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Subcellular distribution of gallium-67 in tumor and liver
.Workin Progress -
Inr.J. Nu
Subcellular Distribution of Gallium-67 in Tumor and Liver ATSUSHI ANDO’, ITSUKOANDO’, MASAZUMITAKESHITA’TATSUNOSUKE , HIRAKI’ and KINICHI HISADA~
‘Schools of Paramedicine’ and ‘Medicine, Kanazawa University, 5-l l-80 Kodatsuno Kanazawa City 920. Japan 2Medical College of Oita, Oita. Japan (Received 16 June 1981) Subcellular distribution of 67Ga was quantitatively determined to evaluate the role of the lysosome in accumulation of 67Ga in malignant tumor tissue and the liver using three different tumor models and the host liver. In Yoshida sarcoma and Ehrlich tumor, most of the radioactivity of 67Ga was localized in the supernatant fraction, and only a small amount of radioactivity was localized in the mitochondrial fraction, which contains lysosomes. In the liver, however, most of the radioactivity was concentrated in the mitochondrial fraction. The radioactivity of this fraction increased with time after the administration of 67Ga and reached approximately 50% of total radioactivity within 24 h. In the case of hepatoma AH109A, radioactivity of the mitochondrial fraction increased with time after administration, and about 30?/ of total radioactivity was concentrated in this fraction after 24 h. It is concluded that lysosome does not play a major role in the tumor concentration of 67Ga, although it may play an important role in the liver concentration of 67Ga. In the case of hepatoma AH109A, it is presumed that lysosome plays a considerably important role in the tumor concentration of 67Ga, hepatoma AH109A possessing some residual features of the liver. Introduction
intracellularly and in greater concentration in viable than in necrotic portions of tumor tissue.“’ SWARTZENDRUBER et uI.,(~) using EM-ARG, have shown that intracellular 67Ga present in normal and neoplastic tissue is localized in lysosome-like bodies l-2 days after iv.-administration in a variety of cell types. The subcellular fractionation and enzymatic studies reported by BROWNet a1.‘4,5’provided further evidence for lysosomal nature of the organelles previously identified by EM-ARG. TAKEDA and coworkers’6,7’showed lysosomal accumulation of 67Ga in two kinds of experimental tumors. Contrary to the results described above, DECKNERet ~1.‘~)on Ehrlich ascites cell, oRI1’g’on Yoshida sarcoma, and IT0 et al.“o’ on VX-2 carcinoma have indicated that little or no 67Ga is associated with lysosomes in tumor tissues. In the present communication. distribution of 67Ga was determined by subcellular fractionation of transplanted tumor tissues. Little or no 67Ga is associated with lysosomes in these tumors. Materials and Methods The following animals and transplanted tumors were used: donryu rats subcutaneously implanted with Yoshida sarcoma, and hepatoma AH109A: ddY mice subcutanously implanted with Ehrlich tumor. Carrier-free 67Ga-citrate (Philips-Duphar Cyclotron and Isotopes Laboratory) in water, pH 6.&8.0 (10 &i/O.4 ml), was injected intravenously into the rats and intraperitoneally into the mice. Ten minutes, 1 h, 3 h, 24 h and 48 h after the administration of 67Ga-citrate, these animals were anesthetized by sodium pentobarbital injection, and the tumor tissues and the liver were excised. The tumor tissues and the livers were homogenized in cold (5°C) 0.25 M sucrose containing 0.01 M Tris-HCl buffer, pH 7.6 (109; w/v) in a Potter-Elvehjem type homogenizer. According to the modified method of H~CEB~OMand SCHNEIDER~’ ” (Fig. l), subcellular fractionation was carried out at 4°C. Fractions from the centrifugations were assayed for radioactivity of 67Ga by a well-type scintillation counter. Protein in five samples of each fraction was measured by LOWRY’Smethods”‘) to determine the amount of protein in each fraction. Bovine serum albumin was used as a standard.
