardiovascular Disease:
Congestive Heart Failure

Congestive heart failure = condition

resulting from the heart’s inability to pump
out all the blood that returns to it
Blood backs up in the veins leading to the
heart, causing an accumulation of fluid in
various parts of the body
Caused by high blood pressure, heart attack,
atherosclerosis, birth defects, rheumatic
fever
 Biochemical markers of myocardial
function (congestive heart failure)

Cardiac natriuretic peptides:

(Atrial natriuretic protein, ANP; brain
natriuretic protein, BNP & pro-peptide
forms)
• Family of peptides secreted by cardiac atria
(+ ventricles) with potent diuretic,
natriuretic & vascular smooth muscle
relaxing activity
• Levels of these neuro-hormonal factors can
be measured in blood
Cardiac natriuretic peptides...

• Clinical usefulness (especially BNP/n-terminal

pro-BNP)
– Detection of LV dysfunction
– “Screening” for heart disease
– Differential diagnosis of dyspnea
– Stratification of CHF patients
• New generation markers currently under
development