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THE ROLE OF IRON IN HEMOGLOBIN SYNTHESIS CLEMENT A. FINCH In comparison with the preceding elegant chemistry, this discussion cannot help but seem both diffuse and obscure. Having made the obvious statement that iron is essential for heme formation and after a few random observations on the behavior of the cell in accomplishing this synthesis, I find that most of my own thinking on this subject extrapolates to hemoglobin production in the intact animal. An attempt will be made to summarize briefly the information relating to the role of iron in hemoglobin synthesis at both a cellular level and as it functions in the total erythropoiesis of the intact organism. At a cellular level the pathway of iron incorporation into hemoglobin may be visualized as starting with the presentation of iron to the cell. In vitro various soluble ferrous or ferric salts of iron are easily assimilated by immature erythrocytes. Some iron chelates such as iron ascorbate or citrate permit uptake, whereas others such as ethylenediamine tetraacetate do not. In viva the transport vehicle is an iron-binding plasma protein called trans- ferrin or siderophilin.~ While this transport globulin may be considerably reduced in such diseases as nephrosis and infection, this decrease has never been demonstrated to be a limiting factor in the delivery of iron to the marrow. All immature red cells through the reticulocyte stage are able to take up iron, while mature erythrocytes do note 3 The pronormoblast and baso- philic normoblast have the greatest capacity to assimilate iron as determined by autoradiograph.4 5 This uptake of radioiron continues in situations where heme synthesis within the cell is arrested by lowering the temperature, or by addition of cyanide or deoxypyridoxine.6 Iron transfer from transferrin to the immature erythrocyte in vitro is reported to be decreased when the per cent of saturation of the iron binding protein drops below 30~.7 The iron assimilated by the developing red cell is either converted to heme, temporarily stored, or remains permanently as a non-heme f faction within the erythrocyte. Storage iron may be demonstrated as specific granules within the developing red cell. Such cells have been referred to as sidero- blasts when nucleated, and siderocytes when non-nucleated.S 9 These iron stores may be useful to insure adequate available iron during the phase of maximal heme synthesis within the cell. There is evidence that their amount may be influenced by the level of plasma iron, and that they may in turn in- fluence the amount of residual non-heme iron found in the adult circulating erythrocyte.6 In avian and mammalian red cells this fraction may be as much asSor10~.6 it The red cell nucleus would seem to be intimately concerned with hemo- globin synthesis. Appreciable amounts of radioiron are found in the nucleus as

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96 PART II. BIOSYNTHESIS OF HEMOGLOBIN shortly after its uptake by the cell.)' i~ i4 At the time when the nucleus dis- appears from the cell, heme synthesis is nearly complete. The biochemical aspects of heme synthesis have already been discussed at some length from the standpoint of the porphyrin moiety. Granick has pre- sented evidence from studies of porphyrin synthesis by bacteria that vinyl groups of the porphyrin ring are essential for insertion of iron.~5 Goldberg et al. from studies employing a hemolysate system of erythrocytes suggest that iron enters at the protoporphyrin stage of pyrrole synthesis. General observations of conditions required for iron incorporation into heme in vitro with intact cells have been presented by Sharpest and by fensen and asso- ciates: which serve to distinguish this process by its enzymatic character from the uptake of iron by the cell. In man there are both hereditary anomalies and acquired defects in red cell production which, if understood, should do much to clarify the mechanics of heme synthesis and the role of iron in this process. Most helpful in ap- praising heme synthesis in To has been the cell content of hemoglobin pre- cursors either in the marrow or in circulating erythrocytes. Normally these are nicely regulated so that stockpiles of porphyrin, iron and globin become depleted as hemoglobinization of the cell is completed (fig. 1~. In specific NORMOBLAST ~ RETI CU LOCYTE Pro Baso Eosino Nucleic Acid Iron Granules Porphyri n Globin Hemoglobi n FIG. 1.