The determination of protein bound iodine by neutron activation analysis

International Journal of Applied Radiation and Isotopm, 1970, Vol. 21, pp. 225--236. Pergamon PreL Printed in Northern Ireland

The Determination of Protein Bound Iodine by Neutron Activation Analysis J. C. V E S E L S K Y , M . N E D B A L E K a n d O. S U S C H N Y International Atomic Energy Agency, Laboratory Seibersdorf, Austria

(Received 1 January 1969) Conventional methods of PBI determination are reviewed and a method for its determination by activation analysis described. PBI is separated from inorganic iodine in plasma samples by gel filtration in a phosphate buffer medium. It is then determined by an activation analytical procedure including chemical separation of I~8I, simple measurement of this isotope in an ordinary liquid beta-counting tube followed by a determination of the chemical recovery making use of an iodometric procedure. Commercial standards analysed by this method showed a PBI value which was consistently higher (by about 20 per cent) than the corresponding values obtained by a purely chemical method. Analyses carried out on rat plasma did not show such a difference. The effect is believed to be due to the composition of the standards used. LA DI~,TERMINATION PAR ANALYSE A A C T I V A T I O N NEUTRONIQ,UE DE L ' I O D E LI]~ A PROTI~INE (PBI) O n passe en revue les mOhodes conventionnelles de mesurer le PBI et on d~cHt une m~thode de le mesurer par l'analyse ~ activation neutronique. On s~pare le PBI de l'iode inorganique dam les dchantillons de plasma par filtration en gel dam un milieu tamponn~ de phosphate. Le dosage se fait ensuite par un procddd analytique h activation qui comporte la st'paration chimique du 1~I, la mesure simple de cet isotope dam u n tuyau h comptage b~ta comme est normal pour les liquides suivies d'une ddtermination du recouvrement chimique en employant une m~thode iodom~triqu¢. Des ~talons de commerce doses par cette m~thode montr~rent une valeur de PBI r~guli~rement plus forte par environ 20 pour cent que la valeur correspondante obtenue par une m~thode purement chimique. Des analyses faites sur du montr~rent une valeur de PBI r~guli~rement plus forte par environ 20 pour cent que la valeur plasma de rat ne rendirent pas de telles differences. O n croit que l'effet suit de la composition des ~talons employ¢~. O I I P E ~ E J I E H H E CBH3AHHOFO BEJIHAMH HO~A IIOCPE~CTBOM HEITITPOHHOFO AHTHBAI~HOHHOPO AHAJIH3A ~aoTcH o6aop 06~2qH0 HpHHHTr~IX MeT0~0B oHpeKe~eHHH CB~aaHHOrO 6exlICaM~ no~a x~ onHca-BaeTc~ MeT0~ OTOP0 onpe~eJtoH~ nyTeg aRTHBa~HOHH0r0 aHaaHaa. CnHSaHH~ 6eagaM~ zo~ o~ea~eTc~ 0T HeopraHn~ec~oro ~o~a B npo6ax Haa3M~ ~HJ~hTpOBaHHeMr e ~ B cpe~e ~oc~aTHOr0 6y~epa. 3a~eM OH onpe~eJI~eTc~ aHTHBa/~HOHH/~IM aHaJIHSOM; B Hpo~Ie~ypy BX0~HT XHMH~ecH0e 0~IeJ~eHHe xa*I, IIp0CT0e H3MepeHHe 3T0r0 H30TOIIa B 05I~IqH0~ 5eTa-ctie'rHO~ ~py6~e, aa qeM cae~yeT XnMnqec~oe B0cc~aHOBJ~eH~e H0~OMeTpHqeCHHM cnoco6o~. B HpoM~mJIeHH~tXCTaH~apTax npoaHaJmaHpoBaHHkrX8THMMeT0~OM, 06HapymHBaeTcH HOCTOHHHO8Ha~eHHe CBHSaHHOrO 6eHHaMH Ho~a 6oaee s~cogoe (HpH6HHSHTeJ~HO H a 20~/o),

HeM C00TBeTCTByIOI~He BeHHqHHt~, I / 0 H y q e H H ~ e

qHCTO XHMHqeCHHM MeTO~OM.

