Eur J Nucl Med (1987) 12:567-572 European I~11 inlt:~Jr Journal of I ~l l l~l l l . / l~.ttE4/

Medicine © Springer-Verlag 1987

Biodistributions of 201TI in tumor bearing animals and inflammatory lesion induced animals Atsushi Ando ~, Itsuko Ando ~, Masaharu Katayama ~, Shigeru Sanada t, Tatsunosuke Hiraki ~, Hirofumi Mori 2, Norihisa Tonami 3, and Kinichi Hisada 3 School of Allied Medical Professions 1, Radioisotope Center 2 and School of Medicine 3 Kanazawa University, Kanazawa, Japan

Abstract. The accumulation of 2°1T1 in tumor and inflam- matory tissues were small. However, this nuclide showed a high concentration in viable tumor tissue, less in connec- tive tissue (containing inflammatory tissue), and was not seen in necrotic tumor tissue regardless of the time after administration of 2°lTl(I)-chloride. In inflammatory le- sions, 2°1T1 accumulated in subcutaneous tissue infiltrated with neutrophils and macrophages, and quite large amounts of this nuclide were accumulated in subcutaneous tissue and sites where neutrophils were crowded. Most 2°1T1 ex- isted in a free form in the fluid of tumor and inflammatory tissues regardless of the time after administration. A small amount of this nuclide was localized in the nuclear, mito- chondrial and microsomal fractions in these tissues, and the nuclide was bound to protein in these fractions. The distribution of 2°lTl(III)-chloride in tumor bearing animals was essentially the same as that of 2°lTl(I)-chloride.

Key words: Thallium-201 - Tumor - Inflammatory lesion

Kawana et al. (1970) showed that Z99Tl can serve as a myo- cardial scanning agent because of its similarity to the in vivo behavior of potassium. 2°aTl, which is of a lower ener- gy than 199T1, has come to be widely used in the diagnosis of myocardial infarction. Cox et al. (1976); Salvatore et al. (1976); Tonami et al. (1977, 1978) and Hisada et al. (1978), in the course of heart imaging of patients with tumors, discovered the affinity of 2°1T1 in the form of thallous chlo- ride to tumor. Clinically, these reports indicated the usefull- ness of 201Tl(i)_chloride in the diagnosis of lung and thyroid c a n c e r ,

The accumulation mechanisms of 2°1T1 in tumor tissue have been investigated, but are not adequately understood. 67Ga-citrate is widely used for tumor scanning (Edwards and Hayes 1969; Higashi et al. 1972), and shows stronger affinity for inflammatory lesions than for malignant tumor (Ando et al. 1984, 1985). The present study was carried out to clarify the biodistribution of 2°lTl(I)-chloride in tu- mor bearing and inflammatory lesion induced animals, and to elucidate the accumulation mechanism in tumor tissue and inflammatory lesions.

Offprint requests to: Atsushi Ando, Ph.D., School of Allied Medi- cal Professions, Kanazawa University, 5-11-80, Kodatsuno, Kana- zawa City, 920, Japan

Materials and methods

Materials

Male Donryu rats (body weights 150 ~200 g) underwent subcutaneous implantation of Yoshida sarcoma (2 x 108 cells/0.5 ml) in the right thigh. Six days to 7 days later, an appropriate amount of radioactive nuclide was admin- istered, at which time the tumor grew to 1.5 cm-2.0 cm in diameter. Male wistar rats (body weight 181 g_+12 g) were each treated by the subcutaneous injection of 0.2 ml turpentine oil (Wako Pure Chemical Ind., Japan) into the right thigh to induce an inflammatory lesion. Five days later an appropriate amount of radioactive nuclide was ad- ministered. Male ddY mice (body weight 35g_+3 g) re- ceived subcutaneous transplantation of Ehrlich tumor (5 × 107 cells/0.1 ml) into the right thigh. These mice were used 7 days to 10 days later at which time the tumor had grown to about 1 cm in diameter.

Carrier free 2°lTl(I)-chloride solution (10~tCi/ml- 200 IxCi/ml) was prepared from 2°lTl(I)-chloride (Daiichi Radioisotope Laboratories Ltd., Japan) and physiological salt solution.

Carrier-free 2°lTl(III)-chloride solution (10 ~tCi/ml) was prepared by oxidizing the Z°lTl(I)-chloride solution with K3Fe(CN)6 (Ishidate 1959). It was ensured that Z°lT1 in 2°~Tl(III)-chloride solution was in the positive trivalent form at the time of administration to the animals, using the fact that a solution containing trivalent thallium ion produced brown precipitate[Tl(OH)3] by NaOH. Pronase E (Protease from streptmyces griseus, Kaken Chemical Co. Japan).

