Rapid Determination of Radiocarbon in Animal Tissues ELI M. PEARCEI, FRANK DEVENUTO, WALTER M. FITCH2, HlLLlARD E. FIRSCHEINE, and ULRICH WESTPHAL Pratein and Steroid Section, Biochemistry Department, A r m y M e d i c a l Research Laboratory, Fort Knox, K y .
tribution of cortiso1-4-C14 in the xhite rat. This procedure makes use of the finding by Tabern and Lahr ( I d ) that xhole organs or tissues form clear colloidal solutions in formamide.
A simple technique was developed for solid counting of radiocarbon samples prepared from animal tissues. The dried tissues and organs containing cortisol-4-C14 were dissolved in hot formamide, and aliquots of the solutions were dried on aluminum planchets. The self-absorption of these cortisol-4-C14muscle preparations was found to be linearly proportionalto the sample thickness. Use of lens paper improved the consistency of the counts. The method is considered well suited for a comparatively rapid determination of radioactivity in animal material.
EXPERIMENTAL
Dissolution of Tissues in Formamide. The various organs and tissues were removed from the experimental animal (rat, injected with cortis01-4-C~~)~ cut into small pieces, and dried to constant weight (2 or 3 days) in a ventilated oven a t 80" C. The dry tissueswere placed in beakers and covered completelywithformamide (Fisher reagent grade, or Matheson technical grade). The mixture was heated on a hot plate a t about 105" C. for half an houl and the dissolving of the tissue was completed by raising the temperature to about 210" C. during an additional hour (hood J . The mixture was stirred throughout these operations. Fresh formamide was added to replace loss by evaporation. The solution was brought t o the desired volume with 95% ethyl alcohol. This procedure also dissolves skin and hair, but not bone.
I C compounds labeled with radiocarbon have become important tools in studies on the distribution and metabolism of these substances in the living organism. I n the determination of carbon-14-containing material, highest accuracy is generally obtained by counting carbon-14 as carbon dioxide or as barium carbonate ( 1 ) . On this basis, Skipper, Bryan, White, and Hutchison ( 1 0 )have described techniques for combustion of animal tissues for carbon-14 assay. Riegel, Hartop, and Kittinger (8) supplemented these methods by the use of extraction and saponification procedures. The radioactive carbon injected was accounted for with an over-all accuracy within
The solutions obtained were homogeneous and in general clear. Liver and gastrointestinal tract sometimes did not dissolve entirely; in this case the insoluble material was removed and extracted thoroughly with additional formamide and 95% ethyl alcohol. The extracts m-ere added to the original solutions; the residue was found to be free of radioactivity and, therefore, was discarded. The color of the colloidal solutions varied from light to dark brown. It was found essential for rapid dissolution to have the tissue in a completely dry state. Separate expeiiments showed that heating the radioactive material (cortisol-4C14) in formamide a t 210" C. for 60 t o 90 minutes and drying under an infrared lamp did not cause any loss of radioactivity. Bttempts to dissolve the animal tissues in dimethylformamide were unsuccessful.
= k l O % (10).
To survey the distribution of a carbon-14-tagged compound in a great variety of organs and in a large number of experimental animals, any procedure involving such steps as combustion, solvent extraction, or saponification is very time-consuming. Therefore, a simple technique for direct measurement of the radioactivity in tissues is needed. The present report describes a solid-counting method that was developed for a study of the dis1
Determination of Radioactivity. The solutions were plated on aluminum planchets which were 30 mm. in diameter and had a 2-mm. rim. To obtain a uniform radioactive layer, the planchets were prewashed with petroleum ether or ether, acetone, a mild detergent solution, for 45 minutes a t about 40" C., and finally with distilled water; the planchets then were dried. Care must br taken during these operations that the planchets do not stick together. Satisfactory washing was accomplished when a drop of
Present address, Chemistry Department, Polytechnic Institute of Brook-
lyn, Brooklyn, N. Y . 2 Present address, Department of Biochemlatry, University of California, Berkeley, Calif. 3 Present address, Atomic Energy Project, School of Medicine and Dentistry, University of Rochester, Rochester, N. Y.
