Estimation of Radioactive Calcium-45 by Liquid Scintillation Counting LEO LUTWAK Metabolic Diseases Branch, National Institute o f Arthritis and Metabolic Diseases, National lnstifutes o f Health, Bethesda, Md.
A technique for the estimation of radioactive calcium-45 in biological fluids such as serum, urine, and stool extracts is based on liquid scintillation counting. The most convenient and applicable procedure for preparing the samples is based on the formation of stable tertiary solutions of waterabsolute ethyl alcohol-toluene. The efficiency of counting is about 6570; counts are reproducible with a standard deviation of 17.3670.
T
radioactive isotope of calcium with the mass number 45 is increasingly important in physiological and clinical studies of bone and endocrine metabolism (I,$, 6). This isotope decays with the emission of a beta particle of maximum energy 0.25 mer. The radiation is usually estimated by precipitation of the calcium-45, in the presence of added carrier calcium, as calcium oxalate, plating of the precipitate onto an appropriate planchet, and subsequent counting in a thin endwindo\$- Geiger-”Iller counter, or in a continuous gas flow proportional counter. Because of the relatively low energy of the emission, self-absorption correction factors must be applied, and the over-all final efficiency of counting is approximately 20% a t one-half infinite thickness. In 1950 Reynolds, Harrison, and Salvini ( 8 ) and Kallmann and Furet ( 7 ) reported that dilute solutions of certain substances in aromatic solvents produced fluorescent emissions in the presence of radiation. This led to the development of sensitive techniques for the counting of many low-energy, betaemitting isotopes used in biochemical research ( 3 ) . The method uses a solution of the radioactive material in HE
Table I.
Solution Water Serum Serum Urine Urine Urine
340
an aromatic solvent, such as toluene, containing an appropriate scintillating phosphor, such as 2,5-diphenyloxazole. This is an advantage because the radioactive substance is dissolved in the same homogeneous medium as the scintillating phosphor and a 4~ geometry is obtained for counting. Inorganic salts are relatively insoluble in aromatic solvents which has been detrimental to the estimation of calcium-45 by scintillation counting. Numerous m-ays may be suggested, however, for the direct incorporation of salts into a counting system. A scintillating gel can be prepared, directly incorporating the salt, using aluminum stearate (4, 5 ) or Thixcin (Baker Castor Oil Co.) (9). Another approach is the solution of the salt in water and the formation of a homogeneous tertiary liquid mixture such as toluene-absolute ethyl alcohol-water. A third technique is the formation of a toluene-soluble organic salt of calcium (3). This paper reports two such procedures, including the counting of calcium-45 as calcium 2-ethylhexanoate in a solution of toluene-phosphor; and the counting as calcium chloride in a tertiary mixture of absolute ethyl alcohol-hydrochloric acid-toluene-phosphor. EXPERIMENTAL
Reagents. Calcium-45. A calcium45 solution, supplied by Oak Ridge National Laboratory as calcium chloride in hydrochloric acid solution, was suitably diluted. Calcium chloride, 0.0451V, was prepared by dissolving ACS grade calcium carbonate, dried to constant weight at l l O o , in 5N hydrochloricacid. Calcium acetate, citrate, lactate, acid phosphate, chloride, and hydroxide, C.P. or reagent grade.
Extraction of Calcium-45 with 2-Ethylhexanoic Acid
Ca’s, C.P.M.
Added
163 6620 9120 8650 9880 10310
ANALYTICAL CHEMISTRY
No. of
Recovered
Extractions
88 6144 10680 14 1480 28
1
4 4 4 4 3
Recovery, 54.2 92.8 117.1 0 15.0 0.3
70
Ammonium oxalate solution, 4%. A reagent grade aqueous solution. 2-Ethylhexanoic acid (Union Carbide Chemicals Co.), technical grade, was redistilled twice in vacuo; Eastman Chemical Products, Inc., technical grade, was used without further purification. Toluene - diphenyloxazole so 1u t i o n , 2.00 grams of diphenyloxazole (Xuclear Enterprises, Ltd.) were dissolved in 500 ml. of toluene, analytical reagent grade. Equipment. The instrument used was a Tri-Carb liquid scintillation spectrometer (Packard Instrument Co., LaGrange, Ill.). The samples were placed in No Sol-Vit glass vials of 5-dram capacity with polyethylene snap caps (T. C. Wheaton Co., Millville, N . J.). RESULTS
2-Ethylhexanoic Acid.
