Introduction

Breast cancer (BC) is the most common malignancy in females. In 2020, more than 2 million new cases were diagnosed worldwide [1, 2]. The prevalence of breast cancer (BC) is high among cancers in Egypt, ranking it the most common cause of cancer mortality in women [3, 4].

Several risk factors for BC are identified. Advanced age, female sex and family history are among the most common factors [5]. In addition, mutations of BRCA 1 and 2 genes account for a significant number of BC cases [6].

BRCA1 and BRCA2 proteins are tumor suppressors that play an important role in DNA damage response and repair. BRCA1 is also needed for estrogen receptor (ER) gene transcription [7]. Upregulated expression of BRCA1 and BRCA2 genes was reported in BC and ovarian cancer, furthermore, high BRCA1 gene expression was demonstrated to increase the risk of early distant metastasis in ER + breast cancer patients [8, 9].

Although BC survival rates have significantly improved recently, however, they remain considerably low in Egypt. This may be attributed to deficient screening mechanisms among Egyptian females where most patients are reported to being diagnosed at late stages with subsequent poor outcomes [10,11,12,13].

Several efforts have been made recently to develop low cost, rapid, easy-to-use cancer diagnostic methods with minimal invasiveness using peripheral blood or urine samples [14]. Metabolome analysis has emerged as an effective tool in cancer diagnosis and prognosis [14, 15].

Amino acids (AA) are an integral part of most metabolomics analysis [16]. Amino acid profiling has demonstrated significant differences in AA levels between cancer patients and healthy controls [14]. Several studies reported a decrease in AA plasma levels in cancer cells probably attributed to their high requirements in cancer cells [17, 18]. However, other studies showed an increase of amino acids levels in tumors which could be a result of the increased cellular proliferation rate [17, 19].

To date, amino acid profiling of Egyptian women with BC hasn’t been well investigated, so we aimed in the present study to examine the alteration of the plasma amino acids profiles and investigate their association with BRCA1 and 2 circulating levels in Egyptian females diagnosed with BC and in females with family history of BC in order to establish potential novel non-invasive screening strategies which will help in the early detection of BC and provide insight into the progression of the disease using a safe, low cost and easy method.

Materials and methods

Patients

The present study is a case control study conducted in the departments of Biochemistry, Faculty of Medicine, Assiut university and General Surgery, Assiut University Hospital, Assiut, during the period from March 2021 to December 2022.

The sample size was calculated using the Steven K. Thompson equation (Thompson, S.K., 1987) to be a total size of 86 samples. We adjusted the sample size to attain 80% power and 5% confidence level of significance (type 1 error)

$$N = \frac{{N \times P\left( {1 - P} \right)}}{{\left[ {N - 1 \times \left( {{d^2} \div {z^2}} \right) + P\left( {1 - P} \right)} \right]}}$$

N = population size, d = margin of error, p =  p value; 0.05 and z = confidence level at 96% is 1.96.

The study included 86 participants that were subdivided into three groups as follows:

Group I

consisted of 26 females who were diagnosed with BC.

Group II

consisted of 22 females who have a family history of BC (relatives of group I).

Group III

consisted of 38 healthy females (control).

An informed consent was obtained from each subject and all study procedures were approved by the Medical Ethics Committee, Faculty of Medicine, Assiut University (IRB no: 04-2023-200140). The aim of the study was explained to each participant before filling data. Personal data including age, weight, height, and BMI were collected during time of admission. Medical history including duration of symptoms, medication used, and presence of other complications associated with symptoms were also collected from patients’ profiles.

Exclusion criteria included females diagnosed with any other type of cancer (benign or malignant), females that past received a chemotherapy or radiotherapy, females with hypertension or diabetes and females who have a history of coronary artery disease, stroke or myocardial infarction.

Sample collection and handling

Samples were collected from patients, relatives and controls. Samples of five milliliters of antecubital venous blood were collected and divided into: Three milliliters of blood which were placed in a tube containing heparin for plasma separation for AAs profile assessment. Another two milliliters were placed in a plain test tube and centrifuged at 3000 rpm for 10 min, then serum was separated and stored at -80˚C until time of ELISA analysis.

