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Troponin: a biomarker for the detection of cardiac necrosis

Introduction

Necrosis, arising from the Greek word neckros for ‘corpse’, refers to irreversible cell death occurring in a specific tissue or organ. It is primarily characterized by cellular swelling, or oncosis, followed by loss of membrane integrity and subsequent rupture of cellular contents into the cellular interstitial space [18]. It is a phenomenon that has vast pathological implications; significant necrotic insult can ultimately result in tissue or organ dysfunction and even death.
Figure 1: Cardaic Necrosis


In clinical practice, leakage of intracellular proteins from necrotic cells into the circulation provides a means of detecting tissue-specific necrosis. Such irreversible cell injury and its extent, is reflected by increased blood or serum samples of such proteins that are unique to that tissue.


One example of a protein that can be used as a biomarker for necrosis is cardiac troponin. Released from necrotic cardiac myocytes, its presence in the blood as a specific isoform unique to cardiac muscle enables diagnosis of myocardial infarction [3].



Background

Figure 2: Cardaic Sarcomere
Troponin (Tn) is a protein complex essential for calcium regulation of skeletal and cardiac muscle contraction [9]. It is located on the actin filament of a muscle sarcomere. Muscle contraction occurs when calcium binds to troponin. This causes a conformational change in the troponin complex, moving aside tropomyosin, another regulatory protein which in the relaxed state covers up actin’s myosin-binding sites. Exposure of actin’s myosin-binding sites thereby facilitates the binding of myosin to actin, and thus the generation of force [14].


Troponin consists of three globular protein subunits, TnT, TnC and TnI, each of which perform a specific function in the regulation of muscle contraction [15]. Both TnT and TnI each have distinct isoforms that are unique for cardiac and skeletal muscle. This difference arises from the nature of their amino-acid sequence. The isoforms of TnC however, do not discriminate between cardiac and skeletal muscle [6].



Z Troponin Table.JPG

Links: OMIM - TnI OMIM - TnT OMIM - TnC



Troponin as a biomarker

In 2000, the American College of Cardiology and the European Society of Cardiology issued a joint statement declaring cardiac troponin as the "preferred biomarker for diagnosis of a myocardial infarction" [10, 16]. Its detection in the blood following cardiac injury is of marked clinical value as cardiovascular disease is a major cause of morbidity and mortality in the western world [13].


A myocardial infarction, commonly known as a heart attack, reflects necrosis of cardiac myocytes caused by prolonged ischemia [16]. Following the loss of membrane integrity after the myocyte dies, its intracellular contents, including troponin, are released into the cardiac interstitium, before eventually entering the circulation. Such is the rationale upon which detection of troponin in the blood can be used as a biomarker for cardiac myocyte injury and thus the diagnosis of a myocardial infarction [16].


Troponin is regarded as the preferred biomarker for cardiac necrosis due to its high tissue-specificity and sensitivity [3,16]. This makes it superior to other biomarkers which include creatine kinase, lactate dehydrogenase and myoglobulin [3]. Accounting for its almost absolute myocardial tissue-specificity is the fact that cardiac muscle contains isoforms of TnI and TnT unique to it only. Either cardiac TnI or TnT can be used to detect cardiac necrosis (clinical studies have shown that they equally effective) [17] but not TnC as no cardiac-specific isoforms of it exist. Cardiac troponin is additionally very sensitive as a biomarker; it is able to detect even microscopic zones of cardiac necrosis [1]. This sensitivity may be related to its high abundance in cardiac myocytes, as the primary function of the heart is contraction and troponin is essential in mediating contraction. By being an abundant protein in the cell follows that it may be more likely to be detected in the blood.


In addition to the diagnosis of myocardial infarction, measurement of cardiac troponin in the blood also aids in its prognosis, including the patient’s risk for future cardiac events as well as predicting the extent of myocardial damage [2]. Higher levels are associated with both increased myocardial damage and risk for future cardiac events. As levels in a healthy person are negligible, an increase is easily detected. The degree of elevation also guides the type of treatment necessary [3].


