59Fe In Blood Research Articles

Intestinal Inflammation Differentially Influences Iron Homeostasis in WT versus Hepcidin KO Rats

IntroductionThe chronic anemia associated with IBD in humans may result from trans‐activation of the the iron‐regulatory hormone (Hamp) by pro‐inflammatory cytokines in hepatocytes. High hepcidin decreases serum iron by blunting intestinal iron absorption and impairing release of storage iron, thus decreasing iron delivery to the erythroid marrow and causing anemia. Pharmacological targeting of hepcidin offers an attractive therapeutic approach to mitigate IBD‐associated anemia. Testing of such interventions has mainly utilized mouse models of hereditary hemochromatosis (HH). Collectively, these mice have inappropriately low (or absent) hepcidin expression, and concomitant increases in intestinal iron absorption leading to iron overload. However, since iron metabolism in mice and humans varies notably, development of complementary experimental models is imperative.ObjectiveWe created a novel model of HH, the Hamp KO rat, and utilized the DSS model of colitis for this investigation. We hypothesized that Hamp levels would increase in rats with colitis (due to pro‐inflammatory cytokine signaling), which would then blunt intestinal iron absorption and cause anemia (thus modeling human IBD). We further postulated that iron absorption would be unaffected by inflammation in Hamp KO rats, thus preventing the development of anemia.MethodsIntestinal inflammation was induced by providing DSS (4% w/v) in drinking water for 7 days. Standard bioassays were utilized to track iron loading. Iron absorption was assessed by oral‐intragastric gavage of an 59Fe transport solution.ResultsHamp−/− rats displayed multi‐visceral iron overload, and intestinal iron absorption was increased (≈2.5 fold). DSS treatment caused similar pathological changes in the duodenum and colon of Hamp KO rats and WT littermates. Systemic inflammation was not observed in any experimental animals. DSS exposure decreased TSAT, and serum and splenic nonheme iron in both genotypes. In WT animals, DSS exposure: 1) decreased blood hemoglobin levels by ≈15%; 2) reduced liver hepcidin expression by ≈60%; 3) and increased intestinal iron absorption by >2‐fold. Conversely, in Hamp−/− rats, DSS treatment increased blood hemoglobin levels and decreased iron absorption by ≈2.5‐fold. Moreover, the distribution of absorbed 59Fe in blood and many tissues was different between genotypes after DSS txt.ConclusionsDespite the lack of systemic inflammation, iron homeostasis was perturbed in WT rats by DSS‐induced enteritis. Reduced hepcidin expression likely resulted from anemia and/or iron depletion of serum and spleen. This may explain why iron absorption increased, since a relative state of iron deficiency may have existed after DSS exposure. In the KOs, intestinal inflammation altered iron homeostasis in distinct ways. This differential response to DSS may relate to the iron‐overload phenotype of the KO rats, or alternatively, it could reflect a role for hepcidin in mediating the pathophysiologic response to intestinal inflammation. Future studies in iron‐depleted Hamp KO rats may allow for a better understanding of the role of hepcidin in regulating iron homeostasis in IBD.Support or Funding InformationNIH Grant R01 DK074867 and R01 DK109717 (to JFC)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Intestinal divalent metal‐ion transporter‐1 is critical for intestinal iron absorption but not that of copper (996.3)

Divalent metal‐ion transporter‐1 (DMT1) is a widely expressed iron transporter serving iron absorption and erythroid iron uptake. DMT1 can also transport certain other transition metal ions; however, questions have arisen as to the role that DMT1 plays in the absorption of copper. We tested the hypothesis that intestinal DMT1 is essential for the absorption of Fe and Cu by examining (1) hematological variables and Fe status, (2) expression of Fe‐related genes (by using RT‐qPCR), and (3) radiotracer metal absorption in a mouse model lacking intestinal DMT1 (i.e. DMT1int/int, generated by crossing floxed DMT1 and villin‐Cre transgenic lines). The DMT1int/int mouse (at age ≍ 120 d) exhibited a profound hypochromic–microcytic anemia (hemoglobin concentration was 1.8 ± SD 0.5 g/dL in DMT1int/int mice cf. 14.6 ± 0.7 g/dL in wildtype, n = 7–9), and severely depleted serum Fe and liver nonheme Fe stores. mRNA expression of Cybrd1 (ferrireductase) was increased to (30 ± 3)‐fold (mean ± propagated SE, n = 3) in enterocytes of DMT1int/int mice cf. wildtype mice and that of the basolateral Fe exporter ferroportin, (2.7 ± 1.0)‐fold. Liver Hamp1 (hepcidin) expression was depressed by 97% ± 48%. Expression of the apical Cu transporter Ctr1 was increased to (2 ± 1)‐fold in enterocytes of DMT1int/int cf. wildtype mice, consistent with other models of iron deficiency. Parenteral Fe injection corrected the hematological and serum‐Fe variables, Fe stores, and gene expression levels in the DMT1int/int mouse. We measured the absorption of 59Fe and 64Cu in mice fed by intragastric gavage. Loss of intestinal DMT1 decreased the 0–4‐hour appearance of 59Fe in blood by 88% ± 24% cf. wildtype. Enterocyte 59Fe content at 4 h was 93% ± 42% lower in DMT1int/int cf. wildtype mice, indicating that the defect was at the level of apical uptake. Intestinal handling of 64Cu in the DMT1int/int mouse was no different from wildtype (P = 0.26). We conclude that DMT1 is critical for intestinal absorption of Fe but not Cu.Grant Funding Source: Supported by PHS Grant DK080047

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.

