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An improved method for the simultaneous determination of iron-55 and iron-59 in blood by liquid scintillation counting
International Journal of Applied Radiation and Isotopes, 1966, Vol. 17, pp. 391-397.
Pergamon Press Ltd. Printed in Northern Ireland
An Improved Method for the Simultaneous Determination of Iron-55 And Iron-59 In Blood by Liquid Scintillation Counting J. Health
Physics
D.
EAKINS
and Medical
and D. A. BROWN
Division,
A.E.R.E.,
Harwell,
Didcot,
Berks
(First received 22 December 1965 and inJina1form 11 January 1966) After A method for the simultaneous determination of Fe55 and Fe5g in blood is described. wet oxidation of the blood sample the iron is precipitated as ferric hydroxide. It is then converted to an insoluble white ferriphosphate complex by dissolving the hydroxide in phosphoric acid and adding a solution of ammonium chloride in absolute ethanol. The resulting precipitate is counted as a gel in a two-channel, liquid-scintillation spectrometer, set to count Fesg alone in one channel and Fess plus a small percentage of Fe5g in the other channel. The counting efficiencies obtained are 19.4 per cent for Fe55 and 33.4 per cent for Fe5g with chemical yields of 95 per cent. The counting efficiency for Fe55 is greater by a factor 3 than the best value previously reported for liquid scintillation counting, with sources containing comparable amounts of inactive iron. The nature of the ferriphosphate complex is discussed. The results of an analysis of the compound suggest that it is an ammonium salt of a ferriphosphoric acid with formula NH,H,[Fe(PO,)s]2H,O. The procedure described may be used for the analysis of materials other than blood. currently being used for the determination of Fe55 and Fe5g in bread samples doubly labelled these isotopes. UNE Fe-55
METHODE ET
DU
AMELIOREE Fe-59
DANS DE
LE
POUR
LE
DOSAGE
SANG
AU
MOYEN
SCINTILLATION
SIMULTANE DU
It is with
DLI
COMPTAGE
LIQUIDE
On dtcrit une methode pour le dosage simultane du Fe55 et du Fr5g dans le sang. Suivant l’oxidation mouillte de l’echantillon de sang le fer est precipite comme hydroxyde ferrique. 11 est en suite converti en un complexe blanc et insoluble de ferriphosphate par la dissolution de l’hydroxyde dans de I’acide phosphorique et par l’addition d’une solution de chlorure d’ammonium en ethanol absolu. Le precipite ainsi forme est compte, en Ctat de gel, dans un spectromttre de scintillation liquide a deux cannelures, ajuste a compter le Fe5g seul dans une cannelure et le Fe55 avec une petite proportion pour cent de Fr5g dans l’autre cannelure. Les efficacites de compte obtenues sont 19,4 pour cent pour le Fe55 et 33,4 pour cent pour le Fe59 avec des rendements chimiques de 95 pour cent. L’efficacitt de comptage pour le Fe55 est suptrieur par un facteur de 3 que la meilleure valeur prtalablement repartee pour le comptage de scintillation liquide, avec des sources qui contiennent des ‘quantites cornparables de fer instinctif. On discute la nature du complexe de ferriphosphate. Les resultats d’une analyse du compose indiquent que ce serait un se1 d’ammonium d’un acide ferriphosphorique ayant la formule
NJWWFeWd212
. 40.
Le procede ici decrit peut servir a l’analyse des materiaux autres que le sang. 11 sert a present au dosage du Fe55 et du Fe5g dans des Cchantillons de pain marques en double facon de ces isotopes. 391
392
J. D. Eakins
C)nncaHnwii LiOCTOHHHO
npoqrcc
MOHieT
WXiO;rb:l~C’TCFi
IIcnoJn~3oBaIi IIc’
Yr)I,ITL
UJiff
and D. A. Brown
Oii~l~~CXOHiLH
1 Tx55, t.
.Fe5”
TOJll~IiO
u
;I;IH
o@a:3qax
aHamlS01~
xac+a.
Icponri.
( )I1
;iI3o~fFo vwf2~IIII~Ix
3Tmm Li:JoTonanILI. EI?;E
VERBESSERTE
BESTIMMUNG
METHODE VON
Fe-55
FiiR
UND
DIE
Fe-59
SZINTILLXTIONSZ.&HLEN
DER
GLEICHZEITIGE
IN
BLUT
DURCH
FLUSSIGKEI’T
Eine Methodc zur gleichzeitigen Bestimmung von Fe”j und Fe”” in Blut wird beschriebcn. Nach nasser Oxydation der Blutprobe wird das Eisen als Ferrihydroxyd ausgefkllt. Es wird dann in eine weisse liisliche FerriphosphatVerbindung umgcwandelt durch Liisung des Hydroxyds in Phosphor&me und durch Zugabe von in absolutem Athylalkohol geliistcm .L\mmoniumchlorid. Der crzielte Niederschlag wird als ein Gel in einem Zweikanal-Szintillationsspektromcter fiir Fliissigkeiten ausgez2ihlt, wobei in eincm Kanal die Zghlung van Fe5” allein erfolgt und in dem anderen die von Fess zuztiglich eines kleinen Teils des Fc”“. Die ZBhlausbeute ist fiir Fe5j dreimal griisser als der bestc jemals fdr Fltissigkeit-Szintillationszghlung gemeldete W’ert, wenn die Quellen vergleichbarc Mengen an instinktivem Eiscn enthalten. Die Beschaffcnheit der Ferriphosphat-Verbindung wird dann besprochen. Die Ergebnisse einer Analyse der Verbindung deuten an, dass es ein Ammoniumsalz einer Ferriphosphatstiurc mit der Formel NH,H,[Fe(P0,)2]2 H,O ist. Das beschriebene \‘erfahren kann such fiir die .jnalyse von anderen Substanzen als Blut verwendet werden. Es wird zur Zeit beniitzt fiir die Bestimmung von Fe”j und FesY in Brotproben, die mit diesen Isotopen zweifach markiert sind.
