neutron activation analysis of magnesium, calcium, strontium, barium, manganese, cobalt
TRANSCRIPT
JOURNAL OF NUCLEAR MEDICINE 7:917-927, 1966
Neutron Activation Analysis of Magnesium,Calcium, Strontium, Barium, Manganese,
Cobalt, Copper, Zinc, Sodium, and Potassiumin Human Erythrocytes and Plasma1
D. A. Olehy,2 R. A. Schmitt,3 and W. F. Bethard
San Diego, California
Except forstrontiumand barium,the elementsconsideredin thisstudyareof known biologicalimportance.Strontiumand barium are of toxicologicinterest. The method presented was developed in conjunction with studies on thechanges in concentrations of selected cations in human erythrocytes duringstorage (1).
Samsahi,Brune,and Wester (2,3,4,5) have developedexcellention exchange procedures for separating many radioactivated elements in biologicalmedia. These procedures involve group separations, using more than one type ofresin, i.e., anion, cation, and mixed bed. It was desired that a rapid and simplifiedion exchange method be devised for selected cations which would have gooddecontamination factors with a minimum of chemistry. Parr and Taylor (6) developed an ion exchange method for Co, Cu, Fe, and Zn, using an anion resinequilibrated with hydrochloric acid. Co, Cu, and Fe were separated as a groupfrom Zn after decontaminating for sodium-24.
Using Kraus and Moore's data (7) on the adsorption coefficients of elementsfrom hydrochloric acid as a guide, the selective elemental elution method wasformulated.
It is conceded that atomic absorption, emission spectrometry, and othermethods of analysis (8, 9, 10) may be just as accurate and more convenient forcertain elements, such as sodium, potassium, and group II A metals; however,for some elements radioactivation has certain advantages, depending on thematrix and trace element concentrations.
1These studies were supported in part by the Office of Naval Research, Department ofthe Navy, under Contract Nonr-4397(00).
2Present address: National Reactor Test Site, Idaho Falls, Idaho.3Present address: Department of Chemistry, Oregon State University, Corvallis, Oregon.
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918 OLEHY, SCHMITr, BETHARD
APPARATUS AND REAGENTS
Columns used were 1 cm2 in cross sectional area by 10 cm with a coarsesintered-glass filter, stopcock. Resin used was Bio-Rads Dowex 1 x 10, 200 to400 mesh in Chlorideform. MallinckrodtTransistAR grade hydrochloricandnitric acids were used together with Mallinckrodt reagent-grade sodium hydroxide, calcium carbonate, magnesium metal, zinc, cobalt nitrate, strontiumcarbonate, barium nitrate, and manganese dioxide. Copper was from 99.999%pure copper foil. These reagents were used to make up the combined carriersolution.
Resin was prepared for use by rinsing with a 2:1 ratio, by volume, of deionized water to resin allowing the resin to settle, then decanting the liquid. Thisprocedure was repeated three to four times until the liquid was clear above theresin.
Elementalstandardswere made from Johnson,Matthey and Co.,Ltd.,Specpurereagents.
EXPERIMENTAL
Blood samples were collectedintravenouslywith polyethylenesyringesorfrom blood-bankstores.Sample volumes averaged 15 ml of whole blood,fromwhich 5 ml aliquots of plasma were taken, and 5.0 to 6.0 ml of red blood cellswere used for the analyses. Specimens were transferred to clean Vycor cruciblesand placedinan oven at 1100 to 120°Cto evaporatethe water.Afterthe specimens were thoroughly dry, they were placed in a muffle furnace at 200°to 250°Cand the temperature was gradually increased to a maximum of 550°C over an
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919NEUTRON ACTIVATION ANALYSIS IN ERYTHROCYTES AND PLASMA
eight-hour period; then they were allowed to stand overnight for complete ashing.However, the plasma samples may require a much longer period for ashing inorder to avoid fusing the samples to the Vycor crucibles.
