mRNA biomarkers sensitive and specific to micro-dose erythropoietin treatment at sea level and altitude.

Recombinant human erythropoietin (rhEPO) is prohibited by the World Anti-Doping Agency. rhEPO abuse can be indirectly detected via the athlete biological passport (ABP). However, altitude exposure challenges interpretation of the ABP. This study investigated whether 5'-aminolevulinate synthase 2 (ALAS2) and carbonic anhydrase 1 (CA1) in capillary dried blood spots (DBSs) are sensitive and specific markers of rhEPO treatment at altitude. ALAS2 and CA1 expression was monitored in DBS collected weekly before, during, and after a 3-week period at sea level or altitude. Participants were randomly assigned to receive 20 IU kg bw-1 epoetin alpha (rhEPO) or placebo injections every second day for 3 weeks while staying at sea level (rhEPO, n = 25; placebo, n = 9) or altitude (rhEPO, n = 12; placebo, n = 27). ALAS2 and CA1 expression increased up to 300% and 200%, respectively, upon rhEPO treatment at sea-level and altitude (P-values <0.05). When a blinded investigator interpreted the results, ALAS2 and CA1 expression had a sensitivity of 92%. Altitude did not confound the interpretation. Altitude affected ALAS2 and CA1 expression less than actual ABP markers when compared between sea level and altitude results. An individual athlete passport-like approach simulation confirmed the biomarker potential of ALAS2 and CA1. ALAS2 and CA1 were sensitive and specific biomarkers of micro-dose rhEPO treatment at sea level and altitude. Altitude seemed less a confounding factor for these biomarkers, especially when they are combined. Thus, micro-dose rhEPO injections can be detected in a longitudinal blinded setting using mRNA biomarkers in DBS.

Duplicate measures of hemoglobin mass within an hour: feasibility, reliability, and comparison of three devices in supine position.

Duplicate measure of hemoglobin mass by carbon monoxide (CO)-rebreathing is a logistical challenge as recommendations prompt several hours between measures to minimize CO-accumulation. This study investigated the feasibility and reliability of performing duplicate CO-rebreathing procedures immediately following one another. Additionally, it was evaluated whether the obtained hemoglobin mass from three different CO-rebreathing devices is comparable. Fifty-five healthy participants (22 males, 23 females) performed 222 duplicate CO-rebreathing procedures in total. Additionally, in a randomized cross-over design 10 participants completed three experimental trials, each including three CO-rebreathing procedures, with the first and second separated by 24 h and the second and third separated by 5-10 min. Each trial was separated by >48 h and conducted using either a glass-spirometer, a semi-automated electromechanical device, or a standard three-way plastic valve designed for pulmonary measurements. Hemoglobin mass was 3 ± 22 g lower (p < 0.05) at the second measure when performed immediately after the first with a typical error of 1.1%. Carboxyhemoglobin levels reached 10.9 ± 1.3%. In the randomized trial, hemoglobin mass was similar between the glass-spirometer and three-way valve, but ∼6% (∼50 g) higher for the semi-automated device. Notably, differences in hemoglobin mass were up to ∼13% (∼100 g) when device-specific recommendations for correction of CO loss to myoglobin and exhalation was followed. In conclusion, it is feasible and reliable to perform two immediate CO-rebreathing procedures. Hemoglobin mass is comparable between the glass-spirometer and the three-way plastic valve, but higher for the semi-automated device. The differences are amplified if the device-specific recommendations of CO-loss corrections are followed.

Determinants and reference values for blood volume and total hemoglobin mass in women and men.

