Walther-Straub-Institut für Pharmakologie und Toxikologie, Ludwig-Maximilians-Universität, D-80336 Munich, Germany
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ABSTRACT |
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Duodenal segments from iron-deficient and iron-adequate rats were luminally perfused ex vivo with solutions containing 1, 10, 50, 100, 200 and 500 µmol 59Fe/l. When duodenal tissue load and mucosal-to-serosal transport had reached a steady state, perfusion was continued without luminal 59Fe supply. Mobilization of 59Fe from the duodenal tissue into the serosally released absorbate followed first-order rate kinetics, which permitted calculation of the asymptotic maximum, the rate constant, and the initial mobilization rate for tissue-to-absorbate transfer. There was no evidence for adaptation of 59Fe tissue binding in iron-deficient segments. 59Fe tissue-to-absorbate transfer increased in proportion to the mobilizable fraction of recently absorbed iron in the tissue, which is indicative of simple diffusion or carrier-mediated transport below saturation. Regulation of the mucosal uptake step appears to determine the mobilizable 59Fe fraction and thus the adaptation of the overall iron absorption process to the demand. Iron retention in the duodenal tissue and iron transfer from here into the body appear not to be either regulated or rate limited.
iron; transport; adaptation
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INTRODUCTION |
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THE BODY'S STATE of iron repletion is determined by the equilibrium between intestinal iron absorption and iron excretion. Because iron is highly conserved by the body (11), intestinal absorption is the primary regulator of total body iron. Duodenum and proximal jejunum adapt their iron absorption capacity in response to changes in body iron stores. More distal regions of small intestine show no regulation and are markedly less efficient in transporting iron (2, 33). The process of intestinal iron transport encompasses three steps: iron is taken up from the lumen, it is retained in the mucosa, and a fraction is transferred out of the mucosa into the organism. The role of each of these steps in the overall process is the subject of this report, particularly as to which of these steps is regulated or rate limited.
On the basis of functional studies with duodenal brush-border membrane vesicles from iron-adequate and severely iron-deficient mice, it has been proposed that regulation of the uptake step alone may be responsible for the increased iron absorption in iron deficiency (27). Accordingly, specific iron transport proteins were described at the brush-border membrane (e.g., see Refs. 8, 17, 30, 39, 40). A transport protein was also described at the basolateral membrane (38), and some earlier findings argue for the basolateral transfer step as being rate limiting (24, 42). This would lead to an accumulation of recently absorbed iron in the intestinal mucosa.
The mucin-mobilferrin-integrin pathway (4) describes a sequence of iron-binding proteins at different locations from the intestinal lumen to the plasma. Iron binds to mucin under acidic pH conditions, e.g., in the stomach, and thus is kept available under the more alkaline conditions in the duodenum. Here, labeled iron was found to be associated with integrins, which are proposed to facilitate the transit of iron through the microvillous membrane. In enterocytes, integrin binds mobilferrin (5), which binds iron and is supposed to shuttle it from the brush-border membrane to the basolateral side of the cell. Here again, an integrin may facilitate iron transfer into the plasma (4). According to this concept, iron binding to intracellular proteins like mobilferrin may also influence the availability of recently absorbed iron for transfer across the basolateral membrane. Indeed, a variety of working groups found ~50% of 59Fe in homogenates from rat small intestinal mucosa in the particle-free supernatant after 10-60 min of luminal perfusion with 59Fe (4, 16, 43). This percentage gives an estimate of the iron fraction that may be available for basolateral transfer. Quantitative analysis of the binding behavior of such recently absorbed 59Fe in mucosal homogenates, however, may lead to artifacts due to possible redistribution of 59Fe after the disruption of tissue and cells (18). Therefore, the present study presents a kinetic approach that was developed to determine the behavior of recently absorbed 59Fe in intact, native duodenal tissue from iron-deficient and iron-adequate rats ex vivo. This approach permits quantification of the mucosal 59Fe load when the absorptive process shows a steady state and to determine the size of the fraction that is available for basolateral transfer. In the same experiment, the rates of basolateral 59Fe transfer and of the overall 59Fe mucosal-to-serosal transport can be determined in parallel. Comparison of these parameters allows a kinetic analysis of the relative contribution of mucosal uptake, retention, and transfer to the overall absorption process for iron in native duodenal tissue.
