NOTE 

Annals of Nuclear Medicine Vol. 6, No. 1, 59-62, 1992 

Application of 67Ga for the estimation of reticulocyte production 

Yasuhito OHKUBO,* Shigeto TAKASU,* Midori ITAKURA,* Shinsuke KATOH,* Akiko SASAYAMA,* Hiroyuki KOHNO* and Akiko KUBODERA** 

*Department of Radiopharmacy, Tohoku College of Pharmacy 
**Department of Radiopharmacy, School of Pharmaceutical Science, Science University of Tokyo 


In order to estimate the production of reticulocytes, which have a larger number of transferrin receptors than erythrocytes, we used 67Ga which is exclusively bound to transferrin in the blood. The pattern of uptake of 67Ga by reticulocytes was quite similar to the time course of transglutaminase activity which might be involved in receptor-mediated endocytosis. The preinjection of Fe3+ decreased the uptake of 67Ga by reticulocytes. These results suggested that 67Ga in a transferrin-bound form was taken up by reticulocytes via receptor-mediated endocytosis. It was showed that the application of 67Ga is very easy and useful for the estimation of reticulocyte production. 

Key words: 67Ga uptake, transferrin, reticulocyte production 


INTRODUCTION 

IT HAS BEEN PRODUCED that 67Ga, a diagnostic agent of tumor1 and inflammation,2,3 was exclusively bound to transferrin in the blood.4,5 It is well known that transferrin is the major serum iron-transport protein.6 Transferrin provides the developing erythroid cells, which require a large amount of iron for heme synthesis, with iron.7-9 Reticulocytes have a larger number of transferrin receptors than erythrocytes.10 Reticulocytes have been used for the study of receptor-mediated endocytosis,11 cell proliferation, and cell differentiation.10 In order to extend these studies further, it is necessary to estimate reticulocyte production, that is to evaluate the number of transferrin receptors produced. Ordinarily radio-labeled transferrin is employed for these studies. For radio-labeling of transferrin, the method of iodination12 with 125I or reductive methylation13 with 3H has been employed. In the present study, we used 67Ga by which transferrin was labeled in vivo instead of 125I- or 3H-labeled transferrin. Consequently we attempted 

 Received June 10, 1991, revision accepted October 25, 1991. 
 For reprints contact: Yasuhito Onkubo, Department of Radiopharmacy Tohoku College of Pharmacy, 4-4-1 Komatsushima, Aoba-ku Sendai, 981, JAPAN. 

to more readily estimate reticulocyte production with 67Ga. 


MATERIALS AND METHODS 

Animals: Male Wistar rats weighing 150-200g were purchased from Shizuoka Laboratory Animal Center (Hamamatsu, Japan), and were housed in wire mesh cages at a room temperature of 23+-1deg.C and a relative humidity of 55+-5%. 
 Chemicals: Phenylhydrazine chloride and ferric chloride were purchased from Nakarai Tesque (Japan). [1, 4-14C] Putrescine dihydrochloride (14C-PUT, specific activity=3.87 GBq/mmol) was obtained from New England Nuclear (USA). N, N,-Dimethylcasein was purchased from Sigma (USA). All other reagents were of analytical grade. 
 Production of reticulocytes: Rats were intraperitonealy administered with phenylhydrazine hydrochloride (40 mg/kg body weight/day) for 3 days. One to five days after the last (the third) administration, blood was collected from the abdominal vein, with heparin as an anticoagulant. 
 Administration of 67Ga: Gallium-67 citrate (kindly supplied by Daiichi Radioisotope Laboratory Ltd, Tokyo, Japan) was diluted with saline to 185 kBq/ml. Each rat was intravenously injected with 67Ga in a dose of 37 kBq (200ul). 
 Preparation of 125I-transferrin: 125I-transferrin (125I-Tf) was prepared by the method described previously.14 Each rat was intravenously injected with 125I-Tf in a dose of 5 kBq (200 ul). 
 Administration of cold-FeCl3: Each rat was intraveneously injected with 100 ul of cold-FeCl3 (2.50 umole/ml saline) 5 min before the administration of 67Ga. 
 Determination of radioactivity: Four hours after the injection of 67Ga or 125I-Tf, the reticulocytes were collected from the abdominal vein of rats given phenylhydrazine, were washed twice with ice-cold saline and the buffy coat was removed during the washing. The radioactivity of the cells was determined with a well-type NaI-scintillation counter (Aloka, ARC-300). The ratio of uptake of 67Ga in reticulocytes was expressed in the following formula: Uptake ratio=A/B 
  A=sample activity (cpm)/sample weight (g) 
  B=total activity administered (cpm)/rat body weight (g). 
 Fractionation of reticulocyte cytosol: After hemolyzing cells with hypo-osmotic buffer (3 mM Tris, 1 mM EDTA, 0.5 mM dithiothreitol), the lysate was centrifuged at 20,000xg for 40 min. This process was repeated twice. Supernatants were collected and then used as the cell cytosol fraction. 
 Determination of transglutaminase activity: The transglutaminase activity of cell cytosol fraction from red blood cells was assayed by the incorporation of 14C-PUT into N, N'-dimethylcasein by the filter paper technique described by Lorand et al.15 with minor modifications. The final assay reaction mixture contained 50 mM Tris-HCl (pH 7.4), 10 mM dithiothreitol, 10 mM (CaCl2, 0.5 mg of N, N'-dimethylcasein, 1 mM PUT (including 1.85 kBq of 14C-PUT), and 10 ul of the sample solution. The reaction was initiated by the addition of a sample solution of cell cytosol fraction, and was carried out at 37deg.C in a total volume of 100 ul. The enzyme reaction was terminated by spotting a 20 ul volume of the mixtures onto a Whatman 3MM filter paper which was immersed in 10% trichloroacetic acid (TCA) solution and fixed on a multi vacuum-filter unit, and the filter paper was washed twice with 1 ml volume of 10% TCA solution. The filters were transferred to scintillation counting vials and 5 ml of scintillation counting fluid (ASC II, Amersham) was added. Radioactivity was determined with a liquid scintillation counter (Beckman, LS-7800). Determination of protein content of red blood cell cytosol fraction was carried out by the method of Bradford et al.16 with bovine serum albumin used as a standard. The enzyme activity was expressed as nmol PUT incorporation into dimethylcasein min-1 mg protein-1. 


