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Radioarsenic labelling of chromium compounds
738
Technical notes
11. BROWN D. \Y. and ~VALL L. ,2. J. Phys. Chem. 65, 915 (1961). and JOIINSON, Ref. 12 in United States 12. WARMAN Atomic Energy Commission, Division of Technical Information, JLl-2901-0-l (1963). 13. KIRCIIER J. I:., RICNULTY J. S., ~~CI;~RI.ING J. L. and LEVY A. R&at. Res. 13, 452 (1960). 14. SHAH J. and J’lI~xm E. C. Int. J. aj$l. Rndiat. Itotofies 17, 155 (1966). by D. JACKSON) United 15. KUKI Z. (Translated Kingdom r\tomic Energy I\uthority, AERETR_ZT\;S 905 (1962). See also KIJRI Z., Snrn~ S. and H~RUI~ARA S. Oak Ridge National Laboratory, ORNL-tr-1774.
Radioarsenic
Labelling
of Chromium
Compounds* (Received 17 Afxil 1969)
Introduction SEVERAL chromium compounds, labelled with 51Clr or 32P, are being used extensively for diagnostic or therapeutic purposes. Particulate (colloidal) chromic phosphate (32P) is perhaps one of the most widely used therapeutic radiopharmaceuticalstt,2). It has been difficult to determine the zonal distribution of this radiocolloid after intracavitary administration. By labelling these particles with “%r it is possible to determine their distribution externally by scanning, but unfortunately because of the poor physical characteristics of 51Cr (low efficiency: nine photons per hundred disintegrations) a relatively large amount of radioactivity is required. Recently, a colloidal form of CrPO, has been demonstrated to concentrate in the mediastinal lymph nodes after intraperitoneal injection(s). After both intracavitary or intralymphatic administration, tagging the radiocolloid with a gamma emitter makes it possible to estimate the area irradiated and allows quantitative evaluation of lymph node uptake. This labelling with other nuclides is not restricted 51&@_ to the insoluble chromium compounds. glycerophosphate complex, a metabolic tumor localizing agent developed recently, presents the same drawbacks due to the Wr radiation noted * Supported
by U.S.P.H.S.
Grant
No. GLI
10548.
above and in addition the half-life is undesirably long for a diagnostic agent. In order to solve these problems %r-/I-glycerophosphate has been labelled with radioarsenic ( 73;2sT,/, = 18 d. and ‘“.\s TI,2 = 26.5 hr)t”). This labelling has been accomplished without altering the in vivo behavior of the original complex significantly. ‘I‘hc basis of this tagging is the property of many anions to replace some of the water molecules in the chromic-hexaaquo complex ion ( Cr(H,0)63+) by a process termed anion penctrationts). iVhen these anions are incorporated into the chromic complex before being precipitated as an insoluble compound, or before a stable complex (as Cr-P-glyccrophosphate) is formed, most of the physicochemical characteristics of the original chromium compound are retained. On the other hand, when the concentration of anions which normally form insoluble chromium salts, i.e. CrP04, CrAsO,, etc. is increased, the resulting complex will undergo more drastic changes, indicating that the specific activity of the radioactive anion used is a limiting factor in complex preparation. In this experimental work the feasibility of tagging various chromium compounds with radioarscnic has been evaluated. In order to determine the degree of change caused by the incorporation of the anionic studies of the physicochemical and radioarsenic, biological properties have been undertaken. Material
and Methods
The preparation of the was done as follows:
assayed
radiocompounds
‘To 0.4 ml of CM>, solution (10 “g/ml), 0.1 ml of H,PO, solution (10 mg/ml) containing the ‘“As as sodium arsenate was added. The mixture was placed in a water bath at 60-70°C and 1 ml of fresh sulfiteacid solution (1.9 sa,SO, + 2.5 ml of cont. HCl in 100 ml of H,O) was added. The volume brought to 8-10 ml with H,O and the solution was neutralized with 1 >i NaHCO,. Finally, the mixture was hcatcd in a boiling water bath (15 min), cooled to room temperature, centrifuged, washed 5 times with distilled H,O and suspended in 0.5 per cent gelatin. (b)
‘“*\sC:rP04
(colloidal)
This technique is similar to that used for the preparation of the particulate form(‘). The only difference is that 02 ml of 20 per cent gelatin was added together with the H,PO,. After cooling it was passed through the anion exchange resin Dowex l-X8 (Cl- form) and through the cation exchange resin Dowex 50-XZ(Ic‘a+ form). Table 1 shows the amount of radioactivity retained by the anion and cation exchange resins from this preparation.
