Instrumental radioactivation analysis of selenium in biological materials

International Journal of Applied Radiation and Isotopes, 1967, Vol. 18, pp. 153-159.

Instrumental Selenium

Pergamon PressLtd. Printed in NorthernIreland

Radioactivation Analysis in Biological Materials and

R. C. DICKSON McMaster

University,

R.

of

H. TOMLINSON

Hamilton,

Ontario,

Canada

(Receiwd 10 Auglut 1966) A correction procedure has been established for the instrumental radioactivation analysis of selenium in dialyzed biological materials, by means of the 19-set isotope Se77nh. Based on the oxygen content of samples, the procedure has enabled a more accurate and sensitive determination than previous methods utilizing Se “* for analysis of selenium in such a matrix. The amounts ofselenium bound to protein in various fractions of human blood and other tissues were investigated using the technique. L’ANALYSE

INSTRUMENTALE DANS

LES

A RADIOACTIVATION MATERIAUX

DU

SELENIUM

BIOLOGIQUES

On a Ctabli un procedt de correction pour l’analyse instrumentale a radioactivation dustlenium dans les mattriaux biologiques dialyzes, utilisant l’isotope “%e de 19 sec. Base sur le contenu en oxygene des Cchantillons, le procede a permis un dosage plus exact et plus sensible que les mtthodes precedentes qui utilisaient le 77mSe pour le dosage du selenium dans une telle matrice. On rechercha les quantites de selenium likes a la prottine dans de diverses fractions de sang humain et d’autres tissus en employant la mtthode. AHAJIIBB

CEJIEHA

B

METOAOM BLtna

ycTauosneHa

ucnpasneuuan

EBOJIOI’II4ECWlX

MATEPMAJIAX

PAAIIOAKTIIBA~IIM MeTOAHKa ~na

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cejleria

c rtoM0tqbto

paAt40-

aKT%iBaqHH B AHaJlH3OBaHHLIX 6uonoruqecKux MaTepEaJlaX, IIOCpeACTBOM IIpkIMeHeHIlcI H30Tona Se”m c nepuoAoM nonypacnwa 19 ceK. OCHOB3HHaR Ha COAepmaHIlH KHCJIOpOA3 B o6pasqax, MeTOAHKa AaeT BO3MO?KHOCTb 6onee TOYHOPO II YYBCTBHTeJIbHOPO OIlpe~e.ileH~H CeJIeHa B 3THX MaTepHaJlaX,=leM IIpeAbIAyqHe MeTOAbI, OCHOBaHHbfe Ha IlpHMeHeHHH S0"". KonmecTBoceneHa,cm3aHHoro c 6enKaMa B pa3nwnmx~paK~mxuenoBesecKoti K~~BH EI ApJ'I'HXTKaHHX, 6~10 ElCCJleAOBaHO llCIIOJlb3JW3TyMeTOJJHKY.

INSTRUMENTELLE

RADIOAKTIVIERUNGSANALYSE BIOLOGISCHEN

VON

SELEN

IN

STOFFEN

Es wurde ein Korrektionsverfahren ausgearbeitet fur die instrumentelle Radioaktivierungsanalyse von Selen in dialysierten biologischen Stoffen mit Hilfe des 19 s-Isotops Se77m. Das auf dem Sauerstoffgehalt der Proben beruhende Verfahren hat eine genauere und empfindlichere Bestimmung ermiiglicht als friihere Methoden, die Se 77m fur die Selen Analyse in solch einem Mutterstoff verwenden. Es wurden die an Protein in verschiedenen Fraktionen von menschlichem Blut und anderen Geweben gebundenen Selenmengen unter Verwendung dieses Verfahrens untersucht. 153

