Galligan Thomas M, Schwacke Lori H, Houser Dorian S, Wells Randall S, Rowles Teri, Boggs Ashley S P
Medical University of South Carolina, College of Graduate Studies, Hollings Marine Laboratory, 331 Fort Johnson Road, Charleston, SC 29412, USA; JHT, Inc. Under Contract to National Oceanic and Atmospheric Administration, National Centers for Coastal Ocean Science, Hollings Marine Laboratory, 331 Fort Johnson Road, Charleston, SC 29412, USA.
National Marine Mammal Foundation, 2240 Shelter Island Drive Suite 200, San Diego, CA 92106, USA.
Gen Comp Endocrinol. 2018 Jul 1;263:80-91. doi: 10.1016/j.ygcen.2018.04.003. Epub 2018 Apr 6.
Systemic steroid hormone measurements are often used in the assessment of reproductive, developmental, and stress physiology in vertebrates. In protected wildlife, such as the common bottlenose dolphin (Tursiops truncatus), these measures can provide critical information about health and fitness to aid in effective conservation and management. Circulating steroid hormone concentrations are typically measured by immunoassays, which have imperfect specificity and are limited to the measurement of a single hormone per assay. Here we demonstrate that reverse phase solid phase extraction (SPE) coupled to liquid chromatography tandem mass spectrometry (LC-MS/MS) allows for the simultaneous, precise (<15% relative standard deviation), and accurate (between 70% and 120% recovery of spiked quantities) measurement of at least seven steroid hormones in dolphin plasma. These seven steroid hormones include three hormones that have been measured previously in bottlenose dolphin blood (progesterone, testosterone, and cortisol) and three hormones which have never been quantified in dolphin blood (17-hydroxyprogesterone, androstenedione, cortisone, and corticosterone). While 17β-estradiol was not detected endogenously, we were able to accurately and precisely measure spiked quantities estradiol. Measures from plasma were more precise (i.e., lower RSD) than serum, and thus we recommend plasma as the preferred matrix for this analytical method. In order to facilitate comparison of current and future plasma-based studies to previous serum-based studies, we characterized the relationships between hormone measurements in matched plasma and serum, and found that measurements across matrices are significantly and positively correlated. Lastly, to demonstrate potential applications of this method, we examined how steroid hormone profiles vary by pregnancy, sexual maturity, and stress status - pregnancy was associated with elevated progesterone, adult males had higher testosterone, and capture stress was associated with elevated corticosteroids. Overall, we conclude that this method will enable investigators to more thoroughly and efficiently evaluate steroid hormone homeostasis in bottlenose dolphins compared to immunoassay methods. These methods can potentially be applied to the assessment of sexual maturity/seasonality, pregnancy status, and stress in free-ranging bottlenose dolphins as well as those maintained under human care, and potentially other marine mammals.
全身类固醇激素测量常用于评估脊椎动物的生殖、发育和应激生理学。在受保护的野生动物中，如普通宽吻海豚（Tursiops truncatus），这些测量可以提供有关健康和适应性的关键信息，以帮助进行有效的保护和管理。循环类固醇激素浓度通常通过免疫测定法测量，该方法特异性不完善，每次测定仅限于测量单一激素。在此，我们证明反相固相萃取（SPE）与液相色谱串联质谱（LC-MS/MS）联用能够同时、精确地（相对标准偏差<15%）和准确地（加标量回收率在70%至120%之间）测量海豚血浆中至少七种类固醇激素。这七种类固醇激素包括先前已在宽吻海豚血液中测量过的三种激素（孕酮、睾酮和皮质醇）以及三种从未在海豚血液中定量的激素（17-羟孕酮、雄烯二酮、可的松和皮质酮）。虽然内源性未检测到17β-雌二醇，但我们能够准确且精确地测量加标量的雌二醇。血浆测量比血清更精确（即相对标准偏差更低），因此我们建议血浆作为该分析方法的首选基质。为了便于将当前和未来基于血浆的研究与先前基于血清的研究进行比较，我们表征了匹配血浆和血清中激素测量之间的关系，发现不同基质的测量显著正相关。最后，为了证明该方法的潜在应用，我们研究了类固醇激素谱如何因怀孕、性成熟和应激状态而变化——怀孕与孕酮升高相关，成年雄性睾酮较高，捕获应激与皮质类固醇升高相关。总体而言，我们得出结论，与免疫测定法相比，该方法将使研究人员能够更全面、有效地评估宽吻海豚的类固醇激素稳态。这些方法有可能应用于评估自由放养的宽吻海豚以及人工饲养的宽吻海豚的性成熟/季节性、怀孕状态和应激，也可能应用于其他海洋哺乳动物。
