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BACKGROUND: Over the last half-century, different stoichiometric equations for calculating the energy cost of exercise based upon the combustion of mixtures of carbohydrates, fats, and proteins have been proposed and modified. With the means of indirect calorimetry, while measuring oxygen uptake, carbon dioxide production, and urinary urea nitrogen excretion, the contribution of specific substrates to overall energy production can be estimated. However, even with their long history of application, no previous studies have evaluated whether the use of different stoichiometric equations provides similar or distinct maximal fat oxidation rate (MFO) responses and information regarding MFO location (FA Tmax) in male athletes. METHODS: Twenty healthy male athletes performed graded exercise testing (GXT) cycle ergometry using breath by breath gas analysis to assess fat oxidation and maximal oxygen uptake. Analysis of variance followed by within-equation effects, within- equation factors, and post hoc pairwise comparisons were used to examine within- equation differences. RESULTS: Compared stoichiometric equations demonstrated significant differences in the mean and maximal fat oxidation rates, varying up to nearly 7%. FA Tmax differences, however, were not noticed. CONCLUSIONS: Our findings suggest that for within- study designs, the equation used appears to be less important, but when inter-study comparisons are planned, caution is in order due to the presence of inter-equation differences.

This study evaluated dynamics of testosterone, cortisol and alpha-amylase during a handball match in high-level male players. Ten male professional players (24.1 ± 3.1 years, 188.2 ± 6.4 cm, 94.6 ± 9.6 kg) were tested on salivary- testosterone, - cortisol and -alpha-amylase levels before (prematch), at halftime, and immediately after the game. Analysis of variance for repeated measurements (ANOVA) with consecutive post-hoc analyses and effect-size differences were calculated to identify differences between measurements. The associations among biomarkers were determined by Pearson's product moment correlation (Pearson's r). The ANOVA indicated significant differences in testosterone (F: 14.31, p < 0.01 ; significant post-hoc differences between prematch and remaining two measurements), and alpha-amylase (F: 9.78, p < 0.01 ; significant post-hoc differences between all measurements). Significant correlations were evidenced between: (i) alpha-amylase- and testosterone-changes during 1st halftime (Pearson's r: 0.81, p < 0.01), and (ii) alpha- amylase- and cortisol- changes during 2nd halftime (Pearson's r: 0.76, p < 0.05). The results indicated specific dynamics of biomarkers during the handball match, with significant increase in alpha-amylase during the entire match and significant increase in the testosterone level during the first half of the match. Further studies are needed to evaluate the associations between real-game performance and changes in hormonal responses.

Background: There is a shortage of longitudinal surveys in highly trained adolescent table tennis athletes reporting typical changes in performance related, hematological, and biochemical parameters. Objectives: The purpose of the study was to: (1) examine yearly variations of performance related parameters, and (2) report the variability of selected hematological and biochemical markers utilized to ascertain health status, and training influences in a group of young Arab table tennis athletes. Methods: Data were collected from ten male adolescent players with a mean age of 14.2 ± 1.3 years and a mean table tennis training experience of 6 years. Physical and biochemical measures were carried out at three important time points throughout the training season: a baseline measure (PRE - September); a mid-season (transition) measure (MID - February); an end of season measure (END - May). Results: Performance related parameters tended to significantly improve over time (P = 0.05), with lowest values displayed during PRE-measure. Hematocrit (P = 0.05), serum testosterone (P = 0.01), mean cell volume (MCV) (P = 0.001) and reticulocytes (RET) (P = 0.017) significantly increased at the END training period compared to PRE. Moreover, 25(OH)D levels (P = 0.001) showed a drop during MID to then significantly increase at the END whilst sex hormone binding globulin (SHBG; P = 0.005) decreased at the END period compared to MID. Conclusions: In conclusion, based on our findings, changes in certain physical parameters and, hematological and biochemical markers, take place during the training period. Therefore, coaches, sports scientists and nutritionists should take into consideration these fluctuations and plan and alter their training programs accordingly.