Hp0Be~eHHt~e H a Hp/~ICHH0~IIJIa3Me aHaHHS~I He BI~HBJIHIOTTaH0~ paSHHI~. IIpe~iioJiaraeTCH~qTO pas~II4qHe 8aBHCHTOT C0CTaBa HCHOHI~yeM~XCTaH~apTOB. DIE B E S T I M M ~ N G VON P R O T E I N G E B U N D E N E M J O D D U R C H N E U T R O N E N - A K T I V I E R U N G S ANALYSE Es wlrd ein l~berblick fiber die gebrAuchlichen Methodcn zur PBI-Besfimmung gegeben mad ein aktivicrungsanalytischcs Verfahren beschriebcn. Das proteingebundene Jod wird 225

226

J. G. Varehk~, .hi. Nedbalek and O. Sua¢hny

vom anorganischen Anteil durch Gelfiltration in einem Phosphatpufl'er-Medium abgetrennt. Die PBI-Bestimmung erfolgt dann mittels einer aktivierurtgsanalytischen Technik, welche eine chemi~he Abtrennung des lssj, eine einfache Me~ung dieses Isotol~ in dnem gewflmlicheu Fliissigkeitsz~ihlrohr und eine chemische Ausbeutebestimmung dutch jodometrL~he Titration einschliellt. Nach diesem Verfahren wurden handelsiibliche PBI-Stmadards analyfiert und an diesen ein um etwa 20% htherer PBI-Wert gefunden als mit einer rein chemischen Methode. Analysen yon Rattenplasmen zeigten keinen derartigen Unterschied. Es ist anztmehmen, daIl die Differenz ihre Ursache in der Zusammemetzung der Standards hat. 1. I N T R O D U C T I O N IN T ~ course of an international intercomparison of chemical iodine determinations in blood serum, which was carried out under the sponsorship of the IAEA ~1}, the result (PBI) obtained by a neutron activation technique was found to be considerably higher (22 per cent) than the mean of the other results. Experience of this kind was also reported by other authorsJ ~ In the following we want to report on our investigations concerning the development of a PBI determination method based upon neutron activation w h i c h might be used to control the results obtained by purely chemical techniques. Analysis of PBI can be roughly divided into the following individual steps:

form of the "Schoeniger-method" (combustion of the sample in oxygen using the "Schoenigerflask") tts,~°~ or as oven-ashing in the presence of alkali, ts,ls-17,41~ in the latter case losses of iodine of 20 percent or more have sometimes been observed, ta't4-16,~) The addition of an oxidizing agent is of advantage, cl°~ For wet ashing, mixtures of chromic and sulfuric acids, ta,4,9.m chloric acid, tt-~'ls'~ or mixtures containing nitric and perchloric acids t49,5°~ are used. In our experiments the chromic-sulfuric acid mixture was found to give best results; the mixture was, therefore, used for the oxidation of PBI-samples. The most important techniques for the isolation of iodine from the reaction mixture are distillation and isothermal diffusion; other (1) Separation of PBI from inorganic iodine, authors use anion exchange to separate iodine (2) Mineralization of the organic material recoil atoms arising from iodine-bearing proteins during neutron irradiation. ~4s~ In the distil(without loss of iodine), lation method iodate generated in the ashing (3) Measurement of iodine. process is reduced with phosphorous acid and Methods for PBI-separation are based mainly the iodine distilled (sometimes with the aid of a upon precipitation, ion exchange, dialysis or carrier gas). In this process some iodine may be gel filtration. Precipitation of PBI is usually lost by adsorption on the walls of the still. A accomplished with trichloroacetic acid, ~4,5,4°~ variety of different distillation procedures ~,II,19~ perchloric acid t6,7) or zinc compounds; tll't7~ and distillation conditions have been usedJ tg'~l~ however, in the presence of large excess of in- T h e isothermal diffusion method, introduced by organic iodine this type of PBI-separation is Spitzy st al., for the separation of micro-amounts frequently a source of difficulties (see Ref. (8)). of iodine from reaction mixtures, t4~ the PBIFor an intercomparison of ion exchange separation by gel filtration and the utilization of methodstg.9.10.~s) with precipitation by trichloro- the Sandell-Kolthoff reaction for the final acetic acid and dialysis see Ref. (12), for a brief determination of the iodine have greatly critical discussion of precipitation, dialysis and facilitated the clinical PBI-determination. gel filtration~S) and of precipitation, gel filtration Ion exchangers are now used for PBI isoand ion exchangeJ ~ O f all these methods gel lation, especially in automated procedures (see filtration ts,4°,~ seems to be the most suitable below), but under certain circumstances (high technique which permits the rapid and safe concentration of inorganic iodine) they appear removal of large quantities of inorganic iodine less attractive, c~6~ (up to 20,000 #g/lOOmltS)); therefore, this The determination of very small amounts of procedure was adopted for the present work. iodine is possible in different ways: wet Mineralization can be carried out by wet or chemistry, X-ray fluorescence, activation analydry ashing procedures; the latter may take the sis or tracer methods may be employed. T h e

