**6. Biochemical markers of myocardial necrosis**

Myocardial cell death can be documented by the appearance in the blood of different proteins released into the blood circulation from the ischemically injured myocytes: including myoglobin, cardiac troponin T and I, CK, LDH, besides many others (e.g. heart fatty acid binding protein). Myocardial infarction is diagnosed when blood levels of sensitive and specific cardiac biomarkers such as cardiac troponin or CK-MB are elevated in the clinical setting of acute myocardial ischemia [33, 49, 50]. Even though elevated biomarkers reflect myocardial damage and necrosis, they do not designate its underlying mechanism. Hence, an elevated value in the absence of clinical findings of ischemia triggers a need to search for other causes of cardiac damage, for example myocarditis [43, 45, 48].

balance ratio of 1:1. Release of tissue CK-MB2 increases its fraction in plasma; a change in the ratio of CK-MB2:CK-MB1 from 1:1 to 2:1 can be identified using high-voltage gel electrophoresis, even though there is no noticeable change in the plasma concentration of CK-MB [56, 57]. Significant fluctuations in the ratio of both the isoforms in plasma can be detected between 2 and 4 h after myocardial injury. Systematic prospective studies have confirmed that CK-MB isoforms act as an early marker of myocardial injury, and have also established a CK-MB2:CK-MB1 ratio above 1.5:1 as a diagnostic criterion [57–59]. The isoform ratio returns to normal within 18–30 h after injury. It has been proposed that a normal 1:1 isoform ratio in a sample collected at least 6 h after an event effectively excludes a diagnosis of myocardial infarction. The rapid return to normal values makes the CK-MB isoforms the best available laboratory investigation for the confirmation of re-infarction. Unfortunately, the analytical procedure used (high-voltage gel electrophoresis) requires specially designed equipment and a great deal of technical expertise, and is therefore unfeasible for daily/routine use. CK-MB is a sensitive as well as specific marker for myocardial infarction. CK-MB usually becomes abnormal 3–4 h after an event of myocardial infarction, peaks in 10–24 h, and returns to nor-

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Besides, skeletal muscle contains trace amounts of CK-MB, so an elevated serum CK-MB may be observed in people with severe skeletal muscle damage and/or renal failure. In such cases, the CK index that is CK-MB divided by total CK is very useful. If the index is lower than 4%,

The troponins are regulatory proteins found in both cardiac and skeletal muscles. They have 3 subunits; troponin I (TnI), troponin T (TnT), and troponin C (TnC). The genes that code for the skeletal and cardiac isoforms of troponin C (TnC) are similar. The skeletal and cardiac subforms for troponin I (TnI) and troponin T (TnT) are distinct, and immunoassays have been developed to distinguish subtypes [63, 64]. Skeletal TnI and TnT are structurally diverse. No cross-reactiv-

Troponin is adhered to the protein tropomyosin and structurally lies within the groove between actin filaments in muscular tissue. In a relaxed muscle, tropomyosin blocks the site of attachment for the myosin cross-bridge thereby consequently preventing contraction. When the muscle cell is triggered to contract by an action potential, calcium channels get open in the sarcoplasmic reticulum hence releasing calcium into the sarcoplasm. A portion of this calcium gets attach to troponin resulting in conformational change that displaces tropomyosin so that

Troponin can originate from both skeletal and cardiac muscles, but the specific forms of troponin vary between types of muscle. The main difference is that the TnC in skeletal muscle has four binding sites for calcium ion, whereas in cardiac muscle there are only three. The process of contraction in both cardiac and skeletal muscles is controlled by variation in the intracellular calcium concentration. When calcium level rises the muscles contract, and when

ity arises between skeletal and cardiac TnI and TnT with the current assays [63, 64].

the cross bridges can attach to actin and ensue muscle contraction [63, 64].

calcium drops the muscles relax. Smooth muscle does not contain troponin [65].

a non-myocardial etiology of a high CK-MB should be suspected [60–62].

mal within 72 h [60–62].

**6.2. Troponin T**

#### **6.1. Creatine kinase and CK-MB isoenzyme**

Creatine kinase is a regulator of high-energy phosphate production, that is utilized in contractile tissues [51]. In addition it also has a more general role in shuttling high-energy phosphate bonds via creatine phosphate from the site of ATP production in the mitochondria to the site of utilization within the cytoplasm [51].