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MATERIALS AND METHODS |
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Animals
The experiments were performed according to the rules of animal care and were approved by the local animal protection committee (Tierschutzkommission der Regierung von Oberbayern: AZ 211-2531-185/87 and AZ 211-2531-25/93). Conventionally bred male Sprague-Dawley rats (Interfauna, Tuttlingen, Germany) with an initial weight of 180 ± 11 g were housed in stainless steel cages on stainless steel grids [3-5 animals/cage, 12:12-h light-dark cycle (0600-1800), 22 ± 1°C, 60 ± 5% humidity]. After an acclimatization period of 1 wk, the animals were randomly divided into two groups. Iron deficiency was induced by feeding an iron-deficient diet (diet C1038, Altromin, Lage, Germany; total iron content of 6 mg Fe/kg) and distilled water ad libitum during fast growth for 10 days (34). During this time, controls were fed an iron-adequate diet of the same composition (diet C1000, Altromin, Lage, Germany; total iron content of 180 mg Fe/kg, iron added as FeSO4).Preparation and Perfusion of Isolated Duodenal Segments
The abdomen was opened under ether anesthesia. The duodenum between the pylorus and the Treitz ligament was removed and luminally perfused ex vivo as described earlier (33, 35, 36). Briefly, the perfusion conditions were as follows: recirculating luminal perfusion with 30 ml of bicarbonate-buffered Tyrode solution (37°C, pH 7.2), which was equilibrated with 95% O2-5% CO2, 25 cmH2O hydrostatic pressure, and 50 ml/min flow rate. The segments were kept in a moist chamber. The serosal absorbate was sampled in 15-min intervals and was quantitated gravimetrically. The mucosal-to-serosal water transport thus determined amounted to 0.771 ± 0.141 and 0.846 ± 0.071 µl · cmTo determine mucosal-to-serosal iron transport, [59Fe]FeCl3 (0.2 µCi/ml) was added to the perfusate after water transport had reached a steady state (~10 min after start of luminal perfusion). Iron was chelated with NTA in a twofold molar excess over iron (37) to prevent the formation of iron hydroxides. 59Fe radioactivity in perfusates, duodenal tissues, and absorbates was determined in a gamma-counter. Corresponding iron quantities were calculated assuming that the same specific 59Fe activity existed in perfusate, tissue, and absorbate.
Analysis of Duodenal 59Fe Tissue Retention by a Two-Step Perfusion Procedure
During the loading period (step 1), the Fe-NTA complex was added to the perfusate at six concentrations (1, 10, 50, 100, 200, and 500 µmol Fe/l). After 30 min, the cumulative 59Fe mucosal-to-serosal transport became linear (Fig. 1). The 59Fe tissue load reached a steady state after 45 min (see Control Experiments). Therefore, after this time, the segment was removed from the perfusion apparatus, flushed with plain oxygenated Tyrode solution (37°C, 25 cmH2O column, 50 ml/min for 1 min), and remounted in a second perfusion apparatus. The rate of the mucosal-to-serosal 59Fe transport under steady-state conditions was determined in the last fraction collected during the loading period.
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In the second perfusion apparatus (step 2), perfusion was continued under identical conditions, except that no 59Fe was added to the perfusate. Thus all 59Fe that was detected in the absorbate during the second period must have been mobilized from the duodenal tissue (mobilization period). During mobilization, one group of segments was perfused with plain Tyrode solution. In a second and third group, the Tyrode solution contained NTA or unlabeled Fe-NTA complex at concentrations corresponding to those used during the loading period.
During the mobilization period, only negligible amounts of
59Fe (<1%) returned into the
luminal perfusate (data not shown). Therefore, the steady-state
59Fe tissue content at the end of
the loading period was assumed to be equal to the sum of the
59Fe quantities that were retained
in the segment plus the 59Fe
quantities that had been released into the absorbate at the end of the
mobilization period. During mobilization,
59Fe was determined in eight
consecutive absorbate samples over 2 h. The mobilization of
59Fe from the duodenal tissue was
assumed to follow first-order rate kinetics (Fig.
