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Establishing dihydropyrimidine dehydrogenase (DPD) activity is highly important in determining the correct starting dose of fluoropyrimidines such as 5-fluorouracil and capecitabine. The concentration ratio of endogenous uracil with its metabolite dihydrouracil (DHU) is a well-known parameter that is linked to DPD activity. Concentration ratios such as thymine over its DPD-converted metabolite dihydrothymine (DHT) is less described and may serve as an alternative diagnostic biomarker for DPD activity. In this study, we describe the development and validation of an ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) assay for the quantification of uracil, DHU, thymine, and DHT in human plasma. In addition, stability experiments were performed. Uracil and thymine were quantified up to 80.0 ng/mL and DHU and DHT up to 800 ng/mL. Intra- and inter-assay precision were maximum 8.0 % and 7.6 %. respectively. Also, recovery was adequate and significant matrix-effects and carry-over were excluded. Stability experiments showed that uracil concentrations increased with 27-52 % when stored for 1 or 2 h at ambient temperatures compared to cold storage. Thymine, DHU, and DHT concentrations remained stable, thymine after 1 h in plasma excluded, showing the DHT:T ratio might be a more robust marker for DPD activity than DHU:U. In conclusion, we present here a novel assay capable of quantifying uracil, thymine, DHU and DHT in a single analytical run. We provide additional data showing increased stability for DHU, thymine and DHT compared to uracil. This assay may be used as a diagnostic test in future studies, establishing the association of these endogenous biomarker concentrations with DPD activity and safety to treatment with fluoropyrimidines. In addition, future research should also be focused on reducing pre-analytical instability. Standardization in this field is essential to set proper reference values and to allow inter-study comparison on clinical outcomes.

Objective Severe cases of coronavirus disease 2019 (COVID-19) can require continuous renal replacement therapy (CRRT) and/or extracorporeal membrane oxygenation (ECMO). Unfractionated heparin (UFH) to prevent circuit clotting is mandatory but monitoring is complicated by (pseudo)-heparin resistance. In this observational study, we compared two different activated partial thromboplastin time (aPTT) assays and a chromogenic anti-Xa assay in COVID-19 patients on CRRT or ECMO in relation to their UFH dosages and acute phase reactants. Materials and Methods The aPTT (optical [aPTT-CS] and/or mechanical [aPTT-STA] clot detection methods were used), anti-Xa, factor VIII (FVIII), antithrombin III (ATIII), and fibrinogen were measured in 342 samples from 7 COVID-19 patients on CRRT or ECMO during their UFH treatment. Dosage of UFH was primarily based on the aPTT-CS with a heparin therapeutic range (HTR) of 50–80s. Associations between different variables were made using linear regression and Bland–Altman analysis. Results Dosage of UFH was above 35,000IU/24 hours in all patients. aPTT-CS and aPTT-STA were predominantly within the HTR. Anti-Xa was predominantly above the HTR (0.3–0.7 IU/mL) and ATIII concentration was >70% for all patients; mean FVIII and fibrinogen were 606% and 7.5 g/L, respectively. aPTT-CS correlated with aPTT-STA (r 2 = 0.68) with a bias of 39.3%. Correlation between aPTT and anti-Xa was better for aPTT-CS (0.78 ≤ r 2 ≤ 0.94) than for aPTT-STA (0.34 ≤ r 2 ≤ 0.81). There was no general correlation between the aPTT-CS and ATIII, FVIII, fibrinogen, thrombocytes, C-reactive protein, or ferritin. Conclusion All included COVID-19 patients on CRRT or ECMO conformed to the definition of heparin resistance. A patient-specific association was found between aPTT and anti-Xa. This association could not be explained by FVIII or fibrinogen.

