## 4 Types of Salivary Metabolomics Experiments
### Abstention from Oral Activities Prior to Saliva Sample Donation: How Long Is Really Necessary?
Notably, significant inter-participant and inter-sampling day variabilities were observed, and these results concur with those reported by us, both previously [3] and in this report (Section 10). Following sample collection, no modifications were observed by these researchers during storage at \(-\)20 \({}^{\circ}\)C for a period of at least 28 days, whereas, as expected, ambient temperature storage gave rise to decreases in the concentrations of selected metabolites. Therefore, the above study concluded that following collection, saliva samples may be stored at ambient temperature, or in the refrigerator at +4 \({}^{\circ}\)C, for periods of up to 6 h, and at \(-\)20 \({}^{\circ}\)C for at least 28 days. When prepared for NMR analysis, samples were found to remain stable at 25 \({}^{\circ}\)C for up to 8 h, and at +4 \({}^{\circ}\)C for up to 48 h Sodium azide addition averted potential early concentration changes in fucose, proline (6-8 h), and xylose (24 h). However, it should also be noted that a number of Gram-positive bacteria, for example _Streptococci_ and _Lactobacillus_, have been reported to be somewhat resistant to the bactericidal actions of sodium azide [106; 112]; these genera are prevalent in cariogenic and saccharolytic dental biofilms. Of critical importance, in 2008, [PERSON] et al. [113] investigated the stability and longevity of the protein content of human WMS in samples provided from the time-point of collection both with and without added inhibitors of bacterial metabolism and proteases. Results acquired showed that protein degradation took place within 30 min of specimen collection and commenced during the actual sample donation process. Although protease inhibitors such as phenylmethylsulphonyl fluoride (PMSF) were found to partially suppress this process, blockers of bacterial metabolism did not. Stable protein deterioration products, which were peptides with molecular masses of 2937, 3370, and 4132 Da, were discovered, and the authors suggested that these may be employed as markers of saliva sample quality. In view of these observations, we should perhaps expect some increases in the salivary concentrations of free amino acids from the point of collection until the analysis of samples; however, with the exception of small increases in salivary proline in samples untreated with sodium azide at ambient temperature or at +4 \({}^{\circ}\)C, the study documented in [60] did not find such storage- and laboratory processing-dependent upregulation. Moreover, we still find evidence for the retention of selected salivary protein species in samples following our standard collection, sample preparation, and storage phases, the latter involving prolonged biobank depository episodes at a temperature of \(-\)80 \({}^{\circ}\)C (Figure 1). Although we may expect species of molecular mass up to 4000 Da to have \({}^{1}\)H NMR resonances with slightly or only negligibly higher \(\Delta\upsilon_{1/2}\) values than those of low-molecular-mass metabolites (ca. 1.5-2.5 Hz), they would certainly not be as large as those of the broad aromatic signals noted in Figure 1 (ca. 55 Hz). In any case, the authors of [113] recommended the freezing of saliva samples immediately following collection, for example, in liquid N\({}_{2}\), in order to minimize protein deterioration. However, the processing of specimens at only +4 \({}^{\circ}\)C, together with the use of protease inhibitors, can also facilitate protein retention therein. Nevertheless, exposure of salivary protein to freeze-thaw episodes may give rise to significant levels of their denaturation, and hence appropriate caution should be applied where this phenomenon is observed. Interestingly, fluoride has the ability to inhibit human salivary amylase activity when present at added levels of \(\geq\)50 mmol./L [114], and this process has been speculated to occur via a mechanism involving its complexation of the critical Ca\({}^{2+}\) ion required for this enzyme's activity. Indeed, this metal ion may form intramolecular cross-links with the protein.