Duodenal Absorption and Tissue Utilization of Dietary Heme and Nonheme Iron Differ in Rats

Chang Cao; Carrie E Thomas; Karl L Insogna; Kimberly O O'Brien

doi:10.3945/jn.114.197939

. 2014 Sep 10;144(11):1710–1717. doi: 10.3945/jn.114.197939

Duodenal Absorption and Tissue Utilization of Dietary Heme and Nonheme Iron Differ in Rats^¹^,^²^,^³

Chang Cao ^5,⁴, Carrie E Thomas ^6,⁴, Karl L Insogna ⁷, Kimberly O O'Brien ^5,^*

PMCID: PMC4195416 PMID: 25332470

Abstract

Background: Dietary heme contributes to iron intake, yet regulation of heme absorption and tissue utilization of absorbed heme remains undefined.

Objectives: In a rat model of iron overload, we used stable iron isotopes to examine heme- and nonheme-iron absorption in relation to liver hepcidin and to compare relative utilization of absorbed heme and nonheme iron by erythroid (RBC) and iron storage tissues (liver and spleen).

Methods: Twelve male Sprague-Dawley rats were randomly assigned to groups for injections of either saline or iron dextran (16 or 48 mg Fe over 2 wk). After iron loading, rats were administered oral stable iron in the forms of ⁵⁷Fe-ferrous sulfate and ⁵⁸Fe-labeled hemoglobin. Expression of liver hepcidin and duodenal iron transporters and tissue stable iron enrichment was determined 10 d postdosing.

Results: High iron loading increased hepatic hepcidin by 3-fold and reduced duodenal expression of divalent metal transporter 1 (DMT1) by 76%. Nonheme-iron absorption was 2.5 times higher than heme-iron absorption (P = 0.0008). Absorption of both forms of iron was inversely correlated with hepatic hepcidin expression (heme-iron absorption: r = −0.77, P = 0.003; nonheme-iron absorption: r = −0.80, P = 0.002), but hepcidin had a stronger impact on nonheme-iron absorption (P = 0.04). Significantly more ⁵⁷Fe was recovered in RBCs (P = 0.02), and more ⁵⁸Fe was recovered in the spleen (P = 0.01).

Conclusions: Elevated hepcidin significantly decreased heme- and nonheme-iron absorption but had a greater impact on nonheme-iron absorption. Differential tissue utilization of heme vs. nonheme iron was evident between erythroid and iron storage tissues, suggesting that some heme may be exported into the circulation in a form different from that of nonheme iron.

Introduction

Iron is an essential micronutrient for humans and plays a critical role in a variety of processes, such as oxygen transport and energy production. Because humans have no regulated mechanism of iron excretion, iron balance relies primarily on tight regulation at the level of intestinal iron absorption (1). Dietary iron is present in 2 distinct forms: heme iron (animal products) and nonheme iron (plants, animal products, and supplements). The absorption of nonheme iron is tightly controlled by body iron status and the liver-derived peptide hormone hepcidin (2). Hepcidin is thought to affect iron absorption by inducing the degradation of the basolateral iron transporter ferroportin (FPN)⁸, thus limiting iron export into the circulation (3). However, this classic model of hepcidin action has been challenged by accumulating cell culture and animal data showing mixed results regarding the relation between hepcidin and duodenal FPN (4–7). Recent studies in mice and Caco-2 cells further suggest that the brush border iron transporter divalent metal transporter 1 (DMT1), not FPN, is the primary target of hepcidin regulation (5). Clearly, in vivo experiments that explore the interrelations between hepcidin, intestinal iron transporter expression, and nonheme-iron transport are needed to clarify the complex mechanisms by which hepcidin modulates the absorption of dietary heme and nonheme sources of iron.

Heme iron constitutes ∼10% of total dietary iron intake in a typical Western diet but is thought to provide nearly one-third of absorbed iron because of its substantially higher absorption and bioavailability (8). Despite the importance of heme in iron nutrition, the mechanisms responsible for heme-iron absorption remain largely uncharacterized. In contrast to nonheme iron, heme-iron absorption is less stringently regulated in response to iron status and dietary inhibitors and enhancers. Apical heme uptake is thought to occur either via receptor-mediated endocytosis (9) or may, in part, be mediated by a low-affinity heme importer, proton coupled folate transporter (PCFT) (10), or to an as yet identified heme importer. It is believed that once in the enterocyte, the majority of heme iron is converted to inorganic iron and enters a common intracellular iron pool with nonheme iron to be exported out to the circulation by FPN. An alternative heme enterocyte export pathway was described in guinea pigs whereby heme is exported intact into the plasma (11). The presence of the heme exporter feline leukemia virus C receptor 1 (FLVCR1) in the human duodenum supports this hypothesis (12). Little is known about the relative impact of hepcidin on heme- vs. nonheme-iron absorption and the degree to which iron overload differentially influences duodenal and tissue expression of heme- and nonheme-iron transporters.

The goal of this study was to use a rat model of iron overload and labeled heme and nonheme iron to examine the impact of hepcidin on heme- vs. nonheme-iron transport across the enterocyte in vivo and to evaluate associations between iron loading and hepcidin expression on duodenal, hepatic, and splenic expression of proteins involved in the transport of heme [PCFT, heme oxygenase 1 (HO-1)] and nonheme iron [DMT1, FPN, duodenal cytochrome b (DCYTB)]. We hypothesized that iron loading would elevate liver hepcidin production and result in parallel reductions in duodenal FPN and nonheme-iron absorption and that iron loading would have a substantially greater ability to downregulate nonheme-iron trafficking proteins and absorption. We further hypothesized that heme- and nonheme-iron absorption would be differentially affected by hepcidin expression.

