Biochemical Markers in Risk Stratification and Diagnosis of Acute Coronary Syndromes: A Laboratory Perspective
Henry O. Ogedegbe, Ph.D., BB(ASCP), C(ASCP)SC
Assistant Professor
Department of Environmental Health, Molecular and Clinical Sciences, Florida Gulf Coast University, 10501 FGCU Blvd. South, Fort
Myers, Florida 33965-6565
Abstract
The spectrum of myocardial ischemic events, which range from angina, reversible tissue injury, and unstable angina, to myocardial
infarction and extensive myocardial tissue necrosis, is known as acute coronary syndrome. The diagnosis of patients presenting with
chest pain has traditionally been binary – rule in or rule out myocardial injury. The diagnosis is based on the World health
Organization’s recommendations, which requires the fulfillment of two of the following criteria: clinical symptoms suggestive of
myocardial ischemia, evolutionary changes in the electrocardiogram, and a serial rise and fall of serum enzymes suggestive of
myocardial injury. In the past, creatine kinase and its isoenzyme, creatine kinase-MB, have been the “gold standards”, but they are not
specific for the myocardium. Newer biomarkers such as troponin T and troponin I are more sensitive and specific indicators of
myocardial infarction and may be used for diagnosis and risk stratification in acute coronary syndrome patients. Biomarkers including
homocysteine, C-reactive protein, serum amyloid A, P-selectin, soluble fibrin, glycogen phophorylase BB isoenzyme, creatine kinaseMB, myoglobin and troponins T and I, along with clinical indicators and electrocardiogram findings are all very useful tools in risk
stratification and diagnosis of acute coronary syndromes. The combination of myoglobin and creatine kinase-MB with clinical
presentations may be employed to assess reperfusion subsequent to thrombolytic therapy. Some of the newer markers such as
troponins T and I are likely to replace creatine kinase and its isoenzyme, CK-MB as the biochemical markers of choice in the
diagnosis of acute coronary syndromes.
Introduction
Acute coronary syndrome (ACS) is a pathophysiologic continuum that results from rupture of an atherosclerotic plaque, with
subsequent platelet aggregation and thrombus formation.1 It represents a spectrum of clinical presentations of myocardial ischemic
events, ranging from angina, reversible tissue injury, and unstable angina, to myocardial infarction (MI) and extensive myocardial
tissue necrosis.2 Clot lysis and antiplatelet therapies have reduced the morbidity and mortality in these syndromes.3 The use of
thrombolytic agents in patients with persistent ST-segment elevation is well established. Treatment includes administration of
antiplatelet and anti-ischemic agents, thrombolytic therapy, and primary percutaneous transluminal coronary angioplasty (PTCA).
Reperfusion therapy, whether thrombolysis or PTCA works best when given within 4 to 6 hours of the onset of signs and symptoms.4
The investigation is on going on the use of antithrombin and antiplatelet agents in patients with unstable angina and non-Q-wave MI.
The diagnosis of myocardial ischemia has traditionally been based on the World Health Organization’s (WHO) recommendation
which includes fulfilling at least two of the following criteria: evidence of clinical symptoms suggestive of myocardial ischemia of
more than 30 minutes duration, evolution of typical electrocardiogram (ECG) changes consistent with myocardial injury and serial
increase in serum enzyme activities.1,2,5
Clinical symptoms even though very important should be assessed carefully because they may sometimes be non specific especially in
diabetic patients and the elderly who usually present with atypical symptoms of ischemia. 2 An electrocardiogram should be performed
soon after presentation because those with either ST-segment elevation greater than 1 mV in contiguous leads or symptoms of new left
bundle branch block should be treated immediately with reperfusion therapy. 2 However, only about 50% of patients with acute
myocardial infarction (AMI) manifest these characteristic changes. Thus the other 50% are missed if diagnosis is based solely on
clinical history, presence of chest pain, and evolutionary changes in the ECG alone. 6,7 The third aspect of the diagnostic triad is the
release of enzymes from necrotizing myocardium. The cardiac enzymes released following AMI include creatine kinase (CK) and its
isoenzymes, lactate dehydrogenase (LD) and its isoenzymes and aspartate aminotransferase (AST), aldolase, myokinase, and alanine
amino transferase (ALT).8 Creatine kinase is the first to show increased activity following AMI and if this pattern continues, further
necrosis may be occurring, and shorter-lived markers or those markers that are at elevated levels for shorter periods such as CK-MB or
myoglobin (Mb) can be used for confirmation. The importance of measuring CK-MB has been well established and it is considered
