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Background: Current studies suggest that cardiac troponin (cTn) forms in the circulation may vary in different clinical scenarios. Our aim was to design a combination of cTn assays specific to the main cTn forms and to evaluate their analytical performance., Methods: We developed immunoassays specific for measuring (1) long-cTnT cTnI-cTnT-TnC (ITC) ternary complex, with cTnT in long form without cleavage at the C-terminal amino acids residue 189-223, designated "long-cTnT ITC complex assay;" (2) both the long-cTnT ITC complex plus short-cTnT ITC complex, designated "hs-total ITC complex assay;" (3) the central part of cTnT of both the long-cTnT ITC complex and free cTnT, designated "hs-cTnT assay." Sex-specific 99th percentile upper reference limits (URLs) were determined. High-sensitivity performance was assessed by examining the imprecision and detectable results above limit of detection (LoD) in the healthy population., Results: Both complex immunoassays exhibited excellent analytical sensitivity, precision, and specificity. The 99th percentile URLs were as follows: long-cTnT ITC complex: male 0.90 ng/L, female 0.87 ng/L; hs-total ITC complex: male 16.15 ng/L, female 10.08 ng/L; hs-cTnT: male 15.57 ng/L, female 14.28 ng/L. The total imprecision at or below the sex-specific 99th percentile URLs was <5% for all assays. The hs-total ITC complex and the hs-cTnT assays showed >50% of measurable concentrations above the LoD. However, <20% were measurable for the long-cTnT ITC complex assay., Conclusions: The cTn assays detected concentrations of major cTn forms in the circulation with high sensitivity, precision, and specificity, supporting their use for monitoring cTn complex and fragmentation forms during myocardial injuries., (© Association for Diagnostics & Laboratory Medicine 2024. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

Immunodetection of cardiac isoforms of troponin I (cTnI) and troponin T (cTnT) in blood samples is widely used for the diagnosis of acute myocardial infarction. The cardiac troponin complex (ITC-complex), comprising cTnI, cTnT, and troponin C (TnC), makes up a large portion of troponins released into the bloodstream after the necrosis of cardiomyocytes. However, the stability of the ITC-complex has not been fully investigated. This study aimed to investigate the stability of the ITC-complex in blood samples. A native ITC-complex was incubated in buffer solutions, serum, and citrate, heparin, or EDTA plasma at various temperatures. Western blotting and gel filtration were performed, and troponins were detected using specific monoclonal antibodies. The ITC-complex dissociated at 37 °C in buffers with or without anticoagulants, in citrate, heparin, and EDTA plasmas, and in serum, into a binary cTnI-TnC complex (IC-complex) and free cTnT. In plasma containing heparin and EDTA, the IC-complex further dissociated into free TnC and cTnI. No dissociation was found at 4 °C or at room temperature (RT) in all matrices within 24 h except for EDTA plasma. After incubation at 37 °C in EDTA plasma and serum, dissociation was accompanied by proteolytic degradation of both cTnI and cTnT. The presence of anti-troponin autoantibodies in the sample impeded dissociation of the ITC-complex. The ITC-complex dissociates in vitro to form the IC-complex and free cTnT at 37 °C but is mostly stable at 4 °C or RT. Further dissociation of the IC-complex occurs at 37 °C in plasmas containing heparin and EDTA.