154

R. C. Dickson and R. H. Tomlinson 1.

INTRODUCTION

RADIOACTIVATICN analysis for selenium in various matrices has been described in the literature.‘1-4) Analysis can be carried out by making use of the prodnction of a radionuclide from one of several stable isotopes. Table 1 summarizes the nuclear characteristics of selenium relevant to activation with thermal neutrons.(l) ? he nuclear reactions occurring upon bombardment of selenium with thermal neutrons,

1. Nuclear data for thermal neutron activation of selenium

TABLE

Target nuclide

Abundance %

SE’4

Se76 Se78 Seso

0.87

Isotopic activation cross section (barns) __~ ~___ 26 + G

9.02 23,52 49.82

Sea2

with rapid radiochemical separations, to determine the selenium content of blood and other biological material.t2) Quantities of selenium as small as 5 x 10es g were determined with an accuracy of -&5 per cent. Sesl has not been extensively used because of difficulties associated with counting the nuclide, which is virtually a pure beta emitter, Analysis for selenium utilizing the very shortlived species Se 77m has been carried out instrumentally on raw sulphur, where little interfering

Product on thermal neutron irradiation Se’”

&77in

713

se79m

0.03 & 0.01 0.5 -+ 0.1 0.050 & 0.025

9.19

G&31??&

Se*l Ses3 se=

which can lead to the production with high specific activity, are:t2)

of nuclides

Se74(n, y)Se75

T& = 120 days

Se76(n, y)Se””

TJ

Se*O(,, y)Se*l

TQ = 18.6 min

= 18.8 sect5)

Since Se76 has a half-life more than adequate to allow post-irradiation radiochemistry and separation from all other activities prior to counting, this nuclide has been most commonly used for activation analysis of selenium.(1*3) Most analysts, however, have irradiated samples for only a small fraction of the time required to achieve saturation, and thus have incurred losses of sensitivity. Irradiation for 12 days in a flux of 1Ol2 neutrons/cm2/sec and subsequent radiochemical isolation of Se75 enabled the analysis of platinum, for amounts of selenium as low as 2 x 10-s g.(l) SeB1 was used in conjunction

Radiation and energy (MeV)

Half-life

~‘0.265, 0.136, 121 da) 0.280, 0.402, others IT e- y 0.162 (QIT) 17.5 set 3.91 min IT 0.096 (QIT) 56.8 min IT e- (y) 0.103 (Q) 18.2 min B- 1.38 (Q) B_ 3.4; y 1.01, 2.02 70 set 0.65, O-35 /Y 1.5; y 0.35 25 min (both Ses3 radionuclides decay independently to yield Br83, T42.3 hr) EC;

radioactivity was produced.‘**@ Although the sensitivity of these analyses was estimated to be low2 ppm of selenium, the reproducibility of results was only f 10 per cent on amounts of the order of a few pg. The short length of time available after irradiation does not readily permit separations to be carried out, and attempts toperformdirect instrumental selenium analyses on biological materials using Se”“’ have shown that the production of Ols activity (T* = 29 set) limits the accuracy and sensitivity of the technique.(‘*s) WAINERDI and coworkers”) estimated the limit of sensitivity of the analysis for selenium in 1 g liver samples to be 0.15 pug, using a flux of 1.2 X 1012 neutrons/ cm2/sec. The reproducibility of analyses, however, due to the oxygen interference, was only f20 per cent. This is unfortunate in that instrumental activation analysis using such a short-lived species, is very rapid.

Instrumental radioactivation analysis of selenium in biological materials The present work was undertaken in an attempt to improve the accuracy and sensitivity of the instrumental method to a degree which would allow significant intercomparison of analyses for selenium at the level at which it occurs in reqonably-sized samples of normal human tissue. 2. METHODS 2.1

