J Kanazawa Med Univ 41：25 − 31, 2016

Rapid Increase in Serum Iron Level After Oral Iron Intake

as an Indicator of Duodenal Iron Absorption
and Inverse Regulation of Iron Absorption by Hepcidin Expression

Shaofu Ma1), Yoshitaka Koshino2), Satoko Yamamoto1),

Takeo Shimasaki1), NaohisaTomosugi1)

Abstract: Objective: Hepcidin is a hepatocyte-derived peptide that is thought to be involved in the

regulation of intestinal iron absorption. Ferric citrate is a phosphate binder with additional effects on iron
absorption. Better understanding of iron absorption under various levels of hepcidin may improve ferric
citrate supplementation in individuals with renal anemia. We provide a new method to predict the
individual iron absorption ability of the duodenum. Methods: Rats on an ordinary diet were given 10 mg
of ferric citrate, and the serum concentrations of hepcidin and iron were monitored for 24 hours. Rats with
hepcidin levels induced by using alternative methods such as bloodletting or intravenous iron loading
were also given ferric citrate, and serum iron level was measured at 2 hours after oral iron intake (2-hour
oral iron absorption test). Results: Serum iron level increased constantly within 2 hours after oral iron
intake, and serum hepcidin level peaked 4 hours after the iron level peaked. In the oral iron absorption
test, the hepcidin levels inversely correlated with increased serum iron levels, and hepcidin expression
levels of >80 ng/ml completely inhibited the increase in iron absorption. Conclusion: This study suggests
that hepcidin expression may be a strong mediator to regulate iron absorption and that performing an oral
iron absorption test with hepcidin may help improve oral iron dosing schedules in patients undergoing
hemodialysis.
Key Words: hepcidin, ferroportin, divalent metal transporter 1, iron absorption, ferric citrate

Almost all patients on maintenance hemodialysis in humans are characterized by the absence of active
have iron deficiency anemia because of blood loss. excretory mechanisms for iron and the regulation of
The amount of iron loss has been estimated to be 1.5 body iron content by modulation of iron absorption
to 2.0 g/year (1). Thus, adequate iron supplementation, in the duodenum (6). Iron is absorbed by an active
typically requiring non-physiological intravenous iron transport mechanism comprised of two steps, namely
administration, has become routine for hemodialysis mucosal uptake of iron from the lumen and mucosal
patients (2), because oral iron supplementation has transfer of iron to the bloodstream (7). Both steps are
been shown to be unable to maintain adequate iron regulated by the body’s demand for iron. The
stores (3-5). However, the adequate iron stores in phenomenon is supported by the transporter
patients and the efficacy of the physiological iron molecules divalent metal transporter (DMT-1) and
absorption route in the intestine are controversial. ferroportin (FPN) (8). Iron influx from the intestinal
The physiological properties of iron metabolism lumen to cells is induced by DMT-1 expression, and
iron efflux from cells to the bloodstream is induced
1)
Depar tment
1)Kohzaburo of Advanced Medicine, Kanazawa Medical
Fujikawa-Yamamoto by FPN expression. These unique mechanisms for
University Graduate
Division of Cell School
Medicine, of Medical
Research InstituteScience, Uchinada,
of Medical Science, cellular iron uptake and transport have evolved to
Ishikawa
Kanazawa920-0293,
Medical Japan
University, Uchinada, Ishikawa 920-0293, prevent the inherent toxicity of iron in the promotion
2)
Department
Japan of Internal Medicine, Mizuho Hospital, Ishikawa, of oxidation due to iron overloading (9). However,
Japan
Corresponding author:Kohzaburo Fujikawa-Yamamoto many broad variations in iron status have been
Accepted:
Accepted: November
March 14, 30,
2016
2006 observed among individuals, and accurate