and HAYES reported in 1969 that i.v.administrated 67Ga often concentrates in soft tissue tumors in humans in sufficient quantity to permit external visualization of neoplastic lesions by diagnostic scintiscanning and camera technique!” This initial communication and a subsequent more detailed clinical report on the affinity of 67Ga for tumor tissue have been confirmed by many other institutions. Elucidation of the mechanism of “Ga uptake by tumor tissue should reveal those factors that control its localization and possibly lead to the development of methods to enhance its affinity for cancers. Early tissue distribution studies in experimental animals indicated that 67Ga was deposited EDWARDS
Results When radioactivity of nuclear fraction, mitochondrial fraction. microsomal fraction and supernatant fraction, is expressed as A (cpm), E (cpm), C (cpm) and D (cpm). respectively, radioactivity (percentage) of the nuclear fraction can be calculated by the following formula: A
A+B+C+D
lOO(“,)
Radioactivity of mitochondrial fraction. microsomal fraction and supernatant fraction was calcu65
N M.R. 911-0
x
Work In Progress
66
Tumor or Liver Homogenize in 0.25 M sucroseTri? 0.01 M, pH 7.6
I
filter(nylon mesh) and centrifuge at 2000rpm 15 min
I
Nuclei CA)
50009 15 min
II
1
105,000g 60 min
Mitochondria
(B)
I
I
I
Supernatant (0)
Micrdsomes (C)
FIG. 1. Preparation of subcellular fractions. lated by substitution of A with B, C and D in the numerator. Radioactivity of each fraction of three different tumor and the liver samples are shown in Tables 1 and 2. In the cases of Yoshida sarcoma and Ehrlich tumor, most of the radioactivity was localized in the supernatant fraction, and a small amount of radioactivity was localized in the mitochondrial fraction (lysosome is contained in this fraction), nuclear fraction and microsomal fraction. But in the liver of rats and mice, the radioactivity in the mitochondrial fraction increased with the passage of time after the administration of 67Ga-citrate. Twenty-four h later, about 50% of total radioactivity was accumulated in this fraction. The amounts of 67Ga concentrated in this fraction reached a plateau at this time. Contrary to data for the mitochondrial ‘fraction, radioactivity of the supernatant fraction decreased with time until 24 h after the administration.
In the case of hepatoma AH109A, radioactivity of the mitochondrial fraction increased with time until 24 h after administration and about 30% of the total radioactivity was concentrated in this fraction. Contrary to data for the mitochondrial fraction, radioactivity of the supernatant decreased with time until 24 h after administration. In the cases of Ehrlich tumor, the host liver and hepatoma AH109A, the relation between 67Ga (%) in mitochondrial fraction and 67Ga (%) in the supernatant fraction is indicated in the Fig. 2. It is shown in this figure that 67Ga was transposed from supernatant fraction to the mitochondrial fraction. Transposition of 67Ga from supernatant to mitochondrial fraction occurred readily in the liver but only slightly in Ehrlich tumor. Total protein (mg) and subcellular distribution (%) of protein in experimental tumors and host livers are shown in Table 3. In these tumors, the greater
TABLE 1. Subcellular distribution (%) of 67Ga in experimental tumors. Each value is expressed as a mean of three experiments Nuclear fraction
Mitochondrial fraction
Microsomal fraction
Supernatant fraction
Yoshida sarcoma 10 min 60 min 3h 24 h 48h
15.6 11.7 15.5 21.4 16.6
9.7 16.5 15.8 16.4 17.1
14.8 23.6 14.8 19.1 19.3
59.9 48.2 53.9 43.1 47.0
Ehrlich tumor 10 min 60 min 3h 24h 48 h
10.9 15.5 12.8 17.1 20.2
6.6 10.9 9.7 16.2 15.8
15.4 15.1 11.9 16.2 15.6
67.1 58.5 65.6 50.5 48.4
Hepatoma AH109A 10 min 60 min 3h 24 h 48 h
14.4 13.6 14.2 24.9 22.9
10.1 13.3 19.4 31.0 29.6
20.4 18.8 20.4 .23.3 21.4
55.1 54.3 46.0 20.8 26.1
6-l
Work in Progress TABLE
2. Subcellular distribution
(“A)of 67Ga in host liver. Each value is expressed as a mean of three experiments
Nuclear fraction The rats transplanted 10 min 60 min 3h 24h 48 h The mice transplanted 10 min 60 min 3h 24 h 48 h The rats transplanted 10 min 60 min 3h 24h 48 h
Ehrlich
6oc
Normal
Mitochondrial fraction
Yoshida sarcoma 16.8 15.5 22.1 22.4 19.2 Ehrlich tumor 26.1 19.6 15.6 20.8 18.5 hepatoma AH109A 22.0 15.2 17.7 18.8 29.0
tumor
liver
h
FIG. 2. Distribution of 67Ga (“/,) between mitochondrial (mit. f.) and supernatant fractions (sup. f.). It is shown that 67Ga was transposed from supernatant fraction to mitochondrial fraction. Transposition of 67Ga from supernatant to mitochondrial fraction readily occurred in the liver but only slightly in Ehrlich tumor.
