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We investigated the effects of recombinant human erythropoietin (rHuEPO) administration on exercise endurance, maximal aerobic performance and total hemoglobin mass (tHb). We hypothesized that frequent, small intravenous injections of epoetin β would increase time trial performance, peak oxygen uptake (V̇O2peak) and tHb in both males and females.We included 48 healthy, recreational to trained males (n = 24, mean ± standard deviation V̇O2peak of 55 ± 5 ml O2 × kg-1 × min-1) and females (n = 24; V̇O2peak of 46 ± 4 ml O2 × kg-1 × min-1) in a counter-balanced, double-blind, randomized, placebo-controlled study design stratified by sex. Time trial performance, V̇O2peak, and tHb were determined before and after intravenous injections of either rHuEPO (9 IU × kg bw-1 epoetin β) or saline (0.9% NaCl) three times weekly for 4 weeks.A time × treatment effect (P0.05) existed for time trial performance. Within the rHuEPO group, mean power output (MPO) increased by 4.1 ± 4.2% (P0.001). Likewise, a time × treatment effect (P0.001) existed for V̇O2peak, where the rHuEPO group improved V̇O2peak and peak aerobic power by 4.2 ± 6.1% (P0.001) and 2.9 ± 4.0% (P0.01), respectively. A time × treatment effect (P0.001) existed for tHb, where the rHuEPO group increased tHb by 6.7 ± 3.4% (P0.001). A main effect of 'sex' alone was also evident (P0.001) but no sex-specific interactions were found. No changes were observed in the placebo group for MPO, V̇O2peak, peak aerobic power or tHb.Micro-doses with intravenous rHuEPO provide a sufficient erythropoietic stimuli to augment tHb and enhance aerobic-dominated performance in both trained males and females.

Background: Indirect detection of recombinant human erythropoietin (rhEPO) abuse in Anti-Doping world is done by hematological module of athlete biological passport (ABP). However, actual markers (reticulocytes (%RET) and hemoglobin (HGB)) of this module are not enough specific and have difficulties to distinguish recombinant human erythropoietin (rhEPO) administration from altitude sojourn especially if both condition are merged. Previous study have demonstrated that erythropoiesis-related RNAs, genes 5’-aminolevulinate synthase 2 ( ALAS2) and carbonic anhydrase 1 ( CA1) in capillary dried blood spots (DBS) could be potential biomarkers to increased specificity and sensitivity of the ABP in case of hypoxic conditions. Study design and methods: In the present study, we monitored ALAS2 and CA1 expression in DBS after injection of 20 IU kg bw-1 epoetin alpha at sea level and the effect merged with altitude exposure. For this purpose, DBSs samples collected from 39 volunteers’ males and females were analyzed. Participants were divided in two groups (EPO and placebo) and subjects from both groups sojourned at sea level and at altitude. The purpose was to evaluate the performance of RNAs biomarkers for the detection of rHEPO injection in a longitudinal blinded setting. Results: Our results illustrated a clear significant increase of ALAS2 until to 300% when treated at sea level and at altitude. Regarding CA1 the increase is up to 200% in both conditions. Interestingly, our results suggested no combined effect of altitude and rhEPO injection on ALAS2 and CA1 biomarkers as previously demonstrated with %RET values. In placebo group. Moreover, no effect of altitude on ALAS2 and CA1 was observed in contrast to %RET parameter. Results from blind setting test demonstrated a performance of interpretation of 92% regarding ALAS2 and CA1 monitoring. Conclusion: Erythropoiesis-related RNAs in capillary DBS such as ALAS2 and CA1 were sensitive and specific biomarkers for low-dose rhEPO misuse at sea level and altitude. Interestingly, altitude seemed not to be a confounding factors for ALAS2 and CA1 biomarkers in contrast to %RET. Furthermore, this investigation provide evidence that low-dose of rhEPO injections can be detected in a longitudinal blinded setting with RNA biomarkers in DBS. World Anti-Doping Agency (WADA) This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Purpose: We investigated whether hepcidin and erythroferrone (ERFE) could complement the Athlete Biological Passport (ABP) in indirectly detecting a 130 mL packed red blood cells (RBCs) autologous blood transfusion. Endurance performance was evaluated.Methods: Forty-eight healthy men (n = 24) and women (n = 24) participated. Baseline samples were collected weekly followed by randomization to a blood transfusion (BT, n = 24) or control group (CON, n = 24). Only the BT group donated 450 mL whole blood from which 130 mL RBCs was reinfused four weeks later. Blood samples were collected 3, 7, 14, 21 and 28 days after donation, and 3, 6, and 24 hours and 2, 3, and 6 days following reinfusion. In the CON group samples were collected with the same frequency. Endurance performance was evaluated by a 650-kCal time trial (n = 13) before and one and six days after reinfusion.Results: A time×treatment effect existed (P < 0.05) for hepcidin and ERFE. Hepcidin was increased (P < 0.01) ~110 and 89% six and 24 hours after reinfusion. Using an individual approach (99% specificity, e.g. allowing 1:100 false-positive), sensitivities, i.e. true positives, of 30% and 61% was found for hepcidin and ERFE, respectively. For the ABP, the most sensitive marker was Off-hr score ([Hb] (g·L-1) - 60 × √RET%) (P < 0.05) with a maximal sensitivity of ~58% and ~ 9% following donation and reinfusion, respectively. Combining the findings for hepcidin, ERFE and the ABP yielded a sensitivity across all time-points of 83% following reinfusion in BT. Endurance performance increased 24 hours (+6.4%, P < 0.01) and six days following reinfusion (+5.8%, P < 0.01).Conclusions: Hepcidin and ERFE may serve as biomarkers in an anti-doping context following an ergogenic, small-volume blood transfusion.