Tecfmical notes TABLE
1. Distribution
-
739
of the radioarsenic after preparation compounds Percentage Colloidal 71.AsC:rI’0,
Retained by Dowex 1-X8 (Cl-) Retained by Dowex 50-X2 (Naf) Final product
(c)
8.1 40.0
11.8 72.0
07 98.0
74AsCr-P-gLycerophosphate
of the chromium
74_~s-Cr-bglycerophosphate
$-‘;\s-C:r--,4lburnin _____
77.9
16 mg of CI+ (as CrDls) were added to 74As sodium arsenate (0.4 pg As) in a volume of 2 ml. After heating 5 min in a boiling water bath, 100 mg of Na-/I-glycerophosphate were added and the pH adjusted to 3.5. The mixture was then heated in a boiling water bath for 15 min. After cooling 5-IO’C, the solution was neutralized to pH 7.2 with 1 M NaHCO, and filtered through a Millipore filter (0.45 (I). The effect of the variation of the atomic ratio Cr/As on the characteristics of the 74AsCr-/Y-glycerophosphate complex was assayed in the following way: In preparation (d) Cr/As ratios of 1 to 2, 1 to 1,2 tol, 2. Effects
to arsenic
4 to 1 and 8 to 1 were utilized. After neutralization to pH 7.2 the preparations were centrifuged for 20 min at 2000 rev/min, washed 3 times with distilled HsO and the radioactivity of the supernatant and the residue was measured. Table 2 shows these values as well as the ratios &/As found in each fraction. The effects of different ratios Cr/As on the physicochemical characteristics of the complex was also tested by a 35 min electrophoresis on Whatman 3 MM paper, using 0.02 M NaHCO, as a buffer and a voltage gradient of 18 V/cm. Figure 1 shows the radioactivity distribution of both 74As and Wr. This 74Ass1Cr glycerophosphate was doubled labelled prepared in a similar way that the ‘“AsCr-,&glycerophosphate, but adding 51Cr as CrCls. The radioactivity was measured in a well counter counting 74As at the O-60-0.64 MeV and 51Cr at the 0.32 nleV energy peaks respectively. The in viva behavior of these compounds was determined injecting intravenously the equivalent to 50 ,LJ~of Cr and 0.05 /lg of As of each radiocompound in a volume of 0.2 ml into groups of white mice and performing whole body counting at different intervals. Figure 2 shows the corresponding values. At the end of the whole body counting experiment (14 days) the animals were sacrificed and the radioactivity counted in blood, liver, spleen, kidney, muscle, femur and
ratio during complex
the preparation
of 74As-Cr-P-glycerophosphate
Soluble
Insoluble Preparation ratio Cr/As 1:2 1:l 2:l 4:l 8:l
74As
16.2
74AW2r-albumirl
TABLE
of the original
chromium
51.9
0.8 mg of Crs+ (as CrCl,) was heated for 5-10 min with the 7”As sodium arsenate (0.4 fig As). Then, 0.2 ml of 25 per cent human serum albumin _t 0.2 ml of 0.1 M sodium acetate buffer (pH 4.0) were added and the mixture incubated at 37’C for 30 mm. Finally the solution was passed through the anion exchange Dowex l-X8 (Cl- form) and through the cation exchange resin Dowex 50-X2 (Na’ form) (‘Table 1). When the pH was finally adjusted to 7.6 a slight turbidity appeared and it was filtered through Millipore filter (0.45 ,LC). (d)
of different
51Cr
74‘4s
(%)
(%)
85.2 64.9 24.2 11.6 3.7
58.4 62.6 14.1 8.8 4.0
Ratio Cr/As 1.0: 1.0: 3.6: 6.6: 8.8:
1.8 1.1 1.0 1.0 1.0
51Cr
74AS
(?I)
(7’0)
Ratio Cr/As
14.8 35.1 75.8 88.4 96.3
41.6 37.4 85.9 91.2 96.0
1 L7.4 1:1.2 1.8: 1.0 4.9: 1.0 9.6: 1.0
Technical
740
Starling
+
notes
Rot10
Cr/As
07iAsCrP04( Particulate) •‘4A~CrP04(CoIloldal)
( 12)
074AsCr-~-glycerophosphale ‘74AsCr-album~n
I
I
( 1.1)
FIG. 2. LVhole body counts of various radioarsenic labellcd chromium compounds injcctcd intravenously into mice.
