The non-destructive INAA-LLR was used in this research study because this method has many definite advantages over other analytical methods, particularly, in the clinical chemistry. For example, after non-destructive INAA-LLR there is a possibility to check the results for some trace elements and to receive additional information about other trace element contents by destructive analytical methods such as atomic absorption spectrometry, inductively coupled plasma atomic emissionspectrometry, inductively coupled plasma massspectrometry and so on, using the same bone samples. Moreover, if a deep-cooled channel of nuclear reactor is available, the non-destructive INAA-LLR allows determining trace element contents in the fresh bone/tumor samples and combining trace element study with histological investigation. It is also necessary to keep in mind that the non-destructive methods are the current gold-standard solution to control destructive analytical techniques.2 The destructive analytical methods are based on measurements of processed tissue. In such studies tissue samples are ashed and/or acid digested before analysis. There is evidence that certain quantities of chemical elements are lost as a result of such treatment.2, 34, 38 There is no doubt that every method available for the measurement of trace element contents in bone and tumor samples can be used. However, when using destructive analytical methods it is necessary to control for the losses of trace elements, for complete acid digestion of the sample, and for the contaminations by trace elements during sample decomposition, which needs adding some chemicals.
In our previous study it was shown that the results of mean values for all representative elements of CRM IAEA H-5 (Animal Bone) and SRM NIST1486 (Bone Meal) were in the range of 95% confidence interval (M±2SD) of the certificates’ values.22, 24, 25, 36 Good agreement with the certified data of CRM and SRM indicate an acceptable accuracy for the trace element mass fractions obtained in the study of intact bone and chondrosarcoma tissue presented in Tables 1-5.
In the control group the mass fractions of Co, Fe and Zn were measured in all samples, but the mass fraction of Rb – in 11 samples and mass fractions of Ag, Cr, Hg, Sb, and Se – in 10 samples Figure 1. In the chondrosarcoma group the mass fraction of all nine trace elements were determined in all samples Figure 1.
Table 2 shows that in chondrosarcoma tissue the mean mass fraction of Ag, Co, Cr, Fe, Hg, Sb, Se, and Zn is higher while the mean mass fraction of Rb is lower than in the normal bone tissues. However, in chondrosarcoma only the mean mass fractions of Co (p ≤ 0.0022), Fe (p ≤ 0.0055), and Se (p ≤ 0.0055) are significantly increased and the mean mass fraction of Rb (p ≤ 0.00076) is significantly decreased when compared with those in normal bone. Different directions of mass fraction changes suggest potential use of mass fraction ratios of these trace elements as chondrosarcoma markers. A simple ratio of two trace element mass fractions, which change in two direction, can improve the difference between such characteristics of intact bone and chondrosarcoma tissues. These conclusion was the main reason for calculating Co/Zn, Cr/Zn, Fe/Zn, Hg/Zn, Sb/Zn, Co/Rb, Cr/Rb, Fe/Rb, Hg/Rb, Sb/Rb, and Se/Rb mass fractions ratios (Table 3). It was found that higher mean values of all selected mass fraction ratios were typical of chondrosarcoma tissue compared with intact bone (Table 4). However, in chondrosarcoma only the mean mass fractions ratio of Co/Zn (p ≤ 0.0224), Fe/Zn (p ≤ 0.0488), Co/Rb (p ≤ 0.00292), Cr/Rb (p ≤ 0.0141), Fe/Rb (p ≤ 0.00253), Sb/Rb (p ≤ 0.00615), and Se/Rb (p ≤ 0.0497) are significantly increased when compared with those in normal bone.
In the control group a statistically significant direct correlation was found, for example, between the Fe and Se (r = 0.60, p ≤0.05), Fe and Co (r = 0.55, p ≤0.01), Co and Hg (r = 0.79, p ≤0.01), Rb and Ag (r = 0.62, p ≤0.05), and between Rb and Cr (r = 0.56, p ≤0.05) mass fractions (Table 5). In the same group a pronounced (p ≤ 0.01) inverse correlation was observed between the Fe and Ag (r = - 0.80, p ≤0.05). If some positive correlations between the trace elements were predictable (e.g., Fe–Co), the interpretation of other observed relationships requires further study for a more complete understanding.
In the chondrosarcoma tissue many significant correlations between trace elements found in the control group are no longer evident, for example, direct correlation between Fe and Se, etc. (Table 5). However, direct correlations between Co and Cr (r = 0.61, p ≤0.05), Hg and Rb (r = 0.75, p ≤0.01) and Hg and Zn (r = 0.64, p ≤0.01) were observed (Table 5). Thus, if we accept the levels and relationships of trace element mass fraction in the intact bone samples of control group as a norm, we have to conclude that with a chondrosarcoma transformation the levels and relationships of trace elements in bone significantly change. No published data referring to correlations between trace elements mass fractions in chondrosarcoma tissue were found.
The changes in trace element contents of cancerous tissues in comparison with non-cancerous tissues may be attributed to a cause or effect of malignant transformation. Bone is a mineralized connective tissue. It is formed by osteoblast, that deposit collage and release Ca, Mg, and phosphate ions that combine chemically within the collagenous matrix into a crystalline mineral, known as bon hydroxyapatite. On average, bone tissue contains about 10-25% water, 25% protein fibers like collagen, and 50% hydroxyapatit Ca10(PO4)6(OH)2. Many trace elements are bone-seeking elements and they are closely associated with hydroxyapatit.24, 25, 26, 27, 28 Chondrosarcoma is classified as a bone tumor. Our previous findings showed that the means of the Ca and P mass fraction in the chondrosarcoma tissue are lower than in normal bone, but the mean of Ca/P ratio is similar.6 It suggested that chondrosarcoma continues to form bon hydroxyapatit but to a lesser degree than normal bone. Our findings show that the mean of the Fe mass fraction in chondrosarcoma tissue samples was 3.48 times greater than in normal bone tissues (Table 2). It is well known that Fe mass fraction in sample depends mainly from the blood volumes in tissues. Chondrosarcoma at least in grades II and III show increased microvascularity.39 Thus, it is possible to speculate that chondrosarcoma is characterized by an increase of the mean value of the Fe mass fraction because the level of tumor vascularization is higher than that in normal bone. As we found, there is a direct correlation between Fe and Co mass fractions (Table 5). Therefore an increased level of Co in the chondrosarcoma may be closely connected with a high Fe content in tumor tissue (Table 2).
In the chondrosarcoma tissue the mean Se mass fractions is 10.1 times higher (p ≤ 0.00069) than in normal bone (Table 2). The high Se level was reported in malignant tumors of ovary,40 lung,41 prostate,42 breast43, 44 intestine,45 and in gastric cancer tissue.46 The role played by Se in those tumors remains unknown, but in general it is accepted that certain proteins containing Se can mediate the protective effects against oxidative stress. The literature-based analysis found the association of malignant tissue transformation with local oxidative stress. Studies have shown that oxidative stress conditions play an important role in both the initiation and the progression of cancer by regulating molecules such as DNA, enhancers, transcription factors, and cell cycle regulators.47 However the cause of increased Se in cancerous tissue and particularly in the chondrosarcoma is not completely understood and requires further studies.