25

regulations according to physiological demand
remain unknown. Assays for DMT-1 and FPN
expression in individual patients are not available in
clinical settings.
Hepcidin is the main hormone/peptide that
regulates plasma iron levels by controlling the iron
export function of FPN, thereby regulating iron
absorption and distribution (10). Measurement of
hepcidin expression level is progressively being
validated (11-13). Currently, its clinical value needs to
be assessed in different physiological situations.
Based on these molecular properties, we aimed to
clarify the iron absorption in individual rat models. In
previous ferrokinetic studies, iron stable isotopes were
used in the analyses of iron absorption in the intestine
(7, 14-16). However, in the clinical field, isotopes are
of no practical use; thus, an alternative method that
does not use isotopes is expected to detect iron
absorption in individual cases.
The aims of this study were to quantify the
magnitude of iron absorption under different hepcidin
levels and to develop a new method to predict the
bioavailability of oral iron in individual cases without
isotope-labeled iron. In this study, we focused on
basic serum hepcidin levels and the changes in serum
iron concentration 2 hours after iron intake.
Materials and methods
1. Animals
Rats (male Sprague-Dawley rats aged 7 to 8
weeks) were obtained from Sankyo Laboratory (Japan)
and maintained on a standard rodent diet (iron content:
20 mg/100 g). Synthetic human hepcidin-25 was
purchased from Peptide Institute (Japan) and dissolved
to appropriate concentrations in phosphate-buffered
saline (PBS). Rats received a single intravenous
injection of 100 μg of human hepcidin-25. Sucrose
iron (40 mg/2 ml; Fesin, Nichi-Iko Pharmaceutical
Co., Japan) was used for the injection. Sucrose iron
was diluted in 4 mg/ml PBS. Rats received a 4 to 12
mg of iron through intravenous injections of 0.5 ml of
iron solution 2 to 6 times a week. A relatively mild
protocol of iron overload was chosen to evaluate the
relationship between iron absorption and hepcidin
expression level under physiological conditions. The
control rats received PBS only at the same time points.
For inhibiting endogenous hepcidin expression, iron
deficiency rats were induced by 1 ml of bloodletting 2
to 6 times a week. For the iron absorption study, the
rats were given 10 mg of ferric citrate (FC), including
2.3 mg of iron (Wako, Japan) in 0.5 ml of PBS, by oral
gavage at 8:00 am. All the experiments were approved
by the animal research committee of Kanazawa
Medical University.
26
Ma/Koshino/et al.
2. Blood parameter measurement
The rats were anesthetized by using 30% (v/v)
isoflurane and killed by exsanguination. Whole blood
samples were collected by abdominal aortic puncture
for measurements of serum iron level, unsaturated
iron-binding capacity (UIBC) and serum hepcidin
level. Serum iron level and UIBC were measured by
using 2-nitroso-5(N-sulfopropylamino)
phenolhydrochloride and ascorbic acid. Serum
hepcidin level was measured by using liquid
chromatography coupled with tandem mass
spectroscopy with reversed phase extraction (Medical
Care Proteomics Biotechnology Ltd., Japan) (11).
3. Statistical analysis
Statistical analyses were performed by using
Statistical package for Social Science(SPSS) Statistics
22 (IBM, USA). The Student t test was used to
compare groups of parametric data. Simple linear
regression analysis was used to examine the
relationship between serum hepcidin levels and
changes in serum iron level 2 hours after iron intake.
Variables that were not normally distributed were log
transformed before the analysis. Data are presented
