Publications by authors named "Will K W H Wodzig"

56 Publications

Cardiac Troponin T: The Impact of Posttranslational Modifications on Analytical Immunoreactivity in Blood up to the Excretion in Urine.

Adv Exp Med Biol 2021 ;1306:41-59

Unit of Clinical Chemistry, Central Diagnostic Laboratory, Maastricht University Medical Center, Maastricht, The Netherlands.

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.
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http://dx.doi.org/10.1007/978-3-030-63908-2_4DOI Listing
May 2021

Biotin interference in high-sensitivity cardiac troponin T testing: a real-world evaluation in acute cardiac care.

Cardiovasc Res 2019 Dec;115(14):1950-1951

Central Diagnostic Laboratory, Department of Clinical Chemistry, Maastricht University Medical Center, Maastricht, The Netherlands.

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http://dx.doi.org/10.1093/cvr/cvz277DOI Listing
December 2019

Multi-Site Coronary Vein Sampling Study on Cardiac Troponin T Degradation in Non-ST-Segment-Elevation Myocardial Infarction: Toward a More Specific Cardiac Troponin T Assay.

J Am Heart Assoc 2019 07 4;8(14):e012602. Epub 2019 Jul 4.

2 Central Diagnostic Laboratory Maastricht University Medical Center Maastricht The Netherlands.

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.
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http://dx.doi.org/10.1161/JAHA.119.012602DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6662151PMC
July 2019

Leucine coingestion augments the muscle protein synthetic response to the ingestion of 15 g of protein following resistance exercise in older men.

Am J Physiol Endocrinol Metab 2019 09 21;317(3):E473-E482. Epub 2019 May 21.

NUTRIM School of Nutrition and Translational Research in Metabolism, Maastricht University Medical Centre, Maastricht, The Netherlands.

Older adults have shown an attenuated postexercise increase in muscle protein synthesis rates following ingestion of smaller amounts of protein compared with younger adults. Consequently, it has been suggested that older adults require the ingestion of more protein to increase postexercise muscle protein synthesis rates compared with younger adults. We investigated whether coingestion of 1.5 g of free leucine with a single 15-g bolus of protein further augments the postprandial muscle protein synthetic response during recovery from resistance-type exercise in older men. Twenty-four healthy older men (67 ± 1 yr) were randomly assigned to ingest 15 g of milk protein concentrate (MPC80) with (15G+LEU; = 12) or without (15G; = 12) 1.5 g of free leucine after performing a single bout of resistance-type exercise. Postprandial protein digestion and amino acid absorption kinetics, whole body protein metabolism, and postprandial myofibrillar protein synthesis rates were assessed using primed, continuous infusions with l-[-H]phenylalanine, l-[-H]tyrosine, and l-[1-C]leucine combined with ingestion of intrinsically l-[1-C]phenylalanine-labeled milk protein. A total of 70 ± 1% (10.5 ±0.2 g) and 75 ± 2% (11.2 ± 0.3 g) of the protein-derived amino acids were released in the circulation during the 6-h postexercise recovery phase in 15G+LEU and 15G, respectively ( < 0.05). Postexercise myofibrillar protein synthesis rates were 16% (0.058 ± 0.003 vs. 0.049 ± 0.002%/h, < 0.05; based on l-[-H]phenylalanine) and 19% (0.071 ± 0.003 vs. 0.060 ± 0.003%/h, < 0.05; based on l-[1-C]leucine) greater in 15G+LEU compared with 15G. Leucine coingestion further augments the postexercise muscle protein synthetic response to the ingestion of a single 15-g bolus of protein in older men.
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http://dx.doi.org/10.1152/ajpendo.00073.2019DOI Listing
September 2019

Dose-Dependent Increases in Whole-Body Net Protein Balance and Dietary Protein-Derived Amino Acid Incorporation into Myofibrillar Protein During Recovery from Resistance Exercise in Older Men.

J Nutr 2019 02;149(2):221-230

NUTRIM School of Nutrition and Translational Research in Metabolism.

Background: Age-related decline in skeletal muscle mass is at least partly attributed to anabolic resistance to food intake. Resistance exercise sensitizes skeletal muscle tissue to the anabolic properties of amino acids.

Objective: The present study assessed protein digestion and amino acid absorption kinetics, whole-body protein balance, and the myofibrillar protein synthetic response to ingestion of different amounts of protein during recovery from resistance exercise in older men.

