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Abstract Background Autologous blood transfusion is one of the illicit strategies, banned by the World Anti-Doping Agency, to increase the levels of hemoglobin, with a consequent improvement in the delivery of oxygen to tissues. At present, this practice is detectable exclusively by the individual, longitudinal monitoring of hematological biomarkers, as in the hematological module of the Athlete Biological Passport; but this indirect approach may suffer from different confounding factors. We are presenting a multi-parametric, analytical strategy to detect autologous blood transfusions by targeting the modification of the red blood cells during storage. We focused on the assessment of “storage lesions”, targeting (i) membrane proteins: Glycophorin-A and Band 3 complex, (ii) biomarkers of oxidative stress: Peroxiredoxin-2, (iii) biomarkers of senescence: CD47 and Phosphatidylserine, (iv) erythrocytes microparticles. Results All of the above markers were monitored, by immunological and flow cytofluorimetric methods, on samples of stored whole blood collected at different time intervals, and on fresh blood samples, collected for official doping control tests, mixed “ex vivo” to simulate an autotransfusion. Although anonymized before the delivery to the laboratory, it was possible to mix samples belonging to the same subject based on the “athlete biological passport” code. Our results showed that the irreversible alteration of RBCs morphology, the loss of membrane integrity, the occurrence of hemolysis phenomena, and, more in general, the “aging” of the erythrocytes during storage are closely related to: (i) the reduced concentration, on the erythrocyte membrane, of Band 3 protein (decrease of 19% and of 39% after 20 and 40 days of storage respectively) and of glycophorin A (− 47% and − 63% respectively); (ii) the externalization of phosphatidyl serine (with a five-fold increase after 20 days and a further 2× increase after 40 days); (iii) the reduced concentration of CD47; and (iv) increased levels of erythrocyte microparticles. Conclusions The most promising method to detect the presence of transfused blood in whole blood samples can be based on a multi-parametric strategy, considering jointly both protein expression on RBCs membranes and micro-vesiculation phenomena.

We considered in this study the possibility of developing an indirect procedure for detecting myostatin inhibition/suppression, a practice that is prohibited as doping in sport. We have specifically considered the potential diagnostic utility of human serum myokines as indirect markers of myostatin inhibition. Myostatin, its main antagonist follistatin, and other myokines (follistatin-like 1, musclin, oncostatin, osteonectin, irisin, brain derived neurotrophic factor, and insulin-like growth factor-1) were selected as a panel of potential biomarkers whose levels may be altered following myostatine suppression. The serum levels of myostatin and of the nine myokines were measured in elite athletes of different age, sex, and sport discipline, and their cross correlation assessed by multivariate analysis. All myokines resulted to be measurable in human serum, except for musclin and irisine, whose levels were below the limits of quantitation in a reduced number of samples. Serum concentrations varied of different orders in magnitude (musclin and osteonectin 10 ng/mL), while no significant differences were found between female and male subjects, with the exceptions of follistatin-like 1 and musclin, showing a higher concentrations in females ( p < 0.05). Levels of insulin-like growth factor 1 and brain derived neurotrophic factor were significantly higher in power athletes than in endurance ones. Multivariate statistics showed that musclin, follistatin-like 1 and oncostatin are more clustered and correlated to myostatin than other myokines, suggesting they could be considered as potential biomarkers of doping by myostatin inhibitors.

