Hungerford Natasha L, Sortais Benoît, Smart Corrine G, McKinney Andrew R, Ridley Damon D, Stenhouse Allen M, Suann Craig J, Munn Kellie J, Sillence Martin N, McLeod Malcolm D
School of Chemistry, F11, The University of Sydney, NSW 2006, Australia.
J Steroid Biochem Mol Biol. 2005 Aug;96(3-4):317-34. doi: 10.1016/j.jsbmb.2005.03.007.
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.
由于在赛马运动中广泛使用合成代谢类固醇存在被滥用的可能性，通过一类常见代谢物来检测多种化合物的筛查测试将是一种有价值的法医工具。已开发出一种酶联免疫吸附测定法（ELISA）来检测马尿中的17α-烷基合成代谢类固醇代谢物。16β-羟基美睾酮（16β,17β-二羟基-17α-甲基-5α-雄甾烷-3-酮）由市售表雄酮（3β-羟基-5α-雄甾烷-17-酮）分六步合成。以美睾酮（17β-羟基-17α-甲基-5α-雄甾烷-3-酮）或通过3-羧甲基肟接头与人血清白蛋白偶联的16β-羟基美睾酮作为抗原，在绵羊体内产生多克隆抗体。通过评估54种代表性类固醇文库竞争性结合抗体的能力来确定抗体交叉反应性。针对16β-羟基美睾酮产生的抗体对所有分析的16β,17β-二羟基-17α-甲基类固醇均表现出优异的交叉反应性，并且已开发出一种ELISA来检测这些类固醇代谢物。使用这种16β-羟基美睾酮测定法，对用司坦唑醇（17α-甲基-吡唑并[4',3':2,3]-5α-雄甾烷-17β-醇）处理过的马的尿液样本进行了分析，分析了原始样本、β-葡萄糖醛酸酶水解后的样本以及固相萃取（SPE）程序后的样本。观察到的吸光度抑制与代谢物16β-羟基司坦唑醇的检测结果一致。通过与标准液相色谱-质谱分析进行比较，确认了阳性筛查结果。针对美睾酮产生的抗体也用于开发ELISA，该ELISA用于检测在给予甲二氢睾酮（17α-甲基雄甾-5-烯-3β,17β-二醇）后保留母体D环结构的代谢物。所开发的ELISA方法可作为检测新的和已知合成代谢类固醇代谢物的主要筛查工具。