8-
LL U E
d
b
-
7-
z s1
// b
d
0
0
8
0
a u 5-
Figure 1. Self-absorption of cortisol-4C14-musclepreparations without lens paper
v1
m
g
4
L
Levels of constant activity: 3000 ;.p.m. 400 c p.m. 40 c.p m. A . No," = 2.04 f 0.34 t
4-
0 W
SIZ 3 2
--/8
1
& '0
I
I
I
I
I
I
I
I
I
I
I
IO
20
30
40
50
60
70
80
90
100
110
1.4
2.8
4.9
5.6
11.3
12.7
7.0 8.4 9.8 MG. PER SO. CM., t
14.1
1762
15.3
V O L U M E 28, N O . 11, N O V E M B E R 1 9 5 6
1763 5o
A
14
distilled water dispersed uniformly over the whole surface of the dried planchet. A dilute solution of silicone stopcock grease in chloroform 1%-asapplied to the inner surface of the rim and in a ring 1 to 2 mm. n-ide to the adjacent horizontal area. A thin film of silicone remained on this surface and helped to prevent the sample from spreading beyond the silicone-free center area. Accumulation of the material around the rim and creeping over it thus was avoided. The consistency of counts obtained in quadruplicate determinations was increased considerably by the use of lens paper (1,4 ) . Circles 29 mm. in diameter were cut out with the aid of a steel punch and placed on the planchets before the solution was applied. As the average weight of a lens paper circle was found to be 7 mg., their thickness was approximately 1 mg. per sq. cm. The volumes of the tissue-formamide solutions mere adjusted so that the material to be counted was contained in 0.5 ml. The solution was pipetted onto the planchet and dried for approximately 12 hours under an infrared lamp. The safety pipetting device used has been described ( 1 4 ) . The dried samples were stored in a desiccator. The radioactivity was measured with a &-gas flow counter (Model D46, Suclear Instrument and Chemical Corp., Chicago 10, Ill.) or in an automatic gas flow counter (Model SC-50, Tracerlab, Inc., Boston 10, Mass.). The counts per minute (c.p.m.) mere corrected for background and for small daily variations in the response of the counters by referring to a standard polystyrene source labeled with carbon-14 which n-as obtained from Oak Ridge Sational Laboratory on allocation from the Isotope Division, U. S. Atomic Energy Commission. Self-Absorption Curves. Self-absorption curves of constant activity n ere prepared using a muscle-formamide-ethyl alcohol stock eolution, the dry weight of which was determined after drying as described above. constant amount of cortisol-4C14 [specificactivity 4.08 pc. per mg.; radiochemical purity higher than 94y0 (fS)]was added to varying quantities of the dissolved muscle material. The cortisol-4-C14solution, the muscle-formamide solution, and 95% ethyl alcohol were mixed in such proportions that the volume of the final solution plated (0.5 ml.) contained betxveen 5 and 100 mg. of dry tissue. The amount of cortiso1-4-C14 in these mixtures was adjusted so that three levels of radioactivity were obtained-Le., approximately 3000, 400, and 40 counts per minute per 0.5 ml. The planchets were prepared in triplicate or quadruplicate without lens paper (Figure 1) and, a t the two lower levels of radioactivity, with lens paper (Figure 2). The reference values (“100%”) for the radioactivity in the experiments without lens paper were determined on a total of 20 planchets with essentially weightless samples (less than 0.14 y of eortisol-4-C14 per sq. cm.). In the experiments with lens paper, the reference value was determined by extrapolating the self-absorption curve to zero
weight. I t x a s observed that the activity values obtained with “weightless” samples (less than 0.1 y per sq. cm.) of cortisol-4-C14 on lens paper fell on this curve when plotted a t the thickness of the lens paper-Le., 1 mg. per sq. cm. (Figure 2). Use of liver tissue instead of muscle in the formamide procedure gave essentially identical self-absorption curves. Statistical Calculations. The counting error was calculated according to established procedures (3). The samples were counted to give standard deviations of counting rates as listed in Table I. The same table gives the over-all standard deviation which was calculated from the experimental data. This expression (Table I line 1) for the significance of the data includes the errors caused by handling, pipetting, plating, etc., in addition to the inherent statistical deviations of the counting rate (Table I, lines 2 and 3). The curves shown in the figures were determined by the method of least squares. The self-absorption values were expressed by Xo/-V as the ordinate with the saniple thickness (milligrams per square centimeter) as the abscissa, where N o = activity in a “weightless” sample, and S = activity observed a t various thicknesses. This method of plotting the “self-absorption factor,’’ J-o/-V, as employed in the figures, is considered useful, because the absorption-free activity can be obtained directly from the observed activity by multiplication with this factor. RESULTS AND DISCUSSION
On the basis of their original observation, Lahr, Olsen, Gleason, and Tabern (6) have developed a method for radioactivity measurements in tissue by homogenizing n-hole organs and dissolving aliquots in formamide. The beta activity of gold-198, phosphorus-32, and yttrium-90 was thus determined by liquid counting at infinite thickness. This procedure could not be utilized in the studies of the present authors, where carbon-14 must be determined in many hundreds of organs and tissues of small laboratory animals (S), making homogenization of the individual samples impractical The results of the self-absorption measurements for the cortisol4-(2’4-muscle-formamide preparations are given in Figures 1 and 2. The abscissas of these figures show that all determina-
