Investigation of keratinase digestion to improve steroid hormone extraction from diverse keratinous tissues

Monitoring the physiology of wild populations presents many technical challenges. Blood samples, long the gold standard of wildlife endocrinology studies, cannot always be obtained. The validation and use of non-plasma samples to obtain hormone data have greatly improved access to more integrated information about an organism's physiological state. Keratinous tissues like skin, hair, nails, feathers, or baleen store steroid hormones in physiologically relevant concentrations, are stable across decades, and can be used to retrospectively infer physiological state at prior points in time. Most protocols for steroid extraction employ physical pulverization or cutting of the sample, followed by mixing with a solvent. Such methods do produce repeatable and useful data, but low hormone yield and detectability issues can complicate research on small or rare samples. We investigated the use of keratinase, an enzyme that breaks down keratin, to improve the extraction and yield of corticosterone from vertebrate keratin tissues. Corticosterone content of keratinase-digested extracts were compared to non-keratinase extracts for baleen from three species of whale (blue, Balaenoptera musculus; bowhead, Balaena mysticetus; southern right, SRW; Eubalaena australis), shed skin from two reptiles (tegu lizard, Salvator merianae; narrow-headed garter snake, Thamnophis rufipunctatus), hair from arctic ground squirrel (AGS; Urocitellus parryii), feathers from Purple Martins (PUMA; Progne subis), and spines from the short-beaked echidna (Tachyglossus aculeatus). We tested four starting masses (10, 25, 50, 100 mg) for each sample; digestion was most complete in the 10 and 25 mg samples. A corticosterone enzyme immunoassay (EIA) was validated for all keratinase-digested extracts. In all sample types except shed skin from reptiles, keratinase digestion improved hormone yield, with PUMA feathers and blue whale baleen having the greatest increase in apparent corticosterone content (100% and 66% more hormone, respectively). The reptilian shed skin samples did not benefit from keratinase digestion, actually yielding less hormone than controls. With further optimization and refinement, keratinase digestion could greatly improve yield of steroid hormones from various wildlife epidermal tissue types, allowing more efficient use of samples and ultimately improving understanding of the endocrine physiology of wild populations.