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Scintillation counting of 14CO2 from in vitro systems: A comparison of some trapping agents
S&kOkT
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COMbiTJNICATl0N.S
5. Technicon Bulletins I lb, c, d, e, Technicon Instrumenta Inc., Ardsley, New York, 10502. 6. BALLENTINE, R, AND Bu~voq D. D., in “Methods in Enzymology,” Vol. III (Colowick, S. P., and Kaplan, N. O., eds.), pp. 1017-1020, 1035-1041. Academic Press, New York, 1964. 7. DIEHL, H., SMITH, G. F., MCBRIDE, L., AND CRYBERG,R., “The Iron Reagents,” 2nd ed., pp. 26, 37. G. Fredrick Smith Chemical Co., 367 McKinley Ave., Columbus, Ohio, 1965. 8. Analytical Methods Committee, Analyst 84,214 (1959). 9. BIDE, R. W., Anal. Biochem. 29,393 (1969). 10. FERRAFLI,A., Ann. N. Y. Acad. Sci. 87, 792 (1960). 11. KERSEN, G., Lundwirtsch. Forsch. 19, 108 (19%). 12. QUABMBY,C., ANDGRIMSHAW, H. M., Analyst 92,305 (1967). 13. RAMSAY,K., B&hem. J. 53, 227 (1954). 14. Technicon Bulletin I lb, Technicon Instruments Inc., Ardsley, New York, 10502.
R. W. BIDE Animal Pathology Division, Health of Animals Branch, Canadcr Department of Agriculture Animal Diseases Research Institute (Western) Lethbridge, Alberta, Canada Received January lb, 1969
Scintillation
Counting A Comparison
of l*CO2
from
of Some Trapping
in Vitro Systems: Agents
The incorporation into a liquid scintillator of 14C0, produced during incubations in Warburg flasks or other closed systems has been carried out by a number of methods. The classic Warburg COz absorber of potassium hydroxide on filter paper has been adapted for radioactive work, the paper being immersed in a scintillator solution either wet (1) or after drying (2). Absorption in NaOH or KOH solution, followed by precipitation as barium carbonate, has also been used, the BaCO, being either count*ed as a suspension with finely divided silica (3) or precipitated on filter paper and counted by immersion (4). In none of these heterogeneous systems can the counting efficiency for individual sampIes be measured. Solutions of NaOH or KOH have also been counted after addition of a water-miscible scintillator solution, either with or without silica powder (5-7). The effective homogeneity of such systems does not seem te have been demonstrated. True homogeneous scintillation counting has been achieved by the use
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of organic bases such as Hyamine, phenethylamine, and ethanolamine, which are miscible wit-h scintillator solutions. It is generally advantageous to use a homogeneous system so that accurate standardization of individual samples, by internal or external methods, can be applied. Information about the efficiency of absorption of CO, by the various methods is scanty, however, and we have accordingly compared a number of them, from the point of view both of absorption efficiency and of possible effects on biological systems in vitro. Table 1 shows the recovery from 10 pmoles of 14C0, released from NaH14C03 in Warburg flasks and absorbed in a number of different trapping agents. It is evident that under these conditions ethanolamine is a very inefficient absorber, even when present in large excess. Both Hyamine and phenethylamine are satisfactory. Aqueous KOH gave low and variable recoveries when counted in a water-miscible scintillator TABLE 1 Efficiency of Some 14CO2 Absorbers Warburg flasks contained 2.5 ml aqueous NaHl4COa (10 pmoles, 3000 dpm). Center wells contained 0.2 ml of indicated absorber. W02 was released by tipping in 0.3 ml 2 N HCl from the side arms, and absorption continued for the time indicated. Center well contents were washed into counting vials with 2 X 0.2 ml water (for KOH) or methanol (for organic bases) and 10 ml scintillator was added. Samples were counted in a Packard Tri-Garb at 8” and standardized with 14C-hexadecane (added at room temperature to ensure complete solution). Recoveries shown are for separate flasks. Radioactivity remaining in the acidified bicarbonate solution at end of absorption period was less than 201, in all cases and controls (bicarbonate not acidified) showed always less than 5% of the radioactivity transferred to the absorber.
