Journal of Trace Elements in Medicine and Biology 84 (2024) 127459
Available online 17 April 2024
0946-672X/© 2024 The Authors. Published by Elsevier GmbH. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).
Interaction and competition for intestinal absorption by zinc, iron, copper,
and manganese at the intestinal mucus layer
Vincent Einhorn
a
,
b
, Hajo Haase
a
,
b
, Maria Maares
a
,
b
,
c
,
*
a
Technische Universit¨
at Berlin, Department of Food Chemistry and Toxicology, Straße des 17. Juni 135, Berlin 10623, Germany
b
Trace Age-DFG Research Unit on Interactions of Essential Trace Elements in Healthy and Diseased Elderly, Potsdam-Jena-Wuppertal, Berlin, Germany
c
Department of Food Chemistry, Institute of Nutritional Science, University of Potsdam, Arthur-Scheunert-Allee 114–116, Nuthetal 14558, Germany
ARTICLE INFO
Keywords:
Trace elements
Bioavailability
Mucus
Intestinal absorption
ABSTRACT
Trace elements such as zinc, manganese, copper, or iron are essential for a wide range of physiological functions.
It is therefore crucial to ensure an adequate supply of these elements to the body. Many previous investigations
have dealt with the role of transport proteins, in particular their selectivity for, and competition between,
different ions. Another so far less well investigated major factor influencing the absorption of trace elements
seems to be the intestinal mucus layer. This gel-like substance covers the entire gastrointestinal tract and its
physiochemical properties can be mainly assigned to the glycoproteins it contains, so-called mucins. Interaction
with mucins has already been demonstrated for some metals. However, knowledge about the impact on the
respective bioavailability and competition between those metals is still sketchy. This review therefore aims to
summarize the findings and knowledge gaps about potential effects regarding the interaction between gastro-
intestinal mucins and the trace elements iron, zinc, manganese, and copper. Mucins play an indispensable role in
the absorption of these trace elements in the neutral to slightly alkaline environment of the intestine, by keeping
them in a soluble form that can be absorbed by enterocytes. Furthermore, the studies so far indicate that the
competition between these trace elements for uptake already starts at the intestinal mucus layer, yet further
research is required to completely understand this interaction.
1. Introduction
Essential trace elements (TE) such as zinc (Zn), copper (Cu), iron
(Fe), and manganese (Mn) are of great importance for a wide variety of
physiological functions. They are critical for the functionality of en-
zymes and other proteins, act as signaling ions or participate in electron
transfer reactions [1–5]. Their relevance is underlined by the broad
spectrum of both physical and mental disorders resulting from, or being
associated with, TE deficiencies [1–6]. However, not only insufficient TE
supply, but also an excess of trace elements can result in serious health
consequences. This applies in particular to redox-active TEs [7]. To
avoid these pathological conditions, it is necessary to strictly control the
homeostasis of each element. This comprises compensation of endoge-
nous losses (e.g., excretion via feces or urine) through uptake from di-
etary sources [8]. The absorption of trace elements mainly takes place at
the brush border in the small intestine. There, a tightly controlled system
of transport proteins is responsible for the uptake from the intestinal
lumen and the subsequent export to the portal blood, complemented by
a basal contribution of unregulated passive transport [5,9–13].
Considering the neutral or even slight alkaline pH of the small intestine
(pH 6.2–7.4) [14], many TEs tend to precipitate under these conditions
due to hydroxypolymerization, significantly lowering their bio-
accessibility. This term describes the quantity of a compound that is
released from its matrix in the intestine and available for absorption
[15]. The primary factor preventing this from happening is the binding
of metal ions by the intestinal mucus, resulting in improved solubility of
the metals, greatly increasing their availability for the absorptive
enterocytes [16–18]. Although this interaction could be of considerable
relevance for the uptake of TEs, the exact molecular events as well as
possible competition of different TEs for interaction with the mucus
layer are poorly understood. For some trace elements there are, so far,
no experimental results regarding this possible interaction, at all.
However, some findings have already been obtained for iron, zinc,
copper, and manganese. Thus, this review aims to summarize these
observations and evaluate a potential influence of intestinal mucus on
trace element absorption.
* Corresponding author at: Technische Universit¨
at Berlin, Department of Food Chemistry and Toxicology, Straße des 17. Juni 135, Berlin 10623, Germany.
E-mail address: [email protected] (M. Maares).
