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Simultaneous determination of copper, manganese and zinc in biological materials by means of neutron activation analysis and chelate extraction
International Journal of Applied Radiation and Isotopes, 1970, Vol. 21, p. 431. Pergamon Press. Printed in Northern Ireland
Rapid Preparation o f 14C-Blood P l a s m a for Liquid Scintillation Counting in D i m e t h y l s u l f o x i d e - T h l x o t r o p i c Gel* (Received 5 November 1969) A NUMBER of procedures have been described for the liquid scintillation counting of x4C or related beta emitting radionuclides in blood plasma. Most of these techniques suffer from one or more disadvantages (such as being expensive, time consuming, requiring equipment for combustion, or else being handicapped by severe quenching). ~x-4) We have developed a rapid procedure for the counting of such samples, based on the solubility of plasma in dimethylsulfoxide, and the ready admixture of the resulting solution with a thixotropic gel. Rats were injected intraperitoneally with 25 pCi of glycine-2-x4C. After 3 days, plasma was harvested. The plasma appears to readily dissolve in dimethylsulfoxide (26"6 ml plasma/100 ml solvent). The solution was added, in varying amounts, to 10 ml of a thixotropic gel (4 per cent Cab-O-Sil, Packard, in prepared dioxane). The dioxane had been prepared by adding to each liter: 7 g 2,5-diphenyloxazole (PPO), 0-05 g 1,4-di (2,5-phenyloxazolyl) benzene (POPOP) and 100 g naphthalene. By means of a4C standards of known activity, a curve of absolute efficiency was constructed as a function of the channels ratio, using a Packard 3375 liquid scintillation spectrometer (red channel: 50-1000, gain 14 per cent; green channel: 150-1000, gain 14 per cent). Addition of radiolabeled plasmadimethylsulfoxide equivalent to 0.5 ml plasma resulted in a counting efficiency of 83 per cent. One ml of plasma could be counted at 75 per cent efficiency.
References I. VON SCHUCHmG S. and E ~ m ~ O F F C. W. A ~ t . Biochem. 5, 93 (1963). 2. KRAGELUIffD E. and DX,~BYE M. Stand J. din. Lab. Invest. 19, 129 (1967). 3. NJ~a)xc~m~x G. D., SINOH B. and JEEJEE~HORY K. N. Int. J. appl. Radiat. Isotopes 19, 685 (1968). 4. ME~a~E R. C. and STIGLITZ R. A. Int. J. appl. Radiat. Isotopes 13, 11 (1962). * Supported by T-492 from the American Cancer Society and by U S P H S Grants C A 06519 and A M 09429.
International Journal of Applied Radiation and Isotopes, 1970, Vol. 2 I, pp. 431--433. Pergamon Press. Printed in Northern Ireland
S i m u l t a n e o u s Detes~mination o f Copper, M a n g a n e s e and Zinc in Biological Materials by Means o f Neutron Activation Analysis and Chelate Extraction (Received 30 September 1969)
1. Introduction
WHEN investigating trace element concentrations in biological materials, one must work up a large number of samples to ensure statistical validity of the results. If the element in question cannot be directly measured (Fig. 2a), activation analysis becomes more difficult because of the required radiochemical separation. It is most important that the disturbing elements Na, K, P, C1 and Br be removed. Processes for the radiochemical separation of Cu, Mn, Zn (and others) are described for example by JERVlS and WONG (1) and TAYLOR et al. c2"3~ The RICHARD P. SPENCER articles deal almost exclusively with column separaMARY ANN BANERJI tions. The process used in our work depends on the use of a metal ion chelation followed by a twoSection of Nuclear Medicine, Department of Radiology phase extraction as described by MULFORDc4). Our Yale University School of Medicine chelating agent is AmmoniumpyrolidindithiocarbNew Haven amate (APDC) and our organic extraction agent is Conn. 06510, U.S.A. 431
432
Technical notes
Methylisobutylketon (MIBK). Thereby, Cu, M n and Zn are extracted quantitatively into the organic phase, while the disturbing elements remain in the aqueous phase or are removed by the wet ashing.
