Using the domestic chicken (Gallus gallus) as an in vivo model for iron bioavailability.

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 (58)Fe (1 mg of Fe in 10 mM ascorbic acid) was injected into the loop. Blood samples were collected every 5 min and for 120 min postinjection and analyzed by inductively coupled argon-plasma mass spectrometry for (58)Fe 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.

Assessing potential effects of inulin and probiotic bacteria on Fe availability from common beans (Phaseolus vulgaris L.) to Caco-2 cells.

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.

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.

Calcium, zinc, and iron bioavailabilities from a commercial human milk fortifier: a comparison study.

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: alpha-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.

A comparison of iron availability from commercial iron preparations using an in vitro digestion/Caco-2 cell culture model.

The objectives of this study were to compare iron availability from commercial preparations of FeSO(4), ferrous gluconate, ferrous fumarate, and a polysaccharide-iron complex using an in vitro digestion/Caco-2 cell culture model. In addition, we sought to determine if calcium carbonate and calcium acetate (common phosphate binding agents) inhibited iron availability from an oral iron supplement when digested simultaneously. Caco-2 cell ferritin formation following exposure to simulated gastric and intestinal digests of the iron supplements was used as a measure of iron uptake and availability. Plates without cell monolayers were included in each replication of the experiment to measure the total amount of soluble iron that resulted from the in vitro digestion. Significantly more iron was taken up from the FeSO(4), ferrous gluconate, and ferrous fumarate than the polysaccharide-iron complex. Similar results comparing FeSO(4) and the polysaccharide-iron complex have been observed in humans. In addition, less iron was taken up from digests with calcium carbonate relative to calcium acetate even though similar amounts of soluble iron were observed in these experiments. The results indicate that when iron supplements and phosphate binders are consumed simultaneously, calcium acetate may be the preferred phosphate binder to maximize iron availability.

Aflatoxicosis alters avian renal function, calcium, and vitamin D metabolism.

Experiments were designed to determine the effects of aflatoxicosis on avian renal function, calcium (CA), inorganic phosphorous (Pi), and vitamin D metabolism, and to determine if the effects of aflatoxin are reversible upon discontinuation of toxin administration. Three-week-old male broiler chickens (n = 12 per treatment) received aflatoxin (AF; 2 mg/kg po) or an equal volume of corn oil, the AF carrier vehicle, for 10 consecutive days. After 10 d of treatment, half of the birds from each treatment group were anesthetized and prepared for renal function analysis, which included a 2-h phosphate loading period. Ten days after discontinuation of AF treatment, the remaining birds in each treatment group were anesthetized and prepared for renal function analysis. AF decreased plasma 25-hydroxy vitamin D [25(OH)D] and 1,25-dihydroxy vitamin D [1,25(OH)2D] levels after 5 d of treatment. After 10 d of treatment, urine flow rate (V), fractional sodium excretion (FENa), and fractional potassium excretion (FEK) were lower in AF-treated birds. In addition, total plasma Ca tended to be lower (p = .10) and fractional Ca excretion (FECa) tended to be higher (p = .10) in the AF-treated birds. Intravenous phosphate loading produced a sharp increase in urine hydrogen ion concentration ([H+]) in the AF-treated birds. Glomerular filtration rate (GFR) was reduced and plasma osmolality was increased in AF-treated birds 10 d after discontinuation of toxin administration. The results indicate that AF directly or indirectly affects Ca and Pi metabolism in avians. At the present time, the effects may be related to altered vitamin D and parathyroid hormone (PTH) metabolism. Aflatoxicosis may decrease endogenous PTH synthesis and the renal sensitivity to PTH. The AF-related increase in urine [H+] during phosphate loading is probably due to increased Na+/H+ counterport, suggesting that AF stimulates sodium reabsorption. Also, the decrease in GFR exhibited 10 d after toxin removal indicates that AF may cause prolonged alteration in renal function.

Altered renal function in broilers during aflatoxicosis.

