Nandrolone decanoate-induced hypogonadism in male rats: Dose- and time-dependent effects on pituitary and testicular hormonal functions.

Anabolic-androgenic steroids (AASs) impairment of reproduction has been reported. We investigated dose- and time-dependent effects of Nandrolone decanoate (ND) on reproductive system in comparison with Testosterone enanthate (TE). Male Wistar rats were administrated with 1, 3, and 9 mg/kg/weeks ND or 1 and 3 mg/kg/weeks TE for 8 weeks, and testicular phenotype and reproductive hormones were assessed at 4 and 8 weeks post-treatments. AASs × treatment period interaction was significant for gonadosomatic index (GSI), testosterone (T), 17ß-estradiol (E2), and luteinizing hormone (LH). At 4 weeks post-treatment, GSI was decreased in rats treated with 3 mg/kg/weeks ND and T was decreased in all ND-treated groups, while no significant changes in LH levels were observed. At 8 weeks post-treatment, GSI was decreased in rats treated with 1 and 3 mg/kg/weeks ND and with 3 mg/kg/weeks TE, T was decreased in all groups, and E2 and LH were increased and decreased, respectively, in rats treated with 9 mg/kg/weeks ND and with 3 mg/kg/weeks TE. The testes showed histopathological defects in both ND- and TE-treated rats suggesting a delay in seminiferous cycle. This study shows AASs-induced hypogonadism at low-dose that coincided with inhibition of T biosynthesis and disruption of T feedback on pituitary.

Impaired vascular function among young users of anabolic-androgenic steroids.

Supraphysiological doses of anabolic-androgenic steroids (AAS) is popular among recreational weightlifters and bodybuilders due to the performance-enhancing properties but is also associated with adverse cardiovascular effects. The knowledge about how AAS affect the vasculature is limited, although results from previous studies suggest alterations in vasoreactivity and morphology. In the present study we investigate the association between long-term use of AAS and vascular function. Hundred and twenty-three males were included in the study, 56 of them current AAS users and 67 weightlifting controls. Vascular function was evaluated by carotid artery reactivity and flow-mediated dilation. AAS users had significantly reduced carotid artery reactivity (p < 0.001) and flow-mediated dilation (p < 0.001) compared to weightlifting controls. Results from the present study indicate that long-term use of AAS affect the cardiovascular system negatively, measured as reduced carotid artery reactivity and flow-mediated dilation. These findings could partly explain sudden cardiovascular events among young long-term users of AAS.

Prevention of bone dehiscence associated with orthodontic tooth movement by prophylactic injection of bone anabolic agents in mice.

Although bone dehiscence may occur during orthodontic tooth movement into the narrow alveolar ridge, a non-invasive prevention method is yet to be fully established. We show for the first time prevention of bone dehiscence associated with orthodontic tooth movement by prophylactic injection of bone anabolic agents in mice. In this study, we established a bone dehiscence mouse model by applying force application and used the granular type of scaffold materials encapsulated with bone morphogenetic protein (BMP)-2 and OP3-4, the receptor activator of NF-κB ligand (RANKL)-binding peptide, for the prophylactic injection to the alveolar bone. In vivo micro-computed tomography revealed bone dehiscence with decreased buccal alveolar bone thickness and height after force application, whereas no bone dehiscence was observed with the prophylactic injection after force application, and alveolar bone thickness and height were kept at similar levels as those in the control group. Bone histomorphometry analyses revealed that both bone formation and resorption parameters were significantly higher in the injection with force application group than in the force application without the prophylactic injection group. These findings suggest that the prophylactic local delivery of bone anabolic reagents can prevent bone dehiscence with increased bone remodelling activity.

The relationship between anabolic androgenic steroid use and body image, eating behavior, and physical activity by gender: A systematic review.

