- Email: [email protected]
Whole-body autoradiography using 11C with double-tracer applications
Int. J. Appl. Radiat. Isot. Vol. 35, No. 2, pp. 129-134, 1984
0020-708X/84$3.00+0.00 Copyright ~ 1984Pergamon Press Ltd
Printed in Great Britain. All rights r'-~rved
Whole-body Autoradiography Using with Double-tracer Applications R. D ' A R G Y l*, S. U L L B E R G , l C.-G. S T A L N A C K E z, and B. L A N G S T R O M ~ Departments of 'Toxicology, ZPhysical Biology and 3Organic Chemistry, University of Uppsala, Sweden (Received I 1 February 1983)
This paper reports that it is possible to obtain good autoradiographic pictures of a "C-labelled compound ([a~C]methionine) in spite of the short half-life of 20 rain by modifying the whole-body autoradiographic (WBA) technique. Double radionuclide autoradiography was performed by utilizing the great difference between the half-lives of ttC and t4C or 35S.The first exposure resulted entirely from I'C, and the second exposure the day after from '(C or 35S, respectively. Satisfactory blackening of the film was obtained by a mean radioactivity of 1,6 x l0~ disinte~ations/cm 2 during the exposure. The "C-WBA technique apparently provides a useful tool in distribution studies on animals, which is valuable in the interpretation of positron emission tomography images of the same "C-compound.
Introduction Whole-body autoradiography (WBA) with long-lived ~-emitters, such as t4C and ~H, is an established technique for studying the distribution of labelled compounds in animals. °) This technique is also of great value in pre-clinical studies of the distribution of radiopharmaceuticals to be used in positron emission tomography (PET). (z) Along with the development of the PET technique, an increasing number of organic compounds have been labelled during the past few years with shortlived positron emitters such as ~3N, "C and ISF (t~/2 = 10, 20 and 110rain, respectively), tSF has been used previously in autoradiographic studies despite its relatively short half-life. 0-5) The application of short-lived nuclides in autoradiographic investigations offers the advantage of obtaining pictures rapidly and also widens the scope of autoradiography. The very high specific activity attainable (theoretically 3.4 x 102°Bq/mol for ItC, compared with 2.3 × 10~ZBq/mol for t4C), allows autoradiography of very small amounts of substance. Exceptionally toxic compounds can thus be used without seriously affecting the animal. Substances like peptide hormones, which are biologically active in very low concentration, might be used for autoradiography without disturbing the receptor affinity, which is often a consequence of radioiodination. (6)
Autoradiography with I~C also opens up the possibility of double labelling with radiocarbon. By utilizing the great difference in half-life between "C and a long-lived nuclide, e.g. '4C, the distribution of a HC-labeUed test substance and a ~"C-labelled reference substance may be compared in the same wholebody section. Biological and other intra-experimental variations are thus eliminated, In this paper a report is given on the modification of the WBA technique to allow the registration of HC. Various kinds of double-tracer autoradiography using methionine as a model tracer have been performed. In some experiments both the L- and o-enantiomers of methionine have also been studied. Materials and Methods Production o f t l C-labelled methionine
The radionuclide "C was produced at the Tandem Accelerator Laboratory in Uppsala by bombarding a nitrogen gas target with protons. The radioactivity was obtained as "CO2 and used to produce ["C]methyl iodide. T h e sulphide anion of homocysteine was alkylated by [UC]methyl iodide in liquid ammonia, yielding ["C]methionine labelled at the S-methyl group. (7.s) The synthesis was used to produce both the o and the t, forms of methionine. (9) Animals
* All corr~pondence should be addressed to: Dr R. d'Argy, Department of Toxicology, Uppsala University, Box 573, $-751 23 Uppsala, Sweden. 129
NMRI mice weighing about 30 g each were used in four different experiments. The animals were kept at room temperature and given a commercial pellet diet
R. D'ARGY et al.
[30
(AB Ewos, S6dertAlje, Sweden) and tapwater ad libitum. They were injected intravenously with 0.2--0.4 mL of the sterile filtered solution. At the start of each experiment, the NC-radioactivity was determined by means of an ionization chamber (Capintec Inc.). The administered radioactivities were in the range of 1.5-5.6 MBq/g (40-150tzCi/g) of ttC and about 9.3kBq/g (0.25/aCi/g) of each [~4C] and [sSS]methionine. The specific activities were 370 GBq/mmol (10 Ci/mmol) of tic 2.10 GBq/mmol (56.TmCi/mmol) and 5.11GBq/mmol (138mCi/mmol) of [~4C] and [sSS]methionine respectively. With the method described in this paper, the (Amersham International), following experiments were performed: I. L-[methyl-t~C]methionine//L-[methyl-" C]methionine II. L-[SSS]methionine//l,-[methyl- HC]methionine III. L-[SSS]methionine//D-[methyl-~ ~C]methionine IV. L-[methyl-NC]methionine (control).