1. INTRODUCTION RADIOISOTOPES years
in studies
disorders. depend
of iron
The upon
absorption
and even
uptake
of different
it is convenient so that vestigated control.
the
of iron form
when
and
is known
given
When
uptake
may be Fe55 and
usually
employed
readily
be
detected
of the
by
such
forms
substance
compared Fesp are in
from
tracer
to it is
in the same day to
investigating
chemical
to use double
blood
in which
may vary considerably
day in the same subject. absorption
absorption
the
of iron,
techniques, to be in-
with that of a the two isotopes studies.
means
Fe5g
can
of its energetic
(E,,,O.27 by
and
(1 .l
y-rays
been used for many
the chemical
administered form,
of iron have
1.3
and 046
electron
capture,
manganese
X-rays
fluorescence
yield
much
MeV)
more
emitting of
0.0059
as a constituent
a number
of workers
methods
step to obtain differ the
only Fe”5
HAL.LBERG
Mev
25 per
is found
the blood,
These
with
cent,
a
and
predominately
of haemoglobin,
is
have
in and
described
methods
of Fess and Fejg
in blood.
consist
of an
initial
the iron in an inorganic
in the technique X-rays.
decays
characteristic
to detect.
iron
for the determination
however,
Fti5
of only
difficult
Metabolized
by its /3-emission
or
Me\‘).
PEACOCK
oxidation form,
and
used for detecting and
EVANS”)
and BRISE(*) use electrodeposition
and of
The simultaneous
determination
of iron-55
and iron-59
the iron and subsequent counting of the X-rays by Geiger-;Lluller methods. DERN and HART(~) use electrodeposition of the iron followed by dissolution and liquid scintillation counting. A more recent technique, developed by PERRY and WARNER@) eliminates the electrodeposition step, the iron being counted as a colourless complex in a mixture of ascorbic acid and hydrochloric acid by a liquid scintillation method. In the above methods a common factor is the low counting efficiency obtained for Fe”“. Geiger-Muller counting of X-rays gives efficiencies of about 5 per cent. The liquid scintillation method of DERN and HART has an efficiency of about 7 per cent for sources containing 5 mg of inactive iron, but the precedure is rather lengthy. The technique developed by PERRY and LVARNER is more rapid but the counting efficiency for Fess is only 2 per cent. However, the liquid scintillation technique has much to recommend it in terms of simplicity and ease of sample preparation. Normally there is considerable quenching of low energy p-emitters when aqueous solutions are added to liquid scintillation systems. The present authors favour the counting technique in which a precipitate containing the active material is held in suspension in the scintillator by a thixotropic silica gel. This method has the advantage of enabling more of the sample to be introduced into the cell, and ifit can be obtained as a pure white precipitate, quenching is often less severe than with conventional liquid scintillation counting. This paper describes a method for obtaining a pure white compound of iron which enables good counting efficiencies to be obtained for Fe55 and Fe5g and permits the two isotopes to be counted simultaneously using a Packard Tricarb 3214 series liquid scintillation spectrometer. A procedure is given for preparing this white compound from whole blood as a means of determining its content of Fe5j and Fe5g in double tracer experiments. The work has been described more fully elsewhere.(j) 2. PREPARATION COMPOUND
OF OF
A WHITE IRON
Few white compounds ofiron are known. The arsenide (FeAs) is reported to be white, but did
in blood by liquid scintillation
393
counting
not seem a suitable material for this application. Ferric phosphate is yellowish white, its colour varying according to the way it is prepared, probably depending on the extent of hydrolysis. TORIBARA et a1.t6) describe the use of phosphoric acid to form a colourless complex of iron in the liquid scintillation counting of Pu’~~. If a solution of ferric hydroxide in phosphoric acid is added to NE 220 liquid scintillator* (the scintillator used in these experiments) the iron is precipitated as a white solid. This is not a suitable procedure for counting Fess as the excess phosphoric acid produces considerable ferric hydroxide is quenching. If however, dissolved in concentrated phosphoric acid, and then absolute ethanol containing a small quantity of ammonium chloride is added with stirring, a pure white precipitate is obtained. This may be centrifuged off, washed with ethanol and is then in a suitable form for gel counting. A convenient quantity of the compound may be made from 5 mg of iron, and in order to prepare calibration sources for counting the following procedure was adopted, using ferric ammonium sulphate as the starting material. The iron is precipitated as ferric hydroxide by the addition of ammonia and centrifuged. The precipitate is dissolved in hydrochloric acid and the iron reprecipitated with ammonia. After centrifuging, the precipitate is washed with water and dissolved in 0.5 ml of concentrated phosphoric acid. 10 ml of absolute ethanol containing 0.5 mg/ml of ammonium chloride is added with thorough stirring, precipitating the iron as the white compound. This is then centrifuged and washed with 10 ml of absolute ethanol. 3 COMPOSITION COMPOUND
OF OF
THE
WHITE
IRON
The compound is a white amorphous powder which shows no crystalline form even at a magnification of 1300. It is almost insoluble in cold water, but water to which the substance has been added gives an acid reaction. In boiling water the compound turns yellow and the solution becomes strongly acid. In alkaline * NE 220 liquid scintillator is a dioxane-based scintillator manufactured by Nuclear Enterprises (G.B.) Ltd., Sighthill, Edinburgh, 11, Scotland.
