Chapter Chapter Determination of inorganic trace elements in ovarian tumor cyst fluid samples by inductively coupled plasma optical emission spectrometry 98 Chapter 5.1 Preface to Chapter Inductively coupled plasma-optical emission spectroscopy was used to study the variations in trace elemental concentrations between benign and malignant human ovarian tumor cyst fluid specimens and to understand the effects of these elements in the progression of ovarian tumor. The samples were prepared by microwave assisted acid digestion prior to ICP-OES analysis. Many of the trace elements were identified and their concentrations were estimated in 30 cyst fluids (15 benign and 15 malignant) samples. The elements Cr, Cd, Cu, Fe, Mn, Pb, Zn and Ni were found to be present in all samples in significant concentration. Among them Fe, Cr, Pb, and Ni were highly elevated in malignant samples compared with benign cyst fluids. The excess levels of trace elements observed in the malignant tumor samples could either be a cause or a consequence of ovarian cancer. Regarding their role in the initiation or promotion of ovarian cancer, one possible interpretation is that the elevated levels of Fe, Cr, Pb and Ni could have led to the formation of free radicals that adversely affect DNA thereby causing ovarian tumor. 99 Chapter 5.2 Introduction Trace elements play a vital role in all biological systems. They take part in all metabolic and physiological processes, being components of different enzymes and catalysts for chemical interactions in living cells [1-3]. Trace elements deficiency or excess amount of trace elements with respect to the human physiological level has been found in patients with certain diseases, including cancer. Study on the trace metals for human health has been attracting much of attention in recent decades. Several elements are usually determined in body fluids and tissues for the diagnosis and monitoring of various disorders, nutritional deficiencies, and occupational or environmental exposure [1-3]. The number of elements of interest has increased in the recent decade, not only the trace metals but the heavy metals as well. The study of the role of the trace elements has been emphasized in the search for the possible causes of cancer. The International Agency for Research on Cancer has proposed trace elements for their carcinogenesis [4, 5]. Some of them or their compounds are now recognized as carcinogenic to humans. Those elements include: Be, Cr, Co, Ni, As, Cd, Sb, Pb, Hg, and Pt. The carcinogenesis of Mn, Fe, Cu, Zn, Se and Sr has not been completely revealed yet, however assessment of their xenobiotic effects associated with disease progress is necessary [6]. Numerous investigations have been carried out in studying the role of trace elements in the development or inhibition of cancer. To understand the cause for metal induced tumors, it is important to understand the mechanism of metal carcinogenicity. Significant differences in the concentration of various trace metals in the blood and tissues of the cancerous patients compared with the healthy personnel have 100 Chapter been reported [7, 8]. Ovarian cancer is one of the common types of cancer worldwide and it is mainly associated with environmental factors rather than genetics. The role of environmental pollutants thus becomes important in the prognosis and screening of the disease. Several studies have been reported in the recent years regarding the trace metal evaluation in the body liquids. Blood is a transport medium for trace metals to and from the tissues and it provides rapid and reliable information about the trace metal metabolism in human body [3, 6]. Consequently, whole blood, serum and plasma have been used in biological research for the determination of trace metal status of individuals and groups [9-11]. Nonetheless, in the case of ovarian tumor, both benign and malignant ovarian cyst fluids are good source of information about tumor marker and other compounds which are associated with the origin and prognosis of tumor. Thus, ovarian cyst fluid analysis will give more reliable information about the metal induced carcinogenicity of trace elements rather than any other body fluids. Determination of metal ions at trace level is difficult for the biological samples, owing to trace amount of these metal ions and complex contents of the matrices. Hence, preconcentration and separation technologies have to be employed to extract analytes from the complex matrix so as to obtain suitable concentrations of the analytes for accurate determination. Sample preparation is an important step for metal analysis of biological samples. The choice of sample preparation depends on the type of sample and the selection of instrument. Commonly used