Assay of Radioiron and Radiosilver in Biological Samples LORETTA CHEONG, GlULlO C. PERRI, and L. M. SHARPE Division o f Ph sics and Biophysics, Sloan-Kettering hstitute, New University o f l a v i a , Pavia, /ta/y
I
h‘ T H E course of experiments on the metabolism of radio-
York, 21, N. Y., and institute of Biochemistry,
then transferred to a 50-ml. graduated cylinder using three 5-ml. portions of 0.1N sulfuric acid for that purpose. The final volume was between 25 and 30 ml.
silver, silver-110, in rats, it was necessary to correct the radiosilver concentration, expressed &s activity per gram of dry weight of tissue, for the activity contributed by the blood of that tissue. This information was obtained indirectly by transfusing blood containing erythrocytes tagged with radioiron, iron-55, from donor rat into the rat containing silver-110 according to thrl technique previously described ( 7 ) . Since each tissue contained both silver-110 and iron-55, it was necessary to devise a method which would permit their activities to be measured independently of each other. This was accomplished by separating the two elements using the cupferron extraction procedure ( 1 , 4 4 ) .
SEPARATIO\
O F IRON-55 AND SILVER-110 BY CUPFERROY EXTRACTION
The digest was diluted to approximately 5N in a separatory funnel and cold freshly prepared cupferron solution and chloroform were added. One milliliter of cupferron per 10 ml. of digest and 10 ml. of chloroform were used. The mixture was shaken for about 30 seconds and the chloroforni was removed. The aqueous phase was extracted twice more, using each time 5 ml. of chloroform. The entire extraction procedure was repeated after addition of 0.5 ml. of cupferron. .411 the iron was considered extracted when addition of 0.5 ml. of cupferron produced only a xhite turhidity. The combined chloroforni extracts were washed three times rising each time 20 ml. of 3 . 6 5 sulfuric acid.
REAGENTS
a a’-Bipyridine, 0.1 70aqueous solution
dupferron (ammonium salt of nitrosophenylhydroxylamine
G. Frederick Smith Chemical Co., Columbus, Ohio), 6% aqueous
TESTS FOR RECOVERY OF IROY-5.5 AND SILVER-110
solution. Prepare a fresh solution daily and keep in refrigeratot until used. Silver-110 nitrate (AgIlO NOa), specific activitl- 15 mc. pet gram of silver; 5.7 X mg. of silver per ml. Carrier iron solution, 1 mg. of iron per ml. Dissolve, nitli warming, chemically pure iron wire in a few milliliters of concentrated nitric acid and hydrochloric acid and dilute to volume This is referred to as the “carrier iron” solution. Iron-55 ammonium citrate, specific activity 258 X in(’ per mg. of iron; 311 X mg. of (ferric) iron per ml. Iron-59 ammonium citrate, specific activity 64 X 10-d in(’ per mg. of iron; 42 X mg. of (feriic-)iron per ml.
Recovery of Silver-I 10 from Rat Liver Digest. Seven milliliters of silver-110 nitrate having an activity of 110 X l o 3 counts per minute were added to a rat liver digest containing 7 ml. of concentrated sulfuric acid and 10 mg. of carrier iron. The solution xas diluted to 5.3’ and was extracted twice with cupferron. The combined chloroform extracts, totaling 40 ml., were washed three times with 20-1111. portions of 3.6N sulfuric acid. The activity of each washing measured: Washing
Volunie of Washing,
Activity of Washing,
1 2 3
20 20 20
3460 360
so.
4PPARATUS FOR COUhTING
All gamma-activity measurements were made by means of a scintillation counter and standard scaler. The detector consisted of a tellurium activated-sodium iodide crystal (Harshaa Chemical Co., Cleveland, Ohio) coupled to an RCA 5819 photomultiplier tube. The signals from the photomultiplier were coupled directly to a Rtandard scaling circuit having a 0.25-volt threshold. Each gamma-activity measurement was made on a 5-ml. portion of the solution contained in a 5-ml. capacity vial. High counting efficiency was attained, as the vial just fitted into a circular well in the crystal S / B inch in diameter and 1.5 inches in depth. No increase in counting rate above background was detected when the scintillation counter was exposed t o the 5.9 k.e.v x-rays from iron-55. The activity of the electroplated iron-55 W&B measured wit11 an argon-filled G-M tube (Tracerlab, Boston, Mass.).
Ml.