Methods: Forty-eight healthy older men [mean ± SEM age: 66 ± 1 y; body mass index (kg/m2): 25.4 ± 0.3] were randomly assigned to ingest 0, 15, 30, or 45 g milk protein concentrate after a single bout of resistance exercise consisting of 4 sets of 10 repetitions of leg press and leg extension and 2 sets of 10 repetitions of lateral pulldown and chest press performed at 75-80% 1-repetition maximum. Postprandial protein digestion and amino acid absorption kinetics, whole-body protein metabolism, and myofibrillar protein synthesis rates were assessed using primed, continuous infusions of l-[ring-2H5]-phenylalanine, l-[ring-2H2]-tyrosine, and l-[1-13C]-leucine combined with ingestion of intrinsically l-[1-13C]-phenylalanine and l-[1-13C]-leucine labeled protein.

Results: Whole-body net protein balance showed a dose-dependent increase after ingestion of 0, 15, 30, or 45 g of protein (0.015 ± 0.002, 0.108 ± 0.004, 0.162 ± 0.008, and 0.215 ± 0.009 μmol Phe · kg-1 · min-1, respectively; P < 0.001). Myofibrillar protein synthesis rates were higher after ingesting 30 (0.0951% ± 0.0062%/h, P = 0.07) or 45 g of protein (0.0970% ± 0.0062%/h, P < 0.05) than after 0 g (0.0746% ± 0.0051%/h). Incorporation of dietary protein-derived amino acids (l-[1-13C]-phenylalanine) into de novo myofibrillar protein showed a dose-dependent increase after ingestion of 15, 30, or 45 g protein (0.0171 ± 0.0017, 0.0296 ± 0.0030, and 0.0397 ± 0.0026 mole percentage excess, respectively; P < 0.05).

Conclusions: Dietary protein ingested during recovery from resistance exercise is rapidly digested and absorbed. Whole-body net protein balance and dietary protein-derived amino acid incorporation into myofibrillar protein show dose-dependent increases. Ingestion of ≥30 g protein increases postexercise myofibrillar protein synthesis rates in older men. This trial was registered at Nederlands Trial Register as NTR4492.
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http://dx.doi.org/10.1093/jn/nxy263DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6374151PMC
February 2019

Mass Spectrometric Identification of Cardiac Troponin T in Urine of Patients Suffering from Acute Myocardial Infarction.

J Appl Lab Med 2018 May;2(6):857-867

Department of Clinical Chemistry, Central Diagnostic Laboratory, Maastricht University Medical Center, Maastricht, the Netherlands.

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.
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http://dx.doi.org/10.1373/jalm.2017.024224DOI Listing
May 2018

Brain tissue plasticity: protein synthesis rates of the human brain.

Brain 2018 04;141(4):1122-1129

Department of Human Biology and Movement Sciences, NUTRIM School of Nutrition and Translational Research in Metabolism, Maastricht University Medical Centre+, Maastricht, The Netherlands.

All tissues undergo continuous reconditioning via the complex orchestration of changes in tissue protein synthesis and breakdown rates. Skeletal muscle tissue has been well studied in this regard, and has been shown to turnover at a rate of 1-2% per day in vivo in humans. Few data are available on protein synthesis rates of other tissues. Because of obvious limitations with regard to brain tissue sampling no study has ever measured brain protein synthesis rates in vivo in humans. Here, we applied stable isotope methodology to directly assess protein synthesis rates in neocortex and hippocampus tissue of six patients undergoing temporal lobectomy for drug-resistant temporal lobe epilepsy (Clinical trial registration: NTR5147). Protein synthesis rates of neocortex and hippocampus tissue averaged 0.17 ± 0.01 and 0.13 ± 0.01%/h, respectively. Brain tissue protein synthesis rates were 3-4-fold higher than skeletal muscle tissue protein synthesis rates (0.05 ± 0.01%/h; P < 0.001). In conclusion, the protein turnover rate of the human brain is much higher than previously assumed.
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http://dx.doi.org/10.1093/brain/awy015DOI Listing
April 2018

Thrombin Activation via Serum Preparation Is Not the Root Cause for Cardiac Troponin T Degradation.

Clin Chem 2017 11 21;63(11):1768-1769. Epub 2017 Sep 21.

Central Diagnostic Laboratory Maastricht University Medical Centre Maastricht, The Netherlands

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http://dx.doi.org/10.1373/clinchem.2017.279182DOI Listing
November 2017

Large Variation in Measured Cardiac Troponin T Concentrations after Standard Addition in Serum or Plasma of Different Individuals.

Clin Chem 2017 07 23;63(7):1300-1302. Epub 2017 May 23.

