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Hyun-tae Kim,1 John P Loftus,1 Jason W Gagné,1 Michael A Rutzke,2 Raymond P Glahn,3 Joseph J Wakshlag1 1Cornell University College of Veterinary Medicine, Department of Clinical Sciences, Ithaca, NY, USA; 2Cornell University, College of Agriculture and Life Sciences, School of Integrative Plant Sciences, Ithaca, NY, USA; 3Robert W. Holley Center for Agriculture and Health, Agricultural Research Service, USDA, Ithaca, NY, USA Purpose: To evaluate the concentrations of chromium, nickel, molybdenum, silica, and aluminum in several commercially available dry dog foods and compare these with current World Health Organization’s (WHO) mean human daily dietary intake. Conversion of dietary intake per megacalorie (Mcal) for both dog foods and human average intake was performed based on the National Research Council recommendation of a 2,900 kcal diet for comparative purposes to average intake and potential toxic exposure.Materials and methods: Forty-nine over-the-counter dry foods formulated for maintenance of healthy dogs yet listed as all life stage foods were analyzed. Concentrations of the ultra-trace minerals were measured via inductively coupled plasma atomic emission and represented per Mcal for comparative purposes as it relates to common intake in dogs in comparison with humans.Results: Chromium, molybdenum, and aluminum concentrations in all of the dog foods were at levels that would be considered above average human daily consumption on a caloric basis. Nickel and silica calculated intakes per Mcal were comparable with human intake patterns, while both trace minerals displayed outliers exceeding at least twofold of the upper range of human daily intake.Conclusion: Overall, ultra-trace minerals found in dog foods were above the expected average daily intake for humans on a caloric basis. There was no evidence of potential chronic toxic exposure based on presumptive intake extrapolated from WHO published toxic intake concentrations for humans or domestic animals. The large range of silica intake from various foods (2.96–83.67 mg/1,000 kcal) may have health implications in dogs prone to silica urolithiasis. Further studies investigating the bioavailability of these ultra-trace minerals and establishing dietary ultra-trace mineral allowance would be ideal; however, based on these findings, consumption of these ultra-trace minerals in over-the-counter dry dog foods appears safe. Keywords: dog food, ultra-trace minerals, food safety, ICP-AES

Inulin, a linear β-fructan, is present in a variety of plants, with relatively high levels of up to 20% in chicory root. It exhibits prebiotic properties and was shown to enhance mineral absorption. Our objectives were to assess the effect of intra-amniotic administration of inulin at 17 d of incubation on the iron status of broiler chicks (at hatch, 21 d) and to continue to monitor iron status with and without dietary inulin on these hatchlings for 42 d. The study included 3 prehatch treatment groups (n = 30): 1) mulin, inulin solution (4% inulin/0.85% saline); 2) control 1, untreated eggs; and 3) control 2, saline solution (0.85% saline). Solutions were injected into the naturally consumed amniotic fluid of 17-d-old chicken embryos (groups 1, 3). Upon hatch (93% hatchability), and from each group, 10 chicks were killed and their small intestine, liver, and cecum were removed for mRNA abundance of intestinal iron-related transporters, liver ferritin amounts, and bacterial analysis of cecal content, respectively. From the remaining chicks of each group, chicks were allbcated to a standard corn-based diet (+/4% inulin, n = 10). During the trial, hemoglobin concentrations and body hemoglobin-Fe values were higher in the inulin group versus controls (P < 0.05). On d 42, birds were anesthetized and their duodenal loops were exposed. A nonocclusive catheter was inserted into the duodenal vein for blood sampling. A solution containing 58Fe (0.1 mg of Fe/10 mM ascorbic acid) added to the digested diet sample was injected into the loop. Blood samples were collected every 5 mm and for 90 mm postinjection and analyzed by inductively coupled argon-plasma mass spectrometry for 58Fe concentrations. At the end of the procedure, animals were killed and cecum contents and sections of the duodenum and liver were removed. Results showed that 58Fe absorption rates were at times higher in the inulin group versus the other groups. Also, mRNA abundance of DMT1 (an Fe transporter) and ferroportin in addition to liver ferritin amounts were higher (P < 0.05) in the inulin group versus controls. Results indicate that intra-amniotic administration and dietary inulin improved the iron status of iron-deficient broilers. [ABSTRACT FROM AUTHOR]

