Insulin-Like Growth Factor-1 (IGF-1) and Its Monitoring in Medical Diagnostic and in Sports

4 Feb 2021

Insulin-Like Growth Factor-1 (IGF-1) and Its Monitoring in Medical Diagnostic and in Sports / Julian Bailes, Mikhail Soloviev. - (Biomolecules 11 (2021) 2 (4 February); p. 1-15)

  • PMID: 33557137
  • PMCID: PMC7913862
  • DOI: 10.3390/biom11020217


Insulin-like growth factor-1 (IGF-1) is the principal mediator of growth hormone (GH), plays a crucial role in promoting cell growth and differentiation in childhood and continues to have an anabolic effect in adults. IGF-1 is part of a wide network of growth factors, receptors and binding proteins involved in mediating cellular proliferation, differentiation and apoptosis. Bioavailability of IGF-1 is affected by insulin-like growth factor binding proteins (IGFBPs) which bind IGF-1 in circulation with an affinity equal to or greater than that of the IGF-1 receptor (IGF-1R). The six IGFBPs serve as carrier proteins and bind approximately 98% of all circulating IGF-1. Other proteins known to bind IGF-1 include ten IGFBP-related proteins (IGFBP-rPs), albeit with lower affinities than the IGFBPs. IGF-1 expression levels vary in a number of clinical conditions suggesting it has the potential to provide crucial information as to the state of an individual's health. IGF-1 is also a popular doping agent in sport and has featured in many high-profile doping cases in recent years. However, the existence of IGFBPs significantly reduces the levels of immunoreactive IGF-1 in samples, requiring multiple pre-treatment steps that reduce reproducibility and complicates interpretation of IGF-1 assay results. Here we provide an overview of the IGF network of growth factors, their receptors and the entirety of the extended family of IGFBPs, IGFBP-rPs, E peptides as well as recombinant IGF-1 and their derivatives. We also discuss issues related to the detection and quantification of bioavailable IGF-1.

Doping Attitudes, Beliefs, and Practices among Young, Amateur Croatian Athletes

9 Feb 2021

Doping Attitudes, Beliefs, and Practices among Young, Amateur Croatian Athletes / Ivan Miskulin, Danijela Stimac Grbic, Maja Miskulin. - (Sports 9 (2021) 2 (9 February); p. 1-10)

  • PMID: 33572386
  • PMCID: PMC7916256
  • DOI: 10.3390/sports9020025


Recent studies revealed that amateur athletes, especially young ones, have an increasing tendency of performance-enhancing drugs (PEDs) usage. The aim of this study was to explore PEDs attitudes, beliefs, and practices among young, amateur Croatian athletes. This cross-sectional study using a specially designed questionnaire as a research tool was done during the August 2019 to January 2020 period among a convenient sample of 400 amateur athletes of median age 18 (interquartile range 15 to 21) years. The prevalence of current PEDs usage was 1.3%, while past PEDs usage prevalence was 3.3%. Current PEDs usage was more frequent among young adults (p = 0.048) and athletes playing individual sports (p = 0.001). Athletes who were engaged in sports from one to five years had more permissive attitudes toward PEDs (p < 0.001) as measured by the Performance Enhancement Attitude Scale. Female athletes had more positive beliefs about PEDs usage (p = 0.008). The study did not establish any correlation between current or past PEDs usage and attitudes toward PEDs as well as beliefs about PEDs usage. However, there was a weak positive correlation between attitudes toward PEDs and athletes' beliefs about PEDs usage (rs = 0.465, p < 0.001). PEDs usage is present among young Croatian amateur athletes. There is a need for interventions directed toward the prevention of PEDs usage in an observed subgroup of athletes.

