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Review
Iron Absorption: Molecular and Pathophysiological Aspects
Margherita Correnti , Elena Gammella, Gaetano Cairo * and Stefania Recalcati

Department of Biomedical Sciences for Health, University of Milan, 20133 Milan, Italy;
margherita.correnti@unimi.it (M.C.); elena.gammella@unimi.it (E.G.); stefania.recalcati@unimi.it (S.R.)
* Correspondence: gaetano.cairo@unimi.it; Tel.: +39-0250315330

Abstract: Iron is an essential nutrient for growth among all branches of life, but while iron is among
the most common elements, bioavailable iron is a relatively scarce nutrient. Since iron is fundamental
for several biological processes, iron deficiency can be deleterious. On the other hand, excess iron
may lead to cell and tissue damage. Consequently, iron balance is strictly regulated. As iron excretion
is not physiologically controlled, systemic iron homeostasis is maintained at the level of absorption,
which is mainly influenced by the amount of iron stores and the level of erythropoietic activity, the
major iron consumer. Here, we outline recent advances that increased our understanding of the
molecular aspects of iron absorption. Moreover, we examine the impact of these recent insights on
dietary strategies for maintaining iron balance.

Keywords: iron; intestine; absorption; regulation; iron homeostasis; gut microbiota

1. Introduction
Iron, the most abundant element on earth [1], in the context of the competition for its
possession between mammalian hosts and bacterial pathogens is a driver of evolution [2].
In fact, iron is an essential microelement for the fundamental biological processes of living
organisms; only two lifeforms not requiring iron are known (Borrelia burgdorferi and Lacto-
bacilli) [3]. Indeed, the role of iron availability on bacterial life and virulence is so important
that an iron-rich environment can re-establish the threat of harmless microorganisms, as
Citation: Correnti, M.; Gammella, E.; shown by the peculiar case of a scientist unknowingly suffering from hereditary hemochro-
Cairo, G.; Recalcati, S. Iron matosis (HH), a genetic disease characterized by iron overload, who died of septicemic
Absorption: Molecular and plague after being exposed to a weakened form of Yersinia pestis [4].
Pathophysiological Aspects. Both iron scarcity and excess may be deleterious; indeed, iron deficiency, which affects
Metabolites 2024, 14, 228. https:// more than a billion people [5], causes anemia and impairs growth, but can protect against
doi.org/10.3390/metabo14040228 some bacterial infections and malaria [6]. On the other hand, insufficient iron availability
Academic Editor: Walter Wahli
impairs immunity; therefore, the “correct” amount of iron is required for health.
Iron participates as a cofactor in several highly conserved biological processes; about
Received: 22 March 2024 400 proteins are numbered in the iron proteome [7]. In mammals, iron is involved in oxygen
Revised: 12 April 2024 transport and storage by heme in hemoglobin and myoglobin, respectively, but also in
Accepted: 13 April 2024
energy metabolism as a cofactor for the heme and iron-sulfur proteins necessary for mito-
Published: 17 April 2024
chondrial function (e.g., Krebs cycle, and electrons transport chain). Moreover, as a cofactor
for ribonucleotide reductase, iron is required for DNA synthesis and cell replication [8].
Interestingly, a recent study showed that iron supplementation, by supplying mitochon-
Copyright: © 2024 by the authors.
drial oxidative metabolism and energy production, was sufficient to promptly restore both
Licensee MDPI, Basel, Switzerland.
muscle function and mass in tumor-bearing mice and in human subjects affected by cancer
This article is an open access article cachexia [9]. Hence, iron is essential for cellular metabolism and growth [10].
distributed under the terms and The importance of iron for life is principally due to its chemical properties, such as
conditions of the Creative Commons the ability to accept or donate electrons, interconverting between the ferrous (Fe2+ ) and
Attribution (CC BY) license (https:// ferric (Fe3+ ) states [11]. This capacity enables iron to bind oxygen and form complexes
creativecommons.org/licenses/by/ with many organic ligands [12]. However, its redox reactivity makes Fe2+ a potentially
4.0/). hazardous biometal involved in reactive oxygen species (ROS) generation through Fenton

Metabolites 2024, 14, 228. https://doi.org/10.3390/metabo14040228 https://www.mdpi.com/journal/metabolites

Metabolites 2024, 14, 228 2 of 15

chemistry [13]. ROS, especially the highly aggressive hydroxyl radical, may damage
proteins, DNA and lipids causing cellular toxicity and organ damage. In particular, the
attack to membrane lipids in the process called lipid peroxidation promotes a specific type of
regulated cell death called ferroptosis [14]. Given the toxicity of iron, particularly in excess,
cells have developed several tight regulatory mechanisms to control the systemic and
intracellular iron homeostasis, thereby maintaining adequate and safe amounts of iron [15].

