## 4 Discussion
Altered levels of tryptophan in urine have been observed in other types of tumors, such as breast and bladder cancer [26]. Tryptophan is an essential amino acid metabolized in tumor microenvironments, immune-privileged, or inflammation sites. Degradation of tryptophan by the enzyme indoleamine-2,3-dioxygenase (IDO) is considered to be an immune defense mechanism, which inhibits the growth of intracellular bacteria, viruses, parasites [27], and malignant tumor cells [28]. Based on different pieces of evidence [29; 30], the catabolic products of tryptophan in cancer are considered important microenvironmental factors that suppress antitumor immune responses. Another metabolite that was increased in our CRC urine samples was histamine. It has been reported that histamine can regulate the proliferation and angiogenesis of cancer cells [31; 32; 33]. Finally, another metabolite decreased in our CRC urine samples was phenylacetylglycine, which is considered a minormetabolite of fatty acids. Increased excretion of phenylacetylglycine has been observed in gastric cancer patients, in which its levels correlated with the cancer T stage [6], and in rats treated with alkylating agents that produced precancerous colorectal lesions possibly related to gut microflora metabolism dysbiosis [34]. Significantly, even if the low number of subjects investigated does represent a limit of the work, a recent study by [PERSON] et al. [11] analyzing the metabolomics profiles of CRC versus healthy patients reported similar results for acetacetate and phenylalanine, a precursor of phenylacetylglycine. In addition, a recent study by [PERSON] et al. [35] identified four metabolites as robust CRC urine biomarkers: proline, diacetylspermine, kynure, and glucose. Because kynure is a tryptophan metabolite, its increased levels correlate well with the decrease of tryptophan found in our CRC samples. In conclusion, metabolomics analysis showed a different urinary metabolic profile between CRC patients and healthy controls undergoing colonoscopy. Altered levels of some metabolites resulted in a significant correlation with colon cancer and were able to distinguish with accuracy CRC patients from C. Both our results and data from literature confirm that urinary metabolic profiling is an effective tool for identifying CRC, and it may be useful in improving diagnosis, prognosis, and response to therapy, representing a noninvasive diagnostic support to be used before other more invasive tests. Though a limit of the study is the number of subjects investigated, a strength of the study is the similar nutritional status of all the subjects undergoing colonoscopy. Our results suggest that for CRC identification, the metabolomics analysis does not need to be associated with colonoscopy considering that similar discriminant results can be observed without it. The next important step is the validation of these data in an independent larger group of patients to transfer this information into the clinic. The following are available online at [[http://www.mdpi.com/2076-3417/10/16/5401/s1](http://www.mdpi.com/2076-3417/10/16/5401/s1)]([http://www.mdpi.com/2076-3417/10/16/5401/s1](http://www.mdpi.com/2076-3417/10/16/5401/s1)), Figure S1: Principal Component Analyses (PCA) scores plot of 1H-NMR spectra of urine samples: C (full circle) and CRC (open circle) subjects. Two outliers are indicated with arrows. Figure S2: [PERSON]'s T2 versus DModX plot: C (full circle) and CRC (open circle) subjects. Table S1: Proton chemical shifts of the metabolites identified in the 500 MHz 1H-NMR spectra of urine samples. [PERSON] and [PERSON] conceived the study and supervised the project. [PERSON], F.C. obtained the samples and clinical details. [PERSON] and [PERSON] performed metabolomics experiments and data analysis. [PERSON], [PERSON], and [PERSON] wrote the first draft of the manuscript, and [PERSON], [PERSON], [PERSON], [PERSON], and [PERSON], contributed to the final version.