I
i
I
I
I
100
2. M e t h o d After a metal-free removal from the animal the samples are freeze dried, homogenized and then, after repeated drying, weighed in polystyrene capsules. T h e y are then irradiated for 2-8 hr with a thermal neutron flux of 2 × 10TM n.cm -2 sec-1. After irradiation the sodium content of the samples is determined by measurement of ~iNa. T h e n the contents of the capsules are wet ashed by means of H 2 S O ~ and I-I~O2. After removal of the excess H 2 0 2 by evaporation a drop of methyl red is added to the clear or light yellow solution. O n e then adds concentrated NaOI-I until the colour changes. T h e changing point is chosen so that no hydroxides precipitate. T h e n with dilute H C I one adjusts the p H of the solution to 3-4.5. F r o m this solution one takes 7.5 ml and again the sodium content is determined. F r o m this figure one can calculate the total amount of sample actually present in the tube. F r o m this point one must work quantitatively, 2 ml of a 2 per cent aqueous A P D C solution are added, as well as 5 ml M I B K . After thorough mixing followed by a 1 rain centrifugation (appr.
x o
13 50
I 2
I
I
a
Zn
•
Cu
[] Mn
3 4 5 6 pH of aqueous solution prior to chelation
FIG. 1. p H
dependency of the extraction of Cu, M n and Zn.
1000 g) one gets a sharp phase-separation. O n e puts 4 ml of the yellow to dark brown organic phase into a tube and measures its activity with a Ge (Li) crystal. T h e aqueous phase should be clear if the wet ashing and pI-t adjustments have been properly done. 3. R e s u l t s Figure 1 shows the p H dependency of the extraction of Cu, M n and Zn. For this determination an
1,37 MeV 2~Na ~
0,51 MeV
I
A
1-75 MeV
10s, O
~-I0~ t/1
S~Cu
SSMn
c o
<-)103
102.
Energy FIG. 2. y spectra of a tumor sample, irradiated for 1 h (thermal neutron flux ~ = 2 × 10lz n.em -2 see-l), measured with a 40 cm s Ge (Li) detector: (a) after wet ashing of the sample but prior to chelation; (b) chelated sample.
Technical notes artificial mixture was used. It consisted of about 200 m g freeze dried non-irradiated liver preparation mixed with a solution of radioactive Cu, M n and Zn. Both phases, organic and aqueous, were measured to determine the activity. Figure 2 shows the results of the separation in the case of a tumor sample which had been irradiated for one hour with a thermal neutron flux of 2 × l0 Is n.cm - s sec-x. Thereby the N a content was reduced to less than 0-01 per cent. Curve (a) has been multiplied by a scale factor to correct for different measuring conditions and to allow comparison to curve (b). I n the biological samples studied the
433
TABLE 1. Max content of a rat liver; Division 2: without chemical separation; Division 3 : determined after the chelation process P No.
/~g M n / g dry fiver
1 2 3 4
8-14 8-20 7.75 7.99
mean 4- SD
8-02 4- 0.2
8.4 8.5 7.7 8.0 8.15 4- 0-37
TABLE 2. Reproducibility of the process as tested on the Cu content of rat plasma
R a t No. I 2 3 4 5
(/~gCu/ml Plasma) Ist test 2nd test 1.31 1-27 1.09 1.11 1-07 1.11 1.19 1.23 1.23
contribution of the elements M n and Zn to the 0.51 M e V line is negligible compared to Cu. Table 1 shows the M n values for a rat liver sample which was divided into four parts before irradiation. As a check on the process the M n content was determined at first immediately after irradiation, without a chemical separation, and then after the chelation process. I n the case of liver samples this is possible because ~ M n can be determined despite the disturbing elements. These results are compared. Table 2 "shows the plasma Cu values for five rats. As a check on the reproduceability of total process the plasma of the individual rats was divided and individually dried, irradiated and worked up at two different days. T h e error of the method, which is composed of weighing, pipetting and statistical errors totals about 5 per cent. U n d e r optimal conditions one person can work up 30 samples within 6 hr in our laboratory. So far we have analysed 500 samples with this process.
1-31 1.07 1-15 1.01
M e a n 4- SD
1.29 -4- 0'02 I.I1 4- 0.03 1.04
1-25
1-18 4- 0"07
1.28 1.34
1-30 4- 0.05
Acknowledgements--This work was carried out as part of an investigation which is supported by the Bundesmlnisterium ftir Wissemchaftliche Forschung.
J.
H. WESCH ZIMMEa~R
J, SCHUHMACHER Institut fiir Nuklearmedizin des Deutschen Krebsforschungszentrums Heidelberg, Germany References 1. JERVIS R. W. and WONG K. Y. Symp. on Nud. Activation Techniques in the L / ~ Sd., p. 137. Amsterd a m (1967). 2. WORWOOD M. and TAYLOR D. M. Int. J. appl. Radiat. Isotopes 19, 753 (1968). 3. CHUECA A., WORWOOD M. and TAYLOR D. M. Int. J. appl. Radiat. Isotopes 20, 335 (1969). 4. MULFOm~ E. Analys. Ber., Vol. 9. Bodenseewerk Perkin-Elmer, l[lberlingen (translated from Atomic Absorption News Lett. 5, N o 4) (1967).
