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Blood, Vol. 94 No. 9 (November 1), 1999:
pp. 3185-3192
Importance of Anemia and Transferrin Levels in the Regulation of
Intestinal Iron Absorption in Hypotransferrinemic Mice
By
K.B. Raja,
D.J. Pountney,
R.J. Simpson, and
T.J. Peters
From the Department of Clinical Biochemistry, King's College School
of Medicine, Denmark Hill, London, UK.
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ABSTRACT |
The hypotransferrinemic mouse (trf hpx) is a mutant
strain exhibiting transferrin deficiency, marked anemia,
hyperabsorption of iron, and elevated hepatic iron stores. We set out
to investigate the relative roles of anemia and of transferrin in the
malregulation of intestinal iron absorption in these animals.
Transfusion of erythrocytes obtained from littermate controls increased
hemoglobin levels and reduced reticulocyte counts in recipient animals.
Although mucosal to carcass 59Fe transfer was reduced,
total duodenal iron uptake was not significantly affected. Iron
absorption in homozygotes, in contrast to littermate controls, was not
reduced by hyperoxia. Mouse transferrin injections, in the short term,
increased delivery of iron to the marrow and raised hemoglobin levels.
Although mucosal transfer and total iron uptake were reduced at the
higher transferrin doses, total uptake was still higher than in
controls. Daily injections of mouse/human transferrin for 3 weeks from
weaning, normalized hemoglobin values, and markedly reduced liver iron
and intestinal iron absorption values in trf hpx
animals. When such daily-injected mice were left for a week to allow
transferrin clearance, iron absorption values were significantly enhanced; hemoglobin or hepatic iron levels were, however, not significantly altered. These data indicate that hyperabsorption of iron
in trf hpx mice is not solely because of the anemia;
transferrin levels per se do affect iron absorption, possibly via a
direct effect on the intestinal mucosa.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
THE REGULATION of iron absorption remains
poorly understood, in spite of the recent cloning of the
hemochromatosis gene (HFe),1 a candidate component of the
regulatory pathway. Subsequent investigations have shown that the
transferrin-receptor and the HFe protein can interact with each
other,2,3 suggesting a role for transferrin in the control
of intestinal iron absorption.
The genetically hypotransferrinemic (trf hpx) mouse,
first described by Bernstein,4 shows cytopathological
features similar to the human conditions, idiopathic
hemochromatosis,5 and congenital atransferrinemia.6,7 The mutant strain exhibits a virtual absence of circulating transferrin as a result of a splicing defect in
transferrin mRNA8 and a hypochromic, microcytic anemia, despite marked hepatic parenchymal iron loading.4,9 Excess liver iron is attributed to the enhanced intestinal absorption of
dietary iron combined with rapid and selective deposition of this iron
in the liver. Although some degree of control over absorption is
exercised by the surplus iron,9 little is known about the pathogenesis of the enhanced iron absorption in these animals.
One study10 claimed that transfusion of "washed"
erythrocytes into trf hpx mice, normalized hematological
parameters (ie, hematocrit and reticulocyte counts) and reduced the
absorption of intragastrically administered iron to values comparable
with those seen in normal balb/c mice. As the transfused homozygous
animals had a marked reduction in serum transferrin, these workers
concluded that anemia alone was responsible for the enhanced absorption
of iron. Our more recent studies using other animal models of chronic
anemia, namely  -thalassaemia11 and
erythropoietin-deficiency,12 have, however, shown only
modest changes in intestinal iron absorption as compared with values in
littermate controls. Moreover, the changes in absorption were mainly
because of increases in the mucosal uptake of radioiron. These findings
thus indicate that factors other than the erythropoietic rate may be
responsible for the enhanced absorption seen in trf hpx
animals. As transferrin is virtually absent in these animals, we
decided to reinvestigate the possibility of whether the anemia or the
lack of circulating transferrin accounts for the malregulation of
intestinal iron absorption in the trf hpx mouse model.
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MATERIALS AND METHODS |
Reagents.
