Kinetic analysis of 59Fe movement across the intestinal wall in duodenal rat segments ex vivo

Klaus Schümann, Bernd Elsenhans, and Wolfgang Forth

Walther-Straub-Institut für Pharmakologie und Toxikologie, Ludwig-Maximilians-Universität, D-80336 Munich, Germany


    ABSTRACT
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Abstract
Introduction
Materials and methods
Results
Discussion
References

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


    INTRODUCTION
Top
Abstract
Introduction
Materials and methods
Results
Discussion
References

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.


    MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and methods
Results
Discussion
References

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 · cm-1 · min-1 in normal and iron-deficient segments, respectively. Water transport at the end of the experiments was between 80 and 100% of that found at the beginning. The glucose concentration in perfusate and absorbate was determined at 15-min intervals. In all absorbate samples, it was more then twice as high as in the perfusate. Such glucose accumulation in the absorbate was taken as evidence for unimpaired viability of the segments throughout the experiment. Addition of the Fe-nitrilotriacetic acid (NTA) complex had no impact on water and glucose transport.

To 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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Fig. 1.   Effect of segment translocation into a second perfusator on the time course of cumulative 59Fe mucosal-to-serosal transport [100 µmol 59Fe-nitrilotriacetic acid (NTA) (1:2), means ± SD; n = 4]. bullet  and , Iron-adequate segments. open circle  and , Iron-deficient segments. Segments in one group (bullet  and open circle ) were continuously perfused in the same perfusor. In the other group ( and ), perfusion was continued in a second perfusator under identical conditions after 45 min, which corresponds to the translocation of segments after the loading period. 59Fe mucosal-to-serosal transport became linear after an equilibration period of ~30 min and was not significantly influenced by the translocation into a second perfusator.

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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Fig. 2.   Kinetics of 59Fe mobilization from duodenal tissue into the absorbate (luminal perfusate contained 10 µmol Fe during loading and mobilization period). Cumulative 59Fe basolateral transfer corresponded to first-order rate kinetics. A function of the type C = A(1 - e-kt) was fitted to the transfer data by nonlinear regression (C = cumulative 59Fe transfer into the absorbate over 2 h, A = asymptotic maximum, k = rate constant, and t = time).

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 gamma -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.


    RESULTS
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Abstract
Introduction
Materials and methods
Results
Discussion
References

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-1) and iron-deficient segments (k = 0.0230 ± 0.0041 min-1).

                              
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Table 1.   59Fe mucosal-to-serosal transport at the end of the loading period (45 min)


                              
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Table 2.   59Fe mucosal-to-serosal transport after 90 min of perfusion and duodenal 59Fe tissue load after 45 and 120 min in control experiments (no translocation of the segments to a second perfusator)

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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Fig. 3.   Changes in duodenal 59Fe steady-state tissue load at different 59Fe-labeled NTA (1:2) concentrations (means ± SD, n = 15). bullet , Iron-adequate segments. open circle , Iron-deficient segments. 59Fe tissue load under steady-state conditions shows saturation characteristics with dissociation constant (Kd) values of 92.5 ± 9.1 and 91.4 ± 7.5 µmol/cm and a maximum tissue load of 15.9 ± 5.4 and 26.5 ± 7.5 nmol/cm in iron-adequate and iron-deficient segments, respectively.

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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Fig. 4.   Duodenal 59Fe tissue load at the end of the loading period and 59Fe tissue retention. A: iron-adequate segments. B: iron-deficient segments. Luminal 59Fe concentrations were 1, 10, 50, 100, 200, and 500 µmol/l (n = 5; for duodenal tissue load, n = 15). 59Fe tissue load under steady-state conditions is symbolized by thick horizontal bars above the columns (means ± SD). 59Fe tissue retention was calculated by subtracting the asymptotic maximum of 59Fe mobilization from the steady-state tissue load. Mobilized means ± SD are represented by vertical arrows. Columns represent the 59Fe quantities that are retained in the tissue; thin horizontal bars give the standard deviations of the values. Solid columns with continuous arrows = no addition during mobilization. Open columns with dashed arrows = perfusate contained NTA during mobilization. Gray columns with dashed arrows = perfusate contained unlabeled Fe-NTA during mobilizations. At concentrations above 1 µmol Fe/l, the 59Fe steady-state tissue load was significantly increased in iron deficiency (P < 0.01). Subsequent perfusion with NTA and Fe-NTA complex mobilized additional 59Fe from the tissue. 59Fe tissue retention showed no significant differences between normal and iron-deficient segments after mobilization without luminal additions (P > 0.05). After mobilization with NTA and Fe-NTA complex, differences were not significant at some concentrations, whereas they were significantly lower (star ) or higher (star ) in iron-deficient segments at other concentrations. The differences, however, showed no systematic pattern and argue for an increased scatter of retention values after mobilization with NTA and Fe-NTA rather than for a decreased retention in iron-deficient segments.