TABLE
Microsomal fraction
Supernatant fraction
14.9 28.2 36.5 40.6 52.3
29.7 24.9 16.8 19.9 14.0
38.6 31.4 24.6 17.1 14.5
13.2 20.0 33.5 48.7 49.9
22.7 19.4 19.6 18.7 16.7
31.4 41.0 31.3 11.8 14.9
19.5 26.4 29.8 49.8 44.5
19.0 21.1 17.4 15.2 13.6
39.5 37.3 35.1 16.2 12.9
amount of protein was in the supernatant fraction, while 8.2-13.7% of total protein was in the mitochondrial fraction. In the livers, the greater amount of protein was in the supernatant fraction, and 23.5-24.9x of total protein was in the mitochondrial fraction. To determine the relative specific activity of 67Ga to protein in each fraction, the radioactivity (%) in each fraction was divided by the quantity (mg) of protein in that fraction. Results of tumor tissues are shown in Table 4 and those of the livers are shown in Table 5. In Yoshida sarcoma and Ehrlich tumor, relative specific activities in microsomal fraction was higher than those in the other fraction. But in hepatoma AH109A, relative specific activity in the mitochondrial fraction increased with time while that in the supernatant fraction decreased with time. The values of relative specific activities tend to reach a plateau at 24 h after administration. In the liver, relative specific activity in each fraction was very similar to those of hepatoma AH109A. Discussion There are two opinions about the mechanism of accumulation of 67Ga in tumor tissues. 0neC3-‘) is the
3. Total protein (mg) and subcellular distribution (%) of the protein in experimental tumor and host liver Nuclear fraction
Yoshida sarcoma Ehrlich tumor Hepatoma AH109A Liver of rat Liver of mouse
38.0 21.6 15.4 17.1 20.6
Mitochondrial fraction 13.7 8.2 10.3 23.5 24.9
Microsomal fraction 8.5 10.2 20.3 18.4 17.2
Supernatant fraction 39.8 60.0 54.0 41.0 37.3
Total protein (weight) in tissues of 100 mg 8.31 6.46 7.90 15.82 15.22
68
Work in Progress
TABLE 4. Relative specific activities (‘/Jmg)* of the protein in the nuclear, mitochondrial. microsomal and supernatant fractions of experimental tumor Nuclear fraction Yoshida sarcoma 10min 60 min 3h 24h 48 h
Mitochondrial fraction
Microsomal fraction
Supernatant fraction
4.9 3.7 4.9 6.8 5.3
8.5 14.5 13.9 14.4 15.0
20.8 33.2 20.8 26.9 27.2
18.1 14.6 16.3 l3.0 14.2
Ehrlich tumor 10 min 60 min 3h 24h 48 h
7.8 11.1 9.1 12.2 14.4
12.5 20.6 18.3 30.6 29.8
23.3 22.9 18.0 24.5 23.6
17.3 15.1 16.9 13.0 12.5
Hepatoma AH109A 10 min 60 min 3h 24h 48 h
11.8 11.1 11.6 20.4 18.8
12.5 16.4 24.0 38.3 36.5
12.8 11.8 12.8 14.6 13.4
12.9 12.7 10.8 4.9 6.1
* Relative specific activities protein (mg) of each fraction.
