THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 238, No. 5, May 1963

Printed in U.S.A.

Relation of Ferritin Iron to Heme Synthesis in Marrow and Reticulocytes*

ABRAHAM MAZUR AND ANNE CARLETON

From the Department of Medicine, Cornell University Medical College, and The New York Hospital, New York 21, New York

(Received for publication, December 31, 1962)

The reaction of protoporphyrin with iron to form heme is usually studied in intact or lysed cells in the presence of salts of ionic iron. Despite the fact that such iron is utilized for heme synthesis, the form of iron available to erythropoietic cells in the intact animal is that which is present in serum in the form of a highly stable iron-protein complex, transferrin. Results obtained by use of ionic iron are misleading to the extent that such studies bypass the biochemical mechanisms which control the entrance of iron into the cell, and lead to concentrations of intracellular iron much higher than are possible in viva. Jandl et al. (1) have demonstrated tha,t binding of iron by serum trans- ferrin controls the entrance of iron into the reticulocyte, and have also shown that the over-a.11 incorporation of such iron into heme of these cells is dependent on oxidative metabolism. It is likely that the incorporation of serum iron into heme in the reticulocyte requires oxidative energy, both for the entrance of iron into the cell and for the synthesis of the protoporphyrin moiety of heme. The only mechanism of biochemical signifi- cance for the release of transferrin-bound iron and its entrance into a cell is that reported by our laboratory (2) for the incor- poration of serum iron into ferritin in the hepatic cell. Although the incorporation of ionic iron into ferritin of surviving liver slices appears not to require oxidative energy, it has been dem- onstrated in vitro as well as in wivo (3) that the corresponding incorporation of serum-bound iron requires the participation of ascorbic acid and adenosine triphosphate.

Although Bessis and Breton-Gorius (4, 5) have demonstrated the presence of ferritin in marrow cells and reticulocytes by use of the electron microscope, these observations do not reveal the origin of such ferritin or the relationship between the ferritin iron and heme which is synthesized in these cells. Falbe-Hansen and Lothe (6) have reported evidence for a short-lived nonheme iron intermediate in the passage of iron from transferrin to hemo- globin in rabbit red cells but have not identified this intermediate. In the present study we report the results of experiments which help to clarify the relationship between ferritin iron, originating from the serum, and heme iron in rat marrow and reticulocytes. The results are consistent with the hypothesis that ferritin iron plays an active role as an intermediate between iron originating from the plasma and the heme synthesized by marrow and reticulocytes.

EXPERIMENTAL PROCEDURE

Rat erythrocytes, rich in reticulocytes, were obtained from animals that had been given subcutaneous injections of 0.5 ml

* Aided by Grant A-1655 from the National Institutes of Health, United States Public Health Service.

of a 1 y0 solution of phenylhydrazine hydrochloride on each of 4 consecutive days. Blood was collected in heparin 3 to 5 days after the last injection, and the cells were washed free of plasma with 0.9 y0 NaCl solution. Human erythrocytes, rich in reticulo- cytes, were obtained through the courtesy of Drs. R. Singer and H. Rappoport of the New York Hospital from patients treated for pernicious anemia. Marrow was aspirated from the hind leg bones of rats, suspended in normal rat serum, and prepared for incubation studies according to the method of Morell, Savoie, and London (7). Partially purified rat ferritin, crystalline horse ferritin, and rabbit antiserum to horse ferritin were prepared by methods described previously (2).

Incubation studies were performed with cells suspended in Krebs-Ringer-phosphate medium, pH 7.4, containing 1 y0 bovine serum albumin. Serum-bound Fe59 was prepared by incubating normal rat serum with trace chemical quantities of FeSgC13 to prevent alteration of the serum iron-binding capacity. After incubation at 37” in an atmosphere of 100% oxygen, the cells were washed several times with NaCl solution, lysed with water, and reconstituted with concentrated Krebs medium together with phosphate buffer, pH 7.4. Aliquots of marrow suspension were removed for total nitrogen determination, and samples of lysed red cell suspensions were analyzed spectrophotometrically for hemoglobin. For isolation of hemin, an aliquot of the cell suspension was mixed with 5 volumes of acetone-glacial acetic acid (3: 1) and brought to incipient boiling in a water bath. Acetone was added to complete precipitation of the proteins. Unlabeled carrier hemin (20 mg), dissolved in pyridine-chloro- form solution, was added when necessary. The mixture was filtered and crystalline hemin was isolated by the method of Labbe and Nishida (8).

The remaining cell suspension was used for the isolation of radioactive ferritin. This was accomplished by heating the mixture in a water bath to 65”, and the chilled suspension was clarified by centrifugation. The insoluble residue was extracted with Krebs-phosphate medium and the clear extract obtained by centrifugation was pooled with the initial extract. Carrier rat ferritin (70 to 100 Hg of Fe) was added, and the total ferritin was precipitated by incubation with excess antiserum. The ferritin-antibody precipitate was collected by centrifugation and washed with NaCl solution several times. The precipitate was assayed for radioactivity and analyzed for chemical iron content. Similarly, the hemin was also counted and assayed spectrophoto- metrically. Fe6g was measured in a well-type scintillation de- tector, and Cl4 in a gas flow counter.