SOLUBILITY SALTS I N %ETHYLHEXT o approximately 100 mg. of salt were added 2.5 ml. of 2-ethylhexanoic acid. Calcium acetate, lactate, and hydroxide dissolved in the acid; the citrate, acid phosphate, and chloride remained insoluble. T o the suspensions of the insoluble salts, 0.5 ml. of 0.3X sodium hydroxide solution was added and the mixture carefully boiled to dissolve the carbonate and chloride. EXTRACTION OF CALCIUM SALTSFROM AQUEOUSSOLUTIOKS.One milliliter of a solution of calcium-45 in water, serum, or urine was treated with 0.5 ml. of 1N potassium hydroxide and extracted with 5 ml. of toluene containing 10% (v./v.) of 2-ethylhexanoic acid. The extraction was repeated up to a total of four times in some instances. The pooled extracts were centrifuged and an aliquot was evaporated to dryness for counting in a gas flow proportional counter. The results are shown in Table I. Four extractions transferred the radioactivity into the toluene layer effectively from serum, but not from urine, Precipitation of the calcium from urine as calcium oxalate, followed by digestion of the precipitate with nitric-perchloric acids, or solution in disodium (ethylenedinitri1o)tetraacetic acid, did not produce solutions extractable with toluene and/or 2-ethylhexanoic acid. It appears, therefore, that use of a soluble salt of calcium does not aid in the counting of calcium-45.
O F CALCIUM ANOIC ACID.
I
80
I
I
I
1
I
I
'\
'.
i' C 600
Figure 2. Background counts in liquid scintillation system as functions of photomultiplier voltages and discriminator settings
4!L Table II. Miscible Mixtures of Toluene-Ethyl Alcohol-Hydrochloric Acid
I
20
800
1
900
1000
1 1100
700
600
h I
700
I
10-10011
I
O
4
1
10-au
800
1000
900
1100
1200
VOLTS
I
1200
1
count, in a system of 0.3 ml. of concentrated hydrochloric acid, 4.0 ml. of Figure 1. Liquid scintillation counting absolute ethyl alcohol, and 6.0 ml. of of calcium-45 0.4y0 diphenyloxazole in toluene over the sanie applied voltage range, for the two discriminator values. The backTertiary Mixtures. MISCIBLE NIX- ground rises markedly beyond 1000 T V R E S OF TOLUENE, ETHYL ALCOHOL, applied volts a t the 10- volts discrimination, but reaches a plateau a t the .4KD HYDROCHLORIC ACID. As s h o n n same value for the 10-100 volts discrimin Table 11, concentrated hydrochloric in ation. acid forms clear miscible solutions Figure 3 shows the effect of increasing n ith toluene-ethyl alcohol mixtures the volume of 0.4% diphenyloxazole in of varying proportions. toluene on the counting efficiency in a COUNTING CHARACTERISTICS. Figure system containing 0.3 ml. of calcium-45 1 demonstrates the efficiency of counting in concentrated hydrochloric acid, 4.0 calcium-45 in a system devised arbiml. of absolute ethyl alcohol, and 5 to trarily t o contain 0.3 ml. of hydro10 nil. of the toluene solution, measured chloric acid, 4.0 nil. of absolute ethyl a t an applied voltage of 910 and 110 alcohol, and 6.0 ml. of 0.4% diphenylvolts a t the two discriminator settings. oxazole in toluene, from a setting of There is a slightly perceptible increase 570 to 1240 volts on the photomultiin efficiency as the volume of tolueneplier circuit with the discriminators a t diphenyloxazole solution is increased 10-100 and 1 0 - a volts. .4t 10-100 from 5 to 8 ml. volts, a peak of efficiency is reached a t Similarly, Figure 4 shows the effect 910 volts; a t 1 0 - a volts, the efficiency of increasing the concentration of approaches plateau values a t about absolute ethyl alcohol in a system con1040 to 1110 volts. taining 0.1 ml. of calcium-45 in concenFigure 2 shoms the magnitude, in trated hydrochloric acid and 6 ml. of counts per minute, of the background 0.4% diphenyloxazole in toluene. The efficiency increases as the concentration of alcohol increases from 14 to 25%, and VOLTS
90
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Ethyl Alcohol, Toluene, Miscibility" HCl, MI. nn. MI. a t 0" C. 0.1 1 1 2 2 1 2 3
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4 6
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1 2
1 1 2 3 4
3
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1 2 4 6 1 1 2 1 2 4 6 1 2 4 6 1 2
++ ++ ++ ++ +t
4 0.4
1 2 3
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1 2 4 1 2 4 6 5 1 2 4 6 6 1 2 4 6 0.5 1 1 2 1 1 3 2 4 1 2 4 Completely miscible. or ar1)arating into layers. fi
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Cloudy
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I 07
R A T I O OF DPO-TOLUENE /TOTAL
08
VOLUME 10
Figure 3. Effect of diphenyloxazoletoluene concentration on counting efficiency
c
01 0
4 1
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10
20
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Figure 4. Effect of ethyl alcohol concentration on counting efficiency VOL. 31, NO. 3, MARCH 1959
341
40001
Table 111.