All chemicals and biochemicals were from either Sigma Chemical Co Ltd
(Poole, Dorset, UK) or BDH Chemicals (Poole). Radio-iron (59FeCl3) was obtained from NEN-Du Pont
(specific activity 0.19 to 2.78TBq/g; Stevenage, Herts, UK).
Animals.
The hypotransferrinemic mice originated from BALB/cJ background.
Homozygous mice (trf hpx), which are phenotypically
distinguishable at birth by their pale appearance, were maintained by
weekly injections of mouse serum (150 µg to 1 mg transferrin) as
described previously.9 Heterozygotes were differentiated
from the wild-type controls by the reduction in serum transferrin
levels. Experiments/manipulations on trf hpx mice were
performed a week after the last serum maintenance dose, thus allowing
for almost complete clearance of exogenously administered transferrin.13 Mice were 6 to 8 weeks of age at the time of study.
Transfusion of erythrocytes.
Control (ie, mixture of heterozygotes and wild types) or
trf hpx animals were injected intraperitoneally
(i.p.) with either 0.15 mol/L NaCl (saline) or washed,
packed erythrocytes (~250 µL) obtained from homozygotes,
heterozygotes or wild-type mice, and studied 3 days post-single or
-double (3-day interval between injections) transfusion. Washing of
erythrocytes with saline 3 times was found to be adequate for removal
of any plasma-associated transferrin. In some experiments, animals were
given an injection of erythrocytes daily for 3 days and studied 2 to 5 days later. Preliminary experiments performed in animals that had been
injected intravenously (IV) with cells/saline showed no differences
compared with the i.p.-injected mice; the 2 groups have therefore been
combined for analysis.
Transferrin injections.
Homozygous mice were injected i.p. with either a single or double dose
of commercially obtained mouse transferrin (Chemicon Int, Temecula,
CA), and studied 4 hours to 1 week later. In addition, some
trf hpx mice were injected daily, starting from weaning,
with a progressively increasing dose (1 to 3 mg) of human or mouse
transferrin for 3 weeks; this injection regime was followed to account
for mouse growth. Iron absorption studies were performed 6 to 18 hours
or 1 week after the last injection. Mice injected on a daily basis for
3 weeks with human albumin received the usual weekly maintenance dose
of transferrin (as serum).
Transferrin determination.
Serum levels were assayed by a radial immunodiffusion
technique14 using 1% agar containing 0.02 mol/L sodium
barbitone buffer (pH, 8.6) and appropriate amounts of either goat
anti-human transferrin (Dynatech Labs, Billinghurst, Sussex, UK) or
sheep anti-mouse transferrin (Chemicon Int). Purified human transferrin
(Sigma) or mouse transferrin (Chemicon Int) was appropriately diluted and assayed at the same time to establish a calibration curve. The
human and mouse antibodies did not cross-react with mouse or human
transferrin, respectively.
Tissue oxygen levels.
Duodenal pO2 levels were determined polarographically in
anesthetized mice by gently inserting a previously calibrated, thin, flexible wire-type electrode into the duodenal mucosa through a slit
made distal to the point where the bile duct joins the small intestine.
The body temperature was maintained by placing mice on a heated pad.
Recordings on a Model pO2-100 monitor (composed of an
ammeter with built-in power supply; Inter-Medical Ltd, Nagoya, Japan)
were not commenced until the electrode was well positioned and secured.
The measurement of pO2 in mammalian tissue via
polarographic electrodes and other means is described in the review by
Vanderkooi et al.15
Iron absorption.