Vycor crucibles were chosen because some of the cations absorb to platinumcrucibles while in the furnace, apparently attributable to the reducing atmospherecreated by carbon monoxide and dioxide from the specimens. This condition wasconfirmed by using radioactive tracers in specimens during ashing in order tocheck on volatility losses. After cooling, the specimens were quantitatively transferred to clean, weighed, 10 ml polyethylene vials for irradiation. The averageweight of ash per unit volume for red blood cells and plasma was 8.9 mg/ml.
Specimen ash and elemental standards were irradiated for 30 minutes at aflux of 2 x 1012 neutrons cm2 sec' in the General Atomic TRIGA reactor.At the end of irradiation, the specimens were removed and transported to thelaboratory as quickly as possible. It should be noted that prior to irradiation theion exchange columns had been equilibrated with concentrated hydrochloricacid and all necessary equipment, carrier solutions, and other apparatus were prepared for immediate use.
cHEMICAL SEPARATION OF ELEMENTS
The irradiated specimen ash was transferred to a test tube containing 1.00ml of mixed carriers having approximate concentrations of magnesium (10.00mg), calcium (5.00 mg), manganese (0.2-0.3 mg), cobalt (5.00 mg), copper(5.00 mg), zinc (5.00 mg), strontium (5.00 mg), and barium (5.00 mg). Thepolyethylene vial was rinsed with concentrated hydrochloric acid to dissolveany remaining ash on the walls of the vial, and this residue was added to the
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920 OLEHY, SCHMiTT, BETHARD
carrier solution. Eight to ten drops of 30%hydrogen peroxide were added to thecarrier solution with enough concentrated hydrochloric acid to make the totalvolume of solutionabout 10 milliliters.The solutionwas heated to boilingtodissolve the ash and to reduce manganese (IV) to manganese (II), as well asto destroyexcesshydrogen peroxide.More hydrochloricacid was added untilthevolume was approximately13 ml, then the solutionwas put throughtheionexchange column. Initialelutionwas performed under lessthan 1 psinitrogenpressurewith a maximum rateof 1 cm3 per mm.
Because of the shorthalflivesof 9.5-min2TMg and 8.8-min49Ca,pressureis necessary to speed the elution. The initial effluent, which contained approximately 50% Mg plus Ca, Sr, and Ba activities, was collected. This effluent wasdilutedwith about 10 ml ofwater and a few dropsof phenolphthaleinindicatorand 19 N sodium hydroxidewas added untilitwas neutralwith slightexcess.Sodium carbonatewas added at0°CtoprecipitatethecarbonatesofBa, Ca, Mg,and Sr.Aftercentrifugingand decantingthe supernate,the precipitatewas dissolvedin a few drops of 6 N nitricacid,dilutedand reprecipitatedwith 6 Nsodium hydroxide,adding 7 to 8 drops in excessof the end pointand againadding1 to2 ml ofsodium carbonateduringcoolinginan icebath.Aftercentrifugingand decanting,the precipitatewas washed once or twicewith deionizedwater,dissolvedin a few drops of 6 N nitricacid,and transferredto a cleanpolyethylenevial.The volume was normalizedwith the standardsof Mg, Sr,Ca,and Ba and counted.
Gamma-ray spectrawere taken of allelementsanalyzed,using a multichannelanalyzerwith3 in.X 3 in.sodium iodidethallium-activatedcrystals,bothwelland solidtypes.Sincethe2TMg,49Ca,sTmSrand 139Baactivitiesareallcom
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NEUTRON ACTIVATION ANALYSIS IN ERYTHROCYTES AND PLASMA 921
bined,two spectraarenecessary.To obtain49Ca and 2TMg, spectrawere takenat20 to 30 keV per channelin a wellcrystal(seeFig.1),then recountedat 5to6 keV perchannelfora longerperiodtoobtaingood spectraofsTmSrand 139Baactivities(seeFig.2).The gamma raysof theseparticularisotopes(‘39Ba,0.16
MeV; 8TmSr,0.39MeV; 27Mg, 0.84-1.0MeV;and 49Ca,3.1MeV) have sufficientenergydifferencesso as not to interfereappreciablywith each other.Spectrumstrippingof the Compton contributiondue to high energy gamma rayswoulddelineate more sharply the ‘39Baand 8rmSr photopeaks and reduce considerably the statistical error in Ba and Sr analysis.
