Biomarkers in acs dr.i.tammi raju

I . TAMMI
RAJU
BIOMARKERS IN ACS

DEFINITION
 Biomarkers are measurable and quantifiable
biological parameters which serve as indices for
health and physiology assessment. This includes
disease risk and diagnosis
[WHO MONICA Project populations. Lancet 2000,
355: 688-700]

 The diagnosis of acute myocardial infarction
(AMI) can be made with the detection of a rise/fall
of cardiac troponin (at least one value above the
99 th percen-tile of the upper reference limit) and
one of
 1) symptoms of ischaemia,
 2) electrocardiogram (ECG) changes of new
ischaemia,
 3) new pathological Q waves or
 4) imaging evidence of new loss of viable
myocardium.

• Acute coronary syndrome (ACS) is an umbrella
term for a wide spectrum of clinical sign and
symptoms suggestive of myocardial ischaemia.
• Diagnosis of acute coronary syndrome (ACS)
encompasses a wide spectrum of myocardial
ischaemia varying from assuredly benign to
potentially fatal.
• Cardiac biomarkers have had a major impact on
the management of this disease and are now the
cornerstone in its diagnosis and prognosis.

BIOCHEMICAL MARKER OF
MYOCARDIAL INJURY
 1. It should be myocardial tissue specific and its
concentration in the myocardium should be high but
should be absent in non-myocardial tissues.
 2. It should be detectable in blood soon after the
myocardial injury i.e. the sensitivity should be high.
 3. It should remain elevated in blood for several days
of the onset of damage so that it can be detected in
patients coming to the hospital quite late after
myocardial infarction.
 4. It could be assayed by simple and quick method
i.e. the turn-around time (TAT) should be low because
the first few hours of myocardial infarction are crucial
for medical intervention.

Thus the appearance of these markers in the blood stream
and their measurable life in the blood following ischaemia
depends on :
 1. Their intracellular location or compartmentation – The
molecules present in cytosol are released first when
myocardial damage occurs whereas the structurally bound
molecules are released later.
 2. Their molecular weight – The larger molecules diffuse at
a slower rate than the smaller molecules.
 3. Their rate of elimination from the blood – The smaller
molecules are eliminated rapidly as compared to larger
molecules.
 4. Blood flow in the necrotic region – The difference in the
circulation of blood in the infarcted area leads to
differences in the release of cytosolic proteins from the
necrotic region while the release of structurally bound

THE CRITERIA FOR NOVEL
MARKERS
An (AHA) scientific statement ;:
 1) the need to demonstrate the degree to which a
novel marker adds to the prognostic information
provided by standard risk markers (both in terms
of discrimination and accuracy);
 2) the clinical value of the marker as measured by
the effect on patient managemen and outcome;
and
 3) the cost-effectiveness of the marker.

PATHOPHYSIOLOGY BASIS OF
BIOMARKERS
 Biomarkers of inflammation .
 C-reactive protein.
 Myeloperoxidase (MPO)
 Soluble fragment CD40 ligand (sCD40L)
 Biomarkers of plaque instability/disruption.
 Pregnancy-associated plasma protein A (PAPP-A)
 Choline
 Placental growth factor
 MMP-9
 Myeloperoxidase (MPO)
 Biomarkers of myocardial ischemia .
 Ischemia-modified albumin (IMA)
 Free fatty acids unbound to albumin (FFAu)
 Heart-type fatty acid binding protein (H-FABP) (PRE
NECROSIS)
 Myocardial necrosis
 Tn ,
 CK-MB
 Pump failure,myocardiac stress
 Nt-PROBNP,GDF-15,ST-2,ET-1

ESTABLISHED AND OLD
BIOMARKERS

ASPARTATE AMINOTRANSFERASE
(AST/GOT)
 The levels of serum AST activity begin to rise 3-8
hours after the onset of the myocardial injury with
peak levels on an average at 24 hours and finally
it returns to normal levels in 3- 6 days.
 It was considered as a very good marker of
cardiac injury as it was found to be normal in
pulmonary embolism, acute abdominal conditions
and other heart conditions such as angina and
pericarditis .
 Agress CM. Evaluation of the transaminase test. Am J
Cardiol 1979; 3:74-93. Kachmar JR. Enzymes, In :
Fundamentals of Clinical Chemistry, NW. Tietz, Editor
Sounders, Philadelphia 1976.
 But later on, its use become limited due to its
elevation in trauma to skeletal muscles and liver
diseases

LACTATE DEHYDROGENASE (LD)
AND ITS ISOENZYME LD1
 An increase in serum LD activity is found
following myocardial infarction beginning within
6 – 12 hours and reaching a maximum at about
48 hours and it remains elevated for 4-14 days
before coming down to normal levels.
 The prolonged elevation makes it a good marker
for those patients admitted to the hospital after
several days of MI.
 However, its use is discouraged due to its non-
specificity as its increased levels are found in
progressive muscular dystrophy, myoglobinuria,
leukaemia, pernicious anaemia, megaloblastic
and hemolytic anaemia, renal disease and in
generalized carcinoma .

 LD has five isoenzymes (LD1, LD2, LD3, LD4
and LD5).
 The cardiac muscles are rich in LD1 and LD2
(LD1>LD2) while in liver LD3, LD4 and LD5 being
plentiful.
 LD1 manifests the greatest catalytic activity in
reducing α-oxobutyrate to α-hydroxybutyrate,
hence it is termed as α-hydroxybutyrate
dehydrogenase and it was considered to be
more sensitive marker of MI than total LD and it
remained elevated even longer than total LD.
 Moreover, α-hydroxybutyrate dehydrogenase
stayed normal or showed small increase in
hepatitis.
Elliot BA, Wilkinson JH, Serum “α-hydroxybutyric

CELLULAR DISTRIBUTION OF
MB,CK-MB,Tn

CREATINE KINASE (CK) AND
ITS ISOENZYME MB (CKMB)
 CK has three isoenzymes namely CKBB, CKMB
and CKMM each consisting of two subunits
named according to main tissue of occurrence : B
(brain) and M(skeletal muscles).
 Myocardium contains 60% CKMB and 40%
CKMM along with traces of mitochondrial CK
(macro CK type II) .
 Being highest in proportion in myocardium
CKMB has been used as the biochemical marker
in patients with suspected acute myocardial
infarction (AMI).
 The ratio of the CK-MB / total CK has also been
proposed for the diagnosis of the origin of raised

 It begins to increase between 3-5 hours after the
onset of infarction and peaking at 16-20 hours.
 The techniques used (electrophoresis and
immunoinhibition) to quantitate CKMB catalytic
activity were not sensitive enough for early use,
being relatively non-specific and long turn-around
time restricted its use primarily for confirming MI
at 24 hours post injury .
 Recently, the measurement of mass
concentrationof CKMB has increased its
sensitivity and specificity enabling to measure
small changes during the early hours following
MI.
 The turn-around time is also low.

 Despite all these advantages of CKMB mass
assay it has two main limitations :
(1) it is not perfectly specific to cardiac injury with
increases occurring in
 large amounts in skeletal muscle and increased
levels found in
 muscular dystrophy ,
 hypothyroidism , hypothermia
 alcoholism ,
 cerebrovascular accidents and
 a variety of myopathies make it unsuitable as a
marker of myocardial injury
(2) the early release pattern limits its use for the
late MI diagnosis.

