Mechanism of tumor and liver concentration of 111In and 169Yb: 111In and 169Yb binding substances in...

Eur J Nucl Med (1982) 7:298-303 European N u c l e a r Journal of Medicine (9 Springer-Verlag 1982

Mechanism of Tumor and Liver Concentration of 111In and 169yb: rain and 169yb Binding Substances in Tumor Tissues and Liver

Atsushi Ando, Itsuko Ando, Tatsunosuke Hiraki, Masazumi Takeshita and Kinichi Hisada Schools of Paramedicine and Medicine, Kanazawa University, Kanazawa, and Medical College of Oita, Oita, Japan

Abstract. Tumor-bearing animals were injected with ~ ~ ~In- and 69yb-citrate. Tumor homogenates, from which the nuclear frac-

tion was removed, and the mitochondrial fractions of the host livers were digested with pronase P. After digestion, the supernatants of the reaction mixtures were applied to Sephadex G-100 columns. The resultant eluates were analyzed for radioactivity, protein, uronic acid, and sialic acids. Three peaks of radioactivity were obtained by gel filtration. The first peak, eluted in the void volume, contained a species whose molecular weight ex- ceeded 40000. The second peak consisted of.substances with molecular weights of 9400-40000. Radioactivity in the third peak was liberated ~laIn and 169yb. These two nuclides in the second peak were bound to acid mucopolysaccharide and/or the sulfated carbohydrate chain of sulfated glycoprotein. It was thought that the nuclides in the first peak might be bound to some acid mucopolysaccharides.

The second peak nuclides seemed to be bound to acid mucopolysaccharide that contained no uronic acids, and/or to the sulfated carbohydrate chain of sulfated glycoprotein. It was concluded that they were bound to the acid mucopolysaccharides and/or the sulfated carbohydrate chain of sulfated glycoprotein in tumor tissues and liver lysosomes.

Introduction

Edwards and Hayes (1969) reported that 67Ga-citrate was con- centrated in soft tissue tumors in humans. Tumor affinity of ~In-chloride was first reported by Hunter and colleagues (Hunter and Dekock 1969; Hunter and Riccobono 1970), and that of ~69yb-citrate was first reported by us (Hisada and Ando 1973). Although the mechanism of localization of 67Ga in malig- nant tumors has been extensively investigated, there have been few such studies on HlIn and 169yb. Takeda and co-workers (1977) reported that lysosomes are the site of accumulation for 11 ~In whether in tumor or normal liver cells. Hagan et al. (1977) indicated that the viable tumor concentration of ~ l I n exceeds the nonviable concentration during the first 48 h. But we (Ando et al. 1981) concluded that the lysosome does not play a major role in the tumor concentration of ~t~In and ~69yb, although it may play an important role in liver concentrations. In the case of hepatoma, it is presumed that lysosome plays an important role in the tumor concentration of these nuclides, hepatoma possessing some residual features of the liver.

Offprint requests to ." Dr. Atsushi Ando, School of Paramedicine, Kana- zawa University, 5-11-80, Kodatsuno, Kanazawa, 920 Japan

We propose that the binding of ~ ~ 1In and 169yb to substances in the body should be considered not physiologically but chemically, because these nuclides are not essential elements for animals. They can be trivalent cations in solution, and naturally, these cations can bind strongly to cation-exchange resins in solution. Among body constituents, the strongest cation-exchange resin-like substances are acid mucopolysaccharides and sulfated glycoprotein which have sulfonic groups in their structures.

The distribution of these nuclides in the tumor tissues determined by macroautoradiogram indicated that their concentration was predominant in connective tissue (especially inflammatory tissue) rather than in viable tumor tissue, regardless of the time after administration (Ando et al. 1977a and b, 1978). It is well- known that fibroblasts produce a large amount of acid mucopolysaccharides as the intercellular substances in inflammatory tissue, and that these mucopolysaccharides have many sulfonic groups and carboxy radicals in their structures.