- Diagram showing depletion of hemoglobin precursors and nucleic acid with normal formation of hemo- globin. abnormalities a deficiency or excess of these may be found. For example, ashen the supply of iron to the marrow is restricted, iron granules (storage irony within the normootast disappear, protoporpnyr~n accumulates and the total hemoglobin production is reduced, resulting in a hypochromic, microcytic erythrocyte. One interesting aspect of chronic iron deficiency anemia is that erythroid hyperplasia is less marked than with acute blood loss anemia or with hemolytic anemias. This raises the possibility that iron may be of importance in multiplication of erythroid cells as well as in heme syn- thesis. In the intact animal a consideration of the role of iron includes a considera- tion of iron supply to the marrow. Normally, this is largely derived from a O 1 1 , At' . 1 1 . 1

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IRON IN HEMOGLOBIN SYNTHESIS FINCH 97 recircuiting of iron from senescent red cells processed by the reticulo-endo- thelial cells and returned through the plasma to the marrow. Studies of the reticulo-endothelial cell indicate that not only is this done within a matter of minutes or a few hours, but also that in the event of an increased need for iron above that provided by erythrocyte catabolism, additional iron is mobilized from body cells. Thus, following acute hemorrhage plasma iron rises at a time when marrow uptake is increasing. While infection would appear to impair reticulo-endothelial mobilization of iron, even here we have been unable to obtain convincing evidence that erythropoiesis is impaired by inadequate iron supply. Thus we conclude that if adequate iron exists in the body, transport to the marrow will be effected. One of the most useful techniques for measuring hemoglobin synthesis in the intact animal or man involves the use of radioiron. The plasma iron turnover, i.e., the amount of plasma iron being fed to tissues, has been shown largely to reflect marrow uptake.~7 The amount of iron appearing in circu- lating erythrocytes indicates how much of this iron was synthesized into hemoglobin within viable erythrocytes. The marrow transit time of radioiron indicates the time required for the process of iron incorporation and red cell maturation.iS Studies of this type indicate that in certain anemiassuch as thalassemia, pernicious anemia and some "refractory" anemias with cellular marrow the iron uptake by the marrow far exceeds the production of viable red cells.~'3 Further study is required to determine to what extent this "ineffective" ery- thropoiesis reflects a defect in fabrication of the cell as compared to abnormality in heme synthesis. Marrow iron transit time is also greatly shortened in certain hemolytic anemias and marrow abnormalities. Part of this is due to the premature delivery of young cells into circulation. Indeed, the total reti- culocyte pool of the marrow may be shifted to the blood under certain con- ditions. It is not known whether or not an acceleration in the rate of heme synthesis within the individual cell also contributes. We might consider what clinical diseases may be associated with abnormali- ties in heme synthesis. This is well documented in lead poisoning where i'2 vitro impairment of porphyrin synthesis has been demonstrated, and where red cells contain unused precursors of heme synthesis, i.e., porphyrin and iron. Likewise, these are isolated case reports in which excess non-heme iron deposits and red cell hypochromia have been demonstrated,20 which strongly imply a primary disturbance in heme synthesis. On the assumption that a decreased hemoglobin concentration within the erythrocyte indicates specifically impaired hemoglobin synthesis, attention has been directed toward thalassemia, pyridoxine and copper deficiencies. Hypochromia may also be seen in patients with hemolytic anemia, adequate iron stores, low plasma iron, but rapid marrow iron turnover. We have encountered this in acquired Coombs-positive hemolytic anemia, sickle-cell anemia and myelofibrosis with myeloid metaplasia.