Department of Clinical Chemistry Maastricht University Medical Centre Maastricht, The Netherlands

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http://dx.doi.org/10.1373/clinchem.2017.272435DOI Listing
July 2017

Food ingestion in an upright sitting position increases postprandial amino acid availability when compared with food ingestion in a lying down position.

Appl Physiol Nutr Metab 2017 Jul 17;42(7):738-743. Epub 2017 Feb 17.

a Department of Human Biology and Movement Sciences, NUTRIM School of Nutrition and Translational Research in Metabolism, Maastricht University Medical Center+, P.O. Box 616, 6200 MD Maastricht, the Netherlands.

Dietary protein digestion and absorption kinetics determine the postprandial increase in muscle protein synthesis. We recently demonstrated that body position during feeding can modulate the postprandial rise in plasma amino acid availability. Here we investigated whether protein ingestion in an upright sitting body position accelerates gastric emptying and improves dietary protein digestion and subsequent amino acid absorption compared with feeding in a supine lying body position. In a crossover design, 8 young males (age, 26 ± 1 years; body mass index, 24.0 ± 0.9 kg·m) ingested 20 g intrinsically l-[1-C]-phenylalanine-labeled milk protein plus 1.5 g paracetamol while sitting in an upright position or lying down in a supine position. Blood samples were collected frequently during a 5-h postprandial period. Gastric emptying rates and dietary protein digestion and absorption were assessed using plasma paracetamol and amino acid concentrations as well as plasma l-[1-C]-phenylalanine enrichments. Peak plasma leucine concentrations were higher when protein was ingested in an upright sitting versus lying position (213 ± 15 vs 193 ± 12 μmol·L, P < 0.05), which was accompanied by a trend for a greater overall leucine response (13 989 ± 720 vs 11 875 ± 1073 AU, respectively; P = 0.05). Peak plasma paracetamol concentrations were higher in the sitting versus lying treatment (11.6 ± 0.5 vs 9.3 ± 0.6 mg·L, P < 0.05). Protein ingestion in an upright sitting position accelerates gastric emptying and increases the postprandial rise in plasma amino acid availability by increasing protein digestion and amino acid absorption rates. Therefore, feeding in an upright body position as opposed to a lying position is an important prerequisite to allow proper postprandial muscle protein accretion.
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http://dx.doi.org/10.1139/apnm-2016-0522DOI Listing
July 2017

Identification and Characterization of Cardiac Troponin T Fragments in Serum of Patients Suffering from Acute Myocardial Infarction.

Clin Chem 2017 Feb 9;63(2):563-572. Epub 2016 Dec 9.

Department of Clinical Chemistry, Central Diagnostic Laboratory, Maastricht University Medical Centre, Maastricht, the Netherlands;

Background: Cardiac 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.

Methods: cTnT 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.

Results: Ten 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.

Conclusions: Using 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.
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http://dx.doi.org/10.1373/clinchem.2016.261511DOI Listing
February 2017

Cardiac troponin T degradation in serum is catalysed by human thrombin.

Biochem Biophys Res Commun 2016 Dec 2;481(1-2):165-168. Epub 2016 Nov 2.

Department of Clinical Chemistry, Central Diagnostic Laboratory, Maastricht University Medical Centre, Maastricht, The Netherlands. Electronic address:

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 proteases 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.
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http://dx.doi.org/10.1016/j.bbrc.2016.10.149DOI Listing
December 2016

Increasing Insulin Availability Does Not Augment Postprandial Muscle Protein Synthesis Rates in Healthy Young and Older Men.

J Clin Endocrinol Metab 2016 Nov 19;101(11):3978-3988. Epub 2016 Jul 19.

Top Institute Food and Nutrition (B.B.L.G., A.M.H.H., H.M.H., J.v.K., L.J.C.v.L.), 6709 PG Wageningen, The Netherlands; Departments of Human Biology and Movement Sciences (B.B.L.G., A.M.H.H., H.M.H., L.J.C.v.L.), Radiology (M.d.H.), Clinical Genetics, and Surgery (M.P.), Laboratory Biochemical Genetics (J.B.), and Central Diagnostic Laboratory (W.K.W.H.W.), Maastricht University Medical Centre, 6200 MD Maastricht, The Netherlands; and Department of Nutrition and Metabolism (B.B.R.), University of Texas Medical Branch, Galveston, Texas 77550.

Context: Skeletal muscle protein synthesis is highly responsive to food intake. It has been suggested that the postprandial increase in circulating insulin modulates the muscle protein synthetic response to feeding.

Objective: The objective of the study was to investigate whether a greater postprandial rise in circulating insulin level increases amino acid uptake in muscle and augments postprandial muscle protein synthesis rates.