Considering the above-mentioned facts, it is reasonable to take acid mucopolysaccharides and sulfated glycoprotein as the substances to which these nuclides could be bound. Anghileri and Heidbreder (1977) obtained evidence indicating that compet- itive binding of 67Ga3 + to Ca 2 +- and M f +-binding sites (calci- um chondroitin sulfate etc.), rather than a metabolic process, is involved in the accumulation of this radioisotope. As 67Ga,

a ~ In, and 169yb are chemically similar in character, their experi- mental results support our view.

For the reasons described above it was presumed that one of the these nuclides binding substances in tumor tissues and liver lysosomes was acid mucopolysaccharide or sulfated glycoprotein. The present study was performed to provide evidence for this hypothesis.

Materials and Methods

Preparation o f Subcellular Fractionation and Digestion

The following animals and transplanted tumors were used : male ddY mice SC implanted with Ehrlich tumor; male Donryu rats SC implanted with Yoshida sarcoma, and hepatoma AH 109A.

Carrier-free ~ ~ tin-citrate solution, pH 6.0-8.0 (approximately 1 ml containing 200 gCi l~In), was prepared from IHIn-chloride solution and 0.08 M sodium citrate solution. ~69yb-citrate solution, pH 6.0-8.0 (approximately 1 ml containing 50 gCi 169yb and 2.5 lag Yb) was prepared from 169yb-chloride solution and sodium citrate solution, aa~In-citrate solution (0.4 ml) was injected IV into the rats and IP into the mice. Twenty-four

0340-6997/82/0007/0298/$01.20

hours after the administration of l~ZIn-, ~69yb-citrate, these animals were anesthetized by sodium pentobarbitaI injection, and the tumor tissues and livers were excised.

The tissues were rinsed in 0.9% NaC1 solution. All manipula- tions were conducted at 4 ° C. The tumor tissues were homogenized with 10 volumes of 0.15 M KC1 containing 0.01 M Tris- HC1 buffer, pH 7.6 in a Potter-Elvehjem type homogenizer. The homogenates were centrifuged for 15 min at 400 g and the sediments (cell debris and nuclear fraction) were discarded. The homogenates (7 ml) from which the nuclear fractions were removed, were adjusted to pH 7.8-8.2 with 0.1 M N a O H and were incubated with 60 mg proteinase (Pronase P, Kaken Chemical Co) at 37 ° C. After 24 h, the reaction mixtures were adjusted again to pH 7.8-8.2 with 0.1 M NaOH, and 30 mg pronase P was added to each. They were further incubated for 24 h at 37 ° C.

The livers were homogenized and nuclear fractions were removed as indicated above. Mitochondrial fractions (lysosomes were contained in these fractions) were collected from the homogenates by centrifuging at 5000 g for 15 min. The mitochondrial fractions were also incubated with pronase P by the same method as that performed on tumor tissue.

169Yb-citrate solution (0.4 ml) was also injected IV into the rats and IP into the mice. These animals were treated by the same procedure used for animals injected with x ~ 1In-citrate.

Gel Filtration

After digestion, the reaction mixtures were centrifuged at 3000 rpm for 20 rain, and the sediments were discarded. The supernatants (5-ml aliquots) were applied to a column of Sepha- dex G-100 medium (1.2 x 75 cm) that had been equilibrated with 0.15 M NaC1 containing 0.1 M acetic ac id+sod ium acetate buffer, pH 5.0. The radioactivity was eluted with the same buffer (120 ml). The flow rate was 0.3 ml/min. Three-ml fractions were collected to measure radioactivity, uronic acids, protein, and sialic acids.

