Thrombin-Mediated Degradation of Human Cardiac Troponin T

AbstractBACKGROUNDCardiac troponin T (cTnT) is an acknowledged biomarker of acute myocardial infarction (AMI) that is known to be prone to proteolytic degradation in serum. Such degradation is usually explained by the action of μ-calpain, although there could be other candidates for that role. In the current study, we explored the hypothesis that thrombin-mediated cTnT cleavage occurs as a result of the serum sample preparation.METHODScTnT degradation was studied by using immunoblotting and mass spectrometry (MS) analysis.RESULTSThe comparison of cTnT isolated from AMI heparin plasma and serum samples showed that cTnT in the plasma samples was mainly present as the full-sized molecule (approximately 35 kDa), while in serum samples it was present as a 29-kDa fragment. The incubation of recombinant cTnT, or native ternary cardiac troponin complex with thrombin or in normal human serum (NHS), resulted in the formation of a 29-kDa product that was similar to that detected in AMI serum samples. No cTnT degradation was observed when thrombin or NHS was pretreated with hirudin, a specific inhibitor of thrombin, or during incubation of troponin in normal heparin plasma. When the products of thrombin-mediated cTnT proteolysis were analyzed by MS, 2 fragments consisting of amino acid residues (aar) 2–68 and 69–288 were identified, which suggests that thrombin cleaves cTnT between R68 and S69.CONCLUSIONSThe results of this study suggest that the 29-kDa fragment of cTnT in AMI serum samples mainly appears due to the cleavage by thrombin during serum sample preparation.

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Lower Cardiac Troponin T and I Results in Heparin-Plasma Than in Serum

Clinical Chemistry ◽

10.1093/clinchem/46.9.1338 ◽

2000 ◽

Vol 46 (9) ◽

pp. 1338-1344 ◽

Cited By ~ 58

Author(s):

Hugo Stiegler ◽

Yuriko Fischer ◽

Jaime F Vazquez-Jimenez ◽

Jürgen Graf ◽

Karsten Filzmaier ◽

...

Keyword(s):

Cardiac Troponin ◽

Heart Surgery ◽

Troponin I ◽

Troponin T ◽

Open Heart Surgery ◽

Cardiac Troponin T ◽

Serum Samples ◽

Coronary Syndrome ◽

Heparin Plasma

Abstract Background: The use of plasma rather than serum for determination of cardiac troponins can improve turnaround time and potentially avoid incomplete serum separation that may produce falsely increased results. We investigated the influence of incomplete serum separation and the effect of heparin-plasma on cardiac troponin concentrations. Methods: Serum and heparin-plasma samples were drawn simultaneously from 100 patients (50 patients with acute coronary syndrome and 50 patients after open heart surgery) and measured on three different analytical systems, two for determination of cardiac troponin I (cTnI; Abbott AxSYM and Bayer ACS:Centaur) and one for cardiac troponin T (cTnT; Roche Elecsys cTnT STAT). Serum samples were reanalyzed after a second centrifugation to assess the influence of incomplete serum separation. Results: Mean results (± 95% confidence interval) in heparin-plasma compared with serum were 101% ± 2% (AxSYM cTnI), 94% ± 3% (ACS:Centaur cTnI), and 99% ± 3% (Elecsys cTnT). Differences >20% were seen in 11% of results on the ACS:Centaur, 9% of results on Elecsys cTnT, and 2% of results on the AxSYM. For the Elecsys cTnT assay, the magnitude of the difference between serum and plasma was independent of the absolute concentration and confined to individual samples, and was reversed by treatment with heparinase. A second centrifugation had no effect on serum results by any of the assays. Conclusion: The concentrations of troponins measured in heparin-plasma are markedly lower than in serum in some cases.

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Plasma or serum samples: measurements of cardiac troponin T and of other analytes compared

Clinical Chemistry and Laboratory Medicine (CCLM) ◽

10.1515/cclm.2004.154 ◽

2004 ◽

Vol 42 (8) ◽

Cited By ~ 8

Author(s):

Roberto Dominici ◽

Ilenia Infusino ◽

Cristina Valente ◽

Irene Moraschinelli ◽

Carlo Franzini

Keyword(s):