Evaluation of dermorphin metabolism using zebrafish water tank model and human liver microsomes

15 Feb 2021

Evaluation of dermorphin metabolism using zebrafish water tank model and human liver microsomes / Juliana de L. Castro, Henrique M.G. Pereira, Valéria de Sousa, Maria Elvira P. Martucci. - (Current Drug Metabolism (2021) 15 February)

  • PMID: 33593255
  • DOI: 10.2174/1389200222666210216095753


Background: Dermorphin is a heptapeptide with an analgesic potential higher than morphine that does not present the same risk for the development of tolerance. These pharmacological features make dermorphin a potential doping agent in competitive sports and is already prohibited for racehorses. For athletes, the development of an efficient strategy to monitor for its abuse necessitates an investigation of the metabolism of dermorphin in humans.

Methods: Here, human liver microsomes and zebrafish were utilized as model systems of human metabolism to evaluate the presence and kinetics of metabolites derived from dermorphin. Five hours after its administration, the presence of dermorphin metabolites could be detected in both models by liquid chromatography coupled to high resolution mass spectrometry.

Results: Although the two models showed common results, marked differences were also observed in relation to the formed metabolites. Six putative metabolites, based on their exact masses of m/z 479.1915, m/z 501.1733, m/z 495.1657, m/z 223.1073, m/z 180.1017 and m/z 457.2085, are proposed to represent the metabolic pattern of dermorphin. The major metabolite generated from the administration of dermorphin in both models was YAFG-OH (m/z 457.2085), which is the N-terminal tetrapeptide previously identified from studies with rats.

Conclusion: Its extensive characterization and commercial availability suggests that it could serve as a primary target analyte for the detection of dermorphin misuse. The metabolomics approach also allowed the assignment of other confirmatory metabolites.

The Cardiac Effects of Performance-Enhancing Medications: Caffeine vs. Anabolic Androgenic Steroids

17 Feb 2021

The Cardiac Effects of Performance-Enhancing Medications: Caffeine vs. Anabolic Androgenic Steroids / Sanjay Sivalokanathan, Łukasz A. Małek, Aneil Malhotra. - (Diagnostics 11 (2021) 2 (17 February); p. 1-14)

  • PMID: 33671206
  • PMCID: PMC7922604
  • DOI: 10.3390/diagnostics11020324


Several performance-enhancing or ergogenic drugs have been linked to both significant adverse cardiovascular effects and increased cardiovascular risk. Even with increased scrutiny on the governance of performance-enhancing drugs (PEDs) in professional sport and heightened awareness of the associated cardiovascular risk, there are some who are prepared to risk their use to gain competitive advantage. Caffeine is the most commonly consumed drug in the world and its ergogenic properties have been reported for decades. Thus, the removal of caffeine from the World Anti-Doping Agency (WADA) list of banned substances, in 2004, has naturally led to an exponential rise in its use amongst athletes. The response to caffeine is complex and influenced by both genetic and environmental factors. Whilst the evidence may be equivocal, the ability of an athlete to train longer or at a greater power output cannot be overlooked. Furthermore, its impact on the myocardium remains unanswered. In contrast, anabolic androgenic steroids are recognised PEDs that improve athletic performance, increase muscle growth and suppress fatigue. Their use, however, comes at a cost, afflicting the individual with several side effects, including those that are detrimental to the cardiovascular system. This review addresses the effects of the two commonest PEDs, one legal, the other prohibited, and their respective effects on the heart, as well as the challenge in defining its long-term implications.

A Cell-Free Bioassay for the Detection of Androgens

11 Mar 2021

A Cell-Free Bioassay for the Detection of Androgens / Elliot R. Cooper, Gillian Hughes, Alexia Kauff, Emma Sutherland, Zoe Ashley, Alison K. Heather. - (Drug Testing and Analysis (2021) 11 March)

