ABSORPTION AND EXCRETION OF IRON*
By ALEXANDER G. LITTLE, JR., M.D., MARSCHELLE H. POWER, P H . D . , and
E. G. WAKEFIELD, M.D., Rochester, Minnesota
CONSIDERING the importance of iron in the body and the extensive work
that has been done on this subject, still relatively little is known concerning
metabolism of iron. During most of the last century many extensive arguments were carried on as to whether or not iron was absorbed at all. The
misconception that it was not absorbed arose from the fact that the small
amounts of iron in the body could not be detected by chemical methods
available at the time. As late as 1893, Stockman l felt it necessary to refute
the theory, which was apparently current then, that the action of iron in
chlorosis arose from the precipitation of poisonous hydrogen sulfide in the
intestine. This he did satisfactorily by successfully treating patients who
had chlorosis with subcutaneous injections of citrate of iron. During the
first part of this century, mainly under the influence of the histologic school,
a theory of metabolism of iron was evolved which has more or less continued
to be in vogue. The most widely accepted current theory has been succinctly stated in Starling's "Human Physiology" 2 : "The absorption of iron
takes place in the duodenum and upper part of the jejunum. Only 1 or 2 mg.
appear in the urine, all the rest being excreted in the large gut and appearing
in the feces, chiefly as sulfide of iron." In other words, this view assumes
that iron is absorbed from the upper part of the intestine regardless of stores
in the body. The excess is then secreted by a specific action of the colonic
mucosa.
Before the present work was undertaken, reports of the previous investigations were thoroughly surveyed and references to them are given in
the thesis which forms the basis of this paper. The main purpose here,
however, is to report any contribution we ourselves may be able to make on
the subject.
RATIONALE AND GENERAL METHOD OF PRESENT WORK
The work reviewed seemed to show fairly definitely that iron is not
excreted by the dog. However, investigations based on human subjects are
open to criticism from three points. First, in experiments that necessitate
administration of iron by mouth, the difficulty is encountered of determining
•Received for publication December 29, 1944.
Abridgment of thesis submitted by Dr. Little to the Faculty of the Graduate School of
the University of Minnesota in partial fulfillment of the requirements for the degree of M.S.
in Surgery.
From the Mayo Foundation (Fellow in Surgery, Dr. Little) (Division of Biochemistry,
Dr. Power) and Mayo Clinic (Division of Medicine, Dr. Wakefield). Since this paper was
written Dr. Little has entered the army and is now an officer of the Medical Corps, Army of
the United States.
627
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A. G. LITTLE, JR., M. H. POWER, AND E. G. WAKEFIELD
many grams of iron in a large volume of stool with accuracy within a few
milligrams. Second, after administration of iron by mouth two to four
weeks are required for the intestine completely to eliminate the dose, as most
of it is precipitated as iron sulfide, which tends to adhere to the mucosa and
is passed slowly. Third, with the administration of iron by mouth, the
additional variable factor of absorption, which is impossible of determination
and control, must be considered.
In the present work we have tried to circumvent these difficulties by intravenous administration of the iron. In this way it is possible to be sure of the
amount of iron that reaches the interior of the body and large quantities do
not have to be determined in the stools over long periods of time. In general,
the iron in the feces was determined during three successive periods. The
first and third periods were used as controls and, during the second period,
iron was injected intravenously. This sequence does not apply to the study
reported as third in this paper and summarized in table 3, which will be
referred to again later.
DETAILS OF PROCEDURE
The metabolic processes of the three subjects used apparently were satisfactory. They had been in an institution for several months on an adequate
diet and could be assumed to have ingested adequate quantities of iron.
Their stools were repeatedly negative for blood by the benzidine test. During the entire time of the study they were kept in a hospital ward where they
could be observed. During the test periods they were placed on diets of
approximately constant daily content of iron, as calculated from accepted
analyses. Urine and stools were collected separately by means of a special,
nonferrous bifurcated commonde, stools were separated into periods by
means of carmine markers. The receptacles were two glass containers which
previously had been rinsed with diluted hydrochloric acid. Collections of
blood were made from veins of the arm, with a stainless steel 18 gauge
needle. To prevent hemolysis, a tourniquet or a syringe was not used. The
first 10 c.c. of blood were allowed to flow into a tube that contained heparin
and were used to ascertain the value for hemoglobin, the number of erythrocytes per cubic millimeter and hematocrit values. The next 10 c.c. were
collected under oil for determinations of serum iron.