herein as mean ± standard error of the mean. Statistical
significance was defined when the p value was <0.05.
Results
1. Serum iron levels after oral iron intake
In a time-course study, the rats (n=45 rats) were
administered 10 mg of FC by oral gavage at 8:00 am
and killed at 0, 0.5, 1, 2, 3, 6, 9, 12, and 24 hours after
FC intake (n = 5 rats in each group). The onset of the
increase in serum iron level after FC intake was quite
rapid (Fig 1). Serum iron and transferrin saturation
levels (TSAT) increased within 30 minutes and
reached the maximum value (488 ± 57.6 μg/dl, 95 ±
2.1%) 2 hours after iron intake (Fig 1A,B). In the rats
on a normal diet, the mean pretreatment serum iron
concentration was 236 ± 39.5 μg/dl. Thereafter, the
serum iron level decreased to its baseline value. On
the other hand, the serum hepcidin level gradually
increased to its maximum value (56.9 ± 10.5 ng/ml) 4
hours after the serum iron level peaked (Fig 1C).
To clarify whether the rats maintained the
circadian rhythm of serum iron, their serum iron
levels were measured at 8:00, 14:00, and 20:00 (n = 5
rats in each group). The serum iron levels were high
in the morning and low in the evening (Fig 2). Under
consideration of the circadian rhythm in the rats, all of
the rats received FC at 8:00 am.
Based on these data, the change in serum iron
level at 2 hours after oral iron intake was substituted
for iron absorption, which was named as 2-hour oral
iron absorption test (2-h OIAT).
 Regulation of Iron Absorption by Hepcidin
Fig 1. The 24-hour Serum Iron Levels After Intake of 10 mg of Ferric
citrate (FC) in the Normal Rats
The rats (n=45 rats) were given 10 mg of FC by oral gavage at 8:00 am
and killed 0, 0.5, 1, 2, 3, 6, 9, 12, and 24 hours after FC intake (n = 5
rats in each group). A, serum iron; B, transferrin saturation levels
(TSAT); C, hepcidin. *Significantly differs from that at the starting
point at 0 hour (p < 0.05).
2. Effects of exogenous hepcidin expression on 2-h
OIAT results
To study the acute effect of exogenous hepcidin
expression, the rats were injected intravenously with
0-, 25-, 50-, and 100-μg doses of human hepcidin-25
at 7:00 am, given 10 mg of FC by oral gavage at
8:00 am, and then killed at 10:00 am (n = 5 rats in
each group). Increasing the hepcidin-25 dose
resulted in a progressive decrease in serum iron
concentration (Fig 3A). In the 2-h OIAT, the rats
given 100 μg of hepcidin-25 showed significantly
lower serum iron levels than the control rats (268 ±
47 μg/dl vs 501 ± 28 μg/dl, p<0.05). Serum human
hepcidin-25 concentration was dependent on the
amount of hepcidin-25 injected (Fig 3B).
Fig 2. Diurnal Variations in Serum Iron Concentration in the Normal
Rats with Free Access to Food
n = 5 rats in each group. *Significantly differs from that at the starting
point at 8:00 am (p < 0.05).
Fig 3. Effect of Exogenous Human Hepcidin-25 on 2-Hour Oral Iron
Absorption Test results
To study the acute effect of exogenous hepcidin, 0-, 25-, 50-, and 100-
mg doses of human hepcidin-25 were injected intravenously at 7:00 am,
10 mg of ferric citrate was administered at 8:00 am, and the rats were
killed at 10:00 am (n = 5 rats in each group). *Significantly differs from
that of the normal controls (p < 0.05).
3. Effects of endogenous hepcidin expression on
2-h OIAT results
To suppress endogenous hepcidin expression, 10
rats were bled 1 ml of whole blood per day, 5 times a
week (iron deficiency group). To stimulate endogenous
hepcidin expression, 20 rats were given 4 or 12 mg of
iron through 2 mg of iron injection per day, 2 or 6 times
a week (mild and severe iron loading groups, n = 10
27

