## 2 Materials and Methods  
### Materials  
We studied NIR spectra of salivary biomarkers: L-fucose [36; 45; 46; 57; 58] (L-(-)-Fucose \(\geq\) 99%, Sigma Aldrich, Saint Louis, MO, USA) and L-proline [36; 45] (L-Proline-Reagent Plus\({}^{*}\)\(\geq\) 99%, Sigma Aldrich, Saint Louis, MO, USA), diluted into distilled water. Both substances have a solubility of 50 mg/mL in water. The measurements were repeated in three independent sequences. For each sequence, four solutions were prepared for both biomarkers with a serial dilution protocol as follows: a mass equivalent to the solubility of the substance, 50 mg/mL for both L-fucose and L-proline, was weighed, and then mixed with 1 mL of Millipore-water (18.2 M\(\Omega\)). This means that the solutions with the highest concentrations have 50 mg of substance in 1 mL of water. The concentrations of solutions in mg/mL and \(\mu\)M are presented in Table 1. The aqueous solutions F1 to F3 and P1 to P3 have a higher concentration of L-fucose and L-proline than human saliva while the F4 and P4 solutions have a concentration similar to that of human saliva [45; 46]. [PERSON] et al. [45] determined the concentrations of L-fucose and L-proline in the saliva of healthy controls (n = 30). For L-fucose, the concentrations were 189.1 (100.6-284.7) \(\mu\)M which is about 0.031 (0.016-0.047) mg/mL. For L-proline, the concentrations were 610.1 (318.5-1244.3) \(\mu\)M which is about 0.070 (0.037-0.143) mg/mL. In the study of [PERSON] et al. [46], salivary L-fucose in OPMDs and oral cancer were determined to be 7.02 mg/dl (0.0702 mg/mL) and 11.66 mg/dL (0.1166 mg/mL), respectively, while saliva from healthy controls had only 3.18 mg/dL (0.0318 mg/mL) of L-fucose. No similar study determining the concentration of L-proline in saliva was found. The concentrations of salivary metabolites identified from patients suffering OSCC [45] and their healthy controls [45] are listed in Table 2.  
Reference samples were prepared by pressing pure L-fucose and L-proline powders into 3 mm thick tablets. These reference samples were used as no previous measurements with L-fucose, or L-proline had been performed with the NIRS measurement setup used in this study. The measurements were performed using three tablets of each substance by repeating the measurements six times on each tablet. In this preliminary study, saliva samples of two OSCC [45; 59] patients, a 73-year-old male patient (sample 1) and a 76-year-old male patient (sample 2), were used. Both samples were collected in Brazil (Piracicaba Dental School, University of Campinas). The samples were collected by [PERSON] et al. [59] and further analyzed by [PERSON] et al. [45]. Patients not diagnosed with OSCC were not included. The age range of the patients in the study by [PERSON] et al. [59] was 52-76 years (mean age 61.6 \(\pm\) 9.6 years). The range for healthy controls was 42-74 years (mean age 54.4 \(\pm\) 9.0 years). The saliva samples were collected unstimulated, meaning that the patients passively droed the saliva into a sterile glass cup for 5 min [45]. The clinical characteristics of the patients are described in a study by [PERSON] et al. [45]. Saliva sample 1 has high L-fucose and L-proline concentrations 694 \(\mu\)M (0.106 mg/mL) and 799 \(\mu\)M (0.092 mg/mL), respectively. Saliva sample 2 has low L-fucose and L-proline concentrations 67 \(\mu\)M (0.011 mg/mL) and 157 \(\mu\)M (0.018 mg/mL), respectively. The collection of saliva and its use in the study was approved by the Ethics Committee for Human Studies, Piracicaba Dental School, Brazil (protocol number 142/2010), and written consent was obtained from every participant.