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Radiochemical determination of short-lived radionuclides in neutron-activated biological samples
ImrrnuriomzlJournal qf Applied Radiarion & hotopus. Vol. 31. pp. 446 lo 441 0 Pcrgamon Press Ltd 1980. Printed in Great Britain 0020708X/L10/0701-0446102.00/0
Radiuchemical Determination of Short-lived Radionuclides in Neutron-activated Biological !Samples CHRISTOPHER
E. CANN* and STANLEY G. PRIJSSIN Department of Nuclear Engineering, University of California, Berkeley, CA, U.S.A.
(Received 25 October 1979; irl rerised.fonn I 1 Decealber 1979)
A radiochemical procedure has been developed for rapid post-irradiation chemistry of neutron-activated biological samples. It is based on destruction of the sample in a sodium permanganatc-nitric acid medium, followed by appropriate radiochemical techniques to separate the elements of interest. Dissolution of most soft tissue samples is complete in 4560 s. The procedure has been used to assay calcium in tissue samples as small as 5 mg (dry-weight basis) with a total separation time of less than 4 min. Mmduction WHILEneutron activation analysis (NAA) has been useful for the measurement of the concentrations of many elements in a wide variety of samples, its use for analysis of biological specimens has been restricted because of the large-quantities of interfering radionuclides produced in the matrix (notablv 15.0 hz4Na. 37.2 min3* Cl and 12.4 h4’ K). The lack bf suitable radiochemical procedures for the isolation of short-lived radionuclides from the bulk matrix has especially hampered the use of NAA to determine concentrations of many trace elements which have short-lived activation products. While pre-irradiation chemistry has been used in some cases, the necessity for carrier-free chemistry in sample preparation has precluded the use of very small specimens. In addition, the contamination problems encountered with pre-chemistry often limit the usefulness of this technique. Post-irradiation chemistry for short-lived radionuclides has been limited primarily by the time taken to dissolve the biological material. In studies of trace-element distributions in tissue samples from animal and human subjects, we were confronted with the need to perform numerous analyses on small samples. The procedure described in this report for the rapid dissolution of biological specimens was found to be quite reliable and easily coupled to simple radiochemical procedures for the assay of trace elements. As illustrative of a complete procedure, we describe the quantitative measurement of calcium concentrations in small samples of human tissue. Methods
and Results
obtained at surgery for hyperparathyroidism at the University of California, San Francisco Medical Center. Fresh tissues were dissected free of the outer layer to remove possible contaminants, placed in precleaned, virgin polyethylene vials?, lyophilized and weighed. After nondestructive analysis by NAA for a variety of other elements, each sample was paired with a standard containing 0.5 mg of calcium and irradiated in the Flexorabbit facility of the Berkeley Research Reactor at a thermal-neutron flux of 1.0 x lOI cm-” s-’ for 10 min. Tissue sample size was 5lOOmg (dry-weight basis). Upon return to the radiochemistry hood the capsule was opened and the sample added with stirring to a 50 ml round-bottom flask containing lO.Oml of 12N HNOs at 95°C and 15.0mg of Ca*+ carrier labeled with 0.2 &i of 4’Ca. 3-5 s later 2.0 ml of saturated (2 M) NaMnO, were added under rapid (3-4 s) but controlled conditions. This is a vigorous reaction with elaboration of gases and special care must be taken to prevent loss of sample at this point. (The preferred procedure for manual addition is the use of a 2.5 or 3ml syringe with the NaMn04 directed down the neck of the iask. The hot solution was kept near boiling and stirred for 30-40s. until the tissue was dissolved. The resultina solution, containing large quantities of MnO,, was filtered through a large Buchner funnel into a 250ml filter flask. The total time from end of irradiation to recovery of filtrate was generally under 60 s. To recover the calcium from the solution, IOml of 12 N NaOH was added with stirring to the hot filtrate again taking care because. of the vigor of this neutralization reaction. Within 5 s, I5 ml of saturated (NH,),CIOd was added to precipitate calcium, the solution was cooled in an ice bath for %5 s and the CaC,04 collected by filtration. Filters were mounted on planchets for counting. The complete procedure was done in a radiochemistry hood and samples were ready to count 3-4min after end of irradiation. Samples were counted with a 35cm’ (active volume) Ge(Li) detector coupled to a 4096-channel analyzer system. Counting time was 10min clock time and pulser electronics were used to determine live time. A comparator standard was counted following the sample count. The 3.084 MeV peak of 49Ca was used for analysis and peak areas were obtained using the computer code SAMPO.