Development of molecular biology-based assays for human selenium status

描述(由申请人提供)：这项研究的总体目标是开发基于分子生物学的人类硒(Se)状态检测方法。我们首次报道了Se依赖的谷胱甘肽过氧化物酶-1(GPX1)的mRNA水平下降到Se充足水平的10%，从而为GPX1是评估人类和动物Se状况的有效生化标志物提供了分子解释，并提出了GPX1表达调控是GPX1作用的一个重要方面的假设。然后，我们使用肝脏中的硒蛋白mRNA水平作为标志物，仔细评估了大鼠的硒需要量，表明分子生物学标志物是评估需要量的有用工具，并且在评估整个生命周期(如怀孕和哺乳期)的需要量时，了解这些分子标志物的调节是重要的。最近，我们进行了一系列研究，调查了血液(一种侵入性较小的组织)的使用情况，发现大鼠全血中GPX1 mRNA的表达与主要组织中的水平相当，可以有效地用于评估硒的状况。因此，我们已经开始评估人类硒蛋白的mRNA水平，以此作为评估人类硒状况的潜在方法。优点是这些水平似乎是由Se状态控制的恒定状态。其次，快速分子生物学检测的出现表明，分子生物学标记物将很快在评估包括人类营养在内的人类健康方面变得重要。我们现在发现，人的血液和啮齿动物的血液一样，有与主要组织相当的硒蛋白mRNA表达水平，这表明这些可能是人类硒状态的有效标记物。在这项拟议的研究中，我们将追求三个具体目标：1.使用我们目前(未获资助的)与英国雷丁公司合作的RPA评估人全血中硒蛋白mRNA的表达，以将这些标记物与血浆GPX3活性和血浆硒水平相关联；2.制定方法并在人全血中使用定量实时聚合酶链式反应(qRT-PCR)来评估阅读样本中许多硒蛋白的硒蛋白基因表达；3.在Madison WI上测定120名成年受试者的全血硒蛋白基因表达水平以及血浆GPX3活性、红细胞GPX1活性和血浆硒浓度，然后测定男性和女性受试者在开始服用安慰剂或每日补充110ug硒后1mo、2mo和4mo时全血硒蛋白基因表达的变化。相关性：这项研究直接专注于提高我们对使用特定分子标记来设定营养需求的理解和能力，因此与人类健康相关。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关于硒的研究建议，包括需要生物标志物来评估硒状况，开发与硒状况的动态平衡调节有关的、有可能用于个体化药物的基于分子生物学的分析。

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.