Cardiovascular diseases (CVD) remain the leading cause of death in most of the industralized world. Myocardial infarction (MI) as a pathologic concept was recognized in the beginning of the 20th century. At autopsy vegetation, due to endocarditis on the aortic valve, was found to have blocked the orifice of the right coronary artery.2 According to the World Health Organization (WHO), the definition of acute MI includes the presence of two of the following three criteria: 1) Characteristic chest pain, usually of more than 30 minutes; 2) diagnostic electrocardiogram (ECG) changes; and 3) a rise and subsequent fall of serial levels of cardiac markers.3 Acute coronary syndromes remain the leading cause of mortality in India and represent an enormous cost to the health care system.4-6 Effective intervention in acute MI is undoubtedly dependent upon early diagnosis. In cases of massive cardiac injury, the above criteria will be met easily. However, in the event of occlusion of small coronary branches or extensive collateral circulation to the ischaemic area, the typical clinical or ECG findings may not be present.7 Thus, diagnosis based on ECG, is a continuing challenge. The concern about missing the diagnosis in patients with acute MI has led to a lower threshold for admission in order to exclude the presence of acute MI. Lee et al reported approximately 60% to 70% of patients, with chest pain, admitted to hospital were eventually diagnosed as not having an MI but rather some different diagnosis.8 According to the criteria for acute MI laid by the WHO,3 cardiac markers can facilitate the diagnosis of an MI. Biochemical markers have long been the cornerstone of diagnosis and continue to play an important role, especially in the group of patients with low medium risk. Use of biochemical markers, to diagnose acute MI, can be dated back to 1954 when aspartate aminotransferase (AST) was first used, which subsequently stimulated a number of investigations on different compounds.9 Creatine phosphokinase (CPK) replaced AST in late 1960’s and Lactate dehydrogenase (LDH) was started to be used as a late marker in 1970’s. Since the early 1980’s, the more specific CK isoenzyme, CK-MB, has become the “gold standard” for the diagnosis of acute MI and additionally, in past few years, the separation of CK-MB into two isoforms by electrophoresis (EP) has played an important role in the development of the automated enzyme immunoassays, which utilize highly sensitive and specific monoclonal antibodies to detect CK-MB and cardiac troponin.7,9 For more than 15 years cardiac form of troponin I is known as a reliable marker of cardiac tissue injury. It is considered to be more sensitive and significantly more specific in diagnosis of MI than the golden markers of last decades CK-MB, as well as myoglobin and LDH isoenzymes. Other biochemical markers such as fatty acid binding protein, glycogen phosphorylase, plasma oxidase and, C-reactive protein have not been accepted widely in the diagnosis of AMI.

Several criteria for the ideal cardiac marker are summarized such as : The ideal cardiac marker should; 1) have sufficient specificity for the diagnosis of myocardial damage, in the presence of skeletal muscle injury, 2) be highly sensitive and capable of detecting even mild myocardial damage, 3) appear in quantities that are in direct proportion to the extent of the injury, 4) be absent or present only in trace amounts, in the circulation, under physiological condition, and have the possibility to be detected as abnormal with even minimal elevation in their levels, 5) be technically easy to measure and should not be very expensive.9,10 Currently none of the available markers meet all these criteria. Therefore continued research on better markers and approaches in the diagnosis of acute coronary syndromes is needed.

Creatinine Phosphokinase (CPK) and its Isoenzyme MB

CK has three isoforms: BB, MB and MM. The characteristics of CK-MB are summarized in Table 1. CK (EC 2.7.3.2) is a cytosolic enzyme that catalyses the reversible transfer of a phosphate group from adenosine triphosphate (ATP) to creatine. The activity of CK is dependent on the muscle mass. CK-MM is predominant in both heart and skeletal muscle but CK-MB is more specific for the myocardium but is also present in tongue, diaphragm, and skeletal muscles, although in minute amounts of only 1% to 3%.15 CK can be elevated in multiple conditions such as hypothyroidism, chronic renal failure, chronic alcoholism, myopathies, following cardioversion, cerebrovascular diseases, intramuscular injections and surgical trauma.9,1215 The specificity of CK-MB is enhanced by the calculation of CK-MB to CK-ratio (CK index).