Introduction The hematological module of the Athlete Biological Passport (ABP) consists of individual and longitudinal monitoring of hematological parameters biomarkers responsive to erythropoiesis alteration. Based on an adaptive Bayesian model, the ABP seeks to identify non-physiological variations that may correspond to different types of blood doping. In this context, the impact of a standard blood donation (500 ml) was evident on the ABP parameters, hence supporting the robustness of the model. However, there is only scarce evidence of single or multiple withdrawals of smaller volumes of blood, closer to a modern blood doping strategy. This study investigated the impact of low-volume blood withdrawal on hematological variables for anti-doping purposes. Methods Twelve healthy subjects (7 women and 5 men; 26 ±6 yrs, 170 ±7 cm; 65 ±7 kg) were recruited. After baseline measurement, 140 ml whole blood was withdrawn with subsequent measures of hematological parameters by flow cytometry (Sysmex XN-1000, Sysmex Corporation, Japan) for 3 weeks. Blood volumes (BV) and total hemoglobin mass (Hbmass) were determined by means of a validated CO-rebreathing method. Ferritin (FERR) concentration was additionally measured by chemifluminescence immunoassay technique. Individual profiles were generated using the official ABP adaptive model using the official software platform of the World Anti-Doping Agency (WADA). Mixed model repeated measures analyses were run for each parameter to determine whether changes in the dependent (hematological variables) variables differed over time (fixed factor). Results 43.2 ±8 g of hemoglobin was removed during the 140 ml blood withdrawal. A significant decrease in Hbmass (726 ±185 vs. 709 ±189 g; p = 0.007) and Red Blood Cell Volume (RBCV; 2190 ±521 vs. 2129 ±552 L; p = 0.028) was observed at D + 7 with a return to basal value at D + 14. No significant variations were observed for the primary and secondary variables of the ABP. No Atypical Passport Finding (ATPF) were generated in the individual ABP profiles. Conversely, FERR was significantly downregulated at D + 7 (95.8 ±76 vs. 87.1 ±76 μg/l; p = 0.018). Discussion/Conclusion While the ABP has demonstrated substantial advantages for traditional doping scenarios, our results challenge the indirect detection of low-volume blood withdrawals in a contemporary blood doping scenario. The impact of low-volume autologous blood transfusion on time trial performance has recently been demonstrated, supporting the need for novel biomarkers with extended detection windows. Hbmass could hence represent an additional marker to screen blood doping while technical issues for the use of CO-rebreathing remain. FERR as a marker of altered iron metabolism was there against shown to be sensitive and possibly suitable for outlining low-volume blood transfusion. The large inter-subject variability underlines the importance of individual longitudinal monitoring to provide sufficient sensitivity in the interpretation of hematological variations. Our study finally confirms that even low-volume blood withdrawals are associated with significant hematological changes while frequent blood samples and additional biomarkers are certainly required to better tackle blood doping strategies using such low-volume transfusions.

The Athlete Biological Passport (ABP) was introduced to complement the direct anti-doping approach by indirectly outlining the possible use of prohibited substances or methods in sports. The ABP proved its effectiveness, at least through a deterrent effect, even though the matrices used for longitudinal monitoring (urine and blood) are subject to many intrinsic (e.g., genetic) and extrinsic (e.g., environmental conditions) confounding factors. In that context, new and more specific biomarkers are currently under development to enhance both the sensitivity and the specificity of the ABP. Multiple strategies are presently being explored to improve this longitudinal monitoring, with the development of the current modules, the investigation of new strategies, or the screening of new types of doping. Nevertheless, due to the variability induced by indirect biomarkers, the consideration of confounding factors should continuously support this research. Beyond tremendous advances in analytical sensitivity, machine learning-based approaches seem inevitable to facilitate an expert interpretation of numerous biological profiles and promote anti-doping efforts. This perspective article highlights the current innovations of the Athlete Biological Passport that seem the most promising. Through different research axes, this short manuscript provides an opportunity to bring together approaches that are more widely exploited (e.g., omics strategies) and others in the early stages of investigation (e.g., artificial intelligence) seeking to develop the ABP.

The hematological module of the Athlete's Biological Passport (ABP) identifies doping methods and/or substances used to increase the blood's capacity to transport or deliver oxygen to the tissues. Recombinant human erythropoietin (rhEPOs) are doping substances known to boost the production of red blood cells and might have an effect on the blood biomarkers of the ABP. However, hypoxic exposure influences these biomarkers similarly to rhEPOs. This analogous impact complicates the ABP profiles' interpretation by antidoping experts. The present study aimed to collect and identify, through a literature search, the physiological effects on ABP blood biomarkers induced by these external factors. A total of 43 studies were selected for this review. A positive correlation (R2 = 0.605, r = 0.778, p < 0.001) was identified between the hypoxic dose and the increase in hemoglobin concentration (HGB) percentage. In addition, the change in the reticulocyte percentage (RET%) has been identified as one of the most sensitive parameters to rhEPO use. The mean effects of rhEPO on blood parameters were greater than those induced by hypoxic exposure (1.7 times higher for HGB and RET% and 4 times higher for hemoglobin mass). However, rhEPO micro-doses have shown effects that are hardly distinguishable from those identified after hypoxic exposure. The results of the literature search allowed to identify temporal and quantitative evolution of blood parameters in connection with different hypoxic exposure doses, as well as different rhEPOs doses. This might be considered to provide justified and well-documented interpretations of physiological changes in blood parameters of the Athlete Biological Passport.