2. Regulation of Cellular Iron Homeostasis

Cellular iron homeostasis in mammalian cells is regulated by balancing iron uptake,
storage, export and utilization [16]. Cells mainly acquire iron from circulating iron-loaded
holo-transferrin (Tf), which is internalized by clathrin-mediated endocytosis after binding
to cell surface transferrin receptor 1 (TfR1) [17]. The binding affinity of Tf for Fe3+ is high
at the physiological pH present at the cell surface and abolished at acidic pH. Therefore,
the acidification of endosomes triggers the release of iron, which is then reduced to Fe2+
by the metalloreductase STEAP3 and transported across the endosomal membrane by
DMT1 (divalent metal transporter 1) or other metal transporters like ZIP8 and ZIP14. The
apoTf-TfR1 complex returns to the plasma membrane, where Tf dissociates from TfR1 [18].
The key role of TfR1 is shown by the severe defects caused by genetic inactivation of TfR1
(reviewed in [17]); however, other forms of uptake independent of the Tf–TfR1 interaction
have been found, including receptor-mediated uptake of ferritin subunits (via TfR1, Scara5
and TIM-2), lipocalin 2 [15] and hyaluronan-CD44 complex [19]. The latter pathways
have been mostly characterized in specific cell lines, but whether they have a general
and functional role in iron uptake is still undetermined. Moreover, under conditions of
iron overload, ZIP14 and ZIP8 may also mediate the uptake of circulating non-Tf-bound
iron (NTBI) [20].
Once inside the cytoplasm, iron enters an incompletely characterized labile iron pool
(LIP) where it loosely binds compounds like glutathione [21] and faces different fates: it
may be stored in ferritin, utilized (e.g., for the synthesis of heme and iron-sulfur clusters)
or released from the cell by ferroportin [22]. Iron export by ferroportin—described in detail
below—is the major route of iron efflux; however, recent findings in experimental models
indicated that secretion of iron-loaded ferritin in exosomes may represent an alternative
pathway that may play a role in pathological conditions [15], such as liver fibrosis [23] and
ferroptosis [24]. Recently, poly-(rC)-binding protein 1 (PCBP1) and PCBP2 were identified
as cytosolic iron chaperones that are responsible for delivering Fe2+ to ferritin for oxidation
and storage, and to ferroportin for export or for other non-heme iron requiring proteins [25].
Ferritin, a heteropolymer formed of 24 subunits of two types (H and L) [26], is able to
accumulate up to 4000 iron atoms in a non-toxic (Fe3+ ) state and thus can have a dual
function: iron storage and the prevention of oxidative damage [27]. In case of necessity, the
iron deposited in ferritin shells can be released in a process involving NCOA4-mediated
autophagic degradation of ferritin [20]. When iron levels are low, NCOA4 binds to ferritin
and delivers it to the nascent autophagosome, which then fuses with the lysosome, thereby
leading to ferritin degradation and iron release (Figure 1A). Given the thousands of iron
atoms present in a ferritin shell, this process is expected to contribute substantially to
cellular iron availability, as also demonstrated by studies showing the important role
of NCOA4-dependent ferritinophagy in processes like erythropoiesis that requires large
amounts of iron for hemoglobin synthesis (reviewed in [28]). When iron is abundant (at
least in cell cultures), NCOA4 can also promote ferritin release in extracellular vesicles
through a CD63-dependent pathway [29].
 Metabolites
Metabolites 2024,
2024, 14,14,
228x FOR PEER REVIEW 3 of3 16
of 15

Figure1.1. Cellular
Figure Cellular and
andsystemic
systemiciron ironmetabolism.
metabolism. (A)(A)Simplified
Simplifiedmodel of IRP-dependent
model of IRP-dependent and in-and
dependent control of intracellular iron metabolism. Under conditions of iron deficiency, active IRP1
independent control of intracellular iron metabolism. Under conditions of iron deficiency, active IRP1
and IRP2 bind iron-responsive elements (IRE) at the 5′ ′untranslated region of ferritin and ferroportin
and IRP2 bind
mRNAs, thusiron-responsive
preventing theirelements (IRE)
translation, andat to
theIREs
5 untranslated
in the 3′ endregionof TfR1of ferritin
and DMT1 and mRNAs,
ferroportin
mRNAs, thus preventing their andtranslation, and to IREs in the ′
3 end of TfR1 and DMT1lead mRNAs,
thus enhancing their stability increasing iron uptake. These combined mechanisms to
thus enhancing
higher theiravailability.
cellular iron stability and increasing
Conversely, iron iron
excess uptake. These
facilitates thecombined
assembly mechanisms lead to
of a 4Fe-4S cluster
in IRP1,
higher whichiron
cellular loses its IRE binding
availability. activity excess
Conversely, and functions as cytoplasmic
iron facilitates aconitase
the assembly of a and triggers
4Fe-4S cluster
inIRP2 proteasomal
IRP1, which loses degradation.
its IRE binding In theactivity
absenceand of active IRPs,asferroportin
functions cytoplasmic andaconitase
ferritin areandactively
triggers
translated, so that superfluous iron is either stored or exported, whereas TfR1 and DMT1 mRNAs
IRP2 proteasomal degradation. In the absence of active IRPs, ferroportin and ferritin are actively
are degraded to prevent additional iron uptake. When cellular iron levels are low, NCOA4 “chap-
translated, so that superfluous iron is either stored or exported, whereas TfR1 and DMT1 mRNAs are
erones” ferritin shells to lysosomal destruction, thus increasing iron availability. Conversely, when
degraded to prevent
iron is abundant, additional
NCOA4 iron uptake.
is targeted When cellular
for degradation and iron
iron levels
is storedare in
low, NCOA4
ferritin. (B) “chaperones”
Hepcidin-
ferritin shells
dependent to lysosomal
regulation destruction,
of body thus increasing
iron homeostasis. Hepcidiniron availability.
expression Conversely,
in the liver is induced when iron is
by body
iron storesNCOA4
abundant, and inflammatory
is targeted forsignals via the BMP/SMAD
degradation and JAK/STAT3
and iron is stored in ferritin.pathways, respectively.
(B) Hepcidin-dependent
Circulating
regulation of hepcidin
body ironinhibits ferroportin-mediated
homeostasis. Hepcidin expressioniron export,
in theprimarily from reticuloendothelial
liver is induced by body iron stores
macrophages and enterocytes, thus resulting in lower levels of
and inflammatory signals via the BMP/SMAD and JAK/STAT3 pathways, respectively. iron in the bloodstream. Conversely,
Circulating
hepcidin synthesis is repressed by iron deficiency or when insufficient oxygen levels stimulate hy-
hepcidin inhibits ferroportin-mediated iron export, primarily from reticuloendothelial macrophages
poxia inducible factor (HIF)-dependent erythropoietin (EPO) production in the kidney. EPO-stim-
and enterocytes,
ulated erythroid thus resulting
precursors in lower
synthesize levels of iron
erythroferrone in thewhich
(ERFE), bloodstream. Conversely,
in turn blocks hepcidin
BMP-mediated
synthesis is repressed by iron deficiency or when insufficient oxygen
hepcidin transcription. This response allows for high ferroportin-mediated iron efflux into plasmalevels stimulate hypoxia
inducible
from the factor
gut and(HIF)-dependent
macrophages. erythropoietin (EPO) production in the kidney. EPO-stimulated
erythroid precursors synthesize erythroferrone (ERFE), which in turn blocks BMP-mediated hepcidin
CellularThis
transcription. ironresponse
homeostasisallows isfor
primarily regulated post-transcriptionally
high ferroportin-mediated iron efflux intoupon plasma binding
from the
of and
gut ironmacrophages.
regulatory proteins (IRP1 and IRP2) to iron-responsive elements (IREs) in the un-
translated regions of the mRNAs for key proteins of iron metabolism [16] (Figure 1A).
When intracellular
Cellular iron levels are
iron homeostasis low, the active
is primarily formspost-transcriptionally
regulated of IRP1 and IRP2 binduponthe IRE in
binding
ofthe 5′ regions
iron of ferritin
regulatory and (IRP1
proteins ferroportin mRNAs,
and IRP2) thereby repressing
to iron-responsive their translation;
elements (IREs) inatthe
untranslated regions of the mRNAs for key proteins of iron metabolism [16] (Figure 1A).
When intracellular iron levels are low, the active forms of IRP1 and IRP2 bind the IRE in
Metabolites 2024, 14, 228 4 of 15