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98 PART II. BIOSYNTHESIS OF HEMOGLOBIN Erythropoiesis does not appear to be influenced in these patients by iron ad- ministration, but heme synthesis may be relatively curtailed by limited red- cell iron stores in similar fashion to that observed in v~tro.6 7' 21 Some caution, however, should be exercised in considering hypoctiromia as synonymous with impaired heme synthesis. Ire recent studies carried out in collaboration with Dr. Phillip Sturgeon, it has been found that iron turn- over, presumably reflecting for the most part heme synthesis, reached maximal levels in thalassemia despite the marked hypochromia of this diseased The relation of microcytosis without hypochromia to heme synthesis is even less clear. In these remarks no attention has been directed to the abundant informa- tion relating to pathogenesis and recognition of the iron-deficient state which after all is the one condition in which the role of iron in heme synthesis is clearly illustrated. This would seem to represent secure ground from which we should look to more unsettled areas. At present we cannot discern any Fraternal breakdown in iron metabolism, either of iron transport or of cellular handling of iron. There are, however, indications of block in heme synthesis in the accumulations of precursors of the heme molecule in a number of diseases. The ferrokinetic studies which have done much to characterize marrow iron turnover in the intact animal now need to be extended to the specific chemical reactions of the cell. Only then will the full significance of the gross chemical and morphologic abnormalities in else red cell be under- stood. REFEREN CES 7. 8. Laurell, C. B.: What is the function of transferrin in plasma? Blood 6: 183-187, 1951. Hahn, P. F., Ross, J. F`., Bale, W. F`., Balfour, W. M. and Whipple, G. H.: Red cell and plasma volumes (circulating and total) as determined by radio iron and by dye, J. Exper. Med. 75: 221, 1942. 3. Lash, R. J., Thomas, E. D., Chow, S. K., F`luharty, R. G. and Finch, C. A.: Iron metabolism. Heme synthesis in Vitro by immature erythrocytes, Science 110: 396-398, 1949. 4. La jtha, L. G. and Suit, H. D.: Uptake of radioactive iron (Fe59) by nucleated red cells in Vitro' Br. J. Haemat. 1: 55-61, 1955. 5. Kraus, L. HI. and Morrison, D. B.: In Vitro incorporation of iron59 into hemo- ~lobin S visualized by autoradiography, Proc. Soc. for Exper. Biol. and Med. 89: 598-602, 1955. 6. Jensen, W. N., Ashenbrucker, H., Cartwright, G. E. and Wintrobe, M. M.: The ur!tal~e in Vitro of radioactive iron by avian erythrs~cytes, J. Lab. Clin. Med. 42: 833-846, 1953. . Jandl, J. H., Inman, J. K. Sirr.mons, R. L.: Transfer of iron and cobalt from serum iron-binding protein to human reticulocytes, Clin. Res. Proc 5: 144, 1957. Douglas, A. S., Dacie, J. V.: The incidence and significance of iron-containing granules in human erythrocytes and their precursors, J. Clin. Path. 6: 307-313, 1953.

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IRON IN HEMOGLOBIN SYNTHESIS FINCH 99 9. Kaplan, E., Zuelzer, W. W. and Mouriquand, C.: Sideroblasts. Study of stainable nonhemoglobin iron in marrow normoblasts, Blood 9: 203-213, 1954. 10. Jensen, W. N., Bush, J. A., Ashenbrucker, H., Cartwright, G. E. and Wintrobe, M. M.: The kinetics of iron metabolism in normal growing swine, J. Exper. Med. 103: 145-159, 1956. 11. Sharpe, L. M., Krishnan, P. S. and Klein, J. R.: Uptake by duck erythrocytes of iron added to blood, Arch. Biochem. and Biophys. 35: 409, 1952. 12. Mills, fI., Huff, R. L., Krupp, M. A. and Garcia, J. F.: Hemolytic anemia sec- ondary to a familial (hereditary) defect in hemoglobin synthesis, Arch. Int. Med. 86: 711-726, 1950. 13. Metcalf, W. K.: A simplified technique of spectrography and its application to the study of intracellular hemoglobin, Blood 6: 1114-1122, 1951. 14. Allfrey, V. and Mirsky, A. E.: The incorporation of Ni5-glycine by avian ery- throcytes and reticulocytes in vitro, J. Gen. Physiol. 35: 841-846, 1952. 15. Granick, S.: The structural and functional relationships between heme and chlorophyll, Harvey Lectures 44: 220-245, 1948, Academic Press, New York. 16. Goldberg, A., Ashenbrucker, H., Cartwright, G. E. and Wintrobe, M. M.: Studies on the biosynthesis of heme in Vitro by avian erythrocytes, Blood 9: 821-833, 1956. 17. Bothwell, T. H., Hurtado, A. N1., Donohue, D. M. and Finch, C. A.: Erythro- kinetics IV. Plasma iron turnover as a measure of erythropoiesis. Blood (in press ) .- 18. Finch, C. A.: Marrow iron turnover. Int'l Cong. of Hemat., 1954. 19. Giblett, E. R., Coleman, D. H., Pirzio-Biroli, G., Donohue, D. M., Motulsky, A. G. and :Finch, C. A.: Erythrokinetics: Quantitative measurements of red cell pro- duction and destruction in normal subjects and patients with anemia, Blood 4: 291-309, 1956. 20. Garby, L., Sjolin, S. and Vahlquist, B.: Chronic refractory hypochromic anaemia with disturbed haem-metabolism, Brit. J. Haemat. ]: 55-67, 1957. 21. Krueger, R. C., Melnick, I. and Klein, J. R.: Formation of heme by broken-cell preparations of duck erythrocytes, Arch. Biochem. and Biophys. 64: 302-310, 1956. 2Z. Sturgeon, P. and Finch, C. A.: Erythrokinetics in Cooley's anemia, Blood 73, 1957. 64-