industry comments on epa opp proposed nanopesticide policy

Intestinal Mucosal Mechanisms Controlling fron Absorption

By MARCEL E. CONRAD, JR. AND WILLIAM H. CROSBY

With the technical assistance of Betty Merrill

T HE QUANTITY OF IRON normally present in the human body is not

great, amounting to about 4 Gm.,2 and it remains constant during life,

reflecting a delicate balance between iron absorbed and lost. Persistent

errors in either direction result in iron deficiency or hemosiderosis.3 It has

been postulated that the iron balance is maintained by controlling iron ab-

sorption.2-4 However, the importance of the intestinal mucosa and its method

of regulating the iron balance have remained unclear.

The present study concerns the mucosa and the mechanics of control of

iron absorption.

METHODS AND MATERIALS

Retention of radioactive iron was measured in humans using iron59 and a whole-body

liquid scintillation counter. The methods and results of absorption studies in 25 normal

and 10 iron-deficient adults have been reported.5 The dose consisted of usc. ferrous59

citrate in 1 mg. of FeSO4 carrier given by mouth to fasting subjects.

Canadian Hooded rats weighing 250-300 Cm. were used in this study. The principles

of laboratory animal care as promulgated by the National Society for Medical Research

were observed. Oral doses of ferrous59 citrate were administered after a 16-hour fast

using a 17-gauge olive-tipped endoesophageal needle. Each animal received 100 � of

iron�9 with 0.25 mg. of carrier iron as ferrous sulfate.

Parenteral doses of rat plasma tagged with 1 mc. d)f ferrous59 citrate (specific actviity:

4.5x 105 mc. per Cmli. of iron) were injected through a polyethylene catheter inserted into

tile juglar vein of albino rats. Each millicurie of iron59 was incubated with 1 ml. of normal

rat plasma for 30 minutes before dosing. The amount of iron59 addedi to the plasma was

insufficient to saturate the iron-l)mding globulin. Rats were killed at predetermined times

after the test dose. The small intestine was removed, opened and washed in 0.9 per cent

saline solution. The tissue was fixed in methanol and! chloroform and embedded in paraffin.

Sections were placed on Lantern Slide Plates or covered with a thick photographic emulsion

(Kodak NTB-3) and were developed after 15-30 days exposure. The tissue was stained

with hematoxylin and eosin. Rats were depleted of iron by weekly blood-letting of 4 ml.Bleeding was accomplished by insertion of a heparinized capillary tube into the medialvenous plexus of the orbit. Rats were iron-loaded by three weekly intramuscular injec-

tions of 25 mg. of iron as dextran-iron.

RESULTS

Gastrointestinal Absorption of iron in Humans (Figs. 1 and 2)

Iron-deficient human subjects absorbed more than 29 per cent of the oral

test dose whereas normal volunteers retained less than 10 per cent of the

radio-iron. Each subject had gradual loss of radioactivity in feces until

From the Departments of Gastroenterology and Hematology, Walter Reed Army Institute

of Research, Walter Reed Army Medical Center, Washington, D. C.

This material was presented in part at time Vilith International Congress of the Interna-

tional Society of Hematology, Tokyo, September 196O.�

Submitted Feb. 12, 1963; accepted for publication Apr. 3, 1963.

406

BLOOD, VOL. 22, No. 4 (OCTOBER), 1963

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INTESTINAL MUCOSAL MECHANISMS 407

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Fig. 1.-Fecal loss of iron59 by a normal iron-replete human subject (A) and anormal human with phlebotomy-induced iron deficiency (B). Both subjects and theiraccumulated feces were counted in a whole-body liquid scintillation counter followingan orally administered dose of iron59. The solid lines represent the amount of radio-activity retained by each subject. The descending limb of the curve reflects loss ofiron59 in the feces, and the final plateau represents the percentage of iron which wasabsorbed. The iron-deficient subject reached this plateau at day 3 to 6, the ironreplete subject at day 10 to 13. In a study of 10 iron-deficient and 25 normal humansthis difference in time required to reach the plateau was observed consistently.5

relative equilibrium occurred. This was achieved in iron-depleted humans

in 3 to 7 days, but in the normal volunteers it was achieved in 6 to 15 (lays

after the test dose. Carmine red dye was administered orally to four normal

and three iron-deficient subjects 4 hours after the test dose of iron. The dye

marker appeared in the feces within 48 hours after ingestion and was gone

by the 5th day in both iron-replete and iron-deficient subjects.