Contents lists available at ScienceDirect
Journal of Trace Elements in Medicine and Biology
journal homepage: www.elsevier.com/locate/jtemb
https://doi.org/10.1016/j.jtemb.2024.127459
Received 9 February 2024; Received in revised form 9 April 2024; Accepted 16 April 2024
Journal of Trace Elements in Medicine and Biology 84 (2024) 127459
2
2. Intestinal mucus
Intestinal mucus is a viscous, gel-like secretion produced by goblet
cells. It lines the entire gastrointestinal tract and is indispensable for its
function and health [19]. It forms an additional physiochemical barrier
protecting the underlying epithelium from pathogens and harmful
chemical or physical influences [20,21]. Besides, mucus plays an
important role in gastrointestinal immunity [22] and, in addition to its
role in absorption of micro- and macronutrients [23], is essential for a
healthy microbiome. Here, it serves as a habitat, significantly influ-
encing the growth, differentiation, and spatial distribution of microor-
ganisms [24–26]. This multitude of functions is enabled by the
composition of the mucus. In addition to water (approx. 90 – 95%), it
comprises lipids (approx. 1 – 2%), salts, and various proteins such as
antibodies and growth factors, as well as one substance that is particu-
larly noteworthy: mucins [27,28]. These glycoproteins make up around
5 – 10% of this physical barrier and are the main structural component,
providing its physiochemical properties and viscoelasticity [29]. To
date, 22 different mucin (MUC) proteins have been described in humans,
with MUC2 being the dominant mucin in the small intestine [30,31]. An
overview of the main MUC proteins in various organs can be found in
Table 1. The different mucins can be broadly classified into two cate-
gories: membrane-bound and secreted [30,32]. While the former are
tightly bound to the plasmalemma by a transmembrane domain, the
latter are free to reassemble after secretion, resulting in the formation of
a complex multimeric network [29,30]. This ability is the result of
intermittent, sometimes described as „knot-like“, cysteine-rich regions
near the C-terminus and several von Willebrand D (VWD)-domains near
the N-terminus of the mucin protein backbone. Their interactions result
in intra- and intermolecular associations through disulfide bonds [33,
34]. Besides these cysteine-rich regions, the protein core contains
another important building block, the so-called PTS-domains. This term
refers to large sequences of proline/threonine/serine tandem repeats
forming the majority of the protein portion of mucins [29,35]. During
their synthesis (see Fig. 1), these segments are the site for extensive
O-glycosylation with different oligosaccharides, carried out by a multi-
tude of different glycosyltransferases in the Golgi. The final
MUC-monomers consist of approximately 80% carbohydrates (w/w)
[27,34,36]. Besides electrically neutral monomers, such as N-ace-
tyl-galactosamine (GalNac), N-acetyl-glucosamine (GlcNAc), mannose
or galactose, the attached saccharide-chains contain negatively charged
sugars, either sialic acid, also called N-acetylneuraminic acid (Neu5Ac),
or O-sulfosaccharides [29,36,37]. After their assembly, the mucins are
stored in the secretory granules of the goblet cell. There, a decreased pH
(approximately 5.2) and an increased calcium concentration cause
conformational changes, leading to a dense packing of the large MUC
structures [31]. Upon secretion, bicarbonate (HCO
3
-
) causes precipita-
tion of Ca
2+
and a rise in pH, resulting in expansion of the mucins up to
1000 times their packed volume [31,38]. The reticular structure
described above and the polyanionic character of the mucus (resulting
from its negatively charged carbohydrate residues) give it a selective
permeability designed to allow nutrients to pass. Foreign bodies or
pathogens, on the other hand, are retained and do not reach the
epithelium due to continuous renewal and shedding of the mucus [39,
40]. Consequently, the two determinants controlling a particle’s ability
to penetrate the mucus are charge and size.
3. Interaction between mucins and metals in the intestinal tract
3.1. General considerations
In the early 1960 s, the mucus layer was considered to primarily
provide mechanical protection for the underlying delicate epithelial
cells [57]. Although this idea is not fundamentally wrong, it had to be
expanded in subsequent years, after it became clear that there are in-
teractions and mutual influences between mucus and different biologi-
cally important compounds in the lumen. These include (metal-)ions
[58,59], enzymes [60], and bacteria [61]. The subsequent increase in
interest led to the identification of sulfate- and sialic-groups in mucins
that were shown to be almost exclusively dissociated at intestinal pH,
giving the mucins their net negative charge [62]. This observation
resulted in the experiments of Crowther and Marriott, who considered
mucins as negatively charged polyelectrolytes. They conducted experi-
ments on the binding capacity of iron, aluminum (Al), calcium (Ca),
cesium (Cs) and sodium (Na) to freeze-dried porcine gastric mucus using
equilibrium dialysis [63]. The authors made some initial assumptions
that were later proven incorrect, namely that trace elements are less
bioavailable and that the mucus layer integrity may be disrupted after
counter ion binding. Still, their experimental findings indicate a clear
correlation between the valence of the metal ions (Me) and the avidity of
mucus for binding them (Me
3+
>Me
2+
>Me
+
) [63]. If this concept is
applied to the absorption processes in the small intestine, valence of the
metal ions should be inversely proportional to their uptake rate, because
a stronger interaction with the mucus layer leads to a decrease in
mobility, consequently lowering the ability of the ion to permeate
through the mucus layer, which is continuously shedding and replacing
[64]. Although this generalization is easy to understand, it is important
to point out that the analysis of actual metal-mucin interactions is much
more complex. Not only are the binding sites pH-dependent [17], but
because mucins contain multiple sulfate- and carboxylic groups [27,65]
they might offer various sites for binding with different affinities for
‚hard‘ and ‚soft‘ metals [17], i.e., metals with different electric charge
density as described by the Hard-Soft Acid-Base (HSAB) concept estab-
lished by Pearson [66]. There are also hurdles in experimental practice
making research on this topic more difficult. While commercial mucin is
readily available, some of its properties are altered during processing
[67–70]. In addition, the quantities of intestinal mucus formed and its
glycosylation pattern can vary. Reasons for this include, for example,
trace element deficiencies [71] or heavy metal contamination of the diet
[72]. Studies performed on the mutual influence of mucins and various
metals are listed in Table 2, while a more detailed insight into the cur-
rent state of knowledge regarding the interaction of the trace elements
iron, copper, zinc, and manganese with mucins is given in the next
chapters.
