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 HNO₃. 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.

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.

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

*T-II = tumor-II, *T-III = tumor and *T-IV = tumor IV.

and tumor-IV with the group of fifteen blood samples from each stage to the ratio of age range between 25 - 73 years has been studied. The research work is accomplished through the effective and efficient co-ordination of participants because they are multi-approached masses and they are counsel and motivated to well co-operation made possible through providing them all details and benefits of studies. This is for their benefits and for society. Participants have multiple questions regarding research, but they were agreed through information mention in questionnaire including age, dietetic routine, physical information, racial origin, smoking along with health conditions. All affected and non-affected have been interviewed face-to-face by authors with the help of staff nurse of concerned hospitals. The situation of patients was actually worse more than 70% in terms of constant illness, deficiencies of nutrition, lack of knowledge about diseases, poor conditions and awareness regarding initial treatments of disease. However physical studies such as blood pressure, weight along with height and biochemical information were also taken from patients admitted in Nuclear Institute of Medicine & Radiotherapy (NIMRA) Jamshoro located in region of Hyderabad Sindh Pakistan. Prior to the sample collection, the protocol of the study was approved by the human ethical committee of the respective hospital and ethical committee of Sindh University, working under the auspices of the Higher Education Commission of Pakistan. The selection of patients with the help of biopsy proved evidence from breast cancer patients through cut-off 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.

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 HNO₃-H₂O₂ (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 laboratory 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].

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.

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].

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.

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.

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 sodium in three different stages of breast cancer patients was observed lower as compared to control females; however, it is inversely proportional to the stages of BC patients. After analyzing it was obtained that the level of sodium was observed low value in fourth stage of breast cancer than other stages of BC, the level of sodium decrease due to dosage of chemotherapy in BC patients. The low level of sodium and potassium were observed in breast cancer patient than control females. Because repeat cycles of chemotherapy may cause dehydration along with diarrhea vomiting and loss of energy through sweating that’s why the level of sodium & potassium was decreased in BC patients. Potassium observed in three different stages of breast cancer was low from tumor-II to tumor-IV due to increase stages of BC cause decrease level of potassium.

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

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].