Methods

Distribution of T M Tl in tumor bearing animals and inflamma- tory lesion induced animals. 2°lTl(I)-chloride solution (4 ~tCi/0.4 ml) was injected intravenously into the tumor bearing rats and intraperitoneally into the tumor bearing mice. Ten rain, 30 rain, 60 rain, 3 h, 24 h and 48 h later, these animals were killed, and about 1 ml of blood was collected from the carotid artery, also the tumor tissue and nine different tissues (Table 1) were excised. These tissues and the blood were weighed immediately and counted in a well type scintillation counter (Aloka, JDC-701) against an appropriate standard to obtain the percentage of injected dose/g of tissue (% dose/g). These values were normalized to a body weight (BW) of 100 g by multiplying by BW/100.

568

2°lTl(III)-chloride solution (4 laCi/0.4 ml) was injected intravenously into the tumor bearing rats. These animals were treated in the same way as the animals injected with 2°lTl(I)-chloride solution. 2°lTl(I)-chloride solution (4 gCi/0.4 ml) was injected intravenously into the inflam- matory lesion induced rats. Ten min, 60 min, 3 h, 24 h and 48 h later, these animals were killed and the blood was collected and the inflammatory lesion and eleven different tissues (Table 2) were excised. These tissues and the blood were assayed by the same procedure used for tumor bearing animals.

Distribution of 2Ol Tl in tumor tissues and inflammatory le- sions. 2°aTl(I)-chloride solution (100 gCi/0.5 ml) was in- jected intravenously into the rats, and intraperitoneally into the mice. The animals were killed, and the tumor tissues and the inflammatory lesion excised at 3 h, 24 h and 48 h after administration of the radiopharmaceutical. These tis- sues were embedded in 2% carboxymethyl cellulose sodium salt and frozen with dry ice and acetone ( - 7 0 ° C) immedi- ately after excision. Following this, the frozen tissues were cut into thin serial sections (10 gm) in a cryostat ( - 2 0 ° C). One of these sections was then placed on X-ray film which was developed after an exposure of several days, a second section was fixed in ethanol, and was then stained with hematoxylin-eosin.

Subcellular distribution of 2 o 1 Tl in tumors and inflammatory lesions. 2°lTl(I)-chloride solution (4 gCi/0.4ml) was in- jected intravenously into the rats and intraperitoneally into the mice. Ten rain, 60 rain, 3 h, 24 h and 48 h after the administration of 2°lTl(I)-chloride, these animals were killed, and the tumor tissues and inflammatory lesions ex- cised. These tissues were homogenized in cold (5 ° C) 0.25 M sucrose containing 0.01 M Tris-HC1 buffer, pH 7.6 (10% w/v) in a Potter-Elvehjem type homogenizer. According to the modified method (Hogeboom 1955) of Hogeboom and Schneider, subcellular fractionation was carried out at 4 ° C. Fractions from centrifugation were assayed for 2°1T1 in a well type scintillation counter.

Preparation of subcellular fractionation, digestion and gel filtration. 2°lTl(I)-chloride solution (4 gCi/0.4 ml) was in- jected intravenously into the rats and intraperitoneally into the mice. Twenty four h after the administration of 2o ~TI(I)- chloride, these anirnals were killed, and the tumor tissues and inflammatory lesions excised.

These tissues were rinsed in 0.9% NaC1 solution, all manipulations were conducted at 4 ° C. The tissues were homogenized seperately with ten volumes of 0.15 M KC1 containing 0.01 M Tris-HC1 buffer, pH 7.6 in a Potter-E1- vehjem type homogenizer. Subcellular fractionation was carried out according to the modified method (Hogeboom 1955) of Hogeboom and Schneider. Five ml aliquots of the supernatant fraction of each homogenate were applied to a column of Sephadex G-50, particle size 50 lam-150 gm (1.2 crux 65 cm), that had been equilibrated with 0.15 M KC1 containing 0.01 M Tris-HC1 buffer, pH 7.6. The radio- activity was eluted with the same buffer (120 ml), the flow rate was 0.3 ml/min and 3 ml fractions were collected to measure radioactivity and protein. After the subcellular fractionation of these homogenates, nuclear, mitochondrial and microsomal fractions from each were mixed. The mix- tures were adjusted to pH 7.8-8.2 with 0.1 M NaOH and were incubated with 60 mg protease (Pronase E) at 37 ° C.

