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

Abstract

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.

Progress in the Detection of Erythropoietin in Blood, Urine, and Tissue / Yukiko Yasuoka, Yuichiro Izumi, Jeff M. Sands, Katsumasa Kawahara, Hiroshi Nonoguchi

• Molecules 28 (2023) 11 (June) 4446, p. 1-17
• PMID: 37298922
• PMCID: PMC10254663
• DOI: 10.3390/molecules28114446

Abstract

Detection of erythropoietin (Epo) was difficult until a method was developed by the World Anti-Doping Agency (WADA). WADA recommended the Western blot technique using isoelectric focusing (IEF)-PAGE to show that natural Epo and injected erythropoiesis-stimulating agents (ESAs) appear in different pH areas. Next, they used sodium N-lauroylsarcosinate (SAR)-PAGE for better differentiation of pegylated proteins, such as epoetin β pegol. Although WADA has recommended the use of pre-purification of samples, we developed a simple Western blotting method without pre-purification of samples. Instead of pre-purification, we used deglycosylation of samples before SDS-PAGE. The double detection of glycosylated and deglycosylated Epo bands increases the reliability of the detection of Epo protein. All of the endogenous Epo and exogenous ESAs shift to 22 kDa, except for Peg-bound epoetin β pegol. All endogenous Epo and exogenous ESAs were detected as 22 kDa deglycosylated Epo by liquid chromatography/mass spectrum (LC/MS) analysis. The most important factor for the detection of Epo is the selection of the antibody against Epo. WADA recommended clone AE7A5, and we used sc-9620. Both antibodies are useful for the detection of Epo protein by Western blotting.

Prohormone supplement 3β-hydroxy-5α-androst-1-en-17-one enhances resistance training gains but impairs user health / Jorge Granados, Trevor L. Gillum, Kevin M. Christmas, Matthew R. Kuennen

• Journal of Applied Physiology 116 (2014) 5 (March), p. 560-569
• PMID: 24381122
• DOI: 10.1152/japplphysiol.00616.2013

Abstract

Prohormone supplements (PS) are recognized not to impart anabolic or ergogenic effects in men, but the research supporting these conclusions is dated. The Anabolic Steroid Control Act was amended in 2004 to classify androstenedione and 17 additional anabolic compounds as controlled substances. The viability of PS that entered the market after that time have not been evaluated. Seventeen resistance-trained men (23 ± 1 yr; 13.1 ± 1.5% body fat) were randomly assigned to receive either 330 mg/day of 3β-hydroxy-5α-androst-1-en-17-one (Prohormone; n = 9) or sugar (Placebo; n = 8) per os and complete a 4-wk (16 session) structured resistance-training program. Body composition, muscular strength, circulating lipids, and markers of liver and kidney dysfunction were assessed at study onset and termination. Prohormone increased lean body mass by 6.3 ± 1.2%, decreased fat body mass by 24.6 ± 7.1%, and increased their back squat one repetition maximum and competition total by 14.3 ± 1.5 and 12.8 ± 1.1%, respectively. These improvements exceeded (P < 0.05) Placebo, which increased lean body mass by 0.5 ± 0.8%, reduced fat body mass by 9.5 ± 3.6%, and increased back squat one repetition maximum and competition total by 5.7 ± 1.7 and 5.9 ± 1.7%, respectively. Prohormone also experienced multiple adverse effects. These included a 38.7 ± 4.0% reduction in HDL (P < 0.01), a 32.8 ± 15.05% elevation in LDL (P < 0.01), and elevations of 120.0 ± 22.6 and 77.4 ± 12.0% in LDL-to-HDL and cholesterol-to-HDL ratios, respectively (both P < 0.01). Prohormone also exhibited elevations in serum creatinine (19.6 ± 4.3%; P < 0.01) and aspartate transaminase (113.8 ± 61.1%; P = 0.05), as well as reductions in serum albumin (5.1 ± 1.9%; P = 0.04), alkaline phosphatase (16.4 ± 4.7%; P = 0.04), and glomerular filtration rate (18.0 ± 3.3%; P = 0.04). None of these values changed (all P > 0.05) in Placebo. The oral PS 3β-hydroxy-5α-androst-1-en-17-one improves body composition and muscular strength. However, these changes come at a significant cost. Cardiovascular health and liver function are particularly compromised. Given these findings, we feel the harm associated with this particular PS outweighs any potential benefit.