the 5′ regions of ferritin and ferroportin mRNAs, thereby repressing their translation; at
the same time, IRPs bind to the IREs at the 3′ of TfR1 and DMT1 mRNAs, protecting these
transcripts from degradation and enhancing their expression [30]. By inhibiting iron storage
and ferroportin-mediated iron export, while simultaneously favoring iron uptake, this
response increases iron availability. Conversely, in iron-replete conditions, the inhibition
of IRP activity reduces the uptake of unnecessary iron because TfR1 and DMT1 mRNAs
are degraded, and allow the translation of ferroportin and ferritin mRNAs, thus favoring
the storage and export of excess iron. Iron levels in the LIP modulate IRP activity by
regulating the stability of IRP2, which in iron-replete conditions undergoes ubiquitination
followed by proteasomal degradation, and controlling the switch of IRP1 between two
different conformations. When iron is scarce, IRP1 is an apo-protein with IRE-binding
activity, whereas, in the presence of sufficient iron, it is able to assemble a 4Fe-4S cluster,
thus acquiring an enzymatic function as cytosolic aconitase [12] (Figure 1A). Notably, other
proteins directly or indirectly linked to iron homeostasis are regulated by IRPs, including
for example hypoxia inducible factor (HIF) 2α [31] (see below) and CD63 [29] (see above).

3. Regulation of Systemic Iron Homeostasis

The majority of the 3–5 g of iron contained in a healthy human body is present in
red blood cells (RBC) as an essential component of hemoglobin and thus serves in oxygen
transport. Significant amounts of iron are also present in the liver, in macrophages and in
the myoglobin of muscles. Mammals do not possess any regulated mechanism for iron
excretion from the body, as iron loss occurs from the sloughing of mucosal and skin cells
or during occasional bleeding. Therefore, the balance is maintained by the tight control
of dietary iron absorption. In duodenal enterocytes, dietary Fe2+ directly internalized
or derived from heme destruction is exported across the basolateral membrane into the
bloodstream by ferroportin. For the binding of iron to Tf, which delivers redox-inert Fe3+
to all body cells, the oxidation of Fe2+ is catalyzed by the membrane-bound ferroxidase
hephaestin and its soluble homolog ceruloplasmin, which is necessary (of which more in
“Mechanisms of iron absorption”).
However, it should be pointed out that Tf-bound iron, whose major role is to sus-
tain erythropoiesis, is derived mostly by the iron recycling activity of reticuloendothelial
macrophages, which clear effete RBC, destroy hemoglobin and heme, and export iron via
ferroportin into the bloodstream where the metal is oxidized by ceruloplasmin and loaded
to Tf [32].
Systemic iron balance is maintained by the hepcidin–ferroportin axis, which coor-
dinates absorption, recycling, utilization and storage [33] (Figure 1B). Hepcidin, a liver-
derived peptide hormone, binds to ferroportin and thus induces its internalization and
degradation, thereby blocking iron efflux (mainly from enterocytes and macrophages) [34].
In a negative-feedback process, elevated hepcidin transcription, which is mainly induced by
high iron availability through the bone morphogenic proteins (BMP)-SMAD1/5/8 pathway
and inflammatory mediators like IL-6 through the JAK-STAT3 pathway, leads to a decrease
in circulating iron levels [35]. Conversely, under conditions of enhanced erythropoiesis,
hepcidin expression is inhibited mainly by erythroferrone (ERFE), which interferes with
the BMP pathway, thus leading to higher ferroportin-mediated iron release into plasma
in order to meet the increased iron demand for RBC synthesis [36] (Figure 1B). Ultimately,
the latter pathway is controlled by the drop in oxygen levels associated with the higher
erythropoietic drive. In fact, ERFE expression in erythroid precursors is induced by ery-
thropoietin [37], which in turn is synthesized by kidney cells in response to the enhanced
activity of HIF, a transcription factor activated by both hypoxia and low levels of iron [38].
Accordingly, it has been recently shown that low oxygen levels in the liver induce the
synthesis of another hepcidin inhibitor, the fractalkine FGL1, which inhibits hepcidin by
binding to BMP6 [39] (Figure 1B). Tough iron, inflammation and erythropoiesis are the
major regulators of hepatic hepcidin expression, it has been shown that other conditions,
e.g., endoplasmic reticulum stress [40], hormones, e.g., leptin [41] or metabolites (e.g.,
Metabolites 2024, 14, 228 5 of 15

lactate) [42] may affect hepcidin-mediated control of iron homeostasis. Moreover, smaller
amounts of hepcidin produced in organs like the lung, the heart, and the intestine, can
modulate iron metabolism locally [33].

