FLAVONOID BIOAVAILABILITY IN HUMANS-CELLULAR STUDIES

描述(申请人提供)：这项研究计划的长期目标是增加我们对细胞运输和新陈代谢如何影响饮食中类黄酮的口服生物利用度的了解，类黄酮是一大类化合物，已被认为在预防人类疾病，特别是心血管疾病和癌症方面发挥重要作用。在具体目标1中，我们将确定SGLT1和MRP2之间的相互关系，包括参与肠道细胞吸收黄酮苷和茶黄酮类的机制，这两类黄酮类是饮食中的主要类黄酮。这些研究将在SGLT1和MRP2转基因细胞以及人类肠道吸收模型Caco-2中进行。潜在最重要的转运蛋白，即MRP2的作用，将在体内直接在MRP2缺陷的Tr-鼠身上进行检测。在特定的目标2中，我们将研究CYPS、UGTS和Sults之间的相互关系，包括鉴定所涉及的主要异构体，以及在肝脏和肠道中类黄酮的代谢。这将在微生物体和完整细胞中完成，例如新鲜的人类肝细胞。这些实验将使我们能够建立黄酮类化合物代谢的主要途径(S)。此外，还将研究类黄酮类代谢的自动诱导，主要集中在细胞色素P450和UGT上。UGT家族酶的重要性将在遗传缺陷的Gunn大鼠体内直接进行检验。在具体目标3中，我们将确定a)细菌和b)过氧化物酶介导的类黄酮分解代谢的作用和机制，包括与蛋白质的共价结合。A)中的实验将在灵知生菌与正常大鼠以及活体人类研究的样本中进行。补充的体外研究将包括鉴定从槲皮素到二氧化碳形成的细菌途径。B)中的实验将在体外进行，使用纯酶和亚细胞组分，然后在完整的细胞系统中进行，在该系统中，可以控制活性氧的产生以及谷胱甘肽的水平。代谢产物的结构鉴定以及共价结合的阐明将是关键因素。这些研究结果有助于我们了解黄酮类化合物的生物利用度，有助于优化这些天然或合成化合物的化学预防作用。

DESCRIPTION (provided by applicant): The long-term goal of this research program is to increase our understanding of how cellular transport and metabolism influence the oral bioavailability of dietary flavonoids, a large class of compounds that has been implicated to play a major role in the prevention of human diseases, in particular cardiovascular disease and cancer. In Specific Aim 1 we will determine the interrelationships between SGLT1 and MRP2, including mechanisms involved, in the enterocyte absorption of flavonoid glycosides and the tea flavonoids, two main classes of dietary flavonoids. These studies will be undertaken in SGLT1- and MRP2-transfected cells and in the human intestinal absorption model Caco-2. The role of the potentially most important transporter, i.e. MRP2, will be directly examined in vivo in the MRP2-deficient Tr- rat. In Specific Aim 2 we will investigate the interrelationships between CYPs, UGTs and SULTs, including the identification of the major isoforms involved, in the hepatic as well as intestinal metabolism of flavonoids. This will be done in microsomes as well as in intact cells, e.g. fresh human hepatocytes. These experiments will allow us to establish the major pathway(s) of metabolism of the flavonoids. In addition, autoinduction of flavonoid metabolism will be examined, mainly focusing on CYPs and UGTs. The importance of the UGT family of enzymes will be directly examined in vivo in the genetically deficient Gunn rat. In Specific Aim 3 we will determine the role and mechanisms of a) bacterial- and b) peroxidase-mediated catabolism of flavonoids, including covalent binding to protein. The experiments in a) will be conducted in gnotobiotic compared to normal rats as well as in samples from an in vivo human study. Complementary in vitro studies will include the identification of the bacterial pathway leading from quercetin to CO2 formation. The experiments in b) will be conducted in vitro, using pure enzymes and subcellular fractions, and then in intact cell systems in which production of reactive oxygen species as well as glutathione levels can be manipulated. Structure identification of metabolites as well as elucidation of covalent binding will be critical factors. The findings from the proposed studies should help us understand the bioavailability of the flavonoids, facilitating optimization of the chemopreventive utility of these natural or synthetic compounds.