After 24 h, the reaction mixtures were again adjusted to pH 7.8-8.2 with 0.1 M NaOH, and 30 mg Pronase E was added to each. They were incubated for 24 h at 37 ° C. After digestion, the reaction mixtures were centrifuged at 3,000 rpm (1,500 g) for 20 min, and the sediments were dis- carded. The supernatants (5 ml aliquots) were applied to a column of Sephadex G-50, particle size 50 gm-150 pm (1 .2cmx65 cm) that had been equilibrated with 0.15 M NaC1 containing 0.1 M acetic acid + sodium acetate buffer, pH 5.0. The radioactivity was eluted with 120 ml of the same buffer. The flow rate was 0.3 ml/min and 3 ml frac- tions were collected to measure radioactivity and protein.

To determine the relationship between the molecular masses of the eluted substance and the number of fractions, a small amount of each of the following marker substances was dissolved in 5 ml of 0.15 M NaC1 containing 0.15 M acetic acid and sodium acetate buffer, pH 5.0: bovine serum albumin (molecular masses 68,000) and 2°lTl(I)-chloride. These solutions were applied to the Sephadex G-50 column and were eluted by exactly the same procedure as for the reaction mixture. The eluates were assayed by measuring 2°1T1 with the above scintillation counter, and protein and amino acid by Lowry's method (Lowry et al. 1951).

Results

Distribution of 2oi TI in tumor bearing animals and inflammatory lesion induced animals

Accumulation of 2°1T1 in tumor, inflammatory lesions and other tissues are shown in Table 1. In Ehrlich tumor bearing mice, the accumulation in the tumor was small, and barely reached 0.69% dose/g 3 h after the administration of g01Tl(i)_chloride ' in contrast the accumulation in the kidney and pancreas was high. In Yoshida sarcoma bearing rats administered 2°lTl(I)-chloride, the accumulation for tumor tissue was also small, and reached 0.88% dose/g 30 rain after the administration of 2°aTl(I)-chloride but the accu- mulation in the kidney, cardiac muscle and pancreas was very large. It was presumed that differences in the distribu- tions of g°IT1 between the above rats and mice were affected by the different methods of injection. In rats administered with 2o 1Tl(iii)_chloride ' the distribution of 201T1 was rather similar to that in rats administered with 2°lTl(I)-chloride. The accumulation of 2°~T1 in inflammatory lesions and other tissues are shown in Table 2, the value for the inflam- matory lesion was 1.85% dose/g 10 min after administra- tion, and decreased with time after administration, the value was larger than those for tumors.

Distribution of 2oi TI in tumor tissues and inflammatory lesions

A typical autoradiogram, hematoxylin + eosin stained sec- tion and sketch illustration of a Yoshida sarcoma excised 24 h after administration of 2°~Tl(I)-chloride, are shown in Fig. 1. For tumor tissue, hematoxylin + eosin stained sec- tions were divided into the following three categories : viable tumor tissue, necrotic tumor tissue and connective tissue which contains inflammatory tissue. As shown in Fig. 1, 2°aT1 accumulated to a large extent in the viable tumor tissue, and less in the connective tissue (containing inflam- matory tissues), regardless of the time after administration and was hardly seen in the necrotic tumor tissue. A similar result was seen in Ehrlich tumor. The autoradiogram, he-

569

Table 1. Mean retention values of 2°1T1 in tissues of tumor bearing animals administered with 2°aTl(I)-chloride and 2°lTl(III)-chloride

Radioactivity of 2°1T1 (% dose/g)

