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Clinical Chemistry 50: 786-787, 2004; 10.1373/clinchem.2003.025924
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(Clinical Chemistry. 2004;50:786-787.)
© 2004 American Association for Clinical Chemistry, Inc.

Letters to the Editor

Time for Troponin T? Implications from Newly Elucidated Structure

Ravinder Sodi1,a, Simon Darn1 and Anthony Stott1

1 Department of Clinical Chemistry, Royal Liverpool and Broadgreen, University Hospitals, Prescot Street, Liverpool L7 8XP, United Kingdom

aAuthor for correspondence. Fax 44-0151-706-4250; e-mail ravsodi{at}yahoo.com.


To the Editor:

The recent publication of the structure of the core domain of the troponin complex in Nature (1) has prompted us to ask: "What is the cardiac troponin T (cTnT) assay actually measuring?"

Roche Diagnostics Corporation has developed an electrochemiluminescence assay for cTnT (Roche E170), which detects free cTnT and binary/ternary troponin complexes released into the serum after a myocardial infarction (2). This assay uses two antibodies (M7 and M11.7) that recognize two specific epitopes (residues 125–131 and 136–147, respectively). The cTnT protein itself consists of 288 amino acids. From the study by Takeda et al. (1), we have been able to ascertain that the amino acid sequences of these epitopes are DRIERRR and EQQRIRNEREKE, respectively, and found that these epitopes are located on the N-terminal "tail" region (TnT1) of cTnT. Unfortunately, the exact conformations of these epitopes remain to be established.

Recently, it has been observed that hemolysis causes interference in the cTnT assay (3). We note that it is possible that when a sample is hemolyzed, intracellular proteases such as cathepsins may be released into the plasma from erythrocytes (4). Furthermore, Roche has recently issued a product alert (March 2002) informing users about the potential interference of hemolysis in the cTnT assay. Whether this interference in the assay is attributable to hemolysis per se or to the released proteases degrading the troponins is not yet known.

The TnT1 region has a high content of {alpha}-helices, rendering it resistant to proteolysis (5). However, residues 183–200, which form a "flexible linker" between TnT1 and the "IT arm" in the core domain of the troponin complex, are less conserved and highly susceptible to proteolysis (6). The study by Takeda et al. (1) indicates that the epitopes are located in the TnT1 region of the protein. We suggest that if the flexible linkers are proteolytically degraded (during hemolysis), it is possible that the fragments formed may not be recognized and detected by the Roche cTnT assay. This could theoretically produce a false-negative result for cTnT in the presence of a myocardial infarction and might explain the decrease in cTnT concentrations in hemolyzed samples.

There are 10 known isoforms of troponin T that are generated by alternative splicing of the TNT2 gene. Isoform 6 (cTnT3) is expressed in the healthy adult heart, whereas isoform 7 (cTnT4) is expressed in the failing adult heart (7). The only difference in amino acid sequence between these two forms is the inclusion of a five-residue peptide (amino acids 15–19) in isoform 6, which is also located on TnT1 (8). This five-amino acid peptide is highly acidic, and its inclusion in the TnT1 structure could add an overall negative charge to the cTnT complex (7). This could in turn increase the likelihood of false-negative results caused by structural changes in the antibody binding sites and proteolytic degradation such that the TnT1 portion is not detected by the assay.

In the light of the work by Takeda et al. (1), we believe it is important to establish exactly what the assay is detecting and how it is being affected. The effect of in vitro proteolysis on cTnT should also be examined. We stress that it is not acceptable to have false-negative results in an assay that is used for critical decision-making. This should reinvigorate research in this area.


References

  1. 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.[CrossRef][Medline] [Order article via Infotrieve]
  2. Wu AHB, Feng Y-J, Moore R, Apple FS, McPherson PH, Buechler KF, et al. Characterization of cardiac troponin subunit release into serum after acute myocardial infarction and comparison of assays for troponin T and I. Clin Chem 1998;44:1198-1208.[Abstract/Free Full Text]
  3. Hammett-Stabler CA, Snyder JA, Chapman JF, Rogers MW, King MS, Phillips JC. Hemolysis interferes with troponin I and troponin T immunoassays [Abstract]. Clin Chem 2003;49:A89.
  4. Yonezawa S, Nakamura K. Species-specific distribution of cathepsin E in mammalian blood cells. Biochim Biophys Acta 1991;23:155-160.[CrossRef]
  5. White SP, Cohen C, Phillips GN, Jr. Structure of co-crystals of tropomyosin and troponin. Nature 1987;325:826-828.[CrossRef][Medline] [Order article via Infotrieve]
  6. Morris EP, Lehrer SS. Troponin-tropomyosin interactions. Fluorescence studies of the binding of troponin, troponin T, and chymotryptic troponin T fragments to specifically labeled tropomyosin. Biochemistry 1984;23:2214-2220.[CrossRef][Medline] [Order article via Infotrieve]
  7. Anderson PAW, Greig A, Mark TM, Malouf NN, Oakeley AE, Ungerleider RM, et al. Molecular basis of human cardiac troponin T isoforms expressed in the developing, adult, and failing heart. Circ Res 1995;76:681-686.[Abstract/Free Full Text]
  8. Research Collaboratory for Structural Bioinformatics. The protein data bank. PDB codes 1J1E and 1J1D. http://www.rcsb.org/pdb (Accessed July 2003)..



The following articles in journals at HighWire Press have cited this article:


Home page
 Clin. Chem.Home page
M. N. Fahie-Wilson, D. J. Carmichael, M. P. Delaney, P. E. Stevens, E. M. Hall, and E. J. Lamb
Cardiac Troponin T Circulates in the Free, Intact Form in Patients with Kidney Failure
Clin. Chem., March 1, 2006; 52(3): 414 - 420.
[Abstract] [Full Text] [PDF]

This Article
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