## 4 Types of Salivary Metabolomics Experiments
### Abstention from Oral Activities Prior to Saliva Sample Donation: How Long Is Really Necessary?
\begin{table}
\begin{tabular}{c c c c c c c} \hline \hline \multirow{2}{*}{**Code**} & \multirow{2}{*}{\begin{tabular}{c} **Disease(s)** \\ **Studied/Notes** \\ \end{tabular} } & \begin{tabular}{c} **Bioanalytical** \\ **Techique(s)** \\ **Employed** \\ \end{tabular} & \begin{tabular}{c} **Sample Sizes:** \\ **Diseased** \\ **(n\({}_{1}\))/Controls (n\({}_{2}\))** \\ \end{tabular} & \
\begin{tabular}{c} **Protocol:** \\ **Pre-Collection** \\ **Abstention/Fasting** \\ **Limit Specified (h)** \\ \end{tabular} & **Reference** & **Date** **Accessed** \\ \hline \multirow{3}{*}{30} & Understanding the \\  & human salivary \\  & metabolome & \({}^{1}\)H NMR &
\begin{tabular}{c} 23 (smokers)/23 \\ (non-smokers) \\ \end{tabular} & 1 & [87] & 20/07/21 \\ \hline \multirow{3}{*}{31} & Females with \\  & temporomandibular disorders & \({}^{1}\)H NMR & 26/27 & 1 & [88] & 20/07/21 \\ \hline \multirow{3}{*}{32} & [PERSON]’s disease & \({}^{1}\)H NMR & 76/37 & 12 & [89] & 20/07/21 \\ \hline \multirow{3}{*}{33} & Salivary \\  & metabolomics & UHPLC-HR-MSLA- \\  & metabolomics & REI-MS & na/140 & 3 & [90] & 20/07/21 \\ \hline \multirow{3}{*}{34} & Effect of experimental \\  & Effect of experimental \\  & gingivitis induction \\  & and erythroid \\ \hline \multirow{3}{*}{35} & [PERSON]’s syndrome. & \({}^{1}\)H NMR & 14/15 & 1 & [92] & 20/07/21 \\ \hline \multirow{3}{*}{36} & Evaluation of sport \\  & performance & \({}^{1}\)H NMR & na/14 & 3 & [93] & 20/07/21 \\ \hline \multirow{3}{*}{37} & Head and neck cancer & \({}^{1}\)H NMR & 45/30 & 1.5 & [94] & 20/07/21 \\ \hline \multirow{3}{*}{38} & Oral cancer & CE-TOF-MS & 26/44 & 1.5 & [95] & 20/07/21 \\ \cline{2-6}  & Pediatric obesity- \\  & related liver disease \\  & and metabolic \\  & syndrome & & & & & \\ \hline \hline \end{tabular}
\end{table} 
Table 2: Cont. In order to achieve an optimal protocol for the post-collection transport (if required) and storage of WMS supernatants, a series of potential interfering and metabolite level-modifying factors should be considered in detail. Firstly, it should be noted that selected biomolecules are susceptible to oxidation on exposure to O2, some substantially so. Thus, free (non-protein-incorporated) salivary thiols (estimated mean concentrations of free cysteine, cysteineylglycine and glutathione (GSH) are \(\leq\)2, \(\leq\)0.4, and \(\leq\)3 umol./L in human saliva [99]) readily oxidize to their corresponding disulfides, the overall process depicted in Equation (1) involving the generation of thiyl radical (RS\({}^{\bullet}\)) and superoxide anion (O2\({}^{\bullet-}\)) intermediates, as does any tissue-derived GSH. However, NMR-based metabolomics researchers need not be too concerned about these processes, since these levels are at or beyond lower limits of quantification (LLOQ) thresholds, and cysteine resonances appear as a resonance height-diminishing complex ABX coupling pattern; such low intensity signals are generally strongly overlapped by those of higher concentration metabolites in the spectral regions involved in any case. However, the generation of any reactive oxygen species (ROS) from such autoxidation reactions, e.g., O2\({}^{\bullet-}\), hydrogen peroxide (H2O2) and ultimately hydroxyl radical (\({}^{\bullet}\)OH), may cause some oxidative damage to other, albeit ROS-scavenging biomolecules. Moreover, salivary pyruvate, and other 2-oxo-carboxylate species such as \(\alpha\)-ketoglutate can be oxidatively decarboxylated by H2O2 directly, with pyruvate being transformed to acetate and CO2 (Equation (2)) [100], and this directly relates to observations of relatively small but nevertheless significant increases in acetate level that we commonly observe following prolonged storage regimens, as have other researchers when samples are stored at 22 \({}^{\circ}\)C for 48 h [60]; however, acetate, along with formate, appear to also be terminal products arising from the radical-mediated decomposition or photodegradation of a wide range of organic biomolecules [101].