A TRACER STUDY OF IRON METABOLISM WITH RADIOACTIVE IRON

I. METHODS: ABSORPTION AND EXCRETION OF IRON

BY D. HAROLD COPP* AND DAVID M. GREENBERG

(From the Division of Biochemistry, University of California Medical School, Berkeley)

(Received for publication, April 1, 1946)

The metabolism of iron is one of the most important subjects in the field of mineral metabolism. ,There have been numerous investigations of its problems, but until recently they have been seriously limited by the methods of study available. The production of radioactive isotopes of iron (1, 2) provided a new research tool which has proved of great value in the investigation of this field. Iron labeled with radioactive isotope may be distinguished from the iron originally present in the body, and so the fate of a given dose of radioiron’ may be determined with some precision.

Previous work with radioiron in the rat (3) and in dogs (4) and humans (5) was handicapped by the low specific activity of the preparations of the radioactive isotope, Fe5g, which were hitherto available. This necessi- tated the use of massive doses of iron beyond the normal physiological limits.

In the present work, use was made of the radioactive isotope Fe55, which is prepared in the cyclotron by bombardment of manganese with deuterons.2 Since the only contamination with inert iron is that oc- casioned by traces of iron in the manganese probe, this isotope may be prepared with a very high specific activity. This made possible the use of very small doses (0.05 mg.) approaching tracer levels. These tracer doses were administered to rats by stomach tube or by injection, and studies were made of the absorption, excretion, storage, utilization, and distribu- tion of iron under certain experimental conditions.

Methods

Preparation of Radioactive I,ron-The radioactive isotope Fe55 was pre- pared by deuteron bombardment (d, 2n) of a manganese probe in the cyclo- tron.2 The Fe55 produced decays by K electron capture with a half life

* The material of this paper was taken from a thesis submitted by D. H. Copp to the Graduate Division of the University of California in partial fulfilment of t,he requirement of the degree of Doctor of Philosophy, June, 1943.

1 Iron labeled by its content of radioactive isotope will be referred to as radioiron, or by the symbol Fe*.

2 Kamen, M. D., personal communication.

377

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378 IRON METAROLISM. I

of approximately 4 years. It emits a soft y-ray and very soft conversion electrons.

Scrapings from the manganese probe were dissolved in 6 M HCI and diluted to 1 M HCl. The copper present was precipitated with hydrogen sulfide and removed. Small amounts of non-radioactive &In, Co, Zn, and phosphate were added as carriers to assist in removing any radioactive isotopes of hhese elements which might be present. A fern mg. of iron were also added before the first precipitation, since only traces of iron were present in the probe. The iron was then precipitated from a chilled solution in 1 M HCl with a chilled 6 per cent aqueous solution of cupferron (6). This compound precipitates iron in the presence of dilute acid, leaving in solution the Mn, Co, Zn, and phosphate which might be radio- active contaminants of the target. The cupferron precipitate was ashed and dissolved in acid. After repeating this procedure four times, no radio- active contaminants could be detected in t,he filtratej and the identity of the Fe5” was further confirmed by the characteristic absorption of its weak radiation.

The Fe* was prepared for administration in neutral isotonic saline as ferric citrate, or iron ammonium citrate. The solution assayed 0.05 mg. of Fe per ml., with a specific activity of approximately 1 to 5 microcuries per mg. The dose administered was 1 ml. per rat.

Dietary Regimens-Rats were rendered iron-deficient by a modification of the procedure of Harris (7). They were weaned at 3 weeks to a diet of powdered whole milk supplemented with 5 mg. of thiamine chloride, 5 mg. of pyridoxine, and 50 mg. of calcium pantothenate per kilo. The diet assayed 5 parts per million of copper,” so that it could not be considered deficient in this element. This may account for the absence of the usual signs of copper deficiency in these animals. They were kept in glass cages with perforated aluminum floors, and were supplied with redistilled water ad Z&turn. When the radioactive iron was administered 5 weeks later, the level of blood hemoglobin was less than half that of the control animals.

Rats weaned to the regular stock colony diet served as controls. Both groups were about 2 months old and still rapidly growing when the dose of Fe* n-as given. Later, experiments were conducted on 6 month-old adult female rats which had been reared and maintained on the stock diet, and which had ceased growing.