Although the role of vitamin D in calcium and bone metabolism is well documented, there is little clarity regarding the implications of low vitamin D status for inflammation, endothelial function, and antioxidant status in adolescent athletes. A prospective cohort study was conducted, and 44 male adolescent athletes, training at a sports academy in the Middle East, were assigned to either the intervention group (VitDs), consisting of vitamin D deficient athletes [twenty-five hydroxyvitamin D (25(OH)D) 30 ng/ml ; n = 22]. Vitamin D status, inflammatory cytokines, endothelium-related variables, and antioxidant enzymes were measured twice during a nine-week training period. At the baseline, the athletes in the VitDs group had significantly lower concentrations of 25(OH)D, vascular endothelial growth factor (VEGF), and glutathione peroxidase (GPx), and higher levels of parathyroid hormone (PTH), interleukin-6 (IL-6), interleukin-1 receptor antagonist (IL-1ra), and nitrite (NO2) (p < 0.05), in comparison to the athletes in the sufficient group. After vitamin D supplementation for the VitDs group, the two cohorts differed considerably in vitamin D binding protein (VDBP) and PTH concentrations (p < 0.05). Our data suggest that the low levels of vitamin D possibly induced alterations in the investigated biochemical parameters of athletes in the VitDs group at the beginning of the monitoring period. Furthermore, while the vitamin D supplementation was effective in increasing 25(OH)D status, it may have concurrently positively influenced variables that are related to inflammation, endothelial function, and enzymatic antioxidants.

Autologous blood transfusion (ABT) has been frequently abused in endurance sport and is prohibited since the mid-1980s by the International Olympic Committee. Apart from any significant performance-enhancing effects, the ABT may pose a serious health issue due to aging erythrocyte-derived “red cell storage lesions.” The current study investigated the effect of blood storage in citrate phosphate dextrose adenine (CPDA1) on the red blood cell (RBC) membrane proteome. One unit of blood was collected in CPDA1 blood bags from 6 healthy female volunteers. RBC membrane protein samples were prepared on days 0, 14, and 35 of storage. Proteins were digested in gel and peptides separated by nanoliquid chromatography coupled to tandem mass spectrometry resulting in the confident identification of 33 proteins that quantitatively change during storage. Comparative proteomics suggested storage-induced translocation of cytoplasmic proteins to the membrane while redox proteomics analysis identified 14 proteins prone to storage-induced oxidation. The affected proteins are implicated in the RBC energy metabolism and membrane vesiculation and could contribute to the adverse posttransfusion outcomes. Spectrin alpha chain, band 3 protein, glyceraldehyde-3-phosphate dehydrogenase, and ankyrin-1 were the main proteins affected by storage. Although potential biomarkers of stored RBCs were identified, the stability and lifetime of these markers posttransfusion remain unknown. In summary, the study demonstrated the importance of studying storage-induced alterations in the erythrocyte membrane proteome and the need to understand the clearance kinetics of transfused erythrocytes and identified protein markers.

Mature red blood cells (RBCs) are the end-stage of a development process that starts in the bone marrow and continues to differentiate, through reticulocyte stage, entering into the circulation with a four-month lifespan. While stored, RBCs undergo different changes. The aim of this study was to evaluate changes occurring in RBC membranes during storage that could be used as possible markers to detect the misuse of blood transfusion in sports. Whole blood was collected from two volunteers in blood bags and stored for 42 days at 4°C. At different times (1, 7, 21, and 42 days of storage) whole blood was extracted under sterile conditions and submitted to RBC membrane ghost preparation and further analysis. Proteomic methods were applied using two strategies: protein oriented using 2-DE gels and peptide oriented using isobaric tags for relative and absolute quantitation (iTRAQ). In both approaches, the goal was to compare detectable changes in RBC membrane proteome before and after standard storage at different times. Some of the changes were confirmed with both methodologies employed, while with others only with one of them. Complementarities of the methods in this case showed to be an advantage. Changes were observed in two different protein complexes. In one of them, changes consisted of proteins decreasing, while increasing in the other during storage of RBCs. They are mostly located in cytoskeleton--spectrin β, band 4.2, ankyrin-1, tropomodulin-1, β adducin, band 4.9 (dematin), tropomyosin, while some changes were also observed in transmembrane proteins (glycophorin C, aquaporin-1, band 3).