Estimation of BRCA1 and BRCA2

BRCA1 and BRCA2 concentrations were determined using commercial ELISA Kits (Catalog Numbers: SG-14,409 and SG-15,000 respectively) purchased from SinoGeneclon Biotech Comp, Hangzhou, China according to the manufacturer’s specifications.

Assay of amino acids profiles

Assay of amino acids was performed in the Metabolic and Genetic Disorder Unit (MGD) of Faculty of Medicine, Assiut University by the Ion exchange separation method through high performance liquid chromatography using a Sykam Automatic Amino Acid Analyzer S433 supplied by Sykam GmbH, Germany (catalog no. 1,120,001). By using an acidic protein precipitation method, free amino acid samples were made from plasma by adding 200 µl of a 10% sulfosalicylic acid solution to 800 µl of plasma, mixing by vortex, and allowing it cool to about 4 °C for 30 min. It was then centrifuged at 14,000 rpm for 10 min. The same amount of sample dilution buffer (catalog no. S000015) was added to the supernatant liquor to dilute it. Direct injections of 100 µl of each of prepared samples and ready to use amino acid physiological standard (Catalog no. 6,006,005) were made. A cation separation column LCAK07/Li was used (catalog no. 5,112,008) with the following specifications: size: 150 mm × 4.6 mm, specification range: methionine (Met) efficiency > 48,000, asymmetry: 0.8–1.5, resolution THR/SER: > 1700, and column pressure: 45–80 bar. Buffer: Sykam Li A, Li B, Li C. The ready to use ninhydrin reagent (catalog no. 8,000,025) and citrate buffers in different pH (2.90, 4.20, and 8.0) were used and we performed the analysis at wavelength 440 nm:570 nm. The sample chromatogram was then compared to the standard measurements curve to obtain various amino acid values, then results were multiplied by a dilution factor of 2.5 (Fig. 1).

Fig. 1
figure 1

Chromatogram of standard amino acids showing their retention times (minutes) and voltage (volt) response peaks. MIN, minute; V, volt; ASP, Aspartate; THR, Threonine; SER, Serine; ASN, Aspargine; GABA, Gamma Amino Butyric Acid; GLY, Glycine; ALA, Alanine; VAL, Valine; MET, Methionine; ILE, Isoleucine; LEU, Leucine; TYR, Tyrosine; PHE, Phenyl Alanine; HIS, Histidine; CARN, Carnitine; TRYP, Tryptophan; ORN, Ornithine; LYS, Lysine; ARG, Arginine

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Statistical analysis

Data entry and analysis were done using SPSS version 22 (Statistical Package for Social Science). Data were presented as mean, standard deviation or median and range. In case of parametric data, one way ANOVA test followed by post-hoc test was used to compare between different groups. While in case of non-parametric data; Kruskal Wallis test was used to compare between different groups and spearman correlation was done to measure correlation between quantitative variables. P-value considered statistically significant when less than 0.05.

Results

Personal data of subjects

The personal data of the three groups of subjects are shown in Table 1. There was no statistically significant difference regarding the age, height, weight, or BMI between the study groups.

Table 1 Personal data of different study groups
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Clinical data of BC patients

Table 2 shows the clinical data of BC patients. The present study included 26 patients diagnosed with BC. 18 of the patients had BC of stage 2 and 8 of them had a stage 3 BC according to TNM staging. 84.5%. of the patients were at grade 2 of the disease and 80.8% did not have any family history of BC. Receptor status percentage differentiated between patients, where luminal A, luminal B and non-luminal cases were 42.3%, 38.5% and 19.2% respectively.

Table 2 Clinical data of BC patients
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BRCA1 and BRCA 2 levels

Table 3 shows the mean levels of BRCA1 and 2 in serum where a significant difference in BRCA1 levels was detected between both (groups 1& II) compared to group III. No statistically significant difference was found in mean BRCA1 level between the patients and their relatives. In addition, a significant difference was found between the mean BRCA2 levels in the three groups as it was significantly higher in patients compared to the relatives’ and control groups. Also, there was a significant increase in mean BRCA2 levels in relatives compared to controls.

