Optimizing hormone extraction protocols for whale baleen: Tackling questions of solvent:sample ratio and variation
Optimizing hormone extraction protocols for whale baleen: Tackling questions of solvent:sample ratio and variation
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DOI：
10.1016/j.ygcen.2021.113828
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发表时间：
2022-01-01
影响因子：
2.7
通讯作者：
Loren Buck, C.
中科院分区：
文献类型：
--
作者：
Fernandez Ajo, Alejandro;Hunt, Kathleen E.;Loren Buck, C.
从鲸须和其他角化组织等替代样本类型获得内分泌数据已被证明是一种有价值的工具，可通过类固醇激素定量研究鲸鱼和其他脊椎动物的生殖和应激生理学，并通过甲状腺激素定量研究代谢应激。这些替代样品类型提供了在结构生长期间血浆水平的综合测量，从而捕获个体内分泌史的数月甚至数年。此外，它们强大而稳定的角蛋白基质允许这些样本储存数年至数十年，从而能够分析和比较过去和现代人群的内分泌模式。然而，从鲸须和其他角化组织中提取和分析激素仍然是新的，需要生物学和分析验证，以确保该方法符合其预期用途的要求。我们利用从南露脊鲸身上尸检得到的鲸须（Eubalaena australis），其在阿根廷的半岛Valde ' s死亡，使用市售的孕酮酶免疫测定（EIA）来解决两个方法学问题：1）用市售EIA分析的鲸须样品的激素含量可靠定量所需的最小样品量是多少，以及2）溶剂体积与样品质量的最佳比率是多少，即，产生最大量的激素的比率，具有高准确性和重复之间的低变异性。我们得出结论，尽可能使用至少20 mg的质量，最好使用80：1的溶剂与样品比例（溶剂体积与样品质量; μ l：mg）进行浸提。这些结果可以帮助研究人员在使用稀有或独特标本的破坏性提取方法时做出明智的方法学决定。
Obtaining endocrine data from alternative sample types such as baleen and other keratinized tissues has proven a valuable tool to investigate reproductive and stress physiology via steroid hormone quantification, and metabolic stress via thyroid hormone quantification in whales and other vertebrates. These alternative sample types provide an integrated measure of plasma levels over the period that the structure was growing, thus capturing months or even years of an individual's endocrine history. Additionally, their robust and stable keratin matrix allows such samples to be stored for years to decades, enabling the analysis and comparison of endocrine patterns from past and modern populations. However, the extraction and analysis of hormones from baleen and other keratinized tissues remains novel and requires both biological and analytical validations to ensure the method fulfills the requirements for its intended use. We utilized baleen recovered at necropsy from southern right whales (Eubalaena australis) that died at Peninsula Valde ' s, Argentina, using a commercially available progesterone enzyme immunoassay (EIA) to address two methodological questions: 1) what is the minimum sample mass required to reliably quantify hormone content of baleen samples analyzed with commercially available EIAs, and 2) what is the optimal ratio of solvent volume to sample mass, i.e., the ratio that yields the maximum amount of hormone with high accuracy and low variability between replicates. We concluded that masses of at least 20 mg should be used whenever possible, and extraction is best performed using an 80:1 ratio of solvent to sample (volume of solvent to sample mass; mu l:mg). These results can help researchers to make informed methodological decisions when using a destructive extraction method with rare or unique specimens.