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The simultaneous determination of zinc and cadmium in biological materials by neutron activation analysis
International Journal of AppliedRadiatior, and Isotopes, 1969,VoL 20, pp. 335-340. PergamonPress. Printed in Northern Ireland
The Simultaneous Determination of Zinc and Cadmium in Biological Materials by Neutron Activation Analysis ANA CHUECA,*
M . W O R , W O O D ~ " a n d D. M . T A Y L O R .
Department of Biophysics, Institute of Cancer Research, Surrey Branch, Belmont, Sutton, Surrey, England
(Received 22 November 1968) A method for the simultaneous determination of zinc and cadmium is described which is suitable for the analysis of small samples of biological material. Following neutron activation and a simple ion exchange separation zinc and cadmium are identified and assayed by gamma ray spectroscopy. The sensitivity of the method is about 3 × 10-s g for cadmium and about 2 x 10-7 g for zinc. The method has been applied to the determination of cadmium and zinc in subcellular fractions prepared from rat testes and kidney. Analysis of a standard kale powder yielded the values of 32.8 4- 3-2/tg/g for zinc and 0.89 4- 0.16/*g/g for cadmium. LE D O S A G E SIMULTANI~ D U Z I N C E T D U C A D M I U M DANS LES M A T E R I A U X B I O L O G I Q U E S P A R L'ANALYSE D ' A C T I V A T I O N N E U T R O N I Q U E On ddcrit une m~thode t~our le dosage simultan6 du zinc et du cadmium lequel est convenable ~ l'analyse de petits 6chantillons de matdriel biologique. Suivant l'activation neutronique et une sdparation simple A 6change d'ions le zinc et le cadmium sont reconnus et dos6s au moyen de la spectroscopie de rayons gamma. La sensibilit6 de la mdthode vient ~ environ 3 × 10-s g pour le cadmium et environ 2 × 10-7 g pour le zinc. On a appuy6 la m6thode au dosage du cadmium et du zinc dans les fractions souscellulaires pr6par6es des testicules et des rognons du rat. L'analyse d'une poudre de thou fris6 servant d'dtalon rendit les valeurs 32,8 ± 3,2/~g]g pour le zinc et 0,89 ± 0,16 pg/g pour le cadmium. O ~ H O B P E M E H H O E O I I P E ~ E J I E H H E I~HHHA H HA~MHH B B H O / I O F H t l E C H H X MATEPHAJIAX AHAYIH3OM A H T H B A ~ H H HE19ITPOHOB Om~cl,iBaeiLiR MeTO~ ~3IH O~HOBpeMe~tH0rO onpe~e~ieHu~ co~epmaHna III4HRa ~I HaAMna ro~nTC~ J~H aHa~ri~OB ne~oJIblnHX 05paa~OB 5noaornqecI~oro MaTepnaJIa. Hoczle aHTI4BaLIH~ tteI~wpOHOB ~I npocworo IlOIIO06MeltHOrO paa~eJienna ImnK u Ka~Mn~t n~enwu/pu~npymTca n ~Io~sepramvc~ HO2IHqecTBeHHOMyaHaanay nocpe~cTnoM ClIeKTpocKoIImI raMMa-n3~Iyqen~tA. LIFBCTBnTeJIbHOCTBMeTO~a OHOJIO3 X 10 -a rp ;Can Ha~MI~I ~I OHOJIO2 X 10-? rp ~SX~lIInHHa. MeTOA npiaMenn~icrt ~aa onpe~eJIeHna coAepmaHn~ I~aAM~I~ rI II~tHHa n cySI~IewoqnhlX t~pammax, IIp~roTomlennl~ix Ha anqeu n noqeu ~p~ic. Ana~Ina cwaHAapTnoro iiopomKa Ka~IycT~,t no~aaa~i Beauqnnu: 32,8-4-3,2 rp/rp ~ i ~ Imnga rt 0,89 ~ 0,16 rp/rp ~irt Ha~MI~l.