2). Therefore, an equation of the type
C = A(1 e
kt)
was fitted to the cumulative 59Fe
mobilization data by means of nonlinear regression analysis (Enzfitter
program; Ref. 20). C stands for the
cumulative 59Fe transfer from the
segment into the absorbate over 2 h; t
stands for time. The equation permits calculation of an asymptotic
maximum (A) for the mobilizable
fraction of the 59Fe tissue load
and a rate constant (k) for the
59Fe mobilization process. The
determined values were distributed evenly above and below the fitted
curve. The variation coefficients for
k and
A were <3% at all concentrations.
The initial rate for 59Fe
mobilization, i.e., the rate of the transfer process at the beginning
of the mobilization process, can be calculated by multiplying the rate
constant k with the mobilizable
59Fe fraction
A.
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Control Experiments
Kinetics of 59Fe mucosal-to-serosal transport and 59Fe steady-state tissue concentration. The 59Fe mucosal-to-serosal transport was compared between segments that remained in the same perfusion apparatus for 165 min and segments that were transferred into a second apparatus after 45 min. To check whether the 59Fe tissue load had reached a steady state at this point in time, it was compared after 45 and 120 min of luminal perfusion with 10, 100, and 500 µmol Fe/l (n = 5). To measure 59Fe distribution between the mucosa and the remaining duodenal tissue, the mucosa was scraped off the segment after perfusion with 100 µmol 59Fe/l (45 min; n = 3).
Mucosal-to-serosal transport of the metal and chelator moieties of the Fe-NTA complex. To investigate to what extent Fe-NTA splits before absorption, the mucosal-to-serosal transport of the complex was investigated. Both moieties of the complex were labeled in separate experiments (59Fe-labeled NTA and Fe-[14C]NTA) and the complex (100 µmol Fe/l) was added to the perfusate of iron-deficient and iron-adequate duodenal and ileal segments (n = 5).
Estimation of isotope dilution during 59Fe mucosal-to-serosal transport. Duodenal segments were perfused for 120 min with plain Tyrode solution. The release of endogenous tissue iron from iron-adequate and iron-deficient duodenal segments into the absorbate was measured after steady-state conditions for mucosal-to-serosal water transport had been reached. Absorbate samples were incubated with 1 N HCl (1:1). After 45 min, protein was precipitated with trichloracetic acid and centrifuged (20 × 103 g for 5 min). The iron concentration was determined in the supernatant by use of atomic absorption spectrometry.
Double labeling with 59Fe and
55Fe.
Iron deficient and iron-adequate duodenal segments were perfused with
59Fe-NTA during the loading period
(1-45 min) and with 55Fe-NTA
during the mobilization period (45-165 min) (100 µmol Fe/l; n = 4). To determine the
59Fe-to-55Fe
ratio in the tissue at different time points, perfusion was stopped at
the beginning of the mobilization period and after 30, 60, 90, and 120 min. At the end of the mobilization period, the segments were flushed,
dried, and solubilized. 59Fe and
55Fe in the solubilized tissue and
in absorbate samples were determined in a beta-counter (window setting:
8-90 keV for 55Fe,
135-185 keV for 59Fe). Values
determined for 59Fe corresponded
well to those determined by -activity measurement before solubilization.
Chromatography of the absorbate samples. An iron-deficient duodenal segment was perfused with 100 µmol/l 59Fe-NTA during the loading period (0-45 min) and with 100 µmol/l unlabeled Fe-NTA during the mobilization period (45-135 min). A second segment was continuously perfused with 100 µmol 59Fe-NTA. In both experiments, an early fraction (0-45 min) and a late fraction (105-135 min) of the absorbate were sampled. After centrifugation (20 × 103 g for 30 min), 300-400 µl of the different absorbate samples were applied to a Sephadex G75 column (2 cm × 100 cm). The column was calibrated with dextran blue 2000, albumin, myoglobin, and vitamin B12 and eluted at a rate of 6 ml/h (0.05 mol/l Tris · HCl + 0.1 mol/l KCl, pH 7.5). Fractions of 2.0 ml were collected; 59Fe was determined in 0.5-ml fractions.