Ca46, M1. 0.2
0.2 0.2 0.2
0.2 0.2 0.2
I
Effect of Variations in Volume of Components of Counting System"
Water, MI. 0 0 0 0 0.1 0 0.2
Ethyl Alcohol, MI. 4.0 4.5 5.0 6.0 6.0 8.0 8.0
Toluene-DPO, hll. 5.0 5.6 6.25 7.5 7.5 10.0 10.0 Average
Relative Efficiency, % 100.0 100.0 95.4 98.3 105.2 103.4 101.6 100.7
a Each value is average of duplicate experiments. Per cent is calculated assuming 100.0% for first set of conditions.
IV. Recovery of Calcium-45 Ca45, C.P.M. Sample Added Found Recovery, Table
% 411.0 411.0 341.8 341.8
155.8 326.0 904.6 62.0
47.5 47.5 78.5 78.5
306.3 145.1 805.8 28.9
Urine 588.4 728.3 1244.9 402.5
113.9 97.3 99.8 97.9 Av. 102.22
Serum 362.7 102.9 175.4 88.2 864.8 97.6 113.3 120.4 Av. 102.27
thereafter changes only slightly as the alcohol concentration increases to 40%. Figure 5 demonstrates the linearity of counting in a system containing 0.3 ml. of hydrochloric acid solution of different calcium-45 concentrations, 4.0 ml. of absolute ethyl alcohol and 6 ml. of 0.4% diphenyloxazole in toluene. Table 111 demonstrates that the efficiency of counting is independent of relatively wide variations in the composition and total volume of the counting system used. Setting the counting rate of the standard system (containing 0.2 ml. of aqueous solution, 40. nil. of ethyl alcohol, and 5.0 ml. of toluene diphenyloxazole solution) a t loo%, variations in the volume of the aqueous phase from 0.3 to 0.4 ml. a t an ethyl alcohol-toluene ratio of 0.8, and with the resulting variation of the watertoluene ratio from 0.02 to 0.04 (final volume ranging from 9.2 to 19.4 ml.), produced an average relative counting efficiency of 100.7% =tstandard deviation 3.61%. The ideal conditions for counting, in a system of 0.3 ml. of calcium-45 in hydrochloric acid, 4.0 ml. of absolute ethyl alcohol, and 6 ml. of 0.4% diphenyloxaBole in toluene, would be to count a t 910 applied volts, discriminating between 10 and 100 volts. This would provide 342
ANALYTICAL CHEMISTRY
Table V.
Reproducibility of Procedure KO.
Sample Standards Serum Urine Stool extracts All samples
of
Duplicate Detns. 53 177 128 30 388
Std. Dev.,
%
I
I 0
I
C.45
Figure 5.
2
3
CONCENTRATION
Linearity of counting
6.08
7 65
7 48
7 23
7 36 7.36
an efficiency of about 65% a t a background of 70 c.p.m.