In the last decade, the market for new psychoactive substances has been enriched by numerous psychedelic phenethylamines, which mimic the psychoactive effect of lysergic acid diethylamide (LSD). In particular, the -NBOMe series, which are more potent than their 2C compounds analogs, are considered worthy substitutes for LSD by users. The purpose of this study was to assess the effects of 25H-NBOMe and its halogenated derivatives (25I-NBOMe and 25B-NBOMe) in comparison to their 2C compounds analogs and LSD on the sensorimotor (visual, acoustic, and overall tactile), reaction time, spontaneous (total distance traveled) and stimulated (drag, accelerod test) motor activity, grip strength test, and prepulse inhibition (PPI) responses in mice. Systemic administration of -NBOMe, 2C compounds analogs, and LSD (0.001–10 mg/kg) differently impaired the sensorimotor, reaction time, motor, and PPI responses in mice. In particular, halogenated (25I and 25B)-NBOMe derivatives appear to be more effective than the entire class of 2C compounds analogs in altering visual and acoustic responses, affecting reaction time, and motor and sensory gating in PPI test. In fact, the specific rank order of compounds potency for nearly all of the experiments showed that (25I and 25B)-NBOMe were more potent than 2C compounds analogs and LSD. -NBOMe and 2C compounds analogs impaired not only the reception of incoming sensory stimuli (visual and acoustic), but their correct brain processing (PPI) in an equal and sometimes stronger way than LSD. This sensory impairment directly affected the spontaneous motor response and reaction time of mice, with no change in performance in stimulated motor activity tests. These aspects should be carefully considered to better understand the potential danger that psychedelic phenethylamines, in particular -NBOMe, may pose to public health, with particular reference to decreased performance in driving and hazardous works that require special sensorimotor skills.

This article presents the results of a study aimed to give new suggestions and strategies for improving the implementation of the flow cytofluorimetry-based method for the detection of homologous blood transfusions in doping control. The method is based on the recognition of the phenotypic mismatch between minority blood group antigens possessed by the donor and the recipient. Two strategies have been followed to reduce the risk of false-negative results: (i) the monitoring of a broader range of erythrocytes surface antigens; and (ii) the application of different surface erythrocyte staining protocols, tailored on the different antigens and the type of antigenic mismatch that had to be detected (whether it is the donor or the recipient who expresses or not the antigen to be detected). Special attention has also been focused on the time factor, to avoid prolonged sample storage, since hemolysis may have a significant impact on the reliability and quality of the results. Our experimental evidence suggests that the risk of false-negative results can be minimized by (i) the expansion of the antigen panel, with the inclusion of four additional targets; (ii) a more accurate selection of the gating area of the red blood cells; (iii) the choice of a better fluorochrome (alexa fluor 488) to be conjugated to the secondary antibody; and (iv) the implementation of different staining protocols depending on the nature of the double population to be detected (donor expressing vs. recipient non-expressing and vice versa). The combination of the above approaches allowed a significant reduction of false-negative results, assessed on samples simulating a homologous blood transfusion between two compatible subjects.

Γ-Valero-lactone (GVL) is a freely marketed organic solvent and food additive. GVL is also an underrated legal substitute for γ-hydroxybutyric acid (GHB), and it is sold under the trade name ''Tranquili-G''. GVL is usually not included in forensic drug testing, even though it has been reported in drug-facilitated sexual assaults (DFSA).To date, few studies verified the effectiveness of GVL as a hypnotic/narcotic substance, and no data are available on its urinary excretion profile. This work aims to fill this knowledge gap and lead to the introduction of GVL in forensic drug testing, especially when DFSA is suspected.To monitor the timeframe of GVL activity in vivo, we have assessed the effects on sensorimotor responses in visual placing tests carried out on CD-1 mice at a dose of 400 mg/kg of GVL, administered by gastric gavage. In parallel, samples of the in vitro and in vivo metabolism studies were analyzed using a rapid and cost-effective analytical procedure based on gas chromatography coupled to mass spectrometry. Our data confirmed that GVL impairs visual placing response in mice in the first 4 h after the intake, and they also show that GVL is a less potent substitute of GHB. The urinary excretion profile of GVL is consistent with the results of the behavioral study, with a maximum of excretion in the first 5 h after the intake. GVL is only detectable in the first 0–8 h after the intake. Our data confirmed the same rapid urinary excretion rate of GHB in urine and the related forensic implications, with the risk of possible false-negative results, especially when DFSA is suspected.