ANALYTICAL CHEMISTRY
1764 Table I.
Standard Deviation of Carbon-14 Determinations with and without Lens Paper
(Compiled from experimental d a t a of Figures 1 and 2) 400 C.P.M. Level, % 40 C . P . N . Level, % With lens Without With Without paper lens paper lens paper lens paper 1. Over-all standard deviation i 7.1 11.2 6.9 14.6 2. Standard deviation of counting r a t e f 1.6 1.6 ... 3. Standard deviation of counting rate & ... ... 3.1-7.5 3.21i0.2 -,
1.
2. 3.
Table 11.
(11).
fi Formula for large counts compared to background (8. E-Tj + TD’Formula for counts not greatly different from background
v2
Self-Absorption Factors for Carbon-14-Labeled Substances
(Factors are given as reciprocals and are accurate within the range of error of the corresponding self-absorption values.) Thickness, f , Mg./Sq. Cm. Substance 5 10 15 20 1.72 2.78 3.70 4.35 “Theoretical” (7) 5.68 1.90 3.04 4.35 Barium carbonate ( 1 6 ) 3.85 2.18 2.98 1.51 Barium carbonate (wet ground) (16) 2.56 1.92 3.17 Barium carbonate (dry ground) (15) 1.49 1.92 2.33 2.78 1.54 Glucose (amorphous) (16) 4.17 2.27 3.17 1.61 Glucose (crystalline) (16) 2.95 1.89 F a t t y acids (16) ‘2; 56 .. 22 55 3 S8 2.39 Wax (17) 3 30 5.60 4.45 2,l8 RIuscle preparations with lens paper 7.04 8 84 5 44. Muscle preparations without lens paper 3 . 7 4
tions were done a t sample thicknesses lower than infinite thickness, which has been found for organic substances to range from 20 mg. per sq. em. (fatty acids) to 25 to 30 mg. per sq. em. (glucose) (16). Figure 1 shows that the self-absorption observed Kith the muscle-formamide procedure appears to approximate a linear proportionality to the sample thickness. It is known that the self-absorption of @-radiation in sample material does not necessarily follow an exponential curve (1, 6 ) . The equation that fitted the experimental observations best \vas found to be N o / N = 2.04 0.34t, where t equals sample thickness in milligrams per square centimeter. This equation and the curve of Figure 1 indicate that the apparent “specific activity” of the samples rises sharply as the sample thickness approaches zero, an effect which presumably is caused by backscattering from the aluminum planchet. I n this range of very low thicknesses (broken line in Figure 1) application of the curve of Figure 1 is not recommended; use of lens paper is considered preferable. The self-absorption curve obtained with the use of lens paper (Figure 2 ) leads through N o / N = 1 a t zero thickness. This is the consequence of the fact that the value of zero thickness was not determined by measuring weightless samples but rather by extrapolating the curve to zero thickness. The weights given on the abscissa in Figure 2 include the weight of the lens paper, which was approximately 7 mg. per planchet. The use of lens paper has the advantage of reducing the spreading of the observations, as indicated by lower standard deviations (Table I). The self-absorption curve in the presence of lens paper followed the equation N o / N = 1 0.23t. A comparison with the exponential curve N o / N = p t / l - e - P t (Z), where fi = 0.31 (Figure 2), indicates better agreement of the observed values with the linear curve. Table I1 shows self-absorption factors for a number of carbon14-labeled compounds. They are given as the reciprocal of S / N o , the percentage of the “zero n-eight”-i.e., self-abaorptionfree activity-observed at certain thicknesses. The “theoretical” values were calculated from the exponential formula given by
+
+
(2).