(‘~ The chemical yield, which averaged 85-90%, was determined 2-3 days later by measurement of the added ‘+‘Ca. This procedure for calcium determination has been used to measure calcium in tissue samples of less than 6 mg (dry-weight basis) with an uncertainty of about 12%. In over 60 samples analyzed (6100 mg 200-800 ppm Ca) measurement uncertainties ranged from 4 to 12%. Calcium in NBS SRM 1577 (Bovine- Liver) was measured as I 15 -+ 12 oom . . (reference value 123 opm) and in SRM 1571 2.01 +-6.18% (certified at (Orchard Leaves) as 2.09 + 0.03%). Optimization of the technique for a given sample size results in uncertainties of 3-4% in measurement of calcium concentrations. Figure I shows the @Ca 3.084MeV ohotoueak in two comparable samoles, one measured without separation from 24Na (1.369 MeV, 2.754 MeV) and ‘*Cl (1.642 MeV. 2.167 MeV). and the other after’separation. Discussion
The tissue samples analyzed in this work consisted mainly of dried parathyroid, thyroid and muscle tissue * Present address: Department of Radiology, University of California, San Francisco, CA 94143, U.S.A. t Olympic Plastics, Los Angeles, Calif. 446
The dissolution procedure was tested with a wide variety of organic materials, including fresh fat and muscle tissue, cartilage, bone and wood. Most of the samples tested dissolved in 30-40s. Cancellous bone, cartilage and NBS SRM 1571 (Orchard Leaves) took 9&120 s, and cortical bone and dry wood required 300s. In some cases, es-
Technical
102-
447
Note
IO’4%
37s
4%
3.004 MeW 3.103 MeV i
3.004 MaV
I
4 n
ii
cd 5 9
1’1
YlO’r 4 8 .
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II
II II
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3280 CHANNEL NUMBER
IO’ 3200
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FIG. 1. Partial y-ray spectra from two samples of parathyroid tissue showing the 3.084 MeV photopeak from %a. Each sample was counted for 10 min beginning 3 min after a 10 min irradiation. Left-hand side no separation from matrix activity, sample mass = 89.9 mg, detector input rate = 1.1 x IO*s-t, source-to-detector distance = 16cm. Right-hand side following radiochemical separation to remove “Na and 3*C1,sample mass = 84.2 mg, detector input rate = 3.3 x IO3 s-‘, source-to-detector distance = 0.3 cm. Note the difference in vertical scales. pecially for the fat tissue, an oil residue was found on top of the aqueous solution of the material. This residue coagulated into a fat-like substance at about 5”C, and was soluble in petroleum ether. Since fat tissue is known to contain calcium, this residue was analtied to determine its content of various elements. Other than bromine (2% of the total in the sample), negligible quantities of the elements of interest were found (less than 0.5% of Ca, Mg, AL Cl and less than 0.01% of Na), indicating that elements bound in the organic matter were released as the cellular matter was destroyed Because of the large quantities of MnO, formed, and the qualities of MnO, as a selective absorbent.“) the filtered MnO, was analyaed for trapped radionuclides. It retained less than 0.5X of the Na. Ca. Me and AL with areater than 99% retention of Mn and I.‘Later tests under iimilar conditions showed retention of less than 1% of Zn, 7% of Cu, 20% of CL and 60% of Fe. The speed with which this procedure can be carried out is its main advantage. In various tests, “V and ‘*Ti radionuclides have been observed, but not quantified. ‘*Al and
“Zn have been measured quantitatively with modification of the separation procedure. In addition to those elements which can only be assayed by use of their short-lived activation products, elements such as cobalt and copper can be assayed without long irradiations by using their short-lived products. Iron analysis, using 57Fe(n,p)s7 Mn, and and iodine analysis are possible because of the quantitative retention of their activation products on the MnO, formed during dissolution. Through increases in sensitivity using larger detectors, the method may be applied to the analysis of very small samples. This will be clinically useful for the analysis.of small tissue biopsies, with detection of such physiologically significant elements as calcium and magnesium at the level of tenths of micrograms. References 1. ROUTTI J. T. and Paussm S. G. Nucl. Instrum. Meth. 72, 125 (1969). 2. GIIWDI F., Pmraa R. and SABBIONI E. J. radioanalyt. Chem. 5, 141 (1970).