Materials and Methods

Rats and treatments.

Twelve adult male Sprague-Dawley rats (8–10 wk old) consumed a standard rodent diet (iron content: 36 μg/g; AIN93-G; Harlan Teklad) for 1 wk before being randomly assigned to 1 of 3 groups: a high iron loading (H-Fe) group, a moderate iron loading (M-Fe) group, or a control group. Rats in the H-Fe and M-Fe groups were administered a total of 48 and 16 mg Fe through i.p. injections of iron dextran twice a week for 2 wk from day 0 to day 13. A relatively mild regimen of iron overload was chosen (∼0.03 and 0.1 mg Fe/g body weight) when compared with other iron loading rodent protocols (0.2–1.5 mg/g body weight) (13–16) to better characterize relations between iron absorption and hepcidin under more physiologic conditions. Rats in the control group were injected with PBS at the same time points during the same period. All study procedures were approved by the Yale Institutional Animal Care and Use Committee.

Isotope preparation.

Stable iron isotopes (⁵⁷Fe at 95% enrichment and ⁵⁸Fe at 93% enrichment) were purchased from Trace Sciences International. The ⁵⁷Fe isotope was converted into ferrous sulfate solution by Anazao Health by using the method of Kastenmayer et al. (17). The ⁵⁸Fe isotope was converted into ferrous citrate and injected into a piglet to intrinsically label porcine RBCs as described elsewhere (18). The ⁵⁸Fe enrichment of the labeled porcine RBCs was 17.3 ± 0.01% as determined by magnetic sector thermal ionization MS (TIMS; ThermoQuest).

Iron absorption studies.

Absorption of heme and nonheme iron was measured 2 wk after iron loading by using orally administered stable iron isotopes. Oral stable iron tracers containing similar amounts of total iron (25.4 μg ⁵⁸Fe as porcine ⁵⁸Fe-hemoglobin; total iron from the hemoglobin dose: 145.0 μg; 172.1 μg ⁵⁷Fe as ⁵⁷FeSO₄; total iron from the FeSO₄ dose: 181.0 μg) were administered by oral gavage on 2 separate days by using a randomized crossover design so that half of the rats were administered the heme-iron tracer on day 14 and the nonheme-iron tracer on day 17 and the remaining rats were administered these 2 forms of iron in the reverse order. Iron tracer doses were separated by 3 d to allow sufficient time for the intestinal epithelium to renew in order to minimize any impact of the first tracer dose on absorption of the second tracer dose. Absorption of the heme and nonheme tracers was assessed by RBC enrichment 10 d after the administration of the second tracer because previous iron tracer studies in rats have established that RBC incorporation of injected iron is complete within 6 d of administration (19, 20). Rats were anesthetized by using 30% (v/v) isoflurane and killed by exsanguination. Whole blood was collected by cardiac puncture and serum was separated and stored at −80°C. Liver, kidney, spleen, heart, and duodenal tissue were harvested, flash-frozen in liquid nitrogen, and stored at −80°C before analysis for tissue iron concentration and iron isotopic enrichment by TIMS, as detailed below.

Blood and tissue iron isotopic enrichment measurement.

Samples of whole blood, liver, and spleen were digested with 4 mL HNO₃ in polytetrafluoroethylene beakers and evaporated to dryness after the solutions were clear. Samples were dissolved in 2 mL 6 mol/L HCl, and iron was extracted by anion exchange chromatography as described previously (21). Isotopic ratios (^57/56Fe and ^58/56Fe) were measured by using TIMS. The natural abundance values used were 0.02317 for ^57/56Fe and 0.00308 for ^58/56Fe. Relative SDs averaged 0.025% and 0.30% for ^57/56Fe and ^58/56Fe, respectively. All acids used were ultrapure (Ultrex; JT Baker).

Calculation of tissue isotope incorporation.

The net amount of ⁵⁷Fe and ⁵⁸Fe incorporated in RBCs was determined by multiplying RBC enrichment of the 2 tracers by the total RBC iron mass assuming a blood volume for male Sprague-Dawley rats of 68.6 mL/kg (22) and an iron content of hemoglobin of 3.47 g/kg (23). A similar approach was used to calculate the net amount of ⁵⁷Fe and ⁵⁸Fe in liver and spleen by using the measured tissue ⁵⁷Fe and ⁵⁸Fe enrichment, tissue iron concentration, and an estimated tissue weight for male Sprague-Dawley rats derived from equations developed on the basis of the age of the rat (24).

Calculation of iron absorption.

Iron absorption was calculated as the total amount of tracer recovered in RBCs, liver, and spleen as a fraction of the oral dose. Because of inadequate liver samples and nickel contamination in some spleen and liver samples that interfered with ⁵⁸Fe enrichment measures, data on total recovered ⁵⁷Fe and ⁵⁸Fe were only available for 11 and 9 of the 12 rats, respectively. From the rats with complete tissue enrichment data, we determined that 80.5% and 71.3% of the total recovered ⁵⁷Fe and ⁵⁸Fe, respectively, were found in RBCs. This allowed us to make assumptions about RBC incorporation of heme- and nonheme-iron tracers for the 3 rats that did not have complete tissue enrichment data. We also calculated absorption on the basis of 80% RBC incorporation, which is frequently used in human studies to estimate iron absorption (25). This method did not change the relations between heme-/nonheme-iron absorption with hepcidin. The values calculated by both methods and their correlations are presented in Supplemental Table 1.

Calculation of iron absorption and tissue iron uptake.

