Trace Elements in Biology

**3**

**EDXRF**

**Chapter 1**

*Daisy Joseph*

**1. Introduction**

Introductory Chapter:

Applications Using Photon

Tracelement Effects in Biological

Induced (EDXRF), Proton Induced

Heavy metals are dangerous to human health. Heavy metals are Sb, As, Bi, Cd, Ce, Cr, Co, Cu, Ga. Trace elements occur in natural environments in small amounts and when present in sufficient concentrations, are toxic to living organisms [1]. Trace elements enter the ecosystems via direct discharges from industrialization processes, sewage sludge, atmospheric deposits and agricultural practices including application of pesticides or fertilizers [2–4]. They can be transferred from sediments to benthic organisms and then become a potential risk to human consumers through the food chain [5]. We have a X-ray emission spectrometer. Energy Dispersive X-ray Fluorescence and Proton Induced X-ray Emission, in which trace

The large number of applications of XRF in our laboratory has been carried out

Concentrations of K, Ca, Mn, Fe, Cu, Zn, Rb and Sr were determined in Indian

radioisotope source induced XRF. The levels of K and Ca were highest in clove and cinnamon. Rubidium and Strontium were found in all spices except cinnamon.

**2.2 Determination of mercury and arsenic in Indian ayurvedic medicines using** 

Elemental concentration in some herbal medical products, produced by different *ayurvedic* pharmacies in India, was determined using Energy Dispersive X-Ray

Spices namely pepper, clove, cardomon, cinnamon, and cumin using Cd 109

(PIXE) and Synchrotron Induced

(EXAFS) X-Ray Spectrometry

elements (micronutrients as well as toxic elements) were anlysed.

**2. Application of EDXRF and PIXE in biosciences**

Chromium and titanium were found only in pepper [6].

in Biosciences. They can be summarized as follows.

**2.1 Detection of trace elements in Indian spices**

#### **Chapter 1**

Introductory Chapter: Tracelement Effects in Biological Applications Using Photon Induced (EDXRF), Proton Induced (PIXE) and Synchrotron Induced (EXAFS) X-Ray Spectrometry

*Daisy Joseph*

## **1. Introduction**

Heavy metals are dangerous to human health. Heavy metals are Sb, As, Bi, Cd, Ce, Cr, Co, Cu, Ga. Trace elements occur in natural environments in small amounts and when present in sufficient concentrations, are toxic to living organisms [1]. Trace elements enter the ecosystems via direct discharges from industrialization processes, sewage sludge, atmospheric deposits and agricultural practices including application of pesticides or fertilizers [2–4]. They can be transferred from sediments to benthic organisms and then become a potential risk to human consumers through the food chain [5]. We have a X-ray emission spectrometer. Energy Dispersive X-ray Fluorescence and Proton Induced X-ray Emission, in which trace elements (micronutrients as well as toxic elements) were anlysed.

## **2. Application of EDXRF and PIXE in biosciences**

The large number of applications of XRF in our laboratory has been carried out in Biosciences. They can be summarized as follows.

#### **2.1 Detection of trace elements in Indian spices**

Concentrations of K, Ca, Mn, Fe, Cu, Zn, Rb and Sr were determined in Indian Spices namely pepper, clove, cardomon, cinnamon, and cumin using Cd 109 radioisotope source induced XRF. The levels of K and Ca were highest in clove and cinnamon. Rubidium and Strontium were found in all spices except cinnamon. Chromium and titanium were found only in pepper [6].

#### **2.2 Determination of mercury and arsenic in Indian ayurvedic medicines using EDXRF**

Elemental concentration in some herbal medical products, produced by different *ayurvedic* pharmacies in India, was determined using Energy Dispersive X-Ray

**Figure 1.** *A typical EDXRF spectrum of an Indian ayurvedic medicine Balguti Kesaria.*

Fluorescence Spectrometry (EDXRF). It is mandatory to look into the effects of these heavy metals being administered in the body functions before taking them over a long period of time (**Figure 1**) [7].

#### **2.3 Drought tolerant and susceptible genotypes of sorghum plants**

Drought tolerant and susceptible genotypes of sorghum plants were analyzed by EDXRF technique to study the correlation of trace elements with drought tolerance capacities for sorghum plants. Samples prepared from mature seeds, young seedlings and old plants were analyzed using 109Cd radioisotope source. The elements such as K, Fe, Cu, Zn, Rb and Sr and Y were seen to be present in different quantities in various samples. K and Fe concentrations were found to be more in the tolerant genotype as compared to the susceptible type. Concentration of Fe decreased with maturity in the tolerant group while it increased with maturity in the susceptible group. The genotype Arfa Gadamak (AG) showed a distinct abnormality in its young seedling with high level of Zn. In conclusion, the drought tolerant and susceptible genotypes of sorghum genotypes (cultivated in Sudan) exhibited varying levels of trace elements. The drought tolerant genotypes of sorghum seeds exhibited high K and Fe concentrations as compared to susceptible genotypes. In seedlings Fe concentration decreased with maturity in the tolerant group while it increased in the susceptible variety [8].

#### **2.4 PIXE studies of blood Pb levels in children of the Dharavi slum areas in Mumbai**

PIXE was used to study lead levels in blood samples of children from Dharavi slum areas. Blood lead levels of children admitted to Sion Hospital, Bombay (India), from the adjoining Dharavi slum areas. Blood samples were collected from 36 children with suspected lead poisoning (indicator was acute anemia) and from 20 control children. The analysis showed that the lead concentration of the patients varied from 0.1 to

**5**

*Introductory Chapter: Tracelement Effects in Biological Applications Using Photon Induced…*

6.0 μg ml−1. In addition to lead, K, Ca, Fe, Cu, Zn, Se, Br and Rb were also detected simultaneously, of which the concentrations of Fe, Cu, Zn, Se, Rb and Pb were determined. The high blood lead levels of the children from this area may be ascribed to environmental pollution due to heavy vehicular traffic and industrial sources. Pb was found to high even in normal children due to vehicular exhaust containing lead in

Cyanobacterial cell organisms grown in Uranyl nitrate and Uranyl Carbonate

were determined for their Uranium uptake by 109Cd induced EDXRF during different time intervals. It was found while elements such as K, Ca, Cr, Mn, Fe and Zn were present in small quantities was seen to be present in significant amounts after 2 hours of uptake and it became saturated in 5 hours after which the uptake

petrol. **Figure 2** shows the PIXE spectrum of a Pb poisoned child [9].

**2.5 Uranium extraction from cynobacterial cells**

reduced and became minimum.