After the initial effluent was collected, an additional 10 ml of concentratedhydrochloric acid was put through the column and the eluent set aside. Manganese was eluted with 10 to 13 ml of 6 N hydrochloric acid and two hydroxideprecipitationswere performed afteradditionof 2 to 3 mg of iron (III)carrier.Aftera waterwash,theprecipitatewas dissolvedwith a few dropsof 6 N nitricacidplus1 to2 dropsof30%hydrogen peroxideand transferredtoa polyethylenevialforcounting(seeFig.3).
Another8 to10 ml of6 N hydrochloricacidwas elutedand setasidebeforeelutingthe°°Coactivity.Cobaltwas elutedwith 10 to 13 ml of4 N hydrochloricaciddirectlyintoa 4-dram polyethylenevialforcounting.This stepisoptionaland canbe usedwhen highcobaltcontentissuspected.'
1The Co sensitivity via counting 5.3-yr 60Ca and with the irradiation time and neutronflux specified above is about 2 micrograms, which is about 20 to 40 times above the Co contentof whole blood. Extension of the irradiation time to 10 hours or increasing the neutron flux bya factor of 20 to 40 will yield the necessary sensitivity. On the other hand, the Co sensitivityvia 10.5-mm 6OmCounder the above irradiation conditions and to the exclusion of the otherelementsisestimatedatabout 0.4micrograms.
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922 OLEHY, SCHMJiI', I3ETHARD
Copper was nextelutedwith 10 ml of 2 N hydrochloricaciddirectlyintoa4-dram polyethylene vial, and spectra were taken (see Fig. 4). After the copperelution, 10 ml of 0.01 N hydrochloric acid was put through the column and discarded to eliminate any iron activity.
Zinc was eluted with 10 to 15 ml of deionized water. If zinc content is high,the eluent can be collected in the same manner as cobalt or copper and thespectra taken directly. However, if the zinc content is low, as it is in bloodplasma,then itisbestto collectthe eluentin a testtube,precipitatezincsulphide from a warm ammoniacal solution with hydrogen sulphide, dissolve theprecipitate with a few drops of 6 N nitric acid, transfer it to a 2-dram polyethylene vial and make the count in a well crystal (see Fig. 5).
Sodium and potassium can be determined instrumentally by collecting allthe discarded eluents and supernates up to the cobalt elution in beakers andevaporating the combined solutions to about 50 ml. In separate beakers dilutethe 24Na and 42K standardsto the same volume as the samples,normalizingalltoone volume.Beakersshouldbe coveredwith Parafilmto preventsplashingorspilling.Spectrashouldbe takenat the appropriategeometry afterallowingforone to two days decay (seeFig.6) and subtractingout 24Na to determinethe42Kcontent(seeFig.7).
cHEMICAL YIELDS
Chemical yields were determined by neutron activation of the recoveredsolutions and comparing them with the appropriate standards, using a flux of7.6 x 1010 neutrons cm2 sec' for 10 minutes.
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NEUTRON ACTIVATION ANALYSIS IN ERYTHROCYTES AND PLASMA 923
Because of the low abundances and crosssectionsof 46Ca and 48Ca,thechemicalyieldsfor calcium could not be determinedwith sufficientaccuracyby thismethod. Therefore,an EDTA titrationprocedurewas used,with calceinasan indicator:(10)
pH 12Ca-calcein + EDTA @‘Ca@+ + calcein
(fluorescent) (brown nonfluorescent)
Under these conditions magnesium does not interfere, but strontium and bariumwill be titrated together with calcium. However, strontium and barium can besubtractedafterdeterminationof theiryields.