The prevalence of anabolic androgenic steroids (AAS) is rising, especially in recreational sports and the general population. While body image significantly influences AAS use, gender differences remain unclear. We examined gender-related connections between AAS use, body image, eating behavior, and physical activity. Following PRISMA guidelines, we analyzed 22 studies: 14 with male-only samples, 5 mixed-gender, 2 with sexual and gender minorities, and 1 with a female-only sample. FINDINGS: confirm body image as a key predictor of AAS use. Though AAS use correlates with eating disorders, outcomes vary by context; for instance, no discernible difference in eating behavior was observed between AAS users and non-users in bodybuilding. Physical activity findings varied, with some studies showing no significant differences between AAS users and non-users. Due to limited gender-comparison studies, conclusive gender-related differences cannot be drawn. This systematic review underscores the complex interplay between AAS use, body image, eating behavior, and physical activity, emphasizing the necessity for further research to develop targeted interventions for diverse populations, addressing AAS-related concerns and promoting overall well-being.

Early Life Androgen Administration Attenuates Aging Related Declines in Muscle Protein Synthesis.

PURPOSE: This study examined the acute and long-term effects of nandrolone decanoate (ND) on fractional synthetic rates (FSR). METHODS: Male C57BL/6 mice were randomized into ND ( n = 20) or sham ( n = 20) groups. ND injections (10 g·kg -1 ·wk -1 ) started at 7 months of ages and continued for 6 wk. Ten animals from each group were randomly separated and examined 1 wk following drug cessation. The remaining animals were examined at 16 months of age. Animals were injected IP with 1.5 mL of deuterated water 24 h before euthanasia. The kidney, liver, heart, gastrocnemius, and soleus were extracted. Samples were analyzed for deuterated alanine enrichment in the bound protein and intracellular fraction by liquid chromatography tandem mass spectrometry to measure estimated FSR (fraction/day (F/D)) of mixed tissue. RESULTS: One-way ANOVA, with treatment and age as fixed factors, indicated that kidney FSR was greater ( P = 0.027) in ND (0.41 ± 0.02 F/D) than sham (0.36 ± 0.014F/D) and higher ( P = 0.003) in young (0.42 ± 0.2 F/D) than old (0.35 ± 0.01 F/D). Liver and heart FSR values were greater ( P ≤ 0.001) in young (0.79 ± 0.06 F/D and 0.13 ± 0.01 F/D, respectively) compared with old (0.40 ± 0.01 F/D and 0.09 ± 0.01 F/D, respectively), but not between ND and sham. Gastrocnemius FSR was ( P ≤ 0.001) greater in young (0.06 ± 0.01 F/D) compared with old (0.03 ± 0.002 F/D), and greater ( P = 0.006) in ND (0.05 ± 0.01 F/D) compared with sham (0.04 ± 0.003 F/D). Soleus FSR rates were greater ( P = 0.050) in young (0.13 ± 0.01 F/D) compared with old (0.11 ± 0.003 F/D), but not between ND (0.12 ± 0.01 F/D) and sham (0.12 ± 0.01 F/D). Old animals who had received ND displayed elevated FSR in the gastrocnemius ( P = 0.054) and soleus ( P = 0.024). CONCLUSIONS: ND use in young adult animals appeared to maintain long-term elevations in FSR in muscle during aging.

Detection of methandienone and its metabolites in equine urine, plasma and hair following a multidose oral administration.