EXTRACTION OF CALCIUM FROM BIOFLUIDS. Serum. Into a 15-ml. centrifuge tube are placed 2.0 ml. of distilled water, 1.0 ml. of 4% ammonium oxalate, and 1.0 to 2 ml. of serum by LOGICAL
pipet, and the solution is mixed by swirling. After a t least 4 hours a t room temperature, the mixture is centrifuged a t 1500 to 2000 r.p.m. for 10 minutes. The supernatant solution is decanted and the tubes are inverted on filter paper for a t least 10 minutes; the lip of the tube is wiped dry with tissue and the precipitate is broken up by a stream of wash mixture (2% ammonia in equal parts of ethyl alcohol, ethyl ether, and water), using a total volume of about 4 ml. The mixture is again centrifuged for 10 minutes at 2000 r.p.m., the supernatant solution is decanted, and the tubes are drained for 10 minutes. The tubes containing the precipitates are dried in a boiling water bath or a constant temperature oven a t 100" C. for 10 to 30 minutes and 0.3 ml. of concentrated hydrochloric acid is added to dissolve the precipitates. Solution may be hastened by placing the tube in boiling water for 30 seconds. Three milliliters of absolute ethyl alcohol, followed by 5 ml. of 0.4% diphenyloxazole in toluene are added to each tube with careful mixing after each addition; the cloudy mixture is immediately decanted into a counting vial and drained for 30 to 60 seconds. To each tube is then added 1.0 ml. of absolute ethyl alcohol, which is decanted again into the counting vial. The vials ?re stored a t 0' C. or below until counting. Urine and Stool Digests. Into a 50ml. centrifuge tube are pipetted 5 to 25 ml. of the urine or stool digest (contain-
ing no more than 50 mg. of calcium). Methyl red is added and the pH adjusted to just alkaline with 1M ammonium hydroxide and 1N hydrochloric acid, as necessary. To each tube is added 1.0 ml. of 4% ammonium oxalate the contents are mixed by swirling, and the mixture is kept a t room temperature for a t least 4 hours. Thereafter, the samples are treated like those of serum. Standards. One-milliliter aliquots from suitable dilutions of a calcium45 solution are pipetted into 15-ml. centrifuge tubes containing 0.5 ml. of 0.045N calcium chloride and 2.0 ml. of distilled water; 1.0 ml. of 4% ammonium oxalate is added with careful swirling. Thereafter, the samples are treated like the serum samples. Recoveries. Table IV lists the average recoveries of calcium-45 added to samples of pooled serum and pooled urine from patients with different bone disorders. It can be presumed from thesevalues that the calcium oxalate precipitation technique does not introduce any significant quenching substances. Reproducibility. Table V lists the standard deviations obtained for a series of duplicate determinations in serum, urine, stool extracts, or standard solutions containing between 2.3 X pc. per nd. of caland 3.4 x cium-45. DISCUSSION
The counting of calcium-45 in biological fluids has been complicated because the estimation of weak beta emission has required the isolation of a purified precipitate; also, the efficiency and therefore, sensitivity of the counting has been of a relatively low order of magnitude, This technique requires only a simple preliminary precipitation to concentrate the calcium and thereafter uses the high sensitivity, precision, and efficiency of the liquid scintillation
counting technique. The present procedure is of particular value in studies requiring the eunrnination of large nunibers of samples over a wide range of radioactive concentrations. LITERATURE CITED
(1) Bauer, G
C. H., Carlsson, hrvid, Lindquist, Bertil, Acta Med. Scand. 158, 143 (1357).
(2) Comar, C. L., Wasserman, R. H., in
“Atomic Energy and Agriculture,” pp. 249-304, Am. Assoc. Advance. of Sci., Washington, 1957. (3) Davidson, J. D., Feigelson, Philip, Inter. J. A p p l . Radiation and Isotopes
2,1(1957). (4) Funt, B. L., flucleonics 14, N o . 8, 83 (1986). (5) Funt, B. L., Hetherington, rlrlene, Science 125, 986 (1957). (6) Heaney, R. P., Whedon, G. D.,
J . Clin. Endocrinol. and Metabolism 18,
1246 11958). (i)Kalimann, Hartmut, Furst, Milton, Phys. Rev. 79, 857 (1950). (8) Reynolds, G. T., Harrison, F. B., Salvini, G . ,Zbid. 78, 488 (1950). (9) White. C. G.. $elf. Samuel. Xucleonics 14, No.’lO, 46 (1956). I
.
RECEIVED for review September 16, 1958. Accepted November 12, 1958.