Chemical Determinations, Chemicals, and Radiochemicals
Glucose and plasma iron were determined by use of the glucose oxidase method and bathophenantroline reaction, respectively (Merckotest nos. 14335 and 3317, Merck, Darmstadt, Germany). Packed cell volume and hemoglobin concentrations were determined by the microhematocrit technique and cyanomethemoglobin method, respectively. Transferrin was quantitated by use of Mancini's radial immunodiffusion (23) [RARa/Trf antiserum, Nordic Immunochemical Laboratories, Tilburg, The Netherlands; rat transferrin (chromatographically purified) was used as standard for calibration (no. 55953, ICN, Eschwege, Germany)]. Iron concentrations in hepatic tissue and in the diet were determined by atomic absorption spectroscopy (Zeeman 3030, Perkin-Elmer, Überlingen, Germany) after wet digestion under pressure (170°C, 2.5 h, ~300 mg tissue in 1 ml nitric acid; Suprapur, Merck, Darmstadt, Germany).59Fe activity was determined in a gamma-counter (GAMAmatic, Kontron, Munich, Germany; window settings of 0.7-1.6 MeV). 14C and 55Fe radioactivity were determined by liquid scintillation counting (LKB 1215 Rack-BETA II, Wallac Oy, Turku, Finland). Tissue was solubilized for beta-counting by addition of tetraethylammoniumhydroxide (1:2 wt/vol, 12 h incubation, Riedel-de-Haen, Seelze, Germany). All chemicals used were of analytical purity and purchased from Merck or Sigma (Munich, Germany). 59Fe and 55Fe were supplied by NEN (Dreieich, Germany). Nitrilotri-[U-14C]acetic acid was purchased from Amersham/Buchler as part of a custom synthesis.
Statistical Treatment of Data
Values of the different groups are presented as means ± SD. Significant differences between more than two corresponding groups were analyzed by one-way ANOVA (5% level). The homogeneity of variances was checked for each ANOVA by means of Hartley's test. When significant differences were found, the Scheffé's test was used (5% level) to test which means were significantly different from each other and which formed homogeneous groups. Differences between corresponding iron-deficient and iron-adequate parameters were assessed by means of Student's t-test (P < 0.05 and P < 0.01).Explanations of Terms
In the context of this work, the term "59Fe tissue load" is the 59Fe content in the duodenal tissue under steady-state conditions of ex vivo perfusion with 59Fe at different concentrations. In contrast, "59Fe tissue retention" is the fraction of 59Fe that is retained in the duodenal tissue after the mobilizable 59Fe fraction has been subtracted from the steady-state 59Fe tissue load."Mucosal-to-serosal transport" is the movement of 59Fe from the luminal perfusate into the serosally released absorbate. One step of overall mucosal-to serosal transport is the 59Fe movement across the enterocytes' basolateral membrane into the submucosal space, from where it enters portal circulation in vivo. In isolated ex vivo segments, absorbed solutes such as 59Fe are channeled from the submucosal space to the serosal surface mainly via lymphatic vessels (21). When mucosal-to-serosal transport reaches a steady state, solutes appear in the absorbate without much dilution (21). Therefore, the term "basolateral transfer" is used for the movement of 59Fe from the duodenal tissue into the absorbate.
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RESULTS |
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Iron deficiency was induced by 10 days of iron-deficient feeding during rapid growth. Hemoglobin concentration showed no significant differences (141 ± 12 for iron deficient vs. 143 ± 9 g/l for iron adequate), whereas plasma transferrin concentration (4.5 ± 0.5 for iron deficient vs. 2.9 ± 0.4 mg/ml for iron adequate), plasma iron concentration (52 ± 12 for iron deficient vs. 167 ± 23 µg/100 ml for iron adequate), and hepatic iron content (41.7 ± 6.2 for iron deficient vs. 128.9 ± 16.6 µg/g wet wt for iron adequate) showed significant changes.