Participants And Design: Forty-eight healthy young (age 22 ± 1 y; body mass index 22.0 ± 0.3 kg/m) and older males (age 68 ± 1 y; body mass index 26.3 ± 0.4 kg/m) ingested 20 g intrinsically L-[1-C]-leucine- and L-[1-C]-phenylalanine-labeled casein protein with or without local insulin infusion. Primed continuous infusions of L-[1-C]-leucine and L-[ring-H]-phenylalanine were applied, with arterial and venous blood samples and muscle biopsies being collected during a 5-hour postprandial period.

Results: Insulin administration did not increase overall leg blood flow (P = .509) but increased amino acid uptake over the leg in both young and older subjects (P = .003). The greater amino acid uptake over the leg did not further increase postprandial muscle protein synthesis rates (0.050% ± 0.006% and 0.037% ± 0.004% per hour vs 0.044% ± 0.004% and 0.037% ± 0.002% per hour in the insulin-stimulated vs control condition in the young and older groups, respectively; P = .804) and did not affect postprandial deposition of dietary protein-derived amino acids in de novo muscle protein (P = .872).

Conclusion: Greater postprandial plasma insulin availability stimulates amino acid uptake over the leg but does not further augment postprandial muscle protein synthesis rates or stimulate the postprandial deposition of protein derived amino acids into de novo muscle protein in healthy young and older men.
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http://dx.doi.org/10.1210/jc.2016-1436DOI Listing
November 2016

Ascorbic acid serum levels are reduced in patients with hematological malignancies.

Results Immunol 2016 12;6:8-10. Epub 2016 Jan 12.

Department of Internal Medicine, Division of Hematology, Maastricht University Medical Center, Maastricht, The Netherlands.

In this paper we demonstrate that patients treated with chemotherapy and/or hematopoietic stem cell transplantation (HSCT) have highly significant reduced serum ascorbic acid (AA) levels compared to healthy controls. We recently observed in in vitro experiments that growth of both T and NK cells from hematopoietic stem cells is positively influenced by AA. It might be of clinical relevance to study the function and recovery of immune cells after intensive treatment, its correlation to AA serum levels and the possible effect of AA supplementation.
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http://dx.doi.org/10.1016/j.rinim.2016.01.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4792862PMC
March 2016

Validation, optimisation, and application data in support of the development of a targeted selected ion monitoring assay for degraded cardiac troponin T.

Data Brief 2016 Jun 3;7:397-405. Epub 2016 Mar 3.

Central Diagnostic Laboratory, Maastricht University Medical Centre, Maastricht, the Netherlands.

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" [1]. 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.
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http://dx.doi.org/10.1016/j.dib.2016.02.051DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4782000PMC
June 2016

Development of a targeted selected ion monitoring assay for the elucidation of protease induced structural changes in cardiac troponin T.

J Proteomics 2016 Mar 6;136:123-32. Epub 2016 Jan 6.

Central Diagnostic Laboratory, Maastricht University Medical Centre, Maastricht, the Netherlands. Electronic address:

Unlabelled: 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 16kDa (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.
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http://dx.doi.org/10.1016/j.jprot.2015.12.028DOI Listing
March 2016

Post-Prandial Protein Handling: You Are What You Just Ate.

PLoS One 2015 10;10(11):e0141582. Epub 2015 Nov 10.

Top Institute Food and Nutrition, Wageningen, The Netherlands.

Background: Protein turnover in skeletal muscle tissue is highly responsive to nutrient intake in healthy adults.

Objective: To provide a comprehensive overview of post-prandial protein handling, ranging from dietary protein digestion and amino acid absorption, the uptake of dietary protein derived amino acids over the leg, the post-prandial stimulation of muscle protein synthesis rates, to the incorporation of dietary protein derived amino acids in de novo muscle protein.

Design: 12 healthy young males ingested 20 g intrinsically [1-13C]-phenylalanine labeled protein. In addition, primed continuous L-[ring-2H5]-phenylalanine, L-[ring-2H2]-tyrosine, and L-[1-13C]-leucine infusions were applied, with frequent collection of arterial and venous blood samples, and muscle biopsies throughout a 5 h post-prandial period. Dietary protein digestion, amino acid absorption, splanchnic amino acid extraction, amino acid uptake over the leg, and subsequent muscle protein synthesis were measured within a single in vivo human experiment.