Prohormones and sport / Frans T. Delbeke, Peter Van Eenoo, Wim Van Thuyne, Noël Desmet

• Journal of Steroid Biochemistry and Molecular Biology 83 (2002) 1-5 (December), p. 245-251
• PMID: 12650722
• DOI: 10.1016/s0960-0760(02)00274-1

Abstract

Several precursors of testosterone and nandrolone introduced on the nutritional supplement market as performance enhancing drugs are banned in sports. Until now they are legally sold without a prescription in the US. Results of excretion studies with related compounds including 7-keto-DHEA and 1-androstenediol are presented. The main metabolites of 7-keto-DHEA are 7-hydroxylated compounds. The commercial 1-androstenediol preparation was contaminated with several other anabolic steroids. Oxidation of 1-androstenediol to 1-androstenedione seems to be the major renal metabolic pathway. Additionally contaminated nutritional supplements containing banned substances not indicated on the label were administered. The results of the excretion studies indicate that after the intake of amounts substantially lower than the recommended dose athletes can fail a doping test for periods up to 120 h.

Prolonged hypogonadism in males following withdrawal from anabolic-androgenic steroids: an under-recognized problem / Gen Kanayama, James I. Hudson, James DeLuca, Stephanie Isaacs, Aaron Baggish, Rory Weiner, Shalender Bhasin, Harrison G. Pope Jr. . - (Addiction 110 (2015) 5 (May); p. 823-831).

• PMID: 25598171.
• PMCID:PMC4398624.
• DOI:10.1111/add.12850

Abstract

AIMS:
To assess the frequency and severity of hypogonadal symptoms in male long-term anabolic-androgenic steroid (AAS) misusers who have discontinued AAS use.

DESIGN:
Cross-sectional, naturalistic.

SETTING:
Out-patient facility.

PARTICIPANTS:
Twenty-four male former long-term AAS users and 36 non-AAS-using weightlifters, recruited by advertisement in Massachusetts, USA. Five of the former users were currently receiving treatment with physiological testosterone replacement, leaving 19 untreated users for the numerical comparisons below.

MEASUREMENTS:
The Structured Clinical Interview for DSM-IV, questions regarding history of AAS use, physical examination, serum hormone determinations and the International Index of Erectile Function (IIEF).

FINDINGS:
Compared with the 36 non-AAS-using weightlifters, the 19 untreated former AAS users displayed significantly smaller testicular volumes [estimated difference, 95% confidence interval (CI) = 2.3 (0.1, 4.5) ml; P = 0.042] and lower serum testosterone levels [estimated difference: 95% CI = 131 (25, 227) dl; P = 0.009], with five users showing testosterone levels below 200 ng/dl despite abstinence from AAS for 3-26 months. Untreated former users also displayed significantly lower scores on the IIEF sexual desire subscale [estimated difference: 95% CI = 2.4 (1.3, 3.4) points on a 10-point scale; P < 0.001]. In the overall group of 24 treated plus untreated former users, seven (29%) had experienced major depressive episodes during AAS withdrawal; four of these had not experienced major depressive episodes at any other time. Two men (8%) had failed to regain normal libidinal or erectile function despite adequate replacement testosterone treatment.

CONCLUSIONS:
Among long-term anabolic-androgenic steroid misusers, anabolic-androgenic steroid-withdrawal hypogonadism appears to be common, frequently prolonged and associated with substantial morbidity.

Prolonged occurrence of cocaine in human saliva and urine after chronic use / Edward J. Cone, William W. Weddington, Jr.