Experiments were conducted to determine the effects of aflatoxicosis on acid-base balance, urine flow rate (V), glomerular filtration rate (GFR), clearance of para-aminohippuric acid (CPAH), plasma osmolality, and the renal handling of Na, K, Ca, and P. Three-week-old broilers were gavaged with aflatoxin at a dose of 2 mg/kg of BW per day for 10 consecutive days. Control birds received an equal volume of corn oil, the aflatoxin carrier vehicle. On the eleventh day, the birds were anesthetized and prepared for renal function analysis. A solution containing inulin, para-aminohippuric acid, and mannitol was infused at a low infusion rate (.1 mL/kg of BW per min) and a high infusion rate (.4 mL/kg of BW per min) to determine if aflatoxin affects the renal response to an acute volume load. Aflatoxicosis decreased the fractional excretion of phosphorous (FEP) and plasma Ca concentration but did not significantly alter any other renal function or acid-base variables. The decrease in FEP and plasma Ca may be a direct result of renal tubular damage, decreased Ca absorption from the gut, or a result of altered circulating levels of parathyroid hormone (PTH), and possibly decreased renal sensitivity to PTH.

The effects of dietary phosphorus and parathyroid hormone (PTH) infusion rates on the avian phosphaturic response to PTH.

Experiments were performed to evaluate the effects of dietary available phosphorus (aP) and PTH infusion rates on avian urinary inorganic phosphate (Pi) excretion. In experiment I, female domestic fowl were fed diets containing low (0.45%) or high (0.83%) aP for 2-4 weeks prior to renal function studies. Pi excretion was significantly higher for birds fed the high-aP diet than for birds fed the low-aP diet. PTH was infused (60-240 units kg body mass-1 h-1) unilaterally into the renal portal system. Para-aminohippuric acid (PAH), included in the unilateral infusate as a marker for effective renal portal perfusion, indicated that PTH must have been delivered to the peritubular surfaces of the infused kidney. However, bilateral but not unilateral phosphaturia occurred, and there were no significant differences in the phosphaturic responses to PTH when low- and high-aP diet treatment groups were compared. In experiment II, PTH was infused at rates of 1-5 units h-1. Infusing PTH at 5 units h-1 caused a unilateral increase in urine flow but the phosphaturic response was still bilateral. It appears unlikely that unilateral renal portal PTH infusions can be used to trigger unilateral phosphaturia in domestic fowl.

Methionine hydroxy analog (free acid) reduces avian kidney damage and urolithiasis induced by excess dietary calcium.

Urinary acidification previously was shown to be an effective treatment for calcium-induced urolithiasis in domestic fowl, but diuresis caused by the acidifying agent (ammonium chloride) was an undesirable side effect. Because supplemental dietary methionine reportedly acidifies mammalian urine, an experiment was conducted to evaluate the efficacy of the free acid form of methionine hydroxy analog (MHA) as an acidifying agent for treating avian urolithiasis. From 5 to 17 wk of age, immature Single Comb White Leghorns were fed diets containing normal calcium (1%) or high calcium (3.5%). Diets were supplemented with 0, 0.3 or 0.6% MHA. Relative to birds fed the normal calcium diets, birds fed the high calcium diet without added MHA were in a state of metabolic alkalosis and excreted more alkaline urine containing high levels of calcium. Birds fed the high calcium diet without MHA also had significantly higher kidney asymmetry ratios, a higher incidence of gross kidney damage, and a higher incidence of urolith formation when compared with birds fed normal calcium diets. When compared with the high calcium diet without MHA, the high calcium diet supplemented with 0.6% MHA significantly acidified the urine without causing detectable metabolic acidosis, significantly reduced kidney asymmetry and gross kidney damage, and reduced the incidence of urolith formation without increasing water consumption or urine flow. These data demonstrate that MHA effectively prevents calcium-induced kidney damage in domestic fowl without causing undesirable side effects. MHA did increase both fractional and absolute calcium excretion during calcium loading.