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.

CRITERIA FOR A TRUE 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 proteins are independent of the blood flow in the infarcted region.

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)CholinePlacental growth factorMMP-9Myeloperoxidase (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 stressNt-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

Sacks HJ, Lanchantin GF. An elevation of serum transaminases in jaundice states. Am J Clin Path. 1960;33:97-108. 10. Elliot BA, Wilkinson.

LACTATE DEHYDROGENASE (LD) AND ITS ISOENZYME

LD1An 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 .

Varley H, Gowenlock AH, Bell M. Enzymes, In : Practical Clinical Biochemistry, Vol. I, 5th edn. William Heinemann Medical Books Ltd. London 1984; p685-770.

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 dehydrogenase” in myocardial infarction and in liver disease. Lancet 1961;1:698-99.

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 CK-MB by some authors.

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. The CKMB mass assay has a diagnostic sensitivity

of 50% at 3 hours and 80% at 6.

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 inlarge 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

MYOGLOBINMyoglobin, 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 2000; 86: 1367-71

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: TROPONINTroponin 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 release of structurally bound troponin resulting in a second peak lasting for several days.

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 diagnostic marker for myocardial infarction and minor cell damage. Eur Heart J 1996; 17: (Suppl. F),

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 cTnIBoth 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 thereby diminishing the signal.

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 hours later is used.

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..

The 12-hour wait for the levels to peak remains the Achilles heel of this bio-marker.

SURVIVAL RATE

ESTIMATION OF INFARCT SIZE

AFTER CABG

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

TROPONIN SANDWITCH-IMMUNOASSAY

Tn IN ESRDThe 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 similar in patients with and without renal failure

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

sequential rise of atleast 20% after 6 hrs.

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 zero and from two to six hours after the onset of symptoms, or from hospital arrival.

Detects <1 pg/ml<10 pg /ml-noncardiac

10-30 pg/ml-mild cardiac injury

BIOMARKERS OF BIOMECHANICAL STRESS

Biomarkers of biomechanical stressBNP/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). MRproANP is emerging therefore to be an important

predictor of adverse events following an AMI.

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 hypertrophic growth following pressure overload .

GDF-15 provides prognostic information following an MI or ACS.

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)

ST2Shimpo 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.

In mouse studies, IL-33 was found to markedly antagonize angiotensin-II and phenylephrine-induced cardiomyocyte hypertrophy.

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 at the bedside.

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 predomi-nantly on endothelial cells mediating nitric oxide release, natriuresis and diuresis .

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

Stewart DJ, Kubac G, Costello KB, Cernacek P: Increased plasma endothelin-1 in the early hours of acute myocardial infarction. J Am Coll Cardiol 1991, 18:38-43.

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 myocardial infarction.

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.

These receptors are now targets for pharmacological therapy .

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-CRPMPOPAPP

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 determinant of long term prognosis after AMI; mortality has been shown to be related to CRP levels independent of left ventricular systolic function.

Suleiman M, Aronson D, Reisner SA, Kapelovich MR, 21. Admission C-

reactive protein levels and 30-day mortality in patients with acute

myocardial infarction. 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 cardiac rupture after a first Q-wave acute myocardial infarction. Circulation 1997, 96:778-784..

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-3Pentraxin-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 .

MPOZhang 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 to ACS.

• 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 Risk Score.

• There was no tabulation of the subsequent rate of adverse events in that report.

• 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, Maseri A. Widespread coronary inflammation in unstable angina. N Engl J Med 2002;347:5–12.

• 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 syndromes. N Engl J Med 2001; 345 :1022-9.

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 not available.

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 to future cardiovascular death.

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 a pathogenic role also in ACS.

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 acute coronary syndromes. Circulation 2003; 108 : 1049-52..

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%)

PLCHO was not a predictor of MI in the follow-up phase, whereas WBCHO was highly predictive .

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 spectrum of ACS.

The biological functions of PlGF are incompletely understood primarily involve initia-tion of the inflammatory

process, which includes therecruitment of circulating macrophages into

atheroscle-rotic lesions, stimulation of smooth muscle cell growth, and

up-regulation of both tissue necrosis factor- andMCP-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 2003;314:5–14.

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).

HEAT SHOCK PROTEINS in CAD

TOLL RECEPTORS

On monocytes/macrophagesActivate 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.

CASPASES IIN CAD

OTHER INFLAMATORY MARKERS

Serum amyloid A

IL-6

IL-18

MCP-1

sICAM-1

Lipo protein assciciated plasma protein A2

MARKERS OF ISCHEMIA

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 ischemic chest pain. Heart Vessels 2008; 23 : 174-80.38.

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 of the occurrence of an isc hemic event but also an indicator of the severity of ischemia. Clinical Chemistry 51,

No. 5, 2005 817

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 ACIDSApple 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 tag (ADIFAB).

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 early detection of acute myocardial infarction in patient samples from the TIMI II trial. J Am Coll Cardiol 2002;39:312A.

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 predictive value to be 65.8% and pooled negative predictive value to be 82.0.

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 death and

7.93-fold increase in risk if Troponin was positive and H-FABP > 5.8 mcg/L

• 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 membrane permeability, which is the case in ischemic myocardial damage.

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 phosphorylase isoenzyme BB in diagnosis of ischemic myocardial injury. Clin Chem 1995;41:966–78..

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.

OSTEOPROTEGERIN

The Year in NonST-Segment Elevation Acute Coronary Syndrome

J. Am. Coll. Cardiol. 2009;54;1544-1555Robert P. Giugliano, and Eugene Braunwald

GDF-15 antihypertrophic effect, prognosticdeath , 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 plaquedeath 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)

A. DIAGNOSIS OF MYOCARDIAL 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 increased risk of death or recurrent ischemic events.

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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