While breast cancer is the second most common type of cancer worldwide, it is the most common type of cancer among women. It is the leading cause of cancer-related death among women in developed and developing countries. Breast cancer risk factors are genetic and epidemiological risk factors such as age, family history, child birth, breastfeeding, and environmental factors [1]. It has been reported that hormones play a role in the development of breast cancers and that breast cancer will not occur without the effect of hormones [2]. Estrogens have an important role in the emergence of the disease, and it has been reported that endogenous estrogens cause cancer by stimulating cell growth and proliferation that cause breast cancer through receptors and their genotoxic metabolites [3]. Hormones are in protein structure and the building blocks of proteins are amino acids. Amino acids play important roles as essential metabolites and physiological regulators. The metabolism of amino acids is closely related to other metabolic networks. In some studies, while the plasma or serum free amino acid concentration remains approximately constant in healthy individuals, it has been reported that free amino acid concentrations are affected in various diseases such as liver and kidney failure, cancer, diabetes and Alzheimer's disease [4-6]. This suggests that free amino acids may be used as biomarkers in the diagnosis of some diseases. Amino acids are strongly regulated in healthy populations to maintain a neutral net protein balance, in spite of the continuous cycling of amino acids for protein synthesis and degradation. In addition, amino acids used to build cells and repairing tissue and also it is the main building material for antibodies to resist invader bacteria and viruses, however it is a basic part of the hormone and enzyme system. Although amino acids have an important role to play in transporting oxygen to various parts of the body, the represent an integral part of the activity of muscles [7]. In cancer patients, the degree of change in the amino acid pattern depends on several factors such as the specific type of cancer, the stage and extension of the disease [8]. The aim of this study is to determine amino acids, in the blood serum sample of women with breast cancer and compare them with the control group.

Material

In the study, blood samples were taken from a total of 77 individuals age between 20 to 65 who applied to Hiwa Cancer Hospital in Slimani, Kurdistan Region of Iraq, between September 2019 and October 2019. 45 Patients with breast cancer, with an average age of (47.3±11.4 years) and 32 healthy controls with an average age of (38.8±11.6 years) were selected. Among the 32 of the breast cancer patients did not receive  treatment, 13 received treatment. While 15 of the cancer patients were breastfeeding, 30 did not breastfeed at all. A personal interview was conducted with all patients using a specially designed full history questionnaire with accurate information.

Exclusion criteria 

These criteria include Patients with diabetes mellitus, pre-diabetes, chronic liver or renal disease, and malignancy other than breast cancer and smoking, and alcohol consumption. 5 mL of blood sample from individuals was taken into a tube and centrifuged at 3500 rpm for 10 minutes. The separated serum samples were taken into Eppendorf tubes and transported to the chemistry laboratory of Fırat University by cold chain and stored at -20 °C until analysis. The study was carried out with the approval of the ethics committee of Hiwa Cancer Hospital, Slimani, Kurdistan Region of Iraq, dated September-2019 and the approval of the ethic committee at Fırat University with the date and number of 22/10/2019-355284.

Determination of Amino Acids

Hydrolysis of blood serum samples was performed according to Elkin and Wasynczuk [9], and derivatization of amino acids in hydrolyzed samples was performed according to Kwanyuen and Burton [10]. The analysis of amino acids carried out by HPLC according to the method applied by Çakmak et al. [11].

Equipment and Chemicals 

Experiments were carried out by SHIMADZU HPLC, Prominence-I LC- 2030C 3D Model equipped with PDA detector. Double distilled (dd H₂O) water was used throughout the work. All the chemical used are reagent or analytical grade and obtained from Merck or Sigma-Aldrich.

Statistical Analysis

The data were evaluated using SPSS 22.0 program compatible with Windows. Comparison of continuous variables between the multiple groups were performed with the One-Way ANOVA test. Results were expressed as mean ± standard deviation p 0<0.05 and 0<0.001 with considered to be statistically significant and highly significant respectively.

77 volunteers divided into two groups; The first group consists of 45 patients with breast cancer and the second one 32 healthy individuals from the same demographic region, as the control group. The experimental findings of amino acids for the breast cancer patients and healthy control groups are given in Table 1.

Amino acids are essential nutrients for the survival of all cell types, have diverse roles within the tumor and in the tumor microenvironment. In addition to their role in biosynthesis, they help maintain the energy source and redox balance. Amino acid derivatives contribute to epigenetic regulation and immune responses associated with tumorigenesis, metastasis [12].

As seen in Table 1 while valine, tryptophan, glycine, histidine, tronin, pyroline, cysteine and asparagine amino acid amounts decreased in breast cancer patients compared to the control group (p<0.001), leucine, isoleucine, phenylalanine, methionine, lysine, alanine, tyrosine, glutamic acid, aspartic acid, arginine, glutamine and serine amino acid amounts were increased (p<0.001).  

Table 1: Amounts Of Amino Acids in Breast Cancer Patients and Control Groups

Note: One-way ANOVA test, were performed for statistical analyses. p<0.001was highly significant

Table 2: Amino Acid Level in the Serum of Breast Cancer Patients and Treated and Untreated Groups

Note: One-way ANOVA test, were performed for statistical analyses. p<0.05 was significant and p<0.001was highly significant

Table 3: Amino Acid Concentrations in the Serum of Women with Breast Cancer by Breastfeeding Status

Note: One-way ANOVA test, were performed for statistical analyses. p<0.05 was significant.