were calculated
view that lysosome plays an important role in the tumor concentration of 67Ga while the other opinion’s-‘O) sustains that lysosome does not play an important role in the tumor concentration of “Ga. From our present study, it was clear that lysosome did not play an important role in the tumor concen-
by dividing
the values of Table
tration of 67Ga in Yoshida sarcoma and in Ehrhch tumor, but the substance (which was contained in mitochondrial fraction) played an important role in the liver concentration of 67Ga and played a considerably important role in the tumor concentration of 67Ga in hepatoma AH109A.
TABLE 5. Relative specific activities (x/g)* of the protein in the nuclear, microsomal and supernatant fractions of the host livers Nuclear fraction The rats transplanted 10 min 60 min 3h 24h 48 h The mice transplanted 10 min 60 min 3h 24h 48 h The rats transplanted 10 min 60 min 3h 24 h 48 h
1 by the
Mitochondrial fraction
Microsomal fraction
mitochondrial,
Supernatant fraction
Yoshida sarcoma 6.2 5.7 8.2 8.3 7.1
4.0 1.6 9.8 10.9 14.1
10.2 8.6 5.8 6.8 4.8
5.9 4.8 3.8 2.6 2.2
Ehrlich tumor 8.5 6.2 5.0 6.6 5.9
3.5 5.3 8.8 12.8 13.2
8.7 7.4 7.5 7.1 6.4
6.6 7.2 5.5 2.1 2.6
5.2 7.1 8.0 13.4 12.0
6.5 1.3 6.0 5.2 4.7
6.1 5.7 5.4 2.5 2.0
hepatoma
* Relative specific activities protein (mg) of each fraction.
AH109A 8.1 5.6 6.5 6.9 10.7 were calculated
by dividing
the values of Table 2 by the
Work in Progress From the paper previously reportedc3-‘), it was thought that the lysosome is the subcellular component which plays a role in the liver and hepatoma AH109A concentration of 67Ga. BROWN et a1.‘4’ pointed out the disruption of lysosome in some phase of the fractionation procedures. Considering Brown’s view, we carried out the subcellular fractionation of the tumor tissue and the host liver by the same procedures at the same time, and the results described above were obtained. We therefore concluded that lysosomes of Ehrlich tumor and Yoshida sarcoma were not disrupted in the fractionation procedures, and that a large amount of 67Ga had not accumulated in lysosome of these tumors before the subcellular fractionation. It is presumed that there is lysosomal affinity of 67Ga in the case of the specific organ such as the liver. Considerably affinity of 67Ga to lysosome in hepatoma AH109A is due to residual nature of the liver possessed in hepatoma AH109A. The extent of the variation of hepatoma from liver may be determined by using this phenomenon as an indicator. References 1.
EDWARDSC. L. and HAYES R. L. J. Nucl. Med. 10, 103-105 (1969).
2.
3.
69
HAYES R. L., NELXIN B., SWARTZENDRUBER D. C., CARLT~NJ. E. and BYRD B. L. Science
167, 289-290 (1970). SWARTZENDRUBER D. C., NELKIN B. and HAYES R. L. J. Nat1 Cancer Inst. 46,941-952 (1971).
4.