Blood volume (BV) is an important clinical parameter and is usually reported per kg of body mass (BM). When fat mass is elevated, this underestimates BV/BM. One aim was to study if differences in BV/BM related to sex, age, and fitness would decrease if normalized to lean body mass (LBM). The analysis included 263 women and 319 men (age: 10-93 years, body mass index: 14-41 kg/m2 ) and 107 athletes who underwent assessment of BV and hemoglobin mass (Hbmass ), body composition, and cardiorespiratory fitness. BV/BM was 25% lower (70.3 ± 11.3 and 80.3 ± 10.8 mL/kgBM ) in women than men, respectively, whereas BV/LBM was 6% higher in women (110.9 ± 12.5 and 105.3 ± 11.2 mL/kgLBM ). Hbmass /BM was 34% lower (8.9 ± 1.4 and 11.5 ± 11.2 g/kgBM ) in women than in men, respectively, but only 6% lower (14.0 ± 1.5 and 14.9 ± 1.5 g/kgLBM )/LBM. Age did not affect BV. Athlete's BV/BM was 17.2% higher than non-athletes, but decreased to only 2.5% when normalized to LBM. Of the variables analyzed, LBM was the strongest predictor for BV (R2 = .72, p < .001) and Hbmass (R2 = .81, p < .001). These data may only be valid for BV/Hbmass when assessed by CO re-breathing. Hbmass /LBM could be considered a valuable clinical matrix in medical care aiming to normalize blood homeostasis.

Validation of a clinically applicable device for fast and accurate quantification of blood volume.

INTRODUCTION: Determination of blood volume (BV) using the dual-isotope (e.g., 99m Tc-labeled red blood cells [99m Tc-RBC] and 125 I-labeled human serum albumin [125 I-HSA]) injection method is limited in medicine due to the long isotope half-life. However, BV has been determined in laboratory settings for 100 years using the carbon monoxide (CO)-rebreathing-based procedure, which allows frequent BV measurements. METHODS: We investigated the reliability and accuracy of a semi-automated CO-rebreathing device by comparing it against the dual-isotope methodology and its ability to detect a known blood removal. In study A, BV was determined three times in ~2 h; twice using the device with rebreathing protocols lasting 2 (CO2min ) and 10 min (CO10min ) and once with the dual-isotope technique. In study B, the accuracy of the device was assessed by its ability to detect a 2% removal of BV. RESULTS: A good correlation was observed between both the CO-rebreathing protocols (r2 = 0.89-0.98; p < 0.001) and the dual-isotope approach (r2 = 0.89-0.95; p < 0.001). In absolute terms BV was 425 ± 263 mL and 491 ± 388 mL lower (p < 0.001) when quantified with the dual-isotope compared to the CO-rebreathing protocols. When reducing BV by 132 ± 25 mL (2%), the device quantified a lower (p < 0.001) BV of 150 ± 45 mL. CONCLUSION: This study emphasizes that the semi-automated device accurately determines small changes (i.e., 2%) in BV and that a high correlation with the dual-isotope methodology exists. The findings are clinically relevant owing to the method's simple and fast nature (the absence of radioactive tracers and reduced time requirements, i.e., ~15 min vs. ~180 min) and the possibility for repeated measurements within a single day.

Changes in Immature Reticulocytes Aid the Indirect Detection of Microdose Recombinant Erythropoietin Use in Men and Women.

PURPOSE: We investigated whether immature reticulocyte fraction (IRF) and the immature reticulocytes to red blood cells ratio (IR/RBC) are sensitive and specific biomarkers for microdose recombinant human erythropoietin (rHuEPO) and whether the inclusion of reticulocyte percentage (RET%) and the algorithm "abnormal blood profile score (ABPS)" increased the athlete biological passport (ABP) sensitivity compared with hemoglobin concentration ([Hb]) and the OFF-hr score ([Hb]-60 × âˆšRET%). METHODS: Forty-eight (♀ = 24, ♂ = 24) participants completed a 2-wk baseline period followed by a 4-wk intervention period with three weekly intravenous injections of 9 IU·kg -1 ·bw -1 epoetin ß (♀ = 12, ♂ = 12) or saline (0.9% NaCl, ♀ = 12, ♂ = 12) and a 10-d follow-up. Blood samples were collected weekly during baseline and intervention as well as 3, 5, and 10 d after treatment. RESULTS: The rHuEPO treatment increased [Hb] (time-treatment, P < 0.001), RET% (time-treatment, P < 0.001), IRF (time-treatment, P < 0.001) and IR/RBC (time-treatment, P < 0.001). IRF and IR/RBC were up to ~58% ( P < 0.001) and ~141% ( P < 0.001) higher compared with placebo, and calculated thresholds provided a peak sensitivity across timepoints of 58% and 54% with ~98% specificity, respectively. To achieve >99% specificity for IRF and IR/RBC, sensitivity was reduced to 46% and 50%, respectively. Across all timepoints, the addition of RET% and ABPS to the ABP increased sensitivity from 29% to 46%. Identification of true-positive outliers obtained via the ABP and IRF and IR/RBC increased sensitivity across all timepoints to 79%. CONCLUSIONS: In summary, IRF, IR/RBC, RET% and ABPS are sensitive and specific biomarkers for microdose rHuEPO in both men and women and complement the ABP.

