Blut 38, 127-134 (1979)

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Blur �9 Springer-Verlag 1979

Capacity of the Mucosal Transfer System and Absorption of Iron after Oral Administration in Rats*

G. Becker, S. Korpilla-Schfifer, K. Osterloh and W. Forth

Institut ftir Pharmakologie und Toxikologie der Ruhr-Universit/it, Im Lottental, Postfach 102148, D-4630 Bochum, Federal Republic of Germany

Die Kapazit~t des mukosalen Transfersystems und die Resorption yon Eisen nach oraler Verabfolgung bei Ratten

Zusammenfassung

1. Die Dosisabh/ingigkeit der Eisenresorption zeigt Sfittigungscharakteristik, wenn Eisen (0,25-5 /~Mol Fe/kg KSrpergewicht) in abgebundene Dfinn- darmschlingen normaler und eisenarmer Ratten appliziert wird.

2. Wird Eisen (2,1-570/~Mol Fe/kg KSrpergewicht) dagegen mit der Schlund- sonde verabfolgt, so ist nur bei normalen Ratten ein/ihnlicher Verlauf der Dosisabhfingigkeit zu beobachten wie nach Applikation in abgebundene Dfinndarmschlingen. Bei eisenarmen Ratten ist der Verlauf der Dosis- abh/ingigkeit ver/indert und zeigt keine eindeutige S/ittigungscharakteristik.

3. Die Unterschiede im Verlauf der Kurven, die die Eisenresorption in Ab- hfingigkeit yon der Dosis beschreiben, werden im Hinblick auf die verschie- denen methodischen Bedingungen diskutiert. Trotz der Tatsache, dab in der Mukosa des Darmes ein Transportsystem f/ir Eisen existiert, ist eine Sfittigungskinetik der Resorption nicht unter allen experimentellen Bedin- gungen zu erwarten.

Schliisselwi~rter: Eisen-Resorption - Eisen-Retention - Sfittigungskurve der Eisen-Resorption - Magenentleerungszeit und Eisen-Resorption

Summary

1. The dose dependence of the iron absorption shows a saturation characteri- stic if iron (0.25-5 #moles Fe/kg body weight) is administered in tied-off intestinal segments of normal and iron-deficient rats.

* Supported by a grant from the Wissenschaftsministerium des Landes Nordrhein-Westfalen. The data were presented at the 18th Spring Meeting of the Deutsche Pharmakologische Gesell- schaft in Mainz 1977. Naunyn-Schmiedeberg's Arch. Pharmacol. 297 (Suppl. II), R1, 4 (1977)

Offprint requests to: Prof. Dr. W. Forth (address see above)

0006-5242/79/0038/0127/$ 1.60

128 G. Becket et al.

2. If the iron doses (2.1-570 #moles Fe/kg body weight) are administered by stomach tube only in normal rats a similar dose dependence curve has been obtained as after administration in tied-off intestinal segments. In iron- deficient rats the shape of the dose dependence curve is changed and shows no clear-cut saturation characteristic.

3. The differences of these dose dependence curves are discussed with respect to the differences of the methodological conditions. A saturation type kinetic for absorption cannot be expected under all experimental condititions despite of the existence of a transfer system with limited capacity for the transport of iron.

Key words: Iron absorption - Iron retention - Saturation type curve of iron absorption - Gastric emptying time and iron absorption

The mucosal epithelium contains a transfer system for iron that enables the body to adapt absorption according to the demand [6]. This transfer systems reveals under certain experimental conditions a saturation type kinetic of the transfer of iron across the mucosal epithelium and the uptake of iron into the mucosal tis- sue [20]. These findings, however, were not unequivocal since by other authors contradictory results were obtained, especially when the investigations were car- ried out on intact living animals feeding iron by stomach tube or in man measuring the oral uptake of radioactively labeled iron [1,7, 8, 10, 11, 15, 16].

Therefore, the investigation of the dose-dependent absorption of iron was repeated using two methods:

a) Tied-off duodenal or jejunal segments of rats of definite length were pre- pared and filled with increasing doses of ~gFe-labeled ionized iron. Ten minutes after the administration of the doses the tied-off segments were removed and the uptake of 59Fe into the organism was measured in a whole body counter for small

animals.

b) 59Fe-labeled iron was administered orally by stomach tube. On the 6th day after the administration the retention of awe in the living animals was meas- ured in a whole body counter for small animals.