Other uses;-
 But it has a definite place for the diagnosis of
reinfarction and has prognostic value in patients
with unstable angina .
 Its potential as an aid in
 non-invasive detection of coronary recanalization
following thrombolytic therapy and also as a
 sensitive marker in detecting myocardial necrosis
following percutaneous coronary intervention has
also been shown

MYOGLOBIN
 Myoglobin, a 18 KD cytosolic protein, appears in
blood earliest after myocardial injury than any
other marker available so far.
 The detectable levels of myoglobin in the blood
are found as early as 2 to 3 hours after the onset.
 Its peak value is obtained at 6 – 12 hours after
the onset of the symptoms and then it normalizes
over the next 24 hours.
 However, it is not cardiac specific as its release
from the skeletal muscles cannot be distinguished
from that released due to cardiac injury
 severe renal insufficiency and in
 alcohol binges .
 Christenson RH, Azzazy HM. Biochemical markers of the
acute coronary syndromes. Clin Chem 1998; 44: 1855-1864

 Several studies have compared the diagnostic
utility of serum myoglobin with other markers like
CKMB, CKMB mass, CKMB isoforms and cardiac
Troponins but the results have been controversial
.
 The high negative predictive value of serum
myoglobin for excluding early infarction has
encouraged its use along with more specific
markers such as CKMB and cardiac troponin and
this two – marker approach has improved the
diagnosis of MI .
 Jernberg T, Lindahl B, James S, Ranquist G, Wallentin
L.Comparison between strategies using creatine kinase –
MB (mass), myoglobin and troponin T in AMI Am J Cardiol

MYOGLOBIN / CARBONIC
ANHYDRASE III RATIO
 Carbonic anhydrase III (CA III) is present in skeletal
muscles and is released into circulation following injury.
 The measurement of myoglobin / carbonic anhydrase III
ratio improves the specificity of myoglobin as an early
marker of MI .
 The ratio was found significantly higher in patient with MI
whereas myoglobin and CA III were released in a fixed
ratio following exercise and showed no significant
difference in the ratio for trauma patients
 This ratio was also used in evaluating the success of
reperfusion after MI
However, its use is limited due to lack of availability of
commercial assay of CA III and it has no use in AMI with a
delayed presentation.
 Beuerle JR, Azzazy HM, Styba G, Duh SH, Christenson
RH. Clin Chim Acta 2000; 294:115-28.

HISTORY: TROPONIN
 Troponin I first described as a biomarker specific for
AMI in 19871; Troponin T in 1989
 Now the biochemical “gold standard” for the
diagnosis of acute myocardial infarction via
consensus of ESC/ACC.
Am Heart J 113: 1333-44
J Mol Cell Cardiol 21: 1349-
53

TROPONINS
 Troponin is a protein complex
located on the thin filament of
striated muscles consisting of
the three subunits namely
Troponin T (TnT), Troponin I
(TnI) and Troponin C (TnC)
each having different structure
and function. Of the three
troponins,
 TnT and TnI are being used as
the biochemical markers for the
diagnosis of myocardial injury.
 The troponins found in cardiac
tissue (cTn) have a different
amino acid sequence than that
present in troponin of skeletal
muscles.
 This makes cTnT and cTnI more
specific for the diagnosis of
myocardial injury.
 These cardiac troponins (cTns)
appear in the blood as early as
3-4 hours of the acute episode
and remain elevated for 4-14
days.

 The pattern of release of
troponin may be
monophasic or biphasic.
 This release kinetics is
related to the distribution of
these proteins within the
myocardial cell.
 About 94-97% of these
troponins is bound to
myofibril and only 3% of
cTnI and 6% of cTnT is free
in the cytoplasm
 When the myocardial
damage occurs the cytosolic
troponins reach the blood
stream quickly resulting in a
rapid peak of serum
troponin observed during the
first few hours.
 This is followed by the

 Studies have shown that cardiac troponins should
replace CKMB The reasons being :
 1. Troponins are highly cardiospecific especially
the TnI (100%).
 2. The prolonged elevation (4-14 days) make it a
good marker for patients admitted to the hospital
after several days of MI.
 3. cTns have greater sensitivity for minor
degrees of myocardial injury due to the
cardiospecificity and their very low concentration in
serum of normal individuals.
 4. These are excellent prognostic indicator in
patients with unstable angina and is a very useful
parameter for stratifying risk in acute coronary
syndrome(ACS).
Rottbauer W, Greten T, Muller-Bard off M et al. Troponin T:A

 5. A single measurement of serum cTnT at the time
corresponding to the slow continuous release after
AMI (~72 hours after onset) can be used as a
convenient and cost effective non-invasive estimate
of infarct size whereas CKMB requires repetitive
sampling
 6. The early serial measurements of cTnI are a
more accurate predictor of early coronary artery
reperfusion after thrombolytic therapy as
compared to CKMB and myoglobin and it also
identifies a subgroup of patients with unstable
coronary syndrome in whom prolonged
antithrombotic treatment with low-molecular
weight heparin can improve the prognosis.

 7.cTn typically increases more than 20 times above the
upper limit of the reference range in myocardial
infarction as compared to creatine kinase-myocardial
band (CK-MB) which usually increases 10 times above
the reference range.
 8.This provides an improved signal - to - noise ratio,
enabling the detection of even minor degree of necrosis
with troponin. The cTn begins to elevate 3 h from the
onset of chest pain in MI. Because of the continuous
release, cTn elevation persists for days (cTnI: 7-10
days, cTnT: 10-14 days).
 9. According to U.S. National Academy of Clinical
Biochemistry (NACB) and Joint European Society of
Cardiology and American College of Cardiology (ESC/
ACC) guidelines cTns are the most specific and
sensitive biochemical markers.

ROC Curve for Tpn T
 Best discriminator
point is 0.2 g/L at 9
h after onset of AMI

cTnT Versus cTnI
 Both cTnT and cTnI are almost equally good markers
and it is difficulty to say which is better because both
have some positive and negative points.
 cTnI is 100% cardiospecific and it is not elevated
in chronic renal disease, trauma and skeletal
muscle disease.
 The overall diagnostic specificity and efficiency of
cTnI is better than cTnT and it (cTnI) is proved to be
the most sensitive marker in detecting myocardial
necrosis following percutaneous intervention .
 Both cTns undergo posttranslational modifications
such as phosphorylation, oxidation, reduction,
proteolysis and form complex with other troponins.
 cTnI is more prone to these modifications and these
modification may prevent some antibodies used in the
assay system from binding to the molecules and

 The other advantage of cTnI may be its greater
specificity in patients of ESRD.
 However, the important advantage of cTnT is that
due to international patent restrictions there is
only one assay for its measurement, thus cTnT
demonstrates a high degree of precision at the low
end of measurement range and a relatively uniform
cut-off concentration.
 In contrast, at least 18 different commercial assays
for cTnI are available leading to considerable
variation in the cut-off concentrations in the definition
of a myocardial infarction by cTnI values.
 Thus, a clinician should be aware of the cTnI cut-off
values specifically associated with the particular
assay used by the laboratory.

 The life-time of cTnT in blood (5- 14 days) is
some what more than that of cTnI (4-10 days).
 Although cardiac troponins are extremely specific
for myocardial necrosis, they do not
discriminate between ischaemic and non-
ischaemic etiologies of myocardial injury.
 Combining troponin with other cardiac biomarkers
may offer complimentary information on the
underlying pathobiology and prognosis in an
individual patient .
 The recommended time course for collection of
blood samples for cTn is at hospital admission, 6
and 12 hours later but when it is used along with
an early marker like myoglobin (two-marker
strategy) then at hospital admission, 4,8 and 12

WASH-OUT PHENOMENON.
 Patients with ST-segment elevation myocardial
infarction who achieve an effective reperfusion have
a greater and earlier peak plasma concentration of
troponin, followed by a faster return to normal –
the so-called “wash-out phenomenon” – compared
with those patients having no significant reperfusion.
 In this event, two blood samples should be collected –
at the time of the patient's admission to hospital,
and 90 min later – and the enzyme plasma
concentrations compared.
 The ratio between the concentrations at these two
points can be used to discriminate between
successful and unsuccessful reperfusion. In general,
the greater the ratio (at least 5), the more likely it
is that reperfusion has occurred.
 If reperfusion has indeed occurred, estimation of
infarct size using peak biomarker concentration may
be not reliable..