The oral iron tracer has 2 primary fates once absorbed across the enterocyte: it is either utilized by RBCs for hemoglobin synthesis or delivered to storage sites (primarily the liver and spleen) (Supplemental Fig. 1). Absorption of heme and nonheme iron was calculated as the total amount of heme (⁵⁸Fe) or nonheme (⁵⁷Fe) tracer recovered in RBCs, liver, and spleen as a fraction of the total oral tracer dose administered. Tissue utilization of absorbed tracers was assessed by using 3 approaches. The first approach presented the Δ percentage excess of each isotope, which reflects the degree to which the natural abundance of isotope in each tissue increased as a result of tissue tracer uptake. The second approach estimated the net quantity (μg) of each iron tracer recovered in the RBC pool, liver, and spleen. The third measure assessed the relative distribution of absorbed tracer in RBCs, liver, and spleen by expressing the amount of tracer in each tissue as a proportion of the total tracer recovered.

Tissue iron determination.

Tissue iron concentration was measured by using a graphite atomic absorption spectrophotometer (Perkin-Elmer 370). Bovine liver standard 1577 (National Bureau of Standards) was analyzed in the same manner to ensure accuracy of the iron measurements.

Western blotting of tissue iron proteins and serum ferritin.

Protein was extracted from liver, spleen, and duodenum by homogenizing tissues in lysis buffer containing protease inhibitors (Sigma Aldrich). Proteins (30 μg) were solubilized in Laemmli buffer and boiled for 5 min at 95°C before SDS-PAGE and subsequent transfer onto polyvinylidene difluoride membranes. Membranes were blocked in Odyssey Blocking Buffer (Li-Cor) for 1 h and incubated with primary antibodies overnight at 4°C. Antibodies and the concentrations used were as follows: anti-FPN (MTP11-A; Alpha Diagnostics) at 1:1000 dilution, anti-transferrin receptor 1 (anti-TFR1; no. 136800; Life Technologies) at 1:5000 dilution; anti-PCFT (ab25134; Abcam) at 1:1000 dilution; anti–HO-1 (SPA-869; Enzo Life Sciences) at 1:500 dilution; and anti-ferritin (ab55077; Abcam) at 1:1000 dilution. After incubation with the primary antibodies overnight at 4°C, membranes were washed 5 times and then incubated with appropriate infrared secondary antibodies (1:5000 dilution; Li-Cor) at room temperature for 1 h. Blots were visualized by using the Odyssey Infrared Imaging System (Li-Cor), and the expression of target proteins was normalized to β-actin (sc47778; Santa Cruz Biotechnology) and expressed as a ratio relative to the control group. Six duodenal lysates from 6 rats appeared to be partially degraded as indicated by substantially lower β-actin concentrations compared with other analyzed duodenal lysates. These lysates were excluded from Western blot analysis of duodenal iron transporters. The degradation likely reflects the known presence of high concentrations of proteases in duodenal tissue.

Serum ferritin was analyzed by Western blotting in serum samples collected on the day of killing. Serum (5 μL) was diluted with deionized H₂O, separated on a 12.5% SDS-PAGE gel, and transferred to a polyvinylidene difluoride membrane. After blocking, the membrane was incubated with a ferritin antibody (1:1000; ab55077; Abcam), probed with an infrared secondary antibody, and analyzed by using the Odyssey imaging system as described above.

qPCR.

Total RNA was extracted from duodenal enterocytes and liver by using TRIzol reagent (Invitrogen), followed by purification with the use of the RNeasy Mini Kit (Qiagen). The cDNA was synthesized by using the iScript cDNA Synthesis Kit (Bio-Rad) and reversed-transcribed by using a PTC-100 PCR machine (MJ Research). qPCR was performed by using TaqMan assays for the following targets: Dmt1 (Rn00565927_m1), Dcytb (Rn01484657_m1), Tfr1 (Rn01474701_m1), hepcidin (Rn00584987_m1), Pcft (Rn01471182_m1), hephaestin (Rn00515970_m1), and Fpn (Rn00591187_m1). β-Actin was used as the endogenous reference (Rn00667869_m1; Applied Biosystems). The PCR reactions were performed by using an iQ2 Optical System (Bio-Rad) with the following protocol: 95°C for 10 min, 40 cycles at 95°C for 20 s, and 60°C for 1 min. Relative quantification of target genes was calculated by using the 2(−ΔΔ C_T) method.

Statistical analysis.

Statistical analyses were performed in JMP 10.0 (SAS Institute). Differences between treatment groups were compared by using 1-factor ANOVA and Tukey’s post hoc test. Variables that were not normally distributed were log-transformed before analysis. Equal variance assumption of ANOVA was checked by Levene’s test. Variables that did not meet the ANOVA assumptions were compared by using nonparametric Wilcoxon’s rank test. A paired t test was used to compare duodenal absorption as well as tissue enrichment of the dietary heme- vs. nonheme-iron tracers. Simple linear regression was used to examine relations between hepcidin, RBC iron incorporation, iron transporter expression, and tissue isotopic enrichment. Residuals of regression analyses were checked for normality by using the Shapiro-Wilk test. An interaction term between iron form (heme vs. nonheme) × hepcidin was used to test whether the effect of hepcidin on iron absorption differed between heme and nonheme iron. Data are expressed as means ± SEMs. Significance was defined when P < 0.05.

Results

Impact of iron loading on tissue iron content, hepcidin, and tissue iron transporter expression.

