Enzyme Immunoassay Analysis for Androgens in Polar Bear (Ursus maritimus) Urine Using Enzyme Hydrolysis

Abstract: Validation of androgen enzyme immunoassays (EIA) using purified antiserum with a high cross-reactivity for testosterone and 5α-dihydrotestosterone in polar bear urine (PBU) has previously been reported. However, the cross-reactivity of this antiserum with urinary testosterone metabolites was not determined. Therefore, our objective was to perform high performance liquid chromatography (HPLC) analysis of PBU to describe the major immunoreactive androgens and metabolites present, and to determine if enzy-matic hydrolysis (EH) of PBU prior to EIA would improve our detection of androgen secretion during different reproductive states. EH of urinary conjugated steroids was performed using ß-glucuroni¬dase-arylsulfatase. Three buffer treatments were tested to determine if buffer type and/or pH negatively influenced EIA parameters. Due to lower matrix interference, PBS (pH 5.0) was selected for subsequent EH. Androgen concentrations of neat and hydrolyzed PBU assayed directly and after extraction were highly correlated (r ≥ 0.954, p < 0.012). HPLC supported these findings, whereby the fraction of neat urine eluting at the same time as the known testosterone-glucuronide standard demonstrated high immunoreactivity after, but not before, EH. Profiles of a breeding and a non-breeding female using parallel fecal, neat PBU, and hydrolyzed PBU samples displayed comparable patterns of androgen secretion, but discrete changes in androgen concentrations were better detected using the latter. These results indicate that accurate analysis of urinary androgen concentrations in the polar bear can be achieved following EH without an extraction step, thus saving substantial time which may critically influence the success of natural or assisted breeding management decisions.
Key Words: enzyme hydrolysis, androgens, polar bear, Ursus maritimus, enzyme immunoassay
Document Type: Research Article
DOI: https://doi.org/10.1578/AM.43.3.2017.245
Page Numbers: 245-253

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