FLAVONOID BIOAVAILABILITY IN HUMANS--CELLULAR STUDIES

最近的流行病学研究强烈表明，类黄酮，一个大的 存在于水果、蔬菜和饮料中的一类植物化学物质， 在人类疾病中具有保护作用，最突出的是在冠状动脉疾病中， 心脏病和癌症。 快速增长的生物化学 研究显示了这些影响的合理机制。 但不 很清楚饮食中的黄酮类化合物，包括它们的糖苷， 进入拟议的细胞作用部位;它们在 人类基本上还是未知的。 该项目的研究提出， 来帮助填补这一重要的信息空白。 我们的一般工作 一种假说是，类黄酮的生物利用度是一个函数， 细胞膜穿透性、蛋白质结合和系统前代谢， 并且随着类黄酮的化学结构而显著变化。 在 目的1：研究人肝癌细胞对黄酮类化合物的摄取 Hep G2和新鲜肝细胞及其相关机制。 这将 包括载体介导转运蛋白的特异性抑制剂的研究。 它还将包括测定血浆蛋白结合的 黄酮类化合物及其对膜渗透的影响。 在目标2中， 用Hep G2细胞测定黄酮类化合物的代谢 作为人肝匀浆。 这将包括HPLC分离 代谢物和结构鉴定使用大气压 化学电离（APCI）质谱（MS）和高场核 磁共振（NMR）光谱法。 所涉及的酶将是 也可通过使用选择性化学和抗体抑制剂进行鉴定 作为同种型特异性表达系统。 在目标3中，我们将确定 类黄酮穿越肠上皮细胞边界的能力， 使用人Caco-2细胞系，作为单层生长在可渗透的 过滤器和切除的大鼠肠作为模型。 和肝细胞一样 在目标1中，我们将确定化学结构对膜的影响 渗透性和运输机制。 这些研究还将 包括P-糖蛋白介导外排以及代谢作为因子 限制净运输。 在目标4中，我们将测试我们的体外观察结果 直接在体内，使用药代动力学方法。 在这些研究中 我们将首先寻求确定类黄酮的生物利用度， 糖苷及其随后水解为糖苷配基， 志愿者通过血浆和尿液测定。

Recent epidemiological studies strongly suggest that flavonoids, a large class of phytochemicals present in fruits, vegetables and beverages, have protective roles in human diseases, most prominently in coronary heart disease and cancer. A rapidly growing number of biochemical studies show plausible mechanisms for these effects. However, it is not clear if the dietary flavonoids, including their glycosides, can gain access to proposed cellular sites of action; their biological fate in humans remains essentially unknown. The studies in this project propose to help fill this essential information gap. Our general working hypothesis is that the bioavailability of flavonoids is a function of cell membrane penetrability, protein binding and presystemic metabolism, and varies markedly with the chemical structure of the flavonoid. In Aim 1 we will focus on flavonoid uptake by the human hepatoma cell line Hep G2 and fresh hepatocytes and the mechanism(s) involved. This will include studies of specific inhibitors of carrier-mediated transporters. It will also include determination of plasma protein binding of the flavonoids and its effect on membrane penetration. In Aim 2 we will determine the metabolism of the flavonoids, using Hep G2 cells as well as human liver homogenates. This will include HPLC isolation of metabolites and structure identification using atmospheric pressure chemical ionization (APCI) mass spectrometry (MS) and high field nuclear magnetic resonance (NMR) spectrometry. The enzymes involved will be identified by use of selective chemical and antibody inhibitors as well as isoform-specific expression systems. In Aim 3 we will determine the ability of flavonoids to traverse the intestinal epithelial cell border, using the human Caco-2 cell line, grown as monolayers on permeable filters, and excised rat intestine, as models. As with the hepatocytes in Aim 1, we will determine the effect of chemical structure on membrane permeability and mechanism(s) of transport. These studies will also include P-glycoprotein-mediated efflux as well as metabolism as factors limiting net transport. In aim 4 we will test our in vitro observations directly in vivo, using pharmacokinetic approaches. In these studies we will in first hand seek to determine the bioavailability of flavonoid glycosides and their subsequent hydrolysis to aglycones in normal volunteers through plasma and urine determinations.