Absorber Phenethylamine” Hyamine* Ethanolaminec KOHd
Absorption time, min
Scintillator
30 60 120 60 60
e
60 60
e II
B I
% WOZ absorbed 99.5 99.5 99.3 99.9 99.0 99.2 101.1 99.9 54.0 57.0 72.3 67.0 86.5 65.0 103.9 100.3
Molar ratio: absorber CO9 released 79 20 110 36
0 Phenethylamine/toluene/methanol 2/1/l (v/v/v) (Woeller, ref. 9). b 1 M solution hydroxide in methanol (Packard Instrument Co. Inc.). 0 Ethanolamine/2-methoxyethanol l/2 (Jeffay and Alvarez, ref. 10). d 10% v/v aqueous solution. * Dioxane based scintillator of Bray (ref. 11). f Toluene/2-methoxyethanol based scintillator of Jeffay and Alvarez (ref. 10). 0 As (e) but containing also 4% w/v finely divided silica (Aerosil, Bush Beach and Segner Bayley, Ltd., London).
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COMMUNICATIONS
with internal standardization using 14C-hexadecane. This was found to be caused by the formation and settling of a gel in the counting vial, resulting in a rapidly falling counting efficiency (Fig. 1). When finely divided silica was incorporated into the scintillator before adding the KOH solution the %O, recovery, using internal standardization, was satisfactory and there was only a small, slow fall in efficiency (Table 1 and
I 100
200
I 300
400
500
600
700
800
MINUTES
FIG. 1. Effect of finely divided silica on liquid scintillation counting rate of “CO2 absorbed in KOH solution and added to water-miscible scintillator: (a) scintillator solution containing 4% w/v silica powder, (6) scintillator solution without silica. Details of scintillator and absorber as in Table 1. Both samples contained the same *% activity.
Fig. 1). The validity of the KOH/silica/internal standard method was confirmed in other experiments in which measured volumes of ‘“CO, of known specific activity were absorbed by KOH solution using vacuum line techniques and counted and standardized as above. Samples of l”CO, up to 166 pmoles in 0.2 ml of KOH solution were counted satisfactorily by this method, the counting rate remaining substantially constant for at least 19 hr. Of the agents tested only Hyamine, phenethylamine, and aqueous KOH (counted with silica) are therefore satisfactory both for quantitative CO2 absorption and for homogeneous scintillation counting. For in vitro systems in which the absorber can be injected at the end of the incubation
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COMMUNICATIONS
period, any of these could be used. However, if conventional manometric measurements are also being carried out, it may be necessary to have the absorber present throughout the incubation, and the possibility arises that interference with the biological system may be caused by volatile organic bases or solvents. This has been tested by using the 3 satisfactory absorbers in Warburg flasks containing rat liver mitochondria oxidizing 1-14C-palmitate (8). It was found that use of the phenethylamine absorber caused almost complete inhibition of the oxidizing system (93.994.7%, 4 experiments), and there was considerable inhibition also with Hyamine (18.1-42%, 4 experiments), taking the 14C02 recovery with KOH absorber as normal for the system. These results show that gross interference with metabolic processes can be caused by the presence of organic CO, trapping agents in the center well of incubation vessels. This method of COZ trapping has been reported a number of times in the literature. The most satisfactory absorber is still aqueous KOH but, as shown here, it is necessary to add finely divided silica to achieve reliable scintillation counting and standardization, even when using a water-miscible scintillator. Further advantages of the KOH are that it is less likely than the organic agents to absorb radioactive volatile metabolites other than CO,, and it is easier to remove quantitatively from the metabolism flask than the organic bases which become rather viscous. REFERENCES 1. BUHLER,
D. R., An&
Biochem. 4, 413 (1962).
2. ONTKO, J. A., AND JACKSON, D., J. BioE. Chem. 238, 3674 3. CLUL~, H. J., Analyst 27, 4 (1962). 4. MORIN, R. J., AND CARRION, M., I&ids 3,349 (1968). 5. SAUERMANN, G., Biochim. Biophys. Acta 118,268 (1966). 6. BHAQAVAN, H. N., COURSIN, D. B., AND DAKSHINXMURTI,
(1964).
K., Arch. B&hem. Biophys. 110, 422 (1965). 7. PASSERON, S., SAVAGEAU, M. A., AND HARAFCY, J., Arch. Biochem. Biophys. I%!?, 124 (1968). 8. LEHNINGER, A. L., in “Methods in Enzymology” (S. P. Colowick and N. 0. Kaplan, eds.), Vol. I, p. 545, Academic Press, New York, 1955. 9. WOELLER, F. H., Anal. Biochem. 2,508 (1961). 10. JEFFAY, H., AND ALVAREZ, J., Anal. Chem. 33,612 (1961). 11. BRAY, G. A., Anal. B&hem. I, 279 (1960).