the “gold standard” for the diagnosis of AMI.7 Creatine kinase-MB has a characteristic release to peak pattern and its concentration is
increased soon after the onset of symptoms, which is very indicative of an AMI. 9,10 When ECG changes are diagnostic, then the
utilization of CK-MB isoenzyme is restricted to that of confirmation of the diagnosis. After AMI, increased levels of AST appear in
serum due to high concentration in heart muscle. The AST level does not become abnormal until 6 to 8 hours after the onset of chest
pain.11 In a typical course for CK and LD isoenzymes, CK-MB peaks first, with LD1 exceeding LD2, 5 to 20 hours later.12 With the
development of newer non-enzymatic biochemical markers such as Mb and cardiac troponins T (cTnT) and I (cTnI) which are not
themselves enzymes the third criterion of the WHO diagnostic triad needs to be revised. 13
The American College of Cardiology and the American Heart Association (ACC/AHA) have published new guidelines on the clinical
and biochemical evaluation of chest pain.14 According to these new recommendations, patients who present with chest discomfort
should undergo early risk stratification that focuses on angina symptoms, physical findings, ECG findings and biomarkers of cardiac
injury. A 12 lead ECG should be obtained immediately in patients with ongoing chest discomfort. Biomarkers of cardiac injury should
be measured in all patients who present with chest discomfort consistent with ACS.14 A cardiac specific troponin is the preferred
biomarker and if available it should be measured in all patients. Creatine kinase-MB by mass assay is also acceptable. In patients with
2
negative cardiac markers within 6 hours of the onset of pain, another sample should be drawn between 6 and 12 hours. 14 C-reactive
protein (CRP) and other markers of inflammation should be measured. and total CK, AST, -hydroxybutyric dehydrogenase and/or
LD should be the marker for the detection of myocardial injury.14 Table 1 shows the comparative time-course of appearance and
disappearance of these markers following the onset of chest pain.
Table 1 Comparative Time Course of Appearance and Disappearance of Biomarkers
Biochemical Marker
Initial elevation after
onset of AMI
1-3 h
3-8 h
3-8 h
2-6 h
8-12 h
10-12 h
6-8 h
3-8 h
3-8 h
Average time until peak Time (days) until return
concentration
to baseline
Mb
6-9 h
1
CK
10-24 h
3-4
CK-MB
10-24 h
2-3
CK-MB subforms
12 h
1-2
LD
72-144 h
8-14
LD1
48-72 h
7-10
AST
18-24
4-5
cTnI and cTnT
24-48 h (1st peak)
3-5
cTnT
72-100 h (2nd peak cTnT 5-10
only)
Key: Mb = myoglobin, CK = creatine kinase, CK-MB = creatine kinase-MB, LD = lactate dehydrogenase, AST = Aspartate
aminotransferase, cTnT = cardiac troponin T, cTnI = cardiac troponin I
In the presence of nonspecific or vague symptoms, the biochemical markers acquire greater significance. 2 It is estimated that 2% to
5% of patients with AMI are discharged and this is the most common cause of malpractice lawsuits against physicians’ today.2 Many
institutions today have dedicated areas within the emergency room (ER) for rapid rule-out of AMI. These areas, which are known by
such names as Chest Pain Evaluation Centers, Chest Pain ER, Chest Pain Center, Chest Pain Evaluation Unit, Short-Stay ED
Coronary Care Unit, and ED Monitored Observation Bed, have as their primary objective the efficient triage of chest pain patients and
improving their care.1,2,15 The pathophysiology of ACS determines the implicated biochemical markers of interest (Table 2). Thus for
optimum assessment of patients’ risks, biochemical markers of plaque formation such as homocysteine and plaque rupture such as Creactive protein (CRP) and serum amyloid A (SAA), and indicators of intracoronary thrombosis such as P-selectin and soluble fibrin
as well as indicators of myocardial ischemia such as glycogen phosphorylase-BB (GP-BB) and biomarkers of myocardial necrosis
such as CK-MB, Mb, cTnT and cTnI could be combined with clinical indicators and ECG findings to provide an accurate diagnosis or
risk assessment.2
Creatine Kinase-MB
Currently, a serial rise and fall in CK and CK-MB is used to confirm the diagnosis of AMI.16 A single CK-MB test used for screening
for AMI is only 50% sensitive when it is performed on a sample taken at the time of arrival of the patient in the ER. A serial test done
over three hours gives more than 90% sensitivity. If the serial testing is carried out within six hours it gives 95% sensitivity.17 CK-MB
is one of three dimeric isoenzymes of CK. All cytosolic CK is composed of M and B subunits. They associate to form CK-MM, CK-
3
Table 2 Biochemical Markers in Risk Stratification and Diagnosis of Acute Coronary Syndrome
Pathophysiology
Plaque Formation
Biochemical Markers
Homocysteine
Advantages/Disadvantages
Elevated levels positively
associated with plaque formation
and CVD.
Plaque Rupture
C-reactive protein
Increased in men and women at
risk for future CVD events. May
be elevated as a result of acute
phase reaction.