*PIXE spectrum of blood of a lead poisoned child.*

**Figure 2.**

*DOI: http://dx.doi.org/10.5772/intechopen.97387*

*Introductory Chapter: Tracelement Effects in Biological Applications Using Photon Induced… DOI: http://dx.doi.org/10.5772/intechopen.97387*

**Figure 2.** *PIXE spectrum of blood of a lead poisoned child.*

*Trace Elements and Their Effects on Human Health and Diseases*

Fluorescence Spectrometry (EDXRF). It is mandatory to look into the effects of these heavy metals being administered in the body functions before taking them

**2.4 PIXE studies of blood Pb levels in children of the Dharavi slum areas in** 

PIXE was used to study lead levels in blood samples of children from Dharavi slum areas. Blood lead levels of children admitted to Sion Hospital, Bombay (India), from the adjoining Dharavi slum areas. Blood samples were collected from 36 children with suspected lead poisoning (indicator was acute anemia) and from 20 control children. The analysis showed that the lead concentration of the patients varied from 0.1 to

Drought tolerant and susceptible genotypes of sorghum plants were analyzed by EDXRF technique to study the correlation of trace elements with drought tolerance capacities for sorghum plants. Samples prepared from mature seeds, young seedlings and old plants were analyzed using 109Cd radioisotope source. The elements such as K, Fe, Cu, Zn, Rb and Sr and Y were seen to be present in different quantities in various samples. K and Fe concentrations were found to be more in the tolerant genotype as compared to the susceptible type. Concentration of Fe decreased with maturity in the tolerant group while it increased with maturity in the susceptible group. The genotype Arfa Gadamak (AG) showed a distinct abnormality in its young seedling with high level of Zn. In conclusion, the drought tolerant and susceptible genotypes of sorghum genotypes (cultivated in Sudan) exhibited varying levels of trace elements. The drought tolerant genotypes of sorghum seeds exhibited high K and Fe concentrations as compared to susceptible genotypes. In seedlings Fe concentration decreased with maturity in the tolerant group while it

**2.3 Drought tolerant and susceptible genotypes of sorghum plants**

*A typical EDXRF spectrum of an Indian ayurvedic medicine Balguti Kesaria.*

over a long period of time (**Figure 1**) [7].

increased in the susceptible variety [8].

**4**

**Mumbai**

**Figure 1.**

6.0 μg ml−1. In addition to lead, K, Ca, Fe, Cu, Zn, Se, Br and Rb were also detected simultaneously, of which the concentrations of Fe, Cu, Zn, Se, Rb and Pb were determined. The high blood lead levels of the children from this area may be ascribed to environmental pollution due to heavy vehicular traffic and industrial sources. Pb was found to high even in normal children due to vehicular exhaust containing lead in petrol. **Figure 2** shows the PIXE spectrum of a Pb poisoned child [9].

#### **2.5 Uranium extraction from cynobacterial cells**

Cyanobacterial cell organisms grown in Uranyl nitrate and Uranyl Carbonate were determined for their Uranium uptake by 109Cd induced EDXRF during different time intervals. It was found while elements such as K, Ca, Cr, Mn, Fe and Zn were present in small quantities was seen to be present in significant amounts after 2 hours of uptake and it became saturated in 5 hours after which the uptake reduced and became minimum.

#### **2.6 Tracelement variation in renal failed patients**

In a separate experiment of application of PIXE in bio-medical research, blood samples of Patients with Chronic renal failure were analyzed for trace element abnormalities and the results showed marked differences in patients before and after dialyses. PIXE being more sensitive in the low Z region due to its higher cross section is an ideal technique for bio-science applications. The knowledge that small amounts of metals which are needed in the diet goes back several hundred years to the discovery of a requirement for iron. It is well established that some elements such as I, Cu, Mn, Zn, Se and Mb in trace quantity are needed in physiology. Very recently, the six "newer trace elements", tin, vanadium, fluorine, silicon, nickel, and arsenic, were discovered to have nutritional requirement. Recommended Dietary Allowances (RDA) and requirements have been set for iron, iodine, and zinc. For copper the issuance of an RDA can be established. Only tentative recommendations for the "newer" trace elements in the form of a range of values can be presently proposed. To establish these recommendations more firmly, knowledge of the content of each of these trace elements in the diet is necessary. A detailed study of trace element abnormalities in serum of patients was carried out by PIXE in the case of non-dialyzed, hemodialysed and posttransplantation. Forty-two patients and eight healthy controls selected for this study were grouped on the basis of their Serum Creatinine (SC) levels. Serum samples of these subjects were excited by protons from the Van de Graff accelerator of 2.5 MeV energy and the Characteristic X-rays were detected by Si (Li) detector (**Table 1**).

In the case of renal failed patients the exact mechanism of trace element disturbance is not known. Reduced renal excretion increased oral intake and Globin Insulin (G.I.) absorption and contamination as well as loss across the hemodialysis membranes have been incriminated Considerable variation in plasma and tissue concentrations of trace elements have been found in different geolgraphical areas due to variation in water and soil content. Patients on dialysis therapy had elevated serum copper levels. Butamante reported similar results and attributed liberation of copper from the dialysis membrane as a cause for hypercupremia. Successful renal transplantation resorted serum copper to normal levels. A well established observation is that serum brooming levels are not different from normal in non dialyzed chronic renal failure patients, but those on dialysis show subnormal levels. Transference of bromine from blood into the


**7**

zinc [10].

analysis of EXAFS.spectra.

*Introductory Chapter: Tracelement Effects in Biological Applications Using Photon Induced…*

dialysate could be responsible for this deficiency. Transplantation resulted in rise of bromine towards normal. As a result of loss of renal function, Sr which depends on the kidney for elimination is probably retained accounting for the significantly elevated serum concentration of Sr in patients of CRF. Patients on hemodialysis and those who received a successful renal allograft had SR concentration within the normal range. Chronic Renal insufficiency did not result in accumulation of lead in our study similar to that observed by Thomson et al. Contamination of the dialysate or dialysate delivery system possibly resulted in elevation of lead concentration while on dialysis. Following renal transplantation the serum lead levels went down as compared to lead levels while on dialysis, but were still significantly elevated when compared to normal. Mobilization of lead which was sequestered in the tissues while the patient was on dialysis into the serum, to be excreted via the kidneys could explain the high lead levels. It could also be possible that the transplanted kidney has not yet attained a normal function with regard to lead excretion and with passage of time normal serum lead level would be achieved. Low and normal serum zinc levels have been reported in non dialysed patients, but in dialyzed patient elevated subnormal and almost normal concentration have been described. An unrestricted dietary protein intake (45–50 g/day) and normo-proteinemia could explain the lack of hypozincemia studied by Mansouri et al., in patients who were on a protein restricted diet (20–30 g/day) and had significant hypoproteinemia. Our study showed a slight, but non significant elevation of serum zinc in hemodialysed patients, probably as a result of contamination of the dialysate or dialysate delivery system with

**2.7 Synchtrotron based EXAFS on Mercury based Indian herbomineral drug.**

An Indian herbomineral drug was characterized for its trace elements by radioisotope induced EDXRF.The drug contains minerals like mercury, sulfur and arsenic disulfide, along with herbs By XRF As, Hg, Fe, Ca were detected. S was not detected as S X-rays get absorbed in the detector window. However AsS and HgS was seen in EXAFS spectra. There is no peak in the experimental data corresponding to the 2nd shell of HgS viz.., the Hg-Hg shell (corresponding to the blue lines). It may happen that Hg is not present in the form of HgS in the samples, instead Hg forms bonds with S, C or O present in the herbs. The peaks near 3 Å for samples 4 & 6 and near 1.5 Å of sample 9 might be due to bond formation of Hg with C or O atoms of the herbs. Sample 6 shows maximum disorder and the Hg-S bond length shows an increasing trend from sample 4 to sample 9. A more comprehensive report is underway regarding its structure after a complete

The XRF results for the herbomineral samples are as follows:

**Sample No Major analyte Trace** Sample 4 Hg Y Sample 5 Hg and As Fe Sample 6 Hg and As Fe Sample 7 Hg and As Fe Sample 8 Hg and As Fe and Ca Sample 9 Hg and As Fe and Cu

