Effects of type of fat in the diet on iron bioavailability assessed in suckling and weanling rats

Differences in iron bioavailability from human milk and milk formulas may in part be due to differences in lipid composition. We investigated the short and long term effects of diets based on different fats [corn, coconut, olive, or soy oil, human milk fat (HMF) and a formula fat blend (FF)] on iron absorption in rats. Suckling rat pups dosed with 59Fe-labeled diets containing different fat sources were killed after 6 h, and blood and individual tissues were counted. Iron availability was estimated by % 59Fe in blood. Pups dosed with a more saturated fat (coconut oil) had a higher % 59Fe in blood than those fed other fat sources. Weanling rats were used to determine iron bioavailability from fat sources using both the hemoglobin repletion method and whole body counting. Hemoglobin regeneration was significantly higher for rats fed the HMF diet (8.4 +/- 0.5 g/dl) than from the FF diet (6.5+/-0.6 g/dl) or the corn oil diet (less saturated) (6.4 +/- 0.3 g/dl). Rats fed diets based on coconut oil (more saturated) had significantly higher % 59Fe retention (61.6 +/- 1.4) than rats fed diets based on FF (49.8 +/- 3.4). There was a significant positive association between oleic acid in the diet and oleic acid in the intestinal mucosa (r = 0.95, p < 0.05) and between linoleic acid in the diet and linoleic acid in the intestinal mucosa (r = 0.97, p < 0.05) suggesting that the dietary treatment altered the fatty acid composition of the brush border membrane. Our results suggest that saturated fats may increase iron absorption and that part of this may be achieved by changes in the fatty acid composition of the intestinal mucosa. Hemoglobin regeneration and % 59Fe retention data suggest that differences in iron absorption from infant diets may in part be due to differences in fat composition. Therefore, lipid composition of infant formulas should also be taken into consideration as a factor influencing iron bioavailability.     

Endogenous iron excretion

The effects of supplemental oral (0, 40, and 400 ppm) and parenteral iron (0 and 2.72 mg Fe iv given initially as a single dose) on iron absorption, excretion, and retention were determined in 30 rats. Endogenous fecal iron excretion was determined by the radioisotope dilution technique after im injection of 80 kBq Fe-59, using blood and certain body tissues as reference sources for the estimation of the specific activity (Bq Fe-59/micrograms Fe) of endogenous iron. The basal diet contained 3.6 ppm Fe. Fe(III)-hydroxide-polymaltose was used as the sole iron source in oral, iv, and im iron treatments. Iron balance as determined from day 14 to 20 of the experiment was not significantly affected by iv iron administration. Nevertheless, a temporarily reduced retention should have occurred, since differences in final body iron contents were lower than 2.72 mg, as compared to the respective untreated groups. Apparent iron absorption and iron retention increased with surplus oral iron, and the efficiency rates were highest with adequate iron supply (40 ppm). True absorption rates of iron were similar without any, and with 40 ppm Fe amounting 40 to 50% of the intake. In the iron deficient rats, half of the actually absorbed iron (about 16 micrograms/d) was lost by endogenous fecal re-excretion, and another 3 micrograms/d by the urinary route. Endogenous loss with feces and with urine increased with further oral iron supply, but at a considerably lower rate as total fecal excretion. Parenterally administered iron did not affect endogenous loss at all. The results indicate that endogenous excretion cannot be regarded as a means to eliminate excessive iron, and might actually be an inevitable loss.     