4. Iron Absorption
Multicellular organisms evolved by recycling nutrients like iron. However, our body,
despite an amount of recycled iron around 25 times greater than the quantity acquired
from the diet, needs to assimilate iron from food on a regular basis. In theory, given that
a balanced diet contains 10–30 mg of iron, the amount of 2 mg iron per day necessary to
compensate for non-specific iron losses should be easily obtained [43]. However, under the
oxidative conditions characterizing the biosphere, environmental iron is insoluble; thus, the
paradoxical contradiction between the great abundance of iron and its poor bioavailability
extends to dietary iron, which indeed is mostly inorganic iron found in food derived from
both plants and animals. This form of iron is absorbed with an efficiency of around 10%
depending on the type of diet, with a range between 14 and 18% of iron absorbed from
mixed diets and 5 and 12% from vegetarian diets. This difference is explained by the
greater absorption efficiency of heme iron, primarily present in hemoglobin and myoglobin
from animal food sources, which is around ~25% [44]. Indeed, though heme consists of
~10–15% of total dietary iron sources in meat-eating populations, it accounts for over 40%
of assimilated iron [45]. The reason for the better absorption of heme is not clear and may
depend on its lipophilic properties. However, the latter explanation is not in line with
evidence that not all mammals show this preference for heme as their major iron source;
in fact, for example, mice absorb dietary heme poorly [46], thereby making this useful
animal model inadequate for the characterization of this still poorly understood mechanism
(see below).
It should be noted that the small fraction of iron absorbed under physiological con-
ditions may increase substantially if body iron stores are depleted or the iron demand is
increased; in an iron-deficient individual a maximum absorption at 20% and 35% for inor-
ganic and heme iron, respectively, has been reported [45]. Similarly, nutritional guidelines
recommendations take into account the higher requirements of infants, menstruating and
pregnant women, who should obtain 2–3 times more daily iron from their diet than the
10 mg value appropriate for adult men.

Mechanisms of Iron Absorption

The transit of iron from the duodenal lumen to the bloodstream can be divided into
three major steps: (a) apical uptake (the transport across the brush border), (b) enterocytic
intracellular phase (iron utilization, storage or export) and (c) basolateral transfer (the release
from the enterocytes to the circulation) (Figure 2). The relative importance of these stages,
which are controlled by pathophysiological signals has been long debated, but recent
findings, including the characterization of the hepcidin/ferroportin axis, seem to indicate
that the basolateral transfer is more critical, as first suggested by studies showing that high
release of iron from the intestine into the bloodstream underlies the inappropriate iron
absorption found in hemochromatosis patients [47].
(a) Apical uptake. The uptake of nutritional nonheme iron, which has been elucidated
at the molecular level, occurs mostly in the first portion of the duodenum and in-
volves the transport of Fe2+ across the apical membrane of enterocytes by DMT1
(Figure 2). However, since Fe3+ is the form of iron mostly present in the diet and the
low pH present in the intestinal lumen is not sufficient to maintain iron in a soluble
form, the previous reduction of iron by ferric reductases, such as Dcytb (duodenal
cytochrome b) is required [15]. Accordingly, the importance of diet composition in
determining the amount of iron absorbed is well recognized. Indeed, the assumption
of reductants like vitamin C can improve iron absorption by making the task of Dcytb
easier. Conversely, food components mainly present in vegetables like phytates, which
are primarily found in cereals and legumes, or tannins, may reduce iron absorption
Metabolites 2024, 14, 228 6 of 15

because of unspecific binding, physical entrapment and decreased intestinal transit