*DOI: http://dx.doi.org/10.5772/intechopen.97387*

**Table 1.** *Values in mean (μg/ml) ± S.D. of concentrations of trace elements in various groups.* *Introductory Chapter: Tracelement Effects in Biological Applications Using Photon Induced… DOI: http://dx.doi.org/10.5772/intechopen.97387*

dialysate could be responsible for this deficiency. Transplantation resulted in rise of bromine towards normal. As a result of loss of renal function, Sr which depends on the kidney for elimination is probably retained accounting for the significantly elevated serum concentration of Sr in patients of CRF. Patients on hemodialysis and those who received a successful renal allograft had SR concentration within the normal range. Chronic Renal insufficiency did not result in accumulation of lead in our study similar to that observed by Thomson et al. Contamination of the dialysate or dialysate delivery system possibly resulted in elevation of lead concentration while on dialysis. Following renal transplantation the serum lead levels went down as compared to lead levels while on dialysis, but were still significantly elevated when compared to normal. Mobilization of lead which was sequestered in the tissues while the patient was on dialysis into the serum, to be excreted via the kidneys could explain the high lead levels. It could also be possible that the transplanted kidney has not yet attained a normal function with regard to lead excretion and with passage of time normal serum lead level would be achieved. Low and normal serum zinc levels have been reported in non dialysed patients, but in dialyzed patient elevated subnormal and almost normal concentration have been described. An unrestricted dietary protein intake (45–50 g/day) and normo-proteinemia could explain the lack of hypozincemia studied by Mansouri et al., in patients who were on a protein restricted diet (20–30 g/day) and had significant hypoproteinemia. Our study showed a slight, but non significant elevation of serum zinc in hemodialysed patients, probably as a result of contamination of the dialysate or dialysate delivery system with zinc [10].

#### **2.7 Synchtrotron based EXAFS on Mercury based Indian herbomineral drug.**

An Indian herbomineral drug was characterized for its trace elements by radioisotope induced EDXRF.The drug contains minerals like mercury, sulfur and arsenic disulfide, along with herbs By XRF As, Hg, Fe, Ca were detected. S was not detected as S X-rays get absorbed in the detector window. However AsS and HgS was seen in EXAFS spectra. There is no peak in the experimental data corresponding to the 2nd shell of HgS viz.., the Hg-Hg shell (corresponding to the blue lines). It may happen that Hg is not present in the form of HgS in the samples, instead Hg forms bonds with S, C or O present in the herbs. The peaks near 3 Å for samples 4 & 6 and near 1.5 Å of sample 9 might be due to bond formation of Hg with C or O atoms of the herbs. Sample 6 shows maximum disorder and the Hg-S bond length shows an increasing trend from sample 4 to sample 9. A more comprehensive report is underway regarding its structure after a complete analysis of EXAFS.spectra.


The XRF results for the herbomineral samples are as follows:

*Trace Elements and Their Effects on Human Health and Diseases*

**2.6 Tracelement variation in renal failed patients**

In a separate experiment of application of PIXE in bio-medical research, blood samples of Patients with Chronic renal failure were analyzed for trace element abnormalities and the results showed marked differences in patients before and after dialyses. PIXE being more sensitive in the low Z region due to its higher cross section is an ideal technique for bio-science applications. The knowledge that small amounts of metals which are needed in the diet goes back several hundred years to the discovery of a requirement for iron. It is well established that some elements such as I, Cu, Mn, Zn, Se and Mb in trace quantity are needed in physiology. Very recently, the six "newer trace elements", tin, vanadium, fluorine, silicon, nickel, and arsenic, were discovered to have nutritional requirement. Recommended Dietary Allowances (RDA) and requirements have been set for iron, iodine, and zinc. For copper the issuance of an RDA can be established. Only tentative recommendations for the "newer" trace elements in the form of a range of values can be presently proposed. To establish these recommendations more firmly, knowledge of the content of each of these trace elements in the diet is necessary. A detailed study of trace element abnormalities in serum of patients was carried out by PIXE in the case of non-dialyzed, hemodialysed and posttransplantation. Forty-two patients and eight healthy controls selected for this study were grouped on the basis of their Serum Creatinine (SC) levels. Serum samples of these subjects were excited by protons from the Van de Graff accelerator of 2.5 MeV energy and the Characteristic X-rays were detected by Si (Li)

In the case of renal failed patients the exact mechanism of trace element disturbance is not known. Reduced renal excretion increased oral intake and Globin Insulin (G.I.) absorption and contamination as well as loss across the hemodialysis membranes have been incriminated Considerable variation in plasma and tissue concentrations of trace elements have been found in different geolgraphical areas due to variation in water and soil content. Patients on dialysis therapy had elevated serum copper levels. Butamante reported similar results and attributed liberation of copper from the dialysis membrane as a cause for hypercupremia. Successful renal transplantation resorted serum copper to normal levels. A well established observation is that serum brooming levels are not different from normal in non dialyzed chronic renal failure patients, but those on dialysis show subnormal levels. Transference of bromine from blood into the

**Elements I II III IV V** Ni .0167 ± .0164 0.034 ± 0.022 0.030 ± 0.033 0.066 ± 0.051 0.042 ± 0.027 Cu .083 ± 0.16 0.83 ± 0.25 0.97 ± 0.33 1.08 ± 0.37 0.71 ± 0.28 Zn 1.12 ± 0.93 1.06 ± 0.25 1.30 ± 0.01 1.38 ± 0.67 1.13 ± 0.47 Se .035 ± 0.017 0.032 ± 0.029 0.035 ± 0.011 0.033 ± 0.018 0.030 ± 0.012 Br 3.66± .154 3.383± 0.241 0.599± 0.417 0.122± 0.127 0.281± 0.255 Rb .178 ± 0.101 0.297 ± 0.177 0.246 ± 0.101 0.172 ± 0.109 0.263 ± 0.114 Sr .029 ± 0.044 0.132 ± 0.088 0.204 ± 0.089 0.111 ± 0.214 0.088 ± 0.082 Pb .173 ± 0.143 0.279 ± 0.137 0.347 ± 0.236 0.797 ± 0.537 0.400 ± 0.260

*Values in mean (μg/ml) ± S.D. of concentrations of trace elements in various groups.*

**6**

**Table 1.**

detector (**Table 1**).

*Trace Elements and Their Effects on Human Health and Diseases*

#### **3. Conclusion**

Though trace elements are required in minimal quantities their presence in the optimal amount is essential for the normal physiological functioning of the body. They are one of the corner stone's in maintenance of biodynamic of the body. Both, excess and the deficiency states lead to initiation, promotion, and progression to various disease processes. The present paper has thoroughly discussed trace elements, as this area is away from the deserved attention. Thus, a comprehensive understanding of these trace elements is essential and significant for disease control and maintaining optimal health and X-ray Emission Techniques such as EDXRF, PIXE and EXAFS have shown to be good diagnostic tools for determining tracelements in Biological samples.

**9**

**Author details**

Nuclear Physics Division, BARC, Trombay, Mumbai, India

© 2021 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium,

\*Address all correspondence to: djoseph@barc.gov.in

provided the original work is properly cited.

Daisy Joseph

*Introductory Chapter: Tracelement Effects in Biological Applications Using Photon Induced…*

*DOI: http://dx.doi.org/10.5772/intechopen.97387*

*Introductory Chapter: Tracelement Effects in Biological Applications Using Photon Induced… DOI: http://dx.doi.org/10.5772/intechopen.97387*

#### **Author details**

*Trace Elements and Their Effects on Human Health and Diseases*

**8**

**3. Conclusion**

ments in Biological samples.