Detection and quantitation of iron in ferritin,transferrin and labile iron pool (LIP) in cardiomyocytes using 55Fe and storage phosphorimaging

Dysregulated iron metabolism has a detrimental effect on cardiac function. The importance of iron homeostasis in cardiac health and disease warrants detailed studies of cardiomyocyte iron uptake, utilization and recycling at the molecular level. In this study, we have performed metabolic labeling of primary cultures of neonatal rat cardiomyocytes with radioactive iron coupled with separation of labeled iron-containing molecules by native electrophoresis followed by detection and quantification of incorporated radioiron by storage phosphorimaging. For the radiolabeling we used a safe and convenient beta emitter ⁵⁵Fe which enabled sensitive and simultaneous detection and quantitation of iron in cardiomyocyte ferritin, transferrin and the labile iron pool (LIP). The LIP is believed to represent potentially dangerous redox–active iron bound to uncharacterized molecules. Using size-exclusion chromatography spin micro columns, we demonstrate that iron in the LIP is bound to high molecular weight molecule(s) (≥5000?Da) in the neonatal cardiomyocytes.     

Fetal monkey absorption of 55Fe from amniotic fluid.

A single dose of 0.5 mCi radioiron (55Fe) was injected directly into the amniotic cavity of each of four pregnant monkeys near term. At 6 or 24 hours after 55Fe adminstration, samples of fetal jejunum, ileum, blood, spleen, liver, lung and fetal membranes were taken for radioautography and scintillation counts. Counts were also made on corresponding maternal tissues. Intestinal radioiron levels and localization indicated that fetuses swallowed and absorbed 55Fe within 6 to 24 hours after injection. Silver grains over the meconium bodies of fetal ileum suggested that intestinal epithelium retained at least some of the absorbed radioiron. However, elevated 55Fe levels in fetal blood and hematopoietic organs indicated that transport of radioiron to the circulation had occurred, presumably through fetal intestine, extraembryonic membranes and/or fetal lungs. Transfer of 55Fe across the placenta from fetus to mother occurred at a slow rate, i.e., samples of maternal organs obtained at 24 hours gave low counts.     

Iron Absorption in Rats Increased by Yeast Glucan

The effects of brewer's yeast cell walls and two of its components, glucan and mannan, on the absorption of ⁵⁹Fe by anemic rats were investigated. After administration of the label, the percentage of ⁵⁹Fe taken up into the blood of group given glucan was generally similar to that of a group given yeast cell walls, both values were higher than in controls. The incorporation of ⁵⁹Fe into the small intestines was higher in the group given glucan than in the controls or a group given a glucan—mannan mixture. Glucan is the main substance in yeast cell walls that increases iron absorption.     

Iron and cadmium uptake by duodenum of hypotransferrinaemic mice

Absorption from food is an important route for entry of the toxic metal, cadmium, into the body. Both cadmium and iron are believed to be taken up by duodenal enterocytes via the iron regulated, proton-coupled transporter, DMT1. This means that cadmium uptake could be enhanced in conditions where iron absorption is increased. We measured pH dependent uptake of ¹⁰⁹Cd and ⁵⁹Fe by duodenum from mice with an in vitro method. Mice with experimental (hypoxia, iron deficiency) or hereditary (hypotransferrinaemia) increased iron absorption were studied. All three groups of mice showed increased ⁵⁹Fe uptake (p<0.05) compared to their respective controls. Hypotransferrinaemic and iron deficient mice exhibited an increase in ¹⁰⁹Cd uptake (p<0.05). Cadmium uptake was not, however, increased by lowering the medium pH from 7.4 to 6. In contrast, ⁵⁹Fe uptake (from ⁵⁹FeNTA₂) and ferric reductase activity was increased by lowering medium pH in control and iron deficient mice (p<0.05). The data show that duodenal cadmium uptake can be increased by hereditary iron overload conditions. The uptake is not, however, altered by lowering medium pH suggesting that DMT1-independent uptake pathways may operate.     