FIG. 1. Electrophoretic study of the effects of the ratio Cr/As on the physicochemical characteristics ofthe 74AsslCr-/Gglycerophosphate complex. carcass. Table 3 includes the values as per cent of injected dose per organ and the ratios liver to kidney, liver to spleen, liver to carcass, blood to muscle and blood to femur. Results
The yield of labelling with radioarsenic is fairly good for all the assayed compounds: 7aAXsCrP04 7JAsCrP0,(colcent; (particulate) - 70-80 per ‘“.4sCr-Albumin = 70-90 loidal) = 40-60 per cent; = 95-99 per cent and 73AsCr-/Sglycerophosphate per cent. The ion exchange analysis indicates that when the radioarsenic is not bound to an insoluble chromium
compound it may be partially exchanged and retained by the anion exchange resin (Table 1). The effects of increasing concentrations of arsenate on the physicochemical characteristics of the ‘“AsCr-/j’-glycerophosphate is clearly demonstrated in ‘Table 2. The larger the Cr/As ratio, the smaller the amount of insoluble Also, the Cr/;\s ratio is almost material formed. similar in both soluble and insoluble fractions, for stoichiometric ratio greater than one. ‘“lZsCrP04 was excreted to a relatively small extent: Particulate 20 per cent and colloidal 48 per cent after 14 days. Almost 65 per cent of 7”AsCr albumin was excreted during the first 24 hr but thereafter its rate of excretion decreased and 15 per cent still remained after 14 days. .\ similar behavior, but with a higher rate of excretion, was observed for 7~XsC:r-,L$ycerophosphatc. In contrast, 87 per cent of ‘.‘,\s-so&urn arsenate was excreted during the first 24 hr and only 2.7 per cent remained in whole body ‘The distribution of the remaining after 14 days. radioactivity 14 days after intravenous injection shows a defined difference bct\cern all these COIIIpounds, and in every case the pattern is completely diff‘crent to t,hat of radioarsenate (‘“As), Table 3. For instance, radioarsenatc does not remain in the
741
Technical notes TABLE
Blood* Liver Spleen Kidney Muscle* Femur* Carcass Injected
3. Radioactivity distribution 14 days after intravenous injection various 74As labelled chromium compounds
dose (cpm)
74As013(4 animals)
74AsCrP0, (particulate) (4 animals)
0.17 0.04 0.02 0.03 0.16 0.42 0.83 8.3
0.03 * 35.7 * 6.45 2 1.75 f 0.39 + 2.63 + 48.5 i 2.9 x
* * & * + & & x
0.01 0.01 0.01 0.01 0.04 0.33 0.19 IO5
0.01 4.33 0.83 0.62 0.09 0.39 3.09 105
into mice of
74AsCrP04 (colloidal) (4 animals)
7”As-Cr-Albumin (4 animals)
74As-Cr-t!?glycerophosphate (5 animals)
0.05 * 0.02 62,5 & 6.96 3.81 + 1.51 0.64 & 0.08 1.26 $- 0.08 4.17 & 0.30 19.1 + 0.89 13.9 x 105
0.04 & 0.01 9.81 & 3.10 0.41 & 0.12 0.27 & 0.01 0.25 i_ 0.20 2.18 + 0.53 5.03 i 2.42 8.8 x IO”
0.71 4 0.27 0.03 & 0.02 0.27 & 0.19 0.18 * 0.05 0.34 & 0.26 8.62 f I.01 4.6 x IO5
98.1 16.4 3.3 0.04 0.01
36.1 23.9 1.9 0.2 0.02
2.6 22.7 0.08 0.01 0.008
’
Ratio Liver to kidney Liver to spleen Liver to carcass Blood* to muscle* Blood* to femur* * Radioactivity
1.6 2.1 0.005 1.1 0.4
20.3 5.5 0.7 0.09 0.01
per g.