Viviperjusion-Because of the very high concentration of iron in the hemoglobin of blood, it is im.portant that the tissues be completely freed of blood before iron analyses are carried out. This is most effectively accomplished by viviperfusion, a procedure \yhereby the living animal is

3 Copper xas assayed by the dilhizoue nxthod through the kindness of Dr. D. I. Arnon, Division of Hant Nutrition, University of California, Berkeley.

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D. H. COPP AND D. M. GREEKBERG 379

perfused with a solution approximat,in, u the composition of blood serum until the blood is almost completely replaced by this fluid.

Whipple (8) first stressed the value of viviperfusion in obtaining reliable analyses of iron in tissues, and described a technique for dogs. A method for use in the rat is given by Austoni, Rabinovitch: and Greenberg (9). A simplification of this latt,er met,hod was developed for the present in- vestigation. It may easily be carried out on any small animal and should prove of value whenever it is desirable to obtain tissues free from blood.

The essential apparatus consists of a burette filled with perfusion fluid warmed to 50”. A modified Tyrode’s solution of the following percentage, composition may be used: T\‘aCl 0.80, KC1 0.02, CaClz 0.02, MgClz 0.01 NaIIsPOd 0.005, NaI-IC03 0.10, and glucose 0.10. The last two com- ponents are added just before use.

The rat is anesthetized wibh nembutnl (4 mg. per 100 gm. of body n-eight) injected int,raperitoneslly. It is tied to an operating board, the abdomen is opened, and the inferior vcna calya is exposed. A large bore needle (18 gage) is inserted in t.he vein, and a blood sample lvithdrawn for determina- tion of blood hemoglobin, total iron, and radioiron. With the needle in place, the syringe is detached and its place is taken by the barrel of a tuber- culin syringe connected by flesible rubber tubing to the tip of the burette, so that warm perfusion fluid flow directly t,hrough the needle into the vein. The needle is ligated in place and the abdomen is closed with clamps. The jugular veins in the neck are next dissected out and opened so that the perfusion fluid flowing into the inferior vena cava may flush out the right auricle and escape with the blood from the jugulars. The veins in the legs are also opened. The animals usually survive from 20 to 40 minutes. At death, the lungs should be white, the liver a light fawn color, and the heart should be full of clear fluid. Presence of clear fluid in the portal vein is a good ind.ication of com.plete perfusion. Occasionally, when this fluid is tinged with blood, it may be necessary t’o perfuse the liver directly through the portal vein to clear it completely of blood.

The organs were dissected out with bright chrome scissors and wet.- ashed. The blood sample n-as corrected for tot,al blood weight from t.he figures given by Donaldson for the rat (10). Muscle stripped from the hind limbs was similarly corrected. Red bone marrow samples (50 to 100 mg.) were obtained by splitting the femurs and tibias and scraping the marrow cavity.

The perfused tissues of a series of normal and iron-deficient rat)s were determined. The result,s are given in Table I. These values show reason- able agreement wit,h those obt.ained by Austoni, Rabinovitch. and Green- berg for rats (9) and by Bogniard and Whipple for dogs (11).

Aslzing Procedure-To avoid the considerable loss of iron which may occur when tissue samples containing chloride are dry-ashed (12), tissue

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380 IRON METABOLISM. I

and excreta samples were wet-ashed in small Erlenmeyer flasks by adding successive portions of concentrated HNO, and cooking to dryness on a hot-plate. 5 to 50 ml. of HNOp were usually adequate for complete diges- tion. A few drops of superoxol aided the process. The total iron in each sample was corrected by subtracting the iron in the acid used for ashing.

The great bulk of the carcass made wet ashing inconvenient. It was accordingly dry-ashed at dull red heat to minimize iron loss. Despite this precaution, the recovery of Fe* from the carcass was often less than that calculated from its blood and muscle content, suggesting a loss of volatile iron during ashing.

The ash was dissolved in 1 M HCI, made to volume, and aliquots were taken for determination of total iron and radioactive iron.

