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Determination of plasma vitamin B12 (B12) is a frequently requested laboratory analysis, mainly employed to establish B12 deficiency. However, an increased level of B12 is a common unexpected finding that may be related to an increased concentration of one of the B12 binding proteins, haptocorrin or transcobalamin. This paper describes the extensive laboratory evaluation of a patient with an elevated level of plasma B12 with various well-established assays. Initial studies suggested the presence of a macromolecule consisting of haptocorrin bound B12. Specific determinations of the B12-binding proteins revealed normal amounts of haptocorrin but a markedly increase in both total and B12 saturated transcobalamin (holo-TC). The results are in accord with the presence of macro-transcobalamin. These experiments reveal that determination of the nature of the B12-macromolecules is troublesome due to differences in assays applied to measure these proteins. In addition, this publication creates awareness of macro-holo-TC as a cause of an unexplained increased B12 level.

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.

To the Editor: Vigorous endurance-type exercise causes cardiac troponin T (cTnT)1 elevations that often exceed the diagnostic cutoff value for acute myocardial infarction (AMI) (1). It remains to be investigated whether exercise-induced cTnT release reflects a physiological or pathological process. Our recent studies utilizing multiple protein analysis techniques allowed determination of circulating cTnT forms, and it appeared that these are different in acute vs chronic pathologies (2, 3). In sera of AMI patients, cTnT is degraded from its intact form (estimated molecular weight by SDS-PAGE; MW est, 40 kDa) to primary ( MW est, 29 kDa) and subsequent secondary ( MW est, 14–18 kDa) fragments in a time-dependent manner (2). Only …

ObjectiveFamilial combined hyperlipidemia (FCHL) is common among survivors of a premature myocardial infarction. FCHL patients are characterized by visceral obesity, fatty liver, and insulin resistance. The aim of the present study was to determine the incidence and determinants of type 2 diabetes (T2D) in a longitudinal cohort of FCHL pedigrees.Research design and methodsFCHL patients, their unaffected relatives and spouses included in our baseline cohort in 1998–2005 (n=596) were re-invited to determine the incidence of self-reported T2D (that was confirmed by medical records), used as the primary outcome measure. The Fatty Liver Index (FLI) and Homeostasis Model Assessment Insulin Resistance (HOMA2-IR) were used as markers of fatty liver and insulin resistance, respectively. A subset of the original cohort underwent ultrasound of the liver, and subcutaneous and visceral fat in 2002–2005 (n=275; ‘ultrasound subcohort’).ResultsFollow-up data (median: 15 years) was acquired for 76%. The incidence rate of T2D was significantly higher in FCHL patients compared with spouses (19.2 per 1000 person-years vs 2.8 per 1000 person-years; HR : 6.3, 95% CI: 2.4 to 16.8), whereas no differences were observed between unaffected relatives and spouses (HR: 0.9, 95% CI: 0.3 to 2.6). Cox’s proportional hazard regression analyses showed that baseline HOMA2-IR and FLI≥60, but not waist circumference, BMI, or the FCHL affected state, were independently associated with incident T2D. Similar results were obtained in the ultrasound subcohort (median follow-up: 11 years), in which baseline HOMA2-IR and fatty liver (assessed by ultrasound) were independently associated with incident T2D.ConclusionThis study further corroborates the suggestion that the liver plays a central role in the pathogenesis of cardiometabolic complications in FCHL. It supports periodical screening for T2D in this high-risk population.

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 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.

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.

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 …

BackgroundA few days of bed rest or immobilization following injury, disease, or surgery can lead to considerable loss of skeletal muscle mass and strength. It has been speculated that such short, successive periods of muscle disuse may be largely responsible for the age-related loss of muscle mass throughout the lifespan.ObjectiveTo assess whether a single intramuscular injection of nandrolone decanoate prior to immobilization can attenuate the loss of muscle mass and strength in vivo in humans.Design, setting and participantsThirty healthy (22 +/- 1 years) men were subjected to 7 days of one-legged knee immobilization by means of a full leg cast with (NAD, n = 15) or without (CON, n = 15) prior intramuscular nandrolone decanoate injection (200 mg).MeasuresBefore and immediately after immobilization, quadriceps muscle cross-sectional area (CSA) (by means of single-slice computed tomography (CT) scans of the upper leg) and one-legged knee extension strength (one-repetition maximum [1-RM]) were assessed for both legs. Furthermore, muscle biopsies from the immobilized leg were taken before and after immobilization to assess type I and type II muscle fiber cross-sectional area.ResultsQuadriceps muscle CSA decreased during immobilization in both CON and NAD (-6 +/- 1% and -6 +/- 1%, respectively; main effect of time PConclusionsThis is the first study to report that nandrolone decanoate administration does not preserve skeletal muscle mass and strength during a short period of leg immobilization in vivo in humans.

