JOURNAL OF NUCLEAR MEDICINE 7:928-934, 1966

in Vivo Reticulocyte Radioiron Assimilation

R. M. Donati,1 M. A. Warnecke' and N. I. Gallagher'

St. Louis, Missouri

INTRODUCTION Investigation of the pathway of iron assimilation by reticulocytes has led to confficting conclusions (1-6). Bessis and Breten-Gorius (6) have suggested that erythrocyte iron is obtained during maturation primarily frem macrophages in the marrow. In contrast, other investigators utilizing in vitro techniques have shown that iron may be directly assimilated by the reticulocyte from the iron binding protein, transferrin (1-5). The quantitative significance of these alternate pathways is uncertain. Moreover, the latter studies utilizing in vitro techniques may not accurately reflect the true in vivo mechanism. This report presents an in vivo technique for reticulocyte study and details observations made on reticu locyte iron dynamics.

MATERIALS AND METHODS Female Sprague-Dawley rats were maintained on Purina Laboratory Chow,2 except as indicated. Reticulocytosis was produced in 300-350 gm rats by with drawal of 3-4 ml of blood by cardiac puncture on four consecutive days. The rats were exsanguinated on the sixth day and reticulocytes in excess of 20% were ob tained. Plasma was separated by centrifugation and the cells were washed and resuspended in an equal volume of 0.9% saline solution. In contrast, a red blood cell pool containing less than 0.1% reticulocytes was prepared by transfusion of 300-350 gm rats with one-half of their total red blood cell mass. Starvation for 96 hours was added for its anti-erythropoietic effect. After starvation, these rats were exsanguinated and the red blood cells washed and resuspended as described above.

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Rats weighing 180-200 gin were initially transfused via the tail vein with packed red blood cellsequivalenttoone-halftheircalculatedtotalred blood cell mass (8); starvation was then initiated. Two mis of the reticulocyte rich suspen sion or 2.0 mis of a reticulocyte poor suspension was injected intravenously on the beginning of the fifth day following the initiation of starvation. Tail vein in jection of 2.0 @sC59Fe/2.0 @g56Fe was made either 20 minutes or six hours after the reticulocyte transfusion.

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Groups of ten animals each were killed at 15, 30, 60, 90 and 120 minutes following the 59Fe injection. The reticulocytes were enumerated and serum iron levels (9 ) and plasma radioiron clearances determined by methods previously described ( 8 ) . Other groups of five animals each were killed at zero hours, 2 hours, 6 hours and 18 hours following the injection of 59Fe and the red blood cell radioiron utilization determined ( 10) prior to and following incubation of cells in 0.5% ethylene diaminetetraacetic acid in a buffered 0.9% saline solution.

RESULTS Results of the erythrocyte radioiron incorporation are presented in Tables I and II. Regardless of the interval between transfusion and the administration of radioiron, the animals transfused with a 20% reticulocyte suspension incorporated more radioiron in the erythrocytes than those transfused with a reticulocytopenic suspension. Comparable reticulocyte counts were attained in both experiments. When radioiron was administered twenty minutes after reticulocyte transfusion (TableI ), thetransfusedreticulocyteshadincorporated11.9±1.5%'of injected 59Fe in two hours. Reticulocytopenic animals had only 0.7 ±0.1%1of the radioiron in their peripheral blood at this time. At six hours, these values were 18.2 ±0.4% and 0.9 ±0.2%' and at 18 hours, 19.8 ±2.9%' and 1.5 ±0.7%' respectively (Table I). When radioiron was administered six hours after transfusion, (Table II), the difference in red blood cell radioiron incorporation between the reticulocyte rich and the reticulocyte poor animals was diminished when compared to the animals given radioiron 20 minutes following transfusion. Serum iron values in the group of animals given 59Fe 20 minutes following transfusion, are presented in Table III. The serum iron level diminished after reticulocyte transfusion, but remained constant at a higher level in the reticulo cytopenic group of animals. Comparison of the radioiron incorporation of eryth rocytes prior to and following incubation with E.D.T.A. solution demonstrated a difference of less than 1.2% in all instances. Animals transfused with reticu locytes exhibited an accelerated (T3@ = 94 mm.) plasma radioiron clearance (Fig. 1) when compared to the reticulocytopenic animals (Th = 124 mm.).

