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Global resource extraction raises concerns about environmental pressures and the security of mineral supply. Strategies to address these concerns depend on robust information on natural resource endowments, and on suitable methods to monitor and model their changes over time. However, current mineral resources and reserves reporting and accounting workflows are poorly suited for addressing mineral depletion or answering questions about the long-term sustainable supply. Our integrative review finds that the lack of a robust theoretical concept and framework for mass-balance (MB)-consistent geological stock accounting hinders systematic industry-government data integration, resource governance, and strategy development. We evaluate the existing literature on geological stock accounting, identify shortcomings of current monitoring of mine production, and outline a conceptual framework for MB-consistent system integration based on material flow analysis (MFA). Our synthesis shows that recent developments in Earth observation, geoinformation management, and sustainability reporting act as catalysts that make MB-consistent geological stock accounting increasingly feasible. We propose first steps for its implementation and anticipate that our perspective as "resource realists" will facilitate the integration of geological and anthropogenic material systems, help secure future mineral supply, and support the global sustainability transition.
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Conservação dos Recursos Naturais , Minerais , Conservação dos Recursos Naturais/métodosRESUMO
INTRODUCTION: Asthma is a heterogeneous disease with poorly defined phenotypes. Patients with severe asthma often receive multiple treatments including oral corticosteroids (OCS). Treatment may modify the observed metabotype, rendering it challenging to investigate underlying disease mechanisms. Here, we aimed to identify dysregulated metabolic processes in relation to asthma severity and medication. METHODS: Baseline urine was collected prospectively from healthy participants (n=100), patients with mild-to-moderate asthma (n=87) and patients with severe asthma (n=418) in the cross-sectional U-BIOPRED cohort; 12-18-month longitudinal samples were collected from patients with severe asthma (n=305). Metabolomics data were acquired using high-resolution mass spectrometry and analysed using univariate and multivariate methods. RESULTS: A total of 90 metabolites were identified, with 40 significantly altered (p<0.05, false discovery rate <0.05) in severe asthma and 23 by OCS use. Multivariate modelling showed that observed metabotypes in healthy participants and patients with mild-to-moderate asthma differed significantly from those in patients with severe asthma (p=2.6×10-20), OCS-treated asthmatic patients differed significantly from non-treated patients (p=9.5×10-4), and longitudinal metabotypes demonstrated temporal stability. Carnitine levels evidenced the strongest OCS-independent decrease in severe asthma. Reduced carnitine levels were associated with mitochondrial dysfunction via decreases in pathway enrichment scores of fatty acid metabolism and reduced expression of the carnitine transporter SLC22A5 in sputum and bronchial brushings. CONCLUSIONS: This is the first large-scale study to delineate disease- and OCS-associated metabolic differences in asthma. The widespread associations with different therapies upon the observed metabotypes demonstrate the need to evaluate potential modulating effects on a treatment- and metabolite-specific basis. Altered carnitine metabolism is a potentially actionable therapeutic target that is independent of OCS treatment, highlighting the role of mitochondrial dysfunction in severe asthma.
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Antiasmáticos , Asma , Corticosteroides/uso terapêutico , Antiasmáticos/uso terapêutico , Asma/genética , Carnitina/uso terapêutico , Estudos Transversais , Humanos , Índice de Gravidade de Doença , Membro 5 da Família 22 de Carreadores de SolutoRESUMO
RATIONALE: Asthma phenotyping requires novel biomarker discovery. OBJECTIVES: To identify plasma biomarkers associated with asthma phenotypes by application of a new proteomic panel to samples from two well-characterised cohorts of severe (SA) and mild-to-moderate (MMA) asthmatics, COPD subjects and healthy controls (HCs). METHODS: An antibody-based array targeting 177 proteins predominantly involved in pathways relevant to inflammation, lipid metabolism, signal transduction and extracellular matrix was applied to plasma from 525 asthmatics and HCs in the U-BIOPRED cohort, and 142 subjects with asthma and COPD from the validation cohort BIOAIR. Effects of oral corticosteroids (OCS) were determined by a 2-week, placebo-controlled OCS trial in BIOAIR, and confirmed by relation to objective OCS measures in U-BIOPRED. RESULTS: In U-BIOPRED, 110 proteins were significantly different, mostly elevated, in SA compared to MMA and HCs. 10 proteins were elevated in SA versus MMA in both U-BIOPRED and BIOAIR (alpha-1-antichymotrypsin, apolipoprotein-E, complement component 9, complement factor I, macrophage inflammatory protein-3, interleukin-6, sphingomyelin phosphodiesterase 3, TNF receptor superfamily member 11a, transforming growth factor-ß and glutathione S-transferase). OCS treatment decreased most proteins, yet differences between SA and MMA remained following correction for OCS use. Consensus clustering of U-BIOPRED protein data yielded six clusters associated with asthma control, quality of life, blood neutrophils, high-sensitivity C-reactive protein and body mass index, but not Type-2 inflammatory biomarkers. The mast cell specific enzyme carboxypeptidase A3 was one major contributor to cluster differentiation. CONCLUSIONS: The plasma proteomic panel revealed previously unexplored yet potentially useful Type-2-independent biomarkers and validated several proteins with established involvement in the pathophysiology of SA.