Though trace elements are required in minimal quantities their presence in the optimal amount is essential for the normal physiological functioning of the body. They are one of the corner stone's in maintenance of biodynamic of the body. Both, excess and the deficiency states lead to initiation, promotion, and progression to various disease processes. The present paper has thoroughly discussed trace elements, as this area is away from the deserved attention. Thus, a comprehensive understanding of these trace elements is essential and significant for disease control and maintaining optimal health and X-ray Emission Techniques such as EDXRF, PIXE and EXAFS have shown to be good diagnostic tools for determining traceleDaisy Joseph Nuclear Physics Division, BARC, Trombay, Mumbai, India

\*Address all correspondence to: djoseph@barc.gov.in

© 2021 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

#### **References**

[1] Adriano DC. Trace elements in terrestrial environments: biogeochemistry, bioavailability, and risks of metals. 2 New York: Springer; 2003.

[2] Han FX, Banin A, Su Y, Monts DL, Plodinec MJ, Kingery WL, Triplett GB. Industrial age anthropogenic inputs of heavy metals into the pedosphere. Naturwissenschaften. 2002;89:497-504. [PubMed]

[3] Han FX, Patterson WD, Xia Y, Sridhar BBM, Su Y. Rapid determination of mercury in plant and soil samples using inductively coupled plasma atomic emission spectroscopy, a comparative study. Water, Air and Soil Pollution. 2006;170:161-171.

[4] Han FX, Su Y, Monts DL, Plodinec MJ, Banin A, Triplett GB. Assessment of global industrial-age anthropogenic arsenic contamination. Naturwissenschaften. 2003;90:395-401. [PubMed]

[5] Soto-Jiménez MF, Arellano-Fiore C, Rocha-Velarde R, Jara-Marini ME, Ruelas-Inzunza J, Páez-Osuna F. Trophic transfer of lead through a model marine four-level food chain: *Tetraselmis suecica*, *Artemia franciscana*, *Litopenaeus vannamei*, and *Haemulon scudderi*. Archives of Environmental Contamination and Toxicology. 2011;61(2):280-291. [PubMed]

[6] D.Joseph, M.Lal, H.N.Bajpai\*and P.K.Mathur,Levels of trace elements of a few Indian spices by EDXRF, J.of Food Science and Technology, Vol.36, No.3, 264-265, (1999)

[7] D.Joseph,Determination of Mercury and Arsenic Content in a few Indian Ayurvedic medicines by EDXRF, International Journal of PIXE, Vol. 19, Nos. 1 & 2 (2009) 77−89,Ó World Scientific Publishing Company

[8] D.Joseph, A.H.AbuAssar,P. Suprasanna, A.Saxena,R.K. Choudhury V. Bapat, Study of trace element correlations with drought Tolerance in different Sorghum Genotypes using Energy Dispersive X-ray Fluorescence (EDXRF) technique. Biol. Trace. Element Res. (2002) Vol. 85, No.3, 252-268

[9] Madan Lal, Daisy Joseph, R.K.Choudhury, H.N.Bajpai , Study of blood levels in children by Proton Induced X-ray Emission, Science of Total Environment, 103(1991), 209

[10] Madan Lal, H.N.Bajpai, Daisy Joseph and P.K.Patra Study of Trace Element Abnormalities in Blood of Chronic Renal Failure (CRF) patients by proton induced X-ray emission (PIXE),International Journal of PIXE, Vol 3, No.3, (1993) 229-236.

**11**

**Chapter 2**

**Abstract**

**1. Introduction**

Techniques

*Gurjeet Singh and Sanjiv Puri*

for analysis of biological samples are also reviewed.

**Keywords:** breast cancer, trace elements and XRF techniques

Role of Trace Elements in Breast

Using X-Ray Fluorescence

Cancer and Their Characterization

*Harpreet Singh Kainth, Deeksha Khandelwal, Ranjit Singh,* 

Breast cancer is the most common serious disease that occurs in the human body. Trace elements have an important function in biological and metabolism processes including activation or inhibition of enzymatic reaction, reactive oxygen species (ROS), competition between trace elements and metal proteins for binding positions and modifications in the permeability of cellular membranes which influence carcinogenic processes. A significant association between the abnormal concentration of trace elements and breast cancer has been found in many studies using XRF techniques like energy dispersive X-ray fluorescence (EDXRF), particle induced X-ray emission (PIXE), total reflection X-ray fluorescence (TXRF), wavelength dispersive X-ray fluorescence (WDXRF) and synchrotron induced X-ray fluorescence (SRIXE). This chapter considers trace elements like Fe, Cu, Zn, Cr, Cl, Ca, P, S, K, Na, Mg, Se, As and Sr. from the standpoint of their role as either inhibitory or causative agents of breast cancer. XRF techniques and sample preparation methods

In the human body, sometimes cells begin to grow out of control and divide into a large number of abnormal cells usually known as cancer cells. Cancer is a multifactorial complex disease. These cancer cells can spread or metastasize from one part of the body to another part and damage the patient's quality of life. Cancer is of various types like kidney cancer, colon cancer and lung cancer, etc. Among all these, breast cancer is the most common that occurs in the human body system. Generally, this type of cancer is commonly found in women. It is widely accepted that breast cancer is hormonally influenced, with most of the risk factors associated with the exposure of breast to stimulatory effects of female reproductive hormones, mostly estrogens, leading to increased cellular proliferation due to which normal cells gets converted into breast cancerous cells. Only 5–10% of all breast cancer cases are due to genetic factors i.e., the inheritance of mutations in breast cancer susceptible genes (BRCA1 and BRCA2). Rest of the cases are having hormonal and

#### **Chapter 2**

## Role of Trace Elements in Breast Cancer and Their Characterization Using X-Ray Fluorescence Techniques

*Harpreet Singh Kainth, Deeksha Khandelwal, Ranjit Singh, Gurjeet Singh and Sanjiv Puri*

#### **Abstract**

Breast cancer is the most common serious disease that occurs in the human body. Trace elements have an important function in biological and metabolism processes including activation or inhibition of enzymatic reaction, reactive oxygen species (ROS), competition between trace elements and metal proteins for binding positions and modifications in the permeability of cellular membranes which influence carcinogenic processes. A significant association between the abnormal concentration of trace elements and breast cancer has been found in many studies using XRF techniques like energy dispersive X-ray fluorescence (EDXRF), particle induced X-ray emission (PIXE), total reflection X-ray fluorescence (TXRF), wavelength dispersive X-ray fluorescence (WDXRF) and synchrotron induced X-ray fluorescence (SRIXE). This chapter considers trace elements like Fe, Cu, Zn, Cr, Cl, Ca, P, S, K, Na, Mg, Se, As and Sr. from the standpoint of their role as either inhibitory or causative agents of breast cancer. XRF techniques and sample preparation methods for analysis of biological samples are also reviewed.

**Keywords:** breast cancer, trace elements and XRF techniques

#### **1. Introduction**

In the human body, sometimes cells begin to grow out of control and divide into a large number of abnormal cells usually known as cancer cells. Cancer is a multifactorial complex disease. These cancer cells can spread or metastasize from one part of the body to another part and damage the patient's quality of life. Cancer is of various types like kidney cancer, colon cancer and lung cancer, etc. Among all these, breast cancer is the most common that occurs in the human body system. Generally, this type of cancer is commonly found in women. It is widely accepted that breast cancer is hormonally influenced, with most of the risk factors associated with the exposure of breast to stimulatory effects of female reproductive hormones, mostly estrogens, leading to increased cellular proliferation due to which normal cells gets converted into breast cancerous cells. Only 5–10% of all breast cancer cases are due to genetic factors i.e., the inheritance of mutations in breast cancer susceptible genes (BRCA1 and BRCA2). Rest of the cases are having hormonal and

**10**

264-265, (1999)

*Trace Elements and Their Effects on Human Health and Diseases*

[8] D.Joseph, A.H.AbuAssar,P. Suprasanna, A.Saxena,R.K.

