Novel assay for determination of androgen bioactivity in human serum.
We have developed a mammalian cell (COS-1) bioassay, which can measure androgen bioactivity directly from a small amount (10 microL) of human serum. The recombinant assay is based on androgen-dependent interaction between the ligand-binding domain and the N-terminal region of the androgen receptor (AR), which were fused to Gal4 DNA-binding domain of Saccharomyces cerevisiae and transcriptional activation domain of herpes simplex VP16 protein, respectively. The interaction is amplified by coexpression of AR-interacting protein 3 in the cells. The reporter plasmid contains 5 Gal4-binding sites upstream of the luciferase gene; luciferase activity in cell lysates is derived from androgen bioactivity in human serum. Saturating concentration of testosterone in FCS induced more than 700-fold induction in relative luciferase activity. The sensitivity was less than 1.0 nmol/L testosterone in FCS. The intra- and interassay coefficients of variation were 8.3% and 21%, respectively. Interaction between the AR termini was blocked by nonsteroidal antiandrogens, and the assay exhibited minimal cross-reactivity with 17 beta-estradiol. Serum androgen bioactivity was studied in 23 boys (13.9--16.8 yr old) with constitutional delay of puberty and in 9 prepubertal boys with cryptorchidism (1.0--6.4 yr old). Androgen bioactivity was detectable in 15 boys with constitutional delay of puberty and in all boys with cryptorchidism during treatment with human CG (range, 1.0-14.5 nmol/L testosterone equivalents). Serum androgen bioactivity measured by the bioassay correlated strongly with serum testosterone concentration (r = 0.93, P < 0.0001, n = 22) but not to 5 alpha-dihydrotestosterone, dehydroepiandrosterone, or androstenedione levels. We conclude that our novel bioassay enables quantitation of mammalian cell response to bioactive androgens in human serum, even in pediatric patients with relatively low androgen levels.
Raivio
T
T
Biomedicum Helsinki, Department of Physiology, Institute of Biomedicine, University of Helsinki, FIN-00014 Helsinki, Finland. taneli.raivio@helsinki.fi
Palvimo
J J
JJ
Dunkel
L
L
Wickman
S
S
Jänne
O A
OA
D000293:Adolescent; D000726:Androgen Antagonists / Q000494:pharmacology; D000728:Androgens / Q000097:blood* / Q000502:physiology; D000818:Animals; D001681:Biological Assay / Q000379:methods*; D019556:COS Cells; D002648:Child; D002675:Child, Preschool; D003456:Cryptorchidism / Q000097:blood; D004967:Estrogens / Q000502:physiology; D004986:Ether; D012739:Gonadal Steroid Hormones / Q000494:pharmacology; D006801:Humans; D007223:Infant; D008297:Male; D012680:Sensitivity and Specificity; D013739:Testosterone / Q000494:pharmacology
D000726:Androgen Antagonists; D000728:Androgens; D004967:Estrogens; D012739:Gonadal Steroid Hormones; D004986:Ether; D013739:Testosterone