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Asma , Qualidade de Vida , Proteínas Sanguíneas , Humanos , Inflamação/metabolismo , Proteômica , Índice de Gravidade de Doença , Esteroides/uso terapêuticoRESUMO
Rationale: New approaches are needed to guide personalized treatment of asthma.Objectives: To test if urinary eicosanoid metabolites can direct asthma phenotyping.Methods: Urinary metabolites of prostaglandins (PGs), cysteinyl leukotrienes (CysLTs), and isoprostanes were quantified in the U-BIOPRED (Unbiased Biomarkers for the Prediction of Respiratory Diseases Outcomes) study including 86 adults with mild-to-moderate asthma (MMA), 411 with severe asthma (SA), and 100 healthy control participants. Validation was performed internally in 302 participants with SA followed up after 12-18 months and externally in 95 adolescents with asthma.Measurement and Main Results: Metabolite concentrations in healthy control participants were unrelated to age, body mass index, and sex, except for the PGE2 pathway. Eicosanoid concentrations were generally greater in participants with MMA relative to healthy control participants, with further elevations in participants with SA. However, PGE2 metabolite concentrations were either the same or lower in male nonsmokers with asthma than in healthy control participants. Metabolite concentrations were unchanged in those with asthma who adhered to oral corticosteroid treatment as documented by urinary prednisolone detection, whereas those with SA treated with omalizumab had lower concentrations of LTE4 and the PGD2 metabolite 2,3-dinor-11ß-PGF2α. High concentrations of LTE4 and PGD2 metabolites were associated with lower lung function and increased amounts of exhaled nitric oxide and eosinophil markers in blood, sputum, and urine in U-BIOPRED participants and in adolescents with asthma. These type 2 (T2) asthma associations were reproduced in the follow-up visit of the U-BIOPRED study and were found to be as sensitive to detect T2 inflammation as the established biomarkers.Conclusions: Monitoring of urinary eicosanoids can identify T2 asthma and introduces a new noninvasive approach for molecular phenotyping of adult and adolescent asthma.Clinical trial registered with www.clinicaltrials.gov (NCT01976767).
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Asma/metabolismo , Biomarcadores/urina , Inflamação/metabolismo , Leucotrieno E4/metabolismo , Leucotrieno E4/urina , Prostaglandinas/metabolismo , Prostaglandinas/urina , Adulto , Asma/fisiopatologia , Feminino , Humanos , Inflamação/fisiopatologia , Masculino , Pessoa de Meia-IdadeRESUMO
Homologous blood transfusion (HBT) is used for doping in endurance sports since the 1960s. The blood comes from a compatible donor, that is, someone with a compatible ABO and rhesus blood group. Despite been prohibited by the IOC in 1985, no detection method was available until 2003. Then came the idea to use red blood cells (RBC) minor blood groups antigens that constitute an "identity" card of someone's RBC to detect the presence of a second RBC population. The method validated for doping control samples uses flow cytometry after incubation of isolated RBC with eight to 12 primary antibodies against specific minor blood groups antigens. The presence of double populations of RBC is revealed by a major and a minor peak in a fluorescence histogram. The sensitivity was estimated sufficient to detect HBT for a few weeks. Despite the complexity and cost of the method, right after its application in 2004, several cases of HBT were identified but the number of cases dropped rapidly over the years. In the 2010s, other ways to detect HBT were developed and evaluated: indirect detection using the Athlete Biological Passport approach, and a few years later forensic DNA analysis to establish the presence of two different DNA in a blood sample after HBT. Despite the high specificity of the latter, the sensitivity was recently questioned in vivo. Nowadays, the flow cytometry method remains the method of choice for HBT detection and recent investigations helped to simplify the method and increase its specificity and sensitivity.