Comment: The availability of a whole-body counter permitted the direct

assay of iron59 in human subjects and provided reliable estimates of the

amount of absorption.5’6 Establishment of a standard test situation provided

a method of differentiating normal and iron-deficient subjects. Iron-depleted

volunteers absorbed increased quantities of the test dose and achieved

equilibrium more rapidly than normal subjects. Prompt loss of carmine red

dye in the stool of both groups demonstrated that the delay in achieving

equilibrium was probably not related to intestinal motility. It is postulated

that delayed loss of iron from normal subjects is due to retention of iron

within the intestinal epithelial cells which are subsequently lost into the

intestinal lumen as they are normally desquamated. Contrariwise, iron-de-

pleted volunteers absorb the iron59 and do not retain it in the intestinal




IRON DEFICIENT

408 CONRAD AND CROSBY

- NORMA�

i1i�>i

Fig. 2.-Gastrointestinal sequestration of iron. Epithelium of the small intestine

is formed in the cr\’pt andi migrates during its life c�scle to the tip of the villus where

it is lost into gut lumen. After an orally administered dose of iron59 a delay in achiev-

ing iron equilibrium (fig. 1) would be observed if a portion of the iron was held in

epithelium and was lost in stool when the irnicosal cell was sloughed. If little iron

was retained in epithelial cells, as postulated in iron deficiency, there would beno delay in achieving iron equilibrium.

mucosa. They, therefore, achieve equilibrium during the time required for

intestinal passage of the unabsorhed Portion of the test dose.

Radioautography of Rat Gut Using iron� (Figs. 3 and 4)

Fasting adult rats were given a large dose of radioactive iron59 through an

endosophageal tube. Normal, iron-depleted and iron-loaded animals were

killed 2 to 48 hours after administration of an oral test dose of iron59. Radio-

autographs of sections from duodenum and jejunum of normal rats showed

radioactivity in the columnar epithelium. Two to 8 hours following the test

dose most of the epithelium covering the villi was radioactive. Gut ob-

tamed 12 to 24 hours after the test dose showed tagging of the cells on the

distal half of the villus. In specimens taken at 36 to 40 hours, only the cells

at the tips of the villi were radioactive. At 48 hours there was radioactivity in

some of the goblet cells hut not in the columnar epithelium. The sections

from iron-depleted and iron-loaded rats showed little radioactive iron con-

centrated within epithelial cells at any time.

Normal and iron-loaded rats were killed 16 hours after receiving an

intravenous injection of iron59. Radioaimtographs of sections from duodenum

and jejunum of these animals showed radioactivity concentrated in columnar

epithelium. The cells in the proximal half of the villi contained more radio-

activity than the older cells near the villous tips. In iron-deficient rats similarly

treated there was less contrast between the radioactivity in epithelial cells and

the radioactivity in the tunica propria, indicating that the amount of radio-

active iron in mucosal cells was small.




rat in a normal state of iron repll )wing riinistered ii

Upper left, animal killed 6 hours after dosing; upper right, 20 hours; lower left, 40hours. These illustrate the progressive loss of radioactive cells replaced by new cells

advancing from the crvpts. Radioactivity becomes fixed in the epithelial cells and islost into the intestinal lumen when the cells are sloughed at the end of their life

cycle. Lower right, at 48 hours; the columnar epithelium contains no iron59 at any

level. Occasional cells, which may be goblet cells or iron-laden macrophages, ex-hibit some residual radioactivity. Similar preparations from iron-deficient and iron-loaded rats showed little or no contrast between the radioactivity in mucosalepithelial cells and that in the tunica propria and other underlying tissues.





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Fig. 4.-Radioautography of mucosa from the rat duodenojejunal junction 16

hours after an intravenous dose of iron59 showed radioactivity in villous epithelium.A. (;reater tagging of the proximal portion of the villus suggests that available bodyiron is incorporated in newly formed cells. B. Note at the base of the villi the clarkareas representing accumulation of radioiron in the crypts of Lieberkiihn. The cells

at the villous tips had been formed before the iron�9 was given; presumably their

iron receptor system had been satisfied with cold iron. With iron-deficient rats, sim-

ilar preparations showed little or no contrast between radioacti�’it� in the mucosal

epithelial cells when compared to underhing intestinal tissue. With iron-loaded rats,

the preparation resembled those obtained from normal rats as illustrated.