W. G. DTJNCOMBE T. J. RISING The Wellcome Research Laboratories, Beckenham, Kent, BRJ SBX, England Received January 98,1969
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COMbiTJNICATl0N.S
5. Technicon Bulletins I lb, c, d, e, Technicon Instrumenta Inc., Ardsley, New York, 10502. 6. BALLENTINE, R, AND Bu~voq D. D., in “Methods in Enzymology,” Vol. III (Colowick, S. P., and Kaplan, N. O., eds.), pp. 1017-1020, 1035-1041. Academic Press, New York, 1964. 7. DIEHL, H., SMITH, G. F., MCBRIDE, L., AND CRYBERG,R., “The Iron Reagents,” 2nd ed., pp. 26, 37. G. Fredrick Smith Chemical Co., 367 McKinley Ave., Columbus, Ohio, 1965. 8. Analytical Methods Committee, Analyst 84,214 (1959). 9. BIDE, R. W., Anal. Biochem. 29,393 (1969). 10. FERRAFLI,A., Ann. N. Y. Acad. Sci. 87, 792 (1960). 11. KERSEN, G., Lundwirtsch. Forsch. 19, 108 (19%). 12. QUABMBY,C., ANDGRIMSHAW, H. M., Analyst 92,305 (1967). 13. RAMSAY,K., B&hem. J. 53, 227 (1954). 14. Technicon Bulletin I lb, Technicon Instruments Inc., Ardsley, New York, 10502.
R. W. BIDE Animal Pathology Division, Health of Animals Branch, Canadcr Department of Agriculture Animal Diseases Research Institute (Western) Lethbridge, Alberta, Canada Received January lb, 1969
Scintillation
Counting A Comparison
of l*CO2
from
of Some Trapping
in Vitro Systems: Agents
The incorporation into a liquid scintillator of 14C0, produced during incubations in Warburg flasks or other closed systems has been carried out by a number of methods. The classic Warburg COz absorber of potassium hydroxide on filter paper has been adapted for radioactive work, the paper being immersed in a scintillator solution either wet (1) or after drying (2). Absorption in NaOH or KOH solution, followed by precipitation as barium carbonate, has also been used, the BaCO, being either count*ed as a suspension with finely divided silica (3) or precipitated on filter paper and counted by immersion (4). In none of these heterogeneous systems can the counting efficiency for individual sampIes be measured. Solutions of NaOH or KOH have also been counted after addition of a water-miscible scintillator solution, either with or without silica powder (5-7). The effective homogeneity of such systems does not seem te have been demonstrated. True homogeneous scintillation counting has been achieved by the use
276
SHORT COMMUNICATIONS
of organic bases such as Hyamine, phenethylamine, and ethanolamine, which are miscible wit-h scintillator solutions. It is generally advantageous to use a homogeneous system so that accurate standardization of individual samples, by internal or external methods, can be applied. Information about the efficiency of absorption of CO, by the various methods is scanty, however, and we have accordingly compared a number of them, from the point of view both of absorption efficiency and of possible effects on biological systems in vitro. Table 1 shows the recovery from 10 pmoles of 14C0, released from NaH14C03 in Warburg flasks and absorbed in a number of different trapping agents. It is evident that under these conditions ethanolamine is a very inefficient absorber, even when present in large excess. Both Hyamine and phenethylamine are satisfactory. Aqueous KOH gave low and variable recoveries when counted in a water-miscible scintillator TABLE 1 Efficiency of Some 14CO2 Absorbers Warburg flasks contained 2.5 ml aqueous NaHl4COa (10 pmoles, 3000 dpm). Center wells contained 0.2 ml of indicated absorber. W02 was released by tipping in 0.3 ml 2 N HCl from the side arms, and absorption continued for the time indicated. Center well contents were washed into counting vials with 2 X 0.2 ml water (for KOH) or methanol (for organic bases) and 10 ml scintillator was added. Samples were counted in a Packard Tri-Garb at 8” and standardized with 14C-hexadecane (added at room temperature to ensure complete solution). Recoveries shown are for separate flasks. Radioactivity remaining in the acidified bicarbonate solution at end of absorption period was less than 201, in all cases and controls (bicarbonate not acidified) showed always less than 5% of the radioactivity transferred to the absorber.