Table 3 BRCA1 and 2 concentrations in serum of different study groups
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Plasma concentrations of amino acids in different study groups

There was a significant statistical difference in mean plasma levels of Phosphoserine, Alanine, Phenylalanine, Glutamate and Cysteine where their plasma levels in patients and relatives were higher compared to levels in the control group. We also noted that the levels of Glutamate were significantly higher in patients compared to females with family history of BC (Table 4). On the other hand, there was a significant statistical decrease in mean plasma levels of Taurine, Threonine, Serine, Glycine, Valine, Methionine and Histidine where their plasma levels were lower in patients and relatives compared to controls (Table 5). There was no statistical significance in mean plasma levels of Aspargine, Aspartate, Leucine, Isoleucine, Tyrosine, Ornithine, Lysine and Arginine between the three groups (Table 6).

Table 4 Amino acids that increased in BC patients compared to controls
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Table 5 Amino acids that decreased in BC patients compared to controls
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Table 6 Amino acids that showed no difference in BC patients compared to controls
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Levels of metabolites

There was no significant difference in mean plasma urea levels among the three groups. However, there was a significant difference decrease in mean ammonia levels in patients compared to controls (Table 7).

Table 7 Levels of metabolites
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Correlation analyses

A significant positive correlation was observed between BRCA1 and Valine levels. In contrast, a significant negative correlation was observed between BRCA2 and Lysine plasma levels (Table 8). Also, a significant positive correlation was detected between the levels of Phosphoserine, Serine, Glycine, Phenylalanine and Ornithine and the hormone receptor status, where the most aggressive non luminal cases showed the highest level of these amino acids (Table 8).

Table 8 Correlation analyses of BRCA1 and 2 levels with amino acid levels and clinical data of BC patients
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Table 9 Correlation analyses of amino acid levels and clinical data of BC patients
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Discussion

Breast cancer (BC) remains a global public health problem and a leading cause of cancer mortality among women [20]. Early diagnosis of BC is limited to the classic screening tools, which have several disadvantages as high radiation risk and high cost, therefore it is necessary to search for novel reliable biomarkers that can be used for its early detection and monitoring of disease progression [21].

An increased interest in the field of metabolomics has been noticed in the last decade due to their potential for clinical applications [22,23,24]. Among the vast types of metabolites, plasma free amino acids (PFAA) present as promising potential disease biomarkers i for different cancers [22,23,24,25].

Under normal conditions, amino acids maintain a state of protein balance, despite continual cycling between protein synthesis and degradation [26]. However, the case differs in cancer cells where PFAAs are in high demand for protein, DNA synthesis and building new blood vessels which may lead to a lower availability of PFAAs in cancer patients [17, 27]. On the other hand, studies reported that the increase in proliferation rate in tumors maybe correlated with an increase in levels of some amino acids [17]. This heterogeneity might be due to the differences in subjects’ demographics, in addition to the different disease stages [18, 19].

The results of the present study demonstrated a significant increase in mean plasma levels of glutamate in patients compared to levels in the control group. We also noted that glutamate was significantly upregulated in patients compared to females with family history of BC. This could possibly be attributed to the fact that tumor cells consume glutamine largely aiming to produce energy through the tricarboxylic acid cycle and to maintain protein synthesis necessary for tumor growth and proliferation [17, 26, 28]. The high rate of glutamine uptake may be also due to the stimulated uptake of essential AA required to maintain the mitochondrial integrity [29]. Furthermore, findings from previous studies demonstrated an impaired ability of the peripheral skeletal muscle to extract glutamate from circulation in patients with gastrointestinal, renal, bronchial and breast cancer leading to accumulating plasma glutamate concentrations [26, 30].

Contradictory findings are reported regarding plasma alanine levels in cancer patients [14, 26, 27]. The increased levels of alanine observed in plasma of cancer patients in our study may be due to its increased production by tumor cells [17, 30]. However, these results are contradictory with those observed by Eniu et al. who showed a downregulation in alanine availability in BC patients which was attributed to its conversion to glucose in the liver [19].