Background The incidence of pulmonary thromboembolism is high in SARS-CoV-2 patients admitted to the Intensive Care. Elevated biomarkers of coagulation (fibrinogen and D-dimer) and inflammation (c-reactive protein (CRP) and ferritin) are associated with poor outcome in SARS-CoV-2. Whether the time-course of fibrinogen, D-dimer, CRP and ferritin is associated with the occurrence of pulmonary thromboembolism in SARS-CoV-2 patients is unknown. We hypothesise that patients on mechanical ventilation with SARS-CoV-2 infection and clinical pulmonary thromboembolism have lower concentrations of fibrinogen and higher D-dimer, CRP, and ferritin concentrations over time compared to patients without a clinical pulmonary thromboembolism. Methods In a prospective study, fibrinogen, D-dimer, CRP and ferritin were measured daily. Clinical suspected pulmonary thromboembolism was either confirmed or excluded based on computed tomography pulmonary angiography (CTPA) or by transthoracic ultrasound (TTU) (i.e., right-sided cardiac thrombus). In addition, patients who received therapy with recombinant tissue plasminogen activator were included when clinical instability in suspected pulmonary thromboembolism did not allow CTPA. Serial data were analysed using a mixed-effects linear regression model, and models were adjusted for known risk factors (age, sex, APACHE-II score, body mass index), biomarkers of coagulation and inflammation, and anticoagulants. Results Thirty-one patients were considered to suffer from pulmonary thromboembolism ((positive CTPA (n = 27), TTU positive (n = 1), therapy with recombinant tissue plasminogen activator (n = 3)), and eight patients with negative CTPA were included. After adjustment for known risk factors and anticoagulants, patients with, compared to those without, clinical pulmonary thromboembolism had lower average fibrinogen concentration of − 0.9 g/L (95% CI: − 1.6 – − 0.1) and lower average ferritin concentration of − 1045 μg/L (95% CI: − 1983 – − 106) over time. D-dimer and CRP average concentration did not significantly differ, 561 μg/L (− 6212–7334) and 27 mg/L (− 32–86) respectively. Ferritin lost statistical significance, both in sensitivity analysis and after adjustment for fibrinogen and D-dimer. Conclusion Lower average concentrations of fibrinogen over time were associated with the presence of clinical pulmonary thromboembolism in patients at the Intensive Care, whereas D-dimer, CRP and ferritin were not. Lower concentrations over time may indicate the consumption of fibrinogen related to thrombus formation in the pulmonary vessels.

Cardiac troponin T (cTnT) is a sensitive and specific biomarker for detecting cardiac muscle injury. Its concentration in blood can be significantly elevated outside the normal reference range under several pathophysiological conditions. The classical analytical method in routine clinical analysis to detect cTnT in serum or plasma is a single commercial immunoassay, which is designed to quantify the intact cTnT molecule. The targeted epitopes are located in the central region of the cTnT molecule. However, in blood cTnT exists in different biomolecular complexes and proteoforms: bound (to cardiac troponin subunits or to immunoglobulins) or unbound (as intact protein or as proteolytic proteoforms). While proteolysis is a principal posttranslational modification (PTM), other confirmed PTMs of the proteoforms include N-terminal initiator methionine removal, N-acetylation, O-phosphorylation, O-(N-acetyl)-glucosaminylation, N(ɛ)-(carboxymethyl)lysine modification and citrullination. The immunoassay probably detects several of those cTnT biomolecular complexes and proteoforms, as long as they have the centrally targeted epitopes in common. While analytical cTnT immunoreactivity has been studied predominantly in blood, it can also be detected in urine, although it is unclear in which proteoform cTnT immunoreactivity is present in urine. This review presents an overview of the current knowledge on the pathophysiological lifecycle of cTnT. It provides insight into the impact of PTMs, not only on the analytical immunoreactivity, but also on the excretion of cTnT in urine as one of the waste routes in that lifecycle. Accordingly, and after isolating the proteoforms from urine of patients suffering from proteinuria and acute myocardial infarction, the structures of some possible cTnT proteoforms are reconstructed using mass spectrometry and presented.

Background Because of its high cardiospecificity, cardiac troponin T (cTnT) is one of the first-choice biomarkers to diagnose acute myocardial infarction (AMI). cTnT is extensively fragmented in serum of patients suffering from AMI. However, it is currently unknown whether all cTnT is completely degraded in the body or whether some cTnT fragments can leave the body via urine. The aim of the present study is to develop a method for the detection of cTnT in urine and to examine whether cTnT is detectable in patient urine. Methods Proteins in urine samples of 20 patients were precipitated using a cTnT-specific immunoprecipitation technique and a nonspecific acetonitrile protein precipitation. After in-solution digestion of the precipitated proteins, the resulting peptides were separated and analyzed using HPLC and mass spectrometry with a targeted selected ion monitoring assay with data-dependent tandem mass spectrometry (t-SIM/dd-MS2). Results The t-SIM/dd-MS2 assay was validated using a synthetic peptide standard containing 10 specific cTnT peptides of interest and with purified human intact cTnT spiked in urine from healthy individuals. Using this assay, 6 different cTnT-specific peptides were identified in urine samples from 3 different patients, all suffering from AMI. Conclusions We show here for the first time that cTnT can be present in the urine of AMI patients using a targeted LC-MS/MS assay. Whether the presence of cTnT in urine reflects a physiological or pathophysiological process still needs to be elucidated.