The tissue CK-MB (MB2 isoform) is first released into the circulation after myocardial injury, and serum CK-MB (MB1 isoform) is formed as a product of CK-MB2, which results from the action of the serum enzyme carboxypeptidase. The proteolytic action of carboxypeptidase removes the terminal positively charged lysine to produce a more negatively charged CK-MB. Puleo16 studied 1110 patients with chest pain, and showed that CK-MB isoform reliably detected MI within the first 6 hours after the onset of symptoms with a sensitivity of 95.7% as compared to only 48% sensitivity of the conventional CK-MB assay. They additionally showed a specificity of 93.9%. The ratio of MB2 to MB1 > 1.5 is indicative of myocardial cell damage. The MB2 and MB2/MB1 ratio increase within 2 hours after the onset of chest pain, and peaks by 4-6 hours, but the sensitivity of the ratio increase with the time interval passed between the onset of symptoms. The sensitivity being 8% at 2 hours, 56% at 4 hours and up to 96% at 6 hours.16,17 However, many false positive results have been observed in patients with urinary tract infections, cholecystitis, pulmonary oedema, congestive heart failure, urosepsis and many types of muscle diseases.9 Although skeletal muscle damage may result in the increase of MB2/MB1 ratio, the CK-MB index should be less than 4.0.18 MB isoform have better sensitivity and specificity within 6 hours of infarct. Furthermore, increased MB2/MB1 ratio has been suggested to be associated with acute rejection in cardiac transplant patients, and also the ratio increased before histological changes of rejection were seen on biopsy.19

Lactate dehydrogenase (LDH)

LD (EC1.1.1.27) is responsible for the interconversion of pyruvate and lactate during glycolysis. It is localized in the cytoplasm, and the highest activities are found in skeletal muscle, liver, heart, kidney and red blood cells. LD is a tetramer composed of two subunits (M or muscle and H or Heart). The two subunits give rise to five Isoenzymes : LD-1 (H4), LD-2 (H3M), LD-3 (H2M2), LD-4 (HM3), LD-5 (M4).7,21 LD-1 is the predominant form in heart but it also found in red blood cells and kidney. LD-2 is also abundant in heart whereas LD-5 is present predominantly in skeletal muscle. Total LD is usually measured from serum as enzymatic activity.7 The use for the diagnosis of acute MI is limited and its use for the diagnosis of “late” (> 24-48 hours) MI has been replaced by cardiac troponins.

Myoglobin

Myoglobin is a 17.8 kDa haeme protein present in the cytosol of skeletal and cardiac muscle but not smooth muscles. Because of its small size, Myoglobin is rapidly released from the areas of ischaemic injury (Table 1). It is rapidly removed from circulation, filtered through the glomerular membrane of kidney, and excreted in the urine.22 The early rise of Myoglobin makes it a marker for early detection of acute MI.22,23 However, Myoglobin is also released in other disease states, including post open-heart surgery, skeletal muscle injury, muscular dystrophy, renal failure, shock and trauma.22,24 Based on a small study by Woo,22 the initial rate of Myoglobin release demonstrates good utility for both early detection and early exclusion of acute MI. Because of its low specificity, proper utilization of this cardiac marker should include the establishment of reference ranges with use of serial determinations on serum samples. The sensitivity and specificity is 90.1% and 74% respectively. If the repeat Myoglobin level doubles within 1 to 2 hours after initial value, it is highly specific for acute MI. However, consistency of sensitivity and specificity is lacking due to several factors summarized in Table 2. The lack of specificity of Myoglobin hampers its utility in the diagnosis of acute MI. Carbonic anhydrase isoenzyme III (CA III), a skeletal muscle specific protein, might be able to improve the specificity of myoglobin. CAIII is found to be present in skeletal muscle but not in cardiac muscle.25,26 By measuring the ratio of Myoglobin to CA III, the source of Myoglobin may be ascertained; myoglobin is increased in MI patients, whereas CAIII is not altered following MI.27 A prospective study of 251 patients by Brogan28 showed that the use of Myoglobin to CAIII ratio was significantly more sensitive than CK-MB, yet as specific for the early (within 3 hours after onset of symptoms) diagnosis of acute MI. Therefore, the use of the ratio can increase the specificity of myoglobin in the diagnosis of AMI.

