Specimens of expressed prostatic fluid (EPF) were obtained from 51 men (mean age 51 years, range 18-82 years) with apparently normal prostates by qualified urologist in the Urological Department of the Medical Radiological Research Centre using standard rectal massage procedure. Subjects were asked to abstain from sexual intercourse for 3 days preceding the procedure. The cytological and bacteriological investigations were used to control the norm conformity of prostatic fluid samples chosen for ¹⁰⁹Cd EDXRF.

The Ethics Committee of the Medical Radiological Research Centre approved the study, and participants gave their informed consent. All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards.

Specimens of EPF were obtained in sterile containers which were appropriately labeled. Twenty μL (microliters) of fluid were taken by micropipette from every specimen for TE analysis, while the rest of the fluid was used for cytological and bacteriological investigations. The chosen 20 μL of the EPF was dropped on 11.3 mm diameter disk made of thin, ash-free filter papers fixed on the Scotch tape pieces and dried in a desiccator at room temperature. Then the dried sample was covered with 4 mm Dacron film and centrally pulled onto a Plexiglas cylindrical frame.

The facility for radionuclide-induced EDXRF included an annular ¹⁰⁹Cd source with an activity of 2.56 GBq, Si(Li) detector and portable multi-channel analyzer combined with a PC. Its resolution was 270 eV at the 6.4 keV line. The facility functioned as follows. Photons with a 22.1 keV ¹⁰⁹Cd energy are sent to the surface of a specimen analyzed inducing the fluorescence K_a X-rays of trace elements. The fluorescence irradiation got the detector through a 10 mm diameter to be recorded.

The duration of the Br, Fe, Rb, Sr, and Zn concentration measurement in one EPF sample was 60 min. The intensity of K_a-line of Br, Fe, Rb, Sr, and Zn for samples and standards was estimated on calculation basis of the total area of the corresponding photopeak in the spectra. The trace element concentration was calculated by the relative way of comparing between intensities of K_a-lines for samples and standards. To determine concentration of the elements by comparison with a known standard, aliquots of solutions of commercial, chemically pure compounds were used for a device calibration. 14 The standard samples for calibration were prepared in the same way as the samples of prostatic fluid. Details of the analytical method and procedures used here for sample preparation were presented in our earlier publications concerning the chemical elements of human prostatic fluid and tissue. 1315161718

(Table 1) depicts our data for Br, Fe, Rb, Sr, and Zn mass fractions in ten sub-samples of CRM IAEA H-4 (animal muscle) and the certified values of this CRM. Of four TE (Br, Fe, Rb, and Zn) with certified values for the CRM we determined contents of all certified elements (Table 1). Mean values (M±SD) for Br, Fe, Rb, and Zn were in the range of 95% confidence interval. Good agreement of the TE contents analyzed by ¹⁰⁹Cd radionuclide-induced EDXRF with the certified data of CRM IAEA H-4 (Table 1) indicate an acceptable accuracy of the results obtained in the study of the prostatic fluid presented in Table 1,Table 2, Table 3, Table 4, Table 5.

(Table 2) presents certain 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 Br, Fe, Rb, Sr, and Zn concentrations as well as of the Zn/Br, Zn/Fe, Zn/Rb, Zn/Sr ratios in EPF of apparently healthy men.

The comparison of our results with published data for TE concentrations in the normal human prostatic fluid 15192021222324252627is shown in Table 3.

To estimate the effect of age on the TE concentrations and Zn/TE ratios in the EPF we examined two age groups: group 1 (aged 18 to 40 years, Mean=27.5 years) and group 2 (aged 41 to 82 years, Mean =59.1 years) (Table 4).

Calculated correlation coefficients between age and TE concentration as well as between age and Zn/TE ratios in the prostatic fluid are collected in Table 5.

(Figure 1) depicts constructed “individual data sets for TE concentrations versus age” or “individual data sets for Zn/TE ratios versus age” diagrams with lines of trend. In our study the best fit in the proportion variance accounted for (i.e. R²) sense maximizes the value of R² using a linear, polynomial, exponential, logarithmic or power law for the approximation.

The mean values and all selected statistical parameters were calculated for five TE (Br, Fe, Rb, Sr, and Zn) concentrations and for four Zn/TE (Zn/Br, Zn/Fe, Zn/Rb, and Zn/Sr) ratios in EPF samples (Table 2).

The mean of Zn concentration obtained for prostatic fluid, as shown in Table 3, agrees well with median of means cited by other researches. 151920212223242526The mean of Rb concentration obtained for EPF agrees well with our data reported 37 years ago. 22 No published data referring to Fe, Br, and Sr concentrations or Zn/TE ratios in EPF were found.

