Investigation of keratinase digestion to improve steroid hormone extraction from diverse keratinous tissues
Investigation of keratinase digestion to improve steroid hormone extraction from diverse keratinous tissues
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DOI：
10.1016/j.ygcen.2021.113795
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发表时间：
2021-04-28
影响因子：
2.7
通讯作者：
Buck, C. Loren
中科院分区：
文献类型：
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作者：
Dillon, Danielle;Ajo, Alejandro Fernandez;Buck, C. Loren
监测野生种群的生理存在许多技术挑战。血液样本长期以来一直是野生动物内分泌学研究的黄金标准，但并不总是能得到。验证和使用非血浆样本来获取激素数据，极大地改进了获取有关生物体生理状态的更综合信息的途径。角质组织，如皮肤、头发、指甲、羽毛或秃头，储存着生理上相关浓度的类固醇激素，几十年来保持稳定，并可用于追溯推断先前时间点的生理状态。大多数类固醇提取方案采用的是样品的物理粉碎或切割，然后与溶剂混合。这种方法确实产生了可重复和有用的数据，但激素产量低和可检测性问题可能会使对小样本或稀有样本的研究复杂化。我们研究了使用角蛋白分解酶来提高脊椎动物角蛋白组织中皮质酮的提取和产量。比较了角蛋白酶消化的提取物中皮质酮的含量，以及三种鲸鱼(蓝鲸，Balaenoptera musculus；弓头鲸，Balaena mystitus；南露脊鲸，SRW；南方真鲸)，两种爬行动物(Tegu lizard，Salator merianae；窄头吊蛇，Thamnopits rufipot tatus)的脱皮，北极松鼠(AGS；Urocitellus parryii)的毛发，紫马丁(PUMA；Progne subis)的羽毛，以及短喙希德纳(Tachyglossus Aculeatus)的刺的皮质酮含量。我们为每个样本测试了四个起始量(10，25，50,100毫克)；10和25毫克的样品消化最完全。皮质酮酶免疫分析(EIA)对所有角蛋白酶消化提取物进行了验证。在所有样本类型中，除了爬行动物脱落的皮肤外，角蛋白酶消化提高了激素产量，其中PUMA羽毛和蓝鲸须毛的表观皮质酮含量增幅最大(分别增加100%和66%)。爬行动物脱落的皮肤样本并没有从角蛋白酶消化中受益，实际上产生的激素比对照组少。通过进一步的优化和细化，角蛋白酶消化可以极大地提高从各种野生动物表皮组织中提取类固醇激素的产量，从而更有效地利用样本，最终提高对野生种群内分泌生理的了解。
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.