59Fe Mucosal-to-Serosal Transport and Rate Constants of Basolateral 59Fe Transfer
Table 1 shows the mucosal-to-serosal transport rates under steady-state conditions at the end of the loading period (n = 15). This parameter showed constant increments after an initial lag phase of 30 min (Fig. 1) and was significantly increased in iron-deficient duodenal segments (P < 0.01). As expected under steady-state conditions, the 59Fe mucosal-to-serosal transport rates after 90 min (Table 2) were not significantly different from those found after 45 min (Table 1). The rate constant for 59Fe mobilization from the segments into the absorbate showed no significant differences between iron-adequate segments (k = 0.0219 ± 0.0046 min
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59Fe Steady-State Tissue Load and 59Fe Tissue Retention
The 59Fe tissue load was significantly increased (P < 0.01) in iron deficiency. This parameter showed no significant differences after 45 and 120 min of perfusion (Table 2). The steady-state tissue load at the end of the loading period was determined retrospectively as the sum of retained 59Fe in the segments plus the cumulative 59Fe release from the segment into the absorbate. These values appear to be correctly calculated as they agreed well with the directly determined 59Fe tissue load after 45 and 120 min (Table 2). The steady-state tissue load showed saturation characteristics (Fig. 3). With the use of the Michaelis-Menten equation, a maximum tissue load of 15.9 ± 5.4 and 26.5 ± 7.5 nmol/cm was calculated for iron-adequate and iron-deficient segments, respectively. The dissociation constant (Kd), however, showed no significant differences between iron-adequate and iron-deficient segments (Kd = 92.5 ± 9.1 and 91.4 ± 7.5 µmol/l). After 45 min of tissue loading (100 µmol 59Fe/l), 78 ± 6% of the radioactivity was found in the duodenal mucosa and 22 ± 6% in the remaining duodenal tissue (n = 3).
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Figure 4 gives the
59Fe tissue load and the
59Fe tissue retention values. In
iron-deficient segments, the 59Fe
steady-state tissue load was significantly increased at all luminal
concentrations except at 1 µmol/l.
59Fe retention in duodenal tissue
showed no significant differences between iron-deficient and
iron-adequate segments after perfusion without any additions during the
mobilization period. Subsequent perfusion with NTA or with unlabeled
Fe-NTA increased 59Fe mobilization
from the duodenal tissue and decreased its retention. The differences
in 59Fe retention between normal
and iron-deficient segments at corresponding luminal iron
concentrations remained small, although they became significant
(P < 0.05) in some cases. The
differences, however, showed no systematic pattern (Fig. 4; no
significant differences in 5 cases, higher retention in iron deficiency
in 2 cases, lower retention in 5 cases; these numbers include the data
at 1 µmol Fe/l, the values for which are too small to be depicted in
Fig. 4). These results argue for increased scatter of retention values after mobilization with NTA and Fe-NTA rather than for a decreased retention in iron-deficient segments.
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Control Experiments
Mucosal-to-serosal transport of the metal and chelator moieties of
the Fe-NTA complex.
Iron as Fe-NTA reaches equilibrium with competing ligands of comparable
affinity within seconds (1). Therefore, the Fe-NTA complex may not pass
the mucosa as such. Because NTA is not metabolically degraded in
mammals (26), 14C radioactivity in
the absorbate represents the intact NTA molecule when offered luminally
as [14C]NTA (100 µmol Fe/l). Mucosal-to-serosal transport of the
[14C]NTA complex
moiety showed no changes in iron deficiency nor were there significant
differences between duodenal and ileal segments (Table
3).
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Estimation of isotope dilution of 59Fe
during mucosal-to-serosal transport.
In iron-adequate and iron-deficient duodenal segments that were
perfused with plain Tyrode solution, an average of 0.41 ± 0.16 and
0.26 ± 0.09 pmol · cm1 · min
1
(n = 4) of endogenous iron was
released from the tissue into the absorbate under steady-state
conditions. Thus, at a luminal concentration of 1 µmol Fe/l, the
59Fe transport is approximately
twice as high as that of endogenous iron in iron-adequate segments and
seven times higher in iron-deficient segments (Table 1). These data
give an estimate of possible isotope dilution effects due to endogenous
iron. As a consequence, concentrations lower than 1 µmol Fe/l were
not applied.
Mucosal-to-serosal water transport.
The steady-state mucosal-to-serosal water transport was neither
significantly influenced by the state of iron repletion (0.771 ± 0.141 for iron deficient vs. 0.846 ± 0.071 µl · cm1 · min
1
for iron adequate) nor by the luminal iron content (data not shown).
Thus differences in duodenal water transfer cannot be held responsible
for differences in the mobilization of
59Fe from the segments.
Double labeling with 59Fe and
55Fe.
