Use of Neutron Activation Analysis and Inductively Coupled Plasma Mass Spectrometry for the Determination of Trace Elements in Pediatric and Young Adult Prostate

The questions about the androgen control and the involvement of trace elements in prostatic reproductive function still remain unanswered. One valuable way to elucidate the situation is to compare the values for the prostatic mass fractions of trace elements in preand post-pubertal boys. The effect of age on the mass fraction of 54 trace elements in intact prostate of 50 apparently healthy 0 30 years old males was investigated by neutron activation analysis and inductively coupled plasma mass spectrometry. Mean values (M ± SΕΜ) for mass fraction (milligram per kilogram, on dry-weight basis) of trace elements were: Ag 0.062 ± 0.008, Al 80 ± 18, Au 0.0092 ± 0.0024, B 5.9 ± 3.5, Be 0.0034 ± 0.0009, Bi 0.018 ± 0.010, Br 26 ± 3, Cd 0.26 ± 0.05, Ce 0.049 ± 0.012, Co 0.035 ± 0.004, Cr 0.49 ± 0.07, Cs 0.036 ± 0.005, Dy 0.0072 ± 0.0018, Er 0.0040 ± 0.0011, Fe 100 ± 10, Gd 0.0065 ± 0.0018, Hg 0.031 ± 0.004, Ho 0.0013 ± 0.0004, La 0.034 ± 0.007, Li 0.064 ± 0.009, Mn 1.69 ± 0.15, Mo 0.54 ± 0.13, Nb 0.013 ± 0.004, Nd 0.025 ± 0.006, Ni 4.1 ± 0.6, Pb 1.3 ± 0.2, Pr 0.0058 ± 0.0015, Rb 14.5 ± 0.8, Sb 0.051 ± 0.006, Sc 0.013 ± 0.002, Se 0.54 ± 0.03, Sm 0.0055 ± 0.0015, Sn 0.22 ± 0.05, Tb 0.0012 ± 0.0004, Th 0.0076 ± 0.0020, Ti 2.8 ± 0.5, Tl 0.0032 ± 0.0009, Tm 0.00064 ± 0.00017, U 0.0025 ± 0.0004, Y 0.036 ± 0.010, Yb 0.0037 ± 0.0012, Zn 281 ± 32, and Zr 0.16 ± 0.04. The upper limit of mean mass fraction of As, Eu, Ga, Hf, Ir, Lu, Pd, Pt, Re, and Ta were: As ≤ 0.069, Eu ≤ 0.0012, Ga ≤ 0.071, Hf ≤ 0.049, Ir ≤ 0.00054, Lu ≤ 0.00063, Pd ≤ 0.014, Pt ≤ 0.0029, Re ≤ 0.0048, and Ta ≤ 0.010. This work revealed that there is a significant tendency for the mass fractions of Cd, Se and Zn in the prostate tissue of healthy individuals to increase with age from the time of birth up to 30 years. It was also shown that high levels of Al, Au, B, Br, Cr, Ga, Li, and Ni mass fraction in prostate tissue do not indicate a direct involvement of these elements in the reproductive function of prostate.


Introduction
The prostate gland is a vital part of the male reproductive system.It produces and excretes much of the liquid portion of semen (about 30% -35% of the semen ejaculate).The prostate mixes its fluids with those from the seminal vesicles to transport the sperm made in the testes.
The prostate of the adult male is known to accumulate high levels of some trace elements, including Zn [1].The reason for the unusually high trace element content in normal prostate gland is not completely understood.The findings of low Zn level in pediatric prostate warranted the conclusion that androgens are the major factors controlling the accumulation and maintenance of a high content of Zn in the prostate [2][3][4][5].
In our previous studies, the high mass fractions of Zn as well as some other trace elements were observed in prostate tissue of adult males when compared with those in nonprostatic soft tissues of the human body [1,[5][6][7][8].However, some questions about the androgen control and the involvement of trace elements in prostatic reproductive function still remain unanswered.One valuable way to elucidate the situation is to compare the values for the prostatic mass fractions of trace elements in pre-pubertal boys with those during early puberty, post-puberty and young adulthood.
The data on trace element mass fractions in pediatric prostate are apparently extremely limited [2,3].There are few studies regarding trace element content in prostate of young adult males, using chemical techniques and instrumental methods [2,3,[9][10][11][12][13][14][15].However, the majority of these data are based on measurements of processed tissue.In many studies tissue samples are ashed before analysis.In other cases, prostate samples are treated with solvents (distilled water, ethanol etc) and then are dried at high temperature for many hours.There is evidence that certain quantities of trace elements are lost as a result of such treatment [16,17].Moreover, only two of these studies employed quality control using certified reference materials (CRM) for determination of the trace element mass fractions [14,15].
The primary purpose of this study was to investigate the possibilities of a non-destructive instrumental neutron activation analysis with high resolution spectrometry of long-lived radionuclides (NAA-LLR) and inductively coupled plasma mass spectrometry (ICP-MS) in the estimation of trace element contents in the samples of prostate tissue.The second aim was to determine reference values for trace element mass fractions in the intact prostate of subjects of different age groups from newborn to young adult males.The third aim was to evaluate the quality of the results making a comparison between NAA-LLR and ICP-MS data obtained.The final aim was to compare the trace element mass fractions in pre-pubertal boys (group 1) with those during early puberty, postpuberty and young adulthood (group 2).
All studies were approved by the Ethical Committee of the Medical Radiological Research Center, Obninsk.