The aim of these experiments was to characterize the methodological differ- ences of these two types of investigations of the intestinal absorption of iron.

Material and Methods

Normal and iron-deficient female Wistar rats (body weight 180-220 g) were used. Iron-deficiency was produced according to the method of Forth and Andres [3]. The degree of iron-deficiency of the rats produced by feeding a low iron diet and by repeated bleeding from the tongue vein is characterized by the data summarized in Table 1. It must be pointed out that the hemoglobin content of the blood of the iron-deficient rats amounted only to 40 % of that of the normal animals indicating a severe anemia. According to the degree of iron-deficiency the iron content in plasma amounts to only 25 % of the normal values and the free iron binding capacity of the plasma increased up to 137% (see Table 1). The iron-deficient animals did not suffer from a protein deficiency despite of the repeated bleeding as can be taken by the values of the protein contents in plasma as well as in the intestinal tissue (Table 1). Protein deficiency was avoided by increasing the protein content of the diet [3].

Capacity of the Mucosal Transfer System of Iron 129

Table 1. Hemoglobin content in blood, iron and protein in plasma and in intestinal tissue of normal and iron-deficient rats

normal iron-deft- N method rats cient rats according to

(No. of reference)

iron in plasma #moles/l 27.5 -+ 2.3

free iron binding #moles/l 59.6 -+ 7.5 capacity of plasma

hemoglobin of blood g/1 144 + 12

protein in plasma g/l 76 -+ 3

iron content in tissue b duodenum nmoles/g wet weight 332 _+ 48 jejunum nmoles/g wet weight 183 _+ 20

protein content in tissue ̀~ duodenum mg/g wet weight 73 + 5 jejunum mg/g wet weight 49 _+ 3

7 ~ + 1.6 30 14

81.5 ~ • 6.3 20 14

61~ +_ 5 20 12

70 + 2 40 9

86 a _+ 48 5 14 98 a _+ 16 5 14

6 6 + 6 5 13 53 -+ 4 5 13

(2 +_ s2); ~ statistically different as compared with b after heart puncture N = number of animals in each group

normal rats, p < 0.01

1. Administration of Iron into Tied-off Segments After an overnight fast during which the animals had free access to water ionized iron 59Fe- (FeC13) was injected into the tied-off segments in physiological saline (pH 2). The length of the tied-off duodenal segments was 5 cm starting from 1 cm distal of the pylorus and that of the jejunal segments was 10 cm starting with the flexura duodenojunalis. The duodenal segments were filled with 0.5 ml and the jejunal segments with 1 ml of a solution containing the same concentra- tion of ionized iron so that per cm length of the intestinal segments the same dose (1, 10, 20, 100 nmoles Fe per cm) was administered. After an exposure period of 10 rain the animals were sacri- fled and the tied-off segments were removed. The 59Fe-activity in the carcasses was determined in a whole body counter for small animals (Packard, type Armac).

2. Administration of Iron by Gastric Tube The ionized ~gFe-labeled iron was administered orally in 2 ml physiological saline (pH 2) by a stomach tube. Six days later 59Fe was measured in a whole body counter for small animals (Packard, type Armac). Since iron, once incorporated, is excreted only poorly the retention allows satisfactorily to evaluate the absorption of iron [4].

The results obtained in these investigations were calculated as nmoles and #moles Fe respecti- vely per kg body weight as well as nmoles and #moles Fe per cm lenght of intestine.

Statistics: The student test (computerized program) was used.

R e s u l t s

1. Absorption of Increasing Doses o f Iron from Tied-off Intestinal Segments

T h e r e su l t s o f t he a b s o r p t i o n o f i r o n a f t e r t he a d m i n i s t r a t i o n i n t o d u o d e n a l a n d

j e j u n a l s e g m e n t s a re s u m m a r i z e d in Fig. 1. I r o n a b s o r p t i o n revea l s a s a t u r a t i o n

k ine t i c in e i t h e r d u o d e n a l a n d j e j u n a l s e g m e n t s : in d u o d e n a l s e g m e n t s t h e s a t u r a -

t i o n dose a m o u n t s to 0 . 5 / ~ m o l e F e p e r kg b o d y weigh t . F u r t h e r i n c r e a s i n g t he d o s e

