Introduction

The prostate gland may be a source of many health problems in men. One of the most serious problems is prostatic carcinoma (PCa). Globally, PCa is the sixth most common cancer. In Western industrialized countries, it is the third most common cancer in males and the second leading cause of cancer death [1–3]. In North America, it is the most common cancer in males and, except for lung cancer, is the leading cause of death from cancer [4, 5].

Cancer is a multietiological and multifactorial complex disease. Epidemiological and laboratory study provided convincing evidence that genetic factors, diet, lifestyle, and environment are major causative factors of prostate cancer. It is well accepted that genetic variation alone does not explain the observed differences in incidence of PCa [6, 7]. A 120-fold difference in rates of PCa among different countries indicates that there is substantial variation in occurrence of this disease and suggests that dietary and environmental factors, including chemical element intake, are of importance [6–12].

Biological systems require not only “bulk” chemical elements but also trace and ultra-trace elements for their functioning. It seems that all chemical elements are more or less involved in various biochemical reactions in cells of the human body [13]. Under normal conditions, the chemical elements are in a state of equilibrium with regard to cellular distribution within tissues. Excessive accumulation, deficiency, or an imbalance of the elements may disturb the cell functions and may result in abnormal cell proliferation and malignant transformation, or in cell degeneration and apoptosis [14–19].

The involvement of trace elements in the etiology of PCa has been debated for almost five decades. It is likely that elevated levels of some metals Ca, Cd, Cr, Cu, Fe, Hg, Mg, Mn, Ni, Pb, V, and Zn and lowered level of Se in prostate tissue possibly initiate and promote PCa [4, 9, 14, 17–28]. The main hypothesis of the molecular mechanisms involved in prostate tumorigenesis is an oxidative DNA damage generated by free radicals of these metals. However, Se compounds have chemopreventive properties for PCa. Metals mentioned above may also be mutagenic through other mechanisms, e.g., by interacting with DNA, and can inhibit zinc finger domains featured in most DNA repair proteins [28–33].

Notably, Zn has been especially highlighted in the literature in relation to PCa. Zn is the second most abundant metal in the human body, serving as a cofactor for more than 300 enzymes with various physiological functions [34]. In the prostate, zinc is accumulated at up to tenfold higher level than in other tissues [35–37] and plays an important role in organ functions [38]. Much of the interest in zinc as an agent for prostate cancer treatment and prevention comes from studies that show a marked reduction of zinc level in prostate cancer tissue versus normal prostate tissue [35, 39]. Proponents of supplemental zinc think that high cellular zinc accumulation is detrimental to the malignant activities of prostate cancer cells. However, at present, there are two diametrically opposite points of view on the role of dietary zinc and supplemental zinc in prostate cancer risk [40].

In our recent study [40], it was shown that not only Zn but also Ca level in the human prostate tissue is almost one order of magnitude higher than in other soft tissues. The unusual high content of Ca in the prostate suggests that Ca may play a role in prostate function and health. The similarity of chemical properties of Ca and rare earth elements (REEs) is well known [40]. Chemical similarity allows ions of REEs to replace not only the ions of Ca and other alkaline earth elements but also transition metal ions such as Fe, Zn, Cu, Mn, Co, Cr, etc. in many macromolecular systems, including enzymes. At the same time, the replacement of REE ions with the ions of alkaline earth elements is impossible. Therefore, the investigation of REE content in the human prostate tissue seems to be especially important.

All current hypotheses that describe the role of trace elements in the etiology of PCa implicate elevated levels of metals in prostate tissue and disturbance in the relationships between elements as the main cause for PCa. Particularly, the focus is on the relationships between trace elements and Zn as well imbalance of trace metals and Se. There is evidence that the complexity of interaction among multiple dietary factors affects the intestinal absorption and assimilation of zinc. For example, the absorption of zinc could be inhibited by iron, calcium, and numerous other ingested micronutrients [40].

In order to confirm or refute these hypotheses, the first step is to investigate the normal levels of trace elements in a healthy prostate and their ratios and correlations with Zn level. To the best of our knowledge, such data are scarce, and the majority of results are based on a nonintact prostate tissue. As a rule, analyzed prostates were obtained from persons who died from different diseases. In some studies, prostatectomy samples were either formalin-fixed or paraffin-embedded. However, there is evidence that these types of tissue treatments lead to loss of some amount of chemical elements [41]. In other studies “histologically normal areas immediately adjacent to tumor” in prostatectomy samples of patients with PCa were used as “healthy” prostate tissues. Moreover, only a few studies used a quality control using certified reference materials for trace element contents.

