Determination of Radioactive S uIf ur in Biological Materials HENRY JEFFAY, FUNS0 0.OLUBAJO, and WILLIAM R. JEWELL Department o f Biochemistry, College of Medicine, University o f Illinois, Chicago, 111.
b A new method for the determination of sulfur-35 in biological tissues, fluids, extracts, and in organic compounds consists of oxidizing the sample to magnesium sulfate using a modified Pirie's reagent, dissolving the oxidation products in glycerol, and diluting with ethyl alcohol and N,N-dimethylformamide. Toluene containing scintillating phosphors is added and the samples are counted in a liquid scintillation counter.
R
WORK indicated that a liquid scintillation method would be most suitable for analyzing the total radioactive sulfur content of a wide variety of substances. However, sulfur compounds occurring in biological materials are not usually soluble in solvents commonly used for liquid scintillation counting methods. Because carbon dioxide and sulfur dioxide have many similar chemical properties, the first attempts t o count sulfur-35 have been modifications of the methods available for counting carbon-14. Passmann, Radin, and Cooper (2) have reported that a methanolic solution of Hyamine, a quaternary amine in the hydroxide form, will complex carbon14-dioxide which is soluble in organic solvents. Vaughan, Steinberg, and Logan (5) have reported that radioactive amino acids and proteins can also be dissolved in Hyamine and counted using a liquid scintillation method. However, as the authors themselves indicated, "quenching is marked with the TCA precipitated tissue proteins frequently having a yellow-brown color. The intensity of the color varies with protein preparation, concentration of protein, and time of heat." The use of an internal standard can overcome these difficulties. However, this laboratory has found that the solubility of crude tissue fractions in Hyamine is somewhat less than reported by other laboratories. Recently Radin and Fried (4) reported a method for counting sulfur-35 as the sulfate salt of the weak base Primene. However, this method may not be suitable for the routine analyses of a large number of tissue S35samples. A biological sample would have to first be oxidized to yield a sulfate salt, ECENT
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ANALYTICAL CHEMISTRY
and the sulfate salt would have to be separated from the cations by ion exchange chromatography before counting. Because the effect of acids other than sulfuric on the efficiency of this counting method has not been described, pure sulfuric acid must be isolated from the oxidation products. This last step may prove difficult for technicians. This paper describes a method readily adaptable to the routine analysis of a large number of samples and applicable to a wide variety of sulfur-3Scontaining substances. The method offers a relatively high counting efficiency (49%). Nonradioactive materials present in the samples do not interfere with the observed counting rate, thus eliminating the necessity of isolating the active compounds. APPARATUS
The liquid scintillation counter is a Tri-Carb liquid scintillation spectrometer, Model 314-DC (Packard Instrument Co., LaGrange, Ill.). The samples and multiplier phototubes are encased in a freezer maintained at 3" C. Samples are counted in 5-dram cylindrical Crystalite vials fitted with 26-mm. polyethylene snap caps (Wheaton Glass Co., Millville, N. J.). MATERIALS
All chemicals used were -4CS reagent grade. The oxidizing reagent used was a modified Pirie's reagent (3) prepared as follows: Three volumes of concentrated nitric acid were added to one volume of 60% perchloric acid. One fourth of this mixture was removed, saturated with magnesium nitrate (50 to 60 grams per 100 ml,), and mixed with the remaining acid mixture. A mixture of absolute ethyl alcohol (distilled over magnesium and iodine) and N,hT-dimethylformamide (redistilled, b.p. 151-153" C.) (1:3 v./v.) was used to obtain a clear solution with toluene and glycerol. The scintillation medium used was a toluene solution (stored in the dark in a n amber bottle) containing 3.0 grams per liter of 2,5-diphenyloxazole and 100 mg. per liter of 1,4 - bis [2 - (5 - phenyloxazolyl)]benzene (both scintillation grade, Arapahoe Chemicals, Inc., Boulder, Colo.). The certified standard solution of sulfuric acid was obtained from Nuclear-Chicago Corp. ?'he radio-
active tissues were prepared by feeding a rat approximately 10 to 20 pc. of yeast ~ r 0 t e i n - S(Abbott ~~ Laboratories, North Chicago, Ill.). PROCEDURE