J. D. Eakins and D. A. Brown
394
solution, particularly on heating, decomposition is rapid and the iron appears to be deposited as ferric hydroxide. It is only slightly soluble in 0.1 N hydrochloric acid, but dissolves readily in 1-O N acid, giving a greenish yellow solution. A sample of the compound was analysed, with the results shown in Table 1. These figures give ratios for Fe:PO,:NH,:H,O:H of 1:2: 1:2:2. WIENLAND and ENSGRABER”) studied the complexes ofiron with phosphoric acid and prepared salts of the ferriphosphoric acids Hs[Fe(PO,),] and H,[Fe(PO,)s]. Two mono-ammonium salts of the former acid, diphosphatoferric acid, were obtained, NH,HJFe(PO,),]7H,O and the anhydrous salt. These, although prepared by a different process (from aqueous solution either after prolonged heating or standing for 6 months) were of similar composition and physical characteristics to the compound obtained. From the evidence available it would seem therefore that the compound is probably an ammonium salt of a complex ferriphosphoric acid and the formula NH,H,[Fe(P0,),]2Hz0 is suggested. The analytical results in Table 1 are compared with the percentage composition of this complex and are in reasonable agreement. 4. PREPARATION COMPOUND FROM 4.1
OF THE WHOLE BLOOD
Isolation of the compound
Blood from human males normally contains from 44 to 56 mg of iron per 100 ml.(s) 10 ml was therefore taken as a convenient volume for analysis, containing about 5 mg of iron. The blood is wet oxidized to obtain the iron in an inorganic form and the complex prepared from ferric hydroxide precipitated from the oxidized 1. Comparison oftheoretical composition of a complex of formula NH,H,[Fe(PO,) ,]ZH,O with results obtained by analysis
TABLE
Analysis
Theoretical
(9{,)
Found (96)
PO, H@ Acidic H
6.0 18.5 62.9 11.9 0.7
5.3 17.8 63.8 11.3 0.7
Total
100.0
98.9
NH, Fe
sample. The procedure is described fully below. Place the aliquot ofblood in a 100 ml Kjeldahl flask and evaporate to dryness under an infrared lamp. Cool and add 4 ml of sulphuric acid (Sp. gr. 1.84) and5mlofnitricacid (Sp. gr. 1.42). Heat the flask on a mantle until brown fumes cease to be evolved and white sulphur trioxide fumes are seen. Allow the flask to cool and add 5 ml of nitric acid (Sp. gr. 1.42). Heat again until white fumes are evolved. Cool the flask and repeat this step. Add 1 ml of perchloric acid (Sp. gr. 1.70) and heat for 15 min. A whitish precipitate is sometimes seen on cooling, probably of ferric sulphate which is insoluble in sulphuric acid. This is dissolved by the addition of 10 ml of 1 M hydrochloric acid and heating to boiling. Cool and transfer the contents of the flask to a centrifuge tube, washing the flask three times with 3 ml aliquots of water. Add 10 M ammonia solution slowly with stirring until a permanent precipitate of ferric hydroxide is obtained, then add 1 ml in excess. Centrifuge and discard the supernate. Dissolve the precipitate in 1 ml of hydrochloric acid (Sp. gr. 1.18) and dilute to 10 ml with water. Reprecipitate the ferric hydroxide by the addition of 10 M ammonia solution and centrifuge off. Wash the precipitate with 10 ml of water. Centrifuge and discard the wash. Dissolve the ferric hydroxide in 0.5 ml of orthophosphoric acid (Sp. gr. 1.75). Add 10 ml of 0.01 M ammonium chloride solution in absolute ethanol slowly with stirring. Centrifuge off the white ferriphosphate complex and discard the supernate. Wash the precipitate with 10 ml of absolute ethanol. Centrifuge and discard the wash. 4.2 Preparation of the compoundfor
counting
of about ‘Aerosil’ silica *, at a concentration 8 per cent (w/v) is used to produce a suitable gel with the iron compound and liquid scintillator. In practice it is found that if the standard glass vials supplied by Packard (20 ml capacity) are filled to the top with ‘aerosil’ after gentle * ‘Aerosil’ standard silica powder is supplied by Bush Beach and Segner Bayley Ltd., Marlow House, Lloyds ;\venue, London, E.C.3.
The simultaneous
determination
of iron-55
and iron-59
tapping to settle the powder, then a satisfactory gel is obtained by the addition of 12 ml of liquid scintillator. The complex is transferred to the vial containing ‘aerosil’ by slurrying the precipitate with aliquots of liquid scintillator and pipetting the slurry into the vial. The vial is shaken thoroughly to obtain thorough mixing and then the contents are swirled gently to assist removal of air bubbles from the gel. The sample is then ready for counting. 5. SELECTION OF OPTIMUM COUNTING CONDITIONS FOR THE SIMULTANEOUS DETERMINATION OF Fe55 AND Fe5g The Packard Tricarb 32 14 liquid scintillation spectrometer is a two channel instrument. It was set up to bring the Fe5g spectrum well to the left of the gain scan and gain curves were obtained for both isotopes. These are shown in Fig. 1. With a gain of 12.5 per cent and a window width of 400-1000, Fe5g was counted
PER
in blood by liquid scintillation
counting
395
free from Fe55 in one channel. Fe55 was counted in the other channel at 100 per cent gain and with a window width of 50-400. Under these conditions the Fe5g contribution in this channel was reduced to 19 per cent. 6. RESULTS
6.1 Measurements
of background
Measurements of the background were made in both channels using phosphate complexes prepared from ferric ammonium sulphate and from whole blood. As shown in Table 2 the background counting rate obtained in the Fe5g channel was the same in each case, but higher values were obtained for the background in the Fe55 channel when the starting material was whole blood. Although the difference is barely significant it corresponds to a level of Fe55 in blood of 0.75 nCi/l. PALMER and BEASLEY'~' have detected similar levels of Fe55 (0.96 nCi/l.) in blood from residents of Richland, Washington
CENT
GAIN
FIG.1. Gain scan of Fe55 and Fe5g with full window 50-1000.