JProduction of lIc
Autoradiographic Technique The time required for the various stages of traditional WBA was reduced. The procedure used is presented schematically in Fig. I, and the time required for each stage is stated. (The suffixes to the subheadings below refer to Fig. 1.) Careful planning at all stages of t~C autoradiography is extremely important, and all equipment has to be ready for use at the different locations. After injection of the tracer and allowing time for distribution (20--90 min), the mouse was killed by inhalation of CO: from dry ice.
Freezing (Step l) The body was frozen by immersion for 2 rain in hexane cooled by dry ice (-70~C), resulting in a temperature of about - 20°C at the centre of a 30 g mouse.
Sawing (Steps 2 and 3) The frozen mouse was attached to a pre-cooled microtome stage by means of a carboxymethyl cellu-
-IAnimal
radi,0pharmaceutical
I !
2. --
.
+
l,ree,ing(? min)
I
T
]Adherlngof intact (animal {2 min)
+ 3~ [ Sawing'{l min)
4.1CMC-'blocksprepared [
--finadvance
4. IAdherlngof sawn
=islices(S min)
I \\x\
,,
8'.)Quantltatlon pieces)~_~Cryosectioning ~l(e.g: punch!ng (IS min)
)
6. lApplicationof sec- l I tions on film (I0 min)I
--
I
picture (15min)
J-
1
[(2 h)
--
'II
1
Application of sections on film "~,
T
~ sections
I
IDeveloping of longllived radionuclide
]'
J-
\
[slices on film (0.5 min;J I
Evaluation:Image' ]
"
\
7'.lExposureof llc
DOUBLETRACEREXPERIMENTS: IFreeze-drying of )
lprocessing,,, etc.
\
[(0.5-15 min)
,,
.
'
I Exposure of Iong-
I lived radionuclide
I)
Fig. 1. Experimental design of "C whole-body autoradiotp'aphy showing the successive stages and a rough estimate of their duration (within brackets). Broken arrows show some alternative stages which, if time allows, can be performed simultaneously.
,]
a
..
;
•
•
Nasal mucosa
;
;
:
•
|
,
Liver Pancreas Kidney Intestines
b
Fig. 2. Autoradiograms of L-[methyl-Uqmethionine illustrating the resolution from Ca) a 2 m m thick sawn slice and (b) a corresponding 20 # m cut section from the same mouse. Light areas correspond, to high concentrations of labelled substance. The exposure times were 5 min and 2 h. respectively. a
'
Salivary g|and ,
"
i/
-
Liver Pancreas intestines •
•
,,
;
:
:Zz,~. . . . . . ":'
"~.'~ .~.r~ ~ ~-.- ~
Fig. 3. Autoradiograms from a mouse showing Ca) the distribution of L-[methyl-nC]methionine and (b) L-[methyl-~aC]methionine in the same section l h after the i.v. injection of the double carbon tracer. Note the high activity in pancreas, liver, and intestinal mucosa. T h e distribution pattern was apparently identical in the two exposures. Exposure time: 2 h and 20 days. respectively. 131
gl
Salivary gland I'
Liver Pancreas Kidney Intestines
"t
:.:
i.
i
b
Fig. 4. Autoradiograms from a mouse showing (a) the distribution of o-[methyl-"C]methionine and (b) L-[35S]methionine in the same section 30 rain after i.v. injection of the double tracer. Note the ve~- high concentration of the D form in the kidney cortex (due to excretion) and the higher incorporation of the L form in the protein synth~izing organs. Exposure time: 2 h and 30 days, respectively.
132
"C-whole-body autoradiography lose (CMC) gel. To facilitate the attachment, the fur of the mouse was moistened by immersion for a few seconds in a detergent. The frozen stage-mounted mouse was then sawn sagittariy into 2 mm thick slices by using a specially designed rotary saw, ('°) located in a cold room at -20°C. With a saw-blade thickness of 2 ram, it proved possible to obtain five slices from each animal.
Cutting (Steps 4 and 5) Frozen CMC blocks were placed in advance in the cutting position on a cryomicrotome (LKB 2250) at -20°C and the sawn slices were then attached to the blocks by a thin layer of CMC gel. Since the sawn slices were plane-parallel and the frozen CMC blocks had been planed, no microtome adjustments were needed and whole-body sections could be cut immediately. To obtain intact sections, Scotch tape (3M:810) was applied to the surface of the block before sectioning. The thickness of the sections was selected at 20, 40 and 60/~m, respectively.
