title: Development of a new gas chromatography-mass spectrometry (GC-MS) methodology for the evaluation of 5α-reductase activity.
abstract: The androgens testosterone (T) and dihydrotestosterone (DHT) play a key role in the function and integrity of prostate tissue, but are also implicated in prostate cancer and benign prostatic hyperplasia (BPH). The reduction of androgen levels can be achieved by the inhibition of 3-oxo-5α-steroid-4-dehydrogenase (5α-reductase), which is responsible for the irreversible conversion of T into its more active metabolite DHT. In fact, the use of 5α-reductase inhibitors (RIs), like finasteride, can be a valuable strategy for the treatment of BPH and in chemoprevention of prostate tumors. In this work a new method based on a dispersive liquid-liquid microextraction (DLLME) procedure, followed by gas chromatography-mass spectrometry (GC-MS), to evaluate the 5α-reductase activity, by measuring the conversion percentage of T into DHT was optimized and validated. Enzymatic assays were carried out in human prostate microsomes, using T as substrate. T and DHT were extracted by the developed DLLME technique and quantified, after silylation, by GC-MS. Variables affecting the extraction efficiency and derivatization of T and DHT were evaluated. The optimized method showed good linearity (with correlation coefficients over 0.9994 for T and 0.9995 for DHT), good recoveries (higher than 80%), and good intra- and inter-day precision (below 13%, 3 levels, n=6). The detection limits for T and DHT were 0.5 nM and the limits of quantification were 5 nM. The new GC-MS method is a good alternative to the already described methods, to evaluate 5α-reductase activity, since it avoids the use of radioactive compounds and corresponds to a fast and sensitive methodology with a good extraction efficiency, accuracy and high recovery. As this method allows the evaluation of 5α-reductase activity, also permits the study of inhibitory efficacy of new molecules as potential RIs.
authors.affiliation: Laboratory of Biochemistry, Department of Biological Sciences, Faculty of Pharmacy, University of Porto, Rua Jorge Viterbo Ferreira, No. 228, 4050-313 Porto, Portugal.
authors.forename: Cristina
authors.initials: C
authors.lastname: Amaral
authors.forename: Sara C
authors.initials: SC
authors.lastname: Cunha
authors.forename: José O
authors.initials: JO
authors.lastname: Fernandes
authors.forename: Elisiário
authors.initials: E
authors.lastname: Tavares da Silva
authors.forename: Fernanda M F
authors.initials: FM
authors.lastname: Roleira
authors.forename: Natércia
authors.initials: N
authors.lastname: Teixeira
authors.forename: Georgina
authors.initials: G
authors.lastname: Correia-da-Silva
mesh_terms: D042944:Cholestenone 5 alpha-Reductase / Q000378:metabolism*; D013196:Dihydrotestosterone / Q000032:analysis* / Q000378:metabolism; D057075:Enzyme Assays / Q000379:methods; D008401:Gas Chromatography-Mass Spectrometry / Q000379:methods*; D006801:Humans; D057230:Limit of Detection; D059627:Liquid Phase Microextraction / Q000379:methods; D008297:Male; D008861:Microsomes / Q000201:enzymology; D011467:Prostate / Q000201:enzymology*; D013739:Testosterone / Q000032:analysis* / Q000378:metabolism
chemical_list: D013196:Dihydrotestosterone; D013739:Testosterone; D042944:Cholestenone 5 alpha-Reductase