Methandienone is an anabolic-androgenic steroid that is prohibited in equine sports due to its potential performance enhancing properties. Metabolism and detection of methandienone in equine urine have been investigated comprehensively in literature; however, there is a limited knowledge about its metabolites in equine plasma and no information about its detection in equine hair. Following a multi-dose oral administration of methandienone to two Thoroughbred horses, 17-epimethandienone, methyltestosterone, two mono-hydroxylated, two di-hydroxylated and three 17α-methylandrostanetriol metabolites were detected in plasma. The majority of these were present as free analytes, whilst the mono-hydroxylated metabolites and one isomer of 17α-methylandrostanetriol were partially conjugated. Estimated peak concentrations of methandienone were 6,000 and 11,100 pg/ml; meanwhile, they were 25.4 and 40.5 pg/ml for methyltestosterone. The most abundant analyte in the post-administration plasma samples of both horses was the mono-hydroxylated metabolite; however, the parent compound provided the longest detection (up to 96 h). Screening analysis of hair enabled the detection of methandienone in mane hair samples only, for up to 3 months. Its mono- and di-hydroxylated metabolites were detected with greater peak responses for up to 6 months post-administration in both mane and tail samples, showing that these metabolites could be better analytical targets for hair analysis when administered orally. A follow-up methodology with an extensive wash procedure confirmed the presence of methandienone and its metabolites in a number of post-administration hair samples. Final wash samples were also analysed to assess the degree of internal incorporation (via bloodstream) against possible external deposition (via sweat/sebum).

Endocrinología del dopaje y los deportes: andrógenos anabolizantes / Doping and sports endocrinology: anabolic-androgenic steroids

El consumo de anabolizantes hormonales afecta no solamente a atletas profesionales, sino también a la población general (culturistas, clientes de gimnasios y adolescentes entre otros). En el primer caso su uso está prohibido y sancionado por la Agencia Mundial Anti-Dopaje y los comités olímpicos. Para los segundos es difícil establecer la prevalencia ya que muchos obtienen los productos a través de compras por Internet. Los motivos para su uso son diversos y se han descrito distintas formas de uso, así como diferentes tipologías de consumidores. Entre los efectos secundarios, el hipogonadismo es la causa más frecuente de consulta endocrinológica. En esta revisión se describen, tras una introducción general al dopaje, los antecedentes históricos de los andrógenos anabolizantes, su clasificación, las formas de uso, los efectos fisiológicos, los efectos adversos en diferentes órganos y sistemas, el tratamiento del hipogonadismo, así como los métodos de detección (AU)

The use of anabolic steroids affects not only professional athletes but also the general population (bodybuilders, gym clients, and adolescents). In the first case, its use is prohibited and sanctioned by the World Anti-Doping Agency and Olympic committees. For the other users, it is difficult to establish its prevalence since many obtain the products via the Internet. The reasons for its use are varied and different forms of use and other types of users have been described. Among the side effects of steroid use, hypogonadism is the most frequent cause for endocrinological consultation. After a general introduction to doping, this review describes the historical background of anabolic–androgenic steroids, their classification, forms of use, physiological effects, adverse effects on different organs and systems, treatment of hypogonadism, as well as detection methods (AU)

NACA and LRP6 Are Part of a Common Genetic Pathway Necessary for Full Anabolic Response to Intermittent PTH.

PTH induces phosphorylation of the transcriptional coregulator NACA on serine 99 through Gαs and PKA. This leads to nuclear translocation of NACA and expression of the target gene Lrp6, encoding a coreceptor of the PTH receptor (PTH1R) necessary for full anabolic response to intermittent PTH (iPTH) treatment. We hypothesized that maintaining enough functional PTH1R/LRP6 coreceptor complexes at the plasma membrane through NACA-dependent Lrp6 transcription is important to ensure maximal response to iPTH. To test this model, we generated compound heterozygous mice in which one allele each of Naca and Lrp6 is inactivated in osteoblasts and osteocytes, using a knock-in strain with a Naca99 Ser-to-Ala mutation and an Lrp6 floxed strain (test genotype: Naca99S/A; Lrp6+/fl;OCN-Cre). Four-month-old females were injected with vehicle or 100 µg/kg PTH(1-34) once daily, 5 days a week for 4 weeks. Control mice showed significant increases in vertebral trabecular bone mass and biomechanical properties that were abolished in compound heterozygotes. Lrp6 expression was reduced in compound heterozygotes vs. controls. The iPTH treatment increased Alpl and Col1a1 mRNA levels in the control but not in the test group. These results confirm that NACA and LRP6 form part of a common genetic pathway that is necessary for the full anabolic effect of iPTH.

