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Background Serum INSL3 is a Leydig cell biomarker, but little is known about the circulating concentration of INSL3 during hypothalamus-pituitary-testicular suppression. Aim To study the concomitant changes in serum concentrations of INSL3, testosterone and LH during experimental and therapeutic testicular suppression. Methods We included serum samples from three different cohorts comprising subjects before and after testicular suppression; 1) Six healthy young men who were treated with androgens (Sustanon®, Aspen Pharma, Dublin, Ireland), 2) Ten transgender girls (male sex assigned at birth) who were treated with three-monthly GnRH agonist injections (Leuprorelinacetat, Abacus Medicine, Copenhagen, Denmark) and 3) Fifty-five patients with prostate cancer who were randomized to surgical castration (bilateral subcapsular orchiectomy) or treatment with GnRH agonist (Triptorelin, Ipsen Pharma, Kista, Sweden). Serum INSL3 and testosterone concentrations were quantified in stored serum samples using validated LC-MS/MS methodologies, and LH was measured by an ultrasensitive immunoassay. Results The circulating concentrations of INSL3, testosterone and LH decreased during experimental testicular suppression in healthy young men by Sustanon injections and subsequently returned to baseline levels after release of suppression. All three hormones decreased during therapeutic hormonal hypothalamus-pituitary-testicular suppression in transgender girls and in prostate cancer patients. Conclusion INSL3 resembles testosterone as a sensitive marker of testicular suppression and reflects Leydig cell function, also during exposure to exogenous testosterone. Serum INSL3 measurements may complement testosterone as a Leydig cell marker in male reproductive disorders, during therapeutic testicular suppression as well as in surveillance of illicit use of androgens.

This study aimed to determine and compare the perception, painfulness, and usability of the minimally invasive Dried Blood Spot (DBS) collections from fingertip vs. upper arm from different athlete populations: males and females representing sports dependent on hand/arm, sports less dependent on hand/arm and para-athletes. To accomplish this, 108 national level athletes from Denmark were recruited (♀ = 49, ♂ = 59, 25 ± 6 years; mean±SD) and 11 Doping Control Officers (DCOs) collected manual fingerprick DBS (HemaSpot HF) and automated upper-arm DBS (Tasso-M20) from each athlete. Athletes and DCOs responded to questionnaires regarding the perception of sample collection procedures. On a 0-10 scale, the athletes reported a low pain score and a very good general experience for both sampling sites but following upper-arm DBS collection the associated pain was rated lower (-0.4 ± 1.6, p < 0.05) and the general experience rated better (+0.6 ± 2.3, p ≤ 0.001) than after the fingerprick DBS collection. The DCOs rated the general experience with the upper-arm DBS collection better (+1.6 ± 1.1, p ≤ 0.01) than the fingerprick DBS collection, partly because problems occurred more frequently during the DBS collection from the fingertip (28%) than from the upper arm (6%). In conclusion, it appears that DBS sampling is affiliated with minimal sensation of pain and is preferred by both DCOs and athletes, independent of gender and discipline, over conventional sample collection methods. Collection of DBS from the upper arm was preferred over fingerprick by both athletes and DCOs.

While misuse of testosterone esters is widespread in elite and recreational sports, direct detection of intact testosterone esters in doping control samples is hampered by the rapid hydrolysis by esterases present in the blood. With dried blood spot (DBS) as sample matrix, continued degradation of the esters is avoided due to inactivation of the hydrolase enzymes in dried blood. Here we have developed the method further for detection of testosterone esters in DBS with focus on robustness and applicability in doping control. To demonstrate the method's feasibility, DBS samples from men receiving two intramuscular injections of Sustanon® 250 (n = 9) or placebo (n = 10), were collected, transported and stored prior to analysis, to mimic a doping control scenario. The presented nanoLC-HRMS/MS method appeared reliable and suitable for direct detection of four testosterone esters (testosterone decanoate, isocaproate, phenylpropionate and propionate) after extraction from DBS. Sustanon® was detected in all subjects for at least five days, with detection window up to 14 days for three of the esters. Evaluation of analyte stability showed that while storage at room temperature is tolerated well for a few days, testosterone esters are highly stable (> 18 months) in DBS when stored in frozen conditions. Collectively, these findings demonstrate the applicability of DBS sampling in doping control for detection of steroid esters. The fast collection and reduced shipment costs of DBS compared to urine and standard blood samples, respectively, will allow more frequent and/or large-scale testing to increase detection and deterrence.

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.

Clenbuterol is a β2 -agonist prescribed for asthmatic patients in some countries. Based on its anabolic and lipolytic effects observed in studies on rodents and in livestock destined for food production, clenbuterol is abused by bodybuilders and athletes seeking leanness. Urinary clenbuterol analysis is part of routine doping analysis. However, the collection of urine samples is time-consuming and can be intimidating for athletes. Dried blood spot (DBS) appears attractive as an alternative matrix, but the detectability of clenbuterol in humans through DBS has not been investigated. This study evaluated if clenbuterol could be detected in DBS and urine collected from six healthy men after oral intake of 80 μg clenbuterol. The DBS and urine samples were collected at 0, 3, 8, 24, and 72 h post-ingestion, with additional urine collections on days 7 and 10. Using LC-MS/MS, it was shown that clenbuterol could be detected in all DBS samples for 24 h post-ingestion and with 50% sensitivity 3 days after ingestion. The DBS method was 100% specific. Evaluation of analyte stability showed that clenbuterol is stable in DBS for at least 365 days at room temperature when using desiccant and avoiding light exposure. In urine, clenbuterol was detectable for at least 7-10 days after ingestion. Urinary clenbuterol concentrations below 5 ng/mL were present in some subjects 24 h after administration. Collectively, these data indicate that DBS are suitable for routine doping control analysis of clenbuterol with a detection window of at least 3 days after oral administration of 80 μg.