The. determination of prot~ bound iodine by neutron activation analysis most frequently used technique for PBI determinations makes use of the Sandell-Kokhoff reaction which is based upon the catalysis by traces of iodine of the Ce ~+ --* Ce s+ reduction with arsenious acid. T h e reaction takes place in sulfuric acid medium or in perchloric acid in the presence of sulfuric acid ~s~ and also in nitric acid. t~l} Numerous authors have used this method ~,°,~) which is frequently applied directly to the mineralized PBI solutions, but various interferences can occur, ts2's~ T h e method has been critically examined by several authors m,~s.~,~ and used as the basis of many automated procedures;t~,2o,~t-~s,~9-~l~ autoanalyzers for PBI-determinations are commercially available. T h e method is suitable for a range of about 0.005-0.2 pg I per sample, ~2~ the use of nitric instead of sulfuric acid as reaction medium makes possible its extension by one additional order of magnitude; an autoanalyzer recently developed by K n a p p and Spitzy, which works on this principle, shows a lower detection limit of 0.0005 pg I. It will perform up to 40 determinations/hr, (~t~ separation of PBI is made by ion exchange, ashing with chloric acid (G. I ~ , e P , private communication). BOOUTH and Scx-m~.o(~s} have used a similar reaction (Mn s+ ~ Mn ~+ reduction) for the determination of iodine in rat thyroids. An X-ray fluoroescence method based on the excitation of the La-radiation of iodine, has also been used to determine iodine in blood serum,(2~} another procedure, making use of the Karadiation of the element, has been applied to the analysis of various animal thyroids; ¢a~) tracer methods have also been proposed for the determination of micro amounts of iodine. (~s,~,a~-~o) Our own method is based on neutron activation analysis. Several nuclear reactions can be used in iodine activation analysis. T h e most important one is the thermal neutron reaction upon I~¢I (abundance I00 per cent, cross-section 6"2 barn leading to ~sI) : lz¢I( n, 7) 12sI $Smln>_ #-, (9s.6%)

> ~SXe (stable)

]zc(~-4%),~+(s.t0-,o/o)>t~STe (stable)

227

According to Mulvey et al. t2~I can be determined analytically also by photon activation :(2~

I~I(7, n)12si12.sg]

p-~ (44%)

l~6Xe (stable) ~"

..... ~o(ss~.),~+(1.s%). 126Te (stable)

The gamma-radiation is produced as bremsstrahlung of an electron beam from a 22 MeV linear accelerator on a cooled aluminium target, c2~} The threshold energy of this reaction is 9.05 -4- 0.15 MeV, (8°~ the maximum reaction cross section 0.231 barn with a gamma-energy of 15-2 McV. cs°~ T h e detection limit of the method given by the authors is 1.25 pg I/l. (2~ Utilization of the (n, 2n) reaction of 127I with reactor neutrons is also possible, but the crosssection of the reaction is only 0.95 × 10-o barn, Cas)* so that the sensitivity is poor. For the determination of iodine in blood fractions the (n, 7)-reaction is the one normally used. This reaction allows the determination o f iodine down to 10-3-10 4 pg but in many cases a chemical isolation of the iodine prior to or after irradiation is required. Prior separation has the advantage that the time consumed by chemical operations is not critical. However, this advantage is more than offset by the continuous danger of contamination of the sample with environmental iodine. T o avoid such effects operations with the sample prior to irradiation must be reduced to a minimum; this can be achieved by employing gel-filtration for the PBI-separation. Direct gamma-spectrometric measurement of 12sI in the irradiated samples has been used(29,ss) and also direct beta-measurement of 12sI after irradiation of rat thyroids with Cd-filtered reactor neutrons,(~7) but this method is rather insensitive (10-7 g I). T h e measurement of lZSI has already been utilized for iodine determinations in various biological objects, such as water,t al) urine,(SZ) iodine-containing hormones on paper chromatograms, (z°'aa~ brain and tumor tissue,(~) tomato seeds,C19) blood fractions,(2,9,~9.as,4s) rice plants(~) etc. These methods have different shortcomings such as lack of chemical recovery measurements, possible uptake of PBI by ion exchangers, * A more recent value is 0.64 4-0"01 × 10-a barn.(St)

