Department of Behavioral Ecology, Johann-Friedrich-Blumenbach Institute for Zoology and Anthropology, University of Goettingen, Goettingen, Germany; Leibniz ScienceCampus Primate Cognition, Goettingen, Germany.
Department of Behavioral Ecology, Johann-Friedrich-Blumenbach Institute for Zoology and Anthropology, University of Goettingen, Goettingen, Germany; Leibniz ScienceCampus Primate Cognition, Goettingen, Germany; Research Group Social Evolution in Primates, German Primate Center, Leibniz Institute for Primate Research, Goettingen, Germany.
Gen Comp Endocrinol. 2019 Sep 15;281:117-125. doi: 10.1016/j.ygcen.2019.05.017. Epub 2019 May 27.
The development of methods to quantify hormones from non-invasively collected samples such as urine or feces has facilitated endocrinology research on wild-living animals. To ensure that hormone measurements are biologically meaningful, method validations are strongly recommended for each new species or sample matrix. Our aim was to validate three commonly used enzyme immunoassays (EIA), one for analysis of cortisol and two for analysis of testosterone, to assess adrenocortical and gonadal endocrine activity, respectively, from the urine of male Barbary macaques. We compared EIA and liquid chromatography-mass spectrometry (LC-MS) results to determine if the EIA measurements truly reflect levels of the target hormone and to determine if antibody cross-reactivities with other steroids were potentially confounding results. Furthermore, we conducted a biological validation of testosterone to ensure that both EIA and LC-MS were able to capture physiologically meaningful differences in hormone levels. We found that cortisol measured by EIA correlated strongly with cortisol measured by LC-MS in both adult and immature males, without the need for deconjugation of steroids in the urine. Both testosterone EIAs correlated strongly with LC-MS in adult males, but only if steroids in the urine were deconjugated by enzymatic hydrolysis prior to analysis. However, in immature males, EIA and LC-MS results did not correlate significantly. Further correlation analyses suggest this is likely due to cross-reactivity of the testosterone antibodies with other adrenal steroids such as cortisol, DHEA, and likely others, which are present at much higher concentrations relative to testosterone in immature males. Testosterone levels were significantly higher in adult compared to immature males as measured by LC-MS but not as measured by EIA. Taken together, our results suggest that the testosterone EIAs are suitable to assess gonadal activity in adult but not immature males, and only if a hydrolysis of the urine is conducted prior to analysis.
从尿液或粪便等非侵入性收集样本中定量激素的方法的发展促进了对野生动物内分泌学的研究。为了确保激素测量具有生物学意义，强烈建议为每个新物种或样本基质进行方法验证。我们的目的是验证三种常用于分析皮质醇的酶联免疫分析（EIA）和两种常用于分析睾酮的 EIA，以分别评估雄性巴巴里猕猴尿液中的肾上腺皮质和性腺内分泌活动。我们比较了 EIA 和液相色谱-质谱（LC-MS）的结果，以确定 EIA 测量是否真实反映了目标激素的水平，以及抗体与其他类固醇的交叉反应是否可能会产生干扰结果。此外，我们还对睾酮进行了生物学验证，以确保 EIA 和 LC-MS 都能够捕捉到激素水平的生理意义上的差异。我们发现，EIA 测量的皮质醇与 LC-MS 测量的皮质醇在成年和未成年雄性中均呈强相关性，而无需对尿液中的类固醇进行去结合。两种睾酮 EIA 在成年雄性中均与 LC-MS 呈强相关性，但前提是在分析前通过酶水解使尿液中的类固醇去结合。然而，在未成年雄性中，EIA 和 LC-MS 的结果没有显著相关性。进一步的相关分析表明，这可能是由于睾酮抗体与其他肾上腺类固醇（如皮质醇、DHEA 等）的交叉反应所致，这些类固醇在未成年雄性中的浓度相对于睾酮要高得多。LC-MS 测量的成年雄性的睾酮水平明显高于未成年雄性，但 EIA 测量的结果并非如此。综上所述，我们的结果表明，睾酮 EIA 适合评估成年雄性的性腺活性，但不适合评估未成年雄性的性腺活性，而且只有在分析前进行尿液水解的情况下才适合。
