ORIGINAL 
Annals of Nuclear Medicine Vol. 2, No. 2, 59-62, 1988 
The effect of FeCl3 on the accumulation of gallium-67 into inflammatory and normal tissues 
Yasuhito OHKUBO,* Sayuri ARAKI,* Kenichi ABE,* Shigeto TAKASU,* Hiroyuki KOHNO* and Akiko KUBODERA** 
*Department of Radiopharmacy, Tohoku College of Pharmacy **Department of Radiopharmacy, School of Pharmaceutical Sciences, Science University of Tokyo 
The effect of FeCl3 on the uptake of 67Ga by inflammatory and normal tissues was studied to clarify the role of transferrin in 67Ga uptake by inflammatory tissue. The administration of FeCl3 5 min before the injection of 67Ga decreased the uptake of 67Ga by liver and spleen, but had little effect on the uptake of 67Ga by the inflammatory tissue. These results suggest that 67Ga is taken up by normal tissues in a transferrin-bound form but in an unbound form by inflammatory tissue. On the other hand, when FeCl3 was simultaneously injected with 67Ga, the uptake of 67Ga by liver and spleen was markedly increased but the uptake by inflammatory tissue was decreased. 
Key words : 67Ga uptake, Inflammation, FeCl3 
INTRODUCTION 
GALLIUM-67 has been used for the detection of various tumors1 and acute and chronic inflammation.2,3 Many hypotheses concerning the mechanism of the uptake of 67Ga by tumors and inflammatory lesions4-5 have been proposed. Tsan et al.8 and Vallabhajosula et al.9 reported that 67Ga was almost all bound to transferrin, iron-binding protein, in blood. It was reported that transferrin played a major role for the mechanism of the uptake of 67Ga by tumors.10-13 On the other hand, it was shown that transferrin transports 67Ga to the tumor tissue but that 67Ga is taken up by tumor cells in an unbound form.7,14-16 The involvement of transferrin in the uptake of 67Ga by inflammatory tissue has not, however, been conclusively demonstrated. We have recently demonstrated that 67Ga densely accumulates in granuloma tissue, that is inflammatory tissue produced by turpentine oil.17 In the present study using FeCl3, we have investigated whether or not transferrin is involved in the uptake of 67Ga by the inflammatory tissue. 

Received March 14, 1988; revision accepted June 1, 1988. 
For reprints contact : Yasuhito Ohkubo, Department of Radiopharmacy, Tohoku College of Pharmacy, 4-4-1, Komatsushima, Sendai 981, JAPAN. 

MATERIALS AND METHODS 
Animals 
Male Wistar rats weighing 150-200 g 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 in relative humidity of 55+-5%. 
Production of inflammatory tissue 
The production of inflammatory tissue, granuloma, was carried out by the same method described in the previous report17 as follows. A paper pellet (size 8 mm) was dipped in turpentine oil and implanted bilaterally in the subcutaneous tissues of the abdomen in each animal. 
67Ga solution 
Gallium-67 citrate (kindly supplied by Nihon Mediphysics Co., Ltd., Takarazuka, Japan) was diluted with saline to 185 kBq (5 uCi)/ml. 
Administration of 67Ga 
Each rat, at 6 days after the administration of turpentine oil, was intravenously injected with 67Ga in a dose of 37 kBq (200 ul). 
Administration of FeCl3 
Each rat, at 6 days after the administration of turpentine oil, was intravenously injected with FeCl3 (6.25-25 umole/ml saline) in a dose of 100 ul 5 min before or 30 min after the administration of 67Ga. The simultaneous administration of FeCl3 and 67Ga was carried out by the intravenous injection of FeCl3 saline solution (12.5 umole/ml) containing 67Ga (185 kBq) in a dose of 200 ul. 
Removal of granuloma and other tissues 
At 4 hrs after the administration of 67Ga, rats were anesthetized with urethane (1.5 g/kg, i.p.) and immediately perfused with cold saline. The inflammatory lesion and other tissues were then removed. The granuloma tissue was obtained from the inflammatory lesion with complete removal of the implanted paper pellet and abscess. 
Determination of radioactivity 
Radioactivity of various tissues removed was determined with a well-type NaI-scintillation counter (Aloka, ARC-300). The uptake ratios of 67Ga in various tissues were expressed in the following formula : Uptake ratio=sample radioactivity (cpm)/ sample weight (g)/total radioactivity administered (cpm)/body weight of rat (g). 
RESULTS 
The effect of various amounts of FeCl3 on the uptake of 67Ga by inflammatory and normal tissues 
To investigate the effect of FeCl3 on the retention of 67Ga in the blood and 67Ga uptake by inflammatory and normal tissues, in vivo experiments were performed (Fig. 1). FeCl3 (0.625, 1.25, and 2.5 umole/rat) was administered 5 min before the injection of 67Ga and the blood, urine, inflammatory and other tissues were removed 4 hrs later. FeCl3 dose dependently reduced 67Ga retention in the blood. Similarly the uptake of 67Ga by the liver and spleen was dose dependently reduced. In contrast to this, FeCl3 (2.5 umole/rat) markedly increased the excretion of 67Ga into the urine. On the other hand, FeCl3 had little effect on the uptake of 67Ga into the inflammatory tissue, granuloma. 