In situ tied-off duodenal segments were used to determine intestinal
iron absorption.9,11 A tracer dose of 59Fe (as
a ferric chelate of nitrilotriacetate, 1:2, in physiological medium)
was injected intraluminally into the tied-off segment of the
anesthetized animal. The duodenal intraluminal contents were gently
flushed-out with warm saline before the injection of radioiron
solution. All experiments were performed under Vmax conditions [Fe(III) = 250 µmol/L] and for an incubation period of
10 minutes. The segment was thereafter removed, flushed with ice-cold
saline, weighed, and counted in a gamma counter (LKB Wallac Model
80000; Turku, Finland). Blood (cardiac puncture) and any other required
tissues were also removed, weighed, and similarly counted. The carcass
was counted in a well-type counter. Aliquots (10 µL) of the radioiron
solution, acting as standard, were counted in both counters to
normalize the 59Fe counts. The activity of 59Fe
in the duodenal segment is referred to as "mucosal retention," whereas the activity in the carcass reflects the "mucosal
transfer." The sum of the 2 parameters represents the "total
mucosal uptake." The activity of 59Fe associated with
the blood was calculated assuming a blood volume of 1.5 mL/25 g body
weight.16 For in vivo 51Cr-EDTA permeability
studies, the physiological medium contained 100 µmol/L
51CrEDTA and 300 µmol/L EDTA, in place of
Fe(NTA)2.
Hematological parameters.
Five microliters of blood obtained by cardiac puncture was mixed with
Drabkin's reagent for hemoglobin determination.17 Reticulocyte counts were performed independently by 2 people on methylene blue-stained blood smears.
Nonheme iron assays.
These were performed on tissue homogenates using a
modification18 of the method of Foy et al.19
Data.
Shown as mean ± SD for (n) determinations. Statistical analysis was
performed using unpaired t-test. Where the variances differed significantly, the Welch's test was used.
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RESULTS |
Figure 1 shows the body weights and
hemoglobin levels, and Table 1 shows the
iron absorption parameters in control and homozygous hypotransferrinemic mice. The heterozygote and wild-type mice have been
combined into a single control group because previous investigations11 have shown no significant differences in
iron absorption between the 2 groups.
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| Fig 1.
Body weights and hemoglobin levels in (a) controls and in
homozygous (trf hpx) mice transfused with (b) nothing,
(c) saline (×2), (d) trf hpx erythrocytes (×2), and
(e through g) control erythrocytes (1 to 3 doses, respectively). All
determinations were performed 3 days after the last transfusion except
for the multiple-transfused group (g) which were performed 5 days
postinjection. *P < .04; **P < .002; ***P < .007 as compared with untreated trf hpx mice (b).
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Homozygous mice had stunted growth as reflected by the reduced body
weights and were markedly anemic with a high degree of reticulocytosis
(23.2 ± 5.5 [6]%) as compared with the control group
(1.3 ± 1.0[6]%). Intestinal iron absorption studies showed a
marked enhancement in trf hpx mice mainly because of
increased transfer of radioiron from the mucosa to the plasma.
Permeability studies performed with 51CrEDTA, a stable
nonabsorbed ecf marker,20 showed no difference between the
control (8.7 ± 4.8 [3] pmol/mg/10 min) and trf hpx
(8.7 ± 4.6 [3]) mice, thus indicating that the enhanced
absorption of iron is specific and not attributable to increased
intestinal permeability. Most of the absorbed radioiron (after a
10-minute incubation in a tied-off duodenal segment) in
trf hpx mice was found in the liver (72 ± 8.1
[6]%), with little being incorporated into erythrocytes
(0.63 ± 0.64 [6]%). In contrast, values for the liver and
erythrocyte 59Fe incorporation in the control group were
16.3 ± 8.0 (10)% and 11.9 ± 12.6 (10)%, respectively. These
values resemble those reported in control/mutant mice studied at 24 hours4 and 3 days5 postadministration of
59FeCl3 in phosphate buffer via the IV and oral
route, respectively. At the time of our study, transferrin could not be
detected immunologically in the serum of trf hpx mice
(ie, <.05 mg/mL).