To determine the relationship between the molecular weight of the eluted substance and the number of fractions, about 5 gg of the following marker substances were dissolved in 5 ml 0. l 5 M NaC1 containing 0.15 M acetic ac id+sod ium acetate buffer, pH 5.0: blue dextran, hyaluronic acid K-salt, chondroitin sulfate A Na-salt, chondroitin sulfate B No-salt, chondroitin sulfate C Na-salt, heparin sodium, dextran T-70, dextran T-40, dextran T-10, inulin, aldolase, albumin from bovine serum, albumin from hen egg, catalase, chymotripsinogen A, ribonuclease A, myoglo- bin, cytochrome C, and cyanocobalamine. These solutions were applied to the above-mentioned Sephadex G-100 and to the below-mentioned Sephadex G-50 columns and were eluted by exactly the same procedures as in reaction mixtures. Fractions (3 ml each) were collected, and each substance in the eluates was assayed by appropriate methods.

Digestion with DNose and RNase

The fractions from the first and second peaks of the Sephadex G-100 column (Figs. 3-6), to which pronase P treated samples of tumor or liver tissues had been applied, were combined respectively and digested with DNase or RNase.

Five-ml of the combined fractions were incubated with 5 mg DNase or RNase at 37 ° C for 3 h. After digestion, the reaction mixture was applied to a column of Sephadex G-50 (1.2 x 65 cm) that had been equilibrated with 0.15 M NaC1 containing 0.1 M acetic ac id+sod ium acetate buffer, pH 5.0. The radioactivity was eluted from the column with the same buffer (120 ml). The

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flow rate was 0.3 ml/min. Fractions (3 ml each) were collected and radioactivity, uronic acid, and protein were determined.

Measurement

Radioactivity of ~11In and 169yb in the eluates was measured by a well-type scintillation counter (Aroka, JDC-701). Protein was determined by the method of Lowry et al. (1951). Uronic acids and acid mucopolysaccharides, dextran, and inulin were measured by the orcinol reaction (Brown 1946). Sialic acids were assayed by the thiobarbituric acid method (Aminoff 1961). Cyano- cobalamine was determined by the absorbance at 361 nm.

In Vitro Binding of 111In and 169yb to Heparin

To three test tubes containing 0.4 ml heparin sodium solution (10 mg/ml), 0.1 ml 1~ l in_nitrat e solution (carrier-free, 20 pCi/ml) was added. Solutions in the three test tubes were adjusted to pH 4.0, 7.0, and 9.0 with 0.01 M N a O H or 0.01 M HC1, respectively. Paper chromatography of the reaction mixtures was carried out on Toyo No. 50 paper with 1% N a H C Q solution by using the ascending method after incubation for 30 rain and for 24 h in room temperature. Paper chromatography of l ~ I n - nitrate solution was carried out simultaneously by the same procedure. Actigrams of paper strips were made with a paper chromatography scanner. Thereafter, paper strips were placed on X-ray film, and this film was developed after an exposure of several days. In addition, the paper strips, on which heparin- ~11In reaction mixtures were chromatographed, were stained with 1% toluidine blue to locate the heparin spot. In the case of 169yb-chloride, an in vitro binding test to heparin was performed by the same procedure used for ~ In -n i t r a t e .

Resul t s

The relationships between molecular weight and the fraction numbers on Sephadex G-50 and G-100 columns are shown in Figs. 1 and 2,

/INk K ..a--n.

• C: Cyanocobalamine (MW 1357.4) D: Albumin from bovine serum (MW 58000)

~J Io / ~ E: Myo~lobi. (,, 17aoo) l I ~,,II!J'i~ \ \ F: Cytoch . . . . C (MW 13500)

~ ] ; / ~ ' ~ I a G: Dextran T-70 (MW 70000) H: Chymetrypsinogen A (MW 25000)

I: Blue Dextran (MW 2000000)

l~/lol t! J: Dextran T-40 ([4!4 40000) K: Heparin Sodium L: Dextran T-IO (MW 9400) M: Inulin (MW 3500-5000) N: Chondroitin Sulfate C, Na-Salt (MW 40000-80000)

O: Chondroitin Sulfate A, Na-Salt (MW 25000-50000)

P: Chondroitin Sulfate B, Na~Salt

?

