Quantitation of Tocopherol Metabolism in Humans

描述（由申请人提供）：越来越多的证据表明，a-生育酚，维生素E的活性形式，可能有助于降低某些慢性疾病的风险，如心血管疾病，高血压和癌症。不过，还需要对维生素E进行更多的研究，以充分了解它的代谢和功能。美国医学研究院/美国国家科学院列出了维生素E的主要研究建议，其中包括量化维生素E代谢；确定质量平衡是否可行，使用稳定同位素标记的维生素E可以估计维生素E需求量；确定维生素E在人体体内的代谢是否可以量化；决定维生素E如何、在哪里、以多快的速度降解；识别代谢过程中的代谢中间体；并确定它们是否具有生物学功能。这项应用的长期目标是量化维生素E代谢的动态和动力学行为，因为它可能发生在人体体内，使用14c -维生素E的真正示踪剂剂量的命运动力学建模，确保维生素E代谢的稳定状态，这与营养有关和有用，并且我们的研究对象不被要求接受比日常生活中经历的风险大得多的风险；即，辐射暴露<飞行1-4小时获得的辐射暴露，这是一种常见的辐射暴露，即使对幼儿和孕妇也是如此。以下具体目标将实现我们的长期目标。具体目标# 1：确定人体内RRR-a-TOC的吸收、增加、分布和消除。为了适应这一点，我们将量化14C-RRR-a-TOC在各种脂蛋白类别及其代谢物之间的转移。具体目标# 2：构建14C-RRR-a-TOC在人体体内可能发生的代谢动力学模型，以检验EHT-a-TOC是否通过“反向a- toc转移”过程被消除。原假设是EHT-a-TOC通过“逆向胆固醇转移”消除。血浆脂蛋白在向EHT输送RRR-a-TOC和从EHT输送RRR-a-TOC的过程中起关键作用。为此，将招募12名志愿者，6名女性和6名男性，并给予单剂量的14c - rrr -a-生育酚（100 nCi），将其添加到一个含有10.5克橄榄油和30克脱脂牛奶的搅打混合物的杯子中。我们将对14C示踪剂进行为期60天的跟踪。该项目的长期目标是通过测试再测试设计来量化人体体内维生素E代谢的动态/动力学行为。此外，使用生育酚代谢的机制模型，我们可以确定生育酚相关酶的单核苷酸多态性（snp）是否影响人类生育酚代谢的动力学行为，正如我们最近在叶酸中所展示的那样。基线血（10ml）将在给药前和给药后60天内以不同的间隔进行，以确保14C标记的a-生育酚完全平衡到a-生育酚池中，这可能会影响研究后期的血浆动力学。脂蛋白分类将使用一种新的方法进行分离。红细胞、淋巴细胞和血小板也将被分离。在给药后30天收集尿液和粪便。每个生物样品的14C含量将在劳伦斯利弗莫尔国家实验室（Livermore， CA）的加速器质谱中心进行测量。采用非放射性高效液相色谱法测定血浆和血浆脂蛋白样品中的14C、14C- rcr -a-生育酚、a-CEHC、a-CPHC以及另外4种未知物质的化学特性。我们将构建a-生育酚代谢的动态/动力学模型，我们已经证明了我们方法的可行性。我们将在给药后相对较长的时间内使用高等离子体采样密度，以获得比任何现有数据集更详细/完整的数据集。如果能规定a-生育酚的特定代谢功能，就有可能确定a-生育酚的最低摄入量，以维持这些关键功能，如防止与动脉粥样硬化有关的脂蛋白氧化，从而将动脉粥样硬化的风险降至最低。公共卫生相关性：维生素E是一组脂溶性化合物的总称，称为生育酚，它们有不同的配方。这些维生素E配方天然存在于许多食物中，作为生物抗氧化剂。先前的研究表明，人类代谢维生素e的方式存在差异。由于人们对维生素e的代谢知之甚少，特别是在正常生理水平下，因此需要在人体生理条件下进行体内研究来了解它。因此，加州大学戴维斯分校的科学家们正在对大约12名健康志愿者进行一项研究，以评估单次口服维生素E的示踪量RRR-（天然形式）的代谢，以了解更多关于人体如何利用这种维生素的信息。我们将特别注意吸收阶段。研究结果有望提供有关维生素E生物效力的信息，并最终改进膳食建议。

DESCRIPTION (provided by applicant): There is growing evidence that a-tocopherol, the active form of vitamin E, may help minimize the risk of certain chronic diseases such as cardiovascular disease, hypertension and cancer. Still, more research on vitamin E is needed to fully understand its metabolism and function(s). The IOM/NAS has listed key research recommendations for vitamin E. The list included quantifying vitamin E metabolism; determining whether mass balance is feasible and vitamin E requirements can be estimated using stable isotope-labeled vitamin E; determining whether vitamin E metabolism as it occurs in vivo in humans can be quantified; determining how, where, and how fast vitamin E is degraded; identifying the metabolic intermediates during metabolism; and determining whether they have biological function. The long term goal of this application is to quantify the dynamic and kinetic behavior of vitamin E metabolism as it might occur in vivo in humans using kinetic modeling of the fate of true tracer doses of 14C-vitamin E that ensure steady state conditions of vitamin E metabolism that are relevant and useful to nutrition, and where our study subjects are not asked to accept risks significantly greater than those experienced in everyday life; i.e., radiation exposures < those obtained in flying for 1-4 h, a common radiation exposure, even for young children and pregnant women. The following specific aims will achieve our long term goal. Specific aim # 1: Determine the absorption, accretion, distribution, and elimination of RRR-a-TOC in humans. To accommodate this, we will quantify the transfer of 14C-RRR-a-TOC among various lipoprotein classes and its metabolites by time since dose. Specific aim # 2: Construct a dynamic and kinetic model of 14C-RRR-a-TOC metabolism