BROWND. H., SWARTZENDRUBER D. C., CARLTONJ. E., BYRDB. L. and HAVESR. L. Cancer Res. 33, 2063-2066 (1973). 5. BROWN D. H., BYRD B. L., CARLTONJ. E., SWARTZENDRUBER D. C. and HAYES R. L. Cancer Res. 36, 956-963 (1976). TAKEDAS., UCHIDAT. and MATSUZAWAT. J. Nucl. Med. 18, 835-839 (1977). TAKEDAS., OKUYAMAS., TAKUSACAWA K. and MATSUZAWA T. Gann 69, 267-211 (1978). DECKNER K., BECKER G., LANG~WSKI U., SCHWERINGH., HORNUNG G. and SCHMIDT C. G. Z. Krebsjorsch. 76, 293-298 (1971). 9. 0~11 H. Strahlentherapie 144, 192-200 (1972). 10. ITO Y., OKUYAMAS., SATO K., TAKAHASHIK., SATOT. and KANNO1. Radiology 100, 357-362
(1971). 11. H~GEB~~MG. H. Methods ofEnzymology, Vol. 1, pp. 16-19. Academic Press, New York (1955). 12. LOWRY0. H., ROSEBROUGH N. J., FARR A. L. and RANDALLR. J. J. Biol. Chem. 193, 265-275 (1951).
Inr.J. Nu
Subcellular Distribution of Gallium-67 in Tumor and Liver ATSUSHI ANDO’, ITSUKOANDO’, MASAZUMITAKESHITA’TATSUNOSUKE , HIRAKI’ and KINICHI HISADA~
‘Schools of Paramedicine’ and ‘Medicine, Kanazawa University, 5-l l-80 Kodatsuno Kanazawa City 920. Japan 2Medical College of Oita, Oita. Japan (Received 16 June 1981) Subcellular distribution of 67Ga was quantitatively determined to evaluate the role of the lysosome in accumulation of 67Ga in malignant tumor tissue and the liver using three different tumor models and the host liver. In Yoshida sarcoma and Ehrlich tumor, most of the radioactivity of 67Ga was localized in the supernatant fraction, and only a small amount of radioactivity was localized in the mitochondrial fraction, which contains lysosomes. In the liver, however, most of the radioactivity was concentrated in the mitochondrial fraction. The radioactivity of this fraction increased with time after the administration of 67Ga and reached approximately 50% of total radioactivity within 24 h. In the case of hepatoma AH109A, radioactivity of the mitochondrial fraction increased with time after administration, and about 30?/ of total radioactivity was concentrated in this fraction after 24 h. It is concluded that lysosome does not play a major role in the tumor concentration of 67Ga, although it may play an important role in the liver concentration of 67Ga. In the case of hepatoma AH109A, it is presumed that lysosome plays a considerably important role in the tumor concentration of 67Ga, hepatoma AH109A possessing some residual features of the liver. Introduction
intracellularly and in greater concentration in viable than in necrotic portions of tumor tissue.“’ SWARTZENDRUBER et uI.,(~) using EM-ARG, have shown that intracellular 67Ga present in normal and neoplastic tissue is localized in lysosome-like bodies l-2 days after iv.-administration in a variety of cell types. The subcellular fractionation and enzymatic studies reported by BROWNet a1.‘4,5’provided further evidence for lysosomal nature of the organelles previously identified by EM-ARG. TAKEDA and coworkers’6,7’showed lysosomal accumulation of 67Ga in two kinds of experimental tumors. Contrary to the results described above, DECKNERet ~1.‘~)on Ehrlich ascites cell, oRI1’g’on Yoshida sarcoma, and IT0 et al.“o’ on VX-2 carcinoma have indicated that little or no 67Ga is associated with lysosomes in tumor tissues. In the present communication. distribution of 67Ga was determined by subcellular fractionation of transplanted tumor tissues. Little or no 67Ga is associated with lysosomes in these tumors. Materials and Methods The following animals and transplanted tumors were used: donryu rats subcutaneously implanted with Yoshida sarcoma, and hepatoma AH109A: ddY mice subcutanously implanted with Ehrlich tumor. Carrier-free 67Ga-citrate (Philips-Duphar Cyclotron and Isotopes Laboratory) in water, pH 6.&8.0 (10 &i/O.4 ml), was injected intravenously into the rats and intraperitoneally into the mice. Ten minutes, 1 h, 3 h, 24 h and 48 h after the administration of 67Ga-citrate, these animals were anesthetized by sodium pentobarbital injection, and the tumor tissues and the liver were excised. The tumor tissues and the livers were homogenized in cold (5°C) 0.25 M sucrose containing 0.01 M Tris-HCl buffer, pH 7.6 (109; w/v) in a Potter-Elvehjem type homogenizer. According to the modified method of H~CEB~OMand SCHNEIDER~’ ” (Fig. l), subcellular fractionation was carried out at 4°C. Fractions from the centrifugations were assayed for radioactivity of 67Ga by a well-type scintillation counter. Protein in five samples of each fraction was measured by LOWRY’Smethods”‘) to determine the amount of protein in each fraction. Bovine serum albumin was used as a standard.
