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A simple method for trapping and measuring expired14CO2
ANALYTICAL BIOCHEMISTRY 64, 2 8 1 - 2 8 3 (1975)
A Simple Method for Trapping and Measuring Expired ~4CO 2 The collection and assay of 14CO2 has been accomplished through the formation of aqueous carbonate salts and the counting of these salts as suspensions (1,2); or alternatively by the use of organic amines to trap the '4CO2 as carbamates (3-5) which can be easily dissolved for efficient counting (6). The difficulty encountered with the use of a suspending material because of its physical nature or the expense of the organic amine in quantities sufficient for trapping the large molar amounts of '4CO2 expired in metabolic experiments of long duration may restrict their usage. Although potassium hydroxide is inexpensive and an efficient CO2 scavenger, its use has been limited by its chemiluminescence, destruction of the scintillation mixture, and insolubility in scintillation cocktails (1,2). We wish to report a method by which '4CO2 trapped in potassium hydroxide solution, may be added directly to the xylene-based commercial scintillation solvent PCS' (Amersham-Searle, Arlington Heights, Illinois) and its radioactivity determined accurately. [~4C] sodium carbonate (supplier indicated 58.9 mCi/mM) was obtained as an aqueous solution from Amersham Searle. PCS was used as described by its manufacturer. Dioxane water solvent scintillation solution was prepared, as described by Snyder (7). A phenethylamine cocktail, as described by Woeller (8) was used to trap and count '4CO2 produced from the acidification of solutions containing '4COa 2. Radioactivity was determined using a Packard series 2425 ambient temperature scintillation spectrometer. Automatic external standard (AES) furnished a correction for quenching. Results obtained using internal standards (['4C,] toluene, Amersham Searle) agreed with the results obtained using AES. A standard solution of Na2'4CO3 of known radioactivity was diluted with 2.5 N potassium hydroxide solution so that 0.5 ml of the final solution contained 2.2 × 103 dpm. Triplicate aliquots of the standard Na.~t4CO3 solution were assayed for radioactivity using the PCS solution and the dioxane solution; or Na2'4COa solutions were acidified, and the '4CO2 trapped was assayed in the phenethylamine solution. The results indicate that the radioactivity added to PCS and the radioactivity trapped by phenethylamine solution were not significantly different (PCS, 2.25 -+- 0.3; phenethylamine, 2.09 -+- .03, t = 0.9191, p = no signifN o t e a d d e d after s u b m i s s i o n : l n s t a - g e l ( P a c k a r d , D o w n e r s G r o v e , I l h n o i s ) , a n o t h e r x y l e n e - b a s e d s c i n t i l l a t i o n s o l v e n t b e h a v e d s i m i l a r l y to P C S in this s t u d y . 281 Copyright © 1975 by Academic Press, Inc. Printed in the United States All rights of reproduction in any form reserved.
282
SHORT COMMUNICATIONS
--5
~4
R=,9974
2 2
u z
1
I 1
I 2
I 3
I 4
I 5
Na214CO3 RadioaclivilyMeasured {,10-3l¢c11
FI6. 1. Linear relationship between radioactivity added a s N a , 2 1 4 C O 3 and radioactivity assayed in PCS scintillation solution. Values reported are means of five determinations of each _+ standard deviation.
icance using Student's t test). 2 The results obtained using the dioxane scintillation mixture demonstrated a large reduction in the assay of radioactivity (7% recovery of radioactivity) probably due to the insolubility of the inorganic salts. Within 48 hr the dioxane solution became colored, perhaps due to the destruction of fluors by the strong alkali. Stable counts were obtained for 72 hr in both the phenethylamine and PCS solutions. Increasing amounts of the standard [14C] sodium carbonate solution were added to PCS, and the radioactivity was assayed. A linear correlation was demonstrated (r = 0.997, p < .001) (Fig. 1) with a slope equal to 1.02 which is close to the theoretical value expected.
ACKNOWLEDGMENT This investigation was supported by National Institutes of Health Grant AM-12626 and GRS-462.
REFERENCES 1. 2. 3. 4. 5. 6.
Funt, B. L., and Hetherington, A. (1958) Science 129, 1429. Husain, S., and Paradise, R. R. (1973) Proc. Soc. Exp. Biol. Med. 142, 316. Passman, J_ M., Radin, N. E., and Cooper, J. A. D. (1956) Anal. Chem. 28, 484. Jeffay, H., and Alvarez, J. (1961) Anal. Chem. 33, 612. Shapira, J., and Perkins, W. H. (1960) Science 131, 414. Kyper, A_ C., and Aghdashi, M. (1972) Anal. Biochem. 45, 341.
2 The mean obtained using the PCS, although not significantly different from that obtained using the phenethylamine, may have been higher due to a small loss of radioactivity through incomplete 14CO2 trapping using phenethylamine.