Samples
Samples of the human prostate were obtained from randomly selected autopsy specimens of 50 males (European-Caucasian) aged 0 day to 30 years.Age ranges for subjects were divided into two groups, with group 1, 0 -13 years (3.3 ± 0.09 years, M ± SEM, n = 29), and group 2, 14 -30 years (24.4 ± 1.0 years, M ± SEM, n = 21).These age groups were selected to reflect the situation before puberty (group 1-infant, childhood, and peripubertal periods) and during and after puberty (group 2-adolescent and young adult periods).The available clinical data were reviewed for each subject.None of the subjects had a history of an intersex condition, endocrine disorder, neoplasm or other chronic disease that would affect the normal development of the prostate.None of the subjects was receiving medications known to affect prostate morphology and prostatic chemical element content.The typical causes of death in most of these patients included sudden infant death syndrome, acute pulmonary etiologies, and trauma.All prostate glands were divided (with an anterior-posterior cross-section) into two portions using a titanium scalpel.One tissue portion was reviewed by an anatomical pathologist while the other was used for the trace element content determination.Only the posterior part of the prostate, including the transitional, central, and peripheral zones, was investigated.A histological examination was used to control the age norm conformity as well as the absence of any microadenomatosis and/or latent cancer.

Sample Preparation
After the samples intended for trace element analysis were weighed, they were transferred to −20˚C and stored until the day of transportation in the Medical Radiological Research Center (MRRC), Obninsk.In the MRRC all samples were freeze-dried and homogenized.The pounded sample weighing about 50 mg was used for chemical element measurement by instrumental NAA-LLR.The samples for NAA-LLR were wrapped separately in a high-purity aluminum foil washed with rectified alcohol beforehand and placed in a nitric acid-washed quartz ampoule.
The samples weighing about 100 mg for ICP-MS were decomposed in autoclaves; 1.5 mL of concentrated HNO 3 (nitric acid at 65%, maximum (max) of 0.0000005% Hg; GR, ISO, Merck) and 0.3 mL of H 2 O 2 (pure for analysis) were added to prostate tissue samples, placed in onechamber autoclaves (Ancon-AT2, Ltd., Russia) and then heated for 3 h at 160˚C -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 (only HNO 3 + H 2 O 2 + deionized water), and the resultant solutions were used as control samples.

NAA-LLR Method
A vertical channel of nuclear reactor was applied to determine the mass fractions of Ag, As, Au, Ba, Br, Cd, Ce, Co, Cr, Cs, Eu, Fe, Gd, Hf, Hg, La, Lu, Nd, Rb, Sb, Sc, Se, Sm, Sr, Ta, Tb, Th, U, Yb, Zn, and Zr by NAA-LLR.The quartz ampoule with prostate samples, standards, and certified reference materials was soldered, positioned in a transport aluminum container and exposed to a 24hour neutron irradiation in a vertical channel with a neutron flux of 1.3 × 10 13 ncm −2 s −1 .Ten days after irradiation samples were reweighed and repacked.
The samples were measured for period from 10 to 30 days after irradiation.The duration of measurements was The element concentrations in aqueous solutions were determined by the quantitative method using multi elemental calibration solutions ICP-MS-68A and ICP-AM-6-A produced by High-Purity Standards (Charleston, SC 29423, USA).Indium was used as an internal standard in all measurements.The next isotope(s) was/were measured and chosen for calculation, for each trace-element (see Table 3).If an element has several isotopes, the concentration of Li,, B, Ti, Ni, Zn, Br, Rb, Mo, Pd, Ag, Cd, Sn, Sb, Te, Nd, Sm, Eu, Gd, Dy, ER, Yb, Hf, Re, Ir, Pt, Hg, Tl, and Pb in a sample was calculated as the mean of the values measured for their different isotopes.
The detection limit (DL) was calculated as:

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where C i is a mean value of the isotope content for measurements in control samples, and SD is a standard deviation of C i determination in control samples.For elements with several isotopes, the DL corresponded to that of the most abundant isotope.
The relative standard deviation (RSD) did not exceed 0.05 for elements with C i > 5 DL and did not exceed 0.20 for elements with C i < 5 DL.

Standards and Certified Reference Materials
For quality control, ten subsamples of the certified reference materials (CRM) IAEA H-4 Animal muscle and IAEA HH-1 Human hair from the International Atomic Energy Agency (IAEA), and also five sub-samples INCT-SBF-4 Soya Bean Flour, INCT-TL-1 Tea Leaves and INCT-MPH-2 Mixed Polish Herbs from the Institute of Nuclear Chemistry and Technology (INCT, Warszawa, Poland) were analyzed simultaneously with the investigated prostate tissue samples.All samples of CRM were treated in the same way as the prostate tissue samples.Detailed results of this quality assurance program were presented in earlier publications [1,7].

Computer Programs and Statistic
A dedicated computer program of NAA mode optimization was used [18].
Using the Microsoft Office Excel program to provide a summary of statistical results, the arithmetic mean, standard deviation, standard error of mean, minimum and maximum values were calculated for all the trace element mass fractions obtained.For elements investigated by two methods the mean of all results was used.The reliability of difference in the results between two age groups was evaluated by Student's parametric t-test.For the construction of "trace element mass fraction versus age" diagrams the Microsoft Office Excel program was also used.

The Possibilities of NAA-LLR
203 Hg has the only line of 279.19 keV which coincides with the 279.54 keV (25%) line of 75 Se.However, 75 Se has more intensive lines 136 (56%) and 265 keV (60%) (See Table 1).Using the information about 75 Se lines 136 keV and 265 keV, the intensity of 279.54 keV line was calculated and the interference with 203 Hg 279.19 keV line was under control.

Precision and Accuracy
The use of two analytical methods allowed us to estimate the mass fractions of 54 trace elements in human prostate tissue.Good agreement was found between the mean values of the Ag, As, Au, Br, Cd, Ce, Co, Cr, Cs, Eu, Gd, Hf, Hg, La, Lu, Nd, Rb, Sb, Se, Sm, Ta, Tb, Th, U, Yb, Zn, and Zr mass fractions determined by NAA-LLR and ICP-MS (Table 4) indicating complete digestion of the prostate tissue samples (for ICP-MS techniques) and correctness of all results obtained by the two methods.The fact that the elemental mass fractions (mean ± SD) of the certified reference materials obtained in the present work were in good agreement with the certified values and within the corresponding 95% confidence intervals [1,7] suggests an acceptable accuracy of the measurements performed on in prostate tissue samples.

Contents of Chemical Elements
The mean values of mass fractions and all selected statistical parameters were calculated for 43 (Ag, Al, Au, B, Be, Bi, Br, Cd, Ce, Co, Cr, Cs, Dy, Er, Fe, Gd, Hg, Ho, La, Li, Mn, Mo, Nb, Nd, Ni, Pb, Pr, Rb, Sb, Sc, Se, Sm, Sn, Tb, Th, Ti, Tl, Tm, U, Y, Yb, Zn, and Zr) trace eleents in the nonhyperplastic prostate gland of males in two age groups 0 -13 and 14 -30 years (Tables 5 and 6, respectively).The contents of these elements were measured in all or a major portion of prostate tissue samples.The As, Eu, Ga, Hf, Ir, Lu, Pd, Pt, Re, and Ta mass fractions were determined in a few samples.The possible upper limit of the mean (≤M) for these trace elements was calculated as the average mass fraction, using the value of the detection limit (DL) instead of the individual value when the latter was found to be below the DL: where C i is the individual value of the trace-element mass fraction in sample −i, n i is number of samples with mass fraction higher than the DL, n j is number of samples with mass fraction lower than the DL, and n = n i + n j is number of samples that were investigated.Generally, the mass fractions of Te in prostate tissue samples were lower than the corresponding DL of ICP-MS (0.003 milligrams per kilogram on a dry mass basis).
The level of Zn in prostate is much higher than contents of other trace element: around an order of magnitude-Fe; two orders of magnitude-Rb; three orders of magnitude-Cr, and Se; four orders of magnitude-Ag, Co, Hg, Sb, and Sc.