Clenbuterol is a beta2 -adrenoceptor agonist marketed as an asthma reliever but is not approved for human use in most countries due to concerns of adverse cardiac effects. Given its demonstrated hypertrophic and lipolytic actions in rodents, clenbuterol is one of the most widely abused doping substances amongt athletes and recreational body-builders seeking leanness. Herein, we examined the effect of clenbuterol ingestion on metabolic rate as well as skeletal muscle mammalian target of rapamycin (mTOR) phosphorylation and protein kinase A (PKA)-signaling in six young men. Before and 140 min after ingestion of 80 μg clenbuterol, resting metabolic rate and contractile function of the quadriceps muscle were measured, and blood samples as well as vastus lateralis muscle biopsies were collected. Clenbuterol increased resting energy expenditure by 21% (P < 0.001), and fat oxidation by 39% (P = 0.006), whereas carbohydrate oxidation was unchanged. Phosphorylation of mTORSer2448 and PKA substrates increased by 121% (P = 0.004) and 35% (P = 0.006), respectively, with clenbuterol. Maximal voluntary contraction torque decreased by 4% (P = 0.026) and the half-relaxation time shortened by 9% (P = 0.046), while voluntary activation, time to peak twitch, and peak twitch torque did not change significantly with clenbuterol. Glycogen content of the vastus lateralis muscle did not change with clenbuterol. Clenbuterol increased circulating levels of glucose (+30%; P < 0.001), lactate (+90%; P = 0.004), insulin (+130%; P = 0.009), and fatty acids (+180%; P = 0.001). Collectively, these findings indicate that clenbuterol is an efficient thermogenic substance that possibly also exerts muscle hypertrophic actions in humans. For these reasons, the restrictions imposed against clenbuterol in competitive sports seem warranted.

Testosterone treatment stimulates the production of red blood cells and alters iron homeostasis. Thus, we investigated whether the 'haematological module' of the athlete biological passport (ABP) used by the World Anti-Doping Agency can be used to indicate misuse of testosterone. Nineteen eugonadal men received intramuscular injections of either 250 mg Sustanon®, a blend of four testosterone esters, or placebo on days 0 and 21 in a randomized, placebo-controlleddouble-blind design. Urine samples and blood samples were collected twice pre-treatment, at least 5 days apart, and on days 1, 3, 5, 10 and 14 post-injections to assess steroidal and haematological biomarkers of the ABP. The steroidal profile was flagged suspicious in all Sustanon®-treated subjects, whereas the haematological profile was flagged suspicious in six out of nine subjects. When both sensitivity and specificity were considered, reticulocyte percentage (RET%) appeared as the best marker of the haematological module for implying testosterone ester misuse. Atypical blood passport samples were used to select time points for further isotope-ratio mass spectrometry (IRMS) analysis of testosterone and its metabolites in simultaneously collected urine. In addition to the testosterone (T) to epitestosterone (E) ratio, the RET% and OFF-Score could help identify suspicious samples for more targeted IRMS testing. The results demonstrate that unexpected fluctuations in RET% can indicate testosterone doping if samples are collected 3-10 days after injection. From an anti-doping perspective, the haematological and steroidal modules of the ABP should complement each other when planning targeted follow-up testing and substantiating likely misuse of testosterone.

The nutritional supplement (NS) industry is one of the fastest growing in the world, and NS use in Denmark is among the highest in Europe. However, the exact use in elite athletes and fitness customers targeted for doping control is unknown. Information from 634 doping control forms obtained in 2014 was evaluated (elite athletes: n = 361; fitness customers: n = 273). The majority of female (92.6%) and male (85.0%) elite athletes and female (100.0%) and male (94.0%) fitness customers declared using one or more NS. The use of non-ergogenic NS was more prevalent in women than in men and in younger (15-34 years) compared with older (35-49 years) subjects, but it was less prevalent in intermittent compared with endurance and power/strength sports. Additionally, fitness customers who tested positive for doping also reported using more NS than subjects testing negative, indicating an association between NS and doping abuse. The present results demonstrate a very high prevalence of NS usage in both elite athletes and fitness customers. This highlights the importance of a strong national regulation of NS to avoid contamination of NS with doping substances.

Purpose: This study tested the hypothesis that autologous blood transfusion (ABT) of ~50% of the red blood cells (RBCs) from a standard 450 ml phlebotomy would increase mean power in a cycling time trial. Additionally, the study investigated whether further ABT of RBCs obtained from another 450 ml phlebotomy would increase repeated cycling sprint ability.Methods: In a randomized, double-blind, placebo-controlled crossover design (3-month wash-out), nine highly trained male subjects donated two 450 ml blood bags each (BT-trial) or were sham phlebotomized (PLA-trial). Four weeks later, a 650 kcal time trial (n=7) was performed three days before and 2 h after receiving either ~50% (135 ml) of the RBCs or a sham transfusion. On the following day, transfusion of RBCs (235 ml) from the second donation or sham transfusion was completed. A 4×30 s all-out cycling sprint interspersed by 4 min of recovery was performed six days before and three days after the second ABT (n=9).Results: The mean power was increased in time trials from before to after transfusion (PConclusion: ABT of only ~135 ml of RBCs is sufficient to increase mean power in a 650 kcal cycling time trial by ~5% in highly trained men. In contrast, a combined high-volume transfusion of ~135 and ~235 ml of RBCs does not alter 4×30 s all-out cycling performance interspersed with 4 min of recovery.