time. The strategy aimed at achieving better iron bioavailability by decreasing the
consumption of food containing these inhibitors should be matched against the re-
cent and warranted trend toward increasing the intake of insoluble fiber. However,
phytases to remove phytic acid from food are increasingly used in food-processing
techniques to reduce these inhibiting effects. Other nutrients, including minerals
like calcium and vitamins, could possibly impair iron absorption (reviewed in [48]).
Dietary heme, originating primarily from meat and seafood, can also be transported
across the apical membrane by a hitherto poorly known mechanism. In fact, heme
carrier protein 1 (HCP1), which was initially identified as an intestinal heme importer,
turned out to transport folate, for which HCP1 has an affinity much higher than for
heme. Alternatively, heme responsive gene (HRG1) that transports heme across the
erythrophagosomal membranes of macrophages during iron recycling from RBC [49]
and is expressed in the human small intestine, could represent a candidate for intesti-
nal heme absorption, but its role in this context is still unknown [44]. In any case, it
is well established that dietary absorbed heme is subsequently catabolized within
intestinal epithelial cells by heme oxygenase 1 (HO-1) to liberate Fe2+ , which then
follows the same destiny of inorganic iron imported by DMT1.
(b) Enterocytic intracellular phase. Internalized Fe2+ enters the LIP in the enterocytic cyto-
plasm and, as in any other cell, is either utilized, incorporated in ferritin, or exported
by ferroportin at the basolateral surface (see below) (Figure 2). Given the function
of the duodenum in body iron absorption, the latter fate is predominant. Recently, a
key role for the chaperone PCBP1 in intestinal iron absorption has been reported [50];
the cell-specific deletion of PCBP1 in mice led to lower iron and ferritin levels in
enterocytes and disrupted iron balance. As already mentioned, iron not used by
the duodenal cells is either reversibly stored in ferritin or exported by ferroportin.
Whether ferritin levels simply reflect the iron status of the enterocyte or play an
active role in the control of absorption has been long discussed. Indeed, we found
that in line with the corresponding IRP binding activity, ferritin expression in duo-
denal biopsies was higher than normal in patients with iron overload and lower in
iron-deficient patients with the exception of the inappropriately low levels found in
patients with genetic hemochromatosis which is characterized by inappropriately
high iron absorption [51,52]. Conversely, the cell-specific deletion of H ferritin in
duodenal cells leads to unrestrained absorption and body iron overload [53], whereas
ferritin overexpression caused by IRP inactivation has the opposite effect [54]. The
current view is that IRP-mediated cell-autonomous regulation of ferritin synthesis sets
a basal level of ferritin, which represents a temporary sink for iron not transferred to
the circulation and is then lost when the apical cells are sloughed. These new findings
provided a novel view of the mucosal block model proposed decades ago [55], but
other control mechanisms, in particular, ferroportin-mediated basolateral transfer and
the discovery of NCOA4-mediated ferritinophagy add complexity to this pathway.
NCOA4 is required to avoid iron trapping in enterocytes when the demand for iron is
high; however, a recent study showed quite surprisingly that in mice with intestine-
specific deletions of NCOA4 iron homeostasis is not altered under normal conditions
or in iron deficiency. In these settings, NCOA4 may be regulated by the HERC2 E3
ubiquitin-protein ligase, which triggers its proteasomal degradation [56]. Conversely,
in a mouse model of genetic iron overload, the silencing of NCOA4 in enterocytes
favored iron retention in the duodenum and mitigated systemic iron loading [57].
These findings, which are in line with the inappropriately low expression of both
H and L ferritin subunits previously detected in the duodenal biopsies of patients
with genetic hemochromatosis [52], suggest that the local inhibition of NCOA4 activ-
ity with consequent iron trapping within enterocytic ferritin may represent a novel
therapeutic approach to limit iron uptake in the clinical conditions characterized by
iron hyperabsorption.
 represented by the efflux of Fe2+ at the basolateral surface, which is accomplished
through conformational changes in ferroportin [58] (Figure 2). The key role of ferro-
portin in dietary iron absorption was shown by the rapid insurgence of anemia in
adult mice in which ferroportin was specifically deleted in intestinal cells [59]. Even-
Metabolites 2024, 14, 228 tually, the combined effect of two multicopper ferroxidases, membrane-bound 7 of 15
haephestin and circulating ceruloplasmin, facilitates iron efflux and allows for Fe3+
loading onto plasma Tf for distribution [45,60]. The characterization of their role in
iron absorption
(c) Basolateral andAs
transfer. mobilization
anticipatedprovided
above, the a molecular
final step basis for the findings
of intestinal of ear-
iron absorption
lier elegant studies
is represented by theshowing
efflux of Fea2+copper-deficient
that at the basolateral diet leads to
surface, iron deficiency
which ane-
is accomplished
mia in pigs
through [61]. Given that
conformational stronginvariations
changes ferroportinin Tf saturation
[58] (Figure 2). do The
not affect ironofab-
key role fer-
sorption
roportin ininboth mouse
dietary ironmodels and patients,
absorption was shown Tf wasbythought
the rapid to be only a passive
insurgence iron
of anemia
acceptor. However,
in adult mice a recent
in which study showing
ferroportin that lamina
was specifically propriainmacrophages,
deleted intestinal cellsin [59].
re-
sponse to inflammation and iron, can produce proteases that
Eventually, the combined effect of two multicopper ferroxidases, membrane-bound degrade Tf locally in
haephestin and circulating ceruloplasmin, facilitates iron efflux and allows for Fe3+
the interstitium, thus impairing ferroportin-dependent iron export [62], suggests that
Tf may play
loading ontoanplasma
unexpectedTf forrole in body iron
distribution absorption.
[45,60]. The characterization of their role
in iron absorption
Interestingly, mutant and mobilization
mice lines were provided
instrumentala molecular basis cloning
in identifying, for the findings
and char-of
earlier elegant studies showing that a copper-deficient diet
acterizing the genes involved in both apical and basolateral stages of iron absorption. leads to iron deficiencyIn
fact, the severe anemia of the microcytic (mk) mouse and the Belgrade (b) rat is dueiron
anemia in pigs [61]. Given that strong variations in Tf saturation do not affect to
absorption
identical missense in mutations
both mouse in models
the DMT1 and patients,
gene Tf was
[63,64]. thought
Similarly, to beand
McKie only a passive
colleagues
iron acceptor.
identified Dcytb [65] However, a recent[66]
and ferroportin study
in showing that lamina
the duodenum propria macrophages,
of hypotransferrinemic (hpx)in
mice,response to inflammation
while positional cloning of andtheiron,
genecan produce
defective in proteases that degrade
the sex-linked Tf locally
anemia (sla) mousein
the interstitium,
resulted thus impairing
in the identification ferroportin-dependent
of hephaestin iron exportof[62],
[67]. The severe phenotypes suggests
all these mutantthat
miceTf may play the
underscore an unexpected
key role of the role in body iron absorption.
corresponding genes in iron metabolism.

