Comparative study between direct analysis in whole blood, oral fluid, and declaration of consumption for the prevalence of nonsteroidal anti-inflammatory drugs and acetaminophen in ultratrail runners.

Ultratrail running is a sport with growing number of adherents. To complete ultratrail despite physical issues such as joint and muscle pain, many runners use nonsteroidal anti-inflammatory drugs (NSAIDs) and acetaminophen. Studies asking participants about their consumption of drugs during ultratrail revealed a prevalence of NSAIDs and acetaminophen up to 70% and 25%, respectively. The aims of the present study were to determine the prevalence of NSAIDs and acetaminophen for 81 runners during the 2021 Ultratrail du Mont Blanc® (UTMB®) using direct analysis of dried blood spots (DBS) and oral fluid (OF) and to compare results with the declaration of consumption by runners; this is to identify the most relevant method to study the prevalence of drugs. Our results show a prevalence of NSAIDs of 46.6% using DBS, 18.5% using OF, and 13.8% based on a questionnaire. Prevalence of acetaminophen were 30.1%, 30.9%, and 22.5% using DBS, OF, and questionnaire, respectively. From this study, we conclude that the analysis of drugs directly in DBS is the most relevant tool to determine the prevalence in ultratrail events.

Simultaneous quantification of 19 nonsteroidal anti-inflammatory drugs in oral fluid by liquid chromatography-high resolution mass spectrometry: Application on ultratrail runner's oral fluid.

Nonsteroidal anti-inflammatory drugs (NSAIDs) are a therapeutic class suspected to be used by ultratrail runners. The use of NSAIDs during ultratrails is known to be associated with various adverse effects. To study the prevalence of NSAIDs intake in ultratrail runners, oral fluid (OF) is a relevant matrix as it is noninvasive and easy to collect. The aim of our work was to develop and validate a liquid-liquid extraction followed by a liquid chromatography (LC)-mass spectrometry (MS)/high resolution mass spectrometry (HRMS) method for the simultaneous quantification of 19 NSAIDs in OF. After a comparison of different liquid-liquid extraction methods, a double step liquid-liquid extraction with chloroform was performed on OF collected with Quantisal®, with extraction recoveries higher than 90%. An Accucore AQ column was selected for the chromatographic separation of NSAIDs. The Q Exactive Plus mass spectrometer operated in full scan and ddms2 mode after positive and negative electrospray ionization. Selectivity, carry-over, matrix effect, and linearity were validated for all NSAIDs. Within-day and between-day accuracy and precision were validated for all NSAIDs (<15% for quality control [QC] samples and <20% for lower limit of quantitation [LLOQ]), except within-day accuracy for the LLOQ of mefenamic acid. A stability study was also performed on OF at room temperature and +4°C. The method was applied on OF from runners who participate to Ultra Trail du Mont Blanc®.

Induction of erythroferrone in healthy humans by micro-dose recombinant erythropoietin or high-altitude exposure.

The erythropoietin (Epo)-erythroferrone (ERFE)-hepcidin axis coordinates erythropoiesis and iron homeostasis. While mouse studies have established that Epo-induced ERFE production represses hepcidin synthesis by inhibiting hepatic BMP/SMAD signaling, evidence for the role of ERFE in humans is limited. To investigate the role of ERFE as a physiological erythroid regulator in humans, we conducted two studies: first, 24 males received six injections of saline (placebo), recombinant Epo (rhEpo) 20 UI kg-1 (micro-dose) or 50 UI kg-1 (low-dose). Second, we quantified ERFE in 22 subjects exposed to high altitude (3800 m) for 15 hours. In the first study, total hemoglobin mass (Hbmass) increased after low- but not after micro-dose injections, when compared to placebo. Serum ERFE levels were enhanced by rhEpo, remaining higher than after placebo for 48 (micro-dose) or 72 hours (low-dose) post-injections. Conversely, hepcidin levels decreased when Epo and ERFE arose, before any changes in serum iron parameters occurred. In the second study, serum Epo and ERFE increased at high altitude. The present results demonstrate that in healthy humans ERFE responds to slightly increased Epo levels not associated with Hbmass expansion and down-regulates hepcidin in an apparently iron-independent way. Notably, ERFE flags micro-dose Epo, thus holding promise as novel anti-doping biomarker.