* P.I.O. Fellow, on leave from the Department of Biochemistry, Faculty of Pharmacy, University of Madrid, Spain. 1" Present address: Department of Biochemistry, University College of South Wales and Monmouthshire, Cardiff, Great Britain. 8 335
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Ana Ghueca, M. Worwoodand D. M. Taylor DIE GLEICHZEITIGE BESTIMMUNG VON ZINK UND CADMIUM IN BIOLOGISCtlEN SUBSTANZEN D U R C H NEUTRONENAKTIVIERUNGSANALYSE Es wird ein Verfahren beschrieben fiir die gleichzeitige Bestimmung von Zink und Cadmium, welches sich ffir die Analyse von kleinen Proben biologischen Materials eignet. Nach Neutronenaktivierung und einfacher Ionenaustauschtrennung werden Zink und Cadmium identifiziert und durch y-Spektroskopie analysiert. Die Empfindlichkeit des Verfahrens ist etwa 3 x 10-s g fiir Cadmium und etwa 2 x 10-7 g ftir Zink. Das Verfahren ist verwendet worden ftir die Bestimmung yon Cadmium und Zink in unterzelligen Fraktionen, die aus Rattenhoden und Nieren hergestellt waren. Die Analyse eines normalen Kohlpulver ergab Werte yon 32, 8 4- 3,2 /~g/g ffir Zink und von 0,89 4- 0,16/~g]g ftir Cadmium. INTRODUCTION
ZINC and c a d m i u m commonly occur together in minerals and in living organisms m and there is increasing evidence to suggest that the biological actions of the essential trace element zinc and the non-essential element c a d m i u m m a y be related to each other. Zinc is known to reverse the necrosis produced by c a d m i u m in the testes ¢2"s) and a protein, metallothionein, containing both zinc and c a d m i u m has been isolated from equine kidneys, c4) For further studies of z i n c - c a d m i u m relationships in biological systems it is desirable to have a sensitive, accurate and fairly rapid method for the determination of these two elements in small samples of biological material. Neutron activation analysis is capable of high sensitivity for the detection of both zinc and c a d m i u m and the technique readily permits simultaneous determination of the two metals. A n u m b e r of methods have been described for the separate determination of zinc 15'6) or c a d m i u m 17's) using activation techniques and one or both of these metals have been included in several procedures for simultaneous multielement analysis, t9-12) This p a p e r describes a simple method for the simultaneous determination of zinc and cadm i u m which is suitable for the analysis of samples containing a few milligrams of biological material. Following neutron activation and ion exchange separation the c a d m i u m and zinc are identified and assayed by g a m m a ray spectroscopy. T h e method has been applied to the determination of zinc and c a d m i u m in subcellnlar fractions of rat testis and kidney. A series of measurements of zinc and c a d m i u m in the standard kale powder of BOWEN113) have also been m a d e in order to provide a comparison between this and other methods.
EXPERIMENTAL Nuclear reactions T h e r m a l neutron b o m b a r d m e n t of c a d m i u m produces three isotopes, nTCd (T1/2 2"9 hr, activation cross section 0.11 barns), n s C d (53 hr, 0.32 barns) and n5'~Cd (43 days, 0.04 barns). O f these isotopes nSCd is the most convenient to use when processing must be carried out at some distance from the reactor. Irradiation of zinc with thermal neutrons also yields three isotopes, 69Zn (2"11~ 55 min), 69mZn (13-8hr) and 65Zn (245 days). I n this method 6SZn is used and measurement of the induced activity is m a d e at least 7 days after irradiation.
Preparation of tissue samples Samples of dried kale powder (kindly supplied by Dr. H. J. M. Bowen, University of Reading, Reading, England) were re-dried at 90°C and approximately 0.1 g samples were weighed out accurately into small polyethylene 'irradiation' tubes (8 m m dia., 15 m m high). Sub-cellular fractions from rat testis or kidney were prepared by homogenising about 4 g of fresh tissue in 0-25 M sucrose and diluting to 50 ml with the same medium. T h e sucrose solution was prepared from 'Analar' sucrose (British Drug Houses Limited, Poole, Dorset, England) and demineralized, distilled water. T h e homogenate was centrifuged successively at 500 g for 10 rain to yield a 'nuclear' fraction; for 17 min at 10,000 g to yield a 'mitochondrial' pellet and finally at 105,000 g for 90 rain to yield a microsomal pellet and a soluble fraction. After separation, the fractions were dried at 75°C under reduced pressure and ground to a coarse powder. Approximately 0-1 g portions
The simultaneous determination of zinc and cadmium in biological materials were accurately weighed out into polyethylene 'irradiation' tubes (9). T h e nitrogen content of the dried samples was determined by a micro-Kjeldahl method.
Preparation of standards Standard solutions of zinc and c a d m i u m were prepared from 'Specpure' Z n O or C d O (Johnson, M a t t h e y and C o m p a n y Limited, London, England), 'Analar' H2SO 4 and demineralised, distilled water. Each solution contained 1000/zg of zinc or eadmium/ml. For activation standards about 0-1 g portions of solution were accurately weighed out into polyethylene 'irradiation' tubes and evaporated to dryness at 75°C. After irradiation the tubes containing the standards were washed in acid, opened and placed in flasks containing 2 ml 10 M HC1, 2 ml concentrated H N O 3 and 10 mg each of zinc and c a d m i u m carrier in a final volume of 20 ml. T h e solutions were boiled for a few minutes then carefully evaporated to dryness and the residue dissolved in 50 ml 1 M HC1. Irradiation U p to 20 irradiation tubes containing samples or standards were packed into a Harwell T y p e A irradiation can and irradiated in B E P O (A.E.R.E., Harwell, England) for 3 days at a neutron flux of 1013 n.cm-2sec -1. A cooling period of 24 hr was allowed to elapse before the can was opened.
Wet ashing After irradiation the tubes were rinsed with dilute hydrochloric acid and distilled water. T h e contents were transferred to 250 ml conical flasks, containing 3 ml concentrated H2SO 4 and 100 # g each of zinc and cadmium, and wet ashed by heating with H2SO 4 and H N O 8 until a clear solution was obtained. T h e remaining H2SO4 was evaporated off and the residues were dissolved in 2-3 ml of 1 M HC1.