Transfusion of trf hpx animals on 2 occassions (with a
3-day gap in between injections), with either saline or erythrocytes
obtained from homozygous animals, did not cause any significant changes in the hemoglobin, mucosal transfer, or total uptake of
59Fe in recipient mice. It is noteworthy that injection of
trf hpx erythrocytes was performed as an additional
control for the transfusion experiments and shows that injection of red
blood cells per se does not affect iron absorption. In contrast, a
single or double transfusion of erythrocytes from control animals
resulted in significant changes in the hemoglobin level in recipient
trf hpx mice, but neither treatment resulted in a
decrease in the total mucosal uptake of iron. The doubly transfused
group, though exhibiting a significant decrease in the percent mucosal
transfer values, showed a significant increase in the mucosal retention
of radioiron (Table 1); thus, no apparent change in overall uptake was
evident. Reticulocyte counts decreased after the single-
(15.1 ± 13.2 [3]%) or double-erythrocyte transfusion
(3.6 ± 3.5[4]%) of control erythrocytes as compared with the
saline-infused group (33.6 ± 7.4 [3]%).
Multiple transfusion of erythrocytes (3 times) induced a further small
increase in the hemoglobin level; the values posttransfusion were
comparable with the values in the control group. As the reduction in
percent mucosal transfer was more prominent and the mucosal retention
values were unaltered in the multiple-transfused group, the total
absorption values showed a reduction, but the values failed to reach
statistical significance (P < .2). It is noteworthy that
the total uptake values were still significantly higher than those in
the control group. Clearance of radioiron by the liver was marginally
increased (82.5 ± 9.0 [6]%), whereas that by the erythrocytes
was reduced (0.10 ± 0.24 [6]%) following the multiple transfusions. Mice studied 2 to 3 days after multiple transfusions also
showed a similar, statistically insignificant reduction in total
mucosal iron uptake (69.8 ± 17.4 [3] pmol/mg/10 min).
Liver and duodenal mucosal non-heme iron levels were unperturbed in the
transfused groups compared with untreated trf hpx mice
(Fig 2). When the hepatic data were
corrected for liver weights, a small but progressive increase in total
iron level was observed with increasing transfusion of control
erythrocytes. Splenic iron levels were also moderately increased
following the triple-transfusion (1.56 ± 0.61[6] nmol/mg
v 1.04 ± 0.71 [6]). When allowance was made for the
spleen weights, the total iron content values were comparable with
those seen in the noninjected group (362 ± 133 [6] nmol v
365 ± 263 [6]). Control animals multiply transfused with
erythrocytes from heterozygote/wild-type littermates had elevated liver
weights and hepatic iron content (2.4 ± 0.5 [5] µmol v
1.9 ± 0.8 [10]) and exhibited small, insignificant, reductions in
mucosal transfer (7.7 ± 1.6 [5] v 10.1 ± 5.4 [10] pmol/mg/10 min), percent mucosal transfer (27.6 ± 4.4 [5]%
v 30 ± 10.5 [10]%), and total mucosal uptake
(27.8 ± 4.6 [5] v 32.3 ± 12.6 [10] pmol/mg/10 min).
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| Fig 2.
(A) Liver iron (total content or tissue concentration)
and (B) duodenal iron in control and homozygous hypotransferrinemic
mice; the latter group was either untransfused or given 1 to 3 transfusions of erythrocytes from control animals. All determinations
were performed 3 days after the last transfusion except for the
multiple-transfused group, which were performed 5 days postinjection.
Data: mean ± SD for 5 to 10 animals in each group except for the
(×1) transfused group where n = 3. *P < .04; **P < .001 as compared with the control group. $,P < .02 as
compared with the nontransfused homozygous group.
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Effect of hyperoxia on iron absorption.
Trf hpx mice exposed to 40% O2 for 3 days
failed to show any marked changes in the degree of reticulocytosis
(18%, 21%, n = 2) or in intestinal iron absorption (total
uptake = 108 ± 33[4] pmol/mg/10 min). Exposure for shorter
periods (ie, 24 and 48 hours) was also without effect on iron
absorption (109 and 168 pmol/mg/10 min, respectively). A statistically
significant reduction in iron absorption was, however, evident in
control mice similarly exposed to 3 days hyperoxia (20.0 ± 6.1
[6] v 32.3 ± 12.6 [10] pmol/mg/10 min,
P < .05), owing to a reduction in both the mucosal
retention (13.4 ± 4.0 [6] v 22.2 ± 8.1 [10]
pmol/mg/10 min) and mucosal transfer (6.6 ± 3.6 [6] pmol/mg/10
min v 10.1 ± 5.4 [10]) of 59Fe.