Two weeks of iron loading with a total of 16 or 48 mg Fe did not significantly influence body weight or hemoglobin concentrations (Table 1). Rats in the H-Fe group had elevated tissue iron concentrations (mg/g dry weight) in liver and spleen, whereas those in the M-Fe group only exhibited significant increases in the iron content of the spleen (Table 1). Iron loading significantly increased liver hepcidin mRNA in the H-Fe group (Fig. 1A). Liver hepcidin did not increase in the M-Fe group (P = 0.2). Hepcidin correlated in logarithmic proportion to liver iron concentrations (r = 0.95, P < 0.0001). Serum ferritin did not differ between the 3 treatment groups (P = 0.1) but was significantly higher in the H-Fe and M-Fe rats combined than in the control rats (P = 0.03) (Fig. 1B).

TABLE 1.

Iron status and tissue iron isotope enrichment in control and iron-loaded rats¹

	Group
	Control	M-Fe	H-Fe	P (ANOVA)
Body weight, g	480 ± 7	466 ± 15	481 ± 18	0.7
Hemoglobin, g/L	171 ± 7	152 ± 15	172 ± 8	0.4
Serum iron, mg/L	1.6 ± 0.2	2.0 ± 0.1	1.9 ± 0.2	0.9
Tissue iron, mg/g dry weight
Liver	0.22 ± 0.02^b	0.55 ± 0.07^b	2.21 ± 0.46^a	0.001
Spleen	1.60 ± 0.27^c	10.2 ± 0.95^b	17.3 ± 2.67^a	0.003
Kidney	0.30 ± 0.03	0.26 ± 0.03	0.26 ± 0.03	0.6
Heart	0.24 ± 0.02^b	0.27 ± 0.01^b	0.39 ± 0.04^a	0.009
⁵⁷Fe excess, %
Blood	14.1 ± 2.6^a	7.6 ± 1.7^a,b	4.1 ± 0.5^b	0.01
Liver	21.2 ± 4.8^a	5.2 ± 2.2²^,b	0.9 ± 0.1^b	0.001
Spleen	3.7 ± 0.9^a	0.8 ± 0.4^b	0.3 ± 0.1^b	0.003
⁵⁸Fe excess, %
Blood	5.7 ± 0.7^a	3.1 ± 0.5^b	2.0 ± 0.3^b	0.002
Liver	8.9 ± 1.6²^,a	1.8 ± 0.5²^,b	0.6 ± 0.1^b	0.004
Spleen	4.0 ± 1.6³^,a	0.5 ± 0.1^b	0.5 ± 0.2^b	0.01

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Values are means ± SEMs; n = 4 unless otherwise noted due to lack of tissue sample or presence of nickel contamination. Labeled means in a row without a common letter differ, P < 0.05. H-Fe, high iron loading; M-Fe, moderate iron loading.

n = 3.

n = 2.

Effect of iron dextran injections on hepcidin, serum ferritin, and liver iron transporter expression in rats. Liver hepcidin mRNA in H-Fe, M-Fe, and control groups (A). Serum ferritin concentrations in the H-Fe and M-Fe groups combined (n = 8) and the control group (n = 4) (B). Liver expression of FPN and TFR1 in rats (C). Values are means ± SEMs; n = 4/group. Means without a common letter differ, P < 0.05. *Different from control, P < 0.05. FPN, ferroportin; HO-1, heme oxygenase 1; H-Fe, high iron loading; M-Fe, moderate iron loading; TFR1, transferrin receptor 1.

The expression of genes involved in iron metabolism in the liver was determined by qPCR (Fpn and Tfr1) and Western blotting (FPN, TFR1, HO-1, and ferritin) (Fig. 1C). Hepatic Tfr1 mRNA expression was significantly lower in rats in the M-Fe (P = 0.01) and H-Fe (P = 0.002) groups than in controls. Iron injections had no impact on hepatic Fpn mRNA (P = 0.8). Similar treatment responses were noted for hepatic FPN protein (P = 0.4). Hepatic expression of ferritin and HO-1 protein was 2 times higher in the H-Fe group than that in control rats, but these differences were not significant (P = 0.08 for ferritin and P = 0.2 for HO-1). The expression of iron proteins in the spleen was assessed by Western blotting. Of all of the proteins assessed, only HO-1 was significantly increased in response to H-Fe injection (P = 0.03). Together, these results indicate the high iron dextran treatment produced the classic phenotype of iron overload, whereas the moderate iron regimen only mildly elevated iron stores in the spleen without significantly affecting hepcidin production or expression of tissue iron transporters.

Impact of iron loading on iron absorption and duodenal iron transporter expression.

Average absorption efficiencies of heme and nonheme iron in control rats were 19.8 ± 1.7% and 45.2 ± 6.0%, respectively. Heme- and nonheme-iron absorption was strongly correlated within rats (r = 0.79, P = 0.002). In contrast to the significantly higher heme- vs. nonheme-iron absorption that is known to occur in humans, the mean heme-iron absorption observed in rats was 60% lower than that observed for nonheme-iron absorption (P = 0.0007; n = 12). Injections of moderate and high doses of iron dextran suppressed nonheme-iron absorption by 46% (24.1% vs. 45.2%; P = 0.01) and 78% (10.0% vs. 45.2%; P = 0.0004), respectively, when compared with the control group. The percentage of heme-iron absorption was similarly decreased in the M-Fe (7.5% vs. 19.8%; P = 0.0002) and H-Fe (4.7% vs. 19.8%; P < 0.0001) groups when compared with the control group (Fig. 2A). Although the degree of suppression of heme- and nonheme-iron absorption was greater with the 48-mg load than with the 16-mg load, these differences did not reach significance (P = 0.08 for nonheme-iron and P = 0.3 for heme-iron absorption).