and HAYES reported in 1969 that i.v.administrated 67Ga often concentrates in soft tissue tumors in humans in sufficient quantity to permit external visualization of neoplastic lesions by diagnostic scintiscanning and camera technique!” This initial communication and a subsequent more detailed clinical report on the affinity of 67Ga for tumor tissue have been confirmed by many other institutions. Elucidation of the mechanism of “Ga uptake by tumor tissue should reveal those factors that control its localization and possibly lead to the development of methods to enhance its affinity for cancers. Early tissue distribution studies in experimental animals indicated that 67Ga was deposited EDWARDS
Results When radioactivity of nuclear fraction, mitochondrial fraction. microsomal fraction and supernatant fraction, is expressed as A (cpm), E (cpm), C (cpm) and D (cpm). respectively, radioactivity (percentage) of the nuclear fraction can be calculated by the following formula: A
A+B+C+D
lOO(“,)
Radioactivity of mitochondrial fraction. microsomal fraction and supernatant fraction was calcu65
N M.R. 911-0
x
Work In Progress
66
Tumor or Liver Homogenize in 0.25 M sucroseTri? 0.01 M, pH 7.6
I
filter(nylon mesh) and centrifuge at 2000rpm 15 min
I
Nuclei CA)
50009 15 min
II
1
105,000g 60 min
Mitochondria
(B)
I
I
I
Supernatant (0)
Micrdsomes (C)
FIG. 1. Preparation of subcellular fractions. lated by substitution of A with B, C and D in the numerator. Radioactivity of each fraction of three different tumor and the liver samples are shown in Tables 1 and 2. In the cases of Yoshida sarcoma and Ehrlich tumor, most of the radioactivity was localized in the supernatant fraction, and a small amount of radioactivity was localized in the mitochondrial fraction (lysosome is contained in this fraction), nuclear fraction and microsomal fraction. But in the liver of rats and mice, the radioactivity in the mitochondrial fraction increased with the passage of time after the administration of 67Ga-citrate. Twenty-four h later, about 50% of total radioactivity was accumulated in this fraction. The amounts of 67Ga concentrated in this fraction reached a plateau at this time. Contrary to data for the mitochondrial ‘fraction, radioactivity of the supernatant fraction decreased with time until 24 h after the administration.