Autoradiographic exposure of sections (Steps 6 and 7) The sections were immediately brought to a cold room at - 2 0 °, equipped with a safe-light, for use as a dark-room. The sections were placed directly onto x-ray film (Kodak Industrex). In some cases thin Mylar foil (6/zm) was placed between the section and film to avoid artefacts due to chemography. To rationalize the dark-room handling, 10-12 seetions were placed onto the same film sheet (24 × 30 cm) and put in an x-ray cassette (Siemens) with or without amplification screens. The exposure time was 2-3 h.
Autoradiographic exposure of sawn slices (Steps 6"and 7") In some instances, x-ray films were also placed against both sides of the sawn slices. A series of exposure times ranging between 0.5 and 15 rain were selected.
Determination of radioactivity (Step 8") The radioactivity of whole mice and sawn slices was determined by measuring photon radiation by means of an ionization chamber (Capintec Inc.). To quantitate the radioactivity in various tissues from the whole-body sections, small circular pieces (3 mm dia.) were punched out of the 60-/~m sections. Pieces were punched from river, pancreas and muscles. Each punched tissue piece was dissolved in 1 mL Soluene 350'~ (Packard) over a period of about I0 rain at 60°C. The pieces were then repeatedly counted in order to separate short- and long-lived radionuclides by ordinary liquid scintillation techniques. The liquid scintillation counter (Packard TRI-CARB 460 CD) was equipped with a luminescence detector, which subtracted chemiluminescence events. Owing to the high fl-energy of HC, the detector efficiency was .~R.I. 3S~--D
133
considered to be very high, and in calculations was therefore assumed to be 100%. On the following day, when the "C had decayed, the sections in the double tracer experiments were dehydrated and placed against x-ray film for exposure of the long-lived radionuclide. The exposure time was 3-4 weeks. In double-tracer experiments after decay of the ~C, a 3-h control exposure was performed in order to prove that no L4C or 3sS interfered with the "C picture. No detectable blackening was obtained during that period of time. Results and Discussion The time aspect is, of course, the most difficult problem in using these short-lived nuclides in WBA, and much attention has been paid to minimizing some of the time-consuming steps. The killed animal was frozen just long enough to ensure that the inner portions had solidified. Sawing into slices reduced the bulk mass and increased the surface, which lessens the time necessary for homogeneous acclimatization to the sectioning temperature (-20°C). In addition, sawing exposes different levels of the animal very rapidly. The sawn slices were attached to pre-planed CMC blocks, so that the microtome could be adjusted before the actual sectioning started. The sections obtained were immediately placed on the x-ray film for exposure, without any time-consuming dehydration. As expected, the autoradiographic resolution was better after the t4C and 3sS exposures as compared to the "C. The difference in resolution was rather small for thin sections (20 ~m) but increased with thickness of section. The maximum energy of "C positrons is 0.98 MeV (95% of the particles penetrate 60~m water-equivalent medium). A sawn slice is thick in relation to the range of the "C-positrons. Figure 2 shows an exposure of L-[methyl-"C]methionine of a sawn 2-mm thick slice (Fig. 2a) and a comparable exposure of a 20/~m cut section (Fig. 2b). The exposure of the sawn slice did not show any fine details but still provided a rough estimate of the relative radioactivity of different organs. The technique of exposing sawn slices is especially rapid and quite simple to perform. An investigation can thus be made with a lower dose of *~C. The most convenient way to obtain a proper exposure of thick slices was to make a sequence of exposures of different durations from each slice (e-g- 0.5, 1, 2 and 5 rain) before developing the film. The resolution of the thin cut sections was somewhat decreased by the x-ray cassette amplification screens. The use of cassettes facilitates the dark-room work, but the amplification screens should be shielded by black paper, to avoid image distortion. The autoradiograms obtained from L-[methyl -UC]methionine and L-[methyl-~4C]methionine, respectively, in the same 20/~m section are shown in Fig. 3. A weak blackening from the intestinal con-
134
R. D'ARGYet aL