Duodenal segments were loaded with
59Fe from the luminal side for 45 min (100 µmol Fe/l). Thereafter, perfusion was continued with
55Fe at the same concentration for
another 2 h. Under steady-state conditions, the total tissue load of
radioactive iron (equal to 59Fe + 55Fe) showed no significant
changes after 30, 60, 90, or 120 min of perfusion with
55Fe (Fig.
5). However, increasing amounts of
59Fe were mobilized from the
duodenal tissue and were substituted by
55Fe during this time. This
replacement showed saturation characteristics. In iron-deficient
segments, a greater percentage of the original 59Fe tissue load was replaced by
55Fe. However, because the
steady-state tissue load with radioactive iron was significantly higher
in iron-deficient segments, the retained
59Fe fraction after 90-120 min of perfusion was
not significantly different from that in iron-adequate segments.
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Chromatography of absorbate samples on Sephadex G75. The amount of endogenous plasma transferrin in the segment is limited. It cannot be replaced when the segment is cut off from the blood supply. During ex vivo perfusion, the segment's transferrin content decreased exponentially due to continuous water transfer (36). When a segment was loaded with 59Fe during the first 45 min of ex vivo perfusion, 15% of the radioactivity in the absorbate was associated with a 80-kDa peak (presumably transferrin). In this situation, the segment's transferrin content is still high and 59Fe is continuously supplied from the lumen.
When the segment is perfused with plain Tyrode solution after 45 min of 59Fe loading, the 59Fe supply is cut off, whereas exponential mobilization of 59Fe from the segment continues. Consequently, total 59Fe activity in 300 µl of absorbate decreased from 33,640 counts/min (cpm) during the first 45 min to 8,850 cpm in the fraction collected during 105-135 min. The percentage of 59Fe associated with the 80-kDa peak (17%), however, was the same as in the early absorbate fraction (15%), which shows that transferrin and 59Fe are mobilized from the tissue at the same rate when the supply is cut off. This finding suggests that basolateral 59Fe transfer out of the enterocytes is much faster than the exponential mobilization of solutes from the interstitial space together with extracellular transferrin, which is a function of intestinal water transfer (36). Indeed, earlier in vivo findings showed that 59Fe is transferred from the intestinal lumen into the blood in <2 min (2, 33). When the segment was continuously perfused with 59Fe, the situation was different. Again, transferrin is mobilized from the segment exponentially (36), i.e., transferrin concentration in the absorbate samples decreased with time. 59Fe absorption rates, however, remained close to constant during continuous 59Fe perfusion (Fig. 1). Therefore, the ratio between transferrin iron binding capacity and 59Fe would decrease during the course of an experiment. Indeed, again, 20% of total 59Fe was found in the 80-kDa peak of the absorbate during the first 45 min of perfusion, when the segment's transferrin content is still high (radioactivity in 300 µl = 32,000 cpm). In the fraction collected during 105-135 min, only 6% of 59Fe was associated with the 80-kDa peak, whereas 94% was found in the low-molecular-weight peak. Total 59Fe radioactivity remained in the same order of magnitude (in 300 µl = 26,020 cpm). Because the mobilizable transferrin quantities are mostly located in the extracellular space of the intestine (31), this shift is evidence for iron leaving the enterocyte in a low-molecular-weight form. It is most likely bound to NTA or to endogenous low-molecular-weight ligands (18, 19) and definitely not to high-molecular-weight proteins. ![]() |
DISCUSSION |
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Comparison Among 59Fe Uptake, the Rates of 59Fe Mucosal-to-Serosal Transport, and 59Fe Basolateral Transfer
As in earlier experiments (33), mucosal-to-serosal 59Fe transport reached a steady state and showed constant increments after an initial lag phase of 30 min (Fig. 1). In vivo, 59Fe appears in the blood a few minutes after the start of luminal 59Fe perfusion (2, 33). This retardation does not argue for impaired 59Fe transport in ex vivo segments, as the steady-state mucosal-to-serosal transport rate was in the same order of magnitude as the rate of mucosal-to-blood transport in vivo; in vivo and ex vivo preparations showed the same adaptation to iron deficiency and the same differences in iron transfer capacity between duodenum and more distal segments (33). The ex vivo retardation is explained by the lack of blood circulation. In ex vivo segments, absorbed solutes form a depot in the interstitial space after their passage through the mucosa. From here, they are drained to the serosal surface of the segment via lymphatic vessels and venules (21). This process needs more time to reach a steady state than that for in vivo, in which the interstitial space is drained via the circulation. The strength of the ex vivo perfusion model is, however, that it permits investigation of the adaptation of intestinal transport to iron deficiency in native duodenal tissue, whereas absorbed solutes in the serosal absorbate are as directly accessible after their basolateral transfer as in monolayer cell cultures.A constant mucosal-to-serosal 59Fe
