β glucuronidase

High-performance liquid chromatography (HPLC) was performed as previously described (36). Briefly, cells were seeded and incubated in 24-well plate for ∼24 h and then treated with the indicated drugs and [³H]-labeled steroids (∼1,000,000 cpm/well; PerkinElmer, Waltham, MA) at 37 °C. Aliquots of medium were collected and treated with β-glucuronidase (Novoprotein Scientific Inc, China) at 37 °C for 2h, extracted with ethyl acetate: isooctane (1:1), and dried in a freeze dryer (Martin Christ Gefriertrocknungsanlagen, Germany). Dried samples were reconstituted in 100 μL of 50% methanol and injected into the HPLC. Metabolites were separated on CORTECS C18 reverse-phase column (Waters, Ireland), using a methanol/water gradient at 40 °C. The column effluent was analyzed using β-RAM model 3 in-line radioactivity detector (LABLOGIC, USA). Results showed the mean and standard deviation (sd) value from one representative experiment. All HPLC studies were run in duplicate and repeated at least three times in independent experiments.

2-3 mg biopsy samples were minced and cultured with DMEM (Invitrogen, Waltham, MA), 10% FBS (ExCell Bio, China), and Penicillin-Streptomycin (100 x; Invitrogen, Waltham, MA, USA) in a 12-well plate at 37°C as previously described.³⁵ (link) Biopsy samples were treated with [³H] labelled pregnenolone (100, 000–200,000 cpm; final concentration was 48 nM) (PerkinElmer, Waltham, MA, USA). 250 μl medium was collected at 84 h for HPLC analysis. Then, samples were treated with β-glucuronidase (Novoprotein Scientific Inc., Shanghai, China) at 37°C for 2 h. Steroids were extracted with a mixture of ethyl acetate and isooctane (1:1), concentrated with a vacuum drier (Martin Christ Gefriertrocknungsanlagen, Osterode, Germany), and resuspended with a mixture of methanol and water (1:1). An Acquity Arc System (Waters, Milford, MA, USA) and a β-RAM model 5 in-line radioactivity detector (LabLogic Systems) were used to analyze metabolites in samples. A mixture of [³H] labelled androgens (AD, DHEA, Prog, Preg PerkinElmer, Waltham, MA, USA) was used as the standard to distinguish metabolites. The percentages of metabolites were calculated based on the area under curve (AUC) for each metabolite. For example, Preg % = (AUC of Preg)/ (AUC of Preg + AUC of all Preg metabolites) x 100 %.

Steroid metabolism and HPLC were performed as described previously.³⁵ (link) Briefly, cells or organoids were treated with [³H]-labeled steroids (pregnenolone, DHEA, AD; 100,000-200,000 cpm per well; PerkinElmer, Waltham, MA). Aliquots of the medium were treated with β-glucuronidase (Novoprotein Scientific Inc., Shanghai, China) and extracted using a mixture of ethyl acetate and isooctane (1:1). Steroids were analyzed using an Acquity Arc System (Waters, Milford, MA, USA) and a β-RAM model 3 in-line radioactivity detector (LABLOGIC, USA). The percentages of metabolites were calculated based on the area under curve (AUC) for each metabolite. For example, pregnenolone% = (AUC of pregnenolone) / (AUC of pregnenolone + AUC of all metabolites) x 100 %.