Fatty Acid-Binding Protein

Fatty acid binding protein (FABP) has a role in the uptake, transport, and metabolism of fatty acids within cells. Heart fatty acid binding protein (hFABP) is a cytoplasmic form of this protein that has been studied for its potential as a new marker of AMI. FABP resembles myoglobin with respect to molecular weight (15 kDa), serum concentration changes and appearance in blood, but has a slightly higher specificity (Table 1). Recent work by Ishii et al showed that FABP is a more sensitive and specific marker than Myoglobin for early diagnosis of acute MI within 6 hours, particularly within 3 hours, after the onset of chest pain. It was reported that perioperative myocardial injury can be diagnosed from the release of cardiac marker at 30 minutes after start of reperfusion, and FABP is a more suitable marker than CK-MB or Myoglobin for early assessment of postoperative myocardial infarction.29 Abe30 also suggested that FABP is useful both in early diagnosis of acute MI, and in discrimination of acute MI from skeletal injury.

But, one potential drawback of hFABP as a cardiac marker is that it is not specific to the heart, being found as well as in high levels in skeletal muscle and the kidneys (and to a lesser extent in other tissues).

Glycogen Phosphorylase

Another potential new cardiac marker is glycogen phosphorylase isoenzyme BB (GPBB, 96 kDa) that is the predominant isoenzyme in human myocardium. The enzyme glycogen phosphorylase is involved in the breakdown of glycogen. It is hypothesized that the release of glycogen phosphorylase into the plasma may be due to the increased glycogenolysis during an acute MI. Mair31 showed that glycogen phosphorylase BB was significantly increased, above the normal range, in most of the patients with unstable angina and transient ST-T changes with the average delay of 4.5 hours from the onset of chest pain to admission. It is released early after AMI onset and can be detected by immunoassays. Increased levels of GPBB can be detected in the serum approximately one to four hours after onset of pain, earlier than current cardiac markers such as CK-MB, myoglobin, and troponin T or troponin I are noted. Thus, use of GPBB as a cardiac marker offers the potential of increased sensitivity combined with specificity for cardiac muscle damage.

Plasma Oxidase

Polymorphonuclear leucocyte (PMN) derived oxyradicals have been known to be important in the tissue injury associated with acute MI, and oxidase is released from PMN’s at the onset of myocardial injury. Lasala32 studied 58 patients in which 50 patients with possible acute MI were admitted to cardiac care unit (CCU) for monitoring compared to 8 patients with pneumonia whose leucocyte counts were greater than 15,000. In addition, 12 healthy volunteers served as controls. In preliminary study, the plasma oxidase activity (POA) was found to be diagnostic for acute MI with sensitivity of 95% while the sensitivity of the corresponding CK-MB was only 54%. However, the POA values were also found to be high in pneumonia patients. The mean time from the onset of chest pain to blood sampling was 3.1 hours, and the POA levels remained elevated longer than that of CK-MB.

Troponins

The troponin complex regulates the calcium-dependent interaction of myosin with actin in muscle contraction. It consists of three subunits, TnT, TnI, and troponin C (TnC), which are located on the thin filament of the contracile apparatus. TnT anchors the troponin complex to tropomyosin, TnC binds calcium ions and initiates the contractile response, and TnI inhibits actin-myosin cross-linking as shown in Fig. 1.