Cardiac troponin can be detected in the blood within 4-6 hours following the onset of chest pain. A peak is reached at approximately 12 hours before beginning to decrease. Levels however can remain elevated for up to 144 hours after the onset of any symptoms [3,9]. This is because release of troponin firstly requires degradation of the structural elements it is bound to within the myofibril [9]. Moreover, in addition to bound troponin subunits, there are also free cytoplasmic components, estimated to be 6–8% for TnT and 3–4% for TnI. It is thought that release of the cytoplasmic troponin accounts for the initial increase of troponin in the blood [17]. This length of time over which troponin can be detected in the blood establishes a large ‘window’ for the diagnosis of myocardial infarction and makes it another reason for being the preferred biomarker. Other available biomarkers do not persist in the blood for up to 6 days like troponin [7].



Testing for Troponin

Figure 3: Testing for Troponin

To diagnose myocardial infarction, a sample of the patient’s blood is obtained and by an immunoassay is used to test for the presence of cardiac troponin (either TnI or TnT) [9]. (Note that diagnosis is made in conjunction with clinical evidence, such as symptoms eg chest pain, or electrocardiogram findings, that the cause of myocardial damage is ischemia [16].)


Two types of cardiac troponin tests exist:

  • a traditional quantitative immunoassay that provides an actual measurement of troponin and takes 45-90 minutes for the result to be obtained;
  • a newer, more rapid qualitative ‘bed-side’ immunoassay that exists in the form of a hand-held device. The test, which takes 15-20 minutes, reports the result as positive or negative and is used commonly in emergency rooms where rapid patient care decisions can be made based on the presence or absence of troponins [3].


The principles of the latter test are provided below in the case of detection of cardiac TnT [5, 6]:

  • a sample of the patient’s blood is put into the well in the device
  • red blood cells are separated from plasma
  • as the antigen, cardiac TnT in the plasma combines with biotinylated anti-troponinT antibodies and gold-labeled anti–cardiac troponinT antibodies
  • the biotin adheres to streptavidin, which is immobilised in a line across the “read” zone of the device
  • if cardiac TnT is present the gold particles produce a redish- purple line


A similar test exists for detection of TnI which also utilizes an immunochromatographic assay with two monoclonal antibodies that are able to recognize two different epitopes on the TnI molecule [4].



Limitations

Whilst cardiac troponin is currently the most specific and sensitive biomarker for cardiac necrosis and the ‘gold standard’ [5] for diagnosis of acute myocardial infarction, it does have some limitations as a biomarker. Firstly, although its presence in the blood indicates cardiac necrosis, it does not indicate the mechanism responsible for the necrosis [3].


Secondly, it is not an early biomarker of cardiac necrosis. It takes approximately 6 hours before it can be detected in the blood so diagnosis before this time is not possible and this does not make it suited to diagnosis in emergency situations [7]. On the other extreme, troponin has a long plasma half life. As its concentration can stay raised for up to 6 days after a myocardial infarction, it is limited for detection of re-infarction during this time [7].


Despite troponin’s specificity for cardiac myocyte injury, sensitive assays of troponin elevation can detect other forms myocyte injury unrelated to myocardial infarction, such as damaged sustained to cardiac myocytes following heart surgery [11] or toxicity from drugs [5, 12]. In this respect it is limited by the fact that its detection is not always synonymous with a myocardial infarction [2].


A final limitation of cardiac troponin testing is that there is currently no standardization of cardiac troponin assays with different commercial assays giving numerically different results. This issue arises from the fact that the detection limits of these assays are unable to characterize normal cardiac troponin levels in healthy controls, as well as different assays using different antibodies targeting other sites on the troponin protein [3,17]. For comparability and to avoid false-positives, the joint committee of the American College of Cardiology and the European Society of Cardiology recommended the following for the diagnosis of myocardial infarction: ‘an increased value for cardiac troponin should be defined as a measurement exceeding the 99th percentile of a reference control group. Reference values must be determined in each laboratory by studies using specific assays with appropriate quality control, as reported in peer-reviewed journals’ [16]. They also recommended that assays have a ‘coefficient of variation of 10% or less’ at this cut-off value [16]. Current research efforts are now driven towards standardization of cardiac troponin assays.



Conclusion

Cardiac troponin is a highly sensitive and specific biomarker of cardiac necrosis. An abnormal blood concentration has clinical significance for cardiac disease; and such a test has ultimately revolutionized the diagnosis of myocardial infarction.