A simplified method for determination of radioactive iron in whole-blood samples

For studies on iron absorption in man radioisotopes represent an easy and simple tool. However, measurement of the orbital electron emitting radioiron, 55Fe, in blood is difficult and insufficiently described in the literature. The present study describes a relatively simple method for simultaneous determination of 55Fe and 59Fe in blood, using a dry-ashing procedure and recrystallization of the remaining iron. The detection limit of the method permits measurements of 0.1 Bq/ml blood thus allowing detection of less than 1% absorption from a 40 kBq dose, which is ethically acceptable in humans. The overall recovery of radioiron from blood is more than 90%, and the coefficient of variation, as judged by the variation in the ratio 55Fe/ 59Fe is in the order of 4%. Combined with whole-body counting of 59Fe and direct γ-counting of 59Fe on blood samples, this method represents a sensitive method for studying the intestinal absorption of 55Fe and 59Fe in man and at the same time allows estimation of the amount of radioiron located in the vascular compartment.

Biochemical States of 59Fe in Blood of Mice Exposed to .GAMMA.-Rays of 4Gy or 10Gy.

In a previous paper, we studied elimination and presence rates of nuclides injected in irradiated mice to investigate metabolic mechanism of several essential metals in injured mice leading to recovery after sublethal irradiation, and it was observed that the elimination and the presence rates of elements in tissues and organs showed a difference between the doses, particularly for 59Fe in liver and blood of mice. In this paper, further observations with biochemical states of 59Fe in the blood of irradiated mice are described as well as with distribution of 59Fe in the blood. An accumulation of 59Fe to erythrocytes was remarkably decreased by the irradiation, and a big radioactive peak (P-1) was seen in a profile of gel filtration of hemolysates in both unirradiation (control) and irradiation samples 2 days after the irradiation. At 8 days after the irradiation, the presence rate of 59Fe in the erythrocytes increased, and in the profile of gel filtration, for 10 Gy irradiation, the peak P-1 decreased and another radioactive peak was observed, but the difference between 4 Gy and control was not clear. These results suggested a metabolic process leading individuals to recovery from injury after irradiation.

New procedure to fortify fluid milk with iron: Metabolic and biochemical study in rats

A comparative study with two groups of 25 rats each was undertaken in order to study fluid milk fortified with SFE-171 (stabilized FeSO 4 by micro encapsulation with phospholipids) or FeSO 4, both labeled with 59Fe. After the administration of either product, its total absorption, biodistribution and incorporation of iron into hemoglobin was determined for both cases. In the first case the total absorption was (28.3 ± 6.0)%, whereas in the second it was (24.1 ± 5.8)%, the difference between both values is statistically significant with a P < 0.01. The biodistribution results show that the iron from SFE-171 follows the same metabolic pathway than that from FeSO 4. The highest amount (approximately 80%) was found in blood, followed by the liver with approximately 11% and bones with a proportion somewhat higher than 4%, the activity in intestine was minimal in both cases. The 59Fe in blood was found 18 days after the administration of either product at a 100% in the red blood cells, which implies that the iron administered in the milk fortified with SFE-171 or with FeSO 4 was incorporated into hemoglobin. These results indicate with a high degree of significance that the fortification of milk with SFE-171 is a procedure which assures the effective absorption of iron and a physiological behavior of this element.

Iron Metabolism in Genetically Obese (ob/ob) Mice

Several reports in the clinical literature suggest that obese children may be at risk for developing iron deficiency. Here the absorption, retention, tissue distribution and tissue levels of iron were compared in lean (+/?) and obese (ob/ob) C57BL/6J mice to examine the impact of obesity on the iron status of this animal model. Obese mice absorbed and retained approximately twice as much 59Fe as lean mice after receiving a solution containing 1 mumol iron per os. This difference was independent of age, severity of obesity and mass of the gastrointestinal tract. Obese mice fed ad libitum had higher levels of 59Fe in blood and fat pads, but lower amounts of 59Fe in the skeletal-muscular system, than lean mice 6 d after subcutaneous injection of 1 mumol of the metal. At least 30% of carcass 59Fe was present in the liver of obese and lean mice 6 d after injection. Despite significantly lower concentrations of iron in liver and bone, blood hemoglobin and hematocrit were significantly higher in obese mice fed ad libitum than in lean mice at 10 wk of age. Plasma iron and transferrin were not affected by chronic obesity. Although several characteristics of iron metabolism differed in obese and lean mice, the results indicate that ob/ob mice were not iron deficient when fed a diet containing an adequate level of this micronutrient. The increased absorption of iron by obese mice probably represents an adaptive response that is required to supply additional micronutrient for the expanded blood volume in these animals.

Iron-donating properties of transferrin

The transferrin molecule has two specific metal-binding sites, each of which may provide iron for the biosynthesis of hemoglobin by reticulocytes. Diferric human transferrin was shown to be a better iron donor, per iron atom, for rabbit reticulocytes, than was monoferric transferrin obtained by isoelectric focusing. The difference in binding of 125I-labeled monoferric and differic transferrin to reticulocytes may be sufficient to account for the difference in iron uptake. In contrast, diferric and monoferric rabbit transferrin both donated iron to reticulocytes at the same rate, per iron atom. In an experiment using 55Fe/59Fe doubly labeled transferrin, one iron binding site of human transferrin was a better iron donor than the other. In rabbit transferrin, the two sites appeared to function equivalently. Care was taken in these experiments to demonstrate that labeled iron added to dilute solutions of transferrin was indeed specifically bound to the protein. A liquid scintillation counting procedure, simpler than existing methods, was developed to quantitate 55Fe and 59Fe in blood.