J. D. Eakins and D. A. Brom
396
2. Background measurements on the ferric phosphate complex
TABLE
Background Source of iron
Ferric ammonium sulphate Whole blood and
attribute
weapons
(countslmin)
F"5
F59
channel
channel
14.6 I- 0.7 17.6 $ 2.1
10.6 + 0.4 10.6 & 0.7
this
to
fallout
from
nuclear
testing.
6.2 Counting eficiencies obtained from Jamples spiked with Fe5s and Fe5s
whole
blood
Sources were prepared from six blood samples spiked with both Fes5 and Fesg. The sources were counted by liquid scintillation counting and also by y-counting the Fejg in order to determine the radiometric recovery. The counting efficiencies were corrected for radiometric yield and the results obtained are presented in Table 3. 6.3
The quenching effect of di$erent amounts of iron
As 10 ml samples of blood are taken for analysis, the total iron content of the sample may vary between 4.4 and 5.6 mg.(*) If differences in the iron content of samples in this range produce a significant effect on quenching then it will be necessary to determine the inactive iron content of the blood and adjust to a constant amount before carrying out the radiochemical analysis. In order to determine the severity of quenching effects over the range concerned, six sources TABLE
were prepared from samples containing various amounts of iron as ferric ammonium sulphate, spiked with both Fess and Fesg. The sources were counted in the liquid scintillation counter and the overall efficiencies determined. The radiometric yields were obtained by y-counting the Fe5g and the corrected liquid scintillation counting efficiencies and inactive iron contents of the sources calculated. The corrected liquid scintillation counting efficiencies are plotted in Fig. 2 against the actual weight of inactive irorl in the sample. As can be seen there is no serious change in quenching over the range of iron normally present in the blood. 6.4 The minimum detectable actioify of Fe55 and Fe”” in blood The minimum detectable activity is often expressed as that activity which will give a count rate equal to twice the standard deviation on the background counting rate. Using the mean backgrounds obtained in Table 2, the efficiencies shown in Table 3 and a 100 min counting period, the following minimum detectable levels are obtained: MDA for Fe”” = 2.1 % lo-’ $X/ml MDA for Fess = 9.0 x 10-s ,uCi/ml It should be noted however that these figures only apply if the isotopes are counted separately. If both are present in the same sample, although the figure for Fesg remains the same, the minimum detectable level for Fe”” rises with the amount of Fe5g present, as the effective background is increased by the contribution of the Fejg to the Fc”s count rate.
3. Counting efficiencies obtained from whole blood samples spiked with Fe”s and Fe””
Sample No.
Radiometric yield by y-counting (” “)
Corrected Fes5 efficiency (Oil)
1 2 3 4 5 6
97.0 92.0 92.3 98.5 95.7 97.5
18.7 19.3 19.9 19.2 18.8 20.3
32.6 32.5 34.5 33.7 33.0 33.9
95.5 & 2.7
19.4 _I 0.6
33.4 1 0.8
Mean
and SD
Corrected efficiency
Fe””
(Oni
The simultaneous determination of iron-55 50
‘; 2
and iron-59 in blood by liquid scintillation counting
397
r
40-
, 1
0
I 2
I 3 MG
FIG. 2. Quenching
7. SUMMARY
AND
OF
I 6
I 5
I 4 IRON
PER
effect of iron as phosphate counting efficiencies.
CONCLUSIONS
The work described in this report demonstrates that FeG5 and Fe5g may be measured simultaneously with good counting efficiencies by gel counting of the ferric phosphate complex in a Packard Tricarb 32 14 liquid scintillation specThe counting efficiency for Fe55 is trometer. increased by about a factor of 3 over previously reported liquid scintillation counting methods and this, combined with the inherently low background of the ‘Tricarb’ spectrometer, gives a minimum detectable level for FeS5 in blood of 0.2 pCi/ml. The method described for the measurement of FeS5 and Fe5g in blood may be used for determining these radionuclides in other materials. Currently measurements are being made on samples of bread which, after the addition of iron carrier, are analysed in the same way as blood samples.
I 7
I 0
, 3
I IO
SOURCE
complex on Fej5 and Fe5g
REFERENCES 1. PEACOCKW. C., EVANSR. D., IRVINEJ. W., GOOD W. N., KIP A. F., WEISS S. and GIBSONJ. G. J. clin. Inaest. 25, 605 (1946). 2. HALLBERC L. and BRISE H. Znt. J. a@$. Radiat. Isot0pe.r 9, 100 ( 1960). 3. DERN J. R. and HART W. L. J. Lab. clin. Med. 57, 322 (1961). 4. PERRY S. W. and WARNER G. T. Int. J. a@l. Radiat. Isotopes 14, 397 (1963). 5. EAKINSJ. D. and BROWND. ‘1. U.K. Atomic EnerD Authority Report AERE-R4945 (1965). C. 6. TORIBARAT. Y., MORKEND. A. and PREDMORE University of Rochester, New York, Report, UR 607 (1962). 7. ~VIENLANDR. F. and ENSGRABERF. Z. anorg. allg. Chem. 84, 340 (1914). 8. GEIGYJ. R. Documenta Gei, ScientiJic Tables. 5th Ed. Basle (1956). 9. PALMERH. E. and BEASLEYT. M. Science 149,43 1 (1965).
