Measurement of long-term iron absorption and loss during iron supplementation using a stable isotope of iron (57 Fe).

We report the first measurements of long-term iron absorption and loss during iron supplementation in African children using a stable isotope of iron (57 Fe). After uniform labelling of body iron with 57 Fe, iron absorption is proportional to the rate of decrease in the 57 Fe tracer concentration, while iron loss is proportional to the rate of decrease in the 57 Fe tracer amount. Anaemic Gambian toddlers were given 2 mg 57 Fe orally to equilibrate with total body iron over 8-11 months. After assignment to the positive control arm of the HIGH study, 22 toddlers consumed a micronutrient powder containing 12 mg iron for 12 weeks followed by 12 weeks without iron supplementation. Their daily iron absorption increased 3·8-fold during the iron supplementation period compared to the control period [median (interquartile range, IQR): 1·00 (0·82; 1·28) mg/day vs. 0·26 (0·22; 0·35) mg/day; P = 0·001]. Unexpectedly, during the supplementation period, daily iron loss also increased by 3·4-fold [0·75 (0·55; 0·87) mg/day vs. 0·22 (0·19; 0·29) mg/day; P = 0·005]. Consequently, most (~72%) of the absorbed iron was lost during supplementation. Long-term studies of iron absorption and loss are a promising and accurate method for assessing and quantifying long-term iron balance and may provide a reference method for evaluating iron intervention programs in vulnerable population groups. This study was registered as ISRCTN 0720906.

Measurement of the enriched stable isotope 58Fe in iron related disorders- comparison of INAA and MC-ICP-MS.

As a safer alternative for the use of radioactive tracers, the enriched stable 58Fe isotope has been introduced in studies of iron metabolism. In this study this isotope is measured with instrumental neutron activation analysis (INAA) in blood samples of patients with iron related disorders and controls after oral ingestion of a 58Fe containing pharmaceutical. Results were compared with those derived from MC-ICP-MS, applied on the same samples, and analytical and practical aspects of the two techniques were compared. Both techniques showed an increased absorption and incorporation in red blood cells of the 58Fe isotope in iron deficient patients in contrast to the controls. In all individuals results of INAA measurements were in good agreement with those of MC-ICP-MS (|zeta| < 2). Uncertainties in INAA are substantially higher than those achievable by MC-ICP-MS but the INAA technique offers a high specificity and selectivity for iron close to 100%. In contrast to INAA, sample preparation before measurement is very critical in MC-ICP-MS and interferences with 58Ni and 54Cr may hamper the measurement of 58Fe and 54Fe respectively. Since it takes at least five days after irradiation to reduce the activity of interfering radionuclides (mainly 24Na), INAA is a more time consuming procedure; the need of a nuclear reactor facility makes it also less accessible than MC-ICP-MS. Costs are comparable. Both INAA and MC-ICP-MS are able to adequately measure changes in iron isotope composition in blood when an enriched stable iron isotope is applied in clinical research. Although MC-ICP-MS is more sensitive, is faster and has easier access, in INAA preparative steps before measurement are simpler and there are hardly demands on the kind and size of the samples. This may be relevant working with biomaterials in a clinical setting.

Iron bioavailability from fresh cheese fortified with iron-enriched yeast.

PURPOSE: An iron-enriched yeast able to lyse at body temperature was developed for iron fortification of chilled dairy products. The aim was to evaluate iron (Fe) absorption from iron-enriched yeast or ferrous sulfate added to fresh cheese. METHODS: Two stable isotope studies with a crossover design were conducted in 32 young women. Fe absorption from fresh cheese fortified with iron-enriched yeast (2.5 mg 58Fe) was compared to that from ferrous sulfate (2.5 mg 57Fe) when ingested with fresh cheese alone or with fresh cheese consumed with bread and butter. Iron absorption was determined based on erythrocyte incorporation of isotopic labels 14 days after consumption of the last test meal. RESULTS: Geometric mean fractional iron absorption from fresh cheese fortified with iron-enriched yeast consumed alone was significantly lower than from the cheese fortified with FeSO4 (20.5 vs. 28.7 %; p = 0.0007). When the fresh cheese was consumed with bread and butter, iron absorption from both fortificants decreased to 6.9 % from the iron-enriched yeast compared to 8.4 % from ferrous sulfate. The relative bioavailability of the iron-enriched yeast compared to ferrous sulfate was 0.72 for the cheese consumed alone and 0.82 for cheese consumed with bread and butter (p = 0.157). CONCLUSIONS: Iron from iron-enriched yeast was 72-82 % as well absorbed as ferrous sulfate indicating that the yeast lysed during digestion and released its iron.