Unoccepted Fe

IRON DEFICIENT


j��ujujjn

Fe Accepted by cell

Fe lost with sloughed cell

NORMAL

IRON LOADED

Fig. 5.-A concept of control of iron absorption by the intestinal mucosa. It is predi-cated that iron absorption is regulated primarily through the columnar epithelium of

the small intestine. In normal, iron-replete subjects the mucosal cells may contain

a variable amount of iron supplied! from the body store. This deposit regulates-

within limits-the quantity of iron which can enter the cell from the gut lumen.After the iron has entered the cell it may proceed into the body to fulfill a require-

ment. Alternatively a portion of the iron may become fixed in the epithelial cyto-

plasm to be lost when the cell is sloughed at the end of its life span. In iron-deficient

subjects there appears to l)e little or no mechanism to inhibit entrance of iron into

the villous epithelial cells or to retain it. Thus dlietarv iron readily proceeds into

the body. In iron-loaded subjects the body iron which is incorporated in the epithe-Hal cells is eventually lost but during the life span of the cells its presence inhibits

the entrance of iron into the cells.

Comment: The large quantity of gamma-emitting radionuclide required

for adequate radioautography prohibits this study- in humans.7 Rats were se-

lected! because the rate of turnover of small intestinal epithelium had been

studied, and it had been shown that dunng their life cycle the columnar

epithelial cells migrate in an orderly fashion from crypts to the tips of villi.8





Radioautography with tritiated thymidine showed the life cycle of these

cells in the rat is about 46 hours.9 The present study in normal iron-replete

rats confirms these observations. Furthermore it supports the hypothesis that

unneeded iron is accepted by the villous epithelium and is retained by these

cells until they are shed by the villus at the end of their life cycle. This

temporary sequestration of iron5#{176}may account for its delayed loss in the

feces. In iron-deficient and iron-loaded animals the iron evidently was either

absorbed and rapidly released to the plasma or it was rejected by the in-

testinal mucosal cells.

The concentration of parenterally administered iron in intestinal epithelium

of normal and iron-loaded animals indicates the existence of a mechanism

whereby a gradient may he established to limit absorption of iron from

the lumen. Further, it provides a mechanism for loss of body iron when these

cells are shed at the end of their lifespan.

DISCUSSION

In 1943 Hahn demonstrated that iron-deficient humans and (logs absorbed

more radioactive iron than did their normal counterparts.10 Thus he demon-

strated that when the iron is not needed it is not absorbed. Hahn postulated

that this “mucosa block” might involve a physiologic receptor, such as the

Protein apoferritin; when the receptor l)eCame filled with absorbed iron, nomore could he accepted by the mucosa until the ferritin had discharged its

iron into the body. Granick’s experiment, which involved the feeding of a

large dose of iron, demonstrated that ferritin could he formed in the guinea

rig’s small intestine, but it did not clarify the way in which the intestinemay accept or reject the small amounts of iron in the diet.11’12

The present study may help to clarify the mechanism whereby unneeded

iron is rejected by the intestinal mucosa. It is concerned with three states of

total body-iron: iron deficiency, iron excess and the normal state of iron

repletion. In iron deficiency the intestine responds by accepting much of an

ingested dose of iron. In iron excess only a trace is absorbed. In the iron-

replete animal enough is accepted to replace the normal, obligated loss.5’0’”’15

The experiments in the human whole-body counter suggested that the col-

umnar epithelial cells might somehow participate in the intestinal control of

iron absorption.5 Iron-deficient subjects lost in their feces portions of the

oral dose of iron59 for 3 to 6 days following its administration. They absorbed

what they needed or what they could get and the rest was promptly defecated.

On the other hand, 6 to 15 days after dosing. the normal iron-replete sub-

jects were still defecating portions of the iron59. Some of the iron had been

sequestered for a time. Location of the area of sequestration was sought.