Libby ( 7 ) . In the last two lines of Table I1 are listed the selfabsorption factors shown in Figures 1 and 2 of the present report. The correction factors observed for the muscle preparations without lens paper are comparatively large. This folloivs from the fact that the “true zero” values in this case (Figure 1) were determined at estremely low thicknesses. It is evident from Table I1 that the self-absorption factors do not shox any consistent relationship with the chemical nature of the compounds. The differences observed are presumably caused by a different physical arrangement of the samples and other variations in the counting conditions. These observations emphasize the necessity of determining experimentally the selfabsorption correction factors for the specific conditions of any experimental setup (9). Application of the procedure in studies on the distribution of cortisol-4-C14in rats ( 3 )shon-ed that the recovery of carbon-14 was essentially quantitative. Ten animals were injected 50,000 to 58,000 c.p.m. (approximately 12.5 to 14.5 y) of this steroid, and 5 minutes later a complete analysis was made of practically all organs and tissues. The average recovery in the 10 rats was The over-all reproducifound to be 105 670, S.D. = * l O . l % . bility of single observations follows from the data of Table I. The weight of the dried organs or tissues that were dissolved in formamide ranged betmeen 3 mg. and about 40 grams. The sample weights on the aluminum planchet5 were as low as 3 mg., but preferably between about 40 and 60 mg. (approximately 5 to 9 mg. per sq. cm.). The outlined procedure permits a comparatively rapid determination of radiocarbon in animal tissues. It was developed for cortisol-4-C14 which did not show any loss of radioactivity when heated in formamide a t 210’ C. or when dried under an infrared lamp. The method appears to be applicable to other carbon-14 compounds, provided a check is made to ascertain that no loss of radioactivity occurs during the various experimental operations. ACKNOWLEDGMENT
The authors are indebted to the Endocrinology Study Section, Department of Health, Education, and Welfare, Public Health Service, National Institutes of Health, for supplying the cortisol4-C14 through Tracerlab, Inc., Boston 10, Mass. LITERATURE CITED (1) Calvin, M., Heidelberger, C., Reid, J. C., Tolbert, B. M., Yankmich, P. F., “Isotopic Carbon,” Wiley, New York, 1949. (2) Comar, C. L., “Radioisotopes in Biology and Agriculture,” McGraw-Hill, New York, 1955.
(3) Firschein, H. E., DeVenuto, F., Fitch, W. M., Pearce, E. M., Westphal, U., AMRL Rept. 257, Fort Knox, Ky. (July 2. 1956). (4) Garrow, J., Piper, E. A., Biochem. J. 60, 527 (1955). (5) Kohman, T. P., ANAL.CHEM.21, 352 (1949).
V O L U M E 28, NO. 11, NOVEMBER 1 9 5 6
1765
(6) Lahr. T. N.. Olsen. R.. Gleason. G. I.. Tabern. D. L.. J. Lab. and Clin. M e d . 45, 66 (1955). (7) Libby, W. F., ANAL.CHEM.19, 2 (1947). (8) Riegel, B., Hartop, W. L., Jr., Kittinger, G. W., Endocrinology 47, 311 (1950). (9) Schweitzer, G. K., Stein, B. R., AT’ucZeonics 7, No. 3, 65 (1950). (10) Skipper, H. E., Bryan, C. E., White, L., Jr., Hutchison, 0. S., J. B i d . Chem. 173, 371 (1948). (11) Snedecor, G. W., “Statistical hIethods,” 4th ed., Iowa State College Press, A4mes,Iowa, 1946.
(12) Tabern. D. L.. Lahr. T. N.. Science 119. 739 11954) (13j Westphal, U., Firschein, H. E., Pearce,‘E. M:, Am. J. Physiol. 185, 54 (1956). AMRL Rept. (14) Westphal, U. F., Firschein, H. E., Pearce, E. 185, Fort Knox, Ky. (April 22, 1955). (15) Wick, A. N., Barnet, H. N., Ackerman, K.,ANAL.CHEM.21, 1511 (1949). (16) Yankwich, P. E., Norris, T. H., Huston, J., Ibid.,19,439 (1947). (17) Yankwich, p. E., Weid, J. Science 107, 651 (194%. RECEIVEDfor review April 18, 1956. Accepted July 16, 1956.
w.,
Determination of Di- and Trialkyl Phosphites in the Presence of Each Other D. N. BERNHART and K. H. RATTENBURY Research Laboratories, Victor Chemical Works, Chicago Heights,
In an alcoholic caustic solution dialliyl phosphites are rapidly hydrolyzed to form sodium monoalkyl phosphites, with no interference from trialkyl phosphites. In an acidic alcoholic solution trialkyl phosphites are readily hydrolyzed to form dialliyl phosphites, which may then be hydrolyzed with caustic. Both reactions are stoichiometric and consume one mole of caustic per mole of phosphite.