Time after injection of 2°1T1

10 rnin 30 min 60 min 3 h 24 h 48 h

Ehrlich tumor bearing mice administered with 2°lTl(I)-chloride

Blood 0.22._ 0.01 0.15 + 0.04 0.15 ± 0.03 0.14 + 0.04 0.07 ± 0.02 0.05._ 0.01 Muscle 0.38._0.16 0.49._0.07 0 . 8 0 _ + 0 . 1 9 1.13-+0.15 0.79-+0.12 0.53±0.07 Liver 2.53 ._ 0.24 3.00 ± 0.37 2.38 -4- 0.57 2.33 _+ 0.31 t .09 ± 0.23 0.67 _ 0.07 Kidney 3.01_+1.37 5.44±0.33 10.09._0.93 22.50±3.58 11.29±1.03 6.13._1.73 Spleen 2.94 + 0.36 2.57 + 0.24 1.84 ± 0.50 1.60-+ 0.22 0.77 ± 0.11 0.63 -4- 0.03 Lung 0.70_+0.06 0.79±0.12 1.14±0.22 0.92-+0.17 0.60±0.15 0.50±0.10 Pancreas 10.63 ± 1.57 10.39-- 1.84 6.54_+ 2.40 5.11 ± 1.04 2.04-- 0.32 1.56-+ 0.33 Cardiac muscle 2.09 + 0.76 1.95 _+ 0.29 2.11 ± 0.20 2.04 _+ 0.18 0.93 -+ 0.21 0.79 ± 0.12 Bone 0.16 -t- 0.08 0.24 ._ 0.03 0.47 -+ 0.05 0.72 ± 0.15 0.68 ._ 0.14 0.48 ._ 0.08 Brain 0.02 _+ 0.01 0.03 _-4- 0.002 0.08 _+ 0.01 0.18 ± 0.05 0.42 _+ 0.05 0.40 ± 0.05 Tumor 0.08 -- 0.04 0.17 _+ 0.05 0.35 ± 0.08 0.69 ± 0.12 0.48 _+ 0.09 0.39 _+ 0.08

Yoshida sarcoma bearing rats administered with 2°aTl(I)-chloride

Blood 0.21 _+ 0.02 0.17 _+ 0.01 0.11 _+ 0.02 0.09 ± 0.01 0.06 ± 0.01 0.03 ± 0.004 Muscle 1.32 _+ 0.40 1.41 ± 0.35 1.20 -+ 0.15 1.25 ± 0.35 1.08 ± 0.10 0.73 + 0.07 Liver 2.35 -- 0.40 3.66 -+ 0.84 2.56 ± 0.05 1.89 ± 0.34 1.03 _+ 0.16 0.58 ± 0.08 Kidney 23.11 ._ 4.17 25.13 ._ 3.34 16.61 ± 1.51 17.63 _+ 2.68 15.44 ± 1.39 9.40 ± 2.11 Spleen 2.89 ± 0.31 3.20 ± 0.64 1.93 + 0.32 1.29 _-4- 0.14 0.91 _+ 0.10 0.52 _+ 0.08 Lung 5.23 _+ 0.54 2.86_+ 0.84 1_93 _+ 0.34 1.41 ± 0.12 0.78 ± 0.14 0.47 ± 0.02 Pancreas 6.52 + 1.26 7.43 -- 1.29 4.33 ± 0.96 3.42 ± 0.29 2.06 -- 0.26 1.12 _+ 0.14 Cardiac muscle 10.51 ± 0.53 7.30 ± 0.81 3.49 ± 0.51 2.38 _+ 0.16 1.23 -- 0.13 0.73 _+ 0.08 Bone 0.79_+ 0.14 1.07-- 0.21 0.75_+ 0.09 0.76_+ 0.12 0.94 + 0.08 0.63_+ 0.07 Brain 0.14--0.02 0.18±0.03 0.12±0.02 0.19±0.03 0.36±0.03 0.27±0.02 Tumor 0.70 -- 0.19 0.88 -- 0.24 0.43 ± 0.09 0.51-4- 0.18 0.36 -+ 0.10 0.23 ± 0.04

Yoshida sarcoma bearing rats administered with 2°lTl(III)-chloride

Blood 0.16_+0.03 0.11 -+ 0 . 0 1 0.09._0.01 0.09._0.05 0.05._0.01 0.03_+0.004 Muscle 0.80_+0.09 0 . 9 6 _ + 0 . 3 0 1 . 0 0 _ + 0 . 1 4 1.00+0.31 0.91 +0.07 0.65_+0.13 Liver 1.71 _+ 0.29 t.89-- 0.14 1.60-- 0.1t 1.58_+ 0.15 0.79 + 0.16 0.68._ 0.13 Kidney 10.11 _+0.95 10.21 +0.72 10 .07-4-1 .01 12.10+0.96 13.54._ 1.43 10.26_+ 1.80 Spleen 1.13 _ 0.08 1.38 -- 0.17 1.05 _+ 0.07 1.03 -- 0.09 0.55 ± 0.05 0.57 ± 0.06 Lung 4.55 -- 0.43 2.54 ._ 0.56 1.28 -4- 0.23 0.92 + 0.14 0.56 -+ 0.09 0.54 + 0.07 Pancreas 2.38 + 0.60 3.00 -- 0.61 2.39 ± 0.27 2.40 + 0.29 1.54 ± 0.15 1.00 ± 0.18 Cardiac muslce 6.90-+ 0.47 4.78_+ 0.18 2.68-+ 0.22 1.31 ± 0.08 0.96-- 0.10 0.76 ± 0.14 Bone 0.59 -+ 0.06 0.73 ± 0.12 0.47 ± 0.08 0.55 ± 0.03 0.75 _+ 0.05 0.56 ± 0.02 Brain 0.09_+ 0.01 0.13 + 0.03 0.07-- 0.01 0.14-- 0.01 0.29-- 0.04 0.29 + 0.03 Tumor 0.47 -- 0.09 0.45 +_ 0.07 0.42 _+ 0.14 0.46 _+ 0.12 0.39 _+ 0.03 0.30 _+ 0.05