Cytochrome P450s (CYPs) are an essential family of enzymes in the human body. They play a crucial role in metabolism, especially in human steroid biosynthesis. Reactions catalyzed by these enzymes are highly stereo- and regio-specific. Lack or severe malfunctions of CYPs can cause severe diseases and even shorten life. Hence, investigations on metabolic reactions and structural requirements of substrates are crucial to gain further knowledge on the relevance of different enzymes in the human body functions and the origin of diseases. One key enzyme in the biosynthesis of gluco- and mineralocorticoids is CYP21A2, also known as steroid 21-hydroxylase. To investigate the steric and regional requirements of substrates for this enzyme, we performed whole-cell biotransformation assays using a strain of fission yeast Schizosaccharomyces pombe recombinantly expressing CYP21A2. The progestogens progesterone, pregnenolone, and their 17α-hydroxy-derivatives were used as substrates. After incubation, samples were analyzed using gas chromatography coupled to mass spectrometry. For progesterone and 17α-hydroxyprogesterone, their corresponding 21-hydroxylated metabolites 11-deoxycorticosterone and 11-deoxycortisol were detected, while after incubation of pregnenolone and 17α-hydroxypregnenolone, no hydroxylated product was observed. Findings were confirmed with authentic reference material. Molecular docking experiments agree with these results and suggest that interaction between the 3-oxo group and arginine-234 of the enzyme is a strict requirement. The presented results demonstrate once more that the presence of an oxo-group in position 3 of the steroid is indispensable, while a 3-hydroxy group prevents hydroxylation in position C-21 by CYP21A2. This knowledge may be transferred to other CYP21A2 substrates and hence help to gain essential insights into steroid metabolism.

3-(1-Naphthalenylmethyl)-1-pentyl-1H-indole (JWH-175) is a synthetic cannabinoid illegally marketed for its psychoactive cannabis-like effects. This study aimed to investigate and compare in vitro and in vivo pharmacodynamic activity of JWH-175 with that of 1-naphthalenyl (1-pentyl-1H-indol-3-yl)-methanone (JWH-018), as well as evaluate the in vitro (human liver microsomes) and in vivo (urine and plasma of CD-1 male mice) metabolic profile of JWH-175. In vitro binding studies showed that JWH-175 is a cannabinoid receptor agonist less potent than JWH-018 on mouse and human CB1 and CB2 receptors. In agreement with in vitro data, JWH-175 reduced the fESPS in brain hippocampal slices of mice less effectively than JWH-018. Similarly, in vivo behavioral studies showed that JWH-175 impaired sensorimotor responses, reduced breath rate and motor activity, and increased pain threshold to mechanical stimuli less potently than JWH-018. Metabolic studies demonstrated that JWH-175 is rapidly bioactivated to JWH-018 in mice blood, suggesting that in vivo effects of JWH-175 are also due to JWH-018 formation. The pharmaco-toxicological profile of JWH-175 was characterized for the first time, proving its in vivo bio-activation to the more potent agonist JWH-018. Thus, it highlighted the great importance of investigating the in vivo metabolism of synthetic cannabinoids for both clinical toxicology and forensic purposes.

Background: Humanin and the mitochondrial open reading frame of the 12S rRNA-c (MOTS-c) are mitochondrial encoded peptides involved in energy metabolism, cytoprotection, longevity, insulin sensitivity and their expression decrease with age. Levels of these molecules have been shown to respond to acute exercise, however little is known about their modulation under different chronic exercise conditions. In this study, we aim to compare levels of Humanin and MOTS-c in non-athletes vs professional (moderate and high endurance) athletes. Methods: Serum samples were collected from 30 non-athlete controls and 75 professional athletes (47 low/moderate endurance and 28 high endurance athletes). Levels of Humanin and MOTS-c were measured by the enzyme linked immunosorbent aaasy (ELISA) and linear models were generated to compare the effect of different levels of endurance exercise on these factors in different age groups. Spearman correlation was used to assess the correlation between these factors in athletes and non-athletes. Results: We showed that professional athletes had lower levels of MOTS-c and higher levels of Humanin than sedentary controls. Within the athletic groups, high endurance athletes had lower levels of Humanin than low/moderate endurance athletes of the same gender/age groups, whereas MOTS-c levels did not change between the subgroups. Humanin and MOTS-c levels were highly correlated in athletes, but not in sedentary controls. Conclusions: This pilot data suggests that serum levels of the mitochondrial proteins MOTS-c and Humanin change in response to chronic exercise with implications on energy metabolism and performance.