Ion exchange separation Dowex 1 × 8-200 anion exchange resin, in the chloride form, (Sigma Chemical C o m p a n y Limited, London, England) was washed several
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times with distilled water. Columns 7.5 cm high by 1 cm dia. were prepared, fitted with a syphoning device (9), and equilibrated by passing 15 ml of 1 M HC1 through the column. T h e sample solution was loaded onto the column and the column was washed with 25 ml of 1 M H C I to remove unwanted elements such as Na, K, Cu, Fe, P etc. Zinc was then eluted by passing 40 ml of 10 -3 M HC1 through the column; the c a d m i u m was eluted with 80 mI of 5 × 10 -4 M HC1. T h e eluates containing the zinc and c a d m i u m were evaporated to about 5 ml, transferred to glass counting vials and diluted to a standard sample height.
Measurement of induced radioactivity T h e g a m m a ray spectra of the samples and standards were measured in a g a m m a ray spectrometer consisting of a 5 in. dia. by 6 in. high, well-type N a I ( T I ) detector coupled to a 400 channel pulse height analyser (Technical, Measurement Corp., North Haven, Conne. U.S.A.). T h e detector is shielded by 4 i n . of steel and placed in a gallery below 4 ft of chalk in order to achieve a low background counting rate(14). Cadmium-115 was measured by the radioactivity in the 0.335 M e V photopeak of its daughter isotope 11stain (half-life 4 hr). Since the n s c d - n 5 I n equilibrium is destroyed during the separation procedure a period of 30 hr was allowed to elapse before the measurements were made. T h e use of the 0-335 M e V photopeak reduces interference from small amounts of 69mZn or 65Zn in the c a d m i u m fraction. Zinc-65 was measured by the radioactivity in the I ' l l M e V photopeak. A decay period of about 7 days was allowed to elapse before the measurements were made. No other isotopes were detected in the zinc and cadmium fractions. RESULTS
Experimental test of the method Suitable aliquots of the standard 65Zn and n s C d solutions were put through the wet ashing process and no losses of radioactivity were observed. Further aliquots of the standard solutions were subjected to the complete procedure and
Ana Ghueea,M. Worwoodand D. M. Taylor
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the results are shown in Table 1. No GSZn or n s C d was found in the 1 M HC1 fraction but similar tests using 22Na or 59Fe showed that 100 per cent of each of these isotopes was recovered in the first 25 ml of 1 M HC1.
TABLE 2. Zinc and cadmium concentrations in 'standard' kale powder
Metal
TABLE 1.
Fraction
I0-2 M HC1 5 x 10-4 M HC1
Mean concentration ( 10 estimations) 4- 1 standard deviation
pg/g dry weight
Percentage of total radioactivity in fraction 65Zn (6 Experiments)
115Cd (7 Experiments)
93.8 4- 1.3 1.4 4- 0.5
0.0 86.0 ± 2.1
Analysis of the 'standard' kale powder One or more samples of the dried kale powder were included with each batch of tissue samples in order to provide a running check of the method. The results of 10 determinations ofzinc and cadmium in the dried kale are shown in Table 2.
Analysis of animal tissue The procedure has been used to determine the concentrations of zinc and cadmium in nuclear, mitochondrial, microsomal and soluble fractions prepared from rat testis and kidney. The results, expressed as mg Zn/g nitrogen and as #g Cd/g nitrogen are listed in Table 3. The 0"25 M sucrose solutions used for the fractionation procedures were also analysed and found to contain 0.4 /,g Zn/g dry weight and 0.07 /,g Cd/g dry weight. DISCUSSION The separation procedure is based on the differential absorption of chloride complexes of metals on anion exchange resins at varying concentrations of HC1 which has been studied extensively by KRAUS and NELSONtlS). I n contrast to other methods in which this ion exchange technique has been used for the simultaneous determination of Co, Cu, Fe, Zn and Mo ~9'16) the present procedure does not give complete recovery of zinc and cadmium.