AFTER CABG
Increase more than 5 x 99 percentile URL during first 72
hrs

TROPONIN SANDWITCH-IMMUNOASSAY

Tn IN ESRD
 The cardiac troponin especially cTnT pose diagnostic
challenges in patients of chronic renal failure.
 Frequent cTnT elevations (30 to 70% of end stage renal
disease (ESRD) patients compared with <5% in similar
patients of cTnI) are seen in patients of renal failure in the
absence of clinical suspicion of ACS .
 The putative mechanisms for chronic elevation of troponin
in chronic renal disease patients include
 endothelial dysfunction,
 acute cardiac stretch,
 microinfarction and
 left ventricular hypertrophy.
 However, it is important to understand that in the setting of
acute coronary syndrome these patients should be treated
as if renal failure were not present as the short term
prognostic value of troponin T for cardiovascular event is

 Increasing evidence suggests that chronically elevated
troponin levels indicate a worse long-term prognosis for
cardiovascular outcomes in this patient population
 False positives have been reported with use of troponin-
T in ESRD patients but not as much with troponin-I
 CK: plasma concentrations are elevated in 30-70% of
dialysis patients at baseline, likely secondary to skeletal
myopathy, intramuscular injections and reduced
clearance.
 CK-MB: 30-50% of dialysis patients exhibit an elevation
in the MB fraction >5% without evidence of myocardial
ischemia
 Therefore, the most specific marker for suspected AMI
in ESRD patients is Troponin-I with an appropriate

DELTA TROPONIN AND ULTRA-SENSITIVE
TROPONINS
 The use of delta
troponin, the change in
the troponin value over
time, has improved the
diagnostic accuracy for
AMI
 Newer ultra-sensitive
troponin assays may be
able to utilise very low
levels of detection, and
employ a „delta
approach‟ that
measures change
between initial and
incremental levels at
Detects <1 pg/ml
<10 pg /ml-noncardiac
10-30 pg/ml-mild cardiac injury

BIOMARKERS OF
BIOMECHANICAL STRESS

Biomarkers of biomechanical stress
BNP/NTproBNP
 One of the best known biomarkers of
biomechanical stress is the B-type Natriuretic
Peptide (BNP).
 Secreted by the ventricles in response to
cardiomyocytes under tension .
 BNP binds and activates receptors causing
reduction in systemic vascular resistance, central
venous pressure and natriuresis.
 This biomarker has a short half-life but is released
with the N-terminal portion of the pro-BNP peptide
(NTproBNP), a peptide much more stable in
serum and can be measured easily.
 In Non ST-elevation acute coronary syndromes
(NSTEACS), this biomarker predicts in-hospital
and 180 day death or heart failure.

 NTproBNP/BNP provides incremental information
on cardiovascular death at one year in the older
population above and beyond GRACE score .
 On its own, it is at least as good as the GRACE
score when predicting inhospital mortality
following AMI .
 it also improves the accuracy of the prognosis
when added to the GRACE score.
 N-terminal pro-B-type natriuretic peptide complements the
GRACE risk score in predicting early and late mortality
following acute coronary syndrome. Clin Sci (Lond) 2009,
117:31-39.

CONDITIONS THAT INFLUENCE (BNP)

MID-REGIONAL PRO-ATRIAL
NATRIURETIC PEPTIDE (MRPROANP)
Khan SQ, Dhillon O, Kelly D, Squire IB, Struck J, Quinn P,
Morgenthaler NG,
comparison with plasma midregional pro-atrial natriuretic
peptide: the LAMP (Leicester Acute Myocardial Infarction
Peptide) study.
J Am Coll Cardiol 2008, 51:1857-1864.
 Like BNP, ANP has similar neurohormonal effects and has
a similar secretory profile post AMI.
 Prior studies have attempted to accurately measure levels
of ANP and N-ANP, with limited success.
 N-ANP has been demonstrated to be associated with late
mortality follow-ing AMI.
 Such early N-ANP assays were often affected by
interferences and instability of analyte.
 Because of disappointing results, ANP was thought to
provide limited prognostic information.

 However, the discovery of the novel MRproANP
fragment a sub-stantially more stable peptide
compared to has led to the finding that
MRproANP is at least as good at predicting
death and heart failure as NTproBNP .
 Immunoluminometric assay for the midregion of pro-atrial
natriuretic peptide in human plasma. Clin Chem 2004,
50:234-236
 When MRproANP levels were divided into
quartiles, the top quartile was associ-ated with a
hazard ratio (HR) of 3.87 (vs. NTproBNP HR
3.25) predicting death at follow-up.
 Both biomarkers had similar AUC of ROC (0.83).

GROWTH DIFFERENTIATION FACTOR-15
(GDF-15)
Wollert KC, Kempf T, Peter T, Olofsson S, James S,
Johnston N, Prognostic value of growth-
differentiation factor- 15 in patients with non-ST-
elevation acute coronary syndrome.
Circulation 2007, 115:962-971.
 GDF-15 is a member of the TGF-Beta cytokine
superfamily.
 It is not normally expressed in the heart, but
under episodes of stress (for example, ischaemia
and reperfusion) its levels go up in a variety of
tissues, including cardiomyocytes.
 It has an antihypertrophic effect, demonstrated in
knockout mice which develop early cardiac

However, GDF-15 is not specific for cardiovascular dis-orders
and has been found to be elevated in a variety of
malignancies (prostate, colon, glial)

ST2
Shimpo M, Morrow DA, Weinberg EO, Sabatine
MS, Murphy SA, Antman EM, Lee RT: Serum
levels of the interleukin-1 receptor family member
ST2 predict mortality and clinical outcome in AMI.
Circulation 2004, 109:2186-2190.
 ST2 is an IL1-receptor-like protein which was
found to be elevated in serum of hearts under
mechanical stress .
 ST2 predicts cardiovascular death following
ACS .
 ST2 turned out to be the target for an Interleukin
called IL-33 which seems to have a
cardioprotective role, and only appears when
myocytes are under biomechanical stress.

 It is thought that ST2/IL33 interaction also
reduces atheroma burden .
 Post AMI though, it correlates somewhat with
NTproBNP , and both these biomarkers predict
death after MI (at six months) or heart failure.
 Investigations into the use of IL33/ST2 pathway
activation as a thera-peutic target are still
ongoing .
 Interleukin-33 prevents apoptosis and improves survival
after experimental myocardial infarction through ST2
signaling. Circ Heart Fail 2009, 2:684-691.
 ST2 is also elevated in
 acute asthma and
 autoimmune disease
 The specificity of ST2 to myocardial tissue stretch
will need to be determined before it can be used

ET1/CTproET1
 Khan SQ, Dhillon O, Struck J, Quinn ; C-terminal pro-
endothelin-1 offers additional prognostic information
in patients after acute myocardial infarction: Leicester
Acute Myocardial Infarction Peptide (LAMP) Study.
Am Heart J 2007, 154:736-742.
 Endothelin-1 or the more stable C-Terminal portion of
pro-Endothelin-1(CTproET1) has also been found to
be predictive of death or heart failure following an
AMI .
 ET1 is a potent vasoconstrictor peptide found
originally in vascular endothelial cells but has
subsequently been isolated in pulmonary, renal and
smooth muscle cells .
 It activates ETA and ETB receptors; ETA receptors
are located predominantly on smooth muscle tissue of
blood vessels, mediating vasoconstriction and sodium
retention, whereas ETB receptors are located

 Endothelin appears to be detrimental post-MI,
 extending the infarct and
 reducing coronary blood flow . It is also
 grossly ele-vated following cardiogenic shock
 ET-1 is very unstable and measuring its levels can be
problematic due to binding with receptors and other
proteins.
 However CTproET1 is a stable by-product of the release
of the pre-cursor which indirectly measures activity of the
endothe-lial system.
 ET1 is increased in proportion to the severity of the
disease post AMI
 Likewise CTproET1 is also elevated post-MI, and levels
above the median pre-dict death or heart failure (HR 5.71,
P= 0.002).
 This variable is independent of age, Killip class and past
medical history.
 Plasma concentration of CTproET-1 peaks at Day 2

2) BIOMARKERS OF
NEUROHORMONAL PATHWAY
ACTIVATION
 Mid-Regional-pro-Adrenomedullin (MRproADM)
 c-terminal-provasopressin (copeptin)

Mid-Regional-pro-Adrenomedullin
(MRproADM)
Khan SQ, O'Brien RJ, Struck J, Quinn P,
LAMP(Leicester Acute Myocardial Infarction
Peptide) study.
J Am Coll Cardiol 2007, 49:1525-1532.
 Adrenomedullin was first identified in human phaeo-
chromocytoma cells .
 It is highly expressed in endothelial cells.
 Adrenomedullin mediates an increase in cAMP with
resultant vasodilatation and hypotension .
 Its other roles have not been well defined, but some
have suggested a cardioprotective role at the time
of the insult.

