Detection and quantification of UV-transparent keto-androgens by dinitrophenylhydrazine derivatization for discontinuous kinetic assays

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.