FLAVONOID BIOAVAILABILITY IN HUMANS-CELLULAR STUDIES

描述（由申请人提供）：本研究计划的长期目标是增加我们对细胞转运和代谢如何影响膳食黄酮口服生物利用度的理解，膳食黄酮是一大类化合物，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SGLT 1和MRP 2之间的相互关系，包括参与类黄酮苷和茶类黄酮（两种主要的膳食类黄酮）的肠细胞吸收的机制。这些研究将在SGLT 1和MRP 2转染细胞和人肠道吸收模型Caco-2中进行。将在MRP 2缺陷型Tr-大鼠体内直接检查潜在最重要转运蛋白（即MRP 2）的作用。在具体目标2中，我们将研究CYP，UGT和SULT之间的相互关系，包括识别肝脏和肠道代谢类黄酮的主要亚型。这将在微粒体以及完整细胞（例如新鲜人肝细胞）中进行。这些实验将使我们能够建立类黄酮代谢的主要途径。此外，将检查类黄酮代谢的自诱导，主要集中在CYP和UGT。UGT家族酶的重要性将在遗传缺陷型古恩大鼠中直接进行体内检查。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催化剂的作用和机制，包括与蛋白质的共价结合。a）中的实验将在与正常大鼠相比的无菌动物中以及在来自体内人类研究的样品中进行。补充的体外研究将包括鉴定从槲皮素到CO2形成的细菌途径。B）中的实验将在体外进行，使用纯酶和亚细胞级分，然后在完整的细胞系统中进行，其中可以操纵活性氧的产生以及谷胱甘肽水平。代谢物的结构鉴定以及共价结合的阐明将是关键因素。拟议研究的结果应该有助于我们了解黄酮类化合物的生物利用度，促进优化这些天然或合成化合物的化学预防效用。

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.