[9] Madan Lal, Daisy Joseph,

R.K.Choudhury, H.N.Bajpai , Study of blood levels in children by Proton Induced X-ray Emission, Science of Total Environment, 103(1991), 209

[10] Madan Lal, H.N.Bajpai, Daisy Joseph and P.K.Patra Study of Trace Element Abnormalities in Blood of Chronic Renal Failure (CRF) patients by

proton induced X-ray emission (PIXE),International Journal of PIXE,

Vol 3, No.3, (1993) 229-236.

No.3, 252-268

Choudhury V. Bapat, Study of trace element correlations with drought Tolerance in different Sorghum Genotypes using Energy Dispersive X-ray Fluorescence (EDXRF) technique. Biol. Trace. Element Res. (2002) Vol. 85,

[1] Adriano DC. Trace elements in

biogeochemistry, bioavailability, and

[2] Han FX, Banin A, Su Y, Monts DL, Plodinec MJ, Kingery WL, Triplett GB. Industrial age anthropogenic inputs of heavy metals into the pedosphere. Naturwissenschaften. 2002;89:497-504.

[3] Han FX, Patterson WD, Xia Y, Sridhar BBM, Su Y. Rapid determination of mercury in plant and soil samples using inductively coupled plasma atomic emission spectroscopy, a comparative study. Water, Air and Soil

Pollution. 2006;170:161-171.

[4] Han FX, Su Y, Monts DL, Plodinec MJ, Banin A, Triplett GB. Assessment of global industrial-age anthropogenic arsenic contamination. Naturwissenschaften. 2003;90:395-401.

[5] Soto-Jiménez MF, Arellano-Fiore C, Rocha-Velarde R, Jara-Marini ME, Ruelas-Inzunza J, Páez-Osuna F. Trophic transfer of lead through a model marine four-level food chain: *Tetraselmis suecica*,

*Artemia franciscana*, *Litopenaeus vannamei*, and *Haemulon scudderi*. Archives of Environmental Contamination and Toxicology. 2011;61(2):280-291. [PubMed]

[6] D.Joseph, M.Lal, H.N.Bajpai\*and P.K.Mathur,Levels of trace elements of a few Indian spices by EDXRF, J.of Food Science and Technology, Vol.36, No.3,

[7] D.Joseph,Determination of Mercury and Arsenic Content in a few Indian Ayurvedic medicines by EDXRF, International Journal of PIXE, Vol. 19, Nos. 1 & 2 (2009) 77−89,Ó World Scientific Publishing Company

terrestrial environments:

Springer; 2003.

**References**

[PubMed]

[PubMed]

risks of metals. 2 New York:

non-hormonal non-genetic risk factors. Non-hormonal cases also indirectly tied to modulation of estrogens exposure.

Breast cancer is the most diagnosed cancer among females worldwide, having recent estimates of 2.1 million new incidence and 630,000 deaths in 2018 [1]. Most incidences of breast cancer are reported in the countries with higher Human Development Index (HDI) alleging westernization of lifestyle linked to menstrual characteristic (age at menarche and menopause and type of menopause), Reproduction factors (older ages at first birth, nulliparity, giving fewer births), exogeneous hormone intake (oral contraceptive pills, Menopausal harmonic therapy), medication (fertility drugs, Diethylstilbesterol), nutrition factors (high fat intake during adolescence, alcohol), anthropometric factors (rapid height growth during childhood and adolescence, high body mass index (BMI), body fat distribution), smoking [2]. Engagement in regular physical activities, to avoid consumption of alcoholic beverages, Breastfeeding with longer duration, balanced diet (fruits and vegetable, soy) is some of the important factors to reduce the risk of breast cancer. Although breast cancer incident rates are highest in economically developed than developing countries, the reverse is true for mortality rates, reflecting limited screening and less effective treatments in such areas. In year 2018, highest incidence rate has been recorded in Australia/New Zealand while highest mortality has been estimated in Melanesia [1].

Trace elements and their role in the cancer process have been a matter of great concern and early reports given by various researchers have proved that there is a relation between trace elements and cancer which play a key role in the biological and metabolic processes in the human body. It is reasonable to assume that the abnormal levels of these trace elements lead to the development of cancer in the human body system. Furthermore, the excess and deficiency of trace elements induce the formation of reactive oxygen species (ROS). It is believed that ROS lead to the formation of almost all types of cancer. Generally, these are divided into two groups: (a) Free oxygen radicals (b) non-radicals. The International Agency for Research on Cancer (IARC) has suggested the list of elements that show the carcinogenetic properties. These elements are Be, Cr, Co, Ni, As, Cd, Sb, Pb, Hg, Pt, Mn, Fe, Cu, Zn, Se and Sr. The abnormal levels of ROS disturb the biological processes and metabolic activities which results in the unchecked normal cells growth into cancerous cells [3]. For the detection of these trace elements, analytical techniques like energy dispersive X-ray fluorescence (EDXRF), total reflection X-ray fluorescence (TXRF), synchrotron induced X-ray fluorescence (SIXRF) and proton induced X-ray fluorescence (PIXE) have been widely used [4–9].

The aim of the present chapter is to discuss the role of trace elements in human breast cancer and the techniques used for quantitative elemental analysis of cancer samples.

#### **2. Methods and materials**

X-ray Fluorescence (XRF) is a well-established non-destructive analytical technique for quantitative as well as qualitative determination of elemental composition in samples independent of their physical and chemical forms. In XRF, either electrons or photons (X-rays/γ-rays) used as the excitation source, are incident on the sample thereby exciting the atoms of the elements present in the sample [10, 11]. The intensity of characteristic X-rays and scattered photons resulting from the photon-atom interaction processes are detected and measured using energy dispersive X-ray fluorescence (EDXRF) spectrometers. In EDXRF, the characteristic X-rays are not diffracted spatially and are detected by a detector with signal processing

**13**

X-ray fluorescence.

*Role of Trace Elements in Breast Cancer and Their Characterization Using X-Ray Fluorescence…*

