Iron from nanostructured ferric phosphate: absorption and biodistribution in mice and bioavailability in iron deficient anemic women

Abstract

Food fortification with iron nanoparticles (NPs) could help prevent iron deficiency anemia, but the absorption pathway and biodistribution of iron-NPs and their bioavailability in humans is unclear. Dietary non-heme iron is physiologically absorbed via the divalent metal transporter-1 (DMT1) pathway. Using radio- iron isotope labelling in mice with a partial knockdown of intestine-specific DMT1, we assessed oral absorption and tissue biodistribution of nanostructured ferric phosphate (FePO4-NP; specific surface area [SSA] 98 m2g-1) compared to to ferrous sulfate (FeSO4), the reference compound. We show that absorption of iron from FePO4-NP appears to be largely DMT1 dependent and that its biodistribution after absorption is similar to that from FeSO4, without abnormal deposition of iron in the reticuloendothelial system. Furthermore, we demonstrate high bioavailability from iron NPs in iron deficient anemic women in a randomized, cross-over study using stable-isotope labelling: absorption and subsequent erythrocyte iron utilization from two 57Fe-labeled FePO4-NP with SSAs of 98 m2g-1 and 188 m2g-1 was 2.8-fold and 5.4-fold higher than from bulk FePO4 with an SSA of 25 m2g-1 (P < 0.001) when added to a rice and vegetable meal consumed by iron deficient anemic women. The FePO4-NP 188 m2g-1 achieved 72% relative bioavailability compared to FeSO4. These data suggest FePO4-NPs may be useful for nutritional applications.

Conflict of interest statement

The authors declare no competing interests.

© 2022. The Author(s).

Figures

Figure 1

Particle characterization. (a–c) TEM micrographs of (a) bulk FePO4, (b) 98 m2 g−1 FePO4 and (c) 188 m2 g−1 FePO4 , scale bars: 100 nm. (d) XRD patterns of 25 m2 g−1, 98 m2 g−1 and 188 m2 g−1 FePO4. The absence of peaks in the X-ray diffraction (XRD) analysis indicates that all compounds were XRD amorphous. X-axis indicates the diffraction angle (2-Theta), Y-axis indicates the counts.

Figure 2

Characterization of the DMT1int/int mouse model. (a) Schematic illustration of the floxed allele and primer positions used to confirm excision by PCR genotyping of intestine knockdown (DMT1int/int, lane 2–4, red box) and control (DMT1fl/fl, lane 5 and 7, blue boxes) mice. Image was cropped and compiled from original images available as Supplementary Figs. 1 and 2. (b) Haemoglobin (Hb) trajectory (PND 24‒42) in DMT1int/int (n = 6 male; n = 4 female) and DMT1fl/fl (n = 10 male; n = 11 female) mice receiving the AIN93G diet containing (i) 35 ppm iron (as ferrous citrate) and (ii) 3 ppm iron (native). Results are shown as means ± SEM. c, Expression of (i) DMT1-exon1A and (ii) DMT1-IRE mRNA in duodenum, colon and liver of 42-day-old DMT1int/int and DMT1fl/fl mice fed an iron deficient (3 ppm iron) diet from PND 24‒42, normalized to 18S and βActin as endogenous reference genes to calculate delta Ct values. Differences in gene expression by genotype (DMT1int/int vs. DMT1fl/fl) were determined by two-sided independent t-tests. The results are shown as means ± SEM and differences were considered significant at p < 0.05.

Figure 3

Eighteen-day feeding study in DMT1int/int and DMT1fl/fl mice. (a) Outline of feeding study. (b) Hemoglobin (Hb) trajectory in (i) DMT1int/int (n = 6 male; n = 9 female) and (ii) DMT1fl/fl (n = 6 male; n = 7 female) mice receiving the AIN93G diet fortified with FePO4-NP (SSA 98 m2 g−1) or FeSO4 (reference compound). Results are shown as means ± SEM. (c) Liver iron concentrations in DMT1int/int and DMT1fl/fl mice fed diets fortified with 35 ppm FeSO4 or FePO4-NP for 18 days (postnatal days [PND] 24‒42). Differences in liver iron concentrations by genotype (DMT1int/int vs. DMT1fl/fl) and by iron compound (FePO4-NP vs. FeSO4) were determined by two-sided independent t-tests. The results are shown as means ± SEM and differences were considered significant at p < 0.05.

Figure 4

Absorption and biodistribution of a single oral dose of radiolabeled FePO4-NP and FeSO4 iron deficient anemic DMT1int/int and DMT1fl/fl mice. (a) Outline of radioisotope study. (b) Biodistribution of 59Fe (% of initial 59Fe/g tissue) from a single dose (~ 50 µg) of FePO4-NP (SSA 98 m2 g−1) after oral gavage in iron deficient anemic DMT1fl/fl (n = 7 male; n = 5 female) and DMT1int/int (n = 7 male, n = 8 female) mice. Differences in tissue 59Fe distribution by genotype (DMT1int/int vs. DMT1fl/fl) were determined by two-sided independent t-tests. The results are shown as means ± SEM and differences were considered significant at p < 0.05. (c) Biodistribution of 59Fe from FePO4-NP and FeSO4 (reference compound) 24 h after oral gavage in iron deficient anemic (i) DMT1int/int mice and (ii) DMT1fl/fl mice. Differences in tissue 59Fe distribution by iron compound (FePO4-NT vs. FeSO4) were determined by two-sided independent t-tests. The results are shown as means ± SEM and differences were considered significant at p < 0.05.

Figure 5

Iron bioavailability of two 57Fe-labeled FePO4-NP in iron deficient anemic women (n = 18). (a) Outline of randomized cross-over study. (b) Fractional iron absorption (%) from rice test meals fortified with FePO4-NPs (SSA 98 m2g−1 and 188 m2 g−1) labeled with a stable isotope (57Fe), and 58Fe-labeled bulk FePO4 (SSA 25 m2 g−1) and FeSO4 as negative and positive reference compounds, respectively. Meal sequence was randomized across all women. Pairwise comparisons were performed using two-sided paired t-tests with Bonferroni adjustment for multiple testing. Boxes indicate median and interquartile ranges, whiskers describe the range of the data (min to max). Differences were considered significant at p < 0.05. CRP C-reactive protein, Hb hemoglobin.