The sample to be counted is placed in a counting vial, and enough modified Pirie's reagent is added to ensure complete oxidation of the sample. Generally, 1 ml. of Pirie's reagent is added for each 0.25 gram ( n e t weight) of tissue or 0.5 ml. of serum and 0.5 ml. of Pirie's reagent for each 0.5 ml. of urine. Correspondingly less can be added if a dilute extract or fraction is to be analyzed (all organic solvents should be evaporated before adding the acid mixture). If the sample is a tissue or other material of large mass, it may be desirable to homogenize the sample before placing it in the counting vial to facilitate oxidation. However, homogenization is not essential. The sample is heated in a sand bath, placed on a large (30 X 60 em.) hot plate. (A Slaco tube heater with ll/sinch holes in the asbestos, and a solid aluminum block may be more convenient.) The temperature is allowed to rise slowly from room temperature to 260" to 280" C. (1 to 2 hours) until a dry residue is obtained. I n the event of incomplete oxidation (the residue in the vial is not completely white), the sample is cooled, an additional 0.5 to 1.0 ml. of Pirie's reagent or 0.5 ml. of nitric acid is added, and the vial is returned to the sand bath. (Caution: if the vial is not cooled, addition of perchloric acid to hot organic material may cause an explosion.) This procedure is repeated until oxidation appears complete. The vial is then heated at 280" C. for i hour or longer. The resulting dry white salts are cooled to approximately 150" C. and immediately dissolved in 1.0 ml. of hot glycerol maintained a t approximatell 100" C. with a boiling water batL This addition is made while the via! is in the hot sand bath (caution should be exercised to prevent charring of the glycerol). To facilitate rapid pipetting of the glycerol, a wide-bore 5-ml. measuring pipet is recommended. ilfter complete solution of the salts the vial is removed from the sand bath, allon-ed to cool, and 6 ml. of the ethyl alcoholN,S-dimethylformamide miyture a r t added from a buret \Tith shaking. To the clear solution, 10 ml. of the toluene solution are added in the absence of direct sun or fluorescent lighting.
2ool----F-
The sample is placed in the freezer a t 3" C. and counted after 1 hour.
SZCOUNTS PER MINUTE 8: BACKGROUND
1100 VOLTS
RESULTS
Operating Voltage. T h e operating voltage was determined in t h e usual manner (1). A sample was counted a t several voltages and various window openings. The simplified expression for the determination of optimum condition was used t o plot the curves shown in Figure 1. The settings a t which the maximal counting rate and miiiinial background noise was observed w r t , T a p 6 (1100 volts) with window openings a t 10-90 (red scale) and 90100 (green scale). Selection of Solvents. It was necessary to find a solvent system capable of dissolving a colorless inorganic sulfate and a n organic scintillator. After considerable experimentation, a solution of 1 ml. of glycerol, 6 ml. of a 1 to 3 (v./v.) mixture of absolute ethyl alcohol-Ar,N-dimethylformamide, and 10 mi. of toluene containing the s:intillators was found to be the most suitable. Concentration of Scintillators. To .-letermine t h e optimum concentration of the scintillators, identical radioactive samples were prepared containing variable amounts of both 2,%diphenyiosazole (DPO) and 1,4i i s [2-(5-phenyloxazolyl)]benzene (POPOP) and counted. The results (Figure 2 ) indicate that a DPO concentration of 3.0 grams per liter and a POPOP concent'ration of 100 mg. per liter would be desirable because any minor errors in solution preparation vould have an insignificant effect on the observed counting rate. Counting Efficiency. Using a certified Nuclear-Chicago Corp. standard, a solution of 7609 d.p.m. of sulfuric acid was prepared and counted by the method described. T h e measured counting rate was 3731 c.p.m., at a background of 45 c.p.m., or 49.Oa/, of the total radioactivity was detected. Linearity of Counting. To determine t h e linearity of counting, 40 counting vials were prepared containing exactly the same quantity of inert salts and solvents, b u t with amounts of from 0 t o 7 ml. of sulfate-35 solution. T h e results of t h e measured counts clearly show a linear relationship between t h e true counts and t h e observed counts over a wide range of activity. Results of a similar experiment with varying amounts of a 10% liver homogenate obtained from an animal receiving a large oral dose of yeast cells-S3j, clearly indicate this same linear increase in the counting rate over a much wider range (up t o 90,000 c.p.m.). Effect of Nonradioactive Materials on Observed Counting Rate. Because oxidation of different samples can
10-40 10-50 10-60 10-70 10-80 10-90 10-100
DISCRIMINATOR SETTING ( R e d S c a l e )
Figure 1. Effect of voltage on counting rate
2a
r
i
mg. POPOP per liter