Each value represents the mean of five animals. These values were normalized to a body weight of 100 g

ma toxy l in+eos in stained section and a sketch illustration for an inf lammatory lesion which was excised 24 h after administrat ion of 2°lTl(I)-chloride, are shown in Fig. 2. The stained section was divided into the following three categories: subcutaneous tissues, subcutaneous tissue infil- trated with neutrophils and macrophages, and sites in which neutrophils were crowded. 2°1T1 accumulated in subcutane- ous tissue infiltrated with neutrophils and macrophages, and quite large amounts accumulated in subcutaneous tis- sue and sites in which neutrophils were crowded.

Subcellular distribution o f 2° l Tl in tumors and inflammatory lesions

When the Z°IT1 in the nuclear fraction, mitochondrial frac- tion, microsomal fraction and supernatant fraction, are ex- pressed as A(cpm), B(cpm), C(cpm) and D(cpm), respec- tively, the Z°lT1 (%) in the nuclear fraction can be calcu-

lated by:

A

A + B + C + D × 100 (%)

The 2°iT1 in the mitochondrial fraction, microsomal frac- tion and supernatant fraction were calculated by substitu- t ion of A with B, C, and D in the numerator. 2°1T1 in each fraction of two different tumors and an inf lammatory lesion is shown in Table 3. In both tumors, most of the 2°1T1 was localized in the supernatant fraction, and a small amount in the nuclear fraction, mitochondrial fraction (ly- sosome is contained in this fraction) and microsomal frac- tion. These values were approximately constant regardless of the time after administration. In inf lammatory lesion, most of the 2°1T1 was localized in the supernatant fraction, and a small amount elsewhere. These results was very simi- lar to those for tumors.

570

Table 2. Mean retention values of 2°1T1 in tissues of inflammatory lesion induced animals administered with Z°lTl(I)-chloride

Radioactivity of Z°lT1 (% dose/g)

Time after injection of 201T1

10 min 60 min 3 h 24 h 48 h

Blood 0.26 _-+ 0.02 0.17 + 0.01 0.14_+ 0.01 0.08 _+ 0.01 0.05 _+ 0.01 Muscle 0.78 + 0.16 1.30 + 0.23 1.29 _+ 0.16 1.20 + 0.12 1.08 _+ 0.06 Liver 2.58_+0.29 2.72_+0.41 2.12___0.24 1.18_+0.19 0.72_+0.09 Spleen 3.44 5- 0.64 2.44_+ 0.19 1.81 _+ 0.02 1.10 ___ 0.09 0.74 ± 0.06 Kidney 20.98 _+ 2.03 20.89 ! 2.62 21.01 _+ 1.24 11.99 _+ 1.28 10.33 _+ 0.77 Lung 4.83 + 1.29 2.48 -+ 0.20 1.74 _ 0.08 1.09 _+ 0.09 0.73 _+ 0.06 Stomach 3.32 ± 0.47 3.18 + 0.30 3.05 -+ 0.23 1.69 i 0.09 1.11 +_ 0.07 Pancreas 7.09 ± 1.50 5.99 -+ 1.70 4.07 _+ 0.23 2.29 _+ 0. t 7 1.51 _+ 0.05 Bone 0.68 -+ 0.11 0.95 + 0.08 1.28 ± 0.10 1.38 _+ 0.07 0.91 _+ 0.08 Cardiac muscle 11.36 -+ 0.75 5.02 _+ 0.82 2.94 i 0.12 1.76 _+ 0.12 0.99 _+ 0.15 Brain 0.12 _+ 0.02 0.18 + 0.01 0.25 _+ 0.01 0.47 _ 0.03 0.39 _ 0.02 Thymus 1.53 + 0.19 2.08 ± 0.43 1.91 ± 0.07 1.05 _+ 0.05 0.68 _+ 0.08 Inflammatory lesion 1.85_+ 0.44 1.17_+ 0.21 0.89_+ 0.07 0.58 ± 0.03 0.43_+ 0.08

