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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; n = 12) or without (15G; n = 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-[ ring-2H5]phenylalanine, l-[ ring-2H2]tyrosine, and l-[1-13C]leucine combined with ingestion of intrinsically l-[1-13C]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 ( P < 0.05). Postexercise myofibrillar protein synthesis rates were 16% (0.058 ± 0.003 vs. 0.049 ± 0.002%/h, P < 0.05; based on l-[ ring-2H5]phenylalanine) and 19% (0.071 ± 0.003 vs. 0.060 ± 0.003%/h, P < 0.05; based on l-[1-13C]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.

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.

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(2)) and older males (age 68 +/- 1 y; body mass index 26.3 +/- 0.4 kg/m(2)) ingested 20 g intrinsically L-[1-C-13]-leucine-and L-[1-C-13]-phenylalanine-labeled casein protein with or without local insulin infusion. Primed continuous infusions of L-[1-C-13]-leucine and L-[ring-H-2(5)]-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 overthe 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.

Background: The age-related decline in skeletal muscle mass is partly attributed to anabolic resistance to food intake. Dietary protein ingestion before sleep could be used as a nutritional strategy to compensate for anabolic resistance. Objective: The present study assessed whether physical activity performed in the evening can augment the overnight muscle protein synthetic response to presleep protein ingestion in older men. Methods: In a parallel group design, 23 healthy older men (mean SEM age: 71 1 y) were randomly assigned to ingest 40 g protein intrinsically labeled with L[1-C-13]-phenylalanine and L-[1-C-13]-leucine before going to sleep with (PRO+EX) or without (PRO) performing physical activity earlier in the evening. Overnight protein digestion and absorption kinetics and myofibrillar protein synthesis rates were assessed by combining primed, continuous infusions of L-[ring-H-2(5)]-phenylalanine, L-[1-C-13]-leucine, and L-[ring-H-2(2)]-tyrosine with the ingestion of intrinsically labeled casein protein. Muscle and blood samples were collected throughout overnight sleep. Results: Protein ingested before sleep was normally digested and absorbed, with 54% +/- 2% of the protein-derived amino acids appearing in the circulation throughout overnight sleep. Overnight myofibrillar protein synthesis rates were 31 % (0.058% +/- 0.002%/h compared with 0.044% +/- 0.003%/h; P

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).

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 …

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.

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.

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 …

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.

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.

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 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

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.

Objective: The aim of the study is to investigate the differential expression of proteins in serum of abdominal aortic aneurysm (AAA) patients in relation to aneurysm size (Dmax) and progression. Methods: Two-dimensional differential in-gel electrophoresis (2D-DIGE) together with tandem mass spectrometry (MS/MS) was used to analyse the serum proteome from patients with small (Dmax 30e54 mm) AAA, either stable (increase Dmax < 5m m year � 1 ; n Z 8) or progressive (increase Dmax �5 mm year � 1 ; n Z 8), and large (Dmax �55 mm; n Z 8) AAA. The identified proteins were quantitatively validated in a larger population (n Z 80). Results: Several proteins were differentially expressed in serum of small stable, small progres- sive and large AAA. Three validated proteins (immunoglobulin G (IgG), a1-antitrypsin (a1-AT) and Factor XII activity) showed strong correlation with Dmax. Size combined with either Factor XII activity or a1-antitrypsin had minimal effect on the prognostic value in predicting aneurysm progression compared with size alone (area under the curve (AUC), 0.85; 95% confidence interval (CI), 0.73e0.97; p < 0.001 and AUC, 0.85; 95% CI, 0.72e0.98; p < 0.001 vs. AUC, 0.83; 95% CI, 0.71e0.96; p < 0.001, respectively). Conclusion: The present study indicates that both Factor XII and a1-antitrypsin are found in increased amounts in the serum of patients with expanding AAA. However, combination of either Factor XII or a1-antitrypsin with aneurysm diameter had little effect on prediction of aneurysm progression versus diameter alone.

