Elad Tako

Dietary inulin affects the expression of intestinal enterocyte iron transporters, receptors and storage protein and alters the microbiota in the pig intestine.

Inulin, a linear beta fructan, is present in a variety of plants including chicory root and wheat. It exhibits prebiotic properties and has been shown to enhance mineral absorption and increase beneficial bacteria in the colon. The aim of the present study was to assess the effect of dietary inulin on the gene expression of selected intestinal Fe transporters and binding proteins. Anaemic piglets at age 5 weeks were allocated to a standard maize-soya diet (control) or the same diet supplemented with inulin at a level of 4 %. After 6 weeks, the animals were killed and caecum contents and sections of the duodenum and colon were removed. Segments of the genes encoding for the pig divalent metal transporter 1 (DMT1) and duodenal cytochrome-b reductase (Dcytb) were isolated and sequenced. Semi-quantitative RT-PCR analyses were performed to evaluate the expression of DMT1, Dcytb, ferroportin, ferritin, transferrin receptor (TfR) and mucin genes. DMT1, Dcytb, ferroportin, ferritin and TfR mRNA levels in duodenal samples were significantly higher in the inulin group (P < or = 0.05) compared with the control. In colon, DMT1, TfR and ferritin mRNA levels significantly increased in the inulin group. Additionally, the caecal content microflora was examined using 16S rDNA targeted probes from bacterial DNA. The Lactobacillus and Bifidobacterium populations were significantly increased in the inulin group (P < or = 0.05) compared with the control group. These results indicate that dietary inulin might trigger an up regulation of genes encoding for Fe transporters in the enterocyte. The specific mechanism for this effect remains to be elucidated.

Biofortified indica rice attains iron and zinc nutrition dietary targets in the field

More than two billion people are micronutrient deficient. Polished grains of popular rice varieties have concentration of approximately 2 μg g−1 iron (Fe) and 16 μg g−1 zinc (Zn). The HarvestPlus breeding programs for biofortified rice target 13 μg g−1 Fe and 28 μg g−1 Zn to reach approximately 30% of the estimated average requirement (EAR). Reports on engineering Fe content in rice have shown an increase up to 18 μg g−1 in glasshouse settings; in contrast, under field conditions, 4 μg g−1 was the highest reported concentration. Here, we report on selected transgenic events, field evaluated in two countries, showing 15 μg g−1 Fe and 45.7 μg g−1 Zn in polished grain. Rigorous selection was applied to 1,689 IR64 transgenic events for insert cleanliness and, trait and agronomic performances. Event NASFer-274 containing rice nicotianamine synthase (OsNAS2) and soybean ferritin (SferH-1) genes showed a single locus insertion without a yield penalty or altered grain quality. Endosperm Fe and Zn enrichment was visualized by X-ray fluorescence imaging. The Caco-2 cell assay indicated that Fe is bioavailable. No harmful heavy metals were detected in the grain. The trait remained stable in different genotype backgrounds.

Biofortified black beans in a maize and bean diet provide more bioavailable iron to piglets than standard black beans.

Our objective was to compare the capacities of biofortified and standard black beans (Phaseolus vulgaris L.) to deliver iron (Fe) for hemoglobin (Hb) synthesis. Two lines of black beans, one standard and the other biofortified (high) in Fe (71 and 106 microg Fe/g, respectively), were used. Maize-based diets containing the beans were formulated to meet the nutrient requirements for swine except for Fe (Fe concentrations in the 2 diets were 42.9 +/- 1.2 and 54.6 +/- 0.9 mg/kg). At birth, pigs were injected with 50 mg of Fe as Fe dextran. At age 28 d, pigs were allocated to the experimental diets (n = 10). They were fed 2 times per day for 5 wk and given free access to water at all times. Body weights and Hb concentrations were measured weekly. Hb repletion efficiencies (means +/- SEM) did not differ between groups and, after 5 wk, were 20.8 +/- 2.1% for the standard Fe group and 20.9 +/- 2.1% for the high Fe group. Final total body Hb Fe contents did not differ between the standard [539 +/- 39 mg (9.7 +/- 0.7 micromol)] and high Fe [592 +/- 28 mg (10.6 +/- 0.5 micromol)] bean groups (P = 0.15). The increase in total body Hb Fe over the 5-wk feeding period was greater in the high Fe bean group [429 +/- 24 mg (7.7 +/- 0.4 micromol)] than in the standard Fe bean group [361 +/- 23 mg (6.4 +/- 0.4 micromol)] (P = 0.034). We conclude that the biofortified beans are a promising vehicle for increasing intakes of bioavailable Fe in human populations that consume beans as a dietary staple.