Creatine Kinase ◽

Cardiac Troponin ◽

Troponin T ◽

Cardiac Troponin T ◽

Cardiac Markers ◽

Serum Samples ◽

Cardiac Marker ◽

Creatine Kinase Mb ◽

Heparin Plasma ◽

Automated Methods

AbstractConflicting data in the literature concern possible differences in the immunochemical measurement of cardiac troponins, either in plasma or in serum. In order to address this specific point, 96 serum and heparin-plasma pairs were obtained for cardiac marker measurement [cardiac troponin T (cTnT); myoglobin (Myo) and creatine kinase-MB isoenzyme (CK-MB)]; 29 additional “common” analytes were measured in 77 such samples. The cardiac markers were measured by electrochemiluminescence (Elecsys 2010, Roche); the other analytes by established automated methods (Modular, Roche). Mean plasma/serum ratios for cTnT (0.95), creatine kinase-MB (1.01) and myoglobin (0.99) were comparable with those of the 29 common analytes (interval of means 0.83–1.05). The distribution of the plasma-serum differences also showed similarities between cardiac markers and other analytes. A few outlier plasma-serum differences (3–5%) were measured for both categories of analytes. Addition of heparin to serum (51 samples) caused decreased cTnT (mean ratio 0.92). In 3 of 51 such samples the cTnT decrease was more marked, but in a second sample from the same subjects (1 week later) such a prominent, heparin-induced loss of cTnT no longer appeared. In conclusion, plasma-serum differences in immuno-reactive cTnT compare with those observed for other analytes. In occasional heparin-plasma samples immunochemical measurement of cTnT may give exceptionally low values. However, in our sample group of 96 patients (cTnT lower or higher than the cut-off in, respectively, 24 and 72 patients), no misclassification occurred if plasma instead of serum cTnT values were considered.

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Development of a Novel Sensor System Based on Magnetic Microspheres to Detect Cardiac Troponin T

International Journal of Polymer Science ◽

10.1155/2020/8855550 ◽

2020 ◽

Vol 2020 ◽

pp. 1-10

Author(s):

Junrong Zhang ◽

Hongxiang Liu ◽

Baofeng Xu ◽

Sijun Huang ◽

Rui Liu ◽

...

Keyword(s):

Cardiac Troponin ◽

Troponin T ◽

Myocardial Damage ◽

Fluorescein Isothiocyanate ◽

High Specificity ◽

Magnetic Microspheres ◽

Cardiac Troponin T ◽

Serum Samples ◽

Magnetic Separator ◽

Specific Antibodies

Acute myocardial infarction (AMI) causes irreversible injury to cardiomyocytes in a short time and may result in various complications, severely threatening patient safety. Therefore, it is necessary to predict the possibility of AMI in the prophase. Prognostic detection of biomarkers that specifically reflect myocardial damage in a patient’s blood has become an essential mediating measure to prevent the serious occurrence of AMI. The present study is aimed at exploring a novel sensing system with high specificity and precision based on magnetic microspheres developed to detect cardiac troponin T (cTnT), which is the most specific diagnostic marker for AMI in cardiovascular diseases. Naive human cTnT protein in serum samples and antigens on functional magnetic microspheres will competitively bind with limited specific antibodies. After rapid removal of heterogeneous elements in the sera using a magnetic separator, fluorescein isothiocyanate-labeled immunoglobulin G is added to react with specific antibodies on the magnetic microspheres. Then, a flow cytometer is used to collect signals of different fluorescence intensities. The results show that the method is characterized by economy, high accuracy, and novelty. It can be used for the detection of cTnT in blood at 1.7–106.1 ng/mL, with a detection limit of 0.5 ng/mL. Thus, the proposed sensor improves the accuracy and efficiency of diagnosis before clinical deterioration of AMI.

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Troponin T and I Assays Show Decreased Concentrations in Heparin Plasma Compared with Serum: Lower Recoveries in Early than in Late Phases of Myocardial Injury

Clinical Chemistry ◽

10.1093/clinchem/46.6.817 ◽

2000 ◽

Vol 46 (6) ◽

pp. 817-821 ◽

Cited By ~ 57

Author(s):

Willie Gerhardt ◽

Gunnar Nordin ◽

Ann-Katrin Herbert ◽

Birgitta Linåker Burzell ◽

Anders Isaksson ◽

...

