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

描述(申请人提供)：进入环境的内分泌干扰性化学物质对人类健康有广泛的影响。人类和鱼类在基因、发育和HPG轴方面有相似之处。鱼类也提供了可观察到的繁殖终点。因此，用斑马鱼等模型鱼进行体内试验是评估这些化学物质对人类健康影响的不可或缺的组成部分。循环中的类固醇激素是内分泌紊乱的关键生物标志物，但在斑马鱼中，类固醇的测量是具有挑战性的，因为有限的血液容量(&lt；5 ul)，费力的样品处理，以及被调查的动物数量。建议的活性基于毛细管电泳法，并创建了一种高通量、无标记的方法来检测血浆中的类固醇。开发了两种分析方法来支持快速分析类固醇的系统方法。在不到5分钟的时间内，无需衍生化就可在单个鱼类中测量类固醇生物标志物，并采用自动化仪器在微尺度水平上处理样品。目的1活性改善血浆中类固醇激素的定量，以确定引起暴露鱼类生理变化的内分泌干扰化学物质。类固醇从低容量的血浆中回收，使用pH介导式堆积在毛细管中浓缩，分离和定量。AIM 2将人工处理样品的方法转化为自动的微尺度提取。商业上可用的磁珠集成在熔融的二氧化硅毛细管中，作为固定用于固相萃取的色谱填料的部件。 毛细管电泳仪中复杂的机器人技术可以在几分钟内处理单个样品。该仪器将样品处理所需的工作量从毫升减少到微升。当两个目标同时使用时，该方法快速且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。这些进展减少了处理和分析样品所需的时间和劳动力，从而使在个体鱼类中测量多种类固醇成为可能。

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.