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Zaichick et al. J Cancer Metastasis Treat 2018;4:60 I http://dx.doi.org/10.20517/2394-4722.2018.52 Page 3 of 15
[35]
All tissue samples were divided into two portions using a titanium scalpel . One was used for
morphological study while the other was intended for chemical element analysis. After the samples intended
[36]
for chemical element analysis were weighed, they were freeze-dried and homogenized .
The pounded samples weighing about 5-10 mg (for biopsy) and 100 mg (for resected materials) were used for
chemical element measurement by INAA-SLR. The samples for INAA-SLR were sealed separately in thin
polyethylene films washed beforehand with acetone and rectified alcohol. The sealed samples were placed in
labeled polyethylene ampoules. The content of Br, Ca, Cl, I, K, Mg, Mn, and Na were determined by INAA-
SLR using a horizontal channel equipped with the pneumatic rabbit system of the water-water-reactor-
special research nuclear reactor (Branch of Karpov Institute, Obninsk). Thyroid samples irradiated by
3
neutrons were measured using a gamma spectrometer. The gamma spectrometer included the 98 cm Ge(Li)
detector with on-line computer-based multichannel analyzer system (NUC 8100, Hungary) and provided a
60
resolution of 1.9 keV on the Co 1332 keV line.
After INAA-SLR investigation the thyroid samples were taken out from the polyethylene ampoules and used
for inductively coupled plasma-atomic emission spectrometry (ICP-AES). The samples were decomposed in
autoclaves. For this 1.5 mL of concentrated HNO (nitric acid at 65%, maximum of 0.0000005% Hg; GR, ISO,
3
Merck, Darmstadt, Germany) and 0.3 mL of H O (pure for analysis) were added to each thyroid samples,
2
2
which were placed in one-chamber autoclaves (Ancon-AT2, Ltd., Moscow, Russia) and then heated for 3 h
at 160-200 °C. After autoclaving, they were cooled to room temperature and solutions from the decomposed
samples were diluted with deionized water (up to 20 mL) and transferred to plastic measuring bottles.
Simultaneously, the same procedure was performed in autoclaves without tissue samples (containing only
HNO + H O + deionized water), and the resultant solutions were used as control samples. Sample aliquots
3
2
2
were used to determine the Al, B, Ba, Ca, Cu, Fe, K, Li, Mg, Mn, Na, P, S, Si, Sr, V, and Zn mass fractions
by ICP-AES using the spectrometer ICAP-61 (Thermo Jarrell Ash, USA). The determination of the ChE
content in aqueous solutions was made by the quantitative method using calibration solutions (High Purity
Standards, USA) of 0.5 and 10 mg/L of each element. The calculations of the ChE content in the probe were
carried out using software of a spectrometer (ThermoSPEC, version 4.1).

Information detailing the NAA-SLR and ICP-AES methods used and other details of the analysis were
presented in our earlier publications concerning chemical element contents in human thyroid, scalp hair,
and prostate [7,23,27,37-42] .

To determine contents of the elements by comparison with a known standard, biological synthetic standards
[43]
(BSS) prepared from phenol-formaldehyde resins were used . In addition to BSS, aliquots of commercial,
chemically pure compounds were also used as standards. Ten sub-samples of certified reference material
(CRM) International Atomic Energy Agency (IAEA) H-4 (animal muscle) and five sub-samples of CRM
of the Institute of Nuclear Chemistry and Technology (INCT, Warszawa, Poland), INCT-SBF-4 Soya Bean
Flour, INCT-TL-1 Tea Leaves, and INCT-MPH-2Mixed Polish Herbs were treated and analyzed in the same
conditions as those for thyroid samples to estimate the precision and accuracy of results.

[44]
A dedicated computer program for INAA mode optimization was used . All thyroid samples were
prepared in duplicate, and mean values of chemical element contents were used. Mean values of chemical
elements contents were used in final calculation for the Br, Fe, Rb, and Zn mass fractions measured by two
methods. Using Microsoft Office Excel, a summary of the statistics, including, arithmetic mean, standard
deviation, standard error of mean, minimum and maximum values, median, percentiles with 0.025 and 0.975
levels was calculated for chemical element contents. The difference in the results between two age groups was
evaluated by the parametric Student’s t-test and non-parametric Wilcoxon-Mann-Whitney U-test.