## 5 Salivary Metabolomics: Techniques and Applications
\begin{table}
\begin{tabular}{c c c c c c} \hline \hline
**Metabolomics** & & & & & **Potential Role in** & \\
**Sequencing** & **Methodologies** & **Strengths** & **Limitations** & **Early Diagnosis of** & **References** \\
**Techniques** & & & & & **Periodontitis** & \\ \hline \multirow{6}{*}{NMR Spectroscopy} & Detects nuclear & - High & - Low sensitivity & Reliable for saliva & \\  & magnetic resonance & reproducibility & - Signal overlap & metabolite & \\  & signals (commonly \({}^{1}\)H, & - Non-destructive & - Costly & fingerprinting and \\  & \({}^{13}\)C); non-destructive & - Absolute & instruments & longitudinal & \\  & and quantitative. & - Good for small & - Requires & Useful for SCFAs & \\  & Separates & - High sensitivity & - Good for small & derivatization & and volatile \\  & volatile/derivatized & - Established & - Limited to & microbial & \\  & composums; detects by & - Protocol & volatile analytes & metabolites. & \\  & & Separates & & & \\  & polar/non-polar & - Broad coverage & - Ion suppression & Gold standard for & \\  & metabolites in liquid & - High sensitivity & - Requires complex & untargeted saliva & [58; 59] \\  & phase; detects ions by & - Versatile & QC & metabolomics. & \\  & MS. & & & & \\  & Measures infrared & & & & Distinguishes \\  & absorption of & - Rapid, label-free & - Lower specificity & healthy from & \\ FTIR Spectroscopy & molecular bonds; & - Minimal & - Mostly qualitative & diseased saliva via & [16; 60; 61] \\  & generates spectral & & preparation & - Sensitive to water & biochemical & \\  & fingerprints. & & & & fingerprinting. \\  & Measures acoustic & - Non-invasive & - Lower metabolite & & \\  & waves generated after & - High sensitivity & specificity & Emerging tool for & \\ PAS (Photoacoustic & absorption of & to molecular & - Limited & real-time screening & \\ Spectroscopy) & modulated light; used & vibrations & commercial & of periodontal & \\  & to assess biochemical & - Can be & systems & changes in saliva. & \\  & changes. & miniaturized & - Sensitive to water & & \\ \hline \hline \end{tabular}
\end{table} 
Table 1: Characteristics of metabolomics sequencing techniques. \begin{table}
\begin{tabular}{c c c c c} \hline \hline
**Biomarkers** & **Molecular Categories** & **Biological Pathways** & **Functions and Significance** & **References** \\ \hline  & & \multicolumn{2}{c}{They result from the fermentation of sugars and proteins from anaerobic bacteria (_Porphyromonas_ & Inducing apoptosis of gingival epithelial cells, impairing fibroblast function, suppressing immune cells, promoting dysbiosis of the principal groups of periodontitis metabolites. & [64; 66; 67; 68; 69; 70; 71] \\  & & \multicolumn{2}{c}{They are energy sources for anaerobic bacteria (_Porphyromonas_ & \\  & & \multicolumn{2}{c}  
The affirmation of Dame et al. [64] was that such differences may be due to differences in ethnicity, oral microflora, the time of sample collection, diet, and other associated factors. Concerning the research about variation in metabolic profile during periodontitis, some preliminary studies have investigated it, giving good potential results [42; 79; 80] (Table 3). Among the cited studies, the most recurrent metabolites that were found after metabolomic untargeted analysis are as follows: short-chain organic acids and amino acids, both detected in six studies [40; 42; 64; 74; 79; 80]. Kim et al. [74] identified five periodontal biomarkers in salivary samples. They conducted a study comparing periodontitis patients to healthy controls and subdivided the periodontitis patients according to the severity of the disease (stage II and stage III periodontitis). According to their study, age does not produce any change in salivary metabolomics, and there is no correlation between the clinical severity of pathology and the metabolites; instead, some of them are indicators of disease. The researchers made both discovery and validation for biomarkers, identifying five metabolites: ethanol, taurine, isovalerate, butyrate, and glucose.