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DOI: 10.1055/s-2005-865824
© Georg Thieme Verlag KG Stuttgart · New York
Rapid Detection of Anabolic Steroids in Urine by Protein Arrays[*]
Publication History
Accepted after revision: May 30, 2005
Publication Date:
30 August 2005 (online)
![](https://www.thieme-connect.de/media/sportsmed/200607/lookinside/thumbnails/10.1055-s-2005-865824-1.jpg)
Abstract
The purpose of this study was to develop a rapid and sensitive method utilizing the state-of-the-art protein arrays technique to detect urinary anabolic steroids (ASs) in athletes. Three experiments were designed to investigate the feasibility of the protein arrays for ASs testing. Firstly, androgen receptor (AR) and estrogen receptor (ER) protein arrays were prepared on polysaccharide-coated slides to investigate whether they can bind to ASs (affinity tests). Secondly, in comparison to adrenergic receptor (the receptor of β-blockers) and opioid receptor (the receptor of narcotic analgesics) arrays, AR and ER protein arrays were used to test whether they can determine the ASs positive urine sample specifically (specific binding tests). At last protein arrays were used to estimate qualitatively the ASs in positive urine samples (qualitative tests). From the results of the affinity tests the shape of the dose-dependence curve suggested a positive cooperative binding of ASs with the protein arrays. The AR and ER protein arrays showed affinities for fluorescence labelled testosterone and estradiol that were similar to those of literatures (0.65 vs. 0.89 nM, 5.96 vs. 10.3 nM, respectively). Based on the data, the sensitivity of testing can reach 0.1 nM that was much better than the World Anti-Doping Code (WADA) standard. Specific binding tests showed that the prohibited substance in positive urine samples belonged to the anabolic estrogenic inhibitor of ASs. From the results of qualitative tests, we could estimate that there were anabolic androgenic steroids in the positive urine samples and their concentration was lower than 50 µM Methyltestosterone. The total time of the test process for ASs in urine needed less than 1 h. In summary, the present study showed that the protein arrays method provided a highly sensitive and rapid alternative to screen urine samples for the detection of the misuse of ASs in athletes and was suitable for testing in both weekly training sessions as well as large-scale competition events.
Key words
Anabolic steroids abuse - urine samples - binding assays - rapid testing
1 The work was undertaken in Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, 1 Xian Nong Tan Street, Beijing 100050, China.
References
- 1 Afanassiev V, Hanemann V, Wolfl S. Preparation of DNA and protein micro arrays on glass slides coated with an agarose film. Nucleic Acids Res. 2000; 28 66
- 2 Benters R, Niemeyer C M, Wohrle D. Dendrimer-activated solid supports for nucleic acid and protein microarrays. Chem Bio Chem. 2001; 2 686-694
- 3 Fang Y, Frutos A G, Lahiri J. Membrane protein microarrays. Am Chem Soc. 2002; 124 2394-2395
- 4 Jenster G, Spencer T E, Burcin M M, Tsai S Y, Tsai M J, O'Malley B W. Steroid receptor induction of gene transcription: a two-step model. Proc Natl Acad Sci USA. 1997; 94 7879-7884
- 5 Karpiesiuk W, Lehner A F, Hughes C G, Tobin T. Preparation and chromatographic characterization of tetrahydrogestrinone, a new “designer” anabolic steroid. Chromatographia. 2004; 60 359-363
- 6 Kenakin T. Principles: receptor theory in pharmacology. Trends Pharmacol Sci. 2004; 25 186-192
- 7 MacBeath G, Schreiber S L. Printing proteins as microarrays for high-throughput function determination. Science. 2000; 289 1760-1763
- 8 Moras D, Gronemeyer H. The nuclear receptor ligand-binding domain: structure and function. Curr Opin Cell Biol. 1998; 10 384-391
- 9 Ohno K, Fukushima T, Santa T, Waizumi N, Tokuyama H, Maeda M, Imai K. Estrogen receptor binding assay method for endocrine disruptors using fluorescence polarization. Anal Chem. 2002; 74 4391-4396
- 10 Renault J P, Bernard A, Juncker D, Michel B, Bosshard H R, Delamarche E. Fabricating microarrays of functional proteins using affinity contact printing. Angew Chem Engl Ed. 2002; 41 2320-2323
- 11 Rich R L, Hoth L R, Geoghegan K F, Brown T A, LeMotte P K, Simons S P, Hensley P, Myszka D G. Kinetic analysis of estrogen receptor ligand interactions. PNAS. 2002; 99 8562-8567
- 12 Roehrborn C G, Zoppi S, Gruber J A, Wilson C M, McPhaul M J. Expression and characterization of full-length and partial human androgen receptor fusion proteins. Implications for the production and applications of soluble steroid receptors in Escherichia coli. Mol Cell Endocrinol. 1992; 84 1-14
- 13 Tsuchihashi H, Yokoyama H, Nagatomo T. Binding characteristics of 3H - CGP12177 to beta- adrenoceptors in rat myocardial membranes. Jpn J Pharmacol. 1989; 49 11-19
- 14 Van Eenoo P, Delbeke F T. The prevalence of doping in Flanders in comparison to the prevalence of doping in international sports. Int J Sports Med. 2003; 24 565-570
- 15 Ward R J, Shackleton C H, Lawson A M. Gas chromatographic - mass spectrometric methods for the detection and identification of anabolic steroid drugs. Br J Sports Med. 1975; 9 93-97
- 16 Yamamoto K R. Steroid receptor regulated transcription of specific genes and gene networks. Annu Rev Genet. 1985; 19 209-252
- 17 Zastawny R L, George S R, Nguyen T, Cheng R, Tsatsos J, Briones-Urbina R, O'Dowd B F. Cloning, characterization, and distribution of a mu-opioid receptor in rat brain. J Neurochem. 1994; 62 2099-2105
- 18 Zhu H, Snyder M. Protein arrays and microarrays. Curr Opin Chem Biol. 2001; 5 40-45
1 The work was undertaken in Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, 1 Xian Nong Tan Street, Beijing 100050, China.
Guan-hua Du
Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College
1 Xian Nong Tan Street
Beijing 100050
China
Phone: + 8601063165184
Fax: + 86 0 10 63 01 77 57
Email: dugh@imm.ac.cn