liver, spleen and kidney (the observed radioactivity is due to the blood pool of each organ), while with the other radioarsenic labelled compounds most of the radioactivity is concentrated in those organs. Discussion Two significant facts can be inferred from these experimental results: (a) The radioarsenic incorporated into the insoluble chromium compounds is very tightly bound to the chromium and presents a very high stability in vivo, and (b) when the radioarsenate is covalently bound to a soluble form of chromium (complex) it may be partially displaced in vitro (anion exchange) and in vim. However, the
amount released in vivo has been shown to be less than 10 per cent(*). From the biological point of view in all these radioarsenic tagged chromium compounds, the tag behaves in the identical way as the original chromium derivatives. Whole body counting (uptake and excretion) and body distribution are completely similar to those of s1CrPOpt6*7), 51Cr-Albununt*) and 51Cr-fi-glycerophosphate tp) (Table 4 and Fig. 3). These complete identifications with the original chromium compounds point out the feasibility and usefulness of this labelling in tracing, delineating and quantifying the distribution of chromium compounds. Special consideration should be given to the fact
TABLE 4. Distribution of radiochromium after intravenous injection of various labelled compounds (as percentage of injected dose per organ) 51CrP04 (particulate) Time Liver Spleen Kidney Femur* Carcass * Per g. t Whole skeleton. -not determined.
14 d. 53.4 2.7 0.77 7.3
51CrP04 (colloidal) 14 d. 45.8 1.0 2.3t 20.1
51Cr
51Cr-/?-glycerophosphate 27 d. 6.6 2.1 3.1 -
30 d. 1.2 0.05 0.25 0.22 8.6
742
Technical notes 4. ;\NGHILERI L.J., REUA R. C. and ~YAGNER I-I.K. Jr. Inuest.Radiol. 4, 91 (1969). 5. GIMBLETT F. G. R. in Inorganic Polymer Chemistt_y, pp. 77-164. Butterworth, London (1963). 6. ANCIIILERI L. J. NIlcl. Afed. 6, 321 (1967). 7. :\NGHILERI L. J. NIzl. Med. 7, 184 (1968). 8. 4NGIIILERI L. J. NIlcl. hfeerl. 4, 364 (1965). 9. ANGHILERI I,. J. to be published in Oncolo~.
Autoradiographic
Detection
Using Polaroid 5’CrP04( Partlculolel b 5’CrP0~(Coll~~doll L5’ Cr ,&glycerophosphofe Cr-olbumtn
of Iodine-131
Film *
0
(Receined 3 Apt-i1 1969)
’
USUAL autoradiographic technique employing a standard X-ray film requires a darkroom, both for preparing the film before csposure and for developing the film; also, a moderate aInount of time is rcquired to process the exposed film. .\ system 01 autoradiography was devised employing Polaroid 4 ;< 5 Film Packets which does not require a darkroom and makes it possible to get a positive print showing the location of the activity within a minute aftrr completing the exposure. The usable picture area of a Polaroid 4 % 5 l’ilm Packet is 3.5 :: 4.5 in. and is located -5% in. from each of the two sides of the packet and -s$ in. from the bottom edge. LVherc necessary to cover a larger area, the usable picture area of two or more lilm packets can be overlapped. The dried chromatography paper, containing the activity, is taped in position on the film packet and the assembly is then sandwiched between two flat surfaces and clamprd or weighted to nIaintain inCare InIIst 1,~. timate contact during exposure. taken not to press the film packet in the arca containing the developer pod. In ordrr to avoid 111~ possibility of contamination of the film packrt 1)) the activity on the chromatoCgram. thr paper may bc wrapped in a picce of Mylar tilm. l‘hc location 01 the chromatogram on the film can bc marked using a ball-point pen with Inodcrate pressure; the pen will make a mark on thr chromatogram and au After removing the paprr indentation on the print. from the film packet the picture is dcvelopcd using a standard Polaroid processing dcvicc. THE
I
2
IO
4 Time,
FIG.
labeled
14
days
3. Whole body count of various compound injected intravenously mice.
51C:r into
that the labelling radioarsenate ( 7”As or “jAs) should have the highest possible specific activity in order not to produce changes in the biological characteristics of the chromium compound. LEOPOLDO J.
ANGHII.ERI*
The Johns Hofikins Medical Iutitutions Department of Radiological Science Baltimore
References CUNNINGIIAY R. F. discussion in ;~NDRE~VS C. I\., KNISELEY J. \V. and \VAGNER H. N. Jr. Rndioactive Pharmaceuticals, U.S. .4tomic Energy Cornmission, p. 687 (1966). BURGER R. H., ASANO hf. and N.YGAIMATSUG. R. Invest. U~oZ. 2, 215 (1964). ANGHILERI L. J. Acta Radiol. 7, 202 (1968). * Present address: University Center, Division of Djuclear Colorado 80220, U.S..\.
of Colorado Medicine,
Medical Denver,
* iYork prrl’ormcd under Xtomic Fnergy Commission.
the auspices
of the U.S.