BACKGROUNDCardiac troponin T (cTnT) is the preferred biomarker for the diagnosis of acute myocardial infarction (AMI). It has been suggested that cTnT is present predominantly in fragmented forms in human serum following AMI. In this study, we have used a targeted mass spectrometry assay and epitope mapping using Western blotting to confirm this hypothesis.METHODScTnT was captured from the serum of 12 patients diagnosed with AMI using an immunoprecipitation technique employing the M11.7 catcher antibody and fractionated with SDS-PAGE. Coomassie-stained bands of 4 patients at 37, 29, and 16 kDa were excised from the gel, digested with trypsin, and analyzed on a Q Exactive instrument set on targeted Selected Ion Monitoring mode with data-dependent tandem mass spectrometry (MS/MS) for identification. Western blotting employing 3 different antibodies was used for epitope mapping.RESULTSTen cTnT peptides of interest were targeted. By using MS/MS, all of these peptides were identified in the 37-kDa, intact, cTnT band. In the 29- and 16-kDa fragment bands, 8 and 4 cTnT-specific peptides were identified, respectively. Some of these peptides were “semitryptic,” meaning that their C-termini were not formed by trypsin cleavage. The C-termini of these semitryptic peptides represent the C-terminal end of the cTnT molecules present in these bands. These results were confirmed independently by epitope mapping.CONCLUSIONSUsing LC-MS, we have succeeded in positively identifying the 29- and 16-kDa fragment bands as cTnT-derived products. The amino acid sequences of the 29- and 16-kDa fragments are Ser79-Trp297 and Ser79-Gln199, respectively.

We read with interest the paper by Schmid et al. [(1)][1], who described elevated cardiac troponin T (cTnT) concentrations in patients with skeletal myopathies in which they suggested cross-reaction of the high-sensitivity (hs-)cTnT immunoassay with skeletal muscle troponin T (skTnT) isoforms as

Cardiac troponin T (cTnT) fragmentation in human serum was investigated using a newly developed targeted selected ion monitoring assay, as described in the accompanying article: "Development of a targeted selected ion monitoring assay for the elucidation of protease induced structural changes in cardiac troponin T" W. This article presents data describing aspects of the validation and optimisation of this assay. The data consists of several figures, an excel file containing the results of a sequence identity search, and a description of the raw mass spectrometry (MS) data files, deposited in the ProteomeXchange repository with id PRIDE: PXD003187. (C) 2015 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/40).

Cardiac troponin T (cTnT) is a highly cardiospecific protein commonly used in the diagnosis of acute myocardial infarction (AMI), but is subject to proteolytic degradation upon its release in the circulation. In this study, a targeted mass spectrometry assay was developed to detect peptides which are differentially present within the different degradation products. cTnT was spiked in human serum and incubated at 37 °C to induce proteolytic degradation. Isolation and fractionation of cTnT and its fragments from serum were performed using immunoprecipitation and SDS-PAGE. Bands migrating to 37 kDa (intact cTnT), 29 kDa (primary fragment), and 19, 18, and 16 kDa (secondary fragments) were excised, digested, and subsequently analysed using targeted selected ion monitoring on a UHPLC-coupled quadrupole-Orbitrap mass spectrometer. Sixteen precursor ions from a total of 11 peptides unique to cTnT were targeted. Precursor ions were detectable up until 1200 ng/L cTnT, which is a typical cTnT concentration after AMI. With tandem-MS and relative quantification, we proved the formation of cTnT fragments upon incubation in human serum and identified differentially present peptides in the fragment bands, indicative of N- and C-terminal proteolytic cleavage. These findings are of importance for the development of future cTnT assays, calibrators, and quality control samples. Biological significance In this study we have developed a gel-based targeted mass spectrometry assay which is able to differentiate between different molecular forms of cTnT. The unravelling of the molecular presentation of cTnT in human serum is of importance in the field of clinical chemistry, where this highly specific and sensitive biomarker is being measured on a routinely basis in patient samples. Knowledge of the amino acid sequence of the different cTnT fragments may aid in the development of improved calibrators and quality control samples. In addition, different fragmentation patterns may be indicative of different underlying pathologies. New antibodies for future assays targeting specific areas of cTnT can thus be created based on this information. This assay will be used in future experiments to assess the fragmentation pattern of cTnT in serum of multiple patient groups in our laboratory.