Cytoplasmic CK is a dimer, composed of both M and/or B subunits, that produce CK-MM, CK-MB and CK-BB iso-enzymes. CK has also a dimeric mitochondrial form consisting of both sarcomeric and non-sarcomeric subunits [52]. Mitochondrial CK is unstable in human serum, and that's why it is difficult to measure. CK-MM is the main isoenzyme found in striated muscle constituting about 97% of the total CK. CK-MB is found principally in cardiac muscle comprising approximately 15–40% of the total CK activity, with the remainder being CK-BB. CK-BB is the predominant iso-enzyme found in brain, intestinal and urinary systems. The skeletal muscle CK-MB produce 2–3% of the total CK activity; the patients with skeletal muscle injury may have increased CK and CK-MB levels [53].

The antibodies in turn inhibit M-subunit activity, with remaining enzyme activity being derived from B-subunits only; CK-BB is not detectable by activity measurement in serum, except the patient has suffered a serious cerebrovascular accident, so the residual activity represents CK-MB activity. Although antibodies had been developed to the B- and M-subunits of CK, it was thought that MB did not have its own unique antigenicity. However, specific antibodies were developed in the mid- 1980s, allowing the development of direct immunological assays for CK-MB. Serum total CK activity and CK-MB concentration rise simultaneously following myocardial injury [54, 55].

For CK-MB, two forms of the MB iso-enzyme were eventually recognized and isolated from plasma; the tissue form is designated CK-MB2; removal of the lysine residue from the carboxy terminus of the single M-subunit, catalyzed by the action of carboxypeptidase-N giving rise to the CK-MB1 isoform. Elimination of the lysine residue, which is positively charged, leaves a more negatively charged isoform thereby leaving a basis for isolation of the isoforms by electrophoresis [56]. The B-subunit is not sensitive to enzymic degradation, so only two isoforms of CK-MB exist. In normal plasma, CK-MB isoforms exist with each other in balance ratio of 1:1. Release of tissue CK-MB2 increases its fraction in plasma; a change in the ratio of CK-MB2:CK-MB1 from 1:1 to 2:1 can be identified using high-voltage gel electrophoresis, even though there is no noticeable change in the plasma concentration of CK-MB [56, 57]. Significant fluctuations in the ratio of both the isoforms in plasma can be detected between 2 and 4 h after myocardial injury. Systematic prospective studies have confirmed that CK-MB isoforms act as an early marker of myocardial injury, and have also established a CK-MB2:CK-MB1 ratio above 1.5:1 as a diagnostic criterion [57–59]. The isoform ratio returns to normal within 18–30 h after injury. It has been proposed that a normal 1:1 isoform ratio in a sample collected at least 6 h after an event effectively excludes a diagnosis of myocardial infarction. The rapid return to normal values makes the CK-MB isoforms the best available laboratory investigation for the confirmation of re-infarction. Unfortunately, the analytical procedure used (high-voltage gel electrophoresis) requires specially designed equipment and a great deal of technical expertise, and is therefore unfeasible for daily/routine use. CK-MB is a sensitive as well as specific marker for myocardial infarction. CK-MB usually becomes abnormal 3–4 h after an event of myocardial infarction, peaks in 10–24 h, and returns to normal within 72 h [60–62].

Besides, skeletal muscle contains trace amounts of CK-MB, so an elevated serum CK-MB may be observed in people with severe skeletal muscle damage and/or renal failure. In such cases, the CK index that is CK-MB divided by total CK is very useful. If the index is lower than 4%, a non-myocardial etiology of a high CK-MB should be suspected [60–62].

#### **6.2. Troponin T**

**6. Biochemical markers of myocardial necrosis**

28 Myocardial Infarction

of cardiac damage, for example myocarditis [43, 45, 48].

muscle injury may have increased CK and CK-MB levels [53].

**6.1. Creatine kinase and CK-MB isoenzyme**

of utilization within the cytoplasm [51].

following myocardial injury [54, 55].