tents caused by accidental warming of the sections can be seen (Fig. 3a). It proved essential to avoid warming the thin non-dehydrated sections with the fingers when placing them against films. If they were carefully handled and kept at -20~'C, chemographic artefacts were avoided. The use of Mylar sheets eliminated these artefacts but also decreased the resolution. The distribution pattern of L-[methyl"C]methionine and L-[methyl-~4C]methionine was apparently identical. The highest activity in the body was found in the pancreas. Other organs with high uptake were the gastrointestinal mucosa, the salivary gland, bone marrow, and the pituitary. These results were also in agreement with previous distribution studies of L-[methyt-~4C]methionineY J.z,~ To achieve a satisfactory blackening from ~C, the sections had to contain a mean radioactivity of 1.6 x 106 disintegrations/cm-' during the exposure. Data obtained from punched pieces of 60-/~m sections indicate that liver, pancreas and muscle contained 1.3, 2.4 and 0.20 cps/MBq, respectively, of the administered L-[methyl-"C]methionine 100 min after the injection. To attain the proper exposure of thin sections, several different thicknesses (e.g., 20, 40 and 60 # m) were placed for exposure simultaneously over a period of about 2 h, a time long enough to acquire 99% of the available tiC-decay events. The absorbed whole body dose to personnel from )]C radiation was less than 0.1 mGy in experiments involving 3-4 animals. The internal fl-dose to the animal was estimated to be about 6.3 x l0 -H Gy g/disintegration. This did not induce any detectable distributional disturbance in these short term experiments. By double-tracer autoradiography, the distributions of L-[methyl2~C]methionine and t-[35S]methionine were compared from the same section. No distribution differences were seen. This indicates that the demethylation of methionine does not influence the overall distribution pattern in the time interval (20-90 min) studied. Autoradiograms of o-[methylHC]methionine and L-[~S]methionine in the same sections are seen in Fig. 4. A clear difference in distribution can be noticed: the lower utilization of the D form, illustrated by higher concentrations in the body fluids, and the very high radioactivity in the inner portions of the kidney cortex, indicating stronger excretion by the kidney (Fig. 4b).
Concluding Remarks Autoradiography of animals larger than mice, such as monkeys, or parts of animals, will take a somewhat longer time but should be feasible by essentially the same procedures as those described here. The animals were sectioned longitudinally (sagittally), but transverse sections, like the pictures obtained by PET, could also be used. In view of the
difficulties of interpreting PET images, ~tC-WBA offers a useful tool in animal studies, which should precede clinical PET work. In this way, the tracer dynamics of various radiopharmaceuticals can be confirmed. In principle such studies can be made with long-lived nuclides, such as t4C or 3H, but this generally involves several weeks exposure time. This is a serious drawback, especially if the preliminary results are not satisfactory, and new variables have to be tried. It is then a great advantage if the results are obtained rapidly in each experiment. Double carbon autoradiography (~tC + t4C) offers an interesting approach by comparing two distribution patterns from the same section, that is without biological variation. In addition, better image separation is obtained as compared with previous autoradiographic double tracer techniques. The possibilities for variation of double tracer autoradiography are numerous: (1) If a molecule is known or suspected to be split in the body: two different preparations of the same substance, labelled in different parts of the molecule, can then be tested. The ~tC and t4C pictures will then be identical initially but will differ with time. (2) Two labelled preparations of the same substance: differences in pattern after administration at time intervals or after using different application routes. (3) Different substances which may be related in some respect. Acknowledgements--We are indebted to Petter Malmborg, Ph.D., and Mr Daniel Latyea for technical assistance. This work forms part of a research project financially supported by the Swedish Natural Science Research Council and the National Swedish Board for Technical Development (No. 79-6790).
References 1. Ullberg S. Science Tools (LKB-produkter, Brornma, 1977). 2. Ter-Pogossian M. M., Raichle M. E. and Sobel B. E. Sci. Am. 243, 141 (1980). 3. Appelgren, L.-E., Ericsson Y. and Ullberg S. Acta Physiol. Scand. 53, 339 (1961). 4. Fukuda H., Matsuzawa T., Abe Y., Endo S., Yamada K., Kubota K., Hatazawa J., Sato T., Ito M., Takahashi T., Iwata g. and Ido T. Eur. J. Nucl. Med. 7, 294 (1982). 5. Ido T., Fukushi K. and Irie T. Biomedical Aspects of Fluorine Chemistry (F_MsFiller R. and Kobayashi Y.) p. 143 (Elsevier, Amsterdam, 1982). 6. Hunter W. M. Br. Med. Bull. 30, 18 (1974). 7. L~.ngstr6m B. and Lundqvist H. Int. J. Appl. Radiat. Isot. 27, 357 (1976). 8. L/mgstr6m B. On the Synthesis of tiC-compounds. Ph.D. Thesis. Acta Universitatis Upsaliensis. No. 555 (1980). 9. Lundqvist H., LAngstrom B. and Malmqvist M. J. Radioanal. Chem. In press. 10. Bersmann K. CRC Crit. Ret'. ToxicoL In press. 11. Hansson E. Acta Physiol. Scand. 46 (Suppl. 161) (1959). 12. Benard P. and Duranel S. Ann. Rech. Vet. 11 201 (1980).