Higher iron pearl millet (Pennisetum glaucum L.) provides more absorbable iron that is limited by increased polyphenolic content

BackgroundOur objective was to compare the capacity of iron (Fe) biofortified and standard pearl millet (Pennisetum glaucum L.) to deliver Fe for hemoglobin (Hb)-synthesis. Pearl millet (PM) is common in West-Africa and India, and is well adapted to growing areas characterized by drought, low-soil fertility, and high-temperature. Because of its tolerance to difficult growing conditions, it can be grown in areas where other cereal crops, such as maize, would not survive. It accounts for approximately 50% of the total world-production of millet. Given the widespread use of PM in areas of the world affected by Fe-deficiency, it is important to establish whether biofortified-PM can improve Fe-nutriture.MethodsTwo isolines of PM, a low-Fe-control (“DG-9444”, Low-Fe) and biofortified (“ICTP-8203 Fe”,High-Fe) in Fe (26 μg and 85 μg-Fe/g, respectively) were used. PM-based diets were formulated to meet the nutrient requirements for the broiler (Gallus-gallus) except for Fe (Fe concentrations were 22.1±0.52 and 78.6±0.51 μg-Fe/g for the Low-Fe and High-Fe diets, respectively). For 6-weeks, Hb, feed-consumption and body-weight were measured (n = 12).ResultsImproved Fe-status was observed in the High-Fe group, as suggested by total-Hb-Fe values (15.5±0.8 and 26.7±1.4 mg, Low-Fe and High-Fe respectively, P<0.05). DMT-1, DcytB, and ferroportin mRNA-expression was higher (P<0.05) and liver-ferritin was lower (P>0.05) in the Low-Fe group versus High-Fe group. In-vitro comparisons indicated that the High-Fe PM should provide more absorbable-Fe; however, the cell-ferritin values of the in-vitro bioassay were very low. Such low in-vitro values, and as previously demonstrated, indicate the presence of high-levels of polyphenolic-compounds or/and phytic-acid that inhibit Fe-absorption. LC/MS-analysis yielded 15 unique parent aglycone polyphenolic-compounds elevated in the High-Fe line, corresponding to m/z = 431.09.ConclusionsThe High-Fe diet appeared to deliver more absorbable-Fe as evidenced by the increased Hb and Hb-Fe status. Results suggest that some PM varieties with higher Fe contents also contain elevated polyphenolic concentrations, which inhibit Fe-bioavailability. Our observations are important as these polyphenols-compounds represent potential targets which can perhaps be manipulated during the breeding process to yield improved dietary Fe-bioavailability. Therefore, the polyphenolic and phytate profiles of PM must be carefully evaluated in order to further improve the nutritional benefit of this crop.

Identification of Black Bean (Phaseolus vulgaris L.) Polyphenols That Inhibit and Promote Iron Uptake by Caco-2 Cells

In nutritional studies, polyphenolic compounds are considered to be inhibitors of Fe bioavailability. Because they are presumed to act in a similar manner, total polyphenols are commonly measured via the Folin-Ciocalteu colorimetric assay. This study measured the content of polyphenolic compounds in white and black beans and examined the effect of individual polyphenols on iron uptake by Caco-2 cells. Analysis of seed coat extracts by LC-MS revealed the presence of a range of polyphenols in black bean, but no detectable polyphenols in white bean. Extracts from black bean seed coats strongly inhibited iron uptake. Examination of the eight most abundant black bean seed coat, non-anthocyanin polyphenols via Caco-2 cell assays showed that four (catechin, 3,4-dihydroxybenzoic acid, kaempferol, and kaempferol 3-glucoside) clearly promoted iron uptake and four (myricetin, myricetin 3-glucoside, quercetin, and quercetin 3-glucoside) inhibited iron uptake. The four inhibitors were present in 3-fold higher total concentration than the promoters (143 ± 7.2 vs 43.6 ± 4.4 μM), consistent with the net inhibitory effect observed for black bean seed coats. The ability of some polyphenols to promote iron uptake and the identification of specific polyphenols that inhibit Fe uptake suggest a potential for breeding bean lines with improved iron nutritional qualities.

