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

个体模型鱼的快速类固醇分析：与疾病暴露相关

• 批准号: 8784218
• 负责人: Lisa A Holland
• 金额: $ 18.5万
• 依托单位: WEST VIRGINIA UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-12-15 至 2016-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8784218
• 关键词: Address; Affect; Androgens; Animal Model; Animals; Antibodies; Automation; Biological Assay; Biological Markers; Blood; Blood Plasma Volume; Blood Volume; Blood capillaries; Blood specimen; Capillary Electrophoresis; Cessation of life; Chemicals; Complex; Computer software; Databases; Development; Disease; Endocrine; Endocrine Disruptors; Endocrine disruption; Endocrine system; Environment; Environmental Exposure; Environmental Health; Environmental Pollution; Epigenetic Process; Equilibrium; Estrogens; Exhibits; Exposure to; Feminization; Fishes; Functional disorder; Gene Proteins; Genes; Health; Hormonal; Hormones; Hour; Human; Immune; Incidence; Individual; Intersex; Investments; Label; Laboratories; Liquid substance; Magnetism; Malignant Neoplasms; Manuals; Maps; Measurement; Measures; Mediating; Methods; Modeling; Monitor; Nature; Obesity; Oocytes; Organism; Pathway interactions; Phase; Physiological; Plasma; Process; Progestins; Protein Binding; Reproduction; Research; Resources; Risk; Rivers; Robotics; Running; Sampling; Silicon Dioxide; Solid; Speed; Steroids; Stream; System; Technology; Testing; Testis; Time; Translating; United States National Institutes of Health; Validation; Water; West Virginia; Work; Zebrafish; analytical method; base; capillary; cognitive development; improved; in vivo; insight; instrument; killings; magnetic beads; male; milliliter; new technology; premature; reproductive; response; screening; small molecule; steroid hormone; tool; toxicant

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.

描述（由申请人提供）：进入环境的内分泌干扰化学物质对人类健康产生广泛的影响。人类和鱼类在基因、发育和 HPG 轴方面有相似之处。鱼类还提供可观察的繁殖终点。因此，斑马鱼等模型鱼的体内测试是评估这些化学物质对人类健康影响的重要组成部分。循环类固醇激素是内分泌干扰的关键生物标志物，但由于血容量有限（<5 uL）、样本处理繁琐以及研究的动物数量，类固醇的测量在斑马鱼中具有挑战性。拟议的活动基于毛细管电泳，并创建一种高通量、无标记的方法来测定血浆中的类固醇。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 分钟的时间内即可测量个体鱼的类固醇生物标志物，无需衍生化，并且自动化仪器适用于在微尺度水平上处理样品。目标 1 活动改进了血浆中类固醇激素的定量，以识别引起暴露鱼类生理变化的内分泌干扰化学物质。类固醇从少量血浆中回收，使用 pH 介导的堆积浓缩在毛细管中，进行分离和定量。目标 2 将手动样品处理方法转化为自动微量提取。市售磁珠集成在熔融石英毛细管中，并用作固相萃取所用色谱填料的玻璃料。 利用毛细管电泳仪器中先进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体积从毫升减少到微升。当两个目标同时使用时，该方法可以快速同时监测每个样本中的多种类固醇。新技术与斑马鱼的微升血浆量兼容。这些进步减少了处理和分析样品所需的时间和劳动力，使得在个体鱼类中测量多种类固醇成为可能。

项目成果

Quantification of circulating steroids in individual zebrafish using stacking to achieve nanomolar detection limits with capillary electrophoresis and UV-visible absorbance detection.

使用堆叠对单个斑马鱼中的循环类固醇进行定量，以通过毛细管电泳和紫外-可见光吸光度检测达到纳摩尔检测限。

• 发表时间: 2015-09
• 影响因子: 4.3
• 作者: Nyakubaya, Vincent T;Durney, Brandon C;Ellington, Marriah C G;Kantes, Amber D;Reed, Paige A;Walter, Shaylyn E;Stueckle, Jennifer Ripley;Holland, Lisa A
• 通讯作者: Holland, Lisa A