RESULTS AND DISCUSSION
The present ashing procedure has been tested for volatility losses at thetemperaturesused with tracesof the elementsbeing analyzed,and lessthan1%loss was the maximum with the exception of potassium. A comparison experiment was made by taking aliquots of red blood cells and determining theirsodium and potassium contents by instrumental neutron-activation analysis. Afterallowingthesodium and potassiumactivitiesto decay away, thesesame aliquotswere ashed and put through the radiochemicalprocedure.The concentrationsdeterminedinthismanner differedby 16% forpotassium,but no change withinexperimentalerrorwas noted forsodium.Sodium and potassiumrecoverywasperformed, using the radiochemical separation procedure by adding knownamounts of 24Na and 42K activitiesand 98% to 99% recoverywas achieved.
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924 OLEHY,ScHM.Wr, BEThARD
A quartz liner was made for the muffle furnace to prevent contamination ofthe samples by flaking from the furnace walls. Error due to neutron flux changeswas averaged by rotation of samples and standards in the rotary rack of theTRIGA reactor at approximately 1 rpm.
When 56Mn is being determined in red blood cells, a correction is necessarybecause of the 56Fe ( n,p ) 56Mn reaction. This reaction has been shown ( 1 ) tocontribute 30% to the total 58Mn activity.
Figures one through seven show typical spectra obtainable with this procedure. For spectra of individual isotopes, see Heath's Catalog (11).
Table I compares the average normal erythrocyte and plasma valuesobtained with the ranges published in the literature. The average values shownfor magnesium, manganese, copper and zinc in red blood cells were from 15individuals, and the plasma values were from 12 individuals. Calcium in redblood cells was from duplicate samples from one individual and calcium inplasma was from four individuals. Strontium and barium in the plasma were determinedfrom fiveindividuals.Sodium and potassiumin red blood cellswerefrom sixindividuals.
Correctionforthe trappedplasma contributionto red cellvalueswas notmade because, for most of the elements analyzed, the amounts are negligiblein relation to the technical errors. The exception would be for sodium and calcium
from plasma, according to Valberg, Ct al., (8). They have shown by two differentcorrection methods that trapped intracellular plasma does contribute substantiallyto sodium and calciumvaluesof erythrocytes.One method was accomplishedby usinga compositecalibrationcurvepreparedwithEvans bluedye fortrappedplasma correction;the second and more accuratemethod involvedthe use of1311, labeled serum albumin (RISA), on each individual blood sample. Both the
mean values and ranges of erythrocyte sodium and calcium were smaller usingRISA measurements than those obtained with a composite calibration curve.Therefore, an average of 40% to 50% correction is necessary; e.g., the corrected Navalue in erythrocytes is 0.20 mg/mI.
All samples analyzed had ACD preservative added. This preservative solution and heparin, another commonly used anticoagulant, were analyzed. It wasfound that the samples with the ACD solution contained negligible amounts ofthe elements concerned, except for sodium, and that heparin contained substantial amounts of manganese, copper and zinc (1). The statistical errors in counting magnesium, manganese, copper, zinc and sodium in red blood cells averaged0.5% to 1.0%; in potassium, 2%to 4%; in strontium, 12%to 15%; in barium, 20% to 50%;and in calcium, 40% to 50%. The statistical counting errors for the elements determined in blood plasma were 3% to 4% for magnesium, 20% to 30% for calcium,5% to 15% for strontium, 30% to 50% for barium, and 1% to 2% for manganese,copper, and zinc.
Weighing errors and transferring losses can be limited to 1.0% if sufficientcare is taken in handling. Chemical yield errors involve only the statistical counting errors, since the recovered carrier is irradiated and counted without otherchemistry or transfer of solution. Since the elements in the solutions are in
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926 OLEHY,ScHMITr, BETHARD
macro amounts, the errors are less than 2.0%. The titrimetric error for calcium isalso less than 2.0%. The percentage range of chemical yields are given in Table II.
Sodium and potassium values for plasma have not been indicated in Table Ibecause they have been well established by other methods. Strontium and bariumin erythrocytes have apparently never been determined. Since values in the literature are only for serum or whole blood, no literature values are listed in Table I.