Tissue oxygen levels.
No difference was evident in the duodenal pO2 levels
between the trf hpx mice (21 mm Hg, n = 2) and the
control group (20.5 ± 1.8 [3] mm Hg). When both groups were,
however, made to inhale 10% O2 for 2 minutes via a small
mouth piece and the measurements performed, a decrease of between 2 and
5 mm Hg was evident as compared with basal values. Conversely,
inhalation of 40% O2 resulted in an increase of 2 to 8 mm
Hg. The mucosal oxygen levels determined with the thin wire electrode
are within the range of values observed in mammalian small intestine by
surface and microelectrode measurements.21-23
Effect of transferrin injection(s) on intestinal iron absorption.
To monitor the effects of transferrin, trf hpx mice were
studied at specific times after injection with various doses of mouse transferrin (Fig 3).
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| Fig 3.
Effect of mouse transferrin injections on circulating
levels, hemoglobin (A) and liver/erythrocyte uptake of the absorbed
radioiron (B). The transferrin (Tf ) dose and sampling time (after
last injection if more than one) are as indicated. Data: mean ± SD
for 4 to 6 animals in each group except for the one studied 3 days post
2.2 mg dose, where n = 3. Detection limit for serum transferrin was
0.05 mg/mL. Any values below this level are therefore not indicated on
the figure. *P < .05; **P < .02; ***P < .004 as compared with untreated homozygous mice.
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Circulating transferrin levels in trf hpx mice were at
the time of study dependent on the dose given and the period
postinjection (Fig 3A). Increased incorporation of absorbed radioiron
by immature red blood cells was apparent 4 to 5 hours after transferrin
injection, and peaked at about 24 hours; thereafter, the red blood cell
59Fe incorporation values decreased and by 7 days the
values had reverted to basal levels (Fig 3B). Alterations in the
incorporation of iron into erythrocytes (and thus in hemoglobin values)
were more striking with the larger transferrin doses. The increased 59Fe incorporation by erythrocytes 24 hours postinjection
with 4.4 mg transferrin was associated with a significant decrease in
radioiron clearance by the liver. Thereafter, as the red blood cell
incorporation of 59Fe decreased, the liver clearance values
equalled or even exceeded values seen in untreated
trf hpx mice. The injected group, however, still
exhibited enhanced intestinal iron absorption (Table
2). Homozygous mice given 8.8 mg
(2 × 4.4 mg) of transferrin however, showed an appreciable
reduction in total uptake, mainly because of reduced transfer of
59Fe from the mucosa to the portal circulation (Table 2).
The uptake values were still higher (P < .04) than those in
wild-type/heterozygotes (total mucosal uptake, 32.3 ± 12.6 [10]
pmol/mg/10 min). It is noteworthy that at the time of experiment there
were no significant alterations in either clearance of radioiron by the
liver or total liver iron content (23.4 ± 6.3 [4] µmol
v 21.2 ± 3.5 [6]) after dosing with 8.8 mg of
transferrin. The fact that circulating transferrin levels 3 days
postinjection with either the single or double dose (with a 3-day gap
in between injections) of 4.4 mg transferrin are similar, indicates
that transferrin clearance is rapid (half-life < 24 hours), and is
in agreement with previous observations.13
Daily injections of commercially available mouse transferrin (1 to 3 mg) over a 3-week period, starting from weaning, normalized hemoglobin
levels, markedly reduced hepatic iron stores, and significantly decreased iron absorption in trf hpx mice (Tables 3 and
4). The iron
absorption values were, however, still higher than the control group.
