Agnoletto, Elisa (2014) Sviluppo di un metodo per la quantificazione dei livelli plasmatici di acidi grassi a media catena (C6-C12) e valutazione del loro valore diagnostico in patologie colorettali mediante fast GC-MS. [Magistrali biennali]
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Colorectal pathologies are diseases that affect the large intestine and they include polyposis, ulcerative colitis and colorectal cancer. In correspondence to these pathologies some authors observed an altered expression of the fatty-acid synthase (FAS), the enzyme appointed to the endogenous biosynthesis of fatty acids. Thus, the investigation of FAS metabolites could give a snap-shot of the physiological state of the organism. From preliminary studies, increased levels of decanoic acid seemed related to the presence of colorectal cancer. For this reason, in this study medium-chain fatty acids have been determined in plasma samples of different kinds of patients using fast gas chromatography coupled with a mass spectrometer with TOF analyzer. In particular, four groups of subjects have been considered: 25 colorectal cancer (CRC) patients, 11 ulcerative colitis patients (RCU), 10 breast cancer patients (MAM) and 17 healthy subjects (C). For every plasma sample, levels of hexanoic, octanoic, decanoic and dodecanoic acids have been quantified in order to highlight some differences in concentrations of some of them in specific groups of subjects. For the quantitation, calibration curves have been built using standard solutions of the analytes and resorting to the internal standard method (using d3-8,8,8-octanoic acid as internal standard). Samples have been added with a known amount of internal standard and, later, fatty acids have been extracted by a mixture of methanol/chloroform 1:2, adding an NaCl solution to favour proteins aggregation. Thereafter, treated samples have been centrifugated in order to separate proteins from liquids and aqueous phase from the organic one. Aqueous phase has been eliminated while organic phase has been collected and the analytes therein have been concentrated. Standard solutions and extracted samples have been derivatized before the GC analysis and every sample has been analyzed twice. The method used for this study, fast GC-MS, has been validated following the guidelines recommended by FDA and EMEA and it has proved accurate, precise, characterized by a wide linear dynamic range and by relatively low values of detection and quantitation limits. From chromatograms and by means of calibration curves, medium-chain fatty acids have been quantified in every plasma sample, and the results have been submitted to statistical tests. Obtained data have showed a statistical difference in decanoic acid concentration in colorectal cancer patients with respect of other groups, suggesting a possible correlation between the increment of this fatty acid and CRC. The study has also pointed out that there is an increment of the concentrations of both hexanoic and dodecanoic acids in correspondence to ulcerative colitis. The obtained results require the analysis of a greater number of samples in order to confirm the observed concentration differences. In addition, a further investigation on the biological causes of these altered lipid profiles is necessary to reach a whole understanding of biochemical processes that occur in the presence of colorectal diseases.
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