Table 1
Selected mucins found in humans classified according to secreted and trans-
membrane types and the organs in which they are most abundant.
Mucin Type Mucin Organs References
Transmembrane MUC1 Respiratory and reproductive
tracts, colon
[32,41]
MUC3A/
B
Small intestine, colon [32,42,43]
MUC4 Esophagus / stomach [42,44]
MUC11 Respiratory tract [30,45]
MUC12 Colon [32]
MUC13 Colon [32]
MUC14 Ovary [46]
MUC15 Spleen, thymus, prostate, lungs [47,48]
MUC16 Stomach [44]
MUC17 Colon [32]
MUC18 Prostate [46]
MUC20 Placenta, colon, prostate [49]
MUC21 Respiratory tract, thymus, colon [49]
Secreted MUC2 Small intestine / colon [31,43,50]
MUC5AC Respiratory and reproductive
tracts
[39]
MUC5B Esophagus [51]
MUC6 Stomach / respiratory tract [31,52]
MUC7 Oral cavity [31,53]
MUC8 Respiratory tract [54,55]
MUC9 Fallopian tubes [49]
MUC19 Lung, liver, kidney, placenta [46]
V. Einhorn et al.
Journal of Trace Elements in Medicine and Biology 84 (2024) 127459
3
3.2. Iron
The existence of two stable oxidation states for iron (Fe
2+
and Fe
3+
)
has led to the utilization in a wide spectrum of biological processes by
most forms of life [102]. In humans, for example, it is involved in oxygen
transport, neurotransmitter synthesis, and energy metabolism [103].
Because of its essentiality it is crucial to maintain a stable iron level and
to replenish any losses. In that context the interest in the interaction
between mucins and trace elements began already in 1967 with a pub-
lication by Davis and colleagues [59]. While examining the iron
chelating properties of gastric juice from volunteers, they found that it
had an iron binding capacity of 0.02 – 0.04 mg per milliliter of gastric
juice and was able to also keep the bound iron soluble at pH 8, pre-
venting its precipitation [59]. They concluded that there are no signif-
icant amounts of iron in ionic form in the intestine, rather being bound
to components of the gastric juice [59]. Further investigation by Rudzki
et al. characterized “gastroferrin”, as the substance was called at the
time, as a glycoprotein consisting of approximately 90% sugar and 10%
protein in which the amino acids serine, proline, threonine, alanine and
glycine dominate [83]. In addition, Rudzki found homology with
blood-group antigens, all of which led to the identification of gastro-
ferrin as gastric mucin [17,83]. While examining the metal-binding
properties of the gastric mucins, Rudzki and Baker found
non-stochiometric binding of iron to mucins. This indicated stabilization
of a colloidal polyhydroxy-iron core by gastroferrin, reducing the ten-
dency for further hydroxypolymerization into larger aggregates at in-
testinal pH [84]. This mechanism can be found for aluminum, another
strong hydrolytic metal, as well [16]. Like iron in other
polyhydroxy-iron chelating systems (i.e., polyhydroxy-iron-fructose
[104]), iron-gastroferrin is expected to be easily dissociable, therefore
increasing the iron bioaccessibility and ultimately improving its ab-
sorption. This point of view is shared by the authors of several studies on
the subject [84–86]. Wien et al. and Conrad et al. injected an iron so-
lution into the small intestine of rats and analyzed the distribution of it
Fig. 1. : Synthesis of MUC2. Schematic representation of MUC2 synthesis. After transcription of the MUC2 gene, the mRNA is translated into a polypeptide chain
with a length of 5179 amino acids. After translation, the MUC2 protein core enters the endoplasmic reticulum, where it is first N-glycosylated and then dimerizes via
the C-terminal cysteine knot. After O-glycosylation in the Golgi and tri-/multimerization in the trans-Golgi-network (TGN), the final MUC2 polymer is densely packed
in secretory granules (see also [31], [56]), which release the MUC polymer by exocytosis if required. ER, endoplasmic reticulum; TGN, trans-golgi-network; PTS,
proline/threonine/serine; vWF-D-domain, von Willebrand factor D domain.
V. Einhorn et al.
Journal of Trace Elements in Medicine and Biology 84 (2024) 127459
4
Table 2
Studies on the interaction between mucins and different metal ions.
Metal Method Main Outcome References
Aluminum −In vivo rat model
(male swiss
albino)
−Rats received
aluminum sulfate
(Al
2
(SO
4
)
3
)
through their diet.
−Soluble mucins and the
adherent mucus layer
retarded
hydroxypolymerization of
Al
3+
by binding it.
−Elements with high
binding strength to mucus
and kinetically slower
ligand exchange rate
migrated more slowly
through the mucus and
were therefore less well
absorbed.