Transcranial Direct Current Stimulation (tDCS) is a non-invasive brain stimulation that may enhance mental and physical performance in sports, representing a potential new form of doping (“brain doping” or “electromagnetic doping”). This study aims to identify diagnostic biomarkers for detecting the possible abuse of tDCS in sport. Brain-Derived Neurotrophic Factor (BDNF) and other neurotrophins (NT, such as beta nerve growth factor, NGF) were pre-selected as potential candidates since their serum values have been observed to change following tDCS. Neurotrophins were measured using ELISA assays in 92 serum samples collected from elite athletes, classified by sex (males = 74; females = 18), age (range 17–25 n = 27, 26–35 n = 36, and over 35 n = 14; age not known n = 15), type of sports practiced (endurance n = 74; power n = 18), and type of sample collection (“in competition” n = 24; “out of competition” n = 68). Single nucleotide polymorphisms (rs6265, rs11030099, and rs11030100) were genotyped on 88 samples to determine their influence on the analytes' basal levels. Athletes older than 35 presented higher BDNF values than younger individuals (p < 0.05). Samples collected “in competition” showed higher BDNF concentrations than those collected “out of competition” (p < 0.05). The studied polymorphisms appeared to affect only on proBDNF, not altering BDNF serum concentrations. NT-3 and NT-4 were poorly detectable in serum. Our results suggest that BDNF can be considered as a first biomarker to detect the abuse of tDCS in sport doping. Further studies are necessary to assess whether proBDNF and beta NGF can also be considered suitable biomarkers to detect the recourse to electromagnetic brain stimulation in sports, especially in the case their serum levels can be monitored longitudinally. To the best of our knowledge, this is the first study aimed to pre-select serum biomarkers to identify the use of tDCS, and represents the first step toward the development of an indirect strategy, preferably based on the longitudinal monitoring of individual data, for the future detection of “brain doping” in sports.

Background: Over the counter preparations containing Saw palmetto and Pygeum africana extracts are commonly used in the treatment of benign prostatic hyperplasia with lower urinary tract symptoms. Their effects are likely due to their anti-androgenic activity, involving the interaction with type 1 and type 2 isoenzymes of the 5α-reductase family. Hypothesis/purpose: For effects similar to the above, the inhibitors of 5α-reductase (e.g., finasteride) are classified as confounding factors in the evaluation of the urinary steroid profile in sport doping. We aimed to verify whether the oral intake of products containing Saw palmetto and Pygeum africana could affect the urinary concentration of a series of steroids, not limited to those included in the “steroidal module” of the Athlete Biological Passport (ABP). Study design: We have followed the effects of a five-day administration of Saw palmetto (320 mg) and Pygeum africana (100 mg) to three non-smokers male subjects on the urinary parameters constituting the “steroidal module” of the ABP, and on the concentration of additional steroid markers. In parallel, we have also measured the urinary concentration of β-sitosterol, which is the active component of Saw palmetto and Pygeum Africana. Methods: Sample collection was performed five days before starting the controlled administration study to obtain the baseline urinary steroid pattern for each subject. After this period, the three volunteers took a dose of Saw palmetto (320 mg) and Pygeum africana (100 mg) formulation orally, once a day, for five consecutive days. Urine samples were collected at different times of the day and stored at −20 °C until analysis. Samples were analyzed by a previously developed and validated method based on gas chromatography coupled to tandem mass spectrometry (GC–MS/MS). Results: Our results show that the intake of a single oral dose of Saw palmetto and Pygeum africana for five consecutive days leads to a broader data dispersion of the target steroids' concentration levels, and, consequently, to a variation of the corresponding individual normality ranges. Conclusion: The intake of dietary supplements containing Saw palmetto and Pygeum africana could be a source of interference in the correct longitudinal evaluation of the individual steroid profile, as described in the framework of ABP. We believe their urinary levels should be monitored as it is presently done for other confounding factors of the steroidal module of the ABP.

ObjectivesCirculating cytokines and oxidative stress markers vary in response to different exercise regimens. This study aims to compare the immune-inflammatory and oxidative stress profiles of elite athletes from different sport disciplines as potential biomarkers of muscle damage, and cardiovascular demand.MethodsSerum samples from 88 consented elite male athletes from different sports disciplines (aquatics, n = 11, athletics, n = 22, cycling, n = 19, football, n = 28 and weightlifting, n = 8) collected at the anti-doping lab in Italy were screened for 38 cytokines and oxidative stress markers. Comparisons were made between different level of power, cardiovascular demand (CD) and endurance, as well as among the sport types.ResultsThe anti-inflammatory interleukin (IL)-10 was higher (p = 0.04) in moderate power compared with the high power group. Conversely, superoxide dismutase (SOD; p = 0.001) and malondialdehyde (MDA; p = 0.007) levels were greater in the higher power groups compared with the lower power counterpart. Among athletes who belong to different CD ranks, IL-1β and monocyte chemoattractant protein-1(MCP1) levels were higher (p = 0.03) in the low CD-rank group compared with high CD counterpart, whereas, SOD levels were higher (p = 0.001) in high and moderate CD-rank groups compared to low counterpart. For endurance groups, IL-10 and macrophage inflammatory protein (MIP)-1beta were increased (p = 0.03) in low/moderate endurance compared with the high endurance group. Finally, MIP1-beta, SOD and catalase varied significantly among the sports groups.ConclusionSpecific markers of inflammation and oxidative stress are associated with different sports disciplines and could be utilized as potential biomarkers of athletes’ health, performance, and recovery from injury.