TABLE II
PERCENTAGE RANGE OF CHEMiCAL YIELDS
Range
Elements (%)
Na 98—100Mg 40—55K 98—100Ca 30—50
1\ln 80—90Cu 80—90Zn 70—90
Sr 8—40Ba 8-40
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NEUTRON ACTIVATION ANALYSIS IN ERYTHROCYTES AND PLASMA 927
SUMMARY
A method has been described for quantitation of Mg, Ca, Sr, Ba, Mn, Co.Cu, Zn, Na and K in human erythrocytes and plasma by thermal neutron activation and rapid ion exchange radiochemical separation. Representative valueshave been presented and compared with previously published ranges, whereavailable in the literature. Limits of error inherent in the method have been given.
REFERENCES
1. BETHARD,W. F., D. A. OLEHY, A1@DR. A. ScHMrrr: The Use of Neutron ActivationAnalysis for the Quantitation of Selected Cations in Human Blood, L'Analyse Par Radio
activation et ses Applications aux Sciences Biologiqves. University Press of France, Paris,France (1964).
2. SAMSAHL,K.: A Fast Radiochemical Method for the Determination of Some EssentialTrace Elements in Biology and Medicine, Aktiebolaget Atomenergie Report AE-168 (December 1964).
3. WESrER, P. 0., D. BRUNE, AND K. SAMSAHL: Radiochemical Recovery Studies of a Separation Scheme for 23 Elements in Biological Material, Intern. I. Appi. Radiation and Isotopes15:59-67 (1964).
4. BRUNE, D., K. SAMSAHL,AND P. 0. WESTER: Determination of Elements in Miii-,Micro-, and Submicrogram Quantities in Human Whole Blood by Neutron Activation Analysis,Atompraxis 9:368 (1963).
5. BRUNE, D., K. SAMSAHL,ANDP. 0. WESTER: “TheAmount.s of As, Au, Br, Cu, Fe,Mo, Se, and Zn in Normal and Uraemic Human Whole Blood. A Comparison by Means ofNeutron Activation Analysis. Aktiebotaget Atomenergie Report AE-134 (1964).
6. PARR, R. M., AND D. M. TAYLOR: The Concentrations of Cobalt, Copper, Iron andZinc in Some Normal Human Tissues as Determined by Neutron Activation Analysis, Biochem.1.94:424 (1964).
7. Ki@us, K. A., AND G. E. MOORE: Anion Exchange Studies, VI. Divalent TransitionElements Manganese to Zinc in Hydrochloric Acid, J. Am. Chem. Soc. 75:1460-1466 (1953).
8. VALBERG, L. S., J. M. H0LT, E. PAULSAN, AND J. SZIVEK: Spectrochemical Analysis
of Sodium, Potassium, Calcium, Magnesium, Copper, and Zinc in Normal Human Erythrocytes,
J. Clin.Invest. 44, No. 3 (1965).9. WACKER, W. E. C., C. ImA, AND K. FuwA: Accuracy of Determinations of Serum
Magnesium by Flame Emission and Atomic Adsorption Spectrometry, Nature, 202:659 (1964).
10. DuniL, HARVEY: Calcein, Calmagite and o,o'-Dihydroxyazobenzene Titrimetric,Colorimetric, and Fluorometric Reagents for Cakium and Magnesium. G. Frederick SmithChemical Company (1964).
11. HE.ATH,R. L.: Scintillation Spectrometry Gamma-ray Spectrum Catalogue, 2nd ed.,USAEC Report TID-4500 (1964).
12. WINTROBE, N. M.: Clinical Hematology, 5th ed., Lea and Febiger (1961).
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1966;7:917-927.J Nucl Med. D. A. Olehy, R. A. Schmitt and W. F. Bethard Cobalt, Copper, Zinc, Sodium, and Potassium in Human Erythrocytes and PlasmaNeutron Activation Analysis of Magnesium, Calcium, Strontium, Barium, Manganese,
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