Zinc Cadmium Zinc/cadmium ratio
32.8 ± 3.2 0.89 ± 0.16 37: I
Nevertheless, the yields of these metals are high and are sufficiently reproducible to obviate the necessity for direct measurement of the chemical yield in each sample. The presence of a small amount of 6nZn in the 115Cd fraction was not found to be serious enough to require a correction to be applied for the eSZn contribution to the radioactivity in the 0"335 M e V photopeak region. However it is possible that some correction might be required if the method was to be applied to samples containing zinc/cadmium ratios very much greater than those measured here. The limits of sensitivity of this method are estimated to be about 3 × 10-Sg for Cd and about 2 × 10-7 g for Zn. The results of the analyses of rat tissues show that the zinc/cadmium ratio is about 3 times greater in the testis than in the kidney as a result of higher zinc levels in the testes and cadmium in the kidney. In general the cadmium results show larger standard deviations than the corresponding values for zinc, but this is probably due to the fact that in these preparations from about 4 g of tissue the cadmium content is close to the limit of detection for this element. The value for the zinc content of the kale powder is in good agreement with the values reported by BOWEN(13), but the concentration of cadmium is higher than the values of 0"38 /zg/g m) and 0"63 /~g/g~le) determined by other workers using activation analysis. A possible explanation of the discrepancies between the values determined by neutron activation analysis may lie in self-shielding effects in the different weights of cadmium irradiated as 'standards. I n the present work the weight of cadmium in the standards was 50-100/zg while LIVINOSTONF., SMIT~Iand STOJANOVlC(x2) used 1-10 #g;
339
The simultaneous determination of zinc and cadmium in biological materials
TABLE 3. Cadmium and zinc concentrations in testis and kidney (Mean 4- 1 standard deviation) Whole tissue
Tissue
Nuclei
Mitochondria Mierosomes 1.16 4- 0.06 1.7 4- 0.5 680
Soluble fraction
Testis (3 estimations)
Zinc mg]gN 1.81 4- 0.26 1.77 + 0.16 1.12 4- 0.04 Cadmium#g/gN 3-0 4- 0.04 1.9 4- 0.7 2-1 4- 0.4 Zn/Cd 600 930 530
1.75 + 0.09 4.2 4- 1.7 420
Kidney (5 estimations)
Zinc mg/gN 1.02 4- 0-14 0"73 4- 0.06 0.56 4-4-0"09 0.95 4- 0.21 1.43 4- 0.34 Cadmium #g/gN 6.0 4- 3.1 2.9 4- 2.1 2"2 4- 0-5 2-9 4- 0.5 10.0 4- 2'9 Zn]Cd 170 250 250 330 140
~ 12°F
ooE 0
I
I
I
I
I
I
I
5
I
I 45
I
I 85
Weight of Cadmium ]rrodiated
I
I )25
FIO. 1. The relationship between induced radioactivity and weight of cadmium after thermal neutron bombardment. Bowen used standards containing 10-20/zg Cd in the form of dilute solutions in order to minimise self-shielding. I n order to assess the possible errors which m a y be caused by self-shielding in the irradiation of microgram quantities of cadmium, 20 standards containing from 0-4 to 123/zg cadmium were irradiated under standard conditions and the activity induced p e r / z g was compared with the weight of metal irradiated. The results, which are plotted in Fig. 1, show that there is a continuing decrease in the activity induced p e r / z g cadmium as the weight of metal irradiated increases. The effect appears to be most marked when less than 1 /zg of cadmium is irradiated. O n the basis of these results it seems likely that the discrepancies between the 3 values for the cadmium content of the kale powder can be explained by the difference in the weight of cadmium in the standards. I n the determination of cadmium it would be preferable to use a number of standards containing different weights of cadmium which will cover the range of weights expected in the unknown samples. An alternative wouId be to
irradiated dilute solutions of cadmium as standards or to disperse the c a d m i u m t h r o u g h o u t an 'inert' matrix to reduce the effects of selfshielding, however this latter approach involves considerable practical difficulty. The procedure described offers a fairly rapid method for the simultaneous determination of zinc and cadmium in biological materials. T h e sensitivity is sufficient to permit the determination of these metals in small samples of tissue or in subcellular fractions prepared from plant or animal tissues.
REFERENCES 1. SCHROEDERH. A., NASONA. P., TIPTON I. H. and BALASSAJ. J. d. Chron. Dis. 20, 179 (1967). 2. PARIZEKJ. J. Endocrinol. 15, 56 (1957). 3. PARIZEK J., BOURSNELL J. C., HAY M. F., BABICKY A. a n d TAYLOR D. M. J. Repro&Fert.
12, 501 (1966). 4. KAGIJ. H. R. and VALLEEB. L. J. Biol. Chem. 235, 3460 (1960). 5. BANKST. E., TUPPER R., WHITE E. M. A. and WORMALLA. Int J. appl. Radiat. Isotopes 4, 222 (1959).
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6. BOWEN H. J. M. Int. J. appl. Radiat. Isotopes 4, 214 (1959). 7. GIBBONSD. Pure appl. Chem. 1, 135 (1960). 8. WESTERMAVa~T. and SJOSTRANDB. Int. J. appl. Radiat. Isotopes 9, 78 (1960). 9. PARR R. M. and TAYLOR D. M. Biochem. J. 91, 424 (1964). 10. SAMSAHLK., BRUNED. and WESTER P. O. Int. J. appl. Radiat. Isotopes 16, 273 (1965). I1. BOWENH . J . M . Analyst92, 118 (1967).
12. LIVINGSTONEH. D., SMITHH. and STOJANOVIC•. Talanta 14, 505 (1967). 13. BOWENH. J. M. Analyst 92, 124 (1967). 14. MAYNEORDW. V., ANDERSONW., BENTLEYR. E., BURTONL. K., CROOKALLJ. O. and TRoa-r N. G. Nature, Lond. 182, 1473 (1958). 15. KRAUS K. A. and NELSON F. Proc. Int. Conf. Peaceful Uses Atom. Energ. Geneva 7, 113 (1955). 16. WORWOOD M. Ph.D. Thesis, University of London, England (1967).