Radiuchemical Determination of Short-lived Radionuclides in Neutron-activated Biological !Samples CHRISTOPHER
E. CANN* and STANLEY G. PRIJSSIN Department of Nuclear Engineering, University of California, Berkeley, CA, U.S.A.
(Received 25 October 1979; irl rerised.fonn I 1 Decealber 1979)
A radiochemical procedure has been developed for rapid post-irradiation chemistry of neutron-activated biological samples. It is based on destruction of the sample in a sodium permanganatc-nitric acid medium, followed by appropriate radiochemical techniques to separate the elements of interest. Dissolution of most soft tissue samples is complete in 4560 s. The procedure has been used to assay calcium in tissue samples as small as 5 mg (dry-weight basis) with a total separation time of less than 4 min. Mmduction WHILEneutron activation analysis (NAA) has been useful for the measurement of the concentrations of many elements in a wide variety of samples, its use for analysis of biological specimens has been restricted because of the large-quantities of interfering radionuclides produced in the matrix (notablv 15.0 hz4Na. 37.2 min3* Cl and 12.4 h4’ K). The lack bf suitable radiochemical procedures for the isolation of short-lived radionuclides from the bulk matrix has especially hampered the use of NAA to determine concentrations of many trace elements which have short-lived activation products. While pre-irradiation chemistry has been used in some cases, the necessity for carrier-free chemistry in sample preparation has precluded the use of very small specimens. In addition, the contamination problems encountered with pre-chemistry often limit the usefulness of this technique. Post-irradiation chemistry for short-lived radionuclides has been limited primarily by the time taken to dissolve the biological material. In studies of trace-element distributions in tissue samples from animal and human subjects, we were confronted with the need to perform numerous analyses on small samples. The procedure described in this report for the rapid dissolution of biological specimens was found to be quite reliable and easily coupled to simple radiochemical procedures for the assay of trace elements. As illustrative of a complete procedure, we describe the quantitative measurement of calcium concentrations in small samples of human tissue. Methods
and Results
obtained at surgery for hyperparathyroidism at the University of California, San Francisco Medical Center. Fresh tissues were dissected free of the outer layer to remove possible contaminants, placed in precleaned, virgin polyethylene vials?, lyophilized and weighed. After nondestructive analysis by NAA for a variety of other elements, each sample was paired with a standard containing 0.5 mg of calcium and irradiated in the Flexorabbit facility of the Berkeley Research Reactor at a thermal-neutron flux of 1.0 x lOI cm-” s-’ for 10 min. Tissue sample size was 5lOOmg (dry-weight basis). Upon return to the radiochemistry hood the capsule was opened and the sample added with stirring to a 50 ml round-bottom flask containing lO.Oml of 12N HNOs at 95°C and 15.0mg of Ca*+ carrier labeled with 0.2 &i of 4’Ca. 3-5 s later 2.0 ml of saturated (2 M) NaMnO, were added under rapid (3-4 s) but controlled conditions. This is a vigorous reaction with elaboration of gases and special care must be taken to prevent loss of sample at this point. (The preferred procedure for manual addition is the use of a 2.5 or 3ml syringe with the NaMn04 directed down the neck of the iask. The hot solution was kept near boiling and stirred for 30-40s. until the tissue was dissolved. The resultina solution, containing large quantities of MnO,, was filtered through a large Buchner funnel into a 250ml filter flask. The total time from end of irradiation to recovery of filtrate was generally under 60 s. To recover the calcium from the solution, IOml of 12 N NaOH was added with stirring to the hot filtrate again taking care because. of the vigor of this neutralization reaction. Within 5 s, I5 ml of saturated (NH,),CIOd was added to precipitate calcium, the solution was cooled in an ice bath for %5 s and the CaC,04 collected by filtration. Filters were mounted on planchets for counting. The complete procedure was done in a radiochemistry hood and samples were ready to count 3-4min after end of irradiation. Samples were counted with a 35cm’ (active volume) Ge(Li) detector coupled to a 4096-channel analyzer system. Counting time was 10min clock time and pulser electronics were used to determine live time. A comparator standard was counted following the sample count. The 3.084 MeV peak of 49Ca was used for analysis and peak areas were obtained using the computer code SAMPO.