electronics. An entire spectrum can be acquired virtually simultaneously in the EDXRF technique so that the detection of most of the elements across the periodic table can be possible within a few seconds. It is mainly a non-destructive chemical analysis technique and a great variety of non-portable and portable experimental set-ups are available. The triaxial geometry of non-portable spectrometers allows radiations (not monochromatic) of selected energy range, depending upon the secondary target. Si (Li) or Ge detectors, cooled by liquid nitrogen [12, 13], are generally used in these types of set-ups. As far as portable spectrometers are concerned, radioactive sources or X-ray tubes are commonly used. To reduce the noise level, detectors are cooled by the Peltier effect. These types of detectors have limitation of poor sensitivity, so trace elements of biological samples were studied less [14]. The wavelength dispersive X-ray fluorescence (WDXRF) technique is very important for the routine elemental analysis for the quality control of various materials. This technique is based on the Bragg's law, nλ = 2dsinθ, where n is an integer determined by the order of diffraction, θ is the scattering angle, d is the inter-planar distance of crystal lattice and λ is the wavelength of impinging radiation. On being incident on a crystalline sample, they are scattered in a peculiar fashion by the atoms undergoing constructive interference, which occurs only if the electromagnetic radiation or subatomic particle waves have the wavelength comparable to the atomic spacing. In this technique, amplification in the signal is obtained due to constructive interference of detected X-rays which obey the Bragg's condition. The crystal monochromator is one of the key parts in WD spectrometers. WDXRF technique uses optimized analyzer crystals and detectors to separate and count the emitted discrete X-ray wavelengths using diffraction from a crystal with a very high degree of resolution. This technique is more stable for analytical accuracy and precision even in performing the chemical analysis as compared to others. Total reflection X-ray fluorescence (TXRF) [15] and micro X-ray fluorescence (μXRF) [16] are the advanced variants of EDXRF. TXRF utilizes the property of total external reflection. In TXRF a fine, collimated and almost parallel X-ray beam from the X-ray tube falls on a smooth polished surface of the target sample in the form of a thin layer of a few nm thickness, at a grazing angle below the critical angle of the surface and gets totally reflected. Due to this condition, a totally reflected beam reduces scattering and absorption of the incident beam in the photon absorption matrix of a sample. This leads to a largely enhanced peak to background ratio, significantly amended fluorescence yield and consequently much better sensitivities to elements present even in ultra-trace levels. The improved detection limits of TXRF make it a valuable tool for trace and ultra-trace element analysis. The TXRF technique is more sensitive due to the use of glancing angle and destructive to some extent as compared to both the EDXRF and WDXRF techniques. **Figures 1** and **2** show the X-ray emission spectra of normal and abnormal breast tissue/blood samples obtained by non-destructive EDXRF, WDXRF and TXRF techniques. In *μ*XRF, X-rays generated by the X-ray tube are converged at a small region ~10 *μ*m on the sample surface by polycapillary lens (an X-ray focusing system) that exploits the phenomenon of multiple total external reflection in array of small hollow glass tubes [17]. The polycapillary lens increases the intensity and spatial resolution of X-rays irradiating on the sample. Also, irradiating the sample with X-rays micro focused only on the target position enhances the signal-to-background ratio by reducing fluorescence X-rays generated from adjacent areas. The photon microprobe is the best technique of the future for material information because of the very low deposit in the matter and its variety of interactions. One can use this technique in various element mapping applications of

Synchrotron induced X-ray fluorescence (SIXRF) offers distinct advantages

over other XRF techniques as the synchrotron radiation has been used as a

*DOI: http://dx.doi.org/10.5772/intechopen.95491*

#### *Role of Trace Elements in Breast Cancer and Their Characterization Using X-Ray Fluorescence… DOI: http://dx.doi.org/10.5772/intechopen.95491*

electronics. An entire spectrum can be acquired virtually simultaneously in the EDXRF technique so that the detection of most of the elements across the periodic table can be possible within a few seconds. It is mainly a non-destructive chemical analysis technique and a great variety of non-portable and portable experimental set-ups are available. The triaxial geometry of non-portable spectrometers allows radiations (not monochromatic) of selected energy range, depending upon the secondary target. Si (Li) or Ge detectors, cooled by liquid nitrogen [12, 13], are generally used in these types of set-ups. As far as portable spectrometers are concerned, radioactive sources or X-ray tubes are commonly used. To reduce the noise level, detectors are cooled by the Peltier effect. These types of detectors have limitation of poor sensitivity, so trace elements of biological samples were studied less [14]. The wavelength dispersive X-ray fluorescence (WDXRF) technique is very important for the routine elemental analysis for the quality control of various materials. This technique is based on the Bragg's law, nλ = 2dsinθ, where n is an integer determined by the order of diffraction, θ is the scattering angle, d is the inter-planar distance of crystal lattice and λ is the wavelength of impinging radiation. On being incident on a crystalline sample, they are scattered in a peculiar fashion by the atoms undergoing constructive interference, which occurs only if the electromagnetic radiation or subatomic particle waves have the wavelength comparable to the atomic spacing. In this technique, amplification in the signal is obtained due to constructive interference of detected X-rays which obey the Bragg's condition. The crystal monochromator is one of the key parts in WD spectrometers. WDXRF technique uses optimized analyzer crystals and detectors to separate and count the emitted discrete X-ray wavelengths using diffraction from a crystal with a very high degree of resolution. This technique is more stable for analytical accuracy and precision even in performing the chemical analysis as compared to others. Total reflection X-ray fluorescence (TXRF) [15] and micro X-ray fluorescence (μXRF) [16] are the advanced variants of EDXRF. TXRF utilizes the property of total external reflection. In TXRF a fine, collimated and almost parallel X-ray beam from the X-ray tube falls on a smooth polished surface of the target sample in the form of a thin layer of a few nm thickness, at a grazing angle below the critical angle of the surface and gets totally reflected. Due to this condition, a totally reflected beam reduces scattering and absorption of the incident beam in the photon absorption matrix of a sample. This leads to a largely enhanced peak to background ratio, significantly amended fluorescence yield and consequently much better sensitivities to elements present even in ultra-trace levels. The improved detection limits of TXRF make it a valuable tool for trace and ultra-trace element analysis. The TXRF technique is more sensitive due to the use of glancing angle and destructive to some extent as compared to both the EDXRF and WDXRF techniques. **Figures 1** and **2** show the X-ray emission spectra of normal and abnormal breast tissue/blood samples obtained by non-destructive EDXRF, WDXRF and TXRF techniques. In *μ*XRF, X-rays generated by the X-ray tube are converged at a small region ~10 *μ*m on the sample surface by polycapillary lens (an X-ray focusing system) that exploits the phenomenon of multiple total external reflection in array of small hollow glass tubes [17]. The polycapillary lens increases the intensity and spatial resolution of X-rays irradiating on the sample. Also, irradiating the sample with X-rays micro focused only on the target position enhances the signal-to-background ratio by reducing fluorescence X-rays generated from adjacent areas. The photon microprobe is the best technique of the future for material information because of the very low deposit in the matter and its variety of interactions. One can use this technique in various element mapping applications of X-ray fluorescence.

Synchrotron induced X-ray fluorescence (SIXRF) offers distinct advantages over other XRF techniques as the synchrotron radiation has been used as a

*Trace Elements and Their Effects on Human Health and Diseases*

modulation of estrogens exposure.

estimated in Melanesia [1].

non-hormonal non-genetic risk factors. Non-hormonal cases also indirectly tied to

Breast cancer is the most diagnosed cancer among females worldwide, having recent estimates of 2.1 million new incidence and 630,000 deaths in 2018 [1]. Most incidences of breast cancer are reported in the countries with higher Human Development Index (HDI) alleging westernization of lifestyle linked to menstrual characteristic (age at menarche and menopause and type of menopause), Reproduction factors (older ages at first birth, nulliparity, giving fewer births), exogeneous hormone intake (oral contraceptive pills, Menopausal harmonic therapy), medication (fertility drugs, Diethylstilbesterol), nutrition factors (high fat intake during adolescence, alcohol), anthropometric factors (rapid height growth during childhood and adolescence, high body mass index (BMI), body fat distribution), smoking [2]. Engagement in regular physical activities, to avoid consumption of alcoholic beverages, Breastfeeding with longer duration, balanced diet (fruits and vegetable, soy) is some of the important factors to reduce the risk of breast cancer. Although breast cancer incident rates are highest in economically developed than developing countries, the reverse is true for mortality rates, reflecting limited screening and less effective treatments in such areas. In year 2018, highest incidence rate has been recorded in Australia/New Zealand while highest mortality has been