Inhaled formoterol impairs aerobic exercise capacity in endurance-trained individuals: a randomised controlled trial.

Background: The 2022 Global Initiative for Asthma guidelines emphasise the inhaled long-acting ß2-agonist formoterol as part of the first treatment step, and therefore formoterol use among athletes will probably increase. However, prolonged supratherapeutic use of inhaled ß2-agonists impairs training outcomes in moderately trained men. We investigated whether inhaled formoterol, at therapeutic doses, imposes detrimental effects in endurance-trained individuals of both sexes. Methods: 51 endurance-trained participants (31 male, 20 female; mean±sd maximal oxygen consumption (V̇ O2 max) 62±6 mL·min-1·kg bw-1 and 52±5 mL·min-1·kg bw-1, respectively) inhaled formoterol (24 µg; n=26) or placebo (n=25) twice daily for 6 weeks. At baseline and follow-up, we assessed V̇ O2 max and incremental exercise performance during a bike-ergometer ramp-test; body composition by dual-energy X-ray absorptiometry; muscle oxidative capacity by high-resolution mitochondrial respirometry, enzymatic activity assays and immunoblotting; intravascular volumes by carbon monoxide rebreathing; and cardiac left ventricle mass and function by echocardiography. Results: Compared to placebo, formoterol increased lean body mass by 0.7 kg (95% CI 0.2-1.2 kg; treatment×trial p=0.022), but decreased V̇ O2 max by 5% (treatment×trial p=0.013) and incremental exercise performance by 3% (treatment×trial p<0.001). In addition, formoterol lowered muscle citrate synthase activity by 15% (treatment×trial p=0.063), mitochondrial complex II and III content (treatment×trial p=0.028 and p=0.007, respectively), and maximal mitochondrial respiration through complexes I and I+II by 14% and 16% (treatment×trial p=0.044 and p=0.017, respectively). No apparent changes were observed in cardiac parameters and intravascular blood volumes. All effects were sex-independent. Conclusion: Our findings demonstrate that inhaled therapeutic doses of formoterol impair aerobic exercise capacity in endurance-trained individuals, which is in part related to impaired muscle mitochondrial oxidative capacity. Thus, if low-dose formoterol fails to control respiratory symptoms in asthmatic athletes, physicians may consider alternative treatment options.

Glucocorticoids Accelerate Erythropoiesis in Healthy Humans-Should the Use in Sports Be Reevaluated?

PURPOSE: The World Anti-Doping Agency prohibits glucocorticoid administration in competition but not in periods out of competition. Glucocorticoid usage is controversial as it may improve performance, albeit debated. A hitherto undescribed but performance-relevant effect of glucocorticoids in healthy humans is accelerated erythropoiesis. We investigated whether a glucocorticoid injection accelerates erythropoiesis, increases total hemoglobin mass, and improves exercise performance. METHODS: In a counterbalanced, randomized, double-blinded, placebo-controlled crossover design (3 months washout), 10 well-trained males (peak oxygen uptake, 60 ± 3 mL O 2 ·min -1 ·kg -1 ) were injected with 40 mg triamcinolone acetonide (glucocorticoid group) or saline (placebo group) in the gluteal muscles. Venous blood samples collected before and 7-10 h, 1, 3, 7, 14, and 21 d after treatment were analyzed for hemoglobin concentration and reticulocyte percentage. Hemoglobin mass and mean power output in a 450-kcal time trial were measured before as well as 1 and 3 wk after treatment. RESULTS: A higher reticulocyte percentage was evident 3 d (19% ± 30%, P < 0.05) and 7 d (48% ± 38%, P < 0.001) after glucocorticoid administration, compared with placebo, whereas hemoglobin concentration was similar between groups. Additionally, hemoglobin mass was higher ( P < 0.05) 7 d (glucocorticoid, 886 ± 104 g; placebo, 872 ± 103 g) and 21 d (glucocorticoid, 879 ± 111 g; placebo, 866 ± 103 g) after glucocorticoid administration compared with placebo. Mean power output was similar between groups 7 d (glucocorticoid, 278 ± 64 W; placebo, 275 ± 62 W) and 21 d (glucocorticoid, 274 ± 62 W; placebo, 275 ± 60 W) after treatment. CONCLUSIONS: Intramuscular injection of 40 mg triamcinolone acetonide accelerates erythropoiesis and increases hemoglobin mass but does not improve aerobic exercise performance in the present study. The results are important for sport physicians administering glucocorticoids and prompt a reconsideration of glucocorticoid usage in sport.