Pergamon Press Ltd. Printed in Northern Ireland
An Improved Method for the Simultaneous Determination of Iron-55 And Iron-59 In Blood by Liquid Scintillation Counting J. Health
Physics
D.
EAKINS
and Medical
and D. A. BROWN
Division,
A.E.R.E.,
Harwell,
Didcot,
Berks
(First received 22 December 1965 and inJina1form 11 January 1966) After A method for the simultaneous determination of Fe55 and Fe5g in blood is described. wet oxidation of the blood sample the iron is precipitated as ferric hydroxide. It is then converted to an insoluble white ferriphosphate complex by dissolving the hydroxide in phosphoric acid and adding a solution of ammonium chloride in absolute ethanol. The resulting precipitate is counted as a gel in a two-channel, liquid-scintillation spectrometer, set to count Fesg alone in one channel and Fess plus a small percentage of Fe5g in the other channel. The counting efficiencies obtained are 19.4 per cent for Fe55 and 33.4 per cent for Fe5g with chemical yields of 95 per cent. The counting efficiency for Fe55 is greater by a factor 3 than the best value previously reported for liquid scintillation counting, with sources containing comparable amounts of inactive iron. The nature of the ferriphosphate complex is discussed. The results of an analysis of the compound suggest that it is an ammonium salt of a ferriphosphoric acid with formula NH,H,[Fe(PO,)s]2H,O. The procedure described may be used for the analysis of materials other than blood. currently being used for the determination of Fe55 and Fe5g in bread samples doubly labelled these isotopes. UNE Fe-55
METHODE ET
DU
AMELIOREE Fe-59
DANS DE
LE
POUR
LE
DOSAGE
SANG
AU
MOYEN
SCINTILLATION
SIMULTANE DU
It is with
DLI
COMPTAGE
LIQUIDE
On dtcrit une methode pour le dosage simultane du Fe55 et du Fr5g dans le sang. Suivant l’oxidation mouillte de l’echantillon de sang le fer est precipite comme hydroxyde ferrique. 11 est en suite converti en un complexe blanc et insoluble de ferriphosphate par la dissolution de l’hydroxyde dans de I’acide phosphorique et par l’addition d’une solution de chlorure d’ammonium en ethanol absolu. Le precipite ainsi forme est compte, en Ctat de gel, dans un spectromttre de scintillation liquide a deux cannelures, ajuste a compter le Fe5g seul dans une cannelure et le Fe55 avec une petite proportion pour cent de Fr5g dans l’autre cannelure. Les efficacites de compte obtenues sont 19,4 pour cent pour le Fe55 et 33,4 pour cent pour le Fe59 avec des rendements chimiques de 95 pour cent. L’efficacitt de comptage pour le Fe55 est suptrieur par un facteur de 3 que la meilleure valeur prtalablement repartee pour le comptage de scintillation liquide, avec des sources qui contiennent des ‘quantites cornparables de fer instinctif. On discute la nature du complexe de ferriphosphate. Les resultats d’une analyse du compose indiquent que ce serait un se1 d’ammonium d’un acide ferriphosphorique ayant la formule
NJWWFeWd212
. 40.
Le procede ici decrit peut servir a l’analyse des materiaux autres que le sang. 11 sert a present au dosage du Fe55 et du Fe5g dans des Cchantillons de pain marques en double facon de ces isotopes. 391
392
J. D. Eakins
C)nncaHnwii LiOCTOHHHO
npoqrcc
MOHieT
WXiO;rb:l~C’TCFi
IIcnoJn~3oBaIi IIc’
Yr)I,ITL
UJiff
and D. A. Brown
Oii~l~~CXOHiLH
1 Tx55, t.
.Fe5”
TOJll~IiO
u
;I;IH
o@a:3qax
aHamlS01~
xac+a.
Icponri.
( )I1
;iI3o~fFo vwf2~IIII~Ix
3Tmm Li:JoTonanILI. EI?;E
VERBESSERTE
BESTIMMUNG
METHODE VON
Fe-55
FiiR
UND
DIE
Fe-59
SZINTILLXTIONSZ.&HLEN
DER
GLEICHZEITIGE
IN
BLUT
DURCH
FLUSSIGKEI’T
Eine Methodc zur gleichzeitigen Bestimmung von Fe”j und Fe”” in Blut wird beschriebcn. Nach nasser Oxydation der Blutprobe wird das Eisen als Ferrihydroxyd ausgefkllt. Es wird dann in eine weisse liisliche FerriphosphatVerbindung umgcwandelt durch Liisung des Hydroxyds in Phosphor&me und durch Zugabe von in absolutem Athylalkohol geliistcm .L\mmoniumchlorid. Der crzielte Niederschlag wird als ein Gel in einem Zweikanal-Szintillationsspektromcter fiir Fliissigkeiten ausgez2ihlt, wobei in eincm Kanal die Zghlung van Fe5” allein erfolgt und in dem anderen die von Fess zuztiglich eines kleinen Teils des Fc”“. Die ZBhlausbeute ist fiir Fe5j dreimal griisser als der bestc jemals fdr Fltissigkeit-Szintillationszghlung gemeldete W’ert, wenn die Quellen vergleichbarc Mengen an instinktivem Eiscn enthalten. Die Beschaffcnheit der Ferriphosphat-Verbindung wird dann besprochen. Die Ergebnisse einer Analyse der Verbindung deuten an, dass es ein Ammoniumsalz einer Ferriphosphatstiurc mit der Formel NH,H,[Fe(P0,)2]2 H,O ist. Das beschriebene \‘erfahren kann such fiir die .jnalyse von anderen Substanzen als Blut verwendet werden. Es wird zur Zeit beniitzt fiir die Bestimmung von Fe”j und FesY in Brotproben, die mit diesen Isotopen zweifach markiert sind.