Discovery of 3-amino-4-{(3S)-3-[(2-ethoxyethoxy)methyl]piperidin-1-yl}thieno[2,3-b]pyridine-2-carboxamide (DS96432529): A potent and orally active bone anabolic agent.

The continuing investigation of SAR of 3-aminothieno[2,3-b]pyridine-2-carboxamide derivatives has been described. In this study, C4-piperidine derivatives with polar functional groups were synthesized to develop orally available bone anabolic agents. The optimized compound 9o (DS96432529), which exhibited the best PK profile and high in vitro activity, showed the highest in vivo efficacy in this series. Moreover, significant synergistic effects were observed following co-administration of DS96432529 and alendronate or parathyroid hormone. The mechanism of action is most likely mediated through CDK8 inhibition.

The impact of different Hormonal Growth Promotants (HGP) on desmin degradation and collagen content of various muscles from pasture and feedlot finished steer carcasses.

The impacts of several hormonal growth promotants (HGP) on Warner-Bratzler Shear Force (WBSF), desmin degradation ratio (DDR) and collagen content (COLL) were assessed. Treatments within feedlot and pasture finished steer carcasses (n = 60, n = 40, respectively) were control (CON-100-F and CON-400-P), oestradiol HGPs (OES-100-F and OES-400-P) and trenbolone acetate/oestradiol HGPs (TBA+OES-100-F only). The longissimus lumborum (LL), gluteus medius (GM), infraspinatus (IS), semitendinosus (ST,) and the LL and biceps femoris (BF) were collected from feedlot and pasture finished steers, respectively. All muscles were aged between 3 and 35 days. The LL from TBA+OES-100-F carcasses had increased WBSF and decreased DDR, which varied in magnitude with ageing (P < 0.05). The GM from OES-100-F steers also had lower DDR (P < 0.05). The feedlot HGP treatments had no impact on the WBSF of the IS, ST or GM and no impact on COLL in the LL. The OES-400-P had no impact on WBSF, DDRor COLL for both muscles (P > 0.05).

Anabolic-Androgenic Steroid Use in Sports, Health, and Society.

This consensus statement is an update of the 1987 American College of Sports Medicine (ACSM) position stand on the use of anabolic-androgenic steroids (AAS). Substantial data have been collected since the previous position stand, and AAS use patterns have changed significantly. The ACSM acknowledges that lawful and ethical therapeutic use of AAS is now an accepted mainstream treatment for several clinical disorders; however, there is increased recognition that AAS are commonly used illicitly to enhance performance and appearance in several segments of the population, including competitive athletes. The illicit use of AAS by competitive athletes is contrary to the rules and ethics of many sport governing bodies. Thus, the ACSM deplores the illicit use of AAS for athletic and recreational purposes. This consensus statement provides a brief history of AAS use, an update on the science of how we now understand AAS to be working metabolically/biochemically, potential side effects, the prevalence of use among athletes, and the use of AAS in clinical scenarios.

Oxandrolone treatment in juvenile rats induced anxiety-like behavior in young adult animals.

AIMS: Oxandrolone (OXA) is a synthetic steroid used for the treatment of clinical conditions associated with catabolic states in humans, including children. However, its behavioral effects are not well known. Our goal was to evaluate the anxiety-like behavior induced in young adult rats after the treatment of juvenile animals with OXA. METHODS: Four-week-old male rats were separated into three groups: Control (CON), therapeutic-like OXA dose (TD), and excessive OXA dose (ED), in which 2.5 and 37.5 mg/kg/day of OXA were administered via gavage for four weeks for TD and ED, respectively. Behavior was evaluated through the elevated plus maze (EPM) and open field (OF) tests. Protein expression of catalase (CAT), superoxide dismutase (SOD), Tumor necrosis factor-α (TNF-α), and dopamine receptor 2 (DrD2) were analyzed in tissue samples of the hippocampus, amygdala, and prefrontal cortex by Western Blot. RESULTS: OXA induced anxiety-like behaviors in both TD and ED animals; it decreased the time spent in the open arms of the EPM in both groups and reduced the time spent in the central zone of the OF in the TD group. In the hippocampus, CAT expression was higher in TD compared with both control and ED animals. No differences were found in the amygdala and prefrontal cortex. TNF-α, SOD, and DrD2 levels were not altered in any of the assessed areas. CONCLUSIONS: Treatment of juvenile rats with OXA led to anxiety-like behavior in young adult animals regardless of the dose used, with minor changes in the antioxidant machinery located in the hippocampus.