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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Table 3.   Mucosal-to-serosal transport of [14C]NTA and 59Fe under steady-state conditions (30-120 min)

The 1:2 complex between iron and NTA is the most stable (complex formation constant of 24.3) (25). Therefore, iron was added to the luminal perfusate in the form of such complexes. Undissociated complexes maintain this stoichiometry and would pass the mucosal barrier in a 1:2 ratio, giving 59Fe transport rates that are only half as high as those for [14C]NTA. However, this is not the case. On a molecular basis, normal duodena transport slightly more 59Fe than [14C]NTA instead of only half as much. In iron-deficient duodenal segments, 59Fe transport was significantly increased and exceeded [14C]NTA transport by 4.5 times (Table 3). This is nine times more than expected for an undissociated complex with a Fe-NTA ratio of 1:2. In ileal segments, 59Fe transport was significantly lower than in the duodenum and showed no adaptation to iron deficiency, which is in accordance with literature (2, 33). In normal and iron-deficient ilea, 59Fe transport was seven and nine times lower, respectively, than that of [14C]NTA. Again, this is different from the ratio of 1:2 expected for the undissociated complex. Thus 59Fe transport responds to iron deficiency and is different between duodenal and ileal segments, whereas [14C]NTA transport shows no response. This finding suggests that both moieties of the Fe-NTA complex pass the mucosal barrier separately. This is in accordance with findings by Snape et al. (38), who found a different subcellular distribution of [14C]NTA and 59Fe in the mouse duodenum. Therefore, 59Fe-NTA can be used to load duodenal segments with 59Fe.

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 · cm-1 · 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 · cm-1 · 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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Fig. 5.   Total 59Fe + 55Fe tissue load in duodenal segments and 59Fe tissue retention after perfusion of 59Fe-loaded segments with 55Fe-NTA. Luminal iron concentration for both isotopes was 100 µmol Fe/l (means ± SD, n = 4). Gray areas = duodenal 59Fe load in iron-deficient segments. Solid areas = duodenal 59Fe load in iron-adequate segments. Open areas = fraction 55Fe load that substituted for the original 59Fe content of the segment during subsequent perfusion with 55Fe. Horizontal bars = SD of retained 59Fe. Percentages shown are duodenal 59Fe retention (percentage of total isotope tissue load). Columns represent total 59Fe + 55Fe tissue load. At time 0, the segment had been loaded with 59Fe for 45 min (steady-state 59Fe tissue load). Although total 59Fe + 55Fe tissue load was significantly increased in all iron-deficient segments investigated (Student's t-test, P < 0.01), it showed no significant differences over time (one-way ANOVA, P < 0.05). In iron-deficient segments, the original 59Fe tissue load was replaced by subsequently absorbed 55Fe to a higher extent than in iron-adequate segments. However, after 90 and 120 min, 59Fe retention showed no significant differences (ns) between corresponding iron-deficient and iron-adequate segments. Although on a percentage basis 59Fe tissue retention differs between iron-deficient and iron-adequate segments, no difference was found in the absolute values of retained 59Fe.

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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Abstract
Introduction
Materials and methods
Results
Discussion
References

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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Fig. 6.   Correlation between initial mobilization rates for 59Fe from the duodenal tissue into the absorbate (= basolateral transfer) and the rates of 59Fe mucosal-to-serosal transport in corresponding experiments. bullet , Iron-adequate segments. open circle , Iron-deficient segments. Correlation coefficient = 0.98; r2 = 0.96. During the mobilization period, the perfusate contained unlabeled Fe-NTA (1:2) in the same concentrations used during the loading period. The initial mobilization rate was calculated from the 59Fe mobilization data. It shows a high correlation with mucosal-to-serosal transport rates, which were directly determined at the end of the loading period of the corresponding experiment. The high degree of correlation supports the assumptions made for the calculation of the initial mobilization rate and shows that first-order rate curves were well fitted to 59Fe mobilization data.

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 alpha -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.


    ACKNOWLEDGEMENTS

The skillful technical assistance of H. Janser, M. Beer, E. Schmid-Loock, and S. Islambouli are gratefully acknowledged.


    FOOTNOTES

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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Results
Discussion
References

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