1. INTRODUCTION RADIOISOTOPES years
in studies
disorders. depend
of iron
The upon
absorption
and even
uptake
of different
it is convenient so that vestigated control.
the
of iron form
when
and
is known
given
When
uptake
may be Fe55 and
usually
employed
readily
be
detected
of the
by
such
forms
substance
compared Fesp are in
from
tracer
to it is
in the same day to
investigating
chemical
to use double
blood
in which
may vary considerably
day in the same subject. absorption
absorption
the
of iron,
techniques, to be in-
with that of a the two isotopes studies.
means
Fe5g
can
of its energetic
(E,,,O.27 by
and
(1 .l
y-rays
been used for many
the chemical
administered form,
of iron have
1.3
and 046
electron
capture,
manganese
X-rays
fluorescence
yield
much
MeV)
more
emitting of
0.0059
as a constituent
a number
of workers
methods
step to obtain differ the
only Fe”5
HAL.LBERG
Mev
25 per
is found
the blood,
These
with
cent,
a
and
predominately
of haemoglobin,
is
have
in and
described
methods
of Fess and Fejg
in blood.
consist
of an
initial
the iron in an inorganic
in the technique X-rays.
decays
characteristic
to detect.
iron
for the determination
however,
Fti5
of only
difficult
Metabolized
by its /3-emission
or
Me\‘).
PEACOCK
oxidation form,
and
used for detecting and
EVANS”)
and BRISE(*) use electrodeposition
and of
The simultaneous
determination
of iron-55
and iron-59
the iron and subsequent counting of the X-rays by Geiger-;Lluller methods. DERN and HART(~) use electrodeposition of the iron followed by dissolution and liquid scintillation counting. A more recent technique, developed by PERRY and WARNER@) eliminates the electrodeposition step, the iron being counted as a colourless complex in a mixture of ascorbic acid and hydrochloric acid by a liquid scintillation method. In the above methods a common factor is the low counting efficiency obtained for Fe”“. Geiger-Muller counting of X-rays gives efficiencies of about 5 per cent. The liquid scintillation method of DERN and HART has an efficiency of about 7 per cent for sources containing 5 mg of inactive iron, but the precedure is rather lengthy. The technique developed by PERRY and LVARNER is more rapid but the counting efficiency for Fess is only 2 per cent. However, the liquid scintillation technique has much to recommend it in terms of simplicity and ease of sample preparation. Normally there is considerable quenching of low energy p-emitters when aqueous solutions are added to liquid scintillation systems. The present authors favour the counting technique in which a precipitate containing the active material is held in suspension in the scintillator by a thixotropic silica gel. This method has the advantage of enabling more of the sample to be introduced into the cell, and ifit can be obtained as a pure white precipitate, quenching is often less severe than with conventional liquid scintillation counting. This paper describes a method for obtaining a pure white compound of iron which enables good counting efficiencies to be obtained for Fe55 and Fe5g and permits the two isotopes to be counted simultaneously using a Packard Tricarb 3214 series liquid scintillation spectrometer. A procedure is given for preparing this white compound from whole blood as a means of determining its content of Fe5j and Fe5g in double tracer experiments. The work has been described more fully elsewhere.(j) 2. PREPARATION COMPOUND
OF OF
A WHITE IRON
Few white compounds ofiron are known. The arsenide (FeAs) is reported to be white, but did
in blood by liquid scintillation
393
counting
not seem a suitable material for this application. Ferric phosphate is yellowish white, its colour varying according to the way it is prepared, probably depending on the extent of hydrolysis. TORIBARA et a1.t6) describe the use of phosphoric acid to form a colourless complex of iron in the liquid scintillation counting of Pu’~~. If a solution of ferric hydroxide in phosphoric acid is added to NE 220 liquid scintillator* (the scintillator used in these experiments) the iron is precipitated as a white solid. This is not a suitable procedure for counting Fess as the excess phosphoric acid produces considerable ferric hydroxide is quenching. If however, dissolved in concentrated phosphoric acid, and then absolute ethanol containing a small quantity of ammonium chloride is added with stirring, a pure white precipitate is obtained. This may be centrifuged off, washed with ethanol and is then in a suitable form for gel counting. A convenient quantity of the compound may be made from 5 mg of iron, and in order to prepare calibration sources for counting the following procedure was adopted, using ferric ammonium sulphate as the starting material. The iron is precipitated as ferric hydroxide by the addition of ammonia and centrifuged. The precipitate is dissolved in hydrochloric acid and the iron reprecipitated with ammonia. After centrifuging, the precipitate is washed with water and dissolved in 0.5 ml of concentrated phosphoric acid. 10 ml of absolute ethanol containing 0.5 mg/ml of ammonium chloride is added with thorough stirring, precipitating the iron as the white compound. This is then centrifuged and washed with 10 ml of absolute ethanol. 3 COMPOSITION COMPOUND
OF OF
THE
WHITE
IRON
The compound is a white amorphous powder which shows no crystalline form even at a magnification of 1300. It is almost insoluble in cold water, but water to which the substance has been added gives an acid reaction. In boiling water the compound turns yellow and the solution becomes strongly acid. In alkaline * NE 220 liquid scintillator is a dioxane-based scintillator manufactured by Nuclear Enterprises (G.B.) Ltd., Sighthill, Edinburgh, 11, Scotland.