Results: 55.3±2.7% of the protein-derived phenylalanine was released in the circulation during the 5 h post-prandial period. The post-prandial rise in plasma essential amino acid availability improved leg muscle protein balance (from -291±72 to 103±66 μM·min-1·100 mL leg volume-1; P<0.001). Muscle protein synthesis rates increased significantly following protein ingestion (0.029±0.002 vs 0.044±0.004%·h-1 based upon the muscle protein bound L-[ring-2H5]-phenylalanine enrichments (P<0.01)), with substantial incorporation of dietary protein derived L-[1-13C]-phenylalanine into de novo muscle protein (from 0 to 0.0201±0.0025 MPE).

Conclusion: Ingestion of a single meal-like amount of protein allows ~55% of the protein derived amino acids to become available in the circulation, thereby improving whole-body and leg protein balance. About 20% of the dietary protein derived amino acids released in the circulation are taken up in skeletal muscle tissue following protein ingestion, thereby stimulating muscle protein synthesis rates and providing precursors for de novo muscle protein synthesis.

Trial Registration: trialregister.nl 3638.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0141582PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4640549PMC
June 2016

Integrating multiple omics to unravel mechanisms of Cyclosporin A induced hepatotoxicity in vitro.

Toxicol In Vitro 2015 Apr 3;29(3):489-501. Epub 2015 Jan 3.

Department of Toxicogenomics, Maastricht University, Maastricht, The Netherlands; Netherlands Toxicogenomics Centre, Maastricht, The Netherlands. Electronic address:

In order to improve attrition rates of candidate-drugs there is a need for a better understanding of the mechanisms underlying drug-induced hepatotoxicity. We aim to further unravel the toxicological response of hepatocytes to a prototypical cholestatic compound by integrating transcriptomic and metabonomic profiling of HepG2 cells exposed to Cyclosporin A. Cyclosporin A exposure induced intracellular cholesterol accumulation and diminished intracellular bile acid levels. Performing pathway analyses of significant mRNAs and metabolites separately and integrated, resulted in more relevant pathways for the latter. Integrated analyses showed pathways involved in cell cycle and cellular metabolism to be significantly changed. Moreover, pathways involved in protein processing of the endoplasmic reticulum, bile acid biosynthesis and cholesterol metabolism were significantly affected. Our findings indicate that an integrated approach combining metabonomics and transcriptomics data derived from representative in vitro models, with bioinformatics can improve our understanding of the mechanisms of action underlying drug-induced hepatotoxicity. Furthermore, we showed that integrating multiple omics and thereby analyzing genes, microRNAs and metabolites of the opposed model for drug-induced cholestasis can give valuable information about mechanisms of drug-induced cholestasis in vitro and therefore could be used in toxicity screening of new drug candidates at an early stage of drug discovery.
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http://dx.doi.org/10.1016/j.tiv.2014.12.016DOI Listing
April 2015

Integrative cross-omics analysis in primary mouse hepatocytes unravels mechanisms of cyclosporin A-induced hepatotoxicity.

Toxicology 2014 Oct 15;324:18-26. Epub 2014 Jul 15.

Department of Toxicogenomics, Maastricht University, Maastricht, The Netherlands; Netherlands Toxicogenomics Centre, Maastricht, The Netherlands. Electronic address:

The liver is responsible for drug metabolism and drug-induced hepatotoxicity is the most frequent reason for drug withdrawal, indicating that better pre-clinical toxicity tests are needed. In order to bypass animal models for toxicity screening, we exposed primary mouse hepatocytes for exploring the prototypical hepatotoxicant cyclosporin A. To elucidate the mechanisms underlying cyclosporin A-induced hepatotoxicity, we analyzed expression levels of proteins, mRNAs, microRNAs and metabolites. Integrative analysis of transcriptomics and proteomics showed that protein disulfide isomerase family A, member 4 was up-regulated on both the protein level and mRNA level. This protein is involved in protein folding and secretion in the endoplasmic reticulum. Furthermore, the microRNA mmu-miR-182-5p which is predicted to interact with the mRNA of this protein, was also differentially expressed, further emphasizing endoplasmic reticulum stress as important event in drug-induced toxicity. To further investigate the interaction between the significantly expressed proteins, a network was created including genes and microRNAs known to interact with these proteins and this network was used to visualize the experimental data. In total 6 clusters could be distinguished which appeared to be involved in several toxicity related processes, including alteration of protein folding and secretion in the endoplasmic reticulum. Metabonomic analyses resulted in 5 differentially expressed metabolites, indicative of an altered glucose, lipid and cholesterol homeostasis which can be related to cholestasis. Single and integrative analyses of transcriptomics, proteomics and metabonomics reveal mechanisms underlying cyclosporin A-induced cholestasis demonstrating that endoplasmic reticulum stress and the unfolded protein response are important processes in drug-induced liver toxicity.
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http://dx.doi.org/10.1016/j.tox.2014.06.003DOI Listing
October 2014

Cardiac troponin in ischemic cardiomyocytes: intracellular decrease before onset of cell death.