Metformin reduces oxandrolone- induced depression-like behavior in rats via modulating the expression of IL-1ß, IL-6, IL-10 and TNF-α.

Oxandrolone (OXA) is an androgen and anabolic steroid (AAS) that is used to reverse weight loss associated with some medical conditions. One of the side effects of OXA is its potential to induce depressive symptoms. Growing evidence suggested that neuroinflammation and cytokines play crucial roles in sickness behavioral and associated mood disturbances. Previous studies showed that metformin attenuated neuroinflammation. This study investigated the potential protective role of metformin against OXA-induced depression-like behavior and neuroinflammation. Twenty- four Wistar male rats were randomly grouped into four groups: the control group (Control) received only vehicle; the oxandrolone group (OXA) received oxandrolone (0.28 mg/kg, i.p); the metformin group (MET) received metformin (100 mg/kg, i.p); and the oxandrolone / metformin group (OXA + MET) received both oxandrolone (0.28 mg/kg, i.p) and metformin (100 mg/kg, i.p). These treatments were administered for fourteen consecutive days. Behavioral tests to measure depression-like behavior were conducted before and after treatments. qRT-PCR was used to measure the relative expression of proinflammatory and anti-inflammatory cytokines in the hippocampus and hypothalamus. The results showed that oxandrolone induced depression-like behavior and dysregulated pro-/anti-inflammatory cytokines, while metformin attenuated these effects. These findings suggest that metformin is a potential treatment to reverse the depressive effects induced by oxandrolone that involve neuroinflammatory effects.

GnRHa/Stanozolol Combined Therapy Maintains Normal Bone Growth in Central Precocious Puberty.

Background: Gonadotropin-releasing hormone agonist (GnRHa) is the gold standard in the treatment of Central Precocious Puberty (CPP) with progressive puberty and accelerative growth. However, GnRHa treatment is reported to result in growth deceleration and prevents growth plate development which leads to a reduction in height velocity. Stanozolol (ST) has been used to stimulate growth in patients with delayed growth and puberty, nevertheless, the effects and mechanisms of ST on CPP with GnRHa treatment are currently unclear. Methods and Results: In the current study, we recorded the following vital observations that provided insights into ST induced chondrogenic differentiation and the maintenance of normal growth plate development: (1) ST efficiently prevented growth deceleration and maintained normal growth plate development in rats undergoing GnRHa treatment; (2) ST suppressed the inhibitory effect of GnRHa to promote chondrogenic differentiation; (3) ST induced chondrogenic differentiation through the activation of the JNK/c-Jun/Sox9 signaling pathway; (4) ST promoted chondrogenic differentiation and growth plate development through the JNK/Sox9 signaling pathway in vivo. Conclusions: ST mitigated the inhibitory effects of GnRHa and promoted growth plate development in rats. ST induced the differentiation of chondrocytes and maintained normal growth plate development through the activation of JNK/c-Jun/Sox9 signaling. These novel findings indicated that ST could be a potential agent for maintaining normal bone growth in cases of CPP undergoing GnRHa treatment.

Oral fucoidan improves muscle size and strength in mice.

