Development of molecular biology-based assays for human selenium status

描述（由申请人提供）：本研究的总体目标是开发基于分子生物学的人体硒（Se）状态测定方法。我们是第一个报告，硒依赖性谷胱甘肽过氧化物酶-1（GPX 1）的mRNA水平下降到硒充足水平的10%，从而提供了一个分子解释，为什么GPX 1是一个有效的生化标志物，用于评估硒在人类和动物的状态，并建议的假设，GPX 1的表达调控是GPX 1的作用的一个重要方面。使用硒蛋白的mRNA水平在肝脏作为标志物，然后，我们仔细评估硒的需求在大鼠中，显示分子生物学标志物是一个有用的工具，用于评估的要求，并了解这些分子标记物的调节是很重要的，在整个生命周期，如在怀孕和哺乳期评估的要求。最近，我们进行了一系列研究，调查血液（一种侵入性较小的组织）的使用，发现大鼠全血中GPX 1 mRNA的表达与主要组织中的水平相当，可有效地用于评估硒状态。因此，我们已经开始评估人类硒蛋白mRNA水平作为一种潜在的方式来评估人体硒状态。其优点是，这些水平似乎是由硒状态的同源平衡控制。第二，快速分子生物学分析的出现表明，分子生物学标记物将很快成为评估人类健康，包括人类营养的重要工具。我们现在已经发现，人的血液，就像在啮齿动物的血液中，硒蛋白mRNA的表达水平相当的主要组织，这表明这些可能是人类硒状态的有效标志物。在这项拟议的研究中，将追求三个具体目标：1。使用RPA评估来自我们目前与英格兰阅读的（未资助的）合作的人全血上的硒蛋白mRNA表达，以将这些标志物与血浆GPX 3活性和血浆Se水平相关联; 2.开发方案并对人全血使用定量实时PCR（qRT-PCR）来评估阅读样品中的许多硒蛋白的硒蛋白mRNA表达; 3.评估威斯康星州麦迪逊市120名成年受试者的全血硒蛋白mRNA水平，沿着血浆GPX 3活性、RBC GPX 1活性和血浆硒浓度，然后确定20名男性和20名女性受试者开始服用安慰剂或每天补充110 μ g硒后1个月、2个月和4个月硒蛋白mRNA表达的变化。相关性：这项研究的重点是提高我们的理解和能力，使用特定的分子标记来设定营养需求，因此与人类健康有关。更具体地说，这项研究将直接解决2000年医学研究所膳食参考摄入量报告中对硒的研究建议，包括需要生物标志物来评估硒的状态，通过开发基于分子生物学的检测方法，这些检测方法与硒状态的稳态调节有关，并有可能用于个体化医学。

DESCRIPTION (provided by applicant): The overall goal of this research is to develop molecular biology-based assays for human selenium (Se) status. We were the first to report that mRNA levels of Se-dependent glutathione peroxidase-1 (GPX1) fall to 10% of Se-adequate levels, thus providing a molecular explanation for why GPX1 is an effective biochemical marker for assessing Se status in humans and animals, and suggesting the hypothesis that regulation of GPX1 expression is an important aspect of the role of GPX1. Using selenoprotein mRNA levels in liver as markers, we then carefully evaluated Se requirements in rats, showing that molecular biology markers were a useful tool for assessing requirements and that understanding the regulation of these molecular markers was important when assessing requirements throughout the life cycle, such as in pregnancy and lactation. More recently, we have conducted a series of studies investigating the use of blood (a less invasive tissue), and found that GPX1 mRNA expression in rat whole blood was comparable to levels in the major tissues and could be used effectively to assess Se status. Thus we have begun to evaluate human selenoprotein mRNA levels as a potential way to assess human Se status. The advantage is that these levels appear to be homoeostatic ally controlled by Se status. Secondly, the advent of rapid molecular biology assays suggests that molecular biology markers will soon become important in assessing human health, including human nutrition. We now have found that human blood, just as in rodent blood, has selenoprotein mRNA expression at levels comparable to major tissues, suggesting that these may be the effective markers of human Se status. In this proposed research, three specific aims will be pursued: 1. To assess selenoprotein mRNA expression using RPA on human whole blood from our current (unfunded) collaboration with Reading, England, to correlate these markers with plasma GPX3 activity and plasma Se levels; 2. To develop protocols and use quantitative real-time PCR (qRT-PCR) on human whole blood to assess selenoprotein mRNA expression for a number of selenoproteins in the Reading samples; 3. To assess whole blood selenoprotein mRNA levels, along with plasma GPX3 activity, RBC GPX1 activity, and plasma Se concentration, in 120 adult human subjects in Madison WI, and then to determine the changes in selenoprotein mRNA expression 1 mo, 2 mo and 4 mo after initiation of placebo or 110 ug Se per day supplementation in 20 male and 20 female subjects. Relevance: This research is directly focused on improving our understanding and ability to use specific molecular markers to set nutrient requirements, and thus is relevant to human health. More specifically, this research will directly address the research recommendations for selenium by the 2000 Institute of Medicine Dietary Reference Intake report, including the need for biomarkers for assessment of selenium status, by developing molecular biology-based assays that are linked to homeostatic regulation of selenium status and that have the potential to be used in individualized medicine.