Exp Mol Pathol 2014 Jun 4;96(3):339-45. Epub 2014 Mar 4.

Department of Clinical Chemistry, Maastricht University Medical Centre, Maastricht, The Netherlands. Electronic address:

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.
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http://dx.doi.org/10.1016/j.yexmp.2014.02.012DOI Listing
June 2014

Classification of hepatotoxicants using HepG2 cells: A proof of principle study.

Chem Res Toxicol 2014 Mar 28;27(3):433-42. Epub 2014 Jan 28.

Department of Toxicogenomics, Maastricht University , Maastricht, The Netherlands.

With the number of new drug candidates increasing every year, there is a need for high-throughput human toxicity screenings. As the liver is the most important organ in drug metabolism and thus capable of generating relatively high levels of toxic metabolites, it is important to find a reliable strategy to screen for drug-induced hepatotoxicity. Microarray-based transcriptomics is a well-established technique in toxicogenomics research and is an ideal approach to screen for drug-induced injury at an early stage. The aim of this study was to prove the principle of classifying known hepatotoxicants and nonhepatotoxicants using their distinctive gene expression profiles in vitro in HepG2 cells. Furthermore, we undertook to subclassify the hepatotoxic compounds by investigating the subclass of cholestatic compounds. Prediction analysis for microarrays was used for classification of hepatotoxicants and nonhepatotoxicants, which resulted in an accuracy of 92% on the training set and 91% on the validation set, using 36 genes. A second model was set up with the goal of finding classifiers for cholestasis, resulting in 12 genes that appeared capable of correctly classifying 8 of the 9 cholestatic compounds, resulting in an accuracy of 93%. We were able to prove the principle that transcriptomic analyses of HepG2 cells can indeed be used to classify chemical entities for hepatotoxicity. Genes selected for classification of hepatotoxicity and cholestasis indicate that endoplasmic reticulum stress and the unfolded protein response may be important cellular effects of drug-induced liver injury. However, the number of compounds in both the training set and the validation set should be increased to improve the reliability of the prediction.
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http://dx.doi.org/10.1021/tx4004165DOI Listing
March 2014

Critical appraisal of 13C breath tests for microsomal liver function: aminopyrine revisited.

Liver Int 2014 Apr 16;34(4):487-94. Epub 2014 Jan 16.

Division of Gastroenterology and Hepatology, Department of Internal Medicine, Maastricht University Medical Center, Maastricht, the Netherlands.

As liver diseases are a major health problem and especially the incidence of metabolic liver diseases like non-alcoholic fatty liver disease (NAFLD) is rising, the demand for non-invasive tests is growing to replace liver biopsy. Non-invasive tests such as carbon-labelled breath tests can provide a valuable contribution to the evaluation of metabolic liver function. This review aims to critically appraise the value of the (13) C-labelled microsomal breath tests for the evaluation of metabolic liver function, and to discuss the role of cytochrome P450 enzymes in the metabolism of the different probe drugs, especially of aminopyrine. Although a number of different probe drugs have been used in breath tests, the perfect drug to assess the functional metabolic capacity of the liver has not been found. Data suggest that both the (13) C(2) -aminopyrine and the (13) C-methacetin breath test can play a role in assessing the capacity of the microsomal liver function and may be useful in the follow-up of patients with chronic liver diseases. Furthermore, CYP2C19 seems to be an important enzyme in the N-demethylation of aminopyrine, and polymorphisms in this gene may influence breath test values, which should be kept in mind when performing the (13) C(2) -aminopyrine breath test in clinical practice.
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http://dx.doi.org/10.1111/liv.12451DOI Listing
April 2014

Posttranslational modifications of cardiac troponin T: an overview.

J Mol Cell Cardiol 2013 Oct 18;63:47-56. Epub 2013 Jul 18.

Department of Clinical Chemistry, Maastricht University Medical Centre, Maastricht, The Netherlands.

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.
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http://dx.doi.org/10.1016/j.yjmcc.2013.07.004DOI Listing
October 2013

Characterization of the perfusate proteome of human donor kidneys.

Ann Clin Biochem 2013 Mar 21;50(Pt 2):140-6. Epub 2013 Feb 21.

Department of Surgery, Maastricht University Medical Center, PO Box 5800, Maastricht 6202 AZ, The Netherlands.

