Cleavage Products of Dietary Carotenoids: Occurrence & Nutritional Function

描述(由申请人提供)：饮食中类胡萝卜素的摄入与心血管疾病和癌症风险的降低有关。人类通过摄入水果和蔬菜来获得饮食中的类胡萝卜素。历史上，人们认为一些类胡萝卜素最重要的作用是通过中央双键裂解必需的微量营养素维生素A(视黄醇)来进行代谢转化。最近，人们对膳食类胡萝卜素“偏心裂解”成脱脂胡萝卜素以及它们可能氧化成脱脂胡萝卜素酸产生了浓厚的兴趣。这些类胡萝卜素代谢产物可能在类胡萝卜素的非维生素A活性中发挥重要作用。目前还没有对人类食物和组织中这些化合物水平的定量分析。关于完整类胡萝卜素对这些化合物的代谢程度及其作用机制的研究还缺乏证据。我们将解决这些知识上的差距。(1)我们确定了两种已知的人类类胡萝卜素代谢酶(BCO1和BCO2)的底物特异性，并对其产物进行了表征。这些研究还将确定细胞视黄醇结合蛋白和类胡萝卜素裂解酶在这些酶的代谢功能中的关系。(2)我们将使用分析化学来证明类胡萝卜素在人类中的相关性，以确定人类血浆、组织和食物中类胡萝卜素和非胡萝卜素的存在和数量。这将通过在受试者食用一份含有营养相关数量的β-胡萝卜素或番茄红素的番茄汁后立即定量测定父母类胡萝卜素和代谢物的血浆浓度，以及通过量化每日食用4周后血浆中的稳态浓度来实现。(3)我们将使用LNCaP细胞，这是一种表现出番茄红素依赖的生长抑制并表达BCO2的人前列腺癌细胞，在番茄红素处理的细胞中寻找脱脂蛋白和脱脂蛋白烯酸，以解决番茄红素在这些细胞中是否代谢以及代谢到什么程度。我们将使用生长抑制并诱导分化的人单核/巨噬细胞U937细胞与类胡萝卜素处理，寻找亲本类胡萝卜素的代谢。(4)我们将通过多种方式探索类胡萝卜素代谢物的生物学(营养)功能：(A)我们将使用我们已经显示出对膳食类胡萝卜素处理具有特有功能反应的细胞系，并询问这些反应是否通过与切割产物直接处理而获得；以及(B)我们将询问这些切割产物和代谢物是否直接激活或拮抗配体依赖的转录因子。(C)我们将喂饲BCO1基因敲除小鼠β-胡萝卜素和番茄红素，以评估BCO2对这些类胡萝卜素的代谢程度，以及类胡萝卜素代谢物在整个动物中的代谢影响。这一结果将加深我们对富含水果和蔬菜的饮食促进健康效果的分子基础的理解。

DESCRIPTION (provided by applicant): Consumption of dietary carotenoids is associated with a decreased risk of cardiovascular disease and cancer. Humans obtain carotenoids in the diet by ingesting fruits and vegetables. Historically, it was thought that the most important role of some carotenoids was their metabolic conversion via cleavage at the central double bond to the essential micronutrient, vitamin A (retinol). There is intense recent interest in the "eccentric cleavage" of dietary carotenoids to apocarotenals and their possible oxidation to apocarotenoic acids. These metabolites of carotenoids may play important roles in the non-vitamin A activities of carotenoids. There are no quantitative analyses of the levels of these compounds in human foods and tissues. Demonstrations of the extent of metabolism of intact carotenoids to these compounds and studies of their mechanisms of action are lacking. We will address these gaps in knowledge. (1) We determine the substrate specificity and characterize the products of the two known human carotenoid-metabolizing enzymes (BCO1 & BCO2). These studies will also establish the relationship of cellular retinol-binding proteins and carotenoid cleavage enzymes in the metabolic function of these enzymes. (2) We will demonstrate the relevance of apocarotenoids in humans using analytical chemistry to characterize the presence and quantify the amounts of apo-carotenals and apocarotenoic acids in human plasma and tissues and in foods. This will be accomplished by quantitation of the immediate post- prandial plasma concentrations of parent carotenoids and metabolites after subjects consume a single serving of tomato juice containing nutritionally relevant amounts of beta-carotene or lycopene as well as by quantitation of steady state concentrations in plasma after 4 weeks of daily consumption. (3) We will use the LNCaP cell, a human prostate cancer cell that demonstrates lycopene-dependent growth inhibition and expresses BCO2 to look for apolycopenals and apolycopenoic acids in lycopene-treated cells to address whether and to what extent lycopene is metabolized in these cells. We will use the human monocyte/macrophage cell, U937, which is growth inhibited and induced to differentiate with carotenoid treatment, to look for metabolism of the parent carotenoids. (4) We will probe the biological (nutritional) function of carotenoid metabolites in multiple ways: (a) we will use cell lines that we have shown have characteristic functional responses to treatment with dietary carotenoids and ask whether these responses are obtained by direct treatment with the cleavage products and (b) we will ask whether these cleavage products and metabolites directly activate or antagonize ligand-dependent transcription factors. (c) We will feed the BCO1 knockout mouse both beta-carotene and lycopene to assess the extent of the metabolism of these carotenoids by BCO2 and the metabolic effects of carotenoid metabolites in the whole animal. The results will enhance our understanding of the molecular basis for the health-promoting effects of diets rich in fruits and vegetables.