When Cr4-glycine was used to measure incorporation into ferri- tin, direct precipitation of the ferritin after addition of carrier,

1817

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as described above, resulted in a ferritin-antibody precipitate contaminated with nonferritin labeled protein. This could be avoided by purification of the ferritin by preliminary precipita- tion twice at 0.5 saturation with ammonium sulfate, followed by dialysis and clarification by high speed centrifugation before addition of antiserum. This method for determination of the Cl4 content of ferritin was especially important in liver, where most of the Cl4 is incorporated into albumin which is adsorbed to some extent by the ferritin-antibody precipitate.

Globin was isolated by the method of Morel1 et al. (7), and free protoporphyrin by the method of Rimington and Tooth (9). To minimize contamination of the isolated compounds with unre- acted isotope, unlabeled serum or glycine was added to the re- spective solutions before isolation.

Incorporation of Serum Fe69into Rat Bone Marrow In Vivo

Control Phenylhydrazine Endotoxin

UBC R.BC RBC 24 hrs = 4160 cpm 24 hrs - 14335 cpm 24 hrs - 1466 cpm 48 hrs - 8100 cpm 48 hrs = 16450 cpm 48 hrs - 3373 cpm

FIG. 1. Incorporation of serum-Fe59 into rat bone marrow in z&o. Serum-bound FeS9 (100,000 c.p.m. per 100 g of body weight) was injected intravenously into three groups of rats. Four ani- mals in each group were killed after various time periods. Results are expressed as the average counts per minute of Fe69 per mg of marrow nitrogen in total cells, heme, and ferritin. Appearance of Fe69 in circulating red blood cells (RBC) 24 and 48 hours after injection of Fe59 is expressed as counts per minute in erythrocytes from 1 ml of blood.

TABLE I

Incorporation of serum-Fe59 in.to heme and ferritin of rat bone marrow suspensions in vitro

Marrow suspensions were incubated with serum-bound Fe59 in an oxygen atmosphere, washed with NaCl solution, and lysed with water before analyses. Results are expressed as counts per minute of Fe69 in total cells, and in total heme and ferritin per 200,000 c.p.m. of serum-FebY added to the incubation mixture, per mg of marrow total nitrogen.

Time of incuba-

tion

min 15 30 60

120 180

I Treated with Treated with phenylhydrazine endotoxin

1,439 412 127 3,205 1,935 790 197 6,310 2,580 1,185 261 10,190 4,410 2,213 416 15,090 5,240 2,475 378 18,850

Heme

1,560 3,495 5,110 8,400

10,280

409 1,107 183 101 760 1,256 324 132 993 2,255 585 214

1,341 2,800 850 287 1,423 3,260 890 410

TABLE II

E$ect of injected endotoxin on incorporation of serum-Fe69 and 2-04-glycine into rat marrow heme

and ferritin in vitro

Marrow prepared from each rat was suspended in Krebs- Ringer-phosphate medium to which was added either serum-Fe59 or 2-Ci4-glycine. The suspensions were incubated at 37” for 1 hour in oxygen, and hemin and ferritin were isolated after addi- tion of respective carriers. Each value represents the average of six values (&standard deviation), each involving the pooled marrow from two rats.

Cont1ols Treated with endotoxin

c.fi.m./mg marrow N

Serum-Fe59 incorpora- tion into:

Heme 720 f 75 (X 103) Ferritin. 806 f 32

2-Cl*-glycine incorpora- tion into:

Heme. 508 f 95 (X 10z) Ferritin 418 f 76

RESULTS

364 f 65 (X103) 491 f 91

211 f 39 (X102) 1242 rt 415

Incorporation of Serum-Fesg into Rat Marrow in Viva-Rats were given intravenous injections of serum-Fe59 (100,000 c.p.m. per 100 g of body weight) and killed at various time intervals. Marrow was removed from the two hind legs of each of four rats, and hemin and ferritin were isolated after the addition of carrier hemin and rat ferritin, respectively. Three groups of rats were studied: normal controls, rats made anemic with phenylhydrazine in order to stimulate marrow erythropoiesis, and rats treated with injections of Escherichia coli endotoxin in an attempt to mimic the anemia associated with chronic infectious disease (3). The results in Fig. 1 represent average values for four rats, ex- pressed as counts per minute per mg of marrow nitrogen to cor- rect for unequal yield of marrow from each rat. In control ani- mals the heme Fe5g content of the marrow rose to a maximum at about 24 hours and then declined. The decline was accom- panied by an increase in FeSg content of the circulating erythro- cytes. Although the total ferritin was less radioactive than the total heme, the FeSg content of the ferritin rose to an earlier maximum, at 4 hours, and fell during the subsequent 20 hours, during a time when the heme-Fe59 content was still rising.