Serum amyloid A
SAA predicts the risk of adverse
outcome in unstable angina.
Thrombus Formation
P-selectin
Elevated levels are indicative of
thrombus formation. Will identify
patients at high thromboembolic
risk from infective endocarditis.
Soluble fibrin
Elevated levels may indicate
likelihood of MI.
Myocardial Ischemia
Glycogen phosphorylase BB
Peak concentrations occur sooner
than CK-MB or cTnT in
perioperative MI. May be
unreliable in the presence of renal
impairment or cerebral injury.
Myocardial Necrosis
Myoglobin
High sensitivity and useful in
early detection of MI. Detection
of reperfusion. Has very low
specificity in setting of skeletal
muscle injury. Rapid return to
normal range limits sensitivity for
later presentations.
Heart-type fatty acid binding
Early biomarker of myocardial
protein
injury. The level in plasma
correlates with infarct size.
CK-MB
Currently the “gold standard” to
confirm the diagnosis of MI.
Rapid, cost-efficient, accurate
assays. Loss of sensitivity in
skeletal muscle disease or injury
including surgery.
CK-MB subforms
Early detection of MI. Released at
2 to 6 hours following an MI.
Specificity profile similar to CKMB. Current assays require
special expertise.
cTnT
Sensitive and specific for MI.
New gold standard for diagnosis
of MI. Low sensitivity in very
early phase of MI. Limited ability
to detect late minor reinfarction
cTnI
Sensitive and specific for MI.
New gold standard. Insufficient
for early diagnosis. Similar to
cTnT in all aspects.
Key: CK-MB = creatine kinase-MB, cTnT= cardiac troponin T, cTnI = cardiac troponin I, SAA = serum amyloid A, CVD = coronary
vascular disease, MI = myocardial infacrtction
4
MB, and CK-BB isoenzymes. CK-MM is found predominantly in striated muscles of both the skeleton and the myocardium.. CK-MB
isoenzyme comprises approximately 20% of total CK in the myocardium, and about 0-3% of CK in the skeletal muscles.2
Various laboratory techniques are used to separate and identify cardiac specific CK-MB isoenzymes from the non-specific CK-MM
and -BB isoenzymes. The concentration of CK-MB released from a necrotizing myocardium can be measured directly or indirectly.
The indirect technique, which includes electrophoresis and immunoinhibition, measures CK-MB enzyme activity in the presence of
substrate and the results are reported in units of activity per liter (U/L). 8,18 Monoclonal antibody techniques have greatly improved
both specificity and sensitivity for the detection of AMI by providing direct measurements of CK-MB mass in g/L.8 The CK-MB
mass concentration is determined by immuno-chemical methods such as the microparticle enzyme immunoassay (MEIA)
technique.8,13 Generally, the mass assay is more sensitive for detection of AMI but both techniques are limited by delayed enzyme
release from damaged myocardial cells.18 Old electrophoresis assays for CK-MB cannot detect AMI as early as the mass
immunoassays.11 Sensitivity for detection of AMI approaches 100% at 10-12 hours, but is only about 57% for the mass assay and
about 32% for CK-MB activity during the first four hours.18
As biomarkers of myocardial injury, both CK and CK-MB have deficiencies, which include the fact that they are present in tissues
other than the myocardium and a rise and fall of these enzymes are associated with conditions other than AMI. 19 It is also recognized
that ischemic cardiac injury can occur without myocardial necrosis and the release of CK and CK-MB can occur without infarction.20
The diagnosis of AMI using increased release of CK and CK-MB has challenged clinicians. Despite its deficiencies, CK-MB is still
the diagnostic marker used in most countries of the world to rule in or rule out AMI. To Make CK-MB measurement more
diagnostically relevant, a CK-MB percent relative index is calculated. The calculation of the percent relative index [CK-MB (in
g/L)/total CK (in U/L) X 100] may assist in the differentiation between myocardial and skeletal muscle causes of increased total
CK.1,21 It has been suggested that CK-MB index values exceeding 2.5% are associated with a myocardial source of the CK-MB.2
However, recent reviews indicate that myocardium related CK-MB have been stated to be as low as 2% or as high 5% depending on
the variability of the numerator and denominator terms in the index.2 The diagnostic cut off depends on the assay due to the lack of
CK-MB standardization among different manufacturers. The percent relative index may not be used for interpretation when total CK
enzyme activity is within reference range. Other investigators including Koch et al22 have suggested that expression of CK-MB as a
percent of total CK degrades efficiency unless total CK is markedly increased and therefore should be abandoned. Creatine kinase MB may also be used in the assessment of re-infarction or infarct extension in patients with a previous MI. 2