Receptor binding techniques have been widely used in different biochemical applications, with isolated membranes being the most used receptor preparation in this type of assays. In this study, intact cells were compared with isolated membranes as receptor support for radioligand receptor binding assay. The growth hormone secretagogue receptor 1a (GHSR-1a) expressed in human embryonic kidney 293 (HEK293) cells was used as a model of G-protein-coupled receptors. Differences between using intact cells in suspension and using isolated membranes were evaluated for different aspects of the receptor binding assay: total binding variations while both receptor preparations remain on ice, modifications in incubation conditions, saturation, and competition using different agonists. Intact cells are more prone to variability. Although under optimized settings both preparations were equivalent, the K(d) value for intact cells was three times higher than that using isolated membranes. However, no significant differences were observed in competition assays obtaining practically identical K(i) values for all ligands tested. For the GHSR-1a, isolated membranes are the better choice if particular incubation conditions are required (less variability), whereas intact cells yield easy, fast, and physiological conditions for receptor binding assays.

Eosinophil cationic protein (ECP) is a component of the eosinophil granule matrix. It shows marked toxicity against helminth parasites, bacteria single-stranded RNA viruses, and host epithelial cells. Secretion of human ECP is related to eosinophil-associated allergic, asthmatic, and inflammatory diseases. ECP belongs to the pancreatic ribonuclease superfamily of proteins, and the crystal structure of ECP in the unliganded form (determined previously) exhibited a conserved RNase A fold [Boix, E., et al. (1999) Biochemistry 38, 16794-16801]. We have now determined a high-resolution (2.0 A) crystal structure of ECP in complex with adenosine 2',5'-diphosphate (2',5'-ADP) which has revealed the details of the ribonucleolytic active site. Residues Gln-14, His-15, and Lys-38 make hydrogen bond interactions with the phosphate at the P(1) site, while His-128 interacts with the purine ring at the B(2) site. A new phosphate binding site, P(-)(1), has been identified which involves Arg-34. This study is the first detailed structural analysis of the nucleotide recognition site in ECP and provides a starting point for the understanding of its substrate specificity and low catalytic efficiency compared with that of the eosinophil-derived neurotoxin (EDN), a close homologue.

More than 30 years ago people have learned that exercise promotes oxidative damage in human tissues. Since then, our knowledge on exercise-induced free radical production and their effect has advanced markedly. In the beginning research has mainly focused on detrimental effects of exercise-induced free radical production (e.g. oxidation of macromolecules); however today a new era of redox biology exist that centers on the cell-signaling effects of exercise-induced free radicals, such as reactive oxygen species (ROS) that were shown to play a crucial role in muscle adaptation to exercise. Erythrocytes are rich in membrane polyunsaturated free fatty acid content and have high cellular concentrations of oxygen and haemoglobin that makes them susceptible to ROS induced damage. However, mild oxidative stress has beneficial effect by increasing endogenous antioxidant mechanisms. Thus, in the present work we have used sensitive mass spectrometry techniques to assess the potentially beneficial oxidative modifications of erythrocyte membrane proteins in response to exercise. We have recruited 3 healthy volunteers and their blood was drawn before exercise, immediately after and few hours after exercise. Erythrocyte membranes ("ghosts") were prepared and membrane proteins isolated. Proteins were then separated by 1-D gel electrophoresis, digested with trypsin and peptides analyzed by Orbitrap ELITE coupled with the Easy n-LC II for nano LC-MS/MS. The acquired data was searched with Proteome Discoverer 1.4 against Homo sapiens database to reveal the changes in the erythrocyte membrane proteome. Furthermore, data acquired was also searched through the database using the comprehensive workflow that included several dynamic modifications of peptides, including N-terminal carboxymethyl, C-terminal oxidation and terminal independent oxidation and trioxidation of peptides. The results obtained revealed several proteins that have translocated from the cytoplasm into the membrane following exercise. Moreover, numerous proteins showed exercise dependent modifications and up/down regulation. Exercise induced proteome changes were demonstrated to play an important role in several signaling pathways related to healthy living and lifestyle.