The Simultaneous Determination of Zinc and Cadmium in Biological Materials by Neutron Activation Analysis ANA CHUECA,*
M . W O R , W O O D ~ " a n d D. M . T A Y L O R .
Department of Biophysics, Institute of Cancer Research, Surrey Branch, Belmont, Sutton, Surrey, England
(Received 22 November 1968) A method for the simultaneous determination of zinc and cadmium is described which is suitable for the analysis of small samples of biological material. Following neutron activation and a simple ion exchange separation zinc and cadmium are identified and assayed by gamma ray spectroscopy. The sensitivity of the method is about 3 × 10-s g for cadmium and about 2 x 10-7 g for zinc. The method has been applied to the determination of cadmium and zinc in subcellular fractions prepared from rat testes and kidney. Analysis of a standard kale powder yielded the values of 32.8 4- 3-2/tg/g for zinc and 0.89 4- 0.16/*g/g for cadmium. LE D O S A G E SIMULTANI~ D U Z I N C E T D U C A D M I U M DANS LES M A T E R I A U X B I O L O G I Q U E S P A R L'ANALYSE D ' A C T I V A T I O N N E U T R O N I Q U E On ddcrit une m~thode t~our le dosage simultan6 du zinc et du cadmium lequel est convenable ~ l'analyse de petits 6chantillons de matdriel biologique. Suivant l'activation neutronique et une sdparation simple A 6change d'ions le zinc et le cadmium sont reconnus et dos6s au moyen de la spectroscopie de rayons gamma. La sensibilit6 de la mdthode vient ~ environ 3 × 10-s g pour le cadmium et environ 2 × 10-7 g pour le zinc. On a appuy6 la m6thode au dosage du cadmium et du zinc dans les fractions souscellulaires pr6par6es des testicules et des rognons du rat. L'analyse d'une poudre de thou fris6 servant d'dtalon rendit les valeurs 32,8 ± 3,2/~g]g pour le zinc et 0,89 ± 0,16 pg/g pour le cadmium. O ~ H O B P E M E H H O E O I I P E ~ E J I E H H E I~HHHA H HA~MHH B B H O / I O F H t l E C H H X MATEPHAJIAX AHAYIH3OM A H T H B A ~ H H HE19ITPOHOB Om~cl,iBaeiLiR MeTO~ ~3IH O~HOBpeMe~tH0rO onpe~e~ieHu~ co~epmaHna III4HRa ~I HaAMna ro~nTC~ J~H aHa~ri~OB ne~oJIblnHX 05paa~OB 5noaornqecI~oro MaTepnaJIa. Hoczle aHTI4BaLIH~ tteI~wpOHOB ~I npocworo IlOIIO06MeltHOrO paa~eJienna ImnK u Ka~Mn~t n~enwu/pu~npymTca n ~Io~sepramvc~ HO2IHqecTBeHHOMyaHaanay nocpe~cTnoM ClIeKTpocKoIImI raMMa-n3~Iyqen~tA. LIFBCTBnTeJIbHOCTBMeTO~a OHOJIO3 X 10 -a rp ;Can Ha~MI~I ~I OHOJIO2 X 10-? rp ~SX~lIInHHa. MeTOA npiaMenn~icrt ~aa onpe~eJIeHna coAepmaHn~ I~aAM~I~ rI II~tHHa n cySI~IewoqnhlX t~pammax, IIp~roTomlennl~ix Ha anqeu n noqeu ~p~ic. Ana~Ina cwaHAapTnoro iiopomKa Ka~IycT~,t no~aaa~i Beauqnnu: 32,8-4-3,2 rp/rp ~ i ~ Imnga rt 0,89 ~ 0,16 rp/rp ~irt Ha~MI~l.