/d i

10 1'5 2~0 2'5 3~0 Fraction number

Fig. 1. Relationship between molecular weight and fraction number. Sephadex G-50

300

A: Aldo lase (MW 158000) I /~l B: Albumin from bovine serum (MW 68000)

I S i C: Albumin from chicken egg (MW45000)

! I D: Ribonuclease A (MW 13700)

odium

in Sulfate B, Na-Salt

in Sulfate A, Na-Salt -50000)

in Su l fa te C, Na-Sal t -80000) c Acid, K-Salt 0-8000000)

tO 15 20 25 30

Fract ion n u m b e r

Fig. 2. Relationship between molecular weight and fraction number• Sephadex G-100

, m

P

: "- rain activity

. . . . Orcinol method

o ........ o Lowry's method

= -" Thiobarbituric acid method [

I first peak 9. 1~ / second :', /

• ; .

# ./ " = 9 \ ", ~/\. i [

2': I \ ': / X& "~,

10 15 20 25 30 35


Fig. 3. Sephadex G-100 chromatography profiles of Ehrlich tumor digested with pronase P at 37 ° C for 48 h

For tumors and livers, radioactivity f rom 3 0 % - 6 0 % of these nuclides remained in the supernatant on centrifugation, after digestion with pronase P. Figures 3-6 indicate the gel fi l tration of the supernatants tha t were produced f rom Ehrlich tumor and the host mouse liver by pronase P treatment . The eluates

o . . . . . . . . o

peak

mln activity

Orcinol method

Lowry's method

Thiobarbituric acid method

second peak

/

i • 6 6 ,: ".

o u c

L .

o m

<

1 : 1 " ... ,.k,

~i ~ / .° .~.~.t ,~° ;I', ", :' A/ :

/ I..- -,~ % ./ . . , , - ~ - . - - . " # " ~ \ "b , , ~ " " , ' - , - . Ig -~- i£ . . . . . . -. . . . . ~ . ' ? ' . ' L ~ _ ,

10 15 20 25 30 35

Fraction n u m b e r

Fig. 4. Sephadex G-100 chromatography profiles of the host liver digested with pronase P at 37 ° C for 48 h

= -" ~ 6 ~ Y b activity

• - - - - - Orc inol method

o ........ o Lowry's method

= "- Thiobarbituric acid method

t ~ second peak

s, ..,k/ l / /.o..

I I : ' / \ i ==

l i ' / Z "° o, ,d] v • I : ', ', .' t h i r d ~i If ~ / J \ ~, .¢ I peak'i

It J : i i

10 15 20 25 30 35


Fig. 5. Sephadex G-100 chromatography profiles of Ehrlich tumor digested with pronase P at 37 ° C for 48 h

of two nuclides produced three peaks. In the cases of Yoshida sarcoma and the host liver, and hepa toma AH109A and the host liver, similar results were obtained. Considering the relation between the molecular weight and the fraction number in the Sephadex G-100 column (Fig. 2), the first peak of these nuclides

.>_

e l

)-

I i

10

-- = ~69Yb act iv i ty

. . . . Orcinol method

o ........ o Lowry 's method

-- -- Th iobarb i tu r ic acid method

~ p e a k

s e c o n d p e a k ~"o

~ : c; "b ,,,: ',,,

c~ ,' ',,

", ,~ t h i r d '.

15 20 25 30 35

@ U e-

R

O

.Q

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

Fig. 6. Sephadex G-100 chromatography profiles of the host liver digested with pronase P at 37 ° C for 48 h

was eluted in the void volume, the third peak was eluted with low molecular weight substances, and the second peak was eluted with intermediate molecular weight substances.

In the Ehrlich tumor, 26.5%, 52.0%, and 10.8% of 111In applied to a Sephadex G-100 column were eluted in the first, second, and third peak, respectively. Those values for the host liver of Ehrlich tumor-bearing mice were 36.2%, 50.7%, and 4.1%, respectively. In the Ehrlich tumor, 32.5%, 57.1%, and 7.5% of 169yb applied to a Sephadex G-100 column were eluted in the first, second, and third peaks, respectively. Those values for the host liver of Ehrlich tumor-bearing mice were 48.8%, 48.6%, and 0.7%, respectively. Uronic acids, protein, and sialic acids in the eluates are also indicated (Figs. 3-6).