Since the first glucagon-like peptide 1 (GLP-1) receptor agonist (GLP-1RA) was approved in 2005 (exenatide twice daily) for type 2 diabetes (T2D), the class has developed with newer compounds having more pronounced effects on glycaemic control and body weight. Also, administration regimes have become more convenient with once weekly injections, and recently an oral administration has become available. Large-scale randomized controlled cardiovascular (CV) outcome trials (CVOTs) have shown that GLP-1RA therapy can reduce the risk of CV disease (CVD) in high-risk individuals with T2D. In addition, GLP-1RAs may have renal benefits driven by new-onset macroalbuminuria, although no effect on hard renal endpoints has been found. Subsequently, the place for GLP-1RA therapy has changed over recent years, with most societies endorsing GLP-1RA therapy in patients with established or high risk of CVD independently of glycaemia. Initiation of GLP-1RA therapy can be complex due to differences in efficacy, side effects and safety profiles as well as administration forms within the class. Implementing guideline recommendations into ideal patient selection may be challenging both in specialty and non-specialty settings. To ensure adequate and proactive use of modern glucose-lowering medications in the treatment of T2D, it is essential to recognize patients with high risk or documented CVD. The present review provides an overview of the efficacy and benefits of the currently available GLP-1RA compounds. Furthermore, we review the results from recent large-scale CVOTs in a clinical context and suggest improving the implementation of GLP-1RA therapy across specialties to improve overall patient selection.

Current markers of iron deficiency (ID) such as ferritin and hemoglobin have shortcomings, and hepcidin and erythroferrone (ERFE) could be of clinical relevance in relation to early assessment of ID. Here, we evaluate whether exposure to altitude-induced hypoxia (2,320 m) alone, or in combination with recombinant human erythropoietin (rHuEPO) treatment, affects hepcidin and ERFE levels before alterations in routine ID biomarkers and stress erythropoiesis manifest. Two interventions were completed, each comprising a 4-wk baseline, a 4-wk intervention at either sea level or altitude, and a 4-wk follow-up. Participants (n=39) were randomly assigned to 20 IU·kg body wt-1 rHuEPO or placebo injections every second day for 3 wk during the two intervention periods. Venous blood was collected weekly. Altitude increased ERFE (P ≤ 0.001) with no changes in hepcidin or routine iron biomarkers, making ERFE of clinical relevance as an early marker of moderate hypoxia. rHuEPO treatment at sea level induced a similar pattern of changes in ERFE (P < 0.05) and hepcidin levels (P < 0.05), demonstrating the impact of accelerated erythropoiesis and not of other hypoxia-induced mechanisms. Compared to altitude alone, concurrent rHuEPO treatment and altitude exposure induced additive changes in hepcidin (P < 0.05) and ERFE (P ≤ 0.001) parallel with increases in hematocrit (P < 0.001), demonstrating a relevant range of both hepcidin and ERFE. A poor but significant correlation between hepcidin and ERFE was found (R2 = 0.13, P < 0.001). The findings demonstrate that hepcidin and ERFE are more rapid biomarkers of changes in iron demands than routine iron markers. Finally, ERFE and hepcidin may be sensitive markers in an anti-doping context.Clinical trial registration: Clinical trials identifier, NCT04227665