T
RIALKYL phosphites are generally prepared by the reaction of alcohol and phosphorus trichloride in the presence of a base (Equation 1); without a base, dialkyl phosphites are formed (Equation 2). I n the commercial preparation of trialkyl phosphites some dialkyl phosphites are usually formed. Therefore, to determine the purity of trialkyl phosphites, it is necessary to determine each of the two components in the mixture. Relatively few such chemical procedures are reported in the literature. One procedure, which is specific for the dialkyl phosphite content, is the nonaqueous titration of weak acids ( 2 ) . Another procedure for purity only is the determination of molecular weight with alcoholic potassium hydroxide (3). Iodine solutions have also been used to indicate purity (6). There are very few published data on the two latter procedures. The method presented here takes advantage of the rapid hydrolysis of alkyl phosphites. I n an alkaline alcoholic medium dialkyl phosphites are instantly hydrolyzed to form sodium monoalkyl phosphites (Equation 3), while trialkyl phosphites react very slowly ( 1 , 4). The rates of reaction of the two components are far enough apart to permit determination of dialkyl phosphites in the presence of trialkyl phosphites by adding an excess of alcoholic sodium hydroxide and immediately titrating the excess caustic nrith standard acid. I n an alcoholic acid medium trialkyl phosphites are very rapidly hydrolyzed to form dialkyl phosphites ( 1 ) (Equation 4). The phosphite is then all present as dialkyl phosphite and may be determined by alkaline hydrolysis. 3ROH
+ PCl, + 3 base
-
+ 3 base. HC1
(RO)3P
(1)
0
3ROH
+ PCl,
I/
-+
(R0)zP-H
+ RC1 + 2HCl
II
EXPERIMENTAL
It was found that in an alcoholic medium 1 mole of dialkyl phosphite consumes 1 mole of sodium hydroxide after 1 minute, and this value remains constant for a t least 1 hour. Trialkyl phosphite, ranging from ethyl to octyl, consumes no sodium hydroxide for as long as 10 minutes in an alcoholic medium. After 10 minutes of hydrolysis in a slightly acidic alcoholic medium, 1 mole of trialkyl phosphite consumes 1 mole of sodium hydroxide after 1 minute, and this value remains constant for 1 hour. All these reactions were carried out a t room temperature. INTERFERENCES
Alcohols, amine hydrochlorides, and dialkyl alkanephosphonates (the rearrangement isomer of trialkyl phosphites) do not interfere with this method. If acidic compounds such as monoalkyl phosphites, or basic compounds such as amines, are present, they can be compensated by neutralizing the alcoholic solution of the sample with 0.LV acid or base. PROCEDURE
Dialkyl Phosphite. Dissolve 1 to 2 grams of trialkyl phosphite in 50 to 100 ml. of dry ethyl alcohol. Neutralize with 0 . 1 s sodium hydroxide to a light pink with phenolphthalein indicator. .4dd 20.0 ml. of 0.1N sodium hydroxide, mix, add about 10 ml. of a 40/,solution of boric acid, and back-titrate with 0.lN hydrochloric acid until the solution is colorless. Each mole of sodium hydroxide consumed is equivalent to 1 mole of dialkyl phosphite. Sample size and excess caustic may be regulated to suit the particular sample that is to be analyzed. Dialkyl phosphites may also be assayed by this procedure. The boric acid is added to buffer the solution, thus preventing local hydrolysis of the trialkyl phosphite when back-titrating with acid.
Table I.
+ NaOH
4
J
(RO)(NaO) -H
Added
1.0 3.0 5.0 10.0 15.0 20.0
?To Tria Found Added Di- and Tributyl Phosphites
0.9 3.0 5.1 9.9 14.8 19.7
99.0 97.0 95.0 90.0 85.0 80.0
Found
99.1 97.0 94.7 89.9 84.8 80.2
Di- a n d Triiso-octyl Bhosphites
+ ROH
(3)
0
(RO)IP
Typical Results with Synthetic Mixtures % Di
0
0
(RO)J’-H
(2)
111.
II + HOH %(RO)*P-H + ROH
7.0 10.0 15.0 20.0
7.0 9.8 14.7 19.9
93.0 90.0 85.0 80.0
Standard deviation of tri-component, 2 parts per thousand.
(4)
92.7 90.2 84.8 79.8
