Extensive Troponin I and T Modification Detected in Serum From Patients With Acute Myocardial Infarction

Abstract

Background-Cardiac troponin I and T (cTnI and cTnT) are specific biochemical serum markers for acute myocardial infarction (AMI). However, cTnI diagnostic assays are plagued by difficulties, resulting in Ն20-fold differences in measured values. These discrepancies may result from the release of the numerous cTnI modification products that are present in ischemic myocardium. The resolution of these discrepancies requires an investigation of the exact forms of cTnI present in the bloodstream of patients after myocardial injury. Methods and Results-A western blot-direct serum analysis protocol was developed that allowed us to detect intact cTnI and a spectrum of up to 11 modified products in the serum from patients with AMI. For the first time, we document both a cTnI degradation pattern and the existence of phosphorylated cTnI in serum. The number and extent of these modifications reflect patterns similar to the time profiles of the routine clinical serum markers of total creatine kinase, creatine kinase-MB, and cTnI (determined by ELISA). Data from in vitro experiments, which were undertaken to study the degradation of human recombinant cTnI and cTnT when spiked in serum, indicate that some modification products present in patient serum existed in the myocardium and that recombinant cTnI alteration dramatically reduces the detectability of cTnI by the Immuno1 assay over time (our assay was unaffected). Key Words: troponin Ⅲ myocardial infarction Ⅲ biological markers Ⅲ diagnosis Ⅲ blotting, western I t is widely accepted that the presence of cardiac troponin I or T (cTnI or cTnT) in blood serum indicates myocardial damage; thus, cTnI and cTnT are considered specific biochemical markers for acute myocardial infarction (AMI). [1][2][3] Despite the widespread use of cTnI and cTnT detection as a diagnostic tool in acute coronary syndromes, problems arise from variations in the sensitivity, selectivity, and specificity of various commercially available diagnostic cTnI immunoassay kits. 4 -7 These differences are due to (1) the lack of mass standardization, 8 -10 (2) the presence of post-translationally modified cTnI in the serum, and (3) variations in antibody cross-reactivities to the various detectable forms of cTnI. 10,11 Although cTnT is thought to be unaffected by such problems, this remains to be proven: thus far, only one manufacturer has marketed a diagnostic cTnT assay. The underlying reason for this controversy is the inability to determine reliably the exact forms and amounts of cTnI and cTnT present in blood. Conclusions-ThisOn the basis of previous findings, some have proposed that only a small amount of free intact cTnI is detectable in blood, with the predominant form being a complex between cTnI and cardiac tropinin C. 11-13 However, post-translational modifications, including selective degradation, covalent complex formation, and phosphorylation of cTnI, occur in the myocardium of ischemic-reperfused rat hearts 14 -16 and human postischemic myocardium. 17,18 In fact, these modification products, and ...

Time-Dependent Degradation Pattern of Cardiac Troponin T in Cases of Death by Burn

“…Wu et al showed the presence of immunoreactive cTnT fragments in the circulation of acute myocardial infarction (AMI) patients (7). Somewhat later, in a study by Labugger et al on troponin I and T degradation in patients with AMI, Western blot data clearly showed the intact protein at 42 kDa, one major (~26 kDa) fragment and two additional minor fragments [~32 kDa] (8). In addition to their presence in serum, cTnT fragments have also been detected in healthy human heart tissue homogenates using the antibodies of the third generation cTnT immunoassay (9,10).…”

Plasma alkaline phosphatase and survival in diabetic patients with acute myocardial infarction

“…A direct interaction at the analytical level (9) could occur. Labugger et al showed that the sera of patients with myocardial infarction has not only intact troponin I, but also a set of modified products and phosphorylated troponin (10). ALP could be involved in dephosphorylation processes involving phosphorylated troponin, with a possible impact in troponin measurements.…”

Myofilament proteins: From cardiac disorders to proteomic changes

“…The N-terminal degradation of cardiac TnI was reported in a tail suspension rat model that simulated microgravity [34]. More interestingly, several groups have reported degradation products of TnI in serum of patients after acute myocardial infarction (AMI) [35][36][37]. Degradation products of TnT were also found both in animal models of ischemia/reperfusion injury [38,39] and in serum of patients with AMI [40,41].…”

“…Protein degradation often involves significant molecular weight (MW) reduction, which can be readily detected by 1-D-gel separation followed by Western blot analysis [31,32,[34][35][36][37][38][39][40][41]. Comparative 2-DE was also used to study proteomic changes on a broader scale, and PMF can be employed to identify changed proteins [46].…”