Each value represents the mean of five animals. These values were normalized to a body weight of 100 g. 201Tl(I)-chloride was administered 5 days after subcutaneous injection of turpentine oil

Table 3. Subcellular distribution of 2°1T1 in tumors and inflamma- tory lesions at various times following administration of Z°~Tl(I)- chloride

Subcellular distribution (%) of 2°iT1

Nuclear Mito- Microsomal Supernatant fraction chondrial fraction fraction

fraction

Fig. 1.A-C. Relationship between morphological specimens of tu- mor tissue and 2°1T1 accumulation. 2°1T1 was greatly accumulated in the viable tumor tissue, less in the connective tissue (containing in the inflammatory tissue) and was hardly seen in the necrotic tumor tissue. A Macroautoradiogram; B Hematoxylin-Eosin stain- ing; C Sketch illustration.

Viable tumor tissue Necrotic tumor tissue

~T~ Connective tissue (contain inflammatory tissue)

Fig. 2A-C. Relationship between morphological specimens of in- flammatory lesion induced with turpentine oil and 2°1T1 accumula- tion. 2o 1T1 was accumulated in subcutaneous tissue infiltrated with neutrophils and macrophages, and quite large amounts were accu- mulated in subcutaneous tissue and at sites where neutrophils were crowded. A Macroautoradiogram; B Hematoxylin-Eosin staining; C Sketch illustration.

Site where neutrophils are crowded Subcutaneous tissue infiltrated with neutrophils and macro- phages

I ~ Subcutaneous tissue

zo l Tl binding substanees in tumors and inflammatory lesions

Supe rna t an t f rac t ion o f h o m o g e n a t e s : In the case o f Ehr l ich tumor , Fig. 3 A indicates the results o f the gel f i l t ra t ion o f the supe rna tan t f rac t ion o f t u m o r h o m o g e n a t e ; 0 .5%

Ehrlich tumor

10 min 6.7 4.6 11.4 77.3 60 min 8.7 3.7 11.7 75.9

3 h 6.1 6.5 9.8 77.6 24 h 8.0 7.0 10.0 75.0 48 h 9.4 10.8 11.8 68.0

Yoshida sarcoma

10 min 7.3 4.4 10.2 78.1 60 rain 5.4 5.5 10.1 79.0

3 h 6.3 4.1 10.0 79.6 24 h 5.8 5.8 8.6 79.8 48 h 7.0 9.9 13.7 69.4

Inflammatory lesion

10 min 9.7 2.2 5.5 82.6 60 min 9.7 2.9 5.8 81.6

3 h 11.1 4.3 5.5 79.1 24 h 12.9 4.0 6.6 76.5 48 h 10.3 6.7 7.8 75.2

Each value is expressed as a mean of three experiments

and 99.3% of 2°aT1 appl ied to a Sephadex G-50 c o l u m n were e luted in the first and second peaks, respectively. La rge a m o u n t s o f p ro te in ( indicated by Lowry ' s me thod ) were e luted in the first peak and a small quan t i ty o f amino acid ( indicated by Lowry ' s m e t h o d ) was e luted in the second peak. In Y o s h i d a sarcoma, similar results were obta ined . In the i n f l a m m a t o r y lesion, Fig. 3 C indicates the results o f the gel f i l t ra t ion o f the supe rna tan t f rac t ion o f the ho- m o g e n a t e ; 1% and 94.8% o f 2°1T1 appl ied to a Sephadex G-50 c o l u m n were e luted in the first and second peaks,

> ;3

.o "o

n,

0 I - - 0 Lowry 's method

o

t

• • 201TI a c t i v i t y

o . . . . . i I p e a k

i f i r s t ~.

l "o o o-o~

10 15 20 25 30 35

F r a c t i o n n u m b e r

i

B

• • 201TI a c t i v i t y / ~

o ~ o Lowry's method

o second [ /

f ko ~

8 "b • -%. ~ f i rs t :" "O-o.o. a, p e a k " / ]~",

\ • a

10 15 20 25 30 35

F r a c t i o n n u m b e r

571

> ;3

.o

= : 201TI a c t i v i t y

o . . . . o L o w r y ' s method

o

f f i rs t \ p e a k ~, ' l \

~ %'o. o / k .W'. ~ : - _ - ' 9 - o - , , ~ . . . . . . . ooo-O ' ,o -o .n .n . , . ~

