Method for Routine Determination of Sulfur-35 in Biological Material SIDNEY S. WALKENSTEIN and CORNELIUS M. KNEBEL Wyeth lnstifute for Medical Research, Radnor, Pa.
b A procedure for determining sulfur35 in biological samples was developed for use in a study on the physiological disposition of sulfur-tagged drugs. The entire analysis, from the oxidation of the organic sulfur to the drying of the barium sulfate precipitate, is performed in a tared centrifuge tube modified to facilitate packing and washing of the precipitate. The results are comparable in reproducibility to those obtained with filtration. The barium sulfate is easily transferred to a planchet for counting. This method, applicable to the simultaneous analysis of large numbers of samples, avoids the danger, inherent in the use of sintered-glass filters, of radioactive contamination of successive samples.
A
consideration of the many methods in use for the conversion of organic sulfur to inorganic sulfate, the FTet combustion of Le Matte, Boinot, and Kahane (7') with niodifications suggested by Pirie (8) and Boursnell, Francis, and Kormall ( 2 ) was selected. This choice was based on the ready applicability of this method to tissue and excreta alike. hforeover, it was free from the cumbersome manipulations and occasional mishaps of the Carius (3) and Folin (6) methods and the requirements for a predried sample of the latter and the Pregl methods (9). As a precipitant for sulfate, barium chloride was preferred to benzidine, as removal of phosphate \vas unnecessary with the former reagent. FTER
APPARATUS
Centrifuge tubes, 40-ml. (A. H. Thomas Go. Catalog KO. 3121-D) modified as in Figure 1 by poking in the flame-softened bottom with a blunt pencil point. Air bath. Steam bath. Centrifuge. METHOD
To 2 grams of tissue, blood, or feces, or 5 ml. of urine in a tared centrifuge tube add 15 ml. of oxidizing agent, a large enough excess to prevent sample loss by flashing. After foaming, if any, subsides, add 50 mg. of copper wire, which is adequate for up t o 22 mg. of sulfur. When the reaction ceases, immerse the centrifuge tube to 4/5 of its height in an air bath a t 180' C. If the solution is not entirely clear after 1 hour, add 5 ml. of oxidant. Take to dryness (3 t o 5 hours) and raise the teniperature of the air bath to 280' C. When fumes no longer appear, remove tubes from air bath, cool below 100' C., and add 5 ml. of 1S hydrochloric acid to the residue, which is either nhite, green, or black a t this stage (if blue, continue heating a t 280' before adding hydrochloric acid), Warm until a clear solution is obtained. To obtain a final pH between 0.5 and 1.0 (8) add 20 ml. of water. If carrier is needed, it is added a t this stage (0.LY sulfuric acid). Heat the solution to boiling and add dropwise an excess of 5% barium chloride solution. Then transfer the tube to a steam bath for 2 hours. After adding 2 drops of 20% Tween 80 to the sides of each tube and washing in with a few drops from a wash bottle, centrifuge the tubes for 10 minutes a t 2500 r.p.m. Carefully
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ANALYTICAL CHEMISTRY
RESULTS AND DISCUSSION
The results of l 0 determinations of the sulfur content of nonlabeled cystine (National Bureau of Standards) are given in Table I. The average deviation from the theoretical value of 26.68 is 0.20%. Table 1.
Sample Weight,
Sulfur Content
Found, 5%
7.5 29.2 31.6 32.7 45.3
26.55 26.29 26.94 26.62 26.42 26.32 26 60 26 48 26 78 26.84
50 9 81 8 95 0
99 9
Figure 1. Modified centrifuge tube
Determination of Sulfur in Cystine
Mg.
49.0
MATERIALS
Oxidant. Three volumes of concentrated nitric acid to 1 volume of 60% perchloric acid. Copper wire. Barium chloride solution, 5%, aged a t least 1 week before use (1, 5 ) . Tween 80, polyoxyethylene sorbitan mono-oleate (Atlas Powder Co., Wilmington, Del.) in two different dilutions: 2 and 20%. Sitric acid, 0.05N. Hydrochloric acid, LI*, Ethyl alcohol. 50%. Sulfuric acid, O . l S , standardized volumetric solution.
pour off the supernatant fluid and add 4 drops of 2% Tween 80 around the circumference of the tube. With a strong jet of 0.05N nitric acid from a wash bottle, resuspend and centrifuge the precipitate. The second wash is identical with the first, except that the suspension is digested for 20 minutes in a steam bath before centrifugation. Hot washes are repeated until the test for chloride is negative, following which the precipitate is washed tm-ice by suspension in 507, ethyl alcohol and centrifugation to remove Tween 80. After the outside of the tube has been carefully rinsed with distilled water it is placed slantwise (to prevent loss of material by bumping) in an oven a t 180' C. for a t least 2 hours. Radioactive samples were plated on cupped nickel-plated planchets (Tracerlab, Boston, Mass.) and counted in a thin end-window Geiger-Muller counter. After subtracting background, the activities of all samples were corrected to infinite thickness (Figure 2 ) .