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The abuse of recombinant erythropoietin (rEPO) and other erythropoietin (EPO) receptor agonists (ERAs) in sports prompted the need for sensitive detection methods of these substances. Dried blood spot (DBS) samples offer an easy solution for simultaneous collection of blood and urine during a doping control, but sensitivity issues are often presented as a challenge for routine EPO analysis from DBS. Its potential use for detecting rEPO micro-doses and the EPO gene c.577del variant thus needed further demonstration. Here, capillary blood collected from the arm skin of 111 athletes with Tasso-M20 (17.5 µL/spot), collected during professional triathlon competitions, were analysed. Also, venous blood samples from healthy volunteers were used to prepare several spots of 20 µL on Mitra VAMS (from an rEPO micro-dose study) and Whatman filter paper (from an EPO gene variant study). Immunopurification of 2 spots with MAIIA EPO Purification Gel Kit and analysis with sodium N-lauroylsarcosinate polyacrylamide gel electrophoresis (SAR-PAGE)/Western blot resulted in sensitive detection of (1) micro-doses of rEPO from Mitra VAMS, (2) endogenous EPO from Tasso-M20 in all in-competition subjects, and (3) the EPO c.577del variant from Whatman filter paper. Additionally, in-competition endogenous EPO was detected in DBS even when matching urine samples had undetectable EPO. In conclusion, this work demonstrated that DBS can be a useful complementary matrix to urine samples for EPO detection.
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Aim: The aim of this study was to examine the association between physical activity (PA) and combined hormonal contraceptive (CHC) on female students' self perceptio of their menstrual cycle symptoms. Methods: Healthy French female students (n = 834) completed an online questionnaire to assess their PA level (Group 1: non-active; Group 2: moderate physical activity; Group 3: high physical activity; Group 4: very high physical activity), menstrual status or contraception use, self-reported diet and medication, impact on engagement in some social activities, and self-assessment of perceived mental and physical symptoms during the week prior to menses (PM) for students with a normal menstrual cycle (NMC), and the week of menses (ME) for normal menstrual cycle students and those using combined hormonal contraception. Results: Whatever the conditions (PM and ME, NMC and CHC), fewer self-perceived symptoms and self-reported alteration in fat intake were reported by the students in Group 4, and more analgesic and anti-inflammatory medication use was reported by Group 1. Fewer self-perceived symptoms were also found in CHC vs NMC female students for all physical activity levels, but in a more marked way when associated with very high physical activity. In addition, less university and sports practice absenteeism was observed with high and very high physical activity. Conclusion: In conclusion, the perception of menstrual cycle symptoms was lower with very high physical activity, as with combined hormonal contraception. Moreover, female students training more than 5 h/week also reported less university absenteeism and impairment in physical activities. Further studies are necessary to establish the causal link of physical activity and combined hormonal contraception on menstrual symptoms.
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Like any athlete, female athletes may be tempted to use prohibited substances during competition or training to enhance their performance. Anti-doping tests performed on female athletes in summer Olympic sports from two geographical areas: Australia/ New Zealand, and France were compared. First, the distribution of sample collections across different sports disciplines, as well as the distribution of substances was investigated. Then the distribution of collections and substances detected in the five sports categories (Strength/Speed, Endurance, Mixed, Motor Skills with High Energy Expenditure, and Motor Skills with Low Energy Expenditure) were studied with consideration of therapeutic use exemptions obtained by the athlete. Australia/New Zealand and France were similar in their overall number of anti-doping collections performed. Likewise, both regions had the same sports disciplines (athletics, aquatics, cycling) and sport categories (Mixed and Endurance) as having the highest number of sample collections. The Motor Skills with High Energy Expenditure, and Motor Skills with Low Energy Expenditure categories had the lowest number of sample collections. However, the number of substances detected was significantly different (p < 0.05) with a greater number of substances found in the French data. There were a few substances in common between the two geographical areas, namely prednisone/prednisolone, carboxy-THC, terbutaline, vilanterol and methylphenidate, but most were different. In-competition tests were the category where most of the AAFs were found.