This work had four aims. The first one was to assess the mass fractions of 52 trace elements in intact prostate of healthy men using inductively coupled plasma mass spectrometry (ICP-MS). The second aim was to evaluate the quality of obtained results. The third aim was to calculate the mean values of the Zn mass fraction/trace element mass fraction ratios for all elements investigated by using individual ratios. The last aim was to estimate the correlations between Zn and other trace elements in normal prostate.

All studies were approved by the Institute of Forensic Medicine, Moscow and the Medical Radiological Research Center, Obninsk Ethical Committees.

Material and Methods

Samples of the human prostate were obtained at postmortems from intact cadavers (64 males, 13–60 years old) within 48 h of death. The majority of deaths were due to traumas. All the deceased were citizens of Moscow. All cadavers had undergone routine autopsy at the Institute of Forensic Medicine, Moscow. Tissue samples were collected from the peripheral zone of prostate dorsal and lateral lobes and then divided into two portions using a titanium scalpel. One of tissue portion was used for morphological study while another was intended for chemical element analysis. A histological examination was used to control the age norm conformity as well as the unavailability of microadenomatosis and latent cancer. None of those who died a sudden death had suffered from any systematic or chronic disorders before.

After the tissue portions intended for chemical element analysis were weighed, they were transferred and stored at −20°C until the day of transportation to the Medical Radiological Research Center (MRRC), Obninsk. In the MRRC, all samples were freeze-dried and homogenized. The sample of homogenized prostate tissue weighing about 50 mg was used for chemical element analysis by ICP-MS.

A concentration of 1.5 mL of HNO3 (nitric acid 65 %, max. 0.0000005 % Hg, GR, ISO, Merck) and 0.3 mL of H2O2 (pure for analysis) were added to prostate tissue samples, placed in one-chamber autoclaves (Ancon-AT2, Ltd., Russia), and then heated for 3 h at 160–200°C to decompose. After autoclaves were cooled to room temperature, 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 prostate tissue samples (only HNO3 + H2O2 + deionized water), and the resultant solutions were used as control samples.

Sample aliquots were used to determine the content of Ag, Al, As, Au, B, Be, Bi, Br, Cd, Ce, Co, Cr, Cs, Dy, Er, Eu, Ga, Gd, Hf, Hg, Ho, Ir, La, Li, Lu, Mn, Mo, Nb, Nd, Ni, Pb, Pd, Pr, Pt, Rb, Re, Sb, Se, Sm, Sn, Ta, Tb, Te, Th, Ti, Tl, Tm, U, Y, Yb, Zn, and Zr by ICP-MS using an ICP-MS Thermo-Fisher “X-7” (Thermo Electron, USA). The measurements were made with the mass-spectrometer parameters shown in Table 1.

Table 1 The spectrometer parameters and the main parameters of mass-spectrum measurements
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The element concentrations in aqueous solutions were determined by the quantitative method using multielemental 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 2). 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 with their different isotopes.

Table 2 The isotope(s) used for determining chemical elements by ICP-MS
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The detection limit (DL) was calculated as:

$$ {\text{DL}} = {C_{\text{i}}} + {3} \times {\text{SD}} $$

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 was the one corresponding to the most abundant isotope. The relative standard deviation 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.

Five subsamples of the Institute of Nuclear Chemistry and Technology (INCT, Warszawa, Poland) certified reference material (CRM) INCT-SBF-4 Soya Bean Flour, INCT-TL-1 Tea Leaves, and INCT-MPH-2 Mixed Polish Herbs were analyzed simultaneously with prostate tissue samples to estimate the precision and accuracy of results. The samples of certified reference materials were treated in the same way as the prostate samples.

Each study specimen was assayed in duplicate using separate weights, and mean values of trace element contents were used in final calculation. Using the Microsoft Office Excel programs, the summary of statistics, arithmetic mean, standard deviation, standard error of mean, minimum and maximum values, median, percentiles with 0.025 and 0.975 levels was calculated for trace element contents and ratios. Standard programs were also used for estimation of intercorrelations of trace element contents in prostate tissue.