SHORT COMMUNICATIONS
283
7. Snyder, F. (1964)Anal. Biochem. 9, 183. 8_ Woeller, F. H. (1961) Anal. Biochem. 2, 508. ROGER A. DAVIS J. PHILIP SHOWALTER FRED KERN, JR. Division of Gastroenterology University of Colorado Medical Center 4200 East Ninth Avenue. Denver, Colorado 80220
Received April 3, 1974: accepted September 20, 1974
A Simple Method for Trapping and Measuring Expired ~4CO 2 The collection and assay of 14CO2 has been accomplished through the formation of aqueous carbonate salts and the counting of these salts as suspensions (1,2); or alternatively by the use of organic amines to trap the '4CO2 as carbamates (3-5) which can be easily dissolved for efficient counting (6). The difficulty encountered with the use of a suspending material because of its physical nature or the expense of the organic amine in quantities sufficient for trapping the large molar amounts of '4CO2 expired in metabolic experiments of long duration may restrict their usage. Although potassium hydroxide is inexpensive and an efficient CO2 scavenger, its use has been limited by its chemiluminescence, destruction of the scintillation mixture, and insolubility in scintillation cocktails (1,2). We wish to report a method by which '4CO2 trapped in potassium hydroxide solution, may be added directly to the xylene-based commercial scintillation solvent PCS' (Amersham-Searle, Arlington Heights, Illinois) and its radioactivity determined accurately. [~4C] sodium carbonate (supplier indicated 58.9 mCi/mM) was obtained as an aqueous solution from Amersham Searle. PCS was used as described by its manufacturer. Dioxane water solvent scintillation solution was prepared, as described by Snyder (7). A phenethylamine cocktail, as described by Woeller (8) was used to trap and count '4CO2 produced from the acidification of solutions containing '4COa 2. Radioactivity was determined using a Packard series 2425 ambient temperature scintillation spectrometer. Automatic external standard (AES) furnished a correction for quenching. Results obtained using internal standards (['4C,] toluene, Amersham Searle) agreed with the results obtained using AES. A standard solution of Na2'4CO3 of known radioactivity was diluted with 2.5 N potassium hydroxide solution so that 0.5 ml of the final solution contained 2.2 × 103 dpm. Triplicate aliquots of the standard Na.~t4CO3 solution were assayed for radioactivity using the PCS solution and the dioxane solution; or Na2'4COa solutions were acidified, and the '4CO2 trapped was assayed in the phenethylamine solution. The results indicate that the radioactivity added to PCS and the radioactivity trapped by phenethylamine solution were not significantly different (PCS, 2.25 -+- 0.3; phenethylamine, 2.09 -+- .03, t = 0.9191, p = no signifN o t e a d d e d after s u b m i s s i o n : l n s t a - g e l ( P a c k a r d , D o w n e r s G r o v e , I l h n o i s ) , a n o t h e r x y l e n e - b a s e d s c i n t i l l a t i o n s o l v e n t b e h a v e d s i m i l a r l y to P C S in this s t u d y . 281 Copyright © 1975 by Academic Press, Inc. Printed in the United States All rights of reproduction in any form reserved.
282
SHORT COMMUNICATIONS
--5
~4
R=,9974
2 2
u z
1
I 1
I 2
I 3
I 4
I 5
Na214CO3 RadioaclivilyMeasured {,10-3l¢c11
FI6. 1. Linear relationship between radioactivity added a s N a , 2 1 4 C O 3 and radioactivity assayed in PCS scintillation solution. Values reported are means of five determinations of each _+ standard deviation.
icance using Student's t test). 2 The results obtained using the dioxane scintillation mixture demonstrated a large reduction in the assay of radioactivity (7% recovery of radioactivity) probably due to the insolubility of the inorganic salts. Within 48 hr the dioxane solution became colored, perhaps due to the destruction of fluors by the strong alkali. Stable counts were obtained for 72 hr in both the phenethylamine and PCS solutions. Increasing amounts of the standard [14C] sodium carbonate solution were added to PCS, and the radioactivity was assayed. A linear correlation was demonstrated (r = 0.997, p < .001) (Fig. 1) with a slope equal to 1.02 which is close to the theoretical value expected.
ACKNOWLEDGMENT This investigation was supported by National Institutes of Health Grant AM-12626 and GRS-462.
REFERENCES 1. 2. 3. 4. 5. 6.
Funt, B. L., and Hetherington, A. (1958) Science 129, 1429. Husain, S., and Paradise, R. R. (1973) Proc. Soc. Exp. Biol. Med. 142, 316. Passman, J_ M., Radin, N. E., and Cooper, J. A. D. (1956) Anal. Chem. 28, 484. Jeffay, H., and Alvarez, J. (1961) Anal. Chem. 33, 612. Shapira, J., and Perkins, W. H. (1960) Science 131, 414. Kyper, A_ C., and Aghdashi, M. (1972) Anal. Biochem. 45, 341.
2 The mean obtained using the PCS, although not significantly different from that obtained using the phenethylamine, may have been higher due to a small loss of radioactivity through incomplete 14CO2 trapping using phenethylamine.
SHORT COMMUNICATIONS
283
7. Snyder, F. (1964)Anal. Biochem. 9, 183. 8_ Woeller, F. H. (1961) Anal. Biochem. 2, 508. ROGER A. DAVIS J. PHILIP SHOWALTER FRED KERN, JR. Division of Gastroenterology University of Colorado Medical Center 4200 East Ninth Avenue. Denver, Colorado 80220
Received April 3, 1974: accepted September 20, 1974