Figure 2.
Figure 2. Dietary
Dietary iron
iron absorption.
absorption. Dietary
Dietary iron
iron isis mainly
mainlycomposed
composedofofpoorly
poorlyabsorbed
absorbedinorganic
inorganic
iron, which is found in food derived from both plants and animals and is mostlyin
iron, which is found in food derived from both plants and animals and is mostly inthe
theferric
ferric(Fe
3+)
(Fe3+ )
form. Heme iron, primarily present in animal food sources like meat, though less abundant is more
bioavailable. The first step of duodenal iron absorption is the transport of ferrous (Fe2+ ) iron by DMT1
at the apical surface of enterocytes. Previous reduction of the predominant Fe3+ by Dcytb is necessary.
Iron bound to heme is internalized by a still unknown importer and iron is then released in the
cytoplasm by the degradative action of heme oxygenase (HO-1). Following the brush border transit,
both the iron imported by DMT1 and that liberated from heme enter the labile iron pool (LIP) and are
either utilized, incorporated in ferritin shells or exported by ferroportin. PCBP1 and PCBP2 function
as chaperones that deliver iron to client proteins. At the basolateral surface, following the efflux of
Fe2+ into the bloodstream by ferroportin, the oxidative action of hephaestin and ceruloplasmin is
required for the binding of Fe3+ to circulating transferrin.
Metabolites 2024, 14, 228 8 of 15

Interestingly, mutant mice lines were instrumental in identifying, cloning and char-
acterizing the genes involved in both apical and basolateral stages of iron absorption. In
fact, the severe anemia of the microcytic (mk) mouse and the Belgrade (b) rat is due to
identical missense mutations in the DMT1 gene [63,64]. Similarly, McKie and colleagues
identified Dcytb [65] and ferroportin [66] in the duodenum of hypotransferrinemic (hpx)
mice, while positional cloning of the gene defective in the sex-linked anemia (sla) mouse
resulted in the identification of hephaestin [67]. The severe phenotypes of all these mutant
mice underscore the key role of the corresponding genes in iron metabolism.

5. Regulation of Iron Absorption

Both local and systemic pathways control duodenal iron absorption. In fact, the three
major players in these settings appear to be the hepcidin/ferroportin axis, the IRE/IRP
regulatory network and the oxygen-iron regulated HIF system. This complex regulation
provides an efficient system to maintain a response to iron requirements that is both fast
(HIF-dependent transcriptional control) and sustained (long-term responses most likely
mediated by the hepcidin/ferroportin axis) [68]. The first two pathways have been de-
scribed above. Regarding the third, it is important to underline that both hypoxia and
iron deficiency activate the HIF response. In fact, the prolyl hydroxylases (PHD) that
catalyze prolyl hydroxylation of HIF, thereby targeting it to ubiquitination and protea-
somal degradation, require oxygen and 2-oxoglutarate as substrates, but also iron as a
cofactor [38]. Notably, though PHD regulates both HIF1α and HIF2α, only the latter is
crucial for iron absorption [69].
These three pathways interact in a complex interplay, as IRPs regulate the translation
of HIF2α mRNA [31], whereas several genes coding for the proteins of iron metabolism
are transcriptional targets of HIF. In the context of duodenal cells, particularly important
targets are DMT1, Dcytb, ferroportin and NCOA4 [70].
Under conditions of low hepcidin levels when iron uptake should be maximized,
ferroportin-mediated iron export leads to a contraction of the LIP and consequent IRP
activation in duodenal enterocytes. This results in higher DMT1 and Dcytb import activity
on the apical membrane and repression of ferritin synthesis, thereby limiting iron storage.
Ferroportin mRNA escapes the negative regulation by IRP because a variant transcript
lacking the IRE is expressed in the duodenum [71]. At the same time, iron scarcity caused
by ferroportin-mediated iron efflux prevents PHD activity and stabilizes HIF2α, which
induces the transcription of DMT1, Dcytb, NCOA4 and ferroportin, thus activating apical
uptake, preventing the accumulation of ferritin and enhancing export. The elucidation
of the crosstalk between the liver and the intestine that is mediated by the hepcidin–
HIF2α axis [72] provides insights into the pathways upregulating iron uptake in response
to body iron deficiency as well as excess iron absorption under pathological conditions
characterized by hepcidin deficiency.
On the contrary, under conditions of iron excess or inflammation, the downregulation
of ferroportin by hepcidin raises enterocyte iron levels, which in turn increases PHD-
dependent degradation of HIF and inactivates IRPs. These mechanisms result in the
down-regulation of all the proteins involved in iron absorption indicated above at the
transcriptional and post-transcriptional levels.
As previously mentioned, a recent study demonstrated that PCBP1 plays an important
role in this context, although the molecular mechanisms remain to be fully elucidated [50].

6. Examples of Diseases Related to Iron Absorption

As stated above, insufficient iron uptake can lead to anemia or nonanemic iron defi-
ciency, which are among the most common diseases worldwide [73]. Often, this occurs
when the physiological needs for iron are increased at certain times in life (e.g., growth,
pregnancy). In some cases, the underlying cause is genetic, like iron refractory iron defi-
ciency anemia (IRIDA), a rare recessive disorder characterized by hypochromic microcytic
anemia and very low transferrin saturation. IRIDA patients bear mutations within the
Metabolites 2024, 14, 228 9 of 15

TMPRSS6 gene, which encodes matriptase-2, a transmembrane serine protease that neg-
atively regulates hepcidin [74]. The resultant high hepcidin levels impair intestinal iron
absorption and iron recycling by macrophages, thus making therapy very difficult, as
oral iron is not absorbed and most intravenous (IV) iron is trapped within the reticuloen-
dothelial system [75]. Interestingly, recent, though still limited, evidence suggests that
polymorphisms in the TMPRSS6 gene may have a more general role in iron deficiency
and anemia [76] (Table 1). However, the most frequent causes of iron deficiency anemia
(IDA) are iron-poor diets, in particular, those lacking animal-source foods that contain
heme iron. In these cases, iron absorption is not intrinsically defective and may be even
increased (Table 1). Iron deficiency is found also in more than 50% of patients with celiac
disease (CD), a chronic immune-mediated pathology affecting the small intestine triggered
by exposure to dietary gluten in genetically predisposed individuals [77]. In these patients,
the proximal duodenum where iron absorption occurs is typically damaged; therefore, only
IV iron therapy can be considered for patients with active CD, whereas oral iron can be
used in patients in which gluten-free diet ameliorated intestinal atrophy. Notably, in some
patients, the normalization of alterations of the intestinal mucosa is sufficient to recover
from iron deficiency and anemia without iron supplementation.