 The activity of = BNP; MRproADM that is, increase of
nitric oxide production causing vasodilata-tion,
natriuresis and diuresis
 Like BNP it is released in proportion to the severity
of heart failure and is inversely related to the left
ventricular ejection fraction (LVEF).
 Adrenomedullin (ADM) is difficult to measure in
plasma as it is partially com-plexed with complement ,
in addition it is also rapidly cleared from the
circulation.
 Indirect quantification of this peptide can be made by
measuring the mid-regional fragment of the
proAdrenomedullin peptide, which is more stable and
secreted in equimolar concentrations as ADM.
 Plasma adrenomedullin concentration in patients with heart
failure. J Clin Endocrinol Metab 1996, 81:180-183.

 However, a recent study using the more stable
MRproADM has shown that post AMI, increased
MRproADM was associated with
 death,
 heart failure or
 both at one year, over and above information
gained from NTproBNP alone.
 Combining the two markers increased the AUC of
the ROC from 0.77 and 0.79 to 0.84.
 MRproADM is very similar to NTproBNP, it is
higher in females than males, and is increased
with age..

C-TERMINAL-
PROVASOPRESSIN
(COPEPTIN)
Reichlin T, Hochholzer W, Stelzig C, Laule K, Freidank
H,
Incremental value of copeptin for rapid rule out of acute
J Am Coll Cardiol 2009, 54:60-68.
 Copeptin is the more stable surrogate of arginine
vaso-pressin (AVP), with well-known effects on
osmoregulation and cardiovascular homeostasis.
 Post AMI, vasopressin is thought to
 (1) increase peripheral vasoconstrictor activity thus
increasing afterload and ventricular stress
 (2) increase protein synthesis in myocytes leading to
hypertrophy.
 (3) vasoconstriction of coronary arteries.
 These effects are mediated via the V1 receptor, whilst
effects on the V2 receptor mediate water retention in
the renal tubules.

 released in stoichiometric proportion to
vasopressin and is stable and easily assayed.
 Copeptin can rule out MI earlier in addition to a
negative Troponin T test .
 At the time of presentation a copeptin level of <
14 pg/ml and a Trop T level of < 0.01 could rule
out a myocardial infarction with an (AUC) of
receiver operating characteristic curve (ROC) of
0.97 (negative predictive value of 99.7%), thus
obviating the need for monitoring and serial blood
tests in a majority of patients.
 Copeptin is a good marker of neurohormonal
stress, making it also useful in risk stratification in
sepsis and other diseases and hence is not
specific to the cardiovascular system.

Biomarkers of plaque instability and
inflammation
 Acute coronary syndromes are caused by
vulnerable plaques.
 It is thought that one of the driving forces causing
atheromatous plaques to rupture or erode,
causing a cascade of events leading to coronary
artery occlusion, is inflammation in the plaques.
 Hs-CRP
 MPO
 PAPP

HSCRP (HIGH-SENSITIVITY C-REACTIVE
PROTEIN)
 C-reactive protein is a
nonspecific inflammatory marker
that is released by the liver in
response to the acute phase
injury.
 CRP can be measured by
multiple assays in acceptable
precisions down to or below 0.3
mg/l and most give comparable
results (designated as high-
sensitive CRP or hsCRP).
 In terms of the association of
CRP and ACS it is important to
distinguish cases without
(unstable angina) and with
necrosis (acute MI).
 In cases of AMI, CRP release is
triggered as an acute phase
reactant secondary to necrosis
and levels of CRP are much
higher and these have been
correlated with infarct size.
 Though infarct size is the major
Suleiman M, Aronson D, Reisner SA,
Kapelovich MR, 21. Admission C-
reactive protein levels and 30-day
mortality in patients with acute
Am J Med 2003; 115 : 695-701.21,22.

The RISCA (recurrence and inflammation in the
acute coronary syndromes) study.
J Am Coll Cardiol 2008; 51 : 2339-46.
 In the absence of infarction, CRP levels correlate
to the extent of atherosclerosis and some studies
have shown that it predicts coronary events in
patients of unstable angina independent of
troponin levels .
 However, a more recent large prospective study
showed only a weak association of CRP levels
and future coronary events in patients of ACS and
even this disappeared once adjusted for other
common clinical variables.
 This study included about two-thirds of AMI
patients and one-third unstable angina patients.

 Another interesting implication of CRP in ACS has
been in terms of treatment: in a study of ACS
patients, those with low CRP levels after statin
therapy had better clinical outcomes than those
with higher CRP levels, regardless of the
resultant level of LDL cholesterol.
 Thus implying that statin therapy in these high
risk patients of ACS should be driven not only by
the target lipid levels but also the CRP levels
achieved.
 Ridker PM, Cannon CP, Morrow D, Rifai N, Rose LM, 26.
McCabe CH, et al. C-reactive protein levels and outcomes
after statin therapy. N Engl J Med 2005; 352 : 20-8.

CRP is elevated post-acute coronary syndrome
almost exclusively in the setting of myocardial
necrosis indicating the level of myocardial
inflammation
 One study found that CRP measurements (taken
between 12 and 24 hours post event) predicted
occurrence of
 heart failure (HR = 2.6, P = 0.04) and
 death (HR = 2.7, P = 0.02) post-MI [89]. Elevated
peak CRP in the early phase of MI was related to
 early mechanical complications, cardiac rupture ,
ventricular aneurysm and thrombus formation
 Anzai T, Yoshikawa T, Shiraki H, Asakura Y, Akaishi M,
Mitamura H, Ogawa S:
C-reactive protein as a predictor of infarct expansion and

 CRP levels post-MI peak at two to four days, then
take 8 to 12 weeks to subside to baseline levels.
 Interestingly, CRP levels post acute MI do not predict
re-infarction.
 Additional acute coronary events can only be
predicted after CRP levels have receded to baseline
levels (after about 12 weeks).
 One of the difficulties with CRP is that it is non-
specific in the presence of other inflammatory
conditions
 rheumatoid arthritis,
 malignancy,
 vasculitis.

PENTRAXIN-3
 Pentraxin-3 (PTX3) is related to classic pentraxins (like
C-reactive protein CRP or serum amyloid P SAP) but is
structurally different.
 It is made in the liver in response to inflammatory
mediators, mainly interleukin-6.
 It is also produced in large amounts by the heart.
 PTX3 is detected inside both normal and hypertrophic
cardiomyocytes, and is increased in AMI.
 Its plasma concentration increases rapidly after the onset
of symptoms, preceding the increase in CRP
concentration, and reaching a peak at 20–24 h after onset
of symptoms .
 In patients with unstable angina, the PTX3 concentration
increases to a lower value than in AMI.
 Accumulating evidence suggests that PTX3, binding with
C1q in the same way that CRP and SAP bind to C1q,
contributes to the mechanism of increase in tissue damage
.