Iron absorption and duodenal iron transporter expression in rats after iron overload treatment. Heme- and nonheme-iron absorption in H-Fe, M-Fe, and control rats; n = 4/group (A). Duodenal mRNA expression of genes involved in iron transport (B). Western blotting of proteins involved in duodenal iron transport; n = 2/group (C). Panels A and B: Values are means ± SEMs. Means without a common letter differ, P < 0.05. *Dcytb*, duodenal cytochrome b; *Dmt1*, divalent metal transporter 1; FPN, ferroportin; HO-1, heme oxygenase 1; H-Fe, high iron loading; M-Fe, moderate iron loading; PCFT, proton coupled folate transporter.

Next we examined duodenal expression of genes involved in heme- and nonheme-iron trafficking (Dmt1, Dcytb, Fpn, hephaestin, Pcft) and explored possible associations with heme- and nonheme-iron absorption. Transcript levels of Dmt1 (P = 0.006) and Dcytb (P = 0.0006) decreased significantly in the H-Fe group compared with the control group (Fig. 2B). No significant differences in mRNA expression of Fpn (P = 0.1), hephaestin (P = 0.3), or Pcft (P = 0.2) were evident between the treatment groups. We also performed Western blotting on selected heme- and nonheme-iron proteins (FPN, PCFT, HO-1, and ferritin) in the 6 rats with available duodenal lysates (Fig. 2C). There were no apparent differences in FPN and PCFT protein expression between iron-loaded and control rats. Although only analyzed in 2 rats/treatment group, average protein expression of HO-1 and ferritin in the H-Fe group was 2 times higher than that in the control group.

Effect of hepcidin on iron absorption and tissue iron transporter expression.

Linear regression analysis showed that liver hepcidin mRNA correlated inversely with absorption of both heme (r = − 0.77, P = 0.003) and nonheme (r = −0.80, P = 0.002) iron. The effect of hepcidin on iron absorption differed significantly between the heme- and nonheme-iron tracers with every unit increase in hepcidin expression, reducing heme absorption by 4.1% and nonheme absorption by 10.0% (P = 0.04). The interaction between dietary iron (nonheme vs. heme iron) and hepcidin is shown in Figure 3.

Correlations of liver hepcidin mRNA with heme- and nonheme-iron absorption in rats; n = 12.

To investigate the molecular basis for the observed difference in the impact of hepcidin on heme- and nonheme-iron absorption, duodenal heme- and nonheme-iron transporter expression was examined in relation to liver hepcidin transcript expression. hepcidin was inversely associated with Dmt1 (r = −0.61, P = 0.04) and Dcytb mRNA (r = −0.72, P = 0.009). There was a nonsignificant negative correlation between hepcidin and Fpn mRNA (P = 0.13). Hepcidin transcript expression did not correlate with duodenal Pcft mRNA expression. In the 6 rats with evaluable duodenal lysates, liver hepcidin mRNA was not associated with any of the iron proteins examined (FPN, HO-1, PCFT, ferritin) except for a nonsignificant positive correlation with ferritin (r = 0.68, P = 0.09).

Relations of liver hepcidin and iron transporter expression in the liver and spleen were also examined. In the liver, hepcidin mRNA was not related to Fpn transcript or protein expression but was strongly correlated with Tfr1 mRNA (r = −0.84, P = 0.0006). An inverse relation between hepcidin mRNA and Tfr1 protein expression approached significance (r = −0.57, P = 0.06). In the spleen, hepcidin was not related to protein expression of TFR1 or FPN.

Tissue utilization of ingested heme and nonheme iron.

To determine whether uptake of absorbed heme (⁵⁸Fe) and nonheme (⁵⁷Fe) by iron storage sites was similar to results obtained in RBCs, the enrichment of ⁵⁷Fe and ⁵⁸Fe in the liver and spleen was compared between treatment groups. Consistent with the RBC enrichment data, the change in the percentage excess of nonheme iron (⁵⁷Fe) in the liver and spleen was significantly lower in iron-loaded rats than in control rats (Table 1). The change in the percentage excess of heme iron (⁵⁸Fe) followed a similar pattern, with rats in the H-Fe and M-Fe groups having significantly lower ⁵⁸Fe enrichment in the liver and spleen than control rats.

To explore whether there were potential differences in tissue utilization of absorbed heme and nonheme iron, the relative recovery of the 2 tracers in RBCs, liver, and spleen was evaluated (Table 2). A significantly higher percentage of nonheme-iron tracer was recovered in the RBC mass than that recovered for heme iron (80.5% vs. 71.3%; P = 0.02). A reverse relation was evident in the spleen, such that significantly more heme-iron tracer was recovered compared with the relative recovery of the nonheme-iron tracer (7.5% vs. 2.7%; P = 0.01). No significant difference in relative recovery of each form of iron was noted in the liver. There was also a differential effect of hepcidin on tissue utilization of absorbed heme and nonheme iron. In the RBC pool, the net amount of both ⁵⁷Fe and ⁵⁸Fe tracer recovered was inversely correlated with liver hepcidin (r = −0.75, P = 0.005, and r = −0.76, P = 0.005, respectively). Hepcidin was not related to the net amount of either the heme- or nonheme-iron tracer recovered in the liver or the spleen.

TABLE 2.