0020-708X/84$3.00+0.00 Copyright ~ 1984Pergamon Press Ltd
Printed in Great Britain. All rights r'-~rved
Whole-body Autoradiography Using with Double-tracer Applications R. D ' A R G Y l*, S. U L L B E R G , l C.-G. S T A L N A C K E z, and B. L A N G S T R O M ~ Departments of 'Toxicology, ZPhysical Biology and 3Organic Chemistry, University of Uppsala, Sweden (Received I 1 February 1983)
This paper reports that it is possible to obtain good autoradiographic pictures of a "C-labelled compound ([a~C]methionine) in spite of the short half-life of 20 rain by modifying the whole-body autoradiographic (WBA) technique. Double radionuclide autoradiography was performed by utilizing the great difference between the half-lives of ttC and t4C or 35S.The first exposure resulted entirely from I'C, and the second exposure the day after from '(C or 35S, respectively. Satisfactory blackening of the film was obtained by a mean radioactivity of 1,6 x l0~ disinte~ations/cm 2 during the exposure. The "C-WBA technique apparently provides a useful tool in distribution studies on animals, which is valuable in the interpretation of positron emission tomography images of the same "C-compound.
Introduction Whole-body autoradiography (WBA) with long-lived ~-emitters, such as t4C and ~H, is an established technique for studying the distribution of labelled compounds in animals. °) This technique is also of great value in pre-clinical studies of the distribution of radiopharmaceuticals to be used in positron emission tomography (PET). (z) Along with the development of the PET technique, an increasing number of organic compounds have been labelled during the past few years with shortlived positron emitters such as ~3N, "C and ISF (t~/2 = 10, 20 and 110rain, respectively), tSF has been used previously in autoradiographic studies despite its relatively short half-life. 0-5) The application of short-lived nuclides in autoradiographic investigations offers the advantage of obtaining pictures rapidly and also widens the scope of autoradiography. The very high specific activity attainable (theoretically 3.4 x 102°Bq/mol for ItC, compared with 2.3 × 10~ZBq/mol for t4C), allows autoradiography of very small amounts of substance. Exceptionally toxic compounds can thus be used without seriously affecting the animal. Substances like peptide hormones, which are biologically active in very low concentration, might be used for autoradiography without disturbing the receptor affinity, which is often a consequence of radioiodination. (6)
Autoradiography with I~C also opens up the possibility of double labelling with radiocarbon. By utilizing the great difference in half-life between "C and a long-lived nuclide, e.g. '4C, the distribution of a HC-labeUed test substance and a ~"C-labelled reference substance may be compared in the same wholebody section. Biological and other intra-experimental variations are thus eliminated, In this paper a report is given on the modification of the WBA technique to allow the registration of HC. Various kinds of double-tracer autoradiography using methionine as a model tracer have been performed. In some experiments both the L- and o-enantiomers of methionine have also been studied. Materials and Methods Production o f t l C-labelled methionine
The radionuclide "C was produced at the Tandem Accelerator Laboratory in Uppsala by bombarding a nitrogen gas target with protons. The radioactivity was obtained as "CO2 and used to produce ["C]methyl iodide. T h e sulphide anion of homocysteine was alkylated by [UC]methyl iodide in liquid ammonia, yielding ["C]methionine labelled at the S-methyl group. (7.s) The synthesis was used to produce both the o and the t, forms of methionine. (9) Animals
* All corr~pondence should be addressed to: Dr R. d'Argy, Department of Toxicology, Uppsala University, Box 573, $-751 23 Uppsala, Sweden. 129
NMRI mice weighing about 30 g each were used in four different experiments. The animals were kept at room temperature and given a commercial pellet diet
R. D'ARGY et al.
[30
(AB Ewos, S6dertAlje, Sweden) and tapwater ad libitum. They were injected intravenously with 0.2--0.4 mL of the sterile filtered solution. At the start of each experiment, the NC-radioactivity was determined by means of an ionization chamber (Capintec Inc.). The administered radioactivities were in the range of 1.5-5.6 MBq/g (40-150tzCi/g) of ttC and about 9.3kBq/g (0.25/aCi/g) of each [~4C] and [sSS]methionine. The specific activities were 370 GBq/mmol (10 Ci/mmol) of tic 2.10 GBq/mmol (56.TmCi/mmol) and 5.11GBq/mmol (138mCi/mmol) of [~4C] and [sSS]methionine respectively. With the method described in this paper, the (Amersham International), following experiments were performed: I. L-[methyl-t~C]methionine//L-[methyl-" C]methionine II. L-[SSS]methionine//l,-[methyl- HC]methionine III. L-[SSS]methionine//D-[methyl-~ ~C]methionine IV. L-[methyl-NC]methionine (control).