Creatine kinase-MB isoforms also termed “subforms”, have been shown to be early markers for AMI. 9 The subforms are CK-MB1
and CK-MB2 and assay values of CK-MB2 greater than 2.6 U/L and CK-MB2 to CK-MB1 ratios greater than 1.7 are indicative of
5
myocardial necrosis.15 These subforms are released simultaneously into the blood at 2-6 hours following AMI and increased subform
ratios can be detected in the serum earlier than CK-MB isoenzyme alone, increasing the sensitivity for early AMI detection and
identification over standard CK-MB assays: at 6 hours, 91% sensitivity for subforms vs 62% for CK-MB.18 Thus subform assay
provides rapid and reliable diagnosis of AMI within 4-6 hours after the onset of symptoms, which is 6 hours before conventional CKMB assays are accurate.23 Unfortunately, these assays are not available at all institutions, and are technically difficult tests requiring
special equipment.18 Currently CK-MB isoforms may be measured by high-voltage electrophoresis and automated stat CK-MB
isoform measurements are being used in some hospitals as an early measure of myocardial injury. 24
A different approach to identifying AMI with serum markers relies on time changes in the serum marker level or delta values as
opposed to an absolute threshold value for normalcy. Because newer assays are becoming ever more sensitive and precise, this
approach has the potential to both reliably identify and reliably exclude AMI if an appropriate time interval and cutoff value is chosen
while the marker value is still in the normal range.25 In a study to assess the critical difference in serial measurements of CK-MB mass
assay and the ability of this critical difference to detect myocardial damage, De Winter et al26 studied 110 patients in whom AMI had
been ruled out. Blood samples were obtained from the patients at 3, 4, 5, 6, 7, 8, 12, 16, 20, 24, hours. They determined that with a
critical difference of 72.6%, an increase of >2.0 g/L between two CK-MB mass measurements would be significant.26 They found
that twenty three of the non-AMI patients had an increase in CK-MB mass >2.0 g/L but five of them had normal cTnT
concentrations. Also among the 110 non-AMI patients, 22 had abnormal cTnT values and 18 of them also had abnormal CK-MB mass
>2.0 g/L. In 20 of the 23 patients with increased CK-MB mass >2.0 g/L, the increase was detected from the values for two samples
collected at 5 and 12 hours after onset of symptoms. They concluded that using critical difference for CK-MB mass >2.0 g/L
detected myocardial damage in patients without AMI.26
Lactate Dehydrogenase
In addition to the heart, LD occurs in many other parts of the body, including the kidneys, red blood cells, brain, stomach, and skeletal
muscle. At least five isoenzymes are known, composed of four subunit peptides designated M and H. The LD 1 isoenzyme is found in
highest concentration in the heart, kidney, and red blood cells. The LD 5 is found in the highest concentration in the liver, and skeletal
muscle.11 The hybrid isoenzymes, LD2, LD3, and LD4 are found in the heart, kidney, red blood cells and several other tissues. 11 Of the
five isoenzyme, LD1 and LD2, are useful in the diagnosis of myocardial ischemia. Level of LD 1 is elevated when myocardial infarction
is present and in other conditions such as leukemia. LD2 is present in all parts of the body except skeletal muscle but is present
predominantly in the heart.27
6
Levels of LD start to increase 24 to 48 hours after occlusion of the coronary artery, peak in 3 to 6 days, and return to normal in 8 to 14
days.27 Levels of LD1 are elevated 10 to 12 hours after the acute myocardial infarction, peak in 2 to 3 days, and return to normal in
approximately 7 to 10 days.4,11 Thus, with the measurement of the level of LD a prolonged retrospective diagnosis of MI can be made.
The amount of LD2 in the blood is usually higher than that of LD1 however patients with AMI have more LD1 than LD2. This "flipped
ratio" usually returns to normal in 7 to 10 days. 28 An elevated level of LD1 with a flipped ratio has a sensitivity and specificity of
approximately 75% to 90% for detection of AMI. 28 In individuals exercising, increases in serum total LD especially LD 1 and a flipped
ratio of LD1 to LD2 1.0, can arise from skeletal muscle as opposed to the myocardium.11
Beta hydroxybutyrate dehydrogenase present in serum represents the LD activity of mostly the LD 1 and LD2 isoenzymes.
Measurement of -hydroxybutyrate dehydrogenase thus indicates the activity of the cardiac LD isoenzymes.11 Because of technical
concerns, measurement of lactate dehydrogenase has largely been replaced by measurement of troponins because of the improved
specificity and duration of elevated levels of the latter biomarkers. Thus use of LD and LD isoenzymes for the detection of AMI is
declining rapidly and only very few if any laboratories are likely to continue to offer these tests for the detection of AMI.