The Study of Menstrual and Other Blood Loss,and Consequent Iron Deficiency,by Fe59 Whole-body Counting

A recently established method of in vivo radioiron investigation in humans, employing a steel-room whole-body counter, has been applied to the study of Fe⁵⁹ absorption and loss in seven menstruating women, six with menorrhagia and hypochromic anemia. All six were found by this method to be iron-deficient, having radioiron absorptions of 53.7-97.5% (normal 5.7-24.7%). With almost 100% radioiron incorporation into the red-cell mass, subsequent drops in Fe⁵⁹ activity, when correlated with monthly menses, revealed estimated menstrual blood losses of 110-550 c.c. The single normal patient absorbed 19.6% of the tracer, with only 33-59 c.c. menstrual blood loss. Additional applications of the technique in assessing episodic (e.g., epistaxis) and continuous (e.g., gastrointestinal) blood loss are also described. The method would appear eminently applicable to the study of any hypochromic anemia of hemorrhagic origin.     

Distribution of55Fe in juvenile adult lampreys,Petromyzon marinus L., following oral intubation of the isotope

Summary The capacity of a sanguivorous lamprey,Petromyzon marinus L., to deal with ingested iron was studied over time using autoradiography and scintillation counting of solubilized tissue samples after intubation of the oesophagus with a single dose of⁵⁵ferrous citrate. A highly efficient mechanism for absorption in the anterior intestine was recognized with 17% of the intubated radioactivity absorbed into the body after only 5 min, 66% by 3 h, and almost 80% by 21 h. Iron concentration in the epithelial cells of the anterior intestine may be a factor in restricting iron absorption during spontaneous feeding. A decline in total body radioactivity over the 15 days following iron intubation probably results from transport of the metal in the blood and release of radioiron from the mucous cells of the posterior intestine. The kidneys appear to play a smaller but still significant role in iron loss. Gradual increases in radioiron concentration (cpm g^–1 wet weight) and percent of total body radioactivity occur in the liver (2 to 26%), carcass (14 to 37%), and integument (4 to 12%) during the course of the experiment, indicating that these are the chief sites of iron storage during times of metal excess. However, eventually integument may also be a site of iron excretion. Significant fluctuations in radioiron concentration (cpm ml^–1) in whole blood during the 15 day period can be correlated with transport of the metal to sites of storage and excretion, and maybe with incorporation into haemoglobin and with erythropoietic activity. Feeding adult lampreys represent a valuable system, with both general and unique characters, for studying iron metabolism in vertebrates.     

Zinc inhibits nonheme iron bioavailability in humans

There is increasing concern about potential negative interactions in combined iron and zinc supplementation. The aim of the present study was to determine the dose-response effect of zinc, given as a solution, on iron bioavailability. Twenty-two healthy adult women were selected to participate in the study. Iron, with or without zinc was given as an aqueous solution on d 1,2,14, and 15 of the study. Iron bioavailability was measured on the basis of erythrocyte incorporation of⁵⁵Fe or⁵⁹Fe 14 d after administration. Subjects received 0.5 mg of iron together with graded zinc concentrations (0-11.71 mg). No significant effect of zinc on iron absorption was found at Zn : Fe molar ratios up to 2 :1. At 5:1,10:1, and 20 :1 molar ratios, a dose-dependent inhibitory effect on iron absorption was observed (28-40% of iron absorption inhibition; one-way repeated-measures ANOVA, F = 4.48,p = 0.02). In conclusion, zinc administration combined with iron in an aqueous solution leads to the inhibition of iron bioavailability, which occurs in a dose-dependent way. This negative interaction should be considered for supplementation programs with both microminerals.     

