Ratios of selected chemical element contents in prostatic tissue as markers of malignancy

The aim of the study was the development of new highly precise testing methods for early diagnosis of prostate cancer. For this purpose, the values of Ca/Al, Ca/B, Ca/Ba, Ca/Mn, Mg/Al, Mg/B, Mg/Ba, Mg/Mn, S/Al, S/B, S/Ba, S/Mn, Zn/Al, Zn/B, Zn/Ba, and Zn/Mn mass fraction ratios in normal (n=37), benign hypertrophic (n=32) and cancerous (n=60) human prostate gland were investigated using a combination of non-destructive and destructive methods: instrumental neutron activation analysis and inductively coupled plasma atomic emission spectrometry, respectively. Mean values ± standard error of mean (M ± SEM) for mass fraction ratios in the normal tissue were as follows Ca/Al 103 ± 21, Ca/B 4320 ± 805, Ca/Ba 2957 ± 577, Ca/Mn 2061 ± 325, Mg/Al 36.1 ± 4.1, Mg/B 1599 ± 239, Mg/Ba 1086 ± 227, Mg/Mn 776 ± 70, S/Al 319 ± 31, S/B 14749 ± 2166, S/Ba 9640 ± 1968, S/Mn 6991 ± 379, Zn/Al 41.3 ± 9.7, Zn/B 1974 ± 559, Zn/Ba 1003 ± 195, and Zn/Mn 875 ± 214, respectively. It was observed that in benign hypertrophic tissues the Ca/B, Mg/B, and S/B mass fraction ratios are lower than normal levels while the Mg/Al and Mg/Mn ratio are significantly higher. In cancerous tissue the values of all ratios investigated were significantly lower than in normal and benign hypertrophic prostate. It was shown that the Ca/Ba, Ca/Mn, Mg/Al, Mg/Ba, Zn/Al, Zn/B, and Zn/Ba mass fraction ratios are the most informative for a differential diagnosis among all investigated mass fraction ratios. Finally, we propose to use the (Ca/Ba)∙(Mg/Al)∙(Zn/Mn) and (Ca/Ba)∙(Mg/Al)∙(S/B)∙(Zn/ Mn) multiplications of mass fraction ratios in a needle-biopsy core as the most informative indicators for distinguishing malignant from benign prostate. Sensitivity, specificity, and accuracy of these tests were 100-9%, 100-2%, and 100-2%, respectively. Further studies on larger number of samples are required to confirm our findings, to study the impact of the chemical element contents on prostate cancer etiology and to examine the long-term pathological outcome. Correspondence to: Vladimir Zaichick, Radionuclide Diagnostics Department, Medical Radiological Research Centre, Koroleva Str-4, Obninsk-249036, Kaluga Region, Russia, Tel: +7 (48439) 60289; Fax: +7 (495) 956 1440; E-mail: vezai@obninsk.com