130 G. Becker et al.

Table 2. Absorption of 59Fe labeled iron from tied-off duodenal and jejunal segments of normal and iron-deficient rats Calculation as nmoles Fe absorbed per cm length of intestinal segment; means of 8rats; :~ • s~. d/n: ratio iron absorbed in iron-deficient rats/iron absorbed in normal rats. On the base of these data the absorption of Fe was calculated per kg body weight (cf. Fig. 1)

intestinal dose iron absorption (nmoles Fe/cm) segment #moles Fe/kg nmoles Fe/cm normal rats iron-deficient rats d/n

duodenum 0.025 1 0.20 • 0.03 0.69 • 0.02 3.4 0.25 10 1.25 • 0.26 4.40 • 0.21 3.5 0.50 20 1.52 4- 0.32 6.26 4- 0.34 4.1 2.50 100 2.43 • 0.63 12.73 • 0.87 5.2 5.00 200 4.52 _+ 0.63 26.94 _+ 2.72 6.0

0.005 0.1 0.003 _+ 0.0006 0.019 • 0.001 6.3 0.050 1 0.016 • 0.005 0.245 _+ 0.027 15.4 0.50 10 0.074 • 0.018 1.05 • 0.15 14.2 1.00 20 0.116 4- 0.027 1.50 _+ 0.20 13 5.00 100 0.240 + 0.080 3.02 • 0.21 12.5

jejunum

1~ 1 g o

o ! . . ~ e

1( o

O

1 0 0 �84

8 1C

r

e-

.g

o

I duodenum e

dose, pmo|es Fe/kg

0.5 1 2.5 5

._.-----~o

O _._.__ @

~~//ere jejunum

/ �9 dose. jumoles Fe/kg

ols i 21s s

Fig. 1. Absorption of increasing doses of 5~Fe-labeled ionized iron from tied-off duodenal and jejunal segments of normal (0) and iron-deficient (O) rats Abscissa: iron dose, #moles Fe per kg body weight. Ordinate: iron absorbed, #moles Fe per kg body weight. Mean of 8 rats. For SEM see Table 2. The dif- ference between the normal and the iron-deficient group is significant for all points (p < 0.005)

beyond the saturat ion is followed only by a moderate increase of the amoun t of i ron absorbed. This holds true for both segments of normal as well as i ron defi- cient rats. Fol lowing the adminis t ra t ion of higher doses (duodenal segments >0.5 #mole Fe per kg; je junal segments > 1 #mole Fe per kg), the amoun t of i ron absorbed apparently increases linearly with the dose (see Fig. 1).

Capacity of the Mucosal Transfer System of Iron 131

100.

Fig. 2. 59Fe-retention in normal (O) and iron-deficient (O) rats fed ~gFe-labeled ionized iron 6 days before the measurement "~ of their 5gFe content Abscissa: iron dose, #moles Fe per kg body ~ 10 weight. Ordinate: iron absorbed,/zmoles Fe .g_ per kg body weight. Mean of 5-8 rats _+ ~ SEM. Above of iron doses of 100 #moles Fe per kg the normal and iron-deficient o group is statistically significantly different (p < 0.005) 1.

.---.--- o o

0 / 6 "0

o

o . dose,/urr~es F.e/kg

6o i~ "~o ~o ~o

2. Absorption after Administration of Increasing Doses by Gastric Tube

I n n o r m a l r a t s t he a m o u n t r e t a i n e d six days a f t e r t he a d m i n i s t r a t i o n i n c r e a s e s

f r o m 2.1 # m o l e s u p to 60 # m o l e s F e p e r k g (Fig. 2). A f u r t h e r i n c r e a s e o f t he d o s e

is n o t f o l l o w e d b y a s t a t i s t i ca l ly s ign i f i can t i n c r e a s e d i r o n r e t e n t i o n . I n i r o n defi-

c i en t a n i m a l s i r o n r e t e n t i o n i nc r ea se s u p to t he dose o f 3 6 0 / z m o l e s F e p e r kg.

B e y o n d th i s d o s e n o f u r t h e r i n c r e a s e o f t he r e t e n t i o n o f 59Fe c o u l d b e o b s e r v e d .

