Radiochemical determination of twelve trace elements in human blood serum

Radiochemical determination of twelve trace elements in human blood serum

Amlytrca Chumca Acta, 257 (1992) 1-5 Elsewer Science Publishers B V , Amsterdam Radiochemical determination of twelve trace elements in human blood...

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Amlytrca Chumca Acta, 257 (1992) 1-5

Elsewer Science Publishers B V , Amsterdam

Radiochemical

determination of twelve trace elements in human blood serum Dana Van

Renterghem and Rota Cornehs *

Laboratory for Analytrcal Chenustty, Rgksunwersttert

Gent, Proefturnstraat 86, B-91300 Ghent (Belgumz)

Raymond Vanholder Nephrologv Department,

Unrverstty Hospital, De Pzntelaan 185, B-9000 Ghent (Belguud

(Received 8th July 1991)

Abstract

Trace and ultra-trace levels of As, Au, Cd, Cs, Cu, Fe, Hg, MO, Rb, Sb, Se and Zn can be determmed sunultaneously m human blood serum by radlochenucal neutron actlvahon analysis Combustton of the lrradlated serum m a Trace-O-Mat apparatus, followed by 1011exchange, allows the detectlon of the y-rays of Interest To calculate the concentrations, the k, standardlzatlon method, adapted for the speclflc sample composltlon and sample-detector cotiguratlon, IS applied The accuracy and preclslon of the method were checked by the analysis of a second-generation reference matenal The results agreed wth the recommended values avallable for most of the elements Ktywordr Acfivatlon analysis, Blood, Serum, Trace elements

Radiochemical neutron activation analysis (RNAA) IS a very sultable technique for studying trace element patterns m human blood serum It 1s a senntlve, precise and accurate multi-element technique provided that post-lrradlatlon chemical separations are applied In this study, the orgamc anion exchanger Dowex l-X8 was employed to eliminate the major radloactlvltles of 82Br CT,,, = 35 3 h), 42K (Tl,2 = 12 36 h) and “Na (Tl,2 = 14 96 h) and to group the elements of interest m such a way as to mmlm1ze y-spectral mterferences This paper describes the u-radlatlon, the development of the destruction-separation procedure and the accuracy of the method

EXPERIMENTAL

Sample a!estructzon After a decay period of 18 h the sample 1s pressed to a pellet and IS decomposed m a TraccO-Mat apparatus (Kurner, Rosenheun) (Fig 1) [1,2] by combustion m an oxygen atmosphere (1 1 mm-‘> with the aid of two halogen infrared reflecting lamps The lamps are turned to the hlghest posltlon and switched on at maximum power for 15 s The volatile elements and the combustion products are deposlted m the cooling system, filled with liquid nitrogen After 2-3 mm, when the sample has been completely ashed, the mtro-

0003-2670/92/$05 00 0 1992 - Elsevter Science Pubhshers B V All rights reserved

2

D VAN

RENTERGHEM

ET AL

rinse with 2 ml of 9 M HBr, invert the sample holder, reflux for 5 mm with 4 ml of 10 M HCl, and rinse twice with 4 ml of 10 M HCl The liquids are brought into the apparatus via the water condenser A penstaltlc pump transfers the sample ahquots to the column via a side-arm at the bottom of the test-tube Separatwn procedure The separation scheme, outlmed m Fig 2, was

_______________ apparatus I, Test-tube, II, bummg chamber, III, coolmg unit, 1, sample holder, 2, lamp, 3, FIN 1 Trace-O-Mat

coolmg Jacket, 4, coohng finger, 5, O2 supply, 6, H,O supply, 7, N, supply

designed on the basis of exlstmg schemes for chromatographlc separations and on systematic surveys of K, values on organic ion exchangers m different media, more speclflcally a table by Kraus and Nelson [3] Preparation of the column The anion exchanger Dowex l-X8 (100-200 mesh, Cl-form) was purchased from Serva As the techmcal-grade product m the batch used exhlblted the same properties as the purified form, the resm could be used urlthout preliminary purlficatlon This mformatlon IS Important, because the quality of the resin drastically influences the behavlour of As, Cu and Se About 2 5 g of resin (dry weight ca 15 g) IS converted from the Cl- mto the Br- form by washmg with 30 ml of 9 M HBr To prevent the resin from bemg displaced, PTFE-wool 1s put on top of the column