Marrow from rats made anemic with phenylhydrazine was much more active than control marrow with respect both to ex- tent of labeling and to the time of appearance of maximal labeling in heme and ferritin. On the other hand, incorporation of serum-Fe59 into heme and ferritin of marrow from rats which had received endotoxin injections was significantly lower than that found for the controls. These effects were confirmed by a lower content of Fe5g in circulating red cells as compared with controls. The effect of endotoxin is ma.de more significant by the finding that marrow from rats which had received endotoxin had a lower nitrogen content (27.4 f 0.9 mg of N per g of marrow, wet weight) than that of controls (31.8 A 1.0 mg of N per g of mar- row). Microscopic examination revealed a hypocellularity and increased fat content of marrow from endotoxin-treated rats as compared with controls1

1 We are indebted to Dr. Ralph L. Engle, The New York Hospi- tal, for the preparation and interpretation of the marrow slides.

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Incorporation of Serum-Fe ( 5g into Rat dfarrow in Vitro-The data in Table I illustrate the results cbtained by incubation of rat marrow suspensions with serum-FeSg. As in the previous experiment, marrow from normal rats was compared with that from rats which had received phenylhydrazine and those which had been given endotoxin. When compared on the basis of equal quantities of marrow total nitrogen, incorporation of serum- bound Fe&g into marrow heme and ferritin from rats which had received phenylhydrazine was much more extensive than that found in controls, whereas incorporation into marrow from endo- toxin-treated rats was lower than that of controls. That endo- toxin had a greater inhibitory effect on heme than on ferritin incorporation can be seen by comparing the ratio of Fesg in heme to Fe5g in ferritin of both series. To clarify further the nature of the effect of endotoxin on marrow, cells taken from rats that had received endotoxin were compared with those from control animals with respect to their ability to incorporate serum-FeSg as well as CWglycine into heme and ferritin. The results in Table II demonstrate a significant inhibition of Fe59 incorpora- tion into heme as well as into ferritin, although whereas there was a decreased incorporation of C?4 into heme, that incorpora.ted into ferritin in endotoxin-treated rats was increased above that of the controls.

Incorporation of Serum-FeSg into Reticulocytes-Rats made anemic by phenylhydrazine injection have a reticulocyte count of 80 to 90%. Such red cells constitute an active system for the incorporation of serum-Fes9 into heme and ferritin, as shown by the results in Table III. That this technique measures the ac- tivity only of the reticulocytes may be presumed from the fact that normal blood contains red cells with very little incorporation activity, and this low activity may be attributed to the small number of reticulocytes normally present in the blood of young growing rats. The results obtained by use of reticulocyte-rich red cells either from anemic rats or from patients with an ab- normally high reticulocyte count demonstrate that serum-Fe59 is incorporated into both heme and ferritin. Although incorpora- tion into heme continues throughout the incubation period, the ferritin Fe59 content reached a maximum and then declined in a manner similar to that described for marrow. Similar results have been obtained by use of reticulocyte-rich red cells from rats made anemic by bleeding.

One example of evidence of a precursor relationship between two compounds is the finding of a higher specific activity of an incorporated isotope in the precursor compound at an early stage of the reaction, followed by a decrease in its specific activity ac- companied by an increase in specific activity of the isotope in the recipient compound. In the experiments described above, specific activities could not be determined because the small quan- tity of cells made it necessary to use carrier ferritin for isolation purposes. Another question raised by the use of carrier ferritin and its isolation as a specific antigen-a.ntibody complex concerns the possibility of the reaction of Fesg nonspecifically with carrier ferritin. To clarify both problems, reticulocyte-rich red cells from 30 rats were pooled and tagged with serum-Fe59 by incuba- tion for 20 minutes. The cells were washed free of excess isotope, resuspended in fresh medium containing unlabeled serum, and incubated for various time periods after one aliquot was removed for analyses. Hemin was isolated directly and ferritin was iso- lated after preliminary purification, as described earlier. In neither case was carrier added.

TABLE III

Incorporation of serum-Fe”9 into rat and human reticulocyte heme and ferritin in vitro

Red cells from rats treated with phenylhydrazine were incu- bated with Fe59 bound to normal rat serum. Red cells rich in reticuloctyes, obtained from patients treated for pernicious anemia, were incubated with Fes9 bound to normal human serum. Carrier rat ferritin or human ferritin was added for isolation of the respective ferritins. Excess antiserum to horse ferritin was used to precipitate rat ferritin, whereas excess antiserum to hu- man ferritin was used to precipitate human ferritin. Results are expressed as counts per minute of Fe69 per 100 mg of hemo- globin in each sample, per 300,000 c.p.m. of Fe69 added to the incubation mixture in the form of serum-bound iron.

Rat cells (SO?& Human cells Human cells Time of reticulocytes) (6.0% reticulocyte) (23.8% reticulocytes) incuba- I / !