Mice treated similarly for 3 weeks with mouse transferrin and then left
for a week (without injections) so that circulating transferrin levels
decreased to undetectable/negligible levels, showed a marked increase
in both the mucosal transfer and total uptake (P < .02)
without any change in liver iron content or hemoglobin values. The
total uptake values were similar to those seen in normally maintained
trf hpx mice. Animals treated with human transferrin on
a daily basis for 3 weeks exhibited a similar increase in the
hemoglobin level and also showed normalized percent mucosal
59Fe transfer and total mucosal uptake values. Liver iron
stores were also reduced, though not to the same extent as in the
mouse-transferrin treated group. As before, mice left untreated for a
week after 3 weeks of daily injections had undetectable circulating
transferrin (ie, <.05 mg/mL) and yielded a significant increase in
both mucosal transfer and total uptake, even though hemoglobin levels
were normal. Circulating transferrin levels at the end of the 3-week period of daily injections were similar in both groups, suggesting that
the protein, though from different sources, is cleared at similar
rates.
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Table 3.
Effect of Daily Transferrin Injections on Circulating
Transferrin, Hemoglobin, and Liver Iron in Homozygous
Hypotransferrinemic Mice
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Table 4.
Iron Absorption in Hypotransferrinemic Mice Injected
Daily for 3 Weeks With Either Mouse or Human Transferrin
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Homozygous mice injected in a similar fashion for 3 weeks, but with
human albumin, exhibited marked anemia, elevated hepatic iron stores,
and enhanced mucosal retention, transfer, and total uptake values;
these characteristics are similar to those seen in mice receiving
maintenance serum injections only, and suggests that the effects of
human transferrin on iron absorption are not because of possible
induction of an immunologic reaction.
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DISCUSSION |
The trf hpx mouse, a genetic strain with a virtual
absence of circulating transferrin, shows similarities to the human
conditions hemochromatosis and atransferrinemia. It is thus a useful
animal model for investigating not only the regulation of iron
absorption but also the mechanism of iron toxicity.24
In spite of the surplus iron, the mutant strain exhibits a marked
anemia of an iron-deficient nature; the sustained hyperabsorption of
iron, though not surprising, is, however, intriguing because (1)
animals lack transferrin, which is necessary for the delivery of iron
as `iron-transferrin,' the primary physiological source of iron for
erythroid cells, and (2) changes in absorption are more marked than
seen in other models with chronic anemia (ie, -thalassemia,11 erythropoietin-deficient12)
or with enhanced reticulocytosis
(phenylhydrazine-treated25). In this study we have
investigated the relative importance of both anemia and transferrin in
the malregulation of intestinal iron absorption in homozygous hypotransferrinemic mice.
The role of anemia/oxygen delivery in the regulation of iron
absorption in trf hpx mice.
Transfusion of erythrocytes from trf hpx animals
resulted in a small, statistically insignificant increase in hemoglobin
levels in recipient mice. Intestinal iron absorption values were
unaffected. The lack of a significant effect on the hemoglobin level is
surprising and may be attributable to the fact that erythrocytes from
trf hpx animals are low in hemoglobin
content.4 Transfusion of washed erythrocytes from
littermate controls (wild-type/heterozygotes), however, caused even
after a single dose, a significant increase in the hemoglobin level and
a decrease in the reticulocyte count. Multiple transfusion of
erythrocytes led to further increases in hemoglobin, with the values
being comparable with those seen in the control group. Even though
mucosal transfer (as percentage of total uptake) decreased appreciably
after the double and triple transfusions, total mucosal uptake failed
to exhibit a significant decrease as compared with the untreated group.
The decrease in mucosal transfer could not be attributable to either
radioiron dilution within the duodenal mucosa (because nonheme iron
levels were unaltered even after the triple transfusion) or to
alterations in 59Fe clearance by the liver. The decrease in
mucosal transfer is in support of the findings of Buys et
al.10 However, our data do not reach the same conclusion
(ie, normalized absorption values posttransfusions). This discrepancy
may be attributable to differences in the absorption method used (ie,
tied-off intestinal segment for 10 minutes v whole-body
retention 4 to 24 hours after gavage) or the iron complex used (FeNTA
v iron-ascorbate). Moreover, our absorption studies were
confined to the duodenum, the primary region where iron absorption
occurs, and were unaffected by effects on intestinal transit.