Robust arm and leg muscle adaptation to training despite ACE inhibition: a randomized placebo-controlled trial.

PURPOSE: 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. METHODS: Healthy, untrained, middle-aged participants (40 ± 7 yrs) completed a randomized, double-blinded, placebo-controlled trial. Participants were randomized to placebo (PLA: CaCO3) or ACE inhibitor (ACEi: enalapril) for 8 weeks and completed a supervised, high-intensity exercise training program. Muscular characteristics in the leg and arm were extensively evaluated pre and post-intervention. RESULTS: Forty-eight participants (nACEi = 23, nPLA = 25) completed the trial. Exercise training compliance was above 99%. After training, citrate synthase, 3-hydroxyacyl-CoA dehydrogenase and phosphofructokinase maximal activity were increased in m. vastus lateralis in both groups (all P < 0.05) without statistical differences between them (all time × treatment P > 0.05). In m. deltoideus, citrate synthase maximal activity was upregulated to a greater extent (time × treatment P < 0.05) in PLA (51 [33;69] %) than in ACEi (28 [13;43] %), but the change in 3-hydroxyacyl-CoA dehydrogenase and phosphofructokinase maximal activity was similar between groups. Finally, the training-induced changes in the platelet endothelial cell adhesion molecule-1 protein abundance, a marker of capillary density, were similar in both groups in m. vastus lateralis and m. deltoideus. CONCLUSION: Eight 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.

Microdoses of Recombinant Human Erythropoietin Enhance Time Trial Performance in Trained Males and Females.

PURPOSE: 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 ß would increase time trial performance, peak oxygen uptake (V̇O 2peak ), and tHb in both males and females. METHODS: We included 48 healthy, recreational to trained males ( n = 24, mean ± SD V̇O 2peak = 55 ± 5 mL O 2 ·kg -1 ⋅min -1 ) and females ( n = 24; V̇O 2peak of 46 ± 4 mL O 2 ·kg -1 ⋅min -1 ) in a counterbalanced, double-blind, randomized, placebo-controlled study design stratified by sex. Time trial performance, V̇O 2peak , and tHb were determined before and after intravenous injections of either rHuEPO (9 IU·kg bw -1 epoetin ß) or saline (0.9% NaCl) three times weekly for 4 wk. RESULTS: A time-treatment effect ( P < 0.05) existed for time trial performance. Within the rHuEPO group, mean power output increased by 4.1% ± 4.2% ( P < 0.001). Likewise, a time-treatment effect ( P < 0.001) existed for V̇O 2peak , where the rHuEPO group improved V̇O 2peak and peak aerobic power by 4.2% ± 6.1% ( P < 0.001) and 2.9% ± 4.0% ( P < 0.01), respectively. A time-treatment effect ( P < 0.001) existed for tHb, where the rHuEPO group increased tHb by 6.7% ± 3.4% ( P < 0.001). A main effect of "sex" alone was also evident ( P < 0.001), but no sex-specific interactions were found. No changes were observed in the placebo group for mean power output, V̇O 2peak , peak aerobic power, or tHb. CONCLUSIONS: Microdoses with intravenous rHuEPO provide a sufficient erythropoietic stimuli to augment tHb and enhance aerobic-dominated performance in both trained males and females.

