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

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.

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

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.

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.

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.

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

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.

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

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.

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

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.

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

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.

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

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.

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

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.

A Low or High Physical Activity Level Does Not Modulate Prostate Tumor Tissue Protein Synthesis Rates.

INTRODUCTION: Physical activity level has been identified as an important factor in the development and progression of various types of cancer. In this study, we determined the impact of a low versus high physical activity level on skeletal muscle, healthy prostate, and prostate tumor protein synthesis rates in vivo in prostate cancer patients. METHODS: Thirty prostate cancer patients (age, 66 ± 5 yr; body mass index, 27.4 ± 2.9 kg·m -2 ) were randomized to a low (<4000 steps per day, n = 15) or high (>14,000 steps per day, n = 15) physical activity level for 7 d before their scheduled radical prostatectomy. Daily deuterium oxide administration was combined with the collection of plasma, skeletal muscle, nontumorous prostate, and prostate tumor tissue during the surgical procedure to determine tissue protein synthesis rates throughout the intervention period. RESULTS: Daily step counts averaged 3610 ± 878 and 17,589 ± 4680 steps in patients subjected to the low and high physical activity levels, respectively ( P < 0.001). No differences were observed between tissue protein synthesis rates of skeletal muscle, healthy prostate, or prostate tumor between the low (1.47% ± 0.21%, 2.74% ± 0.70%, and 4.76% ± 1.23% per day, respectively) and high (1.42% ± 0.16%, 2.64% ± 0.58%, and 4.72% ± 0.80% per day, respectively) physical activity group (all P > 0.4). Tissue protein synthesis rates were nearly twofold higher in prostate tumor compared with nontumorous prostate tissue. CONCLUSIONS: A short-term high or low physical activity level does not modulate prostate or prostate tumor protein synthesis rates in vivo in prostate cancer patients. More studies on the impact of physical activity level on tumor protein synthesis rates and tumor progression are warranted to understand the potential impact of lifestyle interventions in the prevention and treatment of cancer.

Differences in Cardiac Troponin T Composition in Myocardial Infarction and End-Stage Renal Disease Patients: A Blood Tube Effect?

BACKGROUND: Cardiac troponin T (cTnT) is key in diagnosing myocardial infarction (MI) but is also elevated in end-stage renal disease (ESRD) patients. Specific larger cTnT proteoforms were identified for the acute phase of MI, while in serum of ESRD patients solely small cTnT fragments were found. However, others allocated this to a pre-analytic effect due to abundant thrombin generation in serum. Therefore, we investigated the effect of various anticoagulation methods on cTnT composition and concentration and compared the cTnT composition of MI and ESRD patients. METHODS: The agreement of cTnT concentrations between simultaneously collected serum, lithium-heparin (LH) plasma, and ethylenediaminetetraacetic acid (EDTA) plasma was studied using the high-sensitivity (hs-)cTnT immunoassay. cTnT proteoform composition was investigated in a standardized time-dependent manner through spike experiments and in simultaneously collected blood matrixes of MI and ESRD patients. RESULTS: Excellent hs-cTnT concentration agreements were observed across all blood matrixes (slopes > 0.98; 95% CI, 0.96-1.04). Time-dependent degradation (40 kDa intact:29 kDa fragment:15 to 18 kDa fragments) was found in LH plasma and EDTA plasma, and serum in ratios (%) of 90:10:0, 0:5:95, and 0:0:100, respectively (48 h after blood collection). Moreover, gel filtration chromatography (GFC) profiles illustrated mainly larger cTnT proteoforms in MI patients, while in ESRD patients mainly 15 to 18 kDa fragments were found for all matrices. CONCLUSIONS: The extent of cTnT degradation in vitro is dependent on the (anti)coagulation method, without impacting hs-cTnT concentrations. Furthermore, mainly larger cTnT proteoforms were present in MI patients, while in ESRD patients mainly small 15 to 18 kDa cTnT fragments were found. These insights are essential when developing a novel hs-cTnT assay targeting larger cTnT proteoforms.

Posttranslational modifications of cardiac troponin T: an overview.

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.

Low Levels of Serum and Intracellular Vitamin C in Hospitalized COVID-19 Patients.

Vitamin C is a crucial micronutrient for human immune cell function and has potent antioxidant properties. It is hypothesized that vitamin C serum levels decline during infection. However, the precise mechanisms remain unknown. To gain deeper insights into the true role of vitamin C during infections, we aimed to evaluate the body's vitamin C storage during a SARS-CoV-2 infection. In this single-center study, we examined serum and intracellular vitamin C levels in peripheral blood mononuclear cells (PBMCs) of 70 hospitalized COVID-19 patients on the first and fifth days of hospitalization. Also, clinical COVID-19 severity was evaluated at these timepoints. Our findings revealed a high prevalence of hypovitaminosis C and vitamin C deficiency in hospitalized COVID-19 patients (36% and 15%). Moreover, patients with severe or critical disease exhibited a higher prevalence of low serum vitamin C levels than those with moderate illness. Serum vitamin C levels had a weak negative correlation with clinical COVID-19 severity classification on the day of hospitalization; however, there was no correlation with intracellular vitamin C. Intracellular vitamin C levels were decreased in this cohort as compared to a healthy cohort and showed further decline during hospitalization, while serum levels showed no relevant change. Based on this observation, it can be suggested that the reduction of intracellular vitamin C may be attributed to its antioxidative function, the need for replenishing serum levels, or enhanced turnover by immune cells. These data give an incentive to further investigate the role of intracellular vitamin C in a larger and more heterogeneous cohort as well as the underlying mechanisms.

Circulating biomarkers and abdominal aortic aneurysm size.

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.

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

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.

No Effect of Vitamin C Administration on Neutrophil Recovery in Autologous Stem Cell Transplantation for Myeloma or Lymphoma: A Blinded, Randomized Placebo-Controlled Trial.

Vitamin C is an important micronutrient for various immune cells. It increases phagocytic cell function and is necessary for T and natural killer (NK) cell development. Patients in need of an autologous hematopoietic stem cell transplantation (HSCT) are often vitamin C-depleted. We therefore hypothesized that vitamin C supplementation could improve immune recovery in autologous HSCT patients. This blinded, placebo-controlled trial included 44 patients randomized to receive vitamin C or a placebo. The following outcome measures used were clinical and immunological parameters, among others: time to neutrophil recovery, serum, and intracellular vitamin C values. Twenty-one patients received vitamin C, and 23 received a placebo. The time to neutrophil recovery did not differ between the two groups at 11.2 days (p = 0.96). There were no differences in hospitalization time (19.7 vs. 19.1 days, p = 0.80), the incidence of neutropenic fever (57% vs. 78%, p = 0.20), or 3-month overall survival (90.5% vs. 100%, p = 0.13). Bacteremia seemed to occur less in the vitamin C group (10% vs. 35%, p = 0.07). Our study shows no benefit from vitamin C supplementation on neutrophil recovery and hospitalization, despite possible lower rates of bacteremia in the vitamin C group. Therefore, we do not advise vitamin C supplementation in this treatment group.

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

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.

FSH isoform pattern in classic galactosemia.

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.