[16]
Cadmium −In vivo rat model
−Rats received
cadmium nitrate
(Cd(NO
3
)
2
)
through their diet.
−Mucin content in the
intestinal mucus layer as
well as mucus layer
thickness was decreased.
[78]
−In vivo rat model
−Rats received
cadmium chloride
(CdCl
2
) in their
drinking water.
−Accumulation of Cd
2+
in
gastric mucosa
−Decreased mucin content
in the intestinal mucus
layer and decreased
mucus layer thickness led
to mucosal barrier
breakdown.
[72]
Calcium −Mucins isolated
from rat small
intestinal
scrapings.
−Binding studies
using calcium
chloride (CaCl
2
)
−Ca
2+
-binding capacity of
0.14 mol Ca
2+
/kg Mucin.
−Treatment of mucins with
neuraminidase decreased
binding capacity by 90%,
suggesting sialic acid
residues provide most of
the ligands for Ca
2+
.
[79]
−In vivo models
using rats and
chicken
−Animals were fed
a calcium chloride
(CaCl
2
)-enriched
diet.
−Discrete Ca
2+
localizations were found
in the goblet cells, but
only in regions of mucin
storage, indicating an
association between Ca
2+
and intestinal mucins.
[80]
−Commercial
bovine salivary
mucin
−Ca
2+
preferred binding to
carbohydrate portion of
mucin but interacted with
the mucin protein core at
elevated Ca
2+
levels.
[81]
Copper −Recombinant
human MUC2
VWD factor type
D1 region
−In vitro Caco-2 cell
monocultures
−MUC2 bound both Cu
+
and Cu
2+
and
consequently protected
the underlying epithelium
from copper toxicity by
preventing redox cycling.
−MUC2 still could donate
Cu
+
to underlying cells.
−MUC2 probably was found
to have distinct binding
sites for Cu
+
and Cu
2+
.
Identified binding sites in
VWD factor type D1
region (see Fig. 2):
1. For Cu
2+
: 3 histidines, 1
glutamate; log
Kd
= − 12 to
−13; no competition by
Zn
2+
was found
2. For Cu
+
: 3 sulfur ligands;
log
Kd
= − 13
[73]
Chromium −Commercial PGM
(Type unspecified)
was incubated
with different
chromium(III)
complexes.
−Certain Cr
3+
-complexes
bound to mucins and led
to significant
conformational changes.
[82]
Table 2 (continued)
Metal Method Main Outcome References
Iron −Mucins isolated
from human
gastric juice
−Dialysis
experiments using
iron chloride
(FeCl
3
)
−Mucins kept Fe
3+
in
solution at neutral or
alkaline pH.
[59,83,
84]
−In vivo rat model
(Hooded lister
female)
−Rats received iron
sulfate (FeSO
4
)
after fasting for
different intervals
by oral gavage.
−Fe
2+
absorption and Fe
2+
content in the intestinal
mucus layer increased
with duration of fasting
period.
[18]
−In vivo rat model
(Wistar breed)
−Iron chloride
(FeCl
3
) was
injected into the
small intestine.
−Intestinal mucins bound
Fe
3+
reversibly.
−Binding occurred in a
saturable manner; the
average dissociation
constant of the Fe
3+
-
mucin-complex was
9.1 µM.
−Other metals (Zn
2+
, Pb
2+
,
Co
2+
) bound
competitively to the same
binding sites.
[85]
−In vivo rat model
(Sprague-Dawley
breed)
−Rats received
different amounts
of ferric citrate
with their diets.
−The intestinal mucus layer
was able to bind Fe
3+
.
−The sialic acid content of
the intestinal mucus layer
was lower when rats were
fed insufficient levels of
Fe
3+
.
[86]
−In vivo rat model
(Wistar breed)
−Iron chloride
(FeCl
3
) was
injected into the
lumen of the
duodenum.
−Intestinal mucins bound
Fe
3+
.
−Two additional substances
(mobilferrin and integrin)
were isolated from the
intestinal mucus layer and
characterized as Fe
3+
-
binding proteins.
−Multiple Fe
3+
ions were
bound by a single
molecule of mucin.
[87]
−Mucins were
isolated from
intestinal
scrapings of rats.
−Large proportions of the
Fe
2+
-transporters DMT-1
and mobilferrin were
found in goblet cells and
extracellularly associated
with intestinal mucin.
[88]
−In vitro 3D Caco-2
model in combi-
nation with PGM
(type unspecified)
−In vitro digested
food, enriched
with iron chloride
(FeCl
3
), was
applied to the
Caco-2 cells.
−Applying PGM to the
Caco-2 cells led to a sig-
nificant increase of Fe
3+
uptake from heme and
ferritin.
−Fe
3+
uptake from foods
that had a low Fe
3+
content decreased after
applying PGM.
[89]
−In vitro Caco-2
model and mucus-
producing 2D cell
model Caco-2/
HT29-MTX
−In vitro digested
food and iron
chloride (FeCl
3
)
was applied to the
cell models.
−Fe
3+
/ferritin content was
lower in mucus-producing
cell model after incuba-
tion with in vitro digested
food.
−Cells with mucus layer
showed an increase in the
expression of DMT-1
mRNA.