Introduction
The prostate gland may be a source of many health problems in men past middle age, the most common being benign prostatic hyperplasia (BPH), and prostatic carcinoma (PCa). BPH is a noncancerous enlargement of the prostate gland leading to obstruction of the urethra and can significantly impair quality of life [1]. The prevalence of histological BPH is found in approximately 50-60% of males age 40-50, in over 70% at 60 years old and in greater than 90% of men over 70 [2,3]. In many Western industrialized countries, including North America, PCa is the most frequently diagnosed form of noncutaneous malignancy in males and, except for lung cancer, is the leading cause of death from cancer [4][5][6][7][8][9]. Although the etiology of BPH and PCa is unknown, some electrolytes and trace elements have been highlighted in the literature in relation to the development of these prostate diseases [10][11][12][13][14][15][16][17][18][19][20][21][22][23][24][25][26][27][28][29].
Electrolytes and trace elements have essential physiological functions such as maintenance and regulation of cell function and signalling, gene regulation, activation or inhibition of enzymatic reactions, neurotransmission, and regulation of membrane function. Essential or toxic (mutagenic, carcinogenic) properties of chemical elements depend on tissue-specific need or tolerance, respectively [30]. Excessive accumulation, deficiency or an imbalance of the chemical elements may disturb the cell functions and may result in cellular degeneration, death, and malignant transformation [31].
In reported studies, significant changes of chemical element contents in hyperplastic and cancerous prostate in comparison with those in the normal prostatic tissue were observed . Moreover, a significant informative value of Zn content as a tumor marker for PCa diagnostics was shown by us [67,68]. Hence it is possible that besides Zn, some other chemical elements also can be used as tumor markers for distinguish between benign and malignant prostate. Current methods applied for measurement of chemical element contents in samples of human tissue include a number of methods. Among these methods the instrumental neutron activation analysis with high resolution spectrometry of short-lived radionuclides (INAA-SLR) is a non-destructive and one of the most sensitive techniques. It allows measure the chemical element contents in a few milligrams tissue without any treatment of sample that avoids the possibility of changing the content of chemical elements during a sample preparation [69][70][71][72]. However the INAA-SLR allow only determine the mean mass fractions of 6-7 chemical elements in the tissue samples of normal and cancerous prostate glands [15,21,28,65,66]. The inductively coupled plasma atomic emission spectrometry (ICP-AES) is a more power analytical tool than INAA-SLR [18,22,47]  a critical step in elemental analysis by this method. In our previous studies a combination of both analytical methods were offered for using when the results obtained for some chemical elements by ICP-AES were under the control of INAA-SLR data [47].
Analytical studies of the Al, B, Ba, Br, Ca, Cu, Fe, K, Li, Mg, Mn, Na, P, S, Si, Sr, and Zn contents in normal, BPH and PCa tissue were done by us using the combination of INAA-SLR and ICP-AES methods, which allowed for the first time to obtain reliable results [22]. In particular, it was shown that in PCa the mean values of Al, B, Ba, and Mn are higher while those of Ca Mg, S, and Zn are lower than in healthy and hyperplastic prostates [22,[73][74][75][76][77][78]. Obtained results formed the basis for a new method for differential diagnosis of BPH and PCa, the essence of which was to determine the ratios of chemical element contents in the material of needle biopsy of prostate indurated site, because, in comparison with the absolute values of chemical element contents, using their content ratios is more suitable for diagnostics [79].
The present study had three aims. The main objective was to obtain reliable results about the chemical element contents and calculate the values of Ca/Al, Ca/B, Ca/Ba, Ca/Mn, Mg/Al, Mg/B, Mg/Ba, Mg/ Mn, S/Al, S/B, S/Ba, S/Mn, Zn/Al, Zn/B, Zn/Ba, and Zn/Mn mass fraction ratios in intact prostate of healthy men aged over 40 years and in the prostate gland of age-matched patients, who had either BPH or PCa combining in consecutive order non-destructive INAA-SLR with destructive ICP-AES. The second aim was to compare the levels of chemical element ratios in normal, hyperplastic, and cancerous prostate, and the third aim was to evaluate the levels of chemical element ratios for diagnosis of prostate cancer.
All studies were approved by the Ethical Committees of the Medical Radiological Research Centre, Obninsk.

Samples
The patients studied (n=92) were hospitalized in the Urological Department of the Medical Radiological Research Centre. All of them were European-Caucasian, citizens of Moscow and Obninsk (a small city in a non-industrial region 105 km south-west of Moscow). Transrectal puncture biopsy of suspicious indurated regions of the prostate was performed for every patient, to permit morphological study of prostatic tissue at these sites and to estimate their chemical element contents. In all cases the diagnosis has been confirmed by clinical and morphological results obtained during studies of biopsy and resected materials. The age of 32 patients with BPH ranged from 56 to 78 years, the mean being 66 ± 6 (M ± SD) years. The 60 patients aged 40-79 suffered from PCa (stage T1-T4). Their mean age was 65 ± 10 (M ± SD) years.
Intact (Norm) prostates were removed at necropsy from 37 men aged 41-87 who had died suddenly. All deceased were European-Caucasian, citizens of Moscow. Their mean age was 55 ± 11 (M ± SD) years. The majority of deaths were due to trauma. Tissue samples were collected from the peripheral zone of dorsal and lateral lobes of their prostates, within 2 days of death and then the samples were divided into two portions. One was used for morphological study while the other was intended for chemical element analysis. A histological examination was used to control the age norm conformity, as well as to confirm the absence of microadenomatosis and latent cancer [15,21,28].