Delaying Iron Therapy until 28 Days after Antimalarial Treatment Is Associated with Greater Iron Incorporation and Equivalent Hematologic Recovery after 56 Days in Children: A Randomized Controlled Trial.

BACKGROUND: Iron therapy begun concurrently with antimalarial treatment may not be well absorbed because of malaria-induced inflammation. Delaying the start of iron therapy may permit better iron absorption and distribution. OBJECTIVE: We compared erythrocyte iron incorporation in children who started iron supplementation concurrently with antimalarial treatment or 28 d later. We hypothesized that delayed iron supplementation would be associated with greater incorporation and better hematologic recovery. METHODS: We enrolled 100 children aged 6-59 mo with malaria and hemoglobin concentrations of 50.0-99.9 g/L who presented to Mulago Hospital, Kampala, into a randomized trial of iron therapy. All children were administered antimalarial treatment. Children with zinc protoporphyrin (ZPP) ≥80 µmol/mol heme were randomly assigned to start iron supplementation concurrently with the antimalarial treatment [immediate iron (I) group] or 28 d later [delayed iron (D) group]. All children were administered iron-stable isotope (57)Fe on day 0 and (58)Fe on day 28. We compared the percentage of iron incorporation at the start of supplementation (I group at day 0 compared with D group at day 28, aim 1) and hematologic recovery at day 56 (aim 2). RESULTS: The percentage of iron incorporation (mean ± SE) was greater at day 28 in the D group (16.5% ± 1.7%) than at day 0 in the I group (7.9% ± 0.5%; P < 0.001). On day 56, concentrations of hemoglobin and ZPP and plasma ferritin, soluble transferrin receptor (sTfR), hepcidin, and C-reactive protein did not differ between the groups. On day 28, the hemoglobin (mean ± SD) and plasma iron markers (geometric mean; 95% CI) reflected poorer iron status in the D group than in the I group at this intervening time as follows: hemoglobin (105 ± 15.9 compared with 112 ± 12.4 g/L; P = 0.04), ferritin (39.3 µg/L; 23.5, 65.7 µg/L compared with 79.9 µg/L; 58.3, 110 µg/L; P = 0.02), sTfR (8.9 mg/L; 7.4, 10.7 mg/L compared with 6.7 mg/L; 6.1, 7.5 mg/L; P = 0.01), and hepcidin (13.3 ng/mL; 8.3, 21.2 ng/mL compared with 38.8 ng/mL; 28.3, 53.3 ng/mL; P < 0.001). CONCLUSIONS: Delaying the start of iron improves incorporation but leads to equivalent hematologic recovery at day 56 in Ugandan children with malaria and anemia. These results do not demonstrate a clear, short-term benefit of delaying iron. This trial was registered at clinicaltrials.gov as NCT01754701.

Effects of dietary factors on the pharmacokinetics of 58Fe-labeled hemin after oral administration in normal rats and the iron-deficient rats.

Hemin, iron (III) protoporphyrin chloride (IX), as a stable form of heme iron, has been used in iron absorption studies. The aim of the present study was to elucidate the influences of body iron status and three dietary factors (green tea extract, ascorbic acid, and calcium) on the pharmacokinetics of hemin using stable isotope labeling methods followed by ICP-MS measurement. In this study, a rapid, sensitive, and specific ICP-MS method for the determination of (58)Fe originating from hemin in rat plasma was developed and a rat model of iron deficiency anemia was established. It was found that hemin iron absorption increased significantly under iron deficiency anemia status, with AUC0-t and AUC0-∞ showing significant increase in anemic rats compared to normal ones. Green tea extract strongly inhibited hemin iron absorption in both normal rats and iron-deficient rats. In normal rats administered with green tea extract, C max resulted significantly reduced, whereas in anemic rats administered with green tea extract both AUC0-t and AUC0-∞ were reduced. On the other hand, ascorbic acid significantly affected hemin iron absorption only in iron-deficient rats, in which C max showed a significant increase. Interestingly, calcium slowed down the hemin iron absorption rate in normal rats, MRT0-t being significantly different in calcium-treated animals compared to untreated ones. This trend also appeared in the iron-deficient group but it did not reach statistical significance. Our data suggest that the mechanism of hemin iron absorption is regulated by body iron status and dietary factors can influence hemin iron absorption to varying degrees. Moreover, these results may also have general implication in the iron deficiency treatment with iron supplements and fortification of foods.

Nutritional iron supplementation studies based on enriched (57) Fe, added to milk in rats, and isotope pattern deconvolution-ICP-MS analysis.