Background: Troponin composition characterization has been implicated as a next step to differentiate among non-ST elevation myocardial infarction (NSTEMI) patients and improve distinction from other conditions with troponin release. We therefore studied coronary and peripheral troponin compositions in relation to clinical variables of NSTEMI patients., Methods: Samples were obtained from the great cardiac vein (GCV), coronary sinus (CS), and peripheral circulation of 45 patients with NSTEMI. We measured total cTnI concentrations, and assessed both complex cTnI (binary cTnIC + all ternary cTnTIC forms), and large-size cTnTIC (full-size and partially truncated cTnTIC). Troponin compositions were studied in relation to culprit vessel localization (left anterior descending artery [LAD] or non-LAD), ischemic time window, and peak CK-MB value., Results: Sampling occurred at a median of 25 hours after symptom onset. Of total peripheral cTnI, a median of 87[78-100]% consisted of complex cTnI; and 9[6-15]% was large-size cTnTIC. All concentrations (total, complex cTnI, and large-size cTnTIC) were significantly higher in the CS than in peripheral samples (P < 0.001). For LAD culprit patients, GCV concentrations were all significantly higher; in non-LAD culprit patients, CS concentrations were higher. Proportionally, more large-size cTnTIC was present in the earliest sampled patients and in those with the highest CK-MB peaks., Conclusions: In coronary veins draining the infarct area, concentrations of both full-size and degraded troponin were higher than in the peripheral circulation. This finding, and the observed associations of troponin composition with the ischemic time window and the extent of sustained injury may contribute to future characterization of different disease states among NSTEMI patients., (© American Association for Clinical Chemistry 2020.)

Background Cardiac troponin T ( cTnT ) is seen in many other conditions besides myocardial infarction, and recent studies demonstrated distinct forms of cTnT . At present, the in vivo formation of these different cTnT forms is incompletely understood. We therefore performed a study on the composition of cTnT during the course of myocardial infarction, including coronary venous system sampling, close to its site of release. Methods and Results Baseline samples were obtained from multiple coronary venous system locations, and a peripheral artery and vein in 71 non- ST -segment-elevation myocardial infarction patients. Additionally, peripheral blood was drawn at 6- and 12-hours postcatheterization. cTnT concentrations were measured using the high-sensitivity- cTnT immunoassay. The cTnT composition was determined via gel filtration chromatography and Western blotting in an early and late presenting patient. High-sensitivity - cTnT concentrations were 28% higher in the coronary venous system than peripherally (n=71, P<0.001). Coronary venous system samples demonstrated cT n T-I-C complex, free intact cTnT , and 29 kD a and 15 to 18 kD a cTnT fragments, all in higher concentrations than in simultaneously obtained peripheral samples. While cT n T-I-C complex proportionally decreased, and disappeared over time, 15 to 18 kD a cTnT fragments increased. Moreover, cT n T-I-C complex was more prominent in the early than in the late presenting patient. Conclusions This explorative study in non- ST -segment-elevation myocardial infarction shows that cTnT is released from cardiomyocytes as a combination of cT n T-I-C complex, free intact cTnT , and multiple cTnT fragments indicating intracellular cTnT degradation. Over time, the cT n T-I-C complex disappeared because of in vivo degradation. These insights might serve as a stepping stone toward a high-sensitivity- cTnT immunoassay more specific for myocardial infarction.

Background: Increased cardiac troponin T (cTnT) and C-reactive protein (CRP) levels predict mortality in patients with end-stage renal disease (ESRD) on maintenance hemodialysis. Obstructive sleep apnea (OSA) is associated with severe cardiac stress and systemic inflammation. We hypothesized that in patients with ESRD elevated levels of cTnT and CRP are consequences of unrecognized OSA., Methods: After diagnostic polysomnography, serum levels of cTnT and CRP were assessed in two groups of patients. The first group with normal renal function which served as a control group, was recruited from routine patient referrals to the sleep laboratory at the University Hospital Magdeburg. The second group consisted of patients with ESRD on thrice-weekly maintenance hemodialysis treatment (hemodialysis group)., Results: After screening, 15 patients in each group were eligible for inclusion. OSA (apnea hypopnea index (AHI) > or = 10) was associated with significantly elevated serum CRP levels of 5.1 +/- 4.9 mg/l in the control group and 11.6 +/- 10.2 mg/l in the hemodialysis group, compared with patients in the respective groups without OSA. In the control group, cTnT levels were below the lower detection limit, independent of OSA severity. Patients with ESRD but without OSA had low cTnT levels, similar to those of patients in the control group (0.014 +/- 0.01 ìg/l), whereas patients with ESRD and OSA had significantly elevated serum cTnT levels (0.38 +/- 0.3 microg/l, p < 0.05)., Conclusions: OSA is associated with higher CRP levels in patients with normal or impaired renal function, but cTnT is elevated in OSA patients with impaired renal function only. In this pilot study, both parameters suggest an important role of sleep related breathing disorders on cardiac stress and chronic inflammation.