The effect of the time of FeCl3 administration on the uptake of 67Ga in inflammatory and normal tissues 
The administration of FeCl3 (2.5 umole/rat) 30 min after the injection of 67Ga decreased the uptake ratios of 67Ga into the blood more than that 5 min before (Fig. 2). On the other hand, the administration of FeCl3 30 min after the injection of 67Ga increased the uptake of 67Ga by the liver and spleen more than 5 min before. But in all cases, the blood, liver and spleen uptake ratios of 67Ga were greatly decreased by the administration of FeCl3 as compared with 67Ga alone. On the other hand, concerning the uptake ratio of 67Ga into granuloma, there is no difference between the administration of FeCl3 5 min before and 30 min after the injection of 67Ga, although the uptake ratios in both cases were slightly decreased by the administration of FeCl3. 
The effect of the simultaneous administration of FeCl3 and 67Ga on the uptake ratio of 67Ga into inflammatory and normal tissues 
The simultaneous administration of FeCl3 and 67Ga greatly increased the uptake ratios of 67Ga into liver and spleen as compared with 67Ga alone but decreased those into blood and granuloma (Fig. 3). The decrease in the uptake ratio of 67Ga into blood by the simultaneous administration was, however, much smaller than that following the administration of FeCl3 5 min before and 30 min after the injection of 67Ga. 
DISCUSSION 
We used ferric chloride as the iron reagent because ferric citrate poorly disolved in saline, although Hayes et al.6 used ferric citrate to investigate the effect of iron on the distribution of 67Ga. They have reported that ferric citrate slightly increased the blood distribution of 67Ga. The results from Fig. 1 in the present study indicate, however, that FeCl3 inhibits the binding of 67Ga to transferrin, iron binding protein, in blood, so that the retention of 67Ga in blood is greatly reduced by FeCl3 and 67Ga in an unbound form due to FeCl3 is excreted in urine. Additionally FeCl3 can decrease the uptake of 67Ga by liver and spleen without disturbing the uptake of 67Ga by the inflammatory lesions. 
Hayes et al.6 reported that the uptake of 67Ga by normal tissues such as liver or spleen occurred to a major extent by endocytosis in which transferrin is involved, and that the initial entry of 67Ga into inflammatory lesions might occur in the same way as to that of normal soft tissues, while the uptake of 67Ga by tumors occurs mainly by the diffusion of the unbound form. Vallabhajosula et al.7 reported that the uptake of 67Ga into tumors was reduced by transferrin, as compared with 67Ga-citrate alone, i.e., the uptake of 67Ga by tumors occurred in an unbound form. In the present study, the results indicating that transferrin is involved in the uptake of 67Ga by liver and spleen are consistent with those obtained by Hayes et al.,6 although the effect of the ferric citrate used by them on the liver and spleen was weaker than that of the ferric chloride used by us. We think, however, that the uptake of 67Ga into inflammatory tissue occurs in an unbound form in the same way as into tumors. It should be noted, however, that the inflammatory lesions employed by Hayes et al. were abscesses but those employed by us were granuloma tissues, not containing an abscess. Oberhaensli et al.19 have also reported that iron decreased the distribution of 67Ga int the liver and abscess. Both they and Hayes et al.6 injected iron simultaneously with 67Ga. Their results are quite the opposite of ours. In our results the simultaneous injection of iron increased the uptake of 67Ga into liver and spleen. We cannot understand these differences. They must inject iron and 67Ga by separate routes. Moreover, Hayes et al.6 have reported that iron increased the distribution of 67Ga in blood, although that in liver was decreased with iron. These results are unreasonable. The results from the present study show that iron can decrease the amount of 67Ga in blood but cannot remove the 67Ga that has already entered the liver and spleen. Since almost all 67Ga is, however, bound to transferrin in blood,8,9 the decrease in 67Ga in blood due to FeCl3 is the decrease in 67Ga in a bound form. Therefore the decrease in the uptake of 67Ga into liver or spleen when FeCl3 was administered resulted from the decrease in 67Ga binding to transferrin. On the other hand, FeCl3 had little effect on the uptake of 67Ga by granuloma, i.e., 67Ga might accumulate in the inflammatory tissue either in a transferrin bound form or in an unbound form. 
Many workers have expressed confidence that 67Ga is exclusively bound to and transported to various tissues by transferrin in the blood. At the tumor or inflammatory site, 67Ga is then dissociated from the transferrin complex. Vallabhajosula et al.16,18 reported that acidic pH at the tumor site might be one of the factors involved in 67Ga localization in tumors. Moreover, reduction of the pH to 6.8-7.0, below that of normal tissue (7.4), might be due to the accumulation of lactic acid produced by anaerobic glycolysis. We think that the pH at the granuloma site must also be reduced by anaerobic glycolysis. We think that 67Ga is transported to the inflammatory site either in a transferrin bound form or in an unbound form. Gallium-67 in an unbound form directly enters inflammatory tissue, whereas, 67Ga in a bound form is dissociated under acidic conditions at the inflammatory site and then enters the inflammatory tissue. We conclude that transferrin is not involved in the uptake of 67Ga into inflammatory tissue, such as granuloma, and that the uptake of 67Ga uptake into inflammatory tissue may be similar to that occurring in tumor tissues. 
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