Absorber Phenethylamine” Hyamine* Ethanolaminec KOHd
Absorption time, min
Scintillator
30 60 120 60 60
e
60 60
e II
B I
% WOZ absorbed 99.5 99.5 99.3 99.9 99.0 99.2 101.1 99.9 54.0 57.0 72.3 67.0 86.5 65.0 103.9 100.3
Molar ratio: absorber CO9 released 79 20 110 36
0 Phenethylamine/toluene/methanol 2/1/l (v/v/v) (Woeller, ref. 9). b 1 M solution hydroxide in methanol (Packard Instrument Co. Inc.). 0 Ethanolamine/2-methoxyethanol l/2 (Jeffay and Alvarez, ref. 10). d 10% v/v aqueous solution. * Dioxane based scintillator of Bray (ref. 11). f Toluene/2-methoxyethanol based scintillator of Jeffay and Alvarez (ref. 10). 0 As (e) but containing also 4% w/v finely divided silica (Aerosil, Bush Beach and Segner Bayley, Ltd., London).
SHORT
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COMMUNICATIONS
with internal standardization using 14C-hexadecane. This was found to be caused by the formation and settling of a gel in the counting vial, resulting in a rapidly falling counting efficiency (Fig. 1). When finely divided silica was incorporated into the scintillator before adding the KOH solution the %O, recovery, using internal standardization, was satisfactory and there was only a small, slow fall in efficiency (Table 1 and
I 100
200
I 300
400
500
600
700
800
MINUTES
FIG. 1. Effect of finely divided silica on liquid scintillation counting rate of “CO2 absorbed in KOH solution and added to water-miscible scintillator: (a) scintillator solution containing 4% w/v silica powder, (6) scintillator solution without silica. Details of scintillator and absorber as in Table 1. Both samples contained the same *% activity.
Fig. 1). The validity of the KOH/silica/internal standard method was confirmed in other experiments in which measured volumes of ‘“CO, of known specific activity were absorbed by KOH solution using vacuum line techniques and counted and standardized as above. Samples of l”CO, up to 166 pmoles in 0.2 ml of KOH solution were counted satisfactorily by this method, the counting rate remaining substantially constant for at least 19 hr. Of the agents tested only Hyamine, phenethylamine, and aqueous KOH (counted with silica) are therefore satisfactory both for quantitative CO2 absorption and for homogeneous scintillation counting. For in vitro systems in which the absorber can be injected at the end of the incubation
278
SHORT
COMMUNICATIONS
period, any of these could be used. However, if conventional manometric measurements are also being carried out, it may be necessary to have the absorber present throughout the incubation, and the possibility arises that interference with the biological system may be caused by volatile organic bases or solvents. This has been tested by using the 3 satisfactory absorbers in Warburg flasks containing rat liver mitochondria oxidizing 1-14C-palmitate (8). It was found that use of the phenethylamine absorber caused almost complete inhibition of the oxidizing system (93.994.7%, 4 experiments), and there was considerable inhibition also with Hyamine (18.1-42%, 4 experiments), taking the 14C02 recovery with KOH absorber as normal for the system. These results show that gross interference with metabolic processes can be caused by the presence of organic CO, trapping agents in the center well of incubation vessels. This method of COZ trapping has been reported a number of times in the literature. The most satisfactory absorber is still aqueous KOH but, as shown here, it is necessary to add finely divided silica to achieve reliable scintillation counting and standardization, even when using a water-miscible scintillator. Further advantages of the KOH are that it is less likely than the organic agents to absorb radioactive volatile metabolites other than CO,, and it is easier to remove quantitatively from the metabolism flask than the organic bases which become rather viscous. REFERENCES 1. BUHLER,
D. R., An&
Biochem. 4, 413 (1962).
2. ONTKO, J. A., AND JACKSON, D., J. BioE. Chem. 238, 3674 3. CLUL~, H. J., Analyst 27, 4 (1962). 4. MORIN, R. J., AND CARRION, M., I&ids 3,349 (1968). 5. SAUERMANN, G., Biochim. Biophys. Acta 118,268 (1966). 6. BHAQAVAN, H. N., COURSIN, D. B., AND DAKSHINXMURTI,
(1964).
K., Arch. B&hem. Biophys. 110, 422 (1965). 7. PASSERON, S., SAVAGEAU, M. A., AND HARAFCY, J., Arch. Biochem. Biophys. I%!?, 124 (1968). 8. LEHNINGER, A. L., in “Methods in Enzymology” (S. P. Colowick and N. 0. Kaplan, eds.), Vol. I, p. 545, Academic Press, New York, 1955. 9. WOELLER, F. H., Anal. Biochem. 2,508 (1961). 10. JEFFAY, H., AND ALVAREZ, J., Anal. Chem. 33,612 (1961). 11. BRAY, G. A., Anal. B&hem. I, 279 (1960).
W. G. DTJNCOMBE T. J. RISING The Wellcome Research Laboratories, Beckenham, Kent, BRJ SBX, England Received January 98,1969