Experiments in dogs indicate that iron is not absorbed and re-excreted in

bile.13

The present hypothesis places the sequestered iron in the columnar epithe-

hal cells of the small intestine and proposes that the sequestration is involved

with the mechanism of mucosal block (fig. 5). These epithelial cells, moving

along the villus, from crypt to tip, have a lifespan of several days, 2 for





example in rats.9 They are the cells through which dietary iron passes into

the body. Radioautographs of absorptive mucosa in animals killed 6 hours

after an oral dose of iron59 reveal little or no selective concentration of iron

in the case of iron deficiency or iron excess, but the villous epithelium of the

normal animal contained an obvious amount of radioactivity. Evidently these

epithelial cells did not relinquish this iron for it was still present in the radio-

autographs of animals killed 40 hours after dosing.

Within the framework of this hypothesis it is possible to explain the

radioautographic findings. The mucosa of the normal, iron-replete animal re-

jects most of the dietary iron, but accepts some which is needed and some

which is not. The unneeded radioactive iron remains in the epithelial cells,

thereby tagging them. The tagged cells are gradually pushed to the tip of

the villus-and off-by the advancement of newly fonned, untagged cells.

The ability to reject dietary iron, according to Hahn, was a result of satura-

tion of a physiologic receptor such as apoferritin.’#{176} According to the present

hypothesis this receptor is in the columnar epithelial cells. In the normal

animal it is partially saturated by intrinsic iron from the animal’s body, the

degree of saturation reflecting the requirement for iron. The radioautographs

of normal animals given parenteral iron59 demonstrate that intrinsic iron is

incorporated into the villous cells even as they are being formed in the

crypts of Lieberkuhn. The mucosa of the iron-loaded rat blocks almost com-

pletely the incursion of dietary iron into the epithelial cells. It is suggested

that the physiologic receptor is completely saturated with intrinsic iron.

The mucosa of the iron-deficient rat accepts much of the radioiron and be-

cause of a great requirement passes almost all of it into the body; the villous

epithelium remains untagged. These cells may lack the apparatus for re-

jecting and retaining appreciable quantities of iron.

Iron sequestered in the columnar epithelial cells evidently serves not as

an intermediary stage in the absorptive process, but as a means to frustrate

the absorption of iron when it is not needed.

SUMMARY

Radioautographic studies provide evidence to support a concept of the

mechanism whereby the small intestine controls absorption of iron. Three

different states of the body’s iron stores have been considered in this regard:

iron excess, iron deficiency and normal iron repletion. As the columnar

epithelial cells of the duodenal villi are formed they incorporate a portion of

intrinsic iron from the body’s iron store, the amount depending upon the

body’s requirement for new iron. It is predicated that with iron excess the

iron-receptor mechanism in these cells is saturated with intrinsic iron; this

then prevents the cell from accepting dietary iron. In the normal state of

iron repletion the receptor mechanism remains partly unsaturated, allowing

small amounts of dietary iron to enter the cell. Part of this proceeds into the

body to satisfy any metabolic requirement for iron. Part is retained in the

mucosal epithelial cells to complete the saturation of the iron-receptor

mechanism. This bound iron is subsequently lost when the epithelial cells





are sloughed at the end of their life cycle. In iron deficiency it is postulated

that the receptor system is inactive or diminished so that entry of dietary iron

into the body is relatively uninhibited.

SUMMARIO IN INTERLINGUA

Studios radioautographic pro�’icle evidentia in supporto del these de un

rob del intestino tenue in be mechanismo del regulation del absorption de

ferro. Tres differente statos del reservas de ferro del corpore ha essite con-

siderate in iste reguardo: Excesso de ferro, carentia de ferro, e normal reple-

tion die ferro. In tanto que be cellulas columno-epithelial del vilbos duodenal

es formate, ilbos incorpora un portion del ferro intrinsec ab le reservas de

ferro del corpore, in quantitates dependente del requirimento del corpore pro

iiove ferro. Es postulate que in Ic presentia de un excesso de ferro, be mech-

anismo pro be reception de ferro in be mentionate cellulas es saturate con ferro

intrinsec, e isto alora preveni le acceptation de ferro dietari per le ceblulas. In

le stato normal de repletion de ferro le mechanismo de r#{128}�eption remane

partiabmente non-saturate, e isto permitte le entrata de micre quantitates de

ferro dietari ad in Ic cellula. Un parte de iste ferro procede ad in le corpore

pro satisfacer le possil)ilemente existente requirimento pro ferro. Un parte del

ferro, del altere latere, es retenite in le cellulas mucoso-epitheliab, resultante

in le saturation del mechanismo de reception de ferro. Iste ligate ferro es

perdite subsequentemente quando Ic cellulas epithelial es desquamate al fin

de br cyclo vital. In carent-ia de ferro, ii es postulate, Ic systema de reception

de ferro es inactive o reducite, de maniera que le entrata de ferro dietari ad in

Ic corpore es relativemente libere de inhibition.