Contains fulltext : 96918.pdf (Publisher’s version ) (Closed access) Female classic galactosemia patients suffer from primary ovarian insufficiency (POI). The cause for this long-term complication is not fully understood. One of the proposed mechanisms is that hypoglycosylation of complex molecules, a known secondary phenomenon of galactosemia, leads to FSH dysfunction. An earlier study showed less acidic isoforms of FSH in serum samples of two classic galactosemia patients compared to controls, indicating hypoglycosylation. In this study, FSH isoform patterns of five classic galactosemia patients with POI were compared to the pattern obtained in two patients with a primary glycosylation disorder (phosphomannomutase-2-deficient congenital disorders of glycosylation, PMM2-CDG) and POI, and in five postmenopausal women as controls. We used FPLC chromatofocussing with measurement of FSH concentration per fraction, and discovered that there were no significant differences between galactosemia patients, PMM2-CDG patients and postmenopausal controls. Our results do not support that FSH dysfunction due to a less acidic isoform pattern because of hypoglycosylation is a key mechanism of POI in this disease.

Objectives This study investigated the relation between abdominal aortic aneurysm (AAA) wall stress, AAA growth rate and biomarker concentrations. With increasing wall stress, more damage may be caused to the AAA wall, possibly leading to progression of the aneurysm and reflection in up- or downregulation of specific circulating biomarkers. Levels of matrix metalloproteinase-9, tissue inhibitor of matrix metalloproteinase-1, C-reactive protein and alpha 1-antitrypsin were therefore evaluated. Methods Thirty-seven patients (maximum AAA diameter 41–55 mm) with two, three or four consecutive computed tomography angiography (CTA) scans were prospectively included. Diameter growth rate in mm/year was determined between each pair of two sequential CTA scans. AAA wall stress was computed by finite element analysis, based on the first of the two sequential CTA scans only ( n = 69 pairs). Biomarker information was determined in 46 measurements in 18 patients. The relation between AAA diameter and wall stress was determined and the AAA's were divided into three equally sized groups (relative low, medium and high stress). Growth rate and biomarker concentrations were compared between these groups. Additionally, correlation coefficients were computed between absolute wall stress, AAA growth and biomarker concentrations. Results A relative low AAA wall stress was associated with a lower aneurysm growth rate. Growth rate was also positively related to MMP-9 plasma concentration ( r = 0.32). The average MMP-9 and CRP concentrations increased with increasing degrees of relative wall stress, although the absolute and relative wall stress did not correlate with any of the biomarkers. Conclusion Although lower relative wall stress was associated to a lower AAA growth rate, no relation was found between biomarker concentrations and wall stress. Future research may focus on more and extensive biomarker measurements in relation to AAA wall stress.

Abdominal aortic aneurysm (AAA) is an important health problem. Elective surgical treatment is recommended on the basis of an individual's risk of rupture, which is predicted by AAA diameter. However, the natural history of AAA differs between patients and a reliable and individual predictor of AAA progression (growth and expansion rates) has not been established. Several circulating biomarkers are candidates for an AAA diagnostic tool. However, they have yet to meet the triad of biomarker criteria: biological plausibility, correlation with AAA progression, and prediction of treatment effect on disease outcome. Circulating levels of markers of extracellular matrix degeneration, such as elastin peptides, aminoterminal propeptide of type III procollagen, elastase-alpha1-antitrypsin complexes, matrix metalloproteinase 9, cystatin C, plasmin-antiplasmin complexes and tissue plasminogen activator, have been correlated with AAA progression and have biological plausibility. Although studies of these markers have shown promising results, they have not yet led to a clinically applicable biomarker. In future studies, adjustment for initial AAA size, smoking history and the measurement error for determination of AAA size, among other variables, should be taken into account. A large, prospective, standardized, follow-up study will be needed to investigate multiple circulating biomarkers for their potential role in the prediction of AAA progression, followed by a study to investigate the effect of treatment on the circulating levels of biomarkers.