transport implies that 59Fe is
transferred from the submucosal tissue to the serosal surface at the
same rate at which it is taken up from the lumen. This is so as long as
there is no additional buildup of duodenal
59Fe tissue load; indeed, the
59Fe tissue load showed no
significant increases between 45 and 120 min of luminal perfusion
(Table 2). Thus, at the end of a loading period of 45 min,
mucosal-to-serosal transport rates for 59Fe were determined under
steady-state conditions. The end of the loading period is the beginning
of the mobilization period. Therefore, at this point in time, the
mucosal-to-serosal transport rate should be equal to the rate of
59Fe basolateral transfer, which
is expressed by the initial 59Fe
mobilization rate from the segment into the absorbate. Indeed, when
perfusion was continued with a perfusate of the same composition during
the mobilization period (i.e., Tyrode + unlabeled Fe-NTA), the
calculated initial mobilization rate showed a high correlation with the
corresponding mucosal-to-serosal transport rate, which was directly
determined (Fig. 6). The high degree of
correlation supports the assumptions made for the calculation of the
initial 59Fe mobilization rate and
shows that the first-order rate curves were well fitted to the
59Fe mobilization data.
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The rate constants of basolateral 59Fe transfer were independent of the state of iron repletion, which is in agreement with kinetic calculations in beagle dogs (28). Within the range investigated, the rate constants were also independent of the luminal 59Fe concentration. The initial mobilization rate is the product of the rate constant and the asymptotic maximum of 59Fe mobilization from the segment. Because the rate constant does not change, the velocity of 59Fe basolateral transfer at rising luminal concentrations increases in proportion to the mobilizable fraction of the 59Fe tissue load, i.e., it depends linearly on the available 59Fe concentration. This is a feature of simple diffusion. It may also be found in carrier-mediated processes below saturation, which, in this case, would imply that a putative carrier population in the basolateral membrane does not change in response to iron deficiency. Indeed, basolateral transfer of iron has been described as simple diffusion in basolateral membrane vesicle studies (9). Snape et al. (38) have published evidence for a basolateral iron carrier system, which is not adapted in iron deficiency. Both these findings are in accordance with the data of this study.
Duodenal 59Fe Tissue Retention
The 59Fe retention in duodenal segments showed no significant differences between iron-adequate and iron-deficient segments after mobilization with plain Tyrode solution (Fig. 4). Corresponding differences after mobilization with NTA and Fe-NTA were not significant (Fig. 5), or they were small and showed no systematic pattern (Fig. 4). Thus there was no adaptation in duodenal iron binding capacity to explain the changes in mucosal-to-serosal iron transfer in iron deficiency. Accordingly, Conrad and co-authors (3) found no significant differences in the quantity of iron binding substances in duodenal homogenates from iron-deficient and iron-loaded rats. These findings fit well with the observation that duodenal ferritin concentrations and the iron content of mucosal ferritin remained low when enteral iron was given to iron-deficient rats (10). Thus ferritin does not seem to influence duodenal iron retention during the absorption process. Our finding fits also well with the assumption that recently absorbed iron may bind to cytosolic proteins, such as mobilferrin, the binding capacity of which shows no significant differences between iron-deficient, normal, and iron-loaded rats (6).Duodenal Iron Binding During Passage of 59Fe Through the Mucosa
Subsequently absorbed NTA and Fe-NTA complexes mobilized additional 59Fe from duodenal tissue (Fig. 4). In analogy, parenterally applied desferrioxamine (DFO) bound to luminally applied 59Fe in the intestine and reduced the basolateral transfer of 59Fe in vivo (22). Thus complexes between recently absorbed iron and endogenous ligands must have a lower complex formation constant than those formed between iron and NTA or DFO.The kinetic data in undisrupted native duodenal tissue presented here are in accordance with an earlier hypothesis by Conrad and Umbreit (4): as shown by pulse chase experiments (6), iron is supposed to be handed on from the lumen to the plasma along a cascade of endogenous ligands that show an increasing affinity to iron. For this purpose, the complexes formed during the passage of iron through the enterocytes' cytosol must not be too stable. In our experiments, 59Fe appears in the absorbate in a low-molecular-weight fraction as long as it is not bound to the segments' residual transferrin content after basolateral transfer. This finding agrees with the observation that 80% of newly absorbed iron is associated with a low-molecular-weight fraction in intestinal segments from neonatal guinea pigs (19). Again, basolateral transfer of low-molecular-weight iron suggests that the available mucosal 59Fe fraction is not very tightly bound to intracellular proteins.