Separate genes code for the cardiac muscle, fast skeletal muscle and slow skeletal muscle isoforms of TnT and TnI.33 Thus, cardiac TnT and TnI have unique amino acid sequences that bind to specific monoclonal antibodies.34 On the other hand,identical TnC is expressed in cardiac and slow skeletal muscle in addition to a divergent fast skeletal muscle isoform, which prevents its use in the detection of myocardial injury.34 The regulatory troponin complex does not exist in smooth muscle.33

Troponin T

TnT is the tropomyosin binding subunit located on the thin myofilament of the contractile apparatus. In most patients cTnT release was biphasic.35 Some reports have claimed that cTnT has higher sensitivity and negative predictive value in detecting MI than conventionally measured cardiac enzymes.36-38 In contrast to cTnI, cTnT is not totally cardiac specific.32 It is expressed in regenerating muscle as well as in normal skeletal muscle33 and its level increases, in the absence of evidence of myocardial involvement, in patients with polymyositis.34 cTnT is also elevated in confirmed myocarditis, pericarditis and heart contusion following blunt heart trauma.27,29 Moreover, spurious rises in cTnT concentration have been reported in patients with diverse underlying clinical condition such as polymyositis, rhabdomyolysis, chronic muscle disease, and renal failure.39-46

Katus et al47 introduced in 1989 a specific enzyme-linked immunosorbent assay (ELISA) method using two monoclonal antibodies for the detection of cardiac TnT in serum. In the ‘first-generation’ TnT assay only the capture antibody was completely cardiac-specific. However, the detection antibody was only 78% cardiac-specific.48 This assay had about 1-2% cross-reactivity with skeletal muscle TnT.49 The cross-reactivity was found to be immunologic and resulting from unspecific absorption of purified skeletal TnT to the test tubes.49 The unspecific signal-antibody then detected these molecules. Thus, the ‘first-generation’ test could give false-positive results also in patients with severe skeletal muscle injury. The first assays of ‘premarket generation’ had cut-off values as high as 0.5 µg/l.49 The cut-off values for the actual ‘first-generation’ TnT assays were 0.2 µg/l in the earliest studies and 0.1 µg/l in subsequent studies.50

Troponin I

c TnI is a smaller protein with molecular weight of 22.5 kDa. High troponin concentrations persist for at least 5 days, despite its biological half-life of 120 minutes, reflecting a continuing release of this protein from disintegrating myofilaments.51 Cardiac TnI is present in the circulation in three forms: free, as a TnI-TnC complex, and as TnT-TnI-TnC complex.52 Actually, the predominant part of TnI circulates in the form of a complex.53 Furthermore; these three forms circulate in different degrees of proteolytic degradation.52,54 Katrukha et al showed that in necrotic human cardiac tissues and in serum of MI patients, the amino-and carboxy-terminal regions of cardiac TnI are vulnerable to degradation.54 The most stable region was found to be located between amino acid residues 30 and 110, possibly due to its protection by TnC.54 In another study by Shi et al55 the amino terminal region of cardiac TnI molecule was found to be more stable than the carboxyterminal region. These findings are important in explaining the wide variation of values measured with different TnI assays. Some assays has also been reported to be interfered by rheumatoid factor and heterophilic antibodies, which may lead to false increase of TnI.56-58

Human studies have demonstrated the absence of elevated levels of TnI in a variety of clinical conditions such1,9,48-52 as after endurance exercises, skeletal muscle injury, rhabdomyolysis, chronic myopathy, cocaine induced chest pain, hypothyroidism, non-cardiac surgery, and chronic renal failure.

cTnI was proposed to be a gold standard for diagnosis of acute MI. Table 3 and measurement of cTnI would help clarify the diagnosis in patients with concomitant myocardial and skeletal muscle injury.59,60

A small prospective study was performed by Mair61 in order to compare the early sensitivities of myoglobin, CK-MB, Ck-isoform ratios, cTnT and cTnI for acute MI. 37 consecutive patients with acute MI, who were admitted to the CCU within 4 hours after chest pain, were studied. All tested markers had low sensitivity within the 1st 3 hours; and they did not reach 100% sensitivity before 6 to 10 hours after the onset of chest pain. Actually the choice of cardiac marker should be based on the specificity of markers. Subsequently, Pervaiz55 compared cTnI and CK-MB in the diagnosis of AMI in a larger study of 291 patients. They demonstrated that cTnI has higher sensitivity, specificity as well as a wider diagnostic window than CK-MB for diagnosis of acute MI.

