Department of Physiological Sciences and Center for Environmental and Human Toxicology, University of Florida, Gainesville, FL 32611, United States.
Department of Physiological Sciences and Center for Environmental and Human Toxicology, University of Florida, Gainesville, FL 32611, United States.
Gen Comp Endocrinol. 2020 Sep 15;296:113543. doi: 10.1016/j.ygcen.2020.113543. Epub 2020 Jun 27.
Quantification of steroid hormones in fish is an important step for toxicology and endocrinology studies. Among the hormone analysis techniques, liquid chromatography tandem mass spectrometry (LC-MS/MS) has widely been used for measuring hormones in various biological samples. Despite all improvements in the technique, detection of several hormones in a low volume of serum or plasma is still challenging. We developed a robust method for simultaneous quantification of 14 steroid hormones including corticosterone, cortisol, 11-ketotestosterone, progesterone, testosterone, 17OH-progesterone, aldosterone, dihydrotestosterone, estrone, 17β-estradiol, estriol, ethinylestradiol, levonorgestrel and equilin from volumes as low as 10 µL serum or plasma in a short run by LC-MS/MS. The lowest limit of detection in 10 µL serum was 0.012 ng/mL measured for cortisol, progesterone, testosterone, 17OH-progesterone and estrone. Use of high (25 times more) serum volume improved detection limit of hormones by 2-40 times. The method was compared with the radioimmunoassay technique in which testosterone and 17β-estradiol were highly correlated with R of 0.95 and 0.96, respectively. We validated the method by measuring four selected hormones, in low and high plasma volumes of largemouth bass (Micropterus salmoides). In addition, we developed a method to quantify hormones in whole body fish homogenates of small fish and compared the values to plasma concentrations, using fathead minnow (Pimephales promelas). Calculated concentrations of the hormones in plasma were consistent with those in the homogenate and 11-ketotestosterone and 17β-estradiol were significantly different in males and females. The ability to measure hormones from whole body homogenates was further evaluated in two model small fish species, zebrafish (Danio rerio) and juvenile silverside (Menidia beryllina). These results suggest that whole tissue homogenate is a reliable alternative for hormone quantification when sufficient plasma is not available.
鱼类中类固醇激素的定量分析是毒理学和内分泌学研究的重要步骤。在激素分析技术中，液相色谱-串联质谱法（LC-MS/MS）已广泛用于测量各种生物样本中的激素。尽管该技术不断得到改进，但在小体积的血清或血浆中检测几种激素仍然具有挑战性。我们开发了一种强大的方法，可通过 LC-MS/MS 同时定量分析 14 种类固醇激素，包括皮质酮、皮质醇、11-酮睾酮、孕酮、睾酮、17α-羟孕酮、醛固酮、二氢睾酮、雌酮、17β-雌二醇、雌三醇、乙炔雌二醇、左炔诺孕酮和马烯雌酮，最低检测限为 10μL 血清中 0.012ng/mL 的皮质醇、孕酮、睾酮、17α-羟孕酮和雌酮。使用高（25 倍）血清量可将激素的检测限提高 2-40 倍。该方法与放射免疫测定技术进行了比较，其中睾酮和 17β-雌二醇的相关性很高，相关系数分别为 0.95 和 0.96。我们通过测量大口黑鲈（Micropterus salmoides）低和高血浆体积中的四种选定激素验证了该方法。此外，我们开发了一种从小鱼全鱼组织匀浆中定量激素的方法，并与胖头鱼（Pimephales promelas）的血浆浓度进行了比较。计算出的激素在血浆中的浓度与在匀浆中的浓度一致，并且 11-酮睾酮和 17β-雌二醇在雄性和雌性之间存在显著差异。该方法在两种模式小鱼斑马鱼（Danio rerio）和幼银汉鱼（Menidia beryllina）中对全组织匀浆进行了测量激素的能力评估。这些结果表明，当没有足够的血浆时，全组织匀浆是一种可靠的激素定量替代方法。