−Results suggest that the
mucus layer bound Fe
3+
,
thereby buffering spikes in
iron concentration and
delivering it continuously
to the enterocytes.
[90]
(continued on next page)
V. Einhorn et al.
Journal of Trace Elements in Medicine and Biology 84 (2024) 127459
5
within a 10-minute absorption period. Instead of iron precipitation they
found most of the iron bound to mucins, leading the authors to postulate
transfer of inorganic iron from mucins to enterocytes in an absorbable
form [85,86]. In line with this hypothesis is the average dissociation
constant of the mucin-iron complex of 91 µM, which was determined
with ultracentrifugation experiments and isolated duodenal rat mucin,
and lies in a range that is relevant for the luminal iron concentration
[85].
Although these findings have made a significant contribution to the
understanding of iron uptake, in the following years various experi-
mental results suggested that the interaction between iron and mucins is
more complex than just the stabilization of small polyhydroxy-iron
particles. It was shown that the gastrointestinal fluids contained a va-
riety of low molecular weight (LMW) compounds (i.e., lactate, pyruvate,
histidine) that could act as ligands for ionic iron [64]. These LMW
compounds prevent the hydroxypolymerization of iron in the intestine
and finally donate it stoichiometrically to intestinal mucins. This
mechanism has been confirmed, but so far only in in vitro experiments
[85].
Table 2 (continued)
Metal Method Main Outcome References
−In vitro 2D Caco-2
model in combi-
nation with PGM
(type unspecified)
−Mucus-producing
2D cell model
Caco-2/HT29-
MTX
−In vitro digested
food, partly
enriched with iron
chloride (FeCl
3
)
was applied to the
cell models.
−As the ratio of HT29-MTX
to Caco-2 cells increased,
the ferritin formation in
the cell cultures
decreased, especially
when applying Fe
3+
in a
highly bioavailable form.
−Removing the mucus layer
from the HT29-MTX-cells
did not increase ferritin
formation.
[91]
Lead −In vivo
experiments using
male adult
zebrafish
−Fish were exposed
to different
concentrations of
lead acetate
(PbAc
2
).
−Thickness of the intestinal
mucus layer, measured by
histological staining, was
increased.
[92]
Manganese −In vivo sheep
model
−Sheep received
manganese sulfate
(MnSO
4
) or
manganese
glycine hydrate
(Mn-Gly) through
their diet.
−Significant reduction of
mucus layer thickness in
duodenum and jejunum.
[93]
Mercury −In vitro Caco-2
monocultures and
mucus-producing
3D cell model
Caco-2/HT29-
MTX
−Incubation with
solutions of
methylmercury
(MeHg
+
) and
mercuric nitrate
(Hg(NO
3
)
2
).
−Incorporation of HT29-
MTX cells into the model
led to a decrease in
apparent permeability
(P
app
) for both mercury
species.
−High accumulation of
Hg
2+
in the mucus layer.
[94]
−PGM Type II
incubated with
Hg
2+
-solution
−Mucins had a maximum
Hg
2+
binding capacity of
124 mg/g.
−Interaction via strong
electrostatic and
coordinative bonds.
[95]
Zinc −In vivo model
using Cheviot-
Finnish lambs
−Sheep received
zinc-depleted or
zinc-enriched diet
(zinc compound
unspecified).
−Zn
2+
deficiency led to a
decrease in sulphate
content of the intestinal
mucins.
[96]
−In vivo rat model
(Sprague-Dawley
breed)
−Solution of zinc
sulfate (ZnSO
4
)
was applied by
intraperitoneal
injection.
−Dose-related reduction in
stress ulcer incidence
−Increase in gastric wall
mucus
[97]
−In vivo model
using freshwater
rainbow trout
−Zinc sulfate
(ZnSO
4
) was
applied through
catheter into the
small intestine.
−Mucus layer appeared to
regulate Zn
2+
uptake by
enhancing it at low Zn
2+
concentrations and
slowing Zn
2+
absorption
down at higher Zn
2+
concentrations, thereby
acting as a Zn
2+
buffer.
[98]
−In vivo weaned
piglet model
−High dietary Zn
2+
levels
led to an increase in goblet
[99]
Table 2 (continued)
Metal Method Main Outcome References
−Different levels of
zinc oxide (ZnO)
were given with
the diet.
cells and mucin
production measured with
histological staining and
qPCR.
−In vitro mucus-
producing 3D cell
model Caco-2/
HT29-MTX
−Cell-free
measurements
with commercial
PGM II
−Combination of
PGM II and Caco-2
cells
−Zn
2+
-buffering by
intestinal mucins
−Average molar Zn
2+
-binding capacity of 200
−Zn
2+
-binding affinity of
5µM for mucins isolated
from human goblet cell
line HT29-MTX and
6.8 µM for commercial
PGM II
−Transport studies using 3D
intestinal cell models
suggested that intestinal
mucins facilitate Zn
2+
absorption by enterocytes
and acted as a Zn
2+
delivery system for the
intestinal epithelium.
[100]
−Zinc transporter
(ZnT)7 knockout
mice
−ZnT7 knockout led to
decreased number of
mucin-filled goblet cells,
measured with histologi-
cal staining, and altered
microbial composition in
colon of female mice.