15 26,850 5,475 202 1304 225 82 34,600 3,650 1000 30 47,50010,410 1575 2722 523 229 54,700 7,020 1600 60 64,60016,170 1580 4080 906 302 91,80011,900 2350

120 69,00019,900 561 7270 1437 1235 123,00014,500 6275 180 70,00028,850 251 9670 1892 560 139,00020,900 4675

TABLE IV Specific activities of ferritin and heme iron in rat

reticulocytes during incubation in vitro

Reticulocyte-rich red cells from 30 rats were incubated with serum-Fe59 for 20 minutes, and excess isotope then was removed. The cells were reincubated in isotope-free media for varying lengths of time. Hemin and ferritin were isolated directly with- out use of carriers.

Time of reincubation

Hemin specific activity

Ferritin-antibody precipitate

Specific activity

min c.p.m.jcmmole Fe c.p.m./pnole Fe

“0” 1850 x 103 18.5 X 1W 30 1950 x 103 16.4 X lo3 60 2045 X 103 13.1 x 103

120 2195 X IO3 9.9 x 103 -

Fe -

M 37 37 37 29

-

N Fe:N

Irg &e/M 163 0.23 176 0.21 163 0.23 125 0.23

The results in Ta,ble IV demonstrate that after exposure of the cells to Fe5g for 20 minutes, the specific activity of heme Fe was higher than that of ferritin when compared on the basis of equal quantities of iron, although it is clear that whereas the specific activity of heme iron rose, that of ferritin decreased significantly. A comparison of heme and ferritin on the basis of an equal num- ber of iron atoms does not reveal the relative number of Fe59 atoms which entered the hemoglobin molecule as compared with the ferritin molecule. Whereas hemoglobin contains 4 atoms of iron, it can be calculated that horse spleen ferritin contains 2,400 atoms of iron per molecule.2 On this basis, after 20 minutes of exposure of the reticulocytes to the isotope, the specific activity of

2 This calculation is based on a molecular weight of 465,000 and total nitrogen content of 16yo for horse spleen apoferritin, and on a total iron content of 20% and total nitrogen of 11% for horse spleen ferritin. One mole of apoferritin (74,000 g of total N) would be combined with 135,000 g, or 2,420 moles of iron in ferritin.

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1 5.8

24 48 72 Time (hours) 2.3 0.9 0.9 Retie. t%)

FIG. 2. Fate of labeled rat reticulocytes in the circulation. Rat reticulocytes were labeled by incubation with serum-Fe69 or 2-Cl4- glycine, washed free of excess isotope, and injected intravenously into normal rats. Red cells prepared from blood removed from recipient rats were analyzed for Fe69 in heme and ferritin and for Cl4 in heme, globin, and ferritin. Results are expressed as per cent of the counts per minute in each fraction of the l-hour sample. Reticulocyte assays were performed on each blood sample and are expressed as per cent of reticulocytes of the total cells.

Time tminutes)

FIG. 3. Ability of maturing reticulocyte to incorporate serum- Fe69 and 2-CWglycine into heme, globin, and ferritin in vitro. Rats were injected with unlabeled reticulocytes, and the red cells were obtained from blood of recipient rats at various time periods. Cells were incubated with serum-Fe59 or Cl4-glycine for 1 hour at 37” in oxygen, and the heme, globin, and ferritin were analyzed for isotope content.

hemoglobin iron would be 1,850 X 103 x 4, or 7,400 X 103 c.p.m. per pmole, and that of ferritin would be 18.5 x lo3 x 2,400, or 44,400 x lo3 c.p.m. per pmole. That the ferritin isolated from rat reticulocytes has a composition similar to that found for horse spleen ferritin may be concluded by comparing the Fe:N ratio in the antigen-antibody precipitates obtained from rat retic- ulocytes, 0.23, with that found for horse ferritin, 0.29, which contained 20% iron and 11 Y0 total nitrogen.

Fate of Isotopically Labeled Reticulocytes in VivtiA pool of washed rat erythrocytes rich in reticulocytes was separated into three equal aliquots, each of which was incubated in a Krebs phosphate medium at 37” for 1 hour with (a) serum-Fe&g, (b) 2-CP-glycine, and (c) NaCl solution. The cells were washed with NaCl solution several times, resuspended in the Krebs medium, and injected (0.5 ml per 100 g of body weight) into three

groups of normal rats. Blood was withdrawn at suitable time intervals, and the washed cells from rats which had received isotopically tagged cells were analyzed for isotopes in ferritin, heme, and globin. The red cells collected from rats which had received the unlabeled reticulocytes were divided into two ali- quots and incubated with serum-Fe&g and C?4-glycine, respec- tively, for 1 hour. Excess isotope was removed by washing with NaCl solution, and the lysed cells were analyzed for isotopes in heme, ferritin, and globin.

The results in Fig. 2 demonstrate that the injected reticulo- cytes underwent maturation in the circulation of the recipient rats, which was essentially complete by 48 hours, since disap- pearance of reticulocytes, as measured microscopically, was ac- companied by a persistence of labeled heme and globin in the circulating red cells. There was actually a significant increase in the extent of labeling of these compounds in the cells. On the other hand, there was a marked decline in labeled ferritin asso- ciated with the disappearance of the reticulocytes.