Inulin, a prebiotic, may enhance intestinal Fe absorption. Our objective was to assess the effects of supplemental inulin and 2 probiotic bacteria ( B. infantis and L. acidophillus) on Fe availability to Caco-2 cells from common white and red beans ( Phaseolus vulgaris L.). Cooked beans were mixed or not with supplemental inulin (4%, w/w), and then subjected to simulated gastrointestinal digestion (pepsin, pH 2; pancreatin, pH 7.2). Subsequently, the digests were incubated overnight with and without B. infantis or L. acidophilus. Ferritin formation in Caco-2 cells was used to evaluate Fe uptake. Total soluble phenols (Folin–Ciocalteau) and phytate (HPLC-electrochemical detection) were quantified, and the flavonoids profile (HPLC-PDA/UV detection) was monitored in the digests. Supplemental inulin did not affect Fe uptake from white nor red beans. Incubation with B. infantis increased total soluble phenols (TSP) in the digests and decreased Fe uptake. Incubation with L. acidophilus decreased TSP in the digest and increased Fe uptake. Variations in Fe uptake were not associated with soluble phytate concentrations in the digests. The largest change in flavonoids profile were found in the digests incubated with L. acidophilus, which decreased the soluble concentration of astragalin (kaempferol-3-O-glucoside). These results suggest that certain probiotics could increase Fe uptake from common beans. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

We have applied an in vitro digestion/Caco-2 cell culture model to the assessment of iron availability from human milk and a generic cow's milk-based infant formula. Experiments were designed to determine the availability of iron from human milk relative to infant formula and whether known promoters of iron absorption would increase Caco-2 cell iron uptake and availability from the infant formula. In addition, we sought to determine if decreasing the citrate concentration in the infant formula would increase the iron uptake. Although approximately twice as much iron was in solution from digests of the infant formula relative to that of human milk, smaller or equal amounts of iron were taken up from the infant formula relative to the human milk digest. These results are qualitatively similar to in vivo studies. Addition of known iron uptake promoters to infant formula did not enhance Caco-2 cell iron uptake from the infant formula digest, indicating that the iron in the infant formula existed predominantly in a tightly bound unavailable form(s). Enzymatic pretreatment of the infant formula with citrate lyase and oxalacetate decarboxylase decreased the citrate concentration by 67% and resulted in a 64% increase of iron in solution, which corresponded to a 46% increase in the cell iron uptake. Iron uptake from the "low citrate" formula plus cysteine was 102% greater relative to the nontreated formula. The results indicate that too much citrate can reduce iron uptake, particularly if it is present at concentrations greater than promoters such as ascorbic acid and cysteine. [ABSTRACT FROM AUTHOR]

Human and animal studies have shown that amino acids and peptides influence iron absorption from the intestinal lumen. This study was conducted using Caco-2 cell monolayers as the experimental model to determine whether similar effects on iron absorption occur. Conditions were chosen to mimic the pH of the intestinal lumen and the most likely order whereby ferric and ferrous forms of iron would combine with various amino acids and dipeptides resulting from protein digestion. We demonstrated the enhancing effect of cysteine and reduced cysteinyl glycine on iron uptake by Caco-2 cells. The addition of glutathione to the transport media had no effect on uptake from ferrous or ferric iron complexes, nor did it affect iron solubility. Cysteine and reduced cysteinyl glycine increased iron solubility when added to a solution containing insoluble iron. This effect is different from that of ascorbate, which must be combined with soluble ferric iron at pH 2 to reduce and solubilize iron. Taken together, these observations are evidence that cysteine and reduced N-terminal cysteine peptides are capable of enhancing iron uptake from soluble and insoluble ferric iron. These results qualitatively reflect those observed in human studies. Our results indicate that glutathione requires digestion to Cys or Cys-Gly in order to promote iron uptake. The similarity between this study and human studies further reinforces that the Caco-2 cell model is a useful tool in studies of iron absorption and bioavailability. [ABSTRACT FROM AUTHOR]