Myocardial cell death can be documented by the appearance in the blood of different proteins released into the blood circulation from the ischemically injured myocytes: including myoglobin, cardiac troponin T and I, CK, LDH, besides many others (e.g. heart fatty acid binding protein). Myocardial infarction is diagnosed when blood levels of sensitive and specific cardiac biomarkers such as cardiac troponin or CK-MB are elevated in the clinical setting of acute myocardial ischemia [33, 49, 50]. Even though elevated biomarkers reflect myocardial damage and necrosis, they do not designate its underlying mechanism. Hence, an elevated value in the absence of clinical findings of ischemia triggers a need to search for other causes

Creatine kinase is a regulator of high-energy phosphate production, that is utilized in contractile tissues [51]. In addition it also has a more general role in shuttling high-energy phosphate bonds via creatine phosphate from the site of ATP production in the mitochondria to the site

Cytoplasmic CK is a dimer, composed of both M and/or B subunits, that produce CK-MM, CK-MB and CK-BB iso-enzymes. CK has also a dimeric mitochondrial form consisting of both sarcomeric and non-sarcomeric subunits [52]. Mitochondrial CK is unstable in human serum, and that's why it is difficult to measure. CK-MM is the main isoenzyme found in striated muscle constituting about 97% of the total CK. CK-MB is found principally in cardiac muscle comprising approximately 15–40% of the total CK activity, with the remainder being CK-BB. CK-BB is the predominant iso-enzyme found in brain, intestinal and urinary systems. The skeletal muscle CK-MB produce 2–3% of the total CK activity; the patients with skeletal

The antibodies in turn inhibit M-subunit activity, with remaining enzyme activity being derived from B-subunits only; CK-BB is not detectable by activity measurement in serum, except the patient has suffered a serious cerebrovascular accident, so the residual activity represents CK-MB activity. Although antibodies had been developed to the B- and M-subunits of CK, it was thought that MB did not have its own unique antigenicity. However, specific antibodies were developed in the mid- 1980s, allowing the development of direct immunological assays for CK-MB. Serum total CK activity and CK-MB concentration rise simultaneously

For CK-MB, two forms of the MB iso-enzyme were eventually recognized and isolated from plasma; the tissue form is designated CK-MB2; removal of the lysine residue from the carboxy terminus of the single M-subunit, catalyzed by the action of carboxypeptidase-N giving rise to the CK-MB1 isoform. Elimination of the lysine residue, which is positively charged, leaves a more negatively charged isoform thereby leaving a basis for isolation of the isoforms by electrophoresis [56]. The B-subunit is not sensitive to enzymic degradation, so only two isoforms of CK-MB exist. In normal plasma, CK-MB isoforms exist with each other in The troponins are regulatory proteins found in both cardiac and skeletal muscles. They have 3 subunits; troponin I (TnI), troponin T (TnT), and troponin C (TnC). The genes that code for the skeletal and cardiac isoforms of troponin C (TnC) are similar. The skeletal and cardiac subforms for troponin I (TnI) and troponin T (TnT) are distinct, and immunoassays have been developed to distinguish subtypes [63, 64]. Skeletal TnI and TnT are structurally diverse. No cross-reactivity arises between skeletal and cardiac TnI and TnT with the current assays [63, 64].

Troponin is adhered to the protein tropomyosin and structurally lies within the groove between actin filaments in muscular tissue. In a relaxed muscle, tropomyosin blocks the site of attachment for the myosin cross-bridge thereby consequently preventing contraction. When the muscle cell is triggered to contract by an action potential, calcium channels get open in the sarcoplasmic reticulum hence releasing calcium into the sarcoplasm. A portion of this calcium gets attach to troponin resulting in conformational change that displaces tropomyosin so that the cross bridges can attach to actin and ensue muscle contraction [63, 64].

Troponin can originate from both skeletal and cardiac muscles, but the specific forms of troponin vary between types of muscle. The main difference is that the TnC in skeletal muscle has four binding sites for calcium ion, whereas in cardiac muscle there are only three. The process of contraction in both cardiac and skeletal muscles is controlled by variation in the intracellular calcium concentration. When calcium level rises the muscles contract, and when calcium drops the muscles relax. Smooth muscle does not contain troponin [65].