Fucoidan is a sulfated polysaccharide found in a range of brown algae species. Growing evidence supports the long-term supplementation of fucoidan as an ergogenic aid to improve skeletal muscle performance. The aim of this study was to investigate the effect of fucoidan on the skeletal muscle of mice. Male BL/6 mice (N = 8-10) were administered a novel fucoidan blend (FUC, 400 mg/kg/day) or vehicle (CON) for 4 weeks. Treatment and control experimental groups were further separated into exercise (CON+EX, FUC+EX) or no-exercise (CON, FUC) groups, where exercised groups performed 30 min of treadmill training three times per week. At the completion of the 4-week treatment period, there was a significant increase in cross-sectional area (CSA) of muscle fibers in fucoidan-treated extensor digitorum longus (EDL) and soleus fibers, which was accompanied by a significant increase in tibialis anterior (TA) muscle force production in fucoidan-treated groups. There were no significant changes in grip strength or treadmill time to fatigue, nor was there an effect of fucoidan or exercise on mass of TA, EDL, or soleus muscles. In gastrocnemius muscles, there was no change in mRNA expression of mitochondrial biogenesis markers PGC-1α and Nrf-2 in any experimental groups; however, there was a significant effect of fucoidan supplementation on myosin heavy chain (MHC)-2x, but not MHC-2a, mRNA expression. Overall, fucoidan increased muscle size and strength after 4 weeks of supplementation in both exercised and no-exercised mice suggesting an important influence of fucoidan on skeletal muscle physiology.

Potential risk for developing severe COVID-19 disease among anabolic steroid users.

A severe case of COVID-19 was observed in an otherwise healthy 28-year-old man who had taken oxandrolone 40 mg/day as an anabolic steroid. The patient had been taking oxandrolone for enhanced bodybuilding 30 days prior to presenting to an outpatient clinic with COVID-19 symptoms. The patient reported that his symptoms have rapidly worsened over the course of 4 days prior to presenting at the clinic. As part of an experimental antiandrogen treatment for hyperandrogenic men suffering from COVID-19, he was administered a single 600 mg dose of the novel antiandrogen proxalutamide. Twenty-four hours after administration of this dose, marked improvement of symptoms and markers of disease severity were observed. To our knowledge, this is the first case that potentially links anabolic steroid use to COVID-19 disease severity.

Effects of Beef Protein Supplementation in Male Elite Triathletes: A Randomized, Controlled, Double-Blind, Cross-Over Study.

OBJECTIVE: Beef protein extracts are growing in popularity in recent years due to their purported anabolic effects as well as to their potential benefits on hematological variables. The present randomized, controlled, double-blind, cross-over study aimed to analyze the effects of beef protein supplementation on a group of male elite triathletes (Spanish National Team). METHODS: Six elite triathletes (age, 21 ± 3 years; VO2max, 71.5 ± 3.0 ml·kg·min-1) were randomly assigned to consume daily either 25 g of a beef supplement (BEEF) or an isoenergetic carbohydrates (CHO) supplement for 8 weeks, with both conditions being separated by a 5-week washout period. Outcomes, including blood analyses and anthropometrical measurements, were assessed before and after each 8-week intervention. RESULTS: No effects of supplement condition were observed on body mass nor on skinfold thicknesses, but BEEF induced significant and large benefits over CHO in the thigh cross-sectional area (3.02%, 95%CI = 1.33 to 4.71%; p = 0.028, d = 1.22). Contrary to CHO, BEEF presented a significant increase in vastus lateralis muscle thickness (p = 0.046), but differences between conditions were not significant (p = 0.173, d = 0.87). Although a significantly more favorable testosterone-to-cortisol ratio (TCR) was observed for BEEF over CHO (37%, 95% CI = 5 to 68%; p = 0.028, d = 1.29), no significant differences were found for the hematological variables (i.e., iron, ferritin, red blood cell count, hemoglobin or hematocrit). CONCLUSION: Beef protein supplementation seems to facilitate a more favorable anabolic environment (i.e., increased TCR and muscle mass) in male elite triathletes, with no impact on hematological variables.

Detection of the selective androgen receptor modulator GSK2881078 and metabolites in urine and hair after single oral administration.

