Undetectable Free Aromatic Amino Acids in Nails of Breast Carcinoma: Biomarker Discovery by a Novel Metabolite Purification VTGE System

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Abstract
Background: Metabolic reprogramming in breast cancer is depicted as a crucial change in the tumor microenvironment. Besides the molecular understanding of metabolic heterogeneity, appreciable attention is drawn to characterizing metabolite profiles in tumor tissue and derived biological fluids and tissue materials. Several findings reported on the metabolic alterations of free aromatic amino acids (FAAAs) and other metabolites in biological fluids. Furthermore, there is a significant gap in the development of a suitable method for the purification and analysis of metabolite biomarkers in nails of cancer patients. Methods: To address the metabolite alterations, specifically FAAA levels in nails, fingernail clippings of breast cancer patients (N = 10) and healthy subjects (N-12) were used for extraction and purification of metabolites. Here, we reported a novel and specifically designed vertical tube gel electrophoresis (VTGE) system that helped in the purification of metabolites in the range of 100–1,000 Da from nail materials. Here, the VTGE system uses 15% polyacrylamide under non-denaturing and non-reducing conditions, which makes eluted metabolites directly compatible with LC-HRMS and other analytical techniques. Qualitative and quantitative determination of FAAAs in nail lysates was done in positive ESI mode of the Agilent LC-HRMS platform. Results: The analysis on collected data of nail metabolites clearly suggested that FAAAs including tryptophan, tyrosine, phenylalanine, and histidine were undetectable in nail lysates of breast cancer over healthy subjects. This is a first report that showed highly reduced levels of FAAAs in nails of breast cancer patients. Furthermore, the present observation is in consonance with previous findings that showed cancer cachexia and high amino acid catabolism in breast cancer patients that drive metabolite-led cancer growth and proliferation. Conclusion: This paper provides a proof of concept for a novel and specifically developed VTGE process that showed first evidence on the undetectable level of FAAAs in nails of breast cancer patients as metabolite biomarkers. Here, the authors propose the potential use of a VTGE-assisted process to achieve metabolomic discovery in nails of breast cancer and other tumor types.
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