Analysis of anabolic steroids in the horse: development of a generic ELISA for the screening of 17alpha-alkyl anabolic steroid metabolites.
Abstract: Due to the potential for misuse of a wide range of anabolic steroids in horse racing, a screening test to detect multiple compounds, via a common class of metabolites, would be a valuable forensic tool. An enzyme-linked immunosorbent assay (ELISA) has been developed to detect 17alpha-alkyl anabolic steroid metabolites in equine urine. 16beta-Hydroxymestanolone (16beta,17beta-dihydroxy-17alpha-methyl-5alpha-androstan-3-one) was synthesised in six steps from commercially available epiandrosterone (3beta-hydroxy-5alpha-androstan-17-one). Polyclonal antibodies were raised in sheep, employing mestanolone (17beta-hydroxy-17alpha-methyl-5alpha-androstan-3-one) or 16beta-hydroxymestanolone conjugated to human serum albumin, via a 3-carboxymethyloxime linker, as antigens. Antibody cross-reactivities were determined by assessing the ability of a library of 54 representative steroids to competitively bind the antibodies. Antibodies raised against 16beta-hydroxymestanolone showed excellent cross-reactivities for all of the 16beta,17beta-dihydroxy-17alpha-methyl steroids analysed and an ELISA has been developed to detect these steroid metabolites. Using this 16beta-hydroxymestanolone assay, urine samples from horses administered with stanozolol (17alpha-methyl-pyrazolo[4',3':2,3]-5alpha-androstan-17beta-ol), were analysed raw, following beta-glucuronidase hydrolysis, and following solid-phase extraction (SPE) procedures. The suppressed absorbances observed were consistent with detection of the metabolite 16beta-hydroxystanozolol. Positive screening results were confirmed by comparison with standard LCMS analyses. Antibodies raised against mestanolone were also used to develop an ELISA and this was used to detect metabolites retaining the parent D-ring structure following methandriol (17alpha-methylandrost-5-ene-3beta,17beta-diol) administration. The ELISA methods developed have application as primary screening tools for detection of new and known anabolic steroid metabolites.
Publication Date: 2005-07-26 PubMed ID: 16040239DOI: 10.1016/j.jsbmb.2005.03.007Google Scholar: Lookup
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- Journal Article
- Research Support
- Non-U.S. Gov't
- Analytical Methods
- Antibodies
- Biochemistry
- Clinical Study
- Diagnostic Technique
- Enzyme-Linked Immunosorbent Assay (ELISA)
- Equine Health
- Forensic Science
- Horse Racing
- Immunology
- Laboratory Methods
- Metabolic Health
- Metabolites
- Pharmaceuticals
- Physiology
- Steroid Hormones
- Urine Analysis
- Veterinary Care
- Veterinary Medicine
Summary
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The research article focuses on developing a new screening test that can detect a variety of anabolic steroids in horse urine through the identification of a specific class of metabolites.
Research Methodology
To address the problem, the researchers developed and tested an enzyme-linked immunosorbent assay (ELISA) for the detection of 17alpha-alkyl anabolic steroid metabolites in horse urine.
- The process involved synthesizing 16beta-Hydroxymestanolone from a commercially available substance called epiandrosterone.
- They then produced polyclonal antibodies using specific antigens that were raised in sheep.
- To evaluate the cross-reactivity of the antibodies, the researchers tested their ability to bind with 54 representative steroids.
- With the generated antibodies, a new ELISA was defined to identify the 16beta,17beta-dihydroxy-17alpha-methyl steroid metabolites.
Applying the ELISA Test
The ELISA they developed was then used to analyze urine samples collected from horses treated with an anabolic steroid called stanozolol.
- The samples were analyzed in their raw form, post beta-glucuronidase hydrolysis, and after solid-phase extraction procedures.
- The resulting absorbances indicated the detection of a metabolite chemical known as 16beta-hydroxystanozolol.
- The positive screening results were further validated by comparing them with the standard LCMS analyses.
Further Application of Developed Antibodies
The antibodies that were generated against mestanolone were further used to develop a new ELISA to detect metabolites with parent D-ring structures.
- This secondary application was used to determine the presence of these metabolites after administration of methandriol.
Conclusion and Further Application
In conclusion, the ELISA methods that were developed provide valuable tools for screening both new and known anabolic steroid metabolites in horse urine. This has significant potential in enhancing the monitoring process in horse racing and tackling the misuse of anabolic steroids.
Cite This Article
APA
Hungerford NL, Sortais B, Smart CG, McKinney AR, Ridley DD, Stenhouse AM, Suann CJ, Munn KJ, Sillence MN, McLeod MD.
(2005).
Analysis of anabolic steroids in the horse: development of a generic ELISA for the screening of 17alpha-alkyl anabolic steroid metabolites.
J Steroid Biochem Mol Biol, 96(3-4), 317-334.
https://doi.org/10.1016/j.jsbmb.2005.03.007 Publication
Researcher Affiliations
- School of Chemistry, F11, The University of Sydney, NSW 2006, Australia.
MeSH Terms
- Anabolic Agents / administration & dosage
- Anabolic Agents / immunology
- Anabolic Agents / urine
- Androstanols / chemistry
- Androstanols / urine
- Animals
- Antibodies / immunology
- Cross Reactions
- Dihydrotestosterone / analogs & derivatives
- Dihydrotestosterone / immunology
- Enzyme-Linked Immunosorbent Assay
- Estrogenic Steroids, Alkylated / administration & dosage
- Estrogenic Steroids, Alkylated / immunology
- Estrogenic Steroids, Alkylated / urine
- Horses / urine
Citations
This article has been cited 2 times.- Iguchi K, Nagashima K, Mochizuki J, Yamamoto H, Unno K, Miyoshi N. Enokitake Mushroom and Its Active Component, Adenosine, Which Restores Testosterone Production in Impaired and Fatigued Mouse Models.. Nutrients 2023 Apr 29;15(9).
- El-Ansary A, Faddah LM. Nanoparticles as biochemical sensors.. Nanotechnol Sci Appl 2010 Sep 23;3:65-76.
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