BackgroundThe genetic predisposition to elite athletic performance has been a controversial subject due to the underpowered studies and the small effect size of identified genetic variants. The aims of this study were to investigate the association of common single-nucleotide polymorphisms (SNPs) with endurance athlete status in a large cohort of elite European athletes using GWAS approach, followed by replication studies in Russian and Japanese elite athletes and functional validation using metabolomics analysis.ResultsThe association of 476,728 SNPs of Illumina DrugCore Gene chip and endurance athlete status was investigated in 796 European international-level athletes (645 males, 151 females) by comparing allelic frequencies between athletes specialized in sports with high (n = 662) and low/moderate (n = 134) aerobic component. Replication of results was performed by comparing the frequencies of the most significant SNPs between 242 and 168 elite Russian high and low/moderate aerobic athletes, respectively, and between 60 elite Japanese endurance athletes and 406 controls. A meta-analysis has identified rs1052373 (GG homozygotes) in Myosin Binding Protein (MYBPC3; implicated in cardiac hypertrophic myopathy) gene to be associated with endurance athlete status (P = 1.43 × 10−8, odd ratio 2.2). Homozygotes carriers of rs1052373 G allele in Russian athletes had significantly greater VO2max than carriers of the AA + AG (P = 0.005). Subsequent metabolomics analysis revealed several amino acids and lipids associated with rs1052373 G allele (1.82 × 10–05) including the testosterone precursor androstenediol (3beta,17beta) disulfate.ConclusionsThis is the first report of genome-wide significant SNP and related metabolites associated with elite athlete status. Further investigations of the functional relevance of the identified SNPs and metabolites in relation to enhanced athletic performance are warranted.

Selective androgen receptor modulators (SARMs) represent non-steroidal agents commonly abused in human and animal (i.e. equine, canine) sports, with potential for further misuse as growth promoting agents in livestock-based farming. As a direct response to the real and possible implications of illicit application in both sport as well as food production systems, this study incorporated enzymatic hydrolysis (β-glucuronidase/arylsulfatase) into a previously established protocol while maintaining the minimal volume (200 µL) of urine sample required to detect SARMs encompassing various pharmacophores in urine from a range of species (i.e. equine, bovine, human, canine and rodent). The newly presented semi-quantitative UHPLC-MS/MS-based assay is shown to be fit-for-purpose, being rapid and offering high-throughput, with validation findings fulfilling criteria stipulated within relevant doping and food control legislation. • CCβ values determined at 1 ng mL−1 for majority of analytes. • Deconjugation step included in the method led to significantly increased relative abundance of ostarine in analysed incurred urine samples demonstrating the requirement for hydrolysis to detect a total form of emerging SARMs. • Assay amenable for use within routine testing to ensure fair play in animal and human sports and that animal-derived food is free from contamination with SARM residues.

Abstract Background The outstanding performance of an elite athlete might be associated with changes in their blood metabolic profile. The aims of this study were to compare the blood metabolic profiles between moderate- and high-power and endurance elite athletes and to identify the potential metabolic pathways underlying these differences. Methods Metabolic profiling of serum samples from 191 elite athletes from different sports disciplines (121 high- and 70 moderate-endurance athletes, including 44 high- and 144 moderate-power athletes), who participated in national or international sports events and tested negative for doping abuse at anti-doping laboratories, was performed using non-targeted metabolomics-based mass spectroscopy combined with ultrahigh-performance liquid chromatography. Multivariate analysis was conducted using orthogonal partial least squares discriminant analysis. Differences in metabolic levels between high- and moderate-power and endurance sports were assessed by univariate linear models. Results Out of 743 analyzed metabolites, gamma-glutamyl amino acids were significantly reduced in both high-power and high-endurance athletes compared to moderate counterparts, indicating active glutathione cycle. High-endurance athletes exhibited significant increases in the levels of several sex hormone steroids involved in testosterone and progesterone synthesis, but decreases in diacylglycerols and ecosanoids. High-power athletes had increased levels of phospholipids and xanthine metabolites compared to moderate-power counterparts. Conclusions This pilot data provides evidence that high-power and high-endurance athletes exhibit a distinct metabolic profile that reflects steroid biosynthesis, fatty acid metabolism, oxidative stress, and energy-related metabolites. Replication studies are warranted to confirm differences in the metabolic profiles associated with athletes’ elite performance in independent data sets, aiming ultimately for deeper understanding of the underlying biochemical processes that could be utilized as biomarkers with potential therapeutic implications.