Trace elements and their role in the cancer process have been a matter of great concern and early reports given by various researchers have proved that there is a relation between trace elements and cancer which play a key role in the biological and metabolic processes in the human body. It is reasonable to assume that the abnormal levels of these trace elements lead to the development of cancer in the human body system. Furthermore, the excess and deficiency of trace elements induce the formation of reactive oxygen species (ROS). It is believed that ROS lead to the formation of almost all types of cancer. Generally, these are divided into two groups: (a) Free oxygen radicals (b) non-radicals. The International Agency for Research on Cancer (IARC) has suggested the list of elements that show the carcinogenetic properties. These elements are Be, Cr, Co, Ni, As, Cd, Sb, Pb, Hg, Pt, Mn, Fe, Cu, Zn, Se and Sr. The abnormal levels of ROS disturb the biological processes and metabolic activities which results in the unchecked normal cells growth into cancerous cells [3]. For the detection of these trace elements, analytical techniques like energy dispersive X-ray fluorescence (EDXRF), total reflection X-ray fluorescence (TXRF), synchrotron induced X-ray fluorescence (SIXRF) and proton

The aim of the present chapter is to discuss the role of trace elements in human breast cancer and the techniques used for quantitative elemental analysis of cancer

X-ray Fluorescence (XRF) is a well-established non-destructive analytical technique for quantitative as well as qualitative determination of elemental composition in samples independent of their physical and chemical forms. In XRF, either electrons or photons (X-rays/γ-rays) used as the excitation source, are incident on the sample thereby exciting the atoms of the elements present in the sample [10, 11]. The intensity of characteristic X-rays and scattered photons resulting from the photon-atom interaction processes are detected and measured using energy dispersive X-ray fluorescence (EDXRF) spectrometers. In EDXRF, the characteristic X-rays are not diffracted spatially and are detected by a detector with signal processing

induced X-ray fluorescence (PIXE) have been widely used [4–9].

**12**

samples.

**2. Methods and materials**

#### **Figure 1.**

*Insert (a) and (c) show the spectrum of non-healthy (C1) and healthy (H1) blood samples of patients taken by crystal 2. Caption (b) and (d) represent spectrum of non-healthy (C1) and healthy (H1) blood samples of patients taken by crystal 1 of PC-WDXRF spectrometer [32].*

#### **Figure 2.**

*X-ray emission spectra of normal and abnormal breast tissue/blood samples obtained by non-destructive EDXRF and TXRF techniques [31].*

powerful X-ray source [18]. The synchrotron radiation source is characterized by a high degree of polarization and pulse height, high collimation, low emittance, reliability in energy by monochromatized emission. It has become an important tool in various fields of research. The synchrotron source provides the combination

**15**

**Figure 3.**

*technique [38].*

*Role of Trace Elements in Breast Cancer and Their Characterization Using X-Ray Fluorescence…*

of high flux and low divergence which is crucial for the massive success of experiments in the field of SIXRF. An important property of the synchrotron radiation is linear polarization which allows the SIXRF setup to detect the concentrations of specific elements present at trace levels due to a significant reduction in background level produced from Compton scattering. Almost complete repression of Compton scattering can be achieved by locating the detector at 90° with respect to the synchrotron beam in the plane of polarization. Hence the improved detection limit can be obtained using synchrotron radiation. The directionality and brightness of synchrotron radiation provide a superlative capability for micro-beam analysis. **Figure 3** represents the X-ray emission spectra of normal and cancerous

blood serum by non-destructive synchrotron based XRF technique.

Particle induced X-ray emission (PIXE) is well known technique for the elemental analysis and high cross-sections of the elements. In recent years, most of the scientists use this technique for the biological samples. Due to the low level of continuum background, it produced better results than other techniques. PIXE opens up a new era in the field of biological samples where measurements of low Z elements are possible at microscale level. It is also well known that the microbeam PIXE is the technique that offers results in ppm level with high sensitivity and the

*X-ray emission spectra of normal and cancerous blood serum by non-destructive synchrotron based XRF* 

*DOI: http://dx.doi.org/10.5772/intechopen.95491*

#### *Role of Trace Elements in Breast Cancer and Their Characterization Using X-Ray Fluorescence… DOI: http://dx.doi.org/10.5772/intechopen.95491*

of high flux and low divergence which is crucial for the massive success of experiments in the field of SIXRF. An important property of the synchrotron radiation is linear polarization which allows the SIXRF setup to detect the concentrations of specific elements present at trace levels due to a significant reduction in background level produced from Compton scattering. Almost complete repression of Compton scattering can be achieved by locating the detector at 90° with respect to the synchrotron beam in the plane of polarization. Hence the improved detection limit can be obtained using synchrotron radiation. The directionality and brightness of synchrotron radiation provide a superlative capability for micro-beam analysis. **Figure 3** represents the X-ray emission spectra of normal and cancerous blood serum by non-destructive synchrotron based XRF technique.

Particle induced X-ray emission (PIXE) is well known technique for the elemental analysis and high cross-sections of the elements. In recent years, most of the scientists use this technique for the biological samples. Due to the low level of continuum background, it produced better results than other techniques. PIXE opens up a new era in the field of biological samples where measurements of low Z elements are possible at microscale level. It is also well known that the microbeam PIXE is the technique that offers results in ppm level with high sensitivity and the

#### **Figure 3.**

*X-ray emission spectra of normal and cancerous blood serum by non-destructive synchrotron based XRF technique [38].*

*Trace Elements and Their Effects on Human Health and Diseases*

powerful X-ray source [18]. The synchrotron radiation source is characterized by a high degree of polarization and pulse height, high collimation, low emittance, reliability in energy by monochromatized emission. It has become an important tool in various fields of research. The synchrotron source provides the combination

*X-ray emission spectra of normal and abnormal breast tissue/blood samples obtained by non-destructive* 

*Insert (a) and (c) show the spectrum of non-healthy (C1) and healthy (H1) blood samples of patients taken by crystal 2. Caption (b) and (d) represent spectrum of non-healthy (C1) and healthy (H1) blood samples of* 

*patients taken by crystal 1 of PC-WDXRF spectrometer [32].*

**14**

**Figure 2.**

*EDXRF and TXRF techniques [31].*

**Figure 1.**

#### *Trace Elements and Their Effects on Human Health and Diseases*

size of the beam is smaller than the biological sample cell dimensions. In PIXE, the active protons (MeV) excite the target atoms to produce the X-ray spectrum by the inner shell decay process producing X-ray. The emitted X-ray energies are the characteristics of the elements and are proportional to the mass of that element present in the sample for further analysis [19–21]. **Figure 4** illustrates the normal and abnormal breast cancerous tissue obtained from PIXE spectrum. X-Ray absorption spectroscopy is a technique in which a core electron is excited to an empty state of LUMO and continuum, known as X-Ray Absorption Near Edge Spectroscopy (XANES) and extended X-ray absorption fine structure (EXAFS), respectively [22]. Mostly synchrotron is used as X-Ray source, but laboratory based commercial system are also available [23]. XANES spectra reveals average oxidation state, local coordination environment, chemical speciation and symmetry of the metal site while the EXAFS delineates the identity, number, distance of neighboring or adjacent atoms from the excited atom [24, 25]. XAS techniques acquire ascendency over X-Ray crystallographic techniques in the sense that local structural information around an element can be unraveled even from disordered samples such as powders and solutions. XANES spectroscopy has been used to examine the oxidation state of Zn, Fe and Cu in the normal and primary invasive breast cancer tissues [26].