228

J. C. Veselsky, M. Nedbalek and O. Susclmy

complicated processing, necessity of spectrum stripping procedures, etc. 2. EXPERIMENTAL T h e method described below makes use of gel filtration for the separation of PBI. T h e separated PBI solution is subjected to neutron activation followed by destruction o f the organic material and distillation of the iodine. T h e activity induced in the latter is determined by simple beta-counting. A determination of overall chemical recovery provides the basis for an exact calculation of PBI in the original sample. 2.1 The separation of P B I T h e PBI was separated from inorganic iodine on a Sephadex column (Sephadex G-25 coarse; Pharmacia, Uppsala, Sweden) using the method ofSPTiZX"et al. csJ, except that thesodium chloride solution was replaced by phosphate buffer to avoid nuclear and chemical interferences due to chlorine, t~x,4sJ The phosphate buffer (PH = 6.9, see "Procedure") was tested on a PBI standard substance ( I O D O T R O L - S t a n d a r d , Dade Reagents Co., Miami, Florida U.S.A.) and on fresh rat plasma. The Sephadex material (G-25 coarse) was at first equilibrated with buffer medium for at least 16 hr, any air bubbles observed were removed by means of a water pump. Afterwards the gel was transferred to a glass column (200ram high, 10ram dia.), the Sephadex layer (height 160ram above sinter plate) covered with a disc of filter paper and rinsed with buffer solution for 48 hr. For each experiment, the buffer was removed to the level of the filter-disc, and one ml of plasma introduced. When the plasma had moved completely into the column the elution with buffer solution was started (1 ml/min). Rat plasma samples and I O D O T R O L - S t a n d a r d s (though the latter were reported to be free of inorganic iodine) were always subjected to this treatment prior to irradiation (see also Table 5). T o check the gel filtration behaviour of the rat plasma, the latter was labelled with xslI. T h e isotope was injected (in physiological saline solution) into several rats (25 pCi aslI per animal) and after an incubation period of 48 hr the plasma was separated. 1 ml plasma was

then passed through the column eluted and 1 ml fractions were collected in polyethylene ampoules. T h e PBI always appeared in fractions 6-10, inorganic iodine in fractions 1322 (Fig. 1), the sum of lstI in both fractions together amounting to 98 per cent of total lstI. In several independent experiments conducted in parallel the PBI found was reproducibly 90.5 percent, the inorganic fraction 7-5 per cent (see also Ref. (8)). For the gel filtration experiments with the I O D O T R O L - S t a n d a r d s a radioactive labelling of them was required. Direct activation of the iodine with reactorneutrons was not possible because of the danger of denaturation; therefore, an isotope exchange technique was applied; 2 ml of standard were mixed with one drop of a carrier-free solution of 131I (about 1 #Ci) and stored for 48 hr in the refrigerator. The slight isotope exchange was sufficient for an identification of the two fractions (Fig. 2). T o compare the results obtained by gel filtration with another, independent method, some zinc-precipitation experiments were carried out on labelled rat plasma according to the technique applied by BARKER et al. ~xT~ The results are summarized in Table 1. As can be seen from Table 1, the results of the precipitation experiments agree with those obtained by gel filtration within the limits of error. Sephadex fractions 6-10 were collected and sealed in a suitable polyethylene ampoule for activation. 2.2 Activation, mineralization and distillation At first a trial was made to adopt the procedure of BOWEN and CAWSE~xg~,which includes wet-ashing of the activated sample with H N O a - HaPO4, distillation, purification of iodine by an extraction procedure and counting of x2sI in the form of AgI, for our problem using dissolved lyophilized blood plasma as the test material. The sample (1 ml plasma solution + 4 ml buffer) and a standard were irradiated for 20 rain at a thermal neutron flux of 10TM n/sec cmL As irradiation standard we used a solution of ammonium iodide in doubly distilled water (25 #g I/ml). T h e adsorption of the iodine on the walls of the polyethylene ampoule was investigated in several irradiation experiments with the standard solution. 5 ml of the iodine

229

The determination of protein bound iodine by neutron activation analysis

15

It It II ! ! I I I ! I

4-'

