Mikrochim. Acta 122, 67-76 (1996) Mikrochimica Acta

9 Springer-Verlag 1996 Printed in Austria

Determination of Free Iodide in Human Serum: Separation from Other I-Species and Quantification in Serum Pools and Individual Samples

B. Michalke*, P. Schramel, and S. Hasse

GSF-Forschungszentrum fiir Umwelt und Gesundheit, Institut fiir Okologische Chemie, Neuherberg, D-85764 OberschleiBheim, Federal Republic of Germany

Abstract. A method for the determination of free iodide in human serum was developed. For this purpose iodide from pooled serum samples was separated from the organic manner by SEC. The iodide fraction subsequently was freeze- dried and analyzed by ion chromatography for quantification. Investigations for recovery and precision were carried out and were found to show sufficient results. For quality assurance ICP-MS was taken additionally as an total I-detector [1], using native and iodide-spiked serum samples. The iodide results of ICP-MS as well as those of IC were well corresponding. Iodine containing SEC-fractions from iodide-spiked samples showed no increased I-values except that in the iodide fractions, proving that there was no iodide conversion into other I-species (and vice versa) during the whole procedure.

Free iodide from two serum pools of different healthy persons was deter- mined as 2.25 and 2.43 gg I - /L, respectively. The values are related to total iodine levels determined by ICP-MS. For comparative reasons a table of individual iodine and iodide values is presented.

Key words: iodide, iodine, ion chromatography, quality control, human serum.

Iodine is an essential trace element [2, 31. I-containing hormones (thyroxine, tri-iodothyronine [4, 5]) are strongly influencing an extended range of biochemical reactions. Usually different iodine species (e.g. IOn-, I -) are absorbed from the food and reduced to iodide (or remains iodide) in the gastro-intestinal tract [5]. The iodide pool in serum is considered as I-source for the complete synthesis of thyroxine and tri-iodothyronine. The sufficiency of I-supplementation can be seen directly by monitoring iodide in serum or secondarily by monitoring the urinary I--excretion [6]. The latter is state of the art because the direct iodide determination in serum was not possible at low physiological levels with desirable accuracy. A method (pub-

* To whom correspondence should be addressed

68 B. Michalke et al.

l ished in 1966/67) using serum which was labelled with 131I in vivo in considerable amounts [-7, 8] is not pract iced any longer. There, a quant i tat ive separat ion of the iodide f rom other I-species is not realized [7].

The need for a method of I - -determinat ion in serum was the reason for developing a method with the fol lowing features:

a) sufficiently low determinat ion limit; b) high precision and accuracy; c) instrumen- tat ion avai lable in clinical laborator ies; d) the possibi l ity to extend the method to iodine speciat ion invest igat ions by only small methodica l changes.

Material and Methods

1. Sampling and Sample Preparation

Blood was taken from healthy persons by "Vacutainers". The blood samples were stored for 30 rain at room temperature and were then centrifuged with 3000 rpm for 20 min. The supernatant (serum) was carefully collected, pooled and kept frozen at -20~ until further use. In case of individual I--determinations the serum samples were not pooled.

2. SEC-Fraetionation

In principal, the $EC-fractionation was derived from the SEC-separation described in [9]. All sample preparation steps were done in laminar flow benches to avoid iodide contamination from the air [1].

2.5 ml aliquots were taken from thawed serum for further investigations: 0.5 ml were used for total iodine determination [1].

2 ml were given on a LPLC-system, equipped with a SEC-column for the separation of iodide from the organic matrix. Fractions were taken by a fraction collector in 1 rain intervals. The fractions between 38-42 rain (RT of iodide) were pooled, frozen at - 20 ~ and freezedried at a controlled sample temperature of 10 ~

The dry powder was resuspended in 1.5 ml H20 and filtered through a 0.22 gm syringe filter. The sample then was ready for two parallel I--determinations at the IC.

3. LC Parameters for SEC

An "Econo System" (BioRad, Munich, Germany) was used for SEC-separations. The system was combined with a Beckman HPLC pump "114M" for a constant solvent delivery at 4 bar.

Eluent: 0.02 M NaHCO3; flow: 3 ml/min; wavelength: 254 mm Column: 500 x 20 mm, temperature controlled at 20 ~ Stationary phase: TSK HW 40 F (Toso Haas, Stuttgart, Germany) Fractionation: i min intervals = 3 ml by a "Fraction Collector 100" (Phamacia)

Table 1. A "3 step program" for the quality assurance during SEC-separation

Step 1 Step 2 Step 3

Eluent NaC1, 0.5 M H20 NazCO3, 0.02 M Flow 3 ml/min 3 ml/min 3 ml/min Time 120 min 120 min 120 min

Determination of Free Iodide in Human Serum 69

4. Quality-Assurance Concernin9 the SEC-Separation

Just before the separation of I - from the organic matrix the SEC column was rinsed, using a "3-step-program" (Table 1) to get rid of organic molecules, probably adsorbed to the SEC material. Further, a reproducible I - -RT and a quantitative separation is provided.