C.P.M. 120
Added Ag‘lO NO& % 3.2 0.3 0.09
The chloroform in the washed extract was evaporated on a steam bath and the cupferron was oxidized by heating with 2 ml. of concentrated sulfuric and nitric acids, respectively. The residual silver-110 activity of the digest was found to be 52 counts per minute or 4.7 x lo-* % of the original activity. Recovery of Iron-59 from Synthetic Mixture. To test the completeness of extractions of iron by cupferron, a mixture consisting of 0.8 ml. of iron-59 ammonium citrate, having an artivity of 16 X lo3 counts per minute, 10 mg. of carrier iron, and 3.5 nil. of concentrated sulfuric acid was heated to fuming and then diluted to 25 ml. The solution was extracted with chloroform after each of two additions of cupferron. The extracts were combined, the chloroform was evaporated, and the residue was oxidized with concentrated sulfuric and nitric acids. The gamma activity of the residue was found to be 15,680 counts per minute or 97.770 of the original activity. Recovery of Iron-59 from Rat Liver Digest. Iron ammonium citrate (16,440 counts per minute) was mixed with 10 mg. of carrier iron and the digest obtained from a 10-gram rat liver; 16,200 counts per minute or 98.570 of the added activity was recovered in the washed chloroform extract. Iron-59, a gamma emitter, was used in the above experiments because of the simplicity of its measurement in the absence of interfering silver-110.
ELECTROPLATING PROCEDURE AND EQUIYMEVT
The electroplating procedure and equipment were essentially those previously described (3). Electroplating was carried out from a saturated aqueous ammonium oxalate solution at an initial pH of 5 to 6 a t 6 volts, 0.5 ampere. Plating was considered complete when the plating solution contained 1-y (0.35%) or less of iron per ml. of plating solution. The iron was estimated with bipyridine (S),except that ascorbic acid was substituted for sodium bisulfite since it yielded more reproducible results.
From the above data it is ;Lppaient that iron and silver can be quantitatively sepaiated from each other by means of extraction with cupfei ron, pinvided the chloroform extract is washed with :in acid solution.
DIGESTION OF T I S S U E S
All tissues, the largest of which weighed less than 10 grams, were wet-ashed. The tissue was placed in a 300-ml. Kjeldahl flask together with 10 mg. of carrier iron and sulfuric acid and heated until fuming occurred. Two milliliters of concentrated sulfuric acid were used for the first 3 grams and 1 ml. for each additional gram. After fuming occurred, 30% hydrogen peroxide was added, 1 to 2 drops a t a time, until all organic matte1 wm destroyed. Since salts tend to crystallize out of cold concentrated sulfuric acid, thus making transfer of the digest difficult, 25 ml. of water were added and the flask was rrheated until the volume waq rrdiiced to about 15 ml. The solution was
RECOYERk OF S I L I ER-110
\ \ D [ROY-55 FR0.M BIOLOGIC4L SAIlPLES
Various activities of iron-55 and silver-110 were mixed with rat tissues in the amounts usually present in unknown samples. After 10 mg. of carrier iron were added, the tissues were oxidized, the digests mere transferred to separatory funnels, and the iron was extracted with cupferron. The chloroform extract was washed three times with 20-ml. portions of 3.6N sulfuric. siraid and the washings were added t o the aqueous phase. 242
V O L U M E 26, NO. 1, J A N U A R Y 1 9 5 4
a43
Silver-110, T > / ; 270 days, has a complex decay process ( 2 ) ; its beta rays vary in maximum energy Tissue AgliO Fes6 from 0.087 to 2.86 m.e.v. while emitt,ing gammas Recovered Gamma from 0.116 to 1.516 m.e.v. Iron-59, T U Z 47 days, after Activity of emits 0.46 and 0.26 m.e.v. maximum energy betas Activityn cupferron Recovered Activity Wet wt., added, extraction, Radioiron, added&, Recovered. and 1.1 and 1.3 m.e.v. gammas. Iron-55, TI/^ 2.91 grams c.p.m. % C.P.M. c.p.m. 7% years, decays by K-capture resulting in the emisSpleen 0.543 23,500 102 7 480 98 sion of 5.9 k.e.v. x-rays. Kidney 2.32 11,700 97 8 720 97 2,350 102 Heart 0.868 9 240 100 Differential counting of silver-110 and iron-59 in 4.700 99 8 480 98 Lung 1.43 8 240 104 solution based on differences in gamma-ray energy 2,350 97 Brain 1.02 Blood 2 . 0 (ml.) 11,700 98 7= 960 102 requires the use of a gamma-ray spectrometer. Liver 9.31 93,800 100 8 1440 92 Such an instrument is not ordinarily available in e Specific activity: 4.4 X 107 c.p.m./mg. of silver. most biochemical laboratories. Also, separation b Specific activity: I54 X 106 c.p.m./mg. of iron. Gamma activity recovered hefore precipitation of ferric hydroxide from cupferratr means of these two isotopes would by digest. result, in preparations in which the degree of contamination of the one isotope by the other Table 11. Per Cent of Agi'O Electroplated in Presence of 10 could not be ascertained. 3Ig. of Inactive Irona Quantitative electroplating of either mixture of the two isoPlating Agllo topes for the purpose of differential ( a ) beta counting in the Gamma Beta Beta activity activity activity case of silver-110 and iron-59 or ( b ) beta x-ray counting in the Current, Time, addedb, recoveredc, plated, \oltage amp. hours c.p.m. c.p.m. % case of silver-110 and iron-55 would still require the removal of 5 0.5 J 11,900 900 73 calcium and phosphates, as these substances interfere with 9 0.5 5 11,900 510 73 Plectroplating. 