−In male mice an increase
of goblet cells and no
effect on microbiota was
observed.
[101]
−In vitro goblet cell
model HT-29-MTX
−Zinc-deficient
cultivation
−Mucus layer thickness was
reduced (histological
staining) while number of
mucin lakes increased
during Zn
2+
deficiency.
−O-glycan pattern of
secreted mucus from
Zn
2+
-deprived cells
changed, shifting from
predominantly core-1 O-
glycans to core-3 O-
glycans which was also
reflected in deregulation
of glycosyltransferases
responsible for core O-
glycan synthesis.
[71]
3D, three dimensional; PGM, porcine gastric mucin; DMT-1, divalent metal
transporter 1
V. Einhorn et al.
Journal of Trace Elements in Medicine and Biology 84 (2024) 127459
6
All abovementioned observations combined with the isolation of a
water-soluble, iron binding fraction of the intestinal mucus from rats as
well as humans (called “mobilferrin”) led to the proposal of a new
uptake-mechanism for ferric iron by Conrad and Umbreit in 1993. They
termed it the mucin-mobilferrin-integrin-pathway [87,105]. According to
this theory, inorganic iron is solubilized in the stomach at acidic pH.
Next it combines with mucins, keeping the iron soluble as it travels in
the small intestine where its subsequent absorption through the enter-
ocytes´plasma membrane is facilitated by integrins on the cell surface
and carried out by mobilferrin as a carrier [105]. This hypothesis is
supported by the finding that goblet cells contain large amounts of
mobilferrin and secrete the protein along with mucins into the intestinal
lumen [88,89]. In direct contrast are the observations by Laparra et al.
[90]. They studied intestinal iron uptake by measuring cellular ferritin
formation using Caco-2-cells (a common in vitro model for human
enterocytes) after culture in the presence or absence of the
mucin-producing human goblet cell line HT-29-MTX [90], a model
previously developed specifically for iron absorption by Mahler et al.
[91]. Applying digested fish or white beans as a matrix, the iron uptake
by the Caco-2/HT-29-MTX-Co-culture was significantly lower than the
uptake into a Caco-2 monoculture devoid of a mucus layer [90]. In
parallel, however, a marked increase in the mRNA expression of the
divalent metal transporter 1 (DMT-1) was measured in the co-culture
model, but not in the monoculture model [90]. The authors hypothe-
sized that the mucus layer could have lowered the concentration of iron
at the apical side of the enterocytes and delayed the arrival of the added
iron, thereby slowing down the absorption of iron and prompting the
cell to increase the expression of DMT-1 mRNA [90]. This is consistent
with previous observations by Jin et al. where iron uptake from various
in vitro digested iron species into Caco-2 cells was altered in the presence
of extracellularly added commercially available porcine gastric mucins
(PGM) [89].
3.3. Zinc
Due to its multitude of structural, catalytic, and regulatory roles, zinc
provides the basis for a variety of critical cellular functions, underlined
by approximately 10% of all proteins encoded in the human genome
containing zinc as a structural component or in the active site of en-
zymes [106,107]. The first investigations regarding the interplay of zinc
and mucins were conducted in the 1970 s, dealing primarily with the
effect of zinc deficiency on mucins. The intestinal mucus layer from
zinc-deficient sheep showed a decreased total amount and altered
composition of mucins [96,108]. The protein core had higher amounts
of glycine, less histidine and arginine, and a decreased total quantity of
sulfate from sulfonated glycan chains [96,108]. Subsequent cell culture
studies utilizing the in vitro goblet cell model HT-29-MTX confirmed
these results [71]. While Maares et al. did not study the altered amino
acid pattern, it was observed that, besides a reduction in secretion, se-
vere changes in the O-glycan pattern of the secreted mucins occurred
[71]. These observations are of concern because changed O-glycan
composition severely impacts mucus structure, impairing the protective
effect of the mucus barrier and altering the microbiota [33,71]. A critical
role for zinc in mucus synthesis and production is further supported by
the deregulation of MUC2 in zinc-deficient human goblet cells [71] and
by changes of the mucin density in the colon of pigs after feeding
different levels of zinc oxide [99]. Only recently, the impact of mucus on
zinc absorption was investigated. Cell culture experiments using
Caco-2-monocultures and Caco-2/HT-29-MTX-co-cultures showed that
mucus improves zinc uptake by enterocytes and increases its transfer via
the intestinal epithelium into the blood. The underlying mechanisms
could be similar to those observed in the interactions between mucins
and iron [23]. In other words, mucins might be buffering high zinc
concentrations by initially binding large amounts of the metal and then
slowly releasing it to the enterocytes. Even a transport mechanism
similar to that of the mucin-mobilferrin-integrin-pathway is conceivable
[23]. These results are in accordance with the findings Glover and
Hogstrand made when they investigated the uptake of zinc in rainbow
trout. In their experiments, they injected zinc into the anterior portion of
the fish’s intestine through a catheter while observing the effluent
through a small incision at the posterior intestine [98]. They found that
intestinal mucins are an essential factor in the regulation of zinc uptake.