A1 and A2 adenosine receptor regulation of erythropoietin production

The effects of adenosine (ADE) and ADE agonists on erythropoietin (Ep) production were determined using percent (%) 59Fe incorporation in red cells of exhypoxic polycythemic mice. The hemisulfate salt of ADE produced a significant increase in % 59Fe incorporation in response to hypoxia in concentrations of 400 to 1600 nmol/kg/day (i.v.). 5'-N-ethyl-carboxamideadenosine (NECA), a selective A2 receptor agonist, increased radioiron incorporation in a dose-dependent manner (10-100 nmol/kg/day, i.v.). In contrast, N6-cyclohexyladenosine (CHA), a selective A1 receptor agonist, did not affect radioiron incorporation in concentrations up to 1600 nmol/kg/day (i.v.). Albuterol, a beta 2-adrenergic agonist, enhanced % 59Fe incorporation in polycythemic mice and low doses of CHA (50 and 100 nmol/kg/day), which were not effective alone on % 59Fe incorporation in polycythemic mice exposed to hypoxia, inhibited the enhancement in radioiron induced by albuterol (25 and 100 micrograms/kg/day, i.p.) plus hypoxia. Theophylline (20 and 80 mg/kg/day, i.p.), a well-known antagonist of ADE receptors, blocked the ADE and NECA enhancement in radioiron incorporation at a dose of theophylline alone which produced only a slight enhancement of % 59Fe incorporation. These results suggest that ADE may both inhibit through A1 receptor activation and increase via A2 receptor stimulation the production of Ep.     

Voltage-clamp: a useful approach to study in vitro duodenal iron transport in the mouse

The mechanism(s) controlling iron absorption remain(s) uncertain despite the progress in the identification of genes selectively expressed in the duodenum. The availability of experimental models of iron absorption is critical to the clarification of such mechanism(s). In the present study, a simple method for studying in vitro iron absorption in mouse duodenum is described. Short circuit current, open circuit potentials and epithelial conductances were measured in mouse duodenal segments. Also, unidirectional ⁵⁵Fe fluxes at different pH conditions were measured in mice with varying iron status. The findings reinforce evidence for an adaptive response of the iron absorptive process according to the body iron status. Significant differences are demonstrated between iron fluxes measured in normal and parenterally iron loaded mice and at acidic compared to neutral pH environment. Also, a significant difference was observed between transepithelial potential measured in duodenum from iron-loaded compared to untreated mice. Advances in the understanding of the mechanism(s) of iron absorption can be brought by the application of voltage-clamp techniques to the electrophysiologic study of iron overload mouse models.     

The utilization of senescent red cell and hemolysate iron for erythropoiesis.

We report experiments to determine the availability for new hemoglobin production of radioiron from nonviable red cells at various times after deposition in the reticulo-endothelial system and to determine the relative availability of radioiron derived from hemolysates versus that derived from nonviable red cells. When heated nonviable red cells labeled with 59Fe are injected into polycythemic mice the iron is deposited in the reticulo-endothelial system, and less than 1% of it is reutilized for hemoglobin synthesis. If the polycythemic mice are given nonviable red cells 48 hours after exposure to hypoxia, when hemoglobin synthesis is maximal, 25% of the iron is reutilized. When the cells are given 36 hr after exposure to hypoxia, iron reutilization declines to 16%, and when exposure to hypoxia is further delayed, reutilization of the iron falls to a plateau level of 11%. Radioiron from hemolysates, primarily deposited in parenchymal cells of the liver, is less available for new hemoglobin synthesis than is radioiron from nonviable red cells, which is primarily deposited in Kupffer cells of the liver. When transferrin-bound iron is given to polycythemic mice, this iron is also deposited in parenchymal cells of the liver and is also less available for new hemoglobin synthesis. Thus, in relation to an erythropoietic stimulus, the site and time of deposition of iron influence its accessibility for erythropoiesis.     

Iron absorption by Belgrade rat pups during lactation

Divalent metal transporter-1 (DMT1) mediates dietary nonheme iron absorption. Belgrade (b) rats have defective iron metabolism due to a mutation in the DMT1 gene. To examine the role of DMT1 in neonatal iron assimilation, b/b and b/+ pups were cross-fostered to F344 Fischer dams injected with (59)FeCl(3) twice weekly during lactation. Tissue distribution of the radioisotope in the pups was determined at weaning (day 21). The b/b pups had blood (59)Fe levels significantly lower than b/+ controls but significantly higher (59)Fe tissue levels in heart, bone marrow, skeletal muscle, kidney, liver, spleen, stomach, and intestines. To study the pharmacokinetics of nonheme iron absorption at the time of weaning, (59)FeCl(3) was administered to 21-day-old b/b and b/+ rats by intragastric gavage. Blood (59)Fe levels measured 5 min to 4 h postgavage were significantly lower in b/b rats, consistent with impaired DMT1 function in intestinal iron absorption. Tissue (59)Fe levels were also lower in b/b rats postgavage. Combined, these data suggest that DMT1 function is not essential for iron assimilation from milk during early development in the rat.     