Technical notes
11. BROWN D. \Y. and ~VALL L. ,2. J. Phys. Chem. 65, 915 (1961). and JOIINSON, Ref. 12 in United States 12. WARMAN Atomic Energy Commission, Division of Technical Information, JLl-2901-0-l (1963). 13. KIRCIIER J. I:., RICNULTY J. S., ~~CI;~RI.ING J. L. and LEVY A. R&at. Res. 13, 452 (1960). 14. SHAH J. and J’lI~xm E. C. Int. J. aj$l. Rndiat. Itotofies 17, 155 (1966). by D. JACKSON) United 15. KUKI Z. (Translated Kingdom r\tomic Energy I\uthority, AERETR_ZT\;S 905 (1962). See also KIJRI Z., Snrn~ S. and H~RUI~ARA S. Oak Ridge National Laboratory, ORNL-tr-1774.
Radioarsenic
Labelling
of Chromium
Compounds* (Received 17 Afxil 1969)
Introduction SEVERAL chromium compounds, labelled with 51Clr or 32P, are being used extensively for diagnostic or therapeutic purposes. Particulate (colloidal) chromic phosphate (32P) is perhaps one of the most widely used therapeutic radiopharmaceuticalstt,2). It has been difficult to determine the zonal distribution of this radiocolloid after intracavitary administration. By labelling these particles with “%r it is possible to determine their distribution externally by scanning, but unfortunately because of the poor physical characteristics of 51Cr (low efficiency: nine photons per hundred disintegrations) a relatively large amount of radioactivity is required. Recently, a colloidal form of CrPO, has been demonstrated to concentrate in the mediastinal lymph nodes after intraperitoneal injection(s). After both intracavitary or intralymphatic administration, tagging the radiocolloid with a gamma emitter makes it possible to estimate the area irradiated and allows quantitative evaluation of lymph node uptake. This labelling with other nuclides is not restricted 51&@_ to the insoluble chromium compounds. glycerophosphate complex, a metabolic tumor localizing agent developed recently, presents the same drawbacks due to the Wr radiation noted * Supported
by U.S.P.H.S.
Grant
No. GLI
10548.
above and in addition the half-life is undesirably long for a diagnostic agent. In order to solve these problems %r-/I-glycerophosphate has been labelled with radioarsenic ( 73;2sT,/, = 18 d. and ‘“.\s TI,2 = 26.5 hr)t”). This labelling has been accomplished without altering the in vivo behavior of the original complex significantly. ‘I‘hc basis of this tagging is the property of many anions to replace some of the water molecules in the chromic-hexaaquo complex ion ( Cr(H,0)63+) by a process termed anion penctrationts). iVhen these anions are incorporated into the chromic complex before being precipitated as an insoluble compound, or before a stable complex (as Cr-P-glyccrophosphate) is formed, most of the physicochemical characteristics of the original chromium compound are retained. On the other hand, when the concentration of anions which normally form insoluble chromium salts, i.e. CrP04, CrAsO,, etc. is increased, the resulting complex will undergo more drastic changes, indicating that the specific activity of the radioactive anion used is a limiting factor in complex preparation. In this experimental work the feasibility of tagging various chromium compounds with radioarscnic has been evaluated. In order to determine the degree of change caused by the incorporation of the anionic studies of the physicochemical and radioarsenic, biological properties have been undertaken. Material
and Methods
The preparation of the was done as follows:
assayed
radiocompounds
‘To 0.4 ml of CM>, solution (10 “g/ml), 0.1 ml of H,PO, solution (10 mg/ml) containing the ‘“As as sodium arsenate was added. The mixture was placed in a water bath at 60-70°C and 1 ml of fresh sulfiteacid solution (1.9 sa,SO, + 2.5 ml of cont. HCl in 100 ml of H,O) was added. The volume brought to 8-10 ml with H,O and the solution was neutralized with 1 >i NaHCO,. Finally, the mixture was hcatcd in a boiling water bath (15 min), cooled to room temperature, centrifuged, washed 5 times with distilled H,O and suspended in 0.5 per cent gelatin. (b)
‘“*\sC:rP04
(colloidal)
This technique is similar to that used for the preparation of the particulate form(‘). The only difference is that 02 ml of 20 per cent gelatin was added together with the H,PO,. After cooling it was passed through the anion exchange resin Dowex l-X8 (Cl- form) and through the cation exchange resin Dowex 50-XZ(Ic‘a+ form). Table 1 shows the amount of radioactivity retained by the anion and cation exchange resins from this preparation.