Aim: Ankylosing spondylitis (AS) is a chronic systemic inflammatory rheumatic disorder that primarily affects the axial skeleton. To date there is no satisfactory biomarker for diagnosis or prognosis of AS. This study is focused on detection of potential biomarkers for the diagnosis of AS using surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF-MS) with the ultimate goal of developing a progression biomarker for AS. Materials & methods: Protein profiles of sera of 38 AS patients and 38 healthy controls were obtained using ProteinChip arrays. Different arrays and conditions were compared with find out which condition resulted in the optimal discrimination of both groups. Results: Optimal discrimination was reached using weak cation exchange (CM10) and immobilized metal affinity capture coupled with copper (IMAC-Cu2+) arrays. Using a multimarker approach on protein profiles obtained with CM10 arrays resulted in a sensitivity of 66% and a specificity of 74%. Using a multimarker approach on protein profiles obtained with IMAC-Cu2+ arrays resulted in a sensitivity and specificity of 70%. Conclusions: This is the first study that shows that protein profiling in serum using SELDI-TOF-MS can be used as a diagnostic tool for AS. The biomarkers will be validated to confirm if they can be used as progression biomarkers.

BACKGROUND: Sarcoidosis is a multi-systemic inflammatory disorder, which affects the lungs in 90% of the cases. The main pathologic feature is chronic inflammation resulting in non-caseating granuloma formation. Until now there is no satisfying biomarker for diagnosis or prognosis of sarcoidosis. This study is focused on the detection of potential biomarkers in serum for the diagnosis of sarcoidosis using surface-enhanced laser desorption ionization-time of flight-mass spectrometry (SELDI-TOF-MS). METHODS: For detection of potential biomarkers, protein profiles of anion exchange fractionated serum of 35 sarcoidosis patients and 35 healthy controls were compared using SELDI-TOF-MS. Sensitivities and specificities of the potential biomarkers obtained with SELDI-TOF-MS, generated with decision tree algorithm, were compared to the conventional markers angiotensin converting enzyme (ACE) and soluble interleukin-2 receptor (sIL-2R). RESULTS: Optimal classification was achieved with metal affinity binding arrays. A single marker with a mass-to-charge (m/z) value of 11,955 resulted in a sensitivity and specificity of 86% and 63%, respectively. A multimarker approach of two peaks, m/z values of 11,734 and 17,377, resulted in a sensitivity and specificity of 74% and 71%, respectively. These sensitivities and specificities were higher compared to measurements of ACE and sIL-2R. Identification of the peak at m/z 17,377 resulted in the alpha-2chain of haptoglobin. CONCLUSIONS: This study acts as a proof-of-principle for the use of SELDI-TOF-MS in the detection of new biomarkers for sarcoidosis. The peak of the multimarker at m/z 17,377 was identified as the alpha-2chain of haptoglobin.

BackgroundProtein turnover in skeletal muscle tissue is highly responsive to nutrient intake in healthy adults.ObjectiveTo 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.Design12 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.Results55.3±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; PConclusionIngestion 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 registrationtrialregister.nl 3638.

Serum protein profiling by surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF MS) appears to be an important diagnostic tool for a whole range of diseases. Sensitivities and specificities obtained with this new technology often seem superior to those obtained with current biomarkers. However, reproducibility and standardization are still problematic. The present review gives an overview of the diagnostic value of protein profiles obtained with SELDI in studies on prostate and ovarian cancer. To identify aspects important for protein profiling, we compare and discuss differences in pre- and post-analytical conditions presented in the literature supplemented with some of our own data. Further progress in protein profiling as a diagnostic tool requires a more comprehensive description of technical details in all future studies.