Sephadex G-100 filtration indicated that larger molecular weight substances than dextran T-40 (mol wt 40 000) were eluted in the void volume. The first peak of radioactivity was eluted in the void volume, and a large amount of uronic acids appeared in the same fractions as determined by the orcinol method. As the large molecular weight substances composed of uronic acids are acid mucopolysaccharides, the first peak of these nuclides may be bound to acid mucopolysaccharides. The second peak of radioactivity was eluted between dextran T-40 and dextran T-10 (tool wt 9400). The peak for uronic acids and protein could not be observed in the second peak of radioactivity. The third peak of radioactivity was thought to be unbound nuclides, as this fraction was identical to the eluate of ~11In-citrate or

69yb_citrate. Fractions of the first peak of two nuclides obtained from

both Ehrlich tumor and the host liver were digested with DNase or RNase. The digests were applied to Sephadex G-50 columns. The radioactivity was quantitatively eluted in the void volume (Fig. 7). As shown in these four Figures, nuclides were not liberated from large molecular weight substances by digestion with DNase and RNase. In the control experiments, herring sperm D N A was digested with DNase, and yeast R N A was digested with RNase by the above-mentioned procedure. These nucleic acids were completely broken down into low molecular weight

301

T u m o r

10 15 20 25 3t0

Host l iver

tO 15 20 25 30

T u m o r

. . . . . . . . . . . t

10 15 20 25 30


, • H o s t l i ve r

. . . . . . . . . . i

10 15 20 25 30


= ~ t r e a t e d w i t h D N a s e

. . . . . . t r e a t e d w i t h R N a s e

Fig. 7. Sephadex G-50 chromatography" profiles of the eluate (the first peak of Figs. 3-6) digested with DNase and RNase at 37 ° C for 3 h

T u m o r

~A

. . . . . . . . ,

10 15 20 25 30

u

_= E

Host l iver

L . . . . . . . . 15 20 25 30

.= J

T u m o r

15 20 25 30


.=

H o s t l iver

15 20 25 30

F rac t ion n u m b e r

t r e a t e d w i t h D N a s e

. . . . . . t r e a t e d w i t h R N a s e

Fig. 8. Sephadex G-50 chromatography" profiles of the eluate (the second peak of Figs. 3 6) digested with DNase and RNase at 37 ° C f o r 3 h

302

Table 1. Relation between pH and binding rate (%) to heparin

pH 4 pH 7 pH 9

lllIn 30 min 26.6 11.0 0 24 h 48.1 25.0 21.2

169yb 30 rain 14.5 86.3 4.8 24 h 16.6 86.2 20.8

pH and binding rate to heparin is shown in Table 1. The results of 169yb-chloride and heparin-169yb are shown in Fig. 9 and Table 1. As can be seen, considerably large amounts of ~11In and ~69yb were bound to heparin. Among pH 4, pH 7, and pH 9 samples tested, the binding rate of l ~ I n to heparin was at its height at pH 4, and that of ~69yb was highest at pH 7. The binding rate of 111In was enormously increased by incubation for 24 h compared with the 30-min incubation.

Fig. 9. Actigram and autoradlogram

substances. In the cases of Yoshida sarcoma, hepatoma AH109A and those of the host livers, nuclides were not liberated from the large molecular weight substances included in the eluates of first peak with either DNase or RNase. In Ehrlich tumor and the host liver, when eluates of the second peak of nuclides by Sephadex G-100 filtration were digested with DNase or RNase, l l l In and 169yb complex applied to the Sephadex G-50 column was quantitatively eluted in the void volume (Fig. 8). As shown in these Figures, the two nuclides were not liberated from large molecular weight substances in the eluates of the second peak by treatment with DNase or RNase. Neither Yos- hida sarcoma, hepatoma AH109A, nor their host livers released nuclides by the above stated nuclease treatment of the second peak. These results with tumors and their host livers indicated that nuclides were bound to large molecular weight substances other than nucleic acids.