In the case of hepatoma AH109A, radioactivity of the mitochondrial fraction increased with time until 24 h after administration and about 30% of the total radioactivity was concentrated in this fraction. Contrary to data for the mitochondrial fraction, radioactivity of the supernatant decreased with time until 24 h after administration. In the cases of Ehrlich tumor, the host liver and hepatoma AH109A, the relation between 67Ga (%) in mitochondrial fraction and 67Ga (%) in the supernatant fraction is indicated in the Fig. 2. It is shown in this figure that 67Ga was transposed from supernatant fraction to the mitochondrial fraction. Transposition of 67Ga from supernatant to mitochondrial fraction occurred readily in the liver but only slightly in Ehrlich tumor. Total protein (mg) and subcellular distribution (%) of protein in experimental tumors and host livers are shown in Table 3. In these tumors, the greater
TABLE 1. Subcellular distribution (%) of 67Ga in experimental tumors. Each value is expressed as a mean of three experiments Nuclear fraction
Mitochondrial fraction
Microsomal fraction
Supernatant fraction
Yoshida sarcoma 10 min 60 min 3h 24 h 48h
15.6 11.7 15.5 21.4 16.6
9.7 16.5 15.8 16.4 17.1
14.8 23.6 14.8 19.1 19.3
59.9 48.2 53.9 43.1 47.0
Ehrlich tumor 10 min 60 min 3h 24h 48 h
10.9 15.5 12.8 17.1 20.2
6.6 10.9 9.7 16.2 15.8
15.4 15.1 11.9 16.2 15.6
67.1 58.5 65.6 50.5 48.4
Hepatoma AH109A 10 min 60 min 3h 24 h 48 h
14.4 13.6 14.2 24.9 22.9
10.1 13.3 19.4 31.0 29.6
20.4 18.8 20.4 .23.3 21.4
55.1 54.3 46.0 20.8 26.1
6-l
Work in Progress TABLE
2. Subcellular distribution
(“A)of 67Ga in host liver. Each value is expressed as a mean of three experiments
Nuclear fraction The rats transplanted 10 min 60 min 3h 24h 48 h The mice transplanted 10 min 60 min 3h 24 h 48 h The rats transplanted 10 min 60 min 3h 24h 48 h
Ehrlich
6oc
Normal
Mitochondrial fraction
Yoshida sarcoma 16.8 15.5 22.1 22.4 19.2 Ehrlich tumor 26.1 19.6 15.6 20.8 18.5 hepatoma AH109A 22.0 15.2 17.7 18.8 29.0
tumor
liver
h
FIG. 2. Distribution of 67Ga (“/,) between mitochondrial (mit. f.) and supernatant fractions (sup. f.). It is shown that 67Ga was transposed from supernatant fraction to mitochondrial fraction. Transposition of 67Ga from supernatant to mitochondrial fraction readily occurred in the liver but only slightly in Ehrlich tumor.
TABLE
Microsomal fraction
Supernatant fraction
14.9 28.2 36.5 40.6 52.3
29.7 24.9 16.8 19.9 14.0
38.6 31.4 24.6 17.1 14.5
13.2 20.0 33.5 48.7 49.9
22.7 19.4 19.6 18.7 16.7
31.4 41.0 31.3 11.8 14.9
19.5 26.4 29.8 49.8 44.5
19.0 21.1 17.4 15.2 13.6
39.5 37.3 35.1 16.2 12.9
amount of protein was in the supernatant fraction, while 8.2-13.7% of total protein was in the mitochondrial fraction. In the livers, the greater amount of protein was in the supernatant fraction, and 23.5-24.9x of total protein was in the mitochondrial fraction. To determine the relative specific activity of 67Ga to protein in each fraction, the radioactivity (%) in each fraction was divided by the quantity (mg) of protein in that fraction. Results of tumor tissues are shown in Table 4 and those of the livers are shown in Table 5. In Yoshida sarcoma and Ehrlich tumor, relative specific activities in microsomal fraction was higher than those in the other fraction. But in hepatoma AH109A, relative specific activity in the mitochondrial fraction increased with time while that in the supernatant fraction decreased with time. The values of relative specific activities tend to reach a plateau at 24 h after administration. In the liver, relative specific activity in each fraction was very similar to those of hepatoma AH109A. Discussion There are two opinions about the mechanism of accumulation of 67Ga in tumor tissues. 0neC3-‘) is the
3. Total protein (mg) and subcellular distribution (%) of the protein in experimental tumor and host liver Nuclear fraction
Yoshida sarcoma Ehrlich tumor Hepatoma AH109A Liver of rat Liver of mouse
38.0 21.6 15.4 17.1 20.6
Mitochondrial fraction 13.7 8.2 10.3 23.5 24.9
Microsomal fraction 8.5 10.2 20.3 18.4 17.2
Supernatant fraction 39.8 60.0 54.0 41.0 37.3
Total protein (weight) in tissues of 100 mg 8.31 6.46 7.90 15.82 15.22
68
Work in Progress
TABLE 4. Relative specific activities (‘/Jmg)* of the protein in the nuclear, mitochondrial. microsomal and supernatant fractions of experimental tumor Nuclear fraction Yoshida sarcoma 10min 60 min 3h 24h 48 h
Mitochondrial fraction
Microsomal fraction
Supernatant fraction
4.9 3.7 4.9 6.8 5.3
8.5 14.5 13.9 14.4 15.0
20.8 33.2 20.8 26.9 27.2
18.1 14.6 16.3 l3.0 14.2
Ehrlich tumor 10 min 60 min 3h 24h 48 h
7.8 11.1 9.1 12.2 14.4
12.5 20.6 18.3 30.6 29.8
23.3 22.9 18.0 24.5 23.6
17.3 15.1 16.9 13.0 12.5
Hepatoma AH109A 10 min 60 min 3h 24h 48 h
11.8 11.1 11.6 20.4 18.8
12.5 16.4 24.0 38.3 36.5
12.8 11.8 12.8 14.6 13.4
12.9 12.7 10.8 4.9 6.1
* Relative specific activities protein (mg) of each fraction.
