The enzyme 3β-hydroxysteroid dehydrogenase-1 (3βHSD1), encoded by the gene HSD3B1, plays an essential role in the peripheral conversion of 3β-OH, Δ5-steroids to 3-keto, Δ4-steroids. In human physiology, the adrenal produces dehydroepiandrosterone (DHEA) and DHEA-sulfate, which are major precursors for the biosynthesis of potent androgens and estrogens. DHEA is converted by 3βHSD1 and subsequently is converted by steroid-5α-reductase to potent androgens or by aromatase to estrogens. Assessment of 3βHSD1 is therefore critical under various conditions. In this chapter, we detail several approaches to assessing 3βHSD1. First, we describe a genotyping protocol for the identification of a common missense-encoding variation that regulates 3βHSD1 cellular metabolic activity. This protocol distinguishes between the HSD3B1(1245A) and the HSD3B1(1245C) allele which have lower and higher metabolic activity, respectively. Second, we detail mass spectrometry approaches to determining 3βHSD1 activity using stable isotope dilution. Third, we describe methods for using tritiated DHEA and high performance liquid chromatography coupled with a beta-RAM to also determine 3βHSD1 activity. Together, we provide multiple methods of directly assessing 3βHSD1 activity or anticipated 3βHSD1 activity.
Keywords: Androgen; Genotype; HPLC; HSD3B1; LC–MS; Metabolism; Prostate cancer.
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