preparation methods are acid digestion, microwave-assisted digestion. Similarly, 101 Chapter several spectrometric techniques, such as FAAS [12], hydride generation AAS [13], inductively coupled plasma-mass spectrometry [14], graphite furnace AAS [15], capillary electrophoresis [16], atomic fluorescence spectrometry [17] have been investigated for metal determination. ICP-OES is one of the powerful tools for the determination of metals in a variety of different sample matrices. The technique is based upon the spontaneous emission of photons from atoms and ions that have been excited in a RF discharge. Liquid and gas samples may be injected directly into the instrument, while solid samples require extraction or acid digestion so that the analytes will be present in a solution. The sample solution is converted to an aerosol and directed into the central channel of the plasma. At its core the ICP sustains a temperature of approximately 10 000 K, so the aerosol is quickly vaporized. Analyte elements are liberated as free atoms in the gaseous state. Further collisional excitation within the plasma imparts additional energy to the atoms, promoting them to excited states. Sufficient energy is often available to convert the atoms to ions and subsequently promote the ions to excited states. Both the atomic and ionic excited state species may then relax to the ground state via the emission of a photon. These photons have characteristic energies that are determined by the quantized energy level structure for the atoms or ions. Thus the wavelength of the photons can be used to identify the elements from which they originated. The total number of photons is directly proportional to the concentration of the originating element in the sample. In this current study, benign and malignant cyst fluids samples of ovarian tumor patients were studied for the estimation of metal concentration by microwave 102 Chapter assisted acid digestion followed by ICP-OES technique. Moreover, the trace metals are responsible for many complex biological interactions and such relationships are quite complicated, hence reliable prediction of a certain disease based on trace metal analysis requires suitable mathematical or statistical model to distinguish healthy and diseased subjects. 5.3 Materials and methods 5.3.1 Reagents and samples Analytical-reagent grade nitric acid (Merck), Hydrofluoric and hydrochloric acids (Merck), Distilled, de-ionized water (DDW) and ultrapure were used for sample preparation. A total of 30 cyst fluids (15 benign and 15 malignant) samples were analyzed in this study. The collection and storage of the cyst fluid was done as described in chapter 2. 100 µL of each cyst fluid was diluted (10 -fold) to mL with ultrapure prior to the acid digestion. 5.3.2 Microwave-assisted acid digestion The samples are digested prior to analysis with nitric acid in a closed microwave digestion system (Milestone Inc., CT, USA). An aliquot of sample (1 mL) was digested with 1.5 mL of HNO3 diluted with DDW (1+2) for 30 at 300W power. After cooling to room temperature, the digest was poured into poly(propylene) autosampler tubes and diluted to 10 mL with DDW. Prior to use, these tubes were thoroughly cleaned in a sequence with detergent, water, a mixture of HNO3 (1.4 mol L-1) and HCl (1.1 mol L-1) followed by soaking in HNO3 (0.7 mol L-1) and a final 103 Chapter rinse with Milli-Q water. All pipette tips used for blood, plasma, cyst fluid, water and were rinsed in a sequence with HNO3 and DDW. 5.3.3 ICP-OES analysis A Perkin-Elmer dual-view optima 5030 DV ICP-OES system was used for measuring the concentrations of elements. The mode of time scan was chosen to measure the concentration of metal ions due to its low consumption of sample. The operational conditions of the ICP-OES system are given in Table 5.1. Table 5.1 Operational parameters of ICP-OES. Parameters Flush pump rate Parameters value 1.85 mL min−1 Analysis pump rate Pump relaxation time RF power Nebulizer flow Auxiliary gas Light source ICAP view 1.85 mL min−1 s 1350 kw 26.0 psi 1.0 lpm ICAP Dual view 5.4 Results and discussion In the present work, trace metal analysis was carried out in the benign and malignant cyst fluid samples. The metal elements were identified and their concentrations were estimated. Most of the metals were present in all 30 samples and the concentration ranges from ng g-1 to µg g-1 levels. Basic statistical distribution parameters of essential trace metals (Cd, Se, Cu, Fe, Mn, Cr, Ni, Pb and Zn) in the 104 Chapter malignant and benign cyst fluids are shown in Table 5.2, whereas, the average metal concentrations are shown in Figure. 