## 8 Homo- and Heteronuclear Two-Dimensional (2D) NMR Analysis of Human Saliva
### Sample Size Considerations and MV Power Calculations
Moreover, the salivary biomolecules acetoin, alanine, lactate, pyruvate, and succinate are also upregulated following sucrose exposure [20], and results obtained from the carbohydrate-rich food ingestion experiment reported in Section 6.2 are indeed fully consistent with this observation (Figure 2). Moreover. acetoin acts as an external energy store by selected fermentative bacteria, and therefore its determination in saliva may yield some valuable information on the activities of such species. However, this agent is commonly employed as a food flavoring agent and a fragrance, and is also present in a variety of foods, e.g., blackberries, peppers, butter, and cocoa products, along with beer and wine. The relative contributions of the oral microbiome towards the makeup of the human salivary metabolome and the biomolecular composition of WMS have been a subject of conjecture for many years [146]. However, recently some light has been shed on this subject through comparative evaluations of the biomolecular status of this biofluid to that of sterile parotid gland (SPG) saliva [20; 23; 147]. Indeed, these studies have revealed that the SPG fluid lacks a wide range of metabolites present in WMS before entering the oral cavity, and these included bacterially derived organic acid anions, e.g., acetate, propionate, succinate and _n_- and _iso_-butylrates, and methylamines. Bacterial catabolites such as propionate and _n_- and _iso_-butylrates, are predominantly non-NMR-detectable in human plasma, although these metabolites are sometimes visible to us as low-intensity \({}^{1}\)H NMR resonances in spectra of human urine. The investigation reported by [PERSON] et al. in REF. [20] simultaneously explored matched WMS, parotid saliva (PS), and blood plasma in an attempt to differentiate between patterns of metabolites arising from bacterial loads from those arising endogenously. This study found that the bacterial load of WMS was strongly positively correlated with abundant short-chain organic acid anion concentrations therein, along with malodorous amine and some amino acid levels. However, the results acquired also indicated that citrate, lactate and urea entered WMS through PS and the systemic circulation. Curiously, WMS concentrations of urea were found to be inversely correlated with both saccharolytic and proteolytic sources of bacterial load. Hence, in principle, such NMR-based salivary metabolomics datasets can be employed to determine the bacterial load of WMS samples collected from dental patients, and it is likely that a MV consideration of correlations between its organic acid anion (including lactate and citrate), amine, amino acid, and urea concentrations, may serve to provide valuable microbially relevant data, which in turn may lead to reliable diagnostic and perhaps prognostic stratification information for oral diseases and also possibly for dysbiosis-related conditions, for which such metabolomics datasets may complement those acquired on gastrointestinal fluids. Case Study 2: Salivary Phenotype Analysis, and Identification of Differential Metabolite Pools within WMS Supernatants and Their Sources  
Therefore, in this section, we have conducted a case study to further explore (1) between- and within-participant components of variance amongst the salivary \({}^{1}\)H NMR metabolic profiles of a large number of healthy control sample donors, and (2) perform MV factor analysis on these data in order to isolate orthogonal (uncorrelated) components that arise from differential sources and/or pathways, with a particular focus on those arising from host and oral microbiome origins.