Javier de Castro Martinez62 performed a prospective study of 64 patients undergoing CABG. They successfully showed that cTnI had the highest sensitivity of 90.9% and CK-MB had the lowest sensitivity of 72.2% respectively. Hence the authors concluded that cardiac TnI elevation appeared to be an early specific marker for the diagnosis of perioperative MI after CABG.

Kost63 performed a prospective study of 97 patients to compare the use of CK-MB mass, CK-MB isoforms, myoglobin, cTnT and cTnI in the diagnosis of AMI. They successfully showed that cTnI had the highest specificity (100%) and the highest positive predictive value (100%). cTnI, cTnT and CK-MB had the highest sensitivity (90%) and the negative predictive values of cTnI, cTnT and CK-MB were 97.8%, 97.6% and 97.6% respectively. CK-MB isoforms had the lowest sensitivity (70%) and myoglobin had the lowest specificity (75%). The author favoured cTnI due to its high positive predictive value and specificity.

S Ben Ayed64 performed a prospective study of 11 patients, to assess the specificity of biochemical markers, including cTnT and cTnI, during abdominal aortic surgery in a control group of patients without postoperative myocardial infarction. They demonstrated that specificity of myoglobin, CK-MB, cTnI and cTnT was 93%, 92%, 100% and 100% respectively. These authors favoured cTnT and cTnI as highly specific markers for the exclusion of post operative MI in comparison to CK-MB.

Neumayr65 performed a retrospective study of 110 patients admitted to the emergency department for the evaluation of a traumatic chest pain. They demonstrated that cTnI alone had a sensitivity of 90.2%, a specificity of 95.7%, a positive predictive value of 92.5% and a negative predictive value of 94.3%. The combination of CK-Mb and cTnI provided much higher sensitivity (100%) and a much higher negative predictive value (100%), which is particularly important to exclude the diagnosis of acute MI. All these prove that troponins are highly cardiospecific markers.

Troponin T versus I

A few studies have compared troponin T and I directly to each other. Their sensitivities in the diagnosis of myocardial infarction and minor myocardial damage are clinically equal. Troponin I appears to be more specific than T.66 Both give equal prognostic information.68-71 The choice of which marker to be used is institution specific. Emergency physicians should feel confident in the support provided to risk stratification from either assay, and should focus on clinically important turn-around times instead of which test is run.

TnI is a biochemical marker of myocardial injury with a high degree of cardiospecificity. The diagnostic specificity of TnT is lower than that of TnI (92%). This is due to the re-expression of TnT during regeneration and degenerative changes in skeletal muscles. Individual genes encode troponins, but at the level of RNA transcription, one particular gene can give rise to a variety of isoforms owing to the mechanism of alternative splicing. In the skeletal muscles, 15 isoforms of TnT, 2 isoforms of cTnI and 2 isoforms of TnC have been detected. In the human myocardium, 2 isoforms of TnT and 1 isoforms of TnI have been isolated. A myocardium form of TnC has not been found.

TnI is absent in the myocardium and skeletal muscles during embryogenesis and foetogenesis. The synthesis of TnI in the myocardium begins postnatally. On the other hand, the synthesis of both isoforms of TnT (TnT1 and TnT2) is induced during the foetal development of both the myocardium and the skeletal muscles. In adult skeletal muscles, TnT is either absent or present only in minute quantities during degeneration and regeneration.

The different methods for assessment of TnI represent the major disadvantages of its clinical use. There are a variety of kits manufactured by different manufacturers. They usually use a system of two antibodies targeted against distinct epitopes. Thus, the tests differ in their analytical sensitivity as well as in detection limits.

In comparison to TnI, the diagnostic parameters of TnT are identical worldwide. It has also been possible to set up a database of clinically verified data.

It is interesting to note that in literature, describing the role, usefulness, and limitations of various biochemical markers, there is considerable variation with regards to reports of their sensitivity and specificity. This may be attributed to varying reference ranges adopted and the consequent categorization of normal and abnormal.