Fig 4. Effect of Endogenous Hepcidin on 2-Hour Oral Iron Absorption
Test results
To suppress the endogenous hepcidin expression, 10 rats were bled 1 ml
of whole blood per day, 5 times a week (iron deficiency group). To
stimulate 20 rats, 4- or 12-mg of iron was administered through 2 mg of
iron injection per day, 2 or 6 times a week (mild and severe iron loading
groups, n = 10 rats in each group). The controls were given only saline
(n = 10 rats).
*Significantly differs from the 2-hour oral iron absorption test (2-h
OIAT) results of the normal controls (p < 0.05).
rats in each group). The controls had saline only (10
rats). After 2 days of treatment holiday, the 40 rats were
divided into 2-h OIAT and control groups, received 10
mg of FC or saline by oral gavage at 8:00 am, and then
killed at 10:00 am. As expected, the hepcidin levels
were significantly reduced in the iron deficiency group
(2.2 ± 1.5 ng/ml, p<0.05) and significantly increased in
the iron loading groups (67.5 ± 6 ng/ml in the mild
group and 120 ± 24 in the severe group, p<0.05,
p<0.05, respectively) when compared with the control
group (26.3 ± 5.3 ng/ml; Figure 4B). Baseline serum
iron levels were significantly increased in the iron
deficiency group when compared with the severe iron
loading group (Figure 4A). The serum iron level in the
2-h OIAT was significantly reduced in the iron loading
groups when compared with the control group (245.7 ±
22.9 in the mild group, 211.8 ± 17.3 in the severe
group, p<0.05, p<0.05, respectively, and 488.0 ± 57.6
ng/ml in the control group). There was no significant
difference in serum iron levels between the iron
deficiency and control groups.
28
Ma/Koshino/et al.
Fig 5. Effect of Increased Hepcidin Level After the First Oral Iron Intake
on the Second Iron Intake
The rats were given a second 10-mg dose of ferric citrate at 2, 3, or 6
hours after the first iron intake (n = 5 rats in each group).
*Significantly differs from the first 2-hour oral iron absorption test (2-h
OIAT) result (8:00 am; p < 0.05).
4. Effect of endogenous hepcidin expression after
the first oral iron intake on the second iron
intake
The effect of serial iron intake on iron absorption
was examined because increase in hepcidin level
peaked at 6 hours after the first oral intake (Fig 1).
The rats received a second 10-mg dose of FC by oral
gavage at 2, 3, or 6 hours after first iron intake (n = 5
rats in each group). The serum iron levels in the 2-h
OIAT were significantly reduced at 6 hours when
compared with the control at 8:00 am (384 ± 33 μg/dl
vs 488±57.6 μg/dl, p<0.05; Fig 5).
5. Changes in serum iron level induced by 2-h
OIAT in the individual rats
Next, the effects of endogenous hepcidin
expression on iron absorption in iron deficiency,
control and iron loading groups (Fig 5) were
confirmed by using 2-h OIAT in the individual rats,
which had various hepcidin levels. To induce various
hepcidin levels, 14 rats bloodletting (2 ml in 3 rats and
 Regulation of Iron Absorption by Hepcidin
Fig 6. Inverse Correlations Between Serum Hepcidin Levels and Change in Serum Iron Level in the 2-Hour Oral Iron Absorption Test
The inset shows the correlation between serum hepcidin level and the Ln of the change in serum iron level. n = 17, y = −0.054x + 4.99, R2 = 0.91, p < 0.001.
4 ml in another 3 rats) or iron injections ( 2 mg in 3
rats, 4 mg in 2 rats, and 6 mg in 3 rats) was performed
during 7 days, and 3 rats had no treatments. The 0.5-
ml first blood samples at 8:00 am and second blood
samples after 2h-OIAT were collected from all of the
rats. Serum iron levels were compared between the
first and second samples in the individual rats. The
result of the linear regression analysis as shown in
Figure 6 indicate that serum hepcidin levels correlated
inversely with the change in serum iron level (r =
−0.89, p = 0.003). No iron absorption was observed
with 80 ng/ml hepcidin level.
Discussion
The results of our study show that increased serum
iron level 2 hours after iron intake is a useful
alternative to ferrokinetic analysis using radiolabeled
iron for evaluating iron absorption. In addition, our
study shows that the increased serum iron levels
inversely correlated with basic serum hepcidin levels.
The serum iron levels of the rats reflects the
circadian rhythm, and the iron levels were high in the
early morning and low in the late evening, similarly to
those described in previous reports (17, 18).
Accordingly, the rats were administered FC at 8:00
a m i n o u r s t u d y. A l t h o u g h w e d i d n o t u s e a
radioisotope as a ferrokinetic study, the serum iron
concentration was rapidly increased within 30
minutes and peaked constantly at 2 hours after iron
intake apart from the circadian rhythm. Furthermore,
the onset of iron absorption in the duodenum was
extremely rapid. Wheby et al (7) reported that
radiolabeled iron was detected in rat bodies as early as
15 seconds after the iron reached the closed loop of
the duodenum and varied in peak levels at 1 hour.
These findings implied that increased serum iron level
may reflect iron absorption just after influx from the
stomach to the duodenum and that the 2-hour iron
absorption test may be useful for the evaluation of
iron absorption in individual cases. High correlation
was observed between the absorption of isotope-
labeled iron and the change in serum iron level 6
hours after intake of 100 mg of iron in humans (19).
Rivera et al (20) reported that hepcidin accumulated
in the FPN-rich organs, namely the liver, spleen, and
duodenum. They also reported that the rapid action
29