Loss of isotopes from ferritin of the circulating cells might be due either to a loss of ferritin synthesis in maturing red cells or to a very fast turnover (continuing synthesis and breakdown). Furthermore, if the cells had matured, their ability to synthesize heme and globin should have declined. The results of the in- cubation in vitro of red cells collected from rats given injections of unlabeled reticulocytes are helpful in deriving answers to these questions, Fig. 3 demonstrates that the ability of the red cells to incorporate isotopes into heme, globin, or ferritin decreased throughout the experimental period as the reticulocytes disap- peared, showing that a loss of synthesis of these three compounds had taken place. Therefore, during the maturation process fer- ritin protein was degraded without accompanying synthesis, whereas heme and globin synthesis ceased without an accom- panying degradation.

Iron Release Mechanism in Reticulocytes and Hepatic Cell-For ferritin Fe to be a precursor of heme iron, the turnover of Fe in ferritinwould need to be quite rapid. To test this experimentally, rat reticulocytes were labeled with either Fe59 or Cl4 by incuba- tion for 30 minutes. Excess isotopes were removed by washing

l -Heme-Fe59

‘&~--FerritiNe5g

cj-Ferritin-C’4

24 48 12 Time (hours)

5.8 2.3 0.9 0.9 Retie. (%I

FIG. 4. Fate of Fes9 and Cl4 in heme, globin, and ferritin of labeled reticulocytes in vitro. Reticulocytes were labeled by incubation with serum-FeSg or 2-CWglycine, washed free of excess istope, and reincubated in fresh medium containing unlabeled serum or glycine.

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and the cells were resuspended in Krebs-phosphate medium con- taining unlabeled serum or glycine, respectively, for continued incubation. The results in Fig. 4 show a loss of both Fe59 and C?4 from ferritin, although the labeled heme and globin remained intact or rose slightly. These results are consistent with the in- terpretation that the release of Fe and degradation of the apo- ferritin moiety of ferritin are essentially simultaneous and rapid. Control experiments demonstrated that reticulocyte maturation was insignificant during this period of the incubation. Further- more, the results suggest that the release of iron from ferritin in the reticulocyte is associated with degradation of the protein, if the total ferritin content of the cells is assumed to remain con- stant. That this is so may be concluded from the results in Table IV, which demonstrate that the total ferritin content and Fe:N ratio remained constant for at least 1 hour of incubation.

Lw ,--- -A---

-witin-Cl4 100

00 60 120 180 Time (minutes)

FIG. 5. Fate of FeE9 and Cl4 in rat liver ferritin labeled in vitro. Surviving rat liver slices were incubated in Krebs-Ringer-phos- phate solution, pH 7.4, washed free of excess isotope, and reincu- bated in fresh medium with unlabeled serum or glycine.

The suggestion that iron is released from ferritin in the reticulo- cyte as a result of degradation of the protein moiety raises the question of the relative rates of turnover of ferritin iron and pro- tein in the hepatic cell, where the release of iron from this protein has been shown to be mediated via the action of the reduced form of the enzyme, xanthine dehydrogenase (8). Accordingly, aliquots of surviving liver slices from a normal rat were labeled by incubation with either serum-FeSg or CY-glycine, washed free of excess isotopes, and resuspended in fresh media containing un- labeled serum or glycine, respectively. The slices were incubated and specimens were removed for analyses at various time inter- vals. Fig. 5 demonstrates that although the Fesg content of the ferritin declined quite rapidly, the Cl4 content of the ferritin rose somewhat. These results suggest that the turnover of ferritin protein in the liver cell is slower than in the reticulocyte, and confirm our previous finding that the release of iron from hepatic ferritin is a specific reaction unrelated to protein breakdown.

E$ect of Inhibitors-In an attempt to interrupt selectively one of the reactions involved in the incorporation of serum-Fe59 into ferritin and heme, and in this way to show a relationship between these two kinds of incorporation, marrow suspensions and reticu- locytes were studied in the presence of a variety of metabolic in- hibitors. Since the effects of the inhibitors might be simply to prevent the entrance of isotope into the cell rather than to inhibit its incorporation, the cells were washed with NaCl solution after the incubation period, and total radioactivity of the cell was de- termined before separation of ferritin and hemin. The results are expressed as isotope in heme or ferritin as a percentage of that present in the total washed cell. The results in Table V demon- strate that each of the compounds inhibited incorporation of serum-FeSg into heme, whereas inhibition of incorporation into ferritin was produced only by dipyridyl and azide. The remain- ing compounds produced an apparent stimulation of serum-Fe59 incorporation into ferritin. The relative effects of these inhibi- tors are best seen by comparing the ratio of isotope in heme to that in ferritin. In the presence of dipyridyl or azide, incorpora- tion of Fe5g into heme was inhibited to a greater extent than into ferritin.

TABLE V

Effect of inhibitors on relative incorporation of serum-Fe69 into heme and ferritin of rat marrow and reticulocytes in vitro

Equal aliquots of cells were incubated for 1 hour in oxygen in the presence of serum-bound Fe59. Inhibitors were added immedi- ately before addition of the isotope. After incubation, the cells were washed free of excess isotope, and aliquots were removed for total count and for isolation of hemin and ferritin with the aid of carriers. Final concentrations of F- and azide were 5 X 10-a M, and of o-dinitrophenol, Co++, CN-, and a,a’-dipyridyl, 5 X 1OP M. The FeSg in the total cells is expressed as per cent of the Fe59 added to the incubation mixture, whereas heme and ferritin Fe69 are expressed as per cent of the counts in the total cell.