Tecfmical notes TABLE
1. Distribution
-
739
of the radioarsenic after preparation compounds Percentage Colloidal 71.AsC:rI’0,
Retained by Dowex 1-X8 (Cl-) Retained by Dowex 50-X2 (Naf) Final product
(c)
8.1 40.0
11.8 72.0
07 98.0
74AsCr-P-gLycerophosphate
of the chromium
74_~s-Cr-bglycerophosphate
$-‘;\s-C:r--,4lburnin _____
77.9
16 mg of CI+ (as CrDls) were added to 74As sodium arsenate (0.4 pg As) in a volume of 2 ml. After heating 5 min in a boiling water bath, 100 mg of Na-/I-glycerophosphate were added and the pH adjusted to 3.5. The mixture was then heated in a boiling water bath for 15 min. After cooling 5-IO’C, the solution was neutralized to pH 7.2 with 1 M NaHCO, and filtered through a Millipore filter (0.45 (I). The effect of the variation of the atomic ratio Cr/As on the characteristics of the 74AsCr-/Y-glycerophosphate complex was assayed in the following way: In preparation (d) Cr/As ratios of 1 to 2, 1 to 1,2 tol, 2. Effects
to arsenic
4 to 1 and 8 to 1 were utilized. After neutralization to pH 7.2 the preparations were centrifuged for 20 min at 2000 rev/min, washed 3 times with distilled HsO and the radioactivity of the supernatant and the residue was measured. Table 2 shows these values as well as the ratios &/As found in each fraction. The effects of different ratios Cr/As on the physicochemical characteristics of the complex was also tested by a 35 min electrophoresis on Whatman 3 MM paper, using 0.02 M NaHCO, as a buffer and a voltage gradient of 18 V/cm. Figure 1 shows the radioactivity distribution of both 74As and Wr. This 74Ass1Cr glycerophosphate was doubled labelled prepared in a similar way that the ‘“AsCr-,&glycerophosphate, but adding 51Cr as CrCls. The radioactivity was measured in a well counter counting 74As at the O-60-0.64 MeV and 51Cr at the 0.32 nleV energy peaks respectively. The in viva behavior of these compounds was determined injecting intravenously the equivalent to 50 ,LJ~of Cr and 0.05 /lg of As of each radiocompound in a volume of 0.2 ml into groups of white mice and performing whole body counting at different intervals. Figure 2 shows the corresponding values. At the end of the whole body counting experiment (14 days) the animals were sacrificed and the radioactivity counted in blood, liver, spleen, kidney, muscle, femur and
ratio during complex
the preparation
of 74As-Cr-P-glycerophosphate
Soluble
Insoluble Preparation ratio Cr/As 1:2 1:l 2:l 4:l 8:l
74As
16.2
74AW2r-albumirl
TABLE
of the original
chromium
51.9
0.8 mg of Crs+ (as CrCl,) was heated for 5-10 min with the 7”As sodium arsenate (0.4 fig As). Then, 0.2 ml of 25 per cent human serum albumin _t 0.2 ml of 0.1 M sodium acetate buffer (pH 4.0) were added and the mixture incubated at 37’C for 30 mm. Finally the solution was passed through the anion exchange Dowex l-X8 (Cl- form) and through the cation exchange resin Dowex 50-X2 (Na’ form) (‘Table 1). When the pH was finally adjusted to 7.6 a slight turbidity appeared and it was filtered through Millipore filter (0.45 ,LC). (d)
of different
51Cr
74‘4s
(%)
(%)
85.2 64.9 24.2 11.6 3.7
58.4 62.6 14.1 8.8 4.0
Ratio Cr/As 1.0: 1.0: 3.6: 6.6: 8.8:
1.8 1.1 1.0 1.0 1.0
51Cr
74AS
(?I)
(7’0)
Ratio Cr/As
14.8 35.1 75.8 88.4 96.3
41.6 37.4 85.9 91.2 96.0
1 L7.4 1:1.2 1.8: 1.0 4.9: 1.0 9.6: 1.0
Technical
740
Starling
+
notes
Rot10
Cr/As
07iAsCrP04( Particulate) •‘4A~CrP04(CoIloldal)
( 12)
074AsCr-~-glycerophosphale ‘74AsCr-album~n
I
I
( 1.1)
FIG. 2. LVhole body counts of various radioarsenic labellcd chromium compounds injcctcd intravenously into mice.