Keyword(s):

Myocardial Infarction ◽

Chest Pain ◽

Cardiac Troponin ◽

Myocardial Injury ◽

Troponin I ◽

Troponin T ◽

Serum Samples ◽

Heparin Plasma ◽

Time Courses ◽

Serial Samples

Abstract Background: Heparinized plasma samples allow more rapid analysis than serum samples, but preliminary studies showed lower cardiac troponin T (cTnT) results in plasma. We undertook a multicenter study to characterize this effect for cTnT and cardiac troponin I (cTnI). Methods: Blood samples were collected with and without heparin at five hospitals. cTnT was measured by a “third generation” assay (Elecsys®), and cTnI was measured by a commercial immunoassay (IMMULITE®). Results: Mean cTnT was 15% lower in heparin sampling tubes than in serum. Measured concentrations of cardiac troponins also decreased with increasing heparin concentrations added to sera. Heparin-induced losses were greater in early than in late phases after onset of chest pain. Addition of heparin (∼100 IU/mL) to serial samples from nine acute myocardial infarction patients produced mean cTnT losses of 33% at 1–12 h after onset of chest pain, 17% at 13–48 h, and 7% after 48 h. The changing heparin effects were seen for both cTnT and cTnI during time courses of individual patients with myocardial infarction. Conclusion: We suggest that binding of heparin to troponins decreases immunoreactivity, especially in early phases of myocardial injury. The resulting losses may depend on the antibodies used in each troponin assay.

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Time-Dependent Degradation Pattern of Cardiac Troponin T Following Myocardial Infarction

Clinical Chemistry ◽

10.1373/clinchem.2012.200543 ◽

2013 ◽

Vol 59 (7) ◽

pp. 1083-1090 ◽

Cited By ~ 53

Author(s):

Eline PM Cardinaels ◽

Alma MA Mingels ◽

Tom van Rooij ◽

Paul O Collinson ◽

Frits W Prinzen ◽

...

Keyword(s):

Myocardial Infarction ◽

Western Blot ◽

Blot Analysis ◽

Cardiac Troponin ◽

Western Blot Analysis ◽

Troponin T ◽

Time Dependent ◽

Cardiac Troponin T ◽

Serum Samples ◽

Degradation Pattern

BACKGROUNDCardiac troponin T (cTnT) is widely used for the diagnosis of acute myocardial infarction (AMI). However, it is still unclear whether degraded cTnT forms circulate in the patient's blood. We therefore aimed to elucidate which cTnT forms are detected by the clinical assay.METHODSSeparation of cTnT forms by gel filtration chromatography (GFC) was performed in sera from 13 AMI patients to examine cTnT degradation. The GFC eluates were subjected to Western blot analysis with the original antibodies from the Roche immunoassay used to mimic the clinical cTnT assay. To investigate the degradation pattern with time, standardized serum samples of 18 AMI patients collected 0–72 h after admission were analyzed by Western blot analysis.RESULTSGFC analysis of AMI patients' sera revealed 2 cTnT peaks with retention volumes of 5 and 21 mL. Western blot analysis identified these peaks as cTnT fragments of 29 and 14–18 kDa, respectively. Furthermore, the performance of direct Western blots on standardized serum samples demonstrated a time-dependent degradation pattern of cTnT, with fragments ranging between 14 and 40 kDa. Intact cTnT (40 kDa) was present in only 3 patients within the first 8 h after hospital admission.CONCLUSIONSThese results demonstrate that the Roche cTnT immunoassay detects intact as well as degraded cTnT forms in AMI patients' sera during the period of diagnostic testing. Moreover, following AMI, cTnT is degraded in a time-dependent pattern.

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Amino-Functionalization of Carbon Nanotubes by Using a Factorial Design: Human Cardiac Troponin T Immunosensing Application

BioMed Research International ◽

10.1155/2014/929786 ◽

2014 ◽

Vol 2014 ◽

pp. 1-9 ◽

Cited By ~ 12

Author(s):

Tatianny A. Freitas ◽

Alessandra B. Mattos ◽

Bárbara V. M. Silva ◽

Rosa F. Dutra

Keyword(s):

Carbon Nanotubes ◽

Cardiac Troponin ◽

Limit Of Detection ◽

Troponin T ◽

Fractional Factorial ◽

Cardiac Troponin T ◽

Clinical Tool ◽

Serum Samples ◽

Multiwalled Carbon ◽

Amino Functionalization

A simple amino-functionalization method for carbon nanotubes and its application in an electrochemical immunosensor for detection of the human cardiac troponin T are described. Amino-functionalized carbon nanotubes allow oriented antibodies immobilization via their Fc regions, improving the performance of an immunosensor. Herein multiwalled carbon nanotubes were amino-functionalized by using the ethylenediamine reagent and assays were designed by fractional factorial study associated with Doehlert matrix. Structural modifications in the carbon nanotubes were confirmed by Fourier transform infrared spectroscopy. After amino-functionalization the carbon nanotubes were attached to screen-printed carbon electrode and a sandwich-type immunoassay was performed for measuring the cardiac troponin T. The electrochemical measurements were obtained through hydrogen peroxide reaction with peroxidase conjugated to the secondary antibody. Under optimal conditions, troponin T immunosensor was evaluated in serum samples, which showed a broad linear range (0.02 to 0.32 ng mL−1) and a low limit of detection, 0.016 ng mL−1. This amino platform can be properly used as clinical tool for cardiac troponin T detection in the acute myocardial infarction diagnosis.

