title: [Establishment of an in vitro screening model for steroid 5 alpha-reductase inhibitors with the microplate reader].
abstract: OBJECTIVE
  To establish an in vitro screening model for steroid 5 alpha-reductase inhibitors using the microplate reader.
  
  
  METHODS
  Steroid 5 alpha-reductase was obtained from the liver of female rats, an in vitro screening model for steroid 5 alpha-reductase inhibitors established using the 96-well plate and microplate reader after determination of the enzymatic activity, and the reliability of the model verified with the known 5 alpha-reductase inhibitors epristeride and finasteride. Added to the 96-well plate were the final concentrations of testosterone (0-40 micromol/L), NADPH (22 micromol/L), epristeride (0-60 nmol/L) or finasteride (0-60 nmol/ L) and steroid 5 alpha-reductase (20 microl), the total volume of each well adjusted to 200 microl with Tris-Hcl buffer. The 96-well plate was placed in the microplate reader, mixed and incubated at 37 degrees C, followed by detection of the A340nm value at 0 and 10 min and analysis of the data.
  
  
  RESULTS
  The Km value of steroid 5 alpha-reductase was 3.794 micromol/L, with a Vmax of 0.271 micromol/(L. min). The Ki of epristeride was 148.2 nmol/L, with an IC50 of 31.5 nmol/L, and the enzymatic reaction kinetic curve suggested that epristeride was an uncompetitive enzyme inhibitor. The Ki of finasteride was 158. 8 nmol/L, with an IC50 of 13.6 nmol/L. The enzymatic reaction kinetic curve showed that both epristeride and finasteride were competitive enzyme inhibitors, similar to those reported in the published literature.
  
  
  CONCLUSIONS
  A screening model was successfully established, which could rapidly and effectively screen steroid 5 alpha-reductase inhibitors in vitro.
authors.affiliation: National Evaluation Center for the Toxicology of Fertility Regulating Drugs, Shanghai Institute of Planned Parenthood Research, Shanghai 200032, China. wujh_731@163.com
authors.forename: Jian-Hui
authors.initials: JH
authors.lastname: Wu
authors.forename: Zu-Yue
authors.initials: ZY
authors.lastname: Sun
mesh_terms: D058891:5-alpha Reductase Inhibitors / Q000032:analysis*; D000818:Animals; D004353:Drug Evaluation, Preclinical / Q000379:methods*; D005260:Female; D057166:High-Throughput Screening Assays / Q000295:instrumentation / Q000379:methods*; D007124:Immunoenzyme Techniques; D051381:Rats; D017207:Rats, Sprague-Dawley
chemical_list: D058891:5-alpha Reductase Inhibitors