Erythrocyte transfusions had 3 effects, namely increased hemoglobin,
decreased erythropoiesis, and increased liver iron (presumably because
of exogenous/endogenous red cell breakdown). Any of these, independently, could perhaps have resulted in a decrease in mucosal transfer and overall iron absorption. The importance of the change in
liver iron was tested by injecting iron dextran into
trf hpx mice. No significant effect on iron absorption
was seen even though liver iron levels were comparable with those in
erythrocyte-infused trf hpx mice. Increased hemoglobin
is presumed to affect intestinal iron absorption via oxygen delivery to
the mucosa, as oxygen level in the inspired air is known to influence
iron absorption.26,27 However, exposure of homozygous mice
to 40% oxygen for up to 3 days failed to alter iron absorption.
Furthermore, intestinal mucosal pO2 levels in
trf hpx mice did not differ appreciably from those in
the control group. Studies in pigs have shown that intestinal mucosal
pO2 levels remain fairly constant unless the hematocrit
falls below 10%.23 In previous studies, we found that
acute alterations ( 1 week) in reticulocyte levels affected mucosal
transfer more than mucosal uptake,25 whereas chronic
alterations (>1 week) in erythropoiesis affected mucosal uptake more
than transfer.11,12 Mice with chronic anemia and
reticulocytosis comparable with trf hpx animals, namely
-thalassaemic mice, did not show the massively enhanced absorption
of iron seen in trf hpx mice.11 In an
earlier study (unpublished observations, Simpson and Raja, January
1991) we found that exchange transfusion of blood (from the control
group) into trf hpx mice modestly raised the hemoglobin
levels and completely suppressed the reticulocyte response, without
affecting iron absorption in these recipient mice. Based on these
observations, it is unlikely that reticulocytosis is solely responsible
for enhanced mucosal transfer and uptake in the trf hpx mice.
The tendency for the erythropoietic rate and iron absorption in
genetically normal rodents to be decreased by hypertransfusion or
hyperoxia26,27 and increased by bleeding28 or
hypoxia26 is consistent with a regulatory mechanism, which
is related to mucosal oxygen supply. In mice with lifelong anemia, as
in genetic hypotransferrinemia, adaptive changes to the cardiovascular
system and erythrocyte oxygen affinity will have occured to produce an animal able to maintain mucosal oxygen levels, as shown above, despite
the anemia. We have previously shown that duodenal blood flux in
homozygous animals is similar to that in the control
group.29 It is unsurprising that transfusion with
erythrocytes from normal mice may decrease iron absorption, because it
will increase oxygen delivery to the tissues. The finding that
hyperoxia does not depress iron absorption in these homozygous mice
suggests that their adapted mucosal oxygen delivery system is, however,
not responsive to increased inspired oxygen content. This highlights
the fact that responses of trf hpx mice to experimental
manipulation should be interpreted in light of their adaptation to
chronic anemia.
Role of transferrin in the regulation of iron absorption in
trf hpx mice.
Transferrin, though clearly not obligatory for the absorptive process,
may have a role in the regulatory process. Injection of purified mouse
transferrin (1.0 to 4.4 mg) into trf hpx mice markedly
increased delivery of 59Fe to the bone marrow (and thence
to red blood cells) and subsequently led to increases in the hemoglobin
levels. The changes in 59Fe incorporation by erythrocytes,
which were apparent within 4 to 5 hours posttransferrin injection, were
bigger with the higher transferrin doses; the radioiron clearance
values peaked at 24 hours postinfusion and thereafter decreased with
time and by 7 days had reverted to basal levels. Liver 59Fe
clearance values, in all cases but one, showed no significant reduction
after transferrin injection(s). These findings show that the liver in
trf hpx mice has a large capacity for clearance of
nontransferrin bound iron, in support of previous
findings.30 Erythrocytes, however, depend on transferrin as
the main physiological route of iron delivery.