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Analytical validation of a high-sensitivity cardiac troponin T assay in horses

Journal of Veterinary Diagnostic Investigation ◽

10.1177/1040638715593601 ◽

2015 ◽

Vol 27 (4) ◽

pp. 504-509 ◽

Cited By ~ 5

Author(s):

Nicky Van Der Vekens ◽

Marie-Astrid van Dievoet ◽

Hendrik De Puydt ◽

Annelies Decloedt ◽

Sofie Ven ◽

...

Keyword(s):

Cardiac Troponin ◽

Troponin T ◽

Myocardial Damage ◽

High Sensitivity ◽

Cross Reactivity ◽

Cardiac Troponin T ◽

Plasma Samples ◽

Analytical Validation ◽

Serum Samples ◽

Coefficients Of Variation

Although cardiac troponin T (cTnT) assays have been used to detect myocardial damage in horses, a cTnT assay has not been analytically validated, to our knowledge. The aims of this study were to estimate the precision of a high-sensitivity cTnT assay in horses and determine the effect of hemolysis on the measured cTnT concentration. Serum samples from horses were mixed in 3 different pools. Pool 1 consisted of samples from 3 healthy horses, pool 2 from 6 horses with heart failure or atypical myopathy, and pool 3 from 10 horses with atypical myopathy. The within- and between-run coefficients of variation were determined for each pool. Pools 2 and 3 were diluted to estimate linearity. To study the influence of sample hemolysis, serum was collected from 4 horses with a high cTnT concentration, in which hemolysis was mechanically induced. In addition, ethylenediamine tetra-acetic acid blood tubes were collected from 3 other horses, from which hemolysate was prepared and added to plasma at different concentrations. The within- and between-run coefficients of variation of all pools were <10%, and a good linearity was found. Three out of 4 hemolyzed serum samples had a decreased serum cTnT concentration. Plasma samples with a high hemolysis index showed a negative interference, resulting in a lower cTnT concentration. Results of the high-sensitivity cTnT assay were highly reproducible. Because samples from horses with musculoskeletal damage were included, further studies should test the possible cross-reactivity between troponin T of musculoskeletal and cardiac origin before the assay can be used in equine clinical practice.

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Cardiac troponin T composition in normal and regenerating human skeletal muscle

Clinical Chemistry ◽

10.1093/clinchem/43.3.476 ◽

1997 ◽

Vol 43 (3) ◽

pp. 476-484 ◽

Cited By ~ 130

Author(s):

Geza S Bodor ◽

Libby Survant ◽

Ellen M Voss ◽

Stephen Smith ◽

Diane Porterfield ◽

...

Keyword(s):

Skeletal Muscle ◽

Western Blot ◽

Cardiac Disease ◽

Cardiac Troponin ◽

Human Skeletal Muscle ◽

Troponin T ◽

Myofibrillar Protein ◽

Cardiac Troponin T ◽

Muscle Tissues ◽

Normal Human

Abstract Cardiac troponin T (cTnT), measurement of which has been recommended for diagnosing myocardial infarction, was initially believed to be specific for the heart. However, recent publications have reported cTnT in sera of patients without cardiac disease; therefore, we investigated whether cTnT could be found in human skeletal muscle tissues. Using immunohistochemistry, Western blot, and quantitative cTnT ELISA, we assayed human heart (n = 3), normal human skeletal muscle (n = 6), and diseased skeletal muscle samples from patients with polymyositis (PM, n = 13) and Duchenne muscular dystrophy (DMD, n = 6). All heart specimens contained cTnT, but the expression of cTnT in normal skeletal muscle samples varied widely, ranging from no expression (quadriceps femoris) to expression by up to 20% of the muscle fibers (diaphragm). Immunohistochemistry detected cTnT in skeletal muscle of 8 of the PM patients and all of the DMD patients. Mean myofibrillar cTnT concentrations (mg/g myofibrillar protein) were: cardiac = 10.0, normal skeletal = 0.8, PM skeletal = 0.7, and DMD skeletal = 4.37, confirming the results of immunohistochemistry. Western blot analysis also confirmed the expression of cTnT in muscle from DMD patients. These findings provide evidence that cTnT is not 100% cardiac-specific but also is expressed in regenerating (PM and DMD) as well as in normal (nonregenerating) skeletal muscle.