5. IC-Determination

The IC-determinations were carried out on a Dionex 4000 i ion chromatographic system, equipped with a PED.

Parameters: flow: 2.0 ml/min eluent: 4 mM Na2CO3/1.5 mM NaHCO3, He degased PED-potential: + 0.05 V electrode: Ag reference electrode: Ag/AgC1 sample loop: 50 gl precolumn: AG 9-SC, Dionex column: AS 9-SC, Dionex

6. Calibration

The IC was calibrated by two "5 Point calibration cu ryes" (0-10 gg I - / L or 10-50 pg I - /L) for low level or high level determinations.

7. Precision and Recovery

For reasons of quality control and for precision determinations, 2 ml of the pooled serum were mixed with 50 ng iodide. The sample was allowed to stand for 15-30 minutes and then was injected into the LPLC system. The procedure further was following 2. (SEC-separation) and 5. (IC-determination). The whole procedure was repeated 10 times, each with 2 parallel IC-determinations.

The range of IC-determined values was taken for the precision in consecutive I - determinations. The mean of determined values was used for recovery calculations.

8. Quality Control

Aliquots of the SEC-fractions (from native or spiked serum) were used for iodine determinations by ICP-MS according to [1] in parallel to the IC-determinations. The ICP-MS is used as a total iodine detector (independent on the I-species), whereas the IC (PED) is determining only free iodide (species specific detection). The elution pattern of native and spiked samples were monitored and compared, to get information about a possible conversion of free iodide into other I-species (or vice versa) during the SEC separation.

Further, the "redox stability" of iodide was checked by an analytical scheme shown in Fig. 1. There, mass balances were calculated for spiked serum after re-injection into the LPLC system. All fractions were monitored for "isolated" iodide by ICP-MS, obtaining information about possible iodide conver- sion into other species, showing other retention times and being not detectable by IC at the I - potential.

9. Chemicals

NazCO 3, NaHCO3, NaC1 and KI were purchased from Merck (Darmstadt, Germany). The "TSK 40 HW 40 F"-gel was bought from TosoHaas (Stuttgart, Germany) and packed into a glass column

70 B. Michalke et al.

2ml serum -~total iodide~--.~ 50 ng I'spike ~= 55

, - - V i I'-recovery 9 75 % ~ SEC [ 26. % retained / V

~ ,I~ Quantification: ICP-MS : 40 ng

V freeze drying . l~Quantif ication :

(-recovery- 75 %~ ~C ICP-MS : 42 ng 25 % reta ned S [C : 43ngrlg - - -

~ ,I~ Quantification. ICP-MS :31 ng

[C :30 ng

compute due[ to recovery

54 ng

56 ng 57 ng

56 ng

54 ng

Fig. 1. Scheme of the analytical steps of the "re-injection exper- iments". Information is provided about the "redox stability" of iod- ide during SEC-separation as well as about species conversions. Mass balances are calculated

(500 x 20 ram) from Krannich (G6ttingen, Germany). Helium (5.0 quality) was obtained from Messer Griessheim (Munich, Germany).

Results

A. Methodical Aspects

1. Column stability/separations, sample pretreatment. The SEC-pretreatment of the serum before IC analysis improved the possible IC-separations on the analytical column from only few to appr. 400 runs as well as the reproducibility of I - - quantification. This was true for a quantitative SEC-separation of I - from the organic matrix, achieved by an appropriate column handling described in Table 1. When using this procedure a sufficient SEC-separation was guaranteed for 50 runs. Iodide was eluting reproducibly between 38-42 rain without organic interferences. After more applications the I--fractions contained increasingly organic matter, damaging the IC-column.

2. Recovery and precision. When using the described procedures the recovery was found as 75% with a precision of _+ 3% at 50 Lag I - /L in 10 consecutive investiga- tions. The limit of determination of the whole method was calculated (_+ 3o-) as 0.1 ~gI - /L serum. The recovery of the final determination (1C) was 102% _ 7% using an aqueous solution of 1 lag I-/L. The detection limit (IC) was determined at 0.05 gg I-/L.

3. Quality control. For reasons of quality control the recovery and precision investigations were monitored by ICP-MS in parallel to IC. Figure 2 shows the UV-plot of the SEC pretreatment of spiked serum (50ngI-) compared with the iodide-elution (IC determined) and the elution of all iodine species (ICP-MS determined).