Sample

gen supply 1s closed, the coolmg system 1s thawed out by a built-m fan and the lamps are lowered The elements of mterest are collected m the test-tube by refluxmg with an acid, suitable for the subsequent separation During the reflux process the lamps operate at half power and a gentle stream of oxygen 1s maintained to prevent the gases from entering the oxygen mlet The reflux process, resulting m an almost quantitative recovery of the elements of interest, IS as follows reflux for 1 h with the carrier solution, 1 e ,2 ml of HBr + 2 7% H,O,, contammg 100 pg of the elements eluted (As, Cs, Cu, Rb and Se) and 10 pg of the elements retamed on the ion exchanger (Au, Cd, Hg, MO, Sb and Zn), reflux for 15 mm with 2 ml of 9 M HBr,

Br- form 20 ml 10M HCI 2 ml IOM HCI + 1 % H,O,

Cs, Cu, Fe(II), Rb, Br, K, Na

18ml3MHCI+02%H,O,

As, Cu, Fe(II), Se

Fig 2 Separation scheme

DETERMINATION

OF 12 TRACE

ELEMENTS

IN BLOOD

SERUM

Elutwn Control of the oxldatlon state of As, @-, Cu and Se 1s achieved by addltlon of 100 ~1 of a saturated aqueous ammomum iodide solution on top of the column Subsequently, a penstaltlc pump transfers the sample ahquots from the Trace-O-Mat apparatus on to the column The elutmg agents 10 M HCl, 10 M HCl + 1% H,O, and 3 M HCl+ 0 2% H,O, are used m sequence The adsorption and elutlon flow-rates are adjusted to 0 5 ml mm-’ Tracer expenments

The destruction-separation procedure was developed with the aid of the radiotracers 76As (T,,, = 26 32 h), lg8Au CT,,, = 64 68 h), “Br (TI,* = 35 30 h), ‘15Cd (TI,2 = 53 46 h), 13’Cs (TI,2 = 30 1 a), @Cu (TI,2 = 12 70 h) 59Fe CT d), 203Hg (T,,2 = 46 612 d), 4’K (TI,* %3”Q’i; 99Mo (TI,* = 66 02 h), “Na CT,,, = 14 96 h), “Rb (TI,* = 18 66 d), ‘“Sb CT - 60 20 d) 75Se (TI,2 = 119 77 d) and 65Zn ?q 1,2 - = 244 0 ‘d) About lo-20 ~1 of tracer solution were spotted on 500 mg of freeze-dned human blood serum After drymg overnight [4], the doped serum was wrapped m a piece of a preclslon wipe and pressed to a pellet (diameter and height ca 10 mm) with a Parr pellet press of human blood serum A second-generation reference material of human blood serum prepared by Versleck and coworkers [5,6], was analyzed to check the accuracy of the method This reference material contains similar amounts of the elements of interest m the same chemical form as the material studied To avold contammatlon prior to the irradlatlon, all sample handling was done m a dust-poor room and cleaned utensils made from very pure materials were used 171 A 500-mg amount of lyophlhzed serum was placed m a thoroughly cleaned polyethylene container The serum sample was Irradiated for 7 h m a thermal flux of 4 3 X 1Ol2 n cmW2 s-l m channel 17 of the Thetis reactor of the Institute for Nuclear Sciences, Umverslty of Ghent To calculate the concentration, the k, standardlzatlon method [S] was applied, based on the simultaneous xradlatlon of an Au-Al wire (0 5 mg, 0 10% Au,

3

diameter 1 mm, Atec) and a Zr foil (10 mg, thickness 0 125 mm, Goodfellow) as neutron flux monitors The Au also served as the comparator Tivo Ge(Ll) detectors (MK4 and MK7) were used for the y-spectrometrlc measurements The speclflcatlons for the MK4 are 194-keV FWHM with a volume of 85 cm3 and a diameter of active volume of 5 2 cm and for the MK7 2 0 keV, 70 cm3 and 4 65 cm respectively The detectors are calibrated m order to use them for the k, method For each sample the apparent density and the composrtion have to be determined This 1s easily done for eluents, but not for the resm In the resin fraction the radlolsotopes are concentrated on the resin, wthout detectable actmty m the supematant The apparent density of the resin phase, conslstmg of resm particles with 3 M HCl m between, 1s 0 9703 kg me3 The composltlon of the resin, based on chloride determmatlon by mstrumental neutron activation analysis, is 18 7% C1,74%N,647%Cand92%H The monitors were measured at 5 cm from the detector, the comparators and the samples were as close as possible to the detector and at the same distance The measuring times for the monitors and comparators vaned from 5 to 60 mm The samples were counted for 22-60 h