Enriched stable iron isotopes in combination with isotope pattern deconvolution and ICP-MS have been used to study the absorption and bioavailability of iron from supplemented formula milk administrated to lactating rats. The use of two enriched stable isotope tracers, one as the metabolic tracer (here (57) Fe) and the other ((54) Fe) as quantitation tracer, is shown to provide quantitative data about endogenous and exogenous (supplemented) total Fe distribution in rat feces, urine, red blood cells (RBCs), serum, liver, and kidney. The proposed analytical methodology was validated using reference materials (serum, urine, and liver) spiked with both (54) Fe and (57) Fe. Quantitative information about iron absorption/bioavailability and/or metabolism can be obtained from the amounts of endogenous and exogenous iron found in the tissues and fluids analyzed, and about its kinetic after 2 weeks of iron supplementation. The obtained results are discussed in terms of iron exchanged and its half-life in lactating rats and the observed iron levels in serum, RBCs, liver, and kidney comparing nonsupplemented rats and maternal feed rats.

Assessment of iron absorption in mice by ICP-MS measurements of (57)Fe levels.

BACKGROUND: The study of iron metabolism is essential in nutritional sciences as iron deficiency is one of the most common nutritional deficiencies in humans and represents a serious health problem worldwide. The mouse is utilized as a unique and powerful model for the identification and characterization of genes involved in iron metabolism and for studying the pathogenesis of iron disorders. Thus, sophisticated and sensitive techniques have been developed to study iron metabolism in this animal model. In particular, iron absorption has been studied in mice by using the radioisotopes (55)Fe and (59)Fe in tied-off or dissected and everted duodenal segments. Nevertheless, several drawbacks discourage the extended use of these approaches. METHODS AND RESULTS: Here, we report the use of the stable isotope (57)Fe to measure iron absorption in mice. We show that after oral administration of (57)Fe-containing solutions, it is possible to measure both duodenal iron retention and duodenal iron transfer to specific organs, using inductively coupled plasma mass spectrometry (ICP-MS). As (57)Fe is administered orally, no surgical operation is needed before the end of the experiment, thus allowing the measurement of iron absorption under physiologic conditions. Moreover, the use of ICP-MS for (57)Fe detection ensures high sensitivity and provides quantitative data. Finally, the use of a stable isotope enables the measurement of both iron absorption and histologic and/or biochemical analyses in the same animal. CONCLUSIONS: The use of (57)Fe to measure iron absorption in mice, therefore, represents an alternative to radioisotope-based methods, providing a new tool to extend our knowledge on the mechanism of iron absorption.

Nonheme-iron absorption in first-degree relatives is highly correlated: a stable-isotope study in mother-child pairs.

BACKGROUND: Iron absorption in humans is highly variable even after iron status and dietary components that influence iron absorption are controlled for. Inherited factors may help explain this variance. OBJECTIVE: Our objective was to compare nonheme-iron absorption from a noninhibitory, stable-isotope-labeled test meal in preschool-aged children and their mothers. DESIGN: We provided 72 test meals based on degermed maize flour and milk powder and fortified with [(57)Fe]ferrous fumarate or [(58)Fe]ferrous sulfate to healthy Mexican preschool children [n = 18; mean (+/-SD) age: 3.6 +/- 1.0 y] and their mothers [n = 18; mean (+/-SD) age: 28.0 +/- 5.2 y]. Iron absorption was calculated on the basis of incorporation of isotopes into erythrocytes after 14 d and was adjusted for differences in iron status. RESULTS: There was a wide variation in iron absorption from the test meals: in the mothers and children, the median fractional absorption of ferrous sulfate was 22.55% (range: 1.65-54.83%) and 5.51% (range: 2.23-17.20%), respectively (P < 0.0001). After adjustment for serum ferritin, the significant difference in absorption between mothers and their children disappeared. Despite this broad range of iron absorption, corrected fractional iron absorption from the ferrous fumarate-fortified (r(2) = 0.582) and the ferrous sulfate-fortified test meals (r(2) = 0.557) was strongly correlated in mothers and their children (P < 0.0001). There was a striking positive correlation between the mean corrected fractional iron absorption from both test meals in mothers and their children (r(2) = 0.782, P < 0.0001). In regression analyses that included age, sex, and hemoglobin, the only significant predictor of corrected fractional iron absorption in children was corrected fractional iron absorption in their mothers (standardized beta = 0.884, P < 0.001). CONCLUSIONS: Nonheme-iron absorption exhibits a strong familial tendency. After differences in meal matrix and serum ferritin are accounted for, these data suggest that inheritance and/or shared environmental factors explain most of the variance in dietary iron absorption.

Isotope ratio measurements of iron in blood samples by multi-collector ICP-MS to support nutritional investigations in humans.