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Cardiac troponin T quantitative assay failure as a result of antibody interference

African Journal of Laboratory Medicine ◽

10.4102/ajlm.v2i1.23 ◽

2013 ◽

Vol 2 (1) ◽

Cited By ~ 3

Author(s):

Philip H. Fortgens ◽

Fierdoz Omar

Keyword(s):

Immunoglobulin G ◽

Whole Blood ◽

Cardiac Troponin ◽

Troponin T ◽

Protein A ◽

Blocking Agent ◽

Mouse Serum ◽

Cardiac Troponin T ◽

Serum Samples ◽

Heterophile Antibody

Background: Immunoassays are prone to interference by various substances which may cause inaccurate results. This type of interference is difficult to detect analytically.Objective: A case of CARDIAC Troponin T Quantitative reader (Roche Diagnostics) assayfailure was detected and investigated in order to ascertain the likely cause.Method: Patient whole blood was mixed with cardiac troponin T-positive blood, patient and control sera were denuded of immunoglobulin G by protein A-affinity chromatographyand patient sera were mixed with mouse serum. Samples were analysed on a CARDIAC Troponin T Quantitative reader.Results: A mixture of patient whole blood and cardiac troponin T-positive blood resultedin assay failure; removal of immunoglobulin G from patient sera reversed the cardiactroponin T assay failure; the addition of mouse serum as a heterophile antibody blocking agent had no effect.Conclusion: It is proposed that the interference resulting in assay failure may not be because of a heterophile antibody, but rather a result of a circulating autoantibody to cardiactroponin T, which may compete with antibody assay reagents for binding sites.

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P4503A multi-site coronary vein sampling study on cardiac troponin T degradation in non-ST-elevation myocardial infarction

European Heart Journal ◽

10.1093/eurheartj/ehz745.0896 ◽

2019 ◽

Vol 40 (Supplement_1) ◽

Author(s):

G E Cramer ◽

S A J Damen ◽

W H M Vroemen ◽

S T P Mezger ◽

H Suryapranata ◽

...

Keyword(s):

Myocardial Infarction ◽

Cardiac Troponin ◽

Troponin T ◽

Cardiac Troponin T ◽

Peripheral Artery ◽

Serum Samples ◽

Coronary Vein ◽

C Complex ◽

Over Time

Abstract Background High-sensitivity cardiac troponin (hs-cTn) assays have reduced specificity for myocardial infarction (MI). In conditions other than MI, cardiac troponin T (cTnT) elevations have been attributed to small cTnT fragments. In search of improved cTnT assay specificity for MI, determination of the in-vivo molecular appearance of cTnT during MI is pivotal. Purpose To determine cTnT composition close to its site of release and during the course of MI by multi-site coronary venous system (CVS) and peripheral sampling. Methods Lithium-heparinized (LH) plasma and serum samples were obtained from multiple CVS locations in NSTEMI patients. Additionally, samples were drawn from a peripheral artery and vein followed by collections at 6- and 12 hours post-catheterization. cTnT concentrations were measured using the hs-cTnT immunoassay (Roche). The molecular cTnT composition was determined by gel filtration chromatography and Western blotting in an early and late presenting patient. Results CVS hs-cTnT concentrations were 28% higher than in the peripheral artery sample (n=71, p<0.001). In contrast to LH plasma, serum caused pre-analytical cTn T-I-C complex disintegration and cTnT degradation. CVS samples demonstrated presence of cTn T-I-C complex, free intact cTnT, and 29 kDa and 15–18 kDa cTnT fragments in higher absolute concentrations than measured peripherally. While the proportion of cTn T-I-C complex decreased and disappeared over time, 15–18 kDa cTnT fragments increased. Moreover, cTn T-I-C complex was more prominent in the early versus the late presenting patient. Central illustration Conclusions In NSTEMI, cTnT is released as a combination of cTn T-I-C complex, free intact cTnT and multiple cTnT fragments. Over time, the composition of observed cTnT forms changes due to in-vivo degradation. These findings serve as a stepping stone to improve diagnostic accuracy of the hs-cTnT assay for MI.

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