were calculated
view that lysosome plays an important role in the tumor concentration of 67Ga while the other opinion’s-‘O) sustains that lysosome does not play an important role in the tumor concentration of “Ga. From our present study, it was clear that lysosome did not play an important role in the tumor concen-
by dividing
the values of Table
tration of 67Ga in Yoshida sarcoma and in Ehrhch tumor, but the substance (which was contained in mitochondrial fraction) played an important role in the liver concentration of 67Ga and played a considerably important role in the tumor concentration of 67Ga in hepatoma AH109A.
TABLE 5. Relative specific activities (x/g)* of the protein in the nuclear, microsomal and supernatant fractions of the host livers Nuclear fraction The rats transplanted 10 min 60 min 3h 24h 48 h The mice transplanted 10 min 60 min 3h 24h 48 h The rats transplanted 10 min 60 min 3h 24 h 48 h
1 by the
Mitochondrial fraction
Microsomal fraction
mitochondrial,
Supernatant fraction
Yoshida sarcoma 6.2 5.7 8.2 8.3 7.1
4.0 1.6 9.8 10.9 14.1
10.2 8.6 5.8 6.8 4.8
5.9 4.8 3.8 2.6 2.2
Ehrlich tumor 8.5 6.2 5.0 6.6 5.9
3.5 5.3 8.8 12.8 13.2
8.7 7.4 7.5 7.1 6.4
6.6 7.2 5.5 2.1 2.6
5.2 7.1 8.0 13.4 12.0
6.5 1.3 6.0 5.2 4.7
6.1 5.7 5.4 2.5 2.0
hepatoma
* Relative specific activities protein (mg) of each fraction.
AH109A 8.1 5.6 6.5 6.9 10.7 were calculated
by dividing
the values of Table 2 by the
Work in Progress From the paper previously reportedc3-‘), it was thought that the lysosome is the subcellular component which plays a role in the liver and hepatoma AH109A concentration of 67Ga. BROWN et a1.‘4’ pointed out the disruption of lysosome in some phase of the fractionation procedures. Considering Brown’s view, we carried out the subcellular fractionation of the tumor tissue and the host liver by the same procedures at the same time, and the results described above were obtained. We therefore concluded that lysosomes of Ehrlich tumor and Yoshida sarcoma were not disrupted in the fractionation procedures, and that a large amount of 67Ga had not accumulated in lysosome of these tumors before the subcellular fractionation. It is presumed that there is lysosomal affinity of 67Ga in the case of the specific organ such as the liver. Considerably affinity of 67Ga to lysosome in hepatoma AH109A is due to residual nature of the liver possessed in hepatoma AH109A. The extent of the variation of hepatoma from liver may be determined by using this phenomenon as an indicator. References 1.
EDWARDSC. L. and HAYES R. L. J. Nucl. Med. 10, 103-105 (1969).
2.
3.
69
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