Intestinal iron absorption in chickens

Intestinal iron absorption in chickens was studied in vivo, using an intestinal perfusion technique in closed circuit. The results obtained show that iron absorption, at 30 min intervals, is a linear function of test solution iron concentrations of up to 776 μg Fe/20 mL. At higher concentrations, iron saturation occurs. The mucosal epithelial cells seem to be less a limiting factor than in rats. However, in chickens, the binding capacity of plasma might play an important role in the regulation of iron absorption. Iron absorption versus time was analyzed in 15, 30, 60, and 120 min periods for the iron concentration of 14 μg Fe/20 mL. Intestinal iron absorption showed a linear relationship between these two parameters. A period of perfusion of either 30 or 60 min by a solution of 14 μg Fe/20 mL appears suitable since no interference by a saturation process can then occur.     

Iron metabolism in pigeons.

Several haematological parameters such as haemoglobin concentration, haematocrit, erythrocyte number, reticulocyte concentration, plasma iron and total iron binding capacity were determined in 118 urban pigeons of both sexes. No statistically significant sex differences among these parameters were found. In 36 specimens (23 males and 13 females), the plasma iron turnover was determined using 59Fe. The results obtained in this species, expressed per 100 ml-1 blood. day-1 and Kg-1 body weight. day-1, were compared with those of turkeys, ducks and chickens calculated from earlier papers. The highest values versus body weight were observed in pigeons. Organ (liver, spleen, tibia, heart, leg muscle, ribs, sternal keel, gonads and blood) distribution of 59Fe intravenous injection was analyzed during a period from 5 min up to 120 days (19 different times) in groups of 4 pigeons. At the 6 h period, the organs retained the highest dose (20% of total Fe injected), but by the 2nd day period, the radioiron in the blood represented about 98% of the total injected. A fast iron uptake by the circulatory blood was checked and compared with that of other species (domestic fowl, ducks and turkeys). The reticulocyte count in pigeons normally ranged from 4 to 12%, which was consistent with these results. A linear decreasing radioactivity in blood, with an inflexion point on the 40th day was observed. An inverse correspondence between blood and liver was found. Content in other organs decreased uniformly with time, except the heart where the iron content was practically constant during the whole time. Ribs and sternal keel are erythropoietic organs in young pigeons.     

Olfactory ferric and ferrous iron absorption in iron-deficient rats

The absorption of metals from the nasal cavity to the blood and the brain initiates an important route of occupational exposures leading to health risks. Divalent metal transporter-1 (DMT1) plays a significant role in the absorption of intranasally instilled manganese, but whether iron uptake would be mediated by the same pathway is unknown. In iron-deficient rats, blood (59)Fe levels after intranasal administration of the radioisotope in the ferrous form were significantly higher than those observed for iron-sufficient control rats. Similar results were obtained when ferric iron was instilled intranasally, and blood levels of (59)Fe were even greater in the iron-deficient rats compared with the amount of ferrous iron absorbed. Experiments with Belgrade (b/b) rats showed that DMT1 deficiency limited ferric iron uptake from the nasal cavity to the blood compared with +/b controls matched for iron deficiency. These results indicate that olfactory uptake of ferric iron by iron-deficient rats involves DMT1. Western blot experiments confirmed that DMT1 levels are significantly higher in iron-deficient rats compared with iron-sufficient controls in olfactory tissue. Thus the molecular mechanism of olfactory iron absorption is regulated by body iron status and involves DMT1.     