FIG. 1. Electrophoretic study of the effects of the ratio Cr/As on the physicochemical characteristics ofthe 74AsslCr-/Gglycerophosphate complex. carcass. Table 3 includes the values as per cent of injected dose per organ and the ratios liver to kidney, liver to spleen, liver to carcass, blood to muscle and blood to femur. Results
The yield of labelling with radioarsenic is fairly good for all the assayed compounds: 7aAXsCrP04 7JAsCrP0,(colcent; (particulate) - 70-80 per ‘“.4sCr-Albumin = 70-90 loidal) = 40-60 per cent; = 95-99 per cent and 73AsCr-/Sglycerophosphate per cent. The ion exchange analysis indicates that when the radioarsenic is not bound to an insoluble chromium
compound it may be partially exchanged and retained by the anion exchange resin (Table 1). The effects of increasing concentrations of arsenate on the physicochemical characteristics of the ‘“AsCr-/j’-glycerophosphate is clearly demonstrated in ‘Table 2. The larger the Cr/As ratio, the smaller the amount of insoluble Also, the Cr/;\s ratio is almost material formed. similar in both soluble and insoluble fractions, for stoichiometric ratio greater than one. ‘“lZsCrP04 was excreted to a relatively small extent: Particulate 20 per cent and colloidal 48 per cent after 14 days. Almost 65 per cent of 7”AsCr albumin was excreted during the first 24 hr but thereafter its rate of excretion decreased and 15 per cent still remained after 14 days. .\ similar behavior, but with a higher rate of excretion, was observed for 7~XsC:r-,L$ycerophosphatc. In contrast, 87 per cent of ‘.‘,\s-so&urn arsenate was excreted during the first 24 hr and only 2.7 per cent remained in whole body ‘The distribution of the remaining after 14 days. radioactivity 14 days after intravenous injection shows a defined difference bct\cern all these COIIIpounds, and in every case the pattern is completely diff‘crent to t,hat of radioarsenate (‘“As), Table 3. For instance, radioarsenatc does not remain in the
741
Technical notes TABLE
Blood* Liver Spleen Kidney Muscle* Femur* Carcass Injected
3. Radioactivity distribution 14 days after intravenous injection various 74As labelled chromium compounds
dose (cpm)
74As013(4 animals)
74AsCrP0, (particulate) (4 animals)
0.17 0.04 0.02 0.03 0.16 0.42 0.83 8.3
0.03 * 35.7 * 6.45 2 1.75 f 0.39 + 2.63 + 48.5 i 2.9 x
* * & * + & & x
0.01 0.01 0.01 0.01 0.04 0.33 0.19 IO5
0.01 4.33 0.83 0.62 0.09 0.39 3.09 105
into mice of
74AsCrP04 (colloidal) (4 animals)
7”As-Cr-Albumin (4 animals)
74As-Cr-t!?glycerophosphate (5 animals)
0.05 * 0.02 62,5 & 6.96 3.81 + 1.51 0.64 & 0.08 1.26 $- 0.08 4.17 & 0.30 19.1 + 0.89 13.9 x 105
0.04 & 0.01 9.81 & 3.10 0.41 & 0.12 0.27 & 0.01 0.25 i_ 0.20 2.18 + 0.53 5.03 i 2.42 8.8 x IO”
0.71 4 0.27 0.03 & 0.02 0.27 & 0.19 0.18 * 0.05 0.34 & 0.26 8.62 f I.01 4.6 x IO5
98.1 16.4 3.3 0.04 0.01
36.1 23.9 1.9 0.2 0.02
2.6 22.7 0.08 0.01 0.008
’
Ratio Liver to kidney Liver to spleen Liver to carcass Blood* to muscle* Blood* to femur* * Radioactivity
1.6 2.1 0.005 1.1 0.4
20.3 5.5 0.7 0.09 0.01
per g.