Myoglobin
Myoglobin is a low-molecular weight oxygen-binding heme protein that accounts for 5% to 10% of all cytoplasmic proteins. It is
rapidly released from damaged striated muscles of both the skeleton and the myocardium. It is not cardiac specific, therefore the
measurement of Mb should be in the coronary sinus in order to improve specificity when skeletal muscle injury is suspected. It may be
necessary to measure carbonic anhydrase III and Mb simultaneously in order to improve specificity. Skeletal muscle injury results in
the release of Mb and carbonic anhydrase III thus their ratio remains constant in serum. Myocardial injury is associated with a
predominant release of myoglobin.29 Carbonic anhydrase III is not found in cardiac muscle therefore it can be used to differentiate
between skeletal and cardiac muscle damage.30
Myoglobin is important because it is released early into the bloodstream after the onset of chest pain and can be highly effective in the
rule-out of AMI.10 Rapid treatment of patients with chest pain is a major concern in the ER. Dissolution of a clot that is blocking
delivery of oxygen carrying red blood cells to the myocardium can be achieved with such agents as streptokinase (SK) and tissue
plasminogen activator (tPA) or PTCA, which must be performed to have maximum effectiveness in potentially salvaging tissue that
would otherwise be lost. Mb is more sensitive than CK and CK-MB activities during the first few hours after onset of chest pain. 30 It
rises within 1 hours to 3 hours and can be detected in all AMI patients between 6 and 10 hours of chest pain. 26 If the Mb level remains
within the reference range 8 hours after onset of chest pain, AMI is ruled out. 30 However, Mb lacks specificity because Mb released
from skeletal muscle cannot be distinguished from that released from the myocardium. 29
7
Troponins
The troponins I and T are two components of the three member troponin complex which consists of troponin T, troponin I and
troponin C. The three subunits are called regulatory proteins because they are involved with specific roles in the regulation of striated
and cardiac muscle contraction. Troponin T and I are cardiac specific and have different biological functions, amino acid sequences,
and molecular weights than their skeletal muscle forms. Troponin T functions to bind the troponin complex to tropomyosin while
troponin I functions to inhibit the activity of the actomyosin ATPase, and troponin C serves to bind four calcium ions and regulate
contraction.2
In recent years, cTnI cTnT have challenged CK-MB as the "gold standard" for the early biochemical detection of AMI and they appear
also to be superior to LD-1 in the late diagnosis of AMI.11,31,32 The clinical sensitivity of cTnT is similar to that of CK-MB during the
first 48 hours after onset of chest discomfort. Cardiac troponin T shows a clinical sensitivity of 50% to 65% from up to 6 hours after
onset of chest pain therefore like CK-MB, cTnT is not an effective early diagnostic marker. 11 It remains increased for up to 7 to 10
days thus giving a high clinical sensitivity >90% up to 5 to 7 days after the occurrence of an AMI.11 Serum troponins have been
increasingly used in the diagnosis of ACS as studies have shown that they provide greater sensitivity over CK-MB.31 Additionally,
cTnT has the ability to detect myocardial injury in some patients with unstable angina pectoris (UAP). Because these patients bear a
substantial risk for developing adverse events such as AMI, cTnT and, more recently, cTnI have been proposed to be of value in risk
stratification of patients with UAP in view of the possible benefit of an early intervention with antithrombolytic therapy. 32 The
proportion of total cTnT and cTnI, representing the cytosolic pool available for rapid release, and their time-dependent increase after
myocardial necrosis differ.
The concentration of cTnT after an MI increases in serum after 4 hours and achieves an initial peak or plateau at 2 to 5 days. A second
cTnT peak is observed in many patients due to the fact that cTnT has both cytosolic and structurally bound cTnT pools. The first peak
results from the release of the cytosolic pool and second peak reflects the slower release of the structural component later during the
myocardial necrosis process.12 Increased serum cTnT concentrations on day 3 or 4 after AMI are a powerful noninvasive predictor of
poor long-term prognosis, because it reflects residual left ventricular function after AMI. 33 A study to evaluate whether cTnT might be
used for identification of patients with unstable coronary artery disease who might benefit from treatment with low molecular weight
heparin showed that elevation of cTnT identifies a subgroup of patients in whom prolonged antithrombotic treatment was beneficial.34
The very low to undetectable cardiac troponin values in serum in individual without AMI allow the use of lower discriminator values,
compared with CK-MB for determination of MI and risk stratification.11 Cardiac troponins I and T are very specific and should
eliminate false positive diagnosis in AMI in patients with increased levels of CK-MB after skeletal muscle injury.11
8
Cardiac troponin I is restricted to the myocardium within a few months of birth and is thought to be even more specific than cTnT.