REFERENCES

1. Conrad, M. E., Jr., van Hoek, R., and

Crosby, W. H.: Observations on the

natural history of iron deficiency and

the gastrointestinal absorption of iron.

In Proc. Vilith International Congress

of Hematology, Tokyo, Sept., 1980.

Pan Pacific Press, 1962, p. 1151.

2. Cranick, S.: Iron metabolism. Bull. New

York Acad. Med. 30:81, 1954.

3. -: Iron metabolism and hemochroma-

tosis. Bull. New York Acad. Med.

2:403, 1949.

4. NicCance, R. A., and Widdowson, E.

M.: The absorption and excretion of

iron following oral and intravenous

administration. J. Physiol. 94:148,

1938.

5. van Hoek, R., and Conrad, M. E.: Iron

absorption: Measurement of ingested

iron59 by a human whole-body iiquidscintillation counter. J. Clin. Invest.

40:1153, 1961.

6. Anderson, E. C.: Scintillation counters:

The Los Alamos human counter. Brit.

J. Radiol. Suppl. 7, 27, 1957.

7. Morgan, K. Z.: Maximum permissible

internal dose of radionuclides Nuc.

Sci. Eng. 1:477, 1956.

8. Hughes, W. L., Bond, U. P., Brecher,

C., Cronkite, E. P., Painter, R. B.,

Q uastler, H., and Sherman, F. G.:

Cellular proliferation in the mouse asrevealed by radioautography with

tritiated thymidine. Proc. Nat. Acad.

Sci. 44:476, 1958.

9. Qnastler, H.: Some aspects of cell popu-

lation kinetics: In The Kinetics of

Cellular Proliferation. F. R. Stohiman,

Jr., ed. New York, Grune & Stratton,

1959, p. 218.10. Hahn, P. F., Bale, W. F., Ross, J. F.,

Balfour, W. M., and Whipple, C. H.:

Radioactive iron absorption by the

gastrointestinal tract. Influence of





anemia, anoxia and antecedent feed-

ing. Distribution in growing dogs.

J. Exper. Med. 78:169, 1943.

11. Cranick, S.: Ferritin IX. Increase of

the protein apoferritin in the gastro-

intestinal mucosa as a direct response

to iron feeding. The function of fer-

ritin in the regulation of iron absorp-

tion. J. Biol. Chem. 164:737, 1946.

12. Brown, E. B., Dubach, R., and Moore,C. F.: Studies in iron transportation

and metabolism. XI. Critical analysis

of mucosal block by large closes of

inorganic iron in human subjects. J.

Lab. & Clin. Med. 52:335, 1958.

13. Wheby, M. S., Conrad, M. E., Heclberg,

S. E., and Crosby, W. H.: The roleof bile in the control of iron absorp-

lion. Castroenterology 42:319, 1962.

14. Forrester, R. H., Conrad, M. E., and

Crosby, \V. H.: Measurement of total

bodly iron59 in animals using whole-

body liquid scintillation detectors.

Proc. Soc. Exper. Biol. & Mccl. 111:

115, 1962.

15. Pirzio-Biroli, C., and Finch, C. A.: Iron

absorption. III. The influence of iron

stores on iron absorption in the nor-

mal subject. J. Lab. & Clin. Med.

55:216, 1960.

Marcel E. Conrad, Jr., Ala/or, MC, Department of Hematology,

Walter Recd Army Institute of Research, W7ashington D. C.

William H. Crosby, Colonel, MC, Director, Dit�ision of Medi-

cine, and Chief, Department of Hematology� Walter Reed

Army institute of Research, Washington, D. C.




1963 22: 406-415

MARCEL E. CONRAD, JR., Major, WILLIAM H. CROSBY, Colonel and Betty Merrill Intestinal Mucosal Mechanisms Controlling Iron Absorption

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