An AAS Dependent Method for Quantitative Analysis of Essential Trace Elements from Blood Samples of Pakistani Female Breast Cancer Patients

Breast cancer is the second leading cancer in the world. The long-term expo-sure of some metallic compounds induces different forms of cancer, including breast cancer. Trace elements are essential metals for the physiological functions of the cell on a molecular level and also contribute in treatment of many diseases. The aim of study was to compare the level of essential trace elements, sodium, potassium, calcium, iron, and zinc in breast cancer patients with normal healthy adult women. Total forty-five patients (age range from 25 73 years) were included in this study and divided into three groups according to three different stages of breast cancer including tumor-II, tumor-III and tu-mor-IV. Blood was collected from all participants after taking history, clinical data and taking consent. However, about fifteen non-cancer healthy women in age range from 26 - 69 years were subjected to this study. The elemental concentrations were determined through atomic absorption spectrophotometer subsequent to microwave-induced acid digestion. The results of Na, K, Zn, Fe, Ca, were observed to decrease in blood samples of breast cancer patients as compared to non-cancer subjects. The results are reliable with other nu-merous literature reported studies, the efficiency, and deficiency of these trace metals may contribute an important role in the progress of breast cancer. Quantitative of Essential Trace Elements from Blood Samples of Pakistani Female Breast Cancer Patients.


Introduction
Breast cancer is the second leading cause of female cancer mortality [1]. According to the World Health Organization, breast cancer accounts for 16% of cancer deaths globally. The incidence of breast cancer increases with age, a certain lifestyle, genetics, and environmental factors are important risk factors in breast cancer [2]. The early detection of breast cancer is helpful in early diagnosis. There are many trace elements that are part of metalloenzyme and play a vital role in biological functions, for example structure, the composition of macromolecules, oxygen transport hormonal activities and free radical scavenging [3]. For the normal function of all living cells, potassium ion plays an important role in the normal nerve transmits, it is necessary to have K + transfer via nerve cell membrane. The increased concentration of potassium ion or deficiencies can cause adverse effects in biological functions, such as heart function and electrocardiographic change, etc. [4]. The different types of proteins and enzymes that contain potassium ion and decrease concentration of potassium ion can cause death such as gastrointestinal loss which includes (diarrhea and vomiting) and dieresis which is due to renal loss [5]. The decrease in potassium ion concentration symptom can be as decreased reflex response, paralytic illness, muscle weakness and respiratory paralysis which are rare and occur only in severe conditions. Alkalosis and cardiac arrhythmia are also symptoms of decreased (K + ) concentration. On the cellular level, the sodium ion channel activity is involved i.e. secretion proliferation and invasion even though lower levels of sodium involve in cancer progression [6] [7] [8] [9]. Hyponatremia or low concentration of sodium in our body is life threatening condition. Calcium is necessary for the regulation of metabolic energy [10] [11]. Calcium intake decreases the hyperproliferation of females mammary glands which are induced by increased fat diets, calcium can also decrease carcinogenic mammary glands and that is induced through 7,12-dimethylbenz[a]anthrancene [12]. But in some other studies, it has been showed that there is a negative relationship between breast cancer risk and the dietary calcium intake [13]. Calcium is also an important role in the proliferation of the cell. This was seen in prone transgenic mice that have obesity which shows that low level of calcium diet impeded loss in body fat in other hand high calcium diets decreased fat accretion and gain in weight on diet causing obesity and considerably enhanced weight and loss in fat during a caloric restriction regiment. Calcium used in diet caused considerably greater anti-obesity effect than supplemental calcium in these observations. A very important rich source of bioactive compounds is milk that can act as synergistically with suppressed activity of 1, 25-di hydroxyl vitamin D 3 to favorable effect nutrients portioning loss in fat and efficient metabolism [14].
Different metal proteins are made up of iron (Fe) and it has a very important role in different biochemical functions which can be oxygen transport, sensing electron transfer and catalysis. Myoglobin and hemoglobin are the main sources of hem iron in human foods that are obtaining from fish, poultry conserving supplements and dairy products are very important enzymes activities producing intracellular in bile and hepatic enzymes are involved. Patients of cancer are having low level of iron in their blood [15]. Deficiency of zinc (Zn) or change in its metabolism can cause acute or chronic inflammatory intestinal disease that can be due low intake or absorption, high excretions are requirements of metabolism and changed immune system [16]. Zn is primarily available as complex nucleic acids and proteins. It also participates in transmission intermediary metabolism and regulation of genetic information. Further in synthesis, storages, maintenance of chromatin in bio membrane [17]. That's why Zn is considered to act as protector of cellular growth as well as development of neoplastic cells. Recently during animal experiments protective effect of Zn has been demonstrated in which different transplantable tumors were inhibited when they have been implanted into animals maintaining on a diet having decreased Zn [18]. Superoxide dismutase (SOD) is very important enzyme that removes free radicals. Zn is very essential compound of this enzyme and protects against carcinogenesis and it is very necessary for activation of DNA repair enzymes [19]. It has been examined that low plasma Zn concentration was seen after trauma stress and in different malignancies patients. Change in Zn levels has been seen found in lymphatic proliferation along with gastrointestinal tract (GIT) and breast patients [20] [21]. Development or inhibition of cancer due to activity of trace elements is yet unclear although hundreds of investigation that have been performed in this regards [22]. Metal induced oxidative and carcinogenicity has been found in majority of these investigators [23] [24]. Many studies show considerable differences in the concentrations of different trace elements in tissues and blood of healthy persons were compared with cancerous patients [25] [26] [27].
The aim of this study is to understand the level of essential metal (Zn, Fe, Ca, Na, and K) from three different stages of breast cancer (T-II, T-III and T-IV) patients and control healthy females. The level of the correlation between breast cancer patients and control for the selected elements and the degree of impact on each other was compared. Therefore, examination of these elements may suggest any presumptive benefits in the analysis of breast cancer.