Rapid Preparation o f 14C-Blood P l a s m a for Liquid Scintillation Counting in D i m e t h y l s u l f o x i d e - T h l x o t r o p i c Gel* (Received 5 November 1969) A NUMBER of procedures have been described for the liquid scintillation counting of x4C or related beta emitting radionuclides in blood plasma. Most of these techniques suffer from one or more disadvantages (such as being expensive, time consuming, requiring equipment for combustion, or else being handicapped by severe quenching). ~x-4) We have developed a rapid procedure for the counting of such samples, based on the solubility of plasma in dimethylsulfoxide, and the ready admixture of the resulting solution with a thixotropic gel. Rats were injected intraperitoneally with 25 pCi of glycine-2-x4C. After 3 days, plasma was harvested. The plasma appears to readily dissolve in dimethylsulfoxide (26"6 ml plasma/100 ml solvent). The solution was added, in varying amounts, to 10 ml of a thixotropic gel (4 per cent Cab-O-Sil, Packard, in prepared dioxane). The dioxane had been prepared by adding to each liter: 7 g 2,5-diphenyloxazole (PPO), 0-05 g 1,4-di (2,5-phenyloxazolyl) benzene (POPOP) and 100 g naphthalene. By means of a4C standards of known activity, a curve of absolute efficiency was constructed as a function of the channels ratio, using a Packard 3375 liquid scintillation spectrometer (red channel: 50-1000, gain 14 per cent; green channel: 150-1000, gain 14 per cent). Addition of radiolabeled plasmadimethylsulfoxide equivalent to 0.5 ml plasma resulted in a counting efficiency of 83 per cent. One ml of plasma could be counted at 75 per cent efficiency.
References I. VON SCHUCHmG S. and E ~ m ~ O F F C. W. A ~ t . Biochem. 5, 93 (1963). 2. KRAGELUIffD E. and DX,~BYE M. Stand J. din. Lab. Invest. 19, 129 (1967). 3. NJ~a)xc~m~x G. D., SINOH B. and JEEJEE~HORY K. N. Int. J. appl. Radiat. Isotopes 19, 685 (1968). 4. ME~a~E R. C. and STIGLITZ R. A. Int. J. appl. Radiat. Isotopes 13, 11 (1962). * Supported by T-492 from the American Cancer Society and by U S P H S Grants C A 06519 and A M 09429.
International Journal of Applied Radiation and Isotopes, 1970, Vol. 2 I, pp. 431--433. Pergamon Press. Printed in Northern Ireland
S i m u l t a n e o u s Detes~mination o f Copper, M a n g a n e s e and Zinc in Biological Materials by Means o f Neutron Activation Analysis and Chelate Extraction (Received 30 September 1969)
1. Introduction
WHEN investigating trace element concentrations in biological materials, one must work up a large number of samples to ensure statistical validity of the results. If the element in question cannot be directly measured (Fig. 2a), activation analysis becomes more difficult because of the required radiochemical separation. It is most important that the disturbing elements Na, K, P, C1 and Br be removed. Processes for the radiochemical separation of Cu, Mn, Zn (and others) are described for example by JERVlS and WONG (1) and TAYLOR et al. c2"3~ The RICHARD P. SPENCER articles deal almost exclusively with column separaMARY ANN BANERJI tions. The process used in our work depends on the use of a metal ion chelation followed by a twoSection of Nuclear Medicine, Department of Radiology phase extraction as described by MULFORDc4). Our Yale University School of Medicine chelating agent is AmmoniumpyrolidindithiocarbNew Haven amate (APDC) and our organic extraction agent is Conn. 06510, U.S.A. 431
432
Technical notes
Methylisobutylketon (MIBK). Thereby, Cu, M n and Zn are extracted quantitatively into the organic phase, while the disturbing elements remain in the aqueous phase or are removed by the wet ashing.