We investigated whether immature reticulocyte fraction (IRF) and immature reticulocytes to red blood cells ratio (IR/RBC) are sensitive biomarkers for low-dose recombinant human erythropoietin (rhEpo) treatment at sea-level (SL) and moderate altitude (AL) and whether multi (FACS) or single (Sysmex-XN) fluorescence flow cytometry is superior for IRF and IR/RBC determination. Thirty-nine participants completed two interventions, each containing a four-week baseline, a four-week SL or AL (2,230m) exposure and a four-week follow-up. During exposure, rhEpo (20 IU·kg-1) or placebo (PLA) was injected at SL (SLrhEpo n = 25, SLPLA n = 9) and AL (ALrhEpo n = 12, ALPLA n = 27) every second day for three weeks. Venous blood was collected weekly. Sysmex measurements revealed that IRF and IR/RBC was up to ~70% (P < 0.01) and ~190% (P < 0.001) higher in SLrhEpo than SLPLA during treatment and up to ~45% (P < 0.001) and ~55% (P < 0.01) lower post-treatment, respectively. Compared with ALPLA, IRF and IR/RBC was up to ~20% (P < 0.05) and ~45% (P < 0.001) lower post-treatment in SLrhEpo, respectively. In ALrhEpo, IRF and IR/RBC was up to ~40% (P < 0.05) and ~110% (P < 0.001) higher during treatment and up to ~25% (P < 0.05) and ~40% (P < 0.05) lower post-treatment, respectively, compared with ALPLA. Calculated thresholds provided ~90% sensitivity for both biomarkers at SL and 33% (IRF) and 66% (IR/RBC) at AL. Specificity was >99%. Single-fluorescence flow cytometry coefficient of variation was >twofold higher at baseline (P < 0.001), and provided larger or similar changes compared to multi-fluorescence, albeit with smaller precision. In conclusion, IRF and IR/RBC were sensitive and specific biomarkers for low-dose rhEpo misuse at SL and AL.

Dried blood spot (DBS) testing allows fast, easy, and minimally invasive collection of microvolumes of blood. In an anti-doping context, DBS testing has particular relevance for substances prohibited in-competition only such as ephedrine, which is currently detected by urine analysis, since DBS can add information about the blood drug concentrations during the in-competition period. Several collection methods and devices exist for DBS collection from different anatomical sites. Thus, agreements between concentrations of target analytes in DBS samples from different sampling sites, along with between DBS and those in conventional venous plasma samples, need to be evaluated. Herein, we collected matched upper-arm DBS, fingerprick DBS and venous plasma samples from 8 healthy, male subjects in an 8-hour period following oral administrations of 20 mg ('low dose') and 60 mg ('high dose') of ephedrine. We show that the use of alternative sampling sites and matrices are feasible possibilities for ephedrine analysis in doping control. We observed very good agreement between collection sites and that specificity and sensitivity can be upheld despite use of an alternative collection site. However, potential concentration differences between DBS and venous plasma should be considered, and distinct threshold might be necessary if implementing both blood matrices in ephedrine analysis.

Angiotensin-converting enzyme (ACE) inhibitor treatment is widely applied, but the fact that plasma ACE activity is a potential determinant of training-induced local muscular adaptability is often neglected. Thus, we investigated the hypothesis that ACE inhibition modulates the response to systematic aerobic exercise training on leg and arm muscular adaptations.Healthy, untrained, middle-aged participants (40 ± 7 yrs) completed a randomized, double-blinded, placebo-controlled trial. Participants were randomized to placebo (PLA: CaCOForty-eight participants (nEight weeks of high-intensity whole-body exercise training improves markers of skeletal muscle mitochondrial oxidative capacity, glycolytic capacity and angiogenesis, with no overall effect of pharmacological ACE inhibition in healthy adults.