To the Editor: During acute myocardial infarction (AMI), 1 cardiac troponin T (cTnT) is released from the damaged myocardium. One would expect a strong correlation between concentrations of cTnT measured in the blood after AMI and the extent of myocardial damage. However, various studies have shown a rather moderate correlation between infarct size and cTnT concentrations (1, 2). Frequently considered explanations for this phenomenon are methodological or focused on physiological factors associated with differences in release and elimination of cTnT (2). In contrast, less attention has been paid to the possible presence of inherent factors in the blood, such as (auto)antibodies or proteases, that might modify cTnT or interfere with the assay. We hypothesized that these factors may be ubiquitously present in the general population and might affect the measured concentrations of cTnT in individuals. We measured cTnT, cTnI, creatine kinase MB isoenzyme (CK-MB), and myoglobin concentrations in sera from 24 healthy volunteers (age range 22–54 years) before and after the addition of serum from a patient suffering from AMI (age 59 years, cTnT concentration approximately 19.5 μg/L). All participants gave written informed consent and leftover material was used in accordance with the code of proper secondary use of human tissue …

To the Editor: We read with interest the recent article by Katrukha et al. concerning thrombin-mediated degradation of human cardiac troponin T (cTnT) in serum (1). In their study, Katrukha and coworkers confirmed the finding of Streng et al. that human coagulation factor II (thrombin) is a strong mediator of cTnT degradation (2). They compared cTnT degradation in serum and heparin plasma collected simultaneously from the same patients with acute myocardial infarction (AMI), revealing substantial differences in cTnT fragment composition. They also performed in vitro experiments in which troponin T, thrombin, and hirudin were supplemented in various combinations. In addition, the cleavage site in cTnT was localized by use of mass spectrometry and probing of different proteolytic fragments with the use of various antibodies. Their main conclusion was that the primary 29-kDa cTnT fragment was mainly formed because of thrombin activation during serum preparation. We believe that the evidence for thrombin as one of the main proteases for cTnT degradation is convincing. However, we disagree with their final conclusion because there is published evidence suggesting …

Plasma concentrations of cardiac troponins, the preferred biomarkers for the diagnosis of acute myocardial infarction, are often persistently elevated in patients with chronic kidney disease (CKD). The origin of these elevations is unknown: Is it the heart, by increased release, or the kidneys, by decreased renal elimination? In clinical practice, this equivocal view on troponin elevations in patients with reduced glomerular clearance underlies countless clinical discussions among physicians and may delay rapid initiation of adequate treatment when these patients present with chest pain. In the present study, we aimed to discriminate between increased cardiac release and reduced renal elimination as the main process underlying this phenomenon. Specifically, we used the recently demonstrated rhythmic diurnal oscillation pattern of troponin T as a model to assess the contribution of impaired renal elimination to persistently elevated cardiac troponin levels in patients with CKD.1 The diurnal troponin T rhythm is characterized by gradually decreasing concentrations throughout daytime and rising concentrations during nighttime.1 If decreased renal clearance, and not increased production, is the key driver of elevated troponins in patients with CKD, the increased half-life and subsequent accumulation of cardiac troponin T will fade its diurnal rhythm. …

BACKGROUND We have found previously that in acute myocardial infarction (AMI), cardiac troponin T (cTnT) is degraded in a time-dependent pattern. We investigated whether cTnT forms differed in patients with chronic cTnT increases, as seen with renal dysfunction, from those in the acute phase of myocardial infarction. METHODS We separated cTnT forms by gel filtration chromatography (GFC) in end-stage renal disease (ESRD) patients: prehemodialysis (pre-HD) and post-HD (n = 10) and 2 months follow-up (n = 6). Purified (cTnT) standards, quality control materials of the clinical cTnT immunoassay (Roche), and AMI patients' sera also were analyzed. Immunoprecipitation and Western blotting were performed with the original cTnT antibodies from the clinical assay and antibodies against the N- and C-terminal end of cTnT. RESULTS GFC analysis revealed the retention of purified cTnT at 27.5 mL, identical to that for cTnT in quality controls. For all ESRD patients, one cTnT peak was found at 45 mL, pre- and post-HD, and stable over time. Western blotting illustrated that this peak corresponded to cTnT fragments CONCLUSIONS We found that cTnT forms in ESRD patients are small (