MPO
Zhang R, Brennan ML, Fu X, Aviles RJ, Pearse GL,
Penn MS, et al. Association between
myeloperoxidase levels and risk of CAD
JAMA 2001;286:2136–42.
• Infiltrating macrophages and neutrophils participate in
the transformation of stable coronary artery plaques
to unstable lesions with a thin fibrous cap.
• These cells are found more frequently and in higher
concentrations in the culprit lesions of patients with
acute MI and unstable angina (UA) than in patients
with stable coronary disease
• Macrophages secrete matrix metalloproteinases
(MMPs) and metalindependent myeloperoxidase,
which degrade the collagen layer that protects
atheromas from erosion or abrupt rupture .
• As a result, plaques that have been highly infiltrated
with macrophages have a thin fibrous cap and are
vulnerable to erosion or rupture, precipitating events

• Myeloperoxidase activity can be measured in blood and
tissues by assays using hydrogen peroxide and o-
dianisidine dihydrochloride as substrates .
• Recently, mass assays based on an enzyme-linked
immunoassay have been developed for research use
only (Oxis Research and Assay Design)
• Zhang et al. showed that blood and leukocyte
myeloperoxidase activities were higher in patients with
CAD than angiographically verified normal controls, and
that these increased activities were significantly
associated with presence of CAD [odds ratio, 11.9; 95%
confidence interval (CI), 5.5–25.5].
• Result were independent of the patient‟s age; sex;
hypertension smoking, or diabetes status; LDL
concentration; leukocyte count; and Framingham Global

• A key study by Buffon et al. involved 65 patients who
underwent cardiac catheterization with coronary sinus
sampling.
• The myeloperoxidase content of the leukocytes
collected from the arterial circulation and the coronary
sinus effluent were compared
• Not only was there a gradient for myeloperoxidase
across the coronary sinus in patients with ACS, but
that gradient was present even when the culprit lesion
involved with the ACS was in the distribution of the
right coronary artery, a situation in which the venous
effluent from the right coronary artery does not drain
into the coronary sinus .
 Buffon A, Biasucci LM, Liuzzo G, D’Onofrio G, Crea F,

• The potential usefulness for risk stratification of blood
concentrations of myeloperoxidase was examined in
2 recent studies.
• CAPTURE trial ,
• myeloperoxidase mass concentration was measured in
1090 patients with ACS.
• The death and MI rates were determined at 6 months of
follow-up.
• With a cutoff of 350 g/L, the adjusted hazard ratio was
2.25.
• The effects were particularly impressive in patients with
undetectable cardiac troponin T (cTnT; 0.01 g/L), where
the hazard ratio was 7.48
 Brennan et al.
 demonstrated a progressive increase in odds ratios for
major adverse events at 30 days and 6 months with
each quartile increase in myeloperoxidase
concentration.

• In summary, although myeloperoxidase
participates in the inflammatory process of ACS,
neutrophil activation is apparently not induced by
ischemia .
• Thus, myeloperoxidase is more of a marker of
plaque instability an unlike a marker of oxidative
stress and damage.
• Increased myeloperoxidase is not likely to be
specific to cardiac diseases, as activation of
neutrophils and macrophages can occur in any
infectious, inflammatory, or infiltrative disease
process.

PREGNANCY-ASSOCIATED PLASMA
PROTEIN-A
 Pregnancy-associated plasma protein-A (PAPP-
A) is a large, zinc binding proteinase produced by
different cell types, including fibroblasts, vascular
smooth muscle cells, male and female
reproductive tissues and belongs to the insulin-
like growth factor family.
 It is thought to be released when
neovascularization occurs and thus may be a
marker of incipient plaque rupture.
 Its level has been shown to be elevated in
unstable plaques and in circulation in patients of
ACS.
 Bayes-Genis A, Conover CA, Overgaard MT,. Pregnancy-
associated plasma protein A as a marker of acute coronary

 In study of patients with angiographically
confirmed acute coronary syndrome, elevated
serum PAPP-A was a strong independent
predictor of death or recurrent MI, even in
patients with normal serum troponin T
 CAPTURE Study Investigators. Pregnancy-
associated plasma protein-A levels in patients with
acute coronary syndromes:. J Am Coll Cardiol
2005; 45 : 229-37.
 Interestingly, PaPPA > 2.9 mIU/L predicts a 4.6-
fold increase in risk of cardiovascular death, MI
(not predict heart failure) or revascularisation
even without a raised Troponin
 Moreover, standardized assays for PAPP-A are

 Heeschen et al.,
 In a series of 136 consecutive patients presenting to the ED
for suspected ACS (found to be cTnI negative during the
first 24 h after admission), an increase in circulating PAPP-A
appeared to be an independent predictor of future ischemic
cardiac events as well as the need for PCI or coronary
artery bypass graft surgery.

MMP-9
 MMPs are a class of 24 endopeptidases that are
physiologic regulators.
 In the heart, these substances participate in
vascular remodeling, plaque instability, and
ventricular remodeling after cardiac injury.
 MMP-9 is zinc-dependent and is known as
gelatinase Bs of the extracellular matrix .
 Visse R, Nagase H. Matrix metalloproteinases
and tissue inhibitors of metalloproteinases;
structure, function and biochemistry. Circ Res
2003;92:827–39.

 In vascular tissue, MMP-9 and several other
MMPs are localized at the shoulder of a plaque.
 That area, which is thinner, is thought to be the
area prone to rupture.
 In heart tissue, MMP-9 is partially responsible for
the degradation of ground substance after
cardiac injury.
 In other models, that inhibition of MMPs,
including MMP-9, inhibits ventricular remodeling
after acute MI, and there is therapeutic interest in
testing such a strategy clinically.

 The first report was published in 1998 by Kai et al.
 MMP-9 concentrations in presumably healthy controls
was 27 (8) g/L, which was similar to stable angina pts.
 Patients with acute MI had either very increased or
normal MMP-9 concentrations (6 with high and 7 with
normal concentrations) on day 1, whereas patients with
UA all had high concentrations [87 (26) g/L] initially.
 Finnish group correlated blood concentrations
with the extent of CAD in a cohort of 61 patients.
 reference values mean (SD) value of 32.2 (16.1)
g/L.
 Single- or double-vessel disease had a mean value of 40.4
(25.1) g/L
 Triple -vessel disease, a mean of 57.3 (39.1) g/L.
 AetheroGene investigators on 1127 patients
with either stable (n 795) or unstable (n 332)
CAD reported that values of MMP-9 were related

SOLUBLE CD40 LIGAND (sCD40L)
 Soluble CD40 ligand (sCD40L) is expressed on
platelets and released from them on activation.
 It has biological activity that can trigger an
inflammatory reaction in vascular endothelial cells
by the secretion of cytokines and chemokines.
 Membrane bound CD40L and sCD40L forms
interact with the CD40 receptor molecule, which
is present not only on B cells but also on
monocytes, macrophages, and endothelial and
smooth muscle cells in atheroma, leading to
release of matrix MMPs and subsequent
destabilization of the plaque
 Thus upregulation of the CD40L system may play

 Increased sCD40L concentrations have been
demonstrated in other inflammatory disorders,
e.g., autoimmune diseases, multiple sclerosis,
and inflammatory bowel disease, as well as in
stroke, hypercholesterolaemia, and diabetes.
 In OPUS-TIMI16 trial increased sCD40L was
associated with a higher risk for future death
and recurrent myocardial infarction
independent of other variables including cTnI and
CRP.
 Importantly in combination with cardiac troponin I
it significantly improved risk prediction for future
death and MI
 Varo N, de Lemos JA, Libby P, Morrow DA, Murphy SA,
34. Nuzzo R, et al. Soluble CD40L risk prediction after

 Similarly in the CAPTURE study of ACS, increased
sCD40L concentrations were associated with a higher
risk of death and non-fatal MI.
 Notably elevation of soluble CD40 ligand identified
the subgroup of patients likely to benefit from anti-
platelet treatment with abciximab.
 CAPTURE Study Investigators. Soluble CD40 ligand in
acute coronary syndromes. N Engl J Med 2003; 348
:1104-11.
 Therapeutic benefits of sCD40L were also seen in
MIRACL Study wherein patients with acute coronary
syndromes and high sCD40L had a significant
reduction in the risk of recurrent cardiovascular
events with early statin therapy.
 MIRACL Study. Circulation 2004; 110 : 386-91.