Background: Preservation of deceased donor kidneys by hypothermic machine perfusion results in superior transplant outcomes as compared with static cold storage and provides the opportunity to measure biomarkers of cellular injury in perfusate samples. Identification of biomarkers predicting early graft dysfunction so far has met with limited success.

Methods: Two-dimensional difference gel electrophoresis and mass spectrometry were used to explore the proteome of perfusate samples from machine-perfused human donor kidneys (N = 18) and to discover novel biomarkers of ischaemic acute kidney injury.

Results: Thirty-two protein spots were successfully identified, representing 19 unique proteins that were derived from renal tissue and from residual plasma in the renal microcirculation. Two unidentified protein spots were significantly up-regulated, whereas one protein spot--identified as haptoglobin--was significantly down-regulated in the perfusate of ischaemically injured kidneys from donors after cardiac death as compared with kidneys from brain-dead donors who had not suffered warm ischaemic injury. Furthermore, two protein spots were up-regulated in kidneys that never functioned after transplantation, whereas one spot was up-regulated--identified as α1-antitrypsin--in kidneys with delayed graft function.

Conclusions: We provide the first description of the renal perfusate proteome and present preliminary evidence of differentially expressed biomarkers in human donor kidneys with different levels of acute ischaemic injury. Their diagnostic value for the selection of marginal kidneys in clinical transplantation should be determined in future studies.
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http://dx.doi.org/10.1258/acb.2012.011144DOI Listing
March 2013

Protein supplementation during resistance-type exercise training in the elderly.

Med Sci Sports Exerc 2013 Mar;45(3):542-52

Top Institute Food and Nutrition, Wageningen, The Netherlands.

Introduction: Resistance training has been well established as an effective treatment strategy to increase skeletal muscle mass and strength in the elderly. We assessed whether dietary protein supplementation can further augment the adaptive response to prolonged resistance-type exercise training in healthy elderly men and women.

Methods: Healthy elderly men (n = 31, 70 ± 1 yr) and women (n = 29, 70 ± 1 yr) were randomly assigned to a progressive, 24-wk resistance-type exercise training program with or without additional protein supplementation (15 g·d-1). Muscle hypertrophy was assessed on a whole-body Dual-energy X-ray absorptiometry (DXA), limb (computed tomography), and muscle fiber (biopsy) level. Strength was assessed regularly by 1-repetition maximum (RM) strength testing. Functional capacity was assessed with a sit-to-stand and handgrip test.

Results: One-RM strength increased by 45% ± 6% versus 40% ± 3% (women) and 41% ± 4% versus 44% ± 3% (men) in the placebo versus protein group, respectively (P < 0.001), with no differences between groups. Leg muscle mass (women, 4% ± 1% vs 3% ± 1%; men, 3% ± 1% vs 3% ± 1%) and quadriceps cross-sectional area (women, 9% ± 1% vs 9% ± 1%; men, 9% ± 1% vs 10% ± 1%) increased similarly in the placebo versus protein groups (P < 0.001). Type II muscle fiber size increased over time in both placebo and protein groups (25% ± 13% vs 30% ± 9% and 23% ± 12% vs 22% ± 10% in the women and men, respectively). Sit-to-stand improved by 18% ± 2% and 19% ± 2% in women and men, respectively (P < 0.001).

Conclusion: Prolonged resistance-type exercise training increases skeletal muscle mass and strength, augments functional capacity, improves glycemia and lipidemia, and reduces blood pressure in healthy elderly men and women. Additional protein supplementation (15 g·d-1) does not further increase muscle mass, strength, and/or functional capacity.
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http://dx.doi.org/10.1249/MSS.0b013e318272fcdbDOI Listing
March 2013

Circulating biomarkers and abdominal aortic aneurysm size.

J Surg Res 2012 Aug 11;176(2):672-8. Epub 2011 Oct 11.

Department of Surgery, Maastricht University Medical Centre, Maastricht, The Netherlands.

Background: Abdominal aortic aneurysm (AAA) is a degenerative disease of the abdominal aorta leading to progressive dilatation, intra-luminal thrombus (ILT) formation, and rupture. Understanding the natural history of AAA is essential, because different processes and, therefore, different biomarkers, could be involved at each stage of disease progression. The purpose of the present study was to investigate the relationship between systemic expression of biomarkers of inflammation and extracellular matrix remodeling and aneurysm size in AAA patients.