TABLE I

Iron Content* of Tissues of Normal and Iron-Deficient Animals Freed of Blood by Viviperfusion

Tissues -I-

Body weight, gm ........... Blood Hb, gm. per 100 ml . Blood*. .................... Liver* ..................... Spleen*, ................... Bone marrow*. ............ Skeletal muscle*. ..........

Normal rstst

io. of rmpk

24 125 f 8 24 14.1 f 1.1 24 44.5 f 3.2 19 8.3 f 4.0 23 32.7 f 14.0 20 21.3 f 7.2 14 1.4 f 0.4

I 1

s,

-

Iron-depleted ratsi

24 24 24 19 24 12 22

114 f 14 6.4 f 1.3

20.1 f 4.2 2.8 f 0.8

12.2 f 3.8 12.0 f 3.5 0.9 f 0.3

* Iron content is expressed as mg. of Fe per 100 gm. of fresh tissue. t The figures given are the mean values f the standard deviation.

Determination of Total Iron-The total iron was determined on an aliquot from each sample by the o-phenanthroline calorimetric method described by Saywell and Cunningham (13) and critically reviewed by Fortune and Mellon (14). Calorimetric readings were made with a Klett-Summerson photoelectric calorimeter with green Filter 54. This instrument was also used for the determination of hemoglobin by the acid hematin method.

Determination of Radioactive Iron-So soft is t,he radiation of Fe55 that the count on a sample is reduced to half by as little as 10 mg. of ash per sq. cm. This difficulty Iv-as overcome by electroplating the iron in a thin layer for which the absorption of radiation was nil. The method used was a simplified adaptation of that described by Hahn, Bale, and Balfour (15) and by Ross and Chapin (16). The apparatus is shown in Fig. 1. Seam- less tin ointment capsules serve as electrolytic cells. The capsules are held in place on a rack with steel paper clips. These are connected to the

\To. of ample,

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D. H. COPP AND D. M. GREENBERG 381

negative terminal of a source of 110 volts D.C., so that the metal bottom of the ointment capsule becomes the cathode on which the iron is deposited. A plat,inum anode in the center of the capsule is connected through a 15 watt lamp resistance to the other terminal.

The iron sample, or an aliquot, is pipetted into the capsule, and 1 ml. of plating solution is added. This solution, adapted from that used by Hahn et al. (15) is made up in distilled water with the following percentage composition: ferric citrate 1 .l ( = 2 mg. of Fe per ml.), sodium citrate 25.0, ammonium chloride 12.5. The plating is carried out for 5 hours, an additional 1 ml. of plating solution being added as carrier at half time. The solution in the capsule at the end of the process should give little or no iron color with thiocyanate. The capsules are rinsed, dried, and the

FIG. 1. Apparatus for electroplating of radioactive iron

radioiron determined. Since the radiations from Fe55 are too weak to register on the usual metal or glass Geiger counter tubes, use was made of the thin mica window counter tube described by Copp and Greenberg (17).

In ten trials in which standard amounts of Fe* were added to solutions of tissue ash, the recovery by the above method was 99.7 f 1.8 per cent. When considerable quantities of calcium were present (as in the ash from carcass or feces) precipitation of calcium salts interfered with the plating. To avoid this, the calcium was first precipitated with potassium oxalate at pH 4, and the precipitate was filtered off. When this was done, the re- covery was comparable to that with other tissues.

The recovery of the administered Fe* was determined by adding the values for the individual tissues, excreta, and residual carcass, and was

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382 IRON METABOLISM. I

found to be 80 to 90 per cent of the administ.ered dose. Some of the reasons for failure tJo obtain complete recove?y have been mentioned by Hahn et al. (4). Much of the difficulty may be due to loss of iron during dry ash- ing of the carcass.

EXPERIMENTAL

Excretion of Parenterally Administered Radioiron-It has been difficult to obtain conclusive evidence on the excretion of iron from conventional chemical balance studies. However, by injecting labeled radioiron, it is possible to determine exactly how much of the administered iron is ex- creted. A dose of 0.05 mg. of Fe*, equivalent to less than 1 per cent of the total body iron, was given to rat’s on stock diet by intravenous or intra- peritoneal injection. The animals were sacrificed at 12, 23, 48, and 96 hours. The average excretion is shown in Table II. It may be seen that

TABLE II

Excretion* oj Pnrenterally Administered Radioiron

Time following injection

Urine.. 1 Feces. Total.