 However, recent studies have flagged doubts on
the influence of pre-analytical and analytical
conditions on measurement of sCD40L and thus
additional studies are warranted before
implementing wider clinical use.
 Olenchock BA, Wiviott SD, Murphy SA, Cannon CP,
Rafai 37. N, Braunwald E, et al. Lack of association
between soluble CD40L and risk in a large cohort of
patients with acute coronary syndrome in OPUS TIMI-
16. J Thromb Thrombolysis 2007; 26 : 79-84.

CHOLINE
 Choline and phosphatidic acid are major products
generated by phosphodiesteric cleavage of
membrane phospholipids (phosphatidylcholine for
example) catalyzed by phospholipase D
enzymes.
 Whole-blood choline (WBCHO) and plasma
choline (PLCHO) concentrations increase rapidly
after stimulation of phospholipase D (PLD) and
the activation of cell surface receptors in
coronary plaque destabilization and tissue
ischemia.

 WBCHO was a significant predictor of cardiac
death or cardiac arrest, lifethreatening cardiac
arrhythmias, heart failure, and coronary
angioplasty when measured in the first blood
sample on admission.
 cTnI or cTnT and WBCHO were the most
powerful independent predictors in multivariate
analysis, and the combination of WBCHO and
cardiac troponins allowed a superior risk
assessment compared with each test alone.
 WBCHO was not a marker for myocardial
necrosis but indicated high-risk UA in patients
without acute MI (sensitivity, 86.4%; specificity,
86.2%)

PLACENTAL GROWTH
FACTOR
Heeschen C, Dimmeler S, Fichtlscherer S, Hamm
CW, Berger J, Simoons ML, et al. Prognostic
value of placental growth factor in patients with
acute chest pain. JAMA 2004;291:435–41.
 Placental growth factor (PlGF) is one of a family
of platelet-derived proteins that function as potent
chemoatractants for monocytes and are involved
in the regulation of vascular endothelial growth.
 Various tissues express PlGF mRNA, including
thyroid, placenta, and lung
 PlGF ap-pears to be stable in circulation and
must be considered a strong candidate as a
biomarker for plaque instability, myocardial
ischemia, and prognosis of patients in the

 The biological functions of PlGF are incompletely
understood
 primarily involve initia-tion of the inflammatory
process, which includes the
 recruitment of circulating macrophages into
atheroscle-rotic lesions, stimulation of smooth
muscle cell growth, and
 up-regulation of both tissue necrosis factor- and
 MCP-1 by macrophages .
 acti-vates stem cells from a quiescent to
proliferative state and in this way stimulates
hematopoiesis in the bone marrow of mice
 Tjwa M, Luttun A, Autiero M, Carmeliet P. VEGF and PlGF:
two pleiotropic growth factors with distinct roles in
development and homeostasis. Cell Tissue Res

 Furthermore, compelling data have shown that
inhibiting the actions of PlGF suppressed plaque
instability and coronary heart disease .
 Thus, PlGF is not merely a risk marker but also
a disease marker, and may represent a new
therapeutic target for mitigating the disease
process behind ACS.
Hattori K, Heissig B, Wu Y, Dias S, Tejada R, Ferris B, et
al. Nat Med 2002;8:841–9.

 In the CAPTURE cohort, 223 (40.8%) patients
were found to have increased PlGF concentra-
tions, defined as 27.0 ng/L, and were found to
have a markedly increased risk of adverse events
at 30 days (14.8% vs 4.9%).
 The unadjusted hazard ratio for in-creased PlGF
was 34 (95% CI, 1.79 – 6.24) and was statis-
tically significant (P 0.001).
 For the CAPTURE population, the multivariable
modeling showed that increased concentrations
of cTnT ( P 0.03), sCD40L ( P 0.002), and PlGF (
P 0.001) were independent predictors of death or
MI at 30 days, whereas increased CRP was not
(P 0.94).

TOLL RECEPTORS
 On monocytes/macrophages
 Activate proinflammatory NF-KB on endothelial
cells & MAPK pathway---cytokine release and
smooth muscle cell proliferation.
 TLR-4 receptor for endotoxins present in lipid rich
atherosclerotic plaques.
 Leads to uptake of oxidized LDL.---formation of
atherosclerotic plaque.

In conclusion, the TLR4 expression levels on peripheral blood monocytes
in CAD patients were higher than those in non-CAD subjects and
correlated with disease activity, even in low-hsCRP subjects.

OTHER INFLAMATORY
MARKERS
 Serum amyloid A
 IL-6
 IL-18
 MCP-1
 sICAM-1
 Lipo protein assciciated plasma protein A2

 Ischaemia induces a conformational change in
albumin, so that it can no longer bind to
transitional metals such as cobalt or copper.
Using the albumin cobalt binding (ACB) test, the
quantum of ischaemia modified albumin can be
estimated and this serves as an index of
ischaemia.
 Ischaemia-modified albumin (IMA) has been
shown to be an independent predictor of short-
and long-term adverse outcomes over and
above conventional known risk in patients with
ACS.
 Collinson PO, Gaze DC, Kaski JC. Ischemia-modified
albumin predicts short-term outcome and 1-year mortality in
patients attending the emergency department for acute
ISCHAEMIA MODIFIED ALBUMIN

 IMA is not only a marker of the occurrence of
an ischemic event but also an indicator of the
severity of ischemia.
 Bar-Or D, Curtis G, Rao N, Bampos N, Lau E.
Characterization of the Co2 and Ni2 binding amino-acid
residues of the N-terminus of human albumin. Eur J
Biochem 2001;268:42–7.
 Increased IMA values may be found in patients
with cancer, infections, end-stage renal disease,
liver disease, and brain ischaemia also.
 Aslan D, Apple FS. Ischemia modified albumin: clinical and
39. analytical update. Lab Med 2004; 35 : 1-5.
 However, the test‟s assay is cumbersome to use.
 With greater refinement it may be a useful test in
the emergency department (ED) to rule out
ischaemia which is more important at that stage.

 In the study by Bhagavan et al.. Clin Chem
2003;49:581–5 ACB assay results (using an
assay independent of the Ischemia Technology
IMA test) were correlated with final discharge
diagnoses in 75 ED patients with myocardial
ischemia and 92 nonischemic patients.
 The sensitivity and specificity for myocardial
ischemia were 88% and 94%, respectively, and
the positive and negative predictive values were
92% and 91%.
 The ACB test, however, was a poor discriminator
between ischemic patients with and without MI

 Results in the study of Quiles et al. confirmed that
IMA is an early marker of ischemia in the setting
of PCI.
 IMA concentrations in all patients increased
significantly after PCI from baseline to post-PCI (59.9
to 80.9 kilounits/L; P 0.0001).
 IMA was higher in patients with more balloon
inflations, higher pressure inflations, and longer
inflation duration.
 However, because there was some scatter in the
correlations, factors such as the severity and extent of
the lesion and the presence or absence of collateral
blood supply may also play a role in IMA
concentrations.
 The authors suggested that IMA is not only a marker

 IMA concentrations were measured after elective
direct-current cardioversion (DCCV) for atrial
fibrillation to determine whether transient myocardial
ischemia occurred .
 Serum samples for IMA measurement were obtained
before and at 1 and 6 h after DCCV in 24 patients.
 Fourteen patients developed ECG changes (ST-
depression and/or T-wave inversion) after DCCV and
showed IMA concentrations significantly higher than
patients without changes; no significant differences
were demonstrated in CK, CK-MB, and cTnT
concentrations between the 2 groups.
 The results suggested that increased concentrations
of IMA after cardioversion might reflect transient
myocardial ischemia.
 Roy D, Quiles J, Sinha M, Aldama G, Gaze D, Collinson P,
et al. Effect of direct-current cardioversion on
ischemia-modified albumin levels in patients with atrial
fibrillation. Am J
Cardiol 2004;93:366–8.