Individual subunits play different roles:


Cardiac troponin T (cTnT) is a cardio-specific, highly sensitive marker for myocardial injury. Cardiac troponin T rises approximately 3–4 h after acute myocardial infarction (AMI) and may continue up to 2 weeks thereafter [65, 66]. In comparison to ST-elevation myocardial infarction (STEMI), the diagnosis of non-ST elevation myocardial infarction (NSTEMI) mainly relies upon level of cardiac troponin T [66]. The diagnosis of MI can be made when blood levels of cTnT are above the 99th percentile of the accepted limit along with an evidence of myocardial ischemia [67]. Cardiac troponin T is an independent prognostic marker which can forecast the near-, mid-, and even long- term outcome of events in patients with acute coronary syndrome (ACS). Cardiac troponin T is also ideal marker of myocardial injury in the diagnosis and management of non-ST elevation acute coronary syndromes [43, 68] (**Figure 2**).

#### **6.3. Cardiac troponin I**

Cardiac troponin I is the contractile part and it is only present inside the myocardium [69, 70]. It is a part of the troponin complex (I, T, C) that along with the tropomyosin is bound to actin within the thin myofibril filament. cTnI is acquired as free TnI, as well as intricated with troponin C with troponin T termed as binary IT or with both the troponin C and troponin T where it is called as ternary ITC. Its physiological function is to hinder the ATPase activity of the actin-myosin complex during lack of calcium, and thus, to avert muscular contraction [71].

blood in normal circumstances, therefore elevated serum levels of c TnI are more significant for the diagnosis of myocardial necrosis [79]. Data obtained from recent studies specify that the troponin I concentration can be determined within the first 3–6 h after the onset of chest pain. The levels of Troponin I reach the peak level at approximately 12–16 h and remain elevated for 4–9 days after acute MI. The time to attain the peak concentration of cTnI was found to be more among patients who did not underwent thrombolytic therapy [73, 80, 81].

The Diagnostic Value of Biochemical Cardiac Markers in Acute Myocardial Infarction

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**Figure 2.** The troponin regulatory complex; adopted by Roger Cummin.

Recent studies have found that in patients after AMI the predominant form of cTnI exhibited in blood is the binary troponin IC complex with slight concentrations of ternary ITC complex, binary IT complex and free cTnI [82–85]. The release pattern of these forms in MI is still under investigation. Commercially available laboratory methods can identify complexed and free cTnI subforms [82, 86, 87]. Some of the assays have the same responses to different forms of cTnI. The second type may result in over or under estimation of troponin I concentrations in complex biological settings. The equimolar binding characterized as the ability to determine both the complexed and free cTnI forms uniformly leads to an unbiased estimation of the total cTnI found in the samples from same subject in MI. The Access AccuTnI assay identifies the binary troponin IC or IT or ternary troponin ITC complexes and free cTnI evenly. The assay detects both the phosphorylated and dephosphorylated forms of

The American College of Cardiology (ACC) and the European Society of Cardiology (ESC) guidelines advocate that the different laboratories define their own reference range and also

cTnI complex [88].

Three types of tissue isoforms are found. Fast and slow troponin I (19,800 Da) participating in fast and slow twitch skeletal muscle fibers and cTnI (24,000 Da). All the three isoforms of troponin I are encoded by the different genes. The human cTnI reveals merely 54 and 52% amino acid sequence homology with human slow and fast skeletal troponin I, respectively [72]. cTnI specific monoclonal antibody pair is selected. Moreover it is found that skeletal muscles do not express cTnI, neither during development nor in response to a stimuli [72]. cTnIs can differentiate cardiac and skeletal muscle injuries, and facilitates the diagnosis of MI discrete from the skeletal muscle injuries (e.g. rhabdomyolysis, polytraumatism or from the non-cardiac surgery) [72–75]. Increased troponin I levels are also determined in unstable angina [76] and congestive cardiac failure [77]. In acute MI serum concentrations of both cTnI and CK-MB show similar increase and decrease patterns. It is recommended that at least three blood samples should be collected during the early triage period [78]. In the cardiac muscles the level of cTnI is 13 times more than that of CK-MB. Moreover cTnI does not circulate in the The Diagnostic Value of Biochemical Cardiac Markers in Acute Myocardial Infarction http://dx.doi.org/10.5772/intechopen.76150 31

**Figure 2.** The troponin regulatory complex; adopted by Roger Cummin.

Individual subunits play different roles:

complex

30 Myocardial Infarction

complex in place [66].

syndromes [43, 68] (**Figure 2**).