To examine in vitro binding of these two nuclides to heparin, actigrams and autoradiograms of ~ 1 lin_nitrat e and heparin -~l 1In were taken (Fig. 9). The Rf values of ~ 11In-nitrate and heparin- 111In were 0 and 1.0, respectively. The relationship between

Discussion

It is clear that the first and second peaks of 111In and 169yb on Sephadex G-100 filtration were not bound to nucleic acids either in tumors or in the host liver. On Sephadex G-100 filtration, as the substances of molecular weight larger than dextran T-40 were eluted in the void volume, the first peak of these two nuclides would be eluted with acid mucopolysaccharides with molecular weight larger than 40000. It is thought that these nuclides in the first peak may be bound to some of these acid mucopolysaccharides. Uronic acids were only faintly detected in the second peak of radioactivity on Sephadex G-100 filtration, and the protein peak could not be observed in the second peak. Therefore, it was thought that radioactivity in the second peak was bound to the large molecular weight substances other than protein, which contained no uronic acids. Except for nucleic acid and protein, other large molecular weight substances in the body that contain no uronic acids are acid mucopolysaccharides such as kerato sulfuric acid, and the sulfated carbohydrate chain of sulfated glycoprotein. Therefore, it was deduced that radioactivity in the second peak was bound to acid mucopolysaccharides (e.g., kerato sulfuric acid) which contained no uronic acid, or to the sulfated carbohydrate chain of sulfated glycopro- rein. Sialic acids were found between the second and third peaks (Figs. 3 6). They are components of the carbohydrate chain of the glycoprotein. The peak of sialic acids indicated the carbohydrate chains of glycoproteins which were eluted on Sephadex G-100 filtration, though the sialic acids contained no bound radioactivity.

The in vitro binding test indicated that these nuclides were bound to heparin. It is thought that the binding sites of these nuclides in heparin are sulfonic acid groups and carboxyl radicals.

From these results, it may be concluded that these nuclides in the first and second peaks on Sephadex G-100 filtration were bound to sulfonic acid groups and/or carboxyl radicals of acid mucopolysaccharides and/or sulfated carbohydrate chains of sulfated glycoprotein. Moreover it may be presumed that these nuclide binding substances in tumors and liver lysosome are the same substances.

Acknowledgment. We wish to thank Dr. Nobuyuki Ito for his com- ments and suggestions.

303

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Ando A, Sanada S, Hiraki T, Mizukami M, Ando I, Hisada K, Doishita K (1977 a) Study of distribution of 67Ga ' 11 l in and 169yb in tumor tissue by macroautoradiography and histological method (Abstract). Jpn J Nucl Med 14:434-435

Ando A, Doishita K, Ando I, Sanada S, H]raki T, Midsukami M, Hisada K (1977b) Study of distribution of 169yb, 67Ga and lUIn in tumor tissue by macroautoradiography: Comparison between viable tumor tissue and inflammatory infiltration around tumor. Radioisotopes 26:42i 422

Ando A, Doishida K, Ando I, Sanada S, Hiraki T, Midsukami M, Hisada K (1978) Mechanism of tumor affinity of Yb-169, Ga-67 and I n - l l l : Comparison of accumulation between viable tumor tissue and inflammatory infiltranon around tumor by macroautoradiography. Abstracts of the Second International Congress of World Federation of Nuclear Medicine and Biology pp 49, Wash- ington, DC, USA

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Hisada K, Ando A (i973) Radiolanthanides as promising tumor scanning agents. JNM 14:615 617

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Received July 18, 1981

Mechanism of tumor and liver concentration of 111In and 169Yb: 111In and 169Yb binding substances in...

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