UNBOUND FREE FATTY ACIDS
Apple FS, Kleinfeld AM, Adams J III.
Unbound free fatty acid concentrations are
increased in cardiac ischemia.
Clin Proteomics 2004;1:169–72.
 On the basis of preliminary findings, FFAu
concentrations, rather than total FFAs, may
provide a sensitive guide to the pathophysiology
of underlying coronary disease .
 Increased blood catecholamines in association
with ischemia suggest that increased FFAu
concentrations result from increased FFA release
through adipose lipolysis.
 FFAu can be measured with a recombinant fatty-
acid–binding protein labeled with a fluorescent

 Data suggest that in patients presenting with
ischemic symptoms, plasma FFAu monitoring may
provide an early indication of cardiac ischemia.
 Serum FFAu measured in 22 PTCA patients 5 min
before and 30 min after the procedure demonstrated
a 14-fold higher postprocedure value as well as
when compared with healthy volunteers
 (Kleinfeld AM, Prothro D, Brown D, Davis RC,.
Increases in serum unbound free fatty acid
concentrations following coronary angioplasty. Am J
Cardiol 1996;78: 1350–4.89 ).
 FFAu concentrations were significantly higher in the
ECG positive group vs the ECG-negative group.

 FFAu was increased in every instance that cTnI
was increased, and there was a positive
correlation between peak FFAu and cTnI
concentration.
 Additionally, in MI patients, FFAu was increased in
100% of patients at presentation, whereas only 22%
of these patients had increased cTnI at presentation,
indicative of the earlier appearance of this analyte
in the circulation before traditional markers of
myocyte necrosis.
Kleinfeld AM, Kleinfeld KJ, Adams
JE. Serum
levels of unbound free fatty acids reveal high sensitivity for

HEART-TYPE FATTY ACID
BINDING PROTEIN (H-FABP)
Chan CP, Sanderson JE, Glatz JF, Cheng WS, Hempel A,
Renneberg R: A superior early myocardial infarction
marker. Human heart-type fattyacid-binding protein. Z
Kardiol 2004, 93:388-397.
 H-FABP is a low molecular weight protein involved in
myocardial fatty-acid metabolism found in small quantities
in brain, kidney and skeletal tissue.
 It is rapidly released early in myocardial infarction and
necrosis into the cytosol usually peaks within 4 hours
and is generally undetectable after 24-36 hours..
 H-FABP has been shown in mouse studies to be an early
marker of ischae-mia (before morphological evidence of
myocardial necrosis) and can therefore help with
diagnosis of MI earlier .
 However, studies attempting to use H-FABP alone for early
diagnosis of AMI have produced disappointing results.
 One review of six studies found that the pooled positive

 Other more recent studies demonstrated that H-
FABP levels were clearly associated with the
composite end point of death, myocardial
infarction and heart failure at 10 months.
 When levels of H-FABP were measured post-
ACS and divided into quartiles, the top quartile
was asso-ciated with all-cause mortality 6.59
times higher than the lowest quartile, after
adjusting for hsCRP and Troponin.
 In fact, when added to Troponin for risk
stratification,
 a negative troponin and H-FABP level < 5.8 mcg/L
was associated with zero mortality at six months;
 a negative Troponin but H-FABP level > 5.8 mcg/L
was associated with a 4.93-fold increase in risk of

• A 2003 study of 371 consecutive ER patients
presenting with chest pain, in which 37 of 68 patients
arriving within 2 hours of symptom onset had a
diagnosis of myocardial infarction (MI), showed that
H-FABP sensitivity/specificity was 89%/52% (H-
FABP cutoff of 7 ng/ml) in comparison to troponin-T
(Tn-T) sensitivity/specificity of 22%/94% (Tn-T cutoff
not reported).
• Both H-FABP‟s higher sensitivity and Tn-T‟s higher
specificity were statistically significant.
STUDIES

• A similarly designed 2008 multicenter study of
419 ER patients with suspected ACS presenting
within 3 hours of symptom onset demonstrated
that of the 148 patients diagnosed with MI, H-
FABP sensitivity/specificity was 60%/88% (H-
FABP cutoff of 6.2 ng/ml) whereas Tn-T
sensitivity/specificity was 19%/99% (Tn-T cutoff
not reported).
• H-FABP‟s higher sensitivity was again
statistically significant, but Tn-T‟s higher
specificity was not.
• Other similar studies in the literature corroborate
these findings

 H-FABP appears to be a more effective cardiac
biomarker than troponin in early detection of ACS.
 Not withstanding these encouraging preliminary
data, further multicenter studies must be
conducted using standardized H-FABP and
troponin assays before H-FABP can be seriously
considered as a clinical tool for the early
diagnosis of ACS.

GLYCOGEN PHOSPHORYLASE ISOENZYME
BB
Mair J. Glycogen phosphorylase. In: Creighton TE,
ed. The encyclopedia of molecular medicine. New
York: Wiley Publishers, 2002:1489–91
 Glycogen phosphorylase isoenzyme BB (GPBB)
should not be considered a marker of myocardial
ischemia instead, its early release within 2–4 h after
the onset of myocardial damage in parallel with
myoglobin or hearttype fatty-acid–binding protein is
an indicator of irreversible myocardial damage.
 By contrast, delayed release of GPBB after several
hours, e.g., in parallel with lactate dehydrogenase, is
seen after all kinds of myocardial damage (e.g.,
toxic and inflammatory damage or heart contusion).
 The early release of GPBB requires both a burst in
glycogenolysis and concomitantly increased plasma

 Three isoenzymes are found in human tissues:
GPLL (liver), GPMM (muscle), and GPBB (brain).
 The BB and MM isoenzymes are found in the
human heart, but the BB isoenzyme is the
predominant isoenzyme in myocardium.
In clinical studies, GPBB was a very sensitive
marker for the diagnosis of acute MI within 4 h
after chest pain onset. GPBB usually peaks 6 to
20 h after onset of chest pain with early peak
values found in patients with early reperfusion of
the infarct-related coronary artery; it returns to
within the reference interval within 1–2 days after
MI.
Rabitzsch G, Mair J, Lechleitner P, Noll F,
Hofmann U, Krause EG, et al.
Immunoenzymometric assay of human glycogen

 GPBB also increases early in patients with ACS
and reversible ST-T segment alterations in the
resting ECG at hospital admission, which could
be useful for early risk stratification .
 GPBB was found to be sensitive for the detection
of perioperative ischemic myocardial damage and
infarction in patients undergoing coronary artery
bypass grafting, and GPBB more accurately
reflected ischemic myocardial damage than CK-
MB .
(Mair P, Mair J, Krause E-G, Balogh D,. Glycogen
phosphorylase isoenzyme BB mass release after
coronary artery bypass grafting. Eur J Clin Chem
Clin Biochem 1994;32:543–7.98 .

CYSTATIN C
Jernberg T, Lindahl B, James S, Larsson A, Hansson LO,
Cystatin C: a novel predictor of outcome in suspected or confirmed
non-ST-elevation acute coronary syndrome.
Circulation 2004; 110 : 2342-8.45
 Cystatin C is a low molecular weight basic protein
that is freely filtered and metabolized after tubular
reabsorption.
 Some studies have revealed the usefulness of
the cystatin C as a prognostic marker in heart
failure and acute coronary syndrome
 This protein is less influenced by age, gender,
and muscle mass than serum creatinine and thus
may be better indicator of cardiovascular risk than
serum creatinine especially in elderly.