With the use of a high yield prokaryotic expression system, large amounts of human eosinophil cationic protein (ECP) have been obtained. This has allowed a thorough kinetic study of the ribonuclease activity of this protein. The catalytic efficiencies for oligouridylic acids of the type (Up)nU>p, mononucleotides U>p and C>p, and dinucleoside monophosphates CpA, UpA, and UpG have been interpreted by the specific subsites distribution in ECP. The distribution of products derived from digestion of high molecular mass substrates, such as poly(U) and poly(C), by ECP was compared with that of RNase A. The characteristic cleavage pattern of polynucleotides by ECP suggests that an exonucleaselike mechanism is predominantly favored in comparison to the endonuclease catalytic mechanism of RNase A. Comparative molecular modeling with bovine pancreatic RNase A-substrate analog crystal complexes revealed important differences in the subsite structure, whereas the secondary phosphate-binding site (p2) is lacking, the secondary base subsite (B2) is severely impaired, and there are new interactions at the po, Bo, and p-1 sites, located upstream of the P-O-5* cleavable phosphodiester bond, that are not found in RNase A. The differences in the multisubsites structure could explain the reduced catalytic efficiency of ECP and the shift from an endonuclease to an exonuclease-type mechanism.

Eosinophil cationic protein (ECP) is located in the matrix of the eosinophil's large specific granule and has marked toxicity for a variety of helminth parasites, hemoflagellates, bacteria, single-stranded RNA virus, and mammalian cells and tissues. It belongs to the bovine pancreatic ribonuclease A (RNase A) family and exhibits ribonucleolytic activity which is about 100-fold lower than that of a related eosinophil ribonuclease, the eosinophil-derived neurotoxin (EDN). The crystal structure of human ECP, determined at 2.4 A, is similar to that of RNase A and EDN. It reveals that residues Gln-14, His-15, Lys-38, Thr-42, and His-128 at the active site are conserved as in all other RNase A homologues. Nevertheless, evidence for considerable divergence of ECP is also implicit in the structure. Amino acid residues Arg-7, Trp-10, Asn-39, His-64, and His-82 appear to play a key part in the substrate specificity and low catalytic activity of ECP. The structure also shows how the cationic residues are distributed on the surface of the ECP molecule that may have implications for an understanding of the cytotoxicity of this enzyme.

Gene therapy is anticipated as being an important medical development. Essential to its effectiveness is the appropriate activity (protein expression) in the expected target cells. A noninvasive diagnostic procedure of successful gene expression will be of paramount importance to validate its use or its misuse (eg, sports gene doping). Externally detectable labeled oligonucleotide hybridizing with the messenger RNA generated by the transferred gene has been proposed as a possibility to monitor successful gene therapy. The authors selected the erythropoietin gene (Epo) for a pilot study on erythropoietin protein expression in mouse muscle. Oligonucleotides of peptide nucleic acid (PNA) type capable of antisense binding to unique murine Epo-mRNA sequences were synthesized by solid phase methods, and elongated at the N-terminus with the HIV Tat (48–60) cell penetrating peptide. They were labeled with fluorescence and radioactive tags to verify penetration and longer half-life properties in Epo gene transfected C2C12 mouse muscle cells as compared with corresponding wild- type cells. Downregulation of newly expressed erythropoietin protein in such cells additionally confirmed the penetration and hybridizing properties of the selected labeled oligonucleotide. 123I-labeled Tat-PNAs were intravenously injected into mice that had previously received the Epo gene into the right tibialis muscle by DNA electrotransfer. Preferential accumulation of radioactivity in the transferred limb as compared with the contralateral limb was ascertained, especially for 123I-Tat-CTA CGT AGA CCA CT (labeled Tat-PNA 1). This study provides experimental data to support the potential use of external noninvasive image detection to monitor gene therapy. The extension of the approach to more sensitive methods for whole-body external detection such as positron emission tomography appears feasible.

The eosinophil cationic protein (ECP) is a basic secretion proteininvolved in the immune response system. ECP levels in biological fluidsare an indicator of eosinophil-specific activation and degranulationand are currently used for the clinical monitoring and diagnosis ofinflammatory disorders. A polyclonal epitope-specific antibody has beenobtained by immunizing rabbits with a conjugated synthetic peptide. Asequence corresponding to a large exposed loop in the human ECPthree-dimensional structure (D115–Y122) was selected as a putativeantigenic epitope. The antibody was purified on an affinity columnusing recombinant ECP (rECP) as antigen. The antibody (D112–P123 Ab)specifically recognizes rECP and its native glycosylated andnonglycosylated forms in plasma, granulocytes, and sputum. The antibodydetects as little as 1 ng of rECP, can be used both in reducing andnonreducing conditions, and does not cross-react with the highlyhomologous eosinophil-derived neurotoxin or other proteins of thepancreatic ribonuclease superfamily.