## 4 Discussion  
RS was described as a relevant technique for the analysis of biological materials of clinical interest. Since Raman spectrum is composed of sharp bands with distinct characteristics, specific for each molecule, a substance can be easily distinguished from the others due to the intrinsic biochemistry difference [28, 29, 30]. RS can be applied to the analysis of discolored urine, a phenomenon that can be observed in urine bags of hospitalized and long-term bedridden patients. A spectrum of urine colors (white, black, brown, red, pink, orange, yellow, blue-green, and purple) can occur due to changes in urochrome concentration and the presence of other pigments and both endogenous ones, such as uric acid, bilirubin, hemoglobin, or myoglobin, and exogenous causes related to patient diet, medications, and poisons. \begin{table}
\begin{tabular}{c c c c c} \hline \hline
**Element** & **Control (abs)** & **“Blue Bags”** & **Control Wt** & **“Blue Bag” Wt** \\  & & **(abs)** & **[Error] (\%)** & **[Error] (\%)** \\ \hline Chlorine & 25.25 & 33.34 & 16.63 [2.64] & 25.17 [3.47] \\ Carbon & 97.15 & 57.12 & 63.98 [32.64] & 43.13 [20.96] \\ Oxygen & 28.01 & 37.32 & 18.45 [10.09] & 28.18 [13.12] \\ Sodium & - & 1.91 & - & 1.44 [0.46] \\ Calcium & 0.03 & 0.23 & 0.02 [0.08] & 0.18 [0.10] \\ Potassium & - & 0.08 & - & 0.06 [0.08] \\ Nickel & 0.02 & 0.03 & 0.02 [0.08] & 0.02 [0.08] \\ Aluminum & 0.43 & 0.71 & 0.28 [0.14] & 0.54 [0.18] \\ Magnesium & 0.66 & 1.09 & 0.44 [0.19] & 0.82 [0.26] \\ Phosphorus & - & 0.19 & - & 0.14 [0.10] \\ Silicon & 0.16 & 0.30 & 0.10 [0.10] & 0.13 [0.12] \\ Neodymium & 0.13 & 0.12 & 0.08 [0.09] & 0.09 [0.09] \\ \hline \hline \end{tabular}
\end{table} 
Table 2: Results of EDX analysis. Figure 2: Morphological images of urine drainage bag obtained using the scanning electron microscope. On the left side, the clean bag (control); on the right side, the “blue” bag. As can be seen from an inspection of Figure 1, Raman spectra of urine from the bag show the presence of tryptophan (TRP) (1360 cm\({}^{-1}\)) [31]. The TRP is sensitive to the microenvironment and detailed information about the microenvironment of these aromatic side chains can be obtained from variations in intensity of the vibrational modes [9]. The Raman spectrum also revealed the presence of indigo, a metabolite of TRP. TRP is an essential alpha-amino acid found in some foods, especially those high in protein (milk, meat, fish, beans, soybean, eggs, and others). It is needed for the in vivo biosynthesis of proteins and serves as a substrate for several bioactive metabolites, including serotonin, nicotinamide (vitamin B6), tryptamine, and others. In humans, TRP is exclusively obtained from dietary intake [32]. As detailed in Figure 3, the metabolic steps of TRP occur in the large intestine and liver up to the production of indoxyl sulphate (indican), a colorless product that is excreted in the urine tract. In patients, chronically catheterized and chair/bed-bound due to significant disability, like our patient, chronic constipation occurs typically, which causes increased indican excretion in the urinary tract, and, in turn, indigo presence. Figure 3: Metabolic steps of tryptophan (TRP). In the presence of bacterial enzymes such as sulphatases and phosphatases, indican forms two isomeric metabolites: indigo blue (indigotin) and indigo red (indirubin). These metabolites dissolve in or deposit on the surface of urine bag material and are responsible for its blue/purple coloring according to the indigotin/indirubin ratio due to oxidative conditions and the pH value of the urine [33; 34; 35]. We found in the patient's urine both Gram+ and Gram\(-\) bacteria, which produce several types of enzymes, including sulphatases, phosphatases, catalase, and oxidase, capable of sticking to the urine bag surface, and growing and building a matrix to form a community named \"biofilm\" [36].