Abstract Background Iron (Fe) deficiency is the most common micronutrient deficiency worldwide. Iron biofortification is a preventative strategy that alleviates Fe deficiency by improving the amount of absorbable Fe in crops. In the present study, we used an in vitro digestion/Caco 2 cell culture model as the guiding tool for breeding and development of two maize (Zea mays L.) lines with contrasting Fe bioavailability (ie. Low and High). Our objective was to confirm and validate the in vitro results and approach. Also, to compare the capacities of our two maize hybrid varieties to deliver Fe for hemoglobin (Hb) synthesis and to improve the Fe status of Fe deficient broiler chickens. Methods We compared the Fe-bioavailability between these two maize varieties with the presence or absence of added Fe in the maize based-diets. Diets were made with 75% (w/w) maize of either low or high Fe-bioavailability maize, with or without Fe (ferric citrate). Chicks (Gallus gallus) were fed the diets for 6 wk. Hb, liver ferritin and Fe related transporter/enzyme gene-expression were measured. Hemoglobin maintenance efficiency (HME) and total body Hb Fe values were used to estimate Fe bioavailability from the diets. Results DMT-1, DcytB and ferroportin expressions were higher (P Conclusions We conclude that the High Fe-bioavailability maize contains more bioavailable Fe than the Low Fe-bioavailability maize, presumably due to a more favorable matrix for absorption. Maize shows promise for Fe biofortification; therefore, human trials should be conducted to determine the efficacy of consuming the high bioavailable Fe maize to reduce Fe deficiency.

Abstract Background Our objective was to compare the capacities of biofortified and standard colored beans to deliver iron (Fe) for hemoglobin synthesis. Two isolines of large-seeded, red mottled Andean beans (Phaseolus vulgaris L.), one standard ("Low Fe") and the other biofortified ("High Fe") in Fe (49 and 71 μg Fe/g, respectively) were used. This commercial class of red mottled beans is the preferred varietal type for most of the Caribbean and Eastern and Southern Africa where almost three quarters of a million hectares are grown. Therefore it is important to know the affect of biofortification of these beans on diets that simulate human feeding studies. Methods Maize-based diets containing the beans were formulated to meet the nutrient requirements for broiler except for Fe (Fe concentrations in the 2 diets were 42.9 ± 1.2 and 54.6 ± 0.9 mg/kg). One day old chicks (Gallus gallus) were allocated to the experimental diets (n = 12). For 4 wk, hemoglobin, feed-consumption and body-weights were measured. Results Hemoglobin maintenance efficiencies (HME) (means ± SEM) were different between groups on days 14 and 21 of the experiment (P < 0.05). Final total body hemoglobin Fe contents were different between the standard (12.58 ± 1.0 mg {0.228 ± 0.01 μmol}) and high Fe (15.04 ± 0.65 mg {0.273 ± 0.01 μmol}) bean groups (P < 0.05). At the end of the experiment, tissue samples were collected from the intestinal duodenum and liver for further analyses. Divalent-metal-transporter-1, duodenal-cytochrome-B, and ferroportin expressions were higher and liver ferritin was lower (P < 0.05) in the standard group vs. the biofortified group. In-vitro analysis showed lower iron bioavailability in cells exposed to standard ("Low Fe") bean based diet. Conclusions We conclude that the in-vivo results support the in-vitro observations; biofortified colored beans contain more bioavailable-iron than standard colored beans. In addition, biofortified beans seems to be a promising vehicle for increasing intakes of bioavailable Fe in human populations that consume these beans as a dietary staple. This justifies further work on the large-seeded Andean beans which are the staple of a large-region of Africa where iron-deficiency anemia is a primary cause of infant death and poor health status.

Inulin, a linear beta fructan present in a variety of plants, exhibits prebiotic properties. The aim of this study was to assess dietary inulin effects on the expression of genes that encode for selected intestinal iron transporters, on villus surface area and on several microbiota genera. Iron deficient and iron adequate rats were fed a diet containing either 0, 4 or 8% supplemental inulin (n=9). At day 30, animals were killed and cecum contents and sections of tissue from the duodenum were collected for analysis of microbial populations and gene expression, respectively. Semi quantitative RT-PCR used to evaluate relative expression of DMT1, ferritin and ferroportin. DMT1 and ferroportin expressions were higher (P<0.05) in 0% inulin groups compared to inulin supplemented groups. Ferritin expression increased (P<0.05) in 4% and 8% inulin groups compared to 0% inulin. In both iron adequate and deficient rats, dietary inulin increased (P<0.05) intestinal villus surface area. For bacterial analysis, we used cecal contents bacterial DNA and specific primers directed at 4 bacterial genera: Lactobacillus, Bifidobacterium, E. Coli and Clostridium. Lactobacillus populations increased (P<0.05) in the 8% inulin groups compared to others. Bifidobacterium populations increased in the 4% and 8 % inulin groups of the iron adequate rats. Inulin had no effect on E.Coli or Clostridium populations. These results suggest that the inulin effects on bacterial genera, villus surface area and genes encoding for iron transporters are possible mechanisms whereby inulin enhances iron absorption by enterocytes. [ABSTRACT FROM AUTHOR]