I0

1 Q tO

5

/

O~ •

O- - o - e - o - e - o 5

I0

...@... @ " O ~ . o ~ O,--qp @ •'Q

15

20

•

•

•

•

,?5

ml

=

Fio. 1. Gel filtration behaviour of 131I labelled rat plasma in phosphate buffer medium. solution were sealed in an ampoule a n d irradiated for 20 minutes at a neutron flux of 1013 n/sec cmL After the irradiation 11 per cent of the iodine were found to be adsorbed on the walls of the container; several trials to remove it by washing with thiosulfate or other reagents failed. W h e n some concentrated a m m o n i a was added prior to irradiation (4 ml iodine solution + 1 ml conc. ammonia) the adsorption was lowered to a (tolerable) a m o u n t of less than 0"4 per cent. W h e n 0.4 p g I were irradiated under the same conditions in 5 ml phosphate buffer, the adsorption effect was reduced to about 0.1 per cent. After activation the sample was transferred to the distillation apparatus (Fig. 3) together with 20 m g iodine carrier (0-5 ml of a solution of

40 m g I / m l in the form of a m m o n i u m iodide). After addition of concentrated H N O a and H s P O 4 (5 ml each) the iodine was distilled into 5 per cent N a O H . Distillation was assisted by an air-stream. I t was followed by chemical processing (is) and measurement of beta-radioactivity in a liquid-counting tube. T h e measurement in the liquid form was preferred to the counting of 12sI as AgI which frequently forms inhomogeneous layers yielding inconsistent results; the alkaline I2-solution is also suitable for a direct volumetric determination of the chemical recovery. Haft-life measurements on isolated iodine fractions always gave high values (around 30 rain) due t o t h e presence of SSCl. This could be put right b y correcting for the ssCl contribution

230

J. C. Veselsky, M. Nedbalek and O. Susdmy

15.

tO-

T x10

or: 0 ¢,a

5-

•

A

O. 5

10

Fzo. 2. Gel filtration behaviour of a xszI labelled I O D O T R O L control standard in phosphate buffer medium. TABLE I Exp. No. 1

2

% 181I precipitated % latI in solution 93"1 89"4

6"9 10"6

3

93"7

6"4

4 5 6

90"4 92"2 87"6

9"6 7-8 12"4

Average

91"I 4- 2"3

8"9 4- 2"3

to the x2sI spectrum but the procedure appeared rather complicated. T h e chemical processing of the irradiated protein-samples was now investigated by means

Of BeG1 and S~Br tracers. It was possible to improve the separation so that the correction for asCl became unnecessary but the reproducibility of the method remained poor, yielding a standard deviation of 4-20 per cent. Most of this deviation seemed to be attributable to the wet ashing procedure which did not a p p e a r suitable for the materialanalysed. We, therefore, continued our experiments with a wet ashing procedure using chromicsulfuric acid.m) Some experiments with z3tI labelled rat plasma ( + iodate carrier) proved that iodine losses could be practically avoided in this technique (the losses observed amounted to less than 1 per cent). After mineralization the solution was transferred to the distillation

The determination of p'otdn bound iodine by neutron aaivation analy~ Air

25I

Sinte? ptote

•.5"/, NoOH

Go$ burner

~/~ r//4

Fro. 3. Apparatus for the isolation of iodine from the digestion mixture after B o w ~ a n d CAWSE.(19} apparatus (Fig. 3) with some water, the excess of chromic acid and iodate reduced with ferrous sulfate and the iodine expelled by means of an air-stream (the iodine condensing mainly in the horizontal tube of the apparatus); the receiver contained 5 per cent N a O H solution. After dissolving the iodine in the dilute sodium hydroxide (see procedure) the alkaline solution was counted in a liquid-counting tube followed by iodometric determination of the chemical recovery. When applied to activated proteins the procedure yielded radiochemically pure 12sI solutions; numerous gamma-spectroscopic and half-life measurements proved the absence of asCl, ~4Na and other potential contaminants (Fig. 4). 2.3 Procedure (a) Reagents. (Analar reagents are used as far as possible.) Phosphate buffer: 13"6 g KH~PO4 + 35.8 g N a 2 H P O I . 12 HsO in 5 1. aqueous solution; Sephadex G-25 coarse; Sulfuric acid, 20 N: 80 ml water + 100 ml sulfuric acid (d = 1.83, ca. 36 N); Chromium trioxide solution, 10 M : 100 g chromium trioxide in 100ml aqueous solution; Iodine standard, 25 pg I/ml: 28.55 mg ammonium iodide in 1 1. aqueous solution (use twice distilled water) ; Ammonia, conc. (d = 0.91) ; Iodate carrier, 10 mg I/ml: 1"686 g potassium iodate in 100 ml aqueous solution; Ferrous 4