With the perspective of embarking on a human study using a double iron (Fe) stable isotope tracer protocol to assess iron bioavailability, investigations were conducted on Fe isotope ratios in blood samples using a VG Axiom Multi-collector ICP-MS. The factors affecting the precision and accuracy of Fe isotopic ratios, such as spectral- and matrix-induced interferences and Fe recoveries from sample preparation, have been identified and optimized. Major polyatomic interferences (e.g., Ar-O, Ar-OH, and FeH) were significantly reduced by using an Aridus nebulizer and desolvating system. Isobaric metal (e.g., (54)Cr(+) on (54)Fe(+) and (58)Ni(+) on (58)Fe(+)) interferences and Ca-oxides and hydroxides were quantitatively removed during chemical purification of blood samples and selective isolation of Fe by anion-exchange resin, after mineralization of the blood samples by microwave digestion. Quantitative recoveries of Fe from different steps of sample preparation were verified using whole blood reference material. Fe isotopic compositions of the samples were corrected for instrumental mass bias by the standard-sample bracketing method using the certified reference standard IRMM-014. External precisions on the order of 0.008-0.05 (% RSD), 0.007-0.015 (% RSD), and 0.03-0.09 (% RSD) were obtained for (54)Fe/(56)Fe, (57)Fe/(56)Fe, and (58)Fe/(56)Fe, respectively, in the blood for three replicate measurements. The level of precision obtained in this work enables the detection of low enrichments of Fe in blood, which is highly desired in nutrition tracer studies.

Epigallocatechin gallate (EGCG) (TEAVIGO) does not impair nonhaem-iron absorption in man.

A number of studies have shown that tea catechins can inhibit intestinal iron absorption, mostly iron in the nonhaem form. This randomized, double-blind, placebo-controlled, 3-periods cross-over study examined the degree of inhibition of nonhaem iron absorption by pure crystalline epigallocatechin gallate (EGCG). The study was designed to show the maximum inhibitory action of EGCG by selecting 30 healthy women with low iron stores. Treatments were 150 mg, 300 mg EGCG and placebo each for 8 consecutive study days with a wash-out period of 14 days between treatments. Iron incorporation was assessed by supplying 57Fe orally and 58Fe intravenously. Differences in fractional nonhaem iron absorption between the treatments were evaluated by using two-sided ANOVA. Results showed a relative nonhaem iron absorption reduction of 14% with 150mg EGCG and 27% for 300mg EGCG treatment compared to placebo. Differences were statistically significant (p < or = 0.05) between the placebo and the 300mg EGCG treatments and between the 150 and 300 mg EGCG treatments. The inverse relation between EGCG dose and fractional nonhaem iron absorption was linear (p = 0.0002). In this study the magnitude of the inhibitory action of EGCG on nonhaem iron absorption was found to be much lower than that reported in the literature for black tea and similar compounds. The doses of EGCG in supplements, which will be lower than those used in this study, are not expected to have any health relevant effects on iron absorption in subjects with normal iron stores.

Iron absorption by human subjects from different iron fortification compounds added to Thai fish sauce.

OBJECTIVES: (a) To measure iron absorption by human subjects from citric acid stabilized fish sauce fortified with ferrous sulfate, ferric ammonium citrate or ferrous lactate and (b) to identify the effect of added citric acid (3 g/l) on iron absorption from ferrous sulfate fortified fish sauce. DESIGN: Iron absorption from the intrinsically labeled compounds was determined via erythrocyte incorporation of isotopic labels ((57)Fe and (58)Fe) using a randomized crossover design. In three separate absorption studies, 10 adult women each consumed a basic test meal of rice and vegetable soup seasoned with isotopically labeled, iron fortified fish sauce. RESULTS: Iron absorption was significantly lower from ferrous lactate and from ferric ammonium citrate fortified fish sauce than from ferrous sulfate fortified fish sauce. Fractional iron absorption (geometric mean; -1s.d., +1s.d.) was 8.7(3.6; 21.4)% for ferrous lactate compared to 13.0(5.4; 31.4)% from ferrous sulfate, P = 0.003 (study 1) and 6.0(2.5; 14.3)% from ferric ammonium citrate relative to 11.7(4.4; 30.7)% from ferrous sulfate, P < 0.001, in study 2. Citric acid added at a molar ratio of approximately 2.5 to iron had no effect on iron absorption from ferrous sulfate (study 3). Iron absorption in the presence of citric acid was 14.1(6.4; 30.8)% compared to 12.0(5.8; 24.7)% in its absence (P = 0.26). CONCLUSIONS: Iron absorption was 50-100% higher from ferrous sulphate fortified fish sauce than from fish sauce fortified with ferric ammonium citrate or ferrous lactate. In the presence of citric acid as a chelator, ferrous sulfate would appear to be a useful fortificant for fish sauce. SPONSORSHIP: International Atomic Energy Agency (IAEA), Vienna, Austria.