* P.I.O. Fellow, on leave from the Department of Biochemistry, Faculty of Pharmacy, University of Madrid, Spain. 1" Present address: Department of Biochemistry, University College of South Wales and Monmouthshire, Cardiff, Great Britain. 8 335
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Ana Ghueca, M. Worwoodand D. M. Taylor DIE GLEICHZEITIGE BESTIMMUNG VON ZINK UND CADMIUM IN BIOLOGISCtlEN SUBSTANZEN D U R C H NEUTRONENAKTIVIERUNGSANALYSE Es wird ein Verfahren beschrieben fiir die gleichzeitige Bestimmung von Zink und Cadmium, welches sich ffir die Analyse von kleinen Proben biologischen Materials eignet. Nach Neutronenaktivierung und einfacher Ionenaustauschtrennung werden Zink und Cadmium identifiziert und durch y-Spektroskopie analysiert. Die Empfindlichkeit des Verfahrens ist etwa 3 x 10-s g fiir Cadmium und etwa 2 x 10-7 g ftir Zink. Das Verfahren ist verwendet worden ftir die Bestimmung yon Cadmium und Zink in unterzelligen Fraktionen, die aus Rattenhoden und Nieren hergestellt waren. Die Analyse eines normalen Kohlpulver ergab Werte yon 32, 8 4- 3,2 /~g/g ffir Zink und von 0,89 4- 0,16/~g]g ftir Cadmium. INTRODUCTION
ZINC and c a d m i u m commonly occur together in minerals and in living organisms m and there is increasing evidence to suggest that the biological actions of the essential trace element zinc and the non-essential element c a d m i u m m a y be related to each other. Zinc is known to reverse the necrosis produced by c a d m i u m in the testes ¢2"s) and a protein, metallothionein, containing both zinc and c a d m i u m has been isolated from equine kidneys, c4) For further studies of z i n c - c a d m i u m relationships in biological systems it is desirable to have a sensitive, accurate and fairly rapid method for the determination of these two elements in small samples of biological material. Neutron activation analysis is capable of high sensitivity for the detection of both zinc and c a d m i u m and the technique readily permits simultaneous determination of the two metals. A n u m b e r of methods have been described for the separate determination of zinc 15'6) or c a d m i u m 17's) using activation techniques and one or both of these metals have been included in several procedures for simultaneous multielement analysis, t9-12) This p a p e r describes a simple method for the simultaneous determination of zinc and cadm i u m which is suitable for the analysis of samples containing a few milligrams of biological material. Following neutron activation and ion exchange separation the c a d m i u m and zinc are identified and assayed by g a m m a ray spectroscopy. T h e method has been applied to the determination of zinc and c a d m i u m in subcellnlar fractions of rat testis and kidney. A series of measurements of zinc and c a d m i u m in the standard kale powder of BOWEN113) have also been m a d e in order to provide a comparison between this and other methods.
EXPERIMENTAL Nuclear reactions T h e r m a l neutron b o m b a r d m e n t of c a d m i u m produces three isotopes, nTCd (T1/2 2"9 hr, activation cross section 0.11 barns), n s C d (53 hr, 0.32 barns) and n5'~Cd (43 days, 0.04 barns). O f these isotopes nSCd is the most convenient to use when processing must be carried out at some distance from the reactor. Irradiation of zinc with thermal neutrons also yields three isotopes, 69Zn (2"11~ 55 min), 69mZn (13-8hr) and 65Zn (245 days). I n this method 6SZn is used and measurement of the induced activity is m a d e at least 7 days after irradiation.
Preparation of tissue samples Samples of dried kale powder (kindly supplied by Dr. H. J. M. Bowen, University of Reading, Reading, England) were re-dried at 90°C and approximately 0.1 g samples were weighed out accurately into small polyethylene 'irradiation' tubes (8 m m dia., 15 m m high). Sub-cellular fractions from rat testis or kidney were prepared by homogenising about 4 g of fresh tissue in 0-25 M sucrose and diluting to 50 ml with the same medium. T h e sucrose solution was prepared from 'Analar' sucrose (British Drug Houses Limited, Poole, Dorset, England) and demineralized, distilled water. T h e homogenate was centrifuged successively at 500 g for 10 rain to yield a 'nuclear' fraction; for 17 min at 10,000 g to yield a 'mitochondrial' pellet and finally at 105,000 g for 90 rain to yield a microsomal pellet and a soluble fraction. After separation, the fractions were dried at 75°C under reduced pressure and ground to a coarse powder. Approximately 0-1 g portions
The simultaneous determination of zinc and cadmium in biological materials were accurately weighed out into polyethylene 'irradiation' tubes (9). T h e nitrogen content of the dried samples was determined by a micro-Kjeldahl method.
Preparation of standards Standard solutions of zinc and c a d m i u m were prepared from 'Specpure' Z n O or C d O (Johnson, M a t t h e y and C o m p a n y Limited, London, England), 'Analar' H2SO 4 and demineralised, distilled water. Each solution contained 1000/zg of zinc or eadmium/ml. For activation standards about 0-1 g portions of solution were accurately weighed out into polyethylene 'irradiation' tubes and evaporated to dryness at 75°C. After irradiation the tubes containing the standards were washed in acid, opened and placed in flasks containing 2 ml 10 M HC1, 2 ml concentrated H N O 3 and 10 mg each of zinc and c a d m i u m carrier in a final volume of 20 ml. T h e solutions were boiled for a few minutes then carefully evaporated to dryness and the residue dissolved in 50 ml 1 M HC1. Irradiation U p to 20 irradiation tubes containing samples or standards were packed into a Harwell T y p e A irradiation can and irradiated in B E P O (A.E.R.E., Harwell, England) for 3 days at a neutron flux of 1013 n.cm-2sec -1. A cooling period of 24 hr was allowed to elapse before the can was opened.
Wet ashing After irradiation the tubes were rinsed with dilute hydrochloric acid and distilled water. T h e contents were transferred to 250 ml conical flasks, containing 3 ml concentrated H2SO 4 and 100 # g each of zinc and cadmium, and wet ashed by heating with H2SO 4 and H N O 8 until a clear solution was obtained. T h e remaining H2SO4 was evaporated off and the residues were dissolved in 2-3 ml of 1 M HC1.
