Caco-2 Cell Ferritin Formation Predicts Nonradiolabeled Food Iron Availability in an In Vitro Digestion/Caco-2 Cell Culture Model

Abstract

We have adapted an in vitro digestion/Caco-2 cell model to assess Fe availability from foods, by using ferritin formation by Caco-2 cells as an indicator of Fe uptake. Ferritin formation by Caco-2 cells occurs in response to Fe uptake at concentrations of available Fe greater than that of the culture media to which the cells have been adapted. This methodology circumvents the need for using radioactive Fe and thus eliminates the costs and controversies associated with food radiolabeling. To validate this method, we measured ferritin formation in Caco-2 cells exposed to digests containing Fe of relatively high and low availability. Our objective was to determine if ferritin formation would be proportional to Fe uptake and sufficiently sensitive to be an indicator of Fe availability from food digests. Our model uses established in vitro digestion techniques coupled with uptake of Fe by Caco-2 cell monolayers. Measurement of cell ferritin was done by a commercially available RIA. Higher ferritin formation was observed in cells exposed to digests containing FeSO4 plus ascorbic acid vs, digests containing FeSO4 plus citric acid. Additional comparisons of Fe availability from digests of beef, fish, corn and green beans yielded results that demonstrate higher Fe availability (i.e., greater ferritin formation) from beef and fish digests than from digests of corn and green beans. Overall, the results document the promotional effects of ascorbic acid and animal tissue on Fe uptake as measured indirectly by ferritin formation. The results of this study indicate that ferritin formation by Caco-2 cell monolayers is highly sensitive and accurately measures food Fe availability in this in vitro system.

Organic acid bioavailability from banana and sweet potato using an in vitro digestion and Caco-2 cell model

“…The protocol was developed by modifying the method of Glahn et al and Laurent et al [19][20][21][22]. Fresh (4 g) or freeze-dried (1 g) banana and sweet potato were ground with a food chopper from Zyliss Ò (Oxenford, Australia), applying ten pressures for all samples to simulate mastication.…”

“…To assess potential bioavailability, in vitro digestion/Caco-2 cell culture models have been developed in recent years. The model modified from Glahn et al and from Laurent et al has been used in the current study [19][20][21][22]. In vitro digestion simulates the physiochemical and biochemical conditions encountered in the gastrointestinal tract and is used to estimate the rate and extent of release of bioactive compounds from the food matrix into the intestinal lumen (bioaccessibility) and therefore potentially available for uptake.…”

Almond milk fermented with different potentially probiotic bacteria improves iron uptake by intestinal epithelial (Caco-2) cells

“…Cells were maintained with Dulbecco's modified Eagle's medium (DMEM) (Gibco ® , Madrid, Spain) under conditions previously described by Glahn et al (1998). For the assays, Caco-2 cells were seeded at 50,000 cell/cm 2 in collagen-treated 6-well culture plates (Costar, Cambridge, MA, USA), and were grown with DMEM for 12 days.…”

“…50 µmol/L of FeCl 3 were added to the digested almond milk samples and the ferritin formation by Caco-2 cells over a 24 h period was proportional to the cell iron uptake. A latex-enhanced turbidimetric immunoassay (Ferritin-turbilatex; Spinreact, Girona, Spain) was used to measure the Caco-2 cell ferritin content, as Glahn et al (1998) …”

“…Moreover, if the fermentation process is carried out by potentially probiotic bacteria, the developed fermented product could be useful as a means of preventing some immunomodulatory diseases, such as allergies. The Caco-2 cell line, commonly used in conjunction with in vitro digestion techniques, is a useful model for studying intestinal human iron uptake, which it allows to occur simultaneously with food digestion, and is generally regarded as the best available intestinal cell model for studying the mechanisms associated with vectorial iron transport (Glahn, Lee, Yeung, Goldman, & Miller, 1998). In addition, the macrophage-derived RAW 264.7 cell line expresses key genes and proteins of principal pathways for the production of regulatory cytokines (Novak, Babcock, Jho, Helton, & Espat, 2003) and constitutes a cell model used worldwide and a useful tool with which to study the inflammatory response(s) and metabolic activity promoted by food-derived components while still maintaining a rapid and inexpensive system (Deepika, Rastall, & Charalampopoulos, 2011;Kabeerdoss et al, 2011).…”