4,4’-Dimethylaminorex (4,4’-DMAR) is a new synthetic stimulant, and only a little information has been made available so far regarding its pharmaco-toxicological effects. The aim of this study was to investigate the effects of the systemic administration of both the single (±)cis (0.1–60 mg/kg) and (±)trans (30 and 60 mg/kg) stereoisomers and their co-administration (e.g., (±)cis at 1, 10 or 60 mg/kg + (±)trans at 30 mg/kg) in mice. Moreover, we investigated the effect of 4,4′-DMAR on the expression of markers of oxidative/nitrosative stress (8-OHdG, iNOS, NT and NOX2), apoptosis (Smac/DIABLO and NF-κB), and heat shock proteins (HSP27, HSP70, HSP90) in the cerebral cortex. Our study demonstrated that the (±)cis stereoisomer dose-dependently induced psychomotor agitation, sweating, salivation, hyperthermia, stimulated aggression, convulsions and death. Conversely, the (±)trans stereoisomer was ineffective whilst the stereoisomers’ co-administration resulted in a worsening of the toxic (±)cis stereoisomer effects. This trend of responses was confirmed by immunohistochemical analysis on the cortex. Finally, we investigated the potentially toxic effects of stereoisomer co-administration by studying urinary excretion. The excretion study showed that the (±)trans stereoisomer reduced the metabolism of the (±)cis form and increased its amount in the urine, possibly reflecting its increased plasma levels and, therefore, the worsening of its toxicity.

Ecdysterone is a phytosteroid widely discussed for its various pharmacological, growth-promoting, and anabolic effects, mediated by the activation of estrogen receptor beta (ERbeta). Performance-enhancement in sports was demonstrated recently, and ecdysterone was consequently included in the Monitoring Program, to detect potential patterns of misuse in sport. Only few studies on the pharmacokinetics of ecdysterone in humans have been reported so far. In this study, post-administration urine samples in twelve volunteers (single dose of 50 mg of ecdysterone) were analyzed using dilute-and-inject liquid-chromatography–tandem mass spectrometry. Identification and quantitation of ecdysterone and of two metabolites, 14-deoxy-ecdysterone and 14-deoxy-poststerone, was achieved. Ecdysterone was the most abundant analyte present in post-administration urine samples, detected for more than 2 days, with a maximum concentration (Cmax) in the 2.8–8.5 h urine (Cmax = 4.4–30.0 µg/mL). The metabolites 14-deoxy-ecdysterone and 14-deoxy-poststerone were detected later, reaching the maximum concentrations at 8.5–39.5 h (Cmax = 0.1–6.0 µg/mL) and 23.3–41.3 h (Cmax = 0.1–1.5 µg/mL), respectively. Sex-specific differences were not observed. Cumulative urinary excretion yielded average values of 18%, 2.3%, and 1.5% for ecdysterone, 14-deoxy-ecdysterone, and 14-deoxy-poststerone, respectively. Ecdysterone and 14-deoxy-ecdysterone were excreted following first-order kinetics with half-lives calculated with three hours, while pharmacokinetics of 14-deoxy-poststerone needs further evaluation.

Metandienone and methyltestosterone are orally active anabolic-androgenic steroids with a 17α-methyl structure that are prohibited in sports but are frequently detected in anti-doping analysis. Following the previously reported detection of long-term metabolites with a 17ξ-hydroxymethyl-17ξ-methyl-18-nor-5ξ-androst-13-en-3ξ-ol structure in the chlorinated metandienone analog dehydrochloromethyltestosterone (“oral turinabol”), in this study we investigated the formation of similar metabolites of metandienone and 17α-methyltestosterone with a rearranged D-ring and a fully reduced A-ring. Using a semi-targeted approach including the synthesis of reference compounds, two diastereomeric substances, viz. 17α-hydroxymethyl-17β-methyl-18-nor-5β-androst-13-en-3α-ol and its 5α-analog, were identified following an administration of methyltestosterone. In post-administration urines of metandienone, only the 5β-metabolite was detected. Additionally, 3α,5β-tetrahydro-epi-methyltestosterone was identified in the urines of both administrations besides the classical metabolites included in the screening procedures. Besides their applicability for anti-doping analysis, the results provide new insights into the metabolism of 17α-methyl steroids with respect to the order of reductions in the A-ring, the participation of different enzymes, and alterations to the D-ring.