Tissue enrichment of ⁵⁸Fe-heme and ⁵⁷Fe-nonheme in rats after gavage with ⁵⁷FeSO₄ and ⁵⁸Fe-RBCs¹

	Nonheme (⁵⁷Fe)		Heme (⁵⁸Fe)
	Value	n	Value	n	P²
Blood
Iron excess, %	8.6 ± 1.6	12	3.8 ± 0.5	12	0.0007
Net tracer amount, μg	34.9 ± 6.2	12	1.9 ± 0.3	12	0.0001
Recovered tracer, %	80.5 ± 2.4	11	71.3 ± 4.7	9	0.02
Liver
Iron excess, %	9.5 ± 3.3	11	3.5 ± 1.3	10	0.002
Net tracer amount, μg	6.8 ± 1.1	11	0.41 ± 0.04	10	0.0005
Recovered tracer, %	16.8 ± 2.0	11	21.2 ± 3.7	9	0.15
Spleen
Iron excess, %	1.6 ± 0.5	12	1.2 ± 0.5	10	0.4
Net tracer amount, μg	0.9 ± 0.2	12	0.13 ± 0.03	10	0.002
Recovered tracer, %	2.7 ± 0.6	11	7.5 ± 1.8	9	0.01

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Values are means ± SEMs unless indicated otherwise. n < 12 indicates lack of tissue sample or presence of nickel contamination that interfered with ⁵⁸Fe determination; a paired t test was performed for rats with data on both variables.

Paired t test.

Discussion

Few studies have simultaneously assessed the impact of hepcidin on heme- and nonheme-iron absorption and tissue utilization in relation to key iron transport proteins and hepcidin expression. In the rat model of iron loading, hepcidin was found to have a significantly greater ability to regulate absorption of nonheme iron than did heme iron, a difference that was accompanied by significant differences in expression of Dmt1 and Dcytb. Differential tissue utilization of orally ingested heme vs. nonheme iron was also evident, with the RBCs preferentially utilizing iron derived from a dietary nonheme-iron source, whereas the spleen preferentially utilized dietary iron ingested in the form of heme iron.

Iron loading with a moderate iron dose (48 mg) resulted in a 3-fold increase in liver hepcidin and a 78% reduction in nonheme-iron absorption. These changes were accompanied by a marked downregulation in duodenal Dmt1 mRNA, whereas duodenal Fpn mRNA had only a slight, but nonsignificant, decrease in response to iron injection and increased hepcidin production. Similar findings of an inverse association between hepatic hepcidin expression and duodenal iron transporter mRNA were observed in both iron-loaded mice (26) and rats (4, 27). These changes are thought to occur in response to increases in intracellular iron, which could reduce transcript stability via the iron regulatory element/ iron regulatory protein system (28) or inhibit transcription by interfering with hypoxia inducible factor 2 (HIF-2) signaling (29). We found that transcript levels of Dmt1 and Fpn were highly correlated with their functionally associated ferrireductase/ferroxidase, Dcytb and hephaestin, suggesting a coordinated regulation of these genes in the duodenum (30, 31). Contrary to the classic hepcidin-dependent downregulation of FPN protein observed in the macrophage, both FPN protein and transcript expression in the duodenum were unrelated to hepcidin and nonheme-iron absorption. This is in line with recent studies in Caco-2 cells and rodents, which found no impact of hepcidin treatment on duodenal FPN and basolateral iron transport (5, 7, 32). Our results support the accumulating evidence that DMT1, and not FPN, is a primary target for hepcidin regulation in the duodenum. It has been suggested that hepcidin may indirectly affect duodenal FPN protein by downregulating DMT1 and thus decreasing intracellular iron concentrations and hence Fpn transcripts (5). This may explain the low FPN protein concentrations in mice with sustained hepcidin expression (5). It is worth noting that hepcidin remained a significant determinant of nonheme-iron absorption after accounting for variation in duodenal Dmt1 mRNA, suggesting the presence of other absorptive components that mediate the suppression of iron absorption by hepcidin.

Iron loading significantly reduced hepatic expression of TFR1 but had no effect on FPN expression in the liver. The downregulation of liver TFR1 expression by iron injection is consistent with previous studies in iron-loaded rats (33, 34) and in patients with hemochromatosis (35). However, we did not find increased hepatic Fpn mRNA expression previously observed in mice injected with iron dextran (13, 36). The discrepancy between these reports may be explained by the difference in doses used to induce iron overload (0.1 mg Fe/g body weight in our study vs. 1 mg/g body weight in previous studies) or different responses to iron loading between animal strains (36) and sexes (37). Considering the high buffering capacity of the liver for excess iron, it is likely that a certain amount of hepatic iron loading is needed to cause an upregulation of iron export from this organ. Further studies are needed to determine if there is a threshold effect of iron overload on FPN-mediated iron export and whether FPN expression in liver Kupffer cells and hepatocytes respond differently to iron status and hepcidin.

In this study, the absorption of iron from a dietary heme-iron source was, on average, 60% lower than iron absorption from nonheme ferrous sulfate. Our finding of a preferential absorption of nonheme over heme iron has been previously documented in early radiotracer studies in Sprague-Dawley rats in which a 70–90% lower retention of iron from heme-iron sources [hemoglobin (38, 39), myoglobin (40), and beef (41)] was noted compared with iron retention from nonheme-iron sources. However, these data are in direct contrast to human studies that consistently showed a 2–5 times greater relative iron absorption from heme vs. nonheme iron (42–44). The physiologic basis for this species difference in differential heme- vs. nonheme-iron utilization is unknown and perhaps reflects adaptation to habitual diets of primarily animal- vs. plant-based iron sources. Despite the relative differences in heme- vs. nonheme-iron absorption, cellular mechanisms of heme- and nonheme-iron absorption are thought to be similar between these 2 species (45). There is also ample evidence to suggest that rats respond similarly to dietary and physiologic factors known to affect heme-iron absorption in humans, including meat proteins and body iron stores (46). Because of these qualitative similarities, rat models offer important insights on the mechanisms and regulation of heme-iron absorption in humans, although species differences must be considered when making inferences.

