(Circulation. 2001;104:e26.)
© 2001 American Heart Association, Inc.
Correspondence |
Extensive Troponin I and T Modification Detected in Serum From Patients With Acute Myocardial Infarction
Bayer Diagnostics, Tarrytown, NY
To the Editor:
A recent article in Circulation1 characterizes the stability of human recombinant cardiac troponin I spiked into normal human serum. The authors incorrectly concluded on the basis of these studies that the accuracy of the Bayer Immuno 1 troponin I method would decrease with time after the onset of an ischemic event, increasing the risk of a false-negative diagnosis in patients with minor myocardial damage and late admittance to the emergency department.
More than 90% of cardiac troponin I in serum is complexed to troponin C, and little or none is found in a free, uncomplexed form.2,3 Moreover, the apparent stability of cardiac troponin I in serum is greatly enhanced by complexation with troponin C, which is due in part to proteolytic shielding.4 Thus, the stability characteristics of free recombinant cardiac troponin I do not mimic those of the troponin I complex as it exists in patient sera and, therefore, they have little clinical relevance. In a recent study, troponin I measurements by the Bayer Immuno 1 method were performed with actual patient samples incubated at room temperature and at 4°C for up to 48 hours.5 Cardiac troponin I recoveries ranged between 96% and 105%, indicating no decline in recovery. The troponin I epitopes recognized by the antibodies used in the Bayer Immuno 1 method are contained on a proteolytic-resistant region between amino acid residues 30 and 110.4 This accounts for the troponin I stability in samples from patients with acute myocardial infarction when measured by the Bayer Immuno I method. Given that the serial patient profiles of troponin I values closely parallel those of creatine kinase-MB (which is totally independent of the in vitro study), it is highly unlikely that the reduction of troponin I concentrations is due to "some modification of troponin I ... (that) alters its detectability by Immuno 1."1
An additional mechanism contributing to the time-dependent quantitative loss of immunoreactive troponin I observed by the authors when spiking recombinant troponin I in vitro may be adsorption of the free troponin I to the container surfaces. Free troponin I has a strong net positive charge, and it has been established that, without appropriate precautions, significant amounts may be lost to the surface of glass tubes.3 Troponin I–troponin C complexes are less prone to this phenomena. For this reason, manufacturers recommend that glass not be used for the long-term storage of troponin I samples.
References
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Labugger R, Organ L, Collier C, et al. Extensive troponin I and T modification detected in serum from patients with acute myocardial infarction. Circulation. 2000; 102: 1221–1226.
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Katrukha A, Bereznikova A, Esakova T, et al. Troponin I is released in bloodstream of patients with acute myocardial infarction not in free form but as complex. 1997; 43: 1379-1-385.
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Wu A, Feng Y, Moore R, et al. Characterization of cardiac troponin subunit release into serum after acute myocardial infraction and comparison of assays for troponin T and I. Clin Chem. 1998; 44: 1198–1208.
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Katrukha A, Bereznikova A, Filatov V, et al. Degradation of cardiac troponin I: implication for reliable immunodetection. Clin Chem. 1998; 44: 2433–2440.
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Zaman A, Spiegeleer S, Gerits M, et al. Analytical and clinical performance of two cardiac troponin I immunoassays. Clin Chem. 1999; 37: 889–897.
Response
Queen’s University, Kingston, Ontario, Canada
The Heart Center, Division of Cardiology, State University Hospital, Copenhagen, Denmark
The focus of our article was investigating posttranslational modifications to cardiac troponins in patients with acute myocardial infarction (AMI) using Western blot–direct serum analysis and not to test the performance of any diagnostic assay in a system including only recombinant cardiac troponin I as an immunoreactive protein. As part of our investigations, we performed stability studies on recombinant troponin I in serum. In Figure 3 of our article, we demonstrated by Western blot–direct serum analysis that free recombinant cardiac troponin I (as intact and recombinant troponin I1-192) remains relatively stable (in terms of degradation) in serum over 48 hours at 37°C, whereas the concentration determined by immunoassay (Bayer Immuno 1) continuously declined over time. This decrease in recombinant cardiac troponin I in serum cannot be attributable to proteolysis of cardiac troponin I in serum, nor to adsorption of the free cardiac troponin I to the container surfaces, because glass tubes were not used at any stage of our experiment. Katrukha et al1 very thoroughly listed possible modifications to cardiac troponin I that can influence immunogenicity and, as a consequence, detectability by a diagnostic assay. The fact that the antibodies used in the immunoassay are within the "proteolytic-resistant region between amino acid residues 30 and 110" is of minor importance, because proteolysis can now be ruled out as cause for the decline in measured concentration over time. Taking into account the fact that there are at least 2 protein kinase C phosphorylation sites2 and 2 cysteine residues that can be oxidized1 within this region, it is indeed possible that "some as yet unidentified modification ... is responsible for altering the immunogenicity of recombinant cardiac troponin I in the serum, influencing its detectability by the Immuno 1 assay."
Complex formation between cardiac troponin I and other troponin subunits affects its 3D structure and has various consequences for the susceptibility of cardiac troponin I to enzymatic and chemical modification.1 It has been proposed that only a small amount of free cardiac troponin I is detectable in the blood of AMI patients, although the proportions of free cardiac troponin I varied among the patients analyzed and the severity of AMI.1 The exact release pattern of the various forms of cardiac troponin I (whether free or complexed) after an ischemic insult and the correlation between the severity of the insult and the released forms are still unknown.3,4 An increased proportion of free cardiac troponin I in patients with less severe AMI,1 together with cardiac troponin I modification in bypass patients5 and preliminary data from a study on angina patients, could put more weight on the importance of free cardiac troponin I being detectable by a diagnostic assay.
References
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Katrukha AG, Bereznikova AV, Esakova TV, et al. Troponin I is released in bloodstream of patients with acute myocardial infarction not in free form but as complex. Clin Chem. 1997; 43: 1379–1385.
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Noland TS Jr, Gao X, Raynor AL, et al. Cardiac troponin I mutants: phosphorylation by protein kinases C and A and regulation of Ca2+-stimulated MgATPase of reconstituted actomyosin S-1. J Biol Chem. 1995; 43: 25445–25454.
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Datta P, Foster K, Dasgupta A. Comparison of immunoreactivity of five human cardiac troponin I assays toward free and complexed forms of the antigen: implications for assay discordance. Clin Chem. 1999; 45: 2266–2269.
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Giuliani I, Bertinchant JP, Granier C, et al. Determination of cardiac troponin I forms in the blood of patients with acute myocardial infarction and patients receiving crystalloid or cold blood cardioplegia. Clin Chem. 1999; 45: 213–222.
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McDonough JL, Labugger R, Pickett W, et al. Cardiac troponin I is modified in the myocardium of bypass patients. Circulation. 2001; 103: 58–64.
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