Preparation of samples for analysis

All blood and tissue samples were dialyzed in deionized water at 4”C, until a flame test for sodium in the dialysis bath was negative. Each sample was then transferred to a polyethylene beaker lined with 25 gauge Mylar film Type C (Du Pont of Canada Ltd., Montreal, Quebec) and dried at 80”~90°C in a drying oven. Drying of samples was continued until no further moisture could be driven off at the temperature used. The samples were then wrapped in the minimum possible amount of Mylar film and transferred to No. 25 Nalgene polyethylene capsules (The Nalge Co., Rochester, New York, U.S.A.) for irradiation. 2.2

Standards

Desiccated powdered selenium (99-100 per cent) was dissolved in 1:2 H,O-concentrated HNOs, and diluted using pipettes and volumetric flasks to yield standard solutions containing from 1 to 50 ,ug selenium/ml. Several series of selenium standards were prepared for irradiation by pipetting quantities of the standard solutions into Nalgene capsules, which were heat-sealed. Each series of standards contained the same quantity of selenium per capsule, but varying amounts of water.

was begun using a 3 x 3 in. NaI(T1) scintillation crystal and one half of the channels in a Victoreen Pip 400 channel y-ray spectrometer. The y-energy spectrum was observed from 0 to approx 250 kV. The numbers of counts in the Se”* photopeak at 0.16 MeV, and the Olg photopeak at 0.19 MeV were estimated by summing the top five channel counts and subtracting a background consisting of 2.5 times the sum of the first count on each side of the peak, lower than either of its neighbours. Each sample was irradiated twice in the manner described above (or three times if the results of the first two irradiations differed by greater than 4 per cent), and the mean was calculated. For each series of analyses, a plot was made of the logarithm of the selenium count versus the oxygen count for a series of selenium-water standards each containing the same quantity of selenium, and the slope of the straight line obtained, was determined. After irradiation of a tissue sample, a point representing the seIenium and oxygen counts obtained, was plotted on the graph and a line drawn through the point, parallel to that representing the seleniumoxygen standards, was extrapolated to zero selenium This gave the “corrected” oxygen. count for the sample, which was related to that from a standard containing selenium only, and the selenium in the sample was calculated. Although it was not essential in order to obtain selenium analyses, arbitrary cut-off limits were defined (Fig. 4) for the region over which correction plots remained linear, in order that computer calculation of results could be carried out. The extrapolation technique and other computations required, were performed by an IBM 7040 computer using a Fortran programme.

2.3 Irradiation and counting conditions Samples were irradiated in a flux of approximately 1013 neutrons/cms/sec using the 2 MW McMaster University light water-moderated reactor. A rapid-transit pneumatic “rabbit” system, and a specially-constructed quickopening rabbit irradiation vehicle enabled irradiations of short duration, with minimal delay between the end of irradiation and the start of counting of the sample. Each sample was irradiated for 20 sec. Thirty seconds after the end of irradiation, a 30-set live time count

155

3. RESULTS 3.1 Experimental

method

Figure 1 shows the results of pIotting the logarithm of the selenium count, versus the oxygen count,for a series ofstandards containing a fixed amount of selenium and varying amounts Each line represents such of oxygen (as water). a series, containing a different amount of selenium. Figure 2 shows a series of four spectra corresponding to a series as described above, with the Se”* photopeak at 0.16 MeV to the

156

R. C. Dickson and R. H. Ton&son

FIG. 1. Logarithm of the selenium count vs. the oxygen count for series of standards each containing a fixed amount of selenium and varying amounts of oxygen.

-----i -----._

0

8 000

OXYGEN

!6000 COUNT

24000

,

.

.

-_

FIG. 2. Four

spectra

corresponding

to four standards containing the same and varying amounts of oxygen.

* -

amount

of selenium

Instrumentalw&activation analysisof seleniumin biologicalmaterials

157

TABLE 2. Selenium analysis of identical blood samples containing different amounts of oxygen, using the correction procedure Selenium

Sample

in cells

Selenium

olg)

1

1.42

2 3 4 5 6 7 8 9 10

1.38

o-95 1.02

.-

1.39 1.46 1.39 1.46 1.46 l-39 1.45 l-48 1.43 0.04

Mean S.D.

