## 4 Discussion  
Previous studies [6; 9; 10; 11; 12; 19] have suggested the use of a metabolomics approach to identify tumor-associated biological alterations in CRC patients. In the present study, \({}^{1}\)H-NMR spectroscopy coupled with pattern recognition was used to profile urine metabolites of subjects with (CRC) or without (C) CRC. Significantly, metabolic perturbations were evaluated between the urine samples of CRC patients and individuals undergoing colonoscopy for positive FOBT or familial risk of CRC, to whom a similar low-fiber, controlled diet had been administered for few days before they were subjected to the examination. Urine samples were collected just before the colonoscopy. Therefore, both CRC and C groups were evaluated in the same short-term dietary conditions, consisting of a low-fiber diet for a few days. Since diet represents an important factor that contributes to inter-individual variability, our sample collection approach reduced variability related to a different nutritional status. To date, our study, despite the small number of subjects analyzed, is the first to investigate the metabolomics profiles of potential CRC patients under a similarly controlled short-term diet. Based on identification of metabolites with significant statistical variations between CRC and C groups, a ROC curve was constructed to assess the sensitivity and specificity of the biomarker candidates for early detection of CRC. The AUC of the ROC analysis denoted high predictive accuracy of the model. Furthermore, CRC patients showed a urine metabolic profile characterized by a higher sensitivity compared to CEA serum levels. The sensitivity of CEA is well known to be low, but it is often used as a serum biomarker in CRC for integrating diagnosis and monitoring the follow up [20]. Our data confirmed CEA detection limitations, including its relatively low sensitivity and specificity [2], and suggested the possibility of integrating these data with the metabolomics approach. Our study indicates a better sensitivity and specificity of the urine profile test in CRC samples compared to those analyzed with \({}^{1}\)H-NMR spectroscopy by [PERSON] et al. [21] in adenomatous polyp samples. The improved sensitivity and specificity of urine profile tests in CRC samples may be due to a more advanced stage of colon disease in our samples in the CRC group. In our study, the metabolites identified were the end products of several metabolic pathways relating to lipid and amino acid metabolism, which are known to be perturbed during tumor cell proliferation [10]. We found that the levels of acetoacetate, creatinine, and histamine were increased, while those of creatine, phenylacetylglycine, and tryptophan decreased in the urine of the subjects affected by CRC. The increased concentrations of acetoacetate observed in CRC urine may be related to lipid metabolic changes associated with tumor development. The increased content of acetoacetate in our CRC samples could be related to increased \(\beta\)-oxidation due to the strong energy demands accompanying tumor growth. Indeed, in tumor cells, acetyl-CoA is not converted into citrate by the tricarboxylic acid cycle but is processed through an alternative pathway to form ketone bodies, which represent a very efficient fuel source, preferable even to glucose [11; 22]. Cancer cells use some amino acids as an energy source [23]. In our study, we found that creatinine levels increased while those of creatine and tryptophan decreased in the urine of CRC patients. Urinary creatinine increase is normally connected to muscle catabolism, breakdown of proteins, or a combination of the two processes [24]. The increased creatinine levels observed in our tumor samples may be due to the non-enzymatic and irreversible conversion of creatine [25], resulting in significantly decreased levels in CRC urine samples. On the other hand, we found decreased tryptophan levels in CRC urine samples.