Ion exchange separation Dowex 1 × 8-200 anion exchange resin, in the chloride form, (Sigma Chemical C o m p a n y Limited, London, England) was washed several
337
times with distilled water. Columns 7.5 cm high by 1 cm dia. were prepared, fitted with a syphoning device (9), and equilibrated by passing 15 ml of 1 M HC1 through the column. T h e sample solution was loaded onto the column and the column was washed with 25 ml of 1 M H C I to remove unwanted elements such as Na, K, Cu, Fe, P etc. Zinc was then eluted by passing 40 ml of 10 -3 M HC1 through the column; the c a d m i u m was eluted with 80 mI of 5 × 10 -4 M HC1. T h e eluates containing the zinc and c a d m i u m were evaporated to about 5 ml, transferred to glass counting vials and diluted to a standard sample height.
Measurement of induced radioactivity T h e g a m m a ray spectra of the samples and standards were measured in a g a m m a ray spectrometer consisting of a 5 in. dia. by 6 in. high, well-type N a I ( T I ) detector coupled to a 400 channel pulse height analyser (Technical, Measurement Corp., North Haven, Conne. U.S.A.). T h e detector is shielded by 4 i n . of steel and placed in a gallery below 4 ft of chalk in order to achieve a low background counting rate(14). Cadmium-115 was measured by the radioactivity in the 0.335 M e V photopeak of its daughter isotope 11stain (half-life 4 hr). Since the n s c d - n 5 I n equilibrium is destroyed during the separation procedure a period of 30 hr was allowed to elapse before the measurements were made. T h e use of the 0-335 M e V photopeak reduces interference from small amounts of 69mZn or 65Zn in the c a d m i u m fraction. Zinc-65 was measured by the radioactivity in the I ' l l M e V photopeak. A decay period of about 7 days was allowed to elapse before the measurements were made. No other isotopes were detected in the zinc and cadmium fractions. RESULTS
Experimental test of the method Suitable aliquots of the standard 65Zn and n s C d solutions were put through the wet ashing process and no losses of radioactivity were observed. Further aliquots of the standard solutions were subjected to the complete procedure and
Ana Ghueea,M. Worwoodand D. M. Taylor
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the results are shown in Table 1. No GSZn or n s C d was found in the 1 M HC1 fraction but similar tests using 22Na or 59Fe showed that 100 per cent of each of these isotopes was recovered in the first 25 ml of 1 M HC1.
TABLE 2. Zinc and cadmium concentrations in 'standard' kale powder
Metal
TABLE 1.
Fraction
I0-2 M HC1 5 x 10-4 M HC1
Mean concentration ( 10 estimations) 4- 1 standard deviation
pg/g dry weight
Percentage of total radioactivity in fraction 65Zn (6 Experiments)
115Cd (7 Experiments)
93.8 4- 1.3 1.4 4- 0.5
0.0 86.0 ± 2.1
Analysis of the 'standard' kale powder One or more samples of the dried kale powder were included with each batch of tissue samples in order to provide a running check of the method. The results of 10 determinations ofzinc and cadmium in the dried kale are shown in Table 2.
Analysis of animal tissue The procedure has been used to determine the concentrations of zinc and cadmium in nuclear, mitochondrial, microsomal and soluble fractions prepared from rat testis and kidney. The results, expressed as mg Zn/g nitrogen and as #g Cd/g nitrogen are listed in Table 3. The 0"25 M sucrose solutions used for the fractionation procedures were also analysed and found to contain 0.4 /,g Zn/g dry weight and 0.07 /,g Cd/g dry weight. DISCUSSION The separation procedure is based on the differential absorption of chloride complexes of metals on anion exchange resins at varying concentrations of HC1 which has been studied extensively by KRAUS and NELSONtlS). I n contrast to other methods in which this ion exchange technique has been used for the simultaneous determination of Co, Cu, Fe, Zn and Mo ~9'16) the present procedure does not give complete recovery of zinc and cadmium.