Determination of Free Iodide in Human Serum 71

03

0,6

0,5

0,4

< o ,3 0,2

UV up to 2 AU

o,t ]

0 10 20 30 40

minutes

50 60 70 80

0,7

iodide (IC)

0,6

0,5

=L 0,3

0,2

0,1

0 0 10 20 30

~,! Ii , ,,iitlliii~ ,

4O

minutes

50 60 70 80

iodine (ICP-MS)

0,7

0,6

0,5

=1~ 0,4 =1.

0,3

0,2

0,1

0 0 10 20 30 40 50 60 70 80

minutes

Fig. 2. SEC-separation of 2ml spiked (50ng I-) serum: The UV-profile (top) is related to the IC determined iodide elution (middle) and to a iodine monitoring (bottom) by ICP-MS

72

2

15 .=_

, m

10-

B. Michalke et al.

5-

0

13 ' 16

19 , i 22

25 ' q~- -~ 28

31

f rac t ion

/ ' ~ . F ~ ' ~ / / - 4 3 2 ml native serum / i

/ 49 iodide elution

Fig. 3. Comparison of the iodine elution from 2 ml native serum with 2 ml spiked (50 ng) serum. Only the iodide fractions of spiked serum show an elevated I-level, proving that there is no I-conversion into other I-species due to the additional iodide amount

Both, the I-values (ICP-MS) as well as the I--values (IC) were in accordance at the iodide retention time (38-42 min).

Further, I containing SEC-fractions (other than 38-42 min, iodide !) showed the same values in native and spiked samples by ICP-MS (e.g.: fractions 17-23: native: 45ng/ml serum; spiked: 47ng/ml serum). No increased I-values were seen in SEC-fractions of spiked samples except that at the iodide elution time (Fig. 3). Re-injection of the SEC-separated iodide fraction again resulted in a iodide elution only at the I - -retent ion time. The species specific IC-detection proved this iodine species totally to be iodide (Fig. 1),

B. Results About Native I-Values

Figure 4 shows the IC-chromatograms of two individual samples compared to an iodide standard of 1 I~g/L as an example. The iodide and total iodine values of serum pools are compared with individual values of 7 persons in Table 2, each corrected for recovery.

The two different pools (1 and 2) of healthy and not supplemented persons show identical values for total iodine and free iodide. The iodide level is about 3% of total iodine for both pools.

Determination of Free Iodide in Human Serum 73

0,035

0,03

0,025

0,02

1= 0,015

0,01

0,005

0

Standard IHglL

5 6 7 8 9

minutes

0,035 0,03

0,025

1~ 0,02

~" 0,015

0,01

0,005

0 5

Person "A"

6 7 8 9

minutes

Person "D"

0,035

o,03 ~ .~ o,o25 ~ 0,02

~r 0,015

0,01

0,005

5 6 7 8 9

minutes

Fig. 4. IC-chromatogram of a 1 ~g/L standard compared to two individual samples with a "normal" iodide value (middle) and a slightly decreased one (bottom). The determined values are 1.6 gg/L ("A") and 0,75 gg/L (D), which have to be corrected for recovery (75%). The corrected values are given in Table 2

74 B. Michalke et al.

Table 2. Iodine and iodide values of different serum pools and of individual samples

Sample source Sex Total iodine Free iodide I - / I Remarks male/ [pg/L] [pg/L] [%] female

Pool 1 m/fro Pool 2 m/fro

Pool 3 m/fro

Person "A" m Person "B" fm Person "C" m Person "D" fm

Person "E" fm

Person "F" fm

Person "G" fin

82.2 + 8 2.43 _+ 0.3 2.96 From healthy persons 81.3 + 7 2.25 _+ 0.25 2.77 From healthy persons

other than from pool 1 92.4 + 7 9.0 __+ 0.53 9.74 Pooled from resting serum

samples of persons "A, C, F" 78.7 2.1 2.67 Healthy 76.1 4.9 6.44 Diet slightly I supported 45.5 3.3 7.25 - 99.0 1.0 1.01 An increased thyroid gland

activity is known 68.5 42.4 61.9 Strongly I supported 2

times per day. Last supplementation: 3 h before blood sampling

103.8 13.6 13.1 Using I supplemented salt for cooking

128.5 4.2 3.27 I supplementation 1 times per week.