Background Continuous dialysis could provide benefit by constant removal of potassium and phosphate. This study investigates the suitability of specific potassium and phosphate sorbents for incorporation in an extracorporeal device by capacity and regenerability testing. Methods Capacity testing was performed in uraemic plasma. Regenerability was tested for potassium sorbents, with adsorption based on cationic exchange for sodium, with 0.1 M and 1.0 M NaCl. To regenerate phosphate sorbents, with adsorption based on anionic exchange, 0.1 M and 1.0 M NaHCO3 and NaOH were used. Subsequently, sodium polystyrene divinylbenzene sulphonate (RES-A) and iron oxide hydroxide (FeOOH) beads were incorporated in a cartridge for testing in bovine blood using a recirculating blood circuit and a dialysis circuit separated by a high-flux dialyzer (dynamic setup). Preloading was tested to assess whether this could limit calcium and magnesium adsorption. Results In the batch-binding assays, zirconium phosphate most potently adsorbed potassium (0.44 ± 0.05 mmol/g) and RES-A was the best regenerable potassium sorbent (92.9 ± 5.7% with 0.1 M NaCl). Zirconium oxide hydroxide (ZIR-hydr) most potently adsorbed phosphate (0.23 ± 0.05 mmol/g) and the polymeric amine sevelamer carbonate was the best regenerable sorbent (85.7 ± 5.2% with 0.1 M NaHCO3). In the dynamic setup, a potassium adsorption of 10.72 ± 2.06 mmol in 3 h was achieved using 111 g of RES-A and a phosphate adsorption of 4.73 ± 0.53 mmol in 3 h using 55 g of FeOOH. Calcium and magnesium preloading was shown to reduce the net adsorption in 3 h from 3.57 ± 0.91 to -0.29 ± 1.85 and 1.02 ± 0.05 to -0.31 ± 0.18 mmol, respectively. Conclusion RES-A and FeOOH are suitable, regenerizable sorbents for potassium and phosphate removal in dialysate regeneration. Use of zirconium carbonate and ZIR-hydr may further increase phosphate adsorption, but may compromise sorbent regenerability. Use of polymeric amines for phosphate adsorption may enhance sorbent regenerability. Calcium and magnesium preloading considerably reduced net adsorption of these ions.

BACKGROUND: Hypergonadotropic hypoestrogenic infertility is the most burdensome complication for females suffering from classic galactosemia. In contrast, male gonadal function seems less affected. The underlying mechanism is not understood and several pathogenic mechanisms have been proposed. Timing of the lesion, prenatal or chronic post-natal, or a combination of both are not yet clear. METHODS: This review focuses on gonadal function in males and females, ovarian imaging and histology in this disease. It is based on the literature known to the authors and a Pubmed search using the keywords galactosemia, GALT deficiency, (premature) ovarian failure/insufficiency/dysfunction, testicular function, gonadotrophins, FSH, LH (published between January 1971 and April 2009). RESULTS: Male gonads are less affected, boys spontaneously reach puberty, although onset can be delayed. Semen quality has not been extensively studied. Several affected males are known to have fathered a child. Female gonads are invariably affected, although to a varied extent (hypergonadotropic hypoestrogenic ovarian dysfunction). Intriguingly, FSH is often already increased in infancy. Imaging usually shows hypoplastic and streak-like ovaries. Histological findings in some cases reveal the presence of morphologically normal but decreased numbers of primordial follicles, with the absence of intermediate and Graafian follicles. CONCLUSION: Gonads in males seem less affected than in females who exhibit hypergonadotropic hypoestrogenic subfertility. FSH can be elevated in infancy, and ovarian histology sometimes shows the presence of normal primordial follicles with absence of intermediate and Graafian follicles. These findings are similar to other genetic diseases primarily affecting the ovary.