Both heme- and nonheme-iron absorptions were suppressed by iron overload, and both were inversely correlated with liver hepcidin. Liver hepcidin expression explained 59% and 63% of the variation in heme- and nonheme-iron absorption, respectively, but had a greater relative impact on nonheme-iron absorption. Liver total iron concentrations were found to be an equally strong predictor of the absorption of heme and nonheme iron. These relations closely resemble those observed between heme absorption and serum ferritin in humans (42, 44), confirming that heme-iron absorption is responsive to changes in body iron status, although to a lesser extent than nonheme iron. The inhibitory effect of iron status on heme-iron absorption did not seem to be mediated by duodenal PCFT and HO-1, because neither was changed by iron injection or hepcidin. Interestingly, there was a strong inverse relation between heme absorption and duodenal Dmt1 mRNA. This may reflect their shared regulation by body iron status or a role of DMT1 in transporting heme-derived inorganic iron from the endosome after apical endocytosis of heme (9).

The use of stable iron isotopic techniques provided a unique opportunity to simultaneously compare tissue utilization of these 2 types of dietary iron in the same animals in response to body iron stores. Differential tissue utilization of dietary heme vs. nonheme iron was evident, with more nonheme-iron intake being utilized for RBC synthesis, whereas a greater proportion of dietary iron was delivered to the spleen when ingested as heme iron. The relative distribution of absorbed nonheme-iron tracer between the liver, spleen, and RBCs was similar to previous findings obtained after i.v. injection of radio-iron salts in mice (47) and rats (48, 49). Few such data are available for heme iron. Although the majority of absorbed heme was recovered in the RBC iron pool, the fraction of heme tracer recovered in RBCs was significantly lower than that observed for the nonheme-iron tracer. The variable utilization of these 2 forms of iron evident in the RBCs was supported by the significant 2-fold higher enrichment of heme iron in the spleen when compared with enrichment of nonheme iron. There was also a trend for greater uptake of heme iron in the liver, but this difference did not reach significance. The differential tissue utilization of absorbed heme vs. nonheme iron may suggest that a portion of absorbed heme exits the enterocyte in a form distinct from nonheme iron. If heme is exported intact as heme using the FLVCR1 protein found on the basolateral membrane in enterocytes, it should share the same metabolic fate as plasma heme and be taken up by cells that express the heme receptor protein low density lipoprotein receptor-related protein 1 (LRP-1), such as hepatocytes and macrophages in the liver and spleen (50). It was shown in rats that >90% of i.v.-injected heme-hemopexin is recovered in the liver within 2 h of tracer injection (51). This is consistent with our finding that a greater proportion of absorbed heme was delivered to the spleen and liver compared with the relative tissue utilization of nonheme iron.

In conclusion, moderate iron loading in rats significantly increased tissue iron content and hepatic hepcidin expression without affecting serum ferritin or hemoglobin concentrations. Increased hepatic hepcidin expression was associated with significantly lower duodenal absorption of both heme and nonheme iron. The suppressive effects of hepcidin on nonheme- and heme-iron absorption were mediated by DMT1 and were not associated with changes in FPN. Hepcidin more potently downregulated nonheme-iron absorption than heme-iron absorption in rats. There were significant differences in tissue deposition of the absorbed heme- and nonheme-iron tracers, suggesting that some heme may be exported into the circulation in a form different from that of nonheme iron. The cellular and molecular mechanisms underlying this difference require further study.

Supplementary Material

Online Supporting Material

supp_144_11_1710__index.html^{(759B, html)}

Acknowledgments

K.O.O. and C.C. designed the research, analyzed the data, and prepared the manuscript; C.C. and C.E.T. conducted the research; K.L.I. and K.O.O. supervised the experiments and data interpretation; and K.O.O. had primary responsibility for the final content. All authors read and approved the final version of the manuscript.

Footnotes

⁸

Abbreviations used: DCYTB, duodenal cytochrome B; DMT1, divalent metal transporter 1; FLVCR1, feline leukemia virus C receptor 1; FPN, ferroportin; H-Fe, high iron loading; HIF-2, hypoxia inducible factor 2; HO-1, heme oxygenase 1; LRP-1, low density lipoprotein receptor-related protein 1; M-Fe, moderate iron loading; PCFT, proton coupled folate transporter; TFR1, transferrin receptor 1; TIMS, thermal ionization mass spectrometry.

References

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Supplementary Materials

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[bib1] 1.Andrews NC. Forging a field: the golden age of iron biology. Blood 2008;112:219–30. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib2] 2.Nemeth E, Ganz T. Regulation of iron metabolism by hepcidin. Annu Rev Nutr 2006;26:323–42. [DOI] [PubMed] [Google Scholar]

[bib3] 3.Nemeth E, Tuttle MS, Powelson J, Vaughn MB, Donovan A, Ward DM, Ganz T, Kaplan J. Hepcidin regulates cellular iron efflux by binding to ferroportin and inducing its internalization. Science 2004;306:2090–3. [DOI] [PubMed] [Google Scholar]

[bib4] 4.Frazer DM, Wilkins SJ, Becker EM, Vulpe CD, McKie AT, Trinder D, Anderson GJ. Hepcidin expression inversely correlates with the expression of duodenal iron transporters and iron absorption in rats. Gastroenterology 2002;123:835–44. [DOI] [PubMed] [Google Scholar]

[bib5] 5.Brasse-Lagnel C, Karim Z, Letteron P, Bekri S, Bado A, Beaumont C. Intestinal DMT1 cotransporter is down-regulated by hepcidin via proteasome internalization and degradation. Gastroenterology 2011;140(4):1261–71, e1. [DOI] [PubMed]

[bib6] 6.Chung B, Chaston T, Marks J, Srai SK, Sharp PA. Hepcidin decreases iron transporter expression in vivo in mouse duodenum and spleen and in vitro in THP-1 macrophages and intestinal Caco-2 cells. J Nutr 2009;139:1457–62. [DOI] [PubMed] [Google Scholar]