D i s c u s s i o n

T h e d o s e r a n g e s o f i r o n u s e d in p u b l i c a t i o n s d e a l i n g w i t h t he d o s e - d e p e n d e n t

a b s o r p t i o n o f i r o n in a n i m a l s a n d m a n as wel l as in t i ed -o f f i n t e s t i n a l s e g m e n t s o f

r a t s a re s u m m a r i z e d in T a b l e 3. T a b l e 3 does n o t c o n t a i n t he p a p e r s o f T h o m s o n

a n d V a l b e r g [17] a n d T h o m s o n e t al. [18] w h o i n v e s t i g a t e d t he d o s e - d e p e n d e n t

Table 3. Dose range of iron used in publications dealing with dependency of the absorption on the dose

oral administration species body weight ~ iron-dose moles/kg reference

man 70 kg 4.60 • 10 -7 - - 3 . 0 7 • 10 -5 man 70 kg 2.56 • 10 - l ~ 2.56 • 10 .5 rat 200g 4.48 • 10 -7 - - 8 . 9 5 • 10 .5 mouse 14 g 7.67 • 10 -7 - - 3 . 8 4 • 10 -8 man 70 kg 1.43 • 10 -7 - - 1 . 2 8 • 10 -8 rat 250 g 2.00 • 10-~ - - 2 . 0 0 • 10 -4 rat 200 g 2.10 • 10 -6 - - 5.70 • 10 -4

experiments on tied-off duodenal segments

n rat 330 g 2.71 • 10 -r - -2 .71 • 10 .6 n rat 200 g 2.50 • 10 -s - - 5 . 0 0 • 10 -6 d rat 330 g 2.71 • 10 -r - -2 .71 • 10 -5 d rat 200 g 2.50 • 10 -8 - - 5 . 0 0 • 10 -6

[10] Hahn et al., 1951 [16] Smith et al., 1958

[1] Bannerman et al., 1962 [8] Gitlin et al., 1962

[11] Heinrich, 1970 [15] Schade et al., 1970

this paper

[20] Wheby et al., 1964 this paper [20] Wheby et al., 1964 this paper

a weights have been estimated in part n : normal d: iron-deficient

132 G. Becker et al.

absorption of iron on tied-off and perfused duodenal segments of normal and iron deficient rats. Since the total volume of the perfusion fluid is unknown the total dose administered cannot be calculated. Due to the methodological difference of this type of experiments as compared to that of the other ones using the tech- nique of tied-off intestinal segments [20, this paper] these papers were not taken into account here. It must be added that in a concentration range of 0.1-5 • 10 .3 M the transfer of iron from mucosal cells into the organism showed a saturation type kinetic in both normal and iron deficient rats whereas the uptake by the tis- sue increased nearly linearly with the concentration of iron administered on the luminal side [17].

In tied-off intestinal segments the dose dependence of iron absorption shows a saturation characteristic. This holds true for both, uptake of iron into the mucosal epithelium as well as release of iron from the mucosal cells into the organism [20] and is continued by the presented data on duodenal and jejunal segments of both normal and iron deficient rats. However, there are some interest- ing differences between duodenal and jejunal segments:

a) the amount absorbed from duodenal segments both from normal and iron deficient rats is higher than from jejunal segments (see Table 2). This emphasizes the significance of the duodenum for the absorption of iron especially in animals with a balanced iron metabolism.

b) In iron deficiency the body's need is covered by an increased absorption of iron [for lit. see 6]. Furthermore, the area of the mucosal epithelium which is involved in the absorption process is extended into the distal part of the small intestine [2, 5,19].

The results obtained after the administration by stomach tube confirmed the findings of other groups according to which the dose dependent absorption curve of iron can be described by a log-linear or log-log-relationship [8,11, 16]. There are only a few exceptions in literature:

a) Schade et al. [15] obtained a saturation curve for absorption after feeding iron by an "intragastric needle" in rats. However, the saturation type curve of absorption in this paper depends mainly on one of the five points of the investiga- tion of the absorption of increasing iron doses from 5/~moles to 50 #moles Fe per rat. 8-10 animals per dose were used.