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Two doping cases of homologous blood transfusion (HBT) during Tokyo 2020 Summer Olympics have shown that more controls are needed. The method of detection using flow cytometry to evaluate the expression of minor blood group antigens from red blood cells (RBCs) and identify different RBC populations is efficient but still complex to perform with multiple antigens detection. Recently, the interest of using forensic DNA analysis was also highlighted as a potential new method to detect HBT, with possibility to start from dried blood spots (DBS) instead of fresh blood. After a first phase of development, a protocol was validated for HBT detection using DNA analysis after extraction from DBS. Presence of a second DNA was clear down to 2% of donor blood in vitro. A flow cytometry protocol was also developed with preparation and analysis in 96-well plates and detection of two different antigens per well using two secondary antibodies with distinct fluorophores. The objective of the project was to evaluate the window of detection of an HBT performed in vivo with 150 mL of RBC concentrate. Blood samples obtained over 7 weeks post-transfusion were analyzed. DNA profiling from DBS was not sensitive enough to detect the presence of a second DNA even 1 day after transfusion. On the contrary, the flow cytometry protocol was very efficient and allowed identification of several double populations of RBC (expressing/non-expressing several antigens) until day 50 post-transfusion. This protocol can be fully validated for a future application to doping control samples.
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Transfusão de Sangue , Esportes , Transfusão de Sangue/métodos , Eritrócitos , Impressões Digitais de DNA , DNARESUMO
Rationale: Patients with severe asthma are dependent upon treatment with high doses of inhaled corticosteroids (ICS) and often also oral corticosteroids (OCS). The extent of endogenous androgenic anabolic steroid (EAAS) suppression in asthma has not previously been described in detail. The objective of the present study was to measure urinary concentrations of EAAS in relation to exogenous corticosteroid exposure. Methods: Urine collected at baseline in the U-BIOPRED (Unbiased Biomarkers for the Prediction of Respiratory Disease outcomes) study of severe adult asthmatics (SA, n=408) was analysed by quantitative mass spectrometry. Data were compared to that of mild-to-moderate asthmatics (MMA, n=70) and healthy subjects (HC, n=98) from the same study. Measurements and main results: The concentrations of urinary endogenous steroid metabolites were substantially lower in SA than in MMA or HC. These differences were more pronounced in SA patients with detectable urinary OCS metabolites. Their dehydroepiandrosterone sulfate (DHEA-S) concentrations were <5% of those in HC, and cortisol concentrations were below the detection limit in 75% of females and 82% of males. The concentrations of EAAS in OCS-positive patients, as well as patients on high-dose ICS only, were more suppressed in females than males (p<0.05). Low levels of DHEA were associated with features of more severe disease and were more prevalent in females (p<0.05). The association between low EAAS and corticosteroid treatment was replicated in 289 of the SA patients at follow-up after 12-18â months. Conclusion: The pronounced suppression of endogenous anabolic androgens in females might contribute to sex differences regarding the prevalence of severe asthma.
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To achieve optimal sports performances, women and men may show specific doping practices because of the physiological and psychological gender differences, but there are few data on this topic. Here, we report the apparent use of prohibited substances and methods by female athletes based on analyses of the doping tests collected by the French Anti-Doping Agency from 2013 to 2019. We compared the frequency of use and the ergogenic and side effects to those of their male counterparts. The results revealed lower use of prohibited substances in female vs. male athletes, with significantly fewer anabolic agents, hormone and metabolic modulators, and cannabinoids. Gender specificity in utilization of substance classes was also shown. Relatively lower use of hormone modulators and cannabinoids and higher use of beta-2 agonists, diuretics and glucocorticoids were found in the woman cohort compared with men cohort, combined with the different choice of substances, possibly because of the altered ergogenic and/or side effects. However, no impact due to gender regarding the sports disciplines was observed, with both women and men showing similar use of anabolic agents, mainly in the anaerobic sports, and EPO and corticoids, mainly in endurance or mixed sports. Further studies are needed to put these French data into a global perspective, comparing uses across countries and exploring possible new developments in the fight against doping in women.