Inhibitor Total cells

q0 dose

Control. 28.3 o-Dinitrophenol. 14.4 Azide........................ 22.2 F- . . 25.2 CN- . . . . 30.6 co++ . . . . . 20.9 a,a’-Dipyridyl. . . 2.4 I -

70.6 63.1 32.2 63.2

5.6 20.9

6.1

% total 1.62 4.20 1.14 2.90 2.19 2.75 0.40

43.6 15.0 28.3 21.8

2.5 7.6

15.3

Total cells Heme Ferritin Heme Fe? Ferritin Fe69

70 dose

3.0 3.0 1.8 2.5 1.3 2.2 1.0

26.7 22.2 4.1

18.2 7.4

12.2 0.5

qo total 5.7 5.7 3.8 5.9

16.0 7.8 0.2

4.7 3.9 1.1 3.1 0.5 1.6 2.5

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Fig. 6 illustrates the effect of CN- on the time course of the reactions involving incorporation of serum-Fe59 into heme and ferritin of rat reticulocytes. In the presence of this inhibitor the incorporation of isotope into heme is markedly inhibited, whereas the incorporation of Fe5g into ferritin rises to values far above that in the control.

Effect of Lysis on Heme and Ferritin Synthesis-The use of hypotonic or hypertonic media for the incubation of marrow or reticulocytes resulted in marked loss of ability of these suspen- sions to incorporate serum-Fes9 into heme or ferritin. These re- sults are shown in Table VI for rat reticulocytes. In a great many attempts, addition of a large variety of compounds known to be required by homogenates of other cells for metabolic ac- tivity did not suceed in reversing the effect on the incorporation of serum-bound Fe5Q into heme.

Table VII demonstrates the effect of lysis of marrow and reticulocytes on the incorporation of serum-Fe5g as weil as of C14-glycine. Incorporation of Cl4 into free protoporphyrin as well as into heme, globin, and ferritin of reticulocytes and marrow was also markedly inhibited. Sddition of ascorbic acid and ATP was effective in reversing the inhibition of serum-Fe5Q incorpora- tion into ferritin without relieving the inhibition of Cl4 incorpora- tion into this protein, suggesting that this reversal was due to the incorporation of serum-Fe59 into preformed ferritin.

30 bo 120 180

FIG. 6. Effect of CN- (final concentration, 5 X 10m4 M) on the t.ime course of incorporation of serum-Fe5” into heme and ferritin of rat reticulocytes.

TABLE VI

Effect of tonicity of medium on incorporation of serum-Fe69 into heme and ferritin of rat reticulocytes

Reticulocyte-rich red cells from rats were incubated-in Krebs- Ringer-phosphate media in which the NaCl concentration was varied. At the end of I hour the Fe69 content of heme and fer- ritin was determined as described previously. Because of hemolysis in many samples, preliminary washing of cells was eliminated.

Concentration of NaCl Heme Fe” Ferritin Fe59

M c.p.m. c.p.w.

0.01 94 593 0.05 18,200 1,225 0.15 38,550 1,915 0.25 13,150 366 0.50 28 52

TABLE VII

Effect of cell lysis on isotope incorporation into heme, globin, and ferritin of marrow and reticulocytes

Equal aliquots of intact cells, or the equivalent of lysed cells, were incubated with serum-Fes9 or 2-CWglycine for 1 hour at 37” in oxygen, and the various fractions were isolated for isotope assay after addition of carrier. Ascorbic acid, 10 pmoles, and 20 Nmoles of ATP were added where indicated. The results are expressed as per cent of the counts per minute of isotope in the intact cells, for which the values are adjusted to 100.

Fe59 in Cl4 in

/ 1

.-

Reticulocytes : Intact cells.. 100 Lysed cells.. . 19 Lysed + ascor-

bate.. 15 Lysed + ATP. 8 Lysed + ascor-

bate + ATP.. 5 Marrow:

Intact cells. 100 Lysed cells. 10 Lysed + ascor-

bate.. 5 Lysed + ATP. 2 Lysed + ascor-

bate + ATP. 10

F&tin Heme Globin Ferritin

100 100 100 100 6 11 7 24

9 11 11 18 8 6 6 28

87

100 6

c

i

89

7

100 5

3 3

3

7

100 9

12 9

14

10

100 17

20 29

20 -

DISCUSSION

Free 8rotopor- phyrin

100 15

9 11

9

Electron photomicrographs by Bessis and Breton-Gorius (4, 5) reveal the presence in marrow of electron-dense particles charac- teristic of ferritin in reticuloendothelial cells in the vicinity of degraded red cells in the middle of islands of erythroblasts, in the erythroblast cell itself, and in reticulocytes. From these ob- servations it is difficult to assign a function to marrow ferritin, since it is not clear whether the ferritin arises from degraded red cell iron, from circulating plasma iron, or both, and leaves un- settled the question of the utilization of ferritin iron for heme synthesis. The results in Fig. 1 and Table I suggest a relatively rapid turnover for the iron atoms in marrow ferritin. Similarly, reticulocyte ferritin incorporated serum-bound Fes9, reaching a maximum and then declining at a time when the heme Fes9 con- tent was still rising. Specific act,ivities of heme and ferritin FeSQ of reticulocytes after 20 minutes of incubation with serum-Fe59 reveal that the ferritin molecule incorporated many more iron atoms than heme. These results are consistent with the sugges- tion that serum iron is first mostly incorporated into ferritin before its incorporation into heme. Stimulation of marrow eryth- ropoiesis by means of phenylhydrazine produces a marked in- crease in incorporation of serum-Fe59 into both heme and ferritin, in vitro as well as in viva, confirming the close relationship of heme and ferritin iron as well as the association of ferritin synthesis with the same cellular elements in the marrow which are con- cerned with heme synthesis, the erythroid series.

As the reticulocyte circulates in the blood it matures and loses its ability to synthesize heme, globin, and ferritin. At the same time, although heme and globin synthesized during the reticulo- cyte stage remain intact in the mature red cell, ferritin disap-

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May 1963 A. Mazur and A. Carleton 1823

pears. These results suggest that ferritin synthesis ceases and its degradation continues. On the other hand, the reticulocyte itself has an active system for synthesis and breakdown of ferri- tin, and measurement of FeSg and Cl4 during a “chase” experi- ment reveals that the rates of disappearance of these isotopes from reticulocyte ferritin are very similar. These results lead to the conclusion that the release of iron from reticulocyte ferritin is accompanied by a simultaneous breakdown of the protein moiety. This finding is in marked contrast with results obtained with liver slices in which ferritin had been labeled with Fe59 or (74. In the case of the liver cell, ferritin releases its FeSg but the protein moiety is only slightly affected. One may conclude that the re- lease of iron from hepatic ferritin is not associated with protein breakdown but is an independent process. This conclusion is in agreement with our earlier findings (10) that hepatic ferritin iron is released by reaction with reduced santhine dehydrogenase. These results are also of some interest since they present an ex- ample of a protein in which iron and protein moieties “turn over” at different rates in two different tissues of the same ani- mal. Whereas hepatic ferritin may be viewed as a storage form of iron in a dynamic sense, releasing and incorporating iron con- tinuously, marrow ferritin appears to function as an active iron storage protein and is intermediate between serum-bound iron and that which is incorporated into heme.

The results of experiments with metabolic inhibitors demon- strate that heme synthesis, as measured by iron incorporation, is inhibited to a greater extent than is the incorporation of iron into ferritin, leading in many instances to an accumulation of Fe59 in ferritin in amounts far above that in the uninhibited controls. These results are in agreement with the hypothesis that ferritin iron is the precursor for heme iron.

That endotoxin affects the marrow by interfering with the in- corporation of serum-Fesg into ferritin is clear, since the synthesis of the protein moiety, as measured by amino acid incorporation, is not impaired but actually stimulated in marrow taken from endotosin-treated animals. Both Fe5g and CWglycine incorpora- tion into heme are lower after injection of endotoxin than in the control animals. This may be due to an inhibition of proto- porphyrin synthesis or to a direct inhibition of iron incorporation into ferritin, which would normally serve as the precursor for heme iron. The effect of endotosin on rat marrow is in sharp contrast to its effect on the liver and spleen (3), in which iron incorporation from serum into ferritin in these tissues is markedly stimulated. The over-all effect of endotoxin in the rat is, there- fore, (a) an abnormal diversion of serum iron into the liver and spleen, where it is incorporated into ferritin, leading to (b) a de- crease in the quantity of serum iron available to the marrow, where (c) iron incorporation into ferritin and heme is also in- hibited. These effects lead to a decreased incorporation of serum iron into hemoglobin, as is evidenced by a much lower Fe59 content in red cells 24 and 48 hours after serum-Fe59 administra- tion to endotoxin-treated rats as compared to controls. If these effects were to be maintained for some time, one would guess that an anemia would result. However, repeated injections of endo- toxin in rats lead to the prompt appearance of circulating anti- bodies in the plasma (3).

Results of incorporation studies with lysed marrow cells and reticulocytes reveal a marked impairment of synthesis of heme and globin which is not reversed by addition of a large variety of cofactors. Since the incorporation of CWglycine into free proto- porphyrin in reticulocUytes was also inhibited as a result of cell

lysis, it is probable that heme synthesis is inhibited because of a lack of protoporphyrin. On the other hand, although lysis re- sults in a marked decrease in extent of incorporation of Fe59 into ferritin, recovery can be effected by supplying endogenous as- corbic acid and ATP to the lysed cells, suggesting that preformed ferritin protein is still available for combination with iron pro- vided that the latter is liberated from its linkage to transferrin by the action of ascorbate and ATP.

