Center for Excellence in Environmental Toxicology, Department of Systems Pharmacology and Translational Therapeutics, University of Pennsylvania, Philadelphia, PA, United States.
Methods Enzymol. 2023;689:377-385. doi: 10.1016/bs.mie.2023.04.010. Epub 2023 May 5.
Kinetic assays with recombinant enzymes are critical to determine the steady state kinetic parameters for androgen conversion to understand their role in androgen biosynthesis and metabolism. Detection and quantification of 5α-reduced androgens remain difficult to assay because they are UV-transparent compounds. Therefore, radioactive isotopic versions of these compounds are often required to conduct steady-state kinetics. Here we developed a derivatization protocol with dinitrophenylhydrazine (DNPH) to form hydrazones on the ketones of androgens enabling them to be detected by UV-reverse phase high performance liquid chromatography (RP-HPLC). We determined the kinetic parameters for the conversion of 5α-androstane-3,17-dione (5AD) to 5α-dihydrotestosterone (DHT), 11-keto-5α-androstane-3,17-dione (11K-5AD) to 11-keto-5α-dihydrotestosterone (11K-DHT), and 11β-hydroxy-5α-androstane-3,17-dione (11β-OH-5AD) to 11β-hydroxy-5α-dihydrotestosterone (11β-OH-DHT) catalyzed by recombinant aldo-keto reductase 1C3 (AKR1C3) as measured by product formation post DNPH derivatization.
用重组酶进行动力学分析对于确定雄激素向 5α-还原产物转化的稳态动力学参数至关重要，这有助于了解其在雄激素生物合成和代谢中的作用。由于 5α-还原产物是紫外透明化合物，因此检测和定量它们仍然很困难。因此，通常需要这些化合物的放射性同位素版本来进行稳态动力学分析。在这里，我们开发了一种用二硝基苯肼（DNPH）进行衍生化的方案，使雄激素的酮基形成腙，从而能够通过紫外反相高效液相色谱（RP-HPLC）进行检测。我们测定了 5α-雄烷-3,17-二酮（5AD）转化为 5α-二氢睾酮（DHT）、11-酮-5α-雄烷-3,17-二酮（11K-5AD）转化为 11-酮-5α-二氢睾酮（11K-DHT）以及 11β-羟基-5α-雄烷-3,17-二酮（11β-OH-5AD）转化为 11β-羟基-5α-二氢睾酮（11β-OH-DHT）的动力学参数，这些反应是由重组醛酮还原酶 1C3（AKR1C3）催化的，产物形成后通过 DNPH 衍生化进行测量。