of a single 50-μg dose of human hepcidin-25
administered to mice makes human hepcidin-25 an
appealing agent for the prevention of iron release via
FPN from iron-storing organs, which consequently
shows hypoferric effect within 1 hour. Laftah et al (21)
reported that administration of exogenous human
hepcidin-25 significantly reduced the mucosal uptake
and transfer to the body in mice given isotope-labeled
iron. In our study, a single 100-μg dose of exogenous
human hepcidin-25 inhibited the increase in serum
iron in the 2-h OIAT, which might reflect iron
absorption. Although we did not measure the DMT-1
expression, hepcidin may reduce iron absorption by
both inhibiting enterocyte DMT-1 transcription and
binding to FPN on the enterocyte basolateral
membrane (22, 23).
Correlation has been reported between endogenous
serum hepcidin level and iron absorption (15, 16, 24,
25). Cao et al (15) reported that radiolabeled
ferrokinetic studies in rats revealed that liver hepcidin
mRNA expression inversely correlated with nonheme
iron absorption, although their trials were far from
clinical application in individual cases. Others (16, 24,
25) reported that plasma hepcidin is a modest predictor
of dietary iron bioavailability in healthy men and
women with iron statuses ranging from iron deficiency
to iron sufficiency. Eschbach et al (14) reported the
quantitative relationship between serum level of
ferritin, which is a regulator of hepcidin expression via
bone morphogenetic protein (BMP) 6 (26), and iron
absorption in healthy subjects and patients undergoing
long-term dialysis. In all the aforementioned previous
studies, iron absorption was evaluated by measuring
the amount of isotopic iron incorporated into red blood
cells or in whole blood at 2 weeks after ingestion of
isotope-labeled iron. The methods are accurate but
difficult to implement in individual subjects. Although
our method is simple and takes only 2 hours to
perform, our findings about the correlation between
baseline serum hepcidin levels and changes in serum
iron levels in the 2-h OIAT were similar to the results
of the previous studies. Our method is simple and
provides a means for studying iron absorption without
the use of isotope-labeled iron. Furthermore, based on
the inverse correlation curve, we can infer the upper
limit of hepcidin to inhibit iron absorption completly
in patients undergoing hemodialysis. In this regard,
much more studies are necessary in clinical fields.
In the present study, oral iron loading increased
circulating hepcidin level, which in turn affected iron
absorption, followed by a secondary iron intake.
Transferrin receptor (TFR) 2 expression modulates
the signal between iron status and hepcidin expression
via the Bmp6/Smad pathway (27). This might take 4
hours to induce hepcidin expression from TFR2
30
Ma/Koshino/et al.
activation by the alteration of serum iron level.
Reduced efficacy of a second iron supplementation by
the increased hepcidin level may act as a feedback
mechanism to limit iron efflux into plasma.
Furthermore, the amount of oral iron intake is a main
factor of the inhibition of iron uptake via DMT-1 (28).
DMT-1 expression is regulated by intracellular iron-
regulatory proteins, which is regulated by the amount
of intracellular iron. When oral iron is loaded, DMT-1
expression is reduced via the iron-regulatory protein
pathway and then iron uptake is blocked. We did not
perform additional measures after 9 hours and thus
could not clarify whether further decrease in serum
iron level occurred after the 2-h OIAT. Further studies
are needed to analyze the mechanisms of iron
absorption via hepcidin and DMT-1 expressions.
In the previous studies, oral iron supplementation
was unable to maintain adequate iron stores and treat
iron deficiency in patients undergoing hemodialysis
(3-5). Subsequently, the use of intravenous iron
injection in patients with end-stage renal disease has
become widespread. Oral iron supplementation often
causes gastric irritation, nausea, and constipation,
which may decrease treatment adherence and long-
term efficacy. High hepcidin levels induced by severe
ferritinemia block iron absorption. FC was recently
developed as a phosphate binder. Treatment with FC
can induce increases in iron parameters while
maintaining hemoglobin levels, with a safety profile
and without decreased drug adherence. In this
situation, prediction of the bioavailability of oral iron
may be important in making oral iron dosing schedules
for individual patients undergoing hemodialysis who
have low iron absorption, probably because of
excessive iron stores and high hepcidin levels induced
by intravenous iron injection, low levels of
erythropoiesis or both.
Conclusion
Our data suggest that hepcidin level may be the
main regulating factor of iron absorption in the
intestine. Based on the relationship between hepcidin
and serum iron levels 2 hours after iron intake, a new
method for ferrokinetics can be developed in order to
analyze iron absorption without isotope and to
speculate the reason for the lack of iron absorption.
The application of the 2-h OIAT in clinical fields is
expected in the future. At the start of hemodialysis, a
blood sample is drawn for measurement of serum iron
and hepcidin-25 levels before FC ingestion and again
a small amount of blood is drawn for serum iron
measurement 2 hours after oral iron intake.
The present study suggests that inducing high
hepcidin levels (>80 ng/ml) minimizes fractional iron
absorption. The 2-h OIAT may support alternate-day
 Regulation of Iron Absorption by Hepcidin
supplementation. This analytical system can be
applied in clinical fields in order to estimate the
efficacy of oral iron administration in individual
cases. It can also be established as a standard method
for determining the upper-limit hepcidin level for
inhibiting iron accumulation in different physiological
and/or pathological situations.
This study was self-funded by Kanazawa Medical
University. We have greatly benefited from Takanori
Tatsuno, PhD. and professor Yasuhito Ishigaki. We are
also gratefull to the management staff, Ms. Satomi
Okura for her cooperation.
Conflict of interest
One author (N.T.) is the founder of Medical Care
Proteomics Biotechnology Co., Ltd. The other authors
declare that they have no competing interests.
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