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    2020-08-10

    r> Chromatograms of the separation of AQC derivatized amino Cell Counting standards were shown in the Supplementary material (Figs. S1 and S2). Representative chromatograms of the AQC-amino acids in cul-tured cells are shown in Fig. 1. Internal standard calibration curve was constructed for each amino acid. The method was evaluated for linearity, sensitivity, precision, accuracy, and matrix effect accord-ing to US Food and Drug Administration document for bioanalytical method validation. Detailed procedures for method optimization and validation were described previously [17], and results were shown in the Supplementary material (Tables S1–S3). The method developed here is a rapid and sensitive separation method that can complete the analysis of 39 amino acids including l- and d-enantiomers within 15 min with LODs in the sub-pg level. Also, d-enantiomers elute before their corresponding l-enantiomers on Q-Shell column, which is favorable for the quantification of d-amino acids in biological samples due to the lack of interference from the corresponding l-amino acids.
    The starting amino acid levels of uncultured media (prior to any cell growth) were determined after serum supplementation. To determine changes in extracellular amino acid levels over time, uncultured medium was included in the analysis. Data were plot-
    Fig. 1. Representative chromatograms of AQC derivatized amino acids in MCF-7 cancer cells after 48-hour growth in high glucose medium.
    ted as percent change from the uncultured medium, which was
    calculated by the following equation:
    Amino acid level in the cultured medium − amino acid level in the uncultured medium x 100%
    Malignancy indicators (MIs) in were calculated by the following
    equations:
    a. L − amino acids =
    Intracellular L − amino acid levels in MCF − 7 cells
    Intracellular L − amino acid levels in MCF − 10 A cells
    b. D − amino acids =
    Intracellular D − amino acid levels in MCF − 7 cells
    levels
    Intracellular D − amino acid in MCF − 10 A cells
    D − amino acids
    Each experimental condition was performed in triplicate. Aver-age and standard deviations for intracellular and extracellular amino acid levels were calculated from parallel triplicate experi-ments. 
    3. Results and discussion
    3.1.1. Intracellular and extracellular free l-amino acid levels Intracellular free l-amino acid levels were determined for MCF-7 and MCF-10A cells after 24-hours, 48-hours, and 72-hours
    Fig. 2. Intracellular L-Amino Acid Profiles. L-Amino acid levels in MCF-7 and MCF-10A cells after (A) 24-hours, (B) 48-hours, and (C) 72-hours growth in the associated medium. Red bars represent MCF-7 cells grown in high glucose medium, green bars represent MCF-7 cells grown in normal glucose medium, and white bars represent MCF-10A cells grown in the MEGM. * indicates non-chiral amino acids. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
    Extracellular l-amino acid profiles were expressed as percent changes of the amino acid from the uncultured medium (calcu-lated by the equation shown in Materials and methods) and are shown in Fig. 3. The exact values of extracellular l-amino acid levels are shown in Supplementary material Tables S13–S24. Extracel-lular profiles of l-amino acids were divided into two categories: essential and nonessential amino acids, due to the general trends observed in each category. The percent change of the nine essen- 
    tial amino acids from uncultured media showed negative values for both MCF-7 and MCF-10A cells, indicating the uptake of these amino acids from the growth media (Fig. 3A). Regarding nonessen-tial amino acids, as shown in Fig. 3B, cellular uptake (net removal from the growth media) of l-Ser, l-Gln, l-Arg, l-Tyr, and l-Cys was observed for MCF-7 and MCF-10A cells. Cellular release of l-Ala, l-Pro, and l-Glu was observed in both cells lines. Cellular uptake of l-Asp and Gly were observed for MCF-7 cells, but cellular release of l-Asp and Gly was shown for MCF-10A cells. On the other hand, the release of l-Asn was detected for MCF-7 cells, but the uptake of l-Asn was detected for MCF-10A cells.
    3.1.2. Altered l-amino acid profiles and metabolism for MCF-7 breast cancer cells Cancer cells require higher amounts of amino acids and glucose to fulfill the metabolic demands associated with proliferation [1]. Significant increases of Pro, Thr, Glu, Phe, Trp, Met, Asp, Ser, Gln, Leu, His, Val, and Lys have been reported in the saliva from breast cancer patients compared to healthy controls [13]. In our study, l-amino acid concentrations were found to be up to 56 times higher in MCF-7 breast cancer cells compared to non-tumorigenic MCF-10A cells in both high and normal glucose media (Fig. 2). Intracellular high levels of l-amino acids could be one of the reasons that elevated levels of l-amino acids are observed in the saliva and plasma of breast cancer patients.