# Metabolic Profiling of Type 2 Diabetes Patients after Bariatric Surgery by Raman Spectroscopy
## 3 Results
### Raman Analysis of Serum of Patients  
In an attempt to identify a metabolic fingerprint, Raman analysis was conducted on serum samples of Ob-T2D patients before (Ob-T2 DM) and after surgery (BS). We compared these results with those obtained from a control group of healthy, average weight subjects (BMI 21.7 \(\pm\) 2.2 kg/m\({}^{2}\)) matched for age and sex (HC). The analysis of the Raman spectra allowed us to obtain a biochemical overview of the components of the biological sample and the relative amount without the need to target specific pre-defined molecules. Figure 3 shows the overlay of normalized average Raman spectra of all samples analyzed for each group. Each of the Raman peaks represents a different functional group in the sample and thus variations in their intensity reflect a difference in the relative amount in serum. Figure 2: Adiponectin, leptin, ghrelin, and FGF-19 level in obese type 2 diabetes mellitus [Ob-T2 DM; \(n=14\)] patients before and after bariatric surgery [BS; \(n=14\)]. Biomarkers levels were measured in the serum of patients and were compared between groups. \(p\)-values were calculated using the Mann–Whitney test. \({}^{*}p<0.05\); \({}^{**}p<0.005\). A Raman analysis, which obtained differential spectra between each group, revealed the most significant biochemical differences between HC, Ob-T2 DM, and BS (Figure 4). The differential spectrum that subtracted HC signals from Ob-T2 DM evidenced a decrease in 1155, 1408 and 1525 cm\({}^{-1}\) peaks and an increase in 1234 and 1659 cm\({}^{-1}\) peak. These peaks remained consistent when we removed HC signals from BS. However, when we attempted to subtract signals of BS samples from Ob-T2 DM, differential spectra resulted in positive peaks at 1155, 1408, 1525, and 1659 cm\({}^{-1}\), while the peak at 1234 cm\({}^{-1}\) was negative. To better understand the significance of these differences, we analyzed each peak individually. Figure 5 shows the statistically significant variations observed between the three groups. Statistically relevant differences were observed in the peaks corresponding to amide III [1234 cm\({}^{-1}\)], amino acids [1408 cm\({}^{-1}\)], carotenoids [1155 cm\({}^{-1}\) and 1525 cm\({}^{-1}\)], and amide I [1659 cm\({}^{-1}\)]. Specifically, higher Amide III peaks [1234 cm\({}^{-1}\)], associated with an increase in \(\beta\)-folded proteins, and less intense Amide I peaks I [1659 cm\({}^{-1}\)], related to a reduction in \(\alpha\)-helix proteins, were evident in BS group when compared with Ob-T2 DM [42]. In addition, bariatric patients were characterized by less intense peaks of carotenoids [1155 cm\({}^{-1}\) and 1525 cm\({}^{-1}\)] in comparison to HC and Ob-T2 DM. These differences were predictable since micronutrient deficiency, including of carotenoids, is common after BS because this procedure causes anatomical and physiological changes to the gastrointestinal tract that may affect nutrient absorption [43; 44]. Other statistically significant differences were also observed at 1408 cm\({}^{-1}\), the peaks related to amino acids [45]. After BS, amino acid peaks increased when compared with Ob-T2 DM patients, despite remaining statistically lower than HC. The metabolic-biochemical overview obtained by RS appeared to prove that a Raman profile of BS group did not overlap with the Raman signature obtained from HC despite clinical anamnestic and biochemical data suggesting an improvement in the parameter analyzed. Figure 3: Average normalized Raman spectra of all serum samples. Grey spectra corresponded to the average of 23 spectra in the HC patients, while the red and blue signals represent the normalized average of 14 spectra collected from Ob-T2 DM subjects before and after BS, respectively. To confirm the altered metabolic profile of the patients after BS, as observed in the statistical analysis of each peak, spectra of all groups were pooled and further analyzed using principal component analysis (PCA).