At low concentrations of zinc, its absorption was enhanced, while at
higher zinc concentrations an increase in mucus secretion was observed.
This increase in secretion was sequestering considerable amounts of zinc
away from the intestinal epithelia, preventing zinc overload and
possible metal toxicity [98]. Dialysis experiments with commercial PGM
II to investigate the zinc binding capacity of mucins showed multiple
zinc-binding sites within one mucin molecule [71]. The zinc binding
affinity of mucins is in a physiologically relevant range and was deter-
mined to be 0.15 µM and 0.2 µM using PGM II and secreted mucins from
the human goblet cell line HT-29-MTX, respectively [71]. Although the
distinct binding sites have not yet been deciphered, interactions of zinc
with nitrogen and sulfur in the O-glycan branches, for example of Gal-
NAc and Neu5Ac, with the 8–12% free thiols within the mucin protein
core [69,75,76] or even within the VDW D1 region were discussed [23,
73], as zinc forms stronger bonds with nitrogen and sulfur than with
oxygen [74] (Fig. 2).
3.4. Copper
Due to its capability to readily gain or donate electrons, copper is a
cofactor for many metalloenzymes, especially oxidoreductases [11].
Copper deficiency can have severe consequences, most importantly
neurological effects [109]. In addition, copper plays an important role in
iron homeostasis. It acts as a competitor for various enzymes in the iron
transport chain, such as DMT-1 and duodenal cytochrome B (DCYTB).
Additionally, copper is also a component of various copper-dependent
ferroxidases (FOX) that can reduce iron, thereby allowing its binding
to transferrin and the subsequent distribution in the body [110]. The
first comprehensive study dealing specifically with copper and its
binding to mucins was published in 2022. Using recombinant human
MUC2, Reznik et al. found a copper binding site near the protein surface,
distant from the PTS domains. There, copper was coordinated by three
histidines and one glutamate (see also Fig. 2) [73]. Interestingly, zinc
was a poor inhibitor of this binding, even though histidine has a high
affinity for zinc [73,111]. Another important finding by Reznik et al.
were dedicated binding sites for Cu
+
and Cu
2+
. While MUC2-bound
Cu
2+
was reduced by ascorbic acid, the resulting Cu
+
did not undergo
redox cycling in the presence of oxygen. From this observation the au-
thors concluded that MUC2 might help to protect the intestinal epithelia
from copper toxicity by preventing the formation of reactive oxygen
species (ROS) [73].
3.5. Manganese
In the human body, manganese is essential for the function of a va-
riety of enzymes (e.g., transferases, oxidoreductases, lyases) and vita-
mins as well as a normal function of the glucose- and lipid metabolisms
[112]. In the last decade, research on manganese metabolism has seen
some major advances. Particularly noteworthy are the important roles of
the metal transporters Zrt-, Irt-like transporter (ZIP8) (solute carrier
(SLC)39A8), ZIP14 (SLC39A14), and ZnT10 (SLC30A10) in the main-
tenance of manganese homeostasis [113–115]. Although these insights
undeniably represent an advance in our understanding of the handling
of manganese by the human body, the role of the mucus layer in the
intestinal manganese uptake remains to be studied. Based on the ob-
servations for iron, zinc, and copper, it is conceivable that mucus also
has an important influence on the bioaccessibility and absorption of
manganese in the intestine. Unfortunately, no studies are yet available
on the specific influences of the mucus layer on manganese uptake.
However, similar to some findings regarding the interaction of zinc and
V. Einhorn et al.
Journal of Trace Elements in Medicine and Biology 84 (2024) 127459
7
the intestinal mucus layer, there are observations indicating that man-
ganese modulates intestinal mucus production. Makov´
a et al. analyzed
the thickness of the mucus layer of the intestine of sheep after supple-
menting their diet with organic manganese glycine hydrate (Mn-Gly) or
inorganic manganese sulphate (MnSO
4
) [93]. Sheep fed a
manganese-enriched diet showed a reduction in the mucus layer, with
this effect being more pronounced with the organic manganese com-
pound [93]. As an explanation, the authors speculate that the Mn-Gly
passes through the gastrointestinal tract unchanged and then pene-
trates the mucus without any major interaction. In contrast, the inor-
ganic form is dependent on the stabilizing effect of the mucus for
absorption, resulting in an increase of mucus secretion to enhance
manganese uptake [93]. With regard to these explanations, however, it
should be noted that the amount of manganese administered in that
study (approx. 150 ppm) was significantly higher than the amount
required by sheep (approx. 25 ppm) [93,116]. Due to this overload of
manganese, mucus secretion might have been increased in order to keep
excess manganese away from the cell liner and thus avoiding toxicity.