Determination of iron in blood serum using flow injection with luminol chemiluminescence detection.

A simple and rapid fl ow injection method is reported for the determination of iron in blood serum after acid digestion with HNO3 and HClO4, based on luminol CL detection in the absence of added oxidant. The detection limit (3 s) was 1.0 nmol/L with a sample throughput of 120/h. The calibration graph was linear over the range 0.001-1.0 micromol/L (r2 = 0.9974), with relative standard deviations (RSD) (n = 4) in the range 3.2-5%. The effect of interfering cations (Ca(II), Mg(II), Cu(II), Cd(II), Pb(II), Mn(II), Zn(II), Ni(II), Co(II) and Fe(III)) and anions (Cl-, SO4(2-), HCO3-, NO3-, NO2-) were studied using a luminol CL system for Fe(II) determination. The method was applied to normal blood serum and the results (1.32 +/- 0.08-1.74 +/- 0.05 mg/L) were compared with those from a spectrophotometric reference method (1.34 +/- 0.06-1.80 +/- 0.10 mg/L), which agree fairly well with the overall reference range in blood.     

The effect of short-term exposure to low-iron diets on the mucosal processing of ionic iron

Short-term alterations in the amount of iron in the diets of rats caused substantial differences in the distribution of a test dose of radioiron between mucosal transferrin and mucosal ferritin, and also caused a change in the relative amounts of these two proteins in mucosal tissue without resulting in any detectable change in liver iron stores. These differences correlated with changes in the retention of radioiron by the intestinal mucosa and the transport of radioiron to the blood stream. These studies emphasize the importance of local changes in the intestinal mucosa in the regulation of dietary iron absorption.     

Iron absorption in rats increased by yeast glucan

The effects of brewer's yeast cell walls and two of its components, glucan and mannan, on the absorption of 59Fe by anemic rats were investigated. After administration of the label, the percentage of 59Fe taken up into the blood of group given glucan was generally similar to that of a group given yeast cell walls, both values were higher than in controls. The incorporation of 59Fe into the small intestines was higher in the group given glucan than in the controls or a group given a glucan-mannan mixture. Glucan is the main substance in yeast cell walls that increases iron absorption.     

Effects of copper and ceruloplasmin on iron transport in the Caco 2 cell intestinal model

Previous studies have implicated copper proteins, including ceruloplasmin, in intestinal iron transport. Polarized Caco2 cells with tight junctions were used to examine the possibilities that (a) ceruloplasmin promotes iron absorption by enhancing release at the basolateral cell surface and (b) copper deficiency reduces intestinal iron transport. Iron uptake and overall transport were followed for 90 min with 1 &mgr;M 59Fe(II) applied to the apical surface of Caco2 cell monolayers. Apotransferrin (38 &mgr;M) was in the basolateral chamber. Induction of iron deficiency with desferrioxamine (100 &mgr;M; 18 h) markedly increased uptake and overall transport of iron. Uptake increased from about 20% to about 65% of dose, and overall 59Fe transport from <1% to 60% of dose. On the basis of actual iron released into the basal chamber (measured with bathophenanthroline), transport increased 8-fold. Desferrioxamine pretreatment reduced cellular Fe by 55%. The addition of freshly isolated, enzymatically active human ceruloplasmin to the basolateral chamber during absorption had no effect on uptake or transport of iron by the cells. Unexpectedly, pretreatment with three different chelators of copper (18 h), which reduced cellular levels about 40%, more than doubled iron uptake and raised overall transport to 20%. This was so, whether or not cells were also made iron deficient with desferrioxamine. Acute addition of 1 &mgr;M Cu(II) to the apical chamber had no significant effect upon iron uptake, retention, or transport in iron deficient or normal cells, in the presence of absence of ascorbate. We conclude that intestinal absorption of Fe(II) is unlikely to depend upon plasma ceruloplasmin, and that cuproproteins involved in this form of iron transport must be binding copper tightly.