Polyphenolic compounds appear to limit the nutritional benefit of biofortified higher iron black bean (Phaseolus vulgaris L.)

BackgroundOur objective was to determine if a biofortified variety of black bean can provide more bioavailable-iron (Fe) than a standard variety. Two lines of black beans (Phaseolus-vulgaris L.), a standard (DOR500; 59μg Fe/g) and biofortified (MIB465; 88μg Fe/g) were used. The DOR500 is a common commercial variety, and the MIB465 is a line developed for higher-Fe content. Given the high prevalence of Fe-deficiency anemia worldwide, it is important to determine if Fe-biofortified black beans can provide more absorbable-Fe.MethodsBlack bean based diets were formulated to meet the nutrient requirements for the broiler (Gallus-gallus) except for Fe (dietary Fe-concentrations were 39.4±0.2 and 52.9±0.9 mg/kg diet, standard vs. biofortified, respectively). Birds (n=14) were fed the diets for 6-weeks. Hemoglobin-(Hb), liver-ferritin and Fe-related transporter/enzyme gene-expression were measured. Hemoglobin-maintenance-efficiency and total-body-Hb-Fe values were used to estimate Fe-bioavailability.ResultsHemoglobin-maintenance-efficiency values were higher (P<0.05) in the group consuming the standard-Fe beans on days 14, 21 and 28; indicating a compensatory response to lower dietary-Fe. Final total-Hb-Fe body content was higher in the biofortified vs. the standard group (26.6±0.9 and 24.4±0.8 mg, respectively; P<0.05). There were no differences in liver-ferritin or in expression of DMT-1, Dcyt-B, and ferroportin. In-vitro Fe-bioavailability assessment indicated very low Fe-bioavailability from both diets and between the two bean varieties (P>0.05). Such extremely-low in-vitro Fe-bioavailability measurement is indicative of the presence of high levels of polyphenolic-compounds that may inhibit Fe-absorption. High levels of these compounds would be expected in the black bean seed-coats.ConclusionsThe parameters of Fe-status measured in this study indicate that only a minor increase in absorbable-Fe was achieved with the higher-Fe beans. The results also raise the possibility that breeding for increased Fe-concentration elevated the levels of polyphenolic-compounds that can reduce bean Fe-bioavailability, although the higher levels of polyphenolics in the higher-Fe beans may simply be coincidental or an environmental effect. Regardless, Fe-biofortified beans remain a promising vehicle for increasing intakes of bioavailable-Fe in human populations that consume high levels of these beans as a dietary staple, and the bean polyphenol profile must be further evaluated and modified if possible in order to improve the nutritional quality of higher-Fe beans.

Chronic Zinc Deficiency Alters Chick Gut Microbiota Composition and Function

Zinc (Zn) deficiency is a prevalent micronutrient insufficiency. Although the gut is a vital organ for Zn utilization, and Zn deficiency is associated with impaired intestinal permeability and a global decrease in gastrointestinal health, alterations in the gut microbial ecology of the host under conditions of Zn deficiency have yet to be studied. Using the broiler chicken (Gallus gallus) model, the aim of this study was to characterize distinct cecal microbiota shifts induced by chronic dietary Zn depletion. We demonstrate that Zn deficiency induces significant taxonomic alterations and decreases overall species richness and diversity, establishing a microbial profile resembling that of various other pathological states. Through metagenomic analysis, we show that predicted Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways responsible for macro- and micronutrient uptake are significantly depleted under Zn deficiency; along with concomitant decreases in beneficial short chain fatty acids, such depletions may further preclude optimal host Zn availability. We also identify several candidate microbes that may play a significant role in modulating the bioavailability and utilization of dietary Zn during prolonged deficiency. Our results are the first to characterize a unique and dysbiotic cecal microbiota during Zn deficiency, and provide evidence for such microbial perturbations as potential effectors of the Zn deficient phenotype.

Dietary zinc deficiency affects blood linoleic acid: dihomo-γ-linolenic acid (LA:DGLA) ratio; a sensitive physiological marker of zinc status in vivo (Gallus gallus).