Contains fulltext : 88162.pdf ( ) (Closed access) Individuals with a spinal cord injury (SCI) have an increased cardiovascular risk. We hypothesize that (anti)oxidative imbalance is associated with the increased cardiovascular risk in SCI, while exercise can reverse this status. The aim of the study is to compare baseline levels of oxidative stress and antioxidative capacity between individuals with SCI and able-bodied (AB) subjects, and to assess acute and long-term effects of functional electrical stimulation (FES) exercise on oxidative stress and antioxidative capacity in SCI. Venous blood was taken from subjects with an SCI (n = 9) and age- and gender-matched AB subjects (n = 9) to examine oxidative stress through malondialdehyde (MDA) levels, while superoxide dismutase (SOD) and glutathione peroxidase (GPx) enzyme levels represented anti-oxidative capacity. Subsequently, subjects with an SCI performed an 8-week FES exercise training period. Blood was taken before and after the first exercise bout and after the last FES session to examine the acute and chronic effect of FES exercise, respectively. Baseline levels of MDA, SOD and GPx were not different between individuals with SCI and AB subjects. SCI demonstrated a correlation between initial fitness level and MDA (R = -0.83, P = 0.05). MDA, SOD and GPx levels were neither altered by a single FES exercise bout nor by 8 weeks FES training. In conclusion, although individuals with an SCI demonstrate a preserved (anti)oxidative status, the correlation between fitness level and (anti)oxidative balance suggests that higher fitness levels are related to improved (anti)oxidative status in SCI. Nonetheless, the FES exercise stimulus was insufficient to acutely or chronically change (anti)oxidative status in individuals with an SCI. 01 augustus 2010

BACKGROUND: Considerable discrepancy exists in the literature on the proposed benefits of protein supplementation on the adaptive response of skeletal muscle to resistance-type exercise training in the elderly. OBJECTIVE: The objective was to assess the benefits of timed protein supplementation on the increase in muscle mass and strength during prolonged resistance-type exercise training in healthy elderly men who habitually consume adequate amounts of dietary protein. DESIGN: Healthy elderly men (n = 26) aged 72 +/- 2 y were randomly assigned to a progressive, 12-wk resistance-type exercise training program with (protein group) or without (placebo group) protein provided before and immediately after each exercise session (3 sessions/wk, 20 g protein/session). One-repetition maximum (1RM) tests were performed regularly to ensure a progressive workload during the intervention. Muscle hypertrophy was assessed at the whole-body (dual-energy X-ray absorptiometry), limb (computed tomography), and muscle fiber (biopsy) level. RESULTS: The 1RM strength increased approximately 25-35% in both groups (P < 0.001). Dual-energy X-ray absorptiometry and computed tomography scans showed similar increases in leg muscle mass (6 +/- 1% in both groups; P < 0.001) and in the quadriceps (9 +/- 1% in both groups), from 75.9 +/- 3.7 and 73.8 +/- 3.2 to 82.4 +/- 3.9 and 80.0 +/- 3.0 cm2 in the placebo and protein groups, respectively (P < 0.001). Muscle fiber hypertrophy was greater in type II (placebo: 28 +/- 6%; protein: 29 +/- 4%) than in type I (placebo: 5 +/- 4%; protein: 13 +/- 6%) fibers, but the difference between groups was not significant. CONCLUSION: Timed protein supplementation immediately before and after exercise does not further augment the increase in skeletal muscle mass and strength after prolonged resistance-type exercise training in healthy elderly men who habitually consume adequate amounts of dietary protein. This trial was registered at clinicaltrials.gov as NCT00744094.

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.

Using Surface-Enhanced Laser Desorption / Ionization Time-of-Flight Mass Spectrometry (SELDI-TOF-MS), we aimed to detect differences in protein profile in serum samples of two lacunar stroke subtypes. SELDI-TOF-MS, followed by protein identification, was performed in samples of 8 first-ever lacunar stroke patients with MR imaging showing a single symptomatic lacunar lesion (type 1), and 8 with multiple additional "silent" lacunar lesions and extensive white matter lesions (type 2). A 16 kDa protein, identified as alpha-2-chain of haptoglobin (Hp), was found to be overrepresented in type 1 compared to type 2 (peak intensity 12.5 vs. 5.0; p=0.02). As a polymorphism with two alleles, Hp-1 and Hp 2, determines the presence of alpha-1 and/or alpha-2-chains in the Hp-molecule, Hp phenotypic analysis was performed. Hp 1 : Hp-2 allele frequency was 0.562 : 0.438 in type 1 and 0.812 : 0.188 in type 2 (population reference approximately 0.4 : 0.6). We conclude that the overrepresentation of the alpha-2-chain in lacunar stroke type 1 compared to type 2 relates to a higher Hp-2 allele frequency in the former. Yet, compared to population reference, the phenotype distribution in both patient groups deviates towards a high Hp-1 allele frequency. Our findings suggest a role for the Hp gene in the etiology of cerebral small vessel disease. Larger studies are now needed to explore this new candidate gene.