A statistically significant age-related decrease in Br concentration was observed in EPF when two age groups were compared (Table 5). In second group of males with mean age 59.1 years the mean of Br concentration in EPF was 2.2 times lower than in prostatic fluid of the first age group (mean age 27.5 years). A statistically significant decrease in Br concentration was confirmed by the negative Pearson s coefficient of correlation between age and concentration of this element (Table 5, Figure 1). In addition to this a significant decrease in Fe and increase in Zn concentration with increasing of age was shown by the Pearson s coefficient of correlation between age and concentration of the elements (Table 5, Figure 1). A change of Br concentration in the prostatic fluid with age from 18 to 82 years is more ideally fitted by a logarithmic law, Fe - by a linear law, and Zn by a polynomial law (Figure 1). As per author s current information, no published data referring to age-related changes of TE concentration in human EPF is available with the exception of Zn.

Our finding for the Zn age-dependence does not agree with published data. For example, in the first quantitative X-ray fluorescent analysis of Zn concentration in prostatic fluid of 8 apparently healthy men aged 25-55 years no significant variation with age was recognized.19 However, no any statistical treatment of results was done in this investigation. Using Atomic Absorption Spectrophotometry (AAS) for Zn measurement in prostatic fluid specimens obtained from 63 normal male subjects in age from 24 to 76 years Fair and Cordonnier20 did not find any changes in metal level with age. The conclusion was followed from the level of differences between the mean Zn results for three age groups evaluated by parametric Student s t-test. Additionally, Zn, concentration in EPF showed no age relationship in the study of Kavanagh et al.26 when 33 EPF specimens obtained from normal male subjects in age from 15 to 85 years were measured by AAS and the Pearson correlation between age and Zn concentration was used.

A statistically significant age-related increase in Zn/Br and Zn/Fe ratios was observed in EPF when two age groups were compared (Table 4). In addition to this a significant elevation of the Zn/Br and Zn/Fe ratios with increasing of age was shown by the Pearson s coefficient of correlation between age and Zn/TE ratios (Table 5, Figure 1). The changes of Zn/Br and Zn/Fe ratios in the EPF with age from 18 to 82 years were more ideally fitted by a polynomial law (Figure 1). Because the Zn content on one hand and Br and Fe concentrations on the other one in EPF changed in opposite directions with increasing of age the changes of the Zn/Br and Zn/Fe ratios are more sensitive parameters than the absolute values of these TE. If the comparison of concentration of Br, Fe and Zn in EPS of two age group expressed difference as tens percentages, the values of Zn/Br and Zn/Fe ratios in normal EPF of males in the age range 41 to 82 years are almost 5.8 and 2.6 times, respectively, higher than those parameters in the age range 18 to 40 years. No published data on age-related changes of Zn/TE ratios in normal prostatic fluid were found.

Thus, if we accept the levels and relationships of TE concentrations in normal prostatic fluid of males in the age range 18 to 40 years as a norm, we must conclude that after age 40 years the level of Br, Fe and Zn concentrations as well as Zn/Br and Zn/Fe ratios in normal prostatic fluid significantly changed.

The range of means of Zn concentration reported in the literature for normal EPF (from 47.1 to 9870 mg/L) varies widely (Table 3). This can be explained by a dependence of Zn content on many factors, including age, ethnicity, mass of the gland, and others. Not all these factors were strictly controlled in cited studies. Another and, in our opinion, leading cause of inter-observer variability was insufficient quality control of results in these studies. Almost all analytical methods used for TE measurements in EPF were based on investigation of processed fluid with a goal to destroy and remove organic matrix. In such studies prostatic fluid samples were acid digested or dried under high temperature before analysis. There is evidence that by use of these methods some quantities of TE, including Zn, are lost as a result of this treatment. 282930 Thus, when using destructive analytical methods it is necessary to control for the losses of TE, for complete acid digestion of the sample, and for the contaminations by TE during sample decomposition, which needs adding some chemicals. It is possible to avoid these not easy procedures using non-destructive methods, such as the ¹⁰⁹Cd radionuclide-induced EDXRF.

The ¹⁰⁹Cd radionuclide-induced EDXRF developed to determine TE concentrations in EPF is micro method because sample volume 20 μL (one drop) is quite enough for analysis. It is another advantage of the method. Amount of human EPF collected by massage of the normal prostate is usually in range 100-500 μL 2731 but in a pathological state of gland, particularly after malignant transformation, this amount may be significantly lower. Therefore, the micro method of ¹⁰⁹Cd radionuclide-induced EDXRF developed to determine trace element concentrations in prostatic fluid is available for using in clinical studies.