0.1 0.4 0.1 0.4 0.4 1.1 0.3 1.5

- * The figures given are the mean values expressed as per cent, of t,he total dose

of Fe*.

the excretion of Fe* in both urine and feces is small and quite variable. The lack of any significant excretion of even this tracer dose of Fe* confirms t’he observations of Hahn et al. on dogs (I 8). This was in striking contrast to the behavior of cobalt and manganese, elements which are adjacent to iron in the periodic table. When tracer doses of the radioactive isotopes of these elements were injected parenterally, a large part of the dose was excreted within the first’ 2 days, cobalt appearing principally in t’he urine (19), I\-hile manganese was eliminated chiefly in t’he feces (20).

The excretion of Fe* in the bile of rats with a biliary fistula and artificial gallbladder has been reported in a previous publication (21). Only traces of Fe* (0.1 per cent) were excreted in the first 48 hours, in contrast to the significant amount of Co* (2 to 4 per cent) and the large proportion of Mn* (24 to 40 per cent’) which appeared in the bile.

The actual excretion of Fe* into t’he lumen of the intestinal tract was investigated in two groups of rats which were given the radioiron by sub-

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D. H. COPP AND D. M. GREENBERG 383

cutaneous or intraperitoneal injection. After viviperfusion. the small and large intestines were removed separately and the contents were carefully washed out. Both intestines and contents were analyzed for Fe* separately, and the results are presented in Table III. From these values, it appears that, while considerable amounts of Fe” are taken up by the tissues of the intestine, only traces find their way into the lumen.

Absorption of Radioiron from Intestinal Tract-Since only traces of iron are normally excreted, it is evident t,hat the iron in the body must be rcgu- lated by absorption. Here, too, labeled radioiron provides a means of de- termining exactly horn much of a given dose is absorbed. 1 ml. doses containing 0.05 mg. of Fe* were administered by stomach tube to normal and iron-deficient growing 2 month-old rats. This dose, which is much less than the normal daily intake of iron in food, may be expected to follow the same path of metabolism as the normal dietary iron. Such small doses lie within physiological limits, and avoid the possible complicat,ions of

TABLE III

Excretion* of Radioiron into Lumen of Intestinal Tract

I Time following injection

~ 1 12 hrs. 24 hrs. 1 48 hrs.

3 rats

Small intestine (washed out). 2.5 i 1.6 1.7 Contents of small intestine.. 0.2 0.2 0.1 Large intest,ine (washed out), 0.8 0.5 0.8 Contents of large intestine.. 0.2 0.6 0.5

96 hrs.

4 rats

1.6 0.4 0.8 0.7

* The figures given are the mean values expressed as per cent of the total dose of Fe*. The radioiron was administered by intraperitoneal or subcutaneous injection.

massive iron concentrations. After administration of the Fe*! t’he rats mere sacrificed at various time intervals up to 4 days. The stomach, small intestine, large intestine, and feces, including their contents, were analyzed for Fe”. The values, plotted against time, are shown in Fig. 2.

In the normal cont,rol rats, the administ.ered Fe* passed rapidly along the intestinal tract. The stomach emptied quickly, and within 3 hours a considerable part of the dose had passed through the small intestine to reach the large bowel. Significant amounts had appeared in the feces by 12 hours, and almost all of the unabsorbed iron had been excreted within the first 24 hours.

In the iron-deficient rats, the Fe* passed along the intestinal tract at a much slower rate, as was observed by Austoni and Greenberg (3). The poor intestinal tone observed in these anemic animals may account for the

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384 IRON METABOLISM. I

delay. Very little Fe* had reached the large bowel at 3 hours, so that the large amount of Fe* which had already appeared in the body at this time must have been absorbed from the stomach or small mtestine. The most striking delay, however, occurred in the large intestine. Significant

CONTROL - FE= ORAL

FECES

ANEMIC - FE’ ORAL

ISTOMACH IJ 40-: .

