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Novel Assay for Determination of Androgen Bioactivity in Human Serum¹

Biomedicum Helsinki, Department of Physiology (T.R., J.J.P., O.A.J.), Institute of Biomedicine, University of Helsinki, FIN-00014 Helsinki; Hospital for Children and Adolescents (L.D., S.W.), University of Helsinki, FIN-00029 Huch; Institute of Biotechnology (J.J.P.), University of Helsinki, FIN-00014 Helsinki; and Department of Clinical Chemistry (O.A.J.), University of Helsinki, FIN-00014 Helsinki, Finland

Address all correspondence and requests for reprints to: Taneli Raivio, M.D., Ph.D., Biomedicum Helsinki, Department of Physiology, Institute of Biomedicine, University of Helsinki, P. O. Box 63 (Siltavuorenpenger 20 J), FIN-00014 Helsinki, Finland. taneli. raivio{at}helsinki.fi

We have developed a mammalian cell (COS-1) bioassay, which can measure androgen bioactivity directly from a small amount (10 µL) of human serum. The recombinant assay is based on androgendependent 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 17ß-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-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.

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