sulfate, solid; Sodium hydroxide, 5 per cent: 50 g sodium hydroxide in 1 1. aqueous solution; Bromine, saturated solution in water; Glacial acetic acid (d = 1.05) ; Sodium acetate solution 20 percent: 200 g sodium acetate in 1 1. aqueous solution; Formic acid, 80 per cent: 800 ml formic acid (d = 1"22) + 200 ml water; Potassium iodide solution, 5 per cent: 50 g potassium iodide in 1 1. aqueous solution; Sulfuric acid, 2 N: 100 ml 20 Nsulfuric acid + 900 ml water; Sodium thiosulfate, 0"1 At, volumetric solution. (b) Procedure. 1 ml of plasma is passed through the Sephadex column described previously, fractions 6-10 are collected in a suitable polyethylene ampoule, which is sealed with a hot glass rod. 4 ml of iodine standard solution (25 pg I/ml) and 1 ml conc. ammonia are sealed in a second ampoule. Both containers are irradiated for 20 rain. at a thermal neutron flux of 10t2 n/sec cm ~. After irradiation the ampoule containing the standard is opened and aliquot (corresponding to 20/~g I) is transferred to a 100 ml measuring flask containing 100 mg of ammonium iodide dissolved in about 10 ml of water. After the solution is made up to 100 ml, a 10 ml aliquot can be used as standard for counting in the liquid counting tube (the •sample solution is always counted before the standard). With the aid of some water the sample is transferred quantitatively to a 250 ml

252

J. C. Veselsk.y,M. Nedbalek and O. Suschn.y

beaker containing 15 m120 Nsulfuric acid, 1 ml 10 M chromium trioxide solution, 2 ml iodate carrier solution (20 rag I) and two small porcelain fragments (to prevent bumping). T h e mixture is heated on an electric hot-plate until the solution gives off sulfur trioxide and the colour, which is red at the start, is changed to brownish-green; heating should not be continued to a pure green colour (Cra+). The mixture is then transferred (with water) to the distillation apparatus (Fig. 3) and excess chromate and iodate are reduced by addition of 2-3 g of solid ferrous sulfate. 10 ml of 5 per cent sodium hydroxide solution are brought into the test tube serving as a receiver and the air-stream is started and regulated in such a way that the individual gas bubbles in the liquid can be clearly observed separately. T h e sand-bath is heated to 170190°C; condensation of iodine in the sinterplate of the apparatus is avoided by using a second little burner (see Fig. 3). W h e n all the iodine is expelled from the solution, most of it condensing in the horizontal tube of the apparatus the small burner is passed along that tube to drive the condensate over completely into the receiver where it dissolves immediately. T h e alkaline iodine solution from the receiver is transferred to a 15 ml volumetric flask and made up to the mark; a 10 ml aliquot is counted in the liquid counting tube (note time of measurement). Counting takes about 1550 min, depending upon the activity of the sample. The standard is counted immediately afterwards. For determination of the chemical recovery the contents of the liquid counting tube are transferred quantitatively to a 2 5 0 m l Erlenmeyer flask and diluted with water to 100 ml. 5 ml glacial acetic acid and 20 ml 20 per cent sodium acetate solution are added, followed by a slight excess of bromine water which is necessary to partly decolourize the solution (the final solution should be pale yellow). T h e mixture is allowed to stand for 10 min, then formic acid (80 per cent) is added dropwise until the yellow colour has disappeared. After the addition of 5 ml 5 per cent potassium iodide solution and 10 ml 2 N sulfuric acid the iodine is titrated with 0-1 N" thiosulfate using a starch indicator. Chemical recoveries are generally around 70-90 per cent. T h e

result is calculated using the following formula: I =

. ( C s , lC ( v.dv

)s,] .

