br profiles may have great potential
profiles may have great potential for the early detection of can-cer . For example, patients with breast cancer, in both early and advanced stages, showed high levels of amino acids in their saliva compared to a healthy control group . The accumulation of specific amino acids in cancer cells, particularly specific d-amino acids, could be potential oncometabolites, which are defined as the metabolites whose abundance increases markedly and are involved in the development of malignancy 
To date, almost all the cancer studies regarding amino acids focused only on l-amino acids. Questions about d-amino acids and cancer cells have not been asked or answered. One is whether there is cellular uptake or release of d-amino acids during cancer cell proliferation? Second, do d-amino acids exhibit altered profiles in cancer cells as do l-amino acids, and if so, why? This study is the first report of endogenous levels of free l- and d-amino acids in human breast cancer cells (MCF-7) and non-tumorigenic human breast epithelial cells (MCF-10A). Altered profiles and metabolism of free l- and d-amino acids were determined in cultured MCF-7 cells compared to MCF-10A cells. Also, effects of glucose concentra-tion were studied for MCF-7 cell proliferation and their endogenous l- and d-amino 3-Methyladenine levels. Further, a simple test using specific d- and l-amino acid relative levels has been derived and used to produce malignancy indicators (MIs) of cancer cells.
2. Materials and methods
2.1. Chemicals and reagents
Amino acid standards, perchloric acid, and ammonium for-mate were obtained from Sigma-Aldrich (St. Louis, MO,USA). The AccQ·Tag Ultra derivatization kit (AccQ·Tag Ultra reagent pow-der [6-aminoquinolyl-N-hydroxysuccinimide carbamate (AQC)], AccQ·Tag Ultra borate buffer, and AccQ·Tag Ultra reagent diluent) was purchased from Waters Corporation (Milford, MA, USA). All the cell medium and additives were purchased from Sigma-Aldrich. HPLC-MS grade methanol and water were purchased from Sigma-Aldrich, and ultrapure water was obtained from a Milli-Q water system (Millipore, Bedford, MA, USA).
2.2. Cell lines and culture conditions
Human breast cancer cell line (MCF-7) and non-tumorigenic human breast epithelial cells (MCF-10A) were purchased from American Type Culture Collection (ATCC). MCF-7 cells were grown and maintained in normal, or high glucose Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 1% L-glutamine, and 1% penicillin-streptomycin. MCF-10A cells were grown and maintained in Mammary Epithelial Cell Growth Medium (MEGM) that was supplemented with 10% FBS, 100 ng/mL cholera toxin and MEGM kit. All the cells were incubated at 37 ◦ C in a humidified atmosphere of 5% CO2.
Cells were seeded into 150 x 25 mm cell culture dish grown until 80–90% confluency and then split into 9 of 100 x 20 mm cell culture dishes. Triplicate plates were seeded for each experimental condi-tion. Cells were trypsinized and centrifuged at 1000 g for 5 min. The cell pellet was washed twice with phosphate buffer saline (PBS). Cells were counted at the specified time points, i.e., 24 h, 48 h, and 72 h, by conducting the Trypan blue assay using a hemacytometer (Sigma-Aldrich, St. Louis, MO).
2.4. Intracellular and extracellular amino acids extraction and analysis
Amino acids were extracted from the cells or cell media with 0.3 M perchloric acid and 100 M norvaline (internal standard) on ice for 30 s (three 10 s pulses) with a Q-Sonica CL-18 probe (Newtown, CT, USA). After vigorous vortexing, the samples were centrifuged at 4 ◦ C for 20 min at 13,000 rpm. The supernatant was collected, filtered, and derivatized as previously detailed . In brief, 10 L of the extract solution was mixed with 70 L of borate buffer and 20 L of AQC reagent. The sample was vortexed followed by incubation at 55 ◦ C for 10 min.
High performance liquid chromatography-tandem mass spec-trometry (HPLC-MS/MS) analysis was performed on a LCMS-8040 (Shimadzu Scientific Instruments, Columbia, MD, USA), triple quadrupole spectrometer with electrospray ionization (ESI). Two different chiral stationary phases with opposite enantioselectivity were used for all analyses. A Q-Shell column (4.6 x 50 mm), qui-nine based chiral stationary phase, was prepared in-house and utilized for the separation and quantification of amino acids. A gra-dient method was used for the chiral separation of amino acids on the Q-Shell column. Mobile phase A was ammonium formate (100 mM)-methanol (10:90, v/v) (pH* 6), and mobile phase B was ammonium formate (50 mM)-methanol (10:90, v/v) (pH* 5). The following gradient was applied: 0–4 min, 0–100% B; 4–15 min, 100% B; 15–16 min, 100 - 0% B; 16–25 min 100 B%. The second chiral stationary phase was TeicoShell column (4.6 x 150 mm, AZYP, LLC, USA), which was based on macrocyclic glycopeptides. TeicoShell column was used to confirm amino acids peak identity due to its opposite enantioselectivity. For a complex matrix, there is still a chance that an impurity with the same m/z and a similar structure is co-eluting with the analyte of interest on one column. Having a second column with different selectivity can separate the impu-rity that is co-eluting with the analyte on the first column. Indeed this was the case with these samples where L-Hyp overlapped with L-Ile on the Q-Shell column initially but was resolved on the TeicoShell column. Mobile phase A was ammonium formate (5 mM, pH 4), and mobile phase B was acetonitrile. A gradient method for TeicoShell column was applied as following: 0–2 min, 30%–40% B; 2–15 min, 40%–50% B; 15–16 min, 50% to 30% B; 16–30 min, 30% B. The flow rate was 0.65 mL/min for both columns, and a splitter was used before the MS. HPLC-MS/MS was operated in multiple reac-tion monitoring (MRM) mode using positive ESI source. Collision energies and MRM transitions were optimized for each amino acid. Shimadzu LabSolution software was used for data acquisition.