The release characteristics and utilization of cTnI as an early indicator of MI are quite similar to those of cTnT and Ck-MB. Cardiac
troponin I is similar to cTnT in all possible clinical applications and its measurement offers the same advantage over CK-MB as does
cTnT.12 Cardiac troponin T is increased in patients with end stage renal failure and there have been instances of re-expression in
skeletal muscle of the fetal gene for cTnT.32 The claims of non specific elevation of cTnT are based on reports that cTnI is not found
in patients with renal dysfunction.35 However, Direct comparison of cTnI and cTnT measurements in individuals with chronic renal
failure indicate that both troponin values are increased in 10% to 30% of individuals without myocardial injury. 11,36,37 A study of
individuals undergoing hemodialysis showed that increases in cTnT and cTnI predicted poor outcome in cases of MI.11 Since cTnI and
cTnT offer essentially similar information, the choice of which one to use will have to be based on the analytical performance,
convenience, speed, and overall assay cost.12
P-Selectin
An early event in atherogenesis is the demonstration of aggregates of lipid-rich macrophages and T lymphocytes within the intimal
layer of the endothelium. The adhesion of these leukocytes on the endothelial cells and their transendothelial migration are mediated
by adhesion molecules on the endothelial cell membrane that belong to two families of proteins namely the selectins and the
immunoglobulin superfamily.38 P-Selectin plays a critical role in the migration of lymphocytes into tissues. It is found constitutively in
a pre-formed state in the Weibel-Palade bodies of endothelial cells and in the alpha granules of platelets. It is a cell surface adhesion
molecule, expressed on the surface of activated platelets, which is involved in leukocyte rolling and attachment and has been
implicated in the initiation of atherosclerosis.39 Expression of P-selectin is increased on the surface of platelets in patients with
symptomatic coronary artery disease (CAD). 2
P-selectin is mobilized in response to a variety of inflammatory or thrombogenic agents and it is present on the cell surface for only a
few minutes after which it is recycled to intracellular compartments. P-Selectin is involved in the adhesion of macrophages and
lymphocytes to activated endothelium. It causes adhesion of platelets to monocytes and neutrophils and plays a central role in
neutrophil accumulation within thrombi. The adhesion of leukocytes to the endothelium is initiated by weak interactions that produce
a characteristic “rolling” motion of the leukocytes on the endothelial surface. P-Selectin is implicated, along with L-Selectin, in the
mediation of these initial interactions. A stronger interaction, which may involve E-Selectin, follows the initial interactions, and this
leads eventually to extravasation through the blood vessel walls into lymphoid tissues and to sites of inflammation. 38
In a large-scale study of apparently healthy women, baseline P-selectin levels were measured among 115 participants who
9
subsequently developed cardiovascular events and among 250 age and smoking-matched participants who remained free of disease
during 3.5 years of follow-up. The mean level of soluble P-selectin was significantly higher at baseline among women who
subsequently experienced cardiovascular events compared with those who did not. It was concluded that P-selectin levels are elevated
among apparently healthy women at risk for future cardiovascular events. 39
Adhesion molecules mediating leukocyte endothelial interactions are altered subsequent to post ischemic reperfusion and by treatment
with thrombolytic agents and angioplasty. The clinical relevance of these biological changes remains to be determined. 40 E-selectin
and P-selectin may be useful markers with which to identify patients at high thromboembolic risk from infective endocarditis.41
Eriksson et al42 have shown that the transient dynamics of leukocyte-endothelium interactions are important regulators of arterial
leukocyte recruitment and that leukocyte rolling in atherosclerosis is critically dependent on the endothelial selectins
Soluble fibrin
Fibrinogen is an independent risk factor that contributes to thrombosis and cardiovascular disease. The formation of a thrombus is a
prelude to the blockage of an infarct related artery. Consequently, markers of thrombosis, which include soluble fibrin, as well as
fibrin degradation products, may reveal the presence of a recent thrombotic event or risk of an impending one. 2 When fibrin is broken
down, its soluble products are released into the blood stream. Thus, an increased concentration of fibrin degradation products in the
circulation indicates that the body is dissolving a potentially dangerous clot. The formation of a clot is designed to prevent hemorrhage
and if the process in not controlled the clot will expand indefinitely until thrombosis occurs.
Control of thrombosis is regulated by certain coagulation factors including bradykinin as well as various endothelial cell factors. The
coagulation factors function by converting plasminogen to plasmin, which actively degrades fibrin into fibrin degradation products.