  • PMID: 33709622
  • DOI: 10.1002/dta.3024


Androgens remain abused performance-enhancing drugs in sports. Technologies based on mass spectrometry can detect all forms of androgens but fail if the androgen represents a novel structure. A bioassay detects androgens based on function rather than structure. To date, there has been limited adoption of cell-based in vitro bioassays as a screening tool for non-targeted androgen detection because they require expert personnel and specialized equipment to perform. We now describe the development of a cell-free version of an androgen in vitro bioassay. Stage 1 involved in vitro transcription/translation reactions (IVTT) using a DNA template encoding an enhancer/ARE regulatory region upstream of a minimal promoter that drives expression of a reporter protein. The assay detected testosterone across the concentration range of 106.7 to 0.0144 ng/mL (3.7X10-7 -5X10-11 M), with an EC50 of 6.63 ng/mL (23 nM). To reduce complexity, stages 2-4 of development included just in vitro transcription (IVT) reactions, whereby the output was an RNA molecule. Stage 2 involved directly labeling the RNA molecule with fluorophore-labeled nucleotide triphosphates, stage 3 involved reverse transcription-PCR of the RNA molecule and stage 4 utilized an RNA aptamer, Mango II, as its RNA output. The stage 4 product detected testosterone across the range of 106.7-0.0001 ng/mL (3.7X10-7 -5X10-13 M), with an EC50 of 0.04 ng/mL (0.155 nM). Further to this, we showed that the stage 4 product could detect other androgenic molecules. Relative to cell-based bioassays, the Stage 4 product is easy to perform and could be developed into a routine, high-throughput, non-targeted androgen screen.

Profiling of steroid metabolites after transdermal and oral administration of testosterone by ultra-high pressure liquid chromatography coupled to quadrupole time-of-flight mass spectrometry

21 Jun 2013

Profiling of steroid metabolites after transdermal and oral administration of testosterone by ultra-high pressure liquid chromatography coupled to quadrupole time-of-flight mass spectrometry / F. Badoud, J. Boccard, C. Schweizer, F. Pralong, M. Saugy, N. Baume. - (Journal of Steroid Biochemistry and Molecular Biology 138 (2013) November; p . 22-235)

  • PMID: 23796409
  • DOI: 10.1016/j.jsbmb.2013.05.018


The screening of testosterone (T) misuse for doping control is based on the urinary steroid profile, including T, its precursors and metabolites. Modifications of individual levels and ratio between those metabolites are indicators of T misuse. In the context of screening analysis, the most discriminant criterion known to date is based on the T glucuronide (TG) to epitestosterone glucuronide (EG) ratio (TG/EG). Following the World Anti-Doping Agency (WADA) recommendations, there is suspicion of T misuse when the ratio reaches 4 or beyond. While this marker remains very sensitive and specific, it suffers from large inter-individual variability, with important influence of enzyme polymorphisms. Moreover, use of low dose or topical administration forms makes the screening of endogenous steroids difficult while the detection window no longer suits the doping habit. As reference limits are estimated on the basis of population studies, which encompass inter-individual and inter-ethnic variability, new strategies including individual threshold monitoring and alternative biomarkers were proposed to detect T misuse.

The purpose of this study was to evaluate the potential of ultra-high pressure liquid chromatography (UHPLC) coupled with a new generation high resolution quadrupole time-of-flight mass spectrometer (QTOF-MS) to investigate the steroid metabolism after transdermal and oral T administration. An approach was developed to quantify 12 targeted urinary steroids as direct glucuro- and sulfo-conjugated metabolites, allowing the conservation of the phase II metabolism information, reflecting genetic and environmental influences. The UHPLC-QTOF-MS(E) platform was applied to clinical study samples from 19 healthy male volunteers, having different genotypes for the UGT2B17 enzyme responsible for the glucuroconjugation of T. Based on reference population ranges, none of the traditional markers of T misuse could detect doping after topical administration of T, while the detection window was short after oral TU ingestion. The detection ability of the 12 targeted steroids was thus evaluated by using individual thresholds following both transdermal and oral administration. Other relevant biomarkers and minor metabolites were studied for complementary information to the steroid profile, including sulfoconjugated analytes and hydroxy forms of glucuroconjugated metabolites. While sulfoconjugated steroids may provide helpful screening information for individuals with homozygotous UGT2B17 deletion, hydroxy-glucuroconjugated analytes could enhance the detection window of oral T undecanoate (TU) doping.

Keywords: Anabolic androgenic steroids; Phase II metabolism; Quadrupole time-of-flight; Quantification; Testosterone; Ultra-high-pressure liquid chromatography.