DAYS

FIG. 2. Passage of the dose of radioiron along the gastrointestinal tract. Dose of 0.05 mg. of Fe* administered by stomach tube. The control rats were 2 months old, and had been reared and maintained on the stock colony diet. The average weight of the group was 124 f 7 gm., and the mean value for the blood hemoglobin was 14.4 f 1.1 gm. per 100 ml. The anemic rats were weaned at 3 weeks to a milk diet, and were used when 2 months old. The average weight of these animals was 112 f 11 gm., and the mean value for the blood hemoglobin was 5.8 f 1.1 gm. per 100 ml. Stomach A . . . . ) small intestine l - - -, large intestine H -a--. , feces 0 -.

amounts of Fe* did not reach the feces for 24 hours, and a considerable part of the dose was retained for almost 2 days. Since only a third of the Fe* in the large intestine was ultimately excreted in the feces, it would appear that considerable absorption may take place in this organ.

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D. H. COPP AND D. M. GREENBERG 385

The iron-deficient rats absorbed over nine-tenths of the tracer dose of Fe*, indicating a very efficient utilization of dietary iron. While t,he slow rate at which the dose passed along the intestinal tract may have facilitated this efficient, utilization by allowing more time for absorption, it was not the sole factor, since the iron-depleted animals absorbed the Fe* much more rapidly from the very beginning.

The control animals on the other hand absorbed less than a third of the dose. This confirms the observations on dogs (4) and humans (5) for massive doses of Fe*.

DISCUSSION

Since only insignificant amounts of iron are normally excreted, the iron balance in the animal must be regulated by control of absorption. Iron- depleted rats absorbed Fe* much more efficiently than did the controls, even though the latter were still growing actively and therefore required some iron for the synthesis of new hemoglobin. Hahn et al. (4) observed that absorption of large doses of radioiron by normal adult dogs was negli- gible, while iron-depleted animals absorbed considerable amounts.

The difference in absorption was not due to anemia per se, since it does not occur in cases of untreated pernicious anemia (5) nor in acute anem.ia in the dog (22). In the latter case, however, when the level of the blood hemoglof%n had been restored to normal at the expense of depleted iron stores, absorption increased 5 to 10 times. The weight of evidence sup- ports the thesis of Whipple et al. (23) that “absorption of iron is dependent on the need of the body for iron.”

The most efficient absorption of Fe* was obtained when minute doses of Fe* were fed to rats on a milk diet very low in iron. Since the concentra- tion of iron in the lumen of the gut is very low in these animals, it seems highly improbable that the process is one of simple diffusion dependent, on differences in ionic concentration as has been suggested by McCance and Widdowson (24). Indeed the evidence indicates that the absorption of iron may be a specific process involving the intestinal mucosa. The rela- tive uptake of Fe* by the gut and the rapid rate of turnover reported in Paper II of this series lend further support to this view. Hahn et al. (22) observed a “mucosal block” a few hours after feeding iron (but not after injection of colloidal iron) which prevented further absorption. They suggested that this might be due to saturation of the mechanism in- volved. The probable nature of this mechanism has been indicated by Granick (25) who observed the appearance of the iron containing protein ferritin in the wall of the intestine of growing guinea pigs following iron feedings. This presumably reaches equilibrium with the other iron re- serves of the body, and its state of depletion or saturation, reflecting that

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386 IRON METABOLISM. I

of the general body stores, determines the absorption of iron from the intestine.

On continued feeding of iron, he found ferritin in the walls of the small and large intestine, and in sm.aller amounts in the stomach and even the cecum.. This adds some support to our indirect evidence for absorption from the small intestine and large intestine, and for the demonstration by Hahn et al. (22) of absorption from gastric, duodenal, and jejunal pouches. It would appear that iron may be absorbed from any part of the gastro- intestinal tract,.

SUMMARY

1. Iron labeled with the radioactive isotope FeS5 was prepared with a high specific activity compared to that usually obtained with Fe5g. For this reason, small tracer doses (0.05 mg. of Fe*) could be used for adminis- tration to rats. Because of the soft character of the radiations from Fe55, the iron in the samples analyzed was first electroplated, and the radioac- tivity was then measured with a thin mica window Geiger counter tube.