b) Hahn et al. [10] showed in pregnant women depending on the stage of pregnancy a saturation type kinetic of the "uptake" of iron (< 15 weeks of gesta- tion) or a log-linear relationship of the absorption curve (>_25 weeks of gestation). He did not interpret this very interesting difference of the absorption curve except of the assumption of a "mucosal block" for iron absorption indicated by the decreased percentage absorption with increasing doses. When interpreting the saturation type absorption curve in which the saturable term must be attributed to the transport system and the more or less linear term to simple diffusion [15] this would mean that diffusion plays a role for absorption only pregnant women beyond the 25th week of gestation but not for those up the 15 weeks of gestation. Apparently this interpretation makes no sense. It is most difficult to accept that these data represent iron absorption. Hahn et al. [10] themselves pointed out that "uptake" of radioactively labeled iron into the body measured by the radio-

Capacity of the Mucosal Transfer System of Iron 133

activity of blood samples reflects something in between of absorption and utiliza- tion rather than absorption from the G.I. tract.

c) Gitlin and Cruchaud [8] showed with mice that the absorption kinetic of iron can be divided into one process suggesting an enzymatic or carrier reaction being involved and a second one with first order kinetic which is only limited by the availability of iron ions for absorption. These experiments were carried out mainly with normal mice and in part on mice suffering from iron deficiency whose degree is unknown.

There is apparently a discrepancy between the results of the dose-dependent iron absorption obtained on tied-off intestinal segments in situ and on intact living animals. This discrepancy can be understood plausibly taking into account the different experimental conditions. In tied-off segments of definite length the entire dose administered is exposed to the absorbing surface of the mucosal epithelium for a definite time. After administration by stomach tube, however, the total dose does not reach simultaneously the absorbing epithelium because the gastric fluid is emptied stepwise (bolus) into the duodenum.

In contrast to tied-off segments in intact animals the intestinal passage is free and the iron dose is moving along the intestinal tract passing the duodenum, the jejunum and reaching finally the ileum. In contrast to this situation in tied-off intestinal segments the iron load is exposed to one and the same during the entire experimental time. This methodological difference has a consequence, namely, the movement of the non-absorbed parts of the iron dose in intact animals to distally located sections of the intestinal tract. Especially in iron deficiency, the capacity of the transfer system of these segments is remarkably increased [2, 5, 19] so that it is much more difficult to produce a saturation of the uptake of iron into the animals as is possible with tied-off intestinal segments. As to what extent the absorbing surface for absorption of iron by the transfer system is enlarged from proximal to distal parts of the small intestine depends on the degree of iron defi- ciency. This may offer an explanation for the different data in literature concern- ing extent and kinetic of absorption of iron after a single oral iron dose.

Finally, after having fed iron orally i t 'must be taken into account that the exposure time of iron in the gastrointestinal tract is lasting up to 24 h and more since the retention of 591=e is measured not before 6 days following to the admini- stration.

In normal animals in the distal parts of the gastrointestinal tract most probably iron is not absorbed by aid of the transfer system which is located proximally in the duodenum and upper jejunum. How much iron can be absorbed from lower parts of the intestine of iron deficient individuals may depend on the mucosal capacity, the passage time of the ingesta in these sections of the gastrointestinal tract and on the availability of iron for absorption.

The absorption of iron administered as a single oral dose - as the absorption of other substances - apparently depends on (a) the gastric emptying time, (b) the absorbing surface of the mucosa containing the transfer system for iron and (c) the passage time of ingesta along the gastrointestinal tract. These factors can be considered as the reason that a saturation type kinetic for absorption cannot be expected under all experimental conditions despite of the existence of a transfer system with limited capacity for iron in the absorbing mucosal epithelium.

134 G. Becker et al.

Acknowledgement. The experiments on tied-off intestinal segments were carried out in the Labora- torium of Prof. W. Rummel, Homburg/Saar whose many advices and useful discussions dur ing the preparation of this manuscript are gratefully acknowledged.