10 15 2 0 25 3 0 35

F r a c t i o n n u m b e r

: : 201TI ac t iv i ty

o . . . . o Lowr y's method

o., / 1 ,o "o second / \ p,,k

L " " f i rs t ; , o e a k

10 15 2 0 25 30 35

F r a c t i o n n u m b e r

Fig. 3A-D. Sephadex G-50 column chromatography profiles of supernatant. A Supernatant of homogenate of Ehrlich tumor; B Superna- rant which was produced from Ehrlich tumor by Pronase E treatment; C Supernatant of homogenate of inflammatory lesion; D Superna- rant which was produced from inflammatory lesion by Pronase E treatment

respectively. Protein was eluted in the first peak. Consider- ing the relationship between the molecular masses and the fraction number in the Sephadex G-50 column from the previously described reports (Ando et al. 1982, 1983), pro- tein in the first peak was eluted in the void volume and radioactivity in the second peak was free Z°lT1. These re- sults were confirmed by the control experiments in which protein and 2°lTl(I)-chloride were eluted through the above Sephadex G-50 column. From these experimental results, it was concluded that 2°1T1 in the supernatant fraction of the homogenate of tumor and inflammatory tissue was in an almost free form, regardless of the tumor species and inflammatory tissue.

2°lT1 binding substances in nuclear, mitochondrial and

microsomal fractions: In the case of Ehrlich tumor, 2°aT1 mostly remained in the supernatant on centrifugation after digestion with Pronase E. Figure 3B shows the results of the gel filtration of the supernatant which was produced from Ehrlich tumor by Pronase E treatment; 2% and 97.1% of the 2°lT1 applied to a Sephadex G-50 column were eluted in the first and second peaks respectively. Pro- tein (Pronase E) and its debris were eluted between fraction numbers 10 and 24, and amino acid was eluted in the second peak. As shown in Fig. 3 B, Z°lT1 in nuclear, mitochondrial and microsomal fractions was liberated almost completely by digestion with Pronase E, similar results were obtained in Yoshida sarcoma. In inflammatory tissue, 2°lT1 mostly remained in the supernatant on centrifugation after diges-

572

tion with Pronase E. Figure 3 D shows the results of the gel f i l t rat ion of the supernatant which was produced from an in f lammatory lesion by Pronase E t reatment ; 0.4% and 98.5% of 2°1T1 appl ied to the Sephadex G-50 column were eluted in the first and second peaks respectively. Protein (Pronase E) and its debris were eluted between fract ion numbers 10 and 24, and amino acid was eluted in the second peak. 2°1T1 in nuclear, mi tochondr ia l and microsomal frac- tions was also l iberated almost completely by digestion with Pronase E.

F r o m these experimental results, it is obvious that 2°1T1 in nuclear, mi tochondr ia l and microsomal fractions of tu- mors and inf lammatory tissue is bound to protein.

Discussion

As is indicated in Table 1, the accumulat ion of 2°1T1 in tumor, regardless of species, seems less marked than in the case of 67Ga-citrate previously repor ted (Ando et al. 1985). 2°XT1 was greatly accumulated in viable tumor tissue, less in connective tissue (containing in f lammatory tissue), and was hard ly accumulated into necrotic tumor tissue. 6VGa was avidly concentra ted into the connective tissue (contain- ing inf lammatory tissue), was accumulated into the viable tumor tissue, and was scarcely accumulated into necrotic tumor tissue (Ando et al. 1984, 1985). Therefore, 2°1T1 is far bet ter for visualizat ion of viable tumor tissue than 67Ga. These results were identical to the repor t of Ito et al. (1978) that 2°1T1 was avidly accumulated in viable tumor tissue. In rats adminis tered with 2°~Tl(III)-chloride, dis tr ibut ions were ra ther similar to those for rats adminis tered with 2°lTl(I)-chloride. We presumed that the similari ty in distri- but ion of mono and tr ivalent thal l ium was caused by rapid reduction of t r ivalent thal l ium to monovalen t thal l ium after adminis t ra t ion.