Methods And Results: All consecutive patients admitted to the (out-) patient clinic of the surgical department of two large community centers were prospectively included. Patients were divided into three groups based on their aneurysm diameter: small (30-44 mm; n = 59), medium-sized (45-54 mm; n = 64) or large (≥ 55 mm; n = 95) AAA. Linear regression modeling showed that age and serum hsCRP concentration were positively associated, whereas serum HDL and IgG concentrations were negatively associated with aneurysm size. This regression model was corrected for possible bias due to statin use and center of inclusion; and also indicated that in general men have larger aneurysms compared with women.

Conclusions: Different aneurysm sizes showed different expression pattern of HDL, IgG, and hsCRP. These biomarkers may be useful in predicting AAA progression.
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http://dx.doi.org/10.1016/j.jss.2011.09.040DOI Listing
August 2012

Blood profiling of proteins and steroids during weight maintenance with manipulation of dietary protein level and glycaemic index.

Br J Nutr 2012 Jan 23;107(1):106-19. Epub 2011 Jun 23.

Department of Human Biology, NUTRIM School for Nutrition, Toxicology and Metabolism, Maastricht University Medical Centre, PO 616, 6200 MD Maastricht, The Netherlands.

Weight regain after weight loss is common. In the Diogenes dietary intervention study, a high-protein and low-glycaemic index (GI) diet improved weight maintenance. The objective of the present study was to identify (1) blood profiles associated with continued weight loss and weight regain (2) blood biomarkers of dietary protein and GI levels during the weight-maintenance phase. Blood samples were collected at baseline, after 8 weeks of low-energy diet-induced weight loss and after a 6-month dietary intervention period from female continued weight losers (n 48) and weight regainers (n 48), evenly selected from four dietary groups that varied in protein and GI levels. The blood concentrations of twenty-nine proteins and three steroid hormones were measured. The changes in analytes during weight maintenance largely correlated negatively with the changes during weight loss, with some differences between continued weight losers and weight regainers. Increases in leptin (LEP) and C-reactive protein (CRP) were significantly associated with weight regain (P < 0·001 and P = 0·005, respectively), and these relationships were influenced by the diet. Consuming a high-protein and high-GI diet dissociated the positive relationship between the change in LEP concentration and weight regain. CRP increased during the weight-maintenance period only in weight regainers with a high-protein diet (P < 0·001). In addition, testosterone, luteinising hormone, angiotensinogen, plasminogen activator inhibitor-1, resistin, retinol-binding protein 4, insulin, glucagon, haptoglobin and growth hormone were also affected by the dietary intervention. The blood profile reflects not only the weight change during the maintenance period, but also the macronutrient composition of the dietary intervention, especially the protein level.
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http://dx.doi.org/10.1017/S0007114511002583DOI Listing
January 2012

Prolonged leucine supplementation does not augment muscle mass or affect glycemic control in elderly type 2 diabetic men.

J Nutr 2011 Jun 27;141(6):1070-6. Epub 2011 Apr 27.

Top Institute Food and Nutrition, 6700 AN Wageningen, The Netherlands.

The loss of muscle mass with aging has been, at least partly, attributed to a blunted muscle protein synthetic response to food intake. Leucine coingestion has been reported to stimulate postprandial insulin release and augment postprandial muscle protein accretion. We assessed the clinical benefits of 6 mo of leucine supplementation in elderly, type 2 diabetes patients. Sixty elderly males with type 2 diabetes (age, 71 ± 1 y; BMI, 27.3 ± 0.4 kg/m(2)) were administered 2.5 g L-leucine (n = 30) or a placebo (n = 30) with each main meal during 6 mo of nutritional intervention (7.5 g/d leucine or placebo). Body composition, muscle fiber characteristics, muscle strength, glucose homeostasis, and basal plasma amino acid and lipid concentrations were assessed prior to, during, and after intervention. Lean tissue mass did not change or differ between groups and at 0, 3, and 6 mo were 61.9 ± 1.1, 62.2 ± 1.1, and 62.0 ± 1.0 kg, respectively, in the leucine group and 62.2 ± 1.3, 62.2 ± 1.3, and 62.2 ± 1.3 kg in the placebo group. There also were no changes in body fat percentage, muscle strength, and muscle fiber type characteristics. Blood glycosylated hemoglobin did not change or differ between groups and was 7.1 ± 0.1% in the leucine group and 7.2 ± 0.2% in the placebo group. Consistent with this, oral glucose insulin sensitivity and plasma lipid concentrations did not change or differ between groups. We conclude that prolonged leucine supplementation (7.5 g/d) does not modulate body composition, muscle mass, strength, glycemic control, and/or lipidemia in elderly, type 2 diabetes patients who habitually consume adequate dietary protein.
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http://dx.doi.org/10.3945/jn.111.138495DOI Listing
June 2011
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