Zoran Nikolovski

Growth hormone in sport: beyond Beijing 2008.

Human growth hormone (hGH) is a protein endogenously produced predominantly by the anterior pituitary gland. Native hGH and, especially, its recombinant analogue (rhGH), used to treat patients with hormone deficiency, are supposed to be abused by athletes searching its anabolic and lipolytic effects. Hence, hGH use has been prohibited for a long time by the sport authorities, but until recently, hGH abuse could not be detected. Two approaches have been followed when trying to develop methods for GH abuse detection. The direct method identifies an abnormal ratio between GH isoforms-a result of hGH exogenous administration. The time window to find a cheating athlete by this approach is limited by the excretion time of the hormone. The indirect approach measures serum biomarkers directly affected by GH intake (eg, markers of released liver growth factors and of bone and collagen turnover). In this approach, the retrospective power extends further. Alternative possibilities for cheating related to hGH could be the administration of recombinant growth factors themselves, the administration of hGH metabolic precursors such as ghrelin-like GH secretagogues, or the genetic manipulation of muscle growth-related genes (gene doping). In parallel with the new types of abuse, which will surely emerge in the near future, the research and development for the improvement of the analytical detection of GH itself will continue.

Monitoring gene therapy by external imaging of mRNA: pilot study on murine erythropoietin.

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.

Alterations of the erythrocyte membrane proteome and cytoskeleton network during storage – a possible tool to identify autologous blood transfusion

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).

The use of biomarkers to describe plasma-, red cell-, and blood volume from a simple blood test

Plasma volume and red cell mass are key health markers used to monitor numerous disease states, such as heart failure, kidney disease, or sepsis. Nevertheless, there is currently no practically applicable method to easily measure absolute plasma or red cell volumes in a clinical setting. Here, a novel marker for plasma volume and red cell mass was developed through analysis of the observed variability caused by plasma volume shifts in common biochemical measures, selected based on their propensity to present with low variations over time. Once a month for 6 months, serum and whole blood samples were collected from 33 active males. Concurrently, the CO‐rebreathing method was applied to determine target levels of hemoglobin mass (HbM) and blood volumes. The variability of 18 common chemistry markers and 27 Full Blood Count variables was investigated and matched to the observed plasma volume variation. After the removal of between‐subject variations using a Bayesian model, multivariate analysis identified two sets of 8 and 15 biomarkers explaining 68% and 69% of plasma volume variance, respectively. The final multiparametric model contains a weighting function to allow for isolated abnormalities in single biomarkers. This proof‐of‐concept investigation describes a novel approach to estimate absolute vascular volumes, with a simple blood test. Despite the physiological instability of critically ill patients, it is hypothesized the model, with its multiparametric approach and weighting function, maintains the capacity to describe vascular volumes. This model has potential to transform volume management in clinical settings. Am. J. Hematol. 92:62–67, 2017.

A step towards removing plasma volume variance from the Athlete's Biological Passport: The use of biomarkers to describe vascular volumes from a simple blood test

The haematological module of the Athletes Biological Passport (ABP) has significantly impacted the prevalence of blood manipulations in elite sports. However, the ABP relies on a number of concentration-based markers of erythropoiesis, such as haemoglobin concentration ([Hb]), which are influenced by shifts in plasma volume (PV). Fluctuations in PV contribute to the majority of biological variance associated with volumetric ABP markers. Our laboratory recently identified a panel of common chemistry markers (from a simple blood test) capable of describing ca 67% of PV variance, presenting an applicable method to account for volume shifts within anti-doping practices. Here, this novel PV marker was included into the ABP adaptive model. Over a six-month period (one test per month), 33 healthy, active males provided blood samples and performed the CO-rebreathing method to record PV (control). In the final month participants performed a single maximal exercise effort to promote a PV shift (mean PV decrease -17%, 95% CI -9.75 to -18.13%). Applying the ABP adaptive model, individualized reference limits for [Hb] and the OFF-score were created, with and without the PV correction. With the PV correction, an average of 66% of [Hb] within-subject variance is explained, narrowing the predicted reference limits, and reducing the number of atypical ABP findings post-exercise. Despite an increase in sensitivity there was no observed loss of specificity with the addition of the PV correction. The novel PV marker presented here has the potential to improve the ABPs rate of correct doping detection by removing the confounding effects of PV variance.

Can Metabolic Thresholds be used as Exercise Intensity Markers in Adult men with Obesity