Regardless of the time after adminis t ra t ion, most of the 2°1T1 exists in the free form in the fluid o f tumor and in- f l ammatory tissue. A small amount of 2°1T1 was localized in the nuclear, mi tochondr ia l and microsomal fractions of these tissues, and the 2°tT1 was bound to prote in in these fractions. In contrast , Ando (1979) originally determined that 67Ga, HaIn, and 169yb were bound to the acid muco- polysaccharides in two species of tumor tissue and inflam- ma to ry tissue. I t was repor ted by Ando et al. (1980, 1983) that 67Ga binding acid mucopolysacchar ides had been sepa- rated by cellulose acetate electrophoresis from tumor tissue and liver lysosome, and that 67Ga binding acid mucopoly- saccharides in tumor and liver were very similar.

Considering the above results and previously repor ted papers, it is obvious that the accumulat ion mechanisms for 2°iT1 and 67Ga in tumor tissues are very different. In the case of clinical tumor scanning with 2°~Tl(I)-chloride and 67Ga-citrate, these nuclides should be used carefully, having regard for their characteristics, because their biodistr ibu- tions and accumulat ion mechanisms in tumor tissue are very different.

Acknowledgements. This work was supported in part by a Grant in Aid for Scientific Research from the Ministry of Education, Science and Culture.

References

Ando A (1979) Studies on the tumor affinity of 169yb, 167Tm, 67Ga and 111In (in Japanese). PhD Thesis, Kyusyu University, pp 82-99

Ando A, Ando I, Hiraki T, Takeshita M, Tanabe S, Yamazaki R (1980) 6VGa binding substances in the tumor and liver tissues. Radioisotopes 29:250-251

Ando A, Ando I, Hiraki T, Takeshita M, Hisada K (1982) Mecha- nism of tumor and liver concentration of 111In and t69yb; r a i n and 169yb binding substances in tumor tissues and liver. Eur J Nucl Med 7 : 298 303

Ando A, Ando I, Hiraki T, Takeshita M, Hisada K (1983) Mecha- nism of tumor and liver concentration of 6:Ga: 67Ga binding substances in tumor tissues and liver. Int J Nucl Med Biol 10:1-9

Ando A, Ando I, Sanada S, Katayama M, Hiraki T, Doishita K, Midzukami M, Hisada K (1984) Study of the distribution of tumor affinity metal compounds and alkaline metal com- pounds in the tumor tissues by macroautoradiography. Int J Nucl Med Biol 1l:195-201

Ando A, Ando I, Sanada S, Hiraki T, Hisada K (1985) Tumor and liver uptake models of 6VGa-citrate. Eur J Nucl Med 10: 262-268

Cox PH, Belfer AJ, vonder Pompe WB (1976) Thallium 201 chlo- ride uptake in tumors, a possible complication in heart scintig- raphy. Br J Radiol 49 : 767-768

Edwards CL, Hayes RL (1969) Tumor scanning with 67Ga citrate. J Nucl Med 10:103 105

Higashi T, Nakayama Y, Murata A, Nakamura K, Sugiyama T, Suzuki S (1972) Clinical evaluation of 67Ga-citrate scanning. J Nucl Med 13:196-201

Hogeboom GH (1955) Fractionation of cell components of animal tissues. In: Colowick SP, Koplan NO (eds) Methods in Enzy- mology, vol. 1. Academic Press, New York, pp 16-19

Hisada K, Tonami N, Miyamae T, Hiraki T, Yamazaki T, Maeda T, Nakajo M (1978) Clinical evaluation of tumor imaging with 2°lTl-chloride. Radiology 129:497-500

Ishidate M (1959) Qualitative Microanalysis. Nanzando Publ, To- kyo, pp 301 302

Ito Y, Muranaka A, Harada T, Matsuda A, Yokobayashi T, Tera- shima H (1978) Experimental study on tumor affinity of 2°~T1- chloride. Eur J Nucl Med 3:81-86

Kawana M, Krizek H, Porter J, Lathrop KA, Charleston D, Harper PV (1970) Use of 199T1 as a potassium analog in scan- ning. J Nucl Med 11:333

Lowry OH, Rosebrough NJ, Farr AL, Randall RJ (1951) Protein measurement with the folin phenol reagent. J Biol Chem 193:265-275

Salvatore M, Carratu L, Porta E (1976) Thallium-201 as a positive indicator for lung neoplasms : preliminary experiments. Radiol- ogy 121:487-488

Tonami N, Hisada K (1977) Clinical experience of tumor imaging with 2°aTl-chloride. Clin Nucl Med 2:75-81

Tonami N, Bunko H, Michigishi T, Kuwajima A, Hisada K (1978) Clinical application of Z°iT1 scintigraphy in patients with cold thyroid nodules. Clin Nucl Med 3 : 217 221

Received July 31, 1986 / October 11, 1986