Table 1. Major genetic and acquired iron absorption-related disorders. The table summarizes a
list of major genetic and non-genetic iron absorption-related disorders (indicated in bold). Genetic
and non-genetic causes are reported in table. For genetic diseases the gene target of mutation and
the biological function of associated protein are reported. Final alterations of iron absorption with
relative underlying molecular mechanisms are shown.

Non Genetic Function of Altered Molecular Basis of

Disease Mutated Gene Iron Absorption
Cause Target Protein Altered Iron Absorption
Negative regulation
IRIDA - TMPRSS6 Hepcidin increase Reduced ↓
of hepcidin
Diet
IDA - - Hepcidin decrease Increased ↑
(major cause)
ACD Inflammation - - Hepcidin increase Reduced ↓
Modulation of Inappropriate hepcidin
HH - HFE Increased ↑
hepcidin production decrease
Down-regulation of
HAMP Loss of hepcidin
ferroportin
Non-HFE HH - regulation Increased ↑
TFR2 Regulation of
HJV hepcidin expression
SLC40A1 Iron export Hepcidin resistance
HBA1 ERFE-mediated hepcidin
Thalassemia - Hb formation Increased ↑
HBB repression
ERFE-mediated hepcidin
SCD - HBB Hb formation repression Increased ↑
(to be confirmed)

Anemia can also be present in patients with chronic inflammation (ACD) accompanied
by sustained hepcidin production [78] (Table 1). However, in chronic inflammatory bowel
diseases (IBD) like Crohn’s disease, intestine-specific defects, by impairing the absorptive
capacity of the gut, may also hamper to some extent iron uptake, though the associated
anemia may be mainly caused by malabsorption of vitamin B12/folate, blood loss and the
concomitant inflammation [79]. In this regard, it can be speculated that the iron-deficient
anemia affecting patients with another IBD, such as ulcerative colitis (UC) is likely due to the
associated inflammation, as iron is not absorbed in the distal intestine that is affected in UC.
In these settings, an adequate therapeutic response is obtained with IV iron administration
that bypasses the gastrointestinal tract.
Metabolites 2024, 14, 228 10 of 15

On the other hand, there are several pathological conditions that directly or indirectly
cause hyperabsorption, thus leading to iron overload (Table 1). The most common is HH, a
group of genetic disorders in which hepcidin deficiency leads to systemic iron excess [80].
In most cases, hemochromatosis is caused by homozygous mutations in the gene encoding
HFE which has a role in hepcidin regulation. Hepcidin production is impaired and inap-
propriately responding to iron, but still detectable; therefore, the penetrance of the disease
is low. Loss-of-function mutations in other genes encoding hepcidin (HAMP), hemojuvelin
(HJV) and transferrin receptor 2 (TFR2) or gain-of-function mutations in the gene encoding
ferroportin (SLC40A1) cause much rarer forms of hemochromatosis (non-HFE HH), but
in general, iron accumulation proceeds at the higher rate, often leading to juvenile forms
of the disease. In all the forms, the pathophysiological mechanism is unregulated iron
absorption due to defects in the hepcidin/ferroportin axis.
Other widespread genetic diseases affecting iron absorption are hemoglobinopathies
(Table 1). In thalassemia, the imbalance in the production of α and β-globin chains due
to mutations in the α- and β-globin gene clusters (HBA1 and HBB) results in ineffective
erythropoiesis, chronic hemolytic anemia and compensatory hemopoietic expansion [81].
In non-transfusion-dependent thalassemic patients who maintain acceptable hemoglobin
values, despite the presence of excess iron, hypoxia-induced and ERFE-mediated hepcidin
suppression chronically stimulates iron absorption, eventually resulting in massive iron
overload. Patients with more severe forms of thalassemia, which require chronic RBC
transfusions to survive, develop very severe secondary iron overload and iron chelation
therapy is necessary to prevent organ damage. In sickle cell disease (SCD), the premature
breakdown of RBC caused by a single amino acid substitution in β-globin ultimately results
in hemolytic anemia. Chronic hemolysis enhances iron demand for erythropoiesis, thereby
prompting intestinal hyperabsorption of iron; however, iron overload occurs only with
blood transfusion [82]. In fact, in contrast to thalassemia where ineffective erythropoiesis
leads to impaired production and higher destruction of RBC in the bone marrow, in SCD
intravascular hemolysis provides a potential mechanism for iron elimination through in-
creased urinary loss and biliary excretion. Paradoxically, dietary iron restriction ameliorates
anemia and other clinical consequences of SCD [83], possibly by affecting the microbiota,
at least in mouse models [84].
While the pathological consequences of elevated iron stores due to excessive dietary
iron intake have been conclusively demonstrated for conditions like hereditary hemochro-
matosis, which is characterized by an iron absorption rate inappropriate to iron stores [85],
the evidence from epidemiological studies that high dietary iron intake may predispose
to diseases is still not fully convincing [86], and may be jeopardized by the confounding
factors associated with the studies investigating food consumption. However, since high
body iron stores have been associated with pathologies highly relevant to human health
like cancer, metabolic syndrome and cardiovascular diseases [43], further investigation
should be performed.
Iron that is not absorbed in the duodenum eventually arrives at the distal sections
of the intestine where it can fuel microbial growth and possibly influence the intestinal
microbiota, which has been shown to play a relevant role in a broad range of metabolic
and physiologic processes [87]. Since different bacteria have distinct iron requirements for
growth and survival, iron availability can play a role in altering the microbial profile in the
gut. In particular, excess iron can favor potentially pathogenic enterobacteria species at the
expense of protective lactobacilli and bifidobacteria that need little iron [86]. Consequently,
any up-to-date iron therapy needs to consider the effects on the microbiota (see below).
Interestingly, it has also been shown that metabolites produced by various bacterial
species can inhibit duodenal absorption by repressing HIF-mediated expression of iron
transporters and inducing ferritin [88]. Using this strategy, aimed at “stealing” iron from
the host, gut microbes obtain more iron for their own necessities.
Metabolites 2024, 14, 228 11 of 15