J. D. Eakins and D. A. Brown
394
solution, particularly on heating, decomposition is rapid and the iron appears to be deposited as ferric hydroxide. It is only slightly soluble in 0.1 N hydrochloric acid, but dissolves readily in 1-O N acid, giving a greenish yellow solution. A sample of the compound was analysed, with the results shown in Table 1. These figures give ratios for Fe:PO,:NH,:H,O:H of 1:2: 1:2:2. WIENLAND and ENSGRABER”) studied the complexes ofiron with phosphoric acid and prepared salts of the ferriphosphoric acids Hs[Fe(PO,),] and H,[Fe(PO,)s]. Two mono-ammonium salts of the former acid, diphosphatoferric acid, were obtained, NH,HJFe(PO,),]7H,O and the anhydrous salt. These, although prepared by a different process (from aqueous solution either after prolonged heating or standing for 6 months) were of similar composition and physical characteristics to the compound obtained. From the evidence available it would seem therefore that the compound is probably an ammonium salt of a complex ferriphosphoric acid and the formula NH,H,[Fe(P0,),]2Hz0 is suggested. The analytical results in Table 1 are compared with the percentage composition of this complex and are in reasonable agreement. 4. PREPARATION COMPOUND FROM 4.1
OF THE WHOLE BLOOD
Isolation of the compound
Blood from human males normally contains from 44 to 56 mg of iron per 100 ml.(s) 10 ml was therefore taken as a convenient volume for analysis, containing about 5 mg of iron. The blood is wet oxidized to obtain the iron in an inorganic form and the complex prepared from ferric hydroxide precipitated from the oxidized 1. Comparison oftheoretical composition of a complex of formula NH,H,[Fe(PO,) ,]ZH,O with results obtained by analysis
TABLE
Analysis
Theoretical
(9{,)
Found (96)
PO, H@ Acidic H
6.0 18.5 62.9 11.9 0.7
5.3 17.8 63.8 11.3 0.7
Total
100.0
98.9
NH, Fe
sample. The procedure is described fully below. Place the aliquot ofblood in a 100 ml Kjeldahl flask and evaporate to dryness under an infrared lamp. Cool and add 4 ml of sulphuric acid (Sp. gr. 1.84) and5mlofnitricacid (Sp. gr. 1.42). Heat the flask on a mantle until brown fumes cease to be evolved and white sulphur trioxide fumes are seen. Allow the flask to cool and add 5 ml of nitric acid (Sp. gr. 1.42). Heat again until white fumes are evolved. Cool the flask and repeat this step. Add 1 ml of perchloric acid (Sp. gr. 1.70) and heat for 15 min. A whitish precipitate is sometimes seen on cooling, probably of ferric sulphate which is insoluble in sulphuric acid. This is dissolved by the addition of 10 ml of 1 M hydrochloric acid and heating to boiling. Cool and transfer the contents of the flask to a centrifuge tube, washing the flask three times with 3 ml aliquots of water. Add 10 M ammonia solution slowly with stirring until a permanent precipitate of ferric hydroxide is obtained, then add 1 ml in excess. Centrifuge and discard the supernate. Dissolve the precipitate in 1 ml of hydrochloric acid (Sp. gr. 1.18) and dilute to 10 ml with water. Reprecipitate the ferric hydroxide by the addition of 10 M ammonia solution and centrifuge off. Wash the precipitate with 10 ml of water. Centrifuge and discard the wash. Dissolve the ferric hydroxide in 0.5 ml of orthophosphoric acid (Sp. gr. 1.75). Add 10 ml of 0.01 M ammonium chloride solution in absolute ethanol slowly with stirring. Centrifuge off the white ferriphosphate complex and discard the supernate. Wash the precipitate with 10 ml of absolute ethanol. Centrifuge and discard the wash. 4.2 Preparation of the compoundfor
counting
of about ‘Aerosil’ silica *, at a concentration 8 per cent (w/v) is used to produce a suitable gel with the iron compound and liquid scintillator. In practice it is found that if the standard glass vials supplied by Packard (20 ml capacity) are filled to the top with ‘aerosil’ after gentle * ‘Aerosil’ standard silica powder is supplied by Bush Beach and Segner Bayley Ltd., Marlow House, Lloyds ;\venue, London, E.C.3.
The simultaneous
determination
of iron-55
and iron-59
tapping to settle the powder, then a satisfactory gel is obtained by the addition of 12 ml of liquid scintillator. The complex is transferred to the vial containing ‘aerosil’ by slurrying the precipitate with aliquots of liquid scintillator and pipetting the slurry into the vial. The vial is shaken thoroughly to obtain thorough mixing and then the contents are swirled gently to assist removal of air bubbles from the gel. The sample is then ready for counting. 5. SELECTION OF OPTIMUM COUNTING CONDITIONS FOR THE SIMULTANEOUS DETERMINATION OF Fe55 AND Fe5g The Packard Tricarb 32 14 liquid scintillation spectrometer is a two channel instrument. It was set up to bring the Fe5g spectrum well to the left of the gain scan and gain curves were obtained for both isotopes. These are shown in Fig. 1. With a gain of 12.5 per cent and a window width of 400-1000, Fe5g was counted
PER
in blood by liquid scintillation
counting
395
free from Fe55 in one channel. Fe55 was counted in the other channel at 100 per cent gain and with a window width of 50-400. Under these conditions the Fe5g contribution in this channel was reduced to 19 per cent. 6. RESULTS
6.1 Measurements
of background
Measurements of the background were made in both channels using phosphate complexes prepared from ferric ammonium sulphate and from whole blood. As shown in Table 2 the background counting rate obtained in the Fe5g channel was the same in each case, but higher values were obtained for the background in the Fe55 channel when the starting material was whole blood. Although the difference is barely significant it corresponds to a level of Fe55 in blood of 0.75 nCi/l. PALMER and BEASLEY'~' have detected similar levels of Fe55 (0.96 nCi/l.) in blood from residents of Richland, Washington
CENT
GAIN
FIG.1. Gain scan of Fe55 and Fe5g with full window 50-1000.