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The World Anti-Doping Agency (WADA) prohibits athletes from using recombinant growth hormone (GH). The validated method used in antidoping laboratories for the direct detection of exogenous GH in serum requires two immunoluminometric assays (ILMAs): The first mainly measures the concentration of the full-length (22 kDa) form of GH (recGH), and the second measures concentrations of multiple GH fragments produced by the pituitary gland (22 kDa, 20 kDa and other forms) (pitGH). The tube-by-tube analysis is laborious. A recent development opened new possibilities to simplify the detection of recGH in serum: multiplexed immunoassays that detect multiple targets in a single well of a 96-well plate using an ELISA-like procedure with high sensitivity. Our aim was to evaluate this technology by developing a customized assay for GH detection. One pair of antibodies with specificities similar to those of the recGH assay and one pair of antibodies compatible with pitGH detection were selected for a single duplex assay. Forty-eight serum samples (negative athlete samples and positive samples following GH administration) were analyzed using the two methods. The microplate duplex assay discriminated between the negative athlete samples and the positive controls, although the rec/pit ratios from the duplex assay were lower than those obtained with the ILMAs. This new assay would offer a modern alternative to ILMAs, with fewer analytical steps and a smaller sample volume. However, an adaptation of the decision limits seems mandatory.
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Dopagem Esportivo , Hormônio do Crescimento Humano , Hormônio do Crescimento , Humanos , Imunoensaio/métodos , Isoformas de Proteínas , Proteínas Recombinantes , Detecção do Abuso de Substâncias/métodosRESUMO
The World Anti-Doping Agency (WADA) has recently implemented dried blood spots (DBSs) as a matrix for doping control. However, specifications regarding the analysis of the class of prohibited substances called erythropoietin (EPO) receptor agonists (ERAs) from DBSs are not yet described. The aim of this study was to find optimal conditions (sample volume and storage) to sensitively detect endogenous erythropoietin (hEPO) and prohibited ERAs from DBSs and compare detection limits to WADA-stipulated minimum required performance levels (MRPLs) for ERAs in serum/plasma samples. Venous whole blood was spotted onto Whatman 903 DBS cards with primarily 60 µl of blood, but various volumes from 20 to75 µl were tested. All samples were immunopurified with MAIIA EPO Purification Gel kit (EPGK) and analysed with sodium N-lauroylsarcosinate polyacrylamide gel electrophoresis (SAR-PAGE) and Western blot. Sixty-microliter DBSs allowed the detection of the four main ERAs (BRP, NESP, CERA and EPO-Fc) at concentrations close to WADA's MRPLs described for 500 µl of serum/plasma. Different storage temperatures, from -20°C to 37°C, were evaluated and did not affect ERA detection. A comparison of the detection of endogenous EPO from the different anti-doping matrices (urine, serum and DBSs produced from upper arm capillary blood) from five participants for 6 weeks was performed. Endogenous EPO extracted from DBSs showed intra-individual variations in male and female subjects, but less than in urine. Doping controls would benefit from the stability of ERAs on DBSs: It can be a complementary matrix for ERA analysis, particularly in the absence of EPO signals in urine.
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Dopagem Esportivo , Hematínicos , Receptores da Eritropoetina , Detecção do Abuso de Substâncias , Teste em Amostras de Sangue Seco , Eletroforese em Gel de Poliacrilamida , Feminino , Hematínicos/análise , Humanos , Masculino , Receptores da Eritropoetina/agonistas , Detecção do Abuso de Substâncias/métodosRESUMO
Luspatercept (Reblozyl®) is a newly approved anti-anemic drug prohibited by the World Anti-Doping Agency. It promotes erythropoiesis by limiting apoptosis of immature erythroblasts and the risk of misuse by athletes for doping is high. Proposed detection methods have been published recently but only evaluated in vitro. The objective of this study was to perform the first administration of luspatercept in healthy volunteers for antidoping purpose and to evaluate the detectability in serum, dried capillary blood spots (DBS, collected using TASSO M20 device), and urine. Indirect detection was also evaluated by analyzing hematological parameters for the Athlete Biological Passport. Four volunteers (two males, two females) received one subtherapeutic dose of luspatercept (0.25 mg/kg) followed 3 weeks after by a second dose. Samples were collected from before administration until 7 weeks after the second dose. After immunopurification, electrophoretic separation SDS-/SAR-/IEF- polyacrylamide gel electrophoresis (PAGE), and immunodetection, luspatercept was detected at high levels in serum until the end of the collection, sign of a very slow elimination and similarly detected unchanged at lower levels in urine from 2 days after the first administration until 7 weeks postadministration. DBS showed also the same long window of detection. Luspatercept effects were however of limited amplitude on hematological markers, and only two subjects presented atypical points outside the physiological limits during the study. The direct detection method was very efficient, and change of electrophoretic method and detection antibody can be used for confirmation of suspicious samples.