0.94 0.96 0.97 0.96 0.98 O-91 0.97 0.96 0.03

S.D. 2.8% as per cent of mean

0

1.0

2.0 MICROGRAMS

3.0 OF SELENIUM

in plasma

(Pg)

3.1%

4.0

3. The “zero-oxygen” selenium count from the lines in Fig. 1, vs. the selenium content of the standards.

FIG.

left of the Ols photopeak at O-19 MeV, in each spectrum. This photograph was enlarged from a Polaroid print of an experimentally observed series of spectra. In Fig. 3 is shown a plot of the counts representing the SeTTrnphotopeaks given on irradiation by the selenium standards contributing the “zero-oxygen” points on the lines in Fig. 1, versus the actual selenium content of the standards. Figure 4 shows a plot of selenium-water standards and of a series of identical samples of blood cells and plasma which contain various amounts of oxygen (as water), and which after correction procedures described, yielded the data given in Table 2. In Table 3 are given the results of analyses for selenium added to series of samples of blood cells and plasma, identical except for oxygen content. 3.2 Analysis of blood and other human tissues Table 4 shows the results of selenium analyses of the cells and plasma from 15-ml samples of

0

CELLS

0

PLASMA

0

CUT-

I

OFF

1

-CA

I

I

d

-

I

I

I

0

CALIBRATION

8000

4000 OXYGEN

12000

COUNT

FIG. 4. Logarithm of the selenium count vs. the oxygen count for series of identical samples of blood cells and plasma containing various amounts of oxygen.

158

R. C. Dickson and R. H. Tomlinron TABLE

Analysis

Selenium standard added (E!J

Total selenium determined (G)

-%dded selenium determined

Mean of ten samples 1 2 1 2 1 2

6.44 6.44 6.44 6.44 6.44 6.44

1.43 7.96 7.92 7.89 7.77 7.83 7.75

6.53 6.49 646 6.34 6.40 6.32

Mean of nine samples 1 2 1 2 1 2

644 644 6.44 6.44 6.44 644

0.96 7.29 7.47 7.34 7.32 7.25 7.30

6.33 6.51 6.38 6.36 6.39 6.34

Sample No. Cells 1 Cells 2 Cells 3 Cells 4 Plasma 1 Plasma 2 Plasma 3 Plasma 4

3. Analysis for selenium added to identica1 blood samples

Mean Standard deviation Standard deviation (as per cent of mean) whole human blood drawn from 254 normal individuals. A study of the results of selenium analyses is being published. tB) This includes a break down of the selenium content of blood fractions by age, sex and hematocrit, and results of investigations of blood selenium level changes with time and in persons having selected diseases. 4. DISCUSSION 4.1 ExperimentuEmethod A procedure was empirically determined, whereby dialyzed samples of tissue could be TABLE

4. Selenium content of the blood of

254 normal individuals

Blood fraction Cells (from 15 ml whole blood) Plasma (from 15 ml whole blood) Whole blood (15 ml)

Mean selenium content (!%)

Standard deviation (I@)

1.52

0.37

1.21

0.24

2-73

0.55

(&

-

640 0.08 1.3yb

analyzed for selenium in a manner which did not require chemical separations after irradiation. The chief need for dialysis of tissue arose from sodium chloride present, which if allowed to remain during irradiation, led to high activity which obscured the contribution from minor constituents. However, dialysis prior to irradiation also enabled the determination of selenium bound to tissue proteins. All samples contained a residuum of oxygen, even when dried as thoroughly as possible at the temperatures used, and thus necessitated the use of the correction procedure. Analysis of identical blood samples dried for prolonged periods of time, showed that no selenium was lost by volatilization during drying. The reaction Se76(n, y)Se”* has been shown to be the principal nuclear reaction leading to the production of Se77”’ on bombardment of Other selenium with thermal neutrons.(lO) possible modes of productionof the isotope might include Brgs(n,p)Se’7m or Krso(n, a)Se”“. Brs9 is radioactive and could lead to Se”“’ only through a secondary reaction of low probability. Krso is not only of low abundance in biological material, but the cross-sections for n, Mreactions with nuclides in this mass region, are very small.