Reagents and Glassware
The ultrapure water obtained from the ELGA lab water system (Bucks, UK) was used throughout the work. Concentrated nitric acid 65% and hydrogen peroxide 30% were purchased from Merck (Darmstadt, Germany) and were checked for possible trace metal contamination. Standard solutions of Na, K, Ca, Fe, and Zn were prepared by dilution of certified standard solutions (1000 ppm) Fluka Kamica (Bush, Switzerland). Dilute working standard solutions were prepared immediately prior to their use by stepwise dilution of the stock standard solution with 0.2 M HNO 3 . All solutions were stored in polyethylene bottles at 4˚C. All glassware and plastic material used were previously soaked for 24 h in 2 M nitric acid, washed with distilled water, and finally rinsed with de-ionized water and dried and stored in class 100 laminar flow hood.

Apparatus
A Perkin-Elmer model A. Analyst 700 (Norwalk, CT, USA) atomic absorption spectrometer equipped with deuterium background correction was used. The hollow cathode lamps of Na, K, Ca, Fe, and Zn were run under the conditions at optimum values shown in Table 1. Integrated absorbance signals computed by the AA spectrometer were employed throughout. A Pel (PMO23) domestic microwave oven (maximum heating power of 900 W) was used for digestion of the biological samples. Acid washed polytetrafluoroethylene (PTFE) vessels and flasks were used for preparing and storing solutions.

Sample Collection and Pretreatment
At the beginning of work, about fifteen blood samples of non-cancer healthy women have been collected in the age range from 26 -69 years shown in Table   2. Besides this, forty-five blood samples were also collected from affected patients of breast cancer as per stages of breast cancer such as tumor-II tumor-III  affected parts (such as left or right breast cancer) prior to some treatment i.e. radiotherapy, chemotherapy and for last three months they were not taking any supplement minerals. While selection of fifteen non-affected females was fit in approximate same socioeconomic impact, age groups and they were healthy family members and do not use any supplements minerals. However, these were not suffering from any type of cancerous disease and also undergone standard medicinal scheduled examination [28]. The histological factors do not converse in this study.

