title: Invalidation of a commercially available human 5α-dihydrotestosterone immunoassay.
abstract: Enzyme immunoassays (EIA) are commonly utilized for the evaluation of androgens in biological fluids; however, careful consideration must be given to cross-reactivity with other endogenous sex-steroid hormones. Our purpose was to determine the validity of a commonly-utilized commercially-available dihydrotestosterone (DHT) EIA. Serum samples obtained from older hypogonadal men who participated in a 12-month randomized controlled trial evaluating the effects of testosterone-enanthate (125 mg/week) or vehicle in combination with finasteride (5mg/day) or placebo were assayed for DHT via EIA and using a validated gold-standard LC-MS/MS approach. Additionally, commercially-available (DHT-free) buffer containing graded testosterone doses was evaluated by DHT immunoassay. DHT concentrations measured via EIA were 79% to >1000% higher than values obtained by LC-MS/MS (p<0.05), with the largest differences (415-1128%) occuring in groups receiving finasteride. Both LC-MS/MS and EIA indicated that testosterone-enanthate increased serum DHT to a similar magnitude. In contrast, finasteride-induced reductions in DHT were detected by LC-MS/MS, but not EIA (p<0.05). No significant associations were present for DHT concentrations between measurement techniques. Cross-reactivity of testosterone with the immunoassay ranged from 18% to 99% and DHT concentrations measured by EIA were highly associated with the spiked testosterone concentrations in DHT-free buffer (r=0.885, p<0.001). In conclusion, we provide evidence invalidating a commonly-utilized commercially-available DHT immunoassay because significant cross-reactivity exists between testosterone and the EIA and because the changes in DHT observed via EIA were not associated with a validated gold-standard measurement technique. The cross-reactivity of testosterone is particularly concerning because testsoterone is present in 100-fold greater concentrations than is DHT within the circulation.
authors.affiliation: VA Medical Center, Gainesville, FL 32608, United States; Department of Applied Physiology & Kinesiology, University of Florida, Gainesville, FL 32611, United States. Electronic address: jfyarrow@ufl.edu.
authors.forename: Joshua F
authors.initials: JF
authors.lastname: Yarrow
authors.forename: Darren T
authors.initials: DT
authors.lastname: Beck
authors.forename: Christine F
authors.initials: CF
authors.lastname: Conover
authors.forename: Luke A
authors.initials: LA
authors.lastname: Beggs
authors.forename: Bruce A
authors.initials: BA
authors.lastname: Goldberger
authors.forename: Stephen E
authors.initials: SE
authors.lastname: Borst
mesh_terms: D013196:Dihydrotestosterone / Q000097:blood*; D004311:Double-Blind Method; D005189:False Positive Reactions; D018120:Finasteride / Q000008:administration & dosage; D006801:Humans; D007124:Immunoenzyme Techniques / Q000592:standards*; D008297:Male; D008875:Middle Aged; D016032:Randomized Controlled Trials as Topic; D012015:Reference Standards; D053719:Tandem Mass Spectrometry; D013739:Testosterone / Q000008:administration & dosage / Q000097:blood
chemical_list: D013196:Dihydrotestosterone; D013739:Testosterone; D018120:Finasteride
keywords: 5α-Reductase; Dihydrotestosterone; Finasteride; Testosterone