• Journal of Analytical Toxicology 13 (1989) 2 (March-April), p. 65-68
• PMID: 2733393
• DOI: 10.1093/jat/13.2.65

Abstract

Cocaine was detected by immunoassay in saliva and urine of chronic cocaine addicts for 5-10 days during abstinence. Confirmation by a less sensitive but highly specific GC/MS assay of unmetabolized cocaine was successful in saliva through the first 24 h of collection and for the initial 4-5 days in urine. Cocaine saliva concentrations and subject scores for cocaine craving and depression declined during this time and correlated significantly. The presence of unmetabolized cocaine in these biofluids long after the last drug administration suggests that multiple dosing and high exposure to cocaine in man leads to accumulation in deep body compartments and subsequent slow release back into circulation and eventual excretion. The prolonged presence of cocaine in saliva and urine may have implications in testing for cocaine use and in treatment of cocaine withdrawal.

Prolonged stimulation of growth hormone (GH) and insulin-like growth factor I secretion by CJC-1295, a long-acting analog of GH-releasing hormone, in healthy adults / Sam L. Teichman, Ann Neale, Betty Lawrence, Catherine Gagnon, Jean-Paul Castaigne, Lawrence A. Frohman. - (The Journal of Clinical Endocrinology & Metabolism 91 (2006) 3 (1 March); p. 799-805) 

• PMID: 16352683
• DOI: 10.1210/jc.2005-1536

Abstract

Context: Therapeutic use of GHRH to enhance GH secretion is limited by its short duration of action.

Objective: The objective of this study was to examine the pharmacokinetic profile, pharmacodynamic effects, and safety of CJC-1295, a long-acting GHRH analog.

Design: The study design was two randomized, placebo-controlled, double-blind, ascending dose trials with durations of 28 and 49 d.

Setting: The study was performed at two investigational sites.

Participants: Healthy subjects, ages 21-61 yr, were studied.

Interventions: CJC-1295 or placebo was administered sc in one of four ascending single doses in the first study and in two or three weekly or biweekly doses in the second study.

Main outcome measures: The main outcome measures were peak concentrations and area under the curve of GH and IGF-I; standard pharmacokinetic parameters were used for CJC-1295.

Results: After a single injection of CJC-1295, there were dose-dependent increases in mean plasma GH concentrations by 2- to 10-fold for 6 d or more and in mean plasma IGF-I concentrations by 1.5- to 3-fold for 9-11 d. The estimated half-life of CJC-1295 was 5.8-8.1 d. After multiple CJC-1295 doses, mean IGF-I levels remained above baseline for up to 28 d. No serious adverse reactions were reported.

Conclusions: Subcutaneous administration of CJC-1295 resulted in sustained, dose-dependent increases in GH and IGF-I levels in healthy adults and was safe and relatively well tolerated, particularly at doses of 30 or 60 microg/kg. There was evidence of a cumulative effect after multiple doses. These data support the potential utility of CJC-1295 as a therapeutic agent.

Promoting functional foods as acceptable alternatives to doping : potential for information-based social marketing approach / Ricky James, Declan P. Naughton, Andrea Petróczi. - (Journal of the International Society of Sports Nutrition 7 (2010) 37 (10 November); p. 1-11)

• PMID: 21067611
• PMCID: PMC2994790
• DOI: 10.1186/1550-2783-7-37

Abstract

Background: Substances with performance enhancing properties appear on a continuum, ranging from prohibited performance enhancing drugs (PED) through dietary supplements to functional foods (FF). Anti-doping messages designed to dissuade athletes from using PEDs have been typically based on moralising sport competition and/or employing scare campaigns with focus on the negative consequences. Campaigns offering comparable and acceptable alternatives are nonexistent, nor are athletes helped in finding these for themselves. It is timely that social marketing strategies for anti-doping prevention and intervention incorporate media messages that complement the existing approaches by promoting comparable and acceptable alternatives to doping. To facilitate this process, the aim of this study was to ascertain whether a single exposure knowledge-based information intervention led to increased knowledge and subsequently result in changes in beliefs and automatic associations regarding performance enhancements.