In a study, it was reported that the concentration of aspartic acid, glutamic acid, asparagine, serine, glutamine, citrulline, arginine, alanine, tyrosine, valine, phenylalanine isoleucine, leucine, ornithine, lysine amino acids in the serum of breast cancer patients increased compared to the control group [13].

In the study conducted by Eniu et al. [14], the amount of histidine, leucine, isoleucine, lysine, tyronin, valine, alanine, arginine, glutamine, glutamic acid, pyrroline and tyrosine free amino acids in the serum of patients with breast cancer decreased significantly compared to the control, but they reported that the decrease in the amount of methionine, phenylalanine, asparagine, aspartic acid, glycine, serine and cysteine was insignificant. Since free amino acids in serum or plasma are metabolites needed in cells for protein and DNA synthesis during cancer progression, their concentrations decrease [13,15-16]. Increased amino acid concentrations in tumor cells are indicators of high proliferation rate, which is associated with some molecular tumor subtypes [13,16]. It has been reported that the reason for the literature differences in plasma amino acid profile can be attributed to breast cancer stage, age, diet or measurement techniques applied for free amino acid determination [17]. Valine and glycine are known as significant amino acids for the biosynthesis of protein and decreased in cancer groups due to glycine depletion can indicate higher demand of gluconeogenesis or glutathione, caused by oxidative stress in the cancer patient. This finding supports the previous study conducted by Denkert et al. [18] on colon cancer patient. It has been reported that cysteine inhibits tumor growth by regulating redox stabilization, and therefore its concentration is low in cancer patients [19]. Valine and glycine are important amino acids for protein biosynthesis, and it is stated that the amount of glycine decreases to meet the high gluconeogenesis or glutathione demand in cancer patients [18].

The increase in the amount of methionine, glutamine, lysine, and alanine amino acids in the serum of breast cancer patients may be the result of malignant tumors degradation of proteins in normal muscle or tissues [20]. Serine, glutamic acid, aspartic acid, leucine, isoleucine concentrations were higher in patients with cancer. Together with leucine and isoleucine have the greatest potential to activate the mTOR pathway and facilitate protein synthesis in different tissues. Leucine can also stimulate insulin production, which facilitates the activation of the mTOR pathway. It has been reported that there is a significant decrease in the proliferation potential of breast cancer cells in isoleucine and leucine deficiency [21]. Tumors need amino acids to ensure their growth, for example glutamine, aspartic acid and glutamic acid are required for the synthesis of purine and pyrimidine, while serine has a key role in the synthesis of the lipid membrane component, survival and proliferation of various cancer cells in the metabolic network [22]. Increased levels of phenylalanine and tyrosine have been observed in breast cancer patients as a result of increased muscle proteolysis. Phenylalanine is first metabolized in the liver to tyrosine by phenylalanine hydroxylase. In inflammatory conditions such as infection and cancer, increased serum levels of phenylalanine and tyrosine have been reported [22]. Arginine is involved in nitric oxide metabolism, which is one type of free radical that takes part in nitrosative stress, which is a factor in the invasion, and metastasis in the inflammatory process and carcinogenesis. The cause of increased NO has been estimated to be increased levels of arginine, so arginine reduction may offer a potential method of treatment for breast cancer [23].

The serum amino acids levels of the groups treated and untreated with breast cancer patients are given in Table 2.

When the amino acid levels of the untreated group and the treated group were compared, the amount of lysine and aspartic acid decreased significantly (p<0.001), while the amount of tryptophan increased (p<0.037). Changes in other amino acid contents are statistically insignificant (Table 2). 

It is stated that during cancer treatment, the amount of glutamic acid decreases and the level of tryptophan increases. Glutamine and leucine levels decreased after treatment, while other amino acid levels were reported to be higher than control [24]. It has been reported that plasma tryptophan level is elevated after treatment of cancer patients [25]. In the study conducted by Gu et al. [26], it was reported that the plasma amino acid levels of patients with breast, stomach and thyroid gland tumors differed from healthy controls depending on the tumor type.

The isoleucine level, which was increased before treatment, decreased after treatment. In addition, the levels of tryptophan, histidine and valine amino acids decreasing, after treatment, these amino acids were increased to the level of healthy control [27]. The relationship between breastfeeding and breast cancer risk has been reported that lactation causes long-term endogenous hormonal changes, possibly decreased estrogen and increased prolactin production, thus inhibiting the initiation or growth of breast cancer cells [28]. It has been suggested that breastfeeding partially reduces this risk in women from breast cancer, but why breastfeeding reduces the risk of breast cancer has not been explained in detail [29]. 

Amino acids level of patients who have breastfeed and didn't breastfeed that are illustrated in Table 3.

As it can be seen in Table 3, aspartic acid and arginine levels were significantly increased in non-breastfeeding breast cancer patients compared to breast-feeding patients (p<0.05), while the changes in other amino acid levels were statistically insignificant (p>0.05).

It has been observed that the amount of aspartic acid and arginine, which increase in breast cancer patients compared to the control, on the other hand decreased by breastfeeding. There is no literature on the effect of breastfeeding on amino acids in patients with breast cancer.

Statistically significant changes (p<0.001) were observed in the amounts of all amino acids examined in the blood serum of patients with breast cancer. Significant changes were observed in the amounts of lysine, aspartic acid and tryptophan in cancer patients who treated and untreated. Significant changes were observed in the aspartic acid and arginine levels of the patients who breastfeed and did not breastfeed. Our result supports the hypothesis that breast tumor causes changes in serum amino acid profiles.

Acknowledgment

This research was supported by Firat University Scientific Research Center (FUBAP), Project Number FUBAP FF.19.33.

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