Microwave-Assisted Acid Digestion Method
A microwave-assisted digestion (MDM) procedure was used to attain a minimum time for digestion as compared to convection heating on hot plate. The triplicate samples of breast cancer (200 mg) patients and non-affected females have been placed into Teflon PFA flasks. However, two milliliters of newly prepared mixture of concentrated HNO 3 -H 2 O 2 (2:1, v/v) was added to each flask and left for 10 min. Then the flasks were placed in a covered PTFE container. This was then heated following a one-stage digestion program at 80% of total power (900 W) 4 -5 min was required for the decomposition of organic matrix of breast cancer samples. After digestion the flasks were left to cool and the resulting solution was heated to evaporate and remove excess acid then filtered and diluted up 10.0 mL in volumetric flasks with 0.1 M nitric acid. Blank extraction (without samples) was carried out, performing the well-established labora-tory protocols. All digests obtained from both methods were analyzed for Na, K, Ca, Fe and Zn by AAS. The concentrations were obtained directly from calibration graph after correction of the absorbance for the signal of appropriate reagent blank. The validity and efficiency of the (MDM) were checked with those obtained from a conventional wet digestion method [29].

Micronutrients in Control Females
For the determination of five micronutrients such as Na, K, Ca, Fe and Zn from control females, fifteen different blood samples were collected in the age range of 26 -69 years and analyzed by using atomic absorbance spectroscopy. After analyzing it was calculated that the level of sodium was observed slightly high in the age of 69 years as 3353 mg/L while low was observed at the age of 29 years as 3174 mg/L. Similarly, potassium level in control females was analyzed as high in the age of 69 years 209.31 mg/L while low in the age of 26 years as 196 mg/L shown in Table 3. However, level of calcium was calculated within required range as 87.4 mg/L in the age 26 years and 80 mg/L was observed at the age of 69 years. The iron level was obtained 0.59 mg/L at 26 years while 0.82 was analyzed at the age of 35 years. The essential metal zinc was analyzed in lower required range of control as 0.52 mg/L at 26 years however 1.52 mg/L was observed at 60 years. The level of these micronutrients was calculated in required range at same socioeconomic impact, age along life style.

Micronutrients Level in Breast Cancer Patients
The forty-five different blood samples were collected from three stages of breast cancer tumor-II, tumor-III-and tumor-IV in the age range of 25 -73 years. It was calculated that micronutrients level micronutrients such as sodium, potassium, calcium; iron and zinc were decreased as increase stage of breast cancer from T-II to T-IV [1] [3] [12].

Sodium and Potassium
The level of sodium in the second stage of breast cancer patients was observed high as 3085 mg/L at the age of 26 years shown in Table 4 while low value 2945 mg/L at the age of 52 years. Potassium level in 2 nd stage of breast cancer was analyzed high in the range of 158.1 mg/L at the age of 44 years shown in Table 5 while low value was calculated 133.2 mg/L at the age of 61 years, However, calculated results of potassium along with sodium were lower than control females at same socioeconomics impact and age range.

Calcium, Iron and Zinc
Calcium is inversely proportional to the stages of breast cancer. Calcium was analyzed in 2 nd stage of breast cancer high in the range of 78.83 mg/L at the age of 36 years, shown in Table 6 however low value of calcium was calculated 65.27 mg/L at the age of 61 years. The level of Iron in 2 nd stage of breast cancer were found lower than control females, after analyzing it was observed that high   Table 6. Shows the level of calcium in breast cancer patients.  amount of iron observed at the of 0.57 mg/L at 29 years shown in Table 7 while low value was observed at the age of 36 years in the range of 47.0 mg/L. Zinc was also calculated lower level in 2 nd stage of breast cancer patients as compared with control females. However, zinc obtained a high level of 0.75 mg/L at the age of 26 years shown in Table 8. While low level of Zn 0.51 mg/L was observed at the age of 61 years.

Sodium and Potassium
The sodium level in 3 rd stage of breast was calculated in lower than 2 nd stage as well control females, the level which was observed high 2934 mg/L in the age range of 29 years while low 2796 mg/L observed at the age of 65 years. After analyzing it was observed that sodium is inversely proportional to the stages of breast cancer. However, potassium in 3 rd stage of breast cancer was also observed lower than 2 nd stage of breast along with healthy control females. The level of essential micronutrient potassium calculated in the 3 rd stage of breast cancer was high 139.7 mg/L at the age of 25 years while low 134.3 mg/L was observed at the age of 65 years. The level of sodium and potassium are inversely proportional to the stages of breast cancer patients.