Last supplementation: 2 days before blood sampling

Discussion

The methods developed here provide an iodide determination at physiological levels with high reproducibility and precision. The determination limit of the whole procedure (0.1 gg/L) is more than 20 times lower than "normal" physiological I--values enabling monitoring of even decreased I--values (e.g. person "D") with sufficient accuracy. The methodical check by a second, independent method for final determination like ICP-MS gives further evidence of a high accurracy. The stability of iodide during the whole determination method is proven by a) the correspondence of the I-values in SEC-fractions from native and spiked samples (except iodide fractions !), b) the correspondence of iodide results of IC and ICP-MS and c) the "re-injection experiment". There is no iodide conversion into other I-species or vice versa.

The total I - recovery of 75% is sufficient, especially when looking at the low SD (+ 3 %). When using the "high level" calibration curve, elevated iodide levels can be easily determined (e.g. persons "E, F').

The total correspondence of total iodine and free iodide of serum pools from different persons give strong evidence for a marked regulatory mechanism for both, total I and free I-. This is in accordance to the literature [-5].

Determination of Free Iodide in Human Serum 75

This strong regulation may be further proved by the results of person "E". In spite of very high I doses per day (50 tag iodate), the hormone bound I values were conspicuously lower than the (normal) pool values. This is surprising as free I - is available in big amounts. Obviously, here the regulatory mechanism cannot be sufficiently influenced by the increased serum iodide level. Only about 26 lag I/L serum (38%) are bound to I-species of high molecular weight, being probably thyroid gland hormones [5]. This agrees with [11]. There, the I-supplementation is excreted nearly quantitative (92%) via urine.

When looking at person "D", only 1% of total iodine appears to be free iodide. The remaining 99% are obviously hormone bound [5]. This slight decrease of the iodide value may be insignificant, but it principally agrees with the total iodine value being in the upper range and the increased thyroid gland hormone concentration, known from this person. The total iodine values (organic bound + free iodide) and the values of free iodide alone are in good accordance with the literature. [1, 10] report about 80gg iodine/L serum and [4] and [11] give iodide serum values of 7.2_+ 5.8 lag/L (+ 80%) and below 10lag/L, respectively. [8] reports about total serum iodine in the 10 to 100 gg/L range and [7, 8] found 2.8% or 1.8%, respectively, I - from total iodine.

The work of [4] gives iodide values in the physiological range, but the method described there does not fulfill some of our requirements sufficiently. So the detection limit of 1 lag/L (20 pg in 20 lal) was too high for serum samples from the Munich area for an exact determination (e.g. person "D": 1 lag/L). Additionally, our aim was to develop an I--determination method, which can be easily extended for an iodine speciation work. But the sample preparation and the specific chemical mechanism of the post column reaction in [4] avoid the possibility for speciation investigations.

Summarizing, the method described here provides the possibility for monitoring both, physiological ("normal") iodide vahles in serum as well as increased ones. Thus, a more direct information is now available about the sufficiency of iodine supply compared to urinary iodide determinations. The use of ion chromatography for final iodide determination makes the method principally available for clinical laboratories and provides a species specific I--detection. In future, the method may be a further tool for diagnosis purposes, as well as a basis for iodine speciation investigations, when using ICP-MS as the final I-detector.

Abbreviations

IC = ion chromatography, ICP-MS = inductively coupled plasma mass spectrometry, LPLC = low pressure liquid chromatography, PED = pulsed electrochemical detector, SEC= size exclusion chromatography, RT = retention time.

References

[I] P. Schramel, S. Hasse, Mikrochim. Acta 1994, 116, 205. 1-2] E.J. Underwood, Trace Elements in Human and Animal Nutrition, 4th Ed. Academic Press, New

York, 1977. 1-3] A. S. Prasad, Trace Elements and Iron in Human Metabolism, Plenum, New York, 1978. [4] W. Buchberger, J. Chromat. 1988, 439, 129. [5] H. Keller, Klinisch-chemische Labordiagnostikf~ir die Praxis, Thieme, Stuttgart, 1991.

76 Determination of Free Iodide in Human Serum

[6] J.T. Dunn, H. E. Crutchfield, R. Gutekunst, A. D. Dunn, Thyroid, 1993, 3, 119. [7] K. Miiller, Clin. Chim. Acta 1967, 17, 21. [8J E. Makowetz, K. Miiller, H. Spitzy, Microchem. J. 1966, 10, 194. [9] B. Michalke, D. C. Mfinch, P. Schramel, J. Trace Elem. Electrolytes Health Dis. 1991, 5, 251.

[10] G. V. Iyengar, Elemental Analysis of Biological Systems, Vol. 1, CRC, Boca Raton, 1987. [11] W. Buchberger, U. Huebauer, Mikrochim. Acta 1989, 3, 137. [12] S. K. Nath, B. Moinier, F. Thuillier, M. Rongier, J. F. Desjeux, Internat. J. Vitamin Nutr. Res.

1992, 62, 66.

Received February 10, 1995. Revision June 12, 1995.