DISCUSSION

The ability to assimilate iron is characteristic of all red blood cell precursors; however, the degree of iron assimilation appears to be an inverse function of cellular maturity. Thus, the pronormoblast and the basophilic normoblast in corporate the most iron and the reticulocyte the least (11). Extensive investigations of the capacity of the reticulocyte to assimilate iron have been carried out (1-5), yet the mechanism of incorporation remains con troversial. On the basis of electron photomicrographic studies, Bessis and Breten Gorius (6) have suggested that iron is obtained primarily from macrophages by erythrocytic precursors and, thus, iron would have to pass through the marrow compartment prior to assimilation by immature red blood cells. In vitro studies

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by Walsh and associates ( 1), Jandl et al., (2) Pollycove (4) and Morgan and Laurell ( 5 ) have demonstrated that iron may be transferred directly from trans ferrin to the reticulocyte. In vivo studies (4) demonstrated rapid reticulocyte labelling; however, endogenous erythroid marrow activity was not suppressed. In the present study, an in vivo technique in which endogenous erythropoiesis was suppressed was utilized to study reticulocyte iron dynamics. The major variable between groups of animals was a reticulocytosis produced by trans fusion. In all instances, the reticuiocyte replete animals demonstrated increased red blood cell radioiron assimilation as compared to the reticulocyte depleted animals. When the injection of radioiron was delayed until six hours following reticulocyte transfusion, the erythrocyte radioiron incorporation was diminished. This differ ence is apparently a function of reticulocyte maturation into erythrocytes in the circulation and consequent of cessation of red blood cell iron uptake from trans ferrin. The time sequence of reticulocyte iron assimilation is substantially in agreement with in vitro studies. Prior studies ( 11 ) which demonstrated that only 13% of an injected dose of radioiron localized in the marrow of the starved rat and, of even more import, the time sequence of reticulocyte iron incorpo ration demonstrated in the present study, suggest that iron may be assimilated directly by the circulating reticulocytes without traversing the marrow compart.. ment. Coincident with the increase in the reticulocyte radioiron incorporation, the radioiron plasma clearance and the serum iron levels of the reticulocyte frans fused animal diminished (Table III). The serum iron values in the reticulocyto.. penic animals remained stable. These iron changes are small and apparently re lated to erythrocyte radloiron incorporation. The persistence of the lowered serum iron is of interest and its explanation is presently not apparent. Alterations in iron metabolism secondary to transfusion-induced polycythernia and subsequent star vation may explain this inability to restore plasma iron to original levels in this short time interval. On the basisof thesestudies,we suggestthe followingsequence of in vivo reticulocyte iron assimilation. The peripheral reticulocyte picks up iron which does not necessarily have to traverse the marrow compartment in order to be assimilated by the reticulocyte. Coincident with the assimilation of iron by the reticulocyte, the plasma iron levels diminished and the plasma iron clearance rate accelerated.

SUMMARY

An in vivo technique for the study of reticulocytes is presented. The capacity of transfused reticulocytes to assimilate radioiron in vivo was studied. The reticu locyte incorporates iron in vivo which does not necessarily have to traverse the marrow compartment in order to be incorporated. Coincident with reticulocyte iron incorporation the serum iron levels diminish, and the T % plasma clearance rate shortens. IN VIVO RETICULOCYTE RADIOIRON ASSIMILATION 933

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REFERENCES

1. WALSH, R. J., THOMAS, E. D., CHOW, S. K., FLUHARTY, R. C., AND FINCH, C. A.: Iron Metabolism. Heme Synthesisin Vitro by Immature Erythrocytes.Science 110:396, 1949. 2. JANDL, J. H., INMAN, J., SIMMONS, R. L., AND ALLEN, D. W.: Transfer of Iron from Serum Iron-Binding Protein to Human Reticulocytes, I. Gun, invest., 38:161, 159. 3. POLLYCOVE, M. AND MAQSOOD, M.: Existence of an Erythropoietic Labile Iron Pool in Animals. Nature (Lond.), 194:152, 1962. 4. POLLYCOVE, M.: Iron Kinetics, Ed. F. GROSS, Berlin, Springer-Verlag, 1964. 5. MORGAN, E. H. AND LAURELL, C. B.: Studies on the Exchange of Iron Between Trans ferrin and Reticulocytes. Brit. I. Haemat. 9:471, 1963. 6. BEssis, M. C. AND BRETON-GORIUS, J.: Iron Particles in Normal Erythroblasts and Normal and Pathological Erythrocytes. I. Biophys. Biochem. Cytol., 3:503, 1957. 7. BRECHER,C. AND CRONKITE,E. P.: Morphology and Enumeration of Human Blood Platelets. I. Appi. Physiol., 3:365, 1950. 8. TRINDER, P.: The Improved Determination of Iron in Serum. J. Gun. Path., 9:170, 1956. 9. DONATI, R. M., CHAPMAN, C. W., WA.1INEcKE, M. A., AND GALLAGHER, N. I.: Iron Metabolism in Acute Starvation.Proc. Soc. Exper. Biol.Med. 117:50, 1964 10. GALLAGHER, N. I., HAGAN, D. Q., MCCARTHY, J. M., AND LANGE, R. D.: Response of Starved Rats and Polycythemic Rats to Graded Doses of Erythropoietin. Proc. Soc. Exp. Biol. Med., 106:127, 1961. 11. SUIT, H. D., LAJTHA, L. C., OLIVER, R., AND ELLIS, F.: Studies on the 59Fe Uptake by Normoblasts and the Failure of X-Irradiation to Affect Uptake. Brit. I. Haemat. 3:165, 1957.