The results obtained in the present study are consistent with the suggestion that serum-bound iron is released to the marrow by the action of ATP and ascorbic acid in a manner similar to that previously reported for the liver cell (2, 3) and that the iron is mostly incorporated into ferritin. The results of experiments with reticulocytes indicate that the ferritin has a very rapid turnover, releasing its iron as the protein is degraded. As is shown in the following diagram,

ATP ascorbate

Transferrin-Fe+++, ’ Fe++1 protoporphyrin

> heme-Fe++

IL ferritin-Fe+++

the iron released by the ferritin either is incorporated into heme by combination with protoporphyrin or is returned to the serum. The latter conclusion is in agreement with the kinetic studies of Pollycove (II), who used serum-bound Fe59 to demonstrate the presence in the marrow of a “labile iron pool” which returns one- third of its iron to the plasma without being involved in erythro- poiesis. Some of our data support this conclusion when applied to the reticulocyte. One can compare the gain and loss of Fe59 of hemoglobin and ferritin, respectively, from the data in Table IV, for the period of 120 minutes of incubation of tagged reticulo- cytes after removal of excess isotope. Calculated on the basis of counts per minute per pmole, the gain in Fes9 by hemoglobin was 1,380 x 103 c.p.m. and the loss of Fe5g by ferritin was 20,640 x lo3 c.p.m., assuming 2,400 atoms of iron per molecule of ferritin and 4 atoms of iron per molecule of hemoglobin. One may assume that the differences found in the reticulocyte would be greater than that which would exist in the marrow cell, since the reticulocyte has already begun to show signs of loss of ability to synthesize protoporphyrin and globin without impairment of its ability to degrade ferritin. Although the nature of the [Fe++] proposed as a common precursor for ferritin and heme is un- known, the general formulation of this scheme is supported by the experimental data which have been presented.

SUMMARY

The relationship between ferritin iron and heme synthesis has been studied in marrow and reticulocytes in the intact rat and in systems in vitro by use of serum-bound FeSg and 2-CKglycine. The possibility that ferritin iron may be a storage form and pre- cursor for heme iron is supported by the following.

1. Serum-Fe59 is incorporated into marrow ferritin in viva, reaching a maximum at 4 hours, and then declining during a time when the heme Fe59 content continues to rise. Similar re- sults were obtained by incubating marrow cell suspensions or reticulocytes with serum-Fe59.

2. Stimulation of rat marrow erythropoiesis by use of phenyl- hydrazine results in a marked increase in the extent of labeling and a decrease in the time of maximal labeling for both heme and ferritin. On the other hand, treatment of rats with bacterial

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1824 Ferritin Iron and Heme Syndhesis Vol. 238, No. 5

endotoxin results in a marked inhibition in isotope incorporation into both heme and ferritin. Studies in vitro with marrow from rats treated with injections of endotoxin reveal that the inhibition of incorporation of Fe5g into ferritin is not due to an inhibition of ferritin protein synthesis as measured by U4-glycine incorpora- tion.

3. Determination of relative specific activities of Fejg in heme and ferritin 20 minutes after exposure of reticulocytes to serum- Fes9 demonstrates that ferritin contains many more Fesg atoms per molecule than does hemoglobin (calculated from heme).

4. As reticulocytes, labeled with Fe59 and W, circulate in the blood of the rat, they undergo maturation with an accompanying disappearance of both isotopes from ferritin, although the iso- topes in heme and globin persist. Studies in vitro with circulat- ing unlabeled reticulocytes reveal that they lose their ability to incorporate Fe59 or C?4-glycine into heme, globin, or ferritin as they mature.

5. Addition of a variety of metabolic inhibitors to suspensions of marrow cells or reticulocytes results in a more marked inhibi- tion of serum-Fe59 incorporation into heme than into ferritin. Indeed, in several cases, this effect leads to an accumulation of Fe59 in ferritin far above that found in the uninhibited controls.

6. Lysis of marrow or reticulocyte cells results in a loss of ability of these cells to incorporate serum-Fe59 into heme or ferri- tin. Lysis also leads to a loss of incorporation of C14-glycine into heme, globin, and ferritin. Addition of ascorbic acid and adeno- sine triphosphate to the lysed cells stimulates the incorporation

of serum-Fe5g into ferritin but not into heme, suggesting that the damage to the protoporphyrin synthetic mechanism had not been reversed and that the incorporation of Fe59 into ferritin was due to ferritin present at the time of lysis.

Acknowledgments-The skillful assistance of Ann Carlsen and Margaret Toth are gratefully acknowledged.

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3. MAZUR, A., CARLETON, A., AND CARLSEN, A., J. Biol. Chem., 236, 1109 (1961).

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11. POLLYCOVE, M., in R. 0. WALLERSTEIN and S. R. METTIER (Editors), Iron in clinical medicine, University of California Press, Berkeley, 1958, p. 43.

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Abraham Mazur and Anne CarletonRelation of Ferritin Iron to Heme Synthesis in Marrow and Reticulocytes

1963, 238:1817-1824.J. Biol. Chem. 

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