RESULTS 

Figure 1 shows the uptake of 67Ga by reticulocytes at various days after the last administration of phenylhydrazine solution. The uptake of 67Ga by the cells reached the maximum at 3 days after the last administration of phenylhydrazine solution and the uptake ratio was about 6 times that for normal rat erythrocytes. The preinjection of FeCl3 remarkably decreased the uptake of 67Ga by the cells. This result shows that 67Ga had bound to transferrin. 
 Figure 2 shows the uptake of 125I-transferrin by reticulocytes at various days after the last administration of phenylhydrazine solution. The uptake of 125I-transferrin by the cells of rats given phenylhydrazine gradually increased and reached the maximum at 3 days after the last administration of phenylhydrazine solution. 
 Figure 3 shows that transglutaminase (TGase) activity of the cytosol fraction of reticulocytes at various days after the last administration of phenylhydrazine solution. The activity reached the maximum at 3 days after the last administration of phenylhydrazine. 


DISCUSSION 

Tavassoli et al. reported that phenylhydrazine affected cell proliferation in the regenerating marrow stroma implanted.17 It has also been reported that reticulocytes induced by the injection of phenylhydrazine had transferrin receptors and as the reticulocytes matured, the density of the receptors on the surface decreased.10 There must be a close relation between iron uptake and both cell proliferation and differentiation. Therefore, reticulocytes can be used as a good tool for the investigation of cell proliferation and differentiation. It was reported that transferrin might be taken up by rat reticulocytes via receptormediated endocytosis.11 It has been reported that transglutaminase, which is a calcium-dependent enzyme and catalyzes the covalent cross-linking of proteins, might be involved in receptor-mediated endocytosis.18,19 These reports show that transglutaminase must be related to transferrin internalization into reticulocytes. In the present study, transglutaminase activity reached the maximum at 3 days after the last administration of phenylhydrazine solution. This suggests that the receptor-mediated endocytosis on reticulocytes is maximum at 3 days after the last administration of phenylhydrazine solution. The uptakes of 125I-transferrin and 67Ga by reticulocytes also reached the maximum at 3 days after the last administration of phenylhydrazine solution, and the pattern of uptake of 67Ga by reticulocytes was very similar to the time course of transglutaminase activity of the cells. Moreover, 67Ga in a transferrin-bound form might be taken up by reticulocytes since Fe3+ decreased the uptake of 67Ga by reticulocytes. These results suggested that 67Ga in a transferrin-bound form was taken up by the cells via receptor-mediated encocytosis. Consequently, it was suggested that reticulocyte production reached a maxirnum at 3 days after the last administration of the phenylhydrazine. As 67Ga is injected into the blood, 67Ga is immediately bound to transferrin. Therefore, it is not necessary to label transferrin with a radioisotope, e.g., 125I or 3H, in advance in vitro since transferrin is readily labeled with 67Ga in vivo. Moreover, the pattern of 67Ga uptake by reticulocytes is sharper than that of 125I-transferrin uptake. This may be because the 67Ga taken up into reticulocytes in a transferrin-bound form is removed from transferrin at an intracellular site and is accumulated in reticulocytes. The concentration of 67Ga in the blood at 4 h after subcutanous injection is nearly identical to that at 4 h after intravenous injection.20 Therefore 67Ga has the advantage of being easily injected, whereas radiolabeled transferrin must be intravenously injected. Moreover, the determination of 67Ga activity is easier than that of 3H activity, for which preparation of the sample, e.g., oxidation or solubilization, is needed. From the results of this study we think that the application of 67Ga is very easy and useful for the estimation of reticulocyte production. Furthermore, 67Ga will be available for other studies of physiological events in which transferrin is involved. 


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