liver, spleen and kidney (the observed radioactivity is due to the blood pool of each organ), while with the other radioarsenic labelled compounds most of the radioactivity is concentrated in those organs. Discussion Two significant facts can be inferred from these experimental results: (a) The radioarsenic incorporated into the insoluble chromium compounds is very tightly bound to the chromium and presents a very high stability in vivo, and (b) when the radioarsenate is covalently bound to a soluble form of chromium (complex) it may be partially displaced in vitro (anion exchange) and in vim. However, the
amount released in vivo has been shown to be less than 10 per cent(*). From the biological point of view in all these radioarsenic tagged chromium compounds, the tag behaves in the identical way as the original chromium derivatives. Whole body counting (uptake and excretion) and body distribution are completely similar to those of s1CrPOpt6*7), 51Cr-Albununt*) and 51Cr-fi-glycerophosphate tp) (Table 4 and Fig. 3). These complete identifications with the original chromium compounds point out the feasibility and usefulness of this labelling in tracing, delineating and quantifying the distribution of chromium compounds. Special consideration should be given to the fact
TABLE 4. Distribution of radiochromium after intravenous injection of various labelled compounds (as percentage of injected dose per organ) 51CrP04 (particulate) Time Liver Spleen Kidney Femur* Carcass * Per g. t Whole skeleton. -not determined.
14 d. 53.4 2.7 0.77 7.3
51CrP04 (colloidal) 14 d. 45.8 1.0 2.3t 20.1
51Cr
51Cr-/?-glycerophosphate 27 d. 6.6 2.1 3.1 -
30 d. 1.2 0.05 0.25 0.22 8.6
742
Technical notes 4. ;\NGHILERI L.J., REUA R. C. and ~YAGNER I-I.K. Jr. Inuest.Radiol. 4, 91 (1969). 5. GIMBLETT F. G. R. in Inorganic Polymer Chemistt_y, pp. 77-164. Butterworth, London (1963). 6. ANCIIILERI L. J. NIlcl. Afed. 6, 321 (1967). 7. :\NGHILERI L. J. NIzl. Med. 7, 184 (1968). 8. 4NGIIILERI L. J. NIlcl. hfeerl. 4, 364 (1965). 9. ANGHILERI I,. J. to be published in Oncolo~.
Autoradiographic
Detection
Using Polaroid 5’CrP04( Partlculolel b 5’CrP0~(Coll~~doll L5’ Cr ,&glycerophosphofe Cr-olbumtn
of Iodine-131
Film *
0
(Receined 3 Apt-i1 1969)
’
USUAL autoradiographic technique employing a standard X-ray film requires a darkroom, both for preparing the film before csposure and for developing the film; also, a moderate aInount of time is rcquired to process the exposed film. .\ system 01 autoradiography was devised employing Polaroid 4 ;< 5 Film Packets which does not require a darkroom and makes it possible to get a positive print showing the location of the activity within a minute aftrr completing the exposure. The usable picture area of a Polaroid 4 % 5 l’ilm Packet is 3.5 :: 4.5 in. and is located -5% in. from each of the two sides of the packet and -s$ in. from the bottom edge. LVherc necessary to cover a larger area, the usable picture area of two or more lilm packets can be overlapped. The dried chromatography paper, containing the activity, is taped in position on the film packet and the assembly is then sandwiched between two flat surfaces and clamprd or weighted to nIaintain inCare InIIst 1,~. timate contact during exposure. taken not to press the film packet in the arca containing the developer pod. In ordrr to avoid 111~ possibility of contamination of the film packrt 1)) the activity on the chromatoCgram. thr paper may bc wrapped in a picce of Mylar tilm. l‘hc location 01 the chromatogram on the film can bc marked using a ball-point pen with Inodcrate pressure; the pen will make a mark on thr chromatogram and au After removing the paprr indentation on the print. from the film packet the picture is dcvelopcd using a standard Polaroid processing dcvicc. THE
I
2
IO
4 Time,
FIG.
labeled
14
days
3. Whole body count of various compound injected intravenously mice.
51C:r into
that the labelling radioarsenate ( 7”As or “jAs) should have the highest possible specific activity in order not to produce changes in the biological characteristics of the chromium compound. LEOPOLDO J.
ANGHII.ERI*
The Johns Hofikins Medical Iutitutions Department of Radiological Science Baltimore
References CUNNINGIIAY R. F. discussion in ;~NDRE~VS C. I\., KNISELEY J. \V. and \VAGNER H. N. Jr. Rndioactive Pharmaceuticals, U.S. .4tomic Energy Cornmission, p. 687 (1966). BURGER R. H., ASANO hf. and N.YGAIMATSUG. R. Invest. U~oZ. 2, 215 (1964). ANGHILERI L. J. Acta Radiol. 7, 202 (1968). * Present address: University Center, Division of Djuclear Colorado 80220, U.S..\.
of Colorado Medicine,
Medical Denver,
* iYork prrl’ormcd under Xtomic Fnergy Commission.
the auspices
of the U.S.