Results

Table 3 depicts our data for Ag, Al, As, Au, B, Be, Bi, Br, Cd, Ce, Co, Cr, Cs, Dy, Er, Eu, Ga, Gd, Hf, Hg, Ho, Ir, La, Li, Lu, Mn, Mo, Nb, Nd, Ni, Pb, Pd, Pr, Pt, Rb, Re, Sb, Se, Sm, Sn, Ta, Tb, Te, Th, Ti, Tl, Tm, U, Y, Yb, Zn, and Zr mass fractions in five subsamples of INCT-SBF-4 Soya Bean Flour, INCT-TL-1 Tea Leaves, and INCT-MPH-2 Mixed Polish Herbs certified reference materials and the certified (or informative) values of this material.

Table 3 ICP-MS data of chemical element contents in Certified Reference Materials (M ± SD, mg/kg on dry-weight basis)
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Table 4 presents basic statistical parameters (arithmetic mean, standard deviation, standard error of mean, minimal and maximal values, median, percentiles with 0.025 and 0.975 levels) of the Ag, Al, As, Au, B, Be, Bi, Br, Cd, Ce, Co, Cr, Cs, Dy, Er, Eu, Ga, Gd, Hf, Hg, Ho, Ir, La, Li, Lu, Mn, Mo, Nb, Nd, Ni, Pb, Pd, Pr, Pt, Rb, Re, Sb, Se, Sm, Sn, Ta, Tb, Te, Th, Ti, Tl, Tm, U, Y, Yb, Zn, and Zr contents in intact prostate of apparently healthy men.

Table 4 Basic statistical parameters of chemical element mass fractions (in milligrams per kilogram dry-weight basis) in intact human prostate
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Basic statistical parameters (arithmetic mean, standard deviation, standard error of mean, minimal and maximal values, median, percentiles with 0.025 and 0.975 levels) of Zn mass fraction/trace element mass fraction ratios in intact prostate of apparently healthy men are presented in Table 5.

Table 5 Basic statistical parameters of Zn mass fraction/trace element mass fraction ratios in intact human prostate
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The comparison of our results with published data [20, 41–56] for the Ag, Al, As, Au, B, Be, Bi, Br, Cd, Ce, Co, Cr, Cs, Dy, Er, Eu, Ga, Gd, Hf, Hg, Ho, Ir, La, Li, Lu, Mn, Mo, Nb, Nd, Ni, Pb, Pd, Pr, Pt, Rb, Re, Sb, Se, Sm, Sn, Ta, Tb, Te, Th, Ti, Tl, Tm, U, Y, Yb, Zn, and Zr contents in the human prostate is shown in Table 6. Because a number of values for chemical element mass fractions was not expressed on a dry-weight basis in the above works, we calculated these values using published data for water (80 %) [57] and ash (1 % on wet weight basis) [58] contents in prostate of adult men.

Table 6 Median, minimum and maximum value of means of chemical element mass fractions (in milligrams per kilogram dry-weight basis) in prostate according to data from the literature in comparison with our results
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The differences between the mean of trace element mass fractions in the prostate and in the skeletal muscle, liver, and whole blood of reference man [59, 60] is presented in Table 7. The data of reciprocal relationship (values of r, coefficient of correlation) between Zn and other trace element mass fractions are presented in Table 8.

Table 7 The differences between the mean chemical element contents in the prostate and in skeletal muscle, liver, and whole blood of Reference Man (mg/kg, on dry-weight basis)
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Table 8 Correlations (r) of Zn mass fractions and other trace element mass fractions in human prostate
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Discussion

Accurate determination of trace element contents by ICP-MS requires the use of a directly matrix-matched standard, with a similar major chemical composition and mineralogical form to the sample. However, no current standard allows for the quantification of chemical elements in prostate. For this reason, we were forced to evaluate the accuracy of our method using other certified reference materials with the biological matrix, certified for major portion investigated chemical elements—INCT-SBF-4 Soya Bean Flour, INCT-TL-1 Tea Leaves, and INCT-MPH-2 Mixed Polish Herbs (Table 3). The certified values for Ag, Au, Be, Bi, Dy, Er, Ga, Gd, Ho, Ir, Li, Nb, Pd, Pr, Pt, Re, Se, Sn, Te, Ti, Tm, U, Y, and Zr content were not present in these CRMs only (24 chemical elements from 52).