**6.3. Cardiac troponin I**

muscular contraction [71].

• Troponin C binds to calcium ions to create a conformational change in TnI

• Troponin T binds to tropomyosin, interlocking them to constitute a troponin-tropomyosin

• Troponin I binds to actin in thin myofilaments in order to hold the troponin-tropomyosin

Cardiac troponin T (cTnT) is a cardio-specific, highly sensitive marker for myocardial injury. Cardiac troponin T rises approximately 3–4 h after acute myocardial infarction (AMI) and may continue up to 2 weeks thereafter [65, 66]. In comparison to ST-elevation myocardial infarction (STEMI), the diagnosis of non-ST elevation myocardial infarction (NSTEMI) mainly relies upon level of cardiac troponin T [66]. The diagnosis of MI can be made when blood levels of cTnT are above the 99th percentile of the accepted limit along with an evidence of myocardial ischemia [67]. Cardiac troponin T is an independent prognostic marker which can forecast the near-, mid-, and even long- term outcome of events in patients with acute coronary syndrome (ACS). Cardiac troponin T is also ideal marker of myocardial injury in the diagnosis and management of non-ST elevation acute coronary

Cardiac troponin I is the contractile part and it is only present inside the myocardium [69, 70]. It is a part of the troponin complex (I, T, C) that along with the tropomyosin is bound to actin within the thin myofibril filament. cTnI is acquired as free TnI, as well as intricated with troponin C with troponin T termed as binary IT or with both the troponin C and troponin T where it is called as ternary ITC. Its physiological function is to hinder the ATPase activity of the actin-myosin complex during lack of calcium, and thus, to avert

Three types of tissue isoforms are found. Fast and slow troponin I (19,800 Da) participating in fast and slow twitch skeletal muscle fibers and cTnI (24,000 Da). All the three isoforms of troponin I are encoded by the different genes. The human cTnI reveals merely 54 and 52% amino acid sequence homology with human slow and fast skeletal troponin I, respectively [72]. cTnI specific monoclonal antibody pair is selected. Moreover it is found that skeletal muscles do not express cTnI, neither during development nor in response to a stimuli [72]. cTnIs can differentiate cardiac and skeletal muscle injuries, and facilitates the diagnosis of MI discrete from the skeletal muscle injuries (e.g. rhabdomyolysis, polytraumatism or from the non-cardiac surgery) [72–75]. Increased troponin I levels are also determined in unstable angina [76] and congestive cardiac failure [77]. In acute MI serum concentrations of both cTnI and CK-MB show similar increase and decrease patterns. It is recommended that at least three blood samples should be collected during the early triage period [78]. In the cardiac muscles the level of cTnI is 13 times more than that of CK-MB. Moreover cTnI does not circulate in the blood in normal circumstances, therefore elevated serum levels of c TnI are more significant for the diagnosis of myocardial necrosis [79]. Data obtained from recent studies specify that the troponin I concentration can be determined within the first 3–6 h after the onset of chest pain. The levels of Troponin I reach the peak level at approximately 12–16 h and remain elevated for 4–9 days after acute MI. The time to attain the peak concentration of cTnI was found to be more among patients who did not underwent thrombolytic therapy [73, 80, 81].

Recent studies have found that in patients after AMI the predominant form of cTnI exhibited in blood is the binary troponin IC complex with slight concentrations of ternary ITC complex, binary IT complex and free cTnI [82–85]. The release pattern of these forms in MI is still under investigation. Commercially available laboratory methods can identify complexed and free cTnI subforms [82, 86, 87]. Some of the assays have the same responses to different forms of cTnI. The second type may result in over or under estimation of troponin I concentrations in complex biological settings. The equimolar binding characterized as the ability to determine both the complexed and free cTnI forms uniformly leads to an unbiased estimation of the total cTnI found in the samples from same subject in MI. The Access AccuTnI assay identifies the binary troponin IC or IT or ternary troponin ITC complexes and free cTnI evenly. The assay detects both the phosphorylated and dephosphorylated forms of cTnI complex [88].