5.1, for comparative evaluation. Figure 5.1 Comparison of average trace metals in the benign and malignant cyst fluid samples. Large variations were observed among minimum and maximum concentrations of trace metals in all the groups. In the malignant samples, Cu (1.835 µg g-1), Fe (2.252 µg g-1), Pb (1.904 µg g-1), Cr (2.309 µg g-1), Zn (1.059 µg g-1) and Ni (1.352 µg g-1) emerged as major contributors on the average, while; slightly lower mean concentrations were observed for Cd, Se and Mn. Most of the trace metals revealed random distribution in both samples, as shown by large standard deviation (SD) values. Mean concentrations of Fe, Cr, Pb and Ni in the malignant samples were significantly higher than those observed in the benign samples; however, the average concentrations of Cd, Se, Cu and Mn were found to be slightly differing in the malignant cyst fluids compared with the benign samples. 105 Chapter Table 5.2 Distribution of trace elements in benign and malignant ovarian cyst fluid samples. Elements Se Cd Cu Fe Mn Cr Zn Pb Ni Malignant Min 0.076 0.194 0.847 0.274 0.225 0.625 0.072 0.37 0.052 -1 (concentration in µg g ) (n = 15) Max Mean SD 0.996 0.436 0.273 1.05 0.495 0.248 2.643 1.835 0.567 8.183 2.252 2.326 0.984 0.443 0.222 7.853 2.309 2.631 3.695 1.059 0.941 4.525 1.904 1.04 2.738 1.352 0.874 -1 Benign (concentration in µg g ) (n = 15) Min Max Mean SD 0.052 0.726 0.331 0.137 0.152 0.924 0.437 0.192 0.725 2.102 1.725 0.437 0.065 3.167 1.223 0.745 0.189 0.923 0.463 0.199 0.476 5.36 1.08 0.926 0.956 4.491 1.366 0.697 1.627 2.895 0.915 1.073 0.075 2.292 0.586 0.508 106 Chapter 107 Chapter In the present work, significantly elevated levels (p[...]... Chapter 5 Many other trace elements which are known as metal carcinogens such as Be, V and As are widely present in many samples both benign and malignant sample, however there is no significant trend in their quantity Regarding the role of trace elements in the initiation or promotion of tumor, one possible interpretation is that the elevated levels of Fe, Cr, Pb and Ni could have led to the formation of. .. substance Moreover, being an important cofactor for angiogenesis, copper can affect carcinogenesis [31, 32] Without copper, the mediators of angiogenesis cannot function, thereby stopping the growth of new blood vessels The excess copper levels observed in the benign possibly promote the malignant transformation of ovarian tumor The present findings in agreement with the results of earlier studies on... benign and malignant human ovarian cyst fluid specimens and to understand the effects of altered homeostasis of these elements in the etiology of ovarian cancer Almost all the elements identified were found to be elevated in the malignant samples when compared with benign cyst fluids The obtained results indicate that the increase in malignant samples is significant (p< 0.04) for Fe, Cr, Pb and Ni,... while for the elements Se, Cd, Cu and Mn no significant increase is observed The excess levels of trace elements observed in the malignant tumor samples could be a cause of ovarian cancer Considering the possibility of free radical formation which alter the DNA thereby causing ovarian tumor, this probably results in an increased vascularity of malignant cyst fluids, which in turn leads to enhancement of. .. to be high in cancerous tissues [22] Iron is essential for the normal physiological functions in humans, since it is an integral part of many proteins and enzymes It plays a vital role in the regulation of cell growth and in differentiation [23, 24] Physiological maintenance of relatively constant levels of iron is very crucial, since both iron deficiency and iron overload are harmful, and can contribute... free radicals or other reactive oxygen species that adversely affect DNA thereby causing ovarian tumor Tumors, characterized by unregulated multiplication of cells, need an everincreasing supply of essential nutrients including trace elements because of increased cellular activities and requirements 5. 5 Conclusion The present experimental study was undertaken to determine the variations in trace elemental... increase in the incidence of hypertension has been observed following lead exposure in humans [8] In the present study, elevated levels of Pb ((p . for the determination of trace metal status of individuals and groups [9-11]. Nonetheless, in the case of ovarian tumor, both benign and malignant ovarian cyst fluids are good source of information. carried out in studying the role of trace elements in the development or inhibition of cancer. To understand the cause for metal induced tumors, it is important to understand the mechanism of metal. 0.6 25 7. 853 2.309 2.631 0.476 5. 36 1.08 0.926 Zn 0.072 3.6 95 1. 059 0.941 0. 956 4.491 1.366 0.697 Pb 0.37 4 .52 5 1.904 1.04 1.627 2.8 95 0.9 15 1.073 Ni 0. 052 2.738 1. 352 0.874 0.0 75 2.292 0 .58 6 0 .50 8 Malignant