produce salt residues of variable quantity and composition, i t was necessary t o determine the effect various salt mixtures would have on t h e counting rate of a known amount of radioactivity. Large samples of various nomadioactive tissues and urine were oxidized as described above. The dry Oxidation residues were transferred t o a mortar and powdered. Varying quantities of each of the different nonradioactive salt mixtures were placed in counting vials, and a n equal quantity of a radioactive solution was added to each vial. The samples were then dissolved as described and counted. The qualitative composition and the quantity of the oxidation products which would be likely to be present in the routine analysis of biological samples had only a small effect on the observed counting rate. Recovery Analysis and Reproducibility. A sample of a radioactive liver homogenate Kas added t o homogenates of nonradioactive liver, muscle, and kidney. T h e results in Table I show that a recovery of essential 10070 is possible. Triplicate analyses of the various samples indicate that the method yields reproducible results (within 5% error). Other experiments with lower levels of radioactivity indicate the same degree reproducibility. DISCUSSION
0
1
2
3
4
5
Grams DPOper liter
Figure 2. Effect of concentration of scintillators on counting rate
This method is relatively simple and accurate for the determination of the radioactive sulfur content of a wide variety of substances and can be used for the routine handling of a large number of samples. One hundred samples have been analyzed simultaneously. The heating of the oxidation reaction is probably unnecessarily long, but has been found to be most convenient. It is important that all acid be removed and
Table I.
Reproducibility and Recovery Analysis Sample 1 Sample 2 Sample 3 Recovered, Recovered, Recovered, C.p.m.0 Tab C.p.m. yo C.p.m. 70 1 MI. of radioactive 184,905 ,.. 185,560 . .. 186,263 ... liver homogenate (2239) (1370) (1980) (1) (I) +.l MI. of non- 180,231 97.1 187,016 100.8 183,864 99.1 radioactive liver (1905) (1480) (1980) homogenate (I) +.l M1:ofnon- 189,871 102.3 182,206 98.2 183,344 98.8 radioactive kid- (1732) (1370) (1653) ney homogenate (I) +.l Ml: of non- 184,704 99.5 185,797 100.1 radioactive mus- (1416) (880) cle homogenate a Average of 5 determinations (106 counts). Numbers in parenthesis are standard deviations. b Based on average of three radioactive triplicate samples (185,567 c.p.m.).
VOL. 32, NO. 3, MARCH 1960
307
all perchlorates decomposed during the heating. If the salts stand a t room temperatures for any appreciable time, they frequently do not completely dissolve in the glycerol. Preheating the dry salts for approximately 1 hour a t 200" to 280' C. often proved helpful for preparing the glycerol solution. This suggests the possibility that a hydrated form of one of the salts may be formed and it is not readily soluble in the solvent system. The final solution has been found to be stable indefinitely, and the sample may be counted a t any time thereafter. Occasionally, with certain urine and large tissue samples (more than 500 mg.), the oxidation salts did not completely dissolve in the glycerol and complete solvent system. Analysis of smaller quantities of these samples indicated that the presence of this precipitate did not cause any loss of activity in the larger samples. If the specific activity in terms of the sulfur-35 or sulfate-35 content is desired, a sulfate determination can be conveniently made after the sample is counted. For this, the counted sample is transferred to a small separatory
funnel with several portions of water. After separation of the aqueous phase, the insoluble barium or benzidine sulfate can then be precipitated by the addition of the appropriate reagent. K h e n duplicate aliquots of radioactive liver homogenates were oxidized with varying amounts (0.5 t o 3.0 ml.) of modified Pirie's reagent, no significant difference was observed in the counting rate. Furthermore, these results indicated that small errors in the measurement of the volume of glycerol or the ethyl alcohol-dimethylformamide mixture did not change the counting rate. It is absolutely necessary, however, that the sample to be counted be a clear colorless solution. T o obtain the highest counting efficiency, the water impurities in the various solvents should be removed. However, i t may be more convenient to use the commercial grades of absolute ethyl alcohol and N,N-dimethylformamide directly, without further purification. If so, the efficiency would be 4 to 5% lower. Probably the greatest advantage of this method is the direct linearity of the observed counting rate with the true
counting rate over a very large range.