Hair and urine concentrations of the nonsteroidal selective androgen receptor modulator GSK2881078 were examined following single oral administration to investigate its hair incorporation and estimate the general suitability of hair testing for selected androgen receptor modulators. Hair segments were collected following a single dose of 1.5 mg GSK2881078 by repeated shaving of scalp hair at Week 0 (blank), Week 1 (representing the pre-application period), Week 3 (ideally focusing the time of incorporation), and Weeks 5 and 9 (post-administration period). The intact compound and various (at least 4) hydroxy-metabolites exhibited similar elimination profiles. The peak urinary concentration (approximately 920 pg/ml) was observed after 8 h and is reduced to the detection limit (2 pg/ml) on Day 42 following administration of 760 µg GSK2881078. Correspondingly, hair concentrations of GSK2881078 (intact compound only) following a single oral dose of 1.5 mg GSK2881078 reached a peak concentration of 1.7 pg/mg in the segments collected 3 weeks post administration, representing the time of ingestion. The concentration rapidly declined to trace amounts of 0.7 (Week 5) and 0.2 pg/mg (Week 9), respectively. In conclusion, measurement of the intact compound GSK2881078 is feasible for both urine and hair analysis. However, concentrations in hair after single oral administration are in the low pg/mg range and can only be detected, if the segments cover the administration period.

Effect of oxandrolone and timing of pubertal induction on final height in Turner syndrome: final analysis of the UK randomised placebo-controlled trial.

The UK Turner syndrome (TS) study examined the effect on final height of oxandrolone 0.05 mg/kg/day (maximum dose 2.5 mg) versus placebo from 9 years of age; and delaying ethinylestradiol induction of puberty by 2 years from 12 (E12) to 14 (E14) years in growth hormone-treated girls with TS. The study ran from 1999 to 2013. By 2011, eighty-two of 92 participants had reached final height and an interim analysis using the Super-Imposition by Translation And Rotation model showed significant increases in final height with both oxandrolone and E14. The analysis has been repeated now that all 92 patients have reached final height. Oxandrolone still significantly increased final height by 4.1 cm (95% CI 1.6 to 6.6, n=92) compared with 4.6 cm previously. However, the E14 effect was no longer significant at 2.7 cm (95% CI -0.8 to 6.1, n=56) compared with 3.8 cm previously.

Label-free proteomics for discovering biomarker candidates of RAD140 administration to castrated horses.

Selective androgen receptor (AR) modulators (SARMs) are potent anabolic agents with a high potential of misuse in horseracing and equestrian sports. In this study, we applied label-free proteomics to discover plasma protein biomarkers in geldings (castrated horses) after administration with a popular SARM named RAD140. Tryptic peptides were prepared from plasma samples and analyzed by nano-flow ultrahigh-performance liquid chromatography-high-resolution tandem mass spectrometry (nano-UHPLC-HRMS/MS) using data-independent acquisition (DIA) method. Orthogonal projection on latent structure-discriminant analysis (OPLS-DA) has led to the development of a predictive model that could discriminate RAD140-administered samples from control samples and could also correctly classify 18 out of 19 in-training horses as control samples. The model comprises 75 proteins with variable importance in projection (VIP) score above 1. Gene Ontology (GO) enrichment analysis and literature review have identified upregulation of AR-regulated clusterin, and proteins associated with inflammation (haptoglobin, cluster of differentiation 14 [CD14], and inter-alpha-trypsin inhibitor heavy chain 4 [ITIH4]) and erythropoiesis (glycosylphosphatidylinositol-specific phospholipase D1 [GPLD1]) after RAD140 administration. Their changes were confirmed by selected reaction monitoring (SRM) experiments. Similar effects have been reported by the use of androgens and other SARMs. This is the first reported study that describes the use of a proteomic biomarker approach to detect horses that have been administered with RAD140 by applying label-free proteomic profiling of plasma samples. These results support the concept of a biomarker-driven approach to enhance the doping control of RAD140 and potentially other SARMs in the future.