7. Therapy
Various approaches have been considered to address the problem of insufficient
iron availability, which remains the main public health issue related to iron absorption.
Interventions include food processing, as described above for the use of phytase, food
fortification and supplementation [48]. Fortification, i.e., the addition of iron to processed
foods, represents a successful strategy, which, however, requires careful evaluation of
technological, commercial, nutritional and social issues. In general, appropriately designed
and implemented fortification strategies were able to ensure a sufficient iron intake to
prevent the pathological consequences of iron deficiency, primarily anemia [48].
On an individual basis, direct oral supplementation is the most common therapy
involving intestinal absorption, as IV therapy, which remains the therapy of choice for
rapid corrections or for specific types of patients, obviously bypasses the gut route. Iron
preparations usually rely on Fe2+ because it has higher bioavailability than Fe3+ , though
the most commonly used forms, such as ferrous sulfate, sometimes cause gastrointestinal
side effects that may lead to reduced compliance with therapy. Since the usual doses of
oral iron trigger an increase in hepcidin, accompanied by a reduction in iron absorption,
lasting at least 24 h, alternate day dosing may represent a way to maximize absorption [86].
Recently, new oral iron therapies based on Fe3+ have been developed. The maltol–iron
complex proved to be both tolerable and effective [89]. In fact, by making iron stable, iron
bioavailability is increased and the risk of both mucosal toxicity and modifications of the
gut microbiota is reduced. Moreover, high iron bioavailability and excellent gastrointestinal
tolerance characterize sucrosomial iron, an oral iron formulation in which Fe3+ , protected
by a phospholipid bilayer and a sucrester matrix, is absorbed through para-cellular and
trans-cellular routes [90].
An alternative strategy to improve iron status without iron supplementation could
be to improve the efficiency of absorption by exploiting the transmembrane iron gradient
between the gut lumen and the enterocytes. To this regard, hinokitiol, a small molecule
natural product isolated from plants that strongly binds both Fe2+ and Fe3+ was shown
to increase gut iron absorption in two experimental models with impaired apical and
basolateral transfer, the Belgrade (b) rats (with a loss of function mutation of DMT1) and
ferroportin deficient flatiron mice, respectively [91].
As reported above, since eukaryotic cells and microbes compete for iron, nutritional
immunity, i.e., the restriction of available iron by the host [92] is a defense strategy from
infection so important that is widely diffused and is present also in plants. Indeed, it has
been shown that when bacteria threatening plant health enter root tissues, iron acquisition
from the soil is inhibited through the degradation of the iron-deficiency signaling peptide
Iron Man 1 [93]. Therefore, the need to eliminate inadequate iron supply without exceeding
upper tolerable intakes stems from the ample evidence that iron administration can increase
the risk of several infections. To summarize a complex and evolving field, recent evidence
suggests that excessive doses of supplemental iron can indeed increase the risk of bacterial
and protozoal infections (particularly malaria) by excessively enhancing Tf saturation
and thus leading to the formation of NTBI [86]. However, less aggressive interventions,
based for example on fortified food and providing lower amounts of iron, appear generally
safer and represent the best balance of risk and benefits. Moreover, as discussed above,
unabsorbed iron may have a negative impact on health by leading to microbiota dysbiosis.
Thus, accurate dosing should be adopted. In these settings, modern stable iron isotope
techniques are a promising new method to accurately quantify iron absorption and assess
the impact of interventions [94].

8. Conclusions
Thanks to the identification of critical molecules involved in iron transport, our view
of intestinal iron absorption is now more complete and more detailed. However, this highly
regulated process is far from being completely unraveled. For example, the transport of
Metabolites 2024, 14, 228 12 of 15

heme, which is the most important source of dietary iron, still awaits the identification of
the apical transporter.
Traditionally, iron absorption was thought to be regulated by body iron requirements,
with iron stores and erythropoietic activity being the two main players [95]. More recently,
inflammation, which stimulates hepcidin synthesis, and the microbiota, which mutually
interacts with absorptive duodenal cells, have been shown to be other important factors.
The identification of the key proteins involved in intestinal iron absorption and the
increased knowledge about the control of their expression and activity has an important
translational aspect related to iron supplementation, a key global health issue in the context
of the intensive global efforts to combat iron deficiency and the associated anemia.
Available evidence indicates that iron given as supplements in non-physiological
amounts can increase the risk of microbial infections; moreover, the availability of unab-
sorbed iron to the microbial communities of the gut may be dangerous and lead to dysbiosis.
However, lower quantities of iron within a matrix or in highly bioavailable formula-
tions are probably less dangerous, thereby representing an approach that offers the best
balance of risk and advantages. Therefore, also in this context, the yin-yang dual potential
of iron applies and should be considered in public health programs and recommendations.

Author Contributions: Conceptualization, G.C. and S.R.; literature search, G.C., M.C. and E.G.;
writing—original draft preparation, G.C., M.C. and E.G.; writing—review and editing, G.C., M.C.,
E.G. and S.R. All authors have read and agreed to the published version of the manuscript.
Funding: This research received no external funding.
Institutional Review Board Statement: Not Applicable.
Informed Consent Statement: Not Applicable.
Data Availability Statement: Not Applicable.
Acknowledgments: Figures created with BioRender.com (accessed on 15 March 2024).
Conflicts of Interest: The authors declare no conflicts of interest.

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