Analyst

RESULTS

AND DISCUSSION

Tracer expenments The results of the tracer experiments through-

out the radlochemlcal procedure are given m Table 1 Cs, Rb, K, Na, As, Cd, Hg, MO, Sb and Zn are almost quantitatively recovered m one of the three fractions Au 1s more than 95% retamed The combined recoveries of Se account for more than 98% Fe IS dlstrlbuted among the three fractions, with a total recovery of 96-102% In serum its concentration will be calculated, because of gamma spectrometnc conwderatlons, by summmg only the 10 M HCI and the resm fraction, and takmg mto account that the mean recovery of three tracer experiments for these hvo fractions 1s 84 6% * 0 8% (mean f standard devlatlon)

D VAN

4 TABLE 1 Results of the tracer experiments Element

Recovery (%I 10MHCl

3MHCl

Cs Rb Br K Na

>99 > 99 55-70 > 99 > 99 9

0 0 0

    Resm

    As Cu Se

    0 27-31 0

    > 98 68-71 75-78

    o-1 l-2 20-22

    AU Cd Fe Hg MO Sb Zn

    0 0 35-37 0 0 <02 <02

    0 0 13-14 0 0 0 <05

    > 95 100 48-51 100 > 99 > 99 > 98

    0 0

    05-15

      On combmmg the destruction and the separatlon, the recovery of Cu m the 3 M HCl fraction decreases from more than 95% to 69 7 f 14% The remaining 30% m the 10 M HCl eluent cannot be taken mto account because of the contrlbutlon of 24Na to the 511-keV emlsslon of 64Cu @+-emitter) by external pair production Although 30-45% of the Br 1s volatlhzed durmg the destruction, an mterfermg amount of *‘Br actlvlty remains m the sample solution To prevent the adsorptlon of more than 2% Br, the resm 1s converted mto the Br- form and the sample is dissolved m HBr The solvent contains hydrogen peroxide m order to oxldlze remammg orgamc particles The water formed dilutes the HBr so that the salts dissolve better The oxldatlon state of As, Cu and Se 1s controlled by the addrtton of a reductant In 9 M HBr or 10 M HCI, As(II1) 1s retamed, Se preclpltates and Cu(1) 1s eluted Potassmm or sodmm salts cannot be used as reductants because they partly preclpltate m a strongly acldlc medium, so the matrix actlvlty due to 42K and 24Na will be msufficlently removed If ammonium iodide IS used no preclpltatlon of potassium or sodium salts occurs and, especially, Cu 1s adsorbed as a [C&l- complex m the 9 M HBr or 10 M HCl

      RENTERGHEM

      ET AL

      medmm Thus complex IS destroyed by H,Oz and thus Cu 1s eluted as Cu(I1) with 3 M HCI The H,O, also oxldlzes the red Se precipitate and keeps Se m a soluble form [9] Selemum has to be removed as much as possible from the resm 111 order to mmmuze the spectral interference from 75Se (279 5 keV)-203Hg (279 2 keV) so that Hg can be determined accurately The amount of Se eluted (ca 75%) cannot be Improved by mcreasmg the percentage of H,O, or the amount of 10 M HCI + 1% H,O, or of 3 M HCl+ 0 2% H,O, Analyst of reference mated The accuracy of the destruction-separation

      procedure was checked by the analysis of the reference serum prepared by Versleck and coworker [5,6] As a test for the reproduclbrhty of the method, the serum was analysed fivefold Table 2 gives the mean concentrations, the recommended values and the 95% confidence mtervals The data for As, Cs, Fe, Hg, MO, Rb, Se and Zn he wlthm the 95% confidence mterval Cu 1s an exception, as Its mean value 1s slrghtly hrgher than the upper hmlt of the mterval The Au concentration 1s one and two orders of magmtude smaller than the values reported by Iyengar et al [lo] and Jarstad et al [ill, respectively This confirms the general downward trend of mea-

      TABLE 2 Results for twelve trace elements m reference matenal human blood serum Element

      MeanrtS D (n = 5)

