United States Department of Agriculture, Agricultural Research Service, Robert Holley Center for Agriculture and Health;
J Vis Exp. 2022 Apr 28(182). doi: 10.3791/63859.
Knowledge of Fe bioavailability is critical to the assessment of the nutritional quality of Fe in foods. In vivo measurement of Fe bioavailability is limited by cost, throughput, and the caveats inherent to isotopic labeling of the food Fe. Thus, there exists a critical need for an approach that is high-throughput and cost-effective. The Caco-2 cell bioassay was developed to satisfy this need. The Caco-2 cell bioassay for Fe bioavailability utilizes simulated gastric and intestinal digestion coupled with culture of a human intestinal epithelial cell line known as Caco-2. In Caco-2 cells, Fe uptake stimulates the intracellular formation of ferritin, an Fe storage protein easily measured by enzyme-linked immunosorbent assay (ELISA). Ferritin forms in proportion to Fe uptake; thus, by measuring Caco-2 cell ferritin production, one can assess intestinal Fe uptake from simulated food digests into the enterocyte. Via this approach, the model replicates the key initial step that determines food Fe bioavailability. Since its inception in 1998, this model approach has been rigorously compared to factors known to influence human Fe bioavailability. Moreover, it has been applied in parallel studies, with three human efficacy studies evaluating Fe biofortified crops. In all cases, the bioassay correctly predicted the relative amounts of Fe bioavailability from the factors, crops, and overall diet. This paper provides detailed methods on Caco-2 cell culture coupled with the in vitro digestion process and cell ferritin ELISA necessary to conduct the Caco-2 cell bioassay for Fe bioavailability.
铁生物利用度的知识对于评估食物中铁的营养质量至关重要。铁生物利用度的体内测量受到成本、通量和食物铁同位素标记固有的限制。因此，迫切需要一种高通量且具有成本效益的方法。Caco-2 细胞生物测定法就是为了满足这一需求而开发的。铁生物利用度的 Caco-2 细胞生物测定法利用模拟的胃和肠消化，并结合一种称为 Caco-2 的人肠上皮细胞系进行培养。在 Caco-2 细胞中，铁摄取会刺激细胞内铁蛋白的形成，铁蛋白是一种很容易通过酶联免疫吸附测定（ELISA）测量的铁储存蛋白。铁蛋白的形成与铁摄取成正比；因此，通过测量 Caco-2 细胞中铁蛋白的产生，可以评估从模拟食物消化物进入肠细胞的肠内铁摄取。通过这种方法，该模型复制了决定食物铁生物利用度的关键初始步骤。自 1998 年成立以来，该模型方法已经与已知影响人体铁生物利用度的因素进行了严格比较。此外，它还应用于平行研究，其中三项人体功效研究评估了铁强化作物。在所有情况下，生物测定法都正确预测了因素、作物和整体饮食中铁生物利用度的相对量。本文提供了详细的 Caco-2 细胞培养方法，以及体外消化过程和细胞铁蛋白 ELISA，这些都是进行铁生物利用度的 Caco-2 细胞生物测定所必需的。