Anabolic androgenic steroids (AAS) are prohibited as doping substances in sports by the World Anti-Doping Agency. Concentrations and concentration ratios of endogenous AAS (steroid profile markers) in urine samples collected from athletes are used to detect their administration. Certain (non-prohibited) drugs have been shown to influence the steroid profile and thereby sophisticate anti-doping analysis. It was shown in vitro that the non-steroidal anti-inflammatory drug (NSAID) indomethacin inhibits selected steroid-biotransformations catalyzed by the aldo-keto reductase (AKR) 1C3, which plays a key role in the endogenous steroid metabolism. Kinetic parameters for the indomethacin-mediated inhibition of the AKR1C3 catalyzed reduction in etiocholanolone were determined in vitro using two comparing methods. As NSAIDs are very frequently used (not only) by athletes, the inhibitory impact of indomethacin intake on the steroid metabolism was evaluated, and steroid profile alterations were detected in vivo (one male and one female volunteer). Significant differences between samples collected before, during or after the intake of indomethacin for selected steroid profile markers were observed. The presented results are of relevance for the interpretation of results from doping control analysis. Additionally, the administration of NSAIDs should be carefully reconsidered due to their potential as endocrine disruptors.

Background: folliculogenesis is a strictly regulated process that may be affected by endocrine disrupting chemicals (EDCs) through sometimes not so clear molecular mechanisms. Methods: we conducted a multicentric observational study involving six fertility centers across Italy, prospectively recruiting 122 women attending a fertility treatment. Recruited women had age ≤42 years, and normal ovarian reserve. Blood and follicular fluid samples were taken for EDCs measurement using liquid chromatography tandem mass spectrometry and each woman completed an epidemiological questionnaire. Results: The main EDCs found were monobutyl phthalate (MBP) (median blood: 8.96 ng/mL, follicular fluid 6.43 ng/mL), monoethylhexyl phthalate (MEHP) (median blood: 9.16 ng/mL, follicular fluid 7.68 ng/mL) and bisphenol A (BPA) (median blood: 1.89 ng/mL, follicular fluid 1.86 ng/mL). We found that serum MBP concentration was significantly associated with the considered area (p < 0.001, adj. mean: 7.61 ng/mL, 14.40 ng/mL, 13.56 ng/mL; Area 1: Milan–Turin, Area 2: Rome–Naples; Area 3: Catania–Bari, respectively) but negatively with home plastic food packaging (p = 0.004). Follicular MBP was associated with irregular cycles (p = 0.019). No association was detected between EDCs and eating habits and other clinical and epidemiological features. Conclusions: This study represents the first Italian biomonitoring of plastic EDCs in follicular fluid, laying the basis for future prospective evaluation on oocyte quality before assisted reproduction techniques (ART).

Background Supplements are widely used among elite athletes to maintain health and improve performance. Despite multiple studies investigating use of dietary supplements by athletes, a comprehensive profiling of serum supplement metabolites in elite athletes is still lacking. This study aims to analyze the presence of various xenobiotics in serum samples from elite athletes of different sports, focusing on metabolites that potentially originate from nutritional supplements. Methods Profiling of xenobiotics in serum samples from 478 elite athletes from different sports (football, athletics, cycling, rugby, swimming, boxing and rowing) was performed using non-targeted metabolomics-based mass spectroscopy combined with ultrahigh-performance liquid chromatography. Multivariate analysis was performed using orthogonal partial least squares discriminant analysis. Differences in metabolic levels among different sport groups were identified by univariate linear models. Results Out of the 102 detected xenobiotics, 21 were significantly different among sport groups including metabolites that potentially prolong exercise tolerance (caffeic acid), carry a nootropic effect (2-pyrrolidinone), exert a potent anti-oxidant effect (eugenol, ferulic acid 4 sulfate, thioproline, retinol), or originate from drugs for different types of injuries (ectoine, quinate). Using Gaussian graphical modelling, a metabolic network that links various sport group-associated xenobiotics was constructed to further understand their metabolic pathways. Conclusions This pilot data provides evidence that athletes from different sports exhibit a distinct xenobiotic profile that may reflect their drug/supplement use, diet and exposure to various chemicals. Because of limitation in the study design, replication studies are warranted to confirm results in independent data sets, aiming ultimately for better assessment of dietary supplement use by athletes.