[bib7] 7.Mena NP, Esparza A, Tapia V, Valdes P, Nunez MT. Hepcidin inhibits apical iron uptake in intestinal cells. Am J Physiol Gastrointest Liver Physiol 2008;294:G192–8. [DOI] [PubMed] [Google Scholar]

[bib8] 8.Han O. Molecular mechanism of intestinal iron absorption. Metallomics 2011;3:103–9. [DOI] [PubMed] [Google Scholar]

[bib9] 9.West AR, Oates PS. Mechanisms of heme iron absorption: current questions and controversies. World J Gastroenterol 2008;14:4101–10. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib10] 10.Shayeghi M, Latunde-Dada GO, Oakhill JS, Laftah AH, Takeuchi K, Halliday N, Khan Y, Warley A, McCann FE, Hider RC, et al. Identification of an intestinal heme transporter. Cell 2005;122:789–801. [DOI] [PubMed] [Google Scholar]

[bib11] 11.Conrad ME, Weintraub LR, Sears DA, Crosby WH. Absorption of hemoglobin iron. Am J Physiol 1966;211:1123–30. [DOI] [PubMed] [Google Scholar]

[bib12] 12.Keel SB, Doty RT, Yang Z, Quigley JG, Chen J, Knoblaugh S, Kingsley PD, De Domenico I, Vaughn MB, Kaplan J, et al. A heme export protein is required for red blood cell differentiation and iron homeostasis. Science 2008;319:825–8. [DOI] [PubMed] [Google Scholar]

[bib13] 13.Muckenthaler M, Roy CN, Custodio AO, Minana B, deGraaf J, Montross LK, Andrews NC, Hentze MW. Regulatory defects in liver and intestine implicate abnormal hepcidin and Cybrd1 expression in mouse hemochromatosis. Nat Genet 2003;34:102–7. [DOI] [PubMed] [Google Scholar]

[bib14] 14.Nicolas G, Chauvet C, Viatte L, Danan JL, Bigard X, Devaux I, Beaumont C, Kahn A, Vaulont S. The gene encoding the iron regulatory peptide hepcidin is regulated by anemia, hypoxia, and inflammation. J Clin Invest 2002;110:1037–44. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib15] 15.Turbino-Ribeiro SM, Silva ME, Chianca DA, Jr, De Paula H, Cardoso LM, Colombari E, Pedrosa ML. Iron overload in hypercholesterolemic rats affects iron homeostasis and serum lipids but not blood pressure. J Nutr 2003;133:15–20. [DOI] [PubMed] [Google Scholar]

[bib16] 16.Steinbicker AU, Bartnikas TB, Lohmeyer LK, Leyton P, Mayeur C, Kao SM, Pappas AE, Peterson RT, Bloch DB, Yu PB, et al. Perturbation of hepcidin expression by BMP type I receptor deletion induces iron overload in mice. Blood 2011;118:4224–30. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib17] 17.Kastenmayer P, Davidsson L, Galan P, Cherouvrier F, Hercberg S, Hurrell RF. A double stable isotope technique for measuring iron absorption in infants. Br J Nutr 1994;71:411–24. [DOI] [PubMed] [Google Scholar]

[bib18] 18.Young MF, Griffin I, Pressman E, McIntyre AW, Cooper E, McNanley T, Harris ZL, Westerman M, O'Brien KO. Utilization of iron from an animal-based iron source is greater than that of ferrous sulfate in pregnant and nonpregnant women. J Nutr 2010;140:2162–6. [DOI] [PMC free article] [PubMed] [Google Scholar]

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[bib21] 21.Young MF, Glahn RP, Ariza-Nieto M, Inglis J, Olbina G, Westerman M, O'Brien KO. Serum hepcidin is significantly associated with iron absorption from food and supplemental sources in healthy young women. Am J Clin Nutr 2009;89:533–8. [DOI] [PubMed] [Google Scholar]

[bib22] 22.Probst RJ, Lim JM, Bird DN, Pole GL, Sato AK, Claybaugh JR. Gender differences in the blood volume of conscious Sprague-Dawley rats. J Am Assoc Lab Anim Sci 2006;45:49–52. [PMC free article] [PubMed] [Google Scholar]

[bib23] 23.Abrams SA, Wen J, O'Brien KO, Stuff JE, Liang LK. Application of magnetic sector thermal ionization mass spectrometry to studies of erythrocyte iron incorporation in small children. Biol Mass Spectrom 1994;23:771–5. [DOI] [PubMed] [Google Scholar]

[bib24] 24.Mirfazaelian A, Fisher JW. Organ growth functions in maturing male Sprague-Dawley rats based on a collective database. J Toxicol Environ Health A 2007;70:1052–63. [DOI] [PubMed] [Google Scholar]

[bib25] 25. International Atomic Energy Agency. Assessment of iron bioavailability in humans using stable iron isotope techniques. IAEA Human Health Series. Vienna: International Atomic Energy Agency; 2012.

[bib26] 26.Shirase T, Mori K, Okazaki Y, Itoh K, Yamamoto M, Tabuchi M, Kishi F, Jiang L, Akatsuka S, Nakao K, et al. Suppression of SLC11A2 expression is essential to maintain duodenal integrity during dietary iron overload. Am J Pathol 2010;177:677–85. [DOI] [PMC free article] [PubMed] [Google Scholar]

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PERMALINK

Duodenal Absorption and Tissue Utilization of Dietary Heme and Nonheme Iron Differ in Rats1,2,3

Chang Cao

Carrie E Thomas

Karl L Insogna

Kimberly O O'Brien