,) . [(v.JV

)s,I

e

Ys~ : Ysa: Csa: Gst: (Va/VB)s~:

Chemical yield of standard, Chemical yield of sample, Net count-rate of sample, Net count-rate of standard, Ratio of the total volume of purified iodine solution to that of the aliquot counted, (Va/VB)st: Ratio of the total volume of standard solution to that of the aliquot counted, pg Ist: pg I in the standard solution, At: Time difference between the counting of sample and standard (in min). Usually the standard is counted without chemical purification and Yst can be taken as 100 per cent. In some cases a residual activity was observed when the standard was allowed to decay overnight; gamma-spectroscopic and half-life measurements showed the presence of 2~Na (probably from an impurity in the distilled water used in the preparation), so that the standard iodine has also to be oxidized and distilled with addition of carrier. In this procedure, the recovery of standard iodine drops to less than 100 percent; but the radiochemical purity of the standard can be guaranteed. Numerical example: = 91-9 Vo,

Y s , = 77.4%,

Cs~ ~ 1787 c/15 rain,

Cst = 37437 c/15 min =

(V_dVB)s, =

15/10

=

100/10 =

pg I m : 100[5 ---- 20,

1.5, 10,

At ---- 24 rain,

pg I = (91"9177"4). (1787/37431). (1"5/10) X 20. e - 0 " 0 2 7 7 x 2 4 ---- 0.0874 or 8.74 #g PBI/100 ml of sample. From this result a reagent blank has to be subtracted (it was determined on 5 ml buffer solution after passing the Sephadex column; the average of four determinations was 0.0014 0.0001 pg iodine per sample). T h e value calculated above, therefore, was reduced to 8 . 7 4 0.14 -- 8.60 pg PBI/100 ml.

Tb dctaminatbn

ofprotein boundiodhe

Fxo. 4. Gamma-spectrum of *=I isolated from an irradiated IODOTROL

3. RESULTS

AND DISCUSSION

233

by neutron actbation dysis

control standard.

245, 2.35, 2.26, 2.11, 2.43, 2.20, l-83, 194, 194, Z-33, 2.17 Average 2.18 -f 0.20 9.38, 9.16, 8.21, 8.74 Average 8.87 f 0.51 14.1, 13.8, 15.1, 15.3, 14.2, 15.3, 14.6, 14-O Average 14.55 f 061

the manufacturer which are based on chemical determinations. This result is independent of the absolute PBI contents of the standards.* Table 4 shows the results of some PBI-determinations on fresh rat plasmas, the analyses were carried out on two individual plasma pools (Rat I and Rat II}. The values given in Table 4 are comparable with results Corn the literature (see Refi. (1 l), (36)). The discrepancy between the values obtained by NAA and those by chemical analysis gave rise to some additional experiments. The reliability of the PBI-separation by gel filtration (Sephadex) had already been tested (see Section 2.1) ; therefore, the individual

In Table 3 these values are compared with the figures given by the manufkcturer of the standards. The comparison shows that PBI values obtained by activation analysis are consistently higher by about 20 percent than those given by

applied are also reported by the manufacturer of the IODOTROL-standards ( +21*3 per cent when Hycel dry ash method and Tech&on Autoanalyzer results and 3-81 per cent when Hycel and Barker methods are compared in the case of lot No. PBX137A-2).

the way just described three series of commerical PBI control standards were analyzed, the results are shown in Table 2. In

TABLE 2

Standard Series I pg PBL/lOoml Standard Series II fig PBI/lOOml Standard Series III fig PBI/lOOml

* PBI valuesdepending on the analyticaltechnique

TASLE 3. (All ~ahes in ,ug PBI/lOO ml) 1 Value given St. 1 St. 2 St. 3

::: 12.0

2 Value found 2.18 8.87 14.55

3 Value found corr. for blank 2% 8.73 1491

4 Difference (3-l)

5 Diierence (%)

0.34 1.63 241

20.0 22.9 20-l

(3-l )

234

J. C. Veselsky, M . Nedbalek and O. Suschny

TABLE 4 Rat I Rat II

(#g PBI/100 ml) ~ g PBI/100 ml)

3"8, 3"0,

3"6, 2"5,

3"5, 3"0,

3"9 2"4

Average: Average:

3-7 4- 0"18 2"7 4- 0"32

TABLE5. (The number of determinations is given in brackets) Standard (#g PBI/100 ml) (Value given by manufacturer)

/~g PBI/100 ml found using method ~1°~

1.7 6.3 11.5

1.7 (6) 6.3 (4) 11.5 (5)