Comparison with Published Data
The means of Fe and Zn mass fractions obtained for prostate tissue of infant and children (age group 1) as shown in Table 5, agree well with range of mean values reported by Heinzsch et al. (1970) and Leissner et al. (1980) [2,3].No published data referring to other trace element mass fractions in pediatric prostate glands were found.
The obtained values for Ag, As, Cd, Cr, Cs, Fe, Mn, Mo, Ni, Pb, Rb, Se, Ti, Tl, and Zn mass fractions in young adult nonhyperplastic prostate glands as shown in Table 7, agree well with median or range of means cited by other researches for the normal prostate tissue of adult males, including samples received from persons who died from different diseases [14,15,[19][20][21][22][23][24][25][26][27][28][29][30][31][32][33][34].A number of values for chemical element mass fractions were not expressed on a dry weight basis by the authors of the cited references.However, we calculated these values using published data for water-80% [35] and ash-1.0%on wet weight basis [11] contents in prostate of adult men.The means of Al, B, and Br are somewhat higher and of Bi and Sn are somewhat lower than the maximum and minimum mean value of previously reported data, respectively.The means of this work for Au, Hg, Sb, Te, U, and Y is from one to six orders of magnitude lower, than previously reported results.No published data referring to Be, Ce, Dy, Er, Eu, Ga, Gd, Hf, Ho, Ir, La, Li, Lu, Nb, Nd, Pd, Pr, Pt, Re, Sc, Sm, Ta, Tb, Th, Tm, Yb, and Zr mass fractions in prostate gland of adult men were found.

Age-Related Changes
In the histologically normal prostates, we have observed a significant decrease in mass fraction of the Ag, Al, Au, B, Be, Ce, Co, Cr, Dy, Fe, Gd, La, Li Mo, Nb, Nd, Pb, Sc, Se, Sm, Sn, Th, Ti, Tl, Tm, U, and Zr with age from the time of birth up to 30 years, accompanied by an increase

Table 7. Median, minimum and maximum value of means of trace element mass fractions (in milligrams per kilogram dry-mass basis) in prostate tissue of adult males according to data from the literature in comparison with this work results (prostate gland of young adults, 20 -30 years).
Published data [Reference] This M ± SD n = 16 Ag ≤0.1 (2) <0.05 (48) [19] 0.2 (7) [20] 0.06 ± 0.05 Al 27.7 (3) 13 ± 66 (50) [19] 47 (9) [21] 80 ± 98 As 0.045 (1) 0.05 (10) [22] 0.05 (10)  in mass fraction of Cd, Se and Zn (Table 8, Figure 1).In particular, a strongly pronounced (p ≤ 0.001) tendency of  age-related increase in Cd and Zn mass fraction was observed in prostate (Table 8).For example, in prostate of adolescent and young adult (group 2), Cd and Zn mass fraction was 5.2 and 2.9 times, respectively, greater than in prostate of children before puberty (group 1).An increase of Cd and Zn mass fraction in the prostate tissue with age from the time of birth up to 30 years is more ideally fitted by an exponential law than by a linear, polynomial, logarithmic or power law (Figure 1).An increase of Se mass fraction is more ideally fitted by a polynomial law (Figure 1).This work result for age-dependence of Fe and Zn mass fraction is in accordance with earlier findings [2,3].For example, Heinzsch et al. [2] found that Zn mass fraction in normal prostate was higher after the age of 10 (age group 11 -30 years) than before by approximately 1.7 times, and that Fe mass fraction in prostate gland of males aged 11 -30 years was lower than in infant prostate by approximately two times.In accordance with Leissner et al. [3] the mean Zn mass fraction in prostate tissue of 20 -29 years old men was 4.9 times greater than in prostate of 0 -5 years old subjects.