Moderate hypoxic exposure for 4 weeks reduces body fat percentage and increases fat-free mass in trained individuals: a randomized crossover study.

PURPOSE: We evaluated whether or not changes in body composition following moderate hypoxic exposure for 4 weeks were different compared to sea level exposure. METHODS: In a randomized crossover design, nine trained participants were exposed to 2320 m of altitude or sea level for 4 weeks, separated by > 3 months. Body fat percentage (BF%), fat mass (FM), and fat-free mass (FFM) were determined before and after each condition by dual X-ray absorptiometry (DXA) and weekly by a bioelectrical impedance scanner to determine changes with a high resolution. Training volume was quantified during both interventions. RESULTS: Hypoxic exposure reduced (P < 0.01) BF% by 2 ± 1 percentage points and increased (P < 0.01) FFM by 2 ± 2% determined by DXA. A tending time × treatment effect existed for FM determined by DXA (P = 0.06), indicating a reduced FM in hypoxia by 8 ± 7% (P < 0.01). Regional body analysis revealed reduced (P < 0.01) BF% and FFM and an increased (P < 0.01) FFM in the truncus area. No changes were observed following sea level. Bioelectrical impedance determined that BF%, FM, and FFM did not reveal any differences between interventions. Urine specific gravity measured simultaneously as body composition was identical. Training volume was similar between interventions (509 ± 70 min/week vs. 432 ± 70 min/week, respectively). CONCLUSIONS: Four weeks of altitude exposure reduced BF% and increased FFM in trained individuals as opposed to sea level exposure. The results also indicate that a decrease in FM is greater at altitude compared to sea level. Changes were specifically observed in the truncus area.

A semi-automated device rapidly determine circulating blood volume in healthy males and carbon monoxide uptake kinetics of arterial and venous blood.

We examined whether a semi-automated carbon monoxide (CO) rebreathing method accurately detect changes in blood volume (BV) and total hemoglobin mass (tHb). Furthermore, we investigated whether a supine position with legs raised reduced systemic CO dilution time, potentially allowing a shorter rebreathing period. Nineteen young healthy males participated. BV and tHb was quantified by a 10-min CO-rebreathing period in a supine position with legs raised before and immediately after a 900 ml phlebotomy and before and after a 900 ml autologous blood reinfusion on the same day in 16 subjects. During the first CO-rebreathing, arterial and venous blood samples were drawn every 2 min during the procedure to determine systemic CO equilibrium in all subjects. Phlebotomy decreased (P < 0.001) tHb and BV by 166 ± 24 g and 931 ± 247 ml, respectively, while reinfusion increased (P < 0.001) tHb and BV by 143 ± 21 g and 862 ± 250 ml compared to before reinfusion. After reinfusion BV did not differ from baseline levels while tHb was decreased (P < 0.001) by 36 ± 21 g. Complete CO mixing was achieved within 6 min in venous and arterial blood, respectively, when compared to the 10-min sample. On an individual level, the relative accuracy after donation for tHb and BV was 102-169% and 55-165%, respectively. The applied CO-rebreathing procedure precisely detect acute BV changes with a clinically insignificant margin of error. The 10-min CO-procedure may be reduced to 6 min with no clinical effects on BV and tHb calculation. Notwithstanding, individual differences may be of concern and should be investigated further.

Does Hypoxia and Stress Erythropoiesis Compromise Cardiac Function in Healthy Adults? A Randomized Trial.

OBJECTIVES: To investigate whether recombinant human erythropoietin (rHuEPO) injections during an altitude training camp impact heart function. METHODS: Thirty (12 women) moderately trained subjects stayed at 2320 m altitude for 4 weeks while training. Subjects were randomized to placebo (isotonic saline) or rHuEPO (20 IU/kg body weight) i.v. injections. Transthoracic echocardiography imaging was acquired 3 days after arrival to altitude and prior to the first placebo or rHuEPO injection as well as one day after the last rHuEPO injection three weeks later. RESULTS: rHuEPO did not alter cardiovascular morphology parameters, systolic or diastolic function. In the placebo group, altitude exposure improved left ventricle (LV) systolic function due to an increased twist angle but rHuEPO had no additional effects. Pulmonary arterial systolic pressure was unaffected in either group. Notably, rHuEPO hampered LV untwist rate without affecting LV early filling. CONCLUSION: rHuEPO provided during mild altitude exposure does not cause any major effects on heart function. The observed alteration in LV untwist induced by rHuEPO is unlikely to have a meaningful clinical effect. Trial Registration Registered on www. CLINICALTRIALS: gov (NCT04227665).