(‘~ The chemical yield, which averaged 85-90%, was determined 2-3 days later by measurement of the added ‘+‘Ca. This procedure for calcium determination has been used to measure calcium in tissue samples of less than 6 mg (dry-weight basis) with an uncertainty of about 12%. In over 60 samples analyzed (6100 mg 200-800 ppm Ca) measurement uncertainties ranged from 4 to 12%. Calcium in NBS SRM 1577 (Bovine- Liver) was measured as I 15 -+ 12 oom . . (reference value 123 opm) and in SRM 1571 2.01 +-6.18% (certified at (Orchard Leaves) as 2.09 + 0.03%). Optimization of the technique for a given sample size results in uncertainties of 3-4% in measurement of calcium concentrations. Figure I shows the @Ca 3.084MeV ohotoueak in two comparable samoles, one measured without separation from 24Na (1.369 MeV, 2.754 MeV) and ‘*Cl (1.642 MeV. 2.167 MeV). and the other after’separation. Discussion
The tissue samples analyzed in this work consisted mainly of dried parathyroid, thyroid and muscle tissue * Present address: Department of Radiology, University of California, San Francisco, CA 94143, U.S.A. t Olympic Plastics, Los Angeles, Calif. 446
The dissolution procedure was tested with a wide variety of organic materials, including fresh fat and muscle tissue, cartilage, bone and wood. Most of the samples tested dissolved in 30-40s. Cancellous bone, cartilage and NBS SRM 1571 (Orchard Leaves) took 9&120 s, and cortical bone and dry wood required 300s. In some cases, es-
Technical
102-
447
Note
IO’4%
37s
4%
3.004 MeW 3.103 MeV i
3.004 MaV
I
4 n
ii
cd 5 9
1’1
YlO’r 4 8 .
A
‘-
” ., I . If
II
II II
II II
. . . ..
IO0
11 fi il . ..1:I i-1 I I *. ’ . , I..
; .t__.;
I 1 IO211 ! ! * I 1 : _f.:...+I 1 .
-.--lr :* . , I, .
.
.
1 ..__
J
,.,.
3200
3280 CHANNEL NUMBER
IO’ 3200
.:
: :_,. f- . . .;.% . . _.._.’ . -. -
. .
I
I
3280 CHANNEL NUMBER
FIG. 1. Partial y-ray spectra from two samples of parathyroid tissue showing the 3.084 MeV photopeak from %a. Each sample was counted for 10 min beginning 3 min after a 10 min irradiation. Left-hand side no separation from matrix activity, sample mass = 89.9 mg, detector input rate = 1.1 x IO*s-t, source-to-detector distance = 16cm. Right-hand side following radiochemical separation to remove “Na and 3*C1,sample mass = 84.2 mg, detector input rate = 3.3 x IO3 s-‘, source-to-detector distance = 0.3 cm. Note the difference in vertical scales. pecially for the fat tissue, an oil residue was found on top of the aqueous solution of the material. This residue coagulated into a fat-like substance at about 5”C, and was soluble in petroleum ether. Since fat tissue is known to contain calcium, this residue was analtied to determine its content of various elements. Other than bromine (2% of the total in the sample), negligible quantities of the elements of interest were found (less than 0.5% of Ca, Mg, AL Cl and less than 0.01% of Na), indicating that elements bound in the organic matter were released as the cellular matter was destroyed Because of the large quantities of MnO, formed, and the qualities of MnO, as a selective absorbent.“) the filtered MnO, was analyaed for trapped radionuclides. It retained less than 0.5X of the Na. Ca. Me and AL with areater than 99% retention of Mn and I.‘Later tests under iimilar conditions showed retention of less than 1% of Zn, 7% of Cu, 20% of CL and 60% of Fe. The speed with which this procedure can be carried out is its main advantage. In various tests, “V and ‘*Ti radionuclides have been observed, but not quantified. ‘*Al and
“Zn have been measured quantitatively with modification of the separation procedure. In addition to those elements which can only be assayed by use of their short-lived activation products, elements such as cobalt and copper can be assayed without long irradiations by using their short-lived products. Iron analysis, using 57Fe(n,p)s7 Mn, and and iodine analysis are possible because of the quantitative retention of their activation products on the MnO, formed during dissolution. Through increases in sensitivity using larger detectors, the method may be applied to the analysis of very small samples. This will be clinically useful for the analysis.of small tissue biopsies, with detection of such physiologically significant elements as calcium and magnesium at the level of tenths of micrograms. References 1. ROUTTI J. T. and Paussm S. G. Nucl. Instrum. Meth. 72, 125 (1969). 2. GIIWDI F., Pmraa R. and SABBIONI E. J. radioanalyt. Chem. 5, 141 (1970).