Although small decreases were apparent in the mucosal transfer of
59Fe posttransferrin injection, the total mucosal uptake
values did not change significantly in any of the treated groups except for the group given a double dose of 4.4 mg transferrin. The
interpretation of the role of transferrin in the regulation of iron
absorption is, however, hampered by the fact that hemoglobin values
have increased concurrently. We conclude that iron absorption was
reduced, but not normalized by acute correction of anemia, or by
short-term injections of transferrin. We therefore investigated the
involvement of transferrin in the control of iron absorption in
trf hpx mice with long-term correction of anemia.
Transferrin (mouse/human) was injected daily for 3 weeks to normalize
hemoglobin levels. This regime was calculated to produce transferrin
levels which oscillated around a mean value equivalent to that seen in
heterozygote mice (which show normal iron absorption rates11). These transferrin-injected mice exhibited
markedly reduced liver iron stores and had transferrin levels
comparable with values seen in heterozygous mice. Iron absorption
values (especially in the human transferrin-injected group) were
considerably lower than those in the normally maintained, anemic
trf hpx mice. Mice left uninjected thereafter for a
week, thus allowing adequate time for the clearance of
transferrin,13 but not for the development of anemia,
showed markedly increased mucosal transfer and iron absorption values
in spite of no changes being evident in the hemoglobin levels and liver
iron stores; the total uptake values in both the mouse and human
transferrin-treated groups were similar to those seen in normally
maintained trf hpx mice. It is feasible that, in spite
of normalized hemoglobin levels, low transferrin levels may lead to
ineffective (iron-deficient) erythropoiesis, which may in turn
contribute to the enhancement of iron absorption. The failure of daily
mouse transferrin injections to completely normalize iron absorption is
probably attributable to insufficient transferrin being present at all
times throughout the experimental period. The finding that daily
injections of human transferrin were more effective at normalizing
absorption was apparently not attributable to the protein half-life,
nor to a nonspecific effect of a foreign protein. It is possible that human transferrin interacts with transferrin-receptor in a way that
more effectively regulates iron absorption.
These data show that the greatly enhanced intestinal iron absorption in
trf hpx mice, leading to the highest degree of iron
overload reported so far in mice fed a standard rodent diet, is not
solely explained by anemia or increased production of erythrocytes.
Transferrin levels do, however, influence iron absorption (especially
mucosal transfer) independently of effects on hemoglobin levels.
Recently, a transgenic mouse colony with the homologous HFe gene
disrupted has been established.31 These mutant mice were hematologically normal, had almost fully saturated transferrin, and
showed hepatic iron-loading even though their diet was not supplemented
with iron. It has previously been suggested32 that the
amount of iron absorbed via the intestine is a function of the
programming of crypt cells depending on body iron status. As HFe is
reported to be present in the crypts33 and to interact with
transferrin receptors,2,3 it is reasonable that HFe is
involved in the sensing mechanism: Disruption of the gene would result
in the crypt cells not being appropriately programmed, thus resulting
in the malabsorption of iron through over expression of genes for iron
absorption such as the recently described DCT1/Nramp2 transporter
protein.34-36 Our data support the involvement of
transferrin in the control of iron absorption possibly through direct
effects on the intestinal mucosa.
| |
ACKNOWLEDGMENT |
This is a contribution from the King's College Centre for the Study of
Metals in Biology and Medicine, London, UK.
| |
FOOTNOTES |
Submitted November 13, 1998; accepted June 24, 1999.
Supported by a UK Medical Research Council Project Grant.
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address reprint requests to K.B. Raja, PhD, Department of Clinical
Biochemistry, King's College School of Medicine, Denmark Hill, London
SE5 9PJ, UK; e-mail: K.Raja{at}KCL.AC.UK.
| |
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ABSTRACT
INTRODUCTION