Plasmin’s potent fibrinolytic activity is rapidly neutralised by alpha 2-antiplasmin and inflammatory cells digest fibrin degradation
products. Although not a sensitive indicator of MI, when coupled with the typical symptoms of chest pain and shortness of breath,
high concentrations of soluble fibrin may indicate the likelihood of an MI. 2
Glycogen phosphorylase BB
Glycogen 6-phosphorylase (G6P) the main enzyme for glycogenolysis, exists as three isoenzymes; BB (brain), MM (muscle), and LL
(liver). The GP-BB isoenzyme is also found in the myocardium, where it is the predominant phenotype unlike the skeletal muscle,
which contains only the GP-MM isoenzyme. Glycogenolysis is increased in ischemic myocardium and large amounts of GP-BB are
released into the circulation during ischemic events.2 Peak concentrations of GP-BB occurs sooner than CK-MB or cTnT.25 It has been
suggested that G6P and GP-BB concentrations are sensitive markers of perioperative myocardial injury in patients undergoing
10
coronary artery bypass grafting. Glycogen 6-phosphorylase BB demonstrates a rapid rise to detectable levels in patients with
infarction, within 2 to 3 hours of chest pain onset. However the result may be unreliable in the presence of renal impairment or
cerebral injury.29
Fatty-Acid-Binding Protein
Heart-type fatty acid-binding protein (H-FABP), is a low-molecular weight protein, which is involved in lipid homeostasis. It is
abundant in heart muscle and found in lower concentration in skeletal muscle. The amounts in the kidney, liver, and small intestine are
much lower. After myocardial damage, H-FABP is released into the intercellular space and appears in the bloodstream. The amount of
H-FABP released in plasma has been demonstrated to correlate with the size of the infarction. Therefore it has been proposed as an
early biochemical marker for the diagnosis of AMI.43,44 Fatty acid-binding protein like Mb, increases significantly within 3 h after
AMI and returns to normal reference values within 12 to 24 h.45 For the early assessment or exclusion of AMI, H-FABP performs
better than Mb. In addition, the differences in contents of Mb and FABP in heart and skeletal muscles and their simultaneous release
upon muscle injury allow the plasma ratio of Mb/FABP to be applied for discrimination of myocardial muscle injury (ratio 4:5) from
skeletal muscle injury (ratio 20:70).46-48
Homocysteine
There are epidemiological studies which support a positive association between plasma homocysteine (Hcy) concentrations and the
risk for cardiovascular disease (CVD).46 The observation was made by McCully in 1969 that patients with very high concentrations of
plasma Hcy attributable to homocystinuria have an accelerated atherosclerosis. 49 It is postulated that Hcy may cause atherosclerosis by
damaging the endothelium directly or through alteration of oxidative status. Hyperhomocysteinemia, defined as a mildly increased
plasma homocysteine concentration, is positively associated with CVD. Hyperhomocysteinemia, is associated with deficiencies of
vitamin B12, B6, and folate. A study by Ellis et al50 found that patients who were given vitamin B6 for carpal tunnel syndrome and
other degenerative diseases had 27% of the risk of developing acute cardiac chest pain or myocardial infarction, compared with
patients who had not taken vitamin B6. Hyperhomocysteinemia has also been implicated in neural tube defects, inflammatory bowel
disease, Alzheimer’s disease, thrombosis, pregnancy complications, mental disorders, and possibly cancer.51,52
When Hcy concentrations increase there is a direct toxic or irritant effect to the endothelium, which lines the surface of arteries. One
of the potential mechanisms of this damage that has been proposed is "oxidative stress". 46 It has been suggested that
hyperhomocysteinenemia may promote the production of hydroxyl radicals, known peroxidation initiators, through Hcy autooxidation
and thiolactone formation.46 The hydroxyl radicals and peroxidation initiators modify lipoproteins, which are taken up by
macrophages. The macrophages are transformed into foam cells that contribute to the development of atherosclerotic plaque and
11
progression of atherogenesis.53 Homocysteine levels in plasma may be decreased by the administration of vitamin B 12, vitamin B6 and
particularly folic acid in both healthy patients and patients with CVD. 52
Serum Amyloid A
Serum elevation of CRP and SAA protein at the time of hospital admission predicts a poor outcome in patients with unstable angina
and may reflect an important inflammatory component in the pathogenesis of this condition. Serum amyloid A is an acute phase
reactant that transiently binds to high-density lipoprotein (HDL) during an inflammatory response. Serum amyloid A may displace
apolipoprotein A-I, which in turn results in increased catabolism of HDL, or inhibit lecithin-cholesterol acyltransferase activity which
leads to low levels of esterified serum cholesterol. This lipoprotein alteration along with a direct effect of SAA on the endothelium of
the atheromatous plaque is indicative of a potential pathophysiological link between the inflammatory responses expressed by the
serum concentrations of amyloid A and the development of the atherosclerotic process.