Background and aims: Familial combined hyperlipidemia (FCHL) is a complex dyslipidemia associated with premature cardiovascular disease (CVD). The present study was conducted to 1) determine the incidence of CVD in FCHL in this era of protocolled, primary prevention; and 2) examine whether cardiovascular risk estimation based on the Systemic Coronary Risk Estimation (SCORE) chart, as proposed in the 2016 ESC/EAS guidelines for the management of dyslipidemia, is justified in FCHL.Methods: FCHL patients, their normolipidemic (NL) relatives and spouses originally included in our baseline cohort in 1998-2005 (n = 596) were invited for a follow-up visit to determine the incidence of CVD, defined as (non-) fatal coronary artery disease, ischemic stroke and peripheral artery disease requiring invasive treatment.Results: Follow-up data (median: 15 years) was acquired for 85% of the original cohort. The cumulative incidence of CVD was significantly higher in FCHL patients than in spouses (23.6% versus 4.7%; hazard ratio (HR): 5.4, 95% CI: 2.0-14.6; HR after adjustment for risk factors included in SCORE: 4.7, 95% CI: 1.6-13.8), but not in NL relatives compared to spouses (5.8% versus 4.7%). The SCORE chart tended to overestimate CVD risk in the spouses (observed [O]/expected [E] ratio: 0.2, p = 0.01), but not in FCHL patients (O/E: 1.3, p = 0.50).Conclusions: Risk of primary CVD is still substantially increased in FCHL patients, despite preventive measures. The overestimation of CVD risk by the SCORE chart -a nowadays frequently observed phenomenon thanks to improved primary prevention - was not seen in FCHL. These results suggest that more aggressive treatment is justified to avoid excessive CVD in FCHL.

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: 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 …

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.

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.

Background This report describes an in-house developed immunoprecipitation method to isolate insulin-like growth factor binding protein-3 (IGFBP-3) and its isoforms from serum. The method was compared to other existing immunoprecipitation methods. The study of IGFBP-3 isoforms is relevant for further studies on congenital defects in glycosylation (CDG), galactosemia, and alcoholic liver cirrhosis. Methods Monoclonal and/or polyclonal anti-human IGFBP-3 antibodies were covalently immobilised on protein-A Sepharose beads using dimethyl pimelimidate as cross-linker. By incubation with these immobilised antibodies, intact IGFBP-3 and fragments of IGFBP-3 were isolated from serum. Enzyme-linked immunosorbent assay (ELISA) and one-dimensional gel electrophoresis (1-DE) experiments were performed to define the optimal immunoprecipitation method. Isolated proteins were separated by 1-DE and two-dimensional gel electrophoresis (2-DE) and visualised by Western blotting. Results ELISA and 1-DE results illustrated that an optimal isolation was performed using PBS for the incubation with serum. Laemmli sample buffer, containing 2-amino-2-(hydroxymethyl)-1,3-propanediol hydrochloride and sodium dodecyl sulfate, or urea/CHAPS was optimal for the elution. Clinical validation was performed using CDG-Ia serum samples. The 2-DE experiments showed characteristic isoform patterns for CDG-Ia. Conclusions The optimized in-house developed immunoprecipitation method resulted in specific detection of IGFBP-3 isoforms and is suitable for further studies on glycosylation defects.

Objective To characterize the influence of Gelofusine ® (succinylated gelatin) on the performance of point-of-care testing (POCT) for human chorionic gonadotropin (hCG) devices. Design and methods Three brands of urine and urine/serum hCG POCT devices were verified. Results Succinylated gelatin affected the performance of the control band in the QuickVue ® hCG POCT devices. Alternative devices were not affected. The hCG test performance is not influenced. Conclusions Gelofusine ® affects specifically the quality control performance of the QuickVue ® hCG POCT devices.