I
i
I
I
I
100
2. M e t h o d After a metal-free removal from the animal the samples are freeze dried, homogenized and then, after repeated drying, weighed in polystyrene capsules. T h e y are then irradiated for 2-8 hr with a thermal neutron flux of 2 × 10TM n.cm -2 sec-1. After irradiation the sodium content of the samples is determined by measurement of ~iNa. T h e n the contents of the capsules are wet ashed by means of H 2 S O ~ and I-I~O2. After removal of the excess H 2 0 2 by evaporation a drop of methyl red is added to the clear or light yellow solution. O n e then adds concentrated NaOI-I until the colour changes. T h e changing point is chosen so that no hydroxides precipitate. T h e n with dilute H C I one adjusts the p H of the solution to 3-4.5. F r o m this solution one takes 7.5 ml and again the sodium content is determined. F r o m this figure one can calculate the total amount of sample actually present in the tube. F r o m this point one must work quantitatively, 2 ml of a 2 per cent aqueous A P D C solution are added, as well as 5 ml M I B K . After thorough mixing followed by a 1 rain centrifugation (appr.
x o
13 50
I 2
I
I
a
Zn
•
Cu
[] Mn
3 4 5 6 pH of aqueous solution prior to chelation
FIG. 1. p H
dependency of the extraction of Cu, M n and Zn.
1000 g) one gets a sharp phase-separation. O n e puts 4 ml of the yellow to dark brown organic phase into a tube and measures its activity with a Ge (Li) crystal. T h e aqueous phase should be clear if the wet ashing and pI-t adjustments have been properly done. 3. R e s u l t s Figure 1 shows the p H dependency of the extraction of Cu, M n and Zn. For this determination an
1,37 MeV 2~Na ~
0,51 MeV
I
A
1-75 MeV
10s, O
~-I0~ t/1
S~Cu
SSMn
c o
<-)103
102.
Energy FIG. 2. y spectra of a tumor sample, irradiated for 1 h (thermal neutron flux ~ = 2 × 10lz n.em -2 see-l), measured with a 40 cm s Ge (Li) detector: (a) after wet ashing of the sample but prior to chelation; (b) chelated sample.
Technical notes artificial mixture was used. It consisted of about 200 m g freeze dried non-irradiated liver preparation mixed with a solution of radioactive Cu, M n and Zn. Both phases, organic and aqueous, were measured to determine the activity. Figure 2 shows the results of the separation in the case of a tumor sample which had been irradiated for one hour with a thermal neutron flux of 2 × l0 Is n.cm - s sec-x. Thereby the N a content was reduced to less than 0-01 per cent. Curve (a) has been multiplied by a scale factor to correct for different measuring conditions and to allow comparison to curve (b). I n the biological samples studied the
433
TABLE 1. Max content of a rat liver; Division 2: without chemical separation; Division 3 : determined after the chelation process P No.
/~g M n / g dry fiver
1 2 3 4
8-14 8-20 7.75 7.99
mean 4- SD
8-02 4- 0.2
8.4 8.5 7.7 8.0 8.15 4- 0-37
TABLE 2. Reproducibility of the process as tested on the Cu content of rat plasma
R a t No. I 2 3 4 5
(/~gCu/ml Plasma) Ist test 2nd test 1.31 1-27 1.09 1.11 1-07 1.11 1.19 1.23 1.23
contribution of the elements M n and Zn to the 0.51 M e V line is negligible compared to Cu. Table 1 shows the M n values for a rat liver sample which was divided into four parts before irradiation. As a check on the process the M n content was determined at first immediately after irradiation, without a chemical separation, and then after the chelation process. I n the case of liver samples this is possible because ~ M n can be determined despite the disturbing elements. These results are compared. Table 2 "shows the plasma Cu values for five rats. As a check on the reproduceability of total process the plasma of the individual rats was divided and individually dried, irradiated and worked up at two different days. T h e error of the method, which is composed of weighing, pipetting and statistical errors totals about 5 per cent. U n d e r optimal conditions one person can work up 30 samples within 6 hr in our laboratory. So far we have analysed 500 samples with this process.
1-31 1.07 1-15 1.01
M e a n 4- SD
1.29 -4- 0'02 I.I1 4- 0.03 1.04
1-25
1-18 4- 0"07
1.28 1.34
1-30 4- 0.05
Acknowledgements--This work was carried out as part of an investigation which is supported by the Bundesmlnisterium ftir Wissemchaftliche Forschung.
J.
H. WESCH ZIMMEa~R
J, SCHUHMACHER Institut fiir Nuklearmedizin des Deutschen Krebsforschungszentrums Heidelberg, Germany References 1. JERVIS R. W. and WONG K. Y. Symp. on Nud. Activation Techniques in the L / ~ Sd., p. 137. Amsterd a m (1967). 2. WORWOOD M. and TAYLOR D. M. Int. J. appl. Radiat. Isotopes 19, 753 (1968). 3. CHUECA A., WORWOOD M. and TAYLOR D. M. Int. J. appl. Radiat. Isotopes 20, 335 (1969). 4. MULFOm~ E. Analys. Ber., Vol. 9. Bodenseewerk Perkin-Elmer, l[lberlingen (translated from Atomic Absorption News Lett. 5, N o 4) (1967).