In 12 (Al, B, Br, Co, Cs, La, Mn, Mo, Ni, Rb, Th, and Zn) of 22 (Al, As, Br, Cd, Ce, Co, Cr, Cs, Eu, Hg, La, Lu, Mn, Ni, Pb, Rb, Sm, Tb, Th, Tl, Yb, and Zn) and of 25 (Al, As, Br, Cd, Ce, Co, Cr, Cs, Eu, Hf, Hg, La, Lu, Mn, Nd, Ni, Pb, Rb, Sb, Sm, Ta, Tb, Th, Yb, and Zn) chemical elements with certified values for the INCT-SBF-4 Soya Bean Flour, INCT-TL-1 Tea Leaves, and INCT-MPH-2 Mixed Polish Herbs certified reference material, we determined contents of all 28 certified elements Al, As, B, Br, Cd, Ce, Co, Cr, Cs, Eu, Hf, Hg, La, Lu, Mn, Mo, Nd, Ni, Pb, Rb, Sb, Sm, Ta, Tb, Th, Tl, Yb, and Zn (Table 3). Mean values for Al, As, B, Cd, Ce, Co, Cr, Cs, Eu, Hg, La, Lu, Mn, Mo, Nd, Ni, Pb, Rb, Sb, Sm, Tb, Th, Tl, Yb, and Zn were in the range of 95 % confidence interval. Good agreement with the certified data of CRMs indicates an acceptable accuracy of the results obtained in the study of trace elements of the prostate presented in Tables 4, 5, 6, 7, and 8.

The mean values and all selected statistical parameters were calculated for 39 (Ag, Al, Au, B, Be, Bi, Br, Cd, Ce, Co, Cs, Dy, Er, Gd, Hg, Ho, La, Li, Mn, Mo, Nb, Nd, Ni, Pb, Pr, Rb, Sb, Se, Sm, Sn, Tb, Th, Tl, Tm, U, Y, Yb, Zn, and Zr) chemical elements (Table 4). The mass fractions of trace elements were measured in all or a major portion of prostate samples. The content of As, Cr, Eu, Ga, Hf, Ir, Lu, Pd, Pt, Re, Ta, Te, and Ti was 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 DL instead of the individual value when these latter was found below the DL:

$$ \leqslant M = {{{\left( {\sum\limits_i^{{{n_{\text{i}}}}} {{C_{\text{i}}} + DL \cdot {n_{\text{j}}}} } \right)}} \left/ {n} \right.} $$

where C i is the individual value of trace element mass fraction in i-sample, n i is number of samples with the mass fraction higher than the DL, n j is number of samples with the mass fraction lower than the DL, and n = n i + n j is number of investigated samples.

The standard deviation obtained for all trace element mass fractions is particularly large (Table 4). This is due to the very wide individual variation of trace element mass fractions in the human prostate.

Mass fraction of such “trace” element as Zn in prostate tissue is much higher than content of all trace elements investigated (Table 5). Zn level in prostate tissue is higher than the content of Al, Br, and Rb (around an order of magnitude), B, Mn, Ni, and Se (around two order of magnitude), Mo, Pb, and Sn (around three order of magnitude), Ag, Cd, Ce, Co, Cs, Hg, La, Li, Nd, Sb, Y, and Zr (around four order of magnitude), Au, Be, Bi, Dy, Er, Gd, Nb, Pr, Sm, Th, Tl, U, and Yb (around five order of magnitude), and Ho, Tb, and Tm (around six order of magnitude).

The obtained means for Ag, Al, As, B, Bi, Br, Cd, Cr, Cs, Mn, Mo, Ni, Pb, Rb, Se, Sn, Ti, Tl, and Zn as shown in Table 6 agree well with the range of values cited by other researches for the human prostate, including samples received from persons who died from different diseases [20, 41–56]. The means for Au, Hg, Sb, U, and Y are one to two orders of magnitude, and the mean for Te, five orders of magnitude lower than previously reported results. No published data referring to the Be, Dy, Er, Eu, Ga, Gd, Hf, Ho, Ir, La, Li, Lu, Nb, Nd, Pd, Pr, Pt, Re, Sm, Ta, Tb, Th, Tm, Yb, and Zr content in human prostate was found.

The obtained values 913 ± 535 (M ± SD, Table 5) for Zn/Se ratio agrees well with result (M = 804) published by Sapota et al. [26]. No published data referring to ratio of Zn to other chemical element content in human prostate was found.