The American College of Cardiology (ACC) and the European Society of Cardiology (ESC) guidelines advocate that the different laboratories define their own reference range and also an elevated level of cTnI be identified as an amount above the 99th percentile of a normal control group, that is, 99th percentile of the upper reference limit [89, 90].

**Author details**

\*, Arif Malik2

\*Address all correspondence to: shazia.rashid9@gmail.com 1 King Abdulaziz Specialist Hospital, Aljouf, Saudi Arabia

3 Shalamar Medical and Dental College, Lahore, Pakistan

Switzerland: World Health Organization; 2003

the Pakistan Medical Association. 2006;**56**(6):282-285

The American Journal of Cardiology 1984;**54**:902-904

and management. Postgraduate Medical Journal. 2002;**78**:27-30

tions for the therapy. Academic Emergency Medicine. 2002;**9**:1029

4 Women Medical College, Abbotabad, Pakistan

, Rukhshan Khurshid<sup>3</sup>

2 Institute of Molecular Biology and Biotechnology, University of Lahore, Pakistan

5 Institute of Basic Medical Sciences, Khyber Medical University Peshawar, Pakistan

[1] World Health Organization. The World Health Report 2003-Shaping the Future. Geneva,

[2] Iqbal P. Hyperhomocysteinemia and coronary artery disease in Pakistan. The Journal of

[3] Lindsay J Jr, Pichard AD. Acute myocardial infarction with normal coronary arteries.

[4] Osulal S, Bell GM, Hornung RS. Acute myocardial infarction in young adults: Causes

[5] Jr GXB. Bench to bedside: Pathophysiology of acute coronary syndromes and implica-

[6] Juneau M, Roy N, Nigam A, Tardif JC, Larivee L. Exercise above the ischemic threshold and serum markers of myocardial injury. The Canadian Journal of Cardiology.

[7] Karras DJ, Kane DL. Serum markers in the emergency department diagnosis of acute myocardial infarction. Emergency Medicine Clinics of North America. 2001;**19**(2):321-337

[8] Grech ED, Ramdale DR. Clinical review; ABC of interventional cardiology. Acute coronary syndrome: Unstable angina and non-ST segment elevation myocardial infarction.

[9] Davies MJ, Thomas AC. Plaque fissuring—The cause of acute myocardial infarction, sudden ischaemic death, and crescendo angina. British Heart Journal. 1985;**53**(4):363-373

[10] Hort W. Pathology of acute myocardial infarction and the infarct vessel. European Heart

, Uzma Faryal<sup>4</sup>

The Diagnostic Value of Biochemical Cardiac Markers in Acute Myocardial Infarction

and Sumera Qazi<sup>5</sup>

http://dx.doi.org/10.5772/intechopen.76150

33

Shazia Rashid<sup>1</sup>

**References**

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Conversely in patients with unstable angina and acute MI without the evidence of ST segment elevation (NSTEMI) the expectation of suffering from an adverse event is reported to be quite difficult. The advancement as well as commercialization of more specific and more sensitive cardiac troponin I (cTnI) immunoassays have considerably added to the accurate diagnosis of MI and to the risk stratification of NSTEMI/UA patients.

The definition of MI was formally redefined in 2000 by the European Society of Cardiology and the American College of Cardiology to realign evidence of myocardial injury as defined by biomarkers with a particular advocacy on troponin [32]. The 2000/2002 American College of Cardiology (ACC) and the American Heart Association (AHA) Guideline Update evocatively advocate to incorporate the estimation of cTnI for the management of AMI patients and also for the risk stratification of patients presenting with symptoms suggestive of acute coronary syndromes [40, 91]. This definition was updated in 2007 [43] to reflect the progress that had been made in understanding assays. It again relied heavily on a definition based on troponin. Specifically, with rare exception, the diagnosis cannot be made in the absence of elevated biomarkers of cardiac injury [43, 68].

Considering the potential adverse outcomes the estimation of the prognosis should aid clinicians in identification and management of high risk patients. Eventually the evaluation of the prognosis will be helpful in both the identification of site of care as well as in distinguishing patients most likely to get benefit from specific therapeutic interventions.