Effect of Non-Steroidal Anti-Inflammatory Drugs on Sport Performance Indices in Healthy People: a Meta-Analysis of Randomized Controlled Trials.

BACKGROUND: Non-steroidal anti-inflammatory drugs (NSAIDs) are medications that are frequently used by athletes. There may also be some abuse of these substances, although it is unclear whether NSAIDs in fact enhance performance. We performed a systematic review and meta-analysis to evaluate the effect of NSAIDs on sport performance indices. METHODS: We selected randomized trials from the PubMed and Cochrane Library databases investigating the effects of NSAIDs on sport performance. Volunteers could be healthy adult men and women. Any NSAID, administered by any route, taken prior to any type of exercise, and for any duration could be used. The control intervention could be a placebo, an active substance, or no intervention. We included double-blind, single-blind, and open-label studies. The primary outcome was the maximum performance in exercises as defined in each study. The secondary outcomes were the time until self-reported exhaustion and the self-reported pain. RESULTS: Among 1631 records, we retained thirteen parallel-group and ten crossover studies, totaling 366 and 148 subjects, respectively. They were disparate regarding treatments, dose and duration, and the type of exercise. There was neither significant difference in the maximum performance between NSAIDs and control groups nor in the time until exhaustion nor in self-perceived pain. CONCLUSIONS: The existence of an ergogenic effect of NSAIDs on sport performance indices was unable to be concluded, since the level of evidence of the studies is low, the doses tested, and the exercises performed are very heterogeneous and far from those observed in real-life practices. More studies are required.

Low doses of recombinant human erythropoietin does not affect C-terminal FGF23 in healthy men.

Recombinant human erythropoietin (rhEpo) can improve human performance, but misuse remains difficult to detect. C-terminal fibroblast growth factor 23 (cFGF23) was recently demonstrated to increase following injection of a single high dose rhEpo, but the effect of more frequent low doses is unknown. Using a randomized double-blind placebo-controlled design, we investigated whether 2 weeks of subcutaneous injections three times a week of 50 IU/kg Eprex (low-dose) or 20 IU/kg Eprex (micro-dose) increase cFGF23 levels compared with saline (placebo) injections in 24 healthy males. Venous blood was sampled at day -3, 0, 1, 3, 11, 14, 18, and 25 of the treatment and analyzed for cFGF23 and erythropoietin concentration ([EPO]). The level of cFGF23 was similar at days -3, 0, 1, 3, 11, 14, 18, and 25 with the low-dose (23 ± 4, 26 ± 5, 23 ± 7, 27 ± 6, 25 ± 8, 24 ± 10, 22 ± 5, and 24 ± 7 RU/mL, respectively), micro-dose (23 ± 6, 25 ± 5, 23 ± 8, 28 ± 9, 27 ± 7, 25 ± 9, 25 ± 5, and 23 ± 6 RU/mL, respectively) and placebo (23 ± 6, 24 ± 6, 26 ± 7, 26 ± 6, 31 ± 6, 31 ± 7, 24 ± 4, and 27 ± 8 RU/mL, respectively) treatment. The correlation coefficient between plasma [EPO] and plasma cFGF23 levels was R2 = 0.01 and insignificant. The results demonstrate that cFGF23 is not sensitive to low doses of subcutaneous rhEpo injections in healthy males.

Study of a new indirect method based on absolute norms of variation to detect autologous blood transfusion.

The aim of this study was to investigate an indirect method based on a determination of absolute norms of variation in biological markers that could be used to identify autologous blood transfusion within the framework of the fight against doping. The selection of markers was made from experimental variations obtained during different phases including an increase in training volume at sea level, high altitude training, blood withdrawal and autologous blood reinfusion. The global statistical method was then developed in order to fix absolute norms of variation for each selected marker. The markers selected were haematocrit (Hct), haemoglobin concentration ([Hb]), stimulation index (Off-hr) and the absolute norms of variation (normDelta) established for a maximal 15 days period were normDeltaHct(0-15) >6%, normDelta[Hb](0-15) >4% and normDeltaOff-hr(0-15) >20%. From analyses between two blood samples spaced at an interval of maximum 15 days, this method allows to show "abnormal" variation when a variation for one of the selected markers is strictly superior to the absolute norms of variation established. The legal framework for an immediate application of this method could be that of the internal regulations implemented by each international federation in accordance with the health policy in vigour.