Deviation from Theory (26.6893, 70 -0.13 -0.39 +O .26 -0.06
-0.26 -0.36
-0 08 -0 20
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10
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The reproducibility of the method when applied to biological material is indicated by the results listed in Table 11. Two-milliliter aliquots of dog blood containing 1.5 mg. per ml. of sulfur35-labeled 10-( r-dimethylaminopropyl) phenothiazine hydrochloride (Sparine, Wyeth, Inc.), specific activity 109,000 counts per minute per planchet a t in-
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Drying a t 180” C. does not entirely remove adsorbed water and hydrochloric acid and temperatures of more than 500” C. are therefore generally advocated. However, a maximum weight loss from the barium sulfate precipitate of only 2.8y0occurs on heating from 158” to 950” C. (4). Furthermore, as all samples were related to similarly prepared standards with identical selfabsorption corrections, it was felt that its greater convenience warranted the use of the lower drying temperature.
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ACKNOWLEDGMENT
finite thickness, were submitted to the analytical procedure. TKO milliliters of 0.1N sulfuric acid were added prior to precipitation with barium chloride. The deviation from the mean weight of barium sulfate recovered averaged approximately 0.3%. The variation in counting rate was of the same order. Losses in washing were negligible, as collected dried washes had an average of less than 0.4% of the activity of the radioactive barium sulfate itself. Difficulties reported by various investigators, such as incomplete oxidation of fecal matter and tissue, were not encountered with the sample sizes taken for analysis in this study. The use of Tween 80 prevented completely “creeping” of the barium sulfate precipitate. Adhesion of the finer particles t o the sides of the centrifuge tube mas adequately prevented by use of the dilute nitric acid wash solution. The design
Table 11. Determination o f Sulfur-35 in Replicate Blood Samples
Recovered Barium Sulfate“, hlg.
Specific Activity, C.P.M.
44.8 5340 45.0 5290 44.8 5325 44.6 5337 45.1 5324 Includes 23.4 mg. of carrier.
of the indented centrifuge tubes enhanced packing of the precipitate and facilitated removal of maximumamounts of the supernatant layer without disturbing the precipitate. !When ordinary conical centrifuge tubes were used, bumping was excessive and resuspension of the barium sulfate was difficult.
The authors wish to express their gratitude for the interest -shown by Joseph Seifter in this study. LITERATURE CITED
(1) Bo an, E. J., Moyer, H. V., ANAL. &EM. 28, 473 (1956). (2) Boursnell, J. C., Francis, G. E., Wormall, A., Biochem. J . 40, 743 (1946). 3) Carius, L., Ann. 116, 1 (1860). 4) Duval, C., “Inorganic Thermogravi-
metric Analysis,” . Elsevier, Amsterdam, 1953. (5) Erdey, L., Paulik, F., Magyar Tudo-
manyos Akad. 4, 73 (1954). (6) Folin, O., J . Biol. Chem. 1, 131 (1905). ( 7 ) Le Matte, L., Boinot, G., Kahane, E., Compt. rend. soc. biol. 96, 1211 (1923). (8) Pirie, N. W.,Biochem. J . 26, 2041 (1932). (9) Pregl, F., Grant, J., “Quantitative
Organic Microanalysis,” Blakiston, Philadelphia, Pa., 1946. RECEIVED for review November 30, 1956. Accepted March 15, 1957.
Microebulliometer for Determination of Molecular Weight MARTIN DIMBAT and F.
H. STROSS
Shell Developmenf Co., Emeryville, Calif.
b A microebulliometer has been developed which permits relatively convenient and precise determinations of molecular weight on as little as 10 mg. o f sample added to the ebulliometer in 1- to 2-mg. increments. The reduction in sample size over conventional methods was accomplished b y use o f a small solvent volume (5ml.) and a highly sensitive method of measuring temperature. By the use of thermistors, temperature differences can be measured to 6 X 1O P 5 O C. The ebulliometer design permits steadiness o f boiling within the limits of measurement for long periods of time. The sensitivity of temperature measurement also makes
possible extension of the range o f the determinations to molecular weights o f about 20,000.
T
of the small samples commonly obtained by modern separation techniques should often be known. To handle such small samples, ebulliometers have been developed (1, 4, 6-8, 11) which are capable of more sensitive temperature measurement and permit the use of smaller solvent volumes than before. Most recent developments are modifications of the Menzies-Wright ebulliometer (9) using liquid-filled differential thermometers. Ray (11) used a differential HE MOLECULAR WEIGHT
thermopile in a modified Menzies-11-right ebulliometer t o measure temperature difference precisely. Muller and Stolten (IO) reported using thermistors for the temperature observations in a HillBaldes molecular weight apparatus. Thermistors have been used in ebullionieters for service analyses in industry (3). The ebulliometer discussed here is a modification and refinement of this type of instrument. It differs from the hlenzies-Wright principle mainly in the method of measuring the pure solvent boiling point. I n the Menzies-m7right apparatus, sample and reference boiling points are observed by means of the characVOL. 29, NO.
10, OCTOBER 1957
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