Summary Background The value of elevated serum angiotensin-converting enzyme (ACE) activity in the diagnosis and follow-up in sarcoidosis is a matter of ongoing debate. This may be at least related to the insertion (I)/deletion (D) polymorphism in the ACE gene (ACE I/D). ACE activity is influenced by the ACE I/D polymorphism. As a consequence, the use of one reference interval instead of three genotype-specific reference intervals for ACE activity may lead to a less precise interpretation of ACE activity. Methods In order to assess whether determination of ACE activity indeed requires the ACE I/D genotype to be taken into account, we established ACE I/D-corrected reference intervals in healthy, Caucasian volunteers ( n =200). In addition, ACE activities in ACE I/D genotyped patients suspected of or having sarcoidosis ( n =129) were expressed as the Z -score related to ACE I/D-corrected reference intervals. Results Comparison of the Z -score with ACE activity in which ACE I/D is ignored rendered 8.5% misclassification of ‘elevated' versus ‘normal' ACE or vice versa. Conclusions Our data demonstrate a convenient way to circumvent the use of three reference intervals by introducing a Z -score for ACE activity. It also illustrates the need to re-investigating the possible clinical value of serum ACE activity in sarcoidosis by considering ACE I/D.

Background: Protein profiling by surface enhanced laser desorption ionization-time of flight-mass spectrometry (SELDI-TOF-MS) is gaining importance as a diagnostic tool for a whole range of diseases. This report describes a QC procedure, which acts prospectively by checking the calibration before starting profiling experiments. Methods: A well-defined protocol for calibration of the Protein Biosystem IIc instrument was established, using a commercial QC sample containing independent certified standards and by determination of acceptance criteria. Instrument calibration was performed externally every week with the standards provided by the manufacturer. QC was performed for the period of 5 months. Results: According to the acceptance criteria defined in this study, data points should be in the established range of the process mean T2 standard deviations for the mass-to-charge ratios (m/z values), peak intensities, signal-to-noise ratios (S/N), and peak resolutions for insulin and apomyoglobin in the QC sample. Moreover, it was demonstrated that the pipetting variability in the handling of the QC sample significantly contributed to systematic errors and that spotting of a larger volume of QC sample resulted in a better reproducibility. Conclusions: Stringent quality control of the calibration part of SELDI-TOF-MS experiments prevents unreliable data acquisition from the very start. D 2005 Elsevier B.V. All rights reserved.

Recently, the authors of 2 studies have claimed to measure accurately the microheterogeneity of transthyretin (TTR) variants by surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF MS) immunoassays (1)(2). Here we report that the mass spectrometric performance in those studies was not adequate to measure the respective TTR variants. A mass resolution of at least 1000 is required to separate the majority of the currently known posttranslational and mutation-based modifications of TTR (3). Wang et al.(1) performed their analysis at a resolution of 270 [estimated as follows: m /Δ m FWHM = 13828/52 = 266 in Fig. 1A of their report (1)], whereas Schweigert et al. (2) achieved a resolution of 90 [estimated as follows: m /Δ m FWHM = 13851/158 = 88 in panel A2 of Fig. 2 of their report (2)]. We therefore question whether these studies can justify their reported claims. To provide arguments, we performed MALDI-TOF MS analysis on a gold chip ProteinChip® array of a commercially available TTR preparation with an instrument typically used in SELDI-TOF MS (PBS IIc analyzer; Ciphergen). We applied …