Precision Absorptiometry CRAMON M. CRAWFORD’ University o f California, 10s Alamos Scientific laboratory, 10s Alamos,
b To assess the significance of a recent statistical finding which seemed to invalidate differential methods, the rationale of instrumental precision was explored and applied to absorptiometry. Although these statistics were not found to b e germane, pertinent data are too few to prove the validity of published precision methods. The theory here developed stresses the need to recognize the linear scale reading without physical interpretation, and yields the important result that the Ringbom plot is insufficient to specify the range of good precision. The paper describes a new cell correction method, shows that differential methods reduce the effect of scale setting errors, corrects and extends an earlier treatment of ways to attain optimum analyte concentration, and points out the unique ability of the general differential method to use this optimum.
M
attmipts have been made to improxre the ordinary precision of absorptionietry with its 1% error or worse, all based 011 hypotheses about the principal source error. This paper reports a critical exusmination of the ideas involved, prompted partly by Cahn’s finding (16) that the statistics of actual analyses do 1106 wpport the hypothesis (predominariw of scale reading error) on which wid(.ly a w l differential methods depend. ANY
N.M .
the property to be found from the transmittancy-e.g., analyte [the substance determined (22, 49) ] concentration, identity, purity, or structure; reaction end points; photochemical quantum yields; temperature; or pH. There are several methods of improving precision known to precision absorptiometry. Both logic and convenience reserve the name differential for that method which uses reference solutions or their photometric equivalent such as filters. Precision is usually given qualitative definition (BO,22) only. I n this paper, it means also the reciprocal of the standard deviation of the result. Such a measure has the qualitative properties generally associated with precision, which the standard deviation itself does not. The quantity q , which RIandel and Stiehler (38) call sensitivity, is identical with precision as here defined. However, the discussion of the Committee on Balances and Weights (19) concerning the proper meaning of sensitivity can be extended to cover the present case, and provides ground for objection to calling \k a sensitivity.
31i‘
DEFINITIONS
Absorptiometry is here defined as the measurement of transniittancy or some function thereof. Because transmittancy is almost always used to calculate something else, precision absorptiometry is taken to mean precise measurement of Present address. Mississippi State University, State College, Miss.
INSTRUMENTAL PRECISION
Notation. Instrumental measurement consists of calculating a result, y, from a n instrumental reading, R , here considered t o be on a linear but otherwise arbitrary scale. I n turn, R is a function of several experimental variables, or conditions, typified by 5 , . Because y is intended to measure one of the zl, say $1, the experiment is usually arranged so that R is sensitive only to zl. The explicit recognition of R, divorced from any interpretation of its physical meaning, is important, because the physical meaning frequently changes with a change in the manner of using the instrument, and because the significance of any conclusion is more apparent when the quantity of practical interest-the
dial reading-is unambiguously identified as such. For instance, in ordinary absorptiometry, the dial reading measures transmittancy, but in differential absorptiometry the dial reading measures either a ratio or a more complicated function of two or three transmittancies. Assignment of a single symbol for the dial reading in the latter case provides warning that contact with experiment has been lost if these transmittancies are separated algebraically. Improving Precision. The result, y, is calculated from a theoretical or empirical relation y = y(R) whose differential is d g = y’(R) dR. The latter can be interpreted as providing a conversion of the uncertainty, dR (as measured, perhaps, by U R ) , into the corresponding uncertainty, dy, of the result. The curve slope, y’, thus acts as a weight factor, or error coefficient, which by becoming small can make the final uncertainty small. The quantities entering y’ which are susceptible to experimental variations are therefore given values which make y’ small. I n particular, R will be such a quantity. By selecting R so that y’ has its least value, a given error in R will cause least error in y. If it can be shown experimentally that dR does not depend on R-Le., dR is constant-the precision has been optimized with respect to R. The investigation of the dR us. R relation is usually neglected in absorptiometry, but conditions exist in which constancy mould not be expected and cannot be assumed. If dR does depend on R, the least value of d y implies the least value of y’dR, and will not necessarily coincide with least values of either y’ or of dR alone. Whether or not dR depends on R, a decrease of dR may be possible. The uncertainty, dy, may be predicted, for changes whose magnitudes (including sign) are known, by differentiating the assumed relation y = y(R) taking R = R(z,): VOL. 31, NO. 3, MARCH 1959
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