6 0.3 1 15,200 176 11 6 0.3 2 15.200 166 10 Iron-55 was selected because it not only permits measurement 6 0.3 3 15,200 275 17 170 11 6 0.3 4 15,200 of silver activity completely independently of the iron activity 4 0.02 4 8.675 100 11 but also allons an estimate of the degree of contamination of 4 0.02 4 9,750 115 11 the iron by the silver. a Each sample to be electroplated consisted of 10 mg. of inactive iron dissolved in 1 t o 2 ml. of 6 S sulfuric acid t o which was added the indicated Separation of iron from silver by precipitation as ferric hyactivity of Agl10 NOa. droxide, Fe(OH)3,was considered. This method was found to be 6 Snrcific activ efficient for iron precipitation, but the precipitate always included an appreciable amount of silver activity. Furthermore, iron cannot be quantitatively precipitated in the presence of phosphates. The precipitation of silver chloride in acid solution would also yield no advantage, as calcium and phosphates are As indicated in Table I, the recovery of silver-110 was essensoluble under these conditions. tially complete. The chloroform was evaporated from the extract on a steam The cupferron extraction procedure was recently reported bath and the residue was wet-ashed with 5 ml. of concentrated to yield extracts free of substances that interfere with the elecsulfuric acid. The digest was transferred to a 100-ml. centrifuge troplating of iron ( 4 ) . .4lthough cupferron is known to extract tube and concentrated ammonium hydroxide was added until a elements other than iron (1 ), the above experiments demonstrate brown precipitate, Fe( OH)3, appeared. The suspension was centrifuged and the supernatant was discarded after being tested that i t does not combine with silver under the conditions emfor iron as described under the plating procedure above. The ployed. precipitate w m dissolved in 1 ml. of 6N sulfuric acid and was transferred to a 5-ml. capacity counting vial. The volume was CONCLUSIOYS made up to 5 ml. and the gamma activity was measured The cupferron extraction procedure has been applied to the The gamma activity of the cupferrate digest obtained from separation of 10 mg. of iron containing a low iron-55 activity blood was measured before rather than after precipitation of the from as much as 2.13 X 10-3 mg. of silver containing a high ferric hydroxide in order to obtain an estimate of the loss in activity of silver-110. The same extraction process also leaves silver-1 10 gamma activity resulting from the precipitation procalcium and phosphate, found in biological specimens, in the cedure. The data (Tahlc I, fifth column) indicate that less than silver-containing fraction, thus permitting efficient electroplating 10 counts per minute, equivalent to 6 X y of silver, was of the iron-55. The use of iron-55 rather than iron-59 permits a present in each sample irrespective of whether or not the iron check to be made on the efficiency of the separation of the silver was precipitated before counting. from the iron in each simple. While destruction of the cupferrate permits more efficient transfer of the iron, the degradation products obtained tend to LITERATURE C I T E D interfere with the electroplating of iron. These degradation products, however, remain in the supernatant and are discarded (1) F u r m a n , li. H., hIason, IT. B., a n d P e k o l a , J. S., AXAI..CHEY., 21, 1325 (1949). when the iron is precipitated as the hydroxide. (2) K a t l . B u r . S t a n d a r d s , Circ., 499 (1950). After the residual gamma activity of each radioiron sample (3) P e a c o c k , W. C., E v a n s , R. D., I r v i n e , J. W., Jr., G o o d , W.M., was measured, the sample was transferred to an electroplating K i p , A. F., Weiss, S., a n d G i b s o n , J. G . , 2nd.,J. Clin. I n r e s t . , cell and the iron was plated out a t 6 volts. The recovery of 25, 605 (1946). (4) P e t e r s o n , R . E., VAL. CHEX, 24, 1850 (1952). radioiron is listed in the last column of Table I. (5) R o d d y , C. J., "Analytical C h e m i s t r y of t h e M a n h a t t a n P r o j Silver is plated out less eficientlJ-, in the presence of 10 mg. e c t , S e w Yolk, M c G r a w - H i l l Book Co., 1950. of iron, a t 6 volts than a t 9 volts (Table 11). No advantage is (6) S a n d e l l , E. B., a n d C u m m i n g s , P. F., ANAL. CHEM.,21, 1356 gained by reducing the voltage helow 6 volts, as there is no further (1949). (7) S h a r p e , L.II.,C u l h r e t h , G. C., a n d K l e i n , J . R . , Proc. SOC.Exptl. reduction in silver plating. n-hil(a iron plating is markedly proBiol. and Med., 74, 681 (1950). longed as a result of the retluwd current density. Consideration was given to differential counting of mixtures RECEIVED July 9, 1953. Accepted September 19, 1953. Work carried o u t of either iron-59 01 ii-on45 and silver-110 in the liquid or solid under Atomic Energy Commission Contract N o . -4T(30-1)-1451. Giiilio C. state. Perri is a Sloan Foundation F r l l o i r f u r Advancrd R e v a r c h .
Table I. Recovery of Ag"0 and Feb6 from Rat Tissues
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