**17**

*Role of Trace Elements in Breast Cancer and Their Characterization Using X-Ray Fluorescence…*

Breast tissue samples are generally collected from mastectomies, lumpectomies and breast reduction surgery [27–29]. Since, among the healthy population, a significant variation of trace elemental levels is found, generally healthy tissues from areas distant to malignant tissues/neoplastic are also collected from the same individual for comparison. For measurements like SIXRF to be performed directly on the wet tissues, collected tissue Samples are washed with milli-Q water to remove any stain of blood and are stored in formalin (10% formaldehyde in water) at room temperature or kept frozen at <−40°C until the analysis [27, 29, 30]. For EDXRF measurements, tissue samples are lyophilised for at −60°C and low pressure, approximately 10−1 atm, where the low temperature ensures the retention of volatile elements like Arsenic (As) and Mercury (Hg). These dried tissue samples are milled by freezer mill cooled by liquid nitrogen and pressed into pellets without any further chemical treatment or additive [7]. For techniques TXRF, requiring thin sections, tissue samples are cut into small pieces and excised into cylindrical pieces after cooling with liquid nitrogen. The excised tissue is then cut with a cooled microtome to get thin sections. Thin section is positioned in the centre of the sample carrier which is made non-hydrophobic by means of alcoholic silicon solution and then dried. Thin sections are spiked with internal standard solution and are again dried [31]. Various researchers have also used breast cancerous blood and blood serum for trace elements determination. For determination of trace element in human blood samples 4 ml of blood was mixed with 1350 mg cellulose and freeze dried. A fine powder of this mixture was pressed into pellets and used for WD-XRF measurements [32]. Blood serum samples are also lyophilized, and a small pellet is made which has been used for SIXRF in study [33]. For PIXE analysis of these serum samples, to make the sample conducting, graphite powder is also added and and pelletized [34]. **Table 1** shows the trace elements levels reported in the literature for normal and abnormal human breast cancer using XRF

The quantitative elemental analysis of different blood samples (normal and abnormal) was performed by using a commercial WDXRF spectrometer equipped with different anodes X-ray tubes (Rh/Ag/W), a gas flow proportional (FP) counter and a scintillation counter (SC) as photon detectors. The mass concentrations of different elements present in samples were determined using advanced software package available with the spectrometer. The intensity of X-ray lines for the specific

> 1 . ≠

= ×+ *g gg* <sup>∑</sup> *gw w w g*

where, *I*g corresponds to the intensity of the specific element *g*, *C*g refers to the measured concentration of the corresponding element, *D*g is the instrumental calibration coefficient for the given element. The term *m*w denotes the concentration of the other element *w* and *R*gw is the inter-element matrix coefficients. This software enables to evaluate the accurate concentrations of different elements ranging from Be to U present in unknown samples by incorporating corrections due to the matrix effects. The data acquisition time for each target was kept usually as ~20 minutes to collect good statistics under different X-ray peaks arising from

*I DC R m* (1)

element is determined with Lachance-Traill method which is defined as

*DOI: http://dx.doi.org/10.5772/intechopen.95491*

techniques. The values are given in *μ*g/g.

**2.1 Quantification of trace elements**

different elements present in each sample.

*2.1.1 WDXRF*

**Figure 4.** *Normal and abnormal breast cancerous tissue obtained from PIXE spectrum [37].*

#### *Role of Trace Elements in Breast Cancer and Their Characterization Using X-Ray Fluorescence… DOI: http://dx.doi.org/10.5772/intechopen.95491*

Breast tissue samples are generally collected from mastectomies, lumpectomies and breast reduction surgery [27–29]. Since, among the healthy population, a significant variation of trace elemental levels is found, generally healthy tissues from areas distant to malignant tissues/neoplastic are also collected from the same individual for comparison. For measurements like SIXRF to be performed directly on the wet tissues, collected tissue Samples are washed with milli-Q water to remove any stain of blood and are stored in formalin (10% formaldehyde in water) at room temperature or kept frozen at <−40°C until the analysis [27, 29, 30]. For EDXRF measurements, tissue samples are lyophilised for at −60°C and low pressure, approximately 10−1 atm, where the low temperature ensures the retention of volatile elements like Arsenic (As) and Mercury (Hg). These dried tissue samples are milled by freezer mill cooled by liquid nitrogen and pressed into pellets without any further chemical treatment or additive [7]. For techniques TXRF, requiring thin sections, tissue samples are cut into small pieces and excised into cylindrical pieces after cooling with liquid nitrogen. The excised tissue is then cut with a cooled microtome to get thin sections. Thin section is positioned in the centre of the sample carrier which is made non-hydrophobic by means of alcoholic silicon solution and then dried. Thin sections are spiked with internal standard solution and are again dried [31]. Various researchers have also used breast cancerous blood and blood serum for trace elements determination. For determination of trace element in human blood samples 4 ml of blood was mixed with 1350 mg cellulose and freeze dried. A fine powder of this mixture was pressed into pellets and used for WD-XRF measurements [32]. Blood serum samples are also lyophilized, and a small pellet is made which has been used for SIXRF in study [33]. For PIXE analysis of these serum samples, to make the sample conducting, graphite powder is also added and and pelletized [34]. **Table 1** shows the trace elements levels reported in the literature for normal and abnormal human breast cancer using XRF techniques. The values are given in *μ*g/g.

#### **2.1 Quantification of trace elements**

#### *2.1.1 WDXRF*

*Trace Elements and Their Effects on Human Health and Diseases*

breast cancer tissues [26].

size of the beam is smaller than the biological sample cell dimensions. In PIXE, the active protons (MeV) excite the target atoms to produce the X-ray spectrum by the inner shell decay process producing X-ray. The emitted X-ray energies are the characteristics of the elements and are proportional to the mass of that element present in the sample for further analysis [19–21]. **Figure 4** illustrates the normal and abnormal breast cancerous tissue obtained from PIXE spectrum. X-Ray absorption spectroscopy is a technique in which a core electron is excited to an empty state of LUMO and continuum, known as X-Ray Absorption Near Edge Spectroscopy (XANES) and extended X-ray absorption fine structure (EXAFS), respectively [22]. Mostly synchrotron is used as X-Ray source, but laboratory based commercial system are also available [23]. XANES spectra reveals average oxidation state, local coordination environment, chemical speciation and symmetry of the metal site while the EXAFS delineates the identity, number, distance of neighboring or adjacent atoms from the excited atom [24, 25]. XAS techniques acquire ascendency over X-Ray crystallographic techniques in the sense that local structural information around an element can be unraveled even from disordered samples such as powders and solutions. XANES spectroscopy has been used to examine the oxidation state of Zn, Fe and Cu in the normal and primary invasive

**16**

**Figure 4.**

*Normal and abnormal breast cancerous tissue obtained from PIXE spectrum [37].*

The quantitative elemental analysis of different blood samples (normal and abnormal) was performed by using a commercial WDXRF spectrometer equipped with different anodes X-ray tubes (Rh/Ag/W), a gas flow proportional (FP) counter and a scintillation counter (SC) as photon detectors. The mass concentrations of different elements present in samples were determined using advanced software package available with the spectrometer. The intensity of X-ray lines for the specific element is determined with Lachance-Traill method which is defined as

$$I\_{\mathfrak{g}} = D\_{\mathfrak{g}} \mathbf{C}\_{\mathfrak{g}} \times \left(\mathbf{1} + \sum\_{w \bullet \mathfrak{g}} R\_{\mathfrak{g}w} . m\_w \right) \tag{1}$$

where, *I*g corresponds to the intensity of the specific element *g*, *C*g refers to the measured concentration of the corresponding element, *D*g is the instrumental calibration coefficient for the given element. The term *m*w denotes the concentration of the other element *w* and *R*gw is the inter-element matrix coefficients. This software enables to evaluate the accurate concentrations of different elements ranging from Be to U present in unknown samples by incorporating corrections due to the matrix effects. The data acquisition time for each target was kept usually as ~20 minutes to collect good statistics under different X-ray peaks arising from different elements present in each sample.

