John Wiley & Sons, Ltd.

Increased immunoreactive 11‐ketotestosterone concentrations in sheep feces after ACTH challenge

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11‐Oxoetiocholanolone and related substances are important metabolites of cortisol and are excreted via feces in ruminants. To investigate whether 11‐ketotestosterone (11‐KT) or its immunoreactive metabolites are formed and excreted in ruminant feces, an enzyme immunoassay (EIA) was developed and validated. The antibody was raised in rabbits against 11‐KT‐3‐CMO:BSA with biotinylated 11‐KT as a label. The assay showed a sensitivity of 0.3 pg/well. To validate the assay biologically, six rams were injected with a synthetic analogue of the adrenocorticotropic hormone (Synacthen, 2 µg/kg body weight). An aliquot was collected of each fecal portion spontaneously defecated 8 h before Synacthen injection to 24 h after injection and stored at ‐20°C until analysis. Samples (0.5 g) were extracted using 80% methanol and immunoreactive metabolites measured using the 11‐KT EIA and an already established 11,17‐dioxoandrostane (11,17‐DOA) EIA. HPLC separation revealed no peak in the same elution position as authentic 11‐KT; therefore, reacting substances were referred to as 11‐KT equivalents. In the case of 11‐KT immunoreactive substances, the values increased from baseline (median: 136 ng/g feces) to a peak concentration (median: 424 ng/g) 10 to 14 hours after Synacthen injection and declined afterwards. Concentrations of 11,17‐DOA showed the same pattern, but the values were 2‐4 times higher. From this data we conclude that 11‐KT‐like substances, specifically C19O3‐androgens with a 17ß‐hydroxy group, were present in the feces. These substances originate from the adrenals, and are most likely cortisol metabolites. Environ. Toxicol. Chem. © 2013 SETAC

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