Pharmaceutical doses of the banned stimulant oxilofrine found in dietary supplements sold in the USA

7 Apr 2016

Pharmaceutical doses of the banned stimulant oxilofrine found in dietary supplements sold in the USA / Pieter A. Cohen, Bharathi Avula, Bastiaan Venhuis, John C. Travis, Yan-Hong Wang, Ikhlas A. Khan. - (Drug Testing and Analysis 9 (2017) 1 (January); p. 135-142)

  • PMID: 27062112
  • DOI: 10.1002/dta.1976


Oxilofrine (4-[1-hydroxy-2-(methylamino)propyl]phenol) is a pharmaceutical stimulant prescribed in dosages of 16 to 40 mg to stimulate the heart and increase blood pressure. It has never been approved for use in the USA as a prescription drug or as a dietary supplement. Several athletes, however, have been banned from sport for testing positive for oxilofrine and have claimed that they inadvertently consumed oxilofrine in sports supplements. Consumption of supplements containing oxilofrine may also pose serious health risks. For example, one brand of supplements containing oxilofrine has been linked to serious adverse events including vomiting, agitation, and cardiac arrest. We designed our study to determine the presence and quantity of oxilofrine in dietary supplements sold in the USA. A validated ultra-high performance liquid chromatography-quadrupole time of flight-mass spectrometry method was developed for the identification and quantification of oxilofrine. The separation was achieved using a reversed phase column, mass spectrometry detection, and a water/acetonitrile gradient as the mobile phase. The presence of oxilofrine was confirmed using a reference standard. We analyzed 27 brands of supplements labelled as containing a synonym of oxilofrine ('methylsynephrine') and found that oxilofrine was present in 14 different brands (52%) at dosages ranging from 0.0003 to 75 mg per individual serving. Of the supplements containing oxilofrine, 43% (6/14) contained pharmaceutical or greater dosages of oxilofrine. Following instructions on the label, consumers could ingest as much as 250 mg of oxilofrine per day. The drug oxilofrine was found in pharmacological and greater dosages in supplements labelled as containing methylsynephrine.

Plasma and urinary markers of oral testosterone undecanoate misuse

26 Nov 2001

Plasma and urinary markers of oral testosterone undecanoate misuse / Shi Hua Peng, Jordi Segura, Magí Farré, Jose Carlos González, Xavier de la Torre. - (Steroids 67 (2002) 1 (January); p. 39-50)

  • PMID: 11728520
  • DOI: 10.1016/s0039-128x(01)00128-3


Orally administered testosterone undecanoate (TU), an anabolic, androgenic steroid, can potentially be abused by athletes. Indirect evidence for detecting oral TU intake could be deduced from the changes in steroid profile post-administration. Direct evidence could be obtained by detection of unchanged TU in plasma. To this end, both urinary and plasma steroid profiles of six healthy male subjects given a single oral dose of 120 mg of TU were studied by gas chromatography/mass spectrometry (GC/MS) and gas chromatography/tandem mass spectrometry (GC/MS/MS). The increased concentration of glucuronidated testosterone in plasma appears to be the most characteristic sign of oral TU intake. The testosterone glucuronide (TG)/nonconjugated testosterone (T) ratio, TG/17-hydroxyprogesterone (17OHP) ratio, and TG/luteinizing hormone (LH) ratio were observed to be significantly elevated above their basal levels for 10 h, 10 h, and 6 h, respectively. Urinary ratios of TG/epitestosterone glucuronide (EG) were found to be higher than the cut-off value of 6 for the period 4 approximately 8 h post-administration, but only in three subjects. One subject failed to respond with respect to all of the above-mentioned indirect markers, as TG was not significantly increased in either plasma or urine. Unchanged TU was directly detected in plasma of all six subjects from 1 approximately 1.5 h to 4 approximately 6 h after oral TU intake by GC/MS/MS, providing unequivocal proof of exogenous testosterone intake. Distinct and complementary markers for detecting oral TU intake could be obtained from plasma and urine, respectively.