4. Discussion and conclusion
There are two major obstacles affecting absorption and adequate
supply of trace elements: 1) They are soluble in the gastric environment,
but tend to form (poly)hydroxides at higher pH values in the intestine,
causing them to precipitate and become unavailable for absorption by
enterocytes [117]. 2) Despite their importance, care must be taken to
maintain homeostasis, as excessive intake of these elements may lead to
toxicity and thus resulting in serious health consequences [118]. Intes-
tinal mucins are an essential factor to address these two challenges. They
bind the metal cations or pre-existing colloidal polyhydroxy-ions
through electrostatic and covalent interactions, interfering with
further hydroxypolymerization and thus increasing their bio-
accessibility [17,64]. Due to their polyanionic character, mucins also
favor the accumulation of cationic particles at the interface to the
Fig. 2. : Potential binding sites for Zn, Cu, Fe, and Mn within MUC2. Interactions of copper(I) with 3 methionine residues and copper(II) with 3 histidine residues/1
glutamate residue in the vWF D1-domain have been demonstrated [73]. Alternatively, zinc could interact with sulfur atoms at free thiols in the mucins [74], which
account for roughly 15% of the total thiol content [75,76] and are most likely located primarily at the cysteine node of the C-terminus [77]. Another option would be
an interaction of zinc with the nitrogen atoms of the N-acetyl group of Neu5Ac [23,74]. Electrostatic binding with sulphate or carboxyl residues has so far only been
demonstrated for zinc and iron [18,63], but is also likely for other trace elements due to their similar charge. PTS, proline/threonine/serine; vWF-D-domain, von
Willebrand factor D domain; His, Histidine; Glu, Glutamate; Met, Methionine; Cys, Cysteine; Neu5Ac, N-acetylneuraminic acid.
V. Einhorn et al.
Journal of Trace Elements in Medicine and Biology 84 (2024) 127459
8
intestinal content, allowing small amounts of positively charged trace
elements to be more effectively absorbed from the food pulp [119]. At
the same time, the binding of metals to mucin might cause a buffering
effect by the latter. Due to the high binding capacity of mucins for
various metals, these can first be temporarily stored in the mucin and
then be gradually released to the enterocytes [98,120]. Hereby, total
absorption of trace elements can be increased if the amount of available
metals exceeds the current transport capacity of the enterocytes. This
buffer also makes it possible to keep excess or unwanted metals away
from the intestinal epithelium by increased secretion of the mucins,
whose turnover rate is in the range of a few hours [121–124]. This
mechanism would thus complement the relatively rapid metallothionein
response [125] and the change in transporter expression, which is
usually preceded by a lag phase of 1–2 days [126]. In this context, the
different affinity of various metals to mucins mentioned earlier is
especially interesting (in general: Me
3+
>Me
2+
>Me
+
[63]). For the
uptake of the metals to occur, they have to translocate through the
mucus faster than it is secreted and exfoliated. Thus, if the binding be-
tween the mucins and the metal is too strong, the metals would ulti-
mately be excreted via the feces as a mucin-metal complex. While the
dissociation constants for mucin-zinc and mucin-iron with 5–6.8 ×10
−6
[71] and 9.1 ×10
−5
[85], respectively, are in the order of magnitude of
what corresponds to a realistic concentration of these metals in the in-
testine after a meal, it is conceivable that toxic metals such as aluminum
(Al
3+
) are sequestered from the epithelium due to their higher affinity to
the mucins, thus preventing absorption. There might also be a gradient
of affinities for different binding sites throughout the mucus layer that
sieves certain metals. Unfortunately, the data concerning different
metal-mucin binding affinities are not yet sufficient to assess this.
Furthermore, the question arises how specific different binding sites are
for the respective metals. While binding to the protein backbone has
been identified for copper, most of the interaction presumably takes
place between the more accessible O-glycan chains and the metal cat-
ions. The specific binding sites within the highly variable glycan pattern
of mucins, however, still remain to be identified (Fig. 2). Competition for
binding sites could play a role, both between different trace elements
and between trace elements and bulk elements. For example, several
experiments have shown that zinc and calcium reduce each other’s ab-
sorption when administered at the same time [127–129]. Since both
elements have different uptake pathways [130,131], these findings are
likely not a result of competition for transport proteins. In fact, the
presence of calcium was discussed to promote formation of the
zinc-phytate-complex, lowering the intestinal zinc bioaccessibility [132]
even though this has not been confirmed in other human intervention
studies [133–135]. In addition, their interaction could also be due to
competition for binding sites on mucins. As a result, only part of the
calcium or zinc present could reach the enterocytes and be absorbed.
In summary, intestinal mucus has a significant impact on the ab-
sorption of nutrients in general, and trace elements in particular.
Therefore, the role of the mucus layer should be recognized and
considered when planning future studies, especially in the context of
trace element uptake. The hurdles for this have been lowered in the past.
In addition to the use of mucus-forming goblet cell lines, such as HT-29-
MTX,in co-culture models [136] or for the isolation of secreted mucins
[137], there are now methods for the extraction and purification of
native mucins from animals [138,139].
CRediT authorship contribution statement
Maria Maares: Writing – review & editing, Funding acquisition,
Conceptualization. Vincent Einhorn: Writing – original draft, Concep-
tualization. Hajo Haase: Writing – review & editing, Funding acquisi-
tion, Conceptualization.
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
Acknowledgements
Funded by the Deutsche Forschungsgemeinschaft (DFG, German
Research Foundation), Project numbers Research Unit FOR-2558
TraceAge – DFG Research Unit on Interactions of essential trace ele-
ments in healthy and diseased elderly, Potsdam-Berlin-Jena; HA 4318/
4–2; MA 9681/1–1.
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