## 3 Results and Discussion
Figure 2: Signal intensity versus number of data points plotted before (**A**) and after (**B**) sorting by descending signal for isotope \({}^{13}\)C. The cut-off point between two ROIs is marked with a red arrow. Exemplary plots are presented for the same sample as in the last column in Figure 1.  
Only in the case of Zn, in \"W\" ROI, were statistically significant differences noticed, as indicated in Figure 4A. Zinc contents in healthy prostate glands are very high, because this element is required for important prostatic functions [72]. The error bars in Figure 4 represent the differences between biological, not technical, replicates; thus, relatively high values are understandable. Standard deviation values in the case of zinc were higher than in the case of other elements, which was also observed by other authors [15] and in our previous experiment [68]. This inter-group inhomogeneity can be explained by zinc accumulation by prostate gland cells and its metabolic involvement in prostate carcinogenesis [73]. Zn contents in the prostate gland are characterized by very high individual variability [15]. Dietary supplementation with calcium, iron, copper, and selenium could weaken the absorption of zinc at the first stage in the intestinal wall, which may partially explain the effect of dietary supplementation, without such an effect for a non-supplemented, i.e., standard diet. In addition, the implantation of prostate cancer cells could exacerbate zinc deficits in the gland. In men with adenocarcinoma of the prostate, the concentration of zinc in the prostate tissue decreases significantly compared with the surrounding healthy tissues [74]. It has been noticed that the degree of malignancy of prostate cancer may be conditioned by deteriorations in the zinc content. Already in the early stage of carcinogenesis, the concentration of this microelement decreases, and with the development of a tumor, it is further reduced [74,75]. The risk of DNA damage increases, and this leads to a malignant transformation. Moreover, as the prostate epithelial cell transforms into a cancer cell, the ability to accumulate intracellular zinc is lost. In the initial stage of cancer, the expression of zinc uptake transporters in cells (ZIP- and Irr-like proteins) is also reduced, and as the disease progresses, their number continues to decline [76, 77, 78]. Distinguishing ROIs allowed for logical comparison to be drawn, even for the samples which were characterized by inhomogeneous distribution of the elements of interest (Ca, Fe, Zn, and Cu), caused, above all, by the presence of two basically different areas of the prostate gland. Samples were grouped by diet and medical intervention (prostate cancer cell implantation), and the main medical aim of the study was to identify the differences between groups. Comparing the results from bulk analysis was the first step of the study, and some differences were found; however, they did not give a definite answer about the influence of the described interventions. Therefore, more detailed analysis was planned by using LA-ICP-MS with very localized material collection. Results obtained from LA-ICP-MS and ICP-MS analysis (with the working parameters cited in Table 2) were compared (Figure 4). Graphical distribution of the bar plot prepared only with relative signal intensities from LA-ICP-MS measurements showed high correlation in the observed tendencies with bar plots based on quantitative analysis by ICP-MS with solution nebulization after mineral digestion (Figure 4). Figure 4: (**A**) Averaged relative signal intensities \({}^{66}\)Zn/\({}^{13}\)C from “W” ROI (LA-ICP-MS analysis) compared with (**B**) Zn content in the same prostate gland samples quantified by ICP-MS with solution nebulization [68].