## 8 Homo- and Heteronuclear Two-Dimensional (2D) NMR Analysis of Human Saliva
### Sample Size Considerations and MV Power Calculations
For PC2, however, the bacterial catabolites \(n\)-caproate, \(n\)-butyrate, propionate, acetate, 5-aminovalerate, succinate, and trimethylamine were all strong features, as were 3-D-hydroxybutyrate, acetoin, thymine, choline, alanine, and phenylalanine, along with weaker contributions from _iso_-butyrate, pyruvate, DMA, and one of the N-acetyl storage compounds (N-Ac-3). PC3 contains strong loadings from all salivary malodorous amines, valine, and choline, with less strong ones from glycine, histidine, and formate. Moreover, PC4 contained particularly strong loadings from taurine and methanol, with weaker contributions from _iso_-butyrate, 3-D-hydroxybutyrate, lactate, acetate, thymine, all N-acetylated metabolites, glutamate, glutamine, choline, glycine, histidine, and formate. The co-loading of metabolite variables on two or even more PCs, i.e., perhaps a strong loading on one PC but a somewhat weaker one on another, is not at all uncommon in metabolomics experiments in view of probable heterogeneities in their generating sources and/or pathways involved in their production, i.e., for human WMS, either host or microbial ones, or the relative prevalence of bacteria with saccharolytic and/or proteolytic activities within the oral environment. However, from these values, it should be noted that PC1 is mainly strongly loaded with metabolite variables that certainly appear to arise from a host (parotid salivary/blood plasma) source, whereas those robustly loading on PC2 arise exclusively from the salivary microbiome. Indeed, for PC2, selected bacterially derived organic acid anions such as \(n\)-caproate, \(n\)-and _iso_-butyrate, propionate, and 5-aminovalerate, etc., are only detectable in the \({}^{1}\)H NMR profiles of human saliva and not blood plasma nor parotid saliva (although trace levels of these may be detectable therein using more highly sensitive analytical techniques). Indeed, levels of propionate and \(n\)-butyrate in human saliva range from 6-69 and 0-2.9 mmol./L, respectively [3], whereas blood plasma levels of these are very much lower (mean levels in adults from reported studies were found to be only 0.9 and 1.0 mmol./L, respectively) [47]. Although host metabolic pathways also feature lactate, pyruvate, and succinate, they also load significantly upon the microbiome-sourced PC2 since they are also well-known microbial catabolites. Although acetate is certainly \({}^{1}\)H NMR-detectable in blood plasma, its concentrations therein in healthy controls are very much lower than those found in WMS supernatants (Table 5), and therefore this metabolite appears to be predominantly derived from the oral microbiome (PC2) in this study, as might be expected. Since the amino acids alanine and phenylalanine also load strongly on PC2, it appears that such contributions from these species are derived from bacteria with proteolytic activities. Interestingly, although TMA very strongly loaded on PC2, an observation indicating its microbial source, MA and DMA loaded less and insignificantly so. However, all these amines contributed towards PC3 very significantly, along with the amino acid valine, and therefore it appears that these PC3-loading metabolites all arise from a further, albeit orthogonal, source or pathway, which is not simply explicable. However, choline, which is required for the biosynthesis of TMA, loaded marginally but significantly on both PCs 2 and 3. Both MA and DMA are degradation products of TMA and hence may also originate from a choline source. Upregulation salivary choline possibly arises from the diminished clearance of shed buccal epithelial cells throughout the course of sleep periods [33]. Notably, PC2-loading acetate is also derived from choline metabolism [33],and TMA may also be generated from dietary carnitine [34]; additional precursors for it are phosphatidylcholine and betaine. Notwithstanding, since urea is only relatively weakly loaded onto the host source PC1, and less and not significantly so on PCs 2, 3, and 4, its source remains a subject of conjecture.