In our previous studies, it was shown that Zn and Ca levels in the peripheral zone of dorsal and lateral lobes of prostate are almost one order of magnitude higher than in other soft tissues [35–37, 40]. The obtained mean for Al, Au, B, Br, Cd, Cr, Ga, Li, Mn, Ni, Pb, and U mass fraction in human prostate are more than two times higher than mean values of element content in skeletal muscle, liver, and whole blood (Table 7). So, the human prostate accumulates not only for Zn and Ca but also for such trace elements as Al, Au, B, Br, Cd, Cr, Ga, Li, Mn, Ni, Pb, and U. The conclusion for Cd agrees with published data [20, 28, 48, 58].

With the exception of Nb and Rb, we did not find any pronounced correlation between the prostatic zinc and other trace elements (Table 8). This indicates that there is no special relationship between zinc and other trace elements in prostate. The lack of inverse correlation between Zn mass fraction and Cd mass fraction casts doubt on the opinion that Cd has “distinctive agonist effects” with Zn [41, 61, 62]. With the exception of Cd, no published data referring to correlations between Zn and other trace element contents in human prostate were found.

Conclusions

Inductively coupled plasma mass spectrometry is a powerful analytical tool for the determination of chemical element content in the prostate tissue. ICP-MS allows to determine the means of Ag, Al, Au, B, Be, Bi, Br, Cd, Ce, Co, Cs, Dy, Er, Gd, Hg, Ho, La, Li, Mn, Mo, Nb, Nd, Ni, Pb, Pr, Rb, Sb, Se, Sm, Sn, Tb, Th, Tl, Tm, U, Y, Yb, Zn, and Zr (39 elements) and the upper limit of mean for As, Cr, Eu, Ga, Hf, Ir, Lu, Pd, Pt, Re, Ta, Te, and Ti (13 elements). Mean values (M ± SΕΜ) for mass fraction (in milligrams per kilogram on dry-weight basis) of trace elements were as follows: Ag 0.041 ± 0.005, Al 36 ± 4, Au 0.0039 ± 0.0007, B 0.97 ± 0.13, Be 0.00099 ± 0.00006, Bi 0.021 ± 0.008, Br 29 ± 3, Cd 0.78 ± 0.09, Ce 0.028 ± 0.004, Co 0.035 ± 0.003, Cs 0.034 ± 0.003, Dy 0.0031 ± 0.0005, Er 0.0018 ± 0.0004, Gd 0.0030 ± 0.0005, Hg 0.046 ± 0.006, Ho 0.00056 ± 0.00008, La 0.074 ± 0.015, Li 0.040 ± 0.004, Mn 1.53 ± 0.09, Mo 0.30 ± 0.03, Nb 0.0051 ± 0.0009, Nd 0.013 ± 0.002, Ni 4.3 ± 0.7, Pb 1.8 ± 0.4, Pr 0.0033 ± 0.0004, Rb 15.9 ± 0.6, Sb 0.040 ± 0.005, Se 0.73 ± 0.03, Sm 0.0027 ± 0.0004, Sn 0.25 ± 0.05, Tb 0.00043 ± 0.00009, Th 0.0024 ± 0.0005, Tl 0.0014 ± 0.0001, Tm 0.00030 ± 0.00006, U 0.0049 ± 0.0014, Y 0.019 ± 0.003, Yb 0.0015 ± 0.0002, Zn 782 ± 97, and Zr 0.044 ± 0.009, respectively. The upper limit of mean contents of As, Cr, Eu, Ga, Hf, Ir, Lu, Pd, Pt, Re, Ta, and Ti were the following: As ≤0.018, Cr ≤0.64, Eu ≤0.0006, Ga ≤0.08, Hf ≤0.02, Ir ≤0.0004, Lu ≤0.00028, Pd ≤0.007, Pt ≤0.0009, Re ≤0.0015, Ta ≤0.005, and Ti ≤2.6. In all prostate samples, the content of Te was under detection limit (<0.003).

Our data reveal that the human prostate accumulates such trace elements as Al, Au, B, Br, Cd, Cr, Ga, Li, Mn, Ni, Pb, U, and Zn. There is no a special relationship of zinc with other trace elements investigated in the prostate. The lack of inverse correlation between Zn and Cd mass fractions casts doubt on the opinion that Cd has “distinctive agonist effects” with Zn.

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 52 trace element mass fractions in intact human prostate may serve as indicative normal values for urban population of the Russian Central European region.