The iron was administered as ferrous ascorbate. This compound was
first described by Szent-Gyorgyi3 and has been used fairly extensively by
Heilmeyer,4 by Fleischhacker and Schurer-Waldheim 5 and by Friend.6 This
compound was chosen because it can be safely administered in sufficient
quantities. Our supply of ferrous ascorbate was prepared by us after the
method of Maurer and Schiedt.7 Each preparation was analyzed for content
of iron and was kept in a desiccator. At the time of use a quantity was
weighed which would contain the desired amount of iron. This was dissolved in 200 to 300 c.c. of physiologic saline solution and was immediately
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ABSORPTION AND EXCRETION OF IRON
629
injected. The injections contained 30 to 40 mg. of iron each and were given
in a period of 45 to 75 minutes. Faster administration caused mild symptoms, characterized by warmth andflushingof the face. There were no late
symptoms and only one episode of thrombosis. The thrombosis followed
several injections into the same vein.
ANALYTIC PROCEDURE
Determination of iron in blood serum was accomplished by a modification
of the procedure described by Moore.8 The modified procedure was as
follows:
Ashing. Five (or 10) c.c. of serum were measured into a 100 c.c.
Kjeldahl flask, followed by 5 (or 10) c.c. of concentrated nitric acid and
two glass beads. The mixture was heated by means of a microburner over
wire gauze, gently atfirst,then somewhat more vigorously so that the volume
was reduced to 2 to 3 c.c. during about 30 minutes. The flask was allowed
to cool for one to two minutes; then 2 c.c. of 70 per cent perchloric acid were
added, and the heating over wire gauze was continued with the flame adjusted
so that the nitric acid remaining in the digest was distilled off in about 15
to 20 minutes. With the appearance of the white fumes of perchloric acid,
which indicated that the major part of organic matter had been destroyed,
the heat was increased and the solution was heated vigorously over the free
flame for 30 to 40 minutes longer to remove all traces of yellow or green
color. The digest was then allowed to cool, the sides of theflaskwere rinsed
down with 3 to 4 c.c. of iron-free distilled water, and the mixture was boiled
until the added water was distilled off; then the mixture was heated more
vigorously for another 15 to 20 minutes. The digest was again cooled, 10
c.c. of distilled water were added, and the mixture was boiled for one to two
minutes. The flask was then removed from the flame, a drop of concentrated nitric acid was added to the hot solution, followed by 10 c.c. more of
distilled water. Known amounts of iron to serve as standards for color imetric comparison, 5 or 10 gamma in each sample, were carried through the
above procedure, with the same amounts of the various reagents.
Development of Color. To each unknown and standard, prepared as has
been described, were added 6 c.c. of iso-amyl alcohol and 3 c.c. of 20 per cent
solution of sodium thiocyanate. (The flasks containing the solutions were
cooled somewhat below room temperature before the addition of the alcohol.)
After the red iron sulfocyanate complex had been extracted into the alcohol
layer, by shaking the flasks for two to three minutes, the colored layers were
pipetted off directly into 20 mm. absorption cells for determination of light
absorption by means of the Piilfrich photometer, or into the small test tubes
used with the Klett-Summerson photo-electric colorimeter. With the latter
instrument, the green filter number 54, with maximal light transmission at
wave length 540 mm., was used.
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630
A. G. LITTLE, JR., M. H. POWER, AND E. G. WAKEFIELD
Determination of Iron in Feces. The total amount of feces collected in
each period was treated with four SO c.c. portions of concentrated nitric
acid for several days, until the mixture was homogeneous, after which
aliquots of 25 to 30 gm. were weighed into Kjeldahl flasks of a capacity of
300 c.c. These samples were then ashed with nitric and perchloric acids,
and the iron thiocyanate color complex was developed, extracted and determined essentially as described for serum.
RESULTS
Table 1 shows the results obtained with the first subject. This was a
man, 46 years of age, afflicted with syphilis of the central nervous system but
otherwise in good health. The total time of study consisted of 26 consecutive days which were divided into three periods of nine, ten and seven days
respectively. During the second period a total of 311 mg. of iron was
administered intravenously in eight injections.