References

1. Bannermann, M.R., O'Brien, J.P.R., Witts, J.L.: Studies in Iron Metabolism IV. Iron Absorption in Experimental Deficiency. Blood 20, 532-546 (1962)

2. Chirasiri, L., Izak, G.: The Effect of Acute Haemorrhage and Haemolysis on Intestinal Iron Absorption in the Rat. Br. J. Haematol. 12, 611-622 (1966)

3. Forth, W., Andres, H. : Methode zur Erzeugung eines Eisenmangels bei Ratten. Arzneim. Forsch. (Drug Res.) 19, 363-364 (1969)

4. Forth, W., Pfleger, K., Rummel, W., Seifen, E., Richmond, S. J. : Der EinfluB verschiedener Liganden auf Resorption, Verteilung und Ausscheidung yon Eisen nach oraler Verabfolgung. Arch. exptl. Pathol. Pharmakol. 252, 242-257 (1965)

5. Forth, W., Rummel, W. : Zur Frage der Regulation der Eisenresorption durch Gastroferrin, ein eisenbindendes Protein des Magensaftes. Klin. Wochenschr. 46, 1003-1005 (1968)

6. Forth, W., Rummel, W. : Iron Absorption. Physiol. Rev. 53, 724-792 (1973) 7. Gabbe, E. E. : Quantitative Aspects of Iron Deficiency and Iron Therapy. In: Iron Metabolism

and its Disorders. Kiefer, H. (ed.), p. 278-297. Amsterdam, Oxford: Excerpta Medica. New York: American Elsev. Publ. Comp. 1975

8. Gitlin, D., Cruchaud, A.: On the Kinetics of Iron Absorption in Mice. J. Clin. Invest. 41, 344-350 (1962)

9. Gornall, G.A., Bardawill, Ch. J., David, M.M.: Determination of serumproteins by means of the Biuret Reaction. J. Biol. Chem. 177, 751-766 (1949)

10. Hahn, P.F., Carothers, E.L., Darby, W.J., Marin, M., Sheppard, C.W., Cannon, R.O., Beam, A.S., Densen, P.M., Petterson, J.C., McClellan, C.S. : Iron Metabolism in Human Pregnancy as Studied with the Radioactive Isotope 59Fe. Am. J. Obstet. Gynecol. 61,477-486 (1951)

11. Heinrich, H. C. : Intestinal Iron Absorption in Man - Methods of Measurement, Dose Rela- tionship, Diagnostic and Therapeutic Applications. In: Iron Deficiency. Hallberg, L., Har- werth, H.-G., Vannotti, A. (eds.), p. 213-294. London, New York: Academic Press 1970

12. Kleihauer, E., Betke, K. : Zur Hfimoglobinbestimmung mittels Cyanh~miglobin. ~rztl. Lab. 3, 202-205 (1957)

13. Lowry, O.H., Rosebrough, N. J., Farr, A.L., Randall, R. J.: Protein measurement with the Folin Phenol Reagent. J. Biol. Chem. 193, 265-275 (1951)

14. Schade, A. L., Oyama, J., Reinhatz, R.W., Miller, J.R.: Bound Iron and Unsaturated Binding Capacity of Serum. Proc. Soc. Exp. Biol. Med. 87, 443~148 (1954)

15. Schade, St.G., Felsher, B.F., Glader, B.E., Conrad, M.E.: Effect of Cobalt upon Iron Absorption. Proc. Soc. Exp. Biol. Med. 134, 741-743 (1970)

16. Smith, M.D., Pannacciulli, I .M.: Absorption of Inorganic Iron from Graded Doses: Its Significance in Relation to Iron Absorption Tests and the Mucosal Block Theory. Br. J. Haematol. 4, 428-434 (1958)

17. Thomson, A.B.R., Valberg, L.S. : Kinetics of intestinal iron absorption in the rat: effect of cobalt. Am. J. Physiol. 220, 1080-1085 (1971)

18. Thomson, A. B. R., Valberg, L.S., Sinclair, D. G. : Competitive Nature of the Intestinal Trans- port Mechanism for Cobalt and Iron in the Rat. J. Clin. Invest. 50, 2384-2394 (1971)

19. Wack, J.P., Wyatt, J.P.: Studies on Ferrodynamics I. Gastrointestinal Absorption of ~gFe in the Rat under Differing Dietary States. Pathology 67, 237-247 (1959)

20. Wheby, M.S., Jones, L. G., Crosby, W. A. : Studies on Iron Absorption. Intestinal Regulatory Mechanisms. J. Clin. Invest. 43, 1433-1442 (1964)

Received May 2, 1978 / Accepted September 13, 1978