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Dopagem Esportivo , Hematínicos , Masculino , Feminino , Humanos , Voluntários Saudáveis , Receptores de Activinas Tipo II , Fragmentos Fc das Imunoglobulinas , Detecção do Abuso de Substâncias/métodosRESUMO
Jimaixin™ (Jintan Ltd, China) is a biosimilar of recombinant erythropoietin (rEPO) now authorized for therapeutic application in China. With a risk of abuse by athletes, a clear evaluation of its detection using the electrophoretic methods in use in antidoping laboratories was necessary. In a previous work, we showed that Jimaixin™ electrophoretic profile presented slight changes compared with the original drug (first generation rEPO) and that a spike of Jimaixin™ in urine and serum was well identified by SDS-PAGE but with less performance by IEF-PAGE unless a neuraminidase treatment was applied first. The aims of this research were to perform an intravenous administration of Jimaixin™ on three healthy subjects (one microdose [10 IU/kg] and three therapeutic doses [50 IU/kg]) and to evaluate the detection in urine and blood up to 7 days post administration. Analysis of the samples showed that Jimaixin™ detection was complicated by IEF-PAGE due to the loss of the most distinctive basic isoforms. In addition, a neuraminidase treatment did not improve detection (contrary to the observations from spike experiments). On the contrary, Jimaixin™ was very efficiently detected in blood and urine by SDS-PAGE: up to 40 h after a microdose and up to 7 days after the therapeutic doses. The effect of Jimaixin™ on hematological parameters was limited to a clear but transitory increase of the reticulocytes. These data give new elements to better survey a potential misuse of Jimaixin™ by athletes.
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Medicamentos Biossimilares/análise , Dopagem Esportivo/prevenção & controle , Eritropoetina/análise , Detecção do Abuso de Substâncias/métodos , Administração Intravenosa , Adulto , Medicamentos Biossimilares/administração & dosagem , Medicamentos Biossimilares/farmacocinética , Relação Dose-Resposta a Droga , Eletroforese em Gel de Poliacrilamida/métodos , Eritropoetina/administração & dosagem , Eritropoetina/farmacocinética , Humanos , Masculino , Proteínas Recombinantes , Fatores de TempoRESUMO
Continuous erythropoietin receptor activator (CERA) is a third-generation erythropoiesis-stimulating agent that was developed for the treatment of anemia. However, misuse of CERA for doping in endurance sports has been reported. Previous studies have shown blood as the matrix of choice for the detection of CERA, due to its high molecular weight. The use of dried blood spots (DBSs) for anti-doping purposes constitutes a complementary approach to the standard urine and venous blood matrices and could facilitate sample collection and increase the number of blood samples available for analysis due to reduced costs of sample collection and transport. Here, we investigated whether CERA could be indirectly detected in extracts of single DBSs using an erythropoietin-specific immunoassay that is capable of providing results within approximately 2 h. Reconstituted DBS samples were prepared from mixtures of red blood cell pellets and serum samples. The samples were collected in a previous clinical study in which six healthy volunteers were injected with a single, 200 µg dose of CERA. Using a commercially available ELISA kit, CERA was detected in the DBSs with a detection window of up to 20 days post-injection. Furthermore, in order to demonstrate the fitness-for-purpose, three authentic doping control serum samples, which were identified as containing CERA, were analyzed by the presented methodological approach on DBS. The testing procedure described here could be used as a fast and cost-effective method for the detection of CERA abuse in sport.