Background: Elevated concentrations of cardiac biomarkers, such as troponins and natriuretic peptides, have been shown to be predictive of poorer long-term cardiovascular outcomes in stable patients with end-stage renal disease (ESRD). However, little is known about the relationship between elevated concentrations of these cardiac markers and underlying coronary artery pathology in these patients. The aim of the present study was to investigate associations between coronary artery calcification (CAC) and the concentrations of cardiac biomarkers in ESRD patients., Methods: We conducted a cross-sectional study of 38 asymptomatic patients (median age, 54 years; 26 males, 12 females; diabetic, 39%) who were undergoing chronic haemodialysis. In these patients, pre-dialysis circulating concentrations of cardiac troponin T (cTnT), cardiac troponin I (cTnI), creatine kinase-MB (CK-MB) and B-type natriuretic peptide (BNP) were measured. We quantified the level of CAC by multirow spiral computed tomography to obtain a CAC score. CAC scores > or = 400 were defined as being indicative of severe CAC., Results: Severe CAC was detected in 17 patients (45%). The degree of CAC severity was positively associated (P < 0.05) with cTnT concentrations. Thus, 15% of patients had severe CAC in the lowest tertile of cTnT, 50% had severe CAC in the middle third, and 69% in the highest third. Similarly, the degree of severity of CAC was positively associated (P < 0.01) with cTnI concentrations across concentration categories. In contrast, there was no association between the degree of CAC severity and the concentrations of either BNP or CK-MB. A logistic regression analysis revealed that elevated concentrations of cTnT (> or = median vs or = 0.1 ng/ml vs

Background: Cardiac troponins are modified during ischemic injury and are found as a heterogeneous mixture in blood of patients with cardiovascular diseases. We present a strategy to isolate cardiac troponins from human biological material, by use of affinity chromatography, and to provide samples ready for direct analysis by mass spectrometry., Methods: Cardiac troponins were isolated from human left ventricular tissue by affinity chromatography. Isolated troponins were either eluted and analyzed by Western blot or enzymatically digested while bound to affinity beads. The resulting peptide mixture was subjected to mass spectrometry for protein identification and characterization. The same method was used to analyze serum from patients with acute myocardial infarction (AMI)., Results: Affinity chromatography with antibodies specific for one cardiac troponin subunit facilitated the isolation of the entire cardiac troponin complex from myocardial tissue. The three different proteases used for enzymatic digestion increased the total protein amino acid sequence coverage by mass spectrometry for the three cardiac troponin subunits. Combined amino acid sequence coverage for cardiac troponin I, T, and C (cTnI, cTnT, cTnC) was 54%, 48%, and 40%, respectively. To simulate matrix effects on the affinity chromatography-mass spectrometry approach, we diluted tissue homogenate in cardiac troponin-free serum. Sequence coverage in this case was 44%, 41%, and 19%, respectively. Finally, affinity chromatography-mass spectrometry analysis of AMI serum revealed the presence of cardiac troponins in a wide variety of its free and/or complexed subunits, including the binary cTnI-cTnC and cTnI-cTnC-cTnT complexes., Conclusions: Affinity chromatography-mass spectrometry allows the extraction and analysis of cardiac troponins from biological samples in their natural forms. We were, for the first time, able to directly confirm the presence of cardiac troponin complexes in human serum after AMI. This approach could assist in more personalized risk stratification as well as the search for reference materials for cardiac troponin diagnostics.