J. D. Eakins and D. A. Brom
396
2. Background measurements on the ferric phosphate complex
TABLE
Background Source of iron
Ferric ammonium sulphate Whole blood and
attribute
weapons
(countslmin)
F"5
F59
channel
channel
14.6 I- 0.7 17.6 $ 2.1
10.6 + 0.4 10.6 & 0.7
this
to
fallout
from
nuclear
testing.
6.2 Counting eficiencies obtained from Jamples spiked with Fe5s and Fe5s
whole
blood
Sources were prepared from six blood samples spiked with both Fes5 and Fesg. The sources were counted by liquid scintillation counting and also by y-counting the Fejg in order to determine the radiometric recovery. The counting efficiencies were corrected for radiometric yield and the results obtained are presented in Table 3. 6.3
The quenching effect of di$erent amounts of iron
As 10 ml samples of blood are taken for analysis, the total iron content of the sample may vary between 4.4 and 5.6 mg.(*) If differences in the iron content of samples in this range produce a significant effect on quenching then it will be necessary to determine the inactive iron content of the blood and adjust to a constant amount before carrying out the radiochemical analysis. In order to determine the severity of quenching effects over the range concerned, six sources TABLE
were prepared from samples containing various amounts of iron as ferric ammonium sulphate, spiked with both Fess and Fesg. The sources were counted in the liquid scintillation counter and the overall efficiencies determined. The radiometric yields were obtained by y-counting the Fe5g and the corrected liquid scintillation counting efficiencies and inactive iron contents of the sources calculated. The corrected liquid scintillation counting efficiencies are plotted in Fig. 2 against the actual weight of inactive irorl in the sample. As can be seen there is no serious change in quenching over the range of iron normally present in the blood. 6.4 The minimum detectable actioify of Fe55 and Fe”” in blood The minimum detectable activity is often expressed as that activity which will give a count rate equal to twice the standard deviation on the background counting rate. Using the mean backgrounds obtained in Table 2, the efficiencies shown in Table 3 and a 100 min counting period, the following minimum detectable levels are obtained: MDA for Fe”” = 2.1 % lo-’ $X/ml MDA for Fess = 9.0 x 10-s ,uCi/ml It should be noted however that these figures only apply if the isotopes are counted separately. If both are present in the same sample, although the figure for Fesg remains the same, the minimum detectable level for Fe”” rises with the amount of Fe5g present, as the effective background is increased by the contribution of the Fejg to the Fc”s count rate.
3. Counting efficiencies obtained from whole blood samples spiked with Fe”s and Fe””
Sample No.
Radiometric yield by y-counting (” “)
Corrected Fes5 efficiency (Oil)
1 2 3 4 5 6
97.0 92.0 92.3 98.5 95.7 97.5
18.7 19.3 19.9 19.2 18.8 20.3
32.6 32.5 34.5 33.7 33.0 33.9
95.5 & 2.7
19.4 _I 0.6
33.4 1 0.8
Mean
and SD
Corrected efficiency
Fe””
(Oni
The simultaneous determination of iron-55 50
‘; 2
and iron-59 in blood by liquid scintillation counting
397
r
40-
, 1
0
I 2
I 3 MG
FIG. 2. Quenching
7. SUMMARY
AND
OF
I 6
I 5
I 4 IRON
PER
effect of iron as phosphate counting efficiencies.
CONCLUSIONS
The work described in this report demonstrates that FeG5 and Fe5g may be measured simultaneously with good counting efficiencies by gel counting of the ferric phosphate complex in a Packard Tricarb 32 14 liquid scintillation specThe counting efficiency for Fe55 is trometer. increased by about a factor of 3 over previously reported liquid scintillation counting methods and this, combined with the inherently low background of the ‘Tricarb’ spectrometer, gives a minimum detectable level for FeS5 in blood of 0.2 pCi/ml. The method described for the measurement of FeS5 and Fe5g in blood may be used for determining these radionuclides in other materials. Currently measurements are being made on samples of bread which, after the addition of iron carrier, are analysed in the same way as blood samples.
I 7
I 0
, 3
I IO
SOURCE
complex on Fej5 and Fe5g
REFERENCES 1. PEACOCKW. C., EVANSR. D., IRVINEJ. W., GOOD W. N., KIP A. F., WEISS S. and GIBSONJ. G. J. clin. Inaest. 25, 605 (1946). 2. HALLBERC L. and BRISE H. Znt. J. a@$. Radiat. Isot0pe.r 9, 100 ( 1960). 3. DERN J. R. and HART W. L. J. Lab. clin. Med. 57, 322 (1961). 4. PERRY S. W. and WARNER G. T. Int. J. a@l. Radiat. Isotopes 14, 397 (1963). 5. EAKINSJ. D. and BROWND. ‘1. U.K. Atomic EnerD Authority Report AERE-R4945 (1965). C. 6. TORIBARAT. Y., MORKEND. A. and PREDMORE University of Rochester, New York, Report, UR 607 (1962). 7. ~VIENLANDR. F. and ENSGRABERF. Z. anorg. allg. Chem. 84, 340 (1914). 8. GEIGYJ. R. Documenta Gei, ScientiJic Tables. 5th Ed. Basle (1956). 9. PALMERH. E. and BEASLEYT. M. Science 149,43 1 (1965).