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Dopagem Esportivo , Eritropoetina , Hematínicos , Dopagem Esportivo/prevenção & controle , Humanos , Polietilenoglicóis/análiseRESUMO
The modification of gene expression to treat diseases is a field of research with exponential growth. As doping in sport closely follows emerging therapies, a surveillance of the modification of gene expression to enhance performance is needed. The gene coding for erythropoietin (EPO) is one target of interest. Since 2010, several protocols have been proposed to identify EPO gene doping by focusing on the presence in blood of a transgene that differ in size from the endogenous gene sequence, normally found in the human DNA. In this work, our aim was to validate an easily applicable method for EPO gene doping detection in dried blood spots (DBS). We evaluated the detection of EPO transgene in 20-µl DBS after the spike of a plasmid carrying the EPO transgene in whole blood. Three different DBS were compared: Nucleic-Card™, Whatman® 903, and the volumetric 20-µl VAMS™. Detection was performed with real-time polymerase chain reaction (PCR) and validated with two Taqman assays (one commercial and one custom) specific for the EPO transgene. The initial testing procedure could be done using one assay (custom) and the confirmation using the second one (commercial Taqman) with a final check of the size of the PCR product. Starting from 20-µl dried blood, 1000 copies of EPO transgene could efficiently be detected with the three types of DBS, VAMS showing a slightly better sensitivity. No loss of sensitivity was observed after 1-month storage of DBS at room temperature. This method could be applied to DBS collected during doping controls and allows reanalysis.
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Dopagem Esportivo/prevenção & controle , Eritropoetina/genética , Reação em Cadeia da Polimerase em Tempo Real/métodos , Teste em Amostras de Sangue Seco/métodos , Humanos , Temperatura , Fatores de Tempo , TransgenesRESUMO
Erythropoietin receptor agonists (ERAs) are drugs acting on the early erythropoietic stages developed to treat anemia and other erythropoiesis disease and are prohibited by the World Anti-Doping Agency (WADA). As an alternative to ERAs, a new drug, belonging to the transforming growth factor-b inhibitors family, was recently developed to treat diseases linked to ineffective erythropoiesis. This drug, named as Luspatercept (Reblozyl®), is acting on the later stages of erythropoiesis to promote erythrocytes. This drug might be used by cheating athletes either independently or in combination with ERAs. Indeed, it was shown that Luspatercept and recombinant erythropoietin (rEPO) can act synergistically to increase red blood cells production, potentially allowing the use of lower doses for an efficient effect. Our aim was to find a way to combine the detection of ERAs and Luspatercept without impacting the sensitivity and specificity of ERAs detection from the current techniques implemented in antidoping laboratories and to reduce the time of analysis and total sample volume needed. Magnetic beads coated with antibodies were preferred for IP of samples for its potential multiplexing. Then, the following steps of the method were selected considering that SAR/SDS-PAGE are the electrophoretic methods authorized for initial testing procedure by WADA and that biotinylated primary antibodies used for the immunodetection results in the best sensitivity and specificity and is time saving. The method developed in this work for the combined detection of agents affecting erythropoiesis (AAEs) showed specificity, sensitivity, and robustness and is easily and quickly implementable to all antidoping laboratories.
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Receptores de Activinas Tipo II/análise , Dopagem Esportivo/prevenção & controle , Hematínicos/análise , Fragmentos Fc das Imunoglobulinas/análise , Proteínas Recombinantes de Fusão/análise , Detecção do Abuso de Substâncias/métodos , Eletroforese em Gel de Poliacrilamida , Eritropoese/efeitos dos fármacos , Humanos , Sensibilidade e EspecificidadeRESUMO
Aim: Several automated immunoassays have been validated on serum/plasma to evaluate the presence of significant levels of anti-severe acute respiratory syndrome coronavirus 2 (anti-SARS-CoV-2) antibodies, signs of a present or past infection, but the use of dried blood spots (DBS) would facilitate sampling, shipping and storage. Objective: The aim of this project was to give proof of concept of the possibility to use of the automatized Elecsys® anti-SARS-CoV-2 immunoassay with a volumetric DBS device. Results: Linearity and correlation were satisfactory between volumetric DBS and plasma. A cut-off value was suggested and should be validated with more samples. Conclusion: this study strongly support the possibility to work with volumetric DBS instead of serum/plasma to test for anti-SARS-CoV-2 antibodies.