TABLE I
A Man, 46 Years of Age, Who Had Syph ilis of the Central Nervous System. Iron Administered
Intravenously during Second Period. Patient on Constant Diet during Test
Iron, gamma per 100 c.c. Begin
serum
End
Hemoglobin, gm. per 100 c.c. Begin
blood
End
Erythrocytes, millions per Begin
cu. mm. blood
End
Volume packed erythrocytes Begin
(hematocrit)
End
Iron in stools, mg.
Period 2
10 days
Period 3
7 days
216
186
145
186
145
232
13.5
13.5
13.5
13.5
13.5
13.5
4.34
3.99
4.34
43.9
311
Total for period
0.41
7.01
Daily average
0.04
0.70
Total for period
Daily average
4.35
43.9
43.4
0
Iron given in period, mg.
Iron in urine, mg.
Period 1
9 days
46.8
0
159.0
152.2
109.3
17.7
15.2
15.6
The concentration of hemoglobin, the erythrocyte count and the volume
of packed erythrocytes remained approximately constant during the study.
The small variations in the last two might possibly be due to the fact that
the determinations were made on heparinized blood several hours after collection. The value for serum iron was somewhat high, varying from 145
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ABSORPTION AND EXCRETION OF IRON
631
to 232 gamma per 100 c.c. During the period of study the value did not
change significantly, however. The iron in the urine during the first control period totaled 0.41 mg., or an average of 0.04 mg. daily. This increased during the period of administration of iron to a total of 7.01 mg.,
or a daily average of 0.70 mg. The daily fecal iron remained essentially
constant during the three periods, being 17.7 mg., 15.2 mg. and 15.6 mg.,
respectively.
TABLE II
A Man, 36 Years of Age, Who Had Syphilis of the Central Nervous System.
Iron Administered Intravenously during Second Period.
Patient on Constant Diet during Test
Period 1
4 days
Iron, gamma per 100 c.c. Begin
serum
End
115
Volume packed erythrocytes Begin
(hematocrit)
End
Iron given in period, mg.
86
*
11.4
11.6
11.6
10.8
40.5
41.8
41.8
35.6
0
Total for period
Daily average
Period 3
5 days
4(
86
Hemoglobin, gm. per 100 c.c. Begin
blood
End
Iron in stools, mg.
Period 2
8 days
156
0
26.6
48.9
32.6
6.6
6.1
6.5
Table 2 shows the results noted in a study of a paretic man 36 years of
age. He was followed during 17 consecutive days, divided into three periods
of four, eight and five days each. During the second period he was given
156 mg. of iron in four injections.
The concentration of hemoglobin and volume of packed erythrocytes
remained essentially constant. The values for serum iron were somewhat
TABLE
III
A Man, 40 Years of Age, Who Had Schizophrenia. 30 mg. of Iron Administered Intravenously.
Injection Started at 7:15 a.m. and Concluded at 7:45 a.m.
Volume Urine,
c.c.
Total Iron Excreted,
mg.
Average Excretion
per Hour, mg.
Period 1—1 hour, 30 minutes
(7:15 a.m. to 8:45 a.m.)
175
0.19
0.13
Period 2—4 hours, 45 minutes
(8:45 a.m. to 1:30 p.m.)
200
Q.06
0.01
Period 3—3 hours, 30 minutes
(1:30 p.m. to 5 p.m.)
200
0.03
0.01
Total excretion in approximately 10 hours, 0.29 mg.
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632
A. G. LITTLE, JR., M. H. POWER, AND E. G. WAKEFIELD
low, varying from 46 to 115 gamma per 100 c.c. The accuracy of the 46
gamma is probably questionable. The average daily fecal iron remained
fairly constant, being 6.6 mg., 6.1 mg. and 6.5 mg. for the respective periods.
Table 3 shows the results obtained from further study of urinary excretion following intravenous injection. The subject was a man, 40 years of
age, who was suffering from schizophrenia. Iron, 30 mg., was given intravenously and the urine was collected at intervals during the following approximately 10 hours. The total excretion during this period was only 0.29
mg., 0.19 mg. of which appeared during the first 90 minutes.
SUMMARY AND COMMENT
Determination was made of the amount of iron in the feces of two men
whose iron reserve apparently was normal. Injection of relatively large
amounts of iron by vein caused no detectable increase in the fecal iron. This
conclusion is in agreement with the results obtained in dogs by Henriques and
Roland,9 Hahn and associates 10' u » 12 and Quinke.13 It would tend to lend
confirmation to the hypothesis advanced by McCance and Widdowson 14f 15
that the body has no mechanism whereby an excess of iron can be excreted.