Aim Cardiac troponin I (cTnI) and T (cTnT) are the most important biomarkers in the diagnosis of acute myocardial infarction (AMI). Nevertheless, they can be elevated in the absence of AMI. It is unclear if such elevations represent irreversible cardiomyocyte-damage or leakage from viable cardiomyocytes. Our objective is to evaluate whether cTn is released from viable cardiomyocytes in response to ischemia and to identify differences in the release of cTn and its molecular forms. Methods and results HL-1 cardiomyocytes (mouse) were subjected to ischemia (modeled by anoxia with glucose deprivation). The total contents and molecular forms of cTn were determined in culture media and cell lysates. Cell viability was assessed from the release of lactate dehydrogenase (LDH). Before the release of LDH, the intracellular cTn content in ischemic cells decreased significantly compared to control (52% for cTnI; 23% for cTnT) and was not matched by a cTn increase in the medium. cTnI decreased more rapidly than cTnT, resulting in an intracellular cTnT/cTnI ratio of 25.5 after 24 h of ischemia. Western blots revealed changes in the relative amounts of fragmented cTnI and cTnT in ischemic cells. Conclusions HL-1 cardiomyocytes subjected to simulated ischemia released cTnI and cTnT only in combination with the release of LDH. We find no evidence of cTn release from viable cardiomyocytes, but did observe a significant decrease in cTn content, before the onset of cell death. Intracellular decrease of cTn in viable cardiomyocytes can have important consequences for the interpretation of cTn values in clinical practice.

Cardiac troponin T (cTnT) has been shown to be present in fragmented forms in human serum after acute myocardial infarction (AMI). While calpain-1 and caspase-3 have been identified as intracellular pro teases able to cleave the N-terminus of cTnT, it is still unclear which proteases are responsible for the extensive and progressive cTnT fragmentation observed in serum of AMI-patients. In this pilot study we have investigated the possibility that human thrombin may be involved in this process. Purified human cTnT was spiked in unprocessed and deproteinated serum in the presence or absence of either purified human thrombin or PPACK thrombin inhibitor. After immunoprecipitation, SDS-PAGE and Western blotting we observed an increase in cTnT fragmentation when purified thrombin was added to deproteinated serum. Consequently, the addition of thrombin inhibitor to unprocessed serum resulted in a decrease of cTnT fragmentation. Our results suggest that multiple enzymes are involved in cTnT degradation, and that thrombin plays an important role.

The diagnosis of acute myocardial infraction has undergone a noticeable transformation sincethe introduction of the high-sensitivity cardiac troponin assays. The clear shift towards increasingly rapidclinical decision making is reflected in the most recent European Society of Cardiology guidelines, whichinclude a 0-/1-hour algorithm for the diagnosis of non-ST-elevation myocardial infarction. The use of thisalgorithm enables rule-in or rule-out in approximately 70–75% of all patients after a second cardiac troponinmeasurement after one hour. In a minority of patients rule-in or rule-out is achievable even faster; after onlya single blood draw at presentation. Since rapid clinical decision making has several advantages, like fastinitiation of treatment for those who need it and preventing prolonged patient anxiety, various recent studieshave tried to further optimize these cut-off values at presentation.

Cardiac troponin (cTn) is an important sarcomeric protein complex situated on the thin filament and is involved in the regulation of cardiac muscle contraction. This regulation is primarily controlled by Ca(2+) binding to troponin C and in addition fine-tuned by the posttranslational modification of cTnI and cTnT. The vast majority of cTnT modifications involve the phosphorylation by protein kinase C (PKC) or other kinases and the N-terminal cleavage by caspase and calpain. In vitro studies employing reconstituted detergent-skinned fiber bundles and cell culture generally show a detrimental effect of cTnT phosphorylation on muscle contraction, which is backed by some in vivo studies finding increased cTnT phosphorylation in heart failure, but contradicted by others. In addition, N-terminal cleavage of cTnT is thought to be another factor influencing cardiac contraction. Time-dependent degradation of cTnT has been observed in human serum upon myocardial infarction. These molecular changes might influence the immunoreactivity of cTnT in the clinical immunoassay and have consequences for the clinical interpretations of these measurements. No consensus has yet been reached on the occurrence and extent of these observations and their underlying processes are subject of intense scientific debate. This review will focus on discussing these modifications, their implications on physiology and disease and summarizes the complex interplays of different enzymes on the molecular forms of cTnT and their associated effects.