Iron fortification of foods and biofortification of staple food crops are strategies that can help to alleviate Fe deficiency. The broiler chicken may be a useful model for initial in vivo screening of Fe bioavailability in foods due to its growth rate, anatomy, size, and low cost. In this study, we assess the broiler as a model for hemoglobin (Hb) maintenance studies and present a unique duodenal loop technique for direct measurement of intestinal Fe absorption. One-week-old chicks were allocated into Fe-deficient versus Fe-adequate treatment groups. For 6 wk, blood Hb, feed consumption, and BW were measured. At wk 7, birds were anesthetized and their duodenal loops were exposed. The loop was isolated and a nonocclusive catheter was inserted into the duodenal vein for blood sampling. A stable isotope solution containing 58Fe (1 mg of Fe in 10 mM ascorbic acid) was injected into the loop. Blood samples were collected every 5 mm and for 120 mm postinjection and analyzed by inductively coupled argon-plasma mass spectrometry for 58Fe concentrations. In the low-Fe group, Hb concentrations, total body Hb Fe, and BW were lower and Hb maintenance efficiency (indicator for dietary Fe availability) was higher than in the high-Fe group (P < 0.05). Iron absorption was higher in the Fe-deficient birds (P < 0.05). In addition, expression of proteins involved in Fe uptake and transfer [i.e., divalent metal transporter 1 (Fe uptake transporter), ferroportin (involved in Fe transport across the enterocyte), and duodenal cytochrome B reductase (reduces Fe at brush border membrane)] were elevated in the low-Fe group. These results indicate that this model exhibits the appropriate responses to Fe deficiency and has potential to serve as a model for Fe bioavailability. Such a model should be most useful as an intermediate test of in vivo Fe bioavailability observations in preparation for subsequent human studies. [ABSTRACT FROM AUTHOR]

Adding human milk fortifiers (HMF) to human milk (HM) is one way of overcoming the nutrient deficits found in the latter. In this study, the bioavailabilities of calcium, zinc, and iron in S-26/SMA HMF added to HM were compared with those in HM fortified with various bovine milk proteins: α-lactalbumin, colostrum, caseinate, casein phosphopeptides, and whey protein concentrate. The bioavailability of each mineral was assessed using an in vitro digestion/Caco-2 cell culture model. Calcium and zinc uptake by the cells was traced with radioisotopes; iron uptake was assessed via cell ferritin levels. Samples were prepared on an equal protein content basis and with added calcium, but no zinc or iron was added. Results revealed that calcium uptake from HM + S-26/SMA was not different from any of the HM fortified with the bovine milk proteins, except for unfortified HM and HM + colostrum in which calcium uptake was significantly lower (-89 and -38%, respectively). Uptake of zinc and iron were significantly higher for HM + S-26/SMA than for the other HM + fortifiers. [ABSTRACT FROM AUTHOR]

Dry bean is a nutrient-dense food targeted in biofortification programs to increase seed iron and zinc levels. The underlying assumption of breeding for higher mineral content is that enhanced iron and zinc levels will deliver health benefits to the consumers of these biofortified foods. This study characterized a diversity panel of 275 genotypes comprising the Yellow Bean Collection (YBC) for seed Fe and Zn concentration, Fe bioavailability (FeBio), and seed yield across 2 years in two field locations. The genetic architecture of each trait was elucidated via genome-wide association studies (GWAS) and the efficacy of genomic prediction (GP) was assessed. Moreover, 82 yellow breeding lines were evaluated for seed Fe and Zn concentrations as well as seed yield, serving as a prediction set for GP models. Large phenotypic variability was identified in all traits evaluated, and variations of up to 2.8 and 13.7-fold were observed for Fe concentration and FeBio, respectively. Prediction accuracies in the YBC ranged from a low of 0.12 for Fe concentration, to a high of 0.72 for FeBio, and an accuracy improvement of 0.03 was observed when a QTN, identified through GWAS, was used as a fixed effect for FeBio. This study provides evidence of the lack of correlation between FeBio estimated in vitro and Fe concentration and highlights the potential of GP in accurately predicting FeBio in yellow beans, offering a cost-effective alternative to the traditional assessment of using Caco2 cell methodologies., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Izquierdo, Sadohara, Wiesinger, Glahn, Urrea and Cichy.)

