Rapid Steroid Profiling of Individual Model Fish: Relating Exposure to Disease

描述（由申请人提供）：进入环境的内分泌干扰化学品对人类健康具有广泛的影响。人类和鱼类在基因、发育和HPG轴方面有着相似之处。鱼类还提供了可观察到的繁殖终点。因此，用斑马鱼等模式鱼进行的体内试验是评估这些化学品对人类健康影响的一个组成部分。循环类固醇激素是内分泌干扰的关键生物标志物，但由于斑马鱼的血容量有限（<5 uL），样品处理费力，以及研究的动物数量，因此在斑马鱼中测量类固醇具有挑战性。拟议的活动是基于毛细管电泳和创建一个高通量，无标记的方法来测定血浆中的类固醇。两种分析方法的开发，以支持一个系统的方法来快速配置文件类固醇。类固醇生物标志物在不到5分钟的时间内在没有衍生化的情况下在个体鱼中进行测量，并且自动化仪器适于在微量水平上处理样品。目的1：提高血浆中类固醇激素的定量，以确定在暴露的鱼类中引起生理变化的内分泌干扰化学品。从低体积的血浆中回收类固醇，使用pH介导的堆积在毛细管中浓缩，分离并定量。Aim 2将样品处理的手动方法转化为自动化的微量提取。将市售磁珠集成在熔融石英毛细管中，并用作保持用于固相萃取的色谱填料的玻璃料。 利用毛细管电泳仪器中的复杂机器人技术在几分钟内处理单个样品。该仪器将样品处理所需的工作体积从毫升减少到微升。当这两个目标串联使用时，该方法快速并同时监测每个样品中的多种类固醇。这项新技术与斑马鱼相关的微升血浆体积兼容。这些进步使得测量多种类固醇在个别鱼可行的减少所需的时间和劳动力来处理和分析样品。

DESCRIPTION (provided by applicant): Endocrine disrupting chemicals that enter the environment have broad consequences on human health. Humans and fish share similarities in genes, development, and the HPG axis. Fish also provide observable endpoints of reproduction. As a result in vivo tests with model fish, such as the zebrafish, are an integral component to assess the health effects of these chemicals in humans. Circulating steroid hormones are critical biomarkers of endocrine disruption, but measurements of steroids are challenging in zebrafish because of the limited (<5 uL) blood volume, laborious sample processing, and the number of animals investigated. The proposed activities are based on capillary electrophoresis and create a high-throughput, label-free method to assay steroids in plasma. Two analytical methods are developed to support a systematic approach to rapidly profile steroids. Steroid biomarkers are measured in individual fish without derivatization in less than 5 minutes, and an automated instrument is adapted to process samples at the microscale level. Aim 1 activities improve the quantification of steroid hormones in plasma to identify endocrine disrupting chemicals that elicit a physiological change in exposed fish. The steroids are recovered from low volumes of plasma, concentrated in a capillary using pH-mediated stacking, separated, and quantified. Aim 2 translates a manual method of sample processing into an automated microscale extraction. Commercially available magnetic beads are integrated in a fused silica capillary and serve as frits that hold chromatographic packing utilized for solid phase extraction. The sophisticated robotics in the capillary electrophoresis instrument are leveraged to process single samples in minutes. The instrument decreases the working volume needed for sample handling from milliliters to microliters. When both aims are used in tandem, the method rapidly and simultaneously monitors multiple steroids in each sample. The new technology is compatible with the microliter plasma volumes associated with zebrafish. These advances make measurements of multiple steroids feasible in individual fish by reducing the required time and labor to process and analyze samples.