Testosterone/dihydrotestosterone Kit Method for Diagnostic Use Reference Methodology Involving Isotope Dilution/mass Spectrometry

During early fetal life of males, conversion of testosterone (T) to 5a-dihydrotestosterone (DHT) via the action of 5a-reductase in the external genital tissues is essential for complete sexual differentiation of male external genitalia. Measurement of low concentrations of DHT in serum of affected prepubertal males confirms the type of sexual disorder (1,2). Although commercial methodologies have been available for T determinations for many years, to our knowledge no similar diagnostic methods have been available for measuring DHT. Recently, Amersham (Oakville, Ontario) marketed a combination kit (cat. no. TRK.600) for measuring testosterone! dihydrotestosterone by radioimmunoassay, for research use only, and we have compared the performance of this kit with that of our own in-house T method in a clinical setting. In our in-house method, we extract serum samples (0.5 mL male, 1.0 mL female) twice with 3-mL portions of diethyl ether and evaporate the extract in plastic tubes under air. The residue is reconstituted in 1 mL of pH 7.4 phosphate buffer and radioimmunoassayed. The testosterone antiserum is from Diagnostics Biochem Inc. (London, Ontario), the testosterone standard from Bio Ria (Montreal, Quebec), and [1,2,6,7-3H(N)}testosterone from NEN (Lachine, Quebec). After incubation at 4#{176}C overnight, dextran-coated charcoal is added to the tubes, to separate the free and bound fractions. Results by this testosterone assay have compared well in our internal quality-control programs as well as external programs with other laboratories. In the Amershaxn kit method, following the manufacturer’s instructions, we extracted serum samples (0.5 mL male, 1.0 mL female) with two 3mL portions of d.iethyl ether and evaporated the extracts in glass tubes under air. The residue was reconstituted in 1.5 mL of buffer and a 1-mL aliquot was transferred to a glass extraction tube for further extraction of DHT, leaving the remainder for the T analysis. An oxidizing agent was added to the 1-mL aliquot, which destroyed T, leaving only DHT to be extracted with two 3-mL portions of diethyl ether. This extract was dried in glass under air, and the residue was reconstituted in 1 mL of buffer and analyzed for DHT. After incubating for 1 h at room temperature, the assay mixture was placed in an ice bath for 15 mm. Charcoal adsorbant was used to separate the free and bound fractions. In the Amersham kit [3Hltestosterone and antiserum specific for both T and DHT are used. Amersham also stated that DHT cross reacts with the antiserum by about 45% to 50%. Consequently, “total r’ is T plus approximately 45 to 50% DHT (3).