JProduction of lIc
Autoradiographic Technique The time required for the various stages of traditional WBA was reduced. The procedure used is presented schematically in Fig. I, and the time required for each stage is stated. (The suffixes to the subheadings below refer to Fig. 1.) Careful planning at all stages of t~C autoradiography is extremely important, and all equipment has to be ready for use at the different locations. After injection of the tracer and allowing time for distribution (20--90 min), the mouse was killed by inhalation of CO: from dry ice.
Freezing (Step l) The body was frozen by immersion for 2 rain in hexane cooled by dry ice (-70~C), resulting in a temperature of about - 20°C at the centre of a 30 g mouse.
Sawing (Steps 2 and 3) The frozen mouse was attached to a pre-cooled microtome stage by means of a carboxymethyl cellu-
-IAnimal
radi,0pharmaceutical
I !
2. --
.
+
l,ree,ing(? min)
I
T
]Adherlngof intact (animal {2 min)
+ 3~ [ Sawing'{l min)
4.1CMC-'blocksprepared [
--finadvance
4. IAdherlngof sawn
=islices(S min)
I \\x\
,,
8'.)Quantltatlon pieces)~_~Cryosectioning ~l(e.g: punch!ng (IS min)
)
6. lApplicationof sec- l I tions on film (I0 min)I
--
I
picture (15min)
J-
1
[(2 h)
--
'II
1
Application of sections on film "~,
T
~ sections
I
IDeveloping of longllived radionuclide
]'
J-
\
[slices on film (0.5 min;J I
Evaluation:Image' ]
"
\
7'.lExposureof llc
DOUBLETRACEREXPERIMENTS: IFreeze-drying of )
lprocessing,,, etc.
\
[(0.5-15 min)
,,
.
'
I Exposure of Iong-
I lived radionuclide
I)
Fig. 1. Experimental design of "C whole-body autoradiotp'aphy showing the successive stages and a rough estimate of their duration (within brackets). Broken arrows show some alternative stages which, if time allows, can be performed simultaneously.
,]
a
..
;
•
•
Nasal mucosa
;
;
:
•
|
,
Liver Pancreas Kidney Intestines
b
Fig. 2. Autoradiograms of L-[methyl-Uqmethionine illustrating the resolution from Ca) a 2 m m thick sawn slice and (b) a corresponding 20 # m cut section from the same mouse. Light areas correspond, to high concentrations of labelled substance. The exposure times were 5 min and 2 h. respectively. a
'
Salivary g|and ,
"
i/
-
Liver Pancreas intestines •
•
,,
;
:
:Zz,~. . . . . . ":'
"~.'~ .~.r~ ~ ~-.- ~
Fig. 3. Autoradiograms from a mouse showing Ca) the distribution of L-[methyl-nC]methionine and (b) L-[methyl-~aC]methionine in the same section l h after the i.v. injection of the double carbon tracer. Note the high activity in pancreas, liver, and intestinal mucosa. T h e distribution pattern was apparently identical in the two exposures. Exposure time: 2 h and 20 days. respectively. 131
gl
Salivary gland I'
Liver Pancreas Kidney Intestines
"t
:.:
i.
i
b
Fig. 4. Autoradiograms from a mouse showing (a) the distribution of o-[methyl-"C]methionine and (b) L-[35S]methionine in the same section 30 rain after i.v. injection of the double tracer. Note the ve~- high concentration of the D form in the kidney cortex (due to excretion) and the higher incorporation of the L form in the protein synth~izing organs. Exposure time: 2 h and 30 days, respectively.
132
"C-whole-body autoradiography lose (CMC) gel. To facilitate the attachment, the fur of the mouse was moistened by immersion for a few seconds in a detergent. The frozen stage-mounted mouse was then sawn sagittariy into 2 mm thick slices by using a specially designed rotary saw, ('°) located in a cold room at -20°C. With a saw-blade thickness of 2 ram, it proved possible to obtain five slices from each animal.
Cutting (Steps 4 and 5) Frozen CMC blocks were placed in advance in the cutting position on a cryomicrotome (LKB 2250) at -20°C and the sawn slices were then attached to the blocks by a thin layer of CMC gel. Since the sawn slices were plane-parallel and the frozen CMC blocks had been planed, no microtome adjustments were needed and whole-body sections could be cut immediately. To obtain intact sections, Scotch tape (3M:810) was applied to the surface of the block before sectioning. The thickness of the sections was selected at 20, 40 and 60/~m, respectively.