Calcium, Iron and Zinc
Calcium is inversely proportional to the stages of breast cancer, the level of calcium observed in the 3 rd stage of breast cancer was found lower than control females. Calcium which was analyzed in the 3 rd stage was high 72.4 mg/L at the age of 34 years while low 60.1 mg/L observed at the age of 65 years. The level of iron was also decreased as increases the stages of breast cancer, however iron in 3 rd stage of breast cancer analyzed high 0.51 mg/L at the age range of 35 years while low value was also obtained 0.41 mg/L at the age of 65 years. Zinc was also observed lower than control females; after analyzing it was clearly obtained lower level of Zn in 3 rd stage of breast cancer than 2 nd stage of breast cancer. However, the calculated range which was high 0.66 mg/L at the age of 25 years while low value 0.51 mg/L was observed at the age of 65 years.

Sodium and Potassium
Level of sodium in 4 th stage of breast cancer decreased as compared to control females; however, sodium which was observed in 4th stage of breast cancer was observed 2786 mg/L at the age range of 44 years while low value was obtained 2511 mg/L at the age of 70 years. It was observed that the sodium level increased inversely proportional to the stages of breast cancer. The potassium level was also decreasing as it increases stages of breast cancer among women. The level of potassium observed in 4 th stage was high 133 mg/L at the age range of 48 years while low value was calculated 125.3 mg/L at the age of 73 years.

Calcium, Iron and Zinc
The level of calcium decreased by increasing stages of breast cancer patients, however it is inversely proportional to the stages of breast cancer. Calcium level observed in 4 th stage of breast cancer high 59.22 mg/L at the age range of 44 years while low-level value was obtained 49.3 mg/L at the age of 73 years. The iron level was also decreased by increasing the stages of breast cancer patients, level of iron in 4 th stage of breast was calculated high 0.41 mg/L at the age range of 28 years however low level of iron was calculated 0.30 mg/L at the age of 48 years. Zinc observed in 4 th stage of breast cancer was high 0.39 mg/L at the age of 54 years while low level of zinc was also calculated 0.15 mg/L at the age of 73 years. After analyzing it was calculated that zinc is inversely proportional to the stages of breast cancer [30]. The demographic data of the female breast cancer patients and healthy women (hereafter called "controls") Subjects in the two groups were closely matched with age, residential area, profession, and same dietary habits. However, some patients were smokers. Various types of cancer are found in females such as cervical cancer, skin cancer, ovarian cancer, but breast cancer is the leading cause of death all over the world. Incidence rates of breast cancer have been increased over the previous few decades in western countries, particularly among postmenopausal females. The rate of breast cancer is increased in developing countries especially in Asia where the lack of knowledge of basic health such as deficiency of essential macronutrients living style in neighborhood area. The biochemical parameters of cancerous patients were also altered. According to origin of breast cancer it is divided into four types i.e. tumor-I, tumor-II, tumor-III, and tumor-IV.
To determine the micronutrients level in breast cancer patient such as Na, K, Ca, Fe, and Zn in three different stages of breast cancer patients, while the tumor in stage one of breast cancer patient was not found in that area because women's are unaware about it, lack of knowledge about initial arise up of breast cancer [31].

Stage-II
The micronutrient level in stage-II of breast cancer patients was observed low level as compared to control females because in this stage the tumor size II-A ≤ 2 cm and has spread to lymph nodes or 2 -5 cm and has spread to lymph nodes.
While stage II-B 2 -5 cm and has spread to lymph nodes or >5 cm and has not spread to lymph nodes. The level of micronutrients in 2 nd stage of breast cancer is inversely proportional to the stages of breast cancer that why the micronutrients decrease as increases stage of cancer.

Stage-III
Five different micronutrients were analyzed and compared with control females.
After analyzing it was observed that the levels of micronutrients were calculated, however micronutrients level decreased with increase stage of BC.