Methods: In a repeated measure design, 115 male recreational gym users were recruited and provided with a brief information pamphlet on nitrite/nitrate and erythropoietin as a comparison. Measures of knowledge, beliefs and automatic associations were taken before and after the intervention with at least 24 hours between the two assessments. The psychological tests included explicit measures of beliefs and cognitive attitudes toward FF and PED using a self-reported questionnaire and computerised assessments of automatic associations using the modified and shortened version of the Implicit Association Test.

Results: The information based intervention significantly increased knowledge (p < 0.001), changed explicit beliefs in specific FF (p < 0.001) and shifted the automatic association of FF with health to performance (p < 0.001). Explicitly expressed beliefs and automatic associations appear to be independent.

Conclusion: Evidence was found that even a single exposure to a persuasive positive message can lead to belief change and can create new or alter existing associations - but only in the specific domain. Interventions to change outcome expectations in a positive way could be a rewarding avenue for anti-doping. Effective social marketing campaigns for drug free sport should follow appropriate market segmentation and use targeted messages via promoting the natural form as opposed to the purified form of the main active ingredient.

Proof of Gene Doping in a Mouse Model with a Human Erythropoietin Gene Transferred Using an Adenoviral Vector / Takehito Sugasawa, Takuro Nakano, Shin-Ichiro Fujita, Yuki Matsumoto, Genki Ishihara, Kai Aoki, Koki Yanazawa, Seiko Ono, Shinsuke Tamai, Lev Manevich, Haruna Ueda, Noriyo Ishibashi, Kenshirou Tamai, Yasuharu Kanki, Yasuko Yoshida, Koichi Watanabe, Tohru Takemasa, Yasushi Kawakami, Kazuhiro Takekoshi. - (Genes 12 (2021) 8 (16 August); 1249)

• PMID: 34440425
• PMCID: PMC8392868
• DOI: 10.3390/genes12081249

Abstract

Despite the World Anti-Doping Agency (WADA) ban on gene doping in the context of advancements in gene therapy, the risk of EPO gene-based doping among athletes is still present. To address this and similar risks, gene-doping tests are being developed in doping control laboratories worldwide. In this regard, the present study was performed with two objectives: to develop a robust gene-doping mouse model with the human EPO gene (hEPO) transferred using recombinant adenovirus (rAdV) as a vector and to develop a detection method to identify gene doping by using this model. The rAdV including the hEPO gene was injected intravenously to transfer the gene to the liver. After injection, the mice showed significantly increased whole-blood red blood cell counts and increased expression of hematopoietic marker genes in the spleen, indicating successful development of the gene-doping model. Next, direct and potentially indirect proof of gene doping were evaluated in whole-blood DNA and RNA by using a quantitative PCR assay and RNA sequencing. Proof of doping could be detected in DNA and RNA samples from one drop of whole blood for approximately a month; furthermore, the overall RNA expression profiles showed significant changes, allowing advanced detection of hEPO gene doping.

Proportionality and the application of the World Anti-Doping Code / Herman Ram. – (International Sports Law Journal (2012) 3-4 : p. 8-11)

Content:
1.) Introduction
2.) The Athlete’s Position
2.1.) Legal Counsel
2.2.) Confidents and Activists
2.3.) Disciplinary Panels
3.) The NADO’s Position
3.1.) The Position of NADO’s in the Code
3.2.) The Position of the Dutch NADO
4.) The Approach of the Dutch Doping Authority
4.1.) Pre-Hearing Information
4.2.) Legal Opinions
4.3.) Right to Appeal
4.4.) Impartial Advice
5.) Conclusions and Discussion

the present World Anti-Doping Code (the Code) offers relevant possibilities for individual assessment of doping cases. This is not always recognized by the general public and, more importantly, it is not always understood by legal counsel, which acts on behalf of the athlete in doping cases. In other words, there is limited but relevant room for flexibility and proportionality proportionality within the Code, but this room is not always used as it could and should be.

In this article, the Author will try to shed some light on the positions of athletes (paragraph 2) and Anti-Doping Organizations (paragraph 3), in relation to the proportionality issue introduced above. In the fourth paragraph, he will describe the approach of the Dutch Doping Authority, and the fifth and last paragraph provides some conclusions and input for further discussion.