Contemporary blood doping-Performance, mechanism, and detection.

Blood doping is prohibited for athletes but has been a well-described practice within endurance sports throughout the years. With improved direct and indirect detection methods, the practice has allegedly moved towards micro-dosing, that is, reducing the blood doping regime amplitude. This narrative review evaluates whether blood doping, specifically recombinant human erythropoietin (rhEpo) treatment and blood transfusions are performance-enhancing, the responsible mechanism as well as detection possibilities with a special emphasis on micro-dosing. In general, studies evaluating micro-doses of blood doping are limited. However, in randomized, double-blinded, placebo-controlled trials, three studies find that infusing as little as 130 ml red blood cells or injecting 9 IU × kg bw-1 rhEpo three times per week for 4 weeks improve endurance performance ~4%-6%. The responsible mechanism for a performance-enhancing effect following rhEpo or blood transfusions appear to be increased O2 -carrying capacity, which is accompanied by an increased muscular O2 extraction and likely increased blood flow to the working muscles, enabling the ability to sustain a higher exercise intensity for a given period. Blood doping in micro-doses challenges indirect detection by the Athlete Biological Passport, albeit it can identify ~20%-60% of the individuals depending on the sample timing. However, novel biomarkers are emerging, and some may provide additive value for detection of micro blood doping such as the immature reticulocytes or the iron regulatory hormones hepcidin and erythroferrone. Future studies should attempt to validate these biomarkers for implementation in real-world anti-doping efforts and continue the biomarker discovery.

Effect of angiotensin-converting enzyme inhibition on cardiovascular adaptation to exercise training.

Angiotensin-converting enzyme (ACE) activity may be one determinant of adaptability to exercise training, but well-controlled studies in humans without confounding conditions are lacking. Thus, the purpose of the present study was to investigate whether ACE inhibition affects cardiovascular adaptations to exercise training in healthy humans. Healthy participants of both genders (40 ± 7 years) completed a randomized, double-blind, placebo-controlled trial. Eight weeks of exercise training combined with placebo (PLA, n = 25) or ACE inhibitor (ACEi, n = 23) treatment was carried out. Before and after the intervention, cardiovascular characteristics were investigated. Mean arterial blood pressure was reduced (p < 0.001) by -5.5 [-8.4; -2.6] mmHg in ACEi , whereas the 0.7 [-2.0; 3.5] mmHg fluctuation in PLA was non-significant. Maximal oxygen uptake increased (p < 0.001) irrespective of ACE inhibitor treatment by 13 [8; 17] % in ACEi and 13 [9; 17] % in PLA. In addition, skeletal muscle endurance increased (p < 0.001) to a similar extent in both groups, with magnitudes of 82 [55; 113] % in ACEi and 74 [48; 105] % in PLA. In contrast, left atrial volume decreased (p < 0.05) by -9 [-16; -2] % in ACEi , but increased (p < 0.01) by 14 [5; 23] % in PLA. Total hemoglobin mass was reduced (p < 0.01) by -3 [-6; -1] % in ACEi , while a non-significant numeric increase of 2 [-0.4; 4] % existed in PLA. The lean mass remained constant in ACEi but increased (p < 0.001) by 3 [2; 4] % in PLA. In healthy middle-aged adults, 8 weeks of high-intensity exercise training increases maximal oxygen uptake and skeletal muscle endurance irrespective of ACE inhibitor treatment. However, ACE inhibitor treatment counteracts exercise training-induced increases in lean mass and left atrial volume. ACE inhibitor treatment compromises total hemoglobin mass.

Analysis of dried blood spots is a feasible alternative for detecting ephedrine in doping control.

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, because 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 eight healthy male subjects in an 8-h 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 is a feasible possibility 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.