This small acute phase protein is synthesized in response to trauma, infection, inflammation, and neoplasia and their fragments form
tightly ordered fibrillar arrays responsible for clinical amyloidosis. Interleukin (IL)-6 is the major determinant of acute phase reactant
protein synthesis in the liver, and elevations of either IL-6 or SAA can be predictive in determining increased risk of adverse outcomes
in patients with unstable angina.54 Patients who do not have biochemical evidence of myocardial injury but have increased levels of
CRP appear to be at increased risk for adverse outcome especially those whose CRP levels are markedly increased. 15
C-Reactive Protein
C-reactive protein is an acute phase protein originally named by Tillet and Francis in 1930. 55 It is produced by the liver in response to
inflammation and infection. It increases rapidly following many disease conditions such as infections, trauma, and surgery. Persistent
increases in CRP can occur in chronic inflammatory disorders, including autoimmune diseases and malignancy. 56 Measurement of
CRP may be used in the clinical setting to monitor infection as well as post operative complications and the effectiveness of various
treatment modalities.57
The concentrations of CRP have been shown to correlate with markers of endothelial dysfunction.49 Inflammation in the vessel wall is
implicated in the initiation and progression of atherosclerosis and in the erosion or fissure of plaques and eventually in the rupture of
plaques.45 Increased concentrations of CRP and SAA are known to be nonspecific, but may have a role in identifying patients with
unstable coronary plaques.2 There is prominence of macrophages and T lymphocytes in the atheromatous plaque in the walls of the
arteries. Cytokines such as IL-6, which are responsible for the production of CRP by the liver increase in ACS even in the absence of
myocardial necrosis.58
12
There appears to be a relationship between infections and acute systemic inflammation and increased risk for acute cardiovascular
events. Inflammation has been implicated in the initiation and progression of atherosclerosis. It has been shown that the change from
stable to unstable angina is associated with increased plasma levels of CRP, SAA and IL-6, which are indicators of a systemic
inflammatory response.59 The endothelium exerts antithrombotic and vasodilator effects on the vascular wall. When the endothelial
cells are exposed to proinflammatory cytokines, procoagulatant activity is induced. This leads to the expression of cell surface
adhesion molecules and impairs endothelium-dependent vascular relaxation. The result of these altered functions of the endothelial
cells is the promotion of acute events leading to atherosclerotic vascular disease. 59
It has been shown that CRP and SAA have useful prognostic utility in patients with MI, unstable angina, or non-Q wave MI. Studies
have shown that small differences in baseline concentrations of CRP in apparently healthy men and in patients with stable angina
pectoris constitute an independent risk factor for first cardiovascular events. In addition, the increase in CRP after AMI and CRP
concentration during unstable angina and at discharge correlate with risk of recurrent events. 55 In patients with unstable angina,
increased CRP is associated with adverse outcome independent of an increased cTnT or cTnI which are sensitive and specific markers
of myocardial necrosis.60 It has been shown that CRP is a predictor of increased risk for MI, stroke, or peripheral vascular disease in
asymptomatic individuals.61
Conclusion
Clinical history and ECG miss a significant portion of patients with acute cardiac ischemia. It appears that patients with AMI and
some with high-risk "unstable angina" can be identified within 6 hours of hospital presentation using a combination of cardiac
markers. Testing these patients soon after symptom onset or arrival in the ER for Mb, CK-MB subforms, or CK-MB delta appears to
provide the best diagnostic usefulness. For testing later in the clinical course, CK-MB, cTnI, or cTnT are of clear diagnostic and
prognostic value. The markers currently used, however, are unable to identify the significant subset of patients with "non-AMI"
coronary syndromes. These patients require further testing with appropriate noninvasive or invasive diagnostic studies. 45
Several biochemical markers are currently available to the clinical laboratory scientist with which to provide the physician with the
information he or she requires to make an accurate diagnosis in suspected cases of acute coronary syndromes. These biochemical
markers, in addition to the WHO criteria for diagnosing AMI, have gone a long way toward reducing the cost of treating AMI patients
and decreasing the number of malpractice cases resulting from missed diagnosis.
Creatine kinase-MB has long been the “gold standard” for the laboratory diagnosis of AMI. That role is being challenged by the
development, characterization and clinical interpretation of of cTnT and cTnI. 1 Cardiac troponins T and I appear in blood at or near
13
the same time as CK-MB, but remain abnormal for 3 to 10 days.1 The National Academy of Clinical Biochemistry (NACB) committee
recommends that cardiac troponin T or I is the new standard for diagnosis of MI and detection of myocardial cell damage replacing
CK-MB.1 The NACB committee further recommends that there is no longer a role for LD and its isoenzymes in the dianosis of
cardiac diseases. The NACB committee feels that if a hospital is already using cTnT or cTnI then the measurement of LD and hydroxybutyric dehydrogenase should be discontinued. Some cardiologists have expressed concern about completely replacing CKMB with the troponins.1 Many physicians use peak CK-MB for infarct sizing and some physicians have questioned whether serial
troponin measurements can be used for reinfarction because of the prolonged release pattern. Therefore they suggest the continued use
of CK-MB for that purpose.1 The measurement of CK should continue because the marker is inexpensive, readily available and can be
useful in detecting skeletal muscle injury.1i,10 New biomarkers of myocardial injury will continue to be developed and assessed
in patients with ACS as new information is obtained on the pathophysiology of the disease and new therapeutic regimens are found.
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