Over a control period of nine days one of these two men was found to
excrete approximately 0.045 mg. of iron per day in his urine. This is somewhat lower than the determinations of Farrar and Goldhamer 16 who found
0.02 mg. per 100 c.c. and those of Henriques and Roland who estimated 0.08
to 0.32 mg. per day.
As found in study of a third man, following intravenous injection of iron
there is a marked increase in the amount of urinary iron. This has been
found by other observers. The total amount was relatively small, however,
being only 0.29 mg. within 10 hours following the injection of 30 mg. More
than 65 per cent of this appeared within the first 90 minutes. This increased
excretion is apparently due to the sudden and marked increase of iron in
the blood and probably does not take place following its normal absorption
from the intestine.
We are aware that, at postmortem examination in cases of general
paralysis of the insane, pigment is found in the brain. This pigment is
known to be iron and is associated with the local degenerative changes. We
do not believe that this deposition substantially disturbs the systemic metabolism of iron. Nevertheless, the phenomenon should be mentioned here for
its possible significance as knowledge is extended.
BIBLIOGRAPHY
1. STOCKMAN, RALPH: The treatment of chlorosis by iron and some other drugs, Brit.
Med. Jr., 1893, i, 881-885; 942-944.
2. STARLING, E. H.: Human physiology, Ed. 6, 1933, Lea and Febiger, Philadelphia, 1122 pp.
3. SZENT-GYORGYI, A.: Eine Farbenreaktion der Ascorbinsaure, Ztschr. f. physiol. Chem.,
1934, ccxxv, 168.
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ABSORPTION AND EXCRETION OF IRON
633
4. HEILMEYER, L.: Die Behandlung eisenempfindlicher Anamien mit askorbinsaurem Eisen,
zugleich ein Beitrag zum Mechanismus der Eisenwirkung und zur Frage der Eisenmangelkrankheit, Deutsch. Arch. f. klin. Med., 1936, clxxix, 216-231.
5. FLEISCHHACKER, H., and SCHURER-WALDHEIM, F.: Zur peroralen und intravenosen
Therapie mit ascorbinsaurem Eisen, Wien. klin. Wchnschr., 1938, li, 776-780.
6. FRIEND, D. G.: Iron ascorbate in the treatment of anemia, New England Jr. Med., 1938,
ccxix, 910-912.
7. MAURER, KURT, and SCHIEDT, BRUNO: Das Verhalten der d-Arabascorbinsaure und des
Vitamins C gegeniiber Ferrosalzen, Biochem. Ztschr., 1936, cclxxxv, 67.
8. MOORE, C. V.: Studies in iron transportation and metabolism; chemical methods and normal values for plasma iron and "easily split-off" blood iron, Jr. Clin. Invest., 1937,
xvi, 613-626.
9. HENRIQUES, V., and ROLAND, H.: Zur Frage des Eisenstoffwechsels, Biochem. Ztschr.,
1928, cci, 479-485.
10. HAHN, P. F., and WHIPPLE, G. H.: Radioactive iron and its metabolism in anemia,
Jr. Am. Med. Assoc, 1938, cxi, 2285-2286.
11. HAHN, P. F., BALE, W. F., LAWRENCE, E. O., and WHIPPLE, G. H.: Radioactive iron
and its metabolism in anemia, Jr. Exper. Med., 1939, lxix, 739-753.
12. HAHN, P. F., BALE, W. F., HETTIG, R. A., KAMEN, M. D., and WHIPPLE, G. H.: Radio-
active iron and its excretion in urine, bile and feces, Jr. Exper. Med., 1939, lxx, 443-451.
13. QUINKE : Quoted by GOTTLIEB, R.: Ueber die Ausscheidungsverhaltnisse des Eisens,
Ztschr. f. physiol. Chem., 1891, xv, 371-386.
14. MCCANCE,- R. A., and WIDDOWSON, E. M.: Fate of elements removed from blood-stream
during treatment of polycythaemia by acetyl-phenylhydrazine, Quart. Jr. Med., 1937,
vi, 277-286.
15. MCCANCE, R. A., and WIDDOWSON, E. M.: Absorption and excretion of iron, Lancet,
1937, ii, 680-684.
16. FARRAR, G. E., and GOLDHAMER, S. M.: The iron requirement of the normal human
adult, Jr. Nutr., 1935, x, 241-254.
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