Zinc Cadmium Zinc/cadmium ratio
32.8 ± 3.2 0.89 ± 0.16 37: I
Nevertheless, the yields of these metals are high and are sufficiently reproducible to obviate the necessity for direct measurement of the chemical yield in each sample. The presence of a small amount of 6nZn in the 115Cd fraction was not found to be serious enough to require a correction to be applied for the eSZn contribution to the radioactivity in the 0"335 M e V photopeak region. However it is possible that some correction might be required if the method was to be applied to samples containing zinc/cadmium ratios very much greater than those measured here. The limits of sensitivity of this method are estimated to be about 3 × 10-Sg for Cd and about 2 × 10-7 g for Zn. The results of the analyses of rat tissues show that the zinc/cadmium ratio is about 3 times greater in the testis than in the kidney as a result of higher zinc levels in the testes and cadmium in the kidney. In general the cadmium results show larger standard deviations than the corresponding values for zinc, but this is probably due to the fact that in these preparations from about 4 g of tissue the cadmium content is close to the limit of detection for this element. The value for the zinc content of the kale powder is in good agreement with the values reported by BOWEN(13), but the concentration of cadmium is higher than the values of 0"38 /zg/g m) and 0"63 /~g/g~le) determined by other workers using activation analysis. A possible explanation of the discrepancies between the values determined by neutron activation analysis may lie in self-shielding effects in the different weights of cadmium irradiated as 'standards. I n the present work the weight of cadmium in the standards was 50-100/zg while LIVINOSTONF., SMIT~Iand STOJANOVlC(x2) used 1-10 #g;
339
The simultaneous determination of zinc and cadmium in biological materials
TABLE 3. Cadmium and zinc concentrations in testis and kidney (Mean 4- 1 standard deviation) Whole tissue
Tissue
Nuclei
Mitochondria Mierosomes 1.16 4- 0.06 1.7 4- 0.5 680
Soluble fraction
Testis (3 estimations)
Zinc mg]gN 1.81 4- 0.26 1.77 + 0.16 1.12 4- 0.04 Cadmium#g/gN 3-0 4- 0.04 1.9 4- 0.7 2-1 4- 0.4 Zn/Cd 600 930 530
1.75 + 0.09 4.2 4- 1.7 420
Kidney (5 estimations)
Zinc mg/gN 1.02 4- 0-14 0"73 4- 0.06 0.56 4-4-0"09 0.95 4- 0.21 1.43 4- 0.34 Cadmium #g/gN 6.0 4- 3.1 2.9 4- 2.1 2"2 4- 0-5 2-9 4- 0.5 10.0 4- 2'9 Zn]Cd 170 250 250 330 140
~ 12°F
ooE 0
I
I
I
I
I
I
I
5
I
I 45
I
I 85
Weight of Cadmium ]rrodiated
I
I )25
FIO. 1. The relationship between induced radioactivity and weight of cadmium after thermal neutron bombardment. Bowen used standards containing 10-20/zg Cd in the form of dilute solutions in order to minimise self-shielding. I n order to assess the possible errors which m a y be caused by self-shielding in the irradiation of microgram quantities of cadmium, 20 standards containing from 0-4 to 123/zg cadmium were irradiated under standard conditions and the activity induced p e r / z g was compared with the weight of metal irradiated. The results, which are plotted in Fig. 1, show that there is a continuing decrease in the activity induced p e r / z g cadmium as the weight of metal irradiated increases. The effect appears to be most marked when less than 1 /zg of cadmium is irradiated. O n the basis of these results it seems likely that the discrepancies between the 3 values for the cadmium content of the kale powder can be explained by the difference in the weight of cadmium in the standards. I n the determination of cadmium it would be preferable to use a number of standards containing different weights of cadmium which will cover the range of weights expected in the unknown samples. An alternative wouId be to
irradiated dilute solutions of cadmium as standards or to disperse the c a d m i u m t h r o u g h o u t an 'inert' matrix to reduce the effects of selfshielding, however this latter approach involves considerable practical difficulty. The procedure described offers a fairly rapid method for the simultaneous determination of zinc and cadmium in biological materials. T h e sensitivity is sufficient to permit the determination of these metals in small samples of tissue or in subcellular fractions prepared from plant or animal tissues.
REFERENCES 1. SCHROEDERH. A., NASONA. P., TIPTON I. H. and BALASSAJ. J. d. Chron. Dis. 20, 179 (1967). 2. PARIZEKJ. J. Endocrinol. 15, 56 (1957). 3. PARIZEK J., BOURSNELL J. C., HAY M. F., BABICKY A. a n d TAYLOR D. M. J. Repro&Fert.
12, 501 (1966). 4. KAGIJ. H. R. and VALLEEB. L. J. Biol. Chem. 235, 3460 (1960). 5. BANKST. E., TUPPER R., WHITE E. M. A. and WORMALLA. Int J. appl. Radiat. Isotopes 4, 222 (1959).
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6. BOWEN H. J. M. Int. J. appl. Radiat. Isotopes 4, 214 (1959). 7. GIBBONSD. Pure appl. Chem. 1, 135 (1960). 8. WESTERMAVa~T. and SJOSTRANDB. Int. J. appl. Radiat. Isotopes 9, 78 (1960). 9. PARR R. M. and TAYLOR D. M. Biochem. J. 91, 424 (1964). 10. SAMSAHLK., BRUNED. and WESTER P. O. Int. J. appl. Radiat. Isotopes 16, 273 (1965). I1. BOWENH . J . M . Analyst92, 118 (1967).
12. LIVINGSTONEH. D., SMITHH. and STOJANOVIC•. Talanta 14, 505 (1967). 13. BOWENH. J. M. Analyst 92, 124 (1967). 14. MAYNEORDW. V., ANDERSONW., BENTLEYR. E., BURTONL. K., CROOKALLJ. O. and TRoa-r N. G. Nature, Lond. 182, 1473 (1958). 15. KRAUS K. A. and NELSON F. Proc. Int. Conf. Peaceful Uses Atom. Energ. Geneva 7, 113 (1955). 16. WORWOOD M. Ph.D. Thesis, University of London, England (1967).