      Recommended value

      95% confidence Interval

      As (ng g-l) Au (pg is-‘)

      176 f 10 73 rt12 <56 97 f 05 116 f 08 256 k 05 66 * 13 76+07 170* 005 015* 001 102* 005 94 + 03

      196 20 10 0 111 259 66 75 18.5 105 96

      1.56 -23 6

      Cd(ngg-l) Cs (ng g-l)

      cu (pgg-V

      Fe &Lgs-l) Hs (ng g-‘) MO (ngs-‘1 Rb (pg g-‘1 Sb (ng g-‘) Se (pg g-l) Zn (cLg g-9

      17-25 77 -123 107 -115 244 -274 62 - 70 67 - 83 152- 2 18 1 oo- 1 10 94 - 98

      DETERMINATION

      OF 12 TRACE

      ELEMENTS

      IN BLOOD

      5

      SERUM

      sured trace element concentrations at the ng g-’ or sub-ng g-l level m human tissues and body flurds The low concentration of Cd could not be determined For Sb, a reproducible result (0 15 f 0 01 ng g-l) was obtamed m spite of the bad countmg statlstlcs (14-43%) The 95% confidence interval 1s hmlted by the values 0 14 and 0 16 ng g-’ For Sb no recommended value 1s avalable The result 1s compared with a concentration of 0 25 f 0 15 ng g-l obtamed after lrradlatlon for 14 days m a thermal flux of 1014 n cm-’ s-l [121 This value 1s 15 times larger than that determmed m this work, possibly because the quartz containers used for such n-radiations contam measurable amounts of Sb [7] In this study, properly cleaned pure polyethylene contamers were used It can be expected that these containers give rise to neghgrble blank values, because recoil during an nradlatlon of only 7 h m a “low” thermal flux of 4 3 X lOI n cm-’ s-l 1s negligible and because it 1s not necessary to wash the sample out of the container as a consequence of the slight lrradlatlon damage The agreement between the results and the recommended values, If available, confnms thrs assumption The procedure described has been apphed to study the evolution of the concentration of the twelve trace elements m the serum of patients with end-stage renal failure, treated by haemodlafiltratlon 1131 In conclusion, the proposed destructlon-separatlon procedure pemuts the accurate and precise determination of the trace elements As, Au, Cd, Cs, Cu, Fe, Hg, MO, Rb, Sb, Se and Zn m human serum

      Fmanclal support for this work was obtained from the Interumverstty Institute for Nuclear SW ences and the National Fund for Scientific Research (Belgmm) (Research Director Dr R Cornehs)

      REFERENCES

      1 H-B Han, G Kaiser and G Tolg, Anal Chum Acta, 128 (1981) 9 2 G Knapp, SE Raptls, G Kaiser, G Tolg, P Schramel and B Schrelber, Fresenms’ Z Anal Chem, 308 (1981) 97 3 KA Kraus and F Nelson, Proc Int Conf Peaceful Uses Atom Energy, 7 (1956) 118 4 M Rhpelh, Anal Chem , 59 (1987) 116 5 J Versleck, J Hoste, L Vanballenberghe, A De Kesel and D Van Renterghem, J RadIoanal Nucl Chem, Artdes, 113 (1987) 299 6 J Versleck, L Vanballenberghe, A De Kesel, J Hoste, B Wallaeys, J Vandenhaute, N Baeck, H Steyaert, AR Byrne and F W Sunderman, Anal Chum Acta, 204 (1988) 63 7 J Versleck and R Cornells, Trace Elements m Human Plasma or Serum, CRC, Boca Raton, FL, 1989, pp 23-63 8 F De Carte, A Snnomts, A De Wlspelaere and J Hoste, J RadIoanal Nucl Chem , Articles, 113 (1987) 145 9 P S TJloe, J J M De Goel~ and J P W Houtman, J Radloanal Chem , 37 (1977) 511 10 G V Iyengar, W E Kollmer and H J M Bowen, The Elemental Composltlon of Human Tissues and Body Flmds, Verlag Chemle, Wemheun, New York, 1978 11 K Jorstad, B Salbu and AC Pappas, Anal Chem , 53 (1981) 1398 12 D Van Renterghem, Lxence Thesis, Rgksumversltelt Gent, 1984 13 D Van Renterghem, R Cornells and R Vanholder, Lab Clm Med , submltted for pubhcatlon