Zinc is a vital micronutrient used for over 300 enzymatic reactions and multiple biochemical and structural processes in the body. To date, sensitive and specific biological markers of zinc status are still needed. The aim of this study was to evaluate Gallus gallus as an in vivo model in the context of assessing the sensitivity of a previously unexplored potential zinc biomarker, the erythrocyte linoleic acid: dihomo-γ-linolenic acid (LA:DGLA) ratio. Diets identical in composition were formulated and two groups of birds (n = 12) were randomly separated upon hatching into two diets, Zn(+) (zinc adequate control, 42.3 μg/g zinc), and Zn(−) (zinc deficient, 2.5 μg/g zinc). Dietary zinc intake, body weight, serum zinc, and the erythrocyte fatty acid profile were measured weekly. At the conclusion of the study, tissues were collected for gene expression analysis. Body weight, feed consumption, zinc intake, and serum zinc were higher in the Zn(+) control versus Zn(−) group (p < 0.05). Hepatic TNF-α, IL-1β, and IL-6 gene expression were higher in the Zn(+) control group (p < 0.05), and hepatic Δ6 desaturase was significantly higher in the Zn(+) group (p < 0.001). The LA:DGLA ratio was significantly elevated in the Zn(−) group compared to the Zn(+) group (22.6 ± 0.5 and 18.5 ± 0.5, % w/w, respectively, p < 0.001). This study suggests erythrocyte LA:DGLA is able to differentiate zinc status between zinc adequate and zinc deficient birds, and may be a sensitive biomarker to assess dietary zinc manipulation.

Iron bioavailability to piglets from red and white common beans (Phaseolus vulgaris).

Polyphenols in foods may chelate dietary Fe and lower its bioavailability. Concentrations of phenols are higher in red beans than in white beans. The aim of this study was to compare iron bioavailabilities from red and white beans in a piglet hemoglobin repletion model. Fe deficient cross bred piglets (Hampshire x Landrace x Yorkshire) were used. Nutritionally balanced diets (except for Fe) were formulated to contain 50% precooked, dehydrated beans (either small red or Great Northern white). At age 5 weeks, the piglets were assigned to two groups and fed diets containing either red or white beans for 4 weeks. Weight and hemoglobin (Hb) concentrations were monitored weekly. Feed intakes were measured daily. Hemoglobin repletion efficiency (HRE) was calculated as the gain in total body hemoglobin Fe (Hb-Fe) divided by Fe intake. Hb concentrations, Hb-Fe gains, and HRE were not different between the groups at any time point ( p > 0.05). HRE values in the red bean group were 50% in the first week and 30% over the entire 4 week period. In the white bean group, they were 56 and 26%, respectively. Proline-rich protein mRNA concentrations in parotid glands were higher in the red bean group compared to the white bean group. These results show that iron bioavailabilities from red and white beans are similar and suggest that pigs adapt to the inhibitory effects of polyphenols on iron absorption by increasing the secretion of protective proline-rich proteins in the saliva.

Iron and zinc bioavailabilities to pigs from red and white beans (Phaseolus vulgaris L.) are similar.

Common beans contain relatively high concentrations of iron (Fe) and zinc (Zn) but are also high in polyphenols and phytates, factors that may inhibit Fe and Zn absorption. In vitro (Caco-2 cells) and in vivo (pigs) models were used to compare Fe and Zn bioavailabilities between red and white beans, which differ in polyphenol content. Bean/maize diets containing 37% of either white or red cooked beans were formulated. Fe uptake by Caco-2 cells was 14-fold higher from the white bean diet compared to the red bean diet. The diets were fed to anemic piglets (n = 10) for 35 days. On experiment days 7 and 21, pigs were given meals containing beans intrinsically labeled with stable isotopes of Fe and Zn ((58)Fe, (70)Zn), followed by intravenous (iv) injections of (54)Fe and (67)Zn, to assess Fe and Zn absorption. Isotope ratios determined by inductively coupled plasma mass spectrometry in whole blood and plasma samples were used to calculate iron and zinc absorption, respectively. On day 35, animals were killed and duodenal sections were collected for DMT1 gene expression analysis. Fe absorption was 14 and 16% from the first labeled meal and 9 and 10.5% from the second labeled meal for the white and red beans, respectively (P > 0.05). Zn absorption was 28 and 23% from the first meal (P > 0.05) and 31 and 29% from the second meal (P > 0.05) for the white and red beans, respectively. DMT1 gene expression did not differ between treatments. It was concluded that bean color does not affect Fe or Zn bioavailability in vivo and that beans are a good source of bioavailable Fe and Zn.