Autoradiographic exposure of sections (Steps 6 and 7) The sections were immediately brought to a cold room at - 2 0 °, equipped with a safe-light, for use as a dark-room. The sections were placed directly onto x-ray film (Kodak Industrex). In some cases thin Mylar foil (6/zm) was placed between the section and film to avoid artefacts due to chemography. To rationalize the dark-room handling, 10-12 seetions were placed onto the same film sheet (24 × 30 cm) and put in an x-ray cassette (Siemens) with or without amplification screens. The exposure time was 2-3 h.
Autoradiographic exposure of sawn slices (Steps 6"and 7") In some instances, x-ray films were also placed against both sides of the sawn slices. A series of exposure times ranging between 0.5 and 15 rain were selected.
Determination of radioactivity (Step 8") The radioactivity of whole mice and sawn slices was determined by measuring photon radiation by means of an ionization chamber (Capintec Inc.). To quantitate the radioactivity in various tissues from the whole-body sections, small circular pieces (3 mm dia.) were punched out of the 60-/~m sections. Pieces were punched from river, pancreas and muscles. Each punched tissue piece was dissolved in 1 mL Soluene 350'~ (Packard) over a period of about I0 rain at 60°C. The pieces were then repeatedly counted in order to separate short- and long-lived radionuclides by ordinary liquid scintillation techniques. The liquid scintillation counter (Packard TRI-CARB 460 CD) was equipped with a luminescence detector, which subtracted chemiluminescence events. Owing to the high fl-energy of HC, the detector efficiency was .~R.I. 3S~--D
133
considered to be very high, and in calculations was therefore assumed to be 100%. On the following day, when the "C had decayed, the sections in the double tracer experiments were dehydrated and placed against x-ray film for exposure of the long-lived radionuclide. The exposure time was 3-4 weeks. In double-tracer experiments after decay of the ~C, a 3-h control exposure was performed in order to prove that no L4C or 3sS interfered with the "C picture. No detectable blackening was obtained during that period of time. Results and Discussion The time aspect is, of course, the most difficult problem in using these short-lived nuclides in WBA, and much attention has been paid to minimizing some of the time-consuming steps. The killed animal was frozen just long enough to ensure that the inner portions had solidified. Sawing into slices reduced the bulk mass and increased the surface, which lessens the time necessary for homogeneous acclimatization to the sectioning temperature (-20°C). In addition, sawing exposes different levels of the animal very rapidly. The sawn slices were attached to pre-planed CMC blocks, so that the microtome could be adjusted before the actual sectioning started. The sections obtained were immediately placed on the x-ray film for exposure, without any time-consuming dehydration. As expected, the autoradiographic resolution was better after the t4C and 3sS exposures as compared to the "C. The difference in resolution was rather small for thin sections (20 ~m) but increased with thickness of section. The maximum energy of "C positrons is 0.98 MeV (95% of the particles penetrate 60~m water-equivalent medium). A sawn slice is thick in relation to the range of the "C-positrons. Figure 2 shows an exposure of L-[methyl-"C]methionine of a sawn 2-mm thick slice (Fig. 2a) and a comparable exposure of a 20/~m cut section (Fig. 2b). The exposure of the sawn slice did not show any fine details but still provided a rough estimate of the relative radioactivity of different organs. The technique of exposing sawn slices is especially rapid and quite simple to perform. An investigation can thus be made with a lower dose of *~C. The most convenient way to obtain a proper exposure of thick slices was to make a sequence of exposures of different durations from each slice (e-g- 0.5, 1, 2 and 5 rain) before developing the film. The resolution of the thin cut sections was somewhat decreased by the x-ray cassette amplification screens. The use of cassettes facilitates the dark-room work, but the amplification screens should be shielded by black paper, to avoid image distortion. The autoradiograms obtained from L-[methyl -UC]methionine and L-[methyl-~4C]methionine, respectively, in the same 20/~m section are shown in Fig. 3. A weak blackening from the intestinal con-
134
R. D'ARGYet aL