Stage-IV
The level of micronutrients observed in the 4 th stage of BC was at a low level as compared to control females, because low intake of vitamin along with essential minerals may cause deficiency of micronutrients in BC patients. The treatments include radiotherapy means to kill cancer cell through radiation therapy or kill cancerous cell through chemotherapy may cause mostly vomiting along with diarrhea after this very serious condition occur that is why low level of such micronutrients was observed in last stage of breast cancer patients. The level of so- However, one of the most important macronutrients is calcium because it plays an essential role in metabolism. Mostly the functions are relying on the good case of the extracellular Ca with respect to narrow range. The interruption of that firmly ordered homeostatic system cause mess of Ca metabolism and that will predictably affect cellular functions. The low intake of Ca aggregates the risk of BC especially in women having pre-menopausal issues. Ca has been examined in 3 different stages of breast cancer were low than control females. It was seen after checking that when the stage of breast cancer increases from tumor-II to IV the Ca level decreases. The noticeable disease in hemoglobin, hematocrit, packed cell volume, hemoglobin composed of corpuscular and RBC was examined inpatient having cancer than others. The study of biochemical changes in the period of deadly conversation indicated as a form of chemical biopsy due to their facilitation of the diagnosis of organ abnormalities in the chemical eye. Fatigue among cancer patients caused by anemic condition is associated low hemoglobin. Patients having anemia hemoglobin levels are low and thus patients come often tired and they are having lower energy. This is due to fact that there is not enough hemoglobin to carry oxygen to the stationary tissues. So, there is no enough oxygen which can convert nutrients into energy components. The other blood parameter having anemia include RBC, MCH, MCV, and hematocrit could also below. The change in toxic and trace elements in BC patients has got inverse relationship with biochemical parameters. Trace elements act at subcellular or cellular level via various mechanisms. One of such mechanism can be shown as the interaction between hormones and trace elements that maintain metabolism of higher biochemical substrate, therefore, a possible association between etiology of cancer and trace elements might be expected [32] [33] [34]. It was studied that the status of Zn was changed in BC samples of confirmed biopsy cases as compared to non-cancerous females having same age group. Current resulted data shows the considerably decreased levels Ca, Zn, Fe, Na, and K in women having BC in increasing order as breast N, ovary N as compared to control females of same age. Our observation consisted of other epidemiological investigations that show decreased levels of different micronutrients in various cancer patients as shown in Figure 1 [35]. It was also been shown that the content Advances in Breast Cancer Research of Zn in blood serum of tumors was lower than non-cancerous females. Zn is very common to induce the production of metallothionein. That is enriched in cytosine and it is an excellent scavenger of radical having hydroxyl group. Therefore now it has been clear that Zn has got many roles as an antioxidant and is thus an extraordinary candidate for clinical chemoprevention trials in humans [36]. One of major problem in cancer patients is malnutrition but it is not prevented in all cancer cases but is common and serious problems in case of breast and mouth cancer [37]. Commonly it has been seen that cancer patients are having inadequate protein and energy intakes high metabolic rate and several abnormalities in lipid, energy carbohydrate, protein, metabolism. During treatment of cancer through chemotherapy and radiotherapy is also responsible for potential damage to nutrition levels [38]. The risk of certain types of BC, mouth, and oropharyngeal cancer is may be increased due to intake of diet. This is because of lack of essential vitamins and trace elements in poor diet [39].

Conclusion
The result of this investigation showed that patients having BC have got a low level of important trace elements such as (Na, K, Ca, Fe and Zn) when these cancerous patients compared with healthy women. This study also showed that some diet intake and inhibition of toxic elements may increase the risk of cancer and related diseases. Such toxic elements and diet can be smoking, treated unclean water. But this study now provides a significant amount of epidemiologic evidence that role of essential trace elements has got influential effect on cancer risk. Further necessary studies should be carried out to reveal mechanisms of trace elements carcinogenic patients admitted in various hospitals that should be checked for essential elements measurement in their biological sample. So that markers of cancer can be predicated on the earliest possible stage.