Hepcidin and Erythroferrone Complement the Athlete Biological Passport in the Detection of Autologous Blood Transfusion.

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 (RBC) 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 red blood cell was reinfused 4 wk later. Blood samples were collected 3, 7, 14, 21, and 28 d after donation, and 3, 6, and 24 h and 2, 3, and 6 d after 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 1 and 6 d after reinfusion. RESULTS: A time-treatment effect existed ( P < 0.05) for hepcidin and ERFE. Hepcidin was increased ( P < 0.01) ~110 and 89% 6 and 24 h 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 × âˆšRET%) ( P < 0.05) with a maximal sensitivity of ~58% and ~9% after donation and reinfusion, respectively. Combining the findings for hepcidin, ERFE, and the ABP yielded a sensitivity across all time-points of 83% after reinfusion in BT. Endurance performance increased 24 h (+6.4%, P < 0.01) and 6 d after reinfusion (+5.8%, P < 0.01). CONCLUSIONS: Hepcidin and ERFE may serve as biomarkers in an antidoping context after an ergogenic, small-volume blood transfusion.

Reliability and Validity of the SHFT Running Power Meter.

The SHFT device is a novel running wearable consisting of two pods connected to your smartphone issuing several running metrics based on accelerometer and gyroscope technology. The purpose of this study was to investigate the reliability and validity of the power output (PO) metric produced by the SHFT device. To assess reliability, 12 men ran on an outdoor track at 10.5 km·h-1 and 12 km·h-1 on two consecutive days. To assess validity, oxygen uptake (VO2) and SHFT data from eight men and seven women were collected during incremental submaximal running tests on an indoor treadmill on one to four separate days (34 tests in total). SHFT reliability on the outdoor track was strong with coefficients of variance (CV) of 1.8% and 2.4% for 10.5 and 12 km·h-1, respectively. We observed a very strong linear relationship between PO and VO2 (r2 = 0.54) within subjects, and a very strong linear relationship within each subject within each treadmill test (r2 = 0.80). We conclude that SHFT provides a reliable running power estimate and that a very strong relationship between SHFT-Power and metabolic rate exists, which places SHFT as one of the leading commercially available running power meters.

Effects of altitude and recombinant human erythropoietin on iron metabolism: a randomized controlled trial.

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 with 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 antidoping context.

An Abductive Inference Approach to Assess the Performance-Enhancing Effects of Drugs Included on the World Anti-Doping Agency Prohibited List.

Some have questioned the evidence for performance-enhancing effects of several substances included on the World Anti-Doping Agency's Prohibited List due to the divergent or inconclusive findings in randomized controlled trials (RCTs). However, inductive statistical inference based on RCTs-only may result in biased conclusions because of the scarcity of studies, inter-study heterogeneity, too few outcome events, or insufficient power. An abductive inference approach, where the body of evidence is evaluated beyond considerations of statistical significance, may serve as a tool to assess the plausibility of performance-enhancing effects of substances by also considering observations and facts not solely obtained from RCTs. Herein, we explored the applicability of an abductive inference approach as a tool to assess the performance-enhancing effects of substances included on the Prohibited List. We applied an abductive inference approach to make inferences on debated issues pertaining to the ergogenic effects of recombinant human erythropoietin (rHuEPO), beta2-agonists and anabolic androgenic steroids (AAS), and extended the approach to more controversial drug classes where RCTs are limited. We report that an abductive inference approach is a useful tool to assess the ergogenic effect of substances included on the Prohibited List-particularly for substances where inductive inference is inconclusive. Specifically, a systematic abductive inference approach can aid researchers in assessing the effects of doping substances, either by leading to suggestions of causal relationships or identifying the need for additional research.

Immature reticulocytes are sensitive and specific to low-dose erythropoietin treatment at sea level and altitude.

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 4-week baseline, a 4-week SL or AL (2,230 m) exposure, and a 4-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 3 weeks. Venous blood was collected weekly. Sysmex measurements revealed that IRF and IR/RBC were 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 were up to ~20% (P < 0.05) and ~45% (P < 0.001) lower post-treatment in SLrhEpo , respectively. In ALrhEpo , IRF and IR/RBC were 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.