Iron Chlorophyllin Bio-efficacy and Metabolites Following Simulated Digestion and Incubation with Caco-2 Cells

Abstract Objectives Developing countries rely on poorly bioavailable plant-based sources of iron (i.e., FeSO4), leading to iron deficiency anemia. Heme iron is more bioavailable, but predominantly found in red meat. Iron chlorophyllin (IC) utilizes the porphyrin ring of plant-based chlorophyll to bind iron. IC has previously been shown to survive digestion, and to deliver iron to Caco-2 cells (measured as increased ferritin levels). However, the dose-response of IC treatment has not been assessed. We hypothesized that increasing IC concentrations would increase Caco-2 cell iron in a dose dependent manner. We also hypothesized that novel IC metabolites would be observed following in vitro digestion and incubation with Caco-2 cells. Methods In vitro digestion was performed using FeSO4 and hemoglobin as positive controls, deionized water as a negative control, and IC as the treatment (n = 3). Two doses (low and high) of both FeSO4 and hemoglobin were tested, in addition to four doses of IC (i.e., 2, 8, 34, and 81 ppm iron). Gastric and intestinal phases of digestion were mimicked, and digested chyme was centrifuged and filtered before incubation with differentiated Caco-2 human intestinal cells for 4 h. Cell were harvested, and iron concentrations in the chyme, micelles and harvested cells were tested using furnace atomic absorption spectrometry at 248.3 nm, and concentrations compared using one-way ANOVA, followed by Tukey's post-hoc test (P < 0.05). Metabolites of IC following digestion and cell incubation were tested using UHPLC-DAD-HRMS. Results A fraction (6–15%) of the IC was micellarized, with 19%-28% of heme iron micellarized. Cellular iron concentrations increased through the 8 ppm IC dose, but higher doses did not result in greater concentrations of cellular iron. IC delivered as much iron to the cells as heme, and trended toward increased iron delivery relative to FeSO4 (P = 0.068) when comparing across the low dose concentrations. Following digestion, Fe-chlorin e4 and e6 were totally converted to IC derivatives. Dehydrogenated and demethylated IC metabolites were also detected in the cell. Conclusions Results suggest that IC may better deliver iron to Caco-2 cells as compared to FeSO4, and should be further explored for iron supplementation. Funding Sources This project was partially supported by USDA-NIFA-AFRI A1363.