Cysteine is a key contributor to metabolic remodeling associated with cancer [31]. Our results showed increased plasma cysteine levels in BC patients, which comes in line with previous studies [32]. Interestingly, plasma levels of alanine, phenylalanine, glutamate and cysteine were also increased in females with family of BC compared to controls, this could offer these AA profile as important potential early diagnostic tool of BC in Egyptian women. Further follow up is required to confirm these results.

On the other hand, there was a significant decrease in mean plasma levels of taurine, threonine, serine, glycine, valine, methionine and histidine where their plasma levels were lower in patients and relatives compared to controls. Glycine and serine are both classic glycolysis metabolites [24]. It was reported that glycine uptake and metabolism were necessary to promote tumorigenesis and malignancy [33, 34]. Additionally, serine is used for one-carbon metabolism and nucleotide synthesis which is necessary for cancer cell proliferation [35, 36] leading to a decrease in their plasma levels which is in line with our results. On the contrary, several studies reported an increase in serine levels in patients with pancreatic cancer, BC and colorectal cancer [14, 17, 26, 37] which was explained by the increased enzymatic activity involved in serine biosynthesis in tumor cells [38].

Our results showed a significant decrease in valine levels in BC patients compared to control females, however we detected no significant differences in leucine and isoleucine levels. Branched chain AAs (BCAAs) are major contributors in the maintenance of lean body mass, and they play a major role in stimulation of skeletal muscle protein synthesis through activation of mTOR [39, 40]. Lower plasma valine concentrations in BC patients may be due to increased uptake by peripheral muscle tissue. Indeed these results are in agreement with previous studies [41, 42], however, others reported high plasma valine concentrations observed in cancer patients which could be attributed to the enhanced breakdown of body protein shown in those cancer patients [39, 43].

In the present study, we show low levels of taurine in BC patients. Taurine was suggested previously as an early diagnosis biomarker for malignant changes in the breast [44]. We also show low levels of histidine and threonine in BC patients in contradiction with Barnes et al., who reported less histidine uptake in breast cancer patients [26]. Histidine is used to synthesize carnosine, an antioxidant that suppresses cancer proliferation [45]. The increased uptake of histidine in our patients’ tissues may be due to increased tumor induced-oxidative damage in the tissue. As for the lower cancer threonine levels, it was reported that it might be due to increased production of pyruvate from threonine associated with the impaired glutamate uptake as seen in our patients [46].

BRCA1 and BRCA2 genes stand out among DNA repair pathway genes for their specific relationship with BC [47, 48]. Upregulated expression of BRCA1 and BRCA2 genes was reported in BC and high BRCA1 gene expression was demonstrated to increase the risk of early distant metastasis in ER + breast cancer patients [8, 9]. Moreover, a worse overall survival rate was substantially correlated with overexpression of BRCA1/2 [49].

Our study results showed significant elevations in BRCA1 and 2 protein levels in sera of patients and of females with family history compared to healthy females with a significant upregulation of BRCA2 levels in patients compared to females with family history. Also, a significant positive correlation was observed between plasma BRCA1 and Valine levels. In contrast, a significant negative correlation was observed between BRCA2 and Lysine plasma levels. To the best of our knowledge, this is the first study to explore the correlation between BRCA 1&2 and amino acids level in BC.

In conclusion, this study results shows that PFAAs profile can potentially help in early screening for BC patients and for susceptible females. Furthermore, combination of BRCA 1 and 2 assessment and AAs profiling could have a strong diagnostic potential for BC. Further studies are needed to validate this assumption.

Limitations

One of the limitations of this study is the sole assessment of BRCA1 and BRCA2 serum protein levels and not at the gene level. Further research that measures both mRNA and protein levels is essential for a comprehensive understanding of the molecular pathogenesis of breast cancer. Other limitations include lack of follow up in addition to the small sample size. Further studies with larger sample size and follow up are needed to check if the levels of PFAA are associated with risk of development of BC in family subjects and to fully understand the effect of changes in PFAAs particularly in connection to BRCA1,2 genes expression which could provide further insight on their role as prognostic and diagnostic biomarkers of BC.