as it might occur in vivo in humans to test the hypothesis whether EHT-a-TOC is eliminated by a "reverse a-TOC transfer" process. The null hypothesis is that EHT-a-TOC eliminated through "reverse cholesterol transfer." Plasma lipoproteins play key roles in delivering RRR-a-TOC to and from EHT. For this purpose 12 volunteers, 6 women and 6 men, will be recruited and administered a single dose of 14C-RRR-a-tocopherol (100 nCi) that will be added to a cup containing a whipped mix of 10.5 g olive oil and 30 g skim milk. We will follow the 14C tracer over a 60-day period. The long term goal of this project is to quantify the dynamic/ kinetic behavior of vitamin E metabolism in vivo in humans of the RRR-a-TOC and of its 2R stereoisomer forms using a test re-test design. In addition, using a mechanistic model of tocopherol metabolism we can determine whether or not single nucleotide polymorphisms (SNPs) in tocopherol-relevant enzymes affects the kinetic behavior of human tocopherol metabolism as we recently show for folate Clifford et al. 2006. Baseline blood (10 mL) will be taken just before dosing and at various intervals until 60 days post dose to ensure that the 14C labeled a-tocopherol is fully equilibrated into the slowest turning over a-tocopherol pool that can affect the plasma kinetics late in a study. The lipoprotein classes will be separated using a new protocol. Erythrocytes, lymphocytes, and platelets will also be separated. Urine and feces will be collected until 30 days post dose. The 14C content of each biological sample will be measured at the Center for the Accelerator Mass Spectrometry at Lawrence Livermore National Laboratory (Livermore, CA). Chemical identity of the 14C in plasma and plasma lipoprotein samples will also be determined, 14C-RRR-a-tocopherol, a-CEHC, a-CPHC plus 4 more unknowns by using non-radioactive standards in an HPLC. We will construct a dynamic/kinetic model of a-tocopherol metabolism, we have already demonstrated the feasibility of our approach. We will use a high sampling density of plasma over a relatively long time since dosing to acquire a much more detailed/complete dataset than any already existing dataset. Then if specific metabolic functions for a-tocopherol can be stipulated, it will be possible to determine a minimum intake of a-tocopherol to sustain these critical functions, such as, preventing oxidation of lipoproteins, which has been linked to atherogenesis, and therefore minimizing the risk of atherosclerosis. PUBLIC HEALTH RELEVANCE: Vitamin E is a generic name for a group of lipid-soluble compounds known as tocopherols that have different formulas. These Vitamin E formulas occur naturally in many foods and act as biological antioxidants. Previous studies have shown differences in the way humans metabolize vitamin E. Since metabolism of the vitamin is poorly understood especially at normal physiological levels, in vivo studies under physiological conditions are needed in humans to understand it. Therefore, UC Davis Scientists are conducting a study with approximately 12 healthy volunteers to evaluate the metabolism of a single oral administration of tracer amounts of the RRR- (natural form) of vitamin E to learn more about how the human body utilizes this vitamin. Particular attention will be directed to the absorption phase. The results are expected to provide information about vitamin E biopotency and eventually improved Dietary Recommendations.