Field pea is a pulse that delivers high protein content, slowly digestible starch and fiber, and many vitamins and minerals, including iron. Naturally occurring plant phytic acid molecules bind iron, lowering its availability for absorption during digestion. Two low phytic acid ( lpa ) pea lines, 1-2347-144 and 1-150-81, developed by our group had 15% lower yield and 6% lower seed weight relative to their progenitor cultivar. Subsequently, we crossed the two lpa lines and two cultivars, and derived 19 promising lpa pea breeding lines; here we document their agronomic performance based on 10 replicated field trials in Saskatchewan. Seventeen of these lpa lines yielded greater than 95% of the check mean (associated cultivars) and 16 were above 98% of the check mean for 1000 seed weight. The 19 lpa lines showed 27 to 55% lower phytic acid concentration than the check mean. Iron concentrations were similar in all the lpa lines and cultivars, yet the Caco-2 human cell culture assay revealed 14 of the 19 lpa lines had 11 to 55% greater iron bioavailability than check means. Thus, a single round of plant breeding has allowed for closing the gap in performance of low phytic acid pea.

Geophagy, the intentional consumption of earth materials, has been recorded in humans and other animals. It has been hypothesized that geophagy is an adaptive behavior, and that clay minerals commonly found in eaten soil can provide protection from toxins and/or supplement micronutrients. To test these hypotheses, we monitored chimpanzee geophagy using camera traps in four permanent sites at the Budongo Forest Reserve, Uganda, from October 2015-October 2016. We also collected plants, and soil chimpanzees were observed eating. We analyzed 10 plant and 45 soil samples to characterize geophagic behavior and geophagic soil and determine (1) whether micronutrients are available from the soil under physiological conditions and if iron is bioavailable, (2) the concentration of phenolic compounds in plants, and (3) if consumed soils are able to adsorb these phenolics. Chimpanzees ate soil and drank clay-infused water containing 1:1 and 2:1 clay minerals and > 30% sand. Under physiological conditions, the soils released calcium, iron, and magnesium. In vitro Caco-2 experiments found that five times more iron was bioavailable from three of four soil samples found at the base of trees. Plant samples contained approximately 60 μg/mg gallic acid equivalent. Soil from one site contained 10 times more 2:1 clay minerals, which were better at removing phenolics present in their diet. We suggest that geophagy may provide bioavailable iron and protection from phenolics, which have increased in plants over the last 20 years. In summary, geophagy within the Sonso community is multifunctional and may be an important self-medicative behavior.

Improving the nutritional quality of Fe in maize ( Zea mays ) represents a biofortification strategy to alleviate iron deficiency anemia. Therefore, the present study measured iron content and bioavailability via an established bioassay to characterize Fe quality in parts of the maize kernel. Comparisons of six different varieties of maize demonstrated that the germ fraction is a strong inhibitory component of Fe bioavailability. The germ fraction can contain 27-54% of the total kernel Fe, which is poorly available. In the absence of the germ, Fe in the non-germ components can be highly bioavailable. More specifically, increasing Fe concentration in the non-germ fraction resulted in more bioavailable Fe. Comparison of wet-milled fractions of a commercial maize variety and degerminated corn meal products also demonstrated the inhibitory effect of the germ fraction on Fe bioavailability. When compared to beans ( Phaseolus vulgaris ) containing approximately five times the concentration of Fe, degerminated maize provided more absorbable Fe, indicating substantially higher fractional bioavailability. Overall, the results indicate that degerminated maize may be a better source of Fe than whole maize and some other crops. Increased non-germ Fe density with a weaker inhibitory effect of the germ fraction are desirable qualities to identify and breed for in maize.