Instrumental radioactivation analysis

The necessity for correction of instrumentally observed selenium counts apparently derives from the fact that the live time of counting equipment used, varies appreciably over the counting period, which itself extends over more than a half-life of both 019 and Se”“’ which are the main contributors to dead time. Attempts to derive the formof the correction mathematically, resulted in an integral which was soluble only by numerical methods. No solution to this problem was found in the literature. As may be seen from the data in Tables 2 and 3 the correction procedure devised, enabled the determination of selenium in blood samples with a precision of 13 per cent and an accuracy of & l-2 per cent. These figures were calculated from the standard deviation from the mean of the results of anaIyses of identical samples, and samples to which selenium was added, respectively. The sensitivity of the method was estimated from the range over which correction piots remained linear, to be about 0-I pg of Quantities of selenium as small as selenium. O-01 rug could be determined without the aid of computer calculations, with a slight sacrifice in accuracy. This level of sensitivity represented an approximately fifty-fold improvement over the limit of 0.5 ,ug estimated by GUINN, for the instrumental activation analysis of selenium in biological materials, using Se”“’ and a flux comparable to that used in the present studies.ll Quantities of selenium as small as O-005 pug were detectable. It must be admitted that activation analysis for selenium utilizing the longer-lived nuclides previously mentioned, can yield greater sensitivity and accuracy (for these smaller amounts) with the use of a flux comparable to that used in the present work. However, the correction method developed is sufficiently sensitive for analysis of selenium in the majority of biological specimens, and enables determinations to be carried out much more quickly and easily than methods requiring radiochemical separations. The average time for analysis of prepared samples was less than 5 min per sample, including

of selenium

in biological materials

irradiation, counting and computation computer programme.

159

using the

4.2 Analysis of tissue samples The level of accuracy achieved by the correction procedure enabled the meaningful intercomparison of the results of analyses for selenium in human tissues. The method is applicable to other biological materials containing bound selenium in amounts within the limits discussed above. 5. CONCLUSIONS The sensitivity and accuracy provided by the correction method developed, represented a significant improvement over these formerly obtained.(7*11) The procedure enabled the analysis of dialyzed tissue samples for bound selenium in microgram quantities, and allowed meaningful intercomparison of results. authors wish to acknowledge financial support from the National Research Council of Canada.

Acknowledgments-The

REFERENCES 1. MORRIS D. F. C. and KILLICK R. A. Talanta, 10, 279 (1963). 2. BOWEN H. J. M. and CAWSE P. A. Analyst 88, 721 (1963). 3. ERION W. E., Morr W. E. and SHEDLOVSKY.J. P. Trans. Am, nucl. Sot. 3,252 (1960). 4. OKADA M. Tokyo Kogyo Shikensho Hokoku 58, 11 (1963). 5. MALMSKOG S.and KONIJNJ. Nucl. Phys. 38, 196 (1962). 6. OKADA M. Nature Lond. 187, 594 (1960). 7. WAINERDI R. E., MENON M. P. and FITE L. E.

TEES-2671-4,

IX-13

(1965).

8. ALLAWAY W. H. and CARY E. E. Analyt. Chem. 36, 1359 (1964). 9. DICKSONR. C. and TOMLINSONR. H. Clin. Chim. Acta (in press). 10. KRAMER H. H. and WAHL W. H. Nrtcl.Sci. Engng. 22, 373 (1965). 11. GUINN V. P. L’AnaCyse par Radioactivation et ses A@lications aux Sciences Biologiques, p, 74. Presses Universitaires de France, Paris (1964).