Influences of β-HCG administration on carbon isotope ratios of endogenous urinary steroids

17 Feb 2012

Influences of β-HCG administration on carbon isotope ratios of endogenous urinary steroids / Thomas Piper, Norbert Baume, Emanuel Strahm, Caroline Emery, Martial Saugy. - (Steroids 77 (2012) 6 (May); p. 644-654)

  • PMID: 22369868
  • DOI: 10.1016/j.steroids.2012.02.009


Several factors influencing the carbon isotope ratios (CIR) of endogenous urinary steroids have been identified in recent years. One of these should be the metabolism of steroids inside the body involving numerous different enzymes. A detailed look at this metabolism taking into account differences found between steroids excreted as glucuronides or as sulphates and hydrogen isotope ratios of different steroids pointed out possibility of unequal CIR at the main production sites inside the male body - the testes and the adrenal glands. By administration of β-HCG it is possible to strongly stimulate the steroid production within the testes without influencing the production at the adrenal glands. Therefore, this treatment should result in changed CIR of urinary androgens in contrast to the undisturbed pre-treatment values. Four male volunteers received three injections of β-HCG over a time course of 5 days and collected their urine samples at defined intervals after the last administration. Those samples showing the largest response in contrast to the pre-administration urines were identified by steroid profile measurements and subsequent analysed by GC/C/IRMS. CIR of androsterone, etiocholanolone, testosterone, 5α- and 5β-androstanediol and pregnanediol were compared. While pregnanediol was not influenced, most of the investigated androgens showed depleted values after treatment. The majority of differences were found to be statistically significant and nearly all showed the expected trend towards more depleted δ(13)C-values. These results support the hypothesis of different CIR at different production sites inside the human body. The impact of these findings on doping control analysis will be discussed.

Detection of epitestosterone doping by isotope ratio mass spectrometry

1 Apr 2002

Detection of epitestosterone doping by isotope ratio mass spectrometry / Rodrigo Aguilera, Caroline K. Hatton, Don H. Catlin. - (Clinical Chemistry 48 (2002) 4 (1 April); p. 629-636)

  • PMID: 11901061


Background: Epitestosterone is prohibited by sport authorities because its administration will lower the urinary testosterone/epitestosterone ratio, a marker of testosterone administration. A definitive method for detecting epitestosterone administration is needed.

Methods: We developed a gas chromatography-combustion-isotope ratio mass spectrometry method for measuring the delta(13)C values for urinary epitestosterone. Sample preparation included deconjugation with beta-glucuronidase, solid-phase extraction, and semipreparative HPLC. Epitestosterone concentrations were determined by gas chromatography-mass spectrometry for urines obtained from a control group of 456 healthy males. Epitestosterone delta(13)C values were determined for 43 control urines with epitestosterone concentrations > or =40 microg/L (139 nmol/L) and 10 athletes' urines with epitestosterone concentrations > or =180 microg/L (624 nmol/L), respectively.

Results: The log epitestosterone concentration distribution was gaussian [mean, 3.30; SD, 0.706; geometric mean, 27.0 microg/L (93.6 nmol/L)]. The delta(13)C values for four synthetic epitestosterones were low (less than or equal to -30.3 per thousand) and differed significantly (P <0.0001). The SDs of between-assay precision studies were low (< or =0.73 per thousand). The mean delta(13)C values for urine samples obtained from 43 healthy males was -23.8 per thousand (SD, 0.93 per thousand). Nine of 10 athletes' urine samples with epitestosterone concentrations >180 microg/L (624 nmol/L) had delta(13)C values within +/- 3 SD of the control group. The delta(13)C value of epitestosterone in one sample was -32.6 per thousand (z-score, 9.4), suggesting that epitestosterone was administered. In addition, the likelihood of simultaneous testosterone administration was supported by low delta(13)C values for androsterone and etiocholanolone.

Conclusions: Determining delta(13)C values for urinary epitestosterone is useful for detecting cases of epitestosterone administration because the mean delta(13)C values for a control group is high (-23.8 per thousand) compared with the delta(13)C values for synthetic epitestosterones.

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