References

1. Apple, F., Falahati, A., Paulsen, P., Miller, E., & Sharkey, S., Improved detection of minor ischemic myocardial injury with measurement of serum cardiac troponin I, Clinical Chemistry 1997; 43(11):2047-2051.PMID: 9365387


2. Apple, F. & Wu, A., Myocardial Infarction Redefined: Role of Cardiac Troponin Testing, Clinical Chemistry 2001; 47:377-379.Review. PMID: 11238285


3. Babuin, L. & Jaffe, A., Troponin: the biomarker of choice for the detection of cardiac injury, Canadian Medical Association Journal 2005; 173(10): 1191-1202.PMID: 16275971


4. Bodor, G., Porter, S., Landt, Y., & Ladenson J., Development of Monoclonal Antibodies for an Assay of Cardiac Troponin-l and Preliminary Results in Suspected Cases of Myocardial Infarction, Clinical Chemistry 1992; 38(11):2203-2214.PMID: 1424112


5. Collinson, P. & Gaze, D., Biomarkers of Cardiovascular Damage and Dysfunction—An Overview, Heart, Lung and Circulation 2007; 16:71-82..PMID: 9365387 PMID: 17618829


6. Coudrey, L., The Troponins, Arch Intern Med 1998; 158:1173-1180.PMID: 9625396


7. Ferguson, J., Beckett, G., Stoddart, M., Walker, S. & Fox K., Myocardial infarction redefined: the new ACC/ESC definition, based on cardiac troponin, increases the apparent incidence of infarction, Heart 2002; 88:343-347.PMID: 12231588


8. Mair, J., Artner-Dworzak, E., Lechleitner, P., Smidt, J., Wagner, I., Dienst, F. & Puschendorf B., Cardiac Troponin T in Diagnosis of Acute Myocardial Infarction, Clinical Chemistry 1991; 37(6):845-852.


9. Melanson, S., Tanasijevic, M., & Jarolim, P., Cardiac Troponin Assays: A View From the Clinical Chemistry Laboratory, Circulation 2007; 116:501-504.PMID: 17967982


10. Mockel, M., Danne, O Schmidta, A., Goldmanna, M., Muller, C., Dietza, R. & Wu, A., Reference values for cardiac troponins I and T in a goal-oriented concept of health: cardiac marker values in a series of outpatients without acute coronary syndromes, Clinica Chimica Acta 2004; 342: 83–86.PMID: 15026267


11. Nesher, N., Alghamdi, A., Singh, S., Sever, J., Christakis, G., Goldman, B., Cohen, G., Moussa, F. & Fremes, S, Troponin after Cardiac Surgery: A Predictor or a Phenomenon? Annals of Thoracic Surgery 2008; 85:1348-1354.


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13. Sanfilippo, M., Hobbs, S., Knuiman, M.,& Hung. J., Impact of New Biomarkers of Myocardial Damage on Trends in Myocardial Infarction Hospital Admission Rates from Population-based Administrative Data, American Journal of Epidemiology 2008, 168(2) 225-233.PMID: 18468989


14. Stanfield, C. & Germann, W., ‘Principles of Human Physiology Third Edition’, Pearson Benjaminn Cummings, US, 2008.


15. Takeda, S., Yamashita, A., Maeda, K. & Maeda, Y., Structure of the core domain of human cardiac troponin in the Ca2+-saturated form, Nature 2003; 424:35-41.PMID: 12840750


16. The Joint European Society of Cardiology/American College of Cardiology Committee, Myocardial Infarction Redefined—A Consensus Document of The Joint European Society of Cardiology/American College of Cardiology Committee for the Redefinition of Myocardial Infarction; European Heart Journal 2000; 21:1502–1513.PMID: 10973764


17. Wu, A., Feng, Y., Moore, R., Apple, F., McPherson, P., Buechler, F., & Bodor, G., Characterization of cardiac troponin subunit release into serum after acute myocardial infarction and comparison of assays for troponin T and I, Clinical Chemistry 1998, 44(6): 1198-1208.PMID: 9625043


18. Zong, W. & Thompson, C., Necrotic death as cell fate; Genes and Development 2006; 20:1-15. Review. PMID: 16391229


Links: OMIM - TnI OMIM - TnT OMIM - TnC Group 8 Project: Necrosis