As a result, it is not necessary to apply any self-absorption or coincidence correction factors. Because nonradioactive materials present on the samples do not interfere with the counting rate, it is not necessary t'o isolate the radioactive sulfur compound in pure form. Although this determination has been described for the determination of S35 in biological materials, it should be applicable to all oxidizable materials containing S35. LITERATURE CITED
(1) Packard Tri-Carb Liquid Scintillation Spectrometer Operation l l a n u a l , Pack-
ard Instrument Co., LaGrange, Ill. Also, Personal communication from above company officials. (2) Passmann, J. If., Radin, Y S., Cooper, J. A. D., .4xa~.CHEY.28. 484 (1956). (3) Pirie, N. R., Biochern J. 26, 2041 (1932): (4) Radin, N. S.,Fried, R., A N ~ LCHEM. . 30, 1926 (1958). (5) Vaughan, M., Steinberg, D., Logan, J., Science 126, 446 (1957). RECEIVED for review September 10, 1959. Accepted December 23, 1959. Work supported by U. s. Public Health Service Grant -4-1543.
The Absolute Assay of Sulfur-35 by internal Gas Counting W. F. MERRITT and R. C. HAWKINGS Atomic Energy o f Canada limited, Chalk River, Ontario, Canada
b A method is described for measuring the absolute disintegrotion rate of 87.2-day sulfur-35, (j3-€,,, = 0.1 67 m.e.v.). The sulfur to b e assayed is converted to sulfur dioxide by ignition of barium sulfate with red phosphorus in an atmosphere of oxygen. The sulfur dioxide is then mixed with methane and counted in an internal gas proportional counter.
S
ULFUR-35is a pure negatron emitter with a half life of 87.2 days and a maximum beta energy of 0.167 m.e.v. ( 7 ) . ilbsorption losses in solid sources of such a low energy beta emitter are extremely variable and difficult to estimate (6). Internai gas counting (1) is a n-ell established technique for the assay of low energy beta emitters which can be prepared in a gaseous form and do not give poor counting characteristics. Wilkinson has stated (9) that any gas will function in a proportional counter. For the purpose of absolute 308
ANALYTICAL CHEMISTRY
counting, however, one restriction is imposed: The gas used must be such that one, and only one, pulse be recorded for every disintegration which takes place within the sensitive volume of the counter-that is, the gas must neither capture electrons nor release extra electrons in such a way as t o affect the observed number of pulses significantly. Of the two common gaseous compounds of sulfur, hydrogen sulfide, and sulfur dioxide, sulfur dioxide was selected as most desirable. An investigation v a s therefore undertaken of the performance of sulfur dioxide in methane-filled proportional counters, and of a suitabk procedure for the preparation of sulfur dioxide from sulfates without isotopic fractionation. EFFECT O F SULFUR DIOXIDE ON COUNTER EFFICIENCY
Simple tests n ith external sources showed that a proportional gas counter filled n-ith methane would tolera:i> small
quantities of sulfur dioxide nithout apparent, ill effects. A more precise evaluation of possible losses or spurious pulses was made as follows. A gas counter was filled with a mixt,ure of 2 em. of mercury pressure of hydrogen containing tritium (H3) and 74 cm. of mercury pressure of methane. Such a mixture has been shown to haye n high counting efficiency (2). The voltage characteristic for this mixture v a s determined and the plateau counting rate recorded. Successive amounts of inactive sulfur dioxide were then added to the filling and the counting characteristics redetermined. The results of these measurements are shown in Figure 1. The addition. of up to 4.5-mm. Hg pressure of sulfiiv dioxide to 760-mm. Hg pressure n i methane causes no change in t h plateau counting rate to within f 0.1L7;,. In general with increasing concentratior of sulfur dioxide, the plateau region is moved to higher and higher voltages while the voltage region for multiple counting remains essentially const;int.