tents caused by accidental warming of the sections can be seen (Fig. 3a). It proved essential to avoid warming the thin non-dehydrated sections with the fingers when placing them against films. If they were carefully handled and kept at -20~'C, chemographic artefacts were avoided. The use of Mylar sheets eliminated these artefacts but also decreased the resolution. The distribution pattern of L-[methyl"C]methionine and L-[methyl-~4C]methionine was apparently identical. The highest activity in the body was found in the pancreas. Other organs with high uptake were the gastrointestinal mucosa, the salivary gland, bone marrow, and the pituitary. These results were also in agreement with previous distribution studies of L-[methyt-~4C]methionineY J.z,~ To achieve a satisfactory blackening from ~C, the sections had to contain a mean radioactivity of 1.6 x 106 disintegrations/cm-' during the exposure. Data obtained from punched pieces of 60-/~m sections indicate that liver, pancreas and muscle contained 1.3, 2.4 and 0.20 cps/MBq, respectively, of the administered L-[methyl-"C]methionine 100 min after the injection. To attain the proper exposure of thin sections, several different thicknesses (e.g., 20, 40 and 60 # m) were placed for exposure simultaneously over a period of about 2 h, a time long enough to acquire 99% of the available tiC-decay events. The absorbed whole body dose to personnel from )]C radiation was less than 0.1 mGy in experiments involving 3-4 animals. The internal fl-dose to the animal was estimated to be about 6.3 x l0 -H Gy g/disintegration. This did not induce any detectable distributional disturbance in these short term experiments. By double-tracer autoradiography, the distributions of L-[methyl2~C]methionine and t-[35S]methionine were compared from the same section. No distribution differences were seen. This indicates that the demethylation of methionine does not influence the overall distribution pattern in the time interval (20-90 min) studied. Autoradiograms of o-[methylHC]methionine and L-[~S]methionine in the same sections are seen in Fig. 4. A clear difference in distribution can be noticed: the lower utilization of the D form, illustrated by higher concentrations in the body fluids, and the very high radioactivity in the inner portions of the kidney cortex, indicating stronger excretion by the kidney (Fig. 4b).
Concluding Remarks Autoradiography of animals larger than mice, such as monkeys, or parts of animals, will take a somewhat longer time but should be feasible by essentially the same procedures as those described here. The animals were sectioned longitudinally (sagittally), but transverse sections, like the pictures obtained by PET, could also be used. In view of the
difficulties of interpreting PET images, ~tC-WBA offers a useful tool in animal studies, which should precede clinical PET work. In this way, the tracer dynamics of various radiopharmaceuticals can be confirmed. In principle such studies can be made with long-lived nuclides, such as t4C or 3H, but this generally involves several weeks exposure time. This is a serious drawback, especially if the preliminary results are not satisfactory, and new variables have to be tried. It is then a great advantage if the results are obtained rapidly in each experiment. Double carbon autoradiography (~tC + t4C) offers an interesting approach by comparing two distribution patterns from the same section, that is without biological variation. In addition, better image separation is obtained as compared with previous autoradiographic double tracer techniques. The possibilities for variation of double tracer autoradiography are numerous: (1) If a molecule is known or suspected to be split in the body: two different preparations of the same substance, labelled in different parts of the molecule, can then be tested. The ~tC and t4C pictures will then be identical initially but will differ with time. (2) Two labelled preparations of the same substance: differences in pattern after administration at time intervals or after using different application routes. (3) Different substances which may be related in some respect. Acknowledgements--We are indebted to Petter Malmborg, Ph.D., and Mr Daniel Latyea for technical assistance. This work forms part of a research project financially supported by the Swedish Natural Science Research Council and the National Swedish Board for Technical Development (No. 79-6790).
References 1. Ullberg S. Science Tools (LKB-produkter, Brornma, 1977). 2. Ter-Pogossian M. M., Raichle M. E. and Sobel B. E. Sci. Am. 243, 141 (1980). 3. Appelgren, L.-E., Ericsson Y. and Ullberg S. Acta Physiol. Scand. 53, 339 (1961). 4. Fukuda H., Matsuzawa T., Abe Y., Endo S., Yamada K., Kubota K., Hatazawa J., Sato T., Ito M., Takahashi T., Iwata g. and Ido T. Eur. J. Nucl. Med. 7, 294 (1982). 5. Ido T., Fukushi K. and Irie T. Biomedical Aspects of Fluorine Chemistry (F_MsFiller R. and Kobayashi Y.) p. 143 (Elsevier, Amsterdam, 1982). 6. Hunter W. M. Br. Med. Bull. 30, 18 (1974). 7. L~.ngstr6m B. and Lundqvist H. Int. J. Appl. Radiat. Isot. 27, 357 (1976). 8. L/mgstr6m B. On the Synthesis of tiC-compounds. Ph.D. Thesis. Acta Universitatis Upsaliensis. No. 555 (1980). 9. Lundqvist H., LAngstrom B. and Malmqvist M. J. Radioanal. Chem. In press. 10. Bersmann K. CRC Crit. Ret'. ToxicoL In press. 11. Hansson E. Acta Physiol. Scand. 46 (Suppl. 161) (1959). 12. Benard P. and Duranel S. Ann. Rech. Vet. 11 201 (1980).