Comparison with Trace Element Mass Fractions in Liver of Reference Man
For pre-puberty the mean obtained for the Zn mass fraction in prostate tissue is lower than the mean Zn mass fraction in liver of reference man [36,37], but during puberty and postpuberty it is approximately three times higher (Table 9).This implies that the Zn mass fraction in prostate tissue is associated with the male androgen status.Also for post-puberty the means obtained for the Al, Au, B, Br, Cr, Ga, Li, and Ni mass fractions in prostate tissue are higher than their mean values in the liver of reference man.However, mass fractions of these elements in prostate gland of young adult males are lower than in pediatric prostate glands.This implies that the Al, Au, B, Br, Cr, Ga, Li, and Ni mass fractions are an androgen-independent parameter and that these elements are not directly linked to any reproductive function of the prostate.

Conclusions
Both  U 0.0025 ± 0.0004, Y 0.036 ± 0.010, Yb 0.0037 ± 0.0012, Zn 281 ± 32, and Zr 0.16 ± 0.04.The upper limit of mean mass fraction of As, Eu, Ga, Hf, Ir, Lu, Pd, Pt, Re, and Ta were: As ≤ 0.069, Eu ≤ 0.0012, Ga ≤ 0.071, Hf ≤ 0.049, Ir ≤ 0.00054, Lu ≤ 0.00063, Pd ≤ 0.014, Pt ≤ 0.0029, Re ≤ 0.0048, and Ta ≤ 0.010.In all prostate samples, the content of Te was under the detection limit (<0.003).This work result reveals that there is a significant tendency of increase in Cd, Se and Zn mass fraction in the prostate tissue of healthy individuals with age from the time of birth up to 30 years.It means that Cd, Se and Zn mass fractions in prostate tissue are the androgen-dependent parameters.Our finding of positive correlation between the prostatic Zn and Se mass fractions indicates that there is a special relationship of Zn with Se-containing compounds in the prostate.It was also shown that high levels of Al, Au, B, Br, Cr, Ga, Li, and Ni mass fraction in prostate tissue do not indicate a direct involvement of these elements in the reproductive function of prostate.
All the deceased were citizens of Moscow.None of those who died a sudden death had suffered from any systematic or chronic disorders before.The normal state of prostates was confirmed by morphological study.Thus, our data for mass fractions of 54 trace element mass fractions in intact prostate of two groups reflect the infant, childhood, and peripubertal periods (group 1) and adolescent and young adult periods (group 2) may serve as indicative normal values for urban population of the Russian Central European region. i

Figure 1 .
Figure 1.Individual data sets for the Al, Cd, Co, Fe, Hg, Pb, Se, and Zn mass fraction in the nonhyperplastic prostate gland of males between ages 0 -30 years and their trend lines with equations of best fit.

Table 3 . The isotope(s) used for determining chemical ele- ment contents by ICP-MS.
E Isotope(s) E Isotope(s) E Isotope(s) E Isotope(s) E: element.

Table 5 . Basic statistical parameters of trace element mass fraction (in milligrams per kilogram dry-mass basis) in the nonhyperplastic prostate gland of males between ages 0 -13 years (before puberty-the age group 1).
E: Element, M: arithmetic mean, SD: standard deviation, SEM: standard error of mean, Min: minimum value, Max: maximum value; * Titanium tools were used for sampling and sample preparation.

Table 6 . Basic statistical parameters of trace element mass fraction (in milligrams per kilogram dry-mass basis) in the nonhyperplastic prostate gland of males between ages 14 - 30 years (puberty and postpuberty-the age group 2).
E: Element, M: arithmetic mean, SD: standard deviation, SEM: standard error of mean, Min: minimum value, Max: maximum value; * Titanium tools were used for sampling and sample preparation.

Table 8 . Effect of age on mean values (M ± SEM) trace ele- ment mass fraction in pediatric and young adult nonhy- perplastic prostate glands.
* Titanium tools were used for sampling and sample preparation.

Table 9 . The differences between the means of trace element mass fraction in the prostate tissue and in liver of Reference Man (in milligrams per kilogram dry-mass basis).
E: Element, Values in bold have ratio magnitude >2.0.