2. Experiments were conducted on normal 2 month-old rats, and on anem.ic rats which had been depleted of iron by rearing them on a diet of powdered milk.

3. A simplified m.et,hod of viviperfusion was developed to free the tissues and organs of blood.

4. Ko significant escret.ion of Fe* was observed in the bile, urine, or feces following parenteral administration of the tracer dose.

5. Iron-depleted rats absorbed over 90 per cent of the dose of Fe*, while the normal growing rats absorbed less than one-third. Absorption appar- ently took place in both the small and large intestine.

6. There was a relatively high uptake of Fe* by the intestinal wall, al- though only traces were excret#ed into the lumen.

7. Some factors concerned in the absorption of iron are discussed, and a possible mechanism is suggested.

BIBLIOGRAPHY

1. Livingood, J. J., and Seaborg, G. T., Phys. Rev., 64, 51 (1938). 2. Livingood, J. J., and Seaborg, G. T., Phys. Rev., 55, 1268 (1939). 3. $ustoni, M. E., and Greenberg, D. M., J. Biol. Chem., 134, 27 (1940). 4. Hahn, I’. F., Bale, W. F., Lawrence, E. O., and Whipple, G. H., J. Exp. Med., 69,

739 (1939). 5. Balfour, W. M., Hahn, I’. F., Bale, W. F., Pommerenke, W. T., and Whipple,

G. H., J. Exp. afed., 76, 15 (1942). 6. Smith, G. F., Cupferron and.neocupferron, Columbus (193s)” 7. Harris, R. S., science, 76, 495 (1932). 8. Whipple, G. II., Am. J. Physiol., 76, 693 (1926).

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D. H. COPP AND D. hf. GREENBERG 387

9. Austoni, M. E., Rabinovitch, A., and Greenberg, D. AI., J. Viol. Chem., 134, 17 (1940).

10. Donaldson, H. II., The rat, Memoirs of the Wistar Iustitute of Anatomy and Biology, Philadelphia, 2nd edition (1924).

11. Bogniard, R. I’., and Whipple, G. H., J. Ezp. ilIed., 55, 653 (1932). 12. Jackson, S. I-I., Ind. and &g. Chem., Anal. Ed., 10, 302 (1938). 13. Saywell, L. G., and Cunningham, B. B., Id. ad Brig. Chem., 371~1. Ed., 9, 67

(1937). 14. Fortune, W. B., with Mellon, M. G., Ind. and ISny. Chen?., Ard. h’d., 10, 60

(1938). 15. Hahn, P. F., Bale, W. F., and Balfour, \V. M.: Am. J. I’h~~siol., 135, 600 (1942). 16. Ross, J. F., and Chapin, M. A., Rev. Gcient. Insfruments, 13, 77 (1942). 17. Copp, D. I-I., and Greenberg, D. M., Iiev. Scient. Instrwnents, 14, 205 (1943). 18. Hahn, P. F., Bale, W. F., Hettig, 11. A., Kamcn, M. D., and Whipple, G. II.,

J. Exp. Med., 70, 443 (1939). 19. Copp, D. H., and Greenberg, D. M., 1’wc. &-at. Acad. SC., 27, 153 (1941). 20. Greenberg, D. M., and Campbell, W. W., PYOC. Sal. Rcarl. SC., 26, 448 (1940). 21. Greenberg, D. M., Copp, D. I-I., and Cuthbcrtson, E. M., J. Bid. Chem., 147,

749 (1943). 22. Hahn, P. F., Bale, W. F., Ross, J. F., Bnlfour, W. AI., and Whipple, G. II., J.

Exp. Med., 78, 169 (1943). 23. Hahn, P. F., Bale, W. F., Lawrence, E. O., and Whipplc, G. I-I., J. Am. Med.

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D. Harold Copp and David M. GreenbergEXCRETION OF IRON

IRON: I. METHODS: ABSORPTION ANDMETABOLISM WITH RADIOACTIVE

A TRACER STUDY OF IRON

1946, 164:377-387.J. Biol. Chem. 

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