Duodenal 59Fe Uptake
Because the 59Fe tissue retention showed no adaptation to iron deficiency and because the transfer into the absorbate was not rate limiting, the increased 59Fe tissue load in iron deficiency must be caused by an increased duodenal 59Fe uptake. Indeed, the steady-state tissue load showed saturation characteristics with an increased maximum and unaltered dissociation constants in iron deficiency (Fig. 3), which is in accordance to earlier findings in mice, dogs, and humans (13, 28, 32). This implies that iron uptake in iron-deficient duodenum is mediated by an increased number of the same kind of transporters as in the iron-adequate state.The kinetic approach of the present study permits no conclusions on the
molecular nature of mucosal iron-binding ligands or on iron
transporters in the brush-border membrane, a number of which are
described in the literature (7, 8, 17, 29, 30, 39, 40). Some of these
proteins (e.g., Refs. 8, 17) probably bind to mucin; some may be
identical to or may represent aggregates, monomers, or partial
proteolytic digests of others. Thus Teichmann and Stremmel (40)
reported a 160-kDa protein with three identified 54-kDa monomers. This
protein is suspected to be identical to the 56-kDa protein called
mobilferrin and to the 150-kDa -subunit of the integrin to which it
binds (4). A "stimulator of Fe transport" (SFT) was recently
cloned by Gutierrez and co-workers (15). This protein is associated
with endosomes and transports iron across membranes in an
energy-dependent manner. Another new candidate to mediate iron uptake
in the duodenal brush-border is the divalent cation transporter (DCT1),
which was recently cloned by Gunshin and co-workers (14). It is a
product of the Nramp
2 gene (12) and distinct from SFT
(15). This protein shows all the features of a transmembrane protein,
although the location of DCT1 in the brush-border membrane has not yet
been verified. In contrast, brush-border location was evidenced for other iron-binding proteins by fluorescence microscopy (e.g., Refs. 7,
30, 40). A high-affinity 100-kDa iron-binding protein preparation in
rabbits bound significantly more
59Fe when derived from
iron-deficient rabbits (30), supporting its participation in the
adaptation to iron deficiency. Evidence for a role for brush-border
membrane proteins in intestinal iron uptake was provided by the
observation that addition of monospecific antibodies against a 160-kDa
trimeric iron-binding protein decreased the
Fe3+ uptake in human microvillus
membrane vesicles by ~50% (40). 59Fe labeling of a 52-kDa
brush-border protein in rats changed in parallel to brush-border
membrane iron uptake in different states of iron repletion (41). In
these experiments in rats, recently absorbed iron passed by the mucosal
ferritin compartment in vivo. The exchangeable pool of iron within the
mucosa increased, and a greater fraction of a
59Fe test dose was transferred to
the plasma in vivo (41). All these results, the last finding in
particular, support the conclusion that adaptation of mucosal
59Fe uptake is the driving force
for increased iron absorption in iron deficiency.
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ACKNOWLEDGEMENTS |
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The skillful technical assistance of H. Janser, M. Beer, E. Schmid-Loock, and S. Islambouli are gratefully acknowledged.
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FOOTNOTES |
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Address for reprint requests: K. Schümann, Walther-Straub-Institut für Pharmakologie und Toxikologie, Nussbaumstr. 26, 80336 München, Germany.
Received 10 December 1997; accepted in final form 2 November 1998.
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