The Year in NonST-Segment Elevation Acute Coronary Syndrome
J. Am. Coll. Cardiol. 2009;54;1544-1555
Robert P. Giugliano, and Eugene Braunwald

GDF-15 antihypertrophic effect, prognostic
death , hf
ST2 death , hf
C-reactive protein. hf,death,infarct size,atherosclerotic
burden
MPO oxidative stress, plaque rupture,
death
PAPP-P plaque rupture, death,recurrent mi
MYOGLOBIN negative predictive value
MMP-9 plaque instability, and ventricular
remodeling after cardiac injury.
sCD40L destabilization of the plaque
death and recurrent mi
CHOLINE coronary plaque destabilization and
tissue ischemia. death or cardiac
arrest, cardiac arrhythmias, heart
failure, and coronary angioplasty
PLACENTAL GROWTH FACTOR plaque instability, myocardial
ischemia, and prognosis

IMA early marker of the occurrence and
indicator of the severity
UNBOUND FREE FATTY ACIDS early indication of cardiac ischemia
H-FABP Earlier diagnosis of MI
death, myocardial infarction and
heart failure
CYSTATIN C prognostic marker in heart failure
Renal failure

POINT-OF-CARE TESTING (POCT)
Diagnostic Accuracy of Point-of-care Testing for
Acute Coronary Syndromes, in Primary Care
A Cluster-Randomised Controlled Trial Yuki
Tomonaga; Felix Gutzwiller; Thomas F Lüscher;
Walter F Riesen; Markus Hug; Albert Diemand;
Matthias Schwenkglenks;
 POC tests are a simple, rapid and relatively
inexpensive means for reducing hospital stay,
complications and improving adherence to treatment.
 The use of POC tests can lead to a reduction in test
ordering, sample transport to laboratories and data
reporting.
 Decreased TAT(Turn Around Time) is the central
issue in POC testing.
 Several high-throughput automated systems have
enabled the introduction of a wide range of tests to be
performed simply (no requirement for highly trained
personnel) and quickly (without the need for
laboratory processing) implementation.

 The two main types of POC testing formats available
in the clinical setting include- small bench-top
analysers and - hand-
held devices.
 Small bench-top analysers- are basically a
miniaturised version of the mainframe central lab
equipment, except with essential modifications to
prevent operator error and provid rapid, reproducible
results.
 Hand-held devices- are developed using state-of-
the-art microfabrication techniques, which essentially
integrate several key analytical steps i.e. sample
clean-up, separation, analysis and data reporting.
 Devices for cardiac biomarker POC testing are
predominantly based upon immunoassay
methods.

New Development in Biomarker
Discovery
 The traditional approach of biomarker discovery,
which usually focuses on one or a few potential
candidates at a time, has been ineffective and led
to a low rate of biomarker discovery with clinical
utility.
 The pathophysiologic changes in ACS are infl
uenced by many factors, including genetic
andenvironmental factors.
 The complete sequencing of human genome and
recent advances in genomic, transcriptomic,
proteomic, lipidomic, metabolomic, and
bioinformatics technologies offer tremendous
opportunities for novel biomarker discovery.

NATIONAL ACADEMY OF CLINICAL
BIOCHEMISTRY RECOMMENDATIONS FOR
USE OF BIOMARKERS (2007)

INFARCTION
Recommendations for use of biochemical
markers for diagnosis of MI
 Class I
 1. Biomarkers of myocardial necrosis should be
measured in all patients who present with symptoms
consistent with ACS.
 2. The patient’s clinical presentation (history,
physical exam), and ECG should be used in
conjunction with biomarkers in the diagnostic
evaluation of suspected MI.
 3. Cardiac troponin is the preferred marker for the
diagnosis of MI. CK-MB by mass assay is an
acceptable alternative when cardiac troponin is not
available.
 4. Blood should be obtained for testing at hospital
presentation followed by serial sampling with timing
of sampling based on the clinical circumstances.
For most patients, blood should be obtained for
testing at hospital presentation, at 6 to 9 hours, and
again at 12 –24 hours if the earlier samples are
negative and the clinical index of suspicion is high.

 5. In the presence of a clinical history
suggestive of ACS, the following are considered
indicative of myocardial necrosis consistent
with MI:
 a. Maximal concentration of cardiac troponin exceeding
the 99th percentile of values (with acceptable precision)
for a reference control group on at least one occasion
during the first 24 hours after the clinical event
 b. Maximal concentration of CK-MB exceeding the 99th
percentile of values for a gender-specific reference
control group on two successive samples (Values for
CK-MB should rise and fall)
 c. In the absence of availability of a troponin or CK-MB
assay, total CK greater than two times the gender-
specific upper reference limit

 Class IIa
1. For patients who present within 6 hours of the onset
of symptoms, an early marker of myocardial necrosis
may be considered in addition to a cardiac troponin.
Myoglobin is the most extensively studies marker for
this purpose.
 Class IIb
2. A rapid “rule-in” protocol with frequent early sampling
of markers of myocardial necrosis may be appropriate
if tied to therapeutic strategies.
 Class III
1. Total CK, aspartate aminotransferase (AST, SGOT),
beta-hydroxybutyric dehydrogenase, and/or lactate
dehydrogenase should not be used as biomarkers for
the diagnosis of MI.
2. For patients with diagnostic ECG abnormalities on
presentation (e.g. new ST-segment elevation),
diagnosis and treatment should not be delayed while
awaiting biomarker results.

EARLY RISK STRATIFICATION
 Class I
1. Patients with suspected ACS should undergo early
risk stratification based upon an integrated
assessment of symptoms, physical exam findings,
ECG findings, and biomarkers.
2. A cardiac troponin is the preferred marker for risk
stratification and, if available, should be measured in
all patients with suspected ACS. In patients with a
clinical syndrome consistent with ACS, a maximal
concentration exceeding the 99th percentile of values
for a reference control group (with acceptable
precision) should be considered indicative of
increased risk of death and recurrent ischemic events.
3. Blood should be obtained for testing on hospital
presentation followed by serial sampling with timing of
sampling based on the clinical circumstances. For
most patients, blood should be obtained for testing at
hospital presentation, at 6 to 9 hours, and again at 12
–24 hours if the earlier samples are negative and the
clinical index of suspicion is high.

 Class IIa
 1. Measurement of hs-CRP may be useful, in addition
to a cardiac troponin, for risk assessment in patients
with a clinical syndrome consistent with ACS. The
benefits of therapy based on this strategy remain
uncertain.
 2. Measurement of B-type natriuretic peptide (BNP) or
N-terminal pro-BNP (NT-proBNP) may be useful, in
addition to a cardiac troponin, for risk assessment in
patients with a clinical syndrome consistent with ACS.
The benefits of therapy based on this strategy remain
uncertain.
 3. Early repeat sampling of cardiac troponin (e.g. 2 to 4
hours after presentation) may be appropriate if tied to
therapeutic strategies.
 Class IIb
 1. In patients with a high clinical probability of ACS,
maximal concentrations of cardiac troponin exceeding
the 99th percentile (without stringent requirements for
precision) may be recognized as indicative of

2. Measurement of markers of myocardial ischemia,
in addition to cardiac troponin and ECG, may aid
in the short-term risk stratification of patients with
suspected ACS, and in excluding ACS in patients
with a low clinical probability of myocardial
ischemia.
3. A multi-marker strategy that includes
measurement of two or more pathobiologically
diverse biomarkers in addition to a cardiac
troponin, may aid in enhancing risk stratification
in patients with a clinical syndrome consistent
with ACS. BNP and hs-CRP are the biomarkers
best studied using this approach. The benefits of
therapy based on this strategy remain uncertain.
 Class III
Biomarkers of necrosis should not be used for
routine screening of patients with low clinical
probability of ACS.

USE OF BIOCHEMICAL MARKERS
IN THE MANAGEMENT OF NSTE
ACS
A. Clinical decision-making
Class I
 Among patients with a clinical history
consistent with ACS, an increased
concentration of cardiac troponin should
prompt application of ACS management
guidelines for patients with indicators of high
risk.
Class III
 1. Application of management guidelines for ACS
should not be based solely upon measurement of
natriuretic peptides.
 2. Application of management guidelines for
ACS should not be based solely upon
measurement of C-reactive protein.

Biochemical marker measurement
after the diagnosis of acute MI
 Class I
1. Once the diagnosis of acute MI is
ascertained, testing of biochemical markers of
injury at a reduced frequency is valuable to
qualitatively estimate the size of the
infarction, and to detect the presence of
complications such as re-infarction.
 Class IIa
2. CK-MB is the preferred marker for detection of
re-infarction early after the index event when the
concentration of cardiac troponin is still
increased.

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