The first aim of the study was to identify the exercise intensity eliciting the highest (FATmax) and the lowest (FATmin) fat oxidation rate in men with obesity. The second aim was to evaluate if FATmax and FATmin correlate with aerobic (AeT) and anaerobic (AnT) thresholds, which in turn could be used as exercise intensity markers. Nineteen adult sedentary men participated in the study. Breath-by-breath analysis was performed throughout the test to assess maximal oxygen consumption (VO2max) with stoichiometric equations used to calculate fat oxidation rate. Pearson correlation coefficient (r), coefficient of determination (R2) and paired t-test were used to evaluate differences between VO2 at AeT and at FATmax and VO2 at AnT and at FATmin, respectively. FATmax and AeT occurred at 42.80 {+/-} 2.68 % of VO2max and 43.02 {+/-} 2.73 % of VO2max, while FATmin and AnT occurred at 53.40 {+/-} 3.65 % of VO2max and 53.38 {+/-} 3.65 % of VO2max, respectively. A high correlations were found between VO2 at FATmax and at AeT (r = 0.86, p < 0.01) and VO2 at FATmin and at AnT (r = 0.99, p < 0.01). The existing correlations suggest that metabolic thresholds may be used as exercise intensity markers in men with obesity.The first aim of the study was to identify the exercise intensity eliciting the highest (FATmax) and the lowest (FATmin) fat oxidation rate in men with obesity. The second aim was to evaluate if FATmax and FATmin correlate with aerobic (AeT) and anaerobic (AnT) thresholds, which in turn could be used as exercise intensity markers. Nineteen adult sedentary men participated in the study. Breath-by-breath analysis was performed throughout the test to assess maximal oxygen consumption (VO2max) with stoichiometric equations used to calculate fat oxidation rate. Pearson correlation coefficient (r), coefficient of determination (R2) and paired t-test were used to evaluate differences between VO2 at AeT and at FATmax and VO2 at AnT and at FATmin, respectively. FATmax and AeT occurred at 42.80 ± 2.68 % of VO2max and 43.02 ± 2.73 % of VO2max, while FATmin and AnT occurred at 53.40 ± 3.65 % of VO2max and 53.38 ± 3.65 % of VO2max, respectively. A high correlations were found between VO2 at FATmax and at AeT (r = 0.86, p < 0.01) and VO2 at FATmin and at AnT (r = 0.99, p < 0.01). The existing correlations suggest that metabolic thresholds may be used as exercise intensity markers in men with obesity.

Training‐Induced Variations in Haematological and Biochemical Variables in Adolescent Athletes of Arab Origin Throughout an Entire Athletic Season

Abstract The purpose of this study was to observe and report variations in several haematological and biochemical markers throughout an entire athletic season in a large cohort of adolescent athletes of Arab origin. Blood samples were collected from 72 adolescent male athletes at 4 selected time points during their training season. Results expressed in relation to plasma volume were corrected accordingly and significant variations in several variables emerged. Initial uncorrected haematological results revealed that haematocrit (Hct) and mean cell volume (MCV) concentrations noticeably increased at the competitive period (T3) and before the start of the following preseason (T4), whereas reticulocytes equivalent (Ret-He) only rose at T4 phase (p < 0.01). Conversely, corrected red blood cells (RBC), haemoglobin (Hb) and mean cell haemoglobin concentration (MCHC) progressively decreased over the year (p < 0.001). From the electrolytes panel, sodium and chloride considerably reduced at the peak of the training period (T2) to the start of the next preseason (T4), while a significant fall in potassium was mainly observed during the competitive period (T3) (p < 0.001). Coaches and sport scientists could use the results of this study to evaluate typical variations of each age group in order to diagnose potential adverse effects of high training loads, assist in the design of training programs and/or clinical interventions that will safeguard athletes’ health, and consider the important role of plasma volume for the interpretation of results.

P-25 Substrates oxidation at high intensity exercise in sedentary females

Objectives The substrates utilisation at high intensity exercise has been less studied and intensity at which carbohydrates (CHO) remain single energy source while fat utilisation becomes negligible (FATmin), is debatable 4. Since physical activity in aerobic fat burn zone is prescribed to facilitate improvement of fat oxidation and insulin sensitivity while improving health 1, activity equal or above FATmin can’t be considered as beneficial. Therefore, FATmin could become important exercise parameter in sedentary population. The aim of this study was to determine FATmin while performing incremental treadmill test till exhaustion and to assess its correlation with anaerobic threshold (AT), which in turn can be used as an individuals performance marker or last exercise beneficial point. Methodology Thirteen sedentary females (age: 34 ± 3 y, body mass: 62 ± 4 kg, height: 165 ± 4 cm) performed breath-by-breath test to measure maximal oxygen uptake (VO2max;ml·kg-1·min-1) and substrates oxidation (g·min-1). A Pearson correlation coefficient (r), 95% confidence interval (CI) and effect size (R2 ) were used to test connexion between VO2 at AT and at FATmin. Alpha intervals were set at p < 0.05. Substrates oxidation was calculated using a stoichiometric equation 4. The AT was detected by V-slope method 2. Results VO2max was 34.89 ± 4.34 ml·kg-1·min-1 (95% CI 34.02 to 35.52). FATmin and AT occurred at 24.53 ± 4.25 ml·kg-1·min-1 VO2 (95% CI 24.29 to 24.97) and 24.04 ± 4.07 ml·kg-1·min-1 VO2 (95% CI 24.01 to 24.80), respectively. A high correlation was found between VO2 at FATmin and at AT (r = 0.91, p < 0.001, 95 % CI of 0.87 to 0.93). R2 was 0.83. Absolute fat oxidation rate at FATmin was 0.01 ± 0.01 g·min-1 (95% CI 0.00 to 0.01). Conclusions This is the first study to detect FATmin and demonstrate correlation between VO2 at FATmin and at AT in sedentary females. High inter-subjects FATmin variation demonstrates marked degree of individuality and importance of existing correlation should be taken into consideration when aiming to optimise exercise prescription for sedentary population. References American College of Sports Medicine: Position Stand. “The Recommended Quantity and Quality of Exercise for Developing and Maintaining Cardiorespiratory and Muscular Fitness”. Med Sci Sports Exerc 1998;30:975–991. Beaver WL, Wasserman K, Whipp BJ. A new method for detecting anaerobic threshold by gas exchange. J Appl Physiol 1986;60(6):2020–2027. Elia M, Livesey G. Energy expenditure and fuel selection in biological systems: the theory and practice of calculations based on indirect calorimetry and tracer methods. World Rev Nutr Diet 1992;70:68–131. Peric R, Meucci M, Nikolovski Z. Fat utilisationutilization during high intensity exercise: when does it end? Sport Med Open 2016;2(35). DOI: 10.1186/s40798-016-0060-1