Total iodine found (Technicon Autoanalyzer) /~g/100 ml 2.8 (6) 9-4 (6) (15.9)*

Difference (%) +64-7 +49.2 (+38.3)*

* One determination only. parameters of the formula used for the calculation of the results were subjected to a critical consideration. Determinations of the chemical recovery were made by the iodometric titration procedure described above. When recoveries of both sample and standard are determined, a systematic error can be ruled out; when only the recovery of the sample is determined (Yst "~ 100 percent) such an error would influence the result. Therefore, the reliability of the titration procedure was tested. As standard 10 ml of a potassium iodide solution (2 nag I/ml) were used. In nine titrations, the value for iodine obtained by titration was found to be 100"0 -4- 0.24 per cent of theory. This result shows the procedure for the determination of the chemical recovery to be a negligible source of error. T h e figures for V a [ V B are derived from volume measurements (measuring flasks, pipets) which do not show deviations in the order of magnitude observed. The iodine standard was defined by weighing of dry ammonium iodide on a micro balance, here again the error may amount to a fraction of a percent. T h e last parameter to be considered here is Cs,/Cs~: Adsorption losses of iodine during irradiation which would influence the count rate were found to be quite low in standard and sample. Furthermore, after processing the samples were found to contain lasI not significantly contaminated with other activities. T h e influence of slight differences in the densities of the solutions counted (when the standard is not subjected to the chemical processing)

is negligible; the reagent blank low. It is difficult to see any large systematic error which could arise in the determination of PBI by activation analysis according to the method used by us. T o find out possible errors in the chemical determinations ofPBI, a series of such determinations was now carried out on standards and rat plasma samples, use was made of the method of MORANCI°~; PBI was separated by an ion exchange procedure followed by dry ashing and final measurement with a method based on the Sandell-Kolthoff reaction. The reagents required were taken from an original reagent set of Hycel Inc., Houston, Texas (USA). T h e most critical phase of this technique, the dry ashing step, was checked with xstI labelled rat plasma and found to be correct. T h e results of the chemical determination are shown in Tables 5-7. From the values given in Tables 5-7 one can see that the PBI values given by the manufacturer can be reproduced (Table 5) by using method; tl°~ in the case of the rat plasmas there is also good agreement between chemical and activation analytical determinations (Table 7). T h e I O D O T R O L - S t a n d a r d s should only contain PBI and, therefore, the values in columns 1-3 (Table 5) should agree. In fact, however, the differences between PBI and total iodine are even higher than the differences between the values for PBI found by chemical and activation analysis (Table 6); the consistently higher activation analytical values for PBI in the

The determination of protein bound iodine by neutron activation analysis

235

Txnxaz 6. (The number of determinations is given in brackets) Standard (#g PBI/100 ml) (Value given by manufacturer)

#g PBI/100 ml found using method c1°)

pg PBI/100 ml found by neutron activation (corrected for blank)

1.7 7.1 12.0

1.6 + 0.14 (4) 7.1 4- 0.43 (4) 11.2 4- 0.89 (5)

2.04 + 0.21 (11) 8-73 4- 0.51 (4) 14.41 4- 0.61 (8)

TABLE 7. (The number of determinations is given in brackets)

Rat I Rat II

pg PBI/100 ml found using method ~1°)

/~g PBI/100 ml found by neutron activation (corrected for blank)

3"7 + 0"20 (4) 2"7 4- 0"30 (4)

3"7 4- 0"18 (4) 2"7 4- 0"32 (4)

standards might originate from their composition. Some experiments concerning the uptake of iodine compounds by the ion exchanger used (see also Refs. (26), (44)) in thechemical method seemed to indicate such an effect which showed a dependency upon the time of contact between exchanger and solution. Some experiments were made to elute adsorbed compounds from the ion exchanger and to identify them by thin layer chromatography but these were not successful. Acknowledgcments--We are grateful to the initiator of this work, Professor Dr. VE~rrSR (IAEA) for his interest and helpful discussions. We are indebted to Dr. ADAmKEa (SGAE, Biological Division) for the labelling of rat plasma with lslI and to Mr. NEtmAUERand co-workers (SGAE, Reactor Division) for numerous irradiations in the ASTRA-Reactor of the Oesterreichische Studiengesellschaft fuer Atomenergie (SGAE) in Seibersdorf, Lower-Austria. For the total iodine determinations with the Technicon Autoanalyzer of the Tieraerztliche Hochschule Wien we thank Dozent Dr. WEISP.R,for valuable discussions Professor Dr. SPITZY(Technische Hochschule, Graz, Austria).

1. 2. 3. 4. 5. 6.

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