Sample preparation
All tissue samples were divided into two portions. One was used for morphological study while the other was intended for chemical element analysis. After the samples intended for chemical element analysis were weighed, they were freeze-dried and homogenized. The sample weighing about 10 mg (for biopsy materials) and 50-100 mg (for resected materials) was 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 labelled polyethylene ampoules.
After NAA-SLR investigation the prostate samples were taken out from the polyethylene ampoules and used for ICP-AES. The samples 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 one-chamber autoclaves (Ancon-AT2, Ltd., 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 (only HNO 3 +H 2 O 2 + deionized water), and the resultant solutions were used as control samples.

Instrumentation and methods
Information detailing with the NAA-SLR and ICP-AES methods used and other details of the analysis was presented in our previous publication [15,18,21,22,28,47,65,66].

Certified reference materials
For quality control, ten subsamples of the certified reference materials IAEA H-4 Animal muscle 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 [18,22,47].

Computer programs and statistic
A dedicated computer program for INAA mode optimization was used [80] Table 1 depicts certain statistical parameters (arithmetic mean, Table 2.

Discussion
As was shown by us [18,22,47]  The mean values and all selected statistical parameters were calculated for sixteen mass fraction ratios: Ca/Al, Ca/B, Ca/Ba, Ca/Mn, The results presented in Table 1 showed substantial differences between the arithmetic means and the medians, which impeached a normal distribution of the investigated elemental content ratios. These findings were a motive for us to compare the means of mass fraction ratios in normal, BPH and PCa prostate using parametric Student's t-test and nonparametric Wilcoxon-Mann-Whitney U-test. No published data referring to mass fraction ratios of chemical elements in the human prostate was found.
From Table 2, it is observed that in benign hypertrophic tissues the Ca/Al, Ca/Ba, Ca/Mn, Mg/Ba, S/Al, S/Ba, S/Mn, Zn/Al, Zn/B, Zn/ Ba, and Zn/Mn mass fraction ratios do not differ from normal levels while the Mg/Al and Mg/Mn ratios are higher and Ca/B, Mg/B, and S/B ratios are lower. In cancerous tissue all ratios of chemical element mass fractions investigated were significantly lower than in normal parenchyma of the prostate. All these mass fraction ratios show similar variations in cancerous tissues when compared with benign hypertrophic tissues of the prostate.
Analysis of elemental content ratios in prostate tissue could become a powerful diagnostic tool. To a large extent, the resumption of the search for new methods for early diagnosis of PCa was due to experience gained in a critical assessment of the limited capacity of the prostate specific antigen (PSA) serum test [81,82]. In addition to the PSA serum test and morphological study of needle-biopsy cores of the prostate, the development of other highly precise testing methods seems to be very useful. Experimental conditions of the present study were approximated to the hospital conditions as closely as possible. In all cases we analyzed a part of the material obtained from a biopsy of the indurated site in the prostate. Therefore, our data allow us to    evaluate adequately the importance of elemental content ratios for the diagnosis of PCa. As is evident from individual data sets (Figures 1-4) and Table 3, the values of all mass fraction ratios are very informative for a differential diagnosis. For example, if 450 is the value of Ca/Ba mass fraction ratio assumed to be the upper limit for PCa (Figure 1 and Table 3) and an estimation is made for "PCa or intact and BPH tissue", the following values was obtained: The number of prostates investigated was taken into account for calculation of confidence intervals [83]. In other words, if Ca/Ba mass fraction ratio in a prostate biopsy sample does not higher 450, one could diagnose a malignant tumor with an accuracy of (100-2)%. Thus, using the (Ca/Ba)-test makes it possible to diagnose cancer in (100-9)% cases (sensitivity).
It should be noted, that all ratios of chemical element mass fraction investigated in the study are very informative for the diagnosis of PCa and these tests have good levels of sensitivity, specificity and accuracy varied in ranges 89%-100%, 86%-100%, and 89%-100%, respectively (Table 3). However, it is possible to increase a separation distance between the value of "Upper limit" for PCa and the lowest values among normal and BPH results if use a combination of the selected ratios. For example, for this purpose a multiplication of (Ca/Ba)•(Mg/Al)•(Zn/ Mn) or (Ca/Ba)•(Mg/Al)•(S/B)•(Zn/Mn) can be used. If the level 130000 was accepted as the "Upper limit" of (Ca/Ba)•(Mg/Al)•(S/B)•(Zn/Mn) test for the diagnosis of PCa (Figure 5), the sensitivity, specificity and accuracy of this test are 100-9%, 100-3%, and 100-2%, respectively and the lowest value in normal and BPH prostate is almost 30 time higher the highest value in cancerous prostate.
Mass fraction ratios of Ca/Al, Ca/Ba, Ca/Mn, Mg/Ba, S/Al, S/Ba, S/Mn, Zn/Al, Zn/B, Zn/Ba, and Zn/Mn in the needle-biopsy cores could be used as a tool to diagnose PCa and are comparable with characteristics of the Zn mass fraction-test [67,68]. However, it is our opinion that application of the elemental content ratios is more suitable for PCa diagnosis. Elemental mass fraction depends on the sample mass, which decreases with loss of its moisture. The needle-biopsy core is a small piece of tissue with a relatively high "surface/volume" ratio. After sampling, it begins to lose mass very fast. Weight loss of samples depends on the humidity of operating and store rooms [69]. Thus, it is very difficult to determine the fresh mass of needle-biopsy cores and to calculate the precise mass fraction of chemical elements. Sample freeze-dry, storage in air-tight vials until weighing, and then calculating mass fraction on dry mass basis is the only possible method that eliminates the variation in sample mass. Conversely, accuracy of elemental content ratios does not depend on sample mass and changes in moisture content. Therefore, this method does not require dry samples. Moreover, the use of the relations between mass fractions of chemical elements is particularly promising for the development of in vivo diagnostic methods, including the diagnosis of PCa.

Conclusion
The combination of nondestructive INAA-SLR and destructive ICP-AES methods is satisfactory analytical tool for the precise determination of chemical element mass fractions and their ratios in the tissue samples of normal, BPH and carcinomatous prostate glands. In this work, mean values of Ca/Al, Ca/Ba, Ca/Mn, Mg/Ba, S/Al, S/ Ba, S/Mn, Zn/Al, Zn/B, Zn/Ba, and Zn/Mn mass fraction ratios in normal, benign hyperplastic and cancerous prostate were calculated using data of INAA-SLR and ICP-AES. It was observed that in benign hyperplastic prostate the Ca/Al, Ca/Ba, Ca/Mn, Mg/Ba, S/Al, S/Ba, S/ Mn, Zn/Al, Zn/B, Zn/Ba, and Zn/Mn mass fraction ratios do not differ from normal levels while the Mg/Al and Mg/Mn ratios are higher and Ca/B, Mg/B, and S/B ratios are lower. In cancerous prostate the Ca/ Al, Ca/Ba, Ca/Mn, Mg/Ba, S/Al, S/Ba, S/Mn, Zn/Al, Zn/B, Zn/Ba, and Zn/Mn mass fraction ratios are significantly lower than in normal and benign hyperplastic prostate. It was shown that all ratios of chemical element mass fractions investigated in the study are informative for the prostate cancer diagnosis. Finally, we propose to use the estimation of selected elemental mass fraction ratios and some theirs multiplications